Activation by Oxidation: Ferrocene-Functionalized ... - ACS Publications

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/IC

Activation by Oxidation: Ferrocene-Functionalized Ru(II)-Arene Complexes with Anticancer, Antibacterial, and Antioxidant Properties Changhua Mu,† Kathleen E. Prosser, Shane Harrypersad, Gregory A. MacNeil, Rikesh Panchmatia, John R. Thompson, Soumalya Sinha, Jeffrey J. Warren, and Charles J. Walsby* Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby V5A 1S6, British Columbia, Canada

Inorg. Chem. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 11/30/18. For personal use only.

S Supporting Information *

ABSTRACT: Organometallic Ru(II)-cymene complexes linked to ferrocene (Fc) via nitrogen heterocycles have been synthesized and studied as cytotoxic agents. These compounds are analogues of Ru(II)-arene piano-stool anticancer complexes such as RAPTA-C. The Ru center was coordinated by pyridine, imidazole, and piperidine with 0-, 1-, or 2-carbon bridges to Fc to give six bimetallic, dinuclear compounds, and the properties of these complexes were compared with their non-Fc-functionalized parent compounds. Crystal structures for five of the compounds, their Ru-cymene parent compounds, and an unusual trinuclear compound were determined. Cyclic voltammetry was used to determine the formal MIII/II potentials of each metal center of the Rucymene-Fc complexes, with distinct one-electron waves observed in each case. The Fc-functionalized complexes were found to exhibit good cytotoxicity against HT29 human colon adenocarcinoma cells, whereas the parent compounds were inactive. Similarly, antibacterial activity from the Ru-cymene-Fc compounds was observed against Bacillus subtilis, but not from the unfunctionalized complexes. In both cases, the IC50 values correlated quantitatively with the Fc+/0 reduction potentials. This is consistent with more facile oxidation to give ferrocenium, and subsequent generation of toxic reactive oxygen species, leading to greater cytotoxicity. The antioxidant properties of the complexes were quantified by a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. EC50 values indicate that linking of the Ru and Fc centers promotes antioxidant activity.

■

INTRODUCTION

To date, studies of Ru-arene anticancer agents have focused heavily on two general types of compounds. The first of these, developed by Sadler and co-workers, are complexes of the type [Ru(η6-arene)(L)(halide)]+, where L is typically a bidentate nitrogen and/or oxygen donor ligand, and the halide is usually chloride.2b,4 Many of these types of compounds, such as [RuCl(η6-p-cymene)(ethylenediamine)]+, (RuCl(Cym)(En)+, Figure 1),5 show strong cytotoxic activity in vitro.4 Another important family of Ru-arene complexes developed by Dyson and co-workers has the general formula [RuCl2(η6-p-cymene) (pta)], where pta = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane.6 These are known as “RAPTA” complexes, such as RAPTA-C (Figure 1),6b and often exhibit modest cytotoxicity in vitro but have demonstrated promising antimetastatic activity.6b,7 The pta ligand of RAPTA compounds promotes aqueous solubility and may also influence selectivity.6a Modification of pta8 or its replacement with other simple phosphorus9 or nitrogen donor ligands10 generally influences the activity of

The ongoing successes of Ru(III) complexes including NAMIA, KP1019, and NKP-1339 in clinical trials demonstrate that such compounds are legitimate candidates for future chemotherapeutics.1 While these types of octahedral Ru(III) coordination complexes continue to undergo development, organometallic Ru-arene complexes are becoming increasingly predominant in the ruthenium anticancer literature.2 Ru pianostool complexes like those shown in Figure 1 are a particular focus of progress in this area, and numerous derivatives of these compounds have been described.3

Received: September 7, 2018

Figure 1. Archetypal Ru-arene anticancer complexes. © XXXX American Chemical Society

A

DOI: 10.1021/acs.inorgchem.8b02542 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry these complexes only modestly, but the use of this coordination site to append functional molecules has proven successful in some cases.2a,11 For example, coordination of ethacrynic acid, via an imidazole linker, has produced an effective inhibitor of glutathione-S-transferase.12 The resulting complex displays in vitro cytotoxic activity that is among the highest reported for a ruthenium complex.13 The pharmaceutical potential of ferrocene (Fc) compounds has been demonstrated in a variety of applications, including as chemotherapeutics. Well-established synthetic methods for the functionalization of ferrocene enabled the development of an array of compounds with anticancer properties, including: (1) ferrocenium salts, (2) ferrocene conjugates with biologically active molecules, and (3) compounds containing both ferrocenyl groups and other transition metals.14 The role of the ferrocene moiety in chemotherapeutic candidates has been linked to electrochemical activation, where oxidation to generate ferrocenium (Fc+) species can promote the formation of reactive oxygen species (ROS).15 This process likely involves a Haber−Weiss-like cycle ending in the Fenton reaction.15b Notable examples of ferrocene chemotherapeutic candidates include analogues of the breast cancer chemotherapeutic tamoxifen (ferrocifens), which demonstrate enhanced activity against estrogen receptor negative (ER−) tumors.16 Several examples of ferrocene-functionalized ruthenium complexes have also been reported, in some cases showing promising activity in vitro.17 This includes ferrocene-functionalized NAMI-A analogues that exhibit both cytotoxic and antimetastatic activity.18 Furthermore, RAPTA derivatives where pta is replaced by pyridine- or imidazole-based ligands bridged to ferrocene by amide or by ester containing linkers have been described.19 These compounds exhibit moderate to low cytotoxic activity in vitro. In contrast, RAPTA-type complexes with phosphinoferrocene ligands functionalized with amino acids in place of pta show good activity against human ovarian cancer cells. 1 7c Analogues of compounds such as RuCl(Cym)(En)+ in which the ferrocene moiety was attached via a chelating 2,2′-bipyridyl linker have also been reported, but the activity of these compounds has not yet been evaluated.20 In addition to cancer treatment, the pharmacological potential of metal complexes has been investigated extensively in a variety of other applications.21 The current demand for new antimicrobials that target drug-resistant bacterial strains is one such area of development.22 Ruthenium complexes have shown some potential in this area, with examples of both Ru− polypyridyl complexes and RAPTA-type complexes showing promising antibacterial activity.23 Similarly, the application of ferrocene compounds has shown the potential to produce new antibiotics or enhance the properties of existing treatments.24 Notable examples include ferrocenyl penicillin and cephalosporin derivatives, which exhibit activity against drugresistant bacteria with low toxicity in vivo.25 However, development in this area has generally received less attention than Fc derivatives as chemotherapeutics, antimalarials, and biomolecule conjugates.14a,26 In this work, we report a series of new RAPTA derivatives with pta replaced by pyridine, imidazole, or piperidine ligands linked either directly to ferrocene or via alkane or alkene bridges (Figure 2). In each instance, we compare their in vitro cytotoxicity, antibacterial properties, and antioxidant activities with their unfunctionalized parent compounds to assess the

Figure 2. Synthetic route for Ru-cymene-Fc complexes and parent complexes from the dinuclear Ru precursor and N-heterocyclic donor ligands used in this study.

role of both the ruthenium and ferrocene centers in their observed activity. These studies demonstrate the importance of tuning the Fc+/0 reduction potentials (modulating the oxidation of the Fc moiety) for enhancing biological activity and suggest that both metal centers influence antioxidant properties.

■

RESULTS AND DISCUSSION Syntheses and Structures. The Ru-cymene parent complexes and their Fc-functionalized Ru-cymene (Rucymene-Fc) derivatives were synthesized via a two-step procedure. In the first step, the dichloro(η6-p-cymene)ruthenium(II) dimer precursor [RuCl2(η6-p-cymene)]2 was produced according to the literature.27 In the following step (Figure 2), 2 equiv of ligand was reacted with the dimeric Ru(II) precursor to give the Ru-cymene complexes. The Fcfunctionalized ligands 4-ferrocenylpyridine (4-FcPyr), 3ferrocenylpyridine (3-FcPyr), and 4-ferrocenyl(ethyl)pyridine (4-FcEtPyr) were synthesized using our reported protocols,18 and (E)-4-(2-ferrocenylvinyl)pyridine (4-FcVinylPyr)18,28 and 1-ferrocenyl(methyl)imidazole (1-FcMeIm)29 were prepared according to the literature, with some modifications (see Supporting Information). The synthesis of 4-ferrocenylethylpiperidine (4-FcEtPip) is reported for the first time in this work. 4-FcEtPip was identified as a co-product of the synthesis of 4FcEtPyr from the hydrogenation of 4-FcVinylPyr with H2/Pd. It was found that under the same conditions, a 4 h reaction time resulted in 4-FcEtPyr as the major product, whereas a 24 h reaction led to full saturation of both the pyridyl and vinylic groups, giving predominantly 4-FcEtPip. In both cases, the Cp rings of Fc were unaffected, as shown by NMR and X-ray crystallography (see below, Supporting Information, and B


Article

Inorganic Chemistry previous work18). The non-Fc functionalized Ru-cymene parent complexes RuCl2(η6-p-cymene)(pyridine) (Ru-Pyr) and RuCl2(η6-p-cymene)(1-methylimidazole) (Ru-1-MeIm) were prepared according to the literature.10 RuCl2(η6-pcymene)(piperidine) (Ru-Pip) was reported previously as a polymerization catalyst, but its synthesis and characterization were not fully described.30 Details of parent compound syntheses are given in Supporting Information. Single crystals suitable for X-ray diffraction studies were obtained for all Ru-cymene-Fc compounds, except for Ru-4FcEtPyr. The structures determined by X-ray crystallography are shown in Figure 3, with details of crystal data, refinement,

Å, respectively, which are consistent with those of single C−C bonds,31 and the C6−C7 bond length is 1.300 Å, a typical value for a C−C double bond.31 These bond lengths are also commensurate with the values reported for the crystal structure of yjr 4-FcVinylPyr ligand itself (1.48, 1.43, and 1.35 Å).28 The X-ray structure of Ru-4-FcEtPip displays an ethylic linker and a chair conformation for the piperidinyl ring (Figure 3). The bond lengths of the linker, C3−C6, C6−C7, and C7−C8, were found to be 1.525, 1.531, and 1.500 Å, respectively, which are consistent with those of single C−C bonds.31 Furthermore, the average C−C and C−N bond lengths (1.543 and 1.472 Å, respectively) of the coordinated piperidine of Ru-4-FcEtPip correlate well with those of the parent Ru-Pip complex and piperidine itself.32 Finally, the Fe− C bond lengths of all the Fc groups of the Ru-cymene-Fc complexes agree well with those of ferrocene,33 confirming that this series of bimetallic, dinuclear complexes are well described as containing a piano stool-type Ru-cymene center linked to ferrocene. An additional compound, [RuCl(η 6 -p-cymene)(1FcMeIm)2][RuCl3(η6-p-cymene)], was produced unexpectedly during an attempt to crystallize Ru-1-FcMeIm in a mixture of acetone and diethyl ether (1:1, v/v) at −18 °C over two weeks (Figure 4). Under these conditions, Ru-1-FcMeIm is converted to the salt, possibly through an intermolecular ligand-exchange process between its 1-FcMeIm and Cl− ligands. When the crystallization of Ru-1-FcMeIm was conducted in a less polar solvent mixture of CH2Cl2 and hexanes (1:1, v/v), the crystals obtained by evaporating the solvent slowly at room temperature over the course of 3 days were identified as the Ru-1-FcMeIm complex. The crystal structure of [RuCl(η6-p-cymene)(1-FcMeIm)2][RuCl3(η6-p-cymene)] (Figure 4) shows the result of the ligand-rearrangement reaction, which converts two equivalents of Ru-1-FcMeIm to the ionic complex. This salt contains the rarely reported anionic species [RuCl3(η6-p-cymene)]−,10,34 which charge-compensates the [RuCl(η 6 -p-cymene)(1FcMeIm)2]+ cation. Only a limited number of similar salts have been reported previously, in one case under synthetic conditions similar to those shown here, but in other instances using different solvent conditions and ligand ratios.10 The Ru− N bond lengths to the two 1-FcMeIm ligands of [RuCl(η6-pcymene)(1-FcMeIm)2]+, 2.109 and 2.112 Å, are close to that of Ru-1-FcMeIm (2.122 Å), and the Cl bond lengths are also essentially unchanged, demonstrating that the ligand-exchange process does not otherwise change the properties of the Ru(II) cymene centers. To confirm that 1-FcMeIm ligand exchange was not a pathway for decomposition under physiological conditions, 1H NMR were measured after incubation in phosphate-buffered saline (PBS, 10 mM PO43−, 137 mM NaCl, 2.7 mM KCl, pH 7.4) at room temperature for up to 90 h. No evidence of free 1-FcMeIm was observed; nor was free cymene detected. Solution Behavior. While ruthenium complexes are often substitutionally inert,35 ligand-exchange processes under physiological conditions nonetheless mean that Ru anticancer candidates are frequently prodrugs that release ligands to produce active species.36 Ru-cymene complexes with chelating donor ligands, such as RuCl(Cym)(En)+ (Figure 1), undergo exchange of their chloride ligand to generate aqua complexes that are activated toward DNA interactions.2b,37 However, chloride ligand exchange is considered less important to the activity of RAPTA-type complexes.38

