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Page 1 of 30 New Journal of Chemistry
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DOI: 10.1039/C7NJ03138F
A saccharinate-bridged palladacyclic dimer with Pd-Pd

bond: experimental and molecular docking studies of the
interaction with DNA and BSA and in vitro cytotoxicity
activities against human cancer cell lines
New Journal of Chemistry Accepted Manuscript

Published on 28 November 2017. Downloaded by Queen Mary, University of London on 29/11/2017 07:14:47.
Kazem Karami*a, Moloud Alinaghia, Zahra Amirghofranb, Janusz Lipkowskic, Amir Abbas Momtazi-
borojenid,e
a
Department of Chemistry, Isfahan University of Technology, Isfahan, 84156/83111, Iran
b
Immunology Department, Shiraz University of Medical Sciences, Shiraz 71454, Iran
c
Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
d
Department of Medical Biotechnology, School of Medicine, Student Research Committee, Mashhad University of Medical
Sciences, Mashhad, Iran
e
Nanotechnology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran
Abstract
New palladacyclic dimer [Pd2((C,N)L)2(µ-Sac)2] (1), in which L: C14H11NBr and sac:
saccharinate ligand, has been synthesized and completely characterized. X-ray
crystallography has been used to determine the single crystal structure of this Pd(II) complex.
In this dimer, two palladium(II) centers are bridged by saccharinate anion, which is
coordinated to the cyclopalladated units as a bidentate (N- and carbonyl O- atoms) ligand.
According to DNA binding studies (UV-Vis spectroscopy, emission titration and viscosity
measurement), the Pd(II) complex interacts with Calf-thymus DNA (CT-DNA) through
groove binding mode with a binding affinity in the order of 105. Furthermore, UV-Vis and
fluorescence emission spectroscopy have been used to monitor the binding of the complex to
bovine serum albumin (BSA). The complex is mainly located in site I of the protein, based on
the competitive experiments using Warfarin, Ibuprofen and Digoxin as site markers. The
results of molecular docking confirmed the experimental data. Finally, the In vitro
cytotoxicity of sodium saccharin, ligand LH (C14H12NBr), complex 1 and cisplatin against
cervical cancer (HeLa), lung cancer (A549) and breast cancer (MCF-7) cell lines has been
studied. Complexation process has significantly improved the anticancer activity, as IC50
values show. Furthermore, Complex 1 has been tested against NIH normal fibroblast cells.
Therefore based on the SI definition, 1 can be imputed as the selective compound against
cancer cells.
Keywords:
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DOI: 10.1039/C7NJ03138F
Palladacyclic dimer; Saccharinate bridges; DNA & BSA interaction; Molecular docking; In
vitro cytotoxicity
1. Introduction

DNA and proteins are usually regarded as the main molecular targets for drugs and many
drugs exert their effects through binding to DNA or proteins. This is the basis for the design
and discovery of novel and more efficient drugs.1-3 Therefore, synthesis and interaction of
metal complexes with DNA and proteins have been interesting research areas. Metal complex
interactions with specific DNA sequences have been investigated in order to understand the
mechanism of tumor inhibition, design new drugs and recognize specific sites or
conformations targeted on DNA.4
Generally speaking, the three main modes of non‐covalent interactions between most
compounds and DNA are intercalative binding, groove binding and electrostatic attraction.
Intercalations takes place when small molecules intercalate inside the nucleic acid base pairs,
distorting the DNA backbone conformation. Major and minor groove bindings involve
hydrogen bonding or van der Waals interaction of the small molecules with the nucleic acids
with no considerable distortion of the DNA backbone. An electrostatic interaction may occur
between the negatively charged DNA helix phosphate backbone and small molecules with
positive charges.5-8
Moreover, the development of metal based therapeutics as well as analysis of drug protein
interactions affecting the absorption, distribution, metabolism and excretion characteristics of
drugs have been simultaneously concentrated on. Given its considerable binding properties,
serum albumin plays an essential part in drug delivery as a drug carrier. The high cost of
human serum albumin (HSA) has made the cheaper bovine serum albumin (BSA) widely
applicable as an important model protein with regards to the interactions between compounds
and serum albumins.9 The binding mode in the interactions of proteins and drugs is basically
of non-covalent nature. It is necessary to investigate drug protein interaction to understand
the drug action mechanism and design new drugs.10, 11
The serious side effects of cisplatin, a principal metal based drug, which targets DNA,
and its analogues, including nephrotoxicity and drug resistance by the tumor cells have
motivated the discovery of more efficient cytotoxic complexes with other transition metals
and novel ligands. The structural similarities and significant overlap of coordination
chemistry of platinum and palladium closely relate palladium based complexes to their
platinum analogues. The slow dissociation pattern of platinum complexes in comparison with
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palladium (105 times faster) makes them more applicable. The development of palladium
complexes with slower rates of hydrolysis caused by the discreet choice of the ligands has
recently been focused on in palladium based anticancer drugs. Cyclopalladated complexes
containing a strong C-M s-bond, which can overcome all these problems, have proven to be

very promising in this regard.12-17
Since coordinated ligands can modify key parameters such as reactivity and lipophilicity,
they play a significant part in the anticancer activity of metallodrugs.18 Coordination of the
biologically active organic molecules such as ligands to the transition metal ions has been
shown to be promising in this field due to the unique capability of these molecules to bind
with different biological targets.19 Saccharin (1,2-benzisothiazoline-3-(2H)one 1,1-dioxide or
o-sulphobenzimide) and its water soluble alkali and alkali earth salts are presently the most
widely used non-caloric artificial sweeteners throughout the world.20 The first row transition
metal derivatives of the saccharin are protease inhibitors.21 Superoxide dismutase like activity
has been reported for these and other metal saccharinates.22 Saccharin anions are various
polyfunctional ligands due to the presence of several potential donor sites such as nitrogen,
one carbonyl and two sulfonyl oxygen atoms.23 Saccharin complexes have recently been
found to act efficiently as biologically active agents.24, 25 Strong anticancer activity against
MCF-7 and MDA-MB-231 human breast cancer cell lines has been shown by
[Pd(sac)(tpy)](sac)∙4H2O (sac: saccharinate and tpy: 2,2':6',2"-terpyridine) palladium(II)
complex by induction of apoptosis via cell death receptors in vitro.26 Furthermore, trans-
[Pt(2-hmpy)2(sac)2]∙3H2O (2-hmpy: 2-(hydroxymethyl)pyridine) gives rise to relatively
strong anti-growth effect against PC3 for lung cancer cells.27 Excellent anti-proliferative
effect against HeLa cells of human cervix cancer has been shown by another platinum(II)
complex with saccharin, K[Pt(sac)3(H2O)]. IC50 value of 6.8 µM is comparable to that
obtained for cisplatin.28
Synthesis, structural characterization and biological properties of the palladacyclic dimer
with saccharinate bridges [Pd2((C,N)L)2(µ-sac)2] (1), in which L: C14 H11NBr, LH: C14
H12NBr (Scheme 1) and sac: saccharinate ligand, is reported. The investigation of the
biological characteristics of the complex has been focused on (i) the binding properties with
Calf-thymus DNA (CT-DNA) studied through electronic absorption titration, fluorescence
spectroscopy and viscosimetric measurement and (ii) the affinity for bovine serum albumin
(BSA) investigated by UV-Vis and fluorescence spectroscopy. The binding constants (K) and
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number of binding sites (n) have also been calculated. In addition, molecular docking studies
were carried out to obtain detailed binding information of the complex with DNA and BSA.
Ultimately, MTT assay has been used to evaluate the cytotoxic activity of compounds against
cervical cancer (HeLa), lung cancer (A549), breast cancer (MCF-7) and normal fibroblast

cells (NIH).
Ha
Br Hb
N
Ha
Hb
Scheme 1: The molecular structure of ligand (LH).
2. Experimental section
2.1. Materials
All solvents and chemicals were purchased from commercial sources and used
without any further purification. Reagent grade Calf-thymus DNA (CT-DNA), BSA and
methylene blue (MB) were obtained from Sigma Aldrich Chemical Co.
CT-DNA solutions gave a UV absorbance ratio (A260/A280) of over 1.8, showing the
DNA purity.29 Extra pure solvents used in the syntheses and physical measurements were
supplied by Merck Chemical Co. Tris(hydroxymethyl)-aminomethane (Tris) buffer was of
analytical reagent grade and was purchased from Merck Chemical Co. Doubly distilled
deionized water was used in all solution preparations. CT-DNA and BSA stock solutions
were prepared by dissolving them in 5 mM Tris buffer and 50 mM NaCl at pH 7.2.
Absorption spectroscopy was used to determine the DNA concentration per nucleotide by the
molar absorption coefficient (ε = 6600 M–1 cm–1) at 260 nm.30, 31
2.2. Methods and instrumentation
An FT-IR JASCO 680-PLUS spectrophotometer was used to record Fourier transform

infrared spectra in the 400–4000 cm−1 region using KBr pellets. Electronic absorption spectra
were recorded using a JASCO 7580 UV-Vis-NIR double beam spectrophotometer in a 1 cm
path length cell. A Cary Eclipse fluorescence spectrophotometer was used to record
fluorescence spectra in solution. A Bruker spectrometer was used to record NMR spectra at
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400.13 MHz (1H) and 100.61 MHz 13

C {1H}. A Leco, CHNS-932 apparatus was used to
carry out elemental analyses. A Cannon Fenske routine viscometer was used to perform
viscometric titrations at a constant temperature of 298.0 (±0.1) °K in a thermostatic water
bath.

