Synthesis, Characterization, and Antibacterial Studies of Pd(II) and Pt(II) Complexes of Some Diaminopyrimidine Derivatives

Pd(II) and Pt(II) complexes of trimethoprim and pyrimethamine were synthesized and characterized by elemental analysis, UV-Vis, FTIR, and NMR spectroscopy. The complexes are formulated as four coordinate square planar species containing two molecules of the drugs and two chloride or thiocyanate ions. The coordination of the metal ions to the pyrimidine nitrogen atom of the drugs was confirmed by spectroscopic analyses. The complexes were screened for their antibacterial activities against eight bacterial isolates. They showed varied activities with the active metal complexes showing more enhanced inhibition than either trimethoprim or pyrimethamine. The Pd(II) complexes of pyrimethamine showed unique inhibitory activities against P. aeruginosa and B. pumilus, and none of the other complexes or the drugs showed any activity against these bacteria isolates. The MIC and MBC determinations revealed that these Pd(II) complexes are the most active. Structure activity relationship showed that Pt(II) complexes containing chloride ions are more active, while for Pd(II) complexes containing thiocyanate ions showed more enhanced activity than those containing chloride ions.


Introduction
The discovery of potent group of pyrimidines with pronounced antagonistic effect on folic acid in cultures of Lactobacilli [1] led to the development of pyrimethamine and trimethoprim. Pyrimethamine was developed through brilliant feet of organic synthesis guided by biochemical considerations [2]. Additional modifications led to the synthesis of trimethoprim that inhibits bacterial dihydrofolate reductase like other diaminopyrimidines and its consequence selection as antibacterial agent [3][4][5]. Trimethoprim is a broad-spectrum antimicrobial and also exhibits antiparasitic activities [6]. Due to intensive use and misuse, resistance has emerged against trimethoprim [7].

Materials and Physical
Measurements. All reagents and solvents were of analytical grade and used without further purification. Elemental analyses were carried out on a Perkin-Elmer elemental analyzer. Melting point determination was obtained with the Gallenkamp melting point apparatus. Molar conductivity measurement (10 −3 M solutions in dimethylformamide) was obtained on the CON 6/TDS 6 conductivity/TDS Meter. FTIR spectra of the complexes were recorded as KBr pellets on a Perkin-Elmer paragon 2000 FT-IR spectrophotometer in the range 4000-370 cm −1 .

Synthesis of Metal Complexes of the Type [M(L) 2 Cl 2 ].
A solution containing 1 mmol of the respective metal salts (PtCl 2 (COD) 2 , 0.260 g) and (PdCl 2 (CH 3 CN) 2 , 0.374 g) was added to colorless solutions of trimethoprim (2 mmol, 0.508 g) or pyrimethamine (2 mmol, 0.497 g) in 50 mL of methanol. The mixture was refluxed for 4 h and cooled to room temperature, and the solvent was removed in vacuo.
The solid product was dried over CaCl 2 .

Synthesis of Metal Complexes of the Type [M(L) 2 (NCS) 2 ].
A solution containing 1 mmol of the respective metal salts (PtCl 2 (COD) 2 , 0.260 g) and (PdCl 2 (CH 3 CN) 2 , 0.374 g) was added to colorless solutions of trimethoprim (2 mmol, 0.508 g) or pyrimethamine (2 mmol, 0.497 g) in 50 mL of methanol. The mixture was refluxed for 1 h, followed by the addition of a colourless solution of NH 4 NCS (2 mmol, 0.152 g) in methanol and refluxed further for 3 h and cooled to room temperature, and the solvent was removed in vacuo. The solid product was dried over CaCl 2 .

Antibacterial Studies.
The antimicrobial activity of the synthesized compounds as well as their free ligands was studied by the zone of inhibition technique [47,48] [49,50] was used to determine the MIC. The MIC was taken as the lowest concentration of the tested complexes that shows no visible bacterial growth [51]. Samples of organisms were taken from plates which were used for the MIC test that were with no visible growth and subcultured by way of streaking onto a freshly prepared Mueller Hinton agar medium. MBC was carried out with the method of Olorundare et al. [52] and was taken as the lowest concentration of antibiotic at which all bacteria are killed. These plates were incubated at 35-37 ∘ C for 24 h and results taken as the MBC.

