Half-Sandwich Ru(II) Halogenido, Valproato and 4-Phenylbutyrato Complexes Containing 2,2′-Dipyridylamine: Synthesis, Characterization, Solution Chemistry and In Vitro Cytotoxicity

Halogenido and carboxylato Ru(II) half-sandwich complexes of the general composition [Ru(η6-p-cym)(dpa)X]PF6 (1–5) were prepared and thoroughly characterized with various techniques (e.g., mass spectrometry, NMR spectroscopy and X-ray analysis); dpa = 2,2′-dipyridylamine; p-cym = p-cymene; X = Cl− (for 1), Br− (for 2), I− (for 3), valproate(1−) (for 4) or 4-phenylbutyrate(1−) (for 5). A single-crystal X-ray analysis showed a pseudo-octahedral piano-stool geometry of [Ru(η6-p-cym)(dpa)I]PF6 (3), with a η6-coordinated p-cymene, bidentate N-donor dpa ligand and iodido ligand coordinated to the Ru(II) atom. The results of the 1H-NMR solution behaviour studies proved that the complexes 1–5 hydrolyse were in the mixture of solvents used (10% MeOD-d4/90% D2O). Complexes 1–5 were in vitro inactive against the A2780 human ovarian carcinoma cell line, up to the highest tested concentration (IC50 > 100 μM).

In this work, we decided to combine the above-described approaches, and study the effect of the replacement of the chlorido ligand with other halogenido ligands (a known approach utilized, for example, in the reference [3]) as well as by the VP and PB ligands (innovative approach in the field of Ru(II) anticancer complexes) on the in vitro cytotoxicity against the A2780 cells. For this purpose, we chose the easily-obtainable [Ru(η 6 -p-cym)(dpa)Cl]PF6 complex ( Figure 1) [10], recently reported as having moderate, and thus possibly tunable, in vitro cytotoxicity against the MCF-7 cancer cells (IC50 = 40.8 μM) [11]. From the coordination chemistry point of view, this is the first report on Ru(II) complexes containing the VP ligand. Regarding the ruthenium complexes containing the PB ligand, they have been used as either bidentate O,O-coordinated ligand in the [Ru(μ-PB)(CO)2(L 2 )]2 complex, or as η 6 -coordinated 4-phenylbutyric acid (PBA) in the [Ru(η 6 -PBA)(L 3 )Cl] + or [Ru(η 6 -ar)(η 6 -PBA)] 2+ complexes; L 2 = pyridine or triphenylphosphane, L 3 = e.g., 1,10-phenanthroline or benzaldehyde monothiosemicarbazone, and ar = e.g., 1,2,3,4,5,6-hexamethylbenzene [12][13][14]. However, to the best of our knowledge, neither VP nor PB ligand has been reported in the literature to date as the monodentate O-donor ligand of any ruthenium complex.

Synthesis and General Properties
Recently, the preparation [10,11,15,16], X-ray structure [10] and in vitro cytotoxicity [11] of the simple ruthenium(II) complex [Ru(η 6 -p-cym)(dpa)Cl]PF6 were reported. In this work, its structure was modified by either different halogenido ligands (bromido or iodido) or monodentate O-coordinated carboxylato ligands (valproato or 4-phenylbutyrato). The obtained products were thoroughly characterized, their solution chemistry was studied by 1 H-NMR and their in vitro cytotoxicity against the A2780 cells was assessed.

