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Influence of Structural Variation on the Anticancer Activity of RAPTA-Type Complexes: ptn versus pta
Organometallics 2009, 28, 1165–1172
1165
Influence of Structural Variation on the Anticancer Activity of
RAPTA-Type Complexes: ptn versus pta
Anna K. Renfrew,† Andrew D. Phillips,† Alexander E. Egger,†,‡ Christian G. Hartinger,†,‡
Sylvain S. Bosquain,§ Alexey A. Nazarov,† Bernhard K. Keppler,‡ Luca Gonsalvi,§
Maurizio Peruzzini,§ and Paul J. Dyson*,†
Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL),
CH-1015 Lausanne, Switzerland, Institute of Inorganic Chemistry, UniVersity of Vienna, Währinger Strasse
42, 1090 Vienna, Austria, and Istituto di Chimica dei Composti Organometallici, Consiglio Nazionale delle
Ricerche (ICCOM-CNR), Via Madonna del Piano 10, 50019 Sesto Fiorentino, Firenze, Italy
ReceiVed September 16, 2008
A series of compounds of the general formula [M(η6-arene)(ptn)Cl]X (M ) Ru, Os; arene ) p-cymene,
benzene, toluene, hexamethylbenzene; ptn ) 3,7-dimethyl-7-phospha-1,3,5-triazabicyclo[3.3.1]nonane;
X ) Cl-, BF4-) have been prepared and characterized spectroscopically. X-ray diffraction was additionally
used to characterize four of the complexes in the solid state. The hydrolysis of the compounds was
studied, and their cytotoxicity was evaluated in A2780 ovarian cancer cells and found to be comparable
to that of known RAPTA complexes based on 7-phospha-1,3,5-triazatricyclo[3.3.1.1]decane (pta). The
reactivity of the complexes toward double-stranded oligonucleotides and the model protein ubiquitin
was investigated using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and
gel electrophoresis, demonstrating a strong preference for the formation of covalent adducts with the
protein. Correlations among compound structure, hydrolysis, biomolecular interactions, and cytotoxicity
are established.
Introduction
Cisplatin is currently one of the most widely used chemotherapeutic agents for the treatment of cancer, being applied in
over 50% of therapy schemes.1 However, its activity is impeded
by considerable side effects and a high incidence of drug
resistance. A large number of cisplatin analogues have been
developed with improved properties, although secondary effects
continue to present major problems and to date only two
compounds, carboplatin and oxaliplatin, have progressed to
worldwide clinical use.2,3
In recent years, increasing interest has turned to the development of non-platinum inorganic drugs, prominent research being
based on ruthenium, gallium, titanium, cobalt, iron, and gold
complexes.4-10 Ruthenium complexes have emerged as an
* To whom correspondence should be addressed. E-mail: paul.dyson@
epfl.ch.
†
Ecole Polytechnique Fédérale de Lausanne (EPFL).
‡
University of Vienna.
§
Consiglio Nazionale delle Ricerche (ICCOM-CNR).
(1) Jakupec, M. A.; Galanski, M.; Arion, V. B.; Hartinger, C. G.;
Keppler, B. K. Dalton Trans. 2008, 183–194.
(2) Gschwind, A.; Fischer, O. M.; Ullrich, A. Nat. ReV. Cancer 2004,
4, 361–370.
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G. Chimia 2007, 61, 716–724.
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J.; Bischoff, P.; Gaiddon, C.; Loeffler, J.-P.; Pfeffer, M. Eur. J. Inorg. Chem.
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attractive alternative due to their low general toxicity, probable
different mode of action, and diverse synthetic chemistry.
Moreover, certain ruthenium compounds have been found to
display a selectivity toward cancerous cells higher than that of
platinum drugs, leading to reduced side effects, and this
selectivity may be due to their ability to bind to the iron delivery
protein transferrin.1,11,12 Two ruthenium(III)-based drugs,
KP101913 and NAMI-A,14 have currently completed phase I
clinical trials. Both complexes behave quite differently from
cisplatin in vivo; NAMI-A has been shown to be a strong
inhibitor of metastasis while having little effect on primary
tumors,15 and KP1019 effectively reduces colorectal tumors
where cisplatin shows essentially no activity.13
More recently, investigations into the anticancer activity of
organometallic Ru(II) arene compounds have started to gain
attention.16 Different concepts have been followed, including
the development of mono- and bifunctional compounds, targeted
(11) Clarke, M. J.; Zhu, F.; Frasca, D. R. Chem. ReV. 1999, 99, 2511–
2533.
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C. G.; Allmaier, G.; Keppler, B. K. J. Anal. At. Spectrom. 2004, 19, 46–
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Zorbas, H.; Keppler, B. K. J. Inorg. Biochem. 2006, 100, 891–904.
(14) Rademaker-Lakhai, J. M.; van den Bongard, D.; Pluim, D.; Beijnen,
J. H.; Schellens, J. H. Clin. Cancer Res. 2004, 10, 3717–3727.
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Chem. 2004, 4, 1525–1535.
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Commun. 2001, 1396–1397.
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Bregman, H.; Knapp, S.; Meggers, E. Angew. Chem. 2006, 45, 1580–1585.
(18) Ang, W. H.; Daldini, E.; Juillerat-Jeanneret, L.; Dyson, P. J. Inorg.
Chem. 2007, 46, 9048–9050.
10.1021/om800899e CCC: $40.75 2009 American Chemical Society
Publication on Web 01/27/2009
1166 Organometallics, Vol. 28, No. 4, 2009
Renfrew et al.
Scheme 1. Synthesis of [M(η6-arene)(ptn)Cl]X (M ) Ru, Os;
X ) Cl-, BF4-), Including the NMR Numbering Scheme for
the ptn Ligand
Figure 1. Ru(arene)(pta) complexes RAPTA-C, RAPTA-B, and
oxaliRAPTA-C.
approaches and kinase inhibitors,17-22 and the evaluation of
multinuclear ruthenium arene compounds.23-27
RAPTA complexes of the general formula [Ru(η6-arene)(pta)X2] (X ) halide; Figure 1) show relatively low toxicity
in vitro, but in vivo studies on RAPTA-C ([Ru(η6-p-cymene)(pta)Cl2]) and RAPTA-B ([Ru(η6-benzene)(pta)Cl2]) revealed
excellent inhibition of metastasis growth in addition to high
selectivity and extremely low general toxicity.28 The pta ligand
appears to be essential in endowing the function and selectivity
of the RAPTA compound; for example, replacement of pta with
the methylated analogue pta-Me+ greatly increases toxicity in
a healthy cell model relative to cancer cells.28 In contrast, the
chloride ligands can be substituted for a bidentate oxalate ligand
with very little impact on cytotoxicity, although the rate of
reaction with biomolecules is reduced.29 Replacement of a
chloro ligand by a phosphine enables the hydrophobicity and
subsequent biological function to be tuned.30
In this paper the effect of replacing the pta ligand with a
chelating pta analogue, ptn, is described in order to evaluate
the effect of forming a P-N chelate complex without significantly altering the water solubility and acid-base properties of
the resulting complexes. While examples of Ru(II)-arene
complexes containing O or N chelating ligands are known to
display cytotoxicity,31-34 bidentate P,N chelates have not been
studied previously, although Pt drugs with hemilabile P,N
(19) Schmid, W. F.; John, R. O.; Muhlgassner, G.; Heffeter, P.; Jakupec,
M. A.; Galanski, M.; Berger, W.; Arion, V. B.; Keppler, B. K. J. Med.
Chem. 2007, 50, 6343–6355.
