Cytotoxicity of palladium(II) complexes with some alkyl derivates of thiosalicylic acid. Crystal structure of the bis(S-butyl-thiosalicylate)palladium(II) complex, [Pd(S-bu-thiosal)2]

Article abstract of DOI:10.1016/j.poly.2015.01.041

The spectroscopically predicted structure of the obtained bis(S-butyl-thiosalicylate)palladium(II) complex, [Pd(S-bu-thiosal)₂], was confirmed by an X-ray structural study. The asymmetric unit of [Pd(S-bu-thiosal)₂] consists of neutr

Full text of DOI:10.1016/j.poly.2015.01.041

Accepted Manuscript
Cytotoxicity of palladium(II) complexes with some alkyl derivates of thiosali-
cylic acid. Crystal structure of the bis(S-butyl-thiosalicylate)palladium(II) com-
plex, [Pd(S-bu-thiosal) ]
2
Marina Ž. Mijajlović, Miloš V. Nikolić, Verica V. Jevtić, Zoran R. Ratković,
Bojana Simović Marković, Vladislav Volarević, Nebojša N. Arsenijević,
Slađana B. Novaković, Goran A. Bogdanović, Srećko R. Trifunović, Gordana
P. Radić
PII:
S0277-5387(15)00068-6
DOI:
http://dx.doi.org/10.1016/j.poly.2015.01.041
POLY 11179
Reference:
To appear in:
Polyhedron
Received Date:
Accepted Date:
15 October 2014
23 January 2015
Please cite this article as: M. Mijajlović, M.V. Nikolić, V.V. Jevtić, Z.R. Ratković, B.S. Marković, V. Volarević,
N.N. Arsenijević, S.B. Novaković, G.A. Bogdanović, S.R. Trifunović, G.P. Radić, Cytotoxicity of palladium(II)
complexes with some alkyl derivates of thiosalicylic acid. Crystal structure of the bis(S-butyl-
thiosalicylate)palladium(II) complex, [Pd(S-bu-thiosal) ], Polyhedron (2015), doi: http://dx.doi.org/10.1016/j.poly.
2
2
015.01.041
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Cytotoxicity of palladium(II) complexes with some alkyl derivates of
thiosalicylic acid. Crystal structure of the
bis(S-butyl-thiosalicylate)palladium(II) complex, [Pd(S-bu-thiosal)2]
a
a
b
b
Marina Ž. Mijajlović , Miloš V. Nikolić , Verica V. Jevtić , Zoran R. Ratković , Bojana
a
a
a
c
Simović Marković , Vladislav Volarević , Nebojša N. Arsenijević , Slađana B. Novaković ,
c
b
a*
Goran A. Bogdanović , Srećko R. Trifunović and Gordana P. Radić
a
Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovića 69, 34000 Kragujevac, Republic of Serbia;
b
Department of Chemistry, Faculty of Science, University of Kragujevac, Radoja Domanovića 12, 34000 Kragujevac,
Republic of Serbia;
c
VINČA Institute of Nuclear Sciences, Laboratory of Theoretical Physics and Condensed Matter Physics, University of
Belgrade, PO Box 522, 11001 Belgrade, Republic of Serbia;
Abstract
The spectroscopically predicted structure of the obtained bis(S-butyl-
thiosalicylate)palladium(II) complex, [Pd(S-bu-thiosal) ], was confirmed by an X-ray
structural study. The asymmetric unit of [Pd(S-bu-thiosal) ] consists of neutral complex
2
2
molecules, where the Pd(II) ion is placed in a cis-square-planar coordination environment
formed by O and S atoms of two deprotonated S-butyl-thiosalicylic acid ligands. The
cytotoxic effects of the S-alkyl (R = benzyl (L1), methyl (L2), ethyl (L3), propyl (L4) and
butyl (L5)) derivatives of thiosalicylic acid and the corresponding palladium(II) complexes
are reported here. The analysis of cancer cell viability showed that all the tested complexes
are cytotoxic to human colon carcinoma cells (HCT-116 and CaCo-2) and human lung
*
Corresponding author. Tel. : +381 34306800; fax: +381 34306800
e-mail address: vasic_gordana@yahoo.com (G. P. Radić)
1

