Structure, solution chemistry, antiproliferative actions and protein binding properties of non-conventional platinum(ii) compounds with sulfur and phosphorus donors

Article abstract of DOI:10.1039/c0dt00845a

Twelve Pt(ii) complexes with cis-PtP₂S₂ pharmacophores (where P₂ refers to two monodentate or one bidentate phosphane ligand and S₂ is a dithiolato ligand) were prepared, characterized and evaluated as potential

Full text of DOI:10.1039/c0dt00845a

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PAPER
Structure, solution chemistry, antiproliferative actions and protein binding
properties of non-conventional platinum(II) compounds with sulfur and
phosphorus donors†‡
Carolin Mu¨gge,§a Claudia Rothenburger,§a Antje Beyer,a Helmar Go¨rls,a Chiara Gabbiani,b Angela Casini,c
Elena Michelucci,d Ida Landini,e Stefania Nobili,e Enrico Mini,e Luigi Messori*b and Wolfgang Weigand*a
Received 14th July 2010, Accepted 8th December 2010
DOI: 10.1039/c0dt00845a
Twelve Pt(II) complexes with cis-PtP2S2 pharmacophores (where P2 refers to two monodentate or one
bidentate phosphane ligand and S2 is a dithiolato ligand) were prepared, characterized and evaluated as
potential antiproliferative agents. The various compounds were first studied from the structural point
of view; afterward, their solubility properties as well as their solution behaviour were analyzed in detail.
Antiproliferative properties were specifically evaluated against A2780 human ovarian carcinoma cells,
either resistant or sensitive to cisplatin. For comparison purposes similar studies were carried out on
four parent cis-dichloro bisphosphane Pt(II)complexes. On the whole, the cis-PtP2S2 compounds
displayed significant antiproliferative properties while the cis-PtP2Cl2 (cis-dichloro bisphosphane Pt(II))
compounds revealed quite poor biological performances. To gain further insight into the molecular
mechanisms of these bisphosphane Pt(II) compounds, the reactions of selected complexes against the
model protein cytochrome c were investigated by ESI-MS and their adduct formation explored. A
relevant reactivity with cyt c was obtained only for cis-PtP2Cl2 compounds, whereas cis-PtP2S2
compounds turned out to be nearly unreactive. The obtained results are interpreted and discussed in
the frame of the current knowledge of anticancer platinum compounds and their
structure–activity-relationships. The observation of appreciable antiproliferative effects for the
relatively inert cis-PtP2S2 compounds strongly suggests that these compounds will undergo specific
activation within the cellular environment.
available on this issue and the thousands of compounds tested so
far, only very few platinum drugs have successfully reached clinical
Introduction
use.1,2 Strikingly, the approved platinum compounds share some
common structural features such as the presence of two NLG (non
leaving groups, typically nitrogen ligands) and two LG (leaving
groups, e.g. halides or carboxylates) in the cis position.3,4 This
type of configuration allows, in principle, bidentate coordination
to adjacent nucleobases of the DNA double helix.5 Remarkably,
cytotoxic platinum complexes are in most cases prodrugs and
their activation is a normal prerequisite for biological activity.
Activation is normally attained through the release of one or more
labile ligands.6
Platinum compounds play a crucial role in modern anticancer
chemotherapy. However, in spite of the numerous studies now
aInstitute of Inorganic and Analytical Chemistry, Friedrich-Schiller-
University Jena, August-Bebel-Strasse 2, 07743, Jena, Germany. E-mail:
wolfgang.weigand@uni-jena.de; Fax: (+49) 3641 948102
bLaboratory of Metals in Medicine, Department of Chemistry, University
of Florence, Via della Lastruccia 3, 50019, Sesto Fiorentino, Firenze, Italy.
E-mail: luigi.messori@unifi.it; Fax: (+39) 055 4573385
cInstitut des Sciences et Inge´niere Chimiques, Ecole Polytechnique Fe´de´rale
de Lausanne (EPFL)CH-1015, Lausanne, Switzerland
dMass Spectrometry Center (CISM), University of Florence, Via U. Schiff
6, 50019, Sesto Fiorentino, Firenze, Italy
The above observations have prompted scientists since early
times to try to establish the precise SAR (structure–activity-
relationships) for anticancer platinum compounds that might
be useful for the design of new active molecules. These rules
were first reported by Hoeschele et al.7 and rapidly became
very popular within the scientific community. Now, as a far
greater number of platinum compounds are available with known
chemical and biological profiles, it is evident that those original
rules suffer many exceptions and limitations. Indeed, numerous
“rule-breaker” platinum-based drugs have been prepared and
eDepartment of Preclinical and Clinical Pharmacology, University of Flo-
rence, Viale Pieraccini 6, 50139, Firenze, Italy
† This paper is dedicated to Dr. Janina Altman on the occasion of her 80th
birthday.
‡ Electronic supplementary information (ESI) available: 31P{ H} spectra
1
of 7 and 8; molecular structures of 6–10; crystallographic data for 6–10;
ESI-MS spectra of cyt c interacting with compounds 1–4. CCDC reference
numbers: 776102 for 6, 776103 for 7, 776104 for 8, 776105 for 9 and 776106
for 10. For ESI and crystallographic data in CIF or other electronic format
see DOI: 10.1039/c0dt00845a
§ These Authors contributed equally to this work.
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characterized and yet they manifest relevant cytotoxic effects;8
e.g. trans-[PtCl2Py2],9 or the multinuclear compound BBR3464.10
However, the originally devised scheme may still be considered
as a useful starting point for the design of cytotoxic platinum
compounds.
Upon careful analysis of the existing literature, we realized
that the substitution of the original donors in cisplatin (N and
Cl) by either phosphorus or sulfur atoms or both has not been
investigated exhaustively. While several transition metal complexes
bearing phosphane ligands were investigated,11–15 only a few
studies concerned the biological characterization of platinum
compounds where the two ammonia ligands were replaced by
phosphanes. In recent years, a series of additional compounds
of this kind have been prepared and characterized by Longato
et al.16–21 Some related phosphane platinum compounds were
prepared and characterized over the last years in other research
laboratories worldwide.22–30
principle, capable of forming relatively strong coordinative bonds
with Pt(II),40 the P–Pt bond presumably being the more inert. In
addition, both P and S are believed to induce conspicuous trans
effects.41,42
These arguments led us to synthesize and characterize a
series of novel platinum compounds with the above-mentioned
structural features (Fig. 1). The compounds were investigated
concerning their behaviour in solution and their reactivity with
the model protein cyt c (cytochrome c) and were also screened
for their antiproliferative properties in vitro. In particular, we
designed a first series of compounds possessing a cis-type PtP2S2
pharmacophore (where P2 refers to two monodentate or one
bidentate phosphane ligand and S2 is a dithiolato ligand). For
comparison purposes, a few previously described and investigated
The established opinion concerning platinum phosphane com-
plexes, mostly arising from older studies,31 is that they are nearly
devoid of antiproliferative activity. However, this feature is often
due to a poor solubility of the phosphane platinum compounds
and is still controversial and debated.
Recently Romerosa, Bergamini et al. proposed to recon-
sider that issue by associating phosphanes with other bioactive
ligands.32,33 In their studies, the authors showed that Pt(II) com-
plexes containing PPh3 and the N7-coordinated anionic ligand
8-MTT (8-(methylthio)theophyllinate) produce a remarkable an-
tiproliferative activity on some cancer cell lines, which decreases
when PPh3 is replaced by the more hydrophilic PTA ligand.
On both cisplatin-sensitive T2 and cisplatin-resistant SKOV3
cell lines, the complex cis-[PtCl(8-MTT)(PPh3)2] manifested an
activity comparable to cisplatin, whereas the analogous com-
plex cis-[PtCl(8-MTT)(PTA)2], containing PTA ligands instead
of PPh3, displayed only a residual activity. Interesting results
were subsequently obtained with Pt(II) complexes simultaneously
bearing both a PPh3 and a PTA ligand.
