Novel Platinum ( II ) Complexes of Long Chain Aliphatic Diamine Ligands with Oxalato as the Leaving Group . Comparative Cytotoxic Activity Relative to Chloride Precursors

Os complexos de platina são importantes no desenvolvimento de drogas anticancerígenas. Sua citotoxicidade pode ser afetada pelos ligantes abandonadores, devido ao mecanismo da reação de hidrólise que ocorre antes da ligação do complexo ao DNA. Os grupos não-abandonadores, como as diaminas lipofílicas, também podem afetar a velocidade de entrada do composto na célula. Neste trabalho é descrita a síntese de novos complexos de platina(II) contendo ligantes oxalato e derivados de etilenodiamina N-substituída por uma cadeia carbônica longa de tamanho variável. Os produtos foram caracterizados por análise elementar, espectroscopia de absorção na região do infravermelho e ressonância magnética nuclear de H, C e Pt. A atividade biológica dos complexos foi investigada em linhagens de células tumorais (A 549 , B16-F1, B16-F10, MDAMB-231) e não-tumorais (BHK-21 e CHO). O tamanho da cadeia carbônica afetou a citotoxicidade e os complexos com oxalato mostraram-se menos citotóxicos do que os análogos com cloretos.


Introduction
][6] Carboplatin was designed to overcome the severe side effects of cisplatin, and, indeed, the replacement of the labile chloride ligands by a comparatively more stable bidentate O-O leaving group resulted in a modified pharmacodynamic behavior and a more tolerable toxicological profile. 7However, the cross-resistance between cisplatin and carboplatin makes them both ineffective in the treatment of patients who fail to respond to therapy.
Over the last decades, many other platinum drugs have been developed in an attempt to improve the toxicity profile and, in particular, to obtain a drug that is able to overcome resistance.One of these third-generation drugs is oxaliplatin, R,R-1,2-diaminocyclohexane(O-O-oxalato) platinum(II), which has shown a wide and markedly different spectrum of activity and, more importantly, no cross-resistance to cisplatin and carboplatin.The non-hydrolyzable dach ligand makes the complex less polar, which has been proposed to contribute to a better cellular uptake. 8,9Platinum-DNA adducts, which are formed following uptake of the drug into the nucleus of cells, activate several cellular processes that mediate the cytotoxicity of these platinum drugs. 10eaving groups, such as chloride in cisplatin and oxalato in oxaliplatin, can modify both the kinetics of hydrolysis and the reactivity of the drug.Non-leaving groups can confer different characteristics to platinum compounds, improving their cytotoxic activity by modifying cellular uptake and the way the complex interacts with DNA. 11mphiphilic diamines with a long, N-alkyl chain may present surfactant properties.This feature can increase the affinity of the compounds for the cell membrane and thus facilitate their cellular uptake. 12ur research group has already investigated platinum complexes derived from long chain aliphatic diamines.It has been shown that the length of the carbon chain affects cytotoxity and cellular uptake of the chloride complexes. 13he impact of a different leaving group (oxalato) on the cytotoxic activity of the products was investigated in the present work using several cell lines such as breast and lung cancer cells.The toxicity of the complexes was also assessed in non-tumor cell lines such as kidney cells (BHK-21), which allowed us to examine a possible decrease in nephrotoxicity.

