WO2008114054A1 - Protease inhibitors - Google Patents

Protease inhibitors Download PDF

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Publication number
WO2008114054A1
WO2008114054A1 PCT/GB2008/050193 GB2008050193W WO2008114054A1 WO 2008114054 A1 WO2008114054 A1 WO 2008114054A1 GB 2008050193 W GB2008050193 W GB 2008050193W WO 2008114054 A1 WO2008114054 A1 WO 2008114054A1
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WO
WIPO (PCT)
Prior art keywords
azido
benzamide
furo
pyrrol
oxodihydro
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PCT/GB2008/050193
Other languages
French (fr)
Inventor
Jussi Kangasmetsa
Kevin Parkes
Matt Tozer
Tony Johnson
Urszula Grabowska
Lourdes Oden
Christian Sund
Magnus Nilsson
Vladimir Ivanov
Pia Kahnberg
Original Assignee
Medivir Ab
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Publication date
Priority claimed from GB0705213A external-priority patent/GB0705213D0/en
Priority claimed from GB0712182A external-priority patent/GB0712182D0/en
Application filed by Medivir Ab filed Critical Medivir Ab
Publication of WO2008114054A1 publication Critical patent/WO2008114054A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—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

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, C ⁇ thidia 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
  • metabolic bone disease In view of its elevated levels in chondroclasts of osteoarthritic synovium, cathepsin K is implicated in diseases characterised by excessive cartilage or matrix degradation, such as osteoarthritis and rheumatoid arthritis.
  • bisphosphonates have a very long half-life in bone so if effects such as osteonecrosis of the jaw manifest themselves, it is impossible to remove the bisphosphonate from the bone.
  • cathepsin K inhibitors typically have a fast onset and off rate mode of action, which means that if a problem was to be identified, dosing could be halted and there would be no build up of the inhibitor in the bone matrix.
  • UVWXY broadly corresponds to the P3 and P2 (these expressions are explained below) of dipeptide cysteine protease inhibitors
  • Z is inter alia O, S, methylene or -NR-
  • R'i is alkyl, alkylaryl etc
  • Pi and Q are each, inter alia, methylene.
  • cap defines a diverse variety of cyclic structures.
  • the compounds are active inhibitors of cathepsin K, but, as shown below, further modification of the structure yields improvements as regards to pharmacokinetics and/or pharmacodynamics.
  • potent inhibitors of cathepsin K are inbibitors of cathepsin K which show selectivity for cathepsin K over other cathepsins (e.g. selectivity over cathepsin S and/or cathepsin L). Potent inhibitors of cathepsin K which demonstrate properties such as high permeability and/or advantageous metabolic profiles may be expected to be of great value in a clinical setting.
  • R 2 is the side chain of leucine, isoleucine, cyclohexylglycine, O-methyl threonine, 4-fluoroleucine or 3-methoxyvaline;
  • R ,3 3 is H, methyl or fluoro
  • R 4 is C 1- C 6 alkyl; E is a bond or thiazolyl, optionally substituted with methyl or fluoro; n is 0 or 1 ; or a pharmaceutically acceptable salt, N-oxide or hydrate thereof (collectively referred to herein as compounds of the invention).
  • R 2 represents the side chain of leucine. In a second embodiment of the invention R 2 represents the side chain of isoleucine. In a third embodiment of the invention R 2 represents the side chain of cyclohexylglycine. In a fourth embodiment of the invention R 2 represents the side chain of O-methyl threonine. In a fifth embodiment of the invention R 2 represents the side chain of 4- fluoroleucine. In a sixth embodiment of the invention R 2 represents the side chain of 3- methoxyvaline.
  • R 2 presently preferred values include those embodied by the partial structures:
  • R moieties are those represented by the partial structures:
  • R 2 moieties are the side chains of O-methyl threonine, 4- fluoroleucine and 3-methoxyvaline.
  • n 0.
  • n 1
  • R 3 suitably represents methyl or fluoro, especially fluoro.
  • R 3 is suitably positioned as shown by the partial structure:
  • the C 1 -C 6 alkyl definition of R is meant to include both branched and unbranched alkyl moieties containing between one and six carbon atoms in total.
  • R 4 groups are methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, tert- butyl and sec-butyl), pentyl (n-pentyl, isopentyl etc) and hexyl (n-hexyl etc).
  • R 4 represents C 1 -C 4 alkyl (such as C 1 -C 3 alkyl).
  • One R 4 group of particular interest is methyl.
  • a second R 4 group of particular interest is propyl (especially n-propyl).
  • E is a bond.
  • preferred compounds within this embodiment include:
  • R 2 and R 3 are as defined above.
  • Representative examples within this embodiment include:
  • E is thiazolyl which is optionally substituted with methyl or fluoro.
  • Preferred compounds within this embodiment include those of the formula:
  • R 2 and the optional substituent R 3 are as defined above and R 5 is H, methyl or fluoro.
  • R 5 is H, methyl or fluoro.
  • E represents thiazolyl which is unsubstituted.
  • E represents thiazolyl which is substituted with methyl or fluoro, in particular fluoro.
  • E represents thiazolyl which is unsubstituted or substituted by methyl.
  • R 5 is the location of the optional substitution (i.e. R 5 represents H, methyl or fluoro).
  • 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 cartilage or matrix degradation (such as osteoarthritis and rheumatoid arthritis), bone cancers including neoplasia, pain (especially chronic pain).
  • a method for the treatment or prevention of a disorder mediated by cathepsin K comprising the administration of a safe and effective amount of a compound of the invention for the purpose of treating or preventing said disorder which is mediated by cathepsin K.
  • N-protected derivative thereof e.g. Boc-protected
  • the corresponding 1 ,3-dioxolane protected analogue and N-protected derivatives thereof e.g. Boc-protected
  • R 4 represents C h alky! (such as methyl); or a salt or lower alkyl (e.g. C h alky! ester thereof, such as the methyl ester or HCI salt.
  • the invention does not embrace subject matter which is described in international patent application WO2008/007107.
  • the compounds of the invention do not embrace the compounds described on page 37 line 8 to page 38 line 6 of WO2008/007107: N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-(2-(4-methylpiperazin- 1 -yl)thiazol-4-yl)benzamide
  • 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, propionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-
  • the compounds of the invention may in some cases be isolated as the hydrate. Hydrates are typically prepared by recrystallisation from an aqueous/organic solvent mixture using organic solvents such as dioxin, tetrahydrofuran or methanol. Hydrates can also be generated in situ by administration of the corresponding ketone to a patient.
  • the N-oxides of compounds of the invention can be prepared by methods known to those of ordinary skill in the art.
  • N-oxides can be prepared by treating an unoxidized form of the compound of the invention with an oxidizing agent (e.g., trifluoroperacetic acid, permaleic acid, perbenzoic acid, peracetic acid, meta- chloroperoxybenzoic acid, or the like) in a suitable inert organic solvent (e.g., a halogenated hydrocarbon such as dichloromethane) at approximately 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
  • N-oxides of the invention include those with the partial structures:
  • Compounds of the invention in unoxidized form can be prepared from N-oxides of the corresponding compounds of the invention by treating with a reducing agent (e.g., 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 80°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
  • radical positions on any molecular moiety used in the definitions may be anywhere on such moiety as long as it is chemically stable.
  • thiazolyl includes 4-subsituted thiazol-2-yl, 2- substituted thiazol-4-yl, 2-substituted thiazol-5-yl etc (in particular 4-subsituted thiazol- 2-yl or 2-substituted thiazol-4-yl, wherein the optional substituent is at the 5 position).
  • thiazol-4yl wherein the piperazine is substituted at the 2 position of the thiazole and the optional methyl or fluoro substituent is at the 5 position of the thiazole.
  • butyl includes t-butyl, i-butyl, n-butyl etc.
  • each definition is independent.
  • the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms, which said compound may possess. Said mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of said compound. All stereochemically isomeric forms of the compounds of the present invention both in pure form or mixed with each other are intended to be embraced within the scope of the present invention.
  • stereoisomerically pure concerns compounds or intermediates having a stereoisomeric excess of at least 80% (i.e. minimum 90% of one isomer and maximum 10% of the other possible isomers) up to a stereoisomeric excess of 100% (i.e.
  • Compounds of the invention can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomer. While resolution of enantiomers can be carried out using covalent diasteromeric derivatives of compounds of Formula II, 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 the invention in vivo.
  • the active agent While it is possible for the active agent to be administered alone, it is preferable to present it as part of a pharmaceutical formulation.
  • a pharmaceutical formulation will comprise the above defined active agent together with one or more acceptable carriers/excipients and optionally other therapeutic ingredients.
  • the carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.
  • the formulations include those suitable for rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, but preferably the formulation is an orally administered formulation.
  • the formulations may conveniently be presented in unit dosage form, e.g. tablets and sustained release capsules, and may be prepared by any methods well known in the art of pharmacy.
  • Such methods include the step of bringing into association the above defined active agent with the carrier.
  • the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • the invention extends to methods for preparing a pharmaceutical composition comprising bringing a compound of Formula 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, hydroxypropyl-methylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica.
  • Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring or the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may be optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.
  • compositions suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as 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 uM, more preferably 0.01-10 uM, such as 0.1-25 uM 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.
  • group SS is N 3 or a synthon therefor, such as OH or O protected by a conventional protecting group; the two Rb groups define a ketal, such as the bis methyl ketal or together define a cyclic ketal such as 1 ,3-dioxolane; and Rc is an hydroxy protecting group, or less commonly is H or represents the keto function of the end-product inhibitor in cases where the P1 building block as the ketone is elongated with P2 and P3.
  • WO05/066180 describes the preparation of intermediates towards the above P1 building block, including:
  • a synthon for the azide group is typically converted to the azide, prior to coupling with P2, for example by treatment with an azide salt, for example an alkali metal salt such as an aqueous solution of NaN 3 , of the P1 building block dissolved in a solvent such as DMF.
  • an azide salt for example an alkali metal salt such as an aqueous solution of NaN 3 , of the P1 building block dissolved in a solvent such as DMF.
  • P2 is typically an N-protected amino acid such as L-leucine, L-isoleucine, O-methyl-L- threonine, L-3-hydroxyvaline, 4-fluoroleucine or L-cyclohexylglycine
  • P3 typically comprises a capping group such as a benzoic acid derivative with the N-alkyl- piperazinyl-E moiety already introduced or provided with a synthon therefor in the para position.
  • the suitably protected individual building blocks can first be prepared and subsequently coupled together, preferably in the sequence P2+P1 ⁇ P2-P1 followed by N-alkylpiperazinyl-E-benzoic acid*+P2-P1 ⁇ N-alkylpiperazinyl-E-benzoate-P2-P1 , where * denotes an activated form, in order to minimise racemisation at P2.
  • Coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be carried out using standard coupling procedures such as the azide method, mixed carbonic-carboxylic acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active ester (p-nitrophenyl ester, N-hydroxysuccinic imido ester) method, Woodward reagent K-method, carbonyldiimidazole method, phosphorus reagents or oxidation-reduction methods. Some of these methods (especially the carbodiimide method) can be enhanced by adding 1-hydroxybenzotriazole or 4-DMAP. These coupling reactions can be performed in either solution (liquid phase) or solid phase.
  • the coupling step involves the dehydrative coupling of a free carboxyl of one reactant with the free amino group of the other reactant in the present of a coupling agent to form a linking amide bond.
  • coupling agents are found in general textbooks on peptide chemistry, for example, M. Bodanszky, "Peptide Chemistry", 2nd rev ed., Springer-Verlag, Berlin, Germany, (1993) hereafter simply referred to as Bodanszky, the contents of which are hereby incorporated by reference.
  • suitable coupling agents are N,N'-dicyclohexylcarbodiimide, 1- hydroxybenzotriazole in the presence of N, N'- dicyclohexylcarbodiimide or N-ethyl-N'-[ (3-dimethylamino) 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 A- DMAP.
  • Another practical and useful coupling agent is commercially available 2-(IH- benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate. Still another practical and useful coupling agent is commercially available O-(7-azabenzotriazol-1-yl)- N,N,N',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-methylpyrrolidine or 4-DMAP is added to maintain the reaction mixture at a pH of about 8.
  • the reaction temperature usually ranges between 0 °C and 50 °C and the reaction time usually ranges between 15 min and 24 h.
  • the functional groups of the constituent non-natural amino acids generally must be protected during the coupling reactions to avoid formation of undesired bonds.
  • the protecting groups that can be used are listed in Greene, "Protective Groups in Organic Chemistry", John Wiley & Sons, New York (1981 ) and "The Peptides: Analysis, Synthesis, Biology", Vol. 3, Academic Press, New York (1981), hereafter referred to simply as Greene, the disclosures of which are hereby incorporated by reference.
  • the alpha-carboxyl group of the C-terminal residue is usually protected as an ester that can be cleaved to give the carboxylic acid.
  • Protecting groups that can be used include 1 ) alkyl esters such as methyl, trimethylsilyl and t-butyl, 2) aralkyl esters such as benzyl and substituted benzyl, or 3) esters that can be cleaved by mild base or mild reductive means such as trichloroethyl and phenacyl esters.
  • the alpha-amino group of each amino acid to be coupled is typically 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 benzyloxycarbonyls, and 9-fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic carbamate groups such as tertbutyloxycarbonyl (Boc), ethoxycarbonyl, diisopropylmethoxy- carbonyl, 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 °C and room temperature usually 20-22 °C.
  • the first stage in a synthesis of compounds of the invention, such as those of the general formula Il is typically the preparation in solution of a functionalized P1 building block.
  • the P1 building block 14 was prepared as shown above and described in Example 1 or 9.
  • Compound 10 was prepared as described in WO05/066180 and the alcohol functionality oxidized with Dess-Martin Periodinane to provide ketone 9.
  • Treatment of the ketone 9 under the conditions described provided the dimethyl ketal intermediate 10'.
  • Hydrogenolysis of compound 10' removed both the Cbz and benzyl protecting groups to afford the amine 11.
  • Boc protection of the amine functionality of 11 provided the intermediate 12.
  • Displacement of the C-6 mesylate of 13 with sodium azide in DMF provided the required C-6 azide P1 14.
  • intermediate 14 is treated with acetyl chloride in methanol to remove the N-Boc protecting group.
  • azide treatment at elevated temperature directly leads to the removal of the Boc group so the deprotection step could be omitted.
  • the P2 residue is introduced via BocP2-OH using standard coupling conditions such as HATU, DIPEA in
  • Elongation is typically carried out in the presence of a suitable coupling agent e.g., benzothazole-1-yloxytrispyrrolidinophosphonium hexafluorophosphate (PyBOP), O- benzotriazol-l-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate (HBTU), O-(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,N-diisopropylethylamine, triethylamine, N-methyl
  • reaction solvents are inert organic solvents such as halogenated organic solvents (e.g., methylene chloride, chloroform, and the like), acetonitrile, N,N-dimethylformamide, ethereal solvents such as tetrahydrofuran, dioxane, and the like.
  • halogenated organic solvents e.g., methylene chloride, chloroform, and the like
  • acetonitrile e.g., N,N-dimethylformamide
  • ethereal solvents such as tetrahydrofuran, dioxane, and the like.
  • the above elongation coupling step can be carried out by first converting the P3/P2 building block into an active acid derivative such as succinimide ester and then reacting it with the 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, the reaction is carried out in the presence of a suitable base (e.g. triethylamine, diisopropylethylamine, pyridine, and the like).
  • Suitable reaction solvents are polar organic solvents such as acetonitrile, N,N-dimethylformamide, dichloromethane, or any suitable mixtures thereof.
  • L-leucine, L-isoleucine, L-cyclohexylglycine, O-methyl-L threonine and others are available commercially with a number of protecting group variants such as CBz, Boc or Fmoc.
  • Other variants of R 2 are easily prepared from commercially available starting materials.
  • R 2 is - C(CH 3 ) 2 OCH3
  • the P2 side chain alcohol can now be methylated using methyliodide under conventional sodium hydride, imidazole, THF conditions to obtain the desired P2 without substantial racemisation of the alpha centre.
  • This P2-P1 moiety can now be carried through the synthesis as described herein, namely CBz removal and coupling.
  • WO05/565299 describes the preparation of a gamma-fluoroleucine P2 building block.
  • An alternative synthsis of Fmoc and N-Boc-gammafluoroleucine building blocks is shown in Truong et al Syn. Lett. 2005 no 8 1278-1280.
  • the starting material methyl 4-acetylbenzoate
  • Bromination at the ⁇ -position to the ketone is achieved with bromine in acetic acid to provide the desired 4-(2-bromo-acetyl)-benzoic acid methyl ester.
  • 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, ⁇ -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-bromobenzy
  • Favoured N-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl (bz), t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz).
  • Hydroxy and/or carboxy protecting groups are also extensively reviewed in Greene ibid and include ethers such as methyl, substituted methyl ethers such as methoxymethyl, 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-ethoxymethyl, 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
  • 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.
  • Dess-Martin reagent (12.5 g, 30 mmol) was dissolved in DCM (250 ml).
