The CellScan technology for in vitro studies on novel platinum complexes with organoarsenic ligands

Article abstract of DOI:10.1039/b802364f

The present paper examines one of the multiple uses of the CellScan technique, its utilization in the biological evaluation of novel compounds, in order to improve the methods concerning the prediction of their anti-proliferative effects and application as anti-cancer drugs. The CellScan is a laser scanning static cytometer, enabling repetitive spectroscopic measurements in intact living cells. The detection of cell viability and apoptosis is possible, based on the transformations that occur in the cytoplasm matrix of tumour cells influenced by cytotoxic compounds. The measurement of fluorescence changes, due to this phenomenon, is possible with the CellScan system. The potential of this technology to detect the in vitro effects of the inhibitory molecules on tumour cells was demonstrated, making this method a valuable tool for chemosensitivity tests. We synthesized and fully characterised three novel platinum complexes of tertiary arsine ligands: trans-[PtI ₂(2-ⁱPrOC₆H₄AsPh₂) ₂] (1), trans-[PtCl₂(2-MeOC₆H ₄AsPh₂)₂] (2) and cis-[PtCl₂(2- HOC₆H₄AsPh₂)₂] (3). The three compounds are biologically active against tumour cells and their cytotoxicity is comparable with standard drugs. Measurements using the CellScan technology correlate well with the results provided by other bioassay methods.

Full text of DOI:10.1039/b802364f

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PAPER
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The CellScan technology for in vitro studies on novel platinum complexes with
organoarsenic ligands†
Eva Fischer-Fodor,*a Natalia Moldovan,b Piroska Virag,a Olga Soritau,a Ioana Brie,a Peter Lo¨nnecke,c
Evamarie Hey-Hawkinsc and Luminita Silaghi-Dumitrescub
Received 11th February 2008, Accepted 22nd July 2008
First published as an Advance Article on the web 6th October 2008
DOI: 10.1039/b802364f
The present paper examines one of the multiple uses of the CellScan technique, its utilization in the
biological evaluation of novel compounds, in order to improve the methods concerning the prediction
of their anti-proliferative effects and application as anti-cancer drugs. The CellScan is a laser scanning
static cytometer, enabling repetitive spectroscopic measurements in intact living cells. The detection of
cell viability and apoptosis is possible, based on the transformations that occur in the cytoplasm matrix
of tumour cells influenced by cytotoxic compounds. The measurement of fluorescence changes, due to
this phenomenon, is possible with the CellScan system. The potential of this technology to detect the
in vitro effects of the inhibitory molecules on tumour cells was demonstrated, making this method a
valuable tool for chemosensitivity tests. We synthesized and fully characterised three novel platinum
complexes of tertiary arsine ligands: trans-[PtI2(2-iPrOC6H4AsPh2)2] (1), trans-[PtCl2(2-
MeOC6H4AsPh2)2] (2) and cis-[PtCl2(2-HOC6H4AsPh2)2] (3). The three compounds are biologically
active against tumour cells and their cytotoxicity is comparable with standard drugs. Measurements
using the CellScan technology correlate well with the results provided by other bioassay methods.
to study cytotoxicity, genotoxicity, and anti-proliferative effects of
novel compounds.
We synthesized a new group of platinum complexes with the
Introduction
Metal-based chemotherapeutic agents, above all platinum com-
plexes, are an important class of anti-tumour compounds that
have had a significant impact on the treatment of a variety of
tumours for nearly 30 years.
general formula [PtX2L2] (X = I, Cl; L = diphenylarsine ligand) in
order to determine their potential biological activity. These three
platinum complexes have organometallic ligands with structures,
which lead us to presume that they exhibit biological effects: trans-
The unique efficiency observed with standard platinum-based
[PtI2(2-iPrOC6H4AsPh2)2] (1), trans-[PtCl2(2-MeOC6H4AsPh2)2]
(2) and cis-[PtCl2(2-HOC6H4AsPh2)2] (3). Each structure contains
two identical tertiary arsine groups bonded to platin, and the
only difference between these groups being one moiety, which
is isopropyl for compound (1), methyl for (2) and hydroxyl for
compound (3). The molecular structures of (1), (2) and (3) are
analogous.
