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DOI: 10.1039/B822725J (Paper) Dalton Trans., 2009, 3334-3339

Synthesis, X-ray diffraction structure, spectroscopic properties and antiproliferative activity of a novel ruthenium complex with constitutional similarity to cisplatin†

Sanja Grguric-Sipka a, Iryna N. Stepanenko b, Jelena M. Lazic a, Caroline Bartel b, Michael A. Jakupec b, Vladimir B. Arion *b and Bernhard K. Keppler *b
aUniversity of Belgrade, Faculty of Chemistry, Studentski trg 12-16, 11000, Belgrade, Serbia
bUniversity of Vienna, Institute of Inorganic Chemistry, Währinger Str. 42, A-1090, Vienna, Austria. E-mail: vladimir.arion@univie.ac.at (V.B.A.); bernhard.keppler@univie.ac.at (B.K.K.); Fax: +43 1 427752680.

Received 17th December 2008 , Accepted 16th February 2009

First published on 12th March 2009

Abstract

The light-protected reaction of [(η6-p-cymene)RuIICl2]2 with 1-(2-hydroxyethyl)piperazine in dry methanol, followed by addition of excess NH4PF6, afforded the complex [(η6-p-cymene)RuII(NH3)2Cl](PF6) (1) in 47% yield. Attempts to use the same protocol for the synthesis of [(η6-p-cymene)OsII(NH3)2Cl](PF6) led to the isolation of the binuclear triply methoxido-bridged arene-osmium compound [{(η6-p-cymene)Os}2(μ-OCH3)3](PF6) (3). Both compounds were characterised by X-ray crystallography and 1H NMR spectroscopy, and the ruthenium complex also by spectroscopic techniques (IR and UV-vis spectroscopies). The antiproliferative activity of complex 1in vitro was studied in A549 (non-small cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon carcinoma) cells and compared to that of [(η6-p-cymene)RuII(en)Cl](PF6) (2). In contrast to the latter compound, 1 is only modestly cytotoxic in all three cell lines (IC50: 293–542 μM), probably due to the instability of the diammine ruthenium complex in aqueous solution.

Introduction

The first ruthenium complex which showed antiproliferative activity in vitro was [RuIIICl3(NH3)3].1 The synthesis of this compound was prompted by the clinical success of cis-[PtIICl2(NH3)2],2 which is currently one of the most widely and routinely used metal-based anticancer drugs. Further development of [RuIIICl3(NH3)3] was, however, precluded by its low aqueous solubility, which made its administration difficult. These findings led to the synthesis of novel water-soluble platinum and ruthenium complexes with a different number of ammine, aliphatic and aromatic amine or polyamine ligands and investigation of their antiproliferative activity.3–8

The well-established mechanism of cytotoxic action of cisplatin is the alteration of DNA secondary structure via coordination to the N7 atom of a guanine or adenine base, which implies its prior aquation in the cell to the activated complexes [Pt(NH3)2(H2O)Cl]+ and [Pt(NH3)2(H2O)2]2+.9,10

The cytotoxicity of ruthenium compounds correlates with their ability for DNA binding as well. As for cisplatin, aquation is a requisite for the formation of active species. In addition, the activity of Ru(III) complexes, which are relatively inert towards ligand substitution, was found to depend on the ease of reduction to more labile Ru(II) complexes.11 As a consequence, organometallic ruthenium(II) compounds attracted the attention of researchers in their search for novel antiproliferative agents. The use of arene ligands, which stabilise ruthenium in the +2 oxidation state, initiated a new phase in the development of potential ruthenium anticancer drugs.

The structure of the “piano-stool” [(η6-arene)RuII(X)(Y)(Z)] complexes allows for a variation of the three monodentate ligands X, Y and Z. Linking Y and Z to a bidentate chelating ligand (diamine, deprotonated amino acid or β-diketonate) is also well documented.5,12,13 The complexes [(η6-p-cymene)RuII(X)(Y)(Z)] (X, Y or Z = halide, acetonitrile or isonicotinamide), with three monodentate ligands, are inactive in A2780 human ovarian cancer cells.14 At the same time ruthenium complexes [(η6-arene)MII(en)Cl](PF6) (en = ethylendiamine) show cytotoxicity comparable to cisplatin.15–17 Intriguingly, the complexes [(η6-arene)RuII(NH3)2Cl](PF6), which at least constitutionally possess more similarity to cisplatin (three of four ligands are the same as in cisplatin), have not been studied for their antiproliferative activity. The first investigations of the ruthenium/osmium complexes [(η6-benzene)M(NH3)2Cl](PF6) were reported thirty years ago, but were focused mainly on photochemical properties.18–20 Low analytical purity and difficulties with the reproducibility encountered upon synthesis of these compounds were probably the main reasons which prevented their earlier evaluation for cytotoxicity.

