New bioactive 2,6-diacetylpyridine bis(p-chlorophenylthiosemicarbazone) ligand and its Pd(II) and Pt(II) complexes: Synthesis, characterization, cytotoxic activity and DNA binding ability

Article abstract of DOI:10.1016/j.jinorgbio.2014.04.017

Preparation and characterization of 2,6-diacetylpyridine bis( ⁴N-p-chlorophenylthiosemicarbazone) ligand, H₂L, and its palladium(II) and platinum(II) complexes [PdL] and [PtL], is described. The molecular structure of the two new com

Full text of DOI:10.1016/j.jinorgbio.2014.04.017

Journal of Inorganic Biochemistry 138 (2014) 16–23
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
New bioactive 2,6-diacetylpyridine
bis(p-chlorophenylthiosemicarbazone) ligand and its Pd(II) and Pt(II)
complexes: Synthesis, characterization, cytotoxic activity and DNA
binding ability
Ana I. Matesanz a, , Carolina Hernández a,b, Pilar Souza a
⁎
a
Departamento de Química Inorgánica (Módulo 07), Facultad de Ciencias, c/Francisco Tomás y Valiente no. 7, Universidad Autónoma de Madrid, 28049, Madrid, Spain
Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Ciencias del Medio Ambiente, Avd. Carlos III s/n, Universidad de Castilla-La Mancha, 45071, Toledo, Spain
b
a r t i c l e i n f o
a b s t r a c t
Preparation and characterization of 2,6-diacetylpyridine bis(4N-p-chlorophenylthiosemicarbazone) ligand, H2L,
and its palladium(II) and platinum(II) complexes [PdL] and [PtL], is described. The molecular structure of the
two new complexes has been determined by single crystal X-ray diffraction. The ligand acts as dianionic
tetradentate donor coordinating to the metal center in a square planar geometry through the pyridine nitrogen
atom and the azomethine nitrogen and thione sulfur atoms from one thiosemicarbazone arm, the fourth coordi-
nation position is occupied by the hydrazine nitrogen atom of the other arm. New free ligand and its metal com-
plexes have been evaluated for antiproliferative activity in vitro against NCI-H460, T-47D, A2780 and A2780cisR
human cancer cell lines. The cytotoxicity data suggest that these compounds may be endowed with important
antitumor properties, especially H2L and [PtL] since they are capable of not only circumvent cisplatin resistance
in A2780cisR cells but also exhibit high antiproliferative activity in breast cancer T-47D cells. The interaction of
H2L with calf thymus DNA was also investigated and its binding constant (Kb) determined.
Article history:
Received 25 February 2014
Received in revised form 28 April 2014
Accepted 28 April 2014
Available online 9 May 2014
Keywords:
Antitumor activity
Asymmetric coordination
2,6-Diacetylpyridine
Palladium and platinum complexes
Thiosemicarbazone
© 2014 Elsevier Inc. All rights reserved.
1. Introduction
and many efforts have been devoted to the study of the structure–
activity relationship of thiosemicarbazone derivatives. The anticancer
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 also the severe normal
tissue toxicity [1–5]. These disadvantages have driven the development
of improved platinum-based anticancer drugs whose structure and
mode of action differ from that of cisplatin, especially those that interact
with specific molecular targets as for example are the processes associ-
ated to DNA: transcription, replication and repair [6–9].
In this regard, of particular interest are compounds targeting ribonu-
cleotide reductase (RR), enzyme that catalyzes the reduction of ribonu-
cleotides to deoxyribonucleotides and provides the building blocks for
the de novo DNA synthesis in all living cells. Cancer cells require in-
creased RR activity to meet the demand for deoxyribonucleotides that
are needed to support their rapid proliferation. Thus inhibition of RR
activity leads to inhibition of DNA synthesis and repair, and also induces
cell cycle arrest and apoptosis [10–12].
activity of (N)-TSCs is closely related to the nature of the heterocyclic
ring of the parent aldehyde or ketone, metal chelation ability and termi-
nal amino substitution [13–20]. In this sense pyridine ring itself is a part
of many natural and synthetically prepared pharmaceuticals and more-
over it plays a significant role in many biological processes like nicotin-
amide adenine dinucleotide phosphate NADP or the important vitamins
niacin and pyridoxine (vitamins B3 and B6) [21,22].
