Synthesis, spectroscopic characterization, and cytotoxic evaluation of pentasubstituted ruthenocenyl esters

Article abstract of DOI:10.1021/om100645y

This article details the preparation and spectroscopic characterization of a focused library of new 18-electron ruthenocenyl complexes incorporating pentasubstituted Cp ester [C₅(CO₂R)₅] ^- (for R = Me, Et, n-Pr, n-Bu, 2-Pr, 3-Pent, phenyl, and n-thiopropyl), carboxylic acid [C₅(CO₂H)₅] ^-, and carboxylate ligands [C₅(CO₂H) ₄(CO₂)]^2-. Each complex has been characterized using Fourier transform IR and NMR spectroscopy and electrospray mass spectrometry, with single-crystal X-ray structural determinations reported for four complexes: [Ru(η⁵-C₅H₄(C ₅(CO₂CH₃)₅)(η⁵-C ₅(CO₂CH₃)₅)], K[Ru(η ⁵-C₅H₅)(η⁵-C₅(CO ₂H)₄(CO₂))].H₂O, [Ru(η⁵-C₅H₅)(η⁵-C ₅(CO₂H)₅)]·2H₂O, and [Ru(η⁵-C₅H₅)(η⁵-C ₅(CO₂C₆H₅)₅)]. Complexes were also evaluated for in vitro cytotoxic activity against a diverse panel of tumorigenic cell lines and a normal human cell line.

Full text of DOI:10.1021/om100645y

Organometallics 2010, 29, 6237–6244 6237
DOI: 10.1021/om100645y
Synthesis, Spectroscopic Characterization, and Cytotoxic Evaluation
of Pentasubstituted Ruthenocenyl Esters
Leanne S. Micallef,† Bradley T. Loughrey,† Peter C. Healy,† Peter G. Parsons,‡ and
Michael L. Williams*,†
†Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, Australia, and
‡Drug Discovery Group, Queensland Institute of Medical Research, Brisbane, Australia
Received July 4, 2010
This article details the preparation and spectroscopic characterization of a focused library of new
18-electron ruthenocenyl complexes incorporating pentasubstituted Cp ester [C5(CO2R)5]- (for R=
Me, Et, n-Pr, n-Bu, 2-Pr, 3-Pent, phenyl, and n-thiopropyl), carboxylic acid [C5(CO2H)5]-, and
carboxylate ligands [C5(CO2H)4(CO2)]2-. Each complex has been characterized using Fourier trans-
form IR and NMR spectroscopy and electrospray mass spectrometry, with single-crystal X-ray
structural determinations reported for four complexes: [Ru(η5-C5H4(C5(CO2CH3)5)(η5-C5(CO2-
CH3)5)], K[Ru(η5-C5H5)(η5-C5(CO2H)4(CO2))].H2O, [Ru(η5-C5H5)(η5-C5(CO2H)5)] 2H2O, and [Ru-
3
(η5-C5H5)(η5-C5(CO2C6H5)5)]. Complexes were also evaluated for in vitro cytotoxic activity against a
diverse panel of tumorigenic cell lines and a normal human cell line.
Introduction
antitumor,11,12 antimalarial,13 antifungal,14 and DNA cleavage
activity.15 Ruthenocene and its derivatives however have only
received minor interest, but initial studies reveal that these
molecules possess promising antitumor activity.16-20 These
findings, coupled with the recent interest in and highly topical
results achieved by ruthenium(II) arene piano-stool anticancer
complexes,21 prompted our research group to prepare, charac-
terize, and biologically investigate a range of cationic ruthenium-
(II) arene Cp* (η5-C5(CH3)5) metallocenes [(R-Ph)Ru(Cp*)]X,
Biological inorganic chemistry is a discipline of increasing
importance in both therapeutic and diagnostic medicine. It
offers the potential for the design and preparation of novel
complexes capable of treating diseases that are resistant to
conventional therapeutic methods.1-4 Considerable research
has been carried out investigating the therapeutic properties
possessed by inorganic coordination complexes. Organometallic
complexes however have only been sparingly investigated, and
recent studies conclude that these systems hold considerable
potential for use as clinical therapeutic agents due to their unique
array of structural configurations and bonding modes.4-6 Me-
tallocenes have long been recognized to possess a varied range of
biological activity.7,8 In particular, the sandwich complexes
ferrocene and ruthenocene have attracted special attention due
to their neutral, stable, and nontoxic chemical properties.8 A
range of ferrocenyl derivatives display interesting cytotoxic,9,10
where X = BF4-, PF6-, and B(C6H5)4- 22,23 The results from
.
these early studies demonstrated these organoruthenium full-
sandwich complexes to possess potent and selective antipro-
liferative activity toward a range of cancerous cell lines in vitro,
(13) Biot, C.; Francois, N.; Maciejewski, L.; Brocard, J.; Poulain, D.
Bioorg. Med. Chem. Lett. 2000, 10, 839.
(14) Itoh, T.; Shirakami, S.; Ishida, N.; Yamashita, Y.; Yoshida, T.;
Kim, H.-S.; Wataya, Y. Bioorg. Med. Chem. Lett. 2000, 10, 1657.
(15) Baldoli, C.; Maiorana, S.; Licandro, E.; Zinzalla, G.; Perdicchia,
D. Org. Lett. 2002, 4, 4341.
*To whom correspondence should be addressed. E-mail: michael.
williams@griffith.edu.au.
(1) Farrell, N. Bioorganometallics; Jaouen, G., Ed.; Wiley-VCH: Weinheim,
2005.
(2) Storr, T.; Thompson, K. H.; Orvig, C. Chem. Soc. Rev. 2006, 35,
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Scopelliti, R.; Tavernelli, I. Organometallics 2005, 24, 2114.
(17) 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.
(18) Reisner, E.; Arion, V. B.; Guedes, M. F. C.; Lichtenecker, R.;
Eichinger, A.; Keppler, B. K.; Kukushkin, V. Y.; Pombeiro, A. J. L.
Inorg. Chem. 2004, 43, 7083.
(19) Dorcier, A.; Ang, W. A.; Bola∼no, S.; Gonsalvi, L.; Juilelrat-
Jeannerat, L.; Laurenczy, G.; Peruzzini, M.; Phillips, A. D.; Zanobini,
F.; Dyson, P. J. Organometallics 2006, 25, 4090.
(20) Maillard, S.; Gauduchon, J.; Marsaud, V.; Gouilleux, F.; Connault,
E.; Opolon, P.; Fattal, E.; Sola, B.; Renoir, J. K. J. Steroid Biochem. Mol.
Biol. 2006, 100, 67.
(21) Therrien, B. Coord. Chem. Rev. 2009, 253, 493.
(22) Loughrey, B. T.; Healy, P. C.; Parsons, P. G.; Williams, M. L.
Inorg. Chem. 2008, 47, 8589.
