Palladium(II) and platinum(II) bis(thiosemicarbazone) complexes of the 2,6-diacetylpyridine series with high cytotoxic activity in cisplatin resistant A2780cisR tumor cells and reduced toxicity

Article abstract of DOI:10.1016/j.jinorgbio.2013.04.005

Preparation and characterization of four novel 2,6-diacetylpyridine bis(⁴N-tolylthiosemicarbazonato) palladium(II) and platinum(II) complexes, [PdL^1-2] and [PtL^1-2], are described. All compounds have been characterized by elemental analysis and by IR and NMR spectroscopy, and the crystal and molecular structures of complexes [PdL ²] and [PtL²] have been determined by a single crystal X-ray diffraction. The ligands act as dianionic tetradentate donors coordinating to the metal center in a square planar geometry through the N _pyridinic atom and the N_iminic and the S atoms from one thiosemicarbazone arm, the fourth coordination position is occupied by the N_hydrazinic of the other arm. The new compounds synthesized have been evaluated for antiproliferative activity in vitro against NCI-H460, HepG2, MCF-7, A2780 and A2780cisR human cancer cell lines. The cytotoxicity data suggest that [PdL¹], [PdL²] and [PtL²] may be endowed with important antitumor properties since they are capable of not only circumventing cisplatin resistance in A2780cisR cells but also exhibiting high antiproliferative activity in breast cancer MCF-7 cells. Subsequent toxicity study, in LLC-PK1 cells, has also been carried out and shows that none of these compounds are in vitro toxic in the tested concentration range.

Full text of DOI:10.1016/j.jinorgbio.2013.04.005

Journal of Inorganic Biochemistry 125 (2013) 26–31
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Palladium(II) and platinum(II) bis(thiosemicarbazone) complexes of
the 2,6-diacetylpyridine series with high cytotoxic activity in cisplatin
resistant A2780cisR tumor cells and reduced toxicity
1
⁎
Ana I. Matesanz, Inés Leitao , Pilar Souza
Departamento de Química Inorgánica (Módulo 07), Facultad de Ciencias, c/Francisco Tomás y Valiente nº 7, Universidad Autónoma de Madrid, 28049 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Preparation and characterization of four novel 2,6-diacetylpyridine bis(4N-tolylthiosemicarbazonato)
palladium(II) and platinum(II) complexes, [PdL1–2] and [PtL1–2], are described. All compounds have been
characterized by elemental analysis and by IR and NMR spectroscopy, and the crystal and molecular struc-
tures of complexes [PdL2] and [PtL2] have been determined by a single crystal X-ray diffraction. The ligands
act as dianionic tetradentate donors coordinating to the metal center in a square planar geometry through
the Npyridinic atom and the Niminic and the S atoms from one thiosemicarbazone arm, the fourth coordination
position is occupied by the Nhydrazinic of the other arm. The new compounds synthesized have been evaluated
for antiproliferative activity in vitro against NCI-H460, HepG2, MCF-7, A2780 and A2780cisR human cancer
cell lines. The cytotoxicity data suggest that [PdL1], [PdL2] and [PtL2] may be endowed with important
antitumor properties since they are capable of not only circumventing cisplatin resistance in A2780cisR
cells but also exhibiting high antiproliferative activity in breast cancer MCF-7 cells. Subsequent toxicity
study, in LLC-PK1 cells, has also been carried out and shows that none of these compounds are in vitro
toxic in the tested concentration range.
Article history:
Received 21 February 2013
Received in revised form 16 April 2013
Accepted 17 April 2013
Available online 23 April 2013
Keywords:
Antitumor activity
Asymmetric N3S coordination
2,6-Diacetylpyridine
Palladium and platinum complexes
Renal toxicity
Thiosemicarbazone
© 2013 Elsevier Inc. All rights reserved.
1. Introduction
atoms that they possess, among which sulfur is of paramount impor-
tance in the metal-ligand linkage. Particularly, compounds in which
Platinum metallo-drugs are among the most effective agents for
the treatment of cancer however its clinical utility is restricted due
to the frequent development of drug resistance, the limited spectrum
of tumors against which these drugs are active and severe normal tis-
sue toxicity being the nephrotoxicity an important side effect which
interferes with their therapeutic efficiency [1–5].
These disadvantages have driven the development of improved
platinum-based anticancer drugs different from the traditional cisplatin
structure and which could probably have different DNA-binding modes
as well as exhibit different biological profiles [6–10].
In this regard and taking into account that one of the mechanisms
inducing the nephrotoxicity is due to the inactivation of some enzymes
because of the reaction between platinum ions and sulfur containing
proteins, an active area of research focuses on the synthesis of chelate
platinum(II) complexes bearing nitrogen and sulfur mixed donor
atoms, which should prevent the adverse reaction described [11,12].
Thiosemicarbazones (R1R2C_N\NH\C(S)\NR3R4) are an im-
portant and versatile type of ligands due to the potential donor
the thiosemicarbazone side-chain is attached in α position to an N-
heterocyclic ring, namely α-N-heterocyclic thiosemicarbazones
(N-TSCs), are strong metal chelating agents and moreover some of
them have showed antineoplastic activity by themselves. It has
been demonstrated that the biochemical mechanism of action in-
volves, among others, ribonucleotide reductase (RR) inhibition and
non-covalent DNA binding [13–19].
