WO2007041775A1 - Cysteine protease inhibitors incorporating azide groups - Google Patents

Cysteine protease inhibitors incorporating azide groups Download PDF

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Publication number
WO2007041775A1
WO2007041775A1 PCT/AU2006/001478 AU2006001478W WO2007041775A1 WO 2007041775 A1 WO2007041775 A1 WO 2007041775A1 AU 2006001478 W AU2006001478 W AU 2006001478W WO 2007041775 A1 WO2007041775 A1 WO 2007041775A1
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group
cysteine protease
optionally substituted
protease inhibitor
glu
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PCT/AU2006/001478
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French (fr)
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Giang Thanh Le
Giovanni Abbenante
David Paul Fairlie
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The University Of Queensland
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Priority claimed from AU2005905564A external-priority patent/AU2005905564A0/en
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Publication of WO2007041775A1 publication Critical patent/WO2007041775A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/131—Amines acyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C247/00—Compounds containing azido groups
    • C07C247/02—Compounds containing azido groups with azido groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C247/04—Compounds containing azido groups with azido groups bound to acyclic carbon atoms of a carbon skeleton being saturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C247/00—Compounds containing azido groups
    • C07C247/02—Compounds containing azido groups with azido groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C247/12—Compounds containing azido groups with azido groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/40—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of six-membered aromatic rings
    • C07C271/42—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of six-membered aromatic rings with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/54—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of six-membered aromatic rings with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/04—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms
    • C07C275/20—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
    • C07C275/24—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an unsaturated carbon skeleton containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/42—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/22—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the nitrogen-containing ring
    • C07D217/26—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
    • C07D261/06—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members
    • C07D261/10—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D261/18—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
    • C07D307/82—Benzo [b] furans; Hydrogenated benzo [b] furans with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the hetero ring
    • C07D307/84—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C07D307/85—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated

Definitions

  • the present invention relates to methods for producing cysteine protease inhibitors.
  • the present invention also relates to compounds and compositions that modulate the function of cysteine proteases.
  • the compounds described herein may be used for the treatment of ailments, conditions or disease states that are associated with cysteine protease function or would benefit from modulation of the function of a cysteine protease.
  • Proteases also known as proteinases or peptidases, are proteolytic enzymes that break down or hydrolyse proteins. Proteases are essential for the synthesis of all proteins, controlling protein composition, size, shape, turnover and ultimate destruction. There are over 500 human proteases, accounting for 2% of human genes, and similar numbers of proteases occur in every plant, insect, marine organism and in all infectious organisms that cause disease.
  • proteases represent important potential targets for medical intervention because of their important regulatory roles in life. In addition to medical applications of protease research, there is also a potential to improve plant and animal health through enhanced growth and treatment/prevention of parasite infections, crop protection through new herbicides and pesticides, and increased or faster production of food resources.
  • proteases There are five classes of proteases categorized by the catalytic residue that effects enzymatic hydrolysis, namely cysteine, serine, aspartic, metallo and threonine enzymes. All proteases bind their substrates in a groove or cleft, where amide bond hydrolysis occurs.
  • Amino acid side chains of substrates occupy enzyme sub-sites in the groove, designated as S3, S2, S1 , SV, S2', S3', that bind to corresponding substrate/inhibitor residues P3, P2, P1 , PV, P2', P3' with respect to the cleavable amide bond.
  • the substrate amino acid residues N-terminal of the scissile amide bond are designated as ...PA, P3, P2, P1 and residues on the C- terminal side are designated PV P2' etc.
  • Cysteine proteases comprise 3 classes of structurally distinct enzymes that all hydrolyse amide bonds in their substrates via an active site cysteine. They are either papain-like (e.g. lysosomal cathepsins B,S,K,L), ICE-like (e.g. caspases 1-10, 14), or picornaviral-like.
  • Cysteine proteases account for 26% of human endopeptidase enzymes and play crucial roles in diseases, immune defence, inflammation, apoptosis, and bone resorption (Leung-Toung, R.; Li, W. R.; Tarn, T. F.; Karimian, K. C ⁇ rr. Med. Chem. 2002, 9, 979-1002). When over-expressed or unregulated in diseased states cysteine proteases are validated therapeutic targets.
  • molecules that can inhibit cysteine proteases have potential applications as medicines, diagnostics, and pesticides, as well as valuable tools for interrogating and regulating biological and physiological processes.
  • Inhibitors of cysteine proteases usually employ a reactive electrophilic group (alkylating agent, aldehyde, nitrile, ketone, ⁇ -keto-amide, halo-ketone, vinyl sulfone, chloromethane, epoxide, diazomethane, etc.) to covalently bond to the catalytic cysteinyl sulfur of the enzyme (see Powers, J. C; Asgian, J. L.; Ekici, O. D.; James, K. E. Chem. Rev. 2002, 102, 4639-4750 and Otto, H.-H.; Schirmeister, T. Chem. Rev. 1997, 97, 133-171 ).
  • a reactive electrophilic group alkylating agent, aldehyde, nitrile, ketone, ⁇ -keto-amide, halo-ketone, vinyl sulfone, chloromethane, epoxide, diazomethane, etc.
  • cysteine proteases There is a need for improved inhibitors of cysteine proteases and, in particular, for cysteine protease inhibitors that ameliorate one or more of the problems associated with known cysteine protease inhibitors.
  • the present invention arises from the discovery that alkyl azide isosteres confer potent inhibition of cysteine proteases. Inhibition is thought to be competitive, reversible and selective, relying upon multiple non-covalent interactions for enzyme affinity. Thus, incorporation of an azide group into cysteine protease inhibitor molecules provides new, less reactive and/or non- covalent inhibitors of these proteases. Furthermore, replacement of the reactive electrophile of known cysteine protease inhibitors with an azide group may also provide new, less reactive and/or non-covalent inhibitors of these proteases.
  • the present invention provides a method of producing a cysteine protease inhibitor, the method comprising incorporating an azide group into an organic molecule.
  • the azide group may be incorporated into a part of the molecule such that the azide group interacts with the cysteine protease when the molecule is bound thereto.
  • the present invention also provides a cysteine protease inhibitor produced using the aforementioned method.
  • the organic molecule comprises a cysteine protease binding moiety and an end group moiety.
  • the azide group may be incorporated into the end group moiety of the molecule.
  • cysteine protease binding moiety is an amino acid or a peptide.
  • the amino acid or peptide may have the following formula:
  • AA 1 - -AA n - wherein each AA is the same or different and each is an amino acid residue and n is an integer selected from the group consisting of 0, 2, 3, 4, and 5. In specific embodiments n is 0, 2 or 3.
  • R-CO-AA 1 - R-CO-AA 1 -, R-CO-AA 1 -AA 2 -, or R-CO-AA 1 -AA 2 -AA 3
  • AA 1 , AA 2 and AA 3 are the same or different and each is a natural or unnatural amino acid; and R CO- is a nitrogen protecting group.
  • the end group moiety may have the formula: wherein R 1 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted carboxyalkyl. In specific embodiments R 1 is selected from the group consisting of optionally substituted alkyl and optionally substituted carboxyalkyl.
  • the present invention also provides a method of modulating one or more of the activity, specificity or a biological property of a cysteine protease inhibitor containing a functional group that is capable of reacting with a nucleophile, the method comprising replacing the functional group with an azide group.
  • the functional group may be an electrophilic group.
  • electrohilic groups that may be replaced include, but are not limited to, alkylating agents, aldehydes, nitriles, ketones, ⁇ -keto-amides, halo-ketones, vinyl sulfones, chloromethanes, epoxides, diazomethanes, trifluoromethyl ketones, ⁇ -keto amides, fluoromethyl ketones, and diazoketones.
  • the modulation of a biological property of the inhibitor may result in increased efficacy when administered, decreased toxicity, decreased side-effects, increased bioavailability, or increased half-life.
  • the azide group may provide similar binding properties as the functional group it has replaced but it may not be as reactive with nucleophiles in or adjacent the binding site of the target cysteine protease or with other nucleophiles in vivo. As such the azide group may not undergo substantive covalent bonding to the target cysteine protease which may lead to reversible inhibition of the target cysteine protease.
  • the present invention also provides a method for producing a reversible cysteine protease inhibitor, the method comprising replacing a reactive functional group capable of reacting with a target cysteine protease in a inhibitor with an azide group.
  • the azide compounds produced using the methods of the present invention may inhibit the function of cysteine proteases of current pharmaceutical relevance such as, but not restricted to, caspases 1-10 and 14, lysosomal cysteine proteases such as, but not restricted to, cathepsin B, L, S, and K, Rhinovirus 3C protease, and cysteine proteases of pharmaceutical relevance that will be discovered in the future.
  • cysteine proteases of current pharmaceutical relevance such as, but not restricted to, caspases 1-10 and 14
  • lysosomal cysteine proteases such as, but not restricted to, cathepsin B, L, S, and K, Rhinovirus 3C protease, and cysteine proteases of pharmaceutical relevance that will be discovered in the future.
  • the present invention also provides a cysteine protease inhibitor of formula:
  • R 2 is an amino acid or a peptide
  • R 3 is selected from the group consisting of optionally substituted alkyl, optionally substituted carboxyalkyl, optionally substituted aryl, and optionally substituted carboxyaryl
  • R 4 is selected from the group consisting of H, optionally substituted alkyl, and optionally substituted aryl
  • R 5 is selected from the group consisting of optionally substituted alkyl, and optionally substituted aryl.
  • R 2 is an amino acid or peptide having the following formula:
  • AA 1 - -AA n -NR 6 - wherein each AA is the same or different and each is an amino acid residue and n is an integer selected from the group consisting of 0, 2, 3, 4, and 5, and R 6 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, and a nitrogen protecting group. In specific embodiments, n is 0, 2 or 3.
  • R 2 may be a peptide having one of the following formulae:
  • AA 1 , AA 2 and AA 3 are the same or different and each is a natural or unnatural amino acid including, but not restricted to, cyclohexylalanine, t- butylglycine, ⁇ -aminoisobutyric acid, ⁇ -methyl substituted amino-acids, 1- aminocyclohexane carboxylic acid, 2-aminobutyhc acid, cyclohexylalanine, t- butyl glycine, 1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, including the D and L enantiomers of these amino acids; and R 7 CO- is a nitrogen protecting group.
  • R 7 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkyloxy, optionally substituted aryl, optionally substituted aryloxy, optionally substituted amino.
  • suitable R 7 CO- groups include acyl, naphthyl, indoyl, benzyl, biphenyl, and substituted derivatives of these compounds.
  • R 3 may be -CR 8 R 9 -CO 2 R 10 , wherein R 8 , R 9 and R 10 are each independently selected from the group consisting of H, optionally substituted alkyl, and optionally substituted aryl.
  • each R 8 and R 9 is H and R 10 is selected from the group consisting of H, d-do straight chain alkyl, d-do branched chain alkyl, and aryl. More specifically, R 10 is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, benzyl, napthylmethyl, and biphenylmethyl.
  • R 3 may be -(CR 8 R 9 ) n -CH 3 , wherein each R 8 and R 9 in each CR 8 R 9 is independently selected from the group consisting of H, optionally substituted alkyl, and optionally substituted aryl, and n is an integer selected from the group consisting of O, 1 , 2, 3, 4, 5, 6, 7, 8, and 9.
  • R 3 is selected from the group consisting of -(CH 2 )3-CH 3 , and -
  • R 4 may be selected from the group consisting of H, d-do straight chain alkyl, and d-do branched chain alkyl. In specific embodiments, R 4 is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and t-butyl.
  • R 5 may be a bond or -(CR 11 R 12 V, wherein each R 11 and R 12 in each CR 11 R 12 is the same or different and is independently selected from the group consisting of H, and optionally substituted alkyl, and n is an integer from 1 to 10 inclusive. In specific embodiments, n is an integer from 1 to 5 inclusive. In specific embodiments, R 5 is -(CH 2 ) n -- In some embodiments, n is 1.
  • the compound has the formula:
  • R 13 is PG-AA 1 -, PG-AA 1 -AA 2 -, or PG-AA 1 -AA 2 - AA 3 - where AA 1 , AA 2 and AA 3 are the same or different and each is a natural or unnatural amino acid and PG is a nitrogen protecting group.
  • the compound has the formula:
  • R 13 is PG-AA 1 -, PG-AA 1 -AA 2 -, or PG-AA 1 -AA 2 - AA 3 - where AA 1 , AA 2 and AA 3 are the same or different and each is a natural or unnatural amino acid and PG is a nitrogen protecting group, and R 22 is selected from the group - 10 -
  • the peptide is selected from the group consisting of PG-VaI-AIa-, PG-GIu-AIa-, PG-Glu-Leu-, PG-Glu-Pro-, PG-Glu-Thr-, PG-GIu- HiS-, PG-Asp-Leu-, PG-GIu-VaI-, PG-AIa-AIa-, PG-GIy-AIa-, PG-AIa-GIy-, PG- Tbg-Aib-, PG-Phe-Leu-, PG-Cha-Leu-, PG-Tbg-Pro-, PG-Asp-Glu-Leu-, PG- Asp-Glu-Phe-, PG-Asp-Glu-Val-, PG-lle-Glu-Thr-, PG-lle-Glu-Pro-, PG-GIu- Glu-Leu-, PG-Glu-Leu-Leu-, PG-Leu-Leu
  • the nitrogen protecting group is selected from the group consisting of Ac,
  • the compound has one of the following formulae:
  • the azide compounds described herein are non-covalent inhibitors that do not react with the active-site amino acid or any other part of the cysteine protease enzyme. Instead, it is thought that the compounds described herein rely on multiple weak contacts (ionic, H-bonding, ⁇ -stacking, van der Waals interactions) and conformational rigidity (lowering the entropy penalty for protease binding) to impart high affinity and selective inhibition.
  • the present invention also provides a compound of formula:
  • R 19 is amino acid or peptide
  • R 20 is selected from the group consisting of H, optionally substituted alkyl, and optionally substituted aryl
  • R 21 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, and a nitrogen protecting group.
  • R 19 is PG-AA 1 -AA 2 - or PG-AA 1 -AA 2 - AA 3 - where AA 1 , AA 2 and AA 3 are the same or different and each is a natural or unnatural amino acid, and PG is a nitrogen protecting group. Specific peptides include those listed previously. R 20 and R 21 may also be H.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an azide compound described herein, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention further provides the use of an azide compound described herein in the preparation of a medicament for the treatment of a disease state in which the disease pathology may be therapeutically modified by inhibiting a cysteine protease.
  • the present invention further provides a method of inhibiting a cysteine protease comprising administering to a subject an effective amount of an azide compound described herein.
  • the present invention further provides a method of treating a disease state in which the disease pathology may be therapeutically modified by inhibiting a cysteine protease, the method comprising administering to a subject in need thereof an effective amount of an azide compound described herein.
  • the present invention further provides the use of an azide compound described herein in the preparation of a medicament for the treatment of a disease state in which the disease pathology may be therapeutically modified by modulating the activity of a cysteine protease.
  • the present invention further provides a method of modulating the activity of a cysteine protease comprising administering to a subject an effective amount of an azide compound described herein.
  • the present invention further provides a method of treating a disease state in which the disease pathology may be therapeutically modified by modulating the activity of a cysteine protease, the method comprising administering to a subject in need thereof an effective amount of an azide compound described herein.
  • the present invention further provides a method of treating a disease characterised by bone loss including administering to a subject in need thereof an effective amount of an azide compound described herein.
  • the present invention further provides a method of treating a disease characterised by excessive cartilage or matrix degradation including administering to a subject in need thereof an effective amount of an azide compound described herein.
  • references in this specification to a specific active ingredient is also to be understood to include the active ingredient in the form of acid addition salts, solvates and hydrates.
  • the compounds of the present invention may be used in the form of salts derived from inorganic or organic acids. Included among such acid salts, for example, are the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate,
  • This invention also envisions the quaternization of any basic nitrogen- containing groups of the compounds disclosed herein.
  • the basic nitrogen can be quaternized with any agents known to those of ordinary skill in the art including, for example, lower alkyl halides, such as methyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkyl sulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myhstyl and stearyl chlorides, bromides and iodides; and aralkyl halides including benzyl and phenethyl bromides. Water or oil-soluble or dispersible products may be obtained by such quaternization.
  • azide group may be added to an exisiting molecule using reactions (such as substitution reaction) as discussed further herein.
  • the azide group could be part of a molecule or reactant that is used in a synthetic scheme for producing a cysteine protease inhibitor molecule.
  • alkyl as used herein includes both straight and branched chain radicals of up to 12 carbons, preferably 1-8 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, 1-ethylpropyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl and dodecyl.
  • Ci to Cs alkyl as used herein is meant to include substituted and unsubstituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl, pentyl, n-pentyl, isopentyl, and neopentyl.
  • Any alkyl group may be optionally substituted independently by one, two or three halogens, SR', hydroxy, OR', N(R 1 J 2 , C(O)N(R 1 J 2 , where R' is H or Ci to C 6 alkyl, carbamyl, trifluoromethyl, nitro, carboxy, Ci to C 6 alkyl, C 6 to do aryl, Ci to C 6 alkoxy, Ci to C 6 aminoalkyl, Ci to C 6 aminoalkoxy, C 2 to C 6 alkoxycarbonyl, Ci to C 6 hydroxyalkyl, Ci to C 6 hydroxyalkoxy, Ci to C 6 alkylsulfonyl, C 6 to do arylsulfonyl, Ci to C 6 alkylsulfinyl, Ci to C 6 alkylsulfonamido, C 6 to do arylsulfonamido, C 6 to do aryl(Ci to C 6 )alkylsulfonamid
  • alkyl as used herein also includes within its scope saturated cyclic hydrocarbon groups containing 3 to 12 carbons, preferably 3 to 8 carbons.
  • exemplary cycloalkyl groups include, but are not restricted to, substituted and unsubstituted cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, cycloundecane and cyclododecane.
  • the cyclic structure may be substituted with substituents such as halogen, Ci to C 6 alkyl, Ci to C 6 alkoxy, hydroxy groups or amino groups.
  • alkenyl as used herein means an alkyl group wherein a carbon- carbon single bond is replaced by a carbon-carbon double bond.
  • C 2 to C 6 alkenyl includes ethylene, 1 -propene, 2-propene, 1-butene, 2-butene, isobutene and the several isomeric pentenes and hexenes. Both cis and trans isomers are included.
  • alkynyl as used herein means an alkyl group wherein one carbon- carbon single bond is replaced by a carbon-carbon triple bond.
  • C 2 to C 6 alkynyl includes acetylene, 1 -propyne, 2-propyne, 1 -butyne, 2-butyne, 3- butyne and the simple isomers of pentyne and hexyne.
  • aryl and “Ar” as used herein mean monocyclic or bicyclic aromatic groups containing from 6 to 12 carbons in the ring portion, preferably 6-10 carbons in the ring portion. Examples include, but are not restricted to, phenyl, naphthyl and tetrahydronaphthyl.
