Synthesis, biophysical studies, and antiproliferative activity of platinum(II) complexes having 1,2-bis(aminomethyl)carbobicyclic ligands

Article abstract of DOI:10.1021/jm070844u

A selected chemical library of six platinum(II) complexes having 1,2-bis(aminomethyl)carbobicyclic ligands were synthesized after a rational design in order to evaluate their antiproliferative activity and the structure-activity relationships. The cytotoxicity studies were performed using cancer cell lines sensitive (A2780) and resistant (A2780R) to cisplatin. Excellent cytotoxicity was observed for most of complexes, which presented better resistance factors than cisplatin against the A2780R cell line. The interaction of these complexes with DNA, as the target biomolecule, was evaluated by several methods: DNA-platinum binding kinetics, changes in the DNA melting temperature, evaluation of the unwinding angle of supercoiled DNA, evaluation of the interstrand cross-links, and replication mapping. The kinetics of the interaction with glutathione was also investigated to better understand the resistant factors observed for the new complexes.

Full text of DOI:10.1021/jm070844u

424
J. Med. Chem. 2008, 51, 424–431
Synthesis, Biophysical Studies, and Antiproliferative Activity of Platinum(II) Complexes Having
1,2-Bis(aminomethyl)carbobicyclic Ligands
Jordi de Mier-Vinué,†,‡ Marina Gay,†,‡ Ángel M. Montaña,*,† Rosa-Isabel Sáez,† Virtudes Moreno,‡ Jana Kasparkova,§
Oldrich Vrana,§ Pavla Heringova,§ Viktor Brabec,§ Angela Boccarelli,# Mauro Coluccia,# and Giovanni Natile#
Department of Organic Chemistry and Department of Inorganic Chemistry, UniVersity of Barcelona, Martí i Franquès 1-11,
08028 Barcelona, Spain, Institute of Biophysics, Academy of Sciences of the Czech Republic, V.V.i., CZ-61265 Brno, Czech Republic, and
Dipartimento Farmaco Chimico and Dipartimento di Scienze Biomediche e Oncologia Umana, UniVersitá degli Studi di Bari, Bari, Italy
ReceiVed July 11, 2007
A selected chemical library of six platinum(II) complexes having 1,2-bis(aminomethyl)carbobicyclic ligands
were synthesized after a rational design in order to evaluate their antiproliferative activity and the
structure–activity relationships. The cytotoxicity studies were performed using cancer cell lines sensitive
(A2780) and resistant (A2780R) to cisplatin. Excellent cytotoxicity was observed for most of complexes,
which presented better resistance factors than cisplatin against the A2780R cell line. The interaction of
these complexes with DNA, as the target biomolecule, was evaluated by several methods: DNA-platinum
binding kinetics, changes in the DNA melting temperature, evaluation of the unwinding angle of supercoiled
DNA, evaluation of the interstrand cross-links, and replication mapping. The kinetics of the interaction
with glutathione was also investigated to better understand the resistant factors observed for the new
complexes.
Introduction
In the search for new platinum anticancer drugs, great efforts
were devoted to the design of complexes more efficient and
less toxic than the reference drugs already in clinical use. For
this purpose, the rational design of new molecules and the study
of relevant structure–activity relationships (SARa)have been
extended to families of new compounds having high structural
Figure 1. Basic structure of the platinum compounds studied in this
work.
diversity.1
Platinum(II) complexes having a diamino ligand with the two
N-donors attached to a six-membered carbocycle (such as the
1,2-diaminocyclohexane (DACH) ligand present in oxaliplatin)
appear to have interesting anticancer activity.2,3 We have
extended the investigation to the synthesis and biological
evaluation of cisplatinum(II) complexes with ligands containing
a carbobicyclic framework and two methylamino substituents
in 1,2 positions in order to have a 1,4-diaminobutane-like
structure (Figure 1). This type of molecule was designed to
slightly increase the flexibility and the steric hindrance of the
diamine carrier ligand with respect to corresponding complexes
with DACH ligands. Moreover, the stereochemistry and lipo-
philicity of the new ligands were modulated to investigate how
they can influence the interaction with DNA, as the target
molecule, and the pharmacokinetics of the compounds. To this
end, several types of bridges were inserted into the cyclohexane
template. By coordination to platinum, the new ligands generate
a seven-member platinacycle instead of the usual five- or six-
member metallacycles. In the literature there are already a few
examples of seven-member platinacycles having better antipro-
liferative activities than their smaller analogues.4–6 At present,
only chlorides have been used as labile ligands; however, their
substitutions with other anionic ligands, such as carboxylates
or dihydroxylates, are underway in our laboratory. The phar-
macokinetic and the bioavailability of the complexes can depend
very much on the nature of the leaving ligands.
In the present work, a selected chemical library of six
platinum(II) complexes having 1,2-bis(aminomethyl)carbobi-
cyclic ligands have been synthesized and their antiproliferative
activity and structure–activity relationships were investigated.
These compounds were prepared after a rational design of the
changes introduced in the carrier ligand (Figure 1). The cytotoxic
activity of these new platinum complexes was evaluated against
human ovarian cancer cell lines sensitive (A2780) and resistant
(A2780R) to cisplatin. In addition, since DNA is considered
the major pharmacological target of platinum antitumor drugs,
the interaction of these complexes with DNA was also evaluated
by using several biochemical and biophysical methods:
DNA-platinum binding kinetics, changes in the DNA melting
temperature, evaluation of the unwinding angle of supercoiled
DNA, and evaluation of the interstrand cross-links and tran-
scription mapping of the DNA adducts. The kinetics of the
interaction with glutathione was also investigated and put in
relation with the good resistant factors observed for the new
platinum complexes.