carcinoma epithelial cells (A549). The antitumor activities of the above mentioned Pd(II)
complexes are higher in comparison to the corresponding ligands.
Keywords: palladium(II) complexes; crystal structure; cytotoxicity
1. Introduction
Since the seventies when cisplatin, with antitumor activity, was discovered by Rosenberg
and co-workers [1,2], thousands of platinum complexes have been synthesized in order to
obtain new platinum(IV) compounds with improved properties in comparison to the parent
drug cisplatin [3-5]. It has been shown that platinum(II) complexes with antitumor activity are
represented by the general formula [Pt(am)
2
2
X ], having a cis geometry, where (am) is an inert
amine ligand having at least one NH group and X is an easy leaving ligand. The leaving group
should be an anion with a moderately strong coordination to the platinum(II) ion, as well as
with a weak trans effect in order to avoid labilization of the amine ligand bond.
Those discoveries have increased the interest in obtaining efficient ligands and
complexes
cis-diamminedichloridopalladium(II),
cis-1,2-diaminocyclohexanedichloridopalladium(II),
of
other
metals.
The
cis-[PdCl
cis-[PdCl
palladium(II)
(NH ],
(DACH)],
complexes
and
2
3 2
)
2
which
are
analogues to cisplatin, were among the first complexes used in clinical trials as
anti-tumor agents [6].
Platinum(II) complexes are known to be thermodynamically and kinetically more
stable than their palladium(II) analogues. The reactivity of the palladium(II) complexes for
5
hydrolysis and exchange of the ligands is 10 times faster than the corresponding platinum(II)
complexes. Therefore the palladium(II) complexes show lower antitumor activity, but higher
toxicity [6-8].
2

Although the initial results showed no significant antitumor activity of the
palladium(II) complexes, these complexes have been widely studied. Due to their high
reactivity, palladium(II) complexes generally exhibit lower antitumor activity than cisplatin
[
6,7]. It was concluded that the lower activity of the palladium(II) complexes is the result of a
very rapid exchange of the ligands and the inability of the complexes, with an unchanged
structure, to reach biological targets, increasing the risk of adverse effects on biochemical
processes in the cell. In order to overcome these problems some authors [9] suggested that the
palladium(II) ion should be coordinated by chelating ligands, reducing the reactivity of the
palladium center and increasing the stability of the complex.
A palladium(II) complex with a coumarine derivative was synthesized, showing
approximately 7800 times higher activity than carboplatin for A549, K562 and HeLa cells
[
10,11]. This confirms the assumption that complexes with voluminous ligands have better
antitumor activity. Also a large number of palladium(II) complexes with neutral ligands were
tested, such as phosphate derivatives of quinoline [12,13], derivatives of pyridine [14,15] and
pyrazole [16], and these complexes showed significant antitumor activity.
Thiosalicylic acid and its derivatives are used for metal determination [17,18], as
modificators for graphite paste electrodes [19], as photoinitiators for free radical
polymerization [20], in cosmetics [21], in disease treatments (in particular inflammatory,
allergic and respiratory diseases) [22], as well as Ras-tumor growth inhibitors [23]. In our
previously published papers we investigated the antimicrobial activity of some S-alkyl
derivatives of thiosalicylic acids and the corresponding palladium(II) complexes. We
concluded that all the tested compounds showed a low antimicrobial activity, but the
palladium(II) complexes showed higher activity than the corresponding S-alkyl derivatives of
thiosalicylic acid [24].
3

The aim of this paper is to research the in vitro cytotoxic effects of the earlier
synthesized S-alkyl derivatives of thiosalicylic acid and the corresponding palladium(II)
complexes, due to recent great interest in palladium(II) complexes as cytotoxic and anticancer
drugs [25-27]. For the first time, we demonstrate that five newly synthesized palladium(II)
complexes and their corresponding ligand precursors exhibit relevant cytotoxic properties to
three different human cancer cell lines: CaCo-2, HCT116 and A549. The CaCo-2 cell line is a
continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells [28]. The
HCT-116 cell line is an adherent epithelial cell line originating from human colorectal
carcinoma [29], while the human lung carcinoma epithelial cell line A549 is the most usually
used cancer cell line for in vitro research in the field of testing cytotoxicity and metabolism of
new synthesized complexes towards human lung carcinoma epithelial cells [30].
2
. Experimental
2
.1. Materials and measurements
The reagents were obtained commercially and used without further purification. For
1
the infrared spectra, a Perkin-Elmer Spectrum One FT-IR spectrometer was employed. H and
1
3
C NMR spectra were recorded on a Varian Gemini-200 NMR spectrometer using TMS in
DMSO-d as an internal reference, at 22 ºC and with 10 mM solutions of the complexes.
6
Elemental analyses were done on a Vario III CHNOS Elemental Analyzer, Elemental
Analysensysteme GmbH.
2
2
.2. Syntheses
.2.1. General procedure for the synthesis of the S-alkyl thiosalicylic acids (L1)-(L5)
The thioacid ligands L1-L5 were prepared by alkylation of thiosalicylic acid by means
of the corresponding alkyl halogenides in alkaline water-ethanol solution. Thiosalicylic acid
4

