JBCS

INTRODUCTION

Bacterial resistance and the resulting risk for ineffective treatment of infections are serious and growing problems.^1,2 Resistance rates are increasing among several problematic Gram-negative pathogens that are often responsible for serious nosocomial infections, including Acinetobacter spp., Pseudomonasaeruginosa and Enterobacteriaceae.^3,4 Related to the class of Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA) is a well-recognized public health problem throughout the world.⁵

There is clearly a need for the development of new antimicrobials; but more importantly, there is a need for the development of new classes of antimicrobials based on novel chemical structures, mechanisms, and targets of action, which are effective approaches to overcome the resistance of pathogenic bacteria. Due to the success of the platinum anticancer agents, there has been considerable interest in the development of therapeutic agents based upon other transition metals - and in particular ruthenium(II/III) complexes, due to their well-known interaction with DNA. There have been many studies of the antimicrobial properties of a range of ruthenium complexes.^6-8 In the literature,^8,9 the antibacterial activity of ruthenium complexes containing polypyridine ligands that have cis geometry determined by X-ray diffraction is reported. These compounds were tested against the strains Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, among them are the compounds cis-[Ru(bpy)₂(L)(Cl)]⁺, where L = 4-(4-chlorobenzoyl)pyridine or 4-benzoylpyridine, which showed activity against the Gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermidis with MIC (minimum inhibitory concentration) in the ranges of 250-500 and 125-500 µg mL^-1, respectively. In addition to these, the cis-[Ru(bpy)₂(Met)](PF₆)₂ complex, where Met = l-methionine, was also shown to be effective against Gram-positive bacteria, exhibiting MIC values between 62.5-250.0 µg mL^-1. However, none of these compounds demonstrated activity against Pseudomonas aeruginosa. Moreover, the potential therapeutic applications of NO in antibacterial,¹⁰ anti-inflammatory,¹¹ anticancer¹² and wound healing processes¹³ have resulted in an explosion of research interest in NO donor compounds and in related materials capable of delivering NO to desired sites. Therefore, it is also important to prepare compounds that can release NO upon a controlled external stimulation. One suitable stimulus is light, which can be used to promote NO photorelease from metalonitrosyl complexes as described elsewhere.^14,15 Having all this in mind, we prepared and investigated the photochemical behavior of the cis-[Ru(bpy)(phen)L(NO⁺)]ⁿ⁺ complexes, where: L = ImN (complex I) or SO₃^2- (complex II). In addition, we investigated the antimicrobial activity of the compounds against Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa.

 

EXPERIMENTAL

Chemicals

Organic solvents from Sigma-Aldrich and Merck were used without any previous purification. RuCl₃·xH₂O, 2,2'-bipyridine (bpy), sodium nitrite (NaNO₂), sodium sulfite (Na₂SO₃), 1,10'-phenanthroline (phen), imidazole (ImN) and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich and used without any further purification. cis-[Ru(bpy)(phen)Cl₂] was prepared according to the procedure described in the literature.¹⁶ Nitrosyl complexes are soluble in acetone, methanol, dimethyl sulfoxide and partially soluble in water.

Synthesis of the complexes

cis-[Ru(bpy)(phen)(ImN)(NO₂)](PF₆)

The cis-[Ru(bpy)(phen)Cl₂] (0.100 g, 0.190 mmol) precursor complex was dissolved in a 1:1 ethanol/water solution (10 mL) and mixed with imidazole (0.013 g, 0.190 mmol) maintained under reflux for 2 h. After this time, NaNO₂ (0.014 g, 0.190 mmol) was added, allowing to react for more than 2 h, under reflux. The solids (red crystalline solid) were precipitated by the addition of 1.0 g of ammonium hexafluorophosphate (NH₄PF₆). The solid was collected by filtration, washed with cold water, and dried under vacuum. Yield: 75% based on ruthenium(II) for the formula cis-[Ru(bpy)(phen)(ImN)(NO₂)](PF₆). Electronic spectrum in methanol: 222 nm (3.2 × 10⁴ M^-1 cm^-1), 265 nm (3.3 × 10⁴ M^-1 cm^-1), 288 nm (2.5 × 10⁴ M^-1 cm^-1) 318 nm (5.4 × 10³ M^-1 cm^-1), 450 nm (7.2 × 10³ M^-1 cm^-1). Infrared data (KBr pellet, cm^-1): ν(N-H) 3407, ν_sim (C-H) 3076 , ν(C=C) 1602, ν(C=N) 1466, 1445, ν_ass (NO₂^-)1330, ν_sim (NO₂^-) 1284, δ(C-H) 724, 764. Electrochemical data: E_1/2 = 0.79 V (versus Ag/AgCl) for the Ru^III/II redox couple.