drugs, such as cisplatin, cis-diamminecyclobutanedicarboxylato-
platinum(II) (carboplatin) and 1,2-diaminocyclohexaneoxalato-
platinum(II) (oxaliplatin) in cancer treatment, fuelled the devel-
opment of new platinum analogues.1 In the last decade, it was
demonstrated that several newly synthesized platinum compounds
exhibit biological activity against tumour cells, and their anti-
mitotic properties were proven by several procedures.2–8
The CellScan technology is based on a spectrometric method
which allows repetitive multi-parameter measurements to be
performed on intact living cells exposed to different external
stimuli. The equipment is designed to perform fluorescence-based
cytometric studies.9 Using this straightforward and rapid method,
it was possible to establish the cytotoxicity and apoptotic capacity
of chemotherapeutic agents.10,11 Our aim is to establish a procedure
For this purpose, we used the CellScan system, which permits
the detection of fluorescence intensity (FI) and polarization (FP)
of different cell types. There is a strong correlation between
the inhibition of cell growth induced by different chemother-
apeutic agents and the resultant changes in FI and FP. Thus,
changes can be revealed by staining treated cells with fluorescent
dyes such Fluorescein diacetate (FDA) and Rhodamine 123
(Rh123). Hyperpolarization of the fluorescence intensity detected
by the CellScan system can identify apoptotic cells within a
complex cell population; it is a marker of the early apoptotic
processes.12
To verify the accuracy of the new method, we correlated the
results obtained in this way with those determined with customary
procedures, such as the MTT (MTT = 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) viability test13 and one of
the standard methods for assessing DNA damage, namely, the
single-cell gel ‘comet’ assay.14,15
aTumor Biology Laboratory, Research Department, “I. Chiricuta” Cancer
Institute, 34–36 Republici St., 400015, Cluj Napoca, Romania. E-mail:
fischer.eva@iocn.ro; Fax: +40 264 439 260; Tel: +40 262 450 673
bChemistry Faculty, Babes-Bolyai University, Romania
cInstitut fu¨r Anorganische Chemie, Universita¨t Leipzig, Germany
† Electronic supplementary information (ESI) available: POV-Ray repre-
sentations, atomic coordinates and displacement parameters, anisotropic
displacement parameters, bond lengths and angles for 1, 2 and 3. CCDC
reference numbers 693566–693568. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/b802364f
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Tertiary arsine complexes of platinum and other metals have
been recently reported,16–18 but there is no information regarding
their biological activity. Among phenylarsines, the information on
the biological activity was reported for phenylarsine oxide and
dichlorophenylarsine,19–23 and the phenylarsine compounds at-
tached to biomolecules like peptides and enzymes were studied.24,25
Phenylarsine oxide derivates are well known as anti-trypanosome
agents and they are used as drugs.26–28
Results and discussion
Fig. 1 ORTEP representations of the molecular structure of trans-[PtI2(2-
iPrOC6H4AsPh2)2] (1) (left) and trans-[PtCl2(2-MeOC6H4AsPh2)2] (2)
(right); 50% ellipsoids are shown (hydrogen atoms are omitted for clarity).
Synthesis and characterization of Pt complexes with organoarsine
ligands
Complexes of the general formula [PtX2L2], (X = I, L = (2-
isopropoxyphenyl)diphenylarsine, X = Cl, L = (2-methoxy-
phenyl)diphenylarsine, (2-hydroxyphenyl)diphenylarsine) were
prepared by the reaction of the arsine ligands with [PtX2(COD)]
(COD = 1,5-cyclooctadiene) in a molar ratio of 2 : 1 (Scheme 1).
In the case of the complexes with diphenylarsinophenol ethers
(1 and 2) both reagents were dissolved in dichloromethane
with stirring at room temperature, and the final products were
recrystallized from chloroform or from dichloromethane to obtain
crystals suitable for X-ray diffraction.
The diphenylarsinophenol containing platinum complex (3)
was prepared by slow diffusion of solvents; the arsine ligand
was dissolved in toluene and added very slowly as a layer to a
solution of [PtCl2(COD)] in dichloromethane. The final product
was isolated as crystals suitable for X-ray structure determination.
In all complexes, the geometry around the metal center is
square-planar and the organoarsenic ligands are coordinated to
the transition metal through the arsenic atom. Similar complexes
of platinum and palladium with O and P donor ligands have been
reported in the literature.29
Fig.
2
ORTEP representation of the molecular structure of
cis-[PtCl2(2-HOC6H4AsPh2)2] (3), with both independent molecules re-
vealed; 50% ellipsoids are shown (hydrogen atoms are omitted for clarity).
ovarian cancer cell line, the M1–15 highly metastatic melanoma
cell line, and the Hfl-1 normal human lung fibroblast cell line.