Even more recently, Osella et al. showed that PtP2Cl2 (dichloro
bisphosphane Pt(II)) compounds bearing R-(+)- or S-(-)-BINAP
(2,2¢-bis(diphenylphosphane)-1,1¢-binaphthyl) as a bisphosphane
ligand exhibit antiproliferative properties towards A2780 ovarian
carcinoma and HCT116 colon adenocarcinoma cell lines.34 When
compared to analogous dichloro platinum(II) complexes bearing
R-(+)- or S-(-)-DABN (1,1¢-binaphthyl-2,2¢-diamine) as the lig-
and, they proved to be less cytotoxic whilst retaining a comparable
cytotoxicity against both cisplatin-sensitive and resistant A2780
cell lines. All compounds were shown to form adducts with G-
quadruplexes in telomeres, a factor that is believed to positively
affect antitumor activity.35
Thioplatin, on the other hand, provides a quite unique example
of a PtS4 pharmacophore, with the Pt(II) centre being surrounded
by four sulfur atoms. The two chelating units can be opened by
protonation to form a reactive diaqua complex, which is eventually
able to form DNA-crosslinks.36,37
The simultaneous coordination of two phosphorous and two
sulfur donors to the same Pt(II) centre might lead to not-easily-
predictable results and, hopefully, to an innovative Pt(II) chemistry.
For example, Miranda et al. reported on Pt(II) compounds with
a trans-PtP2S2 moiety and revealed promising antiproliverative
properties with this system.38,39 Indeed, both P and S donors are, in
Fig. 1 Compounds 1–14.
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Fig. 2
platinum compounds with a cis-PtP2Cl2 pharmacophore were
taken into consideration as well.
7, compound 8 exhibits an AB spin system, whereas complex
7 gives a single resonance with platinum satellites (Fig. S1‡).
Furthermore, the complexes were characterized by single-crystal
X-ray diffraction analysis (Fig. 3).
The phosphane ligands were chosen on the basis of
precise structural and functional reasons. The chelating
dppm (1,2-bis(diphenylphosphino)methane) and dppe (1,3-
bis(diphenylphosphino)ethane) ligands as well as the monoden-
tate PPh3 may serve as stable, lipophilic ligands. Instead, by
coordinating one or two PTA (1,3,5-phosphatriazaadamantane)
ligands to the platinum centre, hydrophilic complexes are typically
generated.10 The sulfur donor sites were designed to build up to
five- or six-membered chelating units with a variety of functional
groups, i.e. CH2, OH, OEt, O-TBDMS (tert-butyldimethylsilyl).
The variation of the reaction conditions or the use of diverse
solvents, for instance the use of methanol or the addition of
water, cannot avoid the formation of the unexpected asymmetrical
product 8, but do reduce the formation. In contrast, by using cis-
[(dppe)Pt(CO3)] as the starting material only the expected product
7 is generated in a high yield of 71% after purification processes
(Reaction B, Fig. 2). Cis-[(dppe)Pt(CO3)] has the advantage
of exhibiting high solubility in common organic solvents in
contrast to the dichloro complex 2, which is scarcely soluble.
For the substitution reaction with dithiols, starting from bispho-
sphane carbonato compounds, no additional base is necessary
because the coordinated carbonato ligand readily deprotonates
the thiol groups. Another method to suppress the formation of
unfavourable side products was found by introducing a base-stable
silyl protecting group, namely TBDMS, to selectively yield the
O-protected compound 9 (Fig. 1).
For the synthesis of compounds 7 and 9 it was emphasized that
the use of the carbonato Pt(II) complex as the starting material
increased the yield of the compounds. However, it requires an
additional synthetic step, resulting in an overall less covenient
synthesis pathway that would only be applied when necessary.
All the bisphosphane dithiolato Pt(II) complexes 5–14 were
characterized by multinuclear NMR spectroscopy, MS spec-
trometry and elemental analyses. Additionally, the bisphosphane
dithiolato Pt(II) complexes 6–10, bearing two PPh3 or the dppe
ligands, were characterized with single-crystal X-ray diffraction
analysis (Fig. 3 and ESI Fig. S2–S4, Table S1‡).
Results and discussion
The panel of bisphosphane platinum compounds
The bisphosphane Pt(II) complexes that were primarily considered
for this study are represented in Fig. 1. Four compounds bearing
a cis-PtP2Cl2 pharmacophore were prepared and characterized
(1–4). Subsequently, a series of Pt(II) complexes sharing a common
cis-type PtP2S2 scaffold (5–14) were synthesized.
Synthesis and structural characterization
The bisphosphane dithiolato Pt(II) complexes 5–16 were prepared
via substitution reaction of the respective dichloro or carbonato
bisphosphane Pt(II) complexes (e.g. 1–4) with the corresponding
dithiols.43,44
Interestingly, treatment of the dppe complex 2 with 1,3-
dimercaptopropane-2-ol in the presence of K2CO3 in ethanol
under reflux conditions for 6 h afforded not only the expected
symmetrical product 7, but also 8 as the main product (Reaction
A, Fig. 2). The reasons for this are the rearrangement processes
occurring during the synthesis. These observations were not
apparent for the syntheses of similar bisphosphane dithiolato
Pt(II) complexes, e.g. 5 or 14. A possible mode of action and the
factors influencing this rearrangement are the subject of further
investigations.
In the molecular structure of compound 7, the Pt(II) ion
possesses a square planar geometry defined by the two sulfur
atoms of the chelating 2-hydroxypropane-1,3-dithiolato dianion
and two P atoms of the bidentate dppe ligand. The metal atom is
protruded from the least-squares plane of the P2S2 donor set by
˚
0.073 A. The six-membered platinum cycle Pt–S1–C1–C2–C3–S2
possesses a twist-conformation with a disorder at the hydroxyl
group bound to C2. The OH function can be situated either
above or below the Pt–S1 ◊ ◊ ◊ C2 ◊ ◊ ◊ S2 plane. The five-membered
ring formed by the coordination of the dppe ligand to the Pt(II)
centre exhibits an envelope conformation. The two Pt–P distances
Compounds 7 and 8 could be separated via column chromato-
graphy (CHCl3–THF 10 : 0.5). The formation of the products can
1
easily be surveyed via 31P{ H} NMR spectroscopy. In contrast to
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˚
centre. The coordination distances (Pt–P1 = 2.2527(13) A,◦Pt–P2 =
˚
2.2520(13) A) as well as the bite angle P1–Pt–P2 (85.35(5) ) are in
good accordance to complex 7.
Solution chemistry
The behaviour in solution of the novel Pt(II) complexes was
analyzed using UV-vis spectrophotometry. Typically, the solubility
in aqueous media turned out to be a critical issue for these
bisphosphane Pt(II) complexes, as compounds 1–11 and 14 are
poorly soluble in aqueous solutions. Yet, significant differences
in the solubility could be ascertained with the PTA containing
compounds 12 and 13, being soluble in water in concentrations of
at least 10-2 M.
To overcome the problem of poor water solubility, most of
the complexes (1–11 and 14) were initially dissolved in DMSO,
although compounds 12 and 13 were dissolved in water. The
resulting stock solutions of all complexes were diluted in 50 mM
reference phosphate buffer at pH 7.4 as described in the experi-
mental section. Under these conditions, the samples analyzed by
spectrophotometry appeared to be stable for 24 h.
These results inspired the procedure of the antiproliferative
tests reported below. The various compounds were added as small
aliquots of a concentrated DMSO solution, in total amounts that
did not lead to precipitation in the culture medium. The absence of
precipitation was ensured by microscopic inspection of the samples
throughout the incubation process.
