Application of fluorescence microscopy for investigation of cellular distribution of dinuclear platinum anticancer drugs

Article abstract of DOI:10.1021/jm050216h

The dinuclear platinum complexes with aliphatic diamines [{cis-Pt(NH ₃)₂Cl}₂(μ-H₂N(CH ₂)₆-NH₂)](NO₃)₂ (1,1/c,c) and [{trans-Pt(NH₃)₂Cl}₂(μ-H ₂N(CH₂)₄NH₂)](NO₃) ₂ (l,l/t,t), which are known to be highly active in vitro against several cancer cell lines, have been modified with a fluorogenic reporter (carboxyfluorescein diacetate, CFDA) and a hapten (dinitrophenyl, DNP). These labeled complexes have been designed for fluorescence microscopy investigation of cellular pathways of promising dinuclear platinum anticancer drugs and present the first example of labeling biologically active dinuclear platinum complexes with a fluorescent reporter. The modified compounds interact with a guanine model base similarly to the label-free parent complexes. The uptake of the complexes with a fluorescent label and the respective unlabeled complexes in the U2-OS human osteosarcoma cell line and its cisplatin-resistant derivative, U2-OS/Pt cell line has been investigated. Cellular processing of the CFDA- and DNP-modified dinuclear platinum complexes in U2-OS and U2-OS/Pt cells has been studied.

Full text of DOI:10.1021/jm050216h

J. Med. Chem. 2005, 48, 5191-5202
5191
Application of Fluorescence Microscopy for Investigation of Cellular
Distribution of Dinuclear Platinum Anticancer Drugs
Ganna V. Kalayda,† Guofang Zhang,† Tsion Abraham,‡ Hans J. Tanke,*.‡ and Jan Reedijk*,†
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands,
and Laboratory for Cytochemistry and Cytometry, Department of Molecular Cell Biology, Leiden University Medical Center,
Wassenaarseweg 72, 2333 AL Leiden, The Netherlands
Received March 9, 2005
The dinuclear platinum complexes with aliphatic diamines [{cis-Pt(NH3)2Cl}2(µ-H2N(CH2)6-
NH2)](NO3)2 (1,1/c,c) and [{trans-Pt(NH3)2Cl}2(µ-H2N(CH2)4NH2)](NO3)2 (1,1/t,t), which are
known to be highly active in vitro against several cancer cell lines, have been modified with a
fluorogenic reporter (carboxyfluorescein diacetate, CFDA) and a hapten (dinitrophenyl, DNP).
These labeled complexes have been designed for fluorescence microscopy investigation of cellular
pathways of promising dinuclear platinum anticancer drugs and present the first example of
labeling biologically active dinuclear platinum complexes with a fluorescent reporter. The
modified compounds interact with a guanine model base similarly to the label-free parent
complexes. The uptake of the complexes with a fluorescent label and the respective unlabeled
complexes in the U2-OS human osteosarcoma cell line and its cisplatin-resistant derivative,
U2-OS/Pt cell line has been investigated. Cellular processing of the CFDA- and DNP-modified
dinuclear platinum complexes in U2-OS and U2-OS/Pt cells has been studied.
Introduction
cancer agents because the sensitivity of cancer cells to
a given drug varies significantly between tumor cell
types.
Dinuclear and polynuclear cationic platinum com-
plexes present a new, promising class of anticancer
drugs. A number of these complexes exhibit high activity
against various types of tumors, and their cytotoxic
profiles are often different from the profiles of cisplatin
and its analogues.1-4 Many polynuclear platinum com-
plexes overcome intrinsic and acquired resistance to
cisplatin.5-7 Their positive charge provides high affinity
for the presumed cellular target of antitumor drugs, i.e.
nuclear DNA, and additional positive charge introduced
in the bridging ligand appears to result in higher
anticancer activity. Dinuclear platinum complexes with
polyamines spermine and spermidine, and the tri-
nuclear complex BBR3464 that now undergo clinical
trials, serve as good examples.7-10
Different cytotoxic profiles of dinuclear platinum
complexes and their ability to overcome cisplatin resis-
tance are believed to result from structurally different
DNA adducts formed by these compounds.5,11-13 How-
ever, uptake and intracellular transport also play an
important role in the mechanism of action of platinum-
based drugs and in the mechanism of resistance.14-17
Cellular organelles are known to participate in trans-
port of platinum anticancer complexes18-21 and have
been recently found to contribute to cisplatin resistance
and cross-resistance.20 Thus, investigation of cellular
distribution of platinum antitumor drugs might help to
better understand their mechanism of action on an
intracellular level. This knowledge is of great impor-
tance for the development of new tumor-specific anti-
Digital fluorescence microscopy enables visualization
of fluorescent compounds in cells. A big advantage of
this method is that it allows observation of the cells in
a living state. Fluorescence microscopy has been widely
used to study cellular distribution of various organic
drugs22-24 and has been successfully applied to platinum
complexes.18-20 Because most dinuclear platinum anti-
cancer complexes are not intrinsically fluorescent, they
need to be modified with a fluorescent or fluorogenic
tag in order to visualize them in tumor cells.
This paper describes the synthesis and cellular pro-
cessing of new dinuclear platinum complexes modified
with a fluorogenic reporter or a hapten (Figure 1), which
have been designed as model compounds for investiga-
tion of cellular distribution and processing of promising
antitumor dinuclear platinum complexes. Approaches
of labeling dinuclear platinum complexes of cis and
trans geometries with fluorogenic tags are presented.
The pharmacokinetics properties of the labeled model
complexes and their parent dinuclear complexes are
compared. Cellular processing of the new labeled di-
nuclear platinum complexes in cancer cells is described.
Results
Synthesis. The labeled dinuclear platinum complexes
of cis geometry 1,1/c,c/CFDA and 1,1/c,c/DNP (Figure
1), where 1,1/c,c/CFDA is [bis{(1-(5-(and 6-)-carboxy-
fluorescein-diacetate-aminomethyl)-1,2-ethylenedi-
amine)chloroplatinum(II)}(µ-1,6-hexanediamine)] chlo-
ride and 1,1/c,c/DNP is [bis{(1-(2,4-dinitrophenyl-hex-
anoic-aminomethylene)-1,2-ethylenediamine)chloroplat-
inum(II)}(µ-1,6-hexanediamine)] chloride, were designed
similarly to the previously described mononuclear com-
plexes CFDA-Pt and DNP-Pt,18 which represent labeled
* To whom correspondence should be addressed. For H.J.T.: phone,
+31715276190; fax, +31715276180; e-mail: h.j.tanke@lumc.nl.
For J.R.: phone, +31715274459; fax, +31715274671; e-mail:
reedijk@chem.leidenuniv.nl.
† Leiden University.
‡ Leiden University Medical Center.
10.1021/jm050216h CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/16/2005

5192 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Kalayda et al.
Figure 1. Schematic representation of the labeled dinuclear platinum complexes 1,1/c,c/CFDA, 1,1/c,c/DNP, 1,1/t,t/CFDA, and
1,1/t,t/DNP, the parent dinuclear complexes 1,1/c,c and 1,1/t,t, and the compounds used in the control experiments, CFDA-Pt,
DNP-Pt, CFDA-Boc, and DNP-Boc.
Figure 2. Schematic representation of the synthetic route used for preparation of 1,1/c,c/CFDA and 1,1/c,c/DNP.
analogues of cisplatin. A standard method for prepara-
tion of dinuclear platinum complexes was used to
synthesize the dinuclear precursor 2 (Figure 2). It has
been obtained in a good yield and probably represents

Fluorescence Microscopy for Anticancer Drugs
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5193
Figure 3. Schematic representation of the geometric isomers of 2 (R ) Boc, X ) NO3), 3 (R ) H, X ) Cl), 4a (R ) CFDA, X )
Cl), and 4b (R ) DNP, X ) Cl).
a mixture of the three isomers shown in Figure 3.
