WO2007006714A1 - Cysteine protease inhibitors - Google Patents

Cysteine protease inhibitors Download PDF

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WO2007006714A1
WO2007006714A1 PCT/EP2006/063950 EP2006063950W WO2007006714A1 WO 2007006714 A1 WO2007006714 A1 WO 2007006714A1 EP 2006063950 W EP2006063950 W EP 2006063950W WO 2007006714 A1 WO2007006714 A1 WO 2007006714A1
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PCT/EP2006/063950
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French (fr)
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Jussi Kangasmetsa
Stephen Hiscock
Tony Johnson
Bertil Samuelsson
Matt Tozer
Urszula Grabowska
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Medivir Ab
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Definitions

  • This invention relates to inhibitors of cysteine proteases, especially those of the papain superfamily.
  • the invention provides novel compounds useful in the prophylaxis or treatment of disorders stemming from misbalance of physiological proteases such as cathepsin K.
  • the papain superfamily of cysteine proteases is 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 and S, 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.
  • cathepsin K The inappropriate regulation of cathepsin K has been implicated in a number of disorders including osteoporosis, gingival diseases such as gingivitis and periodontitis, Paget's disease, hypercalcaemia of malignancy and metabolic bone disease.
  • gingival diseases such as gingivitis and periodontitis
  • Paget's disease hypercalcaemia of malignancy and metabolic bone disease.
  • cathepsin K is implicated in diseases characterised by excessive cartilege or matrix degradation, such as osteoarthritis and rheumatoid arthritis.
  • Metastatic neoplastic cells typically express high levels of proteolytic enzymes that degrade the surrounding matrix and inhibition of cathepsin K may thus assist in treating neoplasias.
  • UVWXY broadly corresponds to the P3 and P2 of dipeptide cysteine protease inhibitors
  • R 1 is an amide, carbamate or sulphonamide
  • R 3 is H or more speculatively a carbon-linked substituent such as alkyl, alkylaryl etc
  • P1 and P2 are each methylene, or more speculatively speculatively substituted with various carbon chains and cyclic groups.
  • R 1 and R 1 ' are halo; or one of R 1 and R 1 ' is halo, and the other is is H;
  • R 3 is H, -OR 4 , -SR 4 ; R 3' is H; or
  • R 4 is -CrC 6 alkyl, -C 0 -C 3 alkylenecarbocyclyl or -C 0 -C 3 alkyleneheterocyclyl, any of which is optionally substituted with up to three substituents selected from R 7 ;
  • R 5 is -CrC 5 alkyl, -CH 2 CR 5 "C 3 -C 4 -cycloalkyl; R 5 ' is H; or
  • R 5 and R 5 ' together with the carbon to which they are attached define C 4 -C 6 -cycloalkyl
  • R 5 " is H, CrC 2 alkyl, CrC 2 haloalkyl, hydroxyl, OCi-C 2 alkyl, fluoro;
  • R 6 is a stable, optionally substituted, monocyclic or bicyclic carbocycle or heterocycle, wherein the, or each, ring has 4, 5 or 6 ring atoms and 0 to 3 hetero atoms selected from S, O and N and wherein the optional substituents comprise 1 to 3 members selected from R 7 ;
  • R 7 is independently selected from halo, oxo, nitrile, nitro, C1-C4 alkyl, -XNRdRe, -
  • R 8 is independently H, CrC 4 alkyl, C 3 -C 6 cycloalkyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, thiopyranyl, furanyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imid
  • C4alkylR 10 cyano, halo, carboxy, oxo, C1-C4 alkyl, Ci-C4-alkoxy, C1-C4 alkanoyl, carbamoyl;
  • R 10 is 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, any of which is substituted with CrC 4 alkyl, halo, hydroxy, CrC 4 alkoxy
  • X is independently a bond or CrC 4 alkylene
  • Rb is CrC 4 haloalkyl
  • Rc is H, CrC 4 alkyl
  • Re is independently H, CrC 4 alkyl
  • Rd and Re together with the N atom to which they are attached form a morpholine, piperidine, piperazine or pyrrolidine ring optionally substituted with R 9 ; m is independently 0,1 or 2; n is 1 or 2; or a pharmaceutically acceptable salt or prodrug thereof.
  • P1 , P2 and P3 as used herein are provided for convenience only and have their conventional 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.
  • stereochemistry of the proximal end of the P1 group is as depicted in the partial structure below:
  • the halogen of R 1 and/or R 1 is chlorine and most preferably fluorine. It is currently preferred that R 1 is halo, especially fluorine and R 1 is H, but the invention extends to compounds wherein R 1 is halo, especially F and R 1 is H or R 1 and R 1 are each F.
  • R 1 and R 1 are as defined above, but particularly wherein R 1 is F and R 1 is H, especially those with the stereochemistry:
  • R 1 and R 1 are as defined above, especially those with the stereochemistry shown in the partial structure:
  • R 4 in this embodiment include benzyl or C1-C4 alkyl, preferably methyl.
  • compounds within this embodiment may exist as the racemate at the R 3 /R 3 ' centre.
  • the compounds are at least 90%, preferably at least 95%, such as at least 98% enantiomerically pure
  • P1 group may exist in alternative forms, such as and the invention extends to all such alternative forms.
  • R 4 moeity of R 2 comprises C 0 -C 3 alkylenecarbocyclyl C 0 -C 3 alkyleneheterocyclyl
  • preferred R 4 groups include but are not limited to
  • Xi is ethylene, or preferably methylene or more preferebaly a bond
  • D and G are independently chosen from: CR 21 , where R 21 is H, C 0 -Ce alkyl, especially methyl or C 0 -C 3 alkylcarbocyclyl, especially those wherein C 0 or N or N-oxide;
  • E' is chosen from: CH 2 CHR 21 , O. S. SO 2 , NR 21 or N-oxide; J. L, M, R. T. T2, T3 and T4 are independently chosen from: CR 21 or N or N-oxide;
  • R 4 groups for R 2 include where Xi is methylene or preferably a bond and B, D, E', G, J, L, M, R are as defined above.
  • Still more preferred compounds are those in which R 2 is:
  • Xi is methylene or preferably a bond and B, D, E', G, J, L, M, R are as defined above, wherein R 21 is H, methoxy, ethyl, ethyl isopropyl, triflouromethyl, triflouromethyoxy F, CL, S ⁇ 2Me and Ra is H or methyl.
  • R 4 component of R 2 include C 3 -C 6 alkyl, including those which are interrupted with an O or NH as part of the chain, which are unsubstituted or substituted with one or more NH 2 , NHMe, NHC(O)CH 3 , NHMe(C(O)CH3, OH or OMe groups.
  • Particularly favoured values include those which are branched at the alpha position or which include an NH 2 , NHMe, NHC(O)CH 3 , NHMe(C(O)CH 3 , OH or OMe group at the alpha position.
  • R 4 components for R 2 groups include C 3 -C ⁇ cycloalkyl, or the heterocyclic analogues.
  • R 4 components of R 2 include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidine, piperidine, morpholine, tetrahydrofuran, cyclopentene, cyclopentadiene, cyclohexadiene and piperazine.
  • Nitrogen-containing rings may be N-substituted with groups such as C- alkyl, phenyl or benzyl.
  • R 2 comprises a C 3 -C 6 -cycloalkyl group, or a heterocyclic saturated or unsaturated analogue thereof in which the ring system is either connected directly to the remainder of the R 2 moiety or there is one intervening methylene group.
  • a five- or six- membered cyclic ring are most favourable.
  • R 2 groups therefore include: benzoyl; pyridine-2-carbonyl; 1-oxy-pyridine-2-carbonyl; pyridine-3- carbonyl; 1 oxy- pyridine-3-carbonyl; pyridine-4-carbonyl; 1-oxy-pyridine-4-carbonyl; phenyl sulphonyl; pyridine-2-sulphonyl; 1-oxy-pyridine-2- sulphonyl; pyridine-3-sulphonyl; 1-oxy-pyridine- 3-sulphonyl; pyridine-4-sulphonyl; 1-oxy-pyridine-4- sulphonyl; phenylacetyl; phenylcarbamoyl; isobutylcarbamoyl; phenyloxycarbonyl; isobutyloxycarbonyl; pyrrolidine-N-carbonyl; piperidine-N carbonyl; morpholine-N-carbonyl; piperazine-N- carbonyl; 4-methyl-
  • 2-acetylaminoethanoyl 2-acetyl-N methylaminoethanoyl; N 1 N- dimethylaminoacetoyl; 2-aminobutanoyl; N-acetyl- 2 aminobutanoyl; 2-anino-3- methyl butanoyl; N-acetyl-2-amino-3- methylbutanoyl, 2-amino-3,3-dimethylbutanoyl; N- acetyl-2-amino-3,3- dimethylbutanoyl; 2 amino-3-methylpentanoyl; N-acetyl-2-amino-3- methylpentanoyl; pentanoyl; 3 methylpentanoyl; 4-methylpentanoyl; 2- amino-4- methylpentanoyl; N-acetyl-2 amino-4-methylpentanoyl; 2-amino-4,4-dimethylpentanoyl;
  • 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.
  • the stereochemistry of the P2 group preferably corresponds to an L-amino acid as depicted in the partial structure below:
  • the invention also includes all isomers and enantiomers at other chiral centres.
  • R 5 when R 5 ' is H include those embodied by the partial structures:
  • R 5 when R 5 is H includes those with the partial structure: where R 6 is as defined above.
  • R 5 " is H, thus defining a cyclobutylmethyl side chain at P2.
  • R 5 Representative values for R 5 include methyl, hydroxyl, fluoromethyl, difluoromethyl or trifluoromethyl: Accordingly, favoured values of the P2 side chain include,
  • An alternative P2 construction comprises compounds wherein R5 and R5' are cyclised to form cyclopentyl or preferably cyclohexyl.
  • the Ra depicted in formula Il is conveniently hydrogen.
  • E comprises compounds wherein E is -CRbRc-, where Rb is typically halomethyl such as fluoromethyl, difluoromethyl and preferably trifluoromethyl, as illustrated below: where R 6 for the sake of illustration is exemplified with a substituted phenyl and Rc is H.
  • the compound of the invention comprises a high enantiomeric purity, such as more than 80%, preferably more than 95% such as greater than 97% of the S stereoconfiguration at the carbon bearing haloalkyl Rb.
  • a high enantiomeric purity such as more than 80%, preferably more than 95% such as greater than 97% of the S stereoconfiguration at the carbon bearing haloalkyl Rb.
  • the partial structure below represents a typical S-enantiomer with Rb as trifluoromethyl and Rc as H:
  • R 6 is a monocyclic ring with 5 or especially 6 ring atoms, or a bicyclic ring structure comprising a 6 membered ring fused to a 4, 5 or 6 membered ring.
  • R 6 groups include saturated or unsaturated heterocycles or saturated or unsaturated carbocycles, any of which are optionally substituted as described above.
  • R6 generally has an aromatic character, especially in the rign adjacent E.
  • Illustrative variants include C 3 - ⁇ cycloalkyl, phenyl, benzyl, tetrahydronaphthyl, indenyl, indanyl, heterocyclyl such as from azepanyl, azocanyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, thiopyranyl, furanyl, tetrahydrofuranyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl,
  • the saturated heterocycle thus includes radicals such as pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl, piperazinyl, indolinyl, azetidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrofuranyl, hexahydropyrimidinyl, hexahydropyridazinyl, 1 ,4,5,6- tetrahydropyrimidinylamine, dihydro-oxazolyl, 1 ,2-thiazinanyl-1 ,1 -dioxide, 1 ,2,6- thiadiazinanyl-1 ,1 -dioxide, isothiazolidinyl-1 ,1 -dioxide and imidazolidinyl-2,4
  • Preferred monocyclic R 6 groups include substituted pyridyl, substituted pyrimidyl, substituted phenyl, particularly phenyl substituted with a cyclic group such as pyrrolidine-1-yl, piperidine-1-yl, 4-methylpiperidin-1-yl, 4-(piperidin-3-ylmethyl)-piperidin- 1-yl, morpholin-4-yl, 4-methylpiperazin-1-yl, 2-morpholin-4-yl-ethylamino, 2-morpholin- 4-yl-ethyloxy, 1-pyrid-2-ylmethylamino, piperazin-1-yl, piperid-4-yl or N-piperazinyl, N- substituted with Ra or piperidin-1-yl which is 4-substituted with -NRaRb.
  • a phenyl R 6 is conveniently substituted at the 3 or 4 position (para or meta), for example with such a cyclic group.
  • cyclic substituents to a monocyclic R 6 include aryl groups such as phenyl or a 5 or 6 membered heteroaryl group such as thiophene, furyl, triazole, thiazole, diazole, pyrazole or pyrrolidine.
  • Favoured cyclic substituents in this context include thiazol-2-yl, pyrid-3-yl and especially pyrid-2-yl, thien-2-yl or thiazol-5-yl.
  • This cyclic substituent is typically bonded direct to such R 6 species (ie X is a bond), but may also for example comprise an amine spacer such as -NH-, -N(Me), - CH 2 NH, -CH 2 N(Me)-, a CrC 3 alkyl spacer such as -CH 2 - or a CrC 3 -alkyloxy spacer such as ethyloxy
  • any of the cyclic substituents to R 6 in the immediately preceding paragraph may be substituted as described above with R 10 .
  • a heterocycle R 7 group such as thiazolyl can be substituted with d-C 4 alkyl such as methyl or disubstituted such as methyl at one position and a cyclic group at another
  • any of the cyclic substituents to R 6 in the two immediately preceding paragraphs may itself be substituted with a cyclic group (that is R 7 comprises an R 9 moiety) typically a saturated heterocyclic group such as piperidine, piperazine or morpholine, which saturated cyclic group is optionally substituted, for example with d- C 3 alkyl, fluoro, diflouro, CrC 3 alkyloxy or Ci-C 3 alkyloxyCi-C 3 alkyl.
  • this saturated cyclic group (ie R 9 ) may be spaced from the R 6 group by X (eg Ci-C 3 alkyl), amine (eg -NH-), amide, sulphonamide etc, but is typically bonded directly or via methylene.
  • X eg Ci-C 3 alkyl
  • amine eg -NH-
  • amide eg -NH-
  • sulphonamide amide
  • R 9 groups in accordance with the immediately preceding paragraph include heterocycles such as pyrrolidine-1-yl, piperidine-1-yl, 4-methylpiperidin-1-yl, 4- (piperidin-3-ylmethyl)-piperidin-1-yl, morpholin-4-yl, 4-methylpiperazin-1-yl, 2- morpholin-4-yl-ethylamino, 2-morpholin-4-yl-ethyloxy, 1 -pyrid-2-ylmethylamino, piperazin-1-yl, piperid-4-yl or N-piperazinyl, N-substituted with Ra or piperidin-1-yl which is 4-substituted with -NRaRb,
  • heterocycles such as pyrrolidine-1-yl, piperidine-1-yl, 4-methylpiperidin-1-yl, 4- (piperidin-3-ylmethyl)-piperidin-1-yl, morpholin-4-yl, 4-methylpiperazin-1-y
  • R 9 substituents include 4-substituted piperazin-4-yl, such as 4- methyl-piperazin-4-yl or 4-methyloxyethyl-piperazin-4-yl, piperid-1-ylmethyl which is optionally 4-substituted with fluoro or diflouro or morpholinylmethyl.
  • Alternative preferred substituents to a monocyclic R 6 include -NRaRb, -CH 2 NRaRb, CrC 4 straight or branched alkyl or -O-R 9 .
  • R 6 groups thus include:
  • R 6 groups include
  • Rq and Rq' are independently selected from H, C1-C4 alkyl or Ci-C4alkanoyl or together define an unsaturated 5-7 membered ring, such as piperidine, piperazine or morpholine, which may in turn be substituted with groups corresponding to R 10 , particularly CrC 4 alkyl, fluoro or difluoro.
  • R 6 groups include
  • Representative bicyclic groups for R 6 include naphthylenyl, especially naphthylen-2-yl; benzo[1 ,3]dioxolyl, especially benzo[1 ,3]dioxol-5-yl, benzofuranyl, especially benzofuran-2-yl, and especially d-C ⁇ alkoxy substituted benzofuranyl, more especially 5-(2-piperazin-4-carboxylic acid tert-butyl ester- ethoxy) benzofuran-2-yl, 5-(2-morpholino-4-yl-ethoxy)-benzofuran-2-yl, 5-(2-piperazin-l-yl- ethoxy)benzofuran-2-yl, 5-(2-cyclohexyl-ethoxy)-benzofuran-2-yl; 7-methoxy-benzofuran-2-yl, 5-methoxy-benzofuran-2-yl, 5,6-dimethoxy-benzofur
  • Favoured R 6 groups include bicyclic rings such as napthyl, quinoloyl, benzofuranyl, benzothienyl, indolyl and indolinyl, particularly where the linkage is to the 2 position of the ring.
  • Favoured substituents to a bicyclic R 6 group include pyrrolidine-1-yl, piperidine-1-yl, 4-methylpiperidin-1-yl, 4-(piperidin-3-ylmethyl)-piperidin-1-yl, morpholin- 4-yl, 4-methylpiperazin-1-yl, 2-morpholin-4-yl-ethylamino, 2-morpholin-4-yl-ethyloxy, 1- pyrid-2-ylmethylamino, piperazin-1-yl, piperid-4-yl or N-piperazinyl, N-substituted with Ra or piperidin-1-yl which is 4-substituted with -NRaRb.
  • Especially preferred substituents, particularly in conjunction with benzofuranyl include 2-morpholin-4-yl- ethyloxy and N-methyl-piperidin-4-yloxy and those defined below.
  • a currently favoured bicyclic R 6 group is optionally substituted benzothiazol or benzofuryl or benzoxazolyl, including those wherein the substituent is -OR 9 or -NRbR 9 .
  • favoured R 6 groups include benzofur-2-yl, unsubstituted and/or substituted with CrC 4 alkyl, or CrC 4 haloalkyl at the 3 position and/or or substituted in the 5 position with a saturated heterocycle such as piperidine, piperazine or morpholine, which is optionally substituted with C1-C 3 alkyl and/or spaced from the benzofuryl by oxy, methyloxy or ethyloxy.
  • Particularly favoured benzofuryl R 6 groups thus include:
  • X is typically methylene or especially a bond.
  • Halogen or halo includes bromo, chloro and especially fluoro.
  • Haloalkyl means an alkyl group as defined above where at least one carbon atom bears 1 to 3 halogen atoms, preferably fluorine atoms.
  • Representative haloalkyl group includes fluoromethyl, difluoromethyl, trifluoromethyl, 2, fluoroethyl, 2,2difluorethyl, 2,2,2 trifluorethyl and the like.
  • 'Ci-C ⁇ alkyl 1 (also abbreviated as d-C ⁇ alk, or used in compound expressions such as Ci-C 6 alkyloxy etc) as applied herein is meant to include straight and branched chain aliphatic carbon chains such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t- butyl, pentyl, isopentyl, hexyl, heptyl and any simple isomers thereof.
  • the alkyl group may have an unsaturated bond.
  • any C atom in d-C ⁇ alkyl may optionally be substituted by one, two or where valency permits three halogens and/or substituted or the alkylene chain interrupted by a heteroatom S, O, NH. If the heteroatom is located at a chain terminus then it is appropriately substituted with one or 2 hydrogen atoms.
  • CrC n alkyl has the corresponding meaning to CrC 6 alkyl adjusted as necessary for the carbon number.
  • 'Co-Csalkylaryl' as applied herein is meant to include an aryl moiety such as a phenyl, naphthyl or phenyl fused to a C 3 -C 7 cycloalkyl for example indanyl, which aryl is directly bonded (i.e. C 0 ) or through an intermediate methyl, ethyl, propyl, or isopropyl group as defined for CrC 3 alkylene above.
  • aryl moiety such as a phenyl, naphthyl or phenyl fused to a C 3 -C 7 cycloalkyl for example indanyl, which aryl is directly bonded (i.e. C 0 ) or through an intermediate methyl, ethyl, propyl, or isopropyl group as defined for CrC 3 alkylene above.
  • aryl and/or its fused cycloalkyl moiety is optionally substituted with 1-3 substituents selected from halo, hydroxy, nitro, cyano, carboxy, CrC 6 alkyl, CrC 6 alkoxy, Ci-C 6 alkoxyCi-C 6 alkyl, d- C 6 alkanoyl, amino, azido, oxo, mercapto, nitro C 0 -C 3 alkylcarbocyclyl, C 0 - Csalkylheterocyclyl.
  • "Aryl" has the corresponding meaning, i.e. where the C 0 -C 3 alkyl linkage is absent.
  • 'Co-C 3 alkylC 3 C 7 cycloalkyl' 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 methyl, ethyl or proyl group as defined for CrC 3 alkylene above.
  • the cycloalkyl group may contain an unsaturated bond.
  • cycloalkyl moiety is optionally substituted with 1-3 substituents selected from halo, hydroxy, nitro, cyano, carboxy, CrC 6 alkyl, CrC 6 alkoxy, Ci-C ⁇ alkoxyCrC ⁇ alkyl, d-C ⁇ alkanoyl, amino, azido, oxo, mercapto, nitro Co- Csalkylcarbocyclyl, Co-Csalkylheterocyclyl.
  • Co-Csalkylcarbocyclyl as applied herein is meant to include Co-C 3 alkylaryl and Co- C 3 alkylC 3 -C 7 cycloalkyl. Unless otherwise indicated the aryl or cycloalkyl group is optionally substituted with 1-3 substituents selected from halo, hydroxy, nitro, cyano, carboxy, CrC 6 alkyl, CrC 6 alkoxy, Ci-C 6 alkoxyCi-C 6 alkyl, CrC 6 alkanoyl, amino, azido, oxo, mercapto, nitro, C 0 -C 3 alkylcarbocyclyl and/or Co-Csalkylheterocyclyl.
  • Carbocyclyl has the corresponding meaning, i.e. where the Co-C 3 alkyl linkage is absent
  • 'Co-C 3 alkylheterocycylyl' as applied herein is meant to include a monocyclic, saturated or unsaturated, heteroatom-containing ring such as piperidinyl, morpholinyl, piperazinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazinolyl, isothiazinolyl, thiazolyl, oxadiazolyl, 1 ,2,3-triazolyl, 1 ,2,4-triazolyl, tetrazolyl, furanyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazolyl, or any of such groups fused to a phenyl ring, such as quinolinyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazinolyl, benzisothi
  • hetero ring and/or its fused phenyl moeity is optionally substituted with 1-3 substituents selected from halo, hydroxy, nitro, cyano, carboxy, CrC 6 alkyl, CrC 6 alkoxy, Ci-C 6 alkoxyCi-C 6 alkyl, CrC 6 alkanoyl, amino, azido, oxo, mercapto, nitro, Co-C 3 alkylcarbocyclyl, Co-C 3 alkylheterocyclyl.
  • "Heterocyclyl” and “Heteroaryl” have the corresponding meaning, i.e. where the Co-C 3 alkyl linkage is absent.
  • heterocycyl and carbocyclyl moieties within the scope of the above definitions are thus a monocyclic ring with 5 or especially 6 ring atoms, or a bicyclic ring structure comprising a 6 membered ring fused to a 4, 5 or 6 membered ring.
  • Typical such groups include C 3 -C 8 cycloalkyl, phenyl, benzyl, tetrahydronaphthyl, indenyl, indanyl, heterocyclyl such as from azepanyl, azocanyl, 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, benzofuranyl, be
  • the saturated heterocycle moiety thus includes radicals such as pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl, piperazinyl, indolinyl, azetidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrofuranyl, hexahydropyrimidinyl, hexahydropyridazinyl, 1 ,4,5,6- tetrahydropyrimidinylamine, dihydro-oxazolyl, 1 ,2-thiazinanyl-1 ,1 -dioxide, 1 ,2,6- thiadiazinanyl-1 ,1 -dioxide, isothiazolidinyl-1 ,1 -dioxide and imidazolidinyl-2
  • heterocycle may be condensed with a phenyl ring to form a bicyclic ring system.
  • Favoured compounds of the invention include those permutations formed by independent selection of a P3, P2 and P1 member from each of Tables A, B and C:
  • Additional aspects of the invention include a pharmaceutical composition
  • a pharmaceutical composition comprising a compound as defined above and a pharmaceutically acceptable carrier or diluent therefor.
  • a further aspect of the invention is the use of a compound as defined above in the manufacture of a medicament for the treatment of disorders mediated by cathepsin K, such as: osteoporosis, gingival diseases such as gingivitis and periodontitis, Paget's disease, hypercalcaemia of malignancy metabolic bone disease diseases characterised by excessive cartilege or matrix degradation, such as osteoarthritis and rheumatoid arthritis, bone cancers including neoplasia, pain.
  • disorders mediated by cathepsin K such as: osteoporosis, gingival diseases such as gingivitis and periodontitis, Paget's disease, hypercalcaemia of malignancy metabolic bone disease diseases characterised by excessive cartilege or matrix degradation, such as osteoarthritis and rheumatoid arthritis, bone cancers including neoplasia, 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 Formula Il include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, isethionate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, proprionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-
  • the compounds of Formula Il may in some cases be isolated as the hydrate. Hydrates are typically prepared by recrystallisation from an aqueous/organic solvent mixture using organic solvents such as dioxin, tetrahydrofuran or methanol.
  • organic solvents such as dioxin, tetrahydrofuran or methanol.
  • the N-oxides of compounds of Formula (I) can be prepared by methods known to those of ordinary skill in the art.
  • N-oxides can be prepared by treating an unoxidized form of the compound of Formula (I) with an oxidizing agent (e.g., trifluoroperacetic acid, permaleic acid, perbenzoic acid, peracetic acid, meta- chloroperoxybenzoic acid, or the like) in a suitable inert organic solvent (e.g., a halogenated hydrocarbon such as dichloromethane) at approximately O 0 C.
  • an oxidizing agent e.g., trifluoroperacetic acid, permaleic acid, perbenzoic acid, peracetic acid, meta- chloroperoxybenzoic acid, or the like
  • a suitable inert organic solvent e.g., a halogenated hydrocarbon such as dichloromethane
  • the N-oxides of the compounds of Formula (I) can be prepared from the N-oxide of an appropriate starting material.
  • Compounds of Formula (I) in unoxidized form can be prepared from N-oxides of compounds of Formula (I) by treating with a reducing agent (e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus bichloride, tribromide, or the like) in an suitable inert organic solvent (e.g., acetonitrile, ethanol, aqueous dioxane, or the like) at 0 to 8O 0 C.
  • a reducing agent e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus bichloride, tribromide, or the like
  • an inert organic solvent e.g., acetonitrile, ethanol, aqueous dioxane, or the like
  • Compounds of Formula (II) can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomer. While resolution of enantiomers can be carried out using covalent diasteromeric derivatives of compounds of Formula (I), dissociable complexes are preferred (e.g., crystalline; diastereoisomeric salts). Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by taking advantage of these dissimilarities.
  • the diastereomers can be separated by chromatography, for example HPLC or, preferably, by separation/resolution techniques based upon differences in solubility.
  • the optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization.
  • a more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture can be found in Jean Jacques Andre Collet, Samuel H. Wilen, Enantiomers, Racemates and Resolutions, John Wiley & Sons, Inc. (1981 ).
  • the invention extends to prodrugs, solvates, complexes and other forms releasing a compound of formula Il in vivo. 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 Il 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 and the patient 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 ⁇ M, more preferably 0.01-10 ⁇ M, such as 0.1-25 ⁇ M are typically desirable and achievable.
  • Compounds of the invention are prepared by a variety of solution and solid phase chemistries.
  • the compounds are typically prepared as building blocks reflecting the P1 , P2 and P3 moieties of the end product inhibitor.
  • 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 Il are intended to be within the scope of the invention, regardless of binding mode.
  • the P1 building block will be an N-protected- 3-oxo-hexahydro- furo[3,2-b]pyrrole (substituted as necessary), P2 will be an N-protected amino acid, 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.
  • 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 carboniocarboxylic 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.
  • standard coupling procedures such as the azide method, mixed carboniocarboxylic acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active
  • 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.
  • suitable coupling agents are N.N'-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'.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'-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-methyl pyrrolidine or 4-DMAP is added to maintain the reaction mixture at a pH of about 8.
  • the reaction temperature usually ranges between 0 0 C and 50 0 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 N- 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) trialkyl
  • 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 first stage in a synthesis of compounds of the general formula Il is typically the preparation in solution of a functionalized P1 building block.
  • Different nomenclature of compounds according to the present invention can be used. For convenience the carbohydrate nomenclature will generally be used herein.
  • RI-Br, RT, 24 h xix. 10 % Pd on C, ethanol, H 2 , RT, 16 h; xx. CbzLeuOH, EDC, HOBt, Et 3 N; xxi. 10 % Pd on C, ethanol, H 2 , RT, 10 h; xxii. R2COOH, EDC, HOBt, Et 3 N, RT, 18 h; xxiii. SO 3 - pyridine complex, DMSO, THF, Et 3 N, RT, 18 h.
  • the synthetic scheme can then continued as outlined previously to build up the final compound.
  • the amine functionality of 4-benzyl-6-fluoro-octahydro-pyrrolo[3,2- b]pyrrol-3-ol is reacted with the appropriate electrophilic component to obtain a wide variety of groups at the R 2 position.
  • benzyl protection is removed using 4 M HCI in dioxane and the free amine coupled with the appropriate S2 residue, exemplied by L-Leu in the scheme.
  • Subsequent removal of the Cbz moiety and coupling on the P3 residue using the appropriate R 6 acid affords the alcohol which serves as the precursor for the final step. Oxidation of the alcohol affords the required final compound.
  • a typical scheme towards a bicyclic P1 group starts with the ring closure of a suitably protected intermediate which is available in 4 steps from 1 ,2:5,6-di-O-isopropylidene-D- allofuranose, described by Mayer Kurs Reckendorf, Chem. Ber. 101 (1968), 3802-3807, giving a precursor of 3S, 4R stereochemistry.
  • Scheme 1 a) H 2 , Pd/C, methanol, b) benzylchloroformate, pyridine, dichloromethane
  • the azide group of derivative 1 is reduced for example by catalytic hydrogenation using palladium on charcoal or other catalysts suitable, in a suitable solvent such as an alcohol, like ethanol or methanol into the free amine.
  • a suitable solvent such as an alcohol, like ethanol or methanol into the free amine.
  • the obtained nucleophilic nitrogen reacts spontaneously, or optionally in the presence of a suitable base like such as triethyl amine or sodium acetate, with the C-6 position forming a 5,5- bicycle.
  • the leaving group at C-6 is not limited to sulfonate esters, but also other leaving groups such as halogen could be used throughout the synthesis of compounds according to the present invention.
  • the reduction of the azide residue into an amine could also be performed by other methods known from literature, such as treating the azide derivative with a trialkyl- or triarylphosphine followed by hydrolysis of the formed imine derivative.
  • the amine may be N-protected with a suitable protecting group such as a carbamate, like benzyl carbamate of compound 3 or any other similar protecting group which is normally not cleaved with acid.
  • suitable protecting groups which can be found in: Protective groups in organic chemistry, 3 rd edition, 1999, Theodora W. Greene and Peter G. M. Wuts (Wiley&sons).
  • the azide group of derivative 5 is reduced for example by catalytic hydrogenation using palladium on charcoal or other catalysts suitable, in a suitable solvent such as an alcohol, like ethanol or methanol into the free amine.
  • a suitable solvent such as an alcohol, like ethanol or methanol into the free amine.
  • the obtained nucleophilic nitrogen reacts spontaneously, or optionally in the presence of a suitable base like such as triethyl amine or sodium acetate, with the C-6 position forming a 5,5-bicycle which can be N-protected with a suitable protecting group such as its benzyl carbamate (Cbz) to give compound 6.
  • 3-azido-3-deoxy-1 ,2:5,6-di-O-isopropylidene-D-idofuranose (Bull. Chem. Soc. Japan, 57, 7(1984), 237-241) could be a suitable starting material for the 3R, 4S bicycle according to Scheme 3.
  • Compound 6 can be treated with a mild acid, such as diluted acetic acid or similar, which can selectively hydrolyze the 5,6-acetal of compound 6, to obtain a diol.
  • the primary alcohol can be selectively reacted with an alkyl- or arylsulfonyl chloride like p- toluenesulfonyl chloride to give compound 7.
  • the azide group of derivative 7 is reduced for example by catalytic hydrogenation using palladium on charcoal or other catalysts suitable, in a suitable solvent such as an alcohol, like ethanol or methanol into the free amine.
  • nucleophilic nitrogen reacts spontaneously, or optionally in the presence of a suitable base like such as triethyl amine or sodium acetate, with the C-6 position forming a 5,5-bicycle which can be N-protected with a suitable protecting group such as its benzyl carbamate (Cbz) to give compound 8.
  • a suitable base like such as triethyl amine or sodium acetate
  • the ring closure is not limited to the substrates shown above but could also be applied to derivatives as depicted in Scheme 4.
  • Rx in Scheme 4 may be chosen from methyl, trifluoromethyl, p-methylphenyl or similar residues present in readily available alkylsulfonylhalides, preferably a bulky Rx suitable for regioselective reaction on the primary alcohol of a diol as described in Chem. Ber. 101 (1968), 3802-3807.
  • R 1' and R 2' are R 1 and R 2 as defined.
  • Pg could be a suitable protecting group such as a carbamate, like benzyl carbamate or any similar protecting group which is not normally cleaved with acid.
  • Rx in Scheme 5 can be chosen from methyl, trifluoromethyl, p-methylphenyl or similar residues present in readily available alkylsulfonylhalides, preferably a bulky Rx suitable for regioselective reaction on the primary alcohol of a diol as described in Chem. Ber. 101 (1968), 3802-3807.
  • R 1' and R 2' are R 1 and R 2 as defined above.
  • Ry can be hydrogen or a hydroxyl protective group, preferably an ether type protective group.
  • Preferably Ry is hydrogen.
  • PG could be a suitable N-protecting group such as a carbamate, for derivatives in Scheme 5, Ry is typically hydrogen.
  • Compound 9 is protected with a suitable acid stable protecting group such as substituted methyl ether, in particular a benzyl ether, by treating the mono-ol 9 with a base such as sodium hydride or sodium hydroxide in an aprotic solvent such as N 1 N- dimethylformamide (DMF) in the presence of the desired alkylating agent such as the benzyl halide, in particular benzyl bromide.
  • a base such as sodium hydride or sodium hydroxide
  • an aprotic solvent such as N 1 N- dimethylformamide (DMF)
  • the desired alkylating agent such as the benzyl halide, in particular benzyl bromide.
  • the obtained material can then be reduced into compound 10 according to methods described by G. J. Ewing and M. J. Robins, Org. Lett. 1 , 4, 1999, 635-636, or by references therein.
  • the reduction is performed with excess boron trifluoride etherate in the presence of a reducing agent such as trialkylsilane, in particular with excess triethylsilane in a suitable non-protic solvent such as dichloromethane.
  • a reducing agent such as trialkylsilane
  • a suitable non-protic solvent such as dichloromethane.
  • Catalytic hydrogenation of compound 10 using for example palladium-on-charcoal in a suitable solvent or solvent mixture such as ethyl acetate-ethanol in a hydrogen atmosphere, in the presence of di-tert-butyl dicarbonate followed by treatment of the product with acetic anhydride in pyridine gives intermediate 11.
  • the mono-ol 12 is obtained.
  • a fluorine can be introduced on compound 12, and the bicyclic compound then N- deprotected according to Scheme 7.
  • Compound 13 can be treated with a fluorinating agent such as [bis-(2- methoxyethyl)aminosulfur trifluoride] (Deoxo-Fluor®) or with similar fluorinating agents such as diethylaminosulfur trifluoride (DAST) which gives the product 14 with inversion of configuration at C-5.
  • a fluorinating agent such as [bis-(2- methoxyethyl)aminosulfur trifluoride] (Deoxo-Fluor®) or with similar fluorinating agents such as diethylaminosulfur trifluoride (DAST) which gives the product 14 with inversion of configuration at C-5.
  • Compound 14 is then deacetylated by treatment for example with methanolic sodium methoxide, or any similar alkaline solutions with an inorganic base such as sodium hydroxide or sodium carbonate, followed by N-deprotection using acidic conditions such as dichloromethane-trifluoroacetic acid solutions or other methods which could be found in: Protective Groups in Organic Chemistry, 3 rd edition, 1999, Theodora W. Greene and Peter G. M. Wuts (Wiley & Sons).
  • the epimeric fluorine can be obtained by treating derivative 9 above according to Scheme 8.
  • a benzylating agent like benzyl bromide or benzyl chloride in the presence of a base, such as sodium hydride or sodium hydroxide in a aprotic polar solvent, such as N 1 N- dimethylformamide gives derivative 22.
  • a base such as sodium hydride or sodium hydroxide in a aprotic polar solvent, such as N 1 N- dimethylformamide
  • a trialkyl silane such as triethyl silane
  • a Lewis acid such as boron trifluoride etherate or trimethylsilyl trifluoromethanesulfonate
  • the resulting azide can then be selectively reduced by catalytic hydrogenation using for example Palladium on charcoal in the presence of di-tert-butyl carbonate to obtain compound 23.
  • the azide could be reduced with other methods known from literature such as triphenylphosphine-water, followed by protection giving a suitable carbamate.
  • compound 23 could be treated with an acylating agent such as an acyl chloride or acid anhydride, such as benzoyl chloride, in neat organic base such as pyridine or triethyl amine, or in a mixture of an aprotic solvent such as dichloromethane and a base to give compound 24.
  • an excess fluorinating agent such as Deoxo-Fluor®
  • DAST diethylaminosulfur trifluoride
  • an aprotic solvent such as dichloromethane or 1 ,2-dichloroethane.
  • alkali such as methanolic sodium methoxide
  • Selective introduction of a sulfonate ester at the primary alcohol can be accomplished by treating the compound 29 with a small excess of alkyl- or arylsulfonyl chloride in the presence of a base such as pyridine in suitable solvent such as dichloromethane, adding the sufonylating agent at reduced temperature and slowly increase up to room temperature, which gives mono-ol 30.
  • a base such as pyridine
  • suitable solvent such as dichloromethane
  • a derivative such as compound 32 (available as described above or with methods well known in the art) with the substituents at C-3 and C-4 in cis relationship, Lg being a leaving group such as halogen or a sulfonate ester, and with R equal to an azide or a nitrogen protected with a suitable N-protecting group, can be treated with a fluorinating agent such as mentioned above, producing compound 33.
  • a fluorinating agent such as mentioned above
  • the P1 building block is then elongated with the natural or non natural P2 amino acid and the P3 group by conventional solution or solid phase chemistries, such as those outlined or exemplified below, or disclosed in WO00/69855 or WO02/057270.
  • P2 and P3 groups are either commercially available as enantiomers or resolvable from the racemate or obtainable using simple chemical transformations known to one skilled in the art.
  • 4-(methyl-piperazine-1-yl)-benzoic acid can be obtained using Buchwald chemistry (S. L. Buchwald & J. P. Wolfe, Journal of Organic Chemistry, 2000, 65, 1144) and subsequently elaborated.
  • P3 cores such as 4-(1 -piperidin-4-yl)- benzoic acid are prepared from 1-(4-phenyl-piperidine-1-yl)-ethanone using a Friedel- Crafts acylation reaction and subsequently elaborated using standard chemical transformations known to one skilled in the art.
  • P3 moieties such as 5-[2-(4-morpholinyl)ethoxy]-2-benzofuran-2-carboxylic acid, are prepared using Mitsunobu reactions on solid phase as detailed by L. S. Richter & T. R. Gadek in Tetrahedron Lett., 1994, 35, 4705. NMM
  • the P1 building block as the hydroxyl may be elongated and subsequently oxidised as shown in Scheme 14.
  • 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.
  • R 6 and Rc are as defined above and LG is a conventional leaving group such as trifluoromethansulfonate, and the like , with the N-deprotected P1/P2 building block shown above.
  • the reaction is carried out in a suitable organic solvent, including but not limited to, halogenated organic solvents such as methylene chloride, 1 ,2- dibromoethane, and the like, ethereal solvents such as diethyl ether, tetrahydrofuran, acetonitrile, or aromatic solvents such as benzene, toluene, xylene, and the like, or mixtures thereof and optionally in the presence of an organic or inorganic base.
  • halogenated organic solvents such as methylene chloride, 1 ,2- dibromoethane, and the like
  • ethereal solvents such as diethyl ether, tetrahydrofuran, acetonitrile, or aromatic solvents such as benzene
  • the organic base is triethylamine, pyridine, N-methylmorpholine, collidine, diisopropylethylamine, and the like.
  • the inorganic base is cesium carbonate, sodium carbonate, sodium bicarbonate, and the like.
  • the reaction is optionally carried out in the presence of a drying agent such as molecular sieves. Preferably, the reaction is carried out at room temperature.
  • the intermediate can be prepared by methods well known in the art.
  • a compound where R 6 is phenyl or 4- fluorophenyl, Rb is trifluoromethyl, and Rc is hydrogen can be readily prepared from commercially available 2,2,2 trifluoroacetophenone or 2,2,2, 4'-tetrafluoroacetophone respectively, by reducing the keto group to an alcoholic group by suitable reducing agent such as sodium borohydride, lithium aluminum hydride, and the like.
  • suitable reducing agent such as sodium borohydride, lithium aluminum hydride, and the like.
  • the solvent used depends on the type of reducing agent. For example, when sodium borohydride is used the reaction is carried out in an alcoholic organic solvent such as methanol, ethanol, and the like. When lithium aluminum hydride is used the reaction is carried out in an ethereal solvent such as tetrahydrofuran, and the like.
  • Chirally enriched intermediate can be obtained by reduction of the corresponding halogenated acetophenone with a suitable reducing agent such as catechol borane or BH 3 -DMS complex in the presence of a suitable catalyst such as (A or (R) CBS catalyst or (A or (R)-,a -diphenyl-2- pyrrolidine-methanol in the presence of BBN.
  • a suitable reducing agent such as catechol borane or BH 3 -DMS complex
  • a suitable catalyst such as (A or (R) CBS catalyst or (A or (R)-,a -diphenyl-2- pyrrolidine-methanol in the presence of BBN.
  • LG is a suitable leaving group such as trifluoromethansulfonate, and PG a suitable hydroxyl protecting group such as trialkylsilyl, and the like, under the reaction conditions described above.
  • the resulting O-protected hydroxyethylamide is oxidised to the corresponding carboxlic acid and couple to the P1 building block as described below.
  • Suitable hydroxyl protecting groups and reaction conditions for putting them on and removing them can be found in Greene, T.W.; and Wuts, P. G. M. Protecting Groups in Organic Synthesis; John Wiley & Sons, Inc. I 999.
  • the P2 hydroxyethylamine can be prepared from the corresponding natural and unnatural amino acids by methods well known in the art. Some such procedures are described in PCT Application Publication No. WO 03/075836, the disclosure of which is incorporated herein by reference in its entirety.
  • R6 Rb where R 6 is a cyclic group as defined above and Rb is halomethyl, preferably trifluoromethyl with the N-deprotected, carboxy-protected P2 building block or the P1/P2 building block outlined above under reductive amination reaction conditions.
  • the reaction is carried out in the presence of a suitable dehydrating agent such as TiCU, magnesium sulfate, isopropyl trifluoroacetate, in the presence of a base such as diisopropylethylamine, pyridine, and the like and in a suitable organic solvent such as methylene chloride to give an imine.
  • the imine is reduced with a suitable reducing agent such as sodium borohydride, sodium cyanoborohydride, and the like in a suitable organic solvent such as methanol, ethanol, and the like.
  • a suitable oxidizing agent such as Jones oxidizing reagent or H 5 l ⁇ 6 /Cr ⁇ 3 , and the like
  • elongation is typically carried out in the presence of a suitable coupling agent e.g., benzotriazole-1- yloxytrispyrrolidinophosphonium hexafluorophosphate (PyBOP), O- benzotriazol-l-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate (HBTU) , 0-(7-azabenzotriazol-1-yl)-1 ,1 ,3,3-tetramethyl-uronium hexafluorophosphate (HATU), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), or 1 ,3-dicyclohexyl carbodiimide (DCC), optionally in the presence of l-hydroxybenzotriazole (HOBT), and a base such as N 1 N- diisopropylethylamine, triethyl
  • reaction solvents are inert organic solvents such as halogenated organic solvents (e.g., methylene chloride, chloroform, and the like), acetonitrile, N 1 N dimethylformamide, ethereal solvents such as tetrahydrofuran, dioxane, and the like.
  • halogenated organic solvents e.g., methylene chloride, chloroform, and the like
  • acetonitrile e.g., N 1 N dimethylformamide
  • ethereal solvents such as tetrahydrofuran, dioxane, and the like.
  • the above elongation coupling step can be carried out by first converting the P3/P2 building block into an active acid derivative such as succinimide ester and then reacting it with the P1 amine.
  • the reaction typically requires 2 to 3 h to complete.
  • the conditions utilized in this reaction depend on the nature of the active acid derivative. For example, if it is an acid chloride derivative of 4, the reaction is carried out in the presence of a suitable base (e.g. triethylamine, diisopropylethylamine, pyridine, and the like).
  • Suitable reaction solvents are polar organic solvents such as acetonitrile, N 1 N- dimethylformamide, dichloromethane, or any suitable mixtures thereof.
  • haloalkyl hemiacetal is reacted with the protected P2 building block to yield the depicted 2-(1-hydroxymethylamino) acetate intermediate.
  • the reaction is carried out in the presence of a catalytic amount of an acid such as p- toluenesulfonic acid and in an aromatic hydrocarbon solvent such as toluene, benzene, and the like.
  • N-protecting group or "N-protected” as used herein refers to those groups intended to protect the N-terminus of an amino acid or peptide or to protect an amino group against undesirable reactions during synthetic procedures.
  • N- protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t- butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoracetyl, trichloroacetyl, phthalyl, o- nitrophenoxyacetyl, ⁇ -chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4- nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl, and the like, carbamate forming groups such as benzyloxycarbonyl, p- chlorobenzyloxycarbon
  • Favoured N-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz).
  • Hydroxy and/or carboxy protecting groups are also extensively reviewed in Greene ibid and include ethers such as methyl, substituted methyl ethers such as methoxy methyl, methylthiomethyl, benzyloxymethyl, t-butoxymethyl, 2-methoxyethoxymethyl and the like, silyl ethers such as trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS) tribenzylsilyl, triphenylsilyl, t-butyldiphenylsilyl triisopropyl silyl and the like, substituted ethyl ethers such as 1-ethoxy methyl, 1-methyl-1-methoxyethyl, t-butyl, allyl, benzyl, p- methoxybenzyl, dipehenylmethyl, triphenylmethyl and the like, aralkyl groups such as trityl, and pixyl (9-hydroxy-9-phenylxant
  • Ester hydroxy protecting groups include esters such as formate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate, pivaloate, adamantoate, mesitoate, benzoate and the like.
  • Carbonate hydroxy protecting groups include methyl vinyl, allyl, cinnamyl, benzyl and the like.
  • Methanesulfonic acid 5-(2,2-dimethyl-[1 ,3]dioxolan-4-yl)-2,2-dimethyl tetrahydro- furo[2,3-d][1 ,3]dioxol-6-yl ester was dissolved in dimethylformamide (50 ml) and treated with sodium azide (7.3 g, 112.95 mmol).
  • Step c) 1 -(6-Azido-2,2-dimethyl-tetrahydro-furo[2,3-d][1 ,3]dioxol-5-yl)-ethane-1 ,2- diol
  • reaction mixture was then treated dropwise and at O 0 C, with Deoxo-Fluor ([bis(2- methoxyethyl)amino]sulfur trifluoride, 5.00 mmol) and stirred at room temperature overnight.
  • the reaction mixture was quenched with saturated aqueous NaHCO 3 , and partitioned between dichloromethane and water. The organic layer was dried and concentrated, and the residue was purified by column chromatography to yield the title compound (1.37 mmol, 55%).
  • reaction was monitored by MS. The following morning, the reaction mixture was concentrated and the residue suspended in methanol and evaporated several times to remove the boron species. Anhydrous acetone was added to the residue (10 ml) and a catalytic amount of cone, sulfuric acid was added to the reaction mixture (3 drops). The mixture was stirred at room temperature for 3 h, then neutralised with solid sodium carbonate, filtered and concentrated. The residue was purified by column chromatography to afford the title compound.
  • Step h) 1 -Benzyl-2-(2,2-dimethyl-[1 ,3]dioxolan-4-yl)-4-fluoro-pyrrolidin-3S- ol
  • Toluene-4-sulfonic acid 2-(3-azido-1 -benzyl-4-fluoro-pyrrolidin-2-yl)-2-hydroxy-ethyl ester is dissolved in a mixture of methanol and dioxane (1 :5) and treated with triphenylphosphine.
  • the reaction mixture is stirred at room temperature for 3 h, and then treated with water, and stirred at room temperature overnight.
  • the solvents are removed under vaccum, and the residue taken up on diluted aqueous HCI, and washed with ether.
  • the aqueous phase is freeze-dried to afford the title compound.
  • the synthetic scheme can then continued as outlined in Scheme A above to build up the final compound.
  • Olefin metathesis (RCM, ring closing metathesis) is typically performed with a Ru-based catalyst such as the one reported by Miller, S.J., Blackwell, H. E.; Grubbs, R.H. J. Am. Chem. Soc. 118, (1996), 9606-9614, Kingsbury, J. S., Harrity, J. P. A., Bonitatebus, P. J., Hoveyda, A. H., J. Am. Chem. Soc. 121 , (1999), 791-799 and Huang et al., J. Am. Chem. Soc. 121 , (1999), 2674-2678 can alternately be used to effect the metathesis reaction. It will also be recognized that catalysts containing other transition metals such as Mo can be used for this reaction.
  • Step a) ((2R,3R)-3-Vinyl-oxiranyl)-methanol can be synthesised from penta-1 ,4- dien-3-ol in two steps according to the procedure of Alex Romero and Chi-Huey Wong, J. Org. Chem. 2000, 65, 8264-8268.
  • Step b) (1 R,2R,5S)-2-[(S)-1-Acetoxy-2-(toluene-4-sulfonyloxy)-ethyl]-6-oxa-3-aza- bicyclo[3.1.0]hexane-3-carboxylic acid tert-butyl ester; and (1S,2R,5R)-2-[(S)-1-Acetoxy-2-(toluene-4-sulfonyloxy)-ethyl]-6-oxa-3-aza- bicyclo[3.1.0]hexane-3-carboxylic acid tert-butyl ester
  • Step c) (1 R,2R,5S)-2-((R)-1 -Acetoxy-2-azido-ethyl)-6-oxa-3-aza- bicyclo[3.1.0]hexane-3-carboxylic acid terf-butyl ester; and (1S,2R,5R)-2-((R)-1-acetoxy-2-azido-ethyl)-6-oxa-3-aza-bicyclo[3.1.0]hexane-3- carboxylic acid terf-butyl ester.
  • TIc showed complete consumption of starting material and the mixture was concentrated under reduced pressure.
  • pyridine 0.485 ml_, 6.00 mmol
  • dry dichloromethane 2.0 ml_
  • methanesulfonyl chloride 458 ml_, 2.40 mmol
  • the yellow solution was partitioned between saturated sodium hydrogen carbonate solution / chloroform and the aqueous phase was extracted into chloroform (x3).
  • Combined organic extracts were dried (magnesium sulfate), filtered and concentrated under reduced pressure.
  • N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride 0.064 g, 0.34 mmol
  • 1-Hydroxybenzotriazole hydrate 0.051 g
  • benzo[b]furan-2-carboxylic acid 0.052 g, 0.32 mmol
  • DMF 3 ml
  • triethylamine 0.13 ml, 0.9 mmol
  • N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.068 g, 0.36 mmol), 1-hydroxybenzotriazole hydrate (0.055 g) and 4-phenoxybenzoic acid (0.073 g, 0.34 mmol) in DMF (3 ml) was added triethylamine (0.14 ml, 0.97 mmol), then stirred at rt for 24 h. The reaction mixture was then concentrated. The residue was then partitioned between 10% aq. citric acid (30 ml) and ethyl acetate (10 ml).
  • Solid phase synthesis of 8.1 - 8.13 & 8.15 - 8.67 is generally carried out using Murphy's linker methodology using known chemistries as described in WO02/88106.
  • the ketone function of the FmocNH bicycle was derivatised as an acid labile semicarbazone which provided a carboxylic acid for attachment to the aminomethyl functionalised polymer support resin using HBTU, HOBt and NMM. After Fmoc removal the corresponding (1- substituted) cyclobutylmethylalanine Fmoc acid was coupled on where the symmetric anhydride was preformed. Coupling was first carried out for 8 h, and then repeated with fresh reagents overnight. After Fmoc removal the P3 acids were introduced using standard coupling conditions. Washing, drying and cleavage from the resin provided the crude desired material which was purified either by column chromatography or preparative hplc. Compounds which required modified procedures are described below.
  • 6-Fluoro-3-hydroxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic acid tert-butyl ester (0.200 g, 0.81 mmol) was dissolved in DCM (4 ml_) at 0 0 C and TFA (4 ml_) added. After stirring at 0 -4 0 C for 1 h, the solvent was evaporated in vacuo and the residue left under high vacuum for 4 h to afford a brown oil.
  • Trimethoxyethane (6.5 mmol) was dissolved in aqueous hydrochloric acid (1 N, 10 ml) and the reaction heated at 50°C for 1.5 h. The reaction was allowed to cool to RT and was then added to a suspension of 4-(2-piperazin-1-yl- thiazol-4-yl)-benzoic acid (5.0 mmol) in acetonitrile (25 ml) and sodium acetate buffer (1N, pH 5.5, 10ml). The reaction was stirred at RT for 1.5 h. Sodium cyanoborohydride (6.5 mol) was added and the reaction stirred at RT for 16 h.
  • reaction mixture was neutralised to pH 7 with 1M HCI and concentrated by N 2 stream.
  • product was resuspended in water and lyophilised to give 4- ⁇ [(1-methylimidazol-2-yl)methyl]amino ⁇ benzoic acid : m/z 232 in MS ES+ as a white solid which was used directly in the subsequent step.
  • a reaction tube containing a magnetic stirrer bar was charged with ethyl-4- fluorobenzoate (1 mmol), 2-methyl-3-pyridol (1.0 mmol), potassium carbonate (1.08 mmol) and DMF (2 ml). The reaction tube was then sealed and heated by microwave irradiation (100W, 4 mins) to 150 "C and held at that temperature for 80 mins. The solution was filtered to remove the insoluble potassium carbonate and then concentrated in vacuo.
  • Triphelphosphine resin (Fluka, 3 mmolg" ' ' ) in 40 ml of DMF. The solution was gently stirred at 65 ° C for 48 hours. The phosphonium resin was washed with DMF (4x40 ml),
  • a reaction tube containing a magnetic stirrer bar was charged with 1-Methyl-1 H- imidazole-2-carbaldehyde(1.5mmol), potassium carbonate (2.1 mmol), ⁇ 4- (methoxycarbonyl) benzyl(triphenyl) ⁇ phosphonium bromide on polymer support (1.5 mmol) and methanol (4 ml).
  • the reaction tube was then sealed and heated by microwave irradiation (100W, 3 mins) to 150 "C and held at that temperature for 5 mins.
  • reaction mixture was concentrated by N 2 stream, resuspended in water and the product lyophilised to give potassium 4-methyl(pyridin-2-yl)aminomethyl benzoate : m/z 243 in MS ES+ as an off-white solid which was used directly in the subsequent step.
  • Lithium 4-(2-ri(SHdimethylamino)ethyll-5-methyl-1 ,3-thiazol-4-yl)benzoate (Example 8.35) Ethyl 4-(2- ⁇ 1 (S)-[(tert-butoxycarbonyl)(methyl)amino]ethyl ⁇ -5-methyl-1 ,3-thiazol-4- yl)benzoate was prepared as described previously.
  • Triphenyl phosphine polymer bound (8.96 mmol) was suspended in anhydrous dichloromethane (20 ml) then diisopropyl azodicarboxylate (7.76 mmol) was added and the mixture was stirred for 15 minutes at room temperature. Then ethyl 5- hydroxybenzofuran carboxylate (5.97 mmol) was added over 5 minutes followed by the addition of 4-N-boc-3-morpholinecarboxylic acid (5.97 mmol) over 5 minutes too. The mixture was stirred at room temperature over night.
  • the Alloc group was removed with (1 ) DCM (4 x 1 min); (2) borane dimethylamine complex (40 equiv), tetrakis (triphenylphosphine) palladium (0) (0.1 equiv) in anhydrous DCM (2 x 15 min); (3) DCM (3 x 1 min); (4) DMF (3 x 1 min); (5) dioxan-water (9:1 ) (3 x 1 min); (6) DMF (3 x 1 min); (7) MeOH (3 x 1 min); (8) DCM (3 x 1 min) and the peptide resin was treated with dibutyltin dichloride (5 equiv), phenylsilane (5 equiv) and a 37% solution of formaldehyde in water (5 equiv) in THF for 2 hours at room temperature. The reminder of the procedure was carried out as described in the general protocol.
  • Triphenyl phosphine polymer bound (1.37 mmol) was suspended in anhydrous dichloromethane (10 ml) then diisopropyl azodicarboxylate (1.18 mmol) was added and the mixture was stirred for 15 minutes at room temperature. Then ethyl 3-methyl-5- hydroxybenzofuran carboxylate (0.91 mmol) was added over 5 minutes followed by the addition of 4-N-boc-3-morpholinecarboxylic acid (0.91 mmol) over 5 minutes too. The mixture was stirred at room temperature over night.
  • IR 2117cm 1 4-(2-Amino-acetyl)-benzoic acid methyl ester hydrochloride.
  • 4-(2-Azido-acetyl)-benzoic acid methyl ester_(6.53 mmol) was suspended in methanol (30ml) and aqueous hydrochloric acid (6.53 mmol, 1 M) was added.
  • a catalytic amount of palladium on carbon (10% wt) was added and the reaction stirred over an atmosphere of hydrogen for 3h.
  • 4-(2-r2-(S)-(tert-Butoxycarbonyl-methyl-amino)-propionylaminol-acetyl)-benzoic acid methyl ester 4-(2-Amino-acetyl)-benzoic acid methyl ester hydrochloride (2.22mmol), WSCHCI (2.44mmol), Boc-N-methyl-(L)-alanine (2.44mmol) and HOBt (2.77mmol) were suspended in dichloromethane (10ml). NMM (2.44mmol) was added and the reaction stirred for 2h.
  • Boc-L-NMe-Alanine-OH (1.0g, 4.92mmols) was dissolved in dioxan (1OmIs) and to this was added pyridine (0.25mls), di-tert-butyl dicarbonate (1.4g, 6.4mmols) and ammonium hydrogen carbonate (0.49g, 6.2mmols). After stirring for 18 hours the crude reaction mixture was concentrated in vacuo and re-suspended in ethyl acetate. This was washed with 1 M KHSU4 and the organic layer dried over magnesium sulphate. After concentration a clear oil was obtained (0.79g).
  • the ester (0.25g, 1.07mmols) was dissolved in ethanol (1OmIs) and 4-(2-Bromo-acetyl)- benzoic acid methyl ester (1.18mmols, 0.3Og) was added. The reaction was heated to 50 0 C for one hour. The crude product was purified by preparative HPLC (MeCN/H 2 O) to yield 0.138g of a yellow solid. The Boc group was removed via treatment with 4M HCI/dioxan for one hour after which time the reaction mixture was concentrated in vacuo. The free amine (0.093g, 0.265mmols) was then dimethylated.
  • the above methyl ester (564 mg, 1.35 mmol) was heated with 1.35 ml_ 2N NaOH, 5 ml_ THF, and 3.65 ml_ water at 60 0 C for 4 h.
  • the reaction mixture was evaporated, poured into 20 ml_ saturated aqueous NaCI and 20 ml_ CH 2 CI 2 , and then acidified to pH 3 with 5% citric acid, in an ice bath.
  • the layers were separated and the organic phase was extracted further with 2 x 10 ml_ CH 2 CI 2 .
  • This solid was then mixed in dichloromethane-trifluoroacetic acid (2:1 ) and kept at room temperature for 20 minutes. The mixture was concentrated to near dryness and the concentrated once from dichloromethane and once from 1 N HCI in diethylether. The remaining solid was mixed with acetic acid (0.5 ml_), methanol (3 ml_) and tetrahydrofurane (4.5 ml_) and formaldehyde (aq. 37%, 300 ml_) and polystyrene bound cyanoborohydride (2.36 mmol/g, 900 mg) was added. The slurry was then agitated for 16 hours at room temperature.
  • the extracts were evaporated and purified by flashchromathography on silica-gel (packed with ethyl acetate) using ethyl acetate- methanol-triethylamine 20:2:1 as the eluant. Pure fractions were collected and concentrated. The residue was dissolved in 30ml concentrated hydrochloric acid and was refluxed for 12hours. The solution was evaporaded to yield the title compound as a solid.
  • Electrospray ionisation eluting with acetonitrile / ammonium formate buffer.
  • 2R-Bromomethyl-morpholine-4-carboxylic acid tert-butyl ester 12 g of polymer supported triphenylphosphine was suspended in 100 ml dichloromethane at room temperature, 0.8 g alcohol and 2.4 g CBr 4 was added. The mixture was stirred at room temperature for 4 hours, solid supported reagent was removed and residue was concentrated in vacuo. The crude material was dissolved in toluene, treated with 10 g of DARGO G-60, filtered and concentrated in vacuo to yield 0.91 g (88 %) of semi solid 2R-bromomethyl-morpholine-4-carboxylic acid tert-butyl ester.
  • the ethyl ester (0.14g) was dissolved in 10 ml dioxane at room temperature, a solution of 60 mg (4 eq.) LiOH in 3 ml water was added and the mixture was stirred for 16 hours at ambient temperature. The suspension was transferred to a separation funnel, diluted with 20 ml of water and extracted with EtOAc. The aqueous phase was acidified with 30 ml of 1 M citric acid (aq.) and extracted with 2x30 ml EtOAc.
  • Novel enatiomeric P3 building block i. NaOH, 2-aminoethyl hydrogen sulphate, 50 0 C, 1 h; ii. NaOH, H 2 O, 55 0 C, 16 h; iii. BoC 2 O, RT, 5 h; iv. 10 % Pd on carbon, H 2 at 1 atm for 16 h, RT, 3 h; v. Ph 3 P, CBr 4 , RT, 4 h; vi. Carboxy-protected-5-hydroxy-3-methyl-benzofuran-2-carboxylic acid, Cs 2 CO 3 , 4O 0 C, 18 h;
  • Convenient assays for cathepsin K are carried out using human recombinant enzyme, such as that described in PDB. ID BC016058 standard; mRNA; HUM; 1699 BP.
  • the recombinant cathepsin K can be expressed in a variety of commercially available expression systems including E coli, Pichia and Baculovirus systems.
  • the purified enzyme is activated by removal of the prosequence by conventional methods.
  • Standard assay conditions for the determination of kinetic constants used a fluorogenic peptide substrate, typically H-D-Ala-Leu-Lys-AMC, and were determined in either 100 mM Mes/Tris, pH 7.0 containing 1 mM EDTA and 10 mM 2-mercaptoethanol or10OmMNa phosphate, imM EDTA, 0.1%PEG4000 pH 6.5 or 100 mM Na acetate, pH 5.5 containing 5 mM EDTA and 20 mM cysteine, in each case optionally with 1 M DTT as stabiliser.
  • the enzyme concentration used was 5 nM.
  • the stock substrate solution was prepared at 10 mM in DMSO.
  • 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 mM 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. Add 90 ⁇ l assay buffer containing 1 mM DTT and 2 nM cathepsin S to each well of rows B-H and 180 ⁇ l 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 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.
  • 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.
  • 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 1 mM EDTA, pH5.5)
  • the DMSO stock (1OmM in 100%DMSO) is diluted to 10% in assay buffer.
  • V 0 is the velocity of the reaction, Vis the maximal velocity, S is the concentration of substrate with Michaelis constant of K M , and / is the concentration of inhibitor.

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Abstract

Compounds of the formula II wherein R1 and R1, are halo; or one of R1 and R1, is halo, and the other is is H; R2 is R4, -C(=O)R4; -OC(=O)R4, -S(=O)nR4, -S(=O)nNRdRe; R3 is H, -OR4, -SR4; R3' is H; or R3 and R3' together define =O; R4 is -C1C6 alkyl, -C0-C3alkylenecarbocyclyl or -C0-C3alkyleneheterocyclyl, any of which is optionally substituted with up to three substituents selected from R7; R5 is -C1C5 alkyl, -CH2CR5'C3-C4-cycloalkyl; R5' is H; or R5 and R5' together with the carbon to which they are attached define C4-C6-cycloalkyl; R5' is H, C1C2 alkyl, C1C2 haloalkyl, hydroxyl, OC1C2alkyl, fluoro; E is -C(=O)-, -S(=O)m-, -NRaS(=O)m-, -NRaC(=O)-, -OC(=O)-, -CRbRc-; R6 is a stable, optionally substituted, monocyclic or bicyclic carbocycle or heterocycle, wherein the, or each, ring has 4, 5 or 6 ring atoms and 0 to 3 hetero atoms selected from S, O and N; have utility in the treatment of disorders characterized by inappropriate expression or activation of cathepisn K, such as osteoporosis, osteoarthritis, rhuematoid arthritis or bone metastases.

Description

Cysteine Protease Inhibitors
Field of the invention.
This invention relates to inhibitors of cysteine proteases, especially those of the papain superfamily. The invention provides novel compounds useful in the prophylaxis or treatment of disorders stemming from misbalance of physiological proteases such as cathepsin K.
Description of the related art. The papain superfamily of cysteine proteases is 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 and S, 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.
The inappropriate regulation of cathepsin K has been implicated in a number of disorders including osteoporosis, gingival diseases such as gingivitis and periodontitis, Paget's disease, hypercalcaemia of malignancy and metabolic bone disease. In view of its elevated levels in chondroclasts of osteoarthritic synovium, cathepsin K is implicated in diseases characterised by excessive cartilege or matrix degradation, such as osteoarthritis and rheumatoid arthritis. Metastatic neoplastic cells typically express high levels of proteolytic enzymes that degrade the surrounding matrix and inhibition of cathepsin K may thus assist in treating neoplasias.
International patent application no WO04007501 discloses compounds of the formula I
Figure imgf000002_0001
where UVWXY broadly corresponds to the P3 and P2 of dipeptide cysteine protease inhibitors, R1 is an amide, carbamate or sulphonamide, R3 is H or more speculatively a carbon-linked substituent such as alkyl, alkylaryl etc and P1 and P2 are each methylene, or more speculatively speculatively substituted with various carbon chains and cyclic groups.
Brief description of the invention
In accordance with the invention, there is provided compounds of the formula Il
Figure imgf000003_0001
wherein
R1 and R1' are halo; or one of R1 and R1' is halo, and the other is is H;
R2 is R4, -C(=O)R4; -OC(=O)R4, -S(=O)nR4, -S(=O)nNRdRe;
R3 is H, -OR4, -SR4; R3' is H; or
R3 and R3' together define =O;
R4 is -CrC6 alkyl, -C0-C3alkylenecarbocyclyl or -C0-C3alkyleneheterocyclyl, any of which is optionally substituted with up to three substituents selected from R7;
R5 is -CrC5 alkyl, -CH2CR5"C3-C4-cycloalkyl; R5' is H; or
R5 and R5' together with the carbon to which they are attached define C4-C6-cycloalkyl;
R5" is H, CrC2 alkyl, CrC2 haloalkyl, hydroxyl, OCi-C2alkyl, fluoro;
E is -C(=O)-, -S(=O)m-, -NRaS(=O)m-, -NRaC(=O)-, -OC(=O)-, -CRbRc-;
R6 is a stable, optionally substituted, monocyclic or bicyclic carbocycle or heterocycle, wherein the, or each, ring has 4, 5 or 6 ring atoms and 0 to 3 hetero atoms selected from S, O and N and wherein the optional substituents comprise 1 to 3 members selected from R7;
R7 is independently selected from halo, oxo, nitrile, nitro, C1-C4 alkyl, -XNRdRe, -
XNReR8, -NReXR8, NH2CO-, X-R8, X-O-R8, O-X-R8, X-C(=O)R8, X-(C=O)NRdR8, X- NReC(=O)R8, X-NHSOmR8, X-S(=O)mR8, X-C(=O)OR8, X-NReC(=O)OR8; R8 is independently 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, any of which is optionally substituted with up to 3 members selected from R9; R9 is independently selected from hydroxy, XR10, -XNRdRe, -XNReR10, -NRed-
C4alkylR10, cyano, halo, carboxy, oxo, C1-C4 alkyl, Ci-C4-alkoxy, C1-C4 alkanoyl, carbamoyl;
R10 is C3-C6 cycloalkyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, thiopyranyl, furanyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl, phenyl, any of which is substituted with CrC4 alkyl, halo, hydroxy, CrC4alkoxy
X is independently a bond or CrC4 alkylene;
Ra is independently H, CrC4 alkyl or CH3C(=O);
Rb is CrC4haloalkyl; Rc is H, CrC4 alkyl;
Rd is independently H, CrC4 alkyl or CH3C(=O);
Re is independently H, CrC4 alkyl; or
Rd and Re together with the N atom to which they are attached form a morpholine, piperidine, piperazine or pyrrolidine ring optionally substituted with R9; m is independently 0,1 or 2; n is 1 or 2; or a pharmaceutically acceptable salt or prodrug thereof.
Without in any way wishing to be bound by theory, or the ascription of tentative binding modes for specific variables, P1 , P2 and P3 as used herein are provided for convenience only and have their conventional 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.
Preferably the stereochemistry of the proximal end of the P1 group is as depicted in the partial structure below:
Figure imgf000005_0001
Preferably the halogen of R1 and/or R1 is chlorine and most preferably fluorine. It is currently preferred that R1 is halo, especially fluorine and R1 is H, but the invention extends to compounds wherein R1 is halo, especially F and R1 is H or R1 and R1 are each F.
An embodiment of the invention comprises compounds with the partial structure:
Figure imgf000005_0002
where R1 and R1 are as defined above, but particularly wherein R1 is F and R1 is H, especially those with the stereochemistry:
Figure imgf000005_0003
An alternative embodiment comprises compounds with the partial structure:
Figure imgf000005_0004
where R1 and R1 are as defined above, especially those with the stereochemistry shown in the partial structure:
Figure imgf000006_0001
Currently favoured values for R4 in this embodiment include benzyl or C1-C4 alkyl, preferably methyl.
Except for the case where R3 and R3' define a keto function, compounds within this embodiment may exist as the racemate at the R3/R3' centre. Preferably, however, the compounds are at least 90%, preferably at least 95%, such as at least 98% enantiomerically pure
Figure imgf000006_0002
especially the left hand (alpha) configuration, for example when R3 is methoxy.
A favoured embodiment comprises compounds with the partial structure
Figure imgf000006_0003
Especially those with the stereochemistry:
Figure imgf000006_0004
It will be appreciated that the P1 group may exist in alternative forms, such as
Figure imgf000007_0001
and the invention extends to all such alternative forms.
Favoured values for R2 currently include -C(=O)R4; -S(=O)nR4, and -S(=O)nNRdRe; especially where n is 2.
When the R4 moeity of R2 comprises C0-C3 alkylenecarbocyclyl C0-C3 alkyleneheterocyclyl, preferred R4 groups include but are not limited to
Figure imgf000007_0002
where Xi is ethylene, or preferably methylene or more preferebaly a bond;
B. D and G are independently chosen from: CR21, where R21 is H, C0-Ce alkyl, especially methyl or C0-C3alkylcarbocyclyl, especially those wherein C0 or N or N-oxide;
E' is chosen from: CH2 CHR21, O. S. SO2, NR21 or N-oxide; J. L, M, R. T. T2, T3 and T4 are independently chosen from: CR21 or N or N-oxide;
More preferred R4 groups for R2 include
Figure imgf000008_0001
where Xi is methylene or preferably a bond and B, D, E', G, J, L, M, R are as defined above.