The MLS cells were cultivated in a DMEM cell growth media,
supplemented with glutamine, antibiotics and fetal bovine serum.
M1–15 requires a RPMI-1640 medium, while Hfl-1 cells are
cultivated in the nutrient-rich medium F-12. The cell cultures were
◦
maintained at 37 C under a CO2-enriched sterile atmosphere in
an incubator for cell cultures.
IC50 values. The effectiveness of the platinum complexes
against cell proliferation was measured using a quantitative col-
orimetric assay. As described previously,14 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to assess
cytotoxicity. This pale yellow salt is reduced only by the mitochon-
dria of living cells to its blue formazan derivative. Using a multi-
well scanning spectrophotometer (ELISA reader) we measured
the colour intensity, which is proportional to the number of living
cells.
Scheme 1 General procedure for the synthesis of platinum complexes
The test was performed on MLS, M1–15 and Hfl-1 cells. In 96-
welled flat-bottomed microculture plates, cells were suspended in
a concentration of 12 500 cells per well, in 200 ml of cell culture
media. After 24 h incubation in the incubator, each compound
was screened for in vitro anticancer activity by treating the cell
lines with a wide range of concentrations of the platinum complex
solutions. For 1, 2 and 3, we used the concentration range 0.01–
90 mM mL-1, for carboplatin and oxaliplatin the range was
between 0.01–50 mM. The solvent containing the upper limit
1 : 100 ethanol–PBS (phosphate buffered salt solution) was tested
separately and displayed no cytotoxic effect, which could affect
our results. Three different experiments were performed for each
complex.
with the tertiary arsine ligands.
Fig. 1 and Fig. 2 present the molecular structure of the new
platinum compounds.
In cis-[PtCl2(2-HOC6H4AsPh2)2], two independent molecules
are present in the asymmetric unit (Fig. 2) and they have similar
structural properties.
Biological effects
In the order to study the possible anti-proliferative and anti-
mitotic effects of the compounds, we established their chemosensi-
tivity on tumour and normal cell lines. We chose the MLS human
6394 | Dalton Trans., 2008, 6393–6400
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Measurements were made after 48 h of incubation with the
complexes, by removing the cell culture media, staining the
cells with the MTT dye, and then by dissolving the formazan
crystals with dimethylsulfoxyde (DMSO). The chemosensitivity
was expressed as an IC50 value, that is, the concentration of the
compound causing 50% cell death. IC50 values were obtained from
dose-response curves after fitting the data to a linear quadratic
equation using the Graph Pad Prism biostatistics program (95%
confidence intervals, r2 = 0.94, P <0.05).
agents, the cells were stained with the fluorescent dyes Fluorescein
diacetate and Rhodamine 123, and measurements were made with
the CellScan system.
The CellScan apparatus is a laser scanning, static, multi-
parameter cytometer10,30 measuring both fluorescence intensity
(FI) and polarization fluorescence (FP) of individually fluorescent-
labelled cells. The simplicity of this system provides a new
perspective in clinical studies and in the basic research area. The
CellScan system enables a repetitive spectroscopic measurements
on intact living cells within a population under certain physio-
logical conditions. It incorporates a unique ‘cell carrier’, which
allows for repeated high-precision measurements by fixing the
location of the cell in the cell carrier. We can perform repetitive
measurements on the same populations and on the same cells after
various stimulations and different set-ups of the scanner.31
The ‘core’ of the carrier is a 2 mm square grid of precisely
dimensioned conical apertures, 5–8 mm in diameter, spaced at
20 mm, which can hold 10 000 cells, each loaded and maintained in
a separate trap (Fig. 4). Each aperture is dimensioned to hold one
single cell; lymphocytes and human tumour cell dimensions are
compatible with the aperture diameter. We used carriers with 8 mm
sized apertures. The cells were loaded in the grid using a gradient
of pressure. A negative pressure of 1–5 mm H2O, maintained
between the upper and the lower surfaces of the carrier, caused
a rapid fixing of the cells into the orifices. The loaded cells, once
trapped into the conical orifice, can not leave this and remain fixed
during various procedures and manipulations: staining, washing,
microscopic and spectrometric measurements.
The IC50 values obtained are higher against normal Hfl-1 cells
in all cases (Fig. 3), that is, platinum complexes 1, 2 and 3 are more
toxic against MLS ovarian tumour cells. The less toxic is 1 and the
most toxic is 3. IC50 values of carboplatin and oxaliplatin against
MLS ovarian tumour cells are represented for comparison with
novel synthesized compounds.
