Cytotoxic properties of a new organometallic platinum(II) complex and its gold(i) heterobimetallic derivatives

Article abstract of DOI:10.1039/c5dt02714d

A novel platinum(ii) organometallic complex, [Pt(pbi)(Me)(DMSO)], bearing the 2-(2′-pyridyl)-benzimidazole (pbiH) ligand, was synthesized and fully characterized. Interestingly, the reaction of this organometallic platinum(ii) complex with two distinct gold(i) phosphane compounds afforded the corresponding heterobimetallic derivatives with the pbi ligand bridging the two metal centers. The antiproliferative properties in vitro of [Pt(pbi)(Me)(DMSO)] and its gold(i) derivatives as well as those of the known coordination platinum(ii) and palladium(ii) complexes with the same ligand, of the general formula [MCl₂(pbiH)], were comparatively evaluated against A2780 cancer cells, either sensitive or resistant to cisplatin. A superior biological activity of the organometallic compound clearly emerged compared to the corresponding platinum(ii) complex; the antiproliferative effects are further enhanced upon attaching the gold(i) triphenylphosphine moiety to the organometallic Pt compound. Remarkably, these novel metal species are able to overcome nearly complete resistance to cisplatin. Significant mechanistic insight into the study compounds was gained after investigating their reactions with a few representative biomolecules by electrospray mass spectrometry and X-ray crystallography. The obtained results are comprehensively discussed.

Full text of DOI:10.1039/c5dt02714d

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Cytotoxic properties of a new organometallic
platinum(II) complex and its gold(I)
Cite this: DOI: 10.1039/c5dt02714d
heterobimetallic derivatives†
Maria Serratrice,a Laura Maiore,a Antonio Zucca,a,b Sergio Stoccoro,a,b Ida Landini,c
Enrico Mini,c Lara Massai,d Giarita Ferraro,e Antonello Merlino,e,f Luigi Messori*d and
Maria Agostina Cinellu*a,b
A novel platinum(II) organometallic complex, [Pt(pbi)(Me)(DMSO)], bearing the 2-(2’-pyridyl)-benzimid-
azole (pbiH) ligand, was synthesized and fully characterized. Interestingly, the reaction of this organometallic
platinum(II) complex with two distinct gold(I) phosphane compounds afforded the corresponding
heterobimetallic derivatives with the pbi ligand bridging the two metal centers. The antiproliferative pro-
perties in vitro of [Pt(pbi)(Me)(DMSO)] and its gold(I) derivatives as well as those of the known coordination
platinum(II) and palladium(II) complexes with the same ligand, of the general formula [MCl2(pbiH)], were
comparatively evaluated against A2780 cancer cells, either sensitive or resistant to cisplatin. A superior
biological activity of the organometallic compound clearly emerged compared to the corresponding
platinum(II) complex; the antiproliferative effects are further enhanced upon attaching the gold(I) tri-
phenylphosphine moiety to the organometallic Pt compound. Remarkably, these novel metal species are
able to overcome nearly complete resistance to cisplatin. Significant mechanistic insight into the study
compounds was gained after investigating their reactions with a few representative biomolecules by
electrospray mass spectrometry and X-ray crystallography. The obtained results are comprehensively
discussed.
Received 17th July 2015,
Accepted 6th November 2015
DOI: 10.1039/c5dt02714d
www.rsc.org/dalton
congeners of cisplatin, i.e. nedaplatin, lobaplatin and hepta-
platin, were regionally approved for clinical use in Japan,
Introduction
Cisplatin and its second-generation analogues, carboplatin China and Korea, respectively. However, the clinical effective-
and oxaliplatin, are used worldwide for the clinical manage- ness of cisplatin and – to some extent – its analogues is often
ment of a variety of cancers, including bladder, testicular, limited by severe toxic side-effects, due to the rather poor
ovarian, head and neck, and lung cancers.1 Three additional ability of Pt drugs to distinguish between malignant and
normal cells, as well as to the frequent occurrence of intrinsic
and acquired cancer resistance to platinum.2 Such severe draw-
backs have spurred researchers to look for alternatives, both in
the frame of platinum-based compounds3 and in that of other
transition metal derivatives.4 Over the last 50 years, a plethora
of platinum-based compounds were indeed prepared and
screened as candidate antitumor agents. Many variations on
the theme were proposed breaking one or more of the early
established canonical rules of anticancer Pt drugs – i.e. oxi-
dation state +2 of the Pt center, two ammine or amine donor
groups and two anionic leaving groups in a cis geometry –
leading to platinum(IV) complexes,5 trans-platinum(II) com-
plexes,6 monofunctional platinum(II) complexes7 and other
“nonclassical” structures. In addition to mononuclear com-
plexes, also a few multinuclear platinum(II) complexes were
aDepartment of Chemistry and Pharmacy, University of Sassari, via Vienna 2,
07100 Sassari, Italy. E-mail: cinellu@uniss.it
bCIRCC, Consorzio Interuniversitario Reattività Chimica e Catalisi, Università di
Bari, Via Celso Ulpiani 27, 70126 Bari, Italy
cDepartment of Health Sciences, Section of Clinical Pharmacology and Oncology,
University of Florence, viale Pieraccini 6, 50139 Firenze, Italy
dLaboratory of Metals in Medicine, Department of Chemistry, University of Florence,
via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy.
