Synthesis and characterization of two new trans palladium (II) complexes containing benzylamine ligand: DNA/BSA interactions, molecular docking and in vitro cytotoxic activity

Article abstract of DOI:10.1016/j.ica.2017.02.027

Two new trans Pd (II) complexes with formulas Pd(PhCH₂NH₂)₂Cl₂ (1) and [Pd(PhCH₂NH₂)₂(L)₂](NO₃)₂ (2) (L = Letrozole drug; 4,4′-((1H-1,2,4-triazol-1-yl)methylene)dibenzonitrile) have been synthesized and characterized by elemental analysis, FT-IR and NMR spectroscopy. Single crystal X-ray diffractometry has been used to determine the crystal structure of 1. UV–Vis spectroscopy, emission titration, circular dichroism and helix melting methods have been used to study the binding interaction of Pd (II) complexes with Calf Thymus deoxyribonucleic acid (CT-DNA). The analysis results show that 1 can interact with DNA via groove binding and 2 binds to DNA through partial intercalation mode. It was found that the binding constants (K_b) of the complexes toward BSA were (1.4 × 10⁴ M^?1) and (1.68 × 10⁴ M^?1) for 1 and 2, respectively. Competitive binding using Warfarin, Ibuprofen and Digoxin site markers with definite binding sites demonstrated that the complexes bind to more than one site on BSA. In the presence of Ibuprofen, the binding constant was surprisingly not fixed, while a smaller influence was observed in the presence of Warfarin and Digoxin on 1. The results indicated that the binding site for 1 was mainly located within site II of BSA. The result of the competitive titration of 2 was different. In the presence of Warfarin, the K_b value decreased, showing a competition between 2 and Warfarin. In addition, molecular docking studies have been conducted to determine the binding site of the DNA and BSA with 1. Finally, In vitro cytotoxicity of 1, 2 and cisplatin were carried out against leukemia cancer (Raji), lung cancer (A549) and breast cancer (MCF7) cell lines. According to the IC₅₀ values, the cytotoxicity of 2 was more than 1.

Full text of DOI:10.1016/j.ica.2017.02.027

Accepted Manuscript
Research paper
Synthesis and characterization of two new trans palladium (II) complexes con-
taining benzylamine ligand: DNA/BSA interactions, molecular docking and in-
vitro cytotoxic activity
Kazem Karami, Moloud Alinaghi, Zahra Amirghofran, Janusz Lipkowski
PII:
S0020-1693(16)30998-7
http://dx.doi.org/10.1016/j.ica.2017.02.027
ICA 17453
DOI:
Reference:
Inorganica Chimica Acta
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7 December 2016
14 January 2017
24 February 2017
Please cite this article as: K. Karami, M. Alinaghi, Z. Amirghofran, J. Lipkowski, Synthesis and characterization
of two new trans palladium (II) complexes containing benzylamine ligand: DNA/BSA interactions, molecular
docking and in-vitro cytotoxic activity, Inorganica Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica.
2017.02.027
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Synthesis and characterization of two new trans palladium (II) complexes
containing benzylamine ligand: DNA/BSA interactions, molecular docking and in-
vitro cytotoxic activity
Kazem Karami*a, Moloud Alinaghia, Zahra Amirghofranb, Janusz Lipkowskic
aDepartment of Chemistry, Isfahan University of Technology, Isfahan, 84156/83111, Iran
bImmunology Department, Shiraz University of Medical Sciences, Shiraz 71454, Iran
cInstitute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
Abstract
Two new trans Pd (II) complexes with formulas Pd(PhCH2NH2)2Cl2 (1) and
[Pd(PhCH2NH2)2(L)2](NO3)2 (2) (L= Letrozole drug; 4,4'-((1H-1,2,4-triazol-1-
yl)methylene)dibenzonitrile) have been synthesized and characterized by elemental analysis,
FT-IR and NMR spectroscopy. Single crystal X-ray diffractometry has been used to
determine the crystal structure of 1. UV-Vis spectroscopy, emission titration, circular
dichroism and helix melting methods have been used to study the binding interaction of Pd
(II) complexes with Calf Thymus deoxyribonucleic acid (CT-DNA). The analysis results
show that 1 can interact with DNA via groove binding and 2 binds to DNA through partial
intercalation mode.
It was found that the binding constants (Kb) of the complexes toward BSA were (1.4×104M-
1) and (1.68×104 M-1) for 1 and 2, respectively. Competitive binding using Warfarin,
Ibuprofen and Digoxin site markers with definite binding sites demonstrated that the
complexes bind to more than one site on BSA. In the presence of Ibuprofen, the binding
constant was surprisingly not fixed, while a smaller influence was observed in the presence of
Warfarin and Digoxin on 1. The results indicated that the binding site for 1 was mainly
located within site II of BSA. The result of the competitive titration of 2 was different. In the
presence of Warfarin, the Kb value decreased, showing a competition between 2 and
Warfarin. In addition, molecular docking studies have been conducted to determine the

