Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
  • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
  • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• • 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
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
  • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
  • C07D307/26—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
  • C07D307/30—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member 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
  • C07D307/32—Oxygen atoms
• • 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
  • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
  • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
  • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three 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
  • C07D307/68—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
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the invention provides compounds that are useful for the therapeutic treatment of Trypanosoma cruzi infection, to the use of these compounds, and to pharmaceutical compositions comprising them.
• this invention relates to compounds which are inhibitors across a broad range of cysteine proteases, to the use of these 10 compounds, and to pharmaceutical compositions comprising them. Such compounds are useful for the therapeutic treatment of diseases in which participation of a cysteine protease is implicated.
• This hydrolytic action is performed by initially recognising, then binding to, particular three-dimensional electronic surfaces displayed by a protein, which aligns the bond for cleavage precisely within the protease catalytic site.
• Catalytic hydrolysis then commences through nucleophilic attack of the amide bond to be cleaved either via an amino acid side-chain of the protease itself, or through the action of a water molecule that is bound to and activated by the protease.
• Proteases in which the attacking nucleophile is the thiol side-chain of a Cys residue are known as cysteine proteases.
• the general classification of 'cysteine protease' contains many members found across a wide range of organisms from viruses, bacteria, protozoa, plants and fungi to mammals.
• cruzipain a cathepsin L-like cysteine protease
• Chagas' disease (a) Cazzulo, J. J. et al, Curr. Pharm. Des. 7, 1143-1156, 2001; (b) Caffrey, C. R. et al, Curr. Drug Targets, 1, 155-162, 2000).
• cruzipain messenger RNA levels in the epimastigote developmental stage indicate a role in the nutritional degradation of host-molecules in lysosomal-like vesicles (Engel, J. C. et al, J. Cell. Sci, IH, 597-606, 1998).
• the validation of cruzipain as a viable therapeutic target has been achieved with increasing levels of complexity.
• Addition of a general cysteine protease inhibitor, Z- Phe-Ala-FMK to Trypanosoma cruzi-infected mammalian cell cultures blocked replication and differentiation of the parasite, thus arresting the parasite life cycle (Harth, G., et al, Mol.
• cysteine proteases have been classified into five clans, CA, CB, CC, CD and CE (Barrett, A. J. et al, 1998).
• a protease from the tropical papaya fruit 'papain' forms the foundation of clan CA, which currently contains over 80 distinct and complete entries in various sequence databases, with many more expected from the current genome sequencing efforts.
• Proteases of clan C A/family Cl have been implicated in a multitude of disease processes e.g.
• proteases such as cathepsin K (osteoporosis), cathepsin S (autoimmune disorders), cathepsin L (metastases) or parasitic proteases such as falcipain (malaria parasite Plasmodium falciparum), cruzipain (Trypanosoma cruzi infection).
• a bacterial protease, staphylopain S. aureus infection
• X-ray crystallographic structures are available for a range of the above mentioned proteases in complex with a range of inhibitors e.g.
• papain (PDB entries, lpad, lpe6, l ip, lpop, 4pad, 5pad, 6pad, lppp, lthe, lcsb, lhuc), cathepsin K (lauO, lau2, lau3, lau4, latk, lmem, lbgo, layw, layu), cathepsin L (lcs8), cathepsin S (currently on-hold, but published McGrath, M. E. et al, Protein Science, 7, 1294-1302, 1998), cruzain (a recombinant form of cruzipain see Eakin, A. E.
• cysteine protease inhibitors for human use has recently been an area of intense activity.
• CA Cl family members particular emphasis has been placed upon the development of inhibitors of human cathepsins, primarily cathepsin K (osteoporosis), cathepsin S (autoimmune disorders) and cathepsin L (metastases), through the use of peptide and peptidomimetic nitriles (e.g.
• ketoheterocycles e.g. see WO-A-0055144, WO-A-0055124
• monobactams e.g. see WO-A-0059881, WO-A-9948911, WO-A-0109169.
• WO-A-9850533 and WO-A- 0029408 describe compounds that may be referred to as cyclic ketones and are inhibitors of cysteine proteases with a particular reference towards papain family proteases and as a most preferred embodiment, cathepsin K.
• WO-A-9850533 describes compounds subsequently detailed in the literature as potent inhibitors of cathepsin K with good oral bioavailability (Witherington, J., 'Tetrahydrofurans as Selective Cathepsin K Inhibitors', RSC meeting, Burlington House, London, 1999).
• WO-A-9850533 The compounds of WO-A-9850533 were reported to bind to cathepsin K through the formation of a reversible covalent bond between the tetrahydrofuran carbonyl and the active site catalytic cysteine residue (Witherington, J., 1999). Additionally, the same cyclic ketone compounds are described in WO-A-9953039 as part of a wide-ranging description of inhibitors of cysteine proteases associated with parasitic diseases, with particular reference to the treatment of malaria by inhibition of falcipain.
• WO-A-9850533 describes compounds that may also be referred to as cyclic ketones with particular reference towards inhibition of cathepsin S.
• the compounds of WO-A-0069855 are considered to be an advance on compounds of WO-A- 9850533 due to the presence of the ⁇ -substiruent on the cyclic ketone ring system that provides increased chiral stability to the ⁇ -carbon of the cyclic ketone ring system.
• subsequent literature has provided a closely related cyclic ketone series to that described in WO- A-9850533, where an approximately 300-fold loss in inhibitor potency was observed upon introduction of an alkyl group in place of the labile ⁇ -proton (Marquis, R. W. et al, J. Med. Chem., 44, 1380-1395, 2001).
• the present invention provides substituted (2-alkyl-3-alkyl-4-oxo-tetrahydrofuran-3-yl)arnide, (2- alkyl-3-alkyl-4-oxo-tetrahydro-thiophen-3-yl)amide and (1 -alkyl-2-alkyl-5- oxocyclopentyl)amide compounds defined by general formula (I).
