From Synthesis to Antibacterial Activity of Some New Palladium(II) and Platinum(IV) Complexes

Simultaneously with the rapid development of a wide range of antibacterial agents since the 1940s, bacteria have proved extremely adept at developing resistance to each new employed agent. The rapidly increasing incidence of bacterial resistance to antimicrobial agents has become a serious problem worldwide. Resistance mechanisms have been identified and described for all known antibiotics currently available for clinical use (Fluit et al., 2000).


Introduction
Simultaneously with the rapid development of a wide range of antibacterial agents since the 1940s, bacteria have proved extremely adept at developing resistance to each new employed agent.The rapidly increasing incidence of bacterial resistance to antimicrobial agents has become a serious problem worldwide.Resistance mechanisms have been identified and described for all known antibiotics currently available for clinical use (Fluit et al., 2000).
The synthesis and evaluation of the biological activity of the new metal-based compounds is the field of growing interest.Numerous complexes based on palladium(II) and platina(IV)ion have been synthesized and their different biological activities have been documented (Agarwal, 2007;Mishra et al., 2007a;Mishra & Kaushik, 2007).The impact of different palladium and platinum complexes on the growth and metabolism of various groups of microorganisms has been studied.Garoufis et al. (2009) reviewed numerous scientific papers on anti-viral, antibacterial and antifungal activity of palladium(II) complexes with different types of ligands (sulfur and nitrogen donor ligands, Schiff base ligands and drugs as ligands).There are other papers in the literature showing different intensity of palladium(II) and platina(IV) complexes activity on various species of bacteria and fungi (Kovala-Demertzi et al., 2001;Brudzinska et al., 2004;Coombs et al., 2005;Guerra et al., 2005;Ali et al., 2006;Manav et al., 2006;Aghatabay et al., 2007;Kizilcikli et al., 2007;Mishra et al., 2007b;Biyala et al., 2008;Al-Hazmi et al., 2008;Vieira et al., 2009).
The aim of this paper is to describe synthesis of some new palladium(II) and platinum(IV) complexes and in vitro research of their antibacterial activities.The second objective is to evaluate the impact these compounds have on probiotic bacteria.Probiotics are used as supplements and they play significant role in protecting and maintaining the balance of intestinal microflora in antibiotic therapy.

Chemistry
The palladium(II) and platinum(IV) complexes were obtained by direct reaction of the corresponding starting compounds (K 2 PdCl 4 and K 2 PtCl 6 ) and newly synthesized tetradentate or bidentate ligands.The next compounds were synthesized:

The synthesis of the ligands -L1, L2, L3 and corresponding palladium(II) complexes -C1, C2, C3
In 50 mL of dry alcohol (1-propanol, 1-butanol or 1-pentanol), saturated with gas HCl, 1.53 g (7.5 mmol) of H 2 -S,S-eddp was added and the mixture was refluxed for 12 h.The mixture was filtered and left in the refrigerator over night.The obtained white powder was filtered and air-dried.
Complexes were obtained by mixing K 2 [PdCl 4 ] (0.200 g, 0.613 mmol) and equimolar amount of the dpr-S,S-eddp•2HCl During 2 h of stirring 10 cm 3 of water solution of LiOH (0.0294 g, 1.226 mmol) was added in small portions to the reaction mixture.Within this period, pale yellow precipitates of the complexes C1-C3 were obtained, filtered off, washed with cold water, ethanol and ether and air dried (Vasić et al., 2010) (Fig. 1.).