On the other hand, the pyridine ring itself is a part of many natural
and synthetically prepared pharmaceuticals. In addition, the pyridine
moiety plays a significant role in many biological processes like nico-
tinamide adenine dinucleotide phosphate NADP or vitamin B6
[20,21].
Keeping in view the above observations and as part of our sys-
tematic investigation on the coordination chemistry of thiosemi-
carbazone derivatives we planned to construct a series of α-N-
heterocyclic bis(thiosemicarbazone) platinum(II) and palladium(II)
complexes bearing 2,6-diacetylpyridine as heterocyclic ring specifically
using 2,6-diacetylpyridine bis(4N-para-tolylthiosemicarbazone), H2L1,
and 2,6-diacetylpyridine bis(4N-para-tolylthiosemicarbazone), H2L2, li-
gands (Scheme 1).
⁎
Corresponding author. Tel.: +34 914975146; fax: +34 914974833.
E-mail address: pilar.souza@uam.es (P. Souza).
The contributions made by this co-author were performed as part of her
Thus, this work is aimed to describe the synthesis and chemical
characterization of four new 2,6-diacetylpyridine bis(4N-tolylthiosemi-
carbazonato) palladium(II) and platinum(II) complexes. The cytotoxic
1
Research Project at Universidad Autónoma de Madrid (Erasmus Student from
Universidade do Porto).
0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2013.04.005

A.I. Matesanz et al. / Journal of Inorganic Biochemistry 125 (2013) 26–31
27
washed several times with hot water, crystallized from DMSO and finally
dried in vacuo.
2.3.1. [PdL1]
Yield (70%), mp 232 °C (decomposes). Elemental analysis found,
C, 50.50; H, 4.30, N, 15.95; S 10.55; C25H25N7S2Pd requires C, 50.40;
H, 4.30, N, 15.85; S 10.50%. MS (FAB+ with mNBA matrix) m/z
596.07 for [PdL1 + H]+. IR (KBr pellet): n/cm−1 3323 (s, NH); 1582
(s, CN); 1569 (s, CN-thioamide I); 853 (w, CS-thioamide IV); 618,
430 (pyridine ring). 1H NMR (300 MHz, d6-DMSO, ppm), δ = 10.45,
9.40 [s, 4NH, 1H]; δ = 8.40 [d, CH-pyridine, 2H]; 8.25 [t, CH-
pyridine, 1H]; δ = 7.20–7.00 (m, aromatic-thiosemicarbazide, 8H);
δ = 2.50 (s, CH3-thiosemicarbazide, 6H); δ = 2.25 (s, CH3-
diacetylpyridine, 6H). UV/VIS (DMSO): λ/nm 343, 422, 486.
2.3.2. [PtL1]
Yield (55%), mp 216 °C (decomposes). Elemental analysis found, C,
42.35; H, 3.95; N, 13.00; S, 12.25; C25H25N7S2Pt · DMSO requires C,
42.45; H, 4.30; N, 12.85; S 12.60%. MS (FAB+ with mNBA matrix) m/z
683 for [PtL1 + H]+. IR (KBr pellet): n/cm−1 3353 (s, NH); 1586 (s,
CN); 1523 (s, CN-thioamide I); 849 (w, CS-thioamide IV); 617, 428
(pyridine ring). 1H NMR (300 MHz, d6-DMSO, ppm), δ = 10.75, 9.50
[s, 4NH, 1H]; δ = 8.50 [d, CH-pyridine, 2H]; 8.00 [t, CH-pyridine, 1H];
δ = 7.35–7.10 (m, aromatic-thiosemicarbazide, 8H); δ = 2.75 (s,
CH3-thiosemicarbazide, 6H); δ = 2.25, 2.20 (s, CH3-diacetylpyridine,
3H). UV/VIS (DMSO): λ/nm 341, 395, 420.
Scheme 1. General structure of diacetylpyridine bis(4N-tolylthiosemicarbazone)
ligands.
activity of the new compounds synthesized, their parent ligands and cis-
platin (assumed as the reference antitumor drug) against five human
cancer cell lines: NCI-H460 (non-small cell lung cancer), HepG2 (hepato-
cellular carcinoma), MCF-7 (breast cancer), A2780 and A2780cisR
(epithelian ovarian cancer) has been studied. In addition toxicity studies,
on normal renal LLC-PK1 cells, have been carried out as an attempt to pro-
vide an insight into the pharmacological properties of these compounds.
The single-crystal X-ray structures of [PdL2], [PtL2] and H2L1 are also
discussed.
Based on the characterization data, this compound can also be
synthesized by reaction of K2PtCl4 with H2L1 ligand as we described
recently [22].
