Discovery of a highly tumor-selective organometallic ruthenium(II)-arene complex.

Article pubs.acs.org/jmc Discovery of a Highly Tumor-Selective Organometallic Ruthenium(II)−Arene Complex Catherine M. Clavel,† Emilia Păunescu,† Patrycja Nowak-Sliwinska,† Arjan W. Griﬃoen,‡ Rosario Scopelliti,† and Paul J. Dyson*,† † Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland Angiogenesis Laboratory, Department of Medical Oncology, VUMC Cancer Center Amsterdam, 1081 HV Amsterdam, The Netherlands ‡ S Supporting Information * ABSTRACT: A ruthenium(II)−arene complex with a perﬂuoroalkyl-ligand was found to display remarkable selectivity toward cancer cells. IC50 values on several cancer cell lines are in the range of 25−45 μM, and no cytotoxic eﬀect was observed on nontumorigenic (HEK-293) cells at concentrations up to 500 μM (the maximum concentration tested). Consequently, this complex was used as the basis for the development of a number of related derivatives, which were screened in cancerous and noncancerous cell lines. The lead compound was then evaluated in vivo for antiangiogenic activity in the CAM model and in a xenografted ovarian carcinoma tumor (A2780) grown on the CAM. A 90% reduction in the tumor growth was observed. ■ generation of toxic ruthenium(II) species,7,8 and third, at the tumor site reactions (binding) with proteins are preferred to DNA binding, which contrasts with the behavior of platinum(II) complexes such as cisplatin.9−11 Extrapolation of these pathways led to the direct evaluation of ruthenium(II) complexes and, in particular, organometallic ruthenium(II)− arene complexes.12 On the basis of the general formula of halfsandwich ruthenium(II)−arene complexes, i.e., [Ru(η6-arene)XYZ]n+ with X, Y, and Z being a combination of monodentate or bidentate/monodentate ligands, two main families of compounds have emerged with distinct modes of action. Compounds containing the bidentate ethylenediamine (en) ligand and a chloride, i.e., [Ru(η6-arene)(en)Cl]+, are strongly cytotoxic in vitro, 13−15 whereas complexes with three monodentate ligands including a hydrophilic 1,3,5-triaza-7phosphaadamantane (PTA) ligand, i.e., [Ru(η6-arene)(PTA)Cl2] (termed RAPTA), are not cytotoxic16−18 but display relevant antimetastatic16,19,20 and antiangiogenic21 properties in vivo. Although the mechanism of action of this latter class of compounds remains unclear, it seems likely that RAPTA derivatives have a profoundly diﬀerent biochemical mode of INTRODUCTION Platinum-based drugs are widely used to treat cancer, but their therapeutic use can be impaired by intrinsic or acquired resistance and the occurrence of numerous side eﬀects including nephrotoxicity, neurotoxicity, neuropathy, myelosuppresion, thrombocytopenia, and neutropenia.1 The requirement for chemotherapeutic agents that are both active against platinum-resistant tumors and have superior therapeutic windows, i.e., leading to reduced side eﬀects, gave rise to the emergence and rapid development of compounds based on other metals.2 In particular, ruthenium complexes have attracted signiﬁcant attention with two complexes, namely NAMI-A3 and KP1019,4 advancing through clinical trials. The latter compound is active against various primary tumors including those that respond poorly to existing chemotherapy regimens, whereas the ﬁrst displays strong antimetastatic properties, both with relatively mild side eﬀects.5 The mechanism of action of these ruthenium(III) compounds, as well as others, has been widely studied in order to establish the basis for their unique properties, and it would appear that three main features are relevant: ﬁrst, they interact with serum proteins such as albumin and transferrin that endows them with tumor seeking properties,6 second, ruthenium(III) complexes appear to be activated through intracellular reduction to allow © 2014 American Chemical Society Received: February 19, 2014 Published: March 26, 2014 3546 dx.doi.org/10.1021/jm5002748 | J. Med. Chem. 2014, 57, 3546−3558 Journal of Medicinal Chemistry Article Figure 1. Structure of 10 (RAPTA-C) and 1, the latter being used as a structural template for this study. action to classical platinum anticancer agents.22−24 In the nucleosome, for example, it has been shown that RAPTA compounds bind preferentially to the histone core relative to the DNA.25 The identiﬁcation of the full mechanism of action of RAPTA-like molecules is in progress,23,26,27 as a better mechanistic understanding should help to improve the biological and pharmacological proﬁle of these compounds.28−32 RAPTA complexes like RAPTA-C (10) are very hydrophilic and, recently, as part of our ongoing studies,33 we turned our attention to bifunctional hydrophobic complexes, e.g. 1, which carries a long ﬂuorous chain (Figure 1). Indeed, 1 was selected as a promising lead drug candidate because it displays considerable selectivity toward tumor cells relative to model healthy cells. Moreover, in comparison to RAPTA compounds, 1 is substantially more cytotoxic to cisplatin-sensitive and cisplatin resistant ovarian cancer cells.34 Herein, we describe the systematic modiﬁcation of our lead drug candidate 1 and an in vitro and in vivo biological evaluation of this new compound class. Derivatives 3−5 were prepared to study the inﬂuence of the arene ring in comparison to p-cymene in 1 because the substituents on the arene inﬂuence cell uptake14,16,35,36 and dissociation of the arene can take place upon binding of this type of complex to certain biomolecules,37 and both these features inﬂuence the associated antiproliferative activity. Complexes 3−5 contain toluene, hexamethylbenzene, and 1,3,5-tri-iso-propylbenzene, respectively, and were synthesized in 66−97% yield from the pyridine ester ligand 5l33 and the appropriate arene dimers (Scheme 2). Scheme 2. Synthesis of 3−5 ■ RESULTS AND DISCUSSION Compound 1 comprises a ruthenium(II) center with an η6-pcymene ring, a ﬂuorous-functionalized pyridine, and two chloride ligands. Each of these three ligand types was systematically modiﬁed: the p-cymene ligand in 1 was replaced by other arenes with varying steric bulk and the size, type, and position of the linker between the pyridine and the perﬂuorinated chain was also varied (see Scheme 1). The The pyridine ligand was also modiﬁed in a number of ways. A shorter carboxylate linker, acetate, was employed in 6, 7, and 8, with the substitution position corresponding to ortho in 6, meta in 7, and para in 8. The ortho (1l), meta (2l), and para (3l) substituted perﬂuorinated pyridine−acetate ligands were prepared from 1H,1H,2H,2H-perﬂuoro-1-decanol and the appropriate pyridine-acetic acid hydrochlorides in 66−86% yield. Ligand 4l, with a supplementary glycolic ester group as a more hydrolytically cleavable linker between the pyridine moiety and the perﬂuorinated ponytail, was obtained following the three-step route shown in Scheme 3. Complexes 6−9 were obtained in good yield (72−97%) from the direct reaction of [(p-cymene)RuCl2]2 with the corresponding ligands, 1l−4l (Scheme 3). All the new compounds, 1l−4l and 2−9, were fully characterized by 1H, 13C and, where appropriate, 19F NMR spectroscopy, ESI mass spectrometry, IR spectroscopy, and elemental analysis. As in the case of the previously described ligand 5l, the formation of the perﬂuoroalkyl ester ligands 1l−4l is accompanied by a deshielding of ca. 0.4 ppm of the methylene protons at the α position relative to the oxygen atom (as compared to 1H,1H,2H,2H-perﬂuoro-1-decanol). On formation of complexes 2−5, 7, and 9 (containing metasubstituted perﬂuoroalkylpyridine ligands), as well as in the case of complex 8 (which has a para-substituted perﬂuoroalkylpyridine ligand), a deshielding of ca. 0.4 ppm for the two protons at the α position to the nitrogen atom and of a deshielding of ca. 5 ppm for the respective carbon atoms is observed. Complex 6, with an ortho-substituted perﬂuoroalkylpyridine ligand, exhibits some particularities, notably a smaller change in the frequencies of the 1H and 13C resonances Scheme 1. Chemical Modiﬁcations of 1 role of the perﬂuoro-alkyl chain with respect to the observed selectivity was also assessed by replacing it with a methyl group, i.e., in complex 2. Complex 2 was obtained in good yield starting from commercially available methyl 3-(pyridin-3yl)propanoate and the corresponding ruthenium dimer [(pcymene)RuCl2]2. 