Platinum(II) complexes with thiourea derivatives containing oxygen, sulfur or selenium in a heterocyclic ring: Computational studies and cytotoxic properties

Article abstract of DOI:10.1007/s11243-010-9375-9

Platinum(II) complexes with 1-(2-oxolanylmethyl)-2-thiourea, 1-(2-thiolanylmethyl)-2-thiourea and 1-(2-selenolanylmethyl)-2-thiourea were synthesized in order to compare their cytotoxic activities with those of the free thioureas. Their equilibrium geometries and bonding energies in the gas phase and in solution were calculated using density functional theory at the MPW1PW/LanL2DZ level. The IR spectra of the complexes and their free ligands were compared. Stability of the complexes in 0.9% saline aqueous solution was tested by means of HPLC. The cytotoxic activities of the species against five human cell lines were evaluated, as well as the antimicrobial activities against twelve bacterial strains. Springer Science+Business Media B.V. 2010.

Full text of DOI:10.1007/s11243-010-9375-9

Transition Met Chem (2010) 35:639–647
DOI 10.1007/s11243-010-9375-9
Platinum(II) complexes with thiourea derivatives containing
oxygen, sulfur or selenium in a heterocyclic ring: computational
studies and cytotoxic properties
•
L. Fuks E. Anuszewska H. Kruszewska
•
•
•
A. Krowczynski J. Dudek N. Sadlej-Sosnowska
•
´
´
Received: 4 December 2009 / Accepted: 18 May 2010 / Published online: 3 June 2010
Ó Springer Science+Business Media B.V. 2010
Abstract Platinum(II) complexes with 1-(2-oxolanylm-
ethyl)-2-thiourea, 1-(2-thiolanylmethyl)-2-thiourea and 1-
(2-selenolanylmethyl)-2-thiourea were synthesized in order
to compare their cytotoxic activities with those of the free
thioureas. Their equilibrium geometries and bonding
energies in the gas phase and in solution were calculated
using density functional theory at the MPW1PW/LanL2DZ
level. The IR spectra of the complexes and their free
ligands were compared. Stability of the complexes in 0.9%
saline aqueous solution was tested by means of HPLC. The
cytotoxic activities of the species against five human cell
lines were evaluated, as well as the antimicrobial activities
against twelve bacterial strains.
where platinum binds primarily to the adjacent guanine-N7
sites [1–5], thereby preventing cell division and growth.
The most frequently used platinum-containing drugs are
cisplatin and carboplatin (first-generation drugs) or oxa-
liplatin, proplatin and spiroplatin (second generation).
Although they are extremely effective, cancer cells quickly
become resistant to them. Therefore, the search for new
platinum drugs has focused on identifying compounds with
improved tolerability profiles and, in particular, on those
that may overcome the mechanisms of resistance.
Recently, it has been shown that when irradiated even in
small nuclear reactors like the scientific reactor TRIGA,
platinum(II) compounds contain detectable amounts of the
193mPt and 195mPt radionuclides [6]. Both radionuclides
decay by electron capture, so compounds containing these
radionuclides may also be useful for the Auger Electron
Therapy (AET) of cancers.
Introduction
Platinum-based drugs are among the most active anticancer
agents available. They are used for the treatment of a
variety of human solid tumors and form the largest class of
anticancer drugs. They destroy malignant cells by inter-
fering with the DNA, via inter- and intrastrand crosslinks,
In the last two decades, interest in platinum(II) com-
plexes with chelating ligands containing both nitrogen and
sulfur donor atoms has increased, because these complexes
can exhibit either higher anticancer activity or reduced
toxicity in comparison with the known metal-containing
drugs [7–11]. As a result of these studies, a number of
novel Pt(II) complexes sufficiently interesting for clinical
trials have been synthesized. However, only a few of them
are superior to the parent drug in their efficacy [12]. Here,
mention should be made of platinum(II) complexes with
certain thiourea derivatives (in particular containing an
acridine fragment, called ACRAMTUs [13, 14]), which are
potential pharmaceutical agents. Their therapeutic action
differs, however, from that established already for cisplatin
because they interact with DNA in the intercalative mode.
In the same vein, the main objective of this study was to
synthesize and characterize new platinum complexes as
possible chemotherapeutic agents.