Figure 3. Crystal structures of Fc-functionalized Ru-cymene complexes and parent compounds. Structures are drawn at the 30% probability level.

and metrical parameters provided in Tables S1 and S2 in Supporting Information. The Ru(II) centers of each complex exhibit a “piano-stool” geometry with Ru−Cl bond lengths within a small range (2.387−2.431 Å) and similar Ru−N bond lengths for the heterocyclic N-donor ligands (2.092−2.170 Å). Furthermore, comparison of bond lengths demonstrates that the addition of ferrocenyl groups to the parent complexes does not impact significantly on the geometry of the Ru-cymene centers. The crystal structure of Ru-4-FcVinylPyr confirms the presence of the vinylic linker between ferrocene and pyridine. The bond lengths of C3−C6 and C7−C8 are 1.447 and 1.455 C


Article

Inorganic Chemistry

Figure 4. Conversion of Ru-1-FcMeIm to the ionic complex [RuCl(η6-p-cymene)(1-FcMeIm)2][RuCl3(η6-p-cymene)] and crystal structure of the product drawn at the 30% probability level.

at physiological pH for these types of complexes.40 Establishment of equilibrium concentrations was relatively slow for each of the complexes, taking 0.5−2 h, consistent with the overall kinetic inertness of Ru(II) complexes.35 These UV−vis studies were performed in standard PBS with [Cl−] = 139.7 mM, which is near the chloride concentrations in the Dulbecco’s modified Eagle’s medium (DMEM) cellculture medium (125 mM) used in the cancer-cell cytotoxicity studies and the LB Miller broth (171 mM) used for the antibacterial experiments. Consequently, the data indicate that the pyridine- and imidazole-based complexes will be found primarily in their original forms, while the piperidine-based complexes will have significant contributions from monoaqua species in our studies of their anticancer and antibacterial activity. Electrochemical Properties. Redox processes are an important factor in the activity of many metal-containing therapeutics.41 For example, it has been demonstrated that the cytotoxicity of some Fc-functionalized compounds is related to the Fc+/0 reduction potential of the ferrocenyl moieties, with lower E° values resulting in higher cytotoxicity.42 These observations are consistent with the easier-to-oxidize complexes promoting the formation of ferrocenium species, which go on to instigate reactions leading to generation of ROS.15 Therefore, manipulation of the redox thermodynamics of Fc groups conjugated to other metal centers appears to be a promising strategy for the activation of metal-based anticancer agents. The electrochemical properties of all the Ru-cymene-Fc complexes and their parent complexes were studied by cyclic voltammetry (CV) using a glassy-carbon working electrode with 1 mM solutions of the compounds in CH2Cl2 containing the 0.1 M tetrabutylammonium hexafluorophosphate ([NBu4][PF6]) electrolyte. CH2Cl2 was a suitable solvent for performing the experiment because it (1) provides better solubility to the Ru-cymene-Fc complexes, (2) prevents the ligand-exchange of the complexes with solvent molecules, which would be observed in aqueous solutions (see above), and (3) displays a better reversibility for RuIII/II redox couples as compared to PBS. Because of the signal overlap of ferrocene with the ferrocenyl ligands of the complexes, the Fc+/0 couple was used as an external reference. The cyclic voltammograms of the Ru-cymene-Fc complexes show one-electron quasireversible waves assigned to the FeIII/II couple of the Fc group (Fc+/0) and from the RuIII/II couple of the Ru-cymene centers (Figures 6 and S40−S44). Similar electrochemical quasireversibility of the RuIII/II redox process has also been reported from related Ru complexes.19b The

The ligand-exchange reactions of each of the Ru-cymene-Fc compounds and their parent complexes were studied in PBS at 37 °C using time-dependent UV−vis spectroscopy. Under these conditions, the pyridine- and imidazole-based complexes show small time-dependent spectral changes, with distinct isosbestic points (Figures 5, S11−S16). This is consistent with

Figure 5. Time-dependent UV−vis spectra of the solution behavior of Ru-4-FcPyr (200 μM) in PBS with 1% dimethyl sulfoxide (DMSO, v/v) at 37 °C recorded at 2 min intervals over a period of 20 min. Further incubation, up to 3 h, led to only minor spectral changes.

a minimal amount of ligand exchange and is similar to the solution behavior of RAPTA-C, where elevated concentrations of chloride ions inhibit the exchange of Cl− ligands.39 By contrast, the piperidine-based complexes Ru-Pip and Ru-4FcEtPip (Figures S17 and S18) show more distinct timedependent spectral changes. This corresponds to more extensive release of Cl− due to the stronger σ-donor properties of the piperidine ligands, which results in weakening of Ru−Cl bonds. Additional UV−vis measurements of Ru-4-FcEtPip were made in PBS with double the Cl− concentration (274 mM), which led to slower ligand exchange (Figure S19). This is consistent with inhibition of ligand exchange by the higher Cl− concentration, reflecting that loss of chloride is responsible for the time-dependent changes in the spectra of the Rucymene-Fc complexes. Similar behavior was observed for Ru4-FcPyr (Figure S20), which shows smaller spectral changes over time. These observations, and the NMR studies described above for Ru-1-FcMeIm, indicate the formation of equilibrium concentrations of [RuCl(η6-p-cymene)(H2O)(L)]+, as observed for RAPTA-C.39 In principle, subsequent deprotonation of the coordinated water ligand could rapidly yield [RuCl(OH)(η6-p-cymene)(L)]0; however, previous studies of the benzene analogue of RAPTA-C (RAPTA-B) indicate that the aqua complex is the predominant product of ligand exchange D


Article

Inorganic Chemistry

charge can be transferred via the vinylic linker to Fc, resulting in an increase in E1/2(Fc+/0) (Figure S50). This pathway is more limited in Ru-4-FcEtPyr because of the ethylic linker. Ru-4-FcEtPip shows the lowest value of E1/2(Fc+/0) because of the electron-donating properties of the ethyl piperidine ligand. The RuIII/II potential of Ru-4-FcEtPip is 42 mV lower than that of Ru-Pip, possibly because of a through-space interaction with the Fc group.18 As described above, the cytotoxic activity of the Ru-cymeneFc complexes may arise from the generation of ROS.15 If this reactivity is derived from the Fc components, then the ease that ferrocenium species can be generated is expected to be related to the observed activity; a lower value of E1/2(Fc+/0) should correlate with enhanced Fc+ generation. Cytotoxicity Assays in a Human Tumor Cell Line. The anticancer potential of the Ru-cymene-Fc complexes and Rucymene parent compounds was assessed by in vitro cell viability studies using HT29 human colon adenocarcinoma cells. Because of limited aqueous solubility, the cytotoxic properties of the Fc-ligands alone could not be evaluated. Compounds were incubated with HT29 cells for 72 h to assess their cytotoxic activity at concentrations ranging from 0.6 to 200 μM for the Fc-functionalized complexes and from 100 to 1000 μM for the less active parent complexes. In these studies, all wells contained 1% DMSO, which was also tested as the vehicle control. This concentration of DMSO is tolerated by HT29 cells and allowed for sufficient solubility of all compounds tested. The fraction of cells affected by each compound was imaged using a fluorescent cell-permeable nuclear marker (Hoescht 33342 nucleic acid stain) to determine the total cell count, and a cell impermeable nuclear marker (ethidium homodimer I) to count the number of dead cells. The resulting images were analyzed statistically to give sigmoidal dose−response curves (Figure 7a), which were modeled using a four-parameter logistic model to determine 50% inhibitory concentration (IC50) values (Table 1). All the Ru-cymene-Fc complexes described in this work exhibited good cytotoxic activity against HT29 cells with IC50 values in the range of 20.0−57.7 μM, whereas the parent complexes Ru-Pyr, Ru-1-MeIm, and Ru-Pip were inactive (see Supporting Information Figure S21). This comparison demonstrates that the ferrocenyl moieties play a central role in generating cytotoxicity. The observed activity against the HT29 cell line compares favorably to the benchmark Ru(III)

Figure 6. Cyclic voltammogram (scan rate = 100 mV/s) of Ru-4FcEtPip (1 mM) in CH2Cl2 containing the 0.1 M [NBu4][PF6] electrolyte. Arrow indicates the initial scan direction.

RuIII/II and Fc+/0 reduction potentials (vs Fc+/0) are listed in Table 1. The value of E1/2(RuIII/II) for each Ru-cymene center is modulated by contributions from each of the ligands. The effects of the electron-withdrawing chloro ligands43 and the electron-donating cymene ligand19b are offsetting, and thus the electron density on the Ru-cymene centers is determined by the nature of the Fc-functionalized heterocycles in this work. The effects of the Fc-functionalized ligands give RuIII/II potentials that range from 747 to 879 mV versus Fc+/0. Similarly, the nature of the Fc functionalization influences E1/2(Fc+/0) with values spanning −83 to 173 mV. This effect of the heterocycle-Fc ligand is demonstrated by the observation that Ru-4-FcPyr shows a less positive value for E1/2(RuIII/II) and a more positive value for E1/2(Fc+/0) than Ru-3-FcPyr. Analysis of possible resonance structures (Figure S49) suggests that Ru-4-FcPyr has a contributing structure with a negative charge on the N atom, which increases the electron density on the Ru(II) center, decreasing E1/2(RuIII/II), and a corresponding positive charge adjacent to Fc, leading to an increase in the E1/2(Fc+/0). The electron-donating vinylic and ethylic linkers of Ru-4-FcVinylPyr and Ru-4-FcEtPyr44 lead to less positive potentials for both the Ru and Fc centers than Ru-4-FcPyr. Furthermore, the value of E1/2(Fc+/0) of Ru4-FcVinylPyr is 85 mV higher than that of Ru-4-FcEtPyr because of the influence of the conjugated and nonconjugated linkers, respectively. In the case of Ru-4-FcVinylPyr, positive

Table 1. Properties of Ru-Cymene Complexes: (i) MIII/II Reduction Potentials (E1/2) of Ru-Cymene and Fc Moieties vs Fc+/0 As Determined by CV in CH2Cl2, (ii) IC50 Values for Cytotoxicity against HT29 Colorectal Adenocarcinoma Cells, (iii) IC50 Values for Antibacterial Activity against B. subtilis and E. coli Bacteria, and (iv) Antioxidant Activity in Ethanol in Terms of EC50 Values vs 0.1 mM DPPH E1/2 (mV) vs Fc+/0 compounds Ru-Pyr Ru-3-FcPyr Ru-4-FcPyr Ru-4-FcVinylPyr Ru-4-FcEtPyr Ru-1-MeIm Ru-1-FcMeIm Ru-Pip Ru-4-FcEtPip

III/II

Ru

879 872 835 827 827 771 747 839 797

III/II

Fe

100 173 65 −20 80 −83

cytotoxic IC50 (μM)a

antibacterial IC50 (μM)a

HT29 carcinoma

B. subtilis

E. coli

antioxidant EC50 (μM)b

>700 58 (11) 50 (7) 24 (3) 31 (7) >800 34 (2) >1000 20 (4)

>180 56 (6) 78 (17) 34 (7) 32.3 (0.5) >180 44.9 (0.5) >180 21 (6)

>600 >600 >600 54 (6) >600 >600 429 (15) >600 >600

1087 19 67 3 17 1282 3 14 16

a

Uncertainty values are one standard deviation from the mean. bNo activity observed for Fc ligands alone. E


Article

Inorganic Chemistry

We postulated that the origin of the observed activity is generation of ROS by the appended Fc groups. Our previous studies of Fc-functionalized analogues of the Ru(III) anticancer complex NAMI-A showed that ligands of this type can be oxidized to give Fe(III) species.18 Subsequently, these oxidized species could generate toxic ROS via Fenton and Haber−Weiss reactions.15 Importantly, the CV measurements reveal that the E1/2(Fc+/0) values for all the Ru-cymene-Fc complexes are less than 520 mV versus Fc+/0, which is considered to be the threshold for ferrocenyl oxidation inside cells.42a,48 Thus, we anticipate that cytotoxic ferrocenium species are generated in vitro,15a,42b,49 and that the ease of oxidation would be related to the observed activity; a lower value of E1/2(FeIII/II) would correlate with an enhanced ability to generate Fc+. To test this hypothesis, the IC50 values of the Fc-functionalized complexes against the HT29 cell line were plotted against the potentials of the Fc and Ru centers. As shown in Figure 7b, lower E1/2 values (more easily oxidized) of the Fc centers correlate with lower IC50 values (more active), with a linear trend given by IC50 = E1/2(Fc+/0) × 0.12−30. Although the linear correlation is modest (R2 = 0.6), there is a propensity for the complexes with lower E1/2 values to show better cytotoxicity, consistent with a mechanism involving initial generation of Fc+ that can lead to generation of ROS (Figure 8). By contrast, there is no such correlation between E1/2(RuIII/II) values and their anticancer activity. Figure 7. (a) Dose−response data from cytotoxicity testing of Rucymene-Fc complexes against the HT29 human colon adenocarcinoma cell line and fitted sigmoidal curves. (b) Comparison of cytotoxicity IC50 values, determined from sigmoidal fitting, with Fc+/0 and RuIII/II potentials and linear least-squares fit of correlation for Fc moieties.

