Synthesis, characterization and biological activity of a gold(I) triazenide complex against chronic myeloid leukemia cells and biofilm producing microorganisms

*Correspondence: R. Hörner. Laboratório de Bacteriologia, Departamento de Análises Clínicas e Toxicológicas, Centro de Ciências da Saúde, Universidade Federal deSanta Maria (UFSM), Santa Maria,RS. Prédio 26, Sala1201, Campus da UFSM, CEP: 97015-900. Telefax: +55(55) 3220-8751. E-mail: rosmari.ufsm@gmail.com Synthesis, characterization and biological activity of a gold(I) triazenide complex against chronic myeloid leukemia cells and biofilm producing microorganisms

A major breakthrough in the treatment of CML has been achieved with the advent of selective inhibitors of tyrosine kinase such as imatinib mesylate (Glivec) and its derivatives.Despite the good results obtained by these drugs, the lack of response to treatment that occurs when cancer cells develop resistance mechanisms is an important outcome of this disease (Yun et al., 2014;Wang et al., 2015).
Similarly, antimicrobial therapy constitutes a challenge due to the ease with which bacteria acquire resistance genes transmitted by mobile genetic elements (Brown-Jaque, Calero-Cáceres, Muniesa, 2015).Biofilm formation is one of the major virulence factors developed by bacteria.Biofilm is commonly associated with chronic infections because it resists host defense systems and has increased tolerance to antibiotics and disinfectants (Høiby et al., 2010).
The combination of the pharmacophoric radical TZC with metal ions that have biological activity such as ion gold tends to have a synergistic effect, producing more active and stable molecules (Sreedhara, Cowan, 2001).Thus, this study aimed to synthesize and characterize a new triazenide compound complexed with ion gold (I), as well as to evaluate the in vitro antiproliferative and antibacterial activity against the cell line K562 (CML) and bacterial isolates of coagulase-negative staphylococci (CoNS), identified as biofilm producers.

Synthesis of Au(I) triazenide complex {[(1-(2-bromophenyl)-3-(2-nitrophenyl) triazenide](triphenylphosphine)gold(I)}
Under continuous stirring, the free ligand 1-(2-bromophenyl)-3-(2-nitrophenyl)triazene C 12 H 9 N 4 O 2 Br (100 mg, 0.311 mmol) was dissolved in a mixture of 20 mL of methanol and tetrahydrofuran (1:1), resulting in a yellow transparent solution.The free ligand was deprotonated by the addition of 5 drops of an alkaline solution of potassium methoxide, previously prepared by dissolution of potassium hydroxide (1000 mg, 17.82 mmol) in 10 mL of methanol.The reaction mixture changed to transparent deep-red.After stirring of the reaction mixture during 30min, triphenylphosphine-gold(I)chloride (154 mg, 0.311 mmol) was added.The color of the reaction mixture changed to orange and an orange-red precipitate was observed.The reaction mixture was filtered-off and the isolated precipitate was dissolved in 10 mL of pyridine.Transparent orange crystals with a block shape and suitable for X-ray diffraction were obtained after 48h by slow evaporation of the solvent, and vacuum-dried in a Schlenk vessel for three days.

Single-crystal X-ray diffraction
A single-crystal sample was attached to a glass wire and was subjected to the collection of diffraction data at room temperature (20-22 °C) with a Bruker APEX II CCD diffractometer, with an area detector and monochromatized Mo-Kα radiation with graphite monochromator (Bruker, 2004).Data integration and absorption correction were performed with SAINT (Bruker, 2004) and SADABS (Sheldrick, 1996) softwares, respectively.Structures were solved by direct methods (Burla et al., 2005) and refined using the square of the structure factor (F2) and complete matrices, with anisotropic thermal parameters for all non-hydrogenic atoms (Sheldrick, 1997).Fractional coordinates of hydrogen atoms of the phenyl rings were obtained geometrically (C-H = 0.93 Å for the C SP2 atoms), refined and attached to their respective carbon atoms and isotropic thermal parameters with values U iso (H) relating to 1,2U eq C sp2 .Tables with crystallographic parameters and details regarding data collection were generated with the WinGX program (Farrugia, 1999), and graphical representations of structures and/or structural details discussed were generated with the DIAMOND (Brandenburg, 2005) program.

