Synthesis, Characterization and Antibacterial Activities of New Pd(II) And Pt(IV) Complexes of Some Unsymmetrical Tetradentate Schiff Bases

New palladium (II) and platinum (IV) complexes with a series of unsymmetrical tetradentate Schiff bases derived from aromatic 2-hydroxy aldehydes in an ethanolic medium were synthesized and characterized by elemental analyses, melting points, magnetic susceptibility, thermogravimetry, differential scanning calorimetry ( DSC ), infrared ( IR ), and electronic spectral measurements. The purity of the ligands and the metal complexes are confirmed by microanalysis, while the unsymmetrical nature of the ligands was further corroborated by 1 H-NMR spectroscopy. Comparison of the IR spectra of the Schiff bases and their metal complexes confirm that the Schiff bases act as tetradentate bases and their complexes were screened in vitro against 10 human pathogenic bacteria. complexes due to higher molecular mass. The free ligands and metal complexes exhibit pronounced antibacterial activities against some bacterial strains, in a number of cases comparable or even higher with that of chloramphenicol. In general, the antibacterial results show that the majority of the metal complexes were more active than their respective Schiff bases. Chloramphenicol showed the lowest MICs value of less than 0.0098 mg cm -1 against eight bacterial strains used in this study and was more active than all the compounds tested when their MICs values are compared.


Materials and Methods
All reagents and solvents were of the spectroscopic grades and used as received. Ethanol, methanol, chloroform, DMF, DMSO, salicylaldehyde, 5-chlorosalicylaldehyde, 5-nitrosalicylaldehyde, 3-ethoxysalicyl-aldehyde, 2-hydroxy-1-naphthaldehyde, o-phenylenediamine are from Aldrich-Sigma company. Anhydrous Palladium (II) chloride (59% -Merck) and anhydrous Platinum (IV) chloride (57.5% -Merck) were used. Elemental analyses were determined at the Microanalytical Centre, Cairo University, Egypt. IR spectra were recorded on Perkin Elmer (spectrum 1000) FT-IR spectrometer using KBr pellets at the chemistry department, College of Science, King Fahd University for Petroleum and Minerals Saudi Arabia. Conductance of the metal complexes was determined in DMF using a YSI-32 model conductometer. Magnetic measurements were done on solid complexes using the Gouy method. Melting points were recorded on a Gallenkamp apparatus and are uncorrected. Thermogravimetric analyses were measured under nitrogen flow rate: 30 cm 3 min −1 using a Shimadzu TGA-60H thermobalance from room temperature up to 700ºC at the Chemistry Department, College of Science and King Saud University, Saudi Arabia. The differential scanning calorimetry (DSC) analysis was carried out at 20ºC min -1 heating rate up to 450ºC using a Perkin Elmer DSC 4000 series, which was calibrated with indium metal.

Synthesis of the Compounds
The procedure for the preparation of the unsymmetrical Schiff bases was reported elsewhere (Nejo et al., 2009). The complexes were prepared easily and in good yield from an equimolar ratio of the ligands and the metal chloride. So, by the addition of (1.0 mmol) of PdCl 2 dissolved in 20 cm 3 ethanol and (1.0 mmol) PtCl 4 dissolved in 20 cm 3 acetone, to a magnetically stirred warmed (40ºC) solution of the ligand (1.0 mmol) in ethanol (20 cm 3 ). The color of the mixture changed instantly. The mixtures were refluxed for 2.5 h. The complexes were precipitated which, upon cooling, were filtered, washed several times with cold methanol, and allowed to dry in a desiccator over silica gel.

Bacterial strains
The metal complexes and ligands were individually tested against a penal of microorganisms (Gram negative and Gram positive), namely Enterobacter cloacae, Serratia marcescens, Acinetobacter calcaoceuticus, Acinetobacter calcaoceuticus anitratus, Salmonella spp., Staphylococcus aureus, Bacillus pumilus, Bacillus subtilis, Staphylococcus epidermidis, and Micrococcus kristinae. The organisms were obtained from the Department of Microbiology, National Research Centre, Egypt.

