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Mono- and binuclear Ru(II) arene complexes with (fluoro substituted) picolinic acid: Synthesis, characterization and cytotoxicity

Four mono(1e4) and four binuclear Ru(II) arene (5e8) complexes have been isolated from the reaction of [Ru(h-benzene)Cl(m-Cl)]2 or [Ru(h-toluene)Cl(m-Cl)]2 with 2-pyridinecarboxylic acid and 6-fluoro-2pyridinecarboxylic acid. Their structural characterization included IR and NMR spectroscopy and MS spectrometry. The cytotoxic potential of the compounds has been tested by MTT assay in seven human cancer cell lines: alveolar basal adenocarcinoma (A549), large cell lung carcinoma (HTB177), colorectal carcinoma (HCT116), malignant melanoma (A375), prostate adenocarcinoma (PC3), breast carcinoma (MDA-MB-453), cervix adenocarcinoma (HeLa), and one human non-malignant lung fibroblast cell line (MRC-5). Mononuclear complexes 1 and 3 carrying 2-pyridinecarboxylic acid have displayed moderate antiproliferative effect toward HCT116 and HeLa, slightly better in comparison to their binuclear analogues, 5 and 7. The highest activity and cytoselectivity has been observed 1 as it has reduced viability of HCT116 cells 1.5 times more efficiently (IC501⁄4 27.5 mM), than of the MRC-5 cells (IC501⁄4 41.3 mM). In contrast to 1 and 3, compounds 2, 4e8 have been found to exhibit lack of cytotoxicity or mild cytotoxicity with IC50 values ranging from 100 to 300 mM. © 2019 Elsevier B.V. All rights reserved.


Introduction
A history of inorganic medicinal chemistry dates back to the mid-1960s when the discovery of cisplatin (Fig. 1), a powerful antitumor agent, occurred [1]. Although established as a milestone metallodrug, cisplatin's therapeutic value has been limited by significant side effects such as drug resistance and non-specific toxicity [2]. Intensive studies in this field expanded the serie of metallodrugs from platinum to other transition metal complexes such as Ru, Au, Rh, Ir, Os, Re and even Fe and Mo ( Fig. 1) [3]. Among numerous metal-ligand scaffolds, Ru(II/III) complexes singled out as the most promising alternative to platinum-based chemotherapeutics [4]. Two ruthenium derivatives, imidazolium trans-DMSOimidazole-tetrachlororuthenate (NAMI-A, Fig. 1) [5] and indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP-1019) [6] have been tested in clinical trials while the sodium analogue of KP1019, NKP-1339 [7], is set to enter the clinical trials.
The prominent Ru complexes are generally designed to enable high kinetic stability through control ligand substitution and redox reactions allowing the complex to remain intact on the way to the final target [8]. Particular emphasis is given on the functionalized h 6 -arene Ru complexes with three additional coordination sites available for introducing N-, O-, S-or P-donor species and also convenient for fine tuning of their steric and electronic properties [9]. The benefit of the Ru-arene structural flexibility is reflected through their diverse areas of implementations, e.g. catalysis [10], chemical and photochemical sensing [11], supramolecular chemistry [12] and medicinal chemistry [13]. In terms of potential clinical use, advantageous features such as good aqueous solubility and relatively inert arene ligand make them very attractive for structural optimizations aimed for improved in vivo potency [14]. In reactions with two-electron donor ligands mononuclear complexes of general formula [(h 6 -arene)RuCl 2 (L)] are formed [15], while reactions with bidentate ligands result in a formation of binuclear species [(h 6 -arene)RuCl(LÀL)] þ [16]. Reported binuclear assemblies are rare in literature although they exhibit a whole variety of relevant biological effects such as significantly high affinity of binding to DNA [17] or promising cytotoxic effects in human cancer cells gained through cooperatively acting of both Ru centers [18]. Moreover, the appealing structural diversity of Ru complexes can be also functionalized by the effective synergism of both ligands, arene and chelating moiety [19]. In that sense, the literature suggests a rational use of bioinspired molecules with specific roles in certain biological systems. One of the commonly used chelating agent is picolinic acid, a naturally occurring product of tryptophan degradation found in various biological mediums including blood serum, human milk and pancreatic juice [20]. With two different ligator atoms, pyridinecarboxylic acid represents desirable ligand model whose transition metal complexes (e.g. Pt(II), Fe(II), Co(II), Al(III)) have been widely investigated [21e23]. Among them, Cr(III) picolinate derivatives are the most recognizable due to their massive use as nutrition additives [24]. Numerous studies investigated the biological activity of this type of complexes regarding their potential antitumor and antituberculosis activity [25].
Starting from 2011 Grguri c-Sipka and co-workers explored various sets of Ru-arene complexes with 2-pyridinecarboxylic (picolinic) acid and its halogen (fluoro-, chloro-, bromo-) or methyl-substituted derivatives and reported their cytotoxic profile [26]. In order to further contribute the field regarding this interesting model systems, eight new Ru(II)-arene (benzene and toluene) complexes bearing picolinic acid and 6-fluoropicolinic acid were prepared. The first set of compounds included the preparation of mononuclear Ru(II) complexes whose structure was further optimized by reacting them with a suitable linker, N,N 0di(4-pyridinyl)ethanediamide (bpo). The obtained binuclear assembles were primarily inspired with arene ruthenium metallarectangles highly potent towards human ovarian cancer cells with a pronounced selectivity for cancer over healthy cells [27]. The novel compounds were spectroscopically characterized when the coordination mode of N,O ligand was confirmed, identically like in earlier studies. The cytotoxic activity of the reported compounds was investigated by the colorimetric MTT assay in a panel of one human non-malignant cell line , and seven human cancer cell lines (A549, HTB177, PC3, A375, HeLa, HCT116, MDA-MB-453). The obtained results were discussed in terms of comparison between cytotoxic potential of newly synthesized compounds and published Ru(II) picolinato complexes [26] and the examination of the structure-activity correlations between mononuclear species and their corresponding binuclear analogues.

