WO2006064286A1 - Cathepsin s inhibitors - Google Patents

Cathepsin s inhibitors Download PDF

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Publication number
WO2006064286A1
WO2006064286A1 PCT/GB2005/050243 GB2005050243W WO2006064286A1 WO 2006064286 A1 WO2006064286 A1 WO 2006064286A1 GB 2005050243 W GB2005050243 W GB 2005050243W WO 2006064286 A1 WO2006064286 A1 WO 2006064286A1
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WIPO (PCT)
Prior art keywords
alkyl
methyl
ethyl
compound according
hydroxy
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PCT/GB2005/050243
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French (fr)
Inventor
David Hardick
Matt Tozer
Julie Canfield
Michelle Wilson
Alastair Rae
Philip Fallon
Bjorn Classon
Charlotta Lindquist
Susana Ayesa
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Medivir Uk Ltd
Peptimmune, Inc
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Priority claimed from GB0427169A external-priority patent/GB0427169D0/en
Priority claimed from GB0507628A external-priority patent/GB0507628D0/en
Priority claimed from GB0510304A external-priority patent/GB0510304D0/en
Application filed by Medivir Uk Ltd, Peptimmune, Inc filed Critical Medivir Uk Ltd
Publication of WO2006064286A1 publication Critical patent/WO2006064286A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/26—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D307/30—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/32—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • This invention relates to inhibitors of cathepsin S, and their use in methods of treatment for disorders involving cathepsin S such as autoimmune, 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, Trypanosoma cruzei and brucei, Crithidia fusiculata, Schistosoma spp.
  • Cathepsin S is a highly active cysteine protease belonging to the papain superfamily. Its primary structure is 57%, 41% and 45% homologous with that of the human cathepsin L and H and plant cysteine proteases 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.
  • cathepsin S is implicated are asthma, chronic obstructive pulmonary disease, endometriosis and chronic pain.
  • R 1 R', R 1 C(O) , R' C(S), R' SO2 , R' OC(O), R' NHC(O) wherein R' is a monocyclic ring;
  • R 2 , R 4 H, Ci- 7 -alkyl, C 3 - 7 -cycloalkyl;
  • R 3 Ci- 7 -alkyl, C 3 - 7 -cycloalkyl, Ar-Ci - 7 -alkyl;
  • R 5 Ci- 7 -alkyl, Halogen, Ar-Ci - 7 -alkyl, Ci- 3 -alkyl-CONR'",
  • R 6 H, Ci-7-alkyl, Ar-Ci -7-alkyl, Cr 3 -BIkYl-SO 2 -R 1 ", Ci- 3 -alkyl-C(0)-NHR ix or CH 2 XAr,
  • R 3 groups specifically disclosed in WO00/69855 are branched chain alkyl moieties such as n-butyl, t-butyl, 3-(2,2-dimethylpropyl), 4-(2-methylbutyl), 4-(3,3-dimethylbutyl), 4-(3,3-dimethyl- 2-methylbutyl), 4-(3-methyl-2-methylbutyl), or 5-(2-methyl-3-methylpentyl).
  • Page 27, line 13 of WO00/69855 discloses the compound morpholine-4-carboxylic acid [3,3-dimethyl-1S-(2-ethyl-4- oxo-tetrahydrofuran-3-ylcarbamoyl)butyl]amide.
  • R 1 is CrC 4 straight or branched alkyl, optionally substituted with up to three substituents selected from halo and hydroxy;
  • R 2 is halo, hydroxy, methyloxy, or C r C 2 alkyl, which alkyl is optionally substituted with up to three halogens or an hydroxy or a methyloxy;
  • D is -C 3 -C7 alkylene-, thereby defining a cycloalkyl ring;
  • R 3 is a carbocyclic ring selected from C 3 -C 6 cycloalkyl, C 5 -C 6 cycloalkenyl or phenyl, or a heterocyclic ring I selected from azepanyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, thiopyranyl, furanyl, tetrahydrofuranyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl, pyrazolyl, indolyl, which ring is optionally substituted with up
  • R 4 is independently selected from halo, oxo, nitrile, nitro, d-C 4 alkyl, -NRaRb, NH 2 CO-, X-R 5 ,
  • R 5 is H, CrC 4 alkyl, C 3 -C 6 cycloalkyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, thiopyranyl, furanyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl, phenyl, benzyl, any of which is optionally substituted with R 6 ;
  • R 5a is R 5 or -NRaRb
  • R 6 is independently selected from hydroxy, -NH 2 , NHC r C 3 alkyl, N(C r C 3 alkyl) 2 , nitro, cyano, carboxy, oxo, d-C 4 alkyl, Ci-C 4 -alkoxy, d-C 4 alkanoyl, carbamoyl;
  • Ra and Rb are independently selected from H, C r C 4 alkyl and acetyl, or Ra, Rb and the N atom to which they both are joined form a ring selected from morpholine, piperazine, piperidine, pyrrolidine;
  • Ra and Rb are independently selected from H, C r C 4 alkyl and acetyl, or Ra, Rb and the N atom to which they both are joined form a ring selected from morpholine, piperazine, piperidine, pyrrolidine
  • Rc is H, CrC 4 alkyl, C 0 -C 3 alkylcarbocyclyl
  • X is independently a bond or C r C 4 alkylenyl; m is independently 0,1 or 2; and pharmaceutically acceptable salts thereof.
  • R 1 examples include ethyl, 2-fluoroethyl or 2-hydroxyethyl, and methyl, fluoromethyl and hydroxyethyl, especially ethyl and methyl.
  • the stereochemistry at the C-4 and C- 5 positions of the furanone ring is enantiomerically pure, or at least 85%, for example at least 90% or more preferably at least 95% enantiomerically pure 4S, 5S configuration.
  • Preferred P1 groups (as defined below) therefore include:
  • R 10 is conveniently H or a CrC 4 alkyl ether or d-C 4 alkylthioether such as methyloxy, ethyloxy, methylthio- or ethylthio or the corresponding ketals.
  • P1 groups thus include:
  • R 1 ' is H or -CH 3 .
  • R 10 is other than H, it is currently preferred that the stereochemistry at the ring carbon atom which bears R 10 comprises at least 85%, for example at least 90% preferably at least 95% and more preferably 100% enantiomerically pure alpha configuration:
  • D is conveniently pentylene, thereby defining a cyclohexyl ring, or propylene, thereby defining a cyclobutyl ring, but more preferably D is butylene, thereby defining a cyclopentyl ring.
  • R 2 include a halogen such as fluoro, fluoro methyl, difluoromethyl or trifluoromethyl, and most preferably methyl.
  • the side chain comprising D and R 2 ie the P2 group (as defined below) may be in the R or S configuration, or a racemate thereof.
  • the P2 group is substantially, for example greater than 90% and most preferably greater than 95% in the S stereoconfiguration, that is reflecting that of an L-amino acid.
  • Preferred side chains thus include:
  • typical values for R 3 include: unsubstituted or substituted furanyl, especially furan-2-yl or furan-3- yl, or alkyl substituted furanyl such as 2-methylfuran-3-yl, 2,4-dimethylfuran-3-yl, or aryl substituted furanyl, even more especially 5-phenylfuran-2-yl, 5-(2-chlorophenyl)furan-2-yl, 5-(3chlorophenyl)furan-2-yl, 5-(4- chlorophenyl)furan-2- yl, 5-(4-fluorophenyl)furan-2-yl, 5-(4hydroxyphenyl)furan-2-yl, 5-(3- trifluoromethylphenyl)furan-2-yl, 5-(4-trifluoromethylphenyl)furan-2-yl, 5-(3- trifluoromethylphenyl)furan-2-yl, 5-(4-methylphenyl)furan-2-yl,
  • 2H-pyrazolyl unsubstituted or ar ⁇ l-substituted triazolyl, particularly phenyl-substituted triazoles including 3- phenyl-3H-[1 ,2,3]triazol-3-yl; unsubstituted or substituted pyrazinyl, particularly pyrazin-2-yl and 5-methylpyrazin-2-yl; unsubstituted or substituted imidazolyl, particularly 1 -H-imidazol-2-yl, 1 -methyl-1 H-imidazol-4-yl or 1-methyl-IH-imidazol-2-yl; thiophenyl, especially thiophene-3-yl and thiophen-2-yl, more especially heterocycle or aryl substituted C 0 -C 6 alkylthiophenyl, particularly 5-pyridin-2-ylthiophen-2-yl, more especially C r
  • C 6 alkylthiophenyl particularly 5-methylthiophenyl or 3-methylthiophen-2-yl; more especially d- C 6 alkoxythiophenyl, particularly 3-ethoxythiophen-2-yl; phenyl, especially alkyl-substituted phenyl, halogen-substituted phenyl, trihaloalkylsubstituted phenyl, alkoxy-substituted phenyl, or acetoxy-substituted phenyl, especially 4-methylphenyl, 3- chlorophenyl, 4-chlorophenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-chlorophenyl,
  • R 3 include optionally substituted thienyl, pyrazinyl, pyridyl, pyrrolyl, and especially furyl or morpholinyl.
  • R 3 Favoured values for R 3 include fur-3-yl, thien-3-yl, pyrazin-2-yl, pyrid-4-yl, pyrrol-2-yl and especially N-morpholino.
  • R 3 is phenyl particularly phenyl substituted as follows:
  • Ry is halomethyl such as CF 3 or CF 2 or an hydroxylated methyl group, such as HOCH 2 or HO(CH 2 J 2 )C-, any of these preferences being optionally further substituted with an R 4 group such as Rx.
  • R3 comprises phenyl which is substituted with a urea, such as a cyclic urea:
  • a favoured aspect of the invention thus comprises compounds of the formula:
  • R 12 typically comprises a pharmaceutically acceptable ether or ester prodrug which is hydrolysed in vivo to release the parent phenol.
  • R 4 is at the 3, or the 3 and 5 positions of the phenyl ring.
  • Representative values include R 4 as halo, such as 3-fluoro, 3,5-difluoro, 3-chloro or 3,5-dichloro.
  • R 4 values include one or more d-C 4 alkyl, such as methyl, ethyl, i-propyl or t-butyl.
  • Representative values for this aspect of the invention thus include 5-methyl, 5-ethyl, 5-i-propyl, 5-t-butyl, 6-methyl, 5-methyl-3-fluoro.
  • a favoured aspect of the invention comprises compounds of the formula I, wherein R 3 has the partial structure:
  • R 5a is R 5 as defined above, preferably CrC 4 alkyl, such as methyl, ethyl or i-propyl or t-butyl; halogenated CrC 4 alkyl such as trifluoromethyl; C 3 -C 6 cycloalkyl, such as cyclopropyl or cyclohexyl; or phenyl or benzyl, any of which is optionally substituted with R 6 .
  • R 5a may be NRaRb as defined above including cyclic amines, such as -NHMe or -N(Me) 2 , or piperazine, N-methyl piperazine, pyrrolidine, piperidine or morpholine.
  • cyclic amines such as -NHMe or -N(Me) 2
  • piperazine N-methyl piperazine, pyrrolidine, piperidine or morpholine.
  • R 5 include heteroaryl rings such as pyrrolyl, oxazolyl, isoxazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl or indolyl, especially thiazolyl, any of which is substituted with R 6 groups such as d-C 4 alkyl.
  • a currently favoured sulphonamide has the partial structure:
  • Rd' is Me or preferably H
  • R 6 is H or methyl, especially at the ring position, adjacent the N for example:
  • R 5 as defined above, preferably C r C 4 alkyl, such as methyl, ethyl or i-propyl or t-butyl; halogenated CrC 4 alkyl such as trifluoromethyl; C 3 -C 6 cycloalkyl, such as cyclopropyl or cyclohexyl; or phenyl or benzyl, any of which is optionally substituted with R 6 .
  • R 5 together with Rd defines a 3-6 membered N-containing ring such as azidine, pyrrolidine, pyridine, piperidine, morpholine, piperazine or N-methylpiperizine.
  • a sulphonamide substituted phenyl is optionally substituted with an additional substituent R 4 , typically, but not invariably, in the 4 position if the sulphonamide is in the 3 position and vice versa.
  • R 4 groups thus include halo such as chloro or fluoro, CrC 4 alkyl such as methyl (including 2-methyl) and d-C 4 alkoxy such as methoxy.
  • Rd is conveniently d-C 4 alkyl or together with R 5 defines a 3-6 membered N-containing ring such as azidine, pyrrolidine, pyridine, piperidine, morpholine, piperazine or N-methylpiperizine.
  • m is typically 1 (sulphenamide) or preferably 2 (sulphonamide).
  • An alternative phenyl-based R 3 value is phenyl substituted with a pair of R 4 groups which together constitute a nitrogen containing chain of 3 or 4 atoms thereby defining a ring fused to the phenyl such as:
  • R 4 is methyl or especially H.
  • the linkage to E is para to a nitrogen in the fused ring:
  • fused rings for constituting a nitrogen containing ring fused to a phenyl R 3 include
  • Rz is CH; NH or O, especially O and preferably NH
  • R 4 is methyl or especially H.
  • the linkage to E is para to a nitrogen in the fused ring.
  • Still further fused rings for R 3 include variants wherein the fused nitrogen-containing ring defines a saturated or unsaturated 6 membered heterocycle, such as:
  • Rz is NH, O or CH, especially O and preferably NH
  • R 4 and R 4 ' are optional substituents as defined above, preferably H, and O' is absent (ie 2 hydrogen atoms) or keto.
  • the linkage to E is para to a nitrogen in the fused ring.
  • Still further fused rings for R 3 include variants wherein the fused nitrogen containing ring defines an optionally substituted quinoline, isoquinoline, tetrohydroquinoline or tetrahydroisoquinoline moiety, such as
  • R 4 and R 4 ' are H and the linkage to E is para to the nitrogen in the fused ring.
  • R 3 groups include pyrimidyl, such as 2-pyrimidyl, for example 5-OH-pyrimid-2-yl; or pyridyl, such as pyrid-4-yl, for example O ⁇ pyrid-4yl; or pyrid-3-yl, for example 6-hydroxy- pyrid-3-yl.
  • pyrimidyl such as 2-pyrimidyl, for example 5-OH-pyrimid-2-yl
  • pyridyl such as pyrid-4-yl, for example O ⁇ pyrid-4yl
  • pyrid-3-yl for example 6-hydroxy- pyrid-3-yl.
  • a further aspect of the invention comprises a method employing the compounds of the invention for the 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.
  • diseases or conditions include those enumerated in WO 97/40066, such as autoimmune diseases, allergies, such as asthma and hayfever, multiple sclerosis, rheumatoid arthritis and the like.
  • a further example is the treatment of endometriasis, and especially chronic pain, as disclosed in WO0320287.
  • the invention further provides the use of the compounds of formula IV 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 Il MHC-complexes. This association occurs because the structures recognized by T cells, the cells that cause autoimmunity, are complexes comprised of class Il MHC molecules and antigenic peptides.
  • Autoimmune disease can result when T cells react with the host's class Il MHC molecules when complexed with peptides derived from the host's own gene products.
  • any autoimmune disease in which class Il 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 “allergic response” is used herein in accordance with standard usage in the medical field.
  • 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 Il MHC/antigenic peptide complexes. If these class Il MHC/antigenic peptide complexes are inhibited from being formed, the allergic response is reduced or suppressed.
  • 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 Il 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 Il MHC molecules presenting antigenic peptides on the surface of donor cells. To the extent that this response is dependent upon the formation of class Il
  • cathepsin S inhibition of 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.
  • the above embodiments have been illustrated with an MHC class Il mechanism but the invention is not limited to this mechanism of action. Suppression of cathepsin S as a treatment of COPD or chronic pain may not, for example, involve MHC class Il at all.
  • 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 car boxy lie 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 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 3 , or amine functions such as an R 4 sulphonamide amine function.
  • Preferred pharmaceutically acceptable esters include those derived from CrC 6 carboxylic acids such as acetyl or pivaloyl or optionally substituted benzoic acid esters, preferably unsubstituted or substituted with R 6 .
  • Favoured sulphonamide prodrugs include aminoacyls derived from d-C 6 carboxylic acids such as acetyl or pivaloyl or optionally substituted benzoic acid esters, preferably unsubsbstituted or substituted with R 6 .
  • Co-C 3 alkylcarbocyclyl comprises C 0 -C 3 alkylaryl and Co-C 3 alkylC 3 C 7 cycloalkyl.
  • 'Co-C 3 alkylaryr as applied herein is meant to include an ar ⁇ l moiety such as a phenyl, naphthyl or phenyl fused to a C 3 -C 7 CyClOaI kyl (for example indanyl), which aryl is directly bonded (i.e. C 0 ) or through an intermediate methylene, ethylene, or propylene group.
  • 'C 0 -C 3 alkylC 3 C 7 cycloalkyr as applied herein is meant to include a C 3 -C 7 cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, which cycloalkyl is directly bonded (i.e. C o alkyl) or through an intermediate methylene, ethylene, propylene or isopropylene group.
  • the cycloalkyl group may contain an unsaturated bond.
  • the ar ⁇ l or cycloalkyl group is optionally substituted with 1-3 substituents selected from halo, hydroxy, nitro, cyano, carboxy, CrC 6 alkyl, CrC 6 alkoxy, C r C 6 alkoxyCi-C 6 alkyl, CrC 6 alkanoyl, amino, azido, oxo, mercapto, nitro, or C 0 -C 3 alkylR 3 .
  • 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 pharmaceutical composition comprising bringing a compound of Formula IV 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 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.
  • 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 superamily) concentrations of the order 0.01-100 uM, more preferably 0.01-10 uM, such as 0.1-5 uM are typically desirable and achievable. Synthesis of the compounds of the present invention can be performed by different chemical strategies in solution or solid phase or a combination of both. The compounds are typically prepared as building blocks reflecting the P1 , P2 and P3 moieties of the end product inhibitor.
  • the P1 building block will be an N-protected C-5-substituted furan-3-onamine
  • P2 will be an N-protected amino acid in which the side chain comprises the D-containing saturated ring and branched alkyl linker
  • P3 typically comprises a capping group such as a substituted, heteroaroyl or aroyl moiety.
  • the suitably protected individual building blocks can first be prepared and subsequently coupled together i.e. P2+P1 ⁇ P2-P1.
  • precursors of the building blocks can be coupled together and modified at a later stage of the synthesis of the inhibitor sequence.
  • Further building blocks, precursors of building blocks or prefabricated bigger fragments of the desired structure can then be coupled to the growing chain, e.g. R 3 -E-P2*+ P1 ⁇ R 3 -E-P2-P1 or R 3 -E*+P2-P1 ⁇ R 3 -E-P2-P1 , where * denotes an activated form.
  • Coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be carried out using standard coupling procedures such as the azide method, mixed carbonic- car boxy lie acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active ester (pnitrophenyl ester, N-hydroxysuccinic imido ester) method, Woodward reagent K- method, carbonyldiimidazole method, phosphorus reagents or oxidation-reduction methods. Some of these methods (especially the carbodiimide method) can be enhanced by adding 1- hydroxybenzotriazole or 4-DMAP. These coupling reactions can be performed in either solution (liquid phase) or solid phase.
  • the coupling step involves the dehydrative coupling of a free carboxyl of one reactant with the free amino group of the other reactant in the present of a coupling agent to form a linking amide bond.
  • coupling agents are found in general textbooks on peptide chemistry, for example, M. Bodanszky, "Peptide Chemistry", 2nd rev ed., Springer- Verlag, Berlin, Germany, (1993) hereafter simply referred to as Bodanszky, the contents of which are hereby incorporated by reference.
  • suitable coupling agents are N 1 NT- dicyclohexylcarbodiimide, 1-hydroxybenzotriazole in the presence of N 1 N 1 - dicyclohexylcarbodiimide or N-ethyl-N 1 - [ (3dimethylamino) propyl] carbodiimide.
  • a practical and useful coupling agent is the commercially available (benzotriazol-1-yloxy) tris- (dimethylamino) phosphonium hexafluorophosphate, either by itself or in the present of 1-hydroxybenzotriazole or 4-DMAP.
  • Another practical and useful coupling agent is commercially available 2-(IH- benzotriazol-1-yl)-N, N, N 1 , N 1 - tetramethyluronium tetrafluoroborate. Still another practical and useful coupling agent is commercially available 0-(7-azabenzotrizol-1-yl)-N, N 1 N 1 , N 1 - tetramethyluronium hexafluorophosphate.
  • the coupling reaction is conducted in an inert solvent, e. g. dichloromethane, acetonitrile or dimethylformamide.
  • An excess of a tertiary amine e. g. diisopropylethylamine, N- methylmorpholine, N-methylpyrrolidine or 4-DMAP is added to maintain the reaction mixture at a pH of about 8.
  • the reaction temperature usually ranges between 0 °C and 50 °C and the reaction time usually ranges between 15 min and 24 h.
  • the functional groups of the constituent non-natural amino acids generally must be protected during the coupling reactions to avoid formation of undesired bonds.
  • the protecting groups that can be used are listed in Greene, "Protective Groups in Organic Chemistry", John Wiley & Sons, New York (1981) and "The Peptides: Analysis, Synthesis, Biology", Vol. 3, Academic Press, New York (1981), hereafter referred to simply as Greene, the disclosures of which are hereby incorporated by reference.
  • the alpha-carboxyl group of the C-terminal residue is usually protected as an ester that can be cleaved to give the carboxylic acid.
  • Protecting groups that can be used include 1) alkyl esters such as methyl, trimethylsilyl and t.butyl, 2) aralkyl esters such as benzyl and substituted benzyl, or 3) esters that can be cleaved by mild base or mild reductive means such as trichloroethyl and phenacyl esters.
  • the alpha-amino group of each amino acid to be coupled is typically be protected. Any protecting group known in the art can be used. Examples of such groups include: 1) acyl groups such as formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl; 2) aromatic carbamate groups such as benzyloxycarbonyl (Cbz or Z) and substituted bensyloxycarbonyls, and 9- fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic carbamate groups such as tertbutyloxycarbonyl (Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4) cyclic alkyl carbamate groups such as cyclopentyloxycarbonyl and adamantyloxycarbonyl; 5) alkyl groups such as triphenylmethyl and benzyl; 6) trialkylsilyl
  • the alpha-amino protecting group is typically cleaved prior to the next coupling step.
  • Boc group the methods of choice are trifluoroacetic acid, neat or in dichloromethane, or HCI in dioxane or in ethyl acetate.
  • the resulting ammonium salt is then neutralized either prior to the coupling or in situ with basic solutions such as aqueous buffers, or tertiary amines in dichloromethane or acetonitrile or dimethylformamide.
  • the Fmoc group the reagents of choice are piperidine or substituted piperidine in dimethylformamide, but any secondary amine can be used.
  • the deprotection is carried out at a temperature between 0 0 C and room temperature usually 20-22 0 C.
  • any of the natural or non-natural amino acids having side chain functionalities will typically be protected during the preparation of the peptide using any of the above described groups.
  • Those skilled in the art will appreciate that the selection and use of appropriate protecting groups for these side chain functionalities depend upon the amino acid and presence of other protecting groups in the peptide. In the selection of such protecting groups it is desirable that the group is not removed during the deprotection and coupling of the alpha-amino group.
  • Boc when used as the alpha-amino protecting group, the following side chain protecting groups are suitable: p-toluenesulfonyl (tosyl) moieties can be used to protect the amino side chain of amino acids such as Lys and Arg; acetamidomethyl, benzyl (Bn), or tert- butylsulfonyl moities can be used to protect the sulfide containing side chain of cysteine; benzyl (Bn) ethers can be used to protect the hydroxy containing side chains of serine, threonine or hydroxyproline; and benzyl esters can be used to protect the carboxy containing side chains of aspartic acid and glutamic acid.
  • p-toluenesulfonyl (tosyl) moieties can be used to protect the amino side chain of amino acids such as Lys and Arg
  • Fmoc is chosen for the alpha-amine protection
  • usually tert. butyl based protecting groups are acceptable.
  • Boc can be used for lysine and arginine, tert.butyl ether for serine, threonine and hydroxyproline, and tert-butyl ester for aspartic acid and glutamic acid.
  • Triphenylmethyl (Trityl) moiety can be used to protect the sulfide containing side chain of cysteine.
  • the P1 building block may be elongated with the P2 amino acid (or the ready formed P3-P2 intermediate) while the P1 is in the furanone form.
  • elongation with P2/P3 may take place on a furanol which is subsequently oxidised by Dess Martin chemistry in an organic solvent such as DCM.
  • Scheme 1 depicts that a suitable aldehyde 1 , such as cyclopentylaldehyde, can be derivatised into the silyl enol ether 2 using, for example, N-Methyl-N-trimethylsilylacetamide in DMF at room temperature. 2 can then act as a suitable precursor for a number of variations of X.
  • a suitable alkyl halide in the presence of fluoride anion, X can represent suitable alkyl groups.
  • suitable electrophilic fluorinating agents such as SelectfluorTM in a solvent such as DMF or acetonitrile, X can represent fluorine.
  • the conversion of 4 into the chiral amino acid 5 can be achieved with a chiral catalyst, such as [EtDuPHOS-Rh (COD)]+ in a solvent such as methanol under hydrogen pressure of between 1 and 5 bar.
  • a chiral catalyst such as [EtDuPHOS-Rh (COD)]+ in a solvent such as methanol under hydrogen pressure of between 1 and 5 bar.
  • 4 can be converted into the achiral amino acid 6 using a non-chiral catalyst such as that based on a palladium or rhodium-containing species e.g. Wilkinson's catalyst. Resolution of the amino acid will then follow one of many well documented methods, such as enzymatic hydrolysis of the ester, or separation of the racemates by chiral-HPLC.
  • Scheme 2 depicts that the preparation of 3 can also be achieved from direct reaction of a suitable lithium enolate with a suitable electrophilic reagent, such as an alkyl halide. Therefore, treatment of 7 with LDA in THF at -78 0 C followed by quenching of the resultant anion affords 8.
  • the ester group of 8 can be reduced, for example with lithium aluminium hydride to the corresponding alcohol 9.
  • Compound 3 is then prepared by oxidation of the alcohol with a suitable oxidant, such as pyridinium chlorochromate in DCM at room temperature.
  • Scheme 3 shows an alternative synthesis to prepare the C5 alkylene amino acid 16 in homochiral form.
  • Geraniol 10 is converted to the phosphate 11 with diethyl chlorophosphonate and then reacted with a homochiral zinc/copper couple of alanine 12.
  • Compound 13 is obtained from Sn2 reaction of the zinc/copper couple, whilst compound 14 is obtained from the alternative Sn2' mechanism.
  • Ring-closing metathesis reaction of 14 using for example Grubb's catalyst gives the methylcyclopentene derivative 15. Atmospheric pressure hydrogenation of the methylcyclopentene double bond can be achieved with a palladium catalyst in a solvent like methanol to afford the amino acid 16.
  • Enantioselectivity is in excess of 95%.
  • An alternative for substitution at R 2 involves the method shown in Scheme 5.
  • An appropriate cycloalkanone is treated with a zinc/copper couple of alpha-bromomethylacetate in a solvent such as THF at reflux.
  • the hydroxyl group in 24 can be left underivatised and the ester can be reduced to the primary alcohol with a reducing agent such as lithium aluminium hydride.
  • a compound such as 25 can be treated in the same way as compound 20 to afford the desired substituted cycloalkyl alanine.
  • the hydroxyl group in 24 can be derivatised to form the alkyloxy at R 2 , using a reagent such as sodium hydride and an alkyl halide in THF at room temperature or reflux. The same procedure for the synthesis of the achiral amino acid would then apply.
  • a suitable dibromoalkane can be reacted with diethylmalonate with, for example, sodium ethoxide in ethanol to afford the diester 26.
  • This can be converted into the ester/aldehyde 27 in a number of ways, for example with diisobutylaluminium hydride in dichloromethane at -78 deg C.
  • This aldehyde serves as a useful precursor for a number of derivatives.
  • the methylene alcohol 28 can be prepared by reduction of the aldehyde with sodium borohydride in a solvent such as ethanol; the alkyloxy methylene 29 is produced by alkylation of the methylene alcohol 28 with an alkyl halide and a suitable base such as sodium hydride; the methylene fluoride 30 is produced by fluorination of 28 with a suitable fluorinating agent such as DAST or Deoxyfluor.
  • a suitable fluorinating agent such as DAST or Deoxyfluor.
