Novel Pd(ii)-salen complexes showing high in vitro anti-proliferative effects against human hepatoma cancer by modulating specific regulatory genes

Article abstract of DOI:10.1039/c2dt31143g

We have reported the synthesis of a novel salen ligand and its mononuclear Pd-salen complexes derived from 2-{[2-hydroxy-3-{[(E)-(2-hydroxyphenyl) methylidene]amino}propyl)imino]methyl}phenol. The newly synthesized and isolated Pd(ii) complexes have been

Full text of DOI:10.1039/c2dt31143g

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Novel Pd(II)-Salen complexes showing high in vitro anti-proliferative effects
against human hepatoma cancer by modulating specific regulatory genes
Mohammad Azam1*, Zahid Hussain2, Ismail Warad1, Saud I. Al-Resayes1, Mohd. Shahnawaz
Khan3, Mohammad Shakir4, Agata Trzęsowska-Kruszynska5 and Rafal Kruszynski5
1Department of Chemistry, King Saud University, P. O. Box 2455, Riyadh 11451, KSA
2Center of Excellence in Biotechnology Research, King Saud University, Riyadh, 11451, KSA
3PRC, Department of Biochemistry, King Saud University, Riyadh 11451, KSA
4Department of Chemistry, Aligarh Muslim University, Aligarh 202002 India
5Department of X-ray Crystallography and Crystal Chemistry, Institute of General and
Ecological Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924, Lodz, Poland
Author for Correspondence:
Mohammad Azam
Email: azam_res@yahoo.com Tel: +966-596441517
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Abstract:
We have reported the synthesis of a novel Salen ligand and its mononuclear Pd-Salen complexes
derived from 2-{[2-hydroxy-3-{[(E)-(2-hydroxyphenyl)methylidene]amino}propyl)imino]
methyl}phenol. The newly synthesized and isolated Pd(II) complexes have been identified and
fully characterized by various physico-chemical studies viz., elemental analyses, IR, UV–Vis,
1H, 13C NMR spectroscopy, Electron Spray Ionization Mass Spectrometry (ESI-MS) and
TGA/DTA studies. The molecular structure of salen ligand has been ascertained by single crystal
XRD and it is coordinated to Pd(II) ion through two nitrogen and two oxygen atoms. The UV-
Vis data clearly suggest a square planar environment around both the Pd(II) ions. The DNA
binding studies of the synthesized compounds has been investigated by electron spectroscopy
and fluorescence measurements. The results suggest that Pd(II) complexes bind to DNA strongly
as compared to free ligand. The free salen ligand and its Pd(II) complexes have also been tested
against human hepatoma cancer cell line (Huh7) and results manifested exceptional anti-
proliferative effects of Pd(II) complexes. The anti-proliferative activity of Pd(II) complexes has
been modulated by specific regulatory genes.
Keyword: Salen ligand, Pd(II) complexes, Physico-chemical studies, DNA binding, anti-
proliferative, human hepatoma cell, gene expression
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1. Introduction:
Since the revolutionary discovery of cisplatin, chemically named as cis-
(diamminedichloroplatinum(II) as an anticancer drug by Rosenberg, extensive research work
has been carried out on platinum based chemotherapeutic compounds [1-2]. Despite its
remarkable success with high efficacy against human testicular, ovarian, bladder, head and neck
carcinomas, several side effects such as limited water solubility and the dose-dependent
toxicities, mainly nephrotoxicity, neurotoxicity, cytotoxicity, and emetogensis are the major
drawbacks associated with this drug [3-5]. Besides this, prolong use of cisplatin also leads to
cellular resistance in some of the most common cancers [6]. Therefore, to overcome these
problems, attempts are being made to replace this drug with suitable alternatives, and numerous
transition-metal complexes have been synthesized and screened for their anticancer activities [6].
The first non-platinum complex tested in clinical trials was cis-[(CH3CH2O)2(bzac)2Ti(IV)] used
against a wide variety of ascites and solid tumors [7-8]. The significant similarity between the
coordination chemistry of Pd(II) and Pt(II) compounds has advocated studies of Pd(II)
complexes as antitumor drugs, but the higher liability in ligand exchange at Pd centre (105-fold
vs Pt) may cause rapid hydrolysis processes leading to dissociation of complex and formation of
very reactive species unable to reach their pharmacological targets [9]. These problems could be
overcome by using the bulky heterocyclic and chelating ligands. A number of palladium
complexes with aromatic N- and N,N-containing ligands have shown very promising antitumor
characteristics [10,11]. Recent studies demonstrate that some Pd(II) complexes exhibit a
noticeable in vitro cytotoxic activity, comparable to standard platinum- based drugs, cisplatin,
carboplatin and oxaliplatin [12]. Over the years chemists have prepared ligands with a wide
range of features designed to achieve their particular targets. In this context, the chemistry of
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metal complexes containing salen-type ligands has received a lot of attention because of their
versatility and wide range of complexing ability, and are also the key points in the development
of inorganic biochemistry, catalysis, magnetism, medical imaging etc. [13-16]. Some of the salen
complexes have also revealed interesting antioxidant and antitumor properties [17]. Here in this
article, we report the synthesis and characterization of a novel salen ligand derived from 1,3-
diaminopropanol and o-hydroxybenzaldehyde and its Pd(II) complexes. The synthesized ligand
and its complexes have been screened for their in vitro viability and anti-proliferative activities
in human hepatocellular carcinoma (Huh7) cells. We have also analyzed the effect of salen
ligand and its Pd(II) complexes on the expression of the key genes involved in tumor promoting
and tumor suppressing function; such as Wnt-1, Urokinase plasminogen activator (uPA), Ras
association domain family 1A (RASSF1A), and transforming growth factor-β (TGF-β).
