This thesis investigates the design, synthesis and biological assay of cysteine protease inhibitors within the papain superfamily of cysteine proteases. This is achieved by examining the effect of inhibitor design, especially warheads, on IC₅₀ values and structureactivity relationships between cysteine protease inhibitors of the papain superfamily. The representative proteases used are m-calpain, μ-calpain, cathepsin B and papain. Chapter One is an introductory chapter; Chapters Two-Four describe the design and synthesis of cysteine protease inhibitors; Chapter Five discusses assay protocol; and Chapter Six contains the assay results and structure-activity relationships of the synthesised inhibitors. Chapter One introduces cysteine proteases of the papain family and examines the structure, physiology and role in disease of papain, cathepsin B, m-calpain and μ-calpain. The close structural homology that exists between these members of the papain superfamily is identified, as well characteristics unique to each protease. Covalent reversible, covalent irreversible and non-covalent warheads are defined. The generic inhibitor scaffold of address region, recognition and warhead, upon which the inhibitors synthesised in this thesis are based, is also introduced. Chapter Two introduces reversible cysteine protease inhibitors found in the literature and that little is known about the effect of inhibitor warhead on selectivity within the papain superfamily. Oxidation of the dipeptidyl alcohols 2.6, 2.26, 2.29, 2.30, 2.35 and 2.36 utilising the sulfur trioxide-pyridine complex gave the aldehydes 2.3, 2.27, 2.19, 2.2, 2.21 and 2.22. Semicarbazones 2.37-2.40 were synthesised by a condensation reaction between the alcohol 2.3 and four available semicarbazides. The amidoximes 2.48 and 2.49 separately underwent thermal intramolecular cyclodehydration to give the 3-methyl-1,2,4- oxadiazoles 2.41 and 2.50. The aldehydes 2.3 and 2.27 were reacted with potassium cyanide to give the cyanohydrins 2.51 and 2.52. The cyanohydrins 2.51 and 2.52 were separately reacted to give 1) the α-ketotetrazoles 2.43 and 2.55; 2) the α-ketooxazolines 2.42 and 2.58; 3) the esterified cyanohydrins 2.60 and 2.61. A two step SN2 displacement reaction of the alcohol 2.6 to give the azide 2.62, an example of a non-covalent cysteine protease inhibitor. Chapter Three introduces inhibitors with irreversible warheads. The well-known examples of epoxysuccinic acids 3.1 and 3.5 are discussed in detail, highlighting the lack of irreversible cysteine protease specific inhibitors. The aldehydes 2.3 and 2.27 were reacted under Wittig conditions to give the α,β-unsaturated carbonyls 3.14-3.18. Horner- Emmons-Wadsworth methodology was utilised for the synthesis of the vinyl sulfones 3.20- 3.23. The dipeptidyl acids 2.24 and 2.28 were separately reacted with diazomethane to give the diazoketones 3.25 and 3.26. The diazoketones 3.25 and 3.26 were separately reacted with hydrogen bromide in acetic acid (33%) to give the α-bromomethyl ketones 3.27 and 3.28, which were subsequently reduced to give the α-bromomethyl alcohols 3.29-3.32. Under basic conditions the α-bromomethyl alcohols 3.29-3.32 ring-closed to form the peptidyl epoxides 3.33-3.36. Chapter Four introduces the disadvantages of peptide-based inhibitors. A discussion is given on the benefits of constraining inhibitors into the extended bioactive conformation known as a β-strand. Ring closing metathesis is utilised in the synthesis of the macrocyclic aldehyde 4.4, macrocyclic semicarbazone 4.15, the macrocyclic cyanohydrin 4.16, the macrocyclic α-ketotetrazole 4.18 and the macrocyclic azide 4.19. Chapter Five introduces enzyme inhibition