How to obtain Pt(IV) complexes suitable for conjugation to nanovectors from the oxidation of [PtCl(terpyridine)]+

Article abstract of DOI:10.1039/c7dt01706e

Oxidation of [Pt^(II)Cl(terpy)]⁺ (terpy = 2,2′:6′,2′′-terpyridine) has been attempted with several oxidizing agents and under different experimental conditions in order to obtain a Pt(iv) complex suitable for the conjugation to nanovectors to be used in drug delivery targeting for anticancer therapy. The best compromise in terms of yield and purity of the final complex was obtained by microwave-assisted reaction at 70 °C in 50% aqueous H₂O₂ for 2 h. Under these conditions the quantitative formation of [Pt^(IV)Cl(OH)₂(terpy)]⁺ was observed. The subsequent synthetic steps were, (i) functionalization of [Pt^(IV)Cl(OH)₂(terpy)]⁺ in the axial position with succinic anhydride to obtain [Pt^(IV)Cl(OH)(succinato)(terpy)]⁺ and (ii) reaction of the latter with nonporous silica nanoparticles (SNPs) with an external shell containing primary amino groups to obtain a nanovector able to transport the Pt(iv) antitumor prodrug in the form of a conjugate Pt-SNP. Finally, the antiproliferative activity and cell accumulation of [Pt^(II)Cl(terpy)]⁺, [Pt^(IV)Cl(OH)₂(terpy)]⁺, and the Pt-SNP conjugate were measured on three cancer cell lines. Despite highly effective accumulation of Pt-SNP in cells, a modest increase in activity was observed with respect to the molecular species. Further experiments showed that the Pt-SNP conjugate can release [Pt^(II)Cl(terpy)]⁺ upon reduction, but this metabolite may undergo hydrolysis, and the resulting aquo complex could coordinate once again the free amino groups of the SNPs. In the resulting tetraamine form, the Pt(ii) complex conjugated to the SNPs cannot completely perform its antiproliferative activity.

Full text of DOI:10.1039/c7dt01706e

View Article Online
View Journal
Dalton
Transactions
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: E. Gabano, E.
Perin, C. Fielden, J. A. Platts, A. Gallina, B. Rangone and M. Ravera, Dalton Trans., 2017, DOI:
10.1039/C7DT01706E.
This is an Accepted Manuscript, which has been through the
Volume 45 Number
1 7 January 2016 Pages 1–398
Royal Society of Chemistry peer review process and has been
accepted for publication.
Dalton
Transactions
Accepted Manuscripts are published online shortly after
An international journal of inorganic chemistry
www.rsc.org/dalton
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
framework
a Zr-metal–organic
rsc.li/dalton

Please do not adjust margins
Dalton Transactions
Page 1 of 11
View Article Online
DOI: 10.1039/C7DT01706E
Journal Name
ARTICLE
How to obtain Pt(IV) complexes suitable for the conjugation to
nanovectors from the oxidation of [PtCl(terpyridine)]+.
E. Gabano,a E. Perin,a C. Fielden,b J. A. Platts,b A. Gallina,a B. Rangone,a M. Ravera.a,*
Received 00th January 20xx,
Accepted 00th January 20xx
Oxidation of [Pt(II)Cl(terpy)]+ (terpy = 2,2’:6’,2”-terpyridine) has been attempted with several oxidizing agents and in
different experimental conditions in order to obtain a Pt(IV) complex suitable for the conjugation to nanovectors to be
used in drug delivery targeting for anticancer therapy. The best compromise in terms of yield and purity of the final
complex was obtained by microwave-assisted reaction at 70 °C in 50% aqueous H2O2 for 2 h. Under these conditions the
quantitative formation of [Pt(IV)Cl(OH)2(terpy)]+ was observed. Subsequent synthetic steps were i) functionalization of
[Pt(IV)Cl(OH)2(terpy)]+ in the axial position with succinic anhydride to obtain [Pt(IV)Cl(OH)(succinato)(terpy)]+, and ii) reaction
of the latter with nonporous silica nanoparticles (SNPs) with an external shell containing primary amino groups to obtain a
nanovector able to transport the Pt(IV) antitumor prodrug in form of conjugate Pt-SNP. Finally, the antiproliferative
activity and cell accumulation of [Pt(II)Cl(terpy)]+, [Pt(IV)Cl(OH)2(terpy)]+, and Pt-SNP conjugate were measured on three
cancer cell lines. Despite highly effective accumulation of Pt-SNP into cells, a modest increase in activity was observed with
respect to the molecular species. Further experiments showed that the Pt-SNP conjugate can release [Pt(II)Cl(terpy)]+ upon
reduction, but this metabolite may undergo hydrolysis, and the resulting aquo complex could coordinate once again the
free amino groups of the SNPs. In the resulting tetraamine form, the Pt(II) complex conjugated to the SNPs cannot
DOI: 10.1039/x0xx00000x
www.rsc.org/
completely
exert
its
antiproliferative
activity.
significant antitumor properties. Its cellular response is
different from cisplatin-like drugs because DNA intercalation is
associated with monodentate binding. The resulting adducts
inhibit transcription, whereas the low distortion of DNA
significantly eludes repair. Thus, phenanthriplatin proved to be
very effective on several cell lines.9-15
Introduction
The traditional structure-activity relationship (SAR) rules for
antitumor Pt(II) complexes indicated that triamine compounds
are scarcely cytotoxic.1-3 In fact most of these complexes have
very low potency because, in contrast to Pt-diamine
compounds like cisplatin (cis-[PtCl2(NH3)2]), they can only bind
Recently, triamine (or in general monofunctional) Pt(II)
complexes have been re-evaluated as anticancer drugs,16 since
carefully designed ligands allow the resulting Pt compounds to
bind with DNA in a different manner. This could result in
alternative cell death mechanisms and/or circumvention of Pt
resistance. In particular, Pt(II) complexes containing
terpyridine (terpy = 2,2’:6’,2”-terpyridine) derivatives, that can
be regarded as quite bulky triamine complexes,17 are
interesting since some can intercalate into the DNA duplex,
with a preference for 5’-CG sites, due to greater polarity
DNA in
a
monofunctional way.4 However, some cis-
[Pt(A)(Cl)(NH3)2]+ cations, where A is a derivative of pyridine,
pyrimidine, purine, or aniline, inhibit DNA synthesis, block DNA
polymerases and are active against in vivo tumor models.5, 6 In
particular, transcription is strongly inhibited by cis-
[PtCl(NH3)2(py)]+ (py
= pyridine), whereas adducts of
[PtCl(dien)]+ (dien = diethylenetriamine), which do not have
the same steric hindrance, are less effective. Although the
block of transcription by cisplatin and cis-[PtCl(NH3)2(py)]+ is
similar, repair of adducts formed by the latter is reduced so
that they persist longer than those of cisplatin,7 and the
activity of triamine complexes seems to be related to their
steric hindrance.8 Moreover, Lippard et al. showed that,
among several monofunctional platinum(II) complexes,
phenanthriplatin (cis-[PtCl(NH3)2(phenanthridine)]+) displays
19
compared to A-T base pairs.18, The Pt(II)-terpy complexes
also interact with four-stranded G-quadruplex DNA.20-24 G-
quadruplex emerged as a promising anti-cancer target because
their stabilization indirectly inhibits the telomerase enzyme.
