A fluorescent oxaliplatin derivative for investigation of oxaliplatin resistance using imaging techniques

Article abstract of DOI:10.1007/s00775-017-1502-z

Oxaliplatin is the backbone of chemotherapy for advanced colorectal cancer and undergoes clinical trials for treatment of other tumour entities. However, acquired resistance is a major hurdle. Confocal microscopy is a useful tool to get an insight into the mechanisms of resistance but it requires fluorescent compounds. This work describes the synthesis of the novel oxaliplatin derivative (CFDA-oxPt) featuring 5(6)-carboxyfluorescein diacetate and evaluation of its applicability for the investigation of oxaliplatin resistance using imaging techniques. CFDA-oxPt was somewhat less cytotoxic than oxaliplatin in sensitive colorectal cancer cells, with EC₅₀ values of 26 and 5.8?μM, respectively. Nevertheless, the potency of the novel complex was significantly decreased to the EC₅₀ of 711.2?μM in oxaliplatin-resistant cells, as was the case for oxaliplatin (EC₅₀?=?81?μM). After incubation, both nuclear and cytosolic localisation was observed. Over time CFDA-oxPt concentrated near the cell membrane and in the vesicular structures, in contrast to the platinum-free label, which was rapidly excreted. These findings suggest that CFDA-oxPt can be used to study oxaliplatin resistance and open the route to new fluorophore-tethered oxaliplatin derivatives.

Full text of DOI:10.1007/s00775-017-1502-z

J Biol Inorg Chem (2017) 22:1295–1304
DOI 10.1007/s00775-017-1502-z
ORIGINAL PAPER
A fuorescent oxaliplatin derivative for investigation of oxaliplatin
resistance using imaging techniques
Ganna V. Kalayda1 · Maximilian Kullmann1 · Markus Galanski2 · Sabrina Gollos3
Received: 20 August 2017 / Accepted: 13 October 2017 / Published online: 17 October 2017
© SBIC 2017
Abstract Oxaliplatin is the backbone of chemotherapy
for advanced colorectal cancer and undergoes clinical trials
for treatment of other tumour entities. However, acquired
resistance is a major hurdle. Confocal microscopy is a useful
tool to get an insight into the mechanisms of resistance but
it requires fuorescent compounds. This work describes the
synthesis of the novel oxaliplatin derivative (CFDA-oxPt)
featuring 5(6)-carboxyfuorescein diacetate and evaluation
of its applicability for the investigation of oxaliplatin resist-
ance using imaging techniques. CFDA-oxPt was somewhat
less cytotoxic than oxaliplatin in sensitive colorectal cancer
cells, with EC50 values of 26 and 5.8 µM, respectively. Nev-
ertheless, the potency of the novel complex was signifcantly
decreased to the EC50 of 711.2 µM in oxaliplatin-resistant
cells, as was the case for oxaliplatin (EC50 = 81 µM). After
incubation, both nuclear and cytosolic localisation was
observed. Over time CFDA-oxPt concentrated near the cell
membrane and in the vesicular structures, in contrast to the
platinum-free label, which was rapidly excreted. These fnd-
ings suggest that CFDA-oxPt can be used to study oxaliplatin
resistance and open the route to new fuorophore-tethered
oxaliplatin derivatives.
Keywords Platinum complexes · Fluorescent probes ·
Live-cell microscopy · Cellular distribution
Introduction
Oxaliplatin is a third-generation platinum drug with a better
safety profle and lack of cross-resistance with cisplatin. In
combination with 5-fuorouracil and leucovorin, it is used
as standard treatment of advanced colorectal cancer [1].
Besides, oxaliplatin is undergoing clinical trials in gastric,
pancreatic, breast and non-small cell lung cancer. How-
ever, after initial response many patients experience disease
relapse due to the development of tumour cell resistance
[2]. In contrast to cisplatin resistance, the mechanisms of
resistance to oxaliplatin are less well investigated. Cytotoxic
action of oxaliplatin has long been assumed to result from
its binding to genomic DNA, which subsequently triggers
apoptosis in cancer cells, as in the case of other platinum
drugs [1, 2]. A recent report has, however, demonstrated
that oxaliplatin kills cells through the induction of ribosome
biogenesis stress [3]. The role of this process in oxaliplatin
resistance is yet unrevealed. Besides, modifed accumulation
and intracellular transport of the drug contribute to tumor
cell resistance [1, 2]. Identifcation of alternative cellular
and molecular targets of oxaliplatin and understanding their
alterations in oxaliplatin-resistant cells are of great impor-
tance as this information would facilitate the development
of new oxaliplatin-based drugs and formulations as well as
the search for biomarkers.
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-017-1502-z) contains supplementary
material, which is available to authorized users.