Herein we report on (i) the synthesis of [(η6-p-cymene)RuII(NH3)2Cl](PF6) (1) and attempts to prepare its osmium congener, which led to the isolation of [{(η6-p-cymene)OsII}2(μ-OCH3)3](PF6) (3), (ii) the study of their structures, the spectroscopic characterization of 1 and its behaviour in aqueous and alcohol solutions, and (iii) the evaluation of antiproliferative activity of 1in vitro in three human cancer cell lines in comparison with that of [(η6-p-cymene)RuII(en)Cl](PF6) (2).

Result and discussion

Synthesis

We discovered that the reaction of [(η6-p-cymene)RuCl2]2 with 1-(2-hydroxyethyl)piperazine (HEPA) in 1:2.5 molar ratio in dry methanol followed by addition of excess NH4PF6, concentration of the final solution and prolonged standing at 4 °C gave rise to X-ray diffraction quality orange crystals of [(η6-p-cymene)Ru(NH3)2Cl](PF6) (1, Chart 1) in 47.2% yield. The same reaction starting from [(η6-p-cymene)OsCl2]2 produced yellow crystals of [{(η6-p-cymene)Os}2(μ-OCH3)3](PF6) (3, Chart 1). Variation of the reaction stoichiometry, temperature or use of a stronger base, e.g.triethylamine instead of HEPA, did not lead to the osmium analogue of 1. Moreover, the formation of an unidentified black precipitate was observed upon crystallization of 3 at room temperature. Interestingly, the use of triethylamine instead of HEPA in the case of ruthenium also enabled the synthesis of 1. The role of the base used appears to consist in deprotonation of the ammonium cation, which serves as an ammonia source. Examples of ruthenium complexes with piperazine or their derivatives described in the literature are scarce.21–23
Complexes reported in this work; underlined compounds have been characterised by X-ray crystallography.
Chart 1 Complexes reported in this work; underlined compounds have been characterised by X-ray crystallography.

Although the preparation of [(η6-benzene)Ru(NH3)2Cl](PF6) was reported many years ago, this turned out to be unreliable, and later attempts were undertaken to improve the available literature protocols. The reaction was carried out in concentrated aqueous ammonia in methanol, and saturated aqueous NH4PF6 solution was used for the precipitation of the complex as hexafluorophosphate [(η6-benzene)Ru(NH3)2Cl](PF6).18,19 The same experimental conditions were applied to the synthesis of [(η6-benzene)Os(NH3)2Cl](PF6). The synthesis, however, was accompanied by concurrent reactions which led to low yield and unsatisfactory purity of the desired product.20

Attempts to produce [(η6-p-cymene)Os(NH3)2Cl](PF6) by using the protocol optimised for ruthenium complex 1 resulted in the methoxido-bridged arene complex [{(η6-p-cymene)Os}2(μ-OCH3)3](PF6) (3). Although the synthesis of the complex with a [{(η6-benzene)Os}2(μ-OCH3)3]+ cation is well documented in the literature,24X-ray diffraction data on binuclear hydroxido- or alkoxido-bridged osmium-arene compounds were not found in the Cambridge Structural Database.25

Crystal structures of 1 and 3

Complex 1 crystallised in the triclinic centrosymmetric space groupP[1 with combining macron]. The structure of the cation is shown in Fig. 1. Selected bond distances (Å) and angles (deg) are quoted in the legend to Fig. 1. The cation [(η6-p-cymene)Ru(NH3)2Cl]+ has the characteristic “three-leg piano stool” geometry with an η6π-bound p-cymene ring forming the seat and three other monodentate ligands, namely chlorido and two ammonia, as the legs of the stool. The X-ray structure of [(η6-benzene)Ru(NH3)2Cl](PF6)·0.33NH4PF6 determined at room temperature and published in 197826,27 is of poor quality [Ru–Cav 2.185(4), Ru–N 2.129 and Ru–Cl 3.384 Å, N1–Ru–N1i 83.98, N1–Ru–Cl 83.69°] to make a close comparison with geometrical parameters of the cation in 1 (see legend to Fig. 1). The Ru–C(1–6)av in 1 is very similar to that in [(η6-p-cymene)Ru(en)Cl]+ at 2.187(9) Å, while the Ru–Cl bond is markedly shorter than that in [(η6-p-cymene)Ru(en)Cl]+ at 2.442 Å.14,28 The N1–Ru–N2 angle in the ethylendiamine complex at 78.97° is only ca. 3.7° smaller than the corresponding angle in 1.
ORTEP view of the cation [(η6-p-cymene)Ru(NH3)2Cl]+ in 1 with atom labeling scheme. Selected bond lengths (Å) and angles (deg): Ru–C(1–6)av 2.183(7), Ru–Cl 2.4146(4), Ru–N1 2.1504(15), Ru–N2 2.1425(15); N1–Ru–N2 82.70(6), N1–Ru–Cl 84.60(4), N2–Ru–Cl 84.68(4).
Fig. 1 ORTEP view of the cation [(η6-p-cymene)Ru(NH3)2Cl]+ in 1 with atom labeling scheme. Selected bond lengths (Å) and angles (deg): Ru–C(1–6)av 2.183(7), Ru–Cl 2.4146(4), Ru–N1 2.1504(15), Ru–N2 2.1425(15); N1–Ru–N2 82.70(6), N1–Ru–Cl 84.60(4), N2–Ru–Cl 84.68(4).

