Antimicrobial , antioxidant and DNA-binding studies of palladium ( II ) complexes with different chelate ligands containing nitrogen donor atoms

The antimicrobial and antioxidant activities, as well as the DNA-binding of four square-planar Pd(II) complexes, [Pd(terpy)Cl]+ (C1), [Pd(en)Cl2] (C2), [Pd(DMEAIm)Cl2] (C3) and [Pd(dach)Cl2] (C4) (terpy = 2,2′:6′,2′′-terpyridine, en = ethylenediamine, dach = trans-1,2-diaminocyclohexane and DMEAImiPr = N-((1,3-dihydro-1,3-diisopropyl-4,5-dimethyl)-2H-imidazol-2ylidene)-N,N-dimethyl-1,2-ethanediamine are reported. The antimicrobial activities of the Pd(II) complexes with the appropriate ligands were tested using the microdilution method against 18 strains of microorganisms, whereby the minimal inhibitory concentration (MIC) and the minimal microbicidal concentration (MMC) were determined. The antibiofilm activity of [Pd(terpy)Cl]+ and the corresponding ligand were determined on a formed biofilm. The intensity of antimicrobial activity varied depending on the type of microorganism and the tested compound. The C1 complex with the corresponding ligand demonstrated significantly greater overall antimicrobial activity than C2, C3 and C4. The antibacterial activity of the C1 complex was better than its antifungal activity that was overall greater than that of the positive control, fluconazole. The greatest sensitivity for C1 and L1 was with Penicillium italicum (MIC < 0.49 μg mL-1) among the fungi, and with Proteus mirabilis ATCC 12453 (MIC = 0.98 μg mL-1) among the tested bacteria. The tested compounds show low and moderate antibiofilm activity. The complexes showed weak antioxidant properties when tested using the DPPH (1,1-diphenyl-2-picrylhydrazyl) method. The interaction of the metal complexes C1–C4 with calf thymus DNA (CT-DNA) was further examined by absorption (UV–Vis) and emission spectral studies (EthBr displacement studies). Overall, the investigated complexes exhibited good DNA interaction ability.

,N 1 -dimethyl-1,2-ethanediamine are reported.The antimicrobial activities of the Pd(II) complexes with the appropriate ligands were tested using the microdilution method against 18 strains of microorganisms, whereby the minimal inhibitory concentration (MIC) and the minimal microbicidal concentration (MMC) were determined.The antibiofilm activity of [Pd(terpy)Cl] + and the corresponding ligand were determined on a formed biofilm.The intensity of antimicrobial activity varied depending on the type of microorganism and the tested compound.The C1 complex with the corresponding ligand demonstrated significantly greater overall antimicrobial activity than C2, C3 and C4.The antibacterial activity of the C1 complex was better than its antifungal activity that was overall greater than that of the positive control, fluconazole.The greatest sensitivity for C1 and L1 was with Penicillium italicum (MIC < 0.49 μg mL -1 ) among the fungi, and with Proteus mirabilis ATCC 12453 (MIC = 0.98 μg mL -1 ) among the tested bacteria.The tested compounds show low and moderate antibiofilm activity.The complexes showed weak antioxidant properties when tested using the DPPH (1,1-diphenyl-2--picrylhydrazyl) method.The interaction of the metal complexes C1-C4 with calf thymus DNA (CT-DNA) was further examined by absorption (UV-Vis) and emission spectral studies (EthBr displacement studies).Overall, the investigated complexes exhibited good DNA interaction ability.

