Use of perfluorinated phosphines to provide thermomorphic anticancer complexes for heat-based tumor targeting

Article abstract of DOI:10.1021/ic9020433

A series of compounds of general formula [Ru(η⁶-arene)(pta) (PR₃)Cl]BF₄ (arene = p-cymene or 4-phenyl-2-butanol; pta = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane, PR₃ = PPh ₂(p-C₆H₄C₂H₄C ₈F₁₇), PPh(p-C₆H₄C₂H ₄C₈F₁₇)₂, P(pC₆H ₄C₂H₄C₆F₁₃)₃, PPh₃ or P(p-C₆H₄F)₃) have been prepared and characterized by spectroscopic methods. The structure of [Ru(η⁶-p-cymene)(pte)Cl(P(p-C₆H₄F) ₃)]BF4 has also been established in the solid state by X-ray crystallography. The cytotoxicities of the compounds were determined in the A2780 and A2780 cisplatin-resistant cell lines revealing that the fluorinated phosphines significantly increase antiproliferative activity relative to their bis-chloride precursors. Two of the complexes were found to be thermoresponsive, that is, showing poor water solubility at 37 °C and good solubility at 42 °C, the temperature of a heated tumor, providing a method of tumor targeting. Incubation at 42 °C for 2 h resulted in improved cytotoxicities for two of the complexes.

Full text of DOI:10.1021/ic9020433

Inorg. Chem. 2010, 49, 2239–2246 2239
DOI: 10.1021/ic9020433
Use of Perfluorinated Phosphines to Provide Thermomorphic Anticancer
Complexes for Heat-Based Tumor Targeting
Anna K. Renfrew, Rosario Scopelliti, and Paul J. Dyson*
ꢀ
ꢀ ꢀ
Institut des Sciences et Ingenierie Chimiques, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015
Lausanne, Switzerland
Received October 15, 2009
A series of compounds of general formula [Ru(η6-arene)(pta)(PR3)Cl]BF4 (arene = p-cymene or 4-phenyl-2-butanol;
pta = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane, PR3 = PPh2(p-C6H4C2H4C8F17), PPh(p-C6H4C2H4C8F17)2, P(p-
C6H4C2H4C6F13)3, PPh3 or P(p-C6H4F)3) have been prepared and characterized by spectroscopic methods. The
structure of [Ru(η6-p-cymene)(pta)Cl(P(p-C6H4F)3)]BF4 has also been established in the solid state by X-ray
crystallography. The cytotoxicities of the compounds were determined in the A2780 and A2780 cisplatin-resistant cell
lines revealing that the fluorinated phosphines significantly increase antiproliferative activity relative to their bis-chloride
precursors. Two of the complexes were found to be thermoresponsive, that is, showing poor water solubility at 37 °C
and good solubility at 42 °C, the temperature of a heated tumor, providing a method of tumor targeting. Incubation at
42 °C for 2 h resulted in improved cytotoxicities for two of the complexes.
Introduction
based drugs, KP10193 and NAMI-A,4 have currently com-
pleted phase I clinical trials and are in, or set to enter, phase II
trials. Both complexes behave quite differently to cisplatin in
vivo; NAMI-A has been shown to be a strong inhibitor of
metastasis while having little effect on primary tumors,5
whereas KP1019 effectively reduces colorectal tumors where
cisplatin shows limited activity.6
Organometallic compounds are also attracting attention as
anticancer drugs.7 In this context organoruthenium(II) com-
pounds are promising,8 following recent studies that show they
can react with biologically relevant targets.9 We have been
developing a series of organometallic ruthenium (RAPTA)
compounds (Chart 1) with an η6-arene ligand, a monodentate
1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (pta) ligand
and, in most cases, two labile chloride ligands (Figure 1).
These compounds tend to exhibit low general toxicity in vivo,
and depending on the particular compound and tumor
model, are active against metastatic10 and primary tumors.11
One of the main goals in the field of inorganic (or
organometallic) antitumor drugs is to overcome the two
major limitations of cisplatin, namely, poor selectivity and
a high incidence of drug resistance. From the many metal-
based drugs that have been developed, ruthenium has
emerged as an attractive alternative to platinum because of
its excellent activity in tumors where cisplatin is of limited use
and because of its low general toxicity.1,2 Two ruthenium(III)
*To whom correspondence should be addressed. E-mail: paul.dyson@epfl.ch.
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Baker, E. N. J. Bio. Inorg. Chem. 1996, 1, 424. (b) Hartinger, C. G.; Jakupec,
M. A.; Zorbas-Seifried, S.; Groessl, M.; Egger, A.; Berger, W.; Zorbas, H.; Dyson,
P. J.; Keppler, B. K. Chem. Biodiversity 2008, 5, 2140.
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Zorbas, H.; Keppler, B. K. J. Inorg. Biochem. 2006, 100, 891.
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J. Inorg. Chem. 2007, 2803. (b) Schmid, W. F.; John, R. O.; Arion, V. B.; Jakupec,
M. A.; Keppler, B. K. Organometallics 2007, 26, 6643. (c) Habtemariam, A.;
Melchart, M.; Fernandez, R.; Parsons, S.; Oswald, I. D. H.; Parkin, A.; Fabbiani,
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ꢀ
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Eur. J. 2009, 15, 684. (b) Top, S.; Thibaudeau, C.; Vessieres, A.; Brule, E.; Le
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r
2010 American Chemical Society
Published on Web 02/04/2010
pubs.acs.org/IC

2240 Inorganic Chemistry, Vol. 49, No. 5, 2010
Chart 1. Examples of RAPTA-Type Compounds
Renfrew et al.
The nature of the ligand sphere greatly influences the in
vitro activity of the complexes. Increasing hydrophilicity
through hydrogen bonding substituents on the arene ligand
was shown to reduce both cellular uptake and cytotoxicity.12
Accordingly, the replacement of a chloride ligand with
hydrophobic PPh3 was found to increase cellular uptake
and consequently cytotoxicity, but also resulted in increased
toxicity toward a non-tumorigenic cell line,13 which in turn
could be linked to an increased reactivity toward DNA and a
decrease in protein affinity. In related work, Romerosa et al.
demonstrated that binding of [Ru(η5-C5Me5)(pta)Cl2] to
supercoiled DNA is greatly increased on replacement of
the pta ligand by triphenylphospine or triphenylphosphine
monosulfonate,14,15 and a similar effect has been reported for
Pt complexes,16 although the influence on general toxicity
and protein binding was not investigated.
While the increase in cell uptake and consequently in
cytotoxicity shown by the PPh3-derivatized compounds is
promising, it would be necessary to improve selectivity to
reduce potential damage to healthy cells. A number of
strategies have been employed in the design of metal-based
drugs aimed at producing a complex which is preferentially
active in the tumor environment;17,18 here we investigate the
possibility of introducing selectivity through temperature-
controlled solubility.
Figure 1. ORTEP representation of 8b. Ellipsoids are drawn at the 50%
probability level and the BF4- counteranion has been omitted for clarity.
˚
Key bond lengths (A) and angles (deg) include: Ru1-Cl1 2.3819(6),
Ru1-Cl1 2.3165(6), Ru1-P2 2.3424(6), Ru1-Cavg 2.26(2), Cl1-Ru1-
P1 82.19, Cl1-Ru1-P2 87.94(2), P1-Ru1-P2 95.43(2).
cytotoxicity when used with oxaliplatin or nucleoside ana-
logue gemcitabine, no interaction occurs with 5-fluorouracil
or taxane.21 In contrast, a synergistic effect is observed when
the treatment is applied with cisplatin, carboplatin, or alky-
lating agents such as cyclophosphamide and mephalan, with
the degree of synergism increasing as the cell temperature is
raised from 40.5-43.5 °C, although these compounds were
not designed to be thermoresponsive.21 The reason for this
effectis not clearly understood, but for cisplatin itis proposed
to be due to increased DNA platination.19 Exploiting the
difference in temperature between a tumor cell and healthy
cell, a number of specifically designed thermoresponsive
macromolecules have been investigated for application with
thermotherapy. Several types of liposomes have been deve-
loped for the release of chemotherapeutic agents at the heated
tumor temperature,22 and doxorubicin containing liposomes
have entered clinical trials.23 In a different strategy, certain
peptides or proteins become hydrophobic above 40 °C and
aggregate in the heated tumor cells, allowing increased
accumulation of a conjugated drug.24
Phosphines functionalized with perfluorinated chains are
known for their thermomorphic properties with a significant
increase in solubility observed on raising the temperature.25
With the aim of developing thermomorphic complexes, that
is, complexes that show increased solubility at a heated
tumor, a series of RAPTA-type compounds were prepared
that incorporate fluorophosphine ligands. As far as we are
aware these compounds represent the first examples of
thermomorphic small molecule-based drugs.
Thermotherapy is a treatment in which a heat source is
applied to the tumor area causing an increase in temperature.
The compact, disordered structure of tumor cells makes them
less effective at dissipating heat than healthy cells and as
result, the cells become weakened and more sensitive to
chemotherapy.19 A number of chemotherapeutic agents
have been tested in conjunction with thermotherapy, both
in vitro and in vivo, and the results found to vary greatly
between compounds.20 While hyperthermia leads to additive
(12) Scolaro, C.; Geldbach, T. J.; Rochat, S.; Dorcier, A.; Gossens, C.;
Bergamo, A.; Cocchietto, M.; Tavernelli, I.; Sava, G.; Rothlisberger, U.;
Dyson, P. J. Organometallics 2006, 25, 756.
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M.; Keppler, B. K.; Sava, G.; Dyson, P. J. Dalton Trans. 2007, 5065.
(14) Romerosa, A.; Campos-Malpartida, T.; Lidrissi, C.; Saoud, M.;
Serrano-Ruiz, M.; Peruzzini, M.; Garrido-Cardenas, J. A.; Garcıa-Maroto,
F. Inorg. Chem. 2006, 45, 1289.
(15) Romerosa, A.; Saoud, M.; Campos-Malpartida, T.; Lidrissi, C.;
Serrano-Ruiz, M.; Peruzzini, M.; Garrido-Cardenas, J. A.; Garcıa-Maroto,
F. Eur. J. Inorg. Chem. 2007, 18, 2803.
(16) (a) McCaffrey, L. J.; Henderson, W.; Nicholson, B. K.; Mackay,
J. E.; Dinger, M. B. Dalton Trans. 1997, 2577. (b) McCaffrey, L. J.; Henderson,
W.; Nicholson, B. K. Dalton Trans. 2000, 2753.
(17) (a) Kelland, L. R.; Abel, G.; McKeage, M. J.; Jones, M.; Goddard,
P. M.; Valenti, M.; Murrer, B. A.; Harrap, K. R. Cancer Res. 1993, 53, 2581.
(b) Failes, T. W.; Cullinane, C.; Diakos, C. I.; Yamamoto, N.; Lyons, J. G.;
Hambley, T. W. Chem.;Eur. J. 2007, 13, 2974.
(18) Galanski, M.; Baumgartner, C.; Meelich, K.; Arion, V. B.; Fremuth,
M.; Jakupec, M. A.; Schluga, P.; Hartinger, C. G.; von Keyserlingk, N. G.;
Keppler, B. K. Inorg. Chim. Acta 2004, 357, 3237.
(21) Urano, M.; Kuroda, M.; Nishimura, Y. Int. J. Hyperthermia 1999,
15, 79.
(22) (a) Needham, D.; Anyarambhatla, G.; Kong, G.; Dewhirst, M. W.
Cancer Res. 2000, 60, 1197. (b) Lindner, L. H.; Eichhorn, M. E.; Eibl, H.;
Teichert, N.; Schmitt-Sody, M.; Issels, R. D.; Dellian, M. Clin. Cancer Res. 2004,
10, 2168.
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P.; Prosnitz, L.; Kimmick, G.; Blackwell K. (Abstract) In: 10th international
congress of hyperthermic oncology, Munich, Germany, 2008.
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J. Am. Chem. Soc. 2003, 19, 5861.
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Article
Inorganic Chemistry, Vol. 49, No. 5, 2010 2241
Chart 2. Structures of Compounds 1a-9a (top) and 1b- 9b (bottom)
shift presumably because of the insulation provided by the
ethylene spacer.
Structural Characterization of 8b in the Solid State.
Single crystals of 8b suitable for X-ray diffraction were
obtained via slow diffusion (see Experimental Section for
details), and the resulting X-ray structure is shown in
Figure 1. The ruthenium(II) compound exhibits the
piano-stool geometry typical of half-sandwich complexes
and is very similar to that of [Ru(η6-cymene)(pta)-
(PPh3)Cl]BF4, although the Ru-P(C6H4F)3 bond length
13
˚
is slightly shorter [2.3424(6) vs 2.3590(14) A]. Other-
wise, the Ru-arene, Ru-Cl, and Ru-pta bond lengths and
angles are similar to those of RAPTA-C26 and other
RAPTA derivatives.27
Behavior in Aqueous Solution. The hydrolysis of the
water-soluble compound, that is, 4b, was studied by 31P
NMR spectroscopy. Hydrolytic decomposition is central
to the mode of action of cisplatin and is also thought to be
relevant for mono and dichloro ruthenium(II)-arene
based drugs which undergo rapid hydrolysis in water
containing 5 mM NaCl (the approximate salt concentra-
tion inside a cell).28 Hydrolysis is reversed by addition of
100 mM NaCl (corresponding to the salt concentration of
blood plasma). Hydrolysis of RAPTA compounds with
two Cl-ligands occurs readily with the major species
formed being [Ru(η6-arene)(pta)Cl(H2O)]þ. In D2O the
31P NMR spectrum of 4b displays two doublets at 31.9
and -43.7 ppm corresponding to the fluorous phosphine
and pta ligands, respectively. However, following about
5 h of incubation at 37 °C, two new doublets are also
present at 30.4 and -41.2 and are thought to represent the
hydrolysis product, [Ru(η6-4-phenyl-2-butanol)(H2O)-
(pta)(PPh2(p-C6H4C2H4C8F17)]2þ. The new peaks dis-
appear on addition of 100 mM NaCl, and no further
change is observed following 7 days of incubation.
The solubility of phosphines with fluourous alkyl chains
is known to have a strong dependence on temperature.
Gladysz et al. reported a 600-fold increase in the solubility
of P(C2H4C8F17)3 in octane as the temperature changes
from -20 to 80 °C.25 The solubility of 1b-9b in water was
measured over the temperature range 20-45 °C (Figure 2)
with 2b, 3b, 6b, and 8b being insoluble across the entire
temperature range (see Experimental Section for details).
Complexes 4b, 7b, and 9b show moderate solubility at
room temperature (2.5-5 mM), and while the solubility
of 7b and 9b changes only slightly over the temperature
range, the solubility of 4b increases by approximately an
order of magnitude on raising the temperature by 25 °C.
At 37 °C 4b is already soluble to a concentration 3 orders
of magnitude greater than its IC50 concentration (see
below), limiting its potential for heat-based tumor target-
ing. In contrast, 1b and 5b, which are scarcely soluble at
Results and Discussion
Ruthenium(II)-arene complexes with fluorophosphine
ligands 1-6, 8, and 9 (Chart 2) were prepared using the
two-step process shown in Scheme 1. Reaction of the appro-
priate dimers, [Ru(η6-p-cymene)Cl2]2 or [Ru(η6-4-phenyl-2-
butanol)Cl2]2, with a stoichiometric amount of the fluoro-
phosphine ligands, PR3, affords the dichloride complexes
[Ru(η6-arene)(PR3)Cl2] 1a-5a, 8a, and 9a in good yield.
Subsequent reaction of 1a-5a with an excess of NH4BF4,
followed by dropwise addition of the amphiphilic phosphine
pta, results in the formation of the monocationic complexes
[Ru(η6-arene)(pta)(PR3)Cl]BF4 1b-5b. Addition of pta fol-
lowed by the fluorophosphine is also possible, but the yield of
the final product is considerably lower. All complexes show
good solubility in fluorobenzene and chlorinated solvents
and moderate solubility in polar solvents. Complexes 1b-3b
are insoluble in water, whereas the 4-phenyl-2-butanol ana-
logues 4b and 5b are more soluble in polar solvents, and 4b is
also water-soluble. To evaluate the influence of the fluorous
chain on solubility, complexes [Ru(η6-4-phenyl-2-butanol)-
(pta)(PPh3)Cl]BF4 7b, [Ru(η6-p-cymene)(pta)(P(C6H4F)3)-
Cl]BF4 8b, and [Ru(η6-4-phenyl-2-butanol)(pta)(P(C6H4F)3)-
Cl]BF4 9b were prepared for comparison purposes. All
compounds are soluble in chlorinated and polar solvents,
and 7b and 9b are also water-soluble. Compounds with the
p-cymene ligand, that is, 1b-3b and 8b, are obtained as
enantiomers that are indistinguishable by NMR spectro-
scopy. Compounds containing the chiral 4-phenyl-2-butanol
ligand, 4b-7b and 9b, were obtained as two diastereoisomers
that give rise to two sets of peaks in the 31P NMR spectra
in CDCl3, and the isomers were not separated and were
evaluated as a mixture.
