A Pd(II) complex derived from pyridine-2-carbaldehyde oxime ligand: Synthesis, characterization, DNA and BSA interaction studies and in vitro anticancer activity

Article abstract of DOI:10.1016/j.molstruc.2020.128479

A novel Pd(II) complex, [Pd(C₆H₅N₂O)₂], containing pyridine-2-carbaldehyde oxime ligand has been synthesized and characterized using elemental analysis, Fourier-transform infrared, nuclear magnetic resonance and mass spectroscopy. The single crystal structure of this Pd(II) complex has been determined by X-ray crystallography. Based on the DNA binding studies including ultraviolet visible spectrophotometry, fluorescence emission titration and viscosity measurement, the interaction of Pd(II) complex with calf thymus DNA occurs by groove binding. In the absence of an external reductant, the Pd(II) complex cleaves the supercoiled double-stranded DNA under physiological conditions. Moreover, in the presence of Pd(II) complex, the Bovine Serum Albumin microenvironment and secondary structure change. On the basis of the competitive experiments using site markers, the complex is mainly located in site I of the protein. The binding of the Pd(II) complex to DNA was modeled using molecular docking. The antitumor impacts of the ligand and the Pd(II) complex were evaluated in vitro against the mouse colon carcinoma (C26) and melanoma (B16–F0) tumor cell lines. The antitumor activity has been significantly improved by the complexation process. IC₅₀ values smaller than those of cisplatin have been shown by the Pd(II) complex and oxime ligand against cancer cell lines. In addition, Pd(II) complex has been tested against NIH normal fibroblast cells. Consequently, Pd(II) complex may be considered a selective compound against cancer cells, according to the SI definition.

Full text of DOI:10.1016/j.molstruc.2020.128479

Journal Pre-proof
A Pd(II) complex derived from pyridine-2-carbaldehyde oxime ligand: Synthesis,
characterization, DNA and BSA interaction studies and in vitro anticancer activity
Moloud Alinaghi, Kazem Karami, Azar Shahpiri, Akram Kazemi nasab, Amir Abbas
Momtazi-borojeni, Elham Abdollahi, Janusz Lipkowski
PII:
S0022-2860(20)30804-8
DOI:
https://doi.org/10.1016/j.molstruc.2020.128479
MOLSTR 128479
Reference:
To appear in: Journal of Molecular Structure
Received Date: 3 March 2020
Revised Date: 14 May 2020
Accepted Date: 16 May 2020
Please cite this article as: M. Alinaghi, K. Karami, A. Shahpiri, A.K. nasab, A.A. Momtazi-borojeni, E.
Abdollahi, J. Lipkowski, A Pd(II) complex derived from pyridine-2-carbaldehyde oxime ligand: Synthesis,
characterization, DNA and BSA interaction studies and in vitro anticancer activity, Journal of Molecular
Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128479.
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A Pd(II) complex derived from pyridine-2-carbaldehyde oxime
ligand: synthesis, characterization, DNA and BSA interaction
studies and in vitro anticancer activity
b
Moloud Alinaghia, Kazem Karami*a, Azar Shahpiri , Akram Kazemi nasabb, Amir
Abbas Momtazi-borojenic,d , Elham Abdollahid , Janusz Lipkowskie.
aDepartment of Chemistry, Isfahan University of Technology, Isfahan, 84156/83111, Iran
bDepartment of Agricultural Biotechnology, Isfahan University of Technology, Isfahan 84156-83111, Iran
cNanotechnology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran
dDepartment of Medical Biotechnology, School of Medicine, Student Research Committee, Mashhad University of Medical
Sciences, Mashhad, Iran
eInstitute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
Abstract
A novel Pd(II) complex, [Pd(C6H5N2O)2], containing pyridine-2-carbaldehyde oxime ligand
has been synthesized and characterized using elemental analysis, Fourier-transform infrared,
nuclear magnetic resonance and mass spectroscopy. The single crystal structure of this Pd(II)
complex has been determined by X-ray crystallography. Based on the DNA binding studies
including ultraviolet visible spectrophotometry, fluorescence emission titration and viscosity
measurement, the interaction of Pd(II) complex with calf thymus DNA occurs by groove
binding. In the absence of an external reductant, the Pd(II) complex cleaves the supercoiled
double-stranded DNA under physiological conditions. Moreover, in the presence of Pd(II)
complex, the Bovine Serum Albumin microenvironment and secondary structure change. On
the basis of the competitive experiments using site markers, the complex is mainly located in
site I of the protein. The binding of the Pd(II) complex to DNA was modeled using molecular
docking. The antitumor impacts of the ligand and the Pd(II) complex were evaluated in vitro
against the mouse colon carcinoma (C26) and melanoma (B16-F0) tumor cell lines. The
antitumor activity has been significantly improved by the complexation process. IC50 values
smaller than those of cisplatin have been shown by the Pd(II) complex and oxime ligand
against cancer cell lines. In addition, Pd(II) complex has been tested against NIH normal
fibroblast cells. Consequently, Pd(II) complex may be considered a selective compound
against cancer cells, according to the SI definition.
Keywords:
Palladium(II) complex; Pyridine-2-carbaldehyde oxime; DNA interactions; BSA interaction;
Molecular docking; In vitro cytotoxicity

1. Introduction
Metal complexes, which can cleave DNA under physiological conditions, have recently been
of interest both for their possible applications in artificial nucleic acid chemistry and the
development of anticancer agents based on metals [1-3].
The side effects of cisplatin limit its potential efficiency despite its activity in the treatment of
several cancers types [4, 5]. The mechanism of cisplatin action includes binding to DNA in a
covalent manner [6]. Thus, the development of more efficient, target specific, less hazardous
and cytotoxic drugs, which bind non-covalently to DNA, is inevitable.
The most abundant proteins in the circulatory system, serum albumins, are well known for
acting as transporters of several compounds [7], such as drugs and nutrients, mainly by
forming non-covalent complexes as specific binding sites. Thus, the absorption, distribution,
metabolism, excretion properties, stability and toxicity of chemical substances can be
remarkably affected by their binding activities to serum albumins [8]. Bovine Serum
Albumin has been extensively investigated because of its homologous structure to Human
Serum Albumin [9].
Considering the unique capability of biologically active organic ligands to bind with different
biological targets, their coordination to transition metal ions is reportedly promising [10].
Oximes are a group of important functional ligands applied in medicine [11-13], magnetism
[14], catalysis [15] and crystal engineering [16-18]. Oximes are universal ligands easily
designed to form compounds of different properties. In general, there is one active nitrogen in
the neutral oxime group, >C=NOH, which binds to metal cations. Thus, it functions as a
terminal-coordinated monodentate ligand. However, a large number of possibilities are
created to affect the coordination mode by using different substituents in the oxime carbon
depending on the additional coordination sites in the substituents such as those in pyridine
[19-22]. Moreover, the position of the coordinative atoms in the substituent strongly affects
the structure of the material, as observed in the coordination of 2-pyridyl oxime, which
usually generates a chelating mononuclear complex, compared with 3-pyridyl or 4-pyridyl
oxime, which preferably yields polymeric chain like structures via terminal coordination [23].
In addition, complexes with a large variety of nuclearity can be formed by the deprotonation
of the oxime OH group using the negatively charged oxygens for additional coordination [17,
18, 24]. A lot of hydrolytic processes including acyl, phosphoryl or sulfuryl transfer or
cleavage via attacking an electrophilic center, are responsible for the strong nucleophilicity of