compounds KP1019 (IC50 = 20 μM) and NKP-1339 (IC50 = 25 μM).1f Furthermore, comparison with the modest activity of RAPTA-C against HT29 cells (IC50 = 441 μM)45 emphasizes that this type of Ru-arene complex can potentially be activated by replacement of the pta ligand with Fcfunctionalized heterocyclic ligands. Previous studies of RAPTA complexes with pta replaced by structurally related ligands8 or simple nitrogen heterocycles10 demonstrated low activity in each case. However, as shown herein, replacement of pta with functional ligands can generate cytotoxic activity that is on par with clinically relevant Ru complexes. Examples of this strategy include coordination of steroid-functionalized pyridines,46 ethacrynic acid-functionalized imidazole,13 nitric-oxide-donating 4-nitrooxymethylpyridine,47 and phosphinoferrocene amino acid ligands.17c Interestingly, Auzias et al. reported RAPTA-type complexes with pta replaced by pyridine and imidazole ligands bridged to ferrocene via ester and amide linkers.19 However, although these compounds are structurally similar to the complexes reported in this work, they demonstrate relatively low activity with IC50 values of 103−390 μM against A2780 ovarian cancer cells. Because the studies were carried out using a different cell line, comparison of cytotoxicity requires caution. Nonetheless, the contrast in activity of these different classes of Fc-modified Ru complexes may indicate that the potentially cleavable ester and amide groups reduce cytotoxicity. In turn, this suggests that the stable linkers in our compounds are important to their activity, indicating that maintaining the link between the Ru and Fc centers is important to their anticancer properties.

Figure 8. Proposed mechanism for the generation of hydroxyl radicals (ROS) leading to the observed cytotoxic activity by the Ru-cymeneFc complexes.

Antibacterial Activity. The bacterial cell growth inhibition, or death, induced by treatment with current antibiotics is usually associated with inhibition of cellular functions.50 Mechanisms of antibiotic activity involve processes such as inhibition of the synthesis of nucleic acids,51 cell walls,52 and proteins,53 disruption of bacterial membranes,54 and altering of metabolic processes by drug-induced oxidative stresses such as generation of hydroxyl radicals.55 Numerous examples of drugs functionalized with ferrocene for medicinal applications, including antibacterial properties, are reported.16a,25a,49a,56 Ferrocene itself exhibits moderate inhibitory activity against Escherichia coli.57 Furthermore, the ferrocene group provides a hydrophobic component, which increases overall lipophilicity, aiding antibiotic molecules in penetrating through lipophilic bacterial cell membranes.58 F


Article

Inorganic Chemistry The antibacterial activity of the complexes studied in this work was evaluated by a turbidimetric assay. This approach was used to continuously measure the growth of bacteria by relating solution turbidity, evaluated by absorbance at 600 nm, to cell counts during the log phase of growth, the time period during which the number of bacteria increases exponentially.59 Different parameters gleaned from growth curves, such as endabsorbance,60 the slope of the log phase,61 and the area under each growth curve,62 have been used to analyze the effect of antimicrobial agents. However, correlating the area under each growth curve in the log phase to the microbial growth as a function of the concentration of an antimicrobial agent has been demonstrated to provide the most accurate data analysis.63 The inhibitory effect of the Ru-cymene-Fc compounds and their Ru-cymene parent compounds was determined by evaluating the percentage reduction in area under the log phase sections of the bacterial growth curves, as determined by numerical integration. Two strains of bacteria were studied: Bacillus subtilis, which was used as a Grampositive model; and E. coli, which was used as a Gram-negative model. All of the Fc-functionalized Ru-cymene complexes exhibited antibacterial activity against the Gram-positive B. subtilis strain. This correlated with a distinct decrease in turbidity with increasing concentrations of the complexes, as shown in Figure 9a for Ru-4-FcPyr and for the other Ru-cymene-Fc complexes in Supporting Information, Figures S22−S27. The antibacterial activities of the Ru-cymene-Fc complexes were evaluated in terms of IC50 values determined by fitting of a sigmoidal curve to a plot of the area under the log-phase section of the growth curve for each complex against a range of concentrations (Figure 9b). The IC50 values for the inhibition of B. subtilis bacterial growth (Table 1) determined by this method for the Ru-cymene-Fc complexes ranged from 21 to 78 μM, suggesting that the complexes have potential as antibacterial agents. In comparison, RAPTA-C is ineffective against this bacterium,23b as is ferrocene carboxylic acid,64 indicating that the combined effect of the two components generates activity in the Rucymene-Fc compounds. However, it was found that E. coli bacteria (Gram negative) were not sensitive to treatment with the Ru-cymene-Fc complexes. Except for Ru-1-FcMeIm and Ru-4-FcVinylPyr, which exhibited very moderate activities (IC50 = 429 ± 15 μM and IC50 = 514 ± 6, respectively), none of the complexes showed activity at concentrations up to 600 μM (Supporting Information Figures S32−S37). Most importantly, the turbidity assays of the Ru-cymene parent compounds, Ru-1-MeIm, Ru-Pyr, and Ru-Pip, demonstrated no activity against either bacterial strain at concentrations as high as 180 and 600 μM for B. subtilis and E. coli, respectively (Supporting Information Figures S28−S30, S38−S40), demonstrating that the Fc functionalities generate the observed antibacterial properties. The antibacterial properties of the Fcligands alone could not be evaluated under physiological conditions because of limitations in the compounds’ solubility. Similar to the study of the cytotoxic activity against HT29 cells, the IC50 values of the Ru-cymene-Fc complexes against B. subtilis were plotted against the E1/2 values of the Fc and Ru centers (Figure 9c). As shown in Figure 9, there is a strong correlation (R2 = 0.9) between the IC50 values and E1/2(Fc+/0), with a linear trend given by IC50 = E1/2(Fc+/0) × 0.21 + 33, demonstrating that more facile oxidation of the Fc center (lower E1/2 value) corresponds to greater antibacterial activity. No correlation of cytotoxic activity with E1/2(RuIII/II) was

Figure 9. Antibacterial activities of Ru-cymene-Fc complexes against Gram-positive strain B. subtilis. (a) Time-dependent turbidity data for log-phase growth measured by changes in absorbance at 600 nm at 15 min intervals over 2 h for concentrations of Ru-4-FcPyr ranging from 0 to 180 μM. (b) Concentration dependence of cell death determined from turbidity assays of Fc-functionalized complexes, and sigmoidal curve fitting. (c) Comparison of IC50 values, determined from sigmoidal fitting, with Fc+/0 and RuIII/II potentials, and linear leastsquares fit of correlation for Fc moieties.

found, as with the HT29 studies. This suggests that the toxicity in this case may also be attributed to production of ROS following generation of ferrocenium-like species. Previous reports have described the effectiveness of ROS-inducing antibiotics, with direct and indirect pathways leading to reactive Fe species that catalyze these reactions.55a The observed activity in these earlier studies was linked to the formation of hydroxyl radicals, which have been shown to exhibit high antimicrobial efficiency.65 The observation that the Ru-cymene-Fc complexes exhibited more activity against the Gram-positive bacteria B. subtilis than the Gram-negative bacterial E. coli is probably due to the structural differences in the cell walls of these two bacterial strains. The outer membrane of Gram-negative bacteria provides resistance to many antibiotics, and likely also inhibits the transport of the Ru-cymene-Fc complexes into the bacterial cells, consequently reducing their activity.66 G


Article

Inorganic Chemistry Antioxidant Activity. The use of antioxidants for cancer prevention or treatment strategies is controversial.67 ROS can promote tumorigenesis by generating DNA mutations or activating pro-oncogenic signaling pathways.68 Thus, antioxidant supplementation has been proposed for prevention of cancer and to augment chemotherapeutic and radiation treatments.67 On the other hand, elevated levels of ROS are associated with damage to DNA, proteins, and lipids within cells, which can ultimately lead to cell death.68 Consequently, in situ generation of ROS is a well-recognized strategy in chemotherapeutic design.69 Because of altered metabolism, cancer cells tend to have intrinsically higher ROS levels than normal cells, which leads to adaptation through development of increased antioxidant pathways.70 This suggests that antioxidant treatment is not likely to be successful as a cancer treatment strategy, and may actually be detrimental.67 A variety of metal-based antioxidants have been reported71 including ferrocene72 and Ru(II)73 complexes, suggesting that these metal centers could counteract ROS-generating reactions. To test this hypothesis, the antioxidant capabilities of the Ru-cymene-Fc complexes, the ferrocenyl ligands, and the Rucymene parent complexes were evaluated by a 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging assay initially reported by Blois.74 When DPPH accepts a hydrogen atom or an electron from a radical scavenger, there is a resulting solution change from violet to yellow. Consequently, the percentage of DPPH radical scavenged can be monitored by UV−vis measurements of absorbance at 517 nm to give the percentage scavenging activity. The concentration dependence of the of the scavenging activity (Figures S51−S53) was used to calculate EC50 values, which are reported in Table 1. Full details are given in the Supporting Information. DPPH assays show that the Fc-ligands have minimal antioxidant activity (Figure S52), even at a 5:1 ratio with respect to DPPH (0.1 mM). The non-Fc functionalized Rucymene compounds, Ru-Pyr and Ru-1-MeIm, also exhibit little activity, with EC50 values > 1 mM. However, Ru-Pip displays significantly higher antioxidant capacity with EC50 = 14 μM. Greater scavenging activity of Ru-Pip is consistent with a mechanism involving H-atom transfer from the piperidine amine to DPPH (Figure 10). Enhancement of this process for piperidine coordinated to the Ru(II) center reflects a decrease in N−H bond strength upon coordination to Ru(II). Ru-4FcEtPip has a similar EC50 value, also indicating an H-atom transfer from piperidine rather than an Fc-based process.

The Fc-functionalized analogues of Ru-Pyr and Ru-1-MeIm all show significantly higher antioxidant activity than their parent Fc and Ru compounds (Table 1). Here, H-atom transfer is precluded by the lack of an ionizable proton on the ligands, meaning that outer-sphere electron transfer is likely the operative mechanism. Such a mechanism was inferred previously between the Fc moiety of 4-FcVinylPyr and the 4FcEtPyr and a Ru(III) center.18 Although there is no obvious correlation between the EC50 values and the E1/2 values of either metal center (Figure S53), the observed activity demonstrates that both the Ru-cymene and Fc moieties are required for the antioxidant properties of the pyridine and imidazole complexes. Although the antioxidant activity of the Ru-cymene-Fc conjugates is significant, additional experiments are required to probe the mechanism of the pyridine and imidazole complexes in detail. For the purposes of this work, the key observation is that the Ru-cymene-Fc complexes can exhibit antioxidant behavior in addition to cytotoxicity. Furthermore, these observations suggest a potential biological role for the Ru(II) center under physiological conditions, beyond the influence of the arene ligand on transport processes.