L. Fuks (&) Á J. Dudek
Institute of Nuclear Chemistry and Technology, Dorodna 16,
03-195 Warsaw, Poland
e-mail: leon.ichtj@gmail.com
E. Anuszewska Á H. Kruszewska Á N. Sadlej-Sosnowska (&)
National Medicines Institute, Chelmska 30/34,
00-725 Warsaw, Poland
e-mail: sadlej@il.waw.pl
´
´
A. Krowczynski
Department of Chemistry, Warsaw University, Pasteura 1,
02-093 Warsaw, Poland
123

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Transition Met Chem (2010) 35:639–647
In living cells, there is competition for Pt between
and their thio analogs have already been compared in
several papers. For example, it was found that three thio
analogs of Cl-IB-MECA, an agonist at the human A3
adenosine receptor, exhibited higher binding affinities than
Cl-IB-MECA itself [17]. It has also been reported that
2-acetyl-S-octadecyl-rac-1-thioglycero-3-phosphocholine, a
thio analog of a known platelet-activating factor, showed
lower platelet-aggregating potency compared with the
parent ether compound [18]. However, to our knowledge,
no series of compounds containing three consecutive
chalcogens has been investigated to date, and the present
study is thus devoted to this aim.
binding to guanine-N7 and to S-donor ligands. The toxicity
of the Pt complexes is thought to be related to protein
binding, in particular via reactions with thiol groups [15].
Thus, preventing these thiol-Pt interactions should reduce
side effects and so increase the share of the drug that may
react with the guanine-N7 target. One effective way of
perturbing the Pt-amino acid interactions is additional
complexation of the Pt-atom with S-donor ligands, which
are relatively weakly bound to Pt, for example thioethers.
Previous studies of competition for nucleobases and such
weak Pt-containing compounds with S-donor ligands have
shown that easy transfer of Pt from a thioether ligand (but
not from thiolates) to guanine-N7 occurs [15]. Recent
theoretical calculations and experimental competitive
studies showed that platinum forms a weaker bond with the
thioether group of methionine than with the thiol group of
cysteine [16]. These findings resulted in combined che-
motherapeutic procedures consisting of the application of
various Pt(II) complexes together with compounds con-
taining S-donor groups (in this case called chemoprotec-
tants or rescue agents), which have been tested with the
aim of reducing the platinum-based side effects. It can be
hypothesized that the higher anticancer activity or reduced
toxicity of platinum(II) complexes with chelating ligands
containing both nitrogen and sulfur donor atoms is due to
the above-mentioned mechanism.
Experimental
All chemicals and solvents were purchased from Sigma-
Aldrich and used without further purification unless
otherwise noted. 1-(2-Oxolanylmethyl)-2-thiourea (L1),
1-(2-thiolanylmethyl)-2-thiourea (L2), and 1-(2-selenola-
nylmethyl)-2-thiourea (L3) were prepared by the proce-
dures given below.
Preparation of L1
1-(2-Oxolanylmethyl)-2-thiourea was synthesized accord-
ing to the procedure described in Ref. [19]. A mixture of
tetrahydrofurfurylamine (2-oxolanylmethylamine) (7 g,
69 mmol), triethylamine (10 mL, 7.3 g, 72 mmol) and
dioxane (10 mL, 117 mmol) was cooled to -10 °C and
treated with 4.2 mL (70 mmol) of carbon disulfide. The
solution was allowed to warm to room temperature, chilled
again to -10 °C and treated dropwise with 7.5 mL of
ethylchloroformate. After filtering out the precipitated
triethylamine hydrochloride, a solution of 10 mL of trieth-
ylamine in 20 mL of chloroform was added to the filtrate.
When the copious production of carbon oxysulfide had
ceased (after about 5 min at room temperature), the solution
was distilled in vacuo (159–162 °C/32 mm Hg) to furnish
4 g of the product, tetrahydrofurfuryl isothiocyanate.
The compound was stirred with 20 mL of concentrated
NH3 overnight. The precipitate of 1-(2-oxolanylmethyl)-2-
thiourea was recrystallized from water. Colorless crystals of
the product were obtained, yield 4 g (25 mmol, 36%).
A question arises as to whether substitution of the
S-donor groups in the Pt-containing complexes by Se-
donor groups could result in better (or worse) therapeutic
effects. We were especially interested in answering the
question whether substitution of an ether O atom by S
or Se in a chelating ligand could result in greater cyto-
toxicity. To this end, we synthesized the compounds
shown in Scheme 1: 1-(2-oxolanylmethyl)-2-thiourea (L1),
1-(2-thiolanylmethyl)-2-thiourea (L2) and 1-(2-selenola-
nylmethyl)-2-thiourea (L3), and their Pt complexes. The
equilibrium geometries of the complexes were investigated
by quantum theoretical methods, and their cytotoxicities,
the main objective of the present study, were tested on five
cell lines.
