Cellular delivery of pyrenyl-arene ruthenium complexes by a water-soluble arene ruthenium metalla-cage

Article abstract of DOI:10.1039/c2dt30193h

Three pyrenyl-arene ruthenium complexes (M₁-M₃) of the general formula [Ru(η⁶-arene-pyrenyl)Cl₂(pta)] (pta = 1,3,5-triaza-7-phosphaadamantane) have been synthesised and characterised. Prior to the coordination to ruthenium, pyrene was connected to the arene ligand via an alkane chain containing different functional groups: ester (L₁), ether (L₂) and amide (L₃), respectively. Furthermore, the pyrenyl moieties of the M_n complexes were encapsulated within the hydrophobic cavity of the water soluble metalla-cage, [Ru₆(η⁶-p-cymene)₆(tpt) ₂(donq)₃]⁶⁺ (tpt = 2,4,6-tri-(pyridin-4-yl)-1, 3,5-triazine; donq = 5,8-dioxydo-1,4-naphthoquinonato), while the arene ruthenium end was pointing out of the cage, thus giving rise to the corresponding host-guest systems [M_n?Ru₆(η ⁶-p-cymene)₆(tpt)₂(donq)₃] ⁶⁺ ([M_n?cage]⁶⁺). The antitumor activity of the pyrenyl-arene ruthenium complexes (M_n) and the corresponding host-guest systems [M_n?cage][CF₃SO₃] ₆ were evaluated in vitro in different types of human cancer cell lines (A549, A2780, A2780cisR, Me300 and HeLa). Complex M₂, which contains an ether group within the alkane chain, demonstrated at least a 10 times higher cytotoxicity than the reference compound [Ru(η⁶-p- cymene)Cl₂(pta)] (RAPTA-C). All host-guest systems [M _n?cage]⁶⁺ showed good anticancer activity with IC ₅₀ values ranging from 2 to 8 μM after 72 h exposure. The fluorescence of the pyrenyl moiety allowed the monitoring of the cellular uptake and revealed an increase of uptake by a factor two of the M₂ complex when encapsulated in the metalla-cage [Ru₆(η⁶-p- cymene)₆(tpt)₂(donq)₃]⁶⁺.

Full text of DOI:10.1039/c2dt30193h

View Article Online / Journal Homepage / Table of Contents for this issue
Dalton
Transactions
An international journal of inorganic chemistry
www.rsc.org/dalton
Volume 41 | Number 24 | 28 June 2012 | Pages 7165–7432
ISSN 1477-9226
COVER ARTICLE
Therrien et al.
Cellular delivery of pyrenyl-arene ruthenium complexes by a water-soluble
arene ruthenium metalla-cage

Dynamic Article Links
Dalton
View Article Online
Transactions
Cite this: Dalton Trans., 2012, 41, 7201
www.rsc.org/dalton
PAPER
Cellular delivery of pyrenyl-arene ruthenium complexes by a water-soluble
arene ruthenium metalla-cage
Mona Anca Furrer,a Frédéric Schmitt,b Michaël Wiederkehr,b Lucienne Juillerat-Jeanneretb and
Bruno Therrien*a
Received 26th January 2012, Accepted 20th March 2012
DOI: 10.1039/c2dt30193h
Three pyrenyl-arene ruthenium complexes (M1–M3) of the general formula [Ru(η6-arene-pyrenyl)
Cl2(pta)] (pta = 1,3,5-triaza-7-phosphaadamantane) have been synthesised and characterised. Prior to the
coordination to ruthenium, pyrene was connected to the arene ligand via an alkane chain containing
different functional groups: ester (L1), ether (L2) and amide (L3), respectively. Furthermore, the pyrenyl
moieties of the Mn complexes were encapsulated within the hydrophobic cavity of the water soluble
metalla-cage, [Ru6(η6-p-cymene)6(tpt)2(donq)3]6+ (tpt = 2,4,6-tri-(pyridin-4-yl)-1,3,5-triazine; donq =
5,8-dioxydo-1,4-naphthoquinonato), while the arene ruthenium end was pointing out of the cage, thus
giving rise to the corresponding host–guest systems [Mn⊂Ru6(η6-p-cymene)6(tpt)2(donq)3]6+-
([Mn⊂cage]6+). The antitumor activity of the pyrenyl-arene ruthenium complexes (Mn) and the
corresponding host–guest systems [Mn⊂cage][CF3SO3]6 were evaluated in vitro in different types of
human cancer cell lines (A549, A2780, A2780cisR, Me300 and HeLa). Complex M2, which contains an
ether group within the alkane chain, demonstrated at least a 10 times higher cytotoxicity than the
reference compound [Ru(η6-p-cymene)Cl2(pta)] (RAPTA-C). All host–guest systems [Mn⊂cage]6+
showed good anticancer activity with IC50 values ranging from 2 to 8 μM after 72 h exposure. The
fluorescence of the pyrenyl moiety allowed the monitoring of the cellular uptake and revealed an increase
of uptake by a factor two of the M2 complex when encapsulated in the metalla-cage [Ru6(η6-p-
cymene)6(tpt)2(donq)3]6+.
In order to improve the activity of the RAPTA compounds,
Introduction
various functional groups have been attached to the arene ring.12
During the past decades, ruthenium-based compounds have
emerged as a promising alternative to platinum drugs.1 Ruthe-
nium complexes are considered less toxic and able to overcome
resistance mechanisms induced by platinum compounds.2 Arene
ruthenium(II) complexes, especially those coming from the
Sadler and Dyson laboratories (Chart 1), have been investigated
extensively as potential anticancer drugs.3–7 The RAPTA family
of arene ruthenium complexes is characterised by the presence
of a hydrophilic 1,3,5-triaza-7-phosphaadamantane ligand (pta),
a lipophilic capping arene ligand, and two labile chlorido
ligands: these two chlorido ligands being available for aquation.8
Among the first example subjected to biological studies was the
It was found that increased hydrophilicity of the functionalised
arene ligand relative to p-cymene correlates with lower uptake,
while the presence of more lipophilic arene ligands increases the
uptake at the expense of selectivity.13 Tethering a glutathione
transferase inhibitor (ethacrynic acid) to the arene ligand of the
RAPTA framework,14 or incorporation of fluorophosphine
ligands15 to provide thermomorphic anticancer complexes have
also shown promising results by increasing drug efficacy.
Pyrene and pyrenyl derivatives are known to intercalate
between base pairs of DNA, and interfere with transcription,
property that can be exploited to design anticancer drugs.16,17
p-cymene
derivative
[Ru(η6-p-cymene)Cl2(pta)]
coined
RAPTA-C.9 This compound is described as weakly cytotoxic or
inactive in vitro, but very selective and efficient on metastasis
in vivo.10,11 Nevertheless, the main limitation of RAPTA-C is
that high doses are required for effective treatment in vivo.8
aInstitute of Chemistry, University of Neuchatel, Ave de Bellevaux 51,
CH-2000 Neuchatel, Switzerland. E-mail: bruno.therrien@unine.ch;
Fax: +41 32 7182511
bInstitute of Pathology, University Hospital of Lausanne, CHUV-UNIL,
Bugnon 25, CH-1011 Lausanne, Switzerland
Chart 1
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 7201–7211 | 7201

