Highly active neutral ruthenium(II) arene complexes: synthesis, characterization, and investigation of their anticancer properties.

Journal of Inorganic Biochemistry 113 (2012) 77–82 Contents lists available at SciVerse ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio Highly active neutral ruthenium(II) arene complexes: Synthesis, characterization, and investigation of their anticancer properties☆ Gerd Ludwig a, Goran N. Kaluđerović a, b, Martin Bette a, Michael Block a, Reinhard Paschke b, Dirk Steinborn a,⁎ a b Institute of Chemistry, Martin Luther University of Halle-Wittenberg, Kurt-Mothes-Straße 2, D-06120 Halle, Germany Biocenter, Martin Luther University of Halle-Wittenberg, Weinbergweg 22, D-06120 Halle, Germany a r t i c l e i n f o Article history: Received 14 February 2012 Received in revised form 5 April 2012 Accepted 5 April 2012 Available online 12 April 2012 Keywords: Ruthenium(II) complexes P,S ligands Cytotoxic activity a b s t r a c t Reactions of ω-diphenylphosphino-functionalized alkyl phenyl sulﬁdes Ph2P(CH2)nSPh (n = 1, L1; 2, L2; 3, L3), sulfoxides Ph2P(CH2)nS(O)Ph (n = 1, L4; 2, L5; 3, L6) and sulfones Ph2P(CH2)nS(O)2Ph (n = 1, L7; 2, L8; 3, L9) with the dinuclear chlorido bridged ruthenium(II) complex [{Ru(η6-p-cymene)Cl2}2] afforded mononuclear ruthenium(II) complexes of the type [Ru(η 6-p-cymene)Cl2{Ph2P(CH2)nS(O)xPh-κP}] (n/x = 1/ 0, 1; 2/0, 2; 3/0, 3; 1/1, 4; 2/1, 5; 3/1, 6; 1/2, 7; 2/2, 8; 3/2, 9) having the P ∩S(O)x ligands κP coordinated. The complexes were characterized by 1H, 13C and 31P NMR spectroscopy. The crystal structures of complexes 2, 7·CH2Cl2 and 8 were determined by X-ray diffraction analysis. All complexes have been screened for cytostatic activity against cell lines 518A2, 8505C, A253, MCF-7, and SW480. In vitro biological experiments demonstrate that these compounds are active toward the used cell lines. The ruthenium(II) complex [Ru(η 6p-cymene)Cl2{Ph2P(CH2)2SPh-κP}] (2) is the most active compound in the human cancer cell line MCF-7 with the IC50 value 1.4 μM lower than cisplatin (2.0 μM). © 2012 Elsevier Inc. All rights reserved. 1. Introduction Medicinal inorganic chemistry is a ﬁeld of fast growth, increasing prominence, and fascinating opportunities enabled by the design and tuning of metal-based compounds as therapeutic agents [1–5]. Since the discovery of the cytostatic activity of cisplatin by Rosenberg in 1965 [6,7], metal complexes are in common use as anticancer drugs. Cisplatin itself, the prototype of a metal-based anticancer agent, is still the most widely used chemotherapeutic agent in clinical use [8]. The major disadvantage of cisplatin is the high toxicity, causing dosedepending side effects such as neuro-, hepto- and nephrotoxicity and thus limits the clinical application [9]. Another major problem in clinical use of cisplatin is the resistance of some carcinomas against different platinum based drugs [10,11]. Stimulated by the success and also by the disadvantages of cisplatin and related platinum complexes, a wide range of other transition metal complexes was screened for their cytostatic activities where some of them entered clinical trials [12–14]. Ruthenium complexes deserve special interest not only because of anticancer properties, but also because of antimetastatic activity [15]. An advantage of ruthenium complexes is on the one hand their cytotoxic activity, but on the other hand the fact that they only hardly attack normal cells [16–18]. Since the ﬁrst discovery of ruthenium ☆ Dedicated to Professor Wolfgang Beck on the occasion of his 80th birthday. ⁎ Corresponding author. Fax: + 49 34 5 5527028. E-mail address: dirk.steinborn@chemie.uni-halle.de (D. Steinborn). 