  • heterocyclic and "Het” as used herein mean a stable 5- to 7- membered monocyclic or a stable 7- to 10-membered bicyclic heterocyclic ring, which is either saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring.
  • the heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure, and may optionally be substituted with one or more moieties selected from Ci to C 4 alkyl, OR', N(R 1 J 2 , SR', CF 3 , NO 2 , CN, CO 2 R', CON(R'), F, Cl, Br and I, where R' is alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl and acyl, each of which may be optionally substituted.
  • heterocycles include piperidinyl, piperazinyl, 2- oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, pyridyl, pyrazinyl, oxazolidinyl, oxazolinyl, oxazolyl, isoxazolyl, morpholinyl, thiazolidinyl, thiazolinyl, thiazolyl, quinuclidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, benzoxazolyl, furyl, pyranyl, tetrahydrofuryl, tetrahydropyr
  • heteroaryl as used herein means any heterocyclic moiety encompassed by the above definition of Het having 5 to 14 ring atoms, preferably 5, 6, 9 or 10 ring atoms; 6, 10 or 14 ⁇ electrons shared in a cyclic array; and containing carbon atoms and 1 , 2 or 3 oxygen, nitrogen or sulfur heteroatoms.
  • heteroaryl groups are: thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl, xanthenyl, phenoxathiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, tetrazolyl, quinazolinyl, cinnolinyl, pteridinyl,
  • aromatickyl or "arylalkyl” as used herein by itself or as part of another group refers to Ci to C 6 alkyl groups as discussed above having an aryl substituent, such as benzyl, phenylethyl or 2-naphthylmethyl.
  • alkaryl or “alkylaryl” as used herein by itself or as part of another group refers to an aryl group as discussed above having a Ci to C 6 alkyl substituent, such as toluyl, ethylphenyl, or methylnaphthyl.
  • carboxyalkyl refers to a group of formula -alkyl- C(O)O-R', where R' is is alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, and heteroarylalkyl, each of which may be optionally substituted
  • alkoxy refers to the above alkyl groups linked to oxygen.
  • halogen or "halo” as used herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine.
  • amino refers to formylamino, alkylcarbonylamino or arylcarbonylamino.
  • nucleophile and variants thereof as used herein means an atom or substance which is attracted to centres of positive charge and which donates a pair of electrons when it reacts with substrate.
  • nucleophiles include O, S and N atoms that are present in biological molecules.
  • inhibitor and variants thereof as used herein means a reduction or inhibition of a process, including the start, continuation or termination of a process, and in the context of the present invention this term includes an adverse affect on the enzymatic activity of a cysteine protease.
  • a cysteine protease To determine whether or not the activity of a cysteine protease is reduced, one can use any one of the methods described in the examples provided in this specification, or any one of the methods known for that purpose in the art.
  • module means a change or alteration of a process, including the start, continuation or termination of a process, and in the context of the present invention this term includes a change or alteration in the activity of a cysteine protease.
  • subject means any multicellular organism, including a human, plant or an animal subject.
  • the subject organism may be a mammal, a primate, a livestock animal (eg. a horse, a cow, a sheep, a pig, or a goat), a companion animal (eg. a dog, a cat), a laboratory test animal (eg. a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance.
  • a livestock animal eg. a horse, a cow, a sheep, a pig, or a goat
  • a companion animal eg. a dog, a cat
  • a laboratory test animal eg. a mouse, a rat, a guinea pig, a bird
  • the plant may be for example a commercial crop species (eg barley, oat, millet, alfalfa), a leguminous plant (eg soybean, alfalfa, and pea), a non-leguminous plants (e.g., corn, wheat, and cotton), or an angiosperm or cereal.
  • a commercial crop species eg barley, oat, millet, alfalfa
  • a leguminous plant eg soybean, alfalfa, and pea
  • a non-leguminous plants e.g., corn, wheat, and cotton
  • angiosperm or cereal e.g., a commercial crop species (eg barley, oat, millet, alfalfa), a leguminous plant (eg soybean, alfalfa, and pea), a non-leguminous plants (e.g., corn, wheat, and cotton), or an angiosperm or cereal.
  • the present invention includes all hydrates, solvates, complexes and prodrugs of the compounds described herein.
  • Prodrugs are any covalently bonded compounds which release the active parent drug in vivo.
  • isomeric forms including diastereoisomers, enantiomers, tautomers, and geometrical isomers in "E” or “Z” configurational isomer or a mixture of E and Z isomers. It is also understood that some isomeric forms such as diastereomers, enantiomers, and geometrical isomers can be separated by physical and/or chemical methods and by those skilled in the art.
  • Some of the compounds of the disclosed embodiments may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates and mixtures thereof are intended to be within the scope of the subject matter described and claimed.
  • each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.
  • amino acid is a generic designation for groups resulting from replacement of at least one hydrogen atom in the parent structure of carboxylic acid by an amino group, including alpha-, beta-, gamma- and delta-amino acids having a parent structure with 2 to 20 carbon atoms.
  • alpha-amino acids are preferred, including "natural" amino acids that are protein components such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, leucine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; and other "unnatural” or "non-natural” amino acids having a variety of substituents at the ⁇ -carbon in the case of ⁇ -amino acids, or at the ⁇ - or ⁇ -carbon in the case of ⁇ -amino acid, such as norvaline, norleucine, t-butylglycine (tert- leucine), 2-aminoadipic acid, 2-aminobutyric acid, 2-aminoisobutyhc acid, 1 - aminocyclopropanecar
  • amino acid includes within its scope cyclic imino acids.
  • the cyclic imino acid is an optionally substituted cycloalkane carboxylic acid or an optionally substituted cycloalkene carboxylic acid which at least one of the methylene groups is substituted, specifically, such as proline, hydroxyproline, 3,4-dehydroproline, pipecoline acid, adilidine carboxylic acid and 2-azetidine carboxylic acid.
  • amino acid or the abbreviation "AA” includes within its scope protected amino acids.
  • Protected amino acids include N-protected and/or C-protected amino acids. Examples of nitrogen and carboxy protecting groups are found in W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3 rd edition, John Wiley & Sons, New York, 1999.
  • GIy or G Glycine
  • Ala or A Alanine
  • VaI or V Valine
  • Leu or L Leucine
  • He or I Isoleucine
  • Ser or S Serine
  • Thr or T Threonine
  • Cys or C Cysteine
  • Met or M Methionine
  • GIu or E Glutamic acid
  • Asp or D Aspartic acid
  • Lys or K Lysine
  • Arg or R Arginine
  • His or H Histidine
  • Asn or N Asparagine; GIn or Q: Glutamine; Ach: 1 -aminocyclohexane carboxylic acid; Abu: 2-aminobutyric acid; Aib: Aminoisobutyric acid; Cha : cyclohexylalanine; Tbg: t-butyl glycine; Tic: 1 ,2,3,4-tetrahydroisoquinoline-3- carboxylic acid; HCTLJ: 1 H-Benzotriazolium 1-[bis(dimethylamino)methylene] -5chloro-,hexafluorophosphate (1-),3-oxide; Fmoc: 9- fluorenylmethoxycarbonyl; Ac: Acetyl; Cbz or Z: Benzyloxycarbonyl; Boc: t- butoxycarbonyl; BzI: Benzyl.
  • Nitrogen protecting group and "PG” (when bonded to a nitrogen atom) as used herein mean groups known in the art that are readily introduced on to and removed from a nitrogen atom.
  • Nitrogen protecting groups are generally of the formula RCO- wherein R is selected from the group consisting of optionally substituted alkyl, optionally substituted alkyloxy, optionally substituted aryl, optionally substituted aryloxy, optionally substituted amino.
  • nitrogen protecting groups include formyl, acetyl (Ac), trifluoroacetyl, benzyl, benzoyl, benzyloxycarbonyl (Cbz or Z), tert- butoxycarbonyl (Boc), thmethyl silyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted trityl groups, allyloxycarbonyl, 9- fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC) and the like.
  • Other examples of acceptable nitrogen protecting groups are found in W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3 rd edition, John Wiley & Sons, New York, 1999.
  • the present invention arises from the inventors studies in relation to the development of reversible and/or non-covalent inhibitors of cysteine proteases.
  • Inhibitors of cysteine proteases have historically been designed mainly using iterative substrate- and structure-based methodologies (crystal structures and computer-assisted molecular modelling) to derivatise peptide fragments adjacent to the hydrolysable amide bond of substrates.
  • At the heart of the design strategy has been the incorporation of an electrophile at the C-terminus to covalently interact with the nucleophilic thiol of the catalytic cysteine. This covalent reaction generally increases enzyme affinity by 10 3 -10 4 times over unmodified peptide.
  • These reactive electrophilic functionalities include, but are not limited to, aldehydes, ketones, trifluoromethyl ketones, ⁇ -keto amides, nitriles, fluoromethyl ketones, trifluoromethyl ketones and diazoketones.
  • the caspases are a sub-class of cysteine proteases that recognize polypeptide substrates containing aspartate immediately N-terminal to the putative cleavage site. At least eleven human caspases (Caspases 1-10, 14) are known (Pistritto, G. et al., Cell Death Differ 2002, 9, 995-1006).
  • Caspase 1 produces the pro-inflammatory cytokine interleukin-1 ⁇ (IL-1 ⁇ ) from its precursor peptide. When released from monocytes, IL-1 ⁇ stimulates leukocyte adhesion and activation as well as other inflammatory responses. Inhibitors of ICE decrease IL-1 ⁇ production in vitro and in vivo, and have therapeutic potential for inflammatory diseases such as rheumatoid and osteoarthritis (Leung-Toung, R. et al., Current Medicinal Chemistry 2002, 9, 979- 1002).
  • Caspase 3 (apopain, CPP32) is a key executioner in apoptosis. Inhibitors are predicted to be useful therapeutics for aging, neurodegenerative diseases (Alzheimer's disease), stroke and myocardial infarction, and cancer (Talanian, R. V. et al., Journal of Medicinal Chemistry 2000, 43, 3351 -3371 ).
  • Cathepsin K is selectively expressed in osteoclasts where it represents 98% of the total cysteine protease activity and is primarily involved in bone resorption.
  • X-ray structures of inhibitors bound to its active site indicate that the S2 subsite is the most important for selectivity with respect to other lysosomal proteases (cathepsins L, S, B etc.).
  • Inhibitors are currently thought to be promising therapeutics for the treatment of diseases characterized by excessive bone loss such as osteoporosis (McGrath, M. E. Annual Review of Biophysics and Biomolecular Structure 1999, 28, 181 -204).
  • Cathepsin S plays an important role in regulating antigen presentation and immunity. Inhibition of antigen presentation via prevention of invariant chain degradation by cathepsin S may provide a mechanism for immuno-regulation. Control of antigen-specific immune responses has long been desirable as a useful and safe therapy for autoimmune diseases, such as Crohn's disease and arthritis, as well as other T-cell-mediated immune responses. Cathepsin S has also been implicated in a variety of other diseases involving extracellular proteolysis, such as Alzheimer's disease and atherosclerosis.
  • Cathepsin B plays a number of roles inside cells to maintain normal cellular metabolism. Overexpression of cathepsin B has been associated with pathophysiological conditions such as tumor metastasis, inflammation, bone resorption, and myocardial infarction (Yan, S. et al., Biol. Chem. 1998, 379, 113-123).
  • the present invention provides a method of producing a cysteine protease inhibitor, the method comprising incorporating an azide group into an organic molecule.
  • the organic molecule may be a cysteine protease inhibitor molecule, or it may be a molecule that does not have cysteine protease inhibitory properties until the azide group is incorporated in the molecule.
  • the organic molecule may contain a cysteine protease binding moiety which, in itself, shows little or no activity toward the target protease until the azide group is incorporated into the molecule.
  • the azide group may be incorporated into the organic inhibitor molecule by reacting a functional group in a molecule to form an azide.
  • the azide based cysteine protease inhibitor may be formed by substituting a leaving group in a molecule with an azide group. The substitution may be carried out by reacting a molecule containing a leaving group (such as a halide or a functionalised hydroxy) with a source of azide anion (N 3 " ) under appropriate reaction conditions.
  • the azide group may be a part of a precusor molecule which is further reacted to form the cysteine protease inhibitor.
  • the azide group may be incorporated into a part of the molecule such that the azide group interacts with the cysteine protease when the inhibitor molecule is bound thereto. Without intending to be bound by theory, it is possible that the azide group of the azide based cysteine protease inhibitors may be involved in non-covalent electrostatic interactions with cysteinyl-S or imidazole-N in the active site of the target cysteine protease inhibitor.
  • the cysteine protease inhibitor molecule may comprise a cysteine protease binding moiety and an end group moiety.
  • the azide group may be incorporated into the end group moiety of the molecule.
  • AA 1 - -AA n - wherein each AA is the same or different and each is an amino acid residue and n is an integer selected from the group consisting of 0, 2, 3, 4, and 5. In specific embodiments n is 0, 2 or 3.
  • amino acid or peptide is covalently bonded via a carbon atom to the azide group.
  • R-CO-AA 1 - R-CO-AA 1 -, R-CO-AA 1 -AA 2 -, or R-CO-AA 1 -AA 2 -AA 3
  • AA 1 , AA 2 and AA 3 are the same or different and each is a natural or unnatural amino acid; and R CO- is a nitrogen protecting group.
  • the end group moiety may have the formula:
  • R 1 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkenyl, optionally - 26 -
  • R 1 is selected from the group consisting of optionally substituted alkyl and optionally substituted carboxyalkyl.
  • cysteine protease inhibitors are shown in the following table. Modification of any one of these known inhibitors by replacement of an electronhilic group with an azide group may provide a cysteine protease inhibitor having improved properties over the parent inhibitor.
  • the reactive electrophilic group of a cysteine protease inhibitor may be "replaced” by reacting the electrophilic group under appropriate conditions to produce an azide group.
  • the reaction to produce the azide group may be carried out on the protease inhibitor itself, on a derivative of the inhibitor, or on a precursor of the inhibitor.
  • Reactions suitable for converting an electrophilic group to an azide group may be determined by the skilled person having regard to the nature of the electrophilic group. For example, an aldehyde group may be reduced using a reducing agent to produce a secondary alcohol. The secondary alcohol may be converted to a leaving group and subsequently reacted with a source of azide anion (N3 " ). An example of the latter reaction is shown in Example 2.5.1 of this specification. Reference may be made to "Advanced Organic Chemistry” Jerry March 4 th Edn. pp 351-357, Oak Wick and Sons NY (1997).
  • the activity of a cysteine protease inhibitor may be modulated by incorporating an azide group in the inhibitor molecule.
  • the activity of a particular azide inhibitor can be determined using tests that are known for that purpose in the art and/or any one of the tests described in the Examples provided in this specification. Modulation of the activity of a cysteine protease inhibitor may involve an increase or a decrease in the activity relative to a parent or control inhibitor. Preferably, the activity is increased, but in cases where the activity is decreased, there may be other advantages in incorporation of an azide group, such as decreased side effects, increased metabolic stability, etc.
  • the specificity of a cysteine protease inhibitor may be modulated by incorporating an azide group in the inhibitor molecule.
  • the specificity of a particular azide based cysteine protease inhibitor for a particular cysteine protease can be determined using tests that are known for that purpose in the art and/or any one of the tests described in the Examples provided in this specification.
  • cysteine protease inhibitor that may be modulated by preparing azide based cysteine protease inhibitors are toxicity, side-effects, bioavailability, half-life, metabolic stability or solubility.
  • the potency of the azide derivative may be reduced relative to the parent inhibitor.
  • other properties of the inhibitor e.g. bioavailability, solubility, etc
  • any reduced potency of an azide inhibitor may be recovered by appropariate structural variations. For example, in the case of the azide derivative of the known inhibitor 101 , sequential variation at P3 and P4 positions may lead to a more potent inhibitor.
  • cysteine protease inhibitors As discussed, we have prepared a number of azide-based cysteine protease inhibitors and shown that they are active both in vivo and in vitro.
  • the present cysteine protease inhibitors have the general formula:
  • R 2 is an amino acid or a peptide
  • R 3 is selected from the group consisting of optionally substituted alkyl, optionally substituted carboxyalkyl, optionally substituted aryl, and optionally substituted carboxyaryl
  • R 4 is selected from the group consisting of H, optionally substituted alkyl, and optionally substituted aryl
  • R 5 is selected from the group consisting of optionally substituted alkyl, and optionally substituted aryl.
  • R 13 is PG-AA 1 -, PG-AA 1 -AA 2 -, or PG-AA 1 -AA 2 - AA 3 - where AA 1 , AA 2 and AA 3 are the same or different and each is a natural or unnatural amino acid and PG is a nitrogen protecting group.
  • Suitable peptides include PG-VaI-AIa-, PG-GIu-AIa-, PG-Glu-Leu-, PG-Glu-Pro-, PG-Glu-Thr-, PG-Glu-His-, PG-Asp-Leu-, PG-GIu-VaI-, PG-AIa- AIa-, PG-GIy-AIa-, PG-AIa-GIy-, PG-Tbg-Aib-, PG-Phe-Leu-, PG-Cha-Leu-, PG-Tbg-Pro-, PG-Asp-Glu-Leu-, PG-Asp-Glu-Phe-, PG-Asp-Glu-Val-, PG-IIe- Glu-Thr-, PG-lle-Glu-Pro-, PG-Glu-Glu-Leu-, PG-Glu-Leu-Leu-, PG-Leu-Glu- Leu-,
  • the nitrogen protecting group (PG) may be selected from the group consisting of Ac, - 36 -
  • the present invention also provides a compound of formula:
  • R 19 is selected from the group consisting of amino acid, and peptide.
  • R 19 may be PG-AA 1 -AA 2 - or PG-AA 1 -AA 2 - AA 3 - where AA 1 , AA 2 and AA 3 are the same or different and each is a natural or unnatural amino acid and PG is a nitrogen protecting group.
  • the compound may have the formula:
  • R 13 is PG-AA 1 -, PG-AA 1 -AA 2 -, or PG-AA 1 -AA 2 - AA 3 - where AA 1 , AA 2 and AA 3 are the same or different and each is a natural or unnatural amino acid and PG is a nitrogen protecting group, and R 22 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkenyl, and optionally substituted alkynyl.
  • the amino acid- or peptide-based compounds of the present invention may be prepared by coupling the azidomethylene derivative of aspartic acid with a solid phase resin, such as thtylchloride polystyrene resin (TCP resin).
  • TCP resin thtylchloride polystyrene resin
  • the resin loaded with the azidomethylene derivative of aspartic acid can be N-deprotected and acylated with the desired N-protected amino acid. Further rounds of N-deprotection and acylation with suitable amino acids can be used to prepare the desired peptide. Following N-protection with a nitrogen protecting group of choice, the peptide can be cleaved and purified.
  • TCP resin thtylchloride polystyrene resin
  • the intermediates, products and final products obtained by the above synthesis reactions can be isolated and purified by conventional methods for separation and purification, such as chromatography, concentration, vacuum concentration, solvent extraction, crystallization, recrystallization, etc.