All compounds were characterized by H and 31P NMR
1
spectroscopy and electrospray ionization mass spectrometry
(ESI-MS). Table 1 lists the 31P NMR spectroscopic data of
1a-5a and 7a-9a in CDCl3, each of which exhibits a singlet
due to the fluorophosphine ligand. As expected, compounds
1b-9b, that contain a fluorophosphine (or PPh3 in the case of
7b) and pta ligand, display two characteristic doublets
resulting from coupling between the two 31P nuclei. In the
case of 4b-7b and 9b, two diasteroisomers are present in the
31P NMR spectra as two sets of doublets at a ratio of
approximately 1:1, Table 1 describes both isomers. The
number and size of the electron-withdrawing fluorous chains
on the phosphine ligand has little influence on the chemical
(26) Allardyce, C. S.; Dyson, P. J.; Ellis, D. J.; Heath, S. L. Chem.
Commun. 2001, 1396.
(27) (a) Ang, W. H.; Parker, L. J.; De Luca, A.; Juillerat-Jeanneret, L.;
Morton, C. J.; Lo Bello, M.; Parker, M. W.; Dyson, P. J. Angew. Chem., Int.
Ed. 2009, 48, 3854–3857. (b) Casini, A.; Gabbiani, C.; Sorrentino, F.; Rigobello,
M. P.; Bindoli, A.; Geldbach, T. J.; Marrone, A.; Re, Nazzareno, N.; Hartinger,
C. G.; Dyson, P. J.; Messori, L. J. Med. Chem. 2008, 51, 6773. (c) Dorcier, A.; Ang,
W. H.; Bolano, S.; Gonsalvi, L.; Juillerat-Jeannerat, L.; Laurenczy, G.; Peruzzini,
M.; Phillips, A. D.; Zanobini, F.; Dyson, P. J. Organometallics 2006, 25, 4090.
(28) (a) Wang, F.; Chen, H.; Parsons, S.; Oswald, I. D. H.; Davidson, J. E.;
Sadler, P. J. Chem.;Eur. J. 2003, 9, 5810. (b) Scolaro, C.; Hartinger, C. G.;
Allardyce, C. S.; Keppler, B. K.; Dyson, P. J. J. Inorg. Biochem. 2008, 102, 1743.

2242 Inorganic Chemistry, Vol. 49, No. 5, 2010
Scheme 1. General Synthesis of 1-9
Renfrew et al.
Table 1. 31P NMR Data for 1-9 in CDCl3
complex
δ 31P (PR3)
δ 31P (pta)
1a
2a
3a
4a
5a
7a
8a
9a
1b
2b
3b
4b
24.2
23.6
23.6
28.0
27.3
27.7
23.0
24.6
30.0 (2JPP = 52 Hz)
29.7 (2JPP = 55 Hz)
29.8 (2JPP = 55 Hz)
31.3 (2JPP = 54 Hz)
31.4 (2JPP = 54 Hz)
30.8 (2JPP = 53 Hz)
30.9 (2JPP = 54 Hz)
29.2 (2JPP = 54 Hz)
29.4 (2JPP = 54 Hz)
30.4 (2JPP = 53 Hz)
30.5 (2JPP = 53 Hz)
28.8 (2JPP = 54 Hz)
30.3 (2JPP = 54 Hz)
30.5 (2JPP = 53 Hz)
-42.7 (2JPP = 54 Hz)
-43.1 (2JPP = 55 Hz)
-43.3 (2JPP = 55 Hz)
-40.4 (2JPP = 55 Hz)
-40.5 (2JPP = 54 Hz)
-41.4 (2JPP = 54 Hz)
-41.5 (2JPP = 54 Hz)
-42.0 (2JPP = 54 Hz)
-42.2 (2JPP = 55 Hz)
-42.5 (2JPP = 54 Hz)
-42.7 (2JPP = 54 Hz)
-42.5 (2JPP = 54 Hz)
-41.0 (2JPP = 54 Hz)
-41.2 (2JPP = 54 Hz)
5b
6ba
7b
8b
9b
a Recorded in d6-DMSO.
37 °C (40-80 μM), show a 4-fold increase in solubility at
42 °C, and therefore represent much better candidates for
thermal chemotherapy.
In Vitro Anticancer Activity at 37 °C. The in vitro
cytotoxicity of the compounds was evaluated using the
MTT assay which estimates mitochondrial dehydrogen-
ase activity as an indication of cell viability. The com-
pounds were incubated at various concentrations in the
ovarian cancer cell line A2780 and the cisplatin-resistant
variant, A2780cisR, and cell viability measured after 72 h
incubation. Each experiment was performed in triplicate,
repeated twice, and the IC50 values listed in Table 2
were calculated as an average over the two experiments.
Because of the poor aqueous solubility, compounds were
added to medium as DMSO solutions, to give a final
concentration of 0.5% DMSO. Reliable IC50 values could
not be obtained for compounds 2a, 3a, 3b, and 6b as the
complexes formed an oil or precipitated when added to
the cell medium.
Related ruthenium(II)-arene-pta compounds investi-
gated for in vitro activity are generally found to have
low antiproliferative activity in vitro, but nevertheless
display excellent in vivo activities, with the pta ligand
being fundamental to the cytotoxic effect.10,11,29 The
IC50 values of the new RAPTA complexes with fluoro-
phosphine ligands are consistently lower than those of
the dichloro-pta complexes, being up to 2 orders of
magnitude more active. Interestingly, the pta ligand
has little effect on cytotoxicity with the activity of pta
Figure 2. (top) Water solubility ofthe (sparingly) soluble complexes as a
function of temperature; plot of 4b fitted to y = y0 þ Aex/b. (bottom)
Water solubility of thermomorphic complexes 1b and 5b as a function of
temperature.
complexes 1b-9b being very close to that of the ana-
logous dichloro complexes 1a-9a. The nature of the
fluorophosphine ligand, however, appears to have a
considerable influence on the in vitro activity. While
the cytotoxicity of 7b is relatively low (IC50 in A2780 =
184 μM), the analogous mono fluorous-chain complex,
4b shows very good activity (IC50 in A2780 = 6 μM). It
seems unlikely that the cytotoxic effect is due to release
of the fluorophosphine ligand as NMR spectroscopy
indicates that the ligand remains coordinated to the
ruthenium center even after 1 week in solution, and
moreover, the free ligand was found to be completely
inactive. As the fluorous chain has little electronic effect
at the metal, it is probable that the increase in cytotoxi-
city is due to improved uptake resulting from the hydro-
phobic fluorophosphine ligand.
Compound 1b is the most active of the series, with a
cytotoxicity equivalent to cisplatin in the A2780 cells and
(29) Bergamo, A.; Masi, A.; Dyson, P. J.; Sava, G. Int. J. Oncol. 2008, 33,
1281.