E-mail: luigi.messori@unifi.it
eDepartment of Chemical Sciences, University of Naples Federico II, via Cintia,
80126 Napoli, Italy
fCNR Institute of Biostructures and Bioimges, Via Mezzocannone 16, 80100 Napoli,
Italy
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5dt02714d
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successfully developed.8 The basic idea behind this strategy is
that totally different structures from those of cisplatin and its
analogues might provide the opportunity for identifying new
Results and discussion
Synthesis and characterization of the complexes
antitumor drugs with a radically different mechanisms of The ligand 2-(2′-pyridyl)benzimidazole (pbiH) was synthesized
action.9 Nevertheless, in spite of the variety of molecular by condensation of pyridine-2-carboxylic acid and 1,2-phenyl-
mechanisms displayed by the different classes of platinum enediamine in the presence of polyphosphoric acid at 180 °C,
complexes, almost invariantly nuclear DNA turned out to be according to a literature method.21 The palladium complex
the main target for most of them with the consequence that [PdCl2(pbiH)], 1, was prepared by a reaction of the ligand with
resistance processes are often activated after prolonged a stoichiometric amount of Na2PdCl4 in refluxing ethyl alcohol
exposure to Pt drugs. For the above mentioned reasons, in according to
a
literature method,18 while the platinum
order to broaden the spectrum of activity, many researchers complex [PtCl2(pbiH)], 2, was synthesized by a reaction of
focused attention on the potential of anticancer drugs contain- pbiH and [PtCl2(DMSO)2] in a dichloromethane solution. Both
ing non-platinum metal centers.4 Among them, ruthenium-10 compounds 1 and 2 are neutral and both feature the ligand in
and gold-based11 drugs were shown to be a promising alterna- its protonated form, as shown by FT-IR and 1H NMR data
tive or, at least, to be complementary to platinum(II) anticancer which are in accordance with those reported in the literature
drugs. Based on the latter possibility, i.e. the cooperation and/ for these compounds.16–18
or synergism between a Pt-based drug and a cytotoxic non-Pt
Under the same reaction conditions that were employed
metal species, a number of mixed Pt/M systems were develo- for the synthesis of 2, the platinum(II) precursor
ped as well, featuring in some cases improved chemico-physi- [Pt(Me)2(DMSO)2]22 afforded the neutral organometallic deriva-
cal properties and, more interestingly, targeting different tive [Pt(pbi)(Me)(DMSO)], 3, as a yellow solid. This compound
biological molecules.12,13
is soluble in most of the organic solvents and displays a high
Within this frame, we decided to develop a new heterodime- thermal stability (Mp 393 °C). At variance with compounds 1
tallic combination based on Pt(II) and Au(I) by exploiting the and 2, in complex 3 the Pt centre coordinates the pbi ligand
great versatility of 2-(2′-pyridyl)-benzimidazole (pbiH) as a through one iminic nitrogen of the pyridine ring and the
potential bridging ligand. A series of mono- and binuclear AuI deprotonated aminic nitrogen of the imidazole ring. Accord-
and AuIII complexes of this ligand, showing very promising ingly, the 1H NMR spectrum shows the absence in the high fre-
anticancer properties, were recently reported by us.14 An in- quency region of any resonance attributable to the NH proton
depth study of the electronic structures of these compounds (e.g. at δ 13.53 ppm in 2). In the absence of X-ray structural
revealed interesting structure/activity relationships.15 Further- data, complex 3 was characterised in solution by means of
more, the biological properties of various pbiH metal com- bidimensional NMR spectroscopy, namely H–H COSY and
plexes, e.g. [M(pbiH)Cl2] (with M being either Pd or Pt) and of H–H NOESY experiments. The COSY spectrum allowed assign-
1
related compounds have been the subject of various studies. ment of all H pbi resonances and the NOESY spectrum gave
These metal complexes were tested, inter alia, against a variety structural information. In particular the NOESY spectrum
of cancer cell lines including fibroblast and brain tumors,16 showed a NOE cross-peak between the Pt–CH3, at 1.16 ppm,
human RD rhabdomyosarcoma,17 and HeLa-229 and A2780 and the H3′ proton (δ 7.77 ppm), thus indicating that of the
both sensitive and resistant to cisplatin,18 showing on the two possible geometrical isomers the only formed was the one
whole a rather modest inhibition. In light of the poor perform- with the methyl ligand trans to the imino nitrogen of the pyri-
ances of the Pt(II) complex as a potential anticancer agent, and dine ring (Fig. 1), in accordance with the higher trans-influ-
moving in the framework of the so-called “rule breaking” cyto- ence of the deprotonated amino nitrogen with respect to the
toxic platinum agents,19 we developed an organometallic imino nitrogen. A NOE cross-peak is also observed between
version of the above complex, namely [Pt(pbi)(Me)(DMSO)]. the Pt–Me signal and that of the methyl protons of the co-
Indeed, organometallic compounds, i.e. metal complexes con- ordinated DMSO ligand, at 3.34 ppm.
taining at least one direct covalent metal–carbon bond were
The Pt–Me signal at δ 1.16 ppm is flanked by satellites with
recently found to be promising anticancer drug candidates.20
a
2JPt–H of 78.8 Hz; also coupled to 195Pt is the signal of the
3
Moreover, by using the latter species as a metalloligand, we methyl protons of the coordinated DMSO, with a JPt–H of 32.4
could obtain heterodinuclear platinum(II)–gold(I) complexes.
All these new metal complexes, as well as the known platinum(II)
and palladium(II) adducts, have been tested for their anti-
proliferative activities in vitro in A2780 cancer cells, both sensi-
tive and resistant to cisplatin. The results are compared with
those obtained for analogues and parent gold complexes.
In addition, the interactions of the new metal species
with some model biomolecular targets were explored mainly
through ESI MS and X-ray diffraction and valuable information
was gained on the resulting adducts and the inherent
reactivity.
Fig. 1 Complex 3: NOE contacts revealed by NOESY cross-peaks.
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Hz. The H6 signal (δ 9.50 ppm) is strongly de-shielded with Solution studies
respect to the free ligand (Δδ = 0.86 ppm) and coupled to plati-
num (3JPt–H = 21.2 Hz); also downfield shifted is H3′ (Δδ = At variance with the poor solubility of the dichlorido adducts 1
0.28 ppm), in this complex being in the proximity of the co- and 2, the organometallic derivatives 3, 4-PF6 and 5-Cl are well
ordinated nitrogen. At variance, the signals of H3, H4′/H5′ and soluble in both chlorinated and other polar organic solvents.
H6′ are shifted at higher fields.
The complexes were characterized in solution also by absorp-
Reactions of compound 3, acting as a metalloligand, with tion UV-vis spectrophotometry. Spectra were recorded, in some
an equimolar amount of [Au(Ph3P)][PF6] (generated in situ) or cases, in various solvents (see Table S1 and Fig. S1, S2 in the
of [AuCl(TPA)] (TPA
=
1,3,5-triazaphosphaadamantane) ESI†). The spectral profile of the ligand pbiH in the same
afforded the heterobinuclear compounds [(PPh3)Au(μ-pbi)Pt- solvents has been reported elsewhere.14,15 The mononuclear
(Me)(DMSO)][PF6], 4-PF6, and [(TPA)Au(μ-pbi)Pt(Me)(DMSO)]Cl, platinum(II) complexes 2 and 3 and the heterodinuclear PtII/AuI
5-Cl, respectively (Scheme 1). The H NMR spectrum of 4-PF6 complexes 4-PF6 and 5-Cl exhibit, in DMSO, intense transitions
1
in CDCl3 solution shows the signals of the methyl protons in the range 342–345 nm which are assigned as LMCT bands
at δ 1.20 (Pt–Me) and 3.42 ppm (Me2SO) flanked by satellites characteristic of the Pt(II) chromophore. Additional bands in
with Pt–H coupling constants of 73.2 and 30.4 Hz, respectively, the range 291–297 nm are attributed to a metal-perturbed
thus ruling out any transmetalation or ligand exchange reac- intraligand (IL) π–π* transition within the pbi ligand. Besides
tion with the coordinatively unsaturated {(PPh3)Au+} moiety. these, additional bands between 261 and 276 nm are displayed
The latter is coordinated to the imino imidazole nitrogen, as in the spectra of compounds 4-PF6 and 5-Cl, assigned to the
shown by the broadening of the H3 proton of the pyridine phosphane ligands at the gold(I) centres.
