Anti-Mycobacterium tuberculosis and Cytotoxicity Activities of Ruthenium ( II ) / Bipyridine / Diphosphine / Pyrimidine-2-thiolate Complexes : The Role of the Non-Coordinated N-Atom

The [Ru(Spym)(bipy)(P–P)]PF6, [Spym = pyrimidine-2-thiolate anion; P–P = 1 ,2-bis (d iphenylphosphino)e thane , 1 ,3 -bis (d iphenylphosphino)propane and 1,1’-bis(diphenylphosphino)ferrocene] complexes were synthesized and characterized by spectroscopic, electrochemical and elemental analysis, and by X-ray crystallography. The minimal inhibitory concentration (MIC) of the compounds against Mycobacterium tuberculosis and the complex concentration causing 50% tumor cell growth inhibition (IC50) against breast cancer cells, MDA-MB-231, were determined. All three compounds gave promising values in both tests. It is interesting to mention that all three complexes display MICs against Mycobacterium tuberculosis showing higher activity than cycloserine, a second line drug used in the treatment of the illness. The complexes interact weakly with the DNA.


Introduction
2][3][4] In general, ruthenium complexes can have their properties tuned, since slight changes in the coordination environment around the metal center lead to significant alterations of their electrochemical, spectroscopic and chemical behavior, and hence their biological activity, which explains the interest in these compounds. 5,67][18][19][20][21][22][23] Phosphine ligands have both σ-donor and π-acceptor character, being able to stabilize metals in both high and low oxidation states. 24In addition, biphosphine (P-P) ligands also play an important role in catalysis and in bioinorganic chemistry, 25,26 and together with the 2,2'-bipyridine (bipy) ligand, eventually could contribute to increase biological activities and DNA interactions.Therefore, the aim of this work is to synthesize and to characterize ruthenium(II) complexes with general formula [Ru(Spym)(bipy)(P-P)]PF 6 [Spym = pyrimidine-2-thiolate The anti-MTB activity of the compounds was determined by the resazurin microtiter assay (REMA). 27Stock solutions of the test compounds were prepared in DMSO and diluted in Middlebrook 7H9 broth (Difco), supplemented with oleic acid, albumin, dextrose and catalase (OADC enrichment, BBL/Becton Dickinson), to obtain final drug concentrations from 0.15 to 250 μg mL -1 .The serial dilutions were carried out in a Precision XS Microplate Sample Processor (Biotek TM ).Isoniazid was dissolved in distilled water, according to the manufacturers' recommendations (Difco), and used as a standard drug.MTB H 37 Rv (American Type Culture Collection (ATCC) 27294) was grown at 37 o C for 7 to 10 days in Middlebrook 7H9 broth supplemented with OADC, plus 0.05% Tween 80 to avoid clumps.Cultures were centrifuged for 15 min at 3,150 × g, washed twice, suspended in phosphate-buffered saline and aliquots were frozen at -80 °C.After 2 days, the number of colony-forming units (CFU) was determined.MTB H 37 Rv (ATCC 27294) was thawed and mixed in microplate wells with the test compounds and 7H9 broth, yielding a final testing volume of 200 μL with 2 × 10 4 CFU mL -1 .Microplates were incubated for 7 days at 37 °C, after which resazurin was added for the reading.Wells that turned from blue to pink, with the development of fluorescence, indicated growth of bacterial cells, while maintenance of the blue color indicated bacterial inhibition. 27,28The fluorescence was read (530 nm excitation filter and 590 nm emission filter) in a SPECTRAfluor Plus (Tecan ® ) microfluorimeter.The minimal inhibitory concentration (MIC) was defined as the lowest concentration resulting in 90% inhibition of growth of MTB. 28As a standard test, the MIC of isoniazid was determined on each microplate.The acceptable range of isoniazid MIC is from 0.015 to 0.06 μg mL -1 . 27,28Each test was set up in triplicate.

