Redox-Active Antineoplastic Ruthenium Complexes with Indazole

Mar 12, 2005 - Antineoplastic ruthenium(III) complexes are generally regarded as prodrugs, being activated by reduction. Within a homologous series of...
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J. Med. Chem. 2005, 48, 2831-2837

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Redox-Active Antineoplastic Ruthenium Complexes with Indazole: Correlation of in Vitro Potency and Reduction Potential Michael A. Jakupec, Erwin Reisner, Anna Eichinger, Martina Pongratz, Vladimir B. Arion, Markus Galanski, Christian G. Hartinger, and Bernhard K. Keppler* Institute of Inorganic Chemistry, University of Vienna, Waehringer Strasse 42, A-1090 Vienna, Austria Received November 17, 2004

Antineoplastic ruthenium(III) complexes are generally regarded as prodrugs, being activated by reduction. Within a homologous series of ruthenium(III) complexes, cytotoxic potency is therefore expected to increase with increasing ease of reduction. Complexes of the general formula [RuIIICl(6-n)(ind)n](3-n)- (n ) 0-4; ind ) indazole; counterions ) Hind+ or Cl-) and the compound trans-[RuIICl2(ind)4] have been prepared and characterized electrochemically. Lever’s parametrization method predicts that a higher indazole-to-chloride ratio results in a higher reduction potential, which is confirmed by cyclic voltammetry. In vitro antitumor potencies of these complexes in colon cancer cells (SW480) and ovarian cancer cells (CH1) vary by more than 2 orders of magnitude and increase in the following rank order: [RuIIICl6]3- < [RuIIICl4(ind)2]- < [RuIIICl5(ind)]2- , [RuIIICl3(ind)3] < [RuIIICl2(ind)4]+ ≈ [RuIICl2(ind)4]. Thus, the observed differences in potency correlate with reduction potentials largely, though not perfectly, pointing to the influence of additional factors. Differences in the cellular uptake (probably resulting from different lipophilicity) contribute to this correlation but cannot solely account for it. Introduction Ruthenium(III) complex salts with the general formula (HL) trans-[RuIIICl4L2] with two trans-standing azole heterocyclic ligands (L) bound to ruthenium through nitrogen have been recognized for their antitumor activity in animal models for more than a decade.1,2 From this class of compounds, indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], (Hind) trans-[RuIIICl4(ind)2] (KP1019, FFC14A), has been selected for clinical trials because of its remarkable activity in otherwise largely chemoresistant rodent solid tumor models such as autochthonous colorectal carcinoma of the rat.2,3 Knowledge about the mechanisms by which this compound and closely related complexes such as (Him) trans-[RuIIICl4(im)2] (im ) imidazole) (ICR) exert their antitumor effects is only rudimentary to date. Analogous to platinum drugs, interactions with DNA4,5 and nucleotides6 as well as aquation/hydrolysis reactions as preconditions for these interactions7-9 have been subject to investigations. DNA cross-linking efficiencies have been quantified but were found to be generally much lower than that of cisplatin.4,5 Recent studies point to an induction of apoptosis via the mitochondrial pathway.10 The impressive preclinical efficacy of (Hind) trans[RuIIICl4(ind)2] (KP1019, FFC14A) is all the more remarkable as this compound is only moderately cytotoxic in conventional cell culture tests11 and can be administered in vivo in therapeutically active doses over several weeks without any signs of toxicity.2 These findings prompted investigators to assume mechanisms of accumulation in tumor tissue as possible explanations for * To whom correspondence should be addressed. Phone: +43 1 4277 52600. Fax: +43 1 4277 52680. E-mail: [email protected].

this pharmacological behavior, which contrasts sharply with that of antineoplastic platinum drugs. Analytical studies on interactions with transferrin and cellular uptake experiments have provided evidence for a transferrin-mediated transport into tumor cells.12-14 This route is recognized for its capability of mediating a certain tumor selectivity because tumor cells frequently overexpress transferrin receptors as compared to most normal tissues in order to meet their high iron requirement.15,16 Moreover, ruthenium(III) complexes probably act as prodrugs that are preferentially activated under the hypoxic conditions prevailing in many solid tumors. Tumor hypoxia, which results from insufficient tumor vascularization, is a driving factor of the malignant progression and a major factor contributing to failure of radiotherapy or chemotherapy.17,18 Great efforts are being made to exploit tumor hypoxia by utilization of redox-active drugs that are selectively activated by reduction under conditions of low oxygen availability.19,20 The electrochemical potential of the Ru(III)/ Ru(II) couple is physiologically accessible, and glutathione as well as single-electron-transfer proteins in the presence of NADH are capable of reducing ruthenium(III). The resulting ruthenium(II) species are generally less inert, have a higher propensity for ligand exchange reactions and may therefore interact with target molecules more rapidly.21-23 In support of this “activation-by-reduction” hypothesis, ligand exchange and antimetastatic activity of Na trans-[RuIIICl4(im)(Me2SO)] (NAMI) and (Him) trans-[RuIIICl4(im)(Me2SO)] (NAMI-A) is enhanced upon addition of biological reductants such as ascorbic acid, cysteine, or glutathione.24,25 Furthermore, tumor hypoxia is often associated with a slightly acidic extracellular environment,26,27

10.1021/jm0490742 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/12/2005

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Figure 1. Structures of the ruthenium complexes of the general formula [RuCl(6-n)(ind)n](3-n)- (n ) 0-4; ind ) indazole). Counterions (Hind+ or Cl-) are omitted for clarity.

