A Strategy for Imidazotetrazine Prodrugs with Anti-cancer Activity Independent of MGMT and MMR

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. Imidazotetrazine 1 is a fascinating heterocycle, on account of both its Chemistry and the therapeutic applications of its derivatives. It forms the core of the anti-cancer prodrug Temozolomide (1 R = CH3; TMZ). Although TMZ has achieved “blockbuster” status, it remains the lone member of its drug class because of the constraints placed on tumor response to the prodrug by a requirement for DNA mismatch repair (MMR) and resistance mediated through O6-alkylguanineDNA alkyltransferase (MGMT). Herein we report a strategy that achieves remarkably-effective control of the reactive intermediates generated by the imidazotetrazine ring in an aqueous environment, in this example to generate aziridinium ions. This design achieves compounds that react with DNA but exhibit chemosensitivity independently of MMR and MGMT. Scheme 1. Release of electrophiles on hydrolysis of the imidazotetrazine ring. aFor TMZ, R=CH3, MTZ, R=CH2CH2Cl. At neutral or alkaline pH, imidazotetrazines 1 (e.g. TMZ) act as a source of diazonium ions 2, with few exceptions ringopening as shown in Scheme 1. Methyldiazonium is released from TMZ and methylates DNA; lethal interaction of this adduct with MMR causes cell death. There are two kinetic parameters that determine the effectiveness of imidazotetrazine prodrugs. The first is the rate of addition of water (or hydroxide ion) to initiate the ring-opening reaction. This is slow at low pH, so confers acid stability and the convenience of oral dosing. The pH-dependence of this reaction also influences distribution of the prodrug around the body: hydrolysis kinetics at pH 7.4 match closely the uptake rate (peak plasma concentration after 30 min) and metabolic half life (t1⁄2 = 1.29 h) in patients. 5 The other significant kinetic parameter is the reactivity of the latent electrophile. Hydrolysis of methyldiazonium in a purely chemical system has t1⁄2 = 0.39 s. Again, a suboptimal value detracts from clinical effectiveness: ethyldiazonium eliminates or reacts with water before it is able to locate a reactive nucleic acid target site, while longer-lived intermediary electrophiles such as chloronium achieve clinical efficacy. For TMZ, these clinically-useful, if not formally optimized, pharmacokinetic properties were achieved serendipitously. In the design of TMZ analogues with altered spectra of activity, we reasoned that a neighboring group participation (NGP)-based mechanism could be employed to control the incipient alkyldiazonium ions. This would serve the dual functions of directing reactivity and delivering an alternative form of damage to DNA. Since the response of tumors to TMZ is determined by the interaction of covalently modified DNA with DNA repair systems, altering the electrophile would necessarily alter the tumor response. In these respects, the potential of the imidazotetrazine as an acid-stable precursor of aziridines or aziridinium ions was explored. These are reactive intermediates with proven clinical utility, being found widely in, or generated by, synthetic and natural product anti-tumor drugs and prodrugs. Furthermore, O6-aminoethylguanine is known to be refractory to cleavage by MGMT.