Antiproliferative activity of PtII complexes with carboxylated phosphanes in chelated or ring-opened forms

Article abstract of DOI:10.1002/ejic.201200282

The biological activity of four cisplatin-like Pt-phosphane complexes, namely, cis-[PtCl₂(L)₂], L = PPh₃, P(Ph) ₂(p-C₆H₄-COOH), P(Ph)₂(-CH ₂CH₂-COOH) and its succinimidyl ester derivative, has been tested on monolayer cultures of three tumour cell lines (namely, A2780 human ovarian carcinoma and its cisplatin-resistant form A2780Cp8, and human colon adenocarcinoma HCT116). These complexes can undergo intramolecular rearrangements by virtue of their functionalized phosphanes, thereby existing as fully opened (O) or fully closed (C) forms. Our results show that only the opened forms exhibit moderate activity, which, although inferior to the activity exhibited by the archetype metallo-drug cisplatin, is substantially retained in the A2780Cp8 cell line, yielding very low resistance factors. The trend is also maintained on the less cisplatin-sensitive HCT116 line. When the complexes assume the bis-chelated (C) form, the antiproliferative activity is deeply reduced. Two Pt-amine congeners, containing β-alanine and 3-methoxypropylamine, with C and O structures, respectively, were synthesized and their antiproliferative propensity was evaluated for comparison purposes, and a similar scenario was observed. The biological activity of four cisplatin-like Pt-phosphane complexes has been tested. Some of the complexes can undergo intramolecular rearrangement by virtue of the functionalized phosphanes (fully opened/fully closed forms). The results show that only the opened forms exhibit moderate activity, which is substantially retained in the cisplatin-resistant cell lines. Copyright

Full text of DOI:10.1002/ejic.201200282

FULL PAPER
DOI: 10.1002/ejic.201200282
Antiproliferative Activity of PtII Complexes with Carboxylated Phosphanes in
Chelated or Ring-Opened Forms
Mauro Ravera,[a] Elisabetta Gabano,[a] Manuele Sardi,[a] Elena Monti,[b]
Marzia B. Gariboldi,[b] and Domenico Osella*[a]
Keywords: Platinum / Phosphane ligands / Antiproliferation / Intramolecular rearrangement / Biological activity
The biological activity of four cisplatin-like Pt–phosphane
complexes, namely, cis-[PtCl2(L)2], L = PPh3, P(Ph)2(p-C6H4-
COOH), P(Ph)2(-CH2CH2-COOH) and its succinimidyl ester
derivative, has been tested on monolayer cultures of three
tumour cell lines (namely, A2780 human ovarian carcinoma
and its cisplatin-resistant form A2780Cp8, and human colon
adenocarcinoma HCT116). These complexes can undergo in-
tramolecular rearrangements by virtue of their functionalized
phosphanes, thereby existing as fully opened (O) or fully
closed (C) forms. Our results show that only the opened forms
exhibit moderate activity, which, although inferior to the ac-
tivity exhibited by the archetype metallo-drug cisplatin, is
substantially retained in the A2780Cp8 cell line, yielding
very low resistance factors. The trend is also maintained on
the less cisplatin-sensitive HCT116 line. When the com-
plexes assume the bis-chelated (C) form, the antiproliferative
activity is deeply reduced. Two Pt-amine congeners, contain-
ing β-alanine and 3-methoxypropylamine, with C and O
structures, respectively, were synthesized and their antipro-
liferative propensity was evaluated for comparison purposes,
and a similar scenario was observed.
Introduction
Today, more than four decades after the serendipitous
discovery of the anticancer activity of cisplatin {cis-diam-
minedichloridoplatinum(II), cis-[PtCl2(NH3)2]},[1] this
metallo-drug has achieved a key status in the systemic treat-
ment of solid tumours, especially in polychemotherapeutic
regimens.[2] However, its severe side effects due to the non-
specific mode of its action, and the possible therapeutic fail-
ure caused by intrinsic or acquired drug resistance have re-
sulted in a huge effort to develop new platinum complexes
with improved pharmacological properties. Within this
framework, thousands of analogues have been synthesized
(with varying carrier and/or leaving groups) and tested, but
so far only about 35 compounds have entered clinical trials.
Among them, carboplatin and oxaliplatin are in clinical use
world-wide, whereas nedaplatin, lobaplatin, and heptaplatin
have gained limited regional approval in Japan, China and
South Korea, respectively (Figure 1).[3]
Figure 1. Sketch of the platinum-based anticancer drugs that have
gained marketing approval for human use.
tween healthy and cancerous tissues. In this context, the
comparatively more acidic environment found in solid tu-
mours, due to lactic acid fermentation (Warburg effect),[4]
can impact on both drug uptake and activation, thereby
affecting the treatment outcome. Recently, this principle has
been tested with bis-chelate platinum(II) complexes con-
taining amino alcoholato ligands, namely cis-[Pt(NH2-
CH(R)-CH2-O)2].[5] These complexes undergo an interest-
ing intramolecular ligand exchange reaction, whereby they
can exist as O or C bis-chelated forms, with a possible half-
opened structure intermediate. The equilibrium between the
forms is dictated by the nature of the substituents, and de-
pends on biologically relevant parameters such as pH and
chloride concentration. Assuming that these complexes tar-
get DNA, as demonstrated for cisplatin, only the O form
should be able to undergo stepwise hydrolysis and yield an
electrophilic intermediate able to form adducts with DNA,
A strategy to reduce the toxic side effects of Pt-based
compounds relies on their selective activation in tumour
cells, taking advantage of the biochemical differences be-
[a] Dipartimento di Scienze e Innovazione Tecnologica, Università
del Piemonte Orientale “A. Avogadro”,
Viale Michel 11, 15121 Alessandria, Italy
Fax: +39-013-136-0250
E-mail: domenico.osella@mfn.unipmn.it
[b] Department of Theoretical and Applied Sciences, Division of
Biomedical Research, University of Insubria,
Via A. da Giussano 10, 21052 Busto Arsizio (VA), Italy
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201200282.
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whereas the C form would be expected to exhibit signifi-
cantly lower (if any) activity. Indeed, in acidic media mim-
icking the acidic tumour milieu, where O forms prevail, the
antiproliferative activity observed for these complexes is
two- to three-fold higher than at physiological pH.[5,6] Sim-
ilar observations were also reported for a Pt-O-alkyldi-
thiocarbonato series of complexes.[7]
Replacement of the usually employed am(m)ine carrier
ligands in the square-planar PtII structure (described by
Cleare and Hoeschele as the basic requirement for platinum
derivative cytotoxicity)[8] with phosphanes, based on a wide
variety of already synthesized and/or commercially avail-
able bifunctional ligands, could enormously expand the
class of O/C complexes described above, namely cis-
[PtCl2(PPh2(CH2)n-X)2] (X = COOH, OH, OR; n = 2, 3).