Synthesis and General Properties
Recently, the preparation [10,11,15,16], X-ray structure [10] and in vitro cytotoxicity [11] of the simple ruthenium(II) complex [Ru(η 6 -p-cym)(dpa)Cl]PF 6 were reported. In this work, its structure was modified by either different halogenido ligands (bromido or iodido) or monodentate O-coordinated carboxylato ligands (valproato or 4-phenylbutyrato). The obtained products were thoroughly characterized, their solution chemistry was studied by 1 H-NMR and their in vitro cytotoxicity against the A2780 cells was assessed.
The peaks whose mass corresponded to the complex cations, i.e., [Ru(p-cym)(dpa)X] + , of the studied complexes 1-5 were detected in the ESI+ mass spectra (Figure 2). A release of the halogenido (for 1-3) or carboxylato (for 4 and 5) ligands under the used electrospray ionization conditions led to the [Ru(p-cym)(dpa-H)] + species, whose peaks appeared at 406.2 m/z uniformly for all complexes 1-5. The prepared complexes of the general formula [Ru(η 6 -p-cym)(dpa)X]PF6 ( Figure 1) were thoroughly characterized, using elemental analysis, RP-HPLC, FTIR spectroscopy, ESI+ mass spectrometry, 1 H-NMR and 13 C-NMR spectroscopy, and a single crystal X-ray analysis. The composition of the complexes 1-5 was proved by the results of elemental analysis, with up to 0.4% differences between the calculated and found contents of C, H, and N. The RP-HPLC experiments proved >99% purity of the complexes 1-5. FTIR spectra of the complexes 1-5 contained the characteristic peaks of ν(C-H)aliph (2930-2980 cm −1 ) of p-cymene, as well as ν(C-H)ar (3000-3200 cm −1 ), and ν(C-C)ar and ν(C-N)ar (1435, 1470 and 1580 cm −1 ) of the dpa and p-cymene ligands [18]. The maxima detected at ca. 830 cm −1 belong to the ν(P-F) vibrations [19]. The peaks assignable to the characteristic vibrations of the VP (e.g., 2930, 1640 or 1380 cm −1 ) and PB (e.g., 2970, 1640, 1385 or 1300 cm −1 ) ligands [20,21] clearly showed in the FTIR spectra of the complexes 4 and 5. The peaks whose mass corresponded to the complex cations, i.e., [Ru(p-cym)(dpa)X] + , of the studied complexes 1-5 were detected in the ESI+ mass spectra ( Figure 2). A release of the halogenido (for 1-3) or carboxylato (for 4 and 5) ligands under the used electrospray ionization conditions led to the [Ru(p-cym)(dpa-H)] + species, whose peaks appeared at 406.2 m/z uniformly for all complexes 1-5. The 1 H-and 13 C-NMR spectra of the complexes 1-5 (measured in DMSO-d6) contained all the signals of both the p-cym and dpa ligands (Figure 3), and only the N2-H signal was not detected in the 1 H-NMR spectrum of complex 5. The septet of C17-H (i.e., H3C-CH-CH3) was overlapped by the signal of the used solvent for complexes 1-3, but this signal was clearly detected in the 2D NMR spectra, as well as in the 1 H-NMR spectra obtained in different solvents (e.g., MeOD-d4). The signals of C4-H, C5-H and C6-H shifted downfield as a consequence of the coordination of the dpa ligand to the Ru(II) atom, while the signals detected for C3-H showed strong upfield shifts (Table 1). The spectra obtained for the carboxylato complexes 4 and 5 contained, along with the signals of the p-cym and dpa ligands, the signals of the appropriate carboxylato ligands (Figure 3). The 13 C-NMR The 1 H-and 13 C-NMR spectra of the complexes 1-5 (measured in DMSO-d 6 ) contained all the signals of both the p-cym and dpa ligands (Figure 3), and only the N2-H signal was not detected in the 1 H-NMR spectrum of complex 5. The septet of C17-H (i.e., H 3 C-CH-CH 3 ) was overlapped by the signal of the used solvent for complexes 1-3, but this signal was clearly detected in the 2D NMR spectra, as well as in the 1 H-NMR spectra obtained in different solvents (e.g., MeOD-d 4 ). The signals of C4-H, C5-H and C6-H shifted downfield as a consequence of the coordination of the dpa ligand to the Ru(II) atom, while the signals detected for C3-H showed strong upfield shifts (Table 1). The spectra obtained for the carboxylato complexes 4 and 5 contained, along with the signals of the p-cym and dpa ligands, the signals of the appropriate carboxylato ligands (Figure 3). The 13 C-NMR signals of the carboxyl carbon atom (i.e., C21) were detected at ca. 181 ppm (for the VP-containing complex 4) and 179 ppm (for the PB-containing complex 5), and they were shifted by 4.0 ppm, and 4.7 ppm, respectively, as compared with the free carboxylic acids (i.e., VPA and PBA). The positions of these characteristic 13 C-NMR signals of the complexes 4 and 5 were consistent with those reported for trans-[Pt(NH 3 )(py)(VP) 2 ] (182.1 ppm), containing valproate(1−) as the monodentate O-donor ligand [22]. On the other hand, the formerly reported complexes [Ru(µ-PB)(CO) 2 (L 2 )] 2 , containing PB as the O-donor bridging ligand, and [Ru(η 6 -PBA)(L 3 )Cl] + or [Ru(η 6 -ar)(η 6 -PBA)] 2+ , containing PBA as η 6 -coordinated ligand, showed different 13 C-NMR δ values of their COO − signals. In particular, the δ COO-value for [Ru(µ-PB)(CO) 2 (L 2 )] 2 equals 186.5 ppm [12], while it equals 175.0 ppm for [Ru(η 6 -PBA)(L 3 )Cl] + [14]. Czomplexes 1-5 are stable in the used solvent (DMSO-d 6 ), because no changes were detected in their 1 H and 13 C-NMR spectra even after 48 h of standing at ambient temperature.  , given together with the general assignment of the observed signals, as follows; green dots for the dpa signals, blue dots for the p-cym signals and red dots for the VP signals.