Moreover, each of these geometric isomers consists of
three diasteoreomers (R,R; R,S (S,R); S,S). Subsequent
removal of the Boc group did not affect the dinuclear
platinum moiety and resulted in the formation of 3 with
negligible amounts of impurities. The labeled com-
pounds 4a and 4b were obtained by coupling of 3 with
the active esters of CFDA and DNP probes, respectively.
Compounds 2, 3, 4a, and 4b most likely represent
mixtures of isomeric complexes. However, 1H NMR
spectra did not allow us to distinguish among different
isomers for any of the compounds because the chemical
shifts of the protons have been found to be the same,
independent of the ligand location around platinum.
Since all isomers 4a (and also all isomers 4b) represent
equally good fluorogenic models of 1,1/c,c, separation
was not felt to be necessary and the compounds were
used in biological studies as mixtures of isomers. The
purity of the compounds overall was high, as verified
with HPLC/ESI-MS and elemental analysis (as located
amine 5 would provide an opportunity to introduce a
label (CFDA or DNP) relatively far away from the
dinuclear platinum moiety, minimizing possible steric
hindrance. Subsequently, the trifluoroacetyl protection
group was cleaved off by NaOH, while the acid-labile
tert-butoxycarbonyl group was not affected. In such a
way, two amino groups would be available for platina-
tion, and the Boc-protected amino group could later be
replaced with the fluorogenic reporter or the hapten.
Platination of ligand 8 was performed with [trans-Pt-
(NH3)2Cl(dmf)](NO3) species according to the standard
procedure for the synthesis of dinuclear platinum
complexes. Subsequent removal of the Boc group and
reaction with an active ester of CFDA and DNP label
yielded 11a (1,1/t,t/CFDA) and 11b (1,1/t,t,DNP), re-
spectively, which were purified by HPLC. LC/ESI-MS
analysis and 1H and 195Pt NMR spectrometry confirmed
the purity of the products.
Reaction with a Guanine Model Base. The com-
plexes modified with the DNP hapten (1,1/c,c/DNP and
1,1/t,t/DNP) were allowed to react with 9-ethylguanine
(9EtG) at 310 K to investigate how the labeled com-
plexes interact with nucleobases. The parent compounds
1,1/c,c and 1,1/t,t were studied as references. For all four
complexes, the signal of the H8 proton of 9EtG at 7.88
ppm decreases in intensity during the course of the
reaction, while the signals of H8 protons of the products
increase in intensity, indicating platination of the
nucleobase. Only one signal of the product was observed
in the case of a 1,1/c,c/DNP mixture of isomers, indicat-
ing that all of them interact with a guanine model base
identically. Interestingly, the H8 signals of the 9EtG
adducts of 1,1/c,c/DNP and 1,1/c,c appear at 8.30 and
8.31 ppm, respectively, and the H8 signals of the 9EtG
adducts of both 1,1/t,t/DNP and 1,1/t,t appear at 8.27
ppm. This suggests similar conformation of the nucleo-
base in the labeled and label-deprived 9EtG adducts.
The pH titration of the 9EtG adducts of the labeled
and parent complexes was performed. The plots of the
chemical shifts of H8 proton as a function of pH are
presented in Figure 5 and clearly show the absence of
the (de)protonation process at the N7 atom of 9EtG at
low pH, which indicates platinum binding at this
position.26,27 At the same time, (de)protonation at N1
was observed providing additional confirmation for
platinum coordination at N7. Moreover, pH titration
curves for the 9EtG adduct of 1,1/t,t and its DNP-labeled
1
in Supporting Information) and confirmed by H and
195Pt NMR. Any impurities are so small that they are
unlikely to affect the observed biological data. The
relatively large deviation from the theoretical values for
the carbon analyses of 4a and 4b is ascribed to the
presence of the solvent in the samples and will have no
influence on the biological data.
For structural reasons, the dinuclear platinum com-
plexes of trans geometry could not be designed in a
similar fashion. Considering the stability of the result-
ing labeled complexes and possible synthetic routes for
their preparation, introducing the label in the linker
between two platinum atoms appeared to be the best
option. The synthesis of 1,1/t,t/CFDA and 1,1/t,t/DNP
(Figure 1), where 1,1/t,t/CFDA is [bis{trans-diammine-
chloroplatinum(II)}{µ-(2,5-diamino-N-(4-(5-(and 6-)-car-
boxy-fluorescein-diacetate-amino)butyl)valeramide)}] tri-
fluoroacetate and 1,1/t,t/DNP is [bis{trans-diammine-
chloroplatinum(II)}{µ-(2,5-diamino-N-(4-(2,4-dinitrophenyl-
hexanoic-amino)butyl)valeramide)}] trifluoroacetate, is
schematically presented in Figure 4. Ligand 8 was
prepared in three steps from ornithine hydrochloride,
similar to the method previously described by some of
us.25 First, the amino groups of ornithine were protected
by reaction with ethyl trifluoroacetate. This ensured
that no side reactions would take place at the next step
of coupling of amine 5 to the ornithine moiety. Attaching

5194 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Kalayda et al.
Figure 4. Schematic representation of the synthetic route used for preparation of 1,1/t,t/CFDA and 1,1/t,t/DNP.
counterpart almost coincide, again confirming similar
conformation of the guanine molecule in both adducts.
A deviation of the pH titration curve for the 1,1/c,c/DNP
adduct with 9EtG from that for the 1,1/c,c adduct can
be explained by the influence of the chelating ligands
present in the labeled complex.
Thus, these data suggest that the DNP-labeled con-
structs interact with nucleobases in a way similar to
that of the label-free parent compounds.
Uptake of the Complexes by U2-OS and U2-OS/
Pt Osteosarcoma Cells. To compare some of the
pharmacokinetics properties of the labeled and parent
complexes, their uptake by U2-OS and U2-OS/Pt cells
was studied by means of measuring platinum concen-
tration inside the cells after 30 min, 2 h, and 24 h of
incubation.
Figure 6 shows that for all compounds platinum
accumulation in both cell lines increases as a function
of incubation time. Uptake of cisplatin and the fluores-
cent labeled model complexes, CFDA-Pt and DNP-Pt,
is lower in the resistant cell line than in the sensitive
one at any period of incubation. This is in agreement
with previously reported data, which show decreased
cisplatin accumulation in the U2-OS/Pt cell line com-
pared to its sensitive counterpart.28 In contrast, no great
difference in platinum accumulation between U2-OS
and U2-OS/Pt cell lines was found for any of dinuclear
platinum complexes. Thus, the labeled derivatives of
cisplatin and dinuclear platinum complexes mimic the
behavior of the parent compounds. Nevertheless, some
differences in cellular uptake between the labeled and
the label-deprived complexes were observed. Accumula-
tion of 1,1/c,c/CFDA and 1,1/c,c/DNP is significantly
higher than that of the parent complex, 1,1/c,c, most
likely because of the presence of two lipophilic reporters
per molecule (Figure 6b). As is clear from parts a and c
of Figure 6, introducing one label per molecule (as in
the case of CFDA-Pt or 1,1/t,t/CFDA) does not have a
significant influence on the accumulation of the com-
plexes in the cells.

Fluorescence Microscopy for Anticancer Drugs
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5195
Figure 5. Plots of the chemical shift (δ, ppm) of the H8
resonance vs pH for the 9EtG adducts of 1,1/c,c/DNP (O) and
1,1/c,c (4), for the 9EtG adducts of 1,1/t,t/DNP (/) and 1,1/t,t
(0), and for the free 9EtG ligand (×). pH titrations were
performed in 0.1 M NaClO4/D2O solutions at 298 K.