  • Compound 10 from WO05/066180 (7.4 g, 20 mmol) in DCM (50 ml) was added to a stirred solution of oxidant at rt under a nitrogen atmosphere over 45 min. After an additional 90 min stirring the reaction was monitored by TLC (50:50 ethyl acetate:isohexane) Rf 0.21 for alcohol and 0.38 for ketone. Once the reaction was deemed to be complete by TLC, aqueous 10% Na2S2 ⁇ 3 (200 mL) was added and the mixture was stirred at rt for another 15 minutes.
  • Example 1 The product of Example 1 , compound 14, was dissolved in DCM (20 ml) and stirred at rt in presence of Boc-lle-OH (0.7g, 3 mmol), HOBt (0.6g, 4.5 mmol), WSCHCI (1.15g, 6 mmol, and NMM (0.71 ml, 6.5 mmol) for 16 h, diluted with EtOAc (50 ml), washed with aqueous citric acid (1 M, 30 ml), saturated NaHCO3 (30 ml) and brine (30 ml). The organic phase was dried over Na2SO4, filtered and the solvent was evaporated in vacuo, yielding a crude 7 as a clear semi solid (0.81 g,).
  • the resulting amine HCI salt (0.16 g) was treated with the HBr salt of 4-[2-(4-methyl-piperazin-1-yl)-thiazol- 4-yl ⁇ -benzoic acid (0.174 g, 0.37 mmol), HOBt (0.0.51g, 0.37 mmol), WSC HCI (0.11 g, 0.56 mmol) and NMM (0.082 ml, 0.74 mmol) in dry DMF (10 ml) and stirred at rt 2 hours. The mixture was diluted with EtOAc, organic phase was then washed once with saturated NaHCO ⁇ and once with 0.5 M NH3.
  • the C-3 alcohol of the P2-P1 building block of step a) was oxidized with Dess Martin periodinane to the ketone and the ketone protected as the dimethylketal.
  • the Cbz protecting group was then removed from the resulting N-[(S)-1-(3aS,6S,6aS)-(6- fluoro-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]- carbamic acid benzyl ester using standard procedures and the free amine coupled up to 4-(4-propyl-piperazin-1-yl)-benzoic acid.
  • the final step involved cleavage of the dimethylketal to obtain the desired ketone N-[(S)-1-((3aS,6S,6aS)-(6-Fluoro-3-oxo- hexahydrofuro[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-(4-propyl-piperazin-1-yl)- benzamide.
  • the P1 building block was synthesised as shown in WO02/05720, and coupled to N- protected L-leucine and the P3 building block of Example 8 below, and oxidised to the ketone as shown in Examples 2 & 3.
  • step b) 4-Propionylbenzoic acid
  • Methyl 4-propionylbenzoate (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 °C 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 Na 2 S 2 O 5 (20 ml_), saturated aqueous NaCI (20 ml_), and water (2OmL), dried and evaporated to give a yellow oil (1.025 g) that was used directly in the Hantzsch coupling. This material contained 91 % of the desired bromoketone, 5% starting ketone, and 4% 4-bromo-1-butanol, as determined by 1 H NMR.
  • Step h The P3 acid from step g is coupled to the P1-P2 building block from
  • Example 5 and oxidized to the ketone as shown above.
  • reaction mixture was then concentrated onto silica and flash chromatography of the residue using stepwise gradient elution (ethyl acetate in hexane, 20-30%) gave after concentration of the appropriate fractions a colorless syrup (2.03 g, 5.17 mmol, 96 %).
  • step a) To a stirred solution of the product of step a) (1.94 g, 4.94 mmol) in methanol (10 ml) at room temperature was added methanolic sodium methoxide (0.5 M, 10 ml). The reaction mixture was monitored by TLC (hexane-ethyl acetate 7:3 and 1 :1 , visualized by UV-light and staining using ammoniummolybdate-cerium sulfate in aq. 10% sulfuric acid). After 2 h the reaction mixture was neutralized by Dowex (50WX8-100, H + -form, pH monitored by pH-indicator strips), then filtered and concentrated.
  • Dowex 50WX8-100, H + -form, pH monitored by pH-indicator strips
  • the methyl ester (1.76 g, 5.25 mmol) from (d) was heated at 80 °C with 6M HCI (40 ml_) for 5.5 h. More 6M HCI (10 ml_) was added and the mixture was heated at 90 °C for 1 h 15 min. After cooling, the mixture was then evaporated under vacuum and freeze-dried from water to give the final product as cream solids in quantitative yield.
  • 6-Azido-3,3-dimethoxy-hexahydrofuro[3,2-b]pyrrole-4-carboxylic acid tert-butyl ester (256 mg, 0.81 mmol) was deprotected under acidic conditions (methanol/acetyl chloride) to give the crude 6-azido-3,3-dimethoxy-hexahydrofuro[3,2-b]pyrrole hydrochloride, which was coupled with N-Boc-cyclohexyl glycine using conventional DMF/DIPEA/HATU conditions, to give the title compound (315 mg, 85%). MS m/z 454.3(M+H) + .
  • Step b) N-[2-(6-Azido-3,3-dimethoxy -hexahvdrofuro[3,2-blpyrrol-4-yl)-1- cvclohexyl-2-oxo-ethyll-4-r2-(4-methyl-piperazin-1 -yl)-thiazol-4-yll-benzamide (11 b)
  • the tert-butyl ester group was removed from 6-azido-3,3-dimethoxy- hexahydrofuro[3,2-b]pyrrole-4-carboxylic acid tert-butyl ester (100 mg, 0.318 mmol) under acidic conditions.
  • the afforded crude 6-azido-3,3-dimethoxy-hexahydrofuro[3,2- b]pyrrole hydrochloride was coupled with N-Boc-leucine using conventional DMF/DIPEA/HATU conditions which gave the title compound (134 mg, 99%). MS m/z 428.1 (M+H) + .
  • Step b) N-[2-(6-Azido-3,3-dimethoxy-hexahvdrofuro[3,2-blpyrrole-4-carbonyl)-3- methylbutyll-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide (12b)
  • the tert-butyl ester group was removed from [2-(6-Azido-3,3-dimethoxy- hexahydrofuro ⁇ -blpyrrole ⁇ -carbonylJ-S-methylbutyll-carbamic acid tert-butyl ester (134 mg, 0.318 mmol) under acidic conditions (methanol/acetyl chloride).
  • the afforded crude hydrochloride intermediate was then coupled with the HCI salt of 4-[5-methyl-2- (4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzoic acid using conventional
  • Acetyl chloride (0.4 ml_) was added dropwise to a solution of compound 16a (69 mg, 0.154 mmol) in methanol (4 ml_) at 0 °C. The reaction mixture was stirred at rt over night and then concentrated. The residue was dissolved in 1 ,4-dioxane (5 ml) and freeze-dried over night. The solid was dissolved in DMF (4 ml_).
  • Step c) N-M -(6-Azido-3,3-dimethoxv-hexahvdro-furo[3,2-blpyrrole-4-carbonvn-3- methyl-butyl]-3-fluoro-4-[2-(4-methyl-4-oxy-piperazin-1-yl)-thiazol-4-yl]-benzamide
  • 6-Azido-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic acid tert-butyl ester (0.3 mmol) was dissolved in methanol (5 ml), the solution was cooled to 0 °C and acetyl chloride (0.5 ml) was added dropwise during 10 min. The temperature of the reaction mixture was allowed to raise till 22 °C whereafter the mixture was stirred for 6h. The solvent was evaporated and the residue was dissolved in 1 ,4-dioxane and then freeze-dried overnight.
  • Convenient assays for cathepsin K are carried out using human recombinant enzyme, such as that described in PDB.
  • the recombinant cathepsin K can be expressed in a variety of commercially available expression systems including E coli, Pichia and Baculovirus systems.
  • the purified enzyme is activated by removal of the prosequence by conventional methods.
  • Standard assay conditions for the determination of kinetic constants used a fluorogenic peptide substrate, typically H-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.
  • 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 100mM sodium acetate 1 mM EDTA, pH5.5)
  • the DMSO stock (1OmM in 100%DMSO) is diluted to 10% in assay buffer.
  • the compounds of formula Il are thus potent inhibitors of cathepsin K and yet selective over the closely related cathepsin S and L.
  • test compounds (2 ⁇ M) are incubated in pooled human liver cytosol (Xynotech LLC Lenexa US, 1 mg/mL protein in 0.1 M phosphate buffer, pH 7.4) at 37 degrees centigrade over a one hour period.
  • the incubations are initiated by the addition of 1mM NADPH co-factor.
  • Timed sub-samples were taken at 0, 20, 40 and 60 minutes and "crash precipitated" by the addition of 3 volumes ice-cold acetonitrile.
  • the samples were centrifuged at reduced temperature and the supernatants were separated and analyzed by LC-MS-MS
  • Comparative Example 1 employs the down F P1 unit of WO05/066180.
  • Comparative Example 2 employs the down isomer at C6 of the P1 unit of Example 1.
  • Comparative Example 3 employs the preferred P1 and P2 units of WO02/057270 together with a P3 unit within the scope of the present claims (which are outside the scope of WO02/057270).
  • Comparative Example 4 employs the down isomer at C6 of the P1 unit of Example 14.
  • Comparative Example 5 employs the down isomer at C6 of the P1 unit of Example 15.
  • Example 7 (invention) vs Comparative Example 1 and Example 6 (invention) vs Comparative Example 3, that the up-azido of the invention is substantially more metabolically stable than the down- fluoro P1 moiety favoured by prior art WO05/066180, or the unsubstituted P1 moieties favoured by WO02/057270.
  • Example 6 with Comparative Example 2, Example 14 with Comparative Example 4 and Example 15 with Comparative Example 5
  • the enhanced stability shown by the up-azido substituent of the invention is not shown by the otherwise identical compound with down azido at this position.
  • This experiment measures transport of inhibitors through the cells of the human gastroenteric canal.
  • the assay uses the well known Caco-2 cells with a passage number between 40 and 60.
  • the basolateral and the apical wells will contain 1.5 ml_ and 0.4 ml_ transport buffer (TB), respectively, and the standard concentration of the tested substances is 10 ⁇ M. Furthermore all test solutions and buffers will contain 1 % DMSO.
  • TB transport buffer
  • the transport plates Prior to the experiment the transport plates are pre-coated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material. After 21 to 28 days in culture on filter supports the cells are ready for permeability experiments.
  • Transport plate no 1 comprises 3 rows of 4 wells each. Row 1 is denoted Wash, row 2 "30 minutes” and row 3 "60 minutes”. Transport plate no 2 comprises 3 rows of 4 wells, one denoted row 4 "90 minutes”, row 5 "120 minutes and the remaining row unassigned.
  • the culture medium from the apical wells is removed and the inserts are transferred to a wash row (No. 1 ) in a transport plate (plate no.1) out of 2 plates without inserts, which have already been prepared with 1.5 ml_ transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5.
  • HBSS 1.5 ml_ transport buffer
  • the TB in basolateral well also contains 1 % Bovine Serum Albumin.
  • TEER Transepithelial electrical resistance value
  • the transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to the 30 minutes row (No. 2) and fresh 425 ⁇ l_ TB (pH 6.5), including the test substance is added to the apical (donor) well.
  • the plates are incubated in a polymix shaker at 37°C with a low shaking velocity of approximately 150 to 300 rpm.
  • 25 ⁇ l_ samples will be taken from the apical solution after ⁇ 2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.
  • 300 ⁇ l_ will be taken from the basolateral (receiver) wells at each scheduled time point and the post value of TEER is measured at the end the experiment.
  • acetonitrile will be added to a final concentration of 50% in the samples.
  • the collected samples will be stored at -20°C until analysis by HPLC or LC-MS.
  • the basolateral and the apical wells will contain 1.55 mL and 0.4 mL TB, respectively, and the standard concentration of the tested substances is 10 ⁇ M. Furthermore all test solutions and buffers will contain 1 % DMSO.
  • the transport plates Prior to the experiment the transport plates are precoated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material. After 21 to 28 days in culture on filter supports the cells are ready for permeability experiments. The culture medium from the apical wells are removed and the inserts are transferred to a wash row (No.1 ) in a new plate without inserts (Transport plate).
  • the transport plate comprises 3 rows of 4 wells. Row 1 is denoted “wash” and row 3 is the "experimental row”.
  • the transport plate has previously been prepared with 1.5 ml_ TB (pH 7.4) in wash row No. 1 and with 1.55 ml_ TB (pH 7.4), including the test substance, in experimental row No. 3 (donor side).
  • 0.5 ml_ transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts in row No. 1 and the cell monolayers are equilibrated in the transport buffer system for 30 minutes, 37 °C in a polymix shaker. After being equilibrated to the buffer system the TEER value is measured in each well by an EVOM chop stick instrument.
  • the transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to row 3 and 400 ⁇ l_ fresh TB, pH 6.5 is added to the inserts.
  • 250 ⁇ l_ is withdrawn from the apical (receiver) well and replaced by fresh transport buffer.
  • 250 ⁇ l_ samples will be withdrawn and replaced by fresh transport buffer every 30 minutes until the end of the experiment at 120 minutes, and finally a post value of TEER is measured at the end of the experiment.
  • a 25 ⁇ l_ samples will be taken from the basolateral (donor) compartment after ⁇ 2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.
  • acetonitrile will be added to a final concentration of 50% in the samples.
  • the collected samples will be stored at -20°C until analysis by HPLC or LC- MS.
  • FA cum Determination of the cumulative fraction absorbed, FA cum , versus time. FA cum is calculated from:
  • k is the transport rate (min ' i ) defined as the slope obtained by linear regression of cumulative fraction absorbed (FA cum ) as a function of time (min)
  • VR is the volume in the receiver chamber (ml_)
  • A is the area of the filter (cm ⁇ ).
  • fluorination of the benzyl ring or thiazolyl ring typically improves permeability.
  • a fluorine substituent at the meta position of the benzyl ring relative to the amide bond or on the thiazolyl ring can approximately double the compound's cellular permeability relative to the corresponding compound without fluorine substitution (Example 14 vs Example 6, or Example 15 vs Example 6).
  • Greater permeability through the gastrointestinal tissue is advantageous in that it allows for the use of a smaller dose to achieve similar levels of exposure to a less permeable compound administered in a higher dose.
  • a low dose is advantageous in that minimises the cost of goods for a daily dose, which is a crucial parameter in a drug which is taken for protracted time periods.
  • Fluorinated compounds are further believed to produce an advantageous metabolism profile. Less metabolism is observed for the meta-fluorinated benzyl ring analogue than the unsubstituted analogue in pooled, cryopreserved human hepatocytes where the intrinsic clearance for the meta-fluorinated benzyl ring is approximately half that of the unsubstituted analogue.
  • the metabolic processes associated with particular compounds of the invention have been analysed using a Waters Synapt High Definition Mass Spectroscopy Instrument (HDMS).
  • HDMS Waters Synapt High Definition Mass Spectroscopy Instrument
  • an intact ketone on the furopyrrole ring is important for activity against cathepsin K, as this moiety interacts with the catalytic cysteine of the enzyme.
  • compounds which lack a fluorine on the benzyl ring are subject to the normal hepatic and/or other in vivo metabolic processes, (as determined in commercially available liver microsomes, S9 fraction and/or clinical plasma samples) a large proportion of the resulting metabolites are no longer active due to the ketone being reduced.
  • compounds with a benzylic fluorine tend to produce a higher proportion of active metabolites, such as the corresponding piperazine N-oxide (such as shown in Example 17) with an intact ketone on the furopyrrole ring.
  • active metabolites such as the corresponding piperazine N-oxide (such as shown in Example 17) with an intact ketone on the furopyrrole ring.
  • Such an active metabolite can thus assist in maintaining inhibitory pressure on cathepsin K, notwithstanding partial metabolism of the compound.

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Abstract

Compounds of the formula (II): wherein R2 is the side chain of leucine, isoleucine, cyclohexylglycine, O-methyl threonine, 4-fluoroleucine or 3-methoxyvaline; R3 is H, methyl or fluoro; R4 is C1-C6alkyl; E is a bond or thiazolyl, optionally substituted with methyl or fluoro; n is 0 or 1; or a pharmaceutically acceptable salt, N-oxide or hydrate thereof; have utility in the treatment of disorders characterized by inappropriate expression or activation of cathepsinK, such as osteoporosis, osteoarthritis, rheumatoid arthritis or bone metastases.

Description

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, Cήthidia 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 cartilage or matrix degradation, such as osteoarthritis and rheumatoid arthritis.
It is likely that treatment of bone and cartilage disorders such as osteoarthritis and osteoporosis will require life-long administration of a cathepsin K inhibitor, often to a patient population within or nearing the geriatric phase. This places unusually high requirements on the ease of administration of drugs intended for such disorders. For example attempts are underway to stretch the dosage regimes of the current osteoporosis drugs of the bisphosphonate class to weekly or longer administration regimes to aid compliance. However even with improved dosing, other side effects of the bisphosphonates, remain. Bisphosphonates block bone turnover rather than attenuate it as a cathepsin K inhibitor does. For healthy bone it is important to maintain the remodelling process which bisphosphonates block completely. In addition, bisphosphonates have a very long half-life in bone so if effects such as osteonecrosis of the jaw manifest themselves, it is impossible to remove the bisphosphonate from the bone. In contrast, cathepsin K inhibitors typically have a fast onset and off rate mode of action, which means that if a problem was to be identified, dosing could be halted and there would be no build up of the inhibitor in the bone matrix.