Results and Discussion
Pd(II) and Pt(II) complexes, trimethoprim and pyrimethamine, have been synthesized and characterized by elemental analysis, UV-Vis, FTIR, and 1 H and 13 C-nmr spectroscopy. Conductivity measurements on the complexes showed that they are nonelectrolyte in solution. Generally all the complexes are insoluble both in polar and nonpolar solvents except polar coordinating solvents such as DMSO and DMF. The analytical data for the complexes are presented in Table 1 and proposed structures in Figure 1.

Infrared Spectra.
The FTIR spectra of the ligands and metal complexes were compared and assigned on careful comparison. The N-H stretching frequencies of the pyrimidine NH 2 in the free trimethoprim shifted slightly in the metal complexes. It was observed in the same region, 3332-3461 cm −1 , as in the free ligands. The slight shift is ascribed to hydrogen bonding and other noncovalent interactions in the metal complexes. The coordination of the metal ions to trimethoprim affected the v(C=N) stretching vibrations. The v(C=N) that occur at 1635 cm −1 in the free trimethoprim ligand shifted to lower frequencies in all the complexes confirming that the metal ions are coordinated directly to the pyrimidine nitrogen atom. Strong vibrations at 2111 and 2120 cm −1 in [Pd(tmp) 2 (NCS) 2 ] and [Pt(tmp) 2 (NCS) 2 ], respectively, are due to v(NCS) stretching vibrations and may be attributed to the presence of the thiocyanate ion in the coordination sphere of these complexes [53]. The band observed in the complexes in the region 542−502 cm −1 was attributed to v(Pd-N) and v(Pt-N) [54]. Pyrimethamine possesses four potential coordination sites. A comparison of the spectra of pyrimethamine and the metal complexes showed that the bands due to symmetrical and asymmetrical stretching modes of NH 2 in the spectrum of pyrimethamine undergo only very slight changes in the complexes. This indicates that the metal ions bond preferentially to pyrimethamine through the nitrogen atom of pyrimidine. The absorption band at 1629 cm −1 in the spectrum of the pyrimethamine is attributed to the v(C=N) of the pyrimidine ring. It shifted to 1612, 1619, 1639, and 1632 , respectively, which is a good indication that pyrimethamine is coordinated to Pd(II) and Pt(II) ions through the N(1) atom of the pyrimidine ring. The appearance of a prominent absorption bands observed at 2112 and 2107 cm −1 in the complexes [Pd(pyrm) 2 (NCS) 2 ] and [Pt(pyrm) 2 (NCS) 2 ], but absent in the free ligand one has is due to v(NCS) stretching frequency of the thiocyanate ion [55].  [57]. All the four complexes display high energy absorption band around 300 nm which can be attributed to typical charge transfer transitions in the complexes [56,57] confirming the square planar geometries proposed for the metal complexes. The aliphatic region showed multiple signals between 2.20 and 1.00 ppm which support the presence of an ethyl group of the pyrimethamine ligand, integrating for five protons. In [Pt(pyrm) 2 (NCS) 2 ], the chemical shift was observed at (ppm) 7.53 (s, 2H, H-5b, 3b), 7.28 (s, 1H, H-6b), and 6.98 (s, 1H, H-2b). Once again, the aliphatic region showed multiple signals between 2.20 and 1.00 ppm which support the presence of an ethyl group integrating for five protons. The two amino groups of pyrimethamine exhibit characteristic shifts which can be seen as a broad peak at chemical shift value at around 4.00 ppm in both complexes.

