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Journal of Organometallic Chemistry 696 (2011) 1108e1116

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Journal of Organometallic Chemistry

journal homepage: www.elsevier.com/locate/jorganchem

Ruthenium(II) arene complexes with oligocationic triarylphosphine ligands:

Synthesis, DNA interactions and in vitro properties
Dennis J.M. Snelders a, Angela Casini a, Fabio Edafe a, Gerard van Koten b, Robertus J.M. Klein Gebbink b,
Paul J. Dyson a, *
Institut des Sciences et Ingnierie Chimiques, Ecole Polytechnique Fdrale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
Organic Chemistry and Catalysis, Debye Institute for Nanomaterials Science, Faculty of Science, Utrecht University, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: The synthesis, DNA binding properties and cytotoxicity of a series of Ru(II)-arene complexes containing
Received 22 September 2010 oligocationic ammonium-functionalized triarylphosphines, of the type Ru(p-cymene)Cl2(L) (L oligo-
Received in revised form cationic phosphine), are reported. The complexes are highly charged (the overall charge states being 3
16 November 2010
and 6) and circular dichroism spectroscopy and gel electrophoresis studies indicate that the
Accepted 17 November 2010
compounds interact strongly with DNA via electrostatic interactions. Despite forming strong interactions
with DNA, the complexes exhibit only modest cytotoxicities on the human ovarian cancer cell lines
A2780 and A2780cisR.
Bioorganometallic chemistry
2010 Elsevier B.V. All rights reserved.
Phosphine ligands
Metal-based drugs
DNA binding

1. Introduction complexes containing this class of ligands may be envisaged to

occur in a synergistic manner, combining electrostatic binding of
Classical anticancer compounds such as cisplatin and carbo- the cationic organic ligand to the negatively charged phosphate
platin primarily act by direct coordination of the metal ion to DNA groups of DNA, possibly with coordination of the metal ion to
bases [1e4]. Other metal-based compounds may interact with DNA bases.
DNA in different ways, and this may lead to alternative types of In this paper, we describe the synthesis and characterization of
drug activity and selectivity [5,6]. Such interactions are often non- some highly charged (cationic) ruthenium(II)-arene complexes
covalent in nature and include hydrogen bonding, electrostatic, that can potentially interact with DNA via electrostatic and covalent
hydrophobic and p-stacking interactions [7e9]. In cases where binding. Circular dichroism and gel electrophoresis were subse-
a metal ion is coordinated to an active organic ligand, DNA binding quently used to study these interactions.
may occur through a combination of covalent and non-covalent
interactions [10e14].
A family of oligocationic triarylphosphine ligands termed 2. Results
Dendriphos, 1ae4a (Fig. 1) that combine the triphenylphosphine
motif with three (1a) to six (2ae4a) permanent cationic substit- 2.1. Synthesis, characterization and aquation of Ru(p-cymene)Cl2(L)
uents (ammonium groups), have been reported [15e19]. Varia- complexes
tions in the size of these ligands are achieved by altering the
ammonium substituent (R), ranging from a methyl (1a, 2a) or Complexes of the type Ru(p-cymene)Cl2(L) (L 1ae4a) were
benzyl (3a) group, to a Frchet dendron (4a). In view of their prepared by reacting phosphine ligands 1ae4a with 0.5 equiv. of
bifunctional character, these compounds are interesting candi- [Ru(p-cymene)Cl2]2 in acetonitrile at 60  C for 1.5 h. After evapo-
dates in the eld of metal-based drugs. The DNA binding of metal ration of the solvent in vacuo, the products 1be4b were obtained
as orange powders in quantitative yield (Scheme 1). The 1H and
C NMR spectra of 1be4b, recorded in CD3CN, showed minor
* Corresponding author. Fax: 41(0)21 693 98 85. shifts (typically Dd 0.1e0.5 ppm in 1H NMR spectra and
E-mail address: paul.dyson@ep.ch (P.J. Dyson). Dd 0.1e3.0 ppm in 13C NMR spectra) in the signals for the

0022-328X/$ e see front matter 2010 Elsevier B.V. All rights reserved.
D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116 1109

Fig. 1. Oligocationic, ammonium-functionalized phosphine ligands 1ae4a.

phosphine ligand moieties compared to those for the correspond- The water stability of 1b, 2b and 5 was studied by 31P NMR
ing free phosphines 1ae4a. The signals for the p-cymene moieties spectroscopy in D2O. The spectra are given in the supporting
are observed at very similar chemical shifts to those observed in Ru information, Figures S1eS3. The low water solubility of 3b and
(p-cymene)Cl2(PPh3) [20]. The 31P NMR spectra of 1be4b contain 4b precluded such analysis. The 31P NMR spectrum of 1b in D2O,
one singlet around 24e25 ppm, conrming the quantitative recorded after 10 min, shows two major signals at 31.8 and
formation of the desired Ru-phosphine complexes. 26.7 ppm in an approximate ratio of 1:2. After standing at RT in
Extensive investigations into the water stability of Ru(II)(p- solution for 16 h, no changes in the spectrum were observed.
cymene)Cl2(pta) (pta 1,3,5-triaza-7-phosphaadamantane), RAP- Addition of 4 mM NaCl (the approximate chloride concentration
TA-C (6), have been reported. It has been established that in water, 6 in cells) did not lead to a signicant change, but after addition of
undergoes aquation, i.e. substitution of one or both chloride ligands 100 mM NaCl (the approximate chloride concentration in plasma),
by a water molecule. Depending on the pH, the aqua ligands may be the signal at 26.7 ppm remains as the only peak in the spectrum,
deprotonated. At physiologically relevant pH, the dichloride I exists indicating that this signal corresponds to the dichloride form I of
in equilibrium with the mono-aquated species II and the hydroxo- complex 1b. Addition of 1 equiv. AgBF4 led to a decrease in
aqua species III (Scheme 2) [21e24]. intensity of the signal at 26.7 ppm in favor of the signal at

Fig. 2. Tendency towards aquation of 1b, 2b, 5 and 6, expressed as the percentage mono-aquated form II observed by P NMR spectroscopy in D2O at RT.
1110 D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116

Scheme 1. Synthesis of complexes 1be4b bearing oligocationic phosphine ligands and structures of reference compounds 5 and 6.

