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Chapter 1 gives background information on proteases and discusses the concept of protease inhibition as a therapeutic strategy for humans. It introduces the key concept that conformation defines biological activity. It also outlines how proteases almost universally bind their substrate/inhibitors in an extended β-strand conformation. The use of calpain as a prototype protease for the testing of β-strand mimics synthesised later in the thesis is also discussed. Chapter 2 describes how molecular modeling was used to rationalise the structure based activity relationships (SAR) of known calpain inhibitors. Molecular modeling was then used to successfully design a number of acyclic β-strand mimics. The synthesis and testing of eight such inhibitors is described. The most potent β-strand mimic prepared was 2.13. This was determined to have an IC₅₀ of 30 nM against calpain II. Chapter 3 outlines the history and application of ring closing metathesis (RCM) to the synthesis of cyclic compounds. The attempted synthesis of an eight membered cyclic nitrogen to nitrogen conformationally constrained dipeptide is described. The synthesis of a conformationally constrained β-amino acid calpain inhibitor (3.73) is also described. A novel calpain inhibitor motif was designed in Chapter 4. On the basis of this an in-silico combinatorial library of two hundred and eighty eight possible β-strand templates was prepared. Conformational analysis of this library was performed and from this a number of excellent β-strand templates were identified and selected for synthesis. The preparation of ten β-strand templates is described. New microwave irradiation methodology was developed to achieve this. vii The formation of a six-membered catalyst deactivating chelate is also proposed to explain why some dienes fail to undergo RCM. Two methods to circumvent the formation of such a chelate are outlined. The addition of Lewis acid chloro-dicyclohexyl borane to the RCM reaction mixture and chain length alteration are investigated. Chapter 5 describes the design of macrocyclic β-strand mimics using induced fit molecular modelling. The physicochemical properties of these were calculated in-silico. From this analysis a number of Tyr-XX-Gly based and Tyr-XX-Cys based macrocyclic calpain inhibitors were selected for synthesis. The preparation and testing of these are described. In the Tyr-XX-Gly macrocyclic system a number of variables were investigated and numerous SAR implications concluded. Aldehyde 5.14 was identified as the best electrophilic warhead macrocyclic calpain inhibitor with an IC₅₀ against calpain II of 27 nM. The best non-electrophilic warhead macrocycle (5.13) had an IC₅₀ against calpain II of 704 nM. Chapter 6 describes synthetic optimisation for the preparation of calpain inhibitors 2.13, 5.14 and 5.17. Multi-gram quantities of each were prepared. Aldehydes 2.13 and 5.14 were evaluated as anti-cataract agents using in-vivo cataract sheep model. Both of these β-strand mimics were demonstrated to retard cataract development. Macrocycle 5.14 was found to be the most effective, decreasing the rate of cataract development between forty four and forty nine per cent relative to control. Chapter 7 outlines the attempted development of RCM methodology for the chiral synthesis of α-α disubstituted amino acid lactams. In addition, methodology for the stereoselective incorporation of a C-N constrained β-amino acid carbocycle into a peptide or peptidomimetic is described.