* To whom correspondence should be addressed. Phone: +34 934 021
681. Fax: +34-933. 397.878. E-mail: angel.montana@ub.edu.
†
Department of Organic Chemistry, University of Barcelona.
Department of Inorganic Chemistry, University of Barcelona.
Institute of Biophysics, Academy of Sciences of the Czech Republic.
Universitá degli Studi di Bari.
‡
§
Results and Discussion
#
Synthesis of Cisplatinum(II) Complexes. In order to obtain
the desired compounds, six bicyclic diamines were synthesized
by a Diels–Alder reaction7 between fumaronitrile (as dienophile)
aAbbreviations: CT, calf thymus; DPP, differential pulse polarography;
FAAS, flameless atomic absorption spectrophotometry; GSH, glutathione;
SAR, structure–activity relationship; tm, melting temperature.
10.1021/jm070844u CCC: $40.75
2008 American Chemical Society
Published on Web 01/16/2008

Platinum(II) Complexes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 425
Figure 2. Organic synthesis of diamines 1–6.
Table 1. Kinetics of the Binding of 7-12 and Cisplatin to Calf-Thymus
DNA in a Medium of 10 mM NaClO4 at 37 °C Determined by
Differential Pulse Polarographic Assaya
b
compd
t50% (min)
7
8
73
91
9
10
83
64
11
12
cisplatin
110
192
120
a The concentration of DNA was 32 µg/mL and ri ) 0.05. b The times
at which the binding reached 50%.
Biochemical and Biophysical Analysis of DNA Modifica-
tions by Platinum Complexes. DNA Binding. Solutions of
double-helical calf thymus (CT) DNA at 0.1 mg/mL were
incubated with 7-12 at an ri of 0.05 in 10 mM NaClO4 at 37
°C (ri is defined as the molar ratio of added platinum complex
to nucleotide phosphates at the onset of incubation with DNA).
At various time intervals, an aliquot of the reaction mixture
was withdrawn and assayed by differential pulse polarography
(DPP) for platinum not bound to DNA.8 The amount of platinum
bound to DNA, rb (rb is defined as the number of molecules of
the platinum compound bound per nucleotide residue) was
calculated by subtracting the amount of free (unbound) platinum
from the total amount of platinum present in the reaction. The
amount of the platinum compounds bound to DNA increased
with time, and after approximately 10 h all complexes tested
were quantitatively bound. The times at which the binding
reached 50% (t50%) in these binding reactions are shown in Table
1.
Figure 3. Formation of platinum(II) complexes from diamines 1-6.
and an appropriate diene (such as furan, cyclopentadiene, or
cyclohexadiene). In order to prepare the corresponding com-
pounds without a double bond, the intermediate nitriles were
hydrogenated in the presence of Pd/C catalyst. Nitriles were
reduced with LiAlH4 in diethyl ether (or THF in the case of
compound 3) to yield the corresponding diamines 1–6 (Figure
2). Finally, the diamines were reacted with K2PtCl4 in 1:1
ethanol/water to yield the corresponding cisplatinum(II) com-
plexes (Figure 3).
In this synthetic pathway we observe a great difference in
reactivity between the compounds with a hydrocarbon bridge
(1, 2, 5, and 6) and those with an oxygen bridge (3 and 4). The
synthesis of the oxygen-bridged compounds was more difficult
because of the formation of byproducts in nearly all synthetic
steps and the high susceptibility of the cycloadduct to undergo
a retro-Diels–Alder reaction regenerating the starting materials
furan and fumaronitrile.
The values of t50% for all compounds tested were comparable
to that obtained for cisplatin.9 Interestingly, the oxygen bridged
compounds reacted more quickly than cisplatin. In the case of
carbon bridged compounds, there is an interesting relationship
between the values of t50% and the ligand bulkiness. Thus, the
compounds that have smaller bicyclic ligands (7 versus 11 and
In the cases of ligands 5 and 6 the Diels–Alder reaction
between cyclohexadiene and fumaronitrile was rather difficult
and the reactivity was enhanced by addition of BF3 ·OEt2 in
catalytic amounts.
The six diamines were coordinated to platinum, and except
for the oxygen bridged diamines, the platinum complexes were
obtained in medium to good yields (however, complexes 9 and
10 were obtained with yields between 20% and 50%).
8 versus 12) exhibit also smaller values of t50%
.
If we compare the complexes on the basis of the presence or
absence of a double bond (7 versus 8 and 11 versus 12), we
observe significantly smaller values of t50% for compounds
carrying a double bond (especially in the case of 11 versus 12).
In the case of the oxygen-bridged compounds (9 and 10) the
values of t50% were, on the average, smaller than in the other
four cases, indicating that the oxygen atom plays a role in

426 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
de Mier-Vinué et al.
favoring the approach of the compound to the target DNA.
Moreover, in this case the behavior of the compounds having
or lacking a carbon-carbon π-system is reversed (smaller values
of t50% found for the compound lacking the π-system). It is
possible to hypothesize that in the case of oxygen bridged
compounds the approach to DNA is led, to some extent, by the
oxygen atom and therefore the additional effect of a double bond
could be negligible if not detrimental. In contrast, in the case
of compounds lacking an oxygen atom, the approach to DNA
could be favored, to some extent, by the π-electron system of
the CdC double bond when present.
The binding experiments indicate that after 24 h of reaction
time all molecules of the platinum complex are bound to DNA,
therefore making it possible to prepare easily and precisely DNA
samples modified at a preselected value of rb.