(
1 mmol) was added to a 100 mL round bottom flask containing 50 mL of a 30% solution of
ethanol in water and stirred. A solution of NaOH (2 mmol in 5 mL of water) was added to the
acid suspension, whereupon the solution became clear. The corresponding alkyl halogenide (2
mmol) was dissolved in 5 mL of ethanol and transferred to the stirred solution. The resulting
mixture was kept overnight at 60 °C. The reaction mixture was transferred into a beaker and
ethanol was evaporated off on a water bath. Diluted hydrochloric acid (2 mol/L) was added to
the resulting water solution and S-alkyl thiosalicylic acid was precipitated as a white powder.
The free acid was filtered off and washed with plenty of distilled water. The product was
dried under vacuum overnight. Yield: 85-95%.
2
.2.2. Preparation of the bis(S-benzyl-thiosalicylate)–palladium(II) complex, [Pd(S-bz-
thiosal) ] (C1)
The palladium(II) complexes with S-alkyl derivatives of thiosalicylic acid were
obtained by the previously described procedure [24]. K [PdCl ] (0.100 g, 0.3065 mmol) was
2
2
4
dissolved in 10 mL of water on a steam bath and (S-benzyl)-2-thiosalicylic acid (L1) (0.1497
g, 0.613 mmol) was added into the solution. The resulting mixture was stirred for 2 h and
during this time an aqueous solution of LiOH (0.0256 g, 0.613 mmol in 10 mL of water) was
introduced. The complex [Pd(S-bz-thiosal)
2
] (C1), as a yellow precipitate, was filtered,
washed with water and air-dried. Yield: 0.11 g (58.70%). Anal. Calc. for C28
H
22
O
4
S
2
Pd
-
(
r
M = 592.98): C, 56.71; H, 3.74; S, 10.82. Found: C, 56.43; H, 3.85; S, 10.75%. IR (KBr, cm
1
1
)
: 3420, 3057, 1634, 1616, 1562, 1327, 1146, 753, 708, 698. H NMR (200 MHz, DMSO-d ,
6
13
δ ppm): 4.05 (s, 4H, CH
2
), 7.08-8.10 (m, 9H, Ar and bz). C NMR (50 MHz, DMSO-d , δ
6
ppm): 25.9 (CH
2
), 124.1, 125.6, 125.7, 126.2, 126.3, 126.8, 127.3, 127.8, 129.5, 133.2, 136.2,
-
1
39.7 (Ar and bz), 171.5 (COO ).
5

2
.2.3. Preparation of the bis(S-methyl-thiosalicylate)–palladium(II) complex, [Pd(S-met-
thiosal) ] (C2)
The complex [Pd(S-met-thiosal)
using (S-methyl)-2-thiosalicylic acid (L2) (0.103 g, 0.613 mmol) instead of (S-benzyl)-2-
thiosalicylic acid. Yield: 0.08 g (59.80%). Anal. Calc. for C16 Pd (M = 440.672):
2
2
] (C2) was prepared as described in Section 2.2.2.,
-
H
14
O
4
S
2
r
-
1
C, 43.61; H, 3.20; S, 14.52. Found: C, 43.41; H, 3.39; S, 14.21%. IR (KBr, cm ): 3419, 1619,
1
1
597, 1399, 1385, 1332, 1306, 1142, 960, 865, 741, 693, 654. H NMR (200 MHz,
1
3
DMSO-d
6
, δ ppm): 2.35 (s, 6H, CH
3
6
), 7.19-8.08 (m, 8H, Ar). C NMR (50 MHz, DMSO-d ,
-
δ ppm): 14.6 (CH
3
), 123.6, 125.1, 125.2, 129.0, 132.7, 135.7, (Ar), 171.8 (COO ).
2
.2.4. Preparation of the bis(S-ethyl-2-thiosalicylate)–palladium(II) complex, [Pd(S-et-
thiosal) ] (C3)
The complex [Pd(S-et-thiosal)
using (S-ethyl)-2-thiosalicylic acid (L3) (0.1117 g, 0.613 mmol) instead of (S-benzyl)-2-
thiosalicylic acid. Yield: 0.0832 g (57.90%). Anal. Calc. for C18 Pd (M = 468.856):
2
2
] (C3) was prepared as described in Section 2.2.2.,
-
H
18
O
4
S
2
r
-
1
C, 46.11; H, 3.87; S, 13.68. Found: C, 45.97; H, 3.93; S, 13.54%. IR (KBr, cm ): 1436, 1587,
1
1
518, 1393, 752. H NMR (200 MHz, DMSO-d
6
, δ ppm): 1.27 (t, 6H, CH
3
), 2.83 (q, 4H,
1
3
CH
2
), 7.11-8.08 (m, 8H, Ar). C NMR (50 MHz, DMSO-d
6
, δ ppm): 14.4 (CH ), 13.8 (CH ),
3
2
-
1
24.8, 125.3, 126.1, 128.7, 133.2, 135.9 (Ar), 172.0 (COO ).
2
.2.5. Preparation of the bis(S-propyl-2-thiosalicylate)–palladium(II) complex, [Pd(S-pro-
] (C4)
The complex [Pd(S-pro-thiosal)
using (S-propyl)-2-thiosalicylic acid (L4) (0.1203 g, 0.613 mmol) instead of (S-benzyl)-2-
thiosal)
2
2
] (C4) was prepared as described in Section 2.2.2.,
22 4 2 r
thiosalicylic acid. Yield: 0.0889 g (58.40%). Anal. Calc. for C20H O S Pd (M = 496.908):
6