ring; all the pyridine protons are downfield shifted with
Prior to the biological assays, the solution chemistry of 3, 4-
respect to the parent compound 3. The 31P{1H} NMR spectrum PF6 and 5-Cl was analyzed under physiological-like conditions
further confirms the coordination of PPh3 to gold(I): indeed, using UV-vis spectrophotometry. The compounds were first
both the chemical shift of the phosphine (33.2 ppm) and the dissolved in DMSO and the resulting solutions (10−2 M)
absence of satellites ruled out the possibility of coordination diluted in the reference phosphate buffer (PB), at pH 7.4, to
1
to platinum(II).23 In the case of compound 5-Cl, the H NMR the final concentration of 3 × 10−5 M (3) or 10−4 M (4-PF6 and
spectrum in acetone-d6 shows, also in this case, the signals of 5-Cl). Solutions were monitored over 24 h at room temperature
the methyl protons at δ 1.26 (Pt–Me) and 3.54 ppm (Me2SO) (3) or 48 h at 37 °C (4-PF6 and 5-Cl), and representative spec-
2
3
flanked by satellites with JPt–H = 79.2 Hz and JPt–H = 32.8 Hz, tral profiles of 3, 4-PF6 and 5-Cl are shown respectively in
respectively. The N–CH2–N protons of the TPA ligand give rise Fig. S3, S4† and Fig. 2. Contrary to what was observed in the
to an AB spin system centred at δ 4.59 ppm (JAB = 12.2 Hz) and case of the pbi-gold(I) mononuclear compounds, where the
the N–CH2–P a singlet resonance at δ 4.44 ppm. Also in this TPA ligand was found to confer extra stability to the
complex the H6 proton is the most deshielded (δ 9.67 ppm) complex,14 in this case 5-Cl, bearing the Au(TPA) unit, turned
and coupled to 195Pt (3JPt–H = 19.2 Hz). The 31P NMR spectrum out to be the least stable. As shown in Fig. 2, compound 5-Cl is
shows one resonance at δ −52.9 ppm assigned to the phos- quite stable within two hours, while, after this period, the
phane ligand PTA.
intensity of the absorption band at 338 nm began to decrease.
Scheme 1 Chemical structures of the ligand (with the numbering scheme) and complexes.
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Fig. 2 UV-Vis absorption spectral profiles of compounds 4-PF6 and
5-Cl in 10−4 M phosphate buffered solution (10 mM, pH 7.4). Spectra
were recorded at different times over 48 hours at 37 °C.
At variance, the band at 232 nm showed a blue-shift and an
increased intensity. After 10 hours, the spectral profile
remained unchanged. Conversely, compound 4-PF6 was stable
for at least 19 hours; thereafter the intensity of the absorption
band at 339 nm gradually decreased within 30 hours and then
remained stable. In order to gain further insight into the solu-
tion behaviour of compounds 3 and 4-PF6 in a coordinative
solvent, 1H NMR spectra were recorded in CD3CN and moni-
tored over 20 h at room temperature. The proton spectrum of 3
(data reported in the Experimental section) remained
unchanged while that of the cationic complex 4-PF6 showed
some changes in the aromatic region, e.g. broadening of the
H3 and H4 signals. In the case of 4-PF6 31P NMR spectra were
also recorded over 20 h and also in this case a broadening of
the PPh3 signal was observed. Such changes may be due to a
fluxional behaviour of the AuPPh3 fragment.
Fig. 3 UV-Vis absorption spectral profiles of compounds 4-PF6 and
5-Cl in 10−4 M phosphate buffered solution (10 mM, pH 7.4) before and
after the addition of NaAsc and GSH. Spectra were recorded at different
times over 24 hours at 37 °C.
Finally, the stability of compounds 4-PF6 and 5-Cl towards
reducing agents was evaluated (Fig. 3). Sodium ascorbate
(NaAsc) and reduced glutathione (GSH), one of the most
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important intracellular reducing agents, were chosen to mimic towards both the sensitive and the resistant A2780 cell lines,
the biological milieu. Following the addition of 10 : 1 molar with IC50 values of 1.07 and 1.61 μM, respectively. Such a dra-
ratio of NaAsc, compound 4-PF6 was not stable, and the spec- matic difference in the cytotoxic properties between platinum(II)
trum shows that the intensity of the absorption band at coordination and organometallic derivatives of the same
338 nm decreased, probably due to a precipitation phenom- carrier ligand was previously observed, for example, in the case
enon. At variance, complex 5-Cl was quite stable within 1 hour, of [PtCl2(COD)] and [PtMeCl(COD)] (COD = 1,5-cycloocta-
and then the intensity of the band at 336 nm gradually diene);25 this seems to be a general rule not only for platinum(II)26
decreases within 5 hours and thereafter remained stable. A but also for palladium(II) derivatives.27 Most remarkably,
similar behaviour was observed, but more quickly for both the anchoring of the cytotoxic unit Au(Ph3P) to the Pt species
compounds 4-PF6 and 5-Cl, following the addition of GSH: in resulted in a further marked increase of the antiproliferative
both cases a blue-shift of the main absorption band was effects. In fact, compound 4-PF6 showed IC50 values of 0.19
observed which suggests the formation of different species. and 0.37 μM towards A2780/S and A2780/R, respectively, even
However, reduction of the gold(I) center was never observed lower than those observed for the parent compounds 3 and
after the addition of these reducing agents.
[Au(pbiH)(PPh3)][PF6].14 Notably, the 1 : 1 combination of
these two compounds showed similar activity to the com-
pound 4-PF6 against both cell lines, with IC50 values of 0.2 and
0.51 µM, respectively, in A2780/S and A2780/R cells (vs. 0.19
and 0.37 µM for 4-PPF6). Thus, it seems that the two metallic
species present in the heterodimetallic compounds do not
manifest a synergistic effect but rather an additive effect. On
the other hand, it is remarkable that joining the two metallic
species in the same molecular scaffold does not reduce the
biological action of each of them.
Antiproliferative activity of study compounds
The antiproliferative effects of the study compounds were eval-
uated in vitro according to the procedures described by Skehan
et al.,24 and expressed as IC50 values (drug concentrations
required to inhibit cell growth by 50%). All compounds were
tested against the cisplatin-sensitive and cisplatin-resistant
human ovarian cancer cell lines A2780/S and A2780/R,
respectively; data are reported in Table 1.