  • cyclohexylalanines can be prepared as in Scheme 7. Diels-Alder reaction of 1,3-butadiene with appropriately substituted dieneophiles can afford the cyclohexene derivative 32. Reduction of the cyclohexene double bond and manipulation of the ester moiety to the aldehyde 33, as shown in the scheme, provides the precursor to the substituted cyclohexyl alanine amino acids. These final steps can be achieved using the chemistry outlined in Scheme 8.
  • Scheme 8 depicts the synthesis of a methylcyclopentylalanine building block.
  • Commercially available methyl cyclopentane carboxylate 34 is methylated with LDA and iodomethane (i, BuLi, diisopropylamine, MeI) to give 35.
  • Hydrolysis of the ester with LiOH followed by treatment with oxalyl chloride (ii, LiOH, oxalylchloride) gives acid chloride 36.
  • Wolff rearrangement with diazomethane (iii, CH 2 N 2 , Et 3 N) and silver benzoate (iv, silver benzoate, Et 3 N, MeOH) gives the ester 38.
  • a suitable base such as triethylamine or dimethylaminopyridine.
  • the N- protected P1-P2 intermediate is treated with 4M HCI in dioxan.
  • An optionally substituted R 3 - SO 2 CI is added with Et 3 N and a catalytic amount of DMAP.
  • the isocyanate, or equivalent reactive intermediate can be formed by reaction of the amino group of the P2-amino acid with phosgene, or with dinitrophenylcarbonate in the presence of a suitable base, e.g. triethylamine.
  • a suitable base e.g. triethylamine.
  • they can be formed by reaction of the amino group of the P2 amino acid with a suitable chloroformate, e.g. benzylchloroformate.
  • the R3 amine is reacted with the isocyanate of the P2-P1 intermediate under similar conditions.
  • the time, temperature and sequence of addition used depends on the reactivity of the individual reagents.
  • a special case of a urea derivative are compounds wherein R 3 represents an unsaturated ring such as morpholine, piperazine or piperidine which is N-bonded to E as carbonyl.
  • R 3 represents an unsaturated ring such as morpholine, piperazine or piperidine which is N-bonded to E as carbonyl.
  • Such compounds are readily prepared, for example by treating the N-protected P2-P1 intermediate with 4M HCI/dioxane, adding the R3-chloride, for example morpholinyl carbonyl chloride, together with TEA in DCM.
  • R 10 is an hydroxyl, ether, ester or ketone
  • Oxalyl chloride (89 mmol) was added to a solution of 1 -methyl-cyclopentanecarboxylic acid (74 mmol) in DCM at O 0 C. This was followed by a few drops of DMF. The mixture was stirred overnight then the solvents were removed in vacuo to give a pale brown semi-solid which was dissolved 1 :1 THF:MeCN. Triethylamine (96 mmol) was added followed by diazomethane (222 mmol) in diethyl ether. The mixture was stirred overnight then the solvents were removed in vacuo. The residue was dissolved in TBME. The organics were washed (water then NaHCO 3 (sat. aq.)), dried (MgSO 4 ) then concentrated in vacuo to give a yellow oil which was used with no further purification (66.2 mmol, 90%).
  • LiAIH 4 (99.3 mmol) was added portionwise to a solution of (i-methylcyclopentyl)acetic acid methyl ester (66.2 mmol) in THF at O 0 C. The mixture was allowed to warm to room temperature and stirring was continued for 1.5 hours. Ether was added and the mixture cooled to O 0 C. 3.8 ml of water was added followed by 3.8 ml of 15% aqueous NaOH solution then 11.4 ml of water. The mixture was warmed to room temperature and stirred for 15 minutes. Anhydrous MgSO 4 was added and stirring was continued for a further 15 minutes. The mixture was filtered and the filtrate was concentrated in vacuo. Distillation (76 0 C @ 17 mmHg) gave the product as a clear oil (40.7 mmol, 62%).
  • Furan-3-carboxylic acid [(1 S)-((2S)-ethyl-4-oxo-tetrahydrofuran-(3S)-ylcarbamoyl)-2-(1- methyl-cyclopentyl)-ethyl]-amide
  • Electrospray ionisation eluting with acetonitrile / ammonium formate buffer.
  • Electrospray ionisation eluting with acetonitrile / ammonium formate buffer.
  • Triethylamine (26.5 mmol, 3.69 ml, 5.3 eq) was then added (keeping the temperature less than -7O 0 C) and the mixture was then stirred for 1 hour during which time it was allowed to warm to O 0 C. The mixture was quenched with NH 4 CI solution : water (1 :1). The organics were isolated, dried (MgSO 4 ) then concentrated to give the crude aldehyde.
  • Cyclohexane-1,1-dicarboxylic acid diethyl ester,1 was prepared in accordance with JACS 43, 1921, 1368 from diethyl malonate and 1 ,5-dibromopentane.
  • Cyclohexane-1,1-dicarboxylic acid diethyl ester, 1 was taken up in anhydrous THF under nitrogen at room temperature. This was treated with LiAI(O 1 Bu) 3 H (2.5eq) portionwise before refluxing overnight. The reaction mixture was cooled in an ice-bath and treated carefully with 10% KHSO 4 (aq) and allowed to stir for 10 minutes. The resulting precipitate was removed by vacuum filtration and the mother liquors were partitioned between EtOAc and brine. The organic phases were combined, dried over MgS04, filtered and concentrated in vacuo to give a mobile oil.
  • reaction mixture was poured onto ice and the organics were washed with 1M HCI (aq), saturated NaHCO 3 (aq) and brine, then dried over MgSO4, filtered and concentrated in vacuo, to give trifluoro-methanesulfonic acid-1-fluoromethyl-cyclohexylmethyl ester, 5, as an amber oil which was used immediately without further purification in the next step.
  • N-(Diphenylmethylene)glycine ethyl ester was dissolved in DMF and under a nitrogen atmosphere was cooled to O 0 C. This was treated with KO'Bu (1.1eq) and stirred for 20 minutes. To this solution was added trifluoro-methanesulfonic acid-1-fluoromethyl-cyclohexylmethyl ester, 5 dropwise. The reaction mixture was stirred at room temperature under nitrogen overnight then poured into a 1 :1 mixture of diethyl ether : NH 4 CI (aq). The phases were separated and the aqueous phase was washed twice with diethyl ether.
  • Electrospray ionisation eluting with acetonitrile / ammonium formate buffer.
  • Boc protecting group Although the example has been illustrated with a Boc protecting group it will be apparent that other conventional N protecting groups such as those described above in Greene will be amenable to this route and/or the Boc group can be removed and replaced by another conventional N-protecting group such as Fmoc or CBz using conventional protecting group manipulation.
  • Furan-3-carboxylic acid [(1 ft,S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1 ⁇ methyl-cycloheptyl)-ethyl]-amide Data is given for a mixture of diastereoisomers (1 :1 ratio).
  • Morpholine-4-carboxylic acid [(1 f?,S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)- 2-(1-methyl-cycloheptyl)-ethyl]-amide
  • Furan-3-carboxylic acid [1 -(R,S)-(2-(S)-ethyl-4-oxo-tetrahydro-furan-3-(S)-ylcarbamoyl)-2- (1-fluoro-cyclopentyl)-ethyl]-amide
  • step e) The title compound is prepared from the material of step c) above using the same procedure as Example 10, step e).
  • Immobilisation of a P1 buliding block, such as those prepared in WO05/82876, onto a resin via Murphy's linker proceeds as described in Scheme 7 of WO00/69855 and its accompanying text.
  • the Fmoc-protected 5-substituted furan-4-amine is de-protected, extended with the P2 building block of the invention, such as those described at examples 1 and 5, using conventional peptide activation and coupling reagents such as HOBt/HBTU/DMF, as described in WO00/69855.
  • the resin was suspended in a 5% solution of hydrazine in DMF for 1 h. The mixture was filtered, and the resin washed with DMF. The hydrazine treatment and DMF wash was then repeated.
  • the resin was suspended in a 20% solution of piperidine in DMF for 0.5h. The mixture was filtered, and the resin washed with DMF. The piperidine treatment and DMF wash was then repeated.
  • the resin was suspended in a 20% solution of piperidine in DMF for 0.5h. The mixture was filtered, and the resin washed with DMF. The piperidine treatment and DMF wash was then repeated.
  • the resin was suspended in a solution of benzyl chloroformate and ⁇ /-methyl morpholine in DMF, filtered and the residue washed with 1 :1 water: DMF, DMF, THF, DCM and MTBE.
  • the resin was suspended in a 20% solution of piperidine in DMF for 0.5h. The mixture was filtered, and the resin washed with DMF. The piperidine treatment and DMF wash was then repeated.
  • the resin was suspended in a solution of benzylaldehyde in DMF, and a solution of dibutyltin dichloride in THF was added. After 10 minutes, phenyl silane was added and the mixture was shaken overnight. The mixture was filtered and the residue washed with DMF, THF, DCM and MTBE.
  • methods A-F have been illustrated with methyl or ethyl as R 1 , and 1-methyl- cyclopentyl-L-Ala as P2, it will be apparent that corresponding methodology, in conjunction with conventional protection of hydroxyl groups, will be applicable to other P1 and P2 building blocks. Similarly, methods A-F are not limited to the specified classes of P3, but are widely applicable to other species of R 3 , optionally in conjunction with conventional protection of amine, hydroxyl and carboxyl groups.
  • BBr 3 (20 mmol, 5g, 10 eq.) was added to a solution of 4-methoxy-2-methyl benzoic acid (2 mmol, 0.332g) in DCM (20 ml) and the mixture was stirred under argon until HPLC indicated no starting material remained. HCI (0.1 M, 20 ml) was added and the mixture was filtered. The aqueous layer was evaporated then dissolved in methanol. The solvent was evaporated. The dissolution/evaporation protocol was repeated a further 3 times and gave the pure product as a yellow solid (0.24g, 80%).
  • 3-Fluorosalicylaldehyde (117 mg, 0.83 mmol) was dissolved in dry ethyl acetate (15 ml) and Pd/C (12mg, 10 % w/w) was added. The solution was vigorously stirred at room temperature under a hydrogen atmosphere for 6 hrs. Filtration through celite and removal of the ethyl acetate under vacuo afforded the product (70 mg, 67 %) without need for further purification.
  • a buffer solution at pH 5.5 was prepared by the addition of acetic acid to a 1M aqueous sodium acetate solution.
  • Methyl 3-amino-4-hydroxybenzoate (254mg, 1.5 mmol) was dissolved in a mixture of buffer (1ml) and methanol (2ml).
  • Formaldehyde solution (37% by weight in water; 0.75ml, lOmmol) was added, the mixture stirred for 15 minutes, and then sodium cyanoborohydride (283mg, 4.5mmol) was added portionwise. The reaction mixture was stirred for an additional 0.5h and then concentrated. The residual oil was partitioned between water and ethyl acetate.
  • Methanesulfonyl chloride (615 uL) was added to a solution of 4-Amino-3-methoxy-benzoic acid methyl ester (1 g) in dichloromethane (20 ml.) and pyridine (1.5 ml.) and a catalytic amount of DMAP. After 1-16 hrs the mixture was concentrated to near dryness and the product crystallized from added ethanol. This product was hydrolyzed in 2.5 M LiOH (5 ml_), THF (14 ml_), MeOH (7 ml.) in a microwave oven at 110 deg C for 30 min. After cooling, the solution was acidified with aq. HCI and extracted with ethyl acetate, dried with Na 2 SO 4 and concentrated to dryness.
  • the remaining powder was used for coupling to the resin bound P1-P2 building block (described above).
  • the title compound was obtained when cleaved from the resin with 95% TFA in water. After concentration the product was purified on HPLC and freeze dried. The product was characterized by HPLC-MS and NMR.
  • Example 1533, 15.4, 15.6, 15.7, 15.8, 15.9, 15.15, 15.16, 15.17, 15.18, 15.19, 15.22, 15.24, 15.27, 15.28, 15.32, 15.33, 15.36, 15.40, 15.51 , 15.54, 15.64, 15.70 and 15.71 the same procedure as in Example 15.1 was followed.
  • 5-Amino-furan-2-carboxylic acid methyl ester (0.42 g, 3.0 mmol) were mixed together with methyl vinyl ketone (10 ml.) in benzene and heated at reflux for 1 h. Evaporation of solvents were followed by flash chromatography using DCM / MeOH (95:5) as eluent to yield 44% (278 mg. 1.31 mmol) of 5-Acetyl-4-amino-1-hydroxy-cyclohexa-2,4-dienecarboxylic acid methyl ester. This compound were mixed with BF 3 OEt 2 ((284 mg, 2.0 mmol) in benzene (15 ml.) and refluxed for 0.5 h.
  • Trifluoromethane sulfonic anhydride 38OuL was added to polymer supported tiriphenylphosphine oxide (1g) in dichloromethane (15mL). After 1 hrs the mixture was cooled to 0 deg C and a solution of pyridine 3-sulfonic acid (360 mg) as pyridine salt in DCM (4mL) was added. After 30 min. 4-Methanesulfonylamino-benzoic acid methyl ester (318 mg) in dichloromethane (4mL) was added. The mixture was shaken at 25 deg C for 16 hrs. The resin was filtered off and the filtrate concentrated to dryness. The crude was purified by silica column chromatography. Subsequent synthesis was done according to the procedure in Example 1.
  • Example 15.23, 15.35, and 15.52 For the synthesis of Examples 15.23, 15.35, and 15.52 the same procedure as in Example 15.20 was followed.
  • Example 15.29 was synthesized via solid phase synthesis methodology. First coupling of the 4- Amino-benzoic acid to the P1-P2 building block was done followed by washing as described in WO00/69055. Secondly 4-Cyano-benzenesulfonyl chloride (53.2 mg, 0.26 mmol) and a catalytic amount DMAP dissolved in pyridine (2 ml.) and DCM (4 ml.) was added to the P1-P2 building block (220 mg, 0.053 mmol). The reaction was left on agitation at room temperature over weekend. Cleavage from resin was done by addition of 95% TFA (aq, 6 ml.) and agitation for 0.5 h. Toluene (3 ml.) was added after filtration from resin, followed by evaporation.
  • TFA 95% TFA
  • Example 15.38 was synthesized as described in Example 15.1 by successive coupling of the P3 substituent to the P3 building block, hydrolysis of the ester and coupling to the P1-P2 building block. After cleavage from P1 -P2 resin, reduction of nitro group was done by dissolving 4-(4- Nitro-benzenesulfonylamino)-N-[1-(2-ethyl-4-oxo-tetrahydro-furan-3-ylcarbamoyl)-2-(1-methyl- cyclopentyl)-ethyl]-benzamide (13.9 mg, 23.4 ⁇ mol) in MeOH (3ml_) and degassing the solution with N 2 gas. A catalytic amount of palladium on carbon was then added to the reaction solution and a H 2 atmosphere was connected. After 2 hrs, filtration through celite was done, with MeOH as eluent, to yield 11.8 mg (91%) of product after concentration.
  • ⁇ -Fornnyl-thiophene ⁇ -carboxylic acid was coupled to the resin as described in example 1.
  • the resin was swollen in DCM-trimethylortoformate 1 :1 and 4 equiv. of methoxyethylamine was added. After 4 hrs of agitation the resin was washed with DCM and MeOH (2X) and the resin was dried. To this resin in DCM-MeOH-HOAc 2:2:1 borane-pyridine complex was added. After 16 hrs of agitation the resin was washed and cleaved and purified as described in Example 15.1.
  • the assay uses baculovirus-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 ⁇ l/well of 12.5% DMSO are added to rows B - H of two columns of a 96 deep well polypropylene plate. 70 ⁇ l/well of substrate is added to row A. 2 x 250 ⁇ l/well of assay buffer (10OmM Na phosphate, 10OmM NaCI, pH 6.5) is added to row A, mixed, and double diluted down the plate to row H.
  • 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 Kj.
  • the rough Ki is calculated from a preliminary run in which 10 ⁇ l/well of 1 mM boc-VLK-AMC (1/10 dilution of 10 rtiM stock in DMSO diluted into assay buffer) is dispensed to rows B to H and 20 ⁇ l/well to row A of a 96 well Microfluor TM plate. 2 ⁇ l of each 1OmM test compound is added to a separate well on row A, columns 1-10.
  • the second test compound is added to column 6 of the top row, the third to column 1 of the second row etc. Add 1 ⁇ l 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.
  • ⁇ -channel multistepping pipette set to 5 x 10 ⁇ l, distribute 10 ⁇ l/well of substrate to the 384 well assay plate. Distribute the first column of the substrate dilution plate to all columns of the assay plate starting at row A. The tip spacing of the multichannel pipette will correctly skip alternate rows. Distribute the second column to all columns starting at row B.
  • a distributor such as a Multidrop 384, add 30 ⁇ l/well to all wells of the assay plate and read in fluorescent spectrophotomoter such as an Ascent.
  • Fluorescent readings (excitation and emission wavelengths 390nm and 460nm 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.
  • Cathepsin K Ki The procedure of Biological Example 1 with the following amendments is used for the determination of Ki for cathepsin K.
  • the enzyme is E coli expressed human cathepsin K.
  • the substrate is H-D-Ala-Leu-Lys-AMC from Bachem.
  • the assay buffer is 100 rtiM Na phosphate, 1 mM EDTA, 0.1% PEG 4000, pH 6.5.
  • the DMSO stock (see substrate dilutions) is diluted to 10% in assay buffer .
  • 56 ul of substrate is added to row A and 2 x 256 ul of buffer is added to row A.
  • the final cathepsin K concentration is 0.5 nM.
  • the majority of compounds illustrated above provide selectivities over cathepsin K of at least 10-100 fold.
  • 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 10OmM sodium acetate 1mM EDTA, pH5.5)
  • the DMSO stock (1OmM in 100%DMSO) is diluted to 10% in assay buffer.
  • the majority of the compounds illustrated above provide selectivity over cathepsin L of at least 10-100 fold.
  • This example 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.
  • the basolateral 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 ⁇ M. 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.1) 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.
  • TEER 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 ⁇ l_ 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 0 C with a low shaking velocity of approximately 150 to 300 rpm.
  • 25 ⁇ L samples will be 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 ⁇ L 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 50% in the samples.
  • the collected samples will be stored at -2O 0 C until analysis by HPLC or LC-MS.
  • Basolateral to apical transport Generally every compound will be tested in 2-4 wells. The basolateral 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 ⁇ M. 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 HBSS, 25 rtiM MES, pH 6.5
  • HBSS HBSS
  • 25 rtiM MES pH 6.5
  • 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 ⁇ L fresh TB, pH 6.5 is added to the inserts. After 30 minutes 250 ⁇ L is withdrawn from the apical (receiver) well and replaced by fresh transport buffer. Thereafter 250 ⁇ L samples will be withdrawn and replaced by fresh transport buffer every 30 minutes until the end of the experiment at 120 minutes, and finally a post value of TEER is measured at the end of the experiment. A 25 ⁇ L samples will be taken from the basolateral (donor) compartment after ⁇ 2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.
  • acetonitrile will be added to a final concentration of 50% in the samples.
  • the collected samples will be stored at -2O 0 C until analysis by HPLC or LC-MS.
  • FA cum Determination of the cumulative fraction absorbed, FA cum , versus time.
  • FA cum is calculated from: FA - Y ⁇ M. rM cum ⁇ L-I r
  • k is the transport rate (mirr ' ' ) defined as the slope obtained by linear regression of cumulative fraction absorbed (FA cum ) as a function of time (min)
  • VR is the volume in the receiver chamber (ml_)
  • A is the area of the filter (cm 2 ).
  • This example describes procedures for assessing potency of cathepsin S inhibitors on inhibition of in vitro T cell activation by determining concentration of the compound required for reducing 50% of the IL-2 secretion in T cells stimulated with compound-treated antigen presenting cells in an antigen presentation assay using the 19.3 cells and the 9001 cells as the effector cells and the antigen presenting cells, respectively.
  • 19.3 cells are murine T cell hybridomas recognizing type Il collagen (260-272) in the context of HLA-DR1
  • 9001 is an EBV-transformed human B cell line expressing homozygous DR1 molecule.
  • the 9001 cells will be pre-treated with varying concentration of the compounds for 1 hour and then incubated with the T cells in the presence of collagen at a final concentration of 0.1 mg/ml.
  • the cultures will be incubated overnight at 37°C with 5% CO 2 and amount of IL-2 in the supernatant determined with ELISA.
  • the IC 50 -IL-2 values representing the concentration of compounds at which secretion of IL-2 from the T cells is reduced by 50% will be determined by regression analysis
  • MHC class Il molecules bind peptides generated by degradation of endocytosed antigens and display them as MHC class ll-peptide complexes at the cell surface for recognition by CD4+ T cells.
  • MHC class Il molecules are assembled with the assistance of invariant chain (Ii) in the endoplasmic reticulum (ER) and transported to an endocytic compartment where Ii undergoes rapid degradation by endosomal and lysosomal proteases.
  • Ii invariant chain
  • ER endoplasmic reticulum
  • a peptide fragment of Ii, CLIP class ll-associated Invariant chain Peptides
  • cathepsin S In dendritic cells and B cells, cathepsin S is required for complete invariant chain processing and CLIP generation. Inactivating cathepsin S with inhibitors will impair MHC class Il peptide loading and formation of stable MHC/peptide complexes leading to reduced antigen presentation and T cell activation.
  • an antigen presentation assay uses a collagen specific,HLA-DR1 restricted mouse T cell hybridoma (19.3) as effector cells, human EBV-transformed B cells (9001) as antigen presenting cells (APC), and mlL-2 ELISA as the read-out system.
  • Inhibition of Cathepsin S with specific inhibitors will impair the processing and presentation of collagen in APCs which in turn reduces the activation of the T cells.
  • the extent of inhibition on T cells is measured by the degree of reduction in IL-2 secretion.
  • IC 50 -IL-2 represents the concentration of compounds at which secretion of IL-2 from the T cells is reduced by 50% in the antigen presentation assay.
  • Cathepsin S inhibitors Compounds will be dissolved in DMSO to a final concentration of 10 nriM, aliquotted, and stored at -80 C until used.
  • DMEM medium Invitrogen, cat #11995-065
  • fetal bovine serum Hyclone, cat #SH30070.03
  • penicillin 100 ug/ml streptomycin
  • 2 mM L-glutamine Invitrogen, cat #10378-016
  • T cell 19.3, murine DR1 transgenic T cell hybridomas, DR1 restricted, Type Il collagen 260-272 specific
  • Antigen presentation cells (APCs): 9001 , EBV-transformed human B cells expressing homozygous DR1
  • Type Il collagen from chicken sternal cartilage (Sigma, cat. # C-9301) will be dissolved in PBS at 1 mg/ml and stored in aliquots at -80 C.
  • Tissue culture incubator (Forma Scientific, model. #3120) Sorvall centrifuge (Sorvall RC-3B) Plate washer Plate-reader (Tecan, Spectra shell, cat. #20-074)
  • Two-fold serial dilutions of the compounds, starting at 40OuM in AIMV medium, will be transferred to a 96-well round-bottom microtiter plate at a volume of 50ul/well.
  • Antigen-presenting cells will be washed and resuspended in AIMV medium to a density of 0.8x10 6 /ml, and then added to the plates at a volume of 50ul/well, giving the number of cells per well as 40,000.
  • the APCs will be pretreated with compounds for 1 hour at 37C with 5% CO 2 . 4.
  • the T cells will be washed and resuspended in AIMV to a density of 0.8x10 6 /ml.
  • the antigen will be diluted to a 4X concentration in AIMV and mixed 1 to 1 with
  • T cells/antigen mixture will then be added to the assay plates at a volume
  • the plates will be incubated overnight at 37C with 5% CO 2 .
  • Mouse IL-2 ELISA kits will be purchased from Pharmingen (Mouse IL-2 OptEIA set, #2614KI). The ELISA will be performed per manufacturer's instruction.
  • Anti-mlL-2 antibodies will be diluted in carbonate buffer to a final concentration of 2 ug/ml, transferred to an ELISA plate (Costar) at 100 u I/well and then incubated overnight at 4 degreesC.
  • the ELISA plates will be washed 4 times with PBS/0.5% FBS containing 0.05% Tween 20 (wash buffer).
  • the plates will be blocked with the blocking buffer, 10% FBS (fetal bovine serum, Hyclone) for 2 hrs at room temperature (RT) and then washed 4 times with wash buffer.
  • FBS fetal bovine serum, Hyclone
  • the plate will be incubated for 1 hr at RT with a mixture of a biotinylated anti-mlL2 antibody and avidin-HRP prepared in blocking buffer.
  • the plates will be measured at 450 nm with an ELISA plate reader (Spectra, Tecan).
  • a set of purified recombinant mlL-2 with known concentration will be prepared from the stock solution (provided in the kit) with the blocking buffer and assayed in each plate to provide a standard curve for quantification of IL-2.
  • IC 50 represents the concentration of compound at which secretion of IL-2 from the T cells is reduced by 50%.
  • the absorbance at 450 nm from each well will be converted into amount of IL-2 (pg/ml) using the Winselect software (Tecan) based on the standard curve generated from in-plate standards of purified recombinant mlL-2. Means and standard deviations will be calculated from triplicates with Excel.
  • Percent Inhibition average of control wells - average of test wells x 100 average of control wells
  • a dose response curve will be generated by plotting the percent inhibition versus concentration of the compound and the IC 50 -IL-2 value will be calculated with regression analysis.
  • DR-1 transgenic T cell hybridoma has been prepared by E. Rosloniec, University of Tennessee.
  • T + APCs without antigen, without compound treatment, for background signal. We usually get negligible amounts of IL-2 form these wells, and usually don't perform background subtraction. T + APCs, with anti-CD3/CD28, with compounds, for toxicity associated with compounds.
  • T + APCs with antigen, with DMSO (comparable to those received compounds), for toxicity associated with DMSO and for calculation of percent of inhibition.
  • Metabolic stability is determined by commercially available human liver microsome assays, such as XEN 042, assayed in accordace with manufacturer's recommendations.

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Abstract

Compounds of the formula (I) where R1 is C1-C4 straight or branched alkyl, optionally substituted with up to three substituents selected from halo and hydroxy; R2 is halo, hydroxy, methyloxy, or C1-C2 alkyl, which alkyl is optionally substituted with up to three halogens or an hydroxy or a methyloxy; D is - C3-C7 alkylene-, thereby defining a cycloalkyl ring; E is -C(=O)-, -S(=O)m-, -NRdS(=O)m-, -NRaC(=O)-, -OC(=O)-, R3 is an optionally substituted carbocyclic or heterocyclic ring R10 is H, ORc, SRc or together with the gem H is =O or (ORc)2; Ra is independently selected from H, C1-C4 alkyl; have utility in the inhibition of cathepsin S and are thus useful pharmaceuticals against disorders such as autoimmune disorders and chronic pain.

Description

Cathepsin S Inhibitors
Technical Field
This invention relates to inhibitors of cathepsin S, and their use in methods of treatment for disorders involving cathepsin S such as autoimmune, allergy and chronic pain conditions.
Background to the invention and prior art
The papain superfamily of cysteine proteases are widely distributed in diverse species including mammals, invertebrates, protozoa, plants and bacteria. A number of mammalian cathepsin enzymes, including cathepsins B, F, H, K, L, O, S, and W, have been ascribed to this superfamily, and inappropriate regulation of their activity has been implicated in a number of metabolic disorders including arthritis, muscular dystrophy, inflammation, glomerulonephritis and tumour invasion. Pathogenic cathepsin like enzymes include the bacterial gingipains, the malarial falcipains I, II, III et seq and cysteine proteases from Pneumocystis carinii, Trypanosoma cruzei and brucei, Crithidia fusiculata, Schistosoma spp.
In WO 97/40066, the use of inhibitors against Cathepsin S is described. The inhibition of this enzyme is suggested to prevent or treat disease caused by protease activity. Cathepsin S is a highly active cysteine protease belonging to the papain superfamily. Its primary structure is 57%, 41% and 45% homologous with that of the human cathepsin L and H and plant cysteine proteases 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. Moreover, it has been found that destruction of Ii by proteolysis is required for MHC class Il molecules to bind antigenic peptides, and for transport of the resulting complex to the cell surface. Furthermore, it has been found that Cathepsin S is essential in B cells for effective Ii proteolysis necessary to render class Il molecules competent for binding peptides. Therefore, the inhibition of this enzyme may be useful in modulating class ll-restricted immune response (WO
97/40066). Other disorders in which cathepsin S is implicated are asthma, chronic obstructive pulmonary disease, endometriosis and chronic pain.