The result of the X-ray diffraction study of complex 3, which crystallised in the orthorhombic space groupP212121, is shown in Fig. 2. The geometry of both crystallographically independent cations can be described as confacial-bipseudooctahedral with Os1⋯Os2 and Os3⋯Os4 separations of 3.0748(2) and 3.0820(2) Å, respectively, which are larger than those found for the triply hydroxido-bridged arene-ruthenium cations [Ru2(η6-C6H6)2(μ-OH)3]+29 2.9812(7), [Ru2(η6-1,3,5-C6H3Me3)2(μ-OH)3]+30 2.989(3) and [Ru2(η6-p-cymene)2(μ-OH)3]+31 2.990(3) or the triply methoxido-bridged arene-ruthenium cation [Ru2(η6-C6H6)2(μ-OMe)3]+32 and very similar to those found in a tetrameric osmium complex [Os4(η6-C6H6)4(μ2-OH)4(μ4-O)](BPh4)2·2Me2CO30 of 3.0768(25) and 3.0777(23) for Os1⋯Os2 and Os3⋯Os4 distances, correspondingly.


ORTEP view of the cation [{(η6-p-cymene)Os}2(μ-OCH3)3]+ in 3 with atom labeling scheme. Selected bond lengths (Å) and angles (deg): Os1–O1 2.093(3), Os1–O2 2.069(3), Os1–O3 2.068(3), Os2–O1 2.099(3), Os2–O2 2.072(3), Os2–O3 2.073(3) Å; Os1–O1–Os2 94.36(12), Os1–O2–Os2 95.90(12), Os1–O3–Os2 95.88(11).
Fig. 2 ORTEP view of the cation [{(η6-p-cymene)Os}2(μ-OCH3)3]+ in 3 with atom labeling scheme. Selected bond lengths (Å) and angles (deg): Os1–O1 2.093(3), Os1–O2 2.069(3), Os1–O3 2.068(3), Os2–O1 2.099(3), Os2–O2 2.072(3), Os2–O3 2.073(3) Å; Os1–O1–Os2 94.36(12), Os1–O2–Os2 95.90(12), Os1–O3–Os2 95.88(11).

The bond angles for the bridging methoxido ligands Os1–O1–Os2, Os1–O2–Os2, Os1–O3–Os2, Os3–O4–Os4, Os3–O5–Os4 and Os3–O6–Os4 at 94.36(12), 95.90(12), 95.88(11), 94.70(12), 96.40(12) and 95.68(11)° are comparable with those found in [Ru2(η6-C6H6)2(μ-OMe)3]+ 32 with Ru1–O1–Ru2, Ru1–O2–Ru2 and Ru1–O3–Ru2 of 93.9(3), 94.6(3) and 92.6(3)°, respectively.

Stability of 1 in different solvents

We investigated the stability of 1 in dry methanol, water, and 0.1 M NaCl (a similar concentration to that in blood plasma) by UV-vis spectroscopy (Figs. 3, S1–S3†). Fast changes in UV-vis spectra of 1 in H2O and in aqueous 0.1 M NaCl solution indicate that the complex undergoes substitution reactions at the ruthenium centre upon dissolution. The complex does not remain intact even in methanol, and its UV-vis spectra change rapidly with time. Such behaviour of 1 can be explained by solvolysis of Ru–N and Ru–Cl bonds. Evidence of formation of a dimer of the type 3 in methanol is furnished by ESI mass spectrometry, which showed the presence of the peak with m/z 564 assignable to [{(η6-p-cymene)Ru}2(μ-OCH3)3]+. The experimental isotopic pattern, which agrees well with that calculated, is shown in Fig. S4.† Instability of the species generated by reaction of 1 with AgPF6“[(η6-p-cymene)Ru(NH3)2(H2O)]2+” in H2O (Fig. S5†) indicates its further hydrolysis with involvement of Ru–N bonds in further substitution reactions.
UV-vis spectra of 1 in H2O: 24 × 1 h (cycles 1, 2, 4, 8, 12, 16, 20 and 24).
Fig. 3 UV-vis spectra of 1 in H2O: 24 × 1 h (cycles 1, 2, 4, 8, 12, 16, 20 and 24).