Sequence Preference of DNA Adducts. This procedure
involved the extension by TaKaRa Taq DNA polymerase (which
exhibits extreme thermostability) at the 3′ end of a 5′ end
radioactively labeled primer up to the site of platination of the
template strand of pSP73 plasmid (2464 base pairs). The
products of the linear amplification were then examined on DNA
sequencing gels, and the sequence specificity of the platinum
adduct formation was determined to the exact base pair. In vitro
DNA synthesis on double-stranded templates, containing the
adducts of 7-12, generated a population of DNA fragments,
indicating that these adducts terminate duplex synthesis (Figure
4A, lanes 1–6). Sequence analysis of the termination sites
produced by 7-12 (Figure 4) shows that these complexes exhibit
identical sequence dependence of the inhibition and also are
clearly identical to that exhibited by cisplatin (Figure 4A, lane
7). They correspond to guanines (G) and to a considerably lesser
extent to adenines (A). These G and A sites were mostly
contained in GG or AG sequences, which are also the
preferential DNA binding sites for cisplatin. Taken together,
the results of the mapping experiments suggest that the base
sequence selectivity of cisplatin and 7–12 is similar so that the
major adducts formed on DNA by 7–12 and cisplatin are also
identical, presumably 1,2-GG or AG intrastrand cross-links.
Interstrand Cross-Linking. The amounts of interstrand
cross-links formed by 7-12 in linear DNA were measured in
pSP73 plasmid, which was first linearized by EcoRI (EcoRI
cuts only once within pSP73 plasmid) and subsequently
modified by 7–12 at rb ) 0.001. The samples were analyzed
for the interstrand cross-links by agarose gel electrophoresis
under denaturing conditions. An electrophoretic method for
precise and quantitative determination of interstrand cross-
linking by platinum complexes in DNA was described
previously.10–12 Upon electrophoresis under denaturing condi-
tions, 3′-end labeled strands of linearized pSP73 plasmid
containing no interstrand cross-links migrate as a 2464-base
single strand whereas the interstrand cross-linked strands migrate
more slowly as a higher molecular mass species. The band
corresponding to more slowly migrating interstrand-cross-linked
fragments was seen in all experiments where platinum com-
plexes 7–12 were used to modify linearized DNA (Figure 5).
The radioactivity associated with the individual bands in each
lane was measured to obtain estimates of the fraction of non-
cross-linked or cross-linked DNA. The frequency of interstrand
cross-links (the amount of interstrand cross-links per one
molecule of 7–12 bound to DNA) was calculated using the
Poisson distribution from the fraction of non-cross-linked DNA
in combination with the rb values and the fragment size10–12
(Table 2). Compounds 7–12 showed a similar or slightly lower
cross-linking efficiency (2–6%) as parent cisplatin (5–6%).11
Figure 4. (A) Autoradiogram of 6% polyacrylamide/8 M urea
sequencing gel showing inhibition of DNA synthesis by TaKaRa
Taq DNA polymerase on the pSP73 plasmid DNA linearized by
HpaI restriction enzyme and subsequently modified by platinum
complexes. The gel contained the linear amplification products of
control, nonplatinated DNA, and DNA treated with 7-12 or with
cisplatin at rb ) 0.006. Lanes are as follows: (control) unmodified
template; (lanes 1–6) DNA modified by 7–12, respectively; (lane
7) DNA modified by cisplatin; (C, G, T, A) chain-terminated marker
DNAs (note that these dideoxy sequencing lanes give the sequence
complementary to the template strand). The numbers correspond to
the nucleotide sequence numbering of part B. (B) Schematic diagram
showing a portion of the sequence used to monitor inhibition of
DNA synthesis on the template containing adducts of 7. The arrows
indicate the direction of the synthesis. Circles indicate major stop
signals from part A, lane 1. The numbering of the nucleotides in
this scheme corresponds to the numbering of the nucleotides in the
pSP73 nucleotide sequence map.

Platinum(II) Complexes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 427
Table 3. Unwinding of Supercoiled pSP73 DNA by 7-12 and
Cisplatina
unwinding
compd
rb(c)
angleb (deg)
7
8
9
10
11
12
cisplatin
0.065
0.055
0.065
0.065
0.045
0.04
10 ( 2
12 ( 2
10 ( 2
10 ( 2
14 ( 2
16 ( 2
13° 6
Figure 5. Formation of interstrand cross-links by platinum complexes
in linearized pSP73 plasmid. Autoradiogram of denaturing 1% agarose
gels of linearized DNA that was 3′-end labeled. The interstrand cross-
linked DNA appears as the top bands migrating on the gel more slowly
than the single-stranded DNA (contained in the bottom bands). Plasmid
linearized by EcoRI was incubated for 48 h with platinum complexes
at rb values of 0 (lane DNA) or 0.001 (lane CPT, 7–12). Lanes are as
follows: (CPT) DNA modified by cisplatin; (lanes 7–12) DNA modified
by 7–12, respectively.
a Plasmid was incubated with the platinum complex for 48 h in 10 mM
NaClO4 at 37 °C. b The unwinding angle was calculated as described in
the text.
calculated to be -0.036 on the basis of the data relative to cisplatin
for which the rb(c) was determined in this study and Φ was
assumed to be 13°.13 The value of rb(c) at which the supercoiled
and relaxed forms modified by 7–12 comigrate and the corre-
sponding unwinding angles are summarized in Table 3.
Table 2. Frequency of Interstrand Cross-Links (% ICL/Pt) Formed by
7-12 and Cisplatin in Linearized pSP73 Plasmid (2464 Base Pairs), rb
) 0.001a
In these assays we observed a relationship between unwinding
angle and steric bulkiness similar to that observed in the
platinum-DNA binding studies (Table 1). Therefore, ligands
that have a smaller size (7 versus 11 and 8 versus 12) produced
smaller values of unwinding angle. Similarly, ligands containing
a CdC π-system (7 and 11) produced smaller unwinding angles
with respect to compounds lacking the π-system (8 and 12). A
different trend was observed for the oxygen-bridged complexes.
