WO2011070541A1 - Inhibiteurs des cystéine protéases - Google Patents

Inhibiteurs des cystéine protéases Download PDF

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WO2011070541A1
WO2011070541A1 PCT/IB2010/055738 IB2010055738W WO2011070541A1 WO 2011070541 A1 WO2011070541 A1 WO 2011070541A1 IB 2010055738 W IB2010055738 W IB 2010055738W WO 2011070541 A1 WO2011070541 A1 WO 2011070541A1
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alkyl
compound according
halo
methyl
added
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PCT/IB2010/055738
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English (en)
Inventor
Susana Ayesa
Peter Carlquist
Karolina Ersmark
Urszula Grabowska
Ellen Hewitt
Daniel Jönsson
Pia Kahnberg
Björn KLASSON
Peter Lind
Lourdes ODÉN
Kevin Parkes
Daniel Wiktelius
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Medivir Uk Limited
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Priority claimed from GBGB1010084.0A external-priority patent/GB201010084D0/en
Priority to CA2782294A priority Critical patent/CA2782294A1/fr
Priority to US13/514,760 priority patent/US8859605B2/en
Priority to MX2012006652A priority patent/MX2012006652A/es
Priority to CN2010800635673A priority patent/CN102858751A/zh
Priority to BR112012014091A priority patent/BR112012014091A2/pt
Application filed by Medivir Uk Limited filed Critical Medivir Uk Limited
Priority to EP10835594.2A priority patent/EP2509957A4/fr
Priority to RU2012128859/04A priority patent/RU2012128859A/ru
Priority to JP2012542677A priority patent/JP2013513598A/ja
Priority to AU2010329470A priority patent/AU2010329470C1/en
Priority to IN4915DEN2012 priority patent/IN2012DN04915A/en
Publication of WO2011070541A1 publication Critical patent/WO2011070541A1/fr

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Definitions

  • This invention relates to inhibitors of cathepsin S, and their use in methods of treatment for disorders involving cathepsin S such as autoimmune disorders, allergy and chronic pain conditions.
  • the papain superfamily of cysteine proteases are widely distributed in diverse species including mammals, invertebrates, protozoa, plants and bacteria.
  • Pathogenic cathepsin like enzymes include the bacterial gingipains, the malarial falcipains I, II, III et seq and cysteine proteases from Pneumocystis carinii,
  • Cathepsin S is a highly active cysteine protease belonging to the papain superfamily. Its primary structure is 57%, 41% and 45% homologous with human cathepsin L and H and the plant cysteine protease papain respectively, although only 31% homologous with cathepsin B. It is found mainly in B cells, dendritic cells and macrophages and this limited occurrence suggests the potential involvement of this enzyme in the pathogenesis of degenerative disease.
  • R la is H
  • R lb is Ci-Cealkyl, optionally substituted with 1-3 substituents independently selected from: halo, hydroxy, cyano, azido, Ci-Czihaloalkyl, Ci-C4alkoxy, Ci-Czihaloalkoxy, Ci-
  • Ci-C4alkylcarbonyl amine, Ci-C4alkylamine, Ci-C4dialkylamine, Ci- C4alkylsulfonyl, Ci-C4alkylsulfonylamino, aminocarbonyl, aminosulphonyl, Carbocyclyl and Het; or
  • R lb is Carbocyclyl or Het
  • R la and R lb together with the N atom to which they are attached define a saturated cyclic amine with 3-6 ring atoms;
  • Carbocyclyl, Het or cyclic amine is optionally substituted with 1-3 substituents independently selected from halo, hydroxy, cyano, azido, C]-C4alkyl, Ci-C4haloalkyl, d- C4alkoxy, Ci-C4haloalkoxy, Ci-C4alkoxycarbonyl, Ci-C4alkylcarbonyl, amine, Ci- C4alkylamine, Ci-C4dialkylamine, Ci-C4alkylsulfonyl, Ci-C4alkylsulfonylamino, aminocarbonyl, aminosulphonyl, RxOOC-Co-C2alkylene (where Rx is H, C]-C4alkyl or d- C4haloalkyl), phenyl, benzyl or C3-C6cycloalkylCo-C2alkylene;
  • phenyl, benzyl or cycloalkyl moiety is optionally substituted with 1-3 substituents independently selected from halo, Ci-C4alkyl, Ci-C4haloalkyl or d- C4alkoxy);
  • R 2a and R 2b are independently selected from H, halo, Ci-C4alkyl, Ci-C4haloalkyl, Ci-C4alkoxy, or R 2a and R 2b together with the carbon atom to which they are attached form a C 3 -Cecycloalkyl;
  • R 3 is a C5-C10 alkyl, optionally substituted with 1-3 substituents independently selected from halo, Ci-C4haloalkyl, Ci-C4alkoxy, Ci-C4haloalkoxy; or
  • R 3 is a C2-C4alkyl chain with at least 2 chloro or 3 fluoro substituents
  • R 3 is C3-C7cycloalkylmethyl, optionally substituted with 1-3 substituents independently selected from Ci-C4alkyl, halo, Ci-C4haloalkyl, Ci-C4alkoxy, Ci-C4haloalkoxy;
  • R 4 is Ci-Cgalkyl, Ci-Cghaloalkyl, Ci-Cealkylamino, Ci-Cedialkylamino; Het is a stable, monocyclic or bicyclic, saturated, partially saturated or aromatic ring system containing 1-4 heteroatoms independently selected from O, S and N, each ring having 5 or 6 ring atoms;
  • Carbocyclyl is C 3 -C 6 cycloalkyl, Cs-Cecycloalkenyl or phenyl;
  • R la is H and R lb is Ci-C 4 alkyl, such as methyl, ethyl, isopropyl, t-butyl or preferably methyl, optionally substituted with one or more substituents as defined above, preferably 1-3 halo (e.g. F) or a Ci-C 4 alkoxy (e.g. methoxy) group.
  • R lb is Ci-C 4 alkyl, such as methyl, ethyl, isopropyl, t-butyl or preferably methyl, optionally substituted with one or more substituents as defined above, preferably 1-3 halo (e.g. F) or a Ci-C 4 alkoxy (e.g. methoxy) group.
  • R la is H and R lb is methyl, cyclopropyl, 1-phenylethyl, or a 5 or 6 membered heterocyclic ring containing 1-3 nitrogen atoms and 0 or 1 sulphur atoms, the cyclopropyl, phenyl or heterocyclic ring being optionally substituted with up to three substituents independently selected from:
  • Examples of the 5 or 6 membered aromatic heterocyclyl for R lb include pyridyl or pyrimidyl and especially pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, thiadiazolyl, triazolyl or tetrazolyl, optionally substituted with any of which is optionally substituted with Ci-C 4 alkyl (e.g. Me), halo (e.g. F), Ci-C 4 haloalkyl (e.g. CF 3 ), Ci-C 4 alkoxy (e.g. MeO), C 3 -C 6 cycloalkylC 0 -Cialkylene (e.g. cyclopropyl or cyclopropylmethyl, benzyl or Co-C 2 alkyleneCOOH and its Ci-C 4 alkyl esters.
  • An exemplary species is l-methyl-pyrazol-5-yl.
  • the heterocyclic ring is pyrrolyl, pyrazolyl, imidazolyl, triazolyl, thiazolyl or thiadiazolyl, any of which is optionally substituted with Ci-C 4 alkyl, halo, Ci-C 4 haloalkyl, Ci-C 4 alkoxy, C3-C 6 cycloalkyl or C 3 -C 6 cycloalkylmethyl.
  • the heterocyclic ring is pyrazol-l-yl, which is optionally substituted with Ci-C4alkyl, halo, Ci-C/thaloaikyl or cyclopropyl, preferably Ci- C 4 alkyl, such as ethyl or preferably methyl.
  • a preferred value for R lb is pyrazol-l-yl which is N-substituted with Ci-C4alkyl, such as ethyl or methyl.
