WO2007144379A1 - Bicyclic compounds useful as cathepsin s inbhibitors - Google Patents

Abstract

Compounds of formula (I), wherein R1, R2, R3, Ra and E are are defined within, and pharmaceutically acceptable salts, solvates, hydrates and N-oxides thereof having utility in the treatment of disorders mediated by cathepsin S.

Description

BICYCLIC COMPOUNDS USEFUL. AS CATHEPSIN S INBHIBITORS

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

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

In WO 97/40066, the use of inhibitors against Cathepsin S is described. The inhibition of this enzyme is suggested to prevent or treat disease caused by protease activity. Cathepsin S is a highly active cysteine protease belonging to the papain superfamily. Its primary structure is 57%, 41 % and 45% homologous with that of the human cathepsin L and H and plant cysteine proteases papain respectively, although only 31 % homologous with cathepsin B. It is found mainly in B cells, dendritic cells and macrophages and this limited occurrence suggests the potential involvement of this enzyme in the pathogenesis of degenerative disease. Moreover, it has been found that destruction of Ii by proteolysis is required for MHC class Il molecules to bind antigenic peptides, and for transport of the resulting complex to the cell surface. Furthermore, it has been found that Cathepsin S is essential in B cells for effective Ii proteolysis necessary to render class Il molecules competent for binding peptides. Therefore, the inhibition of this enzyme may be useful in modulating class ll-restricted immune response (WO 97/40066). Other disorders in which cathepsin S is implicated are asthma, chronic obstructive pulmonary disease, endometriosis and chronic pain.

According to a first aspect of the invention there is provided a compound of the formula I:

wherein:

one of R¹ and R² is halogen and the other is halogen or H;

E is -C(=O)-, -CH(CrC₃haloalkyl)-, -S(=O)_m-, -OC(=O)-, -NRaC(=O)-NRaS(=O)_m-; where m is "l or 2;

R³ is a stable, optionally substituted, monocyclic or bicyclic, carbocyclic or heterocyclic ring system wherein the or each ring is saturated, partially saturated or aromatic and has 4, 5 or 6 ring atoms and 0 to 3 hetero atoms selected from S, O and N and wherein the optional substituents comprise 1 to 3 members selected from R⁴;

R⁴ is independently selected from halo, hydroxy, oxo, cyano, amino, carboxy, carbamoyl, nitro, sulphonamide, CrC₄ alkyl (optionally substituted with one to three Rb), CrC₄ alkanoyl and XR⁵,

X is a bond or a 1-4 membered linkage comprising 0-4 carbon atoms and/or an amine, amide, sulphonamide, ester, ether, urea or carbamate function;

R⁵ is CrC₄ alkyl or a monocyclic ring selected from C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, phenyl, azepanyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, thiopyranyl, furanyl, tetrahydrofuranyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, tetrazolyl, pyrazolyl, indolyl, any of which alkyl or ring is substituted with one to three Rb:

Ra is independently H, CrC₄ alkyl or -C(=0)Cr₄ alkyl, either alkyl being optionally substituted with 1 to 3 members independently selected from Rb;

Rb is independently halo, hydroxyl, C_rC₄ alkyl, C_rC₄ haloalkyl, -O(C_rC₄ alkyl), -N(C₀-C₄ alkyl)₂, carbamoyl; -NHC(=O)C_rC₄ alkyl;

and pharmaceutically acceptable salts, solvates, hydrates and N-oxides thereof. Without in any way wishing to be bound by theory, or the ascription of tentative binding modes for specific variables, P1 , P2 and P3 as used herein are provided for convenience only and have their conventional meanings and denote those portions of the inhibitor believed to fill the S1 , S2 and S3 subsites respectively of the enzyme, where S1 is adjacent the cleavage site and S3 remote from the cleavage site.

Preferably the stereochemistry of the P1 group is as depicted in the partial structure below:

Preferably the halogen of R¹ and/or R² is chlorine or fluorine. It is currently preferred that R¹ is halo, especially chloro or fluorine and R¹ is H, but the invention extends to compounds wherein R² is halo, especially fluoro or chloro and R² is H.

It will be appreciated that the P1 group may exist in alternative forms, such as

and the invention extends to all such alternative forms.

Representative vaues of E include SO₂ and OC(=O).

Currently preferred values for E invention include carbonyl, thereby defining a peptide bond between R³ and the backbone of the inhibitor. A special case of E as carbonyl are compounds wherein R³ is a nitrogen containing ring, such as morpholine, which is N-bonded to the carbonyl:

A further preferred value for E is -CH(haloalkyl)-, especially where the haloalkyl is trifluoromethyl; and most preferably comprising a high enantiomeric purity, such as more than 80%, preferably more than 95% such as greater than 97% of the S stereoconfiguration:

Substituent R⁵ where present is typically bonded direct to an R³ species (ie X is a bond), but may also include a spacer in the form of a 1-4 membered linkage comprising 0-4 carbon atoms and/or an amine, amide, sulphonamide, ester, ether or carbamate function. Examples of amine spacers include as -NH-, -N(Me)-, -CH₂NH, -CH₂N(Me)- and the like. Other examples of linkages include a C-i-Csalkyl spacer such as -CH₂- Or -CH₂CH₂- or a CrC₃-alkyloxy spacer such as ethyloxy, methyloxy, methyloxymethyl, oxymethyl or oxyethyl. Examples of sulphonamide linkages include -SO₂NMe-, -SO₂NH-, -NHSO₂- and -NMeSO₂-.

Any of the cyclic substituents to R³ described above may itself be substituted as described above with Rb. For example a heterocycle R⁵ group such as thiazolyl can be substituted with C₁-C₄ alkyl such as one or more C₁-C₃ alkyl groups eg methyl or trifluoromethyl or halo.

Typical values for R³ include:

unsubstituted or substituted furanyl, especially furan-2-yl or furan-3-yl, or alkyl substituted furanyl such as 2-methylfuran-3-yl, 2,4-dimethylfuran-3-yl, or aryl substituted furanyl, even more especially 5-phenylfuran-2-yl, 5-(2-chlorophenyl)furan-2-yl, 5-(3chlorophenyl)furan-2-yl, 5-(4- chlorophenyl)furan-2- yl, 5-(4-fluorophenyl)furan-2-yl, 5-(4hydroxyphenyl)furan-2-yl, 5-(3- trifluoromethylphenyl)furan-2-yl, 5-(4-trifluoromethylphenyl)furan-2-yl, 5-(3- trifluoromethylphenyl)furan-2-yl, 5-(4-methylphenyl)furan-2-yl, 5-(4-acetylphenyl)furan-2-yl, or 5- trifluoromethylfuran-2-yl;

unsubstituted or substituted tetrahydrofuranyl, particularly tetrahydrofuran-2-yl or tetrahydrofuran-3-yl;

unsubstituted or substituted morpholinyl;

unsubstituteted or substituted pyrrolyl, particularly pyrrol-2-yl; unsubstituted or substituted piperazinyl, particularly piperazin-1- yl or 4-alkylpiperazinyl, e.g., 4- methylpipeperazin-1 -yl;

unsubstituted or substituted pyrazolyl, particularly IH-pyrazol-2-yl, 1 H-pyrazol-4- yl, 1- or 2- methyl-2H-pyrazol-2-yl or 1- or 2-methyl-2H-pyrazol-3-yl;

unsubstituted or substituted isoxazolyl, particularly isoxazol-5-yl, 3-methylisoxazol-4-yl, 5- methylisoxazol-3-yl, 5-methylisoxazol-4-yl, or 3,5-dimethylisoxazol4-yl;

unsubstituted or substituted thiazolyl, particularly thiazol-2-yl, 2-methylthiazol-2-yl, 2,4- dimethylthiazol-5-yl, or 4-methyl-2-phenylthiazol-5-yl;

unsubstituted or substituted pyrazolyl, particularly alkyl-substituted pyrazolyl including 2-methyl- 2H-pyrazolyl;

unsubstituted or aryl-substituted triazolyl, particularly phenyl-substituted triazoles including 3- phenyl-3H-[1 ,2,3]triazol-3-yl;

unsubstituted or substituted pyrazinyl, particularly pyrazin-2-yl and 5-methylpyrazin-2-yl;

unsubstituted or substituted imidazolyl, particularly 1-H-imidazol-2-yl, 1-methyl-1 H-imidazol-4-yl or 1-methyl-IH-imidazol-2-yl;

thiophenyl, especially thiophene-3-yl and thiophen-2-yl, more especially heterocycle or aryl substituted C₀-C₆alkylthiophenyl, particularly 5-pyridin-2-ylthiophen-2-yl, more especially Q- Cealkylthiophenyl, particularly 5-methylthiophenyl or 3-methylthiophen-2-yl; more especially Cr C₆alkoxythiophenyl, particularly 3-ethoxythiophen-2-yl;

phenyl, especially alkyl-substituted phenyl, halogen-substituted phenyl, trihaloalkylsubstituted phenyl, alkoxy-substituted phenyl, or acetoxy-substituted phenyl, especially 4-methylphenyl, 3- chlorophenyl, 4-chlorophenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-chlorophenyl, 4-fluorophenyl, 4-hydroxyphenyl, or 4-acetylphenyl;

unsubstituted or substituted pyridinyl, particularly pyridine-2-yl;

unsubstituted or substituted cyclobutyl or cyclopentyl.

Convenient values for R³ include optionally substituted thienyl, pyrazinyl, pyridyl, pyrrolyl, and especially furyl or morpholinyl. Favoured values for R³ include fur-3-yl, thien-3-yl, pyrazin-2-yl, pyrid-4-yl, pyrrol-2-yl and especially N-morpholino.

Other preferred embodiments of R³, for example when E is (=0) or -CH(CF₃)-, include optionally substituted, fur-2-yl or thien-2yl:

where R₄' is H, halo (such as F or Cl), OCrC₄ alkyl (such as methoxy), C(=0)NRaRa' (for example dimethylcarbamoyl), NRaC(=0)CrC₄ alkyl such as NHC(=0)Me, ureas, such as NRaC(=O)NRaRa',(for example -NHC(=O)NHCH₃, NHC(=O)N(CH₃)₂ or NHC(=O)NRrRr, where RrRr define a cyclic amine such as pyrrolidine, morpholine, piperidine, piperazine or N- methylpiperazine), and carbamates such as -NRaC(=O)OCi-C₄ alkyl such as NHC(=O)OMe.

A further preferred value for R³ is phenyl particularly phenyl substituted as follows:

where

Ry is -NHC(=O)-Me, -NHC(=O)OMe, F, especially OH and NHAc

and Rx is H, OMe, F, Cl, CN, CF₃, Me.

Other embodiments include those wherein Ry is halomethyl such as CF₃ or CF₂ or an hydroxylated methyl group, such as HOCH₂ or HO(CH₂)₂)C-, any of these preferences being optionally further substituted with an R⁴ group such as Rx.

An alternative embodiment for R³ comprises phenyl which is substituted with a urea, such as a cyclic urea:

Other urea substituent include NRaC(=O)NRaRa', (for example -NHC(=0)NHCH₃, NHC(=O)N(CH₃)₂ or NHC(=O)NRrRr, where RrRr define a cyclic amine such as pyrrolidine, morpholine, piperidine, piperazine or N-methylpiperazine).

Other favoured substituents to a phenyl R³ include 3,5-dichloro, 3,5-difluoro, 3-fluoro-5-cyano, 3-cyano, 4-NHAc-3-Me, 4-NHAC-6-Me, 4-NHAc-3,5-diMe and the like.