To get further initial insight into the possible reactivity in
solution of the reported PtP2S2 complexes, hydrolysis studies were
carried out on compound 10 by NMR spectroscopy. Freshly
prepared solutions of 10 in DMSO/aqueous phosphate buffer
1
(pH 7.4; 1 : 4 ratio) were examined through 31P{ H} NMR
spectroscopy at different temperatures (37 ◦C, 90 ◦C) over several
days.
1
31P{ H} NMR spectroscopy is a convenient method to analyze
the hydrolysis of the compound and has been applied by others
earlier (Fig. 4).24,25,29 The chemical shift is typical of the nature of
the phosphane ligand. The platinum satellites not only confirm
the coordination of P to the Pt(II) centre, but also the coupling
constants give information on the trans influence of the ligand
coordinated trans to P. Any shifting of the P signal would
therefore show a change in the chemical environment of the P
atom. The loss of platinum satellites would additionally indicate
a cleavage of the phosphane ligand from the Pt(II) centre. A
Fig. 3 Molecular structures of compounds 7 (top) and 8 (bottom). The
compounds show disorders at C2, respectively. For clarity, only one motive
is depicted in the pictures. Thermal ellipsoids are given at a 50% probability
level.
1
change in the JP–Pt coupling constant would indicate that the
phosphane ligand is still coordinated, whilst substitution takes
˚
˚
(Pt–P1 = 2.250(2) A, Pt–P2 = 2.250(2) A) are equal and in
good accordance with other dppe dithiolato Pt(II) compounds.45
The bite angle for P1–Pt–P2 (85.79(9)◦) is very similar to other
compounds of this type and typical for the dppe ligand.
In 8, the Pt(II) ion is also coordinated by two S and two
P atoms in a slightly distorted square planar geometry. The
coordination of the dithiol to the Pt(II) centre leads to the
formation of a five-membered ring Pt–S1–C1–C2–S2. Similarly
to 7, the structure exhibits disorders at C2 and C3, presumably
due to the existence of an asymmetric centre at C2. For this
reason, the distances and angles in this moiety cannot be discussed
in detail. A five-membered ring with envelope conformation is
formed through the coordination of the dppe ligand to the Pt(II)
1
Fig. 4 Possible aquation products of 10 in aqueous solution. 31P{ H}
NMR spectra would exhibit characteristic effects: A, change in the 1JP–Pt
coupling constant states substitution trans to P; B, loss of platinum
satellites indicates cleavage of phosphane ligand from the complex.
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place at the sulfur-donor sites. Since the spin system is given by
the shape of the spectrum, the distinction between symmetrically
or unsymmetrically substituted complexes is possible and gives
further information on the aquation process.
analyzed in more detail. Among them, 7 compounds were char-
acterized by a PtP2S2 pharmacophore. The remaining compounds
with an IC50 > 50 mM were classified as inactive. Remarkably, the
two water soluble PtP2S2 compounds 12 and 13 turned out to be
inactive. Additionally, most PtP2Cl2 compounds were discarded,
with the only exception being (dppm)PtCl2 3. Among the inactive
compounds were also 1, which had been tested on A2780 cell lines
recently with comparable results34 and 2, said to be ineffective
in vivo against L1210/0 leukemia earlier.22
It is apparent that a number of compounds with a PtP2S2
scaffold (namely 5–11) show relevant growth inhibition effects
on both cell lines, all being more cytotoxic than cisplatin on
the A2780/R cells. Notably, 3, the only PtP2Cl2 complex that
was tested, resulted to be only moderately active in line with the
behaviour of related PtP2Cl2 compounds.17,27,28,34
Although all the investigated compounds are less active in
the cisplatin resistant phenotype A2780/R than in its cisplatin
sensitive counterpart A2780/S, cross-resistance ratios (r) are
markedly lower (1.5–3.0) than that of cisplatin (17.1) (Table 1).
This observation may suggest that these Pt(II) compounds might
partially overcome some of the mechanisms of resistance to
cisplatin.
The observation of a significant cytotoxic activity for various
PtP2S2 compounds, in spite of their apparent chemical inertness
(vide supra) suggests that these compounds may undergo activation
under biologically relevant conditions (i.e in the cellular milieu)
and thus cause an appreciable cellular damage through platinum
coordination to essential biomolecules. It remains to be established
whether these compounds retain, as cisplatin does, the ability to
bind DNA and to cause the characteristic, often lethal, DNA
intrastrand lesions, or follow a different mode of action.
The tested compound manifested an appreciable stability and
there were neither evident signs of aquation processes nor of sig-
nificant chemical transformations, similar to the inertness of Pt(II)
complexes with tri-n-butylphosphine reported by Kozelka et al.24
Only after heating a solution of 10 to 90 ◦C in DMSO/phosphate
buffer medium for 5 d, the emergence of additional signals in the
1
31P{ H} NMR spectra suggested the formation of several prod-
ucts. All the surveyed main signals exhibited platinum satellites
with varying coupling constants, suggesting that the bisphosphane
ligand stays bound to the Pt centre whilst the sulfur donor sites
might be substituted through several ligands. Due to the variety
of the formed products, most likely due to some decomposition, it
was impossible to isolate and characterize the formed compounds
separately.
To ensure that the presence of chloride ions does not signifi-
cantly influence the hydrolysis of the compounds, the behaviour
of 10 was also tested in the presence of 0.9% NaCl. After 72 h
at 37 ◦C, no reaction was visible in the 31P{ H} NMR spectra,
1
indicating that neither the P nor the S donors are replaced by
chloride.
These results lead to the assumption that the Pt(II) compounds
with the PtP2S2 pharmacophore bear an activation mechanism dif-
ferent from the “classical” drugs cisplatin and analogues, since the
latter compounds were proven to hydrolyze under physiological-
like conditions prior to binding to DNA.46 Yet, binding of the
presented compounds to DNA must not be precluded, as Messere
et al. showed that bisphosphane Pt(II) complexes that wouldn’t
undergo ligand exchange still readily bound to DNA fragments.29
Cytotoxic activity
Reactions with cytochrome c
The antiproliferative effects of the panel of Pt(II) compounds were
carefully determined in vitro against the mentioned A2780 ovarian
carcinoma human cell lines, either sensitive (A2780/S) or resistant
(A2780/R) to cisplatin, according to established protocols. The
cell growth inhibition was measured after 72 h of exposure to the
substances. The resulting IC50 values are shown in Table 1.
Out of an initial screening of the whole panel of 14 substances,
8 compounds manifested relevant antiproliferative actions, with
most of the IC50 values in the low mM range. Those were then
To further characterize the reactivity of the investigated Pt(II)
compounds with biomolecules and their mechanisms of inter-
action, a few representative compounds of the above panel
were reacted with the model protein cyt c and the resulting
products were analyzed through mass spectrometric methods,
using established protocols.47–50 These experiments were performed
with the dichloro complexes 1–4, as well as with the complexes
bearing a 2,3-dimercaptopropanole ligand 6, 10, 11 and 13,
in order to investigate the influence of both the nature of the
phosphane ligand and the presence of Cl or S in the complexes.
In the case of the dithiolato compounds 6, 10, 11 and 13, protein
platination is very scarce, nearly undetectable, after incubation
for 24 and 96 h at 37 ◦C (data not shown). These findings
are in agreement with the relevant chemical inertness of these
compounds (vide supra).
In contrast, the formation of relevant amounts of adducts was
observed in the case of the PtP2Cl2 complexes. Experiments were
performed at 3 : 1 metal complex : cyt c molar ratios, as described
in the experimental section. Representative deconvoluted ESI-MS
spectra are shown in Fig. 5 as well as in the ESI‡.
Cis-[(PPh3)2PtCl2] 1 resulted to be the least reactive PtP2Cl2
compound with cyt c. In fact, incubation for 24 h at 37 ◦C led to
a weak signal (3% r.i.) at 13077.5 Da assigned to a cyt c-Pt(PPh3)2
adduct (Fig. S5 in the ESI‡).