Single Crystal X-ray Analysis
The complex [Ru(η 6 -p-cym)(dpa)I]PF6 (3) was characterized by a single crystal X-ray analysis. The crystal data and structure refinement are given in Table 2, while the selected bond lengths and angles can be found in Table 3.

Single Crystal X-ray Analysis
The complex [Ru(η 6 -p-cym)(dpa)I]PF 6 (3) was characterized by a single crystal X-ray analysis. The crystal data and structure refinement are given in Table 2, while the selected bond lengths and angles can be found in Table 3.
Both the aromatic rings of the dpa ligand form the dihedral angle of 37.00 (8) Table S1 and Figure S1).

1 H-NMR Studies of Solution Chemistry and Interactions with Reduced Glutathione
As it is known for the anticancer complexes of various transition metals (e.g., platinum(II) [28] or ruthenium(II) [29] complexes), hydrolysis of the M-Cl bond/s is an activation step of their action enabling the interaction with the target biomolecules including DNA. Moreover, it is necessary to ensure that the studied complexes do not decompose in water or water-containing solution mimicking physiological conditions, as recently described for similar half-sandwich Ru(II) complexes [30].
After dissolution of the halogenido complexes 1-3 in a mixture of 10% MeOD-d4/90% D2O, the new sets of 1 H-NMR signals of both the dpa and p-cym ligand were detected (e.g., at 7.90 ppm for C4-H or at 2.00 ppm for C20-H in the case of the complex 1). The formation of these new signals is most likely connected with the hydrolysis of the original complexes, showing in the formation of the [Ru(η 6 -p-cym)(dpa)(H2O)] 2+ and/or [Ru(η 6 -p-cym)(dpa)(OH)] + species [2]. The integral intensity ratios of the signals of the initial halogenido complexes (1-3) and their hydrolyzed forms observed after 48 h of standing at ambient temperature ( Figure S2) equaled approximately 1:3 (for 1), 2:3 (for 2) and 3:2 (for 3). In other words, 75% (1), 60% (2) and 40% (3) of the studied halogenido complexes hydrolyzed in the used mixture of solvents after 48 h of standing at ambient temperature. The evidence that the new 1 H-NMR signals detected in the spectra of the complexes 1-3 dissolved in a mixture of 10% MeOD-d4/90% D2O belong to the hydrolyzed complexes was obtained by the addition of 2 molar equivalents of KCl (for 1), KBr (for 2) or KI (for 3) to the appropriate equilibrated solutions, which overturned the hydrolysis progress, resulting in disappearance of the signals of hydrolysates after next 24 h of standing at ambient temperature ( Figure S2).
Glutathione (GSH) is a naturally occurring tripeptide (Glu-Cys-Gly), known to be responsible for the intracellular detoxification of various transition metals [31], including ruthenium [32]. Importantly, it has been reported that the decrease in the GSH level of cancer cells induced by . Non-hydrogen atoms are drawn as thermal ellipsoids at the 50% probability level. The PF 6 − counterion has been omitted for clarity.
Both the aromatic rings of the dpa ligand form the dihedral angle of 37.00(8) • , while the dihedral angles between theses rings and p-cymene ring equal 29.46(8) • (formed by the ring containing the N1 atom) and 27.40 (8) • (formed by the ring containing the N1A atom). The crystal structure of the complex 3 is stabilized by a variety of non-covalent contacts of the N-H···F and C-H···F types (Supplementary Materials Table S1 and Figure S1).