• the first aspect of the invention provides a compound according to formula (I):
• R 2 C 0 - 7 -alkyl, C 3 . 6 -cycloalkyl or Ar-C 0 . 7 -alkyl;
• R 3 -ralkyl, C 3 . 6 -cycloalkyl or Ar-C 0 - 7 -alkyl;
• Y CR 4 R 5 -CO where R 4 , R 5 are independently chosen from C 0 - -alkyl, C - 6 -cycloalkyl and Ar-C 0 - -alkyl;
• (X) 0 CR 6 R 7 , where R 6 and R 7 are independently chosen from C 0 - - alkyl, C - 6 -cycloalkyl and Ar-C 0 - 7 -alkyl and o is a number from zero to three;
• (N) m C(O), C(S), S(O), S(O) 2 , S(O) 2 NH, OC(O), NHC(O), NHS(O), NHS(O) 2 , OC(O)NH, C(O)NH or CR 9 R 10 , where R 9 and R 10 are independently chosen from C 0 - -alkyl, C 3 . 6 -cycloalkyl, Ar-Co- -alkyl and m is a number from zero to three, provided that when m is greater than one, (V) m contains a maximum of one carbonyl or sulphonyl group;
• Z O (in which case compounds of general formula (I) may be named as (2-alkyl-3-alkyl-4-oxo-tetrahydrofuran-3-yl)amides, S (in which case compounds of general formula (I) may be named as (2-alkyl-3-alkyl-4-oxo-tetrahydrothiophen-3-yl)amides or CH 2 (in which case compounds of general formula (I) may be named as and (l-alkyl-2-alkyl-5-oxocyclopentyl)amides;
• U a stable 5- to 7-membered monocyclic or a stable 8- to 11 -membered bicyclic ring which is either saturated or unsaturated and which includes zero to four heteroatoms (as detailed below):
• R 11 is chosen from:
• R 11 may be halogen;
• A is chosen from:
• R 11 is as defined above, or N;
• E is chosen from:
• J, L, M, R, T, T 2 , T 3 and T are independently chosen from: CR 11 and N, where R 11 is as defined above;
• T 5 is chosen from: CH or N;
• q is a number from one to three, thereby defining a 5-, 6- or 7-membered ring.
• B, D, G, J, L, M, R, T, T 2 , T 3 and T 4 may additionally represent an N-oxide (N ⁇ O).
• the present invention includes all salts, hydrates, solvates, complexes and prodrugs of the compounds of this invention.
• the term "compound” is intended to include all such salts, hydrates, solvates, complexes and prodrugs, unless the context requires otherwise.
• Appropriate pharmaceutically and veterinarily acceptable salts of the compounds of general fo ⁇ nula (I) include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, proprionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate, camphorsul
• Prodrugs are any covalently bonded compounds which release the active parent drug according to general formula (I) in vivo.
• a prodrug may for example constitute an acetal or hemiacetal derivative of the exocyclic ketone functionality present in the (2- alkyl-3-alkyl-4-oxo-tetrahydrofuran-3-yl)amide, (2-alkyl-3-alkyl-4-oxo-tetrahydro- thiophen-3-yl)amide or (l-alkyl-2-alkyl-5-oxocyclopentyl)amide scaffold.
• a chiral centre or another form of isomeric centre is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereoisomers, are intended to be covered herein.
• Compounds of the invention containing a chiral centre may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone.
• 'Halogen' as applied herein is meant to include F, Cl, Br, I;
• Heteroatom' as applied herein is meant to include O, S and N;
• 'Co- 7 -alkyl' as applied herein is meant to include stable straight and branched chain aliphatic carbon chains containing zero (i.e. simply hydrogen) to seven carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, heptyl and any simple isomers thereof. Additionally, any C 0 - 7 -alkyl may optionally be substituted at any point by one, two or three halogen atoms (as defined above) for example to give a trifluoromethyl substituent.
• C 0 _ - alkyl may contain one or more heteroatoms (as defined above) for example to give ethers, thioethers, sulphones, sulphonamides, substituted amines, amidines, guanidines, carboxylic acids, carboxamides. If the heteroatom is located at a chain terminus then it is appropriately substituted with one or two hydrogen atoms. A heteroatom or halogen is only present when C 0 - 7 -alkyl contains a minimum of one carbon atom.
• C 1 , 7 -alkyl as applied herein is meant to include the definitions for Co- 7 -alkyl (as defined above) but describes a substituent that comprises a minimum of one carbon.
• 'C 3 - 6 -cycloalkyl' as applied herein is meant to include any variation of 'C 0 -7-alkyl' which additionally contains a carbocyclic ring such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
• the carbocyclic ring may optionally be substituted with one or more halogens (as defined above) or heteroatoms (as defined above) for example to give a tetrahydrofuran, pyrrolidine, piperidine, piperazine or morpholine substituent.
• 'Ar-Co- 7 -alkyl' as applied herein is meant to include any variation of Co-7-alkyl which additionally contains an aromatic ring moiety 'Ar'.
• the aromatic ring moiety Ar can be a stable 5 or 6-membered monocyclic or a stable 9 or 10 membered bicyclic ring which is unsaturated, as defined previously for U in general formula (I).
• the aromatic ring moiety Ar may be substituted by R ⁇ (as defined above for U in general formula (I)).
• R ⁇ as defined above for U in general formula (I)
• C M alkyl is the same as Co- 7 -alkyl except that it contains from one to four carbon atoms.
• preferred R 1 moieties include hydrogen, or a straight or branched alkyl chain, or a straight or branched heteroalkyl chain, or an optionally substituted arylalkyl chain, or an optionally substituted arylheteroalkyl chain.
• R 1 is hydrogen or C M alkyl or Ar-d ⁇ -alkyl and examples of such R 1 substituents include, but are not limited to:
• R 2 is C 0 . 7 -alkyl or Ar-C 0 . -alkyl, for example, hydrogen, straight or branched alkyl chains or heteroalkyl chains or optionally substituted arylalkyl chains.
• Particularly preferred compounds include those in which R 2 is C 0 . 4 -alkyl or Ar-Co ⁇ -alkyl and examples of such R 2 substituents include, but are not limited to:
• R 11 and R 12 are as defined above.
• (2-alkyl-3-alkyl-4-oxo-tetrahydrofuran-3-yl)amide scaffolds including such preferred R 1 and R 2 groups include:
• R 3 is a simple alkyl or arylalkyl group such as methyl.
• Y is CHR 5 CO where R 5 is selected from C 0 . 7 -alkyl or Ar-C 0 . 7 -alkyl, for example hydrogen, a straight or branched alkyl chain, a straight or branched heteroalkyl chain, an optionally substituted arylalkyl chain or an optionally substituted arylheteroalkyl chain. Additionally, in preferred compounds of general formula (I), R 5 is selected from C 3 - 6 -cycloalkyl, for example cyclohexylmethyl.
• R , R and Ar are as defined above. More preferred R 5 groups include C M -alkyl, which may be substituted with OH, NR 12 R 12 , COOR 12 , or CONR 12 or cycloalkylmethyl or Ar-C M -alkyl, where the aryl group may be substituted with R 11 ; wherein each R 11 and R 12 is independently as defined above.
• R 5 groups comprise Ar-CH 2 - ; where the aromatic ring is an optionally substituted phenyl or monocyclic heterocycle. Additionally, even more preferred R 5 groups comprise simple branched alkyl groups such as isobutyl or straight heteroalkyl chains such as benzylsulfanylmethyl or benzylsulphonylmethyl. Furthermore, even more preferred R 5 groups comprise cyclohexylmethyl. Examples of even more preferred Y substituents comprise the following,
• each of R 6 and R 7 is selected from Co- 7 -alkyl or Ar-C 0 - 7 -alkyl, for example hydrogen, a straight or branched alkyl chain, a straight or branched heteroalkyl chain, an optionally substituted arylalkyl chain or an optionally substituted arylheteroalkyl chain.