0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2012.04.003 complexes for cancer research, a large number of ruthenium-based compounds have been investigated and reported in literature by the research groups of Sadler, Dyson and Keppler [19–28]. Thus, octahedral ruthenium(III) complexes [imiH]trans-[Ru(N-imi)(S-dmso)Cl4] (imi = imidazole; type I in Fig. 1) and [indH]trans-[Ru(N-ind)2Cl4] (ind= indazole; type II) have reached clinical trials for cancer treatment [29–34]. Ruthenium complexes of the oxidation state +3 are thought to undergo a reduction into the oxidation state +2 in vivo [35,36]. By introducing a π-bonded arene ligand it is possible to stabilize the oxidation state +2 of ruthenium [37]. Thus, ruthenium(II) arene complexes of the type [Ru(η6-arene)(N∩N)Cl]X (N∩N = diamine chelating ligand; X = Cl, PF6, BPh4; type III in Fig. 1) showed both in vitro and in vivo promising anticancer activity. Complexes of this type exhibited IC50 values (IC50 = concentration of compound that inhibits 50% of cell growth) in vitro in the range of 6–300 μM against human cancer cell lines [37,38]. Recent research focused the synthesis and cytostatic activity of ruthenium(II) complexes with phosphorus ligands such as [Ru(η 6-arene)Cl2(PTA)] (type IV; PTA: 1,3,5-triaza7-phosphaadamantane) [18]. However, only a few examples of ruthenium complexes with mentioned coordination exhibit a cytostatic activity against human cancer cell lines in the range of cisplatin activity [39]. Here, we report on the cytotoxic activity of ruthenium(II) complexes of the type [Ru(η6-p-cymene)Cl2{Ph2P(CH2)nS(O)xPh-κP}], on the inﬂuence of the oxidation state of the sulfur atom (x = 0–2), and the spacer length (n = 1–3) on the cytotoxic activity of these ruthenium(II) complexes, respectively. 78 G. Ludwig et al. / Journal of Inorganic Biochemistry 113 (2012) 77–82 Fig. 1. Examples of ruthenium-based anticancer drugs. 2. Results and discussion 2.1. Syntheses and spectroscopic investigation Table 1 Selected NMR spectroscopic data (δ in ppm, J in Hz) of [Ru(η6-p-cymene)Cl2 {Ph2P(CH2)nS(O)xPh-κP}] (1−9). x Reactions of the dinuclear complex [{Ru(η6-p-cymene)Cl2)}2] with ω-diphenylphosphino-functionalized alkyl phenyl sulﬁdes, Ph2P(CH2)n SPh (n = 1, L1; 2, L2; 3, L3) afforded in toluene with cleavage of the Ru– Cl–Ru bridges the formation of mononuclear complexes of the type [Ru(η 6-p-cymene)Cl2{Ph2P(CH2)nSPh-κP}] (1−3; Scheme 1). Analogously, the ω-diphenylphosphino-functionalized alkyl phenyl sulfoxides Ph2P(CH2)nS(O)Ph (n = 1, L4; 2, L5; 3, L6) and sulfones Ph2P(CH2)nS(O)2Ph (n = 1, L7; 2, L8; 3, L9) were found to react with the starting dinuclear ruthenium complex in methylene chloride and in reﬂuxing methanol, respectively, to yield mononuclear complexes of the type [Ru(η6-p-cymene)Cl2{Ph2P(CH2)nS(O)xPh-κP}] (4−9; Scheme 1). The complexes 1−9 were obtained as orange powders in yields between 67−91%. The solids are stable to air over weeks. All complexes are soluble in dimethylsulfoxide and, except for 7, also in methylene chloride, in which they were found to decompose within 1–2 weeks, even under anaerobic conditions. The complexes 1−9 were characterized by elemental analyses, NMR spectroscopy ( 1H, 13C, 31P), and X-ray single-crystal structure analyses (2, 7, 9). Selected NMR spectroscopic parameters of complexes 1−9 are given in Table 1. Coordination of the ligands to Ru(II) gives rise to strong downﬁeld shifts of the phosphorus resonances δP up to 50 ppm. The resulting singlet resonances between 21.8 and 31.3 ppm were found to be indicative of a κP coordination of Ph2P(CH2)nS(O)xPh to Ru(II) [40]. Coordination-induced