This enzyme is expressed in more than 85% of human tumors
and compensates the loss of telomeres at each round of
replication, thus contributing to cancer survival.25, 26 In another
scenario, Pt(II)-terpy complexes were found to inhibit the
growth of different morphological forms of Trypanosoma cruzi
and Trypanosoma brucei, the parasitic protozoa causative
a.Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte
Orientale, Viale Michel 11, 15121 Alessandria (Italy)
b.School of Chemistry, Cardiff University, Park Place, Cardiff CF10 3AT (UK).
Electronic Supplementary Information (ESI) available: NMR and ESI-MS
characterization of compounds 2 and 3. See DOI: 10.1039/x0xx00000x
agents of Chagas' disease and African trypanosomiasis
(sleeping sickness), respectively.27,
28
Finally, Pt(II)-terpy
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 2 of 11
View Article Online
DOI: 10.1039/C7DT01706E
ARTICLE
Journal Name
derivatives with luminescence properties have been studied (EPR) effect in tumor tissues, by using functionalized polymers,
for bioimaging as DNA staining agents29 and biotinylated Pt(II)- nanoparticles, micelles, liposomes or nanotubes and their
ferrocenylterpyridine complexes were also tested for targeted conjugation with the drugs.37 By this effect, macromolecules
photoinduced cytotoxicity.30
strategy to reduce toxicity and improve uptake vessels are highly disordered and swollen with abundant pores
selectively gather in the tumor interstice, because the tumor
A
properties of antitumor Pt complexes is to turn them into resulting in enlarged gap junctions between endothelial cells
Pt(IV) prodrugs. Such complexes can be reduced in the hypoxic and compromised lymphatic drainage.37 Recently, amino-
tumor environment to the corresponding cytotoxic Pt(II) functionalized silica nanoparticles (NPs) were tested as
metabolite with loss of axial ligands (activation by reduction). efficient carriers for Pt(IV) prodrugs. Controlled drug release
The saturated six-coordinated octahedral geometry of Pt(IV) is from NPs exploits the combination between the unique
generally characterized by kinetic inertness that minimizes off- features of the Pt(IV) prodrugs and those of the tumor
target effects, thus improving the therapeutic properties and environment. The in vitro/in vivo activation by reduction of the
allowing oral administration.31-34 In this context, it is Pt(IV) moiety releases the active square planar Pt(II)
interesting to study Pt(IV) derivatives of Pt(II)-terpy cations, metabolite, with consequent detachment of the axial ligand/s
since only few such derivatives have been already reported, bearing the vector/s.34, 38, 39
where the Pt(II) precursor was oxidized with Cl2,35 or
[KAuCl4].36
In this paper, the synthesis, characterization and in vitro
evaluation of Pt(IV)-terpy complexes (Fig. 1) and the
A further possibility to improve the activity of a drug is the corresponding SNPs conjugate are described. Interestingly,
exploitation of drug targeting and delivery (DTD) methods. most of the traditional synthetic methods failed to give the
Active DTD uses specific molecular interactions between the desired intermediates and microwave-assisted reactions40 had
drugs and cancer cell or tissue. On the contrary, passive DTD to be performed. Such behavior was also theoretically
takes advantage of the enhanced permeability and retention investigated with DFT calculations.
Fig. 1. Sketch of the synthesized complexes 1-3 with numbering scheme for the assignment of NMR signals.
hydrogen peroxide and acetonitrile were used to generate the
reactive peroxyacetimidic acid,46, 47 only 12% oxidation of
1, to
Results and discussion
obtain the acetylamido-hydroxido Pt(IV) complex 2B, was
observed. Syntheses in ethanol with large excess H2O2 to get
the ethoxido-hydroxido derivative 2C gave no reaction with
traditional heating and only up to 15% of oxidized product
with microwave heating. When acetic acid was used as a
solvent, H2O2 was partially successful to give 2A. Traditional
heating (at 30 °C or 50 °C) with 50:1 excess 50% aqueous H2O2
from 1.5 to 48 h gave up to 50% of oxidized product in the
milder conditions, whereas high temperature for a long time
led to about 15% of the diacetato product 2D. On the contrary,
Synthesis of Pt(IV) complexes containing terpyridine.
Complex
1
was synthesized from [PtCl2(η4-cod)] (cod =
cycloocta-1,5-diene) and terpy according to literature
procedures (see experimental section for details).24, 41, 42 The
following step of oxidation of complex
1 was performed with
several oxidizing agents in order to prepare different Pt(IV)
synthons, potentially useful for further coupling reactions
(Scheme 1).36,
43-48
Surprisingly, the most common reaction
conditions failed to get the expected final products.
Iodobenzene dichloride36 and N-chlorosuccinimide45 with both
traditional and microwave-assisted heating gave no oxidation
at all. The use of peroxyacetic acid led to the formation of up
to 5% of the acetato-hydroxido Pt(IV) product 2A. Only H2O2
the microwave-assisted synthesis gave 2A by heating
for 5 min with 50:1 excess 50% aqueous H2O2. Under these
conditions, 50% of was oxidized, and this value did not
1 at 70 °C
1
significantly increase by increasing both excess H2O2 and/or
reaction time.
was efficient in oxidizing complex
1, but the results were
As 2A was the most successful derivative at this stage of
the work, its purification was attempted. The chloride counter
strongly dependent on the reaction conditions. When
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 3 of 11
Journal Name
View Article Online
DOI: 10.1039/C7DT01706E
ARTICLE
ion was exchanged with hexafluorophosphate by using an strong (i.e. 70 °C, 22:1 excess 35% aqueous H2O2, overnight)
44
anion exchange resin to more easily precipitate it. A further traditional methods resulted in oxidation of 1 43,
step on resin was used to re-introduce chloride counter ions to contrary, microwave-assisted reaction at 70 °C in 50% aqueous
gain in water solubility. However, the final product did not H2O2 for 2 h was successful in the quantitative formation of
.
