Palladium (II) complexes based on Schiff base ligands derived from ortho-vanillin; synthesis, characterization and cytotoxic studies

Article abstract of DOI:10.1016/j.ica.2017.11.025

6-Methoxy-2-[(E)-Aryliminomethyl]-phenol (Aryl = Phenyl; 2,6-dimethyl; 2,6-diisopropylphenyl; 2,6-dichlorophenyl), comprising L₁–L₄ ligands, and palladium complexes [Pd(L_n)₂, n = 1–4] have been synthesized. The

Full text of DOI:10.1016/j.ica.2017.11.025

Accepted Manuscript
Research paper
Palladium (II) complexes based on Schiff base ligands derived from ortho-va-
nillin; synthesis, characterization and cytotoxic studies
Zeinab Faghih, Abdollah Neshat, Andrzej Wojtczak, Zahra Faghih, Zahra
Mohammadi, Solmaz Varestan
PII:
S0020-1693(17)31128-3
https://doi.org/10.1016/j.ica.2017.11.025
ICA 17995
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
20 July 2017
10 November 2017
14 November 2017
Please cite this article as: Z. Faghih, A. Neshat, A. Wojtczak, Z. Faghih, Z. Mohammadi, S. Varestan, Palladium
(
II) complexes based on Schiff base ligands derived from ortho-vanillin; synthesis, characterization and cytotoxic
studies, Inorganica Chimica Acta (2017), doi: https://doi.org/10.1016/j.ica.2017.11.025
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Palladium (II) complexes based on Schiff base ligands derived
from ortho-vanillin; synthesis, characterization and cytotoxic
studies
a
b, *
c
a
Zeinab Faghih , Abdollah Neshat , Andrzej Wojtczak , Zahra Faghih , Zahra
b
b
Mohammadi , Solmaz Varestan
a
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz,
Iran
b
Department of Chemistry, Institute for Advanced Studies in Basic, Sciences (IASBS),
Yousef Sobouti Blvd., Zanjan 45137-6731, Iran, a.neshat@iasbs.ac.ir
c
Department of Crystallochemistry and Biocrystallography, Faculty of Chemistry, Nicolaus
Copernicus University in Toruń, Gagarina 7, 87-100 Toruń, Poland
Abstract
6
-Methoxy-2-[(E)-Aryliminomethyl]-phenol (Aryl
diisopropylphenyl; 2,6-dichlorophenyl), comprising L1-
Pd(L
derived from single X-ray crystallography experiments. The central Pd(II) ion is four-
coordinated and surrounded by N environment, adopting a square planar geometry. The
=
Phenyl; 2,6-dimethyl; 2,6-
L
4
ligands, and palladium complexes
[
n
)
2
, n = 1-4] have been synthesized. The geometries of the [Pd(L ) ] complexes were
n
2
2
O
2
ligand is bidentate, coordinating via imine nitrogen and phenolate oxygen atoms to the metal
center. Analysis of the valence geometry within the phenolate rings suggests that in all
1
13
complexes, the O1‒C1 has a double bond character. FT-IR, H, C NMR and single X-ray
crystal structures were reported. The cytotoxicity effect of four Pd(II) complexes was
assessed on three cancerous cell lines: MCF-7 (breast carcinoma), A549 (lung carcinoma)
and SKOV3 (ovarian carcinoma) and compared with that for cis-platin. One out of four metal
1 2
complexes, Pd(L ) , exhibited the highest anti-proliferative activity on three investigated
cancerous cells lines which is more effective than cis-platin. The results showed that this
complex could effectively induce apoptotic death in cancerous cells, probably due to direct
interaction by cellular DNA.
Key words: Metallodrug, cis-platin, ortho-vanillin, apoptotic effect, Annexin V

1
. Introduction
Medicinal inorganic chemistry has grown significantly in the last two decades following
successes with platinum based metallodrugs in clinical tests against certain cancer cells (1-3).
However, side effects of cis-platin has prompted search for alternative metal based drugs in
fight against various types of cancers with less side effects and better cytotoxic properties
compared to cis-platin.
Inorganic and organometallic complexes of copper [4], gold [5] and silver [6] has received
particular attention in this respect. Studies show that both metal and ligand play an important
role in the overall biological activity of metal based drugs. Mechanistic studies have
suggested that each metal has its own unique mechanism of action against cancer cells [7,8].
The coordinated ligand also plays an important role in the anticancer activity of metallodrugs.
Furthermore, ligands surrounding a metal, other than stabilizing the metal ion can modify key
parameters of metal complex such as size, redox states and solubility [9-11].
Schiff base ligands provide a great platform in coordination chemistry for the development of
numerous ligand systems with controllable binding to metal ions [12-14]. Most Schiff bases
are easily accessible using simple synthetic procedures and with careful selection of an amine
precursor and a carbonyl compound, the nature of donor atoms and overall electronic and
steric properties of Schiff bases are tunable [15-17]. Ligands in this class could potentially
stabilize metals in different oxidation states and induce stability in homogeneous and
heterogeneous catalysts, which is particularly useful when they are viewed in catalytic
activity perspectives [18]. Metal complexes derived from Schiff bases have found
applications in different research areas, including and not limited to, molecular magnetism,
catalysis, and medical sciences [19-22]. Despite abundant reports regarding Schiff bases as
ligands in the coordination chemistry of transition metals and their diverse applications, less
research has been reported regarding palladium complexes supported with Schiff base ligands
as anti-tumor agents [23-30]. In this research, the synthesis and cytotoxic properties of four
palladium complexes supported with Schiff bases derived from ortho-vanillin have been
investigated. We implemented ortho-vanillin in the structure of Schiff base ligands (L
1
‒L )
4
because ortho-vanillin is a naturally occurring compound and we sought to explore biological
activity of the resulting Schiff bases in combination with palladium which closely resembles
platinum due to similarities of these two metals in ionic form. In order to study the effects of
ligand size and electronics on the overall cytotoxicity of the palladium complexes, the ligands

