Mitochondrial mode of action of a thymidine-based cisplatin analogue breaks resistance in cancer cells

Article abstract of DOI:10.1002/chem.201000785

Cisplatin analogue complexes with platinum(II) and palladium(II) starting from 3′,5′-diamino-3′,5′-dideoxy-thymidines were synthesized, both with the D-erythro- and D-threo configurations. Complexes of the general formula [MCl₂L] were obtained and characterized. NMR spectroscopic measurements and single crystal X-ray structure analysis showed that the metal centers are coordinated to the ligands by the amino groups in 3′- and 5′-positions and not through the thymine moiety. All ligands and complexes showed no significant in vitro activities except thymiplatin (cis-dichloro(3′,5′-diamino-3′,5′-dideoxy-D-threo- thymidine)platinum(II)). Detailed in vitro studies on the apoptosis pathway in lymphoma (BJAB), leukemia (NALM-6), and melanoma cells (Mel-HO) as well as on transfected or resistant cell lines were carried out. Thymiplatin significantly induced an apoptotic response, which was found to be associated with the loss of mitochondrial membrane potential and with caspase activation. The activity was shown to be independent of Fas-associated protein with death domain (FADD), but dependent on Bcl-2 expression. As a consequence, for thymiplatin a mitochondrial mode of action could be assigned. Moreover, the compound showed activity in cells resistant to common drugs, such as daunorubicin and vincristin, and showed synergistic effects with doxorubicin, vincristin, cytarabin, and daunorubicin. Prove your metal: Cisplatin analogues with platinum(II) and palladium(II) complexes based on 3′,5′-diamino thymidines were synthesized (see figure for an example) and one was found to induce apoptosis mediated by caspase-9 and -3 processing. Thymiplatin was proven to be active on cisplatin, vincristin and daunorubicin resistant leukemia cells, and was synergistic with cytarabin, vincristin, daunorubicin, and doxorubicin in lymphoma cells. Copyright

Full text of DOI:10.1002/chem.201000785

DOI: 10.1002/chem.201000785
Mitochondrial Mode of Action of a Thymidine-Based Cisplatin Analogue
Breaks Resistance in Cancer Cells
[
a]
[b]
[b]
[b]
Liliane A. Onambele, Daniel Koth, Justyna A. Czaplewska, Ulrich S. Schubert,
[
c]
[d]
[e]
[b]
Helmar Gçrls, Shigenobu Yano, Makoto Obata, Michael Gottschaldt,* and
[
a]
Aram Prokop*
Abstract: Abstract: Cisplatin analogue
complexes with platinum(II) and palla-
dium(II) starting from 3’,5’-diamino-
activities except thymiplatin (cis-di-
chloro(3’,5’-diamino-3’,5’-dideoxy-d-
be associated with the loss of mito-
chondrial membrane potential and with
caspase activation. The activity was
shown to be independent of Fas-associ-
ated protein with death domain
(FADD), but dependent on Bcl-2 ex-
pression. As a consequence, for thymi-
platin a mitochondrial mode of action
could be assigned. Moreover, the com-
pound showed activity in cells resistant
to common drugs, such as daunorubicin
and vincristin, and showed synergistic
effects with doxorubicin, vincristin, cy-
tarabin, and daunorubicin.
threo-thymidine)platinum(II)).
De-
3
’,5’-dideoxy-thymidines were synthe-
tailed in vitro studies on the apoptosis
pathway in lymphoma (BJAB), leuke-
mia (NALM-6), and melanoma cells
(Mel-HO) as well as on transfected or
resistant cell lines were carried out.
Thymiplatin significantly induced an
apoptotic response, which was found to
sized, both with the d-erythro- and d-
threo configurations. Complexes of the
general formula [MCl L] were obtained
2
and characterized. NMR spectroscopic
measurements and single crystal X-ray
structure analysis showed that the
metal centers are coordinated to the li-
gands by the amino groups in 3’- and
Keywords: antitumor
agents
·
5
’-positions and not through the thy-
apoptosis · drug resistance · nucleo-
sides · platinum
mine moiety. All ligands and com-
plexes showed no significant in vitro
Introduction
[
a] L. A. Onambele, Dr. Dr. A. Prokop
Childrenꢀs Hospital of the City of Cologne
Amsterdamerstr. 59, 507335 Cologne (Germany)
Fax : (+49)3641948202
Since the discovery of cisplatin as a potent antitumor
[1]
agent, platinum-based anticancer drugs have been con-
[2]
stantly advanced and modified. In current oncology, three
E-mail: prokopa@kliniken-koeln.de
approved platinum drugs play a major role: cisplatin, carbo-
platin, and oxaliplatin. Additionally, combinations of pico-
platin or satraplatin with, for example, paclitaxel, against
certain types of cancer, are under investigation for appro-
[
b] Dr. D. Koth, Dr. J. A. Czaplewska, Prof. U. S. Schubert,
Dr. M. Gottschaldt
Laboratory for Organic and Macromolecular Chemistry (IOMC)
Friedrich-Schiller-University Jena, Humboldtstr. 10
[3]
0
7743 Jena (Germany)
val. The major mechanism of action of these platinum-
based drugs is the formation of DNA adducts by binding to
the N7 sites of purine bases of the double strand to form in-
Fax : (+49)3641948202
E-mail: michael.gottschaldt@uni-jena.de
[
c] Dr. H. Gçrls
[4]
terstrand cross-links. This results in several analogous
pathways for resistance to these compounds, such as reduced
intracellular drug accumulation, inactivation by thiol con-
taining molecules, or enhanced DNA repair, and has led to
an extensive search for compounds with different principles
Institute for Inorganic and Analytical Chemistry
Friedrich-Schiller-University Jena, Lessingstr. 8
0
7743 Jena (Germany)
[
d] Prof. S. Yano
Nara Institute of Science and Technology (NAIST)
8
916-5, Takayama-cho, Ikoma, Nara 630-0192 (Japan)
[5,6]
of operation or enhanced selectivity.
