WO2005082876A1 - C-5 substituted furanone dipeptide cathepsin s inhibitors - Google Patents

Abstract

Compounds of the formula (I), where R is H, F or OH, Q is -(CH2)n- and n is 1, 2 or 3; are inhibitors of cathepsin S and have utility in the treatment of certain immune disorders and chronic pain.

Description

C-5 substituted furanone dipeptide cathepsin S inhibitors

Technical Field

This invention relates to inhibitors of cathepsin S, and their use in methods of treatment for disorders involving cathepsin S such as autoimmune, allergy and chronic pain conditions.

Background to the invention and prior art

The papain superfamily of cysteine proteases are widely distributed in diverse species including mammals, invertebrates, protozoa, plants and bacteria. A number of mammalian cathepsin enzymes, including cathepsins B, F, H, K, L, O, S, and W, have been ascribed to this superfamily, and inappropriate regulation of their activity has been implicated in a number of metabolic disorders including arthritis, muscular dystrophy, inflammation, glomerulonephritis and tumour invasion. Pathogenic cathepsin like enzymes include the bacterial gingipains, the malarial falcipains I, II, III et seq and cysteine proteases from Pneumocystis carinii, Trypanosoma cruzei and brucei, Crithidia fusiculata, Schistosoma spp.

In WO 97/40066, the use of inhibitors against Cathepsin S is described. The inhibition of this enzyme is suggested to prevent or treat disease caused by protease activity. Cathepsin S is a highly active cysteine protease belonging to the papain superfamily. Its primary structure is 57%, 41 % and 45% homologous with that of the human cathepsin L and H and plant cysteine proteases papain respectively, although only 31 % homologous with cathepsin B. It is found mainly in B cells, dendritic cells and macrophages and this limited occurrence suggests the potential involvement of this enzyme in the pathogenesis of degenerative disease. Moreover, it has been found that destruction of li by prateolysis is required for MHC class II molecules to bind antigenic peptides, and for transport of the resulting complex to the cell surface. Furthermore, it has been found that Cathepsin S is essential in B cells for effective li prateolysis necessary to render class II molecules competent for binding peptides. Therefore, the inhibition of this enzyme may be useful in modulating class ll-restricted immune response (WO 97/40066). Other disorders in which cathepsin S is implicated are asthma, chronic obstructive pulmonary disease, endometriosis and chronic pain. International patent application no WO 0069855 describes cathepsin S inhibitors of the formula:

OD wherein:

R¹ = R⁵, R'C(O) , R' C(S), R' S02 , R' OC(O), R' NHC(O)

R' is a monocyclic ring;

R², R⁴ = H, Cι-₇-alkyl, C^-cycloalkyl;

R³ = Cι-₇-alkyl, C₃-₇-cycloalkyl,

R⁵ = C-ι-₇-alkyI, Halogen, Ar-Cι-₇-alkyl, d-₃-alkyl-CONR'"

R⁶ = H, C ₇-alkyl, Ar-C ₇-alkyl, Cι-₃-alkyl-SO₂-R , d-s-alkyl-CCOJ-NHR^ or CH₂XAr,

The R³ groups specifically disclosed in WOOO 69855 are branched chain alkyl moieties such as n-butyl, t-butyl, 3-(2,2-dimethylpropyl), 4-(2-methylbutyl), 4-(3,3-dimethylbutyl), 4-(3,3-dimethyl-2-methylbύtyl), 4-(3-rnethyl-2-methylbutyl), or 5-(2-methyl-3- methylpentyl). Page 27, lines 13 of WOOO 69855 discloses the compound morpholine-4- carboxylic acid [3,3-dimethyl-1 S-(2-ethyl-4-oxo-tetrahydrofuran-3- ylcarbamoyl)butyl]amide.

We have now discovered that a small group of compounds with dramatically improved efficacy and pharmacokinetic properties have the formula I:

where R is H, OH or F, Q is -(CH₂)_n- and π is 1 , 2 or 3; or a pharmaceutically acceptable salt thereof.

Q is thus methylene, thereby defining a cyclohexyl ring, ethylene, thereby defining a cyclohexyl ring, or propylene, thereby defining a cyclooctyl ring.

When R is F or OH, n is typically 2 or 3.

R as H and especially OH is currently favoured, especially when n is 1 or 2.

A further aspect of the invention comprises a method employing the compounds of the invention for the treatment of diseases wherein cathepsin S is a factor, ie diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily.

Examples of such diseases or conditions include those enumerated in WO 97/40066, such as autoimmune diseases, allergies, such as asthma and hayfever, multiple sclerosis, rheumatoid arthritis and the like. A further example is the treatment of endometriasis, and especially chronic pain , as disclosed in WO0320287. The invention further provides the use of the compounds of formula IV in therapy and in the manufacture of a medicament for the treatment of diseases or conditions alleviated or moderated by inhibition of cathepsin S.

In one series of embodiments, the methods are employed to treat mammals, particularly humans at risk of, or afflicted with, autoimmune disease. By autoimmunity is meant the phenomenon in which the host's immune response is turned against its own constituent parts, resulting in pathology. Many human autoimmune diseases are associated with certain class II MHC-complexes. This association occurs because the structures recognized by T cells, the cells that cause autoimmunity, are complexes comprised of class II MHC molecules and antigenic peptides. Autoimmune disease can result when T cells react with the host's class II MHC molecules when complexed with peptides derived from the host's own gene products. If these class II MHC/antigenic peptide complexes are inhibited from being formed, the autoimmune response is reduced or suppressed. Any autoimmune disease in which class II MHC/antigenic complexes play a role may be treated according to the methods of the present invention.

Such autoimmune diseases include, e.g., juvenile onset diabetes (insulin dependent), multiple sclerosis, pemphigus vulgaris, Graves' disease, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis and Hashimoto's thyroiditis.

In another series of embodiments, the methods are employed to treat mammals, particularly humans, at risk of, or afflicted with, allergic responses. By "allergic response" is meant the phenomenon in which the host's immune response to a particular antigen is unnecessary or disproportionate, resulting in pathology. Allergies are well known in the art, and the term "allergic response" is used herein in accordance with standard usage in the medical field.

Examples of allergies include, but are not limited to, allergies to pollen, "ragweed," shellfish, domestic animals (e.g., cats and dogs), bee venom, house dust mite allergens and the like. Another particularly contemplated allergic response is that which causes asthma. Allergic responses may occur, in pan, because T cells recognize particular class II MHC/antigenic peptide complexes. If these class II MHC/antigenic peptide complexes are inhibited from being formed, the allergic response is reduced or suppressed. Any allergic response in which class II MHC/antigenic peptide complexes play a role may be treated according to the methods of the present invention, Although it is not expected that immunosuppression by the methods of the present invention will be a routine prophylactic or therapeutic treatment for common allergies, severe or life- threatening allergic responses, as may arise during asthmatic attacks or anaphylactic shock, may be treated according to these methods.

In another series of embodiments, the methods are employed to treat mammals, particularly humans, which have undergone, or are about to undergo, an organ transplant or tissue graft. In tissue transplantation (e.g., kidney, lung, liver, heart) or skin grafting, when there is a mismatch between the class II MHC genotypes (HLA types) of the donor and recipient, there may be a severe "allogeneic" immune response against the donor tissues which results from the presence of non-self or allogeneic class II MHC molecules presenting antigenic peptides on the surface of donor cells. To the extent that this response is dependent upon the formation of class II MHC/antigenic peptide complexes, inhibition of cathepsin S may suppress this response and mitigate the tissue rejection. An inhibitor of cathepsin S can be used alone or in conjunction with other therapeutic agents, e.g., as an adjunct to cyclosporin A and/or antilymphocyte gamma globulin, to achieve immunosuppression and promote graft survival. Preferably, administration is accomplished by systemic application to the host before and/or after surgery. Alternatively or in addition, perfusion of the donor organ or tissue, either prior or subsequent to transplantation or grafting, may be effective.

The above embodiments have been illustrated with an MHC class II mechanism but the invention is not limited to this mechanism of action. Suppression of cathepsin S as a treatment of COPD or chronic pain may not, for example, involve MHC class II at all.

Currently preferred indications treatable in accordance with t ie present invention include:

Autoimmune indications, including idiopathicthrombocytopertic purpura (ITP), rheumatoid arthritis (RA), multiple schlerosis (MS), myasthen ϊa gravis (MG), Sjδgrens syndrome, Grave's disease and systemic lupus erythematosis (SLE).

Non-automimmune indications include allergic rhinitis, asthma, artherosclerosis, chronic obstructive pulmonary disease (COPD) and chronic pain.

The compounds of the invention can form salts which form an additional aspect of the invention. Appropriate pharmaceutically acceptable salts of the compounds of the invention include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, isethionate, adipate, alginate, aspartate, benzoate, bulyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, lactobionate, pivolate, camphorate, undscanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesulphonate, benzenesulphonate, p-chlorobenzenesulphonate and p-toluesnesulphonate; 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.