Figure 7. Cellular processing of 1,1/c,c/CFDA in U2-OS
human osteosarcoma cells. The images taken 15 min after
internalization are presented in the top row. Images 1 h after
internalization are in the middle row, and images 24 h after
internalization are in the bottom row. Phase contrast images
of the cells at these time points are presented on the left,
corresponding fluorescence images are in the middle, and
superimposed images are on the right.
Figure 8. Cellular processing of 1,1/t,t/CFDA in U2-OS
human osteosarcoma cells. The images taken 15 min after
internalization are presented in the top row. Images 1 h after
internalization are in the middle row, and images 24 h after
internalization are in the bottom row. Phase-contrast images
of the cells at these time points are presented on the left,
corresponding fluorescence images are in the middle, and
superimposed images are on the right.
on cell viability.29,30 The cells were incubated with 1,1/
c,c/CFDA and 1,1/t,t/CFDA and then observed live using
digital fluorescence microscopy. The mononuclear fluo-
rescent-labeled complex CFDA-Pt and metal-free CFDA-
Boc were used in control experiments (images not
shown). The phase contrast and fluorescence images
of the cells after incubation with 1,1/c,c/CFDA and
1,1/t,t/CFDA are presented in Figures 7 and 8, respec-
tively.
As is clear from Figures 7 and 8, cellular distribution
of the dinuclear platinum complexes of cis and trans
geometries is quite similar. Both compounds quickly
enter the cells; several minutes after internalization, the
fluorescence can already be seen throughout the entire
cell. One hour later, fluorescence is mainly concentrated
in the nucleus. Interaction of the platinum complexes
with genomic DNA probably occurs at this stage.
Figure 6. Platinum concentration in U2-OS and U2-OS/Pt
human osteosarcoma cells after 30 min, 2 h, and 24 h of
incubation with the labeled and label-free dinuclear platinum
complexes. The values represent the average of the measure-
ments.
Fluorescence Studies in Living Cells. The CFDA-
labeled dinuclear complexes were used to investigate
cellular processing of potential dinuclear platinum
antitumor drugs in U2-OS human osteosarcoma cells.
CFDA is a fluorogenic label, which is readily taken up
by cells. In the cytoplasm, the acetate esters of CFDA
are cleaved by cellular esterases, yielding a fluorescent
moiety.18 CFDA is known to have a negligible impact

5196 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Kalayda et al.
Figure 9. Combination of 1,1/c,c/CFDA accumulation 24 h
after internalization and staining with Bodipy-TR-ceramide
(a Golgi specific dye) in living U2-OS cells: (A) phase-contrast
image of living U2-OS cells; (B) 1,1/c,c/CFDA (green) fluores-
cence in these cells 24 h after internalization; (C) staining of
the Golgi complex with Bodipy-TR-ceramide (red); (D) super-
imposed representation of the images of 1,1/c,c/CFDA (green)
and Bodipy-TR-ceramide (red).
Figure 11. Cellular processing of 1,1/c,c/DNP in U2-OS
human osteosarcoma cells. The images of the cells fixed 15
min after internalization are presented in the top row, those
of the cells fixed 1 h after internalization are in the middle
row, and images of the cells fixed 24 h after internalization
are in the bottom row. Phase-contrast images of the cells at
these time points are presented on the left, corresponding
fluorescence images are in the middle, and superimposed
images are on the right.
show that the fluorescence of 1,1/c,c/CFDA and 1,1/t,t/
CFDA is localized in the Golgi complex.
Cellular processing of CFDA-Pt and CFDA-Boc, which
were used in control experiments, was found to be
similar to the previously described processing by Mo-
lenaar et al.18 As is clear from the results of the control
experiment, cellular processing of the fluorescent la-
beled dinuclear complexes is very similar to that of
CFDA-Pt. CFDA-Boc is quickly taken up by the cells,
equally distributed throughout the cell, and excreted
within 5-6 h, showing no specific localization in the
nucleus or cellular organelles. This confirms that the
results of fluorescence observations for the CFDA-
labeled dinuclear platinum complexes are induced by a
platinum moiety and not by CFDA label itself.
Figure 10. Combination of 1,1/t,t/CFDA accumulation 24 h
after internalization and staining with Bodipy-TR-ceramide
(a Golgi specific probe) in living U2-OS cells: (A) phase-
contrast image of living U2-OS cells; (B) 1,1/t,t/CFDA (green)
fluorescence in these cells 24 h after internalization; (C)
staining of the Golgi complex with Bodipy-TR-ceramide (red);
(D) superimposed representation of the images of 1,1/t,t/CFDA
(green) and Bodipy-TR-ceramide (red).
To verify that the observed results are label-indepen-
dent, cellular processing of the structurally similar
DNP-modified complexes 1,1/c,c/DNP and 1,1/t,t/DNP
in U2-OS cells was studied. DNP was chosen as a well-
known hapten, which is easily visualized with DNP-
specific antibodies. The cells were incubated with the
DNP constructs under the same conditions as in the
case of the CFDA-labeled complexes. For the observa-
tions, the cells were fixed at different time points after
incubation, and DNP was visualized by indirect immu-
nofluorescence. The images of the cells incubated with
1,1/c,c/DNP and 1,1/t,/DNP are presented in Figures 11
and 12, respectively. Both cis and trans DNP constructs
show accumulation in the nucleus 1 h after internaliza-
tion. Localized accumulation in a cytoplasmic area close
to the nucleus has been observed 24 h after internaliza-
tion with 1,1/c,c/DNP and 1,1/t,t/DNP, similar to the
case of CFDA constructs. No specific accumulation in
U2-OS cells was observed after incubation with DNP-
Boc (images not shown). Thus, cellular distribution of
DNP-labeled dinuclear complexes is not different from
that of structurally similar CFDA-labeled counterparts.
These observations and the results of cellular processing
experiments with platinum-free CFDA-Boc and DNP-
Besides, fluorescence of the trans complex was also
observed in the cytoplasm, next to the nucleus. Four to
five hours after internalization, the fluorescence in the
nucleus becomes weaker and punctuate staining in the
area of cytoplasm close to the nucleus is detected (data
not shown). The same cellular distribution pattern is
observed 24 h after internalization, with the signal in
the nucleus being very weak. On the basis of spatial
considerations, we supposed the cytoplasmic region close
to the nucleus, where the fluorescent complexes ac-
cumulate, to be the Golgi complex. To verify this
hypothesis, a colocalization experiment with a Golgi-
specific fluorescent dye was performed. At 24 h after
internalization, Bodipy-Texas red (TR)-ceramide was
added, and the cells were incubated for 20 min. The
images of the cells incubated with 1,1/c,c/CFDA and the
Bodipy-TR-ceramide are shown in Figure 9, and images
of cells after incubation with 1,1/t,t/CFDA and Bodipy-
TR-ceramide are presented in Figure 10. Green fluo-
rescence of the cis and trans complexes can be seen in
Figures 9B and 10B, respectively, and red fluorescence
of Bodipy-TR-ceramide is shown in Figures 9C and 10C.