2.3.3. [PdL2]
2. Experimental
Yield (35%), mp 226 °C (decomposes). Elemental analysis found, C,
46.80; H, 4.85; N, 13.90; S, 13.50; C25H25N7S2Pd · DMSO · H2O requires
C, 47.00; H, 4.80; N, 14.20; S 13.90%. IR (KBr pellet): n/cm−1 3291 (s,
NH); 1595 (s, CN), 854, 817, 808 (w, CS-thioamide IV band); 638, 419
(pyridine ring). 1H NMR (300 MHz, d6-DMSO, ppm), δ = 10.60, 10.00
[s, 4NH, 1H]; 8.40 [t, CH-pyridine, 1H]; δ = 8.25, 8.10 [d, CH-pyridine,
2H]; δ = 7.55–7.10 (m, aromatic-thiosemicarbazide, 8H); δ = 2.70,
2.60 (s, CH3-thiosemicarbazide, 6H); δ = 2.25 (s, CH3-diacetylpyridine,
6H). UV/VIS (DMSO): λ/nm 268, 293, 405.
2.1. Measurements
Elemental analyses were performed on a LECO CHNS-932 micro-
analyzer. Fast atom bombardment (FAB) mass spectra (MS) were
performed on a VG AutoSpec spectrometer (mNBA: nitrobenzyl alcohol
matrix). All cited physical measurements were obtained by the Servicio
Interdepartamental de Investigación (SIDI) of the Universidad Autónoma
de Madrid.
Melting points were determined with a Stuart Scientific SMP3
apparatus. Infrared spectra (KBr pellets) were recorded on a Bomen–
Michelson spectrophotometer (4000–400 cm−1). Electronic spectra
were recorded on a Thermo Scientific Evolution 260 Bio UV–visible
(UV–VIS) spectrophotometer.
Recrystallization from DMSO led to the isolation of orange crystals
of [PdL2]·0.5DMSO that were suitable for X-ray-diffraction.
2.3.4. [PtL2]
Yield (55%), mp 234 °C (decomposes). Elemental analysis found, C,
41.60; H, 3.90; N, 13.50; S, 9.10; C25H25N7S2Pt · 2H2O requires C,
41.75; H, 4.05; N, 13.65; S, 8.90%. MS (FAB+ with mNBA matrix) m/z
683 for [PtL2 + H]+. IR (KBr pellet): n/cm−1 3250 (s, NH); 1593 (s,
2.2. Materials
CN), 850, 804 (w, CS-thioamide IV band); 638, 420 (pyridine ring). 1
H
NMR (300 MHz, d6-DMSO, ppm), δ = 10.60, 10.15 [s, 4NH, 1H]; δ =
8.50 [d, CH-pyridine, 1H]; 7.80 [t, CH-pyridine, 1H]; δ = 7.45 [d,
CH-pyridine, 1H]; δ = 7.40–7.20 (m, aromatic-thiosemicarbazide,
8H); δ = 2.50 (s, CH3-thiosemicarbazide, 6H); δ = 2.30 (s, CH3-
diacetylpyridine, 6H). UV/VIS (DMSO): λ/nm 255, 310, 400.
Solvents were purified and dried according to standard proce-
dures. Hydrazine hydrate, 2,6-diacetylpyridine, ortho-tolyl isothiocy-
anate, para-tolyl isothiocyanate, PdCl2(PPh3)2 and PtCl2(PPh3)2 were
commercially available.
2.3. Synthesis of compounds
Recrystallization from DMSO led to the isolation of dark red crys-
tals of [PtL2]·DMSO that were suitable for X-ray-diffraction.
The two ligands were synthesized following general procedures as
described in references [22,23]. In support of analytical and spectro-
scopic data, consistent with those previously reported, the X-ray
structure of H2L1 ligand has been determined here for the first time.
Mononuclear neutral complexes of formulae [ML1–2] where M = Pd,
Pt were obtained by reaction of the corresponding MCl2(PPh3)2 metallic
salt with the desired ligand, in toluene, in the presence of Et3N, in
1:1 molar ratios. The reaction mixture was stirred for 2 h at room tem-
perature. The resulting orange solution was filtered and left to stand at
ambient temperature for two days. The solid formed was filtered,
2.4. Crystallography
Data were collected on a Bruker X8 APEX II CCD (compounds H2L1,
PdL2and PtL2). Crystallographic data and selected interatomic dis-
tances and angles are listed in Table 1. For all compounds, the soft-
ware package SHELXTL was used for space group determination,
structure solution, and refinement [24]. The structures were solved
by direct methods, completed with difference Fourier syntheses,
and refined with anisotropic displacement parameters.

28
A.I. Matesanz et al. / Journal of Inorganic Biochemistry 125 (2013) 26–31
Table 1
Crystal data and structure refinement for H2L1, [PdL2] and [PtL2] compounds.