cis-Na[Ru(bpy)(phen)(SO₃)(NO₂)]

The cis-[Ru(bpy)(phen)Cl₂] (0.100 g, 0.190 mmol) precursor complex was dissolved in a 1:1 ethanol/water solution (10 mL) and mixed with Na₂SO₃ (0.025 g, 0.190 mmol) and maintained under reflux for 2 h. After this time, NaNO₂ (0.014 g, 0.190 mmol) was added, allowing to react for more than 2 h, under reflux. The solids (orange crystalline solid) were precipitated by the addition mixture of ethanol/ether solvents and dried under vacuum. Yield: 72% based on ruthenium(II) for the formula cis-Na[Ru(bpy)(phen)(SO₃)(NO₂)]. Electronic spectrum for cis-Na[Ru(bpy)(phen)SO₃(NO₂)] in methanol: 223 nm (2.1 × 10⁴ M^-1 cm^-1), 265 nm (1.7 × 10⁴ M^-1 cm^-1), 286 nm (1.2 × 10⁴ M^-1 cm^-1) 315 nm (2.8 × 10³ M^-1 cm^-1), 440 nm (3.4 × 10³ M^-1 cm^-1). Infrared data (KBr pellet, cm^-1): ν_sim (C-H) 3082, ν(C=C) 1602, ν(C=N) 1470, 1443, ν_ass (NO₂^-)1325, ν_sim (NO₂^-) 1290, ν(SO₃^2-) 1015, 958, 633, δ(C-H) 845, 722. Electrochemical data: E_1/2 = 0.55 V (versus Ag/AgCl) for the Ru^III/II redox couple.

cis-[Ru(bpy)(phen)(ImN)(NO)](PF₆)₃ - complex I

The complex I (Figure 1a) was prepared by dissolving the cis-[Ru(bpy)(phen)(ImN)(NO₂)](PF₆) complex (0.050 g, 0.072 mmol) in methanol (5 mL) under an argon atmosphere. To this mixture, 2.0 mL of concentrated trifluoroacetic acid was added to promote the conversion of NO₂^- to NO⁺.¹⁷ After 2 h, the mixture was concentrated in a rotary evaporator to ca. 8 mL, and the brown amorphous solid was precipitated by the addition of 1.0 g of ammonium hexafluorophosphate (NH₄PF₆), collected by filtration and stored under vacuum. The yield of the precipitated material was 60% based on ruthenium(II) for the formula cis-[Ru(bpy)(phen)(ImN)(NO)](PF₆)₃. Elemental analysis: C₂₅H₂₀F₁₈N₇OP₃Ru: calcd.: C 30.92%, H 2.06%, N 10.10%; found: C 31.05%, H 2.04%, N 9.93%. Infrared data (KBr pellet, cm^-1): ν(N-H) 3416, ν_sim (C-H) 3129, ν(NO⁺) 1942, ν(C=C) 1606, ν(C=N) 1453, 1436, δ(C-H) 769, 725, ν(PF₆^-) 839, 559.