which may further facilitate reduction, as suggested by the pH-dependency of the reduction potential of (Him) trans-[RuIIICl4(im)2] (ICR).7 Thus, active ruthenium(II) species are more likely to be formed in hypoxic as compared to well-oxygenated tissue, and this behavior may contribute to the apparent tumor selectivity of (Hind) trans-[RuIIICl4(ind)2] (KP1019, FFC14A). Any attempt to exploit the reductive environment of solid tumors requires that the reduction potential of the redox-active ruthenium drug lies within a certain window, because at low potentials the compound may not be reduced, while at high potentials it may be reduced under well-oxygenated conditions as well. However, in a simple cell culture system, cytotoxic potency within a homologous series of ruthenium(III) complexes is expected to increase with increasing ease of reduction. To examine this hypothesis, complexes of the general formula [RuIIICl(6-n)(ind)n](3-n)- (n ) 0-4) (Figure 1, compounds 1-5) containing different numbers of indazole vs chloro ligands have been synthesized, characterized electrochemically, and evaluated for their in vitro activity in two human cancer cell lines. The present study reveals a correlation of reduction potentials and antitumor potencies, which can only partially be explained by the different cellular uptakes of the complexes. A direct comparison of the biological activities of ruthenium(III) complexes and their putative ruthenium(II) reduction products is usually impossible due to the instability of the latter, which commonly precludes their isolation in a pure form. Nevertheless, trans-[RuIICl2(ind)4] (6) could be prepared and subjected to biological evaluation in comparison with its ruthenium(III) congener trans-[RuIIICl2(ind)4]Cl (5). Implications for the design of bioreductive ruthenium prodrugs are discussed. Results and Discussion Synthesis. Although ruthenium(II) and ruthenium(III) complexes of the type [RuCl2L4]0/+, where L is a monodentate nitrogen-containing heterocycle, are known, routes of their synthesis are only well-developed when L is pyridine. Thus, trans-[dichlorotetrapyridineruthenium(II)], trans-[RuIICl2(py)4], was obtained by treatment of [RuCln(Ph3E)m]x (n ) m ) 2, x ) 1, E ) P;28 n ) m ) x ) 2, E ) As;28 n ) m ) 3, x ) 1, E ) As;29 or n ) 2, m ) 3, x ) 1, E ) P)30 or [RuCl3(Ph3As)2(MeOH)]30 with an excess of pyridine upon heating. Other methods of preparation leading to trans-[RuIICl2(py)4]‚H2O are based on the reaction of RuCl3 with an excess of pyridine in refluxing aqueous ethanol31 or with zinc powder in a solution of pyridine in hydrochloric

Figure 2. ORTEP drawing of trans-[RuIIICl2(ind)4]+ in 5‚ 2CH3OH with thermal ellipsoids depicted at 50% probability.

acid.32 The corresponding cis-isomer, i.e., cis-[RuIICl2(py)4], was synthesized by heating cis-[RuII(C2O4)(py)4] in hydrochloric acid.33 Ruthenium(III) complexes trans[RuIIICl2L4]+ can be prepared by oxidation of their ruthenium(II) precursors; e.g., trans-[RuIIICl2(py)4][H(ONO2)2] was prepared by dissolution of trans[RuIICl2(py)4] in warm, concentrated HNO3.32 Information on complexes [RuCl2L4] with L other than pyridine is scarce. Crystal structures of [trans-RuIII(OH)2(im)4][trans-RuIIICl4(im)2],34 or trans-[RuIICl2(ind)4]‚ 2(C4H7NO)35 were reported, but both complexes were prepared in a low yield or were poorly characterized. Starting from (Hind) trans-[RuIIICl4(ind)2] and indazole in 1:1.5 molar ratio in refluxing aqueous ethanol, we prepared the complex trans-[RuIICl2(ind)4] in 61% yield. Oxidation of the latter with hydrogen peroxide in methanol in the presence of hydrochloric acid and indazole resulted in the ruthenium(III) species trans[RuIIICl2(ind)4]Cl in 85% yield. Crystal Structures of 5‚2CH3OH and 6‚2(CH3)2CO. Perspective views of the complex cation trans-[RuIIICl2(ind)4]+ in 5‚2CH3OH and of the complex molecule trans[RuIICl2(ind)4] in 6‚2(CH3)2CO with the labeling schemes are shown in Figures 2 and 3, respectively. Cyclic Voltammetry. The cyclic voltammograms of 1 and 2 in DMF at a glassy carbon working electrode closely resemble those of 3, which have been described in detail elsewhere.36 They display one irreversible reduction wave at Ep/2 ) -0.43 V (Table 1). In 1 and 2, an additional wave at Ep ) ca. 1.3-1.4 V starting from an anodic scan, also observed for [Me4N]Cl under the same experimental conditions and found to be consistent with reported observations, was assigned to chloride oxidation.37,38 It is worth mentioning that in 3 this wave appeared only upon reduction of trans-[RuIIICl4(ind)2]with subsequent replacement of chloride by DMF, which is facilitated in the case of Ru(II). Thus, it seems that

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Figure 3. ORTEP drawing of trans-[RuIICl2(ind)4] in 6‚2(CH3)2CO with thermal ellipsoids depicted at 50% probability. Figure 4. Cyclic voltammogram of the reversible Ru(III)/ Ru(II) redox couple of 3 mM [RuCl2(ind)4] (6), in 0.2 M [n-Bu4N][BF4]/DMF at a scan rate of 0.2 V.