H2L1·DMSO·0.5H2O
[PdL2]·0.5DMSO
[PtL2]·DMSO
Molecular formula
C27H34N7O1.5S3
C26H28N7O0.5PdS2.5
C27H31N7OPtS3
Formula weight
576.79
633.10
760.86
Temperature (K)
100 (2)
100 (2)
100 (2)
Wavelength (Å)
0.71073
0.71073
0.71073
Crystal system
Monoclinic
Monoclinic
Monoclinic
Space group
C2/c
P21/c
P21/c
a (Å)
35.225 (3)
14.0884 (13)
13.9098 (12)
b (Å)
7.4795 (7)
7.6059 (6)
7.4227 (7)
c (Å)
21.9895 (16)
27.227 (2)
27.182 (2)
α/°
90
90
90
β/°
98.215 (3)
91.119 (5)
90.439 (4)
γ/°
90
90
90
Volume (Å3)
5734.0 (8)
2917.0 (4)
2806.4 (4)
Z
8
4
4
Density (calculated) (g/cm3)
Absorption coefficient (mm−1
F(000)
1.336
0.295
2440
1.442
0.845
1292
1.801
5.259
1504
)
Crystal size (mm3)
Index ranges
0.40 × 0.15 × 0.10
−47 ≤ h ≤ 47, −10 ≤ k ≤ 10,
−29 ≤ l ≤ 29
35398
0.24 × 0.04 × 0.04
−16 ≤ h ≤ 16, −9 ≤ k ≤ 9,
−32 ≤ l ≤ 31
39446
0.23 × 0.02 × 0.02
−16 ≤ h ≤ 16, −8 ≤ k ≤ 8,
−31 ≤ l ≤ 29
9556
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
R indices (all data)
7429 [R(int) = 0.0629]
7429/0/358
1.002
R1 = 0.0571, wR2 = 0.1598
R1 = 0.1016, wR2 = 0.1970
1.596 and −0.463
5330 [R(int) = 0.1624]
5330/1/352
0.999
R1 = 0.09, wR2 = 0.2526
R1 = 0.2039, wR2 = 0.3237
1.399 and −1.015
4195 [R(int) = 0.062]
4195/0/328
1.011
R1 = 0.085, wR2 = 0.247
R1 = 0.1403, wR2 = 0.2891
2.156 and −2.641
Largest diff. peak and hole, e. Å−3
CCDC 918229, 918230 and 923814 contain the supplementary crys-
tallographic data for compounds H2L1, [PtL2] and [PdL2] respectively.
These data can be obtained free of charge at 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; e-mail: deposit@ccdc.cam.ac.uk].
The normal cells (LLC-PK1) were grown in 199 medium
supplemented with 3% FBS and 1.5 g/L of sodium bicarbonate in an
atmosphere of 5% CO2 at 37 °C. Cell proliferation was evaluated by
the sulforhodamine B assay. Cells were plated in 96-well sterile plates
at a density of 1 · 104 cells per well with 100 μL of medium and were
then incubated for 24 h. After attachment to the culture surface the
cells were incubated with various concentrations of the compounds
tested freshly dissolved in DMSO (1 mg/mL) and diluted in the cul-
ture medium (DMSO final concentration 1%) for 48 h at 37 °C. The
cells were fixed by adding 50 μL of 30% TCA per well. The plates
were treated as described above for cancer cells.
2.5. In vitro antiproliferative activity
The human cancer cells A2780, A2780cisR and NCI-H460 were
grown in RPMI-1640 medium supplemented with 10% fetal bovine
serum (FBS) and 2 mM L-glutamine in an atmosphere of 5% CO2 at
37 °C. The human cancer cells MCF-7 were grown in EMEM (mini-
mum essential medium, Eagle) with 2 mM L-glutamine and Earle's
BSS (adjusted with 1.5 g/L NaHCO3, 0.1 mM nonessential amino
acids and 1 mM sodium pyruvate) and supplemented with 10% FBS
and 0.01 mg/mL bovine insulin in an atmosphere of 5% CO2 at 37 °C.
The human cancer cells Hep-G2 were grown in EMEM medium
supplemented with 10% FBS in an atmosphere of 5% CO2 at 37 °C.
Cell proliferation was evaluated by the sulforhodamine B assay.
Cells were plated in 96-well sterile plates at a density of 1.5 · 104
(for NCI-H460), 1 · 104 (for MCF-7 and Hep-G2) or 4 · 103 (for
A2780 and A2780cisR) cells per well with 100 μL of medium and
were then incubated for 24 h. After attachment to the culture surface
the cells were incubated with various concentrations of the com-
pounds tested freshly dissolved in DMSO (1 mg/mL) and diluted in
the culture medium (DMSO final concentration 1%) for 48 h (for
NCI-H460 and Hep-G2) or 96 h (for A2780, A2780cisR and MCF-7).
The cells were fixed by adding 50 μL of 30% trichloroacetic acid
(TCA) per well.
The effects of compounds were expressed as corrected percentage
inhibition values according to the following equation:
% inhibition ¼ ½1−ðT=CÞꢀ ꢁ 100
where T is the mean absorbance of the treated cells and C the mean
absorbance in the controls.
The inhibitory potential of compounds was measured by calculat-
ing concentration–percentage inhibition curves, these curves were
adjusted to the following equation:
Â
Ã
E ¼ Emax= 1 þ ðIC50=CÞn
where E is the percentage inhibition observed, Emax is the maximal ef-
fects, IC50 is the concentration that inhibits 50% of maximal growth, C
is the concentration of compounds tested and n is the slope of the
semi-logarithmic dose–response sigmoid curves. This non-linear
fitting was performed using GraphPad Prism software [25].
For comparison purposes, the antiproliferative activity of cisplatin was
evaluated under the same experimental conditions. All compounds were
tested in two independent studies with triplicate points. These experi-
ments were carried out at the Unidad de Evaluación de Actividades
Farmacológicas de Compuestos Químicos (USEF), Universidad de Santiago
de Compostela.