1 4
L -L with electron donor methyl and isopropyl substituents as well as electron withdrawing
chloride substituents on the phenyl ring of the imine backbone were synthesized and their
cytotoxicity was tested.
2
. Results and Discussion
2.1. Synthesis and characterization of palladium complexes
Synthesis of Schiff bases, L
1
4
-L , was carried out by refluxing a solution of an aryl amine with
ortho-vanillin in ethanol for 24 h, Scheme 1. The synthesis of Schiff base L
1
provided a
better yield in dichloromethane solvent. Due to better solubility of palladium chloride
precursor in acetonitrile and to avoid side reactions, all complexation reactions in this study
were performed in this solvent by mixing and allowing to react one equivalent of palladium
chloride or palladium acetate with two equivalents of corresponding Schiff bases. It is notable
4 2
that Pd(L ) complex provided higher yields in ether solution. The reactions leading to both
ligand precursors and metal complexes are shown in Scheme 1. The judicious choice of aryl
amine precursor allows for fine tuning of electronic and steric properties of ligands, therefore
influencing the resulting metal complexes' properties. As depicted in Scheme 1, four water-
soluble mononuclear Pd(II) complexes were synthesized. The ligands and metal complexes
1
13
were characterized by spectroscopic methods ( H & C NMR and IR) and in three cases the
molecular structures of metal complexes were studied using single crystal X-ray
crystallography.

R
OH
O
NH
2
O
R
R
OH
N
Ethanol
H
+
O
Reflux, 24 h
R
L1, R = H
L , R = CH
2
3
i
L , R = Pr
3
L4, R = Cl
R
N
O
R
R
N
O
OH
PdCl2
Pd
O
R
R
CH CN, NEt
3
3
O
N
rt, 24 h
R
O
*
Pd(L ) synthesis was carried out using Pd(OAc) in Ether
4 2 2
Pd(L ) , R = H
1
2
Pd(L2)2, R = CH
i
3
Pd(L ) , R = Pr
3
2
*Pd(L ) , R = Cl
4 2
Scheme 1. Synthetic route for Schiff bases (L
1
-L
4
) and corresponding Palladium complexes.
FT-IR spectrum of the free ligands, L
1
4
‒L , displayed a characteristic sharp bands related to
-1
-1
aromatic and aliphatic C‒H stretching vibrations between 2830 cm ‒ 3100 cm as well as
-
1
-1
C=N and C=C and C‒O stretching vibrations from 1300 cm to 1600 cm . As expected, all
these bands underwent a lower or higher frequency shift after coordination of these ligands to
-
1
palladium. For example, the band at ~ 1614 cm associated with stretching frequency of
-1
imine C=N bond in L
1
1 2
shifted to ~ 1604 cm in Pd(L ) , indicative of imine nitrogen
-1
coordination to the metal center. Disappearance of free ligand's OH band at ~ 3200 cm after
coordination of this functional group to metal is a strong indicative of metal complexation
through anionic phenolic oxygen, however in our study this was not observed mainly due to a
broad and weak OH stretching frequency bands observed in the free ligands. Therefore, a
legitimate comparison of the OH vibration frequency in free and coordinated ligand was
improbable.
1
1
H NMR spectra of L
−L
1 4
display similar resonance patterns. H NMR spectrum of L
2
and its
palladium complex, Pd(L
2 2
)
are shown in Figure 1 and Figure 2., respectively. Methoxy

resonance in all free ligands was observed close to δ 4 ppm. Aromatic ring protons resonated
in the range δ 6.5−7.5 ppm. Imine –HC=N resonated in the range δ 8−9 ppm in L and at
δ 10.1 ppm in L . Aromatic hydroxyl (-OH) group resonated in the range δ 13−13.5 ppm in
1 3
−L
4
1
L −
L
3
and an upper filed chemical shift (δ 10.6 ppm) was observed for this group in L .
4
1
Fig. 1. H NMR spectrum of L
2
. The signals from aromatic ring protons are expanded in green.
The resonances related to a hydroxyl (OH) functional group in the free ligand is absent in
metal complexes, indicating coordination of mono anionic oxygen. An upfiled chemical shift
was observed for the imine –HC=N moiety in all complexes compared to free ligands. This is
attributable to a reduced double bond current effects in –HC=N functional group following
coordination of this functional group to metal through nitrogen atom. An upfiled chemical
shift was also observed for methoxy resonance in palladium complexes.

1
2 2
Fig. 2. H NMR spectrum of Pd(L ) . The signals from aromatic ring protons are expanded in green.
The X-ray experimental data and structure refinement for the reported structures are
summarized in Table 1, the selected bond lengths and angles are presented in Table S1
(
ESI†). In Pd(L
1
)
2
3 2 i
and Pd(L ) reported here, the complexes reveal the crystallographic C
symmetry, with the Pd ions located on the center of symmetry. As a consequence, the
asymmetric parts of these structures consist of the half of the molecule, while the other half is
generated by the center of symmetry: [-x,-y+1,-z+1] for Pd(L
1
)
2
3 2
and [-x,-y,-z] for Pd(L ) . In
Pd(L the asymmetric unit of the structure consists of the whole complex molecule. The
2 2
)
Pd(II) coordination sphere is square-planar with the pairs of phenolate O and Schiff base N
occupying the trans positions (Figs. 3‒5).