The synthesis of the ligands -L4, L5, L6, L7 and corresponding palladium(II) complexes -C4, C5, C6, C7
In 50 mL of dry alcohol (ethanol, 1-propanol, 1-butanol or 1-pentanol), saturated with gas HCl, 2.50 g (7.5 mmol) of (H 2 -(S,S)-eddv) was added and the mixture was refluxed for 12 h.The mixture was filtered off and the filtrate was left for a few days in a refrigerator at 4°C.The esters were recrystallized from hot alcohol used for each reaction.Complexes were obtained by mixing K 2 [PdCl 4 ] (0.200 g, 0.613 mmol) and equimolar amount of the L4 (0.241 g, 0.613 mmol), L5 (0.256 g, 0.613 mmol), L6 (0.273 g, 0.613 mmol) or L7 (0.290 g, 0.613 mmol) esters.During 2 h of stirring 10 cm 3 of water solution of LiOH (0.0294 g, 1.226 mmol) was added in small portions to the reaction mixture.Within this period, pale yellow precipitates of the complexes C4-C7 were obtained, filtered off, washed with cold water, ethanol and ether and air dried (Fig. 2.).The crystal structure of C4 was confirmed by X-ray analysis (Radić et al., 2010b;2011a).Thionyl choride (4 cm 3 , 55 mmol) was introduced into a flask containing 50 cm 3 of corresponding ice cooled alcohol (ethyl, n-propyl, n-butyl or n-pentyl; anhydrous conditions) for 1 h.After addition of 2 g (5.54 mmol) [(S,S)-H 4 eddl]Cl 2 the reaction mixture was refluxed for 16 h, filtered off and the filtrate was left for a few days in a refrigerator at 4°C.The esters were recrystallized from the hot alcohol used for each reaction.
Thiosalicylic acid (1 mmol) was added to a 100 cm 3 round bottom flask containing 50 cm 3 of 30% solution of ethanol in water and stirred.A solution of NaOH (2 mmol in 5 cm 3 of water) was added to acid suspension.The solution became clear.The corresponding alkyl halogenide (2 mmol) was dissolved in 5 cm 3 of ethanol and transferred to the stirred solution.The resulting mixture was kept overnight at 60°C.The reaction mixture was transferred into a beaker and ethanol was evaporated off on a water bath.Diluted hydrochloric acid (2 mol/dm 3 ) was added to the resulting water solution and S-alkyl thiosalicylic acid was precipitated as a white powder.The liberated acid was filtered off and washed with plenty of distilled water.The product was dried under vacuum overnight.

The synthesis of the ligand L17 and corresponding palladium(II) complex C17 and corresponding platinum(IV) complex C17a
Benzaldehyde (30 g) was refluxed with ammonium acetate (60 g) for 3 hours.The reaction mixture was cooled and the product was filtered and washed with ethanol.Recrystallization from 1-butanol gave N-benzoyl-N'-benzylidene-meso-1,2-diphenyl-ethylendiamine.Hydrolysis of that compound with 70% sulphuric acid under reflux for 1h gave meso-1,2--diphenyl-ethylenediamine as the basic product of hydrolysis.
3-Chloro-propanoic acid (4.34 g, 0.04 mol) was dissolved in 5 cm 3 of water on ice bath and carefully neutralized with cold water solution of 5 cm 3 NaOH (1.6 g, 0.04 mol).1,2-Diphenyl--ethylenediamine (4.24 g, 0.02 mol) was added to this solution.The mixture was being stirred for 4 hours at 90°C, and during this period 5 cm 3 NaOH water solution (1.6 g, 0.04 mol) was introduced.After that, 5.6 cm 3 6 mol/dm 3 HCl was added and resulting solution was evaporated to the volume of 7 cm 3 ; 6 cm 3 conc.HCl, 6 cm 3 of ethanol and 6 cm 3 of ether were added to the mixture.The white precipitate of H 2 -1,2-dpheddp•2HCl•1.5H 2 O (L17) was separated by filtration and refined with solution water : ethanol = 1 : 2. The crystal structure of L17 was confirmed by X-ray analysis (Radić et al., 2010a).Potassium-hexachloridoplatinate(IV) (0.2 g, 0.411 mmol) was dissolved in 10 cm 3 of water on a steam bath and 1,2-diphenyl-ethylenediamine-N,N'-di-3-propanoic acid (0.1876 g, 0.411 mmol) was added.The reaction mixture was heated for 12 hours and during this period 10 cm 3 of LiOH water solution (0.0394 g, 1.65 mmol) was added in small portions and the solution was filtered and evaporated to small volume.The orange precipitate of s-cis-[PtCl 2 (1,2-dpheddp)] C17b) was separated by filtration, washed with cold water and airdried (Fig. 5).
In 50 cm 3 of dry ethanol, saturated with gas HCl, 1.53 g (7.5 mmol) of H 2 -S,S-eddp was added and the mixture was refluxed for 12 h.The mixture was filtered and left in the refrigerator over night.The obtained white powder of O,O'-diethyl-(S,S)--ethylenediamine-N,N'-di-2-propanoate dihydrochloride, det-S,S-eddp•2HCl (L18) was filtered and air dried.
Fig. 6.Synthesis of the ester det-(S,S)-eddv•2HCl and platinum(IV) complex From Synthesis to Antibacterial Activity of Some New Palladium(II) and Platinum(IV) Complexes 319 ).The crystal structure of L20 was confirmed by X-ray analysis (Dimitrijević et al., 2010).