The plates were incubated at 4 °C for 1 h and then washed five
times with distilled water. The cellular material fixed with TCA was
stained with 0.4% sulforhodamine B dissolved in 1% acetic acid for
10 min. Unbound dye was removed by rinsing with 0.1% acetic acid.
The protein-bound dye was extracted with 10 mM unbuffered Tris
base for determination of optical density (at 515 nm) in a Tecan
Ultra Evolution spectrophotometer.

A.I. Matesanz et al. / Journal of Inorganic Biochemistry 125 (2013) 26–31
29
Table 2
3. Results and discussion
Selected bond distances (Å) for H2L1.
H2L1
3.1. Synthesis and spectroscopic characterization
S(1)\C(16)
S(2)\C(15)
C(2)\N(2)
C(8)\N(1)
C(15)\N(1)
C(15)\N(3)
C(16)\N(2)
C(16)\N(5)
C(17)\N(4)
C(23)\N(6)
N(3)\N(4)
N(5)\N(6)
1.678 (3)
1.683 (3)
1.427 (4)
1.421 (3)
1.344 (3)
1.369 (3)
1.333 (3)
1.362 (4)
1.290 (3)
1.293 (4)
1.374 (3)
1.371 (3)
Reaction of 2,6-diacetylpyridine bis(4N-tolylthiosemicarbazone)
ligands with equimolar amount of MCl2(PPh3)2, where M = Pd(II)
or Pt(II), led to the isolation of the neutral mononuclear complexes
[PdL1–2] and [PtL1–2] in which the corresponding bis(thiosemicarbazone)
behaves as dianionic ligand with deprotonation of hydrazinic (2NH) pro-
tons and [NNNS] donor set.
The new palladium(II) and platinum(II) complexes obtained are
stable to air and moisture and were characterized by elemental anal-
ysis and FAB+ spectrometry and spectroscopic studies (selected IR
and UV–VIS bands are listed in the Experimental section).
The infrared spectral bands most useful for determining the mode of
coordination of the ligands are the υ(C_N) iminic and υ(C_S)
thioamide IV vibrations. These bands are shifted to lower wavenumbers
in the spectra of the complexes suggesting coordination of the imine ni-
trogen and sulfur atoms. The pyridine nitrogen also appears to be in-
volved in coordination based on the in-plane and out-of-plane ring
deformation bands which show shift to upper wavenumbers and fairly
low intensity [26].
On the other hand the sulfur atom S(1) and the imine nitrogen atom
N(6) are in trans position with respect to the C(16)\N(5) bond and the
same configuration is observed for S(2) and N(4) with respect to the
C(15)\N(3) bond, therefore the ligand exists in E configuration
which is often observed in thiosemicarbazones [29]. The C_Nimine
bond distances of 1.290(3) and 1.293(4) are in conformity with a formal
C_N double bond and the C_S bond distances of 1.678(3) and
1.683(3) Å, are very close to a formal C_S double bond length, these
facts confirm the existence of the thiosemicarbazone groups in the
thione form, in the solid state. However, the N(3)\N(4), N(3)\C(15),
C(15)\N(1) and N(5)\N(6), N(5)\C(16), and C(16)\N(2) bond
distances are intermediate between the ideal values of corresponding
single and double bonds which is indicative of some electron delocaliza-
tion along the thiosemicarbazide side chains.
Supramolecular association involves the water of crystallization
molecule, which forms two hydrogen bonds as hydrogen donor
(with sulfur atoms of two DMSO as acceptors) and two more as ac-
ceptor (acting the N(3) atoms of two different ligand molecules as
donor). The packing (Fig. S1, Supporting Information) suggests
weak by π–π stacking interactions (C⋯C distance about 4.1 Å) be-
tween molecules via the terminal phenyl rings.
The electronic absorption spectra of [PdL2] and [PtL2] complexes
exhibit three intense bands in the region 250–485 nm. The two
most energetic bands can be ascribed to ligand-centered n → π*
and π → π* transitions while the third band is assigned to a combina-
tion of ligand to metal (LMCT) and metal to ligand charge transfer
(MLCT) transitions. However for [PdL1] and [PtL1] complexes no tran-
sitions that could be assigned to an n → π* type was observed in the
spectra, probably this band is covered with the more intense π → π*
[27].
3.2. Description of the crystal structures
Good quality crystals suitable for single crystal X-ray diffraction
analysis were obtained for H2L1, [PdL2] and [PtL2] compounds by
recrystallization in dimethyl sulfoxide.
[PdL2]·0.5DMSO and [PtL2]·DMSO were isolated as neutral com-
pounds. The most significant parameters for these complexes are
shown in Table 1. The structures together with the atom labeling
schemes are shown in Fig. 2 and Fig. S2 (Supporting Information). Both
compounds are isostructural hence displaying nearly identical cell pa-
rameters and crystallize in the monoclinic P21/c space group with Z = 4.