On the
2
reach purity higher than 70%, and, for this reason, 2A was (see experimental section for details). For this reason,
2
was
abandoned.
the chosen intermediate for the following work.
When water was used as a solvent, neither mild (i.e. room
temperature, 5:1 excess 35% aqueous H2O2, overnight) nor
Scheme 1. Summary of the attempted oxidation reactions of 1. The heating was performed with traditional hot plates, except where otherwise indicated (MW = microwave).
DFT Calculations
The enthalpy change for reaction “Pt(II)” + H2O2
was calculated in the case of cisplatin and complex
other complexes were added in the calculations, i.e.
[PtCl2(dach)] (dach cyclohexane-1R,2R-diamine) and
→
”Pt(IV)(OH)2”
1. Two
=
[PtCl(dien)]+, for comparison with “PtCl2N2” and “PtClN3”
containing an aliphatic chelating ligands. DFT calculations (see
experimental section for details) predict similar values for
cisplatin, [PtCl2(dach)] and [PtCl(dien)]+ (
184 kJ mol-1, respectively), but a much smaller negative
(-92 kJ mol-1). These data support the lower reactivity of
ꢀ
H = -170, -194 and -
ꢀH for
1
1
with hydrogen peroxide. On the contrary, [PtCl2(dach)] is
49
usually oxidized in the same conditions of cisplatin44, and
[PtCl(dien)]+ can be easily oxidized with 5:1 excess 35%
aqueous H2O2 at room temperature overnight (50% of oxidized
product). To seek explanation for this behaviour, frontier
orbitals were examined (Fig. 2): comparison of
[PtCl(dien)]+ shows that HOMOs are similar in form and
energy, but that the LUMO of is delocalized over the terpy
1 with
1
ligand, with very little density at Pt and none in the Pt—Cl
bond, unlike in [PtCl(dien)]+ where the LUMO has large
contributions from Pt d-orbital and the Pt—Cl bond.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 4 of 11
View Article Online
DOI: 10.1039/C7DT01706E
ARTICLE
Journal Name
with DTD vectors. These anhydrides generate two carboxylic
groups, one of which reacts with an axial OH of , and the
2
other one remaining free for further conjugation. A 5:1 excess
of succinic or glutaric anhydride (70 °C, overnight in DMF) gave
no substitution reactions on 2, but caused its reduction to 1. A
decrease in temperature (40 °C) led to different results: with
glutaric anhydride, 15% of monoglutarato Pt(IV) product was
obtained, together with partial reduction of
succinic anhydride 75% of monosuccinato complex
obtained (see experimental section for details). As gave the
2, whereas with
3
was
3
best compromise in terms of yield and purity, the final step
was limited to it.
Nonporous silica nanoparticles (SNPs) with an external
shell containing primary amino groups were chosen as DTD
vector to transport the Pt(IV) antitumor prodrug. Silica
nanoparticles have attracted significant interest because of
their acceptable biocompatibility, low cost large-scale
synthesis, easy control of surface properties, and the
possibility to modify the external shell with chemical
functionalities needed to introduce the Pt(IV) moiety.
Spherical SNPs containing the 3-aminopropyl arm were
produced according to literature methods (relevant features:
amino group density = 0.283 mmol NH2 g−1 SNPs, dynamic light
scattering diameter = 125
±
3
2 nm, ζ potential = 53
, after activation of the pendant
±
2 mV).39 The
SNPs were loaded with
carboxylic group with the common EDC/NHS (EDC = N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide, NHS N-
hydroxysuccinimide) coupling strategy, through the formation
of amide bonds to form conjugate . The Pt content in was
=
Fig. 2. HOMO and LUMO energies (au) and isosurface plots (at 0.04 au level) for
cisplatin, [PtCl2(dach)], [PtCl(dien)]+ and 1.
4
4
evaluated by inductively coupled plasma optical emission
spectroscopy (ICP-OES) and the results indicated about 2% of
Functionalization of 2 in the axial position and coupling with silica
nanoparticles
coupling, i.e. 6.1
reactions from to 4 is reported in Scheme 2.
ꢁ
mol Pt g-1 SNPs. The complete series of
A subsequent synthetic step consisted of functionalization of
2
1
in the axial position/s. Succinic and glutaric anhydrides were
chosen to introduce a linker necessary for further reaction
Scheme 2. Sketch of the complete series of reactions from 1 to 4. EDC = N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, NHS = N-hydroxysuccinimide; grey balls represent
amino-functionalized nonporous silica nanoparticles (SNPs).
In vitro tests
Compounds 1, 2 and 4 were tested on human ovarian
treat tumor (sarcomatoid MM98 cell line and its cisplatin-
resistant MM98R subline). The reference drug cisplatin was
added for comparison purposes. The results are reported in
Table 1 and Fig. 3.
cancer cells A2780 and two cell lines derived from human
malignant pleural mesothelioma (MPM), a very difficult to
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 5 of 11
View Article Online
DOI: 10.1039/C7DT01706E
Journal Name
ARTICLE
Table 1. Cytotoxicity (IC50), resistance factor (RF), and accumulation ratio (AR) measured for cisplatin, compounds 1 and 2, and conjugate 4, respectively. Data are means ±
standard deviations of at least three independent replicates.
IC50 [µM]a
MM98
ARd
Compounds
RFc
6.1
A2780
MM98R
A2780
MM98
Cisplatin
0.5± 0.1b
3.2 ± 1.0b 19.4 ± 2.8b
1.4 ± 0.6e 2.71±0.18
1
2
4
2.80±0.25 5.41±2.09 4.11±0.66 0.76 0.34±0.03 0.43±0.13
7.01±0.56 10.5±0.1 8.09±0.91 0.77 0.86±0.69 0.17±0.05
0.74±0.05 4.53±0.45 5.78±0.74 1.3 36.9±14.9 57.8±4.5
a
IC50 data were obtained after 72 h of treatment of A2780 ovarian cancer, MM98 malignant pleural mesothelioma and its cisplatin resistant subline MM98R cells; b
50
c
from ref.
concentrations); e from ref. 51
; RF = IC50 (MM98R) / IC50 (MM98); AR = ratio between the intracellular and the extracellular Pt concentration (cells were treated for 4 h with 10 µM Pt
.