31.8 ppm and the appearance of a new signal at 32.3 ppm. After Increasing the concentration of NaCl led to a shift of this ratio in
addition of a second equiv. of AgBF4, the signal at 32.3 remains as favor of the signal at 30.1 ppm, indicating that the peak corresponds
the only peak. Based on comparison with the corresponding pta to form I of complex 5. A relatively high concentration of NaCl
complex RAPTA-C (6) [21,24], the signal at 31.8 ppm may be was required, i.e. 300 mM, for complete disappearance of the
tentatively assigned to the mono-aquated form II of complex 1b signal at 35.2 ppm. Addition of 1 equiv. AgBF4 led to the disap-
and the signal at 32.3 to the hydroxo-aqua form III. For compar- pearance of the signal at 30.1 ppm, leaving the signal at 35.2 as the
ison, the water stability of 2b and 5 was also investigated and only peak in the spectrum, which can be assigned to the mono-
Fig. 2 shows the percentage of the mono-aquated form II aquated form II. Addition of higher amounts of AgBF4 then led to
observed by 31P NMR in D2O, at [NaCl] 0 or 100 mM for each of the appearance of a new signal at 35.5 ppm, which remained as the
the complexes (species of the type III are only observed following only signal after addition of 3 equiv. AgBF4 and can be tentatively
the addition of AgBF4). The hexacationic complex 2b displayed assigned to the hydroxo-aqua form III.
a remarkably different behavior from that of the tricationic
species 1b. The 31P NMR spectrum in D2O of 2b showed only one 2.2. DNA binding studies
signal, at 26.4 ppm. After standing in solution at room tempera-
ture for 24 h, no change in the spectrum was observed. Addition 2.2.1. CD and UV spectroscopy
of 100 mM NaCl did not lead to any change, indicating that the In order to investigate the interaction between complexes
observed signal corresponds to the dichoride form I of 2b. Thus, 1be4b and DNA each compound was incubated for 24 h at room
2b does not undergo aquation in water under the employed temperature with calf-thymus DNA (ctDNA) at various ratios
conditions. Titration of 2b with AgBF4 results in the formation of (r compound:DNA base pairs 0.1, 0.2, 0.3, 0.4 and 0.5). In
two doublets centered around 15.2 ppm, with coupling constants addition, the Ru analogues bearing trianionic tppts (5) and pta (6),
of 799 and 698 Hz, respectively. After addition of 3 equiv. AgBF4, as well as the free phosphine ligands 1ae4a were studied. After
these signals were the only peaks in the spectrum. This pattern of incubation, the ctDNA-complex mixtures were analyzed by CD
signals is characteristic for phosphorus nuclei coordinated to Ag(I) spectroscopy and compared to the spectrum of ctDNA alone. Fig. 3
[25]. shows representative CD spectra obtained for 1be4b (the CD
In the case of 5, the 31P NMR spectrum in D2O showed two major spectra of the other compounds are provided in the Supporting
signals at 35.2 and 30.1 ppm, in an approximate ratio of 3.5 to 1. Information, Figures S4eS8). Polyvalent cations are known to

Scheme 2. Aquation/hydrolysis of complexes of the type Ru(p-cymene)Cl2(L) (L phosphine ligand).

D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116 1111

Fig. 3. CD spectra of calf-thymus DNA following incubation with complexes 1be4b in 20 mM phosphate buffer (pH 7.4). r compound:DNA base pairs 0, 0.1, 0.3 and 0.5.

bind strongly to DNA and can induce DNA condensation or cases. The ctDNA samples incubated with free ligands 1ae4a
precipitation in vitro as a result of phosphate charge neutralization afforded nearly identical CD spectra and very similar UV absorption
[26]. Therefore, to monitor possible DNA precipitation, the UV patterns compared with 1be4b (Fig. 4 and Figs. S4eS7). The cor-
absorption at 260 nm, which is directly related to the concentration responding Ru complex of the trianionic ligand tppts (5) and that
of DNA in solution, was also measured for each CD sample. Fig. 4 of pta (6) did not lead to any change in the CD spectrum of ctDNA
shows the percentage of ctDNA remaining in solution after incu- (Figure S8), nor did these species inuence the UV absorption at
bation with the various complexes and ligands in comparison to 260 nm (Fig. 4). Cisplatin does not lead to any change in the UV
cisplatin treated DNA. absorption (Fig. 4), but it is known to signicantly alter the CD
The CD spectrum of untreated ctDNA shows a positive band spectrum of DNA [27]. The interaction of the hexacationic ligands
around 275 nm and a negative band at about 245 nm [26]. After 2a and 3a with ctDNA at increments of r 0.1 up to r 0.5 was
addition of the tricationic complex 1b, a signicant decrease in studied. For all the solutions, CD spectra and UV absorption at
intensity of the positive CD band at 275 nm was observed at r 0.5, 260 nm were measured (Fig. 5, Figs. S5 and S6). For 2a, not much
along with a decrease in the UV absorption at 260 nm. Increasing change is observed in either the CD spectrum or the UV absorption
r up to 1.5 did not lead to further changes in the CD spectrum. up to r 0.3. At r 0.4, however, an abrupt decrease in the UV
Similar effects were observed for 4b. For the hexacationic absorption is observed with concomitant collapse of the CD spec-
complexes 2b and 3b, only a small change was observed in the CD trum. For 3a, the CD spectrum gradually changes with an increasing
spectrum at r 0.1, but a nearly complete loss of both the CD signal r. The UV absorption shows a similar abrupt change as seen for 2a,
and the UV absorption at 260 nm was observed at r 0.5 in both but it occurs at a higher value of r, i.e. 0.5.