A favoured aspect of the invention thus comprises compounds of the formula:

where R¹, E and R₄ are as defined above and R¹¹ is H, R¹² or -C(=O)R¹² where R¹² is independently d-C₄ alkyl or a monocyclic saturated or unsaturated, hetero or carbocyclic ring or benzyl, (which alkyl, ring or benzyl is optionally substituted with one to three Rb). R¹² typically comprises a pharmaceutically acceptable ether or ester prodrug which is hydrolysed in vivo to release the parent phenol.

Favoured variants of the aspect of the invention in the immediately preceding paragraph include those wherein R₄ is at the 3, or the 3 and 5 positions of the phenyl ring. Representative values include R₄' as halo, such as 3-fluoro, 3,5-difluoro, 3-chloro or 3,5-dichloro. Alternative R₄' values include fluorinated methyl such as trifluormethyl, for example 3-trifluoromethyl, CrC₃alkyloxy, such as methyloxy for example 3-methoxy, or 3,5-dimethoxy, or -C(=0)CrC3 alkyl such as acetyl, for example 3-acetyl. Additional favoured R₄ values include one or more CrC₄ alkyl, such as methyl, ethyl, i-propyl or t-butyl. Representative values for this aspect of the invention thus include 5-methyl, 5-ethyl, 5-i-propyl, 5-t-butyl, 6-methyl, 5-methyl-3-fluoro.

Additional favoured substituents to R³, for example on a phenyl R³, include sulphonamides. Accordingly, a favoured aspect of the invention comprises compounds of the formula I, wherein R³ has the partial structure:

where E is as defined above, preferably -C(=O)- R₄' is as defined above and R¹³ is -NRdSO_mR⁵, convenient values of R⁵ being d-C₄ alkyl, such as methyl, ethyl or i-propyl or t-butyl; halogenated CrC₄ alkyl such as trifluoromethyl; C3-C6 cycloalkyl, such as cyclopropyl, cyclopentyl or cyclohexyl; or phenyl or benzyl, any of which is optionally substituted with 1 to 3 Rb. Alternatively R¹³ is NRaRa' as defined above including cyclic amines, such as -NHMe Or -N(Me)₂, or piperazine, N-methyl piperazine, pyrrolidine, piperidine or morpholine.

Further preferred values for R⁵ include heteroaryl rings such as pyrrolyl, oxazolyl, isoxazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl or indolyl, especially thiazolyl, any of which is substituted with R_b groups such as CrC₄ alkyl. Representative sulphonamides include those with the partial structures:

where E is as defined above R₄' is H or R⁴ as defined above, Rd' is Me or H, and R⁶ is H or methyl, especially at the ring position, adjacent the N for example:

Other representative values of XR⁵ thus include F₃C-S(=O)₂-NH, cyclopropyl-S(=O)₂NH, Me- S(=O)₂)NH-, Et-S(=O)₂NH-, i-Pr-S(=O)₂NH-, Ph-S(=O)₂NH-; MeNH-S(=O)₂NH; (Me)₂S(=O)₂NH- and the like.

Alternatively the sulphonamide may have the other orientation -S(=O)_mNRdR⁵ where R⁵ as defined above, preferably CrC₄ alkyl, such as methyl, ethyl or i-propyl or t-butyl; halogenated CrC₄ alkyl such as trifluoromethyl; C₃-C₆ cycloalkyl, such as cyclopropyl or cyclohexyl; or phenyl or benzyl, any of which is optionally substituted with R⁶. Alternatively R⁵ together with Rd defines a 3-6 membered N-containing ring such as azidine, pyrrolidine, pyridine, piperidine, morpholine, piperazine or N-methylpiperizine.

Representative values of sulphonamide thus include MeNH-S(=O)₂-, (Me)₂N-S(=O)₂- and the like.

As depicted above, a sulphonamide-substituted phenyl is optionally substituted with an additional substituent R⁴, typically, but not invariably, in the 4 position if the sulphonamide is in the 3 position and vice versa. Representative R⁴ groups thus include halo such as chloro or fluoro, CrC₄ alkyl such as methyl (including 2-methyl) and CrC₄alkoxy such as methoxy.

Rd, the N-substituent to a sulphonamide is typically H, CrC₄ alkyl or an acyl moiety such as - C(=O)Ci-C₄ alkyl or optionally substituted benzoyl. Representative values for Rd thus include H, methyl, acetyl, pivaloyl or benzoyl. For sulphonamides in the orientation -S(=O)_mNRdR⁵, Rd is conveniently CrC₄ alkyl or together with R⁵ defines a 3-6 membered N-containing ring such as azidine, pyrrolidine, pyridine, piperidine, morpholine, piperazine or N-methylpiperizine.

In either orientation, m is typically 1 (sulphenamide) or preferably 2 (sulphonamide).

An alternative phenyl-based R³ value is phenyl substituted with a pair of R⁴ groups which together constitute a nitrogen containing chain of 3 or 4 atoms thereby defining a ring fused to the phenyl such as:

where R is an optional substituent selected from R⁴ as defined above, such as H, methyl or halo, and E is also as defined above, Rz is CH, NH or O and the S atom is optionally oxidised to >S=O or preferably >S(=O)₂. Ring nitrogens are optionally substituted with CrC₄ alkyl (such as methyl, ethyl or t-butyl), or C(=0)CrC₄ alkyl (such as acetyl). Typically R⁴ is methyl or especially H.

Preferably the linkage to E is opposed to a nitrogen in the adjacent fused ring:

Other fused rings for constituting a nitrogen containing ring fused to a phenyl R³ include

where Rz is CH; NH or O, especially O and preferably NH, R₄' is H or R⁴ as defened above, especially where R₄' is H, d-C₄ alkyl, NH₂, NHCi-C₄alkyl (such as methylamide), N(CrC₄alkyl)₂ such as dimethylamide), NHC(=O)CrC₄alkyl (such as acetamide). Ring nitrogens are optionally substituted with d-C₄ alkyl (such as methyl, ethyl or t-butyl) , or C(=O)Ci-C₄ alkyl (such as acetyl). R⁴ is an optional substituent R⁴ as defined above, such as H, methyl or halo.

Typically R₄ is methyl or especially H. Preferably the linkage to E is opposed to a nitrogen in the adjacent fused ring.

Still further fused rings for R³ include variants wherein the fused nitrogen-containing ring defines a saturated or unsaturated 6 membered heterocycle, such as:

where Rz is NH, O or CH, especially O and preferably NH and R⁴ is as defined, preferably H, R⁴ is an optional substituent selcted from R⁴ (such as H, methyl or halo) and O' is absent (ie 2 hydrogen atoms) or keto. Preferably the linkage to E is opposed to a nitrogen in the adjacent fused ring.

Still further fused rings for R³ include variants wherein the fused nitrogen containing ring defines an optionally substituted quinoline, isoquinoline, tetrohydroquinoline or tetrahydroisoquinoline moiety, such as

especially wherein one or both R⁴' are H and the linkage to E is opposed to a nitrogen in the adjacent fused ring.

Other favoured R³ groups include pyrimidyl, such as 2-pyrimidyl, for example 5-OH-pyrimid-2-yl; or pyridyl, such as pyrid-4-yl, for example 0→pyrid-4yl; or pyrid-3-yl, for example 6-hydroxy- pyrid-3-yl.

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

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

In one series of embodiments, the methods are employed to treat mammals, particularly humans at risk of, or afflicted with, autoimmune disease. By autoimmunity is meant the phenomenon in which the host's immune response is turned against its own constituent parts, resulting in pathology. Many human autoimmune diseases are associated with certain class Il MHC-complexes. This association occurs because the structures recognized by T cells, the cells that cause autoimmunity, are complexes comprised of class Il MHC molecules and antigenic peptides. Autoimmune disease can result when T cells react with the host's class Il MHC molecules when complexed with peptides derived from the host's own gene products. If these class Il MHC/antigenic peptide complexes are inhibited from being formed, the autoimmune response is reduced or suppressed. Any autoimmune disease in which class Il MHC/antigenic complexes play a role may be treated according to the methods of the present invention. Such autoimmune diseases include, e.g., juvenile onset diabetes (insulin dependent), multiple sclerosis, pemphigus vulgaris, Graves' disease, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis and Hashimoto's thyroiditis.

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

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

In another series of embodiments, the methods are employed to treat mammals, particularly humans, which have undergone, or are about to undergo, an organ transplant or tissue graft. In tissue transplantation (e.g., kidney, lung, liver, heart) or skin grafting, when there is a mismatch between the class Il MHC genotypes (HLA types) of the donor and recipient, there may be a severe "allogeneic" immune response against the donor tissues which results from the presence of non-self or allogeneic class Il MHC molecules presenting antigenic peptides on the surface of donor cells. To the extent that this response is dependent upon the formation of class Il

MHC/antigenic peptide complexes, inhibition of cathepsin S may suppress this response and mitigate the tissue rejection. An inhibitor of cathepsin S can be used alone or in conjunction with other therapeutic agents, e.g., as an adjunct to cyclosporin A and/or antilymphocyte gamma globulin, to achieve immunosuppression and promote graft survival. Preferably, administration is accomplished by systemic application to the host before and/or after surgery. Alternatively or in addition, perfusion of the donor organ or tissue, either prior or subsequent to transplantation or grafting, may be effective. The above embodiments have been illustrated with an MHC class Il mechanism but the invention is not limited to this mechanism of action. Suppression of cathepsin S as a treatment of COPD or chronic pain may not, for example, involve MHC class Il at all.

Assays for the assessment of cathepisn S inhibitors in the treatment of chronic pain, including neuropathic or inflammatory pain are as described in WO 03 20287.

Currently preferred indications treatable in accordance with the present invention include:

Psoriasis;

Autoimmune indications, including idiopathic thrombocytopenic purpura (ITP), rheumatoid arthritis (RA), multiple schlerosis (MS), myasthenia gravis (MG), Sjogrens syndrome, Grave's disease and systemic lupus erythematosis (SLE);

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

The compounds of the invention can form salts which form an additional aspect of the invention. Appropriate pharmaceutically acceptable salts of the compounds of the invention include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, isethionate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesulphonate, benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate; and inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and sulphonic acids. The compounds of the invention may in some cases be isolated as the solvate or hydrate. Certain compounds of the invention form N-oxides which are regarded as within the scope of the invention.

Prodrugs

The compounds of the invention include a number of handles such as OH, NH or COOH groups to which conventional prodrug moieties can be applied. Prodrugs are typically hydrolysed in vivo to release the parent compound in the plasma, liver or intestinal wall. Favoured prodrugs are esters of hydroxyl groups such as a phenolic hydroxyl group at R³, or amine functions such as an R⁴ sulphonamide amine function. Preferred pharmaceutically acceptable esters include those derived from CrCβ carboxylic acids such as acetyl or pivaloyl or optionally substituted benzoic acid esters, preferably unsubstituted or substituted with R⁶. Favoured sulphonamide prodrugs include aminoacyls derived from CrCβ carboxylic acids such as acetyl or pivaloyl or optionally substituted benzoic acid esters, preferably unsubstituted or substituted with R⁴.

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

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

Such methods include the step of bringing into association the above defined active agent with the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing a compound of Formula IV or its pharmaceutically acceptable salt in conjunction or association with a pharmaceutically acceptable carrier or vehicle. If the manufacture of pharmaceutical formulations involves intimate mixing of pharmaceutical excipients and the active ingredient in salt form, then it is often preferred to use excipients which are non-basic in nature, i.e. either acidic or neutral.

Formulations for oral administration in the present invention may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water in oil liquid emulsion and as a bolus etc.

With regard to compositions for oral administration (e.g. tablets and capsules), the term suitable carrier includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring or the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.