While it is possible for the active agent to be administered alone, it is preferable to present it as part of a pharmaceutical formulation. Such a formulation will comprise the above defined active agent together with one or more acceptable carriers/excipients and optionally other therapeutic ingredients. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.

The formulations include those suitable for rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, but preferably the formulation is an orally administered formulation. The formulations may conveniently be presented in unit dosage form, e.g. tablets and sustained release capsules, and may be prepared by any methods well known in the art of pharmacy.

Such methods include the step of bringing into association the above defined active agent with the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing a compound of Formula IV or its pharmaceutically acceptable salt in conjunction or association with a pharmaceutically acceptable carrier or vehicle. If the manufacture of pharmaceutical formulations involves intimate mixing of pharmaceutical excipienfs 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 excipie nts e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvϊ nylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginicacid; 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 gelati n and glycerin, or sucrose and acacia; and mouthwashes comprising the active agerrt in a suitable liquid carrier.

As with all pharmaceuticals, the appropriate dosage for the compounds or formulations of the invention will depend upon the indication, the severity of the disease, the size and metabolic vigour and the patient, the mode of administration and is readily determined by conventional animal trials. Dosages providing intracellular (for inhibition of physiological proteases of the papain superamily) concentrations of the order 0.01-100 uM, more preferably 0.01-10 uM, such as 0.1-5uM are typically desirable and achievable. Certain P1 building blocks represent novel intermediates and define a further aspect of the invention:

where R is F, OH or O-PG" where PG" is an hydroxyl protecting group, PG is H or an N- protecting group and PG' is H, an hydroxyl protecting group or together with the adjacent oxygen defines a keto group.

Preferred stereochemic configurations for the building blocks of the invention include:

Hydroxy protecting group as used herein refers to a substituent which protects hydroxyl groups against undesirable reactions during synthetic procedures such as those O- protecting groups disclosed in Greene, "Protective Groups In Organic Synthesis," (John Wiley & Sons, New York (1981)). Hydroxy protecting groups comprise substituted methyl ethers, for example, methoxymethyl, benzyloxymethyl, 2-methoxyethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, t-butyl and other lower alkyl ethers, such as isopropyl, ethyl and especially methyl, benzyl and triphenylmethyl; tetrahydropyranyl ethers; substituted ethyl ethers, for example, 2,2,2-trichloroethyl; silyl ethers, for example, trimethylsilyl, t-butyldimethylsilyl and t-bulyldiphenylsilyl; and esters prepared by reacting the hydroxyl group with a carboxylic acid, for example, acetate, propionate, benzoate and the like. Favoured hydroxyl protecting groups include acetate, benzyl, methyl and 4-cyanobenzyl.

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, trifluoroacetyl, 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-nitrobenzyIoxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-n^'ιtro-4,5-dimethoxybenzyloxycarbonyl_I 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarboπyl, 2,2,2-trichloraethoxycarbonyl, phenoxycarbonyl, 4- 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, Fmoc, benzyl, t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz).

Various aspects of the invention will now be described byway of example only with reference to the accompanying Examples.

Example 1

(2S)-2-rffe/ -butoxycarbonyl)amino1-3-cvcloheptylpropanoic acid building block

a) Methyl (2S)-2-amino-3-cyclohep1ylpropanoate.

To a stirred solution of R-2,5-dihydro-3,6-dimethoxy-2-isopropyl pyrazine (5 g, 27 mmol) in THF (80 mL) at -78 °C was added dropwise 2.5 M n-butyllithium in hexanes over 40 min, maintaining the temperature of the reaction mixture below -70 °C. To the reaction mixture was added dropwise a solution of iodomethylcycloheptane (Webb et al, J. Med. Chem., 42, 8 (1999), 1415-1421.) (6.8 g, 28.5 mmol) in THF (12 mL) over 50 min, maintaining the temperature of the reaction mixture below -70 °C. The reaction mixture was then stirred at 0 °C for another 75 min, acetic acid was added (2.45 mL) and stirring was continued for another 20 min. The reaction mixture was then diluted with ethyl acetate (200 mLI), washed with brine (2 x 50 mLI), dried (MgSO₄), filtered and concentrated in vacuo. Column chromatography of the residue using stepwise gradient elution (ethyl acetate in hexane 0-5%) gave the pyrazine derivative as an oil (5.4 g, 68%). A mixture of the pyrazine derivative (5.4 g, 18.5 mmol) in acetonitrile (55 mL), water (45 mL) and 1 M aqueous hydrochloric acid (45 mL) was stirred at room temperature for 2 h, then concentrated in vacuo to approximately half the volume. Aqueous NaHCO₃ (1M) was added until just alkaline, then the reaction was extracted with dichloromethane (3 x 50 mL). The extracts were dried (MgSO₄), filtered and concentrated in vacuo onto silica. Column chromatography of the residue using stepwise gradient elution (ethyl acetate in hexane and triethylamine (0.5%) 20-100%) gave pure fractions of (8) which were concentrated in vacuo to a syrup (2.14 g, 58%). Additional material could be obtained by repeated chromatography.

b) (2S)-2-[(tetf-butoxycarbonyl)amino]-3-cycloheptylpropanoic acid

To a stirred solution of the ester from step a) (1.56 g, 7.8 mmol) and di-tert-butyl- dicarbonate (1.88 g, 8.6 mmol) in DMF (15 mL) at 0 °C was added triethylamine (3.25 mL, 23.4 mmol). The reaction mixture was stirred at room temperature overnight then diluted with ethyl acetate and washed with 10% aq. citric acid (3 x 20 mL), dried (MgSO₄), filtered and concentrated in vacuo. Column chromatography using stepwise gradient elution (ethyl acetate in hexane, 0-25%) gave an oil (2.06 g, 88%).

This material was treated with dioxan (35 mL), water (15 mL) and 1M aqueous LiOH at 0 °C for 2 h. To the reaction mixture was then added 10 % aqueous citric acid (50 mL), and this was extracted with dichloromethane (4 x 35 mL). The extracts were dried (MgSO ), filtered and concentrated in vacuo onto silica. Column chromatography using stepwise gradient elution (ethyl acetate in hexane, 50-100%) gave after lyophilization for 2 days (9) as a hard syrup (2 g, 97%).

delta_H (400 MHz, CDCI₃ at 298 K) 4.85 (1 H, d, J 8.0, NH), 4.31 (1 H, m, alpha-H), 1.82- 1.14 (15H, m, CH₂-cycloheptyl).

Example 2

(2S)-2-r(fe/f-butoxycarbonyl)amino1-3-cvclooctylpropanoic acid building block

BocHN

The title compound was prepared analogously to Example 1 with similar yields, starting from hydroxymethylcyclooctane.

deltas (400 MHz, CDCI₃ at 298 K) 4.88 (1 H, d, J 8.0, NH), 4.33 (1 H, m, alpha-H), 1.79- 1.25 (15H, m, CH₂-cyclooctyl).

Example 3 Morpholine-4-carboxylic acid r2-cyclohexyl-1 -(SH2-(S)-ethyl-4-oxo-tetrahvdro-furan-3- (S)-ylcarbamoyl)-ethvπ-amide

, ⁱ B^{oc H}

3a n=2 3b ιv,v

n=1 4a n=1 Example 3 n=24b n=2 Example 4

Scheme 1 :

Reagents and Conditions: (i) NaBH₄, MeOH; (ii) 4MHCI/Dioxane (iii) WSC.HCI, DCM, NMM, Boc-beta-cycloheptyl-ala-OH; (iv) 4M HCI/ Dioxane; (v) morpholinecarbonyl chloride, TEA, DCM; (vi) Dess-Martin, DCM.

(2-(S)-Ethyl-4-oxo-tetrahydra-furan-3-(S)-yl)-carbamic acid fe/f-butyl ester (1) prepared as described in WO00/69855 (0.5 g, 2.2 mmol) was dissolved in methanol (5 mL) and cooled in an ice-bath. Sodium borohydride (91 mg, 2.4 mmol) was added and reaction stirred for 10 minutes. The ice-bath was removed and the reaction stirred for a further 1 h at room temperature. Water was added and the reaction was extracted with ethyl acetate and the organic layer washed with water. The organic layer was dried (MgSO₄) and the solvent removed in vacuo to give (2-(S)-ethyl-4-hydroxy-tetrahydro-furan-3-(S)- yl)-carbamic acid ferf-butyl ester (2) as a white solid (338 mg, 67%). The structure was confirmed by NMR and MS analysis.

Compound (2) (338 mg, 1.5 mmol) was dissolved in 4M hydrochloric acid / dioxan and stirred at room temperature for 1 h. The solvent was removed in vacuo to give the hydrochloride salt as a white solid. This hydrochloride salt was suspended in dichloromethane (5 mL) and WSC.HCI (309 mg, 1.6 mmol) was added followed by BOC-NH-beta-cyclohexyl-L-alanine-OH (517 mg, 1.8 mmol) and NMM (321 uL, 2.9 mmol). The reaction was stirred at room temperature for 2 h, diluted with ethyl acetate and washed with saturated aqueous sodium hydrogen carbonate, water and brine. The organic layer was dried (MgSO₄) and the solvent removed in vacuo to give a tan solid. Purification by column chromatography (10-50% ethyl acetate / heptane) afforded [2- Cyclohexyl-1-(2-(S)-ethyl-4-hydroxy-tetrahydro-furan-3-(S)-ylcarbamoyl)-ethyl]-carbamic acid tert-butyl ester (3a) as a white solid (333 mg, 57%). The structure was confirmed by NMR and MS analysis.