These compounds (9 and 10) gave, on the average, smaller
unwinding angles than the other four compounds, and the
presence or absence of a CdC double bond had practically no
effect. Apparently the oxygen bridge can produce some kind
of additional interaction with DNA, which minimizes unwinding
and offsets any effect of a CdC π-system. In fact, the unwinding
angle was very close to that of cisplatin for all compounds with
the only exception being compound 12, which carries the
bulkiest ligand, lacks a π-system, and produces an unwinding
angle larger than that of cisplatin. From the previous observa-
tions we can conclude that in this series of bicyclic ligands the
unwinding angle is modulated by two factors:
compd
% ICL/Pt
7
8
9
10
11
12
cisplatin
3.8
3.6
3.2
4.3
2.1
5.6
4.8
a Linearized plasmid was incubated with the platinum complex for 48 h
in 10 mM NaClO4 at 37 °C.
Figure 6. Unwinding of supercoiled pSP73 plasmid DNA by
compound 12. The plasmid was incubated with the platinum complex
for 48 h at 37 °C. Lanes are as follows: (lane 1) control, nonmodified
DNA (rb ) 0); (lane 2) rb ) 0.02; (lane 3) rb ) 0.03; (lane 4) rb )
0.04; (lane 5) rb ) 0.05; (lane 6) rb ) 0.06; (lane 7) rb ) 0.07; (lane
8) rb ) 0.08. The top bands correspond to a form of nicked plasmid,
and the bottom bands correspond to the closed, negatively supercoiled
plasmid.
(a) One factor is the presence of an oxygen bridge or an
electron π-system. It causes a lowering of the unwinding angle
(and we have previously observed that it also increases the rate
of DNA binding). It is reasonable to propose that the oxygen
atom or the electron π-system can produce some additional
interactions (electrostatic interactions, hydrogen bonds, or
π-stacking) that can lead to a better fitting of the complex into
the supercoiled DNA structure.
Unwinding Induced in Supercoiled Plasmid DNA. Elec-
trophoresis in native agarose gel was used to quantify the unwinding
induced in pSP73 plasmid by the platinum complexes 7–12 by
monitoring the degree of supercoiling (Figure 6). A compound that
unwinds the DNA duplex reduces the number of supercoils so that
the superhelical density of closed circular DNA decreases. This
decrease of superhelical density induced by binding of unwinding
agents causes a decrease in the rate of migration through the agarose
gel, from which the degree of unwinding can be quantified.13 Figure
6 shows electrophoresis gel results in which increasing amounts
of compound 12 were bound to a mixture of relaxed and
supercoiled pSP73 DNA. Interestingly, the platinum complex
accelerates the mobility of the relaxed form similarly to what is
observed in the case of cisplatin, whose bifunctional binding to
DNA shortens and condenses the DNA helix. The unwinding angle
is given by Φ ) -18σ/[rb(c)] where σ is the superhelical density
and rb(c) is the value of rb at which the supercoiled and relaxed
forms comigrate. Under the present experimental conditions, σ was
(b) Another factor is the steric bulk of the ligands. As the
size of the ligand increased, there is also an increase of Φ (and
also a reduction in the rate of interaction with DNA). This
phenomenon could be interpreted as a consequence of the more
intense distortion produced by bulkier compounds when they
interact with DNA.
DNA Melting. CT DNA was modified by 7–12 to rb ) 0.05
in 10 mM NaClO4. Salt concentration was then further adjusted
by addition of NaClO4 to produce the values of 0.01 or 0.2 M.
The effect on DNA melting temperature (tm) is dependent on
both the amount of platinum bound and the salt concentration.14
All compounds showed a similar behavior. At low ionic
strength there was only a small decrease of tm or no change at
all with respect to free DNA (the observed decrease of tm was
from 0 to 2.7 °C; see Table 4). However, at high ionic strength,
the decrease of tm was greater because of the lower stabilization
imposed on DNA by the positive charge of platinum (the
observed decrease of tm was between 3.4 and 8 °C). Under

428 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
de Mier-Vinué et al.
Table 4. tm Values for Calf-Thymus DNA Modified by 7-12 at rb )
0.05
Table 6. Biological Activity of Compounds 7–12 Tested against
Cisplatin Sensitive (A2780) and Resistant (A2780R) Cell Linesa
tm (°C) (low
tm (°C) (high
IC50 (µM)
resistance cytotoxicity relative
factor
ionic strengtha)
ionic strengthb)
compd
A2780
A2780R
to cisplatinb
compd
7
8
9
10
11
12
noPt
64.4
67.3
65.7
66.5
66.3
66.8
67.1
82.5
84.0
79.0
83.6
81.6
83.3
87.0
7
8
9
10
11
12
1.9 ( 0.9
0.3 ( 0.1
0.07 ( 0.002 0.4 ( 0.007
1.7 ( 0.01 19.7 ( 0.01
0.12 ( 0.05 0.48 ( 0.2
0.07 ( 0.005 0.2 ( 0.05
9.2 ( 3
4.7
8.7
5.6
11.6
4.2
3.5
0.16
1.00
4.28
0.18
2.50
4.28
1.00
2.3 ( 0.2
cisplatin 0.3 ( 0.06
4.5 ( 0.7
17.3
a 0.01 M NaClO4 with 1 mM Tris-HCl/0.1 mM EDTA, pH 7.4. b 0.2 M
NaClO4 with 1 mM Tris-HCl/0.1 mM EDTA, pH 7.4.
a The cytotoxicity of evaluated complexes relative to cisplatin was
estimated as IC50(cisplatin)/IC50(complexes). b Cytotoxicity relative to
cisplatin: IC50(cisplatin)/IC50(compound).