Efforts include the
[
e] Dr. M. Obata
Interdisciplinary Graduate School of Medicine and Engineering
University of Yamanashi, Takeda 4-4-37, Kofu 400-8510 (Japan)
use of polymeric vehicles for target-selective delivery of
[7]
platinum-based anticancer agents, the development of
active complexes of various metal ions as well as their con-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000785.
[8]
jugation with biomolecules.
14498
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Chem. Eur. J. 2010, 16, 14498 – 14505

FULL PAPER
In contrast to the well-explored carbohydrate-based metal
complexes and in particular to their platinum com-
[9–11]
pounds,
the corresponding nucleoside analogues seem to
be virtually unexplored. Only a very few isolated platinum
complexes bearing nucleoside ligands are known, although
studies on the uptake of nucleosides and their derivatives by
different membrane-bound transport proteins have shown
that their uptake is less hindered than for monosaccharide-
[12]
based molecules.
show promising antitumor activities.
Iron containing nucleoside analogues
[13,14]
Compounds based
on N-aminated nucleosides have been studied both in vivo
and in vitro. Their cytotoxic effect against L1210 cells in
vitro was found to be ten-times lower than that of cisplatin,
[15]
and in vivo they did not exhibit any antitumor activity.
cisplatin analogue with an ethylendiamine ligand that is
linked via a propyl group to thymidine was also described.
A
Scheme 1. Schematic representation of the synthesis of platinum(II) and
palladium(II) complexes of 3’,5’-diamino-3’,5’-dideoxy-thymidine with
[16]
The number of studied compounds in which the sugar part
of the nucleoside is used for the complexation of plati-
either the d-threo (1) or d-erythro configuration (2). i) K
reflux, >90%; ii) K [PdCl ], H O, reflux, >90%.
2 4 2
ACHTUNGTRNNEUG[ PtCl ], H O,
2
A
H
U
G
E
N
N
4
2
ACHTUNGTRENNUNGn um(II) is much smaller. All compounds studied so far have
possessed the d-erythro configuration. Among them, a dose
dependent increase in survival time in mice bearing the
L1210 tumor was only observed for the dichlorobis(3’-
spectroscopic measurements. Crystallization of complex 4 by
using ethanol and water resulted in crystals suitable for
single crystal X-ray structure analysis (Figure 1). The asym-
amino-3’-deoxy-d-erythro-thymidine)-platinum(II)
com-
[17]
plex. Although the mode of action could not be assigned
clearly, it has been assumed that the kind of nucleobase and
sugar unit has a strong influence on the antiproliferative ac-
tivity of the corresponding complexes. Inspired by the
strong dependency of biological activity on the configuration
of diaminosaccharides and -nucleosides attached to metal
[
11,17]
ions,
midine with a pseudo-axial arrangement of the amino group
in the 3’-position was used to coordinate platinum(II) in the
present study. For comparison, the analogue d-erythro deriv-
the respective d-threo configured derivative of thy-
AHCTUNGTRENNUNG
[
17]
ative as well as the palladium complexes were also synthe-
sized and tested for their antitumor activity in vitro.
Figure 1. X-ray single crystal structure of a molecule in the crystal of the
palladium complex 4 with numbering according to NMR spectroscopy.
Atomic displacement parameters are drawn at the 50% probability level.
Results and Discussion
Synthesis and structural characterization of the complexes:
The two 3’,5’-diamino-3’,5’-dideoxy-thymidines (1 and 2)
were previously obtained as intermediates for the synthesis
of metal complexes based on their condensation products
metric unit of the crystal contains two symmetrically inde-
pendent molecules of 4 and two water molecules that are
neither involved in the complexation nor bound to the li-
gands. There is only a very small difference between the two
complex molecules within the asymmetric unit. As suspect-
ed, the palladium(II) ion has a square planar coordination
geometry (Table 1). One side of the complex is sterically
shielded by the nucleobase. The nucleobase, which usually
serves to coordinate metal ions, is not involved in the coor-
dination of the metal ion. As expected from the crystal
[18,19]
with salicyliden aldehydes.