• More preferred (X) 0 groups comprise R 6 chosen from hydrogen; R 7 is C ⁇ - -alkyl, which may be substituted with OH, NR 12 R 12 , COOR 12 , or CONR 12 ; or Ar-C M -alkyl, where the aryl group may be substituted with R 11 , wherein each R 11 and R 12 is independently as defined above.
• Examples of preferred (X) 0 groups include the following:
• R 11 and R 12 are as defined previously.
• W is preferably O, S, SO 2 , S(O), C(O) or NR 8 , where R 8 is C 0 . 4 - alkyl; and n is 0 or 1.
• V is preferably C(O), C(O)NH or CHR 10 , where R 10 is C 0 - 4 -alkyl; and m is 0 or 1.
• V and W substituent combinations encompassed by general formula (I) include, but are not limited to:
• U comprises an optionally substituted 5- or 6-membered saturated or unsaturated heterocycle or Ar group or an optionally substituted saturated or unsaturated 9- or 10-membered heterocycle or Ar group.
• Examples of such preferred U rings include the following:
• R 11 is as defined previously.
• More preferred compounds of general formula (I), contain a U group comprising of a bulky alkyl or aryl group at the para position of an aryl Ar. Also, more preferred compounds contain a meta or para-biaryl Ar-Ar, where Ar is as previously defined. Additionally, more preferred compounds contain a 6,6 or 6,5 or 5,6-fused aromatic ring. Examples of more preferred U groups are wherein R 11 , D, E, G, J, L, M, R, T, T 2 , T and T are as defined previously.
• R 11 , D, E, G, J, L, M, R and T are as defined previously
• R , D, E, G, J and L are as defined previously.
• Furan-2-carboxylic acid [1 -(2,3-dimethyl-4-oxo-tetrahydrofuran-3-ylcarbamoyl)-2- (4-hydroxyphenyl)-ethyl] -amide
• Furan-3 -carboxylic acid [ 1 -(2,3-dimethyl-4-oxo-tetrahydrofuran-3-ylcarbamoyl)-2- (4-hydroxyphenyl)-ethyl]-amide
• Morpholine-4-carboxylic acid [1 -(2,3-dimethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)-2-(4-hydroxyphenyl)-ethyl]-amide
• Furan-2-carboxylic acid [l-(2,3-dimethyl-4-oxo-tetrahydrofuran-3-ylcarbamoyl)-3- methyl-butyl]-amide
• Furan-3 -carboxylic acid [ 1 -(2,3 -dimethyl-4-oxo-tetrahydrofuran-3 -ylcarbamoyl)-3 - methyl-butyl]-amide
• Morpholine-4-carboxylic acid [1 -(2,3-dimethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)-3-methyl-butyl]-amide
• Furan-2-carboxylic acid [2-cyclohexyl- 1 -(2,3-dimethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)-ethyl]-amide
• Furan-3 -carboxylic acid [2-cyclohexyl- 1 -(2,3-di ⁇ nethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)-ethyl]-amide
• Morpholine-4-carboxylic acid [2-cyclohexyl-l -(2,3 -dimethyl-4-oxo-tetrahydrofuran- 3-ylcarbamoyl)-ethyl]-amide
• Naphthalene- 1 -carboxylic acid [2-cyclohexyl- 1 -(2,3-dimethyl-4-oxo- tetrahydrofuran-3-ylcarbamoyl)-ethyl]-amide
• Naphthalene-2-carboxylic acid [[2-cyclohexyl-l -(2,3-dimethyl-4-oxo- tetrahydrofuran-3-ylcarbamoyl)-ethyl]-amide
• Furan-2-carboxylic acid [l-(2,3-dimethyl-4-oxo-tetrahydrofuran-3-ylcarbamoyl)-3,3- dimethyl-butyl] -amide
• Naphthalene-2-carboxylic acid 1 -(2,3-dimethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)-3,3-dimethyl-butyl]-amide
• Furan-2-carboxylic acid [2-cyclopentyl-l-(2,3-dimethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)-ethyl]-amide
• Furan-3 -carboxylic acid [2-cyclopentyl-l-(2,3-dimethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)-ethyl]-amide
• Morpholine-4-carboxylic acid [2-cyclopentyl- 1 -(2,3-dimethyl-4-oxo- tetrahydrofuran-3-ylcarbamoyl)-ethyl]-amide
• Furan-2-carboxylic acid [2-benzylsulfanyl-l-(2,3-dimethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)-ethyl] -amide
• Furan-3-carboxylic acid [2-benzylsulfanyl-l -(2,3-dimethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)-ethyl]-amide
• Morpholine-4-carboxylic acid [2-benzylsulfanyl- 1 -(2,3-dimethyl-4-oxo- tetrahydrofuran-3-ylcarbamoyl)-ethyl]-amide
• Furan-2-carboxylic acid [1 -(2,3-dimethyl-4-oxo-tetrahydrofuran-3-ylcarbamoyl)-2- phenylmethanesulfonyl-ethyl]-amide
• Furan-3 -carboxylic acid [l-(2,3-dimethyl-4-oxo-tetrahydrofuran-3-ylcarbamoyl)-2- phenylmethanesulfonyl-ethyl] -amide
• Thiophene-2-carboxylic acid [ 1 -(2,3 -dimethyl-4-oxo-tetrahydro furan-3 - ylcarbamoyl)-2-phenylmethanesulfonyl-ethyl]-amide
• Morpholine-4-carboxylic acid [ 1 -(2,3-dimethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)-2-phenylmethanesulfonyl-ethyl]-amide
• Furan-3 -carboxylic acid [1 -(2,3-dimethyl-4-oxo-tetrahydrofuran-3-ylcarbamoyl)-2- (4-hydroxyphenyl)-ethyl]-amide
• Furan-3 -carboxylic acid [l-(2,3-dimethyl-4-oxo-tetrahydrofuran-3-ylcarbamoyl)-3- methyl-butyl] -amide
• Furan-3 -carboxylic acid [2-cyclohexyl- 1 -(2,3-dimethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)-ethyl]-amide
• Furan-3-carboxylic acid [2-cyclopentyl-l-(2,3-dimethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)-ethyl]-amide
• Thiophene-3-carboxylic acid [2-cyclopentyl-l-(2,3-dimethyl-4-oxo-tetrahydrofuran- 3 -ylcarbamoyl)-ethyl] -amide
• Furan-3 -carboxylic acid [2-benzylsulfanyl-l -(2,3 -dimethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)-ethyl]-amide
• Furan-3 -carboxylic acid [l-(2,3-dimethyl-4-oxo-tetrahydrofuran-3-ylcarbamoyl)-2- phenylmethanesulfonyl-ethyl]-amide
• the first stage in a synthesis of compounds of general formula (I) is the preparation in solution of a functionalised and protected building block.