View Article Online
Moreover, since pyrene possesses fluorescence properties,18 it
allows monitoring of uptake and intracellular localisation of
pyrenyl-functionalised drugs. However, incorporation of the
pyrenyl moiety into the structure of a drug could alter its water
solubility, which is required in order to achieve effective cells
internalisation. Straightforward approaches to circumvent the
possible water solubility problems of the pyrenyl-functionalised
drugs include encapsulating the hydrophobic pyrenyl group into
a water-soluble drug delivery vector.19
[Ru6(η6-p-cymene)6(tpt)2(donq)3]6+. The pyrenyl-arene ruthe-
nium complexes (M1–M3), and their corresponding host–guest
systems [Mn⊂Ru6(η6-p-cymene)6(tpt)2(donq)3]6+ ([Mn⊂cage]6+)
have been evaluated in vitro against human cancer cells of
diverse origins (A549, A2780, A2780cisR, Me300 and HeLa).
The influence of the pyrenyl moiety on the biological activity of
the new complexes and the ability of the water-soluble metalla-
cage to increase cellular uptake of the compounds are also
discussed.
Recently, we have prepared such water-soluble drug delivery
vector: organometallic metalla-cages capable to encapsulate
planar aromatic molecules,19–24 acting as molecular carriers,
and able to release their cargo once inside cancer cells.24 An
example of such drug delivery carrier is the metalla-cage [Ru6(η6-
p-cymene)6 (tpt)2(donq)3]6+ (tpt = 2,4,6-tri-(pyridin-4-yl)-1,3,5-
triazine; donq = 5,8-dioxydo-1,4-naphthoquinonato).22 An impor-
tant feature of this type of metalla-assemblies is their susceptibility
to selectively target cancer cells and increase cellular uptake
through the EPR effect.25–27 The EPR effect (enhanced per-
meability and retention effect) has become a key mechanism for
solid tumour targeting since Matsumura and Maeda experimentally
noticed that in cancer tissues the uptake of large molecules and
proteins was significantly higher compared to healthy tissues.27
Herein, we are presenting multi-functional drugs composed of
a DNA intercalator (pyrene) connected to the arene ligand of
arene ruthenium metal-based drugs analogous to the RAPTA
derivatives, and in order to increase the cellular uptake and
exploit the EPR effect, the pyrenyl group was encapsulated in
the hydrophobic cavity of the large water-soluble metalla-cage
Results and discussion
Synthesis and characterisation
The syntheses of the pyrenyl-arene ruthenium complexes
(M1–M3) are performed in several steps: the first step involves
the reaction of 1,4-cyclohexadiene-1-propanol28 or 1,4-cyclohexa-
diene-1-propanamine29 with 1-pyrenebutyric acid to yield the
dienyl ligands L1 (ester) and L3 (amide), respectively
(Scheme 1). The ether derivative L2 is obtained from L1 follow-
ing a published procedure.30 The spectroscopic and analytical
data are given in the experimental part and they are all consistent
with the proposed structures.
In the second step, ruthenium trichloride hydrate reacts with
these dienyl-ligands (L1–L3) to afford the corresponding dinuc-
lear
arene
ruthenium
complexes
[Ru2(η6-arene-pyre-
nyl)2(μ-Cl)2Cl2] (D1–D3), which upon addition of 2 equivalents
of 1,3,5-triaza-7-phosphaadamantane (pta) forms the pyrenyl-
arene ruthenium complexes [Ru(η6-arene-pyrenyl)Cl2(pta)]
Scheme 1 The synthesis of the pyrenyl-ligands L1–L3 (CDMT = 2-chloro-4,6-dimethoxy-1,3,5-triazine; DCC = dicyclohexylcarbodiimide; DMAP
= 4-(dimethylamino)pyridine; NMM = N-methylmorpholine).
7202 | Dalton Trans., 2012, 41, 7201–7211
This journal is © The Royal Society of Chemistry 2012

View Article Online
Scheme 2 The synthesis of pyrenyl-arene ruthenium complexes M1–M3.
(M1–M3) in good yield (see Scheme 2). The coordination of the
ligands L1–L3 to the ruthenium atom is confirmed by H NMR
complex M1 and the host–guest system [M1⊂cage]6+ in CD3CN
at room temperature.
1
spectroscopy. Three signals between 5 and 6 ppm in a 2 : 2 : 1
ratio corresponding to the protons of the coordinated arene
moiety are observed for all complexes. Moreover, the successful
coordination of pta is clearly demonstrated by 31P NMR, for
which a singlet at −34 ppm is observed, consistent with other
arene ruthenium pta derivatives.9,14
The neutral dichloro pyrenyl-arene ruthenium pta complexes
(M1–M3) have been also encapsulated in the water-soluble
metalla-cage [Ru6(η6-p-cymene)6(tpt)2(donq)3]6+. The host–
It is quite obvious from these 1H NMR spectra that the
pyrenyl moiety is trapped inside the hydrophobic pocket of the
water-soluble metalla-cage, as the pyrenyl proton resonances in
the system [M1⊂cage]6+ are strongly upfield shifted (up to
2.5 ppm) compared to the resonances of the free pyrenyl-arene
complex M1. This strong shift can be explained by the cumulat-
ive effect of the ring current of two tpt panels on the pyrenyl
protons. Additionally, the signals of the CH2 protons of the
alkane chain, connecting the pyrenyl and the arene ruthenium
unit, are strongly influenced upon encapsulation: the proton res-
onance of the CH2 group directly attached to pyrene is shifted
upfield by 1.3 ppm. The 31P chemical shift of the coordinated
pta ligand is not significantly affected as the arene-ruthenium-pta
fragment stands out of the cage and therefore remains unaffected
by the encapsulation process.
guest
systems
[Mn⊂Ru6(η6-p-cymene)6(tpt)2(donq)3]6+
([Mn⊂cage]6+) are synthesised from the dinuclear complex
[Ru2(p-cymene)2(donq)Cl2],22 silver triflate and 2,4,6-tri-
(pyridin-4-yl)-1,3,5-triazine (tpt),31 in the presence of 1 equival-
ent of pyrenyl-arene ruthenium complexes Mn (Scheme 3), fol-
lowing a well established procedure.22
The host–guest systems are isolated as their triflate salts in
excellent yield. They have been characterised by means of infra-
red, UV-visible, fluorescence and NMR spectroscopy as well as
In order to further confirm the formation of the host–guest
systems, a series of diffusion-ordered NMR (DOSY)32 spectra
were recorded. This technique, which is size and shape depen-
dent, provides an estimation of the diffusion coefficient (D) of a
compound in solution. DOSY measurements of the empty cage,
1
by mass spectrometry. Fig. 1 shows the H NMR spectra of the
empty cage [Ru6(η6-p-cymene)6(tpt)2(donq)3]6+, the free
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 7201–7211 | 7203

View Article Online
Scheme 3 Encapsulation of pyrenyl-arene ruthenium complexes M1–M3 in the metalla-cage [Ru6(η6-p-cymene)6(tpt)2(donq)3]6+
.
Fig. 2 400 MHz 2D 1H DOSY NMR spectra recorded at 27 °C in
CD3CN of M1, (bottom), [1]6+ (middle) and [M1⊂cage]6+(top).
Table 1 Diffusion coefficients (D), association constants (Ka) and free
energies (ΔG°) values for the encapsulation of pyrenyl-arene ruthenium
complexes M1–M3 in [Ru6(η6-p-cymene)6(tpt)2(donq)3]6+ (CH2Cl2 at
21 °C)
Fig. 1 The aromatic region of the 1H NMR spectra (RT, CD3CN) of
[Ru6(η6-p-cymene)6(tpt)2(donq)3]6+ (top), [M1⊂cage]6+ (middle) and
M1 (bottom) showing the resonances of the free and encapsulated
pyrenyl moiety, highlighted with red ovals, and the protons of the tpt
panels, blue ovals.
[M1⊂cage]6+
[M2⊂cage]6+
[M3⊂cage]6+
D [m2 s−1
Ka [104 M−1
ΔG° [kcal mol−1
]
5.75 × 10−10
78
−8.03
5.62 × 10−10
7.7
−6.66
5.37 × 10−10
7.2
−6.62
]
]
the host–guest system [M1⊂cage]6+ and complex M1 are pre-
sented in Fig. 2. These experiments show that for [M1⊂cage]6+
each of the proton signals of the host and of the guest possess
the same diffusion coefficient, thus indicating the presence of
only one associated species in solution. The host–guest system
[M1⊂cage]6+ shows a diffusion coefficient of 5.75 × 10−10 m2 s−1
in CD3CN, whereas a diffusion coefficient of 6.16 × 10−10 m2
s−1 is observed for the empty cage, and 11.48 × 10−10 m2 s−1
for the free complex M1. The three host guest systems
[Mn⊂cage]6+ show very similar diffusion values in CD3CN
(Table 1), and these values are consistent with the respective
molecular sizes of the different species, and the proposed
structures.32
Since it was not possible to crystallise the compounds and to
perform an X-ray crystal structure analysis of one of the host–
guest systems, molecular modelling was performed using the
Chem3D software.33 An estimation of the size and shape of the
7204 | Dalton Trans., 2012, 41, 7201–7211
This journal is © The Royal Society of Chemistry 2012

View Article Online
Fig. 3 A Chem3D model of [Mn⊂cage]6+ (Mn in space-filling and metalla-cage in ball and stick representations).
[Mn⊂cage]6+ systems was thus obtained (Fig. 3). As expected,
the simulation shows the pyrenyl moiety being encapsulated
inside the cavity of [Ru6(η6-p-cymene)6(tpt)2(donq)3]6+ and the
arene-ruthenium-pta unit pointing away from the metalla-cage.
The formation of the host–guest systems was further
confirmed by electrospray ionization mass spectrometry
(ESI-MS). The isotopic patterns of all [Mn⊂cage]+ systems are
represented in Fig. 4. Peaks corresponding to the host–guest
systems were assigned at m/z = 1260.78 for [M1 + cage + 3
CF3SO3]3+, m/z = 1256.79 for [M2 + cage + 3 CF3SO3]3+ and
1260.79 for [M3 + cage + 3 CF3SO3]3+ (Fig. 4).
complexes (M1–M3) to the empty metalla-cage [Ru6(η6-p-
cymene)6(tpt)2(donq)3]6+. Consequently, the association con-
stants (Ka) for these host–guest systems [Mn⊂cage]6+ were deter-
mined (eqn (1)). The UV-visible dilution method34 was applied
to evaluate the interaction in CH2Cl2 at 21 °C between the
pyrenyl-functionalised complexes Mn and the metalla-cage
(Table 1). Association constants superior to 7.2 × 104 M−1 were
estimated for these systems and the corresponding binding free
energies (ΔG°) inferior to −6.62 kcal mol−1 were calculated.
These values are comparable to those found for similar host–
guest systems.22
Pyrene and pyrenyl derivatives are well known for their fluor-
escence properties,18 and it was previously shown that encapsu-
lation of 1-(4,6-dichloro-1,3,5-triazin-2-yl)pyrene within
[Ru6(η6-p-cymene)6(tpt)2(donq)3]6+ suppresses its fluor-
escence.24 In a similar manner, the fluorescence of the M1–M3
Host–guest properties
Preparation of the host–guest systems [Mn⊂cage]6+ was also per-
formed in CH2Cl2 by addition of pyrenyl-arene ruthenium
ð1Þ
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 7201–7211 | 7205