Table 5. Half-Times (t1/2) of Reaction of the Reduced Form of
Glutathione with 7-12 and Cisplatina
compd
t1/2 (min)
Assays of Cytotoxicity. The antiproliferative activity of
compounds 7-12 was studied against A2780 and A2780cisR
human ovarian cancer cell lines sensitive and resistant to
cisplatin, respectively. The work with cisplatin-resistant
cancer cell lines was carried out because of the fact that cell
resistance, either intrinsic or acquired, is one of the main
limitations to the clinical use of cisplatin. The capability of
a new compound to overcome cisplatin resistance is expressed
by the resistance factor (RF ) (IC50 resistant cell lines)/(IC50
sensitive cell lines)); the lower the RF value, the better is
the drug. Usually, acquired resistance is accompanied by a
decrease in cellular transport, increase in DNA repair, and
increase in glutathione level.16–18 The IC50 values (concentra-
tions that cause 50% decrease of the cellular growth) for 96 h
of incubation time are quoted in Table 6.
We observe that compounds 9, 11, and 12 show much better
biological activity than cisplatin. In fact, 9 and 12 are 4.28 times
more active than cisplatin and 11 is 2.5 times more active.
Compound 8 has activity similar to that of cisplatin, while
complexes 7 and 10 are less active. It is worth noting that all
compounds present lower resistance factors than cisplatin when
they were tested against the cisplatin resistant cell line, showing
that this family of complexes is able to partially overcome the
cisplatin resistance developed by the A2780R cancer cell line.
This important finding could be related, as mentioned before,
to a lower drug inactivation by glutathione and other sulfur-
containing platinophiles. Some SAR trends between the bicyclic
residue of the ligands and the differences in biological activity
can be established. Thus, for the carbon-bridged complexes (7,
8, 11, and 12), the presence of a CdC π-system appears to
produce a decrease in biological activity with respect to the
complexes lacking such a π-system. Moreover, it is also found
that an increase in steric bulkiness also produces an increase in
biological activity (decrease in IC50 value) so that compounds
with an ethylene bridge (11, 12) afford better results than those
with a methylene bridge (7, 8). In the case of 11 and 12, an
increase in ligand lipophilicity could be the factor responsible
for their higher cytotoxicity. In the case of complexes with an
oxygen bridge (9, 10), the presence of a CdC π-system
(complex 9) causes a relevant increase in biological activity
with respect to the compound lacking such a π-system (complex
10). Once more, the presence of an olefin π-system appears to
have an opposite effect in the ligands containing carbon bridges
with respect to those containing an oxygen bridge.
7
8
9
10
11
12
cisplatin
333
887
61
835
131
22
a The platinum compounds at 23 µM were mixed with 12 mM glutathione
at 37 °C in a medium of 0.1 M Tris-HCl, pH 7.4, plus 5 mM NaCl. For
other details, see the text.
analogous conditions cisplatin produces a tm decrease of 2.5
°C at low ionic strength and a decrease of 4.1 °C at high ionic
strength.14 In aggregate, the melting behavior of DNA modified
by compounds 7-12 is very similar to that of DNA modified
by cisplatin, which is consistent with the hypothesis that the
DNA modifications caused by these complexes are similar to
those caused by cisplatin.
Reactions with Glutathione. Platinum(II) compounds have
a strong thermodynamic preference for binding to S-donor
ligands. Hence, before antitumor platinum(II) drugs reach DNA
in the nucleus of tumor cells or even after they bind to DNA,
they may interact with various compounds including sulfur-
containing molecules. These interactions are generally believed
to play a role in mechanisms underlying tumor resistance to
platinum compounds, their inactivation, and the side effects.
Therefore, the interest in the interactions of platinum antitumor
drugs with sulfur-containing molecules of biological significance
has increased markedly.
An example of endogenous thiols to which platinum(II)
complexes bind when they are administered intravenously or
after they enter the cell is glutathione (GSH). In the present
work, we investigated using UV absorption spectrophotometry15
the reaction of GSH with 7–12. Thus, 7–12 at 23 µM were
mixed with 12 mM GSH (this concentration of GSH represents
a physiologically relevant concentration) at 37 °C in the medium
of 0.1 M Tris-HCl, pH 7.4, and 5 mM NaCl. The half-times of
these reactions are shown in Table 5. There are significant
differences among the compounds, but all compounds bound
GSH with a slower rate than cisplatin. Interestingly, compound
9, containing an oxygen bridge and a CdC π-system, does not
cause an increase in absorption with respect to the blank (a
solution of GSH). Additional work is needed to interpret this
unexpected observation.
These results are rather important because they are consistent
with the better resistant factors observed for the new compounds
in the antiproliferative studies against resistant cancer cell lines
(see below). This is worth noting because one of the mechanisms
of acquired resistance of cancer cells is the enhanced drug
inactivation fostered by greater GSH and metallothionein
contents.16
Conclusion
A series of platinum compounds with promising biological
activities were synthesized by a two- or three-step synthetic
pathway. Four of these compounds showed better antiprolif-
erative activity than cisplatin (up to 4.28 times more active than

Platinum(II) Complexes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 429
cis-{[(3-Aminomethyl-7-oxabicyclo[2.2.1]hept-5-en-2-yl)methyl-
amine]dichlorido}platinum(II) (9). Yellow solid. Yield ) 44%. Mp
) 230–232 °C. IR (KBr) 3459, 3226, 3150 (N-H, st), 2950, 2886,
the clinically relevant drug). It is worth noting that all
compounds exhibit lower resistance factors than cisplatin when
tested against cisplatin resistant cell lines, showing that this
family of complexes is able to partially overcome the cisplatin
resistance developed by the A2780R cancer cell line. Moreover,
the compounds that showed better in vitro biological activities
and lower resistant factors were also the compounds reacting
more slowly with glutathione. Therefore, it is possible to
hypothesize that our compounds are able to overcome resistance
stemming from drug inactivation by glutathione and other sulfur-
containing platinophiles. Some SARs between the bicyclic
ligand moieties and the complex biological activities appear to
involve the steric bulkiness, the presence of an olefinic π-system,
and the presence of a carbon/oxygen bridge. The biophysical
studies showed that this family of compounds behaves like
cisplatin as far as interaction with DNA is concerned.