-
1
C, 48.34; H, 4.46; S, 12.91. Found: C, 48.52; H, 4.11; S, 12.73%. IR (KBr, cm ): 1421, 1589,
1
1
4
1
541, 1520, 1397, 752. H NMR (200 MHz, DMSO-d
6
, δ ppm): 0.98 (t, 6H, CH
3
), 1.76 (m,
), 7.20-8.25 (m, 8H, Ar). C NMR (50 MHz, DMSO-d , δ ppm):
), 27.6 (CH ), 125.1, 126.6, 126.7, 130.5, 134.2, 137.2 (Ar), 172.5
13
H, CH
2
), 2.84 (t, 4H, CH
2
6
3.2 (CH
3
), 22.0 (CH
2
2
-
(
COO ).
2
.2.6. Preparation of the bis(S-butyl-2-thiosalicylate)–palladium(II) complex, [Pd(S-bu-
] (C5)
The complex [Pd(S-bu-thiosal)
using (S-butyl)-2-thiosalicylic acid (L5) (0.1289 g, 0.613 mmol) instead of (S-benzyl)-2-
thiosalicylic acid. Yield: 0.0941 g (58.43%). Anal. Calc. for C22 Pd (M = 524.960):
thiosal)
2
2
] (C5) was prepared as described in Section 2.2.2.,
H
26
O
4
S
2
r
-
1
C, 50.33; H, 4.99; S, 12.22. Found: C, 50.52; H, 4.51; S, 12.56%. IR (KBr, cm ): 3419, 1619,
1
1
597, 1399, 1385, 1332, 1306, 1142, 960, 865, 741, 693, 654. H NMR (200 MHz, DMSO-d
6
,
13
δ ppm): 2.35 (s, 6H, CH
3
6
), 7.19-8.08 (m, 8H, Ar). C NMR (50 MHz, DMSO-d , δ ppm):
-
1
4.6 (CH
3
), 123.6, 125.1, 125.2, 129.0, 132.7, 135.7, (Ar), 171.8 (COO ).
2
.3. X-ray experiment
The diffraction data from a selected single crystal of [Pd(S-bu-thiosal)
2
] were
collected at room temperature with an Oxford Diffraction Xcalibur Gemini S diffractometer
equipped with MoKα radiation (λ = 0.71073 Å). Data integration and scaling of the
reflections were performed with the CRYSALIS software [31]. Empirical absorption
corrections of the diffracted intensities were performed with the SCALE3 ABSPACK [32]
scaling algorithm implemented in the CRYSALIS suite.
The crystal structure was solved by direct methods using SIR2002 [33] and refined
using the SHELXL program [34]. Hydrogen atoms attached to carbon atoms were placed at
7

geometrically idealized positions with C–H distances fixed to 0.96, 0.97 and 0.96 Å from
methyl, methylene and phenyl C atoms, respectively. The isotropic displacement parameters
of the hydrogen atoms were equal to 1.2 Ueq of the parent methylene and phenyl C atoms and
1
.5 Ueq of the methyl C atoms. The crystallographic data are listed in Table 1. The PARST
35] program was used to perform geometrical calculations and the program ORTEP [36] was
employed for molecular graphics.
[
2
2
.4. In vitro cytotoxicity studies
.4.1. Preparation of drug solutions
Stock solutions of the tested palladium(II) complexes and ligand precursors were
made in dimethyl sulfoxide (DMSO) at a concentration of 20 mM, filtered through a 0.22 mm
Millipore filter before use and diluted by a nutrient medium to various working
concentrations. The concentration of DMSO in most concentrated working solutions was 1%
(
-
(
v/v).
The
nutrient
medium
used
was
HEPES
(4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid) buffered RPMI-1640 supplemented with streptomycin
100 mg/mL), penicillin (100 IU/mL) and 10% fetal bovine serum (FBS). MTT,
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide was dissolved (5 mg/mL) in a
phosphate buffer saline (PBS) having a pH of 7.2, and filtered through a 0.22 mm Millipore
filter before use. All reagents were purchased from Sigma Chemicals (Sigma Aldrich,
Munich, Germany).
2
.4.2. Cell lines
The human cancer colon cell line CaCo-2 and the human lung carcinoma epithelial
cell line A549 were purchased from the American Type Culture Collection (ATCC,
Manassas, USA). Human cancer colon cell line HCT-116 was kindly provided by Dr Danijela
8