In vitro cytotoxicity
The in vitro cytotoxicity assays on cultured human tumor cell lines still represent the standard method for the initial screening of antitumor agents.Thus, as a first step in assessing their pharmacological properties, the new ruthenium complexes were assayed against human breast tumor cell lines MDA-MB-231 and L929 (ATCC:CCL 1, mouse fibroblast).The cells were routinely maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), at 37 o C in a humidified 5% CO 2 atmosphere.After reaching confluence, the cells were detached by trypsinization and counted.For the cytotoxicity assay, 5 × 10 4 cells well -1 were seeded in 200 μL of complete medium in 96-well assay microplates (Corning Costar).The plates were incubated at 37 o C in 5% CO 2 for 24 h to allow cell adhesion, prior to drug testing.All tested compounds were dissolved in sterile DMSO (stock solution with maximum concentration of 20 mmol L -1 ) and diluted to 5, 2, 1, 0.5, 0.2, 0.02 and 0.002 mmol L -1 .From each of these dilute samples, 2 μL aliquots were added to 200 μL medium (without FBS) giving a final concentration of DMSO of approximately 1% and a final concentration of the complex diluted about 100×.Attached cells were exposed to the compounds for a 24 h.Cell respiration, as an indicator of cell viability, was then determined by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). 29MTT solution (0.5 mg mL -1 ) was added to cell cultures and incubated for 3 h, after which 100 mL of isopropanol was added to dissolve the precipitated formazan crystals.The conversion of MTT to formazan by metabolically viable cells was monitored in an automated microplate reader at 570 nm.The cell viability percentage was calculated by dividing the average absorbance of the cells treated with the test compounds by that of the control; cell viability percentage was plotted against drug concentration (logarithmic scale) to determine the drug concentration at which 50% of the cells are viable relative to the control (IC 50 ), the error being estimated for the average of 3 trials.

DNA interaction studies
All the measurements on DNA were carried out in Tris-HCl buffer (5 mmol L -1 Tris-HCl and 50 mmol L -1 NaCl, pH 7.4).In order to compare the DNA binding affinities quantitatively, the intrinsic binding constants K b of complexes 1-3 bound to calf thymus (CT)-DNA were found by monitoring the changes in absorbance of the π → π* spectral band (253-275 nm) with increasing concentration of DNA and using equation 1: 30 [DNA] / (ε a - where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficients ε a , ε f and ε b correspond to A obs / [complex], the extinction coefficient for the free ruthenium complex and the extinction coefficient for the free complex in the fully bound form, respectively.In the plot of [DNA] / (ε a -ε f ) vs. [DNA], the value of K b is given by the ratio of slope to intercept.The concentration per nucleotide was determined by absorption spectrophotometric analysis, assuming the molar absorption coefficient 6600 mol -1 L cm -1 at 260 nm. 31

X-Ray crystallography
Crystals of the three compounds were grown by slow evaporation of dichloromethane/methanol or dichloromethane/diethyl ether solutions.The crystals were mounted on a goniometer in an Enraf-Nonius kappa geometry charge-coupled device (CCD) diffractometer with graphite monochromated Mo Kα (λ = 0.71073 Å) radiation.The final unit cell parameters were based on all reflections.Data were collected at room temperature with the COLLECT program, and integration and scaling of the reflections were performed with the HKL Denzo-Scalepack software package. 32,33Absorption correction was carried out by the Gaussian method. 34The structures were solved by direct methods with SHELXS-97. 35The models were refined by full-matrix least squares on F2 by means of SHELXL-97. 36All hydrogen atoms were stereochemically positioned and refined with a riding model.The Oak Ridge thermal ellipsoid plot (ORTEP) views were prepared with ORTEP-3 for Windows. 37Hydrogen atoms on the aromatic rings were refined isotropically, each with a thermal parameter 20% greater than the equivalent isotropic displacement parameter of the atom to which it is bound.