3547 dx.doi.org/10.1021/jm5002748 | J. Med. Chem. 2014, 57, 3546−3558 Journal of Medicinal Chemistry Article Scheme 3. Synthesis of Ligands 1l−4l and Complexes 6−9 of the α position of the ligand with respect to the N atom. The resonances are also broad and poorly resolved, presumably due to the increased steric hindrance that impedes free rotation about the Ru−N bond and/or induces a rapid dissociation/ reassociation process. The 19F NMR spectra of 1l−4l and 3−9 and the very speciﬁc 13 C NMR spectroscopic proﬁle conﬁrm the presence of the ﬂuorous chain. The ﬂuorous chain is also evidenced from the IR spectra by the presence of a strong peak between 1110 and 1250 cm−1. In the case of ligand 4l and complex 9, the second carboxylate group exhibits a shoulder at 1760−1770 cm−1 in the IR spectra, whereas the usual peak appears at 1744 cm−1. The structure of the compounds was also corroborated by ESI-MS. The most abundant peak observed in the spectra of the 1l−4l are those assigned to [M + H]+ ions, whereas the spectra of the complexes with two chlorine ligands were dominated by species assigned to [M − Cl]+ ions. Crystals suitable for X-ray diﬀraction were obtained for 2 by slow diﬀusion of hexane into a chloroform solution of the complex. The structure of 2 is shown in Figure 2, and key structural data are presented in Table 1. Complex 2 adopts the familiar half-sandwich geometry with the bond parameters around the Ru center being remarkably similar to those of 1038,39 (see Table 1), indicating that the coordination sphere is largely preserved on replacing the Pdonor ligand with the N-donor pyridine ligand. In Vitro Antiproliferative Activity. The antiproliferative activity of the ligands 1l−5l and their corresponding complexes, 1−9, was evaluated in A2780 ovarian cancer cells and in the noncancerous human embryonic kidney (HEK-293) cell line (Table 2). Additional antiproliferative activity studies were performed on 1 in a broader panel of cancer cell lines including Figure 2. ORTEP representation of 2 (thermal ellipsoids are 30% equiprobability envelopes and H atoms are spheres of arbitrary diameter). cisplatin-resistant ovarian carcinoma cells, breast cancer MCF-7 and MDA-MB-231 cell lines, and A549 lung cancer cells (Table 3). The ligands are not particularly cytotoxic toward the A2780 cells, whereas the complexes display reasonable IC50 values ranging from 30.5 to 200 μM, all being considerably more cytotoxic than 10.31 Importantly, complexes 3 and 5−7 do not aﬀect noncancerous cell proliferation, with IC50 values exceeding 500 μM (the highest concentration tested). The least active and least selective complex is 2, which does not carry a perﬂuorinated chain. With respect to the other structural modiﬁcations, i.e., the inﬂuence of the arene, the substitution pattern on the pyridine ligand and the length of the alkyl chain 3548 dx.doi.org/10.1021/jm5002748 | J. Med. Chem. 2014, 57, 3546−3558 Journal of Medicinal Chemistry Article Table 1. Key Bond Lengths (Å) and Angles (deg) of 2 and 10 for Comparison Purposes 6 Ru−η Ru−P Ru−N Ru−Clave Cl−Ru−Cl P−Ru−Clave N−Ru−Clave 10a 2 1.692, 1.701 2.296(2), 2.298(3) 1.669(2) 2.112(4) 2.4138(12) 87.64(4) 2.421, 2.426 87.25(8), 88.97(9) 84.01, 85.26 85.22(11) a In the case of 10, there are two independent molecules in the asymmetric unit. Taken from ref 38. Table 2. IC50 Values for Ligands 1l−5l and Complexes 1−10 against the Ovarian Carcinoma A2780 and the Noncancerous HEK-293 Cell Lines after 72 h Determined Using the MTT Assay compd A2780 (μM) HEK-293 (μM) 1l 2l 3l 4l 5l 1 2 3 4 5 6 7 8 9 10 157 ± 9 >500 >500 263 ± 24 263 ± 24 44 ± 1 200 ± 20 87 ± 1 65 ± 9 31 ± 1 177 ± 1 97 ± 3 71 ± 2 40 ± 5 251 ± 14 >500 >500 >500 60 ± 2 >500 >500 >500 >500 86 ± 3 >500 >500 >500 103 ± 12 57 ± 5 >500 Figure 3. (top) Amount of 10, 1, and 3 (pmol in 106 cells) internalized in A2780 cells after 24 h (dose: 250 μM). (bottom) Uptake of 1 (pmol/106 cells) in A2780 and HEK-293 cells after a 24 h incubation (concentration: 250 μM). Table 3. IC50 Values for 1 against Various Cancer Cell Lines Determined Using the MTT Assay after 72 h cell line 1 (μM) A2780cisR MCF-7 MDA-MB-231 A549 25 ± 2 38 ± 2 36 ± 2 43 ± 1 Figure 4. Comparison of DNA proﬁles from A2780 cells in the absence and presence of 1 (50 μM). Flow cytometry analysis of the DNA content after ﬁxation of the A2780 cells in 70% ethanol, a DNA extraction step, and staining with PI. Control−solvent alone (left panel) and 50 μM of 1 (right panel) for 24 h. linker between the pyridine and ester moiety, there are not any clear trends that provide relevant structure−activity relationships, although these diﬀerent structural features signiﬁcantly alter anticancer activity while not changing the selectivity. In comparison to cisplatin, the IC50 in A2780 and HEK-293 cells correspond to 4.3 and 15.3 μM, respectively.40 The uptake of 1 and 3 into A2780 cells was determined following incubation for 24 h (Figure 3). A dose higher than the IC50 value of 1 and 3 was used as the incubation time was reduced. Compared to 10, cell uptake of 1 and 3 is ca. 2-fold greater, which partially explains the superior cytotoxicity of the complexes. Cellular uptake of 1 was also quantiﬁed in the HEK293 cells and is essentially the same as that determined in the A2780 cell line and, therefore, it is not possible to attribute the selective cytotoxicity of the compound to diﬀerences in uptake. DNA proﬁles of propidium iodide (PI) stained A2780 cells treated with 1 were analyzed by ﬂow cytometry in order to establish the mechanism of cell death (see Figure 4 and Table 4). Several reports indicate that ruthenium complexes inhibit the proliferation of cells by preventing cell cycle progression Table 4. Cell-Cycle Changes in A2780 Cells by 1a 1 (μM) DMSO 0.1% 6 12 25 50 G1/G0 (±SD) G2/M (±SD) apoptosis (±SD) 69 ± 3 67.5 ± 0.7 69.0 ± 0.1 77.0 ± 1.4 59 ± 2 16.0 ± 1.4 21 ± 5 17.5 ± 0.7 17.0 ± 1.4 27.0 ± 0.1 2.0 ± 0.1 1.9 ± 0.3 2.2 ± 1.0 2.4 ± 0.4 6.4 ± 0.1 a The cell-cycle alterations were evaluated using PI-FACS analysis of A2780 cells (n = 4) after 24 h incubation of 1 at concentrations ranging from 6 to 50 μM. 3549 dx.doi.org/10.1021/jm5002748 | J. Med. Chem. 2014, 57, 3546−3558 Journal of Medicinal Chemistry Article Figure 5. Assessment of migration inhibition of MDA-MB-231 cells after exposure to 1. Wound closure in MDA-MB-231 cultures after 14 h of incubation with 1 at concentrations between 6 and 50 μM, DMEM culture medium, DMSO 0.1%, and sunitinib at 20 μM as a positive control. **P = 2.6 × 10−7 (for 1) and **P = 8.5 × 10−8 (for sunitinib). Error bars represent standard error of the mean. (B) Typical images of the wound at the beginning of the experiment (0.1% DMSO solution in culture medium as a control) and after 14 h incubation with 1 or sunitinib. Figure 6. In vivo activity of 1 in the CAM model (A,B) and in xenografted ovarian tumors (A2780) grown on the CAM (C,D) using experimental protocol for the CAM only (A) or the tumor-bearing CAM (C), respectively. Complex 1 at 50 μM induced only mild, but statistically signiﬁcant (*P = 1 × 10−3) change in the vasculature architecture, compared to control (DMSO 0.1%), as quantiﬁed by measurement of the branching points (B). Tumor growth curves correspond to the following conditions: CTRL, 1 (50 μM; 100 μL, 1×/day, **P = 1.31 × 10−5) or 1 (25 μM; 100 μL, 2×/day, **P = 7.12 × 10−5). Error bars represent standard error of the mean. Bar stands for 5 mm (A,D) and 400 μm (B). 