the oximate anion (>C=N−O−) [25]. The distinct capability of a family of pyridyl oximes for
reactivation of acetylcholinesterase enzyme (AChE) upon complete inhibition by
organophosphorus compounds as pesticides and chemical warfare agents is particularly
interesting from the pharmaceutical aspect [26-30]. In addition, a wide range of biological
applications of pyridyl oxime derivatives such as sedative, anti-depressant, anti-spasmodic,
analgesic and anti-inflammatory among others in cardiovascular system has been studied
[31]. The studies on the coordination chemistry of pyridine-2-carbaldehyde oxime were first
carried out in the late fifties and early sixties. Single crystal X-ray structures were not
available back then. According to the physical and spectroscopic data based studies,
intramolecular hydrogen bonding in the type of structure can be formed by the square planar
cationic complexes of divalent transition metals [32-34]. [Cu3(OH)(SO4)-{(py)CHNO}3] was
the first structurally characterized pyridine-2-carbaldehyde oxime metal complex [35]. The
2,6-diacetylpyridine dioxime (dapdoH2) monoanion was originally considered a tridentate
chelate in palladium(II) and platinum(II) chemistry. [PdCl(dapdoH)] and [PtCl(dapdoH)]
have been biologically evaluated [36].
In this work, a novel Pd(II) complex, [Pd(C6H5N2O)2], containing pyridine-2-carbaldehyde
oxime ligand has been prepared. Elemental analysis, Fourier-transform infrared, nuclear
magnetic resonance and mass spectroscopy have been used to characterize the complex. The
single crystal structure of this Pd(II) complex was determined by X-ray crystallography.
Electronic absorption titration, fluorescence spectroscopy and viscosimetric measurement
were used to study the DNA and BSA binding behaviors of this compound. Furthermore, the
DNA cleavage activity of the Pd(II) complex and oxime ligand has been measured. In
addition, molecular docking studies have been used to obtain detailed information on the
binding of the complex with DNA and BSA. Finally, the cytotoxic activity of compounds
against mouse colon carcinoma (C26), mouse melanoma (B16-F0) and normal fibroblast cells
(NIH) has been evaluated using MTT assay.
2. Experimental Section
2.1. Materials
Commercial grade reagents in this work were used as received. Reagent grade Calf-thymus
DNA (CT-DNA), BSA and methylene blue (MB) were supplied by Sigma Aldrich Chemical
Co. Extra pure solvents applied in the syntheses and physical measurements and analytical
reagent grade. Tris(hydroxymethyl)-aminomethane (Tris-HCl) buffer were provided by

Merck Chemical Co. Stock solutions of oxime ligand and other solutions were prepared using
doubly distilled deionized water. Stock solutions of concentrated Pd(II) complex were
prepared in DMSO solvent and the appropriate dilutions were prepared using Tris-HCl buffer
at pH 7.2 to obtain the concentrations required throughout the experiments. Maximum
concentration of DMSO was 0.1% v/v. Stock solutions of CT-DNA and BSA were prepared
by dissolution in 5 mM Tris buffer and 50 mM NaCl at pH 7.2. Reagents and cell culture
media were purchased from Gibco Company (Germany).
2.2. Methods and Instrumentation
An FT-IR JASCO 680-PLUS spectrophotometer was used to record FT-IR spectra in the
400-4000 cm−1 region using potassium bromide pellets. A JASCO 7580 UV-Vis-NIR double
beam spectrophotometer was used to record the electronic absorption spectra in a 1 cm path
length cell. Fluorescence spectra were recorded in solution using a Cary Eclipse fluorescence
13
spectrophotometer. NMR spectra were recorded at 400.13 MHz (1H) and 100.61 MHz C
{1H} using a Bruker spectrometer. A Leco CHNS-932 apparatus was used to perform
elemental analyses. An Agillent 6410 QQQ spectrophotometer was used to record mass
spectra. A Cannon Fenske routine viscometer was used to perform viscometric titrations at a
constant temperature of 298.0 (±0.1) K in thermostatic water bath.
2.3. Syntheses
2.3.1. Synthesis of pyridine-2-carbaldehyde oxime ligand (C6H5N2O)
0.276 g of hydroxylamine hydrochloride (4 mmol) was slowly added to 190.2 µL of 2-
pyridinecarboxaldehyde (2 mmol) in ethanol, followed by the dropwise addition of an
aqueous 2 mM solution of sodium hydroxide. The clear solution thus formed was stirred at 50
°C for 2 h. Removal of the solvent by evaporation yielded the ligand as a white precipitate.
(Yield: 93.4%). IR (KBr, cm-1) ν (C-HPh) = 3186-3004, ν (C-Himine) = 2882-2775, ν
1
(C=Nimine) = 1657.ν (C=Npyridine) = 1619. H NMR (DMSO-d6, ppm): δ= 7.38 (ddd, 1H, Hb),
7.78-7.84 (m, 2H, Hc, Hd), 8.07 (s, 1H, HC=N), 8.57 (br d, 1H, Ha), 11.74 (br s, 1H, OH).
2.3.2. Synthesis of Pd(II) complex [Pd(C6H5N2O)2]
0.04 g of the oxime ligand (0.3 mmol) and 0.024 g of sodium acetate (0.3 mmol) were
dissolved in a solvent mixture containing acetonitrile (10 ml) and water (1 ml), and the
mixture obtained was stirred at ambient temperature for about 20 min. A solution of 0.08 g
of Pd(OAc)2 (0.36 mmol) in 5 ml of acetonitrile was added in a dropwise manner to the
system, which was then refluxed at 60 °C for 6 h to obtain a dark green solution. The mixture