The solution behaviour of 1 was also investigated by 1H NMR spectroscopy. 1H NMR spectra were recorded in DMSO-d6and D2O. For the freshly prepared DMSO-d6 solution of 1 a typical spectrum for a metal-coordinated cymene ligand is observed (see Experimental). The spectrum in D2O changes dramatically and five sets of cymene signals can be observed very quickly (Figs. 4, S6–S8†). One cymene set disappears after 1 h, whereas the other four sets are still present with changing intensities after 13 days. Also of note is that signals which can be attributed to metal-free cymene were not observed during the present experiment.


1H NMR spectra of 1 in D2O (CH3 region).
Fig. 4 1H NMR spectra of 1 in D2O (CH3 region).

The 1H NMR spectrum of the elusive “[(η6-p-cymene)Ru(NH3)2(H2O)]2+” species in D2O showed four sets of metal-bound cymene signals (see the CH(CH3)2 region of the spectrum in Fig. S9†). The equilibrium was achieved after 2 days with two sets of signals. These two species were also found in D2O solution of 1 (see Fig. S9†).

Cytotoxicity of 1

In vitro anticancer activity of compounds 1 and 2 was determined in human A549 (non-small cell lung carcinoma), CH1 (ovarian carcinoma) and SW480 (colon carcinoma) cells by means of the colorimetric MTT assay. IC50 values were calculated and are quoted in Table 2, and complete concentration–effect curves are displayed in Fig. 5. CH1 cells were found to be more sensitive to 1, followed by SW480 and A549 cells. In contrast, complex 2 shows the strongest effect in SW480 cells, followed by that in CH1 and A549 cells. Compound 2 has a higher level of antiproliferative activity in all three cell lines, with IC50 values lower than 10 μM. In contrast, compound 1 has very low cytotoxic potential with IC50 values between 293 μM in the cisplatin-sensitive cell line CH1 and 542 μM in the cisplatin-resistant cell line A549. Complex 2 is almost a hundredfold more cytotoxic than its structural analogue, 1. We also performed an MTT test with a shorter exposure time in SW480 cells. It clearly points out that compound 1 requires more than 6 h contact with cells to exert its full cytotoxic effect, indicating at the same time that the species present in the medium after 6 h contribute considerably to total cytotoxic activity. The IC50 value could not be reached within this time and the chosen range of concentration.
Table 1 Crystal data and details of data collection for 1 and 3
Complex 1 3
a R1 = ∑‖Fo| − |Fc‖/∑|Fo|, b w R2 = {∑[w(Fo2−Fc2)2]/∑[w(Fo2)2]}1/2. c GOF = {∑[w(Fo2−Fc2)2]/(n–p)}1/2, where n is the number of reflections and p is the total number of parameters refined.
CCDC no. 707426 707427
empirical formula C10H20ClF6N2PRu C23H37F6O6Os2P
fw 449.77 886.89
space group P[1 with combining macron] (No 2) P212121 (No 19)
a, Å 8.0544(2) 11.2483(3)
b, Å 8.8166(2) 13.6694(4)
c, Å 11.2876(3) 34.2630(9)
α, deg 87.861(2)  
β, deg 88.256(2)  
γ, deg 78.272(2)  
V , Å3 784.07(3) 5268.2(3)
Z 2 8
λ, Å 0.71073 0.71073
ρ calcd, g cm−3 1.905 2.236
crystal size, mm3 0.40 × 0.38 × 0.38 0.38 × 0.28 × 0.22
T, K 100 100
μ, cm−1 13.27 97.65
reflns collected/unique [Rint] 16677/4586 [0.0323] 140298/15486 [0.0601]
R1a 0.0237 0.0228
wR2b 0.0612 0.0489
Flack parameter   0.006(5)
GOFc 1.075 0.993

Table 2 Cytotoxicity of ruthenium complexes 1 and 2 in three human cancer cell lines. Presented are the 50% inhibitory concentrations in A549, CH1 and SW480 cells in the MTT assay. Values are the means ± standard deviations obtained from at least three independent experiments using exposure times of 96 h
  IC50, μM
Compound A549 CH1 SW480
1 542 ± 12 293 ± 24 437 ± 23
2 7.1 ± 1.1 4.4 ± 0.9 3.5 ± 0.5