Chapter 1 gives background information on proteases and discusses the concept of protease inhibition as a therapeutic strategy for humans. It introduces the key concept that conformation defines biological activity. It also outlines how proteases almost universally bind their substrate/inhibitors in an extended β-strand conformation. The use of calpain as a prototype protease for the testing of β-strand mimics synthesised later in the thesis is also discussed. Chapter 2 describes how molecular modeling was used to rationalise the structure based activity relationships (SAR) of known calpain inhibitors. Molecular modeling was then used to successfully design a number of acyclic β-strand mimics. The synthesis and testing of eight such inhibitors is described. The most potent β-strand mimic prepared was 2.13. This was determined to have an IC₅₀ of 30 nM against calpain II. Chapter 3 outlines the history and application of ring closing metathesis (RCM) to the synthesis of cyclic compounds. The attempted synthesis of an eight membered cyclic nitrogen to nitrogen conformationally constrained dipeptide is described. The synthesis of a conformationally constrained β-amino acid calpain inhibitor (3.73) is also described. A novel calpain inhibitor motif was designed in Chapter 4. On the basis of this an in-silico combinatorial library of two hundred and eighty eight possible β-strand templates was prepared. Conformational analysis of this library was performed and from this a number of excellent β-strand templates were identified and selected for synthesis. The preparation of ten β-strand templates is described. New microwave irradiation methodology was developed to achieve this. vii The formation of a six-membered catalyst deactivating chelate is also proposed to explain why some dienes fail to undergo RCM. Two methods to circumvent the formation of such a chelate are outlined. The addition of Lewis acid chloro-dicyclohexyl borane to the RCM reaction mixture and chain length alteration are investigated. Chapter 5 describes the design of macrocyclic β-strand mimics using induced fit molecular modelling. The physicochemical properties of these were calculated in-silico. From this analysis a number of Tyr-XX-Gly based and Tyr-XX-Cys based macrocyclic calpain inhibitors were selected for synthesis. The preparation and testing of these are described. In the Tyr-XX-Gly macrocyclic system a number of variables were investigated and numerous SAR implications concluded. Aldehyde 5.14 was identified as the best electrophilic warhead macrocyclic calpain inhibitor with an IC₅₀ against calpain II of 27 nM. The best non-electrophilic warhead macrocycle (5.13) had an IC₅₀ against calpain II of 704 nM. Chapter 6 describes synthetic optimisation for the preparation of calpain inhibitors 2.13, 5.14 and 5.17. Multi-gram quantities of each were prepared. Aldehydes 2.13 and 5.14 were evaluated as anti-cataract agents using in-vivo cataract sheep model. Both of these β-strand mimics were demonstrated to retard cataract development. Macrocycle 5.14 was found to be the most effective, decreasing the rate of cataract development between forty four and forty nine per cent relative to control. Chapter 7 outlines the attempted development of RCM methodology for the chiral synthesis of α-α disubstituted amino acid lactams. In addition, methodology for the stereoselective incorporation of a C-N constrained β-amino acid carbocycle into a peptide or peptidomimetic is described.

Despite the unfavorable pharmacokinetic properties associated with peptides, they are still of great interest in drug development due to a multitude of interesting biological functions. The development of peptidomimetics strives to maintain or improve the biological activity of a peptide concurrently with removing the unwanted properties. This thesis describes two synthetic approaches to peptidomimetics with particular emphasis on secondary structure mimetics. First the design, synthesis and evaluation of two beta-turn mimetics incorporated in the endorphin Leu-enkephalin is presented. The beta-turn mimetics were stabilized by replacement of the intramolecular hydrogen bond with an ethylene bridge, and the amide bond between Tyr and Gly was replaced with an ether linkage. Linear analogues of the two mimetics were also synthesized. The peptidomimetics and their linear analogues were evaluated in a competitive binding assay at two opiate receptors, my and delta. One of the cyclized beta-turn mimetics was found to be a delta receptor antagonist with an IC50 value of 160 nM. Second a synthetic strategy to a beta-strand mimetic using 2-fluoro-4-iodopyridine as scaffold is described. The synthesis involved a Grignard exchange reaction on the pyridine scaffold using an amino acid derivative as electrophile followed by an SNAr reaction using an amine as nucleophile. The synthesis of a tripeptidomimetic of Leu-Gly-Gly and attempts to introduce chiral building blocks at the C-terminal, as well as studies towards elongated mimetics are presented. Two additional studies deal with the synthesis of two classes of potential thrombin inhibitors based on the pyridine scaffold. The first class contain pyridine as central fragment (P2 residue) substituted with a para-amidinobenzylamine group as P1 residue and various benzoyl groups as P3 residues. Three potential thrombin inhibitors were synthesized and found to be microM inhibitors in an enzymatic assay. In the second class, the pyridine ring serves as P3 residue. This class also lacks a strongly basic group in the P1 position. A small library of eight compounds were synthesized and evaluated in the enzymatic assay. Unfortunately, these compounds lacked inhibitory activity.