The superimposed images (Figures 9D and 10D) clearly

Fluorescence Microscopy for Anticancer Drugs
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5197
As is clear from Table 1, the dinuclear platinum
complex of cis geometry, 1,1/c,c, is as active as cisplatin,
whereas the trans complex 1,1/t,t is significantly less
cytotoxic in the U2-OS osteosarcoma cell line. Such low
activity of the trans complex is surprising because the
trinuclear platinum complex of trans geometry, BBR3464,
has been reported to be much more active than cisplatin
in this cell line.28,31 Both dinuclear platinum complexes
show no cross-resistance with cisplatin. Interestingly,
the U2-OS/Pt cell line has a lower (2.5-fold) degree of
resistance than found earlier by Perego et al. (6-fold).28
Discussion
Labeling dinuclear platinum complexes with a fluo-
rescent or fluorogenic tag gives us an excellent op-
portunity to study cellular pathways of these promising
anticancer drugs using fluorescence microscopy. Time-
lapse digital fluorescence microscopy allows us to follow
the processing of compounds in the cells growing at 37
°C in vitro. Investigation of cellular distribution of
intrinsically fluorescent organic antitumor drugs helped
to reveal the role of cellular organelles, such as lysos-
omes, Golgi apparatus, and mitochondria, in transport
and detoxification of the drugs,22,32 as well as involve-
ment of organelles in resistance mechanisms.24,33 Fluo-
rescence microscopy was also applied to study cellular
behavior of biologically active platinum complexes. To
visualize the latter in cells, antibodies against Pt-DNA
adducts have been developed.34 A cisplatin analogue
labeled with a fluorogenic tag had been synthesized
earlier, and investigation of its cellular processing
revealed involvement of the Golgi apparatus in the
transport of platinum complexes.18 Cellular distribution
of the dinuclear platinum complexes with fluorescent
anthraquinones has also been studied and provided new
insights in the resistance mechanisms in A2780 human
ovarian carcinoma cells.20
The platinum complexes modified with the CFDA
moiety, which are described in this work, present the
first example of labeling dinuclear platinum complexes
with a fluorescent or fluorogenic tag. A label can be
introduced in the bridging ligand as well as in the side
chain. The former approach is especially useful in the
case of trans complexes. The labeled complexes interact
with a guanine model base similarly to the parent
compounds, indicating that modification with a fluoro-
genic tag does not significantly influence binding of the
dinuclear platinum complexes to nucleobases. Cellular
uptake of the labeled compounds is affected by the
presence of a lipophilic fluorescent moiety. It has been
suggested by Hall et al.35 that CFDA-labeled complexes
diffuse in and out of the cells and intracellular compart-
ments at a greater rate compared to the parent com-
pounds. Our results show that cellular accumulation of
the complexes containing two reporters per molecule is
indeed significantly higher. Nevertheless, the modified
complexes appear to mimic the uptake of the parent
compounds. Accumulation of cisplatin and its modified
analogues is lower in cisplatin-resistant cells than in
sensitive cells, which is in agreement with the resistance
profile of the U2-OS/Pt cell line.28 The uptake of the
dinuclear platinum complexes (1,1/c,c and 1,1/t,t) and
the respective model complexes with a fluorescent probe
by sensitive and resistant osteosarcoma cells is the
Figure 12. Cellular processing of 1,1/t,t/DNP in U2-OS
human osteosarcoma cells. The images of the cells fixed 15
min after internalization are presented in the top row, those
of the cells fixed 1 h after internalization are in the middle
row, and images of the cells fixed 24 h after internalization
are in the bottom row. Phase-contrast images of the cells at
these time points are presented on the left, corresponding
fluorescence images are in the middle, and superimposed
images are on the right.
Table 1. In Vitro Cytotoxicity Assay of 1,1/c,c, 1,1/t,t, Cisplatin
and Their Fluorescent-Labeled Derivatives on U2-OS Human
Osteosarcoma Cell Line and Cisplatin-Resistant U2-OS/Pt Cell
Line
IC50 (µM)
complex
U2-OS
U2-OS/Pt
RFa
1,1/c,c/CFDA
1,1/c,c/DNP
1,1/c,c
1,1/t,t/CFDA
1,1/t,t/DNP
1,1/t,t
CFDA-Pt
DNP-Pt
cisplatin
>50
>50
5.1
>50
>50
75.1
>50
>50
2.9
>50
>50
6.1
>50
>50
38.6
>50
>50
7.3
nab
nab
1.2
nab
nab
0.5
nab
nab
2.5
a RF ) the relative ratio of IC50 values in both cell lines ((U2-
OS/Pt)/U2-OS). b na ) not available.
Boc prove that the observed cellular distribution pattern
is induced by a dinuclear platinum moiety.
Cellular processing of the labeled dinuclear platinum
complexes in the cisplatin-resistant U2-OS/Pt cell line
was also investigated. No differences in cellular distri-
bution of the compounds between sensitive and resistant
cells were found (images not shown). The same observa-
tion was previously made in the case of the mononuclear
complex CFDA-Pt18 and was confirmed in our control
experiment.
In Vitro Cytotoxicity Assay in U2-OS and U2-OS/
Pt Cell Lines. The cytotoxicity of the labeled and label-
free parent dinuclear platinum complexes has been
evaluated in U2-OS human osteosarcoma cell line and
its cisplatin-resistant derivative U2-OS/Pt cell line. The
results are given in Table 1. The labeled complexes are
poorly water-soluble and were dissolved in dimethyl-
formamide for the biological tests. Unfortunately, the
toxicity of the solvent at the desired compound concen-
tration did not allow us to precisely determine the IC50
values for the labeled complexes.

5198 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Kalayda et al.
same, which reflects the lack of cross-resistance of 1,1/
c,c and 1,1/t,t with cisplatin in U2-OS/Pt cells. Thus, it
can be concluded that the CFDA- and DNP-labeled
compounds represent good models for the respective
parent dinuclear platinum complexes.
No involvement of lysosomes in the exocytosis of
platinum complexes, as reported for other cell lines,19,41,42
was observed. Also, no accumulation in mitochondria,
as described for other types of cancer cells,43-46 was
found. Apparently, the cellular distribution pattern may
vary significantly between different cancer cell lines.
This has been recently confirmed by investigation of
cellular processing of the dinuclear platinum complexes
with anthraquinones in different cell lines.21
No differences in cellular processing of the labeled
dinuclear platinum complexes in U2-OS and U2-OS/Pt
cells were observed, which is completely in agreement
with the lack of cross-resistance of both 1,1/c,c and 1,1/
t,t with cisplatin. In fact, the U2-OS/Pt cell line has a
relatively low (2.5-fold) degree of resistance. This pro-
vides an explanation for the similar cellular distribution
of CFDA-Pt in sensitive and resistant osteosarcoma cells
as reported by Molenaar et al.18
Cellular processing of the labeled dinuclear complexes
in U2-OS human osteosarcoma cells appeared to be
similar to the previously described cellular distribution
of the mononuclear complex CFDA-Pt.18 Using two
different labels (CFDA and DNP) and the control
experiments with CFDA-Boc and DNP-Boc ensures that
the observed results are induced by a dinuclear plati-
num moiety. Interestingly, no differences in cellular
processing between the complexes with cis and trans
geometries were found. In contrast to cis-configured
dinuclear platinum complexes, complexes with the trans
configuration are known to lose their bridging ligand
upon interaction with glutathione.36,37 Therefore, the
labeled complexes 1,1/t,t/CFDA and 1,1/t,t/DNP could
possibly release their fluorescent linking ligand inside
the cell, resulting in a different cellular distribution
pattern. It must be noted, however, that platinum-free
controls (CFDA-Boc and DNP-Boc) are processed by
cells in a different way compared to the dinuclear
complexes. This finding indicates that the release of the
bridging ligand from 1,1/t,t/CFDA and 1,1/t,t/DNP in
U2-OS cells is negligible, most probably because of the
relatively low GSH content in this cell line.28
The dinuclear complexes enter the cell rapidly, within
minutes. Given their positive charge, this finding indi-
cates active mechanism of uptake for dinuclear com-
plexes. The results of cellular uptake measurements
presented in this paper, as well as some earlier uptake
studies,38 show that dinuclear platinum complexes
accumulate in the cells to the same extent or better than
cisplatin. Within 1 h, they accumulate in the nucleus
where they bind to nuclear DNA. The fluorescence
intensity in the nucleus weakens with time, suggesting
that the labeled complexes are partially removed. In
fact, only a fraction of the complexes binds to DNA, and
as already mentioned, they are likely to diffuse out of
the nucleus. However, removal of platinum-DNA lesions
by nuclear excision repair proteins, as a part of DNA
repair process, also contributes to the overall decrease
of platinum concentration in the nucleus. Several hours
after internalization, the dinuclear platinum complexes
are localized in the Golgi complex, and this localization
is still observed 24 h after internalization. Accumulation
in the Golgi apparatus in sensitive and resistant U2-
OS cell lines has been reported not only for the mono-
nuclear CFDA-labeled analogue of cisplatin18 but also
for the dinuclear platinum complexes with fluorescent
anthraquinones.21 There is mounting evidence indicat-
ing that the Golgi plays an important role in drug
transport.23,24,39 The Golgi complex is probably a site
where platinum complexes accumulate before being
excreted via exocytosis. The platinum compounds might
be exocytosed as a part of general mechanism by which
secretory proteins are transported to the external cell
surface. P-glycoprotein, which is known to be involved
in the efflux of various drugs, is localized on the lumenal
side of Golgi stack membranes in U2-OS cells.40 How-
ever, it is not certain whether P-glycoprotein partici-
pates in the efflux of platinum complexes.