(23) Loughrey, B. T.; Williams, M. L.; Healy, P. C.; Innocenti, A.;
Vullo, D.; Supuran, C. T.; Parsons., P. G.; Poulsen, S.-A. J. Biol. Inorg.
Chem. 2009, 14, 935.
(4) van Rijt, S. H.; Sadler, P. J. Drug Discovery Today 2009, 14, 1089.
(5) Cohen, S. M. Curr. Opin. Chem. Biol. 2007, 11, 115.
(6) Hartinger, C. G.; Dyson, P. J. Chem. Soc. Rev. 2009, 38, 391–401.
(7) Harding, M. M.; Mokdsi, G. Curr. Med. Chem. 2000, 7, 1289.
(8) Fouda, M. F. R.; Abd-Elzaher, M. M.; Abdelsamaia, R. A.;
Labib, A. A. Appl. Organomet. Chem. 2007, 21, 613.
(9) Meunier, P.; Ouattara, I.; Gautheron, G.; Tirouflet, J.; Camboli,
D.; Besancon, J. Eur. J. Med. Chem. 1991, 26, 351.
(10) Top, S.; Tang, J.; Vessieres, A.; Carrez, D.; Provot, C.; Jaouen,
G. Chem. Commun. 1996, 26, 955.
€
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€
€
r
2010 American Chemical Society
Published on Web 11/01/2010
pubs.acs.org/Organometallics

6238 Organometallics, Vol. 29, No. 23, 2010
Scheme 1. Synthesis of Ru(η5-C5H5)(η5-C5(CO2CH3)5) Using Ag(OAc)
Micallef et al.
with the degree of activity dependent on the lipophilicity of the
arene ligand.22,23 As a continuation of our earlier studies, we
endeavored to further ascertain how the alteration of complex
charge, lipophilicity, and the degree of aromatic substitution
impacts the overall biological activity of ruthenium(II) metallo-
cenes. To achieve this aim, we have prepared, characterized, and
assayed a series of neutral 18-electron ruthenocenyl complexes
incorporating pentasubstituted Cp ester, carboxylic acid, and
carboxylate ligands.
the thallium salt of pentakis(methoxycarbonyl)cyclopenta-
diene.32,34 This synthesis proved to be an efficient method for
the preparation of complex 1; however routine handling of
toxic thallium is ultimately unfavorable, and it was therefore of
interest to incorporate a synthetic procedure for complex 1 that
did not involve the use of thallium salts. After a range of trial
reactions, we found that the silver pentakis(methoxycarbony1)-
cyclopentadiene complex originally prepared by Bruce et al.
in 198335 was a suitable replacement for Tl[C5(CO2CH3)5], with
the modified procedure (Scheme 1) affording complex 1 in good
yield. Ag[C5(CO2CH3)5] is prepared through the reaction between
silver acetate and H[C5(CO2CH3)5], providing formation of the
light-sensitive silver salt as a white powder.
Results and Discussion
Synthesis and Characterization. Structurally diverse li-
braries of metallocenes incorporating mono- and disubstituted
Cp ligands have been afforded previously in good yields via a
range of straightforward synthetic procedures.24 Penta- and
decasubstituted metallocenes are less prevalent in the literature,
however, with these complexes often prepared via methods
involving repeated applications of the appropriate substitution
reaction. Each step often results in a diminished yield and
formation of byproducts that are difficult to remove during
workup. Efficient synthetic methods for the preparation of
pentasubstituted metallocenes often involve the use of prepre-
pared pentasubstituted Cp ligands, with this strategy resulting
in the preparation of a range of pentasubstituted alkyl,25-27
aryl,25,28 and halogenated metallocenes.29 Pentasubstituted
metallocenes incorporating electron-withdrawing substituents
such as esters have also been reported,30-33 albeit to a much
lesser extent. An interesting complex prepared during these
initial studies was the ruthenocenyl derivative incorporating
the pentasubstituted Cp ligand (C5(CO2CH3)5)- (pmeCp-).32
Pentamethoxycarbonyl ruthenocene [Ru(η5-C5H5)(η5-C5(CO2-
CH3)5)] (1) is of particular interest to this study due to the
potential it provides for producing a library of pentasubsti-
tuted ruthenocenes through modification using simple orga-
nic procedures. Complex 1 was first prepared in 1983 by Bruce
et al. via a five-step procedure beginning with the preparation of
Addition of RuCl(PPh3)2(η5-C5H5) to this mixture in the
presence of oxygen affords formation of complex 1 with the
silver conveniently removed from the reaction mixture via
filtration in the form of insoluble silver chloride.
During one preparation of complex 1 using Scheme 1,
[Ru(η5-C5H5)(η5-C5(CO2CH3)5)] was isolated from this so-
lution as per normal. However, it was noted that the resulting
mixture was more intensely colored than usual, and pre-
parative TLC of the sample isolated a second compound (2)
in a 0.7% yield. X-ray diffraction studies on crystals of
complex 2 grown by slow evaporation of solvent from a
concentrated chloroform solution showed the structure of 2
to be [Ru(η5-C5H4(C5(CO2CH3)5)(η5-C5(CO2CH3)5)], in
which a 1,2,3,4,40-pentamethoxycarbonyl ring is bonded to
the η5-coordinated cyclopentadiene ring (Figure 1).
The unit cell of 2 consists of discrete molecules with one
molecule comprising the asymmetric unit. The carbon atoms in
the two ruthenium-bound C5 rings are eclipsed. The Ru-C-
(11-15) and Ru-C(21-25) bond lengths of 2.143(4)-2.204(4)
˚
˚
A and 2.174(4)-2.216(3) A, respectively, are comparable to the
˚
˚
values of 2.157(2)-2.178(2) A and 2.178(2)-2.186(2) A re-
ported for (1),32 albeit with a greater range of values, consistent
with the steric and electronic effects of substitution of the
external pentamethylcarbonyl ring (C31-C35) on the Cp ring.
This ring is connected to the Cp ring by a single C-C bond,
˚
C21-C31, with a bond length of 1.467(5) A, with the torsion
(24) Long, N. J. Metallocenes: An Introduction to Sandwich Com-
plexes; Blackwell Science: London, 1998.
angle C22-C21-C31-C32 = -27.5(5)o. Unlike the Ru-coor-
(25) Janiak, C.; Schumann, H. Adv. Organomet. Chem. 1991, 33, 291.
(26) King, R. B.; Bisnette, M. B. J. Organomet. Chem. 1967, 8, 287.
(27) Stein, D.; Sitzmann, H. J. Organomet. Chem. 1991, 402, 249.
(28) Beck, C. U.; Field, L. D.; Hambley, T. W.; Humphrey, P. A.;
Masters, A. F.; Turner, P. J. Organomet. Chem. 1998, 565, 283.
(29) Gassman, P. G.; Winter, C. H. J. Am. Chem. Soc. 1988, 110,
6130.
