The Medicinal Chemistry of Imidazotetrazine Prodrugs

YesTemozolomide (TMZ) is the standard first line treatment for malignant glioma, reaching “blockbuster” status in 2010, yet it remains the only drug in its class. The main constraints on the clinical effectiveness of TMZ therapy are its requirement for active DNA mismatch repair (MMR) proteins for activity, and inherent resistance through O6-methyl guanine-DNA methyl transferase (MGMT) activity. Moreover, acquired resistance, due to MMR mutation, results in aggressive TMZ-resistant tumour regrowth following good initial responses. Much of the attraction in TMZ as a drug lies in its PK/PD properties: it is acid stable and has 100% oral bioavailability; it also has excellent distribution properties, crosses the blood-brain barrier, and there is direct evidence of tumour localisation. This review seeks to unravel some of the mysteries of the imidazotetrazine class of compounds to which TMZ belongs. In addition to an overview of different synthetic strategies, we explore the somewhat unusual chemical reactivity of the imidazotetrazines, probing their mechanisms of reaction, examining which attributes are required for an active drug molecule and reviewing the use of this combined knowledge towards the development of new and improved anti-cancer agents

Antitumour Imidazotetrazines: Past, Present... and Future?

YesIt is 40 years since the publication of the patent that announced the imidazotetrazines temozolomide and mitozolomide to the world and 30 since the discovery that they function as prodrugs of alkyldiazonium reactive intermediates. Temozolomide combined with radiation is established as the first-line treatment for glioma but despite the attentions of the inventors and others, further examples of this intriguing ring system have yet to enter the clinic

Probing Imidazotetrazine Prodrug Activation Mechanisms

yesThe 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

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

The cellular responses to two new temozolomide (TMZ) analogues, DP68 and DP86, acting against glioblastoma multi- forme (GBM) cell lines and primary culture models are reported. Dose–response analysis of cultured GBM cells revealed that DP68 is more potent than DP86 and TMZ and that DP68 was effective even in cell lines resistant to TMZ. On the basis of a serial neurosphere assay, DP68 inhibits repop- ulation of these cultures at low concentrations. The efficacy of these compounds was independent of MGMT and MMR func- tions. DP68-induced interstrand DNA cross-links were dem- onstrated with H2O2-treated cells. Furthermore, DP68 induced a distinct cell–cycle arrest with accumulation of cells in S phase that is not observed for TMZ. Consistent with this biologic response, DP68 induces a strong DNA damage response, including phosphorylation of ATM, Chk1 and Chk2 kinases, KAP1, and histone variant H2AX. Suppression of FANCD2 expression or ATR expression/kinase activity enhanced anti- glioblastoma effects of DP68. Initial pharmacokinetic analysis revealed rapid elimination of these drugs from serum. Collec- tively, these data demonstrate that DP68 is a novel and potent antiglioblastoma compound that circumvents TMZ resistance, likely as a result of its independence from MGMT and mismatch repair and its capacity to cross-link strands of DN

Synthesis and Quantitative Structure–Activity Relationship of Imidazotetrazine Prodrugs with Activity Independent of O6-Methylguanine-DNA-methyltransferase, DNA Mismatch Repair and p53.

The antitumor prodrug Temozolomide is compromised by its dependence for activity on DNA mismatch repair (MMR) and the repair of the chemosensitive DNA lesion, O6-methylguanine (O6-MeG), by O6-methylguanine-DNA-methyltransferase (EC 2.1.1.63, MGMT). Tumor response is also dependent on wild-type p53. Novel 3-(2-anilinoethyl)-substituted imidazotetrazines are reported that have activity independent of MGMT, MMR and p53. This is achieved through a switch of mechanism so that bioactivity derives from imidazotetrazine-generated arylaziridinium ions that principally modify guanine-N7 sites on DNA. Mono- and bi-functional analogs are reported and a quantitative structure-activity relationship (QSAR) study identified the p-tolyl-substituted bi-functional congener as optimized for potency, MGMT-independence and MMR-independence. NCI60 data show the tumor cell response is distinct from other imidazotetrazines and DNA-guanine-N7 active agents such as nitrogen mustards and cisplatin. The new imidazotetrazine compounds are promising agents for further development and their improved in vitro activity validates the principles on which they were designed