There is thus a desire for alternative osteoporosis and osteoarthritis drugs with superior pharmacokinetic and/or pharmacodynamic properties.
International patent application no WO02/057270 discloses compounds of the formula
Figure imgf000003_0001
where UVWXY broadly corresponds to the P3 and P2 (these expressions are explained below) of dipeptide cysteine protease inhibitors, Z is inter alia O, S, methylene or -NR-, R'i is alkyl, alkylaryl etc and Pi and Q are each, inter alia, methylene. Although the generic disclosure in this patent application postulates a very broad range of substituents on Pi and Q, none are individualised or exemplified and no guidance is provided on their synthesis. Indeed - the only synthesis suggestions provided in WO02/05720 do not allow for substitution at Pi or Q at all. The compounds are alleged to be useful, inter alia, for the treatment of protozoal infections such as trypanosomes.
International patent application no WO2005/066180 discloses, inter alia, compounds of the formula:
Figure imgf000004_0001
where "cap" defines a diverse variety of cyclic structures. The compounds are active inhibitors of cathepsin K, but, as shown below, further modification of the structure yields improvements as regards to pharmacokinetics and/or pharmacodynamics.
There remains a need in the art for potent inhibitors of cathepsin K. Of particular benefit are inbibitors of cathepsin K which show selectivity for cathepsin K over other cathepsins (e.g. selectivity over cathepsin S and/or cathepsin L). Potent inhibitors of cathepsin K which demonstrate properties such as high permeability and/or advantageous metabolic profiles may be expected to be of great value in a clinical setting.
Brief description of the invention
According to the present invention, there is provided a compound of formula II:
Figure imgf000004_0002
wherein
R2 is the side chain of leucine, isoleucine, cyclohexylglycine, O-methyl threonine, 4-fluoroleucine or 3-methoxyvaline;
R ,33 is H, methyl or fluoro;
R4 is C1-C6 alkyl; E is a bond or thiazolyl, optionally substituted with methyl or fluoro; n is 0 or 1 ; or a pharmaceutically acceptable salt, N-oxide or hydrate thereof (collectively referred to herein as compounds of the invention).
It will be appreciated that the compounds of the invention can exist as hydrates, such as those of the partial formulae:
Figure imgf000005_0001
and the invention extends to all such alternative forms.
In one embodiment of the invention R2 represents the side chain of leucine. In a second embodiment of the invention R2 represents the side chain of isoleucine. In a third embodiment of the invention R2 represents the side chain of cyclohexylglycine. In a fourth embodiment of the invention R2 represents the side chain of O-methyl threonine. In a fifth embodiment of the invention R2 represents the side chain of 4- fluoroleucine. In a sixth embodiment of the invention R2 represents the side chain of 3- methoxyvaline.
Currently preferred values of R2 include those embodied by the partial structures:
Figure imgf000005_0002
and especially
Figure imgf000005_0003
An alternative group of preferred R moieties are those represented by the partial structures:
Figure imgf000006_0001
Another group of preferred R2 moieties are the side chains of O-methyl threonine, 4- fluoroleucine and 3-methoxyvaline.
In one embodiment of the invention n represents 0. Of particular interest are compounds wherein n represents 1.
R3 suitably represents methyl or fluoro, especially fluoro.
When n represents 1 , R3 is suitably positioned as shown by the partial structure:
Figure imgf000006_0002
The C1-C6 alkyl definition of R is is meant to include both branched and unbranched alkyl moieties containing between one and six carbon atoms in total. Examples of R4 groups are methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, tert- butyl and sec-butyl), pentyl (n-pentyl, isopentyl etc) and hexyl (n-hexyl etc). Suitably R4 represents C1-C4 alkyl (such as C1-C3 alkyl). One R4 group of particular interest is methyl. A second R4 group of particular interest is propyl (especially n-propyl).
In some embodiments of the invention E is a bond. Currently preferred compounds within this embodiment include:
Figure imgf000007_0001
where R2 and R3 are as defined above. Representative examples within this embodiment include:
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4- (4-propyl-piperazin-1 -yl)-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-
4-(4-propyl-piperazin-1-yl)-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-
(4-propyl-piperazin-1-yl)-benzamide; N-[1 -(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methoxy-propyl]-
4-(4-propyl-piperazin-1-yl)-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methoxy-propyl]-
4-(4-propyl-piperazin-1-yl)-benzamide; and pharmaceutically acceptable salts, hydrates and N-oxides thereof.
In other embodiments, E is thiazolyl which is optionally substituted with methyl or fluoro. Preferred compounds within this embodiment include those of the formula:
Figure imgf000007_0002
where R2 and the optional substituent R3 are as defined above and R5 is H, methyl or fluoro. Representative examples within this embodiment include:
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-
[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide; N-[1 -(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1 -cyclohexyl-2-oxo-ethyl]-
4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-
[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methoxy-propyl]- 4-[2-(4-methyl-piperazin-1 -yl)-thiazol-4-yl]-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methoxy-2- methyl-propyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-
[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide; N-[1 -(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1 -cyclohexyl-2-oxo-ethyl]-
4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-
[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methoxy-propyl]- 4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methoxy-2- methyl-propyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-
[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide; N-[1 -(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1 -cyclohexyl-2-oxo-ethyl]-
4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-
[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methoxy-propyl]- 4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
N-[1-(6-azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methoxy-2- methyl-propyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide; and pharmaceutically acceptable salts, N-oxides and hydrates thereof. In one embodiment of the invention E represents thiazolyl which is unsubstituted. In an embodiment of particular interest E represents thiazolyl which is substituted with methyl or fluoro, in particular fluoro. Alternatively E represents thiazolyl which is unsubstituted or substituted by methyl.
Of particular interest are compounds where E represents thiazolyl which is positioned as shown by the partial structure:
Figure imgf000009_0001
wherein R5 is the location of the optional substitution (i.e. R5 represents H, methyl or fluoro).
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 cartilage or matrix degradation (such as osteoarthritis and rheumatoid arthritis), bone cancers including neoplasia, pain (especially chronic pain).
Additionally provided is a method for the treatment or prevention of a disorder mediated by cathepsin K comprising the administration of a safe and effective amount of a compound of the invention for the purpose of treating or preventing said disorder which is mediated by cathepsin K.
Also provided is a compound of the invention for the treatment or prevention of a disorder mediated by cathepsin K.
Further, there is provided as an aspect of the invention novel intermediates (as described herein) which may be of use in the preparation of the compounds of the invention.
In particular there is provided a compound of the formula:
Figure imgf000010_0001
or an N-protected derivative thereof (e.g. Boc-protected). Also provided by the invention is the corresponding 1 ,3-dioxolane protected analogue and N-protected derivatives thereof (e.g. Boc-protected).
Also provided is a compound of the formula:
Figure imgf000010_0002
wherein R4 represents Chalky! (such as methyl); or a salt or lower alkyl (e.g. Chalky!) ester thereof, such as the methyl ester or HCI salt.
In certain embodiments, the invention does not embrace subject matter which is described in international patent application WO2008/007107. For example, the compounds of the invention do not embrace the compounds described on page 37 line 8 to page 38 line 6 of WO2008/007107: N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-(2-(4-methylpiperazin- 1 -yl)thiazol-4-yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(2-(4-methylpiperazin- 1 -yl)thiazol-4-yl)benzamide
N-((S)- 1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3 ,3 -dimethyl- 1 -oxobutan-2-yl)-4-(2-(4-methylpiperazin- 1 -yl)thiazol-4-yI)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-
4(5H,6H, 6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-(2-(4-methylpiperazin- 1 -yl)thiazol-4- yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6ciH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(2-(4-methylpiperazin- 1 -yl)thiazol-4-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(2-(4-methylpiperazin- 1 -yl)thiazol-4-yl)benzamide
N-( 1 -((3aS,6R,6aS)-6-azido-3-oxohexahydro-2H-furo[3,2-b]pyrrole-4- carbonyl)cyclohexyl)-4-(2-(4-methylpiperazin-1-yl)thiazol-4-yl)benzamide
N-((S)- 1 -((3 aS,6R,6aS)-6-azido-3 -oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-(2-(4-(2-methoxyethyl)piperazin- 1 -yl)thiazol-4- yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(2-(4-(2-methoxyethyl)piperazin- 1 -yl)thiazol-4- yl)benzamide pharmaceutically acceptable salts, Ν-oxides or hydrates thereof are also excluded. In certain embodiments, the invention does not embrace subject matter which is described in international patent application WO2008/007114. For example, the compounds of the invention do not embrace the compounds described on page 70 line 6 to page 77 line 8 of WO2008/007114:
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(4-methylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(4-ethylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(4-propy lpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(4-isopropylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(4-(2-methoxyethyl)piperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)- 4-(4-cyclopropylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)- 4-(4-cyclobutylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3 -oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-( 1 -propylpiperidin-4-yl)benzamide N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-( 1 -isopropy lpiperidin-4-yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-( 1 -(2-methoxyethyl)piperidin-4-y l)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-( 1 -cyclopropylpiperidin-4-yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan~2-yl)-4-( 1 -cyclobutylpiperidin-4-y l)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyiτol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-(4-methylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methy 1- 1 -oxopentan-2-y l)-4-(4-ethy lpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-y l)-4-(4-propylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl-l -oxopentan-2-y l)-4-(4-isopropy lpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-(4-(2-methoxyethyl)piperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-y l)-4-(4-cyclopropy lpiperazin- 1 -yl)benzamide N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-(4-cyclobutylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-( 1 -propylpiperidin-4-yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-(l -cyclopropylpiperidin-4-yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-fiiro[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-( 1 -cyclobutylpiperidin-4-yl)benzamide
N-((2S,3S)- 1 -((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yI)-4-(4-methylpiperazin- 1 -yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3-methyl- 1 -oxopentan-2-yI)-4-(4-ethylpiperazin- 1 -yl)benzamide
N-((2S,3S)- 1 -((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3-methyl-1-oxopentan-2-yl)-4-(4-propylpiperazin-1-yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)-3-methyl- 1 -oxopentan-2-yl)-4-(4-isopropylpiperazin- 1 - yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3-methyl-1-oxopentan-2-yl)-4-(4-(2-methoxyethyl)piperazin-1- yl)benzamide N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-(4-cyclopropylpiperazin- 1 - yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-(4-cyclobutylpiperazin- 1 - yl)benzamide
N-((2S,3S)- 1 -((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3-methyl-1-oxopentan-2-yl)-4-(l-methylpiperidin-4-yl)benzamide
N-((2S,3S)-\-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-(l -ethylpiperidin-4-yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3-methyl- 1 -oxopentan-2-yl)-4-(l -propylpiperidin-4-yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-(l -isopropylpiperidin-4-yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- l-oxoρentan-2-yl)-4-(l-(2-methoxyethyl)piperidin-4- yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-( 1 -cyclopropy lpiperidin-4- yl)benzamide
N-((2S,3S)- 1 -((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-( 1 -cyclobutylpiperidin-4- yl)benzamide N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyvrol-4(5H,6H,6aH)-yl)- 3,3 -dimethyl- 1 -oxobutan-2-yl)-4-(4-methylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3 ,3 -dimethyl- 1 -oxobutan-2-yl)-4-(4-ethylpiperazin- 1 -yl)benzamide
N-((S)- 1 -((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3 ,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3 ,3 -dimethyl- 1 -oxobutan-2-yl)-4-(4-propylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3,3-dimethyl- 1 -oxobutan-2-yl)-4-(4-isopropylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-fiiro[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3 ,3 -dimethyl- 1 -oxobutan-2-yl)-4-(4-(2-methoxyethy l)piperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-S-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3 ,3 -dimethyl- 1 -oxobutan-2-yl)-4-(4-cyclopropylpiperazin- 1 -yl)benzamide
N-((.S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3,3-dimethyl- 1 -oxobutan-2-yl)-4-(4-cyclobutylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3 ,3 -dimethyl- 1 -oxobutan-2-yl)-4-( 1 -propy lpiperidin-4-y l)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3,3-dimethyl- 1 -oxobutan-2-yl)-4-(l -cyclopropylpiperidin-4-yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3,3-dimethyl- 1 -oxobutan-2-yl)-4-(l -cyclobutylpiperidin-4-yl)benzamide N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)-1-cyclopentyl-2-oxoethyl)-4-(4-methylpiperazin-1-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1-cyclopentyl-2-oxoethyl)-4-(4-ethylpiperazin-1-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(4-propylpiperazin- 1 -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(4-isopropyIpiperazin- 1 -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(4-(2-methoxyethyl)piperazin- 1 -y l)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(4-cyclopropylpiperazin- 1 -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(4-cyclobutylpiperazin- 1 -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-( 1 -propylpiperidin-4-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopenty l-2-oxoethyl)-4-( 1 -cyclopropylpiperidin-4-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(l -cyclobutylpiperidin-4-yl)benzamide N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(4-methylpiperazin- 1 -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(4-ethylpiperazin- 1 -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(4-propylpiperazin- 1 -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(4-isopropylpiperazin- 1 -yl)benzamide
N-((,S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(4-(2-methoxyethyl)piperazin- 1 -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(4-cyclopropylpiperazin- 1 -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(4-cyclobutylpiperazin- 1 -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(l -methylpiperidin-4-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-( 1 -ethylpiperidin-4-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(l -propylpiperidin-4-yl)benzamide N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(l-isopropylpiperidin-4-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(1-(2-methoxyethyl)piperidin-4-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(1-cyclopropylpiperidin-4-yl)benzamide
N-((S)- 2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(1-cyclobutylpiperidin-4-yl)benzamide
pharmaceutically acceptable salts, Ν-oxides or hydrates thereof are also excluded.
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, propionate, 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 the invention may in some cases be isolated as the hydrate. Hydrates are typically prepared by recrystallisation from an aqueous/organic solvent mixture using organic solvents such as dioxin, tetrahydrofuran or methanol. Hydrates can also be generated in situ by administration of the corresponding ketone to a patient. The N-oxides of compounds of the invention 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 the invention with an oxidizing agent (e.g., trifluoroperacetic acid, permaleic acid, perbenzoic acid, peracetic acid, meta- chloroperoxybenzoic acid, or the like) in a suitable inert organic solvent (e.g., a halogenated hydrocarbon such as dichloromethane) at approximately 0°C. Alternatively, the N-oxides of the compounds of the invention can be prepared from the N-oxide of an appropriate starting material.
Examples of N-oxides of the invention include those with the partial structures:
Figure imgf000020_0001
Compounds of the invention in unoxidized form can be prepared from N-oxides of the corresponding compounds of the invention by treating with a reducing agent (e.g., 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 80°C.
It should be noted that the radical positions on any molecular moiety used in the definitions may be anywhere on such moiety as long as it is chemically stable.
Radicals used in the definitions of the variables include all possible isomers unless otherwise indicated. For instance thiazolyl includes 4-subsituted thiazol-2-yl, 2- substituted thiazol-4-yl, 2-substituted thiazol-5-yl etc (in particular 4-subsituted thiazol- 2-yl or 2-substituted thiazol-4-yl, wherein the optional substituent is at the 5 position). Preferred is thiazol-4yl, wherein the piperazine is substituted at the 2 position of the thiazole and the optional methyl or fluoro substituent is at the 5 position of the thiazole. Similarly butyl includes t-butyl, i-butyl, n-butyl etc.
When any variable occurs more than one time in any constituent, each definition is independent. Unless otherwise mentioned or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms, which said compound may possess. Said mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of said compound. All stereochemically isomeric forms of the compounds of the present invention both in pure form or mixed with each other are intended to be embraced within the scope of the present invention.
Pure stereoisomeric forms of the compounds and intermediates as mentioned herein are defined as isomers substantially free of other enantiomeric or diastereomeric forms of the same basic molecular structure of said compounds or intermediates. In particular, the term "stereoisomerically pure" concerns compounds or intermediates having a stereoisomeric excess of at least 80% (i.e. minimum 90% of one isomer and maximum 10% of the other possible isomers) up to a stereoisomeric excess of 100% (i.e. 100% of one isomer and none of the other), more in particular, compounds or intermediates having a stereoisomeric excess of 90% up to 100%, even more in particular having a stereoisomeric excess of 94% up to 100% and most in particular having a stereoisomeric excess of 97% up to 100%. The terms "enantiomerically pure" and "diastereomerically pure" should be understood in a similar way, but then having regard to the enantiomeric excess, and the diastereomeric excess, respectively, of the mixture in question.
Compounds of the invention can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomer. While resolution of enantiomers can be carried out using covalent diasteromeric derivatives of compounds of Formula II, 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 the invention 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, hydroxypropyl-methylcellulose, 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 uM, more preferably 0.01-10 uM, such as 0.1-25 uM 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 have the formula:
Figure imgf000024_0001
wherein group SS is N3 or a synthon therefor, such as OH or O protected by a conventional protecting group; the two Rb groups define a ketal, such as the bis methyl ketal or together define a cyclic ketal such as 1 ,3-dioxolane; and Rc is an hydroxy protecting group, or less commonly is H or represents the keto function of the end-product inhibitor in cases where the P1 building block as the ketone is elongated with P2 and P3.
WO05/066180 describes the preparation of intermediates towards the above P1 building block, including:
Figure imgf000024_0002
A synthon for the azide group is typically converted to the azide, prior to coupling with P2, for example by treatment with an azide salt, for example an alkali metal salt such as an aqueous solution of NaN3, of the P1 building block dissolved in a solvent such as DMF.