INTRODUCTION
Transition metal complexes in the past few decades have started to play an important role as pharmaceutical and diagnostic agents. 1 Everything started with the discovery of the antitumor characteristics of cisplatin, 2 followed by the discovery of a large number of other metallo-drugs, among which are the Paul Erlich organo-arsenic compound for the treatment of syphilis, antiarthritic gold preparations, and diagnostic agents for magnetic resonance imaging (Gd, Mn, Fe) among others. 3oday a large number of antibiotics and chemotherapeutics are accessible for treatment of infectious diseases.However the resistance of microorganisms to antibiotics and antifungals is a serious problem around the world.This indicates the necessity for the discovery of new compounds endowed with antimicrobial activity as soon as possible.Preferably, these new compounds should have different mechanisms of action, distinct from those of well-known classes of antimicrobial agents to which many clinically relevant pathogens are now resistant.][6][7] The chemical behaviour in solution of structurally analogous Pt(II) and Pd(II) complexes is very similar. 8Since the antitumor activity of certain Pd(II) complexes has been demonstrated, the idea arose to examine several structurally different Pd(II) complexes for their antimicrobial activity as well as their interaction with calf thymus DNA (CT-DNA).The pharmacological activity of metal complexes is highly dependent on the nature of the metal ion and of the ligand, because different ligands exhibit different biological properties.Ideally, the new drug would have an increased spectrum of activity, reduced drug resistance, decreased required dose and reduced toxic side effects.
A review of the current literature showed that Pd(II) complexes with different ligands exhibit significant biological activity, 9 which leaves the possibility for further investigation of the complexes C1-C4 with ligands such as: 2,2′:6′,2′′--terpyridine, ethylenediamine,N 2 The complexes will probably react with a range of different microorganisms relevant for human health.However, the goal of this work was to show whether DNA could be considered as one of the targets.

Material and methods
The reagents were obtained commercially and used without further purification.Elemental analyses were performed on a Vario III C, H, N and S elemental analyser in the CHS mode.Infrared spectra were recorded on a Perkin-Elmer FTIR 31725-X spectrophotometer using the KBr pellet technique.The UV-Vis spectra were obtained on a Perkin-Elmer BIOLOGICAL ACTIVITY OF THE PALLADIUM(II) COMPLEXES 1231 Lambda 35 or 25 double beam spectrophotometer, using 1.0 cm path-length quartz cuvettes (3.0 mL).Fluorescence measurements were run on a RF-1501 PC spectrofluorometer (Shimadzu, Japan).1][12][13] The ligand, DMEAIm iPr , was prepared according to a literature procedure.Synthesis of the palladium(II) complexes C1-C4 [10][11][12][13] [Pd(terpy)Cl]Cl•3H 2 O (C1), [Pd(en)Cl 2 ] (C2) and [Pd(dach)Cl 2 ] (C4).PdCl 2 (0.5 g, 2.8 mmol) was dissolved under reflux in a mixture of 10 mL H 2 O and 3 mL of concentrated HCl.The clear solution was filtered, and a solution of terpy (0.653 g; 2.8 mmol in 10 mL methanol) or en (0.168 g; 2.8 mmol in 10 mL methanol) or dach (2.8 mmol in 10 mL methanol) was added dropwise to the warm solution of [PdCl 4 ] 2-.The pH of the solution was carefully adjusted to 4.5-5.0 by the addition of NaOH.The resulting yellow solution was stirred for a few hours at 50 °C.The obtained solution was left at ambient temperature and a yellow solid was obtained.