Vignjević (Institute Curie, Paris, France). The fibroblasts, isolated as previously described
37], were kindly provided by Dr Biljana Ljujić (Centre for Molecular Medicine and Stem
[
Cell Research, Kragujevac, Serbia) and, as non-cancer cells, were used for studying any
unwanted cytotoxicity of the tested complexes.
CaCo-2 and HCT-116 cells were maintained in RPMI 1640 (Sigma Aldrich, Munich,
Germany) supplemented with 10% fetal bovine serum, FBS (Sigma), penicillin G
(
100 IU/mL), streptomycin (100 µg/mL), and in a humidified atmosphere of 95% air/5% CO
2
at 37 °C. The A549 cells and fibroblasts were cultured in Dulbecco’s Modified Eagle Medium
(
(
DMEM) containing 10% FBS, 100 IU/mL penicillin G and 100 µg/mL streptomycin
Sigma). The cell number and viability were determined by trypan blue staining. CaCo-2,
HCT-116, A549 cells and fibroblasts in passage 3 were used throughout these experiments.
2
.4.3. Assessment of cytotoxicity
The effects of selected complexes on the viability of CaCo-2, HCT-116 and A549
cancer cells and fibroblasts were determined using an MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) test. The CaCo-2, HCT-116 and A549 cells and fibroblasts
4
3
were diluted with medium to 5 x 10 cells/mL, and aliquots (5 x 10 cells/100 µL) were placed
in individual wells in 96-multiplates. After 24 hours, the cells were treated with selected
concentrations of the complexes for 3 days: the medium was exchanged with 100 µL of
different compounds, which had been serially diluted 2-fold in the medium to concentrations
ranging from 250 µM to 3.9 µM. Control wells were prepared by the addition of a culture
medium. The wells containing a culture medium without cells were used as blanks. After
incubation, the drug containing medium was discarded and replaced with serum free medium
containing 15% of MTT (5 mg/mL) dye. After an additional 4 h of incubation at 37°C in a 5%
CO
2
incubator, the medium with MTT was removed and DMSO (150 µL) with glycine buffer
9

(
20 µL) was added to dissolve the blue formazan crystals. The plates were shaken for 10 min.
The optical density of each well was determined at 595 nm. The percentage cytotoxicity was
calculated using the formula:
%
cytotoxicity = 100-((TS-BG0)-E/(TS-BG0) x 100)
where BG0 is the background of the medium alone, TS is the total viability/spontaneous death
of untreated target cells and E is the experimental well.
3
. Results and discussion
.1. Chemistry
3
Palladium(II) complexes with
S-alkyl derivatives of thiosalicylic acid,
[PdCl ] with the S-alkyl
derivatives of thiosalicylic acid (in a molar ratio of 1:2) in water solution, as described earlier
24]. On the basis of the IR and NMR spectra of the ligands and the corresponding Pd(II)
[
Pd(S-alkyl-thiosal)
2
], were obtained by the direct reaction of K
2
4
[
complexes, we concluded that the ligands are bidentately coordinated to the palladium(II) ion,
but claim nothing about the geometry of complexes. Based on S,O-coordination of all the
2 2
ligands and the crystal structure of the [Pd(S-bz-thiosal) ] [24] and [Pd(S-bu-thiosal) ]
complexes, it can be assumed that the other complexes occur in the form of a cis-S-cis-O
geometric isomer.
3
.2. Description of the crystal structure of bis(S-butyl-2-thiosalicylate)-palladium(II) (C5)
The crystal structure of [Pd(S-bu-thiosal) ], together with the atom-labelling scheme,
is presented in Fig. 1. Selected structural parameters are listed in Table 2. The asymmetric
unit of [Pd(S-bu-thiosal) ] consists of neutral complex molecules, where Pd(II) is placed in a
cis-square-planar coordination environment formed by O and S atoms of two deprotonated
2
2
1
0

S-butyl-thiosalicylic acid ligands (Fig. 1). The coordination about the Pd atom is essentially
planar, with a maximum deviation for the O1a donor atom of -0.013(1) Å.
A CSD search [38] reveals that the recently reported crystal structure of the Pd(II)
complex with S-benzyl-thiosalicylate, [Pd(S-bz-thiosal)
Pd(II) complex containing two bidentately coordinated thiosalicylate ligands [24]. The
geometrical properties of the novel [Pd(S-bu-thiosal) ] and [Pd(S-bz-thiosal) ] complexes
2
], represents the only example of a
2
2
could be closely compared. Although both of the complexes coordinate thiosalicylate ligands
in a cis-square-planar fashion, they differ significantly in the spatial orientation of the
thiosalicylate rings and corresponding S-substituents. Namely, in the title compound the same
2 2
type of residues take the same orientation with respect to the PdO S plane, while in the
former compound the same residues occupy opposite sides of the coordination plane. Apart
from this major structural difference, the geometrical features of the two coordination spheres
comprising the S-butyl and S-benzyl [24] derivatives are similar. The Pd–S distances of the
complexes are in good agreement, while the Pd–O bonds are approximately 0.01 Å shorter in
the present compound. The distribution of coordination angles in both complexes is within 4°
from the ideal values, with the largest angle being between the voluminous S atoms (cis-S–
Pd–S). The difference between the trans-O–Pd–S angles is more evident, as in [Pd(S-bu-
thiosal)
bz-thiosal)
complex [24] displays higher deviations of the constituent atoms from the PdO
coordination plane (root-mean-square deviation 0.13 Å) in comparison to the present one
r.m.s. 0.01 Å).
The different spatial distribution of the ligands residues and the less distorted
coordination geometry in [Pd(S-bu-thiosal) ] in comparison to [Pd(S-bz-thiosal) ] suggests a
dissimilar steric influence of the corresponding S-substituents. A comparison of the sets of
2
] they are somewhat closer to linearity [175.59(8) and 177.65(9)°] than in the [Pd(S-
2
] complex [172.52(15) and 172.77(14)°]. Accordingly, the previously reported
2 2
S
(
2
2
1
1