P2 is typically an N-protected amino acid such as L-leucine, L-isoleucine, O-methyl-L- threonine, L-3-hydroxyvaline, 4-fluoroleucine or L-cyclohexylglycine, and P3 typically comprises a capping group such as a benzoic acid derivative with the N-alkyl- piperazinyl-E moiety already introduced or provided with a synthon therefor in the para position.
The suitably protected individual building blocks can first be prepared and subsequently coupled together, preferably in the sequence P2+P1→ P2-P1 followed by N-alkylpiperazinyl-E-benzoic acid*+P2-P1→ N-alkylpiperazinyl-E-benzoate-P2-P1 , where * denotes an activated form, in order to minimise racemisation at P2.
Coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be carried out using standard coupling procedures such as the azide method, mixed carbonic-carboxylic acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active ester (p-nitrophenyl 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 N, N'- dicyclohexylcarbodiimide or N-ethyl-N'-[ (3-dimethylamino) 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 A- DMAP. Another practical and useful coupling agent is commercially available 2-(IH- benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate. Still another practical and useful coupling agent is commercially available O-(7-azabenzotriazol-1-yl)- N,N,N',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-methylpyrrolidine or 4-DMAP is added to maintain the reaction mixture at a pH of about 8. The reaction temperature usually ranges between 0 °C and 50 °C and the reaction time usually ranges between 15 min and 24 h.
The functional groups of the constituent non-natural amino acids generally must be protected during the coupling reactions to avoid formation of undesired bonds. The protecting groups that can be used are listed in Greene, "Protective Groups in Organic Chemistry", John Wiley & Sons, New York (1981 ) and "The Peptides: Analysis, Synthesis, Biology", Vol. 3, Academic Press, New York (1981), hereafter referred to simply as Greene, the disclosures of which are hereby incorporated by reference.
The alpha-carboxyl group of the C-terminal residue is usually protected as an ester that can be cleaved to give the carboxylic acid. Protecting groups that can be used include 1 ) alkyl esters such as methyl, trimethylsilyl and t-butyl, 2) aralkyl esters such as benzyl and substituted benzyl, or 3) esters that can be cleaved by mild base or mild reductive means such as trichloroethyl and phenacyl esters.
The alpha-amino group of each amino acid to be coupled is typically 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 benzyloxycarbonyls, and 9-fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic carbamate groups such as tertbutyloxycarbonyl (Boc), ethoxycarbonyl, diisopropylmethoxy- carbonyl, 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 as phenylthiocarbonyl and dithiasuccinoyl. 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 °C and room temperature usually 20-22 °C.
Once the inhibitor sequence is completed any remaining 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 invention, such as those of the general formula Il is typically the preparation in solution of a functionalized P1 building block.
Figure imgf000027_0001
i. Dess-Martin Periodinane, DCM, 2 h, RT; ii. Trimethylorthoformate, pTsOH, MeOH, 8 h, 60 °C; UiPd(OH)2, H2, MeOH, 48 h, RT; iv. BoC2O, 10% Na2CO3, 16 h, O °C to RT; v. methanesulfonyl chloride, DIPEA, DCM, 2 h, O °C; vi. NaN3, DMF, 2 x 1 h, 130 °C in microwave
The P1 building block 14 was prepared as shown above and described in Example 1 or 9. Compound 10 was prepared as described in WO05/066180 and the alcohol functionality oxidized with Dess-Martin Periodinane to provide ketone 9. Treatment of the ketone 9 under the conditions described provided the dimethyl ketal intermediate 10'. Hydrogenolysis of compound 10' removed both the Cbz and benzyl protecting groups to afford the amine 11. Boc protection of the amine functionality of 11 , provided the intermediate 12. Treatment of the C-6 alcohol of compound 12 with methane sulphonyl chloride and base, provided the mesylate 13. Displacement of the C-6 mesylate of 13 with sodium azide in DMF provided the required C-6 azide P1 14.
Typically to get to the final compound, intermediate 14 is treated with acetyl chloride in methanol to remove the N-Boc protecting group. (In some cases, azide treatment at elevated temperature directly leads to the removal of the Boc group so the deprotection step could be omitted). With the subsequent free amine, the P2 residue is introduced via BocP2-OH using standard coupling conditions such as HATU, DIPEA in
DMF. The terminal Boc protection is again removed with acetyl chloride in methanol and the P3 residue introduced via P3-OH using standard coupling conditions such as
HATU, DIPEA in DMF. Finally the dimethylketal protection is removed with TFA to afford the required final compound.
Elongation is typically carried out in the presence of a suitable coupling agent e.g., benzothazole-1-yloxytrispyrrolidinophosphonium hexafluorophosphate (PyBOP), O- benzotriazol-l-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate (HBTU), O-(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,N-diisopropylethylamine, triethylamine, N-methylmorpholine, and the like. The reaction is typically carried out at 20 to 30 °C, preferably at about 25 °C, 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, N,N-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, the reaction is carried out in the presence of a suitable base (e.g. triethylamine, diisopropylethylamine, pyridine, and the like). Suitable reaction solvents are polar organic solvents such as acetonitrile, N,N-dimethylformamide, dichloromethane, or any suitable mixtures thereof.
P2 building blocks in the form of N-protected L-amino acids are readily available commercially, for example L-leucine, L-isoleucine, L-cyclohexylglycine, O-methyl-L threonine and others are available commercially with a number of protecting group variants such as CBz, Boc or Fmoc. Other variants of R2 are easily prepared from commercially available starting materials. For example compounds wherein R2 is - C(CH3)2OCH3 can be prepared by reacting CBz protected (S)-(+)-2-amino-3-hydroxy- 3-methylbutanoic acid with 3,3-dimethoxy-hexahydro-furo(3,2b)pyrrole to form the desired P2-P1 unit. The P2 side chain alcohol can now be methylated using methyliodide under conventional sodium hydride, imidazole, THF conditions to obtain the desired P2 without substantial racemisation of the alpha centre. This P2-P1 moiety can now be carried through the synthesis as described herein, namely CBz removal and coupling.
WO05/565299 describes the preparation of a gamma-fluoroleucine P2 building block. An alternative synthsis of Fmoc and N-Boc-gammafluoroleucine building blocks is shown in Truong et al Syn. Lett. 2005 no 8 1278-1280.
The preparation of P3 building blocks wherein E is a bond, thiazolyl or substituted thiazolyl, and or the benzoic acid moiety is substituted with R3 are described in WO05/066180 or readily prepared by analogous methods. For example, Scheme E below shows the preparation of a P3 building block wherein E is a fluoro-substituted thiazolyl:
Figure imgf000030_0001
i. HOAc, Br2, RT, 2h, 55% yield; ii. KF, 18-crown-6, CH3CN, 90 °C, 16 h, 31 % yield; iii. HOAc, Br2, 45 °C, 4 h, 100% yield ; iv. 4-methylpiperazine-1-carbothioamide, ethanol, 70 °C, 2 h, 74% yield, v. LiOH, THF, H2O, RT, 16 h, 79 % yield.
Scheme E Synthesis of 4-[5-fluoro-2-(4-methyl-piperazin-1 -yl)-thiazol-4-yl]-benzoic acid
The starting material, methyl 4-acetylbenzoate, is commercially available. Bromination at the α-position to the ketone is achieved with bromine in acetic acid to provide the desired 4-(2-bromo-acetyl)-benzoic acid methyl ester. Subsequent treatment of 4-(2- bromo-acetyl)-benzoic acid methyl ester with potassium fluoride in the presence of 18- crown-6 at 90 °C, provides 4-(2-fluoro-acetyl)-benzoic acid methyl ester after column chromatography. Repeated bromination at the α-position to the ketone is achieved with bromine in acetic acid to provide the desired 4-(2-bromo-2-fluoro-acetyl)-benzoic acid methyl ester. Formation of the thiazole is typically carried out by heating 4-(2-bromo-2- fluoro-acetyl)-benzoic acid methyl ester with 4-methylpiperazine-1-carbothioamide at 70° C for 2 hours. On cooling, the desired 4-[5-fluoro-2-(4-methyl-piperazin-1-yl)- thiazol-4-yl]-benzoic acid methyl ester precipitates out. Deprotection of the methyl ester is carried out using a lithium hydroxide solution and the desired acid, 4-[5-fluoro- 2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzoic acid is generally obtained in good yield as the dihydrochloride salt on workup with hydrochloric acid.
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, A- nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like; alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. Favoured N-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl (bz), t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz).
Hydroxy and/or carboxy protecting groups are also extensively reviewed in Greene ibid and include ethers such as methyl, substituted methyl ethers such as methoxymethyl, 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-ethoxymethyl, 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
Preparation of P1 buildinp block (3aS, 6aS)-6R-azido-3,3-dimethoxy-hexahvdro- furof3,2-blpyrrole-4-carboxylic acid tert-butyl ester 14
Figure imgf000032_0001
Preparation of (3as, 6aS)-6R-benzyloxy-3-oxo-hexahvdro-furor3,2-blpyrrole-4- carboxylic acid benzyl ester 9
Figure imgf000032_0002
Dess-Martin reagent (12.5 g, 30 mmol) was dissolved in DCM (250 ml). Compound 10 from WO05/066180 (7.4 g, 20 mmol) in DCM (50 ml) was added to a stirred solution of oxidant at rt under a nitrogen atmosphere over 45 min. After an additional 90 min stirring the reaction was monitored by TLC (50:50 ethyl acetate:isohexane) Rf 0.21 for alcohol and 0.38 for ketone. Once the reaction was deemed to be complete by TLC, aqueous 10% Na2S2θ3 (200 mL) was added and the mixture was stirred at rt for another 15 minutes. The two phase system was transferred into a separation funnel and extracted twice with EtOAc (200 mL and 100 ml respectively). The combined organic phases were washed once with aqueous saturated NaHCθ3 (100 ml_) and brine (100 ml_), dried over Na2SO4, filtered and the solvent was evaporated in vacuo, yielding the crude product 9 as a clear oil (7.69 g,); ESI+, m/z: 368 (M+ +1 ).
Preparation of (3aS,6aS)-6R-benzyloxy-3,3-dimethoxy-hexahvdro-furor3,2-blpyrrole-4- carboxylic acid benzyl ester 10'
Figure imgf000033_0001
Compound 9 (7.6 g) was dissolved in dry methanol (100 ml). Trimethyl orthoformate (30 ml) and pTsOH (0.2 g) was added at rt under a nitrogen atmosphere. The mixture was heated at 60 °C for 8 hours. The reaction was monitored by TLC (50:50 ethyl acetate:isohexane) Rf 0.38 for ketone and Rf 0.66 for ketal. Once the reaction was deemed to have reached completion, it was cooled to rt and concentrated in vacuo. The crude product was purified by column chromatography over silica gel eluting with ethyl acetate-heptane (1 :4) to ketal 10' as a clear oil (5.9 g, 71 % over 2 steps); ESI+, m/z: 382 (M+ -OMe).
Preparation of (3aS,6aS)-3,3-dimethoxy-hexahvdro-furo[3,2-b] pyrrol-6R-ol 11
Figure imgf000033_0002
A solution of compound 10' (2.5 g, 6.4 mmol) in methanol (60 ml) and Pd(OH)2 (0.7 g) was stirred at rt under H2 atmosphere for 48 hours. The reaction was monitored by TLC (50:50 ethyl acetate:isohexane) for dissappearance of ketal. Once the recation was deemed to have reached completion, the mixture was filtered and concentrated in vacuo to yield a crude compound 11 as a brownish oil (1.15 g); ESI+, m/z: 190 (M+ +1 ).
Preparation of (3aS, 6aS)-6R-hvdroxy-3,3-dimethoxy-hexahvdro-furor3,2-blpyrrole-4- carboxylic acid tert-butyl ester 12
Figure imgf000034_0001
3,3-Dimethoxy-hexahydro-furo[3,2-b] pyrrol-6-ol 11 (2.80 g, 14.8 mmol) was dissolved in 75 ml of a mixture of dioxan/water (2:1 ). A solution of 10% Na2Cθ3 (25 ml) was added drop wise to pH 9-9.5. The mixture was cooled to 0 °C in an ice-water bath and Boc anhydride (amount ?) was added in one portion. The reaction was stirred at rt overnight and the pH of the mixture was maintained at 9-9.5 adding more of the 10% solution of Na2CO3 if necessary. The reaction was monitored by TLC (50:50 ethyl acetate:isohexane). Once completed, the mixture was filtered to eliminate the salts formed and the solvent eliminated in vacuo. The aqueous mixture was extracted with 3 x 100 ml EtOAc, the combined organic phases were washed with 100 ml of water and 100 ml brine, dried over Na2SO4, filtered and the solvent was evaporated in vacuum to afford 3.79 g of carbamate 12 as a clear oil (89%), 94% pure (HPLC), ESI+, m/z: 312 (M++Na).
Preparation of (3aS, 6aS)-6R-methanesulfonyloxy-3,3-dimethoxy-hexahydro-furor3,2- blpyrrole-4-carboxylic acid tert-butyl ester 13
Figure imgf000034_0002
Compound 12 (3.15 g, 10.9 mmol) was dissolved in DCM (50 ml) and cooled to 0 °C . Diisopropylethyl amine (3.9 ml, 21.78 mmol), methanesulfonyl chloride (1.13 ml, 14.48 mmol) and DMAP (0.05g) was added to the solution. The mixture was stirred at 0 °C for 2h, monitored by TLC (50:50 ethyl acetate:isohexane), diluted with EtOAc (75 ml), transferred into a separation funnel and washed with aqueous citric acid (1 M, 30 ml), saturated NaHCO3 (30 ml) and brine (30 ml). The organic phase was dried over Na2SO4, filtered and the solvent was evaporated in vacuo, yielding compound 13 as light brown solid (3.61 g, 91 %). ESI+, m/z: 368 (M+ +1 ).
Preparation of (3aS, 6aS)-6R-azido-3,3-dimethoxy-hexahvdro-furor3,2-blpyrrole-4- carboxylic acid tert-butyl ester 14
Figure imgf000035_0001
To compound 13 (1.1g, 3 mmol) dissolved in DMF (6 ml), an aqueous solution Of NaN3 was added and the mixture was heated in a microwave reactor at 130 °C for 2 x 60 min. The mixture was cooled to rt, saturated with NaCI, transferred into a separation funnel and extracted DCM (4 x 50 ml). The combined organic phases were dried over Na2SO4, filtered and the solvent was evaporated in vacuo, yielding a β-6-azido isomer 14 as a brown oil (0.42 g), ESI+, m/z: 215 (M+ +1 ).
Example 2
P-2 couplinp with L-IIe
[1-((3aS,6aS)-6R-Azido-3,3-dimethoxy-hexahydro-furo[3,2-blpyrrole-4-carbonyl)]-2- methyl-butyll-carbamic acid tert-butyl ester 7
Figure imgf000035_0002
The product of Example 1 , compound 14, was dissolved in DCM (20 ml) and stirred at rt in presence of Boc-lle-OH (0.7g, 3 mmol), HOBt (0.6g, 4.5 mmol), WSCHCI (1.15g, 6 mmol, and NMM (0.71 ml, 6.5 mmol) for 16 h, diluted with EtOAc (50 ml), washed with aqueous citric acid (1 M, 30 ml), saturated NaHCO3 (30 ml) and brine (30 ml). The organic phase was dried over Na2SO4, filtered and the solvent was evaporated in vacuo, yielding a crude 7 as a clear semi solid (0.81 g,). The crude mixture was separated by column chromatography over silica gel eluting with ethylacetate-heptane (1 :4) to yield pure 7 (0.406 g). ESI+, m/z: 428 (M+ +1 ).
Example 3
Couplinp with a P3 function
N-[1-((3aS,6aS)-(6R-Azido-3,3-dimethoxy-hexahvdro-furo[3,2-blpyrrole-4-carbonyl))-2- methyl-butyll-4-r2-(4-methyl-piperazin-1 -yl)-thiazol-4-yll-benzamide 15
Figure imgf000036_0001
[1 -(6R-Azido-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-2-methyl-butyl]- carbamic acid tert-butyl ester 7 (0.4 g, 0.94 mmol) was dissolved in dry MeOH (18 ml) and cooled to 0 °C in an ice-water bath. Acetyl chloride (2 ml) was then added drop wise. The mixture was stirred at rt and the reaction was monitored by TLC (50:50 ethyl acetate:isohexane). When the reaction was completed the solvent was removed in vacuum and the residue freeze dried from H2O: ACN (1 :1) (10 ml). The resulting amine HCI salt (0.16 g) was treated with the HBr salt of 4-[2-(4-methyl-piperazin-1-yl)-thiazol- 4-yl}-benzoic acid (0.174 g, 0.37 mmol), HOBt (0.0.51g, 0.37 mmol), WSC HCI (0.11 g, 0.56 mmol) and NMM (0.082 ml, 0.74 mmol) in dry DMF (10 ml) and stirred at rt 2 hours. The mixture was diluted with EtOAc, organic phase was then washed once with saturated NaHCOβ and once with 0.5 M NH3. The organic solution was dried over Na2SO4, filtered, the solvent was evaporated in vacuum and the crude material was purified by column chromatography over silica gel eluting with acetone to yield pure 15 (0.177 g). ESI+, m/z: 613 (M+ +1 ).