  • the compounds of the present invention are useful for the inhibition of cysteine proteases.
  • the terms “inhibit” and “inhibition” mean having an adverse effect on enzymatic activity.
  • An inhibitory amount is an amount of a compound of the invention effective to inhibit a cysteine protease.
  • the present invention further provides a method of inhibiting a cysteine protease including administering to a patient in need thereof an effective amount of an azide compound described herein.
  • the present invention also provides a method of treating a disease state in which the disease pathology may be therapeutically modified by inhibiting a cysteine protease including administering to a patient in need thereof an effective amount of an azide compound described herein.
  • the compounds of the present invention, or pharmacologically acceptable esters or a salts thereof, having activity of inhibiting cysteine protease is safe with low toxicity, and can be used to treat and prevent various infectious diseases, immune diseases, bone diseases, neurologic diseases, tumors, inflammatory diseases etc., including meningitis, salpingitis, enteritis, inflammatory enteritis, hyperacidic enteritis, sepsis, septic shock, disseminated intravascular coagulation, adult respiratory distress, arthritis, bile duct disease, colitis, encephalitis, endocarditis, glomerular nephritis, hepatitis, myo
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an azide compound described herein, and a pharmaceutically acceptable carrier, diluent or excipient.
  • a pharmaceutically acceptable carrier diluent or excipient.
  • the compound(s) of the present invention, or a pharmaceutically acceptable ester or a salt thereof, formulated with a pharmaceutically acceptable carrier can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the composition may be in the form of solid preparations such as a tablets, capsules, granules or powders, or liquid preparations such as syrups and aqueous suspensions.
  • carriers which are commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried corn starch.
  • the composition may be an injectable preparation in the form of a a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1 ,3-butanediol.
  • suitable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. HeIv or a similar alcohol.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • compositions of this invention may also be administered in the form of suppositories for rectal administration.
  • These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • suitable non-irritating excipient include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
  • Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application.
  • the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier.
  • Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.
  • the pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically- transdermal patches are also included in this invention.
  • compositions of this invention may be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
  • the prophylactic/therapeutic preparation of the present invention can be produced by commonly used methods such as mixing, kneading, granulation, tableting, coating, sterilization and emulsification, depending on preparation form.
  • the content of the azide compounds in the preparation of the present invention is normally about 0.01 to 100% by weight, preferably about 0.1 to 50% by weight, and more preferably about 0.5 to 20% by weight, relative to the entire preparation, depending on preparation form.
  • Pharmaceutically acceptable carriers are various organic or inorganic carrier substances in common use as pharmaceutical materials, including excipients, lubricants, binders and disintegrating agents for solid preparations, and solvents, dissolution aids, suspending agents, isotonizing agents, buffers and smoothing agents for liquid preparations. Other pharmaceutical additives such as preservatives, antioxidants, coloring agents and sweetening agents may be used as necessary.
  • Preferable excipients include lactose, sucrose, D-mannitol, starch, crystalline cellulose and light silicic anhydride.
  • Preferable lubricants include magnesium stearate, calcium stearate, talc and colloidal silica.
  • Preferable binders include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose and polyvinylpyrrolidone.
  • Preferable disintegrating agents include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, crosscarmellose sodium and carboxymethyl starch sodium.
  • Preferable solvents include water for injection, alcohol, propylene glycol, macrogol, sesame oil and corn oil.
  • Preferable dissolution aids include polyethylene glycol, propylene glycol, D- mannitol, benzyl benzoate, ethanol, tris-aminomethane, cholesterol, triethanolamine, sodium carbonate and sodium citrate.
  • Preferable suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride and monosteahc glycerol, and hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose sodium, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose.
  • Preferable isotonizing agents include sodium chloride, glycerol and D-mannitol.
  • Preferable buffers include buffer solutions of phosphates, acetates, carbonates and citrates.
  • Preferable smoothing agents include benzyl alcohol.
  • Preferable preservatives include p-oxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid and sorbic acid.
  • Preferable antioxidants include sulfites and ascorbic acid.
  • the compound of the present invention may be orally or non-orally administered to humans and other animals suffering from various diseases as described above, at doses of, for example, 0.01 to 500 mg/day, preferably 0.1 to 50 mg/day, more preferably 1 to 20 mg/day, per kg body weight.
  • the dose varies depending on kind of compound, route of administration, symptoms, patient age etc., it can be administered at 0.1 to 50 mg/day, preferably 1 to 20 mg/day, per kg of body weight, in 1 to 6 portions, for oral administration to an adult patient.
  • a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • the azide compounds of the present invention may be co-administered with other therapeutically active agents, as required.
  • the inhibitors described herein may also be used to identify and purify cysteine proteases.
  • the inhibitors can be covalently linked to a solid support, such as an affinity column or beads used in batch methods, and used to purify a cysteine protease or enrich a mixture containing the cysteine protease.
  • the inhibitor may be linked to the solid support or bead either directly or via a linker of variable length, such that linkage does not interfere with the binding properties (see, Thomberry, N., Methods in Enz., 244:615-31 (1994)).
  • Figure 1 shows a Lineweaver-Burk plot for varying substrate AcYVAD-AMC (S) and inhibitor AcWEHD-N 3 . Concentrations at pH 7.4 (5OmM HEPES).
  • Figure 2 shows a plot that provides experimental confirmation that inhibitor 13 binds in a reversible manner to caspase-1.
  • the addition of 5OnM of inhibitor 13 completely abolishes caspase-1 processing of the substrate Ac- YVAD-AMC.
  • the activity of the enzyme can be restored by the addition of 2Ox the original concentration of substrate to the fully inhibited enzyme.
  • Figure 3 shows (A) modeled 13 docked in the active site of caspase-1 (pdb 1 1CE), and (B) backbone (C, N, O atoms) supehmposition of 13 on enzyme-bound structure of Ac-YVAD-H.
  • Figure 4 shows selected 1 H-NMR signals for aldehyde 101 (left panel) and azide 13 (right panel) in H 2 O:D 2 O (9:1 , phosphate buffer pH 7.4,
  • Example 1 - Azido based inhibitors Compounds 1 to 46, 49 and 51 to 100 were synthesised using the procedures set out in Example 2 below.
  • Tritylchloride-polystyrene (TCP) resin was obtained from PepChem (Reutlingen, Germany).
  • 0-(1 H-6-Chlorobenzotriazole-1-yl)-1 , 1 ,3,3- tetramethyluronium hexafluorphosphate (HCTLJ) was obtained from Iris Biotech (Marktredwitz, Germany).
  • Fmoc protected amino acids and all other reagents were of peptide synthesis grade and obtained from Auspep (Melbourne, Australia).
  • Mass spectra were obtained on a hybrid quadrupole TOF mass spectrometer (PE SCIEX API QSTAR Pulsar) equipped with an lonspray (pneumatically assisted electrospray) operating at ambient temperature (ISMS). Calibration standard was ammonium adducts of polypropylene glycol (PPG 1000).
  • Fmoc-protected amino acids 4 equivalents of HCTU and two equivalents of DIPEA were employed in each coupling. Fmoc-deprotections and resin neutralization was achieved by 2 x 2 min. treatments with excess 1 :1 piperidine:DMF. Coupling yields were monitored by quantitative ninhydrin assay (Sarin, V. et al., Anal. Biochemistry 1981 , 117, 147) and double couplings were employed for yields below 99.8%. N-terminal acetylation was achieved by treating the fully assembled and protected peptide-resins (after removal of the N-terminal Fmoc group) with HCTU-activated acetic acid as per normal coupling.
  • the peptide was cleaved from resin and protecting groups simultaneously removed by treatment for 2h at room temperature with a solution containing 95% trifluoroacetic acid (TFA):2.5% H 2 O: 2.5% triisopropylsilane (TIPS) (25ml_ solution per 1 gm of peptide-resin).
  • TFA trifluoroacetic acid
  • TIPS triisopropylsilane
  • the TFA solutions were filtered, concentrated in vacuo, diluted with 50% A:50% B, lyophilized, and subsequently purified by semi-preparative rp-HPLC. All products were identified by electron spray mass spectrometer.
  • Acid II (2.2g, 6mmol) was mixed with thtylchloride polystyrene resin (TCP resin; PepChem; product #: PC-01-0010) (4g @ 0.94mmol/g; 3.76mmol) in 9ml of dry DMF.
  • DIPEA (2.08ml, 12mmol) was added and the resin was shaken at room temperature for 16 hours. The solvent was removed and the resin was washed with DMF (three times), methanol (three times) and DCM (three times) then dried under high vacuum.
  • the azidomethylene derivative of L-Fmoc-aspartic acid was prepared by deprotection of the corresponding tert-butyl ester (Boeijen, A; van Ameijde, J; Liskamp R. M. J. J. Org. Chem. 2001 , 6, 8454-8462) and loaded onto TCP resin. After peptide synthesis to achieve the desired sequence, compounds 1- 46, 49 and 51 -83 were cleaved and purified as described above.
  • the hydroxyl derivative VII of protected L-Boc-norleucine acid was prepared as described (Catalano, JG at al. Bioorg. Med. Chem. Lett. 2004, 14, 275- 278).
  • Azide VIII (310mg, 1.28mmol) was dissolved in a solution containing 4.5ml of TFA and 0.5ml of water. The solution was stirred at room temperature for 2 hours and the volatile solvents were removed under vacuum. To the residue 15ml of toluene was added and then evaporated to dryness. The residue was dried until constant weight under high vacuum to give the TFA salt of the norleucine azide derivative.
  • L-Boc-leucine monohydrate (638.2mg, 2.56mmol) was dissolved in DMF (5ml), HBTU (971 mg, 2.56mmol) and DIPEA (330mg, 2.56mmol) was added.
  • TFA salt of LeU-NIe-N 3 derivative was acylated with 2-naphthoic acid (440.3mg, 2.56mmol), which were activated with HBTU (971 mg, 2.56mmol) and DIPEA (660mg, 5.12 mmol).
  • the acylation was allowed to proceed overnight at room temperature, then diluted with 100ml of ethylacetate and 100ml of saturated bicarbonate. After separation the aqueous phase was extracted twice with 50ml of ethylacetate. The combined ethylacetate solution were dried over MgSO 4 and evaporated to dryness. The residue was purified by gravity chromatography using petroleum/ethylacetate (1/1 ) as eluant. Re- crystallization from petroleum/ethylacetate afforded pure compound 86 as white solid (248mg, 47.3%). A less pure fraction of 100mg (19.1 %) was also obtained. - 64 -
  • Boc-protected azide IX was synthesized according to the method of Kokotos et.al.; Journal of Chemical Research, Synopses; 1992, 12, 391.
  • the azide IX (60mg, 0.25mmol) was dissolved in TFA (1 OmL) and stirred for 15 minutes at room temperature.
  • TFA was evaporated off with a stream of nitrogen and to the residue was added Boc-Phe-OH (100mg, 0.38mmol), BOP (168mg, 0.38mmol), DMF (5ml) and finally diisopropylethylamine (0.2ml) and the solution stirred overnight.
  • the solution was quenched with 10% KHSO 4 solution and extracted with ethyl acetate (3x30ml_).
  • the combined ethyl acetate solution was washed with 10% KHSO 4 (1x 3OmL), sat.
  • Inhibitor 13 also occupies very similar conformational space as the known Ac-YVAD-H aldehyde inhibitor (101 ) (Fig. 3B), its backbone atoms (C, N, O) superimposing well (rmsd 0.37A) on the corresponding atoms of 101 in its crystal structure with caspase-1.
  • the backbone adopts the classic extended strand recognized by most proteases ((a) Tyndall, J. D. A.; NaII, T.; Fairlie, D. P. Chem. Rev. 2005, 105, 973-1000. (b) Loughlin, W.
  • the P1 carboxylate side chain of 13 is important, as it is in 101 (Sleath, P. R.; Hendrickson, R. C; Kronheim, S. R.; March, C. J.; Black, R. A. J. Biol. Chem. 1990, 265, 14526-14528.), since conversion to the amide reduced caspase-1 inhibition 50 fold (IC 5 O 230 nM). This supports analogous binding for 14 and 79 at the S1 site in caspase-1.
  • Example 3 Enzyme Inhibition Assays All enzymes were purchased from Merck Pty Ltd.
  • Caspase-1 10OmM NaCI, 5OmM HEPES, 1 OmM DTT, 1 mM EDTA, 10%
  • Caspase-3 10OmM NaCI, 5OmM HEPES, 1 OmM DTT, 1 mM EDTA, 10%
  • Caspase-8 10OmM NaCI, 5OmM HEPES, 1 OmM DTT, 1 mM EDTA, 10%
  • Caspase-1 Recombinant human caspase-1 (3000U) was diluted with 700 ⁇ L of buffer, divided into 70 ⁇ l_ aliquots and stored at -8O 0 C. The diluted solution (4.5 ⁇ l_) was used in each well for individual reactions.
  • Caspase-3 Recombinant human caspase-3 (5000U) was was diluted with 1 ml of buffer, divided into 65 ⁇ L aliquots and stored at -8O 0 C. Each 65uL aliquot was subsequently diluted to 24OuL and this solution used for each well (5 ⁇ L).
  • Caspase-5 Recombinant human caspase-5 (3000U) was diluted with 700 ⁇ L of buffer, divided into 70 ⁇ l_ lots and stored at -80°C. The diluted solution (4.5 ⁇ l_) was used in each well for individual reactions.
  • Caspase-8 Recombinant human caspase-8 (3000U) was diluted with 700 ⁇ L of buffer, divided into 70 ⁇ l_ lots and stored at -8O 0 C. The diluted solution (4.5 ⁇ l_) was used in each well for individual reactions.
  • Cathepsin K Recombinant human cathepsin K (25mg) was purchased from
  • Cathepsin S Recombinant human cathepsin S (25mg) was purchased from Calbiochem. The enzyme solution was diluted with 600ml of buffer, aliquoted into 30 ⁇ L lots and stored at -80°C. Each 3OmL aliquot was subsequently diluted to 15OuL and this solution used for each well (5 ⁇ L).
  • Cathepsin B Cathepsin B from human liver (25ug, cone. 1 .6mg/ml) was dissolved in 700 ⁇ L of buffer. This was divided into 30 ⁇ L portions. Each 3OuI aliquot was further diluted 128X and the subsequent solution divided into 150 ⁇ L aliquots. The 150 ⁇ L aliquots were used to deliver 5 ⁇ L of enzyme to each well.
  • Cathepsin S A 2.OmM DMSO solution of z-V-V-R-AMC (Merck) was diluted 200X in buffer to a final concentration of 1 OmM in each well (K m >100mM).
  • Each inhibitor was dissolved in DMSO to a concentration of 5mM and diluted out to the required concentration range. Seven different concentrations (2 fold dilutions) were used which spanned the IC50 value of the inhibitor and were each repeated in duplicate. The DMSO solution (2 ⁇ l_) of was applied to each well.
  • each well of a 96 well plate was placed 1 ⁇ l_ of substrate, 2 ⁇ l_ of inhibitor and 4.5-5 ⁇ l of the enzyme.
  • the volume was made up to a total of 200 ⁇ L with buffer.
  • the enzyme was incubated with inhibitor for 5 minutes prior to the addition of substrate and reaction monitored on a fluorostar spectrophotometer (BMG Technologies) in a 96-well plate format, using an excitation wavelength of 380nm and emission of 460nm to detect free AMC.
  • the IC50 value for each inhibitor was determined using non-linear regression by fitting the data to a sigmoidal dose-response curve plotting log [I] vs Vi/Vo.
  • a 5mM solution (DMSO) of 13 was diluted to 5 ⁇ M via three 10-fold serial dilutions. 20 ⁇ l_ of this solution was added to 130 ⁇ l_ of buffer and 15 ⁇ l of this solution was applied to each well. Enzyme solution (5 ⁇ l_) was added and the mixture was allowed to incubate for ten minutes at room temperature. To this solution was added a solution of substrate (180 ⁇ l_) in assay buffer. The processing of the Ac-YVAD-AMC substrate was monitored via a fluorescence plate reader with 380nm excitation and 460nm emission wavelengths. The results are shown in Figure 2.
  • aldehyde 101 is unstable in aqueous solutions (Fig.4, left panel), existing initially as a mixture of aldehyde and cyclic hemiacetal (A) followed by rapid aspartate racemization (B, C) in phosphate buffer (Ui 2 4h, 37° C, pH 7.4). No aldehyde is detectable within minutes in 1 OmM Ac-Cys (D, E) or 2OmM glutathione. Azide 13 remained unchanged under all conditions.
  • caspase-1 inhibitors 13 and 15 were assayed against caspase-3, -5, and - 8.
  • Inhibitor 13 was 1000 fold selective for caspase -1 over the closely related caspase 5.
  • 13 does not inhibit caspase-3 and is 100 fold selective as compared to caspase-8.
  • Compound 15 was also found to be 1000 fold selective for caspase-1 as compared to caspase-5. This data proves that the azidomethylene compounds described herein can be modified for selectivity for the target enzyme.
  • R1 -C(CH 3 ) 3
  • R1 -C(CH 3 J 3
  • the ester derivatives of compounds 13 and 14 are potential cell-permeable compounds that could inhibit release of IL-1 ⁇ from human immune cells (Figure 5). With the exception of 2Nap-V-A-D(OEt)-N 3 , the compounds significantly inhibited (decreased) the LPS-induced IL-1 ⁇ production in U937 cells (a pro- monocytic cell line). After 24hrs, there was an increase in IL-1 ⁇ production as measured by ELISA, and this was also seen in the control sample. This was probably due to the cells spontaneously producing IL-1 ⁇ (Control) or cells dying and causing an increase in the background readings.
  • 2Nap-V-A-D(OEt)-N3 had a larger LPS-induced response, although this may have been just due to the compound not entering the cell, and not inhibiting IL-1 ⁇ production.
  • Compounds alone did not elicit a response, although background readings at 24, 48 and 72hrs were high. This is an indication that the caspase-1 inhibitors were blocking IL-1 ⁇ production by human U937 cells. - 73 -
  • Compound 14 was briefly examined in 4 rats to see if it was orally bioavailable.
  • the compound was soluble in water. It was administered orally at 10mg/kg/day over 4 days (Days 10-13 inclusive after administration of Freund's adjuvant) to 4 rats. Signs of arthritis were evaluated on Days 10 and 14 as rear and forepaw inflammation including visual lesions, and weight changes. The data are shown in Table 13. The results indicate that this compound is orally bioavailable.

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Abstract

Disclosed is a method of producing a cysteine protease inhibitor, the method comprising incorporating an azide group into an organic molecule. Also disclosed is a cysteine protease inhibitor containing an azide group.

Description

Cysteine Protease Inhibitors Incorporating Azide Groups
This application claims priority from Australian Provisional Patent Application No. 2005905564 filed on 10 October 2005 and Australian Provisional Patent Application No. 2006904217 filed on 4 August 2006, the contents of which are to be taken as incorporated herein by this reference.