In vitro antimicrobial activity
The tested compounds were first dissolved in dimethyl sulfoxide and then diluted with nutrient liquid medium resulting in 10 % solutions.Antibiotics, tetracycline (Galenika A.D., Belgrade), ceftriaxone (Galenika A.D., Belgrade), and vancomycin (Lek farmaceutska družba, D.D., Slovenia) were dissolved in Mueller-Hinton broth (Torlak, Belgrade), while antifungal drugs fluconazole (Pfizer Inc., USA), ketoconazole (Hemofarm A.D., Serbia) and amphotericin B (Chiesi Pharmaceuticals GmbH, Austria) were dissolved in Sabouraud dextrose broth (Torlak, Belgrade).Antimicrobial activity of palladium(II) complex was tested against 18 microorganisms.All the tested microorganisms are presented in Table I.Clinical isolates of the pathogenic bacteria were donated by the Institute of Public Health, Kragujevac, Serbia.The other microorganisms were provided by the Laboratory of Microbiology, Faculty of Sciences, University of Kragujevac.
The bacterial and fungi suspensions were prepared by taking colonies directly from the surface and suspending them in 5 mL of sterile saline solution.The turbidity of the initial suspension was adjusted using a densitometer (DEN-1, BioSan), and comparing it with 0.5 McFarland's standard. 14When adjusted to the turbidity of the 0.5 McFarland's standard, the bacteria suspensions contained around 10 8 CFU mL -1 and yeast suspension around 10 6 CFU mL -1 (colony-forming unit).Fungi spore suspensions were prepared by careful removal of spores from the mycelium.The initial suspensions were additionally diluted in sterile 0.85 % saline solution in 1:100 ratios for bacteria and yeast, and in 1:1000 for filamentous fungi.
The antimicrobial activities of the ligands and their complexes with palladium(II) were tested by determining the minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) using microtiter plates with resazurin. 15Tetracycline, ceftriaxone, vancomycin, fluconazole, ketoconazole and amphotericin B were used as positive controls.In order to exclude the effect of the solvent (dimethyl sulfoxide) on the growth of the microorganisms, its influence was followed in a concentration of 10 %.
A microtiter plate with 96 wells was filled with 100 μL of nutrient surface in each well.One hundred μL of each tested compound with a starting concentration of 2000 μg mL -1 was added to the first row of the microtiter plate.Twofold dilutions were made in a concentration range from 1000 μg mL -1 in the first row to 7.81 μg mL -1 in the last row of the plate.After that, 10 μL of suspensions of bacteria, yeast, or filamentous fungi spores were added to the appropriate wells.The tested compounds were examined with a bacterial concentration of 10 5 CFU mL -1 and a yeast and fungi spores concentration of 10 3 CFU mL -1 . 16Finally, resazurin, as an indicator of cell growth, was added to each well.The microtiter plates were incubated at 37 °C for 24 h for the bacteria, at 28 °C for 48 h for the yeast, and 72 h for the filamentous fungi.The MIC is defined as the lowest concentration of the tested substance preventing resazurin colour change from blue to pink.With the filamentous fungi, the MIC represents the lowest concentration of the tested substances that prevents mycelium growth.The results were read visually.
The MMC were determined by placing 10 μL of samples from the wells where no indicator colour change was recorded onto nutrient agar medium.At the end of the incubation period, the lowest concentration with no growth (no colony) was defined as the MMC.

Antibiofilm activity
Antibiofilm activities were determined using the method previously described by Christensen et al. 17 It is the most widely used method for examining antibiofilm activity on a formed biofilm.Each test included a biofilm formation control.Bacterial biofilm formation properties were well described by O'Toole and Kolter. 18olystyrene plates with a flat bottom and with 96 wells (Sarstedt, Germany) were prepared by dispensing 100 μL of nutrient broth into each well.Ten μL of a fresh bacterial suspension was added to each well.The inoculated microtiter plates were incubated at 37 °C for 24 h for the Gram-negative bacteria and 48 h for the Gram-positive bacteria.One hundred μL of the stock solution of the tested complexes (concentration of 2000 μg mL -1 ) were added into the first row of the plate.Then, twofold serial dilutions were made for each following row using a multichannel pipette.After 24 h incubation, the content of the plates was removed and ________________________________________________________________________________________________________________________ Available on line at www.shd.org.rs/JSCS/(CC) 2018 SCS.
BIOLOGICAL ACTIVITY OF THE PALLADIUM(II) COMPLEXES 1233 then the wells were washed with 200 μL of buffer solution (0.15 mol L -1 ammonium sulphate, 0.1 mol L -1 potassium dihydrogen phosphate, 0.034 mol L -1 sodium dihydrogen citrate and 0.001 mol L -1 magnesium sulphate) to remove free-floating bacteria.The biofilms formed of adherent cells in the plate were stained with crystal violet (0.1 %) and incubated at room temperature for 20 min.The excess stain was rinsed off with deionised water and the microtiter plates were fixed with 200 μL of ethanol-acetone solution (4:1).The optical densities (OD) of the stained adherent bacteria were determined by a micro ELISA plate reader at a wavelength of 630 nm.The biofilm inhibitory concentration (BIC) was defined as the lowest concentration of each complex that led to the formation of dispersed bacterial biofilms.Broth alone or broth with complexes solutions served as a control for the sterility check and nonspecific binding of the media.All tests were performed in duplicate.Tetracycline, vancomycin and ceftriaxone were used as positive controls.