1 H-NMR Studies of Solution Chemistry and Interactions with Reduced Glutathione
As it is known for the anticancer complexes of various transition metals (e.g., platinum(II) [28] or ruthenium(II) [29] complexes), hydrolysis of the M-Cl bond/s is an activation step of their action enabling the interaction with the target biomolecules including DNA. Moreover, it is necessary to ensure that the studied complexes do not decompose in water or water-containing solution mimicking physiological conditions, as recently described for similar half-sandwich Ru(II) complexes [30].
After dissolution of the halogenido complexes 1-3 in a mixture of 10% MeOD-d 4 /90% D 2 O, the new sets of 1 H-NMR signals of both the dpa and p-cym ligand were detected (e.g., at 7.90 ppm for C4-H or at 2.00 ppm for C20-H in the case of the complex 1). The formation of these new signals is most likely connected with the hydrolysis of the original complexes, showing in the formation of the [Ru(η 6 -p-cym)(dpa)(H 2 O)] 2+ and/or [Ru(η 6 -p-cym)(dpa)(OH)] + species [2]. The integral intensity ratios of the signals of the initial halogenido complexes (1-3) and their hydrolyzed forms observed after 48 h of standing at ambient temperature ( Figure S2) equaled approximately 1:3 (for 1), 2:3 (for 2) and 3:2 (for 3). In other words, 75% (1), 60% (2) and 40% (3) of the studied halogenido complexes hydrolyzed in the used mixture of solvents after 48 h of standing at ambient temperature. The evidence that the new 1 H-NMR signals detected in the spectra of the complexes 1-3 dissolved in a mixture of 10% MeOD-d 4 /90% D 2 O belong to the hydrolyzed complexes was obtained by the addition of 2 molar equivalents of KCl (for 1), KBr (for 2) or KI (for 3) to the appropriate equilibrated solutions, which overturned the hydrolysis progress, resulting in disappearance of the signals of hydrolysates after next 24 h of standing at ambient temperature ( Figure S2). Glutathione (GSH) is a naturally occurring tripeptide (Glu-Cys-Gly), known to be responsible for the intracellular detoxification of various transition metals [31], including ruthenium [32]. Importantly, it has been reported that the decrease in the GSH level of cancer cells induced by co-application of a γ-glutamylcysteine synthetase inhibitor, L-buthionine sulfoximine (L-BSO), led to the cytotoxicity enhancement for similar half-sandwich Ru(II) complexes [3], thus indicating the important role of GSH for cytotoxicity of prospective anticancer ruthenium complexes. That is why similar experiments, as described above, were performed for complexes 1-5 dissolved in 10% MeOD-d 4 /90% D 2 O, with an addition of 2 molar equivalents of GSH. However, no evidence was obtained for the formation of the GS-Ru adducts of the studied halogenido complexes with GSH even after 48 h of standing at ambient temperature, because the 1 H-NMR spectra of the mixtures of complexes 1-3 with GSH contained only the signals of the initial complexes (1-3), their hydrolysates (as described above) and those of free GSH (e.g., at 4.45 ppm and 2.85 ppm for Cys-α CH, and Cys-β CH 2 , respectively) ( Figure 5). co-application of a γ-glutamylcysteine synthetase inhibitor, L-buthionine sulfoximine (L-BSO), led to the cytotoxicity enhancement for similar half-sandwich Ru(II) complexes [3], thus indicating the important role of GSH for cytotoxicity of prospective anticancer ruthenium complexes. That is why similar experiments, as described above, were performed for complexes 1-5 dissolved in 10% MeOD-d4/90% D2O, with an addition of 2 molar equivalents of GSH. However, no evidence was obtained for the