  • R lb is methyl or cyclopropyl.
  • R la is H and R lb is methyl or ethyl which is substituted in the 1-position with a cyclic group such as phenyl, or R lb is a monocyclic heterocyclyl such as pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, thiopyranyl, furanyl, tetrahydrofuranyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, tetrazolyl, pyrazolyl
  • the phenyl or heterocyclyl is optionally substituted, for example with 1-3 substituents independently selected from hydroxy, amino, Ci-C 4 alkyl, halo, Ci- C4haloalkyl, Ci-C4alkoxy, amino, Ci-C4alkylamine, Ci-C4dialkylamine and the like.
  • An exemplary species is 1 -phenylethyl.
  • R la is H and R lb is C3-Cecycloalkyl, preferably cyclobutyl or cyclopropyl, optionally substituted as defined above.
  • the cycloalkyl is unsubstituted or substituted with 1-3 substituents selected from halo (e.g.
  • Ci-C 4 alkyl e.g. 1 or 2 methyl
  • Ci-C4haloalkyl e.g. a CF3 group
  • Ci-C4alkoxy e.g. an MeO group
  • Ci-C4alkylamine e.g. an MeNH- group
  • Ci-C 4 dialkylamine e.g. an (Me) 2 N- group
  • An exemplary species is cyclopropyl, or monofluoro- or gemdifluorocyclopropyl.
  • R la is H and R lb is a 6 or preferably 5 membered aromatic, heterocyclic ring containing 1 -3 nitrogen atoms and 0 or 1 sulphur atoms, optionally substituted as defined above.
  • the heterocyclic ring is linked to the adjacent nitrogen atom of the alpha keto amide group through a carbon atom of the heterocyclic ring.
  • R la , R lb and the N-atom to which they are attached form a 3-6 membered cyclic amine, such as aziridine, azetidine, pyrrolidine, and preferably morpholine, piperazine or piperidine.
  • cyclic amines may be unsubstituted or substituted as described above, preferably with 1-3 substituents selected from halo (e.g. 1 or 2 fiuoro), hydroxy, Ci-C 4 alkyl (e.g. 1 or 2 methyl), Ci-C4haloalkyl (e.g. a CF3 group) Ci-C4alkoxy (e.g. an MeO-group) , Ci- C 4 alkylamine (e.g. an MeNH- group), Ci-C 4 dialkylamine (e.g. an (Me) 2 N- group) and the like.
  • halo e.g. 1 or 2 fiuoro
  • Ci-C 4 alkyl e.g. 1
  • One embodiment of the invention includes compounds of formula I wherein R 2a and R 2b are both hydrogen.
  • At least one of R 2a and R 2b is halo, Ci-C 4 alkyl, Ci-C 4 naloalkyl or Ci-C 4 alkoxy.
  • one of R 2a and R 2b is H, and the other is CI, F, CF 3 or MeO; especially F or MeO.
  • one of R 2a and R 2b is H, and the other is F.
  • Specially preferred according to this embodiment are compounds having the stereochemistry shown in formula (la):
  • R 2a and R 2b together with the carbon atom to which they are attached form a C3-C 6 cycloalkyl.
  • R 3 is cycloalkylalkyl, optionally substituted, for example with halo, (such as F) or alkoxy (such as MeO).
  • exemplary species include 1-methylcyclopentylmethyl, 1- methylcyclohexylmethyl, 1-methylcyclobutylmethyl, l-methyl-3,3-difluorocyclobutylmethyl, 1- methyl-4,4- difluorocyclohexylmethyl, cyclopropylmethyl or l-methyl-3,3- difluorocyclopentylmethyl.
  • R 3 species include t-butylmethyl, cyclobutylmethyl, 1 -methylcyclobutylmethyl and 1 - methylcyclopentylmethyl, any of which is optionally substituted with one or two F or MeO.
  • Representative species are 1-fluorocyclobutylmethyl and 1 -fluorocyclopentylmethyl.
  • Further representative R 3 species include 1-methylcyclopentylmethyl and 1- fluorocyclopentylmethyl,
  • R 3 as a straight or branched alkyl chain of 5-10 C-atoms, optionally substituted with 1-3 halo, (e.g. CI or F), or a Ci-C4alkoxy (e.g. MeO).
  • exemplary species include 2,2-dimethylpropyl, 3,3-dimethylpentyl, 2,2,3,3- tetramethylbutyl.
  • exemplary species of halogenated alkyl include 2,2-dichioroethyl, 3,3,3-trifluoropropyl, 2,2- trifluoromethylethyl, or 2,2,2-trifluoroethyl.
  • One embodiment of the invention include compounds wherein R 4 is Ci-Cgalkyl, such as methyl or ethyl.
  • Another embodiment includes compounds wherein R 4 is Cj-Cehaloalkyl, such as Q- Cechloroalkyl or Ci-Cefluoroalkyl.
  • the compounds of formula I are characterised by various advantageous pharmaceutical properties and exhibit at least one improved property in view of the compounds of the prior art.
  • the inhibitors of the present invention are superior in one or more of the following pharmacological related properties, i.e. potency, decreased cytotoxicity, improved
  • PI, P2 and P3 are provided for convenience only and have their conventional meanings and denote those portions of the inhibitor believed to fill the S 1 , S2 and S3 subsites respectively of the enzyme, where SI is adjacent the cleavage site and S3 remote from the cleavage site.
  • a further aspect of the invention comprises a method employing the compounds of formula I for the prophylaxis or treatment of diseases caused by aberrant expression or activation of cathepsin, i.e. diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily.
  • a further aspect of the invention provides the use of the compounds of formula I in the prophylaxis or treatment of diseases caused by aberrant expression or activation of cathepsin, ie diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily.
  • a further aspect of the invention provides the use of the compounds of formula I for the manufacture of a medicament for the prophylaxis or treatment of diseases caused by aberrant expression or activation of cathepsin S, i.e. diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily.
  • a compound of Formula I for use as a medicament such as in the prophylaxis or treatment of a disorder characterised by inappropriate expression or activation of cathepsin S i.e. diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily.
  • diseases or conditions defined in the immediately preceding four paragraphs include those enumerated in WO 97/40066, such as autoimmune diseases, allergies, such as asthma and hay fever, multiple sclerosis, rheumatoid arthritis and the like.
  • a further example is the treatment of endometriasis, and especially chronic pain, as disclosed in WO03/20287.
  • the invention further provides the use of the compounds of formula I or any subgroup of formula I in therapy and in the manufacture of a medicament for the treatment of diseases or conditions alleviated or moderated by inhibition of cathepsin S.
  • the methods are employed to treat mammals, particularly humans at risk of, or afflicted with, autoimmune disease.
  • autoimmunity is meant the phenomenon in which the host's immune response is turned against its own constituent parts, resulting in pathology.
  • Many human autoimmune diseases are associated with certain class II MHC- complexes. This association occurs because the structures recognized by T cells, the cells that cause autoimmunity, are complexes comprised of class II MHC molecules and antigenic peptides.
  • Autoimmune disease can result when T cells react with the host's class II MHC molecules when complexed with peptides derived from the host's own gene products. If these class II
  • MHC/antigenic peptide complexes are inhibited from being formed, the autoimmune response is reduced or suppressed. Any autoimmune disease in which class II MHC/antigenic complexes play a role may be treated according to the methods of the present invention.
  • autoimmune diseases include, e.g., juvenile onset diabetes (insulin dependent), multiple sclerosis, pemphigus vulgaris, Graves' disease, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis and Hashimoto's thyroiditis.
  • the methods are employed to treat mammals, particularly humans, at risk of, or afflicted with, allergic responses.
  • allergic response is meant the phenomenon in which the host's immune response to a particular antigen is unnecessary or disproportionate, resulting in pathology. Allergies are well known in the art, and the term
  • allergies include, but are not limited to, allergies to pollen, "ragweed,” shellfish, domestic animals (e.g., cats and dogs), bee venom, house dust mite allergens and the like.