The coordination of the second functional group may occur
after halide abstraction, in organic solvents, with silver
salts.[9] Indeed, the chemistry of platinum–phosphanes is
probably one of the most extensively studied in the field of
inorganic chemistry;[10] the number and diversity of phos-
phanes have increased rapidly since the 1960s, with the aim
of obtaining new and more efficient homogeneous cata-
lysts.[11]
In principle, platinum–phosphane complexes may exhibit
some disadvantages with respect to their Pt-amine counter-
parts: (i) the modest water solubility of the phosphane com-
plexes results in poor bioavailability and biodistribution, (ii)
air-and water-stable tertiary phosphanes do not possess any
P–H bonds (a N–H linkage has been shown to be important
in cisplatin-like complexes for establishing hydrogen bonds
with the DNA phosphate backbone in the adduct responsi-
ble for cytotoxic activity), and (iii) the high trans-labilizing
Figure 2. Sketch of the complexes under investigation.
power of P compared to N decreases the stability of the the BINAP compounds appeared to be able to circumvent
Pt–G (G = guanine) adducts. Early disappointing results cisplatin resistance.[17]
regarding the in vivo anticancer activity of the cisplatin an-
A similar activity on cisplatin-resistant cell lines was ob-
alogue cis-[PtCl2(PPh3)2] (O1) (Figure 2)[12] seem to have served very recently for a series of cis-[PtP2S2] pharmacoph-
discouraged extensive investigations of their potential thera- ores (where P2 refers to two monodentate or one bidentate
peutic applications, as indicated by the relative paucity of phosphane ligand and S2 is a dithiolato ligand), suggesting
literature data on these compounds in comparison with the that these compounds will undergo specific activation
platinum-am(m)ine series.[13] More recently it has been sug- within the cellular environment, with possible breaking of
gested that the intramolecular π–π interactions occurring Pt–S or Pt–P bonds.[18]
between the phenyl groups of phosphane and the pyrimid-
In this context, several classes of Pt complexes contain-
ine (or purine) rings assist in stabilizing the Pt–DNA ad- ing functionalized phosphanes with a further donor atom
duct.[14] Indeed, a number of other examples of Pt–phos- (i.e. oxygen from an ether or carboxylate, or nitrogen from
phane complexes interacting with DNA may be found in an amine functionality) in a geometrically suitable position
literature.[13b,15]
for further coordination (i.e. in a β or γ position in an ali-
Interestingly, PPh3 and PPhMe2 have been used as one phatic chain, or in an ortho position in an aromatic ring)
of the two carrier ligands to produce asymmetric platinum have been synthesized. These complexes (as well as their
complexes containing a phosphane and an amine group in Pt–amine counterparts) can exist as O and C forms, with
trans configurations, in order to obtain anticancer drug the half-opened structure as a possible intermediate. Two
candidates that are able to circumvent cisplatin resis- platinum-aminophosphane complexes, cis-[PtCl2{P(Ph)2-
tance.[16]
(CH2CH2NR2)}2], and cis-[PtCl2{P(Ph)2(CH2CH2CH2-
Enantiomeric complexes with the formula cis-[PtCl2L2] NR2)}2] (R = H, Me) exhibited significant cytotoxic effects
{L2 = R-(+)- and S-(–)-BINAP, where BINAP = 2,2Ј-bis(di- against A2780 cells.[19] All forms (half-opened, O, and C)
phenylphosphane)-1,1Ј-binaphthyl}, were tested for their were effective; however, the cytotoxic activity of the half-
antiproliferative activity against three human cancer cell opened and fully opened forms was demonstrated to rely
lines. These complexes were slightly more potent than cis- on adduct formation with DNA,[20] whereas the fully closed
[PtCl2(PPh3)2], but less cytotoxic than cisplatin. However, forms were supposed to act as lipophilic cations disrupting
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Antiproliferative Activity of PtII Complexes with Carboxylated Phosphanes
the mitochondrial membrane potential, as already reported the synthon cis-[Pt(mpa)2I2]. The treatment of this synthon
for AuI–phosphane complexes.[21] Interestingly, the confor- with AgNO3 yielded the diaqua-intermediate that, follow-
mational alterations induced in DNA by the O and half- ing reaction with KCl, yielded the final complex O6.
opened forms differ from that induced by cisplatin, thus
explaining the ability to maintain their cytotoxic activity in
Solution Behaviour of the Pt–Phosphane Complexes
cisplatin-resistant cells.[22]
In the present study, a series of Pt compounds containing
All complexes were stable in pure organic solutions for
carboxylated phosphanes or amines in the O form (O1–O4, at least 24 h, maintaining their original O or C forms, as
O6, Figure 2) have been synthesized de novo (or in some verified by 31P NMR spectra and by conductivity measure-
case re-synthesized), along with some of the corresponding ments. The 31P NMR spectra recorded with the samples in
C forms C3 and C5. The biological activity of these com- pure DMSO were equivalent to those obtained for the sam-
pounds was investigated on monolayer cultures of three dif- ples in DMF, a noncoordinating solvent. This behaviour is
ferent human tumour cells lines that were chosen because in contrast with the well-known higher trans effect exhibited
of their different sensitivity to cisplatin, namely the ovarian by phosphanes with respect to amines, and it has already
carcinoma cell line A2780, together with its cisplatin-resist- been observed for other cisplatin-like Pt–phosphane com-
ant variant A2780Cp8, and the colon carcinoma cell line plexes.[13f] For the formation of DMSO containing species
HCT116, which is known to be less responsive to cisplatin it is necessary to remove Cl– from the complexes with silver
than A2780.
salts.