3550 dx.doi.org/10.1021/jm5002748 | J. Med. Chem. 2014, 57, 3546−3558 Journal of Medicinal Chemistry ■ and inducing apoptosis.21,41,42 As reﬂected by the number of subdiploid cells (Figure 4 and Table 4), a moderate eﬀect on apoptosis induction was observed at the 50 μM dose. Interestingly, incubation of A2780 cells with 1 (50 μM) for 24 h leads to an enhanced number of cells in the G2/M phase and a decrease of cells in the G1/G0 phase of cell cycle. Because 1 inhibits cell proliferation, this result most likely suggests that the compound synchronizes cells in the G2/M phase. Thus, 1 both induces apoptosis and prevents cell cycle progression. At lower doses, 1 did not induce apoptosis, neither did it cause signiﬁcant changes of cells between cell cycle stages. The eﬀect of 1 on the motility of tumor cells was studied in a cell migration assay using invasive MDA-MB-231 human breast adenocarcinoma cells (Figure 5). At a concentration of 50 μM, 1 is able to signiﬁcantly inhibit the migration capacity of tumor cells. The activity is comparable to that of sunitinib (at 20 μM), a clinically used antiangiogenic agent shown before to inhibit cell migration.43 Representative images of the wounds before and after a 14 h incubation with a control solution (0.1% DMSO solution in culture medium), 1, or sunitinib are shown in Figure 5B. On the basis of its combined cellular eﬀect, compound 1 was subsequently investigated in vivo using the chorioallantoic membrane (CAM) assay of the chicken embryo to evaluate the antiangiogenic potential (Figure 6). Initially, 1 was administered via a daily iv injection between EDD 11 and 14, followed by imaging of the CAM vasculature on EDD 15 (Figure 6A). A representative bright-ﬁeld image of the fertilized embryo and the CAM at EDD 11 is shown in Figure 6A, and ﬂuorescence angiographies of the CAM after the 4-day treatment, for 0.1% DMSO (CTRL) and 1 (50 μM), are shown in Figure 6B. A small but signiﬁcant inhibitory eﬀect on the vasculature was observed as avascular zones in the CAM (marked with yellow circles) for 1, as compared to control treated CAMs. It should be noted that this eﬀect is seen at the 50 μM dose, which is at least an order of magnitude lower than the concentration required to inhibit the growth of nontumorigenic HEK-293 cells. A reduction in the number of branching points per mm2 of ca.10% was observed. To investigate the eﬀect of 1 on tumor growth, A2780 ovarian carcinoma cells were inoculated at EDD 7 of the CAM and monitored for 11 days. Established and vascularized tumors were detected 3 days post implantation (EDD 10). Treatment was performed by iv injections on four consecutive days (Figure 6C). Tumors grew to an average size of approximately 150 mm3 by EDD 17 when left untreated. Tumor growth was eﬃciently inhibited following treatment with 1, performed 1×/day (50 μM), or 2×/day (2 × 25 μM, Figure 6D). On the last day of the experiment, the tumor growth was inhibited by approximately 90% (**P = 1.31 × 10−5) at a dose of 1×/day (50 μM), whereas an inhibition of ca. 70% (**P = 7.12 × 10−5) is observed at a dose of 2×/day (2 × 25 μM). The diﬀerence between an eﬀect of 1 administrated once vs twice in a fractionated dose was also statistically signiﬁcant (**P = 1.5 × 10−4). The eﬀect on the vasculature was only marginal (Figure 6B) as compared to the overall tumor growth inhibition (Figure 6D), conﬁrming the antitumor activity of 1 in the absence of a strong antiangiogenic eﬀect. Moreover, no detrimental side eﬀects were observed during the treatment regimen. The dose applied for 1 is considerably lower than that employed with 10 in a engrafted mouse model (CBA mice bearing the MCa mammary carcinoma), which led to a reduction in the number and mass of metastatic tumors in the absence of an eﬀect on the primary tumor.44 Article CONCLUSIONS We disclose a ruthenium(II)−arene complex bearing a perﬂuorinated chain that displays remarkable selectivity toward cancer cells in vitro. At a noncytotoxic dose to healthy cells, the compound was evaluated for antiangiogenic and antitumoral activity in vivo. A modest antiangiogenic eﬀect was observed, whereas a remarkable reduction in tumor growth, i.e., ca. 90%, was observed in the absence of measurable side eﬀects. This behavior is quite distinct from that of 10, in which the hydrophobic perﬂuoroalkyl-modiﬁed ligand in 1 is replaced by an amphiphilic 1,3,5-triaza-7-phosphaadamantane ligand in 10. These diﬀerences in biological activity may therefore be, in part, due to increased uptake of 1 relative to 10, although this does not fully explain the lack of toxicity observed in healthy cells. Nevertheless, further translational development of low molecular weight bifunctional ruthenium(II)−arene complexes is worthwhile as their low toxicity is attractive for development of future anticancer therapies. Moreover, it has been noted that the scarcity of in vivo studies on organometallic compounds in the literature is hindering the development of compounds with genuine clinical potential, as the majority of compounds that are active in vitro tend to be inactive in vivo,45 limiting design strategies based entirely on in vitro data. ■ EXPERIMENTAL SECTION General Procedures. RuCl3·3H2O was obtained from Precious Metals Online. Other chemical reagents were purchased from commercial sources (Aldrich, AlfaAesar, and Acros Chemicals) and used without further puriﬁcation. Reactions were performed in solvents dried using a drying column and collected and used under an inert atmosphere of N2. The dimers [Ru(η6-toluene)Cl2]2, [Ru(η6p-cymene)Cl2]2, [Ru(η6-hexamethylbenzene)Cl2]2, and [Ru(η6-1,3,5tri-iso-propyl-benzene)Cl2]2 were prepared and puriﬁed according to literature procedures. 46−50 The synthesis of 1 and of the corresponding ligand has been reported previously.33 Reactions were performed under N2 using Schlenk technique, and the complexation reactions and manipulation of the ruthenium dimers and complexes were performed in the absence of light. The synthesis of the ligands was monitored by TLC using Merck TLC Silicagel coated aluminum sheets 60 F254, using UV lamp at 254 nm and KMnO4 stain for visualization, and using hexane/EtOAc mixture as eluent. Puriﬁcation of the ligands was carried out by ﬂash column chromatography using a Varian 971-FP Autocolumn puriﬁcation machine and prepacked Silicagel columns (Luknova ﬂash columns (40−60 μm)) using a hexane/EtOAc mixture in gradient as the eluent. 