AR from
activity from
In the attempt to explain these results, the solution
behavior and reduction properties of and (i.e. the Pt(II)
precursor and the complex conjugated to the SNPs,
respectively) were investigated. The stability in solution of
and was studied in HEPES buffer (HEPES 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid, pH = 7.5) at
37 °C with HPLC-MS technique. easily underwent hydrolysis
with the loss of one chloride (about 50% decrease of peak area
of
in 15 min)17 and this is in agreement with the previously
reported propensity to the loss of the ligand trans to terpy in
this kind of complexes.27 On the contrary,
was hydrolyzed
with the loss of the succinato ligand in longer times (about
50% decrease of peak area of in 24 h). was also reacted
with a 10-fold excess of ascorbic acid, as the simplest model of
bio-reductant, in HEPES buffer. The results indicated that is
easily reduced (about 50% of peak area decrease in 20 min);
this data is consistent with the difficulties in the oxidation of
and in the synthesis of often accompanied by reduction.
The fast reduction of should guarantee the release of the
Pt(II) metabolite from the conjugate . However, AR data
showed high accumulation of Pt due to the endocytosis of
without the expected corresponding increase in activity with
respect to . The propensity to hydrolysis of , i.e. the Pt(II)
metabolite of , could explain such strange behavior.
Therefore, to unravel the mystery, complex was dissolved
1
to
4
).38, 39 These data would suggest an increase in
to that, surprisingly, does not occur.
1
4
1
3
1
3
=
1
1
3
3
3
3
Fig. 3. Comparison between IC50 values of cisplatin, compounds 1 and 2, and conjugate
4. See Table 1 for details.
1
3
The Pt(II) precursor
1 was less active than cisplatin except for
3
the cisplatin-resistant MM98R cells, where it maintained about
the same activity shown on sensitive MM98 cells (see
resistance factor, RF, i.e. the ratio between IC50 on MM98R
4
4
and MM98, respectively). In the oxidized form
was uniformly lowered. In the case of the conjugate
activity on A2780 cells was only 4 times better than that of
2
the activity
1
1
4
, the
4
1
1
and almost the same on MM98 and MM98R cells. Cellular
accumulation of all four compounds was evaluated by means
of the intracellular Pt content after 4 h of treatment and
expressed as accumulation ratio (AR, i.e. the ratio between the
intracellular and the extracellular Pt concentration). The
results in Table 1 show that the SNPs are highly effective for
accumulation of Pt into cells (more than 100 times increase in
and maintained for 4 h in an aqueous suspension of the SNPs.
After washing and mineralization, the Pt content on the SNPs
was measured, resulting in the conjugation of 3.9% of the total
available amino groups (11.0 ꢁ
support the hypothesis that conjugate
mol Pt g−1 SNPs). The data
may release upon
4
1
reduction in vitro. After hydrolysis, the aquo derivative of
1
(i.e. [Pt(H2O)(terpy)]2+) could “re-coordinate” the free amino
groups of the SNPs, and, in turn, it can be retained, at least in
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 6 of 11
View Article Online
DOI: 10.1039/C7DT01706E
ARTICLE
Journal Name
part, by the nanoparticles as Pt(II) complex. In the resulting ICP-OES, the Pt 299.797 nm line was selected. Instrumental settings
tetraamine form, the Pt(II) complex conjugated to the SNPs is for ICP-MS were optimized to yield maximum sensitivity for
no longer able to exert its antiproliferative activity. platinum. For quantitative determination, the most abundant
Furthermore, even though the trans orienting effect of terpy isotopes of platinum and indium (used as internal standard) were
could, at least in part, cause the release of hydrolyzed
1 from measured at m/z 195 and 115, respectively.
the amine-decorated nanoparticles, this complex can re-
coordinate the SNPs again.
Synthesis of (SP-4-2)-dichlorido(cycloocta-1,5-diene)platinum(II),
[PtCl2(cod)]
[PtCl2(cod)] was synthesized according to literature
procedures.24,
Experimental section
41
In
particular,
potassium
General procedures
tetrachloroplatinate(II) (1.00 g, 2.41 mmol) was dissolved in 16
mL of ultrapure water and the red solution was filtered to
remove possible solid residues. Then, 24 mL of glacial acetic
acid and cycloocta-1,5-diene (1.00 mL, 8.15 mmol) were added
and the mixture was magnetically stirred for 2 h at 90 °C, in
the dark. During this time, the solution turned from red to pale
yellow and a white solid precipitated. When significant
changes were no longer observed, three quarter of the solvent
was removed under reduced pressure and the white solid was
separated by centrifugation, washed with ultrapure water,
ethanol and diethyl ether and then dried in vacuo. Yield: 703
K2[PtCl4] (Johnson Matthey and Co.) and all other chemicals (Sigma
Aldrich) were used without further purification. Water was purified
(18 Mꢂ cm) using a standard Milli-Q system (Millipore, Bedford,
MA, USA).
The reactions under microwave irradiation were performed
by using a CEM Discover® SP System equipped with a focused
single mode and self-tuning cavity, an air cooling system, an
automated power control based on temperature feedback,
supplying power in 1 W increments from 0 to 300 W.
The NMR spectra were measured on a NMR-Bruker Avance III
spectrometer operating at 500 MHz (1H), 125.7 MHz (13C) and 107.2
MHz (195Pt with a spectral window of 2000 ppm), respectively, and a
JEOL Eclipse Plus spectrometer operating at 400 MHz (1H), 100.6
MHz (13C) and 86.0 MHz (195Pt, with a spectral window of 2000
ppm), respectively. 1H and 13C NMR chemical shifts were reported
in parts per million (ppm) referenced to solvent resonances. 195Pt
NMR spectra were recorded using a solution of K2PtCl4 in saturated
aqueous KCl as the external reference. The shift for K2PtCl4 was
1
mg, 1.88 mmol, 78.0%. H-NMR (500 MHz, CDCl3), δ: 2.27 and
2.71 (m, 8H, -CH2), 5.61 (m, 4H, -CH) ppm. 13C-NMR (500 MHz,
CDCl3), δ: 31.0 (-CH2), 100.2 (-CH) ppm. 195Pt-NMR (500 MHz,
CDCl3), δ: -3342 ppm.