When water was added to the organic solvent to obtain
a final solution with a concentration of 10% v/v DMSO, a
remarkable change was observed in the behaviour of the
complexes. The spectra of the cisplatin-like Pt–phosphane
complexes O1, O2 and O4 showed single peaks (with re-
Results and Discussion
Synthesis and Characterization of PtII Complexes
Phosphane cisplatin-like complexes O1,[23] O2,[24] and lated 195Pt satellites) at higher fields than those observed in
O3, were synthesized by reacting stoichiometric quantities the spectra recorded with the samples in pure DMSO,
of [PtCl2(η4-cod)] (cod = 1,3-cyclooctadiene)[25] and phos- which is compatible with the formation of symmetric cis-
phanes L1–L3 (Figure 2), respectively, in CH2Cl2, accord- diaqua or dihydroxido species (the Pt–H2O species are fairly
ing to published procedures.
acidic with pKa Ͻ 6).[30] The presence of labile Pt–solvent
Complex O4 was prepared in the same manner as O3, species was confirmed in the case of O2 by ESI-MS, where
but required the protection of the carboxylic group of L3 the presence of the [M – Cl – HCl]+ ion was observed. Per-
with hydroxysuccinimide, by using dicyclohexylcarbodi- forming the same experiments in DMSO/phosphate buffer
imide as the coupling reagent.[26] This protection guarantees (in the presence or absence of 5 mm Cl–) did not modify the
that the O form of the complex cannot convert into the overall behaviour of the complexes.
corresponding closed-form under the experimental condi-
tions employed.
Compound O3 showed a different behaviour from those
of O4 and O2. In fact, in this case, the presence of a second
Complex C3 was prepared by reacting O3 with silver tet- coordinating atom (carboxylate oxygen) led to fast intra-
rafluoroborate, in DMF, to remove chlorides. This reaction, molecular chelation, as indicated by the fact that the 31P
which was conducted in a noncoordinating solvent such as NMR spectrum of O3 in DMSO/D2O (pH* ≈ 7) exactly
DMF, allowed us to obtain the bis-chelate form C3 in very overlaps the spectrum obtained for C3 (see Figure S1, Sup-
high yield (Ͼ 80%).
porting Information). Moreover, the –CH2– signals of the
Compounds O6 and C5, the amine-containing analogues ligand, which are completely indistinguishable in the 1H
of O3 and C3, respectively, were added to the series of com- NMR spectrum of the “opened” complex, split into two
pounds studied for comparison purposes. A published broad signals in the spectrum of C3 as a consequence of
modification of Dhara’s method (i.e. the reaction of intramolecular chelation.
K2[PtI4] with β-alanine L5 in the presence of KOH)[27] was
In contrast, the C complex C3 was found intact in
employed to obtain the C compound C5. A considerable DMSO/D2O solution and required strong acidification to
effort was put into getting the O form of this compound; be opened (Figure 3, see also Figure S1 in the Supporting
however, neither Dhara’s method nor the direct reaction of Information).
K2[PtCl4] with L5 (for both methods, with and without
The 31P NMR spectra of C3 in noncoordinating DMF
KOH and ester protection of the carboxylic groups) yielded containing HCl in a molar ratio of 2:1 showed the presence
the O cis-[PtCl2(L5)2] form. Every attempt led to degrada- of the half-opened (ca. 20%) and the O species (ca. 80%)
tion by-products. Indeed, it has been reported that the di- in solution (Figure 4).
rect reaction between K2[PtCl4] and L5 invariably leads to
With a 4:1 acid/complex molar ratio in solution only the
a C form with a trans configuration (after several reaction O species is observed in the spectrum, as a single 31P NMR
steps, including treatment with HCl and KOH).[27] For this signal at the same δ value as that of genuine O3 in [D6]-
reason 3-methoxypropylamine (mpa), L6, was chosen as al- DMSO (see Figure S2, Supporting Information).
ternative model ligand. Dhara’s method was also used in
ESI-MS analysis confirmed the overall behaviour of
this case.[28,29] The reaction between K2[PtI4] and L6 gave C3 in solution: at an acid/Pt ratio of 2:1 the [O/C-H]– and
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FULL PAPER
[O-H]– forms (Figure 3), together with their adducts with
DMF, which are probably produced in the MS source, are
observed in the spectrum. It is necessary to increase the
acid/Pt ratio to 4:1 to obtain the O species (i.e. O3) in solu-
tion.
A similar behaviour was observed for C5: the bis-chelate
complex was strongly resistant to acid treatment, yielding
a complex mixture of derivatives (intact C5, half-opened
intermediates, etc.). Such behaviour is different from that
reported by Sadler et al. for cis-[PtCl2{P(Ph)2(CH2CH2-
NMe2)}2], for which it is not possible to achieve ring-open-
ing even at very low pH.[20]
It is interesting to note that the rates of solvolysis of the
described Pt derivatives in aqueous solutions are higher
than that of cisplatin (taking advantage of the higher trans
labilizing effect of P- with respect to N-donor atoms), but
are considerably lower in DMSO (in the classic associative
mechanism the steric repulsion between the large entering
molecule of DMSO solvent and the bulky phosphanes plays
a major role).[31]
Cytotoxicity
Figure 3. pH- and chloride-dependent intramolecular ligand ex-
change reactions of O3 and C3 that result in half-opened and O
species.
The antiproliferative potential of complexes O1–O4, O6,
C3, and C5 was assessed in human ovarian carcinoma cells
that are sensitive and resistant to cisplatin (A2780 and
A2780Cp8, respectively), as well as the colon adenocarci-
noma HCT116 cell line. For comparison purposes, the cyto-
toxicity of cisplatin was evaluated under the same experi-
mental conditions employed for the complexes. Cellular via-
bility was determined by colorimetric MTT assays [MTT =
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium brom-
ide]. The viability of cells in the presence of the tested com-
pounds was compared to that observed for control cultures
and the inhibition of growth (%) was calculated. The half
maximal inhibitory concentration (IC50) values obtained
following exposure to increasing concentrations of the dif-
ferent Pt complexes for 120 h are shown in Table 1.
The IC50 values of the cisplatin-like phosphane and
amine complexes O1, O2, O4 and O6 range between 20
and 30 μm on the A2780 cell lines. Although less active than
cisplatin, these complexes were found to retain their activity
in the A2780Cp8 cell line and exhibit very low resistance
Figure 4. 31P NMR spectra of a [D7]DMF solution of compound
C3 in the presence of HCl (molar ratio [HCl]:[Pt] = 2:1). The pres-
ence of signals associated with the O (ca. 80%) and half-opened
(ca. 20%) forms is highlighted.
Table 1. IC50 values determined after 5 d of continuous treatment of three different human tumour cell lines (A2780 ovarian carcinoma
and its cisplatin-resistant variant A2780Cp8, and colon adenocarcinoma HCT116) with the compounds under investigation (see Figure 2)
and the reference complex cisplatin.