Article
Inorganic Chemistry, Vol. 49, No. 5, 2010 2243
Table 2. IC50 Values of the Complexes Determined in the A2780 and A2780cisR
Cell Lines
Table 3. IC50 Values in μM of Complexes Determined in the A2780 (above) and
A2780cisR Cell Lines (below) at 37 and 42 °C
compound
A2780/μM
A2780cisR/μM
compound
A2780 (37 °C)
A2780 (42 °C)
PPh2(C6H4C2H4C8F17
)
>400
>400
1b
4b
3 ( 0.2
11 ( 2
2 ( 0.3
9 ( 1
1a
4a
5a
8a
9a
1b
2b
4b
5b
7b
8b
9b
16 ( 2
6 ( 0.5
>200
25 ( 2
11 ( 2
>200
compound
A2780cisR (37 °C)
A2780cisR (42 °C)
77 ( 2
63 ( 5
1.5 ( 0.3
87 ( 6
6 ( 1.5
97 ( 18
184 ( 12
73 ( 2
41 ( 4
>200
1b
4b
3 ( 0.2
14 ( 3
2 ( 0.3
9 ( 1.5
118 ( 7
5 ( 0.5
108 ( 3
13 ( 1
>200
>200
172 ( 6
78 ( 3
trend. It is noteworthy that the incorporation of fluoro-
phosphine ligands in combination with an amphiphilic
phosphine (pta) into the ruthenium(II)-arene moiety
tends to increase antiproliferative activity in the A2780
and A2780cisR cell lines with respect to RAPTA-type
compounds that contain pta alone. The temperature-
dependent solubility of 1b and 4b in water (thermo-
morphic behavior) and modest temperature dependent
in vitro activity, which provides a way of selectively
targeting tumor tissue in vivo by the application of heat
at the tumor site, is quite encouraging. Such a targeting
method is currently undergoing evaluation using macro-
molecular thermomorphic materials. As far as we are
aware, the compounds described herein represent the first
examples deliberately designed of small molecules where
such a strategy has been applied, although small molecule
alkylating agents, including cisplatin, that do not possess
thermoresponsive properties have been evaluated in
thermotherapy.21 While it is likely that the ideal com-
pound has yet to be prepared and evaluated, this app-
roach could prove to be important in the design of future
metal based drugs and potentially small organic mole-
cules with thermomorphic properties. This novel app-
roach also offers a new dimension to the use of fluorine-
containing compounds in medicinal chemistry, where
F-atoms or CF3 groups are usually used to modify the
metabolism of a drug.34
a superior cytotoxicity in the resistant cell line.30 To
exclude the possibility of a DMSO effect, 4b was tested
in both pure medium and a DMSO solution but negligible
difference was found between the resulting values. Typi-
cally, IC50 values determined for the cisplatin resistant
line are about 2-fold higher than for the standard A2780
cells. By comparison, cisplatin is around 20 times less
active in the resistant line.30 Cisplatin resistance in
A2780cisR cells has been attributed to reduced uptake
into the cell,31 increased DNA repair,32 and increased
levels of glutathione transferases and other detoxification
proteins,33 which implies that the mode of uptake and
action of these ruthenium(II)-arene compounds is mark-
edly different to that of cisplatin.
In Vitro Activity at 42 °C. The in vitro activities of
compounds 1b, 4b, and 5b (having shown temperature
dependent solubility in water) were further studied in the
absence of DMSO at 37 and 42 °C. According to an
established procedure,19 the cells were incubated with
various concentrations of the compounds at 42 °C for
2 h, then for a further 72 h at 37 °C. Under these
conditions, negligible reduction of cell viability between
standard and heated cells was observed in control experi-
ments (without the addition of a compound). In both the
A2780 and cisplatin resistant line, 1b and 4b show an
increase in cytotoxicity following the procedure that
includes the 2 h exposure to a temperature of 42 °C
(Table 3). Compound 1b proved to be more active
following 2 h exposure at 42 °C in both cell lines. The
difference in A2780 cells was less marked for 4b, though
still significant in the resistant cell line. The high IC50
value of 5b in the dilution including DMSO (97 μM, see
Table 1) made it impossible to prepare an aqueous
solution or even a suspension at a relevant concentration,
and therefore reliable IC50 values could not be obtained.
The greater cytotoxicity of 1b and 4b at elevated
temperatures is likely to be due to their increased solubi-
lity at the higher temperature, facilitating availability
within the cell medium and ultimately within the cell itself
following uptake. However, at this stage it would be
unwise to rule out other phenomena for the observed
Experimental Section
All manipulations were carried out using standard Schlenk
techniques with all solvents degassed prior to use. The ruthe-
nium dimers, [Ru(η6-cymene)Cl2]2 and [Ru(η6-4-phenyl-2-
butanol)Cl2]2, pta, and 8a were synthesized according to
literature procedures.12,35-37 Fluorophosphine ligands were
purchased from Fluorous Technologies (Pittsburgh, U.S.A.).
1H and 31P NMR spectra were recorded on a Bruker Avance
DPX spectrometer at room temperature. Ho, Hm, and Hp refer
to aromatic protons ortho-H, meta-H, and para-H, respec-
tively. ESI-MS of the complexes were obtained on a Thermo-
finigan LCQ Deca XP Plus quadrupole ion trap instrument set
in positive mode (solvent: methanol; flow rate: 5 μL/mn; spray
voltage: 5 kV; capillary temperature: 100 °C; capillary voltage:
20 V), as described previously.38 Melting points were obtained
on a Stuart Scientific SMP3 melting point apparatus.
Synthesis of [Ru(η6-p-cymene)Cl2(PPh2(p-C6H4C2H4C8F17))]
1a. [Ru(η6-p-cymene)Cl2]2 (100 mg, 0.18 mmol) and PPh2(p-C6H4-
C2H4C8F17) (260 mg, 0.36 mmol) were refluxed in chloroform
(34) Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Chem. Soc.
Rev. 2008, 320.
(35) (a) Zelonka, R. A.; Baird, M. C. Can. J. Chem. 1972, 50, 3063. (b)
Bennett, M. A.; Smith, A. K. Dalton Trans. 1974, 233.
(36) Daigle, D. J.; Pepperman, A. B.; Vail, S. L. J. Heterocycl. Chem.
1974, 11, 407.
ꢁ
€
ꢁ
ꢁ
(30) Auzias, M.; Therrien, B.; Suss-Fink, G.; Stepnicka, P.; Ang, W. H.;
Dyson, P. J. Inorg. Chem. 2008, 47, 578.
(31) Ohmichi, M.; Hayakawa, J.; Tasaka, K.; Kurachi, H.; Murata, Y.
Trends Pharmacol. Sci. 2005, 26, 113.
(32) Masuda, H.; Ozols, R. F.; Lai, G.-M.; Fojo, A.; Rothenberg, M.;
Hamilton, T. C. Cancer Res. 1988, 48, 5713.
(37) Serron, S. A.; Nolan, S. P. Organometallics 1995, 14, 4611.
(38) Dyson, P. J.; McIndoe, J. S. Inorg. Chim. Acta 2003, 354, 68.
(33) Safaei, R. Cancer Lett. 2006, 234, 34.

2244 Inorganic Chemistry, Vol. 49, No. 5, 2010
Renfrew et al.
(50 mL) for 8 h. The solvent volume was reduced in vacuo to
about 10 mL, and the product precipitated with hexane (10 mL).
The resulting red powder was filtered and washed with hexane
(2 ꢀ 20 mL). Yield; 244 mg (67%).
[Ru(η6-4-phenyl-2-butanol)Cl(PPh(p-C6H4C2H4C8F17)2]þ (70%),
m/z = 1170.7 [HO(PPh(p-C6H4C2H4C8F17)2)]þ (100%).
Ru(η6-4-phenyl-2-butanol)Cl2(PPh3)] 7a. [Ru(η6-4-phenyl-2-
butanol)Cl2]2 (100 mg, 0.16 mmol) and PPh3 (84 mg, 0.32
mmol).Yield; 155 mg (83%), red powder.
1H NMR (CDCl3): δ 7.79-7.81 (m, 6H, PPh2Ar), 7.42-7.44
3
(m, 8H, PPh2Ar), 5.22 (d, JHH = 5.6 Hz, 2H, Hm) 5.03 (d,
1H NMR (CDCl3): δ 7.32-7.56 (m, 6H, PPh3), 6.97-7.24
3
3JHH = 6.0 Hz, 2H, Ho), 2.96 (t, JHH = 6.1 Hz, 2H,
(m, 6H, PPh3), 5.66 (d, 3JHH = 5.0 Hz, 2H, Hm), 5.43 (d, 3JHH
=
i
PhCH2CH2C8F17), 2.77-2.79 (m, 1H, Pr), 2.40-2-48 (m,
3.6 Hz, 2H, Ho) 4.75 (d, 1H, Hp), 2.56-2.72 (m, 1H, HCOH),
2.10 (s, 3H arene-CH3), 1.62-1.68 (m, 2H, arene-CH2CH2),
2H, PhCH2CH2C8F17), 1.89, (s, 3H, arene-CH3), 1.13 (d,
3JHH = 6.5 Hz, 6H, CH(CH3)2). 31P NMR (CD2Cl2): δ 24.2
(s, PPh2Ar). ESI-MS (acetone) m/z = 1036.2 [Ru(η6-p-cymene)-
Cl2(PPh2Ph(C2H4C8F17))]Naþ (90%), m/z = 977.6 [Ru(η6-p-
cymene)Cl(PPh2(p-C6H4C2H4C8F17))]þ (75%), m/z = 725.1
[HOPPh2(p-C6H4C2H4C8F17)]þ (100%).