Table 1. Comparison of Reduction Potentials and in Vitro Antitumor Activities of Ruthenium Complexes Differing in Their Numbers of Indazole vs Chloro Ligands E1/2(RuIII/II) (V)b

compound [RuIIICl

(Hind)3 6], 1 (Hind)2[RuIIICl5(ind)], 2 III (Hind)[Ru Cl4(ind)2], 3 [RuIIICl3(ind)3], 4 [RuIIICl2(ind)4]Cl, 5 [RuIICl2(ind)4], 6

-c(-1.36)

-c(-0.87) -0.43 (-0.39) 0.10 (0.10) 0.59 (0.58) 0.58 (0.58)

IC50 (µM)a CH1

SW480

103 ( 13 187 ( 23 35 ( 2 37 ( 2 66 ( 4 63 ( 3 2.6 ( 0.1 2.5 ( 0.3 0.67 ( 0.04 0.69 ( 0.04 0.59 ( 0.04 0.63 ( 0.09

a Inhibitory (50%) concentrations in CH1 and SW480 cells after exposure for 96 h in the MTT assay. Values are the means ( standard errors obtained from at least three independent experiments. b Potentials in V ( 0.02 vs NHE, measured at a scan rate of 0.2 V s-1 in 0.2 M [n-Bu4N][BF4]/DMF (potentials in parentheses were estimated by eq 1). For the irreversible Ru(III)/Ru(II) wave of complex 3, the Ep/2 value is given. c Adequate detection was hampered by rearrangement of the complexes in DMF solution (see the text).

both 1 and 2 undergo conversion into 3 in DMF very rapidly. Possibly the way is the Anderson rearrangement,39 which implies dehydrohalogenation caused by replacement of inner chloride ligand(s) by outer-sphere indazole ligand(s). A similar pathway is presumably involved in the conversion of 3 into 4 in THF35 and that of (Him)2[RuIIICl5(im)] (im ) imidazole) into (Him) trans-[RuIIICl4(im)2] in water.34 It is also worth noting that the cyclic voltammogram of [n-Bu4N][RuIIICl6] in CH2Cl2 shows a one-electron-transfer wave at E1/2 ) -1.44 V vs NHE, providing further evidence in support of the very rapid conversion of 1 and 2 into 3 in DMF.40 This precludes the determination of the genuine redox potentials of 1 and 2. The latter, however, could be estimated by using eq 1 (Table 1, in brackets). The cyclic voltammograms of 4-6 in DMF (Figure 4), at a glassy carbon working electrode, display one redox wave at E1/2 ) 0.10, 0.59, and 0.58 V, respectively, at a scan rate of 0.2 V/s (Table 1). On the basis of the general expression (equation 1) proposed by Lever,41

∑EL + IM

E ) SM‚

(1)

by using the known values of SM (slope) and IM (intercept) for the RuIII/RuII redox couple (SM ) 0.97 and IM ) 0.04)41 and the EL (ligand parameter) values for chloride (EL ) -0.24)41 and indazole (EL ) 0.26),36 it follows that E1/2 increases with increasing number of indazole ligands and decreasing number of chloro ligands. The values found for 3-6 from cyclic voltammograms are in excellent agreement with those calculated by using eq 1 (Table 1).

Figure 5. Cellular uptake of ruthenium(III) complexes containing different numbers of indazole ligands in SW480 cells after exposure for 2 h. Each data point is the mean ( standard error obtained from at least three independent experiments.

It should also be pointed out that the very low aqueous solubility of 4-6 made a cyclic voltammetric study in aqueous solution impossible. Reliable data are only available for complex 3, which shows a reduction potential of E1/2 0.03 V in phosphate buffer at pH 7 36 as compared to Ep/2 -0.43 V in DMF. It has to be noted that the reduction potential of 3 in aqueous solution at pH 7 is considerably higher than those of established or investigational bioreductive drugs, including quinones such as mitomycin C, aromatic N-oxides such as tirapazamine, and nitroimidazoles (all within -0.5 to -0.3 V vs NHE in water, pH 7).20 Cellular Uptake. While no intracellular ruthenium is detectable after 2 h exposure of SW480 cells to the highly charged complex 1 when present in the medium in concentrations up to 5 µM, uptake of the other ruthenium(III) complexes increases linearly with increasing concentration in the medium (Figure 5). Cellular uptake increases in the following rank order (counterions omitted for clarity): [RuIIICl6]3- (1) < [RuIIICl5(ind)]2- (2) < [RuIIICl4(ind)2]- (3) , [RuIIICl2(ind)4]+ (5) < [RuIIICl3(ind)3] (4). Thus, within the more hydrophilic anionic complexes 1-3, cellular uptake is inversely correlated with the charge but remains at a moderate level even in the case of the complex anion of 3. In contrast, the uncharged complex 4 is taken up to a much higher extent (by more than 1 order of magni-

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Figure 6. In vitro antitumor activities of ruthenium complexes containing different numbers of indazole ligands in (A) CH1 cells and (B) SW480 cells after exposure for 96 h. The concentration-effect curves were determined by means of an MTT assay. Each data point is the mean ( standard error obtained from at least three independent experiments.

