Ruthenium ( II ) Complexes Containing Anti-Inflammatory Drugs as Ligands : Synthesis , Characterization and in vitro Cytotoxicity Activities on Cancer Cell Lines

The synthesis, characterization and cytotoxic activity of cis-[Ru(dicl)(dppm)2]PF6 and cis-[Ru(ibu)(dppm)2]PF6, (dppm = 1,1-bis(diphenylphosphine)methane; dicl = diclofenac anion and ibu = ibuprofen anion), are described in this work. Complexes were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), UV-Vis, P{H} nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRESIMS). X-ray structure of cis-[Ru(ibu)(dppm)2]PF6 is also described. Preliminary calf thymus DNA (ct-DNA) binding studies were carried out by UV-Vis and viscosity experiments, with results suggesting the existence of electrostatic interactions between ruthenium complexes and ct-DNA. Cytotoxicity assays were carried out on a panel of human cancer cell lines and a human normal cell line. Complexes displayed a high to moderate cytotoxicity with IC50 ranging from 5 to 47 μmol L . cis-[Ru(ibu) (dppm)2]PF6 was found to be the most active, with IC50 values lower than cisplatin. The degree of cytotoxicity was maintained for the normal cell line, although cis-[Ru(ibu)(dppm)2]PF6 exhibited a similar selectivity to that of cisplatin but with a higher activity for at least two tumor cell lines which evidences a promising anticancer candidate and selects this complex for further experiments.


Introduction
][14][15] Vol. 26, No. 9, 2015 A previous work from our group presented biological results from the diphosphinic ruthenium(II) precursor cis-[RuCl 2 (dppm) 2 ] and its derivative with 2-pyridinecarboxylic acid anion (pic − ), the complex [Ru(pic)(dppm) 2 ]PF 6 , where the pic − ligand coordinates on N,O-bidentate mode.The antimycobacterial activity against MTB H 37 Rv of this complex was evaluated and the minimum inhibitory concentration (MIC) value was in the low micromolar range. 16Some additional studies performed with the analogous [Ru(pic)(dppe) 2 ]PF 6 , dppe = 1,2-bis(diphenylphosphine)ethane, revealed a high antibacterial activity against S. aureus, C. albicans and M. smegmatis. 17This last complex also presented a relatively low acute oral toxicity in mice. 17ue to this background of promising biological results for complexes containing the cis-[Ru(P-P) 2 ] unit, P-P = diphosphine, our current strategy consists in evaluating derivatives with different chelating moiety by replacing the chlorido ligands in order to select new cytotoxic agents against tumor cells.In the present work anti-inflammatory molecules were chosen as coligands to explore the possible synergic effect between the cis-[Ru(dppm) 2 ] unit and these biologically active ligands.][20] In this work the synthesis and characterization of two new ruthenium(II) derivatives with formula [Ru(dicl)(dppm) 2 ]PF 6 (1) and [Ru(ibu)(dppm) 2 ]PF 6 (2) are reported.Furthermore, preliminary binding properties to calf thymus DNA (ct-DNA) and in vitro tests of cytotoxic activities against a panel of human cell lines are presented and discussed.

General
Solvents were purified by standard methods.All chemicals used were of reagent grade or comparable purity.The RuCl 3 •3H 2 O and the ligands 1,1-bis(diphenylphosphino) methane (dppm), sodium diclofenac and racemic sodium ibuprofen were used as received from Aldrich.The ct-DNA was purchased from Sigma Chemical Co. Ltd.The cis-[RuCl 2 (dppm) 2 ] precursor complex was prepared according to the literature method. 21

