Pd(II) and Pt(II) Complexes of Schiff Thiobases Derived From 2-Carbonylpyridine

Pd(ll) and Pt(ll) complexes of three series of Schiff thiobases derived from 2- carbonylpyridine have been synthesized and characterized. The crystal structure of the Pt(ll) derivative of methyl-3-(2-pyridylmethylene)hydrazinecarbodithioate (HFp) was resolved. The ligand coordinates the platinum ion in tridentate fashion by heterocycle and imine nitrogen and thiocarbonyl sulfur. The fourth ligand is a chloride ion. The structure of the complexes is suitable for the formation of monofunctional adducts with DNA. Studies on the interaction of the complexes with Calf thymus DNA by CD reveal modifications in the B form of lineal DNA. Interaction with plasmid DNA was also confirmed in the images obtained by atomic force microscopy.


INTRODUCTION
In recent decades many complexes of transition metal containing an S donor atom have been described as antimalarial, antileukemic or antiviral agents-7. The pyridine derivatives of thiosemicarbazones have been widely studied. Copper(ll), zinc(ll), cadmium(ll), nickel(ll), iron(Ill), manganese(ll) and cobalt(Ill) were described as possible enhancers of the antileukemic properties by coordination to ligands of this kind8-19. Palladium and platinum complexes of dithiocarbazic acid and its derivatives were described20"22. Recently, the crystal structures of palladium(ll) complexes of 2-acetylpyridine N(4)-dimethylthiosemicarbazone have been reported23-24. The presence of only one halide ion in the coordination sphere of the metal enables the molecule to form monofunctional adducts with nucleotides or DNA and thus can explain the possible antitumour or antileukemic activity of the analogous platinum compounds. We have synthesized, characterized and studied the interaction with Calf thymus DNA and plasmid pBR322 DNA of three series of Pd(ll) and Pt(ll) compounds with the Schiff bases HAp (the 2-acetylpyridine Schiff base of Smethyldithiocarbazate), HBp (the 2-benzoylpyridine Schiff base of S-methyldithiocarbazate) and HFp (the 2-formylpyridine Schiff base of S-methyldithiocarbazate). The resolution of the crystal structure of one of the Pt(ll) compounds, the 2-formylpyridine Schiff base of Smethyldithiocarbazate, has confirmed that the molecular structure is analogous to that of palladium complexes reported in the literature.

EXPERIMENTAL Materials and Methods
The complexes were prepared using Johnson Matthey K2[PdCI4] and K2[PtCI4], acetyl, 2-benzoyl and 2-formylpyridine and Carlo Erba CS2 and CH31. Elemental analyses were carried out on a Carlo Erba 1500 microanalyzer at the Serveis Cientffico-Tcnics at the University of Bamelona. The infrared spectra were recorded in solid state (KBr pellets) on an FT-IR Nicolet 5DZ spectrometer in the 4000-400 cm -1 range. 1H{13C}, 13C{1H} and 95pt{1H}NMR spectra were obtained on a Brucker DRX 250 spectrometer using DMSO-d6 as solvent. Chemical shifts were measured relative to TMS in the case of 1H and 13C and to Na2PtCI6 for 195pt. CD spectra were obtained on a JASCO J720 spectropolarimeter with a 450 W xenon lamp. Images of plasmid pBR322 DNA, and adducts of Pt compounds with this DNA were obtained with Extended Nanoscope III (Digital Instruments, Santa Barbara, CA) working in TMAFM mode in about 100 nN. The samples were prepared by incubating the complexes dissolved in a DMSO-Hepes (30:70) mixture with plasmid pBR322 DNA at 37C for 24 hour at several molar ratios complex/DNA (ri). A drop of 2 1 of DNA or DNA-metal complex solution was deposited onto freshly cleaved green mica (Ashville-Schoonmaker Mica Co., Newport New, VA). After adsorption for five minutes at room temperature, the samples were rinsed for ten seconds in a jet of deionized water of 2-Carbonylpyridine 18 MWcm -1 from a Milli-Q water purification system (Millipore, Molshem, France) directed onto the surface with a squeeze bottle. The samples were blow dried with compressed argon over silica gel for 30 minutes before imaging in the AFM.