3
1.26 (d, JHH = 6.7 Hz, 3H, CH3). 31P NMR (CDCl3): δ 24.6
(s, PPh3). ESI-MS (acetone) m/z = 606.9 [Ru(η6-4-phenyl-2-
butanol)Cl2(PPh3)]Naþ (100%), m/z = 549.2 [Ru(η6-4-phenyl-
2-butanol)Cl(PPh3)]þ (74%).
[Ru(η6-p-cymene)Cl2(P(p-C6H4F)3)] 8a. [Ru(η6-cymene)Cl2]2
(150 mg, 0.22 mmol) and P(C6H4F)3 (150 mg, 0.44 mmol) were
refluxed in chloroform for 8 h at 70 °C. Yield; 266 mg (94%), red
powder. Slow diffusion of diethyl ether into a dichloromethane
solution gave crystals suitable for X-ray diffraction.
Compounds 1a-5a and 7a-9a were prepared according
to the procedure described for [Ru(η6-p-cymene)Cl2(PPh2(p-
C6H4C2H4C8F17))] 1a.
[Ru(η6-p-cymene)Cl2(PPh(p-C6H4C2H4C8F17)2)] 2a. [Ru(η6-p-
cymene)Cl2]2 (52 mg, 0.09 mmol) and PPh(p-C6H4C2H4C8F17)2
(213 mg, 0.18 mmol) . Yield; 189 mg (72%). Red powder.
1H NMR (CDCl3): δ 7.76-7.80 (m, 6H, PPhAr2), 7.43-7.45
1H NMR (CDCl3): δ 7.82-7.80 (m, 6H, P(C6H4F)3),
3
7.08-7.19 (m, 6H, P(C6H4F)3), 5.27, (d, JHH = 6.0 Hz, 2H,
Hm) 5.00 (d, 3JHH = 5.8 Hz, 2H, Ho), 2.86-2.95 (m, 1H, iPr),
1.88 (s, 3H arene-CH3), 1.16 (d, 3JHH = 7.2 Hz, 6H, CH(CH3)2).
31P NMR (CDCl3): δ 23.0 (s, P(C6H4F)3). ESI-MS (acetone)
m/z = 645.4 [Ru(η6-p-cymene)Cl2(P(C6H4F)3)]Naþ (100%),
m/z = 587.1 [Ru(η6-p-cymene)Cl(P(C6H4F)3)]þ (80%).
3
(m, 7H, PPhAr2), 5.22 (d, JHH = 6.1 Hz, 2H, Hm) 5.05 (d,
3
3JHH = 5.8 Hz, 2H, Ho), 2.98 (t, JHH = 5.8 Hz, 4H,
i
PhCH2CH2C8F17), 2.76-2-79 (m, 1H, Pr), 2.41-2.50 (m,
4H, PhCH2CH2C8F17), 1.89 (s, 3H arene-CH3), 1.12 (d, 3JHH
=
6.4 Hz 6H, CH(CH3)2). 31P NMR (CD2Cl2): δ 23.6 (s, PPhAr2).
ESI-MS (acetone) m/z = 1482.0 [Ru(η6-p-cymene)Cl2(PPh(p-
C6H4C2H4C8F17)2)]Naþ (60%), m/z = 1424.3 [Ru(η6-p-cymene)-
Cl(PPh(p-C6H4C2H4C8F17)2]þ (70%), m/z = 1170.7 [HO(PPh(p-
C6H4C2H4C8F17)2)]þ (100%).
[Ru(η6-4-phenyl-2-butanol)Cl2(P(C6H4F)3)] 9a. [Ru(η6-4-
phenyl-2-butanol)Cl2]2 (100 mg, 0.16 mmol) and P(C6H4F)3
(100 mg, 0.32 mmol). Yield; 174 mg (73%), red powder.
1H NMR (CDCl3): δ 7.52-7.58 (m, 6H, P(C6H4F)3),
3
7.07-7.24 (m, 6H, P(C6H4F)3), 5.68 (d, JHH = 6.4 Hz, 2H,
[Ru(η6-p-cymene)Cl2(P(p-C6H4C2H4C6F13)3)] 3a. [Ru(η6-p-
cymene)Cl2]2 (52 mg, 0.09 mmol) and P(p-C6H4C2H4C6F13)3
(250 mg, 0.19 mnol). Yield; 223 mg (70%), red powder.
1H NMR (CDCl3): δ 7.76-7.80 (m, 6H, PAr3), 7.43-7.45
Hm), 5.24 (d, 3JHH = 5.9 Hz, 2H, Ho) 4.68-4.72 (m, 1H, Hp),
2.76-2.82 (m, 1H, HCOH), 2.63 (s, 3H arene-CH3), 1.80-1.85
3
(m, 2H, arene-CH2CH2), 1.26 (d, JHH = 6.7 Hz, 3H, CH3).
31P NMR (CDCl3): δ 24.6 (s, P(C6H4F)3). ESI-MS (acetone)
m/z = 660.9 [Ru(η6-4-phenyl-2-butanol)Cl2(P(C6H4F)3)]Naþ
(100%), m/z=602.9[Ru(η6-4-phenyl-2-butanol)Cl(P(C6H4F)3)]þ
(60%).
(m, 6H, PAr3), 5.22, (d, 3JHH = 6.3 Hz, 2H, Hm) 5.05 (d, 3JHH
=
5.8 Hz, 2H, Ho), 2.99-3.03 (m, 6H, PhCH2CH2C6F13),
2.73-2-83 (m, 1H, iPr), 2.46-2.49 (m, 6H, PhCH2CH2C6F13),
1.89 (s, 3H arene-CH3), 1.12 (d, 3JHH = 6.7 Hz, 6H, CH(CH3)2).
31P NMR (CD2Cl2): δ 23.6 (s, PAr3). ESI-MS (acetone) m/z =
1482.0 [Ru(η6-p-cymene)Cl2(P(p-C6H4C6F13)3)]Naþ (60%),
m/z = 1424.3 [Ru(η6-p-cymene)Cl(P(p-C6H4C6F13)3)] (70%),
m/z = 1170.7 [HO(P(p-C6H4C6F13)3)] þ (100%).
Compounds 1b-5b and 8b-9b were prepared according to
the procedure described for 1b.
[Ru(η6-p-cymene)(pta)Cl(PPh2(p-C6H4C2H4C8F17))]BF4 1b.
To a methanol solution (20 mL) of 1a (120 mg, 0.12 mmol)
was added NH4BF4 (64 mg, 0.61 mmol), and the solution stirred
for 1 h, after which a methanol solution of pta (21 mg, 0.13 mmol
in 10 mL) was added dropwise over 30 min and stirred for a
further 2 h. The solvent volume was reduced in vacuo to about
10 mL and product precipitated with hexane (10 mL). The
resulting yellow powder was filtered and washed with hexane
(2 ꢀ 20 mL). The resulting yellow powder was recrystallized
from fluorobenzene and hexane. Yield; 123 mg (83%).
1H NMR (CDCl3): δ 7.55-7.67 (m, 6H, PPh2Ar), 7.39-7.41
[Ru(η6-4-phenyl-2-butanol)Cl2(PPh2(p-C6H4C2H4C8F17))] 4a.
[Ru(η6-4-phenyl-2-butanol)Cl2]2 (116 mg, 0.21 mmol) and PPh2-
(p-C6H4C2H4C8F17) (304 mg, 0.43 mmol).Yield; 389 mg (92%),
red powder.
1H NMR (CDCl3): δ 7.70-7.92 (m, 6H, PPh2Ar), 7.28-7.44
3
(m, 8H, PPh2Ar), 5.28, (br, 2H, Hm) 5.16 (d, JHH = 5.6 Hz,
2H, Ho), 4.59 (m, 1H, Hp), 2.96-3.00 (m, 2H, PhCH2CH2C8F17),
2.74-2-77 (m, 1H, HCOH), 2.42-2-46 (m, 2H, PhCH2-
CH2C8F17), 1.94-1.98 (m, 2H arene-CH2CH2), 1.77-1.80
3
(m, 8H, PPh2Ar), 6.46 (d, JHH = 6.0 Hz, 1H, Hm), 5.89, (d,
3
3JHH = 5.8 Hz, 1H, Ho), 5.76 (d, 3JHH = 6.2 Hz, 1H, Ho’), 5.12
(d, 3JHH = 5.6 Hz, 1H, Hm’), 4.45 (d, 2JHH = 14.2 Hz, 3H, pta),
4.37 (d, 2JHH = 15.0 Hz, 3H, pta), 4.28 (d, 2JHH = 14.8 Hz, 3H,
pta), 3.90 (d, 2JHH = 14.6 Hz, 3H, pta), 3.00 (t, 3JHH = 5.6 Hz,
4H, PhCH2CH2C8F17), 2.80-2.87 (m, 1H, iPr), 2.41-2.45
(m, 2H, PhCH2CH2C8F17), 1.63 (s, 3H, arene-CH3), 1.30 (d,
(m, 2H, arene-CH2CH2), 1.25 (d, JHH = 6.2 Hz, 3H, CH3).