Antioxidant activity
The ability of palladium(II) complexes to scavenge DPPH free radicals was assessed using the method described by Takao et al. 19 The test is based on the exchange of hydrogen atoms or electrons between antioxidant molecules of a chemical complex and DPPH radicals in solution. 20A methanolic DPPH solution (2 mL, 20 μg mL -1 ) was added to sample solutions in methanol (2 mL) at various concentrations (62.5-1000 μg mL -1 ).After 30 min in the dark at room temperature, the absorbance was read in a spectrophotometer at 517 nm.Methanol was used as a blank and ascorbic acid as a positive control.The experiment was performed in triplicate and the results were expressed as arithmetic mean ± standard deviation.Antioxidant activity was expressed as the inhibition percentage, which was calculated using the following equation: The EC 50 value is an effective concentration of a chemical substance in which 50 % of DPPH radicals were scavenged.A low EC 50 value indicates strong ability of a compound to act as a DPPH scavenger.

DNA interactions
A stock solution of CT-DNA was prepared in PBS buffer, which gave a ratio of UV absorbance at 260 and 280 nm (A 260 /A 280 ) of ca.1.8-1.9,indicating that the DNA was sufficiently free of protein and the concentration was determined by the UV absorbance at 260 nm (ε = 6600 dm 2 mol -1 ). 21The UV-Vis spectra were obtained on a Perkin-Elmer Lambda 35 or 25 double beam spectrophotometer, using 1.0 cm path-length quartz cuvettes (3.0 mL).Fluorescence measurements were run on a RF-1501 PC spectrofluorometer (Shimadzu, Japan).The fluorescence spectra were recorded in the range 550-750 nm upon excitation at 527 nm in all cases.The excitation and emission bandwidths were both 10 nm.

UV-Vis absorption studies
In order to compare quantitatively the binding strength of the complexes, the intrinsic binding constants K b were determined by monitoring the changes in absorption at the metal-to--ligand-charge-transfer (MLCT) band with increasing concentration of CT-DNA using Eq. ( 1): 1234 where K b is given by the ratio of slope to the y intercept in plots c DNA /(ε A -ε f ) vs. c DNA , where c DNA is the concentration of DNA in base pairs, ε A = A obsd /[complex], ε f is the extinction coefficient for the unbound complex and ε b is the extinction coefficient for the complex in the fully bound form.

Ethidium bromide (EthBr) displacement studies
The relative binding of complexes to CT-DNA was determined by calculating the quenching constant (K sv ) from the slopes of straight lines obtained from the Stern-Volmer equation (Eq.( 2)): where I 0 and I are the emission intensities in the absence and the presence of the quencher (C1), respectively, [Q] is the total concentration of quencher and K sv is the Stern-Volmer quenching constant, which can be obtained from the slope of the plot of I 0 /I vs.
1][12][13] The structure of these complexes was confirmed by elemental microanalyses and IR, 1 H-NMR and UV-Vis spectroscopy.1][12][13] The antimicrobial activity of C1-C4 complexes was tested against 18 strains of microorganisms determining the MIC and the MMC using the microdilution method.The antibiofilm activity of selected Pd(II) complexes and corresponding ligand were determined on a formed biofilm.The antioxidant activity was tested using the DPPH method.Furthermore, the interactions of these complexes with CT-DNA were investigated using UV-Vis and fluorescence emission spectroscopic techniques.