(30) Bruce, M. I.; Humphrey, P. A.; Skelton, B. W.; White, A. H.
J. Organomet. Chem. 1989, 361, 369.
(31) Bruce, M. I.; Humphrey, P. A.; Williams, M. L. Aust. J. Chem.
1997, 50, 1113.
(32) Bruce, M. I.; Wallis, R. C.; Williams, M. L.; Skelton, B. W.;
dinated rings, this ring consists of two double bonds (C31-
˚
˚
C35, 1.363(5) A, and C34-C35, 1.334(5) A), with the remaining
three bonds single (C31-C32, 1.542(5), C32-C33, 1.512(5),
˚
C34-C35, 1.468(5) A).
Formation of complex 2 indicated the occurrence of a
carbon-carbon coupling reaction between a nonsubstituted
Cp ring and excess Ag[C5(CO2CH3)5] either before or after
pmCp complexation to the ruthenium(II) metal. During this
coupling process a Cp hydrogen is displaced in favor of
[C5(CO2CH3)5]-, thus forming a C-C σ-bond between the
White, A. H. Dalton Trans. 1983, 2183.
(33) Kohl, F. X.; Schlueter, E.; Jutzi, P. J. Organomet. Chem. 1983,
243, C37.
(34) Bruce, M. I.; Walton, J. K.; Williams, M. L.; Hall, S. R.; Skelton,
(35) Bruce, M. I.; Williams, M. L.; Skelton, B. W.; White, A. H.
Dalton Trans. 1983, 4, 799.
B. W.; White, A. H. Dalton Trans. 1982, 2209.

Article
Organometallics, Vol. 29, No. 23, 2010 6239
Figure 1. Molecular projection of [Ru(η5-C5H4(C5(CO2CH3)5)(η5-C5(CO2CH3)5)] (2).
two cyclic species and a 1,5-sigmatropic migration of a
methyl ester group located on the coupled ring. Sigmatropic
migration is an intramolecular process that is generally thermally
induced, whereby a σ-bond adjacent to one or more π-bonds
migrates to a new position in the molecule. There are examples in
the literature of other cyclopentadiene esters undergoing this
process,36,37 although these reactions are commonly performed
in inert solvents (toluene, chlorobenzene) at high temperature
(100 to 150 °C).36 In 1994, the 1,5-sigmatropic rearrangement of
benzyl pentamethoxycarbonylcyclopentadiene was reported to
occur under pressure in a methanol-d4 solvent at 140 °C over a
period of three hours.37 No temperature fluctuations were noted
during the preparation of complex 2, so it appears that the
sigmatropic shift presented here is more facile, occurring at a
temperature of ∼60 °C. To the best of our knowledge, there are
no literature reports of carbon to carbon coupling involving
coordinated Cp ligands as observed here. Attempts to reproduce
the preparation of complex 2 including the use of inert atmo-
sphere, the replacement of H[C5(CO2CH3)5] with K[C5(CO2-
CH3)5], the use of reaction temperatures higher than 60 °C, the
addition of varied stoichiometric ratios of each reagent, and the
direct reaction of 1 with H[C5(CO2CH3)5], K[C5(CO2CH3)5],
and Ag[C5(CO2CH3)5] have not to date resulted in the isolation
of further samples of compound 2.
Following the preparation of complex 1, it was of interest
to pursue the synthetic preparation of a ruthenium penta-
substituted carboxylic acid. The first method incorporated
for the attempted synthesis of this molecule was a potassium
hydroxide catalyzed hydrolysis of complex 1 in H2O. The
pentasubstituted methyl ester was suspended in an aqueous
solution of KOH and then heated under reflux conditions.
The mixture was allowed to cool and then acidified to pH 1.0
through the addition of concentrated hydrochloric acid.
Acidification of the solution resulted in the formation of a
white precipitate, which was found to be highly hydroscopic
under standard atmospheric conditions. Crystals of this
Figure 2. Representative view of the two crystallographically
independent K[Ru(η5-C5H5)(η5-C5(CO2H)4(CO2))] H2O mole-
cules of 3.
3
molecule suitable for X-ray diffraction studies were grown
as the hydrate from a concentrated solution of H2O over a
period of three weeks. The structure of 3 was determined by
single-crystal X-ray diffraction and found to represent the
molecule K[Ru(η5-C5H5)(η5-C5(CO2H)4(CO2))]. A view of
the structure of complex 3 is shown in Figure 2.
The unit cell of 3 consists of two crystallographically inde-
pendent K[Ru(η5-C5H5)(η5-C5(CO2H)4(CO2))] H2O formula
3
units (Figure 2). The potassium cations participate in ion-
dipole interactions with the oxygen atoms of both the carbox-
ylate functional groups and the solvated water molecules
together with an extensive hydrogen-bonding network between
the carboxylate groups and water molecules.
Suprisingly, numerous attempts to fully acidify the complex
failed to protonate the remaining carboxylate anion to yield the
pentasubstituted carboxylic acid [Ru(η5-C5H5)(η5-C5(CO2H)5)]
(4). Preparation of complex 4 was therefore achieved through an
acid-catalyzed hydrolysis of complex 1 using concentrated
hydrochloric acid in a H2O solvent. This reaction afforded pure
(36) Schmidt, P.; Hoffman, R. W.; Backes, J. Angew. Chem., Int. Ed.
Engl. 1972, 11, 513.
(37) Jefferson, E. A.; Warkentin, J. J. Org. Chem. 1994, 59, 463.

6240 Organometallics, Vol. 29, No. 23, 2010
Micallef et al.
A relatively stable and versatile acid halide intermediate
was generated using a modified literature procedure for the
generation of monofluorocarbonyl ferrocene.38 Synthesis of
pentafluorocarbonyl ruthenocene [Ru(η5-C5H5)(η5-C5(COF)5)]
(10) was achieved in a 60% yield under an argon atmosphere
at 0 °C through the reaction between ruthenocenepentacar-
boxylic acid (4), cyanuric fluoride, and pyridine in anhydrous
dichloromethane. Unlike monofluorocarbonyl ruthenocene,
complex 10 does not possess a high level of stability, decom-
posing in a matter of hours if exposed to the atmosphere.
Complex 10 must therefore be stored under an inert atmo-
sphere or prepared prior to use. Due to this instability, accurate
elemental analysis results could not be achieved for complex 10;
however 1H NMR analysis in deuterated chloroform immedi-
ately postsynthesis did confirm the presence of pentafluoro-
carbonyl ruthenocene.