Keeping in view the above observations and as part of our systematic
investigation on the coordination chemistry of thiosemicarbazone deriv-
atives we recently reported palladium(II) and platinum(II) complexes de-
rived of 2,6-diacetylpyridine bis(4N-o-tolylthiosemicarbazone) and 2,6-
diacetylpyridine bis(4N-p-tolylthiosemicarbazone) ligands. The in vitro
antitumor studies have shown that these complexes exhibit important
antiproliferative activity in A2780 and A2780cisR human cancer cell
lines and these results encouraged us to further investigate their cytotoxic
properties as well as those of novel derivatives [23].
This work is aimed to determine if the presence of an aryl ring with
an electron withdrawing substituent (such as p-chlorophenyl group)
results beneficial for the antitumor activity of bis(4N-substituted
thiosemicarbazones) ligands derived from 2,6-diacetylpyridine. There-
fore here we describe the synthesis and chemical characterization of the
new 2,6-diacetylpyridine bis(4N-p-chlorophenylthiosemicarbazone)
α-(N)-heterocyclic thiosemicarbazones, (N)-TSCs, have been
reported to be among the most effective RR inhibitors yet identified
⁎
Corresponding author. Tel.: +34 914973868; fax: +34 914974833.
E-mail address: ana.matesanz@uam.es (A.I. Matesanz).
http://dx.doi.org/10.1016/j.jinorgbio.2014.04.017
0162-0134/© 2014 Elsevier Inc. All rights reserved.

A.I. Matesanz et al. / Journal of Inorganic Biochemistry 138 (2014) 16–23
17
7.45 (d, J = 8.7 Hz, aromatic-thiosemicarbazide, 4H); 2.50 (s, CH3-
diacetylpyridine, 6H). 13C NMR (d6-DMSO, ppm), δ = 178.3 (C_S);
153.78 (C2,C6-pyridine); 149.85 (C_1N); 138.54 (C4-pyridine);
137.17 (C3,C5-pyridine); 130.08 (aromatic-thiosemicarbazide); 128.49
(aromatic-thiosemicarbazide); 128.34 (aromatic-thiosemicarbazide);
127.37 (aromatic-thiosemicarbazide); 121.96 (aromatic-thiosemicar-
bazide); 12.98 (CH3-diacetylpyridine). UV/VIS (DMSO): λ/nm 250, 337.
2.3.2. 2,6-Diacetylpyridine bis(4N-p-chlorophenylthiosemicarbazonato)
palladium(II), [PdL]
Scheme 1. Structure of 2,6-diacetylpyridine bis(4N-p-chlorophenylthiosemicarbazone),
H2L ligand.
The reaction of H2L ligand with PdCl2(PPh3)2, in toluene, in presence
of Et3N, in 1:1 molar ratios over 20 h at room temperature led to the
formation of an orange solution which was filtered and left to stand at
ambient temperature for two days. The brown solid formed was filtered,
washed several times with hot water, diethyl ether and finally dried in
vacuo.
Yield (55%), mp N 250 °C. Elemental analysis found, C, 43.35; H, 3.35,
N, 15.10; S, 10.05; C23H19N7S2Cl2Pd requires C, 43.51; H, 3.02, N, 15.44;
S, 10.10%. MS (FAB+ with mNBA matrix) m/z 636 for [PdL + H]+. IR (KBr
pellet): υ/cm−1 3243 (s, NH); 1595 (s, CN); 830, 804 (vw) (CS-
thioamide IV); 603 (pyridine ring). 1H NMR (300 MHz, d6-DMSO,
ppm), δ = 10.75, 10.18 [s, 4NH, 2H]; 8.43–8.10 [m, CH-pyridine, 3H];
7.71-7.34 (m, aromatic-thiosemicarbazide, 8H); 2.72, 2.62 (s, CH3-
diacetylpyridine, 6H). UV/VIS (DMSO): λ/nm 267, 340, 410, 470.
Recrystallization from DMSO led to the isolation of orange crystals of
[PdL] · DMSO that were suitable for X-ray-diffraction.
ligand, H2L, (Scheme 1) and its palladium(II) and platinum(II) complexes,
[PdL] and [PtL].
The cytotoxic activity of the new compounds synthesized and cis-
platin (assumed as the reference antitumor drug) against four human
cancer cell lines: NCI-H460 (non-small cell lung cancer), T-47D (breast
cancer), A2780 and A2780cisR (epithelial ovarian cancer) has been stud-
ied. The interaction of H2L with calf thymus DNA (CT-DNA) was also in-
vestigated and its binding constant (Kb) determined.
