WO2009112826A1 - Furo [3, 2-b] pyrr0l-3-0nes as cathepsin s inhibitors - Google Patents

Furo [3, 2-b] pyrr0l-3-0nes as cathepsin s inhibitors Download PDF

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Publication number
WO2009112826A1
WO2009112826A1 PCT/GB2009/000653 GB2009000653W WO2009112826A1 WO 2009112826 A1 WO2009112826 A1 WO 2009112826A1 GB 2009000653 W GB2009000653 W GB 2009000653W WO 2009112826 A1 WO2009112826 A1 WO 2009112826A1
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WIPO (PCT)
Prior art keywords
oxoethyl
alkyl
pyrrol
furo
compound
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PCT/GB2009/000653
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French (fr)
Inventor
Martin Quibell
John Paul Watts
Nicholas Sean Flinn
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Amura Therapeutics Limited
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Publication date
Application filed by Amura Therapeutics Limited filed Critical Amura Therapeutics Limited
Priority to EP09720536A priority Critical patent/EP2283022B1/en
Priority to JP2010550257A priority patent/JP5670754B2/en
Priority to ES09720536T priority patent/ES2391609T3/en
Publication of WO2009112826A1 publication Critical patent/WO2009112826A1/en
Priority to US12/853,005 priority patent/US8389737B2/en
Priority to US13/745,413 priority patent/US8552202B2/en
Priority to US14/046,895 priority patent/US8933109B2/en
Priority to US14/535,959 priority patent/US20150164912A1/en
Priority to US14/871,381 priority patent/US20160015685A1/en
Priority to US15/245,968 priority patent/US20160361295A1/en

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    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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    • C07D417/12—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 two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
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    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
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Definitions

  • the present invention relates to compounds that are inhibitors of cysteine proteinases, pharmaceutical compositions containing said compounds, and their use in therapy. More specifically, the invention relates to compounds that are inhibitors of cathepsin S, a cysteine proteinase of the CA clan. Such compounds are particularly useful for the in vivo therapeutic treatment of diseases in which participation of cathepsin S is implicated.
  • Proteinases form a substantial group of biological molecules which to date constitute approximately 2% of all the gene products identified following analysis of several completed genome sequencing programmes. Proteinases have evolved to participate in an enormous range of biological processes, mediating their effect by cleavage of peptide amide bonds within the myriad of proteins found in nature. This hydrolytic action is performed by initially recognising, then binding to, particular three- dimensional electronic surfaces displayed by a protein, which align the bond for cleavage precisely within the proteinase 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 proteinase itself, or through the action of a water molecule that is bound to and activated by the proteinase.
  • cysteine proteinases Proteinases in which the attacking nucleophile is the thiol side-chain of a Cy s residue are known as cysteine proteinases.
  • the general classification of 'cysteine proteinase' contains many members found in a wide range of organisms from viruses, bacteria, protozoa, plants and fungi to mammals.
  • human proteinases such as cathepsin K (osteoporosis, osteoarthritis), cathepsin S (multiple sclerosis, rheumatoid arthritis, autoimmune disorders), cathepsin L (metastases), cathepsin B (metastases, arthritis), cathepsin F (antigen processing), cathepsin V (T-cell selection), dipeptidyl peptidase I (granulocyte serine proteinase activation) or parasitic proteinases such as falcipain (malaria parasite Plasmodium falciparum) and cruzipain ⁇ Trypanosoma cruzi infection).
  • Cathepsin S is a lysosomal cysteine proteinase that is specifically up-regulated under inflammatory conditions. It is highly expressed in the spleen, in professional antigen presenting cells (APCs) and other MHC class II- positive cells and is inducible by IFN- ⁇ .
  • APCs professional antigen presenting cells
  • Intracellular cathepsin S specifically processes invariant chain, a protein involved in the correct loading of MHC-II with antigen (a key step in generating an immune response) (see Shi, G. P. et al., Immunity, 10(2). 197- 206, 1999; Lui, W. and Spero, D. M. Drug News Perspect. 17(6), 357-363, 2004).
  • the MHC-II / antigen complex is then displayed on the surface of the APC, for interaction with and activation of T-cells.
  • Disrupting antigen presentation represents a validated approach to treating diseases with an autoimmune component such as rheumatoid arthritis (e.g.
  • cathepsin S is secreted from macrophages infiltrating sites of inflammation, to aid proteolysis of proteins and facilitate phagocytosis.
  • cathepsin S is responsible for degradation of structural tissue proteins and also mediates pain.
  • Cathepsin S has been implicated in the destruction of articular cartilage in rheumatoid and osteoarthritis (e.g. see Hou, W-S.
  • cathepsin S has been shown to be critical for the maintenance of neuropathic pain and spinal microglia activation in peripheral nerve-injured rats (see Clark, A. K. et al, Proc. Natl. Acad. Sci. USA, 104(25), 10655-10660, 2007; Barclay, J., et al, Pain, 130(3), 225-234, 2007). Therefore inhibition of cathepsin S has therapeutic potential in the treatment of neuropathic pain (e.g. see WO-A-03020287).
  • cysteine proteinase inhibitors for human use has recently been an area of intense activity (e.g. see Deaton, D. N. and Kumar, S., Prog. Med. Chem. 42, 245-375, 2004; Bromme, D. and Kaleta, J., Curr. Pharm. Des., 8, 1639-1658, 2002; Kim, W. and Kang, K., Expert Opin. Ther. Patents, 12(3), 419-432, 2002; Leung-Toung, R. et al. Curr. Med. Chem., 9, 979-1002, 2002; Lecaille, F. et al., Chem. Rev., 102, 4459-4488, 2002; Hernandez, A. A.
  • a first aspect of the invention relates to a compound of formula (T), or a pharmaceutically acceptable salt, hydrate, complex or pro-drug thereof,
  • R 3 and R 4 are H, and the other is selected from Ci ⁇ -alkyl, C 1-6 -haloalkyl, C 1-6 - alkoxy and C 6-12 -aralkyl; or R 3 and R 4 are each independently selected from Ci -6 -alkyl and halo; R 9 is selected from the following:
  • Xi 5 X 2 , X 3 , X4, Xi4, Xi5,Xi6 andX 2 o are each independently selected from:
  • X 9 and X 12 are each independantly selected from:
  • X 1 O and X 11 are each independantly selected from: CH, C-(C 1-6 -alkyl), C-(C w -alkoxy), C-halo, N and R 10 ;
  • X 19 is selected from:
  • X 18 is selected from:
  • X 18 may additionally be selected from C-C(O)NH 2 and C-C(O)N(C 1-6 -alkyl) 2 ;
  • Xi3 and X 17 are each independently selected from:
  • X 23 is selected from:
  • X 25 is selected from: O 5 S, NH and N(C 1-6 -alkyl);
  • X26, X2 7 , X2S and X29 are each independently selected from:
  • X 30 is selected from:
  • X 3I is selected from:
  • X 31 may additionally be
  • X 32 is selected from:
  • X 34 is selected from: NH and NMe
  • R 1O is selected from: wherein:
  • T 1 , T 2 , T 3 and T 4 are each independantly selected from: CH, C-(Cj -6 -alkyl), C-(C 1-6 -alkoxy), C-NH 2 , C-NH(C 1-6 -alkyl), C-N(C 1 .
  • T 1 , T ⁇ , T 3 and T 4 are C-(Ci_ 6 -alkoxy), C-NH 2 , C-NH(C 1- 6-aIkyI), C-N(C w -alkyl) 2 or C-halo;
  • T 5 is selected from:
  • T 6 , T 7 , T 8 , T 9 and T 10 are each Independantly selected from:
  • Tn is selected from: CH 2 , NH and N(Ci -6 -alkyl);
  • T 12 is selected from:
  • T 13 and Ti 4 are each independantly selected from: CH, C-(C 1-6 -alkyl) and C-halo;
  • Tj 5 is selected from:
  • T 16 is selected from:
  • R 10 is selected from: H, C 1-6 -al] ⁇ yl, OH, C 1-6 -alkoxy, NO 2 , halo, CN, C(O)NH 2 , C(O)NH(C 1-6 - alkyl), C(O)N(Ci-6-alkyl)2, C(O)NH(C 3-6 -cycloalkyi ⁇ S(O) 2 NH 2 , S(O) 2 (C 1-6 -alkyl), S(O) 2 NH(C 1-6 -alkyl), S(O) 2 N(C 1-6 -EUCyI) 2 ,
  • n O or 1
  • R 11 is selected from C 1-6 -alkyl, C(O)C 1-6 -alkyl, C(O)(C 3-6 -cycloalkyl), C(O)(aryl), C(O)NH 2 , C(O)NH(C 1-6 -EIlCyI), C(O)N(C 1-6 -alkyl) 2 , C(O)NH(C 3-6 -cycloalkyl), C(O)O(C 1 .
  • R 12 is selected from H and C 1-6 -alkyl.
  • RB is selected from:
  • n O or 1 ; and R 14 is selected from H, C 1-6 -alkyl, C(O)C 1-6 -alkyl, C(O)(C 3-6 -cycloaIkyl), C(O)(aryl), C(O)NH 2 , C(O)NH(C 1-6 -alkyl), C(O)N(C 1-6 -alkyl) 2 , C(O)NH(C 3-6 - cycloalkyl), C(O)O(Ci -6 -aIkyl), C(O)O(C 3 - 6 -cycloalkyl), C(O)O(aryl), S(O) 2 (C 1-6 - alkyl), S(0) 2 (C 3-6 -cycloalkyl), S(O) 2 NH 2 , S(O) 2 NH(C 1-6 -alkyl), S(O) 2 N(C 1-6 -alkyl) 2 , S(O) 2 NH(C 3-6
  • R 15 is selected from H and C 1-6 -alkyl.
  • a second aspect of the invention relates to a pharmaceutical or veterinary composition
  • a pharmaceutical or veterinary composition comprising a compound of formula (I) and a pharmaceutically acceptable or veterinarily acceptable diluent, excipient and/or carrier.
  • a third aspect of the invention relates to a process for preparing a pharmaceutical or veterinary composition as defined above, said process comprising admixing a compound of the invention with a pharmaceutically acceptable or veterinarily acceptable diluent, excipient and/or carrier.
  • a fourth aspect of the invention relates to compounds of formula (I) for use in medicine.
  • a fifth aspect of the invention relates to the use of a compound of formula (I) in the preparation of a medicament for treating a disease selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain.
  • a sixth aspect of the invention relates to a method of inhibiting cathepsin S in a cell, said method comprising contacting said cell with a compound of formula (T).
  • a seventh aspect of the invention relates to method of inhibiting cathepsin S in a subject, said method comprising administering to the subject a pharmacologically effective amount of a compound of formula (I).
  • An eighth aspect of the invention relates to a method of treating a disease selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain, in a subject, said method comprising administering to the subject a pharmacologically effective amount of a compound of formula (I).
  • a disease selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic r
  • a ninth aspect of the invention relates to the use of a compound according to the invention in an assay for identifying further candidate compounds capable of inhibiting one or more cysteine proteinases.
  • a tenth aspect of the invention relates to the use of a compound of formula (I) in the validation of a known or putative cysteine proteinase as a therapeutic target.
  • An eleventh aspect of the invention relates to a process of preparing a compound of formula (I).
  • An eleventh aspect of the invention relates to a compound of formula (I) for treating a disease selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain.
  • a disease selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain.
  • 'alkyP as applied herein includes stable straight and branched chain aliphatic carbon chains which may be optionally substituted. Preferred examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, heptyl and any simple isomers thereof. Suitable substituents include, for example, one or more C 1-6 alkoxy, OH, COOH, COOMe, NH 2 , NMe 2 , NHMe, NO 2 , CN and/or CF 3 groups.
  • alkyl group contains two or more contiguous carbon atoms
  • the alkyl group may optionally contain one or more heteroatoms 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.
  • the group CHa-CH 2 -O-CH 2 -CH 2 - is defined within 'alkyl' as a C 4 alkyl that contains a centrally positioned heteroatom whereas the group CH 3 -CHa-CH 2 -CH 2 - is defined within 'alkyl' as an unsubstituted C 4 alkyl.
  • the alkyl group is a C 1-6 alkyl group, more preferably a C 1-4 group.
  • 'cycloalkyP refers to a cyclic alkyl group (i.e. a carbocyclic ring) which may be substituted (mono- or poly-) or unsubstituted.
  • Suitable substituents include, for example, one or more C 1-6 alkyl, C 1-6 alkoxy, OH, COOH, COOMe, NH 2 , NMe 2 , NHMe, NO 2 , CN 5 CF 3 and/or halo groups.
  • the cycloalkyl group is a C3-6-cycloalkyl, even more preferably a C 3-4 cycloalkyl group.
  • Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
  • the carbocyclic ring itself may optionally contain one or more heteroatoms, for example, to give a heterocycloalkyl group such as tetrahydrofuran, pyrrolidine, piperidine, piperazine or morpholine.
  • 'alkyoxy' refers to the group 'O-alkyl' or 'O-cycloalkyl', wherein alkyl and cycloalkyl are as defined above.
  • 'Halogen' or 'halo' as applied herein encompasses F, Cl, Br, I.
  • the term 'haloalkyl' refers to an alkyl group as defined above substituted by one or more halogen atoms.
  • the term 'aryl' refers to a stable 5 or 6-membered monocytic ring which is unsaturated.
  • the aryl group may optionally include one or more heteroatoms selected from O 5 N and S.
  • the aryl group may be optionally substituted, for example, by one or more C 1-6 alkyl, C 1-6 alkoxy, OH, COOH, COOMe, NH 2 , NMe 2 , NHMe 5 NO 2 , CN, CF3 and/or halo groups. More preferably, the aryl group may be optionally substituted by one or more Me, OMe 5 OEt, OiPr, NO 2 , Cl or F groups.
  • the term 'aralkyl' as applied herein includes an alkyl group as defined above in combination with an aryl group.
  • the aryl group may be an aromatic ring, for example, a stable 5 or 6-membered monocylic or a stable 9 or 10-membered bicyclic ring which is unsaturated.
  • the aryl group may optionally comprise one or more heteroatoms selected from O, N and S.
  • the aryl group may be optionally substituted, for example, by one or more C 1-6 alkyl, C 1-6 alkoxy, OH, COOH, COOMe, NH 2 , NMe 2 , NHMe, NO 2 , CN, CF3 and/or halo groups.
  • the aralkyl group is a Ci-g-alkyl- C 5-1 o-aryl group, even more preferably a Ci. 8 -alkyl-ph.enyl group. More preferably still, the alkyl-aryl group is selected from CH 2 Ph and CH 2 OCH 2 Ph.
  • 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.
  • ketone group of the bicycle core of compounds of formula (I) may exist in alternative forms such as the hydrate (as shown below), and the invention extends to all such alternative forms.
  • one of R 3 and R 4 is H, and the other is selected from Ci- ⁇ -haloalkyl, Cj -6 - alkoxy and
  • R 3 and R 4 are each independently selected from C 1-6 -alkyl and halo;
  • R is selected from the following:
  • X 1 , X 2 , X 3 , X 4 , Xi 4 , X 15 , X 16 and X 2 o are each independently selected from:
  • X 5 , X 6 , X 7 and Xg are each independently selected from:
  • Xp and Xn are each independently selected from:
  • Xio and Xi 1 are each independantly selected from: CH 5 C-(C 1-6 -alkyl), C-(C 1-6 -aUcoxy), C-halo, N and R 10 ;
  • Xi 9 is selected from:
  • X 18 is selected from:
  • X 13 and X 17 are each independently selected from: O, S, NH and N- ⁇ -alkyl);
  • X 22 and X 24 are each independently selected from:
  • X 23 is selected from:
  • X 25 is selected from:
  • X2 6 , X 27 , X28 and X 2 P are each independently selected from:
  • R 1O is selected from: wherein:
  • T 1 , T 2 , T 3 and T 4 are each independantly selected from:
  • T5 is selected from: O, S, NH and N(C 1-6 -alkyl);
  • T 6 , T 7 , T 8 , T 9 and T 10 are each independantly selected from:
  • T 6 -alkyl 2 , C-halo and N; such that a maximum of two of T 6 , T 7 , T 8 , T 9 and T 10 are selected from C-(C 1-6 - alkoxy), C-NH 2 , C-NH(C I-6 -alkyl), C-N(C I-6 -alkyl) 2 , C-halo and N;
  • T 11 is selected from:
  • T 12 is selected from:
  • T 13 and T 14 are each independantly selected from: CH, C-(C 1-6 -alkyl) and C-halo;
  • T 15 is selected from:
  • T 16 is selected from:
  • R 10 is selected from: H 3 Ci-6-alkyl, OH, C ⁇ -alkoxy, NO 2 , halo, CN, C(O)NH 2 , C(O)NH(C 1-6 - alkyl), C(O)N(Ci -6 -alkyl) 2 , and (CHa) n -NR 11 R 12 ; wherein n is O or 1 and R 11 is selected from H, C 1-6 -alkyl, acetyl, C(O)NH 2 , C(O)N(C 1-6 -alkyl) 2 : and R 12 is selected from H and
  • R 3 is H and R 4 is selected from methyl, ethyl, rc-propyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, ethoxy and benzyl: or both R 3 and R 4 are selected from methyl or fluoro or ehloro;
  • X 1 , X 2 , X 3 , X 4 , X 14 , X 15, X 16 and X 2 o are independently selected from: CH, CMe, C-OMe, C-F, C-Cl and N: such that a maximum of two of Xi, X2, X3, Xj, Xw, X 1 5, X 16 and X 20 are chosen as N or
  • X 5 , X 6 , X 7 and X 8 are independently selected from:
  • X 10 and X 11 are independently selected from:
  • X 19 is selected from: CH, CMe, C-OMe, C-C(O)NH 2 , C-C(O)NMe 2 , C-F, C-Cl and N;
  • Xis is selected from:
  • X 18 may additionally be selected from C-C(O)NH 2 and C-C(O)NMe 2 ;
  • X 13 and X 17 are independently selected from:
  • X 22 and X 24 are independently selected from:
  • X 2 5 is selected from: O, S, NH and NMe;
  • X26, X27, X28 and X29 are independently selected from:
  • X 33 is selected from:
  • X 34 is selected from:
  • T 1 , T 2 , T 3 and T 4 are independantly selected from:
  • T 5 is selected from:
  • T 6 , T 7 , T 8 , T 9 and T10 are independantly selected from:
  • T 11 is selected from: CH 2 , NH and NMe;
  • Ti2 is selected from:
  • T 13 and T 14 are independently selected from: CH, CMe, C-F and C-Cl;
  • T 15 is selected from:
  • T 16 is selected from:
  • R 10 is selected from: H, Me, OH, OMe, OEt, OiPr, NO 2 , F, Cl, Br, CN, C(O)NH 2 ,
  • n O or 1 and R 11 is selected from H, Me, acetyl, C(O)NH 2 , C(O)NMe 2 : and R 12 is selected from H and Me;
  • Ri 3 is selected from:
  • R 14 is selected from H, Me, C(O)Me, C(O)(cyclopropyl), C(O)Ph, C(O)NH 2 , C(O)NH(Me), C(O)N(Me) 2 , C(O)NH(cyclopropyl), C(O)O(Me), C(O)O(cyclopropyl), C(O)OPh, S(O) 2 (Me), S(O) 2 (cyclopropyl), S(O) 2 NH 2 , S(O) 2 NH(Me), S(O) 2 N(Me) 2 , S(O) 2 NH(cyclopropyl) and S(O) 2 Ph;
  • R 15 is selected from H and Me.
  • the compound of the invention is of formula Ia
  • R 3 , R 4 and R 9 are as defined above.
  • the compound of the invention is of formula Ib
  • R 3 , R 4 and R 9 are as defined above.
  • R 3 is selected from H and R 4 is selected from methyl, ethyl, propyl, trifluoromethyl and benzyl.
  • both R 3 and R 4 are selected as methyl, fluoro or chloro.
  • both R 3 and R 4 are selected as methyl such that the central amino acid moiety is derived from (iS)-2-amino-2-(4,4- dimethylcyclohexyl)acetic acid (CAS 754178-25-1).
  • R 3 is H and R 4 is methyl such that the central amino acid moiety is derived from the trans configured (5)-2-amino-2-((lr, 45)-4- methylcyclohexyl)acetic acid as shown in formula Ic.
  • R 9 is selected from:
  • R 9 is selected from:
  • X 1 , X 2 , X3, X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X13, Xw, Xis, Xi6, Xn 5 Xi8, X19, X2 5 , X30, X31, R 10 and Ro are as defined above.
  • R 9 is selected from:
  • X 1 , X 2 , X 3 , X 4 , X 7 , X 10 , X 17 , X 18 , X 19 , X 25 , X 30 , X 31 , R 10 and R 14 are as defined above.
  • Rio is selected from:
  • T 1 , T 2 , T 3 , T 4 , T 6 , T 7 , Tg, T 9 and T 10 are as defined above.
  • Rio is: wherein one, two or three of of T 1 , T 2 , T 3 and T 4 are N and the remainder are CH.
  • R 1O is selected from:
  • T 6 , T 7 , T 8 , T 9 and T 10 is N and the remainder are CH.
  • R 9 is selected from:
  • aryl, X 18 , X 19 , X 23 , X 25 are as defined above and;
  • X 2 and X 3 are each independently selected from: CH 5 CMe and C-F;
  • X 30 is selected from:
  • X 31 is selected from:
  • R 14 is selected from C(O)Me, C(O)(cyclopropyl), C(O)NH 2 , C(O)NH(Me), C(O)N(Me) 2 , C(O)NH(cyclopropyI), C(O)O(Me), C(O)O(cyclopropyl), S(O) 2 (Me), S(O) 2 (cyclopropyl), S(O) 2 NH 2 , S(O) 2 NH(Me), S(O) 2 N(Me) 2 , S(O) 2 NH(cyclopropyl) and S(O) 2 Ph.
  • R 9 is selected from:
  • the compound of the invention is selected from the following:
  • the compound of the invention is selected from Examples 1 — 22 described hereinbelow.
  • the compound of the invention is selected from Examples 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 described hereinbelow.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the invention admixed with one or more pharmaceutically acceptable diluents, excipients or carriers.
  • Other active materials may also be present, as may be considered appropriate or advisable for the disease or condition being treated or prevented.
  • the compounds of the present invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy.
  • a pharmaceutical carrier excipient or diluent
  • the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and. the like.
  • suitable diluents include ethanol, glycerol and water.
  • compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • Suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
  • Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • 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.
  • 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.
  • the compounds of the invention can be present as salts or esters, in particular pharmaceutically and veterinarily acceptable salts or esters.
  • salts of the compounds of the invention include suitable acid addition or base salts thereof.
  • suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g.
  • hydrohalic acids such as hydrochloride, hydrobromide and hydroiodide, sulphuric acid, phosphoric acid sulphate, bisulphate, hemisulphate, thiocyanate, persulphate and sulphonic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C 1 - C 4 )-alkyl- or aryl-sul
  • Preferred salts include, for example, 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, camphorsulphonate, 2-naphthalenesulphonate, benzenesulphonate, p-chlorobenzenesulphon
  • Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterif ⁇ ed.
  • Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (Ci-C 4 )-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p
  • Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide.
  • Alcohols include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).
  • the invention includes, where appropriate all enantiomers, diastereoisomers and tautomers of the compounds of the invention.
  • the person skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics.
  • the corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.
  • Enantiomers are characterised by the absolute configuration of their chiral centres and described by the R- and ⁇ -sequencing rules of Cahn, Ingold and Prelog. Such conventions are well known in the art (e.g. see 'Advanced Organic Chemistry', 3 rd edition, ed. March, J., John Wiley and Sons, New York, 1985). 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.
  • Some of the compounds of the invention may exist as stereoisomers and/or geometric isomers — e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms.
  • the present invention contemplates the use of all the individual stereoisomers and geometric isomers of those inhibitor agents, and mixtures thereof.
  • the terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).
  • the present invention also includes all suitable isotopic variations of the agent or a pharmaceutically acceptable salt thereof.
  • An isotopic variation of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature.
  • isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2 H, 3 H, 13 C, 14 C, 15 N, 17 0, 18 0, 31 P, 32 P 5 35 S, 18 F and 36 Cl 3 respectively.
  • isotopic variations of the agent and pharmaceutically acceptable salts thereof are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3 H 5 and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances.
  • the invention includes compounds of general formula (I) where any hydrogen atom has been replaced by a deuterium atom. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.
  • PRODRUGS The invention further includes the compounds of the present invention in prodrug form, i.e. covalently bonded compounds which release the active parent drug according to general formula (I) in vivo.
  • prodrugs are generally compounds of the invention wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject. Reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo. Examples of such modifications include ester (for example, any of those described above), wherein the reversion may be carried out be an esterase etc. Other such systems will be well known to those skilled in the art.
  • a prodrug may for example constitute a ketal or hemiketal derivative of the exocyclic ketone functionality present in the 6-(»S)-chlorotetrahydrofuro[3,2-Z»]pyrrol-3-one scaffold.
  • the invention further relates to the compounds of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation form the solvents used in the synthetic preparation of such compounds.
  • ASSAYS ASSAYS
  • Another aspect of the invention relates to the use of a compound of the invention as defined hereinabove in an assay for identifying further candidate compounds that influence the activity of a cysteine proteinase.
  • the assay is capable of identifying candidate compounds that are capable of inhibiting one or more CACl cysteine proteinases.
  • the assay is a competitive binding assay.
  • the candidate compound is generated by conventional SAR modification of a compound of the invention.
  • conventional SAR modification refers to standard methods known in the art for varying a given compound by way of chemical derivatisation.
  • the identified compound may act as a model (for example, a template) for the development of other compounds.
  • the compounds employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The abolition of activity or the formation of binding complexes between the compound and the agent being tested may be measured.
  • the assay of the present invention may be a screen, whereby a number of agents are tested, hi one aspect, the assay method of the present invention is a high through-put screen.
  • This invention also contemplates the use of competitive drug screening assays in which neutralising antibodies capable of binding a compound specifically compete with a test compound for binding to a compound.
  • Another technique for screening provides for high throughput screening (HTS) of agents having suitable binding affinity to the substances and is based upon the method described in detail in WO 84/03564.
  • the competitive binding assay comprises contacting a compound of the invention with a cysteine proteinase in the presence of a known substrate of said enzyme and detecting any change in the interaction between said cysteine proteinase and said known substrate.
  • a further aspect of the invention provides a method of detecting the binding of a ligand to a cysteine proteinase, said method comprising the steps of: (i) contacting a ligand with cysteine proteinase in the presence of a known substrate of said enzyme; (ii) detecting any change in the interaction between said enzyme and said known substrate; and wherein said ligand is a compound of the invention.
  • One aspect of the invention relates to a process comprising the steps of:
  • Another aspect of the invention provides a process comprising the steps of:
  • the invention also relates to a ligand identified by the method described hereinabove.
  • Yet another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a ligand identified by the method described hereinabove.
  • Another aspect of the invention relates to the use of a ligand identified by the method described hereinabove in the preparation of a pharmaceutical composition for use in the treatment of one or more disorders selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain.
  • disorders selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, athe
  • the above methods may be used to screen for a ligand useful as an inhibitor of one or more cysteine proteinases.
  • the invention therefore provides a method of validating a known or putative cysteine proteinase 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 proteinase, providing a measure of 'potency'.
  • An additional assessment of the binding of a compound of general formula (T) to closely related homologous proteinases of the target and general house-keeping proteinases (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 proteinase activity, in the presence of a compound of general formula (T).
  • a cell-based functional marker of a particular cysteine proteinase activity in the presence of a compound of general formula (T).
  • T a compound of general formula (T)
  • an 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 proteinase 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 proteinase 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 proteinase inhibitors.
  • a third level of complexity may be assessed by monitoring an animal model-based functional marker of a particular cysteine proteinase activity, in the presence of a compound of general formula (I). For example, murine models of Leishmania infection, P.
  • the invention therefore extends to the use of a compound of general formula (I) in the validation of a known or putative cysteine proteinase as a therapeutic target.
  • the compounds of the present invention are structurally distinct from the prior art (e.g. WO-A-02057270; Quibell, M. et. al, Bioorg. Med. Chem. 13, 609-625, 2005; Quibell M 5 et al Bioorg. Med. Chem, 12, 5689-5710, 2004; WO-A-05066180) in that a 6-(S)- chloro substituent and a 4-substituted cyclohexylglycyl moiety form an integral part.
  • This combination of features provides compounds with surprisingly high efficacies for human cathepsin S and high in vitro selectivity versus other mammalian cathepsins, both of which are important properties required for development of an efficacious therapeutic.
  • Prior art compound (38) exhibits a 2.5-fold improvement in in vitro potency against human cathepsin S upon substitution of the (5)-cyclohexylalanyl moiety with (5)-(cyclohexyl)glycyl (Compound 1) but provides an inhibitor that has little selectivity verses cathepsin K. Further addition of a 6-(5)-fluoro substituent to Compound 1 provides a 3.8-fold improvement in in vitro potency against human cathepsin S (Compound 2) but again provides an inhibitor that has little selectivity verses cathepsin K.
  • Compound 6 which contains the 6-(i?)-chloro substituent in place of the 6-(5)-chloro substituent of EXAMPLE 1 is 15- fold less potent verses cathepsin S, but again retains the high selectivity due to presence of the -7- ⁇ «,y-()S)-(4( J S)-methylcyclohexyl)glycyl moiety.
  • Compound 11 which contains the 6-( ⁇ S)-fluoro substituent in place of the 6-( «S)-chloro substituent of EXAMPLE 2 is 11 -fold less potent verses cathepsin S, but retains the high selectivity due to presence of the /r ⁇ r ⁇ -(S)-(4(S)-memylcyclohexyi)grycyl moiety.
  • Compound 12 which contains the 6-( ⁇ !)-chloro substituent in place of the 6-(S)-chloro substituent of EXAMPLE 2 is 25 -fold less potent verses cathepsin S 5 but again retains the high selectivity due to presence of the /r ⁇ /w-( ⁇ S)-(4(S)-methylcyclohexyl)glycyl moiety.
  • the compounds exhibit in vitro inhibition versus human cathepsin S with ICi ⁇ 10 nM, more preferably ⁇ 5 nM 5 even more preferably ⁇ 2 nM and more preferably still ⁇ 1 nM.
  • the compounds of the invention exhibit high selectivity against other mammalian cathepsins displaying little or no inhibitory activity for cathepsins K, L, B and V at 1 ⁇ M compound.
  • THERAPEUTIC USE Compounds of general formula (I) are useful for the in vivo treatment or prevention of diseases in which participation of a cysteine proteinase is implicated.
  • the compound of general formula I is selective for cathepsin S.
  • the term "selective for cathepsin S” means that the inhibitor is selective for cathepsin S over one or more other mammalian CACl cysteinyl proteinases for example cathepsin K, cathepsin L, cathepsin F, cathepsin B and cathepsin V.
  • the inhibitor exhibits a selectivity ratio for cathepsin S over other mammalian CACl cysteinyl proteinases of greater than 2-fold, more preferably greater than 5-fold, more preferably greater than 10-fold, even more preferably greater than 25- fold, more preferably still, greater than 50-fold or 100-fold.
  • 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 proteinase.
  • 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 proteinase.
  • cysteine proteinases 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 proteinases have been implicated in various disease states, including but not limited to, infections by Pneumocystis carinii, Trypsanoma cruzi, Trypsanoma brucei brucei and Crithidia fusiculata; as well as in osteoporosis, osteoarthritis, rheumatoid arthritis, multiple sclerosis, chronic pain, autoimmunity, schistosomiasis, malaria, tumour metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy, and the like (see WO-A-9404172 and EP-A-0603873 and references cited therein).
  • staphylopain a secreted bacterial cysteine proteinase from S. Aureus called staphylopain has been implicated as a bacterial virulence factor (Potempa, J., etal. J. Biol. Chem, 262(6), 2664-2667, 1998).
  • the invention 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 method of treatment or prevention of diseases caused by pathological levels of cysteine proteinases, particularly cysteine proteinases 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 proteinases are implicated, including infections by Pneumocystis carinii, Trypsanoma cruzi, Trypsanoma brucei, Leishmania mexicana, Clostridium histolyticum, Staphylococcus aureus, foot-and-mouth disease virus and Crithidia fiisiculata; as well as in osteoarthritis, rheumatoid arthritis, multiple sclerosis, chronic pain, autoimmunity, schistosomiasis, malaria, tumour metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy.
  • Inhibitors of cathepsin S are useful for the treatment of rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain.
  • the compounds of the invention are particularly useful in the treatment of the above disorders.
  • compositions of the present invention may be adapted for rectal, nasal, intrabronchial, topical (including buccal and sublingual), vaginal or parenteral
  • the formulation is an orally administered formulation.
  • the formulations may conveniently be presented in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
  • the formulations may be in the form of tablets and sustained release capsules, and may be prepared by any method well known in the art of pharmacy.
  • Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, gellules, drops, cachets, pills or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution, emulsion 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.
  • these compositions contain from 1 to 250 mg and more preferably from 10-100 mg, of active ingredient per dose.
  • 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, hydroxypropyhnethylcellulose, 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, polyvin
  • 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.
  • compositions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions.
  • injectable forms typically contain between 10 - 1000 mg, preferably between 10 - 250 mg, of active ingredient per dose.
  • compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
  • the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin.
  • the active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
  • a person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation.
  • a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
  • the dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • an effective amount of a compound of general formula (I) may be administered to inhibit the proteinase 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 (T) is preferred.