Complex 10 was found to be a more versatile starting
material than ruthenocenepentacarboxylic acid (4), with the
substitution reactions between pentafluorocarbonyl rutheno-
cene and either phenol or 1-propanethiol in the presence of
the nucleophilic acylation catalyst 4-dimethylaminopyridine
(DMAP) yielding complexes 11 and 12 in yields of 16% and
33%, respectively (Scheme 3). Complex 11 precipitated from
the reaction mixture as pale yellow crystals suitable for single-
crystal X-ray diffraction studies. Structural analysis confirmed
the molecule as [Ru(η5-C5H5)(η5-C5(CO2C6H5)5)], with one
molecule constituting the asymmetric unit (Figure 4). The
Ru-C(11-15) and Ru-C(21-25) bond lengths of 2.159(3)-
Figure 3. Representative view of [Ru(η5-C5H5)(η5-C5(CO2H)5)]
2H2O (compound 4).
3
Scheme 2. Synthesis of Pentasubstituted Ruthenocenyl Esters
5-9
˚
˚
2.172(3) A and 2.156(6)-1.168(4) A are very similar to those
reported for compound 1.32
In summary, this article details a range of versatile syn-
thetic methods capable of preparing pentasubstituted ruthene-
cenyl derivatives incorporating strong electron-withdrawing
substituents. The library of pure, pentasubstituted ruthenocenyl
esters prepared during this study also afforded us the opportu-
nity to assay these complexes and ascertain their biological
activity. Where possible (stability pending), all prepared com-
plexes (1-12) were characterized using Fourier transform infra-
red and NMR spectroscopy (1H and 13C), electrospray mass
spectrometry, and microanalysis (C, H %) prior to biological
evaluation, with single-crystal X-ray structural determinations
obtained for four complexes: [Ru(η5-C5H4(C5(CO2CH3)5)(η5-
samples of [Ru(η5-C5H5)(η5-C5(CO2H)5)] (4) as a pale yellow
powder in a yield of 95%. Crystals of this compound as the
dihydrate were obtained by slow evaporation of a concentrated
solution in H2O over several weeks, and the single-crystal X-ray
structure was determined. A representative view of complex 4 is
shown in Figure 3.
The crystal structure of 4 consists of discrete molecules of
the compound, with one molecule and two solvated water
molecules constituting the asymmetric unit. The carbon
atoms in the two rings are eclipsed with extensive O-H
O
C5(CO2CH3)5)] (2), K[Ru(η5-C5H5)(η5-C5(CO2H)4(CO2))]
3 3 3
3
hydrogen-bonding networks observed in the crystal lattice
between the carboxylic acid groups and water molecules.
The pentasubstituted carboxylic acid (4) was found to be a
viable intermediate suitable for the preparation of a range of
pentasubstituted ruthenocenyl esters. Complex 4 readily
esterifies in the presence of an alcohol solvent and a catalytic
volume of concentrated hydrochloric acid under reflux con-
ditions (Scheme 2).
H2O (3), [Ru(η5-C5H5)(η5-C5(CO2H)5)] 2H2O (4), and [Ru-
3
(η5-C5H5)(η5-C5(CO2C6H5)5)] (11).
Cell Survival Studies. Ruthenocenepentacarboxylic acid
(4) and the pentasubstituted ruthenocenyl esters (1, 5-9, 11,
and 12) were screened for cytotoxic activity using a SRB
(sulforhodamine B) colorimetric assay of cell survival num-
ber following drug treatment in microtiter wells for 6 days.39
Cell lines chosen for this study included MCF7 (hormone-
dependent breast cancer), DU145 (prostate cancer grade II),
CI80-13S (ovarian cancer), two individual phenotypes of
human melanoma (MM96L and MM418c5), and a control
human fibroblast (NFF, neonatal foreskin fibroblasts).
Results obtained using this assay are listed (Table 1) and
demonstrate the pentasubstituted ruthenocenyl derivatives
to be, on average, relatively weak growth inhibitors of each
tumorigenic cell line. The averaged antiproliferative activ-
ity of these ruthenocenyl derivatives follows the sequence
This method afforded the preparation of a range of novel
pentasubstituted alkyl esters (5-9), each obtained as yellow,
viscous oils in yields in the range 22-89%. Limitations to
this procedure were encountered, however, when attempting
to synthesize esters using poor nucelophiles such as phenol
and 1-propanethiol, with these reactions prompting minimal
(∼1%) to no conversion of the pentacarboxylic acid to the
target esters. It was therefore necessary to convert complex 4
to a more electrophilic intermediate such as an acyl halide
prior to further attempting these syntheses.
(39) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J.; Bokesch, H.; Kenney, S.; Boyd, M. J. Natl.
Cancer Inst. 1990, 82, 1107.
(38) Galow, T. H.; Rodrigo, J.; Cleary, K.; Cooke, G.; Rotello, V. M.
J. Org. Chem. 1999, 64, 3745.

Article
Organometallics, Vol. 29, No. 23, 2010 6241
Scheme 3. Generation of Pentafluorocarbonyl Ruthenocene and Its Subsequent Conversion into Pentasubstituted Ester
Complexes (11 and 12)
respectively. The pentasubstituted methyl ester (1) and car-
boxylic acid (4) are particularly ineffectual under these assay
conditions, with these two complexes achieving IC50 values
exceeding the maximum concentration used during the SRB
assay (1000 μM) against five of the six cell lines tested. The
pentasubstituted propyl ester (6) exhibits a reasonable degree of
activity, achieving low micromolar IC50 values against the CI80-
13S (12.0 μM), MM418c5 (14.0 μM), MM96L (38.0 μM), and
MCF7 (45.0 μM) tumorigenic cell lines, respectively. Cellular
specificity of the propyl ester is also high, with this complex
demonstrating, on average, 37-fold greater growth inhibition of
cancerous cells versus control human fibroblasts (NFF). Com-
plex 9, the pentasubstituted 3-pentyl ester, also achieves a
respectable IC50 value against CI80-13S (12.0 μM) but is
relatively inactive toward the other cell lines. Complex 12, the
pentasubstituted propylthiol ester, demonstrates a unique cyto-
toxic profile compared to the alkyl esters. This complex exhibits
a particular affinity for inhibition of the MM96L tumor cell line,
achieving the lowest IC50 value (11.0 μM) of any complex
screened during this study. Against the CI80-13S (30.0 μM),
MM418c5 (290.0 μM), and MCF7 (404.0 μM) tumor cell lines,
however, complex 12 is shown to be substantially less active than
either complex 6 or 9, respectively.