Synthesis of (SP-4-2)-chlorido(2,2':6',2''-terpyridine)platinum(II)
chloride (1)
Complex
1
was synthesized according to literature
adjusted to -1628 ppm from Na2PtCl6 (δ = 0 ppm).
procedures.42 Briefly, [PtCl2(cod)] (154 mg, 0.412 mmol) was
suspended in 10 mL of ultrapure water. Then, terpy (101 mg,
0.433 mmol) was added to the mixture, which was
magnetically stirred for 30 min at 70 °C. The red mixture was
dried under reduced pressure and the red residue was washed
with diethyl ether and then dried in vacuo. Yield: 170 mg,
RP-HPLC and mass analysis were performed using a Waters
HPLC-MS instrument equipped with Alliance 2695 separations
module, 2487 dual lambda absorbance detector and 3100
mass detector. Chromatographic analyses were carried out
using a C18 Phenomenex Phenosphere-NEXT (5-
mm ID) column. The mobile phase employed was a 70/30 (for
and ) or 90/10 (for ) mixture of 15mM formic acid /
ꢁm, 250×4.6
1
0.341 mmol, 82.7%. H-NMR (400 MHz, MeOD-d4), δ: 7.79 (m,
1
2
3
H2, H14, 2H), 8.40 (m, H1, H15, H3, H13, H7, H9, 6H), 8.47 (m,
H8, 1H), 8.82 (m, H4, H12, 2H) ppm. 13C-NMR (100.6 MHz,
MeOD-d4), δ: 124.1 (C1, C15), 125.3 (C7, C9), 128.7 (C2, C14),
141.7 (C8), 142.4 (C3, C13), 151.4 (C4, C12), 154.9 (C5, C11),
158.8 (C6, C10) ppm. 195Pt-NMR (86.0 MHz, MeOD-d4), δ: -
2734 ppm. ESI-MS: calcd for C15H11Cl2N3Pt [M] 499 m/z, found
(positive ion mode) 464 m/z [M-Cl]+.
methanol; the flow rate was 0.5 mL/min and the UV-visible
detector was set at 210 nm. Electrospray ionization mass
spectra (ESI-MS) were obtained setting the source and
desolvation temperatures to 150 °C and 250 °C, respectively,
and using nitrogen both as a drying and as a nebulizing gas.
The cone and the capillary voltages were usually 30 V or 20 V
and 2.70 kV, respectively. Quasi-molecular ion peaks [M+H]+ or
[M-H]- peaks were assigned on the basis of the m/z values and
of the simulated isotope distribution patterns.
Synthesis of (OC-6-32)-chloridodihydroxido(2,2':6',2''-
Mineralization of Pt-loaded SNPs was performed using
a
terpyridine)platinum(IV) chloride (2)
microwave-assisted acid digestion apparatus Milestone Start D
(Milestone Srl, Sorisole, Italy) with internal temperature sensor.
Platinum was quantified by inductively coupled plasma-optical
emission spectrometry (ICP-OES, Spectro Genesis, Spectro
Analytical Instruments, Kleve, Germany) equipped with a crossflow
nebulizer, or by inductively coupled plasma-mass spectrometry
(ICP-MS, Thermo Optek X Series 2). A platinum standard stock
solution of 1000 mg L-1 was diluted in 1.0% v/v nitric acid to prepare
calibration standards. To quantify the platinum concentration by
Complex
1 (40.0 mg, 0.0801 mmol) was dissolved in 50% w/w
aqueous hydrogen peroxide (2.00 mL, 35.2 mmol) in a
microwave vessel. The vessel was capped and introduced into
the microwave cavity. The microwave unit heated the vessel
content to 70 °C over a 5-min ramp period and then
maintained this temperature for 2 h while stirring the mixture
with a magnetic bar; the power was automatically set at 50 W.
After heating, the vessel was allowed to cool to room
temperature before removing it from the cavity. The solvent
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 7 of 11
Journal Name
View Article Online
DOI: 10.1039/C7DT01706E
ARTICLE
was removed under reduced pressure and the addition of Quantification of the platinum content on conjugate 4
acetone/diethyl ether allowed one to get a yellow solid, that
After microwave acid mineralization of conjugate
4
, the Pt
was washed with diethyl ether and then dried in vacuo. Yield:
1
35.0 mg, 0.0656 mmol, 81.9%. H-NMR (500 MHz, D2O), δ:
content was determined by means of ICP-OES. In particular,
the ethanol suspension of was centrifuged to remove the
4
3
3
8.16 (t, H2, H14, 2H, J = 6.7 Hz), 8.64 (t, H3, H13, 2H, J = 7.8
Hz), 8.73 (d, H7, H9, 2H, 3J = 7.8 Hz), 8.81 (s, H1, H8, H15, 3H),
alcohol and the solid portion was dried. Then, 2-3 mg of dry
product were introduced into quartz cuvettes, together with
800 μL of 70% w/w HNO3 and 200 μL of 35% w/w aqueous
hydrogen peroxide. These devices were put into a closed
vessel in the microwave oven (200 °C, 45 min, 1200 W
microwave power). The vessel was then cooled to room
temperature and the cuvettes were pulled out: their content
was transferred into marked flasks using 1% v/v HNO3 and
sonicated for 1 h at 60 °C. After centrifugation (5 min, 10000
rpm) to remove the silica residue, the quantification of the Pt
content of the supernatant was measured by ICP-OES. The
3
9.35 (d, H4, H12, 2H, J = 5.6 Hz) ppm. 13C-NMR (125.7 MHz,
D2O), δ: 126.9 (C1, C15), 128.0 (C7, C9), 130.6 (C2, C14), 144.9
(C3, C13), 146.0 (C8), 151.6 (C4, C12), 152.2 (C5, C11), 156.7
(C6, C10) ppm. 195Pt-NMR (107.2 MHz, D2O), δ: 387 ppm. ESI-
MS: calcd for C15H13Cl2N3O2Pt [M] 533 m/z, found (positive ion
mode) 498 m/z [M-Cl]+.
Synthesis of (OC-6-43)-(4-
carboxypropanoato)chloridohydroxidoplatinum(IV) (3)
results indicated a Pt loading of 6.23
ꢁ
mol Pt g−1 NPs,
A solution of succinic anhydride (46.9 mg, 0.469 mmol) in 1 mL
corresponding to the conjugation of 2.2% of the total available
amino groups (0.283 mmol NH2 g-1 SNPs).
of DMF was added to a suspension of complex
2 (50.0 mg,
0.0938 mmol) in 2 mL of DMF and the reaction was carried out
at 40 °C overnight. The solvent was then removed under
reduced pressure and an orange solid was precipitated by the Stability and reactions in solution
addition of acetone/diethyl ether. It was purified with an
The solution behavior of complexes
1
and 3 (0.5 mM) was
anionic exchange resin (Amberlite® IRA-400(OH) Supelco, in
studied in 2 mM HEPES buffer (HEPES = 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid, pH = 7.5) at 37 °C. The
which the hydroxido groups were replaced by chloride anions)
in order to remove free succinate and to introduce chlorides as
counter ions. The resulting solution was dried under reduced
pressure and the residue was washed with methanol and dried
reduction of complex
3 (0.5 mM) with ascorbic acid (ascorbic
acid:Pt molar ratio = 10:1) was studied in 2 mM HEPES at 37 °C.