tude) at all concentrations tested. Uptake of the cationic complex 5 is less efficient as compared to that of complex 4 but is still higher than that of 3 by about 1 order of magnitude. In Vitro Antitumor Potency. Compounds 1-6 were evaluated for their capability of inhibiting tumor cell growth in vitro using two human cell lines, CH1 (ovarian carcinoma) and SW480 (colon carcinoma). The resulting concentration-effect curves obtained with continuous exposure for 96 h are depicted in Figure 6. For a more convenient comparison with the reduction potentials of the complexes, potencies (expressed as IC50 values) are listed in Table 1. Overall sensitivities of the two cell lines to the ruthenium complexes are quite similar, and both cell lines reveal the same structure-activity relationships. Potency of the complexes increases in the following rank order (counterions omitted for clarity): [RuIIICl6]3- (1) < [RuIIICl4(ind)2]- (3) < [RuIIICl5(ind)]2- (2) , [RuIIICl3(ind)3] (4) < [RuIIICl2(ind)4]+ (5) ≈ [RuIICl2(ind)4] (6). Within this homologous series of compounds, IC50 values vary by more than 2 orders of magnitude (mainly in the micromolar range). Uncoordinated indazole in concentrations up to 500 µM does not exert any discernible effect in SW480 cells and a less than 10% inhibitory effect in CH1 cells (data not shown). The general tendency of potencies as apparent from the rank order mentioned above is largely consistent with the hypothesized dependency on reduction potentials. However, there is one deviation from the expected rank order, as the monoindazole complex 2 is slightly more active than the bisindazole complex 3. This finding is particularly surprising, since it also contrasts with the cellular uptake and the in vivo activities of these

Jakupec et al.

compounds. In the P388 murine leukemia model, both compounds show comparable therapeutic efficacies in terms of increased survival (T/C ) 160%) at the optimal dose, but the dose of 2 required to obtain this effect is markedly higher than that of 3 (75 vs 45 mg/kg, each administered on three consecutive days).42 Consistent with prior expectations, the tetraindazole complex 5 is markedly more active than the trisindazole complex 4, despite its lower cellular uptake, which may reflect a high reactivity resulting from its easy reducibility. Thus, the observed differences in cellular uptake are neither sufficient to fully explain the correlation of antitumor potencies and reduction potentials nor able to explain the somewhat higher than expected cytotoxicity of 2. With regard to the hypothesized bioreductive activation mechanism, one might expect the ruthenium(II) compound 6 to show a higher potency than its ruthenium(III) congener 5, which is not confirmed by the experimental results, as the concentration-effect curves of both compounds are almost identical. The high reduction potential of 5 might allow a reduction by medium constituents, even in a well-oxygenated environment, so 5 may immediately be converted into 6 under the experimental conditions, and may therefore account for this discrepancy. One must be aware that such an easy reduction may also be accomplished by constituents of body fluids in vivo, irrespective of the oxygenation status of the tissue, and may facilitate undesirable unspecific reactions of the compound with biomolecules before it has reached the hypoxic tumor environment. Simply increasing the tumor-inhibiting potency by increasing the reduction potential might therefore be misleading, because of a concomitant diminution of tumor selectivity. Animal experiments have to be carried out in order to clarify the consequences in terms of therapeutic index and to determine the optimal reduction potential. Furthermore, it remains an open question to be addressed by future experiments whether the transferrin receptor route may also be operative as a mechanism mediating tumor selective accumulation in the case of indazole-containing ruthenium(III) complexes other than (Hind) trans[RuIIICl4(ind)2] (KP1019, FFC14A). Conclusions Consistent with the hypothesis that the antineoplastic activity of ruthenium(III) compounds depends on their reduction to ruthenium(II) species, in vitro potency within a homologous series of ruthenium(III) complexes differing in their numbers of indazole vs chloro ligands increases with increasing ease of reduction. Differences in the cellular uptake of these complexes (probably resulting from their different electric charges) contribute to this correlation but cannot solely account for it. Tuning of reduction potentials by structural modification is a rational strategy to guide the development of ruthenium(III) derivatives designed as tumor-selective (hypoxia-selective) prodrugs, but the influence of additional factors, such as the propensity for activating aquation/hydrolysis reactions in the absence of reducing agents as well as for protein binding reactions, has to be kept in mind. Future studies should determine the optimal electrochemical potential that translates in a

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proper balance between antitumor potency and tumor selectivity.