2. Experimental:
2.1 Materials and Methods
All the reagents and solvents used were of analAR grade quality and were purchased from Merck
and used as received. All complexations reactions were carried out using standard schlenck
techniques under argon. Elemental analyses were recorded on Elementar Varrio EL analyzer.
FT-IR (4000–400 cm-1) spectra were obtained as a KBr pellet using Perkin Elmer 621
spectrophotometer. 1H and 13C NMR spectra of ligand and its Pd(II) complexes were recorded in
CDCl3 using JEOL 400 spectrometer. Chemical shifts are quoted in parts per million from TMS.
Mass spectrometry was performed with a Micromass Quattro Premier tandem MS fitted with an
ESI interface and controlled by MassLynx 4.1 software. MS/MS detection was performed with
electrospray positive ionization mode. Electronic spectra of the complexes were obtained in
dichloromethane on Pharmacia LKB-Biochem, UV-vis spectrophotometer at room temperature.
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Thermal behavior of the synthesized compounds was studied by using SDTQ-600 (TA
o
Instrument) in helium atmosphere (100 mLmin–1) at heating rate of 20 C/min., in the
temperature range from ambient to 800 oC.
2.2 Synthesis:
2.2.1 Synthesis of salen ligand: L
A methanolic solution of o-hydroxybenzaldehyde (2 mmol) was added dropwise to the
methanolic solution of 1,3-diaminopropanol (1 mmol). The reaction mixture was stirred for 5 h
resulting in to a clear yellow colored solution. The resulting colored solution was concentrated to
1 ml followed by addition of 20 ml of diethyl ether to cause precipitation. Precipitate was
removed by filtration. After couple of days, yellow crystals suitable for X-ray diffraction
appeared in filtrate. The crystals were filtered off, washed with hexane and dried in vacuo.
0
1
Yield 70 %, Color: Yellow, Mp.: 138 C; Molecular formula C17H18N2O3; H NMR (CDCl3): δ
(ppm) 13.82 (Ar-OH), 8.35 (s -CH=N), 6.79-7.10 (m, Ar-H), 3.58-3.65 (m 4H –CH2), 3.65 (s,
1H, -CH). 13C NMR (CDCl3): δ (ppm) 163.68 (-CH=N), 62.85 (-CH2), 69.15 (-CH), 160.15,
131.85, 131.91, 116.45, 118.50 and 117.21 (Aromatic carbons), Anal. Cal: C, 68.44; H, 6.08; N,
9.39; O, 16.08 % Found: C, 68.25; H, 5.97; N, 9.28; O, 15.98 % IR, 3270 cm-1 (ν(Ph-OH)), 1655
cm-1 (υ-CH=N), 1278 (υPhenolic C-O).
2.2.2 Synthesis of Complexes: [PdLCl2] 1 and [PdL(OAc)2] 2
A solution of Pd(II) salt (0.50 mmol) dissolved in 10 ml CH2Cl2 was added dropwise in 10 ml
CH2Cl2 solution of ligand (0.50 mmol). The resultant reaction mixture was stirred for half an
hour resulting in to a clear yellow colored solution. The resulting solution was concentrated to 1
ml followed by addition of 10 ml of hexane for precipitation. The resulting colored precipitate
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was isolated and recrystallised in dichloromethane-hexane mixture and obtained in analytically
pure form. No crystal was found suitable for single crystal XRD.
0
1
1 : Yield 70 %, Color: Yellow, Mp.: 138 C; Molecular formula C17H18N2O3PdCl2; H NMR
(CDCl3): δ (ppm) 8.41 (s -CH=N), 6.89-6.97 (m, Ar-H), 3.87 (s, 1H, -CH), 3.86-3.72 (m 4H –
CH2). 13C NMR (CDCl3): δ (ppm) 167.56 (-CH=N), 63.36 (-CH2), 70.58 (-CH), 161.03, 132.71,
131.68, 118.92, 118.72 and 117.07 (Aromatic carbons), Anal. Cal: C, 42.93; H, 3.81; N, 5.89; O,
10.09; Pd, 22.37; Cl, 14.91 % Found: C, 42.88; H, 3.79; N, 5.85; O, 9.98; Pd, 22.32; Cl, 14.85
% IR, 1290 cm-1 (υPd-O), 1625 cm-1 (υ-CH=N
)
0
1
2: Yield 70 %, Color: Yellow, Mp.: 138 C; Molecular formula C21H24N2O7Pd; H NMR
(CDCl3): δ (ppm) 8.56 (s –CH=N), 6.87-7.97 (m, Ar-H), 5.23 (s, 1H, -CH), 3.61-3.75 (m 4H –
CH2), 13C NMR (CDCl3): δ (ppm) 172.25 (-CH=N), 63.85 (-CH2), 70.15 (-CH), 167.85, 161.80,
132.15, 132.25, 117.75, 118.23 (Aromatic carbons), Anal. Cal: C, 48.24; H, 4.62; N, 5.35; O,
21.42; Pd, 20.35 % Found: C, 48.19; H, 4.57; N, 5.31; O, 21.37; Pd, 20.28 % IR, 1282 cm-1
(υPd-O), 1648cm-1 (υ-CH=N
)
2.3 Crystal structure determination
The yellow prism crystal of free L was sealed in glass capillary filled with helium and next it
was mounted on a KM-4-CCD automatic diffractometer equipped with CCD detector, and used
for data collection. X-ray intensity data were collected with graphite monochromated CuKα (λ =
1.54178 Å) radiation at temperature 291.0(3) K, with
ω
scan mode. The 34 seconds exposure
= 68.04°. The unit cell
time was used, and reflections inside Ewald sphere were collected up to
θ
parameters were determined from 3008 strongest reflections. Details concerning crystal data and
refinement are given in Table 1. Examination of reflections on two reference frames monitored
after each 20 frames measured showed no loss of the intensity during measurement. During the
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data reduction Lorentz, polarization and numerical absorption corrections were applied [18]. The
structure was solved by partial structure expansion procedure. All the non-hydrogen atoms were
refined anisotropically using full-matrix, least-squares technique on F2. All the hydrogen atoms
were found from difference Fourier synthesis after four cycles of anisotropic refinement, and
refined as “riding” on the adjacent atom with geometric idealisation after each cycle of
refinement and individual isotropic displacement factors equal 1.2 times the value of equivalent
displacement factor of the parent carbon atoms and 1.5 times of parent oxygen atoms. The
hydroxyl groups were allowed to rotate about the O-C bond axes. The studied crystal was
racemically twinned with the 0.56:0.44 participation of chiral domains. The Flack parameter was
refined as full matrix parameter. The SHELXS97, SHELXL97 and SHELXTL programs were
used for all the calculations [19]. Atomic scattering factors were those incorporated in the
computer programs. Selected interatomic bond distances and angles are listed in Table 2 and
intermolecular interactions are listed in Table 3.