• a typical scheme towards the (2-alkyl-3-alkyl-4-oxo-tetrahydrofuran-3-yl)amide (7) is detailed in Scheme 1.
• FmOC(O) denotes the well known amine protecting group 9-fluorenyl methoxycarbonyl (Fmoc, see Atherton, E. and Sheppard, R. C, 1989) and 'Pg' denotes either a free hydroxyl or an hydroxyl protecting group such as tert-butyl ether.
• synthesis may commence from suitably protected ⁇ -hydroxy- ⁇ - alkyl- ⁇ -aminoacid (4).
• ⁇ -hydroxy- ⁇ -aminoacids are accessible through a variety of literature methods e.g. (a) Adams, Z. M., Jackson, R. F. W., Palmer, N. J., Rami, H. K., Wythes, M. J., J. Chem. Soc, Perkin Trans I, 937-947, 1999, (b) Hubhisten, C, et al, J. Med. Chem, 41, 3972-3975, 1998, (c) Luzzio, F.
• the ⁇ -hydroxy- ⁇ - alkyl- ⁇ -aminoacid (4) is the known (2R, 3SR)- ⁇ -methylthreonine (the 3S isomer corresponds to D-Thr and the 3R isomer corresponds to D- ⁇ // ⁇ Thr).
• Step (b) the solid phase linkage of an aldehyde or ketone, has previously been described by a variety of methods (e.g. see (a) James, I. W., 1999, (b) Lee, A., Huang, L., Ellman, J. A., J. Am. Chem. Soc, 121(43), 9907-9914, 1999, (c) Murphy, A. M., et al, J. Am. Chem.
• Construct (9) is prepared through reaction of the linker molecule (8) and the (2-alkyl- 3-alkyl-4-oxo-tetrahydrofuran-3-yl)amide (7) by reflux in aqueous ethanol / sodium acetate. Although formation of construct (9) is observed at 2hr reaction, optimal formation of construct (9) occurs at 72hr reaction. Standard solid phase techniques (e.g. see Atherton, E. and Sheppard, R. C, 1989) are used to anchor the construct to an amino-functionalised solid phase through the free carboxylic acid functionality of (9), providing the loaded construct (10). Acid mediated cleavage of the fully constructed compounds is optimal at a repeated 24hr reaction.
• Loaded construct (10) may be reacted with a wide range of carboxylic acids available commercially or in the literature, to introduce the left-hand portion 'U-V-W-X-Y' in general formula (I).
• carboxylic acids can be prepared in solution by traditional organic chemistry methods and coupled to construct (10) on the solid phase (Schemes 4-8).
• Scheme 4 treatment in solution of an amino acid, exemplified by (12) with sodium nitrite / H 2 SO , provides the ⁇ -hydroxyacid, exemplified by (13) (Degerbeck, F. et al, J. Chem. Soc, Perkin Trans. , 11-14, 1993).
• Treatment of ⁇ - hydroxyacid, (13) with sodium hydride in a dimethylformamide / dichloromethane mixture followed by addition of benzyl bromide provides 2RS-benzyloxy-3- cyclohexylpropionic acid (14).
• benzylbromide may be replaced by any reasonable Ar-CR 9 R 10 -halide, providing many variations of carboxylic acid (14) following the general conditions detailed.
• the ester intermediate is then simply hydrolysed to acid (14).
• Analogues of (15), exploring a wide range of (V) m and U in general formula (I) may be prepared through the general conditions detailed in Scheme 4. Since the final synthetic step involves a trifluoroacetic acid (TFA) mediated cleavage of the solid phase bound compound, analogues where the substituted ether is labile to TFA may be prepared in solution by an alternative route (see Scheme 11).
• TFA trifluoroacetic acid
• alkylsuccinate esters such as (23) may be prepared and converted to the corresponding substituted alkylsuccinate acid such as (24) following the general conditions detailed.
• morpholine may be replaced by any reasonable amine, providing many variations of carboxylic acid (24) following the general conditions detailed.
• analogues of (25) exploring a wide range of (X) 0 , (N) m and U in general formula (I) may be prepared through the general conditions detailed in Scheme 7.
• biarylalkylacetic acids such as (27) may be prepared by alkylation of the ⁇ -anion of the free acid analogue of (26), which in turn is prepared by Suzuki coupling of phenylboronic acid and 3-bromophenylacetic acid methyl ester.
• Phenylboronic acid may be replaced by a wide range of arylboronic acids in the Suzuki coupling, providing many variations of carboxylic acid (27) following the general conditions detailed.
• analogues of (28) exploring a wide range of group 'U' in general formula (I) may be prepared through the general conditions detailed in Scheme 8.
• a second strategy for the synthesis of compounds of general formula (I) comprises:-
• an inhibitor of general formula (I) can be prepared in solution by traditional organic chemistry methods and coupled to building block (f) (see Scheme 10 exemplified by preparation and use of (2-alkyl-3-alkyl-4-oxo-tetrahydrofuran-3-yl)carbamic acid tert-butyl ester (32)).
• 4-tert-butylbenzyl bromide may be replaced by any reasonable Ar- CR 9 R 10 -halide, providing many variations of carboxylic acid (35) under the conditions shown.
• analogues of (36) exploring a wide range of (V) m and U in general formula (I) may be prepared through the conditions detailed in Scheme 11. '
• the ketone may require protection e.g. as the dimethyl acetal.
• Such a method is detailed and exemplified in Scheme 13 by the preparation and use of (2-alkyl-3-alkyl-4,4-dimethoxy- tetrahydrofuran-3-yl)carbamic acid benzyl ester (41).
• the invention extends to novel intermediates as described above, and to processes for preparing compounds of general formula (I) from each of its immediate precursors. In turn, processes for preparing intermediates from their immediate precursors also form part of the invention.
• Compounds of general formula (I) are useful both as laboratory tools and as therapeutic agents. In the laboratory certain compounds of the invention are useful in establishing whether a known or newly discovered cysteine protease contributes a critical or at least significant biochemical function during the establishment or progression of a disease state, a process commonly referred to as 'target validation'.
• a method of validating a known or putative cysteine protease inhibitor as a therapeutic target comprising: (a) assessing the in vitro binding of a compound as described above to an isolated known or putative cysteine protease, providing a measure of potency; and optionally, one or more of the steps of:
• the invention therefore provides a method of validating a known or putative cysteine protease inhibitor as a therapeutic target. Differing approaches and levels of complexity are appropriate to the effective inhibition and 'validation' of a particular target.
• the method comprises assessing the in vitro binding of a compound of general formula (I) to an isolated known or putative cysteine protease, providing a measure of 'potency'.
• An additional assessment of the binding of a compound of general formula (I) to closely related homologous proteases of the target and general house-keeping proteases (e.g. trypsin) provides a measure of 'selectivity'.
• a second level of complexity may be assessed by monitoring a cell- based functional marker of a particular cysteine protease activity, in the presence of a compound of general formula (I).