2.3. Syntheses
2.3.1. Synthesis of ligand LH: C14 H12NBr
Benzylamine (273 µL, 2.5 mmol) was slowly added to 4-bromobenzaldehyde (0.462
g, 2.5 mmol) dissolved in methanol. The resulting clear solution was stirred at ambient
temperature for 24 h. The ligand was obtained as a white precipitate after evaporating the
solvent. (Yield: 93.7%).
Anal. Calc. for C14H12BrN (%) C, 61.33; H, 4.41; Br, 29.15; N, 5.11. Found C, 61.23;
H, 4.62; Br, 29.08 N, 4.97. IR (KBr, cm-1) ν (C-HPh) = 3081-3027, ν (C-H alph) = 2873-2851,
ν (C=N) = 1644, ν (C=C aromatics) = 1483. 1H NMR (DMSO-d6, ppm): δ= 4.77 (s, 2H,
13
CH2), 7.24-7.38 (m, 5H, CHph), 7.66 (d, 2H, Hb), 7.73 (d, 2H, Ha), 8.5 (s, 1H, HC=N). C
1
{ H} NMR (DMSO-d6, ppm): 63.89 (CH2), 124.21, 126.8, 127.88, 128.34, 129.8, 131.69,
135.16, 139.36 (aromatic rings), 160.69 (HC=N).
2.3.2. Synthesis of complex 1: [Pd2((C,N)L)2(µ-sac)2]
A solution of Pd(OAc)2 (0.025 g, 0.1 mmol) in toluene (5 ml) was added dropwise to
ligand LH (0.06 g, 0.2 mmol) dissolved in toluene (10 ml). The resulting yellow solution was
refluxed at 60 °C for 24 h to obtain complex (a) [Pd2((C,N)L)2(µ-OAc)2]. (Yield: 86.2%).
IR (KBr, cm-1) ν (C-HPh) = 3083-3029, ν (C-H alph) = 2954-2852, ν (C=N) = 1608, ν (µ-
OAc) = 1577, 1418 cm-1.
Excess NaCl was then added to a suspension of (a) in acetone/water solvent and the
resulting mixture was stirred for 24 h at ambient temperature. The yellow precipitate formed
was filtered, washed with water and then air dried to give complex (b) [Pd2((C,N)L)2(µ-Cl)2].
(Yield: 89.9%).
IR (KBr, cm-1) ν (C-HPh) = 3080-3024, ν (C-H alph) = 2956-2850, ν (C=N) = 1608 cm-1.
AgNO3 (0.017 g, 0.1 mmol) was added to a suspension of dimer (b) (0.042 g, 0.05
mmol) in acetone (15 ml) in the dark. Having been stirred for 2 h, the white AgCl precipitate
formed was filtered from the solution. Sodium saccharin salt (0.024 g, 0.1 mmol) was then
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added to the filtrate and the resulting mixture was stirred for 24 h at ambient temperature.
The solvent was removed to about 2 ml and n‐hexane was added to precipitate a yellow solid
of complex 1. Yellow crystals of 1 were obtained from CH2Cl2: n-hexane solvent mixture.

(Yield: 73.7%).
Anal. Calc. for C42H30Br2N4O6Pd2S2 (%) C, 44.9; H, 2.69; Br, 14.22; N, 4.99; S, 5.71. Found
C, 45.23; H, 2.72; Br, 14.08 N, 4.84; S, 5.83. IR (KBr, cm-1) ν (C-HPh) = 3086-3028, ν (C-H
alph) =2955, 2853, ν (C=N) = 1620, ν (C=O) = 1669, ν (S=O) =1154, 1287 cm-1. 1H NMR
(DMSO-d6, ppm): δ= 4.5 (s, 2H, CH2), 6.88-7.35 (m, 8H, aromatic L), 7.71-7.91 (m, 4H,
saccharin bridge), 8.34 (s, 1H, HC=N). 13C {1H} NMR (DMSO-d6, ppm): 59.8 (CH2), 120.2,
123.4, 123.45, 123.5, 127, 127.5, 128, 128.2, 128.4, 128.6, 129.6, 131, 133.1, 133.3, 137,
137.4, 142.5, 145.4 (aromatic rings), 154 (C-Pd), 165.6 (HC=N), 177 (C=O).
2.4. Crystallography
An Agilent SuperNova single crystal diffractometer (Cu K(a) radiation) was used to perform
X-ray diffraction experiments at 100 K. A multi-faceted crystal model based on expressions
derived by R.C. Clark & J.S. Reid was used to make analytical numeric absorption
correction.32 The structures were solved and refined using direct methods by SHELXS97 and
SHELXL (Sheldrick 2008) programs. Hydrogen atoms were added in the calculated positions
and were riding on their respective carbons during the refinement.
2.5. DNA binding studies
A CT-DNA stock solution was prepared in Tris-HCl buffer (5 mM Tris-HCl, 50 mM

NaCl at pH 7.2). Concentrated stock solution of the complex was prepared in DMSO solvent
and then diluted suitably using Tris-HCl buffer at pH 7.2 to obtain the required
concentrations in all the experiments. The ultimate DMSO concentration never exceeded
0.1% v/v. UV spectroscopy was used to study the interaction of 1 with CT-DNA to
investigate the possible binding mode to DNA and calculate the binding constant to DNA
(Kb). Absorption titration experiments were carried out at a constant complex concentration
(60 µM) and various DNA concentrations (0.0-120 µM) in Tris-buffer. Furthermore, the
complex 1 binding mode was further investigated using viscosity measurements. Sample
viscosities were evaluated using the mean values of three replicate measurements. Relative
⁄
specific viscosity ( ⁄ ) values, in which and are the specific viscosity contributions
of DNA in the absence ( ) and presence of each compound (), respectively, were plotted
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against r (r = [complex]/[DNA]).33 The relative viscosities were calculated using the relation
= (t - t0)/t0, in which t0 and t represent the flow times of the blank buffer and the DNA
containing solutions, respectively.34

Fluorescence spectroscopy has been used to perform the competitive study of the
complex with MB to examine the complex capability to displace MB from its DNA-MB
complex. The DNA-MB solution ([DNA]/[MB] = 5) was excited at 630 nm in the absence
and presence of different complex concentrations for MB displacement.35
2.6. Protein binding studies
A BSA stock solution was prepared by dissolving the appropriate quantity of BSA in
the buffer solution (containing 5 mM Tris-HCl/50 mM NaCl at pH 7.2) and stored at 4 °C for
further use. The BSA concentration was obtained by UV-Vis absorption spectroscopy using
ε280 = 44300 M-1cm-1.36
The absorption titration experiments were carried out by keeping BSA concentration
constant (6 µM) while adding various complex concentrations (0.0-12 µM). Equal amounts
of 1 solution were added to both the BSA and the reference solutions during the measurement
of the absorption spectra to eliminate the absorbance due to complex. In the tryptophan
fluorescence quenching experiment, quenching of the tryptophan residues of BSA37 was
performed by keeping BSA concentration constant (6 μM) while varying the complex
(quencher) concentration (0.0-12 µM), giving solutions with different molar ratios of the
quenchers to BSA. Following each addition of the quencher, the fluorescence spectra were
recorded at an excitation wavelength of 280 nm and an emission wavelength of 343 nm in the
fluorometer.
In order to identify the binding location of 1 on BSA, site competitive replacement