Compound
IC50 A2780 [μm]
IC50 A2780Cp8 [μm]
rf[a]
IC50 HCT116 [μm]
cisplatin
O1
0.37Ϯ0.12
22.32Ϯ0.96
60.3
0.69[b]
1.08
1.12
1.04
1.48
1.23
1.00
4.30Ϯ0.90
20.98Ϯ3.50[b]
30.54Ϯ1.75
14.47Ϯ2.14[b]
33.16Ϯ2.31
21.83Ϯ6.10[b]
29.31Ϯ3.19
O2
O3
105.18Ϯ30.93[c]
30.87Ϯ0.70
117.98Ϯ27.12[d]
32.17Ϯ0.01
110.52Ϯ40.69[d]
30.36Ϯ2.11
O4
O6
31.02Ϯ7.04
45.82Ϯ4.84
11.35Ϯ2.6
C3
160.41Ϯ24.22[d]
196.91Ϯ11.63[d]
349.21Ϯ1.01[d,e]
216.48Ϯ51.59[d]
243.44Ϯ7.4[d,e]
C5
348.64Ϯ10.1[d,e]
[a] Resistance factor, rf = (IC50 A2780Cp8)/(IC50 A2780). [b] Data taken from ref.[17]. [c] p Ͻ 0.05 vs. all O form complexes, except O3.
[d] p Ͻ 0.05 vs. all other O forms. [e] p Ͻ 0.05 vs. C3 (meanϮSEM determined from 3–4 independent experiments, where SEM = standard
error of the mean).
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Antiproliferative Activity of PtII Complexes with Carboxylated Phosphanes
factors (rf, defined as [IC50 A2780Cp8]/[IC50 A2780]). The (those performed with and without the presence of the sam-
same trend was observed for the less Pt-sensitive HCT116 ple Pt complex) allowed for an estimation of the relative
cells.
number of guanine sites in DNA that were not modified by
The bis-chelate C complexes C3 and C5 exhibited IC50 platination (S%). Figure 5 shows the data obtained with O2
values Ͼ 100 μm, in other words they were practically de- and C3 and compares them with the data obtained for cis-
void of activity. This is due to their inability to convert into platin. The C complex C5 (not shown in Figure 5) exhibits
the active O form at pH values encountered in normal and the same trend, showing almost unmodified S% values
tumour tissues (7.4–6.0). Interestingly, complex O3 had sig- (around 100%) throughout the whole experimental period
nificantly higher IC50 values compared to the other O form (11 h).
complexes: this can probably be explained by the fast intra-
molecular chelation reaction described above that converts
compound O3 into the closed form C3.
Evaluation of the Interaction between Pt Complexes and
DNA
The interaction between DNA and electrophilic agents
causes chemical and conformational modifications of the
nucleic acid and subsequent changes in its properties that
can be evaluated by electrochemical techniques. Since G has
the highest electron density among the DNA bases, and
therefore the lowest oxidation potential, its oxidation peak
is the easiest parameter to measure. Any electrophilic com-
pound interacting with G, via covalent or intercalating
bonds, causes a decrease in electron density and decreases
the intensity of the G oxidation peak: the stronger the inter-
action, the lower the intensity of the signal.[32] Mascini et
al. have developed biosensors that immobilize DNA (the
biological sensing element) on the surface of screen-printed
electrodes, SPEs, (the signal transducer) to rapidly detect
and measure the oxidation peak of G.[33] Although this
technique represents a very rough approach to the problem,
employing experimental conditions that widely differ from
those encountered in real cells, it has successfully been em-
ployed in comparative studies of the interaction of a series
of metallo-drugs with DNA.[32,33,34] In particular, it is pos-
sible to obtain a reliable description of (i) the timescale over
Figure 5. S% vs. incubation time for 0.25 mm Pt complexes and ds-
DNA ([Pt]:[NP] = 4.5) in 24 mm PB + 5 mm NaCl (pH = 7.4)/2%
of DMSO solutions: triangles = cisplatin, circles = O2 and squares
= C3. Each time point is the mean of three independent experi-
ments (standard deviation Յ 10%). See Experimental Section for
details on the experimental conditions.
In conclusion, complexes that maintain their O form in
solution behave like cisplatin. Conversely, complexes pres-
ent in the closed form in solution reveal no apparent inter-
action with DNA.
Conclusions
The carboxylated phosphane-PtII complexes reported
which the metal complex is hydrolyzed to afford an electro- herein can exist in intramolecular equilibria between C and
philic active intermediate, (ii) the possible mass effect of O forms that are dependant on solution pH and chloride
anions acting as leaving ligands (i.e. chlorides), (iii) the in- concentration.
trinsic affinity of the generated electrophilic agent to DNA,
and (iv) the strength of the perturbation caused by such activity was significantly higher for the O forms (that pre-
metallo-drugs to the DNA chain. vail in solution at acidic pH) than for the C forms. However,
In this series of complexes, the observed antiproliferative
With this method the binding properties of O2 (a model the –COO– functionality in the C forms require very low
for the phosphane based cisplatin-like O complexes), C3 (a pH to be protonated to give the corresponding more active
phosphane based C complex), and C5 (the amine analogue O forms, therefore this intramolecular ligand rearrange-
of C3) with double stranded DNA (ds-DNA) have been ment does not operate under physiopathologic conditions,
evaluated and compared with those of the prototypical as verified by the biological inactivity of complexes C3 and
complex cisplatin that were assessed under the same experi- C5 on monolayer human tumour cell cultures. Moreover,
mental conditions. The Pt complexes were allowed to react the transformation of the original O form O3 (that was ex-
with ds-DNA in solution (24 mm phosphate buffer PB + pected to be active) into the C form C3 (that exhibits poor
5 mm NaCl, pH = 7.4, 2% v/v of DMSO) for up to 11 h. biological activity) in the buffered (pH = 7.4) culture me-
At fixed time points, the DNA (platinated and non-platin- dium during cell treatment results in an increase in the IC50
ated) was adsorbed onto the SPE surface and square wave value, which approaches that of C3. This inactivity also
voltammetry (SWV) scans were performed. The ratio be- suggests that the Pt–P bonds cannot be cleaved under these
tween the heights of the peaks corresponding to guanine experimental conditions, in contrast to a previous sugges-
oxidation in spectra obtained in the two measurements tion.[17]
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nebulizer tip operated at 3000–3500 V and 150 °C, and nitrogen
was both the drying and a nebulizing gas. The cone voltage was
30 V. Quasi-molecular ion peaks [M – H]– (negative ion mode) or
sodiated [M + Na]+ (positive ion mode) peaks were assigned on
the basis of the m/z values and of the simulated isotope distribution
patterns.
Only when ring-closure is hampered by the presence of
an imide functionality (as in O4), the hydrolysis of which
would require harsh conditions, is the O form preserved.
