2.1 Materials and methods

The substances used to obtain the four complexes were of high purity and came from Alfa Aesar (α-ketoglutaric acid—C₅H₆O₅) and Sigma–Aldrich (1-(o-tolyl)biguanide—C₉H₁₃N₅, NiCl₂·6H₂O, NiBr₂, Ni(NO₃)₂·6H₂O, Ni(CH₃COO)₂·4H₂O, and ethanol).

For synthesis of complex combinations, the substances used (nickel salts and two ligands) were dissolved in ethanol. Reactions occurred at approximately 40 °C under agitation. The complexes obtained were vacuum filtered, washed with ethanol, and dried with ethyl ether. The molar ratio of metal salts/α-ketoglutaric acid/1-(o-tolyl) biguanide was 1:1:1 using 1 mmol of each substance (0.1461 g C₅H₆O₅, 0.1913 g C₉H₁₃N_5, 0.2377 g NiCl₂·6H₂O, 0.2185 g NiBr₂, 0,2908 g Ni(NO₃)₂·6H₂O, and 0.2488 g Ni(CH₃COO)₂·4H₂O). Synthesis of complex combinations led to pure compounds, which did not require further purification.

The nitrogen, carbon, and hydrogen content of the synthesized complexes was determined by microcombustion using a Flash 2000 Organic Elemental Analyzer. The presence of chlorine in complex C1 was revealed with AgNO₃, and the percentage of nickel was determined using a Perkin Elmer AAnalyst 400 atomic absorption spectrometer.

Electronic spectra were recorded using a Jasco V670 spectrometer by the diffuse reflection method at room temperature in the range 200–1500 nm, using MgO as a standard.

A Nicolet IS 50 FT-IR spectrophotometer was used for recording FT-IR spectra in the 4000–200 cm⁻¹ range. Thermal analysis was performed with a simultaneous thermogravimetric analysis/differential scanning calorimetry (TGA/DSC) STA 449 F1 Jupiter working in a dynamic air atmosphere at a flow rate of 20 mL/min in the range 25–900 °C with a heating rate of 10 °C/min. The molar electrical conductivity was determined using a CyberScan PCD 6500 conductivity meter in 10⁻³ M of N,N-dimethylformamide solution at 25 °C. Magnetic susceptibility was measured with a Gouy balance using the Faraday method and Hg[Co(SCN)₄] as a calibrant.

Antitumor activity was tested in HeLa tumor cells using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay at a concentration of 500 μg/mL for 24 h incubation at 37 °C in 5% CO₂ atmosphere. The method is based on the following: under certain conditions, NAD(P)H-dependent cellular oxidoreductases may reflect the number of viable cells in a culture. These enzymes may reduce the MTT tetrazolium reagent to violet insoluble formazan (Fig. 1). The color intensity of formazan was evaluated by measuring optical density at a wavelength of 570 nm using a Filter Max F5 Multi-Mode Microplate Reader spectrophotometer.

The tetrazolium dye reduction method is often used to measure the cytotoxicity of some compounds and cell proliferation. Because MTT reagent is sensitive to light, the whole procedure is done in the dark.

The antibacterial activity of ligands and complexes synthesized was determined in vitro against Pseudomonas aeruginosa and Staphylococcus aureus bacterial strains. To determine the minimum inhibitory concentration (MIC), the broth microdilution method was used by successively diluting a 10 mg/mL complex combination solution using 96-well plates. According to the dilution scheme of complex compounds, the influence of a diluted dimethyl sulfoxide (DMSO) solvent was also quantified.

To determine the influence of the test compounds on the adhesion of a microbial biofilm to the inert substrate, the microbial cells were cultivated in the presence of compounds of interest into nutrient broth using 96-well plates. They were incubated at 37 °C for 24 h. The plates were emptied and washed twice with physiological sterile water, and the adhered cells were fixed with 0.1 mL of 80% methanol for 5 min. The methanol solution was removed by overturning, and the adhered cells were stained with 1% crystal violet alkaline solution (0.1 mL/well) for 15 min. The staining solution was removed and then the plates were washed under water stream. The microbial biofilms formed on the plastic plates were converted into suspension by bubbling in 33% acetic acid. The intensity of the suspension color was evaluated by measuring the absorbance at 492 nm using a Max F5 Multi-Mode Microplate Reader.

3.1 Elemental analysis

To establish the formulas of coordination compounds that were synthesized, elemental analysis was performed. A good concordance between calculated and experimental percents of carbon, nitrogen, hydrogen, and nickel was found (Table 1).

3.2 Thermal analysis

On the basis of the interpretation of the results of the TGA, information has been obtained that has contributed to the proposed formulations for coordination complexes, namely, the presence of water molecules and the thermal effects associated with mass loss processes. Thermogravimetric (TG) and DSC curves of the four complexes are presented in Fig. 2(a–d).