Fig. 3 The influence of complexes 1, 2 and 3 on normal (Hfl-1) and
tumour (MLS and M1–15) cell lines, expressed as IC50 values. C1
represents the platinum complex 1, C2 complex 2 and C3 complex 3,
CarboPt is the abbreviation for carboplatin and OxaliPt is the abbreviation
for oxaliplatin.
The cytotoxicity of the platinum complexes studied is compa-
rable with the anti-tumour activity of carboplatin, a widely used
drug in ovarian cancer treatment. The studied compounds are all
more active than oxaliplatin against ovarian tumor cells.
The highly metastatic M1–15 melanoma cell line is more
resistant to the complexes and the IC50 values show a lower
cytotoxicity in comparison with the MLS cells; however, the
compounds are still more active against the melanoma cell line
versus normal Hfl-1 cells, except 3, which shows a lower toxicity
against the highly metastatic M1–15 cells.
Fig. 4 Fluorescent inverse phase microscope image of FDA stained MLS
tumour cells trapped in the orifices of the cell carrier (20-fold magnitude).
The static He–Cd laser cytometer scans each hole and displays
the spread of intensity (FI) on the x axis and polarization (FP)
on the y axis on the screen (Fig. 5). The illumination intensity
of the laser beam is 5 mW, the intensity and polarization values
measured at two wavelengths (527 and 510 nm) characterize each
living cell. The measurements are made using a preset photon-
counting technique, in which the same number of photons is
counted for each cell, regardless of emissions intensity, so that the
measurement has the same precision both for weakly and strongly
emitting cells.
These values helped us to choose the concentrations used for
the CellScan determinations.
CellScan spectrometry. In this experiment, we utilized the
MLS, Hfl-1 and M1–15 cell lines. The cells were suspended in
growth media in 6-well microplates, and were incubated for 24 h,
to allow them to attach to the plate surface. The plates were treated
with 1, 2 and 3 solutions, and also with carboplatin and oxaliplatin.
As a control, we used untreated cells. In each case, we used a
concentration of 5 mM. After 48 h of incubation with the cytotoxic
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Fig. 5 Histogram provided by the CellScan: the polarization and the intensity for each cell, measured at two wavelengths are represented by red and
green dots. In Fig. 5a the untreated (control) MLS cells are shown, (b) and (c) represents FI and FP of MLS cells treated with complex 1, respectively.
The fluorescence polarization (FP) indicates the entropy of the
fluorescent molecules, that is, the relative rotational freedom. The
FP of a fluorochrome increases with increasing viscosity or restric-
tion of molecular rotation. Cell activation and apoptosis involve
conformational changes of cytosolic enzymes and proteins, that
is, intracellular matrix reorganization. The more the molecule’s
rotational movement is restricted, the higher the FP value, and vice
versa. The fluorescence intensity (FI) is expressed as the number
of photon counts per unit of time and the FI of the fluorochrome
reflects intracellular content.
The dyes used for the measurements are FDA and Rh123. FDA
is a non-fluorescent dye, which is converted to its fluorescent
form by enzymatic hydrolysis only in living cells and it is
an indicator of cell viability. Rh 123 is a lipophilic cationic
fluorochrome that selectively accumulates in the mitochondria of
living cells and whose specific uptake or release depends upon
the mitochondrial membrane potential and it therefore reflects
mitochondrial activity. A drop in the FI and an increase in the FP
of Rh123 was attributed to early apoptosis.11,13
A FP value obtained as a result of the measurements using
FDA-stained cells gives us information about the viability of the
cells, and the inhibitory effect of the treatment. The cell growth
inhibition is demonstrated if the treated cell population displays a
high FP value and a high FI value than the untreated control
and no hyperpolarization of FDA or Rh123 was observed in
the resistant cells.9,10 In our experiment, an increase of FP was
observed for the platinum complex 1, 2 and 3 relative to the control
for both the MLS and M1–15 cells (Fig. 5).
display a cytotoxic effect as high as carboplatin and oxaliplatin.
The increase of the FP values ratio is not so prominent in the
experiment with the highly metastatic M1–15 cells.
The Rh123 fluorescent marker gives us information about the
early apoptotic processes, which affect cytoplasmic viscosity and
alterations in cell permeability. The values from Table 1 show us
that 2 displays the most significant capacity to induce apoptosis, in
this case, the FP increase is the biggest, both for MLS and M1–15
cells, although against M1–15 cells we observed a smaller increase
in FP values. Despite its IC50 value, 3 does not induce apoptosis.
