Diamminetetrakis(carboxylato)platinum(IV) Complexes – Synthesis, Characterization, and Cytotoxicity

Abstract A series of eight novel diamminetetrakis(carboxylato)platinum(IV) complexes was synthesized and characterized by multinuclear 1H-, 13C-, 15N-, and 195Pt-NMR spectroscopy. Their antiproliferative potency was evaluated in three human cancer cell lines representing ovarian (CH1), lung (A549), and colon carcinoma (SW480). In cisplatin-sensitive CH1 cancer cells, cytotoxicity was found in the low micromolar range, whereas, in inherently cisplatin-resistant A549 and SW480 cells, the activity was very low or negligible. Astonishingly, raise in lipophilicity of the complexes, as found in the case of cisplatin analogs, did not result in a significant enhancement of the cytotoxic effect.

Cisplatin, carboplatin, and oxaliplatin are administered intravenously against a series of solid tumors. However, some tumors are not accessible due to intrinsic resistance or develop resistance during therapy. Additionally, platinum chemotherapy is accompanied by a set of side effects that are, to some extent, severe and dose-limiting. Despite the development of organic compounds used in that field and the approval of monoclonal antibodies (targeted therapy), Pt-based treatment still builds the basis for combination cancer therapy.
During the last decades, different strategies were pursued with the aim of reducing side effects and accumulating or activating Pt drugs at the tumor site [7]; additionally, oral administration of the complexes would be advantageous with respect to the acceptance of chemotherapy, improving the quality of life and reducing hospitalization costs. Along that line, the development of octahedrally configured Pt IV complexes seems to be most promising [8] [9]. Consequently, it is not surprising that so far four complexes, namely tetraplatin, iproplatin, satraplatin (Fig. 1), and LA-12 (a close analog of satraplatin, but featuring an adamantylamine ligand instead of cyclohexylamine) have been evaluated in phase I -III clinical trials.
Platinum(IV) complexes are kinetically inert. i) Therefore, they can be administered orally via absorption through the gastrointestinal tract, if lipophilic enough. ii) Platinum(IV) complexes act as prodrugs which can be reduced to the Pt II species featuring a higher reactivity/activity (activation by reduction) in the hypoxic (oxygen deficiency) milieu of many solid tumors, accompanied by release of the axial ligands [10] [11]. iii) Platinum(IV) complexes can be derivatized more easily at coordinated OH [12 -15] or peripheral functional groups [16 -18] compared to their Pt II counterparts.
In a more basic approach, we recently reported on a series of diamine(dicarboxylato)dichloridoplatinum(IV) complexes which were investigated with regard to their cytotoxicity, lipophilicity, and cellular accumulation [29 -33]. It was found that, with increasing lipophilicity, the cellular accumulation and the antiproliferative potency were enhanced as well. IC 50 Values in the low nanomolar range, and therefore significantly better compared to those of cisplatin, were observed for the most lipophilic agents.
In case that this behavior is a general characteristic, it should then be possible to improve the cytotoxicity of kinetically more inert diaminetetrakis(carboxylato)platinum(IV) complexes just by increasing their lipophilicity. To validate this hypothesis, eight novel (OC-6-33)-diamminebis(carboxylato)malonatoplatinum(IV) complexes were synthesized, characterized, and their cytotoxicity was evaluated in three human cancer cell lines.
Results and Discussion. -The Pt II precursor, (SP-4-2)-diammine(malonato)platinum(II) (1), was prepared starting from the diamminediiodido complex via reaction with AgNO 3 and subsequent coordination of malonate. Oxidation with 30% H 2 O 2 was performed in aqueous solution at ambient temperature resulting in the octahedrally configured dihydroxido compound 2 (Scheme). The latter was carboxylated with succinic anhydride in DMF as published recently [17]. The terminal and uncoordinated carboxylic acid groups were activated with 1,1'-carbonyldiimidazole (CDI), and converted to the corresponding esters or amides, respectively.
Precursor 3 was not evaluated, since it is known that analogous complexes featuring free carboxylic acid moieties have low antiproliferative potency due to their relatively high solubility in H 2 O [33].
In cisplatin-sensitive CH1 cells, IC 50 values were between 5.9 and 30 mm. However, cytotoxicity in the inherently cisplatin-resistant A549 and SW480 cell lines was negligible or very low. The following structureÀactivity relationships could be drawn from the results in CH1 and SW480 cancer cells: i) IC 50 values of 4a -4d decrease parallel to an increasing lipophilicity (methyl, ethyl, propyl, and butyl ester), ii) an i Pr residue (i.e., 4e) is not favorable compared to Pr (i.e., 4c), iii) exchange of a CH 2 fragment in 4d by oxygen (i.e., 4f) is clearly unfavorable in terms of cytotoxicity due to a lower lipophilicity of the latter, iv) amides 4g and 4h display relatively high IC 50 values.
The finding that IC 50 values in CH1 cells are in the low micromolar range, but not well below 1 mm (cisplatin, 0.16 mm [30]), is not astonishing at first sight, since release of a chelating dicarboxylato ligand (e.g., carboplatin) is rather slow compared to coordinated chloride (e.g., cisplatin). However, it was envisaged to significantly improve the cytotoxic potency by enhancing lipophilic properties and thereby increasing cellular accumulation [32]. In the case of close analogs featuring the same axial ligands, but ethane-1,2-diamine and two chlorido ligands in the equatorial coordination sphere, an improvement of the cytotoxicity by a factor of 40 (0.68 mm vs. 0.018 mm) was achieved comparing the methyl ester derivative with its butyl ester counterpart [30]. On the contrary, in the case of 4a -4d, cytotoxicity could only be raised by a factor of less than 3. Obviously, besides lipophilicity, factors such as the redox potential and the rate of reduction play a crucial role in the mode of action of Pt IV complexes. This assumption was confirmed very recently by Hambley, Gibson and co-workers [34], who showed that coordinated am(m)ines and carboxylates are unfavorable for facilitated electron transfer. They concluded that reduction potentials not necessarily reflect the rates of reduction.
Whether their findings can be generalized will be subject of further investigations. Especially correlation of lipophilicity and reduction potential with cellular accumulation and cytotoxicity of tetrakis(carboxylato)platinum(IV) complexes will be investigated in more detail.

Experimental Part
General. All chemicals and solvents were obtained from commercial suppliers and used without further purification. MeOH and EtOH were dried according to standard procedures, and reverse osmosis water was doubly distilled before use. For column chromatography (CC), silica gel 60 (SiO 2 ; Fluka) was used. (SP-4-2)-Diamminediiodidoplatinum(II) was synthesized by Dharas method [35].