DIAMINE LIGAND RELEASE FROM THE CISPLATIN ANALOG [MESO-1,2-BIS(2,6-DICHLORO-4-HYDROXYPHENYL)ETHYLENEDIAMINE]DICHLOROPLATINUM(II) IN CELL-CULTURE MEDIUM

The stability of the five-membered chelate ring of the cisplatin analogue [meso-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine] dichloroplatinum(II) was investigated under typical cell culture conditions (IMEM-Richter's medium with 10% fetal calf serum, 37-degrees-C). For this purpose, the platinum compound was radiolabeled with tritium in the meta position of the aromatic ring by an acid-catalyzed tritium-exchange reaction, and a reversed-phase HPLC assay with radiochemical detection was developed to monitor for the presence of the free diamine ligand in the cell culture medium. A gradual increase in radioactivity attributed to the free diamine was found in medium containing the dichloroplatinum(II) complex (ca. 25% after 24 h), indicating that the diamine ligand was being released from the metal atom. When 1 mM glutathione (GSH) was included in the incubation medium, the amount of free diamine nearly doubled after 24 h, while the amount of radioactivity attributed to serum protein-platinum adducts decreased relative to incubations without GSH. On the other hand, the omission of serum from the incubations resulted in a dramatic decrease in the amount of radioactivity eluting under the diamine peak, while the concentrations of the two methionine-Pt adducts, which formed in a 1:1 ratio, rose. Through the use of liquid secondary ion mass spectroscopy, the two methionine-Pt adducts were identified as monomethionine metabolites of the title compound, whereby the two chloride ligands have been replaced by the amino acid. These compounds are probably diastereomers since the sulfur of methionine can coordinate to platinum with equal probability either cis or trans to the R-configured benzylamine carbon. On the basis of the chemical shifts of the MeS groups in the 250-MHz H-1 NMR, it is concluded that a S,N-five-membered chelate ring is present in these methionine-Pt adducts.