binding site of the DNA and BSA with 1. Finally, In vitro cytotoxicity of 1, 2 and cisplatin
were carried out against leukemia cancer (Raji), lung cancer (A549) and breast cancer
(MCF7) cell lines. According to the IC50 values, the cytotoxicity of 2 was more than 1.
Keywords
Palladium complexes; Benzylamine; DNA and BSA interaction; Molecular docking; In vitro
cytotoxicity
1. Introduction
Today, the design and synthesis of complexes with medicinal properties; especially
anticancer activity is noticeable [1]. Cancer is the most common disease, which annually
causes the death of many people in industrialized countries [2]. Moreover, the current
treatments such as chemotherapy with cisplatin and other platinum based drugs are
accompanied by many undesirable side effects such as cumulative toxicities of
nephrotoxicity, ototoxicity, tumor resistance, bone and back pain, blood clots, weakness,
fatigue, and hair loss [3, 4]. Hence, significant attempts are being made to replace other
antitumor active transition metal complexes with efficient pharmacological virtues.
Mechanism of function of Pt (II) anticancer drugs demonstrates that the Pd (II) analogs
are appropriate candidates to substitute Pt (II) compounds. Although the structural and
thermodynamic parameters of Pt (II) and Pd (II) are identical, Pd (II) complexes show about
104-105 times higher reactivity [5].
Several workers have concentrated on the application of an eclectic series of palladium
complexes including cis,trans-PdX2L2, palladacycles, dimeric, trimeric, tetrameric as well as
heterobimetallic complexes with anti-cancer potential for the past 10 years [6, 7]. The choice
of the appropriate ligands (using tumor active ligands) and the geometry surrounding the

palladium center are significant in Pd (II) complexes. Most trans palladium complexes have
much higher cytotoxicity values compared with cis-palladium isomers [8] and higher
cytotoxicity than cisplatin, carboplatin and other platinum based analogs according to the
published results [6]. There are many reports on the improved antitumor activity of the trans-
palladium complexes [9, 10]. The in vitro cytotoxic activity of the complexes have been
assessed against both human epidermoid carcinoma derived KB cell line [11], murine
leukemia L1210 cell line [12], P388, L929, K562, MCF7, Hela as well as other cell lines [13-
17]. N-bonded hydrogen suitable for hydrogen bonding, which helps the effective binding of
metal ions to the nucleic acid fragments over those containing heterocyclic N-donor ligands,
seems to be responsible for the improved activity [18, 19]. Therefore, in the present study,
two trans-palladium complexes have been synthesized. Complex 1: [trans-
Pd(benzylamine)2Cl2] and complex 2: [trans-Pd(benzylamine)2(Letrozole)2](NO3)2.
Substitution of chloride ligand with Letrozole, which is an oral non-steroidal aromatase
inhibitor for the treatment of hormonally responsive breast cancer after surgery, provides
context to compare the role of ligands. Palladium acts as a Cell Killing Agent [20] whereas
Letrozole is a Cell Growth Inhibiting Agent [21].
The first step in developing metal based therapeutics is the detailed study of the
interactions between DNA and transition metal complexes [22] because anticancer drugs are
molecules, which bind to DNA of carcinoma cells to prevent the reproduction of the cancer
cell [23]. Small molecules bind to DNA by the following three possible means: (I)
electrostatic interaction between the cationic species and negatively charged DNA phosphate
residues, (II) intercalation between individual base pairs and (III) minor and major DNA
groove binding interaction through hydrogen bonding and van der Waals interaction forces
[24].
On the other hand, understanding the transportation mechanism of drugs in the body
depends on drug-protein binding studies [25]. Moreover, proteins are regarded as the main

cellular targets for anticancer drugs. The most abundant carrier protein in the blood is Human
Serum Albumin (HSA). Since Bovine Serum Albumin (BSA) structure is homologous with
HSA and due to its accessibility and low cost, BSA is often used in biophysical and
biochemical studies [26].
In view of the above considerations, in this report, two new palladium complexes
have been designed, synthesized and thoroughly characterized by IR and NMR spectroscopy.
The detailed structure of 1 was also determined by X-ray single crystal analysis. The
interaction between the complexes and DNA was investigated by molecular docking and
various spectroscopic methods such as UV-Vis absorption spectroscopy, fluorescence
emission spectroscopy, circular dichroism (CD) and thermal denaturation. In addition, the
BSA binding ability has been monitored by UV absorption and tryptophan fluorescence
quenching experiments in the presence of the complexes. The binding constants (K) and
binding site number (n) have also been calculated. Furthermore, the molecular docking
studies have been performed on the structure of BSA. The cytotoxic activity of both
complexes against the leukemia cancer (Raji), lung cancer (A549) and breast cancer (MCF7)
cell lines was evaluated by MTT assay.
2. Materials and methods
2.1. Materials
All solvents and chemicals were obtained from commercial sources and used with no
further purification. As received reagent grade Calf Thymus DNA (CT-DNA), BSA and
methylene blue (MB) were supplied by Sigma Aldrich Chemical Company.
The solutions of CT-DNA gave a UV absorbance ratio (A260/A280) of over 1.8,
indicating the purity of DNA [27]. Extra pure solvents used in the preparation and physical