Field of the Invention
The present invention relates to methods for producing cysteine protease inhibitors. The present invention also relates to compounds and compositions that modulate the function of cysteine proteases. The compounds described herein may be used for the treatment of ailments, conditions or disease states that are associated with cysteine protease function or would benefit from modulation of the function of a cysteine protease.
Background of Invention
Proteases, also known as proteinases or peptidases, are proteolytic enzymes that break down or hydrolyse proteins. Proteases are essential for the synthesis of all proteins, controlling protein composition, size, shape, turnover and ultimate destruction. There are over 500 human proteases, accounting for 2% of human genes, and similar numbers of proteases occur in every plant, insect, marine organism and in all infectious organisms that cause disease.
Proteases represent important potential targets for medical intervention because of their important regulatory roles in life. In addition to medical applications of protease research, there is also a potential to improve plant and animal health through enhanced growth and treatment/prevention of parasite infections, crop protection through new herbicides and pesticides, and increased or faster production of food resources. There are five classes of proteases categorized by the catalytic residue that effects enzymatic hydrolysis, namely cysteine, serine, aspartic, metallo and threonine enzymes. All proteases bind their substrates in a groove or cleft, where amide bond hydrolysis occurs. Amino acid side chains of substrates occupy enzyme sub-sites in the groove, designated as S3, S2, S1 , SV, S2', S3', that bind to corresponding substrate/inhibitor residues P3, P2, P1 , PV, P2', P3' with respect to the cleavable amide bond. According to standard nomenclature, the substrate amino acid residues N-terminal of the scissile amide bond are designated as ...PA, P3, P2, P1 and residues on the C- terminal side are designated PV P2' etc.
Cysteine proteases comprise 3 classes of structurally distinct enzymes that all hydrolyse amide bonds in their substrates via an active site cysteine. They are either papain-like (e.g. lysosomal cathepsins B,S,K,L), ICE-like (e.g. caspases 1-10, 14), or picornaviral-like.
Cysteine proteases account for 26% of human endopeptidase enzymes and play crucial roles in diseases, immune defence, inflammation, apoptosis, and bone resorption (Leung-Toung, R.; Li, W. R.; Tarn, T. F.; Karimian, K. Cυrr. Med. Chem. 2002, 9, 979-1002). When over-expressed or unregulated in diseased states cysteine proteases are validated therapeutic targets.
Thus, molecules that can inhibit cysteine proteases have potential applications as medicines, diagnostics, and pesticides, as well as valuable tools for interrogating and regulating biological and physiological processes.
A number of inhibitors of cysteine proteases have been developed or proposed but none have not yet succeeded in the clinic, mainly due to a common flaw in their design (see Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem. 2000, 43, 305-341 and Abbenante, G.; Fairlie, D. P. Med. Chem. 2005, 1, 71-104). Inhibitors of cysteine proteases usually employ a reactive electrophilic group (alkylating agent, aldehyde, nitrile, ketone, α-keto-amide, halo-ketone, vinyl sulfone, chloromethane, epoxide, diazomethane, etc.) to covalently bond to the catalytic cysteinyl sulfur of the enzyme (see Powers, J. C; Asgian, J. L.; Ekici, O. D.; James, K. E. Chem. Rev. 2002, 102, 4639-4750 and Otto, H.-H.; Schirmeister, T. Chem. Rev. 1997, 97, 133-171 ). Although useful tools in vitro, such inhibitors are problematic as drug leads due to indiscriminate competing reactions in vivo with other nucleophiles (thiols, amines, etc), resulting in low bioavailability, metabolic instability and side-effects (see Rishton, G. M. Drug Discovery Today 2003, 8, 86-96 and Muegge, I. Chem.-Eur. J. 2002, 8, 1976- 1981 ). However, electrophilic isosteres are still thought to be necessary for potent inhibition of cysteine proteases, especially caspases (Talanian, R. V.; Brady, K. D.; Cryns, V. L. J. Med. Chem. 2000, 43, 3351-3371 ).
There is a need for improved inhibitors of cysteine proteases and, in particular, for cysteine protease inhibitors that ameliorate one or more of the problems associated with known cysteine protease inhibitors.
Unless otherwise specifically stated, a reference herein to a patent document, literature article or other matter which is given as prior art is not to be taken as an admission that that document, article or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.
Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.
Summary of the Invention
The present invention arises from the discovery that alkyl azide isosteres confer potent inhibition of cysteine proteases. Inhibition is thought to be competitive, reversible and selective, relying upon multiple non-covalent interactions for enzyme affinity. Thus, incorporation of an azide group into cysteine protease inhibitor molecules provides new, less reactive and/or non- covalent inhibitors of these proteases. Furthermore, replacement of the reactive electrophile of known cysteine protease inhibitors with an azide group may also provide new, less reactive and/or non-covalent inhibitors of these proteases.
The present invention provides a method of producing a cysteine protease inhibitor, the method comprising incorporating an azide group into an organic molecule. The azide group may be incorporated into a part of the molecule such that the azide group interacts with the cysteine protease when the molecule is bound thereto.
The present invention also provides a cysteine protease inhibitor produced using the aforementioned method.
In an embodiment the organic molecule comprises a cysteine protease binding moiety and an end group moiety. The azide group may be incorporated into the end group moiety of the molecule.
In an embodiment the cysteine protease binding moiety is an amino acid or a peptide. The amino acid or peptide may have the following formula:
AA1- -AAn- wherein each AA is the same or different and each is an amino acid residue and n is an integer selected from the group consisting of 0, 2, 3, 4, and 5. In specific embodiments n is 0, 2 or 3.
In specific embodiments the amino acid or peptide has the following formula:
R-CO-AA1-, R-CO-AA1-AA2-, or R-CO-AA1-AA2-AA3
wherein AA1, AA2 and AA3 are the same or different and each is a natural or unnatural amino acid; and R CO- is a nitrogen protecting group.
The end group moiety may have the formula:
Figure imgf000006_0001
wherein R1 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted carboxyalkyl. In specific embodiments R1 is selected from the group consisting of optionally substituted alkyl and optionally substituted carboxyalkyl.
In some embodiments the end group moiety has the formula:
Figure imgf000006_0002
In other embodiments the end group moiety has the formula:
Figure imgf000006_0003
In still other embodiments the end group moiety has the formula:
Figure imgf000006_0004
The present invention also provides a method of modulating one or more of the activity, specificity or a biological property of a cysteine protease inhibitor containing a functional group that is capable of reacting with a nucleophile, the method comprising replacing the functional group with an azide group.
The functional group may be an electrophilic group. Examples of electrohilic groups that may be replaced include, but are not limited to, alkylating agents, aldehydes, nitriles, ketones, α-keto-amides, halo-ketones, vinyl sulfones, chloromethanes, epoxides, diazomethanes, trifluoromethyl ketones, α-keto amides, fluoromethyl ketones, and diazoketones.
The modulation of a biological property of the inhibitor may result in increased efficacy when administered, decreased toxicity, decreased side-effects, increased bioavailability, or increased half-life.
In terms of binding of the inhibitor to a target cysteine protease, the azide group may provide similar binding properties as the functional group it has replaced but it may not be as reactive with nucleophiles in or adjacent the binding site of the target cysteine protease or with other nucleophiles in vivo. As such the azide group may not undergo substantive covalent bonding to the target cysteine protease which may lead to reversible inhibition of the target cysteine protease.
Thus, the present invention also provides a method for producing a reversible cysteine protease inhibitor, the method comprising replacing a reactive functional group capable of reacting with a target cysteine protease in a inhibitor with an azide group.
The azide compounds produced using the methods of the present invention may inhibit the function of cysteine proteases of current pharmaceutical relevance such as, but not restricted to, caspases 1-10 and 14, lysosomal cysteine proteases such as, but not restricted to, cathepsin B, L, S, and K, Rhinovirus 3C protease, and cysteine proteases of pharmaceutical relevance that will be discovered in the future.
Using the methods of the present invention, we have developed a series of new cysteine protease inhibitors incorporating an azide group.
Thus, the present invention also provides a cysteine protease inhibitor of formula:
Figure imgf000008_0001
wherein R2 is an amino acid or a peptide; R3 is selected from the group consisting of optionally substituted alkyl, optionally substituted carboxyalkyl, optionally substituted aryl, and optionally substituted carboxyaryl; R4 is selected from the group consisting of H, optionally substituted alkyl, and optionally substituted aryl; and R5 is selected from the group consisting of optionally substituted alkyl, and optionally substituted aryl.
In an embodiment of the invention, R2 is an amino acid or peptide having the following formula:
AA1- -AAn-NR6- wherein each AA is the same or different and each is an amino acid residue and n is an integer selected from the group consisting of 0, 2, 3, 4, and 5, and R6 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, and a nitrogen protecting group. In specific embodiments, n is 0, 2 or 3.
More specifically, R2 may be a peptide having one of the following formulae:
R7-CO-AA1-, R7-CO-AA1-AA2- or R7-CO-AA1-AA2-AA3 wherein AA1, AA2 and AA3 are the same or different and each is a natural or unnatural amino acid including, but not restricted to, cyclohexylalanine, t- butylglycine, α-aminoisobutyric acid, α-methyl substituted amino-acids, 1- aminocyclohexane carboxylic acid, 2-aminobutyhc acid, cyclohexylalanine, t- butyl glycine, 1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, including the D and L enantiomers of these amino acids; and R7CO- is a nitrogen protecting group.
In specific embodiments, R7 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkyloxy, optionally substituted aryl, optionally substituted aryloxy, optionally substituted amino. Non-limiting examples of suitable R7CO- groups include acyl, naphthyl, indoyl, benzyl, biphenyl, and substituted derivatives of these compounds.
R3 may be -CR8R9-CO2R10, wherein R8, R9 and R10 are each independently selected from the group consisting of H, optionally substituted alkyl, and optionally substituted aryl. In embodiments of the invention, each R8 and R9 is H and R10 is selected from the group consisting of H, d-do straight chain alkyl, d-do branched chain alkyl, and aryl. More specifically, R10 is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, benzyl, napthylmethyl, and biphenylmethyl.
Alternatively, R3 may be -(CR8R9)n-CH3, wherein each R8 and R9 in each CR8R9 is independently selected from the group consisting of H, optionally substituted alkyl, and optionally substituted aryl, and n is an integer selected from the group consisting of O, 1 , 2, 3, 4, 5, 6, 7, 8, and 9. In some embodiments R3 is selected from the group consisting of -(CH2)3-CH3, and -
Figure imgf000009_0001
R4 may be selected from the group consisting of H, d-do straight chain alkyl, and d-do branched chain alkyl. In specific embodiments, R4 is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and t-butyl.
R5 may be a bond or -(CR11R12V, wherein each R11 and R12 in each CR11R12 is the same or different and is independently selected from the group consisting of H, and optionally substituted alkyl, and n is an integer from 1 to 10 inclusive. In specific embodiments, n is an integer from 1 to 5 inclusive. In specific embodiments, R5 is -(CH2)n-- In some embodiments, n is 1.
From the foregoing, it will be appreciated that in some embodiments of the invention the compound has the formula:
Figure imgf000010_0001
wherein R13 is PG-AA1-, PG-AA1 -AA2-, or PG-AA1-AA2- AA3- where AA1, AA2 and AA3 are the same or different and each is a natural or unnatural amino acid and PG is a nitrogen protecting group.
In other embodiments of the invention, the compound has the formula:
Figure imgf000010_0002
wherein R13 is PG-AA1-, PG-AA1 -AA2-, or PG-AA1-AA2- AA3- where AA1, AA2 and AA3 are the same or different and each is a natural or unnatural amino acid and PG is a nitrogen protecting group, and R22 is selected from the group - 10 -
consisting of H, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkenyl, and optionally substituted alkynyl.
In specific embodiments, the peptide is selected from the group consisting of PG-VaI-AIa-, PG-GIu-AIa-, PG-Glu-Leu-, PG-Glu-Pro-, PG-Glu-Thr-, PG-GIu- HiS-, PG-Asp-Leu-, PG-GIu-VaI-, PG-AIa-AIa-, PG-GIy-AIa-, PG-AIa-GIy-, PG- Tbg-Aib-, PG-Phe-Leu-, PG-Cha-Leu-, PG-Tbg-Pro-, PG-Asp-Glu-Leu-, PG- Asp-Glu-Phe-, PG-Asp-Glu-Val-, PG-lle-Glu-Thr-, PG-lle-Glu-Pro-, PG-GIu- Glu-Leu-, PG-Glu-Leu-Leu-, PG-Leu-Glu-Leu-, PG-Phe-Glu-Leu-, PG-Tic-Glu- Leu-, PG-Tyr-Asp-Ala-, PG-Tyr-Glu-Ala-, PG-Tyr-Val-His-, PG-Tyr-Val-Pro-, PG-Tyr-Val-Phe-, PG-Tyr-Val-Leu-, PG-Tyr-Gln-Ala-, PG-Tyr-Phe-Ala-, PG- Tyr-Leu-Ala-, PG-Tyr-Val-Val-, PG-Tyr-Val-Ala-, PG-Tyr-Val-Cha-, PG-Trp-Glu- Aib-, PG-Trp-Glu-Cha-, PG-Trp-Glu-His-, PG-Trp-Glu-Ala-, PG-Trp-Glu-Tbg-, PG-Val-Glu-Thr-, wherein PG is a nitrogen protecting group.
In specific embodiments, the nitrogen protecting group is selected from the group consisting of Ac,
Figure imgf000011_0001
Vo o
Figure imgf000011_0002
Figure imgf000011_0003
- 11 -
Figure imgf000012_0001
In specific embodiments, the compound has one of the following formulae:
Figure imgf000012_0002
It is believed that the azide compounds described herein are non-covalent inhibitors that do not react with the active-site amino acid or any other part of the cysteine protease enzyme. Instead, it is thought that the compounds described herein rely on multiple weak contacts (ionic, H-bonding, π-stacking, van der Waals interactions) and conformational rigidity (lowering the entropy penalty for protease binding) to impart high affinity and selective inhibition.
The present invention also provides a compound of formula:
Figure imgf000012_0003
wherein R19 is amino acid or peptide; R20 is selected from the group consisting of H, optionally substituted alkyl, and optionally substituted aryl; and R21 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, and a nitrogen protecting group.
In some embodiments of the invention, R19 is PG-AA1 -AA2- or PG-AA1 -AA2- AA3- where AA1, AA2 and AA3 are the same or different and each is a natural or unnatural amino acid, and PG is a nitrogen protecting group. Specific peptides include those listed previously. R20 and R21 may also be H.
The present invention also provides a pharmaceutical composition comprising an azide compound described herein, and a pharmaceutically acceptable carrier, diluent or excipient.
The present invention further provides the use of an azide compound described herein in the preparation of a medicament for the treatment of a disease state in which the disease pathology may be therapeutically modified by inhibiting a cysteine protease.
The present invention further provides a method of inhibiting a cysteine protease comprising administering to a subject an effective amount of an azide compound described herein.
The present invention further provides a method of treating a disease state in which the disease pathology may be therapeutically modified by inhibiting a cysteine protease, the method comprising administering to a subject in need thereof an effective amount of an azide compound described herein.
The present invention further provides the use of an azide compound described herein in the preparation of a medicament for the treatment of a disease state in which the disease pathology may be therapeutically modified by modulating the activity of a cysteine protease. The present invention further provides a method of modulating the activity of a cysteine protease comprising administering to a subject an effective amount of an azide compound described herein.
The present invention further provides a method of treating a disease state in which the disease pathology may be therapeutically modified by modulating the activity of a cysteine protease, the method comprising administering to a subject in need thereof an effective amount of an azide compound described herein.
The present invention further provides a method of treating a disease characterised by bone loss including administering to a subject in need thereof an effective amount of an azide compound described herein.
The present invention further provides a method of treating a disease characterised by excessive cartilage or matrix degradation including administering to a subject in need thereof an effective amount of an azide compound described herein.
General Description of the Invention
Various terms that will be used throughout this specification have meanings that will be well understood by a skilled addressee. However, for ease of reference, some of these terms will now be defined.
Reference in this specification to a specific active ingredient is also to be understood to include the active ingredient in the form of acid addition salts, solvates and hydrates. The compounds of the present invention may be used in the form of salts derived from inorganic or organic acids. Included among such acid salts, for example, are the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3- phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.
This invention also envisions the quaternization of any basic nitrogen- containing groups of the compounds disclosed herein. The basic nitrogen can be quaternized with any agents known to those of ordinary skill in the art including, for example, lower alkyl halides, such as methyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkyl sulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myhstyl and stearyl chlorides, bromides and iodides; and aralkyl halides including benzyl and phenethyl bromides. Water or oil-soluble or dispersible products may be obtained by such quaternization.
The terms "azide", "azido" and "-N3" will be understood by a person skilled in the art to refer to a group having the following structure:
4-N=N=N *
The person skilled in the art will appreciate that the structure shown above is a single tautomeric form of the azide group. The azide group also has the tautomeric forms shown below:
Figure imgf000015_0001
When used herein, the terms "azide", "azido", "-N3" and the structure:
Figure imgf000015_0002
are to be interpreted so as to include all possible tautomeric forms of the azide group. The term "incorporating" and variants thereof when used in relation to the preparation of azide based cysteine protease inhibitor molecules means that the azide group may be added to an exisiting molecule using reactions (such as substitution reaction) as discussed further herein. Alternatively, the azide group could be part of a molecule or reactant that is used in a synthetic scheme for producing a cysteine protease inhibitor molecule.
The term "alkyl" as used herein includes both straight and branched chain radicals of up to 12 carbons, preferably 1-8 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, 1-ethylpropyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl and dodecyl. For example, "Ci to Cs alkyl" as used herein is meant to include substituted and unsubstituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl, pentyl, n-pentyl, isopentyl, and neopentyl.
Any alkyl group may be optionally substituted independently by one, two or three halogens, SR', hydroxy, OR', N(R1J2, C(O)N(R1J2, where R' is H or Ci to C6 alkyl, carbamyl, trifluoromethyl, nitro, carboxy, Ci to C6 alkyl, C6 to do aryl, Ci to C6 alkoxy, Ci to C6 aminoalkyl, Ci to C6 aminoalkoxy, C2 to C6 alkoxycarbonyl, Ci to C6 hydroxyalkyl, Ci to C6 hydroxyalkoxy, Ci to C6 alkylsulfonyl, C6 to do arylsulfonyl, Ci to C6 alkylsulfinyl, Ci to C6 alkylsulfonamido, C6 to do arylsulfonamido, C6 to do aryl(Ci to C6)alkylsulfonamido, C6 to Cio aryl(Ci to C6)alkyl, Ci to C6 alkylcarbonyl, C2 to C6 carboxyalkyl, cyano, and trifluoromethoxy and/or carboxy substituents.