1 2
Fig. 3. Representations of the X-ray crystal of Pd(L ) showing thermal ellipsoids for non-hydrogen atoms at
3
0% probability level. Selected geometrical parameters (Å, °): Pd1‒O1A 1.9767(14), Pd1‒O1 1.9768(14),
Pd1‒N1A 2.0179(16), Pd1‒N1 2.0179(16), O1A‒Pd1‒O1 180.00(10), O1A‒Pd1‒N1A 91.66(6),
O1‒Pd1‒N1A 88.34(6), O1A‒Pd1‒N1 88.34(6), O1‒Pd1‒N1 91.66(6), N1‒Pd1‒N1A 180.0.
In the reported complexes, the Pd‒O bonds formed by the phenolate atoms range from
1
3 2
.964(4) to 1.980(4) Å, both found in Pd(L ) , and are significantly shorter than the Pd‒N
distances varying from 2.008(5) to 2.0179(16) Å (Table S1 in ESI†). The N‒Pd‒O angles
involving atoms of the same ligand are larger by approximately 4‒5° than those involving
atoms of different ligands. That observation might be related to the rigidity of the ligand. In
fact, the exocyclic O1‒C1‒C6 and C1‒C6‒C7 angles of almost 126° and 123°, respectively,
are significantly larger than their counterparts O1‒C1‒C2 and C5‒C6‒C7 angles being both
approximately 117°.
Fig. 4. Thermal ellipsoids for non-hydrogen atoms are plotted at 30% probability level. Selected geometrical
parameters (Å, °): Pd1‒O1 1.964(4), Pd1‒O21 1.980(4), Pd1‒N21 2.008(5), Pd1‒N1 2.017(5), O1‒Pd1‒O21
1
9
79.6(2), O21‒Pd1‒N21 92.54(19), O1‒Pd1‒N21 87.19(19), O21‒Pd1‒N1 87.73(19), O1‒Pd1‒N1
2.54(19), N21‒Pd1‒N1 179.3(2).

In all complexes the ligand is a phenolate monoanion. However, analysis of the valence
geometry within the phenolate rings suggests that in all complexes, the O1‒C1 has a double
bond character, with the O‒C distances ranging from 1.299(7) Å in Pd(L
Pd(L . Furthermore, the average distances of the C2‒C3 and C4‒C5 bonds, being 1.364
and 1.352 Å (Table 2), are significantly shorter than other intra-ring distances that range from
.392(10) to 1.443(9) Å. These experimental data suggests that the ligands exist in the
quinone form. Also the methoxy substituents at C2 position reveal C2‒O2 bond distances of
.356(8) Å in Pd(L to 1.374(4) Å in Pd(L
2 2
) to 1.307(2) Å in
1 2
)
1
1
2
)
2
3 2
) .
3 2
Fig. 5. Representations of the X-ray crystal of Pd(L ) with thermal plotted at 30% probability level. Hydrogen
atoms are omitted for clarity. Hydrogen atoms in this strucure are omitted for clarity. Selected geometrical
parameters (Å, °): Pd1‒O1A 1.972(2), Pd1‒O1 1.972(2), Pd1‒N1A 2.017(2), Pd1‒N1 2.017(2), O1A‒Pd1‒O1
1
9
80.00(11), O1A‒Pd1-N1A 92.28(10), O1‒Pd1‒N1A 87.72(10), O1A‒Pd1‒N1 87.72(10), O1‒Pd1‒N1
2.28(10), N1A‒Pd1‒N1 180.0.
In all complexes, the methoxy group is rotated away from the phenolate O1, with the absolute
value of the torsion angles C1‒C2‒O2‒C14 ranging from 166.4(6) in Pd(L to173.6(4)° in
Pd(L
2 2
)
3 2
) .
Furthermore, the Schiff base moiety is co-planar with the phenolate ring, with the torsion
angles C1‒C6‒C7‒N1 ranging from ‒3.6(6) in Pd(L to 0.0(4) ° in Pd(L . The resulting
conformation of the six-membered chelate ring is flat, with the average absolute values of
torsion angles being 4.3, 5.7/4.5 and 3.5° for Pd(L , two rings in Pd(L and in Pd(L
3
)
2
1 2
)
1
)
2
2
)
2
3 2
) ,
respectively. The significant twist is detected around N1-C8 bond of each ligand, that results
in almost perpendicular orientation of the phenolic and phenyl rings, with dihedral angles

between their best planes being 75.99(12), 84.0(4)/89.5(4) and 78.03(19)° for Pd(L
1 2
) ,
Pd(L and Pd(L , respectively.
2
)
2
3 2
)
Table 1. Crystal data and structure refinement for Pd(L
1
)
2
, Pd(L
2
)
2
3 2
and Pd(L )
Identification code
Empirical formula
Formula weight
Temperature; K
Wavelength; Å
Crystal system
Pd(L
1
)
2
Pd(L
2
)
2
3 2
Pd(L )
C28 H24 N2 O4 Pd
558.89
C32 H32 N2 O4 Pd
614.99
C40 H48 N2 O4 Pd
727.20
293(2)
293(2)
293(2)
0.71073
0.71073
0.71073
Orthorhombic
Pbca
Orthorhombic
Triclinic
Space group
P2
1
2
1
2
1
P-1
Unit cell dimensions; Å, °
a = 14.7061(11)
b = 10.2942(8)
c = 16.4768(13)
α = 90
a = 8.2157(7)
b = 17.6417(17)
c = 19.903(2)
α = 90
a = 9.0269(8)
b = 10.1666(9)
c = 10.7579(9)
α = 69.924(8).
β = 89.144(7)
γ = 85.639(8)
924.55(15)
1
β = 90
β = 90
γ = 90
γ = 90
Volume; Å3
2494.4(3)
4
2884.7(5)
4
Z
Density (calculated); Mg/m3
Absorption coefficient; mm-1
F(000)
1.488
1.416
1.306
0.780
0.682
0.543
1136
1264
380
Crystal size; mm
Theta range for data collection
Index ranges
0.559 x 0.411 x 0.137 0.612 x 0.276 x 0.081
0.632 x 0.395 x 0.167
2.139 to 28.432°.
‒11<=h<=7,
2.472 to 28.503°.
2.309 to 28.587°.
‒15<=h<=18,
‒10<=h<=10,
‒
8<=k<=13,
‒23<=k<=21,
‒26<=l<=24
‒13<=k<=13,
‒13<=l<=13
‒21<=l<=21