Synthesis of the ligands
All the ligands were prepared by the same general method12: S-methyldithiocarbazate 22 (10 mmol) in ethanol (40 cm3) was mixed with a solution of 2-acetyl, 2-benzoyl or 2-formylpyridine in the same solvent. The reaction mixture was boiled on a waterbath and its volume was reduced to 30 cm3. The product that formed was collected and dried in vacuo over silica gel. The ligands synthesized were HAp (the 2-acetylpyridine Schiff base of S-methyldithiocarbazate), HBp (the 2benzoylpyridine Schiff base of S-methyldithiocarbazate) and HFp (the 2-formylpyridine Schiff base of S-methyldithiocarbazate).

Spectral studies
The main vibrational bands of the ligands and their complexes are reported in Table 3. The Schiff bases contain the thioamide group and consequently may participate in a thione-thiol tautomerism. The i.r. spectra of the ligands do not show the v(S-H) band at ca 2565 cm-1, but they do show the v(N-H) band at ca 3150 cm-1, indicating that, in the solid state, they are probably in the thione form. However, in the presence of the metal ion, the tautomerization to the thiol and deprotonation is enhanced, and the ligands coordinate in the deprotonated form, as do related thiosemicarbazone ligands. The i.r. spectra of the metal(ll) complexes do not contain either the v(N-H) or the v(S-H) bands, supporting the coordination with the singly negatively charged ligand.
An unambiguous assignment of the v(C=S) is only possible when this group is not linked to a N atom because of the vibrational coupling effects29. In the thioamide system, coupling can take place among C-N stretching, C=S stretching and NH deformation vibrations, and the C=S vibration is not localized. However three bands seem to appear in the regions 1395-1570, 1260-1420 and 940-1140 cm -1 due to the mixed vibrations. The shift of two of the thioamide bands and the disappearance of the third support the deprotonation during the coordination. This third band, the thioamide II, seems to have a significant contribution of the C=S stretching.
The azomethine bands in the i.r. spectra of the complexes appear in a range 1595-1602 cm-1, somewhat higher than those observed for the free ligands. The bands corresponding to the two possible N=C bonds are not resolved in the spectra of these complexes. Some related compounds show a positive shift30,31, but others show a negative shift32,33owing to the fact that they are combination bands, especially in pyridine compounds8. The coordination through the azomethine nitrogen atom is also consistent with the shift of the v(N-N) bands.
The presence of v(M-N) and v(M-S) bands also supports the coordination via the azomethine and pyridine nitrogen atom, and the mercaptide sulfur atom.
The proton NMR spectra of the Schiff bases in d-DMSO do not show any peak near 4 ppm attributable to an S-H proton, but they do show the peak of the N-H proton. In all cases these peaks appear downfield shifted (12-15 ppm) compared with a typical proton of a secondary amine (9 ppm), and they are closer to a protonated heterocyclic nitrogen. Two of the isomers of the ligands in solution show an interaction between the N-H proton and the pyridine nitrogen, although a similar shift could also be caused by an interaction of the H with DMSO. This behaviour has also been observed in similar thiosemicarbazones19. This peak disappears in the spectra of the complexes, which supports the coordination through the thiol form of the ligand. The 13C-NMR spectra of the complexes show an upfield shift of the C=S peak (C7), and downfield shifts of the C=N (azomethine carbon atom, C6) and of the C close to the pyridinic N (C1), according to the coordination through the NNS system. In all the compounds ds-DMSO was used as solvent, but the spectra of the complexes of the HAp ligand were not available due to insufficient solubility.
The 195pt-NMR spectra of the complexes PtBp and PtFp show a shift corresponding to a NNS coordination (again the PtAp spectrum was not available). The signals appear at -3207 and -3208 ppm referred to K2PtCI6.
