nd
(II
ha
(II
atł
Małgorzata Domagała c, Renata Kontek b, Regina Osiecka b, Justyn Ochocki a,*
(non-small cell lung cancer) cell lines as well as normal human peripheral blood lymphocytes. The results obtained indicate that
novel analogues of cis-diamminedichloroplatinum(II) cause inhibition of cells growth which suggest that they could be chemother-
important chemotherapeutic agents used against testicu- CDDP adducts cause bending and unwinding of DNA
double helix and these actions have further important
consequences (e.g. binding HMG proteins, poorly re-
paired CDDP adducts, inhibition of replication, tran-
scription, translation, etc.) [7–9].
.
* Corresponding author. Tel.: +48 42 677 92 16; fax: +48 42 677 92
20.
E-mail address: ochocki@ich.pharm.am.lodz.pl (J. Ochocki).
Journal of Inorganic Biochemistry
JOURNAL OFInorganic0162-0134/$ - see front matter
2005 Elsevier Inc. All rights reservedapeutic drugs in the future.

2005 Elsevier Inc. All rights reserved.
Keywords: Platinum(II) complexes; Pyridine derivatives; cis-and trans-complexes; Cytotoxicity
1. Introduction
cis-Diamminedichloroplatinum(II) (CDDP) was first
synthesized in 1844 and was known as Pyrone
s chloride.
Its antiproliferative properties were described 120 years
later when Rosenberg observed abnormal growth and
inhibition of cell division in Escherichia coli caused by
tetravalent platinum complexes formed in bacterial
medium [1,2]. At present, CDDP is one of the most
lar, ovarian, bladder, head and neck cancer, etc. [3,4].
Clinical application of this drug is limited by many side
effects, e.g. nephrotoxicity, neurotoxicity, myelotoxicity,
ototoxicity, hematological toxicity, peripheral neuropa-
thy, anaphylactic and gastrointestinal reactions [5,6].
CDDP exhibits the ability to bind to DNA and forms
mainly 1,2-d(GpG) and 1,2-d(ApG) intrastrand cross-
links and to a lesser degree 1,3-d(GpXpG) intrastrand
cross-links and interstrand cross-links. As a result,a Department of Bioinorganic Chemistry, Medical University, Muszyn´skiego 1, 90-151 Ło´dz´, Poland
b Department of Cytogenetics and Plant Molecular Biology, University of Ło´dz´, Banacha 12/16, 90-237 Ło´dz´, Poland
c Department of Crystallography and Crystal Chemistry, University of Ło´dz´, Pomorska 149/153, 90-236 Ło´dz´, Poland
Received 25 April 2005; received in revised form 22 June 2005; accepted 24 June 2005
Available online 18 August 2005
Abstract
Preparations of cis- and trans-platinum(II) complexes of diethyl (pyridin-4-ylmethyl)phosphate (4-pmOpe) have been described.
These complexes were identified and characterized by far-IR, 1H NMR, 13C NMR, 31P NMR and 195Pt NMR and microanalyses.
The crystal and molecular structure of trans-platinum(II) complex i.e., trans-[PtCl2(4-pmOpe)2] was determined by the X-ray diffrac-
tion. Novel complexes were assayed for their potential antiproliferative effect against HT 29 (colorectal adenocarcinoma) and A 549Synthesis, spectroscopy a
of cis- and trans-platinum
(pyridin-4-ylmethyl)phosp
of trans-Pt
Urszula Kalinowska a, Ksenia Mdoi:10.1016/j.jinorgbio.2005.06.030antiproliferative activity
) complexes with diethyl
te. X-ray crystal structure
) complex
awska b, Lilianna Che˛cin´ska c,
www.elsevier.com/locate/jinorgbio
99 (2005) 2024–2031
Biochemistry

4-pmOpe was synthesized according to the reported
rganicprocedure [17]. K2[PtCl4] was purchased from Aldrich.
Solvents were purified by standard techniques and were
freshly distilled prior to use. CDCl3 for NMR spectros-
copy was obtained from Dr. Glaser AG Basel.
2.2. Measurements
IR spectra were recorded in a solid state (CsI pellets)
on an ATI Mattson Infinity Series FTIRTM spectrome-
ter in the range 4000–200 cm
1. 1H NMR spectra were
collected at a Varian Mercury-300 spectrometer operat-
ing at a frequency of 300 MHz. 13C NMR spectra were
recorded on a Varian Mercury-300 spectrometer operat-
ing at a frequency of 75.5 MHz. All chemical shifts were
reported using the standard (d) notation in ppm relative
to TMS (1%) as internal standard. 31P NMR spectra
were recorded on a Varian Mercury-300 spectrometer
(121.5 MHz). Chemical shifts were reported relative to
the external reference H3PO4 (85%). The solution
195Pt
NMR data were obtained at 107.495 MHz on a Bruker
DRX-500 spectrometer and referenced to K2PtCl6. All
NMR samples were prepared using CDCl3 as a solvent.