Concluding Remarks
Dinuclear platinum complexes modified with a fluo-
rescent or fluorogenic reporter present a useful tool for
the investigation of cellular distribution of these prom-
ising antitumor drugs. Fluorescence microscopy studies
of their cellular trafficking might provide new insights
in the mechanism of action of this class of anticancer
compounds. Given that many dinuclear platinum com-
plexes overcome cisplatin resistance, labeled complexes
might help to elucidate resistance mechanisms. Using
cell lines with different resistance profiles is especially
interesting because it might reveal new specific features
of a given cell type.
Experimental Section
Instruments. 1H and 195Pt NMR spectra were recorded on
a Bruker DPX 300 MHz spectrometer with a 5 mm multi-
nucleus probe. The temperature was kept constant by a
1
variable-temperature unit. H and 195Pt chemical shifts were
referenced to TSP and Na2PtCl6 (δ ) 0 ppm), respectively. C,
H, and N analyses were performed by the microanalytical
laboratory of Leiden Institute of Chemistry, Leiden University,
The Netherlands.
The mass spectroscopic measurements were performed on
a Finnigan Aqa mass spectrometer equipped with an electro-
spray interface ionization source. Sample solutions were
introduced in the ESI source using an HPLC autosampler and
an acetonitrile/water (50/50) mixture as an eluent running at
0.2 mL/min.
LC/ESI-MS experiments were carried out on a Alltima 3 µm
C18 reversed-phase column (150 mm × 4.6 mm) with a flow
rate of 1 mL/min, sample load of 10-50 µL (1 mg/mL), and
the following gradient conditions: 95% eluent B for 2 min, then
from 95% eluent B to 70% eluent B in 20 min, and subse-
quently from 70% eluent B to 10% eluent B in 8 min (eluent
A, acetonitrile with 0.1% CF3COOH; eluent B, 0.1% aqueous
solution of CF3COOH). After that, the column flow is split.
One part flows through the UV detector (254 nm). The other
part (0.2 mL/min) is directed to the Finnigan Aqa mass
detector operating in electrospray mode.
The samples were purified with reversed-phase chromatog-
raphy using an Alltima 5 µm C18 column (250 mm × 10 mm)
with a flow rate of 4 mL/min and a repetitive sample load of
500 µL using the following gradient: 95% eluent B for 2.4 min,
then from 95% eluent B to 70% eluent B in 23.6 min, and
subsequently from 70% eluent B to 10% eluent B in 9.5 min
(eluent A, acetonitrile with 0.1% CF3COOH; eluent B, 0.1%

Fluorescence Microscopy for Anticancer Drugs
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5199
aqueous solution of CF3COOH). Fractions were collected and
analyzed by mass spectrometry, and the desired fraction was
lyophilized.
(s, 6H, Hj), 1.68 (m, 2H, H2), 1.27 (m, 2H, H3). 195Pt NMR
(DMF-d7): δ -2590.
Bis{(1-(2,4-dinitrophenylhexanoic-aminomethylene)-
1,2-ethylenediamine)chloroplatinum(II)}(µ-1,6-hexanedi-
amine) Chloride (1,1/c,c/DNP, 4b). A solution of 3 obtained
as described above was neutralized with 2 M NaOH until pH
7.5 was attained. Then it was cooled to 0 °C, and a solution of
9.0 mg of DNP-X SE (2.28 × 10-2 mmol) in 1.5 mL of DMF
was added dropwise. The resulting solution was stirred first
for 20 min at 0 °C and then for 30 min more at room
temperature. The solvents were subsequently removed in
vacuo. The residue was stirred vigorously with 4 mL of ice-
cold water, filtered off, and washed three times with 4 mL of
ice-cold water, three times with 4 mL of ethanol, and three
times with diethyl ether. The sample was dried in vacuo.
Yield: 6 mg (35%). Anal. (C36H64N14O10Cl4Pt2). C: calcd, 31.22;
found, 32.22. H: calcd, 4.66; found, 3.86. N: calcd, 14.16;
Synthesis. Materials. CFDA-Pt, DNP-Pt, CFDA-Boc, DNP-
Boc, and {1-(tert-butyloxycarbonylaminomethyl)-1,2-ethylene-
diamine}dichloroplatinum(II) (1) were synthesized as de-
scribed by Molenaar et al.18 The trans-Pt(NH3)2Cl2 compound
was prepared according to the literature procedure.47 [{cis-Pt-
(NH3)2Cl}2(µ-H2N(CH2)6NH2)](NO3)2 (1,1/c,c) and [{trans-Pt-
(NH3)2Cl}2(µ-H2N(CH2)4NH2)](NO3)2 (1,1/t,t) were synthesized
as previously described.48-50 K2PtCl4 was obtained from Johnson
& Matthey. Silver nitrate was purchased from Fisher Scientific
Nederland. Ornithine hydrochloride, ethyl trifluoroacetate,
and dicyclohexylcarbodiimide (DCC) were purchased from
Aldrich. Triethylamine, 1,4-butanediamine, 1,6-hexanedi-
amine, and di-tert-butyl dicarbonate were ordered from Acros.
N-Hydroxysuccinimide (SuOH) was purchased from Nova
Biochem. 5-(and 6-)-Carboxyfluorescein diacetate succinimidyl
ester (5(6)-CFDA SE) and 6-(2,4-dinitrophenyl)aminohexanoic
acid succinimidyl ester (DNP-X SE) were obtained from
Molecular Probes Europe BV, Leiden, The Netherlands. 9-Eth-
ylguanine was purchased from Sigma.
1
found, 14.30. H NMR (DMF-d7): δ 8.96 (s, 1H, Hk), 8.35 (d,
1H, Hj), 7.37 (d, 2H, Hi), 3.61 (m, 4H, Hc + Hh), 3.14 (m, 1H,
Hb), 2.78 (under DMF-CH3, 3H, H1 + Ha), 2.63 (m, 1H, Ha′),
2.29 (t, 2H, Hd), 1.78 (m, 2H, Hg), 1.69 (m, 4H, H2 + He), 1.48
(m, 2H, Hf), 1.34 (m, 2H, H3). 195Pt NMR (DMF-d7): δ -2600.
N-tert-Butoxycarbonyl-1,4-butanediamine (5). To a
solution of 7.5 g (85.08 mmol) of 1,4-butanediamine in 30 mL
of dioxane, a solution of 2.4 g (11.00 mmol) of di-tert-butyl
dicarbonate in 30 mL of dioxane was added dropwise over 1.5
h. The solution was stirred at room temperature for 22 h. The
solvent was evaporated in vacuo, and 50 mL of water was
added. The insoluble material was filtered off, and the filtrate
was extracted with 3 × 45 mL of dichloromethane. The
combined organic phase was dried over MgSO4, filtered, and
evaporated to provide the product. Yield: 1.152 g (60.12%).