Platinum tertiary arsine cytotoxicity (cell growth inhibition) lies
in a range that is comparable with other compounds that have less
toxic ligands. The complexes have poor solubility and are stable
in ethanol and phosphate buffered saline solutions, without the
release of the ligand.
Comet assay. We also performed the alkaline single-cell gel
electrophoresis or ‘comet’ assay, which detects DNA strand
breaks, alkali-labile sites and cross-links in eukaryotic cells. It
is based on electrophoretic mobility and reveals changes in the
tertiary DNA structure of the exposed cells. The procedure is
widely used in testing novel chemicals for genotoxicity, including
platinum complexes and organometallic compounds.32–35
MLS tumour cells were treated with complexes 1, 2 and 3,
carboplatin and oxaliplatin at the same concentrations used for
the polarization measurements. The cells were placed in six-well
plates, and they were incubated in growth media for 24 h, to allow
them to attach to the plate surface. Treatment was made with 1,
2 and 3 solutions, and also with carboplatin and oxaliplatin. As a
control, we used untreated cells. We used the same concentrations
Measurements show the cytotoxic activity of the studied
platinum complexes and the fact that against MLS cell lines they
Table 1 Fluorescence polarization values
FP values obtained with FDA
staining/a.u.
FP values obtained with Rh123
staining/a.u.
Stimulant
Hfl-1
MLS
M1–15
MLS
M1–15
Control
0.409
0.453
0.461
0.420
0.412
0.439
0.316
0.275
0.330
0.332
0.321
0.327
0.263
0.241
0.274
0.275
0.297
0.285
0.362
0.358
0.398
0.371
0.391
0.352
0.415
0.439
0.404
0.419
0.426
0.404
Oxaliplatin
Carboplatin
Complex 1
Complex 2
Complex 3
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Table 2 Lesion scores (LS) and tail factors (TF) for platinum complexes and platin-based drugs
Stage
Control
Oxaliplatin
Carboplatin
Complex 1
Complex 2
Complex 3
0
1
2
72
15
8
2
58
29
13
49
18
20
47
9
13
23
17
19
17
7
3
4
2
3
3
8
0
20
2
22
11
24
1
56
Sum
LS
TF
100
35
7.95
100
157
25.83
100
165
31.35
100
159
34.98
100
186
41.00
100
258
59.98
as for the static cytometry measurements and 48 h effects were
considered.
obtained using this technique are confirmed by literature data
and by convergence with the data acquired by other widely used
methods. It requires only a small number of cells to perform the
in vitro studies, and the measurements can be repeated on the
same cell population because the cell carrier used by the system
has some unique characteristics that permit the positions of living
cells during the experiment to be maintained and revealed.
The new platinum complexes with tertiary arsine ligands exhibit
biological activity. Their activity against normal and tumour cells
is differentiated as all are more active against tumour cell lines.
Genotoxicity is evidenced for complexes 1 and 2. Complex 1 is
less toxic, while 2 has the greatest apoptosis-induction capacity
and the largest gradient, in effect, against normal/malignant cells.
We suppose that this is a consequence of the presence of the
methyl moiety, which leads to methylation of the nucleic acids. The
cytotoxicity of the compounds is analogous to that of standard
anticancer chemotherapeutic platinum drugs and it is comparable
with that of other new platinum complexes that exhibit biological
activity.
The CellScan technology was useful in the analysis of the
three compounds as it has a high sensitivity and selectivity. This
procedure can replace other more difficult and lengthy methods
for assessing biological activity.
The studied compounds are also suitable for testing in vivo. Simi-
lar compounds with improved solubility in biologically compatible
solvents should be synthesized in order to develop anticancer
drugs.
The method used by us was described previously36 and it was
performed according to the alkaline version of the comet assay
test.10,11 Cells incorporated in agarose gel on a microscope slide
are lysed with an alkaline lysis solution (pH > 10) to denude
nucleotides containing supercoiled loops of DNA. After this
operation, gels are placed in a pH > 13 buffer solution for
electrophoretic migration, during which damaged DNA loops
containing breaks lose their supercoils and migrate to the anode.
The damaged cells display an increased electrophoretic migra-
tion of the DNA fragments by forming outlines resembling comets,
in contrast to the round appearance of undamaged DNA. The
length and form of the comet ‘tail’ reveal the levels of damage.