Concentration–effect curves of ruthenium complexes 1 and 2 in the human cancer cell lines A549, CH1 and SW480. Values were obtained by the MTT assay and are means ± standard deviations from at least three independent experiments using exposure times of 96 h, with the exception of the dotted curve of complex 1 in SW480 cells (exposure time 6 h).
Fig. 5 Concentration–effect curves of ruthenium complexes 1 and 2 in the human cancer cell lines A549, CH1 and SW480. Values were obtained by the MTT assay and are means ± standard deviations from at least three independent experiments using exposure times of 96 h, with the exception of the dotted curve of complex 1 in SW480 cells (exposure time 6 h).

Low levels of cytotoxicity in A2780 human ovarian cancer cells with IC50 > 150 μM have been documented for related organometallic compounds with three monodentate ligands, e.g. [(η6-p-cymene)RuII(CH3CN)2Cl](PF6), [(η6-p-cymene)RuII(CH3CN)2Br](PF6), and [(η6-p-cymene)RuII(isonicotinamide)Cl2].14 It was suggested that the observed level of their cytotoxicity, which further decreased with time, is presumably due to their deactivation by components of the cell culture medium and/or the cells before they reach their target sites.5 The formation of the dimer [{(η6-biphenyl)Os}2(μ-OH)3]+ in aqueous solution accounted for the inactivity of [(η6-biphenyl)Os(CH3CN)Cl2] in the human lung A549 cancer cell line.33 In line with these results, we believe that compound 1 has the same fate because of the observed instability in organic and aqueous medium. In contrast to classic platinum(II) complexes, where the presence of ethylenediamine instead of two ammonia ligands is usually disadvantageous for biological activity, the stabilising effect of the bidentate ligand seems to be essential in the case of Ru(II)–arene complexes.

Experimental

Starting materials

RuCl3·3H2O and OsO4 were purchased from Johnson Matthey. [(η6-p-cymene)RuCl2]2 and [(η6-p-cymene)OsCl2]2 were prepared according to literature protocols.34,351-(2-hydroxyethyl)piperazine (HEPA) and triethylamine were purchased from Aldrich. All chemicals were used without further purification. [(η6-p-cymene)RuII(en)Cl](PF6) (2) was prepared as reported by Sadler et al.36

Synthesis of complexes

[(η6-p-cymene)RuII(NH3)2Cl](PF6) (1). To a suspension of [(η6-p-cymene)RuIICl2]2 (0.12 g, 0.2 mmol) in dry methanol (15 mL) a solution of 1-(2-hydroxyethyl)piperazine (0.065 g, 0.5 mmol) in dry methanol (5 mL) was added. The light-protected reaction mixture was stirred at room temperature for 2 h. Then NH4PF6 (0.25 g, 1.6 mmol) was added as a solid, the orange solution was concentrated by rotary evaporation under reduced pressure to 10 mL and allowed to stand at 4 °C for 24 h. The orange crystals formed were filtered off and re-crystallised in ethanol (0.085 g, 47.2%). Found: C, 26.86; H, 4.43; N, 6.12. Calc. for C10H20ClF6N2PRu: C, 26.69; H, 4.45; N, 6.23%. δH (200 MHz; d6-DMSO) 5.60–5.33 (4H, dd, –C6H4–), 2.80 (1H, m, CHMe2), 2.10 (3H, s, C6H4CH3) and 1.20 (6H, d, (CH3)2CH); νmax,cm−1 3381 (m), 3335 (m), 3249 (m), 3193 (m), 1626 (m), 1280 (m), 1255 (m), 1062 (w), 1035 (w), 823 (s) and 553 (s).
Generation of “[(η6-p-cymene)Ru(NH3)2(H2O)]2+” species. AgPF6 (8 mg, 0.03 mmol) was added to a solution of 1 (11.1 mg, 0.025 mmol) in H2O (5 mL). The light-protected mixture was stirred at room temperature for 17 h and then filtered to remove AgCl. The resulting aqueous solution was diluted to 100 mL and used for UV-vis spectroscopy investigation. 10 mL of this solution were lyophilised and studied by NMR spectroscopy.
[{(η6-p-cymene)OsII}2(μ-OCH3)3](PF6) (3). To a suspension of [(η6-p-cymene)OsCl2]2 (60.6 mg, 0.077 mmol) in methanol (15 mL) a solution of 1-(2-hydroxyethyl)piperazine (30.5 mg, 0.23 mmol) in methanol (2 mL) was added. The light-protected reaction mixture was stirred at room temperature for 3.5 h. Then NH4PF6 (92.4 mg, 0.57 mmol) was added, and the yellow solution was concentrated by rotary evaporation under reduced pressure to ca. 5 mL and allowed to stand at −20 °C. The yellow crystals of 3 were studied by X-ray diffraction and NMR spectroscopy. δH (500.10 MHz; DMSO-d6): 6.18–5.97 (8H, dd, –C6H4–), 4.48 (9H, s, μ-OCH3), 2.70 (2H, m, CHMe2), 2.23 (6H, s, C6H4CH3) and 1.23 (12H, d, (CH3)2CH).