angles involving the S atom in two complexes reveals fair differences. In [Pd(S-bu-thiosal)
2
],
similarly to [Pd(S-bz-thiosal) ] and complexes comprising rigid thiosalate chelates [39-44],
2
there is a relatively acute angle at S [the mean value of the Pd–S1–C3 angle is 100.7(3)°], in
contrast to the angle at the O donor [the mean value of the Pd–O1–C1 angle is 129(1)°]. The
C3–S1–C8 angle, which relates the S-substituent to the aromatic part of the ligand, is slightly
smaller in the S-butyl derivative [mean value 103(1)°] than in the S-benzyl derivative [mean
value 106(1)°], suggesting less steric hindrance of residues in the first complex. A major
difference could be observed if one compares the Pd–S1–C8–C9 torsion angle, which
describes the further directionality of the S-butyl and S-benzyl substituents. The mean values
of the Pd–S1–C8–C9 torsion angles are 167(1) and 60(1)° in [Pd(S-bu-thiosal)
bz-thiosal) ] respectively.
Similarly to [Pd(S-bz-thiosal)
are in skew chair conformations. In [Pd(S-bu-thiosal)
2
] and [Pd(S-
2
2
, the six membered chelate rings of [Pd(S-bu-thiosal)
2
]
2
] alone, the chelate rings of the A and B
molecular halves show a significant difference in the C1–C2–C3–S1 torsion angle, which is
equal to 1.2(6) and -9.3(6)°, respectively. In addition the Pd–O1–C1 angle has the value of
1
32.1(3) and 126.5(3)° in the A and B halves, respectively. The two halves further differ in
the level of inclination of their phenyl rings with respect to coordination plane, forming the
different dihedral angles of 50.38(1) and 63.9(1)°.
2
In the crystal packing, molecules of [Pd(S-bu-thiosal) ] associate by means of weak
C–H…O and C–H…π interactions (Table 3). Two equivalent C4–H4…O2 interactions
connect the complex molecules into a two-dimensional sheet parallel to the bc plane, Fig. 2.
The arrangement of the molecules along the a axis is mostly supported by interactions of two
S-butyl groups as C–H donors. The engagement of these fragments in the intermolecular
interactions is dissimilar. Placed approximately in between the two phenyl rings of the
neighbouring molecule, the S-butyl group of fragment A engages in three C–H…π
1
2

interactions. At the same time the S-butyl group of fragment B is placed approximately at the
same level as the oxygen donors of the same neighboring molecule, forming the strongest
C8b−H8b1...O1b interaction (H…O1b = 2.47 Å). The terminal C atoms of the S-butyl group
of fragment B are not engaged in any interactions and, in contrast to those in fragment A,
display noticeable disorder.
3
.3. In vitro cytotoxic activity
Cytotoxicity assay experiments were conducted to determine the effects of the newly
synthesized palladium(II) complexes on the cell viability of human colon and lung carcinoma
cells. The analysis of the cancer cell viability showed that all the tested complexes are
cytotoxic to human colon carcinoma cells (HCT-116 and CaCo-2, Figures 3 and 4) and
human lung carcinoma epithelial cells (A549, Fig. 5). The cytotoxic effect is dose dependent:
a concentration decrease of the tested complexes is followed by a considerable increase of
tumor cell viability.
The tested palladium(II) complexes showed moderate and high cytotoxcities against
human colon and lung carcinoma cells (Figs. 3-5), and low cytotoxicities against fibroblasts
(
Fig. 6). In comparison with the other tested palladium(II) complexes and cisplatin, C3
showed the highest cytotoxicity at all the tested concentrations and against all tested cancer
cell lines (Figs. 3-5). The IC50 value of palladium(II) complex C3 was 3-4 times lower than
the IC50 value of palladium(II) complex C4 and more than 10-15 times lower compared to the
same value of the other tested palladium(II) complexes (Table 4). In addition, the IC50 values
of palladium(II) complex C3 were lower than the IC50 values for cisplatin for all the cancer
cell lines (Table 4). Importantly, the cytotoxicity of C3 complex was above 98% for all
cancer cell lines at the highest tested concentration (250 µL), while at the same concentration
the cytotoxicity of cisplatin was above 98% only against CaCo-2 cells. Additionally, the
1
3