31P NMR (CD2Cl2): δ 28.0 (s, PPh2Ar). ESI-MS (acetone)
m/z = 1052.4 [Ru(η6-4-phenyl-2-butanol)Cl2(PPh2(p-C6H4C2-
H4C8F17))]Naþ (100%), m/z= 994.5 [Ru(η6-4-phenyl-2-butanol)-
Cl(PPh2(p-C6H4C2H4C8F17))]þ (65%), m/z = 725.1 [HOPPh2(p-
C6H4C2H4C8F17)]þ (100%).
3
3JHH = 7.0 Hz, 3H, CH(CH3)2), 1.28 (d, JHH = 6.6 Hz, 3H,
[Ru(η6-4-phenyl-2-butanol)Cl2(PPh(p-C6H4C2H4C8F17)2)] 5a.
[Ru(η6-4-phenyl-2-butanol)Cl2]2 (144 mg, 0.27 mmol) and PPh-
(p-C6H4C2H4C8F17)2) (617 mg, 0.54 mmol).Yield; 611mg (88%),
red powder.
2
CH(CH3)2). 31P NMR (CDCl3): δ 30.0 (d, JPP = 52 Hz,
2
PPh2Ar), -42.7 (d, JPP = 54 Hz, pta). ESI-MS (MeOH)
m/z = 1136.2 [Ru(η6-p-cymene)(pta)Cl(PPh2(Ph(C2H4C8F17))]þ
(100%). Tdecomp. 160 °C.
1H NMR (CDCl3): δ 7.71-7.73 (m, 6H, PPhAr2), 7.35-7.46 (m,
7H, PPhAr2), 5.28, (d, 3JHH = 5.5 Hz, 2H, Hm) 5.18 (d, 2H, 3JHH
=
[Ru(η6-p-cymene)(pta)Cl(PPh(p-C6H4C2H4C8F17)2)]BF4 2b.
5a (180 mg, 0.12 mmol), NH4BF4 (63 mg, 0.60 mmol) and
pta(21 mg, 0.13 mmol in 10 mL.Yield; 156 mg (80%) of yellow
powder.
5.8 Hz, Ho),4.53(m,1H,Hp),2.92-2.95 (m, 4H, PhCH2CH2C8F17),
2.81-2.84 (m, 1H, HCOH), 2.43.2.46 (m, 4H, PhCH2CH2C8F17),
1.94-1.97 (m, 2H arene-CH2CH2), 1.80-1.83 (m, 2H, arene-
CH2CH2), 1.27 (d, 3JHH = 6.5 Hz, 3H, CH3). 31P NMR (CDCl3):
δ27.3(s, PPhAr2). ESI-MS(acetone) m/z= 1499.0 [Ruη6-4-phenyl-
2-butanol)Cl2(PPh(p-C6H4C2H4C8F17)2)]Naþ (60%),m/z= 1441.3
1H NMR (CDCl3): δ 7.59-7.69 (m, 6H, PPhAr2), 7.3-7.41
3
(m, 7H, PPhAr2), 6.66 (d, JHH = 6.0 Hz, 1H, Ho), 6.02 (d,
3JHH = 6.8 Hz, 1H, Hm), 5.78 (d, 3JHH = 5.6 Hz, 1H, Hm’), 5.05

Article
Inorganic Chemistry, Vol. 49, No. 5, 2010 2245
(d, 3JHH = 5.8 Hz, 1H, Hm’), 4.41 (d, 2JHH = 12 Hz, 3H, pta),
4.26 (d, 2JHH = 12 Hz, 3H, pta), 4.16 (d, 2JHH = 16 Hz, 3H, pta),
5.94 (d, 3JHH = 5.7 Hz, 1H, Ho), 5.76 (d, 3JHH = 6.2 Hz, 1H, Ho’),
5.14 (d, 3JHH = 5.8 Hz, 1H, Hm’), 4.70 (d, 2JHH = 12.0 Hz, 3H,
pta), 4.33 (d, 2JHH = 12.8 Hz, 3H, pta), 4.20 (d, 2JHH = 13.8 Hz,
3H, pta), 3.81 (d, 2JHH = 12.4 Hz, 3H, pta), 2.60-2.67 (m, 1H,
2
3
3.89 (d, JHH = 16 Hz, 3H, pta), 3.01 (t, JHH = 6.2 Hz, 4H,
PhCH2CH2C8F17), 2.73-2.79 (m, 1H, iPr), 2.41-2.43 (m, 4H,
PhCH2CH2C8F17), 1.65(s, 3Harene-CH3), 1.32(d,3JHH =6.8Hz,
3
3
iPr), 1.24 (d, JHH = 6.5 Hz, 3H, CH(CH3)2), 1.22 (d, JHH
=
=
3
2
3H, CH(CH3)2), 1.30 (d, JHH2 = 7.0 Hz, 3H, CH(CH3)2).
6.8 Hz, 3H, CH(CH3)2). 31P NMR (CDCl3): δ 28.8 (d, JPP
31P NMR (CDCl3): δ 29.7 (d, JPP = 55 Hz, PPhAr2), -43.1
(d, 2JPP = 55 Hz, pta). ESI-MS (MeOH) m/z = 1588.1 [Ru(η6-p-
cymene)(pta)Cl(PPh(p-C6H4C2H4C8F17)2)] (100%). mp 167-
170 °C.
54 Hz, P(C6H4F)3), -42.5 (d, JPP = 54 Hz, pta). ESI-MS
(MeOH) m/z = 744.1 [Ru(η6-p-cymene)(pta)Cl(P(C6H4F)3)]þ
(100%). Tdecomp. 250 °C.
2
[Ru(η6-4-phenyl-2-butanol)(pta)Cl(P(C6H4F)3)]BF4 9b. 9a
(69 mg, 0.66 mmol), NH4BF4 (64 mg, 0.61 mmol) and pta
(22 mg, 0.15 mmol in 10 mL). The product was isolated as a
racemic mixture. Yield; 101 mg (93%) of yellow powder.
1H NMR (CDCl3): δ 7.54-7.59 (m, 6H, P(C6H4F)3),
[Ru(η6-p-cymene)(pta)Cl(P(p-C6H4C2H4C6F13)3)]BF4 3b. 3a
(200 mg, 0.12 mmol), NH4BF4 (64 mg, 0.61 mmol), and pta
(20 mg, 0.12 mmol in 10 mL). Yield; 171 mg (79%) of yellow
powder.
1H NMR (CDCl3): δ 7.56-7.60 (m, 6H, PAr3), 7.39-7.41 (m,
3
7.07-7.24 (m, 6H, P(C6H4F)3), 6.86 (d, JHH = 5.8 Hz, 1H,
6H, PAr3), 6.68 (d, 3JHH = 5.6 Hz, 1H, Ho), 6.04, (d, 3JHH
=
Ho), 6.19, (d, 3JHH = 6.1 Hz, 1H, Hm), 5.68 (d, 3JHH = 5.8 Hz,
3
3
1H, Hm’), 5.15 (d, JHH = 6.2 Hz, 1H, Ho’), 4.71 (d, JHH
2
5.8 Hz, 1H, Hm), 5.76 (d, JHH = 6.0 Hz, 1H, Hm’), 5.03 (d,
3JHH = 5.6 Hz, 1H, Ho’), 4.44 (d, 2JHH = 13.8 Hz, 3H, pta), 4.36
(d, 2JHH 14.2 = Hz, 3H, pta), 4.07 (d, 2JHH = 12.0 Hz, 3H, pta),
3.90 (d, 2JHH = 10.2 Hz, 3H, pta), 3.00 (t, 3JHH = 5.8 Hz, 6H,
=
=
2
12.4 Hz, 3H, pta), 4.60-4.64 (m, 1H, Hp), 4.39 (d, JHH
2
13.5 Hz, 3H, pta), 4.23 (d, JHH = 13.2 Hz, 3H, pta), 3.90 (d,
2JHH = 12.8 Hz, 3H, pta), 2.79-2.83 (m, 1H, HCOH), 2.63 (s,
3H arene-CH3), 1.79-1.82 (m, 2H, arene-CH2CH2), 1.26 (d,
PhCH2CH2C6F13), 2.78-2.86 (m, 1H, iPr), 2.43-2.44 (m, 6H,
3JHH = 6.5 Hz, 3H, CH3). 31P NMR (CDCl3): δ 30.5 (d, 2JPP
=
3
PhCH2CH2C6F13), 1.63 (s, 3H arene-CH3), 1.30 (d, JHH
=
53 Hz, P(C6H4F)3), 30.3 (d, 2JPP = 54 Hz, P(C6H4F)3), -41.0
(d, 2JPP = 54 Hz, pta), -41.2 (d, 2JPP = 54 Hz, pta).a ESI-MS
(MeOH) m/z= 759.1 [Ru(η6-phenyl-butanol)(pta)Cl(P(C6H4F)3)]þ
(100%). Tdecomp. 162 °C.