Cell culture
The Chronic Myeloid Leukemia K562 cell line, established from the sample of a 53-year-old patient was commercially acquired at the Cell Bank of Rio de Janeiro -BCRJ, Paul Erlich Technical and Scientific Association -APABCAM.Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (Gibco® SBF-), 100 IU/ml penicillin G, 100mg/ml streptomycin for 24 h at 37 °C in a humid atmosphere with 5% carbon dioxide (CO 2 ).

Cytotoxicity assay
The cytotoxic effect of the complex on the cell line K562 was performed using the colorimetric assay based on the reduction of diazonium salt bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Mosmann, 1983).The compound was dissolved in a mixture of ultra-pure water and dimethylsulfoxide DMSO (1:1) and then diluted at different concentrations (100, 50, 25, 12, 6, 3, 1.5 and 0.75 µM).In the cytotoxicity assay, 3 x 10 6 cells/well were used in a 96-well plate exposed to different concentrations of the compound and incubated for 20 h.Control culture containing cells in the absence of the compound was also performed.Imatinib mesylate (Glivec) was the antineoplastic used as reference.After 20h, each well was treated with 20 microliters (uL) of MTT and plates were re-incubated for additional 4h at 37 °C and 5% CO 2 .After incubation, formazan crystals were dissolved by addition of DMSO and the inhibition of cell growth detected by measuring absorbance at 570 nanometers (nm) using an Epoch® microplate reader (Fisher BT2000 Bio-Tek).Each test was performed in triplicate in three independent tests.Results were statistically analyzed by SISVAR software (Ferreira, 1988).For cell death values (%), analysis of data variance was performed and means were compared by Tukey test at 5% error probability.IC 50 values corresponding to the concentration of compound able to inhibit growth of 50% of the cells were analyzed by GraphPad Prism 5.

Isolates and bacterial strains
The in vitro antibacterial activity of the Au (I) triazenide complex was evaluated against seven CoNS bacterial isolates, obtained from an investigation of contamination in platelet concentrates.These microorganisms have been characterized as biofilm producers by adhesion to a borosilicate tube and Congo red agar phenotypic methods (Christensen et al., 1982;Freeman, Falkiner, Keane, 1989), and by screening for icaACD genes by polymerase chain reaction (PCR) (Arciola et al., 2005).Isolates were two Staphylococcus epidermidis, one Staphylococcus saprophyticus, three Staphylococcus haemolyticus and one Staphylococcus warneri.Two strains of international reference standard (American Type Culture Collection -ATCC) of nonbiofilm-producing Staphylococcus epidermidis 12228 and Staphylococcus saprophyticus 15305 were also used.

Determination of the minimum inhibitory concentration (MIC)
Bacterial isolates and ATCC strains, stored in 15% glycerol at -80 °C, were pre-activated using the agar trypticase soy medium (TSA) for 24 h at 35 ± 2 °C.Evaluation of antibacterial activity of the compound was performed using the conventional method of broth microdilution for Minimum Inhibitory Concentration (MIC) based on the Clinical and Laboratory Standards Institute guidelines (CLSI, 2012).The test compound was diluted in DMSO and ultra-pure water (1:1) at a concentration of 2560 µg mL -1 and then successive dilutions were made with concentrations from 128 to 0.25 µg mL -1 .The bacterial inoculum was prepared using a 0.5 McFarland scale, so that each well contained 5 x 10 5 CFU mL -1 .Plates were incubated for 24 h at 35 ± 2 °C and after this period, the MIC was determined visually as the lowest concentration that completely inhibited growth of microorganisms in dilution wells.Each assay was performed in triplicate.

ETHICAL CONSIDERATIONS
This study was approved by the Research Ethics Committee (CEP) of UFSM, under the certificate number of presentation for ethical consideration (CAAE) 0285.0.243.000-09.