Physical measurements

Elemental analyses were carried out with an Elemental Vario EL III microanalyser at the Faculty of Chemistry, University of Belgrade, Serbia. UV-vis spectra were recorded on a Perkin-Elmer Lambda 20 UV-vis spectrophotometer using samples dissolved in dry methanol, water, and 0.1 M NaCl at 298 K. The 1H NMR spectra were recorded with a Bruker FT-NMR spectrometer Avance II™ 500 MHz using standard pulse programs at 500.10 MHz (in D2O, DMSO-d6) and on a Varian Gemini 200 instrument at 200 MHz (in DMSO-d6). Chemical shifts for 1H spectra were referenced to residual 1H present in DMSO-d6 (D2O). Infrared spectra were recorded on a Nicolet 6700 FT-IR spectrometer using the ATR technique. Electrospray ionisation mass spectra of samples dissolved in methanol/acetonitrile were measured with a Finnigan Mat 900S instrument. Expected and experimental isotope distributions were compared.

Crystallographic structure determination

X-ray diffraction measurements were performed on a Bruker X8 APEXII CCD diffractometer at 100 K. Single crystals were positioned at 40 and 60 mm from the detector, and 1033 and 2133 frames were measured, each for 3 and 15 s over 1° scan width for 1 and 3, correspondingly. The data were processed using SAINT software.37Crystal data, data collection parameters, and structure refinement details for 1 and 3 are given in Table 1. Both structures were solved by direct methods and refined by full-matrix least-squares techniques. Non-hydrogen atoms were refined with anisotropic displacement parameters. H atoms were placed at calculated positions and refined as riding atoms in the subsequent least squares model refinements. The isotropic thermal parameters were estimated to be 1.2 times the values of the equivalent isotropic thermal parameters of the non-hydrogen atoms to which hydrogen atoms are bonded. The following computer programs, equipment and table were used: structure solution, SHELXS-97;38 refinement, SHELXL-973;39 molecular diagrams, ORTEP;40 computer, Pentium IV; scattering factors were taken from the literature.41

Cell lines and culture conditions

Human A549 (non-small cell lung carcinoma) and SW480 (adenocarcinoma of the colon) cells were kindly provided by Brigitte Marian (Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Austria). CH1 cells originate from an ascites sample of a patient with a papillary cystadenocarcinoma of the ovary and were a generous gift from Lloyd R. Kelland (CRC Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, UK). Cells were grown in 75 cm2 culture flasks (Iwaki/Asahi Technoglass, Gyouda, Japan) as adherent monolayer cultures in complete medium (Minimum Essential Medium supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 4 mM L-glutamine and 1% non-essential amino acids (100×)). All media and supplements were purchased from Sigma-Aldrich, Vienna, Austria. Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2.

Cytotoxicity test in cancer cell lines

Cytotoxicity was determined by means of a colorimetric microculture assay (MTT assay, MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Fluka). Cells were harvested from culture flasks by trypsinization and seeded in 100 μL aliquots into 96-well plates (Iwaki/Asahi Technoglass, Gyouda, Japan) in densities of 4 × 103 (A549), 1.5 × 103 (CH1) and 2.5 × 103 (SW480) cells per well, respectively, to ensure exponential growth of untreated controls throughout the experiment. Cells were allowed to settle in drug-free complete culture medium for 24 h. Drugs were dissolved and appropriately diluted in complete medium shortly before application. Subsequently 100 μL of the drug dilutions were added per well, and cells were incubated for 96 h. An MTT assay in SW480 cells was also performed with a shorter exposure time. For this purpose, the drug solution was removed after 6 h and cells were incubated with drug-free medium for the remaining 90 h. After exposure, drug solutions or medium, respectively, were replaced by 100 μL/well RPMI 1640 culture medium (supplemented with 10% heat-inactivated fetal bovine serum and 4 mM L-glutamine) plus 20 μL/well MTT solution in phosphate-buffered saline (5 mg/mL). At the end of incubation for 4 h, the medium/MTT mixture was removed and the formazan crystals that were formed in vital cells were dissolved in 150 μL DMSO (dimethyl sulfoxide) per well. Optical densities were measured at 550 nm with a microplate reader (Tecan Spectra Classic), using a reference wavelength of 690 nm. The quantity of vital cells was expressed in terms of T/C values by comparison with untreated control microcultures, and 50% inhibitory concentrations (IC50) were calculated from concentration–effect curves by interpolation. Evaluation is based on means from at least three independent experiments, each comprising three replicates per concentration level.