2.4 DNA binding assays of free L and Complexes 1 and 2
DNA binding experiments were performed on UV-Vis and fluorescence spectroscopy. The
purity of calf thymus DNA was confirmed by taking the ratio of the absorbance values at 260
and 280 nm in Tris EDTA (10mM, pH 7.0) buffer, which was found to be 1.8:1, indicating that
the DNA was sufficiently free of protein and other contaminants. The concentration of DNA for
binding studies was determined by absorption spectroscopy at 260 nm using the molar extinction
coefficient value of 6600 dm3mol-1 cm-1. The novel free L and its complexes 1 and 2 were
dissolved in 5% DMSO. Absorption titration experiments were performed with a fixed
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concentration of DNA (0.1mM) while gradually increasing concentration of free L and its
complexes 1 and 2 from 10-50 µM.
Further, to strengthen the Ligand-DNA binding interaction, fluorescence quenching experiments
were performed. Nucleic acid binding assays of synthesized compounds were carried out in the
presence of ethidium bromide. DNA and ethidium bromide (EB) were dissolved in tris-EDTA
(10 mM, pH 7.0) buffer at the concentrations of 4 and 1 µg/ml, respectively. The concentration
of ligand, L and complexes 1 and 2 was 50 µM. To perform the experiments, DNA was
pretreated with ethidium bromide for 30 min. Then the test solutions were added to this mixture
of EB-DNA, and the change in the fluorescence intensity was measured. Fluorescence
measurements were carried out on a Shimadzu spectrofluorimeter model RF-540 equipped with a
datarecorder DR-3. The fluorescence was recorded at 485–685 nm after exciting the solution at
478 nm. The slits were set at 5 nm for excitation and emission. The path length of the sample
was 1 cm.
To determine the DNA-binding ability of free L and complexes 1 and 2, fluorescence intensity
data were analyzed by the Stern-Volmer equation [20].
F0 /F= 1+Ksv [Q]
Where, F and F0 are the fluorescence intensity with and without the quencher (complex-DNA),
Ksv the Stern-Volmer quenching constant, and Q the concentration of the quencher. The KSV
value of the complexes [EB+DNA+L], [EB+DNA + 1] and [EB+DNA+ 2] were calculated to be
1.23 x 105, 3.85 x 105 and 3.67 x 105 M-1, respectively. A higher KSV value of complex 1
suggests its stronger quenching ability than complex 2 and free L.
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2.5 Cell Culture and Treatment
Human hepatoma cell line Huh7, was grown in Dulbecco’s modified Eagle’s medium (DMEM,
Gibco-BRL) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL) and 10 µg of
gentamycin ml-1 at 37°C in 5% CO2. In the treatment group, Huh7 cells treated for 48 hours with
different test compounds (free L, Complexes 1 and 2 at final concentration of 0.1 µM, along with
vehicle control alone (DMSO).
2.6 Cell Proliferation Assay
Huh7 cells were plated in quadruplicate at a density of 60,000 cells/well in 2 ml of respective
culture media in six well plates. Cells were grown in the regular medium with 5% FBS in the
control and treatment (free L, Complexes 1 and 2) groups. Cell growth curve was analyzed at
dose of (0.1 µM). Cells were trypsinized, and the number of viable cells was determined by 0.4%
trypan blue and counted at different time points starting from day 0 to 48 hours, using a Coulter
Counter (Model ZF; Coulter Electronics, Hertfordshire, UK).
2.7 Quantitative real time RT-PCR of genes involved in cancer
Total cellular RNA from control and treated (free L, Complexes 1 and 2) Huh7 cells were
extracted using Trizol (Invitrogen Life Technologies) according to manufacturer’s protocol. Two
micrograms of total RNA was used for reverse transcription for cDNA preparation. The cDNA
was then used for quantitation of genes involved in cancer by real time PCR (Applied Biosystem,
7500 Real Time PCR system). The primers used to quantify different genes are mentioned in
Table 4.
