Synthesis and Characterization of Platinum ( II ) Complexes from Trifluoromethyl Phenylenediamine , Picoline and N-Benzyl Ethylenediamine Derivatives

Este trabalho descreve a síntese e a caracterização de onze novos complexos de platina(II), contendo como ligantes derivados da 1,2-fenilenodiamina, da N-benzil etilenodiamina, e da 2ou 4-picolina. Os complexos foram preparados em rendimentos satisfatórios pela reação dos ligantes com K 2 [PtCl 4 ]. É descrita também a atividade citotóxica de um dos complexos de platina(II), em sete linhagens de células tumorais de origem humana, que se mostrou muito menos ativo do que a cisplatina.


Introduction
The discovery of the anticancer activity of the coordination compound cis-diaminedichloroplatinum(II) (cisplatin) 1 and its successful clinical use represent important advances for inorganic medicinal chemistry.6][7] Thus, thousands of platinum complexes have been synthesized, aiming to find drugs that have a broader spectrum of activity than that of cisplatin, and reduced side effects.The majority of them followed the set of structureactivity relationships summarized by Cleare and Hoeschele, 8,9 which states that the main condition for a platinum complex to show antitumour activity is that it should have a cis geometry with the general formula cis-[PtX 2 (Am) 2 ], where X is the leaving group (Cl is the most frequently used) and Am is an inert amine (mono or bidentate) with at least one N-H moiety.
According to Bednarski, 10 the "chelate effect" is another important point that must be considered in the synthesis of platinum complexes that contain diamine and related compounds as non-leaving linkers.In the organism, cisplatin can react with some biological molecules such as glutathione, leading to the replacement of the amine group, which could result in inactivity of the complex.For platinum(II) complexes with diamine ligands, this reaction would be less favored due to their higher stability as compared to monodentate nitrogen ligands.][13][14] Thus, Connors et al. 11 have prepared different platinum(II) complexes which contains aromatic amines, such as 1,2phenylenediamine and its derivatives, having different groups in the 4 position, such as chloro, methoxy, nitro and methyl.Later, Meischen et al. 15 reported the antineoplastic activity of a large number of complexes, such as: cis-dichloro (4chloro-1,2-phenylenediamine)platinum(II) (Figure 1, b) and cis-dichloro (1,2-phenylenediamine)platinum(II) (Figure 1, a).Although these compounds were less active than cisplatin, they have shown relevant biological activity against the L1210 leukemic cell line.
It is also important to emphasize that several compounds containing the pyridine ring in their structure are quite important in the medical and biological fields.As examples, one can mention vitamin B6 (pyridoxine), nicotine and chlorpheniramine.Some pyridines can act as painkillers, anti-hystaminic and heart stimulants.They also can show antineoplastic activity in vitro in the cells of skin carcinoma. 16eischen et al. 15 have described the synthesis of many platinum(II) complexes which contain pyridine and picoline derivatives as ligands that showed antineoplasic activity in leukemia lymphoma.
Considering that complexes having diamines as chelated ligands have shown activity against several types of tumors 17,18 and that many aromatic compounds have demonstrated the ability to intercalate between DNA bases, 19 we have recently reported the synthesis of a series of platinum(II) complexes with ethylenediamine derivatives 20 and 1,3-propanediamine derivatives. 21In this paper we report the preparation and characterization of cisplatin analogs having 1,2-diamines or 1,2-aminethiol as ligands, derivatives of 1,2-phenylenediamine, N-benzyl ethylenediamine and picoline.