1H NMR (CDCl3): δ 3.13 (t, 2H, H8), 2.70 (t, 2H, H5), 1.53 (m,
4H, H2 + H3), 1.47 (s, 9H, Boc).
N,N′-Bis(trifluoroacetyl)ornithine (6). To a solution of
4 g (23.72 mmol) of ornithine hydrochloride in 120 mL of
methanol were added 6.67 mL of triethylamine (47.5 mmol)
and 7.05 mL (59.25 mmol) of ethyl trifluoroacetate. The
resulting mixture was stirred at room temperature for 48 h,
filtered, and evaporated to dryness. The white residue was
dissolved in 120 mL of 1 M hydrochloric acid. After evaporation
to dryness, 140 mL of ethyl acetate was added, the solution
was filtered, and the filtrate was concentrated in vacuo. To
the remaining solid, 120 mL of 1 M hydrochloric acid was
added, and the white product was filtered off, washed with 1
M hydrochloric acid, and dried in air. Yield: 6.23 g (81.03%).
Anal. (C9H10N2O4F6) N. C: calcd, 33.34; found, 32.95. H: calcd,
3.11; found, 3.03. 1H NMR (MeOH-d4): δ 4.45 (m, 1H, H1),
3.28 (m, 2H, H4, under MeOH), 1.94 (m, 1H, H2), 1.78 (m, 1H,
H2′), 1.60 (m, 2H, H3).
2,5-Bis(trifluoroacetylamino)-N-(4-tert-butoxycarbo-
nylaminobutyl)valeramide (7). To a solution of 2 g (6.15
mmol) of 6 in 50 mL of tetrahydrofuran were added at 0 °C a
solution of 0.7 g (6.1 mmol) of SuOH in 5 mL of tetrahydro-
furan and a solution of 1.3 g of DCC (6.3 mmol) in 5 mL of
tetrahydrofuran . The mixture was allowed to stand overnight
at 4 °C. The insoluble material was then filtered off, and a
solution of 1.152 g (6.61 mmol) of 5 in 25 mL of ethanol was
added to the filtrate. The resulting solution was stirred at room
temperature for 1 h, filtered, and evaporated to dryness. The
residue was dissolved in 25 mL of ethylene glycol dimethyl
ether, 100 mL of water was added, and the precipitated
product was collected by filtration. The sample was dried in
air. Yield: 2.569 g (84.22%). Anal. (C18H28N4O5F6) C. H: calcd,
5.71; found, 5.58, N: calcd, 11.33; found, 11.14. 1H NMR
(MeOH-d4): δ 4.38 (m, 1H, H1), 3.30 (m, 2H, H4, under MeOH),
3.20 (m, 2H, H8), 3.03 (m, 2H, H5), 1.75-1.85 (m, 2H, H2),
1.55-1.64 (m, 6H, H3 + H6 + H7), 1.40 (m, 9H, Boc).
Bis{(1-(tert-butyloxycarbonylaminomethyl)-1,2-ethyl-
enediamine)chloroplatinum(II)}(µ-1,6-hexanediamine)
Nitrate (2). To a solution of 90.4 mg (0.2 mmol) of 1 in 4.8
mL of dimethylformamide (DMF) was added portionwise over
2 h in the dark a solution of 33 mg (0.19 mmol) of AgNO3 in
1.2 mL of DMF, and the resulting solution was stirred for 5 h
in the dark. The white precipitate of AgCl was then filtered
off, and a solution of 10.4 mg (0.09 mmol) of 1,6-hexanediamine
in 1 mL of DMF was added to the filtrate. The resulting
solution was stirred overnight in the dark and then evaporated
to dryness. The residue was dissolved in 1 mL of methanol.
The product was precipitated with 20 mL of ether, stirred with
ether for several hours to remove traces of DMF, filtered, and
dried in air. Yield: 57 mg (53%). Anal. (C22H54N10O10Cl2Pt2).
C: calcd, 24.47; found, 24.31. H: calcd, 5.04; found, 4.28. N:
calcd, 12.97; found, 12.89. 1H NMR (D2O): δ 3.37 (m, 2H, Hc),
3.02 (m, 1H, Hb), 2.87 (m, 1H, Ha), 2.70 (m, 2H, H1), 2.57 (m,
1H, Ha′), 1.68 (m, 2H, H2), 1.45 (s, 18H, Boc), 1.39 (m, 2H, H3).
195Pt NMR (D2O): δ -2585.
Bis{(1-(aminomethyl)-1,2-ethylenediamine)chloroplat-
inum(II)}(µ-1,6-hexanediamine) Chloride (3). An amount
of 13.4 mg (1.24 × 10-2 mmol) of 2 was dissolved in 1.5 mL of
100 mM hydrochloric acid and heated at 50 °C overnight in
the dark. This resulted in the formation of a solution of 3,
which was directly used for the synthesis of compounds 4a
and 4b. Successful removal of the Boc group under these
conditions was proven by taking an aliquot and performing a
pH titration monitored by 1H NMR. The resonance of Hc
protons showed pH dependence as expected for the protona-
1
tion/deprotonation of the free primary amine. H NMR (D2O,
pH 7.1): δ 3.27 (m, 2H, Hc), 3.01 (m, 1H, Hb), 2.85 (m, 1H,
Ha), 2.75 (m, 3H, H1 + Ha′), 2.57 (m, 1H, Ha′), 1.73 (m, 2H,
H2), 1.43 (m, 2H, H3).
Bis{(1-(5-(and 6-)carboxyfluorescein-diacetate-ami-
nomethyl)-1,2-ethylenediamine)chloroplatinum(II)}(µ-
1,6-hexanediamine) Chloride (1,1/c,c/CFDA, 4a). A solu-
tion of 3 obtained as described above was neutralized with 2
M NaOH until pH 7.5 was attained. Then it was cooled to 0
°C, and a solution of 12.7 mg of 5(6)-CFDA SE (2.28 × 10-2
mmol) in 1.5 mL of DMF was added dropwise. The resulting
solution was stirred first for 20 min at 0 °C and then for 30
min more at room temperature. The solvents were subse-
quently removed in vacuo. The residue was stirred vigorously
with 4 mL of ice-cold water, filtered off, and washed three
times with 4 mL of ice-cold water, three times with 4 mL of
ethanol, and three times with diethyl ether. The sample was
dried in vacuo. Yield: 7 mg (33%). Anal. (C62H66N8O16Cl4Pt2).
C: calcd, 43.52; found, 44.8. H: calcd, 3.89; found, 3.81. N:
calcd, 6.55; found, 6.24. 1H NMR (DMF-d7): δ 8.38 (m, 1H,
Hd), 8.02 (under DMF-HCO, 1H, He), 7.70 (d, 1H, Hf), 7.32
(m, 2H, Hg), 7.03 (m, 4H, Hi + Hh), 3.45 (under HDO, 2H, Hc),
3.13 (m, 1H, Hb), 2.76 (under DMF-CH3, 4H, H1 + Ha), 2.33
2,5-Diamino-N-(4-tert-butoxycarbonylaminobutyl)val-
eramide (8). To a solution of 2.57 g (5.2 mmol) of 7 in 80 mL
of methanol, 58 mL of 0.2 M NaOH (11.6 mmol) was added
dropwise at 0 °C. The solution was stirred overnight at room
temperature, then filtered and extracted with 5 × 35 mL of
CHCl3/MeOH (9:1). The organic phase was dried over MgSO4,

5200 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Kalayda et al.
filtered, and evaporated to provide the product. Yield: 1.57 g
HPLC coupled to MS: gradient, 95% B for 2 min, then from
95% B to 70% B in 20 min and subsequently from 70% B to
10% B in 8 min (eluent A, acetonitrile with 0.1% CF3COOH;
eluent B, 0.1% aqueous solution of CF3COOH); retention time
26.7 and 27.1 min; for both peaks m/z ) 1123 (M - CF3COO),
505 ((M - 2CF3COO)/2).