An interesting observation is made upon comparison of
these results with biological data obtained on a series of prior
published cationic ruthenium(II) arene pentamethylcyclo-
pentadienyl (Cp*) sandwich complexes of the structure [(η5-
C5(CH3)5)Ru(η6-C6H5-R)]þ, where R = n-propyl ester (13)
and n-propyl ketone (14).22 In addition to the literature IC50
values for complexes 13 and 14 obtained against MCF7,
MM96L, and NFF,22 these cationic organoruthenium salts
were also assayed against DU145, CI80-13S, and MM418c5 for
the purpose of this study. The combined series of results for
complexes 13 and 14 are presented (Table 1) and demonstrate
these cationic orgaonruthenium sandwich complexes to be
potent growth inhibitors of each tumorigenic cell line, achieving
low micromolar IC50 values (2.28-8.17 μM). Complexes 13 and
14 demonstrate, on average, 22- and 15-fold greater growth
inhibition of cancerous cells, respectively, compared to the most
active pentasubstituted ruthenocenyl alkyl ester (6). Complexes
6, 13, and 14 are structurally and chemically similar, with the
only major difference between the three molecules being the
presence of the delocalized cationic charge on complexes 13 and
Figure 4. Top-down (above) and representative (below) views
of [Ru(η5-C5H5)(η5-C5(CO2C6H5)5)] (compound 11).
14. It would appear that this cationic charge drastically increases
the biological activity of the ruthenium(II) full-sandwich com-
plexes, a result comparative to that observed for the iron(II) full-
sandwich complexes ferrocene and ferricenium. The ferricenium
salts are delocalized cationic derivatives of ferrocene and, unlike
ferrocene (which is nontoxic), have been reported to inhibit
the growth of Ehrlich ascites tumors, B16 melanoma, colon 38
6 > 9 > 12 > 5 > 8 ≈ 7 > 10 > 1 ≈ 4. Biological activity
appears at least partially governed via complex lipophilicity,
with cytotoxicity increasing noticeably upon increasing the
alkyl chain length of each pentasubstituted complex from the
acid (4) to the methyl (1), ethyl (5), and propyl (6) esters,

6242 Organometallics, Vol. 29, No. 23, 2010
Micallef et al.
Table 1. Inhibitory Concentration That Limits Cellular Proliferation by 50% (IC50) for the Pentasubstituted Ruthenocenyl Complexes
[Ru(η5-C5H5)(η5-C5(CO2R)5)] (1, 5-9, 11, and 12) and Two Cationic Ruthenium Arene Cp* Sandwich Complexes [Ru(η5-C5(CH3)5)-
(η6-C6H5-R)]þ (13 and 14)
IC50 values (μM)a
complex
R
NFF
MCF7
DU145
CI80-13S
MM96L
MM418c5
1
4
5
6
7
8
9
11
12
13
14
Me
H
Et
>1000
>1000
947
>1000
>1000
169
>1000
>1000
330
431
990
237
12.0
61.0
83.0
12.0
361
>1000
>1000
296
>1000
>1000
304
n-Pr
n-Bu
2-Pr
3-Pent
phenyl
n-thiopropyl
n-propyl ester
n-propyl ketone
907
45.0
560
605
206
830
340
38.0
774
264
112
793
11.0
14.0
581
257
100
962
>1000
400
218
>1000
930
10.6
92.2
>1000
>1000
824
312
950
708
4.98
8.17
>1000
404
30.0
2.72
3.86
>1000
290
2.33
4.99
>1000
2.54
2.28
>1000
3.77
6.32
>1000
ruthenocene
a Errors are within the range (5-10% of the reported value. Results are the average of three separate experiments.
carcinoma, Rauscher leukemia, and other experimental solid
tumor systems in vitro.8 It is unclear as yet how the presence of
this cationic charge influences the biological behavior of these
organometallic molecules; however altering the chemical charge
could impart these systems with redox activity, aid in solubility/
cellular uptake, or direct the molecules toward susceptible
biological targets.
The solution was cooled to <5 °C to prompt crystallization of the
product, with the resulting pale yellow-green crystals collected via
vacuum filtration. The product was washed with a 10 mL aliquot of
ice-cold methanol and then dried in vacuo. The complex was
identified by comparison of melting point, IR and NMR spectra
with literature values.32
Yield 0.66 g, 92%; mp 144-145 °C; IR (cm-1) 1726 (s, CdO),
1230 (s, C-O); ESMS (m/z) þve ion, calcd m/z for [M þ Na]þ
544.5, found 544.6, calcd m/z for [2 M þ Na]þ 1065.9, found
1064.5; -ve ion, calcd m/z for [C5(CO2CH3)5]- 355.3, found
355.2; 1H NMR (CDCl3) δ 3.80 (s, 15H, CH3), 4.93 (s, 5H,
C5H5); 13C NMR (CDCl3) δ 52.34 (CH3), 78.77 (C5H5), 82.04
(C5(CO2CH3)5), 165.79 (s, CO2).
Experimental Section
General Procedures. All reactions were undertaken on a
Schlenk line under argon unless stated otherwise. RuCl(PPh3)2(η5-
C5H5),40 K[C5(CO2CH3)5],34 and subsequently H[C5(CO2CH3)5]34
were prepared and isolated using literature methods. All other
starting materials and solvents used while conducting experiments
were commercial products (Aldrich) and used as received. Fourier
transform infrared spectroscopy was conducted on a Thermo
Nicolet-Nexus FT-IR spectrometer with all samples made up as KBr
discs. The following abbreviations apply to the intensity of peaks
found within the spectra: vs, very strong, s, strong, m, medium, w,
weak. Electrospray mass spectrometry experiments were conducted
on a direct injection Waters ZQ 4000 mass spectrometer utilizing
electrospray ionization. All data was processed using Mass Linx
version IV (IBM) software. 1H and 13C NMR spectra were obtained
on a 400 MHz Varian Gemini spectrometer with samples of
complexes 1, 2, and 5-12 being prepared in solutions of CDCl3.
Samples of complexes 3 and 4 were characterized in D2O solutions
due to insolubility in CDCl3. Peaks obtained for the deuterated
solvent were used as the internal reference points for the spectra
(reference peak: CDCl3, 1H, δ 7.26 ppm, 13C δ 77.0 ppm; D2O, 1H,
δ 4.67 ppm, 13C δ 66.5 ppm). All signals have been recorded using
their appropriate chemical shift (δ in ppm), multiplicity, integral
ratio, and coupling constants (Hz). The following abbreviations
apply to the signal multiplicity of peaks within spectra: s=singlet,
d=doublet, t=triplet, m=multiplet. All deuterated solvents were
supplied by Cambridge Isotope Laboratories and were used as
received. Microanalyses were performed by Mr. George Blazak at
the Microanalytical Unit of the University of Queensland.
Synthesis of [Ru(η5-C5H4(C5(CO2CH3)5)(η5-C5(CO2CH3)5)]
(2). On a single occasion, compound 2 was separated from a
preprepared sample of 1 using silica column chromatography
(1:4 EtOAc/Hex). Bright yellow crystals of the product were
then recrystallized from a concentrated solution of chloroform.
