Synthesis, Characterization and Antitumor Activity of cis-bis(acylthioureato) platinum(II) Complexes, cis-[PtL2] [HL1=N,N-Diphenyl-N'-Benzoylthiourea or HL2=N,N-diphenyl-N'-(p-nitrobenzoyl)thiourea]

A low-molecular weight chromium-containing fraction of the material resulting from dichromate reduction by bovine liver homogenate was investigated by NMR and ES-MS. The ES-MS spectrum showed a readily detectable peak at m/z 786.1. The same molecular weight reasonably agreed with the relatively low diffusion coefficient measured by NMR-DOSY experiments on the main species observed in the 1H NMR spectrum. At least two downfield shifted and broad paramagnetic signals were apparent in the 1H NMR spectrum. Temperature dependence of chemical shift was exploited in order to estimate the diamagnetic shift of the signals in the diamagnetic region of the spectrum. 2D TOCSY, NOESY, COSY and 1H-13C HMQC spectra revealed the presence of aromatic protons (which were assigned as His residues), Gly and some other short chain amino-acids. Combinations of the molecular masses of such components together with acetate (which is present in the solution) and chromium atoms allowed a tentative proposal of a model for the compound.


INTRODUCTION
The transition metal complexes perform an important role in Medicinal Inorganic Chemistry due to their pharmacological properties in survival systems. Cis-diaminedichloroplatinum(ll) (cisplatin) is an antineoplastic agent of clinical use, highly effective in treating testicular and ovarian cancers /1/. The biological efficiency of cisplatin and its analogues, carboplatin and oxaliplatin/2,3/, as antitumoral drugs is due to the formation of coordination complexes with nuclear DNA, mainly to form Pt-N7 adducts with two adjacent guanine bases (G-G) on the same nucleotide strand/4/. These conformational changes within the DNA double helix originate the inhibition of DNA replication and RNA transcription and produce finally the cell death/5-8/. Despite its efficacy in various neoplastic diseases, cisplatin has several disadvantages due to its propensity to tumor resistance and by causing several types of dose-limiting toxicity, such as, nephrotoxicity, nausea, neurotoxicity and myelosuppression/9,10/. With the possibility of exploring a new class of anticancer agents, Bierbach et  and a chloride by one monodentate thiourea derivative ligand (L), which coordinates to platinum through a sulfur atom. The cytotoxic activity presented by these complexes was reflected by low values of IC50 for HL- 60  As a part of our continuing efforts for the synthesis and development of new platinum(If) antineoplastic agents with low toxicity and therapeutic improved action, we have prepared platinum(ll) bis-chelate complexes, cis-[PtLz], with acylthiourea ligands, R'-C(O)NHC(S)NRz for their cytotoxic evaluation as possible antitumor agents. In this work, we inform of the synthesis and characterization of novel platinum (II) complexes with the ligands N,N-diphenyl-N'-benzoylthiourea (HL 1) and N,N-diphenyl-N'-(p-nitrobenzoyl) thiourea (HL-) and their in vitro cytotoxic effect on mouse mammary adenocarcinoma TA3. and solvents (Aldrich) were analytical grade and used as supplied, except the acetone which were distilled before use. Elemental analysis were determined on a Fisons-Carlo Erba 1108 elemental microanalyser.
Melting points were determined on a Boetius melting-point apparatus. The infrared (IR) spectra were recorded in solid state (KBr pellets) on a Bruker FT-IR IFS 55 Equinox spectrophotometer in the range 4000 400 cm -The H (300 MHz) and C (75.5 MHz) NMR spectra were recorded on a Bruker advance DRX 300 spectrometer.95pt -NMR spectra were recorded at 86 MHz on a Bruker DRX 400 spectrometer at 300 K using CDCl3 as solvent. The chemical shifts (5) were measured relative to TMS in the case H and 3 C, and to NazPtCI6 (6 (95 Pt) 0 ppm) in the case of 9 Pt-NMR spectra. FAB(+) mass spectra were obtained on a ZAB-HSQ (V.G. Analytical Ltd.) spectrometer.

Synthesis of ligands
The ligands N,N-diphenyl-N'-benzoylthiourea (HL1) and N,N-diphenyl-N'-(p-nitrobenzoyl) thiourea (HL2) were prepared according to the reported methods/20,21/as shown in Scheme 1. For the synthesis of HL2, the used solvent was acetone instead of acetonitrile. To a solution of HL (0.17g, 0.50 retool) in dioxane (40 mL) was added dropwise a solution of KePtCI4 (0.10 g, 0.25 retool) in water (20 mL), followed by sodium acetate (0.041 g, 0.5 retool) in water (2 mL), and stirred for 2 h at 60C The reaction mixture was then stirred for 24 h at room temperature. The yellow precipitate was collected by filtration, washed several times with small portions of water, cold ethanol and dried in vacuo. Recrystallization of the yellow solid from hot dichloromethane gave small pale yellow crystals which were characterized by X-ray crystallography. Yield: 0.124 g (58.0%), m.p. 255-258 C (dec.).

Crystal structure determinations
All single-crystal X-ray measurements were carried out on a CCD SMART APEX diffractometer equipped with a graphite monochromator using MoK radiation (k =0.71073 ). Data were collected at room temperature. The structures were solved by direct methods and refined against F (all data, anisotropic thermal parameters for all non-H atoms, all H atoms located and fully refined with isotropic thermal parameters) using the SHELXS-97 and SHELXL-97 programs/22,23/. Crystal data collection and refinement details for ligand I-IL and cis-[Pt(L )2] complex are summarized in Table 1.  In the IR spectra, the N-H stretching vibrations assigned at 3150-3220 cm for ligands HL and HL disappeared upon complexation due to the deprotonation of (NHCO) amide group in the ligands. The strong absorption bands 1692-1696 cmand 1231-1247 cm corresponding to the vibrations v(C=O and v(C=S), respectively, are shifted to lower frequencies 1640-1650 cm and 1218-1220 cn respectively, upon coordination, which proves that the deprotonated ligands are coordinated to platinum(ll) ion through oxygen and sulfur donor atoms/25-27/.
In the H-NMR spectra, the deprotonation of the ligands in the complexes is confirmed by the disappearance of the N-H signal present in the ligands HL and HL in the range of 8.67-8.69 ppm. For the ligand HL2, the aromatic protons signals were affected by the presence of nitro substituent group in the para position of benzoyl moiety. These signals are shifted to downfield for the protons in the meta ( 0.72 ppm) and ortho (0.17 ppm) positions relative to unsubstituted benzoyl moiety of ligand HLJ.     ICs0 corresponds to the concentration required to inhibit 50 % of the culture growth when cells are exposed to compounds for 48 h. Each value is the mean +/-SD of three independent experiments with each assay performed in triplicate.
In summary, we have prepared the bis-chelate complexes cis-[Pt(Ll)2l and cis-[Pt(L2)2], which are more cytotoxic on the TA3 tumor cell line at micromolar concentration compared to acylthiourea ligands, HL and HL Moreover, it was demonstrated that the participation of the nitro substituent group in the para position of benzoyl moiety in HL slightly increases its cytotoxic activity with respect to ligand HL , whereas, in comparison to complex activities, this substitution is irrelevant. These results could represent an important contribution to establish some mechanism of action of bis-chelate complexes, cis-[PtL2] (not analogous to cisplatin) related to cellular DNA.

SUPPLEMENTARY MATERIAL
Crystallographic data for the structure analysis have been deposited with the Cambridge Crystallographic Data Center as supplementary publication numbers CCDC 214453 and 214454. Copies of the data can be obtained free of charge from the Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk).