Strategy for Imidazotetrazine Prodrugs with Anticancer Activity Independent of MGMT and MMR

Abstract

ABSTRACT: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. KEYWORDS: Imidazotetrazine, aziridinium, Temozolomide, cancer, 13 C NMR, DNA I midazotetrazine 1 is a fascinating heterocycle, on account of both its chemistry and the therapeutic applications of its derivatives. It forms the core of the anticancer prodrug Temozolomide 1 (1, R = CH 3 ; TMZ). Although TMZ has achieved "blockbuster" status, 2 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-alkylguanine-DNA 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.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. 3 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 and, thus, confers acid stability and the convenience of oral dosing. 4 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 (t 1/2 = 1.29 h) in patients. 1,5 The other significant kinetic parameter is the reactivity of the latent electrophile. Hydrolysis of methyldiazonium in a purely chemical system has t 1/2 = 0.39 s. 6 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, 7 while longer-lived intermediary electrophiles such as chloronium achieve clinical efficacy. 8 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 re...

Evaluation of Novel Imidazotetrazine Analogues Designed to Overcome Temozolomide Resistance and Glioblastoma Regrowth

“…For TMZ these are the final reactive intermediates that covalently modify N 7-G, O 6-G, and N 3-A. In contrast, the diazonium ions from DP68 and DP86 undergo an efficient, intra-molecular trapping reaction to form aziridinium ions, and these intermediates react with DNA, predominantly at N 7-G sites (17). The new agents were designed to generate anticancer activity from N 7-G adducts but should adducts occur at O 6-G, these would be resistant to repair by MGMT (18).…”

“…DP68 and DP86 were synthesized as described previously (17), TMZ was from Schering-Plough Corp and Sigma-Aldrich. All 3 agents were prepared as 25 mM stocks in dimethyl sulfoxide (DMSO).…”

“…A disulfide linked imidazotetrazine dimer showed a similar response profile as the established agent busulfan in the NCI60 screening panel (16). The design of the imidazotetrazine compounds investigated herein sought a switch of chemical mechanism with the aims of avoiding known mechanisms of TMZ resistance and generating therapeutic benefit from the major reaction site, N 7-G (70% for TMZ), rather than the minor (5%) O 6-G site (17). In this study, we focus on two of the most promising compounds developed: the mono-functional DP86 and the bi-functional DP68 imidazotetrazines (Figure S1) (17, 18).…”

“…The design of the imidazotetrazine compounds investigated herein sought a switch of chemical mechanism with the aims of avoiding known mechanisms of TMZ resistance and generating therapeutic benefit from the major reaction site, N 7-G (70% for TMZ), rather than the minor (5%) O 6-G site (17). In this study, we focus on two of the most promising compounds developed: the mono-functional DP86 and the bi-functional DP68 imidazotetrazines (Figure S1) (17, 18). These compounds are precursors of aziridinium ions, which are reactive intermediates of proven clinical utility related to those generated by nitrogen mustard drugs (Figure S2C-D).…”

The role of temozolomide in the treatment of aggressive pituitary tumors

“…However, the matching of these two factors is not found in many drugs. Garelnabi et al studied the potential of imidazotetrazine as an acid stable precursor of aziridines [66]. The two compounds they synthesized were not only pH stable but they also achieved formation of the reactive intermediate at 96% efficiency.…”