Antimicrobial activity
The results of testing the antimicrobial activity of the palladium(II) complexes are given in Table I.In the same table, the MIC and MMC values of terpy and dach that were used as ligands for the synthesis of palladium(II) complexes C1 and C4 are given.L2 ligand was not tested due to its volatility, while L3 ligand was not tested due to its air instability.Table S-I of the Supplementary material to this paper shows the values of MIC and MMC for the antibiotics tetracycline, ceftriaxone and vancomycin and the antifungal drugs fluconazole, ketoconazole and amphotericin B that were used as positive controls.A ten percent solution of dimethyl sulfoxide was used as the solvent for conducting the experiments and it was determined that it did not retard the growth of the tested microorganisms.
________________________________________________________________________________________________________________________ Available on line at www.shd.org.rs/JSCS/The intensity of antimicrobial activity varies depending on the type of microorganism and the tested compound.Previous research have shown that palladium(II) complexes have significantly better antimicrobial activity than the appropriate ligands. 5,6,22However, in the present study, the L1 ligand showed the same or slightly better antimicrobial activity, especially towards yeast and filamentous fungi, than the C1 complex.The C4 complex showed better activity than the L4 ligand when tested against bacteria, but the results were reversed for 1236 RAKOVIĆ et al.
the yeast and filamentous fungi.The C2 and C3 complexes could not be compared to the corresponding ligands.
The C1 complex demonstrated significantly greater antimicrobial activity than C2-C4.The antifungal activity of the C1 complex was better than its antibacterial activity.The MIC values for the C1 complex against yeast and filamentous fungi ranged from <0.49 to 15.63 μg mL -1 , while the MIC values for bacteria ranged between 0.98 and 31.25 μg mL -1 .The Gram-positive bacteria were more sensitive to the C1 complex than the Gram-negative bacteria, with an MIC value of 15.62 and 31.25 μg mL -1 , respectively.The exception was P. mirabilis ATCC 12453 with an MIC 0.98 μg mL -1 .The C2-C4 complexes had similar activity towards Gram-positive and Gram-negative bacteria.The results of previous studies showed that Gram-positive bacteria were more sensitive than Gram-negative bacteria. 23,24Others, such as Ali et al. found antibacterial efficacy against Gram-positive and Gram-negative bacteria to be similar. 25f the tested complexes, only C1 demonstrated antimicrobial activity comparable to those of the positive controls.The C1 complex with the corresponding ligand L1 had better antimicrobial activity towards the standard and clinical strain of B. subtilis than ceftriaxone.This compound showed stronger activity towards P. aeruginosa ATCC 27853 and other Gram-negative bacteria than vancomycin (Tables I and S-I).
Apart from the C1 complex, the antifungal activity of which matched that of fluconazole, the other complexes showed moderate and low activity towards most of yeast and filamentous fungi when compared to the positive controls.The most sensitive towards C1 and L1 was P. italicum (MIC < 0.49 μg mL -1 ).Their effects against it matched that of ketoconazole (Tables I and S-I).
Previous research of antimicrobial activity led to different conclusions with most of the studies evidencing markedly lower antifungal activity, 7,26 although a study conducted in 2012 determined that palladium(II) complexes with a derivative of thiosalicylic acid as ligand showed significantly higher antifungal activity towards species from the Aspergillus genus (A.fumigatus, A. flavus and A. restrictus) than the positive control, fluconazole. 6Garoufis et al. explored the antifungal and antibacterial activity of Pd(II) complexes with various ligands (sulphur and nitrogen donor ligands, Schiff bases and ligands used as drugs), and found that they had promising antibacterial but weak antifungal activity. 27tibiofilm activity C1 and L1 were used for examining antibiofilm activity on a formed biofilm because they showed a significantly higher level of antimicrobial activity compared to the other tested complexes.The in vitro antibiofilm activity of palladium(II) complex was tested on three species of bacteria and the results are presented in Table S Based on the given results, it could be concluded that the tested compounds showed low and moderate antibiofilm activity.Palladium(II) complex with terpyridine as ligand showed higher activity than ceftriaxone towards S. aureus ATCC 25923 and vancomycin towards P. mirabilis ATCC 12453.Studies that tested antibiofilm activity of palladium(II) complexes were not found while examining the available literature.