  • Another particularly contemplated allergic response is that which causes asthma. Allergic responses may occur, in man, because T cells recognize particular class II MHC/antigenic peptide complexes. If these class II MHC/antigenic peptide complexes are inhibited from being formed, the allergic response is reduced or suppressed. Any allergic response in which class II MHC/antigenic peptide complexes play a role may be treated according to the methods of the present invention. Immunosuppression by the methods of the present invention will typically be a prophylactic or therapeutic treatment for severe or life-threatening allergic responses, as may arise during asthmatic attacks or anaphylactic shock.
  • the methods are employed to treat mammals, particularly humans, which have undergone, or are about to undergo, an organ transplant or tissue graft.
  • tissue transplantation e.g., kidney, lung, liver, heart
  • skin grafting when there is a mismatch between the class II MHC genotypes (HLA types) of the donor and recipient, there may be a severe "allogeneic" immune response against the donor tissues which results from the presence of non-self or allogeneic class II MHC molecules presenting antigenic peptides on the surface of donor cells. To the extent that this response is dependent upon the formation of class II
  • cathepsin S may suppress this response and mitigate the tissue rejection.
  • An inhibitor of cathepsin S can be used alone or in conjunction with other therapeutic agents, e.g., as an adjunct to cyclosporin A and/or antilymphocyte gamma globulin, to achieve immunosuppression and promote graft survival.
  • administration is accomplished by systemic application to the host before and/or after surgery.
  • perfusion of the donor organ or tissue, either prior or subsequent to transplantation or grafting may be effective.
  • a related aspect of the invention is directed to a method of treating a patient undergoing a therapy wherein the therapy causes an immune response, preferably a deleterious immune response, in the patient comprising administering to the patient a compound of Formula I or a pharmaceutically acceptable salt, n-oxide or hydrate thereof.
  • the immune response is mediated by MHC class II molecules.
  • the compound of this invention can be administered prior to, simultaneously, or after the therapy.
  • the therapy involves treatment with a biologic, such as a protein, preferably an antibody, more preferably a monoclonal antibody.
  • the biologic is Remicade®, Refacto®, ReferonA®, Factor VIII, Factor VII, Betaseron®, Epogen®, Enbrel®, Interferon beta, Botox®, Fabrazyme®, Elspar®, Cerezyme®, Myobloc®, Aldurazyrne®, Verluma®, Interferon alpha, Humira®, Aranesp®, Zevalin® or OKT3.
  • the treatment involves use of heparin, low molecular weight heparin, procainamide or hydralazine.
  • Assays for the assessment of cathepsin S inhibitors in the treatment of chronic pain, including neuropathic or inflammatory pain are as described in WO 03/20287.
  • Psoriasis idiopathic thrombocytopenic purpura (ITP), rheumatoid arthritis (RA), multiple schlerosis (MS), myasthenia gravis (MG), Sjogrens syndrome, Grave's disease and systemic lupus erythematosis (SLE);
  • Non-automimmune indications include allergic rhinitis, asthma, artherosclerosis, chronic obstructive pulmonary disease (COPD) and chronic pain.
  • COPD chronic obstructive pulmonary disease
  • the compounds of the invention can form salts which form an additional aspect of the invention.
  • Appropriate pharmaceutically acceptable salts of the compounds of the invention include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, isethionate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate,
  • the compounds of the invention may in some cases be isolated as the hydrate. Hydrates are typically prepared by recrystallisation from an aqueous/organic solvent mixture using organic solvents such as dioxin, tetrahydrofiiran or methanol.
  • N-oxides of compounds of the invention can be prepared by methods known to those of ordinary skill in the art.
  • N-oxides can be prepared by treating an unoxidized form of the compound of the invention with an oxidizing agent (e.g., trifluoroperacetic acid, permaleic acid, perbenzoic acid, peracetic acid, meta-chloroperoxybenzoic acid, or the like) in a suitable inert organic solvent (e.g., a halogenated hydrocarbon such as dichloromethane) at
  • the N-oxides of the compounds of the invention can be prepared from the N-oxide of an appropriate starting material.
  • Compounds of the invention in unoxidized form can be prepared from N-oxides of the corresponding compounds of the invention by treating with a reducing agent (e.g., sulphur, sulphur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus dichloride, tribromide, or the like) in an suitable inert organic solvent (e.g., acetonitrile, ethanol, aqueous dioxane, or the like) at 0 to 80 °C.
  • a reducing agent e.g., sulphur, sulphur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus dichloride, tribromide, or the like
  • an inert organic solvent e.g., acetonitrile, ethanol, aqueous dioxane, or the like
  • isotopes that maybe incorporated into the compounds of formula I or any subgroup of formula I, include but are not limited to isotopes of hydrogen, such as 2 H and 3 H (also denoted D for deuterium and T for tritium respectively), carbon, such as n C, 13 C and 14 C, nitrogen, such as 13 N and 15 N, oxygen, such as 15 O, 1V O and 18 O, phosphorus, such as 31 P and 32 P, sulphur, such as 35 S, fluorine, such as 18 F, chlorine, such as 36 CI, bromine such as V5 Br, V6 Br, 77 Br and 82 Br, and iodine, such as 12 I, 124 I, 125 I and 131 I.
  • isotopes include but are not limited to isotopes of hydrogen, such as 2 H and 3 H (also denoted D for deuterium and T for tritium respectively), carbon, such as n C, 13 C and 14 C, nitrogen, such as 13 N and 15 N, oxygen
  • isotope included in an isotope-labelled compound will depend on the specific application of that compound. For example, for drug or substrate tissue distribution assays, compounds wherein a radioactive isotope such as 3 H or 14 C is incorporated will generally be most useful. For radio-imaging applications, for example positron emission tomography (PET) a positron emitting isotope such as 11 C, 18 F, 13 N or 15 O will be useful.
  • PET positron emission tomography
  • a heavier isotope such as deuterium, i.e. 2 H, may provide greater metabolic stability to a compound of formula I or any subgroup of formula I, which may result in, for example, an increased in vivo half life of the compound or reduced dosage requirements.
  • 2 H isotope(s) are typically incorporated at position(s) disposed to metabolic liability.
  • suitable positions for incorporation of 2 H isotopes are e.g. as substituents to the cyclobutylene group, i.e. one or both of 2a and R b is 2 H.
  • Isotopically labelled compounds of formula I or any subgroup of formula I can be prepared by processes analogous to those described in the Schemes and/or Examples herein below by using the appropriate isotopically labelled reagent or starting material instead of the corresponding non-isotopically labelled reagent or starting material, or by conventional techniques known to those skilled in the art.
  • the radical positions on any molecular moiety used in the definitions may be anywhere on such moiety as long as it is chemically stable. As used herein, the following terms have the meanings as defined below:
  • C m -C n alkyl used on its own or in composite expressions such as C m -C n halo alkyl, C m - C n alkylcarbonyl, C m -C n alkylamine, C m -C n alkylsulphonyl, C m -C n alkylsufonylamino etc.
  • Ci-C4alkyl means an alkyl radical having from 1 to 4 carbon atoms.
  • Preferred alkyl radicals for use in the present invention are Ci-C4alkyl and includes methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl and isobutyl. Methyl and t-butyl are typically preferred.
  • Ci-C 6 alkyl has a corresponding meaning, including also all straight and branched chain isomers of pentyl and hexyl. Other recitals of C m -C n alkyl, such as C5-C1 0 alkyl have the corresponding meaning.
  • Me means methyl
  • MeO means methoxy
  • Et means ethyl
  • Ac means acetyl
  • Co-C2alkylene used in composite expressions such as C3-C6cycloalkylCo- C2alkylene refers to a divalent radical derived from a methyl or ethyl group, or in the case of Co the term Co-C2alkylene means a bond.
