β-Glucuronidase-cleavable prodrugs of O⁶-benzylguanine and O⁶-benzyl-2′-deoxyguanosine

Glucuronic acid linked prodrugs of O⁶-benzylguanine and O ⁶-benzyl-2′-deoxyguanosine were synthesized. The prodrugs were found to be quite stable at physiological pH and were more than 200-fold less active as inactivators of O⁶-alkylguanine-DNA alkyltransferase (alkyltransferase) than either O⁶-benzylguanine or O ⁶-benzyl-2′-deoxyguanosine. β-Glucuronidase from both Escherichia coli and bovine liver cleaved the prodrugs efficiently to release O⁶-benzylguanine and O⁶-benzyl-2′-deoxyguanosine, respectively. In combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), the prodrugs were not effective adjuvants for HT29 cell killing. However, as expected, incubation of these prodrugs with β-glucuronidase in the culture medium led to much more efficient cell killing by BCNU as a result of the liberation of the more potent inactivators, O⁶-benzylguanine and O⁶-benzyl-2′-deoxyguanosine. These prodrugs may be useful for prodrug monotherapy of necrotic tumors that liberate β-glucuronidase or for antibody-directed enzyme prodrug therapy with antibodies that can deliver β-glucuronidase to target tumor cells.