2. Experimental
2.1. Measurements
Elemental analyses were performed on a LECO CHNS-932 microana-
lyzer. Fast atom bombardment (FAB) mass spectra (MS) were performed
on a VG AutoSpec spectrometer. 1H NMR spectra were recorded on
Bruker AMX-300 spectrometer. All cited physical measurements were
obtained out 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 were recorded on a Bomen–Michelson
spectrophotometer. 13C NMR spectra were recorded on a 400 Advance
Bruker Fourier Transform spectrometer. Electronic spectra were
recorded on a Thermo Scientific Evolution 260 Bio UV–visible (UV–
vis) spectrophotometer.
2.3.3. 2,6-Diacetylpyridine bis(4N-p-chlorophenylthiosemicarbazonato)
platinum(II), [PtL]
It was prepared by the same procedure as described for [PdL] by
reaction of H2L with PtCl2(PPh3)2 and afforded a brown solid.
Yield (35%), mp 192 °C (decomposes). Elemental analysis found, C,
37.90; H, 2.55, N, 13.60; S, 8.60; C23H19N7S2Cl2Pt requires C, 38.18; H,
2.65, N, 13.55; S, 8.86%. MS (FAB+ with mNBA matrix) m/z 724 for
[PtL + H]+. IR (KBr pellet): υ/cm−1 3288, 3208 (s, NH); 1590 (s, CN);
826, 809 (vw) (CS-thioamide IV); 591 (pyridine ring). 1H NMR
(300 MHz, d6-DMSO, ppm), δ = 11.00, 10.30 [s, 4NH, 2H]; δ =
8.56–8.10 [m, CH-pyridine, 3H]; δ = 7.76–7.70 (m, aromatic-
thiosemicarbazide, 8H); δ = 2.78, 2.71 (s, CH3-diacetylpyridine,
6H). UV/VIS (DMSO): λ/nm 249, 267, 366.
2.2. Materials
Solvents were purified and dried according to standard procedures.
Hydrazine hydrate, 2,6-diacetylpyridine, p-chlorophenyl isothiocya-
nate, PdCl2(PPh3)2 and PtCl2(PPh3)2 were commercially available.
Recrystallization from DMSO led to the isolation of orange crystals of
[PtL] · DMSO that were suitable for X-ray-diffraction.
2.4. Crystallography
2.3. Synthesis of compounds
Data were collected on a Bruker X8 APEX II CCD. Crystallographic
data and selected interatomic distances and angles are listed in
Table 1. For all compounds, the software 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.
CCDC 981268 and 981269 contain the supplementary crystallo-
graphic data for compounds [PdL] and [PtL] 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].
2.3.1. 2,6-Diacetylpyridine bis(4N-p-chlorophenylthiosemicarbazone), H2L
An ethanolic solution of hydrazine hydrate (0.250 g, 5 mmol)
was added dropwise with constant stirring to an ethanolic solution
of p-chlorophenyl isothiocyanate (0.848 g, 5 mmol). The reaction
mixture was stirred for one more hour and then the white product
p-chlorophenylthiosemicarbazide formed was filtered, washed with
cold ethanol and diethyl ether, dried in vacuo and recrystallized from
ethanol. An ethanolic solution of the p-chlorophenylthiosemicarbazide
(0.402 g, 2 mmol) was then stirred with 2,6-diacetylpyridine (0.163 g,
1 mmol) for 5 h. The resulting solution was reduced to half volume
and the pale yellow solid formed was filtered, washed with ethanol,
diethyl ether and finally dried in vacuo.
Yield (80%), mp 220 °C (decomposes). Elemental analysis found, C,
52.20; H, 4.25; N, 18.00; S, 12.20; C23H21N7S2Cl2 requires C, 52.07; H,
3.99, N, 18.48; S, 12.09%. MS (FAB+ with mNBA: nitrobenzyl alcohol ma-
trix) m/z 530.0 for [H2L + H]+. IR (KBr pellet): υ/cm−1 3335, 3306, 3210
(w, NH); 1588 (s, CN); 827, 809 (w, CS-thioamide IV); 585 (pyridine
ring). 1H NMR (d6-DMSO, ppm), δ = 10.80 [s, 2NH, 2H]; 10.20 [s, 4NH,
2H]; 8.55 [d, J = 7.9 Hz, CH-pyridine, 2H]; 7.85 [t, J = 7.9 Hz, CH-
pyridine, 1H]; 7.60 (d, J = 8.7 Hz, aromatic-thiosemicarbazide, 4H);
2.5. In vitro antiproliferative activity
The human cancer cells: A2780 and A2780cisR (epithelial ovarian
cancer), T-47D (breast cancer) and NCI-H460 (non-small cell lung can-
cer); 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.