Fig. 4. Percentage of ctDNA remaining in solution after incubation with compounds Fig. 5. Percentage DNA remaining in solution after incubation with compounds 2a or
1e6 or cisplatin at r 0.5, in 20 mM phosphate buffer, measured by UV absorption at 3a at r 0.1e0.5 in 20 mM phosphate buffer, measured by UV absorption at 260 nm
260 nm. r molar ratio of compound vs. DNA base pairs.
1112 D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116

Fig. 6. Gel electrophoresis of pBR322 plasmid DNA treated with 1e6 in compound to DNA base pair ratios (r) of 0.1 and 0.05. B blank (DNA only).

2.2.2. Gel electrophoresis assays (A2780cisR), using the MTT assay (see Experimental Section and
The interaction of 1ae4a, 1be4b, 5 and 6 with DNA was further Table 1).
studied by evaluating the electrophoretic mobility of plasmid Among the cationic complexes, only the trication 1b displayed
pBR322 DNA on agarose gels after incubation for 24 h at 37  C with a moderate cytotoxic effect towards the cell line A2780. The hex-
the compounds (compound:DNA base pair ratio r 0.1 and 0.05) acationic analogue 2b and the dendritic ligand 4a, as well as co-
in comparison to cisplatin treated DNA (Fig. 6). Plasmid DNA exists mplex 4b, are somewhat cytotoxic against the cisplatin-resistant
as two main forms, a supercoiled (SC) form, which migrates fastest cell line. In both cell lines cisplatin is considerably more cytotoxic
(lowest band in Fig. 6) and an open circular form (OC) which than any of the ruthenium complexes.
migrates more slowly. Changes in the mobility of these bands are
generally interpreted as evidence for the occurrence of DNA 3. Discussion
binding [27e30].
In general, at r 0.1, most of the cationic ligands and 3.1. Water stability of the complexes
complexes led to a slight retardation of both the SC and the OC
bands along with some increase in intensity of the OC band and The extent of aquation of the complexes studied herein follows
a decrease in intensity of the SC band. For cisplatin, a more the trend 5 > 6 > 1b >> 2b. The phosphine ligand has a large
pronounced retardation of the SC band is observed. For the free inuence on the positions of the equilibria depicted in Scheme 2,
ligand 3a, the effect was somewhat more pronounced than that i.e. the tendency of the Ru-phosphine complex to undergo aqua-
observed for the corresponding complex 3b. For 2a and 2b, tion. There is no clear correlation between the extent of aquation
dramatic effects were observed, involving extensive retardation of these complexes and the basicity of the corresponding phos-
of both bands. In contrast, for the anionic complex 5 and RAPTA-C phine ligands, which have the following order of decreasing
6, no signicant effects were observed. At r 0.5, extensive DNA basicity, determined through the frequency of the CO stretching
precipitation occurred in many cases, which prevented DNA vibration in the IR spectrum of complexes of the type trans-RhCl
migration. (CO)L2 (L monodentate phosphine ligand): pta (1963 cm1) [33]
>tppts (1968 cm1) [34] >1a (1971 cm1) [18] >2a (1981 cm1)
2.3. Cytotoxicity studies [18]. With the negatively charged phosphine in 5, extensive and
rapid aquation is observed and at the other extreme, for 2b
The cytotoxicities of 1e5 were determined on the human which contains a hexacationic phosphine ligand, aquation is not
ovarian cancer (A2780) cell line and its cisplatin-resistant variant observed. It is not unreasonable to assume that the ability of
a complex to undergo aquation (which results in a change of the
overall charge of the complex by 1) is lower for positively
Table 1
charged complexes (i.e. 1b and 2b), as this leads to an increase of
IC50 values of 1e5 compared with 6 (RAPTA-C) and cisplatin against human ovarian
carcinoma cell lines sensitive (A2780) and resistant (A2780cisR) to cisplatin. the overall positive charge of the complex, which contrasts
with the anionic complex, i.e. 5, which leads to a decrease in the
IC50 (mM)
overall charge.
Compound A2780 A2780cisR
1a >1000 >1000 3.2. DNA interactions of oligocationic phosphines and their Ru
1b 241 >1000
2a >1000 >1000
2b >1000 247
3a >1000 >1000 Circular dichroism spectroscopy is widely used to study the
3b >1000 >1000 solution structure of DNA and the conformational changes that
4a >1000 211
occur upon ligand binding [11,26,27,35e37]. The CD spectrum of
4b >1000 169
5 >1000 >1000 the normal form of DNA (B-DNA) is characterized by a positive
6a 353 n.d. band around 275 nm and a negative band around 245 nm [26].
Cisplatinb 4.3 18.2 After incubation of ctDNA with the tricationic complex 1b, or the
Ref. [31]. corresponding free ligand 1a, a signicant decrease in intensity of
Ref. [32]. the positive CD band at 275 nm was observed at r 0.5. This
D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116 1113