1H (400.13 MHz), 19 F (375.46 MHz), and 13C (100.62 MHz) NMR spectra were recorded on a Bruker Avance II 400 spectrometer at 298 K. The chemical shifts are reported in parts per million (ppm) and referenced to deuterated solvent residual peaks (CDCl3: 1H δ 7.26, 13C δ 77.16 ppm),51 and coupling constants (J) are reported in hertz (Hz). IR spectra were recorded on a PerkinElmer Spectrum One FT-IR spectrometer at room temperature. High resolution electrospray ionization mass spectra (HR ESI-MS) were obtained on a ThermoFinnigan LCQ Deca XP Plus quadropole ion-trap instrument operated in positive-ion mode. Elemental analyses were carried out at the microanalytical laboratory at the Institute of Chemical Sciences and Engineering (EPFL). Melting points were determined using a SMP3 Stuart melting point apparatus and are uncorrected. Compound purity was conﬁrmed by elemental analysis with a minimum percentage of 95%. Synthesis of [Ru(η6-p-cymene)Cl2(methyl-3-(pyridin-3-yl)propanoate)] 2. To a solution of [Ru(η6-p-cymene)Cl2]2 (1 equiv, 0.530 g, 0.865 mmol) in CH2Cl2 (10 mL), a solution of methyl-3(pyridin-3-yl)propanoate (2.1 equiv, 0.300 g, 1.816 mmol) in CH2Cl2 (20 mL) was added, and the resulting mixture stirred at rt in the dark for about 4 days. The reaction mixture was concentrated under reduced pressure almost to dryness. Several drops of Et2O were added 3551 dx.doi.org/10.1021/jm5002748 | J. Med. Chem. 2014, 57, 3546−3558 Journal of Medicinal Chemistry Article IR (ν, cm−1): 3059 (CH-Ar), 2956−2852 (CH2, CH3), 1739 (C O), 1477, 1440, 1418 (CC, CN), 1367, 1329, 1115−1244 (CF2, CF3), 1197 (C−O). ESI-MS(+): m/z found 825.93 [M − Cl]+, calcd for C25H20ClF17NO2Ru 825.99, the experimental isotopic pattern ﬁts well the calculated one. Anal. (%) Calcd for C25H20Cl2F17NO2Ru C 34.86, H 2.34, N 1.63; found C 37.75, H 2.77, N 1.64. [Ru(η6-hexamethylbenzene)Cl2(1H,1H,2H,2H-perﬂuorodecyl-3(pyridin-3-yl)propanoate)] 4. [Ru(η6-hexamethylbenzene)Cl2]2 (0.09 g, 0.135 mmol) and 1H,1H,2H,2H-perﬂuorodecyl-3-(pyridin-3-yl)propanoate (5l) (0.169 g, 0.283 mmol) were used. The product was isolated as an orange solid (0.166 g, η = 66%); mp (°C) 162−163.5. 1 H NMR (CDCl3) δH, ppm: 8.64−8.66 (1H, m, Npy-CH-C, NpyCH-CH), 7.54 (1H, d, Npy-CH-C-CH, 3JH,H = 7.2 Hz), 7.20 (1H, dd overlapped, Npy-CH-CH, 3JH,H = 7.2 Hz), 4.37 (2H, t, O-CH2-CH2, 3 JH,H = 6.4 Hz), 2.93 (2H, t, Py-CH2-CH2-CO, 3JH,H = 7.2 Hz), 2.65 (2H, t, Py-CH2-CH2-CO, 3JH,H = 7.2 Hz), 2.42−2.51 (2H, m, OCH2-CH2), 1.97 (18H, s, 6 × Ar-CH3). 13 C NMR (CDCl3) δC, ppm: 171.7 (1C, CH2-CO), 154.6 (1C, Npy-CH-C), 152.8 (1C, Npy-CH-CH), 137.4 (1C, Npy-CH-C-CH), 136.9 (1C, Npy-CH-C-CH), 124.2 (1C, Npy-CH-CH), 104.8−121.9 (8C, m series, CH2-CF2-CF2, CH2-CF2-CF2, CH2-(CF2)2-CF2, CH2(CF2)3-CF2, CF2-(CF2)2-CF3, CF2-CF2-CF3, CF2-CF2-CF3, CF2-CF2CF3), 91.3 (6C, 6 × C-CH3(Ar)), 56.7 (1C, t, C-i, 3JC,F = 4 Hz, OCH2-CH2), 34.6 (1C, Py-CH2-CH2-CO), 30.5 (1C, t, O-CH2-CH2, 2 JC,F = 22 Hz), 27.6 (1C, Py-CH2-CH2-CO), 15.4 (6C, 6 × CH3C(Ar)). 19 F NMR (CDCl3) δF, ppm: −80.72 (3F, t, CF3, 3JF,F = 9.7 Hz), −113.58 (2F, m, CH2-CF2-CF2), 121.62 (2F, m, CH2-(CF2)2-CF2), −121.87 (4F, m, CH2-(CF2)3-CF2, CH2-(CF2)4-CF2), −122.67 (2F, m, CF2-CF2-CF3), −123.49 (2F, m, CH2-CF2-CF2), −126.07 (2F, m, CF2-CF3). IR (ν, cm−1): 3065−2923 (CH-Ar, CH2, CH3), 1741 (CO), 1430−1474 (CC, CN), 1371, 1115−1242 (CF2, CF3), 1199 (C− O). ESI-MS(+): m/z found 896.07 [M − Cl] + , calcd for C30H30ClF17NO2Ru 896.07, the experimental isotopic pattern ﬁts well the calculated one. Anal. (%) Calcd for C30H30Cl2F17NO2Ru: C 38.68, H 3.25, N 1.50. Found: C 39.94, H 2.95, N 1.53. [Ru(η6-1,3,5-tri-iso-propylbenzene)Cl2(1H,1H,2H,2H-perﬂuorodecyl-3-(pyridin-3-l)propanoate)] 5. [Ru(η6-1,3,5-tri-iso-proylbenzene)Cl2]2 (0.270 g, 0.359 mmol) and 1H,1H,2H,2H-perﬂuorodecyl-3(pyridin-3-yl)propanoate (5l) (0.450 g, 0.753 mmol) were used. The product was isolated as an orange solid (0.689 g, η = 97%); mp (°C) 112.5−113.5. 1 H NMR (CDCl3) δH, ppm: 8.95 (1H, s, Npy-CH-C), 8.91 (1H, d, Npy-CH-CH, 3JH,H = 7.5 Hz), 7.55 (1H, d, Npy-CH-C-CH, 3JH,H = 6.7 Hz), 7.19 (1H, dd overlapped, Npy-CH-CH, 3JH,H = 7.5 Hz), 4.38 (2H, t, O-CH2-CH2, 3JH,H = 6.5 Hz), 2.94 (2H, t, Py-CH2-CH2-CO, 3JH,H = 7.2 Hz), 2.79−2.87 (3H, sept, 3 × Ar-CH(CH3)2, 3JH,H = 6.8 Hz), 2.65 (2H, t, Py-CH2-CH2-CO, 3JH,H = 7.2 Hz), 2.41−2.53 (2H, m, O-CH2-CH2), 1.25 (18H, d, 3 × Ar-CH(CH3)2, 3JH,H = 6.8 Hz). 13 C NMR (CDCl3) δC, ppm: 171.7 (1C, CH2-CO), 155.3 (1C, Npy-CH-C), 153.4 (1C, Npy-CH-CH), 137.6 (1C, Npy-CH-C-CH), 136.7 (1C, Npy-CH-C-CH), 124.0 (1C, Npy-CH-CH), 106.8−120.5 (8C, m series, CH2-CF2-CF2, CH2-CF2-CF2, CH2-(CF2)2-CF2, CH2(CF2)3-CF2, CF2-(CF2)2-CF3, CF2-CF2-CF3, CF2-CF2-CF3, CF2-CF2CF3), 107.5 (3C, 3 × Ar-C-CH(CH3)2), 75.3 (3C, 3 × CH-C(Ar)), 56.8 (1C, t, O-CH2-CH2, 3JC,F = 4 Hz), 34.6 (1C, Py-CH2-CH2-C O), 31.0 (3C, 3 × Ar-CH(CH3)2), 30.6 (1C, t, O-CH2-CH2, 2JC,F = 23 Hz), 27.7 (1C, Py-CH2-CH2-CO), 22.4 (6C, 3 × Ar-CH(CH3)2). 19 F NMR (CDCl3) δF, ppm: −80.73 (3F, t, CF3, 3JF,F = 9.7 Hz), −113.62 (2F, m, CH2-CF2-CF2), −121.62 (2F, m, CH2-(CF2)2-CF2), −121.88 (4F, m, CH2-(CF2)3-CF2, CH2-(CF2)4-CF2), −122.67 (2F, m, CF2-CF2-CF3), −123.49 (2F, m, CH2-CF2-CF2), −126.07 (2F, m, CF2-CF3). IR (ν, cm−1): 2874−3032 (CH-Ar, CH2, CH, CH3), 1747 (CO), 1516, 1467−1416 (Py CC, CN), 1360, 1244−1115 (CF2, CF3), 1195 (C−O). ESI-MS(+): m/z found 938.12 [M − Cl]+, calcd for C33H36ClF17NO2Ru 938.12, the experimental isotopic pattern ﬁts well the calculated one. Anal. (%) Calcd for C33H36Cl2F17NO2Ru: C 40.71, H 3.73, N 1.44. Found: C 40.58, H 3.51, N 1.49. to aﬀord an orange precipitate that was washed with Et2O (3 × 20 mL), hexane (20 mL), and again Et2O (20 mL). The orange solid was removed by ﬁltration and dried under a ﬂow of N2. The procedure was repeated twice to aﬀord the product as an orange solid (0.660 g, η = 77%); mp (°C) 132.5−134. 1 H NMR (CDCl3) δH, ppm: 8.90 (1H, s, Npy-CH-C), 8.87 (1H, d, Npy-CH-CH, 3JH,H = 5.4 Hz), 7.57 (1H, d, Npy-CH-C-CH, 3JH,H = 7.8 Hz), 7.22 (1H, dd overlapped, Npy-CH-CH, 3JH,H = 7.8 Hz, 3JH,H = 5.6 Hz), 5.42 (2H, d, 2 × CH3-C-CH-CH(Ar), 3JH,H = 5.8 Hz), 5.21 (2H, d, 2 × CH3-C-CH(Ar), 3JH,H = 5.8 Hz), 3.66 (3H, s, O-CH3), 2.92− 2.99 (1H, m, Ar-CH(CH3)2), 2.94 (2H, t, Py-CH2-CH2‑CO, 3JH,H = 7.2 Hz), 2.64 (2H, t, Py-CH2-CH2-CO, 3JH,H = 7.2 Hz), 2.07 (3H, s, Ar-CH3), 1.29 (6H, d, Ar-CH(CH3)2, 3JH,H = 7.1 Hz). 13 C NMR (CDCl3) δC, ppm: 172.4 (1C, CH2-CO), 154.9 (1C, Npy-CH-C), 152.8 (1C, Npy-CH-CH), 137.7 (1C, Npy-CH-C-CH), 137.1 (1C, Npy-CH-C-CH), 124.2 (1C, Npy-CH-CH), 103.3 (1C, CH3-C-CH-CH-C(Ar)), 97.2 (1C, CH3-C-CH(Ar)), 82.9 (2C, 2 × CH3-C-CH-CH(Ar)), 82.2 (2C, 2 × CH3-C-CH(Ar)), 51.8 (1C, OCH3), 34.5 (1C, Py-CH2-CH2-CO), 30.6 (1C, Ar-CH(CH3)2), 27.7 (1C, Py-CH2-CH2-CO), 22.3 (2C, Ar-CH(CH3)2), 18.1 (1C, ArCH3). IR (ν, cm−1): 3274 (CH-Ar), 3059, 2874 (CH2,CH, CH3), 1727 (CO), 1473, 1421 (Py CC, CN), 1267 (C−O). ESI-MS(+): m/z found 436.07 [M-Cl]+, calcd for C19H25ClNO2 Ru 436.06, the experimental isotopic pattern ﬁts well the calculated one. Anal. (%) Calcd for C19H25Cl2NO2Ru: C 48.41, H 5.35, N 2.97. Found: C 48.46, H 5.15, N 3.04. General Procedure for the Synthesis of [Ru(η6-arene)Cl2(1H,1H,2H,2H-perﬂuorodecyl-3-(pyridin-3-yl)propanoate)] (Where Arene = Toluene, Hexamethylbenzene, 1,3,5-Tri-isopropylbenzene) 3−5. To a solution (or suspension) of the appropriate ruthenium(II)−arene dimer [Ru(η6-arene)Cl2]2 (1 equiv) in CH2Cl2 (10 mL), a solution of 1H,1H,2H,2H-perﬂuorodecyl-3-(pyridin-3-yl)propanoate (5l) (2.1 equiv) in CH2Cl2 (20 mL) was added and the reaction mixture was stirred at rt in the dark for about 4 days. The reaction mixture was then concentrated under reduced pressure almost to dryness and the product precipitated with Et2O. The resulting precipitate was washed with Et2O (3 × 20 mL), hexane (20 mL), and again Et2O (20 mL), then removed by ﬁltration and dried under a ﬂow of N2. [Ru(η6-toluene)Cl2(1H,1H,2H,2H-perﬂuorodecyl-3-(pyridin-3-yl)propanoate)] 3. [Ru(η6-toluene)Cl2]2 (0.190 g, 0.359 mmol) and 1H,1H,2H,2H-perﬂuorodecyl-3-(pyridin-3-yl)propanoate (5l) (0.450 g, 0.753 mmol) were used. The product was obtained as orange solid (0.566 g, η = 92%); mp (°C) 150−151.5. 