View Article Online
Fig. 4 Excerpts of the ESI-MS spectra corresponding to [M1 + cage + 3 CF3SO3]3+ (left), [M2 + cage + 3 CF3SO3]3+ (middle) and [M3 + cage + 3
CF3SO3]3+ (right) and simulations of the corresponding isotopic patterns in red.
against human A549 pulmonary cancer, A2780 (cisplatin sensi-
tive) and A2780cisR (cisplatin resistant) ovarian cancer cells, as
well as against the Me300 melanoma and HeLa cervix cancer
cells. The IC50 values after 72 h of cell exposure are listed in
Table 2.
Complex M2 shows the highest cytotoxic activity compared to
the reference compound RAPTA-C after 72 h of cell exposure,
suggesting a synergistic effect between the pyrenyl group and
the arene ruthenium unit. The nature of the chemical bond
between both moieties plays an important role in the cytotoxic
effect of the complexes since M2 is bearing the more stable
bond. Therefore, we can hypothesise that it is important to keep
the pyrenyl and the arene ruthenium part together to achieve a
higher cytotoxic effect. The host–guest systems show similar
cytotoxic activity compared to the empty cage. Kinetic studies of
the uptake of M2 and [M2⊂cage]6+ were performed exploiting
the fluorescence of the pyrenyl group (λexc/λem = 312 nm/
Fig. 5 The fluorescence emission titration of [Ru6(η6-p-
378 nm) in HeLa human cervix cancer cells exposed to 2.5 μM
cymene)6(tpt)2(donq)3]6+ in a solution of M2 in CH3CN (5 × 10−6 M) at
concentration of the compounds from 2 to 48 h (Fig. 6). The
21 °C with excitation at 350 nm, showing the decrease in the emission
uptake of both compounds is relatively fast, and reaches a
energy of pyrene upon encapsulation.
plateau after 6 h of incubation. Moreover, this study reveals a
greater intracellular accumulation by a factor of two when the
pyrenyl-derivative M2 is loaded into the metalla-cage as com-
pared to M2 alone, nicely demonstrating the transport capacity of
the water-soluble metalla-cage system and the releasing of the
complexes is suppressed upon encapsulation of the pyrenyl
moiety in the metalla-cage. This phenomenon is illustrated by
the emission spectra of fluorescence titrations. Upon gradual
addition of [Ru6(η6-p-cymene)6(tpt)2(donq)3]6+ (0.2–5 equiv) to
guest after internalisation.
a solution of complexes M1–M3 in acetonitrile (5 × 10−6 M),
strong suppression of the fluorescence was observed (Fig. 5).
The loss of excitation and energy transfer between the pyrenyl
moiety and the metalla-cage lead to a decrease in the emission
Conclusions
Three new pyrenyl-arene ruthenium derivatives comprising a
RAPTA unit appended by a pyrenyl group have been synthesised
and coupled with a water-soluble metalla-cage. The cytotoxicity
of the new compounds has been evaluated in A549, A2780,
A2780cisR, Me300 and HeLa cancer cells. The cytotoxicity of
M2 was at least 10 times higher than the reference compound
RAPTA-C, probably due to higher lipophilic properties induced
by the presence of the pyrenyl appendage, while the
energy of pyrene and, as a consequence, to hypochromism of the
fluorescence.35
Antiproliferative activity
The antitumor activity of the pyrenyl-derivatives M1–M3 and of
the host guest systems [Mn⊂cage][CF3SO3]6 was evaluated
7206 | Dalton Trans., 2012, 41, 7201–7211
This journal is © The Royal Society of Chemistry 2012

View Article Online
Table 2 IC50 values of pyrenyl-arene ruthenium complexes M1–M3, the host–guest systems [Mn⊂cage][CF3SO3]6, RAPTA-C and the empty
metalla-cage [Ru6(η6-p-cymene)6(tpt)2(donq)3]6+ after 72 h exposure of human cancer cells
A549
A2780
A2780cisR
IC50 (μM)
Me30
HeLa
Compound
M1
M2
M3
>25
>25
>25
>25
19.7 6.5
>25
>25
15.8 6.0
>25
>25
17.7 7.0
>25
>25
>25
>25
[M1⊂cage][CF3SO3]6
[M2⊂cage][CF3SO3]6
[M3⊂cage][CF3SO3]6
[cage][CF3SO3]6
RAPTA-Ca
3.0 0.8
2.0 0.4
2.7 1.0
Nd
5.0 2.9
4.7 2.4
3.4 0.6
3.1 1.0
353 14
4.1 3.8
6.9 2.6
2.6 0.5
4.6 0.5
>200
6.4 1.7
2.7 0.6
3.5 0.2
nd
7.0 1.5
5.5 1.1
7.7 2.0
nd
>100
nd
nd
a Values taken from ref. 36.
spectrometer. UV-visible absorption spectra were recorded with
an Uvikon 930 spectrophotometer by using precision cells made
of quartz (1 cm). Electrospray mass spectra were obtained in
positive-ion mode with an LCQ Finigan mass spectrometer. Flu-
orescence spectra were measured on a Varian Cary Eclipse fluor-
escence spectrophotometer equipped with a Varian Cary-block
temperature controller using 1 × 1 cm quartz cells. Elemental
analyses were performed by the Laboratory of Organic Chem-
istry, ETH Zürich (Switzerland).
Synthesis of L1. 1-Pyrene butyric acid (2.0 g, 6.93 mmol),
1,4-cyclohexadiene-1-propanol (1.9 g, 13.87 mmol) and (0.7 g,
5.72 mmol) of 4-(dimethylamino)pyridine (DMAP) in anhy-
drous dichloromethane (40 mL) were cooled at 0 °C and dicyclo-
hexylcarbodiimide (DCC) (1.7 g, 8.24 mmol) was added
gradually. The reaction mixture was stirred for 10 min at 0 °C
then for 12 h at room temperature. The precipitated dicyclohexy-
lurea was removed by filtration over Celite. The filtrate was
washed with HCl (0.5 N) and saturated NaHCO3 aqueous sol-
ution. The organic layer was dried over anhydrous MgSO4 and
filtrated. The filtrate was evaporated in vacuo and the residue
was purified by flash chromatography (dichloromethane) to give
2.2 g of pale-yellow oil.
Fig. 6 Kinetic accumulation studies of pyrenyl-associated fluorescence
in Hela cervix cancer cells exposed to 2.5 μM of [Ru6(η6-p-
cymene)6(tpt)2(donq)3]6+
(
), M2 ( ), and [M2⊂cage]6+
(
).
[M2⊂cages]6+ system was 50 times more cytotoxic than
RAPTA-C. The uptake of M2 was considerably improved upon
encapsulation
within
the
metalla-cage
[Ru6(η6-p-
1
Yield: 2.2 g (77.6%). H NMR (400 MHz, CDCl3, 298 K): δ
cymene)6(tpt)2(donq)3]6+. These new systems could potentially
act as multi-functional drugs: cytotoxicity due to interactions of
the pyrenyl moiety with DNA and the presence of arene ruthe-
nium groups, efficacy against metastasis associated with the
RAPTA unit, and exploiting the EPR effect to better target
cancer cells by using a large carrier.
3
3
(ppm) = 8.31 (1 H, d, J = 9.0 Hz, Hpy), 8.17 (1 H, d, J = 7.6
3
3
Hz, Hpy), 8.16 (1 H, d, J = 7.6 Hz, Hpy), 8.12 (2 H, d, J = 9.0
3
Hz, Hpy), 8.04 (2 H, br s, Hpy), 8.00 (1 H, t, J = 7.6 Hz, Hpy),
7.87 (1 H, d, 3J = 7.6 Hz, Hpy), 5.70 (2 H, br s, CHvCH), 5.43
3
(1 H, br s, CHvC), 4.10 (2 H, t, J = 6.7 Hz, CH2O), 3.40 (2
H, t, 3J = 7.6 Hz, CH2Py), 2.67 (2 H, m, CH2CHv), 2.57 (2 H,
3
m, CH2CHv), 2.47 (2 H, t, J = 7.5 Hz, CH2CO), 2.20 (2 H,
qn, 3J = 7.5 Hz, CH2CH2Py), 2.02 (2 H, t, 3J = 7.61 Hz,
CH2Cv), 1.76 (2 H, qn, 3J = 6.7 Hz, CH2CH2O).
Experimental section
General
Synthesis of L2. To a solution of L1 (1 g, 2.44 mmol) in
freshly distilled chloroform in a pressure Schlenk were added
successively InBr3 (47 mg, 0.13 mmol) and Et3SiH (1.3 g,
10.72 mmol). The reaction mixture was stirred at 60 °C under
nitrogen atmosphere for 4 h. Then water (15 mL) was added and
the resulting orange suspension was stirred continuously until
the disappearance of the colour. The aqueous layer was extracted
with dichloromethane. The combined organic phase was dried
over MgSO4, and filtered. Evaporation of solvent in vacuo
afforded a crude product which was separated by flash
2,4,6-Tri-(pyridin-4-yl)-1,3,5-triazine (tpt),31 [Ru(η6-p-cymene)
Cl2]2,37 [Ru2(η6-p-cymene)2(donq)Cl2],22 1,4-cyclohexadiene-1-
propanol,28 and 1,4-cyclohexadiene-1-propanamine29 were pre-
pared according to published methods. All other reagents were
commercially available (Sigma-Aldrich) and used as received.
The 1H, 13C{1H}, 31P{1H} and DOSY NMR spectra were
recorded with a Bruker Avance II 400 spectrometer using the
residual protonated solvent as internal standard. Infrared spectra
were recorded as KBr pellets with a Perkin-Elmer FTIR 1720 X
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 7201–7211 | 7207