suggests that a signicant interaction occurs between the DNA compounds 2a and 2b, a dramatic effect of the DNA mobility was
and 1a and 1b and that the DNA undergoes some conformational observed, indicating a strong interaction with the DNA. In line
changes due to this interaction. The UV absorption at 260 nm is with the CD studies, the tricationic 1aeb and the dendritic
slightly decreased indicating that DNA precipitation has taken systems 4aeb showed much smaller effects, while anionic 5 and
place. In the case of the hexacationic species 2a, 2b, 3a and 3b, neutral 6 did not interact with the DNA under the conditions. The
the UV absorption at 260 nm was almost reduced to zero at interaction of 6 is known to depend on the pH of the solution and
r 0.5, indicating that nearly all of the DNA has precipitated, occurs only at pH < 7 [29].
explaining the loss of the CD signal in these cases. Similar ob-
servations have been reported upon titration of DNA with cationic
liposomes [37]. 3.3. Cytotoxicity studies
In the case of 3a and 3b, a distinct CD spectrum was observed
at r 0.3e0.4, characterized by a collapse of the positive band at In general all the ligands and complexes are only slightly
275 nm and a strong increase of the negative band. Such CD char- cytotoxic or are not cytotoxic to the human ovarian cancer cell
acteristics have been observed under a variety of conditions and lines A2780 and A2780cisR. Among the class of compounds of the
correspond to a more highly wound form of B-DNA [35]. It thus type Ru(p-cymene)Cl2(L) (L 1e5), only the smallest, tricationic
seems that 3a and 3b induce strong conformational changes up to complex 1b is moderately active against the A2780 cell line,
r 0.3e0.4, while leading to DNA precipitation at r 0.5. For while only the hexacationic complexes 2b and 4b and the ligand
complexes 1be4b, the observed changes in the CD spectra were 4a are active against the A2780cisR cell line. The cytotoxicity
very similar to those observed for the corresponding free ligands values for these compounds fall in the range commonly observed
1ae4a. This indicates that the observed conformational changes for various Ru(h6-arene)-type complexes [31,42,43]. Since the
are ligand-based, rather than metal-based, and that the interaction compounds interact strongly with DNA it seems likely that they
of the cationic complexes and ligands with DNA occurs via elec- do not reach the DNA in vitro. This may be due to reduced
trostatic interactions between the positively charged ammonium cellular uptake although various studies show that highly charged
groups in the ligands and the negatively charged phosphate groups (cationic) ruthenium compounds, albeit based on multiple
of the DNA. ruthenium centers, can enter cells and are cytotoxic [44e46]. It
In contrast, the trianion 5 did not lead to any changes in the should be noted that despite having modest cytotoxicities
CD spectrum, presumably due to repulsive electrostatic interac- a number of ruthenium complexes are highly efcient in vivo
tions between the anionic sulfonate groups in the tppts ligand [22,47,48].
and the phosphate groups of DNA. Addition of RAPTA-C (6) to
DNA did not lead to any spectral change either. Although it is 4. Conclusions
known that various ruthenium compounds, including 6 [38,39],
bind to DNA, for example via coordinative binding of the Ru ion A series of ruthenium(II)-arene complexes with charged
to N atoms of the DNA bases [40], these interactions do not alter monodentate phosphine ligands were prepared and studied. The
the CD spectrum of ctDNA under the employed conditions. This is complexes with charged cationic ligands are quite resistant to
in line with previous investigations that showed no signicant CD aquation and they appear to bind strongly with DNA, mainly via
spectral change upon addition of several ruthenium compounds electrostatic interactions. Depending on the number of charges as
[27]. well as on the steric effects of the substituents at the ammonium
The DNA precipitation observed upon addition of 2a, 2b, 3a groups in the phosphine ligands, the DNA binding afnity of the
and 3b most likely occurs due to charge neutralization through complexes follows the order 2b > 3b > 1b > 4b. Despite forming
strong electrostatic interactions between the ammonium groups of strong interactions with DNA, the compounds display only
these hexacationic species and the phosphate groups of DNA [26]. modest cytotoxic effects on ovarian cancer cells. These studies
It is known that multivalent cation-induced DNA condensation highlight that no single mechanism of action can be attributed to
occurs when 90% of the DNA phosphate charge is neutralized by ruthenium(II)-arene compounds [49] and each compound must
cation binding [41]. Thus, the ability of 2a and 3a to neutralize the be considered separately. The cationic phosphine ligands studied
phosphate charges even at a considerable ionic strength indicates herein may be regarded as effective agents for linking metal ions
a strong interaction with DNA. The slightly higher ratio needed to DNA and as such they might nd application in other elds.
for 3a compared with 2a might be due to an increased steric For example, these systems might be of interest in the nascent
hindrance of 3a due to the added benzyl substituents. Similarly, eld of DNA-based hybrid catalysts for asymmetric synthesis
the relatively weak interaction of 4a and 4b with DNA can be [50,51].
rationalized by their large size and the buried position of the
ammonium groups within the dendritic structure. In the case of 1a
and 1b the weaker DNA binding, compared with 2a and 2b, may 5. Experimental section
be attributed to the presence of only three positive charges in
these species. [RuCl2(p-cymene)]2 [52], 1ae4a [18], 5 [53] and 6 [29] were
The plasmid DNA gel electrophoresis mobility studies are synthesized according to published methods. NMR spectra were
generally in accordance with the CD spectroscopy studies and measured on a Bruker Avance II 400 spectrometer at 25  C unless
showed that the cationic complexes, as well as their correspond- stated otherwise. 1H and 13C{1H} NMR spectra were referenced to
ing ligands, interact with DNA. Most of the compounds promoted residual solvent signals. 31P NMR chemical shifts are given relative
the conversion of supercoiled (SC) DNA into its relaxed open to 85% H3PO4. UV/Vis spectra were recorded on a Jasco V-550
circular form (OC), along with a slight decrease in mobility for spectrophotometer. Elemental analyses were obtained in-house
both forms, although in most cases the effects are less pronounced using a Thermo Fischer Scientic Flash 2000 Organic Elemental
than for cisplatin. In general the ruthenium complexes were Analyzer. ElectroSpray-Ionisation MS data were acquired on a Q-Tof
equally or slightly less effective than the corresponding free Ultima mass spectrometer (Waters) tted with the LockSprayTM
ligands. This is not unexpected due to the low ability of the dual electrospray ion source and operated in positive and ionization
cationic complexes to undergo aquation. For the hexacationic mode.
1114 D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116