and 454 and 376 frames were measured, each for 75 and 70 s over 1.5° and 2° scan for 5‚2CH3OH and 6‚2(CH3)2CO, correspondingly. The data were processed using Denzo-SMN software. The 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 located on difference Fourier maps and isotropically refined (5‚2CH3OH) or placed at calculated positions and allowed to ride (6‚2(CH3)2CO). R-values: R1 ) 0.0424, wR2 ) 0.0879 (for 5‚2CH3OH); R1 ) 0.0260, wR2 ) 0.0642 (for 6‚2(CH3)2CO). The following computer programs were used: structure solution, SHELXS-97;44 refinement, SHELXL97;45 molecular diagrams, ORTEP;46 computer, Intel Pentium II; scattering factors.47 Cyclic Voltammetry. Cyclic voltammograms were measured in a two-compartment three-electrode cell using a 1.0 mm diameter glassy-carbon working electrode probed by a Luggin capillary connected to a silver-wire pseudoreference electrode and a platinum auxiliary electrode. Measurements were performed at room temperature using an EG & G PARC 273A potentiostat/galvanostat. Deaeration of solutions was accomplished by passing a stream of high-purity nitrogen through the solution for 5 min prior to the measurements and then by maintaining a blanket atmosphere of nitrogen over the solution during the measurements. The potentials were measured in 0.2 M [n-Bu4N][BF4]/DMF using the [Fe(η5C5H5)2]0/+ redox couple (E1/2ox ) +0.72 V)48 as the internal standard. All values are quoted relative to NHE and were measured in a ca. 3 mM electrolyte solution of the complex. Cell Lines and Culture Conditions. SW480 cells (adenocarcinoma of the colon) were obtained from the American Type Culture Collection (ATCC) and kindly provided by Brigitte Marian, Institute of Cancer Research, Medical University of Vienna, Austria. CH1 cells originate from an ascites sample of a patient with a papillary cystadenocarcinoma of the ovary and were kindly provided by Lloyd R. Kelland, CRC Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, UK. Cells were grown as adherent monolayer cultures in Minimal Essential Medium (MEM), supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, and 2 mM L-glutamine (all purchased from Gibco/ Invitrogen). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cellular Uptake. SW480 cells were incubated with the ruthenium complexes dissolved in complete culture medium at concentrations ranging from 0.6 to 5 µM for 2 h at 37 °C. The cells were then harvested by trypsinization, and cell suspensions were centrifuged at 1000 rpm for 2 min. The cells were then resuspended in 5 mL of ice-cold PBS (containing 11.5 g of Na2HPO4‚2H2O, 2 g of KH2PO4, 2 g of KCl, and 80 g of NaCl per liter of water), counted with a hemocytometer, and centrifuged at 1000 rpm for 2 min. The cell pellet was dissolved in 100 µL of a 20% aqueous solution of tetramethylammonium hydroxide (TMAH), lyophilized, redissolved in 0.2 M HCl, and stored at 4 °C. Ruthenium contents of the cells were determined by graphite furnace atomic absorption spectroscopy (Perkin-Elmer Zeeman 5100). Furnace parameters were as follows: pretreatment temperature, 1400 °C; atomization temperature, 2500 °C; wavelength, 349.9 nm. Each experiment was performed at least in triplicate. In Vitro Antitumor Assay. In vitro antitumor potency was determined by means of a colorimetric microculture assay (MTT assay). SW480 and CH1 cells were harvested from exponentially growing cultures by trypsinization and seeded into 96-well microculture plates at densities of 1.5 × 104 cells/mL (3.0 × 103 cells/well) and 1.0 × 104 cells/mL (2.0 × 103 cells/well), respectively. After preincubation for 24 h, cells were exposed for 96 h to the test compounds dissolved and serially diluted in complete culture medium. Due to their low aqueous solubility, compounds 4-6 were dissolved in DMSO first and then immediately diluted in complete culture medium to obtain DMSO concentrations of 0.33% or lower. All drug solutions were prepared shortly before use in order to minimize premature hydrolysis. After drug exposure, solutions were

Experimental Section General Synthesis and Characterization. All solvents and reagents were obtained from commercial suppliers and used without further purification. The following compounds were prepared according to previously reported procedures: 1, trisindazolium [hexachlororuthenate(III)], (Hind)3[RuIIICl6];43 2, bisindazolium [pentachloro(1H-indazole)ruthenate(III)], (Hind)2[RuIIICl5(ind)];43 3, indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], (Hind) trans-[RuIIICl4(ind)2];43 and 4, mer-trichlorotris(1H-indazole)ruthenium(III), mer-[RuIIICl3(ind)3].35 Elemental analyses were performed for C, H, N and Cl (Microanalytical Laboratory, Institute of Physical Chemistry, University of Vienna, Austria) and were within (0.4% of the theoretical values. 1H NMR spectra were recorded on a Bruker DPX 400 spectrometer at 298 K. Chemical shifts for 1H were referenced to residual 1H present in deuterated dimethyl sulfoxide. Electrospray ionization mass spectrometry was carried out with a Bruker Esquire3000 instrument (Bruker Daltonics, Bremen, Germany). The given m/z values, originating from the most intense isotopes, were obtained by the mass linearization procedure. Expected and experimental isotope distributions were compared. UV/vis spectra were recorded on a Perkin-Elmer Lambda 20 UV/vis spectrometer. Synthesis of trans-[Dichlorotetra(1H-indazole)ruthenium(III)] Chloride, trans-[RuIIICl2(ind)4]Cl (5). To a solution of trans-[RuIICl2(ind)4] (6) (0.20 g, 0.3 mmol) and indazole (0.06 g, 0.5 mmol) in methanol (400 mL) in the presence of 1 M HCl (20 mL) was added a 30% aqueous solution of hydrogen peroxide (20 mL). The mixture was stirred for 10 min at room temperature. The color of the solution changed from light-green to dark-red. The solution was filtered through filter paper and the solvent was removed by evaporation in a vacuum until the formation of the black precipitate was observed. The product was filtered off, washed with water (100 mL), and dried in a stream of argon at 110 °C. Yield: 0.18 g (85%). Anal. (C28H24N8Cl3Ru): C, H, N, Cl. ESI-MS(+ve): m/z 644, [RuCl2(ind)4]+. UV/vis (CH3OH), λmax, nm (, M-1 cm-1): 452 (2700), 389 (5750), 274 (30 780), 215 (56 130). 1H NMR (400.13 MHz, DMSO-d6): δ ) -22.0 to -29.5 (s, very broad), -0.9 to -4.4 (s, very broad), -2.58 (s, 4H), 0.22 (s, 4H), 4.67 (s, 4H), 5.59 (s, 4H). Synthesis of trans-[Dichlorotetra(1H-indazole)ruthenium(II)], trans-[RuIICl2(ind)4] (6). A mixture of (Hind) trans-[RuIIICl4(ind)2] (3) (0.7 g, 1.2 mmol) and indazole (0.2 g, 1.7 mmol) in ethanol/water 70:30 (140 mL) was heated under reflux for 5 h. The light-green precipitate formed was separated by filtration from the hot solution, washed with water, and dried in a vacuum. Yield: 0.31 g (61%). Anal. (C28H24N8Cl2Ru): C, H, N, Cl. ESI-MS(+ve): m/z 644, [RuCl2(ind)4]+. UV/vis (CH3OH), λmax, nm (, M-1 cm-1): 366 (21 900), 290 (17 660), 218 (25 230). 1H NMR (400.13 MHz, DMSO-d6): δ ) 7.05 (t, J ) 7.5 Hz, 4H), 7.20 (t, J ) 7.7 Hz, 4H), 7.58 (d, J ) 8.4 Hz, 4H), 7.64 (d, J ) 8.1 Hz, 4H), 8.53 (s, 4H), 12.53 (s, 4H). X-ray Crystal Structure Analysis. Single crystals of 5‚ 2CH3OH suitable for an X-ray diffraction study were obtained from methanol and diethyl ether using the vapor phase diffusion technique, whereas those of 6‚2(CH3)2CO were grown from acetone at room temperature. 5‚2CH3OH: C30H32N8Cl3O2Ru, Fw 744.06, red-brown plate, 0.23 × 0.11 × 0.04 mm3, monoclinic, Cc (no. 9), a ) 17.681(4) Å, b ) 11.082(2) Å, c ) 16.738(3) Å, β ) 102.46(3)°, V ) 3202.5(11) Å3, Z ) 4, Dx ) 1.543 g/cm3, µ ) 7.82 cm-1. 6‚2(CH3)2CO: C34H36N8Cl2O2Ru, Fw 760.68, light-green plate, 0.26 × 0.23 × 0.09 mm3, triclinic, P-1 (no. 2), a ) 10.891(2) Å, b ) 13.279(3) Å, c ) 14.399(3) Å, R ) 65.66(3), β ) 69.04(3), γ ) 68.36(3)°, V ) 1710.7(6) Å3, Z ) 2, Dx ) 1.477 g/cm3, µ ) 6.58 cm-1; X-ray diffraction measurements were performed on a Nonius Kappa CCD diffractometer (λ ) 0.71073 Å) at 120 K. Single crystals were positioned at 30 mm from the detector,