Instrumentation
Infrared spectra (IR) were obtained on a PerkinElmer Spectrum Two spectrophotometer equipped with an attenuated total reflectance (ATR) sample holder and ZnSe crystal.The spectra were recorded in the range of 4000-600 cm -1 with a 4 cm -1 resolution.UV-Vis spectroscopy was performed on a Shimadzu UV2501 PC spectrophotometer using cuvettes with a 1 cm path length and methanol as solvent. 31P{ 1 H} nuclear magnetic resonance (NMR) was performed on a Bruker DRX 400 MHz spectrometer with a BBO 5 mm probe at 298 K.The NMR spectra were recorded in CH 2 Cl 2 using a capillary of D 2 O to get the lock and with H 3 PO 4 (85%) as external reference.Conductance data, obtained at 298 K on 1 × 10 -3 mol L -1 methanol solutions of the complexes, were measured with a Tecnopon MCA 150 conductometer.Elemental analyses were performed on a Perkin Elmer 2400 Series II CHNS/O microanalyser.High-resolution mass spectra (HRESIMS) with electrospray ionization were measured on an ultrOTOF (Bruker Daltonics) spectrometer, operating in the positive mode.Methanol was used as solvent system and the samples were infused into the ESI source at a flow rate of 5 µL min -1 .The calculated values for the charged complex ions were made by using ChemBioDraw Ultra 14.0.

Synthesis
The precursor cis-[RuCl 2 (dppm) 2 ] (0.103 mmol; 100 mg) was solubilized in 50 mL of methanol, followed by the direct addition of sodium diclofenac (0.120 mmol; 37.2 mg) or sodium ibuprofen (0.120 mmol; 26.6 mg), respectively, for synthesis of the complexes 1 and 2. The resulting solution was stirred at room temperature for a 6 h period.The final solution was concentrated to ca. 5 mL and an aqueous solution of NH 4 PF 6 (0.150 mmol; 24.4 mg) was added for the precipitation of a yellow solid.The solid was filtered off and washed with water (3 × 5 mL) and diethyl ether (3 × 5 mL) and dried under reduced pressure.

X-ray crystallography
Yellow crystals of the complex 2 were grown by slow evaporation of a dichloromethane solution at room temperature.The data collection was performed using Mo-Kα radiation (λ = 0.71073 Å) on a BRUKER APEX II Duo diffractometer.Standard procedures were applied for data reduction and absorption correction.The structure was solved with SHELXS97 using direct methods 22 and all non-hydrogen atoms were refined with anisotropic displacement parameters with SHELXL97. 23The hydrogen atoms were calculated at idealized positions using the riding model option of SHELXL97. 23Table 1 presents detailed information about the structural determination.

DNA titration and viscosity experiments
A standard solution of ct-DNA was prepared in tris-HCl buffer (5 mol L -1 tris-HCl, pH 7.2).The concentration of this ct-DNA solution was measured from its absorption intensity at 260 nm using the molar absorption coefficient value of 6600 mol -1 L cm -1 .Solutions of ruthenium complexes 1 and 2 used in the experiments were prepared in Tris-HCl buffer containing 2% of dimethyl sulfoxide (DMSO).In the titration experiments, different concentrations of the ct-DNA were used while the ruthenium complex was at 20 µmol L -1 .
Viscosity experiments were carried out using an Ostwald viscometer maintained at a constant temperature of 25 °C in a thermostatic bath.The viscosity of the ct-DNA solution was measured in the presence of increasing amounts of the complexes 1 and 2. The flow times were measured with an automated timer.Each sample was measured three times, and an average flow time was calculated.[26] Human cell lines and culture conditions For the experiments, four different human cell lines from the 4 th through 12 th passages were used: HepG2 (hepatocellular carcinoma), MCF-7 (breast adenocarcinoma), MO59J (glioblastoma) and GM07492A (normal lung fibroblasts).The different cell lines were maintained as monolayers in plastic culture flasks (25 cm 2 ) containing HAM-F10 plus Dulbecco's Modified Eagle Medium (DMEM), 1:1 (Sigma-Aldrich) or only DMEM, depending on the cell line, supplemented with 10% foetal bovine serum (Nutricell) and 2.38 mg mL -1 Hepes (Sigma-Aldrich) at 37 °C in a humidified 5% CO 2 atmosphere.Antibiotics (0.01 mg mL -1 streptomycin and 0.005 mg mL -1 penicillin; Sigma-Aldrich) were added to the medium to prevent bacterial growth.