6.8 Hz, 3H, CH(CH3)2), 1.28 (d, 3JHH = 6.2 Hz, 3H, CH(CH3)2).
31P NMR (CDCl3): δ 29.8 (d, 2JPP = 55 Hz 1P, PAr3), -43.3 (d,
2JPP = 55 Hz, pta). ESI-MS (MeOH) m/z = 1727.2 [Ru(η6-p-
cymene)Cl2(P(p-C6H4C6F13)3)] (100%). mp 172-177 °C.
[Ru(η6-4-phenyl-2-butanol)(pta)Cl(PPh2(p-C6H4C2H4C8F17))]-
BF4 4b. 4a (120 mg, 0.11 mmol), NH4BF4 (64 mg, 0.61 mmol) and
pta (17 mg, 0.11. mmol in 10 mL). The product was isolated as a
racemic mixture. Yield; 102 mg (75%) of yellow powder.
1H NMR (CDCl3): δ 7.55-7.74 (m, 6H, PPh2Ar), 7.43-7.50
[Ru(η6-4-phenyl-2-butanol)(pta)Cl(P(p-C6H4C2H4C6F13)3)]-
BF4 6b. [Ru(η6-4-phenyl-2-butanol)(pta)Cl2] (144 mg, 0.3 mmol)
was refluxed in acetonitrile with 2 mol equiv of NH4BF4 for 1 h.
The solution was filtered, and the solvent removed at reduced
pressure. The resulting solids were extracted with methanol and
precipitated with diethyl ether. The yellow powder was stirred
for 2 days with an excess of P(p-C6H4C2H4C6F13)3 (780 mg,
0.6 mmol) in methanol following which the solution was filtered
to remove unreacted P(p-C6H4C2H4C6F13)3, the solvent volume
reduced, and the product precipitated with hexane as a yellow
solid. The product was isolated as a racemic mixture. Yield;
122 mg (67%).
3
(m, 8H, PPh2Ar), 6.74 (d, JHH = 5.6 Hz, 1H, Ho), 6.22, (d,
3JHH = 4.8 Hz, 1H, Hm), 5.69 (d, 3JHH = 5.4 Hz, 1H, Hm’), 5.28
(d, 3JHH = 5.8 Hz, 1H, Ho’), 4.71 (d, 2JHH = 12.6 Hz, 3H, pta),
4.62-4.65 (m, 1H, Hp), 4.39 (d, 2JHH = 14.8 Hz, 3H, pta), 4.23
(d, 2JHH = 14.2 Hz, 3H Hz, pta), 3.90 (d, 2JHH = 10.6 Hz, 3H,
pta), 3.01 (t, 3JHH = 6.2 Hz, 2H, PhCH2CH2C8F17), 2.73-2.78
(m, 1H, HCOH), 2.46-2.50 (m, 2H, PhCH2CH2C8F17), 1.63
(s, 3H arene-CH3), 1.76-1.79 (m, 2H, arene-CH2CH2), 1.27 (d,
1H NMR (d6-DMSO): δ 7.41-7.62 (m, 6H, PAr3), 6.91-7.10
3JHH = 7.0 Hz, 3H, CH3). 31P NMR (CDCl3): δ 31.4 (d, 2JPP
=
(m, 6H, PAr3), 6.58 (d, 3JHH = 5.5 Hz, 1H, Ho), 6.13, (d, 3JHH
6.0 Hz, 1H, Hm), 5.65 (d, JHH = 5.8 Hz, 1H, Hm’), 5.22 (d,
=
2
3
54 Hz, PPh2Ar), 31.3 (d, JPP = 54 Hz, PPh2Ar), -40.4 (d,
2
2JPP = 55 Hz, pta), -40.5 (d, JPP = 55 Hz, pta).1 ESI-MS
2
3JHH = 5.6 Hz, 1H, Ho’), 4.73 (d, JHH = 12.0 Hz, 3H, pta),
(MeOH) m/z = 1152.2 [Ru(η6-phenyl-butanol)(pta)Cl(PPh2-
(Ph(C2H4C8F17))]þ (100%). Tdecomp. 157 °C.
4.58-4.62 (m, 1H, Hp), 4.23 (d, 2JHH = 12.3 Hz, 3H, pta), 4.10
(d, 2JHH = 12.8 Hz, 3H, pta), 3.90 (d, 2JHH = 13.2 Hz, 3H, pta),
3.00-3.04 (m, 6H, PhCH2CH2C6F13), 2.82-2.85 (m, 1H,
HCOH), 2.43-2.46 (m, 6H, PhCH2CH2C6F13), 1.99-2.02 (m,
2H arene-CH2CH2), 1.84 (m, 2H, arene-CH2CH2), 1.27 (d,
3JHH = 6.8 Hz, 3H, CH3). 31P NMR (d6-DMSO): δ 29.4 (d,
[Ru(η6-4-phenyl-2-butanol)(pta)Cl(PPh(p-C6H4C2H4C8F17)2)]-
BF4 5b. 5a (90 mg, 0.07 mmol), NH4BF4 (37 mg, 0.35 mmol) and
pta (12 mg, 0.8 mmol in 10 mL). The product was isolated as a
racemic mixture. Yield; 91 mg (90%) of yellow powder.
1H NMR (CDCl3): δ 7.55-7.74 (m, 6H, PPhAr2), 7.43-7.50
2
2JPP = 54 Hz, PAr3), 29.2 (d, JPP = 54 Hz, PAr3), -42.0 (d,
3
2
2JPP = 55 Hz, pta), -42.2 (d, JPP = 55 Hz, pta).a ESI-MS
(m, 7H, PPhAr2), 6.67 (d, JHH = 5.6 Hz, 1H, Ho), 6.13, (d,
3JHH = 6.0 Hz, 1H, Hm), 5.65 (d, 3JHH = 6.2 Hz, 1H, Hm’), 5.30
(d, 3JHH = 5.8 Hz, 1H, Ho’), 4.73 (d, 2JHH = 12.0 Hz, 3H, pta),
4.45-4.51 (m, 1H, Hp), 4.23 (d, 2JHH = 13.2 Hz, 3H, pta), 4.10
(d, 2JHH = 13.6 Hz 3H, pta), 3.90 (d, 2JHH = 14.0 Hz, 3H, pta),
3.02 (t, 3JHH = 6.0 Hz, 4H, PhCH2CH2 C8F17), 2.81-2.84 (m,
1H, HCOH), 2.41-2.45 (m, 4H, PhCH2CH2C8F17), 1.96-1.20
(m, 2H arene-CH2CH2), 1.81-1.85 (m, 2H, arene-CH2CH2),
1.27 (d, 3JHH = 7.0 Hz, 3H, CH3). 31P NMR (CDCl3): δ 30.9 (d,
2JPP = 54 Hz, PPhAr2), 30.8 (d, 2JPP = 53 Hz, PPhAr2), -41.4
(d, 2JPP = 54 Hz, pta), -41.5 (d, 2JPP = 54 Hz, pta). [Two Ru,C
configurational diastereomers are present at equal ratio] ESI-
MS (MeOH) m/z = 1594.1 [Ru(η6-phenyl-butanol)(pta)Cl-
(PPh(p-C6H4C2H4C8F17)2)]þ (100%), m/z = 1444.9 [Ru(pta)-
Cl(PPh(p-C6H4C2H4C8F17)2)]þ (40%). Tdecomp. 144 °C.
(MeOH) m/z = 1744.2 [Ru(η6-phenyl-butanol)(pta)Cl(P(p-
C6H4C6F13)3)]þ (100%). Tdecomp. 137 °C.
