Structurally different mono-, bi- and trinuclear Pd(II) complexes and their DNA/protein interaction, DNA cleavage, and anti-oxidant, anti-microbial and cytotoxic studies

Article abstract of DOI:10.1039/c7nj01556a

A series of new structurally different Pd(ii) complexes was obtained by the reactions between K₂[PdCl₄], 3-methoxysalicylaldehyde-4(N)-substituted thiosemicarbazone [H₂L¹-H₂L⁴] and bis(diphenylphosphino)ethane [dppe]. All the complexes were characterized by using various spectral techniques (IR, electronic, ¹H-NMR and mass spectroscopy). The crystal structures of complexes 1, 3, 4 and 3a & 4a were determined using X-ray crystallographic technique. The interactions of the new complexes with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) were evaluated using various spectroscopic methods, which showed the interaction potential of the complexes with CT-DNA and BSA. Further, they cleaved supercoiled DNA pBR322. The anti-oxidant profiles of the complexes were found to be better than the standards against 2,2′-diphenyl-1-picrylhydrazyl radicals (DPPH) and superoxide anion radicals. The antibacterial activity of the complexes against five pathogenic bacteria such as Staphylococcus aureus, Salmonella typhi, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa was found to be better than their parent ligands. The complexes exhibited potential cytotoxicity against human breast cancer cells (MCF-7) in the following order: 4 > 3 > 1 > cisplatin > 2.

Full text of DOI:10.1039/c7nj01556a

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
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Structurally different mono, bi and trinuclear Pd(II) complexes and their
DNA/Protein interaction, DNA cleavage, antiꢀoxidant, antiꢀmicrobial and
cytotoxicity studies
A. Shanmugapriya,aRuchiJain,bD. Sabarinathan,cG. Kalaiarasi,aF. Dallemerd and
R. Prabhakarana*
aDepartment of Chemistry, Bharathiar University, Coimbatore 641 046, India
bDepartment of MRDG, Indian Institute of Science, Bangalore 560012, India
cDepartment of Microbial Biotechnology, Bharathiar University, Coimbatore- 641 046, India
dLab MADIREL CNRS UMR 7246, Aix Marseille University, Saint-Jerome Campus,
MADIREL Bldg., 13397 Marseille Cedex 20, France
Abstract
A series of new structurally different Pd(II) complexes were obtained from the reactions
between K2[PdCl4], 3ꢀmethoxysalicylaldehydeꢀ4(N)ꢀsubstituted thiosemicarbazone [H2L1ꢀ
H2L4] and bis(diphenylphosphino)ethane [dppe]. All the complexes were characterized by
1
various spectral techniques (IR, electronic, HꢀNMR and mass spectroscopy). The crystal
structures of the complexes 1, 3, 4 and 3a & 4a have been determined by Xꢀray
crystallographic technique. The interaction of new complexes with calf thymus DNA (CTꢀ
DNA) and bovine serum albumin (BSA) have been evaluated by various spectroscopic
methods, which showed the interaction potential of the complexes with CTꢀDNA and BSA.
Further, they cleaved supercoiled DNA pBR322. Antiꢀoxidant profile of the complexes found
better than the standards against 2,2'ꢀdiphenylꢀ1ꢀpicrylhydrazyl radical (DPPH) and
superoxide anion radicals. Antibacterial activity of the complexes against five pathogenic
bacteria such as Staphylococcus aureus, Salmonella typhi, Escherichia coli, Bacillus subtilis
and Pseudomonas aeruginosa found better than their parent ligands. The complexes
exhibited potential cytotoxicity against human breast cancer cells (MCFꢀ7) in the following
order 4>3>1>cisplatin>2.
Keywords: Palladium(II) thiosemicarbazone; spectroscopy; Xꢀray crystallography; DNA
binding and DNA cleavage; BSA protein binding; antiꢀoxidant; antiꢀmicrobial; cytotoxicity.
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
Corresponding author. Tel.: +91ꢀ422ꢀ2428319; Fax: +91ꢀ422ꢀ2422387.
Eꢀmail address: rpnchemist@gmail.com (R. Prabhakaran)

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1 Introduction
Metal based compounds offer substantial potential in medicinal chemistry where the
meticulous choice of metal may afford compounds possessing coordination, geometrical and
catalytic properties. The most wellꢀknown metalꢀbased anticancer drug cisplatin arose from
serendipity.1,2Globally, the demand for platinumꢀbased drugs has grown steadily, although
several side effects ranging from nephrotoxicity to drugs resistance of tumour cells.3ꢀ5 Side
effects associated with cisplatin administration, along with limited applicability arising from
specificity shown towards cancer cell lines. One of the alternatives that have shown
considerable promise has been the development of other transition metal based drugs.6ꢀ8 The
combination of transition metals with biologically active molecules has been exploited
showing promising activity due to their unique ability to bind with biological
targets.9,10Palladium(II) complexes are good alternative candidates for metalꢀorganic drugs
due to their thermodynamic and
structural similarities to platinum(II)
complexes.11,12Thiosemicarbazones and their metal complexes have more consideration in the
area of medicinal chemistry, due to their pharmacological properties.13,14The present level of
interest in metal complexes of thiosemicarbazone stems from the fact that the biological
activities are frequently ascribed to the chelation of thiosemicarbazones to a metal ion.15ꢀ
17Metal complexes of salicylaldehydethiosemicarbazones have been considered for their
antitumor activity.18,19 Das and Livingstone suggested that sulphur containing ligands
chelated to palladium(II) are better antitumor agents than those of other metals, as the
palladium(II) chelates possess the proper liability to transport the metal to DNA, its primary
target.20A number of palladium(II) thiosemicarbazone complexes have been synthesised and
examined for their potential as antitumor agents.21ꢀ23Bisphosphines are important ligand
backbones
exhibiting
differential
bite
angle
upon
metal
coordination,
diphenylphosphinoethane (dppe) provides both mono and bidentate coordination mode.24
These ligands were recently employed in the development of antitumor cyclopalladated
complexes.25The cytotoxicity of 1,2ꢀbis(diphenylphosphino)ethane copper(I) complexes was
discovered and the mechanism of activity was also proposed.26 The fact that DNA and
protein biomolecules are electronꢀrich and metal ions electronꢀdeficient compounds to have
strong interactions between metal ions and biomolecules. Therefore, the study of the binding
stuffs of metal complexes with DNA and protein is of great importancein the designing the
new drugs and their application.27 Metal based antioxidants have received recent
responsiveness for their capacity to protect organisms and cell damage induced by oxidative
stress.28In our current article, we attempted to synthesis palladium(II) complexes containing