(20) Schmid, W. F.; John, R. O.; Arion, V. B.; Jakupec, M. A.; Keppler,
B. K. Organometallics 2007, 26, 6643–6652.
(21) Vock, C. A.; Ang, W. H.; Scolaro, C.; Phillips, A. D.; Lagopoulos,
L.; Juillerat-Jeanneret, L.; Sava, G.; Scopelliti, R.; Dyson, P. J. J. Med.
Chem. 2007, 50, 2166–2175.
(22) Ang, W. H.; De Luca, A.; Chapuis-Bernasconi, C.; JuilleratJeanneret, L.; Lo Bello, M.; Dyson, P. J. ChemMedChem 2007, 2, 1799–
1806.
(23) Mendoza-Ferri, M. G.; Hartinger, C. G.; Eichinger, R. E.; Stolyarova, N.; Severin, K.; Jakupec, M. A.; Nazarov, A. A.; Keppler, B. K.
Organometallics 2008, 27, 2405–2407.
(24) Mendoza-Ferri, M. G.; Hartinger, C. G.; Nazarov, A. A.; Kandioller,
W.; Severin, K.; Keppler, B. K. Appl. Organomet. Chem. 2008, 22, 326–
332.
(25) Schmitt, F.; Govindaswamy, P.; Suess-Fink, G.; Ang, W. H.; Dyson,
P. J.; Juillerat-Jeanneret, L.; Therrien, B. J. Med. Chem. 2008, 51, 1811–
1816.
(26) Auzias, M.; Therrien, B.; Suess-Fink, G.; Stepnicka, P.; Ang, W. H.;
Dyson, P. J. Inorg. Chem. 2008, 47, 578–583.
(27) Therrien, B.; Suess-Fink, G.; Govindaswamy, P.; Renfrew, A. K.;
Dyson, P. J. Angew. Chem., Int. Ed. 2008, 47, 3773–3776.
(28) Scolaro, C.; Bergamo, A.; Brescacin, L.; Delfino, R.; Cocchietto,
M.; Laurenczy, G.; Geldbach, T. J.; Sava, G.; Dyson, P. J. J. Med. Chem.
2005, 48, 4161–4171.
(29) Casini, A.; Mastrobuoni, G.; Ang, W. H.; Gabbiani, C.; Pieraccini,
G.; Moneti, G.; Dyson, P. J.; Messori, L. ChemMedChem 2007, 2, 631–
635.
(30) Scolaro, C.; Chaplin, A. B.; Hartinger, C. G.; Bergamo, A.;
Cocchietto, M.; Keppler, B. K.; Sava, G.; Dyson, P. J. Dalton Trans. 2007,
5065–5072.
(31) Habtemariam, A.; Melchart, M.; Fernandez, R.; Parsons, S.; Oswald,
I. D. H.; Parkin, A.; Fabbiani, F. P. A.; Davidson, J. E.; Dawson, A.; Aird,
R. E.; Jodrell, D. I.; Sadler, P. J. J. Med. Chem. 2006, 49, 6858–6868.
ligands have been found to display in vitro cytotoxicity
equivalent to that of cisplatin.35
Results and Discussion
A series of ruthenium(II)- and osmium(II)-arene complexes
with a ptn ligand were prepared in high yield in a single step
(Scheme 1). All complexes are air stable and, with the exception
of 4 · Cl, all complexes are highly water soluble (S(1, 25 °C) )
0.06 g mL-1) with moderate solubility in chlorinated solvents.
The tetrafluoroborate salts, prepared by metathesis of
1 · Cl-5 · Cl with NaBF4, have higher solubility in chlorinated
solvents.
Compounds 1 · Cl-5 · Cl were characterized by 1H and 31P
NMR spectroscopy in D2O and CD2Cl2. 1H-1H COSY and
1
H-13C HETCOR spectra of 1 · Cl were used to assign the
signals corresponding to the ptn ligand (Scheme 1). 1H-15N
correlation and COSY spectra were also recorded for 1 · Cl and
5 · Cl to corroborate peak assignments (see Experimental Section). The 31P NMR spectra of 1-5 exhibit a singlet in D2O
and CD2Cl2 (Table 1). In the 1H NMR spectrum of 1, the signals
of the p-cymene ring are split, indicative of a chiral Ru(II)
center. A closed-ring structure is implied by splitting of the
methylene groups (Scheme 1), also observed for 2-5. 15N NMR
spectra were recorded for complexes 1 · Cl and 5 · Cl; however,
no signal corresponding to the chelating nitrogen was observed,
possibly because the ligand undergoes a rapid, dynamic
opening-closing process involving κ2 T κ1 coordination.
(32) John, R. O.; Arion, V. B.; Jakupec, M. A.; Keppler, B. K.
Ruthenium(II)-arene complex with heterocyclic ligands as prospective
antitumor agent. In Metal Ions in Biology and Medicine; Alpoim, M. C.,
Morais, P. V., Santos, M. A., Cristóvão, A. J., Centeno, J. A., Collery, P.,
Eds.; Editions John Libbey Eurotext: Montrouge, 2006; Vol. 9, pp 4045.
(33) Peacock, A. F. A.; Parsons, S.; Sadler, P. J. J. Am. Chem. Soc.
2007, 129, 3348–3357.
(34) Vock, C. A.; Renfrew, A. K.; Scopelliti, R.; Juillerat-Jeanneret,
L.; Dyson, P. J. Eur. J. Inorg. Chem. 2008, 1661–1671.
(35) Habtemariam, A.; Sadler, P. J. Chem. Commun. 1996, 1785–1786.
Organometallics, Vol. 28, No. 4, 2009 1167
Table 1.
1 · Cl
2 · Cl
3 · Cl
4 · Cl
5 · Cl
ptn
a
P NMR Spectroscopic Data for 1 · Cl-5 · Cl
31
D2O solvent
CD2Cl2 solvent
-27.8
-28.7
-28.0
-31.7
-77.8
-89.0
-33.1a
-31.7
-31.4
-32.4
-80.0b
-91.2
For 1 · BF4. b Solvent CDCl3.
Structural Characterization of 1 · BF4-4 · BF4 in the
Solid State. Crystals of 1 · BF4-4 · BF4 suitable for X-ray
diffraction were obtained via slow diffusion (see the Experimental Section for details). Complex 2 · BF4 crystallized with
two symmetry-independent molecules within the unit cell.
Essentially, both entities have identical metric parameters; the
main difference lies in the rotation of the arene along the
Ru-centroid axis, one molecule featuring an eclipsing of an
arene carbon with the Ru-Cl bond.