The term "alkyl" as used herein also includes within its scope saturated cyclic hydrocarbon groups containing 3 to 12 carbons, preferably 3 to 8 carbons. Exemplary cycloalkyl groups include, but are not restricted to, substituted and unsubstituted cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, cycloundecane and cyclododecane. The cyclic structure may be substituted with substituents such as halogen, Ci to C6 alkyl, Ci to C6 alkoxy, hydroxy groups or amino groups. The term "alkenyl" as used herein means an alkyl group wherein a carbon- carbon single bond is replaced by a carbon-carbon double bond. For example, C2 to C6 alkenyl includes ethylene, 1 -propene, 2-propene, 1-butene, 2-butene, isobutene and the several isomeric pentenes and hexenes. Both cis and trans isomers are included.
The term "alkynyl" as used herein means an alkyl group wherein one carbon- carbon single bond is replaced by a carbon-carbon triple bond. For example, C2 to C6 alkynyl includes acetylene, 1 -propyne, 2-propyne, 1 -butyne, 2-butyne, 3- butyne and the simple isomers of pentyne and hexyne.
The terms "aryl" and "Ar" as used herein mean monocyclic or bicyclic aromatic groups containing from 6 to 12 carbons in the ring portion, preferably 6-10 carbons in the ring portion. Examples include, but are not restricted to, phenyl, naphthyl and tetrahydronaphthyl.
The terms "heterocyclic" and "Het" as used herein mean a stable 5- to 7- membered monocyclic or a stable 7- to 10-membered bicyclic heterocyclic ring, which is either saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure, and may optionally be substituted with one or more moieties selected from Ci to C4 alkyl, OR', N(R1J2, SR', CF3, NO2, CN, CO2R', CON(R'), F, Cl, Br and I, where R' is alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl and acyl, each of which may be optionally substituted. Examples of such heterocycles include piperidinyl, piperazinyl, 2- oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, pyridyl, pyrazinyl, oxazolidinyl, oxazolinyl, oxazolyl, isoxazolyl, morpholinyl, thiazolidinyl, thiazolinyl, thiazolyl, quinuclidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, benzoxazolyl, furyl, pyranyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzoxazolyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and oxadiazolyl.
The term "heteroaryl" as used herein means any heterocyclic moiety encompassed by the above definition of Het having 5 to 14 ring atoms, preferably 5, 6, 9 or 10 ring atoms; 6, 10 or 14 π electrons shared in a cyclic array; and containing carbon atoms and 1 , 2 or 3 oxygen, nitrogen or sulfur heteroatoms. Examples of heteroaryl groups are: thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl, xanthenyl, phenoxathiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, tetrazolyl, quinazolinyl, cinnolinyl, pteridinyl, 4.alpha.H-carbazolyl, carbazolyl, .beta.-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl and phenoxazinyl groups.
The term "arlkyl" or "arylalkyl" as used herein by itself or as part of another group refers to Ci to C6 alkyl groups as discussed above having an aryl substituent, such as benzyl, phenylethyl or 2-naphthylmethyl.
The term "alkaryl" or "alkylaryl" as used herein by itself or as part of another group refers to an aryl group as discussed above having a Ci to C6 alkyl substituent, such as toluyl, ethylphenyl, or methylnaphthyl.
The term "carboxyalkyl" as used herein refers to a group of formula -alkyl- C(O)O-R', where R' is is alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, and heteroarylalkyl, each of which may be optionally substituted
The term "alkoxy" as used herein refers to the above alkyl groups linked to oxygen.
The term "halogen" or "halo" as used herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine.
The term "amino" as used herein refers to formylamino, alkylcarbonylamino or arylcarbonylamino.
The term "optionally substituted" as used herein denotes that the group may or may not be further substituted or fused (so as to form a condensed polycyclic system), with one or more substituent groups. The substituent groups are one or more groups independently selected from the group consisting of halogen, =0, =S, -CN, -NO2, -CF3, -OCF3, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl, cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl, heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxycycloalkyl, alkoxyheterocycloalkyl, alkoxyaryl, alkoxyheteroaryl, alkoxycarbonyl, alkylaminocarbonyl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkyloxy, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, alkylsulfinyl, arylsulfinyl, aminosulfinylaminoalkyl, -COOH, -COR , -C(O)OR , CONHR', NHCOR', NHCOOR', NHCONHR , C(=NOH)R , -SH, -SR , -OR and acyl, where R' is alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl and acyl, each of which may be optionally substituted.
The term "nucleophile" and variants thereof as used herein means an atom or substance which is attracted to centres of positive charge and which donates a pair of electrons when it reacts with substrate. In biological systems, common nucleophiles include O, S and N atoms that are present in biological molecules.
The term "inhibit" and variants thereof as used herein means a reduction or inhibition of a process, including the start, continuation or termination of a process, and in the context of the present invention this term includes an adverse affect on the enzymatic activity of a cysteine protease. To determine whether or not the activity of a cysteine protease is reduced, one can use any one of the methods described in the examples provided in this specification, or any one of the methods known for that purpose in the art.
The term "modulate" and variants thereof as used herein means a change or alteration of a process, including the start, continuation or termination of a process, and in the context of the present invention this term includes a change or alteration in the activity of a cysteine protease.
The term "subject" as used herein means any multicellular organism, including a human, plant or an animal subject.
For example, in the case where the subject is a human or animal, the subject organism may be a mammal, a primate, a livestock animal (eg. a horse, a cow, a sheep, a pig, or a goat), a companion animal (eg. a dog, a cat), a laboratory test animal (eg. a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance.
In the case where the subject is a plant, the plant may be for example a commercial crop species (eg barley, oat, millet, alfalfa), a leguminous plant (eg soybean, alfalfa, and pea), a non-leguminous plants (e.g., corn, wheat, and cotton), or an angiosperm or cereal.
The present invention includes all hydrates, solvates, complexes and prodrugs of the compounds described herein. Prodrugs are any covalently bonded compounds which release the active parent drug in vivo.
It is understood that included in the family of compounds described herein are isomeric forms including diastereoisomers, enantiomers, tautomers, and geometrical isomers in "E" or "Z" configurational isomer or a mixture of E and Z isomers. It is also understood that some isomeric forms such as diastereomers, enantiomers, and geometrical isomers can be separated by physical and/or chemical methods and by those skilled in the art.
Some of the compounds of the disclosed embodiments may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates and mixtures thereof are intended to be within the scope of the subject matter described and claimed.
In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.
Abbreviations and symbols commonly used in the peptide arts are used herein to describe the compounds of the present invention. The term "amino acid" is a generic designation for groups resulting from replacement of at least one hydrogen atom in the parent structure of carboxylic acid by an amino group, including alpha-, beta-, gamma- and delta-amino acids having a parent structure with 2 to 20 carbon atoms. Of these amino acids, alpha-amino acids are preferred, including "natural" amino acids that are protein components such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, leucine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; and other "unnatural" or "non-natural" amino acids having a variety of substituents at the α-carbon in the case of α -amino acids, or at the α - or β-carbon in the case of β -amino acid, such as norvaline, norleucine, t-butylglycine (tert- leucine), 2-aminoadipic acid, 2-aminobutyric acid, 2-aminoisobutyhc acid, 1 - aminocyclopropanecarboxylic acid, 1-aminocyclopentanecarboxylic acid, 1- aminocyclohexanecarboxylic acid, thyronine, ornithine, hydroxyproline and hydroxylysine. The amino acid may be a D- or an L- isomer. The amino acid may also be an amino acid mimetic.
Also, the term "amino acid" includes within its scope cyclic imino acids. The cyclic imino acid is an optionally substituted cycloalkane carboxylic acid or an optionally substituted cycloalkene carboxylic acid which at least one of the methylene groups is substituted, specifically, such as proline, hydroxyproline, 3,4-dehydroproline, pipecoline acid, adilidine carboxylic acid and 2-azetidine carboxylic acid.
Unless otherwise stated, the term "amino acid" or the abbreviation "AA" includes within its scope protected amino acids. Protected amino acids include N-protected and/or C-protected amino acids. Examples of nitrogen and carboxy protecting groups are found in W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York, 1999.
Abbreviations for amino acids, compounds and others used in the present specification are based on abbreviations in common use in relevant fields.
Some examples are: GIy or G: Glycine; Ala or A: Alanine; VaI or V: Valine;
Leu or L: Leucine; He or I: Isoleucine; Ser or S: Serine; Thr or T: Threonine;
Cys or C: Cysteine; Met or M: Methionine; GIu or E: Glutamic acid; Asp or D: Aspartic acid; Lys or K: Lysine; Arg or R; Arginine; His or H: Histidine; Phe or
F; Phenylalanine; Tyr or Y: Tyrosine; Trp or W: Tryptophan; Pro or P: Proline;
Asn or N: Asparagine; GIn or Q: Glutamine; Ach: 1 -aminocyclohexane carboxylic acid; Abu: 2-aminobutyric acid; Aib: Aminoisobutyric acid; Cha : cyclohexylalanine; Tbg: t-butyl glycine; Tic: 1 ,2,3,4-tetrahydroisoquinoline-3- carboxylic acid; HCTLJ: 1 H-Benzotriazolium 1-[bis(dimethylamino)methylene] -5chloro-,hexafluorophosphate (1-),3-oxide; Fmoc: 9- fluorenylmethoxycarbonyl; Ac: Acetyl; Cbz or Z: Benzyloxycarbonyl; Boc: t- butoxycarbonyl; BzI: Benzyl.
The terms "nitrogen protecting group" and "PG" (when bonded to a nitrogen atom) as used herein mean groups known in the art that are readily introduced on to and removed from a nitrogen atom. Nitrogen protecting groups are generally of the formula RCO- wherein R is selected from the group consisting of optionally substituted alkyl, optionally substituted alkyloxy, optionally substituted aryl, optionally substituted aryloxy, optionally substituted amino. Examples of nitrogen protecting groups include formyl, acetyl (Ac), trifluoroacetyl, benzyl, benzoyl, benzyloxycarbonyl (Cbz or Z), tert- butoxycarbonyl (Boc), thmethyl silyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted trityl groups, allyloxycarbonyl, 9- fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC) and the like. Other examples of acceptable nitrogen protecting groups are found in W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York, 1999.
The present invention arises from the inventors studies in relation to the development of reversible and/or non-covalent inhibitors of cysteine proteases. Inhibitors of cysteine proteases have historically been designed mainly using iterative substrate- and structure-based methodologies (crystal structures and computer-assisted molecular modelling) to derivatise peptide fragments adjacent to the hydrolysable amide bond of substrates. At the heart of the design strategy has been the incorporation of an electrophile at the C-terminus to covalently interact with the nucleophilic thiol of the catalytic cysteine. This covalent reaction generally increases enzyme affinity by 103-104 times over unmodified peptide. These reactive electrophilic functionalities include, but are not limited to, aldehydes, ketones, trifluoromethyl ketones, α-keto amides, nitriles, fluoromethyl ketones, trifluoromethyl ketones and diazoketones. The caspases are a sub-class of cysteine proteases that recognize polypeptide substrates containing aspartate immediately N-terminal to the putative cleavage site. At least eleven human caspases (Caspases 1-10, 14) are known (Pistritto, G. et al., Cell Death Differ 2002, 9, 995-1006).
Caspase 1 produces the pro-inflammatory cytokine interleukin-1 β (IL-1 β) from its precursor peptide. When released from monocytes, IL-1 β stimulates leukocyte adhesion and activation as well as other inflammatory responses. Inhibitors of ICE decrease IL-1 β production in vitro and in vivo, and have therapeutic potential for inflammatory diseases such as rheumatoid and osteoarthritis (Leung-Toung, R. et al., Current Medicinal Chemistry 2002, 9, 979- 1002).
Caspase 3 (apopain, CPP32) is a key executioner in apoptosis. Inhibitors are predicted to be useful therapeutics for aging, neurodegenerative diseases (Alzheimer's disease), stroke and myocardial infarction, and cancer (Talanian, R. V. et al., Journal of Medicinal Chemistry 2000, 43, 3351 -3371 ).
Cathepsin K is selectively expressed in osteoclasts where it represents 98% of the total cysteine protease activity and is primarily involved in bone resorption. X-ray structures of inhibitors bound to its active site indicate that the S2 subsite is the most important for selectivity with respect to other lysosomal proteases (cathepsins L, S, B etc.). Inhibitors are currently thought to be promising therapeutics for the treatment of diseases characterized by excessive bone loss such as osteoporosis (McGrath, M. E. Annual Review of Biophysics and Biomolecular Structure 1999, 28, 181 -204).
Cathepsin S plays an important role in regulating antigen presentation and immunity. Inhibition of antigen presentation via prevention of invariant chain degradation by cathepsin S may provide a mechanism for immuno-regulation. Control of antigen-specific immune responses has long been desirable as a useful and safe therapy for autoimmune diseases, such as Crohn's disease and arthritis, as well as other T-cell-mediated immune responses. Cathepsin S has also been implicated in a variety of other diseases involving extracellular proteolysis, such as Alzheimer's disease and atherosclerosis.
Cathepsin B plays a number of roles inside cells to maintain normal cellular metabolism. Overexpression of cathepsin B has been associated with pathophysiological conditions such as tumor metastasis, inflammation, bone resorption, and myocardial infarction (Yan, S. et al., Biol. Chem. 1998, 379, 113-123).
The present studies have shown that inclusion of an azide group in organic molecules provides compounds that inhibit cysteine proteases. Accordingly, the present invention provides a method of producing a cysteine protease inhibitor, the method comprising incorporating an azide group into an organic molecule. The organic molecule may be a cysteine protease inhibitor molecule, or it may be a molecule that does not have cysteine protease inhibitory properties until the azide group is incorporated in the molecule. For example, the organic molecule may contain a cysteine protease binding moiety which, in itself, shows little or no activity toward the target protease until the azide group is incorporated into the molecule.
The azide group may be incorporated into the organic inhibitor molecule by reacting a functional group in a molecule to form an azide. For example, the azide based cysteine protease inhibitor may be formed by substituting a leaving group in a molecule with an azide group. The substitution may be carried out by reacting a molecule containing a leaving group (such as a halide or a functionalised hydroxy) with a source of azide anion (N3 ") under appropriate reaction conditions. Alternatively, the azide group may be a part of a precusor molecule which is further reacted to form the cysteine protease inhibitor.
The azide group may be incorporated into a part of the molecule such that the azide group interacts with the cysteine protease when the inhibitor molecule is bound thereto. Without intending to be bound by theory, it is possible that the azide group of the azide based cysteine protease inhibitors may be involved in non-covalent electrostatic interactions with cysteinyl-S or imidazole-N in the active site of the target cysteine protease inhibitor.
The cysteine protease inhibitor molecule may comprise a cysteine protease binding moiety and an end group moiety. The azide group may be incorporated into the end group moiety of the molecule. We have prepared a number of cysteine protease inhibitors in which the cysteine protease binding moiety is an amino acid or a peptide, such as an amino acid or peptide having the following formula:
AA1- -AAn- wherein each AA is the same or different and each is an amino acid residue and n is an integer selected from the group consisting of 0, 2, 3, 4, and 5. In specific embodiments n is 0, 2 or 3.
In each case the amino acid or peptide is covalently bonded via a carbon atom to the azide group.
In specific embodiments the amino acid or peptide has the following formula:
R-CO-AA1-, R-CO-AA1-AA2-, or R-CO-AA1-AA2-AA3
wherein AA1, AA2 and AA3 are the same or different and each is a natural or unnatural amino acid; and R CO- is a nitrogen protecting group.
The end group moiety may have the formula:
Figure imgf000026_0001
wherein R1 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkenyl, optionally - 26 -
substituted alkynyl, and optionally substituted carboxyalkyl. In specific embodiments R1 is selected from the group consisting of optionally substituted alkyl and optionally substituted carboxyalkyl.
In some embodiments the end group moiety has the formula:
Figure imgf000027_0001
In other embodiments the end group moiety has the formula:
Figure imgf000027_0002
In still other embodiments the end group moiety has the formula:
Figure imgf000027_0003
The present studies have also shown that the azide group can replace reactive electrophilic functionalities present in known cysteine protease inhibitors to provide compounds that inhibit cysteine proteases. - 27 -
For example, we found that replacing the C-terminal aspartic aldehyde in the known caspase-1 inhibitor 101 (Rano, T. A.; Timkey, T.; Peterson, E. P.; Rotonda, J.; Nicholson, D. W., Becker, J. W., Chapman, KT. , Thornberry, N.A., Chem. & Biol. 1997, 4, 149-155) by the azidomethylene derivative of aspartic acid maintains sub-μM inhibition of caspase-1.
Figure imgf000028_0001
101
Some known cysteine protease inhibitors are shown in the following table. Modification of any one of these known inhibitors by replacement of an electronhilic group with an azide group may provide a cysteine protease inhibitor having improved properties over the parent inhibitor.
Known Cysteine Protease Inhibitors
Figure imgf000028_0002
-28-
Figure imgf000029_0001
-29-
Figure imgf000030_0001
R = 2-thienyl
Figure imgf000030_0002
-30-
Figure imgf000031_0001
-31 -
Figure imgf000032_0001
VRT-18858
Figure imgf000032_0002
MX-1013
Figure imgf000032_0003
SB-357114 - 32 -
Figure imgf000033_0001
For example, the pyridazinodiazepine derivative:
Figure imgf000033_0002
has been shown to be a potent caspase inhibitor (DoIIe, R. E.; et al. J. Med. Chem. 1997, 40, 1941-6). Conversion of the aldehyde group into an azide group produces the following azide derivative:
Figure imgf000033_0003
Reference can be made to Abbenante, G. and Fairlie, D. P. Med. Chem. 2005, 1, 71-104 and Abbenante, G. and Fairlie, DP. J. Med. Chem. 2000, 43, 305- 341 (both of which are incorporated herein by reference solely for the purpose of providing details of prior art cysteine protease inhibitors) for further details of the prior art compounds listed above. The azide compounds so produced can be tested for cysteine protease activity using any one of the methods described herein, or any method known for that purpose in the art.
The reactive electrophilic group of a cysteine protease inhibitor may be "replaced" by reacting the electrophilic group under appropriate conditions to produce an azide group. The reaction to produce the azide group may be carried out on the protease inhibitor itself, on a derivative of the inhibitor, or on a precursor of the inhibitor. Reactions suitable for converting an electrophilic group to an azide group may be determined by the skilled person having regard to the nature of the electrophilic group. For example, an aldehyde group may be reduced using a reducing agent to produce a secondary alcohol. The secondary alcohol may be converted to a leaving group and subsequently reacted with a source of azide anion (N3"). An example of the latter reaction is shown in Example 2.5.1 of this specification. Reference may be made to "Advanced Organic Chemistry" Jerry March 4th Edn. pp 351-357, Oak Wick and Sons NY (1997).
It will be appreciated that replacement of a functional group that reacts with nucleophiles with an azide group does not mean that an existing cysteine protease inhibitor is reacted to form the azide group. Rather, the azide group may be introduced at any stage of the synthesis of the cysteine protease inhibitor.