5.1. General procedure for the synthesis of complexes Ru(p-cymene) 5.4. 3b

Cl2(L) (1be4b)

Phosphine ligands 1ae4a and 0.50 equiv. of [Ru(p-cymene)Cl2]2

were dissolved in deoxygenated CH3CN (3e5 mL) and stirred at
60  C for 1.5 h under an inert atmosphere. The solvent was evap-
orated in vacuo affording the products 1be4b as orange powders.

5.2. 1b

Starting materials: 3a (33.0 mg, 19.7 mmol) and [Ru(p-cym-

ene)Cl2]2 (6.0 mg, 9.8 mmol, i.e. 19.6 mmol Ru). Yield: 39 mg (quant.).
H NMR (400.1 MHz, CD3CN): d (ppm) 8.39 (d, 3JP,H 10.0 Hz, 6H,
o-Ar), 7.76 (s, 3H, p-Ar), 7.53 (m, 30H, Ph), 5.40 (d, 3JH,H 5.8 Hz, 2H,
2-Ar), 5.15 (d, 3JH,H 5.9 Hz, 2H, 3-Ar), 4.43 (12H, NCH2), 4.38 (12H,
NCH2), 2.81 (s, 36H, N(CH3)2), 2.25 (sept, 3JH,H 7.0 Hz, 1H), 1.76
Starting materials: 1a (84.2 mg, 0.114 mmol) and [Ru(p- (s, 3H, CH3), 0.90 (d, 3JH,H 6.9 Hz, 6H, C(CH3)2). 13C{1H} NMR
cymene)Cl2]2 (33.7 mg, 55.0 mmol, i.e. 0.110 mmol Ru). Yield: 118 mg (100.6 MHz, CD3CN): d (ppm) 141.3 (d, 2JP,C 8.6 Hz, o-Ar),
(quant.). 1H NMR (400.1 MHz, CD3CN): d (ppm) 7.95 (dd, 140.8 (s, p-Ar), 134.2 (s, Ph), 131.8 (s, Ph), 130.2 (s, Ph), 129.8
JH,H 9.0 Hz, 3JP,H 9.0 Hz, 6H, o-Ar), 7.55 (d, 3JH,H 7.0 Hz, 6H, m- (d, 3JP,C 11.1 Hz, m-Ar), 128.0 (s, Ph), 111.6 (s, 1-Ar), 98.5 (s, 4-Ar),
Ar), 5.31 (d, 3JH,H 6.2 Hz, 2H, 2-Ar), 5.25 (d, 3JH,H 5.8 Hz, 2H, 3-Ar), 91.3 (s, 2-Ar), 87.3 (s, 3-Ar), 69.0 (s, NCH2), 68.4 (s, NCH2), 49.4 (s, N
4.46 (s, 6H, CH2), 3.04 (s, 27H, N(CH3)3), 2.60 (sept, 3JH,H 6.9 Hz,1H), (CH3)2), 31.2 (s, C(CH3)2), 21.9 (s, C(CH3)2), 18.4 (s, Me). 31P{1H}
1.85 (s, 3H), 0.99 (d, 3JH,H 6.9 Hz, 6H, C(CH3)2). 13C{1H} NMR NMR (162.0 MHz, CD3CN): d (ppm) 24.2 (s). HR-ESI MS: (m/z)
(100.6 MHz, CD3CN): d (ppm) 137.2 (d, 1JP,C 43.5 Hz, i-Ar), 136.0 902.3638 {[M-2BF4]2, calc. 902.3677}.
(d, JP,C 9.8 Hz, Ar), 133.2 (d, JP,C 9.9 Hz, Ar), 130.9 (d, 4JP,C 2.4 Hz,
p-Ar),111.0 (s,1-Ar), 97.5 (s, 4-Ar), 90.9 (s, 2-Ar), 88.3 (s, 3-Ar), 69.2 (s, 5.5. 4b
CH2), 53.4 (s, N(CH3)3), 31.2 (s, C(CH3)2), 21.9 (s, C(CH3)2), 17.9 (s, Me).
31 1
P{ H} NMR (162.0 MHz, CD3CN): d (ppm) 24.7 (s). Elem. anal. for
C40H58B3Cl2F12N3PRu (1044.27): calc. (%) C 46.01, H 5.60, N 4.02;
found C 46.01, H 5.25, N 4.31.