■

CONCLUSIONS Heteronuclear bimetallic compounds have the potential to act as multifunctional cytotoxic agents. As we demonstrated here, modification of the linkers between the metal centers of such compounds can change their chemical properties and consequently influence their biological activity. The linker ligands of the Ru-cymene-Fc complexes tune the Fc+/0 reduction potentials of the Fc moieties, which correlates with the anticancer and antibacterial activity of the compounds. While the observed correlation does not explicitly demonstrate a redox cause for this structure−activity relationship, it is consistent with the generation of ferrocenium species and subsequent production of toxic ROS.15 Each of the metal centers of the Ru-cymene-Fc complexes has a specific role to play in the observed activity. At a fundamental level, the Ru(II)-cymene center promotes aqueous solubility, which is necessary, given the relatively low solubility of the ferrocene ligands. Previous studies also indicate that the Ru-arene can influence transmembrane transport processes.75 The correlation of the antibacterial and anticancer activity of these compounds with the relative ease of oxidation of the Fc center suggests that the Fc center acts as a “redox antenna”.49a,76 Therefore, we propose that cytotoxic activity is initiated by the generation of ferrocenium species, which then produce ROS via Fenton chemistry (Figure 8).15 The Fe(III) species responsible for the onset of redox cycling also could arise from degradation products produced following attack by physiological nucleophiles.77 A fundamental component of the structure−activity relationships observed in this work was the use of a small library of Fcbased N-donor ligands. This included a new ligand, 4-FcEtPip, that features a non-aromatic piperidine system for coordination to the Ru(II) center. These ligands provide a straightforward method for installing Fc functionalities into medicinal inorganic frameworks. Furthermore, the availability of different donors (pyridine, imidazole, and piperidine) and different linkers suggest their applicability to modify the activity of many other metal-based therapeutics. In the case of the work described here, access to this full ligand set was key to the

Figure 10. Proposed radical scavenging processes for Ru-cymene-Fc complexes and Ru-Pip. H


Article

Inorganic Chemistry

as a light-yellow solid. Yield: 42%. C17H23Cl2FeN calcd: C, 68.70; H, 7.80; N, 4.71. Found: C, 68.53; H, 8.10; N, 5.10. mp 96−98 °C. 1H NMR (400 MHz, acetone-d6): δ 4.10 (s, 5H, Cp), 4.08 (t, J = 1.8 Hz, 2H, sub-Cp), 4.03 (t, J = 1.8 Hz, 2H, sub-Cp), 2.98 (dt, J = 11.9, 2.7 Hz, 2H, piperidinyl), 2.51 (td, J = 12.0, 2.5 Hz, 2H, piperidinyl), 2.38 (m, 2H, ethylic linker), 1.66 (d, J = 13.3 Hz, 2H, piperidinyl), 1.44 (m, 2H, piperidinyl), 1.35 (m, 1H, piperidinyl), 1.07(qd, 2H, J = 12.0, 4.0 Hz, ethylic linker). [RuCl2(η6-p-cymene)(1-FcMeIm)] (Ru-1-FcMeIm). [RuCl2(η6-pcymene)]2 (0.1225 g, 0.2 mmol) was dissolved in hot toluene (8 mL, 80 °C) and then 2 equiv of 1-FcMeIm (0.1089 g, 0.4 mmol) was added, and the resulting mixture was heated under reflux for 6 h. The reaction solution was allowed to cool down to room temperature, and hexanes (30 mL) were added to precipitate the crude product as a yellow powder, which was isolated by filtration, washed with hexanes (3 × 10 mL), and dried under vacuum. The compound was purified via recrystallization from a mixture of CH2Cl2 and hexanes (6:4 v/v) by slowly evaporating half of the solvent over 3 days at room temperature. The yellow crystals obtained were suitable for study by X-ray diffraction. Yield: 0.127 g, 55.3%. C24H28Cl2FeN2Ru calcd: C, 50.37; H, 4.93; N, 4.89. Found: C, 50.10; H, 4.89; N, 4.64. 1H NMR (400 MHz, acetone-d6): δ 8.00 (t, J = 1.3 Hz, 1H, imidazolyl), 7.33 (t, J = 1.4 Hz, 1H, imidazolyl), 7.14 (t, J = 1.6 Hz, 1H, imidazolyl), 5.47 (d, J = 6.0 Hz, 2H, benzyl, p-cymene), 5.26 (d, J = 6.0 Hz, 2H, benzyl, p-cymene), 5.08 (s, 2H, methylic linker), 4.35 (t, J = 1.9 Hz, 2H, subCp), 4.22 (s, 5H, Cp), 4.20 (t, J = 1.9 Hz, 2H, sub-Cp), 2.89 (septet, J = 6.9 Hz, 1H, isopropyl, p-cymene), 2.08 (s, 3H, methyl, p-cymene), 1.25 (d, J = 6.9 Hz, 6H, isopropyl, p-cymene). [RuCl2(η6-p-cymene)(4-FcPyr)] (Ru-4-FcPyr). [RuCl2(η6-p-cymene)]2 (0.1225 g, 0.2 mmol) was initially dissolved in hot toluene (15 mL, 80 °C), and after that, 2 equiv of 4-FcPyr (0.1052 g, 0.4 mmol) was added, and the resulting mixture was stirred at reflux for 6 h. During the reaction, a red precipitate formed gradually. The solution was allowed to cool to room temperature and more solids were obtained. The solids were isolated by filtration, washed with hexanes (3 × 10 mL), and dried under vacuum. The crude product was then purified via recrystallization from a mixture of CH2Cl2 and hexanes (6:4 v/v) by slowly evaporating half of the solvent over 2 days at room temperature. The pure product was obtained as dark red crystals. Yield: 0.108 g, 47.3%. C25H27Cl2FeNRu calcd: C, 52.74; H, 4.78; N, 2.46. Found: C, 53.10; H, 4.89; N, 2.64. mp 222−224 °C. 1H NMR (400 MHz, acetone-d6): δ 8.83 (dd, J = 5.3, 1.5 Hz, 2H, pyridyl), 7.53 (dd, J = 5.3, 1.5 Hz, 2H, pyridyl), 5.54 (d, J = 5.9 Hz, 2H, benzyl, p-cymene), 5.32 (d, J = 5.9 Hz, 2H, benzyl, p-cymene), 5.00 (t, J = 1.9 Hz, 2H, sub-Cp), 4.57 (t, J = 1.9 Hz, 2H, sub-Cp), 4.10 (s, 5H, Cp), 2.97 (septet, J = 6.9 Hz, 1H, isopropyl, p-cymene), 2.04 (s, methyl, p-cymene), 1.33 (d, J = 6.9 Hz, 6H, isopropyl, pcymene). Crystals suitable for X-ray diffraction were obtained by dissolving the compound in a mixture of CH2Cl2 and hexanes (6:4 v/ v) at room temperature followed by cooling at −18 °C for 1 week. [RuCl2(η6-p-cymene) (3-FcPyr)] (Ru-3-FcPyr). This complex was prepared and purified using the same procedure as for Ru-4-FcPyr but with [RuCl2(η6-p-cymene)]2 (0.1225 g, 0.2 mmol) and 2 equiv of 3-FcPyr (0.1052 g, 0.4 mmol) heated under reflux in toluene for 6 h. The crude product was obtained as a yellow powder, which was purified via recrystallization from a mixture of CH2Cl2 and hexanes (6:4 v/v) by slowly evaporating half of the solvent over 2 days at room temperature. The desired product was obtained as yellow crystals. Yield: 0.116 g, 51.0%. C25H27Cl2FeNRu·H2O calcd: C, 51.12; H, 4.98; N, 2.38. Found: C, 51.41; H, 4.98; N, 2.11. mp 152 °C. 1H NMR (400 MHz, acetone-d6): δ 9.29 (d, J = 2.1 Hz, 1H, pyridyl), 8.85 (dd, J = 5.5, 1.2 Hz, 1H, pyridyl), 7.97 (ddd, J = 7.9, 2.1, 1.4 Hz, 1H, pyridyl), 7.32 (ddd, J = 7.9, 5.7, 0.7 Hz, 1H, pyridyl), 5.59 (d, J = 6.0 Hz, 2H, benzyl, p-cymene), 5.39 (d, J = 6.0 Hz, 2H, benzyl, pcymene), 4.81 (t, J = 1.9 Hz, 2H, sub-Cp), 4.46 (t, J = 1.9 Hz, 2H, sub-Cp), 4.15 (s, 5H, Cp), 3.02 (septet, J = 7.0 Hz, 1H, isopropyl, pcymene), 2.12 (s, 3H, methyl, p-cymene), 1.38 (d, J = 7.0 Hz, 6H, isopropyl, p-cymene). Crystals suitable for X-ray diffraction were obtained by dissolving the compound in a mixture of CH2Cl2 and

discovery of the electrochemical dependence of anticancer and antibacterial activity. The studies of the antioxidant properties of the Ru-cymeneFc complexes show that the Ru center is involved in the observed activity. This demonstrates that the electrochemical properties of both metal centers are likely important to the biological behavior of the compounds. Furthermore, given that the proposed mechanism of these compounds, as with many Fc-containing cytotoxins, is the generation of ROS, 15 antioxidant activity is likely counterproductive.67,78 The cytotoxicity of the Ru-cymene-Fc complexes shows a good correlation with the E1/2(Fc+/0) values, suggesting that antioxidant activity does not have a significant impact in this case. However, this strikes a note of caution in the development of heteronuclear bimetallic cytotoxins, which is that inclusion of a second redox-active metal center could inadvertently induce antioxidant activity that counteracts ROS generation. In summary, by linking a Ru(II)-arene center to Fc via heterocyclic N-donor linkers, we produced new analogues of RAPTA-type complexes that show anticancer and antibacterial activity. The observed activity depends primarily on how readily the Fc center is oxidized, which is tuned by the ligands linking the two metal centers. The electrochemical properties of both metal centers also contribute to antioxidant behavior, demonstrating biologically relevant multifunctional behavior from these heteronuclear bimetallic complexes.

■

EXPERIMENTAL SECTION

Materials. All reagents were purchased from Sigma-Aldrich, except ruthenium(III) chloride hydrate, which was purchased from Pressure Chemical Company, and were used as received. Ferrocenyl pyridine ligands, 4-ferrocenylpyridine (4-FcPyr), 3-ferrocenylpyridine (3FcPyr), and 4-ferrocenyl(ethyl)pyridine (4-FcEtPyr), were synthesized using our previously published protocols.18 (E)-4-(2Ferrocenylvinyl)pyridine (4-FcVinylPyr) was synthesized according to our previously reported method, based on the literature.18,28 1Ferrocenyl(methyl)imidazole (1-FcMeIm) were synthesized based on previously reported methods with some modifications (see Supporting Information).29 The Ru(II) dimeric precursor [RuCl2(η6-pcymene)]227 and the unfunctionalized Ru-cymene parent complexes, RuCl 2 (η 6 -p-cymene)(1-MeIm) (Ru-1-MeIm), 10 RuCl 2 (η 6 -pcymene)(pyridine) (Ru-Pyr),10 and RuCl2(η6-p-cymene)(piperidine) (Ru-Pip)30 were synthesized according to the literature (see Supporting Information). 4-Ferrocenylethylpiperidine (4-FcEtPip). 4-FcVinylPyr (0.5780 g, 2.0 mmol) and 0.40 g of 5% palladium on activated charcoal were added into ethanol (40 mL). The resulting mixture was stirred at room temperature under H2 for 24 h and filtered through diatomaceous earth to remove impurities, and the filtrate was collected and dried by vacuum, which afforded the products 4FcEtPyr and 4-FcEtPip as a pale orange oil. The crude product was purified by column chromatography on silica gel by eluting with a mixture of CH2Cl2/methanol (95:5, v/v). 4-FcEtPyr was obtained as the first yellow band and was then dried under vacuum. The eluent was then changed to methanol only. After flushing down all the other bands, a single immobile yellow band remained in the column. This fraction of the yellow silica gel was transferred into a separating funnel filled with 100 mL of NaOH solution with the pH ≈ 8. The product was deprotonated in this basic solution and became soluble in the organic solvents. CH2Cl2 (20 mL) was subsequently added and the resulting mixture was shaken vigorously to extract the product into the organic layer. This process was repeated about 3 times until the color of silica gel was white. The organic fractions were combined and dried over MgSO4 and solids were removed by filtration. The solvent was evaporated under vacuum to afford the pure 4-FcEtPip product I