  • Ci-C4haloalkyl refers to a Ci-C4alkyl radical, wherein at least one C atom is substituted with a halogen, preferably chloro or fluoro. Trifluoromethyl is typically preferred
  • Ci-C4alkoxy represents a radical Ci-C4alkyl-0 wherein Ci-C4alkyl is as defined above, and includes methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, n-butoxy and isobutoxy. Methoxy and isopropoxy are typically preferred.
  • Ci-Cealkoxy has a corresponding meaning, expanded to include all straight and branched chain isomers of pentoxy and hexoxy. Other recitals of C m - C n alkoxy, such as Cs-Cioalkoxy have the corresponding meaning.
  • Ci-C4haloalkoxy as used herein is meant to include Ci-C4alkoxy wherein at least one C-atom is substituted with one or more halogen atom(s), typically chloro or fluoro. In many cases trifluoromethyl is preferred.
  • Carbocyclyl includes cyclopentyl, cyclohexyl and especially cyclopropyl and cyclobutyl.
  • Carbocyclyl further includes cyclopentenyl and cyclohexenyl, in each case with a single double bond.
  • a frequently preferred value for Carbocyclyl is phenyl.
  • Cyclic amine includes aziridine, azetidine, pyrrolidine, piperidine, piperazine and morpholine.
  • Het is a stable, monocyclic or bicyclic, saturated, partially saturated or aromatic ring system, containing 1-4 hetero atoms independently selected from O, S and N, and each ring having 5 or 6 ring atoms;
  • Exemplary aromatic Het include furan, thiophene, pyrrole, imidazole, pyrazole, triazole, tetrazole, thiazole, oxazole, isoxazole, oxadiazole, thiadiazole, isothiazole, pyridine, pyridazine, pyrazine, pyrimidine, quinoline, isoquinoline, benzofuran, benzothiophene, indole, indazole and the like.
  • Exemplary unsaturated Het include tetrahydrofuran, pyran, dihydropyran, 1,4-dioxane, 1,3- dioxane, piperidine, pyrrolidine, morpholine, tetrahydrothiopyran,
  • the compounds of the invention include a number of handles such as OH, NH or COOH groups to which conventional prodrug moieties can be applied.
  • Prodrugs are typically hydrolysed in vivo to release the parent compound in the plasma, liver or intestinal wall.
  • Favoured prodrugs are esters of hydroxyl groups such as a phenolic hydroxyl group at R 4 , or amine functions such as a sulphonamide amine function.
  • Preferred pharmaceutically acceptable esters include those derived from Ci-C ⁇ 5 carboxylic acids such as acetyl or pivaloyl or optionally substituted benzoic acid esters, preferably unsubstituted or substituted with substituents broadly as described for R la , typically 1-3 halo (e.g.
  • F Ci-C 4 alkyl (e.g. Me), Ci-C 4 haloalkyl (e.g. CF 3 ) or Ci-C 4 alkyloxy (e.g. MeO) groups.
  • Favoured sulphonamide prodrugs include aminoacyls derived from C1-C6 carboxylic acids such as acetyl or pivaloyl or optionally substituted benzoic acid esters, preferably unsubstituted or substituted with substituents broadly as described for variable R la , typically 1-3 halo (e.g. F), Ci-C4alkyl (e.g. Me), Ci-C4haloalkyl (e.g. CF 3 ) or Ci-C4alkyloxy (e.g. MeO) groups.
  • the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms, which said compound may possess. Said mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of said compound. All stereochemically isomeric forms of the compounds of the present invention both in pure form or mixed with each other are intended to be embraced within the scope of the present invention.
  • stereoisomerically pure concerns compounds or intermediates having a stereoisomeric excess of at least 80% (i.e. minimum 90% of one isomer and maximum 10% of the other possible isomers) up to a stereoisomeric excess of 100% (i.e. 100% of one isomer and none of the other), more in particular, compounds or intermediates having a stereoisomeric excess of 90% up to 100%), even more in particular having a stereoisomeric excess of 94% up to 100% and most in particular having a stereoisomeric excess of 97% up to 100%.
  • enantiomerically pure and “diastereomerically pure” should be understood in a similar way, but then having regard to the enantiomeric excess, and the diastereomeric excess, respectively, of the mixture in question.
  • Compounds of the invention can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomer. While resolution of enantiomers can be carried out using covalent diasteromeric derivatives of compounds of Formula I, dissociable complexes are preferred (e.g., crystalline; diastereoisomeric salts). Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by taking advantage of these dissimilarities.
  • the diastereomers can be separated by chromatography, for example HPLC or, preferably, by separation/resolution techniques based upon differences in solubility.
  • the optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization.
  • a more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture can be found in Jean Jacques Andre Collet, Samuel H. Wilen, Enantiomers, Racemates and Resolutions, John Wiley & Sons, Inc. (1981).
  • the active agent While it is possible for the active agent to be administered alone, it is preferable to present it as part of a pharmaceutical formulation.
  • a pharmaceutical formulation will comprise the above defined active agent together with one or more acceptable carriers/excipients and optionally other therapeutic ingredients.
  • the carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.
  • the formulations include those suitable for rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, but preferably the formulation is an orally administered formulation.
  • the formulations may conveniently be presented in unit dosage form, e.g. tablets and sustained release capsules, and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the above defined active agent with the carrier.
  • the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • the invention extends to methods for preparing a
  • composition comprising bringing a compound of Formula I or its
  • pharmaceutically acceptable salt in conjunction or association with a pharmaceutically acceptable carrier or vehicle. If the manufacture of pharmaceutical formulations involves intimate mixing of pharmaceutical excipients and the active ingredient in salt form, then it is often preferred to use excipients which are non-basic in nature, i.e. either acidic or neutral.
  • Formulations for oral administration in the present invention may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in- water liquid emulsion or a water in oil liquid emulsion and as a bolus etc.
  • suitable carrier includes vehicles such as common excipients e.g.
  • binding agents for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica.
  • Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring or 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 gelatine and glycerine, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.
  • the appropriate dosage for the compounds or formulations of the invention will depend upon the indication, the severity of the disease, the size and metabolic vigour and the patient, the mode of administration and is readily determined by conventional animal trials. Dosages providing intracellular (for inhibition of physiological proteases of the papain superfamily) concentrations of the order 0.01-100 ⁇ , more preferably 0.01-10 ⁇ , such as 0.1-5 ⁇ are typically desirable and achievable.
  • a typical first step is the preparation of a PI building block of the formula V
  • PG is a conventional N protecting group such as Boc, CBz or Fmoc and PG* is H or a conventional carboxy protecting group, such as a Ci-C 4 alkyl or benzyl.
  • a suitable starting material is an N-protected cyclo butyl amino acid, of which several are available commercially or can be prepared as shown in the following Examples or as described by Allan et al. in J. Med. Chem., 1990 33(10) 2905-2915.
  • the carboxylic acid (la) is transformed via a Weinreb synthesis to a ⁇ , ⁇ -dimethylhydroxamic acid (lb) which provides the corresponding aldehyde (lc).
  • the aldehyde may also be accessed by reduction of the carboxylic function of a cyclobutyl amino acid and oxidation under Dess Martin conditions.
  • the aldehyde (lc) can be subsequently reacted with the appropriate isocyanide in a Passerini reaction to afford the required a-hydroxy R la R lb amide (Id).
  • t-butylisocyanide can alternatively be used, thus affording the t-butyl amide, which after hydrolysis of the amide, provides the a-hydroxycarboxylic acid PI building block (le).
  • the strongly acidic conditions required to hydrolyse the amide also lead to loss of the NBoc protection, if used, hence, the amine can be used directly to couple to a P2 building block or else if it needs to be stored, the amine can be reprotected.
  • Ci-Cehaloalkyl Ci-Cehaloalkyl
  • R 4* is C.,-C 6 alkyl or C,-C 6 haloalkyl
  • the C terminus is extended first by reaction of the building block of formula V (2a) with an R la* R lb* amine, where R la* and R lb* are R la and R lb respectively or synthons therefor (selected in view of the sensitivity of the R lb function for the P3 elongation conditions outlined below).