As mentioned above, it would be interesting to compare
biological features of a series of compounds containing one
of the three consecutive chalcogens in the five-membered
ring of the ligand, i.e. oxygen, sulfur or selenium. The
biological properties of the oxygen-containing compounds
Preparation of L2
2-Thiolanylmethylamine was synthesized as described in
Ref. [20]. The final product was prepared by a modified
method applied for the preparation of n-butyl isothiocya-
nate [19]: A mixture of 2-thiolanylmethylamine (1.23 g,
10.5 mmol), triethylamine (1 mL, 0.73 g, 7.2 mmol) and
2 mL of dioxane was chilled to -10 °C and treated with
Scheme 1 Thiourea ligands used in this study
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Transition Met Chem (2010) 35:639–647
641
carbon disulfide (0.5 mL, 0.63 g, 8.3 mmol). The solution
was allowed to warm to room temperature, chilled and
treated dropwise with ethyl chlorocarbonate (0.75 mL,
0.85 g, 8 mmol). Then, a solution of triethylamine (1 mL,
7.2 mmol) in chloroform (4 mL) was added at room tem-
perature. After filtering off the precipitate of triethylamine
hydrochloride, the solution was distilled in vacuo (170 °C /
302 mm Hg) to furnish 1 g (6.3 mmol) of the isothiocya-
nate derivative (yield 60%). This was stirred with 5 mL of
concentrated NH3 overnight. The product was recrystal-
lized from water: the colorless crystals (about 240 mg) had
a m.p. of 72 °C. The filtrate was evaporated to dryness and
chromatographed by means of TLC. An additional 340 mg
of the product (L2) was obtained as colorless crystals.
Total yield: 580 mg (3.3 mmol, 54%).
electrospray ionization. All spectra were recorded in the
positive ion mode. The platinum complexes obtained are
labeled Pt(LX)Cl2, (where X stands for 1, 2 or 3, respec-
tively). Numerous attempts to obtain crystals of the com-
plexes suitable for X-ray diffraction studies have failed.
The complexes were insoluble in the organic solvents
commonly used in crystallography, except DMSO. Once
dissolved in the latter, the complexes could be further
diluted in water.
Elemental analyses for C, H, N, S and Cl were per-
formed in the Laboratory of Analytical Chemistry,
Chemical Department (Warsaw Technical University). The
platinum content was established by atomic absorption
spectroscopy (AAS) in the Analytical Department of the
Institute of Nuclear Chemistry and Technology. Analytical
data are presented in Table 1.
Preparation of L3
The compound was synthesized in a similar way to L2 from
2-selenolanylphenylamine. Synthesis of the amine has not
previously been described. It was obtained in a similar way
to 2-methylselenolane [21]: NaBH4 (4.6 g, 121 mmol) was
gradually added (under N2 flux) to 150 mL of an ethanolic
suspension of selenium powder (4.8 g, 61 mmol). After Se
dissolution, 2,5-dichloropentylamine hydrochloride (11.6 g,
60 mmol) was added, and the solution was boiled for 3 h.
Then, ethanol was evaporated off, 200 mL of water was
added and the product was extracted with two 50-mL ali-
quots of diethyl ether. The ether solution was dried with
K2CO3. The product, after evaporation of the ether, was
distilled under vacuum (b.p. 110–120 °C / 302 mm Hg). The
distillate containing the amine was used further without
purification. Colorless crystals of L3 were obtained. Yield
12.0 g (54 mmol, 90%).
Quantum calculations
All calculations were carried out using Gaussian 03 revi-
sion B04 [22]. The ground state of the neutral complexes
was a closed-shell singlet because large ligand field values
lead to low-spin d8 complexes of platinum(II) having tetra-
coordinated planar geometry [23]. The geometry optimi-
zations of the ligands and their complexes were performed
with the MPW1PW91 functional and LanL2DZ basis set,
taking into account relativistic effects [24]. It has already
been shown that calculations performed at the DFT level
give a reasonable description of the structural features of
platinum(II) complexes with thiourea [25]. In addition,
when structures of the Pt(II) and Pt(IV) complexes with
histamine were simulated by using over 20 DFT func-
tionals, the X-ray and theoretical results were sufficiently
consistent for three of them, including the MPW1PW91
functional applied in this study [26].
The purity of the compounds L1–L3 was monitored by
HPLC analysis under gradient conditions.