  • An intravenous infusion of the compound hi 5% dextrose hi 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, hi a manner to maintain the concentration of drug in the plasma at a concentration effective to inhibit a cysteine proteinase.
  • the compounds may be administered one to four tunes 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 hi 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 hemiketals, 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.
  • the one or more compounds of the invention are administered in combination with one or more other active agents, for example, existing drugs available on the market.
  • the compounds of the invention may be administered consecutively, simultaneously or sequentially with the one or more other active agents.
  • Drugs in general are more effective when used in combination.
  • combination therapy is desirable in order to avoid an overlap of major toxicities, mechanism of action and resistance mechanism(s).
  • the major advantages of combining chemotherapeutic drugs are that it may promote additive or possible synergistic effects through biochemical interactions and also may decrease the emergence of resistance.
  • Beneficial combinations may be suggested by studying the inhibitory activity of the test compounds with agents known or suspected of being valuable in the treatment of a particular disorder. This procedure can also be used to determine the order of administration of the agents, i.e. before, simultaneously, or after delivery. Such scheduling may be a feature of all the active agents identified herein.
  • One aspect of the invention relates to a process of preparing a compound of formula (I) as defined above, said process comprising oxidation of a compound of formula (II).
  • Any suitable oxidising agent may be used to convert the secondary alcohol group of (II) into the corresponding ketone (I).
  • Suitable oxidising agents will be familiar to the skilled artisan.
  • the oxidation may be carried out via a Dess-Martin periodinane reaction [Dess, D.B. et al, J. Org. Chem. 1983, 48, 4155; Dess, D.B. et al, J. Am. Chem. Soc. 1991, 113, 7277], or via a Swern oxidation [Mancuso, A. J. et al, J. Org. Chem. 1978, 43, 2480].
  • the oxidation can be carried out using SOs/pyridine/EtsN/DMSO [Parith, J. R. et al, J. Am. Chem. Soc. 1967, 5505; US 3,444,216, Parith, J. R. et al,], P 2 O 5 /DMSO or P 2 O 5 /Ac 2 O [Christensen, S. M. et al, Organic Process Research and Development, 2004, 8, 777].
  • Other alternative oxidation reagents include activated dimethyl sulphoxide [Mancuso, A. J., Swern, D. J., Synthesis, 1981, 165], pyridinium chlorochromate [Piatieatelli, G. et al, Sythesis, 1982,
  • the process comprises treating a compound of formula (II) with Dess- Martin periodinane.
  • the reaction is carried out using dichloromethane as solvent.
  • R 5 is a protecting group or hydrogen
  • protecting group R 5 is selected from benzyloxycarbonyl, tert-butoxycarbonyl, fluoren-9-yhnethoxycarbonyl, 1 -(biphenyl-4-yl)- 1 - methylethoxycarbonyl, ⁇ , ⁇ -dimethyl-3,5-dimethoxylbenzyloxycarbonyl, p- methoxybenzyloxycarbonyl, /Miitrobenzyloxycarbonyl, allyloxycarbonyl and trichloroethoxycarbonyl.
  • R 5 is benzyloxycarbonyl, fert-butoxycarbonyl (Boc) or flouren-9- ylmethoxycarbonyl (Fmoc). In another preferred embodiment R 5 is H.
  • Lg is a leaving group such, as tosylate or mesylate and R 5 is as previously defined.
  • the process of the invention comprises the step of converting a compound of formula (V) into a compound of formula (IV)
  • R 5 is benzyloxycarbonyl (Cbz), and the process comprises hydrogenating a compound of formula (V) in the presence of a palladium catalyst.
  • the process of the invention comprises the step of converting a compound of formula (VI) into a compound of formula (V)
  • the oxidising agent is mCPBA.
  • the oxidising agent is a dioxirane.
  • dioxiranes as oxidising agents is well documented in the literature [see (a)
  • the dioxirane is generated in situ by the reaction of KHS O 5 with a ketone.
  • the oxidation step can also be carried out using an isolated dioxirane, for example a stock solution of the dioxirane formed from acetone.
  • the dioxirane is generated in situ using Oxone®, which is a commercially available oxidising agent containing KHSO 5 as the active ingredient.
  • the claimed process involves the in situ epoxidation of a compound of formula (VI) using Oxone® (2KHSO 5 -KHSO 4 -K 2 SO 4 ) and a ketone co-reactant.
  • the active ingredient of Oxone® is potassium peroxymonosulfate, KHSO 5 [CAS-RN 10058-23-8], commonly known as potassium monopersulfate, which is present as a component of a triple salt with the formula 2KHSO 5 -KHSO 4 -K 2 SO 4 [potassium hydrogen peroxymonosulfate sulfate (5:3:2:2), CAS-RN 70693-62-8; commercially available from DuPont].
  • persulfate reacts with the ketone co- reactant to form a three membered cyclic peroxide (a dioxirane) in which both oxygens are bonded to the carbonyl carbon of the ketone.
  • the cyclic peroxide so formed then epoxidises the compound of formula VI by syn specific oxygen transfer to the alkene bond.
  • the ketone is of formula (XIX)
  • R a and R b are each independently alkyl, aryl, haloalkyl or haloaryl.
  • the alkyl group may be a straight chain or branched alkyl group.
  • the alkyl group is a C 1-20 alkyl group, more preferably a C 1-15 , more preferably still a C 1-I2 alkyl group, more preferably still, a C 1-8 or C 1-6 alkyl group, more preferably a C 1-4 alkyl group.
  • Particularly preferred alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-hutyl, pentyl and hexyl.
  • haloalkyl refers to an alkyl group as described above in which one or more hydrogens are replaced by halo.
  • R a and/or R b are aryl
  • the aryl group is typically a C ⁇ -u aromatic group.
  • Preferred examples include phenyl and naphthyl etc.
  • haloaryl refers to an aryl group as described above in which one or more hydrogens are replaced by halo.
  • R a and R b are as defined above. More preferably, R a and R b are each independently alkyl or haloalkyl.
  • At least one of R a and R b is a haloalkyl, more preferably, CF 3 or CF 2 CF 3 .
  • R a and R are each independently methyl or trifluoromethyl.
  • the ketone is selected from acetone and a 1,1,1 -trifmoroalkyl ketone.
  • the trifluoroalkyl ketone is 1,1,1- trifluoroacetone or l,l,l-trifluoro-2-butanone, more preferably l,l,l-trifmoro-2- butanone.
  • the process of the invention comprises the step of converting a compound of formula (VII) into a compound of formula (VI)
  • the process comprises treating a compound of formula (VII) with tosyl chloride in pyridine.
  • the process comprises treating a compound of formula (VII) with tosyl chloride in dichloromethane and triethylamine.
  • the process of the invention comprises the step of converting a compound of formula (VIH) into a compound of formula (VII)
  • this step comprises the steps of:
  • step (b) converting the product formed in step (a) to a compound of formula (VII).
  • steps (a) and (b) of the above process are a one-pot process.
  • R 5 is benzyloxycarbonyl
  • step (b) comprises treating the mixture formed in step (a) with benzyloxycarbonyl chloride.
  • W is I, Br or OTs, more preferably, Br or OTs, even more preferably OTs.
  • the alcohol is isopropyl alcohol or ethanol.
  • said compound of formula VIII is prepared from a compound of formula TX
  • the above process comprises treating said compound of formula DC with methyl lithium. More preferably, compound of formula DC is compound 47 and compound of formula
  • Isosorbide (43) is converted to the di-tosylate (42) which is obtained following recrystallisation from methanol in 97% yield.
  • Mono-bromination is effected by 2.5eq lithium bromide in DMSO (or DMF) with temperature control 11O 0 C- ⁇ 12O 0 C.
  • the product bromide is isolated following extractive work-up and purification either by column chromatography (74%) or attractive for large scale by recrystallisation from methanol giving a first crop of 55% plus mother liquors containing good quality material that may be pooled from batch runs and purified later.
  • preparation of monobromotosylate (47) with defined stereochemistry by methods in Scheme 15 is attractive for large scale applications.
  • the alcohol functionaUty of (18) may be derivatised as the p ⁇ r ⁇ -toluene sulphonate (Ts) giving (i?)-2-(benzyloxycarbonylamino)-l-((5)-2,5-dihydroiuran-2-yl)ethyl 4- methylbenzenesulfonate (32b) which proceeds through the anti-epoxide (R)-2- (benzyloxycarbonylamino)-l-((15', 2S 5 5S)-3,6-dioxabicyclo[3.1.0]hexan-2-yl)ethyl 4- methylbenzenesulphonate (33b).
  • tosylate (33b) Hydrogenation of tosylate (33b) provides free amine that undergoes intramolecular cyclisation to provide intermediate (74).
  • Intermediate (74) undergoes displacement with an excess of lithium chloride in DMF at 13O 0 C, to give the 6-chloro analogue with inversion of configuration.
  • Urethane protection of the secondary amine of the bicyclic intermediate (69) followed by oxidation to ketone provides intermediate (71) that is particularly useful for solid phase synthesis of compounds of general formula I.
  • the epoxidation to give the desired ⁇ rctz-epoxide is directed by the presence of the tosylate group.
  • intermediate tosylate (74) may be Cbz protected to give protected analogue (34b) that may undergo inversion to chloride (68) through treatment with LiBr in DMF at typically 130 0 C (Scheme 1).
  • novel 4-substituted cyelohexylglycine aminoacids that are an intrinsic feature of compounds of formula I may be prepared following adaptation of a variety of known general literature syntheses of ami ⁇ oacids.
  • a 4-substituted cyclohexane acetic acid e.g. tr ⁇ r ⁇ -4-methylcyclohexane acetic acid CAS 7132-93-6
  • novel chiral aminoacid e.g.
  • tr ⁇ r ⁇ -(4-methylcyclohexyl)acetic acid (131) (CAS 7132-93-6; ABCR GmbH AB168553; Shanghai FWD Chemicals Ltd K7354) is converted into the Evans auxiliary (127) following the general methods detailed in WO98017626 (pg 49).
  • Asymmetric addition of azide is then conducted by deprotonation of (127) and reaction with trisyl azide.
  • Reduction of azide (128) and concomitant Boc amino protection following the general methods detailed in US5128448 provides intermediate (129).
  • hydrolysis of the auxiliary is conducted with hydrogen peroxide and lithium hydroxide following the general methods detailed in US5128448.
  • the final product (130) is obtained following simple aqueous extraction.
  • compounds of general formula (T) may be readily synthesised by a number of chemical strategies, performed either in solution or on the solid phase (see Atherton, E. and Sheppard, R. C. In 'Solid Phase Peptide Synthesis: A Practical Approach', Oxford University Press, Oxford, U.K. 1989, for a general review of solid phase synthesis principles), or a combination thereof.
  • Compounds of general formula (I) may be conveniently considered as a combination of three building blocks (Pl, P2 and P3) that respectively occupy the Sl, S2 and S3 binding sites of the protease (see Berger, A and Schechter, L, Philos. Trans. K Soc.
  • a suitably protected and/or activated building block may then be prepared and subsequently chemically bonded (coupled) together with other building blocks to provide compounds of general formula (T).
  • Compounds of formula (T) may be prepared: (1) by the stepwise addition of P3 and P2 to the bicyclic 6-(5)-chlorotetrahydrofuro[3,2- ⁇ ]pyrrol-3-one core; or (2) by reaction of the bicyclic 6-(jS)-chlorotetrahydrofuro[3,2-5]py ⁇ Ol-3-one core with a P3-P2 prescursor molecule; or (3) by introducing the P3-P2 group prior to formation of the bicyclic 6- (S)-chlorotetrahydrofuro[3,2- ⁇ ]pyrrol-3-one core, i.e. prior to the oxidation step or prior to the intramolecular cyclisation step.
  • each of the Pl, P2 or P3 building blocks may contain additional alternative functionalities that are further transformed following coupling to give the final compound.
  • the ketone functionality of the Pl building block may be protected as a ketal during coupling of building blocks and transformed to the final ketone by hydrolysis following completion of the coupling reactions.
  • the ketone functionality of the Pl building block may be initially introduced via a lower oxidation state such as the corresponding alcohol and following completion of the coupling reactions be re-introduced by oxidation of the alcohol.
  • the ketone functionality of the Pl building block may be protected through a semi-carbazone suitable for solid phase synthesis (e.g. see WO 02/057270 and references cited therein) and following completion of the coupling reactions released from the solid phase by acidolytic reaction.
  • the chemical bond formed by coupling of the building blocks is a secondary amide (P3-P2) or a tertiary amide (P2-P1) that is formed through reaction of an activated carboxylic acid with a primary and secondary amine respectively.
  • P3-P2 secondary amide
  • P2-P1 tertiary amide
  • Many methods are available for activation of a carboxylic acid prior to coupling to an amine and in principle, any of these methods may be used herein.
  • Typical carboxylic acid activation methods are exemplified but not restricted to the azide method, mixed anhydride method (e.g. via isobutylchloroformate), carbodiimide methods (e.g.
  • the coupling reaction may be enhanced by the addition of a further activation catalyst such as 1- hydroxybenzotriazole, or 4-dimemylaminopyridine.
  • a further activation catalyst such as 1- hydroxybenzotriazole, or 4-dimemylaminopyridine.
  • the ⁇ -amino group of the P2 aminoacid building block is usually protected during coupling reactions to the Pl building block to avoid the formation of undesired self- condensation products.
  • the art of ⁇ -amino protection is well known in peptide chemistry (e.g. see Bodanszky, M. 'Principles of Peptide Synthesis', 2 nd rev. ed.
  • example protection groups include, but are not limited to, 9-fluorenyhnethoxycarbonyl (Fmoc), tert- butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) 5 allyloxycarbonyl (Alloc) and trichloroethoxycarbonyl (Treoc).
  • Fmoc 9-fluorenyhnethoxycarbonyl
  • Boc tert- butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • Alloc allyloxycarbonyl
  • Teoc trichloroethoxycarbonyl
  • the Fmoc group is particularly well suited for solid phase syntheses (e.g. see Atherton, E.; Sheppard, R. C.
  • any protecting groups are removed in whatever manner is dictated by the choice of protecting groups (for a general description of protecting groups and their respective stabilities and methods of removal see Greene, T. W. and Wuts, P. G. M. 'Protective Groups in Organic Synthesis' John Wiley and Sons, New York, 1991 and references therein).
  • the entire left hand portion of a compound of general formula (I) i.e. P3-P2 as the carboxylic acid can be prepared in solution by traditional organic chemistry methods and coupled to ketone, alcohol or ketal intermediates such as compounds (lib), (lie) and (lid). Then oxidation of the alcohol intermediate (e.g. Dess- Martin periodinane in DCM) or acidolytic cleavage of the ketal intermediate provides compounds of general formula (I).
  • the alcohol oxidation route is particularly useful when the compound of general formula (I) contains a substituent that is labile to trifluoroacetic acid, this being the final reagent used in each of the solid phase syntheses.
  • one preferred strategy for the synthesis of compounds of general formula (I) comprises:-
  • a second strategy for the synthesis of compounds of general formula (T) comprises:-
  • step (b) Standard organic chemistry methods for the conversion of building block obtained in step (a) towards compounds of general formula (I).
  • compounds of formula (T) may be prepared using conventional solution phase chemistry, for example, as described in Quibell, M et al, Bioorg. Med. Chem, 13, 609-625, 2005 (see in particular, Schemes 3 and 4).
  • the solution phase strategy is attractive in being able to generate larger quantities of preferred analogues, typically on a multi-gram to multi- kilogram scale.
  • compounds of formula (I) may be prepared using conventional solid phase chemistry, for example, as described in Quibell M, et al Bioorg. Med. Chem, 12, 5689-5710, 2004, see in particular, Scheme 3 and Section 3.2, and references cited therein; and Bioorg. Med. Chem, 13, 609-625, 2005, see Scheme 5 and Section 2.2, and references cited therein).
  • the solid phase strategy is attractive in being able to generate many thousands of analogues, typically on a 5-100mg scale, through established parallel synthesis methodologies (e.g. see (a)
  • the synthetic strategy is based on reversible anchorage of the ketone functionality via a hydrazide linker bond using general multipin techniques previously described in the art (Watts J. et al, Bioorg. Med. Chem. 12(11), 2903, 2004; Quibell M., et al, Bioorg. Med. Chem. 5689-5710, 2004; GrabowksaU. et al, J. Comb. Chem. 2000, 2(5), 475).
  • Construct (XVII) is prepared through reaction of the linker molecule and the 6-(S)- chlorotetrahydrofuro[3,2- ⁇ ]pyrrol-3-one (71) by refluxing in aqueous ethanol/sodium acetate. 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 (XVII) 3 providing the loaded construct (XVIII). Loaded construct (XVIII) be reacted with a wide range of carboxylic acids available commercially or in the literature, to introduce the left-hand portion 'P3-P2'.
  • furan-2-carboxylic acid 5- chlorofuran ⁇ -carboxylic acid, thiophene-2-carboxylic acid, 5-chlorothiophene-2- carboxylic acid, furan-3 -carboxylic acid, 5-chlorofuran-3 -carboxylic acid, thiophene-3- carboxylic acid, S-chlorothiophene-S-carboxylic acid, oxazole-2-carboxylic acid, oxazole-5-carboxylic acid, l,3,4-oxadiazole-2-carboxylic acid, thiazole-2-carboxylic acid, thiazole-5-carboxylic acid, l,3,4-thiadiazole-2-carboxylic acid, benzoic acid, 3- methylbenzoic acid, 3-chlorobenzoic acid, 3-fluorobenzoic acid,
  • Solvents were purchased from ROMIL Ltd, U.K. at SpS or Hi-Dry grade unless otherwise stated.
  • 1 H NMR and 13 C NMR were obtained on a Bruker DPX400 (400 MHz 1 H frequency and 100 MHz 13 C frequency; QXI probe) or Bruker Avance 500MHz (TXI probe with ATM) in the solvents 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.
  • AU analytical HPLC were obtained on Phenomenex Jupiter C 4 , 5 ⁇ , 300 A, 250 x 4.6 mm, using mixtures of solvent A (0.1% aq trifluoroacetic acid (TFA)) and solvent B (90% acetonitrile / 10% solvent A) on automated Agilent systems with 215 and / or 254 nm UV detection. Unless otherwise stated a gradient of 10 to 90% B hi A over 25 min at 1.5 mL / min was performed for full analytical HPLC.
  • solvent A 0.1% aq trifluoroacetic acid (TFA)
  • solvent B 90% acetonitrile / 10% solvent A
  • HPLC-MS analysis was performed on an Agilent 1100 series LC/MSD, using automated Agilent HPLC systems, with a gradient of 10 to 90% B in A over 10 min on Phenomenex Luna C 8 , 5 ⁇ , 300 A 5 50 x 2.0 mm at 0.6 mL / min.
  • Semi- preparative HPLC purification was performed on Phenomenex Jupiter C 4 , 5 ⁇ , 300 A, 250 x 10 mm, using a gradient of 10 to 90% B in A over 25 min at 4 mL / min on automated Agilent systems with 215 and / or 254 nm UV detection. Flash column purification was performed on silica gel 60 (Merck 9385) or using isolute SPE flash silica columns (Biotage, Hengoed, UK).
  • OCHCHOTs 5 4.60-4.64 (IH 5 m, CHOTs) 5 4.97-5.01 (1 ⁇ brt, NH), 5.08 (2 ⁇ , brs, CH 2 Ph), 7.29-7.37 (7 ⁇ , aromatic CH 3 CCH and phenyl CH), 7.78 (2 ⁇ , d, J - 8.18 Hz, aromatic OSO 2 CCH); ⁇ c (125 MHz, CDCl 3 ) 21.665 (aryl-CH 3 ), 42.054 (CH 2 NHCbz),
  • Lithium chloride (2.38 g, 56.2 mmol) was added to a stirred solution of (3R, 3aR, 6R, 6aS)-benzyl 3-hydroxy-6-(tosyloxy)tetrahydro-2H-furo[3,2- ⁇ ]pyrrole-4(5H)- carboxylate (34b) (2.435g, 5.62 mmol) in dimethylformamide (75 mL) under an atmosphere of argon.
  • the mixture was heated at 130 0 C for 7 hours then allowed to cool to ambient temperature.
  • the mixture was diluted with dichloromethane (100 mL), then water (50 mL) was added and the mixture filtered through celite (filter cake washed with dichloromethane).
  • aqueous layer was re-extracted with dichloromethane (2 x 100 mL) then the combined organic layers were washed with saturated aqueous sodium hydrogen carbonate solution (100 mL), dried (Na 2 SO 4 ), filtered and reduced in vacuo to leave a yellow oil (6.74 g).
  • Lithium hydroxide monohydrate (165 mg, 3.92 mmol) was added then the mixture was stirred at 0 0 C for 2 hours before adding a solution of sodium sulphite (1.65 g) in water (10 mL) followed by aqueous sodium hydrogen carbonate solution (30 mL). The mixture was stirred for 5 minutes then the volume reduced by half in vacuo (> 25 mbar, external water bath 25 0 C). Water (50 mL) was added then the pH adjusted to ⁇ 2 using 5M hydrochloric acid.
  • the aqueous phase was extracted with dichloromethane (4 x 50 mL), then the combined organic layers were dried (Na 2 SO 4 ), filtered and reduced in vacuo to leave a colourless oil (1.68 g)
  • the oil was partitioned between a solution of sodium carbonate (1.75 g) in water (50 mL) and diethyl ether (40 mL).
  • the aqueous layer was re- extracted with diethyl ether (2 x 40 mL) then the pH adjusted to ⁇ 2 using 5M hydrochloric acid.
  • Solid Phase Chemistry Fmoc-ketone building block (71) may be utilised in a solid phase synthesis of EXAMPLE inhibitors (1-22) of general formula I.
  • the methods used were directly analogous to those described in detail in WO02057270, utilising the 4- ⁇ [(Hydrazinocarbonyl)arnino]methyl ⁇ cyclohexane carboxylic acid trifluoroacetate based linker, solid phase lanterns (ex Mimotopes), standard Fmoc chemistries and acidolytic cleavage followed by semi-preparative HPLC purification (see WO02057270 pg 124-127 for full generic details).
  • Novel compounds (1-22) or prior art compound (38) are detailed for comparison and can readily be prepared by the general methods detailed in WO02057270 or WO0807127 through use of appropriate Fmoc- ketone building blocks (e.g. 6-unsubstituted bicycle (WO02057270), compound 19, pg 134; 6(S)-fluoro bicycle (WO0807127, compound 63, pg 88); 6(S)-chloro bicycle (WO0807127, compound 71, pg 94); 6(i?)-chloro bicycle (WO0807127, compound 79, pg 98)).
  • Fmoc-ketone building blocks are then derivatised as appropriate with a R 9 - COOH carboxylic acid via standard uranium activation techniques.
  • ⁇ PLC-MS Rt 2.39 min, 496.2/498.2 [M + H] + , 514.2/516.2 [M + H + 18] + .
  • Example 20 N-((5)-2-((3 ⁇ 5 r ,65 r ,6fl5)-6-chloro-3-oxodiliydro-2/f-furo[3 ,2-Z?]pyrrol- 4(5H,6H 5 6 ⁇ H)-yl)-l-((lr,45)-4-me1i ⁇ ylcyclohexyl)-2-oxoethyl)- 3-oxo-3,4-dihydro-2H ' - benzof ⁇ ] [ 1 ,4]oxazine-7-carboxamide
  • ⁇ PLC-MS Rt 2.42 min, 512.2/514.2 [M + H] + , 530.2/532.2 [M + H + 18] + .
  • EXAMPLES of the invention may be prepared by traditional solution phase organic chemistry techniques for example from building block (69) (3R, 3aR, 6S, 6aS)-6 ⁇ chlorohexahydro-2H " -furo[3,2-&]pyrrol-3-ol (e.g. following the general methods detailed in WO08007127, pg 103-107).
  • EXAMPLE ketone (free base) (1 mmol) was dissolved in acetonitrile (16.7 mL) and standardised 0.1N HCl (1.3 eq, 13.0 ⁇ mL) was added. The mixture was frozen and lyophilised to leave the EXAMPLE .hydrochloride salt as a solid.
  • 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.
  • Human liver cathepsin B (0.25 nM final; Merck Biosciences), was routinely assayed in 10 mM bis-tris propane; pH 6.5 containing 1 mM EDTA, 5 mM 2-mercaptoethanol, 1 mM CaCl 2 and 60 ⁇ M Z-Phe-Arg-AMC (Bachem, Weil am Rhein, Germany).
  • Vi' is the observed initial rate
  • ' F max app ' is the observed maximum activity at saturating substrate concentration
  • 'J ⁇ yi app ' is the apparent macroscopic binding (Michaelis) constant for the substrate
  • ' [S 0 ]' is the initial substrate concentration.
  • Ki apparent inhibition constant
  • V app LSI v, ⁇ — (2)
  • Equation 2 ' Vi' is the observed residual activity, ' F max app ' is the observed maximum activity (i.e. in the absence of inhibitor), 'J ⁇ M app ' is the apparent macroscopic binding (Michaelis) constant for the substrate, '[S]' is the initial substrate concentration, 'K ⁇ is the apparent dissociation constant and ' [I]' is the inhibitor concentration.
  • Equation 3 In situations where the apparent dissociation constant (K ⁇ ) approached the enczyme concentrations, the K ⁇ app values were calculated using a quadratic solution in the form described by Equation 3 (Morrison, J.F. Trends Biochem. Sci.,_l, 102-105, 1982; Morrison, J.F. Biochim. Biophys. Acta ⁇ 185, 269-286, 1969; Stone, S.R. and Hofsteenge, J. Biochemistry, 25, 4622-4628, 1986).
  • Equation 3 'vf is the observed residual activity, C F' is the difference between the maximum activity ⁇ i.e. in the absence of inhibitor) and minimum enzyme activity, 'E 0 ' is the total enzyme concentration, '!Tj app ' is the apparent dissociation constant and 'I 0 ' is the inhibitor concentration. Curves were fitted by non-linear regression analysis
  • Equation 4 was used to account for the substrate kinetics, where 1 KC is the inhibition constant, '[S 0 ]' is the initial substrate concentration and ⁇ Ku w is the apparent macroscopic binding
  • the concentration dependence of the observed rate of reaction (fc obs ) 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, 61, 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 Eq. 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, 'Ar obs ' 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 £ o b s versus the inhibitor concentration (i.e. k Ob J[ ⁇ ]).
  • Eq. 6 was used, where '[S 0 ]' is the iniitial substrate concentration and 'JKM 3PI " is the apparent macroscopic binding (Michaelis) constant for the substrate.
  • Human and rat liver microsomes were purchased from BD Gentest (Woburn, MA,
  • liver microsome incubations were carried out in 50 mM potassium phosphate buffer at pH 7.4, with a final microsomal protein concentration of 0.5 mg/mL.
  • Compounds were taken from 5 mM DMSO stock solutions and diluted in incubation buffer to give a final concentration of 25 ⁇ M, with a final DMSO concentration of 0.5% v/v. In brief, compounds were added to the incubation buffer along with the liver microsomes and incubated at 37°C for 10 minutes.
  • Human and rat plasma were purchased from Alternative Research Inc. (Southfield. MI, USA). Compounds were taken from 5 mM DMSO stock solutions and added to plasma, which had previously been incubated at 37°C, to give a final concentration of 25 ⁇ M and re-incubated. Aliquots were removed at 2 and 60 minutes and quenched with an equal volume of cold acetonitrile. After mixing vigorously, the precipitated protein matter was removed by filtration (Multiscreen Solvinert filter plates, Millipore, Bedford, MA, USA) and the filtrate analysed by reverse phase HPLC with mass spectrometric detection, using single ion monitoring of the [M+H] species. Metabolic turnover was determined by comparison of peak areas from the ion chromatograms of the parent compound at 2 and 60 minutes and expressed as percent remaining at 1 hour.
  • LogD Determinations were performed in 96 well microtitre plates using a miniaturised "shake-flask" method. In brief, compounds were taken from 10 mM DMSO stock solutions and added to wells containing equal volumes of phosphate buffered saline (10 mM; pH 7.4) (PBS) and 1-octanol (Sigma- Aldrich, Poole, Dorset, UK) to give a final concentration of 50 ⁇ M. The plates were then capped and mixed vigorously for 1 hour on a microtitre plate shaker, after which they were left to stand, allowing the PBS and octanol phases to separate.
  • PBS phosphate buffered saline
  • 1-octanol Sigma- Aldrich, Poole, Dorset, UK
  • the PBS layer was analysed by reverse phase HPLC with mass spectrometric detection, using single ion monitoring of the [M+H] + species.
  • LogD(p ⁇ s ) was determined by comparison of the peak area from the ion chromatograrn of the compound in the PBS phase with that of a 50 ⁇ M standard of the same compound dissolved in acetonitrile/water (50:50) and calculated using the following formula:
  • AUCstd and AUCpbs are the peak areas from the standard and test ion chromatograms respectively.
  • LogD( PB S) determinations were also made using PBS at pH6.9 and 5.5 by adjusting the pH of the buffer prior to the start of the assay, with 0.1 M HCL.

Abstract

A first aspect of the invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, complex or pro-drug thereof, wherein: one of R3 and R4 is H, and the other is selected from C1-6-alkyl, C1-6-haloalkyl, C1-6- alkoxy, and C6-12-aralkyl; or R3 and R4 are each independently selected from C1-6-aIkyl and halo; R9 is a substituted 5 or 6-membered aryl or heteroaryl group or a 6,5- or 6,6-fused biaryl or heterobiaryl group. Compounds of formula (I) exhibit surprisingly high efficacies for human cathepsin S, excellent selectivity verses other mammalian cathepsins and are useful for treatment of diseases such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain.

Description

COMPOUNDS
The present invention relates to compounds that are inhibitors of cysteine proteinases, pharmaceutical compositions containing said compounds, and their use in therapy. More specifically, the invention relates to compounds that are inhibitors of cathepsin S, a cysteine proteinase of the CA clan. Such compounds are particularly useful for the in vivo therapeutic treatment of diseases in which participation of cathepsin S is implicated.
BACKGROUTSD TO THE INVENTION
Proteinases form a substantial group of biological molecules which to date constitute approximately 2% of all the gene products identified following analysis of several completed genome sequencing programmes. Proteinases have evolved to participate in an enormous range of biological processes, mediating their effect by cleavage of peptide amide bonds within the myriad of proteins found in nature. This hydrolytic action is performed by initially recognising, then binding to, particular three- dimensional electronic surfaces displayed by a protein, which align the bond for cleavage precisely within the proteinase 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 proteinase itself, or through the action of a water molecule that is bound to and activated by the proteinase. Proteinases in which the attacking nucleophile is the thiol side-chain of a Cy s residue are known as cysteine proteinases. The general classification of 'cysteine proteinase' contains many members found in a wide range of organisms from viruses, bacteria, protozoa, plants and fungi to mammals.
Cathepsin S and indeed many other crucial mammalian proteinases belong to the papain-like CACl family (see Barrett, AJ et al, in 'Handbook of Proteolytic Enzymes', Eds. Barrett, A. J., Rawlings, N. D., and Woessner, J. F. Publ. Academic Press, 1998, for a thorough discussion). To date, cysteine proteinases have been classified into five clans, CA, CB, CC, CD and
CE (Barrett, A. J. et al, 1998). A proteinase 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. Proteinases of clan CA / family Cl have been implicated in a multitude of house-keeping roles and disease processes, e.g. human proteinases such as cathepsin K (osteoporosis, osteoarthritis), cathepsin S (multiple sclerosis, rheumatoid arthritis, autoimmune disorders), cathepsin L (metastases), cathepsin B (metastases, arthritis), cathepsin F (antigen processing), cathepsin V (T-cell selection), dipeptidyl peptidase I (granulocyte serine proteinase activation) or parasitic proteinases such as falcipain (malaria parasite Plasmodium falciparum) and cruzipain {Trypanosoma cruzi infection).
There currently exists a major unmet need for safe orally administered medications for the treatment of immune-based inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, asthma, atherosclerosis etc. The therapeutic inhibition of cathepsin S has been of great interest to the pharmaceutical industry as a potential target for immune system modulation. Cathepsin S is a lysosomal cysteine proteinase that is specifically up-regulated under inflammatory conditions. It is highly expressed in the spleen, in professional antigen presenting cells (APCs) and other MHC class II- positive cells and is inducible by IFN-γ. Intracellular cathepsin S specifically processes invariant chain, a protein involved in the correct loading of MHC-II with antigen (a key step in generating an immune response) (see Shi, G. P. et al., Immunity, 10(2). 197- 206, 1999; Lui, W. and Spero, D. M. Drug News Perspect. 17(6), 357-363, 2004). The MHC-II / antigen complex is then displayed on the surface of the APC, for interaction with and activation of T-cells. Disrupting antigen presentation represents a validated approach to treating diseases with an autoimmune component such as rheumatoid arthritis (e.g. see Podolin, P.L.,et al., Inflamm Res 50: S159. 2001), multiple sclerosis and myasthenia gravis. As well as its intracellular role in antigen presentation, cathepsin S is secreted from macrophages infiltrating sites of inflammation, to aid proteolysis of proteins and facilitate phagocytosis. However, in chronic inflammatory situations cathepsin S is responsible for degradation of structural tissue proteins and also mediates pain. Cathepsin S has been implicated in the destruction of articular cartilage in rheumatoid and osteoarthritis (e.g. see Hou, W-S. et al, Arthritis and Rheumatism, 46(3), 663-674, 2002 and refs cited therein), vascular tissue damage in atherosclerosis (e.g. see Rodgers, K. J. etal, Arterioscler. Thromb. Vase. Biol. 26, 851-6, 2006) and lung tissue damage in chronic obstructive pulmonary disease (e.g. see Shapiro, S. D. Biochem. Soc. Trans. 30(2), 98-102, 2002 and refs cited therein). Therefore an inhibitior of cathepsin S has the potential to tackle both diseases mediated through antigen presentation and extracellular matrix damage.