* Ganna V. Kalayda
akalayda@uni-bonn.de
1
Department of Clinical Pharmacy, Pharma Centre Bonn,
Institute of Pharmacy, University of Bonn, An der
Immenburg 4, 53121 Bonn, Germany
2
Institute of Inorganic Chemistry, University of Vienna,
Waehringer Str. 42, 1090 Vienna, Austria
3
Department of Pharmaceutical Chemistry, Pharma Centre
Investigation of the intracellular transport and inter-
actions of oxaliplatin remains a challenge. The methods
Bonn, Institute of Pharmacy, University of Bonn, An der
Immenburg 4, 53121 Bonn, Germany
Vol.:(0123456789)
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traditionally used for platinum detection and quantifcation,
e.g. graphite-furnace atomic absorption spectrometry (GF-
AAS) or inductively-coupled plasma mass spectrometry
(ICP-MS) can provide information about cellular localisa-
tion only after the time-consuming isolation of pure orga-
nelles [4]. In addition, cellular fractionation usually does not
allow entire separation of cellular compartments. Moreover,
some platinum may get lost during the process. Radiola-
belling of oxaliplatin with [14C] is possible [5] but subse-
quent cellular studies have similar limitations. Synchrotron
radiation-induced X-ray emission (SRIXE) was applied to
investigate platinum drug localisation in tumour cells, mul-
ticell layer tumour models and spheroids [6–9]. However,
this technique is unsuitable for studies in live cells over time
[4]. The same holds true for nanoscale secondary ion mass
spectrometry (nanoSIMS), in which 300 nm thin sections
of resin-embedded human colon cancer cells treated with
15N-labeled cisplatin were examined [10].
evaluation of its biological activity and investigation of its
cellular distribution using live-cell imaging. The advantage
of the CFDA label is that it is not fuorescent itself, but
becomes fuorescent after being taken up by cells, as the
ester groups are cleaved by intracellular esterases [17]. The
need for hydrolysis may, however, turn into a limitation in
the cells with low esterase levels. CFDA moiety has been
previously introduced to other platinum-based drugs for
cellular imaging but not to oxaliplatin [17, 23–25]. In this
study, a mixture of 5- and 6-carboxyfuorescein diacetate
was used.
Results and discussion
Synthesis of CFDA‑oxPt
For the fluorescent oxaliplatin derivative CFDA-oxPt,
we considered introducing the label at position 4 of the
cyclohexane ring as it ofers maximal distance from the
platinum coordination site. It should be noted, however,
that the mechanism of oxaliplatin interference with ribosome
biogenesis [3] is not yet known, and (1R,2R)-cyclohexane-
diamine may also play a critical role.
Confocal fuorescence microscopy is a versatile and pow-
erful tool to investigate cellular processes. It allows detec-
tion of organelles, macromolecules and fuorescent drugs
inside the cells. Unfortunately, none of platinum drugs is
intrinsically fuorescent, so they need to be modifed with a
fuorescent tag. In the last decades, many cisplatin and some
carboplatin analogues with diferent fuorophores were syn-
thesised and evaluated [4, 11–17]. Modifcation of the struc-
ture resulted in somewhat lower cytotoxicity compared to
the parent drug, however, the fuorescent derivatives showed
cross-resistance with cisplatin [11, 13, 14, 16, 18]. These
fuorophore-tethered platinum complexes have been used for
various purposes, such as investigation of cellular trafck-
ing [11, 15, 17, 19] and transporter relevance [14, 18–20],
identifcation of the interaction partners in the cytosol [21,
22], and for high-resolution imaging of pharmacokinetics in
live animal subjects [16].
(1R,2R)-1,2-diamino-4-cyclohexene 1 (Scheme 1)
appeared a suitable building block because its stereocentres
already feature the same absolute confguration as oxalipl-
atin. The synthesis of CFDA-oxPt is presented in Scheme 1.
First the amino groups of 1 were protected using Boc
(tert-butoxycarbonyl) groups. Then, the acetamide moi-
ety was introduced into the cyclohexane ring of protected
amine 2 following the literature procedure [26, 27]. In brief,
2 was treated with Hg(NO3)2 in acetonitrile and the resulting
product was reduced with NaBH4 [28]. The major diastere-
oisomer (1R,2R,4S)-1,2-bis-(tert-butyloxycarbonylamino)-
4-acetamidocyclohexane (3) could be isolated as described
previously [27] and was used in the next step. After removal
of the Boc groups, which was confrmed by LC–MS, the
resulting compound 4 was deprotonated with N,N-diisopro-
pylethylamine (DIPEA) and subsequently platinated using a
standard method with K2PtCl4 yielding platinum complex 5,
So far, the attention of researchers has been focused
mainly on cisplatin, although very little is known about the
cellular processing of oxaliplatin despite its huge therapeu-
tic potential. Here, we report the synthesis of CFDA-oxPt,
the frst fuorescent oxaliplatin derivative conjugated to
the carboxyfuorescein diacetate (CFDA) moiety (Fig. 1),
Fig. 1 Chemical structures of
O
O
O
oxaliplatin, CFDA-oxPt and
O
platinum-free label CFDA
O
O
O
O
O
O
O
O
H2
N
O
O
O
O
O
O
H2
N
O
O
O
HN
O
O
Pt
O
N
Pt
OH
H2
N
H2
CFDA
CFDA-oxPt
oxaliplatin
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O
HN
NHBoc
NHBoc
NHBoc
NHBoc
NH2
Boc2O
1.