The synthesis pathway ap-
plied is much more efficient than the introduction of two
amino groups into uridine in the 2’,3’-position or into d-
[20,21]
ribose or 2-deoxy-d-ribose,
because the thymine residue
acts as a protecting group of the 3’-position during the syn-
thesis (see Scheme S1 in the Supporting Information). For
the preparation of the platinum (3 and 5) and palladium
complexes (4 and 6) an aqueous solution of potassium tetra-
chloroplatinate or potassium tetrachloropalladinate, respec-
tively, was added dropwise into a boiling solution of the
amine (1 or 2) in water (Scheme 1). After a short time, the
formed complexes precipitated. Uncharged stable com-
[
a]
Table 1. Selected bond lengths (ꢂ) and angles (8) of complex 4.
PdÀN-5’
PdÀN-3’
PdÀCl-1
PdÀCl-2
2.022(11)
2.038(12)
2.301(4)
2.315(4)
N-5ꢀÀPdÀN-3’
N-5ꢀÀPdÀCl-1
N-3ꢀÀPdÀCl-2
Cl-1ÀPdÀCl-2
90.8(4)
87.4(3)
89.4(4)
92.33(13)
II
II
plexes with Pt and Pd of the general formula [MCl L]
2
[a] The dihedral angle at the palladium center between the planes de-
were formed, as shown by mass spectrometry and NMR
fined by PdÀN-5ꢀÀCl-1 and PdÀN-3ꢀÀCl-2 is 0.878.
Chem. Eur. J. 2010, 16, 14498 – 14505
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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14499

M. Gottschaldt, A. Prokop et al.
structure of 4, also the NMR signals of the nucleobases of
the other complexes show no significant shifts compared to
those of the free ligands. These results prove that the thy-
mine is not involved in the coordination of the metals since
coordination to platinum or palladium would result in a
high-field adjustment.
To investigate the stability of the platinum(II) complex 3
compared with cisplatin, AgNO was added to an aqueous
3
solution of compound 3 and the precipitate was filtered off.
Mass spectrometric measurement of the solution showed the
complete disappearance of 3 (m/z 533) and the formation of
a new product with m/z 511 in which one chloride is ex-
changed against a water molecule, analogous to cisplatin
(
Figure S1 in the Supporting Information). Treatment of 3
with 5’-GMP in PBS buffer resulted in the formation of a
+
mono-adduct of the general formula [PtCl ACHUTNGRENNUG( 5’-GMP)L] ,
which was detected by ESI-MS measurements (Figure S2 in
the Supporting Information). This is similar to the treatment
of cisplatin with nucleotides, and shows that platinum com-
plex 3 is able to exchange chloride ions and bind to biologi-
cal targets, and therefore, should exhibit biological activity.
In vitro activities of compounds 1–6: To investigate the bio-
logical activity of the synthesized compounds, their antipro-
liferative and apoptotic properties were screened by using
lymphoma cells (BJAB). The pure ligands (1 and 2) and the
palladium(II) derived compounds (4 and 6) had no signifi-
cant effects (Figure S3 in the Supporting Information). The
Figure 2. Inhibition of proliferation and cell viability. A) BJAB cells were
treated with different concentrations of thymiplatin (3). After incubation
=96 h), prolifera-
tion was measured by using the Casy count system. Values are given as
percentages of inhibition of cell proliferation (error bars are omitted for
clarity). B) BJAB cells were treated with different concentrations of 3
for 2 h. Viability was determined by the LDH release assay. Values are
given as percentages of the control.
for different lengths of time (^ =24,
&
=48, * =72, ~
II
Pt complex with the d-erythro configuration (5) exhibited
comparably small antiproliferative activity (50% after 72 h
incubation at 80 mm). Only thymiplatin (3; cis-dichloro(3’,5’-
diamino-3’,5’-dideoxy-d-threo-thymidine)-platinum(II)) pos-
sessed significant activity (80% growth inhibition after 96 h
incubation at 40 mm and 50% apoptosis after 96 h at 60 mm;
Figures 2A and 3A). Therefore, only the antitumor activity
of the nucleoside analogue thymiplatin (3) was investigated
in more detail with lymphoma (BJAB), leukemia (NALM-
[22]
stasis and elimination of damaged cells. Many anticancer
drugs act by causing the death of tumor cells through the in-
[23]
duction of apoptosis. Apoptosis is a mode of cell death
that appears under normal physiological condition or phar-
macological stimuli. The cell is an active participant in its
own destruction. Apoptosis plays an important role in the
regulation of cell number or in the elimination of damaged
cells. Cells undergoing apoptosis show segmentation of the
cytoplasm and nucleus into apoptotic bodies. In vivo, they
are rapidly recognized and phagocytized, so that no inflam-
matory response is involved in apoptosis. In contrast, necrot-
ic cell death is often associated with extensive tissue damage
resulting in a strong inflammatory response, in vivo.
6), and melanoma cells (Mel-HO) in vitro.
Leukemia is the most common malignant disease in child-
hood. Approximately one third of cancer patients have a
type of leukemia, most commonly acute lymphoblastic leu-
kemia (ALL). Significant research into the causes, diagnosis,
treatment, and prognosis of leukemia is being carried out.
Despite a relatively good prognosis, approximately one
quarter of the patients suffer from relapse and, consequent-
ly, a worse outlook. Furthermore, resistance is a serious
drawback with many existing antileukemia drugs. It has
been suggested that most chemotherapeutics exert their ef-
fects by triggering apoptotic cell death. Accordingly, drug-
induced apoptosis in tumor and leukemia cells predicts the
response of the therapy. BJAB and NALM-6 cells were
chosen because cisplatin shows low activity against them
and cannot be applied in the therapy of leukemia and Bur-
kitt lymphoma leukemia.