18
A.I. Matesanz et al. / Journal of Inorganic Biochemistry 138 (2014) 16–23
Table 1
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
experiments were carried out at the Unidad de Evaluación de Actividades
Farmacológicas de Compuestos Químicos (USEF), Universidad de Santia-
go de Compostela.
Crystal data and structure refinement for [PdL] · DMSO and [PtL] · DMSO.
[PdL] · DMSO
[PtL] · DMSO
Molecular formula
Formula weight
Temperature (K)
Wavelength (Å)
C25H25Cl2N7OPdS3
713.00
100(2)
C25H25Cl2N7OPtS3
801.69
100(2)
0.71073
0.71073
Crystal system
Space group
Triclinic
Pī
Triclinic
Pī
2.6. DNA-binding experiments
a(Å)
b(Å)
c(Å)
α/º
7.4630(5)
13.2509(7)
14.6594(9)
77.351(3)
87.513(3)
81.522(3)
1398.96(15)
2
7.484(3)
Calf thymus DNA (CT-DNA) stock solution was prepared by dissolv-
ing the lyophilized sodium salt in Tris-buffer (NaCl 50 mM, Tris–HCl
5 mM, pH was adjusted to 7.2 with NaOH 0.5 M) by stirring for 5 h.
The CT-DNA solution was standardized spectrophotometrically
[26] by using its known molar absorption coefficient at 260 nm
(6600 M−1 cm−1). The ratio of UV absorbance at 260 and 280 nm,
A260/A280, of ca. 1.9, indicating that the DNA was sufficiently free of
protein. Stock solution was kept frozen until the day of the experiment.
Concentrated stock solutions (5 × 10−3 M and 5 × 10−5 M) of H2L
were prepared dissolving the compound in DMSO. From these stock
solutions, for all experiments the desired concentration of compound
was achieved by dilution with Tris-buffer (NaCl 50 mM, Tris–HCl
5 mM, pH was adjusted to 7.2 with NaOH 0.5 M) to give homogeneous
solutions with DMSO content of less than 2.5%.
13.251(6)
14.680(6)
76.611(14)
87.296(15)
80.818(15)g
1398.0(10)
2
1.905
5.469
784
0.40 × 0.04 × 0.02
−8 ≤ h ≤ 8,
−15 ≤ k ≤ 15,
−16 ≤ l ≤ 17
15003
4672
4672/0/356
1.002
R1 = 0.0450,
wR2 = 0.1074
R1 = 0.0748,
wR2 = 0.1409
1.606 and −2.739
β/º
γ/º
Volume(Å3)
Z
Density (calculated) (g/cm3)
Absorption coefficient (mm−1
F(000)
1.693
1.113
720
)
Crystal size (mm3)
Index ranges
0.57 × 0.09 × 0.03
−8 ≤ h ≤ 8,
−15 ≤ k ≤ 15,
−17 ≤ l ≤ 17
47797
5086
5086/0/356
1.066
R1 = 0.0380,
wR2 = 0.1116
R1 = 0.0498,
wR2 = 0.1297
1.126 and −0.783
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
To investigate the binding mode, spectrophotometric titrations were
performed at a fixed DNA concentration equal to 1.7 × 10−4 M with
increasing concentration of H2L (0–125 μM) and monitoring the absor-
bance change at the wavelength maximum 260 nm after incubation
(10 min at 37 °C).
Final R indices [I N 2σ(I)]
R indices (all data)
Largest diff. peak and hole, e.Å−3
To calculate the binding parameters, the spectrophotometric
titrations were performed with increasing concentration of DNA
(0–40 μM) at a fixed compound concentration equal to 50 μM and
monitoring the absorbance change in one characteristic charge trans-
ference band of the compound after incubation (10 min 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), 4 × 103 (for A2780 and A2780cisR) or 5 × 103 (for T-47D) cells
per well with 100 μL of medium and were then incubated for 24 h
(A2780, A2780cisR and NCI-H460) or 48 h (T-47D). After attachment
to the culture surface the cells were incubated with various concentra-
tions of the compounds tested freshly dissolved in DMSO (1 mg/mL)
and diluted in the culture medium (DMSO final concentration 1%) for
48 h (for NCI-H460) or 96 h (for A2780, A2780cisR and T-47D). The
cells were fixed by adding 50 μL of 30% trichloroacetic acid (TCA) per
well.
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.