 

 

cis-[Ru(bpy)(phen)(SO₃)(NO)](PF₆) - complex II

The complex II (Figure 1b) was prepared by dissolving the cis-Na[Ru(bpy)(phen)(SO₃)(NO₂)] complex (0.050 g, 0.085 mmol) in methanol (4 mL) under an argon atmosphere. To this mixture, 2.0 mL of concentrated trifluoroacetic acid was added to promote the conversion of NO₂^- to NO⁺.¹⁷ After 2 h, the solvent was then concentrated in a rotary evaporator to near 8 mL, and the solid was precipitated by the addition of 1.0 g the ammonium hexafluorophosphate (NH₄PF₆). The brown amorphous solid was collected by filtration, washed with cold water and stored under vacuum. Yield: 65% based on ruthenium(II) for the formula cis-[Ru(bpy)(phen)(SO₃)(NO)](PF₆). Elemental analysis: C₂₂H₁₈F₆N₅O₅PSRu: calcd.: C 37.15%, H 2.53%, N 9.85%, S 4.51%; found: C 36.02%, H 2.45%, N 9.74%, S 4.34%. Infrared data (KBr pellet, cm^-1): ν_sim (C-H) 3125, 3096, ν(NO⁺) 1914, ν(C=C) 1608, ν(C=N) 1450, 1434, ν(SO₃^2-) 1120, 991, 626, 594, δ(C-H) 774, 719, ν(PF₆^-) 843, 558.

Equipment

Electronic spectra (UV-Vis) were recorded on a photodiode-array spectrophotometer, model 8453 (Agilent). The determination equilibrium acid/base and spectrophotometric titration were carried out using the spectrophotometric method (absorbance measurements were performed at 420 and 405 nm for complex I and II, respectively) at 25.0 ± 0.1 in a 0.1 mol L^-1 aqueous solution of HTFA (trifluoroacetic acid). The ionic strength was kept constant at 1.0 mol L^-1 through the addition of KCl and the pH was adjusted by adding NaOH, according to similar studies reported for the Ru^II systems.¹⁶

Infrared (IR) spectra (400-4000 cm^-1) of the compounds dispersed in KBr were recorded on a Shimadzu FTIR-8400S spectrometer.

Electrochemical analysis (cyclic voltammetry and square wave voltammetry) was performed on an Epsilon potentiostat (BASi, Bioanalytical Systems Inc.) using a three-electrode cell. Glassy carbon and a platinum single-wire as used as working and counter electrode, respectively, while an Ag/AgCl (3.5 mol L^-1 KCl) electrode was used as reference. The cyclic voltammograms were recorded in 0.1 mol L^-1 NaTFA (sodium trifluoroacetate), pH = 3.0. Oxygen was removed by purging the solutions with argon. All measurements were performed at 25.0 ± 0.2 ºC.

The ¹H, COSY (correlation spectrometry) and HSQC NMR (heteronuclear single quantum coherence nuclear magnetic resonance) spectra were obtained in a Bruker AVANCE DPX 300 spectrometer in CD₃SOCD₃-d₆. Elementary analyses were performed on a PerkinElmer CHN 2400 analyzer.

Computational studies

Theoretical calculations were performed using the Gaussian 09 program.¹⁸ The DFT (density-functional theory) and TD-DFT (time-dependent density-functional theory) methods were used with the B3LYP (Becke, 3-parameter, Lee-Yang-Parr) functional.^19-22 In the calculations, the polarizable continuum model was used to simulate the effect of the solvent.²¹ The LANL2DZ (Los Alamos National Laboratory 2 Double-Zeta) relativistic effective core potential basis set was used for the Ru atom while the 6-31G(d,p) basis set was used for other atoms. The optimized geometries in a potential energy minimum were confirmed by the absence of any imaginary frequency in vibrational analysis calculations.^23,24 The energy values and the percentage contributions of the orbitals involved in the electronic transitions were obtained using the Multiwfn and GaussSum 3 software.²⁵

Antibacterial activity

Three reference strains from the American Type Culture Collection (ATCC) were used in this study: the Gram-positive bacteria Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228) and the Gram-negative bacteria Pseudomonas aeruginosa (ATCC 9027). The susceptibility of bacteria to complexes I and II was evaluated by determining the MIC of the compounds.