Preclinical anti-cancer activity and multiple mechanisms of action of a cationic silver complex bearing N-heterocyclic carbene ligands

Organometallic complexes offer the prospect of targeting multiple pathways that are important in cancer biology. Here, the preclinical activity and mechanism(s) of action of a silver-bis(N-heterocyclic carbine) complex (Ag8) were evaluated. Ag8 induced DNA damage via several mechanisms including topoisomerase I/II and thioredoxin reductase inhibition and induction of reactive oxygen species. DNA damage induction was consistent with cytotoxicity observed against proliferating cells and Ag8 induced cell death by apoptosis. Ag8 also inhibited DNA repair enzyme PARP1, showed preferential activity against cisplatin resistant A2780 cells and potentiated the activity of temozolomide. Ag8 was substantially less active against non-proliferating non-cancer cells and selectively inhibited glycolysis in cancer cells. Ag8 also induced significant anti-tumour effects against cells implanted intraperitoneally in hollow fibres but lacked activity against hollow fibres implanted subcutaneously. Thus, Ag8 targets multiple pathways of importance in cancer biology, is less active against non-cancer cells and shows activity in vivo in a loco-regional settin

Synthesis and characterisation of novel nopyl-derived phosphonium ionic liquids

A series of novel nopyl-derived chiral phosphonium ionic liquids have been successfully synthesised and characterised. Analysis of each novel ionic liquid was conducted in order to confirm structure, purity and thermal stability

Does an intercalated clinical placement make a difference to learning gain?

noBackground Anecdotally, it has long been felt by academic staff that students on the Bradford 5-year sandwich degree programme (intercalated pre-registration training) performed differently on return to university from those on the continuous 4-year programme. Direct comparisons between cohorts have been difficult to undertake as the two groups were taught separately in their final stage. In 2016-17, a cohort of returning sandwich students was taught alongside a comparable group of continuous students in a final stage module. This study compares the results from these two student cohorts. Method The Pharmacy Special Studies module offered a very broad range of opportunities across laboratory research, systematic and scoping review, product development and care-orientated topics including audit, and analysis of clinical cases, organised in 9 separate “strands.” Students from the sandwich (n=99) and continuous (n=89) courses were offered the same selection of learning experiences. Assessment was by oral presentation and discussion (slides or poster) and written report. The Level 7 marking schemes used were designed so that the highest marks were only available to reward student demonstration of the higher-level critical, analytical and interpretative skills. Results Student performance across all 9 strands of the module was comparable. When module results were split according to cohort, a strong divergence was observed. Sandwich student results displayed an approximately bell-shaped distribution with a mean mark 73.2% (SD 8.1). In contrast, the continuous student results had a lower mean 67.3% (SD 8.5, p<10-5); moreover, the distribution of these marks was distorted with a ‘cliff edge’ in the low 70s and a long tail. Conclusion This analysis shows that students who had completed 6 months pre-registration training achieved, on average, higher grades. Moreover, they demonstrated improved higher-level skills of interpretation and critical analysis compared with the continuous group. Although this is a one-year “snapshot” observation it appears to show that following 6 months preregistration training, students are better able to critically evaluate and interpret data and draw evidence-based conclusions. Such a result could provide evidence for the benefits of intercalated placements and indicate the optimal location of professional training within the academic course. Any impact on pre-registration training itself remains to be determined.Abstract, presented at the Pharmacy Education Conference 2017, Manchester, United Kingdom

Novel, High-yielding Synthesis of meso-Substituted Porphyrins via the Direct Arylation of Porphine.

NoA new method for the synthesis of meso-substituted porphyrins is described: reaction of 5,10,15,20-tetrabromoporphine magnesium complex with aryl or heteroaryl boronic acids in the presence of Pd(PPh3)4 gave meso-substituted porphyrins in yields up to 70%