Synthesis, Characterization, and Evaluation of Antitumor Potential in MCF-7 Cells of Ruthenium-Derived Compounds

To synthesize, characterize and evaluate the antitumor potential derived from ruthenium compounds was generated in this study, from the precursor K[RuCl4(bipy)] a route in a simple and reproducible synthesis for a novel compound of coordinating Ru+3 with bipy and L-trip. The spectroscopic characterization in the middle infrared region (FTIR) shows the interactions between Ru-(L-trip), evidenced by the displacement of the carboxylate ion band for higher energies, and also by the displacements of aliphatic amine bands, suggesting that bidentate coordination of the L-trip ligand occurred. Analysis of the results obtained with thermoanalytical techniques showed that the minimum formula of the compound, [RuCl2(bipy)(L-trip)]1/2H2O. Evaluation of the antitumor potential of precursor K[RuCl4(bipy)] showed the toxic effects on MCF-7 cell line, but did not show selectivity and not reached PBMC cells to the same extent. The evaluation of the antitumor potential of the newly synthesized compound, [RuCl2(bipy)(L-trip)], demonstrated that the insertion of an L-tryptophan molecule into the precursor coordination sphere made it selective when compared to PBMC cells, for MCF-7 type tumor cells.


Introduction
From the studies conducted by Rosenberg and colleagues [1] [2] [3], platinum-based coordination compounds are among the most used drugs in the treatment of cancer. Since studies by Rosenberg and colleagues platinum-derived coordination compounds have been among the most widely used drugs in cancer treatment [4]. However, these compounds exhibit high toxicity, leading patients to present some side effects such as high nephrotoxicity, nausea, vomiting, anorexia, ototoxicity, neurotoxicity and develop resistance to drugs [4] [5] [6].
The toxic effects caused by platinum-derived compounds have prompted several researchers to develop new drugs. Research involving ruthenium-derived compounds as drugs in the treatment of malignant neoplasms has shown promise [7] [8]. This class of compounds presents characteristics such as considerable cytotoxicity, antimetathesis properties and low toxicity when compared to platinum derived compounds.
Like iron, ruthenium is known to have the ability to bind transferrin protein. Due to the high demand for iron, cancer cells tend to increase the numbers of transferrin receptors on their surfaces, capturing them in greater numbers. It is believed that by mimicking iron, a higher concentration of ruthenium is directed to cancer cells, which would explain the lower toxicity of this class of compounds [7] [8].
Considerable cytotoxicity may be related to interactions between the metallic center and the nitrogenous bases of DNA. The results obtained by Gallori [9], suggest that compounds are known as NAMI (sodium trans-(dimethylsulfoxide) (imidazole) tetrachloro ruthenate(III)) and RAP (dichloro-1,2-propylene diamine tetraacetate ruthenium(III)) are capable, in vitro, of interacting with DNA causing changes in its conformation, inhibiting its recognition and cutting by restriction enzymes. However, these compounds only cause considerable DNA damage at relatively high concentrations when compared to cisplatin. Some compounds derived from Ru +3 and Ru +2 with an amine, [10] N-heterocyclic [11] [12], and alkyl sulfoxide linkers [13] also demonstrated potential activity antimetastatic.
Among the compounds with this property stands out NAMI-A [14], this compound has several peculiarities such as excellent selective activity against lung metastases from various solid tumors [14] [15] [16] and very low in vitro cytotoxicity in a panel of 60 strains solid tumor cells [17].
Studies in mice transplanted with S-180 cells and treated with cis-[RuCl 2 (NH 3 ) 4 ] Cl have shown that the compound is well tolerated at therapeutic doses, presenting very low toxicity to the host and extending its life span [20]. Further studies have shown that the compound is selective, exerting significant toxic activity in A-20, SK-Br-3 and S-180 strains, moderate in Jurkat cells and very low toxicity in PBMC cells when compared to A-20, SK-Br-3 and S-180 [23].
All of these properties cited above, prompted us to make a new ruthe-