The evaluation of the antimicrobial activity started with the pre-inoculum, which was prepared by transferring the strain from the stock onto Agar BHI plates, then leaving it in an oven for 24 h at a temperature of 37 ºC. After this period, the inoculum was prepared by resuspending colonies from the pre-inoculum in saline solution (0.9% saline solution) until reaching turbidity of 0.5 on the McFarland scale, corresponding to 108 CFU mL^-1 (colony forming unit per milliliter). Afterwards, 100 μL of the inoculum solution was removed, transferring it to a 9.9 mL saline tube, obtaining a concentration of 10⁶ CFU mL^-1.

Antimicrobial activity screening was obtained using the broth microdilution method. Initially, 100 µL of Mueller-Hinton (MH) broth was added to a 96-well microplate, and 100 µL of the complex at an initial concentration of 800 μmol L^-1 for complex I and 400 μmol L^-1 for complex II, followed by serial concentrations, and finally 100 µL of the inoculum suspension. The microplate was incubated for 18 h at 35 ± 2 ºC under agitation (200 rpm) and the optical density evaluation was performed in a microplate bed (Epoch Biotek, Winooski, USA) with a length of 595 nm.

 

RESULTS AND DISCUSSION

Complexes characterization

The complexes I and II were synthesized from the equimolar ratio between the precursor compound cis-[Ru(bpy)(phen)Cl₂] and the imidazole, sodium sulfite and sodium nitrite ligands to avoid the coordination of another molecule of these ligands to the metal center. Trifluoroacetic acid was used in excess, since this substance is intended to make the pH as low as possible to promote the conversion of NO₂^- to NO⁺. Ammonium hexafluorophosphate has the function of providing the PF₆^- ion necessary for the precipitation of these compounds.

The cis-[Ru(bpy)(phen)L(NO⁺)]ⁿ⁺ complexes, where L = ImN (complex I) or SO₃^2- (complex II), were prepared using an acid treatment of the cis-[Ru(bpy)(phen)L(NO₂)]^+/- aqueous solution. The acid-base equilibrium for the nitrosyl-nitro interconversion in the ruthenium(II) complexes (Equation 1) had been previously studied in similar systems.^15,16

A reversible pH dependence (Equation 1) was observed, as indicated by the changes in UV-Vis spectroscopic results. These changes accompanying this acid-base reaction were collected for cis-[Ru(bpy)(phen)L(NO₂)]^+/- (Figures 1Sa and 1Sb, Supplementary Material, for L = ImN - complex I, SO₃^2- - complex II, respectively), where it was observed the appearance of a new high-intensity band with a maximum at 420 nm (for complex I) and 405 nm (for complex II) at higher pH. The newly formed band is assigned to the dπ(Ru^II) → π*(bpy/phen) MLCT transition of the nitro species formed (Equation 1). Analogous spectroscopic behavior has been observed for other ruthenium nitrosyl complexes.^15,26-29 The spectrophotometric titration curve for this equilibrium (Equation 1) showed only one inflexion, which can be indicative of a unique equilibrium as proposed, with a pH of half-interconversion at 6.3 (complex I) and 8.9 (complex II) (Figure 2). The measured equilibrium constant (K_eq) values for a series of similar type compounds are shown in Table 1. Additionally, this Table includes other parameters such as E_1/2 NO^+/0 and νNO⁺.

 

 

 

 

It is also worth mentioning that the acid-base equilibrium for the nitrosyl-nitro interconversion of the ruthenium(II) complexes has made them possible to be used as synthetic strategy systems.^15,16 Here, the formation of complexes I and II as the product of the acid-base equilibrium described in Equation 1 was confirmed by analysis of the infrared spectrum (Figures 2S and 3S, Tables 1S and 2S, Supplementary Material). It was noticed that bands associated with the nitro ligand at 1330 and 1284 cm^-1 disappeared, followed by the appearance of a new band related to the stretching mode of NO⁺ group at 1942 and 1914 cm^-1 for complexes I and II, respectively.³⁰ Furthermore, vibrational peaks corresponding to the bipyridine ligand from 1610 to 1400 cm^-1 were observed for both complexes attributed to νC=C, νC=N.^8,29,31-33 For complex II were observed peaks assigned to SO₃^2- ligand at 521, 622, 990, and 1155 cm^-1.^16,30,34,35 These latter assignments indicated that the sulfite ion is coordinated through the sulfur atom.^35-38