Preparation and characterization of 1e4
The reaction of [Ru(h 6 -benzene)Cl(m-Cl)] 2 or [Ru(h 6 -toluene) Cl(m-Cl)] 2 with picolinic acid or 6-fluoropicolinic acid in a 1:2 M ratio in ethanol gives rise to 1e4 with more than 70% yields (Scheme 1). The complexes are soluble in DMSO, acetone, chloroform, dichloromethane, and methanol, and insoluble in water, petroleum ether and diethyl ether.
The IR spectra of 1-4 point out the typically intensive C]O vibration observed in the area from 1670 to 1640 cm À1 . The significant difference between wavenumbers for C]O vibration in the spectra of free ligands (ca. 1720 cm À1 ) occurs due to the coordination of the ruthenium(II) center via carboxylic oxygen [26a]. The complexes also display stretching bands at ca. 1600 cm À1 assigned to the C]N vibration.
In 1 H NMR spectra of 1 and 3 (Figs. S1 and S3, ESI) with picolinic acid, the significant shift of the pyridine protons is observed due to the coordination via pyridine nitrogen (C3, C4 and C6 are shifted downfield while C5 is upfield). Additional conformation of the proposed coordination via carboxylic oxygen is noticed in a significant shift of COO À group from 166.22 ppm in free picolinic acid to ca. 170 ppm in 13 C NMR spectra of 1 and 3. 1 H NMR spectra of 2 and 4 (Figs. S2 and S4, ESI) bearing 6-fluoropicolinic acid display quartets for C4 which are shifted downfield in a comparison to the free ligand while resonance of C3 located at ca. 7.74 ppm is shifted upfield. Methyl groups from toluene moiety are dislocated downfield with minor shifts around 0.05 ppm. 13 C NMR spectra confirmed coordination via carboxylic oxygen as the COO À signal is shifted from ca. 164 ppm to ca. 170 ppm.
In the EI-MS spectra of 1-4 the [M þ ] signal was detected. 1 and 2 fragmented in the same manner giving rise to [MePhH] þ ion while 3 and 4 pointed out on [M À CO 2 ] þ fragments.

Preparation and characterization of 5e8
The reaction of two equivalents of previously synthesized complex (1, 2, 3, and 4) with an equivalent of N,N 0 -di(4-pyridinyl) ethanediamide in methanol and the presence of AgCF 3 SO 3 afforded 5e8 with a good yield (Scheme 2). The complexes are soluble in DMSO, acetone, chloroform, dichloromethane, and methanol, and insoluble in water, petroleum ether and diethyl ether. 1 H NMR spectra of 5--8 ( Fig. S5À8, ESI) were assigned due to the shifts of the NH protons and pyridine protons from ethanediamide connector. Due to the formation of Ru assemblies, NH protons marked obvious dislocation from 11.27 ppm to ca. 11.60 ppm. C a protons are also shifted downfield while C b protons are slightly shifted upfield.
In the EI-MS spectra of 5 and 6 the [M þ ] and [MePhH] þ signals were detected, while 7 and 8 suffered total fragmentation.

