In vitro and in vivo antitumor activity of platinum(II) complexes with thiosemicarbazones derived from 2-formyl and 2-acetyl pyridine and containing ring incorporated at N(4)-position: Synthesis, spectroscopic study and crystal structure of platinum(II) c

Article abstract of DOI:10.1016/j.ejmech.2008.08.007

Reactions of thiosemicarbazones of 2-formyl and 2-acetyl pyridine and containing an azepane ring (hexamethyleneiminyl ring) incorporated at N(4)-position, HL¹ (1) and HL² (2) with platinum(II) afforded the complexes, [Pt(L¹

Full text of DOI:10.1016/j.ejmech.2008.08.007

European Journal of Medicinal Chemistry 44 (2009) 1296–1302
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
In vitro and in vivo antitumor activity of platinum(II) complexes with
thiosemicarbazones derived from 2-formyl and 2-acetyl pyridine and
containing ring incorporated at N(4)-position: Synthesis, spectroscopic
study and crystal structure of platinum(II) complexes with thiosemicarbazones,
potential anticancer agents
,
b
a
Dimitra Kovala-Demertzi a , Athanassios Papageorgiou , Leuteris Papathanasis ,
*
Alexandros Alexandratos a, Panagiotis Dalezis b, John R. Miller c, Mavroudis A. Demertzis a
,
*
a Inorganic and Analytical Chemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece
b Symeonidio Research Center, Theagenio Cancer Hospital, Thessaloniki, Greece
c Department of Biological and Chemical Sciences, University of Essex, Colchester CO4 3SQ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 March 2008
Received in revised form 12 August 2008
Accepted 12 August 2008
Available online 31 August 2008
Reactions of thiosemicarbazones of 2-formyl and 2-acetyl pyridine and containing an azepane ring
(hexamethyleneiminyl ring) incorporated at N(4)-position, HL1 (1) and HL2 (2) with platinum(II) afforded
the complexes, [Pt(L1)Cl] (3) and [Pt(L2)Cl] (4). Characterization of the compounds was accomplished by
means of elemental analysis and spectroscopic techniques NMR, UV–vis and IR spectroscopy. The single-
crystal X-ray structure of complex [Pt(L2)Cl] (4) shows that the ligand monoanion coordinates in a planar
conformation to the metal via the pyridyl N atom, the imine-N atom, and thiolato S-atom. Compounds
1–4 have been evaluated for antiproliferative activity in vitro against three human cancer cell lines:
MCF-7 (human breast cancer cell line), T24 (bladder cancer cell line), A-549 (non-small cell lung carci-
noma) and a mouse L-929 (a fibroblast-like cell line cloned from strain L). Ligand 2 exhibited high activity
as anticancer agent against all four cancer cell lines, while ligand 1 exhibited selectivity against MCF-7,
L-929 cell lines and complex 4 against A-549, T-24 cancer cell lines. Also, the acute toxicity and antitumor
activity were evaluated on leukemia P388-bearing mice. Complex 3 afforded five to six cures against
leukemia P388. The in vivo results of the antitumor activity show the two platinum complexes as very
effective chemotherapeutic antileukemic agents.
Dedicated to Professor D.X. West for his
contribution to Chemistry and the Chem-
istry of Thiosemicarbazones.
Keywords:
Platinum(II) complexes
Thiosemicarbazonato complexes
Crystal structure
In vitro and in vivo antitumor activity
Biological evaluation
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
coordination. Firstly, lipophilicity, which controls the rate of entry
into the cell, is modified by coordination and some side effects may
Thiosemicarbazones are among the most potent inhibitors of
ribonucleotide reductase, RR, activity and possess a wide range of
biological activity depending on the parent aldehyde or ketone.
Heterocyclic thiosemicarbazones (TSCs) have aroused considerable
interest in chemistry and biology due to their antibacterial, anti-
malarial, antineoplastic and antiviral activities and represent an
important series of compounds because of potentially beneficial,
biological activity [1].
The chemistry of transition metal complexes of thio-
semicarbazone, TSCs, has been receiving considerable attention
largely because their broad profile of pharmacological activity
affords a diverse variety of compounds with different activities
[2,3]. The biological properties of TSCs are often related to metal ion
decrease upon complexation. In addition, the complex can exhibit
bioactivities which are not shown by the free ligand [4,5]. The
thiosemicarbazone, HL2, has been screened against HSV-1, HSV-2
and leukemia P388 because of its potential for medicinal use [6].
A complex of Pd(II) with HL2 was found to exhibit significant
reduction of toxicity, enhancement of cytogenic damage and
a significant increase of survival time of the drug-treated leukemia
bearing mice (T/C % 166) [14]. The combination of TSCs with agents
like platinum(II) and palladium(II) that damage DNA may lead
to improvements in the effectiveness of cancer chemotherapy
regimens.