measurements were obtained from Merck Chemical Co. Tris(hydroxymethyl)-aminomethane
(Tris) buffer was of analytical reagent grade and was supplied by Merck Chemical Co.
Doubly distilled deionized water was used for preparation of all solutions. Stock solutions of
CT-DNA and BSA were prepared by dissolving them in 5 mM Tris buffer and 50 mM NaCl
at pH 7.2. The DNA concentration per nucleotide was determined by absorption spectroscopy
using the molar absorption coefficient (ε = 6600 M–1 cm–1) at 260 nm [28, 29].
2.2. Methods and instrumentation
Fourier transform infrared spectra were recorded on a FT-IR JASCO 680-PLUS
spectrometer in the region of 4000–400 cm−1 using KBr pellets. A JASCO 7580 UV-Vis-NIR
double beam spectrophotometer was used to record electronic absorption spectra in a 1 cm
path length cell. Fluorescence spectra were recorded in solution on a Cary Eclipse
fluorescence spectrophotometer. NMR spectra were recorded on a Bruker spectrometer at
400.13 MHz (1H), 100.61 MHz 13C–{1H}. Elemental analyses were performed on a Leco,
CHNS-932 apparatus. The Tm spectra were recorded on a Varian BioCary-100 UV-Vis
spectrophotometer using a 1cm path length cell. CD spectra were recorded on an Aviv
Circular Dichroism Spectrometer, model 215 (USA), using a cylindrical cuvette with 0.1 cm
path length.
2.3. Syntheses
2.3.1. Synthesis of complex 1: [trans-Pd(PhCH2NH2)2Cl2]
The complex was synthesized by addition of benzylamine (0.05 ml, 0.4 mmol) to a
solution of Pd(OAc)2 (0.05 g, 0.2 mmol) in 15 ml of toluene solvent. The reaction mixture
was stirred for 24 h at room temperature. After that the reaction product dissolved in acetone/

water (10:1), excess NaCl was added to this mixture, followed by stirring for 12 h to
exchange the acetate ligand with Cl yielding a yellow solid on slow evaporation. The product
was washed with water and dried. CH2Cl2/n-hexane solvent mixture (1:5 ratio) was used to
precipitate a pure yellow solid (Yield: 85%), which was collected and air dried. Yellow
crystals of 1 were obtained from CH2Cl2: n-hexane solvent mixture. Anal. Calc. for
C14H18N2Pd (%) C, 52.43; H, 5.66; N, 8.73. Found C, 52.38; H, 5.59; N, 8.62. IR (KBr, cm-1)
ν (N–H) = 3287, ν (C-HPh) = 3058, ν (C-H alph) = 2924, ν (C=C aromatics) = 1454, ν (Pd-N)
1
= 469. H NMR (CDCl3, ppm): δ= 3.1 (b, 2H, NH2), 4 (m, 2H, CH2), 7.3-7.4 (m, 5H,
13
aromatic ring). C–{1H} NMR (CDCl3, ppm): 49.5 (CH2), 128.2, 128.5, 129, 137 (aromatic
ring).
2.3.2. Synthesis of complex 2: [trans-Pd(PhCH2NH2)2 (L)2](NO3)2
AgNO3 (0.06 g) was added to a suspension of 1 (0.07 g) in acetone (10 ml) with 2:1
molar ratio. The reaction mixture was protected from light and stirred for 2 h at room
temperature. AgCl was removed by filtration to obtain a clear yellow solution. The treatment
of solution with Letrozole (0.1 g) afforded 2 as a grey solid: [trans-Pd(PhCH2NH2)2
(L)2](NO3)2 (Yield: 78.5%). Anal. Calc. for C48H40N14O6Pd (%) C, 56.78; H, 3.97; N, 19.31.
Found C, 56.63; H, 3.92; N, 19.24. IR (KBr, cm-1) ν (N–H) = 3235 ν (C-HPh) = 3061, ν (C-H
alph) = 2926, ν (ꢀ ≡ ꢁꢂLetrozole ligand) = 2231, ν (C=N Letrozole ligand) = 1608, ν (C=C
aromatics) = 1455, ν (Pd-N) = 517, 472. 1H NMR (DMSO-d6, ppm): δ= 1.2 (br S, 1H, CH2),
1.3 (br S, 1H, CH2), 3.46 (S, 2H, NH2), 5.85 (S, 1H, CH), 7.4-8 (m, 13H, aromatic rings),
8.24 (S, 1H, HC=N), 8.83 (S, 1H, HC=N). 13C–{1H} NMR (DMSO-d6, ppm): 42.3 (CH2),
64.2 (CH), 111.3, 118.3, 126.8, 128.5, 128.6, 128.8, 129, 132 (aromatic rings), 143, 145.3
(HC=N), 152.3(ꢀ ≡ ꢁ).
2.4. Crystallography