was concentrated to 2 ml, followed by adding 15 ml of n-hexane to precipitate a dark green
powder, which was then dried at room temperature.
(Yield: 76.9%). Anal. Calc. for C12H10N4O2Pd (%) C, 41.38; H, 2.87; N, 16.09. Found C,
41.27; H, 2.63; N, 16.23. IR (KBr, cm-1) ν (C-HPh) = 3049-3008, ν (C-Himine) =2955, 2852, ν
1
(C=Nimine) = 1612, ν (C=Npyridine) = 1608. H NMR (DMSO-d6, ppm): δ= 7.4 (dd, 1H, Hb),
13
7.54 (d, 1H, Hd), 7.74 (s, 1H, HC=N), 8.02 (dd, 1H, Hc), 9.65 (d, 1H, Ha). C{1H} NMR
(DMSO-d6, ppm): 118.6 (Cb), 120.04 (Cd), 140.02 (C=N), 140.5 (Cc), 146.5 (Ce), 157.4 (Ca).
ESI-MS, m/z (%): 348.8 [M + H].
2.4. Crystallography
X-ray diffraction experiments were performed using an Agilent SuperNova single crystal
diffractometer (Cu K(a) radiation at 150 K. The analytical numeric absorption corrections
were made using a multi-faceted crystal model based on expressions derived by R.C. Clark &
J.S. Reid was used to make [37]. SHELXS97 and SHELXL (Sheldrick 2008) programs were
used to solve and refine the structures by direct methods.
2.5. DNA Binding Experiments
CT-DNA experiments, were carried out in Tris-HCl buffer solution (pH=7.2). CT-DNA
solutions showed a UV absorbance ratio (A260/A280) of > 1.8, indicating the DNA purity [38].
Absorption spectroscopy by the molar absorption coefficient (ε = 6600 M–1 cm–1) at 260 nm
was use to find the DNA concentration per nucleotide [39, 40]. Absorption spectral titration
experiments were performed at constant 60 µM concentration of the Pd(II) complex,
changing CT-DNA concentration in the range of 0.0-120 µM. In addition, viscosity
measurements were used to further study the Pd(II) complex binding mode. The mean values
of three replicate measurements were used in the evaluation of sample viscosities. Relative
⁄
ꢂ ꢃ
⁄
specific viscosity (ꢀ ꢀꢁ)
values, in which ꢀ and ꢀꢁ are the specific viscosity
contributions of DNA in the presence (ꢀ) and absence (ꢀꢁ) of each compound, respectively,
were plotted vs. r (r = [complex]/[DNA]) [41]. The relation ꢀ = (t - t0)/t0, in which t0 and t
indicate the flow times of the blank buffer and DNA containing solutions, respectively, was
used to calculate the relative viscosities [42]. The competitive experiment was performed to
study the ability of the Pd(II) complex to remove methylene blue from DNA methylene blue
mixture. The DNA-methylene blue solution ([DNA]/[methylene blue] = 5) was excited at a
wavelength of 630 nm in the absence and presence of various complex concentrations [43].
2.6. DNA Cleavage Experiments

DNA cleavage was examined using agarose gel electrophoresis carried out by incubation at
37 °C as follows: plasmid PUC57 (0.01 M) in 5 mM Tris buffer (pH=7.2) was subjected to
treatment with oxime ligand or Pd(II) complex, followed by incubation for 3 h. The samples
were then examined by electrophoresis for 1 h at 135 V on 0.07% agarose gel using tris-boric
acid-EDTA buffer. Following electrophoresis, the bands were visualized using UV light and
photographed. The cleavage mechanism of PUC57 was studied in the presence of H2O2,
DMSO and NaN3 as the oxidizing agent, hydroxyl scavenger and singlet oxygen scavenger,
respectively.
2.7. Protein Binding Studies
The stock solution of Bovine Serum Albumin was prepared by dissolving the proper amount
of BSA in the buffer solution, which contained 5 mM Tris-HCl/50 mM NaCl at pH 7.2, and
stored at 4 °C for further application. UV visible absorption spectroscopy was used to obtain
BSA concentration using ε280 = 44300 M-1cm-1 [44]. Absorption titration experiments were
performed by keeping BSA concentration constant at 6 µM while different concentrations of
the complex were added (0.0-12 µM). During the absorption spectral measurements, identical
amounts of Pd(II) complex solution were added to both the BSA and the reference solutions
in order to exclude the absorbance due to the complex. In the tryptophan fluorescence
quenching experiment, the tryptophan residues of BSA [45] were quenched by maintaining
the BSA concentration constant at 6 μM while changing the concentration of the complex
(quencher) in the range of 0.0-12 µM to provide solutions with different molar ratios of
quencher to BSA. Having added the quencher, the fluorescence spectra were recorded at
excitation and emission wavelengths of 280 and 343 nm, respectively, in the fluorometer. Site
competitive replacement tests were performed in the presence of Warfarin, Ibuprofen and
Digoxin site markers by fluorescence titration method to find the binding location of Pd(II)
complex on BSA. Equal concentrations of BSA and site marker (6 µM) were used, followed
by slow addition of Pd(II) complex. The fluorescence spectra were recorded at 298 K with an
excitation wavelength and emission of 280 nm and 300-500 nm, respectively.
2.8. Molecular Docking
Auto Dock 4.2 package was used to carry out molecular docking studies by the Lamarckian
genetic algorithm (LGA) [46, 47]. Conversion of CIF file using Mercury software gave the
PDB format of Pd(II) complex. The initial Bovine Serum Albumin structure was taken from
the Protein Data Bank (PDB ID: 4F5S) at a resolution of 2.47 Å. DNA sequence was also