Reflections collected
16520
20234
6389
Independent reflections
2952 [R(int) =
6662 [R(int) = 0.0666] 4111 [R(int) = 0.0586]
0
.0361]
Completeness to theta
Absorption correction
Max. and min. transmission
Refinement method
25.242° 100.0 %
Analytical
25.242° 99.9 %
Analytical
25.242° 99.9 %
Analytical
0.974 and 0.909
0.955 and 0.725
0.971 and 0.904
Full-matrix least-
Full-matrix least-
squares on F2
Full-matrix least-
squares on F2
2
squares on F
Data / restraints / parameters
Goodness-of-fit on F2
2952 / 0 / 160
1.047
6662 / 0 / 352
1.051
4111 / 0 / 214
1.109
Final R indices [I>2sigma(I)]
R1 = 0.0277, wR2 =
R1 = 0.0520, wR2 =
0.0875
R1 = 0.0533, wR2 =
0.1147
0
.0589
R indices (all data)
R1 = 0.0473, wR2 =
.0682
R1 = 0.0886, wR2 =
0.0981
R1 = 0.0619, wR2 =
0.1232
0
Flack x
n/a
-0.04(2)
n/a
Largest diff. peak and hole;
0.304 and -0.454
1.131 and -1.026
0.660 and -0.702
3
-
e.Å
2
.2 Cytotoxic Effects of Pd(II) Complexes
The cytotoxicity effects of four Pd(II) complexes were assessed on three cancerous cell lines:
MCF-7 (breast carcinoma), A549 (lung carcinoma) and SKOV3 (ovarian carcinoma). As
1 2
shown in Table 2, among four compounds, Pd(L ) demonstrated the best cytotoxic effect on
almost all three cell lines with IC50 of 7.21 ± 1.71, 14.72 ± 5.82 and 21.79 ± 3.07 on MCF-7,
A549 and SKOV3, respectively. This complex also appeared to be more effective than cis-
platin (, 61.56 ± 0.98, 50.81 ± 3.10 and 43.81± 3.79, respectively). Pd(L
2
)
2
4 2
and Pd(L ) have
shown weak cytotoxic effects on three investigated cell lines (IC50>100).

Table 2. In vitro cytotoxicity effect of Pd(II) complexes on cancer cell lines
Pd(II)
IC50
Complex
MCF-7
A549
SKOV3
Pd(L
Pd(L
Pd(L
Pd(L
1
2
3
4
)
)
)
)
2
2
2
2
7.21 ± 1.71
14.72 ± 5.82
>100
21.79 ± 3.07
>100
>100
>100
89.21 ± 3.22
>100
>100
>100
>100
cis-platin
61.56 ± 0.98
50.81 ± 3.10
43.81± 3.79
*
IC50: Inhibitory concentration 50
2
.3 Apoptosis Assay
In the next step, we assessed the capacity of Pd(L
1
)
2
in the induction of apoptosis in MCF-7
cell line by Annexin V/7AAD kit, which is specifically designed for detecting apoptotic and
necrotic cells. Annexin V binds to phosphatidylserine (PS) which is normally found on the
intracellular membrane in live intact cells. During apoptosis, in the early step, the asymmetry
of membrane is lost and PS is transferred to the external layer. To distinguish necrotic cells
from apoptotic cells, 7AAD was also used; as early apoptotic cells will exclude 7AAD,
whereas the apoptotic cells in the late stage as well as necrotic cells absorb 7AAD, which in
turn goes into the nucleus and binds to DNA. As it is observed in Figure 6, Pd(L
1
)
2
could
effectively induce more apoptosis along with an increase in its concentration (early apoptosis
increased from 5.32% at 2 µM to 41.5% at 14 µM).

Fig. 6. Determination of apoptotic effect of Pd(L
1 2
) on MCF-7 cell line. 7AAD stains dead cells. Increased
levels of Annexin V+ apoptotic cells in treated cells comparing to untreated cells is an indicative that Pd(L
1 2
)
-
+
can inhibit tumor cells' growth through induction of apoptosis. Early apoptosis: 7AAD /Annexin V ; late
+
+
+
-
-
-
apoptosis: 7AAD /Annexin V ; necrosis: 7AAD /Annexin V ; viable cells: 7AAD /Annexin V
1 2
Fig. 7. Significantly higher in vitro cytotoxicity of Pd(L ) on MCF-7, A549 and SKOV3 cell lines in
comparison to cis-platin
2
.4 Solution Stability Studies
The stability of palladium complexes Pd(L
1
)
2
, Pd(L
2
)
2
3 2
and Pd(L ) in the aqueous biological
1
media was evaluated by monitoring the H NMR spectra of these compounds (1 mM) in 50
6
mM phosphate buffer over a period of 10 days. Few drops of acetone-d were added to
solubilize the compound in the aqueous media. The obtained spectra are shown in the
Supporting information (Figures S1-S3). In all three compounds, the first spectrum was
recorded after 5 minutes of mixing and shaking solute with NMR solvent. Then, the rest of