1
1593, 1456 cm-1. H NMR (500 MHz, DMSO-d6) δ 1.59–1.67
(m, 1H), 2.07–2.16 (m, 1H), 2.24–2.38 (m, 1H) 2.4–2.45 (m, 1H),
2.75–2.83 (m, 1H), 2.91–2.97 (m, 1H), 4.66 (s, 1H), 4.92 (d, J )
4 Hz, 1H), 5.04 (m, 2NH), 5.15 (s, NH), 5.50 (s, NH), 6.24 (m,
1H), 6.61 (dd, J ) 1.5, 5.5 Hz, 1H) ppm. MS [FAB (+)] m/z 502
(M - 2Cl + NBA), 537 (M - Cl + NBA). Anal. (C8H14ON2Cl2Pt)
C, H, N.
cis-{[(3-Aminomethyl-7-oxabicyclo[2.2.1]hept-2-yl)methyl-
amine]dichlorido}platinum(II) (10). Yellow solid. Yield ) 24%.
Mp ) 248–250 °C. IR (KBr) 3492, 3270, 3226, 3150 (N-H, st),
2969, 2900, 1589, 1456 cm-1. 1H NMR (500 MHz, DMSO-d6) δ
1.42–1.62 (m, 4H), 1.9–2.07 (m, 1H), 2.19–2.27 (m, 1H), 2.28–2.34
(m, 1H), 2.57–2.63 (m, 1H), 2.68-2.75 (m, 1H), 2.85 (m, 1H),
4.2 (d, J ) 4.5 Hz, 1H), 4.5 (t, J ) 4.7 Hz, 1H), 4.85 (s, NH),
4.95–5 (m, NH), 5.12 (s, 2NH) ppm. MS (MALDI-TOF) m/z 351
(M - 2Cl - H), 386 (M - Cl). Anal. (C8H16ON2Cl2Pt) C, H, N.
cis-{[(3-Aminomethylbicyclo[2.2.2]oct-5-en-2-yl)methyl-
amine]dichlorido}platinum(II) (11). Yellow solid. Yield ) 72%.
Mp ) 245–247 °C. IR (KBr) 3444, 3220, 3210 (N-H, st), 3130,
3050, 2940, 2873, 1597, 1456 cm-1. 1H NMR (500 MHz, DMSO-
d6) δ 0.99–1.08 (m, 1H), 1.16–1.26 (m, 1H), 1.48–1.61 (m, 2H),
1.77–1.83 (m, 1H), 1.85–1.94 (m, 1H), 2.34–2.43 (m, 3H), 2.61
(dd, J ) 4.0, 8.0 Hz, 1H), 2.79–2.95 (m, 2H), 4.90–5.00 (m, NH),
5.04–5.18 (s, NH), 5.38–5.46 (m, NH), 5.62–5.82 (m, NH), 6.11
(dd, J ) 1.0, 7.0 Hz, 1H), 6.38 (dd, J ) 1.0, 7.0 Hz, 1H) ppm. MS
(MALDI-TOF) m/z 359.9 (M - 2Cl - H). Anal. (C10H18N2Cl2Pt)
C, H, N.
Because of the interesting properties and the promising
potential as anticancer agents of this series of compounds, further
studies will be carried out to optimize these leads on the basis
of the preliminary SAR results to improve their efficiency,
selectivity, and pharmacokinetic properties. Further studies on
the resistance mechanisms will also be carried out.
Experimental Section
Chemical Synthesis of the Platinum(II) Complexes. Mat-
erials and Methods. NMR spectra were recorded on a Bruker 500
spectrometer using (DMSO-d6) as solvent. Infrared spectra were
recorded by a Nicolet 510 FT-IR spectrophotometer as films on
NaCl crystal plates in the case of oils or as thin plates of the analyte
dispersed in anhydrous KBr in the case of solids. Fast atom
bombardment (FAB) mass spectra were obtained by using a
3-nitrobenzyl alcohol (NBA) matrix and dimethyl sulfoxide (DMSO)
as solvent. Melting points were determined with an Electrothermal
apparatus and are uncorrected. Elemental analyses (combustion
analysis) were obtained with a Carlo Erba EA 1108 apparatus. For
the synthesis and characterization of the diamine ligands 1-6, see
the Supporting Information.
cis-{[(3-Aminomethylbicyclo[2.2.2]oct-2-yl)methylamine]-
dichlorido}platinum(II) (12). Yellow solid. Yield ) 74%. Mp
) 185–187 °C. IR (KBr) 3444, 3270, 3222 (N-H, st), 3125, 2934,
2880, 1600, 1456 cm-1 1H NMR (500 MHz, DMSO-d6) δ
.
1.32–1.51 (m, 12H), 2.36–2.46 (m, 2H), 2.52–2.58 (m, 2H), 4.98
(s, 2NH), 5.16 (s, 2NH) ppm. MS (MALDI-TOF) m/z 355.9 (M -
2Cl - 7H). Anal. (C10H20N2Cl2Pt) C, H, N.
Biochemical and Biophysical Assays. Starting Materials.