While K_eq data have been suggested^11,39-41 as a direct method to evaluate the relative electrophilicity of coordinated nitric oxide in various compounds, there is still no agreement upon standard for expressing the results. According to the electrophilicity parameter, the higher the electron deficiency of the NO moiety resulting from the π-acid character of the [Ru(bpy)(L')(L")]ⁿ⁺ group (L' = phen or bpy and L" = SO₃^2- or ImN), the higher will be the driving force for the reaction of the coordinated NO moiety with OH^- in Reaction 1. Also, in relation to this parameter, the ν(NO⁺) frequency should be observed at higher values with decreasing electron density on the NO fragment. Therefore, one might expect to observe a correlation between the K_eq and ν(NO) frequency values for the [Ru(bpy)(L')(L")]ⁿ⁺ complexes, considering the π-acceptor character of the ImN ligand in the L sequence. The experimental values observed agree with the theoretical order, as can be seen in Table 1.

The UV-Vis spectra of the nitro complexes aqueous medium present two π → π* intraligand (IL) transitions at 264 and 286 nm correlated to the phen and bpy ligands, respectively.^39,42-44 Such transitions are consistent with those observed to [Ru(phen)₂(ppn)]²⁺ and [Ru(bpy)₂(ppn)]²⁺ at 262 and 285 nm, respectively.^40,45-48 Besides, the metal-to-ligand charge-transfer (MLCT) transitions are observed in the range from 405 to 420 nm. In comparison to the nitro complexes, the nitrosyl one presented blue-shifted MLCT transitions because of stabilization of the dπ(Ru) orbitals caused by strong back-bonding interaction with NO⁺ ligand.⁴⁹ The MLCT (dπ(Ru^II) → π*(bpy/phen)) band has a maximum absorption at 330 nm. Furthermore, a band with low molar absorptivity assigned to the MLCT dπ(Ru^II) → π*(NO⁺) transition is observed at 476 nm.^16,41,43,44

TD-DFT calculations are performed for complex I, which the NO ligand is trans to the phen ligand. This result has showed a better correlation with the experimental data. As can be seen in Figure 2, the calculated spectrum presents a great agreement with the experimental one. Thus, the TD-DFT data was used to reinforce the assignment of the electronic transitions.

The most intense band at 265 nm arises from HOMO-3 to LUMO+4 transition. These MO are localized on the phen ligand. The shoulder at ca. 294 nm results from HOMO-2 to LUMO+2 transition associated with the bpy ligand (Figure 3b). These bands correspond to transitions with intraligand (IL) character. On the other hand, the band at 471 nm is dominantly associated with the transition from HOMO-4 localized on Ru to the LUMO+1 that mainly resides on the NO ligand confirming the MLCT character.

 

 

The nitrosyl complexes I and II were further characterized by ¹H NMR, ¹H-COSY (Figures 4S, 5S and Table 3S, Supplementary Material) and HSQC (Figures 6S, 7S and Table 4S, Supplementary Material) spectroscopy. Figure 4 illustrates the hydrogen nuclear magnetic resonance spectra for complexes I and II. The presence of three signals from the N-heterocyclic ring (6.93, 7.08, and 7.38 ppm) suggests that the imidazole ligand is coordinated to the metal center through the nitrogen atom. Additionally, it was observed signals for the hydrogen atoms of phenanthroline and bipyridine suggesting all hydrogen atoms are not diamagnetically equivalent^15,50-52 in agreement with the cis configuration for complexes I and II.