5.3. 2b

Starting materials: 4a (40.0 mg, 7.28 mmol) and a stock solu-

tion of [Ru(p-cymene)Cl2]2 (0.92 mL; 2.36 mg/mL in deoxygenated
CH3CN; 3.54 mmol, i.e. 7.09 mmol Ru). Yield: 42 mg (quant.). 1H NMR
(400.1 MHz, CD3CN): d (ppm) 8.36 (br. s, 6H, o-Ar), 7.72 (br. s, 3H, p-
Ar), 7.4e7.2 (br. m, 120H, Ph), 6.7e6.5 (br. m, 42H, overlapping Ar0,
Ar00 ), 6.48 (s, 12H, o-Ar0 ), 5.34 (br. s, 2H, 2-Ar), 4.90 (br. s, 74H, over-
lapping OCH2, 3-Ar), 4.31 (br. s, 12H, NCH2Ar0 ), 4.20 (br. s, 12H,
NCH2Ar), 2.71 (br. s, 36H, N(CH3)2), 1.76 (s, 3H, CH3), 0.81 (br. s, 6H, C
Starting materials: 2a (56.5 mg, 46.5 mmol) and [Ru(p-cym- (CH3)2) (signal for CH(CH3)2 not resolved). 13C{1H} NMR (100.6 MHz,
ene)Cl2]2 (14.2 mg, 23.2 mmol, i.e. 46.4 mmol Ru). Yield: 71 mg CD3CN): d (ppm) 161.0, (s, m-Ar00 ), 161.0 (s, m-Ar0 ), 140.1 (s, o-Ar0 ),
(quant.). 1H NMR (400.1 MHz, CD3CN): d (ppm) 8.29 (d, 138.0 (s, o-Ar00 ), 129.8 (s, Ph), 129.4 (s, Ph), 128.9 (s, Ph), 128.6 (s, Ph),
JP,H 9.6 Hz, 6H, o-Ar), 7.79 (s, 3H, p-Ar), 5.37 (d, 3JH,H 5.1 Hz, 2H, 113.0 (s, p-Ar0 ), 107.5 (s, p-Ar00 ), 105.2 (s, i-Ar0 ), 102.2 (s, i-Ar00 ), 70.6 (s,
2-Ar), 5.28 (d, 3JH,H 5.2 Hz, 2H, 3-Ar), 4.40 (s, 12H, CH2), 3.02 OCH2), 69.2 (br. s., NCH2), 68.4 (br. s, NCH2), 49.6 (s, N(CH3)2), 30.8 (s,
(s, 54H, N(CH3)3), 2.43 (sept, 3JH,H 6.8 Hz, 1H), 1.84 (s, 3H), 1.03 C(CH3)2), 21.8 (br. s, C(CH3)2), 18.3 (s, Me) (signals for Ar not
(d, 3JH,H 7.7 Hz, 6H, C(CH3)2). 13C{1H} NMR (100.6 MHz, CD3CN): resolved). 31P{1H} NMR (162.0 MHz, CD3CN): d (ppm) 23.8 (s).
d (ppm) 140.9 (d, 3JP,C 10.4 Hz, o-Ar), 140.3 (s, p-Ar), 137.3 Elem. anal. for C340H329B6Cl2F24N6O36PRu (5799.04): calc. (%) C
(d, 1JP,C 43.7 Hz, i-Ar), 130.0 (d, 2JP,C 10.0 Hz, m-Ar), 112.4 (s, 1- 70.42, H 5.72, N 1.45; found C 70.15, H 6.03, N 1.44.
Ar), 99.0 (s, 4-Ar), 90.3 (s, 2-Ar), 88.0 (s, 3-Ar), 68.8 (s, CH2), 53.4
(s, N(CH3)3), 31.3 (s, C(CH3)2), 21.9 (s, C(CH3)2), 18.5 (s, Me). 31P{1H} 5.6. Aquation studies
NMR (162.0 MHz, CD3CN): d (ppm) 24.9 (s). Elem. anal. for
C52H89B6Cl2F24N6PRu (1522.08): calc. (%) C 41.03, H 5.89, N 5.52; Solutions of 1b, 2b and 5 in D2O (10e20 mM) were analyzed
found C 41.21, H 5.99, N 5.52. by 31P NMR spectroscopy. Subsequently, aliquots of stock solutions
D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116 1115

of NaCl or AgBF4 in D2O were added and the resulting mixtures Appendix. Supplementary material
were shaken at room temperature for 1 min, followed by analysis
by 31P NMR spectroscopy. The concentrations of NaCl amounted to Supplementary data related to this article can be found online
4, 100, 200 or 300 mM and the concentrations of AgBF4 amounted at doi:10.1016/j.jorganchem.2010.11.025.
to 1, 2 or 3 equiv with respect to the Ru complex.

5.7. Circular dichroism spectroscopy References

[1] E.R. Jamieson, S.J. Lippard, Chem. Rev. 99 (1999) 2467e2498.