Table 1 IC50 values determined for the most active compounds in the
A2780 cisplatin sensitive (S) and resistant (R) cell lines
Compound
IC50 (mM)
ra
A2780/S
12.0 2.8
5.1 1.5
3.4 1.2
4.6 1.8
3.6 1.8
5.8 1.5
4.6 1.8
2.8 1.4
1.3 0.3
A2780/R
19.7 2.7
7.7 1.5
5.9 0.3
8.5 1.3
8.0 0.2
17.5 3.1
8.3 1.4
6.3 3.0
22.2 4.9
3
1.6
1.5
1.7
1.8
2.2
3.0
1.8
2.3
17.1
5
6
7
8
9
10
11
cisplatin
a r, resistance ratio.
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Fig. S7‡ shows the theoretical and observed isotope patterns of
the [cyt c + 3 Pt(dppe)] adduct at charge state +8.
Cis-[(dppm)PtCl2] 3 shows a similar behaviour, with three Pt
fragments bound to cyt c. Moreover, the platinated cyt c species
become the predominant ones in solution over the unbound
protein after 96 h (Fig. 5b). In detail, the peak at 14108.6 Da in the
spectrum corresponds to the [cyt c + 2 Pt(dppm) + Pt(dppm)H2O]
species, indicating that one Pt(II) has substituted a chlorido ligand
with a water molecule prior to adduct formation. Fig. S8‡ shows
the theoretical and observed isotope patterns of the [cyt c + 2
Pt(dppm) + Pt(dppm)H2O] adduct at charge state +8.
Finally, the PTA-containing complex 4 exhibits a peculiar be-
haviour with respect to the above-mentioned compounds and has
the least tendency to form 3 : 1 Pt:protein adducts. After incubating
the protein with a stoichiometric amount of compound, the [cyt c
+ Pt(PTA)] species at 12709.4 Da are predominant in the sample
(Fig. S9‡). By increasing the metal complex : protein ratio up to
3 : 1, a mixture of adducts is formed, showing protein binding
of free Pt2+ ions as well as a mixture of platinum centres with
varying PTA content (Fig. 5c). The obtained results demonstrate
that among the phosphane ligands under investigation, the PTA
group is the most labile platinum ligand.
In general, for the three most reactive compounds, adducts with
cyt c bear one or more bound Pt-phosphane moieties. This means
that the release of the original chloro ligand takes place upon
protein binding, whilst the phosphane ligand remains bound to
the platinum centre in most cases, with the exception of the PTA
containing complex. In the case of 2, the presence of monodentate
[(dppe)PtCl]+ fragments is observed at 24 h incubation time, which
then convert into bidentate adducts upon chloride loss.
Overall, the reactivity of the PtP2Cl2 compounds with cyt c are
similar to those previously observed for cisplatin and other estab-
lished platinum drugs,47 although all the tested PtP2Cl2 complexes
are much less cytotoxic. It is possible to hypothesize that protein
interactions might account for the reduced antiproliferative effects
of the chloride derivatives in the reported series.
Concluding remarks
In spite of the intense investigations carried out so far on platinum
compounds as potential anticancer agents, only relatively few
studies have considered Pt(II) complexes with phosphane ligands
in the place of amines. This is possibly due to the poor solubility
in aqueous solutions of platinum phosphane complexes and to the
old idea that the replacement of amine with phosphane ligands in
antitumor Pt(II) compounds irreversibly leads to nearly complete
loss of biological activity. However, this latter concept never
achieved any conclusive confirmation and was recently questioned
by several authors. The state of the art on this specific issue was
revisited in detail by Osella et al. in a very recent paper.34
Fig. 5 Deconvoluted ESI-MS spectra of (a) 2, (b) 3 and (c) 4, incubated
with cyt c at a metal complex : protein ratio of 3 : 1 at 37 ◦C, recorded after
96 h.
Representative spectra for compound 2 are shown in Fig. 5a
and S6‡. After 24 h incubation, signals around 14170.6 Da can be
assigned to [cyt c + 2 Pt(dppe) + Pt(dppe)Cl] species and less
intense signals at 14133.6 Da can be attributed to [cyt c + 3
Pt(dppe)] adducts (Fig. S6‡). In addition, low r.i. signals at 13540.5
Da could be attributed to [cyt c + 2 Pt(dppe)] adducts. With time,
the signals of the platinated cyt c species markedly increase, and
the peaks of the adducts containing the [(dppe)PtCl]+ fragment
decrease in favour of the bidentate [Pt(dppe)]2+ adducts (Fig. 5a).
In the present study we have prepared and characterized some
novel Pt(II) complexes bearing the unusual PtP2S2 pharmacophore.
Both P and S ligands are known to be strong ligands for the
Pt(II) centre; thus the resulting complexes are expected to manifest
a high degree of stability (both thermodynamic and kinetic).
The obtained PtP2S2 compounds were thoroughly characterized
from the structural point of view and the hypothesized structures
confirmed. Moreover, their solution behaviour was analyzed in
detail and the extreme stability of the PtP2S2 scaffold towards
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aquation could be demonstrated. Only under very drastic solution
conditions, some initial aquation of a representative PtP2S2
complex was observed.
The following Pt(II) complexes were prepared according to liter-
ature procedures: cis-[(Ph3P)2PtCl2] 1,51 cis-[(dppe)PtCl2] 2,51 cis-
[(dppe)PtCO3],52 cis-[(dppm)PtCl2] 3,53 cis-[(PTA)2PtCl2] 454 and
cis-[(Ph3P)(PTA)PtCl2].54
The novel PtP2S2 compounds were subsequently evaluated
for their antiproliferative potential against the standard A2780
ovarian carcinoma human cell lines, either sensitive or resistant
to cisplatin. For comparison reasons, even a few representative
PtP2Cl2 compounds were tested. Very surprisingly, in spite of their
proved chemical inertness and of the expected poor biological
actions, relevant antiproliferative effects were detected for PtP2S2
compounds, with some of them being only slightly less effective
than cisplatin. Interestingly, the measured cross-resistance ratios
of PtP2S2 compounds are markedly lower than that of cisplatin.
This observation suggests that these Pt(II) compounds might
partially overcome some of the mechanisms of resistance to
cisplatin. Far lower antiproliferative effects were instead evidenced
for PtP2Cl2 compounds. No antiproliferative activity was found for
the water-soluble PtP2S2 compounds bearing PTA ligands.
Afterward, four selected PtP2S2 complexes were challenged
against the model protein cyt c in comparison with a few repre-
sentative PtP2Cl2 compounds to disclose their possible interaction
modes at the molecular level. Reactions were monitored through
an established ESI-MS procedure. Mass spectra revealed a very
poor reactivity of PtP2S2 compounds with cyt c whereas PtP2Cl2
compounds turned out to be far more reactive and to produce
extensive protein platination.
Thus, the reported data prove unambiguously that novel PtP2S2
compounds are very inert both alone, in aqueous solution, and
in their reactions with the model protein cyt c in line with
expectations. On the other hand, the observation of appreciable
cytotoxic effects for several of them against both A2780 cell lines
strongly suggests that these compounds may undergo activation
within the cellular milieu, as chemical activation of the platinum(II)
centre is indeed an essential prerequisite for the antiproliferative
actions. These mechanisms of activation will be the subject of
further investigation.
Structural and purity determination
NMR spectra were recorded with Bruker Avance 200 (operating at
200.13, 50.33 and 81.01 MHz for 1H, 13C and 31P, respectively) and
Bruker Avance 400 (operating at 400.13, 100.63 and 162.00 MHz
for 1H, 13C and 31P, respectively) instruments at room temperature.