experiments were carried out in the presence of three site markers (Warfarin, Ibuprofen and
Digoxin) using the fluorescence titration methods. Equal BSA and site marker concentrations
(6 µM) were used and 1 was then added gradually. The fluorescence spectra were recorded at
298 °K with an excitation wavelength of 280 nm and emission in the 285-500 nm range.
2.7. Molecular docking
Molecular docking was used in the investigation of binding mode and intermolecular
interactions of the complex with DNA and BSA. Auto Dock 4.2 package by the Lamarckian
genetic algorithm (LGA) was applied to perform molecular docking studies.38, 39
The PDB
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format of 1 was obtained by converting CIF file using Mercury software. The initial BSA
structure was taken from the Protein Data Bank (PDB ID: 4F5S) at a resolution of 2.47 Å.
Moreover, d(CGCGAATTCGCG)2 DNA sequence was supplied by the Protein Data Bank
(PDB ID: 3U2N) at a resolution of 1.25 Å. Chain (A) of BSA and all the hetero atoms

including water molecules were removed during DNA preparation and BSA input file. Blind
docking was first used to find the appropriate binding site for the complex interaction with
DNA and BSA.40, 41 Focus docking was then performed on the best location. DNA and BSA
systems were restrained to a grid box with dimensions of 60 × 60 × 60 Å3 and a 0.375 Å grid
spacing, in which almost the entire macromolecules were involved. All other parameters were
maintained at their default values.
2.8. The cell proliferation assay
The growth inhibitory effect of the compounds against HeLa, A549 and MCF-7
cancer cell and NIH normal cell lines were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide (MTT) assay.42 A predetermined concentration of tumor cells
was seeded in the wells of a 96-well plate using different compound concentrations (0-100
µmol dm-3) and incubated at 37 °C with 5% of CO2 and 95% humidity for 48 h. 10 µL of
MTT solution (5 mg mL-1) were then added to each well and the plates were incubated for an
additional 4 h at 37 °C. The insoluble formazan produced was finally dissolved by adding
dimethyl sulfoxide (DMSO) (100 µL/well). Having completely dissolved the dye, the optical
density (OD) was recorded at 570 nm with a reference wavelength of 630 nm in an enzyme
linked immunosorbant assay (ELISA) reader (Bio-Tek’s ELx808, USA). The percentage
inhibition of cells exposed to various treatments was calculated as follows: % Inhibition =
100 - [(test OD/non-treated OD) × 100]. Non-treated cultures in all experiments were
composed of only DMSO solvent at a concentration equal to those in the test wells. The
graph of inhibition percentage against different concentrations was used to determine half
maximal inhibitory concentration (IC50) values.43, 44
The selectivity index (SI) was also
measured based on the IC50 ratio of normal NIH and cancer (HeLa, A549 and MCF-7) cells.
SI values indicate selectivity of the sample to the tested cell lines. Samples with SI values
more than 2 were considered to achieve a high selectivity.45
3. Results and discussion
3.1. Synthesis and spectroscopic characterization

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Ligand (LH: C14 H12NBr) was reacted with palladium acetate (Pd(OAc)2 (2:1 molar
ratio) in toluene at 60 °C to yield the [Pd2((C,N)L)2(µ-OAc)2] dimer (a) containing acetate
bridges. [Pd2((C,N)L)2(µ-Cl)2] chloro bridged dimer (b) was isolated on addition of NaCl to a
acetone/water solvent mixture of (a) in good yield. (b) was reacted with AgNO3 and sodium

saccharin to form [Pd2((C,N)L)2(µ-sac)2] (1) (Scheme 2).
Br Br Br
CH3
Pd(OAc)2 Cl
O O NaCl
Pd Pd
N Toluene Acetone/Water N
24 h N
2 24 h 2
60°C R.T.
(LH) (a) (b)
Br AgNO3
N Acetone
O 2h
O Pd
S O R.T.
N
N
S Na+C7H5NO3S-
O
O Pd O
Br 24 h
N R.T.
(1)
Scheme 2: Synthetic route for the preparation of the Pd(II) complexes.
The complexes isolated as yellow solids were stable at room temperature and soluble
in chlorinated solvents such as CH2Cl2, CHCl3 and polar, aprotic solvents such as DMSO.
The elemental analysis of 1 is in a good agreement with the calculated values. Formation of
the isolated complexes has been confirmed on the basis of characteristic peaks in the IR
spectra. The significant band at 1644 cm-1 in the IR spectrum of the ligand is due to (C=N)
stretching vibrations. ν (C=N) has shifted to a lower frequency in the complexes, indicating
imine nitrogen atom binding to the metal ion.46 Characteristic bands associated with the
bridging acetato ligand at 1577 and 1418 cm-1 disappear when (a) is converted to (b).47
Symmetric and asymmetric (SO2) stretching absorptions of sac appear as strong absorptions
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at about 1154 and 1287 cm-1, respectively. The positions of the peaks corresponding to SO2
vibrations in the complex are almost similar to those of the sodium saccharinate
monohydrate. This suggests that the sulfonyl groups of the sac ligands are not involved in the
coordination with the Pd(II) ions.48 The ν (C=O) vibration, usually characterized by an

extremely good group frequency, has often been exploited for structural studies. This mode
generates a sharp band at 1642 cm−1 in sodium saccharinate 49
, which shifts to lower
frequencies when the carbonyl group participates in metal bonding. The ν (C=O) frequency
does not correlate with the coordination mode of the ligand although an approximate ν (C=O)
frequency limit for the distinction between N-coordinated and uncoordinated saccharinate in
the solid metal complexes is suggested to be 1650 cm−1 50
due to intra and intermolecular
51 -1
interactions. In this complex, ν (C=O) appears at 1669 cm .
The singlet signal corresponding to methylene protons is observed at 4.7 ppm in the
1
H NMR spectrum of ligand LH (Fig. S1, ESI†), which indicates that these two protons are
equal and resonate independently of other nuclei. These two nuclei have become slightly
shielded due to complex formation and resonate at 4.5 ppm (Fig. S2, ESI†). The aromatic
protons of the ligand (Ha and Hb) appear as two doublets at 7.66 and 7.73 ppm, the non-
equivalency of these protons causing splitting and formation of doublets. These protons have
resonated at a lower frequency (6.88-7.35 ppm) in the 1H NMR spectrum of complex. In
addition, a singlet is observed at 8.5 ppm in the ligand spectrum, which corresponds to the
resonance of imine protons of the LH. This signal has also shifted to a lower frequency (8.34
ppm) due to complex formation, as observed in Figure S2. The 1H NMR spectrum of
complex shows a multiplet at 7.71-7.91 ppm, which is not observed in the spectrum of the
ligand. This signal is associated with the aromatic hydrogens of sac ligand, which have
13
resonated at lower frequencies compared with free saccharinate. The C NMR spectrum of
the ligand (Fig. S3, ESI†) shows an aliphatic carbon at 63.9 ppm, eight types of aromatic
13
carbon in the range of 124.21-139.36 ppm and an imine carbon at 160.69 ppm. In the C
NMR spectrum of complex 1 (Fig. S4, ESI†), the signal associated with the aliphatic carbon
appears at 59.8 ppm and those of aromatic carbons are observed in the range of 120.2-145.4
ppm. In addition, the three peaks at 154, 165.6 and 177 correspond to the orthopalladated
carbon, imine carbon of ligand L and carbonyl carbon of sac ligand, respectively. Only one
set of signals is observed in the 1H-and 13
C{1H} NMR spectra of 1 indicating that the
cyclopalladated dimer consists of only one geometrical isomer in the solution 47.
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3.2. Molecular structure of 1
Single crystal X-ray diffraction has been used in the characterization of complex 1 in
the solid phase. Slow diffusion of hexane into a CH2Cl2 solution gave suitable 1 crystals. Fig.