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3ꢀmethoxysalicylaldehydeꢀ4(N)
substituted
thiosemicarbazone
[H2L1ꢀH2L4]
and
diphenylphosphinoethane [dppe] in dichloromethaneꢀmethanol medium. Surprisingly, the
reactions afforded there structurally different products from four of the reactions. The
products were separated and characterised by various spectral, analytical methods. Further,
the structures of them were proved by Xꢀray crystallographic technique. The new complexes
were subjected to study their potential DNA/protein binding, DNA cleavage, antioxidant,
antimicrobial and cytotoxicity.
2 Experimental
1,2ꢀbis(diphenylphosphino)ethane was purchased from Sigma Aldrich Ltd. The ligands
[H2L]1ꢀ4, palladium precursor K2[PdCl4]and palladium(II) complexes were synthesized
according to the standard literature procedures.17a,29,30 All the reagents used were analargrade,
were purified and dried according to the standard procedure.31 Protein free calf thymus DNA
(CTꢀDNA), Ethidium bromide (EB), Bovine serum albumin (BSA) 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀ
yl)ꢀ2,5ꢀdiphenylꢀtetrazoliumbromide (MTT)were purchased from Hi Media. Double distilled
water was used to prepare Tris–HCl buffer and phosphate buffer. Infrared spectra were
measured as KBr pellets on a JASCO FTꢀIR 4100 instrument between 400–4000 cm−1at the
Department of Chemistry, Bharathiar University, Coimbatore, India. Melting points were
measured in a Lab India apparatus. The electronic spectra of the complexes were recorded in
dichloromethane using a JASCO Vꢀ630 Spectrophotometer in the 800ꢀ200 nm range.
1
Emission spectra were recorded by using JASCO FPꢀ6600 Spectrofluorimeter. HꢀNMR
spectra were recorded in DMSO at room temperature with a Bruker 400 MHz instrument,
chemical shift relative to tetramethylsilane. The mass spectrum of the complex 2 was
recorded in DMSO using Waters QꢀTof Micro instrument. Single crystal data collection and
correction for the new Pd(II) complexes 1, 3 and 4 were done at 293 K with CCD kappa
diffractometer using graphite mono chromated Mo Kα (λ = 1.54184 Å) radiation.32 The
structural solution were done by using SHELꢀXTLꢀ9733and refined by full matrix least square
on F2 using SHELꢀXTLꢀ97.34
2.1 Preparation of [(Pd2(Msalꢀtsc)2)(µꢀdppe)] (1)
0.086 g of 3ꢀmethoxysalicylaldehyde 4(N)ꢀthiosemicarbazone [H2ꢀMsalꢀtsc] (0.3063 mmol)
was dissolved in dichloromethane (30 cm3) and added to K2[PdCl4] (0.100 g, 0.3063 mmol)
in hot methanol (30 cm3). The mixture was refluxed for 10 min. To this 0.061 g of
1,2ꢀbis(diphenylphosphino)ethane (0.1531 mmol) was added. After 5 hours refluxing, the

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reaction mixture was allowed to stand for 3 days at room temperature. A reddish brown solid
formed was subjected to thin layer chromatography and a red band was eluted with benzene
:methanol (95:5). The reddish brown solid obtained was recrystallized from toluene/
methanol to yield brown crystals of (1).
Yield: 59 %, M.p. 187 ºC. FTꢀIR (cmꢀ1) in KBr: 1623 (νC=N), 1301 (νCꢀO), 733 (νCꢀS), 1455,
1024, 696 cmꢀ1 (for PPh3); UVꢀVis (CH2Cl2), λmax (nm) (dm3 molꢀ1 cmꢀ1): 244 (46,918) (intraꢀ
1
ligand transition); 304 (20,722), 339 (16,289), 398 (7,614) (LMCT); HꢀNMR (DMSOꢀd6,
ppm, J= Hz): δ 8.219ꢀ8.254 (t, (J= 8), CH=N), δ 6.567ꢀ6.604 (d, (J= 14.8), C4ꢀH), δ 6.502ꢀ
6ꢀ541 (t, (J= 7.8), C5ꢀH), δ 6.824ꢀ6.840 (d, (J= 6.4), C6ꢀH), δ 7.033ꢀ7.051 (d, (J= 7.2), ꢀ
NH2), δ 3.75 (s, OCH3), δ 2.48 (m, (ꢀCH2ꢀ)2), δ 7.220ꢀ7.610 (m, dppe protons).
2.1.1Preparation of [(Pd2(Msalꢀmtsc)2)(µꢀdppe)] (2)
Complex
2
was prepared by the procedure as described for (1), with
3ꢀmethoxysalicylaldehyde 4(N)ꢀmethylthiosemicarbazone [H2ꢀMsalꢀmtsc] (0.085 g, 0.3063
mmol), K2[PdCl4] (0.100 g, 0.3063 mmol) and 1,2ꢀbis(diphenylphosphino)ethane (0.061 g,
0.1531 mmol). A reddish brown solid formed was subjected to thin layer chromatography
and a red band was eluted with benzene : methanol (95:5). The reddish brown solid obtained
was recrystallized from chloroform / methanol to yield brown crystals of (2).
Yield: 60 %, M.p. 196 ºC. FTꢀIR (cmꢀ1) in KBr: 1592 (νC=N), 1306 (νCꢀO), 723 (νCꢀS), 1452,
1027, 671 cmꢀ1 (for PPh3); UVꢀVis (CH2Cl2), λmax (nm) (dm3 molꢀ1 cmꢀ1) : 241 (57,488)
(intraꢀligand transition); 313 (21,051), 347 (15,607), 393 (7,780) (LMCT); 1H NMR (DMSOꢀ
d6, ppm, J= Hz): δ 8.295ꢀ8.316 (t, J= (4.4), CH=N), δ 6.688ꢀ 6.705 (d, (J=6.8), C4ꢀH), δ
6.483ꢀ 6.522 (t, (J=7.8), C5ꢀH), δ 6.962ꢀ 6.982 (d, (J=8), C6ꢀH), δ 6.878ꢀ6.890 (q, –NHCH3),
δ 3.39 (s, –OCH3), δ 2.670ꢀ2.682 (d, (J=4.8), –CH3), δ 2.775 (s, (–CH2–)2), δ 7.325ꢀ 7.593
(m, dppe protons).
2.1.2 Preparation of complex (3)
Complex
3
was prepared by the procedure as described for (1), with
3ꢀmethoxysalicylaldehyde 4(N)ꢀethylthiosemicarbazone [H2ꢀMsalꢀetsc] (0.090 g, 0.3063
mmol), K2[PdCl4] (0.100 g, 0.3063 mmol) and 1,2ꢀbis(diphenylphosphino)ethane (0.061 g,
0.1531 mmol). The resulting reddish orange solution was concentrated. It was subjected to
thin layer chromatography where two spots were identified and isolated by silica gel column
chromatography using benzene–methanol solvent mixture. The light yellow band was eluted