X-ray diffraction experiments were performed at 100 K using an Agilent SuperNova
single crystal diffractometer (Mo K(a) radiation). Analytical numeric absorption correction
was made using a multi-faceted crystal model based on expressions derived by R.C. Clark &
J.S. Reid [30]. Direct methods using the SHELXS97 program were used to solve the
structures and SHELXL (Sheldrick 2008) program was applied to refine it. Hydrogen atoms
were added in the calculated positions and were riding on their respective carbons during the
refinement.
2.5. DNA binding studies
A stock solution of CT-DNA was prepared in Tris-HCl buffer (5 mM Tris-HCl, 50
mM NaCl at pH 7.2). The DNA concentration per nucleotide was determined by absorption
spectroscopy (UV-Vis) using ε260 = 6600 M-1cm-1 [31]. Concentrated stock solutions of metal
complexes were prepared in acetonitrile solvent. The solutions were then diluted suitably by
Tris-HCl buffer at pH 7.2 to obtain the required concentrations in all the experiments. The
DNA-binding studies were performed at room temperature by electronic absorption
spectrometric experiments and luminescence measurement. The thermal denaturation
experiments were performed by measuring the DNA solution absorbance at various
temperatures in the absence and presence of the complexes. Two solutions of DNA and
DNA-Pd (II) complexes were heated continuously in the temperature range of 20-90°C. The
samples were maintained in a holder for 2 minutes at a given temperature during the thermal
experiments and the absorbance was measured at λ = 260 nm and plotted as a function of
temperature (T) from 20 to 90°C. The CD spectra of DNA in the absence and presence of
complexes were recorded in the range of 200–400 nm at 25°C using a bandwidth of 1 nm, a
step interval of 1 nm, an average time of 0.5 s, and a slit width of 0.02 mm. The CD spectrum
of Tris buffer was subtracted from the sample spectra for data analysis.

The competitive study of complexes with MB, which is a planar cationic dye well-
known to intercalate into the CT-DNA independently, has been investigated using
fluorescence spectroscopy to examine the capability of the complex to displace MB from its
DNA-MB complex. The DNA-MB solution ([DNA]/[MB] = 10) was excited at 630 nm in the
absence and presence of different concentrations of complexes for MB displacement [32].
2.6. Protein binding studies
A stock solution of BSA was prepared by dissolving the appropriate amount 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 determined by UV-Vis absorption spectroscopy
using ε280 = 44300 M-1cm-1 [33].
UV-Vis spectra were recorded for a BSA solution titrated with various concentrations
of the complexes at 280 nm and the changes in the BSA absorption were recorded after each
addition. In the tryptophan fluorescence quenching experiment, the fluorescence emission
spectra were recorded in the wavelength range of 300-400 nm for fixed concentrations of
BSA solution in the absence and presence of the complexes (quenchers) at ca. 345 nm
(λex=280 nm).
2.7. Molecular docking
The binding mode and intermolecular interactions of complex 1 with DNA and BSA
were investigated by molecular docking. Molecular docking studies were performed using
Auto Dock 4.2 package by the Lamarckian genetic algorithm (LGA) [34, 35]. The PDB
format of 1 was obtained by converting CIF file using Mercury software. The initial structure
of BSA was taken from the Protein Data Bank (PDB ID: 4F5S) at a resolution of 2.47 Å. In

addition, d(CGCGAATTCGCG)2 DNA sequence was supplied by the Protein Data Bank
(PDB ID: 3U2N) at a resolution of 1.25 Å. During the preparation of the DNA and BSA
input file, chain (A) of BSA and all the hetero atoms including water molecules were
removed. To find the suitable binding site for the interaction of the complexes with DNA and
BSA, the blind docking was first used [36, 37]. The focus docking was then performed on the
best location. The 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 kept at their default values.
2.8. The cell proliferation assay
The growth inhibitory effect of the complexes against Raji, A549 and MCF7 cancer
cell lines were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide
(MTT) assay [38]. A predetermined concentration of tumor cells was seeded in the wells of a
96-well plate with varying concentrations of the synthetic complexes (0-100 µmol dm-3) and
incubated at 37°C with 5% of CO2 and 95% of humidity for 48 h. Afterwards, 10 µL of MTT
solution (5 mg mL-1) were added to each well and the plates were incubated for an additional
4 h at 37°C. Finally, the insoluble formazan produced was dissolved by adding 100 µL/well
of dimethyl sulfoxide (DMSO). After complete dissolution of the dye, the optical density
(OD) was read 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 consisted of only DMSO
solvent at a concentration equal to those in the test wells. The value of half maximal

inhibitory concentration (IC50) was determined from the graph of inhibition percentage
against different concentrations [39, 40].
3. Results and discussion
3.1. Synthesis and characterization:
In the present work, the direct reaction of primary benzylamine (PhCH2NH2) with
palladium acetate (Pd(OAc)2) in a 2:1 molar ratio was carried out in toluene at 25°C to yield
the corresponding bis-amine adduct [Pd(OAc)2(PhCH2NH2)2] (a). Addition of NaCl to a
acetone/water solvent mixture of (a) allowed the isolation of bis(amino)-dichloro
palladium(II) [Pd(Cl)2(PhCH2NH2)2] (1) in good yield. 1 reacted with AgNO3 and Letrozole
(L) to give [Pd(PhCh2NH2)2(L)2](NO3)2 (scheme 1). The results of elemental analysis of the
prepared compounds 1 and 2 are in a good agreement with the calculated values. These
complexes were also characterized by IR and NMR spectroscopy. The crystal structures of 1
have been solved by X-ray diffraction studies.
The coordination mode of the ligand can be reliably followed by IR spectroscopy. In
the IR spectra of the complexes, the ν (N–H) band, which is sensitive to complexation
appears from 3287 to 3229 and 3235 to 3127 cm-1 for asymmetric and symmetric stretching
of 1 and 2, respectively, whereas for N-coordination, a lowering of the ν (N–H) band is
1
expected [41]. In the H NMR spectra of the complexes, the methylene protons are non-
1
equivalent resulting in formation of two separated signals. Moreover, in the H NMR
spectrum of 2, methylene protons are significantly shifted to lower frequencies because of the
anisotropic shielding from the phenyl or cyanide group of Letrozole ligand. 13CNMR shows 4
and 13 signals for 1 and 2, respectively. Single crystal X-ray diffraction method and ORTEP
drawings (Fig. 1) were used to determine the crystal structures of 1. Tables 1 and 2 and
hydrogen bonding geometry (Table 3) give the crystallographic data and bond parameters.