the spectra were recorded with 24 h time gap. No obvious sign of degradation is observed in
1
the H NMR spectra of all three complexes even after 10 days. It is notable that the quality of
signals in the aqueous media is lower than that of pure deuterated acetone.
2
1 2
.5 Interaction of Pd(L ) with DNA
Many chemotherapeutic drugs particularly metal-based drugs target DNA replication, a
crucial event in cell division which is overactive in tumor cells. Most of these compounds act
through binding to DNA and these interaction leads a shift in the mobility of DNA. We used
electrophoresis mobility shift assay to evaluate the interaction of Pd(L
1
)
2
with DNA. As
(B) make an
demonstrated in Figure 8, both cis-platin, as positive control, (A) and Pd(L )
1 2
obvious shift in the mobility of the plasmid in comparison to untreated DNA showing their
interactions with DNA. These results suggested that at least a part of cytotoxic effects of
Pd(L
1
2
) , is exerted through its direct interaction with DNA.
Fig. 8. Electrophoresis mobility shift assay for Pd(L
1
2
) . pET28a plasmid in circular format was incubated with
different concentrations of cis-platin (A) and Pd(L
1 2
) (B).
3
. Conclusion
As demonstrated in Figure 7 and Table 2, among palladium complexes reported in this study,
Pd(L exhibited the highest anti-proliferative activity on three investigated cancerous cells
1 2
)
lines which is even higher than cis-platin. It is quite clear that the cytotoxicity activity of
these complexes are highly dependent on the nature of the substituents present on the phenyl
ring. Single X-ray crystallography results reveal that the nature of the substituents does not
affect the geometry of the complex and all complexes adopt a square planar geometry with no

distortion around metal center. An unsubstituted phenyl ring in ortho positions such as the
one in Pd(L has a reduced steric hindrance compared to other complexes in this study and
1 2
)
this might affect dissociation pattern of the ligand from metal center inside the cell,
influencing overall cytotoxic activity of this complex. The data obtained from apoptosis study
1 2
also confirmed the cytotoxic studies, as Pd(L ) effectively activates apoptosis pathway in
cancer cells. This result is consistent with some recent studies showing that Pd(II) complexes
could induce apoptosis in tumor cells [30, 31]. As represented shift assay results indicated,
similar to cis-platin, Pd(L
part, Pd(L cytotoxic effect is probably mediated through interaction with DNA as cis-
platin does. Overall, initial results represented in this study indicated that the new Pd(II)
complex, Pd(L , can be introduced as a novel potential drug in treating cancer in particular
1 2
) could directly bind to cellular DNA. It implies that, at least in
1 2
)
1 2
)
breast carcinoma. However, through which mechanisms this compound inhibits proliferation
or function of cancerous cells and/or the effect of this compound on other cancerous cells
remains to be elucidated.
4
4
. Experimental
.1. General Procedures and Materials
1
13
1
Reagents and solvents were used as received from commercial suppliers. H, C{ H} NMR
o
spectra were recorded on a Brucker Avance DPX 400 MHz instrument in Acetone-d
6
at 25 C
1
13
using standard pulse programs. H and C shifts are quoted relative to the residual solvent
signals. Infrared spectra were measured on a Bruker Vertex 70 FT-IR spectrometer.
4
4
.2 Synthesis of Ligands and Palladium Complexes
.2.1. Synthesis of L , 6-Methoxy-2-[(E)-phenyliminomethyl]-phenol
1
Ortho-vanillin (0.50 g, 3.3 mmol) was added to a solution of aniline (0.3 g, 3.3 mmol) in
ethanol. The mixture was refluxed for 24 h. The orange precipitate was removed by filtration
o
and washed with n-hexane. Re-crystallization from ethanol solution (20 mL) at 0 C yielded
o
1
analytically pure orange crystals. Yield: 0.645 g, 86%. Mp: 86 C. H NMR (Acetone-d
6
, 400
MHz): δ 13.38 (s, 1H), 8.9 (s, 1H, Ar H), 7.510-7.419 (m, 4H, Ar H), 7.338 (t, 1H, J = 8.0
4
4
Hz, Ar H), 7.214 (dd, 1H, J = 8.0 Hz, J = 4.0 Hz, Ar H), 7.132 (dd, 1H, J = 8.0 Hz, J = 4.0
1
3
1
Hz, Ar H), 6.927 (t, 1H, J = 8.0 Hz, Ar H), 3.893 (s, 3H). C{ H} NMR (Acetone-d
6
, 100
MHz) δ 163.88, 151.64, 148.58, 148.45, 129.47, 126.95, 124.31, 121.29, 119.42, 118.52,
1
1
15.73, 55.64. IR: 3100 (w), 2985 (w), 2972 (w), 2197 (w), 1995 (w), 1900 (w), 1880 (w),
770 (w), 1720 (w), 1614 (s), 1585 (s), 1486 (m), 1466 (s), 1409 (m), 1363 (m), 1329 (w),