International patent application no WO00/69855 describes cathepsin S inhibitors of the formula:
Figure imgf000003_0001
(H)
wherein:
R1 = R', R1C(O) , R' C(S), R' SO2 , R' OC(O), R' NHC(O) wherein R' is a monocyclic ring; R2, R4 = H, Ci-7-alkyl, C3-7-cycloalkyl; R3 = Ci-7-alkyl, C3-7-cycloalkyl, Ar-Ci -7-alkyl; R5 = Ci-7-alkyl, Halogen, Ar-Ci -7-alkyl, Ci-3-alkyl-CONR'", R6 = H, Ci-7-alkyl, Ar-Ci -7-alkyl, Cr3-BIkYl-SO2-R1", Ci-3-alkyl-C(0)-NHRix or CH2XAr,
The R3 groups specifically disclosed in WO00/69855 are branched chain alkyl moieties such as n-butyl, t-butyl, 3-(2,2-dimethylpropyl), 4-(2-methylbutyl), 4-(3,3-dimethylbutyl), 4-(3,3-dimethyl- 2-methylbutyl), 4-(3-methyl-2-methylbutyl), or 5-(2-methyl-3-methylpentyl). Page 27, line 13 of WO00/69855 discloses the compound morpholine-4-carboxylic acid [3,3-dimethyl-1S-(2-ethyl-4- oxo-tetrahydrofuran-3-ylcarbamoyl)butyl]amide.
In accordance with the invention, there is provided novel compounds of the formula I
Figure imgf000003_0002
where
R1 is CrC4 straight or branched alkyl, optionally substituted with up to three substituents selected from halo and hydroxy;
R2 is halo, hydroxy, methyloxy, or CrC2 alkyl, which alkyl is optionally substituted with up to three halogens or an hydroxy or a methyloxy;
D is -C3-C7 alkylene-, thereby defining a cycloalkyl ring; E is -C(=O)-, -S(=O)m-, -NRaS(=O)m-, -NRaC(=O)-, -OC(=O)-,
R3 is a carbocyclic ring selected from C3-C6 cycloalkyl, C5-C6 cycloalkenyl or phenyl, or a heterocyclic ring I selected from azepanyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, thiopyranyl, furanyl, tetrahydrofuranyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl, pyrazolyl, indolyl, which ring is optionally substituted with up to 3 substituents independently selected from R4; or
R4 is independently selected from halo, oxo, nitrile, nitro, d-C4 alkyl, -NRaRb, NH2CO-, X-R5,
X-O-R5, X-0-C(=0)R5, X-C(=O)NRaR5, X-NRaC(=0)R5a, X-NRdSOmR5a, X-SOmNRdR5, X- S(=O)mR5, X-C(=0)0R5, X-NRaC(=0)0R5; or a pair of R4 together define a 5 or 6 membered nitrogen-containing ring fused to R3, optionally substituted with CrC4 alkyl, CrC4 alkyloxy, oxo, hydroxy, halo, NRaRb,;
R5 is H, CrC4 alkyl, C3-C6 cycloalkyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, thiopyranyl, furanyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl, phenyl, benzyl, any of which is optionally substituted with R6;
R5a is R5 or -NRaRb;
R6 is independently selected from hydroxy, -NH2, NHCrC3alkyl, N(CrC3alkyl)2, nitro, cyano, carboxy, oxo, d-C4 alkyl, Ci-C4-alkoxy, d-C4 alkanoyl, carbamoyl; Ra and Rb are independently selected from H, CrC4 alkyl and acetyl, or Ra, Rb and the N atom to which they both are joined form a ring selected from morpholine, piperazine, piperidine, pyrrolidine;
R10 is H, ORc, SRc or together with the gem H is =0;
Ra and Rb are independently selected from H, CrC4 alkyl and acetyl, or Ra, Rb and the N atom to which they both are joined form a ring selected from morpholine, piperazine, piperidine, pyrrolidine
Rc is H, CrC4 alkyl, C0-C3alkylcarbocyclyl;
Rd is H, CrC4 alkyl, C(=O)CrC4 alkyl, C0-C3alkylcarbocyclyl;
X is independently a bond or CrC4 alkylenyl; m is independently 0,1 or 2; and pharmaceutically acceptable salts thereof.
Preferred values for R1 include ethyl, 2-fluoroethyl or 2-hydroxyethyl, and methyl, fluoromethyl and hydroxyethyl, especially ethyl and methyl. Preferably the stereochemistry at the C-4 and C- 5 positions of the furanone ring (ie those from which R1 and the backbone nitrogen extends) is enantiomerically pure, or at least 85%, for example at least 90% or more preferably at least 95% enantiomerically pure 4S, 5S configuration. Preferred P1 groups (as defined below) therefore include:
Figure imgf000005_0001
R10 is conveniently H or a CrC4alkyl ether or d-C4 alkylthioether such as methyloxy, ethyloxy, methylthio- or ethylthio or the corresponding ketals. Embodiments of P1 groups thus include:
Figure imgf000005_0002
especially where R1' is H or -CH3.
Where R10 is other than H, it is currently preferred that the stereochemistry at the ring carbon atom which bears R10 comprises at least 85%, for example at least 90% preferably at least 95% and more preferably 100% enantiomerically pure alpha configuration:
Figure imgf000005_0003
but both alpha and beta configurations produce cathepsin S-active compounds.
D is conveniently pentylene, thereby defining a cyclohexyl ring, or propylene, thereby defining a cyclobutyl ring, but more preferably D is butylene, thereby defining a cyclopentyl ring. Embodiments of R2 include a halogen such as fluoro, fluoro methyl, difluoromethyl or trifluoromethyl, and most preferably methyl.
The side chain comprising D and R2, ie the P2 group (as defined below) may be in the R or S configuration, or a racemate thereof. Preferably, however, the P2 group is substantially, for example greater than 90% and most preferably greater than 95% in the S stereoconfiguration, that is reflecting that of an L-amino acid.
Preferred side chains thus include:
Figure imgf000006_0001
Other embodiments include:
Figure imgf000006_0002
Currently favoured values for E include -O-C(=O)-, -S(=O)2- and especially -C(=O)-.
Returning now to other values of E, typical values for R3 include: unsubstituted or substituted furanyl, especially furan-2-yl or furan-3- yl, or alkyl substituted furanyl such as 2-methylfuran-3-yl, 2,4-dimethylfuran-3-yl, or aryl substituted furanyl, even more especially 5-phenylfuran-2-yl, 5-(2-chlorophenyl)furan-2-yl, 5-(3chlorophenyl)furan-2-yl, 5-(4- chlorophenyl)furan-2- yl, 5-(4-fluorophenyl)furan-2-yl, 5-(4hydroxyphenyl)furan-2-yl, 5-(3- trifluoromethylphenyl)furan-2-yl, 5-(4-trifluoromethylphenyl)furan-2-yl, 5-(3- trifluoromethylphenyl)furan-2-yl, 5-(4-methylphenyl)furan-2-yl, 5-(4-acetylphenyl)furan-2-yl, or 5- trifluoromethylfuran-2-yl; unsubstituted or substituted tetrahydrofuranyl, particularly tetrahydrofuran-2-yl or tetra hyd rof u ra n -3-y I ; unsubstituted or substituted morpholinyl; unsubstituteted or substituted pyrrol yl, particularly pyrrol-2-yl; unsubstituted or substituted piperazinyl, particularly piperazin-1- yl or 4-alkylpiperazinyl, e.g., 4- methylpipeperazin-1 -yl; unsubstituted or substituted pyrazolyl, particularly IH-pyrazol-2-yl, IH-pyrazol-4- yl, 1- or 2- methyl-2H-pyrazol-2-yl or 1 - or 2-methyl-2H-pyrazol-3-yl; unsubstituted or substituted isoxazolyl, particularly isoxazol-5-yl, 3-methylisoxazol-4-yl, 5- methylisoxazol-3-yl, 5-methylisoxazol-4-yl, or 3,5-dimethylisoxazol4-yl; unsubstituted or substituted thiazolyl, particularly thiazol-2-yl, 2-methylthiazol-2-yl, 2,4- dimethylthiazol-5-yl, or 4-methyl-2-phenylthiazol-5-yl; unsubstituted or substituted pyrazolyl, particularly alkyl-substituted pyrazolyl including 2-methyl-
2H-pyrazolyl; unsubstituted or arγl-substituted triazolyl, particularly phenyl-substituted triazoles including 3- phenyl-3H-[1 ,2,3]triazol-3-yl; unsubstituted or substituted pyrazinyl, particularly pyrazin-2-yl and 5-methylpyrazin-2-yl; unsubstituted or substituted imidazolyl, particularly 1 -H-imidazol-2-yl, 1 -methyl-1 H-imidazol-4-yl or 1-methyl-IH-imidazol-2-yl; thiophenyl, especially thiophene-3-yl and thiophen-2-yl, more especially heterocycle or aryl substituted C0-C6alkylthiophenyl, particularly 5-pyridin-2-ylthiophen-2-yl, more especially Cr
C6alkylthiophenyl, particularly 5-methylthiophenyl or 3-methylthiophen-2-yl; more especially d- C6alkoxythiophenyl, particularly 3-ethoxythiophen-2-yl; phenyl, especially alkyl-substituted phenyl, halogen-substituted phenyl, trihaloalkylsubstituted phenyl, alkoxy-substituted phenyl, or acetoxy-substituted phenyl, especially 4-methylphenyl, 3- chlorophenyl, 4-chlorophenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-chlorophenyl,
4-fluorophenyl, 4-hydroxyphenyl, or 4-acetylphenyl; unsubstituted or substituted pyridinyl, particularly pyridine-2-yl; unsubstituted or substituted cyclobutyl or cyclopentyl.
Preferred values for R3 include optionally substituted thienyl, pyrazinyl, pyridyl, pyrrolyl, and especially furyl or morpholinyl.
Favoured values for R3 include fur-3-yl, thien-3-yl, pyrazin-2-yl, pyrid-4-yl, pyrrol-2-yl and especially N-morpholino. Other preferred embodiments of R3, for example when E is (=0), include optionally substituted, fur-2-yl or thien-2yl:
Figure imgf000008_0001
R R
where R4' is H, halo (such as F or Cl), Od-C4 alkyl (such as methoxy), C(=O)NRaRb (for example dimethylcarbamoyl), NRaC(=O)CrC4alkyl such as NHC(=0)Me, ureas, such as
NRaC(=O)NRaRb,(for example -NHC(=O)NHCH3> NHC(=O)N(CH3)2 or NHC(=O)NRrRr, where RrRr define a cyclic amine such as pyrrolidine, morpholine, piperidine, piperazine or N- methylpiperazine), and carbamates such as -NRaC(=0)0Ci-C4 alkyl such as NHC(=0)0Me.
A further preferred value for R3 is phenyl particularly phenyl substituted as follows:
Figure imgf000008_0002
where
Ry is -NHC(=O)-Me, -NHC(=O)OMe, F, especially OH and NHAc and Rx is H, OMe, F, Cl, CN, CF3, Me.
Other embodiments include those wherein Ry is halomethyl such as CF3 or CF2 or an hydroxylated methyl group, such as HOCH2 or HO(CH2J2)C-, any of these preferences being optionally further substituted with an R4 group such as Rx.
An alternative embodiment for R3 comprises phenyl which is substituted with a urea, such as a cyclic urea:
Figure imgf000008_0003
Other urea substituent include NRaC(=O)NRaRb, (for example -NHC(=0)NHCH3, NHC(=O)N(CH3)2 or NHC(=O)NRrRr, where RrRr define a cyclic amine such as pyrrolidine, morpholine, piperidine, piperazine or N-methylpiperazine).
Other favoured substituents to a phenyl R3 include 3,5-dichloro, 3,5-difluoro, 3-fluoro-5-cyano, 3-cyano, 4-NHAc-3-Me, 4-NHAC-6-Me, 4-NHAc-3,5-diMe and the like.
A favoured aspect of the invention thus comprises compounds of the formula:
Figure imgf000009_0001
where R1, R2, D, Rc, E and R4 are as defined above and R11 is H, R12 or -C(=O)R12 where R12 is independently H, d-C6-alkyl which is optionally substituted with R6, C0-C3alkylcarbocyclyl or C0- Csalkylheterocyclyl. R12 typically comprises a pharmaceutically acceptable ether or ester prodrug which is hydrolysed in vivo to release the parent phenol. Currently favoured values of this aspect include those wherein R1 is Me or Et, R10 is H, R2 is Me, D is butylene, Ra is H and E is C(=O).
Favoured variants of the aspect of the invention in the immediately preceding paragraph include those wherein R4 is at the 3, or the 3 and 5 positions of the phenyl ring. Representative values include R4 as halo, such as 3-fluoro, 3,5-difluoro, 3-chloro or 3,5-dichloro. Alternative R4 values include fluorinated methyl such as trifluormethyl, for example 3-trifluoromethyl, CrC3alkyloxy, such as methyloxy for example 3-methoxy, or 3,5-dimethoxy, or -C(=O)Ci-C3 alkyl such as acetyl, for example 3-acetyl. Additional favoured R4 values include one or more d-C4 alkyl, such as methyl, ethyl, i-propyl or t-butyl. Representative values for this aspect of the invention thus include 5-methyl, 5-ethyl, 5-i-propyl, 5-t-butyl, 6-methyl, 5-methyl-3-fluoro.
Additional favoured substituents to R3, for example on a phenyl R3, include sulphonamides. Accordingly, a favoured aspect of the invention comprises compounds of the formula I, wherein R3 has the partial structure:
Figure imgf000010_0001
where E is as defined above, preferably -C(=O)- and R13 is -NRdSOmR5a, where R5a is R5 as defined above, preferably CrC4 alkyl, such as methyl, ethyl or i-propyl or t-butyl; halogenated CrC4 alkyl such as trifluoromethyl; C3-C6 cycloalkyl, such as cyclopropyl or cyclohexyl; or phenyl or benzyl, any of which is optionally substituted with R6. Alternatively R5a may be NRaRb as defined above including cyclic amines, such as -NHMe or -N(Me)2, or piperazine, N-methyl piperazine, pyrrolidine, piperidine or morpholine.
Further preferred values for R5 include heteroaryl rings such as pyrrolyl, oxazolyl, isoxazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl or indolyl, especially thiazolyl, any of which is substituted with R6 groups such as d-C4 alkyl. A currently favoured sulphonamide has the partial structure:
Figure imgf000010_0002
where E and R4 are as defined above, Rd' is Me or preferably H, and R6 is H or methyl, especially at the ring position, adjacent the N for example:
Figure imgf000010_0003
Other representative values of R5 thus include F3C-S(=O)2-NH, cyclopropyl-S(=O)2NH, Me- S(=O)2)NH-, Et-S(=O)2NH-, i-Pr-S(=O)2NH-, Ph-S(=O)2NH-; MeNH-S(=O)2NH; (Me)2S(=O)2NH- and the like.
Alternatively the sulphonamide may have the other orientation -S(=O)mNRdR5 where R5 as defined above, preferably CrC4 alkyl, such as methyl, ethyl or i-propyl or t-butyl; halogenated CrC4 alkyl such as trifluoromethyl; C3-C6 cycloalkyl, such as cyclopropyl or cyclohexyl; or phenyl or benzyl, any of which is optionally substituted with R6. Alternatively R5 together with Rd defines a 3-6 membered N-containing ring such as azidine, pyrrolidine, pyridine, piperidine, morpholine, piperazine or N-methylpiperizine.
Representative values of sulphonamide thus include MeNH-S(=O)2-, (Me)2N-S(=O)2- and the like.
As depicted above, a sulphonamide substituted phenyl is optionally substituted with an additional substituent R4, typically, but not invariably, in the 4 position if the sulphonamide is in the 3 position and vice versa. Representative R4 groups thus include halo such as chloro or fluoro, CrC4 alkyl such as methyl (including 2-methyl) and d-C4alkoxy such as methoxy.
Rd is typically H or an acyl moiety such as -C(=O)CrC4 alkyl or optionally substituted benzoyl. Representative values for Rc thus include H, acetyl, pivaloyl or benzoyl. For sulphonamides in the orientation -S(=O)mNRdR5, Rd is conveniently d-C4 alkyl or together with R5 defines a 3-6 membered N-containing ring such as azidine, pyrrolidine, pyridine, piperidine, morpholine, piperazine or N-methylpiperizine.
In either orientation, m is typically 1 (sulphenamide) or preferably 2 (sulphonamide).
An alternative phenyl-based R3 value is phenyl substituted with a pair of R4 groups which together constitute a nitrogen containing chain of 3 or 4 atoms thereby defining a ring fused to the phenyl such as:
Figure imgf000011_0001
where R4 and E are as defined above, Rz is CH, NH or O and the S atom is optionally oxidised to >S=O or preferably >S(=O)2. Ring nitrogens are optionally substituted with d-C4 alkyl (such as methyl, ethyl or t-butyl) , or C(=O)CrC4 alkyl (such as acetyl). Typically R4 is methyl or especially H. Preferably the linkage to E is para to a nitrogen in the fused ring:
Figure imgf000011_0002
Other fused rings for constituting a nitrogen containing ring fused to a phenyl R3 include
Figure imgf000012_0001
where Rz is CH; NH or O, especially O and preferably NH, R4' is H, CrC4 alkyl, NH2, NHd- C4alkyl (such as methylamide), N(CrC4alkyl)2 such as diethylamide), NHC(=O)CrC4alkyl (such as acetamide). Ring nitrogens are optionally substituted with d-C4 alkyl (such as methyl, ethyl or t-butyl) , or C(=0)CrC4 alkyl (such as acetyl). Typically R4 is methyl or especially H. Preferably the linkage to E is para to a nitrogen in the fused ring.
Figure imgf000012_0002
Still further fused rings for R3 include variants wherein the fused nitrogen-containing ring defines a saturated or unsaturated 6 membered heterocycle, such as:
Figure imgf000012_0003
where Rz is NH, O or CH, especially O and preferably NH, R4 and R4' are optional substituents as defined above, preferably H, and O' is absent (ie 2 hydrogen atoms) or keto. Preferably the linkage to E is para to a nitrogen in the fused ring.
Figure imgf000012_0004
Still further fused rings for R3 include variants wherein the fused nitrogen containing ring defines an optionally substituted quinoline, isoquinoline, tetrohydroquinoline or tetrahydroisoquinoline moiety, such as
Figure imgf000013_0001
especially wherein R4 and R4' are H and the linkage to E is para to the nitrogen in the fused ring.
Other favoured R3 groups include pyrimidyl, such as 2-pyrimidyl, for example 5-OH-pyrimid-2-yl; or pyridyl, such as pyrid-4-yl, for example O→pyrid-4yl; or pyrid-3-yl, for example 6-hydroxy- pyrid-3-yl.
A further aspect of the invention comprises a method employing the compounds of the invention for the 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.
Examples of such diseases or conditions include those enumerated in WO 97/40066, such as autoimmune diseases, allergies, such as asthma and hayfever, multiple sclerosis, rheumatoid arthritis and the like. A further example is the treatment of endometriasis, and especially chronic pain, as disclosed in WO0320287. The invention further provides the use of the compounds of formula IV in therapy and in the manufacture of a medicament for the treatment of diseases or conditions alleviated or moderated by inhibition of cathepsin S.
In one series of embodiments, the methods are employed to treat mammals, particularly humans at risk of, or afflicted with, autoimmune disease. By 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 Il MHC-complexes. This association occurs because the structures recognized by T cells, the cells that cause autoimmunity, are complexes comprised of class Il MHC molecules and antigenic peptides. Autoimmune disease can result when T cells react with the host's class Il MHC molecules when complexed with peptides derived from the host's own gene products. If these class Il MHC/antigenic peptide complexes are inhibited from being formed, the autoimmune response is reduced or suppressed. Any autoimmune disease in which class Il MHC/antigenic complexes play a role may be treated according to the methods of the present invention. Such 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.
In another series of embodiments, the methods are employed to treat mammals, particularly humans, at risk of, or afflicted with, allergic responses. By "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 "allergic response" is used herein in accordance with standard usage in the medical field.
Examples of 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 Il MHC/antigenic peptide complexes. If these class Il MHC/antigenic peptide complexes are inhibited from being formed, the allergic response is reduced or suppressed. Any allergic response in which class Il 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.
In another series of embodiments, the methods are employed to treat mammals, particularly humans, which have undergone, or are about to undergo, an organ transplant or tissue graft. In tissue transplantation (e.g., kidney, lung, liver, heart) or skin grafting, when there is a mismatch between the class Il 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 Il MHC molecules presenting antigenic peptides on the surface of donor cells. To the extent that this response is dependent upon the formation of class Il
MHC/antigenic peptide complexes, inhibition of 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. Preferably, administration is accomplished by systemic application to the host before and/or after surgery. Alternatively or in addition, perfusion of the donor organ or tissue, either prior or subsequent to transplantation or grafting, may be effective. The above embodiments have been illustrated with an MHC class Il mechanism but the invention is not limited to this mechanism of action. Suppression of cathepsin S as a treatment of COPD or chronic pain may not, for example, involve MHC class Il at all.
Assays for the assessment of cathepisn S inhibitors in the treatment of chronic pain, including neuropathic or inflammatory pain are as described in WO 03 20287.
Currently preferred indications treatable in accordance with the present invention include: Psoriasis;
Autoimmune indications, including idiopathic thrombocytopenic purpura (ITP), rheumatoid arthritis (RA), multiple schlerosis (MS), myasthenia gravis (MG), Sjόgrens 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.
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 car boxy lie 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, 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. The compounds of the invention may in some cases be isolated as the hydrate.
Prodrugs
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 R3, or amine functions such as an R4 sulphonamide amine function. Preferred pharmaceutically acceptable esters include those derived from CrC6 carboxylic acids such as acetyl or pivaloyl or optionally substituted benzoic acid esters, preferably unsubstituted or substituted with R6. Favoured sulphonamide prodrugs include aminoacyls derived from d-C6 carboxylic acids such as acetyl or pivaloyl or optionally substituted benzoic acid esters, preferably unsubsbstituted or substituted with R6.
Co-C3alkylcarbocyclyl comprises C0-C3alkylaryl and Co-C3alkylC3C7cycloalkyl. 'Co-C3alkylaryr as applied herein is meant to include an arγl moiety such as a phenyl, naphthyl or phenyl fused to a C3-C7CyClOaI kyl (for example indanyl), which aryl is directly bonded (i.e. C0) or through an intermediate methylene, ethylene, or propylene group. 'C0-C3alkylC3C7cycloalkyr as applied herein is meant to include a C3-C7cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, which cycloalkyl is directly bonded (i.e. Coalkyl) or through an intermediate methylene, ethylene, propylene or isopropylene group. The cycloalkyl group may contain an unsaturated bond.
Unless otherwise indicated the arγl or cycloalkyl group is optionally substituted with 1-3 substituents selected from halo, hydroxy, nitro, cyano, carboxy, CrC6alkyl, CrC6alkoxy, Cr C6alkoxyCi-C6alkyl, CrC6alkanoyl, amino, azido, oxo, mercapto, nitro, or C0-C3alkylR3.
While it is possible for the active agent to be administered alone, it is preferable to present it as part of a pharmaceutical formulation. Such a 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. 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 Formula IV 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.
With regard to compositions for oral administration (e.g. tablets and capsules), the term 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.
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.
As with all pharmaceuticals, 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 superamily) concentrations of the order 0.01-100 uM, more preferably 0.01-10 uM, such as 0.1-5 uM are typically desirable and achievable. Synthesis of the compounds of the present invention can be performed by different chemical strategies in solution or solid phase or a combination of both. The compounds are typically prepared as building blocks reflecting the P1 , P2 and P3 moieties of the end product inhibitor. Without in any way wishing to be bound by theory, or the ascription of tentative binding modes for specific variables, the notional concepts P1 , P2 and P3 as used herein are provided for convenience only and have substantially their conventional Schlecter & Berger meanings and denote those portions of the inhibitor believed to fill the S1, S2, and S3 subsites respectively of the enzyme, where S1 is adjacent the cleavage site and S3 remote from the cleavage site. Compounds defined by Formula I are intended to be within the scope of the invention, regardless of binding mode.
Broadly speaking the P1 building block will be an N-protected C-5-substituted furan-3-onamine, P2 will be an N-protected amino acid in which the side chain comprises the D-containing saturated ring and branched alkyl linker, whereas P3 typically comprises a capping group such as a substituted, heteroaroyl or aroyl moiety.
The suitably protected individual building blocks can first be prepared and subsequently coupled together i.e. P2+P1→ P2-P1. Alternatively, precursors of the building blocks can be coupled together and modified at a later stage of the synthesis of the inhibitor sequence. Further building blocks, precursors of building blocks or prefabricated bigger fragments of the desired structure, can then be coupled to the growing chain, e.g. R3-E-P2*+ P1→ R3-E-P2-P1 or R3-E*+P2-P1→ R3-E-P2-P1 , where * denotes an activated form.
Coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be carried out using standard coupling procedures such as the azide method, mixed carbonic- car boxy lie acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active ester (pnitrophenyl ester, N-hydroxysuccinic imido ester) method, Woodward reagent K- method, carbonyldiimidazole method, phosphorus reagents or oxidation-reduction methods. Some of these methods (especially the carbodiimide method) can be enhanced by adding 1- hydroxybenzotriazole or 4-DMAP. These coupling reactions can be performed in either solution (liquid phase) or solid phase.
More explicitly, the coupling step involves the dehydrative coupling of a free carboxyl of one reactant with the free amino group of the other reactant in the present of a coupling agent to form a linking amide bond. Descriptions of such coupling agents are found in general textbooks on peptide chemistry, for example, M. Bodanszky, "Peptide Chemistry", 2nd rev ed., Springer- Verlag, Berlin, Germany, (1993) hereafter simply referred to as Bodanszky, the contents of which are hereby incorporated by reference. Examples of suitable coupling agents are N1NT- dicyclohexylcarbodiimide, 1-hydroxybenzotriazole in the presence of N1N1- dicyclohexylcarbodiimide or N-ethyl-N1- [ (3dimethylamino) propyl] carbodiimide. A practical and useful coupling agent is the commercially available (benzotriazol-1-yloxy) tris- (dimethylamino) phosphonium hexafluorophosphate, either by itself or in the present of 1-hydroxybenzotriazole or 4-DMAP. Another practical and useful coupling agent is commercially available 2-(IH- benzotriazol-1-yl)-N, N, N1, N1- tetramethyluronium tetrafluoroborate. Still another practical and useful coupling agent is commercially available 0-(7-azabenzotrizol-1-yl)-N, N1N1, N1- tetramethyluronium hexafluorophosphate.
The coupling reaction is conducted in an inert solvent, e. g. dichloromethane, acetonitrile or dimethylformamide. An excess of a tertiary amine, e. g. diisopropylethylamine, N- methylmorpholine, N-methylpyrrolidine or 4-DMAP is added to maintain the reaction mixture at a pH of about 8. The reaction temperature usually ranges between 0 °C and 50 °C and the reaction time usually ranges between 15 min and 24 h.
The functional groups of the constituent non-natural amino acids generally must be protected during the coupling reactions to avoid formation of undesired bonds. The protecting groups that can be used are listed in Greene, "Protective Groups in Organic Chemistry", John Wiley & Sons, New York (1981) and "The Peptides: Analysis, Synthesis, Biology", Vol. 3, Academic Press, New York (1981), hereafter referred to simply as Greene, the disclosures of which are hereby incorporated by reference.
The alpha-carboxyl group of the C-terminal residue is usually protected as an ester that can be cleaved to give the carboxylic acid. Protecting groups that can be used include 1) alkyl esters such as methyl, trimethylsilyl and t.butyl, 2) aralkyl esters such as benzyl and substituted benzyl, or 3) esters that can be cleaved by mild base or mild reductive means such as trichloroethyl and phenacyl esters.
The alpha-amino group of each amino acid to be coupled is typically be protected. Any protecting group known in the art can be used. Examples of such groups include: 1) acyl groups such as formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl; 2) aromatic carbamate groups such as benzyloxycarbonyl (Cbz or Z) and substituted bensyloxycarbonyls, and 9- fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic carbamate groups such as tertbutyloxycarbonyl (Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4) cyclic alkyl carbamate groups such as cyclopentyloxycarbonyl and adamantyloxycarbonyl; 5) alkyl groups such as triphenylmethyl and benzyl; 6) trialkylsilyl such as trimethylsilyl; and 7) thiol containing groups such asphenylthiocarbonyl anddithiasuccinoyl. The preferred alpha-amino protecting group is either Boc or Fmoc. Many amino acid derivatives suitably protected for peptide synthesis are commercially available.