Test substances
The tested compounds were dissolved in DMSO and then diluted into nutrient liquid medium to achieve a concentration of 10%.Antibiotic, doxycycline (Galenika A.D., Belgrade), was dissolved in nutrient liquid medium, a Mueller-Hinton broth (Torlak, Beograd).

Test microorganisms
Antimicrobial activity of twenty-one palladium(II) and platinum(IV) complexes and their ligands was tested against 9 species of bacteria:

Suspension preparation
Bacterial suspensions were prepared by the direct colony method.The colonies were taken directly from the plate and were suspended in 5 mL of sterile 0.85% saline.The turbidity of initial suspension was adjusted by comparing with 0.5 McFarland's standard (0.5 ml 1.17% w/v BaCl 2 ×2H 2 O + 99.5 ml 1% w/v H 2 SO 4 ) (Andrews, 2005).When adjusted to the turbidity of the 0.5 McFarland's standard, bacteria suspension contains about 10 8 colony forming unites (CFU)/mL.Ten-hold dilutions of initial suspension were additionally prepared into sterile 0.85% saline.

Microdilution method
Antimicrobial activity was tested by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) by using microdilution plate method with resazurin (Sarker et al., 2007).The 96-well plates were prepared by dispensing 100 µL of nutrient broth into each well.A 100 µL from the stock solution of tested compound (concentration 2000 µg/mL) was added into the first row of the plate.Then, twofold, serial dilutions were performed by using a multichannel pipette.The obtained concentration range was from 1000 µg/mL to 7.81 µg/mL.A 10 µL of diluted bacterial suspension was added to each well to give a final concentration of 5 x 10 5 CFU/mL.Finally, 10 µL resazurin solution was added to each well inoculated with bacteria.Resazurin is an oxidationreduction indicator used for the evaluation of microbial growth.It is a blue non-fluorescent dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells (Banfi et al., 2003).The inoculated plates were incubated at 37 °C for 24 h.MIC was defined as the lowest concentration of the tested substance that prevented resazurin color change from blue to pink.Doxycycline was used as a positive control.Solvent control test was performed to study an effect of 10% DMSO on the growth of microorganism.It was observed that 10% DMSO did not inhibit the growth of microorganism.Also, in the experiment, the concentration of DMSO was additionally decreased because of the twofold serial dilution assay (the working concentration was 5% and lower).Each test included growth control and sterility control.All tests were performed in duplicate and MICs were constant.Minimum bactericidal concentration was determined by plating 10 µL of samples from wells, where no indicator color change was recorded, on nutrient agar medium.At the end of the incubation period the lowest concentration with no growth (no colony) was defined as minimum bactericidal concentration.