1 shows an ORTEP view of the dinuclear complex 1.
Figure 1: Partially labelled ORTEP view of molecular structure of 1.
Table 1 summarizes the relevant crystallographic data and structural refinement

details. Table 2 lists the selected bond lengths and angles. This compound was crystallized in
the monoclinic space group I2/c with Z=8. The coordination number around each palladium
atom is four in this complex, indicating that Pd(II) atom is bonded to an ortho-carbon atom
and the imino nitrogen atom of L, amido nitrogen and carboxylate oxygen of sac ligand in
distorted square planar geometry for each palladium atom with a Pd-Pd bond between them.
Metal-metal bond length in this compound (2.9 Å), is less than the sum of the van der Waals
52
radii for Pd (3.26 Å) , consistent with the previously reported -NCO-bridged palladium
53, 54
dimers. The sums of angles around palladium atom are 360.42 and 357.02° for Pd1 and
Pd2, respectively, with the distortion most noticeable in the somewhat reduced “bite” angle of
the metalated moiety. The restraints of the five membered rings force the bond angles C36-
Pd1-N5 and C23-Pd2-N3 to 81.4(2) and 81.7(2)°, respectively. The planar coordination
around d8 metallic centers often appears distorted in crystal structures. The distortion shows
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two ‘‘ideal’’ limiting cases: deviation towards tetrahedral coordination and distortion towards
square pyramidal coordination.55 In complex 1, w1 and w2 are 2.33 and 4.24° for Pd1 and -
0.11° and -6.22° for Pd2, respectively. These values are associated with a tetrahedral
distortion from the ideal square planar geometry. The Pd-Cpalladate distances (1.982(6) and

1.962(6) Å) are shorter than the predicted value of 2.081Å (based on the sum of the covalent
radii for C(sp2) and Pd, which are 0.771 and 1.31 Å, respectively).56 However, they are
similar to those observed in a related orthopalladated complex.57, 58 As the molecular structure
shows, the sac ligand bridges the two palladium(II) centers and is coordinated to the
cyclopalladated units as a bidentate (N- and carbonyl O- atoms) ligand. The Pd1-N1 and Pd2-
N2 bond lengths (2.055(5) and 2.035(5) Å), respectively, are longer than Pd1-N5 and Pd2-N3
distances (2.039(5) and 2.020(5) Å, respectively). This lengthening shows a weakening of the
Pd-Nsac bond as a result of the great trans influence of the L imino nitrogen. Furthermore, Pd-
O and C=O bond lengths (due to sac ligand) in this compound lie in the ranges obtained for
related complexes.53, 54, 59
Table 1: Crystallographic data and structure refinement details for complex 1.
1
Empirical formula C84H60Br4N8O12Pd4S4
Formula weight 2246.88
T/K 100 (1) K
Crystal system MONOCLINIC
Space group I2/c
a/Å 43.3833 (10) Å
b/Å 9.7679 (2) Å
c/Å 38.6961 (7) Å
α/° 90°
/° 94.968 (2)°
/° 90°
3
v/ Å 16336.4 (6)
Crystal dimensions / mm3 0.13 × 0.12 × 0.09
Z 8
µ(mm-1) 10.80
Dcalc/ Mg m−3 1.827
F000 8832
range/° 4.1–68.8°
Independent reflections 14931

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Data/restraints/parameters 12923/0/1040
Goodness-of-fit on F2 0.966
Final R indices R1=0.0542, wR2=0.1481
R indices (all data) R1=0.0638,wR2=0.1564

Largest difference peak and hole / 1.74 , −1.71

e Å−3
CCDC number 1522173
Table 2: Selected bond lengths (Å), and angles (°) for complex 1.
Atoms
Bond lengths
Pd1—N5 2.039 (5) Pd2—C23 1.962 (6)
Pd1—N1 2.055 (5) Pd2—N3 2.020 (5)
Pd1—O6 2.176 (4) Pd2—N2 2.035 (5)
Pd1—C36 1.982 (6) Pd2—O1 2.168 (4)
O6—C10 1.258 (7) O1—C1 1.257 (7)
Pd1—Pd2 2.9076 (5)
Bond angles
C36—Pd1—O6 173.3 (2) O6—Pd1—Pd2 83.22 (10)

N5—Pd1—O6 97.00 (18) C23—Pd2—N3 81.7 (2)
N1—Pd1—O6 87.82 (17) C23—Pd2—N2 94.0 (2)
C36—Pd1—N5 81.4 (2) N3—Pd2—N2 175.7 (2)
N5—Pd1—Pd2 105.36 (13) C23—Pd2—O1 170.95 (19)
N1—Pd1—Pd2 78.71 (13) N3—Pd2—O1 94.64 (18)
3.3. DNA binding studies

3.3.1. Electronic absorption titration
An effective technique to study the interactions of CT‐DNA with the metal complexes
is electronic absorption spectrometry. The intensity of the ligand centered π–π* absorption
band (235 and 253 nm) decreases when DNA is added to 1 incrementally (Fig. 2). The
observed hypochromic change with no shift in the UV spectrum of the complex suggests that
it binds to DNA via the groove binding mode.60, 61
Wolfe-Shimmer equation was used to
calculate the intrinsic binding constant, Kb, for the complex:
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DNA/(ε ε ) DNA/(ε ε ) 1/(K (ε ε ) ) (1)
where [DNA] is DNA concentration and ɛa, ɛf and ɛb correspond to Aobs/[complex], extinction
coefficients of the free complex and the complex in the fully bound form, respectively. The

slope and the intercept of the linear fit of a plot of [DNA]/(ɛa − ɛf) vs. [DNA] give 1/(ɛb − ɛf)
and 1/Kb(ɛb − ɛf) (Fig. 2, inset). The ratio of the slope to the intercept can be used to obtain
the intrinsic binding constant (Kb).62 Kb value of 1.05×105M-1 was obtained for 1 using the
above equation. Kb value is close to the previously reported palladium(II) saccharinate
complexes. 63
Figure 2: Electronic spectra of 1 in buffer solution (5 mM Tris-HCl/10 mM NaCl at pH 7.2) upon addition of CT-DNA.
-5 -4
[Complex] = (6×10 M), [DNA] = (0-6×10 M). Arrow shows the absorption intensities decrease upon increasing DNA
concentration. Inset: Plots of [DNA]/( εa-εf) vs. [DNA] for the titration of 1 with CT-DNA.
Compound-DNA complex free energy (∆G) was calculated from the values of binding
constant (Kb) using the following equation:
∆ ! ln $ (2)
Binding constants are measures of compound-DNA complex stability whereas free

energy is an indication of the spontaneity/non-spontaneity of compound DNA binding. Free
energies of 1 were evaluated as a negative value (-6.85 kcal mol-1), indicating the spontaneity
of compound DNA interaction.
3.3.2. Fluorescence studies of competitive DNA binding
Emission titration was performed for further confirmation of the interaction between
the complex and CT-DNA. The fluorescence measurement of the complex with or without
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CT-DNA at room temperature in aqueous solutions showed no emission band. Thus, the
emission spectra do not directly show the binding of compound with CT-DNA. The
competitive binding experiment was carried out using methylene blue (MB) as a probe to
further study the interaction mode of the complex with DNA. MB is a planar cationic dye,

which is well-known for independent intercalation into the CT-DNA. To investigate the
interaction of complexes with DNA, a competitive MB displacement assay was performed.64
It has been previously reported that the fluorescence intensity of MB-DNA system increases
by adding certain complexes.65-67 The complexes are thus bound to the base pairs of DNA by
classical intercalation mode while replacing MB.68 DNA induced emission intensity at 680
nm (630 nm excitation) decreased upon the addition of 1 to DNA pretreated with MB (Fig. 3)
because this complex binds to DNA via non-covalent groove binding.69
Figure 3: Emission spectra of the DNA-MB system in the presence of 1, [DNA] = 5×10-5 M, [complex] = 0-2×10-5 M, [MB] = 1
-5
× 10 M. The arrow shows the emission intensity decrease upon increasing complex concentration.
3.3.3. Viscosity measurements
Viscosity measurements were carried out by keeping the DNA concentration constant
and changing the concentration of 1 (Fig. 4). DNA viscosity is sensitive to DNA length
changes. The relation between the relative viscosity ( ⁄ ) and DNA length (L/L0) is L/L0 =
⁄
(⁄ ) , in which L0 and L are the apparent molecular lengths in the absence and presence
of the compound, respectively.70 The viscosity of DNA solution significantly increases when
a complex binds to DNA via a classical intercalative mode since DNA base pairs are
separated to accommodate the binding complex, giving rise to the lengthening of the DNA
helix and subsequent increase in DNA viscosity.71 However, a small molecule, which binds
exclusively in the DNA grooves under the same conditions, causes little or no change to the
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DNA solution viscosity.34 These results again suggest the interaction of 1 with the groove of
DNA in accordance with the above experimental results.