Complexes 1 · BF4-4 · BF4 represent a unique series of complexes, in which the chelating κ2P,N ligand forms a six-membered
ring with Ru containing no unsaturated bonds. A survey of the
CSD shows that all Ru compounds with the P,N six-membered
ring feature either a substituted pyridine or the incorporation of a
phenyl group or imino functionality into the backbone of the ligand
to enforce rigidity.36 In contrast, the rigidity of the ptn molecule
stems from the incorporation of a tightly constrained CH2 bridgehead in the central core of the ligand, indicated by the small
N-C(H2)-N angle of ca. 112°. The CSD also shows that the
number of η6-arene-bound Ru complexes featuring a six-membered
κ2P,N chelating ligand is significantly lower than the corresponding
five-membered-ring-containing complexes: 53 versus 13. The four
complexes shown in Figure 2 exhibit the typical three-legged pianostool geometry associated with Ru(II)-arene complexes. Not
including the substituents on the arene, the local Cs symmetry is
observable from bisection of the Ru-Cl bond, which divides
through the middle of the ptn ligand where the mirror plane contains
the N-CH2-N bridgehead fragment. The positioning of the ptn
with respect to the arene-Ru-Cl unit is similar in all complexes,
where a slight turning about the P-N(terminal) axis is observed,
and the bulkiest arene, η6-C6Me6, induces the largest degree of
rotation. Metric parameters pertaining to the P center and terminal
nitrogen are unreliable due to a positional disordering of the κ2ptn ligand where the P and terminal N centers overlap with
occupancies ranging from 0.50:0.50 in 4 · BF4 to 0.89:011 in 1 · BF4.
Analysis of the electron density difference maps reveals that the
disorder is restricted to a single plane comprising P, N, and Ru
atoms, and no disorder is present in the orthogonal direction. The
bridgehead carbon center, N-CH2-N, shows no indication of
disorder and is rigidly constrained; thus, the Ru · · · C(H2) distance
is a useful measure of the κ2-ptn-Ru interaction. The bridge
supporting nitrogens also show some degree of bond strain, as
indicated by a relatively planar geometry (sum of C-N-C bond
angles: average of 1 · BF4-4 · BF4 336.3° versus 331.94° in
N(CH3)337 and 330.23° in uncoordinated pta).38 The P-Ru-N
bond angles in complexes 1 · BF4-4 · BF4 range from 79.61(5) to
82.88(9)°. These values represent the lower limit of data relative
to other complexes with six-membered rings containing an arenebound Ru moiety chelated by a κ2-P,N ligand: 79.35-93.03°. The
(36) Allen, F. H. Acta Crystallogr., Sect. B: Struct. Sci. 2002, B58, 380–
388.
(37) Boese, R.; Blaser, D.; Antipin, M. Y.; Chaplinski, V.; de Meijere,
A. Chem. Commun. 1998, 781–782.
(38) Marsh, R. E.; Kapon, M.; Hu, S.; Herbstein, F. H. Acta Crystallogr.,
Sect. B: Struct. Sci. 2002, B58, 62–77.
Figure 2. X-ray structures of the cations in 1 · BF4-4 · BF4 with
50% probability thermal ellipsoids (the BF4-counterions are omitted
for clarity). For 3 · BF4 only one cation is shown.
basic geometry of the complexes is governed by the steric profile
of the coordinating arene. For example, 2 · BF4 with the smallest
arene, η6-C6H6, has the shortest Ru-Cl bond lengths (2.398(2) and
2.400(2) Å), Ru-arene(centroid) length (2.200(8) Å), and
Ru-ptn(bridgehead CH2) distance (4.325(6) Å). The complex
containing the most sterically demanding arene, η6-C6Me6, has the
longest Ru-P (4.400(5) Å) and Ru-Cl distances (2.431(1) Å).
The Ru-arene(centroid) distance in 4 · BF4 is long (1.770(2) Å),
despite the hexamethylbenzene ring being the strongest π-donor
of the series. The positioning of the ptn ligand with respect to the
Cl center shows minor variation (see Table 2), whereas the bond
angle associated with the arene can vary up to 5°, which is again
related to the steric profile of the coordinating arene. In comparison
to the bis-coordinated, nonchelated (η6-C6H5CH2CH2NH2-κ1N)RuCl(pta-κ1P) complex,39 which features both P and N centers bound
to Ru, the arene distance of 1.701(3) Å is considerably shorter
than in 1 · BF4-4 · BF4; however, the Ru-Cl distance in the
tethered complex (2.417(2) Å) is comparable.
Characterization of 1 · Cl-5 · Cl in Aqueous Solution. The
hydrolysis behavior of 1 · Cl-5 · Cl was studied under pseudopharmacological conditions. Hydrolytic decomposition is central
to the mode of action associated with cisplatin40 and is also
thought to be relevant for mono- and dichloro ruthenium(II)-arene
based drugs,41 which undergo rapid hydrolysis in water contain(39) 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, 756–765.
(40) Lippert, B., Cisplatin: Chemistry and Biochemistry of a Leading
Anticancer Drug, 1st ed.; Helvetica Chimica Acta/Wiley-VCH: Zürich/
Weinheim, 1999; p 576.
(41) Scolaro, C.; Hartinger, C. G.; Allardyce, C. S.; Keppler, B. K.;
Dyson, P. J. J. Inorg. Biochem. 2008, 102, 1743–1748.
1168 Organometallics, Vol. 28, No. 4, 2009
Renfrew et al.
Table 2. Selected Bond Lengths (Å) and Angles (deg) for
1 · BF4-4 · BF4
1 · BF4
2 · BF4b
3 · BF4
4 · BF4
Ru-Cl
2.422(2)
2.423(2)
2.431(1)
Ru-arene(centroid)
1.733(2)
1.741(3)
1.786(2)
Ru-Ca
4.377(4)
2.400(2)
2.398(2)
1.724(3)
1.722(3)
4.345(9)
4.325(6)
2.25(2)
2.35(4)
2.20(5)
2.36(6)
2.25(1)
2.27(1)
2.24(3)
2.20(1)
124.0(1)
124.6(1)
92.7(1)
92.2(1)
143.3(1)
143.2(2)
88.7(1)
87.1(3)
90.5(2)
89.7(6)
88.8(12)
86.5(12)
88.5(7)
93.2(7)
79.5(14)
79.9(15)
78.6(6)
80.6(9)
112.2(5)
110.8(7)
4.346(6)
4.398(4)
2.28(3)
2.35(3)
2.24(3)
2.31(3)
2.27(1)
2.20(1)
2.27(1)
2.25(1)
123.3(1)
120.41(6)
91.8(1)
90.79(4)
144.9(1)
148.4(1)
87.9(3)
89.2(3)
90.4(2)
90.1(3)
92.7(7)
89.8(7)
88.6(7)
89.5(10)
81.0(9)
79.8(8)
78.2(9)
77.8(8)
112.4(5)
112.8(3)
c
2.34(2)
2.26(4)
Ru-Pc
2.27(1)
2.25(1)
Cl-Ru-arene(centroid)
121.81(5)
Ca-Ru-Cl
90.0(1)
Ca-Ru-arene(centroid)
148.15(6)
Cl-Ru-Pc
89.0(2)
88.5(2)
Cl-Ru-Nc
86.3(6)
93.9(9)
P-Ru-Nc
80.6(7)
77.1(10)
N-Ca-N
112.0(3)
Ru-N
a
Denotes the bridgehead carbon position. b Two crystallographically
independent molecules are present in the unit cell. c The value should be
treated with caution, due to the disorder of the P1 and N1 positions in
the coordinated ptn ligand.
ing 5 mM NaCl (the approximate salt concentration inside a
cell).42 The process is reversed by addition of 100 mM NaCl
(corresponding to the salt concentration of blood plasma).