The alpha-amino protecting group is typically cleaved prior to the next coupling step. When the Boc group is used, the methods of choice are trifluoroacetic acid, neat or in dichloromethane, or HCI in dioxane or in ethyl acetate. The resulting ammonium salt is then neutralized either prior to the coupling or in situ with basic solutions such as aqueous buffers, or tertiary amines in dichloromethane or acetonitrile or dimethylformamide. When the Fmoc group is used, the reagents of choice are piperidine or substituted piperidine in dimethylformamide, but any secondary amine can be used. The deprotection is carried out at a temperature between 0 0C and room temperature usually 20-22 0C.
Any of the natural or non-natural amino acids having side chain functionalities will typically be protected during the preparation of the peptide using any of the above described groups. Those skilled in the art will appreciate that the selection and use of appropriate protecting groups for these side chain functionalities depend upon the amino acid and presence of other protecting groups in the peptide. In the selection of such protecting groups it is desirable that the group is not removed during the deprotection and coupling of the alpha-amino group.
For example, when Boc is used as the alpha-amino protecting group, the following side chain protecting groups are suitable: p-toluenesulfonyl (tosyl) moieties can be used to protect the amino side chain of amino acids such as Lys and Arg; acetamidomethyl, benzyl (Bn), or tert- butylsulfonyl moities can be used to protect the sulfide containing side chain of cysteine; benzyl (Bn) ethers can be used to protect the hydroxy containing side chains of serine, threonine or hydroxyproline; and benzyl esters can be used to protect the carboxy containing side chains of aspartic acid and glutamic acid.
When Fmoc is chosen for the alpha-amine protection, usually tert. butyl based protecting groups are acceptable. For instance, Boc can be used for lysine and arginine, tert.butyl ether for serine, threonine and hydroxyproline, and tert-butyl ester for aspartic acid and glutamic acid. Triphenylmethyl (Trityl) moiety can be used to protect the sulfide containing side chain of cysteine.
Once the inhibitor sequence is completed any protecting groups are removed in whatever manner is dictated by the choice of protecting groups. These procedures are well known to those skilled in the art.
Preparation of P1 building blocks
The preparation and manipulation of C-5 substituted furanone amines is extensively described in WO0069055 and WO05/082876, whose disclosures are respectively incorporated herein.
The P1 building block may be elongated with the P2 amino acid (or the ready formed P3-P2 intermediate) while the P1 is in the furanone form. Alternatively elongation with P2/P3 may take place on a furanol which is subsequently oxidised by Dess Martin chemistry in an organic solvent such as DCM.
Preparation of P2 building blocks
Scheme 1
Figure imgf000021_0001
Scheme 1 depicts that a suitable aldehyde 1 , such as cyclopentylaldehyde, can be derivatised into the silyl enol ether 2 using, for example, N-Methyl-N-trimethylsilylacetamide in DMF at room temperature. 2 can then act as a suitable precursor for a number of variations of X. For example, by using a suitable alkyl halide in the presence of fluoride anion, X can represent suitable alkyl groups. By employing suitable electrophilic fluorinating agents, such as Selectfluor™ in a solvent such as DMF or acetonitrile, X can represent fluorine. Other electrophilic halogenating reagents, such as N-chlorosuccinimide in the presence of fluoride anion will give derivatives where X is chlorine. Conversion of 3 to 4 can be achieved with N- acetamidophosphonoglycine trimethyl ester in the presence of a suitable base such as potassium tert-butoxide in a solvent such as THF at 0 0C. Alternatively preparation of 4 can be achieved by following the protocol outlined by Schollkopf et al in Liebigs Ann. Chemie 1981 1469-1475. The conversion of 4 into the chiral amino acid 5 can be achieved with a chiral catalyst, such as [EtDuPHOS-Rh (COD)]+ in a solvent such as methanol under hydrogen pressure of between 1 and 5 bar. Alternatively, 4 can be converted into the achiral amino acid 6 using a non-chiral catalyst such as that based on a palladium or rhodium-containing species e.g. Wilkinson's catalyst. Resolution of the amino acid will then follow one of many well documented methods, such as enzymatic hydrolysis of the ester, or separation of the racemates by chiral-HPLC.
Scheme 2
Figure imgf000022_0001
Scheme 2 depicts that the preparation of 3 can also be achieved from direct reaction of a suitable lithium enolate with a suitable electrophilic reagent, such as an alkyl halide. Therefore, treatment of 7 with LDA in THF at -78 0C followed by quenching of the resultant anion affords 8. The ester group of 8 can be reduced, for example with lithium aluminium hydride to the corresponding alcohol 9. Compound 3 is then prepared by oxidation of the alcohol with a suitable oxidant, such as pyridinium chlorochromate in DCM at room temperature. Scheme 3
Figure imgf000023_0001
Scheme 3 shows an alternative synthesis to prepare the C5 alkylene amino acid 16 in homochiral form. Geraniol 10 is converted to the phosphate 11 with diethyl chlorophosphonate and then reacted with a homochiral zinc/copper couple of alanine 12. Compound 13 is obtained from Sn2 reaction of the zinc/copper couple, whilst compound 14 is obtained from the alternative Sn2' mechanism. Ring-closing metathesis reaction of 14 using for example Grubb's catalyst gives the methylcyclopentene derivative 15. Atmospheric pressure hydrogenation of the methylcyclopentene double bond can be achieved with a palladium catalyst in a solvent like methanol to afford the amino acid 16. Enantioselectivity is in excess of 95%.
An alternative method to prepare substituted cycloalkyl alanines of the type shown in Scheme 4, below, is to take a suitable cycloalkanone 17 and treat this with triethylphosphonoacetate in THF at O 0C with potassium tert-butoxide as base. The resultant eneoate 18 can be treated with a variey of nucleophiles, such as organocuprates, substituted amines and substituted thiols to generate compounds of the type shown in 19. Treatment of compounds such as 19 in the manner described for 38 in Scheme 8 below gives the racemic substituted cycloalkyl alanine derivatives. Resolution of the amino acid will then follow one of many well documented methods, such as enzymatic hydrolysis of the ester or separation of the racemates by chiral- HPLC. Scheme 4
Figure imgf000024_0001
Scheme 5
Figure imgf000024_0002
LiAIH4, THF
Figure imgf000024_0003
An alternative for substitution at R2 involves the method shown in Scheme 5. An appropriate cycloalkanone is treated with a zinc/copper couple of alpha-bromomethylacetate in a solvent such as THF at reflux. The hydroxyl group in 24 can be left underivatised and the ester can be reduced to the primary alcohol with a reducing agent such as lithium aluminium hydride. A compound such as 25 can be treated in the same way as compound 20 to afford the desired substituted cycloalkyl alanine. Alternatively, the hydroxyl group in 24 can be derivatised to form the alkyloxy at R2, using a reagent such as sodium hydride and an alkyl halide in THF at room temperature or reflux. The same procedure for the synthesis of the achiral amino acid would then apply.
Scheme 6
Figure imgf000025_0001
A suitable dibromoalkane can be reacted with diethylmalonate with, for example, sodium ethoxide in ethanol to afford the diester 26. This can be converted into the ester/aldehyde 27 in a number of ways, for example with diisobutylaluminium hydride in dichloromethane at -78 deg C. This aldehyde serves as a useful precursor for a number of derivatives. The methylene alcohol 28 can be prepared by reduction of the aldehyde with sodium borohydride in a solvent such as ethanol; the alkyloxy methylene 29 is produced by alkylation of the methylene alcohol 28 with an alkyl halide and a suitable base such as sodium hydride; the methylene fluoride 30 is produced by fluorination of 28 with a suitable fluorinating agent such as DAST or Deoxyfluor. These reagents can also be employed to give the difluorinated compound 31 from the aldehyde 27. Substituted cycloalkyl esters, typified by compound 31 , can be used to prepare the appropriate substituted cycloalkyl alanines 33 in an analogous manner to that outlined in Scheme 8. Scheme 7
Figure imgf000026_0001
homologation and reduction
Figure imgf000026_0002
33
Appropriately substituted cyclohexylalanines can be prepared as in Scheme 7. Diels-Alder reaction of 1,3-butadiene with appropriately substituted dieneophiles can afford the cyclohexene derivative 32. Reduction of the cyclohexene double bond and manipulation of the ester moiety to the aldehyde 33, as shown in the scheme, provides the precursor to the substituted cyclohexyl alanine amino acids. These final steps can be achieved using the chemistry outlined in Scheme 8.
Scheme 8
Figure imgf000026_0003
Figure imgf000026_0004
Scheme 8 depicts the synthesis of a methylcyclopentylalanine building block. Commercially available methyl cyclopentane carboxylate 34 is methylated with LDA and iodomethane (i, BuLi, diisopropylamine, MeI) to give 35. Hydrolysis of the ester with LiOH followed by treatment with oxalyl chloride (ii, LiOH, oxalylchloride) gives acid chloride 36. Wolff rearrangement with diazomethane (iii, CH2N2, Et3N) and silver benzoate (iv, silver benzoate, Et3N, MeOH) gives the ester 38. Reduction of the ester (v, LiAIH4) followed by Dess-Martin periodinane oxidation (vi) gives the aldehyde 40. The aldehyde is treated with KCN and ammonium carbonate (vii, KCN, (NH4J2CO3, HCI), followed by hydrolysis with NaOH and protection of the free amine as its Boc carbamate to give racemic amino acid 16. The enantiomers are separated by conventional chromatographic techniques such as chiral HPLC, before or after coupling to the P1 and/or P3 building blocks. Although the scheme has been illustrated with a cyclopentane variant, the methodology is applicable to other variants of D.
Elongation with P3.
Compounds wherein E is carbonyl are readily prepared by conventional peptide chemistry, from the corresponding (optionally substituted) R3-carboxylic acid. For example the N-protected P2- P1 intermediate is treated with 4M HCI /dioxan and the carboxy protected R3 acid is added together with a coupling mixture such as HBTU/HOBT/DMF/NMM. A substantial number of (substituted) R3 carboxylic acids are commercially available or readily converted from commercially available synthons.
Sulphonamide derivatives i.e. E = S(=0)2- can be prepared by reaction of the amino group of the P2 amino acid with a suitable sulfonyl chloride in a solvent such as dichloromethane in the presence of a suitable base such as triethylamine or dimethylaminopyridine. For example the N- protected P1-P2 intermediate is treated with 4M HCI in dioxan. An optionally substituted R3- SO2CI is added with Et3N and a catalytic amount of DMAP.
Urethane compounds i.e. E is -0C(=0)- can be formed for example by reaction of an R3 alcohol with the isocyanate of the P2 amino acid. The isocyanate, or equivalent reactive intermediate, can be formed by reaction of the amino group of the P2-amino acid with phosgene, or with dinitrophenylcarbonate in the presence of a suitable base, e.g. triethylamine. Alternatively they can be formed by reaction of the amino group of the P2 amino acid with a suitable chloroformate, e.g. benzylchloroformate.
Sulphamide derivatives i.e. E = -NRaS(=O)2- can be prepared by reacting a suitable R3 amine in a sulphonyl chloride solvent followed by reaction of the formed sulfamoyl chloride derivative with the amino group of the above mentioned R4 amino acid in a solvent such as dichloromethane in the presence of a suitable base such as triethylamine. Urea derivatives i.e. E = -NRa-C(=O)- can be prepared by reaction of the corresponding R3 isocyanate with the N-protected amide of the P2-P1 intermediate, typically in an inert organic solvent such as N.N-dimethyl formamide. Conversely, the R3 amine is reacted with the isocyanate of the P2-P1 intermediate under similar conditions. Alternatively the N-protected R3- amine, and N-protected P2-P1 amine are together reacted with L1C(=O)L2, where L1 and L2 are good leaving groups in an inert organic solvent such as N,N-dimethyl formamide, tetrahydrofuran, ethyl acetate or benzene, as shown in J Org Chem 56, 891 (1991). The time, temperature and sequence of addition used depends on the reactivity of the individual reagents.
A special case of a urea derivative are compounds wherein R3 represents an unsaturated ring such as morpholine, piperazine or piperidine which is N-bonded to E as carbonyl. Such compounds are readily prepared, for example by treating the N-protected P2-P1 intermediate with 4M HCI/dioxane, adding the R3-chloride, for example morpholinyl carbonyl chloride, together with TEA in DCM.
Compounds wherein R10 is an hydroxyl, ether, ester or ketone are typically prepared by coupling P1 to P2 & P3 as the 2,3 isopropylidinyl protected building block, such as:
Figure imgf000028_0001
Preparation of the 2,3, P1 building block for a variety of is shown in WO00/69055 and WO05/082876, the content of which is incorporated by reference. For example, acid catalysed removal of the diisopropylidene protecting group, such as with HCI in a suitable solvent such as methanol, produces the alcohol acetal (denoted C1) and the hydroxyl at C2. This hydroxyl can be oxidized to the ketone as described in WO0069055 and WO05/082876 using, for example, Dess-Martin periodination. The alpha anomer at R10 is isolated by preparative HPLC or flash chromatography. Appropriate choice of alcohol and reaction conditions produces other acetal groups as defined by R10 in the invention which can undergo oxidation of the hydroxyl functionality at C2. Suitable protection of the C2 hydroxy function will allow manipulation of the alcohol acetal to the hemi-acetal form, which in turn can be oxidized to the lactone. Preparation of the thioacetal from the hemi-acetal can be achieved by methods as described in LiebigsAnn Chem 1993 p1211-1218 or Journal of Organic Chemistry 1986 p4802-4806. Various aspects of the invention will now be described by way of example only with reference to the accompanying Examples.
Example 1
Preparation of 1-methylcyclobutylalanine P2 bulding block.
(R)- and (S)- N-Boc-d-methylcvclopentvD-alanine benzyl ester
/X ^-CO2Me i /X ^-CO2Me jj /X^-CO2H iϋ, iv
Figure imgf000029_0001
Figure imgf000029_0002
(i) LDA, MeI, -780C, (ii) LiOH, MeOH, (iii) oxalyl chloride, DMF, (iv) Diazomethane, (v) AgOBz, MeOH, (vi) LiAIH4, (vii) Dess - Martin periodinane, (viii) KCN, (NH4J2CO3, (ix) NaOH then BoC2O, (x) BnBr, Cs2CO3, (xi) Chiral HPLC.
a) 1-Methylcyclopentanecarboxylic acid methyl ester
A solution of cyclopentanecarboxylic acid methyl ester (78 mmol) in THF was added to a solution of freshly prepared LDA (78 mmol) in THF at -780C over 5 minutes. The mixture was warmed to O0C and stirred for 30 minutes at which point it was re-cooled to -780C. A solution of methyl iodide (78 mmol) in THF was then added and the mixture was allowed to come to room temperature and stirred overnight. NH4CI (sat. aq.) was added then the mixture was extracted with TBME. The organics were dried (MgSO4) then concentrated in vacuo to give a yellow oil. Column chromatography (silica gel, 1 - 15% EtOAc in heptane) gave the product as a clear oil (42 mmol, 54%).
b) 1 -Methylcyclopentanecarboxyl ic acid
NaOH (88 mmol) was added to a solution of 1 -methyl-cyclopentanecarboxylic acid methyl ester (88 mmol) in MeOH and the mixture was stirred overnight. The resulting solution was then acidified with cone. HCI to pH 2. Water and EtOAc were then added and the organics were separated. The aqueous phase was extracted with EtOAc. The combined organics were dried (MgSO4) then concentrated in vacuo to give a yellow oil which was used with no further purification (74 mmol, 84%).
c) 1-Methylcyclopentane diazoketone
Oxalyl chloride (89 mmol) was added to a solution of 1 -methyl-cyclopentanecarboxylic acid (74 mmol) in DCM at O0C. This was followed by a few drops of DMF. The mixture was stirred overnight then the solvents were removed in vacuo to give a pale brown semi-solid which was dissolved 1 :1 THF:MeCN. Triethylamine (96 mmol) was added followed by diazomethane (222 mmol) in diethyl ether. The mixture was stirred overnight then the solvents were removed in vacuo. The residue was dissolved in TBME. The organics were washed (water then NaHCO3 (sat. aq.)), dried (MgSO4) then concentrated in vacuo to give a yellow oil which was used with no further purification (66.2 mmol, 90%).
d) (1 -Methylcyclopentyl)acetic acid methyl ester
Silver benzoate (11.2 mmol) was added to a solution of 1 -methylcyclopentane diazoketone (22.4 mmol) in methanol containing triethylamine (56 mmol). Gas was seen to be evolved from the mixture which also darkened to black. After 1 hour the mixture was filtered through silica and the filtrate was concentrated in vacuo to give a dark oil. Column chromatography (silica gel, 1 - 10% EtOAc in heptane) gave the product as a clear oil (10.9 mmol, 49%).
e) (I-Methylcyclopentyl)ethanol
LiAIH4 (99.3 mmol) was added portionwise to a solution of (i-methylcyclopentyl)acetic acid methyl ester (66.2 mmol) in THF at O0C. The mixture was allowed to warm to room temperature and stirring was continued for 1.5 hours. Ether was added and the mixture cooled to O0C. 3.8 ml of water was added followed by 3.8 ml of 15% aqueous NaOH solution then 11.4 ml of water. The mixture was warmed to room temperature and stirred for 15 minutes. Anhydrous MgSO4 was added and stirring was continued for a further 15 minutes. The mixture was filtered and the filtrate was concentrated in vacuo. Distillation (760C @ 17 mmHg) gave the product as a clear oil (40.7 mmol, 62%).
f) N-Boc-(1 -methylcyclopentyl)-alanine Dess-Martin periodinane (11.30 mmol) was added to a solution of (i-methylcyclopentyl)ethanol (9.48 mmol) in DCM. The mixture was stirred for 2 hours then diluted with DCM. 1 :1 1M aqueous sodium thiosulfate : saturated aqueous sodium carbonate solution was added and the mixture stirred for 1 hour. The organics were separated, dried (MgSO4) then concentrated in vacuo to give a yellow oil. This was dissolved in 1 :1 ethanohwater then KCN (11.30 mmol) and (NH4J2CO3 (33.20 mmol) were added. The mixture was heated at 6O0C overnight. The ethanol was removed in vacuo and concentrated HCI was added to pH=1. A solid (hydantoin) was formed which was isolated by filtration then dried under vacuum (2.83 mmol, 30%). The solid was dissolved in NaOH (0.7M aqueous) and heated at reflux overnight. The mixture was concentrated to 1/3 original volume and a solution of BoC2O (3.40 mmol) in THF was added. The mixture was stirred overnight then washed with TBME. The aqueous layer was cooled in an ice bath and 10% aqueous KHSO4 was added dropwise to pH = 2. The resulting suspension was extracted with CHCI3. The organics were separated, dried (MgSO4) then concentrated in vacuo to give a white solid (1.48 mmol, 50% based on yield of hydantoin).
g) (R)- and (S)- N-Boc-(1-methylcyclopentyl)-alanine benzyl ester
Benzyl bromide (3.25 mmol) was added to a solution of N-Boc-(1 -methylcyclopentyl) alanine (2.96 mmol) in DMF containing caesium carbonate (5.92 mmol). The mixture was heated at reflux overnight then cooled to O0C and filtered. The filtrate was concentrated in vacuo to give a yellow gum (0.75 mmol, 25%). This was separated on a Chiralpak AD column using an isocratic eluent of 3% 2-propanol in /so-hexane to give the (R)- and (S)- esters as clear gums.
Example 2
2-r3-Ethyl-3-(2-methoxy-ethvn-ureidol-N-(2-ethyl-4-oxo-tetrahvdro-furan-3-vπ-3-(1-methyl- cvclopentvD-propionamide
Figure imgf000031_0001
(i) 4M HCI, (ii) 4-morpholinecarbonyl chloride, TEA, in DCM (iii) H2, 10% Pd/C, (iv) amino ketal, WSCHCI, HOBt, (v) HCI, dioxane, water.
a) 3-(1 -Methyl-cyclopentyl)-2-(S)-[(morpholine-4-carbonyl)-amino]-propionic acid
4M HCI in dioxane was added to (S)-N-Boc-(1-methylcyclopentyl)-alanine benzyl ester and the mixture was stirred for 30 minutes. The solvents were removed in vacuo to give a brown powder. This was dissolved in DCM and cooled to O0C. Triethylamine then 4-morpholine carbonyl chloride were added and the mixture was stirred for 2 hours prior to being washed with
1 M aqueous HCI solution. The organics were dried (MgSO4) then concentrated in vacuo to give a yellow oil. This was dissolved in EtOAc and 2% AcOH (v/v) was added followed by 10% Pd/carbon (catalytic amount). The reaction was stirred under an atmosphere of hydrogen gas overnight. The mixture was filtered and the filtrate concentrated to give the crude acid which was used directly in the next step.
b) N-(S)-(2-(S)-Ethyl-4,4-dimethoxy-tetrahydro-furan-3-yl)-2-(S)-[3-ethyl-3-(2- methoxyethyl)-ureido]-3-(1-methyl-cyclopentyl)-propionamide
WSCHCI (0.20 mmol) and HOBt (0.20 mmol) were added to a solution of 3-(1 -methyl- cyclopentyl)-2-(S)-[(morpholine-4-carbonyl)-amino]-propionic acid (0.18 mmol) in DCM and stirred for 5 minutes. A solution of 1 -(S)-2-(S)-ethyl-4,4-dimethoxy-tetrahydro-furan-3-ylamine (0.18 mmol, 1 M in DCM) was then added and the mixture was stirred overnight. The mixture was diluted with EtOAc then washed (1 M citric acid solution then saturated aqueous NaHCO3), dried (Na2SO4) then concentrated in vacuo to give a yellow oil. This was purified by column (silica gel, 5% methanol in DCM) to give the title compound (0.095 mmol, 53%).
c) 2-[3-Ethyl-3-(2-methoxy-ethyl)-ureido]-N-(2-ethyl-4-oxo-tetrahydro-furan-3-yl)-3-(1- methyl-cyclopentyl)-propionamide
N-(S)-(2-(S)-Ethyl-4,4-dimethoxy-tetrahydro-furan-3-yl)-2-(S)-[3-ethyl-3-(2-methoxyethyl)- ureido]-3-(1-methyl-cyclopentyl)-propionamide (0.095 mmol) was dissolved in a mixture of 4M HCI in dioxane : water (1 :1) and stirred for 1 hour. The solvents were removed in vacuo to give the title compound (0.03 mmol, 32%).
1H NMR (5CDCI3) 6.99 - 7.03 (m, 1H), 4.70 (d, 1H), 4.36 - 4.42 (m, 1H), 4.05 - 4.21 (m, 2H), 3.95 - 4.03 (m, 1 H), 3.85 - 3.92 (m, 1H), 3.65 - 3.75 (m, 4H), 3.30 - 3.42 (m, 4H), 2.05 - 2.12 (m, 1 H), 1.52 - 1.88 (m, 8H), 1.35 - 1.42 (m, 4H), 1.03 (t, 3H), 0.97 (s, 3H), ESMS m/z 418 (MNa+, 100%), 396 (MH+, 45%).
Example 3
2-r3-Ethyl-3-(2-nnethoxy-ethvn-ureidol-N-(2-ethyl-4-oxo-tetrahvdro-furan-3-vπ-3-(1 -methyl- cyclohexyD-propionamide
Figure imgf000033_0001
Using the route outlined in Examples 1 & 2 (excepting steps i and ii in the scheme in Example 1), 1 -methylcyclohexyl carboxylic acid was converted into the title compound.
1H NMR (5CDCI3) 6.99 (d, 1 H), 4.65 (d, 1H), 4.38 - 4.42 (m, 1H), 4.05 - 4.21 (m, 2H), 3.95 - 4.03 (m, 1 H), 3.80 - 3.90 (m, 5H), 3.25 - 3.40 (m, 4H), 1.95 - 2.00 (m, 1 H), 1.70 - 1.85 (m, 2H), 1.23 - 1.60 (m, 10H), 1.05 (t, 3H), 0.95 (s, 3H),
ESMS m/z 432 (MNa+, 30%), 410 (MH+, 100%).
Example 4
The compounds in the table immediately below were prepared analogously to the method disclosed in Example 13.
Example 4.1
Furan-3-carboxylic acid [(1 S)-((2S)-ethyl-4-oxo-tetrahydrofuran-(3S)-ylcarbamoyl)-2-(1- methyl-cyclopentyl)-ethyl]-amide
HPLC retention time of 5.00 min using Synergy Max RP 80 μm 50x4.6mm column, 10→90% 6 min gradient of solution B (solution A = 0.1% TFA in water and solution B = 10% A in acetonitrile) at flow rate of 2ml/min.
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
,
J =
Figure imgf000037_0001
Example 4.12
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydrofuran-(3S)-ylcarbannoyl)-2-(1-nnethyl-cyclopentyl)- ethyl]-4-hydroxy-3-methoxy-benzamide
HPLC retention time of 4.79 min. 1H-NMR (400 MHz, CDCI3) δ 7.40 (s, 1H), 7.24 (d, J = 8.5, 1 H), 6.94 (d, J = 8.5, 1 H), 6.88 (br.d, , J = 8.0, 1 H), 6.39 (br.d, , J = 8.5, 1 H), 4.73-4.68 (m, 1 H), 4.20 (d, J = 17.0, 1 H), 4.06 (d, J = 17.0, 1 H), 3.97-3.85 (m,
Figure imgf000038_0001
2H), 3.95 (s, 3H), 2.19 (dd, J = 5.0 and 14.5, 1 H), 1.85-1.38 (m, 11 H), 1.00 (t, J = 7.5, 3H), 0.99 (s, 3H). Mass spectroscopy : m/z 455 (20, MNa+), 433 (100, MH+).
Example 4.13
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydrofuran-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)- ethyl]-4-hydroxy-benzamide
HPLC retention time of 4.70 min.
1H-NMR (400 MHz, CDCI3) δ 7.56 (d, J = 8.5, 2H),
6.76 (d, J = 8.5, 2H), 6.55 (d, J = 8.5, 1 H), 4.75-4.69
(m, 1H), 4.19 (d, J = 17.0, 1H), 4.08 (d, J = 17.0, 1H),
Figure imgf000038_0002
4.01-3.96 (m, 1 H), 3.89-3.84 (m, 1H), 2.10 (dd, J = 4.5 and 14.5, 1H), 1.79-1.40 (m, 11 H), 1.00 (s, 3H), 0.99
(t, J = 7.5, 3H).
Mass spectroscopy : m/z 425 (20, MNa+), 403 (100,
MH+).
Example 4.14
3-Chloro-Λ/-[(1 S)-((2S)-ethyl-4-oxo-tetrahydrofuran-(3S)-ylcarbamoyl)-2-(1-methyl- cyclopentyl)-ethyl]-4-hydroxy-benzamide HPLC retention time of 5.10 min. 1H-NMR (400 MHz, CDCI3) δ 7.73 (s, 1H), 7.49 (d, J = 8.5, 1 H), 6.98 (d, J = 8.5, 1 H), 6.87 (br.s, 1 H), 6.43 (br.d, J = 8.0, 1 H), 4.66-4.60 (m, 1 H), 4.13 (d, J =
Figure imgf000039_0001
17.0, 1 H), 3.99 (d, J = 17.0, 1 H), 3.92-3.85 (m, 1 H), 3.83-3.79 (m, 1H), 2.08 (dd, J = 4.5 and 14.5, 1H), 1.79-1.31 (m, 11 H), 0.94 (t, J = 7.5, 3H), 0.93 (s, 3H). Mass spectroscopy : m/z 437 (100, MH+).
Example 4.15
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydrofuran-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)- ethyl]-4-hydroxy-3-methyl-benzamide
HPLC retention time of 4.94 min. 1H-NMR (400 MHz, CDCI3) δ 7.52 (br.s, 1H), 7.45 (br.d, J = 9.0, 1 H), 6.76 (br.d, J = 9.0, 1 H), 6.42 (br.d, J = 8.0, 1H), 4.74-4.68 (m, 1H), 4.19 (d, J = 17.0, 1H),
Figure imgf000039_0002
4.06 (d, J = 17.0, 1H), 3.98-3.92 (m, 1 H), 3.89-3.84 (m, 1 H), 2.26 (s, 3H), 2.16 (dd, J = 4.0 and 10.0, 1H), 1.83-1.40 (m, 11 H), 1.00 (t, J = 7.5, 3H), 1.00 (s, 3H). Mass spectroscopy : m/z 439 (20, MNa+), 417 (100, MH+).