Cell viability assay related to human cell lines
Cytotoxic activity on the cell lines was assessed using the Colorimetric Assay in vitro Toxicology-XTT Kit (Roche Diagnostics) according to the manufacturer's instructions.For the experiments, 1 × 10 4 cells were seeded into microplates with 100 µL of culture medium (1:1 HAM F10 + DMEM or DMEM alone) supplemented with 10% fetal bovine serum containing concentrations of the ruthenium complexes ranging from 1.5625 to 1600 µg mL -1 .Negative (no treatment), solvent (0.02% DMSO) and positive (25% DMSO) controls were included.Positive controls comprising cisplatin (Sigma-Aldrich, ≥ 98% purity) were included.After incubation at 36.5 °C for 24 h, the culture medium was removed and cells were washed with 100 µL of phosphate-buffered saline (PBS) to remove the treatments, after which they were exposed to 100 µL of HAM-F10 culture medium without phenol red.Then, 25 µL of XTT were added and the cells were incubated at 36.5 °C for 17 h.The absorbance of the samples was determined using a multi-plate reader (ELISA-Tecan-SW Magellan vs 5.03 STD 2P) at a wavelength of 450 nm and a reference length of 620 nm.

Statistical analysis related to human cell line assays
Cytotoxicity was assessed using the IC 50 response parameter (50% cell growth inhibition) calculated with the GraphPad Prism program, plotting cell survival against the respective concentrations of the treatments.One-way ANOVA was used for the comparison of means (p < 0.05).The selectivity index was calculated by dividing the IC 50 value of the isolated compounds on GM07492-A cells by the IC 50 value determined for human cancer cells.

Synthesis
The reaction of sodium salts of diclofenac and ibuprofen with the ruthenium(II) diphosphine precursor complex cis-[RuCl 2 (dppm) 2 ] resulted in the products 1 and 2 by chlorido exchange under mild conditions as showed in Scheme 1.
The yellow ruthenium(II) complexes 1 and 2 were isolated as pure solids from methanol, in reasonable to good yields.The elemental analyses are described in experimental section and they agreed well with the proposed formulations.The molar conductance values measured in methanol at room temperature range from 98 to 104 S cm 2 mol -1 , revealing the 1:1 electrolytic nature of these complexes. 27Complexes are air stable both in the solid state and in DMSO solutions as evaluated by 31 P{ 1 H} NMR and UV-Vis experiments for a period of 48 h.
The Dν values of 70 cm -1 for complex 1 and 55 cm -1 for 2 are indicative of a η 2 binding mode of the carboxylate group. 21In addition, for complex 1, characteristic vibrational modes of the diclofenac ligand at 3344 and 1100 cm -1 were observed, corresponding to νNH and νPh-Cl, respectively.For both compounds the characteristic P-F stretch of the PF 6 -counterion was seen at 837 cm -1 . 28Most of the vibrational modes observed were characteristic of the dppm ligands occurring practically at same frequencies observed for the precursor cis-[RuCl 2 (dppm) 2 ].
X-ray structure analyses X-ray structure analyses of the complex 2 confirm the IR spectroscopy data.An ORTEP drawing of 2 showing the atom numbering scheme is depicted in Figure 3. Selected bond lengths and angles are presented in Table 2.
Two disordered positions were refined for the fragment which includes the chiral center C(2) as well as the methyl and hydrogen groups attached to it.The two sites are shown together in Figure 3.The solid lines (labeled A) indicate the bonds between the atoms with higher occupation factor (68.4%), whereas the dashed lines (labeled B) represent the species with the lower occupation (31.6%).Plots showing the major and minor components of the disordered structure observed in 2 separately can be found as supplementary information (Figures S1 and S2).The reason for the disorder can clearly be derived from the orientation of the ligand due to the presence of the chiral center C(2), and consequently, two complex species with the ligand in R (major component) and S (minor component) configurations could be detected in the solid structure of the compound.Since the ligand used was a racemic mixture of ibuprofen, it is reasonable that both R and S isomers react with ruthenium(II) precursor.In fact, the 31 P{ 1 H} NMR of a powder of complex 2 also shows two set of signals with relative integration 60:40, revealing that these species are also preserved in solution (see solution studies).Furthermore, the observation of the two types of isomers in the same crystal is not very common.This can be explained as a case of static disorder where 2 presents the R and S configurations distributed among different unit cells. 29his compound crystallizes in the orthorhombic system, space group Pna2(1), with the Ru center adopting a distorted octahedral coordination geometry formed by two cis-chelating diphosphine ligands and the bidentate (η 2 ) carboxylate group of the ibuprofen ligand.The distortions are caused by chelation angles of 70.93(2) and 72.36(2) o imposed by the methylene bridge of dppm ligands and especially by the carboxylate group with an O(1)-Ru-O(2) angle of only 59.34 (9) o .1][32] The Ru-P bond lengths vary from 2.3204(5) to 2.4092(6) Å for mutually trans disposed phosphorus atoms and from 2.2811(7) to 2.2958(7) Å for phosphorus atoms trans positioned to oxygen atoms from de carboxylate group.2][33] The carboxylate ligand is coordinated with a certain degree of asymmetry as illustrated by the Ru-O distances of 2.1676(18) and 2.217(2) Å.1][32][33][34][35] This asymmetry probably is due some weak interactions of the phenyl group of ibuprofen with adjacent phenyl rings of dppm.This kind of asymmetry was previously observed for ruthenium(II) ferrocenylcarboxylates. 34 31 P{ 1 H} Nuclear magnetic resonance (NMR) spectroscopy The 31 P{ 1 H} NMR spectra of complexes 1 and 2 show typical patterns of species containing two cis positioned diphosphines and equal ligands completing the octahedral coordination sphere.For complex 1 a pair of triplets that integrate in 1:1 ratio with chemical shifts at 8.2 and -12.8 ppm was observed.The splitting pattern was consistent with an A 2 X 2 (∆ν / J = 87) assignment similar to those described for analogous complexes. 34For complex 2 a slightly different behavior was observed.In the more deshielded region two triplets with very close chemical shifts (8.4 and 8.2 ppm) appeared, besides one triplet in the shielded region (-12.3ppm), as showed in Figure 4.
The integration of the triplets at 8.4 and 8.2 ppm are in the 1:1 ratio with the triplet at -12.3 ppm.This behavior clearly indicates the presence of a mixture of two very similar species and based on the integration of each line of the signals close to 8 ppm it is found a 60:40 ratio between the species.This ratio is in agreement with the two configurations of ibuprofen ligand observed in crystal structure of the complex 2 as previously discussed.The splitting pattern is also consistent with an A 2 X 2 assignment with ∆ν / J = 88 and 87 for each one of the configurations.In addition, since the PF 6 -counterion was utilized, it was observed the characteristic septet due to the phosphorus-fluorine coupling with chemical shift centered at -144.6 ppm for both complexes.