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

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 I are intended to be within the scope of the invention, regardless of binding mode. Broadly speaking the P1 building block will be an N-protected- 6-fluoro-3-oxo-hexahydro- furo[3,2-b]pyrrole comprising a synthon for the keto group such as hydroxyl or dioxopropylidine, P2 will be an N-protected L-1-methyl-cyclopropylalanine, whereas P3 typically comprises a capping group such as a substituted heteroaroyl or aroyl moiety.

The suitably protected individual building blocks can first be prepared and subsequently coupled together i.e. P2+P1→ P2-P1. Alternatively, precursors of the building blocks can be coupled together and modified at a later stage of the synthesis of the inhibitor sequence. Further building blocks, precursors of building blocks or prefabricated bigger fragments of the desired structure, can then be coupled to the growing chain, e.g. R³-E-P2^*+ P1→ R³-E-P2-P1 or R³-E^*+P2-P1 → R³-E-P2-P1 , where ^* denotes an activated form.

Coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be carried out using standard coupling procedures such as the azide method, mixed carbonic- carboxylic acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active ester (pnitrophenyl ester, N-hydroxysuccinic imido ester) method, Woodward reagent K- method, carbonyldiimidazole method, phosphorus reagents or oxidation-reduction methods. Some of these methods (especially the carbodiimide method) can be enhanced by adding 1- hydroxybenzotriazole or 4-DMAP. These coupling reactions can be performed in either solution (liquid phase) or solid phase.

More explicitly, the coupling step involves the dehydrative coupling of a free carboxyl of one reactant with the free amino group of the other reactant in the present of a coupling agent to form a linking amide bond. Descriptions of such coupling agents are found in general textbooks on peptide chemistry, for example, M. Bodanszky, "Peptide Chemistry", 2nd rev ed., Springer- Verlag, Berlin, Germany, (1993) hereafter simply referred to as Bodanszky, the contents of which are hereby incorporated by reference. Examples of suitable coupling agents are N₁N'- dicyclohexylcarbodiimide, 1-hydroxybenzotriazole in the presence of N₁N'- dicyclohexylcarbodiimide or N-ethyl-N'- [ (3dimethylamino) propyl] carbodiimide. A practical and useful coupling agent is the commercially available (benzotriazol-1-yloxy) tris- (dimethylamino) phosphonium hexafluorophosphate, either by itself or in the present of 1-hydroxybenzotriazole or 4-DMAP. Another practical and useful coupling agent is commercially available 2-(IH- benzotriazol-1-yl)-N, N, N', N'- tetramethyluronium tetrafluoroborate. Still another practical and useful coupling agent is commercially available 0-(7-azabenzotrizol-1-yl)-N, N₁N', 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 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 building block is typically N- protected. Any protecting group known in the art can be used. Examples of such groups include: 1 ) acyl groups such as formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl; 2) aromatic carbamate groups such as benzyloxycarbonyl (Cbz or Z) and substituted bensyloxycarbonyls, and 9- fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic carbamate groups such as tertbutyloxycarbonyl (Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4) cyclic alkyl carbamate groups such as cyclopentyloxycarbonyl and adamantyloxycarbonyl; 5) alkyl groups such as triphenylmethyl and benzyl; 6) trialkylsilyl such as trimethylsilyl; and 7) thiol containing groups such asphenylthiocarbonyl anddithiasuccinoyl. The preferred alpha-amino protecting group is either Boc or Fmoc. Many amino acid derivatives suitably protected for peptide synthesis are commercially available.

The alpha-amino protecting group is typically cleaved prior to the next coupling step. When the Boc group is used, the methods of choice are trifluoroacetic acid, neat or in dichloromethane, or HCI in dioxane or in ethyl acetate. The resulting ammonium salt is then neutralized either prior to the coupling or in situ with basic solutions such as aqueous buffers, or tertiary amines in dichloromethane or acetonitrile or dimethylformamide. When the Fmoc group is used, the reagents of choice are piperidine or substituted piperidine in dimethylformamide, but any secondary amine can be used. The deprotection is carried out at a temperature between 0 °C and room temperature usually 20-22 °C.

Once the inhibitor sequence is completed any protecting groups are removed in whatever manner is dictated by the choice of protecting groups. These procedures are well known to those skilled in the art. The first stage in a synthesis of compounds of the general formula Il is typically the preparation in solution of a functionalized P1 building block. P1 building blocks of both R1/R2 enantiomers are described in detail in WO05/066180, the contents of which are incorporated by reference.

Additional P1 building blocks include those prepared by the scheme below:

i) MsCI, Pyr ii) NaOAc, Ac₂O, DMF, 130 C iii) BF₃XEt₂O, Et₃SiH, DCM iv) TBDPS-CI, Im-H, DMF v) NaOMe vi) SO₂CI₂, Pyr, DCM vii) PPh₃, MeOH, H₂O, then Et₃N, 50 C viii) BoC₂O, Et₃N ix) TBAF, THF x) Dess-Martin Periodinane xi) method a: MeOH, TMOF, p-TsOH,then Et₃N, BoC₂O, then chromatography and finally MeOH, AcCI, method b: MeOH, AcCI, TMOF Although the scheme above has been illustrated with a differential protecting group strategy employing acetyl, mesyl, TBDPS and Boc, it will be apparent that other permutations of conventional protecting groups, as described by Greee (ibid) can be employed. Additionally, it may be convenient to employ the hemiacetal or dimethylketal of the ketone group during coupling of P2 and P3 residues and to regenerate the ketone function in a latter step.

An additional P1 building block is prepared in the scheme below:

Compound 10 in WO05/066180

i. Dess-Martin Periodinane, DCM, 2 h, RT; ii. Trimethylorthoformate, pTsOH, MeOH, 8 h, 60 ⁰C; UiPd(OH)₂, H₂, MeOH, 48 h, RT; iv. BoC₂O, 10% Na₂CO₃, 16 h, O ⁰C to RT; v. HCI, CH₂CH₂/Py, CBzCI vi. CH₂CH₂/Et₃N MsCI vii DMF, LiCI Again, alternative differential protection strategies will be readily apparent to those in the art. The depicted building block may be coupled to P2 and P3 as the dimethyl ketal followed by generation of the active ketone in a later step. Alternatively the ketone can be generated prior to coupling.

A further P1 building block is prepared as described in the scheme below:

i) Tf₂O, pyridine followed by NaN₃ ϋ) AcOH, H₂O ill) TsCI, pyridine, followed by H₂,Pd/C iv) Benzaldehyde, NaCNBH₄ v) Deoxyfluor, DCM, O⁰C, silica chromatography vi) TFA, H₂O vii) HBr, HOAc viii) LiAIH₄ ix) Deprotect with H2/PdC MeOH & couple P2/P3

Alternatively the hydroxyl group of the above building block can be oxidized to the ketone, for example by Dess Martine periodinane, prior to coupling. The thus generated ketone can be protected during coupling as the dimethyl ketal, which is conventionally deprotected to regenerate the active ketone. Any of the above P1 building blocks can then be elongated by coupling to the P2 and P3 (capping) building block as shown below:

1. 4.0M HCI dioxan

2. Boc-L-AA-Ofp. HOBt, DMF, NMM

Typical elongation of a cyclic ketone. This has been illustrated with an example where the P1 ketone function is taen through the elongation procedure. Alterantively, the keto function can be protected, for example as the dimethyl ketal and regenerated, for example by Dess Martin periodinane oxidation after coupling of the P2 and/or P3 building blocks.

Urethane compounds i.e. E is -0C(=0)- can be formed for example by reaction of an R³ alcohol with the isocyanate of the P2 amino acid. The isocyanate, or equivalent reactive intermediate, can be formed by reaction of the amino group of the P2-amino acid with phosgene, or with dinitrophenylcarbonate in the presence of a suitable base, e.g. triethylamine. Alternatively they can be formed by reaction of the amino group of the P2 amino acid with a suitable chloroformate, e.g. benzylchloroformate.

Sulphonamide derivatives i.e. E = S(=0)₂- can be prepared by reaction of the amino group of the P2 amino acid with a suitable R³ sulfonyl chloride in a solvent such as dichloromethane in the presence of a suitable base such as triethylamine or dimethylaminopyridine. Sulphamide derivatives i.e. E = NRaS(=O)₂- can be prepared by reacting a suitable R³ amine in a sulphonyl chloride solvent followed by reaction of the formed sulfamoyl chloride derivative with the amino group of the above mentioned P2 amino acid in a solvent such as dichloromethane in the presence of a suitable base such as triethylamine.

Urea derivatives i.e. E = -NRa-C(=O)- can be prepared by reaction of the corresponding R³ isocyanate with the N-protected amide of the P2-P1 intermediate, typically in an inert organic solvent such as N,N-dimethyl formamide. Conversely, the R³ amine is reacted with the isocyanate of the P2-P1 intermediate under similar conditions. Alternatively the N-protected R³- amine, and N-protected P2-P1 amine are together reacted with L₁C(=O)L₂, where L₁ and L₂ are good leaving groups in an inert organic solvent such as N,N-dimethyl formamide, tetrahydrofuran, ethyl acetate or benzene, as shown in J Org Chem 56, 891 (1991 ). The time, temperature and sequence of addition used depends on the reactivity of the individual reagents.

A special case of a urea derivative are compounds wherein R³ represents an unsaturated ring such as morpholine, piperazine or piperidine which is N-bonded to E as carbonyl. Such compounds are readily prepared, for example by treating the N-protected P2-P1 intermediate with 4M HCI/dioxane, adding the R3-chloride, for example morpholinyl carbonyl chloride, together with TEA in DCM.

Compounds wherein E is -CH(haloalkyl)- are typically elongated by reaction of an intermediate compound of the formula

haloalkyl

where R³ is as defined above and LG is a conventional leaving group such as trifluoromethansulfonate, and the like, with the N-deprotected P1/P2 building block shown above. The reaction is carried out in a suitable organic solvent, including but not limited to, halogenated organic solvents such as methylene chloride, 1 ,2- dibromoethane, and the like, ethereal solvents such as diethyl ether, tetrahydrofuran, acetonitrile, or aromatic solvents such as benzene, toluene, xylene, and the like, or mixtures thereof and optionally in the presence of an organic or inorganic base. Preferably, the organic base is triethylamine, pyridine, N- methylmorpholine, collidine, diisopropylethylamine, and the like. Preferably, the inorganic base is cesium carbonate, sodium carbonate, sodium bicarbonate, and the like. The reaction is optionally carried out in the presence of a drying agent such as molecular sieves. Preferably, the reaction is carried out at room temperature. The intermediate can be prepared by methods well known in the art. For example, a compound where R³ is phenyl or 4- fluorophenyl, the haloalkyl is trifluoromethyl can be readily prepared from commercially available 2,2,2 trifluoroacetophenone or 2,2,2, 4'-tetrafluoroacetophone respectively, by reducing the keto group to an alcoholic group by suitable reducing agent such as sodium borohydride, lithium aluminum hydride, and the like. The solvent used depends on the type of reducing agent. For example, when sodium borohydride is used the reaction is carried out in an alcoholic organic solvent such as methanol, ethanol, and the like. When lithium aluminum hydride is used the reaction is carried out in an ethereal solvent such as tetrahydrofuran, and the like. Reaction of 2,2,2 trifluoro-1-phenylethanol or 222-trifluoro-l-(4- fluorophenyl)ethanol with triflic anhydride provides the desired compound. Chirally enriched intermediate can be obtained by reduction of the corresponding halogenated acetophenone with a suitable reducing agent such as catecholborane or BH₃-DMS complex in the presence of a suitable catalyst such as (A or (R) CBS catalyst or (A or (R)-, a -diphenyl-2- pyrrolidine-methanol in the presence of BBN. Methodology for the synthesis of haloalkyl P3 building blocks (including non-basic P3s) is found in WO2006034006 and WO03075836 and WO05066159.