1
Deuterated chloroform was used as the NMR solvent and H
1
and 13C{ H} NMR spectra were referenced to residual solvent
resonances (7.26 ppm and 77.16 ppm, respectively), whereas
1
31P{ H} NMR spectra were referenced to external H3PO4. The
mass spectra were recorded using SSQ 10 or MAT95XL, using
ESI- or DEI-ionization. IR spectra (KBr pellets) were recorded
on a Perkin Elmer System 2000 FT-IR spectrometer. Elemental
analyses (C, H, N, S) are the results of single measurements and
were carried out with LECO CHNS-932. They confirmed all
compounds to be pure to a degree of ≥95% purity.
Crystal structure determination
Single crystals were obtained from CH2Cl2–pentane. The intensity
data for the compounds were collected on a Nonius KappaCCD
diffractometer using graphite-monochromated Mo-Ka radiation.
Data were corrected for Lorentz and polarization effects but not
for absorption effects.55,56 Crystallographic data as well as structure
solution and refinement details are summarized in Table S1‡. The
structures were solved by direct methods (SHELXS57) and refined
2
by full-matrix least squares techniques against Fo (SHELXL-
9757). All hydrogen atoms were included at calculated positions
with fixed thermal parameters. All non-disordered, non-hydrogen
atoms were refined anisotropically57 and XP (SIEMENS Analyti-
cal X-ray Instruments, Inc.) was used for structure representations.
Overall, the herein reported results are rather intriguing and
make a global interpretation of the biological profile of platinum
phosphane compounds very complex and hard to achieve. The
biological behaviour of this class of compounds had been gener-
alized and oversimplified in early times of platinum compounds
research leading to the (erroneous) idea that platinum phosphane
compounds, on the whole, should be classified as biologically
ineffective. Remarkably, recent and promising results coming from
various laboratories worldwide, and the present investigation,
point out that phosphane ligands may offer new and unexplored
opportunities in the discovery and development of new anticancer
platinum compounds. Even very inert complexes such as those
bearing a PtP2S2 pharmacophore, such as reported here, may
manifest interesting antitumor profiles.
Synthesis of the Pt(II) compounds
cis-Bis(PPh3)2(2-hydroxypropane-1,3-dithiolato)platinum(II) 5.
1,3-Dimercaptopropane-2-ol (27 mg, 0.22 mmol) was added to
a suspension of 1 (158 mg, 0.20 mmol) and K2CO3 (55 mg,
0.40 mmol) in EtOH (50 mL). After stirring at r.t. for 15 h, the
mixture was heated to 78 ◦C for 6 h. The solvent was removed
under reduced pressure and the crude product was extracted with
a CHCl3–H2O mixture, the organic layer was subsequently dried
with Na2SO4, filtered and then the solvent was removed under
reduced pressure. Purification was carried out on silica gel using
a gradient (CH2Cl2/acetone 10 : 0.5). The collected fraction (RF =
0.2) was dried under vacuum and crystallized in CH2Cl2–pentane
◦
to give 5 (90 mg, 54%) as a yellow substance (decomp. 197 C).
Found: C 55.0, H 4.53, S 7.6. Calcd for C39H36OP2PtS2 (841.86):
C 55.6, H 4.3, S 7.6. IR (KBr): nmax/cm-1 3435 (nOH), 3053 (nCH,Ph),
2905 (nas,s CH2), 1480 (nC C), 1435 (nP–Ph), 744 (nC–S). 1H NMR (400
MHz, CDCl3, 25 ◦C): d 7.42 (12 H, m, Ph), 7.26 (8 H, m, Ph),
7.13 (12 H, m, Ph), 4.23–4.34 (1 H, m, CH), 3.10–3.30 (2 H, m,
Experimental
General
All syntheses involving air-sensitive compounds were carried
out using standard vacuum-line and Schlenk techniques under
an Argon atmosphere. All chemicals and solvents used were
obtained from commercial suppliers and were used as received.
1
S–CH2), 2.72–2.97 (2 H, m, S–CH2). 13C{ H} NMR (100 MHz,
CDCl3, 25 ◦C): d 134.8 (m, Ph), 130.9 (m, Ph), 130.1 (m, Ph),
127.5 (m, Ph), 71.0 (s, CH), 32.8 (s w/Pt satellites, 2JC,Pt 16.1 Hz,
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1104 (nC–O–C), 750 (nC–S). H NMR (400 MHz, CDCl3, 25 ◦C):
◦
1
1
S–CH2). 31P{ H} NMR (81 MHz, CDCl3, 25 C): d 27.3 (s w/Pt
3
satellites,1JPPt 2863 Hz). MS (ESI): m/z (%) = 864.0 (M+Na)+.
d 7.76 (8 H, m, Ph), 7.42 (12 H, m, Ph), 3.66 (1 H, t, JHH 9.9
Hz, CH2–O), 3.47–3.52 (1 H, m, CH2–O), 3.39–3.54 (2 H, m,
cis-Bis(PPh3)2(3-hydroxypropane-1,2-dithiolato)platinum(II) 6.
Prepared as described for 5 from 2,3-dimercaptopropane-1-ol
(27 mg, 0.22 mmol), 1 (158 mg, 0.20 mmol) and K2CO3 (55 mg,
0.40 mmol) in EtOH (50 mL) and H2O (1 mL). The purification
was carried out on silica gel using a gradient (CH2Cl2/acetone
10 : 0.25). The collected fraction (RF = 0.1) was dried under
vacuum, crystallized in CH2Cl2–pentane to give 6 (111 mg, 60%)
as a yellow substance (decomp. 196 ◦C). Found: C 52.3, H 4.2, S
6.95. Calcd for C39H36OP2PtS2·CH2Cl2 (926.79): C 51.8, H 4.1, S
6.9. IR (KBr): nmax/cm-1 3436 (nOH), 3053 (nCH,Ph), 2922 (nas,s CH2),
1481 (nC C), 1435 (nP–Ph), 744 (nC–S). 1H NMR (400 MHz, CDCl3,
CH2–CH3), 3.32 (1 H, m, CH), 2.72–3.04 (2 H, m, S–CH2),
3
2.21–2.47 (4 H, m, P–CH2), 1.13 (3 ◦H, t, JHH 7.0 Hz, CH3).
1
13C{ H} NMR (100 MHz, CDCl3, 25 C): d 133.3 (m, Ph), 131.1
(m, Ph), 130.0 (m, Ph), 128.6 (m, Ph), 73.6 (virt. t, 3JCPt 32.9 Hz,
CH2–O), 65.8 (s, CH2–CH3), 50.8 (d, 3JCP 10.6 Hz, CH), 39.6 (d,
1
3JCP 10.7 Hz, S–CH2), 28.1 (m, P–CH2), 15.3 (s, CH3). 31P{ H}
◦
1
NMR (81 MHz, CDCl3, 25 C): d 49.1 (AB spin system, JPPt
2720 Hz, 2JPP 12 Hz), 48.8 (AB spin system, 1JPPt 2778 Hz, 2JPP 12
Hz). MS (ESI): m/z 765.9 (M+Na)+.
2-(tert-butyldimethylsilyloxy)propane-1,3-dithiol58,59
.