1 H NMR (CDCl3) δH, ppm: 8.95 (1H, s, Npy-CH-C), 8.92 (1H, d, Npy-CH-CH, 3JH,H = 5.6 Hz), 7.59 (1H, d, Npy-CH-C-CH, 3JH,H = 7.8 Hz), 7.24 (1H, dd, Npy-CH-CH, 3JH,H = 7.8 Hz, 3JH,H = 5.6 Hz), 5.64 (2H, dd overlapped, 2 × CH3-C-CH-CH (Ar), 3JH,H = 5.5 Hz), 5.53 (1H, dd overlapped, CH3-C-CH-CH-CH (Ar), 3JH,H = 5.5 Hz), 5.27 (2H, d, 2 × CH3-C-CH (Ar), 3JH,H = 5.5 Hz), 4.38 (2H, t, O-CH2CH2, 3JH,H = 6.5 Hz), 2.96 (2H, t, Py-CH2-CH2-CO, 3JH,H = 7.2 Hz), 2.67 (2H, t, Py-CH2-CH2-CO, 3JH,H = 7.2 Hz), 2.41−2.53 (2H, m, O-CH2-CH2), 2.13 (3H, m, Ar-CH3). 13 C NMR (CDCl3) δC, ppm: 171.6 (1C, CH2-CO), 155.1 (1C, Npy-CH-C), 153.2 (1C, Npy-CH-CH), 137.8 (1C, Npy-CH-C-CH), 137.0 (1C, Npy-CH-C-CH), 124.3 (1C, Npy-CH-CH), 104.8−121.7 (8C, m series, CH2-CF2-CF2, CH2-CF2-CF2, CH2-(CF2)2-CF2, CH2(CF2)3-CF2, CF2-(CF2)2-CF3, CF2-CF2-CF3, CF2-CF2-CF3, CF2-CF2CF3), 100.3 (1C, CH3-C-CH(Ar)), 87.3 (2C, 2 × CH3-C-CHCH(Ar)), 81.2 (2C, 2 × CH3-C-CH-CH(Ar)), 79.7 (1C, CH3-C-CHCH-CH(Ar)), 56.6 (1C, t, O-CH2-CH2, 3JC,F = 4 Hz), 34.6 (1C, PyCH2-CH2-CO), 30.5 (1C, t, O-CH2-CH2, 2JC,F = 22 Hz), 27.6 (1C, Py-CH2-CH2-CO), 18.8 (1C, Ar-CH3). 19 F NMR (CDCl3) δF, ppm: −80.76 (3F, t, CF3, 3JF,F = 9.9 Hz), −113.62 (2F, m, CH2-CF2-CF2), −121.64 (2F, m, CH2-(CF2)2-CF2), −121.90 (4F, m, CH2-(CF2)3-CF2, CH2-(CF2)4-CF2), −122.71 (2F, m, CF2-CF2-CF3), −123.52 (2F, m, CH2-CF2-CF2), −126.10 (2F, m, CF2-CF3). 3552 dx.doi.org/10.1021/jm5002748 | J. Med. Chem. 2014, 57, 3546−3558 Journal of Medicinal Chemistry Article General Procedure for the Synthesis of the Ester Ligands 1l−3l. TEA (1 equiv) was added to a suspension of the appropriate 2(pyridinyl)acetic acid hydrochloride (1 equiv) in dry CH2Cl2 (50 mL) at 0 °C, and the mixture was stirred at rt for 20 min. EDCI (1 equiv), 1H,1H,2H,2H-perﬂuoro-1-decanol (1 equiv), and 4-(dimethylamino)pyridine (DMAP) (0.2 equiv) were sequentially added, and the resulting mixture was stirred at rt for ca. 4 days. The mixture was then diluted with CH2Cl2 (100 mL) and washed with H2O (100 mL), and the aqueous phase was re-extracted with CH2Cl2 (2 × 100 mL) and the combined organic phases washed with brine (150 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. Solid deposition on Celite and puriﬁcation by ﬂash chromatography aﬀorded the desired compounds. 1H,1H,2H,2H-Perﬂuorodecyl-2-(pyridin-2-yl)acetate 1l. 2-(Pyridine-2-yl)acetic acid hydrochloride (0.500 g, 2.880 mmol), TEA (0.400 mL, 2.880 mmol), 1H,1H,2H,2H-perﬂuoro-1-decanol (1.337 g, 2.880 mmol), EDCI (0.552 g, 2.880 mmol), and DMAP (0.075 g, 0.576 mmol) were used. The product was isolated as a colorless viscous oil (1.169 g, η = 70%). Rf (Hex/AcOEt 6:4 (v/v)) = 0.33. 1H NMR (CDCl3) δH, ppm: 8.54 (1H, m, Npy-CH-CH, 3JH,H = 4.9 Hz), 7.63 (1H, ddd overlapped, Npy-C-CH-CH, 3JH,H = 7.7 Hz, 4JH,H = 1.8 Hz), 7.25 (1H, d, Npy-C-CH, 3JH,H = 7.7 Hz), 7.17 (1H, m, Npy-CHCH, 3JH,H = 4.9 Hz, 3JH,H = 7.7 Hz), 4.41 (2H, t, O-CH2-CH2, 3JH,H = 6.6 Hz), 3.85 (2H, s, Py-CH2-CO), 2.39−2.52 (2H, m, O-CH2CH2). 13 C NMR (CDCl3) δC, ppm: 170.3 (1C, Py-CH2-CO), 154.1 (1C, Npy-C-CH), 149.7 (1C, Npy-CH-CH), 136.8 (1C, Npy-CH-CCH), 123.9 (1C, Npy-C-CH), 122.3 (1C, Npy-CH-CH), 105.3−122.1 (8C, m series, CH2-CF2-CF2, CH2-CF2-CF2, CH2-(CF2)2-CF2, CH2(CF2)3-CF2, CF2-(CF2)2-CF3, CF2-CF2-CF3, CF2-CF2-CF3, CF2-CF2CF3), 56.9 (1C, t, O-CH2-CH2, 3JC,F = 4 Hz), 43.8 (1C, Py-CH2-C O), 30.6 (1C, t, O-CH2-CH2, 2JC,F = 22 Hz). 19 F NMR (CDCl3) δF, ppm: −81.05 (3F, t, CF3, 3JF,F = 9.6 Hz), −113.78 (2F, m, CH2-CF2-CF2), −121.84 (2F, m, CH2-(CF2)2-CF2, −122.08 (4F, m, CH2-(CF2)3-CF2, CH2-(CF2)4-CF2), −122.90 (2F, m, CF2-CF2-CF3), −123.72 (2F, m, CH2-CF2-CF2), −126.33 (2F, m, CF2-CF3). IR (ν, cm−1): 2970 (CH2), 1742 (CO), 1594, 1477, 1438, (Py CC, CN), 1344, 1242−1116 (CF2, CF3), 1199 (C−O). ESIMS(+): m/z found 584.25 [M + H]+, calcd for C17H10F17NO2 583.24. Anal. (%) Calcd for C17H10F17NO2: C 35.01, H 1.73, N 2.40. Found: C 39.21, H 1.48, N 2.62. 1H,1H,2H,2H-Perﬂuorodecyl-2-(pyridin-3-yl)acetate 2l. 2-(Pyridine-3-yl)acetic acid hydrochloride (0.500 g, 2.880 mmol), TEA (0.400 mL, 2.880 mmol), 1H,1H,2H,2H-perﬂuoro-1-decanol (1.337 g, 2.880 mmol), EDCI (0.552 g, 2.880 mmol), and DMAP (0.075 g, 0.576 mmol, 0.2 equiv) were used. The product was isolated as a white solid (1.902 g, η = 86%); mp (°C) 69.5−70.5. Rf (Hex/AcOEt 5:5 (v/ v)) = 0.32. 1 H NMR (CDCl3) δH, ppm: 8.55 (1H, dd, Npy-CH-CH, 3JH,H = 4.8 Hz, 4JH,H = 1.6 Hz), 8.52 (1H, d, Npy-CH-C, 4JH,H = 1.8 Hz), 7.63 (1H, ddd overlapped, Npy-CH-C-CH, 3JH,H = 7.9 Hz, 4JH,H = 1.6 Hz), 7.28 (1H, dd, Npy-CH-CH, 3JH,H = 4.8 Hz, 3JH,H = 7.9 Hz), 4.42 (2H, t, OCH2-CH2, 3JH,H = 6.5 Hz), 3.66 (2H, s, Py-CH2-CO), 2.41−2.53 (2H, m, O-CH2-CH2). 13C NMR (CDCl3) δC, ppm: 170.4 (1C, CH2CO), 150.5 (1C, Npy-CH-C), 148.9 (1C, Npy-CH-CH), 136.9 (1C, Npy-CH-C-CH), 129.4 (1C, Npy-CH-C-CH), 123.6 (1C, Npy-CH-CH), 104.9−121.9 (8C, m series, CH2-CF2-CF2, CH2-CF2-CF2, CH2(CF2)2-CF2, CH2-(CF2)3-CF2, CF2-(CF2)2-CF3, CF2-CF2-CF3, CF2CF2-CF3, CF2-CF2-CF3), 57.1 (1C, t, O-CH2-CH2, 3JC,F = 4 Hz), 38.3 (1C, Py-CH2-CO), 30.6 (1C, t, O-CH2-CH2, 2JC,F = 22 Hz). 19 F NMR (CDCl3) δF, ppm: −80.74 (3F, t, CF3, 3JF,F = 9.3 Hz), −113.59 (2F, m, CH2-CF2-CF2), −121.64 (2F, m, CH2-(CF2)2-CF2), −121.89 (4F, m, CH2-(CF2)3-CF2, CH2-(CF2)4-CF2), −122.68 (2F, m, CF2-CF2-CF3), −123.52 (2F, m, CH2-CF2-CF2), −126.08 (2F, m, CF2-CF3). IR (ν, cm−1): 2915 (CH2), 1734 (CO), 1575, 1482, 1426, 1407 (Py CC, CN), 1363, 1333, 1245−1115 (CF2, CF3), 1191 (C−O). ESI-MS(+): m/z found 584.25 [M + H]+, calcd for C17H10F17NO2 583.24. Anal. (%) Calcd for C17H10F17NO2: C 35.01, H 1.73, N 2.40. Found: C 39.91, H 2.07, N 2.41. 1H,1H,2H,2H-Perﬂuorodecyl-2-(pyridin-4-yl)acetate 3l. 2-(Pyridine-4-yl)acetic acid hydrochloride (0.500 g, 2.880 mmol), TEA (0.400 mL, 2.880 mmol), 1H,1H,2H,2H-perﬂuoro-1-decanol (1.337 g, 2.880 mmol), EDCI (0.552 g, 2.880 mmol), and DMAP (0.075 g, 0.576 mmol) were used. The product was isolated as a white solid (1.101 g, η = 66%); mp (°C) 76.5−77.5. Rf (Hex/AcOEt 4:6 (v/v)) = 0.33. 1 H NMR (CDCl3) δH, ppm: 8.55 (2H, d, 2 × Npy-CH-CH, 3JH,H = 4.9 Hz), 7.19 (2H, d, 2 × Npy-CH-CH, 3JH,H = 4.9 Hz), 4.40 (2H, t, OCH2-CH2, 3JH,H = 6.4 Hz), 3.62 (2H, s, Py-CH2-CO), 2.38−2.51 (2H, m, O-CH2-CH2). 13 C NMR (CDCl3) δC, ppm: 169.7 (1C, Py-CH2-CO), 150.2 (2C, 2 × Npy-CH-CH), 142.2 (1C, Npy-CH-CH-C), 124.6 (2C, 2 × Npy-CH-CH), 105.3−121.8 (8C, m series, CH2-CF2-CF2, CH2-CF2CF2, CH2-(CF2)2-CF2, CH2-(CF2)3-CF2, CF2-(CF2)2-CF3, CF2-CF2CF3, CF2-CF2-CF3, CF2-CF2-CF3), 57.2 (1C, t, O-CH2-CH2, 3JC,F = 4 Hz), 40.4 (1C, Py-CH2-CO), 30.5 (1C, t, O-CH2-CH2, 2JC,F = 22 Hz). 19 F NMR (CDCl3) δF, ppm: −81.02 (3F, t, CF3, 3JF,F = 9.8 Hz), −113.74 (2F, m, CH2-CF2-CF2), −121.81 (2F, m, CH2-(CF2)2-CF2), −122.07 (4F, m, CH2-(CF2)3-CF2, CH2-(CF2)4-CF2), −122.88 (2F, m, CF2-CF2-CF3), −123.69 (2F, m, CH2-CF2-CF2), −126.31 (2F, m, CF2-CF3). IR (ν, cm−1): 2915 (CH2), 1738 (CO), 1606, 1560, 1421, (Py CC, CN), 1361, 1330, 1116−1241 (CF2, CF3), 1194 (C−O). ESI-MS(+): m/z found 584.25 [M + H]+, calcd for C17H10F17NO2 583.24. Anal. (%) Calcd for C17H10F17NO2: C 35.01, H 1.73, N 2.40. Found: C 35.10, H 1.57, N 2.41. General Procedure for the Synthesis of the [Ru(η6-pcymene)Cl2X] Complexes (with X = 1l−3l) 6−8. To a solution of [Ru(η6-p-cymene)Cl2]2 (1 equiv) in CH2Cl2 (10 mL), a solution of the corresponding ester ligand 1l−3l (2.1 equiv) was added, and the resulting mixture was stirred at rt in the dark for about 4 days. The reaction mixture was concentrated under reduced pressure almost to dryness, and the product was precipitated with Et2O. The precipitate was washed with Et2O (3 × 20 mL), hexane (20 mL), and again Et2O (20 mL) and then removed by ﬁltration and dried under a ﬂow of N2. [Ru(η6-p-cymene)Cl2(1H,1H,2H,2H -perﬂuorodecyl-2-(pyridin-2yl)acetate)] 6. [Ru(η6-p-cymene)Cl2]2 (0.185 g, 0.302 mmol) and 1H,1H,2H,2H-perﬂuorodecyl-2-(pyridin-2-yl)acetate (1l) (0.440 g, 0.754 mmol) were used. The product was isolated as an orange solid (0.385 g, η = 72%); mp (°C) 221−223 (decomp). 