[2-Cyclohexyl-1-(2-(S)-ethyl-4-hydroxy-tetrahydro-furan-3-(S)-ylcarbamoyl)-ethyl]- carbamic acid ferf-butyl ester (3a) (333 mg, 0.83 mmol) was dissolved in 4M hydrochloric acid / dioxan and stirred at room temperature for 1 h. The solvent was removed in vacuo to give the hydrochloride salt as a white solid. The hydrochloride salt was suspended in dichloromethane (3 mL) and moφholine carbonyl chloride (117 uL, 1 mmol) was added followed by triethylamine (266 uL, 1.9 mmol). The reaction was stirred for 5 h at room temperature and then diluted with ethyl acetate. The organic layer was washed with 10% aqueous citric acid, brine and the organic layer dried (MgSO₄) and filtered. The solvent was removed in vacuo to give a residue which was purified by silica chromatography (1-10% methanol / dichloromethane) to afford morpholine-4- carboxylic acid [2-cyclohexyl-1 -(S)-(2-(S)-ethyl-4-hydroxy-tetrahydro-furan-3-(S)- ylcarbamoyl)-ethyl]-amide (4a) (171 mg, 50%) as a white solid. The structure was confirmed by NMR and MS analysis.

Morpholine-4-carboxylic acid f2-cyclohexyl-1 -( S)-(2-(S)-ethyl-4-oxo-tetrahydro-furan-3- ⁽S)-ylcarbamoyl)-ethyπ-amide

Morpholine-4-carboxylic acid [2-cyclohexyl-1 -(S)-(2-(S)-ethyl-4-hydroxy-tetrahydro- furan-3-(S)-ylcarbamoyl)-ethyl]-amide (4a) (171 mg, 0.42 mmol) was dissolved in dichloromethane (2 mL) and Dess-Martin reagent (193 mg, 0.45 mmol) was added. The reaction was stirred for 3 h, diluted with ethyl acetate and washed with aqueous 1M sodium thiosulphate and aqueous 1M sodium bicarbonate solutions. The organic layer was dried (MgSO₄) and the solvent removed in vacuo to give a white residue. The residue was purified by preparative HPLC (30-70% acetonitrile/ water) to give the title compound on lyophilisation, (20mg, 12%) as a white solid. H NMR (CD₃OD, 25 °C, 400MHz): delta 4.3 (1H, m), 4.18 (1H, dd, 1 and 16Hz), 3.99 (1H, d, 16Hz), 3.9 (2H, m), 3.65(4H, m), 3.39(4H, m), 1.5 (13H, m), 1.0(3H, t, 7Hz), 0.9 (2H, m)

LCMS 396 (100%) [M+H]⁺.

Example 4

Morpholine-4-carboxylic acid r2-cycloheptyl-1 -(S)-(2-(S)-ethyl-4-oxo-tetrahydro-furan-3-

(S)-ylcarbamoyl)-ethvπ-amide

[2-Cyclohepty-1 -(2-(S)-ethyl-4-hydroxy-tetrahydro-furan-3-(S)-ylcarbamoyl)-ethyl]- carbamic acid tert-butyl ester (3b), (as depicted in Scheme 1, Example 3) was synthesised in an identical manner to (3a) in Example 3 in 56% yield. The structure was confirmed by NMR and MS analysis.

Morpholine-4-carboxylic acid [2-cycloheptyl-1 -(S)-(2-(S)-ethyl-4-hydroxy-tetrahydro- furan-3-(S)-ylcarbamoyl)-ethyl]-amide (4b) as depicted in Scheme 1 , Example 3 was synthesized in an identical manner to (4a) in Example 3 with 47% yield. The structure was confirmed by NMR and MS analysis. The title compound morpholine-4-carboxylic acid [2-cycloheptyl-1-(S)-(2-(S)-ethyl-4-oxo- tetrahydro-furan-3-(S)-ylcarbamoyl)-ethyl]-amide was synthesised in identical manner to Example 3 above in 11% yield.

1H NMR (CD₃OD, 25 °C, 400MHz): delta 4.3 (1H, m), 4.19 (1H, dd, 1 and 17Hz), 4.0 (1 H, d, 17Hz), 3.95 (2H, m), 3.65(4H, m), 3.4(4H, m), 1.5(17H, m), 1.0 (3H, t, 7Hz)

LCMS 410 (100%), [M+H]⁺.

Example 5

3-{f(2S)-2-(morpholinocarbonyl)amino-3-cvclooctylpropanoyl1amino}-1,4-anhydro-3.5.6- trideoxy-L-erythro-hex-2-ulose

3-{[(2S)-2-(morpholinocarbonyl)amino-3-cyclooctylpropanoyl]amino}-1,4-anhydro-3,5,6- trideoxy-L-erythro-hex-2-ulose was prepared analogously to Example 3 from 1 ,4- anhydro-3-[(tert-butoxycarbonyl)amino]-3,5,6-trideoxy-L-arabino-hexitol (0.11 g, 0.47 mmol) and the building block of Example 2 (0.165 g, 0.55 mmol) to afford the title compound as an amorphous solid (0.080 g, 40% over 3 steps).

delta_H (400 MHz, DMSO-d6 at 298 K) 8.15 (1H, d, J 8, NH-furanone), 6.46 (1H, d, J 8, NH-urea), 4.19-4.09 and 3.95-3.81 (2H+3H, 2 m, alpha-H, H-1a, H-1b, H-3, H^), 3.51 (4H, m, moφholino), 3.28 (4H, m, moφholino), 1.69-1.13 (19H, m, H-5a, H-5b and CH₂- cyclooctyl), 0.91 (3H, t, H-6). LR-MS: Calcd for C^HasNsOg: 424.3. Found: 424 [M+H].

Example 6

C-5 Fluoroethylfuranone P1 building block

BocHN ^/Q a) 3-Azido-3-deoxy-1 ,2-O-isopropylidene-alpha-D-galactofuranose

A mixture of 3-azido-3-deoxy-1 ,2:5,6-di-O-isopropylidene-alpha-D-galactofuranose (T. L. Lowary and O. Hindsgaul, Carbohydr. Res., 251 (1994) 33-67) (3.88 g, 13.6 mmol) in 50% aq. acetic acid (150 mL) was stirred at room temperature for 7 h, then stored at 8 °C overnight. The solution was then concentrated, re-dissolved in dichloromethane (50 mL), and washed with 1M sodium hydrogen carbonate (1x50 mL). The aqueous layer was extracted with dichloromethane (4x40 mL), and the extracts were combined, dried (sodium sulphate), filtered and concentrated in vacuo. Flash chromatography of the residue gave the title compound as a syrup (2.4g, 72%) which crystallized upon standing, and recovered starting material (0.7g, 18%).

b) 3-Azido-6-O-benzyl-3-deoxy-1,2-O-isopropylidene-alpha-D-galactofuranose

A mixture of diol (1) (13.5 g, 55 mmol) and dibutyltin oxide (13.7 g, 55 mmol) in toluene (300 mL) was refluxed for 2 h in a round bottomed flask equipped with a Dean-Stark trap. The solution was then concentrated in vacuo, and to the residue was added caesium fluoride (16.7 g, 110 mmol) and the solids were lyophilized for 2 h. The solid was then suspended in DMF (200 mLI) and benzyl bromide (7.2 L, 61 mmol) was added. The reaction mixture was stirred at room temperature overnight, and then partitioned between ethyl acetate (500 mL) and water (200 mL). The organic layer was then washed with water (3x200 mL), dried (Na₂SO₄), filtered and concentrated in vacuo onto silica. The product was purified by column chromatography (stepwise gradient elution, ethyl acetate in petroleum ether, 20-25%) to afford the title compound as a slight yellow syrup of approximately 90% purity (14.7 g, 80%). Delta_H (400 MHz, CDCI₃ at 298 K) 7.38-7.27 (5H, m, Ar-H), 5.86 (1H, d, 3.9, H-1), 4.61 (1H, dd, J₂,3 1.5, H-2), 4.57 (2H, d, CFfePh), 4.19 (1H, m, H-3), 4.02-3.95 (2H, m, H-4 and H-5), 3.64 (1H, m, H-6b), 3.52 (1H, m, H-6a), 2.63 (1H, d, J₀H, H-₅2.6, OH), 1.56 and 1.34 (6H, 2s, C(CH₃)₂).

c) 3-Azido-6-O-benzyl-3,5-dideoxy-1 ,2-O-isopropylidene-beta-L-ara6/no- hexofuranose