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with 98:2 benzene–methanolwhich afforded yellow crystals of 3a. Followed by 3a, complex
3 was isolated from an orange band by using 90:10 benzene: methanol as eluent and was
crystallized by using chloroformꢀethylacetate mixture to afforded yellowish orange crystals.
[PdCl2(dppe)] (3a). Yield: 21 %,M.p. >250 ºC.Anal.Calcd for C26H24Cl2P2Pd: C, 54.24; H,
4.20. Found: C, 54.17; H, 4.12 %. FTꢀIR (cmꢀ1) in KBr:1456, 1092, 694 cmꢀ1 (for PPh3);
UVꢀVis (DMSO), λmax (nm) (dm3 molꢀ1 cmꢀ1): 272 (22,251) (intraꢀligand transition); 326
(12,020) (LMCT);1HꢀNMR (DMSOꢀd6, ppm): δ 2.34 (s, (–CH2–)2), δ 7.140ꢀ7.288 (m, dppe
protons).
[Pd3(µꢀSꢀMsalꢀetsc)3] (3).Yield: 38 %, M.p. 192 ºC. FTꢀIR (cmꢀ1) in KBr: 1596 (νC=N), 1307
(νCꢀO), 727 (νCꢀS); UVꢀVis (CH2Cl2), λmax (nm) (dm3 molꢀ1 cmꢀ1): 252 (21,868) (intraꢀligand
transition); 319 (13,446) (LMCT); 404 (3,889) (MLCT);1HꢀNMR (DMSOꢀd6, ppm, J= Hz): δ
8.177 (s, CH=N), δ 6.543ꢀ6.582 (t, (J= 7.8), C4ꢀH), δ 6.912ꢀ6.934 (q, C5ꢀH), δ 7.091ꢀ7.112
(t, (J= 4.2), C6ꢀH), δ 7.189ꢀ7.214 (t, (J=5), ꢀNHC2H5), δ 3.750 (s, OCH3), δ 2.486ꢀ2.495 (t,
(J= 3.6), CH2), δ 1.077ꢀ1.095 (t, (J= 7.2), CH3).
2.1.3 Preparation of complex (4)
Complex
4
was prepared by the procedure as described for (1), with 3ꢀ
methoxysalicylaldehyde 4(N)ꢀphenylthiosemicarbazone [H2ꢀMsalꢀptsc] (0.104 g, 0.3063
mmol), K2[PdCl4] (0.100 g, 0.3063 mmol) and 1,2ꢀbis(diphenylphosphino)ethane (0.061 g,
0.1531 mmol). The resulting red coloured solution was concentrated. It was subjected to thin
layer chromatography where two spots were identified and isolated by silica gel column
chromatography using benzene–methanol solvent mixture. The first yellow band was isolated
using a 98:2 benzene–methanol solvent as the eluent which afforded yellow crystals 4a.
Followed by 4a, complex 4 was isolated from a red band by using benzene : methanol (95:5)
as eluent and the obtained orange red solid was recrystallized from chloroformꢀmethanol
mixture to yield red crystals of complex 4.
[PdCl2(dppe)] (4a). Yield: 25 %,M.p. >250 ºC.Anal.Calcd for C26H24Cl2P2Pd: C, 54.24; H,
4.20. Found: C, 54.17; H, 4.12 %. FTꢀIR (cmꢀ1) in KBr:1456, 1092, 694 cmꢀ1 (for PPh3);
UVꢀVis (DMSO), λmax (nm) (dm3 molꢀ1 cmꢀ1): 272 (22,251) (intraꢀligand transition); 326
(12,020) (LMCT);1HꢀNMR (DMSOꢀd6, ppm): δ 2.34 (s, (–CH2–)2), δ 7.140ꢀ7.288 (m, dppe
protons).

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[Pd(Msalꢀptsc)(Msalꢀptaz)] (4). Yield: 63 %, M.p. 202 ºC. FTꢀIR (cmꢀ1) in KBr: 3384 (νOH),
3062 (νNꢀH), 1596 (νC=N), 1316 (νCꢀO), 816 (νC=S), 730 (νCꢀS); UVꢀVis (CH2Cl2), λmax (nm)(dm3
molꢀ1 cmꢀ1): 243 (36,935), 265 (30,997) (intraꢀligand transition); 351(9,373) (LMCT); 405
(3,964) (MLCT); 1HꢀNMR (DMSOꢀd6, ppm, J= Hz): δ 11.45 (s, ꢀOH), δ 6.714ꢀ6.811 (m, C4ꢀ
H & C5ꢀH), δ 7.018ꢀ7.032 (t, (J= 5.6), C6ꢀH), δ 8.533 (s, 1H, CH=N), δ 3.968 (s, 3H, OCH3),
δ 9.439 (s, N(2)HC=S), δ 7.670ꢀ7.689 (d, (J=7.6), NHPh), δ 7.092ꢀ7.463 (m, phenyl protons).
2.2 DNA binding studies
DNA binding studies have been carried out according to the method described in the earlier
reports.17Various concentrations of CTꢀDNA (0.5ꢀ5 µM) in TrisꢀHCl buffer (pH 7.2) were
treated with complexes (1ꢀ4) (10 µM) in 1 % aqueous DMSO. The detailed procedures for
DNA binding experiment was provided in the Supporting Information.
2.2.1 Competitive binding with ethidium bromide
Ethidiumbromide studies have been carried out according to the earlier reported
methods.16,17The detailed procedures for DNA binding experiments were provided in the
Supporting Information.
2.2.2 DNA cleavage studies
The cleavage of DNA was monitored by using agarose gel electrophoresis35 and the detailed
producer was given in supporting information.
2.3 Bovine serum albumin binding studies
Protein binding studies were done according to the earlier reported methods16,17and were
given in the Supporting Information.
2.4. Evaluation of antioxidant activity
The potential antioxidant activity of new palladium(II) complexes was evaluated by DPPH
and superoxide anion radicalꢀscavenging assaysby using standard literature methods.36,37
Further reductive ability of the complexes were assayed by Oyaizu method.38 Further,
estimation of total antioxidant capacity of the complexes has been done by
phosphomolydenum method.39The experimental procedures for above said methods were
given in the Supporting Information.

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2.5 Antibacterial activity studies
The palladium(II) complexes (1ꢀ4) have been screened for their antibacterial activities against
various pathogenic bacteria Staphylococcus aureus, Salmonella typhi, Escherichia coli,
Bacillussubtilis, Pseudomonas aeruginosa and Klebsiellapneumoniae by disc diffusion
method40 and were given in supporting information.
2.6 Cytotoxicity studies
2.6.1 3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) assay41
Cytotoxic activity of new palladium(II) complexes have been evaluated and the IC50 values
were obtained from nonlinear regression using GraphPad Prism 5.42 The experimental
procedure was given in supporting information.
3 Results and discussion
3.1. Synthesis of new palladium(II) complexes
The
reaction
of
two
equivalents
3ꢀmethoxysalicylaldehydeꢀ4(N)ꢀsubstituted
thiosemicarbazone (H2L)1ꢀ4 with two equivalents K2[PdCl4] and one equivalent 1,2ꢀbis
(diphenylphosphino)ethane [dppe] in 1:1 methanolꢀdichloromethane resulted in the formation
of new complexes (Scheme 1). The structure of the complexes (1, 3 and 4) was confirmed by
Xꢀray crystallographic studies. The complex [PdCl2(dppe)]obtained along with complex 3
and 4 was isolated by column chromatography with 98:2 benzene: methanol as eluent. The
identity of the complex has been confirmed by elemental analysis, IR, UVꢀVis and NMR
spectroscopic studies and left without further characterisation. Further, the complexes are
soluble in common organic solvents such as dichloromethane, chloroform, ethanol, methanol,
dimethylformamide and dimethylsulfoxide.