Theoretical calculations
9][40] This approach is further referred to as B3LYP/ Lanl2dz.The geometry optimization was followed by the vibrational frequencies calculation using the same approach.In order to estimate the contributions of specific atoms to the HOMO and LUMO of the complex studied, we performed calculations of fragment densities of states (projected densities of states, PDOS), using the keywords 'Fragment' and 'Population' as implemented in the Gaussian 09 program.For the PDOS calculations we used the B3LYP/ Lanl2dz optimized geometry and the approach designated as B3LYP/[Ru,Fe:CEP-121G; C,H,O,N,P:6-31G*], with the Stevens/Basch/Krauss effective core potential triple-split basis (CEP-121G) on Ru and Fe atoms and the split-valence 6-31G* basis set on the light atoms. 41,42The last approach was shown to provide good agreement with the experimental data.
The 31 P{ 1 H} NMR spectra of the compounds showed typical AX spin systems characterized by the nuclear magnetic nonequivalence of the two phosphorus atoms present in the complexes.The values of the chemical shifts obtained for the complexes 1-3 and for the precursors are given in Table 1.All the compounds exhibited septets whose chemical shift was centered at d -144 ppm, indicating the presence of PF 6 -as the counter-ion.As expected, the doublets shown by the complexes [Ru(Spym)(bipy)(P-P)] PF 6 are upshifted relative to the precursors, indicating that the chlorine ligands in the precursors shield the phosphorus atoms of the phosphines more efficiently than the coordinated Spym -ligand (Table 1).
The 1 H NMR spectra of the compounds show a series of multiplets, ranging from d 7.68 to 6.38, corresponding to the 20 hydrogen atoms of the diphosphine phenyl rings.The eight hydrogen atoms of bipy are observed in the d 9.50-6.87region, and some of the signals overlap with the proton resonances of the phenyl groups.The protons corresponding to the -(CH 2 ) n -groups of the phosphines appeared as multiplets in the d 3.37-2.53region for 1 and d 3.47-1.81for 2. The three protons of Spym show their chemical shifts in the d 8.18-6.20 region.For the [Ru(Spym) (bipy)(dppf)]PF 6 complex 3 the ferrocene hydrogens show resonances between d 6.02 and 3.50, corresponding to eight hydrogen atoms.The six peaks observed for these protons are caused by the non-equivalence of the phosphorus atoms bonded to the cyclopentadienyl rings (Cp), as confirmed by a pair of doublets observed in the 31 P{ 1 H} NMR spectrum of the [Ru(Spym)(bipy)(dppf)]PF 6 complex.
The compounds 1-3 were studied by cyclic voltammetry technique, and it was found that they show one Ru II /Ru III redox pair: complex 1 shows a quasi-reversible process, E 1/2 = 0.99 V, and complex 2 one quasi-reversible redox pair, E 1/2 = 1.00 V (see Table 1).Complex 3, which is a bimetallic compound, exhibits two processes: the first is also quasi-reversible (E 1/2 = 0.77 V) and relates to Ru II /Ru III , while the second, irreversible, with E pa = 1.28 V, belongs to the Fe II /Fe III oxidation process (Figure 1).
To confirm this suggestion, a few drops of the salt NH 4 SCN, dissolved in methanol were added to the electrochemical cell.The typical red color of the "Fe 3+ -SCN" species on the electrode surface appeared only when the potential reached about 1.2 V, showing that at this point, indeed, the iron(II) of the dppf ligand was being oxidized.This also shows that the iron(II) of the dppf ligand is stabilized by its coordination to the ruthenium(II) center.In complexes fac-[RuCl 3 (NO)(dppf)] and Scheme 1. Reaction of syntheses of the [Ru(Spym)(bipy)(P-P)]PF 6 complexes, where P-P means dppe (for complex 1), dppp (for complex 2) and dppf (for complex 3).[Ru(η 6 -C 10 H 14 )(dppf)Cl]PF 6 , the iron(II) was oxidized at l.0 and 0.86 V, respectively. 10,45In the case of [Ru(η 6 -C 10 H 14 )(dppf)Cl]PF 6 there is probably a competitive effect acting to make the oxidation of the iron(II) easier than in the complex [Ru(Spym)(bipy)(dppf)]PF 6 . 46,47he PDOS calculations performed for the complex 3 showed the contributions of the Fe and Ru d-orbitals in the complex, HOMO equal to 48 and 16%, respectively.These results reinforce the assignment of the oxidation processes observed for the complex 3 in its cyclic voltammograms, where the first oxidation process was assigned to the ruthenium(II) and the second oxidation process was assigned to the iron(II).
All the electrochemical processes mentioned above involve one electron, as was shown by electrolytic measurements.