However, the ptn-containing complexes are much less prone to
hydrolysis. ESI-MS of compounds 1 · Cl-3 · Cl and 5 · Cl in
water provide a strong peak corresponding to the parent cation,
[M(η6-arene)(ptn)Cl]+, the most abundant peak in each spectrum, with the aqua (or potentially hydroxo) complex, [M(η6arene)(ptn)(H2O) - H]+, present at ca. 15% relative intensity
for 2 · Cl and 3 · Cl and <5% for 1 · Cl and 5 · Cl.
The stability of 1 · Cl-5 · Cl in D2O (5 mM NaCl) was monitored
by 1H and 31P NMR spectroscopy over a prolonged period (in the
case of 4 · Cl the solution contained 1% v/v DMSO to aid
solubility). Both 1 · Cl and 2 · Cl were found to be remarkably stable
in solution, with no evidence of decomposition after 7 days. In
the case of 3 · Cl and 5 · Cl, a new species, probably corresponding
to the hydrolysis product [M(arene)(ptn)(H2O)]2+, is evident in the
31
P NMR spectra after 24 h. In addition, a small amount of free
arene (<5% intensity) is observed after 3 days, evidenced by 1H
NMR spectroscopy. Only 4 · Cl shows significant decomposition
with complete disappearance of the original compound after 3 days,
accompanied by the formation of two decomposition products in
which the arene ligand has been lost, possibly due to the greater
steric demand of the η6-hexamethylbenzene ligand relative to the
other arenes.
(42) Hartinger, C. G.; Schluga, P.; Galanski, M.; Baumgartner, C.;
Timerbaev, A. R.; Keppler, B. K. Electrophoresis 2003, 24, 2038–2044.
Figure 3. FT-ICR WSIM mass spectra of the reaction mixtures of
RAPTA-C and 1 · Cl with ubiquitin in a complex to protein ratio
of 2:1 after 72 h incubation.
Reactivity of 1 · Cl -5 · Cl toward Model Biomolecular
Targets. Proteins are known to react quickly with metallodrugs
after intravenous administration, and both human serum albumin
and transferrin are considered as important carriers of anticancer
metal complexes.43 In recent years, metal binding to a number
of small proteins with a comparatively low number of potential
binding sites have been studied by mass spectrometry.29,44-46
In particular, the model protein ubiquitin was found useful for
this approach, and metal binding sites can even be determined
by tandem mass spectrometry.46 The reaction of RAPTA-C,
1 · Cl, and 5 · Cl toward ubiquitin was monitored by mass
spectrometry. RAPTA-C and 1 · Cl give essentially the same
type of conjugates after 72 h incubation (Figure 3). However,
initially the reaction of the ptn complex appears to proceed more
quickly, with the Ub-Ru(p-cymene) adduct being the most
abundant peak in the mass spectrum after 24 h. In contrast, for
the Os complex 5 · Cl no assignable adducts were observed even
after incubation for 72 h.
Most metal-based drugs are thought to act by binding to DNA,
thereby preventing replication of the cell.47 The electron-rich
N7 site of guanosine has been identified as the preferred binding
site for most inorganic drugs,48-50 and RAPTA compounds have
previously been found to react with guanosine at the N7 site to
form [Ru(η6-arene)(guanosine)(pta)Cl]+ complexes, while displaying very little reactivity toward other bases.51 In order to
determine whether a similar selectivity is exhibited by the ptn
complexes, D2O solutions of 1 · Cl, 3 · Cl, and 5 · Cl were
incubated at 37 °C with 1 mol equiv of guanosine 5′monophosphate (GMP) and monitored by 1H and 31P NMR
spectroscopy. 1:1 solutions of RAPTA-C in D2O and in 100
mM NaCl in D2O were also studied for comparison.
RAPTA-C reacts rapidly with GMP, with a new species
forming after 10 min, evidenced by the change in frequency of
(43) Timerbaev, A. R.; Hartinger, C. G.; Aleksenko, S. S.; Keppler, B. K.
Chem. ReV. 2006, 106, 2224–2248.
(44) Gibson, D.; Costello, C. E. Eur. Mass Spectrom. 1999, 5, 501–
510.
(45) Hartinger, C. G.; Ang, W. H.; Casini, A.; Messori, L.; Keppler,
B. K.; Dyson, P. J. J. Anal. At. Spectrom. 2007, 22, 960–967.
(46) Hartinger, C. G.; Tsybin, Y. O.; Fuchser, J.; Dyson, P. J. Inorg.
Chem. 2008, 47, 17–19.
(47) Reedijk, J. Chem. ReV. 1999, 99, 2499–2510.
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102, 121–129.
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Organometallics, Vol. 28, No. 4, 2009 1169
Figure 4. FT-ICR-MS peak patterns (top, experimental; bottom, calculated) of the most abundant 7-fold negatively charged adducts (bottom)
resulting from incubation of (from left to right) 1 · Cl, 5 · Cl,and RAPTA-C with ds(5′GTATTGGCACGTA) in a complex to oligonucleotide
ratio of 5:1. The double-stranded oligonucleotide was separated into its complementary single strands (GTATTGGCACGTA and
TACGTGCCAATAC) during MS. Single asterisks indicate a peak also present in the original oligonucleotide sample. Double asterisks
indicate a poorly shaped peak, probably due to overlap with an impurity, and hence the experimental and calculated m/z values are given
for the neighboring peaks.
the H8 proton in the 1H NMR spectrum and of the P atom of
the pta ligand in the 31P NMR spectrum. The reaction reaches
completion after 4 h, and no new species are formed over 72 h.
In the presence of 100 mM NaCl, hydrolysis is suppressed and
no reaction with GMP is observed after 3 days.
Compound 1 · Cl does not react with GMP; following 72 h
of incubation only unreacted 1 · Cl and GMP are visible in the
NMR spectra, with no evidence of hydrolysis or decomposition.
In the case of 3 · Cl and 5 · Cl, a new 31P NMR signal attributed
to the aqua hydrolysis product is visible after 4 h of incubation;
however, there is no evidence for reaction with GMP and the
spectra remain unchanged after 24 h. The experiment was
repeated with adenosine 5′-monophosphate, but no reaction was
observed for 1 · Cl, 3 · Cl, or 5 · Cl over 24 h.