Still more preferred compounds are those in which R 2 is:
Figure imgf000008_0002
where Xi is methylene or preferably a bond and B, D, E', G, J, L, M, R are as defined above, wherein R21 is H, methoxy, ethyl, ethyl isopropyl, triflouromethyl, triflouromethyoxy F, CL, Sθ2Me and Ra is H or methyl.
Favoured values for the R4 component of R2 include C3-C6 alkyl, including those which are interrupted with an O or NH as part of the the chain, which are unsubstituted or substituted with one or more NH2, NHMe, NHC(O)CH3, NHMe(C(O)CH3, OH or OMe groups. Particularly favoured values include those which are branched at the alpha position or which include an NH2, NHMe, NHC(O)CH3, NHMe(C(O)CH3, OH or OMe group at the alpha position.
Examples of Co-C3alkylcarbocyclyl R4 components for R2 groups include C3 -Cβ cycloalkyl, or the heterocyclic analogues. Examples of such R4 components of R2 include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidine, piperidine, morpholine, tetrahydrofuran, cyclopentene, cyclopentadiene, cyclohexadiene and piperazine. Nitrogen-containing rings may be N-substituted with groups such as C- alkyl, phenyl or benzyl.
It is yet more preferred that when R2 comprises a C3-C6-cycloalkyl group, or a heterocyclic saturated or unsaturated analogue thereof in which the ring system is either connected directly to the remainder of the R2 moiety or there is one intervening methylene group. A five- or six- membered cyclic ring are most favourable.
Particularly preferred R2 groups therefore include: benzoyl; pyridine-2-carbonyl; 1-oxy-pyridine-2-carbonyl; pyridine-3- carbonyl; 1 oxy- pyridine-3-carbonyl; pyridine-4-carbonyl; 1-oxy-pyridine-4-carbonyl; phenyl sulphonyl; pyridine-2-sulphonyl; 1-oxy-pyridine-2- sulphonyl; pyridine-3-sulphonyl; 1-oxy-pyridine- 3-sulphonyl; pyridine-4-sulphonyl; 1-oxy-pyridine-4- sulphonyl; phenylacetyl; phenylcarbamoyl; isobutylcarbamoyl; phenyloxycarbonyl; isobutyloxycarbonyl; pyrrolidine-N-carbonyl; piperidine-N carbonyl; morpholine-N-carbonyl; piperazine-N- carbonyl; 4-methyl-piperazine-N-carbonyl; (4-methyl-piperazin-1-yl)- acetoyl; piperazin- 1-yl-acetoyl; furan-2 carbonyl; 5-chlorofuran-2- carbonyl; thiophene-2-carbonyl; 5- chlorothiophene-2 10 carbonyl; furan-3- carbonyl; thiophene-3-carbonyl; cyclopentoyl; cyclohexoyl; cyclopent-3- enoyl; cyclopentylmethylcarbonyl; cyclohexylmethylcarbonyl; pyrrolidine-2-carbonyl; N-acetyl-pyrrolidine-2-carbonyl; piperidine-2-carbonyl; N- acetyl- piperidine-2-carbonyl; tetrahydrofuran-2-carbonyl; 1 aminocyclobutanoyl; 1-aminocyclo- pentanoyl; 1-aminocyclohexanoyl; N-acetyl-1 -aminocyclobutanoyl; N-acetyl-l- aminocyclopentanoyl; N-acetyl-1- ami nocyclohexanoyl; 1-hydroxycyclobutanoyl; 1- hydroxycyclopentanoyl; 1- 1 hydroxycyclohexanoyl; 1-methoxycyclobutanoyl; 1- methoxycyclopentanoyl; 1 methoxycyclohexanoyl; amino cyclopentylacetoyl; amino cyclohexylacetoyl; N acetylaminocyclopentylacetoyl; N-acetylaminocyclohexylacetoyl; 2 acetylaminopropionoyl;. 2-acetylaminoethanoyl; 2-acetyl-N methylaminoethanoyl; N1N- dimethylaminoacetoyl; 2-aminobutanoyl; N-acetyl- 2 aminobutanoyl; 2-anino-3- methyl butanoyl; N-acetyl-2-amino-3- methylbutanoyl, 2-amino-3,3-dimethylbutanoyl; N- acetyl-2-amino-3,3- dimethylbutanoyl; 2 amino-3-methylpentanoyl; N-acetyl-2-amino-3- methylpentanoyl; pentanoyl; 3 methylpentanoyl; 4-methylpentanoyl; 2- amino-4- methylpentanoyl; N-acetyl-2 amino-4-methylpentanoyl; 2-amino-4,4-dimethylpentanoyl; N-acetyl-2-amino 4,4-dimethylpentanoyl; 2- aminopentanoyl; N-acetyl-2- aminopentanoyl; 2-amino 5-methylhexanoyl; N-acetyl-2-amino-5-methylhexanoyl; 2- hydroxy-3- 1 methylbutanoyl; 2- methoxy- 3-methylbutanoyl; 2-hydroxy-3,3- dimethylbutanoyl; 2-methoxy-3, 3- dimethylbutanoyl; 2-hydroxy-3-methylpentanoyl; 2- methoxy-3 methylpentanoyl; 2-hydroxy-4-methylpentanoyl; 2-methoxy-4- methylpentanoyl; 2-hydroxy-4,4-dimethylpentanoyl;2-methoxy-4,4-dimethylpentanoyl; 2- hydroxypentanoyl; 2-methoxypentanoyl; 2-hydroxy-5- methylhexanoyl; 2 methoxy-5- methylhexanoyl;
Where R5' is H, 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. Accordingly, the stereochemistry of the P2 group preferably corresponds to an L-amino acid as depicted in the partial structure below:
Figure imgf000010_0001
but the invention also extends to D-isomers at this position.
The invention also includes all isomers and enantiomers at other chiral centres.
Favoured values for R5 when R5' is H include those embodied by the partial structures:
Figure imgf000010_0002
Further embodiments for R5 when R5 is H includes those with the partial structure:
Figure imgf000011_0001
where R6 is as defined above.
Convenienty, R5" is H, thus defining a cyclobutylmethyl side chain at P2.
Representative values for R5 include methyl, hydroxyl, fluoromethyl, difluoromethyl or trifluoromethyl: Accordingly, favoured values of the P2 side chain include,
Figure imgf000011_0002
Figure imgf000011_0003
particularly those reflecting an L amino acid.
An alternative P2 construction comprises compounds wherein R5 and R5' are cyclised to form cyclopentyl or preferably cyclohexyl.
The Ra depicted in formula Il is conveniently hydrogen.
Preferred E groups include -S(=O)m-, especially -S(=O)2-, and most preferably -C(=O)-.
An alternative embodiment of E comprises compounds wherein E is -CRbRc-, where Rb is typically halomethyl such as fluoromethyl, difluoromethyl and preferably trifluoromethyl, as illustrated below:
Figure imgf000012_0001
where R6 for the sake of illustration is exemplified with a substituted phenyl and Rc is H.
Preferably the compound of the invention comprises a high enantiomeric purity, such as more than 80%, preferably more than 95% such as greater than 97% of the S stereoconfiguration at the carbon bearing haloalkyl Rb. The partial structure below represents a typical S-enantiomer with Rb as trifluoromethyl and Rc as H:
Figure imgf000012_0002
Typically R6 is a monocyclic ring with 5 or especially 6 ring atoms, or a bicyclic ring structure comprising a 6 membered ring fused to a 4, 5 or 6 membered ring.
Typical R6 groups include saturated or unsaturated heterocycles or saturated or unsaturated carbocycles, any of which are optionally substituted as described above. R6 generally has an aromatic character, especially in the rign adjacent E. Illustrative variants include C3-β cycloalkyl, phenyl, benzyl, tetrahydronaphthyl, indenyl, indanyl, heterocyclyl such as from azepanyl, azocanyl, 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, benzofuranyl, benzothienyl, benzimidazolyl, benzthiazolyl, benzoxazolyl, benzisoxazolyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, quinazolinyl, tetrahydroquinazolinyl and quinoxalinyl, any of which may be substituted as described above.
The saturated heterocycle thus includes radicals such as pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl, piperazinyl, indolinyl, azetidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrofuranyl, hexahydropyrimidinyl, hexahydropyridazinyl, 1 ,4,5,6- tetrahydropyrimidinylamine, dihydro-oxazolyl, 1 ,2-thiazinanyl-1 ,1 -dioxide, 1 ,2,6- thiadiazinanyl-1 ,1 -dioxide, isothiazolidinyl-1 ,1 -dioxide and imidazolidinyl-2,4-dione, whereas the unsaturated heterocycle include radicals such as furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, indolyl, isoindolyl. In each case the heterocycle may be condensed with a phenyl ring to form a bicyclic ring system.
Preferred monocyclic R6 groups include substituted pyridyl, substituted pyrimidyl, substituted phenyl, particularly phenyl substituted with a cyclic group such as pyrrolidine-1-yl, piperidine-1-yl, 4-methylpiperidin-1-yl, 4-(piperidin-3-ylmethyl)-piperidin- 1-yl, morpholin-4-yl, 4-methylpiperazin-1-yl, 2-morpholin-4-yl-ethylamino, 2-morpholin- 4-yl-ethyloxy, 1-pyrid-2-ylmethylamino, piperazin-1-yl, piperid-4-yl or N-piperazinyl, N- substituted with Ra or piperidin-1-yl which is 4-substituted with -NRaRb. A phenyl R6 is conveniently substituted at the 3 or 4 position (para or meta), for example with such a cyclic group.
Alternative cyclic substituents to a monocyclic R6 (such as phenyl) include aryl groups such as phenyl or a 5 or 6 membered heteroaryl group such as thiophene, furyl, triazole, thiazole, diazole, pyrazole or pyrrolidine. Favoured cyclic substituents in this context include thiazol-2-yl, pyrid-3-yl and especially pyrid-2-yl, thien-2-yl or thiazol-5-yl. This cyclic substituent (ie R7) is typically bonded direct to such R6 species (ie X is a bond), but may also for example comprise an amine spacer such as -NH-, -N(Me), - CH2NH, -CH2N(Me)-, a CrC3alkyl spacer such as -CH2- or a CrC3-alkyloxy spacer such as ethyloxy
Any of the cyclic substituents to R6 in the immediately preceding paragraph may be substituted as described above with R10. For example a heterocycle R7 group such as thiazolyl can be substituted with d-C4 alkyl such as methyl or disubstituted such as methyl at one position and a cyclic group at another Preferably, any of the cyclic substituents to R6 in the two immediately preceding paragraphs may itself be substituted with a cyclic group (that is R7 comprises an R9 moiety) typically a saturated heterocyclic group such as piperidine, piperazine or morpholine, which saturated cyclic group is optionally substituted, for example with d- C3 alkyl, fluoro, diflouro, CrC3alkyloxy or Ci-C3alkyloxyCi-C3alkyl. As provided in the definition of R7, this saturated cyclic group (ie R9) may be spaced from the R6 group by X (eg Ci-C3alkyl), amine (eg -NH-), amide, sulphonamide etc, but is typically bonded directly or via methylene.
Representative R9 groups in accordance with the immediately preceding paragraph include heterocycles such as pyrrolidine-1-yl, piperidine-1-yl, 4-methylpiperidin-1-yl, 4- (piperidin-3-ylmethyl)-piperidin-1-yl, morpholin-4-yl, 4-methylpiperazin-1-yl, 2- morpholin-4-yl-ethylamino, 2-morpholin-4-yl-ethyloxy, 1 -pyrid-2-ylmethylamino, piperazin-1-yl, piperid-4-yl or N-piperazinyl, N-substituted with Ra or piperidin-1-yl which is 4-substituted with -NRaRb,
Currently preferred R9 substituents include 4-substituted piperazin-4-yl, such as 4- methyl-piperazin-4-yl or 4-methyloxyethyl-piperazin-4-yl, piperid-1-ylmethyl which is optionally 4-substituted with fluoro or diflouro or morpholinylmethyl.
Alternative preferred substituents to a monocyclic R6 (such as phenyl) include -NRaRb, -CH2NRaRb, CrC4 straight or branched alkyl or -O-R9.
Representative R6 groups thus include:
Figure imgf000014_0001
Further representative R6 groups include
Figure imgf000015_0001
especially where Rq and Rq' are independently selected from H, C1-C4 alkyl or Ci-C4alkanoyl or together define an unsaturated 5-7 membered ring, such as piperidine, piperazine or morpholine, which may in turn be substituted with groups corresponding to R10, particularly CrC4 alkyl, fluoro or difluoro.
Currently preferred R6 groups include
Figure imgf000015_0002
Representative bicyclic groups for R6 include naphthylenyl, especially naphthylen-2-yl; benzo[1 ,3]dioxolyl, especially benzo[1 ,3]dioxol-5-yl, benzofuranyl, especially benzofuran-2-yl, and especially d-Cβ alkoxy substituted benzofuranyl, more especially 5-(2-piperazin-4-carboxylic acid tert-butyl ester- ethoxy) benzofuran-2-yl, 5-(2-morpholino-4-yl-ethoxy)-benzofuran-2-yl, 5-(2-piperazin-l-yl- ethoxy)benzofuran-2-yl, 5-(2-cyclohexyl-ethoxy)-benzofuran-2-yl; 7-methoxy-benzofuran-2-yl, 5-methoxy-benzofuran-2-yl, 5,6-dimethoxy-benzofuran-2-yl, especially halogen substituted benzofuranyl, more especially 5-fluoro-benzofuran-2-yl, 5,6-difluoro-benzofuran-2-yl, especially CrC6alkyl substituted benzofuranyl, most especially 3-methyl-benzofuran-2-yl; benzo[b]thiophenyl, especially benzo[blthiophen-2-yl; especially CrC6alkoxy substituted benzo[b]thiopheny], more especially 5,6-dimethoxy- benzo[b]thiophen-2-yl, quinolinyl, especially quinolin-2-yl, quinolin-3-yl, quinolin-4-yl, quinolin-6-yl, and quinolin-S-yl; quinoxalinyl, especially quinoxalin-2-yl; 1 ,8-naphthyridinyl, especially 1 ,8- naphthyridin-2-yl; indolyl, especially indol-2-yl, especially indol-6-yl, indol-5-yl, especially C-i-Cβalkyl substituted indolyl, more especially N-methylindol-2-yl; furo[3,2-b]pyridinyl, especially furo[3,2-b]pyridin-2-yl, and Cι-C6-alkyl substituted furo[3,2-b]pyridinyl, especially 3-methyl-furo[3,2-blpyridin-2-yl; thieno[3,2-b]thiophene, especially thieno[3,2-b]thiophene-2-yl, more especially d- C6alkyl substituted thieno[3,2-b]thiophene-2-yl, more especially 5-tert-buty]-3-methylthieno[3,2-b]thiophene-2-yl. Favoured R6 groups include bicyclic rings such as napthyl, quinoloyl, benzofuranyl, benzothienyl, indolyl and indolinyl, particularly where the linkage is to the 2 position of the ring. Favoured substituents to a bicyclic R6 group include pyrrolidine-1-yl, piperidine-1-yl, 4-methylpiperidin-1-yl, 4-(piperidin-3-ylmethyl)-piperidin-1-yl, morpholin- 4-yl, 4-methylpiperazin-1-yl, 2-morpholin-4-yl-ethylamino, 2-morpholin-4-yl-ethyloxy, 1- pyrid-2-ylmethylamino, piperazin-1-yl, piperid-4-yl or N-piperazinyl, N-substituted with Ra or piperidin-1-yl which is 4-substituted with -NRaRb. Especially preferred substituents, particularly in conjunction with benzofuranyl include 2-morpholin-4-yl- ethyloxy and N-methyl-piperidin-4-yloxy and those defined below.
A currently favoured bicyclic R6 group is optionally substituted benzothiazol or benzofuryl or benzoxazolyl, including those wherein the substituent is -OR9 or -NRbR9. For example, favoured R6 groups include benzofur-2-yl, unsubstituted and/or substituted with CrC4 alkyl, or CrC4 haloalkyl at the 3 position and/or or substituted in the 5 position with a saturated heterocycle such as piperidine, piperazine or morpholine, which is optionally substituted with C1-C3 alkyl and/or spaced from the benzofuryl by oxy, methyloxy or ethyloxy. Particularly favoured benzofuryl R6 groups thus include:
Figure imgf000017_0001
Returning to formula Il in general:
X is typically methylene or especially a bond.
Halogen or halo includes bromo, chloro and especially fluoro.
Haloalkyl means an alkyl group as defined above where at least one carbon atom bears 1 to 3 halogen atoms, preferably fluorine atoms. Representative haloalkyl group includes fluoromethyl, difluoromethyl, trifluoromethyl, 2, fluoroethyl, 2,2difluorethyl, 2,2,2 trifluorethyl and the like.
'Ci-Cβalkyl1 (also abbreviated as d-Cβalk, or used in compound expressions such as Ci-C6alkyloxy etc) as applied herein is meant to include straight and branched chain aliphatic carbon chains such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t- butyl, pentyl, isopentyl, hexyl, heptyl and any simple isomers thereof. The alkyl group may have an unsaturated bond. Additionally, any C atom in d-Cβalkyl may optionally be substituted by one, two or where valency permits three halogens and/or substituted or the alkylene chain interrupted by a heteroatom S, O, NH. If the heteroatom is located at a chain terminus then it is appropriately substituted with one or 2 hydrogen atoms. CrCnalkyl has the corresponding meaning to CrC6alkyl adjusted as necessary for the carbon number.
'Co-Csalkylaryl' as applied herein is meant to include an aryl moiety such as a phenyl, naphthyl or phenyl fused to a C3-C7cycloalkyl for example indanyl, which aryl is directly bonded (i.e. C0) or through an intermediate methyl, ethyl, propyl, or isopropyl group as defined for CrC3alkylene above. Unless otherwise indicated the aryl and/or its fused cycloalkyl moiety is optionally substituted with 1-3 substituents selected from halo, hydroxy, nitro, cyano, carboxy, CrC6alkyl, CrC6alkoxy, Ci-C6alkoxyCi-C6alkyl, d- C6alkanoyl, amino, azido, oxo, mercapto, nitro C0-C3alkylcarbocyclyl, C0- Csalkylheterocyclyl. "Aryl" has the corresponding meaning, i.e. where the C0-C3alkyl linkage is absent.
'Co-C3alkylC3C7cycloalkyl' 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 methyl, ethyl or proyl group as defined for CrC3alkylene above. The cycloalkyl group may contain an unsaturated bond. Unless otherwise indicated the cycloalkyl moiety is optionally substituted with 1-3 substituents selected from halo, hydroxy, nitro, cyano, carboxy, CrC6alkyl, CrC6alkoxy, Ci-CβalkoxyCrCβalkyl, d-Cβalkanoyl, amino, azido, oxo, mercapto, nitro Co- Csalkylcarbocyclyl, Co-Csalkylheterocyclyl.
'Co-Csalkylcarbocyclyl' as applied herein is meant to include Co-C3alkylaryl and Co- C3alkylC3-C7cycloalkyl. Unless otherwise indicated the aryl or cycloalkyl group is optionally substituted with 1-3 substituents selected from halo, hydroxy, nitro, cyano, carboxy, CrC6alkyl, CrC6alkoxy, Ci-C6alkoxyCi-C6alkyl, CrC6alkanoyl, amino, azido, oxo, mercapto, nitro, C0-C3alkylcarbocyclyl and/or Co-Csalkylheterocyclyl. "Carbocyclyl" has the corresponding meaning, i.e. where the Co-C3alkyl linkage is absent
'Co-C3alkylheterocycylyl' as applied herein is meant to include a monocyclic, saturated or unsaturated, heteroatom-containing ring such as piperidinyl, morpholinyl, piperazinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazinolyl, isothiazinolyl, thiazolyl, oxadiazolyl, 1 ,2,3-triazolyl, 1 ,2,4-triazolyl, tetrazolyl, furanyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazolyl, or any of such groups fused to a phenyl ring, such as quinolinyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazinolyl, benzisothiazinolyl, benzothiazolyl, benzoxadiazolyl, benzo-1 ,2,3-triazolyl, benzo-1 ,2,4-triazolyl, benzotetrazolyl, benzofuranyl, benzothienyl, benzopyridyl, benzopyrimidyl, benzopyridazinyl, benzopyrazolyl etc, which ring is bonded directly i.e. (Co), or through an intermediate methyl, ethyl, propyl, or isopropyl group as defined for d-Csalkylene above. Any such non-saturated rings having an aromatic character may be referred to as heteroaryl herein. Unless otherwise indicated the hetero ring and/or its fused phenyl moeity is optionally substituted with 1-3 substituents selected from halo, hydroxy, nitro, cyano, carboxy, CrC6alkyl, CrC6alkoxy, Ci-C6alkoxyCi-C6alkyl, CrC6alkanoyl, amino, azido, oxo, mercapto, nitro, Co-C3alkylcarbocyclyl, Co-C3alkylheterocyclyl. "Heterocyclyl" and "Heteroaryl" have the corresponding meaning, i.e. where the Co-C3alkyl linkage is absent.
Typically heterocycyl and carbocyclyl moieties within the scope of the above definitions are thus a monocyclic ring with 5 or especially 6 ring atoms, or a bicyclic ring structure comprising a 6 membered ring fused to a 4, 5 or 6 membered ring.
Typical such groups include C3-C8cycloalkyl, phenyl, benzyl, tetrahydronaphthyl, indenyl, indanyl, heterocyclyl such as from azepanyl, azocanyl, 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, benzofuranyl, benzothienyl, benzimidazolyl, benzthiazolyl, benzoxazolyl, benzisoxazolyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, quinazolinyl, tetrahydroquinazolinyl and quinoxalinyl, any of which may be optionally substituted as defined herein.
The saturated heterocycle moiety thus includes radicals such as pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl, piperazinyl, indolinyl, azetidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrofuranyl, hexahydropyrimidinyl, hexahydropyridazinyl, 1 ,4,5,6- tetrahydropyrimidinylamine, dihydro-oxazolyl, 1 ,2-thiazinanyl-1 ,1 -dioxide, 1 ,2,6- thiadiazinanyl-1 ,1 -dioxide, isothiazolidinyl-1 ,1 -dioxide and imidazolidinyl-2,4-dione, whereas the unsaturated heterocycle include radicals with an aromatic character such as furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, indolyl, isoindolyl. In each case the heterocycle may be condensed with a phenyl ring to form a bicyclic ring system. Favoured compounds of the invention include those permutations formed by independent selection of a P3, P2 and P1 member from each of Tables A, B and C:
Table A P1 groups
Figure imgf000020_0001
In table A R2a is, foe example, benzoyl, -S(=O)2-pyrid-2-yl, or-S(=O)2N(Me)2
Table B P2 groups
Figure imgf000020_0002
Figure imgf000021_0002
Table C P3 groups
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Additional aspects of the invention include a pharmaceutical composition comprising a compound as defined above and a pharmaceutically acceptable carrier or diluent therefor.
A further aspect of the invention is the use of a compound as defined above in the manufacture of a medicament for the treatment of disorders mediated by cathepsin K, such as: osteoporosis, gingival diseases such as gingivitis and periodontitis, Paget's disease, hypercalcaemia of malignancy metabolic bone disease diseases characterised by excessive cartilege or matrix degradation, such as osteoarthritis and rheumatoid arthritis, bone cancers including neoplasia, 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 Formula Il include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, isethionate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, proprionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate, camphorsulphonate, 2-napthalenesulphonate, 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 Formula Il may in some cases be isolated as the hydrate. Hydrates are typically prepared by recrystallisation from an aqueous/organic solvent mixture using organic solvents such as dioxin, tetrahydrofuran or methanol. The N-oxides of compounds of Formula (I) can be prepared by methods known to those of ordinary skill in the art. For example, N-oxides can be prepared by treating an unoxidized form of the compound of Formula (I) with an oxidizing agent (e.g., trifluoroperacetic acid, permaleic acid, perbenzoic acid, peracetic acid, meta- chloroperoxybenzoic acid, or the like) in a suitable inert organic solvent (e.g., a halogenated hydrocarbon such as dichloromethane) at approximately O0C. Alternatively, the N-oxides of the compounds of Formula (I) can be prepared from the N-oxide of an appropriate starting material.
Compounds of Formula (I) in unoxidized form can be prepared from N-oxides of compounds of Formula (I) by treating with a reducing agent (e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus bichloride, tribromide, or the like) in an suitable inert organic solvent (e.g., acetonitrile, ethanol, aqueous dioxane, or the like) at 0 to 8O0C.
Compounds of Formula (II) can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomer. While resolution of enantiomers can be carried out using covalent diasteromeric derivatives of compounds of Formula (I), dissociable complexes are preferred (e.g., crystalline; diastereoisomeric salts). Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by taking advantage of these dissimilarities. The diastereomers can be separated by chromatography, for example HPLC or, preferably, by separation/resolution techniques based upon differences in solubility. The optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization. A more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture can be found in Jean Jacques Andre Collet, Samuel H. Wilen, Enantiomers, Racemates and Resolutions, John Wiley & Sons, Inc. (1981 ).
It will be appreciated that the invention extends to prodrugs, solvates, complexes and other forms releasing a compound of formula Il in vivo. 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 Il 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.
The appropriate dosage for the compounds or formulations of the invention will depend upon the indication and the patient 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 μM, more preferably 0.01-10 μM, such as 0.1-25μM are typically desirable and achievable.
Compounds of the invention are prepared by a variety of solution and solid phase chemistries. 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 Il 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- 3-oxo-hexahydro- furo[3,2-b]pyrrole (substituted as necessary), P2 will be an N-protected amino acid, 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 carboniocarboxylic 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 N.N'-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, N'.N1- tetramethyluronium tetrafluoroborate. Still another practical and useful coupling agent is commercially available 0-(7-azabenzotrizol-1-yl)- N, N1N1, N'-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-methyl pyrrolidine or 4-DMAP is added to maintain the reaction mixture at a pH of about 8. The reaction temperature usually ranges between 0 0C and 50 0C 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 N- 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.
The first stage in a synthesis of compounds of the general formula Il is typically the preparation in solution of a functionalized P1 building block. Different nomenclature of compounds according to the present invention can be used. For convenience the carbohydrate nomenclature will generally be used herein.
A typical scheme towards the preparation of the compounds of the invention is shown in Scheme A below:
Figure imgf000032_0001
lIl, XIX, XX
Figure imgf000032_0002
i. DCM, pyridine, Tf2O, "5 0C, 1 h; ii. DMF, NaN3, 50 0C, 2 h; iii. AcOH-H2O, 50 0C, 2.5 h; iv. Pyridine, TsCI, 4 0C, 48 h; v. Deoxo-Fluor, pyridine, RT, 18 h; vi. 10 % Pd on C, ethanol, H2, RT, 18 h; vii. PhCHO, NaCNBH3, RT, 18 h; viii. TFA-water (9:1), RT, 1 h; ix. NaBH4, 40 0C, 18 h; x. acetone, cone. H2SO4; RT, 3 h; xi. p-nitrobenzoic acid, Ph3P, 4 0C, 18 h; xii. CH3OH, Et3N, RT, 2 h; xiii. pyridine, Tf2O, "5 0C, 1 h; xiv. DMF, NaN3, 50 0C, 3 h; xv. AcOH-water, 50 0C, 3 h; xvi. pyridine, tosyl chloride, 4 0C, 18 h; xvii. Ph3P, RT, 3 h; xviii. RI-Br, RT, 24 h; xix. 10 % Pd on C, ethanol, H2, RT, 16 h; xx. CbzLeuOH, EDC, HOBt, Et3N; xxi. 10 % Pd on C, ethanol, H2, RT, 10 h; xxii. R2COOH, EDC, HOBt, Et3N, RT, 18 h; xxiii. SO3- pyridine complex, DMSO, THF, Et3N, RT, 18 h.
The synthetic scheme can then continued as outlined previously to build up the final compound. Hence, the amine functionality of 4-benzyl-6-fluoro-octahydro-pyrrolo[3,2- b]pyrrol-3-ol is reacted with the appropriate electrophilic component to obtain a wide variety of groups at the R2 position. Once this functionality is in place, benzyl protection is removed using 4 M HCI in dioxane and the free amine coupled with the appropriate S2 residue, exemplied by L-Leu in the scheme. Subsequent removal of the Cbz moiety and coupling on the P3 residue using the appropriate R6 acid affords the alcohol which serves as the precursor for the final step. Oxidation of the alcohol affords the required final compound.
Although the above scheme has been illustrated with reference to a particular P1 and P2 and employing a peptide bond at E it will be readily apparent that corresponding methodology, starting from various others of the intermediates discussed above, will be applicable to prepare the other compounds of the invention.
A typical reaction scheme towards preparation of compounds of the invention wherein R3 is an ether is shown in Scheme B below:
Figure imgf000033_0001
OMe Vl, VIl, VIII
Figure imgf000033_0002
Figure imgf000033_0003
i. AcOH-H2O, 50 0C, 2.5 h; ii. Ph3P, H2O, THF, RT, 16 h; iii. Dess Martin Periodinane, O 0C, 2 h; iv. THF, H2O, RT, 2 h; v. CH3OH, H2O, c. HCI, 40 0C, 16 h; vi. RI-Br, RT, 24 h; vii. 10 % Pd on C, ethanol, H2, RT, 16 h; viii. CbzLeuOH, EDC, HOBt, Et3N; ix. 10 % Pd on C, ethanol, H2, RT, 10 h; x. R2COOH, EDC, HOBt, Et3N, RT, 18 h; xi. SO3-pyridine complex, DMSO, THF, Et3N, RT, 18 h.
Hydrolysis of ispropylidene in acidic conditions, with aqueous acetic acid yields the corresponding diol. Reduction of the azide group using the Staudinger procedure, with triphenylphosphine and subsequent hydrolysis of the corresponding phosphinimine affords the amine. Oxidation of the primary alcohol provides the aldehyde which in aqueous media cyclises to form the iminosugar. Treatment of this iminosugar with acidic methanol provides the corresponding methyl aza-glycoside. Hence, the amine functionality of 4-benzyl-6-fluoro-2-methoxy-octahydro-pyrrolo[3,2-b]pyrrol-3-ol is reacted with the appropriate electrophilic component to obtain a wide variety of groups at the R2 position. Once this functionality is in place, benzyl protection is removed using 4 M HCI in dioxane and the free amine coupled with the appropriate P2 residue, exemplied by L-Leu in the scheme. Subsequent removal of the Cbz moiety and coupling on the P3 residue using the appropriate acid affords the alcohol which serves as the precursor for the final step. Oxidation of the alcohol affords the required final compound.
Although the above scheme has been illustrated with reference to a particular P1 and P2 and employing a peptide bond at E it will be readily apparent that corresponding methodology, starting from various others of the intermediates discussed above, will be applicable to prepare the other compounds of the invention.
A typical reaction scheme towards preparation of the compounds of the invention wherein R and R define a ketone is shown below
Figure imgf000034_0001
i. AcOH-H2O, 50 0C, 2.5 h; ii. Ph3P, H2O, THF, RT, 16 h; iii. Dess Martin Periodinane, O 0C, 2 h; iv. THF, H2O, RT, 2 h; v. acetone, c. H2SO4 RT 18 h; vi. RI-Br, RT, 24 h; vii. 10 % Pd on C, ethanol, H2, RT, 16 h; viii. CbzLeuOH, EDC, HOBt, Et3N; ix. 10 % Pd on C, ethanol, H2, RT, 10 h; x. R2COOH, EDC, HOBt, Et3N, RT, 18 h; xi. AcOH-H2O, RT, 18 h; xii. Ag2CO3, Celite, benzene, DMF, Δ, 1 h.
Hydrolysis of ispropylidene in acidic conditions, with aqueous acetic acid yields the corresponding diol. Reduction of the azide group using the Staudinger procedure, with triphenylphosphine and subsequent hydrolysis of the corresponding phosphinimine affords the amine. Oxidation of the primary alcohol provides the aldehyde which in aqueous media cyclises to form the iminosugar. Treatment of this iminosugar with anhydrous acetone with catalytic acid provides the corresponding aza-glycoside. Furthermore, the amine functionality of 4-benzyl-6-fluoro-2,2-dimethyl-octahydro-1 ,3- dioxa-4,7-diaza-cyclopenta[a]pentalene is reacted with the appropriate electrophilic component to obtain a wide variety of groups at the R2 position. Once this functionality is in place, benzyl protection is removed using 4 M HCI in dioxane and the free amine coupled with the appropriate P2 residue, exemplied by L-Leu in the scheme. Subsequent removal of the Cbz moiety and coupling on the P3 residue using the appropriate acid affords the isoproylidene. Cleavage of the isopropylidene affords the diol which serves as the precursor for the final step. Oxidation of the alcohols affords the required final compound.
Although the above scheme has been illustrated with reference to a particular P1 and P2 and employing a peptide bond at E it will be readily apparent that corresponding methodology, starting from various others of the intermediates discussed above, will be applicable to prepare the other compounds of the invention.
A typical reaction scheme towards preparation of the compounds of the invention wherein R3 is a thioether is shown in Scheme C below:
Figure imgf000035_0001
i. AcOH-H2O, 50 0C, 2.5 h; ii. Ph3P, H2O, THF, RT, 16 h; iii. Dess Martin Periodinane, O 0C, 2 h; iv. THF, H2O, RT, 2 h; v. RI-Br, RT, 24 h; vi. Ac2O, pyridine, RT, 18 h; vii. Benzenethiol, p-toluene sulphonic acid, RT, 1 h; viii. 10 % Pd on C, ethanol, H2, RT, 16 h; ix. CbzLeuOH, EDC, HOBt, Et3N; x. 10 % Pd on C, ethanol, H2, RT, 10 h; xi. R2COOH, EDC, HOBt, Et3N, RT, 18 h; xii. CH3OH, sodium, RT, 1 h; xiii. SO3- pyridine complex, DMSO, THF, Et3N, RT, 18 h.
Hydrolysis of ispropylidene in acidic conditions, with aqueous acetic acid yields the corresponding diol. Reduction of the azide group using the Staudinger procedure, with triphenylphosphine and subsequent hydrolysis of the corresponding phosphinimine affords the amine. Oxidation of the primary alcohol provides the aldehyde which in aqueous media cyclises to form the iminosugar. Treatment of the amine functionality of 4-benzyl-6-fluoro-octahydro-pyrrolo[3,2-b]pyrrole-2,3-diol with the appropriate electrophilic component provides a wide variety of groups at the R2 position. In turn, treatment with acetic anhydride provides the diacetate. Treatment of the diacetate with benzenethiol in the presence of p-toluenesuphonic acid facilitates the introduction of the phenylthiolate at the anomeric position. Benzyl protection is removed using 4 M HCI in dioxane and the free amine coupled with the appropriate P2 residue, exemplied by L- Leu in the scheme. Subsequent removal of the Cbz moiety and coupling on the P3 residue using the appropriate acid affords the acetate. Cleavage of this O-protecting group and subsequent oxidation of the alcohol affords the required final compound.
A typical scheme towards a bicyclic P1 group starts with the ring closure of a suitably protected intermediate which is available in 4 steps from 1 ,2:5,6-di-O-isopropylidene-D- allofuranose, described by Mayer zum Reckendorf, Chem. Ber. 101 (1968), 3802-3807, giving a precursor of 3S, 4R stereochemistry.
Figure imgf000036_0001
1 3
Scheme 1. a) H2, Pd/C, methanol, b) benzylchloroformate, pyridine, dichloromethane In Scheme 1 the azide group of derivative 1 is reduced for example by catalytic hydrogenation using palladium on charcoal or other catalysts suitable, in a suitable solvent such as an alcohol, like ethanol or methanol into the free amine. The obtained nucleophilic nitrogen reacts spontaneously, or optionally in the presence of a suitable base like such as triethyl amine or sodium acetate, with the C-6 position forming a 5,5- bicycle. The leaving group at C-6 is not limited to sulfonate esters, but also other leaving groups such as halogen could be used throughout the synthesis of compounds according to the present invention. The reduction of the azide residue into an amine could also be performed by other methods known from literature, such as treating the azide derivative with a trialkyl- or triarylphosphine followed by hydrolysis of the formed imine derivative. After the ring closure the amine may be N-protected with a suitable protecting group such as a carbamate, like benzyl carbamate of compound 3 or any other similar protecting group which is normally not cleaved with acid. Suitable protecting groups which can be found in: Protective groups in organic chemistry, 3rd edition, 1999, Theodora W. Greene and Peter G. M. Wuts (Wiley&sons).