0
6
rt
CH
Hg(NO )
3
2,
3CN, ºC, h,
2. NaBH in
0
1
rt
rt
90
NaOH, ºC, h,
min.,
4
NH2
3
1
2
3 M HCl
overnight,
rt
O
O
H2
N
HN
+Cl-
H3N+ Cl-
1.
15
rt
min.,
HN
NH3
Cl
Cl
DIPEA,
2PtCl
3 M
1 M TFA
overnight
Cl
Cl
HCl,
Pt
2. K
rt
4, overnight,
reflux,
+Cl-
N
NH3
H2
4
6
5
O
O
O
O
O
O
=
1. 10 M
7.5
NaOH,
pH
=
CFDA-NHS
O
2.
0
1
rt
CFDA-NHS, ºC, h,
O
O
O
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
1.
rt
H2
N
O
AgNO3, overnight,
2. Na 24 rt
H2
N
O
O
HN
O
HN
Cl
2C2O4,
h,
Pt
Pt
N
O
N
Cl
H2
H2
7
CFDA-oxPt
Scheme 1 Synthetic route used for preparation of CFDA-oxPt
which was fully characterised by multinuclear 1H, 13C, 15N,
and 195Pt NMR spectroscopy (Suppl. Figs. S1–S6). Further,
the acetyl group of 5 was removed using a modifcation of
the literature procedure [26, 27], under refux overnight in
3 M HCl in the presence of 1 M trifuoroacetic acid (TFA).
The cleavage of the acetyl group under these conditions was
confrmed by the LC–MS analysis of the resulting clear solu-
tion containing complex 6. However, compound 6 was not
the only product formed. The dimeric species l-PtCl2PtCl2
and l-PtCl2Pt-L [l=(1R,2R,4S)-cyclohexanetriamine] were
also detected. Nevertheless, 6 was used in the following step
without further purifcation. Coupling of the CFDA tag to
the amino group of the cyclohexane ring yielded the crude
product 7, which was purifed using HPLC (LC–MS, Suppl.
Fig. S7, 1H spectrum, Suppl. Fig. S8). The exchange of the
chloride ligands for oxalate was carried out using a standard
procedure [29]. Chloride ligands were frst exchanged for
the solvent (dimethylformamide) molecules using AgNO3,
subsequently dimethylformamide ligands were replaced by
oxalate to obtain the fnal product (1H spectrum, Suppl. Fig.
S8, LC–MS, Suppl. Fig. S9). The excitation and emission
spectrum of CFDA-oxPt and CFDA for comparison after
hydrolysis in phosphate bufered saline (PBS) overnight at
37°C is shown in Suppl. Fig. S10.
Biological activity
In order to evaluate the applicability of CFDA-oxPt to study
oxaliplatin resistance, the cytotoxicity of the labelled com-
plex in comparison to the parent drug and to the platinum-
free CFDA was assessed in the HCT-8 human ileocecal colo-
rectal adenocarcinoma cell line and its oxaliplatin-resistant
variant HCT-8ox. The platinum-free label (CFDA) was not
cytotoxic up to 1000 µM. As is evident from Table 1 and
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Table 1 Cytotoxicity of oxaliplatin (n = 4), CFDA-oxPt (n = 3) and
the platinum-free CFDA (n = 3)
incubation, the fuorescence close to the membrane became
weaker, and mainly accumulation in vesicular structures
was observed. Interestingly, many more fuorescent vesicles
could be seen in oxaliplatin-resistant cells as compared to
the parent cell line. Twelve hours after incubation, very little
fuorescence was visible in both sensitive and resistant cells.
A substantial fraction of HCT-8 cells appeared to be dead or
dying (cell debris and apoptotic cells are evident from Fig. 2,
HCT-8 cells, 12 h incubation) in contrast to the resistant
counterparts, which still had some fuorescence concentrated
in vesicular structures.
Compound HCT-8
HCT-8ox
pEC50 SEM EC50, µM pEC50 SEM EC50, µM
Oxaliplatin 5.24 0.08
CFDA-oxPt 4.58 0.09
5.8
26.0
> 1000
4.09 0.10
3.15 0.06
nd
81.0
711.2
> 1000
CFDA
nd
nd not determined
growth inhibition curves (Suppl. Fig. S11), CFDA-oxPt was
less potent than oxaliplatin, which is consistent with previ-
ous fndings regarding fuorophore-tethered cisplatin deriva-
tives [13, 14, 16, 18]. In HCT-8 cells, the EC50 values were
26 and 5.8 µM, respectively. Apparently, the introduction
of a large lipophilic fuorophore has a negative impact on
the cytotoxic properties. It has been previously suggested
that fuorescent cisplatin derivatives may use some other
ways to enter the cells than the parent drug [13]. Neverthe-
less, cytotoxicity of CFDA-oxPt was signifcantly lower in
oxaliplatin-resistant cells (p < 0.0001) than in sensitive cells
as in the case of oxaliplatin (p = 0.0001). In HCT-8ox cells,
the EC50 value for oxaliplatin was 81 µM and for CFDA-oxPt
711.2 µM. Thus, oxaliplatin-resistant cells are also resist-
ant to CFDA-oxPt indicating that mechanisms of oxaliplatin
resistance afect the labelled complex as well. As in the case
of other platinum drugs [13, 14, 18], introduction of a fuo-
rophore appears to have little infuence on the intracellular
trafcking pathways relevant for the resistant phenotype. It
should be noted that the growth inhibition curves of CFDA-
oxPt are steeper than those of oxaliplatin indicating a possi-
ble contribution of other mechanisms to cell death compared
to the parent drug). Nevertheless, signifcant reduction of
the complex activity in oxaliplatin-resistant cells suggests
that CFDA-oxPt can be used for investigation of oxaliplatin
resistance.