A solution of (2) (9.5 g, 28.3 mmol) and 1,1'-thiocarbonyldiimidazole (6.56 g, 36.8 mmol) in dichloromethane (100 mL) and pyridine (4.6 mL, 57 mmol) was stirred at room temperature overnight. The reaction mixture was then diluted with dichloromethane (100 mL), washed successively with 1 sulphuric acid (2x 100 mL) and 1M sodium hydrogen carbonate (1x100 mL), then dried (Na₂SO4), filtered and concentrated in vacuo. The solid residue obtained was then re-dissolved in toluene (100 mL), heated to 80 °C, and then a solution of AIBN (2.79 g, 17 mmol) and tri-n-butyltin hydride (9.1 mL, 34 mmol) in toluene (80 mL) was added dropwise over approximately 40 min. The reaction was then stirred for another 40 min at 80 °C before being cooled and concentrated in vacuo onto silica. Flash chromatography of the residue with ethyl acetate in petroleum ether (stepwise gradient elution, 10-15%) gave the title compound as an oil (4.06 g, 45%).

d) 1 ,4-anhydro-3-azido-6-O-benzyl-3,5-dideoxy-L-ara6/t70-hexitol

To a stirred solution of (3) (4.06 g, 12.7 mmol) in dichloromethane (180 mL) at 0 °C was added dropwise a solution of trimethylsilyl trifluoromethane-sulphonate (4.6 mL, 25.4 mmol) in dichloromethane (25 mL) over approximately 30 min. Then triethylsilane (10.1 mL, 63.6 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was then washed with 1M sodium hydrogen carbonate (2x100 mL), and then concentrated in vacuo onto silica. Flash chromatography of the residue with ethyl acetate in petroleum ether (stepwise gradient elution, 20-50%) gave the title compound (4) as an oil (2.14 g, 64%).

d) 1,4-anhydro-6-O-benzyl-3[(tøtf-butoxycarbonyl)amino]-3,5- ideoxy-L-ataύ/no- hexitol

A mixture of (4) (0.78 g, 2.96 mmol), di-tert-butyl-dicarbonate (0.68 g, 3.11 mmol) and palladium on carbon (0.12 g, Acros, 10%) in 1:1 ethyl acetate-ethanol (20 mL), was hydrogenated at approximately atmospheric pressure for 3 h. The reaction mixture was filtered through celite and concentrated in vacuo. Flash chromatography of the residue with ethyl acetate in petroleum ether (stepwise gradient elution, 20-50%) afforded the title compound (5) as a crystalline solid (0.89 g, 89%).

Delta_H (400 MHz, CDCI₃ at 298 K) 7.38-7.27 (5H, m, Ar-H), 5.22 (1H, br s, NH), 4.50 (2H, s, CϋPh), 4.28 (1H, m, H-2), 4.00 (2H, m, H-1b and OH), 3.82 (1 H, dd, J4.4, 9.8, H-1a), 3.72 (1H, m, H-4), 3.62 (2H, m, H-6a and H-6b), 3.51 (1H, m, H-3), 2.06 (1H, m, H-5b), 1.93 (1H, m, H-5a), 1.44 (9H, s, C(CH₃)₃).

e) 1,4-anhydro-2-0-benzoyl-3-[(fetf-butoxycarbonyl)amino]-3,5-dideoxy-L-ara ) r7o- hexitol

To a solution of (5) (1.02 g, 3.03 mmol) and dimethylaminopyridine (0.185 g, 1.51 mmol) in dichloromethane (20 mL) and pyridine (0.73 mL, 9.07 mmol) at 0 °C was added benzoyl chloride (0.39 mL, 3.33 mmol). The reaction mixture was stirred at room temperature for 4 h, then additional benzoyl chloride (0.2 mL) was added and the reaction mixture was stirred overnight. The reaction mixture was then diluted with dichloromethane (80 mL), washed successively with 1 M sulphuric acid (2x50 mL) and 1M sodium hydrogen carbonate (1x50 mL), then dried (Na₂SO₄), filtered and concentrated in vacuo. Flash chromatography of the residue with ethyl acetate in petroleum ether (stepwise gradient elution, 20-40%) afforded a solid (1.25 g, 94%). A mixture of the benzoate (1.23 g, 2.79 mmol) and palladium on charcoal (0.1 g, Acros, 10%) in 2:1 ethyl acetate-ethanol (30 mL) was hydrogenated at approximately atmospheric pressure for 90 min, then filtered through celite and concentrated in vacuo to afford a crystalline solid (0.98 g, quantitative).

Delta_H (400 MHz, CDCI₃ at 298 K) 8.04 (2H, m, Ar-H), 7.58 (1H, m, Ar-H), 7.46 (2H, m, Ar-H), 5.34 (1H, m, H-2), 4.95 (1H, d, NH), 4.16 (1H, dd, J5.4, 10.7, H-1b), 4.06 (2H, m, H-1 a and H-3), 3.94 (1 H, m, H-3), 3.85 (2H, m, H-6a and H-6b), 2.85 (1 H, br s, OH), 2.02 (2H, m, H-5a and H-5b), 1.44 (9H, s, C(CH₃)₃).

f) 1,4-anhydro-3-[(fe/f-butoxycarbonyl)amino]-3,5,6-trideoxy-6-fluoro-L-ara-3/no- hexitol

BocHN .H^/θπ(7)

To a solution'W (6) (0.50 g, 1.42 mmol) in dichloromethane at 0 °C was added bis-(2- methoxyethyl)aminosulphurtrifluoride (0.39 mL, 2.13 mmol). The reaction mixture was stirred at room temperature overnight, then diluted with dichloromethane (40 mL), washed with 1 M sodium hydrogen carbonate (2x30 mL), then dried (Na₂SO₄), filtered and concentrated in vacuo onto silica. Flash chromatography of the residue with ethyl acetate in petroleum ether (stepwise gradient elution, 20-40%) afforded a syrup (0.31 g, 62%). This was then treated with 0.25M methanolic sodium methoxide (6 mL) at room temperature for 45 min, neutralized with Dowex H⁺resin and filtered. The filtrate was made alkaline with triethylamine and concentrated in vacuo. Column chromatography of the residue with ethyl acetate in petroleum ether (stepwise gradient elution, 40-60%) gave a crystalline solid (0.19 g, 85%).

Delta H (400 MHz, CDCI₃ at 298 K) 4.74 (1H, br s, NH), 4.66 (1H, m, H-6b), 4.54 (1H, m, H-6a), 4.29 (1H, m, H-2), 4.00 (1H, dd, J6.3, 9.8, H-1b), 3.84 (1 H, dd, J 3.9, 9.8, H-1 a), 3.74 (1H, m, H-4), 3.62 (1H, m, H-3), 3.52 (1H, br s, OH), 2.06 (2H, m, H-5a and H-5b), 1.46 (9H, s, C(CH₃)₃).

Example 7

3-fr(2S)-2-fmorpholinocarbonyl)amino-3-cvcloheptylpropanoyllamino1-1.4-anhvdro- 3,5,6-trideoxy-6-fluoro-L-ervthro-hex-2-ulose

a) 3-{[(2S)-2-(morpholinocarbonyl)amino-3-cycloheptylpropanoyl]amino}-1,4- anhydro-3,5,6-trideoxy-6-fluoro-L-arabino-hexitol

Compound (16) was prepared by coupling (7) as described in Example 6 (0.096 g, 0.39 mmol) and the building block of Example 1 (0.115 g, 0.40 mmol). Coupling took place in dichloromethane (2 mL) at 0 °C to which was added trifluoroacetic acid (2 mL) and the reaction was stirred at room temperature for 40 min. The solution was then concentrated in vacuo repeatedly from toluene (3x5mL). The residue was then added to N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. HCI (WSC.HCI) (0.081 g, 0.42 mmol), 1-hydroxybenzotriazole hydrate (HOBT) (0.064 g) and (9) (0.123g, 0.40mmol) in DMF (5mL) and triethylamine (0.16mL, 1.15mmol). The reaction was allowed to proceed at room temperature overnight before being concentrated in vacuo. The crude product was dissolved in ethyl acetate (25 mL), washed successively with 10% aqueous citric acid and 1 M sodium hydrogen carbonate, then dried (Na₂SO ), filtered and concentrated in vacuo. Flash chromatography of the residue with 2:3 toluene-ethyl acetate afforded

This compound was N-deprotected in a similar manner to that described for (5) in Example 6. To a solution of the residue in dichloromethane (4 mL) and triethyl amine (0.15 mL, 1.07 mmol) was added morpholine-4-carbonyl chloride (0.054 mL, 0.46 mmol) and the reaction was stirred at room temperature for 48 h. The solution was then concentrated in vacuo and flash chromatography of the residue with methanol in ethyl acetate (stepwise gradient elution, 0-5%) to afford (16) as an amorphous solid (0.083 g, 61%).