Antioxidant activity
DPPH radicals scavenging activity.The results of DPPH radicals scavenging activity, expressed as ЕС 50 values, showed the moderate activity of the tested compounds (Table II).The C2 complex was not tested due to its low solubility in methanol, while the L2 and L3 ligands were not tested for already stated reasons.
The C1 and C3 complexes and the L1 ligand demonstrated no anti-oxidant activity, while the L4 ligand and C4 showed weak antioxidant activity.The С4 complex had significantly better antioxidant activity than L4, but it was lower than that of the positive control, ascorbic acid.Upon examining the literature, it was noticed that there were not many studies that tested the antioxidant activity of palladium(II) complexes.Previous studies showed that palladium(II) complexes have better antioxidant activities than nickel and platinum complexes. 28Others found that Pd(II) complexes have a lower scavenging activity than their parent ligands. 29Moreover, antioxidant activities close to that of ascorbic acid were demonstrated, particularly for palladium chloride complex with two o-chlorobenzylamine ligands, Pd2CBA (C 14 H 15 N 2 Cl 3 Pd). 30Maskovic et al. found that Pd(II) complexes with N,N′-ligands have antioxidant activities higher than that of butylated hydroxytoluene but lower than that of ascorbic acid. 31Pd(II) complex with 2-hydroxy-4-methoxybenzaldehyde-4-phenylthiosemicarbazone shows better antioxidant activity than Ni(II) and Cu(II) compexes with the same ligand, and vitamin C. 32 The Pd(II) complexes tested in the present study did not exhibit significant antioxidant activity, as opposed to the complexes that were investigated in above-mentioned studies.

DNA interactions
This part of the study was focused on the investigation of interactions of complexes C1-C4 with DNA.4][35] Thus, knowledge of whether the studied complexes could interact with DNA to the desired extent enabled DNA to be considered as one of the targets for the complexes in the manifestation of their antimicrobial and antibiofilm activities.

UV-Vis absorption studies
Using complexes C1-C4, UV-Vis titrations were performed.The intrinsic equilibrium binding constant (K b ) was evaluated.The metal complex absorption titration studies were realized at room temperature using a fixed concentration of complex C1-C4 (10 µmol L -1 ) in PBS buffer, and varying the amount of CT-DNA (0-20 µmol L -1 ). 36The absorption intensity of the complex may decrease (hypochromism) or increase (hyperchromism) with a slight increase in the absorption wavelength (bathochromism) upon addition of DNA. 36,37[39]  The interaction of complexes C1-C4 with CT-DNA has also been investigated by EthBr displacement studies, which provided strong evidence about competitive binding of drugs with CT-DNA.Ethidium bromide (EthBr) is a classical intercalator that gives significant fluorescence emission intensity when it intercalates into the base pairs of DNA.DNA-induced EthBr fluorescence emission could be quenched after addition of a complex capable of forming strong interactions with DNA, replacing EthBr. 40The significant decrease in the intensity of the emission band at 612 nm after the addition of increasing amounts of complexes C1-C4 shows competition of the studied complex in binding to DNA, Fig. S-1 of the Supplementary material.The Stern-Volmer quenching constant (K sv ) was calculated from the slopes of the plots I 0 /I vs. [Q] using Eq. ( 2).Thus, complexes C1-C4 are capable of displacing EthBr from the EthBr-DNA complex and could strongly interact with DNA binding sites.
According to the values of constants presented in Table III, good interaction of the studied complexes with CT-DNA is evident.In addition, the results obtained by fluorescence spectroscopy are in excellent agreement with the UV-Vis spectral data, demonstrating that the studied complexes interact with CT-DNA.

CONCLUSIONS
The tested ligands and complexes demonstrated selective and limited antimicrobial activity.The exception is the C1 complex with the corresponding ligand, the effects of which on B. subtilis and P. aeruginosa ATCC 27853 were better than the positive controls.The same complex also showed significant activity towards P. italicum.The C1 complex had higher antibiofilm activity than the positive control towards S. aureus ATCC 25923.Complex C4 showed the best antioxidant activity, while the antioxidant activity of the other complexes was insignificant.Furthermore, the interaction of the Pd(II) metal complexes with calf thymus DNA (CT-DNA) was further examined by absorption (UV-Vis) and emission spectral studies (EthBr displacement studies).Overall, the studied complexes exhibited a good DNA interaction ability.
A control is the absorbance of the control and A sample is the absorbance of the sample.

TABLE II .
The radical scavenging capacity of ligand L4, palladium(II) complex С4 and the positive control (ascorbic acid), expressed as EC 50 values; each value is expressed as the arith-

TABLE III .
The DNA-binding constants (K b ) and Stern-Volmer constants (K sv ) for complexes C1-C4