Synthesis and biological evaluation of novel prodrugs of anthracyclines for selective activation by the tumor-associated protease plasmin

New prodrugs of daunorubicin and doxorubicin designed for selective activat ion by the serine protease plasmin are described. The low toxic prodrugs 3, 4, and 5 are converted to the corresponding cytotoxic drugs upon proteolys is by the tumor-associated protease plasmin. Application of a self-eliminat ing spacer was essential for enzyme activation. A prodrug containing a chlo ro-substituted spacer was synthesized with the aim of enhancing the rate of conversion by plasmin. All prodrugs were highly stable in buffer solution and in serum and on the average 15-fold less cytotoxic than the parent drug s in seven human tumor cell lines. A marked in vitro selectivity was demons trated by incubation of the doxorubicin prodrugs with a plasmin generating MCF-7 breast cancer cell. line transfected with urokinase-type plasminogen activator (u-PA) in comparison with the nontransfected nonplasmin generatin g cell line. Prodrugs 4 and 5 showed the same cytotoxic effect as the free parent drug doxorubicin in the u-PA transfected cells, indicating complete conversion of the prodrug by plasmin. Addition of the plasmin inhibitor Tra sylol drastically increased the ID50 values in the u-PA transfected MCF-7 c ells for both prodrugs 4 and 5.