Yield 0.01 g, 0.7%; mp 219-221 °C; IR (cm-1) 1726 (s, CdO),
1230 (s, C-O); ESMS (m/z) þve ion, calcd m/zfor [M þ Li]þ 882.7,
found 882.3, calcd m/z for [M þ Na]þ 898.8, found 898.3; -ve ion,
calcd m/z for [C5(CO2CH3)5]- 355.3, found 355.2; 1H NMR
(CDCl3) δ 3.70 (s, 6H, CH3), 3.79 (s, 18H, CH3), 3.80 (s, 3H,
CH3), 3.91 (s, 3H, CH3), 5.06 (m, 2H, C5H4 meta), 5.85 (m, 2H,
C5H4 ortho); 13C NMR (CDCl3) δ 52.47, 52.69, 52.87, 53.16, 54.22
(CH3), 74.10 (C5H4), 80.48, 81.86, 83.17, 86.67 (C5(CO2CH3)5),
128.84, 132.19 (C5H4 ipso and C-C5H4), 161.47, 161.69, 164.17,
164.71, 165.37 (CO2).
Synthesis of [Ru(η5-C5H5)(η5-C5(CO2H)4(CO2K))] (3). Com-
plex 1 (0.40 g, 7.68 ꢀ 10-4 mol) and KOH (2.15 g, 3.84 ꢀ 10-2 mol)
were dissolved in 20 mL of H2O, and the resulting solution was
heated at reflux for a period of 48 h. The mixture was then
cooled to <5 °C, and the solution acidified to pH 1.0 through
the addition of concentrated HCl. This prompted the precipita-
tion of a fine microcrystalline precipitate, which was collected
via vacuum filtration and dried in vacuo. Crystals of the product
suitable for X-ray diffraction studies were grown from a con-
centrated solution of H2O.
Yield 0.32 g, 85%; mp 253-254 °C (dec); IR (cm-1) 3447, 2923
(m-br, O-H), 1710 (s, CdO); ESMS (m/z) -ve ion, calcd m/z for
Synthesis. Synthesis of [Ru(η5-C5H5)(η5-C5(CO2CH3)5)] (1). A
solution of H[C5(CO2CH3)5] (0.50 g, 1.40 ꢀ 10-3 mol) and silver
acetate (0.25 g, 1.50 ꢀ 10-3 mol) in methanol (20 mL) under low
light conditions was stirred at 60 °C for one hour in the presence
of O2. To this mixture was carefully added crushed RuCl(PPh3)2-
(η5-C5H5) (1.00 g, 1.38 ꢀ 10-3 mol), and the reaction stirred for a
further 16 h. A hot filtration was performed to remove insoluble
silver chloride, and the solution concentrated to ∼5 mL in vacuo.
1
[M - K]- 450.3, found 451.2; H NMR (D2O) δ 5.01 (s, 5H,
C5H5); 13C NMR (D2O) δ 78.28 (C5H5), 80.97 (C5(CO2H)5),
169.51 (s, CO2).
Synthesis of [Ru(η5-C5H5)(η5-C5(CO2H)5)] (4). Compound 1
(0.40 g, 7.68 ꢀ 10-4 mol) was dissolved in H2O (100 mL) and
heated at reflux for a period of 48 h in the presence of a catalytic
amount of concentrated HCl (0.1 mL). The H2O solvent was
removed in vacuo to yield the product as a pale yellow microcrystal-
line powder. Crystals of the product suitable for X-ray diffraction
studies were grown from a concentrated solution of H2O.
(40) Bruce, M. I.; Hameister, C.; Swincer, A. G.; Wallis, R. C. Inorg.
Synth. 1982, 21, 71.

Article
Organometallics, Vol. 29, No. 23, 2010 6243
Yield 0.33 g, 95%; mp 262-264 °C (dec); IR (cm-1) 3453,
(CDCl3) δ 1.26 (d, J = 6.6 Hz, 30H, CH3), 4.86 (s, 5H, C5H5), 5.06
(m, 5H, OCH); 13C NMR (CDCl3) δ 21.95 (CH3), 69.65 (CH),
78.58 (s, C5H5), 83.06 (C5(CO2CH(CH3)2)5), 164.85 (s, CO2). Anal.
Calcd for C30H40O10Ru: C 54.5, H 6.09. Found: C 54.4, H 6.11.
Synthesis of [Ru(η5-C5H5)(η5-C5(CO2CH(CH2CH3)2)5)] (9).
Compound 4 (0.20 g, 4.43 ꢀ 10-4 mol) was dissolved in 3-pentanol
(100 mL) and heated at reflux for a period of 48 h in the presence of
a catalytic amount of concentrated HCl (0.1 mL). The solvent was
removed in vacuo to yield an oily yellow residue, which was purified
using a TLC prep plate (1:1 ethyl acetate/hexane) to afford the
product as a yellow waxy solid.
2924 (m-br, O-H), 1709 (s, CdO); ESMS (m/z) þve ion, calcd
m/z for [M þ Na]þ 474.3, found 474.9, -ve ion, calcd m/z for
1
[M - H]- 450.3, found 450.8; H NMR (D2O) δ 4.99 (s, 5H,
C5H5); 13C NMR (D2O) δ 79.32 (C5H5), 82.08 (C5(CO2H)5),
170.58 (CO2). Anal. Calcd for C15H10O10Ru 2H2O: C 37.0, H
3
2.90. Found: C 36.9, H 2.86.
Synthesis of [Ru(η5-C5H5)(η5-C5(CO2CH2CH3)5)] (5). Com-
pound 4 (0.20 g, 4.43 ꢀ 10-4 mol) was dissolved in ethanol
(100 mL) and heated at reflux for a period of 48 h in the presence
of a catalytic amount of concentrated HCl (0.1 mL). The solvent
was removed in vacuo to yield an oily yellow residue, which was
purified using a TLC prep plate (2:3 acetone/hexane) to afford
the product as a bright yellow oil.
Yield 0.04 g, 22%; IR (cm-1) 1722 (s, CdO), 1213 (s, C-O);
ESMS (m/z) þve ion, calcd m/z for [M þ H]þ 803.1, found 802.8,
calcd m/z for [M þ Na]þ 825.1, found 824.7, calcd m/z for [2 M þ
Yield 0.23 g, 89%; IR (cm-1) 1727 (s, CdO), 1210 (s, C-O);
ESMS (m/z) þve ion, calcd m/z for [M þ H]þ 593.5, found 592.5,
1
Na]þ 1627.1, found 1626.7; H NMR (CDCl3) δ 0.93 (m, 30H,
CH2CH3), 1.63 (m, 20H, CH2CH3), 4.83 (m, 5H, OCH), 4.88
(s, 5H, C5H5); 13C NMR (CDCl3) δ 9.85 (CH2CH3), 26.10 (CH2-
CH3), 78.47 (OCH), 79.01 (C5H5), 83.15 (C5(CO2CH(CH2-
CH3)2)5), 165.19 (CO2). Anal. Calcd for C40H60O10Ru: C 59.9, H
7.54. Found: C 59.9, H 7.60.
1
calcd m/z for [M þ Li]þ 599.5, found 598.6; H NMR (CDCl3)
δ 1.30 (t, J = 7.2 Hz, 15H, CH3), 4.25 (q, J = 7.2 Hz, 10H, CH2),
4.86 (s, 5H, C5H5);13C NMR(CDCl3) δ14.26 (CH3), 61.93 (CH2),
78.75 (C5H5), 82.62 (C5(CO2CH2CH3)5), 165.36 (CO2). Anal.