Disc diffusion assay
Antibacterial activity of the ligands and metal complexes were carried out using the disc diffusion method described by Bauer et al. (Bauer et al., 1966). Bacteria were maintained on Mueller-Hinton nutrient agar at 4ºC. Molten Mueller-Hinton agar was inoculated with a broth culture of the respective bacterial strains and poured over previously washed and sterilized 90 mm Petri dishes. Palladium (II) chloride, platinum (IV) chloride, metal complexes, and ligands were dissolved in DMSO to a final concentration of 10 mg cm -1 and sterile Whatman filter paper No. 1(6 mm) discs were separately impregnated with each sample to be tested at 5.0 mg cm -1 and placed on the inoculated agar. The plates were incubated at 37ºC for 24 h and the zones of inhibition were measured at the end of the incubation period. Chloramphenicol was used as the standard antibiotic for reference drug.

Minimum inhibitory concentration
The minimum inhibitory concentrations (MICs) of the ligands, metal complexes, and Palladium (II) chloride, platinum (IV) chloride that showed antibacterial activities were determined using the 96-well micro-plate dilution method described (Eloff , 1998). The serial plate concentrations were 5.0, 2.5, 1.25, 0.625, 0.313, 0.157, 0.078 and 0.039 mg cm -1 for the compounds. Standard antibiotic (chloramphenicol) was used as positive control. Bacteria were grown for 18 h in nutrient broth and cultures of 108 colony forming unit (cfu) cm -1 were used and incubated for 24 h at 37ºC. As an indicator of bacterial growth, 40 cm 3 of 0.2 mg cm -1 p-iodonitrotetrazolium (INT) solution was added to each well and incubated at 37ºC for 30-120 min. The colorless tetrazolium salt was reduced to a red-colored product by the biological activity of the organisms, thereby making the inhibition of bacterial growth visible as a clear well. MICs values were recorded as the lowest concentration in which there was no bacterial growth. Each treatment was repeated thrice.

Synthesis
A series of new unsymmetrical tetradentate Schiff base complexes of Pd(II) and Pt(IV) were prepared by refluxing the relevant unsymmetrical Schiff bases with the corresponding metal chloride in methanol at 1:1 molar ratio as in the scheme presented in Fig. 1. All the metal complexes are stable, non-hygroscopic, insoluble in common organic solvents but partially soluble in chloroform and DMSO. The unsymmetrical Schiff base complexes did not melt or decompose when heated to 250ºC. The purity of the Schiff bases and their metal complexes as formulated were established by microanalyses. Although crystal structures are not available, the proposed structures are consistent with all other evidence. The analytical data, color, percentage yields, melting points and magnetic moments of the compounds are presented in Table 1

IR Spectra
The positions of the important bands of the compounds are shown in Table 2. Tentative assignments of the observed bands for the compounds were made by comparing spectra of the metal complexes with those of the Schiff bases. Due to the unsymmetrical nature of the ligands and the complexes two bands were observed for each of the following bonds, υ(C=N), υ(C-O), υ(M-N), and υ(M-O) taking their origin from the different aldehydes (Nejo et al., 2009) The ligands exhibited characteristic C=N stretching frequency at 1610-1621 and 1567-1583 cm -1 , which shift to lower frequencies at 1603-1609 and 1557-1572 cm -1 , respectively, upon complexation. This indicates participation of the azomethine nitrogen in bonding (Aranha et al., 2007). The corresponding phenolic C-O stretching frequency occurs at 1313-1333 and 1276-1289 cm -1 for the ligands and at 1325-1366 and 1287-1317 cm -1 for the complexes. The shift confirms participation of the phenolic O in C-O-M bond formation (Abd-Elzaher, 2001). Thus, it can be concluded that the Schiff bases are tetradentate, coordinating via the azomethine N and the phenolic O. It has also been reported that the metal-ligand vibrational modes are very sensitive to substituent effects from isotopic labeling studies (Hancock and Thornton, 1969;Percy and Thornton, 1972). The substituent effects were based on the position of substitution rather than the nature of the substituents. The υ(M-O) bands are observed to exhibit higher vibrational frequencies than the υ(M-N) bands for the meta-substituents, while the order is reversed for para-substituents regardless of the nature of the substituents. The substituents in the complexes reported in this study are located in the 3-and 5-positions on the salicylaldehyde ring and it is expected that υ(M-O) bands would be greater than υ(M-N) bands, as shown in Table 2

1 H-NMR Spectroscopy
The ligands display signals at δ 6.54, 6.87-7.78 and 9.67 ppm due to -CH=N, aromatic, and -OH protons, respectively. The presence of the phenolic (OH) protons at δ 9.67 ppm that vanished in the spectra of the metal complexes suggested deprotonation and subsequent participation in complexation.