Preparation on solid phase

Compounds of the invention can also be prepared with solid phase chemistry, for example with Murphy's linker methodology using known chemistries as broadly described in WO02/88106. The ketone of the FmocNH bicycle is derivatised as an acid labile semicarbazone which provides a carboxylic acid for attachment to a polymer support (for example furnished with a hyper acid labile Sieber linkage) using HBTU, HOBt and NMM. A After Fmoc removal from the solid-phase, the corresponding P2 Fmoc amino acid, in this case an N-protected L- 1 - methylcyclopropylalanine building block, is coupled with conventional peptide chemistry or using the preformed symmetrical anhydride. Coupling is typically carried out for 16 hours. After Fmoc removal from the solid-phase, the P3 acids are introduced using standard coupling conditions. Washing, drying and cleavage from the resin with 1 % TFA/DCM provides the crude desired material with the semicarbazone linker still attached. Lyophilisation provides a white solid which was treated with a mixture of pyruvic acid, water and acetic acid as described in Steroids 1976, 845-849. The crude cleaved ketone is then purified by preparative reverse-phase HPLC. The P2 bP3 acids were commercially available or accessed through trivial modification of commercially available materials, except as specified under the table.

Method A: General Synthesis of P3 amides using coupling on solid phase

Method B: Synthesis of phenol P3s using solid phase coupling

(includes a hydrazine wash)

After coupling, the resin is suspended in a 5% solution of hydrazine in DMF for 1 h. The mixture was filtered, and the resin washed with DMF. The hydrazine treatment and DMF wash was then repeated.

(Resin cleaved as standard)

Method C: Synthesis of aniline P3s using solid phase coupling

(includes a piperidine treatment to remove FMOC protecting groups)

After coupling, the resin is suspended in a 20% solution of piperidine in DMF for 0.5h. The mixture was filtered, and the resin washed with DMF. The piperidine treatment and DMF wash was then repeated.

(Resin cleaved as standard) Method D: Alternative synthesis of aniline P3s.

DMF

After coupling of 4-FMOC-aminobenzoic acid, the resin is suspended in a 20% solution of piperidine in DMF for 0.5h. The mixture is filtered, and the resin washed with DMF. The piperidine treatment and DMF wash is then repeated. The resin is suspended in a solution of benzyl chloroformate and Λ/-methyl morpholine in DMF, filtered and the residue washed with 1 :1 water: DMF, DMF, THF, DCM and MTBE.

(Resin cleaved as standard)

Method E: Alternative synthesis of anilines

After coupling of 4-FMOC-aminobenzoic acid, the resin is suspended in a 20% solution of piperidine in DMF for 0.5h. The mixture is filtered, and the resin washed with DMF. The piperidine treatment and DMF wash is then repeated. The resin is suspended in a solution of benzylaldehyde in DMF, and a solution of dibutyltin dichloride in THF added. After 10 minutes, phenyl silane is added and the mixture shaken overnight. The mixture is filtered and the residue washed with DMF, THF, DCM and MTBE.

(Resin cleaved as standard)

Method F: General Synthesis of P3 sulfonamides on solid phase

To a suspension of P2-P1 on resin in DMF, is added diisopropylethylamine and sulfonyl chloride. After 16h, the mixture is filtered and resin washed with DMF, THF, DCM and MTBE.

(Resin cleaved as standard)

Methods A-F above are described with reference to a Murphy's linker attached to a solid phase resin such as Novasyn TG resin and cleaved as described. Alternatively, the P1-Murphys linker construct can be attached to Sieber resin as described above and cleaved with the milder 1 % TFA in conjunction to avoid hydrolysis of the amide linkage adjacent the bicyclic P1. Pyruvic acid is used to cleave off the Murphy linker.and the ketone function is generated as described above.

Detailed Description

Various embodiments of the invention or intermediates for their production will now be described by way of example only with reference to the following non-limiting examples. Example 1

Construction of P1 building block

Step a)

A mixture of 54 (See Chem. Ber., 101 (1968), 3802.

& Tetrahedron, 43, 13 (1987), 3095-3108.)

(5.2 g, 13.0 mmol), palladium-on-carbon (10%, Acros, 0.66 g) in methanol was hydrogenated at slight positive pressure. The hydrogen was changed 3 times over a period of 1 h, after TLC (petroleum ether-ethyl acetate 7:3 and dichloromethane-methanol 9:1 , staining with ammonium molybdate-cerium sulfate) indicated complete conversion of the starting material into a major non-UV active spot which colours AMC, and some weaker higher moving spots (dichloromethane-methanol 9:1 ). The reaction mixture was then filtered through Celite and concentrated which gave crude compound 55.

To a suspension of the residue in dichloromethane (60 ml) and pyridine (3.2 ml, 40 mmol) at 0 °C was added benzylchloroformate (0.93 ml, 6.5 mmol). The reaction mixture was stirred at rroom temperature for 2 h after which additional pyridine (3 ml) and benzylchloroformate (0.8 ml) was added at 0 °C. The reaction mixture was then stirred at room temperature overnight, then diluted with dichloromethane (100 ml), washed successively with 1 M aq. sulfuric acid (2 x 50 ml) and 1 M aq. sodium hydrogen carbonate (1 x 50 ml), then dried (sodium sulfate), filtered and concentrated onto silica. Flash chromatography (diameter: 4 cm, YMC-gel: 50 g, packing eluent: ethyl acetate in petroleum ether 1 :4) of the residue using ethyl acetate in petroleum ether 1 :4 (350 ml), 2:3 (250 ml), 1 :1 (250 ml), 3:2 (250 ml) and 3:1 (150 ml) gave compound 56 as a foamy syrup (2.71 g, 8.1 mmol, 62% over 2 steps) after drying in vacuum overnight.

NMR data (400 MHz, CDCI₃): ¹H, 1.33, 1.52 (2 s, 6H, C(CHa)₂), 2.34 (2 d, 1 H, -OH), 3.04 (m, 1 H, H-6a), 3.97 (m, 1 H, H-6b), 4.19 (m, 1 H, H-5), 4.33 (m, 1 H, H-3), 4.68, 4.84 (2 d, 1 H, H-2), 4.79 (t, 1 H, H-4), 5.08-5.24 (m, 2H, CH₂Ph), 5.86 (br s, 1 H, H-1 ), 7.30-7.42 (m, 5H, Ar-H).

Step b)

To a stirred suspension of sodium hydride (60% in mineral oil, Aldrich, 0.34 g, 8.4 mmol) and compound 56 (2.17 g, 6.47 mmol) in dimethylformamide (30 ml) was added benzyl bromide (0.81 mmol, 6.8 mmol) during 5 minutes. After stirring 1 h (TLC: ethyl acetate in petroleum ether 2:3), methanol (approx 2 ml) was added to destroy excess reagent, then immediately partitioned between ethyl acetate (180 ml) and water (150 ml). The organic layer was washed with water (3 x 100 ml), then dried (sodium sulfate), filtered and concentrated onto silica. Flash chromatography (diameter: 4 cm, YMC-gel: 40 g, packing eluent: ethyl acetate in petroleum ether 1 :4) of the residue using ethyl acetate in petroleum ether 1 :4 (100 ml), 3:7 (250 ml) and 2:3 (250 ml) gave a colourless syrup (2.7 g, 6.35 mmol, 98%) after drying in vacuum overnight.

NMR data (400 MHz, CDCI₃): ¹H, 1.31 (s, 3H, C(CH₃)(CH₃)), 1.51 (d, 3H, C(CH₃)(CH₃)), 3.29 (m, 1 H, H-6a), 3.78-3.96 (m, 2H, H-5 and H-6b), 4.22 (dd, 1 H, H-3), 4.64, 4.84 (2 M, 4H, H-2, H-4 and CH₂Ph), 5.07-5.22 (m, 1 H, CH₂Ph), 5.94 (m, 1 H, H-1 ), 7.28-7.39 (m, 1OH, Ar-H).

Step c)

To a stirred solution of compound 7 (2.635 g, 6.19 mmol) in dichloromethane (28 ml) and triethyl silane (9.9 ml, 61.9 mmol) at 0 °C was added borontrifluoride etherate (7.9 ml, 61.9 mmol) in one portion. The reaction mixture was then stirred at rt for 24 h (TLC: petroleum ether-ethyl acetate 4:1 and ethyl acetate-toluene 3:2), then 1 M aq. sodium hydrogen carbonate (40 ml) and some solid sodium hydrogen carbonate was carefully added until bubbling stopped. The resulting mixture was partitioned between dichloromethane (100 ml) and water (100 ml). The organic layer was washed with 1 M aq. sodium hydrogen carbonate (1 x 100 ml) and brine (1 x 100 ml), then dried (sodium sulfate), filtered and concentrated onto silica. Flash chromatography (diameter: 4 cm, YMC-gel: 48 g, packing eluent: ethyl acetate-toluene 3:2) of the residue using ethyl acetate in toluene 3:2 (750 ml) gave a colorless hard syrup (1.38 g, 3.74 mmol, 60%) of about 85-90% purity according to TLC. LR-MS: Calcd for C_2IH₂₄NO₅: 370.2. Found: 370.0 [M+H].

Step d)

A mixture of compound 58 (1.38 g, 3.74 mmol), palladium-on-carbon (Acros, 10%, 0.12 g) and di-tert-butyl-dicarbonate (0.82 g, 3.7 mmol) in ethyl acetate (50 ml) was hydrogenated at slight overpressure. The hydrogen was changed 2 times over a period of 1 h and the reaction was monitored by LC-MS. After 1 h, additional palladium-on-carbon (0.1 g) was added and the reaction mixture was treated with hydrogen for 1 more hour. The reaction mixture was then filtered through Celite and concentrated. The residue was treated with 2:1 pyridine-acetic anhydride (18 ml) overnight, and then concentrated. The residue was redissolved in dichloromethane (60 ml) and was washed successively with 1 M aq. sulfuric acid (2 x 40 ml) and 1 M aq. sodium hydrogen carbonate (1 x 40 ml), and then dried (sodium sulfate) filtered and concentrated. Flash chromatography (diameter: 3 cm, YMC-gel: 20 g, packing eluent: ethyl acetate in toluene 1 :4) of the residue (dissolved in toluene-ethyl acetate 4:1 ) using ethyl acetate in toluene 1 :4 (200 ml) and 1 :3 (150 ml) gave a colourless syrup (1.13 g, 3.0 mmol, 80%) after drying in vacuum overnight.