Results and Discussion
Proligand 1 (Scheme 1) is commercially available and was purified before use by column chromatography (eluent: hexane/ethyl acetate).
Proligands 6, 7 and 8 (Scheme 2) were prepared by treating ethylenediamine with the corresponding benzyl halide in ethanol at room temperature.In the synthesis of these proligands, we have observed the formation of N,N´dibenzylated compounds as well.Thus, during the purification process of proligand 7, we have also isolated the corresponding N,N´-di- (2-nitrobenzyl) All the prepared proligands are new compounds and were purified by column chromatography (eluent: dichoromethane/methanol) with satisfactory yields.They were characterized by analytical and spectroscopic methods (see experimental).The IR spectra of all the proligands showed the characteristic absorptions.For instance, compound 6 shows ν N-H at 3218 cm -1 , ν C-H(aromatic) at 3077 cm -1 and ν C-H(aliphatic) at 2936 cm -1 .In addition to these absorptions, the IR spectrum of proligand 1 showed ν C-F at 1332 cm -1 , the IR spectrum of compounds 6, 7 and 16 showed ν N-O at 1517 and 1346 cm -1 ; for compound 8 one can observe ν C=O at 1728cm -1 .Compounds 19, 20, 24 and 25 showed ν C=N at 1625 cm -1 and ν C-S at 632 cm -1 (for compounds 20 and 25).
In the 1 H NMR spectra, signals were observed in the δ 2.8 -4.0 and 7.0 -7.8, region corresponding to the methylene groups and aromatic hydrogens, respectively.For compound 8, a signal at δ 3.8 attributable to the OCH 3 was also observed.
In the 13 C NMR spectra, signals at the δ 35 -50 region corresponding to the methylenic carbons and signals in the δ 115 to 160 region, due to the aromatic carbons, were observed.For compound 8, the signals corresponding to OCH 3 and C=O were observed at δ 52.1 and 166.2, respectively.
The dichloroplatinum(II) complexes (Schemes 2 and 3) were synthesized by adding the corresponding proligands, previously dissolved in water or methanol to a solution of K 2 [PtCl 4 ] in water at room temperature, and were isolated by simple filtration.
In addition to the absorptions observed for the ligands, the IR spectra of the complexes exhibited absorptions corresponding to ν Pt-N and ν Pt-Cl in the 450 and 320 cm -1 region, respectively.For all complexes, the 1 H NMR spectra showed resonances between δ 6.5-9.3 corresponding to the aromatic hydrogens, and large singlets between δ 5.2-8.5 corresponding to the NH 2 group.Compound 11 also showed a signal at δ 3.7 attribuited to the OCH 3 group.In the 13 C NMR, signals between δ 113.9-166.6 corresponding to the aromatic carbons were observed.For complex 11, one signal at δ 52.2 showed the presence of the methoxy group.The 195 Pt NMR spectra for complex 2 showed only one signal at δ -2194.This chemical shift value can be expected based on data for similar compounds described in the literature.For instance, the spectrum of [Pt(DACH)Cl 2 ] shows a signal at δ -2287 (DACH=1,2-diaminocyclohexane). 22The results of elemental analyses for the complexes are in agreement with the calculated values.
The ID 50 values clearly show that compound 2 is less active in vitro than cisplatin, and much less active than doxorubicin, for instance (Table 1).This result suggests that the presence of the strong electron-withdrawing group in the aromatic ring lead to a decrease in the cytotoxic activity.

Experimental
IR spectra were obtained on a Bomem FT IR MB-102 spectrometer from KBr pellets. 1 H NMR (200 and 400 MHz), 13   C NMR (50 and 100 MHz) and 195 Pt NMR (86 MHz) spectra were recorded on a Bruker Avance DRX 200 and DRX 400 spectrometers at the Federal University of Minas Gerais.Elemental analyses were done at the Mycroanalyses Laboratory at ICNS/CNRS, Gif-sur-Yvette, France and at Central Analítica, USP-Brazil.The progress of all reactions was monitored by thin-layer chromatography which was performed on 2.0 × 6.0 cm aluminium sheets precoated with silica gel 60 (HF-254, Merck) to a thickness of 0.25 mm.The developed chromatograms were viewed under an ultraviolet light.For column chromatography Merck silica gel (70-230 mesh) was used.Solvents were purchased from Vetec Química and were distilled before use.Reagents were purchased from Aldrich and used without further purification.
Test and reference compounds were dissolved to a concentration of 250,000 ng mL -1 in full medium, by 20fold dilution of a stock solution containing 1 mg of compound per 200 μL aqueous solutions containing 1% of dimethylsulfoxide (DMSO) or ethanol using a literature procedure, 23,24 by the microculture sulforhodamine B (SRB) test.The experiment was started on day 0. On day 0, 150 μL of trypsinized tumour cells (1,500-2,000 cells/ well) were plated in 96-wells flat-bottommicrotitre plates (Falcon 3072, BD).The plates were preincubated for 48 h at 37 °C, 8.5% CO 2 , to allow the cells to adhere.On day 2, a threefold dilution sequence of ten steps was made in full medium, starting from the stock solution.Every dilution was used in quadruplicate by adding 50 μL to a column of four wells.On day 7, the incubation was terminated by washing the plate twice with phosphatebuffered saline (PBS).Subsequently, the cells were fixed with 10% trichloroacetic acid in PBS and placed at 4 °C for 1 h.After five washings with tap water, the cells were stained for at least 15 min with 0.4% SRB dissolved in 1% acetic acid.After staining, the cells were washed with 1% acetic acid to remove the unbound stain.The plates were air-dried and the bound stain was dissolved in 150 μL 10 mmol L -1 Tris-base.The absorbance was read at 540 nm using an automated microplate reader (Labsystems Multickan MS).Data were used for the construction of concentration-response curves and determination of the ID50 value by using the Deltasoft 3 software.

Conclusions
This work describes the synthesis and characterization of ten new diamine/ aminethiol proligands and their platinum(II) complexes.These complexes have potential for acting as cytotoxic agents, as was demonstrated by preliminary tests.Further investigation of the biological properties of these compounds is desirable to determine their possible utility as anticancer agents.

Table 1 .
Inhibition doses ID 50 (ng mL -1 ) (doses that reduce by 50% the growth of the cell lines) for compound 2 and other antitumoral drugs g; 81%).