The bonding energy of the complexes was obtained as
the difference between the energy of a given complex and
the energies of the appropriate free ligand and PtCl2. The
energies in aqueous solution were calculated using the
dielectric polarizable continuum model (DPCM), belong-
ing to the self-consistent reaction field (SCRF) models,
together with the united atom topological model for Har-
tree–Fock (UAHF) atomic radii [27], since this method
worked relatively well in calculations of acidic dissociation
constants of a set of molecules [28].
Preparation of the platinum(II) complexes
The title complexes were prepared by the following general
synthetic procedure [22]:
A solution of the appropriate ligand (0.5 mM) in MeOH
(1 mL) was added dropwise at room temperature to an
aqueous solution of K2PtCl4 (0.5 mM; 1 mL) until a molar
ratio of about 1:1 was achieved. The yellow or salmon pink
solid products, which started to precipitate immediately
after adding the first drop, were filtered off, washed several
times with fresh aliquots of warm water and dried in a
vacuum desiccator until a constant mass was reached. The
molecular weights of the Pt complexes with ligands L1 and
L2 were determined by mass spectrometry, courtesy of the
Institute of Inorganic Chemistry, Zurich University, on a
Merck Hitachi M-8000 HPLC/MS spectrometer with
HPLC analysis and spectroscopy
The chromatography was performed using a Merck L7100
chromatograph equipped with an analytical Supelcosil LC-
C18 column and a Shimadzu SPD-10AVP UV-Vis detector.
The purity of the free ligands was checked by the following
123

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Transition Met Chem (2010) 35:639–647
Table 1 Chemical composition, molecular mass and formulae of the compounds
Compounds/formulae
of the ligands
Chemical compositiona
Molecular mass
Color
C
H
N
S
Cl
Pt
Cl:Pt
–
L1 / C6H12N2OS
Pt(L1)Cl2
45.0
7.5
17.5
–
–
–
160
White
Yellow
White
Pink
(45.0)
17.5
(7.6)
2.6
(17.5)
6.6
–
12.8
(12.7)
–
(160.2)
427
34.8
(34.7)
–
2.02
–
(16.9)
41.0
(2.8)
6.8
(6.6)
15.6
(15.9)
6.3
(426.3)
176
L2 / C6H12N2S2
Pt(L2)Cl2
36.0
(40.9)
16.9
(6.9)
2.9
(36.4)
15.3
–
–
(176.3)
442
15.9
(16.1)
–
43.9
(44.1)
–
1.99
–
(16.3)
32.8
(2.7)
5.5
(6.3)
12.6
(12.5)
5.8
(14.5)
14.1
(442.4)
224
L3 / C6H12N2SSe
Pt(L3)Cl2
White
Pink
(32.3)
15.4
(5.4)
2.8
(14.4)
6.9
–
–
(223.2)
489
14.3
(14.5)
39.8
(39.9)
2.03
(14.7)
(2.5)
(5.7)
(6.5)
(489.2)
a
Found—normal text; calculated—in italics
gradient elution: 0–20 min: 20 to 80% A; 20–30 min: 80%
A (where solvent A consisted of acetonitrile with 0.1%
trifluoroacetic acid and solvent B of water with 0.1%
trifluoroacetic acid). The flow rate was 1 mL/min. UV
detection was carried out at 250 nm. Studies of the stability
of the complexes as a function of time were performed by
isocratic elution with a mixture of water and acetonitrile,
vol/vol = 80:20. The UV detector was set at 230 nm.
Infrared spectra were recorded with 1-cm-1 resolution
in the region of 4,000–400 cm-1 on a BRUKER Equinox
55Ò Fourier transform spectrophotometer. The spectra
were recorded as KBr pellets.
Medicines Institute. Cells were grown in MEM supple-
mented with 10% fetal calf serum and antibiotics.
The following microorganisms were obtained from the
ATCC: Enterococcus hirae ATCC 10541, Stenotropho-
monas maltophilia ATCC 12714, Escherichia coli ATCC
10538 and ATCC 8739, Bordetella bronchiseptica ATCC
4616, Bacillus pumilus ATCC 14884, Staphylococcus
epidermidis ATCC 12228, Staphylococcus aureus ATCC
6538P, Pseudomonas aeruginosa ATCC 9027 and ATCC
15442, Candida albicans ATCC 10231 and Aspergillus
brasiliensis 16404. Wild-type Staphylococcus aureus
strains were isolated from clinical material: MRSA 11 from
blood and MRSA 2 from nasal fluid.
Biological studies
Amounts of the complexes equivalent to 12.5 25, 50 or
100 lM were successively dissolved in 0.25 mL of DMSO
and mixed with 4.75 mL of a Mueller-Hinton 2 Agar at
45 °C to obtain solutions with a concentration of 2.5, 5, 10
or 20 mM/mL. Then, 2 lL of a particular cell suspension
of optical density 0.5 unit on the McFarland scale were
applied to the surface of the agar. The lowest concentration
of a tested compound that totally inhibited the growth of
the examined strain was chosen as the MIC value.