Additionally, cathepsin S has been shown to be critical for the maintenance of neuropathic pain and spinal microglia activation in peripheral nerve-injured rats (see Clark, A. K. et al, Proc. Natl. Acad. Sci. USA, 104(25), 10655-10660, 2007; Barclay, J., et al, Pain, 130(3), 225-234, 2007). Therefore inhibition of cathepsin S has therapeutic potential in the treatment of neuropathic pain (e.g. see WO-A-03020287).
In the prior art, the development of cysteine proteinase inhibitors for human use has recently been an area of intense activity (e.g. see Deaton, D. N. and Kumar, S., Prog. Med. Chem. 42, 245-375, 2004; Bromme, D. and Kaleta, J., Curr. Pharm. Des., 8, 1639-1658, 2002; Kim, W. and Kang, K., Expert Opin. Ther. Patents, 12(3), 419-432, 2002; Leung-Toung, R. et al. Curr. Med. Chem., 9, 979-1002, 2002; Lecaille, F. et al., Chem. Rev., 102, 4459-4488, 2002; Hernandez, A. A. and Roush, W. R., Curr. Opin. Chem. Biol., 6, 459-465, 2002; Link, J.O. and Zipfel, S. Curr. Opin. Drug Discov. Dev., 9(4), 471-482, 2006). Considering the CACl family members, particular emphasis has been placed upon the development of inhibitors of human cathepsins, primarily cathepsin K (osteoporosis) and cathepsin S (autoimmune disorders) through the use of covalent-bound but reversible peptide and peptidomimetic nitriles (e.g. see Bekkali, Y. et al, Bioorg. Med. Chem. Lett., 17(9), 2465-2469, 2007; WO-A- 07137738, WO-A-07003056), linear and cyclic peptide and peptidomimetic ketones
(e.g. see Veber, D. F. and Thompson, S. K., Curr. Opiti. Drag Discovery Dev., 3(4), 362-369, 2000; WO-A-02057270, WO-A-04007501, WO-A-06064286, WO-A- 05066180, WO-A-0069855), ketoheterocycles (e.g. see Palmer, J. T. et al, Bioorg. Med. Chem. Lett., 16(11), 2909-2914, 2006, WO-A-04000838), α-ketoamides (e.g. see WO-A-06102243), cyanoamides (WO-A-01077073, WO-A-01068645) and arylnitriles (e.g. see WO-A-07080191, WO-A-07039470, WO-A-06018284, WO-A-05121106, WO-A-04000843). Inhibition of CACl proteases by non-covalent bound compounds has been extensively described in the literature. Particular emphasis has been placed upon inhibition of cathepsin K and cathepsin S by arylammoethylamides (e.g. see Altmann, E., et al, J. Med. Chem., 45(12), 2352-2354, 2002; Chatterjee, A.K. et al, Bioorg. Med. Chem. Lett., 17(10), 2899-2903, 2007; US-20050113356, US- 20050107368, US-20050118568) and substituted pyrazoles or piperidines (e.g. see Wei, J., et al, Bioorg. Med. Chem. Lett., 17(20), 5525-5528, 2007; US-2007117785, US-2003073672, WO-A-02020013).
Thus the extensive prior art describes potent in vitro inhibitors of cathepsin S and inhibitors showing efficacy in numerous animal models of disease. However, the many difficulties in developing a human therapeutic for inhibition of cathepsin S are also evident since presently only one compound is in clinical development (RWJ-445380 for rheumatoid arthritis and psoriasis).
Recently, Quibell, M. (WO-A-02057270) described a new motif for the general inhibition of CACl proteinases based upon a cis-5 ,5-bicyclic ketone (1).
Figure imgf000005_0001
(1) Based upon this motif, highly potent and selective inhibitors of cathepsin K were discovered (see WO-A-0807109, WO-A-0807103, WO-A-0807130, WO-A-0807114, WO-A-0807127, WO-A-0807107, WO-A-0807112). The present inventors have now discovered a small genus of 6-(5)-chlorotetrahydrofuro[3,2-ό]pyrrol-3-ones that exhibit potent and selective in vitro inhibition versus human cathepsin S.
STATEMENT OF INVENTION
A first aspect of the invention relates to a compound of formula (T), or a pharmaceutically acceptable salt, hydrate, complex or pro-drug thereof,
Figure imgf000006_0001
(D wherein: one of R3 and R4 is H, and the other is selected from Ci^-alkyl, C1-6-haloalkyl, C1-6- alkoxy and C6-12-aralkyl; or R3 and R4 are each independently selected from Ci-6-alkyl and halo; R9 is selected from the following:
Figure imgf000007_0001
wherein:
Xi5 X2, X3, X4, Xi4, Xi5,Xi6 andX2o are each independently selected from:
CH, C-(C1-6-alkyl), C-CCμs-alkoxy), C-halo and N; such that a maximum of two Of X1, X2, X3, X4, X14, X1S1X16 and X20 are selected from N, C-halo and C-(C1-6-alkoxy); X5, Xβ, X7 and X8 are each independently selected from:
CH, C-(C1-6-aIkyl), C-(C1-6-alkoxy), C-halo, N and C-OH; such that a maximum of one of X5, X6, X7 and X8 is N, C-halo, C-OH or C-(C1-S- alkoxy);
X9 and X12 are each independantly selected from:
CH, CHCi-6-alkyl), C-(C1-6-alkoxy), C-halo and N;
X1O and X11 are each independantly selected from: CH, C-(C1-6-alkyl), C-(C w-alkoxy), C-halo, N and R10;
X19 is selected from:
CH, C-(Cw-aJJkyl), C-(Ci.6-alkoxy), C-C(O)NH2, C-C(O)NH(C1-6- alkyl), C-C(O)N(C1-6-alkyl)2, C-halo and N;
X18 is selected from:
CH, C-Kd-e-alkyl), C-(C1-6-alkoxy), C-NH2, C-N(C ^e-alkyl^, C-NH(C1- 6-alkyl), C-NHC(O)C1-6-alkyl, C-halo and N; or when X19 is CH, C-(C1-6-alkyl), or C-halo then X18 may additionally be selected from C-C(O)NH2 and C-C(O)N(C1-6-alkyl)2;
Xi3 and X17 are each independently selected from:
O, S, NH and N-(C1-6-alkyl);
X22 and X24 are each independently selected from: CH2, CH-(C1-6-alkyl), O, S, NH, NMe and ) C=O;
X23 is selected from:
CH2, CH-(C1-6-alkyl), C-^-e-alkyTh, NH and NMe; or when either X22 or X24 are other than ) C=O then X23 may additionally be ) C=O or ) S(O)2;
X25 is selected from: O5 S, NH and N(C1-6-alkyl);
X26, X27, X2S and X29 are each independently selected from:
CH5 C-(Ci-6-alkyl), C-(C1-6-alkoxy), C-OH5 C-halo and N; such that a maximum of two of X265 X27, X28 and X29 are selected from C-(C1-6- alkoxy), C-OH5 C-halo and N;
X30 is selected from:
CH2, CH2CH2, NH, NMe, O, S and )C=O;
X3I is selected from:
CH2, NH and NMe; or when X30 is other than )C=O, O or S then X31 may additionally be
) C-O or O;
X32 is selected from:
CH2, CH2CH2, NH, NMe and )C=O;
X33 is selected from: CH2, NH and NMe; or when X32 is other than ) C=O then X33 may additionally be ) C=O or O;
X34 is selected from: NH and NMe;
R1O is selected from:
Figure imgf000010_0001
wherein:
T1, T2, T3 and T4 are each independantly selected from: CH, C-(Cj-6-alkyl), C-(C1-6-alkoxy), C-NH2, C-NH(C1-6-alkyl), C-N(C1.
6-alkyl)2, C-halo and N; such that a maximum of one of T1, T∑, T3 and T4 is C-(Ci_6-alkoxy), C-NH2, C-NH(C1- 6-aIkyI), C-N(C w-alkyl)2 or C-halo;
T5 is selected from:
O, S, NH andN(C1-6-alkyl);
T6, T7, T8, T9 and T10 are each Independantly selected from:
CH, C-(C1-6-alkyl), C-(C1-6-alkoxy), C-NH2, C-NH(C1-6-alkyl), C-N(Ci- e-alkyl)2, C-halo and N; such that a maximum of two of T6, T7, Ts, Tp and T10 are selected from C-(C1-6- alkoxy), C-NH2, C-NH(C1-6-alkyl), C-N(C1-6-alkyl)2, C-halo and N;
Tn is selected from: CH2, NH and N(Ci-6-alkyl);
T12 is selected from:
CH2, NH, N(Ci-6-alkyl) and )C=O; T13 and Ti4 are each independantly selected from: CH, C-(C1-6-alkyl) and C-halo;
Tj 5 is selected from:
O, NH andN(C1_6-alkyl);
T16 is selected from:
CH2 and ) C=O;
or R10 is selected from: H, C1-6-al]<yl, OH, C1-6-alkoxy, NO2, halo, CN, C(O)NH2, C(O)NH(C1-6- alkyl), C(O)N(Ci-6-alkyl)2, C(O)NH(C3-6-cycloalkyiχ S(O)2NH2, S(O)2(C1-6-alkyl), S(O)2NH(C1-6-alkyl), S(O)2N(C1-6-EUCyI)2,
S(0)2NH(C3-6-cycloalkyl) and (CH2VNR11R12;
wherein n is O or 1;
and R11 is selected from C1-6-alkyl, C(O)C1-6-alkyl, C(O)(C3-6-cycloalkyl), C(O)(aryl), C(O)NH2, C(O)NH(C1-6-EIlCyI), C(O)N(C1-6-alkyl)2, C(O)NH(C3-6-cycloalkyl), C(O)O(C1.6-alkyl), C(O)O(C3-6-cycloalkyl), C(O)O(aryl), S(O)2(C 1-6-alkyl), S(O)2(C3- 6-cycloalkyl), S(O)2NH2, S(O)2NH(C1-6-alkyl), S(O)2N(Ci-6-alkyl)2, S(O)2NH(C3-6- cycloalkyl) and S(O)2(aryl);
and R12 is selected from H and C1-6-alkyl.
RB is selected from:
C(O)NH2, C(O)NH(Ci-6-alkyl), C(O)N(C1-6-all<yl)25 C(O)NH(C3-6- cycloalkyl), S(O)2NH2, S(O)2(C1-6-alkyl), S(O)2NH(C1-6-alkyl), S(O)2N(C1-6-alkyl)2, S(O)2NH(C3-6-cycloalkyl) and (CH2)n-NR14Rls;
wherein n is O or 1 ; and R14 is selected from H, C1-6-alkyl, C(O)C1-6-alkyl, C(O)(C3-6-cycloaIkyl), C(O)(aryl), C(O)NH2, C(O)NH(C1-6-alkyl), C(O)N(C1-6-alkyl)2, C(O)NH(C3-6- cycloalkyl), C(O)O(Ci-6-aIkyl), C(O)O(C3-6-cycloalkyl), C(O)O(aryl), S(O)2(C1-6- alkyl), S(0)2(C3-6-cycloalkyl), S(O)2NH2, S(O)2NH(C1-6-alkyl), S(O)2N(C1-6-alkyl)2, S(O)2NH(C3-6-cycloalkyl) and S(O)2(aryl);
and R15 is selected from H and C1-6-alkyl.
Compounds of formula (I) exhibit surprisingly high efficacies for human cathepsin S. In addition, preferred compounds of formula (I) exhibit surprisingly poor in vitro potency verses other human cathepsins.
A second aspect of the invention relates to a pharmaceutical or veterinary composition comprising a compound of formula (I) and a pharmaceutically acceptable or veterinarily acceptable diluent, excipient and/or carrier.
A third aspect of the invention relates to a process for preparing a pharmaceutical or veterinary composition as defined above, said process comprising admixing a compound of the invention with a pharmaceutically acceptable or veterinarily acceptable diluent, excipient and/or carrier.
A fourth aspect of the invention relates to compounds of formula (I) for use in medicine.
A fifth aspect of the invention relates to the use of a compound of formula (I) in the preparation of a medicament for treating a disease selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain. A sixth aspect of the invention relates to a method of inhibiting cathepsin S in a cell, said method comprising contacting said cell with a compound of formula (T).
A seventh aspect of the invention relates to method of inhibiting cathepsin S in a subject, said method comprising administering to the subject a pharmacologically effective amount of a compound of formula (I).
An eighth aspect of the invention relates to a method of treating a disease selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain, in a subject, said method comprising administering to the subject a pharmacologically effective amount of a compound of formula (I).
A ninth aspect of the invention relates to the use of a compound according to the invention in an assay for identifying further candidate compounds capable of inhibiting one or more cysteine proteinases.
A tenth aspect of the invention relates to the use of a compound of formula (I) in the validation of a known or putative cysteine proteinase as a therapeutic target. An eleventh aspect of the invention relates to a process of preparing a compound of formula (I).
An eleventh aspect of the invention relates to a compound of formula (I) for treating a disease selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain. DETAILED DESCRIPTION
The term 'alkyP as applied herein includes stable straight and branched chain aliphatic carbon chains which may be optionally substituted. Preferred examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, heptyl and any simple isomers thereof. Suitable substituents include, for example, one or more C1-6 alkoxy, OH, COOH, COOMe, NH2, NMe2, NHMe, NO2, CN and/or CF3 groups. Additionally, where the alkyl group contains two or more contiguous carbon atoms, an alkene group (-CH=CH-) or alkyne group (-G≡C-) may be present. Furthermore, the alkyl group may optionally contain one or more heteroatoms 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. For example, the group CHa-CH2-O-CH2-CH2- is defined within 'alkyl' as a C4 alkyl that contains a centrally positioned heteroatom whereas the group CH3-CHa-CH2-CH2- is defined within 'alkyl' as an unsubstituted C4 alkyl. Preferably, the alkyl group is a C1-6 alkyl group, more preferably a C1-4 group.
The term 'cycloalkyP as applied herein refers to a cyclic alkyl group (i.e. a carbocyclic ring) which may be substituted (mono- or poly-) or unsubstituted. Suitable substituents include, for example, one or more C1-6 alkyl, C1-6 alkoxy, OH, COOH, COOMe, NH2, NMe2, NHMe, NO2, CN5 CF3 and/or halo groups. Preferably, the cycloalkyl group is a C3-6-cycloalkyl, even more preferably a C3-4 cycloalkyl group. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like. In addition, the carbocyclic ring itself may optionally contain one or more heteroatoms, for example, to give a heterocycloalkyl group such as tetrahydrofuran, pyrrolidine, piperidine, piperazine or morpholine.
The term 'alkyoxy' refers to the group 'O-alkyl' or 'O-cycloalkyl', wherein alkyl and cycloalkyl are as defined above.
'Halogen' or 'halo' as applied herein encompasses F, Cl, Br, I. The term 'haloalkyl' refers to an alkyl group as defined above substituted by one or more halogen atoms.
As used herein, the term 'aryl' refers to a stable 5 or 6-membered monocytic ring which is unsaturated. The aryl group may optionally include one or more heteroatoms selected from O5 N and S. Ih addition, the aryl group may be optionally substituted, for example, by one or more C1-6 alkyl, C1-6 alkoxy, OH, COOH, COOMe, NH2, NMe2, NHMe5 NO2, CN, CF3 and/or halo groups. More preferably, the aryl group may be optionally substituted by one or more Me, OMe5 OEt, OiPr, NO2, Cl or F groups.
The term 'aralkyl' as applied herein includes an alkyl group as defined above in combination with an aryl group. The aryl group may be an aromatic ring, for example, a stable 5 or 6-membered monocylic or a stable 9 or 10-membered bicyclic ring which is unsaturated. The aryl group may optionally comprise one or more heteroatoms selected from O, N and S. In addition, the aryl group may be optionally substituted, for example, by one or more C1-6 alkyl, C1-6 alkoxy, OH, COOH, COOMe, NH2, NMe2, NHMe, NO2, CN, CF3 and/or halo groups. Preferably, the aralkyl group is a Ci-g-alkyl- C5-1o-aryl group, even more preferably a Ci.8-alkyl-ph.enyl group. More preferably still, the alkyl-aryl group is selected from CH2Ph and CH2OCH2Ph.
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.
In particular, the skilled person will appreciate that the ketone group of the bicycle core of compounds of formula (I) may exist in alternative forms such as the hydrate (as shown below), and the invention extends to all such alternative forms.
Figure imgf000016_0001
Hydrate
Abbreviations and symbols commonly used in the peptide and chemical arts are used herein to describe compounds of the present invention, following the general guidelines presented by the IUPAC-IUB Joint Commission on Biochemical Nomenclature as described in Eur. J. Biochem., 158, 9-, 1984. Compounds of formula (T) and the intermediates and starting materials used in their preparation are named in accordance with the IUPAC rules of nomenclature in which the characteristic groups have decreasing priority for citation as the principle group.
In one preferred embodiment of the invention: one of R3 and R4 is H, and the other is selected from
Figure imgf000016_0002
Ci-β-haloalkyl, Cj-6- alkoxy and
Figure imgf000016_0003
or R3 and R4 are each independently selected from C1-6-alkyl and halo;
R is selected from the following:
Figure imgf000017_0001
wherein:
X1, X2, X3, X4, Xi4, X15, X16 and X2o are each independently selected from:
CH, C-(d.6-aikyϊ), C-(Ci_6-alkoxy), C-halo and N; such that a maximum of two of X1, X2, X3, X4, Xi4, X15, X16 and X20 are selected from N, C-halo and C-(C1-6-OIkOXy);
X5, X6, X7 and Xg are each independently selected from:
CH, C-(Ci-6-alkyl), C-Cd^-alkoxy), C-halo5 N and C-OH; such that a maximum of one of X5, X6, X7 and X8 is N, C-halo, C-OH or C-(C1-6- alkoxy);
Xp and Xn are each independently selected from:
CH, C-(C1-6-alkyl), C-(C1-6-alkoxy), C-halo and N;
Xio and Xi1 are each independantly selected from: CH5 C-(C1-6-alkyl), C-(C1-6-aUcoxy), C-halo, N and R10;
Xi9 is selected from:
CH, C-(Ci-6-alkyl), C-(C1-6-alkoxy)5 C-C(O)NH2, C-C(O)NH(CL6- alkyl), C-C(O)N(C ^aUyI)2, C-halo and N;
X18 is selected from:
CH5 C-(C1-6-alkyl), C-(C1-6-alkoxy), C-NH2, C-N(C1-6-alkyl)2, C-NH(C1. 6-alkyl), C-NHC(O)C 1-6-alkyl. C-halo and N; or when X19 is CH, C^C^-alky!)., or C-halo then X18 may additionally be selected from C-C(O)NH2 and C-C(O)N(C 1-6-alkyl)2;
X13 and X17 are each independently selected from: O, S, NH and N-^-alkyl);
X22 and X24 are each independently selected from:
CH2, CH-(C1-6-alkyl), O5 S5 NH and )θ=O;
X23 is selected from:
CH2, CH-(C1-6-alkyl), C-(Ci_6-alkyl)2 and NH; or when either X22 or X24 are other than ) C=O then X23 may additionally be )OO;
X25 is selected from:
O, S, NH and N(C1-6-alkyl);
X26, X27, X28 and X2P are each independently selected from:
CH5 C-(C1-6-alkyl)5 C-(C1-6-alkoxy), C-halo and N; such that a maximum of two of X265 X27, X28 and X29 are selected from C-(C1-6- alkoxy), C-halo and N;
R1O is selected from:
Figure imgf000019_0001
wherein:
T1, T2, T3 and T4 are each independantly selected from:
CH, C-Cd-β-alkyl), C-(C1-6-alkoxy), C-NH2, C-NH(C1-6-alkyl), C-N(Q- 6-alkyl)2, C-halo and N; such that a maximum of one of T1, T2, T3 and T4 is
Figure imgf000019_0002
C-NH2, C-NH(C1- 6-alkyl), C-N(C 1-6-alkyl)2 or C-halo;
T5 is selected from: O, S, NH and N(C1-6-alkyl);
T6, T7, T8, T9 and T10 are each independantly selected from:
CH, C-(d-6-alkyl), C-(C1-^aIkOXy), C-NH2, C-NH(C1-6-alkyl), C-N(C1.
6-alkyl)2, C-halo and N; such that a maximum of two of T6, T7, T8, T9 and T10 are selected from C-(C1-6- alkoxy), C-NH2, C-NH(CI-6-alkyl), C-N(CI-6-alkyl)2, C-halo and N;
T11 is selected from:
CH2, NH and N(C1-6-alkyl);
T12 is selected from:
Figure imgf000019_0003
T13 and T14 are each independantly selected from: CH, C-(C1-6-alkyl) and C-halo;
T15 is selected from:
O, NH andN(Ci-6-alkyl);
T16 is selected from:
CH2 and ) C=O;
or R10 is selected from: H3 Ci-6-alkyl, OH, C^-alkoxy, NO2, halo, CN, C(O)NH2, C(O)NH(C1-6- alkyl), C(O)N(Ci-6-alkyl)2, and (CHa)n-NR11R12; wherein n is O or 1 and R11 is selected from H, C1-6-alkyl, acetyl, C(O)NH2, C(O)N(C 1-6-alkyl)2: and R12 is selected from H and
Figure imgf000020_0001
In one preferred embodiment of the invention:
R3 is H and R4 is selected from methyl, ethyl, rc-propyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, ethoxy and benzyl: or both R3 and R4 are selected from methyl or fluoro or ehloro;
X1, X2, X3, X4, X14, X15, X16 and X2o are independently selected from: CH, CMe, C-OMe, C-F, C-Cl and N: such that a maximum of two of Xi, X2, X3, Xj, Xw, X15, X16 and X20 are chosen as N or
C-Cl or C-OMe;
X5, X6, X7 and X8 are independently selected from:
CH5 CMe, C-OMe, C-F, C-Cl, N and OH; such that a maximum of one of Xs, X6, X7 and X8 is chosen as N or C-Cl or C-OH or
C-OMe; X9 and X12 are independantly selected from:
CH, CMe, C-OMe, C-F, C-Cl and N; X10 and X11 are independently selected from:
CH, CMe3 C-OMe, C-F, C-Cl, N and R10;
X19 is selected from: CH, CMe, C-OMe, C-C(O)NH2, C-C(O)NMe2, C-F, C-Cl and N;
Xis is selected from:
CH, CMe, C-OMe, C-NH2, C-NMe2, C-NHMe, C-NHC(O)Me, C-F, C- Cl and N; or when X19 is CH, CMe or C-F then X18 may additionally be selected from C-C(O)NH2 and C-C(O)NMe2;
X13 and X17 are independently selected from:
O, S, NH and NMe.
X22 and X24 are independently selected from:
CH2, CHMe, O, S, NH, NMe and )C=0;
X23 is selected from: CH2, CHMe, CMe2, NH and NMe; or when either X22 or X24 are other than ) C=O then X23 may additionally be ) C=O or )S(O)2;
X25 is selected from: O, S, NH and NMe;
X26, X27, X28 and X29 are independently selected from:
CH, CMe, C-OMe, C-F, C-Cl3 C-Br and N; such that a maximum of two of X26, X27, X28 and X29 are chosen as C-OMe, C-Cl, C- Br and N; X30 is selected from:
CH2, CH2CH25NH, NMe, O, S and
Figure imgf000022_0001
X31 is selected from: CH2, NH and NMe; or when X30 is other than )C=O, O or S then X31 may additionally be
)C=O or O;
X32 is selected from: CH2, NH5 NMe and ) C=O;
X33 is selected from:
CH2, NH and NMe; or when X32 is other than ) C=O then X33 may additionally be ) C=O or O;
X34 is selected from:
NH and NMe;
T1, T2, T3 and T4 are independantly selected from:
CH, CMe, C-OMe, C-NH2, C-NHMe, C-NMe2, C-F, C-Cl and N: such that a maximum of one of T1, T2, T3 and T4 is chosen as C-OMe, C-NH2, C- NHMe5 C-NMe2, C-F and C-Cl;
T5 is selected from:
O, S, NH and NMe.
T6, T7, T8, T9 and T10 are independantly selected from:
CH, CMe, C-OMe, C-NH2, C-NHMe, C-NMe2, C-F, C-Cl and N: such that a maximum of two of T6, T7, T8, T9 and T10 are chosen as C-OMe, C-NH2, C-
NHMe, C-NMe2, C-F, C-Cl and N;
T11 is selected from: CH2, NH and NMe;
Ti2 is selected from:
CH2, NH, NMe and ) C=O;
T13 and T14 are independently selected from: CH, CMe, C-F and C-Cl;
T15 is selected from:
O, NH and NMe;
T16 is selected from:
CH2 and ) C=O;
or R10 is selected from: H, Me, OH, OMe, OEt, OiPr, NO2, F, Cl, Br, CN, C(O)NH2,
C(O)NHMe, C(O)NMe2, and (CH2VNR11R12:
wherein n = O or 1 and R11 is selected from H, Me, acetyl, C(O)NH2, C(O)NMe2: and R12 is selected from H and Me;
Ri3 is selected from:
C(O)NH2, C(O)NHMe, C(O)N(Me)2, C(O)NH(cyclopropyl), S(O)2NH2, S(O)2(Me), S(O)2NH(Me), S(O)2N(Me)2, S(O)2NH(cyclopropyl) and (CH2)n-NRI4R15; wherein n is 0 or 1 ;
and R14 is selected from H, Me, C(O)Me, C(O)(cyclopropyl), C(O)Ph, C(O)NH2, C(O)NH(Me), C(O)N(Me)2, C(O)NH(cyclopropyl), C(O)O(Me), C(O)O(cyclopropyl), C(O)OPh, S(O)2(Me), S(O)2(cyclopropyl), S(O)2NH2, S(O)2NH(Me), S(O)2N(Me)2, S(O)2NH(cyclopropyl) and S(O)2Ph;
and R15 is selected from H and Me.
In one preferred embodiment, the compound of the invention is of formula Ia
Figure imgf000024_0001
(Ia)
wherein R3, R4 and R9 are as defined above.
In an even more preferred embodiment, the compound of the invention is of formula Ib
Figure imgf000024_0002
(Ib) wherein R3, R4 and R9 are as defined above. In one preferred embodiment, R3 is selected from H and R4 is selected from methyl, ethyl, propyl, trifluoromethyl and benzyl.
In another preferred embodiment, both R3 and R4 are selected as methyl, fluoro or chloro.
In an even more preferred embodiment, both R3 and R4 are selected as methyl such that the central amino acid moiety is derived from (iS)-2-amino-2-(4,4- dimethylcyclohexyl)acetic acid (CAS 754178-25-1).
In a yet even more preferred embodiment, R3 is H and R4 is methyl such that the central amino acid moiety is derived from the trans configured (5)-2-amino-2-((lr, 45)-4- methylcyclohexyl)acetic acid as shown in formula Ic.
Figure imgf000025_0001
(Ic) wherein R9 is as defined above.
In one preferred embodiment, R9 is selected from:
Figure imgf000026_0001
Figure imgf000026_0002
wherein X1, X2, X3, X4, Xs, Xδ, X75 Xs, X9, Xio, Xi n X13, Xi 4, Xi5, Xi6, Xn, Xi8, X19, X22, X23, X24, X25, X30, X31, X34, Rio and R13 are as defined above.
In one preferred embodiment R9 is selected from:
Figure imgf000026_0003
wherein X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X13, Xw, Xis, Xi6, Xn5 Xi8, X19, X25, X30, X31, R10 and Ro are as defined above.
In one preferred embodiment R9is selected from:
Figure imgf000027_0001
wherein X1, X2, X3, X4, X7, X10, X17, X18, X19, X25, X30, X31, R10 and R14 are as defined above.
In one preferred embodiment, Rio is selected from:
Figure imgf000027_0002
wherein T1, T2, T3, T4, T6, T7, Tg, T9 and T10 are as defined above.
In an even more preferred embodiment, Rio is:
Figure imgf000028_0001
wherein one, two or three of of T1, T2, T3 and T4 are N and the remainder are CH. In another preferred embodiment, R1O is selected from:
Figure imgf000028_0002
wherein one of T6, T7, T8, T9 and T10 is N and the remainder are CH.
In one preferred embodiment R9 is selected from:
Figure imgf000028_0003
Figure imgf000029_0001
wherein aryl, X18, X19, X23, X25 are as defined above and;
X2 and X3 are each independently selected from: CH5 CMe and C-F;
X30 is selected from:
CH2, CH2CH2, NH, NMe and O;
X31 is selected from:
CH2, NH and NMe; or when X30 is NH or NMe then X31 may additionally be ) C=O;
and R14 is selected from C(O)Me, C(O)(cyclopropyl), C(O)NH2, C(O)NH(Me), C(O)N(Me)2, C(O)NH(cyclopropyI), C(O)O(Me), C(O)O(cyclopropyl), S(O)2(Me), S(O)2(cyclopropyl), S(O)2NH2, S(O)2NH(Me), S(O)2N(Me)2, S(O)2NH(cyclopropyl) and S(O)2Ph.
In a yet even more preferred embodiment R is selected from:
Figure imgf000030_0001
Figure imgf000030_0002
Figure imgf000030_0003
Figure imgf000030_0004
wherein X18 is as defined above.