shifts of the aliphatic carbon atoms (δC(coord.) – δC(uncoord.)) of the ligands Ph2P(CH2)nS(O)xPh (L1−L9) were detected up to −11 ppm; the highest value (−11.2 ppm) was found in complex 7. The 1JP,C couplings are generally in the range of 20–30 ppm, with the exception of 4 and 7, caused by the direct inﬂuence of the sulﬁnyl or sulfonyl group on the α-carbon atom. All proton resonances of the p-cymene ligand in complexes 1−9 were found to be in a narrow range, largely independent from the type of the S(O)x function. The proton chemical shifts were detected between 4.95−5.22 ppm (aromatic CH), 2.44–2.49/0.78–0.86 ppm (isopropyl CH/CH3), and 1.81–1.87 ppm (methyl CH3). Because of the chirality of the P-functionalized sulfoxide, for the proton and the carbon atoms of the p-cymene ligand in the complexes 4–6 two sets of signals for the diastereotopic atoms (aromatic CH and isopropyl CH3) could be detected. Ph2PCαH2S(O)xPh δα-C (1JP,C) 0 1 27.2 (21.0) 1 4 53.3 (10.3) 2 7 48.2 (11.8) Ph2PCβH2CαH2S(O)xPh Ph2PCγH2CβH2CαH2S(O)xPh δα-C (2JP,C) δβ-C (1JP,C) δP δα-C (3JP,C) δγ-C (1JP,C) δP 31.3 2 28.0 (5.8) 22.6 5 50.7 (4.5) 21.8 8 51.2 25.2 (23.2) 20.0 (27.6) 20.0 (27.0) 22.6 3 34.2 (13.2) 23.7 6 50.6 (11.4) 23.1 9 56.5 (12.1) 22.3 (28.8) 22.0 (22.7) 22.2 (29.1) 24.5 δP 24.7 24.5 2.2. Molecular structures Crystals of [Ru(η 6-p-cymene)Cl2{Ph2PCH2CH2SPh-κP}] (2), [Ru(η6p-cymene)Cl2{Ph2PCH2S(O)2Ph-κP}]·CH2Cl2 (7·CH2Cl2) and [Ru(η6-pcymene)Cl2{Ph2PCH2CH2S(O)2Ph-κP}] (8) suitable for X-ray diffraction analysis were obtained from solutions of methylene chloride/n-pentane at room temperature. The compounds crystallized as isolated molecules without unusual intermolecular interactions (shortest distance between non-hydrogen atoms: 3.173(2) Å, C3···C4’, 2; 3.242(4) Å, C6···O1’, 7·CH2Cl2; 3.233(5) Å, C29···O2’, 8). The molecular structures are shown in Figs. 2–4 and selected structural parameters are given in the ﬁgure captions. All the three complexes have a half sandwich (piano stool) structure, in which ruthenium(II) has coordinated a η 6-cymene ligand, two chlorido ligands as well as a P ∩S-κP (2) and a P ∩SO2-κP (7, 8) ligand, respectively. The angles at the ruthenium(II) atom are close to 90° (85.9(3)–88.5(2)°), so that the structure can be viewed as a distorted octahedron. For all complexes, the Ru–Cl bond lengths (2.408(7)–2.423(7) Å) as also the Ru–P bond length (2.349(6)– 2.371(4) Å) are in the expected range (median Ru–Cl: 2.422 Å, lower/ higher quartile: 2.393/2.469 Å, n = 1153; median Ru–P: 2.316 Å, lower/ higher quartile: 2.275/2.359 Å, n = 1153; n – number of observations). For the ruthenium(II) complexes with the ω-diphenylphosphinofunctionalized alkyl phenyl sulfone ligands the O–S–O (119.6(2)°, 7; 119.4(2)°, 8) and C–S–C (100.5(1)°, 7; 105.4(1)°, 8) angles were found to be as anticipated (median O–S–O: 118.4°, lower/higher quartile: 117.6/119.2°, n = 3010; median C–S–C: 104.7°, lower/higher quartile: 102.8/106.3°, n = 3010). Scheme 1. Synthetic routes to ruthenium(II) complexes bearing Ph2P(CH2)nS(O)xPh-κP ligands (1–9). G. Ludwig et al. / Journal of Inorganic Biochemistry 113 (2012) 77–82 79 Fig. 2. Molecular structure of [Ru(η6-p-cymene)Cl2{Ph2PCH2CH2SPh-κP}] in crystals of 2. The ellipsoids are shown with a probability of 50%. H atoms have been omitted for clarity. Selected structural parameters (distances in Å, angles in °): Ru–Cl1 2.410(4), Ru–Cl2 2.421(4), Ru–P 2.371(4), Cl1–Ru–Cl2 88.5(2), Cl1–Ru–P 87.6(1), Cl2–Ru–P 89.0(2), C24–S–C25 105.7(9). 