1
8
266 (s), 1253 (s), 1195 (s), 1169 (w), 1075 (s), 1000 (w), 995 (s), 920 (w), 856 (w), 831 (w),
11 (s), 782 (s), 766 (s), 734 (s), 690 (s), 590 (w), 495 (w), 380 (w).
4.2.2 Synthesis of L2, 6-Methoxy-2-[(E)-2,6-dimethylphenyliminomethyl]-phenol
Ortho-vanillin (1.0 g, 6.6 mmol) was added to a solution of 2,6-dimethylaniline (0.8 g, 6.6
mmol) in ethanol. The mixture was refluxed for 20 h. The orange precipitate was collected
upon concentration of ethanol solution and removed by filtration and washed with n-hexane.
Re-crystallization from acetone solution (20 mL) at room temperature yielded analytically
o
1
pure orange crystals. Yield: 1.47 g, 88%. Mp: 143 C. H NMR (Acetone-d
6
, 400 MHz): δ
1
3.103 (s, 1H), 8.564 (s, 1H, Ar H), 7.20-7.140 (m, 4H, Ar H), 7.040 (t, 1H, J = 8.0 Hz, Ar
1
3
H), 6.946 (t, 1H, J = 8.0 Hz, Ar H), 3.914 (s, 3H), 2.205 (s, 6H). C NMR (Acetone-d
MHz) δ 168.13, 151.59, 148.62, 148.29, 128.31, 128.04, 124.90, 124.09, 119.01, 118.50,
15.71, 55.58, 17.64. IR: 3450 (br.), 2955 (w), 2931 (w) , 1623 (s), 1590 (w), 1465 (s), 1416
w), 1365 (w), 1330 (w), 1270 (m), 1249 (s), 1185 (s), 1090 (w), 1075 (w), 966 (m), 852 (w),
6
, 100
1
(
8
39 (w), 786 (w), 769 (m), 742 (w), 718 (w).
4
.2.3. Synthesis of L , 6-Methoxy-2-[(E)-2,6-diisopropylphenyliminomethyl]-phenol
3
Ortho-vanillin (1.0 g, 6.6 mmol) was added to a solution of 2,6-diisopropylaniline (1.17 g,
.6 mmol) in ethanol. The mixture was refluxed for 20 h. The orange colored precipitate was
6
formed upon concentration. The red solid was washed with n-hexane solution (2 × 10 mL)
o
and was collected by filtration. Re-crystallization from ethanol solution (20 mL) at 0 C
o
1
yielded analytically pure orange crystals. Yield: 1.78 g, 87%. Mp: 148 C. H NMR
Acetone-d , 400 MHz): δ 13.074 (s, 1H), 8.544 (s, 1H, Ar H), 7.263-7.173 (m, 5H, Ar H),
.961 (t, 1H, J = 8.0 Hz, Ar H), 3.921 (s, 3H), 3.036 (sept, 2H, J = 7.2 Hz), 1.198 (d, 12H, J
(
6
6
1
3
1
=
6
7.2 Hz). C{ H} NMR (Acetone-d , 100 MHz) δ 168.01, 151.54, 148.66, 146.38, 138.42,
1
2
1
7
25.48, 124.05, 123.18, 118.90, 118.64, 115.75, 55.56, 28.01, 22.85. IR: 3040 (w), 2962 (s),
925 (m), 2899 (m), 2866 (m), 1620 (s), 1584 (m), 1457 (s), 1408 (m), 1362 (m), 1326 (m),
254 (s), 1179 (s), 1096 (m), 1074 (m), 967 (s), 910 (w), 855 (m), 832 (m), 780 (m), 762 (m),
37(m).
4
4
.2.4 Synthesis of L , 6-Methoxy-2-[(E)-2,6-dichlorophenyliminomethyl]-phenol
Ortho-vanillin (0.5 g, 3.3 mmol) was added to a solution of 2,6-dichloroaniline (0.53 g, 3.3
mmol) in ethanol. The mixture was refluxed for 20 h. Then, solvent was removed under
vacuum. Viscous yellow liquid was analyzed using thin layer chromatography (3:1,
dichloromethane : n-hexane solvents) and it showed, other than desired product, a side

product and minor unreacted Ortho-vanillin. Attempts to solidify this viscous liquid in polar
and non-polar solvents were not successful. Pure product was obtained using plate
1
chromatography in 3:1 ratio of dichloromethane and n-hexane solvents. Yield: 0.7 g, 72%. H
NMR (Acetone-d
6
, 400 MHz): δ 10.61 (br. s, 1H), 10.12 (s, 1H, Ar H), 7.34 (dd, 1H, J = 8.0
4
4
Hz, J = 1.6 Hz, Ar H), 7.28 (dd, 1H, J = 8.0 Hz, J = 1.6 Hz, Ar H), 7.24 (d, 2H, J = 8.0 Hz,
1
3
1
Ar H), 7.00 (t, 1H, J = 8.0 Hz, Ar H), 6.65 (t, 1H, J = 8.0 Hz, Ar H), 3.90 (s, 3H). C{ H}
NMR (Acetone-d , 100 MHz) δ 195.98, 151.55, 148.36, 128.83, 127.96, 123.58, 121.43,
19.50, 118.89, 118.14, 117.44, 55.72. IR: 3589 (w), 3568 (w), 3547 (w), 3525 (w), 3482
6
1
(
(
w), 3453 (w), 3385 (w), 2980 (w), 2965 (w), 2750 (w), 1654 (m), 1619 (m), 1464 (s), 1390
w), 1325 (w), 1256 (s), 1218 (w), 1073 (w), 954 (w), 777 (w), 764 (w), 737 (w), 720 (w).
4
1 2
.2.5. Synthesis of Pd(L )
In a flask, L
g, 0.22 mmol) was also added to the flask. Triethylamine (NEt
added as base. The mixture stirred at room temperature for 24 h. Then, red precipitate was
collected by filtration and it was washed with n-hexane to remove remaining L . Re-
crystallization of red precipitate from acetone solution resulted in orange crystals suitable for
1
(0.102 g, 0.44 mmol) was dissolved in 10 mL dichloromethane and PdCl
2
(0.04
3
, 0.045 g, 0.44 mmol) was
1
o
1
X-ray diffraction analyses. Yield: 0.066 g, 52%. Mp: 249 C. H NMR (Acetone-d , 400
6
4
MHz): δ 7.91 (s, 2H, Ar H), 7.43- 7.39 (m, 10H, Ar H), 6.99 (dd, 2H, J = 8.0 Hz, J = 4.0 Hz,
4
Ar H), 6.68 (dd, 2H, J = 8.0 Hz, J = 4.0 Hz, Ar H), 6.44 (d, 2H, J = 8.0 Hz, Ar H), 3.34 (s,
1
3
1
6
1
3
1
1
5
6
H). C{ H} NMR (Acetone-d , 100 MHz): δ 163.67, 150.97, 150.06, 128.20, 126.23,
25.90, 124.86, 121.27, 119.95, 115.30, 113.98, 45.45. IR: 3441 (br.), 3081 (w), 3052 (w),
004 (w), 2950 (w), 2924 (w), 2829 (w), 1604 (s), 1590 (s), 1541 (s), 1448 (m), 1462 (s),
441 (s), 1386 (w), 1356 (w), 1326 (s), 1240 (s), 1186 (s), 1110 (m), 1077 (m), 1025 (w),
001 (w), 988 (m), 970 (w), 911 (w), 882 (w), 857 (w), 781 (w), 739 (m), 694 (s), 591 (w),
52 (m).
4
2 2
.2.6. Synthesis of Pd(L )
In a flask, L
.30 mmol) was added to the flask. Triethylamine (NEt
base. The mixture stirred at room temperature for 24 h. Then, red precipitate was collected by
filtration and it was washed with n-hexane to remove any remaining L . Recrystallization of
red precipitate from acetone solution resulted in orange crystals suitable for X-ray diffraction
2
(0.15 g, 0.59 mmol) was dissolved in 10 mL acetonitrile and PdCl
2
(0.052 g,
0
3
, 0.06 g, 0.59 mmol) was added as
2
o
1
analyses. Yield: 0.06 g, 33%. Mp: 308 C. H NMR (Acetone-d , 400 MHz): δ 7.69 (s, 2H,
6