Article

Inorganic Chemistry hexanes (6:4 v/v) at room temperature followed by cooling at −18 °C for 1 week. [RuCl2(η6-p-cymene) (4-FcVinylPyr)] (Ru-4-FcVinylPyr). This complex was prepared and purified using the same procedure as for Ru-4-FcPyr but with [RuCl2(η6-p-cymene)]2 (0.1225 g, 0.2 mmol) and 2 equiv of 4-FcVinylPyr (0.1157 g, 0.4 mmol) heated under reflux in toluene for 6 h. The crude product was obtained as a red powder, which was purified via recrystallization from a mixture of CH2Cl2 and hexanes (6:4 v/v) by slowly evaporating half of the solvent over 2 days at room temperature. The desired product was obtained as red crystals. Yield: 0.162 g, 67.9%. C27H29Cl2FeNRu calcd: C, 54.47; H, 4.91; N, 2.35. Found: C, 54.41; H, 4.87; N, 2.35. mp 208−210 °C (decomp.). 1H NMR (400 MHz, acetone-d6): δ 8.88 (dd, J = 5.0, 1.5 Hz, 2H, pyridyl), 7.49 (d, J = 16.2 Hz, 1H, vinylic linker), 7.46 (dd, J = 5.0, 1.5 Hz, 2H, pyridyl), 6.84 (d, J = 16.2 Hz, 1H, vinylic linker), 5.53 (d, J = 6.0 Hz, 2H, benzyl, p-cymene), 5.32 (d, J = 6.0 Hz, 2H, benzyl, p-cymene), 4.66 (t, J = 1.9 Hz, 2H, subCp), 4.44 (t, J = 1.9 Hz, 2H, sub-Cp), 4.20 (s, 5H, Cp), 2.98 (septet, J = 6.9 Hz, 1H, isopropyl, p-cymene), 2.09 (s, 3H, methyl, p-cymene), 1.34 (d, J = 6.9 Hz, 6H, isopropyl, p-cymene). Crystals suitable for Xray diffraction were obtained by dissolving the compound in a mixture of CH2Cl2 and hexanes (6:4 v/v) at room temperature followed by cooling at −18 °C for 1 week. [RuCl2(η6-p-cymene) (4-FcEtPyr)] (Ru-4-FcEtPyr). This complex was prepared and purified using the same procedure as for Ru-1FcMeIm but with [RuCl2(η6-p-cymene)]2 (0.1225 g, 0.2 mmol) and 2 equiv of 4-FcEtPyr (0.1165 g, 0.4 mmol) heated under reflux in toluene for 6 h. The reaction solution was allowed to cool down to room temperature, and hexanes (30 mL) was added to precipitate the crude product as a yellow powder, which was purified via recrystallization from a mixture of CH2Cl2 and hexanes (6:4 v/v) by slowly evaporating half of the solvent over 2 days at room temperature. The pure product was obtained as yellow crystals. Yield: 0.133 g, 55.6%. C27H31Cl2FeNRu calcd: C, 54.29; H, 5.23; N, 2.34. Found: C, 54.33; H, 5.39; N, 2.53. mp 198−200 °C (decomp.). 1H NMR (400 MHz, acetone-d6): δ 8.90 (dd, J = 5.3, 1.5 Hz, 2H, pyridyl), 7.29 (dd, J = 5.3, 1.5 Hz, 2H, pyridyl), 5.52 (d, J = 6.0 Hz, 2H, benzyl, p-cymene), 5.30 (d, J = 6.0 Hz, 2H, benzyl, p-cymene), 4.14 (s, 5H, Cp), 4.12 (t, J = 1.9 Hz, 2H, sub-Cp), 4.06 (t, J = 1.9 Hz, 2H, sub-Cp), 2.96 (m, septet, J = 7.0 Hz, 1H, isopropyl, p-cymene overlapping with m, 2H, ethylic linker), 2.75 (m, 2H, ethylic linker), 2.01 (s, 3H, methyl, p-cymene), 1.32 (d, J = 6.9 Hz, 6H, isopropyl, pcymene). Crystals suitable for X-ray diffraction were obtained by dissolving the compound in a mixture of acetone and diethyl ether (7:3 v/v) at room temperature followed by cooling at −18 °C for 1 week. [RuCl2(η6-p-cymene) (4-FcEtPip)] (Ru-4-FcEtPip). RuCl2(η6-pcymene)]2 (0.1225 g, 0.2 mmol) and 2 equiv of 4-FcEtPip (0.1189 g, 0.4 mmol) were dissolved in 10 mL of CH2Cl2. After the mixture was stirred at room temperature for 16 h, hexanes (10 mL) were added to the reaction solution under stirring. Half of the solvent was slowly removed by evaporation at room temperature over 3 days, and the desired product was produced as yellow crystals, which were isolated by filtration and washed with hexanes (3 × 3 mL), and dried under vacuum. The crystals as obtained were suitable for study by Xray diffraction. Yield: 0.131 g, 54.2%. C27H37Cl2FeNRu·0.2CH2Cl2 calcd: C, 52.66; H, 6.08; N, 2.32. Found: C, 52.97; H, 6.50; N, 2.69. mp 202−204 °C (decomp.). 1H NMR (400 MHz, acetone-d6): δ 5.46 (d, J = 6.1 Hz, 2H, benzyl, p-cymene), 5.42 (d, J = 6.1 Hz, 2H, benzyl, p-cymene), 4.10 (s, 5H, Cp overlapping with t, J = 1.8 Hz, 2H, subCp), 4.05 (t, J = 1.8 Hz, 2H, sub-Cp), 3.75 (d, J = 15 Hz, 2H, piperidyl), 3.17 (qd, J = 12.7, 2.7 Hz, 2H, piperidyl), 2.96 (m, septet, J = 7.0 Hz, 1H, isopropyl, p-cymene), 2.40 (m, 2H, ethylic linker), 2.16 (s, 3H, methyl, p-cymene), 1.81 (d, J = 13.4 Hz, 2H, piperidyl), 1.67 (m, 1H, piperidyl), 1.47 (m, 2H, piperidyl), 1.30 (d, J = 6.9 Hz, 6H, isopropyl, p-cymene), 1.09 (qd, J = 12.7, 2.7 Hz, 2H, ethylic linker). [RuCl(η6-p-cymene)(1-FcMeIm)2][RuCl3(η6-p-cymene)]. This complex was obtained unexpectedly in the course of crystal growth of Ru1-FcMeIm. The crystallization conditions initially used in an attempt to obtain the structure of Ru-1-FcMeIm involved storing a solution of

the complex in a mixture of acetone and diethyl ether (1:1 v/v) at −18 °C for 2 weeks. However, these conditions converted Ru-1FcMeIm to the ionic complex. The molecule was identified by the Xray crystal structure as shown in Figure 4. X-ray Crystallography. Single crystals suitable for X-ray diffraction analysis were mounted on 100 μm MiTeGen dualthickness micromounts with the temperature regulated by an Oxford Cryosystems Cryostream. X-ray diffraction data were collected using a Bruker SMART DUO diffractometer equipped with an APEX II CCD area detector fixed at a distance of 50 mm from the crystals using Mo Kα radiation filtered with a graphite TRIUMPH-monochromator or Cu Kα radiation (λ(Cu) = 1.54178 Å and λ(Mo) = 0.71073 Å). Structures were solved using the Intrinsic Phasing method refined by a full-matrix least-squares method on F2 using the SHELXL79 software package. Molecular graphics were generated by ORTEP-3 for Windows version 2.0280 and rendered by POV-Ray version 3.7.81 Crystal data, data collection parameters, and analysis statistics are listed in Supporting Information Table S1. Electrochemistry. Cyclic voltammograms were recorded on a Princeton Applied Research potentiostat/galvanostat, equipped with a glassy-carbon working electrode, a Ag/Ag+ reference electrode (0.01 M AgNO3 and 0.1 M tetrabutylammonium hexafluorophosphate in acetonitrile), and a platinum-wire counter electrode. The measurements were performed using 1 mM concentrations of each complex with 0.1 M tetrabutylammonium hexafluorophosphate in 3 mL of CH2Cl2 and recorded using scan rates of 100 mV/s for CV. Because of the signal overlap of ferrocene and ferrocenyl ligands of the complexes in CH2Cl2, the Fc+/0 redox couple was used as an external reference. Cytotoxicity Testing. The HT29 human colon adenocarcinoma cell line was acquired from Dr. M. Bally’s laboratory (BC Cancer Agency Research Center, Vancouver, BC) and were cultured at 37 °C with 5% CO2 in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 2 mM L-glutamine (Gibco). Initially, cells were seeded in quadruplet at 2000 cells/well in 384-well plates (Grener Bio-One) and incubated for 24 h. Compounds to be tested were dissolved in DMSO and diluted with media, and 20 μL aliquots of each complex in media were then added to the seeded cells in each well, to give concentrations in two different ranges: 0−200 μM for the Ru-cymene-Fc complexes and 100−1000 μM for the Ru-cymene parent complexes. The final concentration of DMSO was 1% per well. After 72 h of compound exposure, the cells were stained using 5 μL of 4.8 μM Hoescht 33342 (Life Technologies) to determine the total cell count and 3 μL of 1 μM ethidium homodimer I (Biotium) to determine the dead cell count. After 20 min incubation, the plates were imaged using an IN Cell Analyzer 1000 (GE Healthcare), which is an automated fluorescent microscope platform that enables highcontent screening. Cell counts were determined via the IN Cell Developer Toolbox high-content image analysis software. Cells were classified as “dead” if over 30% overlap of the two stains was detected. The collected data were statistically analyzed using Prism 6.0 (GraphPad software), and half maximal inhibitory concentration (IC50) of each complex was determined from the corresponding fitted dose−response curve. Antibacterial Assay. Antibacterial activities of the Ru-cymene-Fc complexes and their parent Ru-cymene complexes were evaluated against B. subtilis and E. coli using a turbidimetric method in sterile 96well microplates (Thermo Scientific) according to reported protocols.63,82 The growth kinetics of the bacteria were quantified in terms of turbidity monitored at 620 nm83 using a BioTek microplate reader at 15 min intervals over 12 h at 37 °C. The growth of the seed cultures was monitored by a Cary 1E UV−visible spectrophotometer by measuring the optical density at 650 nm (OD650) in a 1 mL cuvette. Luria−Bertani (LB) broth was prepared with deionized water supplied by a Millipore Milli-Q deionization system. All glassware and pipette tips were autoclaved with a wet cycle for 20 min. The assay was started by inoculating 2 μL of single colonies of bacteria into 15 mL of LB broth in a 250 mL Erlenmeyer flask. After 16 h of growth at 37 °C with shaking (200 rpm), an additional 25 mL of LB broth was added. The diluted bacteria J


Article

Inorganic Chemistry Accession Codes

cultures were allowed to continue growing for another 2−3 h until reaching an OD650 of 0.8 and then loaded on microplates with 180 μL of culture in each well. Stock solutions of each compound to be tested were prepared in DMSO and then diluted to required concentrations for testing with LB broth containing 10% by volume DMSO. For B. subtilis treatment, three concentration ranges were used: 0−60 μM in 7.5 μM increments for Ru-1-FcMeIm and Ru-4-FcEtPip, 0−120 μM in 15 μM increments for Ru-4-FcVinylPyr and Ru-4-FcEtPyr, and 0−180 μM in 22.5 μM increments for Ru-4-FcPyr, Ru-3-FcPyr, and all the non-Fc Ru-cymene parent complexes. Higher concentrations were used for E. coli treatment with all of the complexes: 0−600 μM in 100 μM increments. To the microplate wells containing bacterial culture were added 20 μL aliquots of the complexes at each concentration. Blank bacterial suspensions and blank bacterial suspensions with 1% DMSO were prepared as negative controls. All assays were performed in duplicate. DPPH Radical Scavenging Assay. The antioxidant activity of the Ru-cymene-Fc complexes, their corresponding Fc-ligands, and the non-Fc Ru-cymene parent complexes were evaluated using a DPPH radical scavenging assay originally reported by Blois74 with slight modification. A stock solution of DPPH was prepared by dissolving DPPH (0.010 g, 0.025 mmol) in 100 mL of ethanol, resulting in a dark purple solution (0.25 mM). Stock solutions of each compound to be tested were also prepared in ethanol. In Eppendorf microtubes (1.5 mL), the stock solutions were serially diluted with ethanol to give 5 samples, each of which had a final volume of 600 μL. Subsequently, 400 μL of the DPPH stock solution was added to each sample. The solutions were tested with 0.10 mM DPPH in 1 mL of ethanolic solution with concentrations as follows. Concentrations of Ru-1FcMeIm and Ru-4-FcVinylPyr were varied from 0 to 0.01 mM (0− 0.1 equiv) in 0.002 mM increments. For the other Ru-cymene-Fc compounds as well as Ru-Pyr and Ru-1-MeIm, concentrations in the range of 0−0.05 mM (0−0.5 equiv) at 0.01 mM intervals were used. The concentration of Ru-Pip was varied from 0 to 0.05 mM (0−0.5 equiv) in 0.01 mM increments. Antioxidant activity of the Fc-ligands was measured at concentrations of 0−0.5 mM (0−5 equiv) using 0.1 mM increments. The resulting mixtures were shaken vigorously and stored in the dark at 25 °C for 30 min. Thereafter, the absorbance at 517 nm was measured against an ethanol blank using a Cary 1E UV− visible spectrometer to determine the level of discoloration of each sample. EC50 values were then determined by fitting the linear region of the data and solving for the concentration that gave 50% scavenging activity. Optical Spectroscopy. UV−vis spectra were recorded using a Cary 1E UV−visible spectrophotometer equipped with a Haake F3 water bath to maintain all the samples at a temperature of 37 °C. Measurements were performed on 1 mL of solutions of each complex in PBS with 1% DMSO (to increase solubility) at a scan rate of 10 nm/s at 2 min intervals for up to 3 h.