For a 3R, 4S bicycle a similar approach could be used starting from 3-azido-3-deoxy- 1 ,2:5,6-di-O-isopropylidene-D-gulofuranose which can be prepared as described in Tetrahedron Asymmetry, 10 (1999) 1855-1859. This intermediate can then be treated as described in Scheme 2.
Figure imgf000037_0001
6
Scheme 2. a) aq. acetic acid, b) p-toluenesulfonyl chloride, pyridine, DCM, c) H2, Pd/C, methanol, b) benzylchloroformate, pyridine, dichloromethane. Compound 4 can be treated with a mild acid, such as diluted acetic acid or similar, which can selectively hydrolyze the 5,6-acetal of compound 4, to obtain a diol. The primary alcohol can be selectively reacted with an alkyl- or arylsulfonyl chloride like p- toluenesulfonyl chloride to give compound 5. The azide group of derivative 5 is reduced for example by catalytic hydrogenation using palladium on charcoal or other catalysts suitable, in a suitable solvent such as an alcohol, like ethanol or methanol into the free amine. The obtained nucleophilic nitrogen reacts spontaneously, or optionally in the presence of a suitable base like such as triethyl amine or sodium acetate, with the C-6 position forming a 5,5-bicycle which can be N-protected with a suitable protecting group such as its benzyl carbamate (Cbz) to give compound 6.
Alternatively 3-azido-3-deoxy-1 ,2:5,6-di-O-isopropylidene-D-idofuranose (Bull. Chem. Soc. Japan, 57, 7(1984), 237-241) could be a suitable starting material for the 3R, 4S bicycle according to Scheme 3.
Figure imgf000038_0001
Scheme 3. a) aq. acetic acid, b) p-toluenesulfonyl chloride, pyridine, DCM, c) H2, Pd/C, methanol, b) benzylchloroformate, pyridine, dichloromethane.
Compound 6 can be treated with a mild acid, such as diluted acetic acid or similar, which can selectively hydrolyze the 5,6-acetal of compound 6, to obtain a diol. The primary alcohol can be selectively reacted with an alkyl- or arylsulfonyl chloride like p- toluenesulfonyl chloride to give compound 7. The azide group of derivative 7 is reduced for example by catalytic hydrogenation using palladium on charcoal or other catalysts suitable, in a suitable solvent such as an alcohol, like ethanol or methanol into the free amine. The obtained nucleophilic nitrogen reacts spontaneously, or optionally in the presence of a suitable base like such as triethyl amine or sodium acetate, with the C-6 position forming a 5,5-bicycle which can be N-protected with a suitable protecting group such as its benzyl carbamate (Cbz) to give compound 8.
The ring closure is not limited to the substrates shown above but could also be applied to derivatives as depicted in Scheme 4.
Figure imgf000039_0001
Scheme 4. a) reduction of azide into an amine followed by ring closure, b) protection of amine.
Rx in Scheme 4 may be chosen from methyl, trifluoromethyl, p-methylphenyl or similar residues present in readily available alkylsulfonylhalides, preferably a bulky Rx suitable for regioselective reaction on the primary alcohol of a diol as described in Chem. Ber. 101 (1968), 3802-3807. R1' and R2' are R1 and R2 as defined. Pg could be a suitable protecting group such as a carbamate, like benzyl carbamate or any similar protecting group which is not normally cleaved with acid.
Further substrates for the ring closure reaction could be compounds depicted in Scheme 5.
Figure imgf000040_0001
Scheme 5. a) reduction of azide into an amine followed by ring closure, b) protection of amine (optional).
Rx in Scheme 5 can be chosen from methyl, trifluoromethyl, p-methylphenyl or similar residues present in readily available alkylsulfonylhalides, preferably a bulky Rx suitable for regioselective reaction on the primary alcohol of a diol as described in Chem. Ber. 101 (1968), 3802-3807. R1' and R2' are R1 and R2 as defined above. Ry can be hydrogen or a hydroxyl protective group, preferably an ether type protective group. Preferably Ry is hydrogen. PG could be a suitable N-protecting group such as a carbamate, for derivatives in Scheme 5, Ry is typically hydrogen.
Other methodologies to obtain a 5,5-bicycle is disclosed by G. Lin and Z. Shi, Tetrahedron, 53, 4, 1369-1382, 1997.
Further modification of the 5,5-bicyclic compound obtained in scheme 1 is outlined in Scheme 6.
Figure imgf000041_0001
Scheme 6. a) benzyl bromide, sodium hydride, DMF. b) BF3-Et2O, Et3SiH, DCM. c) H2, Pd/C, BoC2O, 1 :1 EtOAc-EtOH. d) pyridine, acetic anhydride, e) H2, Pd/C, EtOAc
Compound 9 is protected with a suitable acid stable protecting group such as substituted methyl ether, in particular a benzyl ether, by treating the mono-ol 9 with a base such as sodium hydride or sodium hydroxide in an aprotic solvent such as N1N- dimethylformamide (DMF) in the presence of the desired alkylating agent such as the benzyl halide, in particular benzyl bromide. The obtained material can then be reduced into compound 10 according to methods described by G. J. Ewing and M. J. Robins, Org. Lett. 1 , 4, 1999, 635-636, or by references therein. Preferably the reduction is performed with excess boron trifluoride etherate in the presence of a reducing agent such as trialkylsilane, in particular with excess triethylsilane in a suitable non-protic solvent such as dichloromethane. Catalytic hydrogenation of compound 10 using for example palladium-on-charcoal in a suitable solvent or solvent mixture such as ethyl acetate-ethanol in a hydrogen atmosphere, in the presence of di-tert-butyl dicarbonate followed by treatment of the product with acetic anhydride in pyridine gives intermediate 11. By repeated catalytic hydrogenation, as described above, the mono-ol 12 is obtained.
A fluorine can be introduced on compound 12, and the bicyclic compound then N- deprotected according to Scheme 7.
Figure imgf000042_0001
Scheme 7. a) Deoxo-Fluor®, dichloromethane. b) methanolic sodium methoxide. c) 1 :1 dichloromethane-trifluoroacetic acid.
Compound 13 can be treated with a fluorinating agent such as [bis-(2- methoxyethyl)aminosulfur trifluoride] (Deoxo-Fluor®) or with similar fluorinating agents such as diethylaminosulfur trifluoride (DAST) which gives the product 14 with inversion of configuration at C-5. Compound 14 is then deacetylated by treatment for example with methanolic sodium methoxide, or any similar alkaline solutions with an inorganic base such as sodium hydroxide or sodium carbonate, followed by N-deprotection using acidic conditions such as dichloromethane-trifluoroacetic acid solutions or other methods which could be found in: Protective Groups in Organic Chemistry, 3rd edition, 1999, Theodora W. Greene and Peter G. M. Wuts (Wiley & Sons).
Alternatively the epimeric fluorine can be obtained by treating derivative 9 above according to Scheme 8.
Figure imgf000043_0001
Figure imgf000043_0002
Scheme 8. a) diisopropylazodicarboxylate, benzoic acid, PPh3, THF. b) methanolic sodium methoxide. c) benzyl bromide, sodium hydride, DMF. d) BF3.Et2θ, Et3SiH, dichloromethane. e) H2, Pd/C, BoC2O, 1 :1 EtOAc-EtOH. f) pyridine, acetic anhydride, g) H2, Pd/C, EtOAc. h) Deoxo-Fluor®, dichloromethane. i) methanolic sodium methoxide. j) 1 :1 dichloromethane-trifluoroacetic acid.
Inversion of configuration at C-5 can be accomplished by reacting compound 16 under Mitsunobo conditions which gives a benzoate ester. Ester hydrolysis with methanolic sodium methoxide followed by treatment of the mono-ol with benzyl bromide provides benzyl protected epimer 17. Reaction steps d-j in Scheme 8 are as described for Schemes 6 and 7.
A further route to a "difluoro derivative" wherein R1 and R2 are fluoro is shown in Scheme 9.
Figure imgf000044_0001
25 26
Figure imgf000044_0002
H 31
Scheme 9. a) benzyl bromide, sodium hydride, DMF. b) EtsSiH, BF3.Et2O or trimethylsilyl trifluoromethanesulfonate, DCM. c) H2, Pd/C, BoC2O, EtOAc-EtOH. d) benzoyl chloride, pyridine, DCM. e) H2, Pd/C, EtOAc. f) Bu2SnO, toluene, reflux, g) benzyl bromide, cesium fluoride, DMF. h) Dess-Martin periodinane. i) Deoxo-Fluor® or diethylaminosulfur trifluoride, DCM. j) methanolic sodium methoxide. k) H2, Pd/C, EtOAc. I) p-toluenesulfonyl chloride, pyridine, DCM. m) DCM, trifluoroacetic acid, n) triethylamine, dichloromethane. The synthesis of the P1 building block can be started from compound 21 (3-azido-3- deoxy-1 ,2-O-isopropylidene-D-allofuranose) which is described by Mayer zum Reckendorf, Chem. Ber. 101 (1968), 3802-3807. Treatment of compound 21 with a benzylating agent like benzyl bromide or benzyl chloride in the presence of a base, such as sodium hydride or sodium hydroxide in a aprotic polar solvent, such as N1N- dimethylformamide gives derivative 22. Compound 22 is then treated with a trialkyl silane, such as triethyl silane, with an excess of a Lewis acid such as boron trifluoride etherate or trimethylsilyl trifluoromethanesulfonate, in a aprotic solvent such as dichloromethane. The resulting azide can then be selectively reduced by catalytic hydrogenation using for example Palladium on charcoal in the presence of di-tert-butyl carbonate to obtain compound 23. Alternatively the azide could be reduced with other methods known from literature such as triphenylphosphine-water, followed by protection giving a suitable carbamate. In order to avoid problems with regioselectivity in the following steps, compound 23 could be treated with an acylating agent such as an acyl chloride or acid anhydride, such as benzoyl chloride, in neat organic base such as pyridine or triethyl amine, or in a mixture of an aprotic solvent such as dichloromethane and a base to give compound 24. Catalytic hydrogenation of compound 24 as described above gives diol 25. Selective benzylation at the primary alcohol of compound 25 can be accomplished by several methods known from the literature. In Scheme 9 the diol is refluxed with dibutyl tin oxide in a suitable solvent such as toluene to form a tin acetal. The tin acetal can then be reacted with a small excess of benzyl bromide and cesium fluoride in DMF giving the desired compound 26. Oxidation of 26 with a suitable oxidizing agent such as Dess-Martin periodinane in dichloromethane converts the secondary alcohol into the keto compound 27 suitable to convert into the difluoride 28. This can be accomplished by treating compound 27 with an excess fluorinating agent such as Deoxo-Fluor®, or with diethylaminosulfur trifluoride (DAST), in an aprotic solvent such as dichloromethane or 1 ,2-dichloroethane. The benzoate ester of compound 28 can be cleaved with alkali such as methanolic sodium methoxide, followed by debenzylation using catalytic hydrogenation to obtain diol 29. Selective introduction of a sulfonate ester at the primary alcohol can be accomplished by treating the compound 29 with a small excess of alkyl- or arylsulfonyl chloride in the presence of a base such as pyridine in suitable solvent such as dichloromethane, adding the sufonylating agent at reduced temperature and slowly increase up to room temperature, which gives mono-ol 30. Treatment of compound 30 under acidic conditions such as mixtures of dichlormethane-trifluoroacetic acid liberates the amine, and treating the product with a base such as triethyl amine promotes the internal ring closure which gives building block 31.
Alternative routes to 5,5-bicycles are shown in Schemes 10 and 11.
Figure imgf000046_0001
Scheme 10. a) fluorinating agent, b) reduction of amine or N-deprotection, optionally followed by N-protection. c) reducing agent.
In Scheme 10 a derivative such as compound 32 (available as described above or with methods well known in the art) with the substituents at C-3 and C-4 in cis relationship, Lg being a leaving group such as halogen or a sulfonate ester, and with R equal to an azide or a nitrogen protected with a suitable N-protecting group, can be treated with a fluorinating agent such as mentioned above, producing compound 33. Upon liberating the masked amine with either reduction of the azide or by a suitable deprotection method, the amine could perform an intramolecular attack at C-6 producing a 5,5- bicycle with structure 34, which could optionally be N-protected (Pg = protecting group or hydrogen). Reduction of C-1 with a suitable reducing agent such as described above or with a similar reducing agent would give building block 35.
In Scheme 11 an alternative route to a difluoro-5,5-bicycle is depicted.
Figure imgf000047_0001
Scheme 11. a) oxidation, b) fluorinating agent, c) reduction of azide or N-deprotection, optionally followed by N-protection. d) reducing agent.
In Scheme 11 compound 36 (available as described above or with methods well known in the art) with the substituents at C-3 and C-4 in cis relationship, Lg being a leaving group such as halogen or a sulfonate ester, and with R equal to an azide or a nitrogen protected with a suitable protecting group, can be oxidized with a Swem-type reaction or other suitable methods which can give compound 37. Treatment of compound 37 according to Scheme 11 with an excess of fluorinating agent such as mentioned above, gives compound 38. Upon liberating the masked amine of 38 with either reduction of the azide or by a suitable deprotection method, the amine could perform an intramolecular attack at C-6 producing a 5,5-bicycle with structure 39, which could optionally be N- protected (Pg = protecting group or hydrogen). Reduction of C-1 with a suitable reducing agent such as described above or with a similar reducing agent gives building block 40.
A convenient route to compounds wherein R1 or R2 is a halogen such as chloro is depicted in Scheme 12
Figure imgf000048_0001
Scheme 12
a) Thionyl chloride, b) methanolic sodium methoxide. c) 1 : 1 dichloromethane-trifluoroacetic acid, d) thionyl chloride, pyridine
The P1 building block is then elongated with the natural or non natural P2 amino acid and the P3 group by conventional solution or solid phase chemistries, such as those outlined or exemplified below, or disclosed in WO00/69855 or WO02/057270. P2 and P3 groups are either commercially available as enantiomers or resolvable from the racemate or obtainable using simple chemical transformations known to one skilled in the art. For example, 4-(methyl-piperazine-1-yl)-benzoic acid can be obtained using Buchwald chemistry (S. L. Buchwald & J. P. Wolfe, Journal of Organic Chemistry, 2000, 65, 1144) and subsequently elaborated. Other P3 cores such as 4-(1 -piperidin-4-yl)- benzoic acid are prepared from 1-(4-phenyl-piperidine-1-yl)-ethanone using a Friedel- Crafts acylation reaction and subsequently elaborated using standard chemical transformations known to one skilled in the art. Alternatively, other P3 moieties, such as 5-[2-(4-morpholinyl)ethoxy]-2-benzofuran-2-carboxylic acid, are prepared using Mitsunobu reactions on solid phase as detailed by L. S. Richter & T. R. Gadek in Tetrahedron Lett., 1994, 35, 4705. NMM
Figure imgf000049_0001
ioxan Et3N, cat DMAP
Figure imgf000049_0002
Scheme 13. Typical elongation of a cyclic ketone
Alternatively the P1 building block as the hydroxyl may be elongated and subsequently oxidised as shown in Scheme 14.
Figure imgf000050_0001
Scheme 14, Typical elongation of an hydroxylated P1 building block
Urethane compounds i.e. E is -OC(=O)- can be formed for example by reaction of an Re 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.
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.
Sulphamide derivatives i.e. E = NRdS(=O)2- can be prepared by reacting a suitable R6 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.
Compounds wherein E is CRbRc are typically prepared by reaction of an intermediate compound of the formula haloalkyl
Figure imgf000051_0001
where R6 and Rc are as defined above and LG is a conventional leaving group such as trifluoromethansulfonate, and the like , with the N-deprotected P1/P2 building block shown above. The reaction is carried out in a suitable organic solvent, including but not limited to, halogenated organic solvents such as methylene chloride, 1 ,2- dibromoethane, and the like, ethereal solvents such as diethyl ether, tetrahydrofuran, acetonitrile, or aromatic solvents such as benzene, toluene, xylene, and the like, or mixtures thereof and optionally in the presence of an organic or inorganic base. Preferably, the organic base is triethylamine, pyridine, N-methylmorpholine, collidine, diisopropylethylamine, and the like. Preferably, the inorganic base is cesium carbonate, sodium carbonate, sodium bicarbonate, and the like. The reaction is optionally carried out in the presence of a drying agent such as molecular sieves. Preferably, the reaction is carried out at room temperature. The intermediate can be prepared by methods well known in the art. For example, a compound where R6 is phenyl or 4- fluorophenyl, Rb is trifluoromethyl, and Rc is hydrogen can be readily prepared from commercially available 2,2,2 trifluoroacetophenone or 2,2,2, 4'-tetrafluoroacetophone respectively, by reducing the keto group to an alcoholic group by suitable reducing agent such as sodium borohydride, lithium aluminum hydride, and the like. The solvent used depends on the type of reducing agent. For example, when sodium borohydride is used the reaction is carried out in an alcoholic organic solvent such as methanol, ethanol, and the like. When lithium aluminum hydride is used the reaction is carried out in an ethereal solvent such as tetrahydrofuran, and the like. Reaction of 2,2,2 trifluoro-1-phenylethanol or 222- trifluoro-l-(4- fluorophenyl)ethanol with triflic anhydride provides the desired compound. Chirally enriched intermediate can be obtained by reduction of the corresponding halogenated acetophenone with a suitable reducing agent such as catechol borane or BH3-DMS complex in the presence of a suitable catalyst such as (A or (R) CBS catalyst or (A or (R)-,a -diphenyl-2- pyrrolidine-methanol in the presence of BBN.
In a corresponding fashion, the intermediate of the formula: haloalkyl
Figure imgf000052_0001
can be reacted with the carboxy-protected P2 building block, which is subsequently deprotected and elongated with the P1 building block as described herein.
Alternatively the above described intermediate is reacted:
Figure imgf000052_0002
LG is a suitable leaving group such as trifluoromethansulfonate, and PG a suitable hydroxyl protecting group such as trialkylsilyl, and the like, under the reaction conditions described above. The resulting O-protected hydroxyethylamide is oxidised to the corresponding carboxlic acid and couple to the P1 building block as described below. Suitable hydroxyl protecting groups and reaction conditions for putting them on and removing them can be found in Greene, T.W.; and Wuts, P. G. M. Protecting Groups in Organic Synthesis; John Wiley & Sons, Inc. I 999. The P2 hydroxyethylamine can be prepared from the corresponding natural and unnatural amino acids by methods well known in the art. Some such procedures are described in PCT Application Publication No. WO 03/075836, the disclosure of which is incorporated herein by reference in its entirety.
Alternatively compounds wherein E is -CRbRc- can be prepared by reaction of a compound of the formula O
R6 Rb where R6 is a cyclic group as defined above and Rb is halomethyl, preferably trifluoromethyl with the N-deprotected, carboxy-protected P2 building block or the P1/P2 building block outlined above under reductive amination reaction conditions. The reaction is carried out in the presence of a suitable dehydrating agent such as TiCU, magnesium sulfate, isopropyl trifluoroacetate, in the presence of a base such as diisopropylethylamine, pyridine, and the like and in a suitable organic solvent such as methylene chloride to give an imine. The imine is reduced with a suitable reducing agent such as sodium borohydride, sodium cyanoborohydride, and the like in a suitable organic solvent such as methanol, ethanol, and the like.
Alternatively compounds wherein E is -CRbRc- can be prepared by reaction of the haloalkylaldehyde with an amine as shown below:
Figure imgf000053_0001
*" formula Il
Figure imgf000053_0002
R5, R5', R6 and Rb are as defined above. Condensation of the haloalkylaldehyde with an aminoethanol (prepared by reducing the corresponding 5/5' alpha amino acid with a suitable reducing agent such as lithium aluminum hydride, and the like under conditions well known in the art), utilizing Dean Stark apparatus provides the depicted cyclic aminal which upon reaction with a Grignard reagent of formula R6MgX (where X is halo) or an organolithium reagent of formula R6Li I provides the depicted hydroxyethylamide. Oxidation of the hydroxyethylamide with a suitable oxidizing agent such as Jones oxidizing reagent or H5lθ6/Crθ3, and the like, then provides the P3/P2 building block which is C-terminal elongated with the P1 building block and oxidised as necessary. As described above elongation is typically carried out in the presence of a suitable coupling agent e.g., benzotriazole-1- yloxytrispyrrolidinophosphonium hexafluorophosphate (PyBOP), O- benzotriazol-l-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate (HBTU) , 0-(7-azabenzotriazol-1-yl)-1 ,1 ,3,3-tetramethyl-uronium hexafluorophosphate (HATU), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), or 1 ,3-dicyclohexyl carbodiimide (DCC), optionally in the presence of l-hydroxybenzotriazole (HOBT), and a base such as N1N- diisopropylethylamine, triethylamine, N-methylmorpholine, and the like. The reaction is typically carried out at 20 to 30 0C, preferably at about 25 0C, and requires 2 to 24 h to complete. Suitable reaction solvents are inert organic solvents such as halogenated organic solvents (e.g., methylene chloride, chloroform, and the like), acetonitrile, N1N dimethylformamide, ethereal solvents such as tetrahydrofuran, dioxane, and the like.
Alternatively, the above elongation coupling step can be carried out by first converting the P3/P2 building block into an active acid derivative such as succinimide ester and then reacting it with the P1 amine. The reaction typically requires 2 to 3 h to complete. The conditions utilized in this reaction depend on the nature of the active acid derivative. For example, if it is an acid chloride derivative of 4, the reaction is carried out in the presence of a suitable base (e.g. triethylamine, diisopropylethylamine, pyridine, and the like). Suitable reaction solvents are polar organic solvents such as acetonitrile, N1N- dimethylformamide, dichloromethane, or any suitable mixtures thereof.
The above method can also be used to prepared compounds where Rc is other than hydrogen utilizing the procedure described above, but substituting R6COH with a ketone of formula R6RbCO and then treating the resulting cyclic aminal with RcLi/RcMgX, followed by oxidation to give the free acid. The free acid is then condensed with under conditions described above.
It will be apparent to a person skilled in the art, that compounds wherein E is CRbRc can also be prepared as follows:
Figure imgf000055_0001
* H → formUla "
Figure imgf000055_0002
In particular conventional O-protection of the above described aminoethanol followed by reaction with the haloalkylhemiacetal provides the depicted haloalkylimine compound, which is treated with an organic lithium compound of formula R6Li where R6 is as defined above. Removal of the oxygen protecting group provides the hydroxyethyamide described in the immediately preceding scheme above which in the corresponding fashion is oxidised to the carboxylic acid and elongated with the P1 building block. Suitable oxygen protecting groups and reaction conditions for putting them on and removing them can be found in Greene, T.W.; and Wuts, P. G. M.; Protecting Groups in Organic Synthesis; John Wiley & Sons, Inc. I 999.
Alternatively, a compound wherein E is CRbRc and R6 is aryl or heteroaryl can be prepared as illustrated below:
Figure imgf000055_0003
In particular the above described haloalkyl hemiacetal is reacted with the protected P2 building block to yield the depicted 2-(1-hydroxymethylamino) acetate intermediate. The reaction is carried out in the presence of a catalytic amount of an acid such as p- toluenesulfonic acid and in an aromatic hydrocarbon solvent such as toluene, benzene, and the like.
Treatment of the 2-(1-hydroxymethylamino)acetate intermediate with R6H under Friedel- Crafts reaction conditions/ BF3-EtT2O provides the carboxy protected P3/P2 building block which is elongated as described above. Similarly, the haloalkyhemiacetal can be reacted with the P2/P1 building block, and oxidised to the ketone, as necessary. The term "N-protecting group" or "N-protected" as used herein refers to those groups intended to protect the N-terminus of an amino acid or peptide or to protect an amino group against undesirable reactions during synthetic procedures. Commonly used N- protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis" (John Wiley & Sons, New York, 1981 ), which is hereby incorporated by reference. N- protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t- butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoracetyl, trichloroacetyl, phthalyl, o- nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4- nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl, and the like, carbamate forming groups such as benzyloxycarbonyl, p- chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1 -(p-biphenylyl)-i -methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4- nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like; alkyl gropus such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. Favoured N-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz).
Hydroxy and/or carboxy protecting groups are also extensively reviewed in Greene ibid and include ethers such as methyl, substituted methyl ethers such as methoxy methyl, methylthiomethyl, benzyloxymethyl, t-butoxymethyl, 2-methoxyethoxymethyl and the like, silyl ethers such as trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS) tribenzylsilyl, triphenylsilyl, t-butyldiphenylsilyl triisopropyl silyl and the like, substituted ethyl ethers such as 1-ethoxy methyl, 1-methyl-1-methoxyethyl, t-butyl, allyl, benzyl, p- methoxybenzyl, dipehenylmethyl, triphenylmethyl and the like, aralkyl groups such as trityl, and pixyl (9-hydroxy-9-phenylxanthene derivatives, especially the chloride). Ester hydroxy protecting groups include esters such as formate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate, pivaloate, adamantoate, mesitoate, benzoate and the like. Carbonate hydroxy protecting groups include methyl vinyl, allyl, cinnamyl, benzyl and the like.
Detailed Description of the Embodiments
Various embodiments of the invention will now be described by way of illustration only with reference to the following Examples.
Example 1-1
Intermediates towards P1 building block
Step a)
Figure imgf000057_0001
A mixture of 54 (5.2 g, 13.0 mmol), palladium-on-carbon (10%, Acros, 0.66 g) in methanol was hydrogenated at slight positive pressure. The hydrogen was changed 3 times over a period of 1 h, after TLC (petroleum ether-ethyl acetate 7:3 and dichloromethane-methanol 9:1 , staining with ammonium molybdate-cerium sulfate) indicated complete conversion of the starting material into a major non-UV active spot which colours AMC, and some weaker higher moving spots (dichloromethane-methanol 9:1 ). The reaction mixture was then filtered through Celite and concentrated which gave crude compound 55.
To a suspension of the residue in dichloromethane (60 ml) and pyridine (3.2 ml, 40 mmol) at 0 0C was added benzylchloroformate (0.93 ml, 6.5 mmol). The reaction mixture was stirred at rroom temperature for 2 h after which additional pyridine (3 ml) and benzylchloroformate (0.8 ml) was added at 0 0C. The reaction mixture was then stirred at room temperature overnight, then diluted with dichloromethane (100 ml), washed successively with 1 M aq. sulfuric acid (2 x 50 ml) and 1M aq. sodium hydrogen carbonate (1 x 50 ml), then dried (sodium sulfate), filtered and concentrated onto silica. Flash chromatography (diameter: 4 cm, YMC-gel: 50 g, packing eluent: ethyl acetate in petroleum ether 1 :4) of the residue using ethyl acetate in petroleum ether 1 :4 (350 ml), 2:3 (250 ml), 1 :1 (250 ml), 3:2 (250 ml) and 3:1 (150 ml) gave compound 56 as a foamy syrup (2.71 g, 8.1 mmol, 62% over 2 steps) after drying in vacuum overnight.
NMR data (400 MHz, CDCI3): 1H, 1.33, 1.52 (2 s, 6H, C(CH3)2), 2.34 (2 d, 1 H, -OH), 3.04 (m, 1 H, H-6a), 3.97 (m, 1 H, H-6b), 4.19 (m, 1 H, H-5), 4.33 (m, 1 H, H-3), 4.68, 4.84 (2 d, 1 H, H-2), 4.79 (t, 1 H, H^), 5.08-5.24 (m, 2H, CH2Ph), 5.86 (br s, 1 H, H-1 ), 7.30- 7.42 (m, 5H, Ar-H).
Step b)
Figure imgf000058_0001
To a stirred suspension of sodium hydride (60% in mineral oil, Aldrich, 0.34 g, 8.4 mmol) and compound 56 (2.17 g, 6.47 mmol) in dimethylformamide (30 ml) was added benzyl bromide (0.81 mmol, 6.8 mmol) during 5 minutes. After stirring 1 h (TLC: ethyl acetate in petroleum ether 2:3), methanol (approx 2 ml) was added to destroy excess reagent, then immediately partitioned between ethyl acetate (180 ml) and water (150 ml). The organic layer was washed with water (3 x 100 ml), then dried (sodium sulfate), filtered and concentrated onto silica. Flash chromatography (diameter: 4 cm, YMC-gel: 40 g, packing eluent: ethyl acetate in petroleum ether 1 :4) of the residue using ethyl acetate in petroleum ether 1 :4 (100 ml), 3:7 (250 ml) and 2:3 (250 ml) gave a colourless syrup (2.7 g, 6.35 mmol, 98%) after drying in vacuum overnight.
NMR data (400 MHz, CDCI3): 1H, 1.31 (s, 3H, C(CH3)(CH3)), 1.51 (d, 3H, C(CH3)(CH3)), 3.29 (m, 1 H, H-6a), 3.78-3.96 (m, 2H, H-5 and H-6b), 4.22 (dd, 1 H, H-3), 4.64, 4.84 (2 M, 4H, H-2, H-4 and CH2Ph), 5.07-5.22 (m, 1 H, CH2Ph), 5.94 (m, 1 H, H-1 ), 7.28-7.39 (m, 10H1 Ar-H).
Example 1-2
4-Benzyl-6-fluoro-octahvdro-pyrrolor3.2-blpyrrol-3-ol P1 building block
Figure imgf000059_0001
Step a) Methanesulfonic acid 5-(2,2-dimethyl-[1 ,3]dioxolan-4-yl)-2,2-dimethyl tetrahydro-furo[2,3-d][1 ,3]dioxol-6-yl ester
5-(2,2-Dimethyl-[1 ,3]dioxolan-4-yl)-2,2-dimethyl-tetrahydro-furo[2,3-d][1 ,3]dioxol-6-ol (9.8 g, 37.65 mmol) was dissolved in dichloromethane (50 ml) and treated with pyridine (6.7 ml, 82.83 mmol). The solution was cooled down to -5 0C, and the triflic anhydride (7.6 ml, 45.18 mmol) was added dropwise. The reaction mixture was stirred at this temperature for about an hour, monitoring by tic (ethyl acetate-heptane 1 :3, Rf 0.42). The reaction was then quenched with ice-water, and the product was extracted in dichloromethane. The extracts were concentrated in vacuum avoiding heating, to afford the title compound (37.65 mmol, assuming quantitative), which was used in the next step without further purification.
Step b) 6-Azido-5-(2,2-dimethyl-[1 ,3]dioxolan-4-yl)-2,2-dimethyl- tetrahydrofuro[2,3d][1 ,3]-dioxole Methanesulfonic acid 5-(2,2-dimethyl-[1 ,3]dioxolan-4-yl)-2,2-dimethyl tetrahydro- furo[2,3-d][1 ,3]dioxol-6-yl ester was dissolved in dimethylformamide (50 ml) and treated with sodium azide (7.3 g, 112.95 mmol). The reaction mixture was stirred at 50 0C for 2h, monitoring by tic (ethyl acetate-heptane 1 :3, Rf 0.60). When the reaction had finished, the solvent was removed under vacuum, and the residue was partitioned between dichloromethane and water, the organic layer was dried and concentrated, an the residue was purified by column chromatography, to afford the title compound (10.35 g, 36.3 mmol, 96.4% in 2 steps).
Step c) 1 -(6-Azido-2,2-dimethyl-tetrahydro-furo[2,3-d][1 ,3]dioxol-5-yl)-ethane-1 ,2- diol
A solution of 6-azido-5-(2,2-dimethyl-[1 ,3]dioxolan-4-yl)-2,2-dimethyl- tetrahydrofuro[2,3d][1 ,3]-dioxole (10.35 g, 36.3 mmol) in acetic acid-water (7:3, 100 ml) was heated to 50 0C for 2.5 h (ethyl acetate-heptane 1 :3, Rf 0.11 ). The solvent was then removed under vacuum, using toluene to co-evaporate traces of acid, to afford the title compound (36.3 mmol, assuming quantitative), which was used without further purification in the next step.
Step d) Toluene-4-sulfonic acid 2-(6-azido-2,2-dimethyl-tetrahydro-furo[2,3- d][1 ,3]dioxol-5-yl)-2-hydroxy-ethyl ester
1 -(6-Azido-2,2-dimethyl-tetrahydro-furo[2,3-d][1 ,3]dioxol-5-yl)-ethane-1 ,2-diol (10.35 g, 36.3 mmol) was dissolved in dichloromethane (100 ml) and treated with pyridine (8.8 ml, 108.9 mmol) followed by tosyl chloride (6.92 g, 36.3 mmol). The reaction mixture was stirred at 4 0C for 48 h (ethyl acetate-heptane 1 :2, Rf 0.23). The mixture was then washed with 1M HCI and saturated NaHCθ3, dried and concentrated. The residue was purified by column chromatography to yield the title compound (13.5 g, 33 mmol, 93.2% in 2 steps).
Step e) Toluene-4-sulfonic acid 2-(6-azido-2,2-dimethyl-tetrahydro-furo[2,3- d][1 ,3]dioxol-5-yl)-2-fluoro-ethyl ester Toluene-4-sulfonic acid 2-(6-azido-2,2-dimethyl-tetrahydro-furo[2,3-d][1 ,3]dioxol-5-yl)-2- hydroxy-ethyl ester (2.50 mmol, Tetrahedron, 43, 3095-3108, 1987) was dissolved in dichloromethane and treated with pyridine (505 μl_, 6.25 mmol). The reaction mixture was then treated dropwise and at O0C, with Deoxo-Fluor ([bis(2- methoxyethyl)amino]sulfur trifluoride, 5.00 mmol) and stirred at room temperature overnight. The reaction mixture was quenched with saturated aqueous NaHCO3, and partitioned between dichloromethane and water. The organic layer was dried and concentrated, and the residue was purified by column chromatography to yield the title compound (1.37 mmol, 55%).
NMR data (400 MHz, CDCI3): 1H, δ 7.78, 7.34 (2d, 4H, p-Ph), 5.90 (d, 1 H, H-1 ), 4.74 (dm, 1 H, H-5), 4.67 (m, 1 H, H-2), 4.32 (m, 1 H, H-6a), 4.30 (m, 1 H, H-4), 4.25 (d, 1 H, H- 3), 3.91 (d, 1 H, H-6b), 2.21 (s, 3H, MePh), 1.46 and 1.30 (2s, 6H, CMe2).
Step f) 7-Benzyl-5-fluoro-2,2-dimethyl-hexahydro-[1 ,3]dioxolo [4',5':4,5] furo[3,2-b]pyrrole.
A solution of toluene-4-sulfonic acid 2-(6-azido-2,2-dimethyl-tetrahydro-furo[2,3- d][1 ,3]dioxol-5-yl)-2-fluoro-ethyl ester (1.47 mmol) in ethanol was treated with Pd(C) (10% w/w, catalytic amount) and stirred under a hydrogen atmosphere overnight, monitoring the reaction by thin layer chromatography (ethyl acetate-heptane 1 :2). When the reaction had finished, the mixture was filtered through a celite cake and concentrated. The residue was dissolved in methanol (10 ml) and treated with benzaldehyde (179 μl_, 1.76 mmol). The mixture was stirred at room temperature for 1 h, then treated with sodium cyanoborohydride (111 mg, 1 ,76 mmol) and stirred at room temperature overnight. The solvent was then removed under vacuum, and the residue partitioned between dichloromethane and water. The organic pahse was dried and concentrated, and the residue was purified by column chromatography to afford the title compound (1.32 mmol, 90%). Rf 0.75 (ethyl acetate-hexane 1 :2).