Calcd for C25H30O10Ru: C 50.8, H 5.11. Found: C 50.0, H 5.23.
Synthesis of [Ru(η5-C5H5)(η5-C5(CO2(CH2)2CH3)5)] (6).
Compound 4 (0.20 g, 4.43 ꢀ 10-4 mol) was dissolved in n-
propanol (100 mL) and heated at reflux for a period of 48 h in the
presence of a catalytic amount of concentrated HCl (0.1 mL).
The solvent was removed in vacuo to yield an oily yellow residue,
which was purified using a TLC prep plate (1:1 ethyl acetate/
hexane) to afford the product as a yellow oil.
Synthesis of [Ru(η5-C5H5)(η5-C5(COF)5)] (10). A suspension
of 4 (0.23 g, 4.30 ꢀ 10-4 mol) and pyridine (0.35 mL, 4.30 ꢀ 10-3
mol) in DCM (25 mL) was cooled to 0 °C. Cyanuric fluoride
(0.37 mL, 4.30 ꢀ 10-3 mol) was added, and the reaction mixture
stirred at 0 °C for two hours. The mixture was poured into a
solution of ice-cold H2O (approximately 30 mL) and filtered,
and the organic layer collected. The solution was concentrated
in vacuo, and the product isolated as a pink crystalline powder
using silica column chromatography (1:5 ethyl acetate/hexane).
Yield 0.06 g, 60%; 1H NMR (CDCl3) δ 5.38 (s, 5H, C5H5).
Synthesis of [Ru(η5-C5H5)(η5-C5(CO2C6H5)5)] (11). Com-
pound 10 (0.07 g, 1.52 ꢀ 10-4 mol), phenol (0.14 g, 1.52 ꢀ
10-3 mol), and DMAP (0.19, 1.52 ꢀ 10-3 mol) were dissolved in
DCM (10 mL) and stirred for 16 h at room temperature. The
solvent was removed in vacuo to yield an oily yellow residue,
which was purified using silica column chromatography (1:1
ethyl acetate/hexane) to afford the product as yellow crystals
suitable for X-ray diffraction studies.
Yield 0.18 g, 61%; IR (cm-1) 1726 (s, CdO), 1206 (s, C-O);
ESMS (m/z) þve ion, calcd m/z for [M þ H]þ 662.8, found 662.5,
calcd m/zfor [M þ Li]þ 668.7, found 668.5, calcd m/zfor [M þ Na]þ
684.8, found 684.5; -ve ion, calcd m/z for [M - (CH2)2CH3]-
618.7, found 619.1, calcd m/z for [M - 2(CH2)2CH3]- 575.6, found
575.5; 1H NMR (CDCl3) δ 0.94 (t, J = 7.4 Hz, 15H, CH3), 1.67 (m,
10H, CH2CH2CH3), 4.13 (t, J = 7.4 Hz, 10H, CH2CH2CH3), 4.88
(s, 5H, C5H5); 13C NMR (CDCl3) δ 10.63 (CH3), 22.10 (CH2CH2-
CH3), 67.62 (CH2CH2CH3), 78.58 (C5H5), 82.63 (C5(CO2(CH2)2-
CH3)5), 165.41 (CO2). Anal. Calcd for C30H40O10Ru: C 54.5, H
6.09. Found: C 54.1, H 6.26.
Yellow crystals, yield 0.03 g, 16%; mp 190-191 °C; IR (cm-1
)
Synthesis of [Ru(η5-C5H5)(η5-C5(CO2(CH2)3CH3)5)] (7). Com-
pound 4 (0.20 g, 4.43 ꢀ 10-4 mol) was dissolved in n-butanol
(100 mL) and heated at reflux for a period of 72 h in the presence of
a catalytic amount of concentrated HCl (0.1 mL). The solvent was
removed in vacuo toyieldanoilyyellowresidue, which was purified
using a TLC prep plate (1:1 ethyl acetate/hexane) to afford the
product as a yellow oil.
1744 (s, CdO), 1592 (m, benzene), 1189 (s, C-O); ESMS (m/z)
þve ion, calcd m/z for [M þ Li]þ 838.8, found 838.6, calcd m/z
for [M þ Na]þ 854.8, found 854.2, calcd m/z for [2 M þ Na]þ
1686.6, found 1685.7; -ve ion, calcd m/z for [C5(CO2C6H5)5]-
1
665.7, found 665.1; H NMR (CDCl3) δ 5.22 (s, 5H, C5H5),
7.16-7.43 (m, 25H, C6H5); 13C NMR (CDCl3) δ 79.86 (C5H5),
82.77 (C5(CO2C6H5)5), 121.59, 126.55, 129.79, 150.69 (C6H5),
163.84 (CO2). Anal. Calcd for C45H30O10Ru: C 65.0, H 3.64.
Found: C 65.1, H 3.79.
Yield 0.17 g, 54%; IR (cm-1) 1732 (s, CdO), 1205 (s, C-O);
ESMS (m/z) þve ion, calcd m/z for [M þ H]þ 732.9, found 732.9,
calcd m/z for [M þ Li]þ 738.9, found 738.9, calcd m/z for [2 M þ
Li]þ 1470.8, found 1470.6, calcd m/z for [M þ Na]þ 754.9, found
755.1, calcd m/z for [2 M þ Na]þ 1486.8, found 1487.6; 1H NMR
(CDCl3) δ 1.01 (t, J = 7.4 Hz, 15H, CH3), 1.46 (m, 10H, (CH2)2-
CH2CH3), 1.71 (m, 10H, CH2CH2CH2CH3), 4.25 (t, J=7.4 Hz,
10H, CH2(CH2)2CH3), 4.95 (s, 5H, C5H5); 13C NMR (CDCl3) δ
13.97 (CH3), 19.36 ((CH2)2CH2CH3), 30.79 (CH2CH2CH2CH3),
65.94 (CH2(CH2)2CH3), 78.65 (C5H5), 82.66 (C5(CO2(CH2)3-
CH3)5), 165.47 (CO2). Anal. Calcd for C35H50O10Ru: C 57.4, H
6.89. Found: C 57.6, H 7.01.
Synthesis of [Ru(η5-C5H5)(η5-C5(CdO(SCH2)CH2CH3)5)]
(12). Compound 11 (0.12 g, 2.61 ꢀ 10-4 mol), 1-propanethiol
(0.24 mL, 2.64 ꢀ 10-3 mol), and DMAP (0.33, 2.64 ꢀ 10-3 mol)
were dissolved in DCM (10 mL) and stirred for 16 h at room
temperature. The solvent was removed in vacuo to yield an oily
yellow residue, which was purified using silica column chroma-
tography (1:9 ethyl acetate/hexane) to afford the product as a
pale yellow microcrystalline material.