Stock solutions of platinum compounds for the biophysical and
biochemical studies were prepared at the concentration of 5 × 10-4
M in 10 mM NaClO4 and stored at 4 °C in the dark. The
concentrations of platinum in the stock solutions were determined
by flameless atomic absorption spectrophotometry (FAAS). CT
DNA (42% G + C, mean molecular mass of about 2 × 107) was
prepared and characterized as described previously.19,20 pSP73
plasmid (superhelical density σ ) 0.036) was isolated according
to standard procedures. Restriction endonucleases EcoRI, HpaI, and
T4 polynucleotide kinase were purchased from New England
Biolabs. Klenow fragment from DNA polymerase I (wild type) was
obtained from Takara (Japan). Acrylamide, bis(acrylamide), and
ethidium bromide were obtained from Merck KgaA (Darmstadt,
Germany). GSH was purchased from Sigma (Prague). Agarose was
from FMC BioProducts (Rockland, ME). Radioactive products were
from MP Biomedicals, (Irvine, CA).
Platination Reactions. DNA was incubated with platinum
complex in 10 mM NaClO4 at 37 °C for 48 h in the dark if not
otherwise stated. The number of molecules of the platinum
compound bound (coordinated) per nucleotide residue (rb values)
was determined by DPP8 or FAAS. For other details, see the text.
Mapping of DNA Adducts. The linear fragment of pSP73 DNA
was obtained as previously described.11,21 A 10 µg amount of
pSP73 was treated with HpaI to obtain linear plasmid (HpaI cuts
only once within this plasmid). After deproteinization by phenol/
chloroform, the modification of this fragment by the platinum
complex was carried out in 10 mM NaClO4 for 48 h at 37 °C to
obtain rb ) 0.006. TaKaRa Taq cycle sequencing kit with TaKaRa
Taq DNA polymerase was used along with the protocol for thermal
cycle DNA sequencing with 5′ end-labeled primer recommended
by the manufacturer with small modifications.22
Synthetic Methodology for the Preparation of the Platinum
Compounds 7–12 Starting from Ligands 1–6. In a typical
experiment a solution of 1 (81 mg, 0.53 mmol) in ethanol (5 mL)
was added to a solution of K2PtCl4 (220 mg, 0.53 mmol) in water
(5 mL). The reaction mixture was stirred in the dark for 24 h, and
then the precipitate was separated from the mother solution by
filtration through a sintered-glass plate (10–16 µm). The yellow
precipitate was washed with water, ethanol, acetone, and dichlo-
romethane and dried under vacuum to obtain 200 mg of product 7
(0.48 mmol, yield ) 90%).
cis-{[(3-Aminomethylbicyclo[2.2.1]hept-5-en-2-yl)methyl-
amine]dichlorido}platinum(II) (7). Yellow solid. Mp ) 240–242
°C. IR (KBr) 3440, 3250, 3210, 3120 (N-H, st), 3075, 2957, 2873,
1
1601, 1454 cm-1. H NMR (500 MHz, DMSO-d6) δ 1.28–1.34
(m, 2H), 1.44 (d, J ) 8.5 Hz, 1H), 1.50 (dd, J1 ) 8.5, 22.1 Hz,
1H), 2.55 (d, J ) 4 Hz, 2H), 2.71–2.77 (m, 1H), 2.81 (s, 2H), 2.90
(d, J ) 9.3 Hz, 1H), 4.92–4.97 (m, NH), 4.99–5.09 (m, 2NH),
5.14–5.21 (m, NH), 5.97 (m, 1H), 6.34 (dd, J ) 3.0, 5.5 Hz, 1H)
ppm. MS (MALDI-TOF) m/z 345.7 (M - 2Cl - H). Anal.
(C9H16N2Cl2Pt) C, H, N.
cis-{[(3-Aminomethylbicyclo[2.2.1]hept-2-yl)methylamine]-
dichlorido}platinum(II) (8). Yellow solid. Yield ) 71%. Mp )
179–182 °C. IR (KBr) 3432, 3255, 3222, 3125 (N-H, st), 2948,
2875, 1603, 1452, 1383 cm-1. 1H NMR (500 MHz, DMSO-d6) δ
1.13 (d, J ) 9.5 Hz, 2H), 1.21–1.27 (m, 2H), 1.37–1.41 (m, 2H),
1.48–1.54 (m, 2H), 2.01–2.08 (m, 1H), 2.11–2.17 (m, 1H),
2.22–2.31 (m, 2H), 2.62–2.74 (m, 2H), 4.82 (s, NH), 4.96 (s, NH),
5.05 (s, NH) ppm. MS (MALDI-TOF) m/z 414 (M - 7H), 385 (M
- Cl), 348 (M - 2Cl - H), 345 (M - 2Cl - 4H). Anal.
(C9H18N2Cl2Pt) C, H, N.
DNA Interstrand Cross-Linking. Platinum complexes were
incubated for 48 h with 1 µg of pSP73 DNA linearized by EcoRI

430 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
de Mier-Vinué et al.
to reach rb ) 0.001. The linear DNA was first 3′-end labeled by
means of Klenow fragment from DNA polymerase I in the presence
of [R-32P]dATP. The platinated samples were precipitated by
ethanol and analyzed for DNA interstrand cross-links by previously
published procedures.10,11 After the platination, the samples were
precipitated by ethanol and the pellet was dissolved in 18 µL of a
solution containing 30 mM NaOH, 1 mM EDTA, 6.6% sucrose
and 0.04% bromophenol blue. The amount of interstrand cross-
links was analyzed by electrophoresis under denaturing conditions
on alkaline agarose gel (1%). After the electrophoresis was
completed, the intensities of the bands corresponding to single
strands of DNA and interstrand cross-linked duplex were quantified
by means of a bioimaging analyzer.
DNA Melting. The melting curves of CT DNA were recorded
by measuring the absorbance at 260 nm. The melting curves of
unplatinated or platinated DNA were recorded after Tris-HCl/EDTA
buffer and NaClO4 were added so that the resulting media contained
0.01 or 0.2 M NaClO4 with 1 mM Tris-HCl/0.1 mM EDTA, pH
7.4. The value of tm was determined as the temperature correspond-
ing to a maximum on the first-derivation profile of the melting
curves. The tm values could be thus determined with an accuracy
of (0.3 °C.