NMR data (400 MHz, CDCI3): 1H, δ 7.3 (m, 5H, Ph), 5.90 (d, 1 H, H-1 ), 4.95 (dt, 1 H, H- 5), 4.82 (dd, 1 H, H-4), 4.05 (d, 1 H, CH2aPh), 3.60 (d, 1 H, CH2ύPh), 3.50 (d, 1 H, H-3), 3.40 (ddd, 1 H, H-6a), 2.60 (ddd, 1 H, H-6b), 1.50 and 1.35 (2s, 6H, CMe2). Step g) 1 -Benzyl-2-(2,2-dimethyl-[1 ,3]dioxolan-4-yl)-4-fluoro-pyrrolidin-3R- ol
7-Benzyl-5-fluoro-2,2-dimethyl-hexahydro-[1.SJdioxolo^'.S'tf.SJfurop^-bJpyrrole (1.32 mmol) was treated with TFA-water (9:1 , 10 ml) at room temperature for 1 h. The mixture was then concentrated, using toluene to co-evaporate the traces of residual acid. The residue was suspended in water and neutralised with aqueous sodium hydroxide, treated with ion exchange resin (DOWEX) and then filtered and concentrated. The residue was dissolved in methanol-water (1 :3, 15 ml) and treated with NaBH4 (2.7 mmol) and stirred at 40 0C overnight. The reaction was monitored by MS. The following morning, the reaction mixture was concentrated and the residue suspended in methanol and evaporated several times to remove the boron species. Anhydrous acetone was added to the residue (10 ml) and a catalytic amount of cone, sulfuric acid was added to the reaction mixture (3 drops). The mixture was stirred at room temperature for 3 h, then neutralised with solid sodium carbonate, filtered and concentrated. The residue was purified by column chromatography to afford the title compound.
NMR data (400 MHz, CDCI3): 1H, δ 7.3 (m, 5H, Ph), 4.84 (dm, 1 H, H-5), 4.35 (m, 2H, Ctf2aPh, H-3), 4.19 (m, 2H, H-1a, H-4), 3.91 (dd, 1 H, H-1 b), 3.44 (d, 1 H, CH2bPh), 3.40 (ddd, 1 H, H-6a), 2.98 (m, 1 H, H-3), 2.64 (m, 1 H, OH), 2.50 (ddd, 1 H, H-6b), 1.47 and 1.37 (2s, 6H1 CMe2).
Step h) 1 -Benzyl-2-(2,2-dimethyl-[1 ,3]dioxolan-4-yl)-4-fluoro-pyrrolidin-3S- ol
1-Benzyl-2-(2,2-dimethyl-[1 ,3]dioxolan-4-yl)-4-fluoro-pyrrolidin-3R-ol is dissolved in THF and treated with p-nitrobenzoic acid and triphenylphosphine. The solution was cooled down in an ice bath, and treated with DIAD, and stirred at 4 0C overnight. The reaction mixture was then diluted with dichloromethane and washed with saturated sodium bicarbonate, dried and concentrated. The residue was purified by column chromatography. The residue was then taken up on methanol and treated with triethylamine, and stirred at room temperature for 2 h, then concentrated and purified by column chromatography to yield title compound. Step i) 3-Azido-1 -benzyl-2-(2,2-dimethyl-[1 ,3]dioxolan-4-yl)-4-fluoro- pyrrolidine
To a solution of 1-benzyl-2-(2,2-dimethyl-[1 ,3]dioxolan-4-yl)-4-fluoro-pyrrolidin-3S-ol in dichloromethane is added pyridine. The solution is cooled down to -5 0C, and triflic anhydride is added dropwise. The reaction mixture is stirred at this temperature for an hour, monitoring by tic. The reaction is then quenched with ice-water, and the product extracted in dichloromethane. The extracts are concentrated in vacuum avoiding heating and the residue dissolved in dimethylformamide and treated with sodium azide. The reaction mixture is stirred at 50 0C for 3 h, monitoring by tic. When the reaction is finished, the solvent is removed under vacuum, and the residue partitioned between dichloromethane and water, the organic layer dried and concentrated, an the residue purified by column chromatography, to afford the title compound.
Step j) Toluene-4-sulfonic acid 2-(3-azido-1-benzyl-4-fluoro-pyrrolidin-2-yl)-2- hydroxy-ethyl ester
A solution of 3-azido-1-benzyl-2-(2,2-dimethyl-[1 ,3]dioxolan-4-yl)-4-fluoro-pyrrolidine in acetic acid-water (7:3) is heated to 50 0C for 3 h. The solvent is then removed under vacuum, using toluene to co-evaporate traces of acid. The residue is dissolved in dichloromethane and treated with pyridine followed by tosyl chloride. The reaction mixture is stirred at 4 0C overnight. The mixture is then washed with 1 M HCI and saturated NaHCO3, dried and concentrated. The residue is purified by column chromatography to yield the title compound.
Step k) 4-Benzyl-6-fluoro-octahydro-pyrrolo[3,2-b]pyrrol-3-ol
Toluene-4-sulfonic acid 2-(3-azido-1 -benzyl-4-fluoro-pyrrolidin-2-yl)-2-hydroxy-ethyl ester is dissolved in a mixture of methanol and dioxane (1 :5) and treated with triphenylphosphine. The reaction mixture is stirred at room temperature for 3 h, and then treated with water, and stirred at room temperature overnight. The solvents are removed under vaccum, and the residue taken up on diluted aqueous HCI, and washed with ether. The aqueous phase is freeze-dried to afford the title compound. The synthetic scheme can then continued as outlined in Scheme A above to build up the final compound. Hence, the amine functionality of 4-benzyl-6-fluoro-octahydro- pyrrolo[3,2-b]pyrrol-3-ol is reacted with the appropriate electrophilic component to obtain a wide variety of groups at the R2 position. Once this functionality is in place, benzyl protection is removed using 4 M HCI in dioxane and the free amine coupled with the appropriate P2 residue, for example the L-Leu depicted in scheme A. Subsequent removal of the Cbz moiety and coupling on the P3 residue using the appropriate R6 acid affords the alcohol which serves as the precursor for the final step. Oxidation of the alcohol (as shown below on a model compound) affords the required final compound.
Example 1-3
An alternative route to a P1 buldinq block :
An alternative route to a P1 building block involves ring closing metathesis as outlined below:
Figure imgf000064_0001
Olefin metathesis (RCM, ring closing metathesis) is typically performed with a Ru-based catalyst such as the one reported by Miller, S.J., Blackwell, H. E.; Grubbs, R.H. J. Am. Chem. Soc. 118, (1996), 9606-9614, Kingsbury, J. S., Harrity, J. P. A., Bonitatebus, P. J., Hoveyda, A. H., J. Am. Chem. Soc. 121 , (1999), 791-799 and Huang et al., J. Am. Chem. Soc. 121 , (1999), 2674-2678 can alternately be used to effect the metathesis reaction. It will also be recognized that catalysts containing other transition metals such as Mo can be used for this reaction.
Step a) ((2R,3R)-3-Vinyl-oxiranyl)-methanol can be synthesised from penta-1 ,4- dien-3-ol in two steps according to the procedure of Alex Romero and Chi-Huey Wong, J. Org. Chem. 2000, 65, 8264-8268.
Step b) Allyl-[(S)-1 -((S)-1 ,2-dihydroxy-ethyl)-allyl]-carbamic acid terf-butyl ester
Figure imgf000065_0001
To a stirred solution of ((2R,3R)-3-vinyl-oxiranyl)-methanol (200 mg, 2.00 mmol) and lithium triflate (312 mg, 2.00 mmol) in dry acetronitrile (0.75 ml_) was added allylamine (0.450 ml_, 6.00 mmol) at room temperature giving a clear colourless solution. The mixture was sealed in a 15 ml_ microwave tube and heated at 120°C for 5 minutes generating a clear orange solution. The mixture was concentrated under reduced pressure and placed under high vacuum to remove all volatile components. To a mixture of the residue and sodium hydrogen carbonate (504 mg, 6.00 mmol) suspended in methanol (8.0 ml_) was added di-terf-butyl dicarbonate (655 mg, 3.00 mmol) at room temperature. The suspension was stirred at this temperature for 48 hours whereupon tic. indicated consumption of starting material. The mixture was concentrated under reduced pressure and partitioned between water / chloroform. The aqueous phase was extracted into chloroform (x3) and the combined organic extracts were dried (magnesium sulfate), filtered and concentrated under reduced pressure. Flash column chromatography eluting with a gradient of 33 % ethyl acetate / /so-hexane to 100 % ethyl acetate provided 372 mg (72 % over 2 steps) of the title compound.
TLC. (ethyl acetate) Rf = 0.44; m/z = 258 (20 % M+H), 202 (100 % M+2H-<Bu), 158 (45 % M+H-Boc) in MS ES+. Step c) (S)-2-((S)-1 ,2-Dihydroxy-ethyl)-2,5-dihydro-pyrrole-1 -carboxylic acid tert- butyl ester
Figure imgf000066_0001
A solution of allyl-[(S)-1-((S)-1 ,2-dihydroxy-ethyl)-allyl]-carbamic acid tert-butyl ester (1.00 g, 3.89 mmol) in dry dichloromethane (70 ml_) was degassed under a stream of nitrogen for 20 minutes before Grubbs 2nd generation catalyst (66 mg, 0.077 mmol) was added at room temperature. The pale brown solution was stirred at room temperature for 7 hours before it was concentrated under reduced pressure. Flash column chromatography eluting with a gradient of 33 % ethyl acetate / /so-hexane to 100 % ethyl acetate provided 778 mg (87 %) of the title compound. TLC. (70 % ethyl acetate / /so-hexane) Rf = 0.18; m/z = 230 (15 % M+H), 174 (100 % M+2H-<Bu), 130 (10 % M+H- Boc) in MS ES+.
Step d) (S)-2-[(S)-1 -Hydroxy-2-(toluene-4-sulfonyloxy)-ethyl]-2,5-dihydro-pyrrole-
1 -carboxylic acid terf-butyl ester
Figure imgf000066_0002
To a stirred solution of (S)-2-((S)-1 ,2-dihydroxy-ethyl)-2,5-dihydro-pyrrole-1 -carboxylic acid tert-butyl ester (460 mg, 2.00 mmol) and pyridine (0.485 ml_, 6.00 mmol) in dry dichloromethane (2.0 ml_) was added p-toluenesulfonyl chloride (458 mg, 2.40 mmol) at 0°C, in 2 portions 30 minutes apart. After 6 hours at 0°C an additional 0.2 equivalents (76 mg) of p-toluenesulfonyl chloride was added and the suspension was left to stand at 2°C overnight. The mixture was partitioned between water / saturated sodium hydrogen carbonate solution / chloroform and the aqueous phase was extracted into chloroform (x3). Combined organic extracts were dried (magnesium sulfate), filtered and concentrated under reduced pressure. Flash column chromatography eluting with a gradient of 5 % ethyl acetate / /so-hexane to 66 % ethyl acetate / /so-hexane provided 660 mg (86 %) of the title compound.
TLC. (70 % ethyl acetate / /so-hexane) Rf = 0.52; m/z = 384 (35 % M+H), 328 (100 % M+2H-'Bu), 284 (10 % M+H-Boc) in MS ES+.
Step e) (S)-2-[(R)-1-Fluoro-2-(toluene-4-sulfonyloxy)-ethyl]-2,5-dihydro-pyrrole-1- carboxylic acid terf-butyl ester
Figure imgf000067_0001
To a stirred solution of (S)-2-[(S)-1-hydroxy-2-(toluene-4-sulfonyloxy)-ethyl]-2,5-dihydro- pyrrole-1-carboxylic acid tert-butyl ester (100 mg, 0.261 mmol) in dry dichloromethane (1.6 ml_) at -78°C was added (diethylamino)sulfur trifluoride (0.344 ml_, 2.61 mmol) dropwise. The reaction mixture was left in the cold bath to slowly warm to room temperature overnight before the mixture was added cautiously to a rapidly stirred mixture of ethyl acetate / water. After stirring for 20 minutes the phases were separated and the aqueous phase was extracted into ethyl acetate (x3). Combined organic extracts were dried (magnesium sulfate), filtered and concentrated under reduced pressure. Flash column chromatography eluting with a gradient of 5 % ethyl acetate / /so-hexane to 66 % ethyl acetate / /so-hexane provided 39 mg (39 %) of the title compound. TLC. (50 % ethyl acetate / /so-hexane) Rf = 0.54; m/z = 386 (22 % M+H), 330 (100 % M+2H-'Bu), 286 (73 % M+H-Boc) in MS ES+.
Example 1-4
Alternative RCM approach to a P1 building block
Figure imgf000068_0001
An alternative end to the scheme in Example 1-3 proceeds as follows:
Figure imgf000068_0002
Figure imgf000068_0003
Step a) (S)-2-[(S)-1 -Acetoxy-2-(toluene-4-sulfonyloxy)-ethyl]-2,5-dihydro-pyrrole-1 - carboxylic acid terf-butyl ester
Figure imgf000068_0004
To a stirred solution of (S)-2-((S)-1 ,2-dihydroxy-ethyl)-2,5-dihydro-pyrrole-1 -carboxylic acid tert-butyl ester (2.00 g, 5.22 mmol) and pyridine (2.11 ml_, 26.1 mmol) in dry dichloromethane (5.5 ml_) was added acetic anhydride (1.55 ml_, 15.6 mmol) dropwise at 0°C. The reaction mixture was allowed to warm to room temperature and stirred overnight before the mixture was partitioned between water / saturated sodium hydrogen carbonate solution / chloroform. The aqueous phase was extracted into chloroform (x3) and the combined organic extracts were dried (magnesium sulfate), filtered and concentrated under reduced pressure. Flash column chromatography eluting with a gradient of 5 % ethyl acetate / /so-hexane to 66 % ethyl acetate / iso- hexane provided 2.06 g (93 %) of the title compound. TLC. (50 % ethyl acetate / iso- hexane) Rf = 0.46; m/z = 426 (30 % M+H), 370 (70 % M+2H-'Bu), 326 (15 % M+H-Boc) in MS ES+.
Step b) (1 R,2R,5S)-2-[(S)-1-Acetoxy-2-(toluene-4-sulfonyloxy)-ethyl]-6-oxa-3-aza- bicyclo[3.1.0]hexane-3-carboxylic acid tert-butyl ester; and (1S,2R,5R)-2-[(S)-1-Acetoxy-2-(toluene-4-sulfonyloxy)-ethyl]-6-oxa-3-aza- bicyclo[3.1.0]hexane-3-carboxylic acid tert-butyl ester
Figure imgf000069_0001
To a rapidly stirred suspension of (S)-2-[(S)-1-acetoxy-2-(toluene-4-sulfonyloxy)-ethyl]- 2,5-dihydro-pyrrole-1-carboxylic acid tert-butyl ester (129 mg, 0.303 mmol), trifluoroacetone (0.299 ml_, 3.33 mmol) and sodium hydrogen carbonate (204 mg, 6.00 mmol) in acetonitrile (2.7 ml_) and Na2EDTA solution (4x104 M, 0.000606 mmol, 1.52 ml_) was added Oxone™ (932 mg, 1.52 mmol) at 0°C, in portions over 1.5 hours. After 2 hours at 0°C additional sodium hydrogen carbonate (102 mg, 1.21 mmol) and Oxone™ (184 mg, 0.299 mmol) was added sequentially and the suspension was stirred at 0°C for 1 additional hour. The mixture was partitioned between water / chloroform and the aqueous phase was extracted into chloroform (x3). Combined organic extracts were dried (magnesium sulfate), filtered and concentrated under reduced pressure. Flash column chromatography eluting with a gradient of 5 % ethyl acetate / /so-hexane to 66 % ethyl acetate / /so-hexane provided 101 mg (75 %) of the title compound as an inseparable 1 : 1 mixture of epoxides.
TLC. (50 % ethyl acetate / /so-hexane) Rf = 0.53; m/z = 442 (50 % M+H), 386 (100 % M+2H-'Bu), 342 (10 % M+H-Boc) in MS ES+.
Step c) (1 R,2R,5S)-2-((R)-1 -Acetoxy-2-azido-ethyl)-6-oxa-3-aza- bicyclo[3.1.0]hexane-3-carboxylic acid terf-butyl ester; and (1S,2R,5R)-2-((R)-1-acetoxy-2-azido-ethyl)-6-oxa-3-aza-bicyclo[3.1.0]hexane-3- carboxylic acid terf-butyl ester.
Figure imgf000070_0001
To a stirred solution of (1 R,2R,5S)-2-[(S)-1-acetoxy-2-(toluene-4-sulfonyloxy)-ethyl]-6- oxa-3-aza-bicyclo[3.1.0]hexane-3-carboxylic acid terf-butyl ester and (1S,2R,5R)-2-[(S)- 1 -acetoxy^-^oluene^-sulfonyloxyJ-ethylJ-β-oxa-S-aza-bicycloβ.1.0]hexane-3- carboxylic acid tert-butyl ester (40 mg, 0.0906 mmol) in dry dimethylformamide (0.6 ml_) was added sodium azide (29 mg, 0.453 mmol) before the mixture was warmed to 40°C for 40 hours. The reaction mixture was diluted with ethyl acetate, washed with water (x1), brine (x1), dried (magnesium sulfate), filtered and concentrated under reduced pressure. Flash column chromatography eluting with a gradient of 5 % ethyl acetate / /so-hexane to 66 % ethyl acetate / /so-hexane provided 21 mg (74 %) of the title compound as an inseparable 1 : 1 mixture of epoxides.
TLC. (50 % ethyl acetate / /so-hexane) Rf = 0.60; m/z = 313 (10 % M+H), 257 (100 % M+2H-'Bu), 213 (80 % M+H-Boc) in MS ES+.
Step d) (3S,3aS,6R,6aS)-6-Acetoxy-3-hydroxy-4-methanesulfonyl-hexahydro- pyrrolo[3,2-ύ]pyrrole-1-carboxylic acid te/if-butyl ester
Figure imgf000070_0002
A solution of (1 R,2R,5S)-2-((R)-1-acetoxy-2-azido-ethyl)-6-oxa-3-aza- bicyclo[3.1.0]hexane-3-carboxylic acid terf-butyl ester and (1S,2R,5R)-2-((R)-1-acetoxy- 2-azido-ethyl)-6-oxa-3-aza-bicyclo[3.1.0]hexane-3-carboxylic acid terf-butyl ester (67 mg, 0.214 mmol) in methanol (20 ml_) was passed over 10 % palladium on carbon (1 ml_/min; H-cube™ cartridge) in the presence of hydrogen (1 atm). TIc showed complete consumption of starting material and the mixture was concentrated under reduced pressure. To a mixture of the residue and pyridine (0.485 ml_, 6.00 mmol) in dry dichloromethane (2.0 ml_) was added methanesulfonyl chloride (458 ml_, 2.40 mmol) at 0°C. After 2 hours at 0°C the yellow solution was partitioned between saturated sodium hydrogen carbonate solution / chloroform and the aqueous phase was extracted into chloroform (x3). Combined organic extracts were dried (magnesium sulfate), filtered and concentrated under reduced pressure. Flash column chromatography eluting with a gradient of 33 % ethyl acetate / /so-hexane to 100 % ethyl acetate provided 16 mg (21 % over 2 steps) of the title compound.
TLC. (ethyl acetate) Rf = 0.43; m/z = 365 (15 % M+H), 309 (100 % M+2H-<Bu), 265 (85 % M+H-Boc) in MS ES+.
Step e) (3aS,6R,6aS)-6-Acetoxy-3-fluoro-4-methanesulfonyl-hexahydro- pyrrolo[3,2-ύ]pyrrole-1-carboxylic acid terf-butyl ester
Figure imgf000071_0001
At at 0°C Deoxofluor™ (0.32 ml_, 1.76 mmol) was added dropwise to (3S,3aS,6R,6aS)- β-acetoxy-S-hydroxy^-methanesulfonyl-hexahydro-pyrrolotS^-ύJpyrrole-i-carboxylic acid tert-butyl ester (16 mg, 0.0439 mmol). The mixture was allowed to warm to room temperature and stirred overnight before it was heated to 45°C for 7 hours. The mixture was added cautiously to a rapidly stirred mixture of chloroform / water. After stirring for 20 minutes the phases were separated and the aqueous phase was extracted into chloroform (x3). Combined organic extracts were dried (magnesium sulfate), filtered and concentrated under reduced pressure. Flash column chromatography eluting with a gradient of 5 % ethyl acetate / /so-hexane to 66 % ethyl acetate / /so-hexane provided 4 mg (25 %) of the title compound as a mixture of epimers. m/z = 367 (5 % M+H), 311 (100 % M+2H-'Bu), 267 (20 % M+H-Boc) in MS ES+.
Step f) (3aS,6R,6aS)-3-Fluoro-6-hydroxy-4-methanesulfonyl-hexahydro- pyrrolo[3,2-ύ]pyrrole-1-carboxylic acid tert-butyl ester
Figure imgf000072_0001
To a stirred solution of (3aS,6R,6aS)-6-acetoxy-3-fluoro-4-methanesulfonyl-hexahydro- pyrrolo[3,2-ύ]pyrrole-1-carboxylic acid tert-butyl ester (36 mg, 0.0982 mmol) in dry methanol (1.5 ml_) room temperature was added sodium methoxide (40 μl_, 0.0196 mmol; 0.5M in methanol) dropwise. The reaction mixture stirred at room temperature for 1 hour before it was and partitioned between saturated ammonium chloride solution / chloroform. The aqueous phase was extracted into chloroform (x3) and the combined organic extracts were dried (magnesium sulfate), filtered and concentrated under reduced pressure to give the title compound 31 mg (97 %). TLC. (50 % ethyl acetate / /so-hexane) Rf = 0.18; m/z = 325 (40 % M+H), 269 (100 % M+2H-*Bu) in MS ES+.
Step g) (3R,3aS,6aS)-6-Fluoro-1 -methanesulfonyl-octahydro-pyrrolo[3,2-ύ]pyrrol-
3-ol
Figure imgf000072_0002
(3aS,6R,6aS)-3-Fluoro-6-hydroxy-4-methanesulfonyl-hexahydro-pyrrolo[3,2-ύ]pyrrole-1- carboxylic acid tert-butyl ester (31 mg, 0.0955 mmol) was dissolved in 4M HCI solutuion in 1 ,4-dioxane (5 ml_) at room temperature. The reaction mixture stirred at room temperature for 1 hour before it was concentrated under reduced pressure to give the title compound as the HCI salt 29 mg (116 %, crude), m/z = 225 (100 % M+H) in MS ES+.
Feasibility Example 2 Elongation with a model P1/P2 system
Step a)
Figure imgf000073_0001
To a solution of compound 62 (0.49 g, 1.7 mmol) in methanol (9.5 ml) was added 0.5 M methanolic sodium methoxide (1 ml), then stirred at rt for 30 min (TLC: Toluene-ethyl acetate 3:2, ninhydrine staining). Methanol washed Dowex W X 8 (50-100 mesh, H+- form) was carefully added (pH was monitored by pH-paper) was added until neutral, then the mixture was filtered and concentrated. The residue was dissolved in dichloromethane and trifluoroacetic acid was added at 0 0C. The reaction mixture was then stirred at rt for 55 min (TLC: dichloromethane-methanol 9:1 , ninhydrine staining), then concentrated. Column chromatography (diameter: 2 cm, silica: 15 g, packing eluent: dichloromethane-methanol 95:5) of the residue (dissolved in dichloromethane- methanol 95:5) using methanol in dichloromethane 5:95 (150 ml), 7:93 (100 ml) and 1 :9 (200 ml) gave a hard syrup which crystallized upon standing (0.39 g, 1.50 mmol, 88%).
NMR data (400 MHz, DMSO-d6): 1H, 3.34, 3.44 (2 dd, 1 H, H-6a), 3.60-3.70 (m, 2H, H- 1a and H-6b), 3.89 (dd, 1 H, H-1 b), 4.15 (d, 1 H, H-3), 4.51 (br s, 1 H, H-2), 4.76 (dd, 1 H, H-4), 5.26 (dd, 2JH,F = 48.3 Hz, H-5).
Step b)
Figure imgf000074_0001
To a stirred solution of compound 64 (0.34 g, 1.30 mmol), N-ethyl-N'-(3- dimethylaminopropyl)carbodiimide hydrochloride (0.28 g, 1.43 mmol), 1- hydroxybenzotriazole hydrate (0.22 g) and Λ/-(tert-Butoxycarbonyl)-L-leucine monohydrate (0.34 g, 1.37 mmol) in DMF (10 ml) was added triethylamine (0.54 ml, 3.9 mmol), then stirred at rt for 24 h. The reaction mixture was the partitioned between 10% aq. citric acid (30 ml) and ethyl acetate (10 ml). The water layer was extracted with ethyl acetate (3 x 10 ml), then the organic layers were combined, and washed successively with water (1 x 20 ml) and 1 M aq. sodium hydrogen carbonate (3 x 20 ml), then dried (sodium sulfate), filtered and concentrated onto silica. Flash chromatography with ethyl acetate in petroleum ether (40-60 %, stepwise gradient elution) of the residue gave 15 (0.35 g, 0.98 mmol, 75%) as a colourless amorphous solid.
LR-MS: Calcd for Ci3H22FN2O5: 305.1. Found: 305.1 [M+2H-f-Butyl].
Feasibility Example 3
Elongation of the model P1/P2 system with a typical P3
Figure imgf000074_0002
To a solution of compound 65 (0.11 g, 0.31 mmol) in dichloromethane (2 ml) at 0 0C was added trifluoroacetic acid (2 ml), then stirred at rt for 45 min. The reaction mixture was then concentrated and co-concentrated with toluene. To a suspension of the residue, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.064 g, 0.34 mmol), 1-Hydroxybenzotriazole hydrate (0.051 g) and benzo[b]furan-2-carboxylic acid (0.052 g, 0.32 mmol) in DMF (3 ml) was added triethylamine (0.13 ml, 0.9 mmol), then stirred at rt for 24 h. The reaction mixture was then concentrated. The residue was then partitioned between 10% aq. citric acid (30 ml) and ethyl acetate (10 ml). The water layer was extracted with ethyl acetate (2 x 10 ml), then the organic layers were combined, and washed successively with water (1 x 10 ml) and 1 M aq. sodium hydrogen carbonate (3 x 10 ml), then dried (sodium sulfate), filtered and concentrated onto silica. Flash chromatography with ethyl acetate in petroleum ether (50-60 %, stepwise gradient elution) of the residue gave 66 (0.11 g, 0.27 mmol, 89 %) as a colourless glassy solid.
LR-MS: Calcd for C2IH26FN2O5: 405.2. Found: 405.1 [M+H].
Feasibility Example 4
Oxidation to P1 ketone (employing a model P1 ).
Figure imgf000075_0001
To a stirred solution of compound 66 (0.10 g, 0.25 mmol) in dichloromethane (4 ml) at rt was added Dess-Martin periodinane (0.12 g, 0.28 mmol). After stirring for 90 minutes the reaction mixture was diluted with dichloromethane (10 ml), washed with 1 :1 1 M aq. sodium hydrogen carbonate- 10 % aq. sodiumthiosulfate (4 x 10 ml), then dried (sodium sulfate), filtered and concentrated onto silica. Flash chromatography with ethyl acetate in petroleum ether (50-60 %, stepwise gradient elution) of the residue gave 67 (0.072 g, 0.18 mmol, 71 %) as a colourless foam. Compound 67 is obtained as a mixture of geometrical isomers (rotamers) and their hydrates.
LR-MS: Calcd for C2i H24FN2O5: 403.2. Found: 403.0 [M+H]. A NMR sample of the ketoforms of 67 was obtained as follows; 5 mg of compound 67 (mixture of geometrical isomers and hydrate forms with the ratio: hydrate/keto 6:4) was dissolved in DMSO-d6, then heated up to 100 0C in the NMR apparatus and then allowed to reach 50 0C upon which NMR indicated only trace amounts of the hydrate forms and the ratio of the rotamers were 2: 1. NMR data (500 MHz, DMSO-d6, 50 0C): 1H, 0.90-1.04 (m, 4 x CH3, major and minor forms), 1.39-1.82 (m, 2 x CH2CH(CH3)2 and 2 x CH2CH(CH3)2, major and minor forms), 3.56 (m, H-6a, minor), 3.82 (m, H-6A, major), 3.97-4.25 (m, 4 x H-1 , major and minor forms and H-6b, minor), 4.37 (dd, H-6b, major), 4.62 (d, H-3, minor), 4.79 (m, H, major), 4.84 (d, H-3, major), 4.94 (m, H-4, major), 5.12 (m, H-4, minor), 5.15-5.34 (m, H-5 major and H-5 minor, H minor, JH,F major = 49.1 Hz, JH,F minor = 49.4 Hz), 7.35 (t, 1 H, Ar-H), 7.47 (t, 1 H, Ar-H), 7.57-7.70 (m, 2H, Ar-H), 7.78 (d, 1 H, Ar-H), 8.18 (d, -NH, minor), 8.70 (d, - NH, major).
Example 5
An alternative P3 (with model PD
Step a)
Figure imgf000076_0001
To a solution of compound 55 (0.11 g, 0.32 mmol) in dichloromethane (2 ml) at 0 0C was added trifluoroacetic acid (2 ml). After stirring for 45 min at rt (TLC: petroleum ether-ethyl acetate 1 :1 and ethyl acetate-methanol-acetic acid-water 40:3:3:2), the reaction mixture was concentrated and co-concentrated from toluene (3 x 5 ml). To a suspension of the residue, 4-(dimethylamino)benzoic acid (0.055 g, 0.33 mmol), N- ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.067 g, 0.35 mmol) and 1-hydroxybenzotriazole hydrate (0.053 g) in DMF (3 ml) was added triethylamine (0.13 ml, 0.95 mmol), then stirred at rt overnight (TLC: petroleum ether-ethyl acetate 2:3 and ethyl acetate-methanol-acetic acid-water 40:3:3:2). The reaction mixture was then concentrated, partitioned between 8% aq. KH2PO4 (30 ml) and ethyl acetate (10 ml). The water layer was extracted with ethyl acetate (3 x 10 ml), and the combined organic layers were washed with water (1 x 10 ml) and 1 M aq. sodium hydrogen carbonate (3 x 10 ml), then dried (sodium sulphate), filtered and concentrated. The residue was redissolved in dichloromethane and concentrated onto silica. Flash chromatography (diameter: 2 cm, Silica: 8 g, packing eluent: petroleum ether-ethyl acetate 1 :1) of the residue (stepwise gradient elution, ethyl acetate in petroleum ether 50-100%) gave a colourless foam (0.10 g, 0.25 mmol, 80%).
LR-MS: Calcd for C2IH31FN3O4: 408.2. Found: 408.1 [M+H].
Step b)
Figure imgf000077_0001
To a stirred solution of the mono-ol 68 (0.096 g, 0.24 mol) in dichloromethane at rt was added Dess-Martin periodinane (0.11 g, 0.26 mmol). The reaction mixture turned red and after stirring for approximately 35 min (TLC: petroleum ether-ethyl acetate 2:3), the reaction mixture was diluted with dichloromethane (10 ml), washed with 1 :1 1 M aq. sodium hydrogen carbonate- 10 % aq. sodiumthiosulfate (4 x 10 ml), then dried (sodium sulfate), filtered and concentrated onto silica. Flash chromatography (diameter: 2 cm, Silica: 7 g, Packing eluent: petroleum ether-ethyl acetate 1 :1 ) of the residue (stepwise gradient elution, ethyl acetate in petroleum ether 50-100%) gave a colourless foam (0.039 g, 0.10 mmol, 41%).
LR-MS: Calcd for C2IH27FN3O4: 404.2. Found: 404.1 [M-H].
Example 6
An alternative P3 (with model P1 )
Step a)
Figure imgf000077_0002
To a solution of compound 55 (0.12 g, 0.32 mmol) in dichloromethane (2 ml) at 0 0C was added trifluoroacetic acid (2 ml), then stirred at rt for 45 min. The reaction mixture was then concentrated and co-concentrated with toluene (3 x 5 ml). To a suspension of the residue, N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.068 g, 0.36 mmol), 1-hydroxybenzotriazole hydrate (0.055 g) and 4-phenoxybenzoic acid (0.073 g, 0.34 mmol) in DMF (3 ml) was added triethylamine (0.14 ml, 0.97 mmol), then stirred at rt for 24 h. The reaction mixture was then concentrated. The residue was then partitioned between 10% aq. citric acid (30 ml) and ethyl acetate (10 ml). The water layer was extracted with ethyl acetate (2 x 10 ml), then the organic layers were combined, and washed successively with water (1 x 10 ml) and 1 M aq. sodium hydrogen carbonate (3 x 10 ml), then dried (sodium sulfate), filtered and concentrated onto silica. Flash chromatography of the residue with 1 :1 ethyl acetate in petroleum ether gave colourless hard syrup (0.14 g, 0.30 mmol, 91%).
LR-MS: Calcd for C25H30FN2O5: 457.2. Found: 457.2 [M+H].
Step b)
Figure imgf000078_0001
To a stirred solution of the mono-ol (0.128 g, 0.28 mmol) in dichloromethane (4 ml) at rt was added Dess-Martin periodinane (0.12 g, 0.28 mmol). After stirring for 90 minutes the reaction mixture was diluted with dichloromethane (10 ml), washed with 1 :1 1 M aq. sodium hydrogen carbonate- 10 % aq. sodiumthiosulfate (4 x 10 ml), then dried (sodium sulfate), filtered and concentrated onto silica. Flash chromatography with ethyl acetate in petroleum ether (50-60 %, stepwise gradient elution) of the residue gave 71 (0.072 g, 0.18 mmol, 71 %) as a colourless foam.
LR-MS: Calcd for C2i H28FN2O5: 455.2. Found: 455.1 [M+H].
Example 8
Additional cathepsin K inhibitors Compounds of the invention are synthesised using the methodology outlined above with the P3 acids tabulated below . 8.1 - 8.13 & 8.15 - 8.20 depicted in the table below are synthesised by successively coupling the N-protected P2 and P3 acids itemised in the table, to a P1 building block using the solid phase methodology outlined below. The construction of P2 and P3 building blocks not readily accessible from commercial sources appears below.
Table 1
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Solid phase synthesis of 8.1 - 8.13 & 8.15 - 8.67 is generally carried out using Murphy's linker methodology using known chemistries as described in WO02/88106. The ketone function of the FmocNH bicycle was derivatised as an acid labile semicarbazone which provided a carboxylic acid for attachment to the aminomethyl functionalised polymer support resin using HBTU, HOBt and NMM. After Fmoc removal the corresponding (1- substituted) cyclobutylmethylalanine Fmoc acid was coupled on where the symmetric anhydride was preformed. Coupling was first carried out for 8 h, and then repeated with fresh reagents overnight. After Fmoc removal the P3 acids were introduced using standard coupling conditions. Washing, drying and cleavage from the resin provided the crude desired material which was purified either by column chromatography or preparative hplc. Compounds which required modified procedures are described below.
1 H-lndole-2-carboxylic acid ri-(6-fluoro-3-oxo-hexahvdrofuror3.2-blpyrrole-4-carbonyl)-
3-methyl-butyll-amide (Example 8.6)
To the resin bound H2N-I_-I_eu-P1 (150 mg, 0.03 mmol) was added a solution of indole- 2-carboxylic acid (24.2 mg, 0.15 mmol) in DMF (1.0 ml_). A solution of 1 ,3- diisopropylcarbodiimide (19 mg, 0.15 mmol) and 1-hydroxybenzotriazole hydrate (23 mg, 0.15 mmol) in DMF (1 ml_) was then added. The reaction was agitated overnight and then washed with DMF (7 x 10 ml_), MeOH (5 x 10 ml_) and TBME (5 x 10 ml_). After drying under vacuum for 17 h, the product was cleaved from the resin by suspension in 10 ml_ of 95: 5 TFA: water for 45 mins. The filtrate was then concentrated under N2 stream, purified by semi preparative HPLC and then freeze dried to give the title compound as a white solid. Compounds were characterised by HPLC, 1H NMR and MS which showed both the ketone and hydrate forms to be present.