obtained from the Protein Data Bank (PDB ID: 3U2N) at a resolution of 1.25 Å. Chain (A) of
BSA and all the other hetero atoms including water molecules were separated during
preparation input files. The suitable binding site for complex-DNA interaction and BSA was
found by blind docking [48, 49]. Afterwards, focus docking was carried out on the best
location. Biomolecule structures were limited to a grid box with 60 × 60 × 60 Å3 dimensions
and a 0.375 Å grid spacing, which involved nearly all the macromolecules. All other
parameters were maintained at their default values.
2.9. Culture Medium and Cell Lines
The National Cell Bank of Pasteur Institute, Tehran, Iran, provided melanoma B16F0 and
colon carcinoma C26 cancer cell lines. Cells were cultured in Dulbecco’s Modified Eagle
Medium (DMEM), which was supplemented with 10% heat inactivated fetal bovine serum
(FBS), 100 U.ml-1 penicillin, 100 μg.ml-1 streptomycin and 5 mM L-glutamine. Afterwards,
the cell lines were grown at the temperature of 37 °C in a humidified atmosphere which
contained 5% CO2.
2.10. MTT Assay
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] colorimetric assay was
employed to study the cytotoxicity of the complex and ligand. 200 μl of cells (5×104 cells.ml-
1) were seeded in 96 well microplates, followed by incubation in 37°C and 5% CO2 for 24 h
to perform the cytotoxicity assay. Afterwards 20 μl of the solutions at specific concentrations
(in DMSO) of the oxime ligand and Pd(II) complex were added to the wells. The control and
treated solutions included the same DMSO concentration [0.5% (v/v)]. 20 μl of MTT solution
(5 mg.ml-1 in phosphate buffer solution) were added 48 h through the incubation, followed by
incubation for another 3 h. Subsequently, the formazan crystals formed were dissolved by
slow replacement of 150 μl of the medium containing MTT with DMSO. An ELISA plate
reader was then used to determine the absorbance at 560 nm. Each test was performed in
triplicate. Non-linear regression of concentration-response curves of the complex against each
tested cell line was used to calculate IC50 (concentration inhibiting the cell growth by 50%)
values.
3. Results and discussion
3.1. Synthesis of the complex and characterization by spectroscopic methods
[Pd(C6H5N2O)2] was prepared by reacting oxime ligand with palladium acetate (1:1.2 molar
ratio) in acetonitrile and water at 60 °C (Scheme 1).

The characteristic bands at 1657 and 1619 cm-1 in the IR spectrum of oxime ligand are
associated with (C=N) stretching vibrations of oxime and pyridine groups. ν (C=N) has been
shifted to a lower frequency in the Pd(II) complex, which shows the nitrogen atom of imine
group has bonded to the metal cation [50].
1
In the H NMR spectrum of the oxime, the aromatic protons (Ha, Hb, Hc and Hd) appear at
7.4-8.5 ppm. Since these protons are non-equivalent, splitting and formation of doublets are
observed (Fig S1). As observed in Scheme 2, Hb has been split by other hydrogens and
appears as a doublet of doublets at 7.38 ppm. Hc and Hd signals have merged and appear at
7.78-7.84 ppm. Ha has resonated as a broad doublet at 8.57 ppm. These protons show up at
different frequencies (7.4-9.65 ppm) in the 1H NMR spectrum of Pd(II) complex (Fig S2). In
the complex spectrum, Ha and Hd appear as doublets. Furthermore, Hb and Hc are represented
4
by a doublet of doublets, which indicates the disappearance of JH-H. Furthermore, a singlet
can be observed at 8.07 ppm in the oxime spectrum, which is associated with the imine
protons of the ligand that has been shifted to a lower frequency (7.74 ppm) because of
1
generation of the complex. As Fig. S1 shows, a broad singlet is observed in the H NMR
spectrum of the ligand at 11.74 ppm. This singlet, which is not observed in the Pd(II)
complex spectrum, corresponds to O-H proton of the oxime ligand. Six types of carbon are
present in the 13C NMR spectrum of the complex (Fig. S3) in the 118.6-157.4 ppm range.
Positive ion Electrospray Ionization Mass Spectrum of the complex shows a major peak at
m/z = 348.8, which is attributed to the [M + H]+ (Fig. S4).
3.2. Molecular Structure of Pd(II) Complex
Single crystal X-ray diffraction in the solid phase has been used to characterize the Pd(II)
complex The slow diffusion of hexane into a CH2Cl2 solution yielded suitable crystals. Fig. 1
shows the ORTEP diagram of the Pd(II) complex.
Table 1 shows the corresponding crystallographic data and structural refinement details and
the selected bond lengths and angles are summarized in Table 2. This complex was
crystallized in the triclinic space group P1 with Z=2. In this complex, the coordination
number around the palladium atom is four, which indicates that Pd(II) atom is bonded to two
oxime ligands from two imino nitrogens and two pyridine nitrogen atoms of the ligands. The
sum of angles around palladium atom is 360.3° for Pd1, the distortion being the most

pronounced in the relatively decreasing “bite” angle of the metalated moiety. The bond angles
of N1-Pd1-N2 and N3-Pd1-N4 are forced to 80.2(4) and 79.7(3)°, respectively, by the
restraints of the five membered ring. Palladium(II) metal square planar geometry is slightly
tetrahedrally distorted and the mean deviations from the least square plane are only 0.030 Å.
The Pd-N bond lengths are 2.031(9) and 2.041 (9) Å for Pd1-N1 and Pd1-N3, respectively,
and 2.035(8) Å for Pd1-N2 and Pd1-N4, which are longer than the sum of the covalent radii
of Pd and N(sp2) atoms (2.011 Å) [51]. In addition, N-O bond lengths in this complex fall in
the ranges found for the corresponding complexes [20, 52].
3.3. DNA Binding Experiments
3.3.1. Electronic Absorption Titration
Molar absorptivity considerably decreases when CT-DNA is added to Pd(II) complex in 5
mM Tris-HCl/50 mM NaCl buffer at pH 7.2 (Fig. 2). The binding of the complex to DNA via
groove binding mode is suggested by the hypochromic change observed without any shift in
its electronic spectrum [53, 54]. The intrinsic binding constants, Kb, of the Pd(II) complex is
obtained by the analysis of the absorption spectral data using Eq. 1 to enable the quantitative
comparison of the DNA binding affinities [55].
[DNA]/(εꢄ − εꢅ) = [DNA]/(εꢆ − εꢅ) + 1/(Kꢆ (εꢆ − εꢅ ) )
(1)
in which [DNA] and ɛa, which is found by calculation of Aobs/[complex], are the
concentration of DNA in base pairs and the apparent extinction coefficient, respectively, and
ɛf and ɛb are the extinction coefficients of the Pd(II) complex in its free form and the complex
in the bound form, respectively. Fitting the set of data into the above equation gives a straight
line with a slope and y-intercept of 1/(ɛb − ɛf) and 1/Kb(ɛb − ɛf), respectively. The slope to
intercept ratio gives Kb (Fig. 2, inset). The value of the obtained intrinsic binding constants,
Kb, (1.44×105 M-1) is lower than those observed for proven intercalators (EtBr and methylene
blue with Kb values of 3.3×105 and 3.45×105 M-1, respectively) [56, 57].
Equation 2 was used to calculate the free energy (∆G) of compound-DNA complex by using
the values of binding constant (Kb):
∆ꢇ = −ꢈꢉ ln ꢊꢆ
(2)