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OCH3
OH
S
K2[PdCl4]
PPh2(CH3)2PPh2
N
N
NHR
H
R= H, CH3, C2H5, C6H5
2:2:1
MeOH/CH2Cl2
5hrs
4) R=C6H5
1) R=H
2) R=CH3
3) R=C2H5
OCH3
C2H5
N
S
H3CO
NH
N
HO
RHN
Pd
O
N
PPh2
N
O
C
OCH3
N
S
N
Pd
OCH3
O
N
N
N
OCH3
S
H
S
PPh2
O
S
H3CO
HN
C2H5
Pd
N
S
O
Pd
S
Pd
N
NH
HN
O
NHR
N
N
C2H5
OCH3
N
N
HPh2
P
Ph2
P
Cl
Cl
Cl
Cl
Pd
Pd
P
P
Ph2
Ph2
4a
3a
Scheme1. Synthesis of new palladium(II) complexes
3.2 Spectroscopic studies
The IR spectra of the ligands (H2L)1ꢀ4 and the corresponding complexes (1ꢀ4) provided
substantial information about the metal ligand bonding (Fig. S1ꢀS5). Strong stretching
frequency observed at 1593ꢀ1539 cmꢀ1 in the ligands corresponding to ν(C=N) was shifted to
1626ꢀ1597 cmꢀ1 in all complexes (1ꢀ4) indicating the coordination of azomethine nitrogen to
metal atom.43 A band in the region 3310ꢀ3457 cmꢀ1 due to the presence of –OH group in the
free ligands (H2L)1ꢀ3 was completely disappeared in the IR spectra of the new complexes (1ꢀ
3) indicating the coordination of phenolic oxygen to palladium after deprotonation. This was
further supported by increase in the phenolic CꢀO stretching frequency from 1260ꢀ1275 to
1307ꢀ1317 cmꢀ1.16b However, in the complex 4, a broad peak corresponding to ν(OH)at 3382
cmꢀ1, suggested that non participation of phenolic OH in bonding. A sharp band observed at

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772ꢀ788 cmꢀ1ascribed to ν(C=S) in the ligands (H2L1ꢀ3) was completely disappeared in the
spectra of new complexes (1ꢀ3) and the appearance of a new band around 726ꢀ727 cmꢀ1 due
to ν(CꢀS) indicating the coordination of the sulphur atom after enolisation followed by
deprotonation.44,17b Whereas in complex 4, there were two signal at 827 cmꢀ1and 728 cmꢀ1,
corresponding to ν(C=S) and ν(CꢀS) respectively and a peak at 3026 cmꢀ1corresponding to NH
stretching added more possibility to predict the presence of thione sulphur. In addition, the
absorption bands in the region 1434 and 1456 cmꢀ1, 1094 and 1097 cmꢀ1, 696 and 689
cmꢀ1 confirms the presence of (diphenylphosphino)ethane in the complexes 1 and 2
respectively.17b However, such signal were absent in the complexes 3 and 4 suggested the
nonꢀparticipation of (diphenylphosphino)ethane in coordination with the metal ions. The
electronic spectra of the complexes (1ꢀ4) have been recorded in chloroform and they
displayed bands in the region around 241ꢀ400 nm (Fig. S6). The bands that appeared at
region 241ꢀ252 nm have been assigned to intra ligand transition and the bands around 304ꢀ
400 nm have been assigned to ligand to metal charge transfer (LMCT) transition.45
1
In the HꢀNMR spectra of ligands (H2L1ꢀ3), a singlet appeared around δ 9.13ꢀ9.14ppm has
been assigned to N(2)HCS proton.46 However, in the spectra of new complexes (1ꢀ3), there
was no resonance attributable to N(2)HCS, indicating the coordination of sulphur in the
anionic form after deprotonation at N(2) (Fig. S7ꢀS11). A sharp singlet appeared at δ 11.34ꢀ
11.40 ppm corresponding to the phenolic –OH group in the free ligands (H2L1ꢀ3)was
completely disappeared in complexes 1ꢀ3confirming the involvement of phenolic oxygen in
coordination.43In complexes 1and 2, a triplet appeared at δ 8.21 and δ 8.29 ppm
corresponding to the azomethine group which may be due to the nuclear quadrupolar effect of
the nitrogen atom44 and in complex 3, a singlet appeared at δ 8.17 for azomethine (CH=N)
proton.16b Moreover in complexes 1 and 2, a multiplet was observed at δ 2.48 andδ 2.77 ppm
respectively due
to the
presence of ethylene protons of
the
1,2ꢀ
bis(diphenylphosphino)ethane.24A sharp singlet corresponding to the phenolic –OH proton
appeared at δ 11.76 ppm in the free ligand H2L4 and the complex 4, the appearance of a
singlet at δ 11.45 ppm indicating the nonꢀparticipation of the phenolic oxygen in
coordination.44 The spectrum of H2L4 showed a sharp singlet atδ 10.00 ppm analogous to
N(2)HCS proton. However, this signal completely disappeared and a new singlet appeared at
δ 9.43 ppm in complex 4 indicating the coordination of the thione form of the sulphur to the
palladium ion.16aIn complex 4, a singlet observed at δ8.53 ppm was assigned to
azomethine(CH=N) proton.17b

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3.3 Xꢀray crystallography
In order to confirm the exact structure, Xꢀray crystallographic studies were done for the new
complexes (1, 3,4and 3a & 4a). The ORTEP diagrams of the complexes are given in Fig. 1ꢀ4.
The crystallographic data, selected bond distances and bond angles are listed in Table S1 and
S2aꢀS2c.Single crystals of [Pd2(Msalꢀtsc)2(µꢀdppe)] (1) complex crystallised in the triclinic
space group Pꢀ1(Fig. 1). In the dimeric complex (1), each palladium atom is coordinated
with a binegative tridentate ligand through phenolic oxygen, N1 hydrazinic nitrogen and
thiolate sulphur atom by forming one six member and another five member ring with a bite
angle of 84.47(7)°[S1ꢀPd1ꢀN1]. The remaining coordination site is occupied by the
phosphorous atom of 1,2ꢀbis(diphenylphosphino)ethane. Inspection of the angles formed
between the metal and the coordinated atoms showed that the metal complex is slightly
distorted from squareꢀplanar geometry. The angles [N1ꢀPd1ꢀS1] 84.47(7)°and [O1ꢀPd1ꢀP1]
84.12(6)° formed between the thiosemicarbazone ligand and the metal are less than 90°. The
angles [O1ꢀPd1ꢀN1] 93.85(9)°and [S1ꢀPd1ꢀP1] 97.58(3)° are therefore greater than 90°.
Collectively the bite angles observed in the molecular structure compare favourably with
those of analogous palladium(II) complexes.47The Pd1ꢀO1 bond distance 2.008(2)Å, PdꢀN1
bond distance 2.023(2)Å and Pd1ꢀS1 bond distance 2.233(8)Å were found as similar to the
reported values.17b,47,48 The strong trans influence of the phosphorous atom is reflected in the
PdꢀN1 bond distance which is slightly longer than the expected length calculated from the
covalent radii of palladium and nitrogen (2.01 Å).49 The trans angles [P1ꢀPd1ꢀN1] and [O1ꢀ
Pd1ꢀS1] were found as 177.52(1)° and 178.29(6)° indicating the considerable deviation from
the ideal symmetry and significant distortion around square planar palladium ion.47,48 Two of
these symmetrical units are bridged through dppe ligand in the complex 1. Complex
1exhibited an intermolecular hydrogen bonding between hydrogen atom H(3A) of amino
group N(3)ꢀH(3A) with hydrazinic nitrogen atom N(2) of a second molecule. In addition, the
hydrogen atom H(3A) of amino group N(3)ꢀH(3A) in the second molecule is involved in
intermolecular hydrogen bonding with hydrazinic nitrogen atom N(2) of the first one. This
intermolecular hydrogen bonding leads to an unremitting 1D chain formation (Fig. S13 and
Table S3).