4-Piperidin-4-yl benzoic acid (Example 8.9)
4-Phenylpiperidine (10.0 g, 62 mmol) and pyridine (5.74 mL, 71 mmol) were dissolved in DCM (80 mL) and cooled to 0 "C. A solution of acetyl chloride (4.00 mL, 71 mmol) in DCM (20 mL) was added drop wise to the above solution. The mixture was then stirred for 2 h at RT and when deemed to be complete by hplc, extracted with water, dried and concentrated in vacuo to afford a light yellow oil (10.6 g, 84%) which solidified on standing and was used without further purification. The yellow oil (10.6 g, 52.2 mmol) was dissolved in DCM and cooled to - 78 "C and treated with oxalyl chloride (18.3 mL, 209 mmol) drop wise followed by the addition of aluminium chloride (20.9 g, 157 mmol) in portions. When the addition was complete, the flask was placed in an ice-salt bath, and the mixture stirred at - 20 "C for 3h and then at RT overnight. The mixture was then poured onto ice-water and extracted with DCM (100 mL x 3), dried and concentrated in vacuo. The residue was dissolved in aq. NaOH (2N) and HCI (6N) was added at 0 "C to acidify the solution to pH 5. The precipitate (7.9 g) was filtered off and washed with water (200 mL). The residue was then suspended in 6N HCI and heated at reflux for 18h. The solvent was evaporated and the residue was recrystallised from ethanol. Crystals were filtered off and provided the title compound (5.05 g, 63%).
4-(5-Piperidin-1-ylmethyl-thiophen-2-yl)benzoic acid (Example 8.15) 5-Bromo-2-thiophenecarboxaldehyde (10 mmol) and piperidine (10 mmol) were mixed in THF (10 ml_) and dibutyltin dichloride (0.2 mmol) was added. After stirring at RT for 5 minutes, phenylsilane (11 mmol) was added and the reaction allowed to stir at room temperature for a further 17 h. The reaction was then concentrated in vacuo and the residue purified by flash chromatography (silica gel, DCM) to give 1-(5-bromo-thiophen- 2-ylmethyl)-piperidine: m/z = 260, 262 in MS ES+ as a golden oil which was used directly in the subsequent step. A reaction tube containing a magnetic stirrer bar was charged with 4-carboxyphenylboronic acid (0.05 mmol), the thiophene bromide (0.05 mmol), Pd(PPh3)4 (0.025 mmol), acetonitrile (2 ml_) and 1 M Na2CO3 (aq) (2 ml_). The reaction tube was then sealed and heated by microwave irradiation (100W, 4 mins) to 150 "C and held at that temperature for 10 mins. After being allowed to cool to room temperature the reaction were acidified to pH 1 with 1 M HCI and the resulting precipitate filtered off. This crude product was then passed through a silica plug to remove any inorganic species and concentrated to give a the titel compound as a brown powder m/z = 304 in MS ES+, which was characterised by hplc and MS and used in the next step without any further purification.
4-(5-Morpholin-4-ylmethyl-thiophen-2-yl)benzoic acid (Example 8.16)
To synthesise 4-(5-morpholin-4-ylmethyl-thiophen-2-yl)benzoic acid, the piperidine was substituted by morpholine in the previous experimental.
5-r2-(4.4-Difluoro-piperidin-1-yl)-ethoxyl-benzofuran-2-carboxylic acid (Example 8.17) To a solution of 4,4-difluoropiperidine hydrochloride (1 g, 6.3 mmol) in THF (20 ml_) was added methylbromoacetate (0.63 ml_, 6.6 mmol) and triethylamine (2.65 ml_, 19.0 mmol). The reaction was heated at reflux for 4 h. The reaction was diluted with water (50 ml_) and the product extracted with ethyl acetate (3 * 20 ml_). The combined organic fractions were washed with brine, dried over magnesium sulphate and concentrated in vacuo to yield (4,4-difluoropiperidin-1-yl)acetic acid methyl ester as a brown oil (1.17g, 96%). MS 194 (M + H)+. To a solution of (4,4-difluoropiperidin-1-yl)acetic acid methyl ester (1.17 g, 6.1 mmol) in THF (15 ml_) at 0 "C was added potion wise lithium aluminium hydride (0.46 g, 12.2 mmol). Once the effervescence had ceased the reaction was heated at 60 "C for 1.5 h. The reaction was quenched with water (10 ml_) followed by sodium hydroxide solution (2N, 10 ml_) then water (10 ml_). The reaction was filtered and the filtrate extracted with ethyl acetate (3 * 20 ml_). The combined organic fractions were washed with brine, dried over magnesium sulphate and concentrated in vacuo to yield 2-(4,4-difluoropiperidin-1-yl)-ethanol as a brown oil (0.99 g, 99%). MS 166 (M + H)+. To a solution of diisopropylazodicarboxylate (0.36 ml_, 1.82 mmol) in DCM (20 ml_) was added polymer supported triphenylphosphine (728 mg, 2.18 mmol). The reaction was stirred at RT for 10 min. 5-Hydroxybenzofuran-2-carboxylic acid ethyl ester (0.25 g, 1.21 mmol) and 2-(4,4-difluoropiperidin-1-yl)-ethanol (210 mg, 1.27 mmol) were added and the reaction stirred at RT for 16 h. The reaction was filtered and the filtrate concentrated in vacuo. The product was purified on silica eluting with 50 % tert-butyl methyl ether in n-heptane to yield 5-[2-(4,4-difluoropiperidin-1- yl)ethoxy]benzofuran-2-carboxylic acid ethyl ester as a yellow solid (375 mg, 88%). MS 354 (M + H)+. To a solution of 5-[2-(4,4-difluoropiperidin-1-yl)ethoxy]benzofuran-2- carboxylic acid ethyl ester (375 mg, 1.06 mmol) in THF (5 ml_) and water (1 ml_) was added lithium hydroxide (34 mg, 2.12 mmol). The reaction was stirred at RT for 16 h. The THF was removed in vacuo and the remaining aqueous solution dried overnight in a freeze dryer to yield the crude title compound as a brown solid. MS 326 (M + H, 5.3 min) and used for coupling onto H2N-I_eu-P1 without any further purification.
5-r2-(4-Trifluoromethyl-piperidin-1-yl)-ethoxyl-benzofuran-2-carboxylic acid (Example
8.18)
To a solution of 4-trifluoromethylpiperidine hydrochloride (1 g, 5.3 mmol) in THF (20 ml_) was added methylbromoacetate (0.52 ml_, 5.5 mmol) and triethylamine (2.2 ml_, 15.8 mmol). The reaction was heated at reflux for 4 h and then diluted with water (50 ml_) and the product extracted with ethyl acetate (3 * 20 ml_). The combined organic fractions were washed with brine, dried over magnesium sulphate and concentrated in vacuo to yield (4-trifluoromethylpiperidin-1-yl)acetic acid methyl ester as a brown oil (1.19 g, 98%). MS 226 (M + H)+. To a solution of (4-trifluoromethylpiperidin-1-yl)acetic acid methyl ester (1.19 g, 5.3 mmol) in THF (15 ml_) at 0 "C was added portion wise lithium aluminium hydride (0.4 g, 10.6 mmol). Once the effervescence had ceased the reaction was heated at 60 "C for 1.5 h. The reaction was quenched with water (10 ml_) followed by sodium hydroxide solution (2N, 10 ml_) then water (10 ml_). The reaction was filtered and the filtrate extracted with ethyl acetate (3 * 20 ml_). The combined organic fractions were washed with brine, dried over magnesium sulphate and concentrated in vacuo to yield 2-(4-trifluoromethylpiperidin-1-yl)-ethanol as a brown oil (1.0 g, 99%). MS 198 (M + H)+. To a solution of diisopropylazodicarboxylate (0.58 ml_, 2.28 mmol) in DCM (20 ml_) was added polymer supported triphenylphosphine (1.14 g, 3.4 mmol). The reaction was stirred at RT for 10 mins. 5-Hydroxybenzofuran-2- carboxylic acid ethyl ester (0.47 g, 2.3 mmol) and 2-(4-trifluoromethylpiperidin-1-yl)- ethanol (0.45 g, 2.28 mmol) were added and the reaction stirred at RT for 16 h. The reaction was filtered and the filtrate concentrated in vacuo. The product was purified on silica eluting with 50 % tert-butyl methyl ether in n-heptane to yield 5-[2-(4- trifluoromethylpiperidin-1-yl)ethoxy]benzofuran-2-carboxylic acid ethyl ester as a yellow solid (548 mg, 62%). MS 386 (M + H)+. To a solution of 5-[2-(4-trifluoromethylpiperidin- 1-yl)ethoxy]benzofuran-2-carboxylic acid ethyl ester (548 mg, 1.42 mmol) in THF (5 ml_) and water (1 ml_) was added lithium hydroxide (45 mg, 2.84 mmol). The reaction was stirred at RT for 16 h. The THF was removed in vacuo and the remaining aqueous solution dried overnight in a freeze dryer to yield the crude title compound as a brown solid. MS 358 (M + H)+ which was used directly for coupling with H2N-I_eu-P1.
4-r2-(4-Methyl-piperazin-1-vn-thiazol-4-yll-benzoic acid (Examples 8.19 & 8.20) To thiocarbonyldiimidazole (2 g, 11.5 mmol) in THF (30 ml_) at RT was added N- methylpiperazine (1.00 g, 10 mmol) drop wise. The reaction was stirred at RT for 2 h and then at 55 "C for 1 h. The reaction was cooled to RT and 20 ml_ of THF was removed in vacuo. 2M NH3 (10 ml_) in MeOH was added and the reaction stirred for 15 h. A further 2M NH3 (10 ml_) in MeOH was added and the reaction maintained at 55 "C for 8 h. A pale yellow precipitate (1.00 g) was observed and filterered off, dried and used directly in the next step. The thiourea (0.84 g, 5.2 mmol) was dissolved in EtOH (30 ml_) and 4-(2-bromo-acetyl)-benzoic acid (1.28 g, 5.2 mmol) was added. The reaction was heated at reflux for 3 h. The reaction was cooled to RT and the solid filtered off. The solid was washed with Et2O and dried thoroughly. This procedure provided the title compound as a pale yellow solid (1.23 g, 77 %).
ri-(6-Fluoro-3-oxo-hexahvdro-furor3.2-blpyrrole-4-carbonyl)-cvclohexyll-carbamic acid 9H-fluoren-9-ylmethyl ester (Example 8.20) Fmoc-i-amino-i-cyclohexane carboxylic acid (0.300 mg, 0.82 mmol) was dissolved in DCM (8 ml_) and DAST (1 ml_, 8.2 mmol) was added. After 1.5 h the starting material was consumed and H2O (5 ml_) was added drop wise with care. The organic layer was removed, dried (Na2SO4) and concentrated in vacuo to afford a pale brown solid (0.287 g, 96 %). This material was used crude in the next step. (1-Fluorocarbonyl-cyclohexyl)- carbamic acid 9H-fluoren-9-ylmethyl ester (0.050 g, 0.135 mmol) was dissolved in DMF (1 ml_) and added to H2N-PI in DMF (1 ml_). NMM (0.027 g, 0.27 mmol) was added and the reaction left overnight. The resin was filtered off to remove spent reagents and fresh reagents were added and the reaction repeated for a further 24 h. After washing with DMF (10 ml_ x 10) and DCM (10 ml_ x 10) the title compound (loading equivalent to 50% yield) was obtained bound to resin.
[^(e-Fluoro-S-oxo-hexahvdro-furorS^-blpyrrole^-carbonvD-S-methyl-butyll-carbamic acid benzyl ester (Example 8.14) with model P2
Figure imgf000090_0001
6-Fluoro-3-hydroxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic acid tert-butyl ester (0.200 g, 0.81 mmol) was dissolved in DCM (4 ml_) at 0 0C and TFA (4 ml_) added. After stirring at 0 -4 0C for 1 h, the solvent was evaporated in vacuo and the residue left under high vacuum for 4 h to afford a brown oil. The residue was dissolved in DMF (5 ml_) and WSCHCI (171 mg, 0.89 mmol), HOBt (137 mg, 1.01 mmol), Cbz-Leu-OH (226 mg, 0.85 mmol) and Et3N (337 μl, 2.43 mmol) added. After stirring at room temperature overnight, the reaction mixture was concentrated in vacuo, dissolved in EtOAc (10 ml_), washed with H2O (5 ml_) and saturated NaHCO3 solution (5 ml_), dried (Na2SO4) and evaporated in vacuo to afford a colourless oil (242 mg; [M+H]+ 395). [1-(6-Fluoro-3- hydroxy-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-carbamic acid benzyl ester (242 mg, 0.62 mmol) was dissolved in dry DCM (8 ml_) and Dess-Martin periodinane (261 mg, 0.62 mmol) added. The reaction immediately turned light brown. After stirring at room temperature for 2.5 h, the yellow solution was diluted with DCM (8 ml_) and washed with saturated NaHCO3 solution (5 ml_), dried (Na2SO4) and evaporated in vacuo to afford a yellow residue. Purification by column chromatography (EtOAc: heptane; 1 :2) yielded the title compound as a colourless oil, 147 mg; [M+H]+ 393.
4-Thiocarbamoyl-piperazine-1-carboxylic acid tert-butyl ester (Example 8.21 ) To a solution of piperazine-1-carboxylic acid tert-butyl ester (32.2 mmol) in tetrahydrofuran (60 ml) was added thiocarbonyldiimidazole (37.0 mmol). The reaction was stirred at RT for 2 h then heated at 55°C for 1 h. The reaction was concentrated in vacuo to approximately half the volume and methanolic ammonia added (7N, 107.4 mmol). The reaction was heated at 55°C for 16 h. The reaction was concentrated in vacuo to approximately half the volume and cooled to 0°C at which point the product precipitated from solution. The product was collected by filtration to yield the title compound as a white solid (3.3 g). 1 H NMR (400MHz, d6-DMSO) 1.40 (9H, s), 3.32 (4H, s), 3.71 (4H, s), 7.42 (1 H,s). 4-[4-(4-Carboxy-phenyl)-thiazol-2-yll-piperazine-1-carboxylic acid tert-butyl ester To a suspension of 4-thiocarbamoyl-piperazine-1 -carboxylic acid tert-butyl ester (13.3 mmol) in ethanol (60 ml) was added 4-(2-bromoacetyl)-benzoic acid (13.3 mmol) and 4- methylmorpholine (13.9 mmol). The reaction was heated at reflux for 2.5 h. The reaction was concentrated in vacuo and the solid washed with water (200 ml) to yield the title compound as a white solid (3.9 g). 1 H NMR (400MHz, CDCI3) 1.45 (9H, s), 3.58 (8H, m), 4.86 (1 H, s), 6.95 (1 H,s), 7.97 (2H, d, J 8 Hz), 8.1 (2H, d, J 8Hz). 4-(2-r4-(2-Methoxy-ethyl)-piperazin-1-vH-thiazol-4-yl)-benzoic acid 4-[4-(4-Carboxy-phenyl)-thiazol-2-yl]-piperazine-1 -carboxylic acid tert-butyl ester (5.0 mmol) was dissolved in hydrochloric acid in dioxane (4N, 25 ml) and the reaction stirred at RT for 2 h. The reaction was concentrated in vacuo to yield 4-(2-piperazin-1-yl- thiazol-4-yl)-benzoic acid. Trimethoxyethane (6.5 mmol) was dissolved in aqueous hydrochloric acid (1 N, 10 ml) and the reaction heated at 50°C for 1.5 h. The reaction was allowed to cool to RT and was then added to a suspension of 4-(2-piperazin-1-yl- thiazol-4-yl)-benzoic acid (5.0 mmol) in acetonitrile (25 ml) and sodium acetate buffer (1N, pH 5.5, 10ml). The reaction was stirred at RT for 1.5 h. Sodium cyanoborohydride (6.5 mol) was added and the reaction stirred at RT for 16 h. The reaction was concentrated in vacuo and the product purified by flash chromatography (silica gel, 10% methanol in dichloromethane) to give the title product as a colourless oil (0.9 g). m/z = 348 (100% M+H) in MS ES+. 4-ri-(5-Bromo-thiophen-2-yl)-ethyll-morpholine (Example 8.22) To a solution of morpholine (1.20 mmol) in titanium (IV) isopropoxide (1.95 mmol) was added 2-acetyl-5-bromothiophene (1.20 mmol). The reaction was heated in a microwave at 150°C for 5 minutes. Sodium borohydride (1.95 mmol) was added and the reaction stirred at RT for 16 h. The reaction was diluted with sodium hydroxide solution (2N, 10 ml) and the solids formed removed by filtration. The filtrate was extracted with ethylacetate (3 x 20 ml), the combined organics were washed with brine and dried over magnesium sulphate. The product was purified by flash chromatography (silica gel, 10- 20% ethylacetate in /so-hexane) to give the title product as a brown oil: m/z = 276 (100%, M+H), 278 (100%, M+H) in MS ES+. 4-[5-(1 -Morpholin-4-yl-ethyl)-thiophen-2-yll-benzoic acid 4-[1-(5-Bromo-thiophen-2-yl)-ethyl]-morpholine (0.36 mmol), 4- methoxycarbonylphenylboronic acid ( 0.43 mmol) and sodium carbonate (1.09 mmol) were suspended in dioxane:water ( 2 ml, 2:1 ). Nitrogen gas was bubbled through the reaction for 5 minutes then tetrakis(triphenylphosphine)palladium(0) (0.04 mmol) added. The reaction was heated in a microwave at 150 °C for 10 min. The reaction was concentrated in vacuo and the product was purified by flash chromatography (silica gel, 10% methanol in dichloromethane) to give the title product as a brown oil: m/z = 318 (50% M+H), 231 (100%, M+H-morpholine) in MS ES+.
4-(r(1-Methylimidazol-2-yl)methyllamino)benzoic acid (Example 8.23) i-Methyl-2-imidazolecarboxaldehyde (5.0 mmol) and methyl-4-aminobenzoate (5.0 mmol) were mixed in MeOH (7 ml_). Acetic acid (0.3 ml_) was added and the mixture stirred for 30 minutes at room temperature. The reaction mixture was cooled, sodium cyanoborohydride (5.0 mmol) was added and the reaction allowed to stir at room temperature for a further 17 h. The reaction mixture was then concentrated under vacuum and partitioned between H2O and EtOAc. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with H2O, brine, dried over MgSO4 and the solvent removed under vacuum. The residue was purified by flash chromatography (silica gel, 5% MeOH in DCM) to give methyl 4-{[(1-methylimidazol-2- yl)methyl]amino}benzoate : m/z = 246 in MS ES+ as a pale yellow solid which was used directly in the subsequent step. To a solution of methyl ester (2.5 mmol) in 1 ,4-dioxane (5 ml_) was added 1 M aqueous KOH solution (5.5 mmol) and the reaction mixture stirred for 18 h. The reaction mixture was neutralised to pH 7 with 1M HCI and concentrated by N2 stream. The product was resuspended in water and lyophilised to give 4-{[(1-methylimidazol-2-yl)methyl]amino} benzoic acid : m/z 232 in MS ES+ as a white solid which was used directly in the subsequent step.
4-r5-π-Morpholin-4-yl-ethyl)-furan-2-yll-benzoic acid (Example 8.25) 2-Acetylfuran (20 mmol) and morpholine (20 mmol) were added to neat titanium isopropoxide (32 mmol) and the reaction stirred under N2 at room temperature for 3h. Methanol (90 ml) was then added followed by the careful portionwise addition of NaBH4 (32 mmol). After stirring at room temperature for 10 mins, the reaction was quenched by addition of 0.1 M NaOH and the resultant mixture filtered through a celite pad. The filtrate was extracted twice with DCM, dried over Na2SO4 and concentrated in-vacuo. Flash chromatography of the residue (silica, 5 to 20% EtOAc in Heptane) yielded pure 4-(1-furan-2-yl-ethyl)-morpholine as a golden oil: m/z in MS ES+ = 182 [M+H]+, 2.76mmol, 14% yield.
4-(1-furan-2-yl-ethyl)-morpholine (1.1 mmol) was taken up in DCM (5 ml) and stirred at O0C. Nitrogen was passed through the reaction vessel and bubbled out through a Dreschel bottle containing a saturated aqueous solution of sodium thiosulphate, whilst bromine (1.54mmol in 2ml DCM) was added dropwise. After addition the reaction was stirred at room temperature for 2h, then diluted with more DCM, washed twice with 2M Na2CO3 solution, dried over Na2SO4 and concentrated in-vacuo. After purification by flash chromatography (silica, 5 to 10% EtOAc in hexane), 4-[1-(5-bromo-furan-2-yl)- ethyl]-morpholine was obtained as a golden oil: m/z in MS ES+ = 260, 262 [M+H]+, 0.46mmol, 42% yield.
4-[1-(5-bromo-furan-2-yl)-ethyl]-morpholine (0.54mmol) was taken up in 7ml toluene and 4-carboxymethylphenylboronic acid (0.54mmol) was added as a solution in 0.7ml of EtOH. 12ml of 2M aqueous Na2CO3 solution was added, followed by Pd(PPh3J4 (0.054mmol). Reaction was stirred at 7O0C for 17h under a nitrogen atmosphere and then cooled to room temperature and extracted with DCM (x2). Combined organic layers were washed with brine, concentrated in vacuo and the residue purified by flash chromatography (silica, 20-50% EtOAc in hexane). This furnished the pure 4-[5-(1- Morpholin-4-yl-ethyl)-furan-2-yl]-benzoic acid methyl ester as a powdery grey solid : m/z in MS ES+ = 316 [M+H]+ , O.Oδmmol, 15% yield.
This ester (O.Oδmmol) was heated to 7O0C in 18% HCI for 2h at which point HPLC showed all the starting material to have been hydrolysed. The reaction was cooled and the product that precipitated out of solution was collected by filtration as a white solid and used directly in the next step.
4-(2-Methyl-pyridin-3-yloxy)-benzoic acid (Example 8.26)
A reaction tube containing a magnetic stirrer bar was charged with ethyl-4- fluorobenzoate (1 mmol), 2-methyl-3-pyridol (1.0 mmol), potassium carbonate (1.08 mmol) and DMF (2 ml). The reaction tube was then sealed and heated by microwave irradiation (100W, 4 mins) to 150 "C and held at that temperature for 80 mins. The solution was filtered to remove the insoluble potassium carbonate and then concentrated in vacuo. The residue was purified by preparative HPLC and freeze dried to give 4-(2-Methyl-pyridin-3-yloxy)-benzoic acid ethyl ester as a white solid which was hydrolysed by 6N aqueous HCI solution heated by microwave irradiation (200W) for 3 mins at 150 "C. The solution was freeze dried to give to 63 mg of hydrochloride salt of the title compound as a white powder m/z = 229 in MS ES+, which was characterised by HPLC and MS.
4-r2-(1-Dimethylamino-ethyl)-thiazol-4-yll-benzoic acid (Example 8.27) 4-(2-ri-(tert-Butoxycarbonyl-methyl-amino)-ethyll-thiazol-4-yl)-benzoic acid methyl ester Boc-L-NMe-Alanine-OH (1.0g, 4.92mmols) was dissolved in dioxan (1OmIs) and to this was added pyridine (0.25mls), di-tert-butyl dicarbonate (1.4g, 6.4mmols) and ammonium hydrogen carbonate (0.49g, 6.2mmols). After stirring for 18 hours the crude reaction mixture was concentrated in vacuo and re-suspended in ethyl acetate. This was washed with 1 M KHSU4 and the organic layer dried over magnesium sulphate. After concentration, a clear oil was obtained (0.79g). This was dissolved in ethylene glycol dimethyl ether (1OmIs) and to this was added Lawesson's reagent (4.31 mmols, 1.74g). After stirring at room temperature for 3 hours the reaction mixture was concentrated in vacuo and the residue re-suspended in ethyl acetate. This was washed with 1M Na2CU3and the organic layer dried over magnesium sulphate. After concentration a yellow oil was obtained. This was purified by flash chromatography (heptane/ethyl acetate) to give a white solid (0.73g). This was dissolved in ethanol (1OmIs) and 4-(2-Bromo-acetyl)-benzoic acid methyl ester (3.34mmols, 0.86g) was added. The reaction was heated to 500C for one hour. The crude product was purified by flash chromatography (heptane/ethyl acetate) to give a white solid (0.39g). ESMS (M + H = 377.23).
4-[2-(1 -Dimethylamino-ethyD-thiazoM-vH-benzoic acid
4-{2-[1-(tert-Butoxycarbonyl-methyl-amino)-ethyl]-thiazol-4-yl}-benzoic acid methyl ester was deprotected with a solution of 4N HCI in dioxan for 1h. The solvent was removed in vacuo and the residue freeze dried to get a white solid which was methylated as followed. 4-[2-(1-Methylamino-ethyl)-thiazol-4-yl]-benzoic acid methyl ester (0.44 mmol) was stirred for one hour with formaldehyde (1.1 equivalent) in methanol (2ml) and sodium acetate buffer (1 N, pH 5.5, 1 ml). Sodium cyanoborohydride (0.49 mmol) was added and the reaction stirred at RT for 2 h. The reaction was concentrated in vacuo and the residue was extracted in EtOAc and washed with a 1 M aqueous solution of sodium carbonate. The organic layer was concentrated in vacuo and the residue was hydrolysed by 6N aqueous HCI solution heated by microwave irradiation (200W) for 3 mins at 150 "C. The solution was freeze dried to give to 134 mg of hydrochloride salt of the title compound as a white powder m/z = 277 in MS ES+, which was characterised by HPLC and MS.
E-4-r2-π H-lmidazol-4-yl)-vinyll-benzoic acid (Example 8.28) {4-(methoxycarbonyl)benzyl(triphenyl)} phosphonium bromide on polymer support. Methyl-4-bromomethyl benzoate (26 mmol) were added to a suspension of 4.4 g of PS-
Triphelphosphine resin (Fluka, 3 mmolg"'' ) in 40 ml of DMF. The solution was gently stirred at 65°C for 48 hours. The phosphonium resin was washed with DMF (4x40 ml),
DCM (4x40 ml) and TBME (2x40 ml) and dried in vacuo for 18 h.
E-4-[2-(1 H-lmidazol-4-yl)-vinyll-benzoic acid
A reaction tube containing a magnetic stirrer bar was charged with 1-Methyl-1 H- imidazole-2-carbaldehyde(1.5mmol), potassium carbonate (2.1 mmol), {4- (methoxycarbonyl) benzyl(triphenyl)} phosphonium bromide on polymer support (1.5 mmol) and methanol (4 ml). The reaction tube was then sealed and heated by microwave irradiation (100W, 3 mins) to 150 "C and held at that temperature for 5 mins.
The solution was filtered to remove the insoluble potassium carbonate and then concentrated in vacuo. The residue was purified by preparative HPLC and freeze dried to give E-4-[2-(1-Methyl-1 H-imidazol-2-yl)-vinyl]-benzoic acid methyl ester as a white solid which was hydrolysed by 6N aqueous HCI solution heated by microwave irradiation (200W) for 3 mins at 150 "C. The solution was freeze dried to give to 90 mg of hydrochloride salt of the title compound as a white powder m/z = 215 in MS ES+, which was characterised by HPLC and MS.
E-4-r2-π-Methyl-1 H-imidazol-2-yl)-vinyll-benzoic acid (Example 8.29) Same as example 8.28. i-Methyl-I H-imidazole-2-carbaldehyde was used as the aldehyde. The title compound was obtained as a white powder m/z = 229 in MS ES+, which was characterised by HPLC and MS.
E-4-r2-(3-Methyl-3H-imidazol-4-yl)-vinyll-benzoic acid (Example 8.30) Same as example 8.28. 3-Methyl-3H-imidazole-4-carbaldehyde was used as the aldehyde. The title compound was obtained as a white powder m/z = 229 in MS ES+, which was characterised by HPLC and MS
4-r2-(1 H-lmidazol-4-yl)-ethyll-benzoic acid (Example 8.31 ) E-4-[2-(1-Methyl-1 H-imidazol-2-yl)-vinyl]-benzoic acid methyl ester was hydrogenated using Pd/C (10% of substrate weight), ammonium formate (5 equivalents) in isopropanol heated by microwave irradiation (200W) for 5 mins at 150 "C. The solution was filtered through celite to remove the insoluble catalyst, diluted with water and freeze-dried to remove the excess of ammonium formate. The obtained solid was hydrolysed by 6N aqueous HCI solution heated by microwave irradiation (200W) for 3 mins at 150 "C. The solution was freeze dried to give to the hydrochloride salt of the title compound as a white powder m/z = 217 in MS ES+, which was characterised by HPLC and MS.
4-r2-(1-Methyl-1 H-imidazol-2-yl)-ethyll-benzoic acid (Example 8.32) Same as example 8.31. 4-[2-(1-Methyl-1 H-imidazol-2-yl)-vinyl]-benzoic acid methyl ester (Example 8.29) was used as the methyl ester. The title compound was obtained as a white powder m/z = 231 in MS ES+, which was characterised by HPLC and MS.
Potassium 4-methyl(pyridin-2-yl)aminomethylbenzoate (Example 8.33) 2-Methylaminopyridine (1.0 mmol) and methyl-4-formylbenzoate (1.0 mmol) were mixed in THF (2 ml_) and dibutyltin dichloride (0.1 mmol) was added. After stirring at RT for 10 minutes, phenylsilane (1.1 mmol) was added and the reaction mixture allowed to stir at room temperature for a further 17 h. The reaction mixture was then concentrated by N2 stream and the residue purified by flash chromatography (silica gel, heptane:EtOAc) to give methyl 4-[methyl(pyridin-2-yl)amino]methylbenzoate : m/z = 257 in MS ES+ as a yellow oil which was used directly in the subsequent step. To a solution of methyl ester (0.27 mmol) in 1 ,4-dioxane (0.6 ml_) was added 1M aqueous KOH solution (0.59 mmol) and the reaction mixture stirred for 18 h. The reaction mixture was concentrated by N2 stream, resuspended in water and the product lyophilised to give potassium 4-methyl(pyridin-2-yl)aminomethyl benzoate : m/z 243 in MS ES+ as an off-white solid which was used directly in the subsequent step.
Sodium 4-(2-(1 (SH(tert-butoxycarbonyl)(methyl)aminolethyl)-5-methyl-1 ,3-thiazol-4- vDbenzoate (Example 8.34)
Ethyl 4-propionylbenzoate (2.0 mmol), pyrrolidinone hydrotribromide (2.1 mmol) and 2- pyrrolidinone (2.2 mmol) were heated in THF (20 ml_) at 5OC for 2.5 h. The mixture was cooled, filtered, concentrated under vacuum and the residual oil partitioned between H2O and MTBE. The aqueous layer was extracted with MTBE, and the combined organic layers were washed with saturated aqueous sodium metabisulfite solution, H2O, brine, dried over MgSO4 and the solvent removed under vacuum. The residue was purified by flash column chromatography (9:1 'Hexane : MTBE) to afford ethyl-4(2'- bromopropionyl)benzoate as a clear oil. Ethyl 4(2'-bromopropionyl)benzoate (0.5 mmol), BOC(Me)AIa thioamide (0.5 mmol) and NMM (0.5 mmol) were heated in EtOH (2 ml_) at 8OC for 3 h. The mixture was cooled, concentrated by N2 stream and the crude product partitioned between H2O and MTBE. The aqueous layer was extracted with MTBE, and the combined organic layers were washed twice with 1 M KHSO4, brine, dried over MgSO4 and the solvent removed under vacuum to give a yellow oil. The residue was purified by flash column chromatography (9:1 'Hexane : EtOAc) to afford an intense yellow fraction. On standing for several hours, this fraction decolorises and ethyl 4-(2-{1 (S)-[(tert- butoxycarbonyl)(methyl)amino]ethyl}-5-methyl-1 ,3-thiazol-4-yl)benzoate was isolated by flash column chromatography (9:1 'Hexane : EtOAc) as a clear oil : m/z = 405 (MH+) and 349 (M-BOC+) in MS ES+. To a solution of ethyl ester (0.24 mmol) in 1 ,4-dioxane (5 ml_) and water (1 ml_) was added 1 M NaOH (0.53 ml_) and the reaction mixture stirred for 18 h. The reaction mixture was concentrated under vacuum, the product resuspended in water and lyophilised to give sodium 4-(2-{1(S)-[(tert-butoxycarbonyl)(methyl)amino]ethyl}-5- methyl-1 ,3-thiazol-4-yl)benzoate : m/z 377 (MH+) and 321 (M-BOC+) in MS ES+ as a white solid which was used directly in the subsequent step.
Lithium 4-(2-ri(SHdimethylamino)ethyll-5-methyl-1 ,3-thiazol-4-yl)benzoate (Example 8.35) Ethyl 4-(2-{1 (S)-[(tert-butoxycarbonyl)(methyl)amino]ethyl}-5-methyl-1 ,3-thiazol-4- yl)benzoate was prepared as described previously.
To a solution of BOC-protected amine (0.25 mmol) in 1 ,4-dioxane (3 ml_) was added 4M HCI in dioxane (4 ml_) and the reaction mixture stirred for 2 h. The reaction mixture was concentrated under vacuum to afford a viscous pale yellow oil. The oil was dissolved in 1 :1 H2O:MeCN and lyophilised to afford ethyl 4-{2-[1(S)-
(methylamino)ethyl]-5-methyl-1 ,3-thiazol-4-yl}benzoate hydrochloride salt. A pH 5.5 buffer was prepared by adding AcOH to 1 M NaOAc until pH 5.5 was reached. The amine hydrochloride (0.28 mmol) was dissolved in 1 :1 buffeπMeOH (4 ml_) and formaldehyde (37 weight % in water ; 0.31 mmol) was added. The mixture was stirred for 1 h and then sodium cyanoborohydride (0.31 mmol) was added portionwise. After 1 h, the reaction mixture was concentrated by N2 stream. The residue was partitioned between saturated aqueous NaHCO3 and EtOAc. The aqueous layer was extracted with EtOAc and the combined organic layers were washed with H2O, brine, dried over Na2SO4 and the solvent removed under vacuum. The residue was purified by preparative HPLC (0.1% TFA in H2O : MeCN). The combined HPLC fractions were partitioned between saturated aqueous NaHCO3 and EtOAc. The aqueous layer was extracted with EtOAc and the combined organic layers were washed with brine, dried over Na2SO4 and the solvent removed under vacuum. The absence of EtOAc was confirmed by 1 H-NMR. The ethyl ester (0.19 mmol) was dissolved in 1 : 1 H20: 1 ,4-dioxane (8 mL) and 1 M LiOH (0.42 mL) was added and the reaction mixture stirred for 17 h. The reaction mixture was adjusted to pH 8 by addition of 1 M HCI. The mixture was concentrated under vacuum, resuspended in 1 :1 H2O:MeCN and lyophilised to give lithium 4-(2-{1 (S)- [(dimethyl)amino]ethyl}-5-methyl-1 ,3-thiazol-4-yl)benzoate : m/z 291 (MH+) in MS ES+ as a white solid and was used directly in the subsequent step.