Related [Ru(η6-arene)(en)Cl]+ compounds have been shown
to bind to DNA oligonucleotides, forming monofunctional
adducts, selectively binding to guanine following loss of the
chloride ligand.52 Oligomer binding studies using the singlestranded oligomer 5′-ATACATGGTACATA-3′ have been carried out on a series of RAPTA complexes and their osmium
analogues, the adducts being analyzed by ESI-MS.53 The
ruthenium compounds were found to form adducts based on
either a Ru-arene-pta fragment following the substitution of
the chloride ligands or a Ru-pta unit following additional loss
of the arene, with a higher percentage of Ru-pta adducts
observed for arenes with electron-withdrawing substituents.39,53
In contrast, the osmium analogues displayed lower reactivity
toward DNA, with only Os-arene-pta adducts formed due to
the stronger Os-arene bond. Loss of the pta ligand has not been
observed, although pta loss on protein binding readily occurs.29,30
To determine the reactivity of the ptn complexes relative to those
containing pta, with respect to the formation of covalent interactions, the reactions of 1 · Cl, 5 · Cl, and RAPTA-C with the doublestranded oligomer 5′-GTATTGGCACGTA-3′ were assessed by
(51) Dorcier, A.; Hartinger, C. G.; Scopelliti, R.; Fish, R. H.; Keppler,
B. K.; Dyson, P. J. J. Inorg. Biochem. 2008, 102, 1066–1076.
(52) Morris, R. E.; Aird, R. E.; Murdoch, P. d. S.; Chen, H.; Cummings,
J.; Hughes, N. D.; Parsons, S.; Parkin, A.; Boyd, G.; Jodrell, D. I.; Sadler,
P. J. J. Med. Chem. 2001, 44, 3616–3621.
(53) Dorcier, A.; Dyson, P. J.; Gossens, C.; Rothlisberger, U.; Scopelliti,
R.; Tavernelli, I. Organometallics 2005, 24, 2114–2123.
FT-ICR-MS and gel electrophoresis. For each complex, samples
were incubated at 1:1 and 5:1 complex to oligomer ratios and
analyzed by nESI-FT-ICR-MS after 24 and 72 h. As was observed
with the single-stranded oligomer, RAPTA-C reacts to form mainly
Ru-arene-pta (up to 70% relative intensity, Figure 4) and smaller
amounts of Ru-pta adducts (up to 10%, referenced to unreacted
single-stranded 5′-GTATTGGCACGTA-3′, the most abundant
peak at charge state 7) with no difference in the spectra after 24 or
72 h at a ratio of 1:1. In comparison 1 · Cl is less reactive, with no
adducts formed at a complex to oligonucleotide ratio of 1:1; the
intensity of unreacted oligonucleotide remains unchanged, although
on increasing the concentration of complex to a 5-fold excess,
adducts of ca. 10% relative intensity corresponding to a monosubstituted Ru-arene-ptn fragment are formed after 24 h (Figure
4). Interestingly, this species is no longer present in spectrum
recorded after 72 h; instead, species in which the arene has been
lost are observed.
Complex 5 · Cl shows very low reactivity toward the oligomer; following 24 h incubation with a 5-fold excess of complex,
only a small proportion of the complex had reacted to give an
Os-arene-ptn adduct (15% relative intensity, Figure 4). After
72 h of incubation the main peaks in the spectrum remained
unchanged. It is worth noting that no signals which could
correspond to arene loss were observed, which is in keeping
with the stronger Os-arene bond. For the Ru complexes the
signal to noise ratio continuously deteriorated, suggesting that
the oligonucleotide steadily degraded with time, an effect that
could also be due to noncovalent interactions between the
complexes and oligonucleotide. Polyacrylamide gel electrophoresis was used to directly determine whether 1 · Cl, 5 · Cl,
and RAPTA-C could degrade the oligonucleotide. In agreement
with the MS data, gel electrophoresis confirmed that RAPTA-C
degraded the oligonucleotide to the greatest extent (Figure 5,
top). In contrast, for 1 · Cl a 5-fold excess of the complex is
required for a significant degradation of the oligonucleotide
(Figure 5, middle) and 5 · Cl is the least reactive (Figure 5,
bottom).
In Vitro Activity. The in vitro activity of 1 · Cl-5 · Cl was
determined using the MTT test in the ovarian cancer cell line
A2780, and antiproliferative activity was found to follow the
1170 Organometallics, Vol. 28, No. 4, 2009
Renfrew et al.
against solid lung metastasis28 and Ehrlich ascites carcinoma54
in the absence of observable side effects.
Conclusions
A series of water-soluble compounds of the general formula
[M(η6-arene)(ptn)Cl]+ (M ) Ru, Os) have been synthesized and
their crystal structures, reactivity with model (potential) biomolecular targets, and cytotoxicities determined. The complexes
are relatively resistant to hydrolysis and are stable even at high
chloride concentrations and at low pH, making the possibility
of a ring-opening activation mechanism under physiological
conditions unlikely. The Ru-ptn complexes show in vitro
activities and reactivity toward ubiquitin comparable to those
of their pta analogues, although their reactivity toward DNA is
lower, possibly due to a much slower rate of hydrolysis. It has
previously been proposed that proteins are a more important
target for RAPTA-type complexes than DNA,55 which is
consistent with the results described herein.
Experimental Section
Figure 5. Gel electrophoresis of RAPTA-C (a), 1 · Cl (b), and 5 · Cl
(c) following incubation for 24 and 72 h with oligonucleotide at
ratios of 1:1 and 5:1. Equal amounts of the oligonucleotide (0.8
µg) were placed on the gel together with standards of 0.8, 0.4, and
0.08 µg. RAPTA-C degrades the oligonucleotide most effectively,
1 · Cl only at a drug to oligonucleotide ratio of 5:1, and 5 · Cl does
not degrade the oligonucleotide.
Table 3. IC50 Values for 1 · Cl-5 · Cl and RAPTA-C in A2780 Cells
complex
IC50 (µM)
1 · Cl
2 · Cl
3 · Cl
4 · Cl
5 · Cl
ptn
RAPTA-C
278 ( 12
179 ( 8
154 ( 11
203 ( 5
>500
>500
353 ( 12
order 3 · Cl > 2 · Cl > 4 · Cl > 1 · Cl . 5 · Cl (Table 3). A similar
trend is observed in RAPTA compounds, with the toluene
derivative also showing the highest activity.28 The osmium
complex is significantly less cytotoxic than its ruthenium
equivalent, possibly the result of slower ligand exchange
kinetics, a feature also observed for other Ru/Os analogues.53
The MS studies suggest that loss of the chloride ligand is the
first step in binding to a potential biomolecular target, and
accordingly the more active compounds, 2 · Cl and 3 · Cl, were
those which showed the highest percentage of aqua species. It
is noteworthy that 1 · Cl-4 · Cl are all more cytotoxic than
RAPTA-C, although RAPTA-C is considerably more reactive
in oligonucleotide and nucleotide binding studies, while showing
an affinity for ubiquitin similar to that of 1 · Cl. Combined, these
data suggest that proteins may be more important as in vitro
targets than DNA for these compounds.