Binding constant shows the stability of the compound-DNA complex while free energy is a
measure of the spontaneity/non-spontaneity of the compound-DNA binding. A negative free
energy was found for the Pd(II) complex (-7.04 kcal mol-1), which indicates that compound-
DNA interaction is spontaneous.
3.3.2. Emission Experiments of Competitive DNA Binding
A comparative binding study was carried out using methylene blue to figure out if the Pd(II)
complex binding with DNA is of the groove or the intercalative type. Methylene blue (MB),
which is a phenothiazinium dye, reportedly bonds with nucleic acids. The binding of
methylene blue to DNA is presumably stabilized by the planar heterocyclic dye via favorable
stacking interactions with its neighboring base pairs [58]. The intercalative binding of MB
with DNA can be used to rationalize the great quenching in the MB emission intensity in the
DNA environment [59], which is expected given the intercalation mechanism [59, 60]. The
DNA bound MB emission spectra have been monitored in the presence of different complex
concentrations (Fig. S5). Based on the experimental results, the complex addition to the DNA
bound methylene blue did not lead to the release of methylene blue molecules whereas the
fluorescence intensity continuously reduced, implying that the two probes, complex and
methylene blue, independently bond with DNA and the binding of one probe was not
influenced by that of the other. The interaction of Pd(II) complex with the DNA was a groove
interaction, as verified experimentally [54, 61].
3.3.3. Viscosity Measurements
DNA viscosity is sensitive to the variations in DNA length. The relationship between the
⁄
relative viscosity (ꢀ ꢀꢁ) and DNA length (L/L0) is shown by L/L0 = (ꢀ ꢀꢁ)ꢂ ꢃ, where L and
⁄
⁄
L0 are the apparent molecular lengths in the presence and absence of the compound,
respectively [62]. The relative specific viscosities of methylene blue and Pd(II) complex
⁄
(ꢀ ꢀꢁ)ꢂ ꢃ, were plotted vs. r (r = [compound]/[DNA]) (fig. S6). As the amount of methylene
⁄
blue increases, the DNA relative viscosity is continuously enhanced, which is in agreement
with the lengthening of the DNA helix. The viscosity of DNA solutions significantly
increases by the intercalation because of DNA helix lengthening when the base pairs are
separated to incorporate the aromatic chromophore of the bound molecule. It is attractive to
associate the increased viscosity observed with the intercalative interaction of methylene blue
[63]. Interestingly, the specific viscosity of DNA slightly decreases for the Pd(II) complex.
Little or no change is observed in the DNA solution viscosity when small molecules

exclusively bind in the DNA grooves under identical conditions [42, 64]. These results again
suggest the interaction of Pd(II) complex with the groove of DNA, as verified by the above
findings.
3.3.4. DNA Cleavage Studies
Metallonucleases have the potential for application in cancer therapy [65]. Some of the
clinically approved anticancer agents aim at the cell death by cleaving DNA [66, 67]. The
DNA cleavage ability of transition metal complexes is an effective means to study the
mechanism of toxicity and artificial nuclease abilities. The DNA-cleavage reactions consist
of three steps: formation of supercoiled, open circular and linear forms (Forms I, II and III,
respectively). The intact supercoil form (Form I) will rather quickly migrate upon exposure of
circular plasmid DNA to electrophoresis. If scission takes place on one strand (nicking), the
supercoil will relax to form a slower moving open circular form (form II). The cleavage of
both strands gives rise to a linear form (form III) migrating between Forms I and II [68].
DNA cleavage without added reductant: The nuclease activity of pyridine-2-carbaldehyde
oxime ligand and its Pd(II) complex in physiological conditions was investigated utilizing
pUC57 plasmid DNA in 5 mM Tris buffer (pH = 7.2) medium without external agents.
Different Pd(II) complex concentrations can cleave a pUC57 plasmid DNA (Form I) into a
nicked form (Form II) (Fig. 3, lines 2–8). No cleavage was observed in the DNA cleavage
experiments carried out with oxime ligand (Fig. 3, lines 9–11). In addition, this shows the
concentration dependence of DNA cleavage activity of the Pd(II) complex.
DNA cleavage with added reductant: Considerable nuclease activity on supercoiled DNA
was shown by the Pd(II) complex in the absence of external agents in a concentration
dependent manner. Pd(II) complex cleaved supercoiled form (Form I) only produced nicked
circular form (Form II) and no linear form (Form III) in the absence of H2O2 (an oxidizing
agent) (Fig. 3, lines 2-8). The activity of DNA cleavage in the absence of an oxidant is often
hydrolytic rather than oxidative [3]. Nevertheless, the most impressive characteristic is that a
higher nuclease activity of the complex and linear form (Form III) is observed in the presence
of H2O2 (Fig. 4, line 3). Pd(II) complex DNA cleavage can be efficiently promoted in the
presence of hydrogen peroxide, according to the results [69]. In addition, oxime ligand
exhibited no significant nuclease in the absence of H2O2 (Fig. 3, lines 9-11). However, in the
presence of H2O2, impressive cleavage of circular plasmid DNA, which converts Form I to
Form II, is shown by the ligand (Fig. 4, line 5), indicating an oxidative cleavage process [70].