1 H NMR (CDCl3, 318 K) δH, ppm: 8.59 (1H, m br, Npy-CH-CH), 7.66 (1H, ddd overlapped, Npy-C-CH-CH, 3JH,H = 7.7 Hz, 4JH,H = 1.4 Hz), 7.28 (1H, d, Npy-C-CH, 3JH,H = 7.7 Hz), 7.19 (1H, dd, Npy-CHCH, 3JH,H = 7.7 Hz, 3JH,H = 4.9 Hz), 5.47 (2H, d, 2 × CH3-C-CHCH(Ar), 3JH,H = 5.8 Hz), 5.33 (2H, d, 2 × CH3-C-CH(Ar), 3JH,H = 5.8 Hz), 4.44 (2H, t, O-CH2-CH2, 3JH,H = 6.6 Hz), 3.90 (2H, s br, Py-CH2CO), 2.93 (1H, sept, Ar-CH(CH3)2, 3JH,H = 6.9 Hz), 2.42−2.55 (2H, m, O-CH2-CH2), 2.16 (3H, s, Ar-CH3), 1.28 (6H, d, ArCH(CH3)2, 3JH,H = 6.9 Hz). 13 C NMR (CDCl3, 318 K) δC, ppm: 170.3 (1C, CH2-CO), 154.4 (1C, br, Npy-C-CH), 150.1 (1C, br, Npy-CH-CH), 136.9 (1C, Npy-CCH-CH), 124.1 (1C, Npy-C-CH), 122.4 (1C, Npy-CH-CH), 107.2− 120.8 (8C, m series, CH2-CF2-CF2, CH2-CF2-CF2, CH2-(CF2)2-CF2, CH2-(CF2)3-CF2, CF2-(CF2)2-CF3, CF2-CF2-CF3, CF2-CF2-CF3, CF2CF2-CF3), 101.5 (1C, CH3-C-CH-CH-C(Ar)), 96.9 (1C, CH3-CCH(Ar)), 81.5 (2C, 2 × CH3-C-CH-CH(Ar)), 80.8 (2C, 2 × CH3-CCH(Ar)), 57.0 (1C, t, O-CH2-CH2, 3JC,F = 5 Hz), 44.0 (1C, Py-CH2CO), 30.9 (1C, t, O-CH2-CH2, 2JC,F = 22 Hz), 30.8 (1C, ArCH(CH3)2), 22.3 (2C, Ar-CH(CH3)2), 19.0 (1C, Ar-CH3). 19 F NMR (CDCl3) δF, ppm: −80.73 (3F, t, CF3, 3JF,F = 9.8 Hz), −113.58 (2F, m, CH2-CF2-CF2), −121.65 (2F, m, CH2-(CF2)2-CF2), −121.89 (4F, m, CH2-(CF2)3-CF2, CH2-(CF2)4-CF2), −122.68 (2F, m, CF2-CF2-CF3), −123.53 (2F, m, CH2-CF2-CF2), −126.06 (2F, m, CF2-CF3). IR (ν, cm−1): 3061 (CH-Ar), 2961, 2867 (CH2, CH, CH3), 1742 (CO), 1567, 1541, 1436 (Py CC, CN), 1362, 1314, 1117− 3553 dx.doi.org/10.1021/jm5002748 | J. Med. Chem. 2014, 57, 3546−3558 Journal of Medicinal Chemistry Article pattern ﬁts well the calculated one. Anal. (%) Calcd for C27H24Cl2F17NO2Ru: C 36.46, H 2.72, N 1.57. Found: C 36.37, H 2.77, N 1.58. Synthesis of 2-(Benzyloxy)-2-oxoethyl-3-(pyridin-3-yl)propanoate (step a before 4l). To a suspension of 3-(pyridin-3yl)propanoic acid (1 equiv, 0.800 g, 5.292 mmol) in dry CH2Cl2 (50 mL), EDCI (1 equiv, 1.014 g, 5.292 mmol) was added, and the resulting mixture was stirred at rt for 15 min. Then benzyl-2hydroxyacetate (1 equiv, 0.751 mL, 5.292 mmol) and DMAP (0.2 equiv, 0.137 g, 1.058 mmol) were added, and the solution was stirred at rt for about 2 days. The mixture was then diluted with CH2Cl2 (200 mL), washed with H2O (150 mL), the aqueous phase re-extracted with CH2Cl2 (2 × 100 mL), and the combined organic phases washed with brine (200 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. Solid deposition on Celite and puriﬁcation by ﬂash chromatography aﬀorded the product as a colorless oil (1.365 g, η = 86%). Rf (Hex/AcOEt 3:7 (v/v)) = 0.28. 1 H NMR (CDCl3) δH, ppm: 8.49 (1H, d, Npy-CH-C, 4JH,H = 1.7 Hz), 8.47 (1H, dd, Npy-CH-CH, 3JH,H = 4.8 Hz, 4JH,H = 1.6 Hz), 7.54 (1H, ddd overlapped, Npy-CH-C-CH, 3JH,H = 7.7 Hz, 4JH,H = 1.7 Hz), 7.32−7.39 (5H, m, 5 × CH(Ph)), 7.22 (1H, dd, Npy-CH-CH, 3JH,H = 4.8 Hz, 4Jd,c = 7.7 Hz), 5.19 (2H, s, O-CH2-Ph), 4.66 (2H, s, O-CH2CO), 2.99 (2H, t, Py-CH2-CH2-CO, 3JH,H = 7.8 Hz), 2.75 (2H, t, Py-CH2-CH2-CO, 3JH,H = 7.8 Hz). 13 C NMR (CDCl3) δC, ppm: 171.8 (1C, CH2-CH2-CO), 167.6 (1C, O-CH2-CO), 149.9 (1C, Npy-CH-C), 148.0 (1C, Npy-CHCH), 135.9 (1C, Npy-CH-C-CH), 135.6 (1C, Npy-CH-C-CH), 135.1 (1C, CH2-C-CH(Ar)), 128.8 (2C, 2 × CH2-C-CH-CH(Ar)), 128.7 (1C, CH2-C-CH-CH-CH(Ar)), 128.5 (2C, 2 × CH2-C-CH-CH(Ar)), 123.5 (1C, C-d), Npy-CH-CH, 67.3 (1C, O-CH2−Ph), 60.9 (1C, OCH2-CO), 34.9 (1C, Py-CH2-CH2-CO), 27.9 (1C, Py-CH2-CH2CO). IR (ν, cm−1): 3031 (CH-Ar), 2957 (CH2), 1763 (CO), 1742 (CO), 1575, 1497, 1480, 1456, 1422 (Py CC, CN), 1222 (C− O). ESI-MS(+): m/z found 300.11 [M + H]+, calcd for C17H17NO4 299.32. Anal. (%) Calcd for C17H17NO4: C 68.21, H 5.72, N 4.68. Found: C 69.33, H 5.80, N 4.72. Synthesis of 2-((3-(Pyridin-3-yl)propanoyl)oxy)acetic Acid (step b before 4l). To a solution of 2-(benzyloxy)-2-oxoethyl-3(pyridin-3-yl)propanoate (1.100 g, 3.645 mmol, 1 equiv) in MeOH (150 mL), Pd/C (10%) (0.05 equiv) was added, and the resulting suspension was degassed for further 15 min. Then H2 gas was passed through the reaction mixture for 8 h and the mixture stirred under a H2 atmosphere overnight. The mixture was ﬁltrated on a Celite pad which was further washed with CH2Cl2/MeOH 50:50 mixture (5 × 60 mL). The ﬁltrate evaporated to dryness, and the resulting solid puriﬁed by ﬂash chromatography to aﬀord a white solid (0.344 g, η = 45%); mp (°C) 126.5−128. Rf (CH2Cl2/MeOH 8:2 (v/v)) = 0.44. 1 H NMR (MeOD-d4) δH, ppm: 8.52 (1H, s, Npy-CH-C), 8.43 (1H, dd overlapped, Npy-CH-CH, 3JH,H = 5 Hz), 7.86 (1H, ddd overlapped, Npy-CH-C-CH, 3JH,H = 7.8 Hz), 7.45 (1H, dd, Npy-CH-CH, 3JH,H = 5 Hz, 4JH,H = 7.8 Hz), 4.60 (2H, s, O-CH2-CO), 3.06 (2H, t, Py-CH2CH2-CO, 3JH,H = 7.3 Hz), 2.83 (2H, t, Py-CH2-CH2-CO; 3JH,H = 7.3 Hz). 13 C NMR (MeOD-d4) δC, ppm: 173.5 (1C, CH2-CH2-CO), 172.6 (1C, O-CH2-CO), 149.3 (1C, Npy-CH-C), 147.0 (1C, NpyCH-CH), 139.6 (1C, Npy-CH-C-CH), 139.0 (1C, Npy-CH-C-CH), 125.5 (1C, Npy-CH-CH), 62.3 (1C, O-CH2-CO), 35.5 (1C, PyCH2-CH2-CO), 28.7 (1C, Py-CH2-CH2-CO). IR (ν, cm−1): 2924 (CH2), 2360 (O−H), 1719 (CO), 1583, 1479 (CC, CN), 1254 (C−O). ESI-MS(+): m/z found 210.05 [M + H]+, calcd for C10H11NO4 209.20. Anal. (%) Calcd for C10H11NO4: C 57.41, H 5.30, N 6.70. Found: C 57.27, H 5.45, N 6.62. Synthesis of 2-((1H,1H,2H,2H-Perﬂuorodecyl)oxy)-2-oxoethyl-3-(pyridin-3-yl)propanoate 4l. To a suspension of 2-((3(pyridin-3-yl)propanoyl)oxy)acetic acid (0.344 g, 1.644 mmol, 1 equiv) in CH2Cl2 (75 mL), EDCI (0.315 g, 1.644 mmol,1 equiv) was added and the mixture was stirred at rt for 15 min. Next, 1H,1H,2H,2H-perﬂuoro-1-decanol (0.763 g, 1.644 mmol, 1 equiv) and DMAP (0.043 g, 0.329 mmol, 0.2 equiv) were added, and the 1239 (CF2, CF3), 1195 (C−O). ESI-MS(+): m/z found 854.04 [M − Cl]+, calcd for C27H24ClF17NO2Ru 854.03, the experimental isotopic pattern ﬁts well the calculated one. Anal. (%) Calcd for C27H24Cl2F17NO2Ru: C 36.46, H 2.72, N 1.57. Found: C 36.41, H 2.67, N 1.53. [Ru(η6-p-cymene)Cl2(1H,1H,2H,2H-perﬂuorodecyl-2-(pyridin-3yl)acetate)] 7. [Ru(η6-p-cymene)Cl2]2 (0.220 g, 0.359 mmol), 1H,1H,2H,2H-perﬂuorodecyl-2-(pyridin-3-yl)acetate (2l) (0.440 g, 0.754 mmol) were used. The product was isolated as an orange solid (0.579 g, η = 91%); mp (°C) 158.2−160. 1 H NMR (CDCl3) δH, ppm: 8.97 (1H, d, Npy-CH-CH, 3JH,H = 5.6 Hz), 8.96 (1H, s, Npy-CH-C), 7.65 (1H, d, Npy-CH-C-CH, 3JH,H = 7.8 Hz), 7.28 (1H, dd overlapped, Npy-CH-CH, 3JH,H = 5.6 Hz, 3JH,H = 7.8 Hz), 5.45 (2H, d, 2 × CH3-C-CH-CH(Ar), 3JH,H = 5.9 Hz), 5.21 (2H, d, 2 × CH3-C-CH-(Ar), 3JH,H = 5.9 Hz), 4.44 (2H, t, O-CH2-CH2, 3 JH,H = 6.5 Hz), 3.67 (2H, s, Py-CH2-CO), 2.96−3.05 (1H, sept, ArCH(CH3)2, 3JH,H = 6.9 Hz), 2.46−2.58 (2H, m, O-CH2-CH2), 2.07 (3H, s, Ar-CH3), 1.30 (6H, d, Ar-CH(CH3)2, 3JH,H = 6.9 Hz). 