Results and discussion
The results of in vitro testing of antibacterial activities of the ligands and corresponding palladium(II) and platinum(IV) complex are shown in Table 1-10.For comparison, MIC and MBC values of doxycycline are listed in Table 11.The solvent (10% DMSO) did not inhibit the growth of the tested microorganisms.
The intensity of antimicrobial action varied depending on the species of microorganism and on the type and concentration of tested compounds.The difference between antimicrobial activity of the ligands and corresponding palladium(II) and platinum(IV) complexes is noticed and, in general, the most active were palladium(II) complexes.
The results of antibacterial testing for the ligands (L1, L2, L3) and corresponding palladium(II) complexes (C1, C2, C3) are shown in Table 1.The results for 3 strains of pathogenic bacteria and 2 species of probiotic bacteria were reported in the paper Vasić et al., (2010).Results for S. enterica, Staphyl.aureus ATCC 25923, S. lutea ATCC 9341 and L. rhamnosus were first presented in this paper.These ligands and complexes, being compared to positive control, showed low to moderate antibacterial activity.MIC and MBC values were in range from <7.81 to >1000 µg/mL, depending on the species of bacteria.Grampositive bacteria showed higher sensitivity.The most sensitive was S. lutea ATCC 9341, where MIC was for C1 and C2 <7.81 µg/mL.The best activity at Gram-negative bacteria was shown by C2 to P. aeruginosa ATCC 27853 and E. coli (MIC was 31.25 µg/mL).The probiotics showed sensitivity similar to the sensitivity of the other bacteria to the tested compounds.Exception is B. animalis subsp.lactis where L2, C2 and L3 inhibited its growth at these concentrations: 7.81 µg/mL, 15,63 µg/mL and <7.81 µg/mL.
The results of testing the ligands (L4, L5, L6, L7) and their palladium(II) complexes (C4, C5, C6, C7) are shown in Table 2 and Table 3.The results of testing for L4 were reported in the paper by Stanković et al., (2011a;2011c) The results of testing the ligands (L12, L13, L14, L15, L16) and corresponding palladium (II) complexes (C12, C13, C14, C15, C16) are shown in Table 6 and Table 7.The results for these testing were accepted for publication in the paper by Radić et al., (2011b).All tested compounds demonstrated selective and moderate antibacterial activity.Tested ligands, with a few exceptions, show very low antimicrobial activity.The activity of corresponding complexes was higher than with the ligands.MICs values for ligands were in range from 250 µg/mL to >1000 µg/mL, and for complexes from 62.5 µg/mL to 1000 µg/mL.The Gram-positive bacteria were more sensitive than the Gram-negative bacteria especially by the activity of the complexes.The best effect was observed in C16 to S. lutea ATCC 9341 were MIC and MBC 62.5 µg/mL.MICs for Gram-negative bacteria were at 500 μg/mL and 1000 μg/mL.The tested complexes (C13) and (C14) exhibited somewhat stronger antibacterial activity towards P. aeruginosa ATCC 27853 (MIC = 250 μg/mL).The probiotics showed sensitivity similar to the sensitivity of the other bacteria (Radić et al., 2011b).MIC, minimum inhibitory concentration (µg/mL), MBC, minimum microbiocidal concentration (µg/mL) Table 9. 6 Antibacterial activity of the ligands (L4, L18) and corresponding complexes (C4a, C18).
Antibacterial activity of the tested platinum(IV) (C4a, C18) complexes and corresponding ligands (L4, L18) are shown in Table 9. Results for these testing was reported in the papers Stanković et al., (2011a,c).The ligands and corresponding platinum(IV) complexes demonstrated low antimicrobial activity.There was no difference in activities between the ligands and corresponding complexes.The ligands and corresponding platinum(IV) complexes showed significant antibacterial activity against S. lutea ATTC 9341.MICs values were in range from 31.25 μg/mL to 62.5 μg/mL, and MBCs values were from 62.5 μg/mL to 125 μg/mL.The tested compounds did not affect the growth of Gram-negative bacteria or their activities were very low (MIC ranged from 500 μg/mL to >1000 μg/mL, MBC from 1000 μg/mL to >1000 μg/mL).Also, probiotic bacteria showed high resistance to the effects of tested substances.MICs were from 125 μg/mL to 1000 μg/mL, and MBCs were from 500 μg/mL to >1000 μg/mL (Stanković et al., 2011a,c).
The results of in vitro testing of antibacterial activities of the ligands (L19, L20, L21) and corresponding platinum(IV) (C19, C20, C21) complex are shown in Table 10.
The difference in action between ligands and corresponding complexes can be seen at Gram-positive bacteria.Ligands have significant antimicrobial effect on probiotic bacteria (L20, L21), and complexes on Gram-positive bacteria (C19, C20, C21).C21 has better antimicrobial effect than two other complexes.The lowest antimicrobial action of compounds was on Gram-negative bacteria, where tested concentrations of ligands almost didn't have the influence, while corresponding complexes had some better action, but still weak and limited.L. rhamnosus also showed similar resistance to the action of tested compounds (none of the tested concentrations had the influence on its growth), while the other probiotic bacteria were more sensitive, especially to the action of ligands, where MIC goes from <31.25 µg/mL to 250 µg/mL.At complexes MIC is in the range from125 µg/mL to 1000 µg/mL.
The gram-positive bacteria were more sensitive than the gram-negative bacteria.The platinum(IV) complexes showed high antibacterial activity against Gram-positive bacteria.MIC values were in range from 7.81 μg/mL to 1000 μg/mL, and MBC values were from 15.63 μg/mL to 1000 μg/mL depending on the species of bacteria.In general, the ligands demonstrated low and selective antimicrobial activity (with few exceptions) and the complexes showed selective and moderate antibacterial activity.MIC values were in range from <7.81µg/mL to >1000 µg/mL and MBC values from 15.625 µg/mL to >1000 µg/mL depending on the species of bacteria.The Gram-positive bacteria were more sensitive than the Gram-negative bacteria.The most sensitive species is S. lutea ATCC 9341.Tested probiotics, with a few exceptions, indicate high resistance toward tested compounds.L. rhamnosus shows the highest resistance among them.The tested complexes C1, C2, C3 and C17a exhibit strong activity towards coli, P. aeruginosa ATCC 27853 and E. faecalis ATCC 29212.The L6, L7 and C6, C7 exhibit strong antibacterial activity towards E. coli.The tested compounds did not affect S. enterica or their activities were low.Some activity showed palladium(II) complexes (C1, C2 , C3, C6 and C17a).At the ligands the most effective antimicrobial activity show L6, L7, L9, L10 and L11 while the most active complexes are C1, C2, C3, C6 and C17a.For eleven ligands (L1 -L11) and corresponding palladium(II) complexes (C1 -C11) antifungal activity is investigated.Palladium(II) complexes showed good antifungal activity opposite to ligands.This study are in keeping with our research to a great extent (Radojević et al., 2010).