2.8 Statistical analysis
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Results were analyzed as the mean SEM, and comparisons of the experimental data were by
two tailed independent sample student’s t-test. P value of <0.05 was considered statistically
significant.
3. Results and Discussions:
The free salen ligand, L has been synthesized by the interaction of o-hydroxybenzaldehyde with
1, 3-diaminopropanol in 2:1 molar ratio in methanolic medium (Scheme). Pd(II) complexes,
synthesized by reaction of free L and Pd(II) salts in 1:1 molar ratio in dichloromethane in
schlenck line under argon atmosphere, are colored microcrystalline solids. All the synthesized
compounds are insoluble in water and soluble in all organic solvents. The formation of ligand
and its Pd(II) complexes were confirmed on the basis of results of elemental analyses, molecular
ion peak in ESI-MS spectra, characteristic absorption bands in FT-IR, and resonance signals in
1
13
the H and C NMR spectra, and single crystal XRD in case of ligand. The geometry around
Pd(II) ions in the complexes was confirmed from the positions of absorption bands observed in
UV–Vis spectra. The in vitro cytotoxic activity of the isolated free L and its Pd(II) complexes
have been tested against human Hepatoma Cancer Cell line, Huh7. Results suggest that Pd(II)
complexes are more prominent in showing anti- proliferative activity when compared with parent
salen ligand.
3.1 X-ray crystallography
A perspective view of free L shows that all atoms occupy the same general positions (Fig 1). The
asymmetric part of the unit cell contains the four molecules of free L, and two of them
(containing N41 and N61 atoms) are related by non-crystallographic pseudosymmetry centre. In
general, the molecules possess different conformations (Table 2), however for the molecules
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related by pseudosymmetry these differences are relatively small (Table 2). Some of the atoms of
free L molecules show the differences in magnitude between components of the anisotropic
displacement parameters along chemical bonds and in the equivalent isotropic displacement
parameters in comparison to those ones of neighbouring bonded atoms. This originates primarily
from twisted character of parts of studied molecules aliphatic chains (for details see torsion
angles in Table 2) and thus more privileged motion and static disorder in directions
perpendicular to the interatomic bonds. The 2-hydroxybenzylideneaminomethyl moieties of all
molecules of free L are close to planarity, and however some obvious deviations of atoms from
the respect weighted least squares planes exist, they are not larger than 0.04 Å. Mentioned
moieties existing in duplicate within each molecule are inclined at 55.08(5), 62.15(5), 77.88(4)
and 75.78(4)°, respectively for molecules containing N1, N21, N41, N61 atoms. The analysis of
C-N bond lengths shows that the double bonds are fully localised within benzylideneamino
moieties.
Each molecule of free L contains the two O—H•••N intramolecular hydrogen bonds within 2-
hydroxybenzylideneamino moieties (Table 3), each forming S(6) motif. The molecules of 1 are
connected by O—H•••O intermolecular interactions (Table 3) to hydrogen bonded tetramers, in
which the hydroxyl substituents of molecule aliphatic part act as hydrogen bond donors, and
hydroxyl substituents of aliphatic and aromatic parts act as hydrogen bond acceptors. The
interactions form the N1DDDD zero-dimensional unitary graph set, which is also zero-
3
3
dimensional on higher graph set levels but it can be described as N3C3 (13) and N4C3 (13)D. In
the structure can be found two intramolecular C—H•••N and one intermolecular C—H•••O short
contacts (Table 3) which can be classified as weak hydrogen bonds [21]. This last interaction
expands the tetramers to the hydrogen-bonded pillar extending along crystallographic [001] axis.
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The stacking interactions are not observed within the crystal structure of free L (the shortest
distance between centroids of close to parallelity aromatic rings is 4.8026).
3.2 IR discussion
The most important IR bands and their assignments are listed in the experimental section. When
comparing the spectroscopic data of Pd(II) complexes with those of free ligand, marked changes
may be noticed in the ligand bands arising from various modes of donor groups involved in
bonding to palladium ions [Fig 2]. The IR spectrum of salen ligand exhibits a characteristic
ν
(CH=N) stretching band at 1655 cm-1 which is shifted to lower frequency and appears at 1630
cm-1 for 1 and 1648 cm-1 for 2 thus, indicating the coordination of azomethine nitrogen to Pd(II)
ion [22-24], which is further confirmed by the appearance of a band at 425-430 cm-1 assigned to
ν
(Pd-N) vibration [25]. The stretching band observed at ca. 3270 cm-1 due to the ν(Ph-OH) group in
the free ligand disappears in Pd(II) complexes due to deprotonation. A band which appeared at
1278 cm-1 (Phenolic C-O) in the free ligand has been shifted to higher frequency and appears at
1290 cm-1 and 1282 cm-1 in 1 and 2, respectively, suggesting the coordination through the
phenolic oxygen atom [24]. The absorption band corresponding to –OH group of
diaminopropanol remain unchanged in Pd(II) complexes concluding that the –OH group of
diaminopropanol doesn’t take part in coordination [26]. The absorption band appeared at 295
cm-1 are assigned to ν(Pd-Cl) vibration in 1, while the 2 shows two absorption bands at 1637 cm-1
and 1430 cm-1, assignable to νa(COO) and νs(COO) stretching vibrations of the carboxylate
group, respectively, indicating the monodentate coordination of the acetate group to Pd(II) ion
[27].