Example 4
N-[1-(3aS,6aS)-6R-Azido-3-oxo-hexahvdro-furo[3,2-blpyrrole-4-carbonyl)-2-methyl- butyll-4-r2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide 16
Figure imgf000037_0001
Compound 15 (39 mg) was dissolved in 2 ml of a mixture of TFA/H2O (97.5:2.5) and stirred at rt between 3 h. Once the reaction was deemed to be complete by HPLC the solvent was removed in vacuo, 5 ml of acetonitrile was added and the mixture was neutralised with solid Na2COs, filter and the solvent removed in vacuum. The crude materials were purified by semi-preparative HPLC to yield 13.4 mg of compound 16, 99% pure (HPLC), ESI+, m/z: 567 (M++H) as a white solid.
Example 5
N-[1-((3aS,6aS)-6R-Azido-3,3-dimethoxy-hexahvdro-furo[3,2-b]pyrrole-4-carbonyl)-3- methyl-butyll-carbamic acid tert-butyl ester 17
Figure imgf000037_0002
The title compound was prepared from compound 14 in a similar manner as Example 1 , last step and Example 2, yielding after column chromatography 86 mg of pure 17. ESI+, m/z: 428 (M+ +1 ).
Example 6
N-ri-(3aS,6aS)-6R-Azido-3-oxo-hexahvdro-furor3,2-blDyrrole-4-carbonyl)-3-methyl- butyll-4-f2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide 18
Figure imgf000038_0001
The title compound was prepared from the amine HCI salt derived from compound 17 in a similar manner as for synthesis of Example 3, yielding 99% pure (HPLC) ketone 18 (12 mg). ESI+, m/z: 567 (M+ +1 ).
Example 7
N-[1-(3aS,6aS)-6R-Azido-3-oxo-hexahvdro-furo[3,2-blpyrrole-4-carbonvπi-3-methyl- butyll-4-(4-propyl-piperazin-1 -yl)-benzamide 21
Figure imgf000038_0002
The title compound was prepared from the amine HCI salt derived from compound 14 in a similar manner as for synthesis of compound 18, yielding 99% pure (HPLC) ketone 21 (11.6 mg). ESI+, m/z: 512 (M+ +1 ).
Comparative Example 1
N-r(S)-1-((3aS,6S,6aS)-(6-Fluoro—3-oxo-hexahvdrofuror3,2-blDyrrole-4-carbonyl)-3- methyl-butyll-4-(4-propyl-piperazin-1-yl)-benzamide
Figure imgf000039_0001
Step a) N-[(S)-1 -(3aS,6S,6aS)-(6-Fluoro-3-oxo-hexahvdro-furo[3,2-blpyrrole-4- carbonvl)-3-methyl-butyll-carbamic acid benzyl ester
Figure imgf000039_0002
The title compound (with the depicted stereochemistry) was prepared analogously to Example 2 of WO05/066180 using Cbz protection.
Step b) N-[(S)-1 -(3aS,6S,6aS)-(6-Fluoro-3-hvdroxy-hexahvdro-furo[3,2-blpyrrole- 4-carbonyl)-3-methyl-butyll-4-(4-propyl-piperazin-1-yl)-benzamide
Figure imgf000039_0003
The C-3 alcohol of the P2-P1 building block of step a) was oxidized with Dess Martin periodinane to the ketone and the ketone protected as the dimethylketal. The Cbz protecting group was then removed from the resulting N-[(S)-1-(3aS,6S,6aS)-(6- fluoro-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]- carbamic acid benzyl ester using standard procedures and the free amine coupled up to 4-(4-propyl-piperazin-1-yl)-benzoic acid. The final step involved cleavage of the dimethylketal to obtain the desired ketone N-[(S)-1-((3aS,6S,6aS)-(6-Fluoro-3-oxo- hexahydrofuro[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-(4-propyl-piperazin-1-yl)- benzamide.
Comparative Example 2
N-r(S)-1-((3aS,6S,6aS)-6-azido-3-oxo)-hexahvdro-furor3,2-blDyrrole-4-carbonyl)-3- methyl-butyll-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yllbenzamide
Figure imgf000040_0001
In the preparation of compound 14, the corresponding 6S-azido isomer - (3aS, 6aS)- 6S-azido-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic acid tert-butyl ester was isolated as the major side product. This isomer was then taken through the remainder of the synthesis as described in Example 6 to afford N-[(S)-1-((3aS,6aS)- 6R-azido-3-oxo)-hexahydro-furo[3,2-ιb]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[2-(4- methyl-piperazin-1-yl)-thiazol-4-yl]benzamide.
Comparative Example 3
N-r(1S)-1-((3aS,6aR)-3-oxo)-hexahvdro-furor3,2-blDyrrole-4-carbonyl)-3-methyl-butvn- 4-f2-(4-methyl-piperazin-1-yl)-thiazol-4-yllbenzamide
Figure imgf000041_0001
The P1 building block was synthesised as shown in WO02/05720, and coupled to N- protected L-leucine and the P3 building block of Example 8 below, and oxidised to the ketone as shown in Examples 2 & 3.
Example 8
An alternative P3 building block
Step a) 4-Cvanopropiophenone
Figure imgf000041_0002
As described for the preparation of 4-cyanoacetophenone (Synth. Commun 1994, 887- 890), a mixture of 4-bromopropiophenone (5.65 g, 26.4 mmol), Zn(CN)2 (1.80 g, 15.3 mmol), and Pd(PPh3)4 (2.95 g, 2.6 mmol) was refluxed at 80 °C in deoxygenated DMF (35 ml_, stored over 4 A molecular sieves, bubbled with Ar before use) for 18 h. The mixture was partitioned between toluene (100 ml_) and 2N NH4OH (100 ml_). The organic phase was extracted with 2N NH4OH (100 ml_), washed with saturated aqueous NaCI (2 x 100 ml_), dried, and evaporated. A 10 mmol scale reaction was done similarly and the crude products were combined. Flash chromatography (330 g silica, 6/1 petroleum ether - EtOAc) gave white solids (5.17 g, 89%).
1 H NMR (CDCI3) δ ppm: 1.22 (t, 3H, J = 7.2 Hz), 3.00 (q, 2H, J = 7.3 Hz), 7.75 (d, 2H, J = 8.8 Hz), 8.03 (d, 2H, J = 8.4 Hz)
13C NMR (CDCI3) δ ppm: 7.8, 32.1 , 116.1 , 117.9, 128.3, 132.4, 139.7, 199.2
step b) 4-Propionylbenzoic acid
Figure imgf000042_0001
4-Cyanopropiophenone (4.67 g, 29.3 mmol) was refluxed with 2N NaOH (90 ml_, 180 mmol) and dioxane (90 ml_) at 95 °C overnight. The mixture was diluted with water (150 ml_), washed with ether (75 ml_), acidified to pH 2 with concentrated HCI, and extracted with ether (3 x 75 ml_). The organic phase was washed with saturated aqueous NaCI (3 x 75 ml_), dried, and evaporated to give yellow solids (5.12 g, 98%).
1 H NMR (CDCI3 + CD3OD) δ ppm: 1.18 (t, 3H, J = 7.2 Hz), 2.99, (q, 2H, J = 7.1 Hz), 7.95 (d, 2H, J = 8.4 Hz), 8.08 (d, 2H, J = 8.8 Hz)
13C NMR (CDCI3) δ ppm: 7.9, 32.1 , 127.7, 130.0, 134.0, 140.0, 168.0, 200.8
Step c) Methyl 4-propionylbenzoate
Figure imgf000042_0002
The benzoic acid above (890 mg, 5 mmol), NaHCO3 (1.26 g, 15 mmol) and iodomethane (935 μl_, 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 (744 mg, 77%).
1 H NMR (CDCI3) δ ppm: 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)
Step d) Methyl 4-(2-bromopropionyl)benzoate
Figure imgf000042_0003
Methyl 4-propionylbenzoate (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 °C 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 Na2S2O5 (20 ml_), saturated aqueous NaCI (20 ml_), and water (2OmL), dried and evaporated to give a yellow oil (1.025 g) that was used directly in the Hantzsch coupling. This material contained 91 % of the desired bromoketone, 5% starting ketone, and 4% 4-bromo-1-butanol, as determined by 1 H NMR.
1 H NMR (CDCI3) δ ppm: 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)
Step e) 4-r2-(4-tert-Butoxycarbonylpiperazin-1-yl)-5-methylthiazol-4-yllbenzoic acid methyl ester
Figure imgf000043_0001
All of the α-bromoketone above and 4-thionocarbonylpiperazine-1-carboxylic acid tert- butyl ester {J. Med. Chem., 1998, 5037-5054, 917 mg, 3.73 mmol) were refluxed in 36 ml_ THF at 70 °C 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 light yellow solids. Chromatography of the precicpitate (silica, 2/1 petroleum ether - EtOAc) gave 32 mg more of compound. Total yield is 44%.
1 H NMR (CDCI3) δ ppm: 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).
Step f) 4-r2-(4-tert-Butoxycarbonylpiperazin-1-yl)-5-methylthiazol-4-yllbenzoic acid
Figure imgf000043_0002
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 °C 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 light yellow solids (537 mg, 98%).
1 H NMR (CDCI3) δ ppm: 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) 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.
Step g) 4-r5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yllbenzoic acid
Figure imgf000044_0001
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 NaCNBHs (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]+).
Step h) The P3 acid from step g is coupled to the P1-P2 building block from
Example 5, and oxidized to the ketone as shown above.
Example 9 Alternative preparation of a P1 buildinp block
Figure imgf000045_0001
Step a)
Figure imgf000045_0002
To a stirred solution of compound 12 from Example 1 (1.55 g, 5.36 mmol), benzoic acid (0.90 g, 8.04 mmol) and triphenylphosphine (1.62 g, 8.04 mmol) in tetrahydrofurane (20 ml) at 0 °C was dropwise added a solution of diisopropyl azodicarboxylate (1.58 ml, 8.02 mmol) in tetrahydrofurane (5 ml) during 30 min. The reaction mixture was slowly allowed to reach room temperature, then stirred overnight. The reaction mixture was then concentrated onto silica and flash chromatography of the residue using stepwise gradient elution (ethyl acetate in hexane, 20-30%) gave after concentration of the appropriate fractions a colorless syrup (2.03 g, 5.17 mmol, 96 %).
NMR data (400 MHz, 298 K, CDCI3): 1H, delta 1.40 and 1.49 (2 s, 9 H), 3.27-3.42 (m, 6H), 3.44-3.51 (2 d, 1 H), 3.69 (m, 1 H), 3.76-3.89 (m, 1 H), 4.01-4.19 (2 d, 1 H), 4.52- 4.78 (m, 2H), 5.26 (brs, 1 H), 7.40-7.46 (m, 2H), 7.54-7.58 (m, 1 H), 7.96 (d, 2H). (2:3 mixture of rotamers)
Step b)
Figure imgf000045_0003
To a stirred solution of the product of step a) (1.94 g, 4.94 mmol) in methanol (10 ml) at room temperature was added methanolic sodium methoxide (0.5 M, 10 ml). The reaction mixture was monitored by TLC (hexane-ethyl acetate 7:3 and 1 :1 , visualized by UV-light and staining using ammoniummolybdate-cerium sulfate in aq. 10% sulfuric acid). After 2 h the reaction mixture was neutralized by Dowex (50WX8-100, H+-form, pH monitored by pH-indicator strips), then filtered and concentrated. Flash chromatography of the residue using stepwise gradient elution (ethyl acetate in hexane, 40-100 %), followed by concentration of the appropriate fractions and drying the residue in high vacuum overnight, gave the product as a colorless crystalline solid (1.23 g, 4.27 mmol, 86 %). In CDCI3 at 25 °C, NMR indicates the product exists as a mixture of rotamers.
NMR data (400 MHz, 298 K, CDCI3): 1H, delta 1.47 (s, 9H), 3.28-3.38 (m, 7 H), 3.62 (d, 1 H), 3.74-3.85 (m, 2H), 4.15 (m, 1 H), 4.45-4.54 (m, 2H).
Step c)
Figure imgf000046_0001
A solution of the product of step b (1.50 g, 5.18 mmol) and pyridine (1.26 ml, 15.6 mmol) in dry dichloromethane (45 ml) was cooled to 0 °C in an ice-water bath. Trifluoromethanesulfonic anhydride (1.74 ml, 10.3 mmol) was added drop wise over 10 minutes under stirring. The reaction mixture was stirred at 0 °C for a further 2 h, and was then washed with chilled 5% aqueous citric acid (150 ml). The aqueous layer was washed with dichloromethane (2 x 25 ml). The combined organic layers were dried (Na2SO4), filtered and concentrated in vacuo. The residue was dissolved in DMF (45 ml) and sodium azide (0.50 g, 7.78 mmol) was added. The reaction mixture was stirred at room temperature for 1 h, and was then concentrated in vacuo. The residue was purified by silica gel column chromatography (using YMC-GEL Silica 6 nM S-50 urn and a step wise gradient of 10-25% ethyl acetate in heptane) to give the N-Boc, dimethyl ketal protected P1 building block as a white solid (0.84 g, 51 %). Example 10
An alternative P3 building block
3-Fluoro- 4-r2-(4-methylpiperazin-1-yl)-thiazol-4-yllbenzoic acid HCI salt
Figure imgf000047_0001
Step a) Methyl 4-bromo-3-fluorobenzoate
4-Bromo-3-fluorobenzoic acid (2.46 g, 11.2 mmol) was dissolved in MeOH (9 ml_) and toluene (4 ml_) and cooled in an ice bath. (Trimethylsilyl)diazomethane (11 ml_, 2.0 M in hexanes, 22 mmol) was added dropwise until the yellow color persisted. The solution was stirred at room temperature for 40 mins and then concentrated in vacuo. A second batch of carboxylic acid (2.43 g) was treated similarly. The crude product from both batches were combined and subjected to flash chromatography (silica, 5/1 pentane - EtOAc) to give the methyl ester as white solids (4.92 g, 95% yield).
1H NMR (400 MHz, CDCI3) delta ppm 7.77 (m, 1 H), 7.71 (m, 1 H), 7.64 (m, 1 H), 3.93 (s, 3H).
Step b) Methyl 4-acetoxy-3-fluorobenzoate AIIyI chloride (105 μl_, 1.28 mmol) and TFA (20 μl_, 0.26 mmol) were added to a suspension of zinc dust (480 mg, 7.34 mmol) and anhydrous cobalt(ll) bromide (96.6 mg, 0.44 mmol) in MeCN (4 ml_), under inert gas. After stirring at room temperature for 10 min, the aryl bromide (1.003 g, 4.30 mmol dissolved in 5 ml_ MeCN) from (a) was added, followed by acetic anhydride (0.45 ml_, 4.79 mmol) and more MeCN (1 ml_). The mixture was stirred overnight, quenched with 1 M HCI (20 ml_), and then extracted with EtOAc (3 x 20 ml_). The organic phase was washed successively with saturated aqueous NaHCO3 (20 ml_) and saturated NaCI (2 x 20 ml_), dried (Na2SO4), and concentrated. Flash chromatography (silica, 6/1 to 4/1 petroleum ether - EtOAc gave recovered bromide (161.1 mg, 16%) and the desired ketone (white solids, 305.5 mg, 36%).
NMR (CDCI3) δ ppm: 1H (400 MHz) 7.94-7.86 (m, 2H), 7.80 (dd, 1 H, J = 11.2, 1.6 Hz), 3.95 (s, 3H), 2.67 (d, 3H, J = 4.4 Hz); 19F (376 MHz) -109.2 (m); 13C (100 MHz) 195.4 (d , J = 3.7 Hz), 165.1 (d, J = 2.2 Hz), 161.6 (d, J = 255 Hz), 135.8 (d, J = 8.1 Hz), 130.7 (d, J = 2.9 Hz), 129.0 ( d, J = 14 Hz), 125.2 (d, J = 3.6 Hz), 117.9 (d, J = 26 Hz), 52.7 (s), 31.4 (d, J = 7.3 Hz).
Step c) Methyl 4-(2-bromoacetoxy)-3-fluorobenzoate
THF (10 ml_) and 2-pyrrolidinone (91 μl_, 1.20 mmol) were added to a mixture of the ketone from b) (198 mg, 1.01 mmol) and pyrrolidone hydrotribromide (532 mg, 1.07 mmol). After heating at 60-65 °C for 2 h, the mixture was concentrated under vacuum and then partitioned between EtOAc (20 ml_) and saturated Na2S2O3 (10 ml_). The aqueous phase was extracted with EtOAc (10 ml_). The organic phases were combined, washed with saturated NaCI (2 x 10 ml_), dried (Na2SO4), and concentrated. Flash chromatography (silica, 7/1 petroleum ether - EtOAc) gave white solids (0.2634 g) containing 84% of the desired bromide (as determined by integration of 19F NMR peaks).
NMR (CDCI3) δ ppm: 1H (400 MHz) 7.93 (m, 1 H), 7.88 (m, 1 H), 7.79 (dd, 1 H, J = 11.2, 1.6 Hz), 4.50 (d, 2H, J = 2.4 Hz), 3.94 (s, 3H); 19F (376 MHz) -108.4 (m).
