Coordination Compounds of Platinum and Palladium with Mixed Ligands (Usnic Acid and 1-(o-Tolyl) Biguanide) – Synthesis, Spectral Characterization and Biological Activity

Four coordination compounds of Pd(II), Pt(II) and Pt(IV) with usnic acid (H3AU) and 1-(otolyl)biguanide (TB) as ligands have been synthesized in view of their potential as antimicrobial, antifungal and antitumor agents. The metal complexes have been characterized by elemental and thermogravimetrical analyses, infrared and electronic spectra. Based on these studies, the following formulas have been proposed for the complexes: [Pd(TB)(H3AU)]PdCl4 (C1), [Pd(TB)(H2AU)] CH3COO (C2), [Pt(TB)(H2AU)Cl2]Cl (C3) and [Pt(TB)(H2AU)]Cl (C4), where H2AU is deprotonated usnic acid. The in vitro biological activities of the new complexes were tested against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Candida albicans ATCC 10231 and HeLa tumor cells. All complexes were found to have good biological properties and therefore they can be further explored in therapeutic applications.

Thus, some coordination compounds of palladium and platinum with biguanides, such as , have been synthesized and tested for their biological activity [12][13][14][15].
In addition, the compound obtained from acyl hydrazone and usnic acid has good activity against Mycobacterium tuberculosis (Koch's bacillus) [36].
Usnic acid (-) showed moderate activity in the P388 (murine leukemia) assay and in vitro cytotoxic activity against L1210 cell lines [37].
Copper(II) complexes synthesized with ligands obtained by condensation of the usnic acid with acyl hydrazides of -naphthoic, caprylic and oxamic acids, as well as thiosemicarbazone derivatives were tested against Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus). The antibacterial activity proved to be higher for the complexes than that for the ligands and the Cu(II) ion [33].
Moreover, new synthesized Pd(II) and Cu(II) complexes with ligands obtained by condensation of usnic acid and hydrazide showed significant biological activity against Aspergillus niger, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis strains as well as cytotoxic activity against HeLa cells of human cervical carcinoma [32].
Most bacteria are very resistant and adaptive microorganisms to environmental changes mainly due to their defense mechanism based on the efflux pumps which eliminate the toxic compounds from inside cells. Bacteria could have different defense response based on the presence of harmful compounds in the environment [38,39]. Various bacterial strains (Pseudomonas aeruginosa, Staphylococcus warneri and Pseudomonas aurantica) proved to be very effective in Reldan EC40 pesticide (Nita-Lazar et al. [40]) and pharmaceutical compounds metabolisation (Nita-Lazar et al. [41]).
In this paper we report the synthesis of four coordination compounds of palladium and platinum with mixed ligands: usnic acid and 1-(o-tolyl)biguanide, as well as their analytical and spectral characterization and biological activity screening.
Synthesis of complexes [Pd(TB)(H2AU)]CH3COO (C2) and [Pt(TB)(H2AU)Cl2]Cl (C3): the ligands were dissolved in ethanol, under slight heating; the required metal salt, i.e., Pd(CH3COO)2 and H2PtCl6, respectively, dissolved in a minimal amount of ethanol, was added slowly under stirring, to the solution of the ligands. After two hours of stirring, the complexes were obtained as a precipitate.
The synthesized complexes were filtered off, washed with ethyl ether and dried under air. The compounds had a high degree of purity, so they did not require further purification.

Physical-chemical characterization of the complexes
The percentages of C, N and H in the composition of the four complexes were analyzed using Flash 2000 Elemental Analyzer. Palladium and platinum content was determined using a Perkin Elmer Aanalyst 400 atomic absorption spectrophotometer. Thermal analyses were performed with a simultaneous thermogravimetric analysis/differential scanning calorimetry (STA/DSC) 449 F1 Jupiter working in a dynamic air atmosphere at a flow rate of 20 mL/min and at a heating rate of 10°C/min up to 900°C.
The UV-Vis diffuse reflectance spectra were recorded on a Jasco V670 spectrometer, in the range of 200-800 nm, using MgO as standard.
The infrared spectra were recorded on a Nicolet IS 50 FT-IR spectrophotometer, in the range 4000-200 cm -1 .