2.3. Biological studies In vitro cytotoxic studies of synthesized ruthenium(II) complexes were performed against 518A2 (melanoma), 8505C (anaplastic thyroid tumor), A253 (head and neck tumor), MCF-7 (breast), and SW480 (colon) cells lines. The cells were cultured in the presence of various concentrations of L1–L9 and of corresponding ruthenium(II) Fig. 3. Molecular structure of [Ru(η6-p-cymene)Cl2{Ph2PCH2S(O)2Ph-κP}] (7) in crystals of 7·CH2Cl2. The ellipsoids are shown with a probability of 50%. H atoms have been omitted for clarity. Selected structural parameters (distances in Å, angles in °): Ru–Cl1 2.408(7), Ru–Cl2 2.423(7), Ru–P 2.360(7), Cl1–Ru–P 86.0(2), Cl2–Ru–P 85.9(3), Cl1−Ru−Cl2 87.7(3), C24–S–C23 100.5(1), O1−S−O2 119.6(2). Fig. 4. Molecular structure of [Ru(η6-p-cymene)Cl2{Ph2PCH2CH2S(O)2Ph-κP}] in crystals of 8. The ellipsoids are shown with a probability of 50%. H atoms have been omitted for clarity. Selected structural parameters (distances in Å, angles in °): Ru–Cl1 2.417(6), Ru–Cl2 2.410(6), Ru–P 2.349(6), Cl1–Ru–P 87.5(2), Cl2–Ru–P 86.5(2), Cl1−Ru− Cl2 87.4(2), C24–S–C23 105.4(1), O1−S−O2 119.4(2). complexes (1−9) for 96 h. The cell viability was analyzed by a sulforhodamine-B (SRB) microculture colorimetric assay [41]. Cell viability was determined by quantitative measurement of SRB incorporation in the cell and the results were normalized with respect to the viability rate in untreated cells. After a 96-h incubation period, increasing concentrations of L1–L9 and 1–9 (0–300 μM) were able to inhibit cell growth of 518A2, 8505C, A253, MCF-7, and SW480 cells in a dose-dependent manner (Fig. 5). The results are shown in Table 2 in which, for comparison, the activity of cisplatin is included. Generally, with the exception of L7, ligands (L1–L9) show lower in vitro activities than the corresponding ruthenium(II) complexes on almost all cell lines. The most effective ligand was found to be L2 (2.8– 8.0 times less active than 2). The ligands with the lowest inﬂuence on cell inhibition are L6 and L9. In almost every case, with exception of complex 6, the ruthenium(II) compounds with sulﬁnyl- (4–6) or sulfonyl-functionalized ligands (7–9) exhibited lower cytotoxicity than cisplatin. Only the ruthenium complexes [Ru(η 6-p-cymene)Cl2 {Ph2P(CH2)nSPh-κP}] (1–3; n=1–3) having a pendant thioether group in the ligand show IC50 values in the same order of magnitude or, in some cases, even lower than cisplatin. The most active compound of the series examined in the current study is compound 2 with an IC50 value of 1.4 μM against cell line MCF-7. Contrary, ruthenium(II) complex 7 with a sulfonyl-functionalized ligand on the same cell line showed IC50 value of 115.0 μM. The ruthenium(II) complex 1 (n/x=1/0) which has a κP coordinated ligand and a sulﬁde group is four times more active against the cell line MCF-7 than the ruthenium(II) complex 4 (n/x = 1/1) having a κP coordinated ligand with a pendant sulﬁnyl group is eighteen times more active against the cell line MCF-7 than the ruthenium(II) complex 7 (n/x = 1/2) with a κP coordinated ligand having a pendant sulfonyl group. In general, the cytotoxic activity was found to increase with decreasing oxidation of the pendant S(O)xPh (x =0–2) group of the ligand. Furthermore, within the ruthenium(II) complexes [Ru(η 6-pcymene)Cl2{Ph2P(CH2)nS(O)xPh-κP}] having pendant sulﬁnyl (x = 1) and sulfonyl (x = 2) groups, a relationship between the spacer length (n = 1–3) and cytotoxic activity could be derived: With increasing 80 G. Ludwig et al. / Journal of Inorganic Biochemistry 113 (2012) 77–82 Fig. 5. Representative graphs showing survival (in %) of cells grown for 96 h in the presence of increasing concentrations of compounds 2 and 3. Table 2 IC50 values (in μMa)) of new ruthenium(II) derivatives (1−9) and the corresponding ligands (L1−L9). The values for cisplatin are given for comparison. Compound n/x L1 L2 L3 L4 L5 L6 L7 L8 L9 1 2 3 4 5 6 7 8 9 cisplatin 518A2 8505C A253 MCF-7 SW480 1/0 93.9 ± 1.9 76.9 ± 3.0 76.5 ± 3.8 64.8 ± 2.6 99.4 ± 1.9 2/0 20.5 ± 1.9 11.0 ± 1.2 9.5 ± 1.1 6.3 ± 0.2 11.3 ± 1.2 3/0 26.8 ± 3.8 18.3 ± 3.7 25.1 ± 2.7 10.7 ± 1.8 23.5 ± 1.9 1/1 50.8 ± 2.9 72.2 ± 3.5 55.0 ± 1.4 31.7 ± 2.0 78.5 ± 0.7 2/1 33.9 ± 4.0 32.7 ± 3.7 27.1 ± 3.2 55.6 ± 3.4 35.6 ± 1.5 3/1 153.3 ± 5.8 96.8 ± 2.0 105.8 ± 6.3 23.0 ± 0.9 151.2 ± 2.5 1/2 5.5 ± 2.0 28.2 ± 2.1 50.4 ± 2.4 46.8 ± 7.2 37.1 ± 2.6 2/2 29.8 ± 1.5 33.5 ± 4.2 27.8 ± 2.7 31.1 ± 2.8 38.1 ± 2.6 3/2 147.6 ± 2.0 128.0 ± 3.3 114.7 ± 3.4 85.3 ± 0.9 149.7 ± 1.9 1/0 1.8 ± 0.5 2.9 ± 0.1 1.7 ± 0.3 6.6 ± 0.5 3.5 ± 0.1 2/0 2.6 ± 0.1 3.9 ± 0.4 3.0 ± 0.1 1.4 ± 0.3 2.6 ± 0.1 3/0 3.0 ± 0.1 3.6 ± 0.1 3.9 ± 0.1 1.8 ± 0.5 2.7 ± 0.1 1/1 26.1 ± 1.2 27.2 ± 4.5 12.7 ± 0.8 31.7 ± 2.0 19.7 ± 1.6 2/1 18.6 ± 0.4 28.6 ± 0.8 26.8 ± 0.4 10.5 ± 1.2 19.5 ± 1.4 3/1 14.3 ± 3.6 12.0 ± 1.0 13.9 ± 1.5 1.8 ± 0.4 11.9 ± 3.3 1/2 26.1 ± 1.3 44.6 ± 4.4 30.9 ± 3.1 115.0 ± 0.6 25.2 ± 3.3 2/2 22.5 ± 2.1 21.1 ± 1.6 20.1 ± 1.7 21.3 ± 1.1 12.5 ± 1.0 3/2 11.7 ± 1.2 11.8 ± 2.5 11.6 ± 2.4 14.1 ± 2.3 10.9 ± 1.1 1.5 ± 0.2 5.0 ± 0.2 0.8 ± 0.1 2.0 ± 0.1 3.2 ± 0.2 lines, but when coordinated to the Ru(η6-p-cymene)Cl2 moiety activity is strongly increased in most cases. Furthermore, the functionalization of the ligands on the metal complex play a signiﬁcant role in modulating the activity. It was found that ruthenium(II) complexes containing the ligands L1, L2, and L3 are potent inhibitors of cancer cell growth. In vitro anticancer activity tests revealed that the most active ruthenium complex is compound 2 against the MCF-7 cell line with an IC50 value of 1.4 μM. Furthermore, the inﬂuences on the cytotoxic activity of the κP coordinated phosphorous ligands have been pointed out. The elongation of the spacer from a methylene to a trimethylene spacer has a positive effect on the cytotoxic activity of the ruthenium(II) complexes 4−9 especially against the cell lines 518A2, 8505C, MCF-7, and SW480. On the other hand, the reduction of the sulfur functionalization, from the sulfonyl via sulﬁnyl to a sulﬁde group, increased the anticancer activity of the ruthenium(II) complexes 1−9 against all the used cell lines. 3. Experimental part 3.1. General comments a) Mean value ± standard deviation. spacer length, an increasing cytotoxic activity against the cell lines 518A2, 8505C, MCF-7, and SW480 was observed. Thus, the cytotoxic activity against MCF-7 cell line of the ruthenium(II) complex 6 with the trimethylene spacer (n = 3; x = 1) is about 18 times higher than that of complex 4 having only the methylene spacer (n = 1; x = 1). An overview about the cytotoxic activity of the ruthenium(II) complexes, in comparison with cisplatin, is given in Fig. 6. 2.4. Conclusion In summary, the in vitro toxicity of a series of half sandwich ruthenium(II) complexes, containing ligands with variable hydrophobic and polar groups, of the type [Ru(η 6-p-cymene)Cl2{Ph2P(CH2)n S(O)xPh-κP}] (n/x = 1/0, 1; 2/0, 2; 3/0, 3; 1/1, 4; 2/1, 5; 3/1, 6; 1/2, 7; 2/2, 8; 3/2, 9) having the P∩S(O)x ligands κP coordinated, has been assessed. The noncoordinated ligands are active against the studied cell All reactions and manipulations were carried out under argon using standard Schlenk techniques. Diethyl ether, toluene, and npentane were dried over Na/benzophenone and methylene chloride over CaH2 and freshly distilled prior to use. NMR spectra ( 1H, 13C, 31P) were recorded at 27 °C on Varian Gemini 200 and VXR 400 spectrometers. Chemical shifts are relative to solvent signals (CDCl3, δΗ 7.24, δC 77.0) as internal references; δ( 31P) is relative to external H3PO4 (85%). Microanalyses (C, H) were performed in the Microanalytical Laboratory of the University of Halle using a CHNS-932 (LECO) elemental analyzer. [{RuCl2(η 6-p-cymene)}2] and the starting compounds L1−L9 were prepared according to literature procedure [40,42]. 