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Fig. 1. ORTEP diagram of [(Pd2(Msalꢀtsc)2)(µꢀdppe)] (1)
The palladium complex 3 was crystallized by chloroformꢀethylacetate mixture. From the unit
cell dimensions, it is clear that the complex3 is crystallised in triclinic system with Pꢀ1 space
group (Fig. 2). The complex 3 consists of sixꢀmember ring of alternating Pd(II) and sulphur
atoms in a chairꢀlike configuration. The remaining two sites of each square planar Pd(II)
centres are occupied by the hydrazinic nitrogen and the phenolic oxygen. The whole structure
formed like a bowl type with (3Pdꢀ3S) three palladium(II)ꢀsulphur ring as the base. This type
of complex has been reported previously for palladium complexes.49 The distance between
the palladium centres [Pd1ꢀPd2] 3.376 Å; [Pd2ꢀPd3] 3.887 Å and [Pd3ꢀPd1] 4.106 Å indicate
that there is no direct bond between the palladium atoms.50The three bond angles involving
bridging sulphur atoms are as follows: [Pd1ꢀS1ꢀPd2] 95.56(3)°; [Pd2ꢀS2ꢀPd3] 117.53(4)° and
[Pd3ꢀS3ꢀPd1] 127.34(4)°. The PdꢀO bond distances 2.003(3) to 2.014(3) Å and PdꢀN bond
distances 1.988(3) to 1.994(3)Å and PdꢀS bond distances 2.244(10) to 2.336(10)Å are
similar to those found in other palladium complexes.50 An intramolecular N(9)ꢀH(9)ꢀO(1)
hydrogen bonding between the terminal nitrogen N(9) and phenolic oxygen O(1) of the
ligand and Complex 3 also exhibited an intermolecular N(3)ꢀH(3)ꢀO(7) hydrogen bonding
between the terminal nitrogen N(3) and oxygen atom of the ethyl acetate moiety which is
present in the crystal lattice (Fig. S14 and Table S3).

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Fig. 2. ORTEP diagram of [Pd3(µꢀSꢀMsalꢀetsc)3] (3)
Though the spectroscopy characterisations of the complex 4 gave some idea about the
composition of the complex, where they failed to indicate the definite binding mode of the
thiosemicarbazone. Hence, the identity of the complex 4 was determined by Xꢀray
crystallography. The unit cell dimensions clearly showed that the crystal of complex 4 is
monoclinic belonging to P21/c space group (Fig. 3). The structure of the complex 4consistsof
ONS chelated thiosemicarbazone ligand and the fourth coordinate site is occupied by sulphur
atom
of
5ꢀ(2ꢀhydroxyꢀ3ꢀmethoxyꢀphenyl)ꢀ4ꢀphenylꢀ2,4ꢀdihydro[1,2,4]triazoleꢀ3ꢀthione
which is formed by the cyclisation of the H2L4 ligand. The [Pd1ꢀO1] bond distance
2.0337(16) Å, [Pd1ꢀN1] bond distance 1.9911(18) Å and [Pd1ꢀS1] bond distance 2.2327(7) Å
and [Pd1–S2] bond distances of 2.3167(6) Å were found as similar to the reported
values.49Theelongation of Pd(1)ꢀS(2) bond may be due to the strong trans influence of N(1)
nitrogen on thione sulphurꢀpalladium in Pd(1)ꢀS(2). This is further evident from the shorter
bond distance of (1.698(2) Å) C16ꢀS2 bond of coordinated ligand.51The trans angles [N1ꢀ
Pd1ꢀS2] and [O1ꢀPd1ꢀS1] were found as 170.54(6)° and 176.81(5)° respectively indicating
the significant distortion around square planar palladium ion.47

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Fig.3. ORTEP diagram of [Pd(Msalꢀptsc)(Msalꢀptaz)] (4)
Single crystals of [PdCl2(dppe)] (3a and 4a) complex crystallised in the monoclinic space
group P1 21/C1(Fig. 4). In this complex, palladium atom is coordinated with two chloride
atoms and third and fourth site was occupied by phosphours atom of 1,2ꢀ
bis(diphenylphosphino)ethane. The [Pd1ꢀC30] bond distance 2.3552(11) Å, [Pd1ꢀC30] bond
distance2.3618(12) Å and [Pd1ꢀP8] bond distance 2.2355(10) Å and [Pd1–P11] bond
distances of 2.2377(11) Å were found as similar to the reported values.38The trans angles
[P8ꢀPd1ꢀCl30] and [P11ꢀPd1ꢀCl31] were found as 175.43(4)° and 172.35(4)° indicating the
considerable deviation from the ideal symmetry and significant distortion around square
planar palladium ion.52

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Fig.4. ORTEP diagram of [PdCl2(dppe)] (3a&4a)
Repeated attempts to get good crystals of complex 2 suitable for the Xꢀray single
crystallographic studies were unsuccessful. Hence, the stoichiometry of complex 2 was
further confirmed by Electro spray Ionization Mass Spectroscopy (ESIꢀMS), the m/z value is
1085.0372 (Fig. S12).
3.4 DNA binding studies
3.4.1 Electronic absorption titration
The investigation of the binding of metal complexes to DNA is of major importance in the
development of drugs. The transition metal complexes interact with DNA via both covalent
and/or nonꢀcovalent interactions. The electronic absorption spectra of complexes1ꢀ4
exhibited two to three absorption bands at 256ꢀ268 nm and 305ꢀ408 nm, which were assigned
to intra ligand transition (IL) and ligand to metal charge transfer transition (LMCT). The
absorption spectra of the complexes (1ꢀ4) at constant concentration (10 µM) in the absence
and the presence of different concentrations of nucleotide CTꢀDNA (0.5ꢀ5 µM) are shown in
Fig. 5. Upon increasing the concentration of CTꢀDNA, hyperchromism was observed in the
complexes 1 and 2 with a small blue shift of 2 nm and 5 nm in the wavelength. However, in
the complexes 3 and 4, hyperchromism without shift in the wavelength was observed in the
intra ligand bands. The LMCT bands exhibited modest hyperchromism with and without blue
shifts from 1ꢀ7 nm for complexes (1ꢀ4) was observed, which indicated that the new

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palladium(II) complexes (1ꢀ4) bind to CTꢀDNA.53 However, the exact mode of binding can
be established only after calculating the intrinsic binding constant Kb values (Table 1) and
confirmation studies by either EBꢀdisplacement studies or thermal denaturation or viscosity
measurement or recording circular dichroism. Even though, hyperchromic effect was
observed, the very high binding constant values suggested that the binding may be an
intercalative.46 This has been further verified by doing EBꢀdisplacement studies. From the
intrinsic binding constant (Kb) values (Table 1), it is inferred that all the complexes (1ꢀ4) bind
with CTꢀDNA efficiently (Fig. 6). Among the four complexes, complex 4 binds more
strongly with CTꢀDNA as compared to trinuclear complex (3) and binuclear complexes (1
and2), which may be due to the presence of 5ꢀ(2ꢀhydroxyꢀ3ꢀmethoxyꢀphenyl)ꢀ4ꢀphenylꢀ2,4ꢀ
dihydro[1,2,4]triazoleꢀ3ꢀthione.Trinuclear complex 3 exhibited better binding than the
binuclear complexes 1 and 2, owing to the presence of palladiumꢀsulphur bridge and the
order of binding affinity is 2<1<3<4. The binding constants of the complexes are sufficiently
high (106 Mꢀ1). This is may be due to the strong supramolecular interaction between cationic
palladium complexes and negatively charged phosphodiester backbone of DNA.54
0.15
1
2
0.15
0.10
0.10
0.05
0.05
0.00
0.00
250
300
350
400
250
300
350
400
Wavelength (nm)
Wavelength (nm)
0.25 3
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
4
0.20
0.15
0.10
0.05
0.00
250
300
350
400
250
300
350
400
Wavelength (nm)
Wavelength (nm)
Fig.5. Absorption titration of fixed concentration (10 µM) of complexes 1ꢀ4 with increasing
concentrations (0.5ꢀ5 µM) of CTꢀDNA (TrisHCl, pH 7.2).