3. Results and discussion
3.1. Synthesis and spectroscopic characterization
A new 2,6-diacetylpyridine bis(4N-monosubstituted thiosemicarba-
zone) ligand has been synthesized with high purities and acceptable
yields. The yellow compound obtained is stable to air and moisture and
was characterized by elemental analysis, FAB+ spectrometry and IR and
NMR (1H and 13C) spectroscopy.
Reaction of 2,6-diacetylpyridine bis(4N-p-chlorophenylthiosemi-
carbazone) ligand with equimolar amount of MCl2(PPh3)2, where M =
Pd(II) or Pt(II), led to the isolation of neutral mononuclear complexes
[PdL] and [PtL] in which the bis(thiosemicarbazone) behaves as dianionic
ligand with deprotonation of hydrazine (2NH) protons and [NNNS] donor
set.
The effects of compounds were expressed as corrected percentage
inhibition values according to the following equation:
Both complexes were characterized by routine analytical and spec-
troscopic techniques. Analytical data are consistent with the formula-
tion given, thus the FAB+ mass spectra exhibited a very weak ion at
m/z = 636 for [PdL] and m/z = 724 for [PtL] which corresponded to
the predicted molecular weight of the [M + H]+ ions and moreover
the isotopic patterns of this signal fit well with the theoretical isotopic
distributions.
% 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 calculating
concentration–percentage inhibition curves, these curves were adjusted
to the following equation:
The significant IR vibrational bands and the 1H chemical shift values
of the free ligand and its complexes are listed in the Experimental sec-
tion and Scheme 1 shows the numbered structure of the free ligand.
As the X-ray study has shown, during metal complexation, the ligand
behaves as a tetradentate dianionic forming two five-membered and
one six-membered chelate rings around the metal center. The high
delocalization and the asymmetric coordination hinder the IR analysis.
The stretching vibration υ(C = N) and the in-plane pyridine deforma-
tion bands are slightly shifted to higher wavenumbers which are consis-
tent with the implication of imine and pyridine nitrogen atoms in the
Â
Ã
E ¼ Emax= 1 þ ðIC50=CÞn
where E is the percentage inhibition observed, Emax is the maximal
effects, 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].

A.I. Matesanz et al. / Journal of Inorganic Biochemistry 138 (2014) 16–23
19
13C NMR spectrum of the free ligand shows carbon signals supporting
the 1H NMR assignments however due to the low solubility of the com-
plexes it was not possible to get 13C NMR spectra of reasonable quality.
The electronic absorption spectra of both [H2L] ligand and [PdL] and
[PtL] complexes exhibit two intense band in the region 250–400 nm,
which can ascribed to ligand-centered n → π* and π → π* transitions.
In addition the spectra of metal complexes exhibit other less energetic
bands assigned to a ligand to metal (LMCT) and metal to ligand charge
transfer (MLCT) transitions [27].
Table 2
Selected bond distances (Å) and angles (°) for [PdL] · DMSO and [PtL] · DMSO.
[PdL] · DMSO
[PtL] · DMSO
Bond lengths (Å)
S(1)\C(8)
1.673(5)
1.779(4)
1.291(6)
1.389(6)
1.373(6)
1.406(6)
1.304(6)
1.297(6)
1.367(6)
1.402(6)
2.029(4)
2.044(4)
1.979(4)
2.2842(11)
S(1)\C(17)
S(2)\C(10)
C(2)\N(5)
C(8)\N(2)
C(10)\N(3)
C(10)\N(4)
C(11)\N(4)
C(17)\N(6)
C(17)\N(7)
C(23)\N(7)
Pt(1)\N(1)
Pt(1)\N(3)
Pt(1)\N(5)
Pt(1)\S(1)
1.804(10)
1.672(11)
1.299(14)
1.298(14)
1.424(14)
1.347(13)
1.421(13)
1.290(14)
1.348(14)
1.411(13)
2.017(9)
S(2)\C(17)
C(6)\N(2)
C(8)\N(3)
C(8)\N(4)
C(9)\N(4)
C(15)\N(5)
C(17)\N(6)
C(17)\N(7)
C(18)\N(7)
Pd(1)\N(1)
Pd(1)\N(3)
Pd(1)\N(5)
Pd(1)\S(2)
3.2. Description of the crystal structures
[PdL] · DMSO and [PtL] · DMSO were isolated as neutral compounds.
The most significant crystallographic data for these complexes are
shown in Table 1, whereas selected bond lengths and bond angles are
presented in Table 2.