It was thought of interest to explore the platinum chemistry of
TSCs, HL1 andHL2, asa continuation of ourstudiesonTSCs and inorder
to widen the scope of investigations on the coordination behaviour of
TSCs, towardsplatinum(II), we carried out systematic studies with the
final goal to develop new biologically active pharmaceuticals.
* Corresponding authors. Fax: þ30651 98786.
E-mail address: dkovala@cc.uoi.gr (D. Kovala-Demertzi).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.08.007

D. Kovala-Demertzi et al. / European Journal of Medicinal Chemistry 44 (2009) 1296–1302
1297
Here, we report the synthesis and chemical characterization of
new platinum(II) complexes. The single-crystal X-ray structure of
complex [Pt(L2)Cl] (4) and the electronic, IR, UV–vis, and NMR
spectroscopic data of the two complexes are reported. The results
of the cytotoxic activity of 1–4 have been evaluated for anti-
proliferative activity in vitro against cancer cell lines: MCF-7, T24,
A-549 and a mouse L-929. Also, the acute toxicity and antitumor
activity were evaluated on leukemia P388-bearing mice.
frequencies are as follows: v(Pt–Nimine), 420–430 cmꢀ1; v(Pt–Cl),
338– 346; v(Pt–S), 400–370 cmꢀ1; and v(Pt–Npy), 320–300 cmꢀ1
.
2.2.2. NMR spectroscopy
In the 1H NMR spectra of the ligands 1 and 2, H–N(3) resonated
at 15.00–13.00 ppm, indicating that this H-atom is involved in
H-bonding [7]. This resonance was absent in the spectra of the
corresponding complexes, which indicated deprotonation of
H–N(3) and, thus, anionic thiosemicarbazonato moieties. In the 1H
NMR spectra of the Pt(II) complexes 3 and 4, H–C(1) was downfield
shifted upon coordination, indicating decreased electron density at
position 1. The pyridyl hydrogens are shifted upon coordination
which indicated variations in the electron density in the pyridyl
ring. The C]S resonances of the thiosemicarbazone moieties in the
free ligands resonated at 183.0 ppm. In the complexes, downfield
shifts were observed, indicating decreased electron density from
the thiolato C-atoms [8–10].
2. Results and discussion
2.1. Chemistry
The two thiosemicarbazones HL1, 1 and HL2, 2, were prepared
according to the method described in the literature [7,16]. Trans-
amination of 4-methyl-4-phenyl-3-thiosemicarbazide (A) in the
presence of azepane and the requisite aldehyde or ketone gives the
two ligands HL1 and HL2, Scheme 1 [7,16]. To synthesize complexes
3 and 4, HL1 and HL2 were added to an aqueous solution of
Na2[PtCl4], and the mixture was stirred for 24 h, Scheme 2. The
structures were confirmed by, IR, UV–vis, 1H and 13C NMR spec-
troscopy and X-ray diffraction data for 4.
2.3. X-ray crystallography
Crystal data and experimental details are listed in Table 1.
Interatomic distances and bond angles are compiled in Table 2. The
crystal structure of compound 4 is shown in Fig. 1. The anionic L2
acts as a tridentate ligand which coordinates to platinum(II)
through the pyridyl nitrogen atom, N(1), the azomethine nitrogen
atom, N(2) and the thiolato sulfur atom. The tridentate ligand
shows a Z, E, Z configuration for the donor centres nitrogen,
nitrogen and sulfur atoms, respectively. The stronger coordination
of the metal to the azomethine nitrogen atom compared to the
pyridyl nitrogen atom is attributed to its higher basicity of N(2). The
negative charge of the monoanionic ligand is delocalized over the
L2 moiety and the S–C bond distance is consistent with increased
single bond character, while the imine C–N distances and both
thioamide C–N distances indicate considerable double bond char-
acter. The tridentate ligand is nearly planar. The displacement from
co-planarity is indicated by the dihedral angle between the pyridyl
2.2. Spectral studies
2.2.1. Infrared spectroscopy
Coordination of the azomethine nitrogen atom to platinum(II) is
suggested by the shift of the
n(C]N) band to lower frequencies along
with the occurrence of the (N–N) band to higher frequency in the IR
n
spectra of the complexes compared to that in the ligand [7–11]. The
breathing motion of the pyridine ring is shifted to a higher frequency
upon complexation and is consistent with pyridine ring nitrogen
atom coordination. The thioamide band, which contains consider-
able v(CS) character, is less intense in the complexes and is found at
a lower frequency, suggesting coordination of the metal through
sulfuratom afterdeprotonation. The platinum donoratom stretching
Scheme 1. The reaction scheme for synthesis of ligands 1 and 2.