From the analysis of the C1 complex TG and DSC curves, the first decomposition step (<180 °C) indicates the loss of two water molecules (experimental loss, 8.12%; calculated loss, 7.72%). In the range of 180–280 °C, chlorine anion is lost as HCl (experimental loss, 7.67%; calculated loss, 7.82%). Ligand decomposition occurs in the third and fourth steps, with strong exothermic effects in the 280–450 °C range.

In case of complex C2 up to approximately 200 °C, we have a mass loss of 7.50%, indicating the presence of two coordinating water molecules. In the range of 200–270 °C, bromine anion is lost as HBr (experimental loss, 16.25%; calculated loss, 15.85%). Above this temperature, the mass loss corresponds to the decomposition of the ligand being accompanied by exothermic effects.

Analyzing the TG and DSC curves of the C3 complex, it is observed that in the first step (<180 °C) the crystallization water molecule is lost (experimental loss, 3.74%; calculated loss, 3.79%). Subsequently, the removal of nitrate ions as nitrogen oxides and oxidative degradation of the ligand takes place. Consequently, in the temperature range 400–500 °C, a strong exothermic effect can be observed.

The thermal analysis curves of the C4 complex indicate that the acetate ion was eliminated as CO₂ and H₂O up to about 250 °C (experimental loss, 13.50%; calculated loss, 13.00%) and the ligand degradation takes place up to 500 °C.

For all four complexes analyzed, the residue obtained is NiO (greenish gray), from which the percentage of nickel was determined, namely, for C1 (experimental, 12.73%; calculated, 12.58%), for C2 (experimental, 11.28%; calculated 11.49%), for C3 (experimental, 11.98%; calculated, 12.36%), and for C4 (experimental, 13.42%; calculated, 13.00%). A good agreement was observed between the spectrophotometrically determined metal content and the corresponding value of the residue from thermal analysis.

3.3 Molar conductivity

All molar conductivities were determined at 25 °C using N,N-dimethylformamide as a solvent and concentrations of 10⁻³ M. The measured values for complexes are as follows: C1, 79.5; C2, 81.7; C3, 72.6; and C4, 77.2 S cm² mol⁻¹. These values indicate a 1:1 electrolyte type for all four complexes [22].

3.4 Magnetic susceptibility

Magnetic susceptibility determinations have led to the following magnetic moment values: 3.19 μΒ for C1, 3.27 μB for C2, and zero for both C3 and C4. Consequently, the first two complexes are paramagnetic, whereas C3 and C4 complexes are diamagnetic.

3.5 Ultraviolet–visible–near-infrared spectra

The stereochemistry of synthesized coordination complexes has been assessed using ultraviolet, visible, and near-infrared (UV–vis–NIR) spectrometry by comparing their spectra to those of the ligands (α-ketoglutaric acid and 1-(o-tolyl)biguanide). The electron spectra of the solid state C1C4 complexes are shown in Fig. 3. The bands and the maxima assigned to the d–d transitions for the four synthesized complexes are shown in Table 2.

All complexes have absorption bands in the range of 230–345 nm that may me attributed to the π–π* and n–π* transitions of organic ligands H₂A and TB, slightly displaced by coordination of the ligand to metal ions.

The electronic spectra of the C1 and C2 complexes are very similar and correspond, by the position and intensity of the visible range bands, to a rhombic distorted octahedral symmetry [23].

The broad cleavage of the first and second octahedral bands cannot be explained on the basis of a tetragonal deformed octahedral symmetry. The existence of two different bidentate ligands in the plane and two water molecules in axial positions, that is, a chromophore NiO₂N₂O₂/2NiO (α-ketoglutaric acid), 2NiN (biguanide), and 2NiO(H₂O), leads to a rhombic deformation of the octahedron and a symmetry that corresponds, with good approximation, to C_2v [23]. Under these conditions, the bands of 8330/8660 and 13,330/13,420 cm⁻¹ correspond to the transitions from the fundamental state ³B₁ to the terms ³A₁ and ³B₂ of the term ³T_2g in the octahedral symmetry.

The other observed peaks that follow in the spectra correspond to the transitions to terms ³B₂ and ³A₂ from the ³T_1g(F) term of the octahedral symmetry. The cleavage of the third octahedral band that involves transitions to terms derived from the ³T_1g(P) level is not observed in any of the complexes. The position of all the observed bands is in agreement with the literature data regarding complexes having oxygen and nitrogen donor atoms and analogous symmetry [24–27].

C3 and C4 complexes do not have bands below 10,000 cm⁻¹ (which are characteristic of octahedral symmetry), their first bands being at 14,600 and 14,710 cm⁻¹, respectively.