3.2. Preparation of [RuCl2(η 6-p-cymene){Ph2P(CH2)nSPh-κP}] (1–3) To a toluene solution (30 mL) of [{RuCl2(η 6-p-cymene)}2] (0.1 g, 0.16 mmol) the respective ligand (0.32 mmol) was added while Fig. 6. Overview about the cytotoxic activity of the ruthenium(II) complexes (1−9) in comparison with cisplatin. [Ru] = Ru(η6-p-cymene)Cl2. G. Ludwig et al. / Journal of Inorganic Biochemistry 113 (2012) 77–82 stirring. The solution was stirred at r.t. overnight, yielding an orange powder that was ﬁltered off, washed with n-pentane (3 × 2 mL), and dried in vacuum. 1 (n = 1). Yield: 165 mg (84%). Anal. Found: C, 56.43; H, 4.82. Calcd for C29H31Cl2PRuS (614.03): C, 56.68; H, 5.08. 1H NMR (200 MHz, CDCl3): δ 0.78 (d, 3JH,H = 6.95 Hz, 6H, CH(CH3)2), 1.87 (s, 3H, CH3), 2.33−2.56 (m, 1H, CH(CH3)2), 4.15 (d, 2JP,H = 3.63 Hz, 2H, PCH2), 5.20 (AA'BB’ spin system, 4H, Ccym-H), 6.80−7.85 (m, 15H, HPh). 13C NMR (50 MHz, CDCl3): δ 17.2 (s, CCH3), 21.2 (s, CH(CH3)2), 27.2 (d, 1JP,C = 21.0 Hz, CH2PPh2), 29.9 (s, CH(CH3)2), 85.7 (d, 2JP,C =5.7 Hz, 2/6-Ccym), 90.4 (d, 2JP,C =4.3 Hz, 3/5-Ccym), 94.1 (s, CCH3), 108.0 (s, CCH(CH3)2), 125.6−136.3 (CPh). 31P NMR (80 MHz, CDCl3): δ 31.3 (s). 2 (n = 2). Yield: 179 mg (89%). Anal. Found: C, 57.98; H, 5.20. Calcd for C30H33Cl2PRuS (628.60): C, 57.94; H, 5.49. 1H NMR (200 MHz, CDCl3): δ 0.84 (d, 3JH,H = 6.93 Hz, 6H, CH(CH3)2), 1.84 (s, 3H, CH3), 2.53−2.92 (m, 5H, CH(CH3)2 + SCH2 + CH2PPh2), 5.12 (AA'BB’ spin system, 4H, Ccym-H), 7.01−7.82 (m, 15H, HPh). 13C NMR (50 MHz, CDCl3): δ 17.3 (s, CCH3), 21.4 (s, CH(CH3)2), 25.2 (d, 1JP,C = 23.2 Hz, CH2PPh2), 28.0 (d, 2JP,C = 5.8 Hz, SCH2), 29.9 (s, CH(CH3)2), 85.6 (d, 2JP,C = 5.8 Hz, 2/6-Ccym), 90.2 (d, 2JP,C = 4.1 Hz, 3/5Ccym), 94.3 (s, CCH3), 108.9 (s, CCH(CH3)2), 125.8−135.6 (CPh). 31P NMR (80 MHz, CDCl3): δ 22.6 (s). 3 (n = 3). Yield: 162 mg (0.79%). Anal. Found: C, 57.34; H, 5.16. Calcd for C31H35Cl2PRuS (642.62): C, 57.88; H, 5.45. 1H NMR (200 MHz, CDCl3): δ 0.78 (d, 3JH,H = 6.95 Hz, 6H, CH(CH3)2), 1.27−1.46 (m, 2H, CH2PPh2), 1.85 (s, 3H, CH3), 2.44−2.73 (m, 5H, CH(CH3)2 + SCH2 + CH2CH2CH2), 5.14 (AA'BB’ spin system, 4H, Ccym-H), 7.04−7.85 (m, 15H, HPh). 13C NMR (50 MHz, CDCl3): δ 17.2 (s, CCH3), 21.2 (s, CH(CH3)2), 22.3 (d, 1JP,C = 28.8 Hz, CH2PPh2), 23.0 (d, 2JP,C = 7.6, CH2CH2CH2), 29.9 (s, CH(CH3)2), 34.2 (d, 2JP,C = 13.2 Hz, SCH2), 85.6 (d, 2JP,C = 5.8 Hz, 2/6-Ccym), 90.4 (d, 2JP,C = 4.3 Hz, 3/5-Ccym), 93.7 (s, CCH3), 108.1 (s, CCH(CH3)2), 125.6−136.1 (CPh). 31P NMR (80 MHz, CDCl3): δ 24.5 (s). 3.3. Preparation of [RuCl2(η 6-p-cymene){Ph2P(CH2)nSOPh-κP}] (4–6) To a methylene chloride solution (25 mL) of [{RuCl2(η6-p-cymene)}2] (0.1 g, 0.16 mmol) the respective ligand (0.32 mmol) was added while stirring. The solution was stirred at r.t. overnight, yielding an orange powder that was ﬁltered off, washed with n-pentane (3× 2 mL), and dried in vacuum. 4 (n = 1). Yield: 135 mg (0.67%). Anal. Found: C, 54.90; H, 4.69. Calcd for C29H31Cl2PRuSO (630.57): C, 55.24; H, 4.96. 1H NMR (200 MHz, CDCl3): δ 0,70 (d, 3JH,H = 6.88 Hz, 3H, CH(CH3)2), 0,99 (d, 3 JH,H = 6.97 Hz, 3H, CH(CH3)2), 1.84 (s, 3H, CH3), 2.42−2.55 (m, 1H, CH(CH3)2), 3.88−4.19 (m, 2H, CH2), 5.16 (AA'BB’ spin system, 4H, Ccym-H), 7.30−8.22 (m, 15H, HPh). 