Incubation of BJAB cells with 10 to 100 mm of 3 led to a
statistically significant decrease in cell viability after 48 h
(data not shown). There was up to 100% time- and concen-
tration-dependent inhibition of cell proliferation after 72 h
incubation (Figure 2A). A decrease in cell counts might be
[24]
due to cell necrosis or apoptosis.
To show that the de-
crease in proliferation was due to apoptosis and not to ne-
crosis, lactose dehydrogenase, which is an enzyme that is re-
leased by plasma membrane lysis and is characteristic of cell
necrosis while apoptotic cells maintain their membrane in-
[25]
Determination of apoptosis induced by thymiplatin: Apop-
tegrity, was shown to be absent in culture supernatants of
tosis represents an important factor in maintaining homeo-
BJAB cells incubated for 2 h with 10–100 mm of 3 (Fig-
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Cisplatin Analogue Nucleosides
FULL PAPER
(
Fas-associated protein with death domain). The intrinsic or
mitochondrial pathway of apoptosis (type II) is associated
with changes in the permeability of the outer mitochondrial
[26]
membrane and the collapse of the membrane potential.
Caspase-9 is the initiator of this pathway and caspase-3 is a
[
27,28]
key executioner of apoptosis.
brane permeabilization is governed by anti- and pro-apop-
Mitochondrial outer mem-
[29]
totic members of the Bcl-2 protein family.
To investigate the involvement of CD95/Fas (type I apop-
tosis), BJAB cells over-expressing a dominant-negative
FADD (FADD-dn) mutation were used alongside control
cells. Treatment of both mutant and control cells with differ-
ent concentrations of 3 indicates that with up to 50 mm the
apoptosis produced is independent of CD95/Fas, as shown
by DNA fragmentation analysis (Figure 4). However, at
concentrations above 50 mm, thymiplatin-induced apoptosis
is partly dependent on FADD-dn expression; this implies
the partial involvement of the extrinsic apoptosis pathway
with higher concentrations.
Figure 3. Apoptosis induction: A) BJAB cells were treated with different
concentrations of 3. After incubation for different lengths of time (& =
4
8,
&
=72,
&
=96 h), DNA fragmentation was measured by flow cytomet-
ric analysis of cellular DNA content. Values are given as percentages of
cells with hypodiploid DNA ÆS.D. (n=3). B) BJAB cells were incubated
with different concentrations of 3 for 48 h. Early and late apoptosis was
measured after annexin V/PI staining by using flow cytometry. Values are
given as percentages of annexin V+/PIÀ cells ÆS.D. (n=3).
ure 2B). Additionally, effects of 3 were studied with respect
to DNA fragmentation, a hallmark of apoptotic cells, which
[
25]
occurs at the end of apoptosis. This was shown to increase
in a time- and dose-dependent manner in the range of con-
centrations of 3 tested. Thus, 3 significantly induces DNA
fragmentation in up to 60% of cells with a half maximal
concentration (AC ) of 50 mm (Figure 3A). Remarkably, the
Figure 4. Dependence and independence of the apoptosis induced by 3
on the over-expression of FADD-dn in BJAB cells. Vector- (gray) and
FADD-dn-transfected (black) BJAB cells were incubated with different
concentrations of the agent for 72 h. Subsequently, DNA fragmentation
was measured by flow cytometric analysis of cellular DNA content.
Values are given as percentages of cells with hypodiploid DNA ÆS.D.
(n=3).
50
most significant effects occurred after 96 h of incubation,
which is a comparably long time for platinum-based agents.
Distinct early- and late-phase apoptotic BJAB cells were
found after treatment with 3, as shown by annexin V/propi-
dium iodide (PI) staining (Figure 3B). A significant induc-
tion in necrosis was not found. Taken together, these data
indicate that 3 specifically induces apoptosis in the BJAB
lymphoma cell line (in contrast to the very low activity of
cisplatin with 14% apoptotic BJAB cells after 72 h incuba-
tion at 100 mm; see Figure S4 in the Supporting Informa-
tion).
To determine whether apoptosis induced by 3 is mediated
through the mitochondrial pathway (type II apoptosis),
BJAB cells treated with different concentrations of 3 were
stained with JC-1 and mitochondrial activation was evaluat-
ed. Flow cytometric analysis of mitochondrial permeability
transition revealed a concentration-dependent disruption of
membrane potential in up to 28% of the cell population
(Figure 5). Furthermore, we could show by Western blot
analysis of BJAB cells treated with 3 that caspases-3 and -9
are processed, respectively, to the active p17 subunit and the
37 kDa product (Figure 6). To determine the role of Bcl-2
(an antiapoptotic protein in the intrinsic pathway) in apop-
tosis induced by 3, Mel-HO/bcl-2 melanoma cell lines trans-
fected to over-express Bcl-2 were incubated with different
concentrations of 3. The results show that apoptosis induced
by the compound is dependent on Bcl-2 levels at 72 and
96 h (data shown for 72 h of incubation in Figure 7).