The moderate antiproliferative activity of the O forms
(which are similar for phosphane and amine congeners)
does not significantly decrease on passing from the cispla-
tin-sensitive ovarian to the less sensitive colon cancer cell
line, and most notably the complexes are able to bypass the
resistance mechanisms developed by the ovarian resistant
subline.
Synthesis of Dichlorido-bis[3-(diphenylphosphanyl)propanoic acid]-
platinum(II) (O3): Ligand L3 (0.938 g, 3.63 mmol) was dissolved in
CH2Cl2 (10 mL) in a three-necked flask in the dark at room temp.
and in an Ar atmosphere. A CH2Cl2 (60 mL) solution of cis-
[PtCl2(cod)] (0.712 g,1.91 mmol) was added dropwise to the former
solution. After 4 h the reaction mixture changed from yellow to
pale yellow and a white powder started to precipitate. This precipi-
tate was then isolated by centrifugation, washed with CH2Cl2 and
Experimental Section
1
diethyl ether and dried in vacuo. Yield: 0.437 g, 81.2%. H NMR
All chemicals (analytical grade) were obtained from Aldrich, except
K2PtCl4 that was obtained from Alfa Aesar, and used as received.
Ligand L4 was prepared by coupling L3 with N-hydroxysuc-
cinimide (NHS) by use of dicyclohexylcarbodiimide (DCC) and tri-
ethylamine (TEA).[26] Compounds cis-[PtCl2(cod)],[25] O1,[23] and
O2[24] were prepared according to literature procedures. Complexes
O6 and C5 were synthesized according to Dhara’s method,[28] with
some modification in the case of C5.[27]
([D7]DMF): δ = 7.60 (t, J = 9.2 Hz, 8 H, H2Ј), 7.50 (m, 4 H, H4Ј),
7.37 (m, 8 H, H3Ј), 2.60 (m, 8 H, H1, H2) ppm. 13C{1H} NMR
([D7]DMF): δ = 172.8 (C3), 133.4 (C2Ј), 131.3 (C4Ј), 129.6 (C1Ј),
128.7 (C3Ј), 54.9 (C2), 24.5 (C1) ppm. 195Pt NMR ([D7]DMF): δ =
–4397 (t, JPt-P = 3640 Hz) ppm. 31P{1H} ([D7]DMF): δ = 7.77 (sat-
ellite bands at –3.47 ppm and 19.01 ppm, JP-Pt = 3640 Hz) ppm.
ESI-MS (CH3OH, 2% DMF): m/z (%) = (100) [M – H]–; calcd. for
[C30H31Cl2O4P2Pt – H]– 782.1; found 782.3. IR (CsI): ν = 1735,
˜
1710 (COOH) cm–1.
Elemental analyses were carried out with a EA3000 CHN Elemen-
tal Analyzer (EuroVector, Milano, Italy). Platinum concentrations
were quantified by means of a Spectro Genesis ICP-MS spectrome-
ter (Spectro Analytical Instruments, Kleve, Germany) equipped
with a crossflow nebulizer. In order to quantify the platinum con-
centration the Pt 299.797 line was selected. A platinum standard
stock solution of 1000 mgL–1 was diluted in 1.0% v/v nitric acid
to prepare calibration standards. The elemental analyses and Pt
contents of the complexes were within Ϯ0.4% absolute of the theo-
retical values. The multinuclear NMR spectra were measured on a
JEOL Eclipse Plus operating at 400 MHz (1H), 161.9 MHz (31P),
100.5 MHz (13C), and 85.9 MHz (195Pt with a spectral window of
Synthesis of Bis[3-(diphenylphosphanyl)propanoate]platinum(II)
(C3): Complex O3 (0.4020 g, 0.513 mmol) was dissolved in DMF
(10 mL) in a three-necked flask in the dark at room temp. AgBF4
(0.199 g, 1.16 mmol) was dissolved in DMF (5 mL) and added to
the former solution. After 2 h the solution was centrifuged to re-
move AgCl. After addition of water to the supernatant solution,
complex C3 precipitated. This product was then dried in vacuo.
Yield: 0.437 g, 81.2%. 1H NMR ([D7]DMF): δ = 7.51 (t, J =
9.5 Hz, 8 H, H2Ј), 7.49 (m, 4 H, H4Ј), 7.36 (m, 8 H, H3Ј), 2.85 (t, J
= 11.0 Hz, 4 H, H2), 2.29 (m, 4 H, H1) ppm. 13C{1H} NMR ([D7]-
DMF): δ = 162.9 (C3), 139.9 (C2Ј), 134.5 (C4Ј), 132.2 (C1Ј), 129.2
(C3Ј), 65.5 (C2), 15.6 (C1) ppm. 195Pt NMR ([D7]DMF): δ = –4286
(t, JPt-P = 3740 Hz) ppm. 31P{1H} ([D7]DMF): δ = 5.36 (satellite
bands at –3.85 ppm and 14.62 ppm, JP-Pt = 3740 Hz) ppm. ESI-
MS (CH3OH, 2% DMF): m/z (%) = (100) [M + Na]+; calcd. for
1
1200 ppm), respectively. H and 13C NMR chemical shifts were re-
ported in parts per million referenced to solvent resonances.
31P{1H} and 195Pt NMR spectra were recorded with 85% w/w
H3PO4 (δ = 0 ppm) and a solution of K2PtCl4 in saturated aqueous
KCl as external references, respectively. The shift for K2[PtCl4] was
adjusted to –1628 ppm relative to Na2[PtCl6] (δ = 0 ppm). The
numbering scheme used to assign the NMR signals in the newly
synthesized complexes is reported in Figure 6.
[C H O P Pt + Na]+ 732.1; found 732.7. IR (CsI): ν = 1735, 1710
˜
30 28
(COOH) cm–1.
4 2
Synthesis of Dichlorido-bis[succinimid-1-yl 3-(diphenylphosphanyl)-
propanoate] platinum(II) (O4): Complex O3 (0.321 g, 0.409 mmol)
was dissolved in acetonitrile (30 mL). TEA (0.203 mL, 2 mmol),
NHS (0.103 g, 0.9 mmol) and DCC (0.186 g, 0.9 mmol) were added
to the solution whilst stirring. After 24 h the suspension was centri-
fuged to remove the precipitate (dicyclohexylurea) and evaporated
to the dryness. The powder was dissolved in dichloromethane and
precipitated with ethyl ether. This product was then dried in vacuo.