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Table 1 Binding constant for interaction of complexes with CTꢀDNA
System
Kb (× 106 Mꢀ1)
2.024±0.17
1.921±0.12
3.612±0.15
3.975±0.18
CTꢀDNA + 1
CTꢀDNA + 2
CTꢀDNA + 3
CTꢀDNA + 4
0.0035
0.0030
0.0025
0.0020
0.0015
0.0010
0.0005
1
2
3
4
0.0000
0
1
2
3
4
5
[DNA]
Fig.6. Plot of [DNA] versus [DNA] / (Ɛaꢀ Ɛf)
3.4.2 Competitive studies with ethidium bromide
From the electronic absorption titration studies, it is inferred that the complexes can bind with
the DNA. Whereas, the observed hyperchromic effect with slight red shift showing the
intrinsic binding constant values in the magnitude of 106, may not be proposed for
electrostatic interaction. Hence, in order to confirm the binding interaction of the complexes
to CTꢀDNA, competitive studies of the palladium(II) complexes with ethidium bromide were
undertaken. EB displacement technique provides indirect evidence for the DNA binding
mode. The fluorescence intensity of EBꢀDNA was gradually decreased with increasing
concentrations of the complexes (1ꢀ4) indicating that the complexes bound to DNA by
competing with EB(Fig. 7). From the quenching plots and quenching constant values, it is
clear that EB was replaced by complexes 1ꢀ4 from the EBꢀDNA system (Fig. 8, Table 2).
Such a characteristic change is an indication that all the complexes interacted with DNA
through intercalation mode.16a,55 The calculated value of the quenching constant (Kq) and
binding constant (Kapp) are listed in Table 2. The quenching constants and binding constants

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of the palladium(II) complexes suggest that the interactions of allof the complexes with DNA
should be intercalation.55
120
2
120 1
100
100
80
80
60
60
40
40
20
20
0
0
550
600
650
700
750
550
600
650
700
750
Wavelength (nm)
Wavelength (nm)
100
80
60
40
20
0
3
120
100
80
60
40
20
0
4
550
600
650
700
750
550
600
650
700
750
Wavelength (nm)
Wavelength (nm)
Fig. 7. Fluorescence quenching curves of ethidium bromide bound to DNA by complexes
1ꢀ4. [DNA] = 10 µM, [EB] = 10 µM and [compound] = (0ꢀ80) µM.
1.22
1.20
1
1.18
2
3
1.16
4
1.14
1.12
1.10
1.08
1.06
1.04
1.02
1.00
0
10
20
30
40
50
60
70
80
90
[Q]
Fig. 8.Plot of [Q] versus I0/I

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Table 2 Quenching constant and Binding constant for interaction of complexes with DNA
System
Kq (× 103Mꢀ1)
1.89±0.10
Kapp (× 106 Mꢀ1)
1.90
1.99
1
2
3
4
1.83±0.09
2.55±0.13
3.15±0.16
1.94
1.63
3.4.3 DNA cleavage studies
To assess the DNA cleavage ability of complexes 1ꢀ4, supercoiled (SC) pBR322 DNA was
incubated with 50 µM concentration of the complexes in a 5 mMTrisꢀHCl/50 mMNaCl
buffer at pH 7.2 for 2 h without the addition of a reductant (Fig. 9). Upon gel electrophoresis
of the reaction mixture, DNA cleavage was observed during which the SC DNA was
converted into nicked circular (NC) DNA and linear DNA. The complexes did not require
any addition of external agents to cleave the DNA. Moreover, the metal precursors
K2[PdCl4], free ligands (H2L1ꢀ4) and 1,2ꢀbis(diphenylphosphino)ethane (dppe) did not show
any cleavage activity. From this result, it is clear that the palladium(II) complexes (1ꢀ4) has
the potential to cleave the supercoil DNA and the new complexes (1ꢀ4) alone are responsible
for the cleavage of DNA.56 Among the four complexes, binuclear complexes 1 and 2
exhibited better DNA cleavage as compare to the other trinuclear 3 and cyclised mononuclear
4 complexes. The highest activity of the binuclear complexes 1 and 2 may be due to the
presence of two planar metal centres which are separated by bridged
bis(diphenylphosphino)ethane can undergo greater interaction as compare with other
complexes. Complex 4 showed good DNA cleavage as compared to trinuclear complex 3,
which may be due to the presence of 5ꢀ(2ꢀhydroxyꢀ3ꢀmethoxyꢀphenyl)ꢀ4ꢀphenylꢀ2,4ꢀ
dihydro[1,2,4]triazoleꢀ3ꢀthione. Oxidative cleavage is usually mediated by the presence of
additives and photo induced DNA cleaving agents i.e. an external agent like light or H2O2 is
required to initiate cleavage. Photo cleavage of nucleic acids allows the use of light to trigger
nuclease activity. However, the general mechanism of hydrolytic cleavage is the hydrolysis
reaction is facilitated by the presence of metal ions, acting as Lewis Acids. These Lewis acids
can activate the phosphate group towards nucleophilic attack, activate water or hydroxide as
nucleophile or increase the leaving group ability of the departing alcohol. The general
accepted mechanism of the DNA hydrolysis reaction is a nucleophilic attack at the DNA

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phosphate backbone, to form a five coordinate intermediate, which can be stabilized by the
catalyst. Thus hydrolytic cleavage mechanism was found in our complexes.57
Fig. 9. Gel electrophoresis diagram showing the cleavage of supercoiled pBR322 DNA by
complexes 1ꢀ4 in 5% DMSO and 95% 5 mMTris−HCl/50 mMNaCl buffer at pH 7.2 and 37
°C with an incubation time of 2 h. Lane B: Buffer+plasmid; Lane P: Plasmid alone ; Lane M:
DNA ladder; Lane 1: Complex 1 (50 µM); Lane 2: Complex 2 (50 µM); Lane 3: Complex 3
(50 µM); Lane 4: Complex 4 (50 µM). Forms SC, NC, and LC are supercoiled, nicked,
circular and linear circular DNA, respectively.
3.5 Protein binding studies
The crucial step in accessing a drug’s bioavailability is assigned to its interaction to plasma
protein. In screening potential therapeutic agents, plasma protein binding is required.58Bovine
serum albumin (BSA) is the most extensively studied serum albumin, which is able to bind a
variety of substrates including metal cations, hormones, and most therapeutic drugs. BSA
possesses three fluorophores(tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe)).
3.5.1 UV absorption spectra of BSA
UV absorption spectrometric titration is useful techniqueto distinguish between static and
dynamic quenching based on the absorption spectra of BSA in the presence of
complexes.59The UV absorption spectra of BSA in the absence and presence of four
complexes (1ꢀ4) (Fig. 10) revealed that the absorption intensity of BSA was enhanced as
concentration of the compounds increased, and there was a blue shift for all the compounds.
The changes in the absorption spectra for BSA + complexes indicate that the test compounds
interact with BSA and altering the secondary state of protein. Based on the enhancement of
the absorption intensity of BSA with blue shift suggesting the static type of quenching of
BSA by the complexes.16a,46