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D. Kovala-Demertzi et al. / European Journal of Medicinal Chemistry 44 (2009) 1296–1302
Table 2
Selected bond lengths (Å) and angles (ꢁ) for 4
Pt–Cl
Pt–S
2.310(2)
2.257(2)
2.047(5)
1.942(2
1.767(6)
1.342(8)
1.353(7)
N(2)–N(3)
N(2)–C(6)
N(3)–C(8)
N(4)–C(8)
N(4)–C(9)
N(4)–C(14)
1.385(7)
1.307(7)
1.322(7)
1.346(7)
1.448(8)
1.471(9)
Pt–N(1)
Pt–N(2)
S–C(8)
N(1)–C(1)
N(1)–C(5)
Cl–Pt–S
S–Pt–N(1)
Cl–Pt–N(2)
97.20(7)
165.64(14)
175.96(15)
Cl–Pt–N(1)
S–Pt–N(2)
N(1)–Pt–N(2)
97.15(14)
85.40(15)
80.30(19)
HAc4Et were tested in a panel of human tumor cell lines of different
origins (breast, colon, and ovary cancers), and cisplatin-refractory/
resistant cell lines and were found to exhibit very remarkable growth
inhibitory activities with mean IC50 values of 0.9–0.5 nM and support
the hypothesis that both [Pt(Ac4Et)2] and [Pd(Ac4Et)2] complexes can
be characterized by cellular pharmacological properties distinctly
different from those of cisplatin [13].
Compounds 1–4 were tested for their antiproliferative activity in
vitro against the cells of three human cancer cell lines: MCF-7
(human breast cancer cell line), T24 (bladder cancer cell line),
A-549 (non-small cell lung carcinoma) and a mouse fibroblast
L-929 cell line. The results of cytotoxic activity in vitro are expressed
Scheme 2. The reaction scheme for synthesis of platinum(II) complexes 3 and 4.
ring and the plane defined by the five-membered chelate ring
Pt–N(2)–C(6)-C(5)–N(1), and between the pyridyl ring and the
plane defined by Pt–S–C(8)–N(3)–N(2), 1.4(1) and 2.3(7), respec-
tively. The dihedral angle between the pyridyl ring and the azepane
ring (hexamethyleneiminyl ring) is 68.2(9). There is a weak inter-
action between the metal atom and the C(7), the methyl carbon,
and its attached hydrogen, H(7C), the metal–hydrogen distance
being 3.04 Å.
The crystal packing is determined by Pt–C, Pt–
interactions, as shown in Fig. 2.The monomers are arranged in
dimers, in a head to tail fashion and are connected by strong
interactions. The most significant double interaction is that
p and p–p,
as IC50 – the concentration of compound (in
mM) that inhibits
p–p
a proliferation rate of the tumor cells by 50% as compared to control
untreated cells, Table 3.
p–p
between the pair of rings Pt–N(2)–C(6)–C(5)–N(1) and N(1)–C(1)–
C(2)–C(3)–C(4)–C(5), (Cg2)–Cg(3) and Cg(3)–Cg(2), respectively;
symmetry operation: 2 ꢀ x, ꢀy, 1 ꢀ z} at a centre to centre separa-
tion of 3.517(4) Å.
The ligand 2 is in the same
mM range compared to cisplatin
against the four cell lines, less cytotoxic against L-929 and A-549
cell lines and more cytotoxic against MCF-7 and T-24 cancer cell
lines. Ligand 2 is 3.5 and 1.6 times more active than cisplatin against
the T24 and MCF-7 cell lines, respectively. The platinum complex 4
is less cytotoxic against MCF-7 and L-929 cell lines and in the same
2.4. Pharmacology: antiproliferative activity in vitro
mM range compared to cisplatin and the parent ligand against T-24
Platinum(II) complexes of N4-ethyl 2-formyl and 2-acetyl pyridine
thiosemicarbazones showed cytotoxicity and were found to be able
to overcome the cisplatin resistance of A2780/Cp8 cells [12]. Pt(II) or
Pd(II) complexes with 2-acetyl pyridine N(4)-ethyl-thiosemicarbazone,
and A-549 cancer cell lines.
The IC50 values for 1 against A-549 and T-24 cell lines are 27.31
and 74.32
mM, respectively, and against MCF-7 and L-929 cell lines
are 2.23 and 2.27
mM, respectively. Ligand 1 is 3.6 more active than
cisplatin against MCF-7 cell line and 3.8 times less active than
cisplatin against L-929 cell line.
Selectivity was exhibited from the platinum(II) complex, 4,
which was found active against A-549 and T-24 cancer cell lines.