13 C NMR (CDCl3) δC, ppm: 169.9 (1C, CH2-CO), 155.6 (1C, Npy-CH-C), 153.6 (1C, Npy-CH-CH), 138.8 (1C, Npy-CH-C-CH), 130.4 (1C, Npy-CH-C-CH), 124.2 (1C, Npy-CH-CH), 105.2−121.8 (8C, m series, CH2-CF2-CF2, CH2-CF2-CF2, CH2-(CF2)2-CF2, CH2(CF2)3-CF2, CF2-(CF2)2-CF3, CF2-CF2-CF3, CF2-CF2-CF3, CF2-CF2CF3), 103.5 (1C, CH3-C-CH-CH-C(Ar)), 97.4 (1C, CH3-C-CH(Ar)), 83.1 (2C, 2 × CH3-C-CH-CH(Ar)), 82.2 (2C, 2 × CH3-C-CH(Ar)), 57.3 (1C, m, O-CH2-CH2, 3JC,F = 4 Hz), 37.7 (1C, Py-CH2-CO), 30.7 (1C, Ar-CH(CH3)2), 30.5 (1C, t, O-CH2-CH2, 2JC,F = 22 Hz), 22.3 (2C, Ar-CH(CH3)2), 18.1 (1C, Ar-CH3). 19 F NMR (CDCl3) δF, ppm: −80.72 (3F, t, CF3, 3JF,F = 9.8 Hz), −113.59 (2F, m, CH2-CF2-CF2), −121.59 (2F, m, CH2-(CF2)2-CF2), −121.85 (4F, m, CH2-(CF2)3-CF2, CH2-(CF2)4-CF2), −122.66 (2F, m, CF2-CF2-CF3), −123.45 (2F, m, CH2-CF2-CF2), −126.06 (2F, m, CF2-CF3). IR (ν, cm−1): 3042 (CH-Ar), 2960, 2868 (CH2, CH, CH3), 1741 (CO), 1572, 1503, 1431 (Py CC, CN), 1353, 1330, 1113− 1232 (CF2, CF3), 1199 (C−O). ESI-MS(+): m/z found 853.92 [M − Cl]+, calcd for C27H24ClF17NO2Ru 854.03, the experimental isotopic pattern ﬁts well the calculated one. Anal. (%) Calcd for C27H24Cl2F17NO2Ru: C 36.46, H 2.72, N 1.57. Found: C 36.69, H 2.73, N 1.53. [Ru(η6-p-cymene)Cl2(1H,1H,2H,2H-perﬂuorodecyl-2-(pyridin-4yl)acetate)] 8. [Ru(η6-p-cymene)Cl2]2 (0.220 g, 0.359 mmol) and 1H,1H,2H,2H-perﬂuorodecyl-2-(pyridin-4-yl)acetate (3l) (0.440 g, 0.754 mmol) were used. The product was isolated as an orange solid (0.544 g, η = 85%); mp (°C) 156.5−157.5. 1 H NMR (CDCl3) δH, ppm: 8.97 (2H, d, 2 × Npy-CH-CH, 3JH,H = 6.4 Hz), 7.23 (2H, d, 2 × Npy-CH-CH, 3JH,H = 6.4 Hz), 5.42 (2H, d, 2 × CH3-C-CH-CH(Ar), 3JH,H = 5.9 Hz), 5.21 (2H, d, 2 × CH3-CCH(Ar), 3JH,H = 5.9 Hz), 4.43 (2H, t, O-CH2-CH2, 3JH,H = 6.3 Hz), 3.69 (2H, s, Py-CH2-CO), 2.97−3.04 (1H, sept, Ar-CH(CH3)2, 3 JH,H = 6.9 Hz), 2.43−2.55 (2H, m, O-CH2-CH2), 2.10 (3H, s, ArCH3), 1.30 (6H, d, Ar-CH(CH3)2, 3JH,H = 6.9 Hz). 13 C NMR (CDCl3) δC, ppm: 169.0 (1C, CH2-CO), 154.7 (2C, 2 × Npy-CH-CH), 144.6 (1C, Npy-CH-CH-C), 125.5 (2C, 2 × Npy-CHCH), 105.5−121.4 (8C, m series, CH2-CF2-CF2, CH2-CF2-CF2, CH2(CF2)2-CF2, CH2-(CF2)3-CF2, CF2-(CF2)2-CF3, CF2-CF2-CF3, CF2CF2-CF3, CF2-CF2-CF3), 103.6 (1C, CH3-C-CH-CH-C(Ar)), 97.1 (1C, CH3-C-CH(Ar)), 82.7 (2C, 2 × CH3-C-CH-CH(Ar)), 82.3 (2C, 2 × CH3-C-CH(Ar)), 57.3 (1C, t, O-CH2-CH2, 3JC,F = 4 Hz), 39.8 (1C, Py-CH2-CO), 30.7 (1C, Ar-CH(CH3)2), 30.4 (1C, t, O-CH2CH2, 2JC,F = 22 Hz), 22.2 (2C, Ar-CH(CH3)2), 18.2 (1C, Ar-CH3). 19 F NMR (CDCl3) δF, ppm: −80.72 (3F, t, CF3, 3JF,F = 9.8 Hz), −113.57 (2F, m, CH2-CF2-CF2), −121.60 (2F, m, CH2-(CF2)2-CF2), −121.86 (4F, m, CH2-(CF2)3-CF2, CH2-(CF2)4-CF2), −122.67 (2F, m, CF2-CF2-CF3), −123.49 (2F, m, CH2-CF2-CF2), −126.07 (2F, m, CF2-CF3). IR (ν, cm−1): 3058 (CH-Ar), 2963, 2873 (CH2, CH, CH3), 1732 (CO), 1502, 1472, 1426 (Py CC, CN), 1351, 1321, 1236− 1117 (CF2, CF3), 1224 (C−O). ESI-MS(+): m/z found 854.03 [M − Cl]+, calcd for C27H24ClF17NO2Ru 854.03, the experimental isotopic 3554 dx.doi.org/10.1021/jm5002748 | J. Med. Chem. 2014, 57, 3546−3558 Journal of Medicinal Chemistry Article m, CF2-CF2-CF3), −123.46 (2F, m, CH2-CF2-CF2), −126.07 (2F, m, CF2-CF3). IR (ν, cm−1): 3052 (CH-Ar), 2958−2872 (CH2, CH, CH3), 1744 (CO), 1473, 1425 (CC, CN), 1384, 1331, 1115−1234 (CF2, CF3), 1198 (C−O). ESI-MS(+): m/z found 925.92 [M − Cl]+, calcd for C30H28ClF17NO4Ru 926.05, the experimental isotopic pattern ﬁts well the calculated one. Anal. (%) Calcd for C30H28Cl2F17NO4Ru: C 37.48, H 2.94, N 1.46. Found: C 37.49, H 2.99, N 1.46. X-ray Diﬀraction. The diﬀraction data for compound 2 were measured at low temperature [140(2) K] using Mo Kα radiation on a mar345dtb system in combination with a Genix Hi-Flux small focus generator (marμX system). The data reduction was carried out by automar.52 The solution and reﬁnement were performed by SHELX.53 The structure was reﬁned using full-matrix least-squares based on F2 with all non-hydrogen atoms anisotropically deﬁned. Hydrogen atoms were placed in calculated positions by means of the “riding” model. Cell Culture. Human A2780 and A2780cisR ovarian carcinoma cells were obtained from the European Centre of Cell Cultures (ECACC, UK). Adenocarcinomic human alveolar basal epithelial cells (A549), human breast adenocarcinoma cells (MDA-MB-231 and MCF7), and nontumorigenic HEK-293 cells were provided by the Institute of Pathology, CHUV, Lausanne, Switzerland. A2780, A2780cisR, and A549 cells were routinely grown in RPMI 1640 medium supplemented with GlutaMAX (Gibco), while MDA-MB-231, MCF-7, and HEK-293 were grown in DMEM medium, both containing heat-inactivated fetal calf serum (FCS, Sigma, USA) (10%) and antibiotics (penicillin/streptomycin) at 37 °C and CO2 (5%). Cell Proliferation Inhibition. Cytotoxicity was determined using the MTT assay (MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide). Cells were seeded in 96-well plates as monolayers with 100 μL of cell solution (approximately 20000 cells) per well and preincubated for 24 h in medium supplemented with 10% FCS. Compounds were prepared as DMSO solution that were rapidly dissolved in the culture medium and serially diluted to the appropriate concentration to give a ﬁnal DMSO concentration of 0.5% NMR spectroscopy indicated that the Ru−N bond in the complexes is stable in DMSO−water solution. Then 100 μL of the drug solution was added to each well and the plates were incubated for another 72 h. Subsequently, MTT (5 mg/mL solution) was added to the cells and the plates were incubated for a further 4 h. The culture medium was aspirated, and the purple formazan crystals formed by the mitochondrial dehydrogenase activity of vital cells were dissolved in DMSO. The optical density, directly proportional to the number of surviving cells, was quantiﬁed at 540 nm using a multiwell plate reader and the fraction of surviving cells was calculated from the absorbance of untreated control cells. Evaluation is based on means from two independent experiments, each comprising three microcultures per concentration level. Cell Uptake Measurements. Cells were seeded in 6-well plates, grown to approximately 50% conﬂuency, and incubated with the corresponding compound for the required incubation time. After incubation, cells were detached using an enzyme free dissociation solution (Millipore) and pelleted for 10 min at 100g and 4 °C and washed twice with ice-cold PBS. Cell lysis was achieved using a freeze−thaw technique. All samples were analyzed for their protein content prior to ICP-MS determination using a bicinchoninic acid (BCA) assay (Sigma-Aldrich). Quantitation of cells was then possible, with a correlation established for this cell line between the sample total protein content and its number of cells.54 All determinations described above were carried out as at least two independent experiments. Sample digestion was carried out in concentrated nitric acid for 3 h. Samples were then ﬁlled to a total volume of 8 mL with water. Indium was added as an internal standard at a concentration of 0.5 ppb. Determination of internalized metal content was achieved on an Elan DRC II ICP-MS instrument (PerkinElmer, Switzerland) equipped with a Meinhard nebulizer and a cyclonic spray chamber. The ICP-MS instrument was tuned using a solution provided by the manufacturer containing 1 ppb of each element Mg, In, Ce, Ba, Pb, and U. External standards were prepared gravimetrically in an identical matrix to the resulting mixture was stirred at rt for about 4 days. The mixture was then diluted with CH2Cl2 (100 mL), washed with H2O (100 mL), the aqueous phase re-extracted with CH2Cl2 (2 × 100 mL), and the combined organic phases were washed with brine (150 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. Deposition of the solid on Celite and puriﬁcation by ﬂash chromatography aﬀord the product as a colorless viscous oil (0.614 g, η = 57%). Rf (Hex/AcOEt 4:6 (v/v)) = 0.35. 