Conclusion

A novel ruthenium compound, namely [(η6-p-cymene)RuII(NH3)2Cl](PF6) (1), which had remained elusive for preparative organometallic chemists engaged in the development of antitumour drugs for quite a long time, was prepared in good yield and comprehensively characterised. However, the presence of two ammonia ligands instead of ethylenediamine results in a tremendous loss of cytotoxicity, probably due to increased susceptibility to inactivating reactions leading to formation (among others) of [{(η6-p-cymene)Ru}2(μ-OH)3]+ species.

Attempts to apply an analogous approach for the synthesis of the osmium counterpart [(η6-p-cymene)OsII(NH3)2Cl](PF6) failed. Instead a novel binuclear triply methoxido-bridged arene–osmium(II) compound, [{(η6-p-cymene)Os}2(μ-OCH3)3](PF6) (3), was isolated and studied by X-ray crystallography and 1H NMR spectroscopy. This is a further example that the synthesis of osmium analogues cannot be performed by simply following the protocols for ruthenium species, and very often non-trivial approaches, particular skills, experience and knowledge of the systems are required.

References

  1. M. J. Clarke, Metal Ions Biol. Syst., 1980, 11, 231–283 CAS.
  2. B. Rosenberg, L. VanCamp, J. E. Trosko and V. H. Mansour, Nature, 1969, 222, 385–386 CrossRef CAS.
  3. W. H. Ang and P. J. Dyson, Eur. J. Inorg. Chem., 2006, 20, 4003–4018 CrossRef.
  4. M. A. Jakupec, M. Galanski, V. B. Arion, C. G. Hartinger and B. K. Keppler, Dalton Trans., 2008, 2, 183–194 Search PubMed.
  5. L. Ronconi and P. J. Sadler, Coord. Chem. Rev., 2007, 251, 1633–1648 CrossRef CAS.
  6. M. Galanski, V. B. Arion, M. A. Jakupec and B. K. Keppler, Curr. Pharm. Des., 2003, 9, 2078–2089 CrossRef CAS.
  7. C. A. Vock, W. H. Ang, C. Scolaro, A. D. Phillips, L. Lagopoulos, L. Juillerat-Jeanneret, G. Sava, R. Scopelliti and P. J. Dyson, J. Med. Chem., 2007, 50, 2166–2175 CrossRef CAS.
  8. C. A. Vock, C. Scolaro, A. D. Phillips, R. Scopelliti, G. Sava and P. J. Dyson, J. Med. Chem., 2006, 49, 5552–5561 CrossRef CAS.
  9. Y. Jung and S. J. Lippard, Chem. Rev., 2007, 107, 1387–1407 CrossRef CAS.
  10. S. van Zutphen and J. Reedijk, Coord. Chem. Rev., 2005, 249, 2845–2853 CrossRef CAS.
  11. M. J. Clarke, Coord. Chem. Rev., 2003, 236, 209–233 CrossRef CAS.
  12. Y. K. Yan, M. Melchart, A. Habtemariam and P. J. Sadler, Chem. Comm., 2005, 38, 4764–4776 RSC.
  13. A. Habtemariam, M. Melchart, R. Fernandez, S. Parsons, I. D. H. Oswald, A. Parkin, F. P. A. Fabbiani, J. E. Davidson, A. Dawson, R. E. Aird, D. I. Jodrell and P. J. Sadler, J. Med. Chem., 2006, 49, 6858–6868 CrossRef CAS.
  14. R. E. Morris, R. E. Aird, P. S. Murdoch, H. Chen, J. Cummings, N. D. Hughes, S. Parsons, A. Parkin, G. Boyd, D. I. Jodrell and P. J. Sadler, J. Med. Chem., 2001, 44, 3616–3621 CrossRef CAS.
  15. F. Wang, H. Chen, S. Parsons, I. D. H. Oswald, J. E. Davidson and P. J. Sadler, Chem. Eur. J., 2003, 9, 5810–5820 CrossRef CAS.
  16. H. Chen, J. A. Parkinson, S. Parsons, R. A. Coxall, R. O. Gould and P. J. Sadler, J. Am. Chem. Soc., 2002, 124, 3064–3082 CrossRef CAS.
  17. H. Chen, J. A. Parkinson, R. E. Morris and P. J. Sadler, J. Am. Chem. Soc., 2003, 125, 173–186 CrossRef CAS.