The ability of CFDA-oxPt to accumulate in the nucleus
was then analysed using DAPI (4′,6-diamidino-2-phenylin-
dole dihydrochloride) staining after fxation. Directly after
incubation, some nuclear accumulation of the fluores-
cent oxaliplatin derivative was observed in both cell lines
(Fig. 3). Much fuorescence was also located in the cyto-
plasm. Five hours after incubation, CFDA-oxPt could only
be found close to the cell membrane of HCT-8 and HCT-
8ox cells and additionally in vesicular structures in resistant
cells. No vesicular accumulation in the sensitive cells could
be detected after fxation (Fig. 3).
We also studied cellular distribution of the platinum-free
label. CFDA was taken up by the cells to a much higher
extent than the platinum complex with no specifc accu-
mulation in cellular compartments. The fuorescence got
weaker with time, and no CFDA (i.e. its fuorescent hydroly-
sis product) could be detected within the cells already 1 h
after incubation (Fig. 4). These fndings clearly show that
the fuorescent dye is processed diferently compared to the
platinum complex underscoring that cellular distribution of
CFDA-oxPt is determined by the oxaliplatin moiety and not
by the carboxyfuorescein diacetate tag.
Similarly to the fuorophore-tethered cisplatin derivatives,
we observed accumulation of CFDA-oxPt in both nucleus
and cytosol [7, 13, 16]. The distribution pattern changed
over time showing predominant localisation near the cell
membrane and in vesicular structures. Accumulation of fuo-
rescent platinum complexes in vesicles is well documented
[14, 15, 19, 30]. In contrast, localisation close to the mem-
brane has not been reported and may represent a distinct
feature of oxaliplatin-like complexes due to their higher lipo-
philicity. In the studies of cellular processing of fuorescent
cisplatin analogues, no remarkable diferences in cellular
distribution pattern in sensitive and cisplatin-resistant cells
were found. In the oxaliplatin-resistant cell line HCT-8ox,
clearly more fuorescent vesicles were detected compared
to the sensitive HCT-8 cells. Increased number of vesicular
compartments may, however, simply be a feature of HCT-
8ox cells and thus unrelated to the specifc processing of
CFDA-oxPt by these cells.
Cellular distribution of CFDA‑oxPt and a plati‑
num‑free CFDA
In order to study the cellular processing of the novel oxalipl-
atin derivative, cells were incubated with the compound and
then monitored over time using live-cell confocal laser scan-
ning microscopy. Directly after incubation, CFDA-oxPt (i.e.
its fuorescent hydrolysis product) was distributed through-
out the sensitive HCT-8 cells with some accumulation in
vesicular structures visible. In oxaliplatin-resistant HCT-8ox
cells, the fuorescent oxaliplatin derivative showed punc-
tate staining in the perinuclear area with some fuorescence
also observed in the nucleus. One hour after incubation,
CFDA-oxPt concentrated on the cell periphery near the cell
membrane and in vesicular formations. Several hours after
Fluorescent drug analogues enable valuable insights into
the mechanism of action on cellular level. However, changes
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Fig. 2 Representative confocal images of live HCT-8 (a) and HCT-8ox (b) cells after incubation with 50 µM CFDA-oxPt for 30 min and subse-
quent incubation with the drug-free medium. Scale bar, 25 µm
in drug structure inevitably lead to the reduction in activity.
Another common problem of fuorescence labelling is the
possibility quenching depending on cellular environment.
Fluorescein-based compounds have their own limitations
such as photobleaching and relatively low fluorescence
quantum yields, which make them unsuitable for applica-
tions such as intravital imaging. It has also been suggested
that CFDA-labelled platinum complexes exhibits lower cyto-
toxicity compared to the BODIPY-tagged counterparts [13].
Nevertheless, CFDA-oxPt can be used to study the mecha-
nisms of oxaliplatin resistance, e.g. alterations in drug trans-
port or in ribosome biogenesis stress in resistant colorectal
cancer cells.
CFDA-oxPt can be employed for investigation of oxalipl-
atin resistance. Furthermore, our work paves a path to other
fuorophore-tagged oxaliplatin-like complexes.