  • the reaction proceeds with conventional peptide chemistries as discussed below.
  • the thus prepared P 1 -prime side unit (2b) is thereafter deprotected at the N terminus and elongated with the P2 building block, providing the N-protected intermediate amine 2c, and subsequently P3 building block, providing the amide 2d, using conventional peptide chemistries.
  • a P2 residue can be introduced via BocP2-OH using standard coupling conditions such as HATU, DIPEA in DMF.
  • the terminal Boc protection is again removed with acetyl chloride in methanol or equivalent and the P3 residue is introduced using standard peptide coupling techniques, such as by reaction with the appropriate P3-acid using conditions like HATU, DIPEA in DMF or by reaction with a P3-acid halide such as the acid chloride or the like.
  • the final steps will generally comprise conversion of the R /R synthons (if present) to their final form and finally oxidation of the alpha hydroxy amide function using Dess Martin conditions to provide the desired alpha keto amide compound 2e.
  • L-amino acids suitable for P2 building blocks and carboxylic acids, carboxylic acid halides and carbamoyl halides suitable for P3 building blocks are commercially available or accessed by simple chemistries or as shown in WO06/064286.
  • the P3 and P2 building blocks may alternatively be coupled first and then reacted with the PI -prime side unit. Elongation is typically carried out in the presence of a suitable coupling agent e.g.,
  • benzotriazole-l-yloxytrispyrrolidinophosphonium hexafluorophosphate PyBOP
  • O- benzotriazol-l-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate HBTU
  • HBTU O- benzotriazol-l-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate
  • HATU 0-(7- azabenzotriazol-l-yl)-l,l,3,3-tetramethyl-uronium hexafluorophosphate
  • EDC l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • DCC 1,3-dicyclohexyl carbodiimide
  • HOBT 1-hydroxybenzotriazole
  • a base such as ⁇ , ⁇ -diisoprop
  • reaction solvents are inert organic solvents such as halogenated organic solvents (e.g., methylene chloride, chloroform, and the like), acetonitrile, N,N- dimethylformamide, ethereal solvents such as tetrahydrofuran, dioxane, and the like.
  • halogenated organic solvents e.g., methylene chloride, chloroform, and the like
  • acetonitrile e.g., N,N- dimethylformamide
  • ethereal solvents such as tetrahydrofuran, dioxane, and the like.
  • the above elongation coupling step can be carried out by first converting the P3/P2 building block into an active acid derivative such as succinimide ester and then reacting it with the PI amine.
  • the reaction typically requires 2 to 3 h to complete.
  • the conditions utilized in this reaction depend on the nature of the active acid derivative. For example, if it is an acid chloride derivative, the reaction is carried out in the presence of a suitable base (e.g. triethylamine, diisopropylethylamine, pyridine, and the like).
  • Suitable reaction solvents are polar organic solvents such as acetonitrile, N,N-dimethylformamide, dichloromethane, or any suitable mixtures thereof.
  • N-protecting group or “N-protected” as used herein refers to those groups intended to protect the N-terminus of an amino acid or peptide or to protect an amino group against undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis” (John Wiley & Sons, New York, 1981), which is hereby incorporated by reference. N-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl,
  • cyclopentyloxycarbonyl adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like; alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like.
  • Favoured N-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl (bz), t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz).
  • Hydroxy and/or carboxy protecting groups are also extensively reviewed in Greene ibid and include ethers such as methyl, substituted methyl ethers such as methoxymethyl,
  • silyl ethers such as trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS) tribenzylsilyl, triphenylsilyl, t-butyldiphenylsilyl triisopropyl silyl and the like, substituted ethyl ethers such as 1- ethoxymethyl, 1 -methyl- 1-methoxy ethyl, t-butyl, allyl, benzyl, p-methoxybenzyl,
  • Ester hydroxy protecting groups include esters such as formate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate, pivaloate, adamantoate, mesitoate, benzoate and the like.
  • Carbonate hydroxy protecting groups include methyl vinyl, allyl, cinnamyl, benzyl and the like.
  • NMR Nuclear Magnetic Resonance
  • multiplicities are denoted s, d, t, m, br and app for singlet, doublet, triplet, multiplet, broad and apparent, respectively.
  • MS Mass Spectrometry
  • LC-MS was obtained with a Waters 2790 LC-system equipped with a Waters XterraTM MS Cs 2.5 ⁇ 2.1x 30 mm column, a Waters 996 Photodiode Array Detector and a Micromass ZMD.
  • HPLC High pressure liquid chromatography
  • BBl-b (1.75 g, 8.78 mmol) was dissolved in CH2CI2 (18 mL) and cooled in an ice bath, under inert gas. Pyridine (2.85 mL) was added, followed by t-butyl isocyanide (1.50 mL, 13.3 mmol). Trifluoroacetic acid (1.35 mL, 17.5 mmol) was then added dropwise over 30 min. The yellow solution was stirred at RT overnight. The mixture was concentrated, diluted with EtOAc (100 mL) and washed successively with IN HC1 (50 mL), saturated NaHCC>3 (50 mL) and saturated NaCl (2 x 50 mL).
  • BBl-c (1.30 g, 4.33 mmol) was refluxed with 6N HC1 (40 mL) until amide hydrolysis was complete as monitored by LCMS. The mixture was evaporated, co-evaporating several times with water. 1M NaOH (15 mL) was added to the residue and the basic solution was stirred under vacuum for 15 min. B0C2O (1.92 g, 8.80 mmol) in dioxane (10 mL) was added, keeping pH at 10 - 11, and the mixture was stirred at RT overnight. The mixture was diluted with water (50 mL), acidified with IN HC1 to pH 3, in an ice bath, and then extracted with EtOAc (2 x 50 mL, then 30 mL). The organic phase was washed with saturated NaCl (50 mL), dried (Na 2 S04) and evaporated to give crude PI building block BB1 (0.649 g).
  • Compound BB2-j (1 g) was dissolved in 6N HC1 (40 mL), and heated to reflux for 24 h after which TLC showed that the reaction had reached completion.
  • the reaction mixture was concentrated in vacuo and residue was dissolved in THF; H 2 0 (7; 3, 50 mL), and TEA (1.8 mL, 0.012 mol) and Boc anhydride (2.6 g, 0.012 mol) were both added.
  • the mixture was stirred at RT for 8 h when TLC confirmed the reaction had reached completion.
  • Step a) ((l-Bromo-3-chloropropan-2-yloxy)methyl)benzene (BB3-a)
  • benzyl bromide (46.0 g, 0.269 mol) and epichlorohydrin (24.9 g, 0.269 mol) and mercurous chloride (0.04 g, 0.085 mmol) was heated for 12 h at 150 °C.
  • the crude product was purified by column chromatography (silica gel 60-120 mesh, eluent 1% EtOAc in pet ether) which afforded the title compound as a viscous liquid (50 g, yield 70 %).
  • trimethyloxonium borontetrafluoride was added in one portion as a solid under vigorous stirring.
  • the reaction mixture was stirred for 3h and diluted with DCM (50 mL) and brine (20 mL), added under vigorous stirring.
  • the organic phase was washed with sodium bicarbonate, brine, dried over sodium sulphate, evaporated and purified on short silica column (DCM as an eluent).
  • DCM sodium bicarbonate
  • brine dried over sodium sulphate
  • the resulting product was dissolved in THF(5 mL), and a solution of tetrabutylammonium fluoride in THF (1M, 4.5 mL) was added, and the reaction was stirred at room temperature for 4.5 h.