• a 'human osteoclast resorption assay' has been utilised as a cell-based secondary in vitro testing system for monitoring the activity of cathepsin K and the biochemical effect of protease inhibitors (e.g. see WO-A-9850533).
• An 'MHC-II processing - T-cell activation assay' has been utilised as a cell-based secondary in vitro testing system for monitoring the activity of cathepsin S and the biochemical effect of protease inhibitors (Shi, G-P., et al, Immunity, 10, 197-206, 1999).
• a marker could simply be a functional assessment of viral (e.g. count of mRNA copies) or bacterial loading and assessing the biochemical effect of protease inhibitors.
• a third level of complexity may be assessed by monitoring an animal model-based functional marker of a particular cysteine protease activity, in the presence of a compound of general formula (I). For example, murine models of Leishmania infection, E.
• the invention therefore extends to the use of a compound of general formula (I) in the validation of a known or putative cysteine protease inhibitor as a therapeutic target.
• Compounds of general formula (I) are useful for the in vivo treatment or prevention of diseases in which participation of a cysteine protease is implicated.
• a compound of general formula (I) for use in medicine especially for preventing or treating diseases in which the disease pathology may be modified by inhibiting a cysteine protease.
• a compound of general formula (I) in the preparation of a medicament for preventing or treating diseases in which the disease pathology may be modified by inhibiting a cysteine protease.
• cysteine proteases function in the normal physiological process of protein degradation in animals, including humans, e.g. in the degradation of connective tissue. However, elevated levels of these enzymes in the body can result in pathological conditions leading to disease. Thus, cysteine proteases have been implicated in various disease states, including but not limited to, infections by Pneumocystis carinii, Trypsanoma cruzi, Tiypsanoma brucei brucei and Crithidia fusiculata; as well as in osteoporosis, autoimmunity, schistosomiasis, malaria, tumour metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy, and the like.
• staphylopain a secreted bacterial cysteine protease from S. Aureus called staphylopain has been implicated as a bacterial virulence factor (Potempa, J., et al. J. Biol. Chem, 262(6), 2664-2667, 1998).
• the mvention is useful in the prevention and/or treatment of each of the disease states mentioned or implied above.
• the present invention also is useful in a methods of treatment or prevention of diseases caused by pathological levels of cysteine proteases, particularly cysteine proteases of the papain superfamily, which methods comprise administering to an animal, particularly a mammal, most particularly a human, in need thereof a compound of the present invention.
• the present invention particularly provides methods for treating diseases in which cysteine proteases are implicated, including infections by Pneumocystis carinii, Trypsanoma cruzi, Trypsanoma brucei, Leishmania mexicana, Clostridium histolyticum, Staphylococcus aureus, foot-and-mouth disease virus and Crithidia fusiculata; as well as in osteoporosis, autoimmunity, schistosomiasis, malaria, tumour metastasis, metachromatic leukodystrophy, muscular dystrophy and amytrophy.
• Inhibitors of cruzipain are useful for the treatment of Chagas' disease.
• an effective amount of a compound of general formula (I) may be administered to inhibit the protease implicated with a particular condition or disease.
• this dosage amount will further be modified according to the type of administration of the compound.
• parenteral administration of a compound of general formula (I) is preferred.
• An intravenous infusion of the compound in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful.
• the parenteral dose will be about 0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg, in a manner to maintain the concentration of drug in the plasma at a concentration effective to inhibit a cysteine protease.
• the compounds may be administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 400 mg/kg/day.
• the precise amount of an inventive compound which is therapeutically effective, and the route by which such compound is best administered, is readily determined by one of ordinary skill in the art by comparing the blood level of the agent to the concentration required to have a therapeutic effect.
• Prodrugs of compounds of the present invention may be prepared by any suitable method. For those compounds in which the prodrug moiety is a ketone functionality, specifically ketals and/or hemiacetals, the conversion may be effected in accordance with conventional methods.
• the compounds of this invention may also be administered orally to the patient, in a manner such that the concentration of drug is sufficient to inhibit bone resorption or to achieve any other therapeutic indication as disclosed herein.
• a pharmaceutical composition containing the compound is administered at an oral dose of between about 0.1 to about 50 mg/kg in a manner consistent with the condition of the patient.
• the oral dose would be about 0.5 to about 20 mg/kg.
• the compounds of this invention which may have good bioavailability, may be tested in one of several biological assays to determine the concentration of a compound which is required to have a given pharmacological effect.
• a pharmaceutical or veterinary composition comprising one or more compounds of general formula (I) and a pharmaceutically or veterinarily acceptable carrier.
• Other active materials may also be present, as may be considered appropriate or advisable for the disease or condition being treated or prevented.
• the carrier, or, if more than one be present, each of the carriers, must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.
• the formulations include those suitable for rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, but preferably the formulation is an orally administered formulation.
• the formulations may conveniently be presented in unit dosage form, e.g. tablets and sustained release capsules, and may be prepared by any methods well known in the art of pharmacy.
• Such methods include the step of bringing into association the above defined active agent with the carrier.
• the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
• the invention extends to methods for preparing a pharmaceutical composition comprising bringing a compound of general formula (I) in conjunction or association with a pharmaceutically or veterinarily acceptable carrier or vehicle.
• Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water in oil liquid emulsion; or as a bolus etc.
• the term "acceptable carrier” includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica.
• vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinyl
• Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring and the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.
• a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent.
• Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
• the tablets may be optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.
• compositions suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.
• Parenteral formulations will generally be sterile.
• a process for the preparation of a pharmaceutical or veterinary composition as described above comprising bringing the active compound(s) into association with the carrier, for example by admixture.
• HPLC-MS analysis was performed on an Agilent 1100 series LC/MSD, using automated Agilent HPLC systems, with a gradient of 10 - 90% B in A over 10 minutes on Phenomenex Columbus C 8 , 5 ⁇ , 300A, 50 x 2.0mm at 0.4mL / min.
• Nuclear magnetic resonance (NMR) were obtained on a Bruker DPX400 (400MHz IH frequency; QXI probe) in the solvents and temperature indicated. Chemical shifts are expressed in parts per million ( ⁇ ) and are referenced to residual signals of the solvent. Coupling constants (J) are expressed in Hz.
• Example inhibitors (1-37) were prepared through a combination of solution and solid phase Fmoc-based chemistries (see 'Solid Phase Peptide Synthesis', Atherton, E. and Sheppard, R. C, IRL Press Ltd, Oxford, UK, 1989, for a general description).
• An appropriately protected and functionalised building block was prepared in solution (e.g. compound (7), Scheme 1), then reversibly attached to the solid phase through an appropriate linker. Rounds of coupling / deprotection / chemical modification e.g. oxidation were then performed until the full length desired molecule was complete (Scheme 2).
• Example inhibitors (1-37) were then released (cleaved) from the solid phase, analysed, purified and assayed for inhibition verses a range of proteases.