 

 

The electrochemical studies of the nitrosyl complexes were performed by using cyclic voltammetry and square wave voltammetry techniques. Figure 8S (Supplementary Material) presents the cyclic voltammogram of I and II complexes in an aqueous medium at pH = 3.0. When a scan in negative potential direction was applied to the working electrode, starting from +0.60 V, only one reversible electrochemical process was observed at 0.219 and -0.102 V for complexes I and II, respectively. This process is characteristic of the {RuNO}^6/7 redox couple. On the other hand, when a scan in positive direction, a second electrochemical process is observed at 0.592 and 0.745 V for complexes I and II, respectively. This process is assigned to the Ru^III/II redox process of the cis-[Ru(bpy)(phen)(L)(H₂O)]ⁿ⁺ complexes, which are generated by NO dissociation from the cis-[Ru(bpy)(phen)(L)(NO⁺)]ⁿ⁺ compounds. A reasonable interpretation of the electrochemical results is proposed in Reactions 2, 3, and 4.

Aiming to reinforce this assignment, the square wave voltammograms for the nitrosyl complexes were acquired by a change in the time polarization of the working electrode, at -0.10 for complex I and -0.30 V for complex II (Figures 9Sa and 9Sb, respectively, Supplementary Material). For both nitrosyl complexes, it was observed that the peak current related to the Ru^III/II redox process increases with an increase in time polarization. A similar behavior is found for other similar complexes of the type cis-[Ru(bpy)₂(M)(NO)](PF₆)₃ in which M are the ligands isonicotinamide, imidazole, sulfite and 4-benzoylpyridine that presented these processes at 0.72, 0.70, 0.62 and 0.73 V, respectively, as reported in the literature.^15,16,30,52

Photochemical studies

Irradiation with blue LED (light-emitting diode) in complexes I and II was performed to qualitatively determine the release of nitric oxide from the coordinating sphere. Photolysis was accompanied by electronic spectroscopy at different times of exposure to the LED.

When analyzing the electronic spectra of the complexes, it is verified that by increasing the exposure time, the intensity of the load transfer transition in the region between 400 and 500 nm increases (Figure 5). Thus, changes in the spectrum suggest that the nitrosyl ligand was released. The irradiation of nitrosyl ruthenium complexes in an aqueous solution with energy photons results in the reduction of nitric oxide and subsequently its release, so that in its place a water molecule is coordinated, forming the aqua complex.^16,30,53,54

 

 

Rose and Mascharak⁵⁵ have proposed two types of mechanisms for NO release in a photochemical form. The first produces the NO complex and [Ru³⁺-solvent], while the second gives NO and [Ru²⁺-solvent] as final products (Equation 5).

It is found that the first mechanism occurs when the nitrosyl ruthenium complexes present ligands such as amine, carboxamide, thiolate, heme and phenolate, while in the second, the complexes have polypyridinic ligands and Schiff base in the second sphere of coordination. In this mechanism, the return of the Ru²⁺ species, after photolysis, seems to proceed from a spontaneous reduction of the transient species Ru³⁺. Analyzing complexes I and II, the irradiations presented a profile compatible with the final product of the second mechanism, due to the similarities of the metal, oxidation state, and binders.^26,55

Antimicrobial activity

The antibacterial activity of the nitrosyl complexes was tested against the Gram-positive bacteria Staphylococus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228) and the Gram-negative bacteria Pseudomonas aeruginosa (ATCC 9027) (Figure 6 and Table 2). The activity of complex I against the Gram-positive strains Staphylococus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 12228) was shown to be moderate, since the MIC values of 500 μmol L^-1 were higher compared to similar ruthenium systems reported in the literature, which presented MIC values of 125 and 7.8 μmol L^-1 for the compound [Ru(mctz)(bpy)(dppf)]PF₆ and 250 and 62.5 μmol L^-1 for the compound [Ru(mcbtz)(bpy)(dppf)]PF₆, respectively.⁵⁶ In addition to these, other similar ruthenium compounds⁵⁷ of the cis-[Ru(bpy)(dppz)Cl₂] and cis-[Ru(bpy)(dppz)(SO₃)(NO)]PF₆ type demonstrated greater activity with MIC values of 19.63 and 39.26 μmol L^-1, in the order indicated, against the strains Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 12228). In turn, complex II showed no antibacterial activity against these Gram-positive strains.