1
3.3 H and 13C NMR Spectroscopic analyses
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The 1H NMR spectrum of the free ligand exhibits a singlet at 13.82 assigned to the aromatic-OH
proton, which is absent in Pd(II) complexes indicating the coordination of phenolic oxygen to
Pd(II) ion. A significant azomethine proton due to –CH=N was observed at 8.35 ppm, which
upon coordination to Pd(II) ion showed a downfiled shift and occurred at 8.41 and 8.56 ppm for
1
1 and 2, respectively [Fig 3] [Fig 1S supporting information]. H NMR spectrum of ligand, L
also shows a series of mutiplets due to aromatic protons in the 6.79–7.10 ppm range. As
expected, upon coordination to Pd(II) ion, these signals show small shifts due to the ligand
electronic redistribution attributed due to interaction of ligand to Pd(II) ion. In addition,
Chemical shifts for -CH (s 1H) and –CH2 (d 4H) protons in free ligand appear at 3.65 ppm and
3.58-3.65 ppm, respectively. The findings of 1H NMR spectra are further supported by 13C NMR
spectra [Fig 4] [Fig 2S]. The 13C NMR spectrum of salen ligand shows a number of signals
corresponding to various carbon atoms in the proposed ligand, expected due to the non-
equivalence of the various carbon atoms. The free L shows sharp signal corresponding to
azomethine carbon at 163.68 ppm while the signal for –CH2, and -CH carbon appears at 62.85
and 69.15 ppm, respectively. The 13C NMR signals for aromatic carbon appear at160.15, 131.85,
131.91, 116.45, 118.50 and 117.21 ppm. These values undergo downfield shift upon
coordination to Pd(II) ion. The complex 2 shows a sharp signal at 172.25 ppm attributed to –
CH=N carbon while the signals for –CH2, -CH carbon appear at 63.85, 70.15 ppm. The signals
for aromatic carbon appear at 160.75, 161.80, 132.15, 132.25, 117.75 and 118.23 ppm [Fig 4].
Similarly, sharp signal for azomethine carbon appears at 167.57 ppm in complex 1. The signals
for –CH2 and –CH carbon appear at 63.36 ppm and 70.58 ppm, respectively, while The ignals for
aromatic carbon appear at 161.03, 132.71, 131.68, 118.92, 118.72 and 117.07 [Fig 2S].
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3.4 Mass spectrometry
The structure of the complexes was also supported by mass spectrometry under positive ion ESI
condition. The ESI-MS spectra of the synthesized ligand and its Pd(II) complexes show the
presence of the molecular ion as sodium adduct [M+Na]+. The ESI-MS spectra showed
molecular ion peaks [M+Na]+ , m/z at 299.34, 476.67, 522.84 corresponding to their molecular
formulae, L, [PdLCl2] and [PdL(OAc)2], while their corresponding calculated m/z being 298.34,
475.67, 522.85, respectively [Fig 5].
3.5 UV-Vis studies
UV–Vis absorption spectra of Pd(II) complexes have been recorded in the 200–800 nm range in
dichloromethane [Fig 6]. The bands below 321 nm are attributed to π-π* and n-π* transitions of
the ligands [28,29]. All Pd(II) complexes are diamagnetic with a square-planar geometry, for
which three spin-allowed d–d transitions are expected, corresponding to transitions from the
three lower lying d-levels to the empty dx2−y2 orbitals [30]. The most prominent absorption
bands appears at 401 nm and 397 nm for 1 and 2, respectively, ascribed to the metal-to ligand
charge transfer transitions [28, 29].
3.6 Thermogravimeric analyses
Thermal stabilities of 1 and 2 were carried out by thermogravimetric analyses (TGA and DTA)
0
in N2 atmosphere at heating rate of 20 C min-1 in the temperature range 20-800 ºC. These
compounds undergo three steps of weight losses [Fig 7]. The TGA of 2 shows first weight loss of
11.45 % corresponding to coordinated water and acetate ion in the temperature range of 30-252
0
0C. Further elevation in temperature from 252-485 C leads to rapid weight loss ca. 48.55 %
attributed to the dissociation of the salen ligand at the imine bond. A gradual weight loss in the
0
temperature range 485-800 C leads to the complete decomposition of organic moiety, until the
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final residue, Pd-O, is left. Thus the total weight loss upto 800 0C is found to be 60 %. The TGA
0
0
data is further supported by DTA, which shows three main steps at 20-200 C, 200-452 C and
452-800 0C. The maximum weight loss ca. 14 %/min occurred in the second stage at 200-452 0C.
The result of TGA and DTA clearly indicated that the second stage is the main degradation
stage.
3.7 DNA binding studies
UV–Vis absorption studies were performed to further ascertain the interaction of ligand and its
Pd(II) complexes with DNA. The UV absorbance showed an increase in absorbance at 260 nm
with increasing concentrations of free L and complexes 1 and 2 [Fig 8]. The rise in the DNA
absorbance is indicative of the interaction between DNA and synthesized compounds. The
obtained results showed that free L and its complexes exhibited hyperchromism (increase in
absorbance) but of varied degree. Complexes 1 and 2 showed greater affinity for DNA as
compared to free L. Interestingly, both the complexes, 1 and 2 showed approximately equal
binding with DNA. Hypochromism and hyperchromism are both spectral features of DNA
connected with its double helix structure. Hypochromism means the DNA-binding mode of the
complex is electrostatic or intercalative which can stabilize the DNA duplex [31, 32]. Contrary to
this hyperchromism means the breakage of the secondary structure of DNA. So we speculate that
our synthesized compounds (free L and its Pd(II) complexes 1 and 2) interact with the secondary
structure of calf thymus DNA resulting in its breakage and perturbation.