Materials and methods
Instruments
NMR spectra were recorded using a Bruker Ascend™
600 MHz instrument at 600.19 (1H) and 150.93 MHz (13C)
or with a Bruker Avance III 500 MHz NMR spectrometer
at 500.32 (1H), 125.82 (13C), 50.70 (15N), and 107.31 MHz
(
195Pt) in DMF-d7, or CDCl3 at 298 K. 195Pt or 15N chemi-
cal shifts were referenced relative to external K2[PtCl4] or
NH4Cl, respectively.
Conclusions
C, H and N analysis was performed by the microanalyti-
cal laboratory of the Institute of Pharmacy, University of
Bonn, Germany.
This is the frst report of a fuorophore-tethered oxaliplatin
derivative. The introduction of the lipophilic fuorescent
tag leads to a decrease in the complex’s potency in HCT-8
cells. Nevertheless, oxaliplatin-resistant HCT-8ox cells are
highly resistant to CFDA-oxPt implying that this platinum
complex is susceptible to the resistance mechanisms operat-
ing in HCT-8ox cell line. In general, our fndings confrm
(for the case of oxaliplatin) previous reports [13, 14, 16, 18]
that platinum-fuorophore complexes are less cytotoxic than
the parent FDA-approved platinum drugs but exhibit cross-
resistance in platinum-resistant cell lines. Taken together,
The high resolution mass spectroscopic measurements
were carried out on a micrOTOF-Q mass spectrometer
(Bruker) with an ESI source coupled to a HPLC Dionex
Ultimate 3000 (Thermo Scientifc) using a EC50/2 Nucleo-
dur C18 Gravity 3 µm column (Macherey–Nagel) with a
fow rate of 300 µL/min. HPLC started with 90% water con-
taining 2 mM CH3COONH4. The gradient began after 1 min
and went to 100% acetonitrile in 9 min following by fushing
with 100% acetonitrile for the next 5 min.
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J Biol Inorg Chem (2017) 22:1295–1304
with 2 mM CH3COONH4). The UV absorption was detected
in the range 190–900 nm using a diode array detector. Mass
spectra were recorded on an API 2000 (Applied Biosystems,
Darmstadt, Germany) mass spectrometer (turbo ion spray
ion source) coupled to an Agilent 1100 HPLC system (Agi-
lent, Böblingen, Germany).
Synthesis
Materials
(1R,2R)-1,2-diamino-4-cyclohexene (1), 5(6)-carboxyfuo-
rescein diacetate (CFDA, a mixture of 5-carboxyfuorescein
diacetate and 6-carboxyfuorescein diacetate) and its N-suc-
cinimidyl ester (CFDA-NHS), sodium borohydride solution
(ca. 12 wt%) in 14 M NaOH, mercury(II) nitrate monohy-
drate, sodium hydroxide, trifuoroacetic acid and potassium
tetrachloroplatinate(II) were purchased from Sigma-Aldrich,
Steinheim, Germany. Di-tert-butyl dicarbonate was ordered
from Alfa Aesar, Karlsruhe, Germany. Hydrochloric acid,
magnesium sulfate, sodium chloride and potassium hydrox-
ide were from VWR International, Langenfeld, Germany.
Silver nitrate and oxalic acid dihydrate were obtained from
Merck KGaA, Darmstadt, Germany. N,N-diisopropylethyl-
amine (DIPEA) was ordered from Acros Organics, Geel,
Belgium. All solvents were reagent grade and were used as
purchased.
Fig. 3 Representative confocal images of HCT-8 and HCT-8ox cells
incubated with 50 µM CFDA-oxPt for 30 min and stained with DAPI
after subsequent incubation with the drug-free medium. Scale bar,
25 µm
(1R,2R)‑1,2‑bis(N,N’‑tert‑butyloxycarbonylamino)‑4‑cy‑
clohexene (2)
LC–MS experiments were carried out using a EC50/2
Nucleodur C18 Gravity 3 µm column (Macherey–Nagel)
with a fow rate of 300 µL/min and the following gradient
conditions: 90% A for 10 min, then from 90% A to 100% B
in 10 min followed by fushing with 100% B for 10 min (elu-
ent A: water with 2 mM CH3COONH4/eluent B: methanol
To a solution of 1 (1.5 g, 13.4 mmol) in ethanol (25 mL), a
solution of di-tert-butyl dicarbonate (7.296 g, 33.4 mmol) in
ethanol (35 mL) was added, and the resulting mixture was
stirred for 90 min. The solvent was then removed in vacuo.
The product was purifed by recrystallisation from ace-
tonitrile and dried in air. Yield: 3.626 g (86.6%). 1H NMR
Fig. 4 Representative confocal images of live HCT-8 (a) and HCT-8ox (b) cells after incubation with 20 µM CFDA for 20 min and subsequent
incubation with the drug-free medium. Scale bar, 25 µm
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J Biol Inorg Chem (2017) 22:1295–1304
1301
(600 MHz, CDCl3): δ = 5.55 (d, J = 3 Hz, 2H, H4/H5), 4.86
(br, 2H, NH), 3.63 (m, 2H, H1/H2), 2.45 (d, J = 17 Hz, 2H,
to dryness under reduced pressure. The residue was further
dried by redissolving in absolute ethanol and evaporating
to dryness. This was repeated twice more yielding a white
residue 4. LC–MS: retention time 0.58 min, m/z = 172.15
([M−H]+).