■

CCDC 1863408−1863416 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kathleen E. Prosser: 0000-0003-3035-5024 Jeffrey J. Warren: 0000-0002-1747-3029 Charles J. Walsby: 0000-0003-3194-8227 Present Address †

Department of Bioengineering, University of California, Berkeley, 94720, CA, USA.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Financial support for this work was provided by The Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN312575 to C.J.W. and RGPIN05559 to J.J.W.) and Simon Fraser University (SFU). K.E.P. acknowledges additional support from NSERC through a Vanier Canada Graduate Scholarship. The authors are very grateful to Dr. Marcel Bally in the Department of Experimental Therapeutics at the BC Cancer Agency for access to cellculture and analysis facilities used in the HT29 cytotoxicity studies. The authors also wish to thank Prof. Robert Young (SFU) for access to the BioTek microplate reader and GraphPad software.

■

REFERENCES

(1) (a) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast, B.; Zorbas, H.; Keppler, B. K. From bench to bedside - preclinical and early clinical development of the anticancer agent indazolium trans[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A). J. Inorg. Biochem. 2006, 100, 891−904. (b) Trondl, R.; Heffeter, P.; Kowol, C. R.; Jakupec, M. A.; Berger, W.; Keppler, B. K. NKP-1339, the first ruthenium-based anticancer drug on the edge to clinical application. Chem. Sci. 2014, 5, 2925−2932. (c) Rademaker-Lakhai, J. M.; van den Bongard, D.; Pluim, D.; Beijnen, J. H.; Schellens, J. H. M. A phase I and pharmacological study with imidazolium-trans-DMSOimidazole-tetrachlororuthenate, a novel ruthenium anticancer agent. Clin. Cancer Res. 2004, 10, 3717−3727. (d) Antonarakis, E. S.; Emadi, A. Ruthenium-based chemotherapeutics: are they ready for prime time? Cancer Chemother. Pharmacol. 2010, 66, 1−9. (e) Hartinger, C. G.; Jakupec, M. A.; Zorbas-Seifried, S.; Groessl, M.; Egger, A.; Berger, W.; Zorbas, H.; Dyson, P. J.; Keppler, B. K. KP1019, A New RedoxActive Anticancer Agent - Preclinical Development and Results of a Clinical Phase I Study in Tumor Patients. Chem. Biodiversity 2008, 5, 2140−2155. (f) 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, 101−110. (g) Levina, A.; Mitra, A.; Lay, P. A. Recent developments in ruthenium anticancer drugs. Metallomics 2009, 1, 458−470. (h) Jakupec, M. A.; Arion, V. B.; Kapitza, S.; Reisner, E.; Eichinger, A.; Pongratz, M.; Marian, B.; Keyserlingk, N. G.; Keppler, B. K. KP1019 (FFC14A) from bench to bedside: preclinical and early clinical developmentan overview. Int. J. Clin. Pharmacol. Ther. 2005, 43, 595−596. (i) Lentz, F.; Drescher,

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b02542. Synthetic methods for 1-FcMeIm, Ru-1-MeIm, and RuPyr; 1H NMR spectra 4-FcEtPip ligand and Ru-cymeneFc complexes; crystal data and details of data collection and refinement and selected bond lengths; UV−vis spectra of time-dependent solution behavior of complexes in PBS; cytotoxicity assays of Ru-1-MeIm, RuPip, and Ru-Pyr; antibacterial assays of all complexes against B. subtilis and E. coli; cyclic voltammograms of complexes; and DPPH radical scavenging assay data (PDF) K


Article

Inorganic Chemistry

S. A New Age for Iron: Antitumoral Ferrocenes. Organometallics 2013, 32, 5626−5639. (c) Ornelas, C. Application of ferrocene and its derivatives in cancer research. New J. Chem. 2011, 35, 1973−1985. (d) Snegur, L. V.; Babin, V. N.; Simenel, A. A.; Nekrasov, Y. S.; Ostrovskaya, L. A.; Sergeeva, N. S. Antitumor activities of ferrocene compounds. Russ. Chem. Bull. 2010, 59, 2167−2178. (15) (a) Osella, D.; Ferrali, M.; Zanello, P.; Laschi, F.; Fontani, M.; Nervi, C.; Cavigiolio, G. On the mechanism of the antitumor activity of ferrocenium derivatives. Inorg. Chim. Acta 2000, 306, 42−48. (b) Tabbì, G.; Cassino, C.; Cavigiolio, G.; Colangelo, D.; Ghiglia, A.; Viano, I.; Osella, D. Water Stability and Cytotoxic Activity Relationship of a Series of Ferrocenium Derivatives. ESR Insights on the Radical Production during the Degradation Process†. J. Med. Chem. 2002, 45, 5786−5796. (16) (a) Hillard, E. A.; Vessières, A.; Jaouen, G. Ferrocene functionalized endocrine modulators as anticancer agents. Top. Organomet. Chem. 2010, 32, 81−117. (b) Top, S.; Tang, J.; Vessières, A.; Carrez, D.; Provot, C.; Jaouen, G. Ferrocenyl hydroxytamoxifen: a prototype for a new range of oestradiol receptor site-directed cytotoxics. Chem. Commun. 1996, 955−956. (c) Top, S.; Dauer, B.; Vaissermann, J.; Jaouen, G. Facile route to ferrocifen, 1-[4(2-dimethylaminoethoxy)]-1-(phenyl-2-ferrocenyl-but-1-ene), first organometallic analogue of tamoxifen, by the McMurry reaction. J. Organomet. Chem. 1997, 541, 355−361. (17) (a) Wu, C.-H.; Wu, D.-H.; Liu, X.; Guoyiqibayi, G.; Guo, D.D.; Lv, G.; Wang, X.-M.; Yan, H.; Jiang, H.; Lu, Z.-H. Ligand-Based Neutral Ruthenium(II) Arene Complex: Selective Anticancer Action. Inorg. Chem. 2009, 48, 2352−2354. (b) Von Poelhsitz, G.; Bogado, A. L.; de Araujo, M. P.; Selistre-de-Araújo, H. S.; Ellena, J.; Castellano, E. E.; Batista, A. A. Synthesis, characterization, X-ray structure and preliminary in vitro antitumor activity of the nitrosyl complex fac[RuCl3(NO)(dppf)], dppf=1,1′-bis(diphenylphosphine)ferrocene. Polyhedron 2007, 26, 4707−4712. (c) Tauchman, J.; Süss-Fink, G.; Š těpnička, P.; Zava, O.; Dyson, P. J. Arene ruthenium complexes with phosphinoferrocene amino acid conjugates: Synthesis, characterization and cytotoxicity. J. Organomet. Chem. 2013, 723, 233−238. (18) Mu, C.; Chang, S. W.; Prosser, K. E.; Leung, A. W. Y.; Santacruz, S.; Jang, T.; Thompson, J. R.; Yapp, D. T. T.; Warren, J. J.; Bally, M. B.; Beischlag, T. V.; Walsby, C. J. Induction of Cytotoxicity in Pyridine Analogues of the Anti-metastatic Ru(III) Complex NAMIA by Ferrocene Functionalization. Inorg. Chem. 2016, 55, 177−190. (19) (a) Auzias, M.; Gueniat, J.; Therrien, B.; Süss-Fink, G.; Renfrew, A. K.; Dyson, P. J. Arene-ruthenium complexes with ferrocene-derived ligands: Synthesis and characterization of complexes of the type [Ru(η6-arene)(NC5H4CH2NHOC-C5H4FeC5H5)Cl2] and [Ru(η6-arene)(NC3H3N(CH2)2O2C-C5H4FeC5H5)Cl2]. J. Organomet. Chem. 2009, 694, 855−861. (b) Auzias, M.; Therrien, B.; Süss-Fink, G.; Š těpnička, P.; Ang, W. H.; Dyson, P. J. Ferrocenoyl Pyridine Arene Ruthenium Complexes with Anticancer Properties: Synthesis, Structure, Electrochemistry, and Cytotoxicity. Inorg. Chem. 2008, 47, 578−583. (20) Auzias, M.; Therrien, B.; Sü ss-Fink, G. Heteronuclear complexes containing the N,N′-di(2-pyridyl)amidocarboxylferrocene ligand. Inorg. Chim. Acta 2012, 387, 446−449. (21) (a) Mukherjee, A.; Sadler, P. J. Metals in Medicine: Therapeutic Agents; John Wiley & Sons, Inc., 2009; pp 80−126. (b) Guo, Z.; Sadler, P. J. Metals in medicine. Angew. Chem., Int. Ed. 1999, 38, 1512−1531. (22) (a) Lemire, J. A.; Harrison, J. J.; Turner, R. J. Antimicrobial activity of metals: mechanisms, molecular targets and applications. Nat. Rev. Microbiol. 2013, 11, 371−384. (b) Hindi, K. M.; Panzner, M. J.; Tessier, C. A.; Cannon, C. L.; Youngs, W. J. The Medicinal Applications of Imidazolium Carbene−Metal Complexes. Chem. Rev. 2009, 109, 3859−3884. (c) Rocha, D. P.; Pinto, G. F.; Ruggiero, R.; de Oliveira, C. A.; Guerra, W.; Fontes, A. P. S.; Tavares, T. T.; Marzano, I. M.; Pereira-Maia, E. C. Coordenaçaõ de metais a antibióticos como uma estratégia de combate à resistência bacteriana. Quim. Nova 2011, 34, 111−118. (d) Medici, S.; Peana, M.; Nurchi, V.

A.; Lindauer, A.; Henke, M.; Hilger, R. A.; Hartinger, C. G.; Scheulen, M. E.; Dittrich, C.; Keppler, B. K.; Jaehde, U. Pharmacokinetics of a novel anticancer ruthenium complex (KP1019, FFC14A) in a phase I dose-escalation study. Anti-Cancer Drugs 2009, 20, 97−103. (2) (a) Nazarov, A. A.; Hartinger, C. G.; Dyson, P. J. Opening the lid on piano-stool complexes: An account of ruthenium(II)-arene complexes with medicinal applications. J. Organomet. Chem. 2014, 751, 251−260. (b) Yan, Y. K.; Melchart, M.; Habtemariam, A.; Sadler, P. J. Organometallic chemistry, biology and medicine: ruthenium arene anticancer complexes. Chem. Commun. 2005, 4764−4776. (3) (a) Singh, A. K.; Pandey, D. S.; Xu, Q.; Braunstein, P. Recent advances in supramolecular and biological aspects of arene ruthenium(II) complexes. Coord. Chem. Rev. 2014, 270−271, 31− 56. (b) Süss-Fink, G. Water-soluble arene ruthenium complexes: From serendipity to catalysis and drug design. J. Organomet. Chem. 2014, 751, 2−19. (c) Mu, C.; Walsby, C. J. Ruthenium Anticancer Compounds with Biologically-derived Ligands. Ligand Design in Medicinal Inorganic Chemistry; Storr, T., Ed.; John Wiley & Sons, Ltd., 2014; pp 405−437. (4) Morris, R. E.; Aird, R. E.; Murdoch, P. d. S.; Chen, H.; Cummings, J.; Hughes, N. D.; Parsons, S.; Parkin, A.; Boyd, G.; Jodrell, D. I.; Sadler, P. J. Inhibition of Cancer Cell Growth by Ruthenium(II) Arene Complexes. J. Med. Chem. 2001, 44, 3616− 3621. (5) Dougan, S. J.; Sadler, P. J. The design of organometallic ruthenium arene anticancer agents. Chimia 2007, 61, 704−715. (6) (a) Ang, W. H.; Casini, A.; Sava, G.; Dyson, P. J. Organometallic ruthenium-based antitumor compounds with novel modes of action. J. Organomet. Chem. 2011, 696, 989−998. (b) Scolaro, C.; Bergamo, A.; Brescacin, L.; Delfino, R.; Cocchietto, M.; Laurenczy, G.; Geldbach, T. J.; Sava, G.; Dyson, P. J. In Vitro and in Vivo Evaluation of Ruthenium(II)−Arene PTA Complexes. J. Med. Chem. 2005, 48, 4161−4171. (7) Nowak-Sliwinska, P.; van Beijnum, J. R.; Casini, A.; Nazarov, A. A.; Wagnières, G.; van den Bergh, H.; Dyson, P. J.; Griffioen, A. W. Organometallic Ruthenium(II) Arene Compounds with Antiangiogenic Activity. J. Med. Chem. 2011, 54, 3895−3902. (8) Renfrew, A. K.; Phillips, A. D.; Egger, A. E.; Hartinger, C. G.; Bosquain, S. S.; Nazarov, A. A.; Keppler, B. K.; Gonsalvi, L.; Peruzzini, M.; Dyson, P. J. Influence of Structural Variation on the Anticancer Activity of RAPTA-Type Complexes: ptn versus pta. Organometallics 2009, 28, 1165−1172. (9) Berger, I.; Hanif, M.; Nazarov, A. A.; Hartinger, C. G.; John, R. O.; Kuznetsov, M. L.; Groessl, M.; Schmitt, F.; Zava, O.; Biba, F.; Arion, V. B.; Galanski, M.; Jakupec, M. A.; Juillerat-Jeanneret, L.; Dyson, P. J.; Keppler, B. K. In vitro anticancer activity and biologically relevant metabolization of organometallic ruthenium complexes with carbohydrate-based ligands. Chem.Eur. J. 2008, 14, 9046−9057. (10) Vock, C. A.; Scolaro, C.; Phillips, A. D.; Scopelliti, R.; Sava, G.; Dyson, P. J. Synthesis, Characterization, and in Vitro Evaluation of Novel Ruthenium(II) η6-Arene Imidazole Complexes. J. Med. Chem. 2006, 49, 5552−5561. (11) (a) Vock, C. A.; Ang, W. H.; Scolaro, C.; Phillips, A. D.; Lagopoulos, L.; Juillerat-Jeanneret, L.; Sava, G.; Scopelliti, R.; Dyson, P. J. Development of Ruthenium Antitumor Drugs that Overcome Multidrug Resistance Mechanisms. J. Med. Chem. 2007, 50, 2166− 2175. (b) Komeda, S.; Casini, A. Next-generation anticancer metallodrugs. Curr. Top. Med. Chem. 2012, 12, 219−235. (12) Ang, W. H.; Parker, L. J.; De Luca, A.; Juillerat-Jeanneret, L.; Morton, C. J.; Lo Bello, M.; Parker, M. W.; Dyson, P. J. Rational Design of an Organometallic Glutathione Transferase Inhibitor. Angew. Chem., Int. Ed. 2009, 48, 3854−3857. (13) Ang, W. H.; De Luca, A.; Chapuis-Bernasconi, C.; JuilleratJeanneret, L.; Lo Bello, M.; Dyson, P. J. Organometallic ruthenium inhibitors of glutathione-S-transferase P1-1 as anticancer drugs. ChemMedChem 2007, 2, 1799−1806. (14) (a) Fouda, M. F. R.; Abd-Elzaher, M. M.; Abdelsamaia, R. A.; Labib, A. A. On the medicinal chemistry of ferrocene. Appl. Organomet. Chem. 2007, 21, 613−625. (b) Braga, S. S.; Silva, A. M. L