The test was performed in the presence of DMSO and NaN3 as the hydroxyl radical scavenger
and singlet oxygen quencher, respectively, to determine the DNA cleavage mechanism by
Pd(II) complex and oxime ligand. The efficiency of DNA cleavage by the complex was not
significantly reduced in the presence of DMSO (Fig. 4, line 2). However, the cleavage
activity of the Pd(II) complex was significantly inhibited by the addition of NaN3 singlet
quencher (Fig. 4, line 4). Singlet oxygen causes the DNA cleavage, based on the results [71].
The experimental results also show that NaN3 and DMSO significantly inhibit the cleavage
activity of the oxime ligand (Fig. 4, lines 6 and 7), indicating the important parts of hydroxyl
radical and singlet oxygen in the DNA cleavage in the presence of H2O2.Oxime ligand
cleaves DNA by oxidative cleavage, according to the results [72].
3.4. BSA Binding Experiments
3.4.1. Absorption Spectral Studies
In order to study the nature of the quenching process, UV-Visible absorption titration of BSA
with the Pd(II) complex was carried out. The quenching mechanism may be of dynamic or
static type. Dynamic quenching takes place in the case of an interaction between a quencher
and the fluorophore in a short lived excited state whereas static quenching occurs upon the
complex formation between the fluorophore and a quencher in the ground state [73]. In the
UV spectrum of BSA, two absorption bands are observed; a strong band corresponding to the
protein backbone and a weak band associated with aromatic amino acids at 200 and 278 nm,
respectively (Fig. 5) [74]. Absorption intensity decreases on the addition of the Pd(II)
complex to BSA without influencing the absorption band position. This verifies that the
interaction of Pd(II) complex with BSA changes the microenvironment of the three aromatic
amino acid residues [1]. In addition, this indicates that a complex-BSA system formation in
the ground state leads to a static interaction between the complex and BSA [75].
3.4.2. Fluorescence Spectroscopic Studies
The interaction between the Pd(II) complex with BSA by tryptophan emission quenching
experiments has been examined in this study. BSA itself shows emission at 345 nm due to the
presence of tryptophan residue in its amino acid sequence [76]. The fluorescence intensity
progressively decreases on the addition of successive amounts of the Pd(II) complex to BSA
(Fig. 6). Eq. 3 is used after correction for the inner-filter effect [77]
)
/ꢖ
ꢒꢕ
ꢋꢌꢍꢎꢎ = ꢋꢍꢆꢏ ꢐ(
(3)
ꢑ
ꢔꢑ
ꢒꢓ

Icorr and Iobs are the corrected and measured fluorescence, respectively, and Aem and Aex are
the absorbance of the complex at the emission and excitation wavelengths, respectively. The
corrected fluorescence quenching data were analyzed using the Stern-Volmer equation [78]
ꢗ
ꢘ
[ ]
[ ]
= 1 + ꢊꢙꢚ ꢛ = 1 + ꢊꢜꢝꢁ ꢛ
(4)
ꢗ
I and I0 are the fluorescence intensities in the presence and absence of the complex,
respectively, Ksv, [Q], kq and τ0 are the Stern-Volmer quenching constant, quencher
concentration, bimolecular quenching rate constant and average lifetime of the fluorophore
without quencher, respectively. The slope of the plot of I0/I vs. [Q] can be used to obtain KSV
(Fig. 6 insets). The calculated Ksv value for Pd(II) complex is 3.51 ×104M-1. The τ0 value for
tryptophan fluorescence in proteins is 2×10-8 s [79], the following equation can be used to
calculate the quenching rate constant (kq):
ꢞꢜ = ꢊꢙꢚ/ꢝꢁ
(5)
The quenching constant (kq) obtained for Pd(II) complex was 1.75 × 1012 M-1s-1. This is
greater than the value anticipated for a purely dynamic quenching mechanism (2.0 × 1010M-
1s-1). This verifies that a static quenching mechanism has happened [80]. The quenching
interaction and the number of binding sites (n) can be calculated using the following equation
assuming similar, independent binding sites in the biomolecule for the static binding constant
(Kb) [81]:
Log ꢟꢠ
(6)
– ꢠꢡ
ꢘ
[ ]
= Log Kꢢ + n Log Q
ꢠ
where n and Kb are the number of binding sites and binding constant, respectively. Log[(I0 −
I)/I] vs. log[Q] plots are linear (Fig. S7). Kb can be used to calculate the intercept and slope of
such plots. An n value near unity indicates a single binding site type for the complex on BSA
(Table 3). The binding free energy of Pd(II) complex to BSA is −6.23 kcal mol−1.
3.4.3. Site Selective Binding
The free and bound forms of the complex can also be affected by the competition of drugs for
binding sites on serum albumin. Thus, the identification of the complex binding site in BSA
is important. The crystal structure of Bovine Serum Albumin indicates that it consists of three
similar domains, referred to as I, II, and III, each one including A and B sub-domains [82].
Site marker competitive tests using markers specifically binding to a specific site on BSA, are

carried out to find the binding site of the complex on BSA. When two or more drugs are used
in any biological system, each one tries to tie itself to binding sites faster than the other. The
drug with a closer resemblance to the protein can replace the other one. In this work, the
specificity of the drug binding was recognized by carrying out competition experiments using
Warfarin, Ibuprofen and Digoxin. In these titrations, the BSA to probe concentration ratio
was 1:1 (6 × 10−6 M: 6 × 10−6 M) (Fig. S8). When the site markers were added into the BSA
solution, the fluorescence intensity remarkably decreased. Plots of log[(I0 − I)/I] vs.
log[complex] were obtained for comparison of the effect of Warfarin, Ibuprofen and Digoxin
on the binding of the complex to BSA (Table 3). The complex-BSA binding constant is 3.71
×104M-1, which is decreased in the presence of Warfarin. This indicates the competition
between Warfarin and the complex. Therefore, Pd(II) complex binds to site I of serum
albumin. Docking studies further verify this observation, as will be subsequently discussed.
3.4.4. The Fluorescence Resonance Energy Transfer (FRET) from BSA to the Pd(II)
Complex
Bovine Serum Albumin emission is quenched in the presence of the Pd(II) complex,
according to the fluorescence spectral changes. This indicates the major overlap of the
tryptophan (Trp) emission spectrum in BSA and the Pd(II) complex absorption spectrum,
resulting in the transfer of energy from the excited state BSA to the Pd(II) complex [83]. In
general, radiative and non-radiative energy transfers are possible. Förster’s resonance energy
transfer (FRET) theory, which is applied in the determination of the distance between the
acceptor (i.e. drug’s binding site) and the donor (i.e. tryptophan residue of BSA), clarifies
non-radiative energy transfer [84]. Eq. 7 may be applied to find the energy transfer efficiency
between the acceptor (A) and the donor (D), E, based on the FRET theory [85]:
ꢂ
ꢣ = 1 − ꢤꢗꢗ ꢥ =
(7)
ꢎ
ꢨ
ꢂꢔ(
)
ꢧ
ꢘ
ꢦ
ꢘ
where I and I0 are the BSA fluorescence intensities measured in the presence and absence of
the Pd(II) complex, r is the distance between A and D and R0, which is obtained suing Eq. 8,
is the critical distance when the energy transfer efficiency is 50% [86]:
ꢈꢁꢩ = 8.79 × 10ꢪꢖꢫꢊꢖꢬꢪꢭΦꢮ
(8)
where K2, N, Φ and J are the dipole orientation factor, medium (solution) refractive index,
fluorescence quantum yield of D in the absence of A, and overlap integral of the absorption
spectrum of A with the fluorescence emission spectrum of D, respectively (Fig. 7).