In one highly preferred embodiment, R9 is selected from:
Figure imgf000031_0002
Figure imgf000031_0003
Figure imgf000031_0004
In one highly preferred embodiment, the compound of the invention is selected from the following:
N-((S)-2-((3aSβSβaS)-6-cUoτo-3-oxodϊhydτo-2Η.-faxo[3,2-b]pyτrol-4(5H,6H,6aH)- y I)- 1 -(( 1 r,45)-4-methylcyclohexyl)-2-oxoethyl)-3 -( 1 H-tetrazol- 1 -yl)benzamide
iV-((S)-2-((3a5,6^6a^-6-cMoro-3-oxodmydro-2H-furo[3.2-b]pyrrol-4(5H,6H;6αH)- yl)-l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-3-(lH-imidazol-l-yl)benzamide N-((5)-2-((3a5,6S56aS)-6-chloro-3-oxodihydro-2H-furo[3,2-&]pyrrol-4(5H;6H,6αH)- yl)- 1 -(( 1 r,4S)-4-methylcyclohexyl)-2-oxoethyl)-3 -(4H- 1 ,2,4-triazol-4-yl)benzamide
N-((^-2-((3a^,65,6a^-6-cMoro-3-oxodihydro-2H-furo[3,2-o]pyrrol-4(5if.6H56a/i)- yl)- 1 -((1 r,4S)-4-methylcyclohexyl)-2-oxoethyl)-3 -(I Η-pyrazol- 1 -yl)benzamide
JV-((5)-2-((3a^,6S,6a^-6-cMoro-3-oxodihydro-2Η-furo[352-&]pyrrol-4(5/f,6H;6αH)- yl)-l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-3-(4Η-l,2,4-triazol-4-yl)benzamide
iV-((5)-2-((3a^56536aS)-6-cMoro-3-oxodihyd^o-2H-furo[3,2-ά]pyπ-ol-4(5H,6H56αH)- yl)-l-((lr.4S)-4-methylcyclohexyl)-2-oxoethyl)nicotinamide
N-((5)-2-((3aiS',61S;6a^-6-cWoro-3-oxodmydro-2H-furo[3,2-6]pyn-oI-4(5H56/f,6aH)- yl)- 1 -((lr^^^-metiiylcyclohexy^^-oxoethy^isonicotinamide
N-((5)-2-((3a5,6556a^-6-cMoro-3-oxodihydro-2H-furo[3,2-6]pyrrol-4(5H,6H;6aH)- yl)- 1 -(( 1 r^^^-methylcyclohexy^^-oxoethy^furan^-carboxamide
N-((^-2-((3a5,65'56a^-6-cMoro-3-oxodihydro-2Η-furo[332-6]pyrrol-4(5H;6H,6αiϊ)- yl)-l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-3-(pyridin-3-yl)benzainide
iV-((5)-2-((3aSr 565r,6a^-6-cMoro-3-oxodihydro-2H-flu:o[352-6]pyrrol-4(5H,6H56αH)- yl)-l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-lH-benzo[c(I[l,253]triazole-6- carboxamide
N-((6)-2-((3a^,65,6a^-6-cMoro-3-oxodihydro-2H-fiu-o[3,2-έ]pyrrol-4(5H,6H;6aH)- yl)- 1 -(( 1 r,4iS)-4-methylcyclohexyl)-2-oxoethyl)benzo [cfJthiazole-6-carboxamide
N-((5)-2-((3a^565,6a^-6-cMoro-3-oxodmydro-2H-ftu:o[352-&]pyτrol-4(5H,6H56αH)- yl)-l-((lr:>45)-4-methylcyclob.exyl)-2-oxoethyl)benzo[c][l,255]oxadiazole-5- carboxamide N-((5)-2-((3a5565,6a^-6^cωoro-3-oxodihydro-2H-furo[3,2-ό]pyiτol-4(5H;6H" 56flH)- yl)-l-((lr,4S)-4-methylcyclohexyl)-2-oxoethyl)-lΗ-indole-5-carboxamide
N-((5)-2-((3aS',65,6a5)-6-cmoro-3-oxodmydro-2H-furo[3,2-ό]pyrrol-4(5H;6H,6flH)- yl)-l-((lr,4S)-4-methylcyclohexyl)-2-oxoetlαyl)-6-hydroxypicolinainide
AL((S)-2-((3aS,6S,6aS)-6-chloro-3-oxodihydro-2H-furo[3,2-&]pyrrol-4(5H;6H,6flH)- yl)- 1 -(( 1 r,4S)-4-methylcyclohexyl)-2-oxoethyl)benzo [d\ [ 1 ,3 ]dioxole-5-carboxamide
N-((5)-2-((3α^65,6αS)-6-cmoro-3-oxodihydro-2H-furo[3,2-έ]pyirol-4(5H,6/f,6βH)- yl)-l-((lr,4S)-4-methylcyclohexyl)-2-oxoethyl)-2,3-dioxo-l,2,354- tetrahydroquinoxaline-6-carboxamide
N-((S)-2φaSβSβaS)-6-chloio-3-oxodihyάio-2H-&xro[3,2-b]pyτrol-4(5H,6HβaH)- yl)- 1 -((1 r,4,S)-4-methylcyclohexyl)-2-oxoethyl)-2-oxo- 1 ,2,3 ,4-tetrahydroquinoline-6- carboxamide
N<(5)-2-((3α5'565,6α^-6-cMoro-3-oxodihydro-2H-furo[352-ό]pyrrol-4(5H,6H;6αH)- yl)-l-((lr,4S)-4-methylcyclohexyl)~2-oxoethyl)-3-oxo-3,4-diliydro-2H- benzo[6][l,4]oxazine-7-carboxainide
N-((5)-2-((3α5',651,6α5)-6-cMoro-3-oxodihydro-2H-faro[3,2-έ]pyτrol-4(5/f,6H;6flH)- yl)- 1 -((1 r,45)-4-methylcyclohexyl)-2-oxoethyl)-2-oxo-2,3 ,4,5-tetrahydro- IH- benzo[6]azepine-7-carboxamide
N-((^-2-((3a5,65,6^5)-6-cUoro-3-oxodmydro-2H-foro[332-Z>]pyiTol-4(5H,6H,6«H)- yl)- 1 -((1 r,45)-4-metb.ylcyclohexyl)-2-oxoetiiyl)-4-(niethylsulfonainido)benzainide
N-((5)-2-((3a5',65,6a^-6-cMoro-3-oxodihydro-2Η-furo[3,2-b]pyrrol-4(5H,6H,6αH)- yl)-l-(4,4-dimethylcyclohexyl)-2-oxoethyl)-3-(lΗ-tetrazol-l-yl)benzamide N-((5)-2-((3a5,6556a^-6-chloro-3-oxodihydro-2H-furo[3,2-6]pyrrol-4(5H,6H,6aH)- yl)-l-(4,4-dime1iιylcyclohexyl)-2-oxoethyl)-3-(lH-iinidazol-l-yl)benzainide
N-((5)-2-((3aS',65'56a^-6-cMoro-3-oxodihydro-2H-furo[3,2-o]pyxrol-4(5H56H;6aH)- yl)-l-(4,4-dimethylcyclohexyl)-2-oxoethyl)-3-(4H-l,2,4-triazol-4-yl)benzajtnide
N-((S)-2-((3a5565,6aS)-6-cMoro-3-oxoduiydro-2H-furo[3,2-Z)]pyrrol-4(5H,6H;6flH)- yl)-l-(4,4-dimethylcyclohexyl)-2-oxoethyl)-3-(lΗ-pyrazol-l-yl)benzamide
N-((5)-2-((3a^,65'56a5)-6-cWoro-3-oxodihydro-2H-furo[3,2-Z)]pyrrol-4(5H,6/f,6aH)- yl)-l-(4,4-dimethylcyclohexyl)-2-oxoethyl)-3-(4Η-l,2,4-1xiazol-4-yl)benzamide
N-((^-2<(3a5565'56a^-6-cmoro-3-oxodihydro-2H-furo[3,2-ό]pyrrol-4(5//,6H,6αi7)- yl)- 1 ^^-dimethylcyclohexy^^-oxoethy^nicotinamide
N-((5)-2-((3a5,6^6a^-6-cMoro-3-oxodihydro-2H-furo[352-6]pyrrol-4(5H;6H56flH)- yl)- 1 -(4,4-diinethylcycloliexyl)-2-oxoethyl)isonicotinainide
N-((5)-2<(3a5,6S'56a^-6-chloro-3-oxodihydro-2Η-furo[3,2-δ]pyrrol-4(5H,6iϊ;6αH)- yl)- 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)furan-2-carboxainide
^-((^^-((S^ό^όa^-β-cmoro-S-oxodihycko^Η-furoP^-^pyrrol^CSH.ό^όfl/f)- yl)-l-(4,4-dimethylcyclohexyl)-2-oxoethyl)-3-(pyridin-3-yl)benzamide
N-((5)-2-((3a5',65'56a^-6-cωoro-3-oxodihydro-2Η-furo[3,2-έ]pyiτol-4(5H36H56α/f)- yl)-l-(4,4-dimethylcyclohexyl)-2-oxoethyl)-lΗ-bertzo[d][l,2,3]tτiazole-6-carboxamide
N-((^-2-((3a^565,6a5)-6-cmoro-3-oxodihydro-2H-furo[352-δ]pyrrol-4(5H56/f,6αH)- yl)-l-(4,4-dimethylcycloliexyl)-2-oxoetliyl)benzo[J]thiazole-6-carboxainide iV^((5)--2-((3aS,6S,6aJS)-6-cmoro-3-oxodihydro-2H-furo[352-δ]pyrrol-4(5H;6H56αH)- yl)-l-(4,4-dimethylcyclohexyl)-2-oxoethyl)benzo[c][l,2,5]oxadiazole-5-carboxamide
N-((5)-2-((3a^565,6aS)-6-cWoro-3-oxodihydro-2H-furo[3,2-o]pyrrol-4(5F,6H,6flf0- yl)- 1 -(4,4-dimethylcyclohexyl)-2-oxoeth.yl)- 1 H-indole-5-carboxamide
N-((S)~2φaSβSβaS)-6-άύoτo-3-oxodihydτo-2Α-faro[3,2-b]pynol-4(5H,6H,6aH)- yl)- 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)-6-hydroxypicolinamide
iV^((5)-2-((3aS'565I,6a^-6-cMoro-3-oxodmydro-2H-ftiro[3,2-δ]pyrrol-4(5i/,6H;6βH)- yl)- 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)benzo \d\ [ 1 ,3] dioxole-5 -carboxamide
N-((5)-2-((3α5,65',6α5)-6-cmoro-3-oxodihydro-2H-furo[3,2-ό]pyπ:ol-4(5i7,6H;6αH)- yl)-l-(4,4-dimetiiylcyclob.exyl)-2-oxoetliyl)-233-dioxo-l,2J3,4-tetrahydroquinoxaline-6- carboxamide
N-((5)-2-((3fl5,65,6α5)-6-cMoro-3-oxodihydro-2//-foro[3,2-έ]pyrrol-4(5i7,6//56β/i)- yl)- 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)-2-oxo- 1 ,2,3 ,4-tetrahydroquin.oline-6- carboxamide
N-((5)-2-((3β^6556αS)-6-cmoro-3-oxodihydro-2H-føo[3,2-&]p)ττol-4(5H,6H,6ΛH)- yl)-l-(4,4-dimeth.ylcyclohexyl)-2-oxoethyl)-3-oxo-3,4-diliydro-2//- benzo[ό][l ,4]oxazine-7-carboxamide
N-((5)-2-((3flS'56556α5)-6-cMoro-3-oxodihydro-2H-furo[3,2-δ]pyiτol-4(5H56H56αH)- yl)-l-(4,4-dimetb.ylcyclohexyl)-2-oxoethyl)-2-oxo-2,354,5-tetrahydro-lH- benzo [Z>]azepine-7-carboxamide
N-((5)-2-((3^,6^6α5)-6-cωoro-3-oxodihydro-2H-furo[3,2-6]pyiτol-4(5H,6H,6αH)- yl)- 1 -(4,4-dimethylcyclohexyl )-2-oxoetibLyl)-4-(methylsulfonamido)benzamide In one particularly preferred embodiment, the compound of the invention is selected from Examples 1 — 22 described hereinbelow.
Even more preferably, the compound of the invention is selected from Examples 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 described hereinbelow.
PHARMACEUTICAL COMPOSITIONS
A further aspect of the invention relates to a pharmaceutical composition comprising a compound of the invention admixed with one or more pharmaceutically acceptable diluents, excipients or carriers. Other active materials may also be present, as may be considered appropriate or advisable for the disease or condition being treated or prevented.
Even though the compounds of the present invention (including their pharmaceutically acceptable salts, esters and pharmaceutically acceptable solvates) can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the "Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and PJ Weller. 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.
Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and. the like. Examples of suitable diluents include ethanol, glycerol and water.
The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
According to a further aspect of the invention, there is provided a process for the preparation of a pharmaceutical or veterinary composition as described above, the process comprising bringing the active compound(s) into association with the carrier, for example by admixture.
In general, 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.
SALTS/ESTERS The compounds of the invention can be present as salts or esters, in particular pharmaceutically and veterinarily acceptable salts or esters.
Pharmaceutically acceptable salts of the compounds of the invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g. hydrohalic acids such as hydrochloride, hydrobromide and hydroiodide, sulphuric acid, phosphoric acid sulphate, bisulphate, hemisulphate, thiocyanate, persulphate and sulphonic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C1- C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Salts which are not pharmaceutically or veterinarily acceptable may still be valuable as intermediates.
Preferred salts include, for example, 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, camphorsulphonate, 2-naphthalenesulphonate, benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate; and inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and sulphonic acids.
Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterifϊed. Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (Ci-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).
ENANTIOMERS/TAUTOMERS
In all aspects of the present invention previously discussed, the invention includes, where appropriate all enantiomers, diastereoisomers and tautomers of the compounds of the invention. The person skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.
Enantiomers are characterised by the absolute configuration of their chiral centres and described by the R- and ^-sequencing rules of Cahn, Ingold and Prelog. Such conventions are well known in the art (e.g. see 'Advanced Organic Chemistry', 3rd edition, ed. March, J., John Wiley and Sons, New York, 1985). 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.
STEREO AND GEOMETRIC ISOMERS
Some of the compounds of the invention may exist as stereoisomers and/or geometric isomers — e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of all the individual stereoisomers and geometric isomers of those inhibitor agents, and mixtures thereof. The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).
The present invention also includes all suitable isotopic variations of the agent or a pharmaceutically acceptable salt thereof. An isotopic variation of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2H, 3H, 13C, 14C, 15N, 170, 180, 31P, 32P5 35S, 18F and 36Cl3 respectively. Certain isotopic variations of the agent and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as 3H or 14C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3H5 and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. For example, the invention includes compounds of general formula (I) where any hydrogen atom has been replaced by a deuterium atom. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.
PRODRUGS The invention further includes the compounds of the present invention in prodrug form, i.e. covalently bonded compounds which release the active parent drug according to general formula (I) in vivo. Such prodrugs are generally compounds of the invention wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject. Reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo. Examples of such modifications include ester (for example, any of those described above), wherein the reversion may be carried out be an esterase etc. Other such systems will be well known to those skilled in the art.
A prodrug may for example constitute a ketal or hemiketal derivative of the exocyclic ketone functionality present in the 6-(»S)-chlorotetrahydrofuro[3,2-Z»]pyrrol-3-one scaffold.
SOLVATES The present invention also includes solvate forms of the compounds of the present invention. The terms used in the claims encompass these forms.
POLYMORPHS
The invention further relates to the compounds of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation form the solvents used in the synthetic preparation of such compounds. ASSAYS
Another aspect of the invention relates to the use of a compound of the invention as defined hereinabove in an assay for identifying further candidate compounds that influence the activity of a cysteine proteinase.
Preferably, the assay is capable of identifying candidate compounds that are capable of inhibiting one or more CACl cysteine proteinases.
More preferably, the assay is a competitive binding assay.
Preferably, the candidate compound is generated by conventional SAR modification of a compound of the invention.
As used herein, the term "conventional SAR modification" refers to standard methods known in the art for varying a given compound by way of chemical derivatisation.
Thus, in one aspect, the identified compound may act as a model (for example, a template) for the development of other compounds. The compounds employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The abolition of activity or the formation of binding complexes between the compound and the agent being tested may be measured.
The assay of the present invention may be a screen, whereby a number of agents are tested, hi one aspect, the assay method of the present invention is a high through-put screen.
This invention also contemplates the use of competitive drug screening assays in which neutralising antibodies capable of binding a compound specifically compete with a test compound for binding to a compound. Another technique for screening provides for high throughput screening (HTS) of agents having suitable binding affinity to the substances and is based upon the method described in detail in WO 84/03564.
It is expected that the assay methods of the present invention will be suitable for both small and large-scale screening of test compounds as well as in quantitative assays.
Preferably, the competitive binding assay comprises contacting a compound of the invention with a cysteine proteinase in the presence of a known substrate of said enzyme and detecting any change in the interaction between said cysteine proteinase and said known substrate.
A further aspect of the invention provides a method of detecting the binding of a ligand to a cysteine proteinase, said method comprising the steps of: (i) contacting a ligand with cysteine proteinase in the presence of a known substrate of said enzyme; (ii) detecting any change in the interaction between said enzyme and said known substrate; and wherein said ligand is a compound of the invention.
One aspect of the invention relates to a process comprising the steps of:
(a) performing an assay method described hereinabove;
(b) identifying one or more ligands capable of binding to a ligand binding domain; and (c) preparing a quantity of said one or more ligands.
Another aspect of the invention provides a process comprising the steps of:
(a) performing an assay method described hereinabove;
(b) identifying one or more ligands capable of binding to a ligand binding domain; and
(c) preparing a pharmaceutical composition comprising said one or more ligands. Another aspect of the invention provides a process comprising the steps of:
(a) performing an assay method described hereinabove;
(b) identifying one or more ligands capable of binding to a ligand binding domain; (c) modifying said one or more ligands capable of binding to a ligand binding domain;
(d) performing the assay method described hereinabove;
(e) optionally preparing a pharmaceutical composition comprising said one or more ligands.
The invention also relates to a ligand identified by the method described hereinabove.
Yet another aspect of the invention relates to a pharmaceutical composition comprising a ligand identified by the method described hereinabove.
Another aspect of the invention relates to the use of a ligand identified by the method described hereinabove in the preparation of a pharmaceutical composition for use in the treatment of one or more disorders selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain.
The above methods may be used to screen for a ligand useful as an inhibitor of one or more cysteine proteinases.
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 proteinase 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'. According to a further aspect of the invention, there is provided a method of validating a known or putative cysteine proteinase as a therapeutic target, the method comprising:
(a) assessing the in vitro binding of a compound as described above to an isolated known or putative cysteine proteinase, providing a measure of potency; and optionally, one or more of the steps of:
(b) assessing the binding of the compound to closely related homologous proteinases of the target and general house-keeping proteinases (e.g. trypsin) to provide a measure of selectivity;
(c) monitoring a cell-based functional marker of a particular cysteine proteinase activity, in the presence of the compound; and (d) monitoring an animal model-based functional marker of a particular cysteine proteinase activity in the presence of the compound.
The invention therefore provides a method of validating a known or putative cysteine proteinase as a therapeutic target. Differing approaches and levels of complexity are appropriate to the effective inhibition and 'validation' of a particular target. In the first instance, the method comprises assessing the in vitro binding of a compound of general formula (I) to an isolated known or putative cysteine proteinase, providing a measure of 'potency'. An additional assessment of the binding of a compound of general formula (T) to closely related homologous proteinases of the target and general house-keeping proteinases (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 proteinase activity, in the presence of a compound of general formula (T). For example, an 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 proteinase 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 proteinase inhibitors (Shi, G-P., et al, Immunity, 10, 197-206, 1999). When investigating viral or bacterial infections such 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 proteinase inhibitors. A third level of complexity may be assessed by monitoring an animal model-based functional marker of a particular cysteine proteinase activity, in the presence of a compound of general formula (I). For example, murine models of Leishmania infection, P. vinckei infection, malaria (inhibition of falcipain) and T. cruzi infection (cruzipain), indicate that inhibition of cysteine proteinases that play a key role in pathogen propagation is effective in arresting disease symptoms, 'validating' said targets.
The invention therefore extends to the use of a compound of general formula (I) in the validation of a known or putative cysteine proteinase as a therapeutic target.
BIOLOGICAL ACTIVITY
The compounds of the present invention are structurally distinct from the prior art (e.g. WO-A-02057270; Quibell, M. et. al, Bioorg. Med. Chem. 13, 609-625, 2005; Quibell M5 et al Bioorg. Med. Chem, 12, 5689-5710, 2004; WO-A-05066180) in that a 6-(S)- chloro substituent and a 4-substituted cyclohexylglycyl moiety form an integral part. This combination of features provides compounds with surprisingly high efficacies for human cathepsin S and high in vitro selectivity versus other mammalian cathepsins, both of which are important properties required for development of an efficacious therapeutic. If either of these intrinsic moieties is removed from compounds of formula I, then a surprisingly large loss in potency and I or a, significant loss in selectivity is observed. Indeed, all of the compounds of the present invention prepared to date exhibit potent and selective in vitro inhibition for human cathepsin S with Ki < 25 nM. In contrast, the majority of the eighty-two prior art compounds detailed in WO-A- 02057270 are significantly less potent against human cathepsin S than the compounds of the present invention, and hi the majority of examples, greater than 100-fold less potent. The closest prior art, compound (38) (see WO- A-02057270, pg 151), exhibits a 111- fold improvement in in vitro potency against human cathepsin S upon addition of a 6- (<S)~chloro substituent and substitution of the (£)-cyclohexylalanyl moiety with trans- (S)-(4(5)-methylcyclohexyl)glycyl (EXAMPLE 1). The surprising synergistic relationship between these two intrinsic changes is clearly seen when comparing prior art compound (38) with novel compounds (1-6) and EXAMPLE 1. Prior art compound (38) exhibits a 2.5-fold improvement in in vitro potency against human cathepsin S upon substitution of the (5)-cyclohexylalanyl moiety with (5)-(cyclohexyl)glycyl (Compound 1) but provides an inhibitor that has little selectivity verses cathepsin K. Further addition of a 6-(5)-fluoro substituent to Compound 1 provides a 3.8-fold improvement in in vitro potency against human cathepsin S (Compound 2) but again provides an inhibitor that has little selectivity verses cathepsin K. Alternatively, addition of a 6-(<S)-chloro substituent to Compound 1 provides a 27 -fold improvement in in vitro potency against human cathepsin S (Compound 3) but again provides an inhibitor that has only a modest 4-fold selectivity verses cathepsin K. However, whilst substitution of the (S)-cyclohexylglycyl moiety of Compound 1 with trans-(S)-(4(S)- methylcyclohexyl)glycyl (Compound 4) only provides a modest 3 -fold improvement in in vitro potency against human cathepsin S, the selectivity verses other mammalian cathepsins, in particular cathepsin K, is dramatically increased to > 65-fold, hi contrast, addition of a 6-(5)-chloro substituent to Compound 1 and substitution of the (^-cyclohexylglycyl moiety with trα«i'-(5)-(4(5)-methylcyclohexyl)glycyl (EXAMPLE 1) not only provides a 45-fold improvement in potency but also gives an inhibitor with high selectivity against other mammalian cathepsins. The importance of both of these modifications for compounds of formula I is clearly seen when comparing EXAMPLE 1 with Compounds 5 and 6. Compound 5 which contains the 6- (»S)-fluoro substituent in place of the 6-(iS)-chloro substituent of EXAMPLE 1 is 5.6- fold less potent verses cathepsin S, but retains the high selectivity due to presence of the /rarø,y-(S)-(4(<S)-methylcyclohexyl)glycyl moiety. Compound 6 which contains the 6-(i?)-chloro substituent in place of the 6-(5)-chloro substituent of EXAMPLE 1 is 15- fold less potent verses cathepsin S, but again retains the high selectivity due to presence of the -7-α«,y-()S)-(4(JS)-methylcyclohexyl)glycyl moiety.
By way of further comparison, consider novel compounds (7-12) and EXAMPLES 2 and 3. Addition of a 6-(5}-fluoro sύbstituent to Compound 7 provides a 3.4-fold improvement in in vitro potency against human cathepsin S (Compound 8), an inhibitor with a modest 6.8-fold selectivity verses cathepsin K. Alternatively, addition of a 6- (S)-chloro substituent to Compound 7 provides a 47-fold improvement in in vitro potency against human cathepsin S (Compound 9), an inhibitor with a modest 7.5-foId selectivity verses cathepsin K. However, whilst substitution of the (S)- cyclohexylglycyl moiety of Compound 7 with trørøs-(S)-(4(S)-memylcyclohexyl)glycyl (Compound 10) only provides a modest 2.2-fold improvement in in vitro potency against human cathepsin S, the selectivity verses other mammalian cathepsins, in particular cathepsin K, is dramatically increased to > 125-fold. La contrast, addition of a 6-(iS)-chloro substituent to Compound 7 and substitution of the (S^cyclohexylglycyl moiety with a /rαw-(S)-(4(5)-methylcyclohexyl)glycyl (EXAMPLE 2) or (S)-(4,4- dimethylcyclohexyl)glycyl (EXAMPLE 3) not only provides a significant improvement in potency verses cathepsin S (77-fold and 15-fold respectively) but also gives an inhibitor with high selectivity against other mammalian cathepsins. Again the importance of both of these modifications for compounds of formula I is clearly seen when comparing EXAMPLE 2 with Compounds 11 and 12. Compound 11 which contains the 6-(<S)-fluoro substituent in place of the 6-(«S)-chloro substituent of EXAMPLE 2 is 11 -fold less potent verses cathepsin S, but retains the high selectivity due to presence of the /rørø-(S)-(4(S)-memylcyclohexyi)grycyl moiety. Compound 12 which contains the 6-(ϋ!)-chloro substituent in place of the 6-(S)-chloro substituent of EXAMPLE 2 is 25 -fold less potent verses cathepsin S5 but again retains the high selectivity due to presence of the /rα/w-(ιS)-(4(S)-methylcyclohexyl)glycyl moiety.
Preferably, the compounds exhibit in vitro inhibition versus human cathepsin S with ICi < 10 nM, more preferably < 5 nM5 even more preferably < 2 nM and more preferably still < 1 nM. The compounds of the invention exhibit high selectivity against other mammalian cathepsins displaying little or no inhibitory activity for cathepsins K, L, B and V at 1 μM compound.
THERAPEUTIC USE Compounds of general formula (I) are useful for the in vivo treatment or prevention of diseases in which participation of a cysteine proteinase is implicated.
Preferably, the compound of general formula I is selective for cathepsin S. As used herein, the term "selective for cathepsin S" means that the inhibitor is selective for cathepsin S over one or more other mammalian CACl cysteinyl proteinases for example cathepsin K, cathepsin L, cathepsin F, cathepsin B and cathepsin V. Preferably, the inhibitor exhibits a selectivity ratio for cathepsin S over other mammalian CACl cysteinyl proteinases of greater than 2-fold, more preferably greater than 5-fold, more preferably greater than 10-fold, even more preferably greater than 25- fold, more preferably still, greater than 50-fold or 100-fold.
According to a further aspect of the invention, there is provided 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 proteinase.
According to a further aspect of the invention, there is provided the use of 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 proteinase.
Certain cysteine proteinases 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 proteinases have been implicated in various disease states, including but not limited to, infections by Pneumocystis carinii, Trypsanoma cruzi, Trypsanoma brucei brucei and Crithidia fusiculata; as well as in osteoporosis, osteoarthritis, rheumatoid arthritis, multiple sclerosis, chronic pain, autoimmunity, schistosomiasis, malaria, tumour metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy, and the like (see WO-A-9404172 and EP-A-0603873 and references cited therein). Additionally, a secreted bacterial cysteine proteinase from S. Aureus called staphylopain has been implicated as a bacterial virulence factor (Potempa, J., etal. J. Biol. Chem, 262(6), 2664-2667, 1998).
The invention 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 method of treatment or prevention of diseases caused by pathological levels of cysteine proteinases, particularly cysteine proteinases 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 proteinases are implicated, including infections by Pneumocystis carinii, Trypsanoma cruzi, Trypsanoma brucei, Leishmania mexicana, Clostridium histolyticum, Staphylococcus aureus, foot-and-mouth disease virus and Crithidia fiisiculata; as well as in osteoarthritis, rheumatoid arthritis, multiple sclerosis, chronic pain, autoimmunity, schistosomiasis, malaria, tumour metastasis, metachromatic leukodystrophy, muscular dystrophy, amytrophy.
Inhibitors of cathepsin S, particularly cathepsin S-specific compounds, are useful for the treatment of rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain. The compounds of the invention are particularly useful in the treatment of the above disorders.
Preferred features for each aspect of the invention are as for each other aspect mutatis mutandis. ADMINISTRATION
The pharmaceutical compositions of the present invention may be adapted for rectal, nasal, intrabronchial, topical (including buccal and sublingual), vaginal or parenteral
(including subcutaneous, intramuscular, intravenous, intraarterial and intradermal), intraperitoneal or intrathecal administration. Preferably the formulation is an orally administered formulation. The formulations may conveniently be presented in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose. By way of example, the formulations may be in the form of tablets and sustained release capsules, and may be prepared by any method well known in the art of pharmacy.
Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, gellules, drops, cachets, pills or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution, emulsion 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. Preferably, these compositions contain from 1 to 250 mg and more preferably from 10-100 mg, of active ingredient per dose.
For compositions for oral administration (e.g. tablets and capsules), 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, hydroxypropyhnethylcellulose, 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. 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.
Other formulations 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.
Other forms of administration comprise solutions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. Injectable forms typically contain between 10 - 1000 mg, preferably between 10 - 250 mg, of active ingredient per dose.
The pharmaceutical compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required. DOSAGE
A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
hi accordance with this invention, an effective amount of a compound of general formula (I) may be administered to inhibit the proteinase implicated with a particular condition or disease. Of course, this dosage amount will further be modified according to the type of administration of the compound. For example, to achieve an "effective amount" for acute therapy, parenteral administration of a compound of general formula (T) is preferred. An intravenous infusion of the compound hi 5% dextrose hi water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful. Typically, the parenteral dose will be about 0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg, hi a manner to maintain the concentration of drug in the plasma at a concentration effective to inhibit a cysteine proteinase. The compounds may be administered one to four tunes 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 hi 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 hemiketals, 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. Typically, 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. Preferably the oral dose would be about 0.5 to about 20 mg/kg.
No unacceptable toxicological effects are expected when compounds of the present invention are administered in accordance with the present invention. 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.
COMBINATIONS
In a particularly preferred embodiment, the one or more compounds of the invention are administered in combination with one or more other active agents, for example, existing drugs available on the market. In such cases, the compounds of the invention may be administered consecutively, simultaneously or sequentially with the one or more other active agents.
Drugs in general are more effective when used in combination. In particular, combination therapy is desirable in order to avoid an overlap of major toxicities, mechanism of action and resistance mechanism(s). Furthermore, it is also desirable to administer most drugs at their maximum tolerated doses with minimum time intervals between such doses. The major advantages of combining chemotherapeutic drugs are that it may promote additive or possible synergistic effects through biochemical interactions and also may decrease the emergence of resistance. Beneficial combinations may be suggested by studying the inhibitory activity of the test compounds with agents known or suspected of being valuable in the treatment of a particular disorder. This procedure can also be used to determine the order of administration of the agents, i.e. before, simultaneously, or after delivery. Such scheduling may be a feature of all the active agents identified herein.
SYNTHESIS
Synthesis of 5,5-Bicyclic Core One aspect of the invention relates to a process of preparing a compound of formula (I) as defined above, said process comprising oxidation of a compound of formula (II).
Figure imgf000055_0001
(H) (I)
Any suitable oxidising agent may be used to convert the secondary alcohol group of (II) into the corresponding ketone (I). Suitable oxidising agents will be familiar to the skilled artisan. By way of example, the oxidation may be carried out via a Dess-Martin periodinane reaction [Dess, D.B. et al, J. Org. Chem. 1983, 48, 4155; Dess, D.B. et al, J. Am. Chem. Soc. 1991, 113, 7277], or via a Swern oxidation [Mancuso, A. J. et al, J. Org. Chem. 1978, 43, 2480]. Alternatively, the oxidation can be carried out using SOs/pyridine/EtsN/DMSO [Parith, J. R. et al, J. Am. Chem. Soc. 1967, 5505; US 3,444,216, Parith, J. R. et al,], P2O5/DMSO or P2O5/Ac2O [Christensen, S. M. et al, Organic Process Research and Development, 2004, 8, 777]. Other alternative oxidation reagents include activated dimethyl sulphoxide [Mancuso, A. J., Swern, D. J., Synthesis, 1981, 165], pyridinium chlorochromate [Piatieatelli, G. et al, Sythesis, 1982,
245] and Jones' reagent [Vogel, A, L, Textbook of Organic Chemistry, 6th Edition].
More preferably, the process comprises treating a compound of formula (II) with Dess- Martin periodinane. Preferably, the reaction is carried out using dichloromethane as solvent.
In one preferred embodiment, the process of the invention comprises the step of converting a compound of formula (III) into a compound of formula (II) through standard amide bond formation between R9CONHCH(C6H9R3R4)COOH and the compound of formula (III; R5 = H) with a suitable carboxylic acid activating agent.
Figure imgf000056_0001
(III) (II)
where R5 is a protecting group or hydrogen.
In one preferred embodiment, protecting group R5 is selected from benzyloxycarbonyl, tert-butoxycarbonyl, fluoren-9-yhnethoxycarbonyl, 1 -(biphenyl-4-yl)- 1 - methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxylbenzyloxycarbonyl, p- methoxybenzyloxycarbonyl, /Miitrobenzyloxycarbonyl, allyloxycarbonyl and trichloroethoxycarbonyl.
More preferably, R5 is benzyloxycarbonyl, fert-butoxycarbonyl (Boc) or flouren-9- ylmethoxycarbonyl (Fmoc). In another preferred embodiment R5 is H.
In a more preferred embodiment the process of the invention comprises the step of converting a compound of formula (TV) into a compound of formula (III; R5 = H)
Figure imgf000057_0001
(rv) (πi) where Lg is a leaving group such, as tosylate or mesylate and R5 is as previously defined.
In an even more preferred embodiment the process of the invention comprises the step of converting a compound of formula (IVa; R5 = H) into a compound of formula (Ilia) or a compound of formula (IVb; R5 = Cbz) into a compound of formula (HIb)
Figure imgf000057_0002
Figure imgf000057_0003
For compounds of formulae (Ilia) and (Illb) the displacement of tosylate is typically performed using an excess of lithium chloride in DMF at 13O0C. Displacement proceeds with inversion of configuration.
In one preferred embodiment the process of the invention comprises the step of converting a compound of formula (V) into a compound of formula (IV)
Figure imgf000058_0001
(V) (IV)
More preferably the intra-molecular cyclisation of compound (V) is induced by removal of the protecting group R . Preferably, for this embodiment, R5 is benzyloxycarbonyl (Cbz), and the process comprises hydrogenating a compound of formula (V) in the presence of a palladium catalyst.
In one preferred embodiment the process of the invention comprises the step of converting a compound of formula (VI) into a compound of formula (V)
Figure imgf000058_0002
(VI) (V) (ami)
In one preferred embodiment, the oxidising agent is mCPBA.
In another preferred embodiment, the oxidising agent is a dioxirane. The use of dioxiranes as oxidising agents is well documented in the literature [see (a)
Hodgson, D. M. et al, Synlett, 310 (2002); (b) Adam, W. et al, Ace. Chem. Res. 22, 205, (1989); (c) Yang, D. et al, J. Org. Chem., 60, 3887, (1995); (d) Mello, R. et al, J. Org. Chem., 53, 3890, (1988); (e) Curci, R. et al, Pure & Appl. Chem., 67(5), 811 (1995); (f) Emmons, W. D. et al, J. Amer. Chem. Soc. 89, (1955)].
Preferably, the dioxirane is generated in situ by the reaction of KHS O5 with a ketone. However, the oxidation step can also be carried out using an isolated dioxirane, for example a stock solution of the dioxirane formed from acetone.
More preferably, the dioxirane is generated in situ using Oxone®, which is a commercially available oxidising agent containing KHSO5 as the active ingredient.
Thus, in one preferred embodiment, the claimed process involves the in situ epoxidation of a compound of formula (VI) using Oxone® (2KHSO5-KHSO4-K2SO4) and a ketone co-reactant.
As mentioned above, the active ingredient of Oxone® is potassium peroxymonosulfate, KHSO5 [CAS-RN 10058-23-8], commonly known as potassium monopersulfate, which is present as a component of a triple salt with the formula 2KHSO5-KHSO4-K2SO4 [potassium hydrogen peroxymonosulfate sulfate (5:3:2:2), CAS-RN 70693-62-8; commercially available from DuPont]. The oxidation potential of Oxone® is derived from its peracid chemistry; it is the first neutralization salt of peroxymonosulfuric acid H2SO5 (also known as Caro's acid). K+ O-S(=O)2(-OOH)
Potassium Monopersulfate
Under slightly basic conditions (pH 7.5-8.0), persulfate reacts with the ketone co- reactant to form a three membered cyclic peroxide (a dioxirane) in which both oxygens are bonded to the carbonyl carbon of the ketone. The cyclic peroxide so formed then epoxidises the compound of formula VI by syn specific oxygen transfer to the alkene bond.
Preferably, the ketone is of formula (XIX)
Figure imgf000060_0001
(XTX) wherein Raand Rb are each independently alkyl, aryl, haloalkyl or haloaryl.