Delta_H (400 MHz, CDCI₃ at 298 K) 7.16 (1H, d, J 3.9, NH-furanol), 4.79 (1H, d, J 7.8, NH-urea), 4.64 (1 H, m, H-6b), 4.52 (1 H, m, H-6a), 4.34 (1 H, m, alpha-H), 4.26 (1 H, m, H-2), 4.02 (1H, dd, J 5.9, 9.8, H-1b), 3.84 (2H, m, H-1 a and H-4), 3.75 (1H, m, H-3), 3.69 (4H, m, moφholino), 3.35 (4H, m, morpholino), 2.11-1.98 (2H, m, H-5), 1.79-1.11 (15H, m, CH₂-cycloheptyl).

b) 3-{[(2S)-2-(moφholinocarbonyl)amino-3-cycloheptylpropanoyl]amino]-1,4- anhydro-3,5,6-trideoxy-6-fluoro-L-θ/yf 7ro-hex-2-ulose

To compound (16) (0.083 g, 0.19 mmol) in dichloromethane (5 mL) was added Dess- Martin periodinane (0.116 g, 0.27 mmol) and the reaction was stirred at room temperature for 2 h. The solution was then diluted with dichloromethane (2 mL) and washed with 1:1 10% aqueous sodium thiosulphate-1 M sodium hydrogen carbonate (3x10 mL), dried (Na₂SO4), filtered and concentrated in vacuo. Column chromatography of the residue using ethyl acetate afforded the title compound (17) as an amorphous solid (0.057 g, 69%).

Delta_H (400 MHz, CDCI₃ at 298 K) 7.39 (1 H, d, J 7.3, NH-furanone), 4.90 (1 H, d, J 7.3, NH-urea), 4.69 (1 H, m, H-6b), 4.58 (1H, m, H-1a), 4.36 (1H, m, alpha-H), 4.24-4.07 (3H, m, H-1 a, H-1 b, H-4), 3.83 (1 H, dd, J 7.8, 9.8, H-3), 3.69 (4H, m, moφholino), 3.35 (4H, m, morpholino), 2.28-2.02 (2H, m, H-5a and H-5b), 1.77-1.12 (15H, m, CH₂- cycloheptyl).

Deltac (100 MHz, CDCI₃ at 298 K, inter alia) 210.6 (C=O), 173.9, 157.8 (amide- and urea carbonyls), 80.5 (C-6, J_C,_F 165), 76.6 (O ), 71.0 (C-1), 66.6 (moφholino), 60.1 (C- 3), 52.6 (alpha-C), 34.7 (C-5). LR-MS: Calcd for C₂ι H₃₅FN₃O₅: 428.3. Found: 428.1 [M+H].

Example 8

3-(r(2S)-2-(morpholinocarbonyl)amino-3-cvclooctylpropanovnamino}-1.4-anhvdro-3,5,6- trideoxy-6-fluoro-L-erythro-hex-2-ulose

a) 3-{[(2S)-2-(morpholinocarbonyl)amino-3-cyclooctylpropanoyl]amino}-1 ,4- anhydro-3,5,6-trideoxy-6-fluoro-L-arai3/A70-hexitol

This was prepared in a similar manner to Example 7, starting from the Example 3 building block (0.082g, 0.33mmol) and the Example 2 building block (0.103g, 0.35mmol), to afford (18) as an amorphous solid (0.088g, 73%).

Delta_H (400 MHz, CDCI3 at 298 K) 7.28 (1 H, d, J 3.9, NH-furanoI), 4.85 (1 H, d, J 7.8, NH-urea), 4.64 (1H, m, H-6b), 4.52 (1 H, m, H-6a), 4.37 (1 H, m, alpha-H), 4.26 (1H, m, H-2), 4.01 (1H, dd, J 5.8, 9.8, H-1b), 3.84 (2H, m, H-1 a and H-4), 3.75 (1H, m, H-3), 3.68 (4H, m, moφholino), 3.35 (4H, m, morpholino), 2.11-1.95 (2H, m, H-5a and H-5b), 1.76-1.23 (17H, m, CH₂-cyclooctyl).

b) 3-{[(2S)-2-(morpholinocarbonyl)amino-3-cyclooctylpropanoyl]amino}-1,4- anhydro-3,5,6-trideoxy-6-fluoro-L-ery 7ro-hex-2-ulose

Compound (18) (0.088g, 0.20mmol) was oxidized similarly to what is described above, to afford the title compound (19) as an amorphous solid (0.067g, 76%).

Delta_H (400 MHz, CDCI₃ at 298 K) 7.69 (1H, d, J6.8, NH-furanone), 5.14 (1H, d, J7.3, NH-urea), 4.69 (1H, m, H-6b), 4.57 (1H, m, H-6a), 4.38 (1H, m, alpha-H), 4.21 (1H, m, H-4), 4.13 (2H, d, J 4.4, H-1 a and H-1 b), 3.82 (1 H, dd, J 7.3, 9.8), 3.68 (4H, m, morpholino), 3.34 (4H, m, morpholino), 2.27-2.02 (2H, m, H-5a and H-5b), 1.73-1.21 (17H, m^l,OH₂-cyclooctyl).

Deltac (100 MHz, CDCI₃ at 298 K, selected signals) 210.5 (C=O), 174.2, 157.8 (amide- and urea carbonyls), 80.4 (C-6, J_C,_F 165), 76.6 (C-4), 71.0 (C-1), 66.6 (morpholino), 60.1 (C-3), 52.7 (C), 34.9 (C-5). LR-MS: Calcd for CszHsrFNsOs: 442.3. Found: 442.2 [M+H].

Example 9

3-{r(2S)-2-(morpholinocarbonyl)amino-3-cvclohexylpropanovnamino1-1.4-anhydro-3.5_<6- trideoxy-6-fluoro-L-ervthro-hex-2-ulose

The title compound was prepared analogously to Example 7 using commercially available cyclohexylalanine which was N-protected with Boc.

LC-MS: Calculated for C^H^FNsOs: 413.23. Found 414.1 [M+H]⁺\ 412.2 [M-H]\

Example 10 P1 building block

a) 3-Azido-3-deoxy-1 ,2-O-isopropylidene-alpha-D-galactofuranose

A mixture of 3-azido-3-deoxy-1 ,2:5,6-di-O-isopropylidene-alpha-D-galactofuranose (T. L. Lowary and O. Hindsgaul, Carbohydr. Res., 251 (1994) 33-67) (3.88 g, 13.6 mmol) in 50% aq. acetic acid (150 mL) was stirred at room temperature for 7 h, then stored at 8 °C overnight. The solution was then concentrated, re-dissolved in dichloromethane (50 mL), and washed with 1M sodium hydrogen carbonate (1x50 mL). The aqueous layer was extracted with dichloromethane (4x40 mL), and the extracts were combined, dried (sodium sulphate), filtered and concentrated in vacuo. Flash chromatography of the residue gave the title compound as a syrup (2.4g, 72%) which crystallized upon standing, and recovered starting material (0.7g, 18%).

b) 3-Azido-6-O-benzyl-3-deoxy-1,2-O-isopropylidene-alpha-D-galactofuranose

A mixture of diol (1) (13.5 g, 55 mmol) and dibutyltin oxide (13.7 g, 55 mmol) in toluene (300 mL) was refluxed for 2 h in a round bottomed flask equipped with a Dean-Stark trap. The solution was then concentrated in vacuo, and to the residue was added caesium fluoride (16.7 g, 110 mmol) and the solids were lyophilized for 2 h. The solid was then suspended in DMF (200 mLI) and benzyl bromide (7.2 mL, 61 mmol) was added. The reaction mixture was stirred at room temperature overnight, and then partitioned between ethyl acetate (500 mL) and water (200 mL). The organic layer was then washed with water (3x200 mL), dried (Na₂SO ), filtered and concentrated in vacuo onto silica. The product was purified by column chromatography (stepwise gradient elution, ethyl acetate in petroleum ether, 20-25%) to afford the title compound as a slight yellow syrup of approximately 90% purity (14.7 g, 80%).