-5
Figure 4: Effect of increasing amounts of 1 on the viscosity of CT-DNA (5×10 M) in 5 mM Tris buffer (r = 0, 0.1, 0.2, 0.3, 0.4
and 0.5).
3.4. Protein binding experiments
Proteins are major targets for therapeutically active complexes. Since the absorption,
metabolism and distribution of the drugs are greatly affected by drug protein interactions 72,
the investigation of drug-protein interaction is an active field of interest in terms of
understanding drug action mechanism and the possibility of designing new and useful
medicines. Consequently, UV-Vis and fluorescence techniques were used to investigate the
binding properties of 1 with BSA.
3.4.1. Electronic absorption titration
Protein structural changes and protein drug complex formation can be understood by
UV-Vis absorption spectroscopy. BSA absorption spectra in the absence and presence of
different concentrations of 1 have been recorded at ambient temperature (Fig. 5). BSA has
two main absorption bands: one located in the 210-240 nm range, which is the skeleton
absorption peak and the other at 280 nm associated with the π → π* transition of the aromatic
amino acids (Trp, Tyr, and Phe).73 Addition of 1 obviously decreases BSA absorption peak in
228 nm with shifted toward longer wavelength, indicating a perturbation of α-helix caused by
a specific interaction between the complex and BSA.74 In addition, maximum absorption
changes at 280 nm show that 1 interacts with BSA molecule (static interaction and the
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microenvironment of the three aromatic acid residues changes in the presence of the
complex.75

-6 -
Figure 5: UV absorption spectra of [BSA] = (6×10 M) in the absence and presence of different concentration of 1 (0-6×10
5
M).
3.4.2. Fluorescence quenching studies
Three are three fluorophores in BSA, tryptophan, tyrosine and phenylalanine residues.
The intrinsic BSA fluorescence is usually contributed by tryptophan alone since
phenylalanine has a very low quantum yield and tyrosine fluorescence is almost completely
quenched if it is ionized.76 BSA has a strong fluorescence emission peak at 345 nm (excited
at 280 nm) (Fig. 6). BSA fluorescence intensity regularly decreases with increased
concentration of 1, whereas the emission maximum wavelengths and shape of the peaks
remain almost unchanged. This indicates that 1 binds to BSA and quenches its intrinsic
fluorescence. After correction for the inner-filter effect using equation (3) 77:
%&'(( %') * (+,-.+,/ )/0 (3)
where Icorr and Iobs are the corrected and measured fluorescence, respectively, and Aex and Aem
are the absorbance of the complex at excitation and emission wavelengths, respectively, the
corrected fluorescence quenching data were analyzed using the Stern-Volmer equation 78:
12
1 $34 5 1 $6 7 5 (4)
1
where I0 and I are the fluorescence intensities in the absence and presence of the complex,
respectively, Ksv is the Stern-Volmer quenching constant, [Q] is the quencher concentration,
kq is the bimolecular quenching rate constant and τ0 is the average lifetime of the fluorophore
without quencher. KSV can be obtained from the slope of a plot of I0/I vs. [Q] (Fig. 6 insets).
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The Ksv value for 1 is 1.08 ×105M-1 in this work. Since the τ0 value for tryptophan
fluorescence in proteins is 2810-8 s 79
, the quenching rate constant, kq, can be calculated
using the following equation:
96 $34 /7

(5)
The quenching constant (kq) obtained for 1 was 5.4 × 1012M-1s-1. Quenching mechanisms are
generally classified as dynamic and static. Dynamic quenching is the process in which the
fluorophore and the quencher come into contact during the transient existence of the exited
state. Static quenching refers to a fluorophore quencher complex formation in the ground
state. The kq value (ca 1012 M−1 s−1) of complex 1 is greater than the maximum scatter
collision quenching constant, i.e. 2.0 × 1010 M−1 s−1. This indicates the existence of a static
interaction between BSA and the added complex.80, 81
-6 -5
Figure 6: Emission spectra of BSA upon the titration of 1. [BSA] = (6×10 M), [complex] = (0-6×10 ). Arrow shows the change
upon the increasing complex concentration. Inset: Plots of I0/I vs [Q]×106.
If similar and independent binding sites are assumed in the biomolecule for the static
binding constant (Kb), quenching interaction and the number of binding sites (n) can be
calculated using the following equation 82:
=>2 – >@
Log Log K A n Log Q (6)
>
where Kb and n are the binding constant and number of binding sites, respectively. Plots of
log[(I0 − I)/I] vs. log[Q] are linear (Fig. 7). The intercept and slope of such plots can be used
to calculate Kb. The values of n close to 1 indicate that there is a single class of binding site
for the complex on BSA (Table 3). The binding free energy of 1 to BSA is −8.06 kcal mol−1.
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Figure 7: Scatchard plots of log [(I0−I)/I] vs log [Q] for determination of the complex-BSA binding constant and the number
of binding sites on BSA for 1.
Table 3: Estimated binding constants for site marker competitive experiments of the complex-BSA system.
System Kb (M-1) n R
Complex-BSA 8.23 8 10D 1.17 0.9983
E
Complex-BSA-Warfarin 6.66 8 10 0.98 0.9980
Complex-BSA-Ibuprofen 7.18 8 10D 1.16 0.9992
Complex-BSA-Digoxin 7.94 8 10D 1.20 0.9960
3.4.4. The Fluorescence Resonance Energy Transfer (FRET) between the complex 1 and
BSA
Energy transfer between two molecules is an important physical phenomenon with

considerable interest for the understanding of some biological systems.83 The mechanism of
fluorescence resonance energy transfer involves a donor fluorophore in an excited electronic
state, which may transfer its excitation energy to a nearby acceptor chromophore in a non-
radiative fashion through long-range dipole-dipole interactions.84 The effective energy
transfer from a protein residue (donor) to a complex (acceptor) occurs under the following
pre-conditions: (i) the donor can produce fluorescence light; (ii) the overlap between the
fluorescence emission spectrum of the donor and UV-Vis absorption spectrum of the acceptor
is enough; (iii) the donor and the acceptor must be in the close proximity to each other
(typically 1 to 10 nanometer).85 The overlap of the absorption spectrum of 1 with the
fluorescence emission spectrum of BSA in the wavelength range of 285-500 nm is shown in
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Fig. 8. The energy transfer efficiency (E) is related not only to the donor–acceptor distance
(r), but also to the critical energy transfer distance (R0). This is described by equation (7) 86:
1
F 1G H (7)
12 .((JI )K
2

where E is the energy transfer efficiency, I0 and I are the fluorescence intensities of BSA in
the absence and presence of the complex, r is the acceptor-donor distance and R0 is the
critical energy transfer distance when the transfer efficiency is 50%. The value for R0 can be
calculated by Equation (8) 87:
L
8.79 8 10Q0D $ 0 R QE ΦT (8)
where K2 is the dipole orientation factor, N is refractive index of the medium, Φ is the
fluorescence quantum yield of the donor and J is the spectral overlap integral between the
fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor
calculated as 87:
U V (W)XY (W)WZ [W
T (9)
U V (W)[W
Where \] (^) is the donor emission spectrum and _+ is the acceptor molar extinction
coefficient normally obtained from an absorption spectrum. In the present case, K2=2/3,
N=1.336 and Φ=0.15. Hence, using Eqs. (7)–(9) above, the following parameters can be
calculated: J = 6.372 × 10−14 cm3 M-1 , R0=3.47 nm, E=0.38 and r=3.76 nm for 1. The
obtained value for the distance between complex and BSA, r < 8 nm, and 0.5R0 < r < 1.5R0,
indicates that the energy transfer from BSA to complex occurred with high probability. The
binding of the complex to BSA was formed through energy transfer, which quenched the
fluorescence of BSA molecules, implying the presence of static quenching interaction.88
Furthermore, the greater r value compared with R0 confirms the static quenching
mechanism.89
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Figure 8: Spectral overlaps of the absorption spectra of 1 with the fluorescence spectra of BSA.
3.4.5. Site selective binding
BSA protein contains three domains (I-III) with two subdomains (A and B) on each
domain.90 Sites I, II and III of serum albumin show attraction for Warfarin, Ibuprofen and
Digoxin, respectively, according to Sudlow et al.91 Competition experiments were carried out
using Warfarin, Ibuprofen and Digoxin to recognize the specificity of the drug binding. The
concentration ratio of BSA and probe was 1:1 (6 × 10−6 M: 6 × 10−6 M) in these experiments
(Fig. 9). The fluorescence intensity significantly decreased upon the addition of site markers
into BSA solution. Plots of log[(I0 − I)/I] vs. log[complex] in the presence of site markers
were prepared to compare Warfarin, Ibuprofen and Digoxin influence on the complex
binding to BSA (Table 3). The binding constant for the complex-BSA system is 8.23 ×105M-1
and it is surprisingly variable in the presence of Warfarin. This shows the simultaneous
competition of Warfarin and 1. Thus, 1 binds to site I of serum albumin. This observation is
further supported by docking studies, as will be discussed later.
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Figure 9: Influence of selected site markers; A: Warfarin, B: Ibuprofen and C: Digoxin on the fluorescence of 1 bound to BSA.
-6 -5
[BSA] = [site markers] = 6×10 M; [complex] = (0-6×10 M). Insets: molecular structures of site markers.
3.5. Molecular docking
3.5.1. Docking with DNA
Molecular docking is generally an attractive procedure for understanding drug-DNA

interactions for rational drug design and discovery. Complex 1 has been successively docked
with DNA to predict the appropriate binding sites in this study. The results show that the
binding sites were obtained with the best binding free energy (∆Gb), (-6.5 kcal mol-1) for the
interaction of 1 with DNA. This shows that the results obtained in the absorption titration
experiments (-6.85 kcal mol-1) are reasonably comparable with those calculated by the
computational method. According to Fig. 10, 1 interacts with DNA into the minor groove. In
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addition, this is stabilized by hydrogen bonding between the sulfonyl oxygen of sac ligand
and guanine base of DNA.