NMR
The 13 C-nmr spectra of the metal complexes of trimethoprim were compared with that of free trimethoprim ligand [61] with a significant shift in the area that has been affected by the coordination to the metal ions. The signal at 155.91 ppm in the ligand has been shifted upfield to a value of 153.60 ppm in [Pt(tmp) 2 Cl 2 ] assigned to C(2a, C6a), both of C=N carbon atom of the pyrimidine ring of trimethoprim. Other 13 Cnmr signal of trimethoprim were observed at 136.69 ppm assigned to the quaternary carbon C(5a, C5b), 106.59 ppm C(1b, 5b), 60.99 ppm assigned to C7a, the peaks at 56.64 and 32.99 ppm assigned to C(2b, 3b, 4b) and CH 3  The 13 C-nmr for both platinum complexes of pyrimethamine gave more information in assigning the necessary signals and affirming the probable structure of the complexes. In the spectra of [Pt(pyrm) 2 Cl 2 ], the singlet resonance at 163.83 is assigned to carbon atom of C=N of the pyrimidine ring; there was a significant upfield shift which is probably due to the effect of coordination to the Pt(II) ion. The peak at 159.43 ppm was assigned to C5a. The phenyl ring carbon C1b-C6b can be seen in the range of 133.58-107.10 ppm. The aliphatic region consists of two peaks at 26.44 and 13.59 ppm assigned to the methylene and the methyl groups, respectively, from the ethyl of the pyrimethamine ligand. [Pt(pyrm) 2 (NCS) 2 ] show a similar trend in the 13 C-nmr spectrum; the singlet peak at 164.34 ppm was assigned to C(2a) of the pyrimethamine. The thiocyanate carbon is observed at 134.02 ppm [62,63]. The carbons of the pyrimethamine phenyl ring were found in the range of 133.34-107.65 ppm. The aliphatic region consists of complex peaks at between 29.93 and 25.62 ppm, indicative of the presence of a methylene group.

Antibacterial Screening of the Metal Complexes.
The complexes showed varied antibacterial activities against both gram-positive and gram-negative bacterial isolates ( Table 2). The highest zone of inhibition of 34 mm was recorded for [Pd(tmp) 2 Cl 2 ] B. cereus. All Pd and Pt complexes of trimethoprim are active against E. coli and P. vulgaris. Their zones of inhibition varied between 28 and 32 mm as against 16 mm shown by trimethoprim drug. Trimethoprim and all its complexes are inactive against P. aeruginosa and B. pumilus. Of all the trimethoprim complexes, [Pd(tmp) 2 (NCS) 2 ] appear to be the least active, inhibiting only E. coli, S. faecalis, and P. vulgaris. It must also be noted that, it might be least active but it is the only complex of trimethoprim that shows a zone of inhibition of 27 mm against S. faecalis whereas the other three complexes did not show any visible inhibition. For the trimethoprim complexes, the structure activity relationship showed that both Pd(II) and Pt(II) complexes containing chloride ions are the most active. They are active against two gram positive bacteria and three gram negative bacteria isolates with the Pt complexes, [Pt(tmp) 2 Cl 2 ], showing relatively   . It must be noted however that high zone of inhibition may not reveal for certain that a particular compound is a stronger antibacterial agent since this may be attributed to factors such as the rate of diffusion of the antibacterial agents and the amount of bacterial isolates present in a certain amount of agar solution [64]. The inhibition of the bacteria isolates by the pyrimethamine complexes are relatively fewer than those of complexes of trimethoprim (Table 2). However, the results showed that Pd(II) complexes of pyrimethamine, [Pd(pyrm) 2 Cl 2 ] and [Pd(pyrm) 2 (NCS) 2 ], have zones of inhibition of 30 and 33 mm against P. aeruginosa and 14 and 33 mm against B. pumilus. None of the complexes of trimethoprim and neither trimethoprim nor pyrimethamine showed any visible activity against these bacteria isolates. The highest zones of inhibition of 33 mm were observed for [Pd(pyrm) 2 (NCS) 2 ] against P. aeruginosa and K. pneumonia. Pyrimethamine inhibited the growth of six bacteria isolates whereas three of its metal complexes inhibited four bacteria isolates while [Pt(pyrm) 2 Cl 2 ] inhibited only three bacteria isolates. In the trimethoprim metal complexes, the complexes containing the chloride ions are generally showed better antibacterial activities than those with NCS ions. In the pyrimethamine metal complexes, the NCS ion is the anion of choice for enhanced activity. The minimum inhibition concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the compounds were evaluated and presented in Tables  3 and 4, respectively. It shows that the Pd(II) complexes of pyrimethamine are more active than the Pt(II) complexes of either trimethoprim or pyrimethamine.

Conclusions
We report the synthesis, characterization, and antibacterial studies of Pd(II) and Pt(II) complexes of trimethoprim and pyrimethamine. The complexes formulated as four coordinate square planar species consisting of two molecules of either trimethoprim or pyrimethamine and two chloride or thiocyanate ions. The complexes were characterized by elemental analysis, electronic, FTIR, and NMR spectroscopy. Spectroscopic analyses confirmed the coordination of the metal ions to the drug through the pyrimidine nitrogen atom. The antibacterial screening of the complexes showed 6 Bioinorganic Chemistry and Applications