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replaced by mixtures of complete RPMI 1640 culture medium and aqueous MTT solution (5 mg/mL) (MTT ) 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; purchased from Fluka). After incubation for 4 h, the medium/MTT mixtures were removed, and the precipitated formazan crystals formed by mitochondrial dehydrogenases of living cells were dissolved in DMSO. Optical densities at 550 nm were measured with a microplate reader (Tecan Spectra Classic), and the quantity of living cells was expressed in terms of T/C values by comparison to untreated controls. To rule out solvent effects, additional controls received the highest DMSO concentration employed. Evaluation is based on means from at least three independent experiments, each comprising six microcultures per concentration level.

Acknowledgment. The authors are indebted to the FWF (Austrian Science Fund) and COST (European Cooperation in the Field of Scientific and Technical Research) for financial support. We also thank Prof. Gerald Giester for X-ray data collection as well as Prof. Armando J. L. Pombeiro and Dr. Gu¨nter Trettenhahn, both for providing electrochemical equipment and for valuable discussions. Supporting Information Available: X-ray crystallographic files in CIF format for 5‚2CH3OH and 6‚2(CH3)2CO and details of the crystal structures. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Keppler, B. K.; Henn, M.; Juhl, U. M.; Berger, M. R.; Niebl, R.; Wagner, F. E. New Ruthenium Complexes for the Treatment of Cancer. Prog. Clin. Biochem. Med. 1989, 10, 41-69. (2) Keppler, B. K.; Lipponer, K.-G.; Stenzel, B.; Kratz, F. New Tumor-Inhibiting Ruthenium Complexes. In Metal Complexes in Cancer Chemotherapy; Keppler, B. K., Ed.; VCH: Weinheim, 1993; pp 187-220. (3) Berger, M. R.; Garzon, F. T.; Keppler, B. K.; Schma¨hl, D. Efficacy of New Ruthenium Complexes Against Chemically Induced Autochthonous Colorectal Carcinoma in Rats. Anticancer Res. 1989, 9, 761-766. (4) Fru¨hauf, S.; Zeller, W. J. New Platinum, Titanium, and Ruthenium Complexes with Different Patterns of DNA Damage in Rat Ovarian Tumor Cells. Cancer Res. 1991, 51, 2943-2948. (5) Malina, J.; Novakova, O.; Keppler, B. K.; Alessio, E.; Brabec, V. Biophysical Analysis of Natural, Double-Helical DNA Modified by Anticancer Heterocyclic Complexes of Ruthenium(III) in CellFree Media. J. Biol. Inorg. Chem. 2001, 6, 435-445. (6) Ku¨ng, A.; Pieper, T.; Keppler, B. K. Investigations into the Interaction Between Tumor-Inhibiting Ruthenium(III) Complexes and Nucleotides by Capillary Electrophoresis. J. Chromatogr. B 2001, 759, 81-89. (7) Ni Dhubhghaill, O. M.; Hagen, W. R.; Keppler, B. K.; Lipponer, K.-G.; Sadler, P. J. Aquation of the Anticancer Complex trans[RuCl4(Him)2]- (Him ) Imidazole). J. Chem. Soc., Dalton Trans. 1994, 1994, 3305-3310. (8) Chatlas, J.; van Eldik, R.; Keppler, B. K. Spontaneous Aquation Reactions of a Promising Tumor Inhibitor trans-ImidazoliumTetrachlorobis(imidazole)ruthenium(III), trans-Him[RuCl4(Im)2]. Inorg. Chim. Acta 1995, 233, 59-63. (9) Anderson C.; Beauchamp, A. L. 1H NMR Study of the Solvolysis of the Paramagnetic Tetrachloro-Bis(imidazole)ruthenium(III) Anion in Water, Methanol, and Dimethyl Sulfoxide. Can J. Chem. 1995, 73, 471-482. (10) Kapitza, S.; Pongratz, M.; Jakupec, M. A.; Heffeter, P.; Berger, W.; Lackinger, L.; Keppler, B. K.; Marian, B. Heterocyclic Complexes of Ruthenium(III) Induce Apoptosis in Colorectal Carcinoma Cells. J. Cancer Res. Clin. Oncol. 2005, 131, 101110. (11) Galeano, A.; Berger, M. R.; Keppler, B. K. Antitumor Activity of Some Ruthenium Derivatives in Human Colon Cancer Cell Lines in Vitro. Arzneim.-Forsch./Drug Res. 1992, 42, 821-824. (12) Kratz, F.; Hartmann, M.; Keppler, B. K.; Messori, L. The Binding Properties of Two Antitumor Ruthenium(III) Complexes to Apotransferrin. J. Biol. Chem. 1994, 269, 2581-2588. (13) Smith, C. A.; Sutherland-Smith, A. J.; Keppler, B. K.; Kratz, B.; Baker, E. N. Binding of Ruthenium(III) Antitumor Drugs to Human Lactoferrin Probed by High-Resolution X-ray Crystallographic Structure Analyses. J. Bioinorg. Chem. 1996, 1, 424431.