  • Lithium borohydride (4.3 g, 0.195 mol) was added in portions to a stirred solution of BB5-a (42 g, 0.177 mol) in EtOH-THF 9: 1 at 0 °C under argon atmosphere. The reaction was stirred for 8h at room temperature then quenched with a saturated solution of ammonium chloride (20 mL). The product was extracted with EtOAc (2x300 mL). The combined organic layers was washed with brine (300 mL) and dried over anhydrous sodium sulphate. The solvent was evaporated under reduced pressure and the afforded crude product was purified by chromatography on a silica column eluted with 15% EtOAc in pet ether, which gave the title compound (27.5 g, 74%).
  • n-BuLi (10 mL, 0.016 mol, 1.6 M solution in hexanes) was added drop-wise to a stirred solution of compound BB5-C (2 g, 5.88 mmol) in THF (170 mL) at -78 °C under argon atmosphere. The stirring was continued for 15 min, followed by drop-wise addition of LiNp (104 mL, 0.42 M solution in THF, 0.044 mol) over 5 min. The dark solution was stirred at -78 °C for lh and then cyclobutanone (0.88 mL, 11.77 mmol) was added drop-wise.
  • the reaction mixture was stirred at -78 °C for 16h then quenched with a saturated solution of NH 4 CI (50 mL) and allowed to warm to room temperature.
  • the reaction was diluted with ether (100 mL) and a saturated solution of NH4CI (10 mL). The layers were separated and the aqueous layer was extracted with ether (2x100 mL). The combined organic layers was dried (Na 2 SC>4) and the solvent was evaporated under reduced pressure.
  • the crude product was purified by chromatography on a silica column eluted with heptane:ether 3:2, which gave the title compound (1.54 g, 70%, ).
  • Step e) ⁇ 2-( 1 -Fluoro-cyclobutyl)- 1 -(2-trimethylsilanyl-ethoxymethoxymethyl)-ethyll -carbamic acid tert-butyl ester (BB5-e)
  • BB5-d (0.5 g, 1.33 mmol), 50% Deoxofluor in THF (excess) and pyridine (excess) were mixed in DCM (10 mL). The resulting mixture was stirred at rt over night. The reaction mixture was washed with 10 % citric acid (aq) and sat. NaHCCh (aq). The organic phase was dried (Na 2 S04) and evaporated. The afforded crude product was purified by chromatography on a silica column using hexane:EtOAc (7: 1 to 2: 1) as eluent, which gave the title compound (192 mg, 38%).
  • Step f) [2-(l-Fluoro-cyclobutyl)-l-hydroxymethyl-ethyl]-carbamic acid tert-butyl ester (BB5-f) A solution of BB5-e (1 2 mg, 0.51 mmol) in 0.1 M HC1 in MeOH (20 mL) was stirred for 3 hours, then triethylamine (1 mL) was added and the solution was concentrated. The afforded crude product was purified by chromatography on a silica column using hexane:EtOAc (2: 1) as eluent, which gave the title compound (69.3 mg, 55%) as a white solid.
  • Step c) (l-rHvdroxy-(4-methyl-thiazol-2-ylcarbamoyl)-methyll-cvclobutyl
  • Step d) (l-tert-Butoxycarbonylamino-3-methoxy-cyclobutyl)-hydroxy-acetic acid (BB8-b)
  • Compound DJ14 (850 mg, 2.57 mmol) was refluxed with 6N HC1 (60 mL) for 16 h until the amide hydrolysis was complete. The solvent was evaporated under reduced pressure and co- evaporated with water. The product was dissolved in THF:H 2 0 (7:3 v/v, 50 mL), cooled to 0 °C and Et 3 N (1.4 mL, 10.2 mmol) was added followed by di-tert-b tyl dicarbonate (2.25 g, 10.2 mol). The mixture was stirred at room temperature overnight.
  • Building block 9 - a PI- prime side building block (BB9)
  • Step a (3-Fluoro- 1 -[hydroxy-(l -methyl-lH-pyrazol-3-ylcarbamoyl)-methyl]-cyclo butyl ⁇ - carbamic acid tert-butyl ester (Al-a)
  • Step c) N- ⁇ 3 -Fluoro- 1 -[hydroxy-( 1 -methyl- 1 H-pyrazol-3-ylcarbamoyl)-methyll -cyclobutyl) -3 - ( 1 -methyl-cyclopentyl -2-propionylamino-propionamide (Al -c)
  • Step b Removal of the Boc group in step b was performed using 4 M HCl in dioxane and/or MeOH. Removal of the Boc group in steps b and c was performed using 4 M HCl in dioxane and/or MeOH.
  • the P2 building block was coupled to the PI -PI '-building block using DMF as solvent and HATU as coupling reagent.
  • the ⁇ amine was coupled to the PI -building block using HATU as coupling agent.
  • Step a (3 -Difluoro-l-[hydroxy-(l-methyl-lH-pyrazol-3-ylcarbamoyl)-methyll-cyclobutyl
  • Step b [ 1 - ⁇ 3.3 -Difluoro - 1 - [hydroxy-( 1 -methyl- 1 H-pyrazo 1-3 -ylcarbamoylVmethyl] - cyclobutylcarbamoyl ⁇ -2-(l-methyl-cyclopentylVethyl]-carbamic acid tert-butyl ester (Al 1-b)
  • Al 1-a (0.629 mmol) was dissolved in MeOH (3mL).
  • a solution of HC1 in dioxane (4M, 1.5 mL) was added. The solution was stirred at RT for 16h, then concentrated and co-evaporated with toluene.
  • Step c) [ 1 - [3 ,3 -Difluoro- 1 -( 1 -methyl- 1 H-pyrazol-3 -ylaminooxalyD-cyclobutylcarbamoyll -2-( 1 - methyl-cyclopentyD-ethyll-carbamic acid tert-butyl ester (Al 1-c)
  • the P3 moiety was introduced using the appropriate acid anhydride.
  • Step b) 2-[l-(2-Acetylamino-2-cyclohexyl-acetylamino -cyclobutyll-2-hydroxy-N-(l-methyl- lH-pyrazol-3-yl)-acetamide (A28-b)
  • step a (prepared according to the method described in Example 1, step a (50 mg, 0.154 mmol) was treated with 2 mL 4 M HC1 in dioxane to yield the dihydrochloric salt of the free amine after freeze-drying. This was reacted with compound A28-a according to the coupling procedure described in Example C9 step b and the product was purified by gradient column
  • Step c) 2-[l -(2 -Acetylamino-2-cyclohexyl-acetylaminoVcyclobutyl]-N-(l -methyl- lH-pyrazol-3- yl)-2-oxo-acetamide (A28)
  • Step b 2-(3,3-Diethyl-ureidoV3-(l-methyl-cyclopentylVpropionic acid lithium salt fUl-b)
  • Compound Ul-a (19 mg, 86 ⁇ ) was dissolved in THF (1 mL) and triethylamine (3 eq) and diethylcarbamoyl chloride (1 eq) were added. The reaction was heated to 50 °C in a sealed tube for 16h. LC/MS analysis showed 90% conversion. EtOAc was added to the reaction solution and the organic phase was washed with 01M HC1 (aq) (3x). The organic layer was dried (MgSO/t), filtered and the solvent removed in vacuo.
  • the organic layer was eluted through a hydrophobic Phase Separator and concentrated in vacuo.
  • the P2 building block was coupled to the PI -PI '-building block using DMF as solvent and HATU as coupling reagent.
  • the ⁇ amine was coupled to the PI -building block using HATU as coupling agent.
  • step c the P3 building block was introduced in a HATU-promoted coupling with the appropriate acid.