Further, ethydium bromide displacement assay for DNA binding study with free L and
complexes 1 and 2 were executed using fluorescence spectroscopy. Nucleic acid bound to some
molecules generally exhibit marked changes in fluorescence properties and this phenomenon is
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used in the studies with DNA [33]. Ethidium bromide, a polycyclic aromatic dye, is the most
widely used fluorescence probe for DNA structure. EB displays very weak fluorescence in
aqueous solution. However, in the presence of DNA, it exhibits intense fluorescence because of
the intercalation to base pairs in DNA [34]. Our results showed that the addition of free L and
complexes 1 and 2 to DNA being complexed with EB causes appreciable reduction in emission
intensity, indicating that synthesized compound have the ability of competitive binding with the
EB for DNA and thus reaffirm the binding of these novel synthesized molecules with DNA.
Moreover, results inflicted complexes 1 and 2 more quenching as compared to free L, thus
indicating greater affinity of both the complexes when compared to ligand. On the basis of above
mentioned results, we suggest that free L and complexes 1 and 2 have the ability to interact with
DNA. However, the affinity for DNA is more in case of complexes 1 when compared with
complex 2 and free L. The higher KSV value implicates the higher binding affinity of complex 1
in comparison to complex 2 and free L. This difference in their DNA binding ability could be
due to the presence of an electron deficient center in the charged Pd(II) complex where an
additional interaction between the complex and phosphate rich DNA back bone may occur [35].
It has been reported that the enhanced fluorescence of the EB– DNA complex can be quenched
at least partially by the addition of a second molecule and this could be used to assess the relative
affinity of the molecule for DNA [Fig 9] [36].
3.8 Effect of free L, complexes 1 and 2 on cell proliferation
To test in vitro anti-proliferative effect of synthesized componds (free L, complexes 1 and 2) on
hepatocellular carcinoma cell lines (Huh7), treatment dose was optimized to avoid cell
cytotoxicity. Huh7 cells were seeded at the same density and subjected to optimum dose of these
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compounds along with control (CTL) in six well plates. The optimized dose was used to treat at
0 hour and anti-proliferative effect was analyzed at different time 0, 12, 24 and 48 hours. As
shown in Fig 1, number of cells at 12 hours of treatment was significantly less in complexes 1
and 2 treated wells. The effect of free L at 12 hours of treatment was similar to that of control. At
24 and 48 hours, the cells treated with complexes 1 and 2 showed significant anti-proliferative
effects compared to free L and control (Fig 10 a and Fig 10 b) The complexes 1 and 2
significantly reduce the cell doubling time as well it showed limited cytotoxicity. Thus, our anti-
proliferative data confirmed the role of complexes 1 and 2 in reducing the cell doubling time and
hence proliferation.
3.9 Effect of free L, complexes 1 and 2 on tumor promoting genes
The cells treated with optimized concentration of free L, complexes 1 and 2 along with vehicle
control alone were subjected to total cellular RNA isolation. The total cellular RNA was
collected and analyzed for the expression of Wnt-1 and uPA mRNA levels. Our data indicate that
Huh7 cells treated with complexes 1 and 2 significantly reduce the expression of Wnt-1 mRNA
levels (Fig 11 a). The reduction was not significantly observed in alone treated Huh7 cells
compared to control. Similar effects were not observed with the level of urokinase plasminogen
activator (uPA) mRNA, when cells were treated with free L, complexes 1 and 2. As shown in
Fig 11b, uPA levels was almost similar to that of the vehicle control only. Our result
demonstrated that complexes 1 and 2 reduce the tumor promoting gene Wnt-1 but not
prometastatic gene uPA.
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3.10. Effect of free L, 1 and 2 on tumor suppressor gene (RASSF1A)
Tumor suppressor gene (RASSF1A) has been studied in several cancers, and reported that large
number of patients have inactivated RASSF1, while similar number have a tumor in which
RASSF1A pathway is partially abrogated by inactivation of other signaling components. Since, it
influences large number of proteins, the effect of compounds on the mRNA expression of
RASSF1Ahave been investigated. Total cellular RNA (treated and control) was analyzed for the
expression ofRASSF1A. Our results show that complexes 1 and 2 treated Huh7 cells
significantly enhance RASSF1A mRNA level compared to control group [Fig 11c]. This effect
was not observed in free L treated cells, and the mRNA expression level was almost similar to
that of control (Fig 11c).
3.11 Effect of free L, 1 and 2 on transforming growth factor-β(TGF-β)
Transforming growth factor-beta (TGF-β) is a multifunctional cytokine involved in the
regulation of growth and differentiation of both normal and transformed cells. Therefore, we
have investigated how these compounds effect the mRNA expression in hepatocellular
carcinoma cell line. We found that all of our synthetic compounds insignificantly enhanced the
mRNA expression of TGF-β (Fig 11d).