Crystal structure
Glassy crystals with orange prismatic habit were obtained from slow evaporation of the stock solution.
Values of the unit cell parameters were obtained based on the refinement of reflections obtained in different quadrants of Ewald sphere, related to the angular region and refined by the least squares method at the end of the collection of intensity data.Crystallographic parameters and further details regarding the collection of data and refinement of the complex are listed in Table I.
In the solid state, the structure of the complex is formed by non-classic aromatic hydrogen bonds type C-H ... M (where M represents the centroid point of the aromatic ring).These interactions involve a polarized hydrogen atom of a C-H group donor of a phenyl ring of triphenylphosphyne in a unit of a molecular complex, and an acceptor ring of another phenyl of triphenylphosphyne, resulting in the formation of centrosymmetric dimers.These dimers are reproduced by the same type of interaction, forming a supramolecular arrangement 1-D.
T h i s c o m p l e x f r a g m e n t s i n v o l v e d i n t h e determination of these interactions are C31-C36 / C56'-C51' for the formation of centrosymmetric dimers, as represented in Figure 3.
In addition to the previous determinations, it is necessary to determine the angles and distances between the two rings.These distances and angles are listed in Table II.The centrosymmetric dimers of the complex, formed by aromatic interactions, are projected in Figure 4.       -1 + x, y, z]. Figure 5 shows the considered fragments with all denominations, which are essential to determine interactions, and these values are described in Table III.

Biological activity
When produced by microorganisms, biofilm protects against host defense mechanisms, preventing or hindering the diffusion of antimicrobials (Høiby et al., 2010).The concentration of antibiotic required to combat infections caused by biofilm-producing microorganisms can be up to a thousand times higher compared to infections caused by their planktonic counterparts (non-biofilm producers) (Musk, Hergenrother, 2006;Høiby et al., 2010).
The tested compound showed significant antibacterial activity when compared to non-biofilm-producing strains.The results of this activity are shown in Table IV.It can be observed that among the seven biofilm-producing clinical isolates, all had MICs lower or equal to those of strains that do not produce biofilm.A study by Presterl and colleagues (2009) has shown that bacterial isolates of biofilm-bactericidal effect (Presterl et al., 2009).The studied Au (I) triazenide complex proved to be more active, compared to these antimicrobial agents, being capable of inhibiting all isolates of biofilm-producing S. epidermidis, at a concentration four times lower (32 µg/mL).
Several studies using TZC compounds have shown that these compounds have broad antimicrobial activity (Hörner et al. 2008;Domingues et al., 2010;Ombaka et al., 2012;Paraginski et al., 2014;Tizotti et al., 2016).Studies suggest that the antibacterial action of TZCs is due to the chelating activity of metal ions that are constituents of the bacteria cell wall, hindering their synthesis and leading to cell death (Hörner et al. 2008;Ombaka et al., 2012).According to Paraginski and colleagues, complexation of TZC compounds with metal ions such as gold (I) can provide potent antibacterial activity (Paraginski et al., 2014).However, this is, to the best of our knowledge, the first study to use such compounds complexed with metals like gold against biofilm-producing microorganisms isolated from platelet concentrates.
In a study using gold nanoparticles against multidrug-resistant bacteria, it has been observed that they act altering membrane potential and decreasing the synthesis of adenosine triphosphate (ATP), causing a general decline in bacterial metabolism (Cui et al., 2012).Barreiro et al. (2012) reported that this metal acts inhibiting bacterial proliferation by blocking cell cytokinesis (Barreiro et al., 2012).Domingues et al. (2010), obtained MIC values>128 mg/mL using TZCs not complexed with metal compounds against an S. epidermidis clinical isolate, as well as S. epidermidis ATCC 12128 strain (Domingues et al., 2010).In addition, another study using 15 TZC compounds not associated with metal showed MIC values=64 mg/mL for hospital isolates of S. epidermidis and S. saprophyticus (Horner et al., 2008).In both studies, the compounds showed lower activity compared to Au (I) triazenide.Thus, we believe that part of the significant antibacterial activity demonstrated is due to the complexation of TZC with metal, which potentiates its action.
As for the anti-proliferative activity, we can see the percentage of cell death obtained by the compound, as shown in Table V.We observed that the compound