The activity of a cysteine protease inhibitor may be modulated by incorporating an azide group in the inhibitor molecule. The activity of a particular azide inhibitor can be determined using tests that are known for that purpose in the art and/or any one of the tests described in the Examples provided in this specification. Modulation of the activity of a cysteine protease inhibitor may involve an increase or a decrease in the activity relative to a parent or control inhibitor. Preferably, the activity is increased, but in cases where the activity is decreased, there may be other advantages in incorporation of an azide group, such as decreased side effects, increased metabolic stability, etc. The specificity of a cysteine protease inhibitor may be modulated by incorporating an azide group in the inhibitor molecule. The specificity of a particular azide based cysteine protease inhibitor for a particular cysteine protease can be determined using tests that are known for that purpose in the art and/or any one of the tests described in the Examples provided in this specification.
Among the biological properties of a cysteine protease inhibitor that may be modulated by preparing azide based cysteine protease inhibitors are toxicity, side-effects, bioavailability, half-life, metabolic stability or solubility.
In some instances, the potency of the azide derivative may be reduced relative to the parent inhibitor. However, other properties of the inhibitor (e.g. bioavailability, solubility, etc) may have been improved by conversion to an azide. Furthermore, any reduced potency of an azide inhibitor may be recovered by appropariate structural variations. For example, in the case of the azide derivative of the known inhibitor 101 , sequential variation at P3 and P4 positions may lead to a more potent inhibitor.
As discussed, we have prepared a number of azide-based cysteine protease inhibitors and shown that they are active both in vivo and in vitro. The present cysteine protease inhibitors have the general formula:
Figure imgf000035_0001
wherein R2 is an amino acid or a peptide; R3 is selected from the group consisting of optionally substituted alkyl, optionally substituted carboxyalkyl, optionally substituted aryl, and optionally substituted carboxyaryl; R4 is selected from the group consisting of H, optionally substituted alkyl, and optionally substituted aryl; and R5 is selected from the group consisting of optionally substituted alkyl, and optionally substituted aryl.
In an embodiment of the present invention the compound has the formula:
Figure imgf000036_0001
wherein R13 is PG-AA1-, PG-AA1 -AA2-, or PG-AA1-AA2- AA3- where AA1, AA2 and AA3 are the same or different and each is a natural or unnatural amino acid and PG is a nitrogen protecting group.
Examples of suitable peptides include PG-VaI-AIa-, PG-GIu-AIa-, PG-Glu-Leu-, PG-Glu-Pro-, PG-Glu-Thr-, PG-Glu-His-, PG-Asp-Leu-, PG-GIu-VaI-, PG-AIa- AIa-, PG-GIy-AIa-, PG-AIa-GIy-, PG-Tbg-Aib-, PG-Phe-Leu-, PG-Cha-Leu-, PG-Tbg-Pro-, PG-Asp-Glu-Leu-, PG-Asp-Glu-Phe-, PG-Asp-Glu-Val-, PG-IIe- Glu-Thr-, PG-lle-Glu-Pro-, PG-Glu-Glu-Leu-, PG-Glu-Leu-Leu-, PG-Leu-Glu- Leu-, PG-Phe-Glu-Leu-, PG-Tic-Glu-Leu-, PG-Tyr-Asp-Ala-, PG-Tyr-Glu-Ala-, PG-Tyr-Val-His-, PG-Tyr-Val-Pro-, PG-Tyr-Val-Phe-, PG-Tyr-Val-Leu-, PG-Tyr- GIn-AIa-, PG-Tyr-Phe-Ala-, PG-Tyr-Leu-Ala-, PG-Tyr-Val-Val-, PG-Tyr-Val-Ala- , PG-Tyr-Val-Cha-, PG-Trp-Glu-Aib-, PG-Trp-Glu-Cha-, PG-Trp-Glu-His-, PG- Trp-Glu-Ala-, PG-Trp-Glu-Tbg-, PG-Val-Glu-Thr-, wherein PG is a nitrogen protecting group.
The nitrogen protecting group (PG) may be selected from the group consisting of Ac, - 36 -
Figure imgf000037_0001
Specific compounds of the invention are numbered 1 to 100 and can be found in Tables 1 to 12.
The present invention also provides a compound of formula:
Figure imgf000037_0002
wherein R19 is selected from the group consisting of amino acid, and peptide.
R19 may be PG-AA1 -AA2- or PG-AA1 -AA2- AA3- where AA1, AA2 and AA3 are the same or different and each is a natural or unnatural amino acid and PG is a nitrogen protecting group. Alternatively, the compound may have the formula:
Figure imgf000038_0001
wherein R13 is PG-AA1-, PG-AA1 -AA2-, or PG-AA1-AA2- AA3- where AA1, AA2 and AA3 are the same or different and each is a natural or unnatural amino acid and PG is a nitrogen protecting group, and R22 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkenyl, and optionally substituted alkynyl.
The compounds of the present invention can be prepared using any suitable methodology. For example, the amino acid- or peptide-based compounds of the present invention may be prepared by coupling the azidomethylene derivative of aspartic acid with a solid phase resin, such as thtylchloride polystyrene resin (TCP resin). The resin loaded with the azidomethylene derivative of aspartic acid can be N-deprotected and acylated with the desired N-protected amino acid. Further rounds of N-deprotection and acylation with suitable amino acids can be used to prepare the desired peptide. Following N-protection with a nitrogen protecting group of choice, the peptide can be cleaved and purified. Other suitable synthetic methodologies will be evident to the person skilled in the art. Reference may also be made to Atherton E. and Sheppard R,C. in Solid phase peptide synthesis : a practical approach, Oxford, England ; New York : IRL Press at Oxford University Press, 1989 and Lloyd-Williams, P., Albehcio, F. and Giralt, E in Chemical approaches to the synthesis of peptides and proteins, Boca Raton : CRC Press, c1997 for further details on suitable methods of peptide synthesis.
The intermediates, products and final products obtained by the above synthesis reactions, as necessary, can be isolated and purified by conventional methods for separation and purification, such as chromatography, concentration, vacuum concentration, solvent extraction, crystallization, recrystallization, etc.
The compounds of the present invention are useful for the inhibition of cysteine proteases. As used herein, the terms "inhibit" and "inhibition" mean having an adverse effect on enzymatic activity. An inhibitory amount is an amount of a compound of the invention effective to inhibit a cysteine protease. Thus, the present invention further provides a method of inhibiting a cysteine protease including administering to a patient in need thereof an effective amount of an azide compound described herein.
The present invention also provides a method of treating a disease state in which the disease pathology may be therapeutically modified by inhibiting a cysteine protease including administering to a patient in need thereof an effective amount of an azide compound described herein. The compounds of the present invention, or pharmacologically acceptable esters or a salts thereof, having activity of inhibiting cysteine protease (ICE, cathepsin B, shock cathepsin L, etc, preferably ICE, etc.) is safe with low toxicity, and can be used to treat and prevent various infectious diseases, immune diseases, bone diseases, neurologic diseases, tumors, inflammatory diseases etc., including meningitis, salpingitis, enteritis, inflammatory enteritis, hyperacidic enteritis, sepsis, septic shock, disseminated intravascular coagulation, adult respiratory distress, arthritis, bile duct disease, colitis, encephalitis, endocarditis, glomerular nephritis, hepatitis, myocarditis, pancreatitis, pericarditis, reperfusion disorder, angitis, acute and delayed allergies, graft rejection, psoriasis, asthma, type I diabetes mellitus, multiple sclerosis, allergic dermatitis, acute and chronic myelocytic leukemia, tissue calcium deficiency, rheumatism, rheumatoid arthritis, arthrosteitis, senile and climacteric osteoporosis, immobile and traumatic osteoporosis, arteriosclerosis, periodontitis, spatial pulmonary fibrosis, hepatic cirrhosis, systemic sclerosis, keloid, Alzheimer's disease and IL-1 -producing tumors, in humans and other mammals (e.g., mice, rats, rabbits, dogs, cats, monkeys, bovines, swines). The present invention also provides a pharmaceutical composition comprising an azide compound described herein, and a pharmaceutically acceptable carrier, diluent or excipient. Thus, the compound(s) of the present invention, or a pharmaceutically acceptable ester or a salt thereof, formulated with a pharmaceutically acceptable carrier can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
For oral administration, the composition may be in the form of solid preparations such as a tablets, capsules, granules or powders, or liquid preparations such as syrups and aqueous suspensions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
Alternatively, the composition may be an injectable preparation in the form of a a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. HeIv or a similar alcohol. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically- transdermal patches are also included in this invention.
The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
The prophylactic/therapeutic preparation of the present invention can be produced by commonly used methods such as mixing, kneading, granulation, tableting, coating, sterilization and emulsification, depending on preparation form.
The content of the azide compounds in the preparation of the present invention is normally about 0.01 to 100% by weight, preferably about 0.1 to 50% by weight, and more preferably about 0.5 to 20% by weight, relative to the entire preparation, depending on preparation form.
Pharmaceutically acceptable carriers are various organic or inorganic carrier substances in common use as pharmaceutical materials, including excipients, lubricants, binders and disintegrating agents for solid preparations, and solvents, dissolution aids, suspending agents, isotonizing agents, buffers and smoothing agents for liquid preparations. Other pharmaceutical additives such as preservatives, antioxidants, coloring agents and sweetening agents may be used as necessary. Preferable excipients include lactose, sucrose, D-mannitol, starch, crystalline cellulose and light silicic anhydride. Preferable lubricants include magnesium stearate, calcium stearate, talc and colloidal silica. Preferable binders include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose and polyvinylpyrrolidone. Preferable disintegrating agents include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, crosscarmellose sodium and carboxymethyl starch sodium. Preferable solvents include water for injection, alcohol, propylene glycol, macrogol, sesame oil and corn oil. Preferable dissolution aids include polyethylene glycol, propylene glycol, D- mannitol, benzyl benzoate, ethanol, tris-aminomethane, cholesterol, triethanolamine, sodium carbonate and sodium citrate. Preferable suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride and monosteahc glycerol, and hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose sodium, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose. Preferable isotonizing agents include sodium chloride, glycerol and D-mannitol. Preferable buffers include buffer solutions of phosphates, acetates, carbonates and citrates. Preferable smoothing agents include benzyl alcohol. Preferable preservatives include p-oxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid and sorbic acid. Preferable antioxidants include sulfites and ascorbic acid.
The compound of the present invention may be orally or non-orally administered to humans and other animals suffering from various diseases as described above, at doses of, for example, 0.01 to 500 mg/day, preferably 0.1 to 50 mg/day, more preferably 1 to 20 mg/day, per kg body weight.
Although the dose varies depending on kind of compound, route of administration, symptoms, patient age etc., it can be administered at 0.1 to 50 mg/day, preferably 1 to 20 mg/day, per kg of body weight, in 1 to 6 portions, for oral administration to an adult patient.
Upon improvement of a patient's condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
An attending physician or a person skilled in the art will appreciate, lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the infection, the patient's disposition to the infection and the judgment of the treating physician.
Depending on the condition to be treated, the azide compounds of the present invention may be co-administered with other therapeutically active agents, as required.
The inhibitors described herein may also be used to identify and purify cysteine proteases. The inhibitors can be covalently linked to a solid support, such as an affinity column or beads used in batch methods, and used to purify a cysteine protease or enrich a mixture containing the cysteine protease. The inhibitor may be linked to the solid support or bead either directly or via a linker of variable length, such that linkage does not interfere with the binding properties (see, Thomberry, N., Methods in Enz., 244:615-31 (1994)).
Description of the Figures
In the figures,
Figure 1 shows a Lineweaver-Burk plot for varying substrate AcYVAD-AMC (S) and inhibitor AcWEHD-N3. Concentrations at pH 7.4 (5OmM HEPES).
[I] = 0, Vmax 43, Km 11.4; [I] = 125nM, Vmax= 45.4, Km= 26.1 ; [I] =
25OnM, Vmax= 40.3, Km= 28.6; [I] = 375nM, Vmax = 36.4, Km = 35.2; [I]
= 50OnM, Vmax = 34.3, Km = 48.8.
Figure 2 shows a plot that provides experimental confirmation that inhibitor 13 binds in a reversible manner to caspase-1. — a — [S] = 14uM, [I] = 0; — •- [S] = 14uM, [I] = 50 nM; -Δ- [S] = 14OnM, [I] = 5OnM; — ■— [S] = 28OnM, [I] =50nM. The addition of 5OnM of inhibitor 13 completely abolishes caspase-1 processing of the substrate Ac- YVAD-AMC. The activity of the enzyme can be restored by the addition of 2Ox the original concentration of substrate to the fully inhibited enzyme.
Figure 3 shows (A) modeled 13 docked in the active site of caspase-1 (pdb 1 1CE), and (B) backbone (C, N, O atoms) supehmposition of 13 on enzyme-bound structure of Ac-YVAD-H.
Figure 4 shows selected 1H-NMR signals for aldehyde 101 (left panel) and azide 13 (right panel) in H2O:D2O (9:1 , phosphate buffer pH 7.4,
37°C) at 5 min (A), 3h (B), 24h (C) or with 10 fold Ac-Cys for 5min (D), 3h (E) or 3 weeks (F). New signals in B, C, E were due to respective protons of D-isomer. 13 remained unchanged under all these conditions (right panel).
Figure 5 shows a plot of IL-1 β concentration vs time for a number of cell permeable compounds. All compounds were tested at 10 μM - compound alone (dashed lines) and with LPS (solid coloured lines) against U937 cells. Controls were DMSO (<0.01 % final volume) and Ac-YVAD-CHO which has a reported EC50 = 5μM in a cell-based assay. The compounds inhibited 11-1 β release vs LPS alone.
Description of Embodiments of the Invention
So that the present invention may be more fully understood, the following examples are provided. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.
Example 1 - Azido based inhibitors Compounds 1 to 46, 49 and 51 to 100 were synthesised using the procedures set out in Example 2 below.
The inhibitory properties of the compounds were then tested using the procedures set out in Example 3 below.
The structures of compounds 1 to 100 as well as the IC50 or % inhibition values are shown in Tables 1 to 12.
- 46 -
Tablθ 1 . Azidomethylene based inhibitors of Caspase-1
Figure imgf000047_0001
R IC50 (nM)
Ac-Asp-Glu-Leu- 5700
Ac-Tyr-Asp-Ala- 1150
Ac-Trp-G Iu-Ai b- 675
Ac-Trp-Glu-His- 696
Ac-Tyr-Val-Ala- 226
Ac-Tyr-Glu-Ala- 310
Ac-Trp-Glu-Cha- 288
Ac-Trp-Glu-Ala- 123
Figure imgf000047_0002
- 47 -
Tablθ 2. Azidomethylene based inhibitors of Caspase-1
Figure imgf000048_0001
R IC50 (nM)
7.9
^■Tbg-Aib-
Figure imgf000048_0002
>50000
Figure imgf000048_0003
- 48 -
Tablθ 3. Azidomethylene based inhibitors of Caspase-1
Figure imgf000049_0001
% Inhibition at
R 50OnM 25OnM
Ac-Tyr-Val-His- 27%
Ac-Tyr-Val-Pro- 20%
Ac-Asp-GI u-Val- 32%
Ac-Tyr-Val-Phe- 38%
Ac-Tyr-Val-Leu- 43%
Ac-Tyr-Leu-Ala- 43%
Ac-Tyr-Val-Val- 48%
Ac-Tyr-Val-Cha- 53%
Ac-Tyr-Gln-Ala- 53%
Ac-Tyr-Phe-Ala- 55%
Ac-Trp-Glu-Tbg- 55%
46%
,G Iu-AIa-
Figure imgf000049_0002
Cl
Ck 92%
^GIu-AIa- -49-
Tablθ 4. Azidomethylene based inhibitors of Caspase-1
Figure imgf000050_0001
R % Inhibition
Ac-Glu-His- 13% at 1000OnM
Z-Glu-His- 56% at 100OnM
Ac-Glu-Leu- 88% at 100OnM
Figure imgf000050_0002
-50-
Table 5. Structural variants of inhibitors of Caspase-1
% IC50
Inhibition
Figure imgf000051_0001
- 51 -
Table 6. Azidomethylene based inhibitors of Caspase-3
Figure imgf000052_0001
% Inhibition at
R IC50 (nM) 500OnM 50OnM
Ac-Asp-Glu-Val- 857
Ac-Asp-Glu-Leu- 393
Ac-Glu-Leu-Leu- 3%
Cbz-Glu-Leu- 383
Cbz-Asp-Leu- 0%
Figure imgf000052_0002
Z-homoGlu-Leu- 0%
Z-IIe- 0%
Z-VaI- 0% - 52 -
Tablθ 7. Azidomethylene based inhibitors of Caspase-3
Figure imgf000053_0001
IC50 % inhibition % inhibition
R= (nM) (500OnM) at 50OnM
Figure imgf000053_0002
o - ov^GI u-Leu-
Figure imgf000053_0003
Benzoyl-Asp-Glu-Leu — 10
Ac-Asp-Glu-Phe^ 10
Cbz-GI u-Ala - 3
Ac-Leu-Glu-Leu — 0 Ac-Phe-Glu-Leu — 0 Ac-Tic-Glu-Leu — 0
- 53 -
Tablθ 8. Azidomethylene based inhibitors of Caspase-8
Figure imgf000054_0001
Ac-Asp-Glu-Leu- 23 Ac-Asp-Glu-Val- 22 Ac-Glu-Glu-Leu- 32
Cbz-Glu-Leu- 128 71 o U-Glu-Leu- 44
O2N- ,0 ipGlu-Leu- 35 o
Figure imgf000054_0002
Ac-lle-Glu-Thr- 28
Ac-VaI-G lu-Thr- 35 Ac-lle-Glu-Pro- 23 - 54 -
Tablθ 9. Azidomethylene based inhibitors of Caspase-8
Figure imgf000055_0001
Benzoyl-Asp-Glu-Leu —
Ac-Asp-Glu-Phe— 17
Cbz-Glu-Ala - 4
Ac-Leu-Glu-Leu — 0
Ac-Phe-Glu-Leu — 10
Ac-Tic-Glu-Leu — 0 Z-HomoGlu-Letι — 0 - 55 -
Tablθ 10. Azidomethylene based inhibitors of Cathepsin K
Figure imgf000056_0001
% Inhibition at
R = 125OnM 25nM IC50(nM)
Figure imgf000056_0002
98 Cbz-Phθ- LeU-N3 Not yet tested
99 BiPhenyl-Phe-Leu-N3 Not yet tested
100 Biphenyl-Cha-I_eu-N3 Not yet tested - 56 -
Tablθ 1 1 . Azidomethylene based inhibitors of Cathepsin S
Figure imgf000057_0001
% Inhibition at
R = 50OnM IC50(nM)
Figure imgf000057_0002
98 Cbz-Phe-Leu-N3 35 99 BiPhenyl-Phe-Leu-N3 1 -6
100 Biphenyl-Cha-Leu-N3 1.6 - 57 -
Tablθ 12. Azidomethylene based inhibitors of Cathepsin B
Figure imgf000058_0001
% Inhibition at
R = 50OnM IC50(nM)
Figure imgf000058_0002
98 Cbz-Phe-Leu-N, 40 Example 2 - Synthesis of Azidomethylene Inhibitors
2.1 General Synthesis
Tritylchloride-polystyrene (TCP) resin was obtained from PepChem (Reutlingen, Germany). 0-(1 H-6-Chlorobenzotriazole-1-yl)-1 , 1 ,3,3- tetramethyluronium hexafluorphosphate (HCTLJ) was obtained from Iris Biotech (Marktredwitz, Germany). Fmoc protected amino acids and all other reagents were of peptide synthesis grade and obtained from Auspep (Melbourne, Australia).
