Antitumor imidazo[5,1-d]-1,2,3,5-tetrazines: compounds modified at the 3-position overcome resistance in human glioblastoma cell lines

Synthetic routes to 3-substituted imidazo[5,1-d]-1,2,3,5-tetrazines structurally related to temozolomide were explored. Interaction of 4-diazoimidazole-5-carboxamide with an isocyanate afforded high product yields when the isocyanate was available in acceptable purity. Alternatively, alkylation of the nor-temozolomide anion afforded high yields of new imidazotetrazines. Several compounds, evaluated against a panel containing matched MGMT± glioma cell lines, showed equal inhibitory activity irrespective of MGMT status; the N3-propargyl-imidazotetrazine (10m) was prioritised as an alternative to temozolomide able to bypass drug-resistance mechanisms. In Taq polymerase assays 10m, like temozolomide and its ring-opened counterpart MTIC, alkylated DNA at clusters of three and five guanine residues; covalent modification of N-7 sites of guanine were detected in piperidine cleavage assays. Compound 10m did not cross-link DNA but induced double-strand breaks evidenced by γ-H2AX detection. Propargyl-substituted imidazotriazene (13g), showed comparable activity to 10m indicating that ring-opening of the bicyclic nucleus of novel imidazotetrazine is probably required for activity.