 

 

 

 

However, against the Gram-negative bacteria Pseudomonas aeruginosa, both complexes I and II demonstrated more selective action, since an MIC corresponding to 57.23 µmol L^-1 (55 µg mL^-1) and 200.8 µmol L^-1 (139 µg mL^-1) was presented, respectively, being more active against these strains. In comparison, a series of ruthenium nitrosyl complexes⁵⁸ tested against Pseudomonas aeruginosa of the fac-[Ru(NO)Cl₃(P,P-L)] type, where P,P-L = bis[(2-diphenylphosphino)phenyl]ether and trans-(NO,OMe)-[RuCl(OMe)(NO)(P,P-L)(L)]PF₆ where L = pyridine, 4-methylpyridine, 1-methylimidazole and 1H-benzimidazole demonstrated MIC > 500 µg mL^-1. This indicates that the compounds under study are promising, considering that Pseudomonas aeruginosa is a Gram-negative bacterium that has an outer membrane with lipopolysaccharides (LPS) and is generally more resistant to antibacterial treatments.^59,60 Additionally, reports in the literature^8,57,61-63 demonstrate that bipyridine, phenanthroline, imidazole and free coordination sulfite ligands have a higher minimum inhibitory concentration than compounds I and II. Thus, the ability to inhibit the growth of the tested bacteria is due to characteristics presented by the complexes formed, not being attributed separately to each of the ligands.

According to the literature,^56,64-66 there are some factors that are important regarding the metallic complexes presenting antimicrobial activity, among these are the chelate effect and the total charge of the complexes. Complexes formed by ligands that coordinate to the metallic centers in a bidentate manner, such as 1,10'-phenanthroline, 2,2'-bipyridine and 2,2'-dipyridylamine, present greater antimicrobial efficiency when compared to complexes that have monodentate ligands, such as pyridine. In the case of the total charge of the complexes, it is observed that the antimicrobial activity decreases in the following order: cationic > neutral > anionic.⁶⁴ In the literature,⁶⁷ antimicrobial peptides have been studied in which electrostatic interactions have been observed. In bacteria, due to the presence of negatively charged components such as lipoteichoic acid associated with the peptidoglycan wall (Gram-positive bacteria) and lipopolysaccharides associated with the outer membrane (Gram-negative bacteria), an initial electrostatic interaction of the cationic antimicrobial peptides occurs, promoting the adsorption of these molecules and a disturbance in the surface layer of the microorganism. In addition, interactions occur in the deeper layers of the bacterial membrane, either by electrostatic interactions due to the presence of anionic components in the membrane, such as phosphatidylglycerol and cardiolipin, or by hydrophobic interactions due to the phospholipid bilayer present in these unicellular organisms.^68,69

 

CONCLUSIONS

The results obtained through spectroscopic and electrochemical techniques indicated the formation of nitrosyl complexes, since the appearance of characteristic bands of the ligands was observed in both the infrared and UV-Vis spectra and in electrochemistry. These complexes exhibit photo (blue light) and redox-induced NO release. Furthermore, the pH values in the interconversion between NO⁺ and NO₂^- exhibited more favorable characteristics in complex II, as the conversion pH was higher than the physiological pH of 7.4. Upon analyzing the antibacterial assays against Pseudomonas aeruginosa bacteria, it was noted that both compounds demonstrated the capacity to inhibit the growth of these strains. However, only complex I exhibited activity against the strains Staphylococcus aureus and Staphylococcus epidermidis. Based on MIC values, complexes are classified as bacteriostatic agent.

 

SUPPLEMENTARY MATERIAL

Supplementary material is available free of charge at http://quimicanova.sbq.org.br as PDF file.

 

ACKNOWLEDGMENTS

All the authors are thankful to CAPES and CNPq for the financial support. We are all thankful to CENAPAD-SP (Centro Nacional de Processamento de Alto Desempenho) for computational resources to execute DFT calculations and to CENAUREMN (Centro Nordestino de Aplicação e Uso da Ressonância Magnética Nuclear) of Federal University of Ceará for NMR acquisitions. The Analytical Center of the Chemistry Institute of UFRN for the use of the equipment used in this work.

 

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Associate Editor handled this article: Marcela M. Oliveira