From the in vitro experimental results, it is indicated that the two
ligands HL1 and HL2 are more active than their corresponding
Table 1
Crystal data and structure refinement for 4
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C14H19ClN4PtS
505.93
291(2) K
0.71073 Å
Monoclinic
P21/n
Unit cell dimensions
a ¼ 7.258(4) Å
a
¼ 90ꢁ
b ¼ 24.869(5) Å,
b
¼ 104.27(2)ꢁ
c ¼ 9.150(4) Å,
1600.7(11) Å3
4
g
¼ 90ꢁ
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
2.099 Mg/m3
9.061 mmꢀ1
968
Crystal size
Theta range for data collection
Index ranges
Reflections collected
Independent reflections
Completeness to theta ¼ 26.01ꢁ
Absorption correction
Refinement method
Data/restraints/parameters
Goodness-of-fit
0.20 ꢂ 0.25 ꢂ 0.75 mm
1.64–26.01ꢁ
0 ꢃ h ꢃ 8, 0 ꢃ k ꢃ 30, ꢀ10 ꢃ l ꢃ 11
3376
3215 [Rint ¼ 0.025]
99.4%
Refined empirical
Full matrix least squares on F2
3.125/0/192
1.079
Final R indices [I > 1.5
R indices (all data)
Extinction coefficient
s
(I)]
R1 ¼0.0292, wR2 ¼ 0.0823
R1 ¼0.0416, wR2 ¼ 0.0879
0.0043(3)
Largest difference peak and hole
1.307 and ꢀ0.884 e Åꢀ3
Fig. 1. Perspective view of 4.

D. Kovala-Demertzi et al. / European Journal of Medicinal Chemistry 44 (2009) 1296–1302
1299
Table 3
The in vitro activity of 1–4 (expressed as IC50
L-929 cancer cell lines
(m
M)) against MCF-7, T-24, A-549 and
Compounds
MCF-7
T-24
L-929
A-549
HL1 (1)
2.23 ꢄ 0.31
5.02 ꢄ 0.10
20.30 ꢄ 2.10
25.70 ꢄ 3.12
8.00 ꢄ 0.50
74.32 ꢄ 6.70
11.80 ꢄ 0.12
19.83 ꢄ 2.11
12.83 ꢄ 0.09
41.7 ꢄ 2.10
2.67 ꢄ 0.22
3.04 ꢄ 0.34
37.70 ꢄ 3.10
77.10 ꢄ 5.10
0.70 ꢄ 0.05
27.31 ꢄ 0.10
6.55 ꢄ 0.21
89.30 ꢄ 6.10
6.92 ꢄ 0.35
1.53 ꢄ 0.15
HL2 (2)
[Pt(L1)Cl] (3)
[Pt(L2)Cl] (4)
Cisplatin
causes marked differences on the biological results a significant
increase of survival time of the drug- treated leukemia bearing
mice, T/C % 147 and 118 for HL1 and HL2, respectively. Probably the
Pt(II) complexes, [Pt(L1)Cl], 3 and [Pt(L2)Cl], 4, may interact better
with DNA and/or proteins than the other compounds. In conclu-
sion, these experiments showed that the activity of the metal
complexes 3 and 4 is different compared with the activity of the
ligands alone and probably represent independent cytotoxic
entities.
Complex 4 was proved to be the most potent antileukemic
agent, comparing the T/C % value and almost non-toxic, producing
low level of toxicity. It is important to remark that complex 3 gave
a high T/C % value of 298 and afforded additionally five of six cures
in leukemia P388. The results of their in vivo biological tests showed
that platinum(II) complexes 3 and 4 proved less toxic than the free
ligands 1 and 2, while their antileukemic potency was significantly
improved. The most potent compound
4 had significantly
decreased toxicity and compound 3 exhibited impressive potency
(5/6 cures against leukemia P388). The in vivo results of the anti-
tumor activity show the two platinum complexes as very effective
chemotherapeutics, antileukemic agents.
These compounds 1–4 may be considered as polyfunctional
molecules, possessing an azepane ring, theiosemicarbazonato
portion, N–N–CS–N and a pyridyl ring. Obviously, these groups are
capable of hydrophobic, azepane ring (hexamethyleneiminyl ring),
hydrogen bonding (thiosemicarbazonato portion, N–N–CS–N) and
p–p interactions (pyridyl ring) with the corresponding bio-
receptor. The molecules reach to the receptor microenvironment
Fig. 2. (a) Packing diagram of 4 viewed along
a and (b) c axis.
Table 4
platinum complexes, which indicates that the mechanism of action
is different.
Ligand 2 exhibited high activity as anticancer agent against all
four cancer cell lines, while ligand 1 exhibited selectivity against
MCF-7, L-929 cell lines and the complex 4 against A-549, T-24
cancer cell lines.