Other ruthenium drug candidates, such as NAMI-A, exhibit
similar cytotoxicities, and while they are much less active in
vitro than, for example, cisplatin, they show excellent activities
in vivo.15,28,54 For example, the least cytotoxic ruthenium
compound in vitro is RAPTA-C; nevertheless, in vivo it is active
The ptn ligand was prepared by sodium-mediated reduction of
[Me-pta]+, as previously described, and sublimed under vacuum
prior to use.56 The ruthenium and osmium chloro-bridged dimers
and RAPTA-C were synthesized according to literature procedures.16,57,58 All solvents were degassed prior to use. 1H, 13C,
15
N, and 31P NMR spectra were recorded on a Bruker Avance II
400 spectrometer at room temperature. Spectra were referenced to
the 1H signal of the NMR solvent or by inclusion of an external
reference: H3PO4 for 31P NMR. ESI-mass spectra of the compounds
were obtained in water or acetonitrile on a ThermoFinnigan LCQ
Deca XP Plus quadrupole ion-trap instrument operated in positive
ion mode over a mass range of m/z 150-1000. The ionization
energy was set at 5.0 V and the capillary temperature at 150 °C.
General Method for the Preparation of Complexes 1-5.
[M(η6-arene)Cl2]2 (M ) Ru, Os; 0.29 mM) and ptn (0.58 mM)
were refluxed in chloroform for 5 h. The resulting solution was
filtered and the solvent volume reduced to 25%. The product was
triturated with pentane and the resulting powder filtered and washed
with pentane and diethyl ether. [M(η6-arene)(ptn)Cl]BF4 complexes
1 · BF4-5 · BF4 were obtained by addition of 1 equivalent NaBF4
to a CH2Cl2 solution of the chloride analogues. The formed NaCl
was filtered off and the product was precipitated by addition of
pentane, filtered and dried in Vacuo.
[Ru(η6-p-cymene)(ptn)Cl]Cl (1 · Cl). Yield: 231 mg (85%), red
powder. Slow diffusion of pentane into a CH2Cl2 solution of [Ru(η6p-cymene)(ptn)Cl]BF4 (1 · BF4) afforded crystals suitable for structural analysis by X-ray diffraction.
1
H NMR (400.13 MHz, D2O, 25 °C; δ, ppm): 6.23 (m, 2H, Ar
CH), 5.06 (m, 2H, Ar CH), 4.78 (d, 1H, 2JHH ) 12.0 Hz, bridgehead
N-CHH-N), 4.19-3.74 (m, 6H, CH2), 3.49 (dd, 2H, 2JPH ) 12.0
Hz, 2JHH ) 8.0 Hz, N-CHH-P), 2.97 (d, 1H, 2JHH ) 12.0 Hz,
bridgehead N-CHH-N), 2.89 (s, 3H, N-CH3), 2.70 (sept, 1H,
3
JHH ) 6.8 Hz, Ar CH(CH3)2), 2.14 (s, 3H, Ar CH3), 1.83 (d, 3H,
2
JPH ) 12.0 Hz, P-CH3), 1.21 (d, 3H, 3JHH ) 6.8 Hz, Ar
CH(CH3)2), 1.11 (d, 3H, 3JHH ) 6.8 Hz, Ar CH(CH3)2). 31P{1H}
NMR (D2O, 161.98 MHz; δ, ppm): -27.9. ESI-MS (H2O, positive
ion mode; m/z): 443.9 [Ru(η6-p-cymene)(ptn)Cl]+ (100%).
(54) Chatterjee, S.; Kundu, S.; Bhattacharyya, A.; Hartinger, C. G.;
Dyson, P. J. J. Biol. Inorg. Chem. 2008, 13, 1149-1155.
(55) Dyson, P. J.; Sava, G. Dalton Trans. 2006, 1929–1933.
(56) Assmann, B.; Angermaier, K.; Paul, M.; Riede, J.; Schmidbaur,
H. Chem. Ber. 1995, 128, 891–900.
(57) Zelonka, R. A.; Baird, M. C. Can. J. Chem. 1972, 50, 3063–3072.
(58) Bennett, M. A.; Smith, A. K. J. Chem. Soc., Dalton Trans. 1974,
233–241.
Organometallics, Vol. 28, No. 4, 2009 1171
[Ru(η -C6H6)(ptn)Cl]Cl (2 · Cl). Yield: 161 mg (70%), orangebrown powder. Slow diffusion of pentane into a CH2Cl2 solution
of [Ru(η6-C6H6)(ptn)Cl]BF4 (2 · BF4) afforded crystals suitable for
structural analysis by X-ray diffraction.
6
1
H NMR (400.13 MHz, CD2Cl2, 25 °C; δ, ppm): 5.94 (s, 6H,
Ar CH), 4.79 (d, 1H, 2JHH ) 12.0 Hz, bridgehead N-CHH-N),
4.25-3.75 (m, 6H, CH2), 3.53 (dd, 2H, 2JPH ) 14.0 Hz, 2JHH )
12.0 Hz, N-CHH-P), 3.40 (d, 1H, 2JHH ) 12.0 Hz, bridgehead
N-CHH-N), 2.99 (s, 3H, N-CH3), 1.92 (d, 3H, 2JPH ) 14.0 Hz,
P-CH3). 31P{1H} NMR (161.98 MHz, D2O, 25 °C; δ, ppm): -28.7.
ESI-MS (H2O, positive ion mode; m/z): 352.9 [Ru(η6-C6H6)(ptn)]+
(10%), 369.9 [Ru(η6-C6H6)(H2O)(ptn) - H]+ (10%), 387.9 [Ru(η6C6H6)(ptn)Cl]+ (100%).
[Ru(η6-C6H5CH3)(ptn)Cl]Cl (3 · Cl). Yield: 227 mg (87%),
orange powder. Slow diffusion of diethyl ether into a methanol
solution of [Ru(η6-C6H5CH3)(ptn)Cl]BF4 (3 · BF4) afforded crystals
suitable for structural analysis by X-ray diffraction.
H NMR (400.13 MHz, D2O, 25 °C; δ, ppm): 6.27 (m, 2H, Ar
o-CH), 6.04 (m, 1H, p-CH), 5.85 (m, 2H, m-CH), 4.87 (d, 1H,
2
JHH ) 5.6 Hz, bridgehead N-CHH-N), 4.25-3.78 (m, 6H, CH2),
3.54 (dd, 2H, 2JPH ) 14.0 Hz, 2JHH ) 9.0 Hz, N-CHH-P), 3.15
(d, 1H, 2JHH ) 5.6 Hz, bridgehead N-CHH-N), 2.91 (s, 3H,
N-CH3), 2.09 (s, 3H, Ar CH3), 1.98 (d, 3H, 2JPH ) 9.0 Hz,
P-CH3). 31P{1H} NMR (161.98 MHz, D2O, 25 °C; δ, ppm): -28.0.
ESI-MS (H2O, positive ion mode; m/z): 367.1 [Ru(η6C6H5CH3)(ptn)]+ (5%), 383.8 [Ru(η6-C6H5CH3)(H2O)(ptn) - H]+
(15%), 401.9 [Ru(η6-C6H5CH3)(ptn)Cl]+ (100%).