View Article Online
chromatography (dichloromethane–hexane 1 : 1) to give 700 mg
100.2 (Car), 84.6 (CHar), 79.9 (CHar), 77.8 (CHar), 64.1
(CH2O), 33.9 (CH2CO), 32.9 (CH2Py), 30.0 (CH2Ph), 28.6
(CH2CH2O), 27.6 (CH2CH2CO). Elemental analysis (%) Calc.
for C58H52Cl4O4Ru2·1.5 CH2Cl2 (1284.38): C, 55.64; H, 4.32;
Found: C, 55.67; H, 4.37. ESI-MS (m/z): 1109.4 [M − 2 Cl +
Na]+.
of colourless oil.
1
Yield: 700 mg (72.7%). H NMR (400 MHz, CDCl3, 298 K):
δ (ppm) = 8.29 (1 H, d, 3J = 9.3 Hz, Hpy), 8.16 (1 H, d, 3J = 7.6
3
Hz, Hpy), 8.16 (1 H, d, J = 7.6 Hz, Hpy), 8.11 (2 H, m, Hpy),
3
8.02 (2 H, AB system, Hpy), 7.98 (1 H, t, J = 7.6 Hz, Hpy),
7.88 (1 H, d, 3J = 7.6 Hz, Hpy), 5.69 (2 H, br s, CHvCH), 5.41
3
Synthesis of D2. Ligand L2 (394 mg, 0.95 mmol) in 4 mL
dichloromethane was added to a solution of RuCl3·nH2O
(125 mg, 0.47 mmol) in 20 mL ethanol. The reaction mixture
was refluxed for 15 h. The orange precipitate was filtered,
washed with ethanol, diethyl ether and pentane and dried
in vacuo.
(1 H, br s, CHvC), 3.48 (2 H, t, J = 6.7 Hz, CH2O), 3.41 (2
3
3
H, t, J = 6.7 Hz, CH2O), 3.37 (2 H, t, J = 7.6 Hz, CH2Py),
2.72–2.52 (4 H, m, CH2CH=), 2.01 (2 H, t, 3J = 7.61 Hz,
CH2Cv), 1.91 (2 H, m, CH2), 1.78 (2 H, m, CH2), 1.70 (2 H,
q, 3J = 6.7 Hz, CH2).
Yield: 242 mg (91%). UV-vis [1.0 × 10−5 M−1, CH2Cl2] λmax
/
Synthesis of L3. 1-Pyrene butyric acid (500 mg, 1.73 mmol),
2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) (364 mg,
2.07 mmol) and N-methylmorpholine (NMM) (524 mg,
5.19 mmol) in anhydrous tetrahydrofuran (12 mL) were stirred at
room temperature for 1 h. The resulting white suspension was
subsequently treated with 1,4-cyclohexadiene-1-propanamine
(237 mg, 1.73 mmol). The reaction mixture was stirred for an
additional 6 h and then quenched with water and extracted two
times with 10 mL of dichloromethane. The combined organic
phases were washed two times with 10 mL of saturated Na2CO3
and brine. The organic layer was dried over anhydrous MgSO4,
filtrated and concentrated in vacuo. The crude product was separ-
ated by flash chromatography (dichloromethane) to yield 450 mg
of white powder.
nm (ε/M−1 cm−1) = 345 (105 330), 328 (76 636), 318 (33 041),
277 (131 405), 266 (73 864), 244 (180 884), 235 (123 065). IR
(KBr): ν = 3044 (CHaryl), 2936 (s, CH2), 1117 (m, CO). 1H
3
NMR (400 MHz, CDCl3, 298 K): δ (ppm) = 8.29 (2 H, d, J =
9.3 Hz, Hpy), 8.19 (4 H, m, Hpy), 8.15 (2 H, br s, Hpy), 8.13 (2
3
H, br s, Hpy), 8.05 (4 H, ABsystem, Hpy), 8.01 (2 H, t, J = 7.6
3
3
Hz, Hpy), 7.88 (2 H, d, J = 7.6 Hz, Hpy), 5.54 (4 H, t, J = 5.5
3
3
Hz, Har), 5.43 (2 H, t, J = 5.5 Hz, Har), 5.34 (4 H, d, J = 5.7
Hz, Har), 3.42 (8 H, m, CH2O), 3.37 (4 H, t, 3J = 7.6 Hz,
CH2Py), 2.62 (4 H, t, 3J = 7.6 Hz, CH2Ph), 1.87 (8 H, m,
CH2CH2Py, CH2CH2Ph), 1.71 (4 H, m, CH2CH2CH2). 13C{1H}
NMR (101 MHz, CDCl3, 298 K) δ (ppm) 136.8 (Cpy), 131.5
(Cpy), 131.0 (Cpy), 129.8 (Cpy), 128.7 (Cpy), 127.6 (CHpy),
127.4 (CHpy), 127.3 (CHpy), 126.6 (CHpy), 125.9 (CHpy), 125.2
(Cpy), 125.1 (Cpy), 124.9 (CHpy), 124.9 (CHpy), 124.8 (CHpy),
123.5 (CHpy), 101.2 (Car), 83.9 (CHar), 80.5 (CHar), 79.6
(CHar), 70.8 (CH2O), 69.7 (CH2O), 33.3 (CH2Py), 30.7
(CH2Ph), 29.8 (CH2), 29.5 (CH2), 28.5 (CH2). Elemental analy-
sis (%) Calc. for C58H56Cl4O2Ru2 (1129.02): C, 61.70; H, 5.00;
Found: C, 61.42; H, 4.98. ESI-MS (m/z): 1081.4 [M − 2 Cl +
Na]+.
1
Yield: 450 mg (63.8%). H NMR (400 MHz, CDCl3, 298 K)
δ (ppm) = 8.31 (1 H, d, 3J = 9.3 Hz, Hpy), 8.17 (2 H, d, 3J = 7.6
3
Hz, Hpy), 8.11 (2 H, d, J = 7.6 Hz, Hpy), 8.03 (2 H, br s, Hpy),
3
3
8.00 (1 H, t, J = 7.6 Hz, Hpy), 7.86 (1 H, d, J = 7.7 Hz, Hpy),
5.67 (2 H, br s, CHvCH), 5.41 (1 H, br s, CHvC), 5.30 (1 H,
3
br s, NH), 3.40 (2 H, t, J = 7.0 Hz, CH2Py), 3.25 (2 H, q,
3J = 7.0 Hz, CH2NH), 2.66–2.61 (2 H, m, CH2CHv),
2.59–2.51 (2 H, m, CH2CHv), 2.35–2.13 (4 H, m, CH2CO,
3
CH2CH2CO), 1.98 (2 H, t, J = 7.4 Hz, CH2Cv), 1.60 (2 H, q,
Synthesis of D3. Ligand L3 (560 mg, 1.37 mmol) was added
to a solution of RuCl3·nH2O (173 mg, 0.68 mmol) in 40 mL
ethanol. The reaction mixture was refluxed for 15 h. The dark-
green precipitate was filtered, washed with ethanol, dichloro-
methane, diethyl ether and pentane and dried in vacuo.
3J = 7.3 Hz, CH2CH2NH).
Synthesis of D1. Ligand L1 (1.4 g, 3.42 mmol) in 6 mL
dichloromethane was added to a solution of RuCl3·nH2O
(430 mg, 1.64 mmol) in 30 mL ethanol. The reaction mixture
was refluxed for 15 h. The brown precipitate was filtered,
washed with ethanol, several drops of dichloromethane and
diethyl ether and dried in vacuo.
Yield: 350 mg (88%). UV-vis [1.0 × 10−5 M−1, CH2Cl2] λmax
/
nm (ε/M−1 cm−1) = 345 (76 900), 328 (55 279), 277 (93 787),
267 (53 752), 244 (132 342), 235 (90 580). IR (KBr): ν = 3319
(s, NH), 3044 (CHaryl), 2928 (s, CH2), 1643 (s, CO), 1050 (s,
CN). 1H NMR (400 MHz, CDCl3, 298 K): δ (ppm) = 8.32 (2 H,
Yield: 750 mg (78%). UV-vis [1.0 × 10−5 M−1, CH2Cl2] λmax
/
nm (ε/M−1 cm−1) = 345 (149 512), 328 (103 926), 314 (45 752),
277 (152 691). IR (KBr): ν = 3060 (w, CHaryl), 2958 (s, CH2),
1730 (s, CO), 1173 (m, CO). 1H NMR (400 MHz, DMF,
298 K): δ (ppm) = 8.49 (2 H, d, 3J = 9.3 Hz, Hpy). 8.24–8.34 (8
3
d, J = 9.0 Hz, Hpy), 8.20–8.05 (7 H, m, Hpy), 8.05–7.92 (7 H,
3
m, Hpy), 7.89 (2 H, d, J = 7.7 Hz, Hpy), 5.91 (2 H, br s, NH),
3
3
5.47 (2 H, t, J = 5.3 Hz, Har), 5.40 (4 H, t, J = 5.3 Hz, Har),
5.16 (4 H, d, 3J = 5.3 Hz, Har), 3.39 (4 H, t, 3J = 7.2 Hz,
CH2Py), 3.24 (4 H, m, CH2NH), 2.43 (4 H, t, 3J = 7.4 Hz,
CH2Ph), 2.35–2.27 (4 H, m, CH2CO), 2.25–2.19 (4 H, m,
CH2CH2CO), 1.73 (4 H, m, CH2CH2Ph). Elemental analysis
(%) Calc. for C58H54Cl4N2O2Ru2·1.25 CH2Cl2 (1261.18): C,
56.43; H, 4.52; N 2.22 Found: C, 56.30; H, 4.19, N, 2.33.
ESI-MS (m/z): 1106.2 [M − 2 Cl + Na]+.
3
H, m, Hpy), 8.18 (4 H, AB system, Hpy), 8.09 (2 H, t, J = 7.6
3
Hz, Hpy), 8.01 (2 H, m, Hpy), 5.76 (4 H, dd, J = 5.7 Hz, Har),
3
5.60–5.63 (6 H, m, Har), 4.20 (4 H, t, J = 6.5 Hz, CH2O), 3.45
(4 H, t, 3J = 7.2 Hz, CH2Py), 2.68 (4 H, t, 3J = 7.6 Hz, CH2Ph),
3
2.55 (4 H, t, J = 7.2 Hz, CH2CO), 2.14 (4 H, m, CH2CH2CO),
2.06 (4 H, m, CH2CH2O). 13C{1H} NMR (101 MHz, DMF,
298 K) δ (ppm) = 173.6 (COO), 137.1 (Cpy), 132.0 (Cpy), 131.5
(Cpy), 130.5 (Cpy), 129.2 (Cpy), 128.3 (CHpy) 128.2 (CHpy),
128.0 (CHpy), 127.3 (CHpy), 126.8 (CHpy), 125.9 (Cpy), 125.5
(Cpy), 125.7 (CHpy), 125.6 (CHpy), 125.5 (CHpy), 124.2 (CHpy),
Synthesis of M1. The dimeric complex (D1) (50 mg,
0.04 mmol) was refluxed with pta (13 mg, 0.08 mmol) in
1 : 1 methanol/dichloromethane (20 mL) for 6 h. The reaction
7208 | Dalton Trans., 2012, 41, 7201–7211
This journal is © The Royal Society of Chemistry 2012