Complex 1 crystallizes in a tetragonal system with space group P42/n with 4 independent
molecules within the unit cell. The trans configuration assumed by the amino ligands is the
normal geometry observed for bis(amino)-palladium(II) complexes [42].
3.2. DNA binding studies
3.2.1. Electronic absorption titration
The binding mode of DNA with metal complexes can be efficiently examined by
electronic absorption spectroscopy. The absorption spectra of complexes were recorded for a
fixed concentration of free metal complex [4 × 10-5 M] with increasing concentrations of CT-
DNA [0–4.0 × 10-4 M] (Fig. 2). The absorption peaks are 206 nm for 1 and 207 and 236 nm
for 2, which can be attributed to intraligand π–π* transition. Addition of DNA resulted in a
reduction in absorbency (hypochromism) without any shift in the absorption maxima in the
UV spectrum of 1 and with bathochromic shift (2 nm red shift) in spectrum of 2. This
suggests that 2 interacts with CT-DNA basically through the intercalative mode because
intercalation leads to hypochromism and bathochromism in UV absorption spectra due to the
intercalative mode, which involves a strong interaction between the complex molecule and
the base pairs of DNA [43]. The intrinsic binding constant, Kb, for the complexes were
calculated from Wolfe-Shimmer Equation:
[DNA]/(εꢃ − εꢄ)ꢂ=ꢂꢂ[DNA]/(εꢅ − εꢄ) + ꢂꢂ1/(Kꢅꢂ(εꢅ − εꢄꢂ)ꢂ)ꢂꢂꢂꢂꢂ (1)
The absorption coefficients εa, εf, and εb correspond to Aobs/[DNA], the extinction
coefficients for the free complex and the complex in the fully bound form, respectively. In
particular, εf was determined by a calibration curve of the isolated Pd (II) complexes in an
aqueous solution, following Beer's law. A plot of [DNA]/[εa-εf] vs. [DNA] gave a slope and

an intercept equal to 1/[εa-εf] and 1/Kb[εb-εf], respectively (Fig. 2, inset) [44]. Intrinsic
binding constant, Kb, values of 4.13×105M-1 for 1 and 5.22×106 M-1 for 2 were obtained
following the above equation. 2 binds more strongly than 1. This may be due to the
intercalation of planar heterocyclic ring and bulky benzonitrile groups of Letrozole ligands,
between the planes of DNA nucleic bases. DNA has several hydrogen bonding sites in the
major and minor grooves. Since the complexes contain amine NH groups, there could be
hydrogen bonding between the complexes and insertion of base pairs in DNA.
Free energy (∆G) of compound-DNA complex was calculated from the values of
binding constant (Kb) using the following equation (2):
∆G = – RT ln Kb
(2)
Binding constants are measures of compound-DNA complex stability while free
energy indicates the spontaneity/non-spontaneity of compound-DNA binding. Free energies
of 1 and 2 were evaluated as negative values (-7.66 and -9.16 Kcal mol-1, respectively),
which shows the spontaneity of compounds-DNA interaction. These results, however, show
that 2 binds to DNA more spontaneously compared with 1.
3.2.2. Fluorescence emission titration
To further confirm the interaction between the complexes and CT DNA, emission
titrations were performed. The emission spectra for these complexes emit luminescence in the
buffer at room temperature with a maximum at 359 nm (ꢆꢇꢈ = 286 nm) for 1 and 443 nm
(ꢆꢇꢈ = 385nm) for 2. The luminescent properties of the complexes were perturbed when
DNA was added to palladium complex solution and binding of the complex to DNA was
found to decrease the luminescence intensity for 1 and increase it for 2, (Fig. 3). Excited state
reactions, molecular rearrangement, energy transfer, ground state complex formation, and

collisional quenching can result in quenching. This is normally referred to as non-radiative
energy transfer from excited species to other molecules [45]. The interaction between
naproxen and ds-DNA by a groove binding mode causes a strong fluorescence quenching
[46]. DNA intercalators act as DNA molecular light switches. Therefore, the emission
enhancement of the complex in the presence of DNA is observed [47, 48]. Reduction of the
vibrational modes caused by restricting the mobility of complex at the binding site leads to
higher emission intensity [49]. The observed emission quenching for 1 demonstrates that this
compound can be a non-intercalation mode with DNA [50]. The emission intensity of peak
increases in the spectrum of 2 indicates that it may interact with DNA as an intercalator [51].
A competitive methylene blue (MB) displacement assay was carried out to investigate
the interaction of complexes with DNA. Previously, it has been reported that the fluorescence
intensity of MB-DNA system is increased by adding certain complexes [52-54]. This
indicates that the complexes are bound to the base pairs of DNA by classical intercalation
mode while replacing the MB [55]. When the complexes were added to DNA pretreated with
MB, the DNA induced emission intensity at 680 nm (630 nm excitation) was decreased for 1
and a partial replacing of MB by 2 from the MB–DNA system was observed (Fig. 4), as these
complexes bind to DNA via non-covalent groove binding or partial intercalation [7, 56].
3.2.3. Circular Dichroism Studies
Circular dichroic studies are useful in diagnosing changes in the morphology of DNA
during the complex-DNA interactions. The sign and shape of the CD spectra are different
between B-DNA with a right handed double helical structure and Z-DNA with a left handed
double helical structure. In particular, B-form oligo nucleotides are characterized by a
positive long wavelength band at about 260-280 nm due to base stacking and a negative band
around 245 nm due to right handed helicity, which is opposite to that of Z-DNA [57]. The