2.032(9)
1.985(9)
2.279(3)
Bond angles (º)
The structures together with the atom labeling schemes are shown
in Figs. 1 and 2. Both compounds are isostructural hence displaying
nearly identical cell parameters, crystallize in the triclinic Pī space
group with Z = 2 and the asymmetric units contain one molecule of
the neutral complex and one dimethyl sulfoxide solvent molecule.
The metal ion presents a square planar geometry where the
bis(thiosemicarbazonate) ligand is coordinated to the metal ion
through the pyridine nitrogen atom and the azomethine nitrogen and
thione sulfur atoms from one thiosemicarbazone arm and being the
fourth coordination position occupied by the hydrazine nitrogen atom
of the other thiosemicarbazone arm generating two typical five mem-
bered (PdSCNN and PdNCCN or PtSCNN and PtNCCN) and one six
membered (PdNNCCN or PtNNCCN) chelate rings. Coordination by
hydrazine nitrogen atom instead of the azomethine nitrogen atom,
although uncommon, has been found in the bibliography for some d8
bis(thiosemicarbazone) complexes [23,28–31].
The M\N and M\S bond distances are similar to those found in
other palladium(II) and platinum(II) complexes. It is important to
note that the two thiosemicarbazone moieties, which are symmetrically
deprotonated, coordinate in a different fashion. 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 1.779(4) for
[PdL] · DMSO and 1.804(10) for [PtL] · DMSO while the monodentate-
thiosemicarbazone [N(3), hydrazine nitrogen atom] arm maintains
its thione form as reflects in their C\S bond length of 1.673(5) for
N(1)\Pd(1)\N(3)
N(1)\Pd(1)\N(5)
N(3)\Pd(1)\S(2)
N(5)\Pd(1)\S(2)
N(1)\Pd(1)\S(2)
N(3)\Pd(1)\N(5)
91.73(15)
80.58(15)
104.2(11)
83.43(11)
163.95(11)
172.25(15)
N(1)\Pt(1)\N(3)
N(1)\Pt(1)\N(5)
N(3)\Pt(1)\S(2)
N(5)\Pt(1)\S(2)
N(1)\Pt(1)\S(2)
N(3)\Pt(1)\N(5)
91.8(3)
80.5(4)
103.7(2)
83.0(3)
164.5(3)
172.2(4)
coordination however this induces only minor changes in υ(C_S)
thioamide IV band which decreases slightly in intensity.
In the 1H NMR spectrum of the double-armed H2L ligand, two
independent singlets at δ = 10.80 and 10.20 ppm are observed for
NC_N\2NH\ and \C(S)\4NH\ protons respectively. A doublet at
δ = 8.55 ppm and a triplet at δ = 7.85 ppm are assigned to pyridyl
ring protons. Aromatic p-chlorophenyl protons appear as a multiplet
at δ = 7.60–7.45 ppm and a sharp singlet at δ = 2.50 is assigned to
the methyl protons. In [PdL] and [PtL] complexes, the anionic coordina-
tion of the ligand is evidenced by the disappearance of the signal
corresponding to the NC_N\2NH\ protons. On the other hand, the
\C(S)\4NH\ protons appear two independent signals (10.75 and
10.18 ppm for [PdL] and 11.00 and 10.30 ppm for [PtL]) due to the
asymmetric coordination. The pyridyl ring protons appear as a multiplet
at δ = 8.43–8.10 ppm for [PdL] and δ = 8.56–8.10 ppm for [PtL] and
the rest of the proton signals appear at nearly identical positions if
they are compared with the corresponding ligand signals.
Fig. 1. Molecular structure of [PdL]. The displacement ellipsoids are drawn at the 50% probability.

20
A.I. Matesanz et al. / Journal of Inorganic Biochemistry 138 (2014) 16–23
Fig. 2. Molecular structure of [PtL]. The displacement ellipsoids are drawn at the 50% probability.
[PdL] · DMSO and 1.672(3) Å for [PtL] · DMSO, typical of double C_S
bond.
cancers. For comparison purposes, the cytotoxicity of cisplatin was al-
ways evaluated under the same experimental conditions.
Comparison of C\N and N\N bond distances with typical lengths of
single and double bonds [C\N 1.47, C_N 1.28, N\N 1.45, N_N 1.25 Å]
suggests extensive charge delocalization over the thiosemicarbazone
moieties [32,33] and also agrees with the thiolate tautomeric form of
the bidentate thiosemicarbazonate arm and the thione tautomeric
form of the monodentate one (Scheme 2).