Conclusion
The intensity of antimicrobial action varied depending on the species of microorganism and on the type of tested compounds.The tested ligands, with few exceptions, show low antimicrobial activity.The difference between antimicrobial activity of the ligands and corresponding palladium(II) and platinum(IV) complexes is noticed and, in general, the most active were palladium(II) complexes.The Gram-positive bacteria were more sensitive than the Gram-negative bacteria.The most sensitive species is Sarcina lutea ATCC 9341 and the most resistant is Salmonella enterica where the tested compounds did not affect or their activities were low.Tested probiotics, with a few exceptions, also indicate high resistance toward tested compounds.

Fig. 1 .
Fig. 1.The preparation of some alkyl esters of H 2 -S,S-eddp and corresponding palladium(II) complexes

Fig. 2 .
Fig. 2. The preparation of some alkyl esters of H 2 -S,S-eddv and corresponding palladium(II) complexes

Fig. 3 .
Fig. 3.The preparation of some alkyl esters of H 2 -S,S-eddl and corresponding palladium(II) complexes

Fig. 8 .
Fig. 8.The synthesis of: a) esters (R 2 -S,S-eddba•2HCl); b) complexes [PtCl 4 (R 2 -S,S-eddba)] . The tested ligands, with few exceptions, show very low antimicrobial activity, while palladium(II) complexes show selective and moderate activity.Interestingly, L6, L7 and C6, C7 exhibit strong antibacterial activity towards E. coli, Staphyl.aureusATCC25923 and S. lutea ATCC 9341, MIC ranged <7.81 µg/mL to 31.25µg/mL.Probiotic bacteria showed high resistance to the effects of tested substances.The most sensitive was B. subtilis IP 5832 to C5 and C4 (MIC was 7.81µg/mL and 15.63 µg/mL).areshown in Table4 and Table 5.The ligands and complexes, being compared to positive control, with few exceptions, showed low antibacterial activity.MIC and MBC values were in range from <7.8 to >1000 µg/mL, depending on the species of bacteria.L9, L10 and L11 showed excellent results to S. lutea ATCC 9341 (MIC and MBC <7.81 µg/mL) and L10 and L11 to S. lutea ATCC 9341, Staphyl.aureus ATCC 25923 and L. rhamnosus (MIC <7.81 µg/mL).In this case the ligands acted better than corresponding complexes and it is an exception.The complexes have weak antimicrobial activity and some better influence was seen on B. subtilis IP 5832 were MIC was in range from 39.06 to 312.5 µg/mL.

Table 11 .
Antibacterial activity of the positive control -doxycycline