formation of the GS-Ru adducts of the studied halogenido complexes with GSH even after 48 h of standing at ambient temperature, because the 1 H-NMR spectra of the mixtures of complexes 1-3 with GSH contained only the signals of the initial complexes (1-3), their hydrolysates (as described above) and those of free GSH (e.g., at 4.45 ppm and 2.85 ppm for Cys-α CH, and Cys-β CH2, respectively) ( Figure 5). Regarding the carboxylato complexes 4 and 5, their hydrolysis was connected with a release of the appropriate O-donor ligands, resulting in the complex species [Ru(η 6 -p-cym)(dpa)(H2O)] 2+ and free carboxylates ( Figure 6). The positions of 1 H-NMR signals of the formed complex species (e.g., at 7.89 ppm for C4-H or at 2.01 ppm for C20-H for the hydrolyzed form of the complex 4) were consistent with those of the hydrolyzed halogenido complexes (see above), which strongly suggested the same composition of the complex species (most likely aqua complexes [Ru(η 6 -p-cym)(dpa)(H2O)] 2+ ) formed by the hydrolysis of all the complexes 1-5. Further, the signals of the released carboxylato ligands appeared in the 1 H-NMR spectra with the δ values well-correlating with free carboxylate anions ( Figure 6). As for the valproato complex 4, the triplet of the terminal methyl groups (i.e., C25-H) of the VP ligand showed at 0.62 ppm in the 1 H-NMR spectra recorded on the fresh 10% MeOD-d4/90% D2O solutions. Together with this signal, another triplet was detected at 0.78 ppm, whose integral intensity increased in time and whose position correlated with that of free valproate(1-) anion (0.78 ppm in the same mixture of solvents), thus this signal can be unambiguously assigned to the released VP ligand. Regarding the 4-phenylbutyrato complex 5, the 1 H-NMR multiplets of the aliphatic hydrogens of the PB ligand appeared at 1.65, 2.17 and 2.25 ppm, for the fresh 10% MeOD-d4/90% D2O solutions of complex 5 and its mixture with GSH. A hydrolytic release of the PB ligand led to the changes of these positions to 1.77, 2.10 and 2.54 ppm, which correlated well with the positions of these signals detected in the spectrum of free 4-phenylbutyrate (the experiment performed with PBA in 10% MeOD-d4/90% D2O). Interestingly, the carboxylato complexes 4 and 5 differed markedly one from another, in connection with the hydrolysis process. It has been observed that ca. 90% of the PB-containing complex 5 hydrolyzed after 48 h of standing at ambient temperature, while the valproato complex 4 was more stable and only ca. 25% hydrolyzed (Figure 7). Remarkably, although no signs of interactions of the carboxylato complexes (or their hydrolysates) with GSH were observed in the acquired 1 H-NMR spectra, the presence of GSH in the mixtures with complexes 4 or 5 enhanced the release of the appropriate carboxylato ligand up to ca. 98% for both complexes 4 and 5 after 48 h of standing at ambient temperature (Figure 7). Interestingly, the carboxylato complexes 4 and 5 differed markedly one from another, in connection with the hydrolysis process. It has been observed that ca. 90% of the PB-containing complex 5 hydrolyzed after 48 h of standing at ambient temperature, while the valproato complex 4 was more stable and only ca. 25% hydrolyzed (Figure 7). Remarkably, although no signs of interactions of the carboxylato complexes (or their hydrolysates) with GSH were observed in the acquired 1 H-NMR spectra, the presence of GSH in the mixtures with complexes 4 or 5 enhanced the release of the appropriate carboxylato ligand up to ca. 98% for both complexes 4 and 5 after 48 h of standing at ambient temperature (Figure 7).