Acute toxicity and antitumor activity of of 1–4 and related TSCs and Pd(II) complexes
on leukemia P388-bearing BDF1
MST ꢄ SDb (days)
T/Cb (%)
Cures
a
a
Compound
LD50
LD10
Controls
–
Saline
40
10.00 ꢄ 0.41
15.30 ꢄ 0.47
12.30 ꢄ 0.44
31.00 ꢄ 0.51
43.40 ꢄ 0.49
10 90 þ 0.19
11.25 ꢄ 0.21
9.40 ꢄ 0.22
10.80 ꢄ 0.25
11.50 ꢄ 0.19
13.00 ꢄ 0.25
100
147
118
298*
417
106
129
111
127
132
149
166
138
0/6
0/6
0/6
5/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
HL1 (1)
63
60
80
107
25
HL2 (2)
37
53
76
10
20
50
40
38
[Pt(L1)Cl] (3)
[Pt(L2)Cl] (4)
HAcTscc
2.5. In vivo antitumor experiments
HFoTscc
The acute toxicity and antitumor activity were evaluated on
leukemia P388-bearing mice for compounds 1–4. The acute toxicity
and the antitumor activity of these compounds were correlated
with the related complexes of 2-formyl pyridine and 2-acetyl
pyridine thiosemicarbazone, HFoTsc and HAcTsc, respectively
[5,14,15].
The ligands 1 and 2 cause acute toxicity and display some
antitumor activity, while the complexes 3 and 4 show reduction of
the toxicity, and very high increase of survival time of the drug-
treated leukemia bearing mice, Table 4. The ligand 2 is achieving
a T/C % value of 118 and the Pt(II) complex, 4, of 417. The ligand 1 is
achieving a T/C % value of 147 and the Pt(II) complex, 3, of 298. Also,
five of the six mice were cured and were considered as long-term
survivors. The long-term survivors are defined as mice alive 90 days
after tumor inoculation. The replacement of a methyl group at
position C(7) (HL2) by a C(7)H group (HL1) in the 2-pyridyl position
[Pd(AcTsc)Cl]c
[Pd(AcTsc)2]c
[Pd(FoTsc)Cl]c
[Pd(FoTsc)2]c
[Pd(L2)2]d
Cisplatine
80
65
150
30
37
22.60 ꢄ 6.5
a
LD50 and LD10 (dosage mg/g body wt)values were estimated graphically: percent
deaths due to the toxicity of each dose are shown on the ordinate, while the
administered doses are indicated on the abscissa on semi-logarithmic paper. LD50
and LD10 ¼ lethal doses for 50 and 10%, respectively, of the mice used (10 animals per
dose).
b
LD10 was given as a single dose on the first day after tumor transplantation for
antitumor evaluation. MST, mean survival time; T/C %, median survival time of the
drug-treated animals versus corn oil-treated controls (C).
c
Where HFoTsc and HAcTsc is the related pyridine-2-carbaldehyde and 2-acetyl
pyridine Tsc, respectively [14,15].
d
Ref. [14].
Ref. [28]; dose 3 mg/kg.
e

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D. Kovala-Demertzi et al. / European Journal of Medicinal Chemistry 44 (2009) 1296–1302
and then must interact with its receptor. The receptor may have
a lipophilic pocket, a hydrogen bonding surface and a system to
was refluxed for 15 min and then left standing in a refrigerator for
1 h. The resulting yellow precipitate was filtered off, washed with
cold CH3CN, recrystallized from CH3CN and dried in vacuo over
silica gel, and then at 40 ꢁC in vacuo over P4O10 for 3 h [7,16]; m.p.
159 ꢁC. Yield 50%. Elemental analysis calcd for C14H20N4S: C, 60.9;
H, 7.2; N, 20.3; S, 11.6. Found: C, 61.7; H, 7.6; N, 20.7; S, 11.7; m.p.
p
interact with the lipophilic, hydrogen bonding moieties and pyri-
dine ring of compounds 1–4. The goal of reducing toxicity while
maintaining therapeutic efficiency can be accomplished by
improving the solubility of the complexes, by slowing down
degradation processes through shielding of the platinum with
bulky ligands, and by increasing membrane permeability with
more lipophilic ligands. In complexes 3 and 4 it is anticipated that
extra lipophilicity and extra bulk to shield the platinum are both
introduced by the azepane ring and the molecular shape of
compounds.