View Article Online
mixture was filtered and concentrated. Diethyl ether was added
to afford M1 as an orange solid which was dried in vacuo.
(ppm)
=
−33.9. Elemental analysis (%) Calc. for
C35H40Cl2N3OPRu·0.25 CH2Cl2 (742.89): C, 56.99; H, 5.49, N,
5.66; Found: C, 57.18; H, 5.57; N, 5.81. ESI-MS (m/z): 746.3
[M + Na]+.
Yield: 46 mg, (72%). UV-vis [1.0 × 10−5 M−1, CH2Cl2] λmax
/
nm (ε/M−1 cm−1) = 344 (35 129), 328 (24 776), 314 (11 562),
277 (42 964), 266 (25 446), 244 (62 443), 235 (43 365).
IR (KBr): ν = 3060 (CHaryl), 2920 (s, CH2), 1728 (s, CO), 1240
Synthesis of M3. The dimeric complex (D3) (45 mg,
0.04 mmol) was refluxed with pta (12 mg, 0.08 mmol) in 1 :
1 methanol/dichloromethane (30 mL) for 5 h. The reaction
mixture was filtered and concentrated. Diethyl ether was added
to afford M3 as an orange solid which was dried in vacuo.
1
(s, CN). H NMR (400 MHz, CD3CN, 298 K): δ (ppm) = 8.39
(1 H, d, J = 9.3 Hz, Hpy), 8.24 (2 H, t, J = 7.0 Hz, Hpy), 8.21
(1 H, s, Hpy), 8.19 (1 H, d, J = 1.3 Hz, Hpy), 8.10 (2 H, AB
system, Hpy), 8.04 (1 H, t, J = 7.6 Hz, Hpy) 7.94 (1 H, d, J =
7.8 Hz, Hpy), 5.48 (2 H, dt, J = 5.8 Hz, J = 1.0 Hz, Har), 5.37
(2 H, d, J = 5.8 Hz, Har), 5.02 (1 H, tt, J = 5.8 Hz, J = 1.0
Hz, Har), 4.44 (6 H, s, CH2NCH2), 4.17 (6 H, s, CH2PCH2),
3
3
4
3
3
Yield: 46 mg (81%). UV-vis [1.0 × 10−5 M−1, CH2Cl2] λmax
/
3
4
nm (ε/M−1 cm−1) = 345 (57 719), 328 (41 142), 277 (70 800),
266 (43 230), 244 (102 499), 235 (72 946). IR (KBr): ν = 2928
(s, CH2), 1638 (s, CO), 1012 (s, CN). 1H NMR (400 MHz,
3
3
4
3
3
4.08 (2 H, t, J = 6.3 Hz, CH2O), 3.38 (2 H, t, J = 7.6 Hz,
CH2Py), 2.41 (2 H, t, 3J = 7.3 Hz, CH2Ph) 2.40 (2 H, t, 3J = 7.3
Hz, CH2CO), 2.09 (2 H, m, CH2CH2CO), 1.89 (2 H, m,
CH2CH2O). 13C{1H} NMR (101 MHz, CD3CN, 298 K) δ
(ppm) = 174.3 (COO), 137.4 (Cpy), 132.3 (Cpy), 131.9 (Cpy),
131.3 (Cpy), 130.7 (Cpy), 128.7 (CHpy), 128.5 (CHpy), 128.3
(CHpy), 127.6 (CHpy), 127.1 (CHpy), 126.0 (CHpy), 126.0
(CHpy), 125.8 (CHpy), 125.4 (Cpy), 125.3 (Cpy), 124.5 (CHpy),
110.5 (Car), 88.1 (d, J (P-C) = 5.8 Hz, CHar), 87.2 (d, J (P-C) =
1.8 Hz, CHar), 78.8 (CHar), 73.6 (d, 2J (P-C) = 6.8 Hz,
CH2NCH2), 64.3 (CH2O), 53.3 (d, 1J (P-C) = 18.0 Hz,
CH2PCH2), 34.4 (CH2CO), 33.2 (CH2Py), 30.5 (CH2Ph), 29.0
(CH2CH2Ph), 27.7 (CH2CH2Py). 31P{1H} NMR (161 MHz,
CD3Cl, 298 K) δ (ppm) = −33.5. Elemental analysis (%) Calc.
for C35H38Cl2N3OPRu·0.25 CH2Cl2 (756.88): C, 55.94; H, 5.13,
N, 5.55; Found: C, 55.76; H, 5.11; N, 5.65. ESI-MS (m/z): 760.2
[M + Na]+.
3
CD3CN, 298 K): δ (ppm) = 8.44 (1 H, d, J = 9.3 Hz, Hpy),
8.28 (1 H, d, J = 6.9 Hz, Hpy), 8.26 (1 H, d, J = 6.9 Hz, Hpy),
8.23 (1 H, s, Hpy), 8.21 (1 H, d, J = 1.9 Hz, Hpy), 8.13 (2 H,
3
3
4
3
AB system, Hpy), 8.07 (1 H, t, J = 7.6 Hz, Hpy), 7.98 (1 H, d,
3J = 7.8 Hz, Hpy), 6.54 (1 H, br s, NH), 5.56 (1 H, tt, J = 5.8
3
4
Hz, J = 1.6 Hz, Har), 5.46 (2 H, d, J = 5.8 Hz, Har), 5.18 (2 H,
3
t, J = 5.8 Hz, Har), 4.46 (6 H, s, CH2NCH2), 4.22 (6 H, s,
3
3
CH2PCH2), 3.45 (2 H, t, J = 7.7 Hz, CH2Py), 3.27 (2 H, q, J
= 6.4 Hz, CH2NH), 2.43 (2 H, t, J = 7.4 Hz, CH2Ph), 2.30
3
3
(2 H, t, J = 7.2 Hz, CH2CO), 2.15 (2 H, m, CH2CH2CO), 1.83
(2 H, qn, J = 7.0 Hz, CH2CH2NH). 13C{1H} NMR (101 MHz,
3
CD3CN, 298 K): δ (ppm) = 172.1 (CONH), 136.5 (Cpy), 131.1
(Cpy), 130.6 (Cpy), 129.5 (Cpy), 128.3 (Cpy), 127.3 (CHpy),
127.2 (CHpy), 126.9 (CHpy), 126.2 (CHpy), 125.8 (CHpy), 124.7