Electronic Spectra
Electronic absorption spectra of the Pd(II) and Pt(IV) complexes were recorded in 10 -5 mol L -1 solutions of each complex in DMSO from 11111.11 to 52631.58 cm -1 at room temperature and their results are presented in Table 3. Solution spectra of all the Pd(II) complexes in DMSO show two bands at 20619-20877 and 25974-26178 cm -1 . These two transitions are assignable to square-planar geometry (Kolawole and Osowole, 2009;Boghaei and Lashanizadegan, 2000). At higher energy, two more intense absorptions are observed in the range 30395-39063 cm -1 , which are likely due to charge transfer or intra-ligand transitions. The assignment of square-planar geometry is supported by the diamagnetism of the complexes. The electronic spectra of Pt(IV) complexes consist of two energy bands at 20106-20713 cm -1 and 26654-26821 cm -1 which is assignable to square-planar geometry of Pt(IV) complexes. One strong high energy band at 30211-34961 cm -1 is also observed, assigned to metal-ligand charge transfer. The electronic spectral data are given in Table 3.  (482) a The spectra were noisy in the UV region and some of the bands could not be extracted

Magnetic Susceptibility Measurements
Pd(II) complexes have 4d 8 electronic configuration and should exhibit a magnetic moment higher than expected for two unpaired electrons in octahedral (2.8-3.2 B.M.) and tetrahedral (3.4-4.2 B.M.) complexes. Deviations from spin-only moment of 2.83 B.M. is due to two unpaired electrons are attributed to orbital contributions. However, Pd(II) square-planar complexes are diamagnetic (Boghaei and Lashanizadegan, 2000). The observed diamagnetism confirms the square planar environment for the Pd(II) complexes, Table 1. At room temperature Pt(IV) complexes observe diamagnetism. These results, Table 1 support the square planar geometry for Pt(IV) complexes.

Thermal Analysis
The thermal decomposition characteristics of the Pd(II) and Pt(IV) complexes were studied using thermal analytical techniques (TGA and DSC) under nitrogen. Transition temperatures, enthalpy changes, and decomposition temperatures of complexes are tabulated in Table 4.
The results show good agreement with the formulas suggested from the analytical data, Table 1. The TGA data indicate that H 2 L 1 , H 2 L 2 , H 2 L 3 , and H 2 L 4 start decomposition at 244ºC, 239ºC, 266ºC and 246ºC, respectively, indicating H 2 L 3 has the greatest stability. The thermograms of the Pd(II) and Pt(IV) complexes exhibited no mass loss up to 362.4ºC, indicating the absence of coordinated solvent and high thermal stability of the complexes. A single decomposition step which involved partial loss of the same set of organic moieties was observed for the metal complexes.
In the TGA curve of the PdL 1 complex an estimated mass loss of 25.64% (Calc.=25.72%) was observed at 402.5ºC, attributed to the loss of C 6 H 4 and HCN from the organic moiety. The decomposition curve of PdL 2 showed single-step degradation with an onset temperature of 382ºC and an estimated mass loss of 20.4% (Calc.=20.26%). The weight loss is attributed to loss of C 6 H 4 and HCN. For this step, an exothermic peak with t max =428.6ºC in the DSC was observed. The PdL 3 complex was stable up to 362.4ºC where the decomposition began. The thermogram of the PdL 3 complex showed a mass loss of 30.93% (Calc.=31.04%), which corresponds to the loss of C 6 H 4 , 2HCN, and C 2 H 5 . The onset of decomposition of the PdL 4 complex occurs at 385.5ºC with an estimated mass loss of 21.99% (Calc.=21.89%), which may be attributed to the loss of C 6 H 4 and HCN. The order of thermal stability of the Pd(II) complexes, PdL 1 > PdL 2 > PdL 4 > PdL 3 , showed that the complexes with electronwithdrawing substituents on the salicylaldehyde were more stable than the other complexes.
In the TGA curve of the PtL 1 complex proceeded with a single degradation step. The onset degradation temperature begins at 422.9ºC with an estimated mass loss of 22.03% (Calc.=21.82%), accompanied by an exothermic peak with t max =432.9ºC on the DSC curve which reasonably accounted for the combined loss of C 6 H 4 and 2HCN groups. The thermogram of the PtL 2 complex showed a mass loss of 13.18% (Calc.=12.60%) at the onset degradation temperature of 415.4ºC and an exothermic DSC peak in this region, t max =430.8ºC, which also corresponds the loss of C 6 H 4 . The thermogram of the PtL 3 complex shows an estimated mass loss of 17.24% (Calc.=17.08%) with a corresponding exothermic peak with t max =423.3ºC on the DSC curve which could be attributed to the loss of C 6 H 4 and HCN. The TGA curve of the PtL 4 complex showed single-step decomposition. The onset decomposition temperature begins at 413.2ºC with an estimated mass loss of 13.86% (Calc.=13.61%), attributed to the loss of C 6 H 4 . An exothermic peak with t max =418.1ºC on the DSC curve was observed for this step. The order of thermal stability of the Pt(IV) complexes, PtL 2 > PtL 1 > PtL 4 > PtL 3 , corresponds to the reverse order for that of the free ligands, thus coordination seemed to have stabilized the ligands.