Figure 10: (A) The interaction of complex with DNA using the Autodock 4.2 docking software. (B) The bases of DNA
interactions with 1 in the active site.
3.5.2. Docking with BSA
The molecular docking technique was also used to study the drug protein interactions
of 1 with the most probable site of BSA and verify our experimental results. The best value of
∆Gb for the interaction of the complex with BSA was found to be -7.64 kcal mol-1. The
obtained value is in agreement with the binding free energy obtained from the experimental
value of Kb (-8.05 kcal mol-1). Fig. 11 shows the docked conformations. 1 binds to BSA by
the residues such as Trp 134, Pro 281, Leu 282, Leu 283, Glu 284, His 18, Leu 154 and Glu
17, as observed.
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Figure 11: (A) Results of docking procedure of 1 in the interaction with BSA. (B) The BSA amino acid residues in interaction
with the complex, which results in quenching.
3.6. In vitro selective cancer cell cytotoxic activity

MTT dye reduction method, as a widely accepted bioassay, was used in the evaluation
of the in vitro cytotoxic activity of sodium saccharin, LH and 1 against HeLa cervical cancer,
A549 lung cancer and MCF-7 breast cancer cells. Furthermore, complex 1 was tested against
NIH normal fibroblast cells. Cytotoxicity is the degree to which an agent has a specific
destructive action in certain cells and is the quality of toxicity to cells. Cells were incubated
in the presence of increasing concentrations of the complexes for 48 h after which anti-
proliferation activity was measured according to the procedure described in the materials and
methods section. Cisplatin, a common anticancer drug, was used for comparison purposes
under identical experimental conditions. 1 showed significant anti-proliferation activity
against the cancer cell lines in the 0-100 µmol dm-3 range (Fig. S5, ESI†). Table 4 shows the
cytotoxicity data, expressed as IC50 values, which are the concentrations of a compound
inhibiting 50% of the cell growth. The in vitro cytotoxicity of 1 against the MCF-7 and A549
cell lines is higher than that of widely used drug, cisplatin, under identical conditions,
according to the IC50 values. Complexation process, on the other hand, significantly improves
the anticancer activity. These results suggest that this complex may be a potential anticancer
agent and further investigation is necessary to achieve better efficiency and find out the mode
of action. Complex 1 exhibits low cytotoxic activity (IC50: 165 µM) against normal NIH cells
after 48 h (Fig. S6, ESI†), although cisplatin as standard cytotoxic compound showed
significant toxicity. The selective effect of the compound was verified by SI values which
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were 7.43, 7.14, and 5.42 for MCF-7, A-549, and HeLa cell lines, respectively, in comparison
by the normal NIH cells. Therefore based on the SI definition, complex 1 can be imputed as
the selective compound against cancer cells. The results are supported with the other studies
in which more cytotoxicity was found against cancer cells compared with the normal cells92-

97
with regards the sensitivity of cancer cells towards the death compounds.
Table 4: Selective cytotoxicity data IC50 (µM) of the compounds against cancer and normal cell lines.
Compound IC50 (µM) ± SD

HeLa A549 MCF-7 NIH
Sodium saccharin >200 >200 >200 -

Ligand LH >200 93.8±4 >200 -
Complex 1 30.4±2 23.1±1 22.2±1 165±1
Cis-platin 17.6± 1 24.3 ± 1 26.3± 1 38.67± 4
4. Conclusion
A new Pd(II) complex has been synthesized and characterized by conventional

spectroscopic techniques in this study. Single crystal X-ray diffraction studies have also been
use to determine the molecular structures of the complex, showing that the Pd(II) ion is in a
distorted square planar coordination environment. The DNA binding properties of the
complex have been investigated by fluorescence spectroscopy, electronic absorption and
viscosity measurements. The results suggest that 1 interacts with CT-DNA via groove
binding. The results also show that this compound binds to BSA and changes its structure and
the quenching of BSA fluorescence by the complex is of static type. Moreover, the bound
complex is mainly located in site I, based on the competitive site marker experiments.
Molecular docking studies verified interaction mode of 1 with DNA and BSA. In addition,
the in vitro anticancer activity of the free ligands and Pd(II) complex against the three cancer
cell lines shows that 1 has more toxicity than the free ligands. The IC50 values indicate that
the complex is a potential anticancer candidate against the MCF-7 and A549 cell lines, but no
significant viability reductions were observed in normal cells.
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Electronic Supplementary Information (ESI)
Fig. S1: 1H NMR spectra of ligand LH in DMSO-d6. Fig. S2: 1H NMR spectra of complex 1
in DMSO-d6. Fig. S3: 13C {1H} NMR spectra of ligand LH in DMSO-d6. Fig. S4: 13C {1H}
NMR spectra of complex 1 in DMSO-d6. Fig. S5: In vitro cytotoxic activity of sodium

saccharin (A), ligand LH (B) and complex 1 (C) against HeLa, A549 and MCF-7 tumor cell
lines. Fig. S6: In vitro cytotoxic activity of complex 1 against NIH normal fibroblast cells.
CCDC 1522173 contains the supplementary crystallographic data for complex. These data
can be obtained free of charge from the Cambridge Crystallographic Data Center via
www.ccdc.cam.ac.uk/data_request/cif.
Acknowledgement
We are grateful to the Isfahan University of Technology (IUT) for financial support.
References:
1. N. P. Barry and P. J. Sadler, Chem. Commun. 2013, 49, 5106-5131.

2. G. Sava, G. Jaouen, E. A. Hillard and A. Bergamo, Dalton Trans. 2012, 41, 8226-8234.
3. K. Zheng, F. Liu, X.-M. Xu, Y.-T. Li, Z.-Y. Wu and C.-W. Yan, New J. Chem. 2014, 38,
2964-2978.
4. C. Rajarajeswari, R. Loganathan, M. Palaniandavar, E. Suresh, A. Riyasdeen and M. A.
Akbarsha, Dalton Trans. 2013, 42, 8347-8363.
5. M. A. Husain, S. U. Rehman, H. M. Ishqi, T. Sarwar and M. Tabish, RSC Adv. 2015, 5,
64335-64345.
6. T. Sarwar, H. M. Ishqi, S. U. Rehman, M. A. Husain, Y. Rahman and M. Tabish, Int. J.Biol.
Macromol. 2017, 98, 319-328.
7. M. Sirajuddin, S. Ali and A. Badshah, J. Photochem. Photobiol. B, Biol. 2013, 124, 1-19.
8. T. Sarwar, M. A. Husain, S. U. Rehman, H. M. Ishqi and M. Tabish, Mol. Biosyst. 2015, 11,
522-531.
9. S. Dasmandal, A. Kundu, S. Rudra and A. Mahapatra, RSC Adv. 2015, 5, 79107-79118.
10. Y. Bai, S. Sun, H. Zhang and T. Zhao, Anal. Methods. 2013, 5, 7036-7041.
11. J. Haribabu, K. Jeyalakshmi, Y. Arun, N. S. Bhuvanesh, P. T. Perumal and R. Karvembu,
RSC Adv. 2015, 5, 46031-46049.
12. A. R. Kapdi and I. J. Fairlamb, Chem. Soc. Rev. 2014, 43, 4751-4777.
13. L. Kelland, Nat. Rev. Cancer, 2007, 7, 573-584.
14. Y. P. Ho, S. C. Au‐Yeung and K. K. To, Med. Res. Rev. 2003, 23, 633-655.
15. M. M. Shoukry, A. A. Shoukry and M. N. Hafez, J. Coord. Chem. 2010, 63, 652-664.
16. K. Karami, M. Hosseini-Kharat, H. Sadeghi-Aliabadi, J. Lipkowski and M. Mirian, Eur.
J.Med. Chem. 2014, 73, 8-17.
17. E. G. Rodrigues, L. S. Silva, D. M. Fausto, M. S. Hayashi, S. Dreher, E. L. Santos, J. B.
Pesquero, L. R. Travassos and A. C. Caires, Int. J. Cancer, 2003, 107, 498-504.
18. B. B. Zmejkovski, A. Savić, J. Poljarević, N. Pantelić, S. Aranđelović, S. Radulović, S.
Grgurić-Šipka, G. N. Kaluđerović and T. J. Sabo, Polyhedron, 2014, 80, 106-111.
19. G. Gasser, I. Ott and N. Metzler-Nolte, J. Med. Chem. 2010, 54, 3-25.
20. D. J. Ager, D. P. Pantaleone, S. A. Henderson, A. R. Katritzky, I. Prakash and D. E. Walters,
Angew. Chem. Int. Ed. 1998, 37, 1802-1817.
21. C. T. Supuran, G. Loloiu and G. Manole, Rev. Roum. Chim. 1993, 38, 115-115.
22. M. C. Apella, R. Totaro and E. J. Baran, Biol. Trace Elem. Res. 1993, 37, 293-299.
23. V. T. Yilmaz, S. Guney and C. Thöne, Z. Anorg. Allg. Chem. 2002, 628, 1406-1410.
View Article Online
DOI: 10.1039/C7NJ03138F
24. E. J. Baran and V. T. Yilmaz, Coord. Chem. Rev. 2006, 250, 1980-1999.
25. Y. I. Binev, C. T. Petkov and L. Pejov, Spectrochim. Acta Mol. Biomol. Spectrosc. 2000, 56,
1949-1956.
26. E. Ulukaya, F. Ari, K. Dimas, E. I. Ikitimur, E. Guney and V. T. Yilmaz, Eur. J. Med. Chem.
2011, 46, 4957-4963.
27. F. Ari, N. Aztopal, C. Icsel, V. T. Yilmaz, E. Guney, O. Buyukgungor and E. Ulukaya,