1
(99.92%). H NMR (MeOH-d4): δ 3.24 (m, 1H, H1), 3.19 (m,
2H, H8), 3.03 (m, 2H, H4), 2.62 (m, 2H, H5), 1.65 (m, 2H, H2),
1.58 (m, 6H, H3 + H6 + H7), 1.40 (m, 9H, Boc). ESI-MS: m/z
303.17 (M + H), 325.08 (M + Na).
Bis{trans-diamminechloroplatinum(II)}{µ-(2,5-diamino-
N-(4-tert-butoxycarbonylaminobutyl)valeramide)} Ni-
trate (9). To a solution of 150 mg (0.50 mmol) of trans-
[PtCl2(NH3)2] in 5 mL of DMF, a solution of 81 mg (0.475
mmol) of AgNO3 in 5 mL of DMF was added. The solution was
stirred in the dark overnight at room temperature. The
precipitated AgCl was filtered off. To the remaining yellow
filtrate, 68 mg (0.225 mmol) of ligand 8 in 3 mL of DMF was
added dropwise. After the mixture was stirred in the dark for
24 h, DMF was evaporated in vacuo. The remaining solid was
dissolved in a minimal amount of methanol. After that, the
excess diethyl ether was added, resulting in the formation of
a white precipitate. The suspension was stirred for several
hours to remove DMF, and the white product was filtered off,
washed with diethyl ether, and dried in air. The complex was
purified by recrystallization from a mixture of ethanol and
water (3:2), washed with ethanol and diethyl ether, and
subsequently lyophilized. Yield: 115 mg (48%). Anal. (C14H42N10-
O9Cl2Pt2). C: calcd, 17.60; found, 17.14. H: calcd, 4.43; found,
4.32. N: calcd, 14.66; found, 15.00. 1H NMR (D2O): δ 3.82 (m,
1H, H1), 3.38 (m, 2H, H4), 3.12 (m, 2H, H8), 2.65 (m, 2H, H5),
1.74 (m, 2H, H2), 1.60-1.70 (m, 6H, H3 + H6 + H7), 1.43 (m,
9H, Boc). 195Pt NMR (D2O): δ -2401.
Reaction of 1,1/c,c/DNP and 1,1/t,t/DNP with 9EtG. The
reactions of the complexes 1,1/c,c/DNP, 1,1/c,c, 1,1/t,t/DNP, and
1,1/t,t (2 mM) with 2 equiv of 9EtG (4 mM) were monitored
1
over 48 h at 310 K by H NMR spectrometry in a mixture of
D2O and DMF-d7 (1:1) in the case of 1,1/c,c/DNP and 1,1/t,t/
DNP and in 0.1 M NaClO4 D2O solution (pD ∼7.5) in the case
of the label-free complexes. The pH titration of the products
was subsequently performed by adjustment of pD with 0.1 and
1 M DCl and with 0.1 and 1 M NaOD without using a buffer.
pD values were measured at 298 K using a PHM 80 pH meter
(radiometer) before and after each 1H NMR measurement. The
pH values were corrected for the H/D isotope effect.51
Measurements of Platinum Accumulation in Cancer
Cells. The 12-well culture plates containing exponentially
growing U2-OS or U2-OS/Pt cells in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal calf
serum (Gibco, Paisley, Scotland), penicillin (100 units/mL,
Duchefa, The Netherlands), and streptomycin (100 µg/mL,
Duchefa, The Netherlands) (2 × 105 cells/well) were exposed
to 20 µM of the labeled complexes (CFDA-Pt, DNP-Pt, 1,1/c,c/
DNP, 1,1/c,c/CFDA, 1,1/t,t/CFDA, and 1,1/t,t/DNP) and the
parent compounds (1,1/c,c, 1,1/t,t, and cisplatin) dissolved in
DMEM for 30 min, 2 h, and 24 h (the complexes were added
in duplicate). After incubation, the cells were washed twice
with PBS, and 350 µL/well lysis buffer containing 10 mM Tris,
pH 8.0, 150 mM NaCl, and 0.4% Triton X-100, was added. The
content of each well was collected in an Eppendorf tube, and
100 µL of 10% SDS (sodium dodecyl sulfate) was added to each
sample. After that, the samples were treated for 30 min at 50
°C with 20 µg/mL of proteinase K (BV Sphaero Q, Gorinchem,
The Netherlands). The samples were diluted with 20% HNO3
to a final volume of 3 mL for measurement of platinum content.
Platinum concentration was measured on a Varian Vista-MPX
charge-coupled simultaneous ICP-OES (inductively coupled
plasma optical emission spectrometer). The uptake experi-
ments were carried out with duplicate cultures.
Cellular Processing Experiments. The U2-OS human
osteosarcoma cell line (HTB 96, American Type Culture
Collection) was originally derived from a sarcoma of the tibia.
The cisplatin-resistant U2-OS/Pt cell line was kindly provided
by Paola Perego of the Instituto Nazionale per lo Studio e la
Cura dei Tumori, Milan, Italy. U2-OS cells and its cisplatin-
resistant U2-OS/Pt cells were grown in Dulbecco’s modified
minimal essential medium without phenol red (Life Technolo-
gies) supplemented with 10% fetal bovine serum and antibiot-
ics in a humidified 5% CO2, 95% air atmosphere. For living
cell observations, the cells were plated in 35 mm culture dishes
with a glass coverslip incorporated at the bottom (Mattek Corp.
Ashland, MA), which is directly in contact with a 40× NA 1.30
oil-immersion objective (Neofluor) (Zeiss, Jena, Germany)
during observations.
The cells were allowed to grow to 40-50% confluence before
incubation. The CFDA-labeled compounds were diluted in
serum-free medium and added to the cells at a final concentra-
tion of 10 µM. Then the cells were incubated for 30 min and
subsequently washed with serum-free medium, and full growth
medium was added to the cells, which were monitored over
48 h on an inverted microscope. The DNP-labeled compounds
were observed after immunocytochemical staining (see below).
Immunocytochemical Staining of the DNP-Labeled
Compounds. For immunohistochemical detection of the DNP-
labeled compounds, the cells were fixed at different time points
after internalization with 2% formaldehyde in CSK buffer (10
mM Pipes, pH 6.8, 300 mM sucrose, 100 mM NaCl, 3 mM
MgCl2, 1 mM EGTA, 0.5% Triton X-100). In the fixation
procedure, the cells were washed twice with PBS. The cells
were fixed for 10 min at room temperature. Then the cells were
washed twice with PBS before immunohistochemical staining.