Jakupec et al. (14) Pongratz, M.; Schluga, P.; Jakupec, M. A.; Arion, V. B.; Hartinger, C. G.; Allmaier, G.; Keppler, B. K. Transferrin Binding and Transferrin-Mediated Cellular Uptake of the Ruthenium Coordination Compound KP1019, Studied by Means of AAS, ESIMS and CD Spectroscopy. J. Anal. At. Spectrom. 2004, 19, 4651. (15) Kratz, F.; Beyer, U. Serum Proteins as Drug Carriers of Anticancer Agents: A Review. Drug Deliv. 1998, 5, 281-299. (16) Qian, Z. M.; Li, H.; Sun, H.; Ho, K. Targeted Drug Delivery via the Transferrin Receptor-Mediated Endocytosis Pathway. Pharmacol. Rev. 2002, 54, 561-587. (17) Ho¨ckel, M.; Vaupel, P. Tumor Hypoxia: Definitions and Current Clinical, Biologic, and Molecular Aspects. J. Natl. Cancer Inst. 2001, 93, 266-276. (18) Shannon, A. M.; Bouchier-Hayes, D. J.; Condron, C. M.; Toomey, D. Tumour Hypoxia, Chemotherapeutic Resistance and HypoxiaRelated Therapies. Cancer Treat. Rev. 2003, 29, 297-307. (19) Brown, J. M. The Hypoxic Cell: A Target for Selective Cancer Therapy-Eighteenth Bruce F. Cain Memorial Award Lecture. Cancer Res. 1999, 59, 5863-5870. (20) Wardman, P. Electron Transfer and Oxidative Stress as Key Factors in the Design of Drugs Selectively Active in Hypoxia. Curr. Med. Chem. 2001, 8, 739-761. (21) Clarke, M. J.; Bitler, S.; Rennert, D.; Buchbinder, M.; Kelman, A. D. Reduction and Subsequent Binding of Ruthenium Ions Catalyzed by Subcellular Components. J. Inorg. Biochem. 1980, 12, 79-87. (22) Clarke, M. J. Ruthenium Chemistry Pertaining to the Design of Anticancer Agents. Prog. Clin. Biochem. Med. 1989, 10, 2539. (23) Clarke, M. J. Ruthenium Metallopharmaceuticals. Coord. Chem. Rev. 2003, 236, 209-233. (24) Mestroni, G.; Alessio, E.; Sava, G.; Pacor, S.; Coluccia, M.; Boccarelli, A. Water-Soluble Ruthenium(III)-Dimethylsulfoxide Complexes: Chemical Behavior and Pharmaceutical Properties. Met.-Based Drugs 1994, 1, 41-63. (25) Sava, G.; Bergamo, A.; Zorzet, S.; Gava, B.; Casarsa, C.; Cocchietto, M.; Furlani, A.; Scarcia, V.; Serli, B.; Iengo, E.; Alessio, E.; Mestroni, G. Influence of Chemical Stability on the Activity of the Antimetastasis Ruthenium Compound NAMI-A. Eur. J. Cancer 2002, 38, 427-435. (26) Griffiths, J. R. Are Cancer Cells Acidic? Br. J. Cancer 1991, 64, 425-427. (27) Gerweck, L. E. Tumor pH: Implications for Treatment and Novel Drug Design. Semin. Radiat. Oncol. 1998, 8, 176-182. (28) Poddar, R. K.; Agarwala, U. Triphenylarsine and Phosphine Complexes of Ruthenium(II) and Their Reactions with Pyridine. J. Inorg. Nucl. Chem. 1973, 35, 567-575. (29) Poddar, R. K.; Khullar, I. P.; Agarwala, U. Ruthenium(III) Complexes with Triphenylarsine. Inorg. Nucl. Chem. Lett. 1974, 10, 221-227. (30) Ruiz-Ramı´rez, L.; Stephenson, T. A.; Switkes, E. S. New Ruthenium(III) and Ruthenium(II) Complexes Containing Triphenylarsine and -phosphine and Other Ligands. J. Chem. Soc., Dalton Trans. 1973, 1973, 1770-1982. (31) El-Hendawy, A. M.; Griffith, W. P.; Taha, F. I.; Moussa, M. N. Studies on Transition-Metal Oxo and Nitrido Complexes. Part 10. New Oxo-Ruthenium and Oxo-Osmium Pyridine Complexes, and Use of the Former as Catalysts for Oxidation of Alcohols. J. Chem. Soc., Dalton Trans. 1989, 1989, 901-906. (32) Al-Zamil, N. S.; Evans, E. H. M., Gillard, R. D.; James, D. W.; Jenkins, T. E.; Lancahire, R. J.; Williams, P. A. Adducts of Coordination Compounds-12. New Hydrogen Dinitrates and Their Structures. Polyhedron 1982, 1, 525-534. (33) Raichart, D. W.; Taube, H. Synthesis of cis-Dihalotetrapyridineruthenium(II) Complexes (Halide ) Chloride, Bromide, Iodide) and Verification of Their cis Stereochemistry. Inorg. Chem. 1972, 11, 999-1002. (34) Anderson, C.; Beauchamp, A. L. Reactions of the Tetrachlorobis(imidazole)ruthenium(III) and Pentachloro(imidazole)ruthenium(III) Anions with Imidazole and N6,N6-Dimethyladenine. Inorg. Chem. 1995, 34, 6065-6073. (35) Pieper, T.; Sommer, M.; Galanski, M.; Keppler, B. K.; Giester, G. [RuCl3ind3] and [RuCl2ind4]: Two New Ruthenium Complexes Derived from the Tumor-Inhibiting RuIII Compound Hind (OC6-11)-[RuCl4ind2] (ind ) indazole). Z. Anorg. Allg. Chem. 2001, 627, 261-265. (36) Reisner, E.; Arion, V. B.; Guedes da Silva, F. M. C.; Lichtenecker, R.; Eichinger, A.; Keppler, B. K.; Kukushkin, V. Yu.; Pombeiro, A. J. L. Tuning of Redox Potentials for the Design of Ruthenium Anticancer Drugs-An Electrochemical Study of [transRuCl4L(DMSO)]- and [trans-RuCl4L2]- Complexes, where L ) imidazole, 1,2,4-triazole, indazole. Inorg. Chem. 2004, 43, 70837093.