160 ꢁC. Yield 43%. IR (KBr): 1583 (C]N), 1014
n(N–N), 880 n(C]S),
594 15.07 (s, 1H, NH), 8.79 (d,
n
(py) cmꢀ1 1H NMR (DMSO-d6):
;
d
J ¼ 5.0 Hz, H1), 7.58 (t, J ¼ 9.2 Hz, H2), 7.80 (t, J ¼ 7.7 Hz, H3), 7.99 (d,
J ¼ 7.7 Hz, H4), 2.60 (s, CH3); 13C NMR (DMSO-d6):
d 148.6(C1),
124.1(C2), 136.5 (3), 121.8(C4), 152.8(C5),146.7(C6), 13.0 (CH3),
182.8(C8).
3. Experimental section
3.2.3. [Pt(L1)Cl] (3)
To a solution of 1 (0.55 mmol) in methanol (10 ml) was added
the stock solution of Na2[PtCl4] (15 mL, 0.6 mmol). The reaction
mixture was stirred for 3 days at 24 ꢁC and then left at 3–4 ꢁC for 1
day. The dark-red powder was filtered off, recrystallized from hot
methanol and washed with cold methanol and ether, dried in vacuo
over silica gel, and re-dried at 70 ꢁC in vacuo over P4O10. Colour
dark-red and m.p. 230 ꢁC (with decomposition). Yield 70%.
Elemental analyses calcd for C13H17N4SPtCl: C, 31.8; H, 3.5; N, 11.4;
3.1. Measurements
Solvents were purified and dried according to standard proce-
dures. The two ligands were prepared according to the method
described in the literature [7,16]. [Pd(FoTsc)Cl], [Pd(FoTsc)2],
[Pd(AcTsc)Cl] and [Pd(AcTsc)2] (where HFoTsc and HAcTsc is pyri-
dine-2-carbaldehyde and 2-acetyl pyridine thiosemicarbazone,
respectively) were prepared according to Refs. [17,18]. [Pd(L2)2] was
prepared according to Ref. [19]. For the platinum(II) compounds
a stock 0.04 M solution of Na2[PtCl4] was prepared by dissolving
PtCl2 (2.66 g, 10 mmol) in conc. HCl under reflux, filtering to
remove a turbidity of undissolved material, neutralizing with
Na2CO3 and diluting with distilled water up to 250 ml (pH ¼ 6.0–
6.5). Infrared and far-infrared spectra were recorded on a Perkin–
Elmer Spectrum GX FT-IR System spectrophotometer using KBr
pellets (4000–400 cmꢀ1) and nujol mulls dispersed between
polyethylene disks (400–40 cmꢀ1). NMR experiments were per-
S, 6.5. Found: C, 31.8; H, 2.6; N, 10.9; S, 7.0. %. FT-IR (KBr):
1595; (N–N), 1025; (C]S), 767, 757 (py), 618, 583; v(Pt–N), 422;
v(Pt–Cl), 346; v(Pt–S), 372; v(Pt–Npy), 318 cmꢀ1 1H NMR (DMSO-
n(C]N),
n
n
d
;
d6):
d
8.89 (d, J ¼ 5.0 Hz, H1), 7.43 (t, J ¼ 3.7 Hz, H2), 8.02 (t,
J ¼ 2.5 Hz, H3), 7.73 (d, J ¼ 7.5 Hz, H4), 8.56 (s, H6); 13C NMR (DMSO-
d6):
d 159.8(C1), 129.4(C2), 139.8(C3), 124.0(C4), 147.5(C5),
145.4(C6), 187.5(C7),
3.2.4. [Pt(L2)Cl] (4)
A solution of 2 (0.55 mmol) in methanol (10 ml) was added to
the stock solution of Na2[PtCl4] [(30 mL, 0.6 mmol). The reaction
mixture was stirred for 3 days at 24 ꢁC and then left at 3–4 ꢁC for 1
day. The orange powder was filtered off, washed with cold meth-
anol and ether, dried in vacuo over silica gel, and re-dried at 70 ꢁC in
vacuo over P4O10. Yield 30%; m.p. 285 ꢁC (with decomposition).
Elemental analyses calcd for C14H19N4SPtCl: C, 33.2; H, 4.0; N,11.1; S
formed using
a Bru¨ker AMX-400 MHz NMR spectrometer.
Elemental analyses, C, H, N and S were performed on a Carlo Erba
EA (model 1108).