Investigation of the induced apoptotic pathway: The process
of apoptosis is controlled by a diverse range of cell signals,
which can originate either extracellularly (extrinsic pathway,
type I apoptosis) or intracellularly (intrinsic pathway, type II
apoptosis). The type I apoptosis cascade is triggered by the
membrane-anchored Fas-receptor (CD95/Fas) and involves
the proapoptotic protein for the extrinsic pathway FADD
Chem. Eur. J. 2010, 16, 14498 – 14505
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M. Gottschaldt, A. Prokop et al.
Figure 7. Dependence of thymiplatin-induced apoptosis on the over-ex-
pression of Bcl-2 in Mel-HO cells. Vector–bcl-2-transfected Mel-HO cells
were treated with different concentrations of 3 for 72 h. DNA fragmenta-
tion was measured by flow cytometric analysis of cellular DNA content.
Values are given as percentages of cells with hypodiploid DNA ÆS.D.
Figure 5. Inhibition of mitochondrial permeability transition. BJAB cells
were incubated with different concentrations of 3 for 48 h. Subsequently,
mitochondrial permeability transition was measured by flow cytometry
on a single cell level. Values of mitochondrial permeability transition are
given as percentages of cells with low mitochondrial membrane potential
ÆS.D. (n=3).
(
n=3).
ble in clone cells that over-express the anti-apoptotic protein
Bcl-2 by 30-fold (Figure 7).
Activity of thymiplatin against resistant cells and synergetic
effects: Drug resistance is a serious drawback for many ex-
[30]
isting anticancer drugs. One way to overcome the problem
[31]
of drug resistance is to develop new drugs. However, an
attractive alternative is the production of drugs that are syn-
[31]
ergistic to existing ones. In order to investigate whether
thymiplatin (3) could be effective with vincristin and dau-
norubicin resistance, different concentrations of 3 were used
to treat resistant cell lines (NALM-6/VCR and Nalm-6/
DAUNO) for 72 h. Interestingly, thymiplatin was effective
in inducing apoptosis in these cell lines as shown by DNA
fragmentation analysis (Figure 8). Compared to cisplatin
Figure 6. Compound 3 induces caspase-9 and caspase-3 activation. BJAB
cells were incubated either with 3 (80 mm) or epirubicin (5 mm) as the pos-
itive control. After 40 and 52 h of incubation, the cells were collected
and lysed. Cytosolic protein (40 mg) was separated by sodium SDS-PAGE
and subjected to Western blot analysis. Immunoblots probed with anti-
caspase-3, anti-caspase-9, and anti-b-actin antibodies are shown. The po-
sition of the 17 kDa active subunit of caspase-3 and the 37 kDa active
subunit of caspase-9 are indicated. Equal loading and blotting was veri-
fied by detection of b-actin.
(
see Figure S4 in the Supporting Information) thymiplatin
possesses a higher activity against NALM-6 as well as their
resistant variants. Subsequently, we investigated whether 3
could have any synergistic effects with other common drugs.
Therefore, BJAB cells were incubated with low concentra-
tions of 3 in combination with low concentrations of cytara-
bin, vincristin, daunorubicin, and doxorubicin. Thymiplatin
was synergistic with all drugs tested as shown by DNA frag-
mentation analysis at 72 and 96 h (Figure 9).
We could demonstrate a dose- and time-dependent inhibi-
tion of the proliferation (Figure 2B) of cancer cells after
treatment with 3. The processing of caspase-3 (Figure 6)
could be visualized and the rate of DNA fragmentation de-
termined (Figure 3A). These results suggest that 3 induces
apoptosis in tumor cells. The activation of caspase-3 corre-
lated with the activation of caspase-9 (Figure 6). Consistent
with an early disruption of the mitochondrial membrane po-
tential (Figure 5), we have demonstrated that the intrinsic
pathway probably mediates thymiplatin-induced apoptosis.
The dependence of thymiplatin-induced apoptosis on Bcl-2
over-expression and its independence of FADD expression
up to a concentration of 40 mm (Figure 4) further implicates
the intrinsic pathway in thymiplatin-induced apoptosis. At
higher concentrations (>40 mm), 3 is able to induce apopto-
sis through the extrinsic pathway (Figure 4), which is possi-
Conclusion
In conclusion, we have synthesized new platinum(II) and
palladium(II) thymidin-based complexes of the general for-
mula [MCl L]. The coordination of the metal ions is realized
2
by the amino groups introduced in the 3’- and 5’-positions
for both classes with the d-erythro and d-threo configura-
tions, whereas the nucleobase is not involved. The ligands as
well as the palladium(II) complexes were found to possess
no significant cytotoxicity. In contrast, the absolute configu-
ration at the 3’-position of the platinum(II) complexes has a
major impact on their biological activity. Only for the plati-
num complex with the d-threo configuration (3, thymiplatin)
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Cisplatin Analogue Nucleosides
FULL PAPER
1
1
2
H; H-6), 6.21–5.89 (m, 5H; H-1’, NH
H; H-3’), 3.10–2.83 (m, 2H; H-5’, H-5’’), 2.70–2.64 and 2.59–2.53 (2m,
H; H-2’, H-2’’), 1.83 ppm (s, 3H; ÀCH
]DMSO): d = 163.7 (C-4), 150.4 (C-2), 136.2 (C-6), 110.1 (C-5), 82.24
C-4’), 75.3 (C-1’), 52.5 (C-3’), 40.9 (C-5’), 38.0 (C-2’), 12.2 ppm (ÀCH
);
2
), 4.28 (m, 1H; H-4’), 3.78 (m,
1
3
3
); C NMR (100 MHz,
[
D
6
(
3
IR (ATR): n˜ max =3489, 3196, 3109, 2826, 1673, 1469, 1369, 1267, 1109,
À1
+
+
1
047, 958, 761, 666 cm ; MS (ESI): m/z: 528.1 [M+Na] , 543.0 [M+K] ;
elemental analysis calcd (%) for C10 Pt: C 23.73, H 3.19, N
1.07; found: C 23.13, H 3.38, N 10.48.