Delta_H (400 MHz, CDCI₃ at 298 K) 7.38-7.27 (5H, m, Ar-H), 5.86 (1 H, d, J3.9, H-1), 4.61 (1H, dd, J₂,₃ 1.5, H-2), 4.57 (2H, d, CHP ), 4.19 (1H, m, H-3), 4.02-3.95 (2H, m, H-4 and H-5), 3.64 (1H, m, H-6b), 3.52 (1H, m, H-6a), 2.63 (1H, d, J_OH, _H-52.6, OH), 1.56 and 1.34 (6H, 2s, C(CH₃)₂).

c) 3-Azido-6-O-benzyl-3,5-dideoxy-1,2-O-isopropylidene-beta-L-ara6//70- hexofuranose

A solution of (2) (9.5 g, 28.3 mmol) and 1,1'-thiocarbonyldiimidazole (6.56 g, 36.8 mmol) in dichloromethane (100 mL) and pyridine (4.6 mL, 57 mmol) was stirred at room temperature overnight. The reaction mixture was then diluted with dichloromethane (100 mL), washed successively with 1 M sulphuric acid (2x 100 mL) and 1 M sodium hydrogen carbonate (1x100 mL), then dried (Na₂SO₄), filtered and concentrated in vacuo. The solid residue obtained was then re-dissolved in toluene (100 mL), heated to 80 °C, and then a solution of AIBN (2.79 g, 17 mmol) and tri-n-butyltin hydride (9.1 mL, 34 mmol) in toluene (80 mL) was added dropwise over approximately 40 min. The reaction was then stirred for another 40 min at 80 °C before being cooled and concentrated in vacuo onto silica. Flash chromatography of the residue with ethyl acetate in petroleum ether (stepwise gradient elution, 10-15%) gave the title compound as an oil (4.06 g, 45%).

d) 1 ,4-anhydro-3-azido-6-O-beπzyl-3,5-dideoxy-L-arad/fJθ-hexitol

To a stirred solution of (3) (4.06 g, 12.7 mmol) in dichloromethane (180 mL) at 0 °C was added dropwise a solution of trimethylsilyl trifluoromethane-sulphonate (4.6 mL, 25.4 mmol) in dichloromethane (25 mL) over approximately 30 min. Then triethylsilane (10.1 mL, 63.6 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was then washed with 1 M sodium hydrogen carbonate (2x100 mL), and then concentrated in vacuo onto silica. Flash chromatography of the residue with ethyl acetate in petroleum ether (stepwise gradient elution, 20-50%) gave the title compound (4) as an oil (2.14 g, 64%).

d) 1 ,4-anhydro-6-0-benzyl-3[(fert-butoxycarbonyl)amino]-3,5-dideoxy-L-ata6/t70- hexitol

A mixture of (4) (0.78 g, 2.96 mmol), di-tert-butyl-dicarbonate (0.68 g, 3.11 mmol) and palladium on carbon (0.12 g, Acros, 10%) in 1:1 ethyl acetate-ethanol (20 mL), was hydrogenated at approximately atmospheric pressure for 3 h. The reaction mixture was filtered through celite and concentrated in vacuo. Flash chromatography of the residue with ethyl acetate in petroleum ether (stepwise gradient elution, 20-50%) afforded the title compound (5) as a crystalline solid (0.89 g, 89%).

Delta_H (400 MHz, CDCI₃ at 298 K) 7.38-7.27 (5H, m, Ar-H), 5.22 (1H, br s, NH), 4.50 (2H, s, CJ±>Ph), 4.28 (1 H, m, H-2), 4.00 (2H, m, H-1 b and OH), 3.82 (1 H, dd, J 4.4, 9.8, H-1a), 3.72 (1H, m, H-4), 3.62 (2H, m, H-6a and H-6b), 3.51 (1H, m, H-3), 2.06 (1H, m, H-5b), 1.93 (1H, m, H-5a), 1.44 (9H, s, C(CH₃)₃).

Example 11

3-{r(2S)-2-(morpholinocarbonyl)amino-3-cycloheptylprooanoyllamino}-1.4-anhvdro-3.5- dideoxy-L-eryf/7/O-hex-2-ulose

a) 3-{[(2S)-2-(te/f-butoxycarbonyI)amino-3-cycloheptylpropanoyl]amino}-1,4- anhydro-6-O-benzyl-3,5-dideoxy-L-ara6/πo-hexitol.

1)

To a solution of P1 building block (5) (0.13 g, 0.38 mmol) in dichloromethane (2 mL) at 0 °C was added trifluoroacetic acid (2 mL) and the reaction was stirred at room temperature for 40 min. The solution was then concentrated in vacuo repeatedly from toluene (3x5mL). The residue was then added to N-ethyl-N'-(3- dimethylaminopropyl)carbodiimide. HCI (WSC.HCI) (0.081 g, 0.42 mmol), 1- hydroxybenzotriazole hydrate (HOBT) (0.064 g) and P2 building block from Example 1 (0.123g, 0.40mmol) in DMF (5mL) and triethylamine (0.16mL, 1.15mmol). The reaction was allowed to proceed at room temperature overnight before being concentrated in vacuo. The crude product was dissolved in ethyl acetate (25 mL), washed successively with 10% aqueous citric acid and 1 sodium hydrogen carbonate, then dried (Na₂SO₄), filtered and concentrated in vacuo. Flash chromatography of the residue with 2:3 toluene-ethyl acetate afforded (11) as a foam (0.18 g, 93%).

b) 3-{[(2S)-2-(morpholinocarbonyl)amino-3-cycloheptylpropanoyl]amino}-1 ,4- anhydro-6-O-benzyl-3,5-dideoxy-L-arajb//70-hexitol

Compound (11) (0.18 g, 0.36 mmol) was N-deprotected in a similar manner to that described for (5) above. To a solution of the residue in dichloromethane (4 mL) and triethyl amine (0.15 mL, 1.07 mmol) was added morpholine-4-carbonyl chloride (0.054 mL, O.46 mmol) and the reaction was stirred at room temperature for 48 h. The solution was then concentrated in vacuo and flash chromatography of the residue with methanol in ethyl acetate (stepwise gradient elution, 0-5%) afforded a hard syrup (0.13 g, 70%).

Delta_H (400 MHz, CDCI₃ at 298 K) 7.38-7.27 (5H, m, Ar-H), 7.14 (1H, d, J2.9, NH- "furanol"), 4.82 (1H, d, J7.8, NH-"urea"), 4.55 (2H, 2d, PhCϋ>), 4.26 (2H, m, H-2 and alpha-H), 4.01 (1H, dd, J6.8, 9.8, H-1b), 3.84 (2H, m, H-1 a and H-4), 3.72-3.59 (7H, m, H-6a+b, H-3, moφholino), 3.37-3.26 (4H, m, moφholino), 2.06 (1H, m, H-5b), 1.92 (1 H, m, H-5a), 1.73-1.07 (15H, m, CH₂-cycloheptyl).

c) 3-{[(2S)-2-(morphoIinocarbonyl)amino-3-cycloheptylpropanoyl]amino}-1,4- anhydro-3,5-dideoxy-L-e/ _ ?ro-hex-2-ulose.

To a solution of (12) (0.13 g, 0.25 mmol) in dichloromethane (5 mL) was added Dess- Martin periodinane (0.116 g, 0.27 mmol) and the reaction was stirred at room temperature for 2 h. The solution was then diluted with dichloromethane (2 mL) and washed with 1:1 10% aqueous sodium thiosulphate-1 M sodium hydrogen carbonate (3x10 mL), dried (Na₂SO4), filtered and concentrated in vacuo. Column chromatography of the residue using ethyl acetate afforded a solid (0.11 g, 86%). The keto compound (0.098 g, 0.19 mmol) in 1:1 ethyl acetate-ethanol (10 mL) was hydrogenated in the presence of palladium on carbon (0.037 g, Acros 10%) at approximately atmospheric pressure for 1 h. The suspension was then filtered through celite and concentrated in vacuo. Column chromatography of the residue using ethyl acetate-methanol (9:1 ) afforded (13) as an amoφhous solid (0.067 g, 83%).

Delta_H (400 MHz, CDCI₃ at 298 K) 7.20 (1 H, d, J 7.8, NH-furanone), 4.97 (1 H, d, J 7.8, NH-urea), 4.31-4.18 (3H, m, H-1b, H-4 and alpha-H), 4.13-4.03 (2H, m, H-1 a and H-3), 3.78 (2H, m, H-6a and H-6b), 3.68 (4H, m, moφholino), 3.35 (4H, m, morpholino), 2.94 (1H, br s, OH), 2.10 (1H, m, H-5b), 1.96 (1 H, m, H-5a), 1.78-1.13 (15H, m, CH₂- cydoheptyl). Delta_c (100 MHz, CDCI₃ at 298 K, inter alia) 221.2 (C=O), 174.1 , 158.0 (amide and urea carbonyls), 78.8 (C-4), 71.0 (C-1 ), 66.6 (morpholino), 59.6 (C-3), 59.0 (C-6), 53.2 (alpha-C), 44.3 (moφholino), 36.3 (C-5). LR-MS: Calcd for C^H^Nc .: 426.3. Found: 426.5 [M+H].

Example 12 3-([(2S)-2-(morpholinocarbonyl)amino-3-cycloheptylpropanoynamino}-1.4-anhvdro-3.5- dideoxy-L-er /7ro-hex-2-ulose

a) 3-{[(2S)-2-(morpholinocarbonyl)amino-3-cyclooctylpropanoyl]amino}-1,4- anhydro-6-O-benzyl-3,5-dideoxy-L-arad/no-hexitol

Compound 14 was prepared in a similar manner to Example 11 starting from P1 building block (5) (0.15 g, 0.44 mmol) and the P2 building block of Example 2 (0.14 g, 0.47 mmol), to afford (14) as a foam (0.16 g, 69% over 2 steps).