5-(4-Methyl-morpholin-2-ylmethoxy)-benzofuran-2-carboxylic acid (Example 8.36) Ethyl 5-methoxybenzofuran carboxylate (22.7 mmol) was dissolved in dichloromethane (20 ml) and a 1.0 M solution of boron tribromide methyl sulphide complex in dichloromethane (68.1 mmol) was added. The mixture was heated at reflux over night. The solvent was evaporated under vacuo and the residue purified by flash chromatography to obtain ethyl 5-hydroxybenzofuran carboxylate as a white solid. Triphenyl phosphine polymer bound (8.96 mmol) was suspended in anhydrous dichloromethane (20 ml) then diisopropyl azodicarboxylate (7.76 mmol) was added and the mixture was stirred for 15 minutes at room temperature. Then ethyl 5- hydroxybenzofuran carboxylate (5.97 mmol) was added over 5 minutes followed by the addition of 4-N-boc-3-morpholinecarboxylic acid (5.97 mmol) over 5 minutes too. The mixture was stirred at room temperature over night. The solvent was evaporated under vacuo and the residue purified by flash chromatography to obtain ethyl 5-(4-Boc- morpholin-2-ylmethoxy)-benzofuran-2-carboxylate: m/z = 406 in MS ES+, as clear oil. Ethyl 5-(4-Boomorpholin-2-ylmethoxy)-benzofuran-2-carboxylate (2.47 mmol) was dissolved in 30 ml of a 4.0 M solution of hydrochloric acid in dioxan and stirred at room temperature for 1 hour. After removing the solvent under vacuo the resulting amine was dissolved in anhydrous dichloromethane and N-methylmorpholine (5.67 mmol) was added and stirred at room temperature for 5 minutes. Then allylchloroformate (2.71 mmol) was added and the mixture was stirred at room temperature over night under an inert atmosphere. The mixture was washed with a 1.0 M solution of hydrochloric acid, water, dried over Na2SU4 and the solvent was evaporated in vacuo. The residue was purified by flash chromatography to yield ethyl 5-(4-alloc-morpholin-2-ylmethoxy)- benzofuran-2-carboxylate: m/z = 390 in MS ES+, as a white solid. Ethyl 5-(4-alloc-morpholin-2-ylmethoxy)-benzofuran-2-carboxylate (2.09 mmol) was dissolved in 3 ml of tetrahydrofuran. Then 3 ml of a 1.0 M solution of lithium hydroxide were added and the mixture stirred at room temperature over night. After removing the tetrahydrofuran under vacuo the mixture was acidified with a 1 M solution of hydrochloric acid to Congo red, extracted with dichloromethane, washed with water, dried over Na2SU4 and the solvent was removed under vacuo to yield 5-(4-alloc- morpholin-2-ylmethoxy)-benzofuran-2-carboxylic acid as a white solid. 5-(4-Alloc-morpholin-2-ylmethoxy)-benzofuran-2-carboxylic acid (3 equiv) was then incorporated on the peptide as described previously (600 mg; 0.32 mmol/g) with HBTU (3 equiv), HOBt (3 equiv) and NMM (6 equiv) in DMF over night at room temperature. The Alloc group was removed with (1 ) DCM (4 x 1 min); (2) borane dimethylamine complex (40 equiv), tetrakis (triphenylphosphine) palladium (0) (0.1 equiv) in anhydrous DCM (2 x 15 min); (3) DCM (3 x 1 min); (4) DMF (3 x 1 min); (5) dioxan-water (9:1 ) (3 x 1 min); (6) DMF (3 x 1 min); (7) MeOH (3 x 1 min); (8) DCM (3 x 1 min) and the peptide resin was treated with dibutyltin dichloride (5 equiv), phenylsilane (5 equiv) and a 37% solution of formaldehyde in water (5 equiv) in THF for 2 hours at room temperature. The reminder of the procedure was carried out as described in the general protocol.
3-Methyl-5-(4-methyl-morpholin-2-ylmethoxy)-benzofuran-2-carboxylic acid (Example 8.37) 4-Methoxyphenol (0.119 mol) was dissolved in dry toluene and treated with sodium hydride (0.120 mol) at room temperature for 60 h. The sodium phenolate solution was heated to +100 0C and α-chloroacetoacetate (0.09 mol) was added. After stirring at +110 0C for a further 4 hours, the mixture was cool to room temperature, washed with water and brine, dried over Na2SO4 and the solvent was evaporated in vacuo to yield a crude α-(4-methoxyphenoxy)acetoacetate as a dark brown oil. Phosphoric acid (0.22 mol) was mixed with P2Os (0.35 mol) at room temperature and stirred at +130 0C for 4 hours. The mixture was allowed to cool to +100 0C, the acetoacetate was slowly added, and held at that temperature for 2 hours. After cooling to room temperature, ice was carefully added to the stirred solution, extracted with toluene, concentrated in vacuo and purified by flash chromatography on silica to yield ethyl 3-methyl-5-methoxybenzofuran carboxylate: m/z = 235 in MS ES+, as a white solid.
Ethyl 3-methyl-5-methoxybenzofuran carboxylate (8.53 mmol) was dissolved in dichloromethane (10 ml) and a 1.0 M solution of boron tribromide methyl sulphide complex in dichloromethane (25.59 mmol) was added. The mixture was heated at reflux over night. The solvent was evaporated under vacuo and the residue purified by flash chromatography to obtain ethyl 3-methyl-5-hydroxybenzofuran carboxylate as a white solid.
Triphenyl phosphine polymer bound (1.37 mmol) was suspended in anhydrous dichloromethane (10 ml) then diisopropyl azodicarboxylate (1.18 mmol) was added and the mixture was stirred for 15 minutes at room temperature. Then ethyl 3-methyl-5- hydroxybenzofuran carboxylate (0.91 mmol) was added over 5 minutes followed by the addition of 4-N-boc-3-morpholinecarboxylic acid (0.91 mmol) over 5 minutes too. The mixture was stirred at room temperature over night. The solvent was evaporated under vacuo and the residue purified by flash chromatography to obtain ethyl 3-methyl-5-(4- Boc-morpholin-2-ylmethoxy)-benzofuran-2-carboxylate: m/z = 419 in MS ES+, as clear oil.
Ethyl 3-methyl-5-(4-Boc-morpholin-2-ylmethoxy)-benzofuran-2-carboxylate (1.05 mmol) was dissolved in 30 ml of a 4.0 M solution of hydrochloric acid in dioxan and stirred at room temperature for 1 hour. After removing the solvent under vacuo the resulting amine was dissolved in 20 ml of a mixture 2 to 1 of methanol and a buffered solution of acetic acid and sodium acetate at pH=5.3. A 37% solution of formaldehyde in water (1.16 mmol) was added and the mixture was stirred at room temperature for 1 hour. Then sodium cyanoborohydride (1.16 mmol) was added and the mixture was stirred over night at room temperature. The methanol was removed under vacuo and the water was eliminated by liophylisation. The solid obtained was purified by flash chromatography to yield ethyl 3-methyl-5-(4-methyl-morpholin-2-ylmethoxy)- benzofuran-2-carboxylate: m/z = 334 in MS ES+, as a white solid. Ethyl 3-methyl-5-(4-methyl-morpholin-2-ylmethoxy)-benzofuran-2-carboxylate (0.12 mmol) was dissolved in 300 ul of tetrahydrofuran. Then 300 ul of a 1.0 M solution of lithium hydroxide were added and the mixture stirred at room temperature for 3 hours. The tetrahydrofuran was removed under vacuo and the water eliminated by lyophilisation to yield the tilted compound as a white solid: m/z = 304 in MS ES-.
4-r2-(1-Dimethylamino-ethyl)-thiazol-5-yll-benzoic acid lithium salt (Example 8.38) 4-(2-Azido-acetyl)-benzoic acid methyl ester.
4-(2-Bromo-acetyl)-benzoic acid methyl ester (15.5mmol) was dissolved in ethanol (120ml) and acetic acid (4.8ml). Sodium azide (31 mmol) was added and the reaction stirred at 40C overnight. The ethanol was removed and the mixture diluted with ethyl acetate (100ml). The organic layer was washed with saturated sodium hydrogen carbonate (2x50ml) and dried (MgSO4). The solvent was removed in vacuo to give a yellow solid, which was re-crystallized from ethanol to give the title compound as a pale yellow solid (2.6g). IR 2117cm 1 4-(2-Amino-acetyl)-benzoic acid methyl ester hydrochloride. 4-(2-Azido-acetyl)-benzoic acid methyl ester_(6.53 mmol) was suspended in methanol (30ml) and aqueous hydrochloric acid (6.53 mmol, 1 M) was added. A catalytic amount of palladium on carbon (10% wt) was added and the reaction stirred over an atmosphere of hydrogen for 3h. The reaction was filtered and the solvent removed in vacuo to give the title compound (1.3g) as a yellow solid m/z = 194 in MS ES+, which was used in the next step without purification.
4-(2-r2-(S)-(tert-Butoxycarbonyl-methyl-amino)-propionylaminol-acetyl)-benzoic acid methyl ester. 4-(2-Amino-acetyl)-benzoic acid methyl ester hydrochloride (2.22mmol), WSCHCI (2.44mmol), Boc-N-methyl-(L)-alanine (2.44mmol) and HOBt (2.77mmol) were suspended in dichloromethane (10ml). NMM (2.44mmol) was added and the reaction stirred for 2h. The reaction was diluted with ethyl acetate (50ml) and washed with 10% citric acid (2x25ml) and saturated sodium hydrogen carbonate (2x25ml). The organic layer was dried (MgSO4) and the solvent removed in vacuo to give a brown oil residue. Purification by silica chromatography eluting with 10-50% ethyl acetate/ iso-hexane gave the title compound (620mg) as a pale yellow oil m/z = 379 in MS ES+. 4-(2-(S)-H -(tert-Butoxycarbonyl-methyl-amino)-ethyll-thiazol-5-yl)-benzoic acid methyl ester. 4-{2-[2-(S)-(tert-Butoxycarbonyl-methyl-amino)-propionylamino]-acetyl}-benzoic acid methyl ester (1.65mmol) was dissolved in dry THF and Lawesson's reagent (2.5mmol) was added. The reaction was heated at reflux for 5h and cooled to room temperature. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate (100ml). The organic layer was washed with 10% citric acid (2x50ml) and saturated sodium hydrogen carbonate (2x50ml) and dried (MgSO4). The solvent was removed in vacuo to give a yellow oil residue, which was purified by silica chromatography to give the title compound (570mg) as a pale yellow solid.1 H NMR (CDCI3, 400MHz) 1.5(s, 9H), 1.6(d, 7Hz), 2.8 (brs, 3H), 3.9(s, 3H), 5.6(brm, 1 H), 7.6(m, 2H), 7.9(s, 1 H), 8.0(m, 2H). 4-(2-(S)-ri-(tert-Butoxycarbonyl-methyl-amino)-ethyll-thiazol-5-yl)-benzoic acid. 4-{2-(S)-[1 -(tert-Butoxycarbonyl-methyl-aminoJ-ethylJ-thiazol-δ-ylJ-benzoic acid methyl ester (0.75mmol) was dissolved in methanol (10ml) and lithium hydroxide (10ml, 1 M) was added. The reaction was stirred at room temperature overnight and the methanol removed in vacuo. The aqueous solution was taken to pH=3 with 1 M hydrochloric acid and extracted with ethyl acetate (2x50ml). The organic layer was dried (MgSO4) and the solvent removed in vacuo to give an off-white powder, which was purified by silica chromatography eluting with 50-80% ethyl acetate/ iso-hexane. The title compound was obtained as a white powder (252mg) m/z = 363 in MS ES+. 4-[2-(S)-H -Dimethylamino-ethyl)-thiazol-5-vH-benzoic acid methyl ester. 4-{2-(S)-[1-(tert-Butoxycarbonyl-methyl-amino)-ethyl]-thiazol-5-yl}-benzoic acid (0.75mmol) was dissolved in 50% TFA/ DCM (2ml) and stirred for 1 h. The solvent was removed in vacuo and the residue placed under high vacuum for 3h to give a light brown oil residue. The residue was dissolved in methanol (2ml) and buffer (1 ml, 1 M sodium acetate/ acetic acid, pH=5.5) was added. Formaldehyde (0.83mmol, 37wt% in water) was added and the reaction stirred for 30 minutes. Sodium cyanoborohydride (0.83mmol) was added and the reaction stirred overnight at room temperature. The methanol was removed in vacuo and the aqueous diluted with saturated sodium hydrogen carbonate (25ml). The aqueous layer was extracted with ethyl acetate (2x25ml) and the organic layer dried (MgSO4). The solvent was removed in vacuo and the residue purified by silica chromatography to give the title compound (158mg) as an off-white solid 1 H NMR (CD3OD, 400MHz) 1.5(d, J 7Hz), 2.3(s, 6H), 3.9(s, 3H), 3.95(q, J 7Hz), 7.7(m, 2H), 8.0(m, 3H).
4-r2-(1-Dimethylamino-ethyl)-thiazol-5-yll-benzoic acid lithium salt. 4-{2-(S)-[1-(tert-Butoxycarbonyl-methyl-amino)-ethyl]-thiazol-5-yl}-benzoic acid (0.54mmol) was dissolved in methanol (2ml) and lithium hydroxide (0.54mmol, 1 M) was added. The reaction was stirred overnight and the methanol removed in vacuo. The residue was diluted with water (5ml) and the aqueous layer extracted with ethyl acetate. The aqueous layer was freeze-dried to give the title compound as an off-white solid (143mg) which was used in the next step without further purification m/z = 277 in MS ES+.
δ-N.N-Dimethylamino-I H-indole^-carboxylic acid (Example 8.39)
5-Amino-1 H-indole-2-carboxylic acid ethyl ester.
5-Nitro-1 H-indole-2-carboxylic acid ethyl ester (14.9 mmol) was suspended in acetone
(50 ml) and added to a mixture of titanium(lll) chloride (91ml, >10% in 2M hydrochloric acid) and ammonium acetate (265ml, 4M). The reaction was stirred for 2h and neutralized with saturated sodium hydrogen carbonate. The mixture was extracted with ethyl acetate (100ml) and the organic layer dried (MgSO4). The solvent was removed in vacuo to give a light brown solid which was purified by silica chromatography to give the title compound as an off-white solid (1.57g) m/z = 205 in MS ES+. 5-N.N-Dimethylamino-1 H-indole-2-carboxylic acid ethyl ester.
5-Amino-1 H-indole-2-carboxylic acid ethyl ester (7.7mmol) was dissolved in acetonitrile (30ml) and formaldehyde (19.2mmol, 37%wt in water) was added. Sodium cyanoborohydride (7.7mmol) was added and the reaction stirred at room temperature overnight. The acetonitrile was removed in vacuo and the residue was purified by silica chromatography to give the title compound was a pale yellow solid (244mg). m/z = 233 in MS ES+.
5-N.N-Dimethylamino-1 H-indole-2-carboxylic acid 5-N,N-Dimethylamino-1 H-indole-2-carboxylic acid ethyl ester (1.Oδmmol) was suspended in ethanol (1 ml) and lithium hydroxide (1.2ml, 1 M in water) was added. The reaction was stirred at room temperature overnight. The solution was taken to pH=7 with 1 M hydrochloric acid and the ethanol removed in vacuo. The aqueous layer was extracted with ethyl acetate and the organic layer dried (MgSO4). The ethyl acetate was removed in vacuo to give the title compound as a yellow powder (75mg), which was used in the next step without purification, m/z = 205 in MS ES+.
4-12-H -(tert-Butoxycarbonyl-methyl-amino)-ethyll-thiazol-4-yl)-benzoic acid (Example
8.40)
Boc-L-NMe-Alanine-OH (1.0g, 4.92mmols) was dissolved in dioxan (1OmIs) and to this was added pyridine (0.25mls), di-tert-butyl dicarbonate (1.4g, 6.4mmols) and ammonium hydrogen carbonate (0.49g, 6.2mmols). After stirring for 18 hours the crude reaction mixture was concentrated in vacuo and re-suspended in ethyl acetate. This was washed with 1 M KHSU4 and the organic layer dried over magnesium sulphate. After concentration a clear oil was obtained (0.79g). This was dissolved in ethylene glycol dimethyl ether (1 OmIs) and to this was added Lawesson's reagent (4.31 mmols, 1.74g). After stirring at room temperature for 3 hours the reaction mixture was concentrated in vacuo and the residue re-suspended in ethyl acetate. This was washed with 1M Na2CU3and the organic layer dried over magnesium sulphate. After concentration a yellow oil was obtained. This was purified by flash chromatography (heptane/ethyl acetate) to give a white solid (0.73g). This was dissolved in ethanol (1OmIs) and 4-(2-Bromo-acetyl)-benzoic acid methyl ester (3.34mmols, 0.86g) was added. The reaction was heated to 500C for one hour. The crude product was purified by flash chromatography (heptane/ethyl acetate) to give a white solid (0.39g). ESMS (M + H = 377.23) which was subsequently hydrolysed to the corresponding acid. 4-r2-(1-Dimethylamino-2-methoxy-ethyl)-thiazol-4-yll-benzoic acid (Example 8.41 ) Boc-L-Serine(OMe)-OH (2.4g, β.Ommols) was dissolved in dioxan (2OmIs) and to this was added pyridine (0.31 mis), di-tert-butyl dicarbonate (1.7g, 7.8mmols) and ammonium hydrogen carbonate (0.62g, 7.2mmols). After stirring for 18 hours the crude reaction mixture was concentrated in vacuo and re-suspended in ethyl acetate. This was washed with 1 M KHSO4 and the organic layer dried over magnesium sulphate. After concentration the crude product was purified by flash chromatography to yield 0.55g of a clear oil. This was dissolved in ethylene glycol dimethyl ether (2OmIs) and to this was added Lawesson's reagent (2.78mmols). After stirring at room temperature for 3 hours the reaction mixture was concentrated in vacuo and the residue purified by flash chromatography (silica gel, DCM) to give a yellow oil (2-Methoxy-1-thiocarbamoyl- ethyl)-carbamic acid tert-butyl ester (0.49g). The ester (0.25g, 1.07mmols) was dissolved in ethanol (1OmIs) and 4-(2-Bromo-acetyl)- benzoic acid methyl ester (1.18mmols, 0.3Og) was added. The reaction was heated to 500C for one hour. The crude product was purified by preparative HPLC (MeCN/H2O) to yield 0.138g of a yellow solid. The Boc group was removed via treatment with 4M HCI/dioxan for one hour after which time the reaction mixture was concentrated in vacuo. The free amine (0.093g, 0.265mmols) was then dimethylated. The crude HCI salt was dissolved in 5mls of methanol and buffered with 2.5mls pH 5.5 Sodium acetate/acetic acid. Formaldehyde was added (0.58mmols) and the reaction stirred for one hour. Sodium cyanoborohydride was then added (0.58mmols, 0.036g) and the reaction stirred for a further thirty minutes. The reaction mixture was concentrated in vacuo and purified by preparative HPLC to yield 60mg of a yellow solid. Finally, the methyl ester was hydrolysed with 1 M LiOH (5mls) and dioxan (5mls) at room temperature for two hours. The reaction mixture was concentrated in vacuo and lyophilised from water to yield 62mg of the desired acid as the lithium salt. ESMS (M + H = 307.04)
4-r2-(4-Fluoro-1-methyl-pyrrolidin-2-yl)-thiazol-4-yll-benzoic acid (Example 8.42)
4-Fluoro-2-[4-(4-methoxycarbonyl-phenyl)-thiazol-2-yl]-pyrrolidine-1 -carboxylic acid tert- butyl ester (0.1g) was treated with 4M HCI/dioxan (1OmIs) for 2 hours. The reaction mixture was then concentrated in vacuo and lyophilised from water to yield a yellow solid (0.8g). The crude HCI salt was dissolved in 5mls of methanol and buffered with 2.5mls pH 5.5 Sodium acetate/acetic acid. Formaldehyde was added (0.38mmols, 0.0032mls) and the reaction stirred for one hour. Sodium cyanoborohydride was then added (0.38mmols, 0.024g) and the reaction stirred for a further thirty minutes. The reaction mixture was concentrated in vacuo and the residue re-suspended in ethyl acetate. This was washed with 1 M Na2CU3 and the organic layer dried over magnesium sulphate. After concentration a yellow solid was obtained (0.075g). Finally, the methyl ester was hydrolysed with 1 M LiOH (5mls) and dioxan (5mls) at room temperature for two hours. The reaction mixture was concentrated in vacuo and lyophilised from water to yield 62mg of the desired acid as the lithium salt. ESMS (M + H = 306.88)
2-r4-(4-Carboxy-phenyl)-thiazol-2-yll-4-fluoro-pyrrolidine-1 -carboxylic acid (Example
8.43)
2-r4-(4-Carboxy-phenyl)-thiazol-2-vH-4-fluoro-pyrrolidine-1 -carboxylic acid tert-butyl ester The amide (0.32g, 1.37mmols) was dissolved in ethylene glycol dimethyl ether (1OmIs) and to this was added Lawesson's reagent (0.61 g, 1.δmmols). After stirring at room temperature for 3 hours the reaction mixture was concentrated in vacuo and the residue re-suspended in ethyl acetate. This was washed with 1 M Na2CO3and the organic layer dried over magnesium sulphate. After concentration a yellow oil was obtained. This was purified by flash chromatography (heptane/ethyl acetate) to give a white solid (0.36g). This was dissolved in ethanol (1OmIs) and 4-(2-Bromo-acetyl)-benzoic acid methyl ester (0.41 g ,1.59mmols) was added. The reaction was heated to 500C for one hour. The crude product was purified by flash chromatography (heptane/ethyl acetate) to give a white solid (0.34g). The methyl ester was then treated with 1 M LiOH (1OmIs) and dioxan (1OmIs) for 3 hours. After quantitative hydrolysis, the crude product was concentrated in vacuo and the residue re-suspended in ethyl acetate. This was washed with 1 M KHSO4 and the organic layer dried over magnesium sulphate. After concentration in vacuo the product was lyophilised from acetonitrile/water to yield the title compound as a white solid (0.32g). ESMS (M + H = 393.03).
Lithium 5-(4-N-methylmorpholino-2S-methyloxy)benzofuran carboxylate (Example 8.36 as single enantiomers)
Ethyl 5-(4-boc-morpholino-2R-methyloxy)benzofuran carboxylate. 4-Boc-2R-hydroxymethylmorpholine was prepared according to method described in Heterocycles, 1993 35, 105-109. To a mixture of polymer supported triphenylphosphine (2.4 mmol) and the hydroxymethylmorpholine ( 1.2 mmol) in dry dichloromethane (5 ml) ethyl-5-hydroxybenzofuran-2-carboxylate (1.2 mmol) and DIAD (1.2 mmol) was added at room temperature. The mixture was stirred further 16 hours, fitrated, diluted in dry dichloromethane (5 ml) and stirred at room temperature another 16 hours. Filtrated, concentrated in vacuo and purified by y flash chromatography on silica (ethylacetate, hexane)to yield ethyl-5-(4-boc-morpholino-2R-methyloxy)benzofuran carboxylate (0.3 mmol), m/z = 406 in MS ES+, as an oil. Lithium 5-(4-N-methylmorpholino-2R-methyloxy)benzofuran carboxylate.
Ethyl-5-(4-boc-morpholino-2R-methyloxy)benzofuran carboxylate (0.3 mmol) was dissolved in HCI in dioxane (4M, 15 ml), stirred at room temperature for 4 hours, concentrated in vacuo to a pale yellow oil. The crude benzofuran hydrochloride (0.3 mmol) and formaldehyde (0.35 mmol) were mixed in THF (5 ml_) and dibutyltin dichloride (0.05 mmol) was added. After stirring at RT for 5 minutes, phenylsilane (0.6 mmol) was added and the reaction allowed to stir at room temperature for a further 17 h. The reaction was then concentrated in vacuo and the residue purified by flash chromatography (silica gel, ethyl acetate, isopropanol, triethylamine) to give ethyl-5-(4- N-methylmorpholino-2R-methyloxy)benzofuran carboxylate: m/z = 320 in MS ES+ as a clear oil.
To ethyl-5-(4-N-methylmorpholino-2R-methyloxy)benzofuran carboxylate (0.6 mmol) in 5 ml dioxane was added LiOH (0.6 mmol) in 1 ml of water. The mixture was refluxed for 16 hours, concentrated in vacuo to give lithium 5-(4-N-methylmorpholino-2R- methyloxy)benzofuran carboxylate: m/z 292 in MS ES+ as a white solid. Lithium 5-(4-N-methylmorpholino-2S-methyloxy)benzofuran carboxylate.
S-isomer: m/z 292 in MS ES+ was prepared as a white solid white solid according to method used to prepare R-isomer but substituting 4-Boc-2R-hydroxymethylmorpholine by 4-Boc-2S-hydroxymethylmorpholine.
4-(3-Methyl-5-morpholino-4-ylmethyl-thiophen-2-yl)-benzoic acid (Example 8.44)
5-Bromo-4-methyl-thiophene-2-carboxylic acid methyl ester (8.51 mmol) was dissolved in EtOH (100ml) and NaOH (42.5 mmol) added, as a 1M solution in water. Reaction was heated to 8O0C for 2h, after which time all starting material had been consumed. Reaction was then concentrated in vacuo and the residue taken up in DCM and shaken with 1 M HCI. The resulting biphasic mixture was then filtered and the filtrant washed with hexane and dried under vacuum. This gave 5-Bromo-4-methyl-thiophene-2- carboxylic acid as off white solid : m/z in MS AP- = 219, 221 [M-H]" , 6.79 mmol, 80%. 5-Bromo-4-methyl-thiophene-2-carboxylic acid (6.79 mmol) was taken up in 30ml DMF and morpholine (7.47 mmol), WSCHCI (7.47 mmol) and HOBt (7.47 mmol) were added. Reaction was stirred at room temperature for17h and then diluted with EtOAc, washed with 1 M HCI and brine, dried over Na2SO4 and concentrated in vacuo. Flash chromatography of the residue (silica, 33-50% EtOAc in hexane) gave (5-Bromo-4- methyl-thiophen-2-yl)-morpholin-4-yl-methanone as a pale golden oil : m/z in MS ES+ = 290, 292 [M+H]+, 4.67mmol, 69%.
(5-Bromo-4-methyl-thiophen-2-yl)-morpholin-4-yl-methanone (1.78 mmol) was added to a flask containing 1 M THF-BH3 complex in THF (4.45mmol). Reaction was stirred at reflux for 2.5h under N2. Methanol was then added until gas evolution ceased, followed by 10ml of 1 M NaOH and the reaction stirred at reflux for a further 7h. The mixture was cooled to room temperature and extracted with EtOAc. This extract was concentrated in vacuo and the residue taken up in 1 M HCI and washed with EtOAc. The acid layer was then basified with 1 M NaOH and extracted back into EtOAc. Removal of solvent gave 4- (5-Bromo-4-methyl-thiophen-2-ylmethyl)-morpholine as a colourless oil : m/z in MS ES+ = 276, 278 [M+H]+, 0.98 mmol, 55%. 4-(5-Bromo-4-methyl-thiophen-2-ylmethyl)-morpholine (0.98mmol) was taken up in 10ml toluene and 4-carboxymethylphenylboronic acid (0.98mmol) was added as a solution in 1 ml of EtOH. 6ml of 2M aqueous Na2CO3 solution was added, followed by Pd(PPh3J4 (0.098mmol). Reaction was stirred at 7O0C for 17h under a nitrogen atmosphere and then cooled to room temperature and extracted with DCM (x2). Combined organic layers were washed with brine, concentrated in vacuo and the residue purified by flash chromatography (silica, 33-99% EtOAc in hexane). This furnished the pure 4-(3-Methyl- 5-morpholi-4-ylmethyl-thiophen-2-yl)-benzoic acid methyl ester as a waxy white solid : m/z in MS ES+ = 332 [M+H]+ , 0.090mmol, 9%. This ester (0.09mmol) was heated to 7O0C in 18% HCI for 2h at which point HPLC showed all the starting material to have been hydrolysed. The reaction was cooled and the product that precipitated out of solution was collected by filtration as a white solid. With no further purification this was coupled using the standard procedure.
3-Methyl-4-(5-morpholin-4-ylmethyl-furan-2-yl)-benzoic acid (Example 8.45) A three necked flask was charged with methyl 4-bromo-3-methylbenzoate (2.18mmol), bis(pinacolato)diboron (2.29mmol), palladium acetate (0.065mmol), potassium acetate (6.54mmol) and DMF (10ml). The solution was degassed by bubbling through N2 gas for 30mins and was then heated to 8O0C under N2 for 3h. Reaction was then cooled to room temperature and 4-(5-Bromo-furan-2-ylmethyl)-morpholine (2.18mmol), cesium carbonate (3.27mmol) and Pd(PPh3)4 (0.065mmol) added. The reaction was heated to 8O0C and stirred for a further 17h. Mixture was then diluted with EtOAc and water and filtered through a celite pad to remove black particulates. The organic layer was separated, washed with brine and dried over Na2SO4 and concentrated in vacuo. Flash chromatography of the residue (silica, 10-99% EtOAc in hexane) gave 3-Methyl-4-(5- morpholin-4-ylmethyl-furan-2-yl)-benzoic acid methyl ester as a grey powdery solid: m/z in MS ES+ = 316 [M+H]+, 0.51 mmol, 23%.
This ester (0.51 mmol) was heated to 7O0C in 18% HCI for 2h at which point HPLC showed all the starting material to have been hydrolysed. The reaction was cooled and the product that precipitated out of solution was collected by filtration as a white solid. With no further purification this was coupled using the standard procedure.
4-r5-Methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-vH-benzoic acid (Example 8.46)
4-propionylbenzoic acid (890 mg, 5 mmol), NaHCO3 (1.26 g, 15 mmol), and iodomethane (935 uL, 15 mmol) in DMF (10 ml_) were stirred at RT overnight. The mixture was diluted with saturated aqueous NaCI (50 ml_) and extracted with ether (3 x
50 ml_). The organic phase was washed with water (50 ml_), dried, and evaporated.
Flash chromatography (90 g silica, 2/1 petroleum ether - EtOAc) gave white solids of 4-
Propionyl-benzoic acid methyl ester (744 mg, 77%).
1 H NMR (CDCI3, 400MHz) δ 1.24 (t, 3H, J = 7 Hz), 3.03 (q, 2H, J = 7 Hz), 3.95 (s, 3H),
8.0 and 8.12 (ABq, 4H)
4-Propionyl-benzoic acid methyl ester (744 mg, 3.87 mmol), pyrrolidone hydrotribromide (1.98 g), and 2-pyrrolidinone (380 mg, 4.5 mmol) in THF (38 ml_) were heated at 50 0C under nitrogen for 3 h. The mixture was cooled, filtered, concentrated, and then redissolved in ether (50 ml_). The ether solution was washed successively with water (20 ml_), saturated aqueous sodium thiosulphate (20 ml_), saturated aqueous NaCI (20 ml_), and water (2OmL), dried and evaporated to give crude 4-(2-Bromo- propionyl)-benzoic acid methyl ester as a yellow oil (1.025 g) that was used directly in the Hantzsch coupling. This material contained 91% of the desired bromoketone, 5% starting material, and 4% 4-bromo-1-butanol, as determined by 1 H NMR.
1 H NMR (CDCI3, 400MHz) δ 1.92 (d, 3H, J = 7 Hz), 3.96 (s, 3H), 5.28 (q, 1 H, J = 7 Hz), 8.07 and 8.14 (ABq, 4H)
All of the 4-(2-Bromo-propionyl)-benzoic acid methyl ester above and piperazine-1- carboxylic acid terf-butyl ester (J. Med. Chem., 1998, 5037-5054, 917 mg, 3.73 mmol) were refluxed in 36 ml_ THF at 70 0C for 2 h, under N2. The precipitate was filtered and the filtrate evaporated to give yellow solids. Flash column chromatography (silica, 5/1 petroleum ether - EtOAc) gave 624 mg of 4-[4-(4-Methoxycarbonyl-phenyl)-5-methyl- thiazol-2-yl]-piperazine-1 -ca rboxylic acid tert-butyl ester as a light yellow solid. Chromatography of the precipitate (silica, 2/1 petroleum ether - EtOAc) gave 32 mg more of compound. Total yield is 44%.
1 H NMR (CDCI3, 400MHz) δ 1.46 (s, 9H), 2.43 (s, 3H), 3.42, (m, 4H), 3.54 (m, 4H), 3.90 (s, 3H), 7.68 and 8.04 (ABq, 4H).
The above methyl ester (564 mg, 1.35 mmol) was heated with 1.35 ml_ 2N NaOH, 5 ml_ THF, and 3.65 ml_ water at 60 0C for 4 h. The reaction mixture was evaporated, poured into 20 ml_ saturated aqueous NaCI and 20 ml_ CH2CI2, and then acidified to pH 3 with 5% citric acid, in an ice bath. The layers were separated and the organic phase was extracted further with 2 x 10 ml_ CH2CI2. The organic phases were combined, washed with water (10 ml_), dried, and evaporated to give 4-[4-(4-Carboxy-phenyl)-5-methyl- thiazol-2-yl]-piperazine-1-carboxylic acid tert-butyl ester as a light yellow solid (537 mg, 98%).
1 H NMR (CDCI3, 400MHz) δ 1.48 (s, 9H), 2.47 (s, 3H), 3.47 (m, 4H), 3.57 (m, 4H), 7.74 and 8.12 (ABq, 4H).
13C NMR (CDCI3, 100MHz) δ ppm: 12.6, 28.3, 42.8, 48.1 , 80.3, 119.1 , 127.8, 128.2, 130.1 , 140.5, 145.6, 154.6, 167.2, 171.4. LCMS: (M + H)+ 404, (M - H)" 402. 4-[4-(4-Carboxy-phenyl)-5-methyl-thiazol-2-yl]-piperazine-1-carboxylic acid tert-butyl ester (0.421 mmol) was dissolved in 4M HCI in 1 ,4-dioxane, and stirred at room temperature for 1 h. The solvent was then removed under vacuum, and the residue 4-(5-Methyl-2-piperazin-1-yl-thiazol-4-yl)-benzoic acid was suspended in methanol (10 ml) and treated with AcOH/AcONa buffer (pH ~5.5, 5 ml), and formaldehyde (0.547 mmol). The reaction mixture was stirred at room temperature for 1 h, then treated with NaCNBH3 (0.547 mmol) and stirred at room temperature overnight. The solvent was then removed under vacuum, and the residue was purified by column chromatography to afford the title compound (0.403 mmol, 95%). MS(ES) m/z 318 (100%, [M+H]+).
4-(2-Morpholin-4-yl-thiazol-4-yl)-benzoic acid (Example 8.47) 4-(2-bromoacetyl)benzoic acid (1.23 mmol) and 1-morpholinethiocarboxamide (1.23 mmol, J. Med. Chem 1998, 41, 5037-5054) were mixed in THF (10 ml_), then refluxed for 3.5 h. The reaction mixture was then allowed to reach room temperature and the obtained precipitate was collected by filtration and washed with 4 portions of diethyl ether. The crude product was crystallized from hot 1 :1 EtOH-EtOAc to give a first harvest of colorless needles (0.16 g, 0.55 mmol). 1 H NMR (DMSO-d6, 400 MHz) δ 7.94 (4H, m), 7.49 (1 H, s), 3.72 (4H, m), 3.44 (4H, m).
4-(2-Piperidin-1-yl-thiazol-4-yl)-benzoic acid (Example 8.48) 4-(2-bromoacetyl)benzoic acid (1.23 mmol) and 1-piperidinethiocarboxamide (1.23 mmol) were mixed in THF (10 ml_), then refluxed for 3 h. The reaction mixture was then allowed to reach room temperature and the obtained precipitate was collected by filtration and washed with 3 portions of diethyl ether. The crude product was crystallized from hot 1 :1 EtOH-EtOAc to give a first harvest of colorless needles (0.28 g, 0.95 mmol). 1 H NMR (DMSO-d6, 400 MHz1) δ 7.93 (4H, m), 7.40 (1 H, s), 3.48 (4H, m), 1.60 (6H, m).
4-(2-Dimethylamino-thiazol-4-yl)-benzoic acid (Example 8.49)
To a stirred mixture of thiocarbonyldiimidazole (44.9 mmol) in THF (40 ml_) at room temperature was added portionwise 2 M Dimethylamine in THF (44 mmol) and a temperature increase was observed. 40 min after final addition the reaction mixture was heated to 55 0C for 1 h, then allowed to reach room temperature again. The reaction was then concentrated in vacuo and the residue purified by flash chromatography (silica gel, Petroleum ether-EtOAc) to give the intermediate lmidazole-1-carbothioic acid dimethylamide. This material was treated with freshly prepared sat. ammonia in methanol (40 ml) for 60 h, then concentrated in vacuo and the precipitated residue was suspended in diethyl ether and collected by filtration. The precipitate was washed with diethyl ether and air-dried to give a slight yellow solid (1.71 g, 16.4 mmol) which was used in the subsequent step. 4-(2-bromoacetyl)benzoic acid (1.23 mmol) and 1- piperidinethiocarboxamide (1.23 mmol) were mixed in THF (10 ml_), then refluxed for 3 h. The reaction mixture was then allowed to reach room temperature and the obtained precipitate was collected by filtration and washed with 3 portions of diethyl ether. The crude product was crystallized from hot 1 :1 EtOH-EtOAc to give a first harvest of colorless needles (0.1 g, 0.40 mmol). 1 H NMR (DMSO-d6,400 MHz) δ 7.94 (4H, m), 7.37 (1 H, s), 3.11 (6H, m).