Structural sudy
X-Ray structures of the compounds are represented in Figure 2. Data collection and experimental details are summarized in Table 2, and selected bond distances and angles are presented in Table 3.All the compounds show distorted geometry around the ruthenium center with one phosphorus atom positioned trans to the nitrogen atom of the ligand HSpym (P1 trans to N1) and the other phosphorus atoms trans to one nitrogen of the bipyridine (P2 trans to N4).Thus, in all three complexes the sulfur atom is positioned trans positioned to the nitrogen atom (N3) of the 2,2'-bipyridine ligand.
9][50] In the new complexes the Ru-P (trans to the nitrogen atom of Spym) bond lengths are shorter than the Ru-P distances (trans to the nitrogen atoms of bipy).Ru-P distances of P-P follow the order: dppf > dppp > dppe.The P-Ru-P angles are comparable to the values previously observed for this kind of complex. 49,50o stabilize the crystal structure of the complexes 1-3, there are weak C-H…F-P and C-H…N-C intermolecular interactions, as well as π-π stacking and van der Waals forces.Interestingly, the crystal structure of the complex 3 forms infinite hydrophobic channels formed by the dppf  moiety (Figure 3).This channel is filled by disordered methanol molecules that act as guest solvent, displaying an important role for crystal self-assembly stabilization.
In general, the host-guest systems are stabilized by noncovalent interactions such as depicted in Figure 3.

Vibrational spectra
The IR spectra of all three complexes confirm the presence of the Spym -ligand coordinated to the metal.The band assigned to the ν(N-H) vibration, which appears at 3200-3100 cm -1 in free thiones, is absent in the spectra of the complexes, indicating that the ligand is coordinated to the metal in the deprotonated form. 51,52Bands from the coordinated Spym -ligand appear at approximately 1570 and 1549 cm -1 (ν(C=C) + ν(C=N)), 1434 cm -1 (ν(N=C) + d(CH)) and at 1375 and 1159 cm -1 (ν(C=S)) for all three complexes.[55][56] Electronic spectra Electronic spectra of the three complexes were obtained in dichloromethane and in DMSO solutions and the results are listed in Table 4.The transitions found for all three complexes are at about 290 nm (ε ca.30000 mol -1 L cm -1 ) and 420 nm (ε ca.4000 mol -1 L cm -1 ), corresponding to intraligand (IL) and metal-ligand charge transfer (MLCT) transitions, respectively.[59][60] Cytotoxicity against MDA-MB-231 cell lines The MDA-MB-231 tumor cancer cells and L929 mouse cells were exposed to each complex and to the free ligands, for a period of 24 h, in order to allow them to reach the DNA or any other biological target.As a positive control, the cytotoxicity of cisplatin was assessed under the same experimental conditions.The IC 50 values, calculated from the dose-survival curves generated by the MTT assays performed after the drug treatment, are shown in Table 5.It can be seen that the free ligands were inactive against either the tumor cell or the mouse cell line.
All three new complexes are very active against MDA-MB-231 breast human tumor cells showing low values of IC 50 and high values of IC 50 against normal L929 cells.The high activity of complexes 1-3 gives support to our previous suggestion that a non-coordinated atom, in this case, the nitrogen (N2) atom of the Spym -ligand, can allow interaction of the complexes with DNA or another biomolecule, increasing the cytotoxicity of the complexes.The good results obtained for similar complexes with 4,6-dimethyl-2-mercaptopyridine also support this suggestion. 53