Step d) Methyl 3-fluoro- 4-r2-(4-methylpiperazin-1 -yl)-thiazol-4-yllbenzoate
EtOH (5.0 ml_) was added to the bromoketone above (193 mg, 0.70 mmol) and 4- methyl-piperazine-1-carbothioic acid amide (113 mg, 0.71 mmol) and the mixture was heated at 70 °C for 2h 15 min. The precipitates were filtered, washed with cold EtOH, and dried under vacuum and characterized. The procedure was repeated in a larger scale for 1.75 g bromoketone (6.36 mmol).
NMR (1/1 CDCI3 - CD3OD) δ ppm: 1H (400 MHz) 8.20 (m, 1 H), 7.86 (dd, 1 H, J = 8.4, 1.6 Hz), 7.76 (dd, 1 H, J = 11.4, 1.8 Hz), 7.38 (d, 1 H, J = 2.4 Hz), 4.23 (br, 2H), 3.95, (s, 3H), 3.65 ( br, 4H), 3.32 (br, 2H), 2.98 (s, 3H); 19F (376 MHz) -114.0 (m). LCMS [M+H]+ = 336.
The precipitates from both preparations were combined and suspended in saturated NaHCO3 (50 ml_). The mixture was extracted with EtOAc. The organic phase was washed with water, dried (Na2SO4), and evaporated to give the title compound as cream solids (1.76 g).
Step e) 3-fluoro- 4-r2-(4-methylpiperazin-1-yl)-thiazol-4-yllbenzoic acid HCI salt
The methyl ester (1.76 g, 5.25 mmol) from (d) was heated at 80 °C with 6M HCI (40 ml_) for 5.5 h. More 6M HCI (10 ml_) was added and the mixture was heated at 90 °C for 1 h 15 min. After cooling, the mixture was then evaporated under vacuum and freeze-dried from water to give the final product as cream solids in quantitative yield.
NMR (DMSO-c/6) δ ppm: 1H (400 MHz) 11.60 (br, 1 H), 8.18 (t, 1 H, J = 8.0 Hz), 7.82 (dd, 1 H, J = 8.4, 1.6 Hz), 7.72 (dd, 1 H, J = 12.0, 1.6 Hz), 7.48 (d, 1 H, J = 2.8 Hz), 4.11 (m, 2H), 3.58 (m, 2H), 3.49 (m, 2H), 3.19 (m, 2H), 2.80 (d, 3H, J = 4.4 Hz); 19F (376 MHz) -113.5 (m); 13C (100 MHz) 168.9, 166.0, 159.0 (d, J = 250 Hz), 143.4, 131.4 (d, J = 8 Hz), 129.8, 125.8 (d, J = 11 Hz), 125.6, 116.6 (d, J = 24 Hz), 111.1 (J = 15 Hz), 51.1 , 45.0, 41.9. LCMS [M+H]+ = 322.
Example 11
N-[2-(6-Azido-3-oxo-hexahydrofuro[3,2-blDyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[2-(4- methyl-piperazin-1-yl)-thiazol-4-yll-benzamide
Figure imgf000050_0001
Step a) [2-(6-Azido-3,3-dimethoxy-hexahvdrofuror3,2-blpyrrol-4-yl)-1-cvclohexyl-
2-oxo-ethyll-carbamic acid tertbutyl ester (11a)
6-Azido-3,3-dimethoxy-hexahydrofuro[3,2-b]pyrrole-4-carboxylic acid tert-butyl ester (256 mg, 0.81 mmol) was deprotected under acidic conditions (methanol/acetyl chloride) to give the crude 6-azido-3,3-dimethoxy-hexahydrofuro[3,2-b]pyrrole hydrochloride, which was coupled with N-Boc-cyclohexyl glycine using conventional DMF/DIPEA/HATU conditions, to give the title compound (315 mg, 85%). MS m/z 454.3(M+H)+.
Step b) N-[2-(6-Azido-3,3-dimethoxy -hexahvdrofuro[3,2-blpyrrol-4-yl)-1- cvclohexyl-2-oxo-ethyll-4-r2-(4-methyl-piperazin-1 -yl)-thiazol-4-yll-benzamide (11 b)
[2-(6-Azido-3,3-dimethoxy-hexahydrofuro[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]- carbamic acid tertbutyl ester (310 mg, 0.68 mmol) was deprotected under acidic conditions in the same way as the method described above, and the crude pyrrole hydrochloride intermediate was then coupled with the HBr salt of 4-[2-(4-methyl- piperazin-1-yl)-thiazol-4-yl]-benzoic acid using using conventional DMF/DIPEA/HATU conditions, to give the title compound (421 mg, 97%).
MS m/z 639.3 (M+H)+.
1H-NMR (400MHz, CDCI3): 7.90-7.80 (m, 4H), 6.96 (d, 1 H), 6.88 (s, 1 H), 4.90 (m, 1 H), 4.78 (d, 1 H), 4.68 (t, 1 H), 4.32 (m, 1 H), 3.95 (d, 1 H), 3.73 (d, 1 H), 3.70 (m, 1 H), 3.60 (m, 4H), 3.47 (s, 3H), 3.45 (t, 1 H), 3.26 (s, 3H), 2.58 (m, 4H), 2.38 (s, 3H), 1.98-1.65 (m, 6H), 1.36-1.10 (m, 5H). Step c) N-r2-(6-Azido-3-oxo-hexahvdrofuror3,2-blpyrrol-4-yl)-1-cvclohexyl-2-oxo- ethyll-4-r2-(4-methyl-piperazin-1 -yl)-thiazol-4-yll-benzamide (11c)
N-[2-(6-Azido-3,3-dimethoxy -hexahydrofuro[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo- ethyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide (200 mg, 0.313 mmol) was hydrolyzed under acidic conditions (TFA/H2O) and preparative HPLC chromatography (C8, gradient 10 - 90%MeCN/H2O) gave pure title product 89 mg (48%), as a mixture of 2 rotamers of both the ketone (20%) [MS m/z 593.5 (IvRH)+] and the hydrate (80%)
[MS m/z 611.6 (M+H2O+H)+].
Example 12
N-[2-(6-Azido-3-oxo-hexahvdrofuro[3,2-blpyrrole-4-carbonyl)-3-methylbutyll-4-[5- meth yl-2-(4-meth yl-piperazin-1-yl)-thiazol-4-yll-benzamide
Figure imgf000051_0001
Step a) [2-(6-Azido-3,3-dimethoxy-hexahvdrofuro[3,2-blpyrrole-4-carbonyl)-3- methylbutyll-carbamic acid tertbutyl ester (12a)
The tert-butyl ester group was removed from 6-azido-3,3-dimethoxy- hexahydrofuro[3,2-b]pyrrole-4-carboxylic acid tert-butyl ester (100 mg, 0.318 mmol) under acidic conditions. The afforded crude 6-azido-3,3-dimethoxy-hexahydrofuro[3,2- b]pyrrole hydrochloride was coupled with N-Boc-leucine using conventional DMF/DIPEA/HATU conditions which gave the title compound (134 mg, 99%). MS m/z 428.1 (M+H)+.
Step b) N-[2-(6-Azido-3,3-dimethoxy-hexahvdrofuro[3,2-blpyrrole-4-carbonyl)-3- methylbutyll-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide (12b) The tert-butyl ester group was removed from [2-(6-Azido-3,3-dimethoxy- hexahydrofuroβ^-blpyrrole^-carbonylJ-S-methylbutyll-carbamic acid tert-butyl ester (134 mg, 0.318 mmol) under acidic conditions (methanol/acetyl chloride). The afforded crude hydrochloride intermediate was then coupled with the HCI salt of 4-[5-methyl-2- (4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzoic acid using conventional
DMF/DIPEA/HATU conditions, to give the title compound (166 mg, 84%). MS m/z 627.3 (M+H)+.
1H-NMR (400MHz, CDCI3): 7.82-7.65 (m, 4H), 6.96 (d, 1 H), 4.90 (m, 1 H), 5.02 (m, 1 H), 4.75 (dd, 1 H), 4.68 (dt, 1 H), 4.40 (dt, 1 H), 3.90 (d, 1 H), 3.75 (d, 1 H), 3.74 (m, 1 H), 3.65 (m, 4H), 3.43 (s, 3H), 3.41 (t, 1 H), 3.26 (s, 3H), 3.00 (m, 4H), 2.73 (s, 3H), 2.42 (s, 3H), 1.85 (m, 1 H), 1.70 (m, 2H), 1.05 (d, 3H), 1.00 (d, 3H).
Step c) N-[2-(6-Azido-3-oxo-hexahvdrofuro[3,2-blpyrrole-4-carbonv0-3- methylbutyll-4-r5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide (12c)
N-[2-(6-Azido-3,3-dimethoxy-hexahydrofuro[3,2-b]pyrrole-4-carbonyl)-3-methylbutyl]-4- [5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide (160 mg, 0.255 mmol) was hydrolyzed under acidic conditions (TFA/H2O) and preparative HPLC chromatography (C8, gradient 10 - 90%MeCN/H2O) gave pure title product 93 mg (63%), as a mixture of 2 rotamers of both the ketone (20%) [MS m/z 581.4 (M+H)+] and the hydrate (80%) [MS m/z 599.5 (M+H2O+H)+].
Example 13
N-f2-(6-Azido-3-oxo-hexahvdrofurof3,2-blpyrrole-4-carbonyl)-3-fluoro-3-methylbutyll-4- [2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide
Figure imgf000053_0001
Step a) r2-(6-Azido-3,3-dimethoxy-hexahydrofuro[3,2-blpyrrole-4-carbonyl)-3- fluoro-3-methylbutyll-carbamic acid tertbutyl ester (13a)
The tert-butyl ester group was removed from 6-azido-3,3-dimethoxy- hexahydrofuro[3,2-b]pyrrole-4-carboxylic acid tert-butyl ester (63 mg, 0.20 mmol) under acidic conditions (methanol/acetyl chloride). The afforded crude 6-azido-3,3- dimethoxy-hexahydrofuro[3,2-b]pyrrole hydrochloride was coupled with N-Boc-γ- fluoroleucine (preparation in Truong et al Syn. Lett. 2005 No 8 using conventional DMF/DIPEA/HATU conditions to give the title compound (84mg, 94%). MS m/z 446.2 (M+H)+.
Step b) N-r2-(6-Azido-3,3-dimethoxy-hexahydrofuro[3,2-blpyrrole-4-carbonyl)-3- fluoro-3-methylbutyll-4-r2-(4-methyl-piperazin-1 -yl)-thiazol-4-yll-benzamide (13b)
The tert-butyl ester group was removed from [2-(6-azido-3,3-dimethoxy- hexahydrofuro[3,2-b]pyrrole-4-carbonyl)-3-fluoro-3-methylbutyl]-carbamic acid tertbutyl ester (84 mg, 0.188 mmol) under acidic conditions. The afforded crude hydrochloride intermediate was then coupled with the HBr salt of 4-[2-(4-methyl-piperazin-1-yl)- thiazol-4-yl]-benzoic acid using conventional DMF/DIPEA/HATU conditions which gave the title compound (110 mg, 93%). MS m/z 631.2 (M+H)+.
1H-NMR (400MHz, CDCI3): 7.90-7.78 (m, 4H), 6.96 (d, 1 H), 6.90 (s, 1 H), 5.06 (m, 1 H), 4.76 (d, 1 H), 4.70 (m, 1 H), 4.52 (dt, 1 H), 3.90 (d, 1 H), 3.80 (m, 1 H), 3.75 (d, 1 H), 3.62 (m, 4H), 3.42 (s, 3H), 3.42 (t, 1 H), 3.25 (s, 3H), 2.65 (m, 4H), 2.45 (s, 3H), 2.15 (m, 1 H), 1.52 (d, 3H), 1.48 (d, 3H).
Step c) N-r2-(6-Azido-3-oxo-hexahydrofuro[3,2-blpyrrole-4-carbonyl)-3-fluoro-3- methylbutyll-4-r2-(4-methyl-piperazin-1 -yl)-thiazol-4-yll-benzamide (13c) N-[2-(6-Azido-3,3-dimethoxy-hexahydrofuro[3,2-b]pyrrole-4-carbonyl)-3-fluoro-3- methylbutyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide (105 mg, 0.166 mmol) was hydrolyzed under acidic conditions (TFA/H2O). The residue was purified by preparative HPLC chromatography (C8, gradient 10 - 90% MeCN/H2O) which gave the pure title compound 70 mg (68%), as a mixture of 2 rotamers of both the ketone (20%) [MS m/z 585.5 (M+H)+] and the hydrate (80%) [MS m/z 603.6 (M+H2O+H)+].
Example 14
N-[2-(6-Azido-3-oxo-hexahydrofuro[3,2-b]pyrrole-4-carbonyl)-3-methylbutyl]-3-fluoro-4- [2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide
Figure imgf000054_0001
Step a) [2-(6-Azido-3,3-dimethoxy-hexahvdrofuro[3,2-blpyrrole-4-carbonyl)-3- methylbutyll-carbamic acid tertbutyl ester (14a)
The tert-butyl ester group was removed from 6-azido-3,3-dimethoxy- hexahydrofuro[3,2-b]pyrrole-4-carboxylic acid tert-butyl ester (63 mg, 0.20 mmol under acidic conditions (MeOH/acetyl chloride). The afforded crude 6-azido-3,3-dimethoxy- hexahydrofuro[3,2-b]pyrrole hydrochloride was coupled with N-Boc-leucine using conventional DMF/DIPEA/HATU conditions which gave the title compound (83 mg, 97%). MS m/z 428.1 (M+H)+.
Step b) N-[2-(6-Azido-3,3-dimethoxy-hexahvdrofuro[3,2-blpyrrole-4-carbonyl)-3- methylbutyll-3-fluoro-4-[2-(4-methylpiperazin-1-yl)-thiazol-4-yll-benzamide (14b)
The tert-butyl ester group was removed from [2-(6-azido-3,3-dimethoxy- hexahydrofuro[3,2-b]pyrrole-4-carbonyl)-3-methylbutyl]-carbamic acid tertbutyl ester (83 mg, 0.194 mmol) under acidic conditions. The afforded crude hydrochloride intermediate was then coupled with the HCI salt of 3-fluoro-4-[2-(4-methyl-piperazin-1- yl)-thiazol-4-yl]-benzoic acid using conventional DMF/DIPEA/HATU conditions which gave the title compound (110 mg, 93%). MS m/z 631.2 (M+H)+.
1H-NMR (400MHz, CDCI3): 8.18 (t, 1 H), 7.52 (m, 2H), 7.20 (s, 1 H), 7.08 (d, 1 H), 4.98 (m, 1 H), 4.78 (d, 1 H), 4.70 (m, 1 H), 4.40 (dt, 1 H), 3.90 (d, 1 H), 3.85 (d, 1 H), 3.85 (m, 1 H), 3.65 (m, 4H), 3.43 (s, 3H), 3.42 (t, 1 H), 3.26 (s, 3H), 2.80 (m, 4H), 2.58 (s, 3H), 1.83 (m, 1 H), 1.70 (m, 2H), 1.05 (d, 3H), 1.00 (d, 3H).
Step c) N-[2-(6-Azido-3-oxo-hexahvdrofuro[3,2-blpyrrole-4-carbonyl)-3- methylbutyll-3-fluoro-4-r2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide (14c)
N-[2-(6-Azido-3,3-dimethoxy-hexahydrofuro[3,2-b]pyrrole-4-carbonyl)-3-methylbutyl]-3- fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide (105 mg, 0.166 mmol) was hydrolyzed under acidic conditions (TFA/H2O) and preparative HPLC chromatography (C8, gradient 10 - 90% MeCN/H2O) gave pure title product 72 mg (74%), as a mixture of 2 rotamers of both the ketone (28%) [MS m/z 585.5 (M+H)+] and the hydrate (72%) [MS m/z 603.5 (M+H2O+H)+].
Example 15
N-[1-(6-Azido-3-oxo-hexahvdro-furo[3,2-blpyrrole-4-carbonyl)-3-methyl-butyl]-4-[5- fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide
Figure imgf000055_0001
Step a) [2-(6-Azido-3,3-dimethoxy-hexahvdrofuro[3,2-blpyrrole-4-carbonyl)-3- methylbutyll-carbamic acid tertbutyl ester (15a) The tert-butyl ester group was removed from compound 14 (63 mg, 0.20 mmol) under acidic conditions (methanol/acetyl chloride). The afforded crude 6-azido-3,3- dimethoxy-hexahydrofuro[3,2-b]pyrrole hydrochloride was coupled with N-Boc-leucine using conventional DMF/DIPEA/HATU conditions, which gave the title compound (81 mg, 95%). MS m/z 428.3 (M+H)+.
Step b) N-[2-(6-Azido-3,3-dimethoxy-hexahvdrofuro[3,2-blpyrrole-4-carbonyl)-3- methylbutyll-4-r5-fluoro-2-(4-methyl-piperazin-1 -yl)-thiazol-4-yll-benzamide (15b)
The tert-butyl ester group was removed from compound 15a (81 mg, 0.189 mmol) under acidic conditions (methanol/acetyl chloride). The afforded crude hydrochloride intermediate was coupled with the HCI salt of 4-[5-fluoro-2-(4-methylpiperazin-1-yl)- thiazol-4-yl]-benzoic acid using conventional DMF/DIPEA/HATU conditions, which gave the title compound (105 mg, 88%). MS m/z 631.2 (M+H)+.