Eq. 9 is used to obtain the J value [86]:
ꢴ
ꢅ
(ꢱ)ꢲ (ꢱ)ꢱ ꢵꢱ
ꢯ
ꢰ
ꢳ
ꢮ =
(9)
( )
ꢱ ꢵꢱ
ꢅ
ꢯ
ꢰ
where ꢶ (ꢸ) and ꢹꢑ(ꢸ) are the fluorescence intensity of D and the molar absorption
ꢷ
coefficient of A at wavelength ꢸ, respectively. The J value was found to be 8.262 × 10–14 cm3
L mol–1 for the Pd(II) complex. The E, R0, and r values found for the binding of the Pd(II)
complex to BSA, obtained using K2 = 2/3, N = 1.336, and Φ = 0.15, were 0.25, 3.63 nm, and
4.36 nm, respectively. The D-to-A distances (r) calculated were below 8 nm and the r values
were bigger than the corresponding value for R0 (0.5R0 < r < 1.5R0). The energy transfer
from BSA to complex takes place with a great chance and the quenching mechanism is static
for the interaction, according to the results [87].
3.5. Molecular Docking
3.5.1. Docking with DNA
Pd(II) complex binds to the minor groove of DNA, AT-rich sequence, as shown by the
molecular docking analysis (Fig. 8). A hydrogen bond is formed between the Pd(II) complex
(oxime ligand oxygen) and the adenine base of DNA. The relative binding energy of the
docked structure for the Pd(II) complex with the DNA molecule (∆Gb) was calculated to be -
7.7 kcal mol-1. This indicates the reasonable comparability of the results of the absorption
titration tests (-7.04 kcal mol-1) with those found using the computational method.
3.5.2. Docking with BSA
The Pd(II) complex is situated in domain I, as Fig. 9 shows. A hydrogen bond between the
Pd(II) complex oxygen atom and Glu 125 (2.2 Å) is observed. The amino acid residues of
BSA, which interacted with the complex, are Phe 36, Pro 113, Lys 114, Leu 115, Lys 116,
Leu 122, Glu 125, Phe 133, Lys 136, Tyr 139 and Tyr 160. Furthermore, the distance between
the Pd(II) complex and Trp 134 residue is 7.0 Å. The best ∆Gb value for the interaction of
BSA with the complex determined was found to be -7.44 kcal mol-1 which is in conformity
with the binding free energy found using the experimental value of Kb (-6.23 kcal mol-1).
3.6. In vitro Selective-Cytotoxic Activity
Melanoma B16F0 and colon carcinoma C26 cell lines were treated with Pd(II) complex and
oxime ligand at the concentration range of 1.25-10 µM to assess the in vitro cytotoxicity of
the compounds against cancer cells. The cytotoxicity of the compounds after 48 h was
measured using MTT-assay method, which is a well-documented bioassay. The cytotoxic

activity of Cis-platin, a standard anti-cancer drug, was investigated for the sake of
comparison under identical experimental conditions. Concentration dependent cytotoxicity
against both cancer cell lines is shown by the compounds (Fig. S9). The high and low Pd(II)
complex and oxime ligand concentrations (10 and 1.25 μM, respectively) against C26 cancer
cells showed (93.56 and 84.32%) and (21.09 and 14.28%) cytotoxicity, respectively. The
corresponding cytotoxicity values in the case of B16F0 cancer cell line were (97.36% and
91.56%) and (39.98% and 33.8%), respectively. While important and high cytotoxic activity
was shown by the compounds against both B16F0 and C26 cancer cell lines, low toxicity was
identified against NIH normal cells in comparison with cisplatin (Table 4). Therefore, Pd(II)
complex shows higher cytotoxicity against both cancer cell lines compared with oxime
ligand. Remarkably low cytotoxic activity was shown by both compounds against normal cells
with IC50 values of >100 µM. Thus, the cytotoxicity of the compounds against normal cells
was lower compared with cisplatin. The results are in compliance with the other experimental
data, which indicate the inhibitory effect of palladium complexes on cancer cells. Palladium
complexes were found to have significant cytotoxicity against various cancer cells such as
MCF-7 breast cancer cell line [88]. According to other in vitro studies, saccharinate and/or
terpyridine complexes of palladium inhibit the concentration dependent growth of MCF-7
and MDA-MB-231 breast cancer cell lines at IC50 values of up to 3.05 and 0.09 μM,
respectively [89, 90]. In vivo study on Walker tumor bearing rats further verify these results.
Therefore, natural biocompounds derived palladium complexes inhibit tumor growth by 90%.
[91]. The SI values, found by comparing the IC50 of the complexes against each cancer cell
with that of the normal cell line to assess the selective effect of the compounds against cancer
cells, are some of the other significant results of this in vitro cytotoxicity study [92]. The SI
values of both compounds exceeded 2. Consequently, both compounds have potential
selective cytotoxicity on cancer cells. The results are further confirmed by the previous
reports on more cytotoxic activity to cancer cells compared with the normal cells [93-99]
arising from the cancer cell sensitivity to the death complexes. To summarize, important
selective concentration dependent cytotoxic activity on proliferation and viability of breast
and colon cancer cells, in vitro, has been shown by both compounds. However, it is necessary
to confirm anticancer effects using validated in vivo studies.
4. Conclusion
A novel Pd(II) complex bearing pyridine-2-carbaldehyde oxime ligand has been prepared in
the present work. X-ray crystallographic study of the Pd(II) complex indicates a square planar