NMR data (400 MHz, CDCI₃): ¹H, 1.45 (s, 9H, C(CHa)₃), 2.08 (s, 3H, COCH₃), 3.10 (m, 1 H, H- 6a), 3.74-3.99 (m, 3H, H-1 a, H-5 and H-6b), 4.1 1 (m, 1 H, H-1 b), 4.16-4.74 (m, 4H H-3, H-4 and CH₂Ph), 5.31 (m, 1 H, H-2), 7.28-7.40 (m, 5H, Ar-H). Step e)

A mixture of compound 60 (1.08 g, 2.86 mmol) and palladium-on-carbon (10%, 0.15 g) in ethyl acetate (30 ml) was hydrogenated at slight over pressure for 2 h (TLC: toluene-ethyl acetate 4:1 and 1 :1 ), then filtered through Celite and concentrated. The mixture was concentrated from dichloromethane (3 x 10 ml), then dissolved in dichloromethane and to the solution was added bis-(2-methoxyethyl)aminosulphur trifluoride (50% in THF, 2.12 ml, 2 eq.) at 0 °C. After stirring at rt overnight additional bis(2-methoxyethyl)aminosulphur trifluoride (50% in THF, 2 ml) was added and the reaction mixture was stirred at rt for another night (TLC: toluene-ethyl acetate 1 :1 , ninhydrine staining), then 1 M aq. sodium hydrogen carbonate was added carefully until bubbling stopped. The resulting mixture was diluted with dichloromethane (50 ml), and the organic layer was washed once with 1 M aq. sodium hydrogen carbonate (40 ml), then dried (sodium sulfate), filtered and concentrated. Flash chromatography (diameter: 3 cm, Silica: 25 g, packing eluent: toluene-ethyl acetate 4:1 ) of the residue (dissolved in toluene-ethyl acetate 4:1 ) using toluene- ethyl acetate 4:1 gave compound 62 (0.49 g, 1.7 mmol, 59 %) as a colourless syrup after drying in vacuum overnight. Some starting material and sulphur intermediate could be recovered from the reaction mixture.

LR-MS: Calcd for C₉H₁₃FNO₅: 234.1. Found: 234.0 [M+2H-f-Butyl].

The P1 building block can be used in fluid phase synthesis, or coupled to solid phase as described below.

Example 2

Preparation on solid phase

Solid phase synthesis was carried out as outlined above using Murphy's linker via a hyper acid labile Sieber linkage. After Fmoc removal from the P1-P1 -solid phase, the P3 acids are introduced using standard coupling conditions. Washing, drying and cleavage from the resin with 1 % TFA/DCM provides the crude desired material with the semicarbazone linker still attached. Lyophilisation provides a white solid which was treated with a mixture of pyruvic acid, water and acetic acid as described in Steroids 1976, 845-849. The crude cleaved ketone was then purified by preparative reverse-phase HPLC. The P2 acids was as described in Example 3 below. The P3 acids are commercially available or accessed through trivial modification of commercially available materials, except as specified under the table.

Example 3

Homochiral 1-methylcvclopentylanine P2 building block

Diethyl geranyl phosphate

To a solution of the geraniol (15 g, 97.3 mmol) and pyridine (31.5 ml, 389.2 mmol) in dichloromethane (100 ml) was added the phosphorochloridric acid diethyl ester (16.9 ml, 1 16.8 mmol) at OC under a nitrogen atmosphere, and the mixture stirred for 1 hour at OC and then overnight at RT. The mixture was diluted with ether (200 ml) and washed with 1 M hydrochloric acid (3 x), brine and saturated sodium bicarbonate and dried over anhydrous magnesium sulphate. The solvent was removed under reduced pressure and the product purified by column chromatography (silica gel, 20 - 40% EtOAc in iHexane) to give the desired product as a colourless oil (14.72g, 50.7 mmol, 51 %.

δ_H (400 MHz, CDCI₃) 1.30 (6H, t), 1.58 (3H, s), 1.63 (3H, s), 1.69 (3H, s), 2.00-2.17 (4H, m), 4.04-4.15 (4H, m), 4.58 (2H, t), 5.03-5.10 (1 H, m), 5.36-5.41 (1 H, m);

Λ/-(terf-butoxycarbonyl)-3-iodo-L-alanine methyl ester

Iodine (1.25 eq., 70.70 mmol, 17.94 g) was added portionwise to a mixture of PPh₃ (1.25 eq., 70.70 mmol, 18.54 g) and imidazole (1.25 eq., 70.70 mmol, 4.81 g) in DCM (250 ml) at 0 deg C over 15 minutes. The cooling bath was removed and the mixture was stirred for 20 minutes. A solution of Boc-Ser-OMe (1 eq., 56.6 mmol, 12.40 g) in DCM (50 ml) was then added dropwise over 45 minutes at 0 deg C. The mixture was stirred at this temperature for a further 1 hour then warmed to room temperature. The mixture was filtered through silica (50% tBuOMe in ihexane eluant) then concentrated. 50% tBuOMe in ihexane was added and the precipitate formed was filtered off. The solution was concentrated and the residue purified by column (silica, 15% -> 50% tBuOMe in ihexane) to give the product as a pale yellow oil which solidified on standing. The compound was stored in the dark until ready for use.

¹H NMR (400 MHz, CDCI₃) δ; 5.38 (brd, 1 H), 4.50-4.56 (m, 1 H), 3.79 (s, 3H), 3.50-3.60 (m, 2H), 1.43 (s, 9H).

2-tert-Butoxycarbonylamino-4,8-dimethyl-4-vinyl-non-7-enoic acid methyl ester (2)

NH

Zinc dust (31.4 mmol, 2.0 g, 6 eq) was heated under vacuum for 5 - 10 minutes then allowed to cool under a nitrogen atmosphere. A solution of Λ/-(tert-butoxycarbonyl)-3-iodo-L-alanine methyl ester (see below) (1.74 mmol, 1.7 g, 1 eq.) in DMF (4 ml) was then added and the mixture stirred at OC for 20 minutes or until zinc insertion was complete. This can be monitored by TLC.

Anhydrous LiCI (2.6 equivalents based on iodo serine) and CuCN (1.3 equivalents) were mixed in a separate flask and DMF (8 ml) was added. The resulting white suspension was cooled to - 3OC. The solution containing the organometallic was then added dropwise. After addition was complete, the green solution was warmed to OC and stirred at this temperature for 10 minutes. It was then cooled to -30C again and a solution of diethyl geranyl phosphate (6.9 mmol, 2.0 g, 1.3 eq.) in DMF (5 ml) was added dropwise. The mixture was then stirred overnight slowly warming from -30C to room temperature.

Ethyl acetate was added to precipitate the copper salts and the mixture was filtered. The solvents were removed in vacuo to remove the DMF. The residue was then dissolved in EtOAc then washed (saturated NH₄CI then brine), dried (Na₂SO₄) then the solvents were removed in vacuo. The residue was purified by column (silica gel, 1 % - 10% EtOAc in isohexane) to give the product (1.20 mmol, 0.41 g, 23%). This was identified to be predominantly the S_N2' compound.

NMR δ_H (400 MHz, CDCI₃) 1.04 (3H, s), 1.42 (9H, s), 1.50-1.60 (4H, m), 1.66 (3H, s), 1.80-2.10 (5H, m), 3.70 and 3.72 (3H, 2 x s), 4.25-4.35 (1 H, m), 4.80-4.85 (1 H), 4.92-5.13 (4H, m), 5.68- 5.78 (1 H, m). 2-tert-Butoxycarbonylamino-3-(1-methyl-cyclopent-2-enyl)-propionic acid methyl ester

Grubbs 2^nd generation catalyst

8O⁰C, Toluene

A solution of 2-tert-butoxycarbonylamino-4,8-dimethyl-4-vinyl-non-7-enoic acid methyl ester (2.35 mmol, 0.80 g) in toluene (20 ml) was de-gassed with a stream of nitrogen gas for 15 minutes then Grubbs 2^nd generation catalyst was added (0.1 Og, 0.12 mmol, 5 mol%). The mixture was heated at 8OC overnight then the solvent was removed in vacuo. The residue was purified by column (silica, 2% - 10% EtOAc in isohexane) to give the required product (0.53g, 1.86 mmol, 80%).

NMR δ_H (400 MHz, CDCI₃) 1.10 (3H, s), 1.42 (9H, s), 1.60-2.40 (6H, m), 3.70 and 3.72 (3H, 2 x s), 4.31-4.40 (1 H, m), 4.79-4.83 (1 H, m), 5.50-5.58 (1 H, m), 5.61-5.68 (1 H,m).

2-tert-Butoxycarbonylamino-3-(1-methyl-cyclopentyl)-propionic acid methyl ester.

10%Pd/C was added to a solution of X (0.51 mmol, 0.14g) in MeOH (5 ml). The mixture was stirred under an atmosphere of hydrogen overnight. The mixture was filtered through celite, washing with methanol and the filtrate was concentrated in vacuo. The residue was purified by column (silica, 5%-10% EtOAc in isohexane) to give the product as a clear oil (0.094 g, 0.33 mmol, 65%).

NMR δ_H (400 MHz, CDCI₃) 0.98 (3H, s), 1.36-1.42 (13H, m), 1.46-1.65 (5H, m), 1.80-1.84 (1 H, m), 3.68 (3H, s), 4.31-4.40 (1 H, m), 4.82-4.88 (1 H, m). Example 4 Preparation of P3 building blocks

The building blocks described in this example are coupled to P1-P2 units on solid or fluid phase as described above, and where necessary deprotected/reduced to restore the active cyclic ketone functionality at P1.

The P1 is the characteristic halogenated pyrrolofuranone described above, such as those described in Examples 1 , 5, 6 or 7. The P2 unit is the L-1-methylcyclopentylalanine building block of Example 3.

4.1 3-Dimethylaminomethyl-4-hvdroxy-benzoic acid

A solution of formaldehyde (37% in water, 1 1.28 mmol, 0.914 g, 0.94 eq.) was added to a solution of ethyl 4-hydroxybenzoate (12.00 mmol, 2 g, 1 eq.) and dimethylamine (40% (water), 12.75 mmol, 1.43 g, 1.06 eq.) in water (5 ml). The solution turned cloudy and was stirred overnight by which time it had cleared. The mixture was then heated to 9O⁰C for 2 hours during which time it turned orange. This was poured into water and extracted with EtOAc. The organics were dried (MgSO₄) and concentrated in vacuo to give a viscous orange oil (2.57 g, 96%). This was dissolved in wet methanol (20 ml) and NaOH (23 mmol, 0.92 g, 2 eq.) was added. The mixture was heated at reflux for 2 hours then it was cooled and acidified (1 M HCI (aq), pH 5). The acidic mixture was lyophilized and the resulting solid extracted with methanol. The solvents were evaporated to give the product as a white solid (1.47 g, 61 %).

4.2 3-tert-Butyl-4-hvdroxy-benzoic acid

MeI (66.6 mmol, 4.14 ml, 2 eq.) and K₂CO₃ (59.9 mmol, 8.28 g, 1.8 eq.) were added to a solution of 2-terfbutyl phenol (33.3 mmol, 5 g) in DMF (60 ml) and the mixture heated at 8OC for 24 hours. The mixture was cooled, diluted with diethyl ether then washed with water. The organics were dried (MgSO₄) and the solvents removed in vacuo to give a yellow oil. This was purified by silica column (isohexane → 10% EtOAc in isohexane) to give the product as a pale yellow oil (1.79 g, 33%).

This oil was dissolved in acetonitrile (55 ml) and N-bromo-succinimide (10.9 mmol, 1.94 g, 1 eq.) was added. The mixture was stirred overnight then the solvent was removed in vacuo. The residue was partitioned between water and EtOAc. The solvents were evaporated to give the product as a yellow oil (1.79 g, 61 %).

A portion of this product (4.1 1 mmol, 1.00 g) was dissolved in THF (5 ml) and added dropwise to a suspension of magnesium (8.22 mmol, 0.20 g, 2 eq.) in THF (5 ml) containing 1 crystal of iodine. This mixture was heated at reflux for 1 hour and then allowed to cool to room temperature whereupon it was poured onto vigorously stirred solid CO₂. This was stirred until the mixture came to room temperature. 1 MHCI (aq) (10 ml) was added and the organics separated. The aqueous layer was extracted with diethyl ether. The solvents were evaporated to give the product as a pale brown solid (0.37 g, 43%). This was dissolved in DCM (20 ml) and BBr₃ (20 mmol, 5 g, 11 eq.) was added. The mixture was stirred for 3 days whilst being monitored by HPLC. The mixture was treated with HCI solution (0.1 M) then filtered. The aqueous layer was evaporated then dissolved in methanol. The solvent was evaporated. The dissolution/evaporation protocol was repeated a further 3 times and gave the pure product as a white solid (0.04 g, 12%).