H3/H6), 1.95 (m, 2H, H3′/H6′), 1.41 (s, 18H, CH3) ppm. 13
C
NMR (100 MHz, CDCl3): δ = 156.49 (C=O), 125.02 (C4/
C5), 79.30 (C(t-Bu)), 51.34 (C1/C2), 32.85 (C3/C6), 28.38
(CH3) ppm. LC–MS: retention time 11.38 min, m/z = 313.2
([M+H]+). Anal. Calcd for C16H28N2O4: C 61.51, H 9.03,
N 8.97, found: 61.65, H 9.02, N 8.98.
Residue 4 was dissolved in water (50 mL) and fltered
through a Millipore flter. To this solution, ethanol (75 mL)
and subsequently N,N-diisopropylethylamine (DIPEA,
0.75 mL, 4.3 mmol) were added and stirred for 10 min
at room temperature. Then ethanol was removed under
reduced pressure. To the resulting aqueous solution, K2PtCl4
(560 mg, 1.35 mmol) in water (50 mL) was added and stirred
for 24 h in darkness. The yellow precipitate formed was fl-
tered of, washed with water, absolute ethanol and ether to
yield complex 5. Yield: 294 mg (50%). 1H NMR (500 MHz,
DMF-d7): δ = 7.68 (d, J = 7.2 Hz, 1H, C4-NH), 5.87 (m,
1H, C1-NH), 5.68 (m, 1H, C2-NH), 5.22 (m, 2H, C1-NH,
C2-NH), 3.97 (m, 1H, H4-eq), 3.34 (m, 1H, H2-ax), 2.82 (m,
1H, H1-ax), 2.19 (m, 1H, H3-eq), 1.96 (m, 1H, H6-eq), 1.91
(s, 3H, CH3), 1.85 (m, 1H, H6-ax), 1.79 (td, J = 12.7, 4 Hz,
1H, H3-ax), 1.64 (m, 1H, H5-eq), 1.48 (tt, J = 14, 4 Hz, 1H,
H5-ax) ppm. 13C NMR (126 MHz, DMF-d7): δ = 168.88
(C=O), 63.37 (C1), 59.42 (C2), 44.50 (C4), 35.99 (C3),
28.78 (C5), 27.25 (C6), 22.40 (CH3) ppm. 15N NMR
(50 MHz, DMF-d7): δ = − 19.3 (C1-N), − 20.6 (C2-N) ppm.
195Pt NMR (107 MHz, DMF-d7): δ = − 670 ppm. HRMS
m/z [M+H]+ calcd for C8H18Cl2N3OPt: 438.0451, found:
438.0429; [M+NH4]+ calcd for C8H21Cl2N4OPt: 455.0716,
found: 455.0698; [2 M]+ calcd for C16H34Cl4N6O2Pt2:
875.0833, found: 875.0810.
(1R,2R,4S)‑1,2‑bis(N,N′‑tert‑butyloxycarbonylamino)‑4
‑acetamidocyclohexane (3)
A suspension of Hg(NO3)2 × H2O (6.42 g, 18.74 mmol) in
acetonitrile (25 mL) was cooled down to 0 °C under Ar.
To this suspension, compound 2 (3.437 g, 11 mmol) was
added slowly in small portions over 30 min. The reaction
mixture was allowed to warm up to room temperature and
stirred for 6 h. The reaction was then cooled down to 0 °C
and quenched by slow addition of 10 M NaOH (17.2 mL,
172 mmol) and NaBH4 solution (ca. 12 wt%, 13.85 mL,
60.4 mmol) in 14 M NaOH. The mixture was allowed to
warm up to room temperature and was stirred for 1 h. Then,
NaCl (9.65 g, 165.1 mmol) and water (13.8 mL) were added,
and the resulting slurry was stirred for additional 30 min.
Subsequently, the slurry was fltered through a Celite plug
and the plug was washed with 150 mL dichloromethane.
After adding 45 mL water to the fltrate, the organic layer
was separated and the aqueous layer washed with 75 mL
dichloromethane. The organic layers were combined, dried
over MgSO4 and evaporated to obtain a crude product. The
product was purifed by column chromatography (silica
gel, CH2Cl2/CH3OH 20:1) yielding a waxy solid 3. Yield:
1.532 g (37.5%). 1H NMR (600 MHz, CDCl3): δ = 6.98 (br,
1H, C4-NH), 4.97 (m, 2H, C1-NH/C2-NH), 4.17 (m, 1H,
H4-eq), 3.45 (m, 1H, H2-ax), 3.31 (m, 1H, H1-ax), 2.08 (m,
1H, H3-eq), 1.95 (s, 3H, CH3), 1.91 (m, 1H, H6-eq), 1.84
(m, 1H, H5-eq), 1.55 (tt, J = 14, 3 Hz, 1H, H5-ax), 1.43
(td, J = 13, 3 Hz, 1H, H3-ax), 1.42–1.32 (s (CH3(t-Bu)) + s
(CH3(t-Bu)) + m (H6-ax), 19H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 169.79 (CO(CH3)), 156.61 (CO(Boc)), 156.34
(CO(Boc)), 79.71 (C(t-Bu)), 79.51 (C(t-Bu)), 54.62 (C1),
50.77 (C2), 44.80 (C4), 36.37 (C3), 28.45 (C5), 28.35
(CH3(Boc)), 28.33 (CH3(Boc)), 27.58(C6), 23.41 (CH3)
ppm. HRMS m/z [M+H]+ calcd for C18H34N3O5: 372.2493,
found: 372.2507.