Where Ra and/or Rb are alkyl, the alkyl group may be a straight chain or branched alkyl group. Preferably, the alkyl group is a C1-20 alkyl group, more preferably a C1-15, more preferably still a C1-I2 alkyl group, more preferably still, a C1-8 or C1-6 alkyl group, more preferably a C1-4 alkyl group. Particularly preferred alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-hutyl, pentyl and hexyl.
As used herein, the term "haloalkyl" refers to an alkyl group as described above in which one or more hydrogens are replaced by halo.
Where Ra and/or Rb are aryl, the aryl group is typically a Cβ-u aromatic group.
Preferred examples include phenyl and naphthyl etc.
As used herein, the term "haloaryl" refers to an aryl group as described above in which one or more hydrogens are replaced by halo.
By way of example, the reaction of KHSO5 (Oxone®) with a ketone of formula XVI would form a dioxirane of formula:
Figure imgf000060_0002
wherein Raand Rb are as defined above. More preferably, Raand Rb are each independently alkyl or haloalkyl.
In a highly preferred embodiment, at least one of Ra and Rb is a haloalkyl, more preferably, CF3 or CF2CF3.
In one preferred embodiment, Ra and R are each independently methyl or trifluoromethyl.
In one preferred embodiment of the invention, the ketone is selected from acetone and a 1,1,1 -trifmoroalkyl ketone.
In a more preferred embodiment of the invention, the trifluoroalkyl ketone is 1,1,1- trifluoroacetone or l,l,l-trifluoro-2-butanone, more preferably l,l,l-trifmoro-2- butanone.
In one preferred embodiment the process of the invention comprises the step of converting a compound of formula (VII) into a compound of formula (VI)
Figure imgf000061_0001
(VII) (VI)
Preferably the process comprises treating a compound of formula (VII) with tosyl chloride in pyridine. Alternatively the process comprises treating a compound of formula (VII) with tosyl chloride in dichloromethane and triethylamine.
In one preferred embodiment the process of the invention comprises the step of converting a compound of formula (VIH) into a compound of formula (VII)
Figure imgf000062_0001
(VIII) (VII)
where W is halogen or tosyl. Preferably, this step comprises the steps of:
(a) reacting a compound of formula (VIII)5 where W is halogen or OTs, with aqueous ammonia and alcohol; and
(b) converting the product formed in step (a) to a compound of formula (VII).
Preferably, steps (a) and (b) of the above process are a one-pot process.
In one particularly preferred embodiment, R5 is benzyloxycarbonyl, and step (b) comprises treating the mixture formed in step (a) with benzyloxycarbonyl chloride.
Preferably, W is I, Br or OTs, more preferably, Br or OTs, even more preferably OTs.
Preferably, the alcohol is isopropyl alcohol or ethanol.
In one preferred embodiment of the invention, said compound of formula VIII is prepared from a compound of formula TX
Figure imgf000062_0002
IX vπi
Preferably, the above process comprises treating said compound of formula DC with methyl lithium. More preferably, compound of formula DC is compound 47 and compound of formula
VIII is compound 14. Treatment of monobromotosylate 47 with zinc dust at room temperature in organic / aqueous mixtures (most preferably an isopropanol, tetrahydrofuran, water, ammonium chloride mixture) provides alcohol 14 respectively in high yield. Additionally, completion of the one-pot conversion gives alcohol VII and with defined stereochemistry and in high yield.
Figure imgf000063_0001
(47) (14) (VII)
Commencing from the commercially available sugar isosorbide, the present invention also provides facile preparation of monobromotosylate 47. One highly preferred preparation is shown below in Scheme 15
Figure imgf000063_0002
Isosorbide
Figure imgf000063_0003
(14) Scheme 15: (a) TsCl5 trieώylamine, DCM, 250C -» 5O0C5 2Oh under Ar; (b) LiBr5
DMSO, HO0C^ 12O0C, 1Oh under Ar; (c) Zn5 1PrOH5 THF, H2O, NH4Cl, RT, 16h; (d) (i) NH4OH5 NH3 in 1PrOH, 750C5 16h; (ii) Cbz-Cl, Na2CO3, dioxane, water.
Isosorbide (43) is converted to the di-tosylate (42) which is obtained following recrystallisation from methanol in 97% yield. Mono-bromination is effected by 2.5eq lithium bromide in DMSO (or DMF) with temperature control 11O0C-^ 12O0C. The product bromide is isolated following extractive work-up and purification either by column chromatography (74%) or attractive for large scale by recrystallisation from methanol giving a first crop of 55% plus mother liquors containing good quality material that may be pooled from batch runs and purified later. Thus, preparation of monobromotosylate (47) with defined stereochemistry by methods in Scheme 15 is attractive for large scale applications. Treatment of monobromotosylate (47) with zinc dust at room temperature in organic / aqueous mixtures (most preferably an isopropanol, tetrahydrofuran, water, ammonium chloride mixture) provides alcohol (14) which is derivatised as the Cbz compound (18) through one pot conversion. In one highly preferred embodiment of the invention, the 6-Cl-555-bicylic core is prepared in accordance with the steps set forth in Scheme 1 below:
The alcohol functionaUty of (18) may be derivatised as the pαrø-toluene sulphonate (Ts) giving (i?)-2-(benzyloxycarbonylamino)-l-((5)-2,5-dihydroiuran-2-yl)ethyl 4- methylbenzenesulfonate (32b) which proceeds through the anti-epoxide (R)-2- (benzyloxycarbonylamino)-l-((15', 2S5 5S)-3,6-dioxabicyclo[3.1.0]hexan-2-yl)ethyl 4- methylbenzenesulphonate (33b). Hydrogenation of tosylate (33b) provides free amine that undergoes intramolecular cyclisation to provide intermediate (74). Intermediate (74) undergoes displacement with an excess of lithium chloride in DMF at 13O0C, to give the 6-chloro analogue with inversion of configuration. Urethane protection of the secondary amine of the bicyclic intermediate (69) followed by oxidation to ketone provides intermediate (71) that is particularly useful for solid phase synthesis of compounds of general formula I. Advantageously, the epoxidation to give the desired αrctz-epoxide is directed by the presence of the tosylate group.
Figure imgf000065_0001
Scheme 1: (a) TsCl5 pyridine: (b) (i) mCVBA, DCM or (ii) OXONE®, NaHCO3, 1,1,1- trifmoroacetone, CH3CN3 H2O, Na2-EDTA O 0C or (iii) 30% H2O2, CH3CN, MeOH5 NaHCO3; (c) Pd-C5 H2, ethanol; (d) LiCl, DMF; 13O0C; (e) Cbz-Cl, Na2CO3, dioxane, H2O; (f) Fmoc-Cl, Na2CO3, dioxane, H2O; (g) Dess-Martin periodinane, anhydrous DCM, RT.
Alternatively, intermediate tosylate (74) may be Cbz protected to give protected analogue (34b) that may undergo inversion to chloride (68) through treatment with LiBr in DMF at typically 130 0C (Scheme 1).
Preparation of Novel Aminoacids
The novel 4-substituted cyelohexylglycine aminoacids that are an intrinsic feature of compounds of formula I may be prepared following adaptation of a variety of known general literature syntheses of amiαoacids. In one such method, a 4-substituted cyclohexane acetic acid (e.g. trørø-4-methylcyclohexane acetic acid CAS 7132-93-6) is converted to the novel chiral aminoacid (e.g. (£)-2-(fer^butoxycarbonylamino)-2-((l/*, 4S)-4-methylcyclohexyl)acetic acid (130)) following Scheme 18 (general method is detailed in WO-A-98017626 (pg 49)).
Figure imgf000066_0001
Scheme 18. (a) (i) Pivaloyl chloride, THF, triethylamine; (ii) (S)-4-benzyl-2- oxazolidinone (CAS 90719-32-7), n-BuLi, hexanes; (b) (i) KHMDS5 THF, toluene, N2, -780C; (ii) Trisyl azide, THF; (c) PdVC, H2, DMF, Boc2O; (d) 30% H2O2, LiOH, THF, H2O.
For example, commercially available trørø-(4-methylcyclohexyl)acetic acid (131) (CAS 7132-93-6; ABCR GmbH AB168553; Shanghai FWD Chemicals Ltd K7354) is converted into the Evans auxiliary (127) following the general methods detailed in WO98017626 (pg 49). Asymmetric addition of azide is then conducted by deprotonation of (127) and reaction with trisyl azide. Reduction of azide (128) and concomitant Boc amino protection following the general methods detailed in US5128448 provides intermediate (129). Finally, hydrolysis of the auxiliary is conducted with hydrogen peroxide and lithium hydroxide following the general methods detailed in US5128448. The final product (130) is obtained following simple aqueous extraction.
Alternative 4-substituted cyclohexane acetic acids may be used following Scheme 18 to provide alternative analogues of compounds of formula I. For example, trans-(4- ethylcyclohexyl)acetic acid (CAS 125533-06-4) provides compounds of formula I where R3 = H and R4 = Et; frαrø-(4-methoxycyclohexyl)acetic acid (CAS 879877-61-9) provides compounds of formula I where R = H and R4 = OMe; 4- (trifluoromethylcyclohexyl)acetic acid (CAS 803736-46-1) provides compounds of formula I where R3 = H and R4 = CF3; trans-(4-n-propylcyclohexyi)acetic acid (CAS 71458-18-9) provides compounds of formula I where R3 = H and R4 = w-propyl; trans- (4-isopropylcyclohexyl)acetic acid (CAS 882658-76-6) provides compounds of formula I where R3 = H and R4 = isopropyl; (4,4-dimethylcyclohexyl)acetic acid (CAS 681448- 25-9, see WO-A-04037769, compound 39C, pg42) through the known aminoacid (S)-2- amino-2-(4,4-dimethylcyclohexyl)acetic acid (CAS 754178-25-1, see WO-A- 03062265, example XVII, pg 197), provides compounds of formula I where R3, R4 = Me; (4,4-difluorocyclohexyl)acetic acid (CAS 915030-40-9, see WO-A-06124490) through the known aminoacid 2-amino-2-(4,4-difiuorocyclohexyl)acetic acid (CAS 769169-46-2), provides compounds of formula I where R3, R4 = F.
Other novel 4-substituted cyclohexane acetic acids may readily be prepared from the corresponding 4-substituted cyclohexanone following the general methods detailed by Bennani, Y. L. et al (WO-A-04037769).
Figure imgf000067_0001
Scheme 19. (a) (i) Triethylphosphoacetate NaH5 THF; (b) Pd/C, H2, EtOH; (c) NaOH3
EtOH
Synthesis of Compounds of Formula (I)
To those skilled in the practices of organic chemistry, compounds of general formula (T) may be readily synthesised by a number of chemical strategies, performed either in solution or on the solid phase (see Atherton, E. and Sheppard, R. C. In 'Solid Phase Peptide Synthesis: A Practical Approach', Oxford University Press, Oxford, U.K. 1989, for a general review of solid phase synthesis principles), or a combination thereof.
Compounds of general formula (I) may be conveniently considered as a combination of three building blocks (Pl, P2 and P3) that respectively occupy the Sl, S2 and S3 binding sites of the protease (see Berger, A and Schechter, L, Philos. Trans. K Soc.
Lond. [Biol.], 257, 249-264, 1970 for a description of the designation of enzyme S- subsites and substrate P-subsites within enzyme-substrate or enzyme-inhibitor complexes). The notional concepts of Pl, P2 and P3 are used herein for convenience only and the above-mentioned compounds are intended to be within the scope of the invention regardless of binding mode.
Figure imgf000068_0001
A suitably protected and/or activated building block may then be prepared and subsequently chemically bonded (coupled) together with other building blocks to provide compounds of general formula (T).
Compounds of formula (T) may be prepared: (1) by the stepwise addition of P3 and P2 to the bicyclic 6-(5)-chlorotetrahydrofuro[3,2-ό]pyrrol-3-one core; or (2) by reaction of the bicyclic 6-(jS)-chlorotetrahydrofuro[3,2-5]pyπOl-3-one core with a P3-P2 prescursor molecule; or (3) by introducing the P3-P2 group prior to formation of the bicyclic 6- (S)-chlorotetrahydrofuro[3,2-ό]pyrrol-3-one core, i.e. prior to the oxidation step or prior to the intramolecular cyclisation step.
Thus, alternative orders of coupling of the building blocks are possible, for example P2 + Pl -* P2-P1 then addition of P3 -» P3-P2-P1 or P3 + P2 -> P3-P2 then addition to Pl -> P3-P2-P1. Within each of these combinations each of the Pl, P2 or P3 building blocks may contain additional alternative functionalities that are further transformed following coupling to give the final compound. For example the ketone functionality of the Pl building block may be protected as a ketal during coupling of building blocks and transformed to the final ketone by hydrolysis following completion of the coupling reactions. Alternatively, the ketone functionality of the Pl building block may be initially introduced via a lower oxidation state such as the corresponding alcohol and following completion of the coupling reactions be re-introduced by oxidation of the alcohol. Alternatively, the ketone functionality of the Pl building block may be protected through a semi-carbazone suitable for solid phase synthesis (e.g. see WO 02/057270 and references cited therein) and following completion of the coupling reactions released from the solid phase by acidolytic reaction.
The chemical bond formed by coupling of the building blocks is a secondary amide (P3-P2) or a tertiary amide (P2-P1) that is formed through reaction of an activated carboxylic acid with a primary and secondary amine respectively. Many methods are available for activation of a carboxylic acid prior to coupling to an amine and in principle, any of these methods may be used herein. Typical carboxylic acid activation methods are exemplified but not restricted to the azide method, mixed anhydride method (e.g. via isobutylchloroformate), carbodiimide methods (e.g. via dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1 -ethyl-3-(3'-dimetb.ylamino propyl)carbodiirnide), active ester method (e.g. via p-nitrophenyl ester, N- hydroxysuccinic imido ester, pentafluorophenyl ester), uronium method (e.g. via addition of HBTU5 PyBop, BOP)5 carbonyldiimidazole method or via pre-formation of acyl fluorides or acyl chlorides. IB some instances the coupling reaction may be enhanced by the addition of a further activation catalyst such as 1- hydroxybenzotriazole, or 4-dimemylaminopyridine. A general description of carboxylic acid activation techniques and the use of activation additives may be found in Bodanszky, M. 'Principles of Peptide Synthesis', 2nd rev. ed., Springer- Verlag, Berlin, 1993 and references cited therein.
The α-amino group of the P2 aminoacid building block is usually protected during coupling reactions to the Pl building block to avoid the formation of undesired self- condensation products. The art of α-amino protection is well known in peptide chemistry (e.g. see Bodanszky, M. 'Principles of Peptide Synthesis', 2nd rev. ed.5 Springer- Verlag, Berlin, 1993 and references cited therein) and example protection groups include, but are not limited to, 9-fluorenyhnethoxycarbonyl (Fmoc), tert- butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz)5 allyloxycarbonyl (Alloc) and trichloroethoxycarbonyl (Treoc). The Fmoc group is particularly well suited for solid phase syntheses (e.g. see Atherton, E.; Sheppard, R. C. in 'Solid Phase Peptide Synthesis A Practical Approach', IRL Press, Oxford, U.K., 1989) typically being removed by treatment with 20% v/v piperidine in dimethylformamide or 1% v/v 1,8- diazabicyclo[5.4.0]undec-7-ene in dimethylformamide. The Boc group is particularly well suited to solution phase syntheses typically being removed by treatment with trifluoroacetic acid based mixtures or HCl in dioxane or ethyl acetate. The Cbz group is also particularly well suited for solution phase syntheses typically being removed by catalytic hydrogenation with hydrogen and palladium catalysis or by treatment with HBr in acetic acid. Once the coupling sequence is complete, any protecting groups are removed in whatever manner is dictated by the choice of protecting groups (for a general description of protecting groups and their respective stabilities and methods of removal see Greene, T. W. and Wuts, P. G. M. 'Protective Groups in Organic Synthesis' John Wiley and Sons, New York, 1991 and references therein).
In the simplest example, the entire left hand portion of a compound of general formula (I) (i.e. P3-P2) as the carboxylic acid can be prepared in solution by traditional organic chemistry methods and coupled to ketone, alcohol or ketal intermediates such as compounds (lib), (lie) and (lid). Then oxidation of the alcohol intermediate (e.g. Dess- Martin periodinane in DCM) or acidolytic cleavage of the ketal intermediate provides compounds of general formula (I). The alcohol oxidation route is particularly useful when the compound of general formula (I) contains a substituent that is labile to trifluoroacetic acid, this being the final reagent used in each of the solid phase syntheses.
Figure imgf000071_0001
Examples of these different coupling tactics have been detailed previously (see (i) Quibell, M. et. al., Bioorg. Med. Chem. 13, 609-625, 2005. (ii) Wang, Y. et. al, Bioorg. Med. Chem Lett. 15, 1327-1331, 2005) and the optimum synthetic route is dependant upon the specific substituent combinations of the target compound of general formula (I).
In more detail, one preferred strategy for the synthesis of compounds of general formula (I) comprises:-
(a) Preparation of an appropriately functionalised and protected bicyclic ketone or bicyclic alcohol building block in solution; (b) Attachment of the building block (a) to the solid phase through a linker that is stable to the conditions of synthesis, but readily labile to cleavage at the end of a synthesis (see James, I. W., Tetrahedron, 55 (Report If 489), 4855-4946, 1999, for examples of the 'linker' function as applied to solid phase synthesis); (c) Solid phase organic chemistry (see Brown, R. D. J. Chem. Soc, Perkin Trans.1, 19, 3293-3320, 1998), to construct the remainder of the molecule;
(d) Compound cleavage from the solid phase into solution; and
(e) Cleavage work-up and compound analysis.
A second strategy for the synthesis of compounds of general formula (T) comprises:-
(a) Preparation of an appropriately functionalised and protected bicyclic intermediate building block in solution. Preferred protecting groups for solution phase chemistry are the 9-fluorenylmethoxycarbonyl (Fmoc), Nα-tert-butoxycarbonyl (Boc), Nα-benzyloxycarbonyl (Cbz) and Nα-allyloxycarbonyl group (Alloc).
(b) Standard organic chemistry methods for the conversion of building block obtained in step (a) towards compounds of general formula (I).
As mentioned above, in one preferred embodiment of the invention, compounds of formula (T) may be prepared using conventional solution phase chemistry, for example, as described in Quibell, M et al, Bioorg. Med. Chem, 13, 609-625, 2005 (see in particular, Schemes 3 and 4). The solution phase strategy is attractive in being able to generate larger quantities of preferred analogues, typically on a multi-gram to multi- kilogram scale.
In an alternative preferred embodiment of the invention, compounds of formula (I) may be prepared using conventional solid phase chemistry, for example, as described in Quibell M, et al Bioorg. Med. Chem, 12, 5689-5710, 2004, see in particular, Scheme 3 and Section 3.2, and references cited therein; and Bioorg. Med. Chem, 13, 609-625, 2005, see Scheme 5 and Section 2.2, and references cited therein). The solid phase strategy is attractive in being able to generate many thousands of analogues, typically on a 5-100mg scale, through established parallel synthesis methodologies (e.g. see (a)
Bastos, M.; Maeji, N. J.; Abeles, R. H. Proc. Natl. Acad. ScI USA, 92, 6738-6742, 1995).
The synthetic strategy is based on reversible anchorage of the ketone functionality via a hydrazide linker bond using general multipin techniques previously described in the art (Watts J. et al, Bioorg. Med. Chem. 12(11), 2903, 2004; Quibell M., et al, Bioorg. Med. Chem. 5689-5710, 2004; GrabowksaU. et al, J. Comb. Chem. 2000, 2(5), 475).
Compounds of formula (III; R = Fmoc) may be oxidised to the corresponding ketone (e.g. XVI, Scheme 3) and utilised in a solid phase synthesis of inhibitor molecules (I). 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. Soc, 114, 3156-3157, 1992). A suitable method amenable to the reversible linkage of an alkyl ketone functionality is through a combination of the previously described chemistries. The semicarbazide, A-
[[(hydrazinocarbonyl)amino]methyl]cyclohexane carboxylic acid, trifluoroacetate (Murphy, A. M., et al, J. Am. Chem. Soc, 114, 3156-3157, 1992), may be utilised as illustrated in Scheme 3, exemplified by linkage of the Fmoc protected 6-(S)- chlorotetrahy drofuro [3 ,2-6]pyrrol-3 -one (71).
Figure imgf000074_0001
General
Figure imgf000074_0002
(xvπi)
Scheme 3: (a) (71) in 90% EtOH / H2O / 1.5eq NaOAc / 4-[[(hydrazinocarbonyl)- amino]meth.yl]- cyclohexane carboxylic acid.trifluoroacetate, 2hr reflux, (b) 3eq construct (XVII) / 3eq HBTU / 3eq HOBt / 6eq NMM3 NH2-SOLID PHASE5 DMF3 RT, o/n. (c) 20% piperidine / DMF, 30mins. (d) Range of chemistries to introduce P3- P2 (e) TFA / H2O (95:5, v/v), RT3 2hr.
Construct (XVII) is prepared through reaction of the linker molecule and the 6-(S)- chlorotetrahydrofuro[3,2-έ]pyrrol-3-one (71) by refluxing in aqueous ethanol/sodium acetate. 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 (XVII)3 providing the loaded construct (XVIII). Loaded construct (XVIII) be reacted with a wide range of carboxylic acids available commercially or in the literature, to introduce the left-hand portion 'P3-P2'.
Preferred carboxylic acids for the introduction of the [R9-CO] synthon are known in the literature with the following representative examples; furan-2-carboxylic acid, 5- chlorofuran^-carboxylic acid, thiophene-2-carboxylic acid, 5-chlorothiophene-2- carboxylic acid, furan-3 -carboxylic acid, 5-chlorofuran-3 -carboxylic acid, thiophene-3- carboxylic acid, S-chlorothiophene-S-carboxylic acid, oxazole-2-carboxylic acid, oxazole-5-carboxylic acid, l,3,4-oxadiazole-2-carboxylic acid, thiazole-2-carboxylic acid, thiazole-5-carboxylic acid, l,3,4-thiadiazole-2-carboxylic acid, benzoic acid, 3- methylbenzoic acid, 3-chlorobenzoic acid, 3-fluorobenzoic acid, 3-bromobenzoic acid, 3-metboxybenzoic acid, 3-nitrobenzoic acid, 3,5-difluorobenzoic acid, nicotinic acid (CAS 59-67-6), 5-fluoronicotinic acid, 5-chloronicotinic acid, 5-methoxynicotinic acid, 5-nitraαicotinic acid, pyrimidine-5-carboxylic acid (CAS 4595-61-3), isonicotinic acid, 2-fluoroisonicotinic acid, 3-(lH-pyrrol-l-yl)benzoic acid (CAS 61471-45-2), 3-(1H- pyrazol-l-yl)benzoic acid (CAS 264264-33-7), 3-(lH-imidazol-l-yl)benzoic acid (CAS 108035-47-8), 3-(lH-l,2,3-triazol-l-yl)benzoic acid (CAS 335255-82-8), 3-(4H-l,2,4- triazol-4-yl)benzoic acid (CAS 335255-80-6), 3-(lH-l,2,4-triazol-l-yl)benzoic acid (CAS 167626-64-4), 3-(lH-tetrazol-l-yl)benzoic acid (CAS 204196-80-5), 3-(2H- l,2,3-triazol-2-yl)benzoic acid (CAS 90556-58-4), 3-(lH-imidazol-5-yl)benzoic acid (CAS 912569-71-2), 3-(lH-imidazol-2-yl)benzoic acid (CAS 391668-62-5), 3-(4H- l,2,4-triazol-3-yl)benzoic acid (CAS 876715-37-6), 3-(lH-tetrazol-5-yl)benzoic acid (CAS 73096-39-6), 3-(lH-pyrazol-3-yl)benzoic acid (CAS 850375-11-0), 3-(furan-2- yl)benzoic acid (CAS 35461-99-5), 3-(thiophen-2-yl)benzoic acid (CAS 29886-63-3), 3-(isoxazol-5-yl)benzoic acid (852180-44-0), 3-(isothiazol-5-yl)benzoic acid (CAS 904085-98-9), 3-(oxazol-5-yl)benzoic acid (CAS 252928-82-8), 3-(thiazol-5-yl)benzoic acid (CAS 252928-84-0), 3-(oxazol-2-yl)benzoic acid (CAS 473538-18-0), 3-(thiazol- 2-yl)benzoic acid (CAS 847956-27-8), 3-(&ran-3-yl)benzoic acid, (CAS 168619-07-6), 3-(tbiophen-3-yl)benzoic acid (CAS 20608-89-3), 3-(2-methyltbiazol-4-yl)benzoic acid (CAS 28077-41-0), 3-(l,2,4-oxadiazol-3-yl)benzoic acid (CAS 912577-30-1), 3-(l- methyl-lH-pyrazol-3-yl)benzoic acid (CAS 915707-39-0), 3-(2-methyl-lH-imidazol-l- yl)benzoic acid (CAS 898289-59-3), 3-(3-methyl-l,2,4-oxadiazol-5-yl)benzoic acid (CAS 915707-45-8), 3-(l-methyl-lH-pyrazol-5-yl)benzoic acid (CAS 628297-55-2), 3- (pyridin-4-yl)benzoic acid, 3-(pyrimidin-4-yl)benzoic acid, 3-(pyridin-3-yl)benzoic acid (CAS 4385-77-7), 3-(pyrimidin-5-yl)benzoic acid (CAS 852180-74-6), 3-(pyridin- 2-yl)benzoic acid (CAS 4467-07-6), 3-(pyrimidin-2-yl)benzoic acid (CAS 579476-26- 9), benzofdjthiazole-ό-carboxylic acid (3622-35-3), lH-indole-5-carboxylic acid (1670-81-1), lH-benzo[d][l,2,3]triazole-6-carboxylic acid (CAS 23814-12-2), benzo[c][l,2,5]oxadiazole-5-carboxylic acid (CAS 19155-88-5), 6-hydroxypicolinic acid (CAS 19621-92-2), 2,3-dioxo-l,2,3,4-tetrahydroqumoxdine-6-carboxylic acid, 2- oxo-l,2,3,4-tetrahydroquinoline-6-carboxylic acid (CAS 70639-77-9), 2-oxoindoline-5- carboxylic acid (CAS 102359-00-2), 2-oxoindoline-6-carboxylic acid (CAS 334952- 09-9), 2,3-dioxoindoline-5-carboxylic acid (CAS 25128-32-9), 3-oxo-3,4-dihydro-2H- benzo[b][l,4]oxazine-7-carboxylic acid (CAS 214848-62-1), A-
(methylsulfonamido)benzoic acid (CAS 7151-76-0), 2-oxo-2,3,4,5-tetrahydro-lH- benzo[b]azepine-7-carboxylic acid (CAS 117030-69-0).
The present invention is further described by way of example.
EXAMPLES
General procedures
Solvents were purchased from ROMIL Ltd, U.K. at SpS or Hi-Dry grade unless otherwise stated. 1H NMR and 13C NMR were obtained on a Bruker DPX400 (400 MHz 1H frequency and 100 MHz 13C frequency; QXI probe) or Bruker Avance 500MHz (TXI probe with ATM) in the solvents indicated. Chemical shifts are expressed in parts per million (δ) and are referenced to residual signals of the solvent. Coupling constants (J) are expressed in Hz. AU analytical HPLC were obtained on Phenomenex Jupiter C4, 5 μ, 300 A, 250 x 4.6 mm, using mixtures of solvent A (0.1% aq trifluoroacetic acid (TFA)) and solvent B (90% acetonitrile / 10% solvent A) on automated Agilent systems with 215 and / or 254 nm UV detection. Unless otherwise stated a gradient of 10 to 90% B hi A over 25 min at 1.5 mL / min was performed for full analytical HPLC. HPLC-MS analysis was performed on an Agilent 1100 series LC/MSD, using automated Agilent HPLC systems, with a gradient of 10 to 90% B in A over 10 min on Phenomenex Luna C8, 5μ, 300 A5 50 x 2.0 mm at 0.6 mL / min. Semi- preparative HPLC purification was performed on Phenomenex Jupiter C4, 5μ, 300 A, 250 x 10 mm, using a gradient of 10 to 90% B in A over 25 min at 4 mL / min on automated Agilent systems with 215 and / or 254 nm UV detection. Flash column purification was performed on silica gel 60 (Merck 9385) or using isolute SPE flash silica columns (Biotage, Hengoed, UK).
Preparation of Benzyl (Ry2-((5)-2,5-dihvdrofttran-2-yr)-2-hvdro%vethyl carbamate (18V (D Preparation of (3i?, 3aS, 65, 6a5Vhexahvdrofurof 3,2-6] furan- 3,6-diyl bis(4-methylbenzenesuifonate) (42). A stirred solution of j?-toluenesulfonyl chloride (57.4 g, 301 mmol) and isosorbide (43) (20 g, 137 mmol) in pyridine (315 mL) was heated at 95 0C for 4.5 hours under an atmosphere of argon then stood at ambient temperature for 16 hours before being poured onto iced-water (1 L). The aqueous was extracted with dichloromethane (2 x 500 mL), then the combined organic layers were washed with water (2 x 500 mL), then dried (Na2SO4), filtered then reduced in vacuo to leave a viscous oil (65.22 g). The oil was crystallized from hot methanol (350 mL). The white solid was collected by filteration in vacuo, then washed with methanol (100 mL) and dried in vacuo to obtain ditosylate (42) as a white solid (45.87 g, 74%). TLC (Rf= 0.30, EtOAc : heptane 2 : 3), analytical HPLC single main peak, R1 = 20.219 min., HPLC-MS 455.1 [M + H]+, 931.2 [2M + Na]+, [α] ∞+57.2° (c = 10.2, CHCl3); δH (500 MHz, CDCl3) 2.44 (6H, s, CH3), 3.68 (IH, dd, J= 9.80 and 6.46 Hz, CH2), 3.82-3.87 (2Η, m, CH2), 3.94 (1Η, d, J= 11.28 Hz, CH2), 4.46 (IH, d, J= AAA Hz, CHCHOTs), 4.58 (IH, t, J = 4.74 Hz, CHCHOTs), 4.82-4.86 (2H9 m, CHOTs), 7.32-7.36 (4Η, m, aromatic CH3CCH), 7.74-7.80 (4H, m, aromatic OSO2CCH).
(ii) Preparation of (3S, 3aS, 6S, 6aiS')-6-bromohexahvdrofiιroF3,2-61furan-3-vI 4- methylbenzenesulfonate (47). Lithium bromide (9.6 g, 110.1 mmol) was added to a stirred solution of ditosylate (42) (20.0 g, 44.05 mmol) in dimethylformamide (100 mL) under an atmosphere of argon. The mixture was heated at 110 °C for 5 hours then stood at ambient temperature for 3 days, then heated at 90 0C for 3.5 hours. The mixture was diluted with water (250 mL) extracted with tert-bntyl methyl ether (4 x 125 mL) then the organic phase washed with water (3 x 125 mL), brine (125 mL), dried (MgSO4), filtered and reduced in vacuo to leave a brown oil (16.8 g). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 0 : 100 to 30 : 70 gave bromotosylate (47) (11.88 g, 74%) as a pale yellow solid. TLC (R/ = 0.20, EtOAc : heptane 1 : 3); analytical HPLC main peak, Rt = 18.050 min; HPLC-MS 381.0 / 383.0 [M + H2O + H]+, 385.0 / 387.0 [M + Na]+; [α] 1J +51.0° (c = 5.0, CHCl3); δH (500 MHz, CDCl3) 2.45 (3H5 s, CH3), 3.84 (IH, dd, J= 11.19 and 3.51 Hz, CH2), 4.05-4.15 (3Η, m5 CH2), 4.28 (1Η, d, J= 3.40 Hz, CHBr), 4.78 (1Η, d, J= 3.37 Hz, CHCH), 4.84 (IH, d, J= 3.42 Hz, CHOTs), 4.90 (1Η, d, J= 3.37 Hz, CHCH), 7.36 (2Η, brd, J= 7.98 Hz, aromatic CH3CCH), 7.79 (2Η, brd, J= 8.32 Hz, aromatic OSO2CCH).
fiii) Preparation of f5Vl-ff5)-2.5-dihvdrofuran-2-vD-2-hvdro:^ethyl 4-methvI benzenesulfonate (14). Ammonium chloride (20 mg, 0.37 mmol) then zinc dust (20 mg, 0.31 mmol) were added to a solution of bromotosylate (47) (100 mg, 0.28 mmol) hi ethanol (1.5 mL) under argon. The mixture was stirred for 16 hours before filtering the suspension through celite in vacuo. The filter cake was washed with ethanol (20 mL) then the filtrate reduced in vacuo to leave a residue (111 mg). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 20 : 80 to 40 : 60 gave alcohol (14) (53 mg, 68%) as a white solid. TLC (Rj-= 0.15, EtOAc : heptane 1 : 2); analytical ΗPLC main peak, Rt = 12.543 min; ΗPLC-MS 285.1 [M + H]+, 302.1, 591.2 [2M + Na]+; [α]^5 -86.8° (c - 5.3, CHCl3); δH (500 MHz, CDCl3) 2.12 (IH, brs, OH), 2.44 (3Η, s, aryl-CH3), 3.77 (2Η, d, J = 4.85 Hz, CH2OH), 4.54-4.58 (3H, m, CH2OCH)5 4.94-4.98 (IH5 m5 CHOTs), 5.64-5.67 and 5.97-6.00 (2Η total, m, CH2CH=CH), 7.33 (2Η, brd, J= 8.23 Hz, aromatic CH3CCH)5 7.79 (2Η, brd, J= 8.31 Hz, aromatic OSO2CCH); δc (125 MHz5 CDCl3) 21.660 (CH3), 62.303 (CH2OH)5 75.940 (OCH2CH=CH), 82.720 and 85.221 (OCHCHOTs), 124.792, 127.977, 129.479 and 129.749 (OCH2CH=CH and aromatic CH), 133.496 (CHOSO2C quaternary), 144.973 (CH3C quaternary).