Biological activity
The biological activity of the ligands and C1-C4 complexes was tested in vitro against two Grampositive and Gram-negative bacterial strains (Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853), one fungal strain (Candida albicans ATCC 10231) and HeLa cells.
The antimicrobial and antifungal activities were quantified based on bacterial and fungal growth inhibition monitored by spectrophotometric determination at 620 nm. A quantitative method was used, based on the production of binary serial microdilutions. The working range was 1.00 -0.0019 mg/mL and the solvent used was DMSO.
This process was NAD(P)H-dependent cellular oxidoreductases and spectrophotometrically monitored at a wavelength of 570 nm.
The detection of biological activity was performed according with the methods described by Mihalache et al. [42].

Elemental analysis
To determine the molecular formulas of the four complexes, the percentages of carbon, nitrogen, hydrogen were determined by elemental analysis and the metal content by molecular absorption spectrometry. The differences between the percentages obtained experimentally and those calculated were very small. [

Thermal analysis
The thermograms obtained from the thermal decomposition of complexes C1-C4 gave information's regarding the mass losses and the thermal effects that accompany them.
The TG curves of the palladium (II) complexes indicate that these compounds are stable to 220°C, when it starts their decomposition. This indicates that neither of the two complexes has water or alcohol in the molecule. Above this temperature, oxidative degradation of the complexes takes place. The final residue resulting from the thermal decomposition of the two complexes is palladium [43].
As for the previous two compounds, the C3 and C4 complexes are stable over a large temperature range (30-200 o C), which confirms that they do not contain water or alcohol. Above this temperature the complexes undergo decomposition. For the two complexes, the slightly endothermic process, https://doi.org /10.37358/Rev. Chim.1949 Rev. Chim., 71 (4) without mass loss, at 210°C, could be attributed to their melting. The large, highly exothermic process, occurring between 400 and 500°C is due to oxidative degradation and elimination of the organic ligands. The final residue is platinum [44].

Ultraviolet-visible spectra
Based on the UV-Vis spectra of the complexes compared to the ligands used in the synthesis, their stereochemistry was established. The electronic d-d transitions observed in the spectra of the complexes with their assignments and the corresponding symmetry are given in Table1.
The electronic spectra of the complexes C1 and C2 exhibited a strong absorption band, with maximum at 25975 cm -1 and 26315 cm -1 , respectively, that can be attributed to 1 A1g → 1 B1g transition.
In the case of C1 complex the band at 19230 cm -1 corresponds to the transition 1 A1g → 1 A2g. The same assignment corresponds to the shoulder in the spectrum of the C2 complex located at 19610 cm -1 .
According to the experimental data and to the literature reports, the symmetry proposed for these two complexes is square planar [45,46]. C3 and C4 complexes contain platinum in different oxidation states, namely +IV in C3 and +II in C4. It was found that platinum maintained its valence in the substances used in synthesis: H2PtCl6 for C3 and PtCl2 in C4.
In the UV-Vis spectrum of the C3 complex the three bands observed at 17095 cm -1 , 22990 cm -1 and 28570 cm -1 can be attributed to the d-d transitions from the ground state 1 A1g to 1 T1g, 1 T2g and 3 T1g, respectively, according to an octahedral symmetry.
For the C4 complex the symmetry proposed is square-planar, as confirmed by the bands at 20830 cm -1 and 25640 cm -1 which are assigned to 1 A1g → 1 A2g and 1 A1g → 1 B1g transitions.