Dichlorido[(1R,2R,4S)‑4‑(5(6)‑carbonylaminofuores‑
cein‑diacetate)cyclohexane‑1,2‑diamine]platinum(II) (7)
In a typical experiment, 3 M HCl (2 mL, 6 mmol) and tri-
fuoroacetic acid (TFA, 150 µL, 1.96 mmol) were added
to complex 5 (40 mg, 0.091 mmol). The suspension was
refuxed overnight (100 °C) yielding a clear yellow solu-
tion of crude complex 6. LC–MS: retention time 0.51 min;
m/z = 396.24 ([M+H]+), 413.28 ([M+NH4]+).
The pH of the solution was frst adjusted to 6 with 10 M
NaOH. The solution was then fltered through a Millipore
flter and the pH was further adjusted to 7.5 with 10 M
NaOH. The solution was cooled down to 0 °C and 5(6)-car-
boxyfuorescein diacetate, N-succinimidyl ester (CFDA-
NHS, 25 mg, 0.045 mmol) in dimethylformamide (4 mL)
was added dropwise. The resulting mixture was stirred for
20 min at 0 °C and subsequently for 1 h at room tempera-
ture. The solvents were removed in vacuo. The residue was
quickly washed with ice-cold water, absolute ethanol and
ether. The synthetic procedure was repeated four times.
The crude products obtained were combined and purifed
with HPLC (System Gold, Beckman Coulter, Germany)
Dichlorido[(1R,2R,4S)‑4‑acetamidocyclohexane‑1,2‑di‑
amine]platinum(II) (5)
To a solution of 3 (0.5 g, 1.35 mmol) in acetone (10 mL)
cooled to 0 °C, 3 M HCl (6.5 mL, 19.5 mmol) was added.
The resulting solution was warmed up to room tempera-
ture, stirred at room temperature overnight, and evaporated
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J Biol Inorg Chem (2017) 22:1295–1304
using a Nucleodur C18 HTec column (5_m; 250 × 10 mm;
Macherey–Nagel, Germany). The mobile phase consisted
of ultrapure water (A) and acetonitrile (B). A gradient
method was established (0–5 min 20% B, 5–10 min 30%
B, 10–30 min 60% B, 30–37 min 95% B, 37–44 min 20%
B). Due to the use of a mixture of 5- and 6-carboxyfuo-
rescein diacetate a double peak of the product and of unre-
acted CFDA was observed. The retention time of 7 was
30.8 min, and of CFDA 36.4 min. Yield: 14.65 mg (9.8%).
195Pt NMR (107 MHz, DMF-d7): δ = − 653 ppm. LC–MS
(DMF solution): retention time 7.42 min; m/z = 838.3
([M+H]+), 855.2 ([M+NH4]+). HRMS m/z [M+H]+ calcd
for C31H30Cl2N3O8Pt: 838.1043, found: 838.1039.
was obtained by incubation with stepwise increasing oxali-
platin concentrations. The cells were grown as monolayers
in RPMI-1640™ medium supplemented with 10% foetal
calf serum, 100 I.E./ml penicillin and 0.1 mg/ml strepto-
mycin at 37 °C in a humidifed atmosphere containing 5%
CO2. Every 10 passages, a new backup of cells was thawed
to ensure that resistance mechanisms remained unchanged
during cultivation.
Growth inhibition assays in HCT‑8 and HCT‑8ox cells
Growth inhibition by CFDA-oxPt, free label CFDA and
oxaliplatin was determined using an MTT-based assay
(MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetra-
zolium bromide) [31]. Due to the limited solubility of 5, the
cytotoxicity of this complex could not be assessed. The cells
(3000 cells/well in 100 μL growth medium) were seeded in
96-well microtiter plates and allowed to attach overnight.