(CHpy), 124.6 (CHpy), 124.5 (CHpy), 124.4 (Cpy), 124.3 (Cpy),
123.4 (CHpy), 109.1 (Car) 87.0 (d, J (P-C) = 5.5 Hz, CHar), 85.6
(d, J (P-C) = 1.7 Hz, CHar), 77.6 (CHar), 72.3 (d, 2J (P-C) = 6.8
1
Hz, CH2NCH2), 52.0 (d, J (P-C) = 17.8 Hz, CH2PCH2), 37.7
Synthesis of M2. The dimeric complex (D2) (71 mg,
0.06 mmol) was refluxed with pta (20 mg, 0.12 mmol) in 1 :
1 methanol/dichloromethane (30 mL) for 6 h. The reaction
mixture was filtered and concentrated. Diethyl ether was added
to afford M2 as a yellow solid which was dried in vacuo.
(CH2NH), 35.1 (CH2CO), 32.1 (CH2Py), 29.5 (CH2Ph), 28.3
(CH2CH2NH), 27.3 (CH2CH2CO). 31P{1H} NMR (161 MHz,
CD3CN, 298 K) δ (ppm) = −33.5. Elemental analysis (%) Calc.
for C35H39Cl2N4OPRu·0.5 CH2Cl2 (777.13): C, 54.87; H, 5.19,
N, 7.21; Found: C, 55.35; H, 5.47; N, 7.36. ESI-MS (m/z):
759.2 [M + Na]+.
Yield: 68 mg (74%). UV-vis [1.0 × 10−5 M−1, CH2Cl2] λmax
/
nm (ε/M−1 cm−1) = 345 (42 893), 328 (30 529), 315 (14 842),
277 (53 543), 266 (32 311), 244 (77 794), 235 (55 462). IR
(KBr): ν = 3044 (CHaryl), 2936 (s, CH2), 1117 (m, CO), 840 (m,
CO), 1240 (s, CN). 1H NMR (400 MHz, CD3CN, 298 K): δ
Encapsulation of functionalised complexes (M1–M3) in metalla-
cage [Ru6(η6-p-cymene)6(tpt)2(donq)3]6+
3
(ppm) = 8.40 (1 H, d, J = 9.3 Hz, Hpy), 8.27 (2 H, m, Hpy),
3
The dinuclear complex [Ru2(η6-p-cymene)2(donq)Cl2] (60 mg,
0.08 mmol) reacted with AgCF3SO3 (42 mg, 0.16 mmol) in
dichloromethane (15 mL) for 2 h. After removal of the resulting
AgCl salt, tpt (16 mg, 0.05 mmol), and the pyrenyl-derivative
(M1–M3) (0.03 mmol) were added and refluxed for 15 h. The
reaction mixture was filtrated and concentrated in vacuo. The
residue was dissolved in 2 mL of dichloromethane and triturated
in diethyl ether (40 mL) to precipitate the product which was
filtered and dried in vacuo.
8.22 (2 H, d, J = 8.5 Hz, Hpy), 8.12 (2 H, AB system, Hpy),
3
3
8.07 (1 H, t, J = 7.5 Hz, Hpy), 7.98 (1 H, d, J = 7.7 Hz, Hpy),
3
3
5.47 (2 H, t, J = 5.6 Hz, Har), 5.39 (2 H, d, J = 5.6 Hz, Har),
5.00 (1 H, t, 3J = 5.6 Hz, Har), 4.45 (6 H, s, CH2NCH2), 4.18 (6
3
H, s, CH2PCH2), 3.44–3.49 (4 H, m, CH2O), 3.40 (2 H, t, J =
7.6 Hz, CH2Py), 2.42 (2 H, t, J = 7.7 Hz, CH2Ph), 1.93–1.80
3
(4 H, m, CH2), 1.72–1.65 (2 H, m, CH2). 13C{1H} NMR
(101 MHz, CDCl3, 298 K): δ (ppm) = 137.1 (Cpy), 131.5 (Cpy),
131.0 (Cpy), 129.88 (Cpy), 128.7 (Cpy), 127.7 (CHpy), 127.5
(CHpy), 127.3 (CHpy), 126.7 (CHpy), 126.1 (CHpy), 125.3
(CHpy), 125.1 (Cpy), 125.2 (CHpy), 125.0 (Cpy), 124.9 (CHpy),
123.7 (CHpy), 111.3 (d, J (P-C) = 5.0 Hz, Car), 87.5 (d, J (P-C)
= 5.8 Hz, CHar), 85.2 (CHar), 77.3 (d, J (P-C) = 6.8 Hz, CHar),
73.4 (d, 2J (P-C) = 6.6 Hz,CH2NCH2), 70.9 (CH2O), 70.2
(CH2O), 52.9 (d, 1J (P-C) = 17.5 Hz, CH2PCH2), 33.5
(CH2Py), 30.7 (CH2Ph), 29.9 (CH2CH2O), 29.3 (CH2CH2CH2),
29.0 (CH2CH2Ph). 31P{1H} NMR (161 MHz, CD3Cl, 298 K) δ
Synthesis of [M1⊂cage][CF3SO3]6. This complex was syn-
thesised according to the procedure described above using com-
pound M1 (20 mg, 0.03 mmol). Yield: 98 mg (85%). UV-vis
[1.0 × 10−5 M−1, CH2Cl2] λmax/nm (ε/M−1 cm−1) = 693
(11 147), 437 (39 967), 344 (76 141), 328 (77 825), 277
(94 127), 244 (225 063). IR (KBr): ν = 3064 (CHaryl), 2925 (s,
CH2), 1631 (s, COdonq), 1574 (s, CCtpt), 1536 (s, CNtpt), 1224
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 7201–7211 | 7209