Delta_H (400 MHz, CDCI₃ at 298 K) 7.38-7.27 (5H, m, Ar-H), 7.13 (1H, d, J2.9, NH- furanol), 4.81 (1 H, d, J 7.8, NH-urea), 4.55 (2H, 2d, PhChU), 4.26 (2H, m, H-2 and alpha-H), 4.00 (1H, dd, 6.3, H-1b), 3.84 (2H, m, H-1 a and H-4), 3.65 (7H, m, morpholino, H-3 and H-6), 3.32 (4H, m, morpholino), 2.07 (1H, m, H-5b), 1.93 (1H, m, H-5a) 1.71-1.17 (17H, m, CH₂-cyclooctyl).

b) 3-{[(2S)-2-(morpholinocarbonyl)amino-3-cycloheptylpropanoyl]amino}-1,4- anhydro-3,5-dideoxy-L-e/ -Λro-hex-2-ulose

The title compound was prepared in a similar manner to Example 11 , starting from (14) (0.16 g, 0.30 mmol) which gave first the benzyl protected keto-compound (0.114 g, 72%). Hydrogenation of this benzyl protected keto-compound (0.100 g, 0.19 mmol) gave the title compound (15) as an amoφhous solid (0.070 g, 82% over 2 steps).

Delta_H (400 MHz, CDCI₃ at 298 K) 7.25 (1 H, d, J 7.3, NH-furanone), 5.02 (1 H, d, J 7.8, NH-urea), 4.30 (1H, m, alpha-H), 4.22 (2H, H-1 b and H-4), 4.06 (2H, m, H-1 a and H-3), 3.78 (2H, m, H-6), 3.68 (4H, m, morpholino), 3.35 (4H, m, morpholino), 2.94 (1H, brs, OH), 2.09 (1H, m, H-5b), 1.96 (1H, m, H-5b), 1.78-1.23 (17H, m, CH2-cyclooctyl).

Deltac (100 MHz, CDCI₃ at 298 K, inter alia) 211.2 (C=O), 174.1 , 158.0 (amide and urea carbonyls), 78.8 (C-4), 70.8 (C-1), 66.6 (morpholino), 59.9 (C-3), 59.1 (C-6), 52.8 (alpha-C), 44.3 (moφholino), 36.0 (C-5). LR-MS: Calcd for C₂iHs6N₃O6: 440.3. Found: 440.5 [M+H]. Example 13

3-{[(2S)-2-(morpholinocarbonyl)amino-3-cyclohexylpropanovπamino}-1.4-anhydro-3,5- dideoxy-L-e/v-ftro-hex-2-ulose

The title compound was prepared as a white powder, analogously to Example 12 using commercially available cyclohexylalanine N-protected with Boc.

LC-MS: Calcd for C2oH₃₃N₃O₆: 411.24. Found: 412.2 [M+H]⁺, 410.2 [M-H]\

Biological Examples

Compounds of the invention and comparative prior art compounds are assayed as follows:

Biological Example 1 Cathepsin S Ki determination

The assay uses baculovirus-expressed human cathepsin S and the boc-Val-Leu-Lys- AMC fluorescent substrate available from Bachem in a 384 well plate format, in which 7 test compounds can be tested in parallel with a positive control comprising a known cathepsin S inhibitor comparator.

Substrate dilutions

280μl/well of 12.5% DMSO are added to rows B - H of two columns of a 96 deep well polypropylene plate. 70μl/well of substrate is added to row A. 2 x 250μl/well of assay buffer (1 OOmM Na p osphate, 10OmM NaCI, pH 6.5) is added to row A, mixed, and double diluted down the plate to row H. Inhibitor dilutions.

10Oμl/well of assay buffer is added to columns 2-5 and 7-12 of 4 rows of a 96 well V bottom polypropylene plate. 200μl/well of assay buffer is added to columns 1 and 6.

The first test compound prepared in DMSO is added to column 1 of the top row, typically at a volume to provide between 10 and 30 times the initially determined rough Kj. The rough Ki is calculated from a preliminary run in which 10 μl/well of 1mM 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 10mM 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 and180 μl to row A.Mix row A using a multichannel pipette and double dilute to row G. Mix row H and read in the fluorescent spectrophotometer. The readings are Prism data fitted to the competitive inhibition equation, setting S = 100μM and K_M = 100μM to obtain an estimate of the Kj, up to a maximum of 100μM.

The second test compound is added to column 6 of the top row, the third to column 1 of the second row etc. Add 1 μl of comparator to column 6 of the bottom row. Mix column 1 and double dilute to column 5. Mix column 6 and double dilute to column 10.

Using an 8-channel multistepping pipette set to 5 x 10μl, distribute 10μl/well of substrate to the 384 well assay plate. Distribute the first column of the substrate dilution plate to all columns of the assay plate starting at row A. The tip spacing of the multichannel pipette will correctly skip alternate rows. Distribute the second column to all columns starting at row B.

Using a 12-channel multistepping pipette set to 4 x 10μl, distribute 10μl/well of inhibitor to the 384 well assay plate. Distribute the first row of the inhibitor dilution plate to alternate rows of the assay plate starting at A1. The tip spacing of the multichannel pipette will correctly skip alternate columns. Similarly, distribute the second, third and fourth rows to alternate rows and columns starting at A2, B1 and B2 respectively. Mix 20ml assay buffer and 20μl 1 M DTT. Add sufficient cathepsin S to give 2nM final concentration.

Using the a distributor such as a Multidrop 384, add 30μ 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.

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.

Biological Example 2 Cathepsin K Ki

The procedure of biological example 1 with the following amendments is used for the determination of Ki for cathepsin K.

The enzyme is E coli expressed human cathepsin K. The substrate is H-D-Ala-Leu-Lys- AMC from Bachem. The assay buffer is 100 mM Na phosphate, 1mM EDTA, 0.1% PEG 4000, pH 6.5. The DMSO stock (see substrate dilutions is 10%. 56 ul of substrate is added to row A and 2 x 256 ul of buffer is added to row A. The final cathepsin K concentration is 0.5 nM.

Biological Example 3 Permeability

This example measures transport of inhibitors through the cells of the human gastroenteric canal. The assay uses the well known Caco-2 cells with a passage number between 40 and 60.

Apical to basolateral transport Generally every compound will be tested in 2-4 wells. The basolateral and the apical wells will contain 1.5 mL and 0.4 mL transport buffer (TB), respectively, and the standard concentration of the tested substances is 10 μM. Furthermore all test solutions and buffers will contain 1% DMSO. Prior to the experiment the transport plates are pre- coated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material. After 21 to 28 days in culture on filter supports the cells are ready for permeability experiments.

Transport plate no 1 comprises 3 rows of 4 wells each. Row 1 is denoted Wash, row 2 "30 minutes" and row 3 "60 minutes". Transport plate no 2 comprises 3 rows of 4 wells, one denoted row 4 "90 minutes", row 5 "120 minutes and the remaining row unassigned.

The culture medium from the apical wells is removed and the inserts are transferred to a wash row (No. 1) in a transport plate (plate no.1) out of 2 plates without inserts, which have already been prepared with 1.5 mL transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5. In A→B screening the TB in basolateral well also contains 1% Bovine Serum Albumin.

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts and the celf^; 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), see Figure 1 and 2. 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, FA^m, versus time. FA^m is calculated from:

^FAcum ⁼ - TT^"

*-DI

Where CRJ is the receiver concentration at the end of the interval i and CQJ 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:

where k is the transport rate (min^-'' ) 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), and A is the area of the filter (cm²). Reference compounds

Example 4

Cellular cathepsin S Ki

This example describes procedures for assessing potency of cathepsin S inhibitors on inhibition of in vitro T cell activation by determining concentration of the compound required for reducing 50% of the IL-2 secretion in T cells stimulated with compound- treated antigen presenting cells in an antigen presentation assay using the 19.3 cells and the 9001 cells as the effector cells and the antigen presenting cells, respectively. 19.3 cells are murine T cell hybridomas recognizing type II collagen (260-272) in the context of HLA-DR1 , and 9001 is an EBV-transformed human B cell line expressing homozygous DR1 molecule. The 9001 cells will be pre-treated with varying concentration of the compounds for 1 hour and then incubated with the T cells in the presence of collagen at a final concentration of 0.1 mg/ml. The cultures will be incubated overnight at 37°C with 5% CO₂ and amount of IL-2 in the supernatant determined with ELISA. The ICso-IL-2 values representing the concentration of compounds at which secretion of IL-2 from the T cells is reduced by 50% will be determined by regression analysis Major histocompatibility complex (MHC) class II molecules bind peptides generated by degradation of endocytosed antigens and display them as MHC class ll-peptide complexes at the cell surface for recognition by CD4+ T cells. MHC class II molecules are assembled with the assistance of invariant chain (li) in the endoplasmic reticulum (ER) and transported to an endocytic compartment where li undergoes rapid degradation by endosomal and lysosomal proteases. A peptide fragment of li, CLIP (class ll-associated Invariant chain Peptides) remains bound in the class II peptide binding groove, until removed by the chaperone molecule H-2M in mouse or HLA-DM in humans. This allows peptides derived from proteolytic degradation of foreign and self proteins to bind class II molecules and subsequently to be presented to T cells in the context of MHC molecules. In dendritic cells and B cells, cathepsin S is required for complete invariant chain processing and CLIP generation. Inactivating cathepsin S with inhibitors will impair MHC class II peptide loading and formation of stable MHC/peptide complexes leading to reduced antigen presentation and T cell activation.