Synthesis
Synthesis of K[RuCl 4 (bipy)]: The entire synthesis procedure was performed as described by James [25], with some changes. In a flat-bottomed flask, 0.5014 g RuCl 3 •nH 2 O was solubilized in 25.0 mL of methanol, the resulting solution was stirred at 200 rpm for 30 minutes for further addition of 0.3047g solubilized bipy in 25.0 mL of methanol. After the addition of bipy, the system was stirred and heated to 100˚C under reflux. After 2 hours, 400.0 mL of methanol and 0.3059 g of KCl were added thus ensuring the excess counter ion K + . The system was kept under the same stirring conditions and temperature for a further 20 hours. After 20 hours the red solution had its temperature reduced to 60˚C and was maintained until the volume was reduced to 15 mL. The brown precipitate was separated by vacuum filtration and desiccated for 24 hours. Yield: 94.24%.
Purification: In a 1000.0 mL beaker, 0.7918 g of K[RuCl 4 (bipy)] and a KCl spatula tip in 1000.0 mL of a 36.5% -38% HCl/1: 1 water solution were solubilized. The system then remained under stirring and bathing at 100˚C for 30 minutes. After 30 minutes, the system was hot filtered by gravity and the orange-colored supernatant was placed in a 100˚C bath to reduce its volume to ~30.0 mL. Slow cooling of the solution generated red, needle-shaped crystals.
Synthesis of [RuCl 2 (bipy)(L-trip)]: In a 25.0 mL beaker, 0.0478 g L-trip, 0.0195 g NaHCO 3 and ~5.0 mL distilled water were added. The system was stirred for 30 minutes for further addition of 0.1012 g of K[RuCl 4 (bipy)]. After 1 hour, the system was vacuum filtered. The precipitate was dried with ether and desiccated with silica for 24 hours. At the end of this procedure, 0.0680 g was obtained.
The same procedure was repeated twice, and in one of them the system was heated to 60˚C and in the other, the synthesis time was extended by another 3 hours.  (Table 1).

Preparation of Ruthenium Compound
Cell viability assay by acridine orange staining method: At the end of the incubation period, the falcon tubes were centrifuged at 1500 rpm for 10 minutes and had their supernatant discarded. The pellet formed was stained with 200.0 µl of freshly prepared acridine orange solution (concentration 14.4 mg/mL) and allowed to stand for 1 minute for dye action. The resulting solution was resuspended in medium 199, after which the tubes were centrifuged and washed with PBS a further 2 times. Stained cells were placed on microscope slides (26 × 76 mm) and, after mounting with coverslips (24 × 24 mm), were analyzed by blind fluorescence microscopy (Nikkon Eclipse E200). The cell viability index was obtained by counting. For each treatment at least 100 cells were analyzed. Green Tukey test. Differences between treatments were considered significant when the p-value was less than 0.05 (p < 0.05).

Characterization Methods
The synthesized compounds were characterized by UV-VIS and FTIRmed spectroscopy, and TG-DTA thermal analysis.
The measurements in the FTIRmed region were obtained in a Perkin Elmer