1 H NMR (CDCl3) δH, ppm: 8.48 (1H, d, Npy-CH-C, 4JH,H = 2.1 Hz), 8.45 (1H, dd, Npy-CH-CH, 3JH,H = 4.8 Hz, 4JH,H = 1.7 Hz), 7.52 (1H, ddd overlapped, Npy‑CH-C-CH, 3JH,H = 7.8 Hz, 4JH,H = 1.7 Hz), 7.20 (1H, dd, Npy-CH-CH, 3JH,H = 4.8 Hz, 4JH,H = 7.8 Hz), 4.61 (2H, s, O-CH2-CO), 4.45 (2H, t, O-CH2-CH2, 3JH,H = 6.5 Hz), 2.98 (2H, t, Py-CH2-CH2-CO, 3JH,H = 7.6 Hz), 2.75 (2H, t, Py-CH2-CH2-CO, 3 JH,H = 7.6 Hz), 2.40−2.53 (2H, m, O-CH2-CH2). 13 C NMR (CDCl3) δC, ppm: 171.8 (1C, CH2-CH2-CO), 167.4 (1C, O-CH2-CO), 150.0 (1C, Npy-CH-C), 148.1 (1C, Npy-CHCH), 135.9 (1C, Npy-CH-C-CH), 135.6 (1C, Npy-CH-C-CH), 123.5 (1C, Npy-CH-CH), 105.3−121.9 (8C, m series, CH2-CF2-CF2, CH2CF2-CF2, CH2-(CF2)2-CF2, CH2-(CF2)3-CF2, CF2-(CF2)2-CF3, CF2CF2-CF3, CF2-CF2-CF3, CF2-CF2-CF3), 60.7 (1C, O-CH2-CO), 57.3 (1C, t, O-CH2-CH2, 3JC,F = 4 Hz), 34.9 (1C, Py-CH2-CH2-C O), 30.6 (1C, t, O-CH2-CH2, 2JC,F = 22 Hz), 27.9 (1C, Py-CH2-CH2CO). 19 F NMR (CDCl3) δF, ppm: −80.76 (3F, t, CF3, 3JF,F = 9.8 Hz), −113.64 (2F, m, CH2-CF2-CF2), −121.65 (2F, m, CH2-(CF2)2-CF2), −121.88 (4F, m, CH2-(CF2)3-CF2, CH2-(CF2)4-CF2), −122.60 (2F, m, CF2-CF2-CF3), −123.48 (2F, m, CH2-CF2-CF2), −126.07 (2F, m, CF2-CF3). IR (ν, cm−1): 2956 (CH2), 1746 (CO), 1576, 1480, 1425 (Py CC, CN), 1370, 1331, 1246−1116 (CF2, CF3), 1197 (C−O). ESI-MS(+): m/z found 656.07 [M + H]+, calcd for C20H14F17NO4 655.30. Anal. (%) Calcd for C20H14F17NO4: C 36.66, H 2.15, N 2.14. Found: C 36.66, H 2.16, N 2.11. Synthesis of [Ru(η 6 -p-cymene)Cl 2 (2-((1H,1H,2H,2Hperﬂuorodecyl)oxy)-2-oxoethyl-3-(pyridin-3-yl)propanoate)] 9. To a solution of [Ru(η6-p-cymene)Cl2]2 (0.165 g, 0.269 mmol, 1 equiv) in CH2Cl2 (10 mL), a solution of 2-((1H,1H,2H,2Hperﬂuorodecyl)oxy)-2-oxoethyl-3-(pyridin-3-yl)propanoate (4l) (0.370 g, 0.565 mmol, 2.1 equiv) in CH2Cl2 (20 mL) was added, and the resulting mixture was stirred at rt in the dark for about 4 days. The reaction mixture was concentrated under reduced pressure almost to dryness, and the obtained viscous orange oil was washed with Et2O (3 × 20 mL), hexane (20 mL), and again Et2O (20 mL). The resulting orange oil was dried under high vacuum to aﬀord an orange solid (0.514 g, η = 97%); mp (°C) = 89.5−91. 1 H NMR (CDCl3) δH, ppm: 8.93 (1H, s, Npy-CH-C), 8.89 (1H, d, Npy-CH-CH, 3JH,H = 4.3 Hz), 7.61 (1H, d, Npy-CH-C-CH, 3JH,H = 6.5 Hz), 7.22 (1H, m, Npy-CH-CH), 5.43 (2H, d, 2 × CH3-C-CHCH(Ar), 3JH,H = 4.9 Hz), 5.21 (2H, d, 2 × CH3-C-CH(Ar), 3JH,H = 4.9 Hz), 4.63 (2H, s, O-CH2-CO), 4.46 (2H, t, O-CH2-CH2, 3JH,H = 6.3 Hz), 2.96−3.01 (1H, m, Ar-CH(CH3)2), 3.01 (2H, t, Py-CH2-CH2CO, 3JH,H = 6.5 Hz), 2.78 (2H, t, Py-CH2-CH2-CO, 3JH,H = 6.5 Hz), 2.43−2.54 (2H, m, O-CH2-CH2), 2.08 (3H, s, Ar-CH3), 1.31 (6H, d, Ar-CH(CH3)2, 3JH,H = 7.1 Hz). 13 C NMR (CDCl3) δC, ppm: 171.5 (1C, CH2-CH2-CO), 167.4 (1C, O-CH2-CO), 155.1 (1C, Npy-CH-C), 153.0 (1C, Npy-CHCH), 137.8 (1C, Npy-CH-C-CH), 136.8 (1C, Npy-CH-C-CH), 124.3 (1C, Npy-CH-CH), 105.3−121.9 (8C, m series, CH2-CF2-CF2, CH2CF2-CF2, CH2-(CF2)2-CF2, CH2-(CF2)3-CF2, CF2-(CF2)2-CF3, CF2CF2-CF3, CF2-CF2-CF3, CF2-CF2-CF3), 103.5 (1C, CH3-C-CH-CHC(Ar)), 97.3 (1C, CH3-C-CH(Ar)), 82.8 (2C, 2 × CH3-C-CHCH(Ar)), 82.3 (2C, 2 × CH3-C-CH(Ar)), 60.7 (1C, O-CH2-CO), 57.3 (1C, t, O-CH2-CH2, 3JC,F = 4 Hz), 34.4 (1C, Py-CH2-CH2-C O), 30.7 (1C, Ar-CH(CH3)2), 30.5 (1C, t, O-CH2-CH2, 2JC,F = 22 Hz), 27.7 (1C, Py-CH2-CH2-CO), 22.4 (2C, Ar-CH(CH3)2), 18.2 (1C, Ar-CH3). 19 F NMR (CDCl3) δF, ppm: −80.72 (3F, t, CF3, 3JF,F = 9.3 Hz), −113.59 (2F, m, CH2-CF2-CF2), −121.62 (2F, m, CH2-(CF2)2-CF2), −121.88 (4F, m, CH2-(CF2)3-CF2, CH2-(CF2)4-CF2), −122.68 (2F, 3555 dx.doi.org/10.1021/jm5002748 | J. Med. Chem. 2014, 57, 3546−3558 Journal of Medicinal Chemistry ■ samples (with regard to internal standard and nitric acid) with single element standards obtained from CPI International (Amsterdam, The Netherlands). Apoptosis Assay. A2780 cells were seeded on 6-well plates (2 × 105 cells/well) and grown for 24 h in complete medium before treatment. Compound 1 was freshly dissolved in DMSO, diluted in complete medium, and added to the cells at the ﬁnal concentrations indicated in Table 4. After incubation for 24 h, apoptosis was measured by ﬂow cytometric determination of subdiploid cells after DNA extraction and subsequent staining with propidium iodide (PI) as described previously. 55 Brieﬂy, cells were harvested and subsequently ﬁxed in 70% ethanol at 20 °C. After 2 h, the cells were resuspended in DNA extraction buﬀer (45 mM Na2HPO4, 2.5 mM citric acid, and 1% Triton X-100, pH 7.4) for 20 min at 37 °C. PI was added to a ﬁnal concentration of 20 μg/mL, and log scale red ﬂuorescence was analyzed on a FACS Calibur (BD Biosciences, NJ, U.S.). Wound Assay (Migration). The migration capability of cells was measured using the wound assay.56 Human breast adenocarcinoma (MDA-MB-231) cells were grown to conﬂuence and cells were labeled with calcein AM (Molecular Probes, C3100MP, Carlsbad, USA) for 15 min (1:2000, Molecular Probes), and “scratch wounds” (with an approximate width of 350 μm) were made in the monolayer by removing cells with a sterile scratch tool (Peira Scientiﬁc Instruments, Belgium). Cultures were washed with PBS, and the medium was replaced by fresh medium and incubated with 1 at doses between 6 and 50 μM for 14 h. Plates were scanned using an Acumen eX3 laser scanner cytometer (TTP LabTech Ltd., UK) to record images for computational analysis of scratch sizes using UGR Scratch Assay 6.2 software (DCI Laboratories, Peira Scientiﬁc Instruments, Belgium). Developmental CAM Model. Antiangiogenic eﬃcacy of 1 was tested in the physiologically developing chicken embryo chorioallantoic membrane (CAM) model between embryo development days (EDDs) 11 and 14. Complex 1 was applied by iv injection (50 μM, 100 μL/day for four consecutive days), at EDDs 11, 12, 13, and 14. The control eggs were treated with (100 μL/day of 0.9% NaCl 100 μL/day for four consecutive days). At EDD 15, the CAMs were visualized in ovo using FITC-dextran (20 kDa, 20 μL, 25 mg/mL, Sigma-Aldrich) epiﬂuorescence angiography and subsequently analyzed by the image-processing quantiﬁcation method described previously.57 Brieﬂy, on the basis of the FITC-dextran ﬂuorescence angiography, the skeleton of the vascular network is built, and deﬁned descriptors, i.e., branching points (mm2), give information on the vascular architecture. Five to six eggs were tested per condition. Errors bars represent the standard error of the mean. Human Ovarian Carcinoma in the CAM. First, 1 × 106 A2780 cells were prepared as a spheroid in a 25 μL hanging drop and 3 h later were transplanted on the surface of the CAM (EDD 7) as described previously.58 At EDD 11, a solution of 1 in 0.1% DMSO was injected iv at a concentration of 50 μM (100 μL/day) or 2 × 25 μM (2 × 50 μL/day, administered with 6 h interval) for four consecutive days. Tumors were measured daily for 8 days. At EDD 17, the experiment was terminated. Statistical Analysis. Values are given as mean values ± standard deviations (in vitro) or the standard error of the mean (in vivo). Data are represented as averages of independent experiments. Statistical analysis was done using the t test (developmental CAM) or two-way Anova (tumor growth curves). *P indicating p-values lower than 0.05 were considered statistically signiﬁcant. ■ Article AUTHOR INFORMATION Corresponding Author *Phone: +41216939854. E-mail: paul.dyson@epﬂ.ch. 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