FT-IR Spectra
Comparative analysis of the IR spectra of the usnic acid and 1-(o-tolyl)biguanide and their metal complexes allows to establish the donor atoms of the ligands in coordination to the metal ions. The assignments of the spectral bands characteristic of the ligands and the complexes C1-C4 are presented in Table 2.
The strong band appearing at 1676 cm -1 in the IR spectrum of the usnic acid may be assigned to the stretching vibration (C=O) of the ketone function [47,48]. This band shifts to lower wave numbers in the IR spectra of all complexes, indicating the coordination of the usnic acid through the ketone oxygen atoms.
The very strong band, with maximum at 1191 cm -1 , may be assigned to the stretching vibration of C-Ophenolic [49]. Its position is practically unchanged in the spectrum of the complex C1, but it is shifted to higher wavenumbers in the spectra of the complexes C2-C4. These observations suggest the deprotonation of the usnic acid in the complexes C2-C4 and coordination through phenolic oxygen linked to carbon 3. Thus, the usnic acid acts as neutre bidentate OO donor in the complex C1 and as a monobasic bidentate OO donor in the complexes C2-C4.
The band due to the stretching vibration of the imine group, (C=N), appearing at 1610 cm -1 in the IR spectrum of 1-(o-tolyl)biguanide, shows a downward shift in the spectra of the complexes C2-C4 https://doi.org/10.37358/RC. 20.4.8073 and an upward shift in the spectrum of the complex C1. These observations are in according to the involvement of imine nitrogen atoms in coordination to the metal ions [49].
In the IR spectrum of the complex C2 a new strong absorption band at 1395 cm -1 may be assigned to sym(COO) of acetate group. The strong absorption band at 1552 cm -1 in the spectrum of the same complex is due to asym(COO), the value of  = asym(COO) -sym(COO) = 157 cm -1 being in the range of ionic acetate [50]. Table 2. IR bands and their assignments for the synthesized complexes and ligands (ῡmax, cm -1 ) Based on these data, the four coordination complexes are proposed to have the conformations shown in Figure 1.  Figure 1. Proposed formulas for C1-C4 complexes 3.5. Antimicrobial activity C1-C4 complexes, H3AU and TB were tested in vitro against Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 strains to determine antibacterial activity. Following the tests performed it was observed that the antimicrobial activity against the Gram positive species Staphylococcus aureus was very good for complexes and the usnic acid while for 1-(o-tolyl) biguanide was much weaker. Platinum complexes have the lowest minimum inhibitory concentration, 0.0019 mg/mL, four times lower than that of usnic acid.
In the case of Gram negative bacteria Pseudomonas aeruginosa both ligands have low antimicrobial activity. All complexes have a good activity against this strain, the tetravalent platinum complex having the lowest MIC of 0.0625 mg/mL. The solvent used in dilutions did not influence the antimicrobial activity of the tested compounds at working concentrations.
The minimum inhibitory concentrations against the two bacterial strains for the usnic acid, 1-(otolyl)biguanide and the complexes synthesized are shown in Figures 2 and 3.

Antifungal activity
Antifungal activity of the ligands and the metal complexes was tested against Candida albicans ATCC 10231. The results indicate that the ligands have a poor activity, C1 and C2 complexes have a moderate one, while the platinum complexes have the best antifungal activity. The latter have a MIC of 0.125 mg/mL.
The minimum inhibitory concentrations for ligands and C1-C4 complexes are shown in Figure 4.

Antitumor activity
Antitumor activity was tested on HeLa cells for the complexes and the ligands. The percentages of viability of these cells in the presence of the tested compounds were calculated relative to the untreated control sample. The solutions used for these samples had a concentration of 500 µg/mL and were incubated at 37°C for 24 hours.
Platinum complexes have reduced the viability of HeLa cells by 35% C3 and 32% C4, respectively, thus showing a good cytotoxic effect. The ligands and palladium complexes had a weaker effect on this type of tumor cells. Figure 5 shows the viability of HeLa cells in the presence of ligands and complexes.

Conclusions
Four new complexes of Pd(II), Pt(II) and Pt(IV) were obtained using usnic acid and 1-(otolyl)biguanide as ligands. The composition and the stereochemistry around the metal ions were established based on the analytical and spectral data. Pd(II) complexes have square planar symmetry, C3 complex contains tetravalent platinum and it has octahedral symmetry, while the complex C4 is square planar with divalent platinum. C1 is of the Vauquelin type, having a complex anion PdCl4 2-.
The good antibacterial activity of the complexes against S. aureus is mainly due to the usnic acid found in their composition. Platinum complexes have a better activity against all tested strains compared to palladium complexes.
The antitumor activity of the C1 complex is better than the C2 and can be explained by the number of palladium ions in the molecule, namely, two for the first versus one for the second complex. As a general observation, the biological activity of the ligands is improved by complexation to the metal ions.