percentage of dead HCT-116, CaCo-2 and A549 cells were above 20% after 72h treatment
with the lowest tested concentration (3.9 µL) of C3, suggesting that this complex has potent
anti-tumor activity. The lowest activity against all the tested cancer cell lines was observed
for the complex C5. The order of the cytotoxic activity of the palladium(II) complexes and
cisplatin, C3 > cisplatin > C4 > C2 > C1 > C5, was the same against all the tested cancer
cells.
Further, we also demonstrated that the cytotoxicities of the palladium(II)
complexes against cancer cell lines were significantly higher than those of their ligand
precursors. The ligand precursors showed moderate to low cytotoxicities against HCT-116,
CaCo-2 and A549 cancer cell lines (Figs. 3-5), and very low cytotoxicities against fibroblasts
(
Fig. 6). Cisplatin showed a significantly higher cytotoxicity than the ligand precursors at all
tested concentrations and against all cancer cell lines (Figs. 3-5). The cytotoxic activity of the
ligand precursors against all the cell lines correlated with cytotoxicities of the palladium(II)
complexes (cisplatin> L3 > L4 > L2 > L1 > L5).
Fig. 1 shows that the palladium(II) ion is "blocked" by coordination of two ligands,
but the molecules are more opened on the Pd-S side than the Pd-O side. This might be a
reason for an easier interaction with a DNA molecule, breaking of the Pd-S bonds and finally
binding to DNA, knowing that Pd-S is kinetically labile [45].
It is important to note that all the tested palladium(II) complexes and their ligand
precursors as well as cisplatin showed low cytotoxicity against fibroblasts. At the highest
tested concentration (250 µL) the percentage of dead fibroblasts was below 50% for all the
tested palladium(II) complexes and their ligand precursors, and at the lowest tested
concentration (3.9 µL) none of the tested compounds were cytotoxic against fibroblasts.
1
4

Due to the obtained results, the complex C3 seems to be a good candidate (among the
tested palladium(II) complexes) for future pharmacological investigation in the field of colon
and lung cancer research.
Both palladium(II)complexes characterized by single crystal X-ray analysis (C1 and
C5) generally exhibit the lowest cytotoxic activity. Since these two compounds display
significant conformational differences it is not possible to find any connection between their
structural properties and level of activity (different conformational properties versus low
activity in both cases). However, since C1 and C5 possess the most voluminous S-
substituents, it seems that the size of the substituent has some influence on the cytotoxic
activity (where less voluminous S-substituents are favorable).
4
. Conclusion
The cytotoxicities of the reported palladium(II) complexes were significantly higher
than those of their ligand precursors. The tested palladium(II) complexes showed moderate
and high cytotoxicities against human colon and lung carcinoma cells and low cytotoxicities
against fibroblasts. In comparison with other tested palladium(II) complexes and cisplatin,
palladium(II) complex C3 showed the highest cytotoxicity at all tested concentrations and
against all tested cancer cell lines.
2
The molecular structure of the mononuclear complex [Pd(S-bu-thiosal) ] was
determined by the single-crystal X-ray diffraction method. Although two deprotonated S-
butyl-thiosalicylic acid ligands form a symmetric cis-square-planar coordination around the
Pd(II) metal atom, they show a significant difference in their conformation. The major
differences between the two molecular halves are: (i) the value of the C1–C2–C3–S1 torsion
angles in the chelate rings [1.2(6) and -9.3(6)°], (ii) the spatial orientation of the S-
substituents and (iii) the inclination angle of the two phenyl rings with respect to the
1
5

coordination plane (dihedral angles of 50.38(1) and 63.9(1)°). The engagement of the two S-
bu-thiosal ligands in intermolecular interactions is also dissimilar.
Acknowledgment
This work was supported by the Ministry of Education, Science and Technological
Development of Republic of Serbia (Projects No 172014, 172016, 172034, 175069 and
1
75103).
Appendix A. Supplementary data
CCDC 1027150 contains the supplementary crystallographic data for this paper. These
data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or
from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ,
UK; fax: (+44) 1223 336 033; or e-mail: deposit@ccdc.cam.ac.uk.
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1
9

Figures
Table 1. Crystallographic data for [Pd(S-bu-thiosal)2].
Empirical formula
Formula weight
C H O PdS
22 26 4 2
524.95
Color, crystal shape
Crystal size (mm )
yellow, prism
0.18 x 0.15 x 0.14
293(2)
0.71073
Orthorhombic
Pbca
3
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
10.3458(3)
20.5915(6)
20.7746(6)
b (Å)
c (Å)3
V (Å )
Z
Dcalc (Mg/m )
μ (mm )
4425.7(2)
8
1.576
3
-1
1.053
θ range for data collection (°)
Reflections collected
2.95 to 29.10
16607
Independent reflections, Rint
Completeness (%) to θ = 26°
Refinement method
5257, 0.0405
99.9
Full-matrix least-squares on F
5257 / 0 / 264
1.095
2
Data / restraints / parameters
2
Goodness-of-fit on F
Final R /wR indices [I >2σ(I)]
0.0552/ 0.1014
1
2
-3
Largest diff. peak and hole (e Å ) 0.682 /−0.560