• multipins polyamide 1.2 - lO ⁇ mole loadings, see www.mimotopes.com
• the 1.2 ⁇ mole gears were used to provide small scale crude examples for preliminary screening, whilst the lO ⁇ mole crowns were used for scale-up synthesis and purification of preferred examples.
• Standard coupling and Fmoc deprotection methods were employed (see Grabowska, U. et al, J. Comb. Chem. 2(5). 475-490, 2000. for a thorough description of solid phase multipin methodologies).
• Building Block-linker constructs were carboxyl activated with 2-(lH- benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluoro phosphate (HBTU, 1 mole equivalent), 1-hydroxybenzotriazole.hydrate (HOBT, 1 mole equivalent) and N-methylmorpholine (NMM, 2 mole equivalents) in dimethylformamide (DMF, typically 1 to lOmL) for 5 minutes.
• HBTU 2-(lH- benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluoro phosphate
• HOBT 1-hydroxybenzotriazole.hydrate
• NMM N-methylmorpholine
• DMF dimethylformamide
• Activated species were dispensed to the appropriate wells of a polypropylene 96-well plate (Beckman, lmL wells, 500 ⁇ L solution per well for crowns or 250 ⁇ L solution per well for gears) in a pattern required for synthesis. Loaded free amino Building Block-linker constructs (e.g.(l ⁇ )) were added and the coupling reaction left overnight. Following overnight coupling, crowns / gears were taken through standard cycles washing and Fmoc deprotection (see Grabowska, U. et al). Identical activation and coupling conditions were used for the coupling of a range of carboxylic acids (R-COOH). Alternatively, chloroformates e.g. morpholine-4-carbonylchloride (lOmole equivalent), were coupled in DMF with the addition of NMM (lOmole equivalents).
• a mixture of 95% TFA / 5% water was pre-dispensed into two polystyrene 96-well plates (Beckman, lmL wells, 600 ⁇ L solution per well for crowns or 300 ⁇ L solution per well for gears) in a pattern corresponding to that of the synthesis.
• the completed multipin assembly was added to the first plate (mother plate), the block covered in tin foil and cleaved for 2 x 24hours.
• the cleaved multipin assembly was then removed from the first plate and added to the second plate (washing plate) for 15 minutes.
• the spent multipin assembly was then discarded and the mother / washing plates evaporated on an HT-4 Gene Vac plate evaporator.
• Etheral diazomethane [generated from addition of diazald (4.7 g, -15 mmol) in diethyl ether (75 mL) onto sodium hydroxide (5.25 g) in water (7.5 mL) / ethanol (15 mL) at 60 °C] was added to the activated amino acid solution at -20 °C. The mixture was then allowed to warm to room temperature and stirred for 20 h. A few drops of acetic acid were added to the mixture. tert-Butylmethylether (100 mL) was then added to the mixture. The ethereal layers were then washed with water (3 x 75 mL), dried ( ⁇ a 2 SO 4 ) and the solvents removed under reduced pressure to give a yellow residue (2 g).
• ⁇ c (100 MHz, CDC1 3 ) 14.5 (2-CH 3 ), 16.3 (3-CH 3 ), 47.5 (u, CHCH 2 O, Fmoc), 61.4 (q, C ⁇ ), 67.3 (d, CHCH 2 O, Fmoc), 70.7 (d, COCH 2 O), 77.5 (C ⁇ ), 120.4, 125.4, 127.5, 128.2 (8 x fluorenyl CHs'), 141.7, 1483.8 and 144.2 (4 x fluorenyl quaternary Cs'), 155.1 (OCON) and 214.1 (C-4).
• building block-linker construct (9) was attached to the solid phase providing loaded building block-linker construct (10) as follows: Building block-linker construct (9) (0.146mmoles), 2-(lH-benzotriazole-l-yl)- 1,1,3,3-tetramethyluroniumhexafluoro phosphate (HBTU, 55mg, 0.146mmole), 1- hydroxybenzotriazole.hydrate and (HOBT, 22mg, 0.146mmole) were dissolved in dimethylformamide (3mL) and N-methylmorpholine (NMM, 64 ⁇ L, 0.29mmole) added.
• HBTU 2-(lH-benzotriazole-l-yl)- 1,1,3,3-tetramethyluroniumhexafluoro phosphate
• HOBT 1- hydroxybenzotriazole.hydrate and (HOBT, 22mg, 0.146mmole) were dissolved in dimethylformamide (3mL) and N-methylmorpholine (NMM, 64 ⁇ L,
• HPLC Rt 12.86mins (> 95%), HPLC-MS 480.1 [M + H] + , 959.3 [2M + Na] + .
• HPLC Rt 9.97mins (> 90%), HPLC-MS 481.1 [M + H] + , 961.3 [2M + Na] + .
• HPLC Rt 19.80mins (> 85%), HPLC-MS 403.3 [M + H] + , 827.5 [2M + ⁇ a .
• HPLC Rt 19.23mins (> 80%), HPLC-MS 423.3 [M + H] + , 867.4 [2M + ⁇ a .
• HPLC Rt 18.77mins (> 90%), HPLC-MS 429.2 [M + H] + , 879.3 [2M + Na] + .
• HPLC Rt 15.79mins (> 80%), HPLC-MS 417.0 [M + H] + , 855.1 [2M + Naf .
• Trifluoroacetic acid (77 mL) was added to a solution of (2R) 2-tert- butoxycarbonylamino-3-phenylpropionic acid l-methyl-2-oxopropyl ester (7.99 g, 23.8 mol) in anhydrous dichloromethane (140 mL) at 0 °C under a nitrogen atmosphere. The mixture was stirred for 1 h at 0 °C then evaporated under reduced pressure to afford a residue. Toluene (40 mL) was then added to the residue and evaporated under reduced pressure. This procedure was repeated twice, to remove excess trifluoroacetic acid. The residue was then dissolved in anhydrous 2-propanol (390 mL) under a nitrogen atmosphere.
• Methylmorpholine (56 ⁇ L) was added then the unsealed pressure vessel allowed to warm to ambient temperature. The mixture was diluted with saturated aqueous sodium hydrogen carbonate solution (100 mL) then the product extracted into dichloromethane (3 x 100 mL). The combined dichloromethane layers were washed with brine (50 mL), dried ( ⁇ a 2 SO 4 ) and evaporated under reduced pressure to afford a residue.
• Ethereal diazomethane [generated from diazald ( ⁇ 15 mmol) addition in diethyl ether (75 mL) to sodium hydroxide (5.25 g) in water (7.5 mL)/ethanol (15 mL) at 60°C and dried over potassium hydroxide pellets] was added to a stirred solution of (IR, 2S) (2-tert-butoxy-l-fluorocarbonyl-l-methylpropyl)- carbamic acid 9H-fluoren-9-ylmethyl ester (138 mg, ⁇ 0.316 mmol) in dichloromethane (3 mL) at 0 °C. The mixture was then allowed to warm to ambient temperature and stirred for 20 hours.