3.2. Syntheses of TSCs and platinum(II) complexes
3.2.1. (E)-N0-(pyridin-2-ylmethylene)azepane-1-
6.3. Found: C, 31.1; H, 4.0; N, 11.1; S, 6.3%. IR (KBr):
(N–N), 1023; v(C]S), 770; (py), 619; v(Pt–Cl), 338; v(Pt–N), 429;
v(Pt–S), 419; v(Pt–Npy), 301 cmꢀ1 1H NMR (DMSO-d6):
9.05 (d,
n(C]N), 1599;
carbothiohydrazide, HL1 (1)
n
d
4-Methyl-4-phenyl-3-thiosemicarbazide was prepared accord-
ing to the method described by Scovil [16]. The crude product,
4-methyl-4-phenyl-3-thiosemicarbazide, was recrystallized from
a mixture of EtOH and distilled water (3:1). To a solution of
4-methyl-4-phenyl-3-thiosemicarbazide (1.000 g, 5.52 mmol) in
CH3CN (1.50 ml), azepane (0.63 mL, 5.52 mmol) and 2-formyl
pyridine (0.55 mL, 5.52 mmol) were added. The mixture was
refluxed for 45 min and then left standing in a refrigerator for 12 h.
The resulting light-orange precipitate was filtered off, washed with
cold CH3CN, recrystallized from CH3CN and dried in vacuo over
silica gel, and then at 40 ꢁC in vacuo over P4O10 for 3 h. [7,16]; m.p.
109 ꢁC. Yield 53%. Elemental analysis calcd for C13H18N4S: C, 59.5;
H, 6.9; N, 21.4; S 12.2. Found: C, 60.5; H, 5.4; N, 21.7; S, 12.4%. m.p.
;
d
J ¼ 5.2 Hz, H1), 7.41 (t, J ¼ 6.7 Hz, H2), 7.93 (d, J ¼ 7.7 Hz, H3), 8.90
(d, J ¼ 7.0 H4), 2.32 (s, CH3); 13C NMR (DMSO-d6):
d 147.7(C1),
124.8(C2), 139.2(C3), 122.9(C4), 152.9(C5), 149.9(C6),184.1(C8).
Suitable crystals for X-ray study were obtained by crystallization
from a fresh solution of DMF.
3.3. X-ray crystallography
A crystal of dimensions 0.75 ꢂ 0.25 ꢂ 0.20 mm. was glued to
a glass fiber and mounted on the head of an Enraf–Nonius CAD4
diffractometer. Graphite-monochromated Mo K
radiation was used. The unit cell was found to be monoclinic from
analysis of 25 centred reflections in the range 23.9ꢁ < < 26ꢁ. Space
group P21/n was assigned, with (Mo K
) 9.061 mmꢀ1, d(calc)
2.099 g cmꢀ3
temperature 291(1) K; 3376 reflections were
collected in
mode in the range 1.6ꢁ < < 26.0ꢁ. Three standard
a (l 0.71073 Å)
108 ꢁC. Yield 53.0%. IR (KBr): 1591
n
(C]N), 1002
n
(N–N), 879
q
n
(C]S), 580
n
(py) cmꢀ1 1H NMR (DMSO-d6):
;
d
13.38 (s, 1H, NH),
m
a
8.56 (d, J ¼ 5.0 Hz, H1), 7.35 (t, J ¼ 2.5 Hz, H2), 7.60 (t, J ¼ 9.9 Hz, H3),
,
7.45 (d, J ¼ 7.5 Hz, H4), 7.85 (s, H6); 13C NMR (DMSO-d6):
u
-2
q
q
d
144.3(C1), 129.4(C2), 137.4(C3), 128.4(C4), 152.2(C5), 146.7(C6),
reflections were collected every 2 h. Decay of 28% ca. was observed
and corrected during processing [17]. Lorentz and polarisation
effects were corrected. An empirical absorption correction was
183.1(C7).
3.2.2. (E)-N0-(1-(pyridin-2-yl)ethylidene)azepane-1-
carbothiohydrazide, HL2 (2)
made using
j
-scans of 9 reflections (Tmax ¼ 0.0181, Tmin ¼ 0.0075).
The structure was solved by the Patterson heavy atom method and
successive cycles of full matrix refinement and Fourier difference
syntheses [18]. An empirical absorption correction was made using
the program ‘‘DIFABS’’ [19]. All non-hydrogen atoms were refined
anisotropically on F by full matrix least squares. Hydrogen atoms
Compound 2 was prepared as described in literature [7,16]. To
a
solution of 4-methyl-4-phenyl-3-thiosemicarbazide (1.000 g,
5.52 mmol) in CH3CN (5 mL), azepane (0.63 ml, 5.52 mmol) and
2-acetyl pyidine (0.61 mL, 5.52 mmol) were added. The mixture

D. Kovala-Demertzi et al. / European Journal of Medicinal Chemistry 44 (2009) 1296–1302
1301
were placed in calculated positions and used in structure factor
calculations but not refined. Molecular graphics were performed
from PLATON2001 [20,21].
Each compound in given concentration was tested in triplicates
in each experiment, which was repeated three times.