Ov) Alternative preparation of dS^-l-f^-Z^-dihvdrofiiran^-vD^-hvdroxyethvI 4- methyl benzenesulfonate (14). A solution of ammonium chloride (200 mg, 3.7 mmol) in water (2.5 mL) then zinc dust (200 mg, 3.1 mmol) were added to a solution of bromotosylate (47) (1 g, 2.75 mmol) in tetrahydrofuran (10 mL) and propan-2-ol (5 mL) under argon. The mixture was stirred for 16 hours before filtering the suspension through celite in vacuo. The filter cake was washed with diethyl ether (20 mL). Hydrochloric acid (IM5 20 mL) was added to the filtrate then the organic phase separated. The aqueous layer was extracted with diethyl ether (20 mL) then the combined organic phase was washed with brine (20 mL), then dried (MgSO4), filtered and reduced in vacuo to leave a residue (1.06 g). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 20 : 80 to 50 : 50 gave alcohol (14) (528 mg, 68%) as a white solid, [α] 1^ -82.7° (c = 11.3, CHCl3).
(V) Preparation of benzyl (iΩ-2-(r5V2.5-dihvdrofuran-2-vIV2- hydroxyethylcarbamate (18). Zinc and 'One-pot' procedure. A solution of ammonium chloride (600 mg, 11.2 mmol) in water (7.5 mL) was added to a solution of bromotosylate (47) (3.0 g, 8.26 mmol) in propan-2-ol (15 mL) under argon. Zinc dust (600 mg, 9.2 mmol) was then added in portions over 4 minutes and the mixture was stirred for 16 hours before filtering the suspension through celite in vacuo. The filter cake was washed with diethyl ether (60 mL). Hydrochloric acid (IM, 60 mL) was added to the filtrate then the organic phase separated. The aqueous layer was extracted with diethyl ether (60 mL) then the combined organic phase was washed with brine (60 mL), then dried (MgSO4), filtered and reduced in vacuo. The residue was dissolved in ammonium hydroxide (18 mL) and a solution of ammonia in propan-2-ol (12 mL, 2.0M, 24 mmol), then divided into two equal portions and heated in sealed tubes at 75 0C for 16 hours. The mixtures were combined using methanol then the solvents were removed in vacuo. The residue was azeotroped with diethyl ether (3 x 10 mL) to obtain (i?)-2-amino-l-((<S)-2,5-dihydrofuran-2-yl)ethanol which was used without further purification.
A solution of sodium carbonate (1.84 g, 17.4 mmol) in water (16 mL) was added whilst stirring to a suspension of (J?)-2-amino-l-((5)-2,5-dihydrofuran-2-yl)ethanol (assumed to be 8.26 mmol) in 1,4-dioxane (20 mL). The mixture was cooled to 0 0C then benzylchloroformate (1.77 mL, 12.4 mmol) was added dropwise over 5 minutes. The mixture was stirred at 0 °C for 55 minutes then dichloromethane (75 mL) and water (100 irtL) added. The organic phase was separated and the aqueous extracted with dichloromethane (2 x 50 mL). The organic phase was washed with brine (50 mL), then dried (Na2SC>4), filtered and reduced in vacuo to leave a residue (3.7 g). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 20 : 80 to 70 : 30 gave alcohol (18) (1.26 g, 58%). [α] 1J -62.0° (c = 5.0, CHCl3).
Preparation of (i?)-2-fbenzvIoxycarbonvIammo)-l-(6->>-2.,5-dihvdrofuran-2-vI) ethyl 4-methyl benzenesulfonate (32tΛ. A solution of p-toluenesulfonyl chloride (368 mg, 2.03 mmol) in pyridine (1.5 mL) was added to alcohol (18) (333 mg, 1.27 mmol). The mixture was stirred at 14 °C for 16 hours and at 24 0C for 3.5 hours then diluted with tert-butyl methyl ether (35 mL). The organic layer was washed with water (15 mL), brine (15 mL), then dried (MgSO4), filtered and reduced in vacuo to leave a pale yellow oil (0.712 g). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 15 : 85 to 30 : 70 gave tosylate (32b) (429 mg, 81%) as a white solid. TLC (J?/= 0.75, EtOAc : heptane 3 : Y), analytical HPLC single main peak, Rt = 18.93 min., HPLC-MS 374.2, 418.2 [M + H]+, 857.3 [2M + Na]+; [a]™ -30.2° (c = 1.326,
CHCl3); δH (500 MHz, CDCl3) 2.39 (3H5 s, aryl-CH3), 3.29-3.37 and 3.53-3.62 (2H total, m, CF2NH), 4.44-4.50 and 4.52-4.57 (2H total, m, OCH2CH-CH), 4.59-4.65 (IH, m, OCHCH=CH), 4.87-4.92 (IH, m, CHOTs), 5.05 (2H, m, OCH2Ph), 5.03 (1Η, brs, NH), 5.69-5.73 and 5.94-5.98 (2Η total, m, CR2CH=CH), 7.28 (2H, d, J= 8.10 Hz, aromatic CH3CCH), 7.29-7.37 (5Η, phenyl CH), 7.77 (2Η, d, J = 8.10 Hz, aromatic OSO2CCH); δc (125 MHz, CDCl3) 21.627 (aryl-CH3), 41.119 (CH2NHCbz), 66.856 (CH2Ph), 75.987 (OCH2CH=CH), 82.352 (CHOTs)5 85.622 (OCHCH=CH), 124.792, 127.825, 128.027, 128.126, 128.504, 129.357 and 129.537 (OCH2CH=CH and aromatic CH), 133.674 (CHOSO2C quaternary), 136.348 (Cbz quaternary), 144.941 (CH3C quaternary), 156.273 (Cbz C=O).
Epoxidation studies with 6R)-2-(T>enzyloxycarbonvIamino)-l-ffiSr)-2,5-dihvdro furan-2-vDethvI 4-methvIbenzenesuIfonate (32b). (a) 3-Chloroperbenzoic acid (97 mg, ≤ 77%, 0.43 mmol) was added to a stirred solution of alkene (32b) (36 mg, 0.086 mmol) in dichloromethane (1.5 mL). The mixture was stirred for 20 hours at ambient temperature then 3-chloroperbenzoic acid (97 mg, <
77%, 0.43 mmol) was added and stirring continued for 1 day at 24 0C then diluted with dichloromethane (15 mL). The organic phase was washed with aqueous sodium hydroxide solution (5%, 10 mL), water (10 mL). then dried (Na2SO4), filtered and reduced in vacuo to leave a residue (0.038 mg). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 10 : 90 to 50 : 50 gave (in order of elution) anti-(33b) (16 mg, 43%) as a colourless viscous oil and ^yn-epoxide (9 mg, 24%) as a white solid. Data for anti-(33b); TLC (Rf = 0.50, EtOAc : heptane 1 : 1), analytical HPLC single main peak, Rt = 17.999 min., HPLC-MS 434.1 [M + H]+, 456.1 [M + Na]+, 889.2 [2M + Na]+; [α] ^7 +25.6° (c = 2.54, CHCl3); δH (500 MHz, CDCl3) 2.41 (3H, S5 aryl-CHO, 3.31-3.38 and 3.60-3.66 (2H total, m, CH2NH), 3.67 (IH, d, J = 10.46 Hz, OCH2CH)5 3.75 and 3.81 (each IH5 d, J = 2.50 and 2.75 Hz respectively,
OCH2CHCH), 3.94 (IΗ, d, J = 10.57 Hz, OCH2CH), 4.07 (IH, d, J = 6.90 Hz,
OCHCHOTs)5 4.60-4.64 (IH5 m, CHOTs)5 4.97-5.01 (1Η brt, NH), 5.08 (2Η, brs, CH2Ph), 7.29-7.37 (7Η, aromatic CH3CCH and phenyl CH), 7.78 (2Η, d, J - 8.18 Hz, aromatic OSO2CCH); δc (125 MHz, CDCl3) 21.665 (aryl-CH3), 42.054 (CH2NHCbz),
56.175 and 57.048 (OCH2CHCH), 67.031 (CH2Ph), 67.672 (OCH2CH), 76.732
(OCHCHOTs), 79.388 (CHOTs)5 127.776, 128.108, 128.222, 128.544 and 130.043
(aromatic CH), 133.249 (CHOSO2C quaternary), 136.192 (Cbz quaternary), 145.487 (CH3C quaternary), 156.224 (Cbz C=O).
(b) To a solution of alkene (32b) (262 mg, 0.63 mmol) in acetonitrile (4 mL) and aqueous Na2-EDTA (4 mL, 0.4 mmol solution) at 0 0C was added 1,1,1- trifluoroacetone (0.67 mL, 7.54 mmol) via a pre-cooled syringe. To this solution was added in portions a mixture of sodium bicarbonate (0.44 g, 5.28 mmol) and OXONE® (1.20 g, 1.95 mmol) over a period of 55 minutes. The mixture was stirred for 2.5 hours then diluted with water (25 mL) and the product extracted into dichloromethane (2 x 25 rnL). The combined organic layers were washed with brine (12.5 mL) then dried (Na2SO4), filtered and reduced in vacuo to leave a residue (310 mg). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 15 : 85 to 50 : 50 gave anti-(33b) as a viscous white oil (216 mg, 79%). Preparation of (3R, 3aR. 6R, 6a-t)-3-hvdroxyhexahvdro-2K-furor3,2-61pyrrol-6-yl
4-methylbenzenesulfonate (74V Ethanol (1.5 niL) was added dropwise to a mixture of
10% palladium on charcoal (20 mg) and αnti~(33b) (100 mg, 0.25 mmol) under an atmosphere of argon. The argon was replaced by hydrogen then the suspension was stirred for 4.5 hours before filtering the mixture through celite in vacuo. The filter cake was washed with ethanol (10 mL) then the solvents removed in vacuo from the filtrate.
The residue was azeotroped with toluene (2 x 3 mL) to obtain (3R, 3ai?5 6R, 6aS)-3- hydroxyhexahydro-2H-furo[3,2-δ]pyrrol-6-yl 4-methylbenzenesulfonate (74) which was used without further purification. TLC (Rf= 0.01, EtOAc : heptane 1 : 1), HPLC- MS 300.1 [M + H]+, 621.2 [2M + Na]+.
Preparation of (3JR, 3ai?, 6R, 6aS)- benzyl 3-hvdroxy-6-(tosyloxy)tetrahvdro-2Jg- fnrof3,2-^pyrroIe-4(5iyVearboxyIate (34b). A solution of sodium carbonate (6.2 mg, 0.058 mmol) in water (0.15 mL) was added whilst stirring to a solution of aminoalcohol (74) in 1,4-dioxane (0.3 mL). Benzylchloroformate (5.9 μL, 0.042 mmol) was added then the mixture stirred for 2 hours. Water (5 mL) was added and the product extracted into dichloromethane (2 x 5 mL). The organic layer was washed with brine (5 mL), then dried (Na2SO4), filtered and reduced in vacuo to leave a residue (10.6 mg). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 20 : 80 to 50 : 50 gave bicyclic alcohol (34b) (6.6 mg, 54%) as a white solid. TLC (Rf= 0.20, EtOAc : heptane 1 : 1), analytical HPLC single main peak, Rt = 17.32 min., HPLC-MS 434.1 [M + H]+, 889.2 [2M + Na]+; [α] ™ -25.7° (c = 2.53, CHCl3); δH
(500 MHz, CDCl3) mixture of rotamers major : minor 2 : 1; 2.01 (0.33H, brs, OH minor), 2.43 (3H, s, aryl-CH3), 2.77 (0.66H, brs, OH major), 3.18-3.24 (0.33H, m, CbZNCH2 minor), 3.33-3.38 (0.66H, m, CbzNCH2 major), 3.79-3.85 (1Η, m, OCH2CHOH), 3.86-3.91 (IH, m, CbzNCH2), 3.92-3.96 (0.33Η, m, OCH2CHOH minor), 3.96-4.01 (0.66H, m, OCH2CHOH major), 4.13-4.16 (IH, m, CbzNCH), 4.35 (0.33Η, m, OCH2CHOH minor), 4.45 (0.66H, m, OCH2CHOH major), 4.56 (0.33H, t, J = 4.64 Hz, TsOCHCH, minor), 4.64 (0.66Η, t, J= 4.36 Hz, TsOCHCH, major), 4.71- 4.78 (1Η, m, TsOCHCΗ ), 5.06-5.17 (2Η, m, CH2Ph), 7.31-7.38 (7Η, m, phenyl CH and aromatic CH3CCH), 7.80 (2Η, d, J= 8.33 Hz, aromatic OSO2CCH); δc (125 MHz, CDCl3) 21.683 (aryl-CH3), 47.384 / 47.855 (CbzNCH2), 67.636 / 67.717 (CH2Ph),
68.042 / 68.817 (CbzNCH), 75.525 / 75.967 (OCH2CHOH). 75.967 / 76.836 (OCH2CHOH), 76.068 / 76.401 (TsOCHCH), 79.342 / 80.208 (TsOCHCH), 127.965, 128.107, 128.382, 128.510, 128.605, 128.753, 129.940 and 129.997 (aromatic CH), 132.991 (CHOSO2C quaternary), 135.779 / 135.869 (Cbz quaternary), 145.319 (CH3C quaternary), 153.862 / 154.751 (Cbz C=O).
Preparation of (3aS. 6S. 6aiSV(9#-FIuoren-9-vI)methvI 6-chloro-3-oxotetrahvdro-
2ff-furor3.2-61pyrroIe-4(5#VcarboxyIate (71V Following Scheme 17.
(i) Preparation of (3 R, 3aR, 6S, 6a5)-Benzyl 6-chloro-3-hydroxytetrahydro-27J- furo[3,2-5]pyrrole-4(5H)-carboxylate (68).
Figure imgf000083_0001
Scheme 17. (a) LiCl, DMF5 130 °C; (b) Pd-C, H2, ethanol; (c) Fmoc-Cl, Na2CO3, dioxane, H2O; (d) Dess-Martin periodinane, anhydrous DCM; (e) LiCl, DMF5 130 °C; fb) Cbz-Cl, Na2CO3, dioxane, H2O.
Lithium chloride (2.38 g, 56.2 mmol) was added to a stirred solution of (3R, 3aR, 6R, 6aS)-benzyl 3-hydroxy-6-(tosyloxy)tetrahydro-2H-furo[3,2-έ]pyrrole-4(5H)- carboxylate (34b) (2.435g, 5.62 mmol) in dimethylformamide (75 mL) under an atmosphere of argon. The mixture was heated at 130 0C for 7 hours then allowed to cool to ambient temperature. The mixture was diluted with dichloromethane (100 mL), then water (50 mL) was added and the mixture filtered through celite (filter cake washed with dichloromethane). The filtrate was separated then the organic phase washed with water (2 x 50 mL), then dried (Na2SO4), filtered and reduced in vacuo to leave a residue (1.54 g). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 20 : 80 to 60 : 40 gave alcohol (68) (1.28 g, 77%) as an orange-brown solid. TLC {Rf= 0.40, EtOAc : heptane 2 : 1), analytical HPLC single main peak, R1 = 11.47 min., HPLC-MS 298.1 / 300.1 [M + H]+, 617.1 [2M + Na]+; [α] ^30 -72.8° (c =
2.61, CHCl3); δH (500 MHz, CDCl3) mixture of rotamers major : minor 2 : 1; 1.78 and 2.24 (approx. IH total, each brs, OH)5 3.58-3.63 (IH, m, 1 x CbzNCH2), 3.83-3.88 (2H, m, OCH2CHOH), 3.91 (0.66H, d, J= 13.08 Hz3 1 x CbZNCH2, major), 4.02 (0.33H5 J=
13.09 Hz, 1 x CbZNCH2, minor), 4.24-4.26 (1Η, m, CHCl), 4.39-4.42 (0.66Η, m,
CbzNCH minor and OCH2CHOH minor), 4.43 (0.66H, d, J = 4.33 Hz, CbzNCH major), 4.52 (0.66Η, brs, OCH2CHOH major), 4.72-4.75 (IH, m, CHCHCl)5 5.11-5.16 (1.66H, m, 2 x CH2Ph major and 1 x CH2Ph minor), 5.24 (0.33Η, d, J= 12.29 Hz 1 x
CH2Ph minor), 7.29-7.37 (5Η, m, phenyl CH ); δc (125 MHz5 CDCl3) 53.57 / 53.74
(CbZNCH2), 57.91 / 58.38 (CHCl)5 67.53 / 67.58 (CH2Ph), 67.69 / 68.64 (CbzNCH),
75.06 / 75.93 (OCH2CHOH), 75.12 / 75.18 (OCH2CHOH)5 86.66 / 87.59 (CHCHCl),
127.85, 127.90, 128.24, 128.32, 128.56 and 128.69 (aromatic CH)5 135.97 / 136.15 (Cbz quaternary), 154.41 / 154.96 (Cbz C=O).
(ii) (3R, 3aR, 68, 6aS)-(9H-Fluoren-9-yl)methyl 6-chloro-3-hydroxytetrahydro-2H- furo[352-δ]pyrrole-4(5H)~carboxylate (70). Ethanol (8.5 mL) was added dropwise to a mixture of 10% palladium on charcoal (55 mg) and alcohol (68) (550 mg, 1.85 mmol) under an atmosphere of argon. The argon was replaced by hydrogen then the suspension was stirred for 1 hour 35 minutes before filtering the mixture through celite in vacuo. The filter cake was washed with ethanol (45 mL) then the solvents removed in vacuo from the filtrate. The residue was azeotroped with toluene (3 x 5 mL) to obtain (3R, 3aR, 6S, 6aS)-6-chlorohexahydro-2H-furo[3,2-6]pyrrol-3-ol (69) which was used without further purification. A solution of sodium carbonate (0.49 g, 4.63 mmol) in water (7.5 mL) followed by a solution of 9-fluorenylmethoxycarbonyl chloride (0.55 g, 2.13 mmol) in 1,4-dioxane
(2.5 mL) was added dropwise over 15 minutes whilst stirring to a solution of aminoalcohol (69) in 1,4-dioxane (5 mL). The mixture was stirred for 60 minutes then water (50 mL) was added and the product extracted into dichloromethane (3 x 25 mL), then dried (Na2SO4), filtered and reduced in vacuo to leave a colourless oil. Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 10 : 90 to 45 :
55 gave alcohol (70) (623 mg, 87%) as a white solid. TLC (Rf= 0.45, EtOAc : heptane
1 : 1), analytical HPLC single main peak, Rt = 16.54 min., HPLC-MS 386.1 / 388.1 [M + H]+, 408.1 / 410.1 [M + Na]+; [α] ^75 -51.9° (c = 2.3I5 CHCl3); (proton complex) δc
(125 MHz, CDCl3) 47.21 / 47.41 (Fmoc CH), 53.30 / 53.43 (FmocNCH2), 57.74 / 58.36 (CHCl), 66.04 / 67.42 (Fmoc CH2), 67.87 / 68.52 (FmocNCH), 74.81 / 75.09 (OCH2CHOH), 74.92 / 75.51 (OCH2CHOH), 86.57 / 87.24 (CHCHCl), 119.80 / 119.82 / 120.00 / 120.64 / 124.55 / 124.63 / 124.90 / 127.04 / 127.08 / 127.40 / 127.51 / 127.78 / 127.80 / 127.87 and 127.91 (aromatic CH), 141.21 / 141.29 / 141.38 / 143.44 / 143.70 / 143.88 and 143.91 (aromatic quaternary), 154.13 / 154.79 (Fmoc C=O).
(iii) (3aS, 6S, 6aS)-(9H-Fluoren-9-yl)methyl 6-chloro-3-oxotetrahydro-2H"-furo[3,2- δ]ρyrrole-4(5iϊ)-carboxylate (71). Dess-Martin periodinane (1.32 g, 3.11 mmol) was added to a stirred solution of alcohol (70) (600 mg, 1.56 mmol) in dichloromethane (15 mL) under an atmosphere of argon. The mixture was stirred for 19 hours then diluted with dichloromethane (50 mL) then washed with a mixture of saturated aqueous sodium bicarbonate and 0.25M sodium thiosulphate solution (1 : 1, 30 mL), saturated aqueous sodium bicarbonate (25 mL), brine (25 mL), then dried (Na2SO4), filtered and reduced in vacuo to obtain a white solid (935 mg). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 15 : 85 to 100 : 0 gave ketone (71) (506 mg, 85%) as a white solid contaminated with 2-iodosylbenzoic acid (< 5%). TLC (Rf= 0.35, EtOAc : heptane 1 : 1), analytical HPLC single main peak, Rt = 15.81 min., HPLC-MS 384.1 / 386.1 [M + H]+, 406.1 / 408.1 [M + Na]+ 424.1 / 426.1 [M + H2O + Na]+, 789.1 / 791.2 [2M + Na]+; [α]^55 -144.6° (c = 2.18, CHCl3); δH (500 MHz, CDCl3) mixture of rotamers major : minor 0.55 : 0.45; 3.75-3.89 (IH, m3 1 x FmocNCH2), 3.93-4.03 (1.55Η, m, 1 x OCH2C=O and 1 x FmOcNCH2 major), 4.12- 4.22 (1.45Η, m, 1 x OCH2C=O and 1 x FmocNCH2 minor), 4.25 (0.55Η, brt, J= 6.72 Hz5 Fmoc CH major), 4.30-4.44 (2.45Η, m, CHCl, 1 x FmocNCH2 and Fmoc CH minor), 4.45 (0.45Η, d, J= 4.46 Hz5 FmocNCH minor), 4.50-4.58 (1.55Η, m, 1 x Fmoc CH2 and FmocNCH major), 4.85 (0.55Η, d, J= 4.44 Hz, CHCHCl major), 4.90 (0.45H, d, J= 4.41 Hz, CHCHCl minor), 7.27-7.76 (8H, aromatic CH); δc (125 MHz, CDCl3) 47.09 / 47.13 (Fmoc CH), 53.43 / 53.66 (FmocNCH2), 57.60 / 58.09 (CHCl), 60.47 / 60.87 (FmocNCH), 67.86 / 68.56 (Fmoc CH2), 70.75 (OCH2C=O), 86.32 / 87.32 (CHCHCl), 119.93 / 119.99 / 120.08 / 124.87 / 124.94 / 125.17 / 125.36 / 127.09 / 127.71 and 127.74 (aromatic CH), 141.28 / 141.32 / 143.51 / 143.63 and 144.16 (aromatic quaternary), 154.88 / 154.94 (Fmoc C=O), 206.45 / 206.64 (OCH2C=O).
Alternative preparation of (3R, 3ai?, 6S. βaSl-Benzyl 6-chIoro-3- hvdroxytetrahvdro-2Hr-furor3,2-&1pyrroIe-4f5Jy)-carboxyIate (68). Lithium chloride (142 mg, 3.34 mmol) was added to a stirred solution of (32?- 3aR, 6R, 6aS) 3- hyclroxyhexahydro-2H-furo[3,2-δ]pyrrol-6-yl 4-methylbenzenesulfonate (74) (100 mg, 0.33 mmol) in dimethylformamide (3 mL) under an atmosphere of argon. The mixture was heated at 130 0C for 2.75 hours then allowed to cool to ambient temperature to give a solution containing 6-chloroaminoalcohol (69). A solution of sodium carbonate (89 mg, 0.84 mmol) in water (1.5 mL) was added followed by benzylchloroformate (0.105 mL, 0.74 mmol). The mixture was stirred for 35 minutes then dichloromethane (10 mL) and water (15 mL) added. The organic phase was separated and the aqueous extracted with dichloromethane (2 x 5 mL). The combined organic phase was washed with brine (5 mL), then dried (Na2SO4), filtered and reduced in vacuo to leave a black residue (97 mg). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 5 : 95 to 50 : 50 gave 6-chloroalcohol (68) (48 mg, 48%) as a pale yellow oil. TLC (Rf= 0.30, EtOAc : heptane 3 : 2), analytical ΗPLC single main peak, Rt = 11.47 min., ΗPLC-MS 298.0 / 300.0 [M + H]+, 617.1 / 619.1 [2M + Na]+; [α] £ -76.9° (c =
4.81, CHCl3). Preparation of (SV4-benzyl-3-f2-(flr.4S)-4-methylcvclohexyI>acetyl)oxazolidin-2- one (127); Scheme 18 Trans-(4-methylcyclohexyl)acetic acid (2.Og, 12.8mmol, ABCR GmbH, AB 168553) was dissolved in anhydrous THF (10OmL), stirred and cooled to -780C. Triethylamine (2.36mL, 16.0mmol) was added followed by pivaloyl chloride (1.78mL, 14.4mmol) and the mixture was stirred at O0C for Ih. The mixture was then re-cooled to -780C. (S)-4-benzyl-2-oxazolidinone (4.38g, 24.6mmol) was dissolved in anhydrous THF with stirring, cooled to -780C and n-BuLi (2.5M, 9.95mL, 24.9mrnol) added. This mixture was added via cannula to the pre-activated acid mixture over 5mins. The reaction was stirred at O0C for Ih, ambient temperature for 4h, then reduced in vacuo. The resultant slurry was dissolved in DCM (10OmL) and washed with potassium phosphate (0.1M, pH7, 10OmL). The aqueous was back-washed with DCM (2 x 10OmL) and the combined organics washed with 5% Na2CO3 (10OmL), then brine (10OmL) and dried over Na2SO4. Filtration and reduction in vacuo gave a crude yellow wax (6.9g). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 0 : 100 to 15 : 85 gave a mixture of product (127) and (S)-4-benzyl-3- pivaloyloxazolidin-2-one as an off-white waxy solid (3.7g). TLC (R/ ~ 0.50, EtOAc : heptane 1 : 2), analytical HPLC, Rt = 15.70 (pivaloyl-auxilliary 32.5%), 19.00 min (desired 67.5%)., HPLC-MS 262.1 [M + H]+ (pivaloyl-auxilliary), 316.2 [M + H]+, 653.2 [2M + Na]+.
By δH (500 MHz, CDCl3) NMR integration of product (127) CH3 (d, 0.83) and pivaloyl-auxilliary (CHs)3 (s, 1.38), shows (127) is present at ~ 60%.
Preparation of (5V3-((5)-2-Azido-2-((lr. 4SV4-methylcvclohexyr>acetyr)-4- benzyIoxazoIidin-2-one (128) A solution of potassium bis(trimethylsilyl)amide (0.5M in toluene, 30.6 mL, 15.29 mmol) was added over 5 minutes to a stirred solution containing a 3 : 2 mixture of (5)-4-benzyl-3-(2-((lr, 45)-4- methylcyclohexyl)acetyl)oxazolidin-2-one (127) and (5)-4-benzyl-3- pivaloyloxazolidin-2-one (3.7 g, (127) estimated to be 7.05 mmol) in tetrahydrofuran (70 mL) at -70 0C under an atmosphere of argon. The solution was stirred at -70 °C for
20 minutes then a solution of trisyl azide (5.62 g, 18.18 mmol) in tetrahydrofuran (40 mL, precooled to -70 °C) was added via cannula over 8 minutes. The mixture was stirred at -70 0C for 1 hour then glacial acetic acid (1.85 mL) was added. The cooling bath was removed and the mixture stirred for 45 minutes at ambient temperature then at 30 0C for 2 hours. A saturated aqueous solution of sodium hydrogen carbonate (100 mL) was added then the majority of solvents were removed in vacuo. The residue was partitioned between dichloromethane (300 mL) and brine (300 mL). The aqueous layer was re-extracted with dichloromethane (2 x 100 mL) then the combined organic layers were washed with saturated aqueous sodium hydrogen carbonate solution (100 mL), dried (Na2SO4), filtered and reduced in vacuo to leave a yellow oil (6.74 g). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 0 : 100 to 20 : 80 gave a 1 : 3 mixture of (5)-4-ben2yl-3-pivaloyloxazolidin-2-one and (S)-3-((S)-2- azido-2-((lr, 45)-4-methylcyclohexyl)acetyl)-4-benzyloxazolidin-2-one (4) as a pale yellow oil (2.815 g, estimated yield of (128) 84%). Data for (5)-3-((5)-2-azido-2-((l5, 4JR)-4-methylcyclohexyl)acetyl)-4-benzyloxazolidin-2-one (128): TLC {Rr = 0.45, EtOAc : heptane 1 : 3), analytical HPLC5 Rt = 20.181 min., HPLC-MS 329.2 [M - N2 + H]+, 735.4 [2M + Na]+.
Preparation of tert-Butyl fASV2-ff£)-4-benzvI-2-oxooxazoIidm-3-vIVl-f(lr. 4SV4- methylcyelohexylV2-oxoethylcarbamate (129). 10% Palladium on charcoal (500 mg) was added to a 1 : 3 mixture of (<S)-4-benzyl-3-pivaloyloxazolidin-2-one and (β)-3-((S)~ 2-azido-2-((lr, 4S)-4-methylcyclohexyl)acetyl)-4-benzyloxazolidin-2-one (128) (2.75 g) followed by a solution of di-tert-bυtyl dicarbonate (6.06 g, 27.8 rnmol) in NJV- dimethylformamide (30 mL) under an atmosphere of argon. The argon was replaced by hydrogen then the mixture was stirred for 4 hours before filtering through celite in vacuo. The filter cake was washed with ΛyV-dimethylformamide (50 mL) then the solvents removed in vacuo from the filtrate (water bath temperature <50 0C). The residue was dissolved in ethyl acetate (400 mL) then washed with brine (3 x 100 mL), dried (MgSO4), filtered and reduced in vacuo to leave a pale brown oil (6.9 g). Flash chromatography over silica, eluting with ethyl acetate : heptane mixtures 0 : 100 to 30 : 70 gave tert-butyl (5)-2-((S)-4-benzyl-2-oxooxazolidin-3-yl)-l-((lr, 45)-4- methylcyclohexyl)-2-oxoethylcarbamate (129) as a white oily solid (1.415 g). TLC (R/ = 0.35, EtOAc : heptane 1 : 3), analytical HPLC5 Rt = 19.760 min., HPLC-MS 331.2 [M - Boc + 2H]+, 375.2 [M + 2H - 'Bu]+, 883.4 [2M + Na]+; [α] £ +72.5° (c = 2.35, CHCl3).
Preparation of (S)-2-(tert-Bxito^\carbonylajmao)-2-((lr, 4SV4- methylcvcIohexyDaeetic acid fl30). Aqueous hydrogen peroxide solution (30% 1.52 mL) was added to a stirred solution of tert-butyl (£)-2-((<S)-4-benzyl-2-oxooxazolidin- 3-yl)-l-((lr, 45)-4-methylcyclohexyl)-2-oxoethylcarbamate (129) (1.36 g, 3.16 mmol) in a mixture of tetrahydrofuran (40 mL) and water (12 mL) at 0 °C. Lithium hydroxide monohydrate (165 mg, 3.92 mmol) was added then the mixture was stirred at 0 0C for 2 hours before adding a solution of sodium sulphite (1.65 g) in water (10 mL) followed by aqueous sodium hydrogen carbonate solution (30 mL). The mixture was stirred for 5 minutes then the volume reduced by half in vacuo (> 25 mbar, external water bath 25 0C). Water (50 mL) was added then the pH adjusted to < 2 using 5M hydrochloric acid. The aqueous phase was extracted with dichloromethane (4 x 50 mL), then the combined organic layers were dried (Na2SO4), filtered and reduced in vacuo to leave a colourless oil (1.68 g) The oil was partitioned between a solution of sodium carbonate (1.75 g) in water (50 mL) and diethyl ether (40 mL). The aqueous layer was re- extracted with diethyl ether (2 x 40 mL) then the pH adjusted to < 2 using 5M hydrochloric acid. The acidified aqueous layer was then extracted with diethyl ether (3 x 50 mL) then the combined organic layers dried (MgSO4), filtered and reduced in vacuo to leave (£)-2-(fer/-butoxycarbonylamino)-2-((lr, 45)-4-methylcyclohexyl)acetic acid (130) which was contaminated with approximately 10% of (5)-4- benzyloxazolidin-2-one as an oily white solid (858 mg). HPLC-MS 172.1 [M - Boc + 2H]+, 216.1 [M + 2H - 'Bu]+, 257.1 [M - CH3 + H]+, 565.4 [2M + Na]+; [α] £ +29.6° (c
= 3.205, CHCl3). δH (500 MHz, CDCl3) mixture of rotamers major : minor 3 : 1; 0.85 (3H, d, J= 6.51 Hz, CH3CH), 0.86-0.98 (2H, m, 1 x cyclohexyl-CH2), 1.05-1.33 (3Η, m, CH3CH and 1 x cyclohexyl-CH2)5 1.43 (9H5 s, (CHs)3C)5 1.59-1.80 (5Η, m, NCHCH and 2 x cyclohexyl-CH2)5 4.02 (0.25Η, brs, NCH), 4.18-4.26 (0.75H5 m, NCH), 5.03 (0.75H5 d5 J = 8.82, NH), 6.05 (0.25Η, brd, J = 4.14 Hz, NH); δc (125 MHz, CDCl3) 22.403 (CH3CH), 27.718, 29.304, 34.576 and 34.653 (cyclohexyl-CH2), 28.297 ((CH3)3C), 32.202 (CH3CH), 40.386 (NHCHCH)5 58.012 (NHCH), 80.000 ((CH3)3Q, 155.754 (NHC=O), 177.156 (CHC=O).