Example 4.16
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydrofuran-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)- ethyl]-3-fluoro-4-hydroxy-benzamide
HPLC retention time of 4.89 min.
1H-NMR (400 MHz, CDCI3) δ 7.51-7.49 (m, 1 H), 7.41-
7.38 (m, 1 H), 7.00-6.96 (m, 1H), 6.48 (br.d, J = 8.0,
1H), 4.72-4.66 (m, 1 H), 4.20 (d, J = 17.0, 1H), 4.07 (d,
Figure imgf000039_0003
J = 17.0, 1 H), 4.00-3.95 (m, 1 H), 3.88-3.84 (m, 1 H),
2.12 (dd, J = 4.5 and 14.5, 1 H), 1.85-1.39 (m, 11 H),
1.01 (t, J = 7.5, 3H), 1.00 (s, 3H).
Mass spectroscopy : m/z 443 (40, MNa+), 421 (100, (d,
(d, J
= H),
Figure imgf000040_0001
Example 5
Preparation of 1-methylcvclobutylalanine building block XO0Et
Figure imgf000041_0001
1
PCC DCM
Figure imgf000041_0002
*(+)-1 ,2-bis(2S,5S)-diethylphosphonolanbenzene (cyclooctadiene)rhodium (l)triflate
1-Methyl-cyclobutanecarboxylic acid ethyl ester 1 was prepared from ethyl cyclobutanecarboxylate by the method described in J. Am. Chem. Soc, Vol. 103 No.2 1981 436-442.
1-Methyl-cyclobutanecarboxylic acid ethyl ester 1 (1eq) was stirred under a nitrogen atmosphere at O0C in anhydrous THF. To this solution was added portionwise lithium aluminium hydride (1.5eq) and the suspension was stirred at room temperature for 3 hours. The reaction mixture was cooled on ice, treated with 1 M HCI (aq) and stirred at O0C 20 minutes. The solution was passed through a pad of celite and the filtrate extracted into diethyl ether. The organic phases were dried over MgSO4, filtered and concentrated in vacuo to give (1 -methyl- cyclobutyl)-methanol, 2.
Pyridinium chlorochromate (1.25eq) and the same weight of celite were taken up as a suspension in anhydrous dichloromethane. To this was added dropwise a solution of compound 2 (1eq) in anhydrous dichloromethane and the resulting heterogeneous mixture was stirred at room temperature for 3 hours. The reaction mixture was passed through a pad of silica, eluting with 19:1 isohexanes: ethyl acetate to give 1-methylcyclobutanecarboxaldehyde, 3.
Compound 3 (1eq) was dissolved with stirring in anhydrous dichloromethane, and to this was added Boc-phosphoglycine trimethyl ester (0.5eq) and 1 ,8-diazabicyclo[5.4.0]undec-7-ene (1.2eq). The resulting solution was stirred at ambient temperature under nitrogen overnight. The reaction mixture was partitioned between dichloromethane and successively 1M HCI (aq), sat. NaHCO3 (aq) and sat. NaCI (aq). The organic layer was dried over MgSO4, filtered and concentrated in vacuo. The resulting oil was purified by flash column chromatography, eluting with 1%methanol in dichloromethane to give 2-tert-butoxycarbonylamino-3-(1-methyl- cyclobutyl)-acrylic acid methyl ester, 4.
Compound 4 was dissolved in anhydrous methanol and degassed with nitrogen. (+)-1 ,2-bis (2S,5S)-diethylphosphonolanbenzene (cyclooctadiene)rhodium (I) triflate was added and degassing was continued for a further 10 minutes. The reaction was shaken under a hydrogen atmosphere (4 bar) for 48 hours. The solution was concentrated, in vacuo and purified by flash chromatography, eluting with dichloromethane, to give 2S-tert-butoxycarbonylamino-3-(1- methyl-cyclobutyl)-propionic acid methyl ester, 5.
HPLC retention time 5.88min (monitored at 215 and 254 nm) HPLC using Synergy Max RP 80 μm 50x4.6mm column, 10→90% 6 min gradient of solution B (solution A = 0.1 % TFA in water and solution B = 10% A in acetonitrile) at flow rate of 2ml/min.
MS [M+H]+ 272.08 (20%) [M-Boc+H]+ 172.06 (100%)
Electrospray ionisation, eluting with acetonitrile / ammonium formate buffer.
1H NMR (400 MHz, CDCI3) δ 4.89-4.79 (1H, m) 4.33-4.27 (1H, m) 3.71 (3H, s) 1.98-1.62 (8H, m) 1.42 (9H, s) 1.22 (3H, s)
Example 6
Furan-3-carboxylic acid Td SM(2S)-ethyl-4-oxo-tetrahvdrofuran-(3S)-ylcarbamoyl)-2-(1-methyl- cyclopentvD-ethyll-amide
Figure imgf000042_0001
The title compound was prepared analogously to example 2 employing the P2 building block of Example 5.
MS [M+H]+ 363.02 (70%) [M+Na]+ 385.03 (100%)
Electrospray ionisation, eluting with acetonitrile / ammonium formate buffer.
Example 7 Preparation of 1 -ethylcyclobutylalanine P2 Building Block
Figure imgf000043_0001
1 -Hydroxymethyl-1 -ethyl cyclobutane
OH
"-Butyl lithium (1.6 M (hexanes), 100 mmol, 62.5 ml, 2 eq.) was added dropwise to a solution of 1Pr2NH (100 mmol, 14.13 ml, 2 eq.) in THF (100 ml) at O0C. This was stirred for 15 minutes then cyclobutane carboxylic acid (50 mmol, 4.78 ml, 1 eq.) was added dropwise at O0C. The solution was stirred for 1.5 hours. EtI (50 mmol, 4.08 ml, 1 eq.) was added dropwise and the solution was stirred overnight, allowing it to come to room temperature. HCI (2M, 150 ml) was added and the resulting mixture was extracted with EtOAc. The organics were dried (MgSO4) then concentrated in vacuo to give the crude product as a semi-solid (6.98g). This was dissolved in THF (70 ml), cooled in an ice-bath and LiAIH4 (75 mmol, 2.84g) was added portionwise. This was stirred at room temperature for 3 hours then cooled in an ice-bath and diluted with diethyl ether. Water (2.84 ml), 10% NaOH(aq) (2.84 ml) and further water (8.5 ml) were added sequentially. The mixture was stirred for 15 minutes then MgSO4 was added. After a further 30 minutes stirring, the mixture was filtered and the organics evaporated to give a clear oil. Distillation (68 - 7OC at 10 mmHg) gave the product as a clear oil (4.24 g, 74%).
1H NMR (400 MHz) (δ, CDCI3) 3.50 (s, 2H), 1.65 - 1.85 (m, 6H), 1.49 (q, J 7.0, 2H), 0.82 (t, J 7.0, 3H).
2-tert-Butoxycarbonylamino-3-(1-ethyl-cvclobutyl)-acrylic acid methyl ester
Figure imgf000043_0002
A solution of DMSO (15 nnnnol, 0.57g, 3 eq.)) in DCM (2.5 ml) was added dropwise to a solution of oxalyl chloride (7.5 nnnnol, 0.65 ml, 1.5 eq.) in DCM (10 ml) keeping the temperature at less than -7O0C. 1-Hydroxymethyl-1 -ethyl cyclobutane (5 mmol, 0.57 g) in DCM (2.5 ml) was added after 10 minutes again keeping the temperature less than -7O0C. The mixture was then warmed to -5O0C for 30 minutes and then re-cooled to -780C. Triethylamine (26.5 mmol, 3.69 ml, 5.3 eq) was then added (keeping the temperature less than -7O0C) and the mixture was then stirred for 1 hour during which time it was allowed to warm to O0C. The mixture was quenched with NH4CI solution : water (1 :1). The organics were isolated, dried (MgSO4) then concentrated to give the crude aldehyde. This was dissolved in DCM (2.5 ml) and added dropwise to a solution of phosphonate (5.5 mmol, 1.63 g, 1.1 eq.) in DCM (2.5 ml) to which DBU (11.00 mmol, 1.64 ml, 2.2 eq.) had been added at O0C and had been stirred for 10 minutes. The mixture was stirred overnight at room temperature then washed (cold 1M HCI solution then NaHCO3 solution), dried (Na2SO4) and concentrated in vacuo to give a yellow solid. The was purified by silica column (isohexane → 10% EtOAc in ihexane) to give the product as a white solid (0.42g, 30%).
1H NMR (400 MHz) (δ, CDCI3) 6.44 (s, 1H), 3.76 (s, 3H), 2.14 - 2.24 (m, 2H), 1.90 - 1.98 (m, 3H), 1.78 - 1.83 (m, 1 H), 1.73 (q, J 7.5 2H), 1.45 (s, 9H), 0.86 (t, J 7.5, 3H)
ESMS m/z(%) 306 (MNa+, 100), 284 (MH+, 65), 184 (MH-BoC+, 80).
N-ferfButoxycarbonyl-fethvDcvclobutyl alanine methyl ester
Figure imgf000044_0001
(S, S)-Et-DuPhOS (0.0148 mmol, 10.69 mg, 1 mol%) was added to a solution of 2-tert- Butoxycarbonylamino-3-(1-ethyl-cyclobutyl)-acrylic acid methyl ester in methanol (10 ml). The solution was agitated under 4 atm. pressure of hydrogen for 72 hours. The solvents were then evaporated and the residue passed through silica (eluting with DCM) to give the product as a clear gum (0.35g, 82%).
1H NMR (400 MHz) (5, CDCI3) 4.84 (br d, 1 H), 4.18 - 4.30 (m, 1 H), 3.71 (s, 3H), 1.58 - 1.92(m, 10H), 1.43 (s, 9H), 0.84 (t, J 7.5, 3H),
CIMS m/z(%) 306 (MNa+, 100), 286 (MH+, 80). N-ferfButoxycarbonyl-fethvDcvclobutyl alanine
Figure imgf000045_0001
IMLiOH (aq) (1.81 nnnnol, 1.81 ml, 1.5 eq.) was added to a solution of N-fertButoxycarbonyl- (ethyl)cyclobutyl alanine methyl ester (1.21 nnnnol, 0.35g) in THF (10 ml) at O0C. This was stirred overnight then diluted with water and extracted with EtOAc. The organics were dried (MgSO4) and concentrated to give the product as clear oil (0.32g, 97%).
1H NMR (400 MHz) (δ, CDCI3) 4.84 (br d, 1 H), 4.22 - 4.30 (m, 1 H), 1.54 - 2.02 (m, 10H), 1.44 (S, 9H), 0.84 (t, J 7.5, 3H)
Although this example has been illustrated with a Boc protecting group it will be apparent that other conventional N-protecting groups such as those described above in Greene, including Fmoc, CBz etc will be amenable to this route and/or the Boc group can be removed and replced with an alternative N-protecting groups using conventional protecting group manipulations.
Example 8
Preparation of 1-fluorocvclobutylalanine P2 Building Block
Figure imgf000045_0002
Cvclopentylidenemethoxy-trimethyl-silane
Figure imgf000045_0003
A solution of cyclopentane carboxaldehyde (129.5 mmol, 13.18g) in DCM (100 ml) was added to a solution of trimethylsilyl trifluoromethanesulfonate (155.4 mmol, 28.09 ml, 1.2 eq.) and diisopropylethylamine (155.4 mmol, 27.54 ml, 1.2 eq.) in DCM (700 ml) at O0C. The mixture was stirred for 1.5 hours then concentrated in vacuo. The residue was suspended in isohexane then filtered. The organics were washed (water), dried (Na2SO4) then concentrated in vacuo to give a pale brown oil. This was distilled (53 - 540C at 9 mmHg) to give a clear oil (13.21g, 60%).
1H NMR (400 MHz) (δ, CDCI3) 6.17 (m, 1 H), 2.21 - 2.26 (m, 2H), 2.13 - 2.18 (m, 2H), 1.55 - 1.67 (m, 4H), 0.17 (s, 9H).
2-(Benzhvdrylidene-amino)-3-(1-fluoro-cvclopentyl)-propionic acid ethyl ester
Figure imgf000046_0001
Selectfluor (19.36 mmol, 6.85g, 1.1 eq.) was added to a solution of cyclopentylidenemethoxy- trimethyl-silane (17.6 mmol, 3.0Og) in DMF (50 ml) at O0C. The was allowed to warm to room temperature and stirred for 1.5 hours. The mixture was diluted with water then extracted with isohexane. The organics were dried (MgSO4) and carefully concentrated to give a pale yellow oil. This was dissolved in THF (20 ml) and a solution of LiBH4 (2M (THF), 21.12 mmol, 10.56 ml, 1.2 eq.) was added dropwise at O0C. The mixture was stirred for 30 minutes. Water was added and the mixture stirred for a further 10 minutes. The mixture was extracted with diethyl ether. The organics were dried (MgSO4) and the solvent removed by distillation (360C at 750 mmHg) to give a yellow oil (17.6 mmol, 2.08g). This was dissolved in DCM (10 ml) and triethylamine (19.4 mmol, 2.70 ml, 1.1 eq.) was added. The solution was cooled to -2O0C and trifluoromethanesulfonic anhydride (19.4 mmol, 3.21 ml, 1.1 eq.) was added dropwise over 5 minutes. The solution was warmed to O0C and stirred for 1 hour then poured onto ice. The organics were washed (cold 1 M HCI solution then 10% Na2CO3), dried (MgSO4) was concentrated by distillation (380C at 750 mmHg) to give a black solution which was shown to be the triflate by 1H and 19F NMR. This was added to a solution in which potassium fertbutoxide (1 M (THF), 19.4 mmol, 19.4 ml) had been added to glycine imine ethyl ester (17.6 mmol, 4.7Og, 1 eq.) dissolved in DMF (25 ml) and stirred for 20 minutes at O0C. The resulting mixture was stirred at room temperature for 5 hours at room temperature then poured into a diethyl ether : saturated ammonium chloride solution mixture. The organics were washed (water then brine), dried (MgSO4) and concentrated in vacuo to give a brown oil. This was purified by silica chromatography (1% EtOAc in isohexane → 33% EtOAc in isohexane) to give a yellow solid (0.61g, 9% (4 steps)).
1H NMR (400 MHz) (δ, CDCI3) 7.14 - 7.69 (m, 10H), 4.36 - 4.41 (m, 1H), 4.13 - 4.21 (m, 2H), 2.58 - 2.68 (m, 1 H), 2.21 - 2.34 (m, 1 H), 1.45 - 2.00 (m, 8H), 1.22 - 1.30 (m, 3H).
19F NMR (376 MHz) (δ, CDCI3) -144.19 - -144.67 (m)
CIMS m/z (%) 368 (MH+, 100).
N-(FluorenylmethoxycarbonylMfluoro)cvclopentyl alanine
Figure imgf000047_0001
2MNaOH (aq), 3.30 mmol, 1.65 ml, 2 eq.) was added to a solution of 2-(benzhydrylidene- amino)-3-(1-fluoro-cyclopentyl)-propionic acid ethyl ester (1.65 mmol, 0.608g) in dioxane at O0C. The mixture was allowed to warm to room temperature then stirred overnight. 1M HCI (aq) was then added to pH 0.5 and the mixture was stirred for 6 hours then extracted with diethyl ether. The aqueous phase was neutralised with 1 M NaOH(aq) solution. Dioxane (5 ml) then 10%Na- 2CO3 (aq), 4.12 mmol, 4.25 ml, 2.5 eq.) were added and the mixture was cooled in an ice bath. Fluorenylmethylchloroformate (1.65 mmol, 0.43g, 1 eq.) was then added portionwise with stirring over 30 minutes. The ice bath was removed and the mixture stirred overnight and was acidifed to pH 3 with 1M HCI(aq). The aqueous layer was evaporated and the residual solid washed with EtOAc. The organics were concentrated in vacuo to give the product as a cream solid (0.54g, 81%).
1H NMR (400 MHz) (δ, CDCI3) 7.25 - 7.81 (m, 8H), 4.31 - 4.42 (m, 2H), 4.19 - 4.24 (m, 1H), 2.32 -2.43 (m, 1 H), 1.54 - 2.12 (m, 9H).
19F NMR (376 MHz) (δ, CDCI3) -145.25 - -145.74 (m)
CIMS m/z 378 (MH+, 45%) Although this example has been illustrated with an Fmoc protecting group it will be apparent that other conventional N-protecting groups such as those described above in Greene, including Boc, CBz etc, will be amenable to this route and/or the Fmoc group can be removed and replaced with another N-protecting group using conventional protecting group manipulations. As with other non-natural amino acids, the L and D diastereomers are separated by conventional chiral HPLC, or as described in Advanced Organic Chemistry: 3rd Edition: author J March, pp 104-107 including for example the formation of diastereomeric derivatives having convenient optically active auxiliary species followed by separation and then cleavage of the auxiliary species.
Example 9
Preparation of 1-fluoromethylcvclohexylalanine P2 building block
Figure imgf000048_0001
Figure imgf000049_0001
2
(iii)
Figure imgf000049_0002
>=^co2a h (Vi)
Figure imgf000049_0003
(i) NaOEt, EtOH, 80°C, (ii) LiAI(O1Bu)3H, THF, reflux, (iii) Deoxofluor, (iv) LiAIH4, THF O0C, (v) Tf2O, Et3N, DCM (vi) KO1Bu, DMF, (vii) NaOH (aq, 1 ,4-dioxan, (viii) HCI(aq), (ix) FmocCI Na2CO3, 1 ,4-dioxan
Cyclohexane-1,1-dicarboxylic acid diethyl ester,1 , was prepared in accordance with JACS 43, 1921, 1368 from diethyl malonate and 1 ,5-dibromopentane.
Cyclohexane-1,1-dicarboxylic acid diethyl ester, 1 , was taken up in anhydrous THF under nitrogen at room temperature. This was treated with LiAI(O1Bu)3H (2.5eq) portionwise before refluxing overnight. The reaction mixture was cooled in an ice-bath and treated carefully with 10% KHSO4(aq) and allowed to stir for 10 minutes. The resulting precipitate was removed by vacuum filtration and the mother liquors were partitioned between EtOAc and brine. The organic phases were combined, dried over MgS04, filtered and concentrated in vacuo to give a mobile oil. This was purified by flash column chromatography to give 1-hydroxymethyl- cyclohexanecarboxylic acid ethyl ester, 2 as a colourless oil (51%). 1-Hydroxymethyl-cyclohexanecarboxylic acid ethyl ester, 2 was taken up in [bis (2- methoxyethyl) amino] sulphur trifluoride and heated overnight at 7O0C. The reaction was then allowed to cool to O0C and carefully treated with saturated NaHCO3 (aq) dropwise. This was stirred at room temperature for 30 minutes. The mixture was washed twice with DCM and the combined organics were washed with saturated NaHCO3 (aq) and brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by flash column chromatography to give 1 -fluoromethyl-cyclohexane carboxylic acid ethyl ester, 3, as a colourless oil (34%)
1-Fluoromethyl-cyclohexane carboxylic acid ethyl ester, 3, was dissolved in anhydrous THF and cooled under a nitrogen atmosphere to O0C. This was treated portionwise with LiAIH4 (2eq) and warmed to room temperature for 4 hours. After this time the reaction was cooled to O0C and carefully treated with 2M HCI(aq) and stirred for 20 minutes. The reaction was filtered through a pad of celite and the pad was washed with diethyl ether. The collected solution was partitioned between brine and diethyl ether. The combined organic phases were dried over MgSO4, filtered and concentrated in vacuo to give an oil. This was purified by flash chromatography to give (1- fluoromethyl-cyclohexyl) methanol, 4, as a colourless oil (67%).
(1 -Fluoromethyl-cyclohexyl) methanol, 4, was dissolved in anhydrous DCM under N2 and cooled to -2O0C. NEt3 (1.1eq) was added and the reaction was stirred for 5 minutes. This was then treated dropwise with triflic anhydride (1 1eq) and the solution was stirred at O0C for 1hour. The reaction mixture was poured onto ice and the organics were washed with 1M HCI (aq), saturated NaHCO3 (aq) and brine, then dried over MgSO4, filtered and concentrated in vacuo, to give trifluoro-methanesulfonic acid-1-fluoromethyl-cyclohexylmethyl ester, 5, as an amber oil which was used immediately without further purification in the next step.
N-(Diphenylmethylene)glycine ethyl ester was dissolved in DMF and under a nitrogen atmosphere was cooled to O0C. This was treated with KO'Bu (1.1eq) and stirred for 20 minutes. To this solution was added trifluoro-methanesulfonic acid-1-fluoromethyl-cyclohexylmethyl ester, 5 dropwise. The reaction mixture was stirred at room temperature under nitrogen overnight then poured into a 1 :1 mixture of diethyl ether : NH4CI (aq). The phases were separated and the aqueous phase was washed twice with diethyl ether. The organic phases were combined and washed several times with brine, dried over MgSO4, filtered and concentrated in vacuo. The resulting residue was purified by flash column chromatography to give 2-(benzhydrylidene- amino)-3-(1 fluoromethyl-cyclohexyl) propionic acid ethyl ester, 6 (28%).
2- (Benzhydrylidene-amino)-3-(1-fluoromethyl-cyclohexyl)propionic acid ethyl ester, 6, was taken up in 1,4-dioxan and treated with 2 M NaOH (aq) (2eq) with stirring. After the starting material had been consumed (tic), the reaction mixture was acidified by addition of 2M HCI (aq) and stirred overnight at room temperature. The solution was concentrated in vacuo and the residue was partitioned between TBME and water. The pH of the aqueous phase was adjusted to p H 7 by careful addition of 2M NaOH (aq) prior to lyophilisation. The resulting residue was then suspended in 10% Na2CO3 (aq) and dioxan until a homogeneous solution was obtained. Fmoc chloride was added portion wise to the ice-cooled solution over 12 hrs and this was allowed to stir at room temperature overnight. The reaction mixture was washed with TBME and the resulting aqueous was acidified with 2M HCI (aq) and allowed to freeze dry. The resulting solid was triturated with methanol and the mother liquors were collected by filtration and concentrated in vacuo. To remove last traces of salts the resulting solids were partitioned between water and ethyl acetate. The organics were dried over MgSO4, filtered and concentrated in vacuo to give 2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-(10fluoromethyl- cyclohexyl)-propionic acid ethyl ester, compound 7.
HPLC retention time β.OOmin (monitored at 215 and 254 nm) HPLC using Synergy Max RP 80 μm 50x4.6mm column, 10→90% 6 min gradient of solution B (solution A = 0.1 % TFA in water and solution B = 10% A in acetonitrile) at flow rate of 2ml/min.
MS [M+H]+ 426.26 [M=Na]+ 449.25
Electrospray ionisation, eluting with acetonitrile / ammonium formate buffer.
Although this example has been illustrated with a Boc protecting group it will be apparent that other conventional N-protecting groups such as those described above in Greene, such as Fmoc, CBz etc will be amenable to this route and/or the Boc group can be removed and replaced with a conventional N-protecting group using conventional protecting group manipulations. The diastereomers are isolated by conventional amino acid chiral HPLC.
Example 10
Preparation of 1 -methylcvcloheptylalanine P2 Building Block
Figure imgf000051_0001
Scheme 4 above provides an effective route to this building block as follows: Step a)
Figure imgf000052_0001
Potassium fert-butoxide (10.5 g, 0.09 mol) was suspended in anhydrous THF (200 ml) and the solution was cooled in an ice-bath. Ethyl phosphonoacetate (17.6 ml, 0.09 mol) was dissolved in anhydrous THF (30 ml) and added dropwise to the cooled solution. After the addition was complete, cycloheptanone (10 g, 0.09 mol) was dissolved in anhydrous THF (40 ml) and added dropwise to the ylide. The reaction was allowed to reach room temperature and stirring was continued overnight. The reaction was concentrated in vacuo and the residue was taken up in diethyl ether and extracted with water and saturated brine. The organic fraction was then dried (Na2SO4), filtered and concentrated. Purification on silica gel eluting with heptane only gave the product as a clear oil (9 g, 55 %).
Step b.
Figure imgf000052_0002
As reference: Tetrahedron Lett. 2003, 44, 4265.
Copper (I) iodide (2.090 g, 10.98 mmol) was suspended in dry diethyl ether (5 ml) and stirred at 0 0C under argon. A 1.4M solution of methyl lithium in diethyl ether (15.7ml, 21.96 mmol) was slowly added until the initial yellow colour turned white. The reaction was stirred for a further 10 min before the solvent was thoroughly removed in vacuo at O0C. Ice-cooled dry dichloromethane (30 ml) was added to the residue under argon and this solution was then further cooled to -780C. Ice-cooled trimethylsilyl chloride (1.38 ml, 10.98 mmol) and cycloheptylidene-acetic acid ethyl ester (1 g, 5.49 mmol) were added. The solution was stirred at -780C for a further 3 hrs and allowed to warm-up to O0C prior to being quenched with saturated ammonium chloride solution and ammonium hydroxide (1 :1). The organic layer was separated, dried (Na2SO4) and the solvent was removed in vacuo to afford the product (856 mg, 79 %), without need for further purification.
Step c.
Figure imgf000053_0001
(i-Methyl-cycloheptyl)-acetic acid ethyl ester (500 rng, 2.53 mmol) was slowly added to a stirring 1 M solution of lithium aluminium hydride in tetrahydrofuran (5.1 ml) at room temperature. The solution was heated to reflux for 2hrs until complete by TLC. 1M NaOH (aq) was then added dropwise to the ice-cooled solution, until the salts precipitated. The solution was filtered through celite and extracted with ethyl acetate. The combined organics were dried (MgSO4) and the solvent was removed in vacuo to afford the product (352 mg, 89 %), without need for further purification.
Step d.
Figure imgf000053_0002
2-(1-Methyl-cycloheptyl)-ethanol (352 mg, 2.26 mmol) was dissolved in dry dichloromethane and stirred at room temperature while Dess-Martin periodinane (959 mg, 2.26 mmol) was added. The solution was stirred for a further 4 hrs when TLC indicated the reaction had gone to completion. Then 10 % sodium thiosulphate solution and saturated sodium bicarbonate solution (1 :1) were added and the solution was stirred at room temperature for 15 mins. After filtration, the organic layer was separated, dried (MgSO4) and the solvent was removed in vacuo to afford the product (340 mg, 98 %). This material was used immediately in the next step.
Step e.
Figure imgf000053_0003
Freshly prepared (1 -methyl-cycloheptyO-acetaldehyde (1.980 g, 12.86 mmol) was dissolved in ethanol (25 ml) and water (25 ml) and stirred at room temperature while potassium cyanide (921 mg, 14.14 mmol) and ammonium carbonate (3.331 g, 34.67 mmol) were added. The solution was then heated to 6O0C for 24 hrs. The next day, the ethanol was removed in vacuo to aid the precipitation of the hydantoin intermediate, 5-(1-methyl-cycloheptylmethyl)-imidazolidine-2,4- dione, which was subsequently removed by filtration and dried in vacuo, (1.919 g, 67 %).
1H-NMR (400 MHz, DMSO) δ 10.60 (br.s, 1 H), 7.80 (br.s, 1 H), 4.00-3.90 (m, 1 H), 1.80-1.20 (m, 14H), 0.90 (s, 3H).
5-(1-Methyl-cycloheptylmethyl)-imidazolidine-2,4-dione (1.919 g, 8.57 mmol) was dissolved in 0.7 M sodium hydroxide solution (40 ml) and stirred at 1000C overnight. The following day, the solution was cooled to room temperature and the volume was reduced in vacuo by half. The di- fert-butyl dicarbonate (2.054 g, 9.42 mmol) in tetrahydrofuran (25 ml) was added and the solution was stirred at room temperature for 48 hrs. The solution was then acidified to pH 3 using 1 M potassium hydrogen sulphate solution and extracted with ethyl acetate. The combined organics were dried (MgSO4) and the solvent was removed in vacuo. Purification by column chromatography (isohexane: ethyl acetate; 1 :1) afforded the product.