Semi-preparative rp-HPLC purification of the linear peptides was performed using a Waters Delta 600 chromatograpy system fitted with a Waters 486 tunable absorbance detector with detection at 214 nm. Purification was performed by eluting with solvents A (0.1 % TFA in water) and B (9:1 CH3CN:H2θ, 0.1 % TFA) on a Phenomenex Luna Ci 8 15μ 250 x 21.2 mm steel jacketed column run at 20 mL/min. Analytical rp-HPLC analyses were performed using a Waters 600 chromatography system fitted with a Waters 996 photodiode array detector and processed using Waters Millenium software. Analytical analyses were performed using gradient elutions with solvents A and B on a Phenomenex Luna C-| 8 5 μ 250 x 4.6 mm column run at 1.0 mL/min.
Mass spectra were obtained on a hybrid quadrupole TOF mass spectrometer (PE SCIEX API QSTAR Pulsar) equipped with an lonspray (pneumatically assisted electrospray) operating at ambient temperature (ISMS). Calibration standard was ammonium adducts of polypropylene glycol (PPG 1000).
2.2 Synthesis and Purification of Peptides
Peptides were synthesized manually by standard solid phase methods using HCTU/DIPEA activation for Fmoc chemistry on TCP resin (substitution 0.72 mmol.g"1, 0.25 mmol scale syntheses, 350 mg resin). Four equivalents of - 59 -
Fmoc-protected amino acids, 4 equivalents of HCTU and two equivalents of DIPEA were employed in each coupling. Fmoc-deprotections and resin neutralization was achieved by 2 x 2 min. treatments with excess 1 :1 piperidine:DMF. Coupling yields were monitored by quantitative ninhydrin assay (Sarin, V. et al., Anal. Biochemistry 1981 , 117, 147) and double couplings were employed for yields below 99.8%. N-terminal acetylation was achieved by treating the fully assembled and protected peptide-resins (after removal of the N-terminal Fmoc group) with HCTU-activated acetic acid as per normal coupling. The peptide was cleaved from resin and protecting groups simultaneously removed by treatment for 2h at room temperature with a solution containing 95% trifluoroacetic acid (TFA):2.5% H2O: 2.5% triisopropylsilane (TIPS) (25ml_ solution per 1 gm of peptide-resin). The TFA solutions were filtered, concentrated in vacuo, diluted with 50% A:50% B, lyophilized, and subsequently purified by semi-preparative rp-HPLC. All products were identified by electron spray mass spectrometer.
2.3 Synthesis of compound 13
Figure imgf000060_0001
DIPEA
Figure imgf000060_0002
II
Peptide
Figure imgf000060_0003
synthesis
Figure imgf000060_0004
The azidomethylene derivative I of protected L-Fmoc-aspartic acid was prepared as described (Boeijen, A; van Ameijde, J; Liskamp R. M. J. J. Org. Chem. 2001 , 6, 8454-8462).
Tert-butyl ester I (2.7g, 6.4mmol) was stirred at room temperature in a cocktail containing 4.75ml of TFA, 0.125ml of water and 0.125ml of triisopropylsilane for 2 hours. All volatile solvents were removed under vacuum and the residue was dissolved in dichloromethane (200ml), washed with water. The organic phase was dried over MgSO4, filtered and evaporated to dryness. The last trace of solvents was removed by drying under high vacuum for 2 hours. The obtained product (II) was brownish solid and was pure by HPLC. Yield 2.2g, 94%. CaIc. Mw (M+H) = 367.14; obs Mw = 367.1 1
Acid II (2.2g, 6mmol) was mixed with thtylchloride polystyrene resin (TCP resin; PepChem; product #: PC-01-0010) (4g @ 0.94mmol/g; 3.76mmol) in 9ml of dry DMF. DIPEA (2.08ml, 12mmol) was added and the resin was shaken at room temperature for 16 hours. The solvent was removed and the resin was washed with DMF (three times), methanol (three times) and DCM (three times) then dried under high vacuum.
TCP resin loaded with acid II (200mg, 0.198mmol) was deprotected and acylated with Fmoc-Ala-OH, Fmoc-Val-OH and 2-naphthoic acid, cleaved and purified by rp-HPLC as described in general method. Fractions containing desired products were identified by mass spectrometer and their purity was ascertained by analytical rp-HPLC. Pure fractions were combined and freeze- dried to afford the final product 13 as white solid (12.8mg; 13.8%). Mass spectroscopy and NMR characterizations were described above. A less pure fraction of 15mg (16%) was also obtained. - 61 -
2.4 Synthesis of compounds 1 -46, 49 and 51 -83
Compounds 1 -46, 49 and 51 -83
Figure imgf000062_0001
The azidomethylene derivative of L-Fmoc-aspartic acid was prepared by deprotection of the corresponding tert-butyl ester (Boeijen, A; van Ameijde, J; Liskamp R. M. J. J. Org. Chem. 2001 , 6, 8454-8462) and loaded onto TCP resin. After peptide synthesis to achieve the desired sequence, compounds 1- 46, 49 and 51 -83 were cleaved and purified as described above.
2.5 Synthesis of compounds 84-100
BocNH
Figure imgf000062_0002
V: R1 = (CH2)3CH3 VI: R1 = (CH2CH(CH3)2 84-100
Compounds 84-100 were prepared from the appropriate azide derivatives V or VI by standard solution phase methods using HCTU/DIPEA or HBTU/DIPEA activation for Boc peptide synthesis. The compounds were purified by rp-HPLC or gravity column chromatography to purity of greater than 95%. As examples, synthesis of compounds 86 and 105 will be described below. - 62 -
2.5.1 Synthesis of compound 86
Figure imgf000063_0001
VII VIII
Figure imgf000063_0002
The hydroxyl derivative VII of protected L-Boc-norleucine acid was prepared as described (Catalano, JG at al. Bioorg. Med. Chem. Lett. 2004, 14, 275- 278).
Under a nitrogen atmosphere, diisopropyl azodicarboxylate (DIPADC, 9 mmol, 1.7.4 ml_, 1.5 equiv) was added dropwise to a cooled (O0C) solution of PPh3 (9 mmol, 2.36 g) in dry THF (27 ml_). Subsequently, a 1.5 M solution of HN3 in toluene (12 mmol, 7.9 ml_, 2 equiv) was added dropwise, whereupon Boc- protected amino alcohol (6 mmol, 1 equiv) was added in one portion. The reaction mixture was stirred overnight at room temperature, and was concentrated in vacuo. After gravity column chromatography with petroleum ether/ethylacetate (4/1 ), the azide VIII was obtained as an oily residue (1.31 g, 90%) which crystallized upon standing at 40C.
Azide VIII (310mg, 1.28mmol) was dissolved in a solution containing 4.5ml of TFA and 0.5ml of water. The solution was stirred at room temperature for 2 hours and the volatile solvents were removed under vacuum. To the residue 15ml of toluene was added and then evaporated to dryness. The residue was dried until constant weight under high vacuum to give the TFA salt of the norleucine azide derivative. In a separate flask, L-Boc-leucine monohydrate (638.2mg, 2.56mmol) was dissolved in DMF (5ml), HBTU (971 mg, 2.56mmol) and DIPEA (330mg, 2.56mmol) was added. The solution was stirred for 5 minutes and added to the above TFA salt of the norleucine azide derivative followed by addition of DIPEA (330mg, 2.56mmol). The reaction mixture was stirred for 3 hours at room temperature then diluted with 100ml of ethylacetate and 100ml of saturated bicarbonate. After separation the aqueous phase was extracted twice with 50ml of ethylacetate. The combined ethylacetate solutions were dried over MgSO4 and evaporated to dryness. The residue was dried until constant weight under high vacuum and subjected to TFA cleavage as described above for compound VIII. The obtained TFA salt of LeU-NIe-N3 derivative was acylated with 2-naphthoic acid (440.3mg, 2.56mmol), which were activated with HBTU (971 mg, 2.56mmol) and DIPEA (660mg, 5.12 mmol). The acylation was allowed to proceed overnight at room temperature, then diluted with 100ml of ethylacetate and 100ml of saturated bicarbonate. After separation the aqueous phase was extracted twice with 50ml of ethylacetate. The combined ethylacetate solution were dried over MgSO4 and evaporated to dryness. The residue was purified by gravity chromatography using petroleum/ethylacetate (1/1 ) as eluant. Re- crystallization from petroleum/ethylacetate afforded pure compound 86 as white solid (248mg, 47.3%). A less pure fraction of 100mg (19.1 %) was also obtained. - 64 -
2.5.2 Synthesis of compound 105
B
Figure imgf000065_0001
105
The Boc-protected azide IX was synthesized according to the method of Kokotos et.al.; Journal of Chemical Research, Synopses; 1992, 12, 391.
The azide IX (60mg, 0.25mmol) was dissolved in TFA (1 OmL) and stirred for 15 minutes at room temperature. The TFA was evaporated off with a stream of nitrogen and to the residue was added Boc-Phe-OH (100mg, 0.38mmol), BOP (168mg, 0.38mmol), DMF (5ml) and finally diisopropylethylamine (0.2ml) and the solution stirred overnight. The solution was quenched with 10% KHSO4 solution and extracted with ethyl acetate (3x30ml_). The combined ethyl acetate solution was washed with 10% KHSO4 (1x 3OmL), sat. NaHCO3 (2x 50ml) and water (2x50ml). This gave 120mg of crude product that was dissolved in TFA (10ml) and stirred for 15 minutes. The TFA was evaporated off with nitrogen. The residue was dissolved in DMF (10ml) and to this solution was added biphenyl carboxylate (100mg, O.δmmol), BOP (0.223g, 0.5mmol) and DIPEA (0.2ml). The solution was stirred at room temperature for two hours. The solution was then quenched with 10% KHSO4 solution and - 65 -
extracted with ethyl acetate (3x30ml_). The combined ethyl acetate solution was washed with 10% KHSO4 (1x 3OmL), sat. NaHCO3 (2x 50ml) and water (2x50ml). The crude product was purified by radial chromatography using a 30% ethyl acetate/ 70% light petroleum mixture as eluant. The product crystallized from the eluant upon cooling and was filtered from solution. This gave 35mg, of pure tripeptide 105 as a white solid in 30% yield.
2.6 High Resolution mass spectroscopy characterisation and retention time of representative azidomethylene inhibitors
Figure imgf000066_0001
2.7 Docking azide inhibitor 13 in active site of cysteine proteases
Modelling of 13 in the substrate-binding active site of caspase-1 using GOLD (Fig. 3) showed that the naphthoyl substituent (P4), and side chains of VaI (P3), Ala (P2) and pseudo-aspartate (P1 ) of 13 occupied expected sites (Fig. 3A: S4- S1 respectively) in caspase-1 (crystal structure of (Ac-YVAD-H )-enzyme complex, pdb: 1 ICE, Wilson, K. P.; Black, J. A. F.; Thomson, J. A.; Kim, E. E.; Griffith, J. P.; Navia, M. A.; Murcko, M. A.; Chambers, S. P.; Aldape, R. A.; Raybuck, S. A.; Livingston, D. J. Nature, 1994, 370, 270-275.). Inhibitor 13 also occupies very similar conformational space as the known Ac-YVAD-H aldehyde inhibitor (101 ) (Fig. 3B), its backbone atoms (C, N, O) superimposing well (rmsd 0.37A) on the corresponding atoms of 101 in its crystal structure with caspase-1. The backbone adopts the classic extended strand recognized by most proteases ((a) Tyndall, J. D. A.; NaII, T.; Fairlie, D. P. Chem. Rev. 2005, 105, 973-1000. (b) Loughlin, W. A.; Tyndall, J. D. A.; Glenn, M. P.; Fairlie, D. P. Chem. Rev. 2004, 104, 6085-6117. (c) Fairlie, D. P.; Tyndall, J. D. A.; Reid, R. C; Wong, A. K.; Abbenante, G.; Scanlon, M. J.; March, D. R.; Bergman, D. A.; Chai, C. L. L.; Burkett, B. A. J. Med. Chem. 2000, 43, 1271-1281. (d) Tyndall, J.; Fairlie, D. P. J. MoI. Rec. 1999, 12, 363-370.) Corresponding side chains also align closely. The P1 carboxylate side chain of 13 is important, as it is in 101 (Sleath, P. R.; Hendrickson, R. C; Kronheim, S. R.; March, C. J.; Black, R. A. J. Biol. Chem. 1990, 265, 14526-14528.), since conversion to the amide reduced caspase-1 inhibition 50 fold (IC5O 230 nM). This supports analogous binding for 14 and 79 at the S1 site in caspase-1.
Example 3 - Enzyme Inhibition Assays All enzymes were purchased from Merck Pty Ltd.
3.1 Assay Buffers
Caspase-1 : 10OmM NaCI, 5OmM HEPES, 1 OmM DTT, 1 mM EDTA, 10%
Glycerol, 0.1 % CHAPS, pH=7.4 Caspase-3: 10OmM NaCI, 5OmM HEPES, 1 OmM DTT, 1 mM EDTA, 10%
Glycerol, 0.1 % CHAPS, pH=7.4
Caspase-8: 10OmM NaCI, 5OmM HEPES, 1 OmM DTT, 1 mM EDTA, 10%
Glycerol, 0.1 % CHAPS, pH=7.4
Caspase-5: 10OmM NaCI, 5OmM HEPES, 1 OmM DTT, 1 mM EDTA, 10% Glycerol, 0.5% CHAPS, pH=7.4
Cathepsin K: 10OmM Na acetate, 1OmM DTT, 12OmM NaCI, pH=5.5
Cathepsin S: 10OmM Na acetate, 1 OmM DTT, 12OmM NaCI, pH=5.5
Cathepsin B : 10OmM Na acetate, 1 OmM DTT, 12OmM NaCI, pH=5.5
3.2 Enzymes Concentrations
Caspase-1 : Recombinant human caspase-1 (3000U) was diluted with 700μL of buffer, divided into 70μl_ aliquots and stored at -8O0C. The diluted solution (4.5μl_) was used in each well for individual reactions. Caspase-3: Recombinant human caspase-3 (5000U) was was diluted with 1 ml of buffer, divided into 65μL aliquots and stored at -8O0C. Each 65uL aliquot was subsequently diluted to 24OuL and this solution used for each well (5μL).
Caspase-5: Recombinant human caspase-5 (3000U) was diluted with 700μL of buffer, divided into 70μl_ lots and stored at -80°C. The diluted solution (4.5μl_) was used in each well for individual reactions.
Caspase-8: Recombinant human caspase-8 (3000U) was diluted with 700μL of buffer, divided into 70μl_ lots and stored at -8O0C. The diluted solution (4.5μl_) was used in each well for individual reactions. Cathepsin K: Recombinant human cathepsin K (25mg) was purchased from
Calbiochem. The enzyme solution was diluted with 600ml of buffer, divided into
65μl_ aliquots and stored at -800C. Each 65uL aliquot was subsequently diluted to 24OuL and this solution used for each well (5μL).
Cathepsin S: Recombinant human cathepsin S (25mg) was purchased from Calbiochem. The enzyme solution was diluted with 600ml of buffer, aliquoted into 30μL lots and stored at -80°C. Each 3OmL aliquot was subsequently diluted to 15OuL and this solution used for each well (5μL).
Cathepsin B: Cathepsin B from human liver (25ug, cone. 1 .6mg/ml) was dissolved in 700μL of buffer. This was divided into 30μL portions. Each 3OuI aliquot was further diluted 128X and the subsequent solution divided into 150μL aliquots. The 150μL aliquots were used to deliver 5μL of enzyme to each well.
3.3 Substrates
Caspase-1 : A 2.8mM DMSO solution of Ac-YVAD-AMC (Merck) was diluted 200X in buffer to a final concentration of 14mM in each well (Km=14nM). Caspase-3: A 1.94m M DMSO solution of Ac-DEVD-AMC (Merck) was diluted 200X in buffer to a final concentration of 1 OmM in each well (Km=9.7nM). Caspase-5: A 2.OmM DMSO solution of Ac-WEHD-AMC (Merck) was diluted 200X in buffer to a final concentration of 1 OmM in each well (Km=10nM).
Caspase-8: A 2mM DMSO solution of Ac-IETD-AMC (Merck) was diluted 200X in buffer to a final concentration of 1 OmM in each well (Km= 6mM). Cathepsin K: A 2.OmM DMSO solution of z-F-R-AMC (Merck) was diluted 200X in buffer to a final concentration of 1 OmM in each well (Km=70mM). Cathepsin S: A 2.OmM DMSO solution of z-V-V-R-AMC (Merck) was diluted 200X in buffer to a final concentration of 1 OmM in each well (Km>100mM). Cathepsin B: A 2.OmM DMSO solution of z-F-R-AMC (Merck) was diluted 200X in buffer to a final concentration of 1 OmM in each well (Km= 40OmM).
3.4 Inhibitors
Each inhibitor was dissolved in DMSO to a concentration of 5mM and diluted out to the required concentration range. Seven different concentrations (2 fold dilutions) were used which spanned the IC50 value of the inhibitor and were each repeated in duplicate. The DMSO solution (2μl_) of was applied to each well.
3.5 Fluorescence
In each well of a 96 well plate was placed 1 μl_ of substrate, 2μl_ of inhibitor and 4.5-5μl of the enzyme. The volume was made up to a total of 200μL with buffer. The enzyme was incubated with inhibitor for 5 minutes prior to the addition of substrate and reaction monitored on a fluorostar spectrophotometer (BMG Technologies) in a 96-well plate format, using an excitation wavelength of 380nm and emission of 460nm to detect free AMC. The IC50 value for each inhibitor was determined using non-linear regression by fitting the data to a sigmoidal dose-response curve plotting log [I] vs Vi/Vo. The assays for caspases-1 , -3 & 8 were done at room temperature, assays for cathepsin K and S and B were at 300C. Example 4 - Evidence of Competitive Binding For Azide-Based Cysteine Protease Inhibitors
Lineweaver burke analysis of Km and Vmax values for caspase-1 against various concentrations of inhibitor Ac-WEHD-N3 4 (Figure 1 ) showed that Vmax remained essentially constant whereas Km increased with increasing inhibitor concentrations. This result shows that the azide inhibitors of caspase-1 are binding competitively in the active site of this enzyme and are reversible inhibitors.
Example 5 - Competitive Binding Study of Inhibitor 13
A 5mM solution (DMSO) of 13 was diluted to 5μM via three 10-fold serial dilutions. 20 μl_ of this solution was added to 130 μl_ of buffer and 15 μl of this solution was applied to each well. Enzyme solution (5μl_) was added and the mixture was allowed to incubate for ten minutes at room temperature. To this solution was added a solution of substrate (180μl_) in assay buffer. The processing of the Ac-YVAD-AMC substrate was monitored via a fluorescence plate reader with 380nm excitation and 460nm emission wavelengths. The results are shown in Figure 2.