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0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
BSA
BSA+1
BSA+2
BSA+3
BSA+4
200
250
300
Wavelength (nm)
Fig.10.UV absorption spectra of BSA (10 µM) in the presence of complexes (10 µM).
3.5.2 Fluorescence quenching studies of BSA
The intrinsic fluorescence of BSA will provide considerable information on their structure
and is often utilized in the study of protein folding and association reaction. The interactions
of BSA with the complexes (1ꢀ4) were studied by emission titration at room temperature. A
solution of BSA (10 µM) was titrated with various concentrations of complexes (0ꢀ100 µM).
The effects of the complexes on the fluorescence emission spectra of BSA are shown in
Fig. 11. The addition of the complexes (1ꢀ4) to a solution of BSA resulted in a significant
decrease in the fluorescence intensity of BSA at 348 nm, up to 57.86, 57.06, 70.65 and
72.86 % from the initial fluorescence intensity of BSA accompanied by a blue shift of 1ꢀ3 nm
for all the complexes (1ꢀ4). This is mainly due to the active site in protein is buried in a
hydrophobic environment.60The results suggested a definite interaction of all the complexes
with BSA.46The quenching constants (Ksv) were calculated from slope of the plot I0/I versus
[Q], the concentration of complexes (1ꢀ4) resulted in a linear plot (Fig. 12) and this linear fit
plot represents a single quenching mechanism for all the complexes (1ꢀ4). The value of
binding constant (K) can be determined from the slope of the plot log [F0 – F/F] versus
log[Q] (Fig. 13). The calculated value of the quenching constant (Ksv), binding constant (Kb)
and the number of binding sites (n) are provided in Table 3. Complex 3 has a high degree of
binding than other complexes.

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1
400
300
200
100
0
2
400
300
200
100
0
300
350
400
450
300
350
450
Wavelength (nm4)00
Wavelength (nm)
3
400
300
200
100
0
4
400
300
200
100
0
300
350
400
450
500
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)
Fig. 11. Fluorescence quenching of BSA (1 × 10ꢀ5 M; λexi = 280; λemi = 346 nm) in the
absence and presence of various concentration of complexes (0ꢀ100 µM).
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
1
2
3
4
0
20
40
60
80
100
[Q]
Fig. 12.SternꢀVolmerPlot of BSA

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1
2
3
4
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
-5.0
-4.8
-4.6
-4.4
-4.2
-4.0
log [Q]
Fig. 13. Plot of log [Q] versus log (F0ꢀF/F)
Table 3 Quenching constant (Ksv) binding constant (Kb) and number of binding sites (n) for
interaction of complexes (1ꢀ4) with BSA.
System
BSA + 1
Ksv( × 103Mꢀ1)
5.48±0.20
6.11±0.13
7.33±0.24
6.40±0.39
Kb( × Mꢀ1)
n
4.92× 102 ±0.21
1.98× 102 ±0.12
4.69× 103 ±0.29
3.98× 104 ±0.32
0.703
0.601
0.952
1.207
BSA + 2
BSA + 3
BSA + 4
3.5.3 Synchronous fluorescence spectroscopic studies of BSA
The synchronous fluorescence method is usually applied to find out the conformation
changes in the active site of the protein, that is around tryptophan and tyrosine.61 Hence, the
synchronous fluorescence spectra of BSA were measured before and after the addition of the
complexes to get valuable information on the structural changes and molecular microꢀ
environment. Synchronous fluorescence spectra displays tyrosine residue of BSA at
wavelength interval ∆λ of 15 nm whereas tryptophan residues of BSA at ∆λ of 60 nm. As the
concentration of complexes (0ꢀ100 µM) added to BSA (10 µM) is increased, a decrease in the
fluorescence intensity was observed for all the complexes in both tryptophan and tyrosine but
the tryptophan spectra were accompanied by a blue shift (Fig. S15). It reveals that the binding
around tryptophan residues was strengthened. The results clearly indicate that all the
complexes bind to the active site of protein and brought conformational change in the

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secondary structure of protein, which makes them potential molecules for biological
applications.
3.6 Antioxidant activity
Free radicals play an important role in the inflammatory process. Number of
thiosemicarbazone derivatives are reported to act either as inhibitors of free radical
production or as radical scavengers.62ꢀ65,16aThus, the compounds possessing antioxidant
properties might play a vital role against inflammation and lead to potentially effective drugs.
Since the experiments conducted so far revealed that the Pd(II) complexes have good DNA
and protein binding ability, it is considered worthwhile to investigate their antioxidant
activity.
3.6.1 DPPH· scavenging assay
Antioxidants with DPPH radical scavenging activity are receiving much attention since they
can explore interesting anticancer, antiꢀageing and antiꢀinflammatory activities.16aThe free
radical scavenging activity of the compounds increased with an increase in the concentration
of the compounds17b,64and it is measured in terms of IC50 values (Table 4 and Fig. S16). The
results of the DPPH test showed that the new complexes exhibited a remarkable radical
scavenging activity than the wellꢀestablished standard (STD) vitamin C17a and also found to
be significantly higher than the parent ligands and metal precursors.16,17 Complexes4and 3
being good DPPH scavenger by comparing with the other complexes (Fig. 14).The observed
antioxidant activity of the palladium complex may be due to the neutralization of freeꢀradical
character of DPPH by transfer of either an electron or a hydrogen atom.65All the complexes
exhibited significant antioxidant activity than the reported complexes.16,17b,67

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350
300
250
200
150
100
50
DPPH
Superoxide
0
STD H2L1 H2L2 H2L3 H2L4 Pd(II) dppe
1
2
3
4
Fig. 14. Antioxidant activity (IC50 values) of ligands, bis(diphenylphosphino)ethane,
K2[PdCl4] and new Pd(II) complexes (1ꢀ4).Error barsrepresent the standard deviation of the
−
mean (n=3)
.
STD (Standard) = Vitamin C (DPPH˙) and Butylatedhydroxytoluene (O2 ˙)
3.6.2 O2־·scavenging assay
Reactive oxygen species (ROS) are generated during cellular metabolic process and assist in
ꢀ.
different biochemical reactions. The superoxide anion O2 may lead to heavy effects including
ꢀ.
tissue injury and inflammation.68Superoxide (O2 ) is highly reactive among the reactive
oxygen species (ROS). We have tried to examine the antioxidant potential of K2[PdCl4],
ligands, 1,2ꢀbis(diphenylphosphino)ethane and new Pd(II) complexes and their corresponding
IC50 values were shown in Table 4. From the values, it can be concluded that the Pd(II)
complexes exhibited better scavenging activity than thefree ligands, metal precursorsand the
standard (STD) Butylatedhydroxytoluene (BHT)(Fig. S17 & 14).67 Among the four
complexes, complex 4 showed the better scavenging ability in bleach out the free radicals
than other complexes. The order of the activity follows in the order 4>3>1>2. The lower IC50
values observed in antioxidant assays have demonstrated that these complexes have strong
potential to be applied as scavengers for eliminating radicals.