The cytotoxicities of the complexes were estimated in vitro
by the MTT1 assay. The complexes were dissolved in
DMSO and diluted in minimum essential medium (MEM)
to the required concentrations; the final DMSO concen-
tration did not exceed 1%. Five human cell lines: normal
(WS1) and neoplastic (ME18, ME18/R, HeLa, KB-V1),
sensitive (ME18, HeLa) and resistant to cytostatics (ME18/
R, KB-V1), were tested. The IC50 values were determined
in three independent experiments as the concentration of
the complexes required for 50% inhibition of cell growth.
Cell lines: WS1, HeLa and KB-V1 were obtained from
the American Type Culture Collection (ATCC), ME18 cell
line was a gift from the Institute of Oncology, Warsaw, and
ME18/R line was obtained experimentally by the National
Results and discussion
Structures of the free ligands and complexes
and ligand-bonding energies
As expected, the main differences in the structures of the
free ligands occur within the five-membered ring that
includes the oxygen, sulfur, or selenium heterocyclic
atoms. Both theoretical [29] and experimental data [30]
prove that the conformation of the free tetrahydrofuran ring
1
Test for measuring cell growth with colorimetric assay using yellow
colorimetric indicator: MTT (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide.
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Transition Met Chem (2010) 35:639–647
643
Fig. 1 Structure of minimum
energy calculated for L2 (left)
compared with that found
experimentally for L2 (right;
thermal ellipsoids are drawn at
the 30% probability level
Table 2 Calculated bond lengths and angles in the free ligands
(Scheme 1) at the MPW1PW91/LanL2DZ level
(L1), X=O (L2), X=S (L3), X=Se
˚
Bond lengths (A)
C(2)–S(1)
1.732
1.370
1.357
1.478
1.466
1.537
1.543
1.533
1.731
1.368
1.363
1.907
1.895
1.536
1.539
1.534
1.730
1.368
1.364
2.024
2.013
1.536
1.539
1.534
C(2)–N(3)
C(L2)–N(4)
C(11)–X(14)
C(18)–X(14)
C(13)–C(11)
C(13)–C(17)
C(17)–C(18)
Angles (°)
N(3)–C(2)–N(4)
N(3)–C(2)–S(1)
C(13)–C(11)–X(14)
C(11)–X(14)–C(18)
115.62
120.46
105.78
109.82
115.36
120.36
105.01
93.03
115.47
120.47
104.78
89.43
Scheme 2 Postulated coordination modes of 1-(2-oxolanylmethyl)-
2-thiourea (L1) with the PtCl2 synthon
were taken into consideration for complexes containing
ligands L2 and L3, respectively. The corresponding com-
plexes with L2 are denoted as Pt-(L2-I), Pt-(L2-II), and
Pt-(L2-III), while those with L3 are denoted as Pt-(L3-I),
Pt-(L3-II), and Pt-(L3-III).
Dihedral C11–C13–C17–C18 35.05
48.84
52.07
is an envelope structure with a dihedral angle u (calculated
for the four carbon atoms) of 52.5°. In the tetrahydrofuran
ring, which is a fragment of L1, the ring structure is
modified. Namely, it takes on a twisted conformation
characterized by the coplanarity of three adjacent ring
atoms (C11–O–C18) and the midpoint between the oppo-
site (C13–C17) bond. The conformation of the five-mem-
bered rings in L2 and L3 is similar to that in L1. The
calculated structure of L2 when compared with the
experimental structure obtained by X-ray diffraction is
shown in Fig. 1. The corresponding bond lengths and
angles in free ligands are shown in Table 2.
The structures shown in Scheme 2 assume that the
ligands remain in the thione tautomers. This is most
probably true in the gas phase as well as in the solid state
structures of the complexes. This hypothesis is supported
by the calculations of the relative energies of the thiol and
thione forms of the ligands and of three forms of the
complexes. The thiol tautomers are higher in energy by
tens of kcal/mol. Bonding energies calculated in the gas
phase for the thione tautomers of the complexes are shown
in Table 3, and their geometric parameters are displayed in
Table 4.