To solutions of calf-thymus DNA (base pair concentration: [2] E. Wong, C.M. Giandomenico, Chem. Rev. 99 (1999) 2451e2466.
60 mM) in sodium phosphate buffer (20 mM, pH 7.4, no other [3] J. Reedijk, Chem. Rev. 99 (1999) 2499e2510.
added salts), appropriate aliquots of 103 M solutions of 1ae4a, [4] T.W. Hambley, J. Chem. Soc. Dalton Trans. (2001) 2711e2718.
[5] P.C.A. Bruijnincx, P.J. Sadler, Curr. Opin. Chem. Biol. 12 (2008) 197e206.
1be4b, 5 or 6 were added to give compound:base pair ratios (r) of [6] J. Reedijk, Eur. J. Inorg. Chem. 2009 (2009) 1303e1312.
up to 0.5 in a total volume of 0.5 mL. The mixtures were incubated [7] S. Komeda, T. Moulaei, K.K. Woods, M. Chikuma, N.P. Farrell, L.D. Williams,
for 24 h at room temperature. CD spectra were recorded on a Jasco J. Am. Chem. Soc. 128 (2006) 16092e16103.
[8] I.M. Dixon, F. Lopez, A.M. Tejera, J.-P. Estve, M.A. Blasco, G. Pratviel,
J-810 spectropolarimeter using a quartz cuvette with a path length B. Meunier, J. Am. Chem. Soc. 129 (2007) 1502e1503.
of 1 cm with 10 accumulations per spectrum. [9] A. Oleksi, A.G. Blanco, R. Boer, I. Usn, J. Aymam, A. Rodger, M.J. Hannon,
M. Coll, Angew. Chem. Int. Ed. Engl. 45 (2006) 1227e1231.
[10] F.-J.K. Rehmann, L.P. Cuffe, O. Mendoza, D.K. Rai, N. Sweeney, K. Strohfeldt,
5.8. DNA electrophoresis W.M. Gallagher, M. Tacke, Appl. Organomet. Chem. 19 (2005) 293e300.
[11] S. Roy, K.D. Hagen, P.U. Maheswari, M. Lutz, A.L. Spek, J. Reedijk, G.P. van
Wezel, ChemMedChem 3 (2008) 1427e1434.
Samples with pBR322 plasmid DNA were prepared by adding [12] C. Scolaro, T.J. Geldbach, S.b. Rochat, A. Dorcier, C. Gossens, A. Bergamo,
the required volume of freshly prepared solutions of 1ae4a, 1be4b, M. Cocchietto, I. Tavernelli, G. Sava, U. Rothlisberger, P.J. Dyson, Organome-
5, 6 or cisplatin in MilliQ water. The concentration of plasmid in tallics 25 (2005) 756e765.
[13] H. Chen, J.A. Parkinson, S. Parsons, R.A. Coxall, R.O. Gould, P.J. Sadler, J. Am.
the reaction mixture was 50 ng L1 and the concentration of the Chem. Soc. 124 (2002) 3064e3082.
compounds was varied to give different compound:base pair ratios [14] O. Novakova, H. Chen, O. Vrana, A. Rodger, P.J. Sadler, V. Brabec, Biochemistry
(r 0.5, 0.1 and 0.05). The mixtures were incubated at 37  C for 42 (2003) 11544e11554.
[15] R. Kreiter, R.J.M. Klein Gebbink, G. van Koten, Tetrahedron 59 (2003) 3989e3997.
24 h. The mobility of the pBR322 samples was analyzed by gel
[16] D.J.M. Snelders, R. Kreiter, J.J. Firet, G. van Koten, R.J.M. Klein Gebbink, Adv.
electrophoresis on a 0.8% (w/v) agarose gel (Boehringer-Mannheim, Synth. Catal. 350 (2008) 262e266.
Germany) at 100 V cm1 at 25  C for 1 h in Tris-acetate/EDTA buffer. [17] D.J.M. Snelders, G. van Koten, R.J.M. Klein Gebbink, J. Am. Chem. Soc. 131
The gel was stained for 30 min in 0.5 g mL1 (w/v) ethidium (2009) 11407e11416.
[18] D.J.M. Snelders, K. Kunna, C. Mller, D. Vogt, G. van Koten, R.J.M. Klein Geb-
bromide and the bands were analyzed with a UVP gel scanner. bink, Tetrahedron: Asymmetry 21 (2010) 1411e1420.
[19] D.J.M. Snelders, C. van der Burg, M. Lutz, A.L. Spek, G. van Koten, R.J.M. Klein
Gebbink, ChemCatChem 2 (2010) 1425e1437.
5.9. Cytotoxicity assay [20] E. Hodson, S.J. Simpson, Polyhedron 23 (2004) 2695e2707.
[21] C. Scolaro, C.G. Hartinger, C.S. Allardyce, B.K. Keppler, P.J. Dyson, J. Inorg.
Human A2780 and A2780cisR cells were obtained from the Biochem. 102 (2008) 1743e1748.
[22] C. Scolaro, A. Bergamo, L. Brescacin, R. Delno, M. Cocchietto, G. Laurenczy,
European Centre of Cell Cultures (ECACC, Salisbury, UK). All cell T.J. Geldbach, G. Sava, P.J. Dyson, J. Med. Chem. 48 (2005) 4161e4171.
culture reagents were obtained from Gibco-BRL (Basel, Switzer- [23] H. Horvth, G. Laurenczy, Kath, J. Organomet. Chem. 689 (2004) 1036e1045.
land). Cells were grown in RPMI 1640 medium containing 10% [24] C. Gossens, A. Dorcier, P.J. Dyson, U. Rothlisberger, Organometallics 26 (2007)
fetal calf serum (FCS) and antibiotics. The cytotoxicity of the [25] E.C. Alyea, J. Malito, J.H. Nelson, Inorg. Chem. 26 (1987) 4294e4296.
compounds was determined using the MTT assay according to the [26] V.A. Bloomeld, D.M. Crothers, I. Tinoco, Nucleic Acids: Structures, Properties,
method described by Alley et al. [54]. In brief, cells (100 mL) were and Functions. University Science Books, Sausalito, CA, 2000.
[27] E. Gallori, C. Vettori, E. Alessio, F.G. Vilchez, R. Vilaplana, P. Orioli, A. Casini,
added to a 96-well cell culture plates (Corning, NY) at the density
L. Messori, Arch. Biochem. Biophys. 376 (2000) 156e162.
of 25$103 cells per well and incubated at 37  C for 24 h. The stock [28] H.M. Ushay, T.D. Tullius, S.J. Lippard, Biochemistry 20 (1981) 3744e3748.
solutions of the compounds were prepared by dissolving the [29] C.S. Allardyce, P.J. Dyson, D.J. Ellis, S.L. Heath, Chem. Commun. (2001)
compounds in DMSO to reach a concentration of 102 M. They 1396e1397.
[30] A. Romerosa, M. Saoud, T. Campos-Malpartida, C. Lidrissi, M. Serrano-Ruiz,
were then diluted in RPMI medium and added to the wells (100 mL) M. Peruzzini, J.A. Garrido, F. Garca-Maroto, Eur. J. Inorg. Chem. 2007 (2007)
to obtain a nal concentration ranging between 0 and 1000 mM. 2803e2812.
DMSO at comparable concentrations did not show any effects on cell [31] A.K. Renfrew, A.D. Phillips, A.E. Egger, C.G. Hartinger, S.S. Bosquain,
A.A. Nazarov, B.K. Keppler, L. Gonsalvi, M. Peruzzini, P.J. Dyson, Organome-
cytotoxicity. A negative control (RPMI medium only) and a positive tallics 28 (2009) 1165e1172.
control (cisplatin) were run for all the assays. Quadruplicate cultures [32] A. Casini, M.C. Diawara, R. Scopelliti, S.M. Zakeeruddin, M. Gratzel, P.J. Dyson,
were established for each condition tested. After 72 h incubation at Dalton Trans. 39 (2010) 2239e2245.
[33] F.P. Pruchnik, P. Smolen  ski, Appl. Organomet. Chem. 13 (1999) 829e836.
37  C, 10 mL of a solution of MTT (3-(4,5-dimethylthiazole-2-yl)-2,5- [34] P. Serp, M. Hernandez, B. Richard, P. Kalck, Eur. J. Inorg. Chem. 2001 (2001)
diphenyltetrazolium bromide) in PBS (5 mg mL1) was added to 2327e2336.
each well, and the plates were then incubated for 3 h at 37  C. The [35] W.C. Johnson, in: G.D. Fasman (Ed.), Determination of the Conformation of
Nucleic Acids by Electronic CD, Plenum Press, New York, 1996, pp. 433e468.
medium was then aspirated and DMSO (100 mL) was added to [36] A.M. Tamburro, L. Celotti, D. Furlan, V. Guantieri, Chem. Biol. Interact 16
dissolve the precipitate. The absorbance of each well was measured (1977) 1e11.
at 580 nm using a 96-well multiwell-plate reader (iEMS Reader MF, [37] L. Ciani, A. Casini, C. Gabbiani, S. Ristori, L. Messori, G. Martini, Biophys. Chem.
127 (2007) 213e220.
Labsystems, Bioconcept, Switzerland) and compared to the values of
[38] A. Dorcier, P.J. Dyson, C. Gossens, U. Rothlisberger, R. Scopelliti, I. Tavernelli,
control cells incubated without complexes. The percentage of cell Organometallics 24 (2005) 2114e2123.
survival was then determined. [39] C.S. Allardyce, P.J. Dyson, D.J. Ellis, P.A. Salter, R. Scopelliti, J. Organomet. Chem.
668 (2003) 35e42.
[40] W. Han Ang, P.J. Dyson, Eur. J. Inorg. Chem. 2006 (2006) 4003e4018.
Acknowledgements [41] R.W. Wilson, V.A. Bloomeld, Biochemistry 18 (1979) 2192e2196.
[42] M. Auzias, J. Gueniat, B. Therrien, G. Sss-Fink, A.K. Renfrew, P.J. Dyson,
J. Organomet. Chem. 694 (2009) 855e861.
The authors thank Ecole Polytechnique Fdrale de Lausanne [43] M. Gras, B. Therrien, G. Sss-Fink, P. Stepnicka, A.K. Renfrew, P.J. Dyson,
(EPFL) for nancial support. J. Organomet. Chem. 693 (2008) 3419e3424.
1116 D.J.M. Snelders et al. / Journal of Organometallic Chemistry 696 (2011) 1108e1116