Then medium was discarded and ten subsequent dilutions
prepared from the stock solutions of oxaliplatin in ultrapure
water and of CFDA and CFDA-oxPt in dimethylformamide,
were added to the cells in triplicates (100 µl/well). Some
control wells contained 0.5% dimethylformamide, which
corresponded to the highest solvent concentration in the
dilutions of CFDA-oxPt and CFDA. After 72 h of incuba-
tion, 50 μL of a 5 mg/mL MTT solution in PBS was added
to each well and incubated at 37 °C for 60 min. After this,
medium was discarded and 100 μL dimethylsulfoxide was
added to each well, yielding purple solutions. Absorbance of
the converted dye was measured at 570 nm with background
subtraction at 690 nm using a Multiscan Ascent® microti-
ter plate reader (Thermo Fisher Scientifc, Langenselbold,
Germany). The results were analysed and the pEC50 values
(pEC50 = − log EC50 (mol/L), EC50 is the drug concentra-
tion that produces 50% of the maximum possible response)
were determined for each independent experiment with the
GraphPad Prism™ analysis software package (GraphPad
Software, San Diego, USA) using non-linear regression (sig-
moidal dose response, variable slope). As the data of growth
inhibition experiments can be assumed to be log-normally
distributed, the mean pEC50 values were calculated from the
results of three to four independent experiments and used for
the determination of the respective EC50 values. The signif-
cance of diferences was evaluated using Student’s t test. p
values < 0.05 were considered statistically signifcant.
[(1R,2R,4S)‑4‑(5(6)‑carbonylaminofuorescein‑diac‑
etate)cyclohexane‑1,2‑diamine]oxalatoplatinum(II)
(CFDA‑oxPt)
AgNO3 (7.1 mg, 0.0418 mmol) was added to a solution of 7
(17.8 mg, 0.0213 mmol) in dimethylformamide (0.452 mL).
The resulting solution was stirred overnight at room tem-
perature in the darkness. The precipitate of silver chloride
was centrifuged of at 18,620×g for 10 min. To the remain-
ing solution, an in situ prepared solution of potassium oxa-
late from oxalic acid dihydrate (2.57 mg, 0.0204 mmol) in
dimethylformamide (0.2 mL) and KOH (0.0204 mL of 1 M
KOH solution in methanol, 0.0204 mmol) was added. The
reaction mixture was stirred for 24 h at room temperature in
the darkness and the solvents were subsequently removed
in vacuo. The product was purifed on a HPLC system as
described above to obtain a white solid of CFDA-oxPt. Due
to the use of a mixture of 5- and 6-carboxyfuorescein diac-
etate, a double peak of the product was observed. The reten-
tion time was 25.7 min. Yield: 13.31 mg (73.3%). 195Pt NMR
(107 MHz, DMF-d7): δ = − 357 ppm. LC–MS (DMF solu-
tion): retention time 7.13 min; m/z = 855.5 ([M+H]+), 871.6
([M+NH4]+). HRMS m/z [M+H]+ calcd for C33H30N3O12Pt:
855.1475, found: 855.1461.
Excitation and emission spectra
CFDA-oxPt and CFDA were diluted in PBS each at the fnal
concentration of 50 nM and incubated overnight at 37° C.
The excitation and emission spectra of the resulting fuores-
cent solution were recorded at room temperature on the Per-
kin Elmer luminescence spectrometer LS 55 (Beaconsfeld,
UK) at λex = 488 nm.
Cellular processing experiments
Cell lines and cell culture
One day prior to treatment, the cells were plated in 35 mm
culture dishes with a glass cover slip incorporated in the
bottom (Willco Wells B.V., Amsterdam, the Netherlands)
and allowed to grow. Then CFDA-oxPt was added to the
cells in serum-free medium at the fnal concentration of
The human ileocecal colorectal adenocarcinoma cell lines
HCT-8 and HCT-8ox were kindly provided by Dr. R. A.
Hilger, University of Essen, Germany. The resistant variant
1 3

J Biol Inorg Chem (2017) 22:1295–1304
1303
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50 μM and incubated for 30 min, subsequently washed, and
the full growth medium was added to the cells, which were
monitored over time at 37 °C in an atmosphere containing
5% CO2 on the inverted Nikon Eclipse Ti A1 laser scanning
confocal microscope (Nikon Corporation, Tokyo, Japan)
using a Plan-Apo VC 60 × oil immersion objective (NA 1.4,
Nikon). Control cells were incubated with CFDA at the fnal
concentration of 20 μM in serum-free medium for 20 min,
washed and observed live as described above.
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For the co-localisation experiments, cells were seeded
on cover slips one day before incubation. Then the cells
were treated with 50 μM CFDA-oxPt in serum-free medium
for 30 min. Subsequently, the cells were washed and fxed
with methanol and 5 μg/mL DAPI in methanol was added
to the cells and incubated for 5 min at room temperature.
After subsequent washing with methanol, the cover slips
were mounted in GelMount™ medium for microscopic
observations.
11. Benedetti BT, Peterson EJ, Kabolizadeh P, Martinez A, Kipping
R, Farrell NP (2011) Efects of noncovalent platinum drug-pro-
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Images were scanned sequentially using 405 and 488 nm
lasers in combination with a DM 405/488/543/640 dichroic
beam splitter. The fuorescence signals were collected using
bandpass flters 425–475 nm and 500–550 nm (Nikon). The
images were analysed with NIS-Elements AR 3.2 software
(Nikon).
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Acknowledgements This work was supported by the Deutsche
Forschungsgemeinschaft (JA 817/4-1). The authors are grateful to Prof.
Dr. Ulrich Jaehde for helpful discussions and to Prof. Dr. Christa E.
Müller for providing laboratory facilities.
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