observed CD spectrum of CT DNA consists of a positive band at 277 nm due to base stacking
and a negative band at 247 nm due to helicity, which is characteristic of DNA in right handed
B-form in the UV region. The variations in DNA CD signals, which are observed on
interaction with drugs, may often be associated with the corresponding changes in DNA
structure. Therefore, less or no perturbation are exhibited by simple groove binding and
electrostatic interaction of small molecules on the base stacking and helicity bands, while the
intensities of both bands, which stabilize the right handed B conformation of CT-DNA, are
enhanced by intercalation, as observed in the classical intercalator methylene blue [58-60].
For 1 and 2, the CD spectra of DNA showed conformational changes by addition of
complexes, without any changes in the wavelength (Fig. 5). According to the description, 1
binds to the DNA via groove mechanism [7]. The decrease of negative bands and increase of
positive bands shows that 2 has been intercalated into the base pairs of DNA. The structural
basis of this transition may be related to the closeness of the two sugar-phosphate backbones
along the grooves in A-DNA, the negative charge of which can be effectively neutralized by
the positively charged palladium of 2 through intercalation [61].
3.2.4. Thermal denaturation studies
During thermal denaturation of DNA, the duplex DNA is denatured into single strand
components in the presence or absence of drugs. Melting DNA can be used to distinguish
those molecules binding via intercalation and groove mechanism. In the presence of metallo-
intercalators, Tm rises sharply until all the intercalating sites are saturated, after which the
stabilization due to electrostatic binding Tm increases less steeply [62, 63]. Melting
temperature (Tm) of CT-DNA was 72.5 °C under our experimental conditions. Tm values of

the complex-DNA solutions were also measured at a [Complex]: [DNA] ratio of 1:2. The
temperature was raised from 40 to 95° C (Fig. 6). The present complexes show ∆Tm values
(2.9 ± 1° C), (4±1 °C) for 1 and 2, respectively. ∆Tm values of complexes are characteristic
of an electrostatic groove binding for 1 and a partial intercalation behavior for 2 [56, 58, 64,
65].
3.3. BSA binding experiment
Since serum albumins are well known to bind to small aromatics, the possible binding
interactions of 1 and 2 with BSA have been investigated by absorption and emission titration
experiments at room temperature.
3.3.1. Electronic absorption titration
BSA has two main absorption bands in UV spectra: one located in the range of 210-
240 nm, which is the skeleton absorption peak, and the other at 278 nm, which is the aromatic
amino acid (Trp, Tyr, and Phe) absorption band [66]. By adding the two complexes, the BSA
skeleton absorption intensity in the range of 210-240 nm is decreased, indicating a
perturbation of α-helix induced by a specific interaction between complexes and BSA (Fig. 7)
[67].
3.3.2. Fluorescence quenching studies
The fluorescence properties of BSA arise mainly from the presence of tryptophan,
tyrosine and phenylalanine residues [68]. Quenching can occur by either dynamic or static
mechanism. Dynamic quenching refers to a process in which fluorophore and the quencher

come in contact during the transient existence of the excited state while static quenching is
the fluorophore quencher complex formation in the ground state. Quenching of the emission
intensity of tryptophan residues of BSA at 345 nm (excitation wavelength at 280 nm) was
monitored using 1 and 2 as quenchers with increasing complex concentration. According to
Fig. 8, the fluorescence emission intensities of BSA show moderate decreasing trend with
increasing concentration of the two complexes, which indicates that the complex interactions
with BSA change protein secondary structure resulting in changes in BSA tryptophan
environment [69].
The ability of complexes to quench the emission is associated with the extent of their
interaction with BSA, which follows the Stern-Volmer equation (3):
I0/I= 1 + Ksv [Q] = 1 + kqτ0 [Q] (3)
where I0 and I are the fluorescence intensities in the absence and presence of the
complexes, respectively, Ksv is a linear 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. In the present work, the value of Ksv for Pd complexes are
2.4 ×104 and 2.5 ×104 M−1 for 1 and 2, respectively.
Since the value of τ0 for tryptophan fluorescence in proteins is 2×10-8 s [70], the
quenching rate constant, kq, can be calculated using the fallowing equation:
kq= Ksv/τ0
(4)
The obtained values of quenching constants (kq) were 1.2 × 1012 and 1.25 × 1012
M−1s−1 for 1 and 2, respectively. However, the maximum scatter collision quenching constant,
kq, for various quenchers with the biopolymer is 2 × 1010 M−1s-1 [71]. Thus, the rate constant
calculated by protein quenching procedure is greater than kq of scatter procedure. This
indicates that a static quenching mechanism is operative [72].