At variance with what was observed for 4-PF6, the presence
of the Au(TPA) moiety in compound 5-Cl caused a significant
decrease in the antiproliferative activity towards both cell
lines. Surprisingly, the IC50 values reported for compound 5-Cl
are even higher than those observed for the parent compounds
3 and [Au(pbi)(TPA)],14 and comparable to those of the free
ligand.
While the free ligand showed a moderate cytotoxicity with
IC50 values of 45.3 and 60.0 μM against the sensitive and the
resistant cell lines, respectively, coordination of at least one
metal centre typically resulted in greater cytotoxic effects. Of
the two mononuclear adducts [MCl2(pbiH)], the palladium
complex 1 showed a remarkable activity against both cell lines,
showing IC50 values in the low micromolar range, thus being
comparable to the gold(III) adduct [Au(pbi)Cl2],14 while the
activity of the platinum complex 2 was only moderate. Further-
Biomolecular interactions: ESI MS studies
more, compound 1 was more active against the cisplatin-resist- The interactions of the lead compounds 3 and 4-PF6 with three
ant line (RI = 0.5), while 2 showed a positive RI value (1.7). At selected model proteins – hen egg white lysozyme (lysozyme),
variance, the organometallic platinum(II) mononuclear com- bovine pancreatic ribonuclease (RNase A) and horse heart cyto-
pound 3 manifested a remarkable antiproliferative activity chrome c (cyt c) – and one oligonucleotide (ODN4: 5′-CGC
Table 1 Drug sensitivity profiles of cisplatin-sensitive and -resistant human ovarian carcinoma cell lines (A2780/S and A2780/R) towards the study
compounds. For comparison purposes the values obtained with cisplatin are reported
IC50 (μM) SD
Compounds
A2780/S
A2780/R
RI
1.3
0.8
0.5
1.7
1.5
2
1.3
1.5
—
1.2
2.53
pbiH
45.30 1.40
6.60 4.01
4.01 0.91
22.51 4.37
1.07 0.13
0.19 0.03
1.50 0.10
0.60 0.05
35.24 9.42
13.30 2.61
0.20 0.08
1.81 0.68
60.00 3.40
5.31 0.66
1.86 0.62
38.01 6.95
1.61 0.48
0.37 0.05
2.00 0.10
0.90 0.02
>50
[Au(pbi)Cl2]a
[PdCl2(pbiH)] (1)
[PtCl2(pbiH)] (2)
[Pt(pbi)(Me)(DMSO)] (3)
[(PPh3)Au(μ-pbi)Pt((Me)(DMSO)][PF6] (4-PPF6)
[Au(pbiH)(PPh3)][PF6]a
[Au(pbi)(PPh3)]a
[(TPA)Au(μ-pbi)Pt(Me)(DMSO)]Cl (5-Cl)
[Au(pbi)(TPA)]a
28.83 1.04
0.51 0.06
27.54 5.02
3 + [Au(pbiH)(PPh3)][PF6]
CDDP
15.2
Results are the mean of at least three independent experiments; SD, standard deviation; RI, resistance index. a Data from ref. 14.
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Fig. 4 Deconvoluted LTQ-Orbitrap ESI mass spectra of compound
4-PF6 in the presence of cytochrome c (A) and RNase (B) with a metallo-
drug–protein molar ratio of 3 : 1.
GCG-3′) were analysed by ESI-MS as the latter method appears
to be highly suitable, direct and informative to monitor the
binding behaviour of these biomolecules in solution. The
resulting deconvoluted ESI-MS spectra are shown respectively
in Fig. S6, S7† and Fig. 4, 5. Lysozyme does not interact with
the two tested compounds; in contrast, formation of stable
metal–protein adducts with cyt c and RNase A is clearly docu-
mented by the appearance of peaks of higher molecular mass
than those of the native proteins. Remarkably, formation of
adducts with cyt c and RNase A implies disruption of the start-
ing metal complexes and/or loss of one or more ligands.
Fig. 5 LTQ-Orbitrap ESI mass spectra of 4-PF6 in the presence of
ODN4 with a metallodrug–oligonucleotide molar ratio of 3 : 1.
In the case of compound 3, the predominant adduct with
both proteins (cyt c and RNase A) is straightforwardly assigned
to protein binding of [Pt(pbi)(Me)] fragments; however, a
second intense peak, corresponding to a different stoichio- different way; the ESI MS spectrum shows two peaks at
metry – i.e. 2 : 1 Pt/protein – was observed with cyt c (Fig. S6†). 13 878.22 Da and 14 071.55 Da, which represent the adducts
Interestingly, compound 4-PF6 interacts with RNase A in a with bare Au(I) (Fig. 4B). It seems that RNase A shows some
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kind of selectivity for gold over platinum, which is not shown fragments bound to one or two ODN4 molecules were
by cytochrome c. Nevertheless, when the ODN4-4PF6 solutions detected. In particular, only one peak was found associated
were analyzed (Fig. 5), new peaks corresponding to the metal with the [Pt(pbi)(Me)] fragment, while peaks corresponding to
Fig. 6 Details of the metal binding sites in the RNase A/3 and RNase A/4-PF6 adducts. A Pt ion is bound to His105 (panels A and C) and His119
(panels B and D) side chains in the molecule A of RNase A/3 (panels A–D), whereas a Au ion is bound to the His105 side chain in molecule A of
RNase A/4-PF6 (panels E–F). In panels A, B and E, 2Fo − Fc electron density maps are contoured at the 1.0σ level (cyan) and 3.5σ level (red). In panels
C, D and F an anomalous electron density map (green) is contoured at the 3.0σ level. Close to the Au binding site, the interpretation of the electron
density map of possible Au ligands is complicated by the presence of a number of well-structured water molecules, which are modeled as an
alternative (with occupancy 0.7) to the metal (occupancy 0.3).
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ODN4/AuPPh3 or ODN4/Au(I) adducts were identified with binding sites the electron density map is too poor to allow the
different stoichiometries. This finding suggests that the modelling of metal ligands.
complex may breakdown releasing bare Au(I) or AuPPh3
The finding that, under the same experimental conditions,
species during adduct formation. With compound 3 the spec- a Pt containing fragment from 3 is able to bind the catalyti-
trum shows only one peak related to the ODN4/[Pt(pbi)(Me)] cally important residue His119 of RNase A whereas no frag-
adduct (Fig. S7†).
ments from 4-PF6 are observed in the protein active site of the
The presence of different metal centers in the same mole- RNase A/4-PF6 adduct prompted us to perform in solution
cule, as in 4-PF6, may give the opportunity to obtain different catalytic activity studies of the enzyme in the absence and pres-
biomolecular adducts which could lead to enhanced cytotoxic ence of the two compounds, separately. The catalytic activity of
effects owing to the additive effect of coupled metals.