cis-Dichloro(3’,5’-diamino-3’,5’-dideoxy-d-threo-thymidine)palladium(II)
H
2 4 3
16Cl N O
1
(
(
4): With ligand 1 and K
2
A
H
U
G
R
N
U
G
4
] (203.8 mg, 0.624 mmol); yield 246 mg
1
6
1
3
1
4
3
3
1
2
1
3
.80 ppm (s, 3H; ÀCH
), 148.7 (C-2), 137.1 (C-6), 110.4 (C-5), 82.8 (C-4’), 80.2 (C-1’), 52.1 (C-
’), 40.8 (C-5’), 35.7 (C-2’), 12.0 ppm (ÀCH
); IR (ATR): n˜ max =3491,
221, 3114, 2828, 1696, 1599, 1569, 1472, 1368, 1284, 1267, 1157, 1108,
6
); C NMR (62 MHz, [D ]DMSO): d = 165.2 (C-
3
3
À1
+
+
045, 958, 760 cm ; MS (ESI): m/z: 417.9 [M+H] , 555.9 [M+K] ; ele-
Pd·H O: C 27.57, H 4.17, N
mental analysis calcd (%) for C10
2.86; found: C 26.86, H 3.92, N 12.31.
cis-Dichloro(3’,5’-diamino-3’,5’-dideoxy-d-erythro-thymidine)-platinu-
H
16Cl
2
N
4
O
3
2
1
[
17]
m(II) (5): With ligand 2 and K
2
2
A
H
U
G
R
N
N
4
] (259.15 mg, 0.624 mmol); yield
]DMSO): d 11.33 (s, 1H;
), 3.62–3.40 (m, 2H;
1
6
=
2
1
3
ÀCH ); C NMR (100 MHz, [D ]DMSO): d = 163.71 (C-4), 150.14 (C-
3
6
2
5
1
), 136.7 (C-6), 110.1 (C-5), 83.6 (C-4’), 80.1 (C-1’), 55.9 (C-3’), 44.9 (C-
’), 35.9 (C-2’), 11.9 (ÀCH
); IR (ATR): n˜ max =3478, 3194, 3114, 2825,
3
À1
659, 1470, 1368, 1268, 1209, 1076, 1030, 952, 770, 697 cm ; MS (micro-
Figure 8. Overcoming: A) vincristin, and B) daunorubicin resistance in
NALM-6 cells. Wild-type and resistant cells were treated with different
concentrations of thymiplatin for 72 h. Then, DNA fragmentation was
measured by flow cytometry of cellular DNA content. Values are given
as percentages of cells with hypodiploid DNA ÆS.D. (n=3).
+
+
ESI): m/z: 529.1 [M+Na] , 544.1 [M+K] ; elemental analysis calcd (%)
for C10 Pt: C 23.73, H 3.19, N 11.07; found: C 22.75, H 3.57, N
0.18.
cis-Dichloro(3’,5’-diamino-3’,5’-dideoxy-d-erythro-thymidine)-palladiu-
m(II) (6): With ligand 2 and K [PdCl ] (203.8 mg, 0.624 mmol); yield
46 mg (94%); H NMR (250 MHz, [D ]DMSO): d 11.28 (s, 1H;
NH), 7.40 (s, 1H; H-6), 5.96 (dd, J=3.1, 5.3 Hz, 1H; H-1’), 4.8 4.65, 4.55,
4.43 (m, 4H; NH ), 3.41–3.18 (m, 4H; H-3’, H-4’, H-5’, H-5’’), 2.27–2.13
m, 2H; H-2’, H-2’’), 1.78 ppm (s, 3H; ÀCH
MeOD): d = 163.4 (C-4), 149.9 (C-2), 136.2 (C-6), 109.9 (C-5), 82.97 (C-
); IR
H
2 4 3
16Cl N O
1
2
A
H
U
G
R
N
N
4
1
2
6
=
notable antiproliferative and cytotoxic activity could be ob-
served.