All these reactions were followed by monitoring the decrease
of the area of the chromatographic peaks of the Pt complexes
in HPLC-UV-MS and the increase of possible new peaks. The
mobile phase was a mixture of 15 mM aqueous HCOOH and
1
in vacuo. Yield: 44.8 mg, 0.0707 mmol, 75.4%. H-NMR (500
MHz, D2O), δ: 2.62 (s, H17, H18, 4H), 8.13 (m, H2, H14, 2H),
3
3
8.60 (t, H3, H13, 2H, J = 7.9 Hz), 8.69 (d, H7, H9, 2H, J = 7.9
Hz), 8.75 (s, H1, H8, H15, 3H), 9.34 (d, H4, H12, 2H, 3J = 5.7 Hz)
ppm. 13C-NMR (125.7 MHz, D2O), δ: 29.9 (C17, C18), 126.5 (C1,
C15), 127.6 (C7, C9), 130.3 (C2, C14), 144.9 (C3, C13), 146.0
(C8), 151.7 (C4, C12), 152.8 (C5, C11), 157.3 (C6, C10), 177.2
CH3OH in ratios of 70/30 for 1 and 90/10 for 3.
DFT calculations
(C19), 180.3 (C16) ppm. 195Pt-NMR (107.2 MHz, D2O), δ: 524 All DFT calculations were carried out using Gaussian0952 at the
54
ppm. ESI-MS: calcd for C19H17Cl2N3O5Pt [M] 633 m/z, found B3LYP53, level, using a basis set consisting of LANL2DZ on
(positive ion mode) 598 m/z [M-Cl]+.
Pt55, 56 and 6-31G(d)57 on all remaining atoms. All species were
fully optimized without any symmetry constraint, and
confirmed as minima by harmonic frequency calculations. The
same calculations were used to estimate thermal corrections
to electronic energy to obtain enthalpy changes, and to extract
HOMO/LUMO plots.
Loading of complex 3 on silica nanoparticles (conjugate 4)
A
mixture of
3
(12.1 mg, 0.0191 mmol) and N-(3-
hydrochloride
dimethylaminopropyl)-N′-ethylcarbodiimide
(EDC, 4.4 mg, 0.0229 mmol) in 1 ml of ultrapure water was
magnetically stirred for 5 min at room temperature, then N-
hydroxysuccinimide (NHS, 2.6 mg, 0.0229 mmol) was added
Cytotoxicity
and the reaction was performed for 30 min. In the meanwhile, Compounds 1, 2 and 4 were tested on three cancer cell lines:
an ethanol suspension of amino-functionalized silica the human ovarian carcinoma cell line A2780, purchased from
nanoparticles SNPs (13.67 mg mL−1), prepared according to ECACC (European Collection of Cell Cultures, UK), a primary
previously published procedures,39 was centrifuged to remove malignant pleural mesothelioma (MPM) cancer cell line,
the solvent, washed with ultrapure water, centrifuged and derived of pleural effusion of previously untreated patients
resuspended in water. The resulting suspension of SNPs was suffering from MPM, namely MM98 (sarcomatoid), and on a
added to the previously prepared solution of activated
the mixture was magnetically stirred for 2 h at room exposure to sub-lethal concentrations of cisplatin for several
temperature to get conjugate
. The product was collected by months, called MM98R.58 The last two cell lines were obtained
centrifugation (10 min, 10000 rpm), washed several times with from the Hospital of Alessandria (Pathology Unit).
3 and cisplatin-resistant cell line derived from wild type MM98 by
4
water and, then, resuspended in ethanol.
A2780 were grown in RPMI1640, whereas DMEM (Sigma-
Aldrich) was used for MPM cells. All media were supplemented
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 8 of 11
View Article Online
DOI: 10.1039/C7DT01706E
ARTICLE
Journal Name
with L-glutamine (2 mM), penicillin 100 IU mL-1, streptomycin quantitative determination, the most abundant isotopes of
(100 mg L-1) and 10% fetal bovine serum (FBS). Cell culture and platinum and indium (used as internal standard) were
the treatments were carried on at 37 °C in a 5% CO2 humidified measured at m/z 195 and 115, respectively.
chamber. Cell were challenged with the compounds under
study for 72 h continuous treatment (CT).
The level of Pt found in cells after the treatment was
normalized to the cell number and the cellular volume,
Cisplatin and complex
aqueous solution brought to pH 3 with HCl (final stock every sample, to obtain the intracellular Pt concentration. The
concentration 1 mM), complex was dissolved in water (final ratio between the intracellular and the known extracellular
stock concentration 5 mM), whereas conjugate
was concentration of offered Pt is defined accumulation ratio, AR.60
1 were dissolved in 0.9% w/v NaCl calculated from the actual mean cell diameter measured for
2
4
suspended in absolute ethanol (final Pt stock concentration
0.125 mM). The mother solutions were diluted in complete
medium to required concentration range. In the case of co-
solvent the total absolute ethanol concentration never
exceeded 0.2% (this concentration was found to be non-toxic
to the cell tested).
Conclusions
In summary, this research has shown that, differently from
Pt(II) complexes containing aliphatic di- or triamines, the
oxidation of Pt(terpy) compounds is much more difficult and
leads to Pt(IV) complexes somewhat problematic to handle. In
To assess the growth inhibition of the compounds under
investigation, a cell viability test, i.e. the resazurin reduction
assay was used.59 Briefly, cells were seeded in black sterile
tissue-culture treated 96-well plates. At the end of the
its intermediate form 2, the Pt(IV)-terpy was reasonably easy
to use in the functionalization of silica nanoparticles.
Surprisingly, the cytotoxic features observed in similar Pt(IV)-
SNPs conjugates were not repeated, despite increased cellular
accumulation by endocytosis. The possible explanation of such
unsatisfactory result may be traced back to the reactivity of
treatment, viability was assayed by 10 ꢁ
g mL-1 resazurin (Acros
Chemicals, France) in fresh medium for 1 h at 37 °C, and the
amount of the reduced product, resorufin, was measured by
means of fluorescence (excitation 535 nm, emission 595 nm)
with a Tecan Infinite F200Pro plate reader (Tecan Austria). In
each experiment, cells were challenged with the drug
candidates at different concentrations and the final data were
calculated from at least three replicates of the same
experiment performed in triplicate. The fluorescence of 8 wells
containing medium without cells were used as blank.