1
Yield: 0.319 g, 80%. H NMR (CD2Cl2): δ = 7.46 (m, 12 H, H3Ј,
H4Ј), 7.30 (m, 8 H, H2Ј), 2.80 (s, 8 H, H2ЈЈ, H3ЈЈ), 2.94 (m, 4 H, H2),
2.61 (m, 4 H, H1) ppm. 13C{1H} NMR (CD2Cl2): δ = 168.9 (C4ЈЈ
,
C1ЈЈ), 167.3 (C3), 133.3 (C3Ј, C4Ј), 128.9 (C2Ј), 127.6 (C1Ј), 30.9 (C2),
27.3 (C1) 25.6 (C2ЈЈ, C3ЈЈ) ppm. 195Pt NMR (CD2Cl2): δ = –4410 (t,
JP-Pt = 3640 Hz) ppm. 31P{1H} (CD2Cl2): δ = 7.41 (with satellite
bands at –3.83 ppm and 18.65 ppm, JP-Pt = 3640 Hz) ppm. ESI-
MS (CH3OH, 2% DMF): m/z (%) = (100) [M + Na]+; calcd. for
[C38H36Cl2N2O8P2Pt + H]+ 977.1; found 977.4.
Figure 6. Numbering scheme used to assign the NMR signals in
the spectra of complexes containing ligands L3, L4 and L6.
Electrospray ionization mass spectra (ESI-MS) were obtained with
a Micromass ZMD mass spectrometer. Typically, a dilute solution
of the compound in DMF/methanol (1:99) was delivered directly
to the spectrometer source at 0.01 mLmin–1 with a Hamilton
microsyringe controlled by a single-syringe infusion pump. The
Synthesis of Dichlorido-bis(3-methoxypropylamine)platinum(II)
(O6): K2PtCl4 (0.250 g, 0.6 mmol) was dissolved in water (5 mL)
whilst stirring, and KI (0.597 g, 1.16 mmol) was added at room
temp. After 1 h, 3-methoxypropylamine (0.184 mL, 1.8 mmol) was
3446
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Eur. J. Inorg. Chem. 2012, 3441–3448

Antiproliferative Activity of PtII Complexes with Carboxylated Phosphanes
added dropwise to the solution and the reaction was maintained
for 2 h. The orange-brown precipitate {cis-[diodidobis(3-methoxy-
propylamine)PtII]} was removed from solution by centrifugation,
washed with water, ethanol and dried in vacuo. The resultant pow-
der (0.2 g, 0.318 mmol) was added to a solution of AgNO3 (0.108 g,
0.637 mmol) in H2O at 40 °C. After 24 h the solution was centri-
fuged to remove AgI and evaporated to 7 mL. KCl (0.514 g,
6.678 mmol) was added to the solution and, after 24 h, the yellow
precipitate the formed was removed by centrifugation, washed with
cold water, ethanol and dried in vacuo. Yield: 0.095 g, 67%. 1H
NMR ([D7]DMF): δ = 3.44 (m, 4 H, H3), 3.27 (s, 6 H, H4), 2.88
tions containing ds-DNA and the platinum complex (sample test)
or only ds-DNA (blank test). Then, the sensor was immersed in
acetate buffer, and a SWV scan was recorded to measure the height
of the oxidation peak of the non-platinated G residues (at +0.95 V
vs. Ag pseudoreference electrode) on the DNA attached to the elec-
trode surface. Interaction of the platinum complexes with DNA
was evaluated by monitoring changes in the magnitude of the oxi-
dation peak for G. The extent of DNA modification was evaluated
by the value of the percentage decrease (S%) in the signal, which
is the ratio between the height of the G peak of the platinated
DNA adsorbed onto the SPE at different reaction times (Ssample
)
(m, 4 H, H1), 1.93 (m, 4 H, H2) ppm. 13C{1H} NMR ([D7]DMF): and the height of the G peak of the DNA in the buffer solution
δ = 70.5 (C3), 58.0 (C4), 45.1 (C2), 30.8 (C1) ppm. 195Pt NMR ([D7]- without any complex (Sblank), S% = (Ssample/Sblank)ϫ100. Analyte
DMF): δ = –2220 ppm. ESI-MS (CH3OH, 2% DMF): m/z (%) =
(100) [M + Na]+; calcd. for [C8H22Cl2N2O2Pt + Na]+ 467.1; found
467.5.
solutions of the platinum complexes were prepared in DMSO, then
diluted with phosphate buffer (10 mm, pH = 7.4), containing NaCl
(5 mm), to give solutions comprising 2% of DMSO and analyte
concentrations of 0.25 mm. After adding the ds-DNA to the solu-
tions ([Pt]/[NP] = 4.5), the resulting mixtures were gently stirred at
25 °C, and the electrochemical activity of the G residues on DNA
were measured at various times with the sensor. The SWV param-
eters were: frequency, 200 Hz; step potential, 15 mV; amplitude,
40 mV; potential range 0.2–1.4 V vs. Ag pseudoreference electrode.
All experiments were done in triplicate, and the mean values were
calculated. The relative standard deviation is not reported on the
graphs presented herein for the sake of clarity, nevertheless, it was
Յ 10% for all analyses.
Cytotoxicity Assays: Three human tumour cell lines were used for
the present study: A2780 (ovarian carcinoma) and HCT116 (colon
adenocarcinoma) were obtained from ATCC (American Type Cul-
ture Collection, Manassas, VA, USA); A2780Cp8 cells (so-called
because of their ability to grow in medium containing 8 μm cispla-
tin) were developed by chronic exposure of the parent cisplatin-
sensitive line to increasing concentrations of cisplatin, and were
originally obtained from Dr. R. Ozols (Fox Chase Cancer Center,
Philadelphia, PA, USA). A2780Cp8 cells have a number of features
that differ from the parental cell line that contribute to the resistant
phenotype, including alterations in mismatch and nucleotide ex-
cision DNA repair systems and increased glutathione levels.[35] The
three cell lines were maintained in RPMI-1640 (Sigma, Italy) sup-
plemented with 10% fetal calf serum (Celbio, Italy), 2 mm l-gluta-
mine and 1% antibiotic mixture (streptomycin/penicillin; Sigma,
Italy) under standard culture conditions (95% O2 and 5% CO2 at
37 °C in a humidified atmosphere). Cell survival following exposure
to the platinum complexes was evaluated by MTT assays, which
are based on the reduction of MTT by living cells.[36] Briefly,
1ϫ103 cells per well were plated onto 96 well sterile plates and
allowed to attach and grow for 24 h. All Pt complexes were dis-
solved in DMSO to obtain 10 mm stock solutions that were diluted
with complete medium for cell treatment. The final range of Pt
concentrations was 10–500 μm, and each concentration was tested
on 6 replicate wells; the DMSO concentration never exceeded
0.1%. After five days, MTT was added to each well (final concen-
tration: 0.4 mgmL–1) and the plates were incubated for 3 h at
37 °C. Cell viability was determined by measuring the absorbance
(λ = 570 nm) in individual wells with a Universal Microplate
Reader (Bio-Tek Instruments, Winooski, VT, USA). For each cell
line 3–4 independent experiments were performed.