2
1
3
(
3
); C NMR (60 MHz,
Thymiplatin inhibits proliferation and specifically induces
apoptosis in lymphoma cell lines. The induced apoptosis is
mediated by caspase-9 and -3 processing, which is character-
ized by a loss of mitochondria membrane potential and is
dependent on Bcl-2. Further, it is independent of CD95/Fas
signaling. Therefore, a mitochondrial mode of action can be
assigned to thymiplatin. Moreover, it is active on vincristin
and daunorubicin resistant leukemia cells. Additionally, syn-
ergistic effects with cytarabin, vincristin, daunorubicin, and
doxorubicin could be shown with lymphoma cells.
4
’), 81.0 (C-1’), 54.2 (C-3’), 42.3 (C-5’), 36.1 (C-2’), 12.1 ppm (ÀCH
3
(
ATR): n˜ max =3475, 3198, 3112, 2830, 1662, 1561, 1468, 1362, 1269, 1074,
À1
1034, 953, 774, 705 cm
Pd·H
;
elemental analysis calcd (%) for
C
10
H
16Cl
2
N
4
O
3
2
O: C 27.57, H 4.17, N 12.86; found: C 27.12, H 4.23,
N, 12.22.
X-ray crystal structure of 4: The intensity data for the compounds were
collected on a Nonius KappaCCD diffractometer by using graphite mon-
ochromated Mo-K
larization effects, but not for absorption effects. The structure was
solved by direct methods (SHELXS ) and refined by full-matrix least-
a
radiation. Data were corrected for Lorentz and po-
[32,33]
[
34]
2
[35]
squares techniques against Fo (SHELXL-97).
All hydrogen atoms
were included at calculated positions with fixed thermal parameters. All
[
35]
nonhydrogen atoms were refined anisotropically. A XP (Siemens Ana-
lytical X-ray Instruments, Inc.) was used for structure representations.
Experimental Section
[
36]
À1
Crystal data for 4:
prism, size 0.05ꢃ0.05ꢃ0.05 mm , orthorhombic, space group P2 2 2
C
10
H
16Cl
2
3
N
4
O
3
Pd·H
2
O, M
w
=434.56 gmol , yellow
, a=
7.9191(7), b=9.7204(10), c=40.738(4) ꢂ, V=3135.9(5) ꢂ , T=À908C,
Z=8, 1calcd. =1.832 gcm , m (MoKa)=15.43 cm , F(000)=1720, 6491 re-
flections in h (À11/11), l(À51/52), measured in the range 2.158ꢀ
(À9/6), k
Vꢀ27.458, completeness Vmax =71.1%, 4101 independent reflections,
int =0.0676, 2903 reflections with F >4s(F ), 379 parameters, 0 re-
General procedure for the synthesis of the metal complexes: K
[PdCl ] (0.624 mmol) dissolved in water (5 mL) were added dropwise
to a solution of the respective 3’,5’-diamino-3’,5’-dideoxythymidine (1 or
; 150 mg, 0.625 mmol) in boiling water (20 mL). Within a few minutes a
precipitate was formed. To complete the complex formation, the reaction
mixture was stirred for 5 h and stored, overnight, in a refrigerator. The
precipitate formed was filtered off. To increase the yield of the target
compound, the filtrate was concentrated and the precipitates formed
were combined and washed successively with cold water, cold ethanol,
and diethyl ether, and finally dried under vacuum.
2
A
H
N
T
E
N
N
[PtCl
4
] or
1 1 1
3
K
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
4
À3
À1
AHCTUNGTRENNUNG
2
A
H
U
G
R
N
N
A
H
U
T
E
N
N
ACHTUNGTRENNUNG
R
o
o
straints, R1obs =0.0647, wR2obs =0.1313, R1all =0.1108, wR2all =0.1522,
GOOF=1.053, Flack parameter À0.03(8), largest difference peak and
À3
hole: 1.079/À1.222 eꢂ
.
Cell culture: The following human cell lines and their chemoresistant
cis-Dichloro(3’,5’-diamino-3’,5’-dideoxy-d-threo-thymidine)platinum(II)
sublines were used: control vector and FADD-dn-transfected Burkitt-like
[
37]
(
3): With ligand 1 and K
2
A
T
N
T
E
N
N
4
] (259.15 mg, 0.624 mmol); yield 301 mg
6
]DMSO): d = 11.38 (s, 1H; NH), 7.90 (s,
lymphoma (BJAB) cells,
which stably express a dominant negative
1
(
FADD mutant lacking the N-terminal death effector domain (kindly do-
Chem. Eur. J. 2010, 16, 14498 – 14505
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
14503

M. Gottschaldt, A. Prokop et al.
Figure 9. Synergistic effects. BJAB cells were treated with thymiplatin (15/20 mm) as well as with: A) doxorubicin (2/3 nm), B) daunorubicin (2.5/3 nm),
C) cytarabin (5/10 nm), and D) vincristin (0.3/0.5 nm). DNA fragmentation was determined after 96 h (A, B, C) and 72 h (D). Combination of thymiplatin
with these drugs resulted in an increase in DNA fragmentation. Values are given as percentages of cells with hypodiploid DNA ÆS.D. (n=3) (black
areas=addition, white areas=synergy).
nated by Prof. Dr. S. Fulda, University of Ulm); control vector and Bcl-2
transfected melanoma cells (Mel-HO), which stably over-express the
anti-apoptotic protein Bcl-2 (kindly provided by Dr. Eberle, Charitꢄ
Berlin); the leukemia B-cell precursor (Nalm-6) and its vincristin and
daunorubicin resistant sublines. All cell lines were cultured in
RPMI 1640 supplemented with fetal calf serum (FCS; 10%), except for
Mel-HO cells, which were cultured in DMEM with FCS (10%).