Crystallographic data for the structure reported in this paper
have been deposited with the Cambridge Crystallographic Data
Centre as supplementary publication no. CCDC-21939. Copies of the
data can be obtained free of charge on application to CCDC, 12
Union Road, Cambridge CB2 1EZ, UK (fax: (þ44)1223-336-033;
e-mail: deposit@ccdc.cam.ac.uk).
3.4.2. In vivo antitumor experiments
Mice. BALB/c, DBA/2 and BDF1 mice of both sexes, weighting 20–
23 g, 6–8 weeks old were used for toxicity studies and antitumor
evaluation. Mice obtained from the experimental section of the
Research Center of Theagenion Anticancer Hospital, Thessaloniki,
Greece, were kept under conditions of constant temperature and
humidity, in sterile cages, with water and food. Leukemia P388-
bearing BDF1 (DBA/2$C57BL) mice were used to evaluate the
cytostatic effect. Lymphocytic P388 leukemias were maintained in
ascitic form by injection of 106 cell at 6-day intervals, into the
peritoneal cavity of DBA/2 mice.
3.4. Biological experiments
3.4.1. Antiproliferative assay in vitro
Compounds. Test solutions of the compounds tested (1 mg/ml)
Compounds. For intraperitoneal (ip) treatment, stock solutions of
the compounds used in this study were prepared immediately
before use. They were suspended in corn oil in the desired
concentration following initial dissolution in 10% dimethylsulf-
oxide (DMSO). This concentration by itself produced no observable
toxic effects
Estimation of acute toxicity. The acute toxicity of the compounds
was determined following a single ip injection into BDF1 mice in
groups of 10 mice per dose at three different dosages. The mice
were observed for 30 days and the therapeutic dose of the
compounds was determined after graphical estimation of the LD50
(30-day curves). The dose used for a single treatment was equal to
the LD10.
Antileukemic evaluation. For the survival experiments, the anti-
leukemic activity of the tested compounds against Leukemia P388
was assessed from the oncostatic parameter T/C %, that is, the
increase in median life span of the drug-treated animals (T)
excluding long-term survivors versus corn oil-treated controls (C)
was expressed as a percentage. The other index of the antileukemic
activity used was the number of long-term survivors defined as
mice alive for 90 days after tumor inoculation. Each drug-treated
group consisted of six mice while the tumor control group included
eight mice; in each group, equal numbers of male and female mice
were used. Experiments were initiated by implanting mice with
tumor cells according to the protocol of the National Cancer Insti-
tute, USA [27]. The experiments were terminated on day 90.
Statistical evaluation of the experimental data was made by the
Wilcoxon test.
were prepared by dissolving the substance in 100
completed with 900 l of tissue culture medium. Afterwards, the
tested compounds were diluted in culture medium to reach the
final concentrations of 100, 40, 10, 1, and 0.1 ng/ l. The solvent
ml of DMSO
m
m
(DMSO) in the highest concentration used in test did not reveal any
cytotoxic activity.
Cells. The established in vitro cancer cell lines are maintained in
the Cell Culture Collection of the University of Ioannina. Twenty-
four hours before addition of the tested agents, the cells were
plated in 96-well plates at a density of 104 cells per well. The MCF-7
and T24 cells were cultured in the D-MEM (Modified Eagle’s
Medium) medium supplemented with 1% antibiotic and 10% fetal
calf serum. L-929 cells were grown in Hepes-buffered RPMI 1640
medium supplemented with 10% fetal calf serum, penicillin (50 U/
ml) and streptomycin (50 mg/ml). A-549 cells were grown in F-12 K
Ham’s medium supplemented with 1% glutamine, 1% antibiotic/
antimycotic, 2% NaHCO3 and 10% fetal calf serum. The cell cultures
were maintained at 37 ꢁC in a humid atmosphere saturated with 5%
CO2. Cell number was counted by the Trypan blue dye exclusion
method. MCF-7, L-929 and A-549 cells were determined by the
sulforhodamine
[10,25,26].
B assay, while T24 cells by the MTT assay
SRB assay. The details of this technique were described by
Skekhan et al. [22]. The cytotoxicity assay was performed after
72-hour exposure of the cultured cells to varying concentrations
(from 0.1 to 100 ng/ml) of the tested agents. The cells attached to the
plastic were fixed by gently layering cold 50% trichloroacetic acid
(TCA) on the top of the culture medium in each well. The plates
were incubated at 4 ꢁC for 1 h and then washed five times with tap
water. The background optical density was measured in the wells
filled with culture medium, without the cells. The cellular material
fixed with TCA was stained for 30 min with 0.4% sulforhodamine B
dissolved in 1% acetic acid. Unbound dye was removed by rinsing
with 1% acetic acid. The protein-bound dye was extracted with
10 mM unbuffered Tris base for determination of optical density (at
540 nm) in a computer-interfaced, 96-well microtiter plate.
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