Chemistry
The platinum complexes with chloride and ethylenediamine derivatives (1, 2, 3 and 4) were prepared by a procedure analogous to that reported earlier by our research group for the propanodiamine derivatives. 13,14he platinum complexes with oxalato (5, 6, 7 and 8), in their turn, were obtained from the chloride precursors (Scheme 1) using an extension of Dhara`s method. 15In this case, the reaction with silver nitrate was performed first and the resulting insoluble silver chloride was filtered off.Then, sodium oxalate was added to produce the desired complexes.Considering that the substituted nitrogen of the diamine is pro-chiral, the products are enantiomeric mixtures and no attempt was made to separate them.
For all complexes, the IR spectra (Table 1) show absorptions corresponding to n(N-H) at 3200-3100 cm -1 and n(C-H) around 2920 cm -1 .The IR spectra of the chloride compounds show absorptions corresponding to n(Pt-Cl) around 320 cm -1 , characteristic of cis platinum compounds.For the oxalato complexes, the disappearance of the absorption due to n(Pt-Cl) at 320 cm -1 is noticeable, as well as the appearance of absorptions for n(Pt-O) around 550 cm -1 (Figure 1).Other bands were recorded at 1670 and 810 cm -1 and were assigned to n(C=O) and n(C-C=O), respectively.
NMR spectra were acquired using dmso-d 6 as the solvent.The chemical shifts are shown in Table 2.In the 1 H NMR spectra of the diamines, signals around d 0.9 (CH 3 ) as a triplet, 1.2 (CH 2 ), 2.6 (CH 2 -NH) and 2.8 (CH 2 -NH 2 ) as multiplets were observed.In the d 1.4 to 2.5 region, the signals corresponding to the NH 2 and NH hydrogens could be seen as triplet resonances.In the 1 H NMR spectra of the complexes, a high frequency shift was observed for the signals relative to the NH and NH 2 hydrogens.They appeared as three broad signals in the d 5.2 to 6.2 region for the chloride complexes and 5.6 to 6.5 for the oxalato analogues.
No significant difference was observed when the The 195 Pt NMR spectra (Figure 2) for complexes 1-4 showed only one signal around d -2350, while for complexes 5-8 only one signal, around d -2050, was observed.7][18] As expected, a shift to high frequency was observed, caused by the replacement of the chloride by oxalato, which altered the coordination sphere around the platinum ion.
The results of elemental analysis obtained for the complexes are in good agreement with the calculated values.

Cytotoxic activity
To analyze the potential of the compounds as antitumor agents, their cytotoxicity was evaluated in comparison to cisplatin and carboplatin in a panel of four tumor cell lines: lung cancer (A 549 ), melanoma (B16-F1 and B16-F10) and breast adenocarcinoma (MDA-MB-231), Table 3.The complexes were also examined for their cytotoxic properties on two types of non-tumor cell lines, kidney (BHK-21) and ovarian (CHO).The IC 50 values, calculated from the dose survival curves obtained after 120 h of drug exposure (MTT test), are shown in Table 3. 19 We observed that, in all cell lines assessed, the cytotoxicity was influenced by the leaving group, with oxalato complexes showing decreased activity compared to chloride precursors.Especially with the B16-F10 cell line, IC 50 values vary from 6.3 to 3.6 µmol L -1 for chloride complexes (1-4) and from 38.0 to 16.6 µmol L -1 for the oxalato analogues (5-8).With the A 549 cell line, while the IC 50 value for chloride complexes is around 10 µmol L -1 , those for the oxalatos vary from 60.6 to 13.5 µmol L -1 .
The nature of the non-leaving group also affects cytotoxicity.The cytotoxic activity of the compounds in tumor cell lines exhibits an increasing trend with the increase of the carbon chain length when comparing complexes having 8, 10 and 12 carbons in the alkyl moiety.
The analogues containing 14 carbons show less or similar activity.This structure/activity relationship is confirmed by log P values for the corresponding chloride complexes (Table 3), which were determined from the partition coefficients in n-octanol/water.This parameter is used to determine the lipophilic character of drugs: hydrophobic compounds will have a high log P value whereas hydrophilic compounds will have it low.We have found that as log P increases IC 50 values decrease, i.e., increasing complex hydrophobicity increases cytotoxic activity. 20,21his correlation has been observed before by our group for similar platinum(II) complexes with ligands derived from 1,3-propanediamine; it can be explained based on the lipophilicity of the compounds. 13The more lipophilic the  compound is, the more rapidly it enters cells and the higher is its cytotoxic activity.The time the drug takes to enter the cells can explain why the chloride complexes were, in general, more cytotoxic than the corresponding oxalato complexes.Pereira-Maia and Garnier-Suillerot 22 have shown that carboplatin is less toxic than cisplatin because carboplatin enters cells at a slower rate, and that may be what is occurring for the complexes reported herein.As another possible explanation, the rate of hydrolysis of the leaving group may differ between the chloride and oxalato complexes.Hydrolysis of the oxalato complexes requires initial ring-opening, ligand displacement by chloride and subsequent conversion to the aquo and/or hydroxy species, thus requiring more time to bind with the DNA bases. 23,24isplatin and carboplatin are more cytotoxic than the amphiphilic complexes, except when compared to carboplatin in A 549 cell line, against which six of the amphiphilic complexes were more cytotoxic.
It is interesting to note that all the compounds exhibited lower cytotoxicity than cisplatin against non-tumor kidney cell (BHK-21), since nephrotoxicity is one of the most severe side effects caused by cisplatin.Also, compounds 1-4 were less toxic than cisplatin and carboplatin against non-tumor ovarian cell (CHO).A good alternative platinum drug to be used in cancer chemotherapy should be toxic in tumor cells and less aggressive in normal cells to avoid the various side effects.