Redox-Active Antineoplastic Ruthenium Complexes (37) Arion, V. B.; Reisner, E.; Fremuth, M.; Jakupec, M. A.; Keppler, B. K.; Kukushkin, V. Yu.; Pombeiro, A. J. L. Synthesis, X-ray Diffraction Structures, Spectroscopic Properties, and in Vitro Antitumor Activity of Isomeric (1H-1,2,4-Triazole)Ru(III) Complexes. Inorg. Chem. 2003, 42, 6024-6031. (38) Serli, B.; Zangrando, E.; Iengo, E.; Mestroni, G.; Yellowlees, L.; Alessio, E. Synthesis and Structural, Spectroscopic, and Electrochemical Characterization of New Ruthenium Dimethyl Sulfoxide Nitrosyls. Inorg. Chem. 2002, 41, 4033-4043. (39) Davis, J. A.; Hockensmith, C. M.; Kukushkin, V. Yu.; Kukushkin, Yu. N. Synthetic Coordination Chemistry: Principles and Practice; World Scientific: Singapore, 1996. (40) Duff, C. M.; Heath, G. A. From [RuX6] to [Ru(RCN)6]: Synthesis of Mixed Halide-Nitrile Complexes of Ruthenium, and Their Spectroelectrochemical Characterization in Multiple Oxidation States. J. Chem. Soc., Dalton Trans. 1991, 1991, 24012411. (41) Lever, A. B. P. Electrochemical Parametrization of Metal Complex Redox Potentials, Using the Ruthenium(III)/Ruthenium(II) Couple to Generate a Ligand Electrochemical Series. Inorg. Chem. 1990, 29, 1271-1285. (42) Keppler, B. K. unpublished results.

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2837 (43) Lipponer, K.-G.; Vogel, E.; Keppler, B. K. Synthesis, Characterization, and Solution Chemistry of trans-Indazoliumtetrachlorobis(indazole)ruthenate(III), a New Anticancer Ruthenium Complex. IR, UV, NMR, HPLC Investigations and Antitumor Activity. Crystal Structures of trans-1-Methyl-indazoliumtetrachlorobis-(1-methylindazole)ruthenate(III) and its Hydrolysis Product trans-Monoaquatrichlorobis-(1-methylindazole)-ruthenate(III). Met.-Based Drugs 1996, 3, 243-260. (44) Sheldrick, G. M. SHELXS-97, Program for Crystal Structure Solution; University Go¨ttingen: Go¨ttingen, 1997. (45) Sheldrick, G. M. SHELXL-97, Program for Crystal Structure Refinement; University Go¨ttingen: Go¨ttingen, 1997. (46) Johnson, C. K. Report ORNL-5138; Oak Ridge National Laboratory: Oak Ridge, 1976. (47) International Tables for X-ray Crystallography; Kluwer Academic Press: Dordrecht, 1992; Vol. C, Tables 4.2.6.8 and 6.1.1.4. (48) Barette, W. C.; Johnson, H. W.; Sawyer, D. T. Voltammetric Evaluation of the Effective Acidities (pKa′) for Broensted Acids in Aprotic Solvents. Anal. Chem. 1984, 56, 1890-1898.

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