Bis{trans-diamminechloroplatinum(II)}{µ-(2,5-diamino-
N-(4-(5-(and 6-)carboxyfluorescein-diacetate-amino)bu-
tyl)valeramide)} Trifluoroacetate (1,1/t,t/CFDA, 11a). A
solution of 38.2 mg (0.040 mmol) of 9 in 2 mL of 0.1 M
hydrochloric acid was stirred in the dark overnight at 50 °C
to obtain a solution of 10, which was directly used for the
synthesis of 11a. After this solution was neutralized to pH 8.0
with 2 M NaOH, a solution of 21.2 mg (0.038 mmol) of 5(6)-
CFDA SE in 2 mL of DMF was added dropwise at 0 °C. The
resulting solution was stirred at 0 °C for 15 min and then for
30 min at room temperature. The solvents were removed in
vacuo, and the residue was dissolved in 2 mL of a mixture of
acetonitrile and water (1:1) and purified by high-performance
liquid chromatography as described above. The purified prod-
1
uct was lyophilized and characterized by H and 195Pt NMR
and LC/ESI-MS. Yield: 2.1 mg (3.8%). 1H NMR (D2O): δ 8.22
(s, 1H, Hd), 8.09 (d, 1H, He), 7.61 (d, 1H, Hf), 7.28 (s, 2H, Hi),
7.08(d, 2H, Hg), 6.98 (d, 2H, Hh), 3.49 (m, 1H, H1), 3.38 (m,
2H, H4), 3.24 (m, 2H, H8), 3.02 (m, 2H, H5), 2.38 (s, 6H,Hj),
1.95-1.45 (m, 8H, H2 + H3 + H6 + H7). 195Pt NMR (D2O): δ
-2410. Analytical HPLC coupled to MS: gradient, 95% B for
2 min, then from 95% B to 70% B in 20 min and subsequently
from 70% B to 10% B in 8 min (eluent A, acetonitrile with
0.1% CF3COOH; eluent B, 0.1% aqueous solution of CF3-
COOH); retention time 27.3 and 27.7 min; for both peaks m/z
) 1287 (M - CF3COO), 587 ((M - 2CF3COO)/2).
Bis{trans-diamminechloroplatinum(II)}{µ-(2,5-diami-
no-N-(4-(2,4-dinitrophenylhexanoicamino)butyl)valer-
amide)} Trifluoroacetate (1,1/t,t/DNP, 11b). A solution of
38.2 mg (0.040 mmol) of 9 in 2 mL of 0.1 M hydrochloric acid
was stirred in the dark overnight at 50 °C to obtain a solution
of 10, which was directly used for the synthesis of 11b. After
this solution was neutralized to pH 8.0 with 2 M NaOH, a
solution of 15.0 mg (0.038 mmol) of DNP-X SE in 2 mL of DMF
was added dropwise at 0 °C. The resulting yellow solution was
stirred at 0 °C for 15 min and subsequently for 30 min at room
temperature. The solvents were removed in vacuo, and the
residue was dissolved in 2 mL of the mixture of acetonitrile
and water (1:1) and purified by high-performance liquid
chromatography as described above. The purified product was
1
lyophilized and characterized by H and 195Pt NMR and LC/
1
ESI-MS. Yield: 2.1 mg (4.2%). H NMR (D2O): δ 9.17 (s, 1H,
Hk), 8.34 (d, 1H, Hi), 7.20 (d, 1H, Hj), 3.58 (m, 3H, Hh + H1),
3.26 (m, 2H, H4), 3.19 (m, 2H, H8), 2.71 (m, 2H, H5), 2.27 (m,
2H, Hd), 1.73 (m, 6H, He + Hg + H2), 1.65 (m, 6H, H3 + H6 +
H7), 1.46 (m, 2H, Hf). 195Pt NMR (D2O): δ -2430. Analytical

Fluorescence Microscopy for Anticancer Drugs
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5201
After fixation, the cells were pretreated with 0.1% Triton
X-100 in PBS for 15 min to improve target accessibility. The
cells were then rinsed with TBS buffer (100 mM Tris-HCl, pH
7.5, 150 mM NaCl), incubated for 45 min with the primary
antibody, a rabbit anti-DNP antibody (Sigma, St. Louis, Mo.),
washed twice with TBS buffer, and incubated for 30 min with
the secondary antibody, a goat anti-rabbit ALEXA 488 anti-
body (Molecular Probes, Leiden, The Netherlands). Antibody
solutions were diluted in 100 mM Tris-HCl, 150 mM NaCl,
and 0.5% blocking reagent (Boehringer). After the final wash-
ing steps, the cells were gradually dehydrated in an ethanol
series of 70%, 90%, 100% for 1 min each. The coverslips were
mounted in CytoFluor/DAPI (4,6-diamidino-2-phenylindole) for
microscopic observation.
Golgi Staining. Staining of the Golgi complex was per-
formed with N-({4-[4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-
diaza-s-indacen-3-yl]phenoxy}acetyl)sphingosine (Bodipy-TR-
ceramide, Molecular Probes, Leiden, The Netherlands). Twenty-
four hours after incubation with 1,1/c,c/CFDA and 1,1/t,t/
CFDA, Bodipy-TR-ceramide was added to the cells to a final
concentration of 100 nM in serum-free medium. After 20 min
of incubation with this dye, the cells were rinsed with serum-
free medium and observed live using digital fluorescence
microscopy. The CFDA-labeled complexes, emitting in green,
and Bodipy-TR-ceramide, emitting red light, were sequentially
visualized and digitally recorded.
Digital Fluorescence Microscopy. Fluorescence micros-
copy experiments were performed on a Axiovert 135 TV (Zeiss,
Jena, Germany) inverted microscope equipped with a 100 W
mercury arc lamp for fluorescence excitation and bright field
illumination for phase contrast images. The filter set to detect
CFDA fluorescence consisted of an hq 480/40 nm band-pass
excitation filter, an hq 535/50 band-pass emitter filter, and a
Q505 long-pass beam splitter. Bodipy-TR-ceramide was de-
tected using a filter set composed of an hq 560/55 band-pass
excitation filter, an hq 645/75 band-pass emitter filter, and a
Q595 long-pass beam splitter. The temperature of the culture
medium was controlled between 36 and 37 °C by a BIOPTECHS
(Butler, PA) objective heater and a heated ring surrounding
the culture chamber. Digital images were taken with a cooled
CCD camera (Photometrix PLX, Tucson, AZ) using SCILL
Image software (Multihouse, The Netherlands).
Growth Inhibition Assays in U2-OS and U2-OS/Pt
Cells. Growth inhibition by the compounds was determined
using an MTT-based assay (MTT, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-2H-tetrazolium bromide).52 The U2-OS and U2-
OS/Pt cells were grown as monolayers in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal calf
serum (Gibco, Paisley, Scotland), penicillin (100 units/mL,
Duchefa, The Netherlands), and streptomycin (100 µg/mL,
Duchefa, The Netherlands). After trypsinization, cells were
divided in 96-well plates at concentrations of (3-5) × 103 cells/
well in 100 µL growth medium. The cells were allowed to
attach overnight. After 24 h, fresh stock solutions (1 mg/mL)
of 1,1/c,c, 1,1/t,t, and cisplatin in Millipore water and of the
labeled compounds in dimethylformamide were diluted in
medium. Five subsequent dilutions of each compound were
added to the cells in quadruplicate, yielding a total volume of
200 µL in each well. Dimethylformamide diluted in medium
at different concentrations (0.2-10%) was added to the cells
in a control experiment. After 72 h of incubation, 50 µL of a 5
mg/mL MTT solution in PBS was added to each well and
allowed to develop in the incubator (usually 120 min). After
this, the medium was discarded and 100 µL of DMSO was
added to each well, yielding purple solutions. The optical
density was measured at 590 nm using a Biorad 550 micro-
plate reader. The IC50 values (drug concentration that inhibits
cell growth for 50% with respect to control) were determined
graphically using the Graphpad Prism analysis software
package. In the cytotoxicity tests of the labeled complexes,
dimethylformamide was added to the control cells.
0002/00, and D20/0003/01 (Metal Compounds in the
Treatment of Cancer and Viral Diseases), are kindly
acknowledged. The authors are also indebted to a
Training Network Grant from the EU in the 5th
Framework program (MEDICINOR, Grant No. HPMT-
CT-2000-00192). The authors thank Johnson & Matthey
(Reading, U.K.) for their generous gift of K2PtCl4. This
work was performed under the auspices of the joint
BIOMAC Research Graduate School of Leiden Univer-
sity and Delft University of Technology.
Supporting Information Available: Results from el-
emental analysis for 2, 4a, 4b, 6, 7, and 9 and HPLC spectra
for 11a and 11b.
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