Statistical Analysis: The cytotoxic effects of the Pt complexes were
quantified by calculating the drug concentration that inhibited tu-
mour cell growth by 50% (IC50), based on nonlinear regression
analysis of dose-response data performed with the Calcusyn soft-
ware (Biosoft, Cambridge, UK). Data in Table 1 are expressed as
meanϮSEM of 3–4 independent experiments. Differences in the
IC50 values for the different complexes in the different cell lines
were analyzed, with the Prism 4.03 software 4.03 (GraphPad Soft-
ware, Inc., La Jolla, CA), by two-way analysis of variance, ANOVA
with Bonferroni’s test for multiple comparisons.
Supporting Information (see footnote on the first page of this arti-
cle): NMR spectra displaying the solution behaviour of compounds
C3 and O3.
Acknowledgments
This research was carried out within the framework of the Euro-
pean Cooperation COST D39 Action (Metallo-Drug Design and
Action) and Inter-University Consortium for Research on the
Chemistry of Metal Ions in Biological Systems (CIRCMSB, Bari,
Italy).
Electrochemical Biosensor Measurements: Electrochemical mea-
surements on screen-printed electrodes (SPEs) were made with an
Autolab PGSTAT12 electrochemical analyzer (Eco Chemie,
Utrecht, The Netherlands) interfaced with a personal computer
running GPES 4.9 electrochemical software. The planar screen-
printed electrochemical cell consisted of a graphite working elec-
trode, a graphite counterelectrode, and a silver pseudoreference
electrode. SPEs were printed as previously reported; each electrode
was disposable.[37] The electrode surface was pretreated by applying
a potential of +1.6 V for 2 min and +1.8 V for 1 min while it was
immersed in acetate buffer (0.25 m, pH 4.75) containing KCl
(10 mm); this procedure is necessary to activate the electrode sur-
face and make it conducive to the immobilization of DNA. After
pre-treatment of the electrode, the ds-DNA was immobilized at a
fixed potential (+0.5 V vs. Ag pseudoreference electrode for 300 s)
onto the SPE surface by dipping the electrode strip into test solu-
[1] a) B. Rosenberg, L. Van Camp, T. Krigas, Nature 1965, 205,
698–699; b) B. Rosenberg, L. Van Camp, J. E. Trosko, V. H.
Mansour, Nature 1969, 222, 385–386.
[2] L. Kelland, Nat. Rev. Cancer 2007, 7, 573–584.
[3] N. J. Wheate, S. Walker, G. E. Craig, R. Oun, Dalton Trans.
2010, 39, 8097–8340.
[4] a) O. Warburg, Science 1956, 123, 309–314; b) G. Helmlinger,
F. Yuan, M. Dellian, R. K. Jain, Nat. Med. 1997, 3, 177–182.
[5] M. Galanski, C. Baumgartner, V. Arion, B. K. Keppler, Eur. J.
Inorg. Chem. 2003, 2619–2625.
[6] M. Galanski, C. Baumgartner, K. Meelich, V. B. Arion, M.
Fremuth, M. A. Jakupec, P. Schluga, C. G. Hartinger, N.
Graf v. Keyserlingk, B. K. Keppler, Inorg. Chim. Acta 2004,
357, 3237–3244.
[7] a) E. Amtmann, M. Zoller, H. Wesch, G. Schilling, Cancer
Chemother. Pharmacol. 2001, 47, 461–466; b) W. Friebolin, G.
Eur. J. Inorg. Chem. 2012, 3441–3448
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
3447

M. Ravera, E. Gabano, M. Sardi, E. Monti, M. B. Gariboldi, D. Osella
FULL PAPER
Schilling, M. Zoeller, E. Amtmann, J. Med. Chem. 2004, 47,
2256–2263.
[18]
C. Mügge, C. Rothenburger, A. Beyer, H. Görls, C. Gabbiani,
A. Casini, E. Michelucci, I. Landini, S. Nobili, E. Mini, L.
Messori, W. Weigand, Dalton Trans. 2011, 40, 2006–2016.
A. Habtemariam, P. J. Sadler, Chem. Commun. 1996, 1785–
1786.
A. Habtemariam, B. Watchman, B. S. Potter, R. Palmer, S. Par-
sons, A. Parkin, P. J. Sadler, J. Chem. Soc., Dalton Trans. 2001,
1306–1318.
P. J. Barnard, S. J. Berners-Price, Coord. Chem. Rev. 2007, 251,
1889–1902.
K. Neplechová, J. Kasˇparková, O. Vrána, O. Nováková, A.
Habtemariam, B. Watchman, P. J. Sadler, V. Brabec, Mol.
Pharm. 1999, 56, 20–30.
P. B. Chock, J. Halpern, F. E. Paulick, Inorg. Synth. 1973, 14,
90–92.
V. Ravindar, H. Schumann, H. Hemling, J. Blum, Inorg. Chim.
Acta 1995, 240, 145–152.
D. Drew, J. R. Doyle, Inorg. Synth. 1972, 13, 47–49.
M. Santimaria, T. Maina, U. Mazzi, M. Nicolini, Inorg. Chim.
Acta 1995, 240, 291–297.
L. F. Krylova, L. M. Kovtunova, G. V. Romanenko, Bioinorg.
Chem. Appl. 2008, Article ID 983725; DOI: 10.1155/2008/
983725.
[8] a) M. J. Cleare, J. D. Hoeschele, Platinum Met. Rev. 1973, 17,
2–13; b) M. J. Cleare, J. D. Hoeschele, Bioinorg. Chem. 1973, 2,
187–210; c) M. J. Cleare, Coord. Chem. Rev. 1974, 12, 349–405.
[9] G. K. Anderson, R. Kumar, Inorg. Chem. 1984, 23, 4064–4068.
[10] D. W. Allen in Organophosphorus Chemistry (Eds.: D. W. Allen,
J. C. Tebby), Royal Society of Chemistry, Cambridge, 2006, vol.
35, pp. 1–91.