The ligand, L and its complexes 1 and 2 are tested for in vitro antitumor and anti-proliferative
activity and expression of different genes involved in cancer. On the basis of our experimental
results, complexes 1 and 2 showed a strong dose dependent growth inhibition of Huh7 cells
compared to DMSO (control) treated cells. The free L exhibits least inhibitory effect compared
to complexes 1 and 2. The anti-proliferative effect of complexes 1 and 2 corroborate earlier
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findings of palladium complexes derivatives that demonstrate a strong dose dependent growth
inhibition [37, 38]. To determine whether these compounds effect key players in cancer, we
measured the level of expression of Wnt-1, uPA, RASSF1 and TGF-β by real time PCR. In
mammals, Wnt signaling is complex and regulates diverse functions including initiation and
progression of cancer [39-42]. Therefore, we have tested the effect of our synthetic compounds
on the mRNA expression of Wnt-1. Complexes 1 and 2 demonstrated significant inhibition of
Wnt-1 expression compared to DMSO treated control. The free L treated cells showed decrease
Wnt-1 mRNA expression but not significant like that of complexes 1 and 2. These findings
establish that the Pd(II) complexes have anti-proliferative and anticancer effects compared to
parent ligand. Another important gene (uPA) analyzed in our study involves in metastatic
cascade and implicate the degradation of the extracellular matrix and subsequent invasion by
cancer cells [43-45]. Due the importance of this gene in metastasis, we analyzed its mRNA
expression in the presence of the synthetic compounds. Surprisingly, none of the compounds
suppress or activate the expression of uPA mRNA in Huh7 cell line. In most of the cancers, uPA
is epigenetically regulated, as the promoter region of this gene hypomethylated [46]. The level of
uPA mRNA expression is regulated by the methylation pattern (epigenome), it may a reason by
these compounds had no effect on the expression of mRNA.
The Ras-association domain family 1A (RASSF1A) gene is a new candidate tumor suppressor
gene (TSG) and has been reported to play an important role in the malignant progression of
many tumors [47-49]. Due to its role in development of HCC, we have tested the expression of
this gene in presence of our compounds. The finding was remarkable and showed enhanced
RASSF1A expression in presence of complexes 1 and 2, although free L expression was similar
to the control group. We speculate that complexes 1 and 2 may involve in genetic alternations
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and hence gain in RASSF1A expression. How these compounds alter the genetic structure needs
to be analyzed in detail. Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that
regulates cell proliferation, angiogenesis, metastasis, and immune suppression [50, 51]. TGF-β
has biphasic effect in tumor growth; carcinogenesis and early tumor growth are suppressed by
TGF-β whereas this growth factor apparently accelerates tumor progression in more advanced
aggressive tumors [49, 50]. Therefore, the effect of these compounds on the mRNA expression
has been investigated. There was insignificant increase in the level of mRNA expression
compared to control when Huh7 cells were treated with free L and complexes 1 and 2.
4. Conclusion:
A series of novel mononuclear Pd(II) complexes derived from salen ligand has been synthesized
and characterized by elemental analyses, and various spectroscopic studies. UV/Vis data reveal a
square planar geometry around Pd(II) ion. The structure of ligand has been confirmed by single
crystal XRD. The ligand and its complexes have been tested against human hepatoma cancer cell
line and results suggest that Pd(II) complexes have significant anti-proliferative effects.
5. Supplementary material available:
CCDC876116 contains the supplementary crystallographic data for this paper. This data can be
obtained free of charge from the Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk./data_request/cif
.
6. Acknowledgements:
This work is supported by Research Centre, College of Science, King Saud University, Riyadh
KSA
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7. References:
1. N.J. Wheate, S. Walker, G.E. Craig, R. Oun, Dalton Trans. 39 (2010) 8113–8127.
2. A.G. Quiroga, F.J. Ramos-Lima , A. Alvarez-Valdés, M. Font-Bardía, A. Bergamo, G.
Sava C. Navarro-Ranninger, Polyhedron 30 (2011) 1646–1650.
3. S.J. Lippard, P. Pil, Encyclopedia of cancer, cis-platin and related drugs. San Diego,
California: Academic Press, (1997) 392-410
4. F.J. Ramos-Lima, A.G. Quiroga, Jose M. Perez, Carmen Navarro-Ranninger, Polyhedron
22 (2003) 3379–3381
5. B. Lippert (Ed.), Cisplatin, 2006.
6. S. van Zutphen, J. Reedjik, Coord. Chem. Rev. 249 (2005) 2845–2853.
7. M.J. Clarke, F. Zhu, D.R. Frasca, Chem. Rev., 99 (1999) 2511
8. T. Schilling, B.K. Keppler, M.E. Heim, Invest, New Drugs, 13 (1996) 327
9. A.S. Abu-Surrah, M. Kettunen, Curr. Med. Chem. 13 (2006) 1337–1357.
10. G. Zhao, H. Lin, Curr. Med. Chem. Anticancer Agents 5 (2005) 137–147.
11. D.Kovala-Demertzi, A. Bocarelli,M.A.Demertzis,M. Coluccia, Chemotherapy 53 (2007)
148–152
12. N.J. Wheate, S. Walker, G.E. Craig, R. Oun, Dalton Trans. 39 (2010) 8113–8127.
13. E.C. Niederhoffer, J.H. Timmons, A.E. Martell, Chem. Rev. 84 (1984) 137
14. K. Srinivasan, P. Michaud, J.K. Kochi, J. Am. Chem. Soc. 108 (1986) 2309
15. W. Zhang, J.L. Loebach, S.R. Wilson, E.N. Jacobsen, J. Am. Chem. Soc., 112 (1990)
2801.
16. J. Tisato, F. Refosco, F. Bandoli, Coord. Chem. Rev. 135 (1994) 325.
21

Dalton Transactions
Page 22 of 42
View Online
17. J. Fonseca, J. Martinez, Luís C.-Silva, A. L. Magalhães, M. T. Duarte, C. Freire, Inorg.
Chimica Acta, 363 (2010) 4096-4107
18. X-RED. Version 1.18. STOE & Cie GmbH, Darmstadt, Germany (1999).
19. G.M. Sheldrick, A short history of SHELX, Acta Crystallogr. A64 (2008) 112-122.
20. A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar, N.J. Turro, J.K. Barton, J. Am.
Chem. Soc. 111 (1989) 3051–3058.