To assess the potency of the cathepsin S inhibitors, an antigen presentation assay uses a collagen specific, HLA-DR1 restricted mouse T cell hybridoma (19.3) as effector cells, human EBV-transformed B cells (9001) as antigen presenting cells (APC), and mlL-2 ELISA as the read-out system. Inhibition of Cathepsin S with specific inhibitors will impair the processing and presentation of collagen in APCs which in turn reduces the activation of the T cells. The extent of inhibition on T cells is measured by the degree of reduction in IL-2 secretion. ICso-IL-2 represents the concentration of compounds at which secretion of IL-2 from the T cells is reduced by 50% in the antigen presentation assay.

MATERIALS

Cathepsin S inhibitors

Compounds will be dissolved in DMSO to a final concentration of 10 mM, aliquotted, and stored at -80 C until used.

Cells All the cells will be cultured in DMEM medium (Invitrogen, cat #11995-065) supplemented with 10% fetal bovine serum (Hyclone, cat #SH30070.03), 100 U/ml penicillin, 100 ug/ml streptomycin and 2 mM L-glutamine (Invitrogen, cat #10378-016).

T cell: 19.3, murine DR1 transgenic T cell hybridomas, DR1 restricted, Type II collagen 260-272 specific

Antigen presentation cells (APCs): 9001, EBV-transformed human B cells expressing homozygous DR1

Antigen

Type II collagen from chicken sternal cartilage (Sigma, cat. # C-9301) will be dissolved in PBS at 1 mg/ml and stored in aliquots at -80 C.

EQUIPMENT

Tissue culture incubator (Forma Scientific, model. #3120) Sorvall centrifuge (Sorvall RC-3B) Plate washer Plate-reader (Tecan, Spectra shell, cat. #20-074)

PROCEDURES

Antigen presentation assay

1. Two-fold serial dilutions of the compounds, starting at 400uM in AIMV medium, will be transferred to a 96-well round-bottom microtiter plate at a volume of 50ul/well. 2. Antigen-presenting cells will be washed and resuspended in AIMV medium to a density of 0.8x10⁶/ml, and then added to the plates at a volume of 50ul/well, giving the number of cells per well as 40,000.

3. The APCs will be pretreated with compounds for 1 hour at 37C with 5% CO₂.

4. The T cells will be washed and resuspended in AIMV to a density of 0.8x10⁶/ml. 5. The antigen will be diluted to a 4X concentration in AIMV and mixed 1 to 1 with the

T cells. 6. The T cells/antigen mixture will then be added to the assay plates at a volume of 100ul/well.

7. The plates will be incubated overnight at 37C with 5% CO₂.

8. Supernatant will be carefully removed from each well and measured for amount of IL-2 with ELISA.

IL-2 ELISA

Mouse IL-2 ELISA kits will be purchased from Pharmingen (Mouse IL-2 OptEIA set, #2614KI). The ELISA will be performed per manufacturer's instruction.

1. Anti-mlL-2 antibodies will be diluted in carbonate buffer to a final concentration of 2 ug/ml, transferred to an ELISA plate (Costar) at 100 ul/well and then incubated overnight at 4°C.

2. The ELISA plates will be washed 4 times with PBS/0.5% FBS containing 0.05% Tween 20 (wash buffer).

3. The plates will be blocked with the blocking buffer, 10% FBS (fetal bovine serum, Hyclone) for 2 hrs at room temperature (RT) and then washed 4 times with wash buffer.

4. 100 μL of supernatants from each well of the assay plates will be transferred to the ELISA plate and incubated for 2 hrs at RT.

5. After washing 4 times, the plate will be incubated for 1 hr at RT with a mixture of a biotinylated anti-mlL2 antibody and avidin-HRP prepared in blocking buffer. 6. Following 8 washes with wash buffer, the substrate (TMB) will be added to the plate and incubated at RT for 15-30 minutes until the color develops.

7. Color development will be terminated by the addition of 2N sulfuric acid.

8. The plates will be measured at 450 nm with an ELISA plate reader (Spectra, Tecan). 9. A set of purified recombinant mlL-2 with known concentration will be prepared from the stock solution (provided in the kit) with the blocking buffer and assayed in each plate to provide a standard curve for quantification of IL-2.

DETERMINATION OF IC50-IL-2 OF CATHEPSIN S INHIBITORS The potency of each compound will be measured by the IC50 value derived from this assay. IC₅₀ represents the concentration of compound at which secretion of IL-2 from the T cells is reduced by 50%.

The absorbance at 450 nm from each well will be converted into amount of IL-2 (pg/ml) using the Winselect software (Tecan) based on the standard curve generated from in- plate standards of purified recombinant mlL-2. Means and standard deviations will be calculated from triplicates with Excel.

The average amounts of IL-2 (pg/ml) from triplicates of both the test and the control wells (received comparable amount of DMSO) will be used to generate the percent inhibition using the following formula.

Percent Inhibition = average of control wells - average of test wells x 100 average of control wells

A dose response curve will be generated by plotting the percent inhibition versus concentration of the compound and the IC₅₀-IL-2 value will be calculated with regression analysis.

DR-1 transgenic T cell hybridoma has been prepared by E. Rosloniec, University of Tennessee.

Following controls are included and analyzed as appropriate:

T + APCs, without antigen, without compound treatment, for background signal. We usually get negligible amounts of IL-2 form these wells, and usually don't perform background subtraction.

T +- APCs, with anti-CD3/CD28, with compounds, for toxicity associated with compounds. T + APCs, with antigen, with DMSO (comparable to those received compounds), for toxicity associated with DMSO and for calculation of percent of inhibition.

Comparative Trial

Compounds of the invention and the closest prior art were assayed in the assays above and produced the results tabulated below.

Table 1 Comparative trial

It will thus be apparent that the compounds of the invention show 4 to 8 fold (R=H), 9.4 to 40 fold (R = F) or 4.5 to 14.2 fold (R= OH) better activity against cathepsin S measured as Ki relative to the prior art. When cathepsin S activity is measured as IC₅₀, the compounds of the invention show 4.2 to 18 fold, 10.6 to 60 fold or 11-15 fold, respectively, better potency than the prior art.

However it will also be apparent that not only is potency significantly improved by the compounds of the invention, but selectivity over the closely related cathepsin K enzyme is also enhanced. In particular the compounds of the invention can be dosed some 6 to 137 times (R = H), 6 or 7 fold (R = F) or 14-23 fold (R = OH) greater than the prior art before imparting the same undesirable inhibitory effect on cathepsin K as the prior art (defined as a reduction by of cathepsin K activity by half).

Additionally, the compounds of the invention typically display at least as good or better pharmacokinetics than the prior art. For example, the cell permeability of the presently claimed R = H compounds, as assessed in the Caco-2 system, is 5.8 to 6.6 fold better than the prior art.

All references including patent and patent applications referred to in this application are incoφorated 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 or step or group of integers but not to the exclusion of any other integer or step or group of integers or steps.

Claims

Claims

1. A compound of the formula I:

where R is H, F or OH, Q is -(CH₂)_n- and n is 1 , 2 or 3; or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 , wherein Q is ethylene.

3. A compound according to claim 1 , wherein Q is propylene.

4. A compound according to claim 1 , wherein Q is methylene.

5. A compound according to any one of claims 1 to 4, wherein R is H.

6. A compound according to any one of claims 1 to 3, wherein R is F.

7. A compound according to any one of claims 1 to 3, wherein R is OH.

8. A compound according to claim 4, wherein R is F or OH.

9. A compound according to claim 1 which is any one of Examples 1 to 13, or a pharmaceutically acceptable salt thereof.

10. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 9 and a pharmaceutically acceptable carrier or diluent therefor.

11. Use of a compound according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment or prophylaxis of disorders dependent on cathepsin S.

12. Use according to claim 11 , wherein the disorder is an autoimmune disorder such as MS, RA, juvenile diabetes or asthma.

13. Use according to claim 11 , wherein the disorder is chronic pain.

14. A method for the treatment or prophylaxis of disorders dependent upon cathepsin S comprising the administration of an effective amount of a compound as defined in any one of claims 1 to 9 to an individual suffering from or threatened with the disorder.

15. A method according to claim 14, wherein the disorder is an autoimmune disorder such as MS, RA, juvenile diabetes or asthma.

16. A method according to claim 14, wherein the disorder is chronic pain.

17. A compound of the formula:

PG-HN °-^pG' where R is F, OH or OPG";

PG is an N protecting group or H;

PG' is H, an hydroxyl protecting group or together with the adjacent oxygen defines a keto group; and

PG" is an hydroxyl protecting group.