For the static quenching interaction, if it is assumed that there are similar and
independent binding sites in the biomolecule, the binding constant (Kb) and the number of
binding sites (n) can be calculated from the following equation: [73]
–ꢂꢊꢌ
Log ꢉꢊ
ꢋꢂ
[ ]
= LogꢂKꢍ + nꢂLogꢂ Q (5)
ꢊ
where n is the average number of binding sites per albumin molecule and Kb is the
binding constant for the complex-protein interaction in the present case. The double
logarithmic plot of log [I0-I/I] vs. log [Q] is shown in Fig. 9. For the system complexes and
BSA, the values of Kb and n at 298K were (1.4×104 M-1, 1.3) for 1 and (1.68×104 M-1, 1.2)
for 2. Moreover, the linear correlation coefficient was calculated to be 0.99, which indicates
that the assumptions underlying the derivation of equation (5) were satisfied. Complex 2
exhibits a higher binding constant for BSA than 1 (n > 1). This shows that there are more
binding sites on BSA.
3.3.3. Site selective binding
The crystal structure of BSA contains three domains (sites) (I, II, III), which have
similar 3D structures. Each domain is composed of two subdomains (A and B) [74]. Among
these, IIA and IIIA subdomains with hydrophobic nature function as principle binding sites
[75]. Subdomain IIA corresponds to the drug site I, which is known to be a Warfarin binding
site and subdomain IIIA is referred to as drug site II, to which Thyroxin, Ibuprofen,
Diazepam and some other drugs bind [76]. Digoxin binding is independent of sites I and II
[77]. Digoxin binding site is also denoted as site III [78].
To determine the specificity of the drug binding, competition experiments were
performed with Warfarin, Ibuprofen and Digoxin. In these experiments, 1 or 2 (0-6.0×10−5
mol L−1) were gradually added to a solution of BSA and site markers held in equimolar
concentrations (6.0×10−6 mol L−1). As shown in Fig. 10, with the addition of site markers into

BSA solution, the fluorescence intensity became significantly lower than that without the site
markers. Equation (5) was used for the analysis of the binding constant in the presence of site
markers to simplify the comparison of Warfarin, Ibuprofen and Digoxin influence on the
complex binding to BSA (Table 4). The binding constant is surprisingly variable in the
presence of Ibuprofen, while a smaller influence is observed in the presence of Warfarin and
Digoxin for 1. The results indicate that the binding site for 1 is mainly located within site II
of BSA. Competitive titration result of 2 is different. In the presence of Warfarin, Kb value
decreased. This indicates that 2 binds to the same site in BSA as Warfarin [79]. Docking
studies also support this observation, as will be discussed later.
3.4. Molecular docking
3.4.1. Docking with DNA
In the molecular docking studies, 1 was successively docked into DNA to predict the
appropriate binding site and preferred orientation inside DNA. The lowest energy
conformations of the docked structures are illustrated in Fig. 11. Complex 1 interacts with
DNA near the A/T rich sequence and the almost planar parts of the complex fit into the minor
groove of the DNA. 1 is stabilized by hydrogen bonding through the NH2 group of the
benzylamine ligand. The calculated free binding energy (∆Gb) of the interaction of 1 with
DNA was found to be -9.4 kcal mol-1. The binding free energy indicates a high binding
affinity between the complex and DNA.
3.4.2. Docking with BSA
The experimental observations were substantiated through molecular docking studies
and the preferred binding sites of complex on the BSA molecule have been analyzed. The
docked conformations are shown in Fig. 12. As shown, 1 binds to BSA by the residues such
as ASP 255, ASP 258, ASP 236, TYR 262, LEU 259, LEU 233 and LYS 232. Some of these
residues are related to site I (IIA) and some are related to site II (IIIA) of BSA [80-82].

Results for docking show that 1 can bind to BSA via more than one site. This is confirmed
with n value (n=1.3) from equation (5). Complex 1 and Warfarin can bind cooperatively at
the same binding site. On the contrary, there is a competition between 1 and Ibuprofen for
binding at site II, which results in the decrease of Kb in related competitive titration.
However, site I is large enough to accommodate Warfarin and 1 molecules simultaneously
[83], as observed by no perceptible reduction in Kb value.
3.5. In vitro selective cancer cell cytotoxic activity
Numerous palladium complexes have been investigated for their anticancer activity
[84-86]. In the present study, the anticancer activity of the synthetic palladium complexes on
Raji leukemia cell line, A549 lung cancer and MCF7 breast cancer cells were evaluated 48 h
after the treatment using MTT-dye reduction method, as a widely used cytotoxicity assay.
Cytotoxicity is the degree to which an agent has a specific destructive action in certain cells
and is the quality of toxicity to cells. Cancer cells were incubated in the presence of
increasing concentrations of the complexes for 48 h and 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 the same
experimental conditions. As shown in Fig. 13, these complexes revealed significant anti-
proliferation activity against the cancer cell lines in the range of 0-100 µmol dm-3. The
cytotoxicity data, expressed as IC50 valuess are presented in (Table 5). IC50 values are defined
as the concentration of a compound, which inhibits 50% of the cell growth. According to the
IC50 values, the cytotoxicity of 2 was more than 1. The stronger activity of 2 could be
attributed to the presence of Letrozole ligands [21]. These results suggest that these
complexes may be potential anticancer agents and further investigation are required to
achieve better efficiency and find more on their mode of action.
4. Conclusion