Biological Properties
Antibacterial activity of the ligands and complexes were tested in vitro against 10 human pathogenic bacteria. The activities were compared with that of chloramphenicol, a standard broad-spectrum antibiotic for bacterial strains. The compounds were tested at a concentration of 5 mg cm -1 in DMSO using the paper disc diffusion method. The growth inhibition zones were measured in diameter (mm) and the results are reproduced in Table 5. The susceptibility zones measured were clear zones around the discs killing the bacteria. All the Schiff bases and their complexes exhibit varying degrees of inhibitory effects on the growth of the tested bacterial species.
H 2 L 1 and H 2 L 2 were effective against all the bacteria, with inhibitory zones of 8-12 and 9-14 mm, respectively. H 2 L 3 was effective against eight organisms, with inhibitory zones of 8-10 mm, whereas H 2 L 4 was effective against five organisms with inhibitory zones of 9-10 mm. The Pd(II) and Pt(IV) complexes of all the Schiff bases were highly effective against all the bacterial strains with zones of inhibition of 10-24 mm. The increased activity of the metal complexes can be explained on the basis of Overtones concept (Dharmaraj et al., 2001) and Tweedy's chelation theory (Tweedy, 1964). Metal complexes also disturb the respiration process of the cell and thus block the synthesis of proteins, which restricts further growth of the organism (Dharmaraj et al., 2001). A general observation is that the Pt(IV) complexes were more active than the Pd(II) complexes while the standard drug, chloramphenicol, exhibited higher antimicrobial activity than the Schiff bases and their metal complexes, with inhibitory zone of 18-30 mm.
MICs values of the 10 bacteria strains used in this study are presented in Table 6. Antibacterial MICs less than 1 mg cm -1 is considered to be an acceptable antibacterial activity (Aligiannis et al., 2001). When the Schiff bases and the corresponding complexes have the same MICs, the complexes are considered to be more active because of the lower molar concentration of the complexes due to higher molecular mass. The free ligands and metal complexes exhibit pronounced antibacterial activities against some bacterial strains, in a number of cases comparable or even higher with that of chloramphenicol. In general, the antibacterial results show that the majority of the metal complexes were more active than their respective Schiff bases. Chloramphenicol showed the lowest MICs value of less than 0.0098 mg cm -1 against eight bacterial strains used in this study and was more active than all the compounds tested when their MICs values are compared.

CONCLUSION
Four unsymmetrical tetradentate Schiff bases and their Pd(II) and Pt(IV) complexes were prepared, characterized and were screened in vitro against 10 human pathogenic bacteria. The free ligands and the metal complexes exhibit antibacterial activities against some strains of the microorganisms, which in a number of cases were comparable with that of chloramphenicol. In the future, this might open the door to their use as vital and effective antibiotics.