4.3 3-Acetyl-4-hydroxy-benzoic acid

Mg(CIO₄)₂ (0.602 mmol, 0.134 g, 2 mol%) was added to a solution of ethyl 4-hydroxybenzoate (30.1 mmol, 5 g) in Ac₂O (45.15 mmol, 4.25 ml) and the mixture was stirred overnight. This was diluted with DCM then washed with water. The organics were dried (MgSO₄) and the solvent evaporated to give a clear oil. This was azeotroped twice with toluene to give the product as a clear oil (5.73 g, 92%). This was mixed with AICI₃ (82.5 mmol, 10.99 g, 3 eq.) and KCI (28.9 mmol, 2.15 g, 1.05 eq.) and heated to 15O⁰C for 1.5 hours during which time a dark foam was formed. This was cooled in an ice bath and ice cold 2MHCI (aq) (100 ml) was added. The solution was stirred for 5 minutes then ethanol (20 ml) was added. This was heated at reflux for 45 minutes then cooled in an ice bath. The solid formed was collected by filtration then purified by recrystallisation from THF/EtOH to give the product as a cream solid (1.30 g, 26%).

4.4 4-Hvdroxy-2-methyl-benzoic acid

BBr₃ (20 mmol, 5 g, 10 eq.) was added to a solution of 4-methoxy-2-methyl benzoic acid (2 mmol, 0.332 g) in DCM (20 ml) and the mixture was stirred under argon until HPLC indicated no starting material remained. HCI (0.1 M, 20 ml) was added and the mixture was filtered. The aqueous layer was evaporated then dissolved in methanol. The solvent was evaporated. The dissolution/evaporation protocol was repeated a further 3 times and gave the pure product as a yellow solid (0.24 g, 80%).

4.5 3-Ethyl-4-hydroxy-benzoic acid

As reference: J. Am. Chem. Soc, 1984, 106, 174.

A sodium hydroxide solution (5 ml, 20 % w/v) was added to alfa-cyclodextrin (371 mg, 0.33 mmol) and copper powder (26 mg, 0.41 mmol). Then 2-ethylphenol (0.48 ml, 4.09 mmol) was added followed by the dropwise addition of carbon tetrachloride (0.77 ml, 7.98 mmol). The reaction was stirred at 80 ⁰C under nitrogen for 6 hrs. It was then allowed to cool to room temperature, ethyl acetate was added (10 ml) and the solution was acidified using 1 MHCI (aq). The solution was extracted with more ethyl acetate and the combined organics were dried (MgSO₄) and the solvent was removed in vacuo. Purification by column chromatography (isohexane: ethyl acetate; 1 :1 ) afforded the product (204 mg, 30 %).

HPLC retention time of 3.70 min. Mass spectroscopy: m/z 166 (100, MH⁺). 4.6 4-Hydroxy-3-propyl-benzoic acid

The title compound was prepared as described for the preparation of 4.5, but using 2- propylphenol instead of 2-ethylphenol.

HPLC retention time of 4.13 min. Mass spectroscopy: m/z 181 (100, MH⁺).

4.6a 4-Hydroxy-3-isopropyl-benzoic acid

The title compound was prepared as described for the preparation of 4.5, but using 2- isopropylphenol instead of 2-ethylphenol.

HPLC retention time of 4.03 min. Mass spectroscopy: m/z 181 (100, MH⁺).

4. 7 2-Fluoro-6-methyl-phenol

3-Fluorosalicylaldehyde (1 17 mg, 0.83 mmol) was dissolved in dry ethyl acetate (15 ml) and Pd/C (12 mg, 10 % w/w) was added. The solution was vigorously stirred at room temperature under a hydrogen atmosphere for 6 hrs. Filtration through celite and removal of the ethyl acetate under vacuo afforded the product (70 mg, 67 %) without need for further purification.

HPLC retention time of 4.09 min. 4.8 3-Fluoro-4-hvdrc>xy-5-methyl-benzoic acid

The title compound was prepared as described for the preparation of 4.5, but using 2-fluoro-6- methyl-phenol (14.8) instead of 2-ethylphenol.

HPLC retention time of 3.17 min.

4.9 2-Oxo-1 ,2,3,4-tetrahvdro-quinazoline-6-carboxylic acid

Prepared as described in Eur. J. Org. Chem. 22 2000 3755-62.

4.10 3- Methyl -2-oxo-1 ,2,3,4-tetrahydro-quinazoline-6-carboxylic acid

Prepared as described in Chem. Pharm. Bull. 36 (6) 2253-2258.

4.1 1 4-(Difluoromethyl)benzoic acid

4-Cyanobenzaldehyde (655 mg, 5 mmol) was dissolved in [bis(2-methoxyethyl)amino]sulfur trifluoride (2 ml) - exothermic! The reaction was then heated to 6O⁰C and monitored by HPLC for the disappearance of starting aldehyde (typically 24 to 36 h). After this time, DCM (20 ml) was added and the reaction was poured onto ice. The organic fraction was separated off, dried (Na₂SO₄), filtered and concentrated. Purified on silica (5 % ethyl acetate / iso-hexane) to give 4- (difluoromethyl)benzonitrile as a clear, colourless oil, 345 mg, 45%.

4-(Difluoromethyl) benzonitrile (600 mg, 3.9 mmol) was heated to reflux in 2M aqueous sodium hydroxide solution (20 ml) for 2h. During this time the initial suspension of starting material dissolved. The reaction was cooled and acidified with aqueous 2M HCI solution to give a white precipitate. This was collected by filtration, washed with water and then dried in vacuo. 4- (Difluoromethyl) benzoic acid was obtained as a white powder, 543 mg.

4.12 4-{ethyl[(9H-fluoren-9-ylmethoxy)carbonyl1amino)benzoic acid

To a mixture of 4-ethylaminobenzoic acid (1.0 g, 6.05 mmol) in 1 ,4-dioxane (12 ml) and 0.5M aqueous sodium hydroxide solution (12 ml) was added 9-fluorenylmethoxycarbonyl chloride (1.72 g, 6.66 mmol). The mixture was partitioned between 1 M HCI (aq) and dichloromethane. The aqueous layer was extracted with dichloromethane and the combined organic extracts were washed with water, brine and dried over sodium sulfate. The solvent was removed and the crude product purified by flash column chromatography on silica to give 4-{ethyl[(9H-fluoren-9- ylmethoxy)carbonyl]amino}benzoic acid as a cream solid.

4.13 Lithium 4-[methyl(phenylsulfonyl)amino1benzoate

To a mixture of methyl 4-(methylamino)benzoate (1 mmol), 4-dimethylaminopyridine (2 mg), and diisopropylethylamine (1.1 mmol) in acetonitrile (3 ml) was added benzenesulfonyl chloride (1.1 mmol). The reaction mixture was stirred for 16h, and the mixture concentrated by nitrogen stream. The crude product was partitioned between 1 M HCI (aq) and ethyl acetate. The aqueous layer was extracted with ethyl acetate and the combined organic extracts were washed with 1 M HCI (aq), water, brine and dried over sodium sulfate. The solvent was removed and the crude product was purified by flash column chromatography on silica to give methyl 4- [methyl(phenylsulfonyl)amino]benzoate as a white solid (249 mg, 81 %).

Methyl 4-[methyl(phenylsulfonyl)amino]benzoate (0.80 mmol) was dissolved in 1 ,4-dioxane (5 ml) and 1 M LiOH (aq) (0.80 mmol) and water (1 ml) were added. After stirring 16h, the sample was concentrated under vacuum, the residue dissolved in 1 : 1 water-acetonitrile and the mixture lyophilized to give lithium 4-[methyl(phenylsulfonyl)amino]benzoate as an off-white solid (238 mg, 100%).

4.14 3-(dimethylamino)-4-hvdroxybenzoic acid

3-Amino-4-hydroxybenzoic acid (459 mg, 3 mmol) was dissolved in methanol (12 ml) and toluene (36 ml) was added. A 2.0 M solution of (trimethylsilyl)diazomethane in hexanes (1.5 ml, 3.0 mmol) was added dropwise and the mixture stirred for 0.5h. The reaction mixture was concentrated under vacuum and the residue purified by flash column chromatography on silica to afford methyl 3-amino-4-hydroxybenzoate as a pink solid (254 mg, 51 %).

A buffer solution at pH 5.5 was prepared by the addition of acetic acid to a 1 M aqueous sodium acetate solution. Methyl 3-amino-4-hydroxybenzoate (254 mg, 1.5 mmol) was dissolved in a mixture of buffer (1 ml) and methanol (2 ml). Formaldehyde solution (37% by weight in water; 0.75 ml, 10 mmol) was added, the mixture stirred for 15 minutes, and then sodium cyanoborohydride (283 mg, 4.5 mmol) was added portionwise. The reaction mixture was stirred for an additional 0.5h and then concentrated. The residual oil was partitioned between water and ethyl acetate. The aqueous layer was extracted with ethyl acetate and the combined organic extracts were washed with water, brine and dried over sodium sulfate. The solvent was removed and the crude product was purified by flash column chromatography on silica to give methyl 3-dimethylamino-4-hydroxybenzoate as a yellow gum (213 mg, 73%).

To a solution of methyl 3-dimethylamino-4-hydroxybenzoate (210 mg, 1.1 mmol) in 1 ,4-dioxane (2 ml) was added 1 M LiOH (aq) (4 mmol). After stirring for 4Oh, the mixture was acidified to pH 2 by addition of 1 M HCI (aq). The mixture was partitioned between water and ethyl acetate, and the aqueous layer was lyophilized to give a mixture of sodium chloride and 3-dimethylamino-4- hydroxybenzoic acid as a brown semi-solid (378 mg). The crude material was used in the subsequent reaction. 4.15 1-[(9H-fluoren-9-ylmethoxy)carbonyl1-1 ,2,3,4-tetrahydroquinoline-6-carboxylic acid

To a mixture of 1 ,2,3,4-tetrahydroquinoline-6-carboxylic acid (1.03 g, 5.8 mmol) in 1 ,4-dioxane (12 ml) and 0.5M aqueous sodium hydroxide solution (12 ml) was added 9- fluorenylmethoxycarbonyl chloride (1.68 g, 6.5 mmol). The mixture was partitioned between 1 M HCI (aq) and dichloromethane. The aqueous layer was extracted with dichloromethane and the combined organic extracts were washed with water then brine and dried over sodium sulfate. The solvent was removed in vacuo and the crude product purified by flash column chromatography on silica to give 1-[(9H-fluoren-9-ylmethoxy)carbonyl]-1 , 2,3,4- tetrahydroquinoline-6-carboxylic acid as a white solid (2.Og; 86%).

4.16 4-[(2,2,2-trifluoroethyl)amino1benzoic acid

To a suspension of methyl-4-aminobenzoate (302 mg, 2.0 mmol) in dichloromethane (3 ml) was added trifluoroacetic anhydride (0.31 ml, 2.2 mmol). The mixture was stirred for 1 hour and then partitioned between 1 M NaHCO₃ (aq) and dichloromethane. The aqueous layer was further extracted with dichloromethane, and the combined organic extracts washed with water then brine and dried over sodium sulfate. The solvent was removed to give methyl 4- [(trifluoroacetyl)amino]benzoate as a white solid (525 mg, 100%).

