Synthesis, Characterization and in Vitro Antitumor Activity of Platinum(II) Oxalato Complexes Involving 7-Azaindole Derivatives as Coligands

The platinum(II) oxalato complexes [Pt(ox)(naza)2] (1–3) were synthesized and characterized by elemental analysis (C, H, N), multinuclear NMR spectroscopy (1H, 13C, 15N, 195Pt) and electrospray ionization mass spectrometry (ESI-MS); naza = 4-chloro-7-azaindole (4Claza; 1), 3-bromo-7-azaindole (3Braza; 2) or 4-bromo-7-azaindole (4Braza; 3). The prepared substances were screened for their in vitro antitumor activity on the osteosarcoma (HOS) and breast adenocarcinoma (MCF7) human cancer cell lines, where 2 showed moderate antitumor effect (IC50 = 27.5 μM, and 18.3 μM, respectively). The complex 2 was further tested on a panel of six others human cancer cell lines, including the malignant melanoma (G361), cervix carcinoma (HeLa), ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R), lung carcinoma (A549) and prostate adenocarcinoma (LNCaP). This substance was found to be moderate antitumor effective against G361 (IC50 = 17.3 μM), HeLa (IC50 = 31.8 μM) and A2780 (IC50 = 19.2 μM) cell lines. The complex 2 was also studied by NMR for its solution stability and by ESI-MS experiments for its ability to interact with biomolecules, such as cysteine, glutathione or guanosine 5'-monophosphate.


Introduction
Platinum carboxylates represent a notable group of transition metal complexes, which have been used for the treatment of various types of cancer for many years [1][2][3]. Various carboxylate anions involved in the structures of the clinically used or studied platinum-based metallotherapeutics can be mentioned, particularly the cyclobutane-1,1-dicarboxylate dianion (involved in carboplatin), glycolate dianion (involved in nedaplatin), lactate dianion (involved in lobaplatin), malonate dianion (involved in heptaplatin), acetate anion (involved in satraplatin) or neodecanoate anion (involved in aroplatin) [4][5][6][7][8][9]. One of the carboxylate-based leaving groups is the oxalate dianion involved in the well-known substance oxaliplatin clinically used mainly for the treatment of colorectal tumours [10]. In other words, the platinum(II) oxalato complexes are, thanks to the mentioned oxaliplatin, biologically relevant and worth studying group of compounds.

General Properties
The platinum(II) oxalato complexes [Pt(ox)(naza) 2 ] (1-3; Scheme 1) were prepared by a one-step synthetic procedure using the bis(oxalato)platinate(II) salt as a starting compound [17]. The composition of the products was proved by the results of elemental analysis. The electrospray ionization mass spectra obtained in the positive mode (ESI+) of the studied complexes contained the molecular peaks detected at 589.   [23] or its derivatives [24].