13C NMR (50 MHz, CDCl3): δ 17.3 (s, CCH3), 20.5 (s, CH(CH3)2), 22.4 (s, CH(CH3)2), 30.1 (s, CH(CH3)2), 53.3 (d, 1JP,C = 10.3 Hz, CH2PPh2), 84.1 (d, 2/6-Ccym), 91.9 (d, 3/5Ccym), 95.0 (s, CCH3), 108.9 (s, CCH(CH3)2), 123.7−145.4 (CPh). 31P NMR (80 MHz, CDCl3): δ 22.6 (s). 5 (n = 2). Yield: 148 mg (0.72%). Anal. Found: C, 55.67; H, 4.90. Calcd for C30H33Cl2PRuSO (644.04): C, 55.90; H, 5.16. 1H NMR (200 MHz, CDCl3): δ 0,85 (d, 3JH,H = 6.93 Hz, 3H, CH(CH3)2), 0,92 (d, 3 JH,H = 6.94 Hz, 3H, CH(CH3)2), 1.82 (s, 3H, CH3), 2.44−2.95 (m, 5H, CH(CH3)2 + SOCH2 + CH2PPh2), 5.09 (AA'BB’ spin system, 4H, Ccym-H), 7.30−7.81 (m, 15H, HPh). 13C NMR (50 MHz, CDCl3): δ 17.4 (s, CCH3), 20.0 (d, 1JP,C = 27.6 Hz, CH2PPh2), 21.4 (s, CH(CH3)2), 21.6 (s, CH(CH3)2), 30.0 (s, CH(CH3)2), 50.7 (d, 2JP,C = 4.5 Hz, SOCH2), 85.6 (d, 2 JP,C = 5.5 Hz, Ccym), 86.1 (d, 2JP,C = 5.9 Hz, Ccym), 89.7 (d, 2JP,C = 3.9 Hz, Ccym), 90.2 (d, 2JP,C = 4.4 Hz, Ccym), 95.2 (s, CCH3), 109.5 (s, CCH(CH3)2), 128.1−133.5 (CPh). 31P NMR (80 MHz, CDCl3): δ 23.7 (s). 6 (n = 3). Yield: 143 mg (68%). Anal. Found: C, 56.48; H, 5.27. Calcd for C31H35Cl2PRuSO (658.06): C, 56.53; H, 5.36. 1H NMR (200 MHz, CDCl3): δ 0,77 (d, 3JH,H = 6.87 Hz, 3H, CH(CH3)2), 0,80 (d, 3 JH,H = 6.87 Hz, 3H, CH(CH3)2), 1.22−1.63 (m, 2H, CH2PPh2), 1.84 (s, 81 3H, CH3), 2.41−2.71 (m, 5H, CH(CH3)2 + CH2CH2CH2 + SOCH2), 5.15 (AA'BB’ spin system, 4H, Ccym-H), 7.37−7.89 (m, 15H, HPh). 13C NMR (50 MHz, CDCl3): δ 16.6 (d, 2JP,C = 7.4 Hz, CH2CH2CH2), 17.4 (s, CCH3), 21.1 (s, CH(CH3)2), 21.3 (s, CH(CH3)2), 22.0 (d, 1JP,C = 22.7 Hz, CH2PPh2), 30.0 (s, CH(CH3)2), 50.6 (d, 3JP,C = 11.4 Hz, SOCH2), 85.4 (d, 2JP,C =5.6 Hz, Ccym), 85.8 (d, 2JP,C =5.5 Hz, Ccym), 90.1 (d, 2JP,C =3.9 Hz, Ccym), 90.6 (d, 2JP,C =4.3 Hz, Ccym), 95.1 (s, CCH3), 109.2 (s, CCH(CH3)2), 124.4−142.5 (CPh). 31P NMR (80 MHz, CDCl3): δ 24.7 (s). 3.4. Preparation of [RuCl2(η 6-p-cymene){Ph2P(CH2)nSO2Ph-κP}] (7–9) A solution of [{RuCl2(η6-p-cymene)}2] (0.1 g, 0.16 mmol) and the respective ligand (0.32 mmol) in MeOH (15 mL) were heated to reﬂux for 3 h, during which time the initial orange-red solution became red in color. The solution had been concentrated under reduced pressure before n-pentane (5 mL) was added. The resulting precipitate was ﬁltered off, washed with n-pentane (3× 2 mL), and dried in vacuum. 7 (n = 1). Yield: 177 mg (86%). Anal. Found: C, 54.08; H, 5.09. Calcd for C29H31Cl2PRuSO2 (646.02): C, 53.87; H, 4.83. 1H NMR (200 MHz, CDCl3): δ 0.78 (d, 3JH,H = 6.87 Hz, 6H, CH(CH3)2), 1.83 (s, 3H, CH3), 2.44 −2.51 (m, 1H, CH(CH3)2), 4.66 (d, 2JP,H = 6.25 Hz, 2H, PCH2), 5.16 (AA'BB’ spin system, 4H, Ccym-H), 7.28−8.15 (m, 15H, HPh). 13C NMR (50 MHz, CDCl3): δ 17.2 (s, CCH3), 21.1 (s, CH(CH3)2), 29.9 (s, CH(CH3)2), 48.2 (d, 1JP,C = 11.8 Hz, CH2PPh2), 85.6 (d, 2JP,C = 6.2 Hz, 2/ 6-Ccym), 90.5 (d, 2JP,C = 4.4 Hz, 3/5-Ccym), 94.3 (s, CCH3), 108.9 (s, CCH(CH3)2), 126.9−142.0 (CPh). 31P NMR (80 MHz, CDCl3): δ 21.8 (s). 8 (n = 2). Yield: 192 mg (91%). Anal. Found: C, 55.07; H, 5.38. Calcd for C30H33Cl2PRuSO2 (660.04): C, 54.54; H, 5.04. 1H NMR (200 MHz, CDCl3): δ 0.88 (d, 3JH,H = 6.95 Hz, 6H, CH(CH3)2), 1.81 (s, 3H, CH3), 2.42−2.56 (m, 1H, CH(CH3)2), 2.79−3.06 (m, 2H, SO2CH2), 3.46 (d, 2JP,H = 5.29 Hz, 2H, PCH2), 5.09 (AA'BB’ spin system, 4H, Ccym-H), 7.41−7.76 (m, 15H, HAr). 13C NMR (50 MHz, CDCl3): δ 17.4 (s, CCH3), 20.0 (s, 1JP,C = 27.0 Hz, CH2PPh2), 21.6 (s, CH(CH3)2), 30.0 (s, CH(CH3)2), 51.2 (s, SO2CH2), 85.9 (d, 2JP,C = 5.6 Hz, 2/6-Ccym), 89.8 (d, 2JP,C = 4.1 Hz, 3/5-Ccym), 95.2 (s, CCH3), 109.5 (s, CCH(CH3)2), 128.1−133.5 (CPh). 31P NMR (80 MHz, CDCl3): δ 23.1 (s). 9 (n = 3). Yield: 187 mg (87%). Anal. Found: C, 55.16; H, 5.29. Calcd for C31H35Cl2PRuSO2 (674.05): C, 55.19; H, 5.23. 