Given that the analytically determined Pt : Cl molar
ratio in the platinum(II) complexes is close to 1:2
(Table 1), we searched for the most probable structure of
these complexes. Three tentative configurations were pro-
posed as starting points for the calculations. Scheme 2
presents the configurations for the first compound in the
series, namely for the platinum(II) complex with 1-(2-
oxolanylmethyl)-2-thiourea. The acyclic species with L1
bound monodently to the PtCl2 synthon is denoted below
as Pt-(L1-I). The two cyclic structures are denoted as
Pt-(L1-II) and Pt-(L1-III), respectively. The structure of
Pt-(L1-II) contains a seven-membered ring, whereas that
of Pt-(L1-III) has a four-membered ring. Similar structures
The data shown in Table 3 show that in the gas phase,
structure II, containing a 7-membered ring, is the most
stable for all three complexes. On that basis, we may
assume that the complexes mainly, if not exclusively, take
the form of structure II (Scheme 2) in the solid state as
well. The higher energy of structures III seems to be
consistent with the lower stability of four-membered rings
caused by the relatively large ring strains and angle
deformations. However, this structure has been experi-
mentally found in thioplatin—a drug proposed for cancer
therapy in 2001 [31]. The energy differences between
structures II and I or III are significantly greater for the
123

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Transition Met Chem (2010) 35:639–647
Table 3 Calculated bonding energies and relative bonding energies
(kcal/mol) of three structures of Pt(L1)Cl2, Pt(L2)Cl2 and Pt(L3)Cl2
in the gas phase
Gas phase
Ebond
Ebond (rel)
Pt(L1)Cl2
Pt-(L1-I)
Pt-(L1-II)
Pt-(L1-III)
Pt(L2)Cl2
Pt-(L2-I)
Pt-(L2-II)
Pt-(L2-III)
Pt(L3)Cl2
Pt-(L3-I)
Pt-(L3-II)
Pt-(L3-III)
-50.05
-56.38
-49.01
6.33
0
Fig. 2 The lowest energy structures of the platinum(II) complexes
with 1-(2-oxolanylmethyl)-2-thiourea, optimized at the MPW1PW91/
LanL2DZ level
7.37
-51.35
-69.34
-43.15
18.0
0
˚
˚
(1.594 A) and C–HÁÁÁCl (2.932 A). However, for each of
the structures, a set of conformers of higher energy was
found. Hydrogen bonds in the higher energy conformers
were longer, which suggests weaker energy stabilization
due to these H-bonds.
26.2
-51.83
-74.42
-41.66
22.6
0
In aqueous solutions, however, the energy consider-
ations lead to different results. Here, the energy difference
between the thiol and thione tautomers is sufficiently high
to make the existence of the unionized thiol forms unlikely.
Nevertheless, after proton dissociation, the energy of the
system composed of the anionic form of the complex
(thiolate) and the solvated proton significantly decreases
relative to the energy of unionized thiol and thione struc-
tures (the aqueous solvation energy of the proton, of
-265.9 kcal/mol, was taken from ref. [32]). However, for
conformers III, the energies of the thione structures remain
the lowest, whereas for the conformers I, II, and for the
free ligands, the energy differences between the thiolates
and thiones and between thiolates and thiols are strongly
negative (of the order of -30 to -100 kcal/mol).
32.8
complexes containing sulfur and selenium than the oxygen
heteroatom. Figure 2 presents the structures of Pt-(L1-II)
(complex containing a seven-membered ring) and Pt-(L1-
III) (containing a four-membered ring) optimized at the
MPW1PW91/LanL2DZ level.
The data shown in Table 4, as well as the structures
shown in Fig. 2, refer to the lowest energy conformers that
were found in the process of energy minimization. Two
intramolecular hydrogen bonds were found for these most
stable conformers of structures II and III. For example, for
˚
Pt-(L1-II), the bonds are N–HÁÁÁCl (2.644 A) and C–HÁÁÁCl
˚
(2.557 A) (Table 4). For Pt-(L1-III), they are N–HÁÁÁO
Table 4 Calculated bond
˚
Pt-(L1-II), X=O
Pt-(L2-II), X=S
Pt-(L3-II), X=Se
lengths (A) and angles (°) in the
platinum complexes having the
lowest energy conformation
˚
Bond lengths (A)
Pt–S
2.444
2.359
2.396
2.656
1.347
1.357
2.644
2.557
2.145
2.448
2.389
2.389
2.819
1.343
1.352
2.492
2.503
2.408
2.450
2.403
2.387
2.829
1.342
1.351
2.423
2.409
2.480
Pt–Cl(1)
Pt–Cl(2)
Pt–C
C–NH2
C–NH
H(1)ÁÁÁCl(1)
H(2)ÁÁÁCl(2)
Pt–X
Angles (o)
Cl(1)–Pt–Cl(2)
90.07
119.24
119.36
88.79
90.66
118.73
119.73
86.25
90.33
118.79
119.91
86.71
S–C–NH2
NH–C–NH2
X–Pt–S
C–X–C
102.95
17.62
89.35
87.54
C–C–C–C dihedral in 5-membered ring
31.72
48.49
123

Transition Met Chem (2010) 35:639–647
645
-1
Fig. 3 Spectra of L1 (upper)
and Pt(L1)Cl2 (bottom); KBr
pellet
0,75
0,50
0,25
0,00
1075 cm
L1
1,5
L1
1,0
0,5
1000
1025
1050
1075
1100
0,0
500
1000
1500
2000
2500
3000
3500
4000
Frequency, cm-1
0,50
-1
1071 cm
0,2
0,1
0,0
Pt(L1)Cl
2
0,25
0,00
Pt(L1)Cl2
500
1000
1500
2000
2500
3000
3500
4000
Frequency, cm-1
1000
1025
1050
1075
1100
Frequency, cm-1
Therefore, it can be assumed that in aqueous solutions, the
complexes might exist in the form of thiolates and take also
the linear conformations.