RNase A, free and in the presence of 3 and 4-PF6, on yeast RNA
was evaluated by the Kunitz method (see the Experimental
section for further details). The presence of 4-PF6 does not
Biomolecular interactions: X-ray crystallography
To gain deeper insight into how compounds 3 and 4-PF6 alter the catalytic activity of the protein, even at a high metal
might interact with proteins, we have grown crystals of the compound to protein ratio (Fig. S9†). On the contrary, when
adducts that they form with RNase A and solved the X-ray the protein is incubated in the presence of 3, a slight decrease
structures of both RNase A/3 and RNase A/4-PF6 complexes. in the catalytic power of the protein was observed, in particular
Monoclinic RNase A crystals with two molecules in the asym- at a high metal compound to protein ratio (Fig. S9†). These
metric unit were prepared following a procedure previously data are in full agreement with crystallographic results indicat-
described28 (see the Experimental section for further details). ing that a fraction of the molecules of RNase A/3 adduct are
To prepare the crystals of the adducts, native RNase A crystals characterized by an occluded active site cleft.
were soaked in solutions containing 2.5–5 mM of the com-
pound. The structures of the resulting RNase A/3 and RNase
A/4-PF6 crystals were refined to an R-factor of 0.195 and 0.183
Experimental section
(R-free of 0.254 and 0.234) at 1.98 and 1.68 Å resolution, respecti-
General remarks
vely. Crystallographic data and refinement statistics are listed
All the solvents were purified and dried according to standard
procedures. The ligand 2-(2-pyridyl)benzimidazole (pbiH) was
synthesized by a condensation reaction of pyridine-2-carboxylic
acid with 1,2-phenylenediamine in the presence of polyphos-
phoric acid at 180 °C, according to a literature method.21
cis-[Pt(Me)2(DMSO)2] was synthesized according to ref. 22.
Complex 1 was obtained as described in ref. 18. Elemental ana-
lyses were performed with a Perkin-Elmer elemental analyser
in Table S2 of the ESI.† The metal binding sites were deter-
mined by a comparative inspection of 2Fo − Fc (Fig. 6A, B, E),
Fo − Fc and anomalous difference Fourier (Fig. 6C, D, F)
electron density maps. The root mean square deviations of
the carbon alpha atoms of molecules A and B in the adducts
from those of the same molecules in the free RNase A struc-
tures (PDB code 1JVT) are within the range 0.34–1.33 Å
(0.34–0.55 Å excluding residues 16–22 of molecule A in
RNase A/4-PF6, which adopt a different conformation), indi-
cating that the overall structure of RNase A is preserved upon
the binding of the two metal compounds. Analysis of the
crystal structures of RNase A/3 and RNase A/4-PF6 indicates
that in both cases the binding occurs only in molecule A
(Fig. S8†). This is not surprising since it was already shown
that the accessibility of the protein active site in molecule A
of monoclinic crystals of RNase A is higher than that of
molecule B.29
1
240B. H and 31P{1H} NMR spectra were recorded with Varian
VXR 300 and Bruker Avance III 400 spectrometers at 298 K.
Chemical shifts are given in ppm relative to internal TMS for
1H and external 85% H3PO4 for 31P; J values are given in Hz.
1H–1H COSY and 1H–1H NOESY experiments were performed
by means of standard pulse sequences. UV-vis spectra were
recorded on a Varian Cary 50 or a Hitachi U-2010 UV-vis
spectrophotometer.
Spectroscopic data of 2-(2-pyridyl)benzimidazole (pbiH)
RNase A/3 has two Pt binding sites located on the surface of
the protein: close to the His105 side chain and to the His119 Selected IR bands (νmax/cm−1): 3060 ν(N–H), 1593, 1568, 1400,
side chain, i.e. in the protein active site (Fig. 6A–D). The Pt 1314, 1280, 744, 703. 1H NMR (300 MHz, CDCl3): δ 10.77
center coordinates to the NE2 atom of His105 and the ND1 (broad s, 1H; NH), 8.64 (dd, 1H, JH–H = 4.8, 1.7 Hz; H6), 8.44
atom of His119 with an unrestrained bond length of about (dt, 1H, JH–H = 7.9, 1.1 Hz; H3), 7.88 (td, 1H, JH–H = 8.4, 1.7, Hz;
2.3 Å and the occupancy values of 0.5 and 0.4, respectively. At H4), 7.86 (m, 1H, JH–H = 9.1, 1.6, 1.3 Hz; H6′), 7.49 (m, 1H,
variance, inspection of the electron density maps reveals that JH–H = 9.3, 5.6, 1.9 Hz; H3′), 7.38 (ddd, 1H, JH–H = 7.5, 4.8,
RNase A/4-PF6 presents a single metal binding site, close to 1.2 Hz; H5), δ 7.30 (m, 2H, JH–H = 9.3, 6.7, 5.4, 1.2, 0.8 Hz; H4′, H5′).
the His105 side chain (Fig. 6E and F). On the basis of mass 1H NMR (300 MHz, acetone-d6): δ 12.11 (broad s, 1H; NH), 8.67
spectrometry data collected on the RNase A/4-PF6 system, it (d, 1H, JH–H = 4.8 Hz; H6), 8.40 (d, 1H, JH–H = 7.8 Hz; H3), 7.98
could be inferred that a Au atom is bound to this site, (td, 1H, JH–H = 8.0, 1.7 Hz; H4), 7.69 (broad m, 2H, JH–H
=
although with low occupancy (occupancy value = 0.3). The Au 5.4 Hz; H3′, H6′), 7.47 (ddd, 1H, JH–H = 7.5, 4.8, 1.2 Hz; H5),
coordinates to the NE2 atom of His105 with a bond length of 7.26 (broad m, 2H; H4′, H5′). 1H NMR (300 Mz, DMSO-d6):
2.2 Å. Unfortunately in both structures, close to the metal δ 13.09 (broad s, 1H; NH), 8.72 (d, 1H, JH–H = 4.7 Hz; H6),
Dalton Trans.
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8.31 (d, 1H, JH–H = 7.9 Hz; H3), 7.99 (td, 1H, JH–H = 7.8, 1.7 Hz; H4),
[(PPh3)Au(μ-pbi)Pt(Me)(DMSO)][PF6] (4-PF6). A solution of
7.69 (m, 1H, JH–H = 9.3, 7.0, 2.1 Hz; H6′), 7.51 (m, 2H; H5 + H3′), [AuCl(PPh3)] (241.7 mg, 0.5 mmol) and AgPF6 (126.4 mg,
7.21 (m, 2H, JH–H = 7.4, 1.5 Hz; H4′, H5′).