4
Ar H), 7.22-7.13 (m, 6H, Ar H), 6.95 (dd, 2H, J = 8.0 Hz, J = 4.0 Hz, Ar H), 6.67 (dd, 2H, J
4
=
8.0 Hz, J = 4.0 Hz, Ar H), 6.42 (t, 2H, J = 8.0 Hz, Ar H), 3.41 (s, 6H), 2.383 (s, 6H).
1
3
1
C{ H} NMR (Acetone-d
6
, 100 MHz): δ 164.07, 156.96, 151.07, 148.06, 131.71, 127.82,
1
2
1
9
25.84, 125.58, 120.24, 114.58, 113.70. 54.65, 18.38. IR: 3500 (br.), 2975 (m), 2937 (m),
758 (w), 2738 (w), 2676 (m), 2622 (m), 2603 (m), 2500 (w), 2495 (m), 1605 (s), 1538 (w),
468 (s), 1439 (s), 1397 (w), 1328 (m), 1253 (s), 1233 (w), 1157 (m), 1029 (w), 1035 (w),
86 (w), 856 (w), 734 (w).
4
3 2
.2.7. Synthesis of Pd(L )
In a flask, L
.14 mmol) was also added to the flask. Triethylamine (NEt
as base. The mixture stirred at room temperature for 48 h. Then, red precipitate was collected
by filtration and it was washed with n-hexane to remove any remaining L . Due to solubility
of the metal complex, Pd(L , in acetonitrile, the red colored supernatant was left in ambient
3
(0.088 g, 0.28 mmol) was dissolved in 10 mL acetonitrile and PdCl
2
(0.025 g,
0
3
, 0.029 g, 0.28 mmol) was added
3
3 2
)
temperature to slowly evaporate and to solidify. The collected solid was dissolved in acetone
to remove any remaining trimethylamine hydrochloride salt. Finally, red solution in acetone
was slowly evaporated to yield suitable crystals for X-ray analysis. Yield: 0.07 g, 69%. Mp:
o
1
2
84 C. H NMR (Acetone-d
6
, 400 MHz): δ 7.75 (s, 2H, Ar H), 7.37 (t, 3H, J = 8.0 Hz, Ar
4
H), 7.25 (d, 4H, J = 8.0 Hz, Ar H), 6.99 (dd, 2H, J = 8.0 Hz, J = 4.0 Hz, Ar H), 6.68 (dd, 2H,
4
J = 8.0 Hz, J = 4.0 Hz), 6.41 (t, 2H, J = 8.0 Hz), 3.67 (sept, 4H, J = 7.2 Hz), 3.29 (s, 6H),
1
3
1
1
.33 (d, 12H, J = 7.2 Hz), 1.19 (d, 12H, J = 7.2 Hz). C{ H} NMR (Acetone-d
δ 168.01, 151.54, 148.66, 146.38, 138.42, 125.48, 124.05, 123.18, 118.90, 118.64, 115.75,
5.56, 28.01, 22.85. IR: 3085 (w), 2959 (w), 2924 (w), 2866 (w), 1603 (s), 1540 (w), 1464
6
, 100 MHz):
5
(
(
m), 1437 (m), 1382 (w), 1362 (w), 1326 (m), 1244 (m), 1220 (w), 1172 (w), 1106 (w), 1077
w), 990 (w), 858 (w), 772 (w), 730 (w), 526(w).
4
.2.8. Synthesis of Pd(L
4 2
)
In a flask, L (0.053 g, 0.18 mmol) was dissolved in 10 mL ether and Pd(OAc) (0.02 g, 0.09
4
2
mmol) was added to the flask. The mixture stirred at room temperature for 24 h. Then, the
precipitate was collected by filtration and it was washed small amount of ether. Dark red
supernatant from reaction and solution from precipitate wash was combined and left in a
4 2
small glass vial at ambient temperature for crystallization. Due to low solubility of Pd(L )
1
3
complex in deuterated solvents, a suitable C NMR spectrum was not obtained for this
o
1
6
compound. Yield: 0.035 g, 56%. Mp: 186 C. H NMR (Acetone-d , 400 MHz): δ 7.88 (s,