Table 3 shows that both the p-chlorophenyl substituted free ligand
H2L and its platinum(II) complex [PtL] present important antiprolifera-
tive activity in the low-micromolar range, against ovarian (A2780, cis-
platin sensitive, and A2780cisR, cisplatin resistant) and breast (T-47D)
cancer cells. It is remarkable to note that both compounds exhibit better
cytotoxic effects against T-47D cells than cisplatin by comparing their
IC50 values.
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 being the bond
angles between adjacent coordinating atoms in the 80.5–104.2° range.
The crystal structures are stabilized by intermolecular hydrogen inter-
actions involving the N(7) atom of the bidentate thiosemicarbazonate
arms and the oxygen atom of DMSO solvent molecule being the
N(7)\H(7)⋯O(1) contact distance 2.83 Å for both complexes and
b(NHO) angle 170.4° for [PdL] and 169.2° for [PtL]. Further stabilization
of the crystal packing is provided by intermolecular π–π stacking inter-
actions involving the whole planar bis(thiosemicarbazone)-metal skel-
eton (Fig. 3), with an interplane separation about ≈3.5 Å.
The A2780cisR cell line encompasses all of the known major mecha-
nisms of resistance to cisplatin: reduced drug transport, enhanced DNA
repair/tolerance, and elevated GSH levels. The ability of H2L and [PtL]
compounds to circumvent cisplatin-acquired resistance was confirmed
from the resistance factor values, RF (defined as IC50 in A2780cisR/IC50
in A2780) since both have a much better RF value than cisplatin. An
RF value of b2 was considered to denote non-cross-resistance and
therefore these compounds are able to circumvent cisplatin resistance
[34,35].
From a chemical point of view, analysis of these data together with
those of our previous study [23] in which the related ligand 2,6-
diacetylpyridine bis(4N-p-tolylthiosemicarbazone), H2L2, resulted
inactive in both A2780 and A2780cisR cell lines and its [PtL2] com-
plex showed a slightly lower antiproliferative activity than [PtL] complex
(see Table 3) suggests that the presence of one electron withdrawing
group attached to the 4N atom of the thiosemicarbazone moiety results
beneficial for the antiproliferative activity of the bis(thiosemicarbazone)
ligands of the 2,6-diacetylpyridine series.
3.3. In vitro antiproliferative activity
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 cells lines containing examples of
lung (NCI-H460), breast (T-47D) and ovarian (A2780 and A2780cisR)
Scheme 2. Delocalization system in the thiosemicarbazonate moiety.

A.I. Matesanz et al. / Journal of Inorganic Biochemistry 138 (2014) 16–23
21
Fig. 3. Crystal packing view of [PdL] · DMSO along a axis, dashed lines denote π⋯π interactions.
3.4. DNA interaction studies
the change in the absorption intensity of the typical CT-DNA spectral
band at 260 nm. As Fig. 4 shows, when the concentration of H2L is
gradually increased a significant increase in absorption of the
DNA band occurs being the percentage of hyperchromism observed
[% hyperchromism = (ADNA bound − ADNA free) / ADNA bound] about
40%.
In order to initially address if any direct interaction with DNA is part
of the mechanism of action of the compounds, UV–visible absorption
spectra in absence and presence of CT-DNA were carried out for H2L
ligand, which have a significant effect against the tested cell lines.
These characteristics suggest non-covalent surface (major or
minor groove) binding along outside of DNA helix. The above obser-
vations are comparable to those reported earlier for various neutral
bis(thiosemicarbazone) palladium and platinum complexes [37,38].
The H2L ligand exhibit, in DMSO:Tris buffer (2.5:100) mixture, one
broad intense band of intraligand π–π* transition at 250 nm and other
less intense of intraligand n–π* transition at 337 nm and any interaction
with DNA could perturb it.
Thus, in order to determine the intrinsic binding constant (Kb), ab-
sorption titration experiments were performed by maintaining a constant
H2L concentration (50 μM) while gradually increasing the concentration
of DNA (0–40 μM) and monitoring the change in the absorption intensity
of the intraligand charge transfer band. While measuring the absorption
spectra, an equal amount of DNA was added to both the test solution
and the reference solution to eliminate the absorbance of DNA itself.
The data were then fitted to the following equation, that is only valid
for low compound:DNA ratios (i.e., far from the DNA saturation) and as-
sumes no binding cooperativity [39]:
3.4.1. Absorption spectral studies
The binding affinity between DNA and H2L ligand can be detected by
UV–vis absorption spectroscopy by measuring the changes in the
absorption properties of: a) DNA (for a variable H2L concentration) or
b) H2L ligand molecule (for a variable DNA concentration).