The symmetry of these complexes is square-planar, in agreement with the observed diamagnetism and the fundamental term is ¹A_1g. In agreement with the literature data, the observed peaks correspond to the first and second bands in D_4h symmetry (square-planar) [28–30].

The electronic spectra of the four complexes dissolved in DMSO were also recorded. It has been observed that the positions of the bands due to the metal ion do not change from those in the solid state, which means that the proposed symmetries are maintained. The electronic spectra of C1C4 complexes dissolved in DMSO are shown in Fig. 4.

3.6 FT-IR spectra

To determine the coordination mode of the ligands to the metal ion, the FT-IR spectra of the C1C4 complexes were compared with those of the H₂A and TB ligands [31]. The characteristic spectral bands and their assignments are shown in Table 3. To determine the coordination pattern of the H₂A ligand, the IR spectrum of sodium α-ketoglutarate was also analyzed, the difference between the wavelengths corresponding to the bands ν(COO⁻)_asym and ν(COO⁻)_sym being 181 cm⁻¹.

In the spectra of the four complex combinations, a band shift due to the valence vibration of the imine group, ν(CN), from 1610 cm⁻¹ to higher values is observed. This is in agreement with the coordination of the TB ligand to the metal ions through the lone pair of imine nitrogen. The displacement of this band to larger wavelengths is explained by the destruction of the π electron delocalization [32]. Coordination of 1-(o-tolyl)biguanide by iminic nitrogen atoms is also supported by the band displacement due to δ(NH) + ν(CN) coupled vibration.

The displacement of the ν(CO) band below 1720 cm⁻¹ confirms that the keto group in the α position of the H₂A ligand is involved in the coordination for all the analyzed complexes.

From the difference between the corresponding wavenumbers of ν(COO⁻)_asym and ν(COO⁻)_sym bands (<181 cm⁻¹), it was established that the H₂A ligand coordinates with the metal ion in its deprotonated form HA⁻, through α oxygen atom of the ketone group and the oxygen from the hydroxyl of the adjacent carboxyl group.

The presence of a single absorption band for the characteristic vibration modes ν₁–ν₄ in the C3 complex spectrum indicates the presence of a nitrate anion in the ionization sphere.

In the C4 complex, the intense bands at 1570 and 1410 cm⁻¹ can be attributed to the out of the coordination sphere ionic acetate.

Bands between 260 and 270 cm⁻¹ and 432 and 450 cm⁻¹, respectively, were assigned to the formation of NiO and NiN bonds.

The coordination water in the C1 and C2 complexes is confirmed by the presence of the three characteristic bands located at approximately 3400, 600, and 700 cm⁻¹. In the case of C3 complex, the stretched band from 3370 cm⁻¹ is attributed to the crystallization water.

3.7 Biological activity

Metabolism of HeLa cells varies depending on the type of the material used. Thus, ligands and C1 and C2 complexes show a very weak effect on HeLa tumor cells under the tested conditions, whereas complex C3 and C4 combinations have demonstrated a moderate cytotoxic effect on them. Fig. 5 shows the absorbance readings at 570 nm for the analyzed samples (at a concentration of 500 μg/mL for 24 h incubation at 37 °C) and untreated control sample indicating the metabolic activity of HeLa cell cultures by the MTT method.

Evaluation of the antimicrobial activity of the ligands and synthesized complexes was done on S. aureus and P. aeruginosa species. It has been observed that DMSO (the solvent used in dilutions) does not influence the antimicrobial activity of the tested compounds at the chosen working concentrations.

In the case of Gram-positive S. aureus, C2 has shown identical activity to ligands, whereas C1, C3, and C4 complexes show a better activity. Thus, C4 has the best activity, whose MIC value is 0.75 mg/mL.

The activity of complexes synthesized (except C2) against Gram-negative bacteria P. aeruginosa is better for synthesized complexes than for H₂A ligand but similar to TB ligand. C4 complex has the best activity among the tested substances against these bacteria (MIC is 1.00 mg/mL). MICs for the ligands and C1C4 complexes against the two bacterial strains are shown in Figs. 6 and 7.

Regarding the ability to inhibit the adhesion of the microbial biofilm to the inert substrate, in S. aureus, all the test compounds (ligands and complexes obtained) inhibit this process in a dose-dependent manner to a minimum biofilm eradication concentration of 0.04 mg/mL (C3, C4), 0.09 mg/mL (TB, H₂A, C2), and 0.18 mg/mL (C1). Against P. aeruginosa, all compounds have inhibitory capacity up to a minimum biofilm eradication concentration of 0.04 mg/mL (TB, C1C4) and for H₂A of 0.09 mg/mL.

The influence of ligands and complexes on adhesion to inert substrate capacity is shown in Figs. 8 and 9.