Although the example has been illustrated with a Boc protecting group it will be apparent that other conventional N protecting groups such as those described above in Greene will be amenable to this route and/or the Boc group can be removed and replaced by another conventional N-protecting group such as Fmoc or CBz using conventional protecting group manipulation.
Example 11
Solution phase preparation of inhibitors
The compounds in the table immediately below were prepared analogously to the method disclosed in Example 13 using the appropriate commercially available P3 acid, and the respective P2 building block of Example 6, 7 or 10:
Example 11.1
Furan-3-carboxylic acid [(1 ft,S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1 ■ methyl-cycloheptyl)-ethyl]-amide Data is given for a mixture of diastereoisomers (1 :1 ratio).
HPLC retention time of 5.67 min.
1H-NMR (400 MHz, CDCI3) δ 8.18 (br.d, J = 6.0,
0.5H), 7.98-7.96 (m, 0.5H), 7.92-7.91 (m, 0.5H),
Figure imgf000055_0001
7.71 (br.d, J = 8.0, 0.5H), 7.61 (br.d, J = 7.5, 0.5H),
7.40 (app.t, J = 1.5, 0.5H), 7.38 (app.t, J = 1.5,
0.5H), 7.30 (br.d, J = 8.0, 0.5H), 6.69-6.67 (m,
0.5H), 6.65-6.63 (m, 0.5H), 4.80-4.70 (m, 1 H), 4.20-
3.80 (m, 4H), 1.90-1.64 (m, 4H), 1.50-1.25 (m,
12H), 1.02 (t, J = 7.0, 1.5H), 0.97 (t, J = 7.0, 1.5H),
0.91 (s, 1.5H), 0.89 (s, 1.5H).
Mass spectroscopy: m/z 810 (15, 2MH+), 427 (20,
MNa+), 405 (100, MH+).
Example 11.2
Morpholine-4-carboxylic acid [(1 f?,S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)- 2-(1-methyl-cycloheptyl)-ethyl]-amide
Data is given for a mixture of diastereoisomers (1 :1 ratio).
1H-NMR (400 MHz, CDCI3) δ 7.15 (br.d, J = 6.0,
1 H), 4.73 (br.d, J = 8.0, 1H), 4.36-4.26 (m, 1H),
4.16-3.80 (m, 4H), 3.66-3.58 (m, 4H), 3.35-3.22 (m,
Figure imgf000055_0002
4H), 1.93-1.65 (m, 4H), 1.50-1.28 (m, 12H), 0.96 (t,
J = 7.0, 3H), 0.84 (s, 3H).
Mass spectroscopy: m/z 424 (100, MH+).
Example 11.3
Furan-3-carboxylic acid [1 -(R,S)-(2-(S)-ethyl-4-oxo-tetrahydro-furan-3-(S)-ylcarbamoyl)-2- (1-fluoro-cyclopentyl)-ethyl]-amide
Figure imgf000056_0001
Example 12
Preparation of 1-Hydroxycvclopentyl-(D,L)-alanine P2 Building block
Step a) 1 -(1 -hydroxy-cyclopropyljethanoic acid, ethyl ester
Figure imgf000056_0002
See scheme 5 in conjunction with Tetrahedron, 1994, 50, 11709. Preparation of the Zn-Cu couple : To a vigorously stirring and hot (nearly refluxing) solution of copper(ll) acetate (750 mg, 4.13 mmol) in glacial acetic acid (100 ml) was added granulated zinc (7.5 g, 114.73 mmol). The solution was stirred for 10 min. The solution was then allowed to cool to room temperature and decanted. The residue was washed with diethyl ether (6 x 50 ml) and the washes were repeatedly decanted off.
Experimental: The freshly prepared Zn-Cu couple was suspended in dry diethyl ether (50 ml) and vigorously stirred at room temperature. Ethylbromoacetate (7.08 ml, 63.85 mmol) was very carefully added until the reaction was initiated (this may take a little gentle heating). Once this careful addition had been completed the process was repeated for the addition of cyclopentanone (4.51 ml, 51.06 mmol). The mixture was then heated to 4O0C for 12 hrs. The cooled solution was acidified with 5M sulphuric acid solution and extracted with diethyl ether. The combined organics were dried (Na2SO4) and the solvent was removed in vacuo. Purification by column chromatography (isohexane: ethyl acetate; 10:1) afforded the product.
Step b)
Figure imgf000057_0001
The title product is prepared analogously to Example 10, step c) from 1-(1-hydroxy- cyclopropyl)ethanoic acid, ethyl ester
Step c)
Figure imgf000057_0002
The title product is prepared analogously to Example 10 step d) from 1 -(1 -hydroxy- cyclopentyl)ethanol.
Step d)2-ferf-Butoxycarbonylamino-(3f?,S)-(1 -hydroxy-cyclopentyl)-propionic acid
Figure imgf000058_0001
The title compound is prepared from the material of step c) above using the same procedure as Example 10, step e).
Although this example has been illustrated with a Boc protecting group it will be apparent that other conventional N-protecting groups such as those described above in Greene, such as Fmoc, CBz etc will be amenable to this route and/or the Boc group can be removed and replaced with a conventional N-protecting group using conventional protecting group manipulations. As is conventional with non-natural amino acids, the D and L diasteromers are isolated by chiral HPLC.
Example 13
Preparation on solid phase
Immobilisation of a P1 buliding block, such as those prepared in WO05/82876, onto a resin via Murphy's linker proceeds as described in Scheme 7 of WO00/69855 and its accompanying text. The Fmoc-protected 5-substituted furan-4-amine is de-protected, extended with the P2 building block of the invention, such as those described at examples 1 and 5, using conventional peptide activation and coupling reagents such as HOBt/HBTU/DMF, as described in WO00/69855.
Method A: General Synthesis of P3 amides using coupling on solid phase
Figure imgf000058_0002
Method B: Synthesis of phenol P3s using solid phase coupling
(includes a hydrazine wash) Et
Figure imgf000059_0001
After coupling, the resin was suspended in a 5% solution of hydrazine in DMF for 1 h. The mixture was filtered, and the resin washed with DMF. The hydrazine treatment and DMF wash was then repeated.
(Resin cleaved as standard)
Method C: Synthesis of aniline P3s using solid phase coupling
(includes a piperidine treatment to remove FMOC protecting groups)
in DMF
Figure imgf000059_0002
Figure imgf000059_0003
After coupling, the resin was suspended in a 20% solution of piperidine in DMF for 0.5h. The mixture was filtered, and the resin washed with DMF. The piperidine treatment and DMF wash was then repeated.
(Resin cleaved as standard)
Method D: Alternative synthesis of aniline P3s.
Method D
Figure imgf000059_0004
DMF
Figure imgf000059_0005
After coupling of 4-FMOC-aminobenzoic acid, the resin was suspended in a 20% solution of piperidine in DMF for 0.5h. The mixture was filtered, and the resin washed with DMF. The piperidine treatment and DMF wash was then repeated.
The resin was suspended in a solution of benzyl chloroformate and Λ/-methyl morpholine in DMF, filtered and the residue washed with 1 :1 water: DMF, DMF, THF, DCM and MTBE.
(Resin cleaved as standard)
Method E: Alternative synthesis of anilines
Method E
Figure imgf000060_0001
4. T TFFAA:: H2O
Figure imgf000060_0003
Figure imgf000060_0002
After coupling of 4-FMOC-aminobenzoic acid, the resin was suspended in a 20% solution of piperidine in DMF for 0.5h. The mixture was filtered, and the resin washed with DMF. The piperidine treatment and DMF wash was then repeated.
The resin was suspended in a solution of benzylaldehyde in DMF, and a solution of dibutyltin dichloride in THF was added. After 10 minutes, phenyl silane was added and the mixture was shaken overnight. The mixture was filtered and the residue washed with DMF, THF, DCM and MTBE.
(Resin cleaved as standard)
Method F: General Synthesis of P3 sulfonamides on solid phase
Figure imgf000061_0001
To a suspension of P2-P1 on resin in DMF, was added diisopropylethylamine and sulfonyl chloride. After 16h, the mixture was filtered and resin was washed with DMF, THF, DCM and MTBE.
(Resin cleaved as standard)
Although methods A-F have been illustrated with methyl or ethyl as R1, and 1-methyl- cyclopentyl-L-Ala as P2, it will be apparent that corresponding methodology, in conjunction with conventional protection of hydroxyl groups, will be applicable to other P1 and P2 building blocks. Similarly, methods A-F are not limited to the specified classes of P3, but are widely applicable to other species of R3, optionally in conjunction with conventional protection of amine, hydroxyl and carboxyl groups.
The synthesis of P3 building blocks which were not commercially available is described at the foot of the table.
Example 14.1
N-[ (1 S) -((2S)-Ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)- ethyl]-4-hydroxy-2-methyl-benzamide
By following the procedure described in Method B, the title compound was prepared in 45% yield from P2-P1 on resin and 4-hydroxy-2-methyl-benzoic acid
MS/ES: m/z 417 (100%, MH+)
Figure imgf000061_0002
Example 14.2
3-Ethyl-Λ/-[(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbannoyl)-2-(1-nnethyl- cyclopentyl)-ethyl]-4-hydroxy-benzamide
By following the procedure described in Method B, the title compound was prepared in 44% yield from P2-P1 on resin and 2-ethyl-4-hydroxy-benzoic acid.
MS/ES: m/z 431 (100%, MH+)
Figure imgf000062_0001
Example 14.3
4-Difluoromethyl-Λ/-[(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl- cyclo pe ntyl )-ethyl] -be nza m ide
By following the procedure described in Method B, the title compound was prepared in 38% yield from P2-P1 on resin and 4-difluoromethyl-benzoic acid.
MS/ES: m/z 437 (100%, MH+)
Figure imgf000062_0002
Example 14.4
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)- ethyl]-4-hydroxy-3-propyl-benzamide
By following the procedure described in Method B, the title compound was prepared in 35% yield from P2-P1 on resin and 4-hydroxy-3-propyl-benzoic acid. MS/ES: m/z 445 (100%, MH+)
Figure imgf000063_0001
Example 14.5
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbannoyl)-2-(1-nnethyl-cyclopentyl)- ethyl]-4-hydroxy-3-isopropyl-benzamide
By following the procedure described in Method B, the title compound was prepared in 42% yield from P2-P1 on resin and 4-hydroxy-3-isopropyl-benzoic acid.
MS/ES: m/z 445 (100%, MH+)
Figure imgf000063_0002
Example 14.6
3-fert-Butyl-Λ/-[(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl- cyclopentyl)-ethyl]-4-hydroxy-benzamide
By following the procedure described in Method B, the title compound was prepared in 11% yield from P2-P1 on resin and 3-fert-butyl-4-hydroxy-benzoic acid.
MS/ES: m/z 459 (90%, MH+), 381 (100%), 330 (42%, M-C6H10NO2)
Figure imgf000063_0003
Example 14.7
4-Ethylamino-Λ/-[(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1 -methyl- cyclo pe ntyl )-ethyl] -be nza m ide
By following the procedure described in Method C, the title compound was prepared in 41% yield from P2-P1 on resin and 4-[ethyl-(9H-fluoren-9-ylmethoxycarbonyl)-amino]- benzoic acid.
MS/ES: m/z 430 (100%, MH+)
Figure imgf000064_0001
Example 14.8
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)- ethyl]-3-fluoro-4-hydroxy-5-methyl-benzamide
By following the procedure described in Method B, the title compound was prepared in 23% yield from P2-P1 on resin and 3-fluoro-4-hydroxy-5-methylbenzoic acid.
MS/ES: m/z 435 (100%, MH+)
Figure imgf000064_0002
Example 14.9
5-Chloro-Λ/-[(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl- cyclopentyl)-ethyl]-6-hydroxy-nicotinamide
By following the procedure described in Method B, the title compound was prepared in 25% yield from P2-P1 on resin and 5-chloro-6-hydroxynicotinic acid. 26%
25%
Figure imgf000065_0001
37%
18%
16%
Figure imgf000066_0001
20%
20%
20%
Figure imgf000067_0001
20%
20%
16%
Figure imgf000068_0001
16%
12%
Figure imgf000069_0001
Example 14.23
4-Benzenesulfonylamino-Λ/-[(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbannoyl)-2- (1-methyl-cyclobutyl)-ethyl]-benzamide
By following the procedure described in Method A, the title compound was prepared in 19% yield from P2-P1 on resin and 4-[(phenylsulfonyl)amino]benzoic acid.
MS/ES: m/z 528 (100%, MH+)
Figure imgf000070_0001
Example 14.24
2-Amino-benzothiazole-6-carboxylic acid [(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)- ylcarbamoyl)-2-(1-methyl-cyclobutyl)-ethyl]-amide
By following the procedure described in Method A, the title compound was prepared in 21% yield from P2-P1 on resin and 2-[(tert-butoxycarbonyl)amino]-1,3-benzothiazole-6- car boxy lie acid.
1H-NMR (400 MHz, CD3CN) δ 8.18-8.19 (m, 1 H), 7.82-7.85 (m, 1H), 7.46-7.48 (m, 1 H), 7.30-7.32 (m, 1H), 7.14-7.16 (m, 1 H), 4.58-
Figure imgf000070_0002
4.64 (m, 1 H), 3.95-4.15 (m, 3H), 3.85-3.89 (m, 1 H), 3.60 (s, 3H), 1.88-2.06 (m, 4H), 1.60-1.86 (m, 6H), 1.21 (3H, s), 0.99 (t, J=7.4, 3H) MS/ES: m/z 445 (87 %, MH+)
Example 14.25
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclobutyl)-ethyl]- 4-methanesulfonylamino-3-nnethyl-benzannicle
By following the procedure described in Method A, the title compound was prepared in 24% yield from P2-P1 on resin and 3-methyl-4-[(methylsulfonyl)amino]benzoic acid.
MS/ES: m/z 480 (100%, MH+)
Figure imgf000071_0001
Example 14 26
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclobutyl)-ethyl]- 4-(propane-2-sulfonylamino)-benzamide
By following the procedure described in Method A, the title compound was prepared in 23% yield from P2-P1 on resin and 4-[(isopropylsulfonyl)amino]benzoic acid.
MS/ES: m/z 494 (100%, MH+)
Figure imgf000071_0002
Example 14.27
5-Hydroxy-pyridine-2-carboxylic acid [(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)- ylcarbamoyl)-2-(1-methyl-cyclopentyl)-ethyl]-amide
By following the procedure described in Method B, the title compound was prepared in 44% yield from P2-P1 on resin and 5-hydroxypyridine-2-carboxylic acid. MS/ES: m/z ΛOΛ (100%, MH+)
Figure imgf000072_0001
Example 14.28
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbannoyl)-2-(1-nnethyl-cyclopentyl)- ethyl]-2-hydroxy-isonicotinamide
By following the procedure described in Method B, the title compound was prepared in 41% yield from P2-P1 on resin and 2-hydroxyisonicotinic acid.
MS/ES: m/z 404 (100%, MH+)
Figure imgf000072_0002
Example 14.29
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)- ethyl]-2-hydroxy-6-methyl-isonicotinamide
By following the procedure described in Method B, the title compound was prepared in 37% yield from P2-P1 on resin and 2-hydroxy-6-methylisonicotinic acid.
MS/ES: m/z 418 (100%, MH+)
Figure imgf000072_0003
Example 14.30
2-Oxo-2,3-dihydro-1 H-benzoimidazole-5-carboxylic acid [(1 S)-((2S)-ethyl-4-oxo-tetrahydro- furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)-ethyl]-annicle
By following the procedure described in Method A, the title compound was prepared in 39% yield from P2-P1 on resin and 2-oxo-2,3-dihydro-1H-benzimidazole-5-carboxylic acid.
MS/ES: m/z 443 (100%, MH+)
Figure imgf000073_0001
Example 14.31
4-(Benzenesulfonyl-methyl-amino)-Λ/-[(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)- ylcarbamoyl)-2-(1-methyl-cyclopentyl)-ethyl]-benzamide
By following the procedure described in Method A, the title compound was prepared in 49% yield from P2-P1 on resin and lithium 4-[methyl(phenylsulfonyl)amino]benzoate.
MS/ES: m/z 556 (100%, MH+)
Figure imgf000073_0002
Example 14.32
4-Acetylamino-Λ/-[(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl- cyclopentyl)-ethyl]-benzamide
By following the procedure described in Method A, the title compound was prepared in 48% yield from P2-P1 on resin and 4-(acetylamino)benzoic acid. MS/ES: m/z 444 (100%, MH+)
Figure imgf000074_0001
Example 14.33
2-Oxo-i ^S^-tetrahydro-quinazoline-θ-carboxylic acid [(1 S)-((2S)-ethyl-4-oxo-tetrahydro- furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)-ethyl]-annide
By following the procedure described in Method A, the title compound was prepared in 35% yield from P2-P1 on resin and 2-oxo-1,2,3,4-tetrahydroquinazoline-6-carboxylic acid.
MS/ES: m/z 457 (100%, MH+)
Figure imgf000074_0002
Example 14.34
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)- ethyl]-4-thioureido-benzamide
By following the procedure described in Method A, the title compound was prepared in 18% yield from P2-P1 on resin and 4-[(aminocarbonothioyl)amino]benzoic acid.
MS/ES: m/z 461 (100%, MH+)
Figure imgf000074_0003
Example 14.35
2-Benzylamino-thiazole-5-carboxylic acid [(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)- ylcarbamoyl)-2-(1-methyl-cyclopentyl)-ethyl]-amide
By following the procedure described in Method A, the title compound was prepared in 39% yield from P2-P1 on resin and 2-(benzylamino)-1,3-thiazole-5-carboxylic acid.
MS/ES: m/z 499 (100%, MH+)
Figure imgf000075_0001
Example 14.36
4-Amino-3-chloro-Λ/-[(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1- methyl-cyclopentyl)-ethyl]-benzamide
By following the procedure described in Method A, the title compound was prepared in 10% yield from P2-P1 on resin and 4-amino-3-chlorobenzoic acid.
MS/ES: m/z 436 (100%, MH+)
Figure imgf000075_0002
Example 14.37
Thiophene-2,5-dicarboxylic acid 2-amide 5-{[(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)- ylcarbamoyl)-2-(1-methyl-cyclopentyl)-ethyl]-amide}
By following the procedure described in Method A, the title compound was prepared in 33% yield from P2-P1 on resin and 5-(aminocarbonyl)thiophene-2-carboxylic acid.
Figure imgf000076_0001
Figure imgf000077_0001
28% yield from P2-P1 on resin and 4-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-3- methyl benzoic acid.
MS/ES: m/z 416 (100%, MH+)
Figure imgf000078_0001
Example 14.43
4-Amino-Λ/-[(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl- cyclopentyl)-ethyl]-3-methoxy-benzamide
By following the procedure described in Method C, the title compound was prepared in 9% yield from P2-P1 on resin and 4-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-3- methoxybenzoic acid.
MS/ES: m/z 432 (100%, MH+)
Figure imgf000078_0002
Example 14.44
{4-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)- ethylcarbamoyl]-phenyl}-carbamic acid benzyl ester
By following the procedure described in Method D, the title compound was prepared in 5% yield from P2-P1 on resin, 4-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-benzoic acid and benzyl chloroformate.
Figure imgf000079_0001
MS/ES: m/z 363 (100%, MH+)
Figure imgf000080_0001
Example 14.46
Thiophene-3-carboxylic acid [2-(1-methyl-cyclopentyl)- (1 S)-((2S)-methyl-4-oxo-tetrahydro- furan-(3S)-ylcarbamoyl)-ethyl]-amide
By following the procedure described in Method A, the title compound was prepared in 59% yield from P2-P1 on resin and thiophene-3-carboxylic acid.
MS/ES: m/z 379 (100%, MH+)
Figure imgf000080_0002
Example 14.47
4-Hydroxy-3-methyl-Λ/-[2-(1 -methyl-cyclopentyl)- (1 S)-((2S)-methyl-4-oxo-tetrahydro-furan- (3S)-ylcarbamoyl)-ethyl]-benzamide
By following the procedure described in Method B, the title compound was prepared in 64% yield from P2-P1 on resin and 4-hydroxy-3-methylbenzoic acid.
MS/ES: m/z 403 (100%, MH+)
Figure imgf000080_0003
Example 14.48
3-Fluoro-4-hydroxy-Λ/-[2-(1 -methyl-cyclopentyl)- (1 S)-((2S)-methyl-4-oxo-tetrahydro-furan- (3S)-ylcarbamoyl)-ethyl]-benzamide
By following the procedure described in Method B, the title compound was prepared in 67% yield from P2-P1 on resin and 3-fluoro-4-hydroxybenzoic acid.
1H-NMR (400 MHz, CD3CN) δ 7.61-7.64 (m, 1 H), 7.54-7.57 (m, 1H), 7.11-7.16 (m, 2H), 7.02-7.07 (m, 1H), 4.58-4.63 (m, 1 H), 3.96-
Figure imgf000081_0001
4.14 (m, 3H), 3.74-3.79 (m, 1H), 1.99-2.04 (m, 1 H), 1.75-1.81 (m, 1H), 1.61-1.67 (m, 4H), 1.41-1.50 (m, 3H), 1.32-1.38 (m, 4H), 0.99 (s, 3H) MS/ES: m/z 407 (100%, MH+)
Example 14.49
4-Benzenesulfonylamino-Λ/-[2-(1 -methyl-cyclopentyl)- (1 S)-((2S)-methyl-4-oxo-tetrahydro- furan-(3S)-ylcarbamoyl)-ethyl]-benzamide
By following the procedure described in Method A, the title compound was prepared in 76% yield from P2-P1 on resin and 4-[(phenylsulfonyl)amino]benzoic acid.
MS/ES: m/z 528 (100%, MH+)
Figure imgf000081_0002
Example 14.50
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1 -methyl-cyclopentyl )- ethyl]-benzamide
By following the procedure described in Method A, the title compound was prepared in 55% yield from P2-P1 on resin and benzoic acid.
Figure imgf000082_0001
Figure imgf000083_0001
MS/ES: m/z 446 (100%, MH+)
Figure imgf000084_0001
Example 14.55
5-Methyl-4-morpholin-4-ylmethyl-furan-2-carboxylic acid [(1 S)-((2S)-ethyl-4-oxo-tetrahydro- furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)-ethyl]-annide
By following the procedure described in Method A, the title compound was prepared in 56% yield from P2-P1 on resin and 5-methyl-4-(morpholin-4-ylmethyl)-2-furoic acid.
MS/ES: m/z 490 (100%, MH+)
Figure imgf000084_0002
Example 14.56
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)- ethyl]-4-hydroxy-3-morpholin-4-ylmethyl-benzamide
By following the procedure described in Method B, the title compound was prepared in 35% yield from P2-P1 on resin and 4-hydroxy-3-(morpholin-4-ylmethyl)benzoic acid.
MS/ES: m/z 502 (100%, MH+)
Figure imgf000084_0003
Example 14.57
4-Benzylamino-Λ/-[(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbannoyl)-2-(1 -methyl- cyclopentyl)-ethyl]-benzamide
By following the procedure described in Method E, the title compound was prepared in 27% yield from P2-P1 on resin, 4-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}benzoic acid and benzaldehyde.
MS/ES: m/z 492 (100%, MH+)
Figure imgf000085_0001
Example 14.58
Quinoline-6-carboxylic acid [(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1- methyl-cyclopentyl)-ethyl]-amide
By following the procedure described in Method A, the title compound was prepared in 54% yield from P2-P1 on resin and quinoline-6-carboxylic acid.
1H-NMR (400 MHz, CDCI3) δ 9.28 (s, 1 H), 8.77 (s, 1 H), 8.40 (br s,
H
L 1 H), 8.24 (br s, 1 H), 8.06 (s, 2H),
H
O V 7.83-7.88 (m, 2H), 4.85-4.89 (m,
N O 1H), 4.11-4.28 (m, 3H), 3.82 (t, 1 H, J=8.4), 1.80-1.98 (m, 4H), 1.64(s, 4H), 1.48-1.55 (m, 3H), 1.36-140 (m, 1H), 1.25 (s, 1 H), 1.11 (t, 3H, J = 7.4), 1.02 (s, 3H) MS/ES: m/z 438 (100%, MH+)
Example 14.59
1 ,2,3,4-Tetrahydro-quinoline-6-carboxylic acid [(1 S)-((2S)-ethyl-4-oxo-tetrahydro-furan- (3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)-ethyl]-annicle
By following the procedure described in Method C, the title compound was prepared in 49% yield from P2-P1 on resin and 1-[(9H-fluoren-9-ylmethoxy)carbonyl]-1 ,2,3,4- tetrahydroquinoline-6-carboxylic acid.
MS/ES: m/z 442 (100%, MH+)
Figure imgf000086_0001
Example 14.60
3-Methyl-2-oxo-1 ,2,3,4-tetrahydro-quinazoline-6-carboxylic acid [(1 S)-((2S)-ethyl-4-oxo- tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)-ethyl]-amide
By following the procedure described in Method A, the title compound was prepared in 37% yield from P2-P1 on resin and S-methyl^-oxo-I^S^-tetrahydroquinazoline-θ-carboxylic acid.
MS/ES: m/z 471 (100%, MH+)
Figure imgf000086_0002
Example 14.61
Λ/-[(1 S)-((2S)-Ethyl-4-oxo-tetrahydro-furan-(3S)-ylcarbamoyl)-2-(1-methyl-cyclopentyl)- ethyl]-4-(2,2,2-trifluoro-ethylamino)-benzamide
By following the procedure described in Method A, the title compound was prepared in 51% yield from P2-P1 on resin and 4-[(2,2,2-trifluoroethyl)amino]benzoic acid.
Figure imgf000087_0001
ethyl]-benzamide
By following the procedure described in Method A, the title compound was prepared in 31% yield from P2-P1 on resin and benzoic acid.
MS/ES: m/z 373 (100% ,MH+)
Figure imgf000088_0001
Example 14.65
4-Hydroxy-Λ/-[2-(1 -methyl-cyclopentyl)- (1 S)-((2S)-methyl-4-oxo-tetrahydro-furan-(3S)- ylcarba moyl )-ethyl] -be nza m ide
By following the procedure described in Method B, the title compound was prepared in 56% yield from P2-P1 on resin and 4-hydroxybenzoic acid.
MS/ES: m/z 389 (100%, MH+)
Figure imgf000088_0002
Example 14.66
3-Chloro-4-hydroxy-Λ/-[2-(1 -methyl-cyclopentyl)- (1 S)-((2S)-methyl-4-oxo-tetrahydro-furan- (3 S)-ylcarba moyl )-ethyl] -be nza m ide
By following the procedure described in Method B, the title compound was prepared in 59% yield from P2-P1 on resin and 3-chloro-4-hydroxybenzoic acid.
Figure imgf000089_0001
ylcarbamoyl)-ethyl]-3-propyl-benzamide
By following the procedure described in Method B, the title compound was prepared in 29% yield from P2-P1 on resin and 4-hydroxy-3-propylbenzoic acid.
MS/ES: m/z 431 (100%, MH+)
Figure imgf000090_0001
Example 14.70
3-Dimethylamino-4-hydroxy-Λ/-[2-(1 -methyl-cyclopentyl)- (1 S)-((2S)-methyl-4-oxo- tetrahydro-furan-(3S)-ylcarbamoyl)-ethyl]-benzamide
By following the procedure described in Method B, the title compound was prepared in 34% yield from P2-P1 on resin and 3-dimethylamino-4-hydroxybenzoic acid.
MS/ES: m/z 432 (100%, MH+)
Figure imgf000090_0002
Example 14.71
3-Dimethylaminomethyl-4-hydroxy-Λ/-[2-(1 -methyl-cyclopentyl)- (1 S)-((2S)-methyl-4-oxo- tetrahydro-furan-(3S)-ylcarbamoyl)-ethyl]-benzamide
By following the procedure described in Method B, the title compound was prepared in 27% yield from P2-P1 on resin and 3-dimethylaminomethyl-4-hydroxy-benzoic acid. 17%
30%
Figure imgf000091_0001
ylcarbamoyl)-ethyl]-3-morpholin-4-ylmethyl-benzamide
By following the procedure described in Method B, the title compound was prepared in 39% yield from P2-P1 on resin and 4-hydroxy-3-(morpholin-4-ylmethyl)benzoic acid.