[Ru(η6-4-phenyl-2-butanol)(pta)Cl(PPh3)]BF4 7b. [Ru(η6-4-
phenyl-2-butanol)(pta)Cl2] (80 mg, 0.17 mmol), NH4BF4
(90 mg, 0.86 mmol), followed by triphenylphosphine (50 mg,
0.19 mmol). The solution was stirred at room temperature for
5 h, the solvent volume reduced, and hexane added to induce
precipitation. The resulting yellow solid was washed with
hexane (2 ꢀ 20 mL) and diethyl ether (2 ꢀ 20 mL). Yield;
123 mg (91%).
1H NMR (CDCl3): δ 7.59-7.68 (m, 6H, PPh3), 7.30-7.35
(m, 9H, PPh3), 6.66 (d, 3JH3H = 5.8 Hz, 2, Ho), 6.13, (d, 3JHH
=
6.2 Hz, H, Hm), 5.71 (d, JHH = 5.5 Hz, 1H, Hm’), 5.40 (d,
3JHH = 6.0 Hz, 1H, Ho’), 4.69 (d, 2JHH = 12.8 Hz, 3H, pta), 4.62
(d, 2JHH = 15.5 HZ, 3H, pta), 4.56-4.60 (m, 1H, Hp), 4.24 (d,
[Ru(η6-p-cymene)(pta)Cl(P(C6H4F)3)]BF4 8b. 8a (222 mg,
0.31 mmol), NH4BF4 (164 mg, 1.56 mmol) and pta (50 mg,
0.32 mmol in 10 mL). Yield; 101 mg (93%) of yellow powder.
1H NMR (CDCl3): δ 7.52-7.595 (m, 6H, P(C6H4F)3),
7.04-7.19 (m, 6H, P(C6H4F)3), 6.58 (d, 3JHH = 6.0 Hz, H, Hm),
2
2JHH = 14.8 Hz, 3H, pta), 3.92 (d, JHH = 13.0 Hz, 3H, pta),
2.78-2.82 (m, 1H, HCOH), 1.78-1.81 (m, 2H arene-CH2CH2),
1.68 (m, 2H, arene-CH2CH2), 1.26 (d, 3JHH = 6.8 Hz, 3H, CH3).
2
31P NMR (CDCl3): δ 30.5 (d, JPP = 53 Hz, PPh3), 30.4 (d,

2246 Inorganic Chemistry, Vol. 49, No. 5, 2010
Table 4. Solubility of 1b, 4b, 5b, 7b, and 9b in H2O
Renfrew et al.
Table 5. Selected Crystallographic Data for 8b
S (mM) S (mM) S (mM) S (mM) S (mM) S (mM)
42 °C
parameter
formula
8b
20 °C
30 °C
37 °C
40 °C
45 °C
C34H38BClF7N3P2Ru
830.94
monoclinic
P21/c
14.4292(3)
14.2971(3)
17.6308(4)
90
109.367(2)
90
1b
4b 2.6
5b
7b 3.9
9b 5.1
0
0
11
0
3.9
5.1
0.1
19
0.1
5.0
5.3
0.2
23
0.1
5.4
5.3
0.3
26
0.3
5.6
5.3
0.7
31
0.4
6.1
5.4
fw (g mol-1
)
crystal system
space group
0
˚
a (A)
˚
b (A)
˚
c (A)
2
2JPP = 53 Hz, PPh3), -42.5 (d, JPP = 54 Hz, pta), -42.7 (d,
R (deg)
β (deg)
γ (deg)
2JPP = 54 Hz, pta).a Tdecomp. 180 °C.
Hydrolysis Study. A 1 mM solution of 4b in D2O was
incubated over 3 days at 37 °C. Aliquots were taken after 1, 2,
5, 24, 48, 72, and 168 h and analyzed by 31P NMR spectroscopy.
Solubility Measurements. Samples of a known mass were
suspended in H2O (2 mL) and incubated for 30 min at tempera-
tures ranging between 20 and 45 °C. The sample was filtered to
remove any insoluble material, and 990 μL transferred to an
NMR tube containing 10 μL of a known concentration of
triethylphosphate. The concentration of compound in solution
was determined by integration of the 31P NMR spectra to give
the ratio of compound to triethylphosphate. The mass of the
sample in the initial suspension and the volume of triethlypho-
sphate were adjusted according to the solubility of the com-
pound. For example, for 1b at 20 °C 0.8 mmol (2 mg of a 20 mg/
20 mL solution) of the complex with 11 mmol (10 μL from a
1:9 v/v solution of triethylphosphate/H2O) of triethylpho-
sphate, for 4b at 20 °C 10 mmol (25 mg/2 mL) of the complex
with 22 mmol (10 μL from a 2:8 v/v solution of triethylpho-
sphate/H2O) of triethyl phosphate. Concentrations at each
temperature measured are given in Table 4.
3
˚
volume (A )
Z
3431.35(12)
4
1.608
Dcalc (g cm-3
)
μ (mm-1
F(000)
temp (K)
)
0.697
1688
140(2)
22576
6949
measured reflns
unique reflns
θ range (deg)/completeness (%)
no. of data/parameters/restraints
GoFa
2.99 to 26.37/99.1
6949/483/92
1.039
Rb [I > 2σ(I)]
0.0290
0.0723
wR2b (all data)
largest diff. peak/hole (e A
-3
˚
)
1.566/-0.546
P
a GoF is defined as { [w(Fo - Fc2)2]/(n - p)}1/2 where n is the
2
P
number of data and p is the number of parameters refined. b R = ||Fo|
P
P
P
- |Fc||/ |Fo|, wR2 = { w(Fo2 - Fc2)2/ w(Fo2)2}1/2
.
plates as monolayers with 100 μL of cell solution (approximately
20,000 cells) per well and preincubated for 24 h in medium
supplemented with 10% FCS. With the exception of 4b and 9b,
which were dissolved directly in the culture medium, com-
pounds were added as DMSO solutions and serially diluted to
the appropriate concentration (to give a final DMSO concen-
tration of 0.5%). A 100 μL portion of drug solution was added
to each well, and the plates were incubated for another 72 h.
Subsequently, MTT (5 mg/mL solution in phosphate buffered
saline) was added to the cells, and the plates were incubated for a
further 2 h. The culture medium was aspirated, and the purple
formazan crystals formed by the mitochondrial dehydrogenase
activity of vital cells were dissolved in DMSO. The optical
density, directly proportional to the number of surviving cells,
was quantified at 540 nm using a multiwell plate reader (iEMS
Reader MF, Labsystems, U.S.), and the fraction of surviving
cells was calculated from the absorbance of untreated control
cells. Evaluation is based on means from 2 independent experi-
ments, each comprising 3 microcultures at each concentration
level.
X-ray Structure Determination. Data for 8b was collected on a
KUMA CCD diffractometer system using graphite-monochro-
mated Mo KR radiation. Data reduction was performed with
CrysAlis RED.39 The structure was solved by direct methods
with SHELXS-9740 and refined by least-squares fit on F2 using
SHELXL-97.40 The absorption correction was applied using the
multiscan procedure SADABS.41 All non-hydrogen atoms were
refined anisotropically with hydrogen atoms placed in geome-
trically calculated positions and refined using a riding model.
ORTEP342 was used to produce the graphical representation of
8b. Relevant data is given in Table 5.
Cell Line and Culture Conditions. The human A2780 ovarian
cancer cell line was obtained from the European Collection of
Cell Cultures (Salisbury, U.K.). Cells were grown routinely in
RPMI medium containing glucose, 5% fetal calf serum (FCS),
and antibiotics at 37 °C and 5% CO2.
Cytotoxicity Test (MTT Assay). Cytotoxicity was determined
using the MTT assay (MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide. Cells were seeded in 96-well
Acknowledgment. We thank the Swiss National Science
Foundation and EPFL for financial support and Severine
Moret for preparing 1a-3a.
(39) CrysAlis PRO; Oxford Diffraction Ltd.: Abingdon, Oxfordshire, U.K., 2009.
€
€
(40) Sheldrick, G. M. SHELX97; University of Gottingen: Gottingen,
Germany, 1997.
Supporting Information Available: The crystallographic in-
formation file (cif) of complex 8b. This material is available free
of charge via the Internet at http://pubs.acs.org.
€
(41) Sheldrick, G. M. SADABS, version 2006; University of Gottingen:
€
Gottingen, Germany, 1997.
(42) Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.