Figures
Table 2. Selected bond lengths (Å) and angles (º) for [Pd(S-bu-thiosal)2].
Bond
Pd1−O1
A
2.013(3)
2.2526(11)
1.784(4)
1.818(4)
1.284(5)
1.216(5)
1.517(6)
1.392(6)
90.75(8)
132.1(3)
102.62(14)
119.7(4)
121.9(4)
125.4(4)
123.3(3)
B
2.013(3)
2.2572(11)
1.777(4)
1.822(5)
1.292(5)
1.216(5)
1.503(6)
1.401(6)
88.76(9)
126.5(3)
98.88(14)
118.4(4)
121.3(4)
124.7(4)
123.5(3)
Pd1−S1
S1−C3
S1−C8
O1−C1
O2−C1
C1−C2
C2−C3
O1−Pd1−S1
Pd1−O1−C1
Pd1−S1−C3
O1−C1−C2
O1−C1−O2
C1−C2−C3
C2−C3−S1
O1a−Pd1−O1b
S1a−Pd1−S1b
O1a−Pd1−S1b
O1b−Pd1−S1a
86.92(11)
93.58(4)
175.59(8)
177.65(9)

Figures

Figures

Figures
Fig. 3. Representative graphs of HCT-116 cell survival after 72 h cell growth in the presence of
palladium(II)-complexes, cisplatin and ligand precursors.

Figures
Fig. 4. Representative graphs of CaCo-2 cell survival after 72 h cell growth in the presence of
palladium(II)-complexes, cisplatin and ligand precursors.

Figures
Fig. 5. Representative graphs of A549 survival after 72 h cell growth in the presence of
palladium(II)-complexes, cisplatin and ligand precursors.

Figures
Fig. 6. Representative graphs of fibroblasts survival after 72 h cell growth in the presence of
palladium(II)-complexes, cisplatin and ligand precursors.

Figures
Table 3. Geometrical parameters (Å, °) of the hydrogen bonds and C−H… π interactions.
D−H…A
D−H
0.93
0.93
0.97
D…A
3.345(6)
3.324(6)
3.422(6)
H…A
2.59
2.58
2.47
D−H…A
139
137
168
i
C4a−H4a...O2b
C4b−H4b...O2a
C8b −H8b1...O1b
ii
iii
C−H… π
C−H
H…Cg
H-Perp
C−H…Cg
iii
C8a−H8a1...Cg1
C8a−H8a2...Cg1
C9a−H9a2...Cg2
0.97
0.97
0.97
2.82
3.20
3.21
2.78
3.02
2.68
124
98
137
iii
iii
Cg1 and Cg2 are the centroids of the phenyl rings in fragments A and B, respectively. Symmetry codes: (i) −x+1,
y+1/2, −z+3/2; (ii) −x+1/2, −y, z+1/2; (iii) x−1/2, y, −z+3/2.

Figures
a
Table 4. IC (μM) values after 72 h of action of the investigated complexes, cisplatin and ligand
5
0
precursors on HCT116, CaCo2, A549 cells and fibroblasts, as determined by an MTT assay.
Compound HCT-116 cells
CaCo-2 cells
116.48 +/- 20.4
91.42 +/- 15.8
19.69 +/- 3.8
38.47 +/- 10.1
110.62 +/- 19.8
21.11+/- 3.28
182.68 +/- 27.7
186.21 +/- 26.5
180.61 +/- 28.4
182.12 +/- 27.1
236.6 +/- 30.3
A549 cells
77.12 +/- 14.7
66.07 +/- 12.4
2.14 +/- 0.6
43.07 +/- 11.9
171.95 +/- 25.4
3.12+/- 0.26
204.58 +/- 29.9
198.39 +/- 27.4
157.34 +/- 22.1
181.74 +/- 26.5
288.94 +/- 41.1
fibroblasts
C1
C2
105.71 +/-18.2
30.31 +/-6.2
301.85 +/- 39.1
277.69+/- 28.7
217.12 +/- 24.8
230.12 +/- 31.5
343.37 +/- 48.2
218.25+/-28.12
415.75 +/- 50.7
400.26 +/- 51.8
324.19 +/- 45.2
384.75 +/- 55.7
579.94 +/- 65.9
C3
2.24 +/- 0.3
C4
9.65 +/- 1.4
C5
cisplatin
L1
128.16 +/- 21.4
3.06 +/- 0.25
184.97 +/- 28.4
158.51 +/- 23.1
144.31 +/- 20.4
147.99 +/- 20.5
224.81 +/- 32.3
L2
L3
L4
L5
a
Mean values +/- SD (standard deviation) from three experiments.