Results and Discussion
Thermal Analysis TG-DTA Curves: The TG-DTA curves shown in Figure 1 show    ion as well as the residue formed. Thermal characterization and qualitative tests with the observation of water outflow in the test tube, absence of precipitate in AgNO 3 tests, and the possible formation of RuO 2 residue, suggest stoichiometry [RuCl 2 (bipy)(L-trip)]•1/2H 2 O. FTIR med Spectroscopy: Figure 2 illustrates the FTIR med spectra of the compounds studied. A significant difference can be observed between the spectrum of [RuCl 2 (bipy)(L-trip)], its precursor K[RuCl 4 (bipy)], and the free L-trip ligand. The most relevant changes occur in the carboxylate ion and aliphatic amine regions.
The presence of characteristic bands such as ν (NH 3 + ) and δ (NH 3 + ), as well as the position of the carboxylate ion stretching vibrations in the free L-trip ligand, may indicate whether it is in zwitterion form [27] [28]. In the spectrum in the above spectrum these bands are observed at 3078 cm −1 for ν as (NH 3 + ), 2073 cm −1 for torsional oscillations of the group (NH 3 + ), 1659 cm −1 for δ (NH 3 + ), 1582 cm −1 for νas (COO − ) and 1410 cm −1 for νs (COO − ) thus confirming the zwitterion form Advances in Biological Chemistry Ni +2 , Cu +2 and Zn +2 compounds with L-trip, these bands are present between 3344 and 3270 cm −1 [29]. It is noted that the presence of water in the compound masks the indole ν (NH) band which can be observed at 3401 cm −1 in the free ligand. The decrease in intensity followed by overlap indicates that indole participates in intramolecular interactions. Despite the presence of water, it is still possible to observe the ν (CH) at 3073 cm −1 . According to the literature, the carboxylate ion is very versatile and can coordinate in many ways [30].  [30]. These shifts increase the distance between the frequencies relative to ν (COO − ) compared to the free binder in the form of sodium or potassium salt [30]. In the spectrum of the new compound, it is possible to observe a shift of the νas (COO − ) band from 1582 cm −1 to 1602 cm −1 . This displacement, for higher energies, corroborates the expected for νas (COO − ). However, it seems that νs (COO − ) at 1410 cm −1 had a slight displacement to the region of 1419 cm −1 , overlapping with the band at 1421 cm −1 , initially observed in the starting compound, diverging than expected [28] [30]. Despite the unexpected behavior of νs (COO − ) at 1410 cm −1 , the carboxylate ion is unlikely to be chelated or bridged to the metal [30]. In addition to indicative of Ru-N bond formation, obtained in FTIR med analysis, qualitative tests on silver nitrate test tube and absence of precipitate confirm the presence of two chlorine atoms in the coordination sphere. The presence of these two chlorine atoms in the coordinating sphere makes it impossible for any type of carboxylate ion to bind other than the monodentate form. Even though νs (COO − ) has not shifted to lower energies, it is possible to observe an increase in the distance between frequencies relative to ν (COO − ) compared to the free binder in salt form. For the new compound, synthesized in this paper, the distance between the frequencies referring to ν (COO − ) is 183 cm −1 . This result coincides with that expected for a monodentate carboxylate ion coordination [30]. In addition to what has already been discussed, it is possible in the spectrum of the new compound to observe bands that were not originally present in the spectrum of the precursor compound. These bands refer to indole ν (CN) at 1342 cm −1 and p (CH 2 ) at 878 and 801 cm −1 . In the free ligand are observed, respectively, in 1355, 858, 849, and 803 cm −1 . It is possible to notice small displacements that may be related to intramolecular interactions. This type of interaction is observed for the compound. [Ru(bipy) 2 (L-trip)]ClO 4 [26]. Amino acids can coordinate with transition metals in two ways, monodentate or bidentate, with the most frequent bidentate form being [31] [32]. Analysis of the above spectra gives strong evidence of the formation of the Ru-O and Ru-N bond with lymphatic amine nitrogen. FTIR med analysis allows us to evaluate that the L-trip molecule coordinates in a bidentate manner to the metallic center. This type of coordination fits well with that described for ruthenium L-trip compounds, [26] [32] covers [28] [33], platinum [34] [35], palladium [35], among others.
Cytotoxic evaluation of ruthenium compounds: The acridine orange staining assay allowed the distinction between living and dead cells in each of the experimental groups, thus allowing to evaluate if the tested compounds presented toxicity to the PBMC and MCF-7 cells. The degeneration processes, as well as their probable mechanisms, will be evaluated in further tests.
The data obtained by analysis of variance (ANOVA) followed by the multiple comparisons test (Tukey) give us results with 95% reliability. Thus, it is possible to state that there are no significant differences in cell death between the PBMC/MCF-7 groups, showing us that the rate of dead cells was similar in both strains. (Figure 3  ruthenium-derived compounds [7] [8]. Some ruthenium-derived compounds show some selectivity for tumor cells as compared to PBMC cells. This is the case of the cis-[RuCl 2 (NH 3 ) 4 ]Cl compound which has relevant toxicity for A-20, SK-Br-3, and S-180 strains, moderate in Jurkat cells and very low toxicity in PBMC cells when compared to strains. A-20, SK-Br-3, and S-180 [23].

Conclusion
From the precursor K[RuCl 4 (bipy)] a simple and reproducible synthesis route for a new Ru +3 coordination compound with bipy and L-trip was generated in the present work. Due to the thermal instability of the new compound, it was not possible to develop a purification process for it. Spectroscopic characterization gives us strong indications of Ru-(L-trip) interaction. The displacement of the carboxylate ion band at higher energies in FTIR med and the absence of aliphatic amine bands indicate bidentate coordination of the L-trip ligand to the ruthenium metal. With studies in thermoanalytical techniques, it is possible to suggest the minimum formula of the compound, [RuCl 2 (bipy)(L-trip)]•1/2H 2 O. The new compound was thermally unstable, qualitative test tube testing and heat synthesis synthesize thermal instability below 100˚C with the release of an odor similar to that generated by L-trip burning. The evaluation of the antitumor potential of precursor K[RuCl 4 (bipy)] showed that it has considerable toxic effects on the MCF-7 cell line, but it did not show selectivity and not reached PBMC cells in the same proportion. Evaluation of the antitumor potential of [RuCl 2 (bipy)(L-trip)]•1/2H 2 O showed selectivity for MCF-7 cells as compared to PBMC cells.