We considered five possible stages based on the distance of DNA
migration ‘tail length’. The migration of the DNA fragments from
the nucleus is visualized by manual microscopic analysis, which is
equal in sensitivity to computerized analysis.37
We evaluated the DNA damage by means of two parameters:
lesion score (LS) and tail factor (TF), both expressed in arbi-
trary units. The lesion score represents the product of the cells
ratio and stage. The tail factor quantifies the global damage in the
cells by associating the average proportion of DNA migrated in
the comet tail and it represents the percentage of DNA in the tail
relative to the amount of DNA in the entire comet. We considered
the values obtained for untreated cells as a ‘control’ reference for
each case.
In every case, we obtained a positive result, that is, a significant
increase in LS and TF versus the control. The comet assay
results show that DNA damage induced by the commonly used
platinum-based drugs and the complexes 1 and 2 are comparable,
while compound 3 induces the most strand lesions among the
studied compounds (Table 2). Massive amounts of stage 1 and
stage 2 lesions support the genotoxicity of platinum-based drugs
described in the literature. In the case of complex 3, the huge
number of stage 4 lesions is confirmation of the cytotoxic effect.
Complexes 1 and 2 show genotoxicity and an increase in stage
2 lesions is also observed. Complex 2 also exhibits many stage 3
lesions, which confirm its cytotoxic effect.
Experimental
Materials
Complex synthesis. All reactions were carried out under an
atmosphere of dry nitrogen using standard Schlenk or vacuum
line techniques. The solvents were purified (toluene refluxed
over Na–benzophenone; CH2Cl2 and n-hexane refluxed over
powdered CaH2) and distilled under nitrogen. [PtX2(COD)] (X =
Cl, I) was prepared according to the literature procedure.38 All
chemicals were of reagent grade and were used as received: 1,5-
cyclooctadiene was purchased from Merck and H2[PtCl6]·6H2O
was generously donated by Umicore AG & Co KG, Hanau-
Wolfgang, Germany.
Conclusions
The CellScan system has already proved its utility in a number of
important biochemical, immunological, kinetic, pharmacological
and physicochemical applications and is an appropriate tool
for analysing newly synthesized compounds. It is a simple and
rapid method to assess cytotoxicity and genotoxicity. The results
Biological effects. For this purpose we performed cell cultures
on both tumour and normal cell lines.
The Hfl-1 cell line was acquired from Sigma Aldrich Corpora-
tion from the European Collection of Cell Cultures; The M1–15
melanoma cell line was a generous gift from Prof. Falus Andras,
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Semmelweiss University, Budapest, Hungary, whereas the MLS
ovarian cell line was a donation from Dr Yael S. Schiffenbauer
from MedisEl Company, Lod, Israel.
(G. M. Sheldrick, SADABS, Program for Scaling and Correction
of Area-Detector data, Go¨ttingen, 1997) for empirical absorption
correction.
The cells were defrosted carefully, cultivated in special culture
flasks with adequate cell growth media, under sterile conditions.
Growth conditions were assured using incubators with a stable
temperature (37 ◦C), 5% CO2 atmosphere and constant humidity.
Several cell passages were performed using enzymatic pro-
cedures. Experiments were made when confluence of the cells
achieved 80% on the flask surface. Cell lines were adherent,
and they were seeded on 96-well cell culture plates for the IC50
measurements, and on 6-well plates for the cytometric and comet
assay tests.
The cells were incubated for 24 h before adding the platinum
complexes. After the treatment with sterile solutions with differing
concentrations of complexes 1, 2 and 3 and platinum drugs,
respectively, the culture plates were kept in incubator for another
24 and 48 h before the measurements were taken.
We made 3 independent experiments for the MTT test and
measurements were made directly using the 96-well plates.
For the cytometric and comet assay tests, a removal from the 6-
well plates was made mechanically and not enzymatically to avoid
accidental cell damage, which could be caused by the trypsine.
For the cytometric assay we made 3 different experiments, and
for the comet assay, for each compound, we performed 3 different
electrophoretic migrations on 4 separate microscope slides.
Cell cultures were performed using Dulbecco’s modified eagles
medium, RPMI 1640 and F-12 cell growth media from Sigma
Aldrich Corporation. Phosphate buffered salt solution, trypsine
EDTA, MTT salt, ethidium bromide, low melting point agarose,
type I agarose, FDA and Rh 123 fluorescent dyes were also pro-
vided by Sigma Aldrich. NaCl p.a., NaOH p.a. and EDTA·2HCl
p.a. were obtained from Merck & Co. Inc., and TrizmaHCl from
Fluka Analytics.