Pale yellow microcrystals, yield 0.11 g, 33%; mp 91-92 °C; IR
(cm-1) 1656 (s, CdO), 1006 (s, C-S); ESMS (m/z) þve ion, calcd
m/z for [M þ Na]þ 765.1, found 765.3, calcd m/z for [2 M þ Na]þ
Synthesis of [Ru(η5-C5H5)(η5-C5(CO2CH(CH3)2)5)] (8). Com-
pound 4 (0.20 g, 4.43 ꢀ 10-4 mol) was dissolved in 2-propanol
(100 mL) and heated at reflux for a period of 48 h in the presence of
a catalytic amount of concentrated HCl (0.1 mL). The solvent was
removed in vacuo toyieldanoilyyellowresidue, which was purified
using a TLC prep plate (1:1 ethyl acetate/hexane) to afford the
product as a yellow oil.
1
1507.2, found 1507.9; H NMR (CDCl3) δ 0.98 (t, J = 7.4 Hz,
15H, (CH2)2CH3), 1.64 (m, 10H, CH2CH2CH3), 2.92 (t, J = 7.4
Hz, 10H, CH2CH2CH3), 5.06 (s, 5H, C5H5); 13C NMR (CDCl3)
δ13.65 ((CH2)2CH3), 22.72 (CH2CH2CH3), 33.31 (CH2CH2CH3),
82.15 (C5H5), 89.87 (C5(COS(CH2)2CH3)5), 189.41 (COS). Anal.
Calcd for C30H40O5S5Ru: C 48.6, H 5.43. Found: C 48.6, H 5.49.
Crystal Structure Determinations. Unique data sets for com-
pounds 2, 3, 4, and 11 were measured at 295(2) K within the
specified 2θmax limit using a Rigaku AFC 7R four-circle diffrac-
tometer [θ-2θ scan mode, monochromatized Mo KR radiation
Yield 0.08 g, 27%; IR (cm-1) 1726 (s, CdO), 1219 (s, C-O);
ESMS (m/z) þve ion, calcd m/z for [M þ H]þ 662.8, found 662.5,
calcd m/z for [M þ Li]þ 668.7, found 668.6, calcd m/z for [2 M þ
Li]þ 1330.5, found 1330.7, calcd m/z for [M þ Na]þ 684.8, found
684.5, calcd m/z for [2 M þ Na]þ 1346.5, found 1346.6; 1H NMR
˚
(λ = 0.71073 A), from a 12 kW rotating anode source], yielding

6244 Organometallics, Vol. 29, No. 23, 2010
Micallef et al.
N independent reflections, No with I > 2.0σ(I) being considered
“observed” and used in the expression of the conventional refine-
ment residual R. The structures were solved by direct methods and
refined by full-matrix least-squares using SHELXL9741 after
semiempirical absorption corrections based on ψ-scans. Aniso-
tropic thermal parameters were refined for all non-hydrogen atoms,
solution of ruthenocenepentacarboxylic acid (4) was prepared
by dissolving ∼10 mg of the complex in milli Q water (1 mL).
These stock solutions were diluted as necessary for testing. Cells
were seeded in 96-well microtiter plates at approximately 5000
cells per 100 μL (NFF), 3000 cells per 100 μL (MCF7, DU145,
CI80-13S, MM418c5), and 1000 cells per 100 μL (MM96L).
Seven dilutions of each drug were added to triplicate wells. The
plates were incubated for a period of 6 days prior to incorpora-
tion of the SRB staining method.39 The culture medium was
removed from the plates, and each plate was washed with
phosphate-buffered saline (PBS). The plates were fixed with
methylated spirits for 15 min, then washed with tap water. SRB
solution (50 μL, 0.4% sulforhodamine B dye (w/v) in 1% (v/v)
acetic acid) was added to each well and left at room temperature
for 45 min. The SRB solution was removed, and the plates were
washed quickly, once with tap water and twice with 1% (v/v)
acetic acid solution. In the case of the NFF cell assay, these
plates were washed three times with 1% (v/v) acetic acid solu-
tion. Tris base (100 μL, 10 mM, unbuffered, pH > 9) was added
to each well to solubilize the protein-bound dye. Plates were left
for 5 min, and then the absorbance was measured on a multiwell
plate reader at 564 nm. The percentage of surviving cells was
calculated from the absorbance of untreated control cells. The
IC50 values for the inhibition of cell viability were determined by
fitting the plot of the percentage of surviving cells against drug
concentration with a sigmoidal function.
while (x, y, z, Uiso)H were included and constrained at estimated
values. Neutral atom complex scattering factors were employed,
while computation used the TeXsan crystallographic software
package of Molecular Structure Corporation,42 ORTEP-3,43 and
PLATON.44 Crystal data for compounds 2, 3, 4, and11 are listed in
the Supporting Information of this article.
Crystallographic data for the structural analyses have been
deposited with the Cambridge Crystallographic Data Centre:
CCDC Nos. 782233-782236 for compounds 2, 3, 4, and 11,
respectively. Copies of this information may be obtained free of
charge from The Director, CCDC, 12 Union Road, Cambridge,
CB2 1EZ, UK, fax: þ44 1223 366 033; e-mail: deposit@ccdc.ac.uk;
or on the web: http://www.ccdc.cam.ac.uk.
Cell Survival Studies. All cell lines were cultured in heat-
inactivated fetal calf serum (10%, CSL, Australia) in RPMI
1640 medium supplemented with penicillin (100 U/mL), strep-
tomycin (100 μg/mL), and HEPES (3 mM) at 5% CO2, 99%
humidity at 37 °C. Primary human fibroblasts were obtained
from neonatal foreskin and cultured in the above medium.
Culture media was replaced every three days, and cell mono-
layers were split when 70-80% confluent. Routine mycoplasma
tests were performed using Hoescht stain and were always
negative.
Acknowledgment. We thank Griffith University, the
Queensland Institute of Medical Research, and the Eskitis
Institute for Cell and Molecular Therapies for support of
this work.
Stock solutions of 1, 5-9, 11, and 12 were prepared by
dissolving the complexes (∼10 mg) in DMSO (10 μL). A stock
(41) Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112.
(42) TeXsan for Windows, Version 1.06; Molecular Structure Corpora-
tion: The Woodlands, TX, USA, 1997-2001.
(43) Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
(44) Spek, A. L. J. Appl. Crystallogr. 2003, 36, 7.
Supporting Information Available: Crystallographic data
(including cif files) for compounds 2, 3, 4, and 11 (CCDC
782233-782236). This material is available free of charge via
the Internet at http://pubs.acs.org.