Solid Phase Chemistry Fmoc-ketone building block (71) may be utilised in a solid phase synthesis of EXAMPLE inhibitors (1-22) of general formula I. The methods used were directly analogous to those described in detail in WO02057270, utilising the 4- {[(Hydrazinocarbonyl)arnino]methyl}cyclohexane carboxylic acid trifluoroacetate based linker, solid phase lanterns (ex Mimotopes), standard Fmoc chemistries and acidolytic cleavage followed by semi-preparative HPLC purification (see WO02057270 pg 124-127 for full generic details). Novel compounds (1-22) or prior art compound (38) are detailed for comparison and can readily be prepared by the general methods detailed in WO02057270 or WO0807127 through use of appropriate Fmoc- ketone building blocks (e.g. 6-unsubstituted bicycle (WO02057270), compound 19, pg 134; 6(S)-fluoro bicycle (WO0807127, compound 63, pg 88); 6(S)-chloro bicycle (WO0807127, compound 71, pg 94); 6(i?)-chloro bicycle (WO0807127, compound 79, pg 98)). Fmoc-ketone building blocks are then derivatised as appropriate with a R9- COOH carboxylic acid via standard uranium activation techniques.
Example 1. N-((^-2-((3aSr 56S,6aS)-6-chloro-3-oxodihydro-2H-furo[3,2-&]ρyrrol- 4(5H;6H;6αH)-yl)-l-((lr,41S)-4-methylcyclohexyl)-2-oxoethyl)benzamide
Figure imgf000090_0001
HPLC-MS Rt = 3.05 min, 419.2/421.2 [M + H]+, 437.2/439.2 [M + H + IS]+.
Example 2. N-((6)-2-((3a5',6<S',6a5)-6-chloro-3-oxodihydro-2H-furo[3,2-&]pyrrol- 4(5H,6H:,6αH)-yl)-l-((lr,4S)-4-methylcyclohexyl)-2-oxoethyl)-3-(lH-tetrazol-l- yl)benzamide
Figure imgf000091_0001
HPLC-MS Rt = 2.87 min, 487.2/489.2 [M + H]+, 505.2/507.2 [M + H + IS]+.
Example 3. N-((S)-2-((3aS6S6aS)-6-ch[oτo-3-oxo<&wdro-2H-fwo\32-b~\OYττol- 4(5H,6H,6aH)-yϊ)~ 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)-3-( 1 H-tetrazol- 1 - yl)benzamide
Figure imgf000091_0002
HPLC-MS Rt = 3.00 min; 501.2/503.2 [M + H]+, 519.2/521.2 [M + H + IS]+.
Example 4. JV-((,S)-2-((3a556S',6aS)~6-chloro-3-oxodiliydro-2H-furo[3,2-&]pyrrol- 4(5H,6H,6«H)-yl)-l-((lr,4,S)-4-methylcyclohexyl)-2-oxoethyl)-3-(lH-iinidazol-l- yl)benzamide
Figure imgf000091_0003
HPLC-MS Rt - 2.31 min, 485.2/487.2 [M + H]+, 503.2/505.2 [M + H + 18]+.
Example 5. N-((.S^-2-((3aS,65',6a^-6-chloro-3-oxodihvdro-2H-furor3,2-6bvrrol-
4(5H56/f,6αF)-yl)-l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-3-(4H-lJ2,4-triazol-4- yl)benzamide
Figure imgf000092_0001
HPLC-MS Rt = 2.53 min, 486.2/488.2 [M + H]+, 504.2/506.2 [M + H + IS]+.
Example 6. N-((8)-2-((3aS£S6aS)-6-cUoτo-3-oxodϊhv&o-2R-faio\32-b'\OYπol- 4(5H,6H;6«H)-yl)-l-((lr,41S)-4-methylcyclohexyl)-2-oxoethyl)-3-(lH-pyrazol-l- yl)benzamide
Figure imgf000092_0002
HPLC-MS Rt = 3.11 min5 485.2/487.2 [M + H]+, 503.2/505.2 [M + H + 18]+.
Example 7. N-fr^-2-rr3aS,6&6a^-6-chloro-3-oxodihvdro-2H-furor3,2-61pvrrol- 4(5H,6H,6aH)-yϊ)- 1 -((lr545)-4-methylcyclohexyl)-2-oxoethyl)-3-(4H- 1 ,2,4-triazol-4- yl)benzamide
Figure imgf000092_0003
HPLC-MS Rt = 2.80 min, 486.2/488.2 [M + H]+, 504.2/506.2 [M + H + 18]+.
Example 8. N-(($)-2-((3aS6S,6aS)-6-cMoro-3~oxo<miv<ko-2H-futo{32-b'lOvrtol- 4(5H,6H,6αi^-yl)-l-((lr,4^-4-methylcyclohexyl)-2-oxoethyl)nicotinamide
Figure imgf000093_0001
HPLC-MS Rt = 2.49 inin, 420.2/422.2 [M + EQ+, 438.2/440.2 [M + H + 18]+.
ExamEk9. iV-((»S)-2-((3aS,6^6aiS)-6-chloro-3-oxodihydro-2H-furo[3,2-6]pyrrol- 4(5H,6H,6αH)-yl)- 1 -((1 r,45)-4-methylcyclohexyl)-2-oxoethyl)isonicotinamide
Figure imgf000093_0002
HPLC-MS Rt = 2.45 min, 420.2/422.2 [M + H]+, 438.2/440.2 [M + H + 18]+.
E^mE|eJ0. N-((5)-2-((3a5'565,6a^-6-cMoro-3-oxodihydro-2H-furo[332-ό]pyrrol- 4(5i7,6H36α/i)-yl)-l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)furan-2-carboxamide
Figure imgf000093_0003
HPLC-MS Rt = 2.81 min, 409.1/411.1 [M + H]+, 427.2/429.2 [M + H + 18]+.
Example 11. N-ff^-2-rr3^6S.6a^-6-chloro-3-oxodihvdro-2H-furo[3.2-61ρvτrol- 4(5H56H,6aH)-yl)-l-((lr,4S)-4-methylcyclohexyl)-2-oxoethyl)-3,5-difluorobenzainide
Figure imgf000094_0001
HPLC-MS Rt = 3.30 min, 455.2/457.2 [M + H]+, 473.1/475.1 [M + H + 18]+.
Example 12. N-rr^-2-rf3a5'.65'.6a^-6-cMoro-3-oxodihvdro-2H-fυror3.2-&1pvrrol-
4(5/J,6H,6αiϊ)-yl)-l-((lr545)-4-methylcyclohexyl)-2-oxoethyl)-3-(pyriciin-3- yl)benzamide
Figure imgf000094_0002
ΗPLC-MS Rt = 2.39 min, 496.2/498.2 [M + H]+, 514.2/516.2 [M + H + 18]+.
Example 13. iV-((5}-2-((3aS1,65'56aS)-6-chloro-3-oxodihydro-2H-furo[352-έ]pyπOl- 4(5H,6H,6αiϊ)-yl)-l-((lr,4S)-4-methylcyclohexyl)-2-oxoethyl)-lH- benzo[ύf][l,2,3]triazole -6-carboxamide
Figure imgf000094_0003
HPLC-MS JJt = 2.61 min, 460.2/462.2 [M + H]+, 478.2/480.2 [M + H + 18]+.
Example 14. N-(f^-2-(('3a$',65'.6a$r)-6-chloro-3-oxodihγdro-2H-furor3.2-6lDvrrol- 4(5H,6//,6αH)-yl)-l-((l/%45)-4-methylcyclohexyl)-2-oxoethyl)berLzo[^]thiazole-6- carboxamide
Figure imgf000095_0001
HPLC-MS i?t = 2.91 min, 476.1/478.1 [M + H]+, 494.1/496.1 [M + H + 18]+.
Example 15. N-((S)-2-((3aSβSβaS)-6~chloτo-3-oxodΑydro-2Η.-faxo[3,2-b)pynol- 4(5iJ56H;6flH)-yl)-l-((lr,45)-4-metliylcyclohexyl)-2-oxoethyl)benzo[c][l32,5] oxadiazole-5-carboxamide
Figure imgf000095_0002
ΗPLC-MS Rt = 3.23 min, 461.2/463.2 [M + H]+, 479.2/481.2 [M + H + 18]+.
ExaffiE]eJi. N-((5)-2-((3a5s65'56a^-6-cmoro-3-oxodihydro-2H-furo[352-ό]pyrrol-
4(5H,6H,6αH)-yl)-l-((lr545)-4-metliylcyclohexyl)-2-oxoeth.yl)-lH-indole-5- carboxamide
Figure imgf000095_0003
HPLC-MS Rt = 2.95 min, 458.2/460.2 [M + H]+ 476.2/478.2 [M + H + 18]+.
Example 17. N-r(^)-2-rπaS',66'.6a^)-6-chloro-3-oxodilivdro-2H-fiiror3.2-Z'lpvrrol- 4(5H56H,6αH)-yl)-l-((lr,45)-4-me1hylcyclohexyl)-2-oxoetiiyl)-6-hydroxypicolinaiiiide
Figure imgf000096_0001
HPLC-MS Rt = 2.51 min, 436.2/438.2 [M + H]+, 454.2/456.2 [M + H + 18]+.
Example 18. N-((S)-2-((3aS,6S,6aS)-6-dύoτo--3~oxoάϊhydro-2H-faxo[352-6]pyrrol-
4(5H;6H;6a/i)-yl)-l-((lr,4JS)-4-inethylcyclohexyl)-2-oxoethyl)-2,3-dioxo-l5253,4- tetrahydroquinoxalήie-6-carboxamide
Figure imgf000096_0002
HPLC-MS R1 = 2.00 min, 503.2/505.2 [M + H]+, 521.2/523.2 [M + H + IS]+.
Example 19. N-((^-2-(r3α5'.6&6g^-6-chloro-3-oxodihvdro-2iJ'-furo[3.2-61pvrrol-
4(5H,6H,6αH)-yl)-l-((lr545)-4-methylcyclohexyl)-2-oxoethyl)-2-oxo-l,2,3,4- tetrahydroquinoline-6-carboxamide
Figure imgf000096_0003
HPLC-MS Rt = 2.28 min, 488.2/490.2 [M + H]+, 506.2/508.2 [M + H + 18]+.
Example 20. N-((5)-2-((3α5r,65r,6fl5)-6-chloro-3-oxodiliydro-2/f-furo[3 ,2-Z?]pyrrol- 4(5H,6H56αH)-yl)-l-((lr,45)-4-me1iιylcyclohexyl)-2-oxoethyl)- 3-oxo-3,4-dihydro-2H'- benzofέ] [ 1 ,4]oxazine-7-carboxamide
Figure imgf000097_0001
HPLC-MS Rt = 2.31 min, 490.2/492.2 [M + H]+, 508.2/510.2 [M + H + 18]+.
Example 21. N-((8)-2-((3aS,6S,6a$)-6-dύoro-3 -oxodihydro-2//-furo [3 ,2-ό]pyrrol-
4(5H,6iT,6αH)-yl)-l-((lr,4JS)-4-methylcyclohexyl)-2-oxoetliyl)-4-
(methylsulfonamido)benzamide
Figure imgf000097_0002
ΗPLC-MS Rt = 2.42 min, 512.2/514.2 [M + H]+, 530.2/532.2 [M + H + 18]+.
Example 22. iV-((5)-2-((3α»S',65',6α5)-6-chloro-3-oxodihydro-2//'-furo[3 ,2-ό]pyrrol-
4(5H,6H56αH)-yl)-l-((lr,41S)-4-methylcyclohexyl)-2-oxoethyl)-2-oxo-253,455- tetrahydro-lHr-benzo[δ]azepine-7-carboxamide
Figure imgf000097_0003
HPLC-MS Rt = 2.48 min, 502.2/504.2 [M + H]+, 520.2/522.2 [M + H + 18]"1
Solution Phase Syntheses Alternatively, EXAMPLES of the invention may be prepared by traditional solution phase organic chemistry techniques for example from building block (69) (3R, 3aR, 6S, 6aS)-6~chlorohexahydro-2H"-furo[3,2-&]pyrrol-3-ol (e.g. following the general methods detailed in WO08007127, pg 103-107).
Formation of EXAMPLE .hydrochloride salt.
EXAMPLE ketone (free base) (1 mmol) was dissolved in acetonitrile (16.7 mL) and standardised 0.1N HCl (1.3 eq, 13.0^mL) was added. The mixture was frozen and lyophilised to leave the EXAMPLE .hydrochloride salt as a solid.
EXAMPLE A. Assays for Cysteine Protease Activity
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.
In vitro Cathepsin Ki Inhibition measurements
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). The equilibrium inhibition constants (Kfs) for each compound were measured under steady-state conditions monitoring enzyme activity as a function of inhibitor concentration. The values were calculated on the assumption of pure competitive behaviour (Cornish-Bowden, A. Fundamentals of enzyme kinetics Portland Press; 1995, 93-128.).
Human recombinant cathepsin K (0.25 nM final; B. Turk, Josef, Stefan Institute, Ljubljana, Slovenia), was routinely assayed in 100 mM sodium acetate; pH 5.5 containing 1 mM EDTA, 10 mM L-cysteine and 1.8 μM Z-Leu-Arg-AMC ([S]=KM). Human recombinant eathepsin S (0.25nM final, Merck, E.coli cat # 219343) was routinely assayed in 1OmM Bis Tris Propane; pH 6.5 containing ImM EDTA, 5mM β mercaptoethanol, ImM CaCl2 and 45μM Boc-Val-leu-Lys-AMC ([S] = KM) (Sigma Chemical Company, Poole, U.K.).
Human liver cathepsin B (0.25 nM final; Merck Biosciences), was routinely assayed in 10 mM bis-tris propane; pH 6.5 containing 1 mM EDTA, 5 mM 2-mercaptoethanol, 1 mM CaCl2 and 60 μM Z-Phe-Arg-AMC
Figure imgf000099_0001
(Bachem, Weil am Rhein, Germany).
Human recombinant cathepsin V (0.25 nM final, Merck Biosciences) was routinely assayed in 100 mM sodium acetate; pH 5.5 containing 10 mM L-cysteine, 0.001% (vol./vol.) zwittergent 3-12 (Merck Biosciences) and 5 μM Z-Leu-Arg-AMC (Ku= 0.5 μM) (Amura).
Human liver cathepsin L (0.25 nM final, Athens Research and Technology, GA, USA) was routinely assayed in 10 mM bis-tris propane; pH 6.5 containing 1 mM EDTA, 5 mM 2-mercaptoethanol, 1 mM CaCl2 and 4 μM Ac-Phe-Arg-AMC ([S] = Ku) (Bachem).
Measurement of the apparent macroscopic binding (Miehaelis) constants 6£iuapp) for substrates
The apparent macroscopic binding constant (Ku^) 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.). Vm "PP r p -[
V, = ■
[so] + κ app (1)
M
In Equation 1 Vi' is the observed initial rate, ' Fmax app' is the observed maximum activity at saturating substrate concentration, 'Jζyiapp' is the apparent macroscopic binding (Michaelis) constant for the substrate, ' [S0]' is the initial substrate concentration.
Measurement of the inhibition constants
The apparent inhibition constant (Kϊ) for each compound was determined on the basis that inhibition was reversible and occurred by a pure-competitive mechanism. The Ki values were calculated, from the dependence of enzyme activity as a function of inhibitor concentration, by direct regression analysis (Prism v 3.02) using Equation 2 (Comish-Bowden, A., 1995.).
V app LSI v, = ≡≡ — (2)
[S] + (K^XlI]ZK1)]
In Equation 2 ' Vi' is the observed residual activity, ' Fmax app' is the observed maximum activity (i.e. in the absence of inhibitor), 'JΪM app' is the apparent macroscopic binding (Michaelis) constant for the substrate, '[S]' is the initial substrate concentration, 'K\ is the apparent dissociation constant and ' [I]' is the inhibitor concentration.
In situations where the apparent dissociation constant (K^) approached the enczyme concentrations, the Kιapp values were calculated using a quadratic solution in the form described by Equation 3 (Morrison, J.F. Trends Biochem. Sci.,_l, 102-105, 1982; Morrison, J.F. Biochim. Biophys. Acta^ 185, 269-286, 1969; Stone, S.R. and Hofsteenge, J. Biochemistry, 25, 4622-4628, 1986).
F(E0 -i0 ~κr +V(E0 -i0 -κ:ppf +4.κr-E0 > K^ = KXl + [S0]I KM opp) (4)
In Equation 3 'vf is the observed residual activity, CF' is the difference between the maximum activity {i.e. in the absence of inhibitor) and minimum enzyme activity, 'E0' is the total enzyme concentration, '!Tjapp' is the apparent dissociation constant and 'I0' is the inhibitor concentration. Curves were fitted by non-linear regression analysis
(Prism) using a fixed value for the enzyme concentration. Equation 4 was used to account for the substrate kinetics, where 1KC is the inhibition constant, '[S0]' is the initial substrate concentration and ςKu w is the apparent macroscopic binding
(Michaelis) constant for the substrate (Morrison, 1982).
The second-order rate of reaction of inhibitor with enzyme
Where applicable, the concentration dependence of the observed rate of reaction (fcobs) 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, 61, 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.
Figure imgf000101_0001
The activity fluorescence progress curves were fitted by non-linear regression analysis (Prism) using Eq. 5 (Morrison, 1969; Morrison, 1982); where 'F' is the fluorescence response, 't' is time, 'v0' is the initial velocity, 'vs' is the equilibrium steady-state velocity, 'Arobs' 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 £obs versus the inhibitor concentration (i.e. kObJ[ϊ]). To correct for substrate kinetics, Eq. 6 was used, where '[S0]' is the iniitial substrate concentration and 'JKM3PI" is the apparent macroscopic binding (Michaelis) constant for the substrate.
Kinact
Figure imgf000102_0001
Compounds of the invention when tested by the above described assays exhibit cathepsin K inhibitory activity with an in vitro Ki inhibitory constant of less than or equal to 10OnM.
Liver Microsomal Incubations:
Human and rat liver microsomes were purchased from BD Gentest (Woburn, MA,
USA) and β-nicotmamide adenine dinucleotide 2'-phosphate reduced tetrasodium SaIt (NADPH) was purchased from Sigma- Aldrich (Poole, Dorset, UK). All liver microsome incubations were carried out in 50 mM potassium phosphate buffer at pH 7.4, with a final microsomal protein concentration of 0.5 mg/mL. Compounds were taken from 5 mM DMSO stock solutions and diluted in incubation buffer to give a final concentration of 25 μM, with a final DMSO concentration of 0.5% v/v. In brief, compounds were added to the incubation buffer along with the liver microsomes and incubated at 37°C for 10 minutes. The reaction was then initiated by the addition of NADPH, previously dissolved in incubation buffer, to give a final concentration of 1 mM and re-incubated at 370C. Aliquots were removed at 2 and 60 minutes and quenched with an equal volume of cold acetonitrile. After mixing vigorously, the precipitated protein matter was removed by filtration (Multiscreen Solvinert filter plates, Millipore, Bedford, MA, USA) and the filtrate analysed by reverse phase HPLC with mass spectrometric detection, using single ion monitoring of the [M+H]+ species. Metabolic turnover was determined by comparison of peak areas from the ion chromatograms of the parent compound at 2 and 60 minutes and expressed as percent remaining at 1 hour. Plasma Incubations:
Human and rat plasma were purchased from Innovative Research Inc. (Southfield. MI, USA). Compounds were taken from 5 mM DMSO stock solutions and added to plasma, which had previously been incubated at 37°C, to give a final concentration of 25 μM and re-incubated. Aliquots were removed at 2 and 60 minutes and quenched with an equal volume of cold acetonitrile. After mixing vigorously, the precipitated protein matter was removed by filtration (Multiscreen Solvinert filter plates, Millipore, Bedford, MA, USA) and the filtrate analysed by reverse phase HPLC with mass spectrometric detection, using single ion monitoring of the [M+H] species. Metabolic turnover was determined by comparison of peak areas from the ion chromatograms of the parent compound at 2 and 60 minutes and expressed as percent remaining at 1 hour.
LogD Determinations: LogD(PBS) determinations were performed in 96 well microtitre plates using a miniaturised "shake-flask" method. In brief, compounds were taken from 10 mM DMSO stock solutions and added to wells containing equal volumes of phosphate buffered saline (10 mM; pH 7.4) (PBS) and 1-octanol (Sigma- Aldrich, Poole, Dorset, UK) to give a final concentration of 50 μM. The plates were then capped and mixed vigorously for 1 hour on a microtitre plate shaker, after which they were left to stand, allowing the PBS and octanol phases to separate. The PBS layer was analysed by reverse phase HPLC with mass spectrometric detection, using single ion monitoring of the [M+H]+ species. LogD(pβs) was determined by comparison of the peak area from the ion chromatograrn of the compound in the PBS phase with that of a 50 μM standard of the same compound dissolved in acetonitrile/water (50:50) and calculated using the following formula:
Figure imgf000103_0001
Where AUCstd and AUCpbs are the peak areas from the standard and test ion chromatograms respectively. LogD(PBS) determinations were also made using PBS at pH6.9 and 5.5 by adjusting the pH of the buffer prior to the start of the assay, with 0.1 M HCL.
Various modifications and variations of the described aspects 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 of carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.
Table 1: Biological properties for EXAMPLE compounds, prior art compound (38)
(WO-A-02057270, pg 151) and novel Compounds 1 - 15.
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt, hydrate, complex or pro-drug thereof,
Figure imgf000110_0001
(I) wherein: one of R3 and R4 is H, and the other is selected from C1-6-alkyl, C1-6-haloalkyl, C1-6- alkoxy and C6-12-aralkyl; or R3 and R4 are each independently selected from Ci-6-alkyl and halo; R9 is selected from me following:
Figure imgf000111_0001
wherein:
Xi, X2, X3, X4, X14, X15jX16 andX2o are each independently selected from:
CH, C-(C1-6-alkyl), C-(Ci-6-alkoxy), C-halo and N; such that a maximum of two of X1, X2, X3, X4, X14, X15, X16 and X20 are selected from N, C-halo and C-(Ci-6-alkoxy); X5, X6, X7 and Xs are each independently selected from:
CH, C-(Ci.6-alkyl), C^d-e-alkoxy), C-halo, N and C-OH; such that a maximum of one of X5, X6, X7 and X8 is N, C-halo, C-OH or C-(C1-O- alkoxy);
Xp and Xi2 are each independantly selected from:
CH, C-(d-e-alkyl), C-(C1-6-alkoxy), C-halo and N;
X1O and X11 are each iiidependantly selected from:
CH, C-(Ci-6-alkyl), C-(d_6-alkoxy)5 C-halo, N and R10;
Xi9 is selected from:
CH, C-(C1-6-alkyl), C-(C1-6-alkoxy), C-C(O)NH2, C-C(O)NH(C1-6-alkyl), C-C(O)N(C1-6-alkyl)2, C-halo and N;
X18 is selected from:
CH, C-(C1-6-alkyl), C-(C1-6-alkoxy), C-NH2, C-N(C1-6-alkyl)2, C-NH(Ci- 6-alkyl), C-NHC(O)C1-6-allcyl, C-halo and N; or when X19 is CH, C-(C1-6-alkyl), or C-halo then Xi8 may additionally be selected from C-C(O)NH2 and C-C(O)N(C1-6-alkyl)2;
Xi3 and X17 are each independently selected from: O, S, NH and N-(C1-6-alkyl);
X22 and X24 are each independently selected from:
CH2, CH-(Ci-6-allsyl), O, S, NH, NMe and ) C=O;
X23 is selected from:
CH2, CH-(Ci-6-alkyl), C-(C1-6-alkyl)2, NH and NMe; or when either X22 or X24 are other than ) C=O then X23 may additionally be ) C=O or ) S(O)2; X25 is selected from:
O, S, NH and N(C1-6-alkyl);
X26, X27, X28 and X29 are each independently selected from:
CH3 C-(C1-6-alkyl), C-(C1-6-alkoxy), C-OH, C-halo and N; such that a maximum of two of X26, X27, X28 and X29 are selected from C-(C1-6- alkoxy), C-OH, C-halo and N;
X30 is selected from:
CH2, CH2CH2, NH, NMe, O, S and )C=0;
X31 is selected from:
CH2, NH and NMe; or when X30 is other than )C=0, O or S then X31 may additionally be
)C=O or O;
X32 is selected from:
CH2, CH2CH2, NH, NMe and ) C=O;
X33 is selected from:
CH2, NH and NMe; or when X32 is other than ) C=O then X33 may additionally be ) C=O or
O;
X34 is selected from:
NH and NMe;
Rio is selected from:
Figure imgf000114_0001
wherein:
Ti, T2, T3 and T4 are each independantly selected from:
CH5 C-(C1-6-alkyl), C-(C1-6-alkoxy), C-NH2, C-NH(Ci-6-alkyl), C-N(C1.
6-alkyl)2, C-halo and N; such that a maximum of one of Ti, T2, T3 and T4 is C-(Ci-6-alkoxy), C-NH2, C-NH(C1- 6-alkyl), C-N(C1-6-alkyl)2 or C-halo;
T5 is selected from:
O, S, NH and N(Cϊ-6-alkyl);
T6, T7, T8, T9 and Ti0 are each independantly selected from:
CH, C-(C1-6-alkyl), C-(C1-6-alkoxy), C-NH2, C-NH(C1-6-alkyl), C-N(C1- 6-alkyl)2, C-halo and N; such that a maximum of two of T6, T7, T8, T9 and T1O are selected from C-(C1-6- alkoxy), C-NH2, C-NH(C1-6-aIkyl), C-N(C1-6-aIkyl)2, C-halo and N;
Tn is selected from:
CH2, NH and N(C1-6-alkyl);
Ti2 is selected from:
CH2, NH, N(Ci-6-alkyl) and )C=O; Ti3 and T14 are each independantly selected from:
CH, C-(C1-6-alkyl) and C-halo;
T15 is selected from:
0, NH andN(Ci-6-alkyl);
T] 6 is selected from:
CH2 and ) C=O;
or R10 is selected from:
H, Ci-6-alkyl, OH, Ci-6-alkoxy, NO2, halo, CN, C(O)NH2, C(O)NH(C1-6- alkyl), C(O)N(C1-6-alkyl)2, C(O)NH(C3-6-cycloalkyl)5 S(O)2NH2, S(O)2(C1-6-alkyl), S(O)2NH(C1-6-alkyl), S(O)2N(C1-6-alkyl)2,
S(O)2NH(C3-6-cycloalkyl) and (CH2)H-NR11R12;
wherein n is O or 1;
and R11 is selected from C1-6-alkyl, C(O)C1-6-alkyl, C(O)(C3-6-cycloalkyl), C(O)(aryl), C(O)NH2, C(O)NH(C1-6-alkyl), C(O)N(C1-6-alkyl)2, C(O)NH(C3-6-cycloalkyl), C(0)0(Ci-6-alkyl), C(O)O(C3.6-cycloalkyl), C(O)O(aryl), S(O)2(C1 -6-alkyl), S(O)2(C3- 6-cycloalkyl), S(O)2NH2, S(O)2NH(Ci-6-alkyl),
Figure imgf000115_0001
S(O)2NH(C3-6- cycloalkyl) and S(O)2(aryl);
and R12 is selected from H and C1-6-alkyl.
R13 is selected from:
C(O)NH2, C(O)NH(C1-6-alkyl), C(O)N(C1.6-alkyl)2, C(O)NH(C3-6- cycloalkyl), S(O)2NH2, S(O)2(C1-6-alkyl), S(O)2NH(C1.*;^!), S(O)2N(C1-6-alkyl)2, S(O)2NH(C3-6-cycloalkyl) and (CH2)n-NR14Rls;
wherein n is O or 1 ; and R14 is selected from H, C^-alkyl, C^d-e-alkyl, C(0)(C3-6-cycloalkyl),
C(O)(aryl), C(O)NH2, C(O)NH(Ci.6-alkyl), C(O)N(C1-6-alkyl)2, C(O)NH(C3-6- cycloalkyl), C(O)O(Ci.6-alkyl), C(O)O(C3-6-cycloaLkyl)3 C(O)O(aryl), S(O)2(C1-6- alkyl), S(O)2(C3.6-cycloalkyl)5 S(O)2NH2, S(O)2NH(C1-6-alkyl), S(O)2N(C1-6-alkyl)2, S(O)2NH(C3-6-cycloalkyl) and S(O)2(aryl);
and R15 is selected from H and Ci-6-alkyl.
2. A compound according to claim 1 wherein:
R3 is H and R4 is selected from methyl, ethyl, ^-propyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, ethoxy and ben2yl: or both R3 and R4 are selected from methyl or fluoro or chloro;
X1, X2, X3, X4, X14, X15, Xi6 and X2o are independently selected from:
CH, CMe, C-OMe, C-F, C-Cl and N: such that a maximum of two OfX1, X2, X3, X4, Xi4, X1S1Xi6 and X20 are chosen as N or C-Cl or C-OMe;
X5, X6, X7 and X8 are independently selected from:
CH, CMe, C-OMe, C-F, C-Cl, N and OH; such that a maximum of one of X5, X6, X7 and X8 is chosen as N or C-CI or C-OH or C-OMe;
X9 and Xi2 are independently selected from:
CH5 CMe, C-OMe, C-F, C-Cl and N;
Xio and Xπ are independently selected from:
CH, CMe, C-OMe, C-F, C-Cl, N and R10;
Xi9 is selected from:
CH5 CMe, C-OMe, C-C(O)NH2, C-C(O)NMe2, C-F, C-Cl and N; Xi 8 is selected from:
CH, CMe5 C-OMe5 C-NH2, C-NMe2, C-NHMe5 C-NHC(O)Me5 C-F5 C- Cl and N; or when X19 is CH5 CMe or C-F then X1 g may additionally be selected from C-C(O)NH2 and C-C(O)NMe2;
XB and X]7 are independently selected from: O5 S5 NH and NMe.
X22 and X24 are independently selected from:
CH2, CHMe5 O5 S5 NH5 NMe and ) C=O;
X23 is selected from:
CH2, CHMe5 CMe2, NH and NMe; or when either X22 or X24 are other than ) C=O then X23 may additionally be ) C=O or )S(O)2;
X25 is selected from:
O5 S5 NH and NMe;
X26, X27, X28 and X29 are independently selected from:
CH, CMe, C-OMe, C-F, C-Cl5 C-Br and N; such that a maximum of two of X26, X27, X28 and X29 are chosen as C-OMe5 C-Cl, C-Br and N;
X30 is selected from:
CH2, CH2CH25NH5 NMe5 O5 S and }θ=0;
X3! is selected from:
CH2, NH and NMe; or when X30 is other than )C=O, O or S then X31 may additionally be )C=O or O;
X32 is selected from:
CH2, NH3 NMe and ) C-O;
X33 is selected from:
CH2, NH and NMe; or when X32 is other than )C=O then X33 may additionally be )C=O or O;
X34 is selected from:
NH and NMe;
T1, T2, T3 and T4 are independantly selected from:
CH, CMe, C-OMe, C-NH2, C-NHMe, C-NMe2, C-F, C-Cl and N: such that a maximum of one of T1, T2, T3 and T4 is chosen as C-OMe, C-NH2, C- NHMe, C-NMe2, C-F and C-Cl;
T5 is selected from:
O, S, NH and NMe.
T6, T7, T8, T9 and T10 are independantly selected from:
CH, CMe, C-OMe, C-NH2, C-NHMe, C-NMe2, C-F, C-Cl andN: such that a maximum of two of T6, T7, T8, T9 and Ti0 are chosen as C-OMe, C-NH2, C- NHMe, C-NMe2, C-F, C-Cl andN;
Tn is selected from:
CH2, NH and NMe; T12 is selected from:
CH2, NH, NMe and ) C=O;
TB and T14 are independently selected from: CH, CMe3 C-F and C-Cl;
T15 is selected from:
O, NH and NMe;
Tj6 is selected from:
CH2 and ) C=O;
or R10 is selected from:
H, Me, OH, OMe, OEt, OiPr, NO2, F, Cl, Br, CN, C(O)NH2, C(O)NHMe, C(O)NMe2, and (CH2VNR11R12:
wherein n = O or 1 and R11 is selected from H, Me, acetyl, C(O)NH2, C(O)NMe2:
11 and R is selected from H and Me;
Ri3 is selected from:
C(O)NH2, C(O)NHMe, C(O)N(Me)2, C(O)NH(cyclopropyl), S(O)2NH2, S(O)2(Me), S(O)2NH(Me), S(O)2N(Me)2, S(O)2NH(cyclopropyl) and (CH2VNR14R15;
wherein n is O or 1 ;
and R14 is selected from H5 Me, C(O)Me, C(O)(cyclopropyl), C(O)Ph, C(O)NH2, C(O)NH(Me), C(O)N(Me)2, C(O)NH(cyclopropyl), C(O)O(Me), C(O)O(cyclopropyl), C(O)OPh, S(O)2(Me), S(O)2(cyclopropyl), S(O)2NH2, S(O)2NH(Me), S(O)2N(Me)2, S(O)2NH(cyclopropyl) and S(O)2Ph; and R is selected from H and Me.
3. A compound according to claim 1 or claim 2 which is of formula Ia,
Figure imgf000120_0001
(Ia)
wherein R3, R4 and R9 are as defined in claim 1.
4. A compound according to claim 1 or claim 2 which is of formula Ib,
Figure imgf000120_0002
(Ib)
wherein R , R >4 and R are as defined in claim 1.