they move from 1,075 (L1) to 1,071 cm-1 (Pt(L1)Cl2),
from 935 (L2) to 928 cm-1 (Pt(L2)Cl2) and from
922 cm-1 (L3) to 913 cm-1 (Pt(L3)Cl2). This suggests
that in the solid phase, the oxygen/sulfur/selenium het-
eroatoms of the five-membered rings are also involved in
the metal complexation (as in the structure II shown in
Scheme 2).
Vibrational spectra
Representative IR spectra, namely of L1 and Pt(L1)Cl2, are
shown in Fig. 3. The spectra of L2 and Pt(L2)Cl2 as well as
of L3 and Pt(L3)Cl2 appeared not to differ significantly
from these of the oxygen ones.
Stabilities of the complexes
The absence of any band in the 2,450–2,650 cm-1
region, which might be assigned to the m(S–H) vibrations of
the ligands [33], suggests the absence of any thiol tautomer
in the solid state. On the other hand, both m(C=S) bands at
ca. 1,350 cm-1 and different NH-stretching vibrational
bands at above 3,150 cm-1 [33] can be observed. This
indicates that the species remain mostly as the thione
tautomers.
The stabilities of Pt(L1)Cl2, Pt(L2)Cl2 and Pt(L3)Cl2 in
standard aqueous physiological solution (0.9% NaCl; pH
4.5) were checked by recording their UV–Vis spectra
(200–900 nm) vs. time. No changes in the spectra were
observed within about 1 week. The HPLC chromatograms
of samples withdrawn occasionally from the saline solu-
tions of the complexes did not exhibit noticeable changes
either. The same was observed for the aqueous solutions of
the complexes to which glutathione (GSH), a tripeptide
being one of the main donors of the SH groups in the
cytoplasmic environment, was added.
The m(C=S) bands are weakened and shifted to higher
wavenumbers in all the complexes when compared to
the corresponding free ligands (60, 20 and 30 cm-1 for the
Pt(L1)Cl2, Pt(L2)Cl2 and Pt(L3)Cl2, respectively). On the
other hand, bands in the region of 3,440–3,270 cm-1
,
attributed to different stretching modes of both amine
groups in the spectra of the free ligands, show appreciable
differences to the spectra of the complexes. All these dif-
ferences in the spectra may be a consequence of the metal
coordination via the sulfur donor atom from the thiourea
moiety, as reported earlier [22]. Of special interest are the
bands related to the C-X-C ring vibrations of the ligands (in
the region 1,500–750 cm-1). Comparison of the spectra
registered for Pt(L1)Cl2, Pt(L2)Cl2 and Pt(L3)Cl2 with
those of the appropriate free ligands reveals that the rele-
vant bands change their position and intensity. Namely
Cytotoxicities of the complexes
Results obtained for the cytotoxicities of complexes,
measured by the half-maximal inhibitory concentration
(IC50), are shown in Table 5. It can be concluded that the
cytotoxicities of Pt(L1)Cl2 and Pt(L2)Cl2 are similar for all
types of cells examined. However, for Pt(L3)Cl2, the IC50
was lower than those for Pt(L1)Cl2 and Pt(L2)Cl2 for three
cell lines. The effectiveness of Pt(L3)Cl2 in inhibiting the
growth of HeLa cancer cells was comparable to that of
carboplatin (IC50 48 vs. 23 lg/mL [34]).