To a stirred solution of methyl 4-[(trifluoroacetyl)amino]benzoate (124 mg, 0.5 mmol) in an. THF(I ml) at 0 ⁰C under an argon atmosphere was added borane-dimethyl sulfide complex (57 mg, 0.75 mmol) and the mixture heated at reflux for 2h. The mixture was allowed to cool, methanol (approx. 0.1 ml) was added dropwise until effervescence ceased. The mixture was partitioned between water and 1 :1 ethyl acetate-MTBE. The aqueous layer was extracted three times with ethyl acetate-MTBE, and the combined organic extracts washed with water, brine and dried over sodium sulfate. The solvent was removed and the crude product was purified by flash column chromatography on silica to give methyl 4-[(2,2,2-trifluoroethyl)amino]benzoate as a white crystalline solid (47 mg, 32%).

To a solution of methyl 4-[(2,2,2-trifluoroethyl)amino]benzoate (130 mg, 0.56 mmol) in 1 ,4- dioxane (2 ml) was added 1 M LiOH (aq) (0.61 mmol). After stirring at 4OC for 4h, further 1 M LiOH (aq) (0.30 mmol) was added. The mixture was stirred at room temperature until all ester was hydrolysed. The mixture was concentrated and the residue partitioned between 1 M HCI (aq) and ethyl acetate. The aqueous layer was further extracted with ethyl acetate and the combined organic extracts were washed with water, brine and dried over sodium sulfate. The solvent was removed to give 4-[(2,2,2-trifluoroethyl)amino]benzoic acid as an off-white solid (1 19 mg, 97%).

4.17 4-Methanosulphonylamino-3-methoxy-benzoic acid

Methanesulfonyl chloride (615 μL) was added to a solution of 4-amino-3-methoxy-benzoic acid methyl ester (1 g) in dichloromethane (20 imL) and pyridine (1.5 imL) and a catalytic amount of DMAP. After 1-16 hrs the mixture was concentrated to near dryness and the product crystallized from added ethanol. This product was hydrolyzed in 2.5 M LiOH (5 imL), THF (14 imL), MeOH (7 mL) in a microwave oven at 1 10 ⁰C for 30 min. After cooling, the solution was acidified with aq. HCI and extracted with ethyl acetate, dried with Na₂SO₄ and concentrated to dryness.

4.18 4-(Methanesulfonyl-ιmethyl-aιmino)-benzoic acid

A mixture of 4-Methanesulfonylamino-benzoic acid methyl ester (0.5 g), methyl iodide (0.4 mL) and potassium carbonate (0.9 g) in acetonitrile (10 mL) was kept in a microwave oven at 120 ⁰C for 10 min. The cooled mixture was filtered and concentrated to dryness. The remains were precipitated by addition of DCM and the solid was collected and dried under vacuum. Hydrolysation of the methyl ester as described fo the preparation of 4.17 gave the title compound.

4.19 4-Methanesulphonylamino-3-methylbenzoic acid

The title compound was prepared according to the procedure described for the preparation of 4.77, but using 4-amino-3-methylbenzoic acid methyl ester instead of 4-amino-3-methoxy- benzoic acid methyl ester.

4.20 4-Methanesulphonylaminobenzoic acid

The title compound was prepared according to the procedure described for the preparation of 4.17, but using 4-amino-benzoic acid methyl ester instead of 4-amino-3-methoxy-benzoic acid methyl ester and isopropanesulphonyl chloride instead of methanesulphonyl chloride.

4.21 4-Amino-2-methyl-benzoic acid methyl ester

4-Acetylamino-2-methyl-benzoic acid methyl ester was kept in cone. HCI/MeOH 1 :1 in a microwave oven at 70 ⁰C for 2 hrs. After cooling the solid was collected and dried under vacuum. Hydrolysation of the methyl ester as described fo the preparation of 4.17 gave the title compound.

4.22 4-Methanesulphonylamino-2-methyl-benzoic acid methyl ester

Methanesulfonyl chloride was added to a solution of 4-amino-2-methyl-benzoic acid methyl ester in dichloromethane, pyridine and a catalytic amount of DMAP. After 1-16 hrs the mixture was concentrated to near dryness and the product crystallized from added ethanol. This product was hydrolyzed in 2.5 M LiOH (5 ml_), THF (14 ml_), MeOH (7 ml.) in a microwave oven at 110 ⁰C for 30 min. After cooling, the solution was acidified with aq. HCI and extracted with ethyl acetate, dried with Na₂SO₄ and concentrated to dryness.

4.23 3-Chloro-4-methanesulphonylaminobenzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 3-chloro-4-aminobenzoic acid methyl ester instead of 4-amino-3-methoxy- benzoic acid methyl ester.

4.24 4-Methanosulphonylamino-2-methoxy-benzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-amino-2-methoxy-benzoic acid methyl ester instead of 4-amino-3-methoxy- benzoic acid methyl ester.

4.25 4-Methanesulphonylaminobenzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-amino-benzoic acid methyl ester instead of 4-amino-3-methoxy-benzoic acid methyl ester and cyclopropanesulphonyl chloride instead of methanesulphonyl chloride.

4.26 S-Fluoro^-methanesulphonylaminobenzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-amino-3-fluoro-benzoic acid methyl ester instead of 4-amino-3-methoxy- benzoic acid methyl ester.

4.26a 3-Acetyl-4-amino-benzoic acid methyl ester

δ-Amino-furan^-carboxylic acid methyl ester (0.42 g, 3.0 mmol) were mixed together with methyl vinyl ketone (10 ml.) in benzene and heated at reflux for 1 h. Evaporation of solvents were followed by flash chromatography using DCM / MeOH (95:5) as eluent to yield 44% (278 mg. 1.31 mmol) of 5-Acetyl-4-amino-1-hydroxy-cyclohexa-2,4-dienecarboxylic acid methyl ester. This compound were mixed with BF₃ OEt₂ (284 mg, 2.0 mmol) in benzene (15 ml.) and refluxed for 0.5 h. The reaction mixture was quenched with NaHCO₃ (sat) and extracted with dichloromethane. The precipitation formed in the organic phase was collected and confirmed to be the product by characterization with LC-MS and ¹H NMR. Yield: 127 mg (50%).

4.27 3-Acetyl-4-methanesulphonylamino-benzoic acid

Methanesulfonyl chloride was added to a solution of 3-acetyl-4-amino-benzoic acid methyl ester (4.87) in dichloromethane, pyridine and a catalytic amount of DMAP. After 1-16 hrs the mixture was concentrated to near dryness and the product crystallized from added ethanol. This product was hydrolyzed in 2.5 M LiOH, THF, MeOH in a microwave oven at 1 10 ⁰C for 30 min. After cooling, the solution was acidified with aq. HCI and extracted with ethyl acetate, dried with Na₂SO₄ and concentrated to dryness.

4.28 4-(3-Fluoro-benzenesulphonylamino)-benzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-amino-benzoic acid methyl ester instead of 4-amino-3-methoxy-benzoic acid methyl ester and 3-fluoro-benzenesulphonyl chloride instead of methanesulphonyl chloride.

4.29 4-(2-Fluoro-benzenesulphonylamino)-benzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-amino-benzoic acid methyl ester instead of 4-amino-3-methoxy-benzoic acid methyl ester and 2-fluoro-benzenesulphonyl chloride instead of methanesulphonyl chloride.

4.30 4-(1-Methyl-1 H-imidazole-2-sulphonylamino)-benzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-amino-benzoic acid methyl ester instead of 4-amino-3-methoxy-benzoic acid methyl ester and 1-methyl-1-H-imidazole-2-sulphonyl chloride instead of methanesulphonyl chloride. 4.31 4-(Toluene-2-sulphonylamino)-benzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-amino-benzoic acid methyl ester instead of 4-amino-3-methoxy-benzoic acid methyl ester and toluene-2-sulphonyl chloride instead of methanesulphonyl chloride.

4.32 4-(2-Fluoro-benzenesulphonylamino)-benzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-amino-benzoic acid methyl ester instead of 4-amino-3-methoxy-benzoic acid methyl ester and 2-fluorobenzenesulphonyl chloride instead of methanesulphonyl chloride.

4.33 4-(Pyridine-3-sulphonylamino)-benzoic acid

Trifluoromethane sulphonic anhydride (380 μl_) was added to polymer supported tiriphenylphosphine oxide (1 g) in dichloromethane (15 ml_). After 1 h the mixture was cooled to 0 ⁰C and a solution of pyridine 3-sulphonic acid (360 mg) as the pyridinium salt in DCM (4 ml.) was added. After 30 min 4-methanesulphonylamino-benzoic acid methyl ester (318 mg) in dichloromethane (4 ml.) was added. The mixture was shaken at 25 ⁰C for 16 h. The resin was filtered off and the filtrate was concentrated to dryness followed by purification using column chromatography on silica. Hydrolysis of the methyl ester as described for the preparation of 4.17 gave the titled compound. 4.34 4-Benzenesulphonylamino-3-methylbenzoic acid

The title compound was prepared according to procedure described for the synthesis of 4- butane-1-sulphonylamino)-benzoic acid methyl ester (15.68) but using 4-amino-3-methyl- benzoic acid instead of 4-amino-benzoic acid and phenylsulphonyl chloride instead of butylsulphonyl chloride. Hydrolysis of the methyl ester as described for the preparation of 4.17 gave the titled compound.

4.34 4-(3-Chloro-benzenesulphonylamino)-benzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-amino-benzoic acid methyl ester instead of 4-amino-3-methoxy-benzoic acid methyl ester and 3-chlorobenzenesulphonyl chloride instead of methanesulphonyl chloride.

4.35 4-(Pyridine-2-sulphonylamino)-benzoic acid

The title compound was prepared according to the procedure described for the preparation of compound 4.33 but using pyridine 2-sulphonic acid instead of pyridine 3-sulphonic acid. 4.36 4-Methylsulphamoyl-benzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using methylamine instead of 4-amino-3-methoxy-benzoic acid methyl ester and 4- chlorosulphsulphonyl benzoic acid methyl ester instead of methanesulphonyl chloride.

4.37 4-(2,4-Dimethyl-thiazole-5-sulphonylamino)-benzoic acid

The title compound was prepared according to procedure described for the synthesis of 4- (butane-i-sulphonylamino)-benzoic acid methyl ester but using 2,4-dimethyl-thiazole-5- sulphonyl chloride instead of butylsulphonyl chloride. Hydrolysis of the methyl ester as described for the preparation of 4.17 gave the titled compound.

4.38 4-(3-Methoxy-benzenesulphonylamino)-benzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-amino-benzoic acid methyl ester instead of 4-amino-3-methoxy-benzoic acid methyl ester and 3-methoxybenzenesulphonyl chloride instead of methanesulphonyl chloride. 4.39 4-(3-Methoxy-benzenesulphonylamino)-benzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-amino-benzoic acid methyl ester instead of 4-amino-3-methoxy-benzoic acid methyl ester and 4-methoxybenzenesulphonyl chloride instead of methanesulphonyl chloride.

4.40 6-Benzenesulphonylamino-nicotinic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-amino-nicotinic acid methyl ester instead of 4-amino-3-methoxy-benzoic acid methyl ester and benzenesulphonyl chloride instead of methanesulphonyl chloride.

4.41 3-Methylsulphamoyl-benzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.39), but using methylamine instead of 4-amino-3-methoxy-benzoic acid methyl ester and 3- chlorosulphsulphonyl benzoic acid methyl ester instead of methanesulphonyl chloride.