5. A compound according to any preceding claim wherein R3 is H, and R4 is selected from methyl, ethyl, propyl, trifluoromethyl and benzyl.
6. A compound according to any one of claims 1 to 4 wherein R3 and R4 are each independently selected from methyl, fiuoro and chloro.
7. A compound according to claim 6 wherein R3 and R4 are both, methyl.
8. A compound according to any one of claims 1 to 5 which is of formula Ic,
Figure imgf000121_0001
(Ic) wherein R is as defined in claim 1.
9. A compound according to any preceding claim wherein R9 is selected from:
Figure imgf000121_0002
Figure imgf000121_0003
wherein Xi, X2, X3, X4, X5, X6, X7, Xs, X9, Xi0, Xn3 Xi3, Xw, Xi5, Xi6;. Xi7, Xi8, Xi95 X22, X23, X24, X25, X30, X31, X34, Rio and R13 are as defined in claim 1.
10. A compound according to any preceding claim wherein R is selected from:
\
X-io
\^°
X 19 χ(
^17
/
^3D
X31 X
Figure imgf000122_0001
wherein Xj, X2, X3, XA, XJ, X\O, XM, X\%, X\% X25, X30, X31, Rio and R14 are as defined in claim 1.
11. A compound according to any preceding claim wherein R9 is selected from:
Aryl \ Λ HCL \ \
X 19
Xi9 X ■ y/ J \
/
X N-
Λ25 \
O
Figure imgf000122_0002
X23- -o
Figure imgf000123_0001
wherein aryl, X18, X19, X23, X25 are as defined in claim 1 and;
X2 and X3 are each independently selected from: CH, CMe and C-F;
X3O is selected from:
CH2, CH2CH25NH, NMe and O;
X31 is selected from:
CH2, NH and NMe; or when X30 is NH or NMe then X31 may additionally be ) C=O;
and R14 is selected from C(O)Me, C(O)(cyclopropyl), C(O)NH2, C(O)NH(Me), C(O)N(Me)2, C(O)NH(cyclopropyl), C(O)O(Me), C(O)O(cyclopropyl)5 S(O)2(Me), S(0)2(cyclopropyl), S(O)2NH2, S(O)2NH(Me), S(O)2N(Me)2, S(O)2NH(cyclopropyl) and S(O)2Ph.
12. A compound according to any preceding claim wherein R9 is selected from:
Figure imgf000124_0001
Figure imgf000124_0002
Figure imgf000124_0003
Figure imgf000124_0004
wherein X18 is as definedin claim 1.
13. A compound according to any preceding claim wherein R9 is selected from:
Figure imgf000125_0001
Figure imgf000125_0002
Figure imgf000125_0003
Figure imgf000125_0004
14 A compound according to any preceding claim which is selected from the following:
N-((5)-2-((3a5,6^6a5)-6-cMoro-3-oxodmydro-2H-furo[3,2-6]pyrrol-4(5H56H,6flii0-yl)- l-((lr,4S)-4-memylcyclohexyl)-2-oxoethyl)-3-(lH-tetrazol-l-yl)benzarnide
N-((5)-2-((3aS56S,6a^-6-cωoro-3-oxodmydro-2H-furo[3,2-δ]pyrrol-4(5iϊ,6H,6αiϊ)-yl)- l-((lr,4^-4-memylcyclohexyl)-2-oxoethyl)-3-(lH-imidazol-l-yl)berizarnide N-((5)-2-((3a5,6^6a^-6-chloro-3-oxodihydro-2H-fco[352-ό]pyirol-4(5H,6H;6α/i)-yl)- l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-3-(4H-l,2,4-triazol-4-yl)benzamide
N-((5)-2-((3a5,6556a-?)-6-cHoro-3-oxodihydro-2H-furo[3s2-5]pyrrol-4(5H,6H,6αH)-yl)- 1 -(( 1 r,45)-4-methylcyclohexyl)-2-oxoethyl)-3 -( 1 Η-pyrazol- 1 -yl)benzamide
N-((5}-2-((3a5,65'56a^-6-cΗoro-3-oxodihydro-2Η-furo[3,2-δ]pyrrol-4(5H56H,6αH)-yl)- l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-3-(4H-l,2,4-triazol-4-yl)benzamide
N-((^-2-((3a556S36a^-6-cMoro-3-oxodihydro-2H-too[3,2-&]pyrrol-4(5H,6H,6flH)-yl)- l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)nicotinamide
N-((5)-2-((3a^,6^6a^-6-cMoro-3-oxodihydro-2H-furo[3,2-6]pyrrol-4(5H,6H,6flH)-yl)- 1 -((1 r,45)-4-methylcyclohexyl)-2-oxoethyl)isonicotinainide
N-((^-2-((3a5,65,6a^-6-cMoro-3-oxodihyώ:o-2H-furo[332-έ]pyrrol-4(5H56Zr,6αH)-yl)- 1 -(( 1 r,45)-4-methylcyclohexyl)-2-oxoethyl)furan-2-carboxamide
N-((S)-2-((3a536S',6aS)-6-cMoro-3-oxodihydro-2H-tøo[352-&]pyrrol-4(5H,6H,6αH)-yl)- l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-3-(pyridiii-3-yl)benzamide
N-((S)-2-((3a5,65',6a^-6-cMoro-3-oxod%clro-2Η-furo[352-&]pyrrol-4(5H,6H36flH)-yl)- l-((lrs45)-4-methylcyclohexyl)-2-oxoethyl)-lΗ-benzo[<i][l3233]triazole-6-carboxainide
N-((5)-2-((3aS,6536a^-6-cWoro-3-oxodihydro-2H-furo[332-Z)]pyiTol-4(5H36H;6flH}-yl)- 1 -(( 17',4,S)-4-methylcyclohexyl)-2-oxoethyl)benzo [<i]thiazole-6-carboxaniide
N-((5)-2-((3aS365l 36a^-6-cHoro-3-oxodmydro-2H-furo[332-Z»]pyrrol-4(5H,6i7,6αJ?)-yl)- 1 -(( 1 r34S)-4-methylcyclohexyl)-2-oxoethyl)benzo [c] [1 ,2,5] oxadiazole-5 -caxboxamide N<(^-2-((3a5,6556a-5)-6-cMoro-3-oxodihydro-2H-furo[3,2-δ]pyrrol-4(5JfJ6/i6αH)-yl)-
1 -(( 1 r,4S)-4-methylcyclohexyl)-2-oxoethyl)- 1 H-indole-5-carboxamide
N-((5)-2-((3a^56556a5)-6-chloro-3-oxodihydro-2H-foro[3,2-ά]pyrrol-4(5/i6H,6αH)-yl)- 1 -((1 r^^^-methylcyclohexy^^-oxoethy^-β-Iiydroxypicolinamide
N-((5)-2-((3a5,6S36a^-6-cldoro-3-oxodihydro-2H-furo[3,2-&]pyrrol-4(5H;6H,6«H)-yl)- l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)benzo[^][l,3]dioxole-5-carboxamide
N-((S)-2φaSβSβaS)-6~cMoτo-3-oxodjhyόio-2H-faro[3,2-b]ρyττol-A(5H,6H,6aH)-y\)- 1 -(( 1 r,4S)-4-methylcyclohexyl)-2-oxoethyl)-253-dioxo- 1 ,2,3 ,4-tetraliydroquinoxalhie-6- carboxamide
^((5}-2-((3α5.65l,6fl^-6-cMoro-3-oxodihydro-2H-furo[3,2-&]pynol-4(5H56H36fl^ 1 -(( 1 r,4S)-4-methylcyclohexyl)-2-oxoethyl)-2-oxo- 1 ,2,3 ,4-tetrahydroquinoline-6- carboxamide
N-((5)-2-((3α5',6^6α5)-6-cMoro-3-oxodihydro-2H-furo[352-ό]pyrrol-4(5H,6H;6αH)-yl)- l-((lr54S)-4-methylcyclohexyl)-2-oxoethyl)-3-oxo-3,4-dihydro-2H- benzo [5] [ 1 ,4]oxazine-7-carboxamide
N-((5)-2-((3α5s65'56^-6-cMoro-3-oxodihydro-2H-flu-o[3,2-&3pyirol-4(5H56H,6^-yl> l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-2-oxo-2,3,4,5-tetrab.ydro-lH"- benzo[ό]azepine-7-carboxamide
N-((5)-2-((3αS56^6α^-6-chloro-3-oxodihydro-2H-furo[3,2-δ]pyrrol-4(5H,6H;6αi?)-yl)- l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-4-(methylsulfonaiiiido)benzainide
^((^^-((S^ό^όa^-β-chloro-S-oxodihydro^Η-furoP^-δjpyrroMCSH^^βαJ^-yl)- 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)-3 -(I Η-tetrazol- 1 -yl)benzamide N-((5)-2-((3a5,6Sr,6aS)-6~chloro-3-oXodihydro-2H-furo[3,2-δ]pyrrol-4(5H56H56flH)-yl)-
1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)-3 -(I Η-imidazol- 1 -yl)benzamide
N-((5}-2-((3a51 565f,6a5)-6-cMoro-3-oxodihydro-2Η-furo[3,2-ό]pyrrol-4(5H,6H56αH)-yl)- 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)-3 -(4H- 1 ,2,4-triazol-4-yl)benzamide
N-((^-2-((3a556S,6a^-6-chloro-3-oxodihydro-2H-furo[3;2-6]pyrrol-4(5H,6H;6flH)-yl)- l-(434-dimethylcyclohexyl)-2-oxoethyl)-3-(lH-pyrazol-l-yl)benzamide
N-((^-2-((3a5565r,6a^-6-cWoro-3-oxodihydro-2H-furo[3,2-Z>]pyrrol-4(5H,6F,6aH)-yl)- 1 -(4,4-dimeth.ylcyclohexyl)-2-oxoethyl)-3 -(4H- 1 ,2,4-triazol-4-yl)benzamide
N-((^-2-((3a5f 365r,6aS)-6-cMoro-3-oxodihyciro-2H-furo[352-έ]pyrrol-4(5iy56i7,6^i7)-yl)- l-(4,4-dimethylcyclohexyl)-2-oxoethyl)nicotinamide
N-((S)-2-((3a556S,6a5)-6-cmoro-3-oxodihydro-2H-furo[352-δ]pyrrol-4(5H.6iϊ,6flH)-yl)- l-(4,4-dimethylcyclohexyl)-2-oxoetb.yl)isonicotinamide
N-((5)-2-((3a5,6^6a^-6-cmoro-3-oxod%dro-2H-furo[3,2-&]pyrrol-4(5H56H36αH)-yl)- l-(4,4-dimethylcyclohexyl)-2-oxoethyl)furajα-2-carboxamide
iV-((^-2-((3a5,65,6a5)-6-cHoro-3-oxodihydro-2H-foro[352-έ]pyrrol-4(5H,6H,6βi?)-yl)- 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)-3 -(pyridin-3 -yl)benzamide
N-((5)-2-((3a^565'J6a5)-6-cWoro-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H36H,6flH)-y0 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)- 1 H-benzo [ d\ [ 1 ,2,3]triazole-6-carboxamide
N-((5)-2-((3a556^6aS)-6-cMoro-3-oxodmydro-2H-furo[3,2-ό]pyrrol-4(5/i6H;6flH)-yl)- l-(4,4-dimethylcyclohexyl)-2-oxoethyl)benzo[c(]thiazole-6-carboxainide N-((5)-2-((3a5,65,6a5)-6-clύoro-3-oxodihydro-2H-furo[352-ό]pyrrol-4(5H36H;6flH)-yl)- l-(4,4-dimethylcycloliexyl)-2-oxoethyl)benzo[c][l;,2.5]oxadia2;ole-5-carboxainide
Λr-((5)-2-((3aS,65',6aS)-6-cΗoro-3-oxodihydro-2Η-furo[332-ό]pyrrol-4(5H,6H56αiϊ)-yl)- 1 -(4,4-dimethylcycIohexyl)-2-oxoetliyl)- 1 H-indole-5-carboxamide
^-((^^-((S^β^βa^-ό-cωoro-S-oxodihydro^H-furotS^-δlpyrroMCSH^H^α/^-yl)- l-(4,4-dimethylcyclohexyl)-2-oxoethyl)-6-hydroxypicolinamide
N-((5)-2-((3a^,65,6a^-6-cWoro-3-oxodihydro-2H-furo[352-6]pyrrol-4(5/i6H,6aH)-yl)- 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)benzo [J] [1 ,3] dioxole-5 -carboxamide
N-((S)-2φaSβSβaS)-6-cMoro-3-oxodϊhydτo-2H-faro[3,2-b]pynol-4(5H,6ff,6aH)-yϊ)- 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)-2,3 -dioxo- 1 ,2,3 ,4-tetrahydroquinoxaline-6- carboxamide
N-((5)-2-((3α5',65',6fl^-6-cMoro-3-oxodmycko-2/f-furo[3,2-έ]pyiτol-4(5H,6if,6αH)-yl)- 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)-2-oxo- 1 ,2,3 ,4-tetrahydroquinoline-6- carboxamide
N-((5)-2-((3αS,65r,6α5)-6-cMoro-3-oxodihydro-2H-ftιro[352-b]pyiτol-4(5H,6H56βH)-yl)- l-(4,4-dimethylcyclohexyl)-2-oxoethyl)-3-oxo-3,4-dihydro-2H-beri2:o[έ][l,4]oxazine-7- carboxamide
N-((S)-2-((3aS,6S,6aS)-6-chloio-3-oxodihydτo-2H-fmo[3,2-b]pynolA(5HMMH)-yiy 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)-2-oxo-2,3 ,4,5 -tetrahydro- 1 i7-benzo [b] azepine- 7-carboxamide
N-((5)-2-((3fl5,65,6«5)-6-cMoro-3-oxodmydro-2H-furo[3,2-&]pyrrol-4(5H,6H,6fl/i)-yl)- 1 -(4,4-dimethylcycloh.exyl )-2-oxoetb.yl)-4-(methylsulfonamido)benzamide
15. A compound according to claim 14 which is selected from the following:
N<(5)-2-((3a5,65',6a^-6-cmoro-3-oXodmydro-2H-furo[3,2-δ]pyrrol-4(5i756H;6flH)-yl)- l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)benzamide [I];
N-((^-2-((3aS,6^6aS)-6-cmoro-3-oxodmydro-2H-furo[352-6]pyrrol-4(5H,6H56αH)-yl)- l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-3-(lH-tetrazol-l-yl)berizamide [2];
N-((^-2-((3a5565,6a5)-6-cmoro-3-oxodmydro-2H-fco[352-b]pyiτol-4(5H,6H36flH)-yl)- l-(4,4-dimethylcyclohexyl)-2-oxoethyl)-3-(lΗ-tetrazol-l-yl)benzaπiide [3];
N-((5)-2-((3a5565,6a^-6-cWoro-3-oxodihydro-2H-fiu:o[352-6]pyrrol-4(5H,6H,6aH)-yl)- 1 -((1 r,4iS)-4-methylcyclohexyl)-2-oxoethyl)-3 -(I H-imidazol- 1 -yl)benzamide [4] ;
^((5}-2-((3a^,65,6a5)-6-cMoro-3-oxodmydro-2H-furo[352-ό]pyrrol-4(5/i6H,6flH)-yl)- l-((lrs45)-4-methylcyclohexyl)-2-oxoe1hyl)-3-(4H-l,2,4-tria2ol-4-yl)benzamide [5];
N<(^-2-((3a5,65,6a5)-6-cMoro-3-oxodmycko-2H-iuro[352-&]pyrrol-4(5H;6H;6«H)-yl)- 1 -((1 r,46)-4-methylcyclohexyl)-2-oxoethyl)-3 -(I H-pyrazol- 1 -yl)benzamide [6] ;
ΛT-((S)-2-((3a536S,6a5)-6-cMoro-3-oxodmydϊo-2H-furo[352-5]pyrrol-4(5H,6H,6αH)-yl)- l-((lr54S)-4-methylcyclohexyl)-2-oxoethyl)-3-(4Η-l,2,4-triazol-4-yl)benzainide [7];
N-((^-2-((3a5365',6a6)-6-cMoro-3-oxodihydro-2H-furo[352-ό]pyrrol-4(5/i;6H;6αH)-yl)- l-((lr,4,S)-4-methylcyclohexyl)-2-oxoethyl)nicotinamide [8];
N-((^-2-((3a5r 56S,6a5)-6-cWoro-3-oxodihydro-2H-furo[3}2-&]pyrrol-4(5H,6H,6aH)-yl)- 1 -(( 1 r,45)-4-methylcyclohexyl)-2-oxoethyl)isonicotinamide [9] ;
N-((5)-2-((3a5'565's6a^-6-cHoro-3-oxodmydro-2H-furo[352-δ]pyrrol-4(5H,6/f,6αH)-yl)- 1 -(( 1 r,4S)-4-methylcyclohexyl)-2-oxoethyl)furan-2-carboxamide [10]; N-((^-2-((3a5565,6a-5)-6-cWoro-3-oxodihydro-2H-too[3,2-5]pyrrol-4(5H56H,6«H)-yl)- l-((lr.45)-4-methylcyclohexyl)-2-oxoethyl)-3.5-difluorobenzamide [H];
N-((5)-2-((3a5',6^6a^-6-cMoro-3-oxodihydro-2Η-furo[352-ό]pyrrol-4(5/f,6H;6αH)-yl)- l-((lr545)-4-methylcyclohexyl)-2-oxoethyl)-3-(pyridin-3-yl)benzamide [12];
N-((^-2-((3a5,65,6a^-6-cWoro-3-oxodihydro-2H-furo[352-&]pyrrol-4(5H,6H;6flH)-yl)- l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-lH-benzo[^][l,2,3]triazole -6-carboxamide [13];
ΛH(^-2-((3a£,6S,6aS)-6-cMoro-3-oxodihydro-2H-to^
1 -(( 1 r,45)-4-methylcyclob-exyl)-2-oxoethyl)benzo [d]thiazole-6-carboxamide [14];
N-((^-2-((3a556^6a6)-6-cMoro-3-oxodmycko-2H-furo[3,2-%yrrol-4(5H,6H,6αH)-yl)- 1 -((1 r,45)-4-methylcyclob.exyl)-2-oxoethyl)benzo [c] [ 1 ,2,5] oxadiazole-5 -carboxamide [15];
^-((^^-((S^ό^όa^-ό-cMoro-S-oxodmydro^H-furoP^-έJpyiTol^CS/iό/ζeα/^-yl)- 1 -(( 1 r,45)-4-methylcyclohexyl)-2-oxoethyl)- 1 H-indole-5-carboxamide [16];
N-((5)-2-((3a5',6556a^-6-cmoro-3-oxodihydro-2H-foro[3,2^]pyiτol-4(5H,6/i6βH)-yl)- 1 -((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-6-hydroxypicolinamide [17] ;
N-((5)-2-((3αS,6S'56α5)-6-cMoro-3-oxodihydro-2H-furo[352-&]pyrrol-4(5/f,6H,6fliϊ)-yl)- l-((lr,41S)-4-methylcyclohexyl)-2-oxoethyl)-23-dioxo-l,2,3,4-tetrahydroquinoxaline-6- carboxamide [18];
N-((^-2-((3a5,6S',6a5)-6-chloro-3-oxodihydro-2H-furo[352-6]pyrrol-4(5H56//;6aH)-yI)- l-^lr^^^-methylcyclohexy^^-oxoethy^^-oxo-l^^^-tetrahydroquinoline-ό- carboxamide [19]; iV^-((S)-2-((3flS's6556aS)-6-cMoro-3-oxodmydro-2H-foo[3,2-6]pyirol-4(5H,6H;6flH)-yl)- l-((lr,4S)-4-methylcycloh.exyl)-2-oxoethyl)-3-oxo-3,4-dihydro-2/i'- benzo[&] [ 1 ,4] oxazine-7-carboxamide [20] ;
N-((^-2-((3α5'56556α^-6-cmoro-3-oxodihydro-2H-furo[352-ό]pyrrol-4(5H,6H56fl/i)-yl)- 1 -(( lr,4,S)-4-methylcyclohexyl)-2-oxoethyl)-4-(methylsulfonajaiido)benzainide [21]; and
N-((5)-2-((3α5565',6α6)-6-cMoro-3-oxodihydro-2H-furo[3,2-6]pyrrol-4(5/J36/ζ6αH)-yl)- l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-lH- benzo[&]azepine-7-carboxamide [22].
16. A compound according to claim 14 which is selected from the following:
JV-((,S)-2-((3aS,6S,6a^-6-cli[oro-3-ox^ l-((lr,45)-4-methylcyclohexyl)-2-oxoethyl)-3-(lΗ-tetrazol-l-yl)benzamide [2];
N-((5)-2-((3a^,65,6a5)-6-cMoro-3-oxodmydro-2H-furo[352-δ]pyrrol-4(5if,6/f,6flH)-yl)- 1 -(4,4-dimethylcyclohexyl)-2-oxoethyl)-3 -(I H-tetrazol- 1 -yl)benzamide [3 ] ;
N-((5)-2-((3a5565,6a5)-6-cWoro-3-oxod%dro-2H-ftu:o[3,2-&]pyrrol-4(5H,6H36aH)-yl)- l-((lr545)-4-meώylcyclohexyl)-2-oxoethyl)-3-(lΗ-iirdda2;ol-l-yl)benzamide [4];
N-((^-2-((3aS,6S,6a^-6-cmoro-3-oxodmydro-2H-furo[3,2-&]pyrrol-4(5H,6H,6aH)-yl)- l-((lr,4^-4-metiiylcyclohexyl)-2-oxoethyl)-3-(4H-l,2,4-triazol-4-yl)benzamide [5];
N-((5)-2-((3aS,65,6a^-6-cmoro-3-oxod%dro-2H-furo[3,2-ό]pyrrol-4(5H;6H,6αH)-yl)- 1 -(( 17-,45)-4-methylcyclohexyl)-2-oxoethyl)-3 -( 1 H-pyrazol- 1 -yl)benzamide [6] ;
N-((5)-2-((3a5,6556a^-6-cMoro-3-oxodmydro-2H-furo[3,2-&]pyrrol-4(5/i6H,6αH)-yl)- 1 -((1 r,45)-4-methylcyclohexyl)-2-oxoethyl)-3 -(4H- 1 ,2,4-triazol-4-yl)benzamide [7] ; ^-((^^-((SaxS^^όa^-ό-cmoro-S-oxodUiydro^H-furoP^-όlpyrrol^CSH^^όfliO-yl)-
1 -(( 1 r,45)-4-methylc5'clohexyl)-2-oxoethyl)nicotinamide [8] ;
.V-((S)-2-((3a-S;6&6aφ-6-cWoro-3-oxodihy^
1 -(( 1 r,4iS)-4-methylcyclohexyl)-2-oxoethyl)isonicotinamide [9] ;
N-((S}-2-((3a5?6S,6aS)-6-cMoro-3-oxodihydro-2H-furo[3>2-&]pyrrol-4(5H,6H,6flH)-yl)- 1 -(( 1 r^i^^-meiJiylcyclohexy^^-oxoethy^furaQ^-carboxamide [10];
N-((5}-2-((3a5,65's6a^-6-cMoro-3-oxodihydro-2H-furo[3,2-ό]pyrrol-4(5H,6H,6flH)-yl)- l-((lr?45)-4-methylcyclohexyl)-2-oxoethyl)-3-(pyridin-3-yl)benzamide [12];
ΛH(S)-2-((3aS,6_S;6aS)-6-cMoro-3-oxodiLhyd^
1 -((1 r,45)-4-methylcyclohexyl)-2-oxoethyl)- 1 H-benzo[d][ 1 ,2,3]triazole -6-carboxamide
[13];
N-((.S)-2-((3aS,65',6a5)-6-cWoro-3-oxodihycko-2H-ftiro[3}2-3]pyrrol-4(5H,6^ 1 -(( 17*,4<S)-4-methylcyclohexyl)-2-oxoethyl)benzo [£z]thiazole-6-carboxainide [14];
N-((S)-2-((3a5565.6aS)-6-cmoro-3-oxodihydro-2H-furo[3J2-6]pyrrol-4(5H,6H.6αH)-yl)- 1 -(( 1 /•^^^-methylcyclohexyD^-oxoethyObenzotc] [ 1 ,2,5] oxadiazole-5-carboxamide [15];
-V-((5)-2-((3aS',65r,6aS)-6-cmoro-3-oxodihydro-2H-furo[3,2-&]pyrrol-4(5H,6H,6flH)-yl)- l-((l/*,4)S)-4-methylcyclohexyl)-2-oxoethyl)-lΗ-indole-5-carboxamide [16];
N-((-S)-2-((3a^,65,6aS)-6-cMoro-3-oxodihydro-2H-fliro[3,2^]pyiTol-4(5H,6H,6a/^ l-((lr,4.S)-4-metliylcyclohexyl)-2-oxoethyI)-6-hydroxypicoIinainide [17]; N-((^-2-((3αS'565',6fl^-6-cmoro-3-oxodihydro-2H-fiu:o[3,2-6]pyrrol-4(5H56H.6αH)-yl)-
1 -(( 1 r,45)-4-methylcycloliexyl)-2-oxoeth.yl)-2-oxo- 1,2,3 ,4-tetrahydroquuioline-6- carboxamide [19];
N-((5)-2-((3α5'565',6fl5)-6-chloro-3-oxodihydro-2i7-furo[3,2-ό]pyτrol-4(5H,6i^6αH)-yl)- 1 -((1 r,45)-4-methylcyclohexyl)-2-oxoethyl)-3-oxo-3 ,4-dihydro-2H- benzo [b] [ 1 ,4] oxazine-7-carboxamide [20] ;
^-((^^-((Sα^ό^β^-β-cbloro-S-oxodihydro^H-furoP^-^pyrrol^CS/iόi^όfl/O-yl)- 1 -(( 1 r,45)-4-methylcyclohexyl)-2-oxoethyl)-4-(metiiylsulfonamido)benzamide [21]; and
N-((5)-2-((3α5,6S',6α5)-6-chloro-3-oxodmydro-2H-furo[3,2-ό]pyrrol-4(5HJ6H,6αH)-yl)- l-((lr545)-4-inethylcyclohexyl)-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-liy- benzo[ό]azepine-7-carboxamide [22].
17. A pharmaceutical or veterinary composition comprising a compound according to any one of claims 1 to 16 and a pharmaceutically acceptable or veterinarily acceptable diluent, excipient and/or carrier.
18. A process for preparing a pharmaceutical or veterinary composition according to claim 17, said process comprising admixing a compound according to any one of claims 1 to 16 with a pharmaceutically acceptable or veterinarily acceptable diluent, excipient and/or carrier.
19. A compound according to any one of claims 1 to 16 for use in medicine.
20. Use of a compound according to any one of claims 1 to 16 in the preparation of a medicament for treating a disease selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain.
21. Use of a compound according to any one of claims 1 to 16 for inhibiting a cysteine proteinase.
22. Use according to claim 21 wherein the cysteine proteinase is a CACl cysteine proteinase.
23. Use according to claim 22 wherein the CACl cysteine proteinase is cathepsin S.
24. A method of inhibiting a cysteine proteinase in a cell, said method comprising contacting said cell with a compound according to any one of claims 1 to 16.
25. A method of inhibiting a cysteine proteinase in a subject, said method comprising administering to the subject a pharmacologically effective amount of a compound according to any one of claims 1 to 16.
26. A method according to claim 24 or claim 25 wherein the cysteine proteinase is a CACl cysteine proteinase.
27. A method according to claim 26 wherein the CACl cysteine proteinase is cathepsin S.
28. A method of treating a disease selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain in a subject, said method comprising administering to the subject a pharmacologically effective amount of a compound according to any one of claims 1 to 16.
29. Use of a compound according to any one of claims 1 to 16 in an assay for identifying further candidate compounds capable of inhibiting one or more cysteine proteinases.
30. Use according to claim 29 wherein said assay is a competitive binding assay.
31. Use according to claim 30 wherein said competitive binding assay comprises contacting a compound according to any one of claims 1 to 16 with a cysteine proteinase and detecting any change in the interaction between the compound according to any one of claims 1 to 16 and the cysteine proteinase.
32. A method of validating a known or putative cysteine proteinase as a therapeutic target, the method comprising:
(a) assessing the in vitro binding of a compound according to any one of claims 1 to 16 to an isolated or known putative cysteine proteinase, providing a measure of potency; and optionally, one or more of the steps of:
(b) assessing the binding of a compound according to any one of claims 1 to 16 to closely related homologous proteinases of the target and general housekeeping proteinases (e.g. trypsin) to provide a measure of selectivity;
(c) monitoring a cell-based functional marker of a particular cysteine proteinase activity in the presence of a compound according to any one of claims 1 to 16; and
(d) monitoring an animal model-based functional marker of a particular cysteine proteinase activity in the presence of a compound according to any one of claims 1 to 16.
33. Use of a compound according to any one of claims 1 to 16 in the validation of a known or putative cysteine proteinase as a therapeutic target.
34. A compound according to any one of claims 1 to 16 for treating a disease selected from rheumatoid arthritis, multiple sclerosis, myasthenia gravis, transplant rejection, diabetes, Sjogrens syndrome, Grave's disease, systemic lupus erythematosis, osteoarthritis, psoriasis, idiopathic thrombocytopenic purpura, allergic rhinitis, asthma, atherosclerosis, obesity, chronic obstructive pulmonary disease and chronic pain.
35. A process of preparing a compound according to any one of claims 1 to 16, said process comprising treating a compound of formula (II) with an oxidizing agent,
Figure imgf000137_0001
(H) (I) wherein R3, R4 and R9 are as defined in claim 1.
36. A process according to claim 35 wherein the oxidizing agent is Dess-Martin periodinane.
37. A process according to claim 35 or claim 36 which comprises the step of converting a compound of formula (III), where R5 is a protecting group or hydrogen, into a compound of formula (II), by treating a compound of formula (HIa) (R5 = H) with a compound of formula R9CONHCH(C6H9R3R4)COOH
Figure imgf000137_0002
where R is a protecting group or hydrogen.
38. A process according to claim 37 wherein protecting group R5 is selected from benzyloxycarbonyl, tert-butoxycarbonyl, fluoren-9-ylmethoxycarbonyl, l-(biphenyl-4- yl)- 1 -methylethoxycarbonyl, α,α-dimethyl-3 ,5-dimethoxylbenzyloxycarbonyl, p- methoxybenzyloxycarbonyl,
Figure imgf000138_0001
allyloxycarbonyl and trichloroethoxycarbonyl.
39. A process according to claim 37 or claim 38 which comprises the step of converting a compound of formula (IV) into a compound of formula (III; R5 = H)
Figure imgf000138_0002
where Lg is a leaving group such as tosylate or mesylate and R5 is as defined in claim
37.
40. A process according to claim 39 which comprises the step of converting a compound of formula (IVa; R5 = H) into a compound of formula (Ilia) or a compound of formula (IVb; R5 = Cbz) into a compound of formula QlIb)
Figure imgf000138_0003
Figure imgf000139_0001
41. A process according to claim 39 or claim 40 which comprises the step of converting a compound of formula (V) into a compound of formula (IV)
Figure imgf000139_0002
(V) (IV)
42. A process according to claim 41 wherein R5 is benzyloxycarbonyl (Cbz), and the process comprises hydrogenating a compound of formula (V) in the presence of a palladium catalyst.
43. A process according to claim 41 or claim 42 which comprises the step of converting a compound of formula (VI) into a compound of formula (V) by treating said compound of formula (VI) with an oxidizing agent
Figure imgf000139_0003
(VI) (V) (anti)
44. A process according to claim 43 wherein the oxidising agent is mCPBA or a dioxirane.
45. A process according to claim 43 or claim 44 which comprises the step of converting a compound of formula (VII) into a compound of formula (VI)
Figure imgf000140_0001
(VII) (VI)
46. A process according to claim 45 which comprises treating a compound of formula (VII) with (a) tosyl chloride in pyridine, or (b) tosyl chloride in dichloromethane and triethylamine.
47. A process according to claim 45 or claim 46 which comprises the step of converting a compound of formula (VIII) into a compound of formula (VII)
Figure imgf000140_0002
(VIII) (VII) where W is halogen or tosyl.
48. A process according to claim 47 which comprises the steps of:
(c) reacting a compound of formula (VIH)5 where W is halogen or OTs, with aqueous ammonia and alcohol; and
(d) converting the product formed in step (a) to a compound of formula (VII).
49. A process according to claim 48 wherein steps (a) and (b) are carried out in a one-pot process.
50. A process according to claim 47 wherein said compound of formula VIII is prepared from a compound of formula IX
Figure imgf000141_0001
IX VIII
51. A process according to claim 50 which comprises treating said compound of formula IX with zinc in aqueous isopropanol.
PCT/GB2009/000653 2008-03-13 2009-03-11 Furo [3, 2-b] pyrr0l-3-0nes as cathepsin s inhibitors WO2009112826A1 (en)

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US12/853,005 US8389737B2 (en) 2008-03-13 2010-08-09 Furo[3, 2-B] pyrrol-3-ones as cathespin S inhibitors
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US14/046,895 US8933109B2 (en) 2008-03-13 2013-10-04 Furo[3, 2-B] pyrrol-3-ones as cathespin S inhibitors
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US14/871,381 US20160015685A1 (en) 2008-03-13 2015-09-30 Furo [3, 2-b] pyrr0l-3-0nes as cathespin s inhibitors
US15/245,968 US20160361295A1 (en) 2008-03-13 2016-08-24 Furo[3, 2-b] pyrr0l-3-ones as cathespin s inhibitors

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