Article

Inorganic Chemistry

(36) (a) Clarke, M. J. Ruthenium metallopharmaceuticals. Coord. Chem. Rev. 2003, 236, 209−233. (b) Süss-Fink, G. Arene ruthenium complexes as anticancer agents. Dalton Trans. 2010, 39, 1673−1688. (c) Melchart, M.; Sadler, P. J. Ruthenium Arene Anticancer Complexes; Wiley-VCH Verlag GmbH & Co. KGaA, 2006; pp 39−64. (37) Chen, H.; Parkinson, J. A.; Morris, R. E.; Sadler, P. J. Highly Selective Binding of Organometallic Ruthenium Ethylenediamine Complexes to Nucleic Acids: Novel Recognition Mechanisms. J. Am. Chem. Soc. 2003, 125, 173−186. (38) Ang, W. H.; Daldini, E.; Scolaro, C.; Scopelliti, R.; JuilleratJeannerat, L.; Dyson, P. J. Development of Organometallic Ruthenium−Arene Anticancer Drugs That Resist Hydrolysis. Inorg. Chem. 2006, 45, 9006−9013. (39) Scolaro, C.; Hartinger, C. G.; Allardyce, C. S.; Keppler, B. K.; Dyson, P. J. Hydrolysis study of the bifunctional antitumour compound RAPTA-C, [Ru(η6-p-cymene)Cl2(pta)]. J. Inorg. Biochem. 2008, 102, 1743−1748. (40) Gossens, C.; Dorcier, A.; Dyson, P. J.; Rothlisberger, U. pKa Estimation of Ruthenium(II)−Arene PTA Complexes and their Hydrolysis Products via a DFT/Continuum Electrostatics Approach. Organometallics 2007, 26, 3969−3975. (41) (a) Reisner, E.; Arion, V. B.; Keppler, B. K.; Pombeiro, A. J. L. Electron-transfer activated metal-based anticancer drugs. Inorg. Chim. Acta 2008, 361, 1569−1583. (b) Romero-Canelón, I.; Sadler, P. J. Next-Generation Metal Anticancer Complexes: Multitargeting via Redox Modulation. Inorg. Chem. 2013, 52, 12276−12291. (c) Zhang, P.; Sadler, P. J. Redox-Active Metal Complexes for Anticancer Therapy. Eur. J. Inorg. Chem. 2017, 1541−1548. (42) (a) Swarts, J. C.; Vosloo, T. G.; Cronje, S. J.; Du Plessis, W. C.; Van Rensburg, C. E. J.; Kreft, E.; Van Lier, J. E. Cytotoxicity of a series of ferrocene-containing β-diketones. Anticancer Res. 2008, 28, 2781−2784. (b) Shago, R. F.; Swarts, J. C.; Kreft, E.; Van Rensburg, C. E. J. Antineoplastic activity of a series of ferrocene-containing alcohols. Anticancer Res. 2007, 27, 3431−3434. (c) Fourie, E.; Erasmus, E.; Swarts, J. C.; Jakob, A.; Lang, H.; Joone, G. K.; Van Rensburg, C. E. J. Cytotoxicity of ferrocenyl-ethynyl phosphine metal complexes of gold and platinum. Anticancer Res. 2011, 31, 825−830. (43) Govender, P.; Lemmerhirt, H.; Hutton, A. T.; Therrien, B.; Bednarski, P. J.; Smith, G. S. First- and Second-Generation Heterometallic Dendrimers Containing Ferrocenyl-Ruthenium(II)Arene Motifs: Synthesis, Structure, Electrochemistry, and Preliminary Cell Proliferation Studies. Organometallics 2014, 33, 5535−5545. (44) Vollhardt, K. P. C. Organic Chemistry Structure and Function, 6th ed.; W.H.Freeman: New York, 2011. (45) Dorcier, A.; Ang, W. H.; Bolaño, S.; Gonsalvi, L.; JuilleratJeannerat, L.; Laurenczy, G.; Peruzzini, M.; Phillips, A. D.; Zanobini, F.; Dyson, P. J. In Vitro Evaluation of Rhodium and Osmium RAPTA Analogues: The Case for Organometallic Anticancer Drugs Not Based on Ruthenium. Organometallics 2006, 25, 4090−4096. (46) Schobert, R.; Seibt, S.; Effenberger-Neidnicht, K.; Underhill, C.; Biersack, B.; Hammond, G. L. (Arene)Cl2Ru(II) complexes with N-coordinated estrogen and androgen isonicotinates: Interaction with sex hormone binding globulin and anticancer activity. Steroids 2011, 76, 393−399. (47) Zhao, J.; Prosser, K. E.; Chang, S. W.; Zakharia, S. P.; Walsby, C. J. Combining a Ru(II)-arene complex with a NO-releasing nitrateester ligand generates cytotoxic activity. Dalton Trans. 2016, 45, 18079−18083. (48) Swarts, J. C.; Swarts, D. M.; Maree, D. M.; Neuse, E. W.; La Madeleine, C.; Van Lier, J. E. Polyaspartamides as water-soluble drug carriers. Part 1: Antineoplastic activity of ferrocene-containing polyaspartamide conjugates. Anticancer Res. 2001, 21, 2033−2037. (49) (a) Patra, M.; Gasser, G. The medicinal chemistry of ferrocene and its derivatives. Nat. Rev. Chem. 2017, 1, 0066. (b) Neuse, E. W.; Kanzawa, F. Evaluation of the activity of some water-soluble ferrocene and ferricenium compounds against carcinoma of the lung by the human tumor clonogenic assay. Appl. Organomet. Chem. 1990, 4, 19− 26.

M.; Lachowicz, J. I.; Crisponi, G.; Zoroddu, M. A. Noble metals in medicine: Latest advances. Coord. Chem. Rev. 2015, 284, 329−350. (23) (a) Li, F.; Collins, J. G.; Keene, F. R. Ruthenium complexes as antimicrobial agents. Chem. Soc. Rev. 2015, 44, 2529−2542. (b) Allardyce, C. S.; Dyson, P. J.; Ellis, D. J.; Salter, P. A.; Scopelliti, R. Synthesis and characterisation of some water soluble ruthenium(II)-arene complexes and an investigation of their antibiotic and antiviral properties. J. Organomet. Chem. 2003, 668, 35−42. (24) (a) Scutaru, D.; Tataru, L.; Mazilu, I.; Vata, M.; Lixandru, T.; Simionescu, C. Contributions to the synthesis of some ferrocenecontaining antibiotics. Appl. Organomet. Chem. 1993, 7, 225−231. (b) Simionescu, C.; Lixandru, T.; Scutaru, D.; Vâta̧ ,̌ M. Mono- and heterodi-substituted derivatives of ferrocene; β-Lactamic antibiotics. J. Organomet. Chem. 1985, 292, 269−273. (c) Chohan, Z. H. Antibacterial and antifungal ferrocene incorporated dithiothione and dithioketone compounds. Appl. Organomet. Chem. 2006, 20, 112− 116. (d) Chohan, Z. H.; Praveen, M. Synthesis, characterization and antibacterial properties of symmetric 1,1′-ferrocene derived Schiffbase ligands and their Co(II), Cu(II), Ni(II) and Zn(II) chelates. Appl. Organomet. Chem. 2000, 14, 376−382. (e) Kowalski, K.; Skiba, J.; Oehninger, L.; Ott, I.; Solecka, J.; Rajnisz, A.; Therrien, B. Metallocene-Modified Uracils: Synthesis, Structure, and Biological Activity. Organometallics 2013, 32, 5766−5773. (f) Tiwari, K. N.; Monserrat, J.-P.; Hequet, A.; Ganem-Elbaz, C.; Cresteil, T.; Jaouen, G.; Vessières, A.; Hillard, E. A.; Jolivalt, C. In vitro inhibitory properties of ferrocene-substituted chalcones and aurones on bacterial and human cell cultures. Dalton Trans. 2012, 41, 6451−6457. (25) (a) Edwards, E. I.; Epton, R.; Marr, G. Organometallic derivatives of penicillins and cephalosporins a new class of semisynthetic antibiotics. J. Organomet. Chem. 1975, 85, C23−C25. (b) Edwards, E. I.; Epton, R.; Marr, G. 1,1′-Ferrocenyldiacetic Acid Anhydride and its Use in the preparation of heteroannularly substituted ferrocenyl-penicillins and -cephalosporins. J. Organomet. Chem. 1976, 122, C49−C53. (c) Scutaru, D.; Mazilu, I.; Vâta̧ ,̌ M.; Tǎtaru, L.; Vlase, A.; Lixandru, T.; Simionescu, C. Heterodisubstituted derivatives of ferrocene. Ferrocene-containing penicillins and cephalosporins. J. Organomet. Chem. 1991, 401, 87−90. (26) van Staveren, D. R.; Metzler-Nolte, N. Bioorganometallic Chemistry of Ferrocene. Chem. Rev. 2004, 104, 5931−5986. (27) Bennett, M. A.; Smith, A. K. Arene ruthenium(II) complexes formed by dehydrogenation of cyclohexadienes with ruthenium(III) trichloride. J. Chem. Soc., Dalton Trans. 1974, 233−241. (28) Bhadbhade, M. M.; Das, A.; Jeffery, J. C.; McCleverty, J. A.; Badiola, J. A. N.; Ward, M. D. Redox-responsive Bi- and Tri-nuclear iron/molybdenum complexes incorporating the ferrocenyl unit as a redox spectator. J. Chem. Soc., Dalton Trans. 1995, 2769−2777. (29) Forgie, J. C.; El Khakani, S.; MacNeil, D. D.; Rochefort, D. Electrochemical characterisation of a lithium-ion battery electrolyte based on mixtures of carbonates with a ferrocene-functionalised imidazolium electroactive ionic liquid. Phys. Chem. Chem. Phys. 2013, 15, 7713−7721. (30) Simal, F.; Sebille, S.; Hallet, L.; Demonceau, A.; Noels, A. F. Evaluation of ruthenium-based catalytic systems for the controlled atom transfer radical polymerisation of vinyl monomers. Macromol. Symp. 2000, 161, 73−86. (31) Haynes, W. M. CRC Handbook of Chemistry and Physics; CRC Press/Taylor and Francis: Boca Raton, FL, 2015; Vol. 133. (32) Parkin, A.; Oswald, I. D. H.; Parsons, S. Structures of piperazine, piperidine and morpholine. Acta Crystallogr., Sect. B: Struct. Sci. 2004, 60, 219−227. (33) Dunitz, J. D.; Orgel, L. E.; Rich, A. The crystal structure of ferrocene. Acta Crystallogr. 1956, 9, 373−375. (34) Cabeza, J. A.; da Silva, I.; del Río, I.; García-Granda, S. Crystallographic report: [N,N′-Bis-(6-methylpyrid-2-ylium)-(1R,2R)1,2-diaminocyclohexane] bis-[(p-cymene)-trichlororuthenate(II)]. Appl. Organomet. Chem. 2005, 19, 209−210. (35) Helm, L.; Merbach, A. E. Water exchange on metal ions: experiments and simulations. Coord. Chem. Rev. 1999, 187, 151−181. M