1,3-Di-
◦
mercaptopropane-2-ol (1.58 g, 12.72 mmol) and imidazole
(1.98 g,19.15 mmol) were dissolved in dry DMF (5 mL) at
25 C): d 7.41 (12 H, m, Ph), 7.26 (8 H, m, Ph), 7.14 (12 H, m,
Ph), 3.73–3.78 (1 H, m, CH2–O), 3.52–3.58 (1 H, m, CH2–O),
0
◦C. The mixture was slowly dropped into a solution of tert-
3.02–3.24 (1 H, m, CH), 2.89–2.99 (1 H, m, S–CH2), 2.53–2.74
1
(1 H, m, S–CH2), 2.23 (1 H, m, OH). 13C{ H} NMR (100 MHz,
butyldimethyl-chlorosilane (TBDMSCl, 2.28 g, 15.16 mmol) in
5 mL of dry DMF, leading to a colour shift from colourless to pale
pink. After stirring at r.t. for 15 h, extraction was carried out with
hexane, followed by washing with 10% NaHCO3 and water. The
organic extracts were dried with Na2SO4, filtered and evaporated
to dryness to yield 2-(tert-butyldimethylsilyloxy)propane-1,3-
dithiol (2.3 g, 75% yield) as a colourless oil, which was used
without further purification. IR (Nujol): nmax/cm-1 2939 2925
2855 (nas,s CH3/CH2), 1362 (nsC(CH3)3), 1254 (nSi–C), 837 (nSi–C), 743
CDCl3, 25 ◦C): d 134.7 (m, Ph), 131.0 (m, Ph), 130.2 (m, Ph),
127.5 (m, Ph), 65.3 (virt. t, 3JCPt 18.7 Hz, CH2–O), 55.8 (dd, 3JCP(A)
11.1 Hz, 3JCP(B) 12.6 Hz, CH), 40.6 (dd, 3JCP(A) 12.2 Hz, 3JCP(B) 14.4
◦
1
Hz, S–CH2). 31P{ H} NMR (81 MHz, CDCl3, 25 C): d 24.6 (AB
spin system, 1JPPt 2855 Hz, 2JPP 24 Hz), 23.4 (AB spin system, 1JPPt
2929 Hz, 2JPP 24 Hz). MS (ESI): m/z 863.9 (M+Na)+.
cis-Bis(dppe)(2-hydroxypropane-1,3-dithiolato)platinum(II) 7.
Method A: Prepared as described for 5 from 1,3-dimercaptop-
ropane-2-ol (27 mg, 0.22 mmol), 2 (133 mg, 0.20 mmol) and
K2CO3 (55 mg, 0.40 mmol) in EtOH (50 mL). Method B:
1,3-Dimercaptopropane-2-ol (27 mg, 0.22 mmol) was added
to a solution of cis-[(dppe)Pt(CO3)] (120 mg, 0.18 mmol) in
acetone (40 mL). The mixture was stirred at room temperature
for 15 h, then the solvent was removed under reduced pressure.
Purification was carried out on silica gel using a gradient
(CHCl3–THF, 10 : 0.5), the collected fraction (RF = 0.2) was dried
under vacuum, crystallized in CH2Cl2–pentane to give a pale
yellow substance of 7 (Method A: 13 mg, 9%; Method B: 101 mg,
71%, decomp. 206 ◦C). Found: C 48.6, H 4.4, S 8.9. Calcd for
C29H30OP2PtS2 (715.70): C 48.7, H 4.2, S 9.0. IR (KBr): nmax/cm-1
3435 (nOH), 3051 (nCH, Ph), 2908 (nas,s –CH2), 1483 (nC C), 1435 (nP–Ph),
1
(nC–S). H-NMR (200 MHz, CDCl3): d 0.07 (6H, s, Si(CH3)2),
0.88 (9H, s, C(CH3)3), 1.38 (2H, t, SH, 3JHH 8.5 Hz) 2.71 (4H, m,
1
S–CH2), 3.80 (1H,m, CH). 13C{ H}-NMR (50 MHz, CDCl3): d
-4.5 (Si(CH3)2), 18.0 (C(CH3)3), 25.8 (C(CH3)3), 29.2 (S–CH2),
73.8 (CH).
cis-Bis(dppe)(2-(tert-butyldimethylsilyloxy)propane-1,3-dithio-
lato)platinum(II) 9. Prepared as described for 7 (Method B)
from 2-(tert-butyldimethylsilyloxy)propane-1,3-dithiol (52 mg,
0.22 mmol) and cis-[(dppe)Pt(CO3)] (120 mg, 0.18 mmol) in
acetone (40 mL). The purification was carried out on silica
gel using a gradient (CH2Cl2–acetone, 10 : 0.2). The collected
fraction (RF = 0.4) was dried under vacuum and crystallized
in CH2Cl2–pentane to give 9 (114 mg, 76%) as a pale yellow
substance (decomp. 201 ◦C). Found: C 50.85, H 5.5, S 7.6. Calcd
for C35H44OP2PtS2Si (829.96): C 50.65, H 5.3, S 7.7. IR (KBr):
◦
1
750 (nC–S). H NMR (400 MHz, CDCl3, 25 C): d 7.77 (8 H, m,
Ph), 7.43 (12 H, m, Ph), 4.33 (1 H, m, CH), 3.15–3.33 (2 H, m,
S–CH2), 2.91–3.11 (2 H, m, S–CH2), 2.85 (1 H, d, 3JHH 10.11 Hz,
n
max/cm-1 3053 (nCH,Ph), 2956 (nasCH3), 2926 (nasCH2), 2855 (nsCH3/CH2),
1
1
OH), 2.28 (4 H, m, P–CH2). 13C{ H} NMR (100 MHz, CDCl3,
1471 (nC C), 1436 (nP–Ph), 1254 (nSi–C), 835 (nSi–C), 748 (nC–S). H
NMR (400 MHz, CDCl3, 25 C): d 7.79 (8 H, m, Ph), 7.42 (12
◦
◦
25 C): d 133.4 (m, Ph), 131.2 (m, Ph), 128.7 (m, Ph), 128.6 (m,
3
2
Ph), 71.6 (s w/Pt satellites, JCPt 26.7 Hz, CH), 30.7 (virt. t JCPt
H, m, Ph), 4.05–4.17 (1 H, m, CH), 2.77–3.32 (4 H, m, S–CH2),
21.3 Hz, S–CH2), 28.7 (m, P–CH2). 31P{ H} NMR (81 MHz,
2.28 (4 H, m, P–CH2), 0.82 (9 H, s, C(CH3)3), -0.02 (6 H, s,
1
◦
CDCl3, 25 ◦C): d 48.4 (s w/Pt satellites, 1JPPt 2748 Hz). MS (EI):
m/z 715 (M+).
Si(CH3)2). 13C{ H} NMR (100 MHz, CDCl3, 25 C): d 133.5 (m,
Ph), 131.1 (m, Ph), 129.3 (m, Ph), 128.6 (m, Ph), 76.4 (s w/Pt
satellites, 3JCPt 21.0 Hz, CH), 31.8 (s w/Pt satellites, 2JCPt 20.9 Hz,
S–CH2), 28.6 (m, P–CH2), 26.0 (s, C(CH3)3), 18.3 (s,◦C(CH3)3),
1
cis-Bis(dppe)(3-ethoxypropane-1,2-dithiolato)platinum(II)
8.
Prepared as described for 5 from 1,3-dimercaptopropane-2-ol
(27 mg, 0.22 mmol), 2 (133 mg, 0.20 mmol) and K2CO3 (55 mg,
0.40 mmol) in EtOH (50 mL). The purification was carried out
on silica gel using a gradient (CHCl3–THF 10 : 0.5), the collected
fraction (RF = 0.4) was dried under vacuum and crystallized in
CH2Cl2–pentane to give 8 (68 mg, 46%) as a pale yellow substance
(decomp. 183 ◦C). Found: C 49.9, H 4.55, S 8.55. Calcd for
C31H34OP2PtS2 (743.76): C 50.1, H 4.6, S 8.6. IR (KBr): nmax/cm-1
3053 (nCH,Ph), 2969 (nasCH3), 2913 (nasCH2), 1483 (nC C), 1436 (nP–Ph),
-4.6 (s, Si(CH3)2). 31P{ H} NMR (81 MHz, CDCl3, 25 C): d 47.9
1
(s w/Pt satellites, 1JPPt 2738 Hz). MS (ESI): m/z 851.9 (M+Na)+.
cis-Bis(dppe)(3-hydroxypropane-1,2-dithiolato)platinum(II) 10.