2
6
1
8
H), 7.58-7.55 (m, 4H), 7.50-7.48 (t, 2H), 7.00 (d, 2H, J = 8.0 Hz), 6.71 (d, 2H, J = 8.0 Hz),
.46 (t, 2H), 3.47 (s, 6H). IR: 3441 (br.), 3000 (w), 2928 (w), 2820 (w), 1606 (m), 1577 (s),
537 (m), 1460 (w), 1370 (w), 1327 (m), 1249 (s), 1203 (w), 1108 (w), 1076 (w), 969 (w),
57 (w), 784 (w), 736 (m), 646 (w), 464 (w).
4
4
.3 Biological Assay
.3.1. Cell Lines and Cell Culture
Three human cell lines including MCF-7 (breast cancer), A549 (lung cancer), SKOV3
ovarian cancer) were purchased from the National Cell Bank of Iran (NCBI, Pasteur
(
Institute, Tehran, Iran). All cell lines were cultured in complete culture medium containing
Roswell Park Memorial Institute (RPMI) 1640 medium (Biosera, UK), 10 % fetal bovine
serum (FBS; Gibco, USA) and 1 % penicillin-streptomycin (Biosera, UK) and were
2
incubated at 37 °C in a CO incubator with 95% humidity. The cytotoxic effects of four
Pd(II) complexes on these cell lines were assessed using the 3-(4,5-dimethylthiazolyl)-2,5-
diphenyltetrazolium bromide (MTT) standard assay as described before [32,33]. Briefly, the
cells were detached, counted and 10,000 cells were then seeded in 96-well microplate in 100
µl complete culture medium per well. The cells were incubated for 24 h and then treated with
different concentrations of each compound in triplicate manner. The compounds were solved
in DMSO (Sigma, Germany) and then diluted in culture medium to make different
concentrations (1-100 µM). cis-platin with different concentrations was also used as positive
control. Culture medium were added to three wells with and without cells considered as
negative controls and blanks, respectively. The cells were incubated for 48 h at 37 °C in a
humidified CO incubator. The medium was completely removed and replaced by 100 µl
2
RPMI containing MTT (0.5 mg/ml; Sigma, Germany) and incubated for more 3 h to
formazan complexes were formed. The medium containing MTT was then completely poured
out and 100 µl DMSO was added to each well to dissolve the formazan crystals and were
incubated for 30 min. The absorbance of each well was read at 495 nm with a microplate
ELISA reader. The data were analyzed and 50 % inhibitory concentration (IC50) for each
compound were obtained by CurveExpert 4.1. Data are presented as mean ± SD. Each
experiment was independently repeated three times.

4
.3.2 Apoptosis Detection
The induction of apoptosis by Pd(L
Annexin V Apoptosis Detection Kit (Biolegend, USA) according to manufactured
instruction. Briefly, the cells were treated with three different concentrations of Pd(L
1 2
) on MCF-7 cell line was determined by BioLegend's
)
1 2
including IC50 concentration (2, 7 and 14 µM) for 48 h. The cells were then trypsinized and
washed twice with cold BioLegend's Cell Staining Buffer and then re-suspended in Annexin
V Binding Buffer. Two microliter FITC-conjugated Annexin V and then 2 µl of 7-AAD
3
Viability Staining Solution were added to each tube containing 200 × 10 cells. The cells
were gently vortexed and incubated for 15 min in the dark at room temperature and analyzed
directly by four-color FACSCalibur flow cytometer (BD Biosciences, USA). At least 50,000
cells were acquired. The experiment was repeated two times and the data was analyzed by
FlowJo software.
4
1 2
.3.3 Interaction of Pd(L ) with DNA
pET28a plasmid (5369 bp) in circular format was used to investigate the interaction of
Pd(L with DNA as previously described [34]. The same aliquots of circular form of
pET28a plasmid (1250 ng) were prepared in a buffer containing Tris-HCl, pH = 8.5 (Roche,
Germany) and were incubated with different concentrations of compound Pd(L (100, 400
)
1 2
1 2
)
and 800 µM) at 37 °C for 48h. Untreated DNA and cis-platin treated DNA (in the same
concentrations) were also included as negative and positive controls, respectively. Then, 8 µl
of each sample was mixed with 3 µl KBC loading dye (Kawsar Biotec, Iran) and loaded onto
1
% agarose gel (Invitrogen, USA) and electrophoresed for 90 min at 70 V in 0.5% TEA
buffer. The gel was then visualized by a UV detector.
4
.3.4 X-ray Crystal Structure Determination and Refinement
Crystals of Pd(L and Pd(L were grown from the acetone solutions. The X-ray
1
)
2, Pd(L
2
)
2
3 2
)
diffraction data were collected with an Oxford Sapphire CCD diffractometer using MoKα
radiation λ = 0.71073 Å, at 293(2) K, by ω-2θ method. Structures were solved by the direct
2
methods and refined with the full-matrix least-squares method on F with the use of
SHELX2014 program package [35]. Analytical absorption corrections were applied with
2 2
CrysAlisPro 1.171.38.43 (Rigaku OD, 2015) [36] (Table 1). The absolute structure of Pd(L )
was determined with the Flack method [37], with the Flack x being -0.04(2). Hydrogen atoms
were located from the electron density maps and their positions were constrained in the

refinement with the appropriate AFIX commands as implemented in SHELX. The structural
data have been deposited with Cambridge Crystallographic Data Centre, the CCDC numbers
1
554780, 1554781 and 1554782 for Pd(L
1 2 2 2 3 2
) , Pd(L ) and Pd(L ) , respectively.
Acknowledgement
Funding for this work by the Institute for Advanced Studies in Basic Sciences (IASBS)
Research Council and the Iran National Science Foundation (Grant no. 93039954) is
acknowledged. Thanks are also due to Mr. Biglari, the operator of Bruker NMR spectroscopy
instrument at IASBS, for taking time to record the NMR spectra of these compounds.
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Palladium (II) complexes based on Schiff base ligands derived
from ortho-vanillin; synthesis, characterization and cytotoxic
studies
*
*
Highly stable four coordinate palladium complexes
Higher anti-proliferative activity on three cancerous cell lines than cis-
platin
*Higher anti-proliferative activity on three
cancerous cell lines than cis-platin*