  18. D. R. Robertson, T. A. Stephenson and T. Arthur, J. Organomet. Chem., 1978, 162, 121–136 CrossRef CAS.
  19. W. Weber and P. C. Ford, Inorg. Chem., 1986, 25, 1088–1092 CrossRef CAS.
  20. Y. Hung, W.-J. Kung and H. Taube, Inorg. Chem., 1981, 20, 457–463 CrossRef CAS.
  21. G. Sanchez, C. Bifano and H. Krentzien, Acta Cientifica Venezolana, 1984, 35, 44–47 Search PubMed.
  22. K. S. Siddiqi, P. Khan, N. Singhal and S. A. A. Zaidi, Indian J. Chem., 1980, 19A, 265–267 Search PubMed.
  23. S. Grguric-Sipka, M. Al. A. M. Alshtewi, D. Jeremic, G. N. Kaluderovic, S. Gomez-Ruiz, Z. Zizak, Z. Juranic and T. J. Sabo, J. Serb. Chem. Soc., 2008, 73, 619–630 CrossRef CAS.
  24. T. Arthur, D. R. Robertson, D. A. Tocher and T. A. Stephenson, J. Organomet. Chem., 1981, 208, 389–400 CrossRef CAS.
  25. CSD version 5.29, November 2007.
  26. R. O. Gould, C. L. Jones, D. R. Robertson and T. A. Stephenson, Cryst. Structure Comm., 1978, 7, 27–32 Search PubMed.
  27. CSD version 5.29, November 2007 (ABZRUP code).
  28. CSD version 5.29, November 2007 (CAFPOP code).
  29. T. D. Kim, T. J. McNeese and A. L. Rheingold, Inorg. Chem., 1988, 27, 2554–2555 CrossRef CAS.
  30. R. O. Gould, C. L. Jones, T. A. Stephenson and D. A. Tocher, J. Organomet. Chem., 1984, 264, 365–378 CrossRef CAS.
  31. V. Artero, A. Proust, P. Herson and P. Gouzerh, Chem. Eur. J., 2001, 7, 3901–3910 CrossRef CAS.
  32. R. O. Gould, T. A. Stephenson and D. A. Tocher, J. Organomet. Chem., 1984, 263, 375–384 CrossRef CAS.
  33. A. F. A. Peacock, A. Habtemariam, S. A. Moggach, A. Prescimone, S. Parsons and P. J. Sadler, Inorg. Chem., 2007, 46, 4049–4059 CrossRef CAS.
  34. M. A. Bennett and A. K. Smith, J. Chem. Soc., Dalton Trans., 1974, 2, 233–241 Search PubMed.
  35. W. A. Kiel, R. G. Ball and W. A. G. Graham, J. Organomet. Chem., 1990, 383, 481–496 CrossRef CAS.
  36. PCT Int. Appl., WO 2001030790, 2001.
  37. SAINT-Plus (Version 7.06a) and APEX2. Bruker-Nonius AXS Inc. 2004, Madison, Wisconsin, USA Search PubMed.
  38. G. M. Sheldrick, SHELXS-97, Program for Crystal Structure SolutionUniversity of Göttingen, Göttingen, Germany, 1997 Search PubMed.
  39. G. M. Sheldrick, SHELXS-97, Program for Crystal Structure Refinement, University of Göttingen, Göttingen, Germany, 1997 Search PubMed.
  40. G. K. Jonson, Report ORNL-5138, Oak Ridge National Laboratory, Oak Ridge, TN, 1976 Search PubMed.
  41. International Tables for X-ray Crystallography, ed. A. J. C. Wilson, Kluwer Academic Press, Dodrecht, The Netherlands, 1992; Vol. C, Tables 4.2.6.8 and 6.1.1.4 Search PubMed.

Footnote

† Electronic supplementary information (ESI) available: UV-vis spectra of 1 in CH3OH, H2O and 0.1 M NaCl (Figures S1–S3), mass spectra of 1 after 4 days in MeOH (Figure S4), UV−vis spectra of “[(η6-p-cymene)Ru(NH3)2(H2O)]2+” species in H2O (Figure S5), 1H NMR spectra of 1 in D2O (Figures S6–S8), 1H NMR spectra of “[(η6-p-cymene)Ru(NH3)2(H2O)]2+” species in D2O (Figure S9). CCDC reference numbers 707426 (for 1) and 707427 (for 3). For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/b822725j
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