The metal ion presents a square planar geometry being the
bis(thiosemicarbazone) ligand attached through the Npyridinic atom,
the Niminic and the S atoms from one thiosemicarbazone arm and
being the fourth coordination position occupied by the Nhydrazinic of
the other thiosemicarbazone arm generating two typical five mem-
bered (PdSCNN and PdNCCN or PtSCNN and PtNCCN) and one six
The molecular structure of H2L1 free ligand together with the
atomic numbering scheme is shown in Fig. 1. Crystallographic data
is shown in Table 1 and selected bond lengths are listed in Table 2.
The crystal structure of H2L1 consists of discrete H2L1·DMSO·0.5H2O
molecules with the two thiosemicarbazone arms adopting a syn-open
arrangement in order to minimize unfavorable electronic interactions
between the two tolyl groups. Therefore to achieve the coordinative
disposition found in d8 metal complexes, syn-close conformation, a
rearrangement of the molecule structure, by twisting around the C\C
bonds adjacent to the pyridine ring, is necessary [28].
Fig. 1. Molecular structure of H2L1 ligand.

30
A.I. Matesanz et al. / Journal of Inorganic Biochemistry 125 (2013) 26–31
Fig. 2. Molecular structure of [PdL2] complex.
membered (PdNNCCN or PtNNCCN) chelate rings. Coordination by
Nhydrazinic instead of Niminic, although uncommon, has been found
in the bibliography for some d8 bis(thiosemicarbazone) complexes
[22,30–32].
(less than 90°). The angles N(4)\M(1)\N(2) and N(2)\M(1)\S(2)
are therefore greater than 90°.
In both compounds the crystal structure is stabilized by inter-
molecular hydrogen interaction involving the N(7) atom of the
bidentate thiosemicarbazone arm and the oxygen atom of DMSO sol-
vent molecule.
It is important to note that the two thiosemicarbazone moieties
coordinate in a different fashion and so upon coordination the
bidentate-N∧S arm undergoes significant evolution from the thione
to the thiol form which is reflected in C\S distance of 1.735(15) for
[PdL2]·DMSO and 1.80(2) for [PtL2]·DMSO while the monodentate-
Nhidrazinic thiosemicarbazone arm presents a shorter C\S bond
length [1.641(16) for [PdL2]·DMSO and 1.68(3) Å for [PtL2]·DMSO].
On the other hand The C\N and N\N bond distances, listed
in Table 3, are intermediate between formal single and double
bonds, pointing to extensive delocalization over the entire 2,6-
diacetylpyridine bis(thiosemicarbazone) skeleton [33].
3.3. Antiproliferative activity against tumor and normal cells
To assess the antitumor potential of the synthesized compounds,
its antiproliferative activity (in powder solid form) was tested in
vitro against a panel of human cancer cell lines containing examples
of lung (NCI-H460), breast (MCF7), liver (Hep-G2) and ovarian
(A2780 and A2780cisR) cancers. For comparison purposes, the cyto-
toxicity of cisplatin was always evaluated under the same experimen-
tal conditions.
Inspection of the angles formed between the metal ion (M = Pd2+
,
Pt2+) and the coordinated atoms shows that the metal is contained
within a slightly distorted square-planar environment. The distortion
is caused by the restricted bite angle of the N(4), N(5), S(2) donor set
as reflected in the S(2)\M(1)\N(5) and N(4)\M(1)\N(5) angles
The results indicate that the two free ligands, H2L1 and H2L2, and
platinum complex [PtL1] showed at 100 μM concentration, very low
cellular growth inhibition (b50%) and therefore had no evaluable cyto-
toxicity (IC50 > 100 μM). However [PdL1], [PdL2] and [PtL2] complexes
displayed high antiproliferative activity against epithelian ovarian
(A2780, cisplatin sensitive, and A2780cisR, cisplatin resistant) and
breast (MCF-7) cancer cells (Table 4). It is remarkable to note that pal-
ladium complex [PdL1] exhibited better cytotoxic effects against MCF-7
Table 3
Selected bond distances (Å) and angles (°) for [PdL2] and [PtL2].
and A2780cisR cells than cisplatin by comparing their IC50
.
[PdL2]
[PtL2]
The A2780cisR cell line encompasses all of the known major
mechanisms of resistance to cisplatin: reduced drug transport, en-
hanced DNA repair/tolerance, and elevated GSH levels. The ability of
S(1)\C(18)
S(2)\C(10)
C(2)\N(3)
1.641 (16)
1.735 (15)
1.275 (17)
1.333 (17)
1.328 (18)
1.335 (18)
1.46 (2)
1.400 (18)
1.366 (17)
1.436 (17)
2.304 (4)
2.062 (12)
2.017 (10)
1.976 (12)
91.9 (5)
1.68 (3)
1.80 (2)
1.24 (3)
1.37 (3)
1.26 (3)
1.36 (3)
1.47 (3)
1.34 (3)
1.43 (3)
1.49 (3)
2.307 (6)
1.99 (2)
2.04 (2)
1.98 (2)
90.3 (8)
173.2 (7)
83.0 (8)
104.4 (5)
165.3 (6)
82.4 (5)
C(8)\N(5)
C(10)\N(6)
C(10)\N(7)
C(11)\N(7)
C(18)\N(1)
C(18)\N(2)
C(19)\N(1)
M\S(2)
M\N(2)
M\N(4)
M\N(5)
N(2)\M\N(4)
N(2)\M\N(5)
N(4)\M\N(5)
N(2)\M\S(2)
N(4)\M\S(2)
N(5)\M\S(2)
Table 4
In vitro antiproliferative activity of the bis(thiosemicarbazone) compounds and cis-
platin, evaluated in human MCF-7 (breast cancer), A2780 and A2780cisR (epithelian
ovarian cancer) cell lines.