[44] N.P.E. Barry, O. Zava, J. Furrer, P.J. Dyson, B. Therrien, Dalton Trans. 39 (2010) [49] P.J. Dyson, Chimia 61 (2007) 698e703.
5272e5277. [50] S. Park, H. Sugiyama, Angew. Chem. Int. Ed. Engl. 49 (2010) 3870e3878.
[45] J. Mattsson, P. Govindaswamy, A.K. Renfrew, P.J. Dyson, P. Stepnicka, [51] A.J. Boersma, R.P. Megens, B.L. Feringa, G. Roelfes, Chem. Soc. Rev. 39 (2010)
G. Sss-Fink, B. Therrien, Organometallics 28 (2009) 4350e4357. 2083e2092.
[46] B. Therrien, G. Sss-Fink, P. Govindaswamy, A.K. Renfrew, P.J. Dyson, Angew. [52] M.A. Bennett, A.K. Smith, J. Chem. Soc. Dalton Trans. (1974) 233e241.
Chem. Int. Ed. Engl. 47 (2008) 3773e3776. [53] P.J. Dyson, D.J. Ellis, G. Laurenczy, Adv. Synth. Catal. 345 (2003) 211e215.
[47] A. Bergamo, A. Masi, P.J. Dyson, G. Sava, Int. J. Oncol. 33 (2008) 1281e1289. [54] M.C. Alley, D.A. Scudiero, A. Monks, M.L. Hursey, M.J. Czerwinski, D.L. Fine,
[48] S. Chatterjee, S. Kundu, A. Bhattacharyya, C.G. Hartinger, P.J. Dyson, J. Biol. B.J. Abbott, J.G. Mayo, R.H. Shoemaker, M.R. Boyd, Cancer Res. 48 (1988)
Inorg. Chem. 13 (2008) 1149e1155. 589e601.