[11] a) J. H. Downing, M. B. Smith in Comprehensive Coordination
Chemistry II (Eds.: J. A. McCleverty, T. J. Meyer), Elsevier,
Amsterdam, 2003, vol. 1, pp. 253–296; b) F. Joò, Aqueous Or-
ganometallic Catalysis, Kluwer, Dordrecht, 2001.
[19]
[20]
[21]
[22]
[12] S. J. Berners-Price, P. J. Sadler, Struct. Bonding (Berlin) 1988,
70, 27–102.
[23]
[24]
[13] a) V. Scarcia, A. Furlani, B. Longato, B. Corain, G. Pilloni,
Inorg. Chim. Acta 1988, 153, 67–70; b) G. Trovò, G. Bandoli,
U. Casellato, B. Corain, M. Nicolini, B. Longato, Inorg. Chem.
1990, 29, 4616–4621; c) A. R. Khokhar, Q. Xu, Z. H. Siddik,
J. Inorg. Biochem. 1990, 39, 117–123; d) F. Sampedro, A. M.
Molins-Pujol, J. Bonal, J. Barbé, S. Garrido, M. Pueyo, M.
Llagostera, F. Sànchez-Ferrando, Eur. J. Med. Chem. 1991, 26,
539–543; e) J. Kozelka, E. Segal, C. Bois, J. Inorg. Biochem.
1992, 47, 67–80; f) G. Trovò, G. Valle, B. Longato, J. Chem.
Soc., Dalton Trans. 1993, 669–673; g) G. Trovò, B. Longato, B.
Corain, A. Tapparo, A. Furlani, V. Scarcia, F. Baccichetti, F.
Bordin, M. Palumbo, J. Chem. Soc., Dalton Trans. 1993, 1547–
1550; h) N. Margiotta, A. Habtemariam, P. J. Sadler, Angew.
Chem. 1997, 109, 1233; Angew. Chem. Int. Ed. Engl. 1997, 36,
1185–1187; i) H. Yuge, T. K. Miyamoto, Inorg. Chim. Acta
1998, 279, 105–110; j) G. Puxty, H. Bjelosevic, T. Persson,
S. K. C. Elmroth, Dalton Trans. 2005, 3032–3038; k) H. Bjelo-
sevic, C. Spégel, A. S. Snygg, L. Gorton, S. K. C. Elmroth, T.
Persson, Tetrahedron 2006, 62, 4519–4527; l) A. Messere, E.
Fabbri, M. Borgatti, R. Gambari, B. D. Blasio, C. Pedone, A.
Romanelli, J. Inorg. Biochem. 2007, 101, 254–260; m) D. Mon-
tagner, E. Zangrando, B. Longato, Inorg. Chem. 2008, 47,
2688–2695; n) P. Bergamini, E. Marchesi, V. Bertolasi, M.
Fogagnolo, L. Scarpantonio, S. Manfredini, S. Vertuani, A.
Canella, Eur. J. Inorg. Chem. 2008, 529–537.
[25]
[26]
[27]
[28] S. C. Dhara, Indian J. Chem. 1970, 8, 193–194.
[29] a) R. C. Harrison, C. A. McAuliffe, Inorg. Chim. Acta 1980,
46, L15–L16; b) F. D. Rochon, L. M. Gruia, Inorg. Chim. Acta
2000, 306, 193–204.
[30] S. J. Berners-Price, T. G. Appleton in Platinum-Based Drugs in
Cancer Therapy (Eds.: L. R. Kelland, N. Farrell), Humana
Press Inc., Totowa, New Jersey, 2000, pp. 3–35.
[31] J. D. Atwood, Inorganic and Organometallic Reaction Mecha-
nisms, Brooks/Cole, Monterey, California, 1985, pp. 46–75.
[32] G. Bagni, D. Osella, E. Sturchio, M. Mascini, Anal. Chim. Acta
2006, 573–574, 81–89.
[33] M. Ravera, G. Bagni, M. Mascini, J. C. Dabrowiak, D. Osella,
J. Inorg. Biochem. 2007, 101, 1023–1027.
[34] a) M. Ravera, S. Baracco, C. Cassino, D. Colangelo, G. Bagni,
G. Sava, D. Osella, J. Inorg. Biochem. 2004, 98, 984–990; b) G.
Bagni, M. Ravera, D. Osella, M. Mascini, Curr. Pharm. Anal.
2005, 1, 217–224; c) M. Mascini, G. Bagni, M. L. Di Pietro,
M. Ravera, S. Baracco, D. Osella, Biometals 2006, 19, 409–418.
[35] B. C. Behrens, T. C. Hamilton, H. Masuda, K. R. Grotzinger,
J. Whang-Peng, K. G. Louie, T. Knutsen, W. M. McKoy, R. C.
Young, R. F. Ozols, Cancer Res. 1987, 47, 414–418.
[36] M. C. Alley, D. A. Scudiero, A. Monks, M. L. Hursey, M. J.
Czerwinski, D. L. Fine, B. J. Abbott, J. G. Mayo, R. H. Shoe-
maker, M. R. Boyd, Cancer Res. 1988, 48, 589–601.
[37] M. Ravera, G. Bagni, M. Mascini, D. Osella, Bioinorg. Chem.
Appl. 2007, Article ID 91078; DOI: 10.1155/2007/91078.
Received: March 20, 2012
[14] D. Montagner, E. Zangrando, B. Longato, Inorg. Chem. 2008,
47, 2688–2695.
[15] a) L. Schenetti, A. Mucci, B. Longato, J. Chem. Soc., Dalton
Trans. 1996, 299–303; b) B. Longato, L. Pasquato, A. Mucci,
L. Schenetti, Eur. J. Inorg. Chem. 2003, 128–137; c) B. Longato,
L. Pasquato, A. Mucci, L. Schenetti, E. Zangrando, Inorg.
Chem. 2003, 42, 7861–7871; d) B. Longato, D. Montagner, E.
Zangrando, Inorg. Chem. 2006, 45, 8179–8187; e) D. Mon-
tagner, B. Longato, Inorg. Chim. Acta 2008, 361, 1676–1680.
[16] F. J. Ramos-Lima, A. G. Quiroga, B. García-Serrelde, F.
Blanco, A. Carnero, C. Navarro-Ranninger, J. Med. Chem.
2007, 50, 2194–2199.
[17] S. Bombard, M. B. Gariboldi, E. Monti, E. Gabano, L. Gavig-
lio, M. Ravera, D. Osella, J. Biol. Inorg. Chem. 2010, 15, 841–
850.
Published Online: June 13, 2012
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Eur. J. Inorg. Chem. 2012, 3441–3448