In vitro reactions of a cyanocobalamin-cisplatin conjugate with nucleoside monophosphates

Cisplatin (CP) is a widely used anticancer drug characterized by toxic side effects that could be alleviated using novel delivery systems including CP prodrugs. The in vitro incubation of a putative prodrug, obtained from cyanocobalamin (CNCbl) and cis-diamminemonochloroplatinum(II) (mCP), with nucleoside monophosphates (NMPs) was investigated.

The in vitro reactions between the putative prodrug CNCbl-mCP and the NMPs of adenosine (AMP), guanosine (GMP), cytidine (CMP) and uridine (UMP) were carried out in slightly acidic water-methanol solutions at 37°C for 24 h. Each sample was examined using reversed-phase liquid chromatography coupled with electrospray ionization in positive ion mode and tandem mass spectrometry (RPLC/ESI-MS/MS) by collision-induced dissociation in a linear ion-trap mass spectrometer.

Seven adducts were recognized as formed by substitution reactions of the chloride ligand in planar CP. Comparison between observed and theoretical isotopic patterns together with MS/MS fragmentation pathways revealed the presence of single or multiple binding sites depending on the NMP involved. The CNCbl-mCP conjugate was found to interact with N⁷ or O⁴ atoms of GMP and UMP, respectively, generating single adducts, while two isomeric adducts were observed for CMP. Finally, AMP gave rise to three isomeric adducts.

In agreement with literature data relevant to the interaction between CP and NMPs, the most reactive nucleotides were AMP and GMP. The present RPLC/ESI-MS/MS approach is very promising for investigation of the reactions of CP conjugates with ribonucleotides not only in vitro but also in vivo.