21. G.R. Desiraju, T. Steiner, The Weak Hydrogen Bond in Structural Chemistry and
Biology, Oxford University Press, Oxford, 1999.
22. L.J. Baucher, J. Inorg. Nucl. Chem. 36 (1974) 531-536.
23. J. Uttamachandani, R.N. Kapoor, Trans. Met. Chem. 3 (1978) 79-81.
24. N. Dharmaraj, P. Viswanathamurthi, K. Natarajan, Trans. Met. Chem. 26 (2001) 105-
109.
25. K. I. Goldberg, J. V.-Martinez, G.E.-Perez, L.J. Ackerman, D.X. West, Polyhedron, 18,
1999, 1177-1182
26. L.D. Popov, Y.P. Tupolova, V.V. Lukov, I.N. Shcherbakov , A.S. Burlov, S.I.
Levchenkov, V.A. Kogan, K.A. Lyssenko, E.V. Ivannikova, Inorganica Chimica Acta
362 (2009) 1673–1680.
27. K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds
(Wiley, New York, 1986; Mir, Moscow 1991).
28. A.B.P. Lever, Inorganic Electronic Spectroscopy, second ed., Elsevier, New York, 1984.
29. M. Ulusoy, O. Birel, O. Sahin, O. Buyukgungor, B. Cetinkya, Polyhedron (2012) In
press
22

Page 23 of 42
Dalton Transactions
View Online
30. H.H. Jaffe, M. Orchin, Theory and Applications of Ultraviolet Spectroscopy, J. Wiley,
New York, 1962.
31. E.C. Long, J.K. Barton, Acc. Chem. Res. 23 (1990) 271.
32. R. Sujata, S. Rona, S. Munna, J. Inorg. Biochem. 100 (2006) 1320.
33. C. Liu, J. Zhou, J. Inorg. Biochem. 71 (1998) 1–6.
34. F. Leng, D. Graves, J.B. Chaires, Biochim. Biophys. Acta 1442 (1998) 71–81.
35. J. Liu, T. Zhang, T. Lu, L. Qu, H. Zhou, Q. Zhang, L. Ji, J. Inorg. Biochem. 91 (2002)
269–276
36. S. Yellappa, J. Seetharamappa, L.M. Rogers, R. Chitta, R.P. Singhal, F. D’Souza,
Bioconjugate Chem., 17 (2006) 1418-1425.
37. L. Giovagnini, L. Ronconi, D. Aldinucci, D. Lorenzon, S. Sitran, D. Fregona, J Med
Chem. 48 (2005) 1588 -1595.
38. H. Cheng, F. Huq, P. Beale, K. Fisher, Eur J Med Chem. 41(2006) 896-903.
39. W. Wei, M.S. Chua, S. Grepper, Mol Cancer. 8 (2009) 76
40. Y. Takigawa, A.M. Brown, Curr Drug Targets. 9 (2008) 1013-1024.
41. S.B. Nambotin, J.R. Wands, M. Kim, Anticancer Agents Med Chem. 11(2011) 549-559.
42. H.C. Lee, M. Kim, J.R. Wands, Front Biosci. 11 (2006) 1901-1915
43. P.A. Andreasen , L. Kjoller, L. Christensen, M.J. Duffy, Int J Cancer. 72 (1997) 1-22
44. S.A. Rabbani, In Vivo. 12 (1998) 135-142.
45. S.A. Rabbani, A.P. Manzar, SurgOncol Clin N Am. 10 (2001) 393-415
46. N. Shukeir, P. Pakneshan, G. Chen, M. Szyf, S.A. Rabbani, Cancer Res. 66 (2006) 9202-
9210.
23

Dalton Transactions
Page 24 of 42
View Online
47. R. Dammann, C. Li, J.H. Yoon, P.L. Chin, S. Bates, G.P. Pfeifer, Nat Genet. 25 (2000)
315–19.
48. K. Dreijerink, E. Braga, I. Kuzmin, ProcNatlAcad Sci. USA 98 (2001) 7504–7509.
49. L. Shivakumar, J. Minn, T. Sakamaki, R. Pestell, M.A. White, Mol Cell Biol. 22 (2002)
4309–4318.
50. A. Nawshad, D. Lagamba, A. Polad , E.D. Hay, Cells Tissues Organs 179 (2005) 11–23
51. R.L. Elliott, G.C. Blobe, J Clin Oncol 23 (2005) 2078–2093.
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OH
OH
Stirring
CHO
N
N
Methanol
OH
H2N NH2
HO
OH
o-hydroxybenzaldehyde
1,3-diaminopropanol
Salen ligand, L
CH2Cl2
inert atmosphere
Stirring
PdX2
OH
N
N
Pd
X
X
O
O
X = Cl, OOCCH3
Scheme: Schematic representation of synthesis of salen ligand and its Pd(II) complexes
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Fig 1: Ortep diagram of free Ligand, L plotted with 50% probability of displacement ellipsoids.
The part of atoms labels and hydrogen atoms were omitted for clarity.
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Fig 2: IR Spectra of ligand, L, Complex 1 and 2
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Fig 3: 1H NMR Spectrum of Complex 1
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Fig 4: 13C NMR spectrum of Complex 2
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