MS/ES: m/z 488 (100%, MH+)
Figure imgf000092_0001
Example 14.76
Thiophene-2-carboxylic acid [2-(1-methyl-cyclopentyl)- (1 S)-((2S)-methyl-4-oxo-tetrahydro- furan-(3S)-ylcarbamoyl)-ethyl]-amide
By following the procedure described in Method B, the title compound was prepared in 29% yield from P2-P1 on resin and thiophene-2-carboxylic acid.
MS/ES: m/z 379 (100%, MH+)
Figure imgf000092_0002
Example 14 Continued: Non-commercial P3 Building Blocks
3-Dimethylaminomethyl-4-hvdroxy-benzoic acid
Figure imgf000092_0003
A solution of formaldehyde (37% (water), 11.28 mmol, 0.914g, 0.94 eq.) was added to a solution of ethyl 4-hydroxybenzoate (12.00 mmol, 2g, 1 eq.) and dimethylamine (40% (water), 12.75 mmol, 1.43g, 1.06 eq.) in water (5 ml). The solution turned cloudy and was stirred overnight by which time it had cleared. The mixture was then heated to 9O0C for 2 hours during which time it turned orange. This was poured into water and extracted with EtOAc. The organics were dried (MgSO4) and concentrated in vacuo to give a viscous orange oil (2.57 g, 96%). This was dissolved in wet methanol (20 ml) and NaOH (23 mmol, 0.92 g, 2 eq.) was added. The mixture was heated at reflux for 2 hours then it was cooled and acidified (1M HCI(aq), pH 5). The acidic mixture was lyophilized and the resulting solid extracted with methanol. The solvents were evaporated to give the product as a white solid (1.47g, 61%).
3-tert-Butyl-4-hvdroxy-benzoic acid
Figure imgf000093_0001
MeI (66.6 mmol, 4.14 ml, 2 eq.) and K2CO3 (59.9 mmol, 8.28g, 1.8 eq.) were added to a solution of 2-ferfbutyl phenol (33.3 mmol, 5g) in DMF (60 ml) and the mixture heated at 8OC for 24 hours. The mixture was cooled, diluted with diethyl ether then washed with water. The organics were dried (MgSO4) and the solvents removed in vacuo to give a yellow oil. This was purified by silica column (isohexane → 10% EtOAc in isohexane) to give the product as a pale yellow oil (1.79g, 33%).
This oil was dissolved in acetonitrile (55ml) and N-bromo-succinimide (10.9 mmol, 1.94g, 1eq.) was added. The mixture was stirred overnight then the solvent was removed in vacuo. The residue was partitioned between water and EtOAc. The solvents were evaporated to give the product as a yellow oil (1.79g, 61%).
A portion of this product (4.11 mmol, 1.00 g) was dissolved in THF (5 ml) and added dropwise to a suspension of magnesium (8.22 mmol, 0.2Og, 2 eq.) in THF (5 ml) containing 1 crystal of iodine. This mixture was heated at reflux for 1 hour and then allowed to cool to room temperature whereupon it was poured onto vigorously stirred solid CO2. This was stirred until the mixture came to room temperature. 1MHCI (aq) (10 ml) was added and the organics separated. The aqueous layer was extracted with diethyl ether. The solvents were evaporated to give the product as a pale brown solid (0.37 g, 43%). This was dissolved in DCM (20 ml) and BBr3 (20 mmol, 5g, 11 eq.) was added. The mixture was stirred for 3 days whilst being monitored by HPLC. The mixture was treated with HCI solution (0.1M) then filtered. The aqueous layer was evaporated then dissolved in methanol. The solvent was evaporated. The dissolution/evaporation protocol was repeated a further 3 times and gave the pure product as a white solid (0.04 g, 12%).
3-Acetyl-4-hvdroxy-benzoic acid
Figure imgf000094_0001
Mg(CIO4J2 (0.602 mmol, 0.134g, 2 mol%) was added to a solution of ethyl 4-hydroxybenzoate (30.1 mmol, 5g) in Ac2O (45.15 mmol, 4.25 ml) and the mixture was stirred overnight. This was diluted with DCM then washed with water. The organics were dried (MgSO4) and the solvent evaporated to give a clear oil. This was azeotroped twice with toluene to give the product as a clear oil (5.73g, 92%). This was mixed with AICI3 (82.5 mmol, 10.99g, 3 eq.) and KCI (28.9 mmol, 2.15g, 1.05 eq.) and heated to 15O0C for 1.5 hours during which time a dark foam was formed. This was cooled in an ice bath and ice cold 2MHCI (aq) (100 ml) was added. The solution was stirred for 5 minutes then ethanol (20 ml) was added. This was heated at reflux for 45 minutes then cooled in an ice bath. The solid formed was collected by filtration then purified by recrystallisation from THF/EtOH to give the product as a cream solid (1.3Og, 26%).
4-Hvdroxy-2-methyl-benzoic acid
Figure imgf000094_0002
BBr3 (20 mmol, 5g, 10 eq.) was added to a solution of 4-methoxy-2-methyl benzoic acid (2 mmol, 0.332g) in DCM (20 ml) and the mixture was stirred under argon until HPLC indicated no starting material remained. HCI (0.1 M, 20 ml) was added and the mixture was filtered. The aqueous layer was evaporated then dissolved in methanol. The solvent was evaporated. The dissolution/evaporation protocol was repeated a further 3 times and gave the pure product as a yellow solid (0.24g, 80%).
3-Ethyl-4-hvdroxy-benzoic acid
Figure imgf000095_0001
As reference: J. Am. Chem. Soc, 1984, 106, 174.
A sodium hydroxide solution (5 ml, 20 % w/v) was added to β-cyclodextrin (371 mg, 0.33 mmol) and copper powder (26 mg, 0.41 mmol). Then 2-ethyl phenol (0.48 ml, 4.09 mmol) was added followed by the dropwise addition of carbon tetrachloride (0.77 ml, 7.98 mmol). The reaction was stirred at 8O0C under nitrogen for 6 hrs. It was then allowed to cool to room temperature, ethyl acetate was added (10 ml) and the solution was acidified using 1MHCI (aq). The solution was extracted with more ethyl acetate and the combined organics were dried (MgSO4) and the solvent was removed in vacuo. Purification by column chromatography (isohexane: ethyl acetate; 1 :1) afforded the product (204 mg, 30 %).
HPLC retention time of 3.70 min.
Mass spectroscopy: m/z 166 (100, MH+).
4-Hvdroxy-3-propyl-benzoic acid
Figure imgf000095_0002
Prepared via the same method as above.
HPLC retention time of 4.13 min. Mass spectroscopy: m/z 181 (100, MH+).
4-Hvdroxy-3-isopropyl-benzoic acid
Figure imgf000096_0001
Prepared via the same method as above.
HPLC retention time of 4.03 min.
Mass spectroscopy: m/z 181 (100, MH+).
2-Fluoro-6-methyl-phenol
Figure imgf000096_0002
3-Fluorosalicylaldehyde (117 mg, 0.83 mmol) was dissolved in dry ethyl acetate (15 ml) and Pd/C (12mg, 10 % w/w) was added. The solution was vigorously stirred at room temperature under a hydrogen atmosphere for 6 hrs. Filtration through celite and removal of the ethyl acetate under vacuo afforded the product (70 mg, 67 %) without need for further purification.
HPLC retention time of 4.09 min.
3-Fluoro-4-hvdroxy-5-methyl-benzoic acid
Figure imgf000096_0003
Prepared via the same method outlined previously. HPLC retention time of 3.17 min.
2-Oxo-i ,2,3,4-tetrahydro-quinazoline-6-carboxylic acid
Figure imgf000097_0001
Prepared as described in Eur. J. Org. Chem. 22 2000 3755-62.
3- Methyl ^-oxo-i ^^^-tetrahydro-quinazoline-e-carboxylic acid
Figure imgf000097_0002
Prepared as described in Chem. Pharm. Bull. 36 (6) 2253-2258.
4-(Difluoromethyl)benzoic acid
Figure imgf000097_0003
4-Cyanobenzaldehyde (655 mg, 5 mmol) was dissolved in [bis(2-methoxyethyl)amino]sulfur trifluoride (2 ml) - exothermic! The reaction was then heated to 6O0C and monitored by HPLC for the disappearance of starting aldehyde (typically 24 to 36 h). After this time, DCM (20 ml) was added and the reaction was poured onto ice. The organic fraction was separated off, dried (Na2SO4), filtered and concentrated. Purified on silica (5 % ethyl acetate / iso-hexane) to give 4- (difluoromethyl)benzonitrile as a clear, colourless oil, 345 mg, 45%.
4-(Difluoromethyl) benzonitrile (600 mg, 3.9 mmol) was heated to reflux in 2M aqueous sodium hydroxide solution (20 ml) for 2h. During this time the initial suspension of starting material dissolved. The reaction was cooled and acidified with aqueous 2M HCI solution to give a white precipitate. This was collected by filtration, washed with water and then dried in vacuo. 4- (Difluoromethyl) benzoic acid was obtained as a white powder, 543 mg
4-(ethvir(9H-fluoren-9-ylmethoxy)carbonyllamino}benzoic acid
Figure imgf000098_0001
To a mixture of 4-ethylaminobenzoic acid (1.0g, 6.05mmol) in 1,4-dioxane (12ml) and 0.5M aqueous sodium hydroxide solution (12ml) was added 9-fluorenylmethoxycarbonyl chloride (1.72g, 6.66mmol). The mixture was partitioned between 1 M HCI (aq) and dichloromethane. The aqueous layer was extracted with dichloromethane and the combined organic extracts were washed with water, brine and dried over sodium sulfate. The solvent was removed and the crude product purified by flash column chromatography on silica to give 4-{ethyl[(9H-fluoren-9- ylmethoxy)carbonyl]amino}benzoic acid as a cream solid.
Lithium 4-rmethyl(phenylsulfonyl)aminolbenzoate
Figure imgf000098_0002
To a mixture of methyl 4-(methylamino)benzoate (1 mmol), 4-dimethylaminopyridine (2 mg), and diisopropylethylamine (1.1 mmol) in acetonitrile (3 ml) was added benzenesulfonyl chloride (1.1 mmol). The reaction mixture was stirred for 16h, and the mixture concentrated by nitrogen stream. The crude product was partitioned between 1M HCI (aq) and ethyl acetate. The aqueous layer was extracted with ethyl acetate and the combined organic extracts were washed with 1 M HCI (aq), water, brine and dried over sodium sulfate. The solvent was removed and the crude product was purified by flash column chromatography on silica to give methyl 4- [methyl(phenylsulfonyl)amino]benzoate as a white solid (249 mg, 81%).
Methyl 4-[methyl(phenylsulfonyl)amino]benzoate (0.80 mmol) was dissolved in 1,4-dioxane (5 ml) and 1M LiOH (aq) (O.δOmmol) and water (1 ml) were added. After stirring 16h, the sample was concentrated under vacuum, the residue dissolved in 1 :1 water-acetonitrile and the mixture lyophilized to give lithium 4-[methyl(phenylsulfonyl)amino]benzoate as an off-white solid (238mg, 100%).
3-(dimethylamino)-4-hvdroxybenzoic acid
Figure imgf000099_0001
3-Amino-4-hydroxybenzoic acid (459mg, 3 mmol) was dissolved in methanol (12ml) and toluene (36ml) was added. A 2.0M solution of (trimethylsilyl)diazomethane in hexanes (1.5ml, 3.0 mmol) was added dropwise and the mixture stirred for 0.5h. The reaction mixture was concentrated under vacuum and the residue purified by flash column chromatography on silica to afford methyl 3-amino-4-hydroxybenzoate as a pink solid (254mg, 51%).
A buffer solution at pH 5.5 was prepared by the addition of acetic acid to a 1M aqueous sodium acetate solution. Methyl 3-amino-4-hydroxybenzoate (254mg, 1.5 mmol) was dissolved in a mixture of buffer (1ml) and methanol (2ml). Formaldehyde solution (37% by weight in water; 0.75ml, lOmmol) was added, the mixture stirred for 15 minutes, and then sodium cyanoborohydride (283mg, 4.5mmol) was added portionwise. The reaction mixture was stirred for an additional 0.5h and then concentrated. The residual oil was partitioned between water and ethyl acetate. The aqueous layer was extracted with ethyl acetate and the combined organic extracts were washed with water, brine and dried over sodium sulfate. The solvent was removed and the crude product was purified by flash column chromatography on silica to give methyl 3-dimethylamino-4-hydroxybenzoate as a yellow gum (213mg, 73%).
To a solution of methyl 3-dimethylamino-4-hydroxybenzoate (210mg, 1.1 mmol) in 1,4-dioxane (2ml) was added 1 M LiOH (aq) (4mmol). After stirring for 4Oh, the mixture was acidified to pH 2 by addition of 1 M HCI (aq). The mixture was partitioned between water and ethyl acetate, and the aqueous layer was lyophilized to give a mixture of sodium chloride and 3-dimethylamino-4- hydroxybenzoic acid as a brown semi-solid (378mg). The crude material was used in the subsequent reaction.
1 -r(9H-fluoren-9-ylmethoxy)carbonyll-1 ,2,3,4-tetrahydroquinoline-6-carboxylic acid
Figure imgf000100_0001
To a mixture of I^S^-tetrahydroquinoline-θ-carboxylic acid (1.03g, 5.8mmol) in 1 ,4-dioxane (12ml) and 0.5M aqueous sodium hydroxide solution (12ml) was added 9- fluorenylmethoxycarbonyl chloride (1.68g, 6.5mmol). The mixture was partitioned between 1 M HCI (aq) and dichloromethane. The aqueous layer was extracted with dichloromethane and the combined organic extracts were washed with water then brine and dried over sodium sulfate. The solvent was removed in vacuo and the crude product purified by flash column chromatography on silica to give 1-[(9H-fluoren-9-ylmethoxy)carbonyl]-1, 2,3,4- tetrahydroquinoline-6-carboxylic acid as a white solid (2.Og; 86%).
4-r(2,2,2-trifluoroethyl)aminolbenzoic acid
Figure imgf000100_0002
To a suspension of methyl-4-aminobenzoate (302mg, 2.0mmol) in dichloromethane (3ml) was added trifluoroacetic anhydride (0.31 ml, 2.2mmol). The mixture was stirred for 1 hour and then partitioned between 1 M NaHCO3 (aq) and dichloromethane. The aqueous layer was further extracted with dichloromethane, and the combined organic extracts washed with water then brine and dried over sodium sulfate. The solvent was removed to give methyl 4- [(trifluoroacetyl)amino]benzoate as a white solid (525mg, 100%).
To a stirred solution of methyl 4-[(trifluoroacetyl)amino]benzoate (124mg, 0.5mmol) in an. THF(I ml) at O0C under an argon atmosphere was added borane-dimethyl sulfide complex (57mg, 0.75mmol) and the mixture heated at reflux for 2h. The mixture was allowed to cool, methanol (approx. 0.1 ml) was added dropwise until effervescence ceased. The mixture was partitioned between water and 1 :1 ethyl acetate-MTBE. The aqueous layer was extracted three times with ethyl acetate-MTBE, and the combined organic extracts washed with water, brine and dried over sodium sulfate. The solvent was removed and the crude product was purified by flash column chromatography on silica to give methyl 4-[(2,2,2-trifluoroethyl)amino]benzoate as a white crystalline solid (47mg, 32%).
To a solution of methyl 4-[(2,2,2-trifluoroethyl)amino]benzoate (130mg, 0.56 mmol) in 1,4- dioxane (2ml) was added 1 M LiOH (aq) (0.61 mmol). After stirring at 4OC for 4h, further 1 M LiOH (aq) (0.30mmol) was added. The mixture was stirred at room temperature until all ester was hydrolysed. The mixture was concentrated and the residue partitioned between 1M HCI (aq) and ethyl acetate. The aqueous layer was further extracted with ethyl acetate and the combined organic extracts were washed with water, brine and dried over sodium sulfate. The solvent was removed to give 4-[(2,2,2-trifluoroethyl)amino]benzoic acid as an off-white solid (119mg, 97%).
Example 15
Solid phase synthesis
Compounds depicted in the table below were synthesized on solid phase by successive coupling of the indicated P3 substituent (if present) to the depicted P3 building block, hydrolysis of the ester protecting group (as necessary) and coupling to the P1-P2 building block. Typical coupling conditions and the construction of P3 building blocks not available from commercial sources appears below the table.
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Example 15 Continued: Reaction conditions and non-commercial building blocks
Solid phase synthesis of compounds in the table above was carried out using methodology as described in Example 13.
After Fmoc removal the P3 acids were introduced using standard coupling conditions which are exemplified by the following procedure for Example 15.65:
4-(5-Methyl-Thiazole-2-sulfonamino)-benzoic acid (39 mg, 0.13 mmol), HOBt (19 mg, 0.12 mmol), HBTU (45 mg, 0.12mmol) and NMM (25μl, 0.24 mmol) were added to the resin bound P1-P2 building block (170mg, 0.025mmol) in DMF (6ml_). The reaction was stirred for 16hrs. After filtration of the resin and washing with DCM and MeOH, the title compound was obtained when cleaved from the resin with 95% TFA in water. After concentration the product was purified on HPLC and freeze dried. The product was characterized by HPLC-MS and NMR. N-ri-(2-Ethyl-4-oxo-tetrahydro-furan-3-ylcarbannoyl)-2-(1-nnethyl-cvclopentyl)-ethyll-4- methanesulfonylamino-3-methoxy-benzannide (Example 15.1)
Methanesulfonyl chloride (615 uL) was added to a solution of 4-Amino-3-methoxy-benzoic acid methyl ester (1 g) in dichloromethane (20 ml.) and pyridine (1.5 ml.) and a catalytic amount of DMAP. After 1-16 hrs the mixture was concentrated to near dryness and the product crystallized from added ethanol. This product was hydrolyzed in 2.5 M LiOH (5 ml_), THF (14 ml_), MeOH (7 ml.) in a microwave oven at 110 deg C for 30 min. After cooling, the solution was acidified with aq. HCI and extracted with ethyl acetate, dried with Na2SO4 and concentrated to dryness. The remaining powder was used for coupling to the resin bound P1-P2 building block (described above). The title compound was obtained when cleaved from the resin with 95% TFA in water. After concentration the product was purified on HPLC and freeze dried. The product was characterized by HPLC-MS and NMR.
For the synthesis of Examples 15.33, 15.4, 15.6, 15.7, 15.8, 15.9, 15.15, 15.16, 15.17, 15.18, 15.19, 15.22, 15.24, 15.27, 15.28, 15.32, 15.33, 15.36, 15.40, 15.51 , 15.54, 15.64, 15.70 and 15.71 the same procedure as in Example 15.1 was followed.
For the synthesis of Examples 15.10, 15.11, 15.13, 15.14, 15.25, 15.34, 15.39, 15.42, 15.43, 15.44, 15.45, 15.46, 15.49, 15.50, 15.53, 15.56, 15.57, 15.58, 15.59, 15.60, 15.61, 15.62, 15.63 and 15.72 the P3 building block came from commercial sources. The rest of the synthesis followed the procedure from Example 15.1.
The P3 building blocks of Examples 15.2, 15.5, 15.12, 15.26, 15.37, 15.48, 15.68, 15.69, 15.71 and the P3 substituents for examples 15.35, 15.41 , 15.65, 15.66, 15.67, 15.70, 15.71 were synthesized according to procedures presented below. Subsequent synthesis followed the procedure from Example 15.1.
4-(Methanesulfonyl-methyl-amino)-benzoic acid methyl ester (P3 building block for Example 15.2)
A mixture of 4-Methanesulfonylamino-benzoic acid methyl ester (0.5 g), methyl iodide (0.4 mL) and potassium carbonate (0.9 g) in acetonitrile (10 mL) was kept in a microwave oven at 120 deg C for 10 min. The cooled mixture was filtered and concentrated to dryness. The remains were precipitated from added DCM and the solid was collected, dried in a vacuum and used in the next step.
4-Amino-2-methyl-benzoic acid methyl ester (P3 building block for Example 15.5) 4-Acetylamino-2-methyl-benzoic acid methyl ester was kept in cone. HCI/MeOH 1 :1 in a microwave oven at 70 deg C for 2 hrs. After cooling the solid was collected, dried in a vacuum and used in the next step.
3-Acetyl-4-amino-benzoic acid methyl ester (P3 building block for Example 15.12)
5-Amino-furan-2-carboxylic acid methyl ester (0.42 g, 3.0 mmol) were mixed together with methyl vinyl ketone (10 ml.) in benzene and heated at reflux for 1 h. Evaporation of solvents were followed by flash chromatography using DCM / MeOH (95:5) as eluent to yield 44% (278 mg. 1.31 mmol) of 5-Acetyl-4-amino-1-hydroxy-cyclohexa-2,4-dienecarboxylic acid methyl ester. This compound were mixed with BF3 OEt2 ((284 mg, 2.0 mmol) in benzene (15 ml.) and refluxed for 0.5 h. The reaction mixture was quenched with NaHCO3 (sat) and extracted with dichloromethane. A precipitation formed in the organic phase was collected and confirmed to be the product by characterization with LC-MS and 1H NMR. Yield: 127 mg (50%).
N-ri-(2-Ethyl-4-oxo-tetrahvdro-furan-3-ylcarbamoyl)-2-(1-methyl-cvclopentyl)-ethyll-4-(pyridine- 3-sulfonylamino)-benzamide (Example 15.20)
Trifluoromethane sulfonic anhydride (38OuL) was added to polymer supported tiriphenylphosphine oxide (1g) in dichloromethane (15mL). After 1 hrs the mixture was cooled to 0 deg C and a solution of pyridine 3-sulfonic acid (360 mg) as pyridine salt in DCM (4mL) was added. After 30 min. 4-Methanesulfonylamino-benzoic acid methyl ester (318 mg) in dichloromethane (4mL) was added. The mixture was shaken at 25 deg C for 16 hrs. The resin was filtered off and the filtrate concentrated to dryness. The crude was purified by silica column chromatography. Subsequent synthesis was done according to the procedure in Example 1.
For the synthesis of Examples 15.23, 15.35, and 15.52 the same procedure as in Example 15.20 was followed.
4-Benzenesulfonylamino-N-ri-(2-ethyl-4-oxo-tetrahvdro-furan-3-ylcarbamoyl)-2-(1-methyl- cyclopentyl)-ethyll-3-methyl-benzamide (Example 15. 21)
For synthesis of P3 cap see procedure for P3 building block for Example 15.68. Subsequent synthesis was done according to Example 15.1.
4-(2,4-Dimethyl-thiazole-5-sulfonylamino)-benzoic acid methyl ester (P3 building block for Example 15.26) See procedure for P3 cap Example 15.68.
4-(4-Cvano-benzenesulfonylamino)-N-ri-(2-ethyl-4-oxo-tetrahvdro-furan-3-ylcarbamoyl)-2-(1- methyl-cvclopentvD-ethyll-benzamide (Example 15.29)
Example 15.29 was synthesized via solid phase synthesis methodology. First coupling of the 4- Amino-benzoic acid to the P1-P2 building block was done followed by washing as described in WO00/69055. Secondly 4-Cyano-benzenesulfonyl chloride (53.2 mg, 0.26 mmol) and a catalytic amount DMAP dissolved in pyridine (2 ml.) and DCM (4 ml.) was added to the P1-P2 building block (220 mg, 0.053 mmol). The reaction was left on agitation at room temperature over weekend. Cleavage from resin was done by addition of 95% TFA (aq, 6 ml.) and agitation for 0.5 h. Toluene (3 ml.) was added after filtration from resin, followed by evaporation.
Subsequent purification and characterization was done according to the procedure in Example 15.1.
For the synthesis of Examples 15.30 and 15.31 the same procedure as in Example 15.29 was followed.
Pyridine 4-sulfonic acid (P3 substituent for Example 15.35)
4-Mercaptopyridine (500 mg) was dissolved in glacial acetic acid (18ml_), followed by the addition of 35% hydrogen peroxide (6ml_). The solution was warmed at 80 deg C for 90 min. and then concentrated to dryness. The product was re-crystallized from methanol :water, dried in a vacuo and used in the next step.
Subsequent synthesis to P3 building block was done according to the procedure in Example 20.
N-ri-(2-Ethyl-4-oxo-tetrahvdro-furan-3-ylcarbamoyl)-2-(1-methyl-cvclopentyl)-ethyll-4-(4-methyl- thiazole-2-sulfonylamino)-benzamide (P3 substituent for Example 15.37)
4-methyl-1 ,3-thiazole (2.0 g, 20.2 mmol) was dissolved in methyl -f-butyl ether (46 ml.) and the solution was cooled to 00C. Addition of isopropyl magnesium chloride (10.1 ml_, 2.0 M) was done drop wise at 0°C. The mixture was then heated to 40°C and sulfur dioxide in dimethoxymethane (4.2 ml_, 6.0 M) was added drop wise and the reaction was then left at this temperature for 1 h. After cooling the reaction mixture to 00C, /V-chlorosuccinimide (4.05 g, 30.3 mmol) was added, and the reaction were kept at 00C for 45 min. After addition of HCI (0.2 M, aq, 50 ml.) at 0°C the reaction was left to warm up to ambient temperature for 2 hrs and extracted with methyl-f-butyl ether. The organic phase was washed with of HCI (0.2 M, aq), water and brine then dried over Na2SO4, filtered and evaporated to yield 2.33 g (59%) of product.
4-(4-Amino-benzenesulfonylamino)-N-ri-(2-ethyl-4-oxo-tetrahvdro-furan-3-ylcarbamoyl)-2-(1- methyl-cvclopentvD-ethyll-benzamide (Example 15.38)
Example 15.38 was synthesized as described in Example 15.1 by successive coupling of the P3 substituent to the P3 building block, hydrolysis of the ester and coupling to the P1-P2 building block. After cleavage from P1 -P2 resin, reduction of nitro group was done by dissolving 4-(4- Nitro-benzenesulfonylamino)-N-[1-(2-ethyl-4-oxo-tetrahydro-furan-3-ylcarbamoyl)-2-(1-methyl- cyclopentyl)-ethyl]-benzamide (13.9 mg, 23.4 μmol) in MeOH (3ml_) and degassing the solution with N2 gas. A catalytic amount of palladium on carbon was then added to the reaction solution and a H2 atmosphere was connected. After 2 hrs, filtration through celite was done, with MeOH as eluent, to yield 11.8 mg (91%) of product after concentration.
N-ri-(2-Ethyl-4-oxo-tetrahvdro-furan-3-ylcarbamoyl)-2-(1-methyl-cvclopentyl)-ethyll-4-(pyridin-2- ylamino)benzamide (Example 15.41)
4-Aminobenzoic acid methyl ester (1 g, 6.6 mmol), 2-fluoropyridine (1.28 g, 13.2 mmol) and potassium carbonate (1.83 g, 13.2 mmol) in DMF was heated in a microwave oven at 2000C for 20 min. The residue was extracted with dichloromethane and water. The organic layer was dried and concentrated, purified on a SiO2 column (Toluene-EtOAc 8:2) to give 4-(Pyridin-2-ylamino)- benzoic acid methyl ester.
Subsequent synthesis was done according to procedure in Example 15.1.
5-r(2-Methoxy-ethylamino)-methvH-thiophene-2-carboxylic acid H -(2-ethyl-4-oxo-tetrahvdro- furan-3-ylcarbamoyl)-2-(1 -methyl-cvclopentvD-ethyll-amide (Example 15.47)
δ-Fornnyl-thiophene^-carboxylic acid was coupled to the resin as described in example 1. The resin was swollen in DCM-trimethylortoformate 1 :1 and 4 equiv. of methoxyethylamine was added. After 4 hrs of agitation the resin was washed with DCM and MeOH (2X) and the resin was dried. To this resin in DCM-MeOH-HOAc 2:2:1 borane-pyridine complex was added. After 16 hrs of agitation the resin was washed and cleaved and purified as described in Example 15.1.
5-(2-Methoxy-ethylcarbamoyl)-thiophene-2-carboxylic acid (P3 building block for Example 15.48) δ-Formyl-thiophene^-carboxylic acid (740 mg, 4.7 mmol), methoxyethylamine (412 μl_, 4.7 nnnnol), HATU (4.5 g 11.8 nnnnol), ethyldiisopropylamine (4 ml) and DMF (2 m) in DCM (20 ml) was stirred for 2 hrs. The mixture was extracted with DCM and aq. bicarbonate. The organic layer was dried and concentrated. This residue was oxidized in t-BuOH (4ml) by the addition of sodium phosphate buffer (0.5M, 2 ml) and aq. KMnO4 (1M, 2ml). After 10 min. addition of sat. aq. Na2SO4 (2ml) was done and pH was adjusted to 3 with aq. HCI. Extracted with EtOAc. Organic layer was dried and concentrated.