Example 6 - Stability of Azides in Human Serum
Compounds 13 and 14 were dissolved directly into serum at a concentration of 1 mg/ml. The serum was incubated at 370C and 20μl aliquots were taken at 0, 30, 70, 120, 150, 210, 300, 360 min and one sample at 24hrs. The 20μl aliquots were diluted with 380 μl of a 3:1 solution of acetonithle /water and centrifuged at 1000Og for 4min to remove serum proteins. Standard solutions of both compounds were made by dissolving the solid into 20% DMSO water to a concentration of 1 mg/ml and diluting this by two fold for 7 iterations. 20μl_ aliquots were then taken from each tube and diluted with 380 μl of 3:1 acetonitrile /water. Standard solutions for both compounds gave a standard curve with r2 = 0.99 or better and LC-MS of the serum samples showed that 100% recovery of the compounds was achieved even after 24 hours incubation in serum at 37°C.
This result indicates that these two compounds are both stable in serum and do not bind to serum proteins.
Example 7 - Stability ofAzides in buffer solutions
Compoundsv 13 and 14 were completely stable in aqueous solutions (>1 month, 37°C), assay buffer (14 h, pH 7.2, 10 mM dithiothreitol) ± enzyme, or 2OmM glutathione (pH 7.2, 9, 11 ), as monitored quantitatively by LCMS.
For comparative studies, compounds 13 or 101 were dissolved in phosphate buffer (pH = 7.4, H2O/D2O 9/1 ) to a concentration of 1 mM. The samples were incubated at 37°C and followed by NMR at indicated time points. For experiments with thiols, Ac-Cys (1 OmM) or glutathione (2OmM) were dissolved in the phosphate buffer, pH was adjusted to 7.4 and the solution was used freshly for stability studies as above. NMR 1 D 1H-NMR spectra were recorded on a Bruker Avance DRX-600 spectrometer. Spectra were processed using Topspin (Bruker, Germany) software.
Under these conditions, aldehyde 101 is unstable in aqueous solutions (Fig.4, left panel), existing initially as a mixture of aldehyde and cyclic hemiacetal (A) followed by rapid aspartate racemization (B, C) in phosphate buffer (Ui2 4h, 37° C, pH 7.4). No aldehyde is detectable within minutes in 1 OmM Ac-Cys (D, E) or 2OmM glutathione. Azide 13 remained unchanged under all conditions.
Example 8 - Selectivity of Caspase-1 Inhibitors for other caspases Selectivity data for Caspase-1 Inhibitors vs Caspase-5, -3, and 8
IC50 (nM) Caspase 1 3 5 8
Figure imgf000072_0001
The caspase-1 inhibitors 13 and 15 were assayed against caspase-3, -5, and - 8. Inhibitor 13 was 1000 fold selective for caspase -1 over the closely related caspase 5. In addition, 13 does not inhibit caspase-3 and is 100 fold selective as compared to caspase-8. Compound 15 was also found to be 1000 fold selective for caspase-1 as compared to caspase-5. This data proves that the azidomethylene compounds described herein can be modified for selectivity for the target enzyme.
Finally, there are various modifications and alterations, that could be adopted or made to the embodiments described herein, that are also within the scope of the present invention.
Example 9 Cysteine protease inhibition in cells
Conversion of compounds 13 and 14 to a cell permeable prodrug forms -72-
Figure imgf000073_0001
13 R1 = -CH(CH3J2, R2 = -CH3, R3 = -H 80 R1 = -CH(CH3J2, R2 = -CH3, R3 = -H
14 R1 = -C(CH3)3 R2 = R3 = -CH3 81 R1 = -C(CH3J3 R2 = R3 = -CH3
Compound 13 (0.5g, 1 .0 mmol) was stirred at room temperature in 30ml of dry ethanol containing 1 M HCI for 48hrs. The volatile solvents were removed in vacuum and the residue recrystallised from 50/50 acetonitrile/water to give 150mg of the ethyl ester 80 as a white crystalline solid in 30% yield.
Compound 14 (341 mg, 0.687mmol) was stirred at room temperature in 30ml of dry ethanol containing 1 M HCI for 48hrs. The volatile solvents were removed in vacuum and the residue was purified by rp-HPLC to purity of greater than 99%. The product was obtained as yellowish solid in 62% yield (225mg).
The ester derivatives of compounds 13 and 14 are potential cell-permeable compounds that could inhibit release of IL-1 β from human immune cells (Figure 5). With the exception of 2Nap-V-A-D(OEt)-N3, the compounds significantly inhibited (decreased) the LPS-induced IL-1 β production in U937 cells (a pro- monocytic cell line). After 24hrs, there was an increase in IL-1 β production as measured by ELISA, and this was also seen in the control sample. This was probably due to the cells spontaneously producing IL-1 β (Control) or cells dying and causing an increase in the background readings. 2Nap-V-A-D(OEt)-N3 had a larger LPS-induced response, although this may have been just due to the compound not entering the cell, and not inhibiting IL-1 β production. Compounds alone did not elicit a response, although background readings at 24, 48 and 72hrs were high. This is an indication that the caspase-1 inhibitors were blocking IL-1 β production by human U937 cells. - 73 -
Example 10 Animal Studies
Compound 14 was briefly examined in 4 rats to see if it was orally bioavailable. The compound was soluble in water. It was administered orally at 10mg/kg/day over 4 days (Days 10-13 inclusive after administration of Freund's adjuvant) to 4 rats. Signs of arthritis were evaluated on Days 10 and 14 as rear and forepaw inflammation including visual lesions, and weight changes. The data are shown in Table 13. The results indicate that this compound is orally bioavailable.
Table 13. Rat data
Figure imgf000074_0001
Finally, it will be appreciated that various modifications and variations of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the art are intended to be within the scope of the present invention.

Claims

Claims:
1. A method of producing a cysteine protease inhibitor, the method comprising incorporating an azide group into an organic molecule.
2. A method according to claim 1 , wherein the organic molecule is a cysteine protease inhibitor molecule.
3. A method according to either claim 1 or claim 2, wherein the azide group is incorporated into a part of the molecule such that the azide group interacts with the cysteine protease when the molecule is bound thereto.
4. A method according to any one of claims 1 to 4, wherein the molecule comprises a cysteine protease binding moiety and an end group moiety.
5. A method according to claim 4, wherein the azide group is incorporated into the end group moiety of the molecule.
6. A method according to either claim 4 or claim 5, wherein the cysteine protease binding moiety is an amino acid or a peptide.
7. A method according to claim 6, wherein the amino acid or peptide is covalently bonded via a carbon atom to the azide group.
8. A method according to either claim 6 or claim 7, wherein the amino acid or peptide has the following formula:
AA1- -AAn- wherein each AA is the same or different and each is an amino acid and n is an integer selected from the group consisting of 0, 2, 3, 4, and 5.
9. A method according to claim 8, wherein n is 0, 2 or 3.
10. A method according according to either claim 8 or claim 9, wherein the amino acid or peptide has the following formula:
R-CO-AA1-, R-CO-AA1-AA2-, or R-CO-AA1-AA2-AA3
wherein AA1, AA2 and AA3 are the same or different and each is a natural or unnatural amino acid; and R CO- is a nitrogen protecting group.
11. A method according to any one of claims 4 to 10, wherein the end group moiety has the formula:
Figure imgf000076_0001
wherein R1 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted carboxyalkyl.
12. A method according to claim 11 , wherein R1 is selected from the group consisting of optionally substituted alkyl and optionally substituted carboxyalkyl.
13. A method according to either claim 1 1 or claim 12, wherein the end group moiety has the formula:
Figure imgf000076_0002
14. A method according to either claim 1 1 or claim 12, wherein the end group moiety has the formula: - 76 -
Figure imgf000077_0001
15. A method according to either claim 1 1 or claim 12, wherein the end group moiety has the formula:
Figure imgf000077_0002
16. A method according to any one of claims 1 to 15, wherein the cysteine protease is ICE-like or papain-like.
17. A method according to claim 16, wherein the cysteine protease is selected from the group consisting of caspase-1 , caspase-3, caspase-8, cathepsin K, cathepsin S, and cathepsin B.
18. A method of modulating one or more of the activity, specificity or a biological property of a cysteine protease inhibitor containing a functional group that is capable of reacting with a nucleophile, the method comprising replacing the functional group with an azide group.
19. A method as in claim 18, wherein the functional group is an electrophilic group.
20. A method as in claim 19, wherein the electrophilic group is selected from the group consisting of an alkylating agent, aldehyde, nitrile, ketone, α- keto-amide, halo-ketone, vinyl sulfone, chloromethane, epoxide, diazomethane, trifluoromethyl ketone, α-keto amide, fluoromethyl ketone, and diazoketone.
21. A method as in any one of claims 18 to 20, wherein the modulation of a biological property results in increased efficacy when administered, decreased toxicity, decreased side-effects, increased bioavailability, or increased half-life.
22. A cysteine protease inhibitor of formula:
Figure imgf000078_0001
wherein R2 is an amino acid or a peptide; R3 is selected from the group consisting of optionally substituted alkyl, optionally substituted carboxyalkyl, optionally substituted aryl, and optionally substituted carboxyaryl; R4 is selected from the group consisting of H, optionally substituted alkyl, and optionally substituted aryl; and R5 is selected from the group consisting of a bond, optionally substituted alkyl, and optionally substituted aryl.
23. A cysteine protease inhibitor according to claim 22, wherein R2 is an amino acid or peptide having the following formula:
AA1- -AAn-NR6- wherein each AA is the same or different and each is an amino acid, n is an integer selected from the group consisting of 0, 2, 3, 4, and 5, and R6 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, and a nitrogen protecting group.
24. A cysteine protease inhibitor according to claim 23, wherein n is 0, 2 or 3.
25. A cysteine protease inhibitor according to any one of claims 22 to 24, wherein R2 is a peptide group having the following formula:
R7-CO-AA1-, R7-CO-AA1-AA2-, or R7-CO-AA1-AA2-AA3
wherein AA1, AA2 and AA3 are the same or different and each is a natural or unnatural amino acid; and R7CO- is a nitrogen protecting group.
26. A cysteine protease inhibitor according to claim 25, wherein R7 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkyloxy, optionally substituted aryl, optionally substituted aryloxy, optionally substituted amino.
27. A cysteine protease inhibitor according to any one of claims 22 to 26, wherein R3 is -CR8R9-CO2R10, wherein R8, R9 and R10 are each independently selected from the group consisting of H, optionally substituted alkyl, and optionally substituted aryl.
28. A cysteine protease inhibitor according to claim 27, wherein R8 and R9 are H.
29. A cysteine protease inhibitor according to either claim 27 or claim 28, wherein R10 is selected from the group consisting of H, d-do straight chain alkyl, d-do branched chain alkyl, and aryl.
30. A cysteine protease inhibitor according to claim 29, wherein R10 is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, t-butyl, benzyl, napthylmethyl, and biphenylmethyl.
31. A cysteine protease inhibitor according to claim 30, wherein R10 is H.
32. A cysteine protease inhibitor according to any one of claims 22 to 26, wherein R3 is -(CR8R9)n-CH3, wherein each R8 and R9 in each CR8R9 is independently selected from the group consisting of H, optionally substituted alkyl, and optionally substituted aryl, and n is an integer selected from the group consisting of 0, 1 , 2, 3, 4, 5, 6, 7, 8, and 9.
33. A cysteine protease inhibitor according to claim 32, wherein R3 is selected from the group consisting of -(CH2)3-CH3, and -(CH2MCHs)2-
34. A cysteine protease inhibitor according to any one of claims 22 to 33, wherein R4 is selected from the group consisting of H, d-do straight chain alkyl, and d-do branched chain alkyl.
35. A cysteine protease inhibitor according to claim 34, wherein R4 is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, and t-butyl.
36. A cysteine protease inhibitor according to claim 35, wherein R4 is H.
37. A cysteine protease inhibitor according to any one of claims 22 to 36, wherein R5 is -(CR11R12V, wherein each R11 and R12 in each CR11R12 is the same or different and is independently selected from the group consisting of H, and optionally substituted alky, and n is an integer from 1 to 10 inclusive.
38. A cysteine protease inhibitor according to claim 37, wherein n is an integer from 1 to 5 inclusive.
39. A cysteine protease inhibitor according to either claim 37 or claim 38, wherein R5 is -(CH2)n-.
40. A cysteine protease inhibitor according to claim 39, wherein n is 1.
41. A cysteine protease inhibitor according to any one of claims 22 to 40, wherein the compound has the formula:
Figure imgf000081_0001
wherein R13 is PG-AA1-, PG-AA1 -AA2-, or PG-AA1-AA2- AA3- where AA1, AA2 and AA3 are the same or different and each is a natural or unnatural amino acid and PG is a nitrogen protecting group.
42. A cysteine protease inhibitor according to any one of claims 22 to 40, wherein the compound has the formula:
Figure imgf000081_0002
wherein R13 is PG-AA1-, PG-AA1 -AA2-, or PG-AA1-AA2- AA3- where AA1, AA2 and AA3 are the same or different and each is a natural or unnatural amino acid and PG is a nitrogen protecting group, and R22 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkenyl, and optionally substituted alkynyl.
43. A cysteine protease inhibitor according to claim 42, wherein R22 is selected from the group consisting of -(CH2)3-CH3, and -(CH2)2-(CH3)2.
44. A cysteine protease inhibitor according to any one of claims 22 to 43, wherein the peptide is selected from the group consisting of PG-VaI-AIa-, PG- GIu-AIa-, PG-Glu-Leu-, PG-Glu-Pro-, PG-Glu-Thr-, PG-Glu-His-, PG-Asp-Leu-, PG-GIu-VaI-, PG-AIa-AIa-, PG-GIy-AIa-, PG-AIa-GIy-, PG-Tbg-Aib-, PG-Phe- Leu-, PG-Cha-Leu-, PG-Tbg-Pro-, PG-Asp-Glu-Leu-, PG-Asp-Glu-Phe-, PG- Asp-Glu-Val-, PG-lle-Glu-Thr-, PG-lle-Glu-Pro-, PG-Glu-Glu-Leu-, PG-Glu-Leu- Leu-, PG-Leu-Glu-Leu-, PG-Phe-Glu-Leu-, PG-Tϊc-Glu-Leu-, PG-Tyr-Asp-Ala-, - 81 -
PG-Tyr-Glu-Ala-, PG-Tyr-Val-His-, PG-Tyr-Val-Pro-, PG-Tyr-Val-Phe-, PG-Tyr- Val-Leu-, PG-Tyr-GIn-Ala-, PG-Tyr-Phe-Ala-, PG-Tyr-Leu-Ala-, PG-Tyr-Val- VaI-, PG-Tyr-Val-Ala-, PG-Tyr-Val-Cha-, PG-Trp-Glu-Aib-, PG-Trp-Glu-Cha-, PG-Trp-Glu-His-, PG-Trp-Glu-Ala-, PG-Trp-Glu-Tbg-, PG-Val-Glu-Thr-, wherein PG is a nitrogen protecting group.
45. A cysteine protease inhibitor according to any one of claims 41 to 44, wherein the nitrogen protecting group is selected from the group consisting of Ac,
Figure imgf000082_0001
46. A cysteine protease inhibitor according to any one of claims 22 to 45, wherein the cysteine protease is ICE-like or papain-like.
47. A cysteine protease inhibitor according to claim 46, wherein the cysteine protease is selected from the group consisting of caspase-1 , caspase-3, caspase-8, cathepsin K, cathepsin S, and cathepsin B.
48. A pharmaceutical composition comprising a cysteine protease inhibitor of any one of claims 22 to 47, and a pharmaceutically acceptable carrier, diluent or excipient.
49. A use of a cysteine protease inhibitor of any one of claims 22 to 47 in the preparation of a medicament for the treatment of a disease state in which the disease pathology may be therapeutically modified by inhibiting a cysteine protease.
50. A use according to claim 49, wherein the disease state is selected from the group consisting of infectious diseases, immune diseases, bone diseases, neurologic diseases, tumors, and inflammatory diseases.
51. A use according to either claim 49 or claim 50, wherein the disease state is selected from the group consisting of meningitis, salpingitis, enteritis, inflammatory enteritis, hyperacidic enteritis, sepsis, septic shock, disseminated intravascular coagulation, adult respiratory distress, arthritis, bile duct disease, colitis, encephalitis, endocarditis, glomerular nephritis, hepatitis, myocarditis, pancreatitis, pericarditis, reperfusion disorder, angitis, acute and delayed allergies, graft rejection, psoriasis, asthma, type I diabetes mellitus, multiple sclerosis, allergic dermatitis, acute and chronic myelocytic leukemia, tissue calcium deficiency, rheumatism, rheumatoid arthritis, arthrosteitis, senile and climacteric osteoporosis, immobile and traumatic osteoporosis, arteriosclerosis, periodontitis, spatial pulmonary fibrosis, hepatic cirrhosis, systemic sclerosis, keloid, Alzheimer's disease and IL-1-producing tumors.
52. A method of inhibiting a cysteine protease in a subject including administering to a subject an effective amount of a cysteine protease inhibitor of any one of claims 22 to 47.
53. A method of treating a disease state in which the disease pathology may be therapeutically modified by inhibiting a cysteine protease including administering to a subject in need thereof an effective amount of a cysteine protease inhibitor of any one of claims 22 to 47.
54. A method according to claim 53, wherein the disease state is selected from the group consisting of infectious diseases, immune diseases, bone diseases, neurologic diseases, tumors, and inflammatory diseases.
55. A method according to either claim 53 or claim 54, wherein the disease state is selected from the group consisting of meningitis, salpingitis, enteritis, inflammatory enteritis, hyperacidic enteritis, sepsis, septic shock, disseminated intravascular coagulation, adult respiratory distress, arthritis, bile duct disease, colitis, encephalitis, endocarditis, glomerular nephritis, hepatitis, myocarditis, pancreatitis, pericarditis, reperfusion disorder, angitis, acute and delayed allergies, graft rejection, psoriasis, asthma, type I diabetes mellitus, multiple sclerosis, allergic dermatitis, acute and chronic myelocytic leukemia, tissue calcium deficiency, rheumatism, rheumatoid arthritis, arthrosteitis, senile and climacteric osteoporosis, immobile and traumatic osteoporosis, arteriosclerosis, periodontitis, spatial pulmonary fibrosis, hepatic cirrhosis, systemic sclerosis, keloid, Alzheimer's disease and IL-1-producing tumors.
56. A cysteine protease inhibitor produced according to the method of any one of claims 1 to 13.
57. A method according to claim 1 and substantially as hereinbefore described with respect to any one or more of the accompanying examples.
58. A cysteine protease inhibitor according to claim 22 and substantially as hereinbefore described with respect to any one or more of the accompanying examples.
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