0.5 mmol) in dichloromethane (30 mL) was stirred in the dark
[PdCl2(pbiH)] (1). An ethanol solution (10 mL) of pbiH until AgCl precipitation was completed. Then, the filtered solu-
(150 mg, 0.77 mmol) was added to a solution of K2PdCl4 tion was added to a solution of 3 (241.7 mg, 0.5 mmol) in the
(250 mg, 0.77 mmol) in the same solvent (30 mL). The result- same solvent (20 mL). The mixture was stirred in the dark for
ing mixture was stirred in the dark for 6 h at 78 °C. The solid 4 h at room temperature. Afterwards the solution was filtered
which formed was filtered off, washed with water, EtOH and and concentrated to a small volume; addition of diethyl ether
Et2O and dried under vacuum. Yield 87%. Mp > 400 °C. Anal. gave a yellow solid which was filtered off and vacuum dried to
Calc. for C12H9N3Cl2Pd: C, 38.69; H, 2.43; N, 11.28%. Found: give the analytical sample. Yield 71%. Mp: 163 °C (dec.). Anal.
C, 38.39; H, 2.50; N, 11.09%. Selected IR bands (νmax/cm−1
,
Calcd for C33H32AuF6N3OP2PtS: C, 36.47; H, 2.97; N, 3.87%.
Nujol): 1612, 1588, 1567, 1481, 1459, 1446, 341 and 324 Found: C, 36.19; H, 3.01; N, 3.68%. Selected IR bands
1
ν(Pd–Cl); H NMR (300 MHz, DMSO-d6): δ 14.86 (broad s, 1H; (νmax/cm−1, Nujol): 1609, 1019 (PPh3), 840 (PF6), 748, 712.
NH), 9.09 (d, 1H, JH–H = 5.6 Hz; H6), 8.68 (d, 1H, JH–H = 8.1 Hz; 1H NMR (400 MHz, CDCl3): δ 9.64 (d, 1H, JH–H = 5.6, Hz; H6),
H3), 8.40 (td, 1H, JH–H = 7.8, 1.6 Hz; H4), 8.32 (d, 1H, JH–H
=
8.82 (broad d, 1H; H3), 7.95 (t, 1H, JH–H = 8.0 Hz; H4), 7.89
7.0 Hz; H6′), 7.81 (ddd, 1H, JH–H = 7.4, 5.7, 1.6 Hz; H5), 7.76 (d, 1H, JH–H = 8.0 Hz; H3′), 7.75 (d, 1H, JH–H = 8.0 Hz; H6′),
(d, 1H, JH–H = 8.3 Hz; H3′), 7.48 (td, 1H, JH–H = 7.0, 1.3 Hz; H5′), 7.56–7.40 (m, 16H; H5 + H-PPh3), 7.26 (t, 2H, JH–H = 7.6 Hz,
7.40 (td, 1H, JH–H = 8.4, 1.3 Hz; H4′).
H4′/H5′), 7.19 (t, JH–H = 7.8 Hz, H5′/H4′), 3.42 (s, 6H, JPt–H
3
[PtCl2(pbiH)] (2). A dichloromethane solution (10 mL) of 30.4 Hz; Me-DMSO), 1.20 (s, 3H, 2JPt–H = 73.2 Hz; Me). 1H NMR
pbiH (97.6 mg, 0.5 mmol) was added to a solution of (400 MHz, CD3CN): δ 9.70 (d, 1H, JH–H = 5.6, Hz; H6), 8.86
cis-[PtCl2(DMSO)2] (211 mg, 0.5 mmol) in the same solvent (broad d, 1H; H3), 7.99 (t, 1H, JH–H = 8.0 Hz; H4), 7.91 (d, 1H,
(30 mL). The mixture was stirred in the dark for 3 h at room JH–H = 8.0 Hz; H3′), 7.84 (broad d, 1H, JH–H = 6.8 Hz; H6′), 7.81
temperature. The yellow solid which formed was filtered off (dt, 1H, JH–H = 8.0, 1.6 Hz; H5), 7.70–7.63 (m, 15H; H-PPh3),
and dried under vacuum. Yield 81%. Mp 239 °C (dec.). Anal. 7.23 (m, 2H, JH–H = 8.0, 6.8 Hz, H4′, H5′), 3.40 (s, 6H, 3JPt–H 34.4
2
Calc. for C12H9N3Cl2Pt: C, 31.25; H, 1.97; N, 9.11%. Found: Hz; Me-DMSO), 1.12 (s, 3H, JPt–H = 78.4 Hz; Me). 31P NMR
C, 31.19; H, 1.85; N, 9.02%. Selected IR bands (νmax/cm−1
,
(161.9 MHz, CDCl3): δ 33.2 (s, PPh3); −144.1 (hept, PF6).
1
Nujol): 1614, 1590, 1496, 1483, 344 and 328 ν(Pt–Cl); H NMR 31P NMR (161.9 MHz, CD3CN): δ 30.9 (s, PPh3); −144.6
(300 MHz, DMSO-d6): δ 13.53 (broad s, 1H; NH), 9.46 (d, 1H, (hept, PF6).
JH–H = 5.7 Hz; H6), 8.78 (d, 1H, JH–H = 8.4 Hz; H3), 8.44 (t, 1H,
[(TPA)Au(μ-pbi)Pt(Me)(DMSO)]Cl (5-Cl). An acetone solu-
JH–H = 7.8 Hz; H4), 8.31 (d, 1H, JH–H = 7.8 Hz; H6′), 7.83–7.78 tion (20 mL) of [Pt(pbi)(Me)(DMSO)] (154.6 mg, 0.32 mmol)
(m, 2H; H3′ + H5), 7.51 (pseudo t, 1H, JH–H = 7.5, 7.2 Hz; H5′), was added to
suspension of [Au(TPA)Cl] (125.2 mg,
7.42 (pseudo t, 1H, JH–H = 8.1, 7.2 Hz; H4′).