IC50
SD (μM)
RF
A2780
A2780cisR
MCF-7
IC50(A2780cisR)/
IC50(A2780)
H2L1
>100
>100
5.97
>100
21
>100
>100
5.80
>100
22
>100
> 100
4.41
H2L2
[PdL1]
[PtL1]
[PdL2]
[PtL2]
Cisplatin
0.06
0.02
0.49
0.45
0.35
0.97
173.9 (5)
82.1 (5)
104.0 (3)
164.0 (4)
82.0 (4)
>100
1
2
1
1
48
2
0.86
1.1
8.68
20
0.83
18
7.21
44
2
0.13
8.75

A.I. Matesanz et al. / Journal of Inorganic Biochemistry 125 (2013) 26–31
31
Table 5
Appendix A. Supplementary data
In vitro antiproliferative activity of the bis(thiosemicarbazone) compounds and cis-
platin, evaluated in normal LLC-PK1 renal cells.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.jinorgbio.2013.04.005.
LLC-PK1
%Inhibition (100 μM)
IC50
SD (μM)
H2L1
2
1
20
2
1
23
90
>100
>100
>100
>100
>100
>100
6.86
References
H2L2
[PdL1]
[PtL1]
[PdL2]
[PtL2]
Cisplatin
[1] N.J. Wheate, S. Walker, G.E. Craig, R. Oun, Dalton Trans. 39 (2010) 8113–8127.
[2] E. Wong, C.M. Giandomenico, Chem. Rev. 99 (1999) 2451–2466.
[3] Z. Guo, P.J. Sadler, Angew. Chem. Int. Ed. 38 (1999) 1512–1531.
[4] M.S. Razzaque, Nephrol. Dial. Transplant. (2007) 1–5.
[5] M. Okuda, K. Masaki, S. Fukatsu, Y. Hashimoto, K. Inui, Biochem. Pharmacol. 59
(2000) 195–201.
0.10
[6] K. van der Schilden, F. García, H. Kooijman, A.L. Spek, J.G. Haasnoot, J. Reedijk,
Angew. Chem. Int. Ed. 43 (2004) 5668–5670.
[7] J.M. Pérez, V. Cerrillo, A.I. Matesanz, J.M. Millán, P. Navarro, C. Alonso, P. Souza,
ChemBioChem 2 (2001) 119–123.
[8] T.W. Hambley, Coord. Chem. Rev. 166 (1997) 181–223.
[9] J. Kasparkova, V. Marini, Y. Najajreh, D. Gibson, V. Brabec, Biochemistry 42 (2003)
6321–6332.
[10] M. Huxley, C. Sanchez-Cano, M.J. Browning, C. Navarro-Ranninger, A.G. Quiroga,
A. Rodger, M.J. Hannon, Dalton Trans. 39 (2010) 11353–11364.
[11] C. Marzano, A. Trevisan, L. Giovagnini, D. Fregona, Toxicol. In Vitro 16 (2002)
413–419.
[12] J.S. Casas, E.E. Castellano, J. Ellena, M.S. García-Tasende, M.L. Pérez-Parallé, A.
Sánchez, A. Sánchez-González, J. Sordo, A. Touceda, J. Inorg. Biochem. 102
(2008) 33–45.
[13] T.S. Lobana, R. Sharma, G. Bawa, S. Khanna, Coord. Chem. Rev. 253 (2009)
977–1055.
[14] J.M. Vila, T. Pereira, A. Amoedo, M. Graña, J. Martínez, M. López-Torres, A.
Fernández, J. Organomet. Chem. 623 (2001) 176–184.
[15] J.S. Casas, M.S. García-Tasende, J. Sordo, Coord. Chem. Rev. 209 (2000) 197–261.
[16] A.I. Matesanz, P. Souza, Mini Rev. Med. Chem. 9 (2009) 1389–1396.
[17] A.C. Sartorelli, K.C. Agrawal, A.S. Tsiftsoglou, E.C. Moore, Adv. Enzyme Regul.
(1977) 117–139.
[18] R.A. Finch, M.C. Liu, A.H. Cory, J.G. Cory, A.C. Sartorelli, Adv. Enzyme Regul. 39
(1999) 3–12.
[19] R.A. Finch, M.C. Liu, S.P. Grill, S.P.W.C. Rose, R. Loomis, K.M. Vasquez, Y.C. Cheng,
A.C. Sartorelli, Biochem. Pharmacol. 59 (2000) 983–991.
[20] E. Lukevits, Chem. Heterocyc. Comp. 31 (1995) 639–650.
[21] L.E. Kapinos, H. Sigel, Inorg. Chim. Acta 337 (2002) 131–142.