1H NMR (400 MHz, CDCl3): δ 0.79 (d, 3JH,H = 6.94 Hz, 6H, CH(CH3)2), 1.43−1.56 (m, 2H, CH2PPh2), 1.85 (s, 3H, CH3), 2.44−2.51 (m, 1H, CH(CH3)2), 2.54−261 (m, 2H, CH2CH2CH2), 2.83−2.87 (m, 2H, SO2CH2), 5.13 (AA'BB’ spin system, 4H, Ccym-H), 7.38−7.81 (m, 15H, HPh). 13C NMR (100 MHz, CDCl3): δ 16.9 (d, 2JP,C = 6.7 Hz, CH2CH2CH2), 17.3 (s, CCH3), 21.3 (s, CH(CH3)2), 22.2 (d, 1JP,C = 29.1 Hz, CH2PPh2), 30.0 (s, CH(CH3)2), 56.5 (d, 3JP,C = 12.1 Hz, SO2CH2), 85.6 (d, 2JP,C = 5.8 Hz, 2/6-Ccym), 90.2 (d, 2JP,C = 4.2 Hz, 3/5-Ccym), 94.2 (s, CCH3), 108.4 (s, CCH(CH3)2), 127.7−138.9 (CPh). 31P NMR (80 MHz, CDCl3): δ 24.5 (s). 3.5. X-ray crystallography Data for X-ray diffraction analyses of single crystals of 2 and 8 were collected on a Stoe-IPDS 2T diffractometer and of 7·CH2Cl2 on a StoeIPDS diffractometer at 200(2) K using Mo-Kα radiation (λ = 0.7103 Å, graphite monochromator). A summary of the crystallographic data, the data collection parameters and the reﬁnement parameters is given in Table 3. Absorption corrections were applied numerically and by multiscan with X-RED32 (Tmin/Tmax: 0.73/0.88, 2; 0.53/0.63, 7·CH2Cl2; 0.85/ 0.90, 8) [43]. The structures were solved with direct methods using SHELXS-97 and reﬁned using full-matrix least-square routines against F2 with SHELXL-97 [44,45]. All non-hydrogen atoms were reﬁned with anisotropic displacement parameters and hydrogen atoms with isotropic ones. H atoms were placed in calculated positions according to the riding model. CCDC 870415 (2), 870416 (7·CH2Cl2), and 870417 (8) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. 82 G. Ludwig et al. / Journal of Inorganic Biochemistry 113 (2012) 77–82 Table 3 Crystallographic data, data collection parameters, and reﬁnement parameters for 2, 7·CH2Cl2, and 8. Empirical formula Mr Crystal system Space group a/Å b/Å c/Å β/° V/Å3 Z Dcal/g·cm− 3 μ(Mo-Kα)/mm− 1 F(000) θ range/° Rﬂn collected Reﬂn observed [I > 2σ(I)] Rﬂn independent Data/restraints/ parameters Goodness-of-ﬁt on F2 R1, wR2 [I > 2σ(I)] R1, wR2 (all data) Largest diff. peak and hole/e Å− 3 2 7·CH2Cl2 8 C30H33Cl2PRuS 628.56 Orthorhombic Pbca 10.4688(4) 19.4369(9) 27.3589(10) C30H33Cl2O2PruS 660.56 Orthorhombic P212121 12.9979(9) 13.3661(9) 16.7576(9) 5567.0(4) 8 1.500 0.906 2576 2.66−28.00 24985 5319 C29H31Cl2O2PRuS.CH2Cl2 731.46 Monoclinic P21/c 9.6747(7) 13.1189(10) 25.491(2) 104.724(8) 3129.1(4) 4 1.553 0.987 1488 2.27–26.05 23670 4930 6695 (Rint = 0.0227) 6695/0/319 6067 (Rint = 0.0614) 6067/0/365 7018 (Rint = 0.0314) 7018/0/337 0.941 1.001 0.847 0.0217, 0.0463 0.0337, 0.0490 0.395/− 0.376 0.0333, 0.0806 0.0451, 0.0846 0.554/− 0.734 0.0231, 0.0420 0.0313, 0.0431 0.289/− 0.477 2911.3(3) 4 1.507 0.875 1352 2.87−28.00 20662 5943 3.6. Biological studies 3.6.1. Cell lines, culture conditions, and preparation of drug solutions The cell lines 518A2, 8505C, A253, MCF-7, and SW480 were kindly provided by Dr. Thomas Müller, Department of Hematology/Oncology, Martin Luther University of Halle-Wittenberg, Halle (Saale), Germany. Cultures were maintained as monolayers in RPMI 1640 (PAA Laboratories, Pasching, Austria) supplemented with 5–10% heat-inactivated fetal bovine serum (Biochrom AG, Berlin, Germany) and penicillin/ streptomycin (PAA Laboratories) at 37 °C in a humidiﬁed atmosphere with 5% CO2. Stock solutions of investigated compounds were prepared in dimethylsulfoxide (DMSO, Sigma Aldrich) at a concentration of 20 mM, ﬁltered through Millipore ﬁlter, 0.22 μm, before use, and diluted by nutrient medium to various working concentrations. Nutrient medium was RPMI-1640 (PAA Laboratories) supplemented with 10% fetal bovine serum (Biochrom AG) and penicillin/streptomycin (PAA Laboratories). 3.6.2. 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