Bioorg. Med. Chem. 2013, 21, 6427-6434.

28. M. Cavicchioli, A. C. Massabni, E. E. Castellano, L. P. Sabeh and C. M. Costa-Neto, Inorg.
Chim. Acta, 2007, 360, 3055-3060.
29. J. Marmur, J. Mol. Biol. 1961, 3, 208IN201-218.
30. M. Reichmann, S. Rice, C. Thomas and P. Doty, J. Am. Chem. Soc. 1954, 76, 3047-3053.
31. P. Sathyadevi, P. Krishnamoorthy, N. S. Bhuvanesh, P. Kalaiselvi, V. V. Padma and N.
Dharmaraj, Eur. J. Med. Chem. 2012, 55, 420-431.
32. R. Clark and J. Reid, Acta Crystallogr., Sect. A: Found. Crystallogr. 1995, 51, 887-897.
33. V. A. Bloomfield, D. M. Crothers and I. Tinoco, Physical chemistry of nucleic acids, Harper
& Row New York, 1974.
34. R. Esteghamat-Panah, H. Hadadzadeh, H. Farrokhpour, J. Simpson, A. Abdolmaleki and F.
Abyar, Eur. J. Med. Chem. 2017, 127, 958-971.
35. N. Nanjundan, P. Selvakumar, R. Narayanasamy, R. A. Haque, K. Velmurugan, R.
Nandhakumar, T. Silambarasan and R. Dhandapani, J. Photochem. Photobiol. B, Biol. 2014,
141, 176-185.
36. E. Alarcon, A. Aspée, M. Gonzalez-Bejar, A. Edwards, E. Lissi and J. Scaiano, Photochem.
Photobiol. Sci. 2010, 9, 861-869.
37. N. S. Quiming, R. B. Vergel, M. G. Nicolas and J. A. Villanueva, J. Health Sci. 2005, 51, 8-
15.
38. G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Goodsell and A. J.
Olson, J. Comput. Chem. 2009, 30, 2785-2791.
39. G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart, R. K. Belew and A. J.
Olson, J. Comput. Chem. 1998, 19, 1639-1662.
40. C. Hetényi and D. van der Spoel, Protein Sci. 2002, 11, 1729-1737.
41. C. Hetényi and D. van der Spoel, FEBS Lett. 2006, 580, 1447-1450.
42. N. Bordbar, M. H. Karimi and Z. Amirghofran, Cell. immunol. 2012, 280, 44-49.
43. M. Asadi, Z. Asadi, L. Zarei, S. B. Sadi and Z. Amirghofran, Spectrochim. Acta Mol. Biomol.
Spectrosc. 2014, 133, 697-706.
44. M. Asadi, Z. Asadi, S. B. Sadi, L. Zarei, F. M. Baigi and Z. Amirghofran, Spectrochim. Acta
Mol. Biomol. Spectrosc. 2014, 122, 118-129.
45. A. Koch, P. Tamez, J. Pezzuto and D. Soejarto, J. Ethnopharmacol. 2005, 101, 95-99.
46. M. A. Ali, A. H. Mirza, R. J. Butcher, M. T. H. Tarafder, T. B. Keat and A. M. Ali, J. Inorg.
Biochem. 2002, 92, 141-148.
47. K. Karami, M. H. Kharat, C. Rizzoli and J. Lipkowski, J. Organomet. Chem. 2013, 728, 16-
22.
48. V. T. Yilmaz, S. Caglar and W. T. Harrison, Z. Anorg. Allg. Chem. 2004, 630, 948-951.
49. O. Quinzani, S. Tarulli, O. Piro, E. J. Baran and E. Castellano, Z. Naturforsch., B: Chem. Sci.
1997, 52, 183-187.
50. P. Naumov and G. Jovanovski, Curr. Org. Chem. 2001, 5, 1059-1077.
51. P. Naumov and G. Jovanovski, J. Mol. Struct. 2001, 563, 335-339.
52. J. Luo, J. R. Khusnutdinova, N. P. Rath and L. M. Mirica, Chem. Commun. 2012, 48, 1532-
1534.
53. M. D. Santana, R. García-Bueno, G. García, G. Sánchez, J. García, A. R. Kapdi, M. Naik, S.
Pednekar, J. Pérez and L. García, Dalton Trans. 2012, 41, 3832-3842.
54. J. L. Serrano, J. Pérez, L. García, G. Sánchez, J. García, K. Tyagi and A. Kapdi, RSC Adv.
2012, 2, 12237-12244.
55. J. Pérez, L. García, E. Pérez, J. L. Serrano, J. F. Martínez, G. Sánchez, G. López, A. Espinosa,
M. Liu and F. Sanz, New J. Chem. 2003, 27, 1490-1496.
View Article Online
DOI: 10.1039/C7NJ03138F
56. L. Pauling, The nature of the chemical bond and the structure of molecules and crystals: an
introduction to modern structural chemistry, Cornell university press, 1960.
57. J. G. Delis, P. G. Aubel, K. Vrieze, P. W. van Leeuwen, N. Veldman and A. L. Spek,
Organometallics, 1997, 16, 4150-4160.
58. K. Karami, M. Hosseini-Kharat, C. Rizzoli, H. Tavakol and J. Lipkowski, J. Organomet.
Chem. 2014, 752, 152-160.