  • Step b) (9H-fluoren-9-yl)methyl l-(2-(l-methyl-lH-pyrazol-3-ylamino)-2- oxoacetyDcyclobutylcarbamate (A39-b)
  • Step c) (Z)-4-((2-( 1 -( 1 -(((9H-fluoren-9-ynmethoxy carbonylamino)cyclobutyl ' )-2-( 1 -methyl- 1 H- pyrazol-3-ylaminoV2-oxoethylidene hydrazinecarboxamido methyl)cyclohexane-carboxylic acid (A39-C)
  • Step d) Resin bound (Z)-4-((2-(l-(l-(((9H-fluoren-9-yl)methoxy)carbonylamino)cyclobutyl)-2- (1 -methyl- lH-pyrazol-3-ylamino)-2-oxoethylidene)hydrazinecarboxamido)methyl)cyclohexane- carboxylic acid (A39-d)
  • Amine resin (TG resin NovaSyn, 01-64-0043, 0.25 mmol/g, 2 g, 0.5 mmol) was added to a solution of acid A39-C (546 mg, 0.85 mmol), HBTU (760 mg, 2 mmol), HOBt (306 mg, 2 mmol) and NMM (550 ⁇ ⁇ , 4 mmol) in DMF (13 mL). The slurry was agitated at room temperature overnight. The resin was filtered and then washed several times alternating with DMF, DCM and MeOH.
  • Step e) Resin bound 4-(((Z)-2-(l-(l-(3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(l- methylcyclopentyl)-2-oxobutyl)cyclobutyl)-2-( 1 -methyl- 1 H-pyrazo 1-3 -ylamino)-2- oxoethylidene)hydrazinecarboxamido)methyl)cyclohexanecarboxylic acid (A39-e)
  • Resin A39-d (0.5 mmol) was treated with a solution of 20% piperidine in DMF (30 mL) for lh, then filtered and washed with DMF (3x 20 mL), and several times alternating with DCM and MeOH.
  • the resulting amine resin suspended in DMF was added to Fmoc-protected 2-amino-3- (l-methyl-cyclopentyl)-propionic acid (294 mg, 0.75 mmol), HBTU (380 mg, 1 mmol), HOBt (153 mg, 1 mmol) and NMM (350 ⁇ , 2 mmol).
  • the slurry was agitated at room temperature over night.
  • the resin was filtered, washed as described above, and dried under vacuum.
  • Resin bound amide A39-e (0.05 mmol) was treated with a solution of TFA:H 2 0 95:5 (4 mL) for 4h.
  • the resin was filtered and washed with DCM (3 x 5 mL) and MeOH (3 x 5 mL).
  • the filtrate and the washes were pooled and concentrated and the afforded crude product was purified by RP-LC-MS (0.1 % NH 4 OH in acetonitrile-0.1 % aq. NH 4 OH, 40-65% over 9 min). Overall yield 4%.
  • Convenient assays for cathepsin K are carried out using human recombinant enzyme, such as that described in PDB.
  • the recombinant cathepsin K can be expressed in a variety of commercially available expression systems including E coli, Pichia and Baculovirus systems.
  • the purified enzyme is activated by removal of the prosequence by conventional methods.
  • Standard assay conditions for the determination of kinetic constants used a fluorogenic peptide substrate, typically H-Z)-Ala-Leu-Lys-AMC, and were determined in either 100 mM Mes/Tris, pH 7.0 containing 1 mM EDTA and 10 mM 2-mercaptoethanol orlOOmMNa phosphate, imM EDTA, 0.1%PEG4000 pH 6.5 or 100 mM Na acetate, pH 5.5 containing 5 mM EDTA and 20 mM cysteine, in each case optionally with 1M DTT as stabiliser.
  • the enzyme concentration used was 5 nM.
  • the stock substrate solution was prepared at 10 mM in DMSO.
  • the assay uses baculo virus-expressed human cathepsin S and the boc-Val-Leu-Lys-AMC fluorescent substrate available from Bachem in a 384 well plate format, in which 7 test compounds can be tested in parallel with a positive control comprising a known cathepsin S inhibitor comparator.
  • 280 ⁇ of 12.5% DMSO are added to rows B - H of two columns of a 96 deep well polypropylene plate. 70 ⁇ 11 of substrate is added to row A. 2 x of assay buffer (100 mM Na phosphate, lOOmM NaCl, pH 6.5) is added to row A, mixed, and double diluted down the plate to row H.
  • assay buffer 100 mM Na phosphate, lOOmM NaCl, pH 6.5
  • assay buffer 100 ⁇ of assay buffer is added to columns 2-5 and 7-12 of 4 rows of a 96 well V bottom polypropylene plate. 200 ⁇ of assay buffer is added to columns 1 and 6.
  • the first test compound prepared in DMSO is added to column 1 of the top row, typically at a volume to provide between 10 and 30 times the initially determined rough 3 ⁇ 4.
  • the rough K is calculated from a preliminary run in which 10 ⁇ of ImM boc-VLK-AMC (1/10 dilution of 10 mM stock in DMSO diluted into assay buffer) is dispensed to rows B to H and 20 ⁇ /well to row A of a 96 well Micro fluorTM plate. 2 ⁇ of each 10 mM test compound is added to a separate well on row A, columns 1-10. Add 90 ⁇ assay buffer containing 1 mM DTT and 2 nM cathepsin S to each well of rows B-H and 180 ⁇ to row A.
  • the second test compound is added to column 6 of the top row, the third to column 1 of the second row etc. Add ⁇ ⁇ ⁇ of comparator to column 6 of the bottom row. Mix column 1 and double dilute to column 5. Mix column 6 and double dilute to column 10.
  • a distributor such as a Multidrop 384, add 30 ⁇ to all wells of the assay plate and read in fluorescent spectrophotometer such as an Ascent.
  • Fluorescent readings (excitation and emission wavelengths 390 nm and 460 nm respectively, set using bandpass filters) reflecting the extent of enzyme cleavage of the fluorescent substrate, notwithstanding the inhibitor, are linear rate fitted for each well.
  • the enzyme is commercially available human cathepsin L (for example Calbiochem).
  • the substrate is H-D-Val-Leu-Lys-AMC available from Bahcem.
  • the assay buffer is 1 OOmM sodium acetate 1 mM EDTA, pH5.5)
  • the DMSO stock (10 mM in 100%DMSO) is diluted to 10% in assay buffer.
  • vo is the velocity of the reaction, Vis the maximal velocity, S is the concentration of substrate with Michaelis constant of KM, and / is the concentration of inhibitor.
  • the compounds of formula I are thus potent inhibitors of cathepsin S and yet selective over the closely related cathepsin K and L.
  • This experiment measures transport of inhibitors through the cells of the human gastroenteric canal.
  • the assay uses the well known Caco-2 cells with a passage number between 40 and 60. Apical to baso lateral transport
  • the baso lateral and the apical wells will contain 1.5 mL and 0.4 mL transport buffer (TB), respectively, and the standard concentration of the tested substances is 10 ⁇ . Furthermore all test solutions and buffers will contain 1% DMSO. Prior to the experiment the transport plates are pre-coated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material. After 21 to 28 days in culture on filter supports, the cells are ready for permeability experiments.
  • TB transport buffer
  • Transport plate no 1 comprises 3 rows of 4 wells each. Row 1 is denoted Wash, row 2 "30 minutes” and row 3 "60 minutes”. Transport plate no 2 comprises 3 rows of 4 wells, one denoted row 4 "90 minutes”, row 5 "120 minutes and the remaining row unassigned.
  • the culture medium from the apical wells is removed and the inserts are transferred to a wash row (No. 1) in a transport plate (plate no.l) out of 2 plates without inserts, which have already been prepared with 1.5 mL transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5.
  • transport buffer HBSS, 25 mM HEPES, pH 7.4
  • the TB in basolateral well also contains 1 % Bovine Serum Albumin.
  • TEEPv Transepithelial electrical resistance value
  • the transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to the 30 minutes row (No. 2) and fresh 425 TB (pH 6.5), including the test substance is added to the apical (donor) well.
  • the plates are incubated in a polymix shaker at 37 °C with a low shaking velocity of approximately 150 to 300 rpm. After 30 minutes incubation in row 2, the inserts are moved to new pre-warmed basolateral (receiver) wells every 30 minutes; row 3 (60 minutes), 4 (90 minutes) and 5 (120 minutes).