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Table 4 The radical scavenging activity of the compounds and the results are expressed as
the mean ± SD (n=3).
Compounds
IC50 values (ꢁM)
−
DPPH˙
O2 ˙
Standard (STD)
327.16±5.68
248.97±2.44
228.83±2.29
213.84±3.76
215.65±4.12
172.01±0.69
133.61±0.51
22.47±0.09
23.97±0.12
21.09±0.11
20.84±0.05
288.55±1.14
225.86±2.00
179.58±1.41
141.72±1.45
170.29±1.24
128.60±0.53
108.29±0.29
18.51±0.07
25.54±0.15
18.32±0.09
18.13±0.10
H2L1
H2L2
H2L3
H2L4
K2[PdCl4]
dppe
1
2
3
4
−
*(Standard) STD= Vitamin C (DPPH˙) and Butylatedhydroxytoluene (O2 ˙)
3.6.3 Reducing ability
Depending on the reducing power of each compound, the test solution changes its colour
from yellow to various shades of blue and green. The presence of reducers (i.e., antioxidants)
reduces the Fe3+/ferricyanide complex to the ferrous form, the Perl’s Prussion blue after the
addition of trichloroacetic acid and ferric chloride that can be monitored at 700 nm. The
reducing power of the standard BHT at various concentrations was higher than that of test
compounds. Increase in concentration of the compounds increases the reducing ability and
the new palladium(II) complexes (1ꢀ4) which have strong reducing ability than the free
ligands, dppe and K2[PdCl4]. The complex 4 has higher reducing ability when compared to
the other complexes (Fig. 15).

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0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
STD
1
H L
2
2
H L
2
3
H L
2
4
H L
2
dppe
K
[PdCl ]
2 4
1
2
3
4
20
40
60
80
100
Concentration
(ꢀg/ml)
Fig. 15.Reductive ability of ligands, bis(diphenylphosphino)ethane, K2[PdCl4] and new
Pd(II) complexes (1ꢀ4). Error barsrepresent the standard deviation of the mean (n=3).
3.6.4 Total antioxidant activity assay
The total antioxidant activity of Pd(II)precursors, free ligands and their corresponding
palladium(II) complexes (1ꢀ4) was assessed by phosphomolybdenum method. This method is
quantitative, since the antioxidant activity is expressed as the number of equivalents of
ascorbic acid. The total antioxidant activities of the complexes were higher than the free
ligands and the values are listed in Table 5. Among the four complexes, complex 4 showed
the good activity.
From the above results, it can be concluded that the scavenging effects ofK2[PdCl4],
1,2ꢀbis(diphenylphosphino)ethane and free ligands are comparatively less than their
corresponding Pd(II) complexes, which is mainly due to the chelation of the organic ligand
with the Pd(II) ion.

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Table 5 Estimation of Total antioxidant capacity of new Pd(II) complexes and the results are
expressed as the mean ± SD (n=3).
Compounds
µg Ascorbic acid equivalents/ml
9.84±0.55
H2L1
H2L2
10.95±0.56
H2L3
9.52±0.61
H2L4
12.26±0.45
K2[PdCl4]
9.52±0.48
dppe
7.87±0.53
39.64±0.44
1
2
3
4
31.37±0.31
47.61±0.37
52.82±0.33
3.7 Antibacterial activity studies
Metal ions are adsorbed on the cell walls of the microorganisms, disturbing the respiration
process of the cells and thus blocking the synthesis of protein required for their growth.
Hence, metal ions are vital for the growthꢀinhibitory effects.69 Antibacterial studies of
synthesised compounds have been carried out with some bacterial microorganisms namely
Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhiand Escherichia
coli. From the result, it is noted that the K2[PdCl4], ligands and dppe did not show any
significant activity at 1 mg/ml concentration. From the bactericidal activity, it is clear that the
complexes (1ꢀ4) were more effective towards gram positive bacterial strains than gram
negative bacterial strains (Fig. 16), due to the different structure of the cell walls.70All the
complexes were found to be good activity on all the pathogens except E. Coli, in particularly,
complex 4 exhibited better activity on B. subtilis, S.aureusand S. typhi. The activities of the
complexes may be explained by chelation theory.71 The variation of activity exhibited by
different complexes against various organisms is dependent on either the differences in
ribosome in microbial cells or the impermeability of the cells of the microbes.72

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1
2
3
4
20
15
10
5
0
B.subtilis
S.aureus
P.aeruginosa
S.typhi
Fig.16. Antiꢀbacterial screening of new Pd(II) complexes (1ꢀ4). Error bars represent the
standard deviation of the mean (n=3).
3.8 In vitro cytotoxicity studies
3.8.1 Cytotoxic activity against human tumour cell lines
The positive results obtained from the DNA binding and DNA cleavage, protein binding,
antibacterial and antioxidant studies encouraged us to test the cytotoxicity of the complexes
against MCFꢀ7 human breast cancer cell line and HeLa human cervical cancer cell line using
the colorimetric MTT assay (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide).
Cells were incubated with each compound for a period of 24 h and cisplatin was used for
comparison. There were no substantial effect was observed in cell proliferation by the
K2[PdCl4], ligands and dppe when compared with control cells (Table 6). All the complexes
exhibited
better
activity
compared
to
ligands,
metal
precursors
and
bis(diphenylphosphinoethane) (dppe). Moreover, on comparing the IC50 of these complexes
with that of cisplatin for the MCFꢀ7 cell line, complexes 1, 3 and 4 showed better activities
with exception of complex 2(Table 6).23Whereas in HeLa cell line, all the complexes showed
very low cellular growth inhibition at the maximum concentration of 100 µM and therefore
had no evaluable cytotoxicity (IC50> 100 µM).HeLa is a cervical cancer cell line. Whereas,
MCFꢀ7 is a breast adenocarcinoma cell line. These two cell lines are originated from distinct
organs. In generally, cytotoxic molecules internalise into cells through various mechanisms
such as endocytosis, diffusion and cell surface receptors mediated. If molecules internalise
into cells through particular receptors and those molecules can only enter into the cells. The

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cytotoxic ability of the compounds has been investigated by using the percentage of cell
viability versus concentration of compounds (Fig. 17& S18). The observed cytotoxic effects
by the new palladium(II) complexes towards the MCFꢀ7 cell line was better than those of
previously reported palladium complexes.23The overall results observed in the cytotoxicity of
the complexes are very similar to that of the DNA/protein binding and antioxidant activity. In
general, the Pd(II) complexes exhibited anticancer activity in the order of 4>3>1>2.Complex
4 exhibited better activity than other complexes and this may be due to the presence of 5ꢀ(2ꢀ
hydroxyꢀ3ꢀmethylꢀphenyl)ꢀ4ꢀphenylꢀ2,4ꢀdihydro[1,2,4]triazoleꢀ3ꢀthione. Complex 3 showed
moderate activity than the bisphosphine bridged binuclear complexes (1 and 2).Further
cytotoxicity of complexes was tested against nonꢀcancerous normal human keratinocyteline
(HaCaT) and the complexes showed less toxic towards the normal cells which is evident from
its higher IC50 values (IC50> 100 µM). These results suggest that the complexes possess
anticancer activities in human breast cancer cells with less toxic against normal cells.
100
Cisplatin
Cell proliferation (MTT) assay
1
90
H L
2
80
2
H L
2
70
3
H L
2
60
4
H L
2
50
K [PdCl ]
2
dppe
1
4
40
30
20
10
0
2
3
4
0
20
40
60
80
100
Concentration (ꢀM)
Fig. 17. MCFꢀ7 cells were treated with different concentrations of complexes (1ꢀ4), ligands,
bis(diphenylphosphino)ethane, K2[PdCl4] and cisplatin (3.125, 6.25, 12.5, 25, 50, 100) for
24 h. cell viability was assessed by cell proliferation (MTT) assay. Error bars represent the
standard deviation of the mean (n=3).