In Vitro Cytotoxicity
In this work, the structure of the recently reported complex [Ru(η 6 -p-cym)(dpa)Cl]PF6 (1 in this work) [10,11] was modified by the replacement of the chlorido ligand by either bromido (2)

Syntheses
[Ru(µ-Cl)(η 6 -p-cym)Cl] 2 was prepared according to the reported synthetic procedure performed in the microwave reactor [35]. Silver valproate (Ag(VP)) and silver 4-phenylbutyrate (Ag(PB)) were obtained by the neutralization of the methanolic solutions of valproic or 4-phenylbutyric acid with the stoichiometric amount of 1 M NaOH (5 min of stirring at ambient temperature) followed by the addition of 1 molar equivalent of silver triflate (5 min of stirring at ambient temperature in the dark).

Methods
1 H, 13 C, 1 H-1 H gs-COSY, 1 H-13 C gs-HMQC and 1 H-13 C gs-HMBC spectra were acquired for DMSO-d 6 solutions at 298 K on a JEOL JNM-ECA 600II device at 600.00 MHz ( 1 H) and 150.86 MHz ( 13 C); gs = gradient selected, COSY = correlation spectroscopy, HMQC = heteronuclear multiple quantum coherence, HMBC = heteronuclear multiple bond coherence. The spectra were calibrated against the residual signals of the used solvent at 2.50 ppm ( 1 H-NMR) and 39.52 ppm ( 13 C-NMR) [36]. The splitting of the 1 H-NMR signals is defined as s = singlet, d = doublet, t = triplet, qui = quintet, dt = doublet of triplets, br = broad band, m = multiplet. Electrospray ionization (ESI) mass spectra of the methanol solutions were obtained of on a LCQ Fleet Ion Trap mass spectrometer (Thermo Scientific; Qual Browser software, version 2.0.7; Waltham, MA, USA) in the positive ionization mode (ESI+). Elemental analysis (C, H, N) was performed using a Flash 2000 CHNS Elemental Analyzer (Thermo Scientific). FTIR spectra were recorded using Nexus 670 FT-IR (Thermo Nicolet) on an ATR diamond plate between 400 and 4000 cm −1 . RP-HPLC experiments were performed using UHPLC-MS (Dionex/Thermo Scientific) mass spectrometer and an ReproSil-Pur Basic C18, 5 µm pore size, 200 × 4.6 mm. Mobile phase used was H 2 O 0.1% Htfa/MeCN at gradients of t = 0 min 10% B, t = 30 min 80% B, t = 40 min 80% B, t = 41 min 10% B, and t = 55 min 10% B over a 55 min period. Flow rate was 1 mL·min −1 , and the detection wavelength was set at 254 nm. H 2 O and acetonitrile (MeCN) of HPLC grade were used for the RP-HPLC experiments with an addition of trifluoroacetic acid (Htfa).
A Monowave 300 (Anton PaarGmbH, Graz, Austria) microwave reactor was used for the synthesis of the starting dimeric Ru(II) compound as well as for the chlorido complexes 1* and 1 (30 mL microwave vials equipped with magnetic stirring bars).
A suitable single crystal of [Ru(η 6 -p-cym)(dpa)I]PF 6 (3) was selected and placed on an D8 QUEST monocrystal diffractometer (Bruker, Billerica, MA, USA) with PHOTON 100 CMOS detector, using the Mo-Kα radiation (λ = 0.71075 Å). The APEX3 software package was used for data collection and reduction [37]. The structures were solved using a direct method and refined using the Bruker SHELXTL Software Package (Bruker) [38]. X-ray crystallographic data have been deposited in the Cambridge Crystallographic Data Centre (Cambridge, United Kingdom) under the accession number CCDC 1515628. The graphics were drawn and additional structural calculations were performed by DIAMOND (Version 4.0.3.; Crystal Impact GbR, Bonn, Germany) [39] and Mercury [40] software (Version 3.0; Cambridge Crystallographic Data Centre, Cambridge, United Kingdom).

1 H-NMR Studies of Aqueous Chemistry and Interactions with GSH
Complexes 1-5 were dissolved in 60 µL of MeOD-d 4 and diluted with 540 µL of D 2 O to get the 1 mM solutions. The 1 H-NMR spectra were recorded immediately after the preparation of the samples (0 h) and after 0.5, 1, 2, 4, 6, 24 and 48 h of standing at ambient temperature. After that, 5 molar equivalent of KCl (for 1), KBr (for 2) or KI (for 3) were added to the solutions and the 1 H-NMR spectroscopy was carried out on the fresh solutions (0 h) and after 1, 6 and 24 h of standing at ambient temperature. Similar experiments were performed with an addition of GSH, as follows: 1-5 (amounts necessary for the final concentration of 1 mM) were dissolved in 60 µL of MeOD-d 4 and 2 molar equivalent of GSH dissolved in 540 µL of D 2 O were added. The 1 H-NMR spectra were acquired right after the preparation of the solutions (0 h) and after 0.5, 1, 2, 4, 6 and 24 h of standing at ambient temperature. The obtained 1 H-NMR spectra were calibrated against the residual signal of D 2 O found at 4.85 ppm.

Cell Culture and In Vitro Cytotoxicity
The A2780 human ovarian carcinoma cells, purchased from the European Collection of Cell Cultures (ECACC), were cultured in RPMI-1640 medium supplemented with 10% of fetal calf serum, 1% of 2 mM glutamine and 1% penicillin/streptomycin, according to the ECACC instructions. The cells were grown at 37 • C and 5% CO 2 in a humidified incubator as adherent monolayers.
The cultured A2780 cells were seated in the 96-well culture plates and pre-incubated in drug-free media at 37 • C for 24 h. After that, the cells were treated with the 0.01-100.0 µM solutions of complexes 1-5 and cisplatin (prepared by appropriate dilution of the fresh 100 mM stock solutions of the tested substances dissolved in DMF) for 24 h (exposure time) at 37 • C. Then, the solutions containing the tested compounds were removed and the cells were washed with drug-free medium and kept under drug-free medium for the next 72 h (recovery time). In parallel, the cells were treated with vehicle (0.1% DMF, v/v) and Triton X-100 (1%, v/v) to assess the minimal (100% viability) and maximal (0% viability) cell damage, respectively.
The in vitro cytotoxicity was assessed using an MTT assay and evaluated spectrophotometrically at 540 nm (Tecan Group Ltd., Männedorf, Switzerland). The cytotoxicity data were received from three independent experiments (each conducted in triplicate) using the cells from three different passages.