Staphylococcus warneri =64
Staphylococcus epidermidis ATCC 12228* =64 Staphylococcus saprophyticus ATCC 15305* =64 *Non-biofilm producing ATCC strains.producing S. epidermidis have increased resistance to antibiotics.These researchers evaluated the combination of azithromycin at a concentration of 512 µg/ml with high concentrations of ceftriaxone, showing a significant  activity was not dose-dependent, being more active in the concentration of 50 uM, which was also evidenced by Domingues et al. (2010) in trials with TZCs against AML cells (Domingues et al., 2010).In addition, concentrations of 50, 25 and 12 mM showed no significant difference through the statistical analysis of results, as well as the concentrations of 100, 3 and 1.5 mM.Imatinib Mesylate (Glivec) showed no significant difference in the established concentrations, except for the concentration of 0.75 mM.Regarding IC50, we found that the Au (I) triazenide complex showed cytotoxicity value lower than imatinib mesylate (Glivec), which is the standard drug used for the treatment of CML.
Primary studies have shown that TZC compounds may have their activity related to the inhibition of the tyrosine kinase enzyme (Matheson et al., 2004), which is directly related to the malignancy of CML (Gora-Tybor, Robak, 2008).Furthermore, it is known that the acquired resistance to tyrosine kinase inhibitors have been a problem for several patients with this malignancy (Yun et al., 2014;Wang et al., 2015).
In clinical practice, drugs bound to this metal have been especially studied in cultured cells systems, where it is possible to assess their cytotoxic potential (Gouvea et al., 2012;Bertrand, Casini, 2014).Gold organometallic compounds play an important role in medicinal chemistry, and these studies were determinant to trigger an enormous interest in the scientific community for new complexes of this metal, aiming their application to antitumor therapy (Bertrand, Casini, 2014), rheumatoid arthritis therapy, and viral and parasitic diseases (Berners-Price, Filipovska, 2011).
According to the results, we suggest that the significant biological activity demonstrated by the Au (I) triazenide complex studied is due to the presence of gold ion which enhances the action of diazoamine group.Moreover, this research is part of an initial study, the first investigation to use Au (I) triazenide complex against K562 cells and biofilm-producing bacteria.As a result, other analyses are being carried out through the synthesis of new TZC compounds complexed with metals, as well as the elucidation of the possible mechanisms of action.

FIGURE 2 -
FIGURE 2 -Triazenide gold complex structural projection (I).Thermal ellipsoids with probability level of 50%.Hydrogen atoms omitted for better viewing.

FIGURE 3 -
FIGURE 3 -Projection of fragments involved in the non classical aromatic hidrogen bondings C-H ••• Ph of the dimers of the C31-C36 / C51'-C56 'complex.[Symmetry Code x-1, y-2, z].Hydrogen atoms are omitted for better layout view.M = centroid point of the aromatic ring.

Figure 6
Figure 6 depicts the projection of the supramolecular arrangement of the complex formed by centrosymmetric dimers reproduced in a 1-D chain via non-classical aromatic centrosymmetric hydrogen bonds (C-H ••• Ph) in the crystallographic direction [100].

FIGURE 6 -
FIGURE 6 -Projection of the supramolecular arrangement of the complex in the crystallographic direction of the axis b, formed by non-classical aromatic hydrogen bondings C54-H54 ••• M '' (where M is the centroid point of the aromatic ring C51 '' -C56 ').Hydrogen atoms are omitted for better visualization.Showing partial cell content.[Symmetry code ('): -1 + x, y, z].

TABLE I -
Data from intensity collection and refinement of the crystal/molecular structure of the Au (I) triazenide complex

TABLE IV -
Antibacterial

TABLE III -
Geometric parameters for the non-classical aromatic hydrogen bonds (C-H ••• Ph) of the 1-D arrangement of the complex