123

646
Transition Met Chem (2010) 35:639–647
Table 5 IC50 values of the investigated platinum(II) complexes
Cell line
Pt(L1)Cl2
Pt(L2)Cl2
Pt(L3)Cl2
IC50 SD
(lg/mL)
IC50
(mM)
IC50 SD
(lg/mL)
IC50
(mM)
IC50 SD
(lg/mL)
IC50
(mM)
WS1 (human embryonic skin fibroblasts)
ME18 (human melanoma cells)
64.6 18.8
81.0 27.3
102.0 20.8
173.0 27.0
114.0 20.0
0.152
0.191
0.239
0.406
0.268
42.3 14.6
100.0 21.0
83.6 9.0
0.096
0.226
0.189
0.370
0.249
78.4 23.3
75.1 6.8
48.5 13.7
56.5 3.6
52.2 12.7
0.160
0.154
0.099
0.116
0.107
ME18/R (human melanoma subline resistant to doxorubicin)
HeLa (human cervix carcinoma cells)
163.6 20.0
110.0 10.5
KB-V1 (HeLa subline resistant to vinblastine)
Antimicrobial activities of the complexes
Conclusions
The antimicrobial activities of Pt(L1)Cl2 and Pt(L2)Cl2
were expressed as their minimal inhibitory concentrations
(MIC). The results of the MIC measurements are presented
in Table 6.
The results of the structure modeling suggest the formation
of Pt complexes with the thiourea derivatives possessing a
seven-membered ring in the solid state. Based on the
thermodynamic stabilities of the complexes, one would
expect platinum(II) to interact most strongly with L3. In
consequence, Pt(L3)Cl2 could be expected to be a better
anticancer agent than Pt(L1)Cl2 and Pt(L2)Cl2, at least
with respect to some cell lines. Of the five cancer cell lines
studied, three displayed enhanced susceptibility to the
complex containing selenium, according to the diminished
IC50 value. The best result was an IC50 of 48 lg/mL for
ME18/R (human melanoma subline resistant to doxorubi-
cin). The results indicate that studies concerning Pt com-
plexes containing a selenium atom in the ligand are worthy
of continuation.
It can be seen that both complexes were particularly
efficient against Staphylococcus epidermidis; Pt(L1)Cl2
inhibited growth of the strain at a concentration of 5 mM,
whereas Pt(L2)Cl2 was active at 2.5 mM. The latter was also
active against Bacillus pumilus at a concentration of 5 mM.
All other bacterial strains were inhibited by Pt(L1)Cl2 at a
concentration of 10 mM, and by Pt(L2)Cl2 at a concentration
of 20 mM. In addition to antimicrobial activities, the activ-
ities of Pt(L1)Cl2 and Pt(L2)Cl2 against two fungal strains
were also tested (Table 6). However, the two strains were
resistant to the Pt complexes up to a concentration of 20 mM.
Table 6 MIC values of the
Strains
MIC (mM)
investigated platinum(II)
complexes
Pt(L1)Cl2
Pt(L2)Cl2
Bacteria
Enterococcus hirae ATCC 10541
Stenotrophomonas maltophilia ATCC 12714
Escherichia coli ATCC 10538
10
10
10
10
10
10
5
20
20
20
20
20
5
Escherichia coli ATCC 8739
Bordetella bronchiseptica ATCC 4616
Bacillus pumilus ATCC 14884
Staphylococcus epidermidis ATCC 12228
Staphylococcus aureus MRSA 11 from the blood
Staphylococcus aureus MRSA 2 from nose
Staphylococcus aureus ATCC 6538P
Pseudomonas aeruginosa ATCC 9027
Pseudomonas aeruginosa ATCC 15442
Fungi
2.5
20
20
20
20
20
10
10
10
10
10
Candida albicans ATCC 10231
Aspergillus brasiliensis ATCC 16404
[20
[20
[20
[20
123

Transition Met Chem (2010) 35:639–647
647
Acknowledgments Financial support from the Polish Ministry of
Science and Higher Education, especially through financing of the
supporting Polish-Portuguese bilateral scientific agreement between
the Institute of Nuclear Chemistry and Technology and the Techno-
logical and Nuclear Institute Transition Metal Complexes as Novel
Agents of Expected Biological Activity directed towards Auger
Electron Therapy (AET), is gratefully acknowledged. The authors are
thankful to the Ministry for Grant No N N204 141437. We are
indebted to the Interdisciplinary Centre for Mathematical and Com-
putational Modeling of Warsaw University for allowing the authors to
use their computational resources. The authors thank Mr. Nikos Ag-
orastos for recording the mass spectra.
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