The crystallographic data for complex 3 were collected on a
Stoe-IPDS imaging plate diffractometer (phi-scan rotation and
˚
MoKa radiation (l = 0.71073 A) and the numerical absorption
correction was performed with X-RED (Version 1.22 (2001),
STOE Data Reduction Program, STOE & Cie GmbH, Darm-
stadt).
Biological effects
The cell culture laboratory is equipped with Heto Holten and
Laminair laminar hoods, with Hettich centrifuges, with humidified
Heto Holten Cellhouse 154 and Uniequip CO2 series incubators
with CO2 admission.
The fluorescence was tested using a Desktop CellScan appa-
ratus [MedisEl Technologies, Lod, Israel]. The cell carriers were
provided by MedisEl. The cytotoxicity was determined using a
Tecan Sunrise ELISA reader with Magellan operating system.
For the single-cell gel assay the electrophoresis was performed
in a Consort electrophoresis chamber and the power source was
Consort type EV231.
Image analysis of microscope slides for the comet assay test
were made using a Nikon fluorescence microscope with CCD
image capture and Lucia analyzer software. The image capture
of the fluorescent-marked tumour cells on the cell carrier surface
was made using a Zeiss Observer D1 optical microscope with an
Axiovision Release 4.3.6/2007 software. The statistical analysis
was performed using the Graph Pad Prism 5 biostatistics program.
Preparation of complexes 1 and 2
Synthesis of ligand solution. (2-isopropoxyphenyl)diphenylar-
sine (0.04 g, 0.11 mmol) for complex 1 and (2-methoxy-
phenyl)diphenylarsine (0.17 g, 0.5 mmol) for complex 2, in
dichloromethane was added to a dichloromethane solution of
[PtI2(COD)] (COD = 1, 5-cyclooctadiene) (0.03 g, 0.053 mmol)
for 1 and [PtCl2(COD)] (0.09 g, 0.025 mmol) for 2. The reaction
mixture was stirred at room temperature until a precipitate
was formed. The precipitate was filtered and then recrystallized
from dichloromethane and n-hexane to give the corresponding
complexes.
The supplier of all the disposables used for the biological
experiments was Nalgene Nunc International.
The chemotherapeutical drugs, carboplatin (Carboplatin) and
oxaliplatin (Eloxantine), were procured from Sindan Pharma.
Instrumentation
Characterization of new compounds. Infrared spectra were
recorded on a Perkin-Elmer System 2000 FTIR spectrometer
scanning between 400–4000 cm-1 using KBr disks and between
200–400 cm-1 using CsI plates, and also on a Bruker-Vector 22
spectrometer scanning between 200–4000 cm-1.
1. Yield: 0.03 g, 46%, orange crystals, mp 271 ◦C. Found%: C
42.4, H 3.5, I 12.5. Calcd% C42H42As2I2O2Pt: C 42.8, H 3.6, I 12.7.
IR/cm-1: 333 (Pt–I), 279 (Pt–As). dH/ppm (300 MHz, CDCl3,
Me4Si) 1.09 (d, 12H, CH3, J = 6.04 Hz), 4.50 (m, 2H, CH), 6.79–
6.88 (m, 4H), 7.35–7.39 (m, 16H), 7.84–7.87 (m, 8H).
1
The H NMR spectra of the transition metal complexes were
recorded on an AVANCE 300 spectrometer (Bruker). The chemical
1
shifts for the H NMR spectra are reported in parts per million
2. Yield: 0.15 g, 64%, pale yellow crystals, mp 274 ◦C. Found:
C 48.4, H 3.4, Cl 7.1. Calcd% C38H34As2Cl2O2Pt: C 48.6, H 3.6,
Cl 7.5%, IR/cm-1: 334 (Pt–Cl), 282 (Pt–As). dH/ppm (300 MHz,
CDCl3, Me4Si): 3.84 (s, 6H, CH3), 6.89–7.04 (m, 6H), 7.38–7.43
(m, 14H), 7.82 (m, 8H).
(ppm) at 300 MHz with tetramethylsilane as the standard. The
assignments were based on those for the unbound ligand and those
for analogous complexes. The elemental analyses were recorded on
a VARIO EL (Heraeus).
The crystallographic data for complexes 1 and 2 were collected
on a Siemens CCD diffractometer (SMART) using MoKa
Preparation of complex 3
˚
radiation (l = 0.71073 A) and omega-scan rotation (Table 3).