0.32 mmol) in the same solvent (30 mL). The mixture was
a
[Pt(pbi)(Me)(DMSO)] (3). A dichloromethane solution stirred for 24 h at room temperature in the dark. Afterwards
(10 mL) of pbiH (97.6 mg, 0.5 mmol) was added to a solution the solution was filtered and concentrated to a small volume;
of cis-[PtMe2(DMSO)2] (190.7 mg, 0.5 mmol) in the same addition of diethyl ether afforded a beige solid that was
solvent (30 mL). The mixture was stirred in the dark for 24 h at filtered off and vacuum dried to give the analytical sample.
room temperature. Afterwards the solution was filtered and Yield 57%. Mp: 165 °C. Anal. Calcd for C21H29AuClN6OPPtS:
concentrated to a small volume; addition of diethyl ether C, 28.92; H, 3.35; N, 9.64%. Found: C, 29.02; H, 3.14;
afforded a yellow solid that was filtered off and vacuum dried N, 9.55%. Selected IR bands (νmax/cm−1, Nujol): 1609, 1279,
to give the analytical sample. Yield 72%. Mp 393 °C. Anal. 1151, 1098, 1014, 948, 741. 1H NMR (400 MHz, acetone-d6):
Calcd for C15H18N3OPtS: C, 37.34; H, 3.55, N, 8.71%. Found: 9.67 (d, 1H, JH–H = 6.0, 3JPt–H = 19.2 Hz; H6), 8.33 (d, 1H, JH–H
=
C, 37.10; H, 3.55; N, 8.82%. Selected IR bands (νmax/cm−1
,
7.6 Hz, H3), 8.17 (t, 1H, JH–H = 8.0, 7.2 Hz, H4), 7.83 (dd, 1H,
1
Nujol): 1613, 1567, 1167, 1117, 888, 740. H NMR (400 MHz, JH–H = 7.2, 1.6 Hz, H3′), 7.64 (dd, 1H, JH–H = 6.0, 2.4 Hz, H6′),
CD2Cl2): δ 9.50 (d, 1H, JH–H = 6.0, JPt–H = 21.2 Hz; H6), 8.24 7.56 (t, 1H, JH–H = 6.0, Hz, H5), 7.08 (m, 2H, H4′, H5′), 4.68
3
(d, 1H, JH–H = 7.6 Hz; H3), 7.89 (t, 1H, JH–H = 7.6 Hz; H4), 7.77 (d, 3H, JAB = 12.4 Hz, 3 N–CHAHB–N), 4.56 (d, 3H, JAB = 12.4 Hz,
(d, 1H, JH–H = 8.0 Hz; H3′), 7.61 (d, 1H, JH–H = 7.6 Hz; H6′), 7.30 3 N–CHAHB–N), 4.45 (s, 6H, 3 N–CH2–P), 3.54 (s, 6H, JPt–H
=
3
(t, broad, 1H, JH–H = 6.8 Hz; H5), 7.05 (m, 2H, JH–H = 7.6 Hz, 32.8 Hz, CH3-DMSO), 1.26 (s, 3H, JPt–H = 79.2 Hz, CH3).
2
H4′, H5′), 3.34 (s, 6H, JPt–H = 32.4 Hz; Me-DMSO), 1.16 (s, 3H, 31P NMR (161.9 MHz, acetone-d6): δ −52.9 (s, TPA).
3
2JPt–H = 78.8 Hz; Me). 1H NMR (400 MHz, CD3CN): δ 9.60
3
UV-visible spectrophotometric studies
(d, 1H, JH–H = 5.2, JPt–H = 20.4 Hz; H6), 8.28 (d, 1H, JH–H
=
7.6 Hz; H3), 8.07 (td, 1H, JH–H = 8.0, 1.6 Hz; H4), 7.85 (dd, 1H, The electronic spectra were recorded by diluting small
JH–H = 7.2, 2.4 Hz; H3′), 7.65 (dd, 1H, JH–H = 6.0, 2.0 Hz; H6′), amounts of freshly prepared concentrated solutions of the
7.49 (ddd, 1H, JH–H = 7.6, 6.0, 1.6 Hz; H5), 7.12 (m, 2H, JH–H
=
individual complexes in DMSO in phosphate buffer at pH 7.4
7.2, 1.6 Hz, H4′, H5′), 3.41 (s, 6H, JPt–H = 32.8 Hz; Me-DMSO), or in ammonium acetate at pH 6.8 (spectral profiles shown in
3
2
1.23 (s, 3H, JPt–H = 80.4 Hz; Me). Assignments based on Fig. S5†). The concentration of each compound in the final
2D-COSY and NOESY spectra.
sample was 10−4 M or 3 × 10−5 M.
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Sample preparation and mass spectrometric analysis
ions) to the coordinates, the structures converged to an R
factor of 0.191 and 0.183 (R-free of 0.253 and 0.234), for RNase
A/3 and RNase A/4-PF6, respectively. Refinement statistics are
reported in Table S2.† Structure validation has been carried
out using Procheck.34 Coordinates and structure factors were
deposited in the Protein Data Bank under accession codes
5E5E and 5E5F for RNase A/3 and RNase A/4-PF6, respectively.
Metal complex/protein samples were prepared by mixing, in
20 mM ammonium acetate buffer (pH 6.8), protein (i.e. cyt c,
lysozyme, RNase A) at 100 µM concentration and the metal
complex in a 3 : 1 metal/protein ratio. The solution was incu-
bated at 37 °C for 24 h; after this time, water the final concen-
tration of sample was 25 µM (50% CH3OH).
Metal complex/ODN4 adducts were prepared by mixing
equivalent amounts of the ODN4 (50 μM) and complexes in
water at 37 °C. After 24 h incubation, CH3OH was added to the
reaction mixture before the analysis; the final concentration of
sample was 25 µM (50% CH3OH).
ESI-MS spectra were recorded by direct introduction at
5 μl min−1 flow rate in an Orbitrap high-resolution mass
spectrometer (Thermo, San Jose, CA, USA), equipped with a
conventional ESI source. The working conditions were as
follows: spray voltage 3.1 kV, capillary voltage 45 V and capil-
lary 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
RNase catalytic assay
The catalytic activity of RNase A and of the adducts formed in
the reaction of the protein with 3 and 4-PF6 towards yeast RNA
was determined by the Kunitz spectrophotometric assay,35 as
was previously done in other studies,36 using 0.5 mg mL−1 of
RNA in 50 mM sodium acetate/acetic acid buffer, pH 5.0, at
298 K. Spectrophotometric measurements were performed
with a Jasco spectrophotometer. The enzyme concentration
was 0.1 μg mL−1. Experiments were repeated three times and
performed after 24 h of incubation, with the protein to metal
ratios of 1 : 2, 1 : 5, and 1 : 10.
were obtained by using the integrated Xtract tool. For spec- Conclusions
trum acquisition a nominal resolution (at m/z 400) of 100 000
was used.