The UV–vis absorption spectrum of the typical β-form DNA exhibits
a characteristic π → π* band at 260 nm as consequence of the chromo-
phoric groups in purine and pyrimidine moieties. Compounds binding
with DNA through intercalation are consistent with hypochromism
(decrease in DNA band absorption), resulted of a stacking interaction
between the aromatic ligand chromophore and the base pair of DNA.
In case of compounds binding with DNA through external contact
(including groove binding and electrostatic attraction) usually
hyperchromism (increase in DNA band absorption) is observed
which is attributable of a contraction and overall damage of the
secondary structure of DNA [36].
Thus, the absorption spectrum of CT-DNA in presence of H2L was
recorded, by keeping constant CT-DNA concentration (1.7 × 10−4 M)
in diverse [CT-DNA]/[H2L] mixing ratios (R = 0.5–1.5) and monitoring
½DNAꢀ=ðεa−εf Þ ¼ ½DNAꢀ=ðεb−εf Þ þ 1=fKbðεb−εf Þg
Table 3
In vitro antiproliferative activity of H2L, [PdL] and [PtL] complexes and H2L2, [PtL2] and cisplatin, evaluated in human T-47D (breast cancer), A2780 and A2780cisR (epithelial ovarian can-
cer) cell lines.
IC50
SD (μM)
A2780
A2780cisR
T-47D
RF IC50(A2780cisR)/IC50(A2780)
H2L
7.16
24
7.12
N100a
20
0.14
0.21
13
1
7.42
N100
9.17
ND
ND
12
0.14
0.07
1.8
N 4.2
1.3
–
[PdL]
[PtL]
1
N100
9.13
N100a
18
0.07
H2L2
[PtL2]
Cisplatin
2a
0.01
1a
0.10
1.1
8.8
0.88
7.77
1
The IC50 values are averages of two independent determinations.
ND = not determined.
a
Values taken from Ref. [23].

22
A.I. Matesanz et al. / Journal of Inorganic Biochemistry 138 (2014) 16–23
Wavelength (nm)
Fig. 4. UV absorption spectrum of CT-DNA in the absence (black curve) and presence of increasing amounts of compound H2L. The data were collected for [CT-DNA] = 1.7 × 10−4 M and
[H2L] = 0, 2.5 × 10−6, 6.0 × 10−5, 8.0 × 10−5, 1.25 × 10−4 M (the arrow shows the changes upon increasing amounts of complex).
where [DNA] is the concentration of the nucleic acid in base pairs, εa is the
apparent absorption coefficient obtained by calculating Aobs/[compound],
and εf and εb are the absorption coefficients of the free and the fully
bound compound, respectively.
A plot (Fig. 5) of [DNA] / (εb − εf) versus [DNA], gives a slope
of 1 / (εb − εf) and a Y-intercept equal to 1 / {Kb(εb − εf)}. The intrinsic
binding constant Kb is calculated as the ratio of the slope to the
Y-intercept.
On titration of CT-DNA a slight increase in the absorptivity of this
band is observed which is indicative of interaction between the
electronic states of the ligand chromophore with that of DNA bases.
The magnitude of intrinsic binding constant was calculated to be
7.03 × 104 M−1 (correlation coefficient R2 = 0.99) which is modest,
however it should be kept in mind that the biological activity of α-
(N)-heterocyclic thiosemicarbazones is not only due to their non-
covalent DNA binding but they are also potent inhibitors of DNA synthesis
100
80
60
40
20
0
0,5
0,45
0,4
15
20
25
30
35
0,35
0,3
[DNA]x106 (M)
0,25
0,2
0,15
0,1
240
260
280
300
320
340
360
380
400
Wavelength (nm)
Fig. 5. UV absorption spectrum of H2L in the absence (black curve) and presence of increasing amounts of compound CT-DNA. The data were collected for [H2L] = 5 × 10−5 M and
[CT-DNA] = 0, 20 × 10−6, 60 × 10−6, 70 × 10−6, and 75 × 10−6 M. The insert shows a fitting of the absorbance data used to obtain the binding constants.

A.I. Matesanz et al. / Journal of Inorganic Biochemistry 138 (2014) 16–23
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an electron withdrawing substituent (p-chlorophenyl group) has been
successfully prepared and characterized.
This study has identified both the free ligand H2L and the Pt(II) com-
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also exhibit high antiproliferative activity against breast (T-47D) cancer
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Appendix A. Supplementary data
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