coordination around the Pd(II) ion with a little tetrahedrally distorted. The DNA binding
properties of the complex were investigated using electronic absorption, fluorescence
spectroscopy and viscosity measurements. The interaction of the Pd(II) complex with DNA is
of groove binding type, based on the results. In addition, the Pd(II) complex could cleave the
supercoiled plasmid under physiological conditions without any additional reductants. The
reactivity towards BSA showed a static emission quenching by the complex. The binding
distances of Pd(II) complex with BSA were calculated to be 4.36 nm, based on the Foster’s
theory. According to the competitive studies using Warfarin, Ibuprofen and Digoxin site
markers, and the complex was mainly located in site I of the protein. The binding of the
Pd(II) complex to DNA and BSA was modeled by molecular docking. The cytotoxic
investigations indicate that the Pd(II) complex has high cytotoxic activity against various cell
lines tested. Moreover, the cytotoxicity results indicated the higher efficiency of Pd(II)
complex compared with the corresponding free ligand under the same experimental
conditions.
Acknowledgments
The authors gratefully acknowledge the financial support of this research by the Isfahan
University of Technology (IUT).
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Figure Captions:
Fig 1: ORTEP diagram for Pd(II) complex.
Fig 2: Electronic spectra of Pd(II) complex in buffer solution (5 mM Tris-HCl/10 mM NaCl
at pH 7.2) upon addition of CT-DNA. [Complex] = (6×10-5M), [DNA] = (0-6×10-4M). Arrow
shows the absorption intensities decrease upon increasing DNA concentration. Inset: Plots of
[DNA]/( εa-εf) vs. [DNA] for the titration of Pd(II) complex with CT-DNA.
Fig 3: Gel electrophoresis diagrams showing the cleavage of pUC57 DNA (0.01 M) at
different concentrations of the Pd(II) complex and the oxime ligand in Tris–HCl/NaCl buffer
(pH = 7.2) at 37°C for 3 h. Lane 1: marker;Lane 2: DNA control, Lane 3: DNA + Pd(II)
complex (50 µM); Lane 4: DNA + Pd(II) complex (100 µM); Lane 5: DNA + Pd(II) complex
(300 µM); Lane 6: DNA + Pd(II) complex (500 µM); Lane 7: DNA + Pd(II) complex (700
µM); Lane 8: DNA + Pd(II) complex (900 µM); Lane 9: DNA + oxime ligand (100 µM);
Lane 10: DNA + oxime ligand (300 µM); and Lane 11: DNA + oxime ligand (500 µM).
Fig 4: Gel electrophoresis diagrams showing the cleavage of pUC57 DNA (0.01 M) by the
Pd(II) complex and oxime ligand (700 μM) in Tris-HCl buffer (pH = 7.2) at 37ºC for 3 h in

the presence of DMSO, H2O2 and NaN3 (700 μM). Lane 1: DNA control; Lane 2: DNA +
Pd(II) complex + DMSO; Lane 3: DNA + Pd(II) complex + H2O2; Lane 4: DNA + Pd(II)
complex + NaN3; Lane 5: DNA + oxime ligand + H2O2; Lane 6: DNA + oxime ligand +
H2O2 + DMSO; and Lane 7: DNA + oxime ligand + H2O2 + NaN3.
Fig 5: UV absorption spectra of [BSA] = (6×10-6M) in the absence and presence of different
concentration of Pd(II) complex (0-6×10-5M).
Fig 6: Emission spectra of BSA upon the titration of Pd(II) complex. [BSA] = (6×10-6M),
[complex] = (0-6×10-5). Arrow shows the change upon the increasing complex concentration.
Inset: Plots of I0/I vs [Q]×106.
Fig 7: Spectral overlaps of the absorption spectra of Pd(II) complex with the fluorescence
spectra of BSA.
Fig 8: (A) Results of docking procedure of Pd(II) complex with the minor groove binding to
the DNA, (B) The bases of DNA interactions with Pd(II) complex in the active site.
Fig 9: (A) Results of docking procedure of Pd(II) complex in the interaction with BSA
residues, which results in quenching, (B) The BSA amino acid residues in interaction with Pd(II)
complex.

Table 1: Crystallographic data and structure refinement details for Pd(II) complex.
Pd(II) complex
Empirical formula
C12H14N4O4P0Pd
384.67
Formula weight
T/K
150 (1)
Crystal system
Triclinic
Space group
a/Å
P1
7.4056 (6)
8.9741 (8)
10.6709 (9)
109.898 (3)
96.053 (4)
90.052 (4)
b/Å
c/Å
α/°
ꢀ/°
ꢁ/°
v/ Å3
662.60 (10)
Crystal dimensions / mm3 0.12 × 0.11 × 0.08
Z
2
µ(mm-1)
Dcalc/ Mg m−3
11.53
1.928
384
F000
4.4–54.4
1609
ꢂ range/°
Independent reflections
Data/restraints/parameters 12650/61/192
Goodness-of-fit on F2
1.118
R1= 0.0518, wR2=
0.1384
Final R indices
R1= 0.0576,wR2=
0.1455
R indices (all data)
Largest difference peak
and hole / e Å−3
CCDC number
1.69, −0.92
1827296

Table 2: Selected bond lengths (Å), and angles (°) for Pd(II) complex.
Bond lengths
Bond angels
N1—Pd1—N2
Pd1-N1
Pd1-N2
Pd1-N3
Pd1-N4
O1-N1
O2-N3
2.031 (9)
2.035 (8)
2.041 (9)
2.035 (8)
1.283 (11)
1.272 (11)
80.2 (4)
99.9 (3)
179.5 (3)
178.9 (3)
100.3 (4)
79.7 (3)
N3—Pd1—N2
N4—Pd1—N2
N3—Pd1—N1
N4—Pd1—N1
N4—Pd1—N3
Table 3: Estimated binding constants for site marker competitive experiments of the complex-BSA system.
System
Kb (M-1)
n
R
Complex-BSA
3.71 × 10ꢃ
2.50 × 10ꢃ
3.59 × 10ꢃ
3.58 × 10ꢃ
1.025
0.91
1.08
0.99
0.9974
0.9910
0.9914
0.9961
Complex-BSA-Warfarin
Complex-BSA-Ibuprofen
Complex-BSA-Digoxin
Table 4: Selective cytotoxicity data IC50 (µM) of the compounds against cancer and normal cell lines.
Compound
IC50 (µM) ± SD
B16-F0
C26
NIH
Oxime ligand
Pd(II) complex
Cis-platin
4.2±1
2.7±2
17.0± 1
2.9 ± 4
>100
2.0±1
>100
26.0 ± 1
38.67± 4

Hd
O
OH
NH2OH.HCl
NaOH
Hc
N
H
N
2 h
50°C
Hb
N
Ha
pyridine-2-carbaldehyde oxime ligand
2-pyridine carboxaldehyde
Pd(OAc)2
NaOAc
Hd
Hc
Hb
6 h
60°C
N O-
N
Pd
N
Ha
-O N
Pd(II) complex
Scheme 1: Synthetic route for the preparation of pyridine-2-carbaldehyde oxime ligand and Pd(II) complex.
Hb
3Jbc=6.9 Hz
3Jba=4.8 Hz
4Jbd=1.6 Hz
Scheme 2: splitting pattern of Hb in 1HNMR spectra of oxime ligand.