Data reduction was performed with SAINT (area-detector in-
tegration software, version 6.01 (1999), Siemens Industrial Au-
tomation, Inc., Madison, WI) including the program SADABS
A
solution of (2-hydroxyphenyl)diphenylarsine (0.03 g,
0.09 mmol) in toluene was added as a layer over a solution
of [PtCl2(COD)] (0.04 g, 0.044 mmol) in dichloromethane. As
6398 | Dalton Trans., 2008, 6393–6400
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Table 3 Crystallographic data for single-crystal X-ray structures of compounds 1, 2 and 3
Compound
1
2
3
Empirical formula
Formula weight
Temperature/K
l/pm
C42H42As2I2O2Pt
1177.49
213(2)
71.073
Monoclinic
P21/c
930.16(16)
1821.6(3)
1215.6(2)
90
106.261(3)
90
1.9773(6)
2
C42H42As2Cl10O2Pt
1278.19
210(2)
C36H30As2Cl2O2Pt
910.43
213(2)
71.073
71.073
Crystal system
Space group
a/pm
Triclinic
Triclinic
¯
¯
P1
P1
919.1(3)
1124.10(7)
b/pm
1169.0(3)
1344.4(4)
114.160(4)
92.344(4)
105.966(4)
1.2476(6)
1
1.701
4.697
624
0.30 ¥ 0.20 ¥ 0.20
2.02–29.24
-12 ≤ h ≤ 12, -15 ≤ k ≤ 15,
-17 ≤ l ≤ 18
13 002
1594.26(12)
1861.95(12)
83.834(8)
84.727(8)
85.694(9)
3.2963(4)
4
1.835
6.443
1760
0.20 ¥ 0.10 ¥ 0.10
2.21–28.00
-14 ≤ h ≤ 14, -18 ≤ k ≤ 21,
-24 ≤ l ≤ 24
22 550
c/pm
a/◦
b/◦
g /◦
V/nm3
Z
D
calcd/Mg m-3
1.978
6.805
1120
Absorption coefficient/mm-1
F(000)
Crystal size/mm3
0.20 ¥ 0.10 ¥ 0.10
2.07–29.40
-12 ≤ h ≤ 12, -21≤ k ≤ 23,
-15 ≤ l ≤ 10
12 871
Theta range for data collection (Hmin–Hmax)/◦
Index ranges
Reflections collected
Independent reflections
Completeness to Hmax
Absorption correction
Min./max. transmission
Refinement method
Restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2s(I)]
R indices (all data)
4868 [Rint = 0.0259]
6044 [Rint = 0.0230]
14 616 [Rint = 0.0410]
89.3%
89.2%
91.8%
SADABS
SADABS
Numerical
0.5494/0.3431
Full-matrix least-squares on F2
4868/0/225
0.4535/0.3331
Full-matrix least-squares on F2
6044/0/260
1.080
R1 = 0.0332, wR2 = 0.0789
R1 = 0.0384, wR2 = 0.0809
2.527 and -1.441
0.5651/0.3590
Full-matrix least-squares on F2
14 616/0/775
0.706
R1 = 0.0326, wR2 = 0.0551
R1 = 0.0763, wR2 = 0.0605
1.134 and -1.420
1.041
R1 = 0.0271, wR2 = 0.0645
R1 = 0.0395, wR2 = 0.0685
1.014 and -1.436
-3
˚
Largest diffraction peak and hole/e A
a result of the slow diffusion of the two solutions, complex 3
was obtained (0.02 g, 49%) as pale yellow crystals suitable for
X-ray analysis, mp 239 ◦C. Found: C 47.9, H 3.2, Cl 8.0. Calcd%
C36H30As2Cl2O2Pt: C 47.5, H 3.3, Cl 7.8%, IR/cm-1: 317, 325
(Pt–Cl), 277 (Pt–As). dH/ppm (300 MHz, CDCl3, Me4Si): 6.34
(dd, 2H, J1,2 = 6.98, J1,3 = 1.13), 6.87–7.21 (m, 6H), 7.39–7.45 (m,
12H), 7.74 (m, 8H). An OH resonance was not observed.
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We gratefully acknowledge financial support from the EU Marie
Curie Training Site HPMT-CT-2000-00110 and the International
Postgraduate Programm (IPP ‘Research at the Frontiers of Chem-
istry’) at the University of Leipzig (N. M.). We thank Umicore
for the generous donation of H2[PtCl6]·6H2O. The authors from
‘I. Chiricuta’ Cancer Institute Cluj Napoca want to express their
gratitude to Dr Yael S. Schiffenbauer for all her help and support
during this study.
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