Unwinding of Negatively Supercoiled DNA. Unwinding of
closed circular supercoiled pSP73 plasmid DNA was assayed by
an agarose gel mobility shift assay.13 The unwinding angle Φ,
induced per platinum-DNA adduct, was calculated upon the
determination of the rb value at which the complete transformation
of the supercoiled to the relaxed form of the plasmid was attained.
Samples of plasmid DNA were incubated with platinum complexes
at 37 °C in the dark for 48 h. All samples were precipitated by
ethanol and redissolved in the TAE (Tris-acetate/EDTA) buffer.
An aliquot of the precipitated sample was subjected to electro-
phoresis on 1% agarose gels running at 25 °C in the dark with
TAE buffer and the voltage set at 30 V. The gels were then stained
with ethidium bromide, followed by photography with a transillu-
minator. The other aliquot was used for the determination of rb
values by FAAS.
Reactions with Reduced Form of Glutathione. Reactions of
GSH with platinum complexes were investigated by monitoring
UV absorption at 260 nm of solutions containing the platinum
complex and GSH exactly as described in the previous work;15,23
the absorbance at 260 nm reflects the presence of platinum-sulfur
and disulfide bonds. The platinum compounds were mixed with
GSH at 37 °C in a medium of 0.1 M Tris-HCl, pH 7.4, plus 5 mM
NaCl, in the dark under nitrogen atmosphere. Reactions were
initiated by mixing the platinum complex with the buffer followed
by immediate addition of GSH.
seeded into 96-well microtiter plates in 100 µL of the appropriate
culture medium at a plating density of 10 000 cells/well. After
seeding, microtiter plates were incubated at 37 °C for 24 h prior to
addition of the compounds. After 24 h, several samples of each
cell line were fixed in situ with cold trichloroacetic acid (TCA) to
represent a measurement of the cell population at the time of
compound addition. Experimental compounds were freshly dis-
solved in culture medium and stepwise diluted to the desired final
concentrations, following the addition of different compound
concentrations to quadruplicate wells. The plates were further
incubated at 37 °C for 96 h. Cells were fixed in situ by the gentle
addition of 50 µL of cold 50% (w/v) TCA (final concentration,
10%) and incubated for 1 h at 4 °C. The supernatant was discarded,
and the plates were washed four times with tap water and air-dried.
SRB solution (100 µL) at 0.4% (w/v) in 1% acetic acid was added
to each well, and plates were incubated for 30 min at room
temperature. After the samples were stained, unbound dye was
removed by washing five times with 1% acetic acid and the plates
were air-dried. Bound stain was then dissolved in 10 mM TRIZMA
[tris(hydroxymethyl)aminomethane] base and the absorbance was
read on an automatic plate reader at 515 nm. The compound
concentration able to inhibit cell growth by 50% (IC50 ( SD) was
then calculated from semilogarithmic dose-response plots obtained
from three separate experiments.
Acknowledgment. We thank the Spanish Ministry of
Education and Science for financial support (Grants CTQ-2002-
00601-BQU, CTQ-2005-01834-BQU, and CTQ-2007-64843-
BQU) and the University of Bari and the Italian Ministry for
University and Research (Prin Grant 2005 032730). This
research was also supported by the Grant Agency of the Czech
Republic (Grant 305/05/2030), the Grant Agency of the Ministry
of Health of the Czech Republic (Grant NR8562-4/2005),
Ministry of Education of the Czech Republic (Grant MSMT
LC06030), and the Academy of Sciences of the Czech Republic
(Grants 1QS500040581 and KAN200200651). We thank mem-
bers of COST Action D39 for stimulating discussions. J.
Kasparkova is an international research scholar of the Howard
Hughes Medical Institute. Fellowships to J. de Mier and to M.
Gay from the Generalitat de Catalunya and from The University
of Barcelona, respectively, are also gratefully acknowledged.
Supporting Information Available: Synthesis and characteriza-
tion data of diamine ligands 1-6 and elemental analysis (combus-
tion analysis) data for complexes 7-12. This material is available
free of charge via the Internet at http://pubs.acs.org.
Other Physical Methods. Absorption spectra were measured
with a Varian Cary 4000 UV–vis spectrophotometer equipped with
a thermoelectrically controlled cell holder and quartz cells with a
path length of 1 cm. FAAS measurements were carried out with a
Varian AA240Z Zeeman atomic absorption spectrometer equipped
with a GTA 120 graphite tube atomizer. For FAAS analysis DNA
was precipitated with ethanol and dissolved in 0.1 M HCl. The
gels were visualized by using the BAS 2500 Fujifilm bioimaging
analyzer, and the radioactivities associated with bands were
quantitated with the AIDA image analyzer software (Raytest,
Germany).
Cytotoxicity Studies. The growth inhibitory effect of compounds
under investigation was evaluated on the A2780/A2780cisR pair
of human ovarian cancer cell lines (parent line from an untreated
patient and derived cisplatin-resistant line). Tumour cells were
kindly supplied by Dr. L. Kelland (The Institute of Cancer Research,
Surrey, U.K.). A2780 and A2780cisR cells were maintained at 37
°C in a 10% CO2 humidified air in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% heat-inactivated fetal bovine
serum, 2 mM glutamine, 10 µg/mL insulin, 0.5 µg/mL hydrocor-
tisone, 2.5 µg/mL amphoterycin B, and 50 µg/mL gentamicin. All
culture media and reagents were from Euroclone (Paignton, U.K.).
The growth inhibitory effect of platinum compounds was evaluated
by using the sulforhodamine-B (SRB) assay. Briefly, cells were
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