studies. The BODIPY-casein fluorogenic assay used for establishing inhibitor potency against m-calpain and μ-calpain is validated. Assay protocols are also established and validated for cathepsin B, papain, pepsin and α- chymotrypsin. A discussion of the effect of solvent on enzyme activity is also included as part of this study. Chapter Six presents the assay results for all the inhibitors synthesised throughout this thesis and an extensive structure-activity relationship study between inhibitors is included. The alcohols 2.26 and 2.30 are unprecedented examples of non-covalent, potent, cathepsin B inhibitors (IC₅₀ = 0.075 μM selectivity 80-fold and 1.1 μM, selectivity 18-fold). The macrocyclic semicarbazone 4.15 is an unprecedented example of a potent macrocyclic cysteine protease inhibitor (m-calpain: IC₅₀ = 0.16 μM, selectivity 8-fold). The cyanohydrin 2.51 contains an unprecedented cysteine protease warhead and is a potent and selective inhibitor of papain (IC₅₀ = 0.030 μM, selectivity 3-fold). The O-protected cyanohydrin 2.61 is a potent and selective inhibitor of pepsin (IC₅₀ = 1.6 μM, selectivity 1.5-fold). The top ten warheads for potent, selective cathepsin B inhibition are: carboxylic acid, methyl ester, diazoketone, esterified cyanohydrin, α-bromomethyl ketone, α,β- unsaturated aldehyde, vinyl sulfones, α-bromomethyl-C₃-S,R-alcohol, alcohol and α,β- unsaturated ethyl ester. The selectivity of these warheads was between 5- and 130-fold for cathepsin B. The best inhibitors for cathepsin B were the α-bromomethyl ketone 3.26 (IC₅₀ = 0.075 μM, selectivity 16-fold), the α,β-unsaturated aldehyde 3.18 (IC₅₀ = 0.13 μM, selectivity 13-fold) and the esterified cyanohydrin 3.59 (IC₅₀ = 0.35 μM, selectivity 22- fold). Chapter Seven outlines the experimental details and synthesis of the compounds prepared in this thesis.

This thesis investigates the design, synthesis and biological assay of cysteine protease inhibitors within the papain superfamily of cysteine proteases. This is achieved by examining the effect of inhibitor design, especially warheads, on IC₅₀ values and structureactivity relationships between cysteine protease inhibitors of the papain superfamily. The representative proteases used are m-calpain, μ-calpain, cathepsin B and papain. Chapter One is an introductory chapter; Chapters Two-Four describe the design and synthesis of cysteine protease inhibitors; Chapter Five discusses assay protocol; and Chapter Six contains the assay results and structure-activity relationships of the synthesised inhibitors. Chapter One introduces cysteine proteases of the papain family and examines the structure, physiology and role in disease of papain, cathepsin B, m-calpain and μ-calpain. The close structural homology that exists between these members of the papain superfamily is identified, as well characteristics unique to each protease. Covalent reversible, covalent irreversible and non-covalent warheads are defined. The generic inhibitor scaffold of address region, recognition and warhead, upon which the inhibitors synthesised in this thesis are based, is also introduced. Chapter Two introduces reversible cysteine protease inhibitors found in the literature and that little is known about the effect of inhibitor warhead on selectivity within the papain superfamily. Oxidation of the dipeptidyl alcohols 2.6, 2.26, 2.29, 2.30, 2.35 and 2.36 utilising the sulfur trioxide-pyridine complex gave the aldehydes 2.3, 2.27, 2.19, 2.2, 2.21 and 2.22. Semicarbazones 2.37-2.40 were synthesised by a condensation reaction between the alcohol 2.3 and four available semicarbazides. The amidoximes 2.48 and 2.49 separately underwent thermal intramolecular