• Building block-linker construct (9) (10.6mg, 0.0193mmole), 2-(lH-benzotriazole-l- yl)-l,l,3,3-tetramethyluroniumhexafluoro phosphate (HBTU, 7.3mg, 0.0193mmole), 1-hydroxybenzotriazole.hydrate and (HOBT, 2.96mg, 0.0193mmole) were dissolved in dimethylformamide (0.5mL) and N-methylmorpholine (NMM, 4.2 ⁇ L, 0.0386mmole) added.
• HBTU 2-(lH-benzotriazole-l- yl)-l,l,3,3-tetramethyluroniumhexafluoro phosphate
• 1-hydroxybenzotriazole.hydrate and (HOBT, 2.96mg, 0.0193mmole) were dissolved in dimethylformamide (0.5mL) and N-methylmorpholine (NMM, 4.2 ⁇ L, 0.0386mmole) added.
• HPLC Rt 19.4mins (>90%), HPLC-MS 403.3 [M + H] + , 827.5 [2M + ⁇ af.
• the compounds of this invention may be tested in one of a number of literature based biochemical assays that are designed to elucidate the characteristics of compound inhibition.
• the data from these types of assays enables compound potency and the rates of reaction to be measured and quantified. This information, either alone or in combination with other information, would allow the amount of compound required to produce a given pharmacological effect to be determined.
• Fluorescence high throughput assays were carried out in either 384-well microtitre plates (Corning Costar 3705 plates, Fisher Scientific) or 96-well 'U' bottomed Microfluor WI microtitre plates (Thermo Labsystems, Ashford, Middlesex, U.K.). Fluorescence assays were monitored using a SpectraMax Gemini fluorescence plate reader (Molecular Devices).
• HEPES Hydroxyethylpiperazine ethanesulfonate
• tris-hydroxylmethyl aminomethane (tris) base bis-tris-propane and all the biological detergents (e.g. CHAPS, zwittergents, etc.) were purchased from CN Biosciences UK, Beeston, Nottinghamshire, U.K.
• Glycerol was purchased from Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, U.K.
• Stock solutions of substrate or inhibitor were made up to 10 mM in 100 % dimethylsulfoxide (DMSO) (Rathburns, Glasgow, U.K.) and diluted as appropriately required. In all cases the DMSO concentration in the assays was maintained at less than 1% (vol./vol.).
• DMSO dimethylsulfoxide
• CaCl 2 10 mM BTP, pH 6.5 containing 1 mM EDTA, 142 mM NaCl, 1 mM DTT, 1 mM CaCl 2 , 0.035 mM Zwittergent 3-16 III: 50mM HEPES pH 7.2, 10% Glycerol, 0.1% CHAPS, 142 mM NaCl, 1 mM
• EDTA, 5 mM DTT IV 100 mM sodium phosphate, pH 6.75 containing 1 mM EDTA and 10 mM L- cysteine V: 50 mM trisacetate, pH 8.4 containing 1 mM EDTA, 10 mM L-cysteine and
• 35 and 1 mM EDTA XI 100 mM sodium acetate; pH 5.5 containing 10 mM L-cysteine; 142 mM sodium chloride and 1 mM EDTA
• Wild-type cruzipain derived from Trypanosoma cruzi Dm28 epimastigotes, was obtained from Dr. Julio Scharfstein (Instituto de Biof ⁇ sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil).
• Activity assays were carried out in 100 mM sodium phosphate, pH 6.75 containing 1 mM ⁇ DTA and 10 mM L-cysteine using 2.5 nM enzyme.
• Ac-Phe-Arg-AMC R M ap ⁇ 12 ⁇ M
• D- Nal-Leu-Lys-AMC Ku pp ⁇ 4 ⁇ M
• Ac-FR- AMC was used at a concentration equivalent to ⁇ - M app and D-Nal-Leu-Lys-AMC was used at a concentration of 25 ⁇ M.
• the rate of conversion of substrate to product was derived from the slope of the increase in fluorescence monitored continuously over time.
• CPB Leishmania mexicana cysteine protease B
• Wild-type recombinant CPB without the C-terminal extention (i.e. CPB2.8 ⁇ CT ⁇ ; Sanderson, S.J., et. al, Biochem. J., 347, 383-388, 2000) was obtained from Dr. Jeremy Mottram (Wellcome Centre for Molecular Parasitology, The Anderson College, University of Glasgow, Glasgow, U.K.). Activity assays were carried out in 100 mM sodium acetate; pH 5.5 containing 1 mM EDTA; 200 mM NaCl and 10 mM DTT (Alves, L.C., et. al, Mol. Biochem. Parasitol, 116, 1-9, 2001) using 0.25 nM enzyme. Pro-Phe-Arg-AMC K w ⁇ 38 ⁇ M) was used as the substrate at a concentration equivalent to Rjy ⁇ app - The rate of conversion of substrate to product was derived from the slope of the increase in fluorescence monitored continuously over time.
• Bovine cathepsin S, human cathepsin L, human cathepsin H and human cathepsin B were obtained from CN Biosciences. Recombinant human cathepsin S, human cathepsin K and human cathepsin X were obtained from Dr. Boris Turk (Josef Stefan Institute, Ljubljana, Slovenia). Unless otherwise stated, all peptidase activity assays were carried out in 10 mM bis-tris-propane (BTP), pH 6.5 containing 1 mM EDTA, 5 mM 2-mercaptoethanol and 1 mM CaCl 2 .
• BTP bis-tris-propane
• Human cathepsin H activity assays were carried out in 10 mM BTP pH 6.5, 142 mM NaCl 2 , 1 mM CaCl 2 , 1 mM EDTA, 1 mM DTT, 0.035 mM Zwittergent 3-16.
• Human cathepsin K assays were carried out in 100 mM sodium acetate; pH 5.5 containing 20 mM L-cysteine and 1 mM EDTA (Bossard, M.J., et. al, J. Biol Chem., 2 ⁇ , 12517-12524, 1996).
• Human cathepsin X assays were carried out in 100 mM sodium acetate; pH 5.5 containing 20 mM L- cysteine; 0.05% (w/v) Brij 35 and 1 mM EDTA (Santamaria, I., et. al, J. Biol. Chem., 273, 16816-16823, 1998; Klemencic, J, et al, Eur. J. Biochem., 267, 5404- 5412, 2000).
• the final enzyme concentrations used in the assays were 0.5 nM bovine cathepsin S, 1 nM cathepsin L, 0.1 nM cathepsin B, 0.25nM Cathepsin K; 1 nM cathepsin X and 10 nM cathepsin H.