[Ru(η6-C6Me6)(ptn)Cl]Cl (4 · Cl). Yield: 215 mg (72%), redbrown powder. Crystals suitable for structural analysis by X-ray
diffraction were obtained by slow diffusion of pentane into a CH2Cl2
solution of [Ru(η6-C6Me6)(ptn)Cl]BF4 (4 · BF4).
1
1
H NMR (400.13 MHz, D2O, 25 °C; δ, ppm): 4.94 (m, 1H,
bridgehead N-CHH-N), 4.45-3.53 (m, 8H, CH2) 3.40 (m, 1H,
bridgehead N-CHH-N), 2.99 (s, 3H, N-CH3), 1.92 (d, 3H, 2JPH
) 14.4 Hz, P-CH3), 1.55 (s, 18H, Ar CH3). 31P{1H} NMR (161.98
MHz, D2O, 25 °C): δ -31.7 ppm. ESI-MS (MeCN, positive ion
mode; m/z): 454.3 [Ru(η6-C12H18)(H2O)(ptn) - H]+ (20%), 472.0
[Ru(η6-C12H18)(ptn)Cl]+ (100%).
[Os(η6-C6H6)(ptn)Cl]Cl (5 · Cl). Yield: 261 mg (86%), yellow
powder. A microcrystalline powder of [Os(η6-C6H6)Cl(ptn)]BF4
(5 · BF4) was obtained by slow diffusion of ether into a methanol
solution; however, the sample diffracted weakly.
H NMR (400.13 MHz, D2O, 25 °C; δ, ppm): 6.02 (s, 6H, Ar
CH), 5.08 (d, 1H, 2JHH ) 12.4 Hz, bridgehead N-CHH-N),
4.42-3.87 (m, 6H, CH2), 3.79 (dd, 2H, 2JPH ) 14.8 Hz, 2JHH )
8.0 Hz, N-CHH-P), 3.14 (d, 1H, 2JHH ) 12.4 Hz, bridgehead
N-CHH-N), 3.08 (s, 3H, N-CH3), 1.85 (d, 3H, 2JPH ) 12.0 Hz,
P-CH3). 31P{1H} NMR (161.98 MHz, D2O, 25 °C; δ, ppm): -77.8.
ESI-MS (H2O, positive ion mode; m/z): 460.0 [Os(η6C6H6)(H2O)(ptn) - H]+ (5%), 477.9 [Os(η-C6H6)(ptn)Cl]+ (100%).
X-ray Structure Determination. Suitable single crystals were
selected and manipulated in a perfluoro poly(alkyl ether) oil matrix
(F06206K, ABCR Co.). The crystals were mounted on the end of a
glass fiber attached to a metal pin fixed to a goniometer head which
was placed in the Euler cradle, while a cold blanket of N2 gas was
maintained. The data for structures 1 · BF4, 3 · BF4, and 4 · BF4 were
collected on a Nonius KappaCDD diffractometer equipped with a
Bruker-Apex II CCD area detector and an Enraf-Nonius FR590 X-ray
generator and the data for 2 · BF4 were collected on an Oxford-Kuma
Kappa diffractometer with a Sapphire CCD area detector. All instruments utilize a graphite-monochromated Mo KR radiation source with
λ ) 0.710 73 Å. The crystals were kept under a 140 or 100 K gaseous
flow of N2 during the collection procedure. For 1 · BF4, 3 · BF4, and
4 · BF4, the unit cell and orientation matrix were determined by
indexing reflections measured from phi/chi scans and analyzed with
1
the program DIRAX,59 while for 2 · BF4 the unit cell was determined
from the entire data set using CrysAlis RED.60 All data sets are based
on collecting reflections using an optimized scanning strategy utilizing
the programs CollectCCD61 and CrysAlis CCD (for 2 · BF4 only). After
data integration with either EvalCCD62 or CrysAlis RED (2 · BF4), a
multiscan absorption correction based on a semiempirical method was
applied using the SADABS63 or ABSPACK (a subprogram of
CrysAlis RED) program. Space group determination was performed
with the XPREP program.64 A structure solution based on the directmethods algorithm was employed with SHELXS-97.65 Afterward,
anisotropic refinement of all non-hydrogen atoms was completed on
the basis of a least-squares full-matrix method against F2 data using
SHELXL-97.66 Hydrogen atoms were added in geometrically calculated positions and refined as a riding model using a scaled thermal
parameter to the connecting atom. In all cases, positional disordering
of the terminal P and N atoms of the ptn was observed, which was
resolved by splitting the atoms over two positions and allowing the
total occupancy of the disordered groups to freely refine (details
described in the CIF files in the Supporting Information). In some cases,
the thermal parameters of some atoms were isotropically restrained.
A small number of reflections in some cases were removed when ∆(Fo2
- Fc2)/σ exceeded 10.0. Important data for all structures are given in
Table 4. Drawings were produced with ORTEP-3.67
Hydrolysis Studies. The hydrolytic stability of compounds
1 · Cl-5 · Cl (1 mM) was determined in D2O at 37 °C by 1H and
31
P NMR spectroscopy. The pHs of the solutions were between
5.4 and 5.8.
Protein and (Oligo)nucleotide Binding Studies. (a) Sample
Preparation. The HPLC-purified double-stranded 13-mer oligonucleotide ds(5′-GTATTGGCACGTA-3′) was bought as an aqueous
solution with a concentration of 0.2 mM from A/S Technology
(Denmark) and checked by polyacrylamide gel electrophoresis (PAGE)
for complete annealing. RAPTA-C, 1 · Cl, and 5 · Cl were dissolved
in water and immediately incubated with the oligonucleotide at
effective complex to oligonucleotide ratios of 10:10 and 50:10 µM in
a total volume of 300 µL in Eppendorf vials in a thermomixer (300
rpm; Eppendorf) at 37 °C. Evaporation was minimized by sealing the
tubes with Parafilm and covering them with several layers of aluminum
foil. Aliquots of 100 µL were taken after 1 and 3 days and stored at
-20 °C until analysis by mass spectrometry or PAGE. The aqueous
samples were thawed completely, and an aliquot of 5 µL was diluted
1:10 with a 1.1 mM solution of ammonium acetate in water-npropanol-methanol (20:5:65), resulting in a final DNA concentration
of 1 µM immediately prior to analysis. DNA was ionized with 1.8-2
kV in negative ion mode and 0.4 psi pressure. The XCalibur software
(version 2.0.5) was used for data analysis and was also applied for
offline recalibration of the full scan mass spectra, using the charge
distributions (4- to 9-fold negatively charged) of single-stranded
TACGTGCCAATAC.
For the protein-binding studies, 1 · Cl, 5 · Cl, and RAPTA-C were
incubated with ubiquitin (from bovine red blood cells, minimum
90%; Sigma) at a molar ratio of 2:1, and samples were taken after
24 and 72 h incubation at 37 °C. The samples were diluted 1:100
(59) Duisenberg, A. J. M. J. Appl. Crystallogr. 1992, 25, 92–96.