NMR and ESI-MS Stability and Interaction Studies
1 H and 195 Pt NMR spectroscopy (solution of 2 in DMF-d 7 and DMF-d 7 /H 2 O mixture, 9:1 v/v) and electrospray ionization mass spectrometry (ESI-MS; solution of 2 in methanol and water/methanol mixture, 1:1 v/v) (the presence of the organic solvents ensured the solubility of the studied complex, because carrying out of the experiments in water was prevented by limited solubility of the mentioned complex in water) were used to investigate the behaviour of the representative complex 2 in the mentioned organic or water-containing solvents. As it is generally accepted for the antitumor active platinum(II) complexes, hydrolysis is a crucial step within the mechanism of action, which lead to the replacement of leaving groups (i.e., the oxalato ligands) and formation of the activated and more reactive aqua-and/or hydroxidoplatinum(II) species, probably with formulas cis-[Pt(H 2 O) 2 (3Braza) 2 ] 2+ and/or cis-[Pt(OH) 2 (3Braza) 2 ] in our case [1,2,27,28]. The hydrolysis of the platinum(II) oxalate complexes should be connected with opening of the PtO 2 C 2 ring and/or substitution of the oxalate dianion by two H 2 O or OH − species as ligands, both resulting in the change of inner coordination sphere (resulting in new peaks in mass spectra) and electron density within the initial complex, which is known to provide different 1 H and 195 Pt NMR chemical shifts [27,28]. Since we did not observe any new signals in both the 1 H and 195 Pt NMR spectra (Figure 1), it can be concluded that the complex 2 is stable and do not undergo any changes within the structure during 5 days in DMF-d 7 as well as in the DMF-d 7 /H 2 O mixture. Similarly it was found that the complex is stable and did not show any change in the composition from the mass spectrometry point of view, because its mass spectra (methanol solutions) recorded after 12 h did not contain any novel peaks as compared with the spectra obtained on the fresh solution of 2. In the case of water/methanol mixture solution of 2, we detected several new peaks in the mass spectra in comparison with the spectra of 2 dissolved in pure methanol, but their isotopic distribution did not correspond to that of platinum-containing species (Figure 2). In other words, no new platinumcontaining species was found in the mass spectra recorded on the water/methanol solution of 2, which, as in the case of NMR, proved that no hydrolytic processes proceeded under the experimental condition used. Thus it can be said that we did not get any evidence of the hydrolysis usually involved within the mechanism of action of the cytotoxic active platinum(II) complexes including the clinically used platinum(II) oxalate complex oxaliplatin [1,2,29]. On the other hand, it is known that the oxaliplatin hydrolysis in water (leading to diaqua-species) and under in vivo conditions has different course, since the latter one provides the carbonato or phosphato adducts (instead of the above mentioned diaqua ones), which consequently enhance the reactivity of such species towards nucleophiles including nucleobases [30][31][32]. It means that although we did not observed any processes usually associated with the action of cytotoxic platinum(II) complexes (see Section 2.3. for the in vitro cytotoxicity of 1-3) under experimental conditions, the cytotoxic action itself is not excluded with respect to different conditions in the cells (cytosol involving various ions) as compared with those used in the herein discussed NMR and ESI-MS experiments.
ESI-MS was also used to study the ability of 2 to interact with sulphur-containing biomolecules (cysteine (cys) and reduced glutathione (GSH)) or guanosine 5'-monophosphate (GMP) in water/methanol mixture (1:1 v/v) (again, the presence of the organic solvent ensured the solubility of the studied complex). It is well-known that ability of the cytotoxic platinum(II) complexes to interact with the intracellular sulphur-containing compounds correlates with their activity as well as with the resistance of the respective tumours in terms of inactivation of the platinum(II) species and their removing from the cell [1,29]. With respect to this phenomena, ability of the studied platinum(II) complexes to interact with the sulphur-containing biomolecules should be investigated by relevant techniques. In the case of this work, we studied an interaction of 2 with the mixture of cys and GSH in water/methanol mixture. We did not observe any adducts of 2 (or its fragments formed during ionization) with cysteine or reduced glutathione assignable to the species formed by their interaction (Figure 2), which corresponds to the above-described reluctance of 2 to undergo hydrolysis. The only exception from this statement is very weak peak of the {[Pt(cys)(ox)(3Braza) 2 ]+H} + species (Figure 2, inset) detected in the ESI+ spectra of the studied complex 2 at 799.5 m/z (calcd. 799.9 m/z), which most probably contains a ring-opened product of the interaction with a monodentate bound oxalate dianion. The mechanism of action itself of the clinically used antitumor active platinum(II) complexes is based on the covalent binding of activated platinum(II) species to the nuclear DNA molecule of the tumour cells [33], which is also expected for most of the platinum(II) complexes having cytotoxic effect. A simple model to study the ability of the platinum(II) complexes to bind DNA molecule is based on binding reactions with various nucleobase-based compounds such as GMP employed in this work. However, we have to state that we did not detect any species whose mass and isotopic distribution would correspond with those of adduct of the studied complex, its fragments or its hydrolysis products with GMP ( Figure 2).
As it is mentioned above, 1-3 follow recently reported [24] analogous oxalato complexes involving different types of 7-azaindole halogeno-derivatives, concretely 3-chloro-7-azaindole (3Claza), 3-iodo-7-azaindole (3Iaza) and 5-bromo-7-azaindole (5Braza), which, similarly to 1 and 3, did not show any effect against both HOS and MCF7 cell lines up to the concentration of 10.0, 25.0, and 0.5 μM, respectively. Concerning all six platinum(II) oxalato complexes with different 7-azaindole derivatives together, it can be said that the biological activity, in terms of bioavailability, is strongly affected by the position of halogeno-substituent of the 7-azaindole moiety, because the solubility of the complexes with 3Claza, 3Braza and 3Iaza (10.0-50.0 μM) is significantly higher than that of the complexes involving the 7-azaindole derivatives substituted in the position 4 (4Claza and 4Braza) or 5 (5Braza), whose solubility did not exceed 1.0 μM in the medium used. Asterisk (*) symbolizes significant difference (p < 0.05) in in vitro antitumour activity of 2 as compared to cisplatin; nt = not tested.

Synthesis of Complexes 1-3
A solution of 1.0 mmol of 4Claza (for 1), 3Braza (for 2) or 4Braza (for 3) in 10 mL of hot (50 °C) ethanol was slowly poured into the solution of K 2 [Pt(ox) 2 ]•2H 2 O (0.5 mmol) in 10 mL of hot (50 °C) distilled water. The reaction mixtures were stirred at 50 °C for two days. The products, which formed, were filtered off, washed (5 mL of distilled water and 5 mL of ethanol) and dried at 40 °C ( Figure 1). The described syntheses followed a procedure reported in our recent works for analogous complexes with different 7-azaindoles [23,24].