1H-NMR (400MHz, CDCI3): 7.92 (d, 2H), 7.82 (d, 2H), 6.84 (d, 1 H), 5.03 (m, 1 H), 4.75 (d, 1 H), 4.70 (m, 1 H), 4.42 (dt, 1 H), 3.90 (d, 1 H), 3.75 (d, 1 H), 3.74 (m, 1 H), 3.47 (m, 4H), 3.43 (s, 3H), 3.42 (t, 1 H), 3.25 (s, 3H), 2.55 (m, 4H), 2.37 (s, 3H), 1.84 (m, 1 H), 1.70 (m, 2H), 1.05 (d, 3H), 1.00 (d, 3H).
Step c) N-[1-(6-Azido-3-oxo-hexahvdro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl- butyll-4-r5-fluoro-2-(4-methyl-piperazin-1 -yl)-thiazol-4-yll-benzamide (15c)
The ketal of compound 16b (100 mg, 0.159 mmol) was hydrolyzed under acidic conditions (TFA/H2O) followed by purification by preparative HPLC chromatography (C8, gradient 10 - 90% MeCN/H2O) which gave the title compound 35.1 mg (38%), as a mixture of 2 rotamers of both the ketone (26%) [MS m/z 585.2 (M+H)+] and the hydrate (74%) [MS m/z 603.2 (M+H2O+H)+].
Example 16
N-f1-(6-Azido-3-oxo-hexahvdro-furof3,2-blpyrrole-4-carbonyl)-3-fluoro-3-methyl-butyll- 3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide
Step a) [1 -(6-Azido-3,3-dimethoxy-hexahvdro-furo[3,2-blpyrrole-4-carbonyl)-3- fluoro-3-methyl-butyll-carbamic acid tert-butyl ester (16a)
Figure imgf000057_0001
Acetyl chloride (0.4 ml_) was added dropwise to a solution of compound 14 (Example 1 step f) (69 mg, 0.221 mmol) in methanol (4 ml_) at 0 °C. The reaction mixture was stirred at rt over night and then concentrated. The residue was redissolved twice in dry DMF (5 ml) and concentrated to dryness, then again dissolved in DMF (3 ml_). y- Fluoro-BOC-Leu-OH (50 mg, 0.201 mmol) and DIEA (133 μl_, 0.802 mmol) was added to the solution before it was cooled to 0 °C and HATU (80 mg, 0.211 mmol) was added. The reaction was stirred for 3 hours at room temperature whereafter the solvent was removed by rotary evaporation. The crude mixture was dissolved in EtOAc (20 ml_) and washed with 10% citric acid (aq) (10 ml_) and NaHCO3 (sat, aq) (10 ml_). The organic phase was dried with Na2SO4, filtered and evaporated. The crude product was purified by flash chromatography (heptane: ethyl acetate 1 :1) to give compound 16a (69 mg, 77 %). [M+H]+ = 446.
Step b) N-H -(6-Azido-3,3-dimethoxy-hexahvdro-furor3,2-blpyrrole-4-carbonyl)-3- fluoro-3-methyl-butyll-3-fluoro-4-r2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide
(16b)
Figure imgf000057_0002
Acetyl chloride (0.4 ml_) was added dropwise to a solution of compound 16a (69 mg, 0.154 mmol) in methanol (4 ml_) at 0 °C. The reaction mixture was stirred at rt over night and then concentrated. The residue was dissolved in 1 ,4-dioxane (5 ml) and freeze-dried over night. The solid was dissolved in DMF (4 ml_). 3-Fluoro-4-[2-(4- methyl-piperazin-1-yl)-thiazol-4-yl]-benzoic acid x HCI (63 mg, 0.177 mmol) and DIEA (102 μl_, 0.617 mmol) was added to the solution before it was cooled to 0 °C and HATU (62 mg, 0.162 mmol) was added. The reaction mixture was stirred for 3 hours at room temperature whereafter the solvent was removed by rotary evaporation. The crude mixture was dissolved in EtOAc (20 ml_) and washed with 2x15 ml_ NaHCO3 (Sat, aq). The organic phase was dried with Na2SO4, filtered and evaporated. The crude product was purified by flash chromatography (ethyl acetate: acetone 1 :1 + 0.1 % TEA) to give the crude title compound (123 mg, >100 %). [M+H]+ = 649. The crude compound was used in the next step without any further purification.
Step c) N-H -(6-Azido-3-oxo-hexahvdro-furor3,2-blpyrrole-4-carbonyl)-3-fluoro-3- methyl-butyll-3-fluoro-4-r2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide (16c)
Figure imgf000058_0001
Compound 16b (123 mg crude, 0.154 mmol) was dissolved in 20 ml_ TFA: H2O (97.5: 2.5) and stirred for 2 hours. The solvent was removed and the crude product was dissolved in ethyl acetate and washed with 2 x10 ml_ NaHCO3 (Sat, aq)- The organic phase was dried with Na2SO4, filtered and evaporated. The crude product was purified by semi-prep. HPLC on a Sunfire C18 column with mobile phases A (90:10 H2O: acetonitrile, 10 mM NH4Ac) and B (10:90 H2O: acetonitrile, 10 mM NH4Ac) going from 40-65% B. The purification was repeated four times (second: 40-60% B, third: 40-45% B, fourth: 30-45% B) to give the title compound as an off-white solid (14 mg, 15 %). [M+H]+ = 603.
Example 17
N-[1-(6-Azido-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-3-fluoro- 4-[2-(4-methyl-4-oxy-piperazin-1-yl)-thiazol-4-yl]-benzamide Step a) 3-Fluoro-4-r2-(4-methyl-4-oxy-piperazin-1-yl)-thiazol-4-yll-benzoic acid methyl ester (17a)
Figure imgf000059_0001
m-Chloroperbenzoic acid (250 mg, 70-75%) was added to a solution of compound 10d (154 mg, 0.46 mmol) in dichloromethane (10 ml), and the mixture was stirred overnight. The mixture was diluted with dichloromethane (50 ml), the organic phase was washed with saturated aqueous sodium bicarbonate followed by back extraction of the water phase with dichloromethane. The combined organic phases were washed with citric acid and brine, dried over sodium sulfate, filtered and evaporated. The obtained N-oxide was used further without additional purification. Yield 89% (143 mg) LC/MS [M+1] 353.
Step b) 3-Fluoro-4-r2-(4-methyl-4-oxy-piperazin-1-yl)-thiazol-4-yll-benzoic acid (17b)
Figure imgf000059_0002
LiOH (1 M, 2 ml) was added to a solution of the ester 17a (143 mg, 0.41 mmol) in THF- water (2:1 ). After stirring 4h at room temperature, the solution was acidified by addition of 1 M HCI to pH 4. Brine (5 ml) was added and the mixture was extracted with EtOAc- acetone (92:7) 3 times, the organic layers were combined, dried over sodium sulfate, filtered and evaporated. The residue was triturated in ether, the solid was filtered off and used without further purification in the next step. Yield 58 mg, 0.17 mmol, 41%, LC/MS [M+1] 339.
Step c) N-M -(6-Azido-3,3-dimethoxv-hexahvdro-furo[3,2-blpyrrole-4-carbonvn-3- methyl-butyl]-3-fluoro-4-[2-(4-methyl-4-oxy-piperazin-1-yl)-thiazol-4-yl]-benzamide
(17c)
Figure imgf000060_0001
Acetyl chloride (0.5 ml) was added dropwise during 10 min to a cooled solution (0 °C) of compound 17 (from Example 5) (49 mg, 0.17 mmol) in methanol (5 ml). The temperature of reaction mixture was allowed to raise till 22 °C and the stirring was continued for 6h, The reaction mixture was concentrated and the residue dissolved in 1 ,4-dioxane and freeze-dried overnight.
The obtained salt was dissolved in DMF (7 ml). Water (0.6 ml), compound 17b (0.17 mmol) and 0.55 ml of diisopropylethylamine was added and the mixture was cooled to 0 °C whereafter HATU (55 mg) was added. The reaction mixture was stirred for 2h, evaporated to a volume of 1.5 ml and purified by prep. HPLC (NH4OAc buffer, 30:80 system (MeCN-water) which gave the title compound (19 mg, 18%) LC/MS [M+1] 647.
Step d) N-[1 -(6-Azido-3-oxo-hexahvdro-furor3,2-blpvrrole-4-carbonvl)-3-methyl- butyl]-3-fluoro-4-[2-(4-methyl-4-oxy-piperazin-1 -yl)-thiazol-4-yl]-benzamide (17d)
Figure imgf000060_0002
Compound 17c (19mg) was dissolved and stirred in a solution TFA97.5% -water 2.5% (2ml) and water. After 2h the solution was concentrated and the residue dissolved in acetonitrile (7 ml) and stirred with solid sodium bicarbonate for 1 h. The formed solid was filtered off, the acetonitrile solution was evaporated, and the residue dissolved in 1 ,4-dioxane and lyophilized on freeze-d her which gave the title compound (15 mg, 86%). LC/MS [M+1] 601 and [M+19] 619 (hydrate form) Comparative Example 4
N-f1-(6-Azido-3-oxo-hexahvdro-furof3,2-blpyrrole-4-carbonyl)-3-methyl-butyll-3-fluoro- 4-f2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide
Step a H -(6-Azido-3,3-dimethoxy-hexahvdro-furor3,2-blpyrrole-4-carbonyl)-3- methyl-butyll-carbamic acid tert-butyl ester (19a)
Figure imgf000061_0001
6-Azido-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic acid tert-butyl ester (0.3 mmol) was dissolved in methanol (5 ml), the solution was cooled to 0 °C and acetyl chloride (0.5 ml) was added dropwise during 10 min. The temperature of the reaction mixture was allowed to raise till 22 °C whereafter the mixture was stirred for 6h. The solvent was evaporated and the residue was dissolved in 1 ,4-dioxane and then freeze-dried overnight.
The obtained salt was dissolved in DMF (3 ml), Boc-Leu-OH (0.3 mmol) and diisopropylethylamine (0.6 ml) was added. The mixture was cooled to 0 °C and then HATU (145 mg) was added. The reaction mixture was stirred for 2h, the solvent was evaporated and the residue was distributed between ethyl acetate and water. The organic phase was washed with water once more, then dried with brine and sodium sulfate, filtered and evaporated. The residue was purified by flash chromatography (ethyl acetate - hexane 1 :1 ) to give the title compound (78 mg, 61%) LC/MS [M+1] 428.
Step b) N-H -(6-Azido-3-oxo-hexahvdro-furor3,2-blpyrrole-4-carbonyl)-3-methyl- butyll-3-fluoro-4-r2-(4-methyl-piperazin-1 -yl)-thiazol-4-yll-benzamide (19b)
Figure imgf000062_0001
Acetyl chloride (0.5 ml) was added dropwise during 10 min to a cooled solution (0 °C) of compound 19a (53 mg, 0.183 mmol) in methanol (5 ml). The temperature of the reaction mixture was allowed to raise to 22 °C and mixture was stirred for 6h. The solvent was evaporated and the residue dissolved in 1 ,4-dioxane and freeze-dried overnight.
The obtained salt was dissolved in DMF (3 ml), compound 10e (0.183 mmol) and diisopropylethylamine (0.6 ml) was added and the mixture was cooled to 0 °C whereafter HATU (60 mg) was added. After stirring for 2h, the solvent was evaporated and the residue was distributed between ethyl acetate and water. The organic phase was washed once more with water, then dried with brine and sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography (5% MeOH in CHCI3, with 0.1 % NEt3 which gave the title compound (87 mg, 75%) LC/MS [M+1] 631.
Comparative Example 5
N-[1-(6-Azido-3-oxo-hexahvdro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[5- fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yll-benzamide (20)
Figure imgf000062_0002
Acetyl chloride (0.5 ml) was added dropwise during 10 min to a cooled solution (0 °C) of compound 19a (42 mg, 0.146 mmol) in methanol (5 ml). The temperature of the reaction mixture was allowed to raise till 22 °C. After stirring for 6h, the reaction mixture was concentrated and the residue dissolved in 1 ,4-dioxane and freeze-dried overnight.
The obtained salt was dissolved in DMF (3 ml), 4-[5-fluoro-2(-4-methylpiperazin-1-yl)- thiazol-4-yl]-benzoic acid (0.146 mmol) and diisopropylethylamine (0.5 ml) was added. The mixture was cooled to 0 °C and then HATU (48 mg) was added. After stirring for 2h, the solvent was evaporated and the residue was distributed between ethyl acetate and water. The organic phase was washed once more with water, then dried with brine and sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography (5% of MeOH in CHCI3, with 0.1% NEt3) which gave the title compound (65 mg, 71 %) LC/MS [M+1] 631.
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
100 μl/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 = 100 μM and KM = 100 μM to obtain an estimate of the Ki, up to a maximum of 100 μM.
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 10 μl, distribute 10 μl/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 20 ml assay buffer and 20 μl 1 M DTT. Add sufficient cathepsin S to give 2 nM final concentration.
Using the a distributor such as a Multidrop 384, add 30 μl/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 100mM 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 1 mM dithiothreitol just before use. 2ul of 10mM inhbitor made up in 100% DMSO is dispensed into row A. 10μl of 50 μM substrate (=1/200 dilution of 10 mM 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. Ki values were calculated according to equation 1.
Figure imgf000066_0001
where vo is the velocity of the reaction, V\s the maximal velocity, S is the concentration of substrate with Michaelis constant of KM, and / is the concentration of inhibitor.
Results are presented as:
A under 50 nanomolar B 50-500 nanomolar C 501 -1000 nanomolar
D 1001 - 5000 nanomolar
E 5001 - 10 000 nanomolar
F in excess of 10 000 nanomolar
TABLE 1
Figure imgf000066_0002
Figure imgf000067_0001
Representative values from specific batches of compound/enzyme/assay occasion are as follows:
Figure imgf000067_0002
The compounds of formula Il are thus potent inhibitors of cathepsin K and yet selective over the closely related cathepsin S and L.
Metabolic Stability
Compounds of the invention and the indicated comparative examples were tested for metabolic stability in a conventional cytosol assay in which the compounds are subjected to a standardized extraction of metabolic enzymes and the disappearance of the compound monitored by HPLC or MS.
In short, test compounds (2μM) are incubated in pooled human liver cytosol (Xynotech LLC Lenexa US, 1 mg/mL protein in 0.1 M phosphate buffer, pH 7.4) at 37 degrees centigrade over a one hour period. The incubations are initiated by the addition of 1mM NADPH co-factor. Timed sub-samples were taken at 0, 20, 40 and 60 minutes and "crash precipitated" by the addition of 3 volumes ice-cold acetonitrile. The samples were centrifuged at reduced temperature and the supernatants were separated and analyzed by LC-MS-MS
Comparative Example 1 employs the down F P1 unit of WO05/066180. Comparative Example 2 employs the down isomer at C6 of the P1 unit of Example 1. Comparative Example 3 employs the preferred P1 and P2 units of WO02/057270 together with a P3 unit within the scope of the present claims (which are outside the scope of WO02/057270). Comparative Example 4 employs the down isomer at C6 of the P1 unit of Example 14. Comparative Example 5 employs the down isomer at C6 of the P1 unit of Example 15.
TABLE 2
Figure imgf000068_0001
Figure imgf000069_0001
It will be apparent from the respective comparisons between Example 7 (invention) vs Comparative Example 1 , and Example 6 (invention) vs Comparative Example 3, that the up-azido of the invention is substantially more metabolically stable than the down- fluoro P1 moiety favoured by prior art WO05/066180, or the unsubstituted P1 moieties favoured by WO02/057270. Additionally, by comparing Example 6 with Comparative Example 2, Example 14 with Comparative Example 4 and Example 15 with Comparative Example 5, it will be apparent that the enhanced stability shown by the up-azido substituent of the invention is not shown by the otherwise identical compound with down azido at this position.
Permeability
This experiment measures transport of inhibitors through the cells of the human gastroenteric canal. The assay uses the well known Caco-2 cells with a passage number between 40 and 60.
Apical to basolateral transport
Generally every compound will be tested in 2-4 wells. The basolateral and the apical wells will contain 1.5 ml_ and 0.4 ml_ transport buffer (TB), respectively, and the standard concentration of the tested substances is 10 μM. Furthermore all test solutions and buffers will contain 1 % DMSO. Prior to the experiment the transport plates are pre-coated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material. After 21 to 28 days in culture on filter supports the cells are ready for permeability experiments.
Transport plate no 1 comprises 3 rows of 4 wells each. Row 1 is denoted Wash, row 2 "30 minutes" and row 3 "60 minutes". Transport plate no 2 comprises 3 rows of 4 wells, one denoted row 4 "90 minutes", row 5 "120 minutes and the remaining row unassigned.
The culture medium from the apical wells is removed and the inserts are transferred to a wash row (No. 1 ) in a transport plate (plate no.1) out of 2 plates without inserts, which have already been prepared with 1.5 ml_ transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5. In A→B screening the TB in basolateral well also contains 1 % Bovine Serum Albumin.