cyclodehydration to give the 3-methyl-1,2,4- oxadiazoles 2.41 and 2.50. The aldehydes 2.3 and 2.27 were reacted with potassium cyanide to give the cyanohydrins 2.51 and 2.52. The cyanohydrins 2.51 and 2.52 were separately reacted to give 1) the α-ketotetrazoles 2.43 and 2.55; 2) the α-ketooxazolines 2.42 and 2.58; 3) the esterified cyanohydrins 2.60 and 2.61. A two step SN2 displacement reaction of the alcohol 2.6 to give the azide 2.62, an example of a non-covalent cysteine protease inhibitor. Chapter Three introduces inhibitors with irreversible warheads. The well-known examples of epoxysuccinic acids 3.1 and 3.5 are discussed in detail, highlighting the lack of irreversible cysteine protease specific inhibitors. The aldehydes 2.3 and 2.27 were reacted under Wittig conditions to give the α,β-unsaturated carbonyls 3.14-3.18. Horner- Emmons-Wadsworth methodology was utilised for the synthesis of the vinyl sulfones 3.20- 3.23. The dipeptidyl acids 2.24 and 2.28 were separately reacted with diazomethane to give the diazoketones 3.25 and 3.26. The diazoketones 3.25 and 3.26 were separately reacted with hydrogen bromide in acetic acid (33%) to give the α-bromomethyl ketones 3.27 and 3.28, which were subsequently reduced to give the α-bromomethyl alcohols 3.29-3.32. Under basic conditions the α-bromomethyl alcohols 3.29-3.32 ring-closed to form the peptidyl epoxides 3.33-3.36. Chapter Four introduces the disadvantages of peptide-based inhibitors. A discussion is given on the benefits of constraining inhibitors into the extended bioactive conformation known as a β-strand. Ring closing metathesis is utilised in the synthesis of the macrocyclic aldehyde 4.4, macrocyclic semicarbazone 4.15, the macrocyclic cyanohydrin 4.16, the macrocyclic α-ketotetrazole 4.18 and the macrocyclic azide 4.19. Chapter Five introduces enzyme inhibition studies. The BODIPY-casein fluorogenic assay used for establishing inhibitor potency against m-calpain and μ-calpain is validated. Assay protocols are also established and validated for cathepsin B, papain, pepsin and α- chymotrypsin. A discussion of the effect of solvent on enzyme activity is also included as part of this study. Chapter Six presents the assay results for all the inhibitors synthesised throughout this thesis and an extensive structure-activity relationship study between inhibitors is included. The alcohols 2.26 and 2.30 are unprecedented examples of non-covalent, potent, cathepsin B inhibitors (IC₅₀ = 0.075 μM selectivity 80-fold and 1.1 μM, selectivity 18-fold). The macrocyclic semicarbazone 4.15 is an unprecedented example of a potent macrocyclic cysteine protease inhibitor (m-calpain: IC₅₀ = 0.16 μM, selectivity 8-fold). The cyanohydrin 2.51 contains an unprecedented cysteine protease warhead and is a potent and selective inhibitor of papain (IC₅₀ = 0.030 μM, selectivity 3-fold). The O-protected cyanohydrin 2.61 is a potent and selective inhibitor of pepsin (IC₅₀ = 1.6 μM, selectivity 1.5-fold). The top ten warheads for potent, selective cathepsin B inhibition are: carboxylic acid, methyl ester, diazoketone, esterified cyanohydrin, α-bromomethyl ketone, α,β- unsaturated aldehyde, vinyl sulfones, α-bromomethyl-C₃-S,R-alcohol, alcohol and α,β- unsaturated ethyl ester. The selectivity of these warheads was between 5- and 130-fold for cathepsin B. The best inhibitors for cathepsin B were the α-bromomethyl ketone 3.26 (IC₅₀ = 0.075 μM, selectivity 16-fold), the α,β-unsaturated aldehyde 3.18 (IC₅₀ = 0.13 μM, selectivity 13-fold) and the esterified cyanohydrin 3.59 (IC₅₀ = 0.35 μM, selectivity 22- fold). Chapter Seven outlines the experimental details and synthesis of the compounds prepared in this thesis.