  • 25 ⁇ samples are taken from the apical solution after ⁇ 2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.
  • 300 ⁇ ⁇ will be taken from the basolateral (receiver) wells at each scheduled time point and the post value of TEER is measured at the end the experiment.
  • acetonitrile will be added to a final concentration of 0% in the samples.
  • the collected samples will be stored at -20 °C until analysis by HPLC or LC-MS.
  • the baso lateral and the apical wells will contain 1.55 mL and 0.4 mL TB, respectively, and the standard concentration of the tested substances is 10 ⁇ . Furthermore all test solutions and buffers will contain 1% DMSO. Prior to the experiment the transport plates are precoated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material.
  • the transport plate comprises 3 rows of 4 wells. Row 1 is denoted “wash” and row 3 is the “experimental row”.
  • the transport plate has previously been prepared with 1.5 mL TB (pH 7.4) in wash row No. 1 and with 1.55 mL TB (pH 7.4), including the test substance, in experimental row No. 3 (donor side).
  • transport buffer 0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts in row No. 1 and the cell monolayers are equilibrated in the transport buffer system for 30 minutes, 37 °C in a polymix shaker. After being equilibrated to the buffer system the TEER value is measured in each well by an EVOM chop stick instrument.
  • the transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to row 3 and 400 fresh TB, pH 6.5 is added to the inserts. After 30 minutes 250 is withdrawn from the apical (receiver) well and replaced by fresh transport buffer.
  • Cpj is the receiver concentration at the end of the interval i and Crjf is the donor concentration at the beginning of interval i.
  • Cpj is the receiver concentration at the end of the interval i
  • Crjf is the donor concentration at the beginning of interval i.
  • Greater permeability through the gastrointestinal tissue is advantageous in that it allows for the use of a smaller dose to achieve similar levels of exposure to a less permeable compound administered in a higher dose.
  • a low dose is advantageous in that it minimizes the cost of goods for a daily dose, which is a crucial parameter in a drug which is taken for protracted time periods.

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Abstract

L'invention concerne des composés représentés par la formule (I), dans laquelle R1a représente H; et R1b représente un alkyle en C1-C6, un carbocyclyle ou Het; ou R1a et R1b définissent ensemble une amine cyclique saturée comportant 3-6 atomes de noyau; R2a et R2b représentent indépendamment H, un halo, un alkyle en C1-C4, un haloalkyle en C1-C4 ou un alcoxy en C1-C4, ou R2a et R2b forment ensemble avec l'atome de carbone auxquels ils sont liés un cycloalkyle en C3-C6; R3 représente une chaîne alkylique en C5-C10 ramifiée, un haloalkyle en C2-C4 ou un cycloalkyle -CH2C3-C7; R4 représente un alkyle en C1-C6, un haloalkyle en C1-C6, un alkylamino en C1-C6 ou un dialkylamino en C1-C6. Ces composés sont destinés à prévenir ou à traiter un trouble caractérisé par une expression ou une activation inappropriées de la cathepsine S.
PCT/IB2010/055738 2009-12-10 2010-12-10 Inhibiteurs des cystéine protéases WO2011070541A1 (fr)

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US13/514,760 US8859605B2 (en) 2009-12-10 2010-12-10 Cysteine protease inhibitors
MX2012006652A MX2012006652A (es) 2009-12-10 2010-12-10 Inhibidores de proteasa de cisteina.
CN2010800635673A CN102858751A (zh) 2009-12-10 2010-12-10 半胱氨酸蛋白酶抑制剂
BR112012014091A BR112012014091A2 (pt) 2009-12-10 2010-12-10 inibidores de cisteína protease.
CA2782294A CA2782294A1 (fr) 2009-12-10 2010-12-10 Inhibiteurs des cysteine proteases
EP10835594.2A EP2509957A4 (fr) 2009-12-10 2010-12-10 Inhibiteurs des cystéine protéases
RU2012128859/04A RU2012128859A (ru) 2009-12-10 2010-12-10 Ингибиторы цистеиновой протеазы
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WO2012168852A1 (fr) * 2011-06-08 2012-12-13 Medivir Uk Ltd Inhibiteurs de cystéine protéase
WO2012172473A1 (fr) * 2011-06-13 2012-12-20 Medivir Uk Ltd Inhibiteurs de la cystéine protéase
EP2582660A1 (fr) * 2010-06-16 2013-04-24 Medivir UK Ltd Nouveaux inhibiteurs de cathepsine s protéase, utiles dans le traitement, par ex., de maladies auto-immunes, d'allergies et de douleurs chroniques

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WO2010070615A1 (fr) * 2008-12-19 2010-06-24 Medivir Uk Ltd Inhibiteurs de cystéine protéase
EA201200866A1 (ru) * 2009-12-10 2012-12-28 МЕДИВИР ЮКей ЛИМИТЕД Ингибиторы цистеиновых протеаз
GB201314503D0 (en) * 2013-08-13 2013-09-25 Medivir Ab Cysteine protease inhibitor salt
CN113332420B (zh) * 2021-06-30 2023-07-28 蚌埠医学院 日本血吸虫半胱氨酸蛋白酶抑制剂在动脉粥样硬化方面的应用

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EP1498411A1 (fr) * 2002-04-25 2005-01-19 Ono Pharmaceutical Co., Ltd. Composes derives de dicetohydrazine et medicaments contenant ces composes comme ingredient actif
WO2006102423A1 (fr) * 2005-03-21 2006-09-28 Celera Genomics Composes alpha cetoamides utilises en tant qu'inhibiteurs des cysteines proteases
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EP2582660A1 (fr) * 2010-06-16 2013-04-24 Medivir UK Ltd Nouveaux inhibiteurs de cathepsine s protéase, utiles dans le traitement, par ex., de maladies auto-immunes, d'allergies et de douleurs chroniques
EP2582660A4 (fr) * 2010-06-16 2013-11-27 Medivir Uk Ltd Nouveaux inhibiteurs de cathepsine s protéase, utiles dans le traitement, par ex., de maladies auto-immunes, d'allergies et de douleurs chroniques
EP2752404A1 (fr) * 2010-06-16 2014-07-09 Medivir UK Ltd Inhibiteurs de la cystéine protéase
WO2012168852A1 (fr) * 2011-06-08 2012-12-13 Medivir Uk Ltd Inhibiteurs de cystéine protéase
WO2012172473A1 (fr) * 2011-06-13 2012-12-20 Medivir Uk Ltd Inhibiteurs de la cystéine protéase

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AU2010329468B2 (en) 2013-08-22
AU2010329470B2 (en) 2013-08-22
WO2011070539A1 (fr) 2011-06-16
RU2012128859A (ru) 2014-01-20
US8859605B2 (en) 2014-10-14
EA201200866A1 (ru) 2012-12-28
EP2509956A4 (fr) 2013-06-19
AU2010329470A1 (en) 2012-06-14
EP2509956B1 (fr) 2014-07-23
JP2013513597A (ja) 2013-04-22
US20120283305A1 (en) 2012-11-08
AU2010329468A1 (en) 2012-06-14
US20120309673A1 (en) 2012-12-06
BR112012014091A2 (pt) 2017-04-04
EP2509957A1 (fr) 2012-10-17
MX2012006653A (es) 2012-11-30
CA2782292A1 (fr) 2011-06-16
EP2509957A4 (fr) 2013-05-22
MX2012006652A (es) 2012-11-30
KR20120110173A (ko) 2012-10-09
IN2012DN04915A (fr) 2015-09-25
EP2509956A1 (fr) 2012-10-17
US8815809B2 (en) 2014-08-26
CN102834383A (zh) 2012-12-19
BR112012013861A2 (pt) 2017-09-26
KR20120093423A (ko) 2012-08-22
IN2012DN04914A (fr) 2015-09-25
CN102858751A (zh) 2013-01-02
CA2782294A1 (fr) 2011-06-16
JP2013513598A (ja) 2013-04-22

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