0.5 ml_ transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts and the cell monolayers equilibrated in the transport buffer system for 30 minutes at 37 °C in a polymix shaker. After being equilibrated to the buffer system the Transepithelial electrical resistance value (TEER) is measured in each well by an EVOM chop stick instrument. The TEER values are usually between 400 to 1000 Ω per well (depends on passage number used).
The transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to the 30 minutes row (No. 2) and fresh 425 μl_ TB (pH 6.5), including the test substance is added to the apical (donor) well. The plates are incubated in a polymix shaker at 37°C with a low shaking velocity of approximately 150 to 300 rpm.
After 30 minutes incubation in row 2 the inserts will be moved to new pre-warmed basolateral (receiver) wells every 30 minutes; row 3 (60 minutes), 4 (90 minutes) and 5 (120 minutes).
25 μl_ samples will be taken from the apical solution after ~2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.
300 μl_ will be taken from the basolateral (receiver) wells at each scheduled time point and the post value of TEER is measured at the end the experiment. To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at -20°C until analysis by HPLC or LC-MS.
Basolateral to apical transport
Generally every compound will be tested in 2-4 wells. The basolateral and the apical wells will contain 1.55 mL and 0.4 mL TB, respectively, and the standard concentration of the tested substances is 10 μM. Furthermore all test solutions and buffers will contain 1 % DMSO. Prior to the experiment the transport plates are precoated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material. After 21 to 28 days in culture on filter supports the cells are ready for permeability experiments. The culture medium from the apical wells are removed and the inserts are transferred to a wash row (No.1 ) in a new plate without inserts (Transport plate).
The transport plate comprises 3 rows of 4 wells. Row 1 is denoted "wash" and row 3 is the "experimental row". The transport plate has previously been prepared with 1.5 ml_ TB (pH 7.4) in wash row No. 1 and with 1.55 ml_ TB (pH 7.4), including the test substance, in experimental row No. 3 (donor side).
0.5 ml_ transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts in row No. 1 and the cell monolayers are equilibrated in the transport buffer system for 30 minutes, 37 °C in a polymix shaker. After being equilibrated to the buffer system the TEER value is measured in each well by an EVOM chop stick instrument.
The transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to row 3 and 400 μl_ fresh TB, pH 6.5 is added to the inserts. After 30 minutes 250 μl_ is withdrawn from the apical (receiver) well and replaced by fresh transport buffer. Thereafter 250 μl_ samples will be withdrawn and replaced by fresh transport buffer every 30 minutes until the end of the experiment at 120 minutes, and finally a post value of TEER is measured at the end of the experiment. A 25 μl_ samples will be taken from the basolateral (donor) compartment after ~2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.
To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at -20°C until analysis by HPLC or LC- MS.
Calculation
Determination of the cumulative fraction absorbed, FAcum, versus time. FAcum is calculated from:
Figure imgf000072_0001
Where CRJ is the receiver concentration at the end of the interval i and CQ\ is the donor concentration at the beginning of interval i. A linear relationship should be obtained.
The determination of permeability coefficients (Papp- cm/s) are calculated from:
Figure imgf000073_0001
where k is the transport rate (min'i ) defined as the slope obtained by linear regression of cumulative fraction absorbed (FAcum ) as a function of time (min), VR is the volume in the receiver chamber (ml_), and A is the area of the filter (cm^).
Reference compounds
Figure imgf000073_0002
Testing of compounds of the invention using the above methodology indicates that fluorination of the benzyl ring or thiazolyl ring typically improves permeability. For example, a fluorine substituent at the meta position of the benzyl ring relative to the amide bond or on the thiazolyl ring can approximately double the compound's cellular permeability relative to the corresponding compound without fluorine substitution (Example 14 vs Example 6, or Example 15 vs Example 6). Greater permeability through the gastrointestinal tissue is advantageous in that it allows for the use of a smaller dose to achieve similar levels of exposure to a less permeable compound administered in a higher dose. A low dose is advantageous in that minimises the cost of goods for a daily dose, which is a crucial parameter in a drug which is taken for protracted time periods.
Metabolism
Fluorinated compounds are further believed to produce an advantageous metabolism profile. Less metabolism is observed for the meta-fluorinated benzyl ring analogue than the unsubstituted analogue in pooled, cryopreserved human hepatocytes where the intrinsic clearance for the meta-fluorinated benzyl ring is approximately half that of the unsubstituted analogue. The metabolic processes associated with particular compounds of the invention have been analysed using a Waters Synapt High Definition Mass Spectroscopy Instrument (HDMS).
Additionally, an intact ketone on the furopyrrole ring is important for activity against cathepsin K, as this moiety interacts with the catalytic cysteine of the enzyme. When compounds which lack a fluorine on the benzyl ring are subject to the normal hepatic and/or other in vivo metabolic processes, (as determined in commercially available liver microsomes, S9 fraction and/or clinical plasma samples) a large proportion of the resulting metabolites are no longer active due to the ketone being reduced. In contrast, compounds with a benzylic fluorine tend to produce a higher proportion of active metabolites, such as the corresponding piperazine N-oxide (such as shown in Example 17) with an intact ketone on the furopyrrole ring. Such an active metabolite can thus assist in maintaining inhibitory pressure on cathepsin K, notwithstanding partial metabolism of the compound.
Abbreviations
DMF dimethylformamide DCM dichloromethane
TBDMS te/t-butyldimethylsilyl RT room temperature
THF tetrahydrofuran Ac acetyl TLC thin layer chromatography DMAPdimethylaminopyridine
EtOAc ethyl acetate uM micromolar
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 II:
Figure imgf000076_0001
wherein
R2 is the side chain of leucine, isoleucine, cyclohexylglycine, O-methyl threonine, 4-fluoroleucine or 3-methoxyvaline;
R3 is H, methyl or fluoro;
R4 is C1-C6 alkyl;
E is a bond or thiazolyl, optionally substituted with methyl or fluoro; n is 0 or 1 ; or a pharmaceutically acceptable salt, N-oxide or hydrate thereof.
2. A compound according to claim 1 , with the proviso that the compound is not:
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrro1-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-(2-(4-methylpiperazin- 1 -yl)thiazol-4-yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(2-(4-methylpiperazin- 1 -yl)thiazol-4-yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3 ,3 -dimethyl- 1 -oxobutan-2-yl)-4-(2-(4-methylpiperazin- 1 -yl)thiazol-4-yI)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-
4(5H,6H, 6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-(2-(4-methylpiperazin- 1 -yl)thiazol-4- yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6ciH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(2-(4-methylpiperazin- 1 -yl)thiazol-4-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(2-(4-methylpiperazin- 1 -yl)thiazol-4-yl)benzamide
N-( 1 -((3aS,6R,6aS)-6-azido-3-oxohexahydro-2H-furo[3,2-b]pyrrole-4- carbonyl)cyclohexyl)-4-(2-(4-methylpiperazin-1-yl)thiazol-4-yl)benzamide
N-((S)- 1 -((3aS,6R,6aS)-6-azido-3 -oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-(2-(4-(2-methoxyethyl)piperazin- 1 -yl)thiazol-4- yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(2-(4-(2-methoxyethyl)piperazin- 1 -yl)thiazol-4- yl)benzamide
3. A compound according to either claim 1 or 2, with the proviso that the compound is not:
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydτo-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(4-methylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(4-ethylpiperazin- 1 -yl)benzamide
N-((S)4-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(4-propy lpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(4-isopropylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-(4-(2-methoxyethyl)piperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)- 4-(4-cyclopropylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)- 4-(4-cyclobutylpiperazin- 1 -yl)benzamide
N-((S)- 1 -((3aS,6R,6aS)-6-azido-3 -oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-( 1 -propylpiperidin-4-yl)benzamide N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-(4-cyclobutylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-( 1 -propylpiperidin-4-yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-(l -cyclopropylpiperidin-4-yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-fiiro[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-( 1 -cyclobutylpiperidin-4-yl)benzamide
N-((2S,3S)- 1 -((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yI)-4-(4-methylpiperazin- 1 -yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3-methyl- 1 -oxopentan-2-yI)-4-(4-ethylpiperazin- 1 -yl)benzamide
N-((2S,3S)- 1 -((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3-methyl-1-oxopentan-2-yl)-4-(4-propylpiperazin-1-yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3-methyl- 1 -oxopentan-2-yl)-4-(4-isopropylpiperazin- 1 - yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-fυro[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3-methyl-1-oxopentan-2-yl)-4-(4-(2-methoxyethyl)piperazin-1- yl)benzamide N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydτo-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-( 1 -isopropy lpiperidin-4-yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-( 1 -(2-methoxyethyl)piperidin-4-y l)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan-2-yl)-4-( 1 -cyclopropylpiperidin-4-yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4,4-dimethyl- 1 -oxopentan~2-yl)-4-( 1 -cyclobutylpiperidin-4-y l)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyiτol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-(4-methylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methy 1- 1 -oxopentan-2-y l)-4-(4-ethy lpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-y l)-4-(4-propylpiperazin- 1 -yl)benzamide
Λr-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl-l -oxopentan-2-y l)-4-(4-isopropy lpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-yl)-4-(4-(2-methoxyethyl)piperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 4-methyl- 1 -oxopentan-2-y l)-4-(4-cyclopropy lpiperazin- 1 -yl)benzamide N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-(4-cyclopropylpiperazin- 1 - yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-(4-cyclobutylpiperazin- 1 - yl)benzamide
N-((2S,3S)- 1 -((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3-methyl-1-oxopentan-2-yl)-4-(l-methylpiperidin-4-yl)benzamide
N-((2S,3S)-\-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-(l -ethylpiperidin-4-yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3-methyl- 1 -oxopentan-2-yl)-4-(l -propylpiperidin-4-yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-(l -isopropylpiperidin-4-yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- l-oxoρentan-2-yl)-4-(l-(2-methoxyethyl)piperidin-4- yl)benzamide
N-((2S,3S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-( 1 -cyclopropy lpiperidin-4- yl)benzamide
N-((2S,3S)- 1 -((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol- 4(5H,6H,6aH)-yl)-3 -methyl- 1 -oxopentan-2-yl)-4-( 1 -cyclobutylpiperidin-4- yl)benzamide N-((S)Λ-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3,3 -dimethyl- 1 -oxobutan-2-yl)-4-(4-methylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3 ,3 -dimethyl- 1 -oxobutan-2-yl)-4-(4-ethylpiperazin- 1 -yl)benzamide
N-((S)- 1 -((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3 ,3 -dimethyl- 1 -oxobutan-2-yl)-4-(4-propylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3,3-dimethyl- 1 -oxobutan-2-yl)-4-(4-isopropylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-fiiro[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3 ,3 -dimethyl- 1 -oxobutan-2-yl)-4-(4-(2-methoxyethy l)piperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-S-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3 ,3 -dimethyl- 1 -oxobutan-2-yl)-4-(4-cyclopropylpiperazin- 1 -yl)benzamide
N-((.S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3,3-dimethyl- 1 -oxobutan-2-yl)-4-(4-cyclobutylpiperazin- 1 -yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3 ,3 -dimethyl- 1 -oxobutan-2-yl)-4-( 1 -propy lpiperidin-4-y l)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3,3-dimethyl- 1 -oxobutan-2-yl)-4-(l -cyclopropylpiperidin-4-yl)benzamide
N-((S)-1-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 3,3-dimethyl- 1 -oxobutan-2-yl)-4-(l -cyclobutylpiperidin-4-yl)benzamide N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(4-methylpiperazin-l -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(4-ethylpiperazin- 1 -y l)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(4-propylpiperazin- 1 -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(4-isopropyIpiperazin- 1 -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(4-(2-methoxyethyl)piperazin- 1 -y l)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(4-cyclopropylpiperazin- 1 -yl)benzamide
N-((S)^-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(4-cyclobutylpiperazin- 1 -yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-( 1 -propylpiperidin-4-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopenty l-2-oxoethyl)-4-( 1 -cyclopropylpiperidin-4-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclopentyl-2-oxoethyl)-4-(l -cyclobutylpiperidin-4-yl)benzamide N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(l-isopropylpiperidin-4-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexy l-2-oxoethyl)-4-(1-(2-methoxyethyl)piperidin-4-yl)benzamide
N-((S)-2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(1-cyclopropylpiperidin-4-yl)benzamide
N-((S)- 2-((3aS,6R,6aS)-6-azido-3-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)- 1 -cyclohexyl-2-oxoethyl)-4-(1-cyclobutylpiperidin-4-yl)benzamide
4. A compound according to any one of claims 1 to 3, wherein R2 is the side chain of leucine or cyclohexylglycine.
5. A compound according to any one of claims 1 to 3, wherein R2 is the side chain of leucine, cyclohexylglycine or 4-fluoroleucine.
6. A compound according to any one of claims 1 to 3, wherein R2 is the side chain of O-methyl threonine, 4-fluoroleucine and 3-methoxy valine.
7. A compound according to any one of claims 1 to 6, wherein n represents 0.
8. A compound according to any one of claims 1 to 6, wherein n represents 1.
9. A compound according to claim 8, wherein R3 is positioned as shown by the partial structure:
Figure imgf000084_0001
10. A compound according to any one of claims 1 to 9, wherein R3 is fluoro or methyl.
11. A compound according to claim 10, wherein R3 represents fluoro.
12. A compound according to any one of claims 1 to 11 , wherein E represents thiazolyl which is unsubstituted.
13. A compound according to any one of claims 1 to 11 , wherein E represents thiazolyl which is substituted with methyl or fluoro.
14. A compound according to claim 13, wherein E represents thiazolyl which is substituted by fluoro.
15. A compound according to any one of claims 12 to 14, wherein E is
Figure imgf000085_0001
wherein R5 represents H, methyl or fluoro.
16. A compound according to claim 15, wherein E represents thiazolyl which is positioned as shown by the partial structure:
Figure imgf000085_0002
wherein R5 represents H, methyl or fluoro.
17. A compound according to either claim 15 or 16, wherein R5 is H or CH3.
18. A compound according to any one of claims 1 to 11 wherein E represents a bond.
19. A compound according to any one of claims 1 to 18, wherein R4 represents Ci- C4 alkyl.
20. A compound according to claim 19, wherein R4 is methyl.
21. A compound according to claim 19, wherein R4 is propyl.
22. A compound according to claim 1 which is selected from:
Figure imgf000086_0001
N-[1-(3aS,6aS)-6R-Azido-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-2-methyl- butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
Figure imgf000086_0002
N-[1-(3aS,6aS)-6R-Azido-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl- butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
Figure imgf000086_0003
N-[1-(3aS,6aS)-6R-Azido-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)]-3-methyl- butyl]-4-(4-propyl-piperazin-1 -yl)-benzamide;
Figure imgf000087_0001
N-[2-(6-Azido-3-oxo-hexahydrofuro[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[2-(4- methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
Figure imgf000087_0002
N-[2-(6-Azido-3-oxo-hexahydrofuro[3,2-b]pyrrole-4-carbonyl)-3-methylbutyl]-4-[5- methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
Figure imgf000087_0003
N-[2-(6-Azido-3-oxo-hexahydrofuro[3,2-b]pyrrole-4-carbonyl)-3-fluoro-3-methylbutyl]-4- [2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
Figure imgf000088_0001
N-[2-(6-Azido-3-oxo-hexahydrofuro[3,2-b]pyrrole-4-carbonyl)-3-methylbutyl]-3-fluoro-4- [2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
Figure imgf000088_0002
N-[1-(6-Azido-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[5- fluoro-2-(4-methyl-piperazin-1 -yl)-thiazol-4-yl]-benzamide; or
Figure imgf000088_0003
N-[1-(6-Azido-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-fluoro-3-methyl-butyl]- 3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;
or a pharmaceutically acceptable salt, N-oxide or hydrate thereof.
23. A compound according to claim 1 which is:
Figure imgf000089_0001
N-(6-Azido-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-3-fluoro-4-[2-(4-methyl-4-oxy- piperazin-1-yl)-thiazol-4-yl]-benzamide, or a pharmaceutically acceptable salt or hydrate thereof.
24. A pharmaceutical composition comprising a compound as defined in any of claims 1 to 23 and a pharmaceutically acceptable carrier or diluent therefor.
25. Use of a compound as defined in any of claims 1 to 23 in the manufacture of a medicament for the treatment or prevention of disorders mediated by cathepsin K.
26. A compound according to any one of claims 1 to 23 for use as a medicament.
27. A compound according to claim 26 for use in the treatment or prevention of a disorder mediated by cathepsin K .
28. A method for the treatment of a disorder mediated by cathepsin K comprising administering a safe and effective amount of a compound according to any one of claims 1 to 23 to a subject in need thereof.
29. The use, method or compound according to claim 25, 27 or 28, 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 cartilage or matrix degradation (such as osteoarthritis and rheumatoid arthritis), bone cancers including neoplasia, pain (especially chronic pain).
30. A compound of the formula:
Figure imgf000090_0001
wherein the Rb groups define a ketal, such as the bis methyl ketal or together define a cyclic ketal such as 1 ,3-dioxolane, or an N-protected derivative thereof.
31. A compound according to claim 30 which is:
Figure imgf000090_0002
32. A compound of the formula:
Figure imgf000090_0003
wherein R4 represents C1-6alky!, or a salt or lower alkyl (e.g. C1-6alky!) ester thereof.
33. A compound according to claim 32 which is:
Figure imgf000090_0004
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