High-resolution mass spectrometry (HRESI)
Mass spectra of complexes containing ruthenium are typical for their isotopic pattern demonstrated by the presence of 96 Ru (5.5%), 98 Ru (1.9%), 99 Ru (12.7%), 100 Ru (12.6%), 101 Ru (17.1%), 102 Ru (31.6%) and 104 Ru (18.6%) isotopes, with the nuclide abundance in parentheses.Furthermore, complex 1 had a ligand with chlorine ( 35 Cl (75.8%) and 37 Cl (24.2%)) which contributes with an additional isotopic pattern (Figure 5).The high-resolution mass spectra of the compounds 1 and 2 were recorded and the obtained data confirm the established pattern (Figures 5a and 5b).In this study, the m/z values listed below in the text refer to the peak of the most abundant element corresponding to the 102 Ru isotope.The HRMS spectra were acquired in the positive mode and the charged complex ions were observed at m/z 1164.1520[M] + (1) and 1075.2660[M] + (2), in agreement with calculated values for C 64 H 54 Cl 2 NO 2 P 4 Ru, 1164.1520 and C 63 H 61 O 2 P 4 Ru, 1075.2660,respectively.Collision-induced dissociation (CID) experiments (MS/MS) with an increasing collisional energy using N 2 as collision gas under the selected ions at m/z 1164.1520[M] + (1) and 1075.2660[M] + (2), showed a fragmentation pathway just for complex 1 even in higher collisional energies.The loss of 295 u was proposed for a neutral elimination of the ligand (diclofenac, acid form) (Figure 5c).In an attempt to study the nature of the ruthenium complexes interactions with ct-DNA, UV-Vis absorption spectra were obtained by titration of the complexes with increasing concentrations of ct-DNA.The electronic spectra of complexes 1 and 2 showed an intense absorption peak around 264 nm, which could be attributed to an intraligand π-π* transition of the coordinated groups in the complex, that has been selected to study the spectral changes with ct-DNA addition.Both complexes displayed the same behavior in which absorption decreases with ct-DNA titration, however, this characteristic is attributed only to dilution effects.This was demonstrated by titration of the complexes with buffer solution (not containing ct-DNA) in which the same absorption decrease was observed.All these spectra are showed in supplementary information (Figure S4).These data showed that these complexes do not exhibit covalent or intercalative interactions with ct-DNA. 36,37ue to the very weak interaction (hypochromism < 3%) was not possible determine the intrinsic binding constant (K b ) between the ruthenium complexes and ct-DNA.
The possible mode of interaction between complexes and ct-DNA was also evaluated by viscosity experiments.It is well known that classical intercalators, such as ethidium bromide, lead to an increase in the viscosity of ct-DNA because separation of the base pairs occurs to accommodate the intercalator.][40] However, complexes 1 and 2, exhibited essentially no effect on the viscosity of ct-DNA as demonstrated in a plot (η/η o ) 1/3 versus [complex]/[DNA] showed in supplementary information (Figure S5).This result is consistent with existence of electrostatic interactions between ruthenium complexes and ct-DNA. 25,26Considering the molecular structure and positive charge of the complexes, electrostatic interactions involving the negatively charged phosphate groups of ct-DNA are expected.

