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.

During this thesis we tried to design, synthesise and analyse some novel devices for the selective delivery of peptides. These systems are based on the enzyme-activated anticancer prodrugs developed by Prof. Gesson in Poitiers and the peptide rotaxanes developed by Prof. Leigh in Edinburgh. The innovative rotaxanes we constructed are devised to protect and selectively release a peptide in response to an enzyme-specific stimulus for the targeted therapy of cancer. In Chapter 1 we tried to expose the main synthetic strategies aimed at improving the stability and permeation features of biologically active peptides. We examined some prodrug approaches and particularly the tumour-activated prodrugs (TAPs), largely investigated for use in anticancer chemotherapy. TAPs are generally three-part molecules composed of trigger, spacer and effector units. We also presented the original methodology developed by Prof. Leigh, namely the hydrogen bond-directed assembly of peptide rotaxanes, to protect a peptide thread from external environment. Finally we presented our project which consists of a combination of the peptide prodrug and rotaxane approaches. Therefore, based on the knowledge of both research groups we tried in Chapter 2 to develop some model systems in order to study the influence of the rotaxane architecture upon prodrug molecules. The first step towards such rotaxane-based peptide prodrugs relied on the efficient design of a spacer which has to be bulky enough to work as a stopper for the macrocycle. Much of the work presented in this chapter is based on the design and synthesis of such self-immolative units. We then explored the response of our model rotaxanes under the action of the activating enzyme. After this detailed study, in Chapter 3 we applied our concept to the biologically active peptide Met-enkephalin. In this chapter we presented a comparison between a rotaxane prodrug of Met-enkephalin and its non-interlocked derivative. Thus both compounds were successfully synthesised and evaluated to release the free peptide after enzymatic activation. The protective effect of encapsulating the peptide within a rotaxane assembly was also studied in human plasma and with different proteases. Finally, in Chapter 4, we introduced the construction of a rotaxane-based molecular machine programmed to synthesise a short peptide unit from the amino acids carried on its thread. We synthesised with success a one-station model rotaxane to study the catalyst effect of the macrocycle. Unfortunately this model machine proved not to work and current research is still ongoing to achieve such a synthetic device.

Yes / Silane-substituted imidazotetrazines 1,2 were investigated for their activity as anticancer prodrugs related to temozolomide (TMZ). The TMS-derivative 1 showed an activity profile against TMZ susceptible and resistant cell lines very similar to TMZ; in contrast, the SEM-derivative 2 showed activity irrespective of MGMT expression or MMR deficiency (Table). Probing the prodrug activation mechanism by NMR kinetic studies determined that the TMS compound 1 follows a reaction pathway and time-course very similar to temozolomide. 1H-NMR spectra of the reaction mixture showed considerable incorporation of deuterium into the final alkylation products of the reaction (methanol and methyl phosphate) as had previously been shown for temozolomide (Wheelhouse, R.T., et al. Chem. Commun. 1993, 15, 1177–1178). The SEM-derivative 2 reacted more rapidly than TMZ or TMS-derivative 1. Somewhat surprisingly, the silane remained intact throughout the experiment and the observed reaction was the hydrolysis of the imidazo-tetrazine to ultimately release formaldehyde hydrate and 2-TMS-ethanol. In conclusion, TMS-derivative 1 is a diazomethane precursor with prodrug activation mechanism, kinetics and anti-cancer activity in vitro similar to TMZ. In contrast, the SEM derivative 2 was more rapidly hydrolysed, a precursor of 2-TMS-ethanol and had activity in vitro different from TMZ. 2-TMS-ethanol was previously reported as a non-toxic compound in mice (Voronkov, M.G., et al. Dokl. Akad. Nauk SSSR 1976, 229, 1011–1013) and is known as a substrate for alcohol dehydrogenase (Zong, M.-H., et al. Appl. Microbiol. Biotechnol. 1991, 36, 40–43) and as a modest inhibitor of acetylcholinesterase (Aberman, A., et al. Biochim. Biophys. Acta 1984, 791, 278–280; Cohen, S.G., et al. J. Med. Chem. 1985, 28, 1309–1313).