A novel organometallic platinum(II) complex (3) bearing the
pbi ligand has been synthesised and characterised showing
remarkable antiproliferative properties and the ability to over-
come cisplatin resistance. This complex may be straight-
forwardly transformed into the corresponding heterobimetallic
gold–platinum complexes by a reaction with gold phosphanes:
Preparation of crystals, X-ray diffraction data collection,
refinement and X-ray structural analysis of RNase A/3 and
RNase A/4-PF6
RNase A crystals were obtained using the procedure previously two distinct heterobimetallic complexes were specifically pre-
described by Vitagliano et al., 2000.28 Briefly, crystals of the pared with either AuPPh3 or AuPTA as attached metallic frag-
protein were grown by the hanging drop vapor diffusion ments to the organometallic Pt(II) complex.
method using a reservoir solution containing 22–24% (w/v)
The new heterobimetallic complexes were extensively
PEG4K and 10 mM sodium citrate at pH 5.1–5.3. The crystals characterised by NMR and UV-vis, manifesting acceptable
are monoclinic, space group C2, with two molecules in the stability and solubility profiles in aqueous environments;
asymmetric unit. The adducts were prepared by a three-day these features render them well amenable for standard in vitro
soaking in a solution that comprised the standard mother pharmacological testing.
liquor used for the crystal growth to which a 2.5–5 mM solu-
Notably, the organometallic complex 3 demonstrated far
tion of each compound was added. Crystals soaked in this enhanced cytotoxic properties when compared to the corres-
solution were mounted for the X-ray data collection without ponding platinum(II) coordination compound 2; in addition it
using a cryoprotectant, as was done in previous studies.30
is able to overcome completely cisplatin resistance. It is
X-ray diffraction data were collected at 100 K using a inferred that the CH3–Pt motif is a key feature of the biological
Saturn944 CCD detector equipped with Cu Kα X-ray radiation profile. Afterwards, the antiproliferative properties of the two
from a Rigaku Micromax 007 HF generator at the CNR Insti- heterobimetallic complexes were also assayed in comparison
tute of Biostructures and Bioimages, Napoli, Italy. Data were with the parent mononuclear platinum(II) and gold(I) com-
processed, scaled and merged using HKL2000.31 Data collec- pounds in a representative cancer cell line, i.e. A2780S/R. Inter-
tion statistics are reported in Table S2.†
estingly, the heterobimetallic Pt–Au complex bearing the
The structures were solved by molecular replacement triphenylphosphine ligand manifested highly enhanced cyto-
method, using molecule A of PDB file 1JVT,29 without water toxic effects compared to the mononuclear Pt complex 3. An
molecules, as the starting model. The refinement was carried additive rather than a synergistic effect of the two different
out with Refmac5.7,32 and model building and map inspec- metal centres is suggested by the comparable inhibitory effects
tions were performed using Coot.33 5% of the data were used displayed by a combination of the parent platinum(II) and gold(I)
for the calculation of the R-free value. After several rounds of compounds and the Pt–Au complex. On the other hand, it is
refinement using the maximum likelihood option in Refmac, remarkable that joining the two metallic species in the same
manual adjustments of side-chain atoms and addition of molecular scaffold does not reduce the biological action of
metals, water molecules and other ligands (acetate and sulfate each of them.
Dalton Trans.
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To gain some deeper insight into the underlying mecha-
nisms and interactions, we analysed the reactivity of the study
compounds with a few representative biomolecular targets, i.e.
cyt c, lysozyme, RNase A and ODN4 by ESI-MS and X-ray crys-
tallography. ESI-MS revealed that a number of adducts are
indeed formed with the model proteins cyt c and RNase A and
with the standard oligonucleotide ODN4 and their nature
could be identified. Furthermore mass spectrometry data
suggest that 4-PF6 presents a different behaviour when reacted
with proteins compared to its monometallic counterpart 3. In
addition, X-ray structures of the adducts formed in the reac-
tion between RNase A and both 3 and 4-PF6 were solved.
Crystal structures point out that Pt and Au ions are bound to
His residues on the surface and in the active site region of
RNase A. Thus a joint analysis of the ESI-MS and crystallo-
graphic data of the protein adducts allows us to state that this
4 (a) N. P. Barry and P. J. Sadler, Chem. Commun., 2013, 49,
5106–5131; (b) N. Muhammad and Z. Guo, Curr. Opin.
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7 T. C. Johnstone, J. J. Wilson and S. J. Lippard, Inorg. Chem.,
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Au–Pt heterodimetallic compound undergoes transformation 10 (a) M. J. Clarke, Coord. Chem. Rev., 2003, 236, 209–233;
and cleavage upon protein binding so that only monometallic
fragments are found to be associated with biomolecules. His
residues appear to be the major binding site for both Pt and
Au metal ions derived from the starting compound.
(b) G. Suss-Fink, Dalton Trans., 2010, 39, 1673–1688;
(c) G. Sava, A. Bergamo and P. J. Dyson, Dalton Trans., 2011,
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To the best of our knowledge, our study offers the second 11 (a) S. J. Berners-Price and A. Filipovska, Metallomics, 2011,
example in the literature of heterodimetallic Pt(II)–Au(I) com-
plexes showing remarkable cytotoxic properties,13 and the first
example featuring an organoplatinum(II) moiety. In addition,
the detailed structural characterization of an adduct formed in
3, 863–873; (b) L. Ronconi and D. Fregona, Dalton Trans.,
2009, 10670–10680; (c) C.-M. Che and R. W.-Y. Sun, Chem.
Commun., 2011, 47, 9554–9560; (d) B. Bertrand and
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the reaction between a protein and a heterodimetallic com- 12 For Pt/Ru complexes see for instance: (a) A. Herman,
pound is provided with relevant mechanistic implications.
J. M. Tanski, M. F. Tibbetts and C. M. Anderson, Inorg.
Chem., 2007, 47, 274–280; (b) C. M. Anderson, I. R. Taylor,
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Acknowledgements
13 For Pt/Au complexes see for instance: M. Wenzel,
E. Bigaeva, P. Richard, P. Le Gendre, M. Picquet, A. Casini
and E. Bodio, J. Inorg. Biochem., 2014, 141, 10–16.
Luigi Messori and Lara Massai gratefully acknowledge Benefi-
centia Stiftung (Vaduz, Liechtenstein), AIRC (IG-16089) and
COST Action CM1105 for generous financial support and Prof. 14 M. Serratrice, M. A. Cinellu, L. Maiore, M. I. Pilo, A. Zucca,
Carla Bazzicalupi for the gift of ODN4. M. A. C. gratefully
acknowledges the Regione Autonoma della Sardegna (RAS) for
C. Gabbiani, A. Guerri, I. Landini, S. Nobili, E. Mini and
L. Messori, Inorg. Chem., 2012, 51, 3161–3171.
the grant “Premialità Regionale 2011”. G. F. and A. M. thank 15 L. Maiore, M. C. Aragoni, C. Deiana, M. A. Cinellu, F. Isaia,
M. Amendola and G. Sorrentino for technical assistance in the
X-ray diffraction data collection. We thank Dr Elena Michelucci
for recording ESI-MS spectra at CISM.
V. Lippolis, A. Pintus, M. Serratrice and M. Arca, Inorg.
Chem., 2014, 53, 4068–4080.
16 (a) C. Mock, I. Puscasu, M. J. Rauterkus, G. Tallen,
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109–106; (b) G. Tallen, C. Mock, S. B. Gangopadhyay,
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