Experimental
IR spectra were obtained on a Bomem FT IR MB-102 spectrometer in KBr pellets. 1 H NMR (300 MHz), 13 C NMR (75 MHz) and 195 Pt NMR (64 MHz) spectra were recorded on a Bruker spectrometer.For 13 C NMR proton decoupling was used.Elemental analyses were performed at the Universidade de São Paulo, Brazil.

Reagents
All chemicals were reagent grade and were used without further purification, except for the reagents used in cytotoxic activities which were filtered (0.2 µm).

Synthesis of chloride complexes 1-4 (Scheme 1)
To a solution of K 2 PtCl 4 (0.415 g, 1 mmol) in water (5 mL), the appropriate diamine (1 mmol) dissolved in water/ methanol (5 mL) was slowly added during 4 h.After stirring for 24 h at room temperature, the pallid yellow solid formed was filtered off, washed with water, methanol and dried.

Synthesis of oxalato complexes 5-8 (Scheme 1)
To the chloride complexes (1 mmol) in acetone (10 mL), AgNO 3 (2.2 mmol) was added.After stirring for 24 h at 60 °C, the white solid formed (AgCl) was filtered off.The filtrate was concentrated to dryness in a rotaevaporator and the yellow solid was dissolved in acetone and filtered to eliminate any excess of AgNO 3 .Na 2 C 2 O 4 (0.303g; 1 mmol) in water (100 mL) was added to the filtrate.After stirring for 24 h at 50 °C, a beige precipitate was formed.The products were isolated by filtration, washed with water, methanol and dried.Yields: (5) 0.239 g (53%), (6) 0.418 g (86%), (7)

Partition coefficients
Partition coefficients for the platinum complexes were determined in duplicate in an n-octanol/water system. 25ach complex was dissolved in n-octanol at 20 µmol L -1 and, subsequently, an equal volume of water was added.The mixtures were shaken mechanically for 24 h to ensure the distribution between the two solvent phases.The platinum concentration was determined in both phases by GFAAS in a THGA-Perkin Elmer spectrophotometer equipped with a graphite tube atomizer and an Analyst 600 auto sampler.Results are expressed as logarithm of P (log P), where P is the total platinum concentration in n-octanol divided by the total platinum concentration in the aqueous layer. 26

13 C
NMR spectra of the diamines was compared with the spectra of the complexes.Signals corresponding to the CH 3 group occurred around d 14.In the d 22-32 region, signals attributable to the carbons from the aliphatic chain were observed.The carbon atoms directly bound to NH 2 and NH gave signals in the d 40-50 region.For the oxalato complexes, signals corresponding to the carboxylato carbon were observed around d 166.