Yes / A series of 3-(benzyl-substituted)-imidazo[5,1-d]-1,2,3,5-tetrazines (13) and related derivatives with 3-heteromethyl groups has been synthesised and screened for growth-inhibitory activity in vitro against two pairs of glioma cell lines with temozolomide-sensitive and -resistant phenotypes dependent on the absence/presence of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). In general the compounds had low inhibitory activity with GI50 values > 50 µM against both sets of cell lines. Two silicon-containing derivatives, the TMS-methylimidazotetrazine (9) and the SEM-analogue (10), showed interesting differences: compound (9) had a profile very similar to that of temozolomide with the MGMT+ cell lines being 5 to 10-fold more resistant than MGMT– isogenic partners; the SEM-substituted compound (10) showed potency across all cell lines irrespective of their MGMT status.

yes / The archetypal prodrug of the imidazotetrazine class is the anticancer agent temozolomide (TMZ). The prodrug activation kinetics of TMZ show an unusual pH dependence: it is stable in acid and rapidly hydrolyses in alkali (Denny, B.J., et al. Biochemistry 1994, 33, 9045–9051). The incipient drug MTIC has the opposite properties—relatively stable in alkali but unstable in acid. In this study, the mechanism of prodrug activation was probed in greater detail to determine whether the reactions are specific or general acid or base catalysed. Three prodrugs and drugs were investigated, TMZ, MTIC and the novel dimeric imidazotetrazine EA27. Hydrolysis in a range of citrate-phosphate buffers (pH 8.0, 7.4, 4.0) was measured by UV spectrophotometry. Reaction of TMZ and MTIC obeyed single-phase, pseudo-first order kinetics (Figure 1). EA27 was more complex, showing biphasic but approximately pseudo-first order kinetics, Figure. General acid or base catalysis indicates that protonation or deprotonation is the rate-limiting step (rls). In biological milieu, the nature and concentration of other acidic or basic solutes may affect the prodrug activation reaction. In contrast, specific acid or base catalysis indicates that protonation or deprotonation occurs before the rls, so catalysis depends only on the local concentration of hydroxide or hydronium ion (i.e., pH) so the reaction kinetics are not expected to change appreciably in a biological medium.

Yes / Glioblastoma multiforme (GBM) is a grade IV brain tumor characterized by a heterogeneous population of cells that are highly infiltrative, angiogenic and resistant to chemotherapy. The current standard of care, comprised of surgical resection followed by radiation and the chemotherapeutic agent temozolomide, only provides patients with a 12–14 month survival period post-diagnosis. Long-term survival for GBM patients remains uncommon as cells with intrinsic or acquired resistance to treatment repopulate the tumor. In this review we will describe the mechanisms of resistance, and how they may be overcome to improve the survival of GBM patients by implementing novel chemotherapy drugs, new drug combinations and new approaches relating to DNA damage, angiogenesis and autophagy.

Yes / The imidazotetrazine ring is an acid-stable precursor and prodrug of highly reactive alkyl diazonium ions. We have shown that this reactivity can be managed productively in an aqueous system for the generation of aziridinium ions with 96% efficiency. The new compounds are potent DNA alkylators and have antitumor activity independent of the O6-methylguanine-DNA methyltransferase and DNA mismatch repair constraints that limit the use of Temozolomide.