Two new palladium compounds bearing benzylamine ligand have been described in
this work. The crystal structure of complex 1 was determined by X-ray crystallography
revealing a (distorted) tetragonal geometry for Pd (II). Electronic absorption, fluorescence
spectroscopy, thermal denaturation and DNA solution circular dichroism (CD) measurements
were used to explore the DNA binding properties of the two complexes. The results
suggested that 1 could interact with CT-DNA via groove binding, 2 showed a different
behavior and binding to DNA as a partial intercalator. The reactivity towards BSA revealed
that the quenching of BSA fluorescence by the two complexes are static quenching and 2
exhibits greater binding affinity than 1. The site marker competitive experiments indicate that
both Pd (II) complexes bind to the BSA molecule in more than one site. Molecular docking
studies restated interaction mode of complex 1 with DNA and BSA. The cytotoxic studies
show that 2 exhibits more cytotoxic activity against different cell lines.
Acknowledgments
Funding of this research by the Isfahan University of Technology (IUT) is gratefully
acknowledged.
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Scheme 1. Synthesis of complexes 1 and 2. (I): direct reaction of primary benzylamine
(PhCH2NH2) with Pd(OAc)2 in a 2:1 molar ratio has been carried out in toluene at 25°C (a),
addition of NaCl to a acetone/water solvent mixture of (a) yielded complex (1). (II): (1)
reacted with AgNO3 and Letrozole (L) in acetone and was protected from light at room
temperature to prepare complex (2).
Fig. 1. ORTEP diagram for complex 1
Fig. 2. Electronic spectra of 1 (A) and 2 (B) in buffer solution (5 mM Tris-HCl/10 mM NaCl
at pH 7.2) upon addition of CT-DNA. [Complex] = (5×10-5M), [DNA] = (5×10-4M). Arrow
shows the absorption intensities decrease upon increasing DNA concentration. Inset: Plots of
[DNA]/(ꢂεꢃ − εꢄ) vs. [DNA] for the titration of 1 and 2 with CT-DNA
Fig. 3. Emission spectra of complexes 1 (A) and 2 (B). [Complex] = (5×10-5M) in Tris-HCl
buffer (pH 7.2) in the presence and absence of [CT-DNA] = (5×10-4M) at room temperature.
Arrow shows change in intensity with increasing concentration of DNA
Fig. 4. Emission spectra of the DNA-MB system in the presence of complexes 1 (A) and 2
(B), [DNA] = 5×10-5 M, [complex] = 0-1×10-5 M, [MB] = 5 × 10-6 M. The arrows show the
emission intensity changes upon increasing complexes concentration
Fig. 5. CD spectra of [CT-DNA] = (1×10-4mol L-1) in the absence and presence of
[complexes] = (5×10-5mol L-1) in 5 MmTrisHCl with 50 Mm NaCl (pH 7.2)
Fig. 6. Plots of the changes of absorbance at 260 nm of [CT-DNA] = (8×10-5mol L-1) on
heating in the absence and the presence of [complex] = (4×10-5mol L-1) in 5 mM TrisHCl
with 50 Mm NaCl
Fig. 7. UV absorption spectra of [BSA] = (5×10-6M) in the absence and presence of
[complexes] = (5×10-5M)
Fig. 8. Emission spectra of BSA upon the titration of complexes 1 (A) and 2 (B). [BSA] =
(5×10-6M), [complex] = (0-5×10-5). Arrow shows the change upon the increasing complex
concentration. Inset: Plots of F0/F vs [Q]×106
Fig. 9. Scatchard plots of log [(F0−F)/F] vs log [Q] for determination of the complex-BSA
binding constant and the number of binding sites on BSA for complex 1 (A), 2 (B)

Fig. 10. Influence of selected site markers on the fluorescence of complex 1 (A), complex 2
(B) bound to BSA (T=298K, λex = 285 nm). [BSA] = [site markers] = 5×10-6M; [complex]
= (0-5×10-5M)
Fig. 11. Results of docking procedure of complex 1 with the minor groove binding to the
DNA.
Fig. 12. Results of docking procedure of complex 1 in the interaction with BSA residues,
which results in quenching
Fig. 13. In vitro cytotoxic activity of 1 and 2 against different tumor cell lines. Raji (A),
A549 (B), MCF7 (C)
Table 1
Crystallographic data and structure refinement details for complex 1
Table 2
Selected bond lengths (Å), and angles (°) for complex 1
Table 3
Hydrogen bond geometry (Å, °)
Table 4
Estimated binding constants for site marker competitive experiments of the complexes-BSA
system

Table 5
Cytotoxicity data (IC50) of the palladium (II) complexes against Raji, A549 and MCF-7
tumor cell lines
Scheme 1

(I)
H
H
Pd(OAc)2
N
CH2ph
X
Pd
X= OAc (a)
X= Cl (1)
X
NH2
phH2C
N
H
H
N
C
(II)
N
N
N
-AgCl
C
N
N
2+
C
N
C
H2N
Pd
N
N
N
N
N
2 NO3
N
NH2
C
N
C
N
(2)
Fig. 1.

Fig. 2.

Fig. 3.