Fluorescence data were normalized to 100% cell viability for
non-treated cells. Half inhibiting concentration (IC50), defined
as the concentration of the drug reducing cell viability by 50%,
was obtained from the dose-response sigmoid using Origin Pro
(version 8, Microcal Software, Inc., Northampton, MA, USA).
the Pt(II) metabolite, obtained in vitro after reduction from
with the free amino groups of the SNPs. However, the
biological features of the conjugate and that of the Pt(II)-
terpy precursor remain interesting in the case of cisplatin-
resistant cell lines.
4,
4
1
Acknowledgements
This research was supported by the Compagnia di San Paolo
within the research project “Biplanes”. We were indebted to
Inter-University Consortium for Research on the Chemistry of
Metals in Biological Systems (CIRCMSB, Bari) for providing
opportunities of stimulating discussion.
Cellular uptake
Cells under investigation (2×106) were seeded in 25 cm2 flasks
and treated for 4 h with 10 µM of the complexes under
Notes and references
investigation (1
ꢁM in the case of 4) in complete medium.
1.
2.
3.
4.
5.
6.
M. J. Cleare and J. D. Hoeschele, Platinum Metals Review,
1973, 17, 2-13.
M. J. Cleare and J. D. Hoeschele, Bioinorganic Chemistry,
1973, 2, 187-210.
M. J. Cleare, Coordination Chemistry Reviews, 1974, 12,
349-405.
N. Farrell, Y. Qu, L. Feng and B. Van Houten, Biochemistry,
1990, 29, 9522-9531.
L. S. Hollis, A. R. Amundsen and E. W. Stern, Journal of
Medicinal Chemistry, 1989, 32, 128-136.
L. S. Hollis, W. I. Sundquist, J. N. Burstyn, W. J.
Heigerbernays, S. F. Bellon, K. J. Ahmed, A. R. Amundsen,
E. W. Stern and S. J. Lippard, Cancer Research, 1991, 51,
1866-1875.
K. S. Lovejoy, R. C. Todd, S. Z. Zhang, M. S. McCormick, J.
A. D'Aquino, J. T. Reardon, A. Sancar, K. M. Giacomini and
S. J. Lippard, Proceedings of the National Academy of
Sciences of the United States of America, 2008, 105, 8902-
8907.
After 4 h, cells were washed two times with PBS (1X), detached
from the flask using 0.05% Trypsin 1X + 2% EDTA (HyClone,
Thermo Fisher) and harvested in fresh complete medium. An
automatic cell counting device (Countess®, Life Technologies),
was used to measure the cell number and the mean diameter
from every cell count. About 5×106 cells were transferred into
a borosilicate glass tube and centrifugated at 1100 rpm for 5
min at room temperature. The supernatant was carefully
removed by aspiration; in order to limit the cellular loss, about
200 µL of the supernatant were left. Cellular pellets were
stored at -80 °C until mineralization. After defrosting, 70% w/w
HNO3 was added and left 1 h at 60 °C in an ultrasonic bath.
Before the measurement, the HNO3 was diluted to a final 3%
v/v concentration. Platinum determination was performed by
inductively coupled plasma-mass spectrometry (ICP-MS,
Thermo Optek X Series 2). Instrumental settings were
optimized to yield maximum sensitivity for platinum. For
7.
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 9 of 11
Journal Name
8. C. Francisco, S. Gama, F. Mendes, F. Marques, I. Cordeiro 32.
View Article Online
DOI: 10.1039/C7DT01706E
ARTICLE
E. Wexselblatt and D. Gibson, Journal of Inorganic
Biochemistry, 2012, 117, 220-229.
Dos Santos, A. Paulo, I. Santos, J. Coimbra, E. Gabano and
M. Ravera, Dalton Transactions, 2011, 40, 5781-5792.
G. Y. Park, J. J. Wilson, Y. Song and S. J. Lippard, 34.
Proceedings of the National Academy of Sciences of the
33.
D. Gibson, Dalton Transactions, 2016, 45, 12983-12991.
T. C. Johnstone, K. Suntharalingam and S. J. Lippard,
Chemical Reviews, 2016, 116, 3436-3486.
9.
United States of America, 2012, 109, 11987-11992.
T. C. Johnstone, S. M. Alexander, W. Lin and S. J. Lippard,
35.
G. T. Morgan and F. H. Burstall, Journal of the Chemical
Society, 1934, 1498-1500.
10.
11.
Journal of the American Chemical Society, 2014, 136, 116- 36.
118.
M. T. Gregory, G. Y. Park, T. C. Johnstone, Y. S. Lee, W.
Yang and S. J. Lippard, Proceedings of the National 37.
Academy of Sciences of the United States of America,
F. G. Baddour, M. I. Kahn, J. A. Golen, A. L. Rheingold and
L. H. Doerrer, Chemical Communications, 2010, 46, 4968-
4970.
T. Lammers, F. Kiessling, W. E. Hennink and G. Storm,
Journal of Controlled Release, 2012, 161, 175-187.
M. Ravera, E. Perin, E. Gabano, I. Zanellato, G. Panzarasa,
K. Sparnacci, M. Laus and D. Osella, Journal of Inorganic
Biochemistry, 2015, 151, 132-142.
M. Ravera, E. Gabano, I. Zanellato, E. Perin, A. Arrais and
D. Osella, Dalton Transactions, 2016, 45, 17233-17240.
E. Gabano, S. Gama, F. Mendes, F. Fregonese, A. Paulo
and M. Ravera, Inorganica Chimica Acta, 2015, 437, 16-
19.
J. X. McDermott, J. F. White and G. M. Whitesides, Journal
of the American Chemical Society, 1976, 98, 6521-6528.
G. Annibale and B. Pitteri, in Inorganic Syntheses, ed. J. R.
Shaple, John Wiley & Sons, Inc., 2004, vol. 34, ch. 2, pp.
76-81.
P. Gramatica, E. Papa, M. Luini, E. Monti, M. B. Gariboldi,
M. Ravera, E. Gabano, L. Gaviglio and D. Osella, Journal of
Biological Inorganic Chemistry, 2010, 15, 1157-1169.
G. Ermondi, G. Caron, M. Ravera, E. Gabano, S. Bianco, J.
A. Platts and D. Osella, Dalton Transactions, 2013, 42,
3482-3489.
M. Ravera, E. Gabano, G. Pelosi, F. Fregonese, S. Tinello
and D. Osella, Inorganic Chemistry, 2014, 53, 9326-9335.
G. Pelosi, M. Ravera, E. Gabano, F. Fregonese and D.
Osella, Chemical Communications, 2015, 51, 8051-8053.