Article

Inorganic Chemistry (50) Antibiotics: Actions, Origins, Resistance; Walsh, C., Ed.; ASM Press, 2003; p 335. (51) (a) Drlica, K.; Malik, M.; Kerns, R. J.; Zhao, X. Quinolonemediated bacterial death. Antimicrob. Agents Chemother. 2008, 52, 385−392. (b) Floss, H. G.; Yu, T.-W. Rifamycin-Mode of Action, Resistance, and Biosynthesis. Chem. Rev. 2005, 105, 621−632. (52) Tomasz, A. The mechanism of the irreversible antimicrobial effects of penicillins: how the beta-lactam antibiotics kill and lyse bacteria. Annu. Rev. Microbiol. 1979, 33, 113−137. (53) Vakulenko, S. B.; Mobashery, S. Versatility of aminoglycosides and prospects for their future. Clin. Microbiol. Rev. 2003, 16, 430− 450. (54) (a) Steinbuch, K. B.; Fridman, M. Mechanisms of resistance to membrane-disrupting antibiotics in Gram-positive and Gram-negative bacteria. MedChemComm 2016, 7, 86−102. (b) Lo, A.; Castaldo, G.; Remaut, H. Bacterial Membranes as Drug Targets; Caister Academic Press, 2014; pp 449−489. (c) Bolla, J.-M.; Alibert-Franco, S.; Handzlik, J.; Chevalier, J.; Mahamoud, A.; Boyer, G.; KiećKononowicz, K.; Pagès, J.-M. Strategies for bypassing the membrane barrier in multidrug resistant Gram-negative bacteria. FEBS Lett. 2011, 585, 1682−1690. (d) Vooturi, S. K.; Firestine, S. M. Synthetic membrane-targeted antibiotics. Curr. Med. Chem. 2010, 17, 2292− 2300. (e) Lohner, K. New strategies for novel antibiotics: peptides targeting bacterial cell membranes. Gen. Physiol. Biophys. 2009, 28, 105−116. (55) (a) Kohanski, M. A.; Dwyer, D. J.; Hayete, B.; Lawrence, C. A.; Collins, J. J. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 2007, 130, 797−810. (b) Dwyer, D. J.; Kohanski, M. A.; Hayete, B.; Collins, J. J. Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli. Mol. Syst. Biol. 2007, 3, 91. (c) Kohanski, M. A.; Dwyer, D. J.; Wierzbowski, J.; Cottarel, G.; Collins, J. J. Mistranslation of membrane proteins and two-component system activation trigger antibiotic-mediated cell death. Cell 2008, 135, 679−690. (56) (a) Dubar, F.; Egan, T. J.; Pradines, B.; Kuter, D.; Ncokazi, K. K.; Forge, D.; Paul, J.-F.; Pierrot, C.; Kalamou, H.; Khalife, J.; Buisine, E.; Rogier, C.; Vezin, H.; Forfar, I.; Slomianny, C.; Trivelli, X.; Kapishnikov, S.; Leiserowitz, L.; Dive, D.; Biot, C. The Antimalarial Ferroquine: Role of the Metal and Intramolecular Hydrogen Bond in Activity and Resistance. ACS Chem. Biol. 2011, 6, 275−287. (b) Long, B.; He, C.; Yang, Y.; Xiang, J. Synthesis, characterization and antibacterial activities of some new ferrocene-containing penems. Eur. J. Med. Chem. 2010, 45, 1181−1188. (c) Dallas, A.; Kuhtz, H.; Farrell, A.; Quilty, B.; Nolan, K. Versatile reagents: ferrocenyl azolium compounds as auxiliary ligands for the Heck reaction and potential antifungal agents. Tetrahedron Lett. 2007, 48, 1017−1021. (d) Howarth, J.; Hanlon, K. Novel N-ferrocenylmethyl, N′-methyl-2substituted benzimidazolium iodide salts with in vitro activity against the P. falciparum malarial parasite strain NF54. Tetrahedron Lett. 2001, 42, 751−754. (e) Howarth, J.; Hanlon, K. N-Ferrocenylmethyl, N′-Methyl-2-substituted benzimidazolium iodide salts with In vitro activity against the Leishmania infantum parasite strain L1. Bioorg. Med. Chem. Lett. 2003, 13, 2017−2020. (f) El Arbi, M.; Pigeon, P.; Top, S.; Rhouma, A.; Aifa, S.; Rebai, A.; Vessières, A.; Plamont, M.-A.; Jaouen, G. Evaluation of bactericidal and fungicidal activity of ferrocenyl or phenyl derivatives in the diphenyl butene series. J. Organomet. Chem. 2011, 696, 1038−1048. (57) Zabic, M.; Kukric, Z.; Topalic-Trivunovic, L. Influence of ferrocene and its derivatives on growth of Escherichia coli (ATTC 25922). Chem. Ind. Chem. Eng. Q. 2009, 15, 251−256. (58) Beagley, P.; Blackie, M. A. L.; Chibale, K.; Clarkson, C.; Meijboom, R.; Moss, J. R.; Smith, P. J.; Su, H. Synthesis and antiplasmodial activity in vitro of new ferrocene-chloroquine analogues. Dalton Trans. 2003, 3046−3051. (59) (a) Stubbings, W. J.; Bostock, J. M.; Ingham, E.; Chopra, I. Assessment of a microplate method for determining the postantibiotic effect in Staphylococcus aureus and Escherichia coli. J. Antimicrob. Chemother. 2004, 54, 139−143. (b) Shapiro, E.; Baneyx, F. Stress-based identification and classification of antibacterial agents:

second-generation Escherichia coli reporter strains and optimization of detection. Antimicrob. Agents Chemother. 2002, 46, 2490−2497. (c) Edberg, S. C.; Miskin, A. Turbidimetric determination of blood aminoglycoside levels by growth curve analysis. J. Pharm. Sci. 1980, 69, 1442−1443. (60) Mattila, T. A modified Kelsey-Sykes method for testing disinfectants with 2,3,5-triphenyltetrazolium chloride reduction as an indicator of bacterial growth. J. Appl. Bacteriol. 1987, 62, 551−554. (61) Adams, M. R.; Hall, C. J. Growth inhibition of food-borne pathogens by lactic and acetic acids and their mixtures. Int. J. Food Sci. Technol. 1988, 23, 287−292. (62) Tiina, M.; Sandholm, M. Antibacterial effect of the glucose oxidase-glucose system on food-poisoning organisms. Int. J. Food Microbiol. 1989, 8, 165−174. (63) Skyttä, E.; Mattila-Sandholm, T. A quantitative method for assessing bacteriocins and other food antimicrobials by automated turbidometry. J. Microbiol. Methods 1991, 14, 77−88. (64) Wenzel, M.; Patra, M.; Senges, C. H. R.; Ott, I.; Stepanek, J. J.; Pinto, A.; Prochnow, P.; Vuong, C.; Langklotz, S.; Metzler-Nolte, N.; Bandow, J. E. Analysis of the Mechanism of Action of Potent Antibacterial Hetero-tri-organometallic Compounds: A Structurally New Class of Antibiotics. ACS Chem. Biol. 2013, 8, 1442−1450. (65) (a) Imlay, J.; Chin, S.; Linn, S. Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science 1988, 240, 640−642. (b) Imlay, J. A.; Linn, S. Bimodal pattern of killing of DNA-repair-defective or anoxically grown Escherichia coli by hydrogen peroxide. J. Bacteriol. 1986, 166, 519−527. (66) Beers, M.; Fletcher, A. J.; Jones, T. V.; Berkow, R.; Merck Publishing Group The Merck Manual of Medical Information: Home Edition, 2nd ed.; Elsevier Health Sciences: London, United Kingdom, 2003. (67) Gorrini, C.; Harris, I. S.; Mak, T. W. Modulation of oxidative stress as an anticancer strategy. Nat. Rev. Drug Discovery 2013, 12, 931−947. (68) Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T. D.; Mazur, M.; Telser, J. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 2007, 39, 44− 84. (69) Lau, A. T. Y.; Wang, Y.; Chiu, J.-F. Reactive oxygen species: current knowledge and applications in cancer research and therapeutic. J. Cell. Biochem. 2008, 104, 657−667. (70) Trachootham, D.; Alexandre, J.; Huang, P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discovery 2009, 8, 579−591. (71) Milaeva, E. R. Metal-based antioxidants - potential therapeutic candidates for prevention the oxidative stress - related carcinogenesis: mini-review. Curr. Top. Med. Chem. 2011, 11, 2703−2713. (72) Liu, Z.-Q. Potential applications of ferrocene as a structural feature in antioxidants. Mini-Rev. Med. Chem. 2011, 11, 345−358. (73) Bonfili, L.; Pettinari, R.; Cuccioloni, M.; Cecarini, V.; Mozzicafreddo, M.; Angeletti, M.; Lupidi, G.; Marchetti, F.; Pettinari, C.; Eleuteri, A. M. Arene-RuII Complexes of Curcumin Exert Antitumor Activity via Proteasome Inhibition and Apoptosis Induction. ChemMedChem 2012, 7, 2010−2020. (74) Blois, M. S. Antioxidant determinations by the use of a stable free radical. Nature 1958, 181, 1199−1200. (75) Romero-Canelón, I.; Pizarro, A. M.; Habtemariam, A.; Sadler, P. J. Contrasting cellular uptake pathways for chlorido and iodido iminopyridine ruthenium arene anticancer complexes. Metallomics 2012, 4, 1271−1279. (76) Gasser, G.; Ott, I.; Metzler-Nolte, N. Organometallic Anticancer Compounds. J. Med. Chem. 2011, 54, 3−25. (77) Prins, R.; Korswagen, A. R.; Kortbeek, A. G. T. G. Decomposition of the ferricenium cation by nucleophilic reagents. J. Organomet. Chem. 1972, 39, 335−344. (78) Perera, R. M.; Bardeesy, N. When antioxidants are bad. Nature 2011, 475, 43−44. N


Article

Inorganic Chemistry (79) Hübschle, C. B.; Sheldrick, G. M.; Dittrich, B. ShelXle: a Qt graphical user interface for SHELXL. J. Appl. Crystallogr. 2011, 44, 1281−1284. (80) Farrugia, L. J. ORTEP-3 for Windows - a version ofORTEP-III with a Graphical User Interface (GUI). J. Appl. Crystallogr. 1997, 30, 565. (81) Persistence of Vision (TM) Raytracer; Persistence of Vision Pty. Ltd: Victoria, Australia. http://www.povray.org/ (accessed 2013). (82) Brown, M. R. W. Turbidimetric method for the rapid evaluation of antimicrobial agents–inactivation of preservatives by nonionic agents. J. Soc. Cosmet. Chem. 1966, 17, 185−192. (83) (a) Brocklehurst, K. R.; Morby, A. P. Metal-ion tolerance in Escherichia coli: analysis of transcriptional profiles by gene-array technology. Microbiology 2000, 146, 2277−2282. (b) Chekmenev, E. Y.; Vollmar, B. S.; Forseth, K. T.; Manion, M. N.; Jones, S. M.; Wagner, T. J.; Endicott, R. M.; Kyriss, B. P.; Homem, L. M.; Pate, M.; He, J.; Raines, J.; Gor’kov, P. L.; Brey, W. W.; Mitchell, D. J.; Auman, A. J.; Ellard-Ivey, M. J.; Blazyk, J.; Cotten, M. Investigating molecular recognition and biological function at interfaces using piscidins, antimicrobial peptides from fish. Biochim. Biophys. Acta, Biomembr. 2006, 1758, 1359−1372. (c) Lauth, X.; Shike, H.; Burns, J. C.; Westerman, M. E.; Ostland, V. E.; Carlberg, J. M.; Van Olst, J. C.; Nizet, V.; Taylor, S. W.; Shimizu, C.; Bulet, P. Discovery and characterization of two isoforms of moronecidin, a novel antimicrobial peptide from hybrid striped bass. J. Biol. Chem. 2002, 277, 5030− 5039.

O


Activation by Oxidation: Ferrocene-Functionalized ... - ACS Publications

Recommend Documents