[22] A.I. Matesanz, P. Souza, Inorg. Chem. Commun. 27 (2013) 5–8.
[23] M. Maji, S. Ghosh, S.K. Chattopadhyay, Transit. Met. Chem. 23 (1998) 81–85.
[24] SHELXTL-NT Version 6.12, Structure Determination Package, Bruker-Nonius AXS,
Madison, Wisconsin, USA, 2001.
[PdL1], [PdL2] and [PtL2] complexes to circumvent cisplatin-acquired
resistance was confirmed from the resistance factor values, RF (de-
fined as IC50 in A2780cisR/IC50 in A2780) since all of them have a
much better RF than cisplatin. An RF of b2 was considered to denote
non-cross-resistance and therefore the three compounds tested are
able to circumvent cisplatin resistance [34,35].
These observations, on the ability of 2,6-diacetylpyridine
bis(4N-tolyl-tolylthiosemicarbazone) complexes to overcome cisplatin
resistance in A2780cisR cells, agree with our previous studies of
bis(4N-substituted thiosemicarbazones) derived from 3,5-diacetyl-1,2,4-
triazol heterocyclic ring [36–38].
Platinum base therapies are frequently associated with cumulative
and irreversible toxicities such as nephropathy. Therefore, in order to
investigate this possible side effect, the compounds investigated and
cisplatin were subsequently tested, in vitro, on normal renal LLC-PK1
cells in the same μM range (1–100). As showed in Table 5, all com-
pounds showed at the maximum concentration tested very low cellular
growth inhibition (b50%) and therefore had no evaluable cytotoxicity
(IC50 > 100 μM) in this normal cell line. This means that none of
these compounds are toxic, in vitro, in the tested concentration range.
4. Conclusions
[25] GraphPad Prism, Version 2.01, GraphPad Software, Inc., San Diego, CA, 1996.
[26] M.A. Ali, A.H. Mirza, A.L. Tan, L.K. Wei, P.V. Bernhardt, Polyhedron 23 (2004)
2037–2043.
[27] A.A. Ali, H. Nimir, C. Aktas, V. Huch, U. Rauch, K. Schäfer, M. Veith, Organometallics
31 (2012) 2256–2262.
[28] M.R. Bermejo, R. Pedrido, A.M. González-Noya, M.J. Romero, M. Vázquez, L.
Sorace, New J. Chem. 27 (2003) 1753–1759.
[29] M. Vázquez, L. Fabbrizzi, A. Taglietti, R.M. Pedrido, A.M. González-Noya, M.R.
Bermejo, Angew. Chem. Int. Ed. 43 (2004) 1962–1965.
[30] C.A. Brown, W. Kamisnsky, K.A. Claborn, K.I. Goldberg, D.X. West, J. Braz. Chem.
Soc. 13 (2002) 10–18.
[31] J.I. Gradinaru, S.T. Malinowski, M.A. Popovici, M. Gdaniec, Crystallogr. Rep. 50
(2005) 217–223.
A new family of Pt(II) and Pd(II) bis(thiosemicarbazone) com-
pounds incorporating the 2,6-diacetylpyridine heterocyclic ring has
been successfully prepared and characterized.
This study has identified [PdL1], [PdL2] and [PtL2] complexes as hav-
ing high antiproliferative activity since they are capable of not only
circumventing cisplatin resistance in A2780cisR cells but also exhibiting
high antiproliferative activity against breast (MCF-7) cancer cells.
Moreover, it is important to note that these compounds exhibit very
low nephrotoxicity with respect to cisplatin which confirmed that
these complexes are very specific on cancer cells.
[32] T.R. Todorović, A. Bacchi, G. Pelizzi, N.O. Juranić, D.M. Sladić, I.D. Brčeski, K.K.
In conclusion, this study indicates that the newly synthesized
complexes valuably lead to the development of new anticancer che-
motherapeutic agents capable of retaining their activity in the
human ovarian carcinoma cell line resistant to cisplatin A2780cisR.
Anelković, Inorg. Chem. Commun. 9 (2006) 862–865.
[33] R. Pedrido, A.M. González-Noya, M.J. Romero, M. Martínez-Calvo, M. Vázquez
López, E. Gómez-Fórneas, G. Zaragoza, M.R. Bermejo, Dalton Trans. (2008)
6776–6787.
[34] L.R. Kelland, C.F.J. Barnard, K.J. Mellish, M. Jones, P.M. Goddard, M. Valenti, A.
Bryant, B.A. Murrer, K.R. Harrap, Cancer Res. 54 (1994) 5618–5622.
[35] J. Ruiz, C. Vicente, C. Haro, D. Bautista, Inorg. Chem. 52 (2013) 974–982.
[36] A.I. Matesanz, C. Hernández, A. Rodríguez, P. Souza, Dalton Trans. 40 (2011)
5738–5745.
Acknowledgments
We are grateful to Ministerio de Economía y Competitividad,
Instituto de Salud Carlos III of Spain (PI080525 and PI1100659) for fi-
nancial support.
[37] A.I. Matesanz, J. Perles, P. Souza, Dalton Trans. 41 (2012) 12538–12547.
[38] A.I. Matesanz, P. Souza, J. Inorg. Biochem. 101 (2007) 245–253.