Microanalyses of C, H and N were performed with a
Perkin–Elmer 2400 analyzer. Melting points were deter-For many years it was believed that trans-platinum
compounds were non-active as antitumour agents be-
cause trans-diamminedichloroplatinum(II) is biologi-
cally inactive although it binds to DNA (forming
mainly monoadducts and interstrand cross-links) [10].
However, since the 1990s many trans-platinum com-
plexes have been discovered with significant anti-tumour
activity against different tumor cells including these
resistant to CDDP [11–15].
In the present paper we describe the synthesis, char-
acteristics of cis and trans-platinum complexes which
contain two diethyl (pyridin-4-ylmethyl)phosphates (4-
pmOpe) as non-leaving ligands. The environments of
Pt(II) atoms are square-planar, formed by two N atoms
from 4-pmOpe ligands and two Cl atoms.
The aim of the studies was also to estimate antiprolif-
erative activity of these new chemical compounds in
three cell lines in vitro. In this regard we used the ability
of viable cells to convert the yellow dye MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphentyltetrazolium bromide]
to purple crystals of formazan by mitochondrial dehy-
drogenases [16]. The IC50 values were evaluated (the
drug concentration inhibiting 50% of the cell growth
after 72 h exposure of cells to the drug).
2. Experimental
2.1. Materials
U. Kalinowska et al. / Journal of Inomined with Bo¨etius apparatus.2.3. X-ray crystallography
Crystals suitable for X-ray analysis were obtained by
crystallization from ethanol–chloroform (4:1). Diffrac-
tion datawere collected usingAFC-5SRigakudiffractom-
eter [18] with graphite-monochromatized MoKa
radiation. Lattice parameters were obtained by least-
square refinement of setting angles of 25 reflections (h
range from 29.14 to 29.85). A total of 3386 reflections
were measured by x scan technique. After each group
of 150 reflections three standard intensities were moni-
tored and 7.5% intensity fluctuation was observed. All
data were corrected for Lorentz and polarization effects
[19]. Analytical absorption correction [20] was also ap-
plied. The structure was solved by Patterson method
(SHELXS97) [21] and refined on F2 by full-matrix least-
squares technique (SHELXL97) [21]. All non-hydrogen
atoms were treated as anisotropic. During the refinement
the ethoxy groups revealed large atomic displacement
parameters. Finally, four atoms (C21, C22, C31 and
C32), appeared to be disordered over two orientations
with equal site occupancies (0.5). Similarity restraints
were used in the refinement of the atomic displacement
parameters of those disordered atoms. Moreover, bond-
length restraints were applied to all C–C bond involving
the disordered atoms. All H-atoms bonded to C atoms
were included in the refinement, at calculated positions,
in the riding-model approximation, with C–H distances
of 0.93 A˚ (aromatic), 0.97 A˚ (CH2) and 0.98 A˚ (CH3).
The isotropic displacement parameters were set at
1.2–1.5 Ueq of the carrier atom.
The final geometrical calculations and drawings were
performed with PLATON [22]. Crystal structure and
refinement data are summarized in Table 1.
2.4. Preparation of the platinum(II) complexes: cis-
[PtCl2(4-pmOpe)2], trans-[PtCl2(4-pmOpe)2]
K2[PtCl4] (0.6880 mmol) was dissolved in 10 ml of
water and added to a methanol solution (2 ml) of ligand
(4-pmOpe) (1.376 mmol). The reaction mixture was stir-
red at 45 C for 6 days in the darkness. After 5 h of
heating a brownish-yellow oil was formed and trans-
formed into the yellow precipitation with further stir-
ring and heating. After removing the water solution
the precipitation became sticky. Acetone (20 ml) was
added to the resulting precipitation dissolving it
partially. The pale yellow solid, which is insoluble in
acetone, was collected by filtration, washed with several
portions of acetone, recrystallized from the mixture:
ethanol–chloroform (4:1) and dried in air. The obtained
compound was trans-[PtCl2(4-pmOpe)2]: yield 17%,
m.p. 199–201 C. The elemental analysis for these
crystals were consistent with the formula [PtCl2
(4-pmOpe)2] Æ H2O requires: C 31.02%; H 4.43%; N
Biochemistry 99 (2005) 2024–2031 20253.62%. Found: C 31.00%; H 3.90%; N 3.65%. Crystals