Anti-mycobacterial activity
The compounds were investigated for their in vitro anti-mycobacterial activity against M. tuberculosis strain H37Rv, by the microplate Alamar Blue assay (MABA) method. 27The MICs found for the ruthenium complexes 1-3, free ligands and isoniazid are shown in Table 5.According to these tests, the compounds exhibited promising activity, with MIC values in the range 1.56-4.12mg mL -1 , lower than those of cycloserine (MIC 12.5-50.0mg L -1 ), a second-line drug used in several schemes of conventional tuberculosis treatment. 58As can be seen in Table 5 the MIC values for free ligands were much higher than those observed for the complexes 1-3.In our previous publication, it was suggested that for the complex [Ru(dppb)(pic)(bipy)]PF 6 (pic = picolinic acid) action against M. tuberculosis may be in the cell wall biosynthesis, considering the differential expression of a cell wall hydrolase. 61Thus, in our laboratory, research is ongoing to figure out if the diphosphine/pyrimidine-2-thiolate complexes studied in this work follow the same mechanism of action against M. tuberculosis previously suggested.

DNA binding studies
Electronic absorption spectroscopy is an effective method of examining the mode and extent of binding of a metal complex with DNA.Thus, in order to shed further light into the mode of interaction of the new compounds with DNA, experiments of absorption titration were performed, yielding the binding constants K b = 1.6 × 10 3 mol -1 L for 1; 1.3 × 10 3 mol -1 L for 2 and 1.1 × 10 3 mol -1 L for 3.All K b values are much lower than those observed for the typical classical intercalator ethidium bromide (K b = 4.94 × 10 5 mol -1 L in 25 mmol L -1 Tris-HCl/40 mmol L -1 NaCl buffer, pH 7.9). 30On the other hand, K b found for 1-3 present the same order of magnitude comparing with some hexacoordinated ruthenium(II) complexes with N-heterocyclic ligands, under similar conditions. 62The K b values tendency of 1 > 2 > 3 agree with the order dppe > dppp > dppf.It suggests that the less sterically-hindered dppe in the complex 1 allows its interaction with DNA through non-coordinated nitrogen atom of the Spym ligand more effectively than 2 and 3.

Conclusions
Three new complexes with general formula [Ru(Spym) (bipy)(P-P)]PF 6 , [Spym = pyrimidine-2-thiolate; bipy = 2,2'-bipyridine; P-P = 1,2-bis(diphenylphosphino) ethane, 1,3-bis(diphenylphosphino)propane or 1,1'-bis(diphenylphosphino)ferrocene)] were synthesized and characterized by spectroscopy, cyclic voltammetry and X-ray crystallography.The complexes exhibit low DNA binding affinity, but the in vitro antitumor activity test utilizing the MDA-MB-231 human tumor cell line indicated a high degree of cytotoxicity for all three complexes, better than cisplatin at the same concentration.Antimicrobial activity assays of the new complexes provided evidence that they are potential agents against M. tuberculosis H37Rv.The MIC values of anti-M.tuberculosis activity obtained for the complexes showed for them an activity higher than for cycloserine, a second line drug used in the treatment of the illness.Thus, these complexes are promising as anti-M.tuberculosis and antitumor drugs.

Figure 3 .
Figure 3. Crystal self-assembly of the complex 3 viewed along the c-axis, showing the hydrophobic channel.

Table 1 .
31P { 1 H} NMR and cyclic voltammetry data of the compounds a Refers to Fe III /Fe II process.d:Chemical shift; 2 J P-P : two bond P-P coupling constant; E pa : anodic peak potential; I pa /I pc : anodic peak current/cathodic peak current ratio.Vol.27,No. 1, 2016

Table 3 .
Selected bond distances and bond angles of complexes 1-3

Table 5 .
IC50and MIC values of complexes 1-3, free ligands and reference drugs, measured in 1.0% DMSO solutions IC 50 : drug concentration at which 50% of the cells are viable relative to the control; H37Rv: M. tuberculosis strain; SI: selectivity index calculated by dividing IC 50 for the mouse cells by the MIC for the pathogen; MIC: minimal inhibitory concentration; N.M.: not measured.