Chemical behavior of the Ru(II)-arenes in DMSO
Numerous studies concerning the stability of the Ru(II)-arene type of complexes were performed in order to predict their fate under the physiological conditions [28]. As water and DMSO are commonly used solvents for biological assay experiments, chemical behavior is usually followed by 1 H NMR spectral changes in deuterated DMSO, as a function of time and later on compared to precursor compounds.
The stability of reported complexes 1e8 were found to be completely stable in DMSO after 72 h long monitoring (representative spectra of 1 and 5 shown as Figs. S9 and S10 in ESI). Since all NMR spectra showed no traces of the ligand dissociation or any other kind of decomposition, it is concluded that this type of Ru(II)arenes surprisingly remains intact in DMSO.

Cytotoxicity analysis
The cytotoxicity of the eight reported complexes 1e8 was investigated by the colorimetric MTT assay, in a panel of one human non-malignant cell line , and seven human cancer cell lines (A549, HTB177, PC3, A375, HeLa, HCT116, MDA-MB-453). Results obtained after 72 h of continuous drug action, are presented as IC 50 values (mM), in Table 1. Well-known chemotherapeutic agent Scheme 2. Synthesis of the binuclear complexes, 5e8.
cisplatin was also tested as reference compound.
Mononuclear Ru(II) complexes 1 and 3 carrying picolinic acid, displayed moderate antiproliferative effect particularly toward colorectal carcinoma (HCT116) and cervix adenocarcinoma cells (HeLa) (Fig. 2). The highest activity and cytoselectivity was observed for mononuclear complex 1 toward HCT116 cells: it was capable of reducing viability of HCT116 cells 1.5 times more efficiently (IC 50 ¼ 27.5 mM), than of the MRC-5 cells (IC 50 ¼ 41.3 mM). In a contrast to these mononuclear species, their binuclear analogues, 5 and 7 did not exhibit much of improvement in antiproliferative activity or selectivity toward cancer cells as the IC 50 values exceeded 100 mM. Moreover, both mono-and binuclear compounds bearing 6-fluoro substituent were found to exhibit lack of cytotoxicity or mild cytotoxicity resulting in IC 50 values from 100 to 300 mM.
Design and synthesis of polynuclear complexes are challenging comparing to the synthesis of mononuclear complexes, regarding the optimization of structure-activity correlations. It is more complicated to predict the effect of stereochemistry of polynuclear metal complexes on their biological properties in vitro, and their reactivity toward biomolecules, such as proteins or nucleic acids, in physiological environment. One successful example of polynuclear metal complex, which reached clinical trials, is trinuclear platinum complex BBR3464 which exhibits different mechanism of action comparing to cisplatin, and is active in human tumor xenografts resistant or poorly responsive to cisplatin, as well as in p53 mutant carcinoma cells [29]. BR 3464 also produces high levels of DNA lesions in the cell, different from cisplatin. However, literature on polynuclear ruthenium complexes and their in vitro antitumor potential is rare. Some previous studies indicated that there was no direct correlation between the number of metal centers and cytotoxicity of the metal complexes, since polynuclear metal core can affect compound solubility, its ability to pass through the cell membrane or to slow or interfere with binding dynamics in the cell [30,31]. In our case, synthesized binuclear Ru(II) analogues 5e8 did not show positive effect in terms of their cytotoxic activity, (IC 50 values were in the wide range of concentrations up to 300 mM, or above). For the most of compounds cytotoxicity against non-tumor (MRC-5) cell line, was found slightly higher compared to the tumor cells, with selectivity index (SI), comparable to cisplatin in tested cell lines, ranging from 0.2 to 1.5 (Table TS1, ESI). The highest SI values were obtained in cervical (HeLa) and colorectal (HCT116) carcinoma cells, however, compounds with SI value < 2 are considered to give general toxicity, i.e., it can also cause cytotoxicity on normal cells. Factors responsible for moderate tumor cells response to binuclear ruthenium complexes may also account to mechanisms associated with multidrug resistance, or elevated cellular glutathione (GSH) content [32]. Tested binuclear complexes may be also able to form different interactions with biomolecules, in comparing to cisplatin, such as cross-links (protein-protein or DNA-protein), though further study is needed, in order to precisely determine their capacity to accumulate in the cell, intracellular drug targets and the mechanism of cell response. Surprisingly, mononuclear Ru(II) complexes, 1 and 3, displayed moderate antiproliferative effect toward HCT116 and HeLa, significantly stronger in comparison to their binuclear analogues, 5 and 7. Our previous study [26b] on mononuclear Ru(II) arene complexes carrying picolinato ligand, indicated the ability of this type of complexes to exhibit inhibitory effect on cell adhesion, migration and angiogenesis, regardless of their antiproliferative effect. On the basis of these considerations, further in vitro and in vivo antitumor study is needed in order to determine whether these novel mono-or polynuclear Ru(II) compounds could potentially affect cellular migratory or invasive properties.
Elemental analyses (C, H and N) were carried out on Elemental Vario EL III microanalyzer (for 2, 6 and 7) and Carlo Erba microanalyser (for 3). The infrared spectra were recorded on a Nicolet 6700 FT-IR spectrometer using ATR technique. The signal intensities are reported in wavenumbers and denoted by the following abbreviations: vs ¼ very strong, s ¼ strong, m ¼ medium and w ¼ weak. The NMR ( 1 H and 13 C) spectra were recorded on a Bruker Avance III 500 instrument in DMSO-d 6 with TMS as the reference. The mass spectra of the mononuclear complexes were obtained with an Agilent Technologies 5975C inert XL MSD instrument using the direct insertion technique while the spectra for binuclear complexes were recorded with ultra performance liquid chromatography and Electrospray ionization mass spectrometry (UPLC-MS) by using an Acquit system from Gilson and a Waters X-Bridge BEH130. Spectral data of 1À8 are given in Supplementary material (ESI) to this paper.