Prepared as described for 5 from 2,3-dimercaptopropane-1-ol
(27 mg, 0.22 mmol), 2 (133 mg, 0.20 mmol) and K2CO3 (55 mg,
0.40 mmol) in MeOH (50 mL). Purification was carried out on
silica gel using a gradient (CH2Cl2–acetone, 10 : 0.5), the collected
fraction (RF = 0.3) was dried under vacuum and crystallized in a
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CH2Cl2–pentane mixture◦to give 10 (120 mg, 79%) as a pale yellow
substance (decomp. 199 C). Found for C 46.5, H 4.1, S 8.2, Cl
4.5. Calcd for C29H30OP2PtS2·0.5CH2Cl2 (758.17): C 46.7, H 4.1,
S 8.5, Cl 4.7. IR (KBr): nmax/cm-1 3435 (nOH), 3051 (nCH, Ph), 2916
(nas,s CH2), 1483 (nC C), 1435 (nP–Ph), 749 (nC–S). 1H NMR (400 MHz,
CDCl3, 25 ◦C): d 7.83 (4 H, m, Ph), 7.67 (4 H, m, Ph), 7.45 (6 H,
m, Ph), 7.42 (6 H, m, Ph), 3.53–3.67 (2 H, m, CH2–O), 3.26 (1 H,
m, CH), 2.75–2.92 (2 H, m, S–CH2), 2.19–2◦.53 (5 H, m, P–CH2,
0.40 mmol) in EtOH (50 mL). The purification was carried out
via Soxhlet extractor in CHCl3, the collected fraction was dried
under vacuum to give 13 (57 mg, 45%) as a colourless substance
(decomp. 220 ◦C). Found: C 28.8, H 5.1, N 12.9, S 10.5. Calcd
for C15H30N6OP2PtS2 (631.59): C 28.5, H 4.8, N 13.3, S 10.15. IR
(KBr): nmax/cm-1 3435 (nOH), 2933 (nas,s CH2), 1483 (nC C), 743 (nC–S).
1H NMR (400 MHz, D2O, 25 ◦C): d 4.49 (6 H, s, N–CH2–N), 4.22
(6 H, s, N–CH2–P), 3.44–3.54 (2 H, m, O–CH2), 2.94–3.15 (1 H,
m, CH), 2.66–2.80 (1 H, m, S–CH2),◦2.54–2.61 (1 H, m, S–CH2).
1
OH). 13C{ H} NMR (100 MHz, CDCl3, 25 C): d 133.6 (m, Ph),
1
3
133.0 (m, Ph), 131.3 (m, Ph), 130.0 (m, Ph), 128.7 (m, Ph), 65.3
13C{ H} NMR (100 MHz, D2O, 25 C): d 70.6 (d, JCP 7.2 Hz,
(virt. t, 3JCPt 19.8 Hz, CH2–O), 54.6 (d, 3JCP 8.9 Hz, CH), 39.2 (d,
N–CH2–N), 63.7 (s w/Pt satellites, JCPt 32.4 Hz, O–CH2), 52.6
3
1
3JCP 10.1 Hz, S–CH2), 28.3 (m, P–CH2). 31P{ H} NMR (81 MHz,
(d, 3JCP 11.4 Hz, CH), 50.6 (m, N–CH2–P),◦37.8 (d, 3JCP 11.8 Hz,
CDCl3, 25 ◦C): d 49.0 (AB spin system, 1JPPt 2720 Hz, 2JPP 12 Hz),
48.9 (AB spin system, 1JPPt 2778 Hz, 2JPP 12 Hz). MS (ESI): m/z
737.8 (M+Na)+.
S–CH2). 31P{ H} NMR (81 MHz, D2O, 25 C): d -55.6 (AB spin
1
1
2
1
system, JPPt 2546 Hz, JPP 59 Hz), -56.3 (AB spin system, JPPt
2568 Hz, 2JPP 59 Hz). MS (ESI): m/z 632.0 (M+).
cis-(PPh3)(PTA)(2-hydroxypropane-1,3-dithiolato)platinum(II)
14. Prepared as described for 12 from 1,3-dimercaptopropane-
2-ol (27 mg, 0.22 mmol), cis-[(PPh3)(PTA)PtCl2] (137 mg,
0.20 mmol) and K2CO3 (55 mg, 0.40 mmol) in EtOH (50 mL).
The purification was carried out by washing of the crude product
with a small portion of H2O, EtOH and Et2O. Subsequently, the
product was dried under vacuum to give 14 (82 mg, 56%) as a
pale yellow powder. Found: C 43.7, H 4.3, N 5.55, S 8.5. Calcd for
C27H33N3OP2PtS2 (736.73): C 44.0, H 4.5, N 5.7, S 8.7. 1H NMR
(400 MHz, CDCl3, 25 ◦C): d 7.69 (6 H, m, Ph), 7.63 (3 H, m, Ph),
7.47 (6 H, m, Ph), 4.51 (1 H, m, CH), 4.19 (6 H, m, N–CH2–N),
3.77 (6 H, s, N–CH2–P), 2.84–2.72 (2 H, m, S–CH2), 2.57–2.45 (2
cis-Bis(dppm)(3-hydroxypropane-1,2-dithiolato)platinum(II) 11.
2,3-Dimercaptopropane-1-ol (21 mg, 0.17 mmol) and K2CO3
(41 mg, 0.30 mmol) was added to a solution of 3 (98 mg,
0.15 mmol) in CH2Cl2 (30 mL). The mixture was stirred at room
temperature for 15 h, followed by extraction with water (3 ¥
20 mL). The organic layer was dried with Na2SO4, filtered and
the solvent was removed under reduced pressure. The purification
was carried out on silica gel using a gradient (CH2Cl2–acetone
10 : 0.25), the collected fraction (RF = 0.2) was dried under vacuum
and crystallized from CH2Cl2–pentane to give 11 (70 mg; 67%) as a
yellow substance (decomp. 204 ◦C). Found: C 48.1, H 3.85, S 8.85.
Calcd for C28H28OP2PtS2 (701.68): C 47.9, H 4.0, S 9.1. IR (KBr):
◦
1
H, m, S–CH2). 13C{ H} NMR (100 MHz, CDCl3, 25 C): d 134.6
n
max/cm-1 3433 (nOH), 3051 (nCH,Ph), 2922 (nas,s CH2), 1483 (nC C),
1436 (nP–Ph), 733 (nC–S). H NMR (400 MHz, CDCl3, 25 ◦C): d
7.80 (8 H, m, Ph), 7.40 (12 H, m, Ph), 4.51 (2 H, t w/Pt satellites,
2JHP 10.5 Hz, 3JHPt 42.5 Hz, P–CH2–P), 3.61–3.73 (2 H, m, CH2–
O), 3.21 (1 H, m, CH), 2.69–2.86 (2 H, m, S–CH2), 2.34 (1 H, dd,
1
(m, Ph), 131.3 (m, Ph), 130.6 (m, Ph), 128.3 (m, Ph), 73.0 (s, CH),
3
72.9 (d, JCP 7.1 Hz, N–CH2–N), 51.3 (m, N–CH2–P), 39.1 (m,
◦
1
S–CH2). 31P{ H} NMR (81 MHz, CDCl3, 25 C): d 21.5 (AB spin
1
2
1
system, JPPt 2872 Hz, JPP 27 Hz), -63.6 (AB spin system, JPPt
2442 Hz, 2JPP 27 Hz). MS (ESI): m/z 736.0 (M+).
3
1
3JHH(1) 3.82 Hz, JHH(2) 3.88 Hz, OH). 13C{ H} NMR (100 MHz,
CDCl3, 25 ◦C): d 132.8 (m, Ph), 131.5 (m, Ph), 129.0 (m, Ph), 65.1
(s w/Pt satellites, 3JCPt 21.1 Hz, CH2–O), 54.4 (dd, 3JCP(A) 6.0 Hz,
3JCP(B) 5.9 Hz, CH), 45.1 (t, 1JCP 30.◦8 Hz, CH2), 39.1 (s, S–CH2).