The mass spectra of the complexes, registered in acetonitrile, show the same pattern in all cases, consistent with the monomeric formulation of the compounds. The strongest peak is due to the [ML(CH3CN)] + ion, in which the lost CI atom has been replaced by a molecule of the solvent. This substitution has been observed in many molecules34. A peak attributable to the [(ML)2(CI)] + ion has also been observed. In Figure 1 the molecular structure of the complex chloro(S-methyl-I-N-(2pyridyl)dithiocarbazate)platinum(ll) is shown. Bond distances and angles are collected in Table 6. The platinum atom is localized on a plane square. The thiocarbazate ligand coordinates in tridentate fashion to the pyridinic nitrogen N(1), the azomethinic nitrogen N(2) and the thiolate sulfur S(1). The fourth coordination site is occupied by a chlorine atom.
The Schiff base is an anion with a single negative charge, which allows tautomerism to the iminothiolate form, as has been described for the analogous thiocarbazate 14 and thiosemicarbazone 15 complexes. So, the negative charge generated in mercaptane deprotonation is delocalized in the C-N-N-C system when the ligand binds to the platinum atom. This is reflected in  Figure 2 shows the monoclinic cell with four molecules of the complex. These are parallel to their symmetry plane, but alternated in reference to the chlorine atoms.  The circular dichroism spectra of Calf Thymus DNA and Calf Thymus DNA incubated with the complexes at 37 C for 24 h with several molar ratios were recorded. The Omax and Omin for ,max and ,min values at different molar ratio ri, are collected in tables 7, 8 and 9. The ligands and the complexes showed a slight modification of the circular dichroism spectra. This behaviour has been observed in another compound with unique position of coordination, [Pt(dien)CI]CI35,36. However, the modification caused by HBp and its complexes is greater that the produced by HAp and HFp.  The CD spectrum of the adduct DNA:ligand shows a slight increase in the ellipticity of the positive band, similar to that of Pd-complex. This behaviour may be due to modifications in the base stacking, which produce an increase in the coiling of the double helix and stabilization of the B form of DNA.
However, the Pt-complex shows a different behaviour, decreasing the ellipticity of this band for the same values of ri. This effect may be due to an instabilization of the B form of DNA.
In the CD spectra of the HBp series slightly higher differences in the ellipticity values can be detected, probably due to the presence of an R group in the position in the pyridine ring of the HBp ligand. This group is larger than the methyl group of the HAp ligand or H of the HFp ligand and, due to its plane geometry, it can intercalate between the base-pairs of DNA causing a greater interaction. The behaviour observed for the ligand HBp and for its complexes is analogous to that of cisplatin. The ellipticity increases in the positive band for small values of ri and decreases when ri increases.
In this case, from the observation of the CD spectra, it can be deduced that the ligand HFp and its Pd complex show the same behaviour, stabilization of the B form of DNA, while the Pt complex causes an instabilization of this form. In conclusion, there is parallel in the interaction of HAp and HFp and their respective derivatives with DNA. The behaviour is similar to that of cisplatin. However, in the HBp series, the changes in the ellipticity of DNA are greater, probably due to the presence of the R group which could allow an additional interaction with the double helix.   Figure 3 shows the images corresponding to plasmid pBR322 (a), the sane DNA incubated for 24 h with PtFp(b) and incubated for 24 h with PtBp (c). The derivative Pt-Fp modifie,'; the relaxed form (.f the free plasmid. The closed circular form observed in (a) for the plasmid DNI\ adopts a supercoiled form, as can be observed in (b). The interaction of the derivative Pt-Bp is rnor{: dramatic, the plasmid DNA is converted into oblates. The effects of the free,-,platinurr liflrds ()n the plasrnid DNA in the same conditions are not significant.

2-Carbonylpyridine
[his is consistent with the results of CD for the same compounds,. In both cases the structure of the compounds allows monofunctional covalent interaction between a platinum atom 2-Carbonylpyridine and DNA, but in the case of the Pt-Bp complex the presence of the R group causes additional interaction and the plasmid DNA is strongly modified. Since both complexes have shown their reactivity against DNA, they are excellent candidates to be tested in antitumor and antiviral probes.