View Article Online
(s, CO), 1275 (s, CN), 1149 (m, CO). 1H NMR (400 MHz,
CD3CN, 298 K) δ (ppm) = 8.66–8.07 (br), 7.72–7.23 (br) 7.09
(2 H, br, Hpy), 6.33 (1 H, d, 3J = 9.0 Hz, Hpy), 6.27 (1 H, d, 3J =
Synthesis of [M3⊂cage][CF3SO3]6. This complex was syn-
thesised according to the procedure described above using com-
pound M3 (20 mg, 0.03 mmol). Yield: 89 mg (78%). UV-vis
[1.0 × 10−5 M−1, CH2Cl2] λmax/nm (ε/M−1 cm−1) = 693
(11 299), 435 (42 383), 344 (79 242), 328 (82 617), 277
(95 836), 243 (242 047). IR (KBr): ν = 3064 (CHaryl), 2967 (s,
CH2), 1643 (s, [COdonq, COamide]), 1574 (s, CCtpt), 1535 (s,
CNtpt). 1H NMR (400 MHz, CD3CN, 298 K) δ (ppm) =
8.65–8.50 (br), 8.40–8.21 (br), 7.67–7.30 (br), 7.31 (1 H, d, 3J =
3
3
7.2 Hz, Hpy), 5.98 (1 H, d, J = 9.0 Hz, Hpy), 5.89 (1 H, d, J =
3
7.2 Hz, Hpy), 5.82 (2 H, d, J = 5.5 Hz, Har-g), 5.71 (12 H + 2
3
H, br, Hcym + Har-g), 5.61 (2 H, d, J = 5.5 Hz, Har-g), 5.50 (12
3
H + 1 H, br, Hcym + Hpy) 5.23 (1 H, t, J = 5.5 Hz, Har-g), 5.15
3
3
(1 H, t, J = 7.2 Hz, Hpy), 4.87 (1 H, d, J = 7.8 Hz, Hpy), 4.46
(6 H, s, CH2NCH2), 4.38 (2 H, t, CH2O), 4.27 (6 H, s,
CH2PCH2), 2.82 (6 H, sept, 3J = 6.8 Hz, CH(CH3)2), 2.70 (2 H,
3
8.6 Hz, Hpy), 7.25 (1 H, br, NH), 7.11 (1 H, d, J = 8.6 Hz,
3
3
Hpy), 6.43 (1 H, br, Hpy), 6.26 (2 H, br, Hpy), 6.01 (1 H, br,
Hpy), 5.79 (1 H, br, Hpy), 5.68 (12 H + 4 H, br, Hcym + Har-g),
5.58 (1 H, br, Hpy), 5.47 (12 H, br, Hcym) 5.36 (1 H, br, Har-g),
5.03 (1 H, br, Hpy), 4.66 (1 H, br, Hpy), 4.45 (6 H, s,
CH2NCH2), 4.27 (6 H, s, CH2PCH2), 3.55 (2 H, q, CH2NH),
t, J = 7.5 Hz, CH2Ph), 2.30 (2 H, t, J = 6.8 Hz, CH2CO),
2.23–2.12 (2 H + 2 H, m, CH2Py + CH2CH2O), 2.05 (18 H, s,
3
CH3), 1.30 (36 H, d, J = 6.8 Hz, CH(CH3)2), 1.17 (2 H, m,
CH2CH2CO). 13C{1H} NMR (101 MHz, CD3CN, 298 K) δ
(ppm) = 173.1 (COO), 170.6 (CO), 152.2 (CHα), 137.4 (CHq),
126.4 (CHpy), 126.2 (CHpy), 126.0 (CHpy), 124.1 (CHpy), 124.0
(CHpy), 123.9 (CHpy), 123.8 (CHpy), 123.6 (CHpy), 123.5
(CHβ), 121.3 (CHpy), 111.2 (Cq), 109.1 (Car-g), 103.3 (Ccym),
99.2 (Ccym), 87.0 (CHar-g), 85.7 (CHar-g), 84.0 (CHar-cym), 82.9
(CHar-cym), 77.2 (CHar-g), 72.5 (d, 2J (P-C) = 6.7 Hz,
CH2NCH2), 63.3 (CH2O), 52.5 (d, 1J (P-C) = 17.6 Hz,
CH2PCH2) 33.4 (CH2CO), 31.2 (CH2Py), 30.2 (CH(CH3)2),
28.9 (CH2Ph), 27.6 (CH2CH2O), 25.2 (CH2CH2CO), 21.1 (CH
(CH3)2), 16.3 (CH3). 31P{1H} NMR (161 MHz, CD3CN,
298 K) δ (ppm) = −33.1. ESI-MS (m/z): 1260.78 [M1 + cage +
3 CF3SO3]3+.
3
2.82 (6 H, m, CH(CH3)2), 2.71 (2 H, t, J = 7.3 Hz, CH2Ph),
2.27 (2 H, m, CH2CO), 2.16 (18 H + 2 H, br, CH3
+
CH2CH2O), 2.08 (2 H, br, CH2CH2NH), 1.30 (36 H, d, 3J = 6.7
Hz, CH(CH3)2), 0.92 (2 H, br, CH2Py). 13C{1H} NMR
(101 MHz, CD3CN, 298 K) δ (ppm) = 173.7 (CONH), 171.8
(CO), 170.7 (Ctpt), 154.1 (CHα), 143.1 (Ctpt), 138.7 (CHq),
127.5 (CHpy), 126.5 (CHpy), 126.3 (CHpy), 124.5 (CHpy), 124.4
(CHpy), 124.2 (CHpy), 124.1 (CHpy), 124.0 (CHβ), 123.7
(CHpy), 121.7 (CHpy), 112.4 (Cq), 109.1 (Car-g), 104.7 (Car-cym),
100.6 (Car-cym), 88.5 (CHar-g), 85.2 (CHar-g), 85.5 (CHar-cym),
83.8 (CHar-cym), 77.8 (CHar-g), 73.4 (d, 2J = 6.8 Hz, CH2NCH2),
1
53.3 (d, J (P-C) = 17.5 Hz, CH2PCH2) 39.0 (CH2NH), 35.9
(CH2CO) 32.0 (CH2CH2CO), 30.9 (CH(CH3)2), 30.1 (CH2Ph),
28.9 (CH2CH2NH), 22.1 (CH(CH3)2), 17.2 (CH3). 31P{1H}
NMR (161 MHz, CD3CN, 298 K) δ (ppm) = −34.6. ESI-MS
(m/z): 1260.79 [M3 + cage + 3 CF3SO3]3+.
Synthesis of [M2⊂cage][CF3SO3]6. This complex was syn-
thesised according to the procedure described above using com-
pound M2 (20 mg, 0.03 mmol). Yield: 98 mg (86%). UV-vis
[1.0 × 10−5 M−1, CH2Cl2] λmax/nm (ε/M−1 cm−1) = 692
(10 712), 640 (8 983), 437 (40 333), 345 (78 239), 328 (80 515),
277 (98 589), 244 (234 100). IR (KBr): ν = 3064 (CHaryl), 2925
(s, CH2), 1631 (s, COdonq), 1574 (s, CCtpt), 1536 (s, CNtpt),
1224 (s, CO), 1275 (s, CN), 1149 (m, CO). 1H NMR (400 MHz,
CD3CN, 298 K) δ (ppm) = 8.65–8.00 (br), 7.64–7.31 (br) 7.15
Cell culture
Human lung (A549) and cervix (HeLa) cancer cells were
obtained from the American Tissue Type Culture Collection
(Manassas, VA, USA). A2780s ovarian cancer cells were
obtained from the ECACC (Salisbury, UK). Human
Me300 melanoma cells were kindly provided by D. Rimoldi,
Ludwig Institute of Cancer Research, Lausanne branch. HeLa
and A549 cells were routinely grown in Dulbecco’s modified
Eagle’s medium containing 4.5 g L−1 glucose, while A2780, and
Me300 were grown in RPMI 1640 medium both supplemented
with 10% heat-inactivated foetal calf serum and antibiotics (all
from Gibco, Basel, Switzerland).
3
3
(1 H, d, J = 8.5 Hz, Hpy), 7.05 (1 H, d, J = 8.5 Hz, Hpy), 6.32
3
3
(1 H, d, J = 8.5 Hz, Hpy), 6.25 (1 H, d, J = 8.5 Hz, Hpy), 5.98
(2 H, br, Hpy), 5.71 (12 H, br, Hcym,), 5.50 (12 H + 2 H + 1 H,
3
br, Hcym + Har-g + Hpy), 5.37 (2 H, d, J = 5.5 Hz, Har-g), 5.21
3
(1 H, t, J = 5.5 Hz, Har-g), 5.15 (1 H, br, Hpy), 4.90 (1 H, br,
Hpy), 4.36 (6 H, s, CH2NCH2), 4.12 (6 H, s, CH2PCH2), 3.57 (2
H, t, 3J = 6.0 Hz, CH2O), 3.52 (2 H, t, 3J = 6.0 Hz, CH2O), 2.82
3
3
(6 H, sept, J = 6.8 Hz, CH(CH3)2), 2.53 (2 H, t, J = 7.5 Hz,
CH2Ph), 2.15 (2 H, m, CH2Py), 2.05 (18H + 2 H, CH3
+
3
CH2CH2Ph), 1.49 (2 H, br, CH2CH2O), 1.30 (36 H, d, J = 6.8
Hz, CH(CH3)2), 1.06 (2 H, br, CH2CH2Py). 13C{1H} NMR
(100 MHz, CD3CN, 298 K) δ (ppm) = 173.1 (COO), 152.5
(CHα), 137.4 (CHq), 127.0 (CHpy), 126.2 (CHpy), 126.1 (CHpy),
124.5 (CHpy), 124.2 (CHpy), 123. (CHpy), 123.8 (CHpy), 123.7
(CHpy), 123.3 (CHβ), 121.5 (CHpy), 111.2 (Cq), 109.6 (Car-g),
103.9 (Car-cym), 99.5 (Car-cym), 87.2 (CHar-g), 85.9 (CHar-g), 84.3
(CHar-cym), 82.8 (CHar-cym), 77.6 (CHar-g), 72.5 (CH2NCH2),
70.4 (CH2O), 69.8 (CH2O) 52.3 (CH2PCH2) 31.9 (CH2Py),
30.3 (CH(CH3)2), 29.7 (CH2Ph), 29.5 (CH2CH2O), 28.9
(CH2CH2Ph), 27.3 (CH2CH2Py), 21.5 (CH(CH3)2), 16.2 (CH3).
31P{1H} NMR (161 MHz, CD3CN, 298 K) δ (ppm) = −35.1.
ESI-MS (m/z): 1256.79 [M2 + cage + 3 CF3SO3]3+.
Evaluation of the cytotoxicity of the complexes
Cells in 48-well plates (Costar, Corning, USA) were exposed at
37 °C to increasing concentrations of complexes in complete
culture medium for 72 h (5 mM stock solution in DMSO). After
washing with phosphate-buffered saline (PBS), the supernatants
were replaced with fresh medium and cell survival was measured
using the 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium
bromide (MTT) test. MTT (Merck) was added at 250 μg mL−1
and incubation was continued for 2 h, as previously described.38
Then the cell culture supernatants were removed, the cell layer
was dissolved in isopropanol/0.04 N HCl, and the absorbance at
7210 | Dalton Trans., 2012, 41, 7201–7211
This journal is © The Royal Society of Chemistry 2012