(60) CrysAlis CCD and CrysAlis RED, Version 1.71; Oxford Diffraction
Ltd, Abingdon, U.K., 2006.
(61) COLLECT: Data Collection Software; Bruker AXIS BV, Delft,
The Netherlands, 1999.
(62) Duisenberg, A. J. M.; Kroon-Batenburg, L. M. J.; Schreurs,
A. M. M. J. Appl. Crystallogr. 2003, 36, 220–229.
(63) Sheldrick, G. M. SADABS: Area Detector Absorption and Other
Corrections, version 2.06; Bruker-AXS, Madison, WI, 2003.
(64) XPREP: Reciprocal Space Exploration, Version 6.14; Bruker-AXS,
Madison, WI, 2003.
(65) Sheldrick, G. M. SHELXS-97, Program for Crystal Structure
Solution; University of Göttingen, Göttingen, Germany, 1997.
(66) Sheldrick, G. M. SHELXL-97, Program for Crystal Structure
Refinement; University of Göttingen, Göttingen, Germany, 1997.
(67) Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
1172 Organometallics, Vol. 28, No. 4, 2009
Renfrew et al.
Table 4. Selected Crystallographic Data for 1 · BF4-4 · BF4
formula
fw
color, habit
cryst syst
space group
a, Å
b, Å
c, Å
R, deg
β, deg
γ, deg
V, Å3
Z
cryst dimens, mm3
dcalcd, g cm-3
µ, mm-1
no. of rflns
R(int)
no. of obsd rflns
min/max transmissn
R1 (I > 2σ(I))
R1 (all data)
wR2 (I > 2σ(I))
wR2 (all data)
no. of restraints
no. of params
goodness of fit on F2
resid electron density, e Å-3
1 · BF4
2 · BF4
3 · BF4
4 · BF4
C17H30RuPN3ClBF4 · CH2Cl2)
687.73
orange, prismatic
orthorhombic
Pna21
12.6112(13)
12.8196(15)
15.4543(11)
90
90
90
2498.5(4)
4
0.430 × 0.174 × 0.113
1.828
1.065
51 795
0.0687
4391
0.6950
0.0277
0.0352
0.0481
0.0510
1
293
1.094
0.474, -0.391
C13H22RuPN3ClBF4
474.64
orange, irregular
triclinic
P1j
12.4204(12)
12.8918(14)
13.3447(18)
113.158(11)
108.410(10)
101.856(9)
1730.0(3)
4
0.27 × 0.25 × 0.24
1.828
1.198
10 310
0.0347
5205
0.778 20
0.0381
0.0592
0.0839
0.0938
6
451
1.018
0.658, -0.744
C14H23RuPN3ClBF4
488.66
orange, prismatic
monoclinic
P21/c
8.7241(9)
15.7830(16)
13.8016(13)
90
103.171(9)
90
1850.4(3)
4
0.417 × 0.261 × 0.189
1.754
1.119
36 207
0.0736
3254
0.5698
0.0511
0.0624
0.1054
0.1099
0
230
1.222
0.790, -0.744
C19H34RuPN3ClBF4
558.79
orange, prismatic
orthorhombic
Pnma
9.5739(16)
12.9609(9)
18.1197(13)
90
90
90
2248.4(4)
4
0.297 × 0.197 × 0.072
1.651
0.932
38 876
0.0962
2104
0.6950
0.0366
0.0673
0.0497
0.0729
0
165
1.140
0.502, -0.500
with H2O-CH3CN-HCOOH (70:30:1) and immediately analyzed
by mass spectrometry. The mass spectra were recalibrated using
the different charge states of ubiquitin in the positive ion mode as
internal standards.
(b) Mass Spectrometry. For electrospray ionization mass
spectrometry, the samples were placed into a 96-well plate in an
Advion TriVersa robot (Advion Biosciences, Ithaca, NY) equipped
with a 5.5 µm nozzle chip. The ESI robot was controlled with
ChipSoft v7.2.0 software employing the following parameters: gas
pressure 0.40 psi; voltage 1.8-2.0 kV; sample volume 10 µL. The
samples were analyzed in negative ion mode using a hyphenated
ion-trap-FT-ICR mass spectrometer comprising an LTQ XL and
an 11 T FT-ICR MS (both ThermoFisher Scientific, Bremen,
Germany). The Xcalibur software bundle was utilized for data
acquisition (Tune Plus version 2.2 SP1; ThermoFisher Scientific)
and data analysis (Qual Browser version 2.2; ThermoFisher
Scientific). Mass spectra were recorded at a resolution of 75 000
at m/z 500 for m/z 350-2000. A single scan consisted of 5
microscans, and the spectrum was averaged over at least 50 scans.
In addition to full scan ion trap and FT-ICR mass spectra, protein
binding data were collected in WSIM mode (m/z 770-870).
(c) Gel Electrophoresis. Samples were thawed, and 10 µL
aliquots (0.8 µg of oligonucleotide) were mixed with 2 µL of 6X
sample buffer and completely loaded on a native 20% polyacrylamide gel. Puc-mix (smallest oligonucleotide 45 bp) was used as
mass ruler in the two outermost lanes, and pure double-stranded
DNA of sequence ds(GTATTGGCACGTA) was used in amounts
of 0.8, 0.4, and 0.08 µg as standards for estimation of the
concentration of the oligonucleotide upon incubation. 1X TBE
buffer was used as electrolyte, and electrophoresis was performed
at a constant current (12 mA) for 3-4 h. Gels were stained with
ethidium bromide (0.5 µg/mL) for 15-20 min and visualized under
UV.
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.
Determination of Cell Viability (MTT Assay). Cytotoxicity
was determined using the MTT assay (MTT ) 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). Cells were seeded
in 96-well 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. Compounds were dissolved
directly in the culture medium to the appropriate concentration,
with the exception of 4 · Cl, which was added as a DMSO solution
and serially diluted to the appropriate concentration (to give a final
DMSO concentration of 0.5%). A 100 µL portion of the drug
solution was added to each well, and the plates were incubated for
another 72 h. Subsequently, MTT solution (0.2 mg/mL) 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, and the fraction of surviving cells was
calculated from the absorbance of untreated control cells. Evaluation
is based on means from two independent experiments, each
comprising three microcultures per concentration level.
Acknowledgment. We are indebted to the EPFL, the
Swiss National Science Foundation, the University of Vienna,
the FWF-Austrian Science Fund (CGH Schrödinger Fellowship J2613-N19), and COST D39 for financial support.
This research was supported by Marie Curie Intra European
Fellowships within the 6th and 7th European Community
Framework Programme projects MEIF-CT-2005-025287CARCAS (A.D.P.) and 220890-SuRuCo (A.A.N.). We thank
Dr. Rosario Scopelliti for collecting the X-ray data and Prof.
Yury O. Tsybin for use of the FT-ICR-MS.
Supporting Information Available: CIF files giving crystallographic data for 1 · BF4-4 · BF4. This material is available free
of charge via the Internet at http://pubs.acs.org.
OM800899E