UV-vis spectrophotometry
UV-vis absorption spectra were recorded on a Varian Cary 50
UV-vis spectrophotometer. Stock solutions (10-2 M) of each
compound were prepared by dissolving the complex under in-
vestigation in DMSO. Stock solutions of 12 and 13 were prepared
in H2O. UV-vis measurements were then carried out by diluting
the compounds’ stock solutions to 10-4 M in phosphate buffer
(50 mM, pH 7.4). Spectra were collected between 800–200 nm for
24 h at r.t., operating in 10 min intervals during the first hour and
in 1 h intervals afterwards.
1
31P{ H} NMR (81 MHz, CDCl3, 25 C): d -40.8 (AB spin system,
1JPPt 2348 Hz, 2JPP 70 Hz), -39.8 (AB spin system, 1JPPt 2329 Hz,
2JPP 70 Hz). MS (ESI): m/z 723.8 (M+Na)+.
cis-Bis(PTA)2(ethane-1,2-dithiolato)platinum(II) 12. Ethane-
1,2-dithiol (20 mg, 0.21 mmol) and K2CO3 (55 mg, 0.40 mmol)
were added to a solution of 4 (114 mg, 0.20 mmol) in EtOH
(50 mL). The mixture was heated to 78 ◦C for 6 h. Upon cooling
down to room temperature the product precipitated as pale yellow
powder. After filtration the product was washed with a small
portion of H2O, EtOH and Et2O. The product 12 was dried under
vacuum (115 mg, 96%). Found C 27.7, H 4.7, N 13.6, S 10.5. Calcd
NMR spectroscopy
Hydrolysis studies were carried out on compound 10. Method
A: 10 was dissolved in DMSO-d6/phosphate buffer (50 mM,
1
for C14H28N6P2PtS2 (601.57): C 27.95, H 4.7, N 14.0, S 10.7. H
NMR (400 MHz, D2O, 25 ◦C): d 4.57 (6 H, s, N–CH2–N), 4.29 (6
1
prepared in D2O) (9 : 1 ratio, 5 ¥ 10-3 M). 31P{ H} NMR spectra of
1
H, s, N–CH2–P), 2.50 (4 H, t, 3JHH 9.0 Hz, S–CH2). 13C{ H} NMR
this solutionwere recorded at 37 ◦C over 24 h (30 min intervals) and
remained identical over the course of the measurement. Method
B: 10 was dissolved in DMSO-d6/phosphate buffer (50 mM,
prepared in D2O) (4 : 1 ratio, 10-2 M) and stirred at 90 ◦C for
(100 MHz, D2O, 25 ◦C): d 70.3 (m, N–CH2–N), 50.3 (d, 3JCP 11.5
1
Hz, N–CH2–P), 36.6 (m, S–CH2). 31P{ H} NMR (81 MHz, D2O,
25 ◦C): d -55.3 (s w/Pt satellites, 1JPPt 2554 Hz). MS (DEI): m/z
601.0 (M+).
1
5 d. 31P{ H} NMR spectra of the solution were recorded over the
cis-Bis(PTA)2(3-hydroxypropane-1,2-dithiolato)platinum(II) 13.
Prepared as described for 12 from 2,3-dimercaptopropane-1-ol
(27 mg, 0.22 mmol), 4 (114 mg, 0.20 mmol) and K2CO3 (55 mg,
course of the reaction until decomposition in favour of specific
hydrolysis was observed, the last after 124 h. Method C: 10 was
dissolved in DMSO-d6/phosphate buffer (50 mM, prepared in
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D2O) (4 : 1 ratio, 10-2 M) with an overall NaCl content of 0.9%.
calculated as the ratio of the IC50 values in the resistant cell line
and the IC50 values in the sensitive one.
◦
1
31P{ H} NMR spectra of this solution were recorded at 37 C over
72 h (2 h intervals) and remained identical over the course of the
measurement.
Cytochrome c interaction studies
Samples were prepared dissolving horse heart cyt c (Sigma C7752)
(100 mM) in tetramethylammonium acetate buffer (TMAA,
25 mM, pH 7.4). Then each of the selected metal complexes
was added to the solutions (3 : 1 or 1 : 1 metal:protein ratio) and
incubated at 37 ◦C for 24 h or 96 h. After a 20-fold dilution
with HCOOH (0.1%), the ESI-MS spectrum was recorded (direct
introduction, flow rate 5 mL min-1) in an LTQ-Orbitrap high-
resolution mass spectrometer (Thermo, San Jose, CA, USA),
equipped with a conventional ESI source. The spectrometer’s
working conditions were the following: spray voltage 3.1 kV,
capillary voltage 45 V and capillary temperature 220 ◦C. The
sheath and the auxiliary gases were set, respectively, at 17
(arbitrary units) and 1 (arbitrary units). For acquisition, Xcalibur
2.0. software (Thermo) was used and monoisotopic and average
deconvoluted masses were obtained by using the integrated Xtract
tool. For spectrum acquisition a nominal resolution (at m/z 400)
of 100,000 was used.
Cell lines
For cytotoxicity studies the representative cisplatin-sensitive ovar-
ian carcinoma A2780/S human cell line and the cisplatin-resistant
A2780/R cell line (produced by repeated 1 h weekly exposure to
50 mM of the sensitive parental cell line60) were used. Cell lines were
maintained in RPMI 1640 medium supplemented with fetal bovine
serum (FBS) and antibiotics at 37 ◦C in a 5% CO2 atmosphere and
subcultured twice weekly.
Cell growth inhibition studies
The cytotoxic effects of these platinum compounds were evaluated
on the growth of A2780/S and A2780/R cell lines. The compounds
were diluted in DMSO as stock solutions. In order to establish
the potential antiproliferative effects of DMSO, its activity was
evaluated using the same experimental conditions as for the
compounds under investigation (vide infra).
Acknowledgements
Exponentially growing cells were inoculated into 96-well mi-
crotitre plates at plating densities of 2 ¥ 103 cells/well. After cell
inoculation, the microtitre plates were incubated under standard
culture conditions (37 ◦C, 5% CO2, 95% air and 100% relative
humidity) for 24 h prior to the addition of experimental drugs.
After 24 h, medium was removed and replaced with appropriate
medium-containing drug concentrations ranging from 0.3 to
100 mM for a continuous exposure of 72 h for all platinum
compounds tested. For comparison purposes the cytotoxicity
effects of cisplatin, measured at the same experimental conditions,
were also determined.
The Authors thank Heraeus Holding GmbH for a generous gift
of K2[PtCl4] and COST Action D39 for financial support (COST-
STSM-D39-3461 (CR) and COST-STSM-D39-5610 (CM)). CM
gratefully acknowledges financial support provided by the Stu-
dienstiftung des deutschen Volkes. AC gratefully acknowledges
the Swiss Confederation (SER project C09.0027 “Interactions
of metal-based anticancer agents with proteins”) and the Swiss
National Science Foundation for financial support (AMBIZIONE
project number PZ00P2_121933/1).
According to the procedure described by Skehan et al.,61 the
assay was terminated by the addition of cold tricholoroacetic
acid (TCA). Cells were fixed in situ by 10% TCA and stained
by sulforhodamine B (SRB) solution at 0.4% (w/v) in 1% acetic
acid. After staining, unbound dye was removed by washing five
times with 1% acetic acid and the plates were air dried. Bound
stain was subsequently solubilized with 10 mM tris base, and the
absorbance was read on an automated plate reader at a wavelength
of 540 nm.
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