View Article Online
13 C. Scolaro, A. B. Chaplin, C. G. Hartinger, A. Bergamo, M. Cocchietto,
B. K. Keppler, G. Sava and P. J. Dyson, Dalton Trans., 2007, 5065.
14 W. H. Ang, L. J. Parker, A. De Luca, L. Juillerat-Jeanneret, C. J. Morton,
M. Lo Bello, M. W. Parker and P. J. Dyson, Angew. Chem., Int. Ed.,
2009, 48, 3854.
540 nm was measured in a 96-well multiwell-plate reader (iEMS
Reader MF, Labsystems, Bioconcept, Switzerland) and compared
with the values of control cells incubated without complexes.
Experiments were conducted in triplicate wells and repeated
three times.
15 A. K. Renfrew, R. Scopelliti and P. J. Dyson, Inorg. Chem., 2010, 49,
2239.
16 L. Hernandez-Folgado, D. Baretić, I. Piantanida, M. Marjanović,
M. Kralj, T. Rehm and C. Schmuck, Chem.–Eur. J., 2010, 16, 3036.
17 K. M. Guckian, B. A. Schweitzer, R. X. F. Ren, C. J. Sheils, D.
C. Tahmassebi and E. T. Kool, J. Am. Chem. Soc., 2000, 122, 2213.
18 M. Baba, M. Saitoh, Y. Kowaka, K. Taguma, K. Yoshida, Y. Semba,
S. Kasahara, T. Yamanaka, Y. Ohshima, Y.-C. Hsu and S. H. Lin, J.
Chem. Phys., 2009, 131, 224318.
19 J. Mattsson, O. Zava, A. K. Renfrew, Y. Sei, K. Yamaguchi, P. J. Dyson
and B. Therrien, Dalton Trans., 2010, 39, 8248.
20 P. Govindaswamy, J. Furrer, G. Süss-Fink and B. Therrien, Z. Anorg.
Allg. Chem., 2008, 634, 1349.
21 (a) J. Mattsson, P. Govindaswamy, J. Furrer, Y. Sei, K. Yamaguchi,
G. Süss-Fink and B. Therrien, Organometallics, 2008, 27, 4346; (b) J.
W. Yi, N. P. E. Barry, M. A. Furrer, O. Zava, P. J. Dyson, B. Therrien and
B. H. Kim, Bioconjugate Chem., 2012, 23, 461.
22 N. P. E. Barry and B. Therrien, Eur. J. Inorg. Chem., 2009, 4695.
23 J. Freudenreich, N. P. E. Barry, G. Süss-Fink and B. Therrien,
Eur. J. Inorg. Chem., 2010, 2400.
24 (a) O. Zava, J. Mattsson, B. Therrien and P. J. Dyson, Chem.–Eur. J.,
2010, 16, 1428; (b) F. Schmitt, J. Freudenreich, N. P. E. Barry,
L. Juillerat-Jeanneret, G. Süss-Fink and B. Therrien, J. Am. Chem. Soc.,
2012, 134, 754.
Evaluation of the uptake of the complexes
HeLa cells were grown in 6-well cell culture plates (Costar,
Corning, USA) until 25% confluent as previously described.39
Then, 2.5 μM of compounds (1 mM stock solution in DMSO)
were added to cell medium and the cells were exposed for
various periods of time. After incubation with the compounds,
the cells were washed with PBS and harvested by enzymatic
digestion (Trypsin-EDTA). Then, the cells were washed in ice-
cold PBS using centrifugation at 100 g for 5 min and re-sus-
pended in 1.5 mL isopropanol followed by ultra-sound treatment
for 3 min. The suspension was centrifuged at 6000 g for 5 min
and the pyrenyl fluorescence of the supernatant was measured
in a spectrofluorometre (LS50, PerkinElmer) at the λexc/λem
312/378 nm, respectively.
=
25 A. Pitto-Barry, N. P. E. Barry, O. Zava, R. Deschenaux, P. J. Dyson and
B. Therrien, Chem.–Eur. J., 2011, 17, 1966.
Acknowledgements
26 A. Pitto-Barry, N. P. E. Barry, O. Zava, R. Deschenaux and B. Therrien,
Chem.–Asian J., 2011, 6, 1595.
27 Y. Matsumura and H. Maeda, Cancer Res., 1986, 46, 6387.
28 J. Hannedouche, G. J. Clarkson and M. Wills, J. Am. Chem. Soc., 2004,
126, 986.
29 R. A. Fujimoto, J. Boxer, R. H. Jackson, J. P. Simke, R. F. Neale, E.
W. Snowhill, B. J. Barbaz, M. Williams and M. A. Sills, J. Med. Chem.,
1989, 32, 1259.
A generous loan of ruthenium chloride hydrate from the Johnson
Matthey Technology Centre is gratefully acknowledged, and we
would like to thank the Swiss National Science Foundation for
financial support (grant 200020-129518). F.S. is financially sup-
ported by the Swiss Commission for Technology and Innovation
CTI (grant no. 10550.2 PFLS-LS).
30 N. Sakai, T. Moriya and T. Konakahara, J. Org. Chem., 2007, 72,
5920.
31 H. L. Anderson, S. Anderson and J. K. M. Sanders, J. Chem. Soc.,
Perkin Trans. 1, 1995, 2231.
References
1 C. G. Hartinger, S. Zorbas-Seifried, M. A. Jakupec, B. Kynast, H. Zorbas
and B. K. Keppler, J. Inorg. Biochem., 2006, 100, 891.
2 M. A. Jakupec, M. Galanski, V. B. Arion, C. G. Hartinger and
B. K. Keppler, Dalton Trans., 2008, 183.
3 C. G. Hartinger and P. J. Dyson, Chem. Soc. Rev., 2009, 38, 391.
4 G. Süss-Fink, Dalton Trans., 2010, 39, 1673.
5 G. S. Smith and B. Therrien, Dalton Trans., 2011, 40, 10793.
6 W. H. Ang and P. J. Dyson, Eur. J. Inorg. Chem., 2006, 2006, 4003.
7 S. J. Dougan and P. J. Sadler, Chimia, 2007, 61, 704.
8 P. J. Dyson, Chimia, 2007, 61, 698.
9 C. S. Allardyce, P. J. Dyson, D. J. Ellis and S. L. Heath, Chem.
Commun., 2001, 1396.
10 (a) C. Scolaro, A. Bergamo, L. Brescacin, R. Delfino, M. Cocchietto,
G. Laurenczy, T. J. Geldbach, G. Sava and P. J. Dyson, J. Med. Chem.,
2005, 48, 4161; (b) B. Wu, M. S. Ong, M. Groessl, Z. Adhireksan, C.
G. Hartinger, P. J. Dyson and C. A. Davey, Chem.–Eur. J., 2011, 17,
3562.
11 A. Bergamo, A. Masi, P. J. Dyson and G. Sava, Int. J. Oncol., 2008, 33,
1281.
12 C. Scolaro, T. J. Geldbach, S. Rochat, A. Dorcier, C. Gossens,
A. Bergamo, M. Cocchietto, I. Tavernelli, G. Sava, U. Rothlisberger and
P. J. Dyson, Organometallics, 2006, 25, 756.
32 C. S. Johnson, Prog. Nucl. Magn. Reson. Spectrosc., 1999, 34, 203.
33 Chem3D Pro 11.0 for PC, CambridgeSoft, Cambridge, MA.
34 H. M. Colquhoun, Z. Zhu, D. J. Williams, M. G. B. Drew, C.
J. Cardin, Y. Gan, A. G. Crawford and T. B. Marder, Chem.–Eur. J.,
2010, 16, 907.
35 N. Bouquin, V. L. Malinovskii and R. Haner, Chem. Commun., 2008,
1974.
36 (a) C. A. Vock, W. H. Ang, C. Scolaro, A. D. Phillips, L. Lagopoulos,
L. Juillerat-Jeanneret, G. Sava, R. Scopelliti and P. J. Dyson, J. Med.
Chem., 2007, 50, 2166; (b) W. H. Ang, A. De Luca, C. Chapuis-Bernas-
coni, L. Juillerat-Jeanneret, M. Lo Bello and P. J. Dyson, ChemMedChem,
2007, 2, 1799; (c) A. Casini, F. Edafe, M. Erlandsson, L. Gonsalvi,
A. Ciancetta, N. Re, A. Ienco, L. Messori, M. Peruzzini and P. J. Dyson,
Dalton Trans., 2010, 39, 5556.
37 R. A. Zelonka and M. C. Baird, Can. J. Chem., 1972, 50, 3063.
38 F. Schmitt, M. Auzias, P. Štěpnička, Y. Sei, K. Yamaguchi, G. Süss-Fink,
B. Therrien and L. Juillerat-Jeanneret, JBIC, J. Biol. Inorg. Chem., 2009,
14, 693.
39 F. Schmitt, L. Lagopulos, P. Käuper, N. Rossi, N. Busso, J. Barge,
G. Wagnières, C. Laue, C. Wandrey and L. Juillerat-Jeanneret, J. Con-
trolled Release, 2010, 144, 242.
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 7201–7211 | 7211