• the substrates used for cathepsin S, cathepsin L, cathepsin B, cathepsin K and cathepsin H were boc-Val-Leu-Lys-AMC (K M app ⁇ 30 ⁇ M), Ac-Phe-Arg-AMC ( ⁇ M a p ⁇ 20 ⁇ M), Z- Phe-Arg-AMC (K u app ⁇ 40 ⁇ M), Z-Leu-Arg-AMC (UC M app ⁇ 2 ⁇ M); Bz-Phe-Nal-Arg- AMC (i ⁇ v ⁇ app - 150 ⁇ M) respectively.
• the substrate concentration used in each assay was equivalent to the Ku pp -
• the rate of conversion of substrate to product was derived from the slope of the increase in fluorescence monitored continuously over time. Trypsin peptidase activity assays
• Clostripain (Sigma) activity assays were carried out in 10 mM BTP, pH 6.5 containing 1 mM EDTA, 5 mM 2-mercaptoethanol and ImM CaCl 2 using 0.3 nM enzyme.
• Z-Gly-Gly-Arg-AMC K M app ⁇ 100 ⁇ M was used as the substrate at a concentration equivalent to -.
• M app - The rate of conversion of substrate to product was derived from the slope of the increase in fluorescence monitored continuously over time.
• Calpain human erythrocyte ⁇ -calpain and porcine kidney m-calpain; CN Biosciences
• activity assays were carried out in 10 mM HEPES, pH 7.5 containing 2 mM 2-mercaptoethanol and CaCl 2 using 25 nM of either enzyme (Sasaki, et. al, J. Biol. Chem., 259, 12489-12494, 1984).
• the buffer contained 100 ⁇ M CaCl 2 and Abz-Ala-Asn-Leu-Gly-Arg-Pro-Ala-Leu-Tyr(NO 2 )- Asp-NH 2 ( T M app ⁇ 20 ⁇ M; Incenta Limited) was used as the substrate.
• the assay buffer contained 200 ⁇ M CaCl 2 and Abz-Lys-Leu- Cys(Bzl)-Phe-Ser-Lys-Gln-Tyr(NO 2 )-Asp-NH 2 (K M app ⁇ 22 ⁇ M; Incenta Limited) was used as the substrate.
• S. aureus V8 was obtained from Prof. S. Arvidson, Karolinska Institute, Sweden. Extracellular S. aureus V8 cysteine peptidase (staphylopain) activity assays were carried out using partially purified 5 aureus V8 culture supernatant (obtained from Dr. Peter Lambert, Aston University, Birmingham, U.K.). Activity assays were carried out in 10 mM BTP, pH 6.5 containing 1 mM EDTA, 5 mM 2- mercaptoethanol and ImM CaCl 2 using two-times diluted partially purified extract.
• FMDV-LP Foot-and-mouth disease leader peptidase
• Recombinant wild-type FMDV-LP was obtained from Dr. Tim Skern (Institut fur Medizinische Biochemie, Abiller fur Biochemie, Universtat Wien, Wien, Austria). Activity assays were carried out in 50 mM trisacetate, pH 8.4 containing 1 mM EDTA, 10 mM L-cysteine and 0.25% (w/v) CHAPS using 10 nM enzyme. For the inhibition assays, Abz-Arg-Lys-Leu-Lys-Gly-Ala-Gly-Ser-Tyr(N0 2 )-Glu-NH 2 (JW P ⁇ 51 ⁇ M, Incenta Limited) was used as the substrate at a concentration equivalent to ⁇ i app . The rate of conversion of substrate to product was derived from the slope of the increase in fluorescence monitored continuously over time.
• Caspases 1-10 were obtained from CN Biosciences or Bio Vision Inc. (Mountain View, CA, USA) and all assays were carried out in 50mM HEPES; pH 7.2, 10% (v/v) glycerol, 0.1% (w/v) CHAPS, 142 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol (DTT) using 0.1-1 U per assay.
• K M &PP The apparent macroscopic binding constant for each substrate was calculated, from the dependence of enzyme activity as a function of substrate concentration. The observed rates were plotted on the ordinate against the related substrate concentration on the abscissa and the data fitted by direct regression analysis (Prism v 3.02; GraphPad, San Diego, USA) using Equation 1 (Cornish-Bowden, A. Fundamentals of enzyme kinetics Portland Press; 1995, 93-128.).
• Equation 1 ' is the observed initial rate
• 'P m a ⁇ app ' is the observed maximum activity at saturating substrate concentration
• '2f M app ' is the apparent macroscopic binding (Michaelis) constant for the substrate
• '[S 0 ]' is the initial substrate concentration.
• K_ apparent inhibition constant
• Equation 2 'vi' is the observed residual activity, ' max app ' is the observed maximum activity (i.e. in the absence of inhibitor), ' ⁇ M app ' is the apparent macroscopic binding (Michaelis) constant for the substrate, '[S]' is the initial substrate concentration, 'Kf is the apparent dissociation constant and '[I]' is the inhibitor concentration.
• K_ app values were calculated using a quadratic solution in the form described by Equation 3 (Morrison, J.F. Trends Biochem. Sci.,_7_, 102-105, 1982; Morrison, J.F. Biochim. Biophys. Act ⁇ 1 185, 269-286, 1969; Stone, S.R. and Hofsteenge, J. Biochemistry, 25, 4622-4628, 1986).
• Equation 3 ' J ' is the observed residual activity, 'F' is the difference between the maximum activity (i.e. in the absence of inhibitor) and minimum enzyme activity,
• Equation 4 was used to account for the substrate kinetics, where 'K is the inhibition constant, '[S 0 ]' is the initial substrate concentration and ' ⁇ M app s the apparent macroscopic binding (Michaelis) constant for the substrate (Morrison, 1982).
• the concentration dependence of the observed rate of reaction ( o s ) of each compound with enzyme was analysed by determining the rate of enzyme inactivation under pseudo-first order conditions in the presence of substrate (Morrison, J.F., TIBS, 102-105, 1982; Tian, W.X. and Tsou, C.L., Biochemistry, 21, 1028-1032, 1982; Morrison, J.F. and Walsh, C.T., from Meister (Ed.), Advances in Enzymol, 6L, 201-301, 1988; Tsou, C.L., from Meister (Ed.), Advances in Enzymol, 61, 381-436, 1988;). Assays were carried out by addition of various concentrations of inhibitor to assay buffer containing substrate. Assays were initiated by the addition of enzyme to the reaction mixture and the change in fluorescence monitored over time. During the course of the assay less than 10% of the substrate was consumed.
• the activity fluorescence progress curves were fitted by non-linear regression analysis (Prism) using ⁇ q. 5 (Morrison, 1969; Morrison, 1982); where 'F' is the fluorescence response, 't' is time, 'v 0 ' is the initial velocity, 'v s ' is the equilibrium steady-state velocity, 'k ob _' is the observed pseudo first-order rate constant and 'D' is the intercept at time zero (i.e. the ordinate displacement of the curve).
• the second order rate constant was obtained from the slope of the line of a plot of k 0 _ s versus the inhibitor concentration (i.e. k 0bS /[ ⁇ ). To correct for substrate kinetics, ⁇ q.

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