59. P. Naumov, G. Jovanovski, S.-Z. Hu, I.-H. Suh, I. A. Razak, S. Chantrapromma, H.-K. Fun
and S. W. Ng, Acta Crystallogr. Sect. C: Cryst. Struct. Commun. 2001, 57, 1016-1019.
60. R. Loganathan, S. Ramakrishnan, M. Ganeshpandian, N. S. Bhuvanesh, M. Palaniandavar, A.
Riyasdeen and M. A. Akbarsha, Dalton Trans. 2015, 44, 10210-10227.
61. K. Karami, M. Alinaghi, Z. Amirghofran and J. Lipkowski, Inorg. Chim. Acta, 2017.
62. A. Wolfe, G. H. Shimer Jr and T. Meehan, Biochemistry (Mosc). 1987, 26, 6392-6396.
63. C. Icsel and V. T. Yilmaz, DNA Cell Biol. 2013, 32, 165-172.
64. Z. Shokohi-pour, H. Chiniforoshan, A. A. Momtazi-borojeni and B. Notash, J. Photochem.
Photobiol. B, Biol. 2016, 162, 34-44.
65. L. Z. Zhang and P. Cheng, J. Inorg. Biochem. 2004, 98, 569-574.
66. M. Bordbar, M. Tabatabaee, M. Alizadeh-Nouqi, Z. Mehri-Lighvan, H. R. Khavasi, A.
YeganehFaal, F. Fallahian and M. Dolati, J. Iran. Chem. Soc. 2016, 1-8.
67. M. Bordbar, M. Tabatabaee, A. Yeganeh Faal, Z. Mehri Lighvan and R. Fazaeli, Synth. React.
Inorg. Met.-Org. Chem. 2015, 45, 1882-1888.
68. L. Z. Zhang and P. Cheng, Inorg. Chem. Commun. 2004, 7, 392-394.
69. K. Karami, Z. M. Lighvan, S. A. Barzani, A. Y. Faal, M. Poshteh-Shirani, T. Khayamian, V.
Eigner and M. Dušek, New J. Chem. 2015, 39, 8708-8719.
70. M. Anjomshoa, H. Hadadzadeh, S. J. Fatemi and M. Torkzadeh-Mahani, Spectrochim. Acta
Mol. Biomol. Spectrosc. 2015, 136, 205-215.
71. W. Villarreal, L. Colina-Vegas, C. Rodrigues de Oliveira, J. C. Tenorio, J. Ellena, F. C.
Gozzo, M. R. Cominetti, A. G. Ferreira, M. A. B. Ferreira and M. Navarro, Inorg. Chem.
2015, 54, 11709-11720.
72. N. Wang, L. Ye, B. Zhao and J. Yu, Braz. J. Med. Biol. Res. 2008, 41, 589-595.
73. R. Esteghamat-Panah, H. Farrokhpour, H. Hadadzadeh, F. Abyar and H. A. Rudbari, RSC
Adv. 2016, 6, 23913-23929.
74. K. Karami, S. Hashemi, J. Lipkowski, F. Mardani, A. A. Momtazi‐borojeni and Z. M.
Lighvan, Appl. Organomet. Chem. 2017.
75. X.-F. Zhao, Y. Ouyang, Y.-Z. Liu, Q.-J. Su, H. Tian, C.-Z. Xie and J.-Y. Xu, New J. Chem.
2014, 38, 955-965.
76. A. Sułkowska, J. Mol. Struct. 2002, 614, 227-232.
77. M. Van De Weert, J. Fluoresc. 2010, 20, 625-629.
78. J. Keizer, J. Am. Chem. Soc. 1983, 105, 1494-1498.
79. Y.-J. Hu, Y. Liu, J.-B. Wang, X.-H. Xiao and S.-S. Qu, J. Pharm. Biomed. Anal. 2004, 36,
915-919.
80. J. R. Lakowicz and G. Weber, Biochemistry (Mosc). 1973, 12, 4161-4170.
81. S. Lehrer, Biochemistry (Mosc). 1971, 10, 3254-3263.
82. R. L. Joseph and R. Lakowicz, Journal, 1999.
83. L. A. Sklar, B. S. Hudson and R. D. Simoni, Biochemistry (Mosc). 1977, 16, 5100-5108.
84. S. A. Hussain, arXiv preprint arXiv:0908.1815, 2009.
85. T. Főrster, Discuss. Faraday Soc. 1959, 27, 7-17.
86. F. Deng and Y. Liu, J. Lumin. 2012, 132, 443-448.
87. Y. Yue, X. Chen, J. Qin and X. Yao, J. Pharm. Biomed. Anal. 2009, 49, 753-759.
88. Y.-J. Hu, Y. Liu, L.-X. Zhang, R.-M. Zhao and S.-S. Qu, J. Mol. Struct. 2005, 750, 174-178.
89. W. He, Y. Li, C. Xue, Z. Hu, X. Chen and F. Sheng, Bioorg. Med. Chem. 2005, 13, 1837-
1845.
90. Y. Song, Y. Liu, W. Liu, F. A. Villamena and J. L. Zweier, RSC Adv. 2014, 4, 47649-47656.
91. I. SJÖHOLM, B. Ekman, A. KOBER, I. LJUNGSTEDT-PÅHLMAN, B. SEIVING and T.
SJÖDIN, Mol. Pharmacol. 1979, 16, 767-777.
92. M. Rakad and A. Jumaily, Iraqi Journal of Cancer and Medical Genetics, 2010, 3, 20-23.
View Article Online
DOI: 10.1039/C7NJ03138F
93. M. P. Heng, S. K. Sinniah, W. Y. Teoh, K. S. Sim, S. W. Ng, Y. K. Cheah and K. W. Tan,

Spectrochim. Acta Mol. Biomol. Spectrosc. 2015, 150, 360-372.
94. R. M. Vaden, K. M. Gligorich, R. Jana, M. S. Sigman and B. E. Welm, Breast Cancer Res.
2014, 16, 472.
95. L. R. Kelland, B. A. Murrer, G. Abel, C. M. Giandomenico, P. Mistry and K. R. Harrap,
Cancer Res. 1992, 52, 822-828.

96. J. L. Hickey, R. A. Ruhayel, P. J. Barnard, M. V. Baker, S. J. Berners-Price and A.

Filipovska, J. Am. Chem. Soc. 2008, 130, 12570-12571.
97. V. Zuco, R. Supino, S. C. Righetti, L. Cleris, E. Marchesi, C. Gambacorti-Passerini and F.
Formelli, Cancer Lett. 2002, 175, 17-25.
View Article Online
DOI: 10.1039/C7NJ03138F
Synthesis, characterization and biological activities of a

saccharinate-bridged palladacyclic dimer are reported in this
work.


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A saccharinate-bridged palladacyclic dimer with Pd-Pd

New Journal of Chemistry Accepted Manuscript

New Journal of Chemistry Accepted Manuscript

New Journal of Chemistry Accepted Manuscript

very promising in this regard.12-17

New Journal of Chemistry Accepted Manuscript

2.2. Methods and instrumentation

An FT-IR JASCO 680-PLUS spectrophotometer was used to record Fourier transform

400.13 MHz (1H) and 100.61 MHz 13

New Journal of Chemistry Accepted Manuscript

2.3.1. Synthesis of ligand LH: C14 H12NBr

2.3.2. Synthesis of complex 1: [Pd2((C,N)L)2(µ-sac)2]

New Journal of Chemistry Accepted Manuscript

2.5. DNA binding studies

A CT-DNA stock solution was prepared in Tris-HCl buffer (5 mM Tris-HCl, 50 mM

New Journal of Chemistry Accepted Manuscript

2.6. Protein binding studies

In order to identify the binding location of 1 on BSA, site competitive replacement

2.7. Molecular docking

New Journal of Chemistry Accepted Manuscript

2.8. The cell proliferation assay

3. Results and discussion

3.1. Synthesis and spectroscopic characterization

New Journal of Chemistry Accepted Manuscript

saccharin to form [Pd2((C,N)L)2(µ-sac)2] (1) (Scheme 2).

(LH) (a) (b)

Scheme 2: Synthetic route for the preparation of the Pd(II) complexes.

New Journal of Chemistry Accepted Manuscript

3.2. Molecular structure of 1

New Journal of Chemistry Accepted Manuscript

1 shows an ORTEP view of the dinuclear complex 1.

Figure 1: Partially labelled ORTEP view of molecular structure of 1.

Table 1 summarizes the relevant crystallographic data and structural refinement

New Journal of Chemistry Accepted Manuscript

Table 1: Crystallographic data and structure refinement details for complex 1.

Formula weight 2246.88

T/K 100 (1) K

Crystal system MONOCLINIC

Space group I2/c

a/Å 43.3833 (10) Å

b/Å 9.7679 (2) Å

c/Å 38.6961 (7) Å

Dcalc/ Mg m−3 1.827

Independent reflections 14931

Final R indices R1=0.0542, wR2=0.1481

R indices (all data) R1=0.0638,wR2=0.1564

New Journal of Chemistry Accepted Manuscript

Largest difference peak and hole / 1.74 , −1.71

C36—Pd1—O6 173.3 (2) O6—Pd1—Pd2 83.22 (10)

3.3. DNA binding studies

DNA/(ε  ε )  DNA/(ε  ε )  1/(K  (ε  ε ) ) (1)

New Journal of Chemistry Accepted Manuscript

Binding constants are measures of compound-DNA complex stability whereas free

3.3.2. Fluorescence studies of competitive DNA binding

New Journal of Chemistry Accepted Manuscript

DNA/(ε ε ) DNA/(ε ε ) 1/(K (ε ε ) ) (1)

%&'(( %') * (+,-.+,/ )/0 (3)