N-n-^-EthvM-oxo-tetrahvdro-furan-S-ylcarbamovn^-n-methyl-cvclopentvn-ethyll^-^^^- trifluoro-ethanesulfonylamino)-benzamide (Example 15.55)
4-Ami no benzoic acid (16.5mg, 0.12 mmol) was used for coupling to the resin bound P1-P2 building block (described above). 2,2,2, trifluoromethane sulfonyl chloride (21.1 μl, 0.192 mmol), Pyridine (31 μl, 0.384 mmol), and catalytic amount of DMAP were added to the resin in DCM (6ml_) and stirred for 16 hrs. After filtration of the resin and washing with DCM and MeOH, the title compound was obtained when cleaved from the resin with 95% TFA in water. After concentration the product was purified on HPLC and freeze dried. The product was characterized by HPLC-MS. Yield: 7.3 mg (44%).
4-(5-Methyl-Thiazole-2-sulfonamino)-benzoic acid (P3 substituent for Example 15.65)
See procedure for P3 substituent Example 15.70
4-(4-Methyl-Thiazole-2-sulfonamino)-benzoic acid (P3 substituent for Example 15.66)
See procedure for P3 substituent Example 15.37
4-(4-lsopropyl-Thiazole-2-sulfonamino)-benzoic acid (P3 substituent for Example 15.67)
See procedure for P3 substituent Example 15.71
4-(Butane-1-sulfonylamino)-benzoic acid methyl ester (P3 building block for Example 15.68)
4-Amino-benzoic acid methyl ester (1.0 g, 6.62 mmol) and a catalytic amount of DMAP were dissolved in pyridine (0.5 mL) and DCM (15 mL). The reaction mixture was cooled to 0 0C and butylsulfonyl chloride (1.01 mL, 6.62 mmol) was added via a syringe. The reaction was left stirring and allowed to warm up to room temperature over night. Evaporation of solvents was followed by addition of DCM and the organic phase was washed with HCI (aq, 1 M), water and brine. After being dried over Na2SO4, the organic phase was filtered and evaporated to yield 1.34 g (74%) of 4-(butane-1-sulfonylamino)-benzoic acid methyl ester.
Subsequent hydrolysis of 4-(butane-1-sulfonylamino)-benzoic acid methyl ester was done according to the procedure in Example 15.1.
4-(4-Ethyl-thiazol-2-ylamino)-benzoic acid (P3 building block for Example 15.69)
4-Thioureido-benzoic acid ethyl ester (0.52 g, 2.23 mmol) and 1 -Bromo-butan-2-one (0.25 ml_, 2.45 mmol) was mixed in dioxane (4 ml.) and microwave heated at 110 °C for 15 min. To the precipitated crystals were added DCM and NaHCO3. The organic phase was after separation washed with water and brine, dried over Na2SO4, filtered and evaporated to yield 0.58 g (93%) of 4-(4-Ethyl-thiazol-2-ylamino)-benzoic acid ethyl ester. Subsequent hydrolysis of 4-(4-Ethyl- thiazol-2-ylamino)-benzoic acid ethyl ester was done according to the procedure in Example 15.1.
5-Methyl-thiazole-2-sulfonyl chloride (P3 substituent for Example 15.70)
5-methyl-1 ,3-thiazole (1.0 g, 10.1 mmol) was dissolved in methyl -f-butyl ether (25 ml.) and the solution was cooled to 00C. Addition of isopropyl magnesium chloride (10.1 ml_, 2.0 M) was done drop wise at 0°C. The mixture was then heated to 40°C and sulfur dioxide in dimethoxyethane (1.64 ml_, 7.7 M) was added drop wise and the reaction was then left at this temperature for 45 min. After cooling the reaction mixture to 00C, Λ/-chlorosuccinimide (2.02 g, 15.2 mmol) was added and the reaction were kept at 00C for 1 h. After addition of HCI (aq, 0.2 M, 25 ml.) at 0°C the reaction was left to warm up to ambient temperature for 2 hrs and extracted with methyl-f-butyl ether. The organic phase was washed with of HCI (aq, 0.2 M), water and brine then dried over Na2SO4, filtered and evaporated to yield 1.82 g (91%) of product.
4-lsopropyl-thiazole-2-sulfonyl chloride (P3 substituent for Example 15.71)
1-Bromo-3-methyl-butan-2-one (1.15 g, 6.95 mmol) and thioformamide ( 0.43 g, 6.95 mmol) were dissolved in dioxane (10 ml.) and heated in microwave at 110 °C for 15 min. Dichloromethane and NaHCO3 was added and after separation the organic phase was washed with NaOH (aq, 1 M) and water. Back-extracted water phase with dichloromethane. The combined organic phases were washed with water and brine, dried over Na2SO4, filtered and evaporated to yield 0.82g (70%) of 4- isopropyl-1 ,3-thiazole. 4-lsopropyl-1 ,3-thiazole (0.62 g, 4.9 mmol) was dissolved in methyl-f-butyl ether (15 ml.) and the solution was cooled to 0°C. Addition of isopropyl magnesium chloride (2.9 ml_, 2.0 M) was done drop wise at 00C. The mixture was then heated to 400C and sulfur dioxide in dimethoxyethane (0.79 ml_, 7.7 M) was added drop wise and the reaction was then left at this temperature for 45 min. After cooling the reaction mixture to 0°C, Λ/-chlorosuccinimide (0.97 g, 7.3 mmol) was added, and the reaction were kept at 0°C for 1 h. After addition of HCI (aq, 0.2 M, 10 ml.) at 00C the reaction was left to warm up to ambient temperature for 2 hrs and extracted with methyl-f-butylether. The organic phase was washed with of HCI (aq, 0.2 M), water and brine then dried over Na2SO4, filtered and evaporated to yield 0.92 g (84%) of product.
Biological Example 1
Cathepsin S Ki determination
The assay uses baculovirus-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.
Substrate dilutions
280μl/well of 12.5% DMSO are added to rows B - H of two columns of a 96 deep well polypropylene plate. 70μl/well of substrate is added to row A. 2 x 250μl/well of assay buffer (10OmM Na phosphate, 10OmM NaCI, pH 6.5) is added to row A, mixed, and double diluted down the plate to row H.
Inhibitor dilutions.
100μl/well of assay buffer is added to columns 2-5 and 7-12 of 4 rows of a 96 well V bottom polypropylene plate. 200μl/well 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 Kj. The rough Ki is calculated from a preliminary run in which 10 μl/well of 1 mM boc-VLK-AMC (1/10 dilution of 10 rtiM stock in DMSO diluted into assay buffer) is dispensed to rows B to H and 20 μl/well to row A of a 96 well Microfluor ™ plate. 2 μl of each 1OmM test compound is added to a separate well on row A, columns 1-10. Add 90 μl assay buffer containing 1mM DTT and 2 nM cathepsin S to each well of rows B-H and180 μl to row A.Mix row A using a multichannel pipette and double dilute to row G. Mix row H and read in the fluorescent spectrophotometer. The readings are Prism data fitted to the competitive inhibition equation, setting S = 100μM and KM = 100μM to obtain an estimate of the Kj, up to a maximum of 100μM.
The second test compound is added to column 6 of the top row, the third to column 1 of the second row etc. Add 1 μl 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.
Using an δ-channel multistepping pipette set to 5 x 10μl, distribute 10μl/well of substrate to the 384 well assay plate. Distribute the first column of the substrate dilution plate to all columns of the assay plate starting at row A. The tip spacing of the multichannel pipette will correctly skip alternate rows. Distribute the second column to all columns starting at row B.
Using a 12-channel multistepping pipette set to 4 x 10μl, distribute 10μl/well of inhibitor to the 384 well assay plate. Distribute the first row of the inhibitor dilution plate to alternate rows of the assay plate starting at A1. The tip spacing of the multichannel pipette will correctly skip alternate columns. Similarly, distribute the second, third and fourth rows to alternate rows and columns starting at A2, B1 and B2 respectively.
Mix 20ml assay buffer and 20μl 1 M DTT. Add sufficient cathepsin S to give 2nM final concentration.
Using the a distributor such as a Multidrop 384, add 30μl/well to all wells of the assay plate and read in fluorescent spectrophotomoter such as an Ascent.
Fluorescent readings, (excitation and emission wavelengths 390nm and 460nm 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.
Fitted rates for all wells for each inhibitor are fitted to the competitive inhibition equation using SigmaPlot 2000 to determine V, Km and Ki values. The majority of the compounds illustrated above provide Ki values of 100 nM or less in this assay.
Biological Example 2
Cathepsin K Ki The procedure of Biological Example 1 with the following amendments is used for the determination of Ki for cathepsin K.
The enzyme is E coli expressed human cathepsin K. The substrate is H-D-Ala-Leu-Lys-AMC from Bachem. The assay buffer is 100 rtiM Na phosphate, 1 mM EDTA, 0.1% PEG 4000, pH 6.5. The DMSO stock (see substrate dilutions) is diluted to 10% in assay buffer . 56 ul of substrate is added to row A and 2 x 256 ul of buffer is added to row A. The final cathepsin K concentration is 0.5 nM. The majority of compounds illustrated above provide selectivities over cathepsin K of at least 10-100 fold.
Biological Example 3
Cathepsin L Ki
The procedure of Biological Example 1 with the following amendments is used for the determination of Ki for cathepsin L.
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 10OmM sodium acetate 1mM EDTA, pH5.5) The DMSO stock (1OmM in 100%DMSO) is diluted to 10% in assay buffer. Enzyme is prepared at δnM concentration in assay buffer plus 1 mM dithiothreitol just before use. 2ul of 1OmM inhbitor made up in 100% DMSO is dispensed into row A. 10ul of 50 uM substrate (=1/200 dilution of 1OmM stock in DMSO.diluted in assay buffer.) The majority of the compounds illustrated above provide selectivity over cathepsin L of at least 10-100 fold.
Biological Example 4
Permeability
This example 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 basolateral transport
Generally every compound will be tested in 2-4 wells. The basolateral 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 μM. 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.
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.1) 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. In A→B screening the TB in basolateral well also contains 1% Bovine Serum Albumin.
0.5 ml. transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts and the cell monolayers equilibrated in the transport buffer system for 30 minutes at 37 0C in a polymix shaker. After being equilibrated to the buffer system the Transepithelial electrical resistance value (TEER) is measured in each well by an EVOM chop stick instrument. The TEER values are usually between 400 to 1000 Ω per well (depends on passage number used).
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 μl_ TB (pH 6.5), including the test substance is added to the apical (donor) well. The plates are incubated in a polymix shaker at 370C with a low shaking velocity of approximately 150 to 300 rpm.
After 30 minutes incubation in row 2 the inserts will be moved to new pre-warmed basolateral (receiver) wells every 30 minutes; row 3 (60 minutes), 4 (90 minutes) and 5 (120 minutes).
25 μL samples will be 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 μL 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. To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at -2O0C until analysis by HPLC or LC-MS.
Basolateral to apical transport Generally every compound will be tested in 2-4 wells. The basolateral 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 μM. 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.
After 21 to 28 days in culture on filter supports the cells are ready for permeability experiments. The culture medium from the apical wells are removed and the inserts are transferred to a wash row (No.1) in a new plate without inserts (Transport plate).
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).
0.5 mL transport buffer (HBSS, 25 rtiM 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 0C 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 μL fresh TB, pH 6.5 is added to the inserts. After 30 minutes 250 μL is withdrawn from the apical (receiver) well and replaced by fresh transport buffer. Thereafter 250 μL samples will be withdrawn and replaced by fresh transport buffer every 30 minutes until the end of the experiment at 120 minutes, and finally a post value of TEER is measured at the end of the experiment. A 25 μL samples will be taken from the basolateral (donor) compartment after ~2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.
To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at -2O0C until analysis by HPLC or LC-MS.
Calculation
Determination of the cumulative fraction absorbed, FAcum, versus time. FAcum is calculated from: FA - Y ^M. rMcum ~ L-I r
DI
Where CRJ is the receiver concentration at the end of the interval i and Crjj is the donor concentration at the beginning of interval i. A linear relationship should be obtained.
The determination of permeability coefficients (Papp. cm/s) are calculated from:
app (A - 60)
where k is the transport rate (mirr'' ) defined as the slope obtained by linear regression of cumulative fraction absorbed (FAcum ) as a function of time (min), VR is the volume in the receiver chamber (ml_), and A is the area of the filter (cm2).
Reference compounds
Figure imgf000128_0001
Biological Example 5
Cellular cathepsin S K1
This example describes procedures for assessing potency of cathepsin S inhibitors on inhibition of in vitro T cell activation by determining concentration of the compound required for reducing 50% of the IL-2 secretion in T cells stimulated with compound-treated antigen presenting cells in an antigen presentation assay using the 19.3 cells and the 9001 cells as the effector cells and the antigen presenting cells, respectively. 19.3 cells are murine T cell hybridomas recognizing type Il collagen (260-272) in the context of HLA-DR1 , and 9001 is an EBV-transformed human B cell line expressing homozygous DR1 molecule. The 9001 cells will be pre-treated with varying concentration of the compounds for 1 hour and then incubated with the T cells in the presence of collagen at a final concentration of 0.1 mg/ml. The cultures will be incubated overnight at 37°C with 5% CO2 and amount of IL-2 in the supernatant determined with ELISA. The IC50-IL-2 values representing the concentration of compounds at which secretion of IL-2 from the T cells is reduced by 50% will be determined by regression analysis
Major histocompatibility complex (MHC) class Il molecules bind peptides generated by degradation of endocytosed antigens and display them as MHC class ll-peptide complexes at the cell surface for recognition by CD4+ T cells. MHC class Il molecules are assembled with the assistance of invariant chain (Ii) in the endoplasmic reticulum (ER) and transported to an endocytic compartment where Ii undergoes rapid degradation by endosomal and lysosomal proteases. A peptide fragment of Ii, CLIP (class ll-associated Invariant chain Peptides) remains bound in the class Il peptide binding groove, until removed by the chaperone molecule H-2M in mouse or HLA-DM in humans. This allows peptides derived from proteolytic degradation of foreign and self proteins to bind class Il molecules and subsequently to be presented to T cells in the context of MHC molecules. In dendritic cells and B cells, cathepsin S is required for complete invariant chain processing and CLIP generation. Inactivating cathepsin S with inhibitors will impair MHC class Il peptide loading and formation of stable MHC/peptide complexes leading to reduced antigen presentation and T cell activation.
To assess the potency of the cathepsin S inhibitors, an antigen presentation assay uses a collagen specific,HLA-DR1 restricted mouse T cell hybridoma (19.3) as effector cells, human EBV-transformed B cells (9001) as antigen presenting cells (APC), and mlL-2 ELISA as the read-out system. Inhibition of Cathepsin S with specific inhibitors will impair the processing and presentation of collagen in APCs which in turn reduces the activation of the T cells. The extent of inhibition on T cells is measured by the degree of reduction in IL-2 secretion. IC50-IL-2 represents the concentration of compounds at which secretion of IL-2 from the T cells is reduced by 50% in the antigen presentation assay.
MATERIALS
Cathepsin S inhibitors Compounds will be dissolved in DMSO to a final concentration of 10 nriM, aliquotted, and stored at -80 C until used.
Cells
All the cells will be cultured in DMEM medium (Invitrogen, cat #11995-065) supplemented with 10% fetal bovine serum (Hyclone, cat #SH30070.03), 100 U/ml penicillin, 100 ug/ml streptomycin and 2 mM L-glutamine (Invitrogen, cat #10378-016).
T cell: 19.3, murine DR1 transgenic T cell hybridomas, DR1 restricted, Type Il collagen 260-272 specific
Antigen presentation cells (APCs): 9001 , EBV-transformed human B cells expressing homozygous DR1
Antigen
Type Il collagen from chicken sternal cartilage (Sigma, cat. # C-9301) will be dissolved in PBS at 1 mg/ml and stored in aliquots at -80 C.
EQUIPMENT
Tissue culture incubator (Forma Scientific, model. #3120) Sorvall centrifuge (Sorvall RC-3B) Plate washer Plate-reader (Tecan, Spectra shell, cat. #20-074)
PROCEDURES
Antigen presentation assay
1. Two-fold serial dilutions of the compounds, starting at 40OuM in AIMV medium, will be transferred to a 96-well round-bottom microtiter plate at a volume of 50ul/well.
2. Antigen-presenting cells will be washed and resuspended in AIMV medium to a density of 0.8x106/ml, and then added to the plates at a volume of 50ul/well, giving the number of cells per well as 40,000.
3. The APCs will be pretreated with compounds for 1 hour at 37C with 5% CO2. 4. The T cells will be washed and resuspended in AIMV to a density of 0.8x106/ml.
5. The antigen will be diluted to a 4X concentration in AIMV and mixed 1 to 1 with
the T cells.
6. The T cells/antigen mixture will then be added to the assay plates at a volume
of 100ul/well.
6. The plates will be incubated overnight at 37C with 5% CO2.
7. Supernatant will be carefully removed from each well and measured for amount of IL-2 with ELISA.
IL-2 ELISA
Mouse IL-2 ELISA kits will be purchased from Pharmingen (Mouse IL-2 OptEIA set, #2614KI). The ELISA will be performed per manufacturer's instruction.
1. Anti-mlL-2 antibodies will be diluted in carbonate buffer to a final concentration of 2 ug/ml, transferred to an ELISA plate (Costar) at 100 u I/well and then incubated overnight at 4 degreesC.
2. The ELISA plates will be washed 4 times with PBS/0.5% FBS containing 0.05% Tween 20 (wash buffer).
3. The plates will be blocked with the blocking buffer, 10% FBS (fetal bovine serum, Hyclone) for 2 hrs at room temperature (RT) and then washed 4 times with wash buffer.
4. 100 μL of supernatants from each well of the assay plates will be transferred to the ELISA plate and incubated for 2 hrs at RT.
5. After washing 4 times, the plate will be incubated for 1 hr at RT with a mixture of a biotinylated anti-mlL2 antibody and avidin-HRP prepared in blocking buffer.
6. Following 8 washes with wash buffer, the substrate (TMB) will be added to the plate and incubated at RT for 15-30 minutes until the color develops. 7. Color development will be terminated by the addition of 2N sulfuric acid.
8. The plates will be measured at 450 nm with an ELISA plate reader (Spectra, Tecan).
9. A set of purified recombinant mlL-2 with known concentration will be prepared from the stock solution (provided in the kit) with the blocking buffer and assayed in each plate to provide a standard curve for quantification of IL-2.
DETERMINATION OF IC50-IL-2 OF CATHEPSIN S INHIBITORS
The potency of each compound will be measured by the IC50 value derived from this assay. IC50 represents the concentration of compound at which secretion of IL-2 from the T cells is reduced by 50%.
The absorbance at 450 nm from each well will be converted into amount of IL-2 (pg/ml) using the Winselect software (Tecan) based on the standard curve generated from in-plate standards of purified recombinant mlL-2. Means and standard deviations will be calculated from triplicates with Excel.
The average amounts of IL-2 (pg/ml) from triplicates of both the test and the control wells (received comparable amount of DMSO) will be used to generate the percent inhibition using the following formula.
Percent Inhibition = average of control wells - average of test wells x 100 average of control wells
A dose response curve will be generated by plotting the percent inhibition versus concentration of the compound and the IC50-IL-2 value will be calculated with regression analysis.
DR-1 transgenic T cell hybridoma has been prepared by E. Rosloniec, University of Tennessee.
Following controls are included and analyzed as appropriate:
T + APCs, without antigen, without compound treatment, for background signal. We usually get negligible amounts of IL-2 form these wells, and usually don't perform background subtraction. T + APCs, with anti-CD3/CD28, with compounds, for toxicity associated with compounds.
T + APCs, with antigen, with DMSO (comparable to those received compounds), for toxicity associated with DMSO and for calculation of percent of inhibition.
Biological Example 6
Human Liver Micrososomes
Metabolic stability is determined by commercially available human liver microsome assays, such as XEN 042, assayed in accordace with manufacturer's recommendations.
Biological Example 7
Comparative Trial
Compounds of the invention and the closest prior art were assayed in the assays above and produced the results tabulated below.
Figure imgf000133_0001
Figure imgf000134_0001
All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.
Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

Claims

Claims
1. A compound of the formula I,
Figure imgf000135_0001
where R1 is CrC4 straight or branched alkyl, optionally substituted with up to three substituents selected from halo and hydroxy;
R2 is halo, hydroxy, methyloxy, or CrC2 alkyl, which alkyl is optionally substituted with up to three halogens or an hydroxy or a methyloxy; D is -C3-C7 alkylene-, thereby defining a cycloalkyl ring; E is -C(=O)-, -S(=O)m-, -NRdS(=O)m-, -NRaC(=O)-, -OC(=O)-,
R3 is a carbocyclic ring selected from C3-C6 cycloalkyl, C5-C6 cyclohexenyl or phenyl, or a heterocyclic ring selected from azepanyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, thiopyranyl, furanyl, tetrahydrofuranyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, tetrazolyl, pyrazolyl, indolyl, which ring is optionally substituted with up to 3 substituents independently selected from R4;; R4 is independently selected from halo, oxo, nitrile, nitro, d-C4 alkyl, -NRaRb, NH2CO-, X-R5, X-O-R5, X-0-C(=0)R5, X-C(=O)R5, X-C(=O)NRaR5, X-NRaC(=O)R5, X- NRdSOmR5a, X- SOmNRdR5, X-S(=O)mR5, X-C(=0)0R5, X-NRaC(=0)0R5; or a pair of R4 together define a 5 or 6 membered, nitrogen-containing ring fused to R3, optionally substituted with d-C4 alkyl, d-C4 alkyloxy, oxo, hydroxy, halo, NRaRb,
R5 is H, CrC4 alkyl, C3-C6 cycloalkyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, thiopyranyl, furanyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl, phenyl, benzyl, any of which is optionally substituted with R6; R5a is R5 or -NRaRb;
R6 is independently selected from hydroxy, -NH2, NHCrC3alkyl, N(Ci-C3alkyl)2, nitro, cyano, carboxy, oxo, CrC4 alkyl, CrC4-alkoxy, CrC4 alkanoyl, carbamoyl; R10 is H, ORc, SRc or together with the gem H is =0 or (ORc)2; Ra is independently selected from H, CrC4 alkyl;
Rb is H, CrC4 alkyl or acetyl, or Ra, Rb and the N atom to which they both are joined form a ring selected from morpholine, piperazine, piperidine, pyrrolidine; Rc is H, CrC4 alkyl, C0-C3alkylcarbocyclyl; Rd is H, CrC4 alkyl, C(=O)CrC4 alkyl, C0-C3alkylcarbocyclyl; X is independently a bond or d-C4 alkylenyl; m is independently 0,1 or 2; and pharmaceutically acceptable salts thereof.
2. A compound according to claim 1, wherein R1 is ethyl, 2-fluoroethyl or 2-hydroxyethyl.
3. A compound according to claim 1, wherein R1 is methyl.
4. A compound according to claim 1, wherein R2 is methyl.
5. A compound according to claim 4, wherein D is butylene, thereby defining a cyclopentyl ring.
6. A compound according to any of claims 1 -5, wherein Ra and/or Rb and/or Rc and/or Rd are H.
7. A compound according to any of claims 1-6, wherein E is -C(=O)-.
8. A compound according to claim 7, wherein R3 is optionally substituted furyl, thienyl, pyrazinyl, pyridyl, pyrrolyl or morpholinyl.
9. A compound according to claim 8 wherein R3-E- is one of the partial structures:
Figure imgf000136_0001
where R4' is H, halo, Od-C4 alkyl, C(=O)NRaRb, NRaC(=O)CrC4 alkyl, NRaC(=O)NRaRb or ■ NRaC(=O)OCrC4 alkyl. NHC(=O)OMe.
10. A compound according to claim 9, wherein R4' is fluoro, methoxy, dimethylcarbamoyl, NHC(=O)Me, -NHC(=O)NHCH3, NHC(=O)N(CH3)2 , NHC(=O)OMe or NHC(=O)NRrRr,where RrRr define a cyclic amine selected from pyrrolidine, morpholine, piperidine, piperazine or N- methylpiperazine.
11. A compound according to claim 1 , wherein R3 is phenyl,
12. A compound according to claim 11, wherein the phenyl is substituted with m-fluoro, p- fluoro, p-hydroxy, p-hydroxy-m-chloro, p-hydroxy-m-fluoro, p-hydroxy-m-methoxy, p-hydroxy-m- methyl, bis-p-chloro-p-hydroxy, m-cyano, p-acetamido or p-pyrimid-2-yl.
13. A compound according to claim 11 , with the formula:
Figure imgf000137_0001
where R1, R2, D, Ra, E and R4 are as defined above and R10 is H, R11 or -C(=O)R11 where R11 is independently H, d-C6-alkyl which is optionally substituted with R6, C0-C3alkylcarbocyclyl or C0- C3al kyl heterocyclyl .
14. A compound according to claim 1, wherein R3 comprises the partial structure:
Figure imgf000137_0002
where R12 is-NRdSOmR5a and E, R4, R5a, Rd' are as defined in claim 1.
15. A compound according to claim 14 wherein E is -(C=O)-.
16. A compound according to claim 15, wherein R5 is d-C4 alkyl, such as methyl, ethyl or i- propyl or t-butyl; halogenated CrC4 alkyl such as trifluoromethyl; C3-C6 cycloalkyl, such as cyclopropyl or cyclohexyl; or phenyl or benzyl, any of which is optionally substituted with R6.
17. A compound according to claim 15, with the partial structure:
Figure imgf000138_0001
where E and R4 are as defined above, Rd' is Me or preferably H, and R6 is H or methyl, especially with the partial structure:
Figure imgf000138_0002
18. A compound according to claim 1, wherein R3 has the partial structure:
Figure imgf000138_0003
where R4 and E are as defined above, Rz is CH, NH or O and the S atom is optionally oxidised to >S=O or preferably >S(=O)2.
19. A compound according to claim 1, wherein R3 has the partial structure:
Figure imgf000138_0004
where R4' is H, CrC4 alkyl, NH2, NHCrC4alkyl (such as methylamide), N(CrC4alkyl)2 such as dimethylamide), NHC(=O)CrC4alkyl (such as acetamide); ring nitrogens are optionally substituted with CrC4 alkyl (such as methyl, ethyl or t-butyl) , or C(=O)CrC4 alkyl (such as acetyl); and R4 is as defined in claim 1.
20. A compound according to claim 1 , wherein a pair of R4 define a nitrogen containing ring fused to R3 with the partial structure:
Figure imgf000139_0001
where
R4' is H, CrC4 alkyl, NH2, NHCrC4alkyl (such as methylamide), N(CrC4alkyl)2 such as dimethylannide), NHC(=O)CrC4alkyl (such as acetamide); ring nitrogens are optionally substituted with d-C4 alkyl (such as methyl, ethyl or t-butyl) , or C(=O)Ci-C4 alkyl (such as acetyl);
R4 is as defined in claim 1 ; O' is absent (ie 2 hydrogen atoms) or keto.
21. A compound according to claim 1 , wherein R10 is H.
22. A pharmaceutical composition comprising a compound as claimed in any of claims 1-21 and a pharmaceutically acceptable carrier or vehicle therefor.
23. Use of a compound as claimed in any of claims 1 to 21 in the manufacture of a medicament for the treatment or prophylaxis of disorders caused by aberrant cathepsin S expression or activation.
24 Use according to claim 23, wherein the disorder is an autoimmune disorder such as MS, RA, juvenile diabetes or asthma.
25. Use according to claim 23, wherein the disorder is chronic pain.
26. A method for the treatment or prophylaxis of disorders caused by aberrant cathepsin S expression or activation comprising the administration of an effective amount of a compound as defined in any of claims 1 -21 to an individual suffering from or threatened with the disorder.
27 A method according to claim 26, wherein the disorder is an autoimmune disorder such as MS, RA, juvenile diabetes or asthma.
28. A method according to claim 26, wherein the disorder is chronic pain or psoriasis.
PCT/GB2005/050243 2004-12-13 2005-12-13 Cathepsin s inhibitors WO2006064286A1 (en)

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