M. Ravera, E. Gabano, I. Zanellato, F. Fregonese, G. Pelosi,
J. A. Platts and D. Osella, Dalton Transactions, 2016, 45,
5300-5309.
2014, 111, 9133-9138.
38.
12.
M. W. Kellinger, G. Y. Park, J. Chong, S. J. Lippard and D.
Wang, Journal of the American Chemical Society, 2013,
135, 13054-13061.
T. C. Johnstone and S. J. Lippard, Journal of the American
Chemical Society, 2014, 136, 2126-2134.
T. C. Johnstone, G. Y. Park and S. J. Lippard, Anticancer
Research, 2014, 34, 471-476.
39.
40.
13.
14.
15.
16.
17.
18.
19.
20.
21.
M. Ravera, E. Gabano, I. Zanellato, E. Perin, A. Arrais and 41.
D. Osella, Bioinorganic Chemistry and Applications, 2016.
S. X. Chong, S. C. F. Au-Yeung and K. K. W. To, Current 42.
Medicinal Chemistry, 2016, 23, 1268-1285.
I. Eryazici, C. N. Moorefield and G. R. Newkome, Chemical
Reviews, 2008, 108, 1834-1895.
43.
A. H. J. Wang, J. Nathans, G. van der Marel, J. H. van Boom
and A. Rich, Nature, 1978, 276, 471-474.
M. Howe-Grant and S. J. Lippard, Biochemistry, 1979, 18, 44.
5762-5769.
K. Suntharalingam, A. J. P. White and R. Vilar, Inorganic
Chemistry, 2009, 48, 9427-9435.
45.
S. Bombard, M. B. Gariboldi, E. Monti, E. Gabano, L.
Gaviglio, M. Ravera and D. Osella, Journal of Biological 46.
Inorganic Chemistry, 2010, 15, 841-850.
C. Y. Wei, L. J. Ren and N. Gao, International Journal of 47.
Biological Macromolecules, 2013, 57, 1-8.
E. Morel, F. Poyer, L. Vaslin, S. Bombard and M. P.
Teulade-Fichou, Inorganica Chimica Acta, 2016, 452, 152- 48.
158.
22.
23.
M. Ravera, E. Gabano, S. Tinello, I. Zanellato and D. Osella,
Journal of Inorganic Biochemistry, 2017, 167, 27-35.
S. R. A. Khan, S. Huang, S. Shamsuddin, S. Inutsuka, K. H.
Whitmire, Z. H. Siddik and A. R. Khokhar, Bioorganic &
Medicinal Chemistry, 2000, 8, 515-521.
24.
S. Gama, I. Rodrigues, F. Mendes, I. C. Santos, E. Gabano, 49.
B. Klejevskaja, J. Gonzalez-Garcia, M. Ravera, R. Vilar and
A. Paulo, Journal of Inorganic Biochemistry, 2016, 160,
275-286.
50.
51.
52.
I. Zanellato, I. Bonarrigo, D. Colangelo, E. Gabano, M.
Ravera, M. Alessio and D. Osella, Journal of Inorganic
Biochemistry, 2014, 140, 219-227.
25.
D. Colangelo and D. Osella, Current Medicinal Chemistry,
2005, 12, 3091-3102.
26.
27.
D. Colangelo and D. Osella, Journal, 2009, 4, 562-586.
G. Lowe, A. S. Droz, T. Vilaivan, G. W. Weaver, L.
Tweedale, J. M. Pratt, P. Rock, V. Yardley and S. L. Croft,
Journal of Medicinal Chemistry, 1999, 42, 999-1006.
S. Bonse, J. M. Richards, S. A. Ross, G. Lowe and R. L.
Krauth-Siegel, Journal of Medicinal Chemistry, 2000, 43,
4812-4821.
V. M. Manikandamathavan, N. Duraipandy, M. S. Kiran, V.
G. Vaidyanathan and B. U. Nair, RSC Advances, 2015, 5,
24877-24885.
K. Mitra, A. Shettar, P. Kondaiah and A. R. Chakravarty,
Inorganic Chemistry, 2016, 55, 5612-5622.
Y. Shi, S. A. Liu, D. J. Kerwood, J. Goodisman and J. C.
Dabrowiak, Journal of Inorganic Biochemistry, 2012, 107,
6-14.
M. Ravera, E. Gabano, I. Zanellato, I. Bonarrigo, M.
Alessio, F. Arnesano, A. Galliani, G. Natile and D. Osella,
Journal of Inorganic Biochemistry, 2015, 150, 1-8.
M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M.
A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B.
Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li,
H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L.
Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J.
Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H.
Nakai, T. Vreven, J. A. Montgomery Jr., J. E. Peralta, F.
Ogliaro, M. J. Bearpark, J. Heyd, E. N. Brothers, K. N.
Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K.
Raghavachari, A. P. Rendell, J. C. Burant, S. S. Iyengar, J.
Tomasi, M. Cossi, N. Rega, N. J. Millam, M. Klene, J. E.
Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R.
Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R.
28.
29.
30.
31.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 10 of 11
View Article Online
DOI: 10.1039/C7DT01706E
ARTICLE
Journal Name
Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K.
Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J.
Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J. B.
Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox, Gaussian
09, Revision C.01, Gaussian, Inc., Wallingford CT, 2010.
A. D. Becke, Journal of Chemical Physics, 1993, 98, 5648-
5652.
C. Lee, W. Yang and R. G. Parr, Physical Review B, 1988,
37, 785-789.
R. Ditchfield, W. J. Hehre and J. A. Pople, Journal of
Chemical Physics, 1971, 54, 724-728.
P. C. Hariharan and J. A. Pople, Theoretica Chimica Acta,
1973, 28, 213-222.
P. J. Hay and W. R. Wadt, Journal of Chemical Physics,
1985, 82, 270-283.
I. Zanellato, C. D. Boidi, G. Lingua, P. G. Betta, S. Orecchia,
E. Monti and D. Osella, Cancer Chemotherapy and
Pharmacology, 2011, 67, 265-273.
53.
54.
55.
56.
57.
58.
59.
60.
E. Magnani and E. Bettini, Brain Research Protocols, 2000,
5, 266-272.
A. Ghezzi, M. Aceto, C. Cassino, E. Gabano and D. Osella,
Journal of Inorganic Biochemistry, 2004, 98, 73-78.
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 11 of 11
Dalton Transactions
View Article Online
DOI: 10.1039/C7DT01706E
Pt(II)-terpyridine complexes were hardly oxidized to Pt(IV) prodrugs, loaded onto silica
nanovectors and in vitro tested for their antiproliferative properties.