4-r2-(lsopropyl-methyl-amino)-5-methyl-thiazol-4-vH-benzoic acid (Example 8.50)
To a solution of 4-propionylbenzoic acid (11.2 mmol), benzyl alcohol (1.1 ml_,10.7mmol) and dimethylaminopyridine (0.14 g, 1.1 mmol) in dichloromethane (90 ml) at 0 0C was added N-Ethyl-N'-(3-dimethylaminopropyl)carbodiimide x HCI (2.4 g, 12.3 mmol), then stirred at room temperature overnight. The obtained solution was then diluted with DCM, washed successively with aq. 10% citric acid and aq. sat. sodium hydrogen carbonate, then dried (Na2SO4), filtered and concentrated in vacuo. Flash chromatography of the residue (silica gel, Petroleum ether-EtOAc) gave a colorless oil which crystallized upon standing (2.67 g). A portion of the benzyl ester from above (1 g, 3.73 mmol) was refluxed with 2-pyrrolidinone (0.37 g, 4.33 mmol) and pyrrolidone hydrotribromide (1.85 g, 3.73 mmol) in THF for 1.5 h. The resulting reaction mixture was allowed to reach room temperature, then diluted with EtOAc, washed successively with water, aq. 10% sodium thiosulphate, aq. sat. sodium hydrogen carbonate and brine, then dried (Na2SO4), filtered and concentrated in vacuo. The obtained bromide from above was directly mixed with isopropylthiocarboxamide (0.44 g, 3.73 mmol) in THF (20 ml_) and refluxed overnight, then concentrated onto silica. Flash chromatography of the residue (silica gel, Petroleum ether-EtOAc-Et3N) gave a light red oil (1.28g, 3.49 mmol). To a stirred solution of the thiazole derivative (0.250 g, 0.68 mmol), obtained above, in acetonitrile (7 ml_), acetic acid (1.3 ml_) and aq. 37% formaldehyde (2 ml_) at 0 0C was added sodium cyanoborohydride (0.09 g), then stirred at room temperature overnight. Additional sodium cyanoborohydride (0.08 g) was added, and after stirring for additional 2 h, the reaction mixture was diluted with water, neutralized using aq. 0.5 M sodium carbonate, then extracted wih dichloromethane. The dichloromethane layers were collected, dried (Na2SO4), filtered and concentrated. Flash chromatography (silica gel, Petroleum ether-EtOAc) of the residue gave a slight yellow crystalline solid (0.115 g). 1 H NMR (CDCI3, 400 MHz,) . 8.09 (2H, d), 7.72 (2H, d), 7.2-7.32 (5H, m), 5.38 (2H, s), 4.27 (1 H, m), 2.92 (3H, s), 2.42 (3H, s), 1.22 (6H, d). The benzyl ester from above (0.25 g, 0.66 mmol) was hydrolysed by treating with aq. 1 M LiOH (1.3 ml_) in THF (2 ml_) at 60 0C overnight. The obtained solution was then made slight acidic with aq. 10% citric acid and then extracted using dichloromethane. The organic layer was then dried
(Na2SO4), filtered and concentrated. Column chromatography of the residue (Silica gel, dichloromethane-methanol) gave the title compound as a crystalline solid (0.19 g) m/z = 304 in MS ES+, which was characterised by hplc and MS.
4-(2-Methylamino-thiazol-4-yl)-benzoic acid (Example 8.51 )
To 25 ml of ethanol were added 4-(2-bromoacetyl)benzoic acid ( 486 mg, 2 mmole) and N-methyl thiourea ( 180 mg, 2 mmole). The reaction mixture was refluxed for 3 hr and the TLC showed the disappearing of the starting materials and the formation of a fluorescent product. The reaction was cooled on ice. The product was collected on filtration and washed with ethanol pre-cooled to 0 0C twice ( 2 x 3ml ), followed by diethyl ether. After drying, 486 mg product was obtained. 1 H NMR (DMSO-d6, 400 MHz) δ 7.97 (2H, d), 7.89 (2H, d), 7.32 (1 H, s), 2.96 (3H, s).
4-r2-(4.4-Difluoro-piperidin-1-yl)-thiazol-4-yll-benzoic acid (Example 8.52) 4,4-difluoropiperidine ( hydrochloride salt, 1.57 g, 10 mmole) and diisopropylethylamine ( 1.74 ml, 10 mmole) in acetone ( 10 ml) was slowly dropped into a mixture of ethoxycarbonylisothiocyanate (1.02 ml, 10 mmole ) in aceton ( 10 ml) at 0 0C. When the addition was completed, the reaction was kept under stirring at room temperature for one hour. 3N hydrochloric acid ( 15 ml ) was added and the reaction mixture was extracted with ethyl acetate. The organic phase was concentrated in vacuo.
To the residue was added concentrated hydrochloric acid ( 20 ml ) and the reaction was kept at 80 0C for 5 hours. Water ( 30 ml ) was added to the reaction. After the neutralization with ammonium carbonate, the reaction mixture was extracted with ethyl acetate. The organic phase was washed with water and dried in vacuo to obtain the crude intermediate 4,4-Difluoro-piperidine-1-carbothioic acid amide (1.21 g). To the residue from above ( 360 mg , 2 mmole ) and 4-(2-bromoacetyl)benzoic acid (486 mg, 2mmole) in THF ( 20 ml ) were refluxed for 5 hours. TLC showed the disappearing of the starting materials and the formation of a fluorescent product. The reaction was cooled on ice. The solid was collected by filtration. The product was recrystallized from ethanol (380mg). 1 H NMR (DMSO-d6, 400 MHz) δ 7.96 (4H, m), 7.51 (1 H, s), 3.66 (4H, m) 2.12 (4H, m).
Yields of the following title compounds in examples 8.53-8.61 and 8.63 were in general between 30 and 90%.
4-(2-lsopropylamino-thiazol-4-yl)-benzoic acid (Example 8.53)
Isopropyl-thiourea (2.47 mmol) and 4-(2-Bromo-acetyl)-benzoic acid (2.47 mmol) were mixed in THF (12 ml_). After stirring at room temperature for 5 minutes the mixture was heated to 80 0C for 2 hours. The volume was reduced to 5 ml_ and the mixture was then cooled to -20 0C and filtered. The solid was washed with a small amount of diethylether and dried, m/z = 263.1 in MS ES+, which was characterized by hplc and MS and used in the next step without any further purification.
3-r2-(4-Methyl-piperazin-1-yl)-thiazol-4-yll-benzoic acid (Example 8.54) 4-Methyl-piperazine-1-carbothioic acid amide (2.47 mmol) and 3-(2-Bromo-acetyl)- benzoic acid (2.47 mmol) were mixed in THF (12 ml_). After stirring at room temperature for 5 minutes the mixture was heated to 80 0C for 2 hours. The mixture was then cooled to room temperature and filtered. The solid was washed with a small amount of diethylether and dried, m/z = 304.1 in MS ES+, which was characterized by hplc and MS and used in the next step without any further purification.
3-(2-lsopropylamino-thiazol-4-yl)-benzoic acid (Example 8.55) Isopropyl-thiourea (2.47 mmol) and 3-(2-Bromo-acetyl)-benzoic acid (2.47 mmol) were mixed in THF (12 ml_). After stirring at room temperature for 5 minutes the mixture was heated to 80 0C for 2 hours. The volume was reduced to 5 ml_ and the mixture was then cooled to -20 0C and filtered. The solid was washed with a small amount of diethylether and dried, m/z = 263.1 in MS ES+, which was characterized by hplc and MS and used in the next step without any further purification.
4-(2-Piperidin-4-yl-thiazol-4-yl)-benzoic acid (Example 8.56) 4-Thiocarbamoyl-piperidine-1-carboxylic acid tert-butyl ester (2.47 mmol) and 4-(2- Bromo-acetyl)-benzoic acid (2.47 mmol) were mixed in THF (12 ml_). After stirring at room temperature for 5 minutes the mixture was heated to 80 0C for 2 hours. The volume was reduced to 5 ml_ and diethylether (5 ml_) was added. The mixture was then cooled to -20 0C and filtered. The solid was washed with a small amount of diethylether and dried, m/z = 289.1 in MS ES+, which was characterized by hplc and MS and used in the next step without any further purification.
4-r2-(1-Methyl-piperidin-4-yl)-thiazol-4-yll-benzoic acid (Example 8.57)
To a solution of 4-(2-Piperidin-4-yl-thiazol-4-yl)-benzoic acid (1 mmol) in acetic acid (0.5 ml_), methanol (3 ml_) and tetrahydrofurane (4.5 ml_) was added formaldehyde (aq. 37%, 300 ml_) and polystyrene bound cyanoborohydride (2.36 mmol/g, 900 mg). The slurry was then agitated for 16 hours at room temperature. The slurry was then filtered and the resin washed with methanol (2 ml_). The solution was concentrated to dryness in vacuo, m/z = 303.1 in MS ES+, which was characterized by hplc and MS and used in the next step without any further purification.
4-r2-(Pyridin-3-ylamino)-thiazol-4-yll-benzoic acid (Example 8.58) Pyridin-3-yl-thiourea (2.06 mmol) and 4-(2-Bromo-acetyl)-benzoic acid (2.06 mmol) were mixed in THF (12 ml_). After stirring at room temperature for 5 minutes the mixture was heated to 80 0C for 2 hours. The mixture was then cooled to room temperature and filtered. The solid was washed with a small amount of diethylether and dried, m/z = 298.0 in MS ES+, which was characterized by hplc and MS and used in the next step without any further purification.
4-r2-(Pyridin-2-ylamino)-thiazol-4-yll-benzoic acid (Example 8.59)
Pyridin-2-yl-thiourea (2.06 mmol) and 4-(2-Bromo-acetyl)-benzoic acid (2.06 mmol) were mixed in THF (12 ml_). After stirring at room temperature for 5 minutes the mixture was heated to 80 0C for 2 hours. The mixture was then cooled to room temperature and filtered. The solid was washed with a small amount of diethylether and dried, m/z = 298.0 in MS ES+, which was characterized by hplc and MS and used in the next step without any further purification.
4-(2-Cvclopentylamino-thiazol-4-yl)-benzoic acid (Example 8.60) Isothiocyanato-cyclopentane (4 g) in ammonia (37% in water, 8 ml_) and methanol (32 ml_) was stirred for 16 hours, filtered of and dried. The Cyclopentyl-thiourea (2.06 mmol) and 4-(2-Bromo-acetyl)-benzoic acid (2.06 mmol) were mixed in THF (12 ml_). After stirring at room temperature for 5 minutes the mixture was heated to 80 0C for 2 hours. The mixture was then cooled to room temperature and filtered. The solid was washed with a small amount of diethylether and dried, m/z = 289.05 in MS ES+, which was characterized by hplc and MS and used in the next step without any further purification.
4-(2-Cvclopropylamino-thiazol-4-yl)-benzoic acid (Example 8.61 ) Isothiocyanato-cyclopropane (4 g) was mixed with ammonia (37% in water, 8 ml_) and methanol (32 ml_) at 0 0C and then stirred for 16 hours at room temperature. The mixture was then cooled to 0 0C, filtered, washed with a little water and dried. The Cyclopropyl-thiourea (2.06 mmol) and 4-(2-Bromo-acetyl)-benzoic acid (2.06 mmol) were mixed in THF (12 ml_). After stirring at room temperature for 5 minutes the mixture was heated to 80 0C for 2 hours. The mixture was then cooled to room temperature and filtered. The solid was washed with a small amount of diethylether and dried, m/z = 261.0 in MS ES+, which was characterized by hplc and MS and used in the next step without any further purification.
4-r2-(Cvclopropyl-methyl-amino)-thiazol-4-vH-benzoic acid (Example 8.62) 4-(2-Cyclopropylamino-thiazol-4-yl)-benzoic acid (1.98 mmol), methyliodide (4.36 mmol) and potassium carbonate were mixed in DMF (20 ml_) and stirred for 72 hours at room temperature. The mixture was concentrated to dryness and partitioned between dichloromethane and water. The organic layer was dried (MgSO4) and concentrated to dryness. This solid was mixed with THF (4 ml_), methanol (2 ml_) and 1 N LiOH (3 mmol) and heated to 50 0C for 1 hour. The mixture was then cooled to room temperature and 1 N HCI was added until pH 4. The mixture was concentrated in vacuo, then the obtained residue was redissolved in dichloromethane-methanol and concentrated onto silica. Flash chromatography of the residue (silica gel, dichloromethane-methanol) gave the title compound as an off-white solid (0.1g), m/z = 275.0 in MS ES+, which was characterised by hplc and MS.
4-r2-(1-Methyl-pyrrolidin-3-yl)-thiazol-5-yll-benzoic acid (Example 8.63) S-Thiocarbamoyl-pyrrolidine-i-carboxylic acid tert-butyl ester (2.47 mmol) 4-(2-Bromo- acetyl)-benzoic acid (2.47 mmol) were mixed in THF (12 ml_). After stirring at room temperature for 5 minutes the mixture was heated to 80 0C for 1 hour. The mixture was then cooled to room temperature and filtered. The solid was washed with a small amount of diethylether and dried, m/z = 304.1 in MS ES+. This solid was then mixed in dichloromethane-trifluoroacetic acid (2:1 ) and kept at room temperature for 20 minutes. The mixture was concentrated to near dryness and the concentrated once from dichloromethane and once from 1 N HCI in diethylether. The remaining solid was mixed with acetic acid (0.5 ml_), methanol (3 ml_) and tetrahydrofurane (4.5 ml_) and formaldehyde (aq. 37%, 300 ml_) and polystyrene bound cyanoborohydride (2.36 mmol/g, 900 mg) was added. The slurry was then agitated for 16 hours at room temperature. The slurry was then filtered and the resin washed with methanol (2 ml_). The solution was concentrated to dryness in vacuo, m/z = 289.0 in MS ES+, which was characterized by hplc and MS and used in the next step without any further purification.
2-(1 -methylpiperazine-4-yl)-6-(4-carboxyphen-1 -vD-pyridine hydrochloride (Example 8.64)
2.6-dibromopyridine (11.8g, 50mmol) was dissolved in dimethylformamide (50ml) and 1- methylpiperazine (5.Og, 50mmol) and sodium iodide (0.6g) was added. The solution was heated to 80 0C for 30 minutes, then allowed to reach room temperature and diluted with ethyl acetate and water. The water layer was carefully extracted and the organic layers were collected, dried (Na2SO4) and concentrated. Flash chromatography of the residue on silica-gel (packed with ethyl acetate) using ethyl acetate-methanol- triethylamine 20:2:1 as the eluant. Pure fractions were collected and concentrated. 1.54g of the residue was dissolved in dimethoxymethane(48ml) and tetrakistriphenylphosphine palladium(O) (5.Og) was added. The solution was degassed and stirred for 15minutes under N2. 4-ethoxycarbonylphenylboronic acid (1.16g) was added followed by 36ml aq. 1 M sodium hydrogen carbonate solution. The solution was degassed one more time and heated to reflux and stirred for 12hours. The solution was filtered and the filtercake was carefully extracted with ethyl acetate and dimethoxymethane. The extracts were evaporated and purified by flashchromathography on silica-gel (packed with ethyl acetate) using ethyl acetate- methanol-triethylamine 20:2:1 as the eluant. Pure fractions were collected and concentrated. The residue was dissolved in 30ml concentrated hydrochloric acid and was refluxed for 12hours. The solution was evaporaded to yield the title compound as a solid. 1 H-NMR (400MHz, DMSO-d6) δ 2.7 (3H, m) 3.3 (4H, m) 4.5 (4H, m) 7.0 (1 H, m) 7.4 (1 H, m) 7.8 (1 H, m) 8.0 (1 H, m) 8.15 (1 H, m) 11.5 (1 H, bs).
4-(6-Morpholin-4-yl-pyridin-2-yl)-benzoic acid hydrochloride (Example 8.65) 2.6-dibromopyridine (2.Og) was dissolved in dimethoxymethane and morpholine (4.0ml) and sodium iodide (0.3g) was added. The solution was heated to reflux for 1 hour, then allowed to reach room temperature. The obtained solution was diluted with ethyl acetate, washed with water, then dried (Na2SO4), filtered and concentrated. Flash chromatography of the residue using stepwise gradient elution (ethyl acetate in hexane 20-33%). Pure fractions were concentrated and then subjected to Suzuki coupling as described in example 8.64, then purified by flash chromatography as described above. Pure fractions where collected and concentrated, then the residue was dissolved in 30ml concentrated hydrochloric acid and was refluxed for 1 hour. The obtained solution was evaporated to yield the title compound as a solid. 1 H-NMR (400MHz, DMSQI6) δ 3.6 (4H, m) 3.7 (4H, m) 6.9 (1 H, m) 7.3 (1 H, m) 7.7 (1 H1M) 7.9 (2H, m) 8.1 (2H, m).
Example 9
Novel P2 building block
Figure imgf000119_0001
PCC DCM
Figure imgf000119_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 (i-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 1 M 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, CDCI35 4.83.79 (1 H, m) 4.33-4.27 (1 H, m) 3.71 (3H, s) 1.98-1.62 (8H, m) 1.42 (9H, s) 1.22 (3H, s)
Example 10.1
Novel enantiomeric P3 building block
Figure imgf000121_0001
Figure imgf000121_0002
i. NaOH, 2-aminoethyl hydrogen sulphate, 50 0C, 1 h; ii. NaOH, H2O, 55 0C, 16 h; iii. BoC2O, RT, 5 h; iv. 10 % Pd on carbon, H2 at 1 atm for 16 h, RT, 3 h; v. Ph3P, CBr4, RT, 4 h; vi. δ-hydroxy-S-methylbenzofuran^-carboxylic acid ethyl ester, Cs2CO3, 40 0C, 18 h; vii. LiOH, RT, 16 h; viii. 2-amino-1-(6-fluoro-3,3-dimethoxy-hexahydro-furo[3,2- b]pyrrol-4-yl)-4-methyl-pentan-1-one, HOBt, WSCHCI, RT, 60 h; ix. 10 % HCI in CH3OH, RT, 2 h; x. Bu2SnCI2, HCOH, phenylsilane, RT, 16 h; xi. TFA, RT, 2 h.
2R-Benzyloxymethyl-morpholine-4-carboxylic acid tert-butyl ester 2g of S-(+)-2-(benzyloxymethyl)-oxirane and 7 g of 2-aminoethyl hydrogen sulfate were weighed into a 100 ml round bottle flask, 2 g of NaOH dissolved in H2O was added and the stirred mixture was heated at 50 0C for 1 hour. 4 g of NaOH dissolved in 10 ml H2O, solution was added to the stirred mixture, which was then heated at 55 0C for 16 h. After cooling the mixture to room temperature, it was diluted with 100 ml H2O and 100 ml dioxane and 2.66 g of di-tert-butyl dicarbonate was added. The mixture was stirred at room temperature for 5 hours, transferred into a separation funnel and extracted with 2 x 75 ml of toluene. Combined organic phases were washed with 2 x 50 ml of 1 M citric acid (aq.), once with brine, dried over Na2SO4 and concentrated in vacuo. The crude material was purified on silica yielding 2R-Benzyloxymethyl-morpholine-4-carboxylic acid tert-butyl ester 1.85 g (49%) as a clear oil.
2R-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl ester The oil (1.80 g) was dissolved in 50 ml ethanol and 100 mg palladium on carbon (10%) was added. The mixture was hydrogenated at 1 atm H2-pressure for 16 h, filtered through silica/alumina (5 cm and 0.5 cm, respectively) and concentrated in vacuo yielding 1.18 g (92%) 2R-hydroxymethyl-morpholine-4-carboxylic acid tert-butyl ester as a white crystalline solid. 2R-Bromomethyl-morpholine-4-carboxylic acid tert-butyl ester 12 g of polymer supported triphenylphosphine was suspended in 100 ml dichloromethane at room temperature, 0.8 g alcohol and 2.4 g CBr4 was added. The mixture was stirred at room temperature for 4 hours, solid supported reagent was removed and residue was concentrated in vacuo. The crude material was dissolved in toluene, treated with 10 g of DARGO G-60, filtered and concentrated in vacuo to yield 0.91 g (88 %) of semi solid 2R-bromomethyl-morpholine-4-carboxylic acid tert-butyl ester.
2R-(2-Ethoxycarbonyl-3-methyl-benzofuran-5-yloxymethyl)-morpholine-4-carboxylic acid tert-butyl ester
The bromide (0.41 g) and 5-hydroxy-3-methylbenzofuran-2-carboxylic acid ethyl ester (0.24 g) was dissolved in 4 ml dry DMF, 1.0 g of CS2CO3 was added, and the mixture was stirred at 40 0C for 16 h. The mixture was diluted with 25 ml EtOAc, washed with water, 1 M citric acid and brine before drying the organic phase over Na2SO4.
Concentration in vacuo yielded a brown oily crude product which was purified on neutral alumina to yield 0.14 g (31%) 2R-(2-Ethoxycarbonyl-3-methyl-benzofuran-5- yloxymethyl)-morpholine-4-carboxylic acid tert-butyl ester as a white solid.
2R-(2-Carboxy-3-methyl-benzofuran-5-yloxymethyl)-morpholine-4-carboxylic acid tert- butyl ester
The ethyl ester (0.14g) was dissolved in 10 ml dioxane at room temperature, a solution of 60 mg (4 eq.) LiOH in 3 ml water was added and the mixture was stirred for 16 hours at ambient temperature. The suspension was transferred to a separation funnel, diluted with 20 ml of water and extracted with EtOAc. The aqueous phase was acidified with 30 ml of 1 M citric acid (aq.) and extracted with 2x30 ml EtOAc. The organic phase was washed with brine, dried over Na2SO4 and concentrated in vacuo to 0.125 g (97%) 2R- (2-Carboxy-3-methyl-benzofuran-5-yloxymethyl)-morpholine-4-carboxylic acid tert-butyl ester as a white solid. MS-ES" (390, 100%).
Example 10.2
Novel enatiomeric P3 building block
Figure imgf000123_0001
i. NaOH, 2-aminoethyl hydrogen sulphate, 50 0C, 1 h; ii. NaOH, H2O, 55 0C, 16 h; iii. BoC2O, RT, 5 h; iv. 10 % Pd on carbon, H2 at 1 atm for 16 h, RT, 3 h; v. Ph3P, CBr4, RT, 4 h; vi. Carboxy-protected-5-hydroxy-3-methyl-benzofuran-2-carboxylic acid, Cs2CO3, 4O 0C, 18 h;
2S-Benzyloxymethyl-morpholine-4-carboxylic acid tert-butyl ester
3g of R-(+)-2-(benzyloxymethyl)-oxirane and 10.5 g of 2-aminomethyl hydrogen sulfate were weight in a 100 ml round bottle flask, 3 g of NaOH dissolved in H2O was added and the stirred mixture was heated at +50 0C for 1 hour. 6 g NaOH was dissolved in 10 ml H2O, solution was added to the stirred mixture, which was then heated at + 550C for 72 h. After cooling the mixture to room temperature, it was diluted with 100 ml H2O and 100 ml dioxane and 4.0 g of di-tert-butyl dicarbonate was added. The mixture was stirred at room temperature for 5 hours, transferred into a separation funnel and extracted with 2x75 ml of toluene. Combined organic phases were washed with 2 x 50 ml of 1 M citric acid (aq.), once with brine, dried over Na2SO4 and concentrated in vacuo. The crude material was purified on silica yielding 2S-Benzyloxymethyl-morpholine-4- carboxylic acid tert-butyl ester 2.55 g (45%) as a clear oil.
2S-Hydroxymethyl-morpholine-4-carboxylic acid tert-butyl ester
The oil (2.55 g) was dissolved in 50 ml ethanol and 100 mg palladium on carbon (10%) was added. The mixture was hydrogenated at 1 atm H2-pressure for 16 h, filtered through silica/alumina (5 cm and 0.5 cm, respectively) and concentrated in vacuo yielding 1.7 g (94%) 2S-hydroxymethyl-morpholine-4-carboxylic acid tert-butyl ester as a white crystalline solid.
2S-Bromomethyl-morpholine-4-carboxylic acid tert-butyl ester
6.5 g of polymer supported triphenylphosphine was suspended in 50 ml dichloromethane at room temperature, 0.42 g alcohol and 1.28 g CBr4 was added. The mixture was stirred at room temperature for 2 hours, solid supported reagent was removed and residue was concentrated in vacuo. The crude material was dissolved in toluene, treated with 10 g of DARGO G-60, filtered and concentrated in vacuo to yield 0.41 g (77 %) of semi solid 2S-bromomethyl-morpholine-4-carboxylic acid tert-butyl ester. This is reacted to form the carboxy-protected 5-hydroxy-3-methylbenzofuran-2- carboxylic acid as shown in the example immediately above to form a P3 building block. Alternatively, the 5-hydroxybenzofuryl-P3 may be built into the P1/P2/P3 sequence and the intermediate of step c) subsequently reacted.
Biological Examples
Determination of cathepsin K proteolytic catalytic activity
Convenient assays for cathepsin K are carried out using human recombinant enzyme, such as that described in PDB. ID BC016058 standard; mRNA; HUM; 1699 BP.
DE Homo sapiens cathepsin K (pycnodysostosis), mRNA (cDNA clone MGC:23107
RX MEDLINE;. RX PUBMED; 12477932.
DR RZPD; IRALp962G1234.
DR SWISS-PROT; P43235;
The recombinant cathepsin K can be expressed in a variety of commercially available expression systems including E coli, Pichia and Baculovirus systems. The purified enzyme is activated by removal of the prosequence by conventional methods.
Standard assay conditions for the determination of kinetic constants used a fluorogenic peptide substrate, typically H-D-Ala-Leu-Lys-AMC, and were determined in either 100 mM Mes/Tris, pH 7.0 containing 1 mM EDTA and 10 mM 2-mercaptoethanol or10OmMNa phosphate, imM EDTA, 0.1%PEG4000 pH 6.5 or 100 mM Na acetate, pH 5.5 containing 5 mM EDTA and 20 mM cysteine, in each case optionally with 1 M DTT as stabiliser. The enzyme concentration used was 5 nM. The stock substrate solution was prepared at 10 mM in DMSO. Screens were carried out at a fixed substrate concentration of 60 μM and detailed kinetic studies with doubling dilutions of substrate from 250 μM. The total DMSO concentration in the assay was kept below 3%. All assays were conducted at ambient temperature. Product fluorescence (excitation at 390 nm, emission at 460 nm) was monitored with a Labsystems Fluoroskan Ascent fluorescent plate reader. Product progress curves were generated over 15 minutes following generation of AMC product.
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.
10Oμl/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 mM 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 1 mM DTT and 2 nM cathepsin S to each well of rows B-H and 180 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 Ki, 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 8-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.
Cathepsin L Ki
The procedure above 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 1 mM EDTA, pH5.5) The DMSO stock (1OmM in 100%DMSO) is diluted to 10% in assay buffer. Enzyme is prepared at 5nM concentration in assay buffer plus 1mM 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)
Inhibition Studies
Potential inhibitors are screened using the above assay with variable concentrations of test compound. Reactions were initiated by addition of enzyme to buffered solutions of substrate and inhibitor. Kj values were calculated according to equation 1
Figure imgf000127_0001
where V0 is the velocity of the reaction, Vis the maximal velocity, S is the concentration of substrate with Michaelis constant of KM, and / is the concentration of inhibitor.
Abbreviations
DMF dimethylformamide DCM dichloromethane
TBDMS tert-butyldimethylsilyl RT room temperature
THF tetrahydrofuran Ac acetyl TLC thin layer chromatography DMAPdimethylaminopyridine
EtOAc ethyl acetate
All references referred to in this application, including patents 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 Il
Figure imgf000128_0001
wherein
R1 and R1' are halo; or one of R1 and R1' is halo, and the other is is H;
R2 is R4, -C(=O)R4; -OC(=O)R4, -S(=O)nR4, -S(=O)nNRdRe;
R3 is H, -OR4, -SR4; R3' is H; or
R3 and R3' together define =O;
R4 is -Ci-C6 alkyl, -Co-Caalkylenecarbocyclyl or -C0-C3alkyleneheterocyclyl, any of which is optionally substituted with up to three substituents selected from R7;
R5 is -C1-C5 alkyl, -CH2CR5"C3-C4-cycloalkyl; R5' is H; or
R5 and R5' together with the carbon to which they are attached define CvrCβ-cycloalkyl;
R5" is H1 Ci-C2 alkyl, Ci-C2 haloalkyl, hydroxyl, OCi-C2alkyl, fluoro;
E is -C(=O)-, -S(=O)m-, -NRaS(=O)m-, -NRaC(=O)-, -OC(=O)-, -CRbRc-;
R6 is a stable, optionally substituted, monocyclic or bicyclic carbocycle or heterocycle, wherein the, or each, ring has 4, 5 or 6 ring atoms and 0 to 3 hetero atoms selected from S1 O and N and wherein the optional substituents comprise 1 to 3 members selected from R7;
R7 is independently selected from halo, oxo, nitrile, nitro, C1-C4 alkyl, -XNRdRe1 -
XNReR8, -NReXR8, NH2CO-, X-R8, X-O-R8, O-X-R8, X-C(=O)R8, X-(C=O)NRdR8, X- NReC(=O)R8, X-NHSOmR8, X-S(=O)mR8, X-C(=O)OR8, X-NReC(=O)OR8;
R8 is independently H, C1-C4 alkyl, C3-C6 cycloalkyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, thiopyranyl, furanyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl, phenyl, any of which is optionally substituted with up to 3 members selected from R9; R9 is independently selected from hydroxy, XR10, -XNRdRe, -XNReR10, -NReC1-
C^alkylR10, cyano, halo, carboxy, oxo, Ci-C4 alkyl, Ci-C4-alkoxy, Ci-C4 alkanoyl, carbamoyl;
R10 is C3-C6 cycloalkyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, thiopyranyl, furanyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl, phenyl, any of which is substituted with C1-C4 alkyl, halo, hydroxy, Ci-C4alkoxy
X is independently a bond or C1-C4 alkylene;
Ra is independently H1 Ci-C4 alkyl or CH3C(=O); Rb is CrC4haloalkyl;
Rc is H, Ci-C4 alkyl;
Rd is independently H, C1-C4 alkyl or CH3C(=O);
Re is independently H, C1-C4 alkyl; or
Rd and Re together with the N atom to which they are attached form a morpholine, piperidine, piperazine or pyrrolidine ring optionally substituted with R9; m is independently 0,1 or 2; n is 1 or 2; or a pharmaceutically acceptable salt or hydrate thereof.
2. A compound according to claim 1 , wherein R1' is halo and R1 is H.
3. A compound according to claim 2, wherein R1' is fluoro.
4. A compound according to claim 4, wherein the stereochemistry is as depicted in the partial structure below:
Figure imgf000129_0001
5. A compound according to claim 1 , wherein R1 and R2 are fluoro.
6. A compound according to claim 1 , with the partial structure:
Figure imgf000130_0001
7. A compound according to claim 6, wherein R4 is benzyl or C1-C4 alkyl, preferably methyl.
8. A compound according to claim 1 with the partial structure:
Figure imgf000130_0002
9. A compound according to claim 1, wherein R2 is-C(=O)R4; -S(=O)nR4, and - S(=O)nNRdRe; especially where n is 2.
10. A compound according to claim 9, wherein R4 is an optionally substituted, 5 or 6 membered, monocyclic, saturated or unsaturated ring containing 0 or 1 nitrogen atom, which is directly bonded to the remainder of R2, or linked vi a methylene group.
11. A compound according to claim 9, wherein R2 is C3-C6 alkyl, optionally interrupted with an O or NH as part of the the chain, which are unsubstituted or substituted with one or more NH2, NHMe, NHC(O)CH3, NHMe(C(O)CH3, OH or OMe groups, especially those which are branched at the alpha position or which include an NH2, NHMe, NHC(O)CH3, NHMe(C(O)CH3, OH or OMe group at the alpha position.
12. A compound according to claim 1 , wherein R5 and R5' are as shown in one of the partial structures:
Figure imgf000131_0001
13. A compound according to claim 1, wherein R5 and R5> are as shown in the partial structure:
Figure imgf000131_0002
14. A compound according to claim 13, wherein R5" is H, F, OH or preferably methyl.
15. A compound according to claim 1, wherein the Ra depicted in formula Il is H.
16. A compound according to claim 1 , wherein E is -CRbRc-.
17. A compound according to claim 16, where Rb is trifluoromethyl.
18. A compound according to claim 1, wherein E is -C(=O)-.
19. A compound according to claim 1, wherein R6 is substituted phenyl.
20. A compound according to. claim 19, wherein the substituent comprises -NRdRe, CH2NRdRe, -NRdR8, -NRe-X-R8, d-C4 straight or branched alkyl or -O-R8.
21. A compound according to claim 20, wherein the substituent comprises -NH-CH2phenyl, -NHCH2pyridyl or -NH-phenyl, wherein each phenyl or pyridyl ring is substituted with Ci-C4-alkyl, -NRdRe, -NRdR8 or -NRe-X-R8.
22. A compound according to claim 19, wherein the substituent comprises C3-C6 cycloalkyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, thiopyranyi, furanyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indolyl, phenyl, any of which is optionally substituted with 1-3 members independently selected from R9.
23. A compound according to claim 22, wherein the substituent is thiazolyl, 5-methyl- thiazolyl or thienyl, optionally substituted with R9.
24. A compound according to claim 23, wherein the substituent is 4-substituted thiazol-2-yl, 4-substituted-5-methylthiazol-2-yl or 5-substituted thien-2-yl, wherein the 4 or 5 substituent is H or R9.
25. A compound according to claim 24, wherein the thiazolyl, 5-methylthiazolyl or theinyl is substituted with morpholinyl, morpholinylmethyl-, piperidinyl, piperidinylmethyl, piperazinyl, piperazinylmethyl, any of which is substituted with CrC3 alkyl, fluoro, difluoro or Ci-C3 alkyl-O-Ci-C3alkyl-.
26. A compound according to claim 25, wherein the substituent to the thiazolyl, 5- methylthiazolyl or thienyl is piperid-4-yl which is substituted with methyl, piperazinyl which is N-substituted with CrC3 alkyl or methyloxyethyl-, -or piperid-1-ylmethyl- which is unsubstituted or 4-substituted with fluoro or di-fluoro.
27. A compound according to claim 19, wherein the substituent comprises a morpholine, piperidine or piperazine ring, optionally substituted with R9.
28. A compound according to claim 27 comprising piperid-4-yl or N-piperazinyl, N- substituted with Ra or piperidin-1-yl which is 4-substituted with -NRdRe.
29. A compound according to claim 1 , wherein R6 is: benzothiazol or benzofuryl, 3- methylbenzofuryl or benzoxazolyl, any of which is substituted with R7.
30. A compound according to claim 29, wherein the substituent is -OR8, -OXR8 , -
NRdR8 or -NRdXR8.
31. A compound according to claim 30, wherein R8 is piperid-4-yl, piperazin-1-yl, piperidin-1-yl or morpholino, any of which is substituted with halo or C1-C3 alkyl.
32. A compound according to claim 31, wherein the optional substituent to R6 is N- morpholinylethyloxy, N-morpholinylmethyloxy, N-methylpiperid-4-yloxy, N- methylmorpholin-3-ylmethyioxy or N-methylmorpholin-3-ylethyloxy.
33. A pharmaceutical composition comprising a compound as defined in any of claims 1 to 32 and a pharmaceutically acceptable carrier or diluent therefor.
34. Use of a compound as defined in any of claims 1-32 in the manufacture of a medicament for the treatment of disorders mediated by cathepsin K.
35. Use according to claim 34, wherein the disorder is selected from: osteoporosis, gingival diseases such as gingivitis and periodontitis,
Paget's disease, hypercalcaemia of malignancy metabolic bone disease diseases characterised by excessive cartilege or matrix degradation, such as osteoarthritis and rheumatoid arthritis. bone cancers including neoplasia, pain.
36. A compound as defined in any of claims 1 to 32 for use as a medicament.
37. A compound as defined in any one of claims 1 to 32 for use in the treatment or prevention of a disorder characterised by inappropriate expression or activation of cathepsin K.
38. A method for the treatment or prevention of a disorder characterised by inappropriate expression or activation of cathepsin K comprising the administration of a safe and effective amount of a compound according to any one of claims 1 to 32 to a subject in need thereof.
PCT/EP2006/063950 2005-07-07 2006-07-06 Cysteine protease inhibitors WO2007006714A1 (en)

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