In vitro cytotoxic activity
The human cell lines were exposed to the ruthenium(II) complexes and cisplatin for a period of 24 h, in order to allow them reach DNA or any other biological target.The IC 50 values, calculated from the dose-survival curves generated by the XTT assays obtained after drug treatment are shown in Table 3.
Complexes 1 and 2 have showed, in general, high cytotoxicity against all the human tumor cell lines assayed ] by diclofenac and ibuprofen resulted in higher cytotoxic activity probably due to the differences in lipophilicity upon complexation influencing the amount of compound that gets inside the cells.Further studies are necessary to verify the biological targets of this class of ruthenium(II) complexes.Although these complexes displayed low selectivity they present potential for the treatment of breast adenocarcinoma and gliobastoma since they present similar SI value to that of cisplatin but a higher activity, so they could be used in lower concentrations.
with IC 50 values ranging from 5 to 9 µmol L -1 , except for complex 1 in MCF-7 cells that showed a moderate cytotoxicity as presented in Table 3. Complex 2 displayed higher activity than 1 in all the tumor cell lines assayed with similar IC 50 values independent of the cell line.This non-selective activity of complex 2 was not observed for complex 1 that was much less cytotoxic in MCF-7 cells.Compared with the reference metallodrug cisplatin, complex 1 displayed approximately the same cytotoxic activity for HepG2 and MCF-7 cells and a three times increased activity for MO59J cells.Complex 2 had similar activity to cisplatin for HepG2 cells and a four times increased activity against MCF-7 and MO59J cells.The selectivity index (SI) (SI = IC 50 GM07492A / IC 50 human tumor cell line) was smaller than 1 for all the cell lines assayed for complex 1, while for complex 2 the SI values are very close to 1, both indicating a low selectivity.Under the same experimental conditions cisplatin also displayed SI values close to 1 for MCF-7 and MO59J tumor cell lines.The precursor complex cis-[RuCl 2 (dppm) 2 ] was less active than the complexes 1 and 2 by factors ranging from 4.1 to 21.6.A similar increase in toxicity was observed against the normal cell line GM07492A.These data clearly indicates that the exchange of two chlorido ligands by a bidentate anti-inflammatory molecule makes the cis-[Ru(dppm) 2 ] 2+ unity complex more cytotoxic, probably due to the different lipophilicity and consequent entrance in the cells.Further experiments concerning the amount of ruthenium complex that gets inside the cells as well as its intracellular targets, beyond DNA, are required to understand details of the observed activity and will be a point of study in a near future.

Conclusions
In this investigation two new ruthenium(II) complexes containing dppm and the anions of antiinflammatory drugs diclofenac and ibuprofen with formula

Table 1 .
Crystallographic data and structural refinement details for complex 2

Table 3 .
Inhibitory activity of ruthenium(II) complexes and cisplatin against normal and tumor cell lines, expressed as IC 50 c MO59J: glioblastoma; d GM07492A: normal lung fibroblasts.