Synthesis of the binuclear Ru(II) complexes, 5e8
Previously synthesized complex (1e4) (0.24 mmol, 2 eqs) was dissolved in methanol and AgCF 3 SO 3 (0.26 mmol, 2.2 eqs) was added. The reaction mixture was left stirring in the following 3 h at room temperature. The obtained precipitate was filtered off while the solution was used to further react with N,N 0 -di(4-pyridinyl) ethanediamide (0.12 mmol, 1 Eq). The reaction mixture was left stirring overnight at room temperature. The obtained solution was evaporated to the half of its volume, and diethylether was slowly added dropwise. Orange-red precipitate was isolated and rinsed with cold ethanol and diethylether (Scheme 2).

Cytotoxicity analysis
For determination of cell viability in cultures, we used the mitochondrial-dependent reduction of 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan as described elsewhere [35,36]. Cells were seeded into 96-well culture plates (Thermo Scientific Nunc™) at cell densities of 5000 c/w (MRC-5, A549, HTB177, HCT116), 3000 c/w (PC3, A375, MDA-MB-453) or 2000 c/w (HeLa) in 100 mL of cell culture medium, and left overnight. Eight tested agents were dissolved in DMSO to the stock concentration of 30 mM immediately before experiment, whereas further dilutions were made in the culture medium, so that the final concentration of DMSO never exceeded 1% (v/v). Triplicate wells were treated with varying concentrations of tested compounds (18.75 mM, 37.5 mM, 75 mM, 150 mM and 300 mM), in the final volume of 150 mL cell culture medium, per well. Cisplatin (cisdiamminedichloridoplatinum(II), CDDP) was used in this study as the reference compound. After 72 h of continuous incubation at 37 C in 5% CO 2 humidified atmosphere, 20 mL of MTT solution (Sigma-Aldrich), was added to each well (5 mg/mL). The culture plates were incubated for the next 4 h at 37 C, and finally 100 mL of 10% sodium dodecyl sulfate (SDS) was added to dissolve formed formazan crystals. Absorbances were measured after 24 h at a wavelength of 570 nm on a microplate reader (Thermo Labsystems Multiscan EX 200e240 V). The modification in cell viability was expressed as percentage of viability in treated samples compared to untreated control samples (taken as 100%), corrected for values obtained for wells without plated cells. The IC 50 values (concentration of investigated compound that cause 50% decrease in the number of viable cells in a treated cell population compared to a non-treated control), were determined from the cell survival diagrams.

Conclusions
New mono-and binuclear ruthenium(II) complexes with picolinic acid and its fluoro derivative were synthesized and characterized by various methods (IR, NMR, MS). Four mononuclear Ru compounds were obtained reacting [Ru(h 6 -benzene)Cl(m-Cl)] 2 or [Ru(h 6 -toluene)Cl(m-Cl)] 2 with the ligand (picolinic acid and 6fluoropicolinic acid). The coordination mode was typical for this type of ligands, via pyridine nitrogen and carboxylic oxygen. Furthermore, the novel compounds were used as precursor complexes for obtaining their corresponding binuclear analogues with N,N 0 -di(4-pyridinyl)ethanediamide as a suitable connector. However, in vitro cytotoxicity of binuclear assemblies has surprisingly revealed the lack of cytotoxicity or mild cytotoxicity. On the other hand, precursor compounds, namely mononuclear benzene and toluene complexes bearing picolinic acid demonstrated moderate antiproliferative effect particularly toward HCT116 and HeLa. In a comparison to the compounds already reported in literature [26a,26f], these complexes displayed improved activity and selectivity. Therefore further investigations regarding their binding modes, sites, and affinities will be the topic of our interest.

Declaration of competing interest
There are no conflicts to declare.