Measurement of DNA fragmentation: Apoptotic cell death was deter-
[
40]
mined by using a modified cell-cycle analysis that detects DNA frag-
mentation at the single-cell level. For measurement of DNA fragmenta-
5
tion, cells were seeded at a density of 1ꢃ10 cells per mL and treated
with different concentrations of thymiplatin (3). After 72 h incubation,
cells were collected by centrifugation at 300g for 5 min, washed with
PBS at 48C and fixed in PBS/formaldehyde (2%, v/v) on ice for 30 min.
After being fixed, the cells were incubated with ethanol/PBS (2:1, v/v)
for 15 min, centrifuged, and resuspended in PBS containing RNaseA
Measurement of cell death with the LDH release assay: Cytotoxicity was
measured by using the release of lactate dehydrogenase (LDH) as de-
[
38]
À1
scribed previously. After incubation with different concentrations of 3
for 2 H; LDH released by BJAB cells was measured in cell culture super-
natants by using a cytotoxicity detection kit from Boehringer Mannheim
(40 mgmL ). After incubation for 30 min at 378C, the cells were centri-
À1
fuged again and finally resuspended in PBS containing PI (50 mgmL ).
Nuclear DNA fragmentation was then quantified by flow cytometric de-
termination of hypodiploid DNA. Data were collected and analyzed by
using a FACScan (Becton Dickinson, Heidelberg, Germany) equipped
with the CELL Quest software. Data are given in %hypoploidy (subG1),
which reflects the number of apoptotic cells.
(
5
Mannheim, Germany). The supernatants were centrifuged at 300g for
min. Cell-free supernatants (20 mL) were diluted with PBS (80 mL) and
the reaction mixture (100 mL) was added. Then, the time-dependent for-
mation of the reaction product was quantified photometrically at 490 nm.
The maximum amount of LDH activity released by the cells was deter-
mined by lysis with Triton X-100 (0.1%) in culture medium and was set
as 100% cell death.
Measurement of the mitochondrial permeability transition: After incuba-
tion with different concentrations of 3, BJAB cells were collected by cen-
trifugation at 300g at 48C for 5 min. The mitochondrial permeability
transition was then determined by staining the cells with 5,5,6,6-tetra-
chloro-1,1,3,3-tetraethyl-benzimidazolylcarbocyanin iodide (JC-1; Molec-
Determination of cell concentration and cell viability: Cell viability was
ꢅ
determined with the CASY Cell Counter+Analyzer System from
[
26,41]
5
Schaerfe System, GmbH (Reutlingen, Germany). Settings were specifi-
cally defined for the requirements of the cells used. With this system the
cell concentration can be analyzed simultaneously in three different size
ranges: cell debris, dead cells, and viable cells can be determined in one
ular Probes, Leiden, The Netherlands) as described.
Cells (1ꢃ10 )
were resuspended in phenol red-free RPMI 1640 (500 mL) without sup-
plements, and JC-1 was added to give
a final concentration of
À1
2.5 mgmL . The cells were incubated for 30 min at 378C under moderate
shaking. Control cells were likewise incubated in the absence of JC-1
dye. The cells were harvested by centrifugation at 300g, 48C for 5 min,
washed with ice-cold PBS and resuspended in PBS (200 mL) at 48C. The
mitochondrial permeability transition was then quantified by flow cyto-
metric determination of cells with decreased fluorescence, that is, those
[
39]
5
measurement. BJAB cells were seeded at a density of 1ꢃ10 cells per
mL and were treated with different concentrations of 3; untreated cells
served as control. After 24 h incubation the cells were resuspended, and
1
00 mL from each well was diluted in CASYton (10 mL; ready-to-use iso-
tonic saline solution) for immediate automated count.
14504
www.chemeurj.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 14498 – 14505

Cisplatin Analogue Nucleosides
FULL PAPER
with mitochondria displaying a lower membrane potential (DY
were collected and analyzed by using a FACScan (Becton Dickinson)
equipped with the CELL Quest software. Data are given in %cells with
m
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the late apoptotic cells. Samples were prepared according to the manu-
facturerꢀs instructions. Flow cytometric analysis was performed by using
a FACS-Calibur cytometer (Becton Dickinson, San Jose, CA, USA). The
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[
[
[
[
[
2
2
Western blot analysis: After incubation for either 40 or 52 h with 3
1
(
80 mm), BJAB cells were washed twice with PBS and lysed in buffer con-
taining Tris-HCl (10 mm), pH 7.5, NaCl (300 mm), Triton X-100 (1%),
MgCl (2 mm), ethylenediamino tetra-acetic acid (EDTA; 5 mm), and one
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2
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After being
3
[
2
1
body for 1 h. The membrane was then washed three times with PBST,
and secondary antibody in PBST was applied for 1 h. Finally, the mem-
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Received: March 29, 2010
Published online: October 27, 2010
Chem. Eur. J. 2010, 16, 14498 – 14505
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
14505