4.42 Pyridine 4-sulphonic acid

4-Mercaptopyridine (500 mg) was dissolved in glacial acetic acid (18 ml), followed by the addition of 35% hydrogen peroxide (6 ml). The solution was warmed at 80 ⁰C for 90 min. and then concentrated to dryness. The product was re-crystallized from methanokwater, dried in a vacuo and used in the next step.

4.43 4-(Pyridine-4-sulphonylamino)-benzoic acid

The title compound was prepared according to procedure described for the synthesis of 4- (pyridine-3-sulphonylamino)-benzoic acid methyl ester but using pyridine 4-sulphonic acid instead of pyridine 3-sulphonic acid.

4.44 6-Methanesulphonylamino-nicotinic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-amino-nicotinic acid methyl ester instead of 4-amino-3-methoxy-benzoic acid methyl ester.

4.45 4-Methylthiazole-2-sulphonyl chloride

4-Methylthiazole (2.0 g, 20.2 mmol) was dissolved in methyl-f-butyl ether (46 ml.) and the solution was cooled to 0 ⁰C. lsopropyl magnesium chloride (10.1 ml_, 2.0 M) was added drop wise at 0°C. The mixture was then heated to 40 ⁰C and sulphur dioxide in dimethoxymethane (4.2 ml_, 6.0 M) was added drop wise and the reaction was then left at this temperature for 1 h. After cooling the reaction mixture to 0 ⁰C, Λ/-chlorosuccinimide (4.05 g, 30.3 mmol) was added, and the reaction was kept at 0 ⁰C for 45 min. After addition of HCI (0.2 M, aq, 50 ml.) at 0 ⁰C the reaction was left to warm up to ambient temperature for 2 hrs and extracted with methyl-f-butyl ether. The organic phase was washed with of HCI (0.2 M, aq), water and brine then dried over Na₂SO₄, filtered and evaporated to yield the title compound (2.33 g, 59%).

4.46 4-(4-Methylthiazole-2-sulphonylamino)-benzoic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 4-aminobenzoic acid methyl ester instead of 4-amino-3-methoxy-benzoic acid methyl ester and 4-methylthiazole-2-sulphonyl chloride instead of methanesulphonyl chloride.

4.47 4-(4-Nitro-benzenesulphonylamino)-benzoic acid

The title compound was prepared according to procedure described for the synthesis of (4.17), but using 4-aminobenzoic acid instead of 4-amino-3-methoxy-benzoic acid methyl ester and 4- nitrobenzenesulphonyl choride instead of methanesulphonyl chloride.

4.48 2-Methanesulphonylamino-thiazole-5-carboxylic acid

The title compound was prepared according to the procedure described for the preparation of (4.17), but using 2-thiazole-5-carboxylic acid methyl ester instead of 4-amino-3-methoxy- benzoic acid methyl ester. 4.49 4-(Pyridin-2-ylamino)-benzoic acid

4-Aminobenzoic acid methyl ester (1 g, 6.6 mmol), 2-fluoropyridine (1.28 g, 13.2 mmol) and potassium carbonate (1.83 g, 13.2 mmol) in DMF was heated in a microwave oven at 200°C for 20 min. The residue was extracted with dichloromethane and water. The organic layer was dried, concentrated and purified on a Siθ₂ column (toluene-EtOAc 8:2). Hydrolysis of the methyl ester as described for the preparation of 4.17 gave the title compound.

4.50 5-(2-Methoxy-ethylcarbamoyl)-thiophene-2-carboxylic acid

δ-Formyl-thiophene^-carboxylic acid (740 mg, 4.7 mmol), methoxyethylamine (412 μl_, 4.7 mmol), HATU (4.5 g 1 1.8 mmol), ethyldiisopropylamine (4 ml) and DMF (2 m) in DCM (20 ml) was stirred for 2 hrs. The mixture was extracted with DCM and aq. bicarbonate. The organic layer was dried and concentrated. This residue was oxidized in t-BuOH (4ml) by the addition of sodium phosphate buffer (0.5M, 2 ml) and aq. KMnO₄ (1 M, 2ml). After 10 min sat. aq. Na₂SO₄ (2 ml) was added and the pH was adjusted to 3 with aq. HCI. Extracted with EtOAc. The organic layer was dried and concentrated.

4.51 4-(2-Chloropyridine-3-sulphonylamino)-benzoic acid

The title compound was prepared according to procedure described for the preparation of (4.77) but using 4-amino-benzoic acid instead of 4-amino-3-methoxy-benzoic acid methyl ester and 2- chlorobenzene-3-sulphonyl choride instead of methanesulphonyl chloride. 4.52 4-(4-Methyl-pyridine-3-sulphonylamino)-benzoic acid

The title compound was prepared according to procedure described for the synthesis of 4- (pyridine-3-sulphonylamino)-benzoic acid methyl ester but using 4-methyl-pyridine-3-sulphonic acid instead of pyridine-3-sulphonic acid.

4.53 4-Ethanesulphonylamino-benzoic acid

The title compound was prepared according to procedure described for the preparation of (4.17) but using 4-amino-benzoic acid instead of 4-amino-3-methoxy-benzoic acid methyl ester and ethanesulphonyl choride instead of methanesulphonyl chloride.

4.54 3-Chloro-4-methanesulphonylamino-benzoic acid

The title compound was prepared according to procedure described for the preparation of (4.17) but using 4-amino-3-chlorobenzoic acid instead of 4-amino-3-methoxy-benzoic acid methyl ester.

4.55 4-Butane-1-sulphonylamino)-benzoic acid

4-Amino-benzoic acid methyl ester (1.0 g, 6.62 mmol) and a catalytic amount of DMAP were dissolved in pyridine (0.5 ml.) and DCM (15 ml_). The reaction mixture was cooled to 0 °C and butylsulphonyl chloride (1.01 ml_, 6.62 mmol) was added via a syringe. The reaction was left stirring and allowed to warm up to room temperature over night. Evaporation of solvents was followed by addition of DCM and the organic phase was washed with HCI (aq, 1 M), water and brine. After being dried over Na₂SO₄, the organic phase was filtered and evaporated gave the methyl ester of the title compound (1.34 g, 74%). Hydrolysis of the methyl ester as described for the preparation of (4.17) gave the title compound.

4.56 4-(4-Ethyl-thiazol-2-ylamino)-benzoic acid

4-Thioureido-benzoic acid ethyl ester (0.52 g, 2.23 mmol) and 1-Bromo-butan-2-one (0.25 ml_, 2.45 mmol) was mixed in dioxane (4 ml.) and microwave heated at 1 10 °C for 15 min. To the precipitated crystals were added DCM and NaHCU3. The organic phase was separated, washed with water and brine, dried over Na₂SO₄, filtered and evaporated to yield 4-(4-Ethyl- thiazol-2-ylamino)-benzoic acid ethyl ester (0.58 g, 93%). Hydrolysis of the ethyl ester as described for the preparation of (4.17) gave the title compound.

4.57 5-Methyl-thiazole-2-sulphonyl chloride

5-Methyl-1 ,3-thiazole (1.0 g, 10.1 mmol) was dissolved in methyl-f-butyl ether (25 ml.) and the solution was cooled to 0°C. Addition of isopropyl magnesium chloride (10.1 ml_, 2.0 M) was done drop wise at 0°C. The mixture was then heated to 40°C and sulfur dioxide in dimethoxyethane (1.64 ml_, 7.7 M) was added drop wise and the reaction was then left at this temperature for 45 min. After cooling the reaction mixture to 0°C, Λ/-chlorosuccinimide (2.02 g, 15.2 mmol) was added and the reaction were kept at 0°C for 1 h. After addition of HCI (aq, 0.2 M, 25 ml.) at 0°C the reaction was left to warm up to ambient temperature for 2 hrs and extracted with methyl-f-butyl ether. The organic phase was washed with of HCI (aq, 0.2 M), water and brine then dried over Na₂SO₄, filtered and evaporated to yield the title compound (1.82 g, 91 %). 4.58 4-(5-Methylthiazole-2-sulphonylamino)-benzoic acid

The title compound was prepared according to procedure described for the preparation of (4.17) but using 4-amino-benzoic acid instead of 4-amino-3-methoxy-benzoic acid methyl ester and 5- methylthiazole-2-sulphonyl chloride (4.1 19) instead of methanesulphonyl chloride.

4.59 4-lsopropylthiazole-2-sulphonyl chloride

1-Bromo-3-methyl-butan-2-one (1.15 g, 6.95 mmol) and thioformamide (0.43 g, 6.95 mmol) were dissolved in dioxane (10 ml.) and heated in microwave at 110 °C for 15 min. Dichloromethane and NaHCO₃ was added and after separation the organic phase was washed with NaOH (aq, 1 M) and water. Back-extracted water phase with dichloromethane. The combined organic phases were washed with water and brine, dried over Na₂SO₄, filtered and evaporated to yield 0.82 g (70%) of 4- isopropyl-1 ,3-thiazole.

4-lsopropyl-1 ,3-thiazole (0.62 g, 4.9 mmol) was dissolved in methyl-f-butyl ether (15 ml.) and the solution was cooled to 0 °C. Addition of isopropyl magnesium chloride (2.9 ml_, 2.0 M) was done drop wise at 0°C. The mixture was then heated to 40 °C and sulfur dioxide in dimethoxyethane (0.79 ml_, 7.7 M) was added drop wise and the reaction was then left at this temperature for 45 min. After cooling the reaction mixture to 0°C, Λ/-chlorosuccinimide (0.97 g, 7.3 mmol) was added, and the reaction were kept at 0°C for 1 h. After addition of HCI (aq, 0.2 M, 10 ml.) at 0 °C the reaction was left to warm up to ambient temperature for 2 hrs and extracted with methyl-f-butylether. The organic phase was washed with of HCI (aq, 0.2 M), water and brine then dried over Na₂SO₄, filtered and evaporated to yield the title compound (0.92 g, 84%). 4.60 4-(4-lsopropylthiazole-2-sulphonylamino)-benzoic acid

The title compound was prepared according to procedure described for the preparation of (4.58) but using 4-amino-benzoic acid instead of 4-amino-3-methoxy-benzoic acid methyl ester and 4- isopropylthiazole-2-sulphonyl chloride instead of methanesulphonyl chloride.

Example 5

Alternative P1 building block

Starting material

See Chem. Ber., 101 (1968), 3802. and Tetrahedron, 43, 13 (1987), 3095-3108.

Step a)

Palladium on carbon was added to a solution of the azide diol starting material (12.5 g, 31.3 mmol) in MeOH. The reaction was stirred under an H₂ atmosphere at RT overnight. The precipitated product was dissolved in EtOH/H₂0 and the reaction filtered. The filtrate was concentrated in vacuo and freeze dried. Yield 9.64 g, 82%

Step b)

The amine of the previous step (2.15 g, 5.76 mmol) was dissolved in MeOH buffer and benzaldehyde added. The reaction was stirred at RT for two hours. NaCNBH₃ (434 mg, 6.9 mmol) was added, causing effervescence. The reaction was stirred at RT overnight. The reaction was concentrated in vacuo and extracted with EtOAc. The product was purified on silica, eluting with 30% EtOAc/Hex. Yield 1.49 g, 88%

Step c)

The N-benzyl, isopropylidine protected mono-alcohol of the previous step (1.49 g, 5.1 mmol) was dissolved in DCM (5 ml) and cooled to O⁰C. Deoxyfluor™ (1.13 ml, 6.1 mmol) was added and the reaction stirred at O⁰C- RT overnight. The reaction was given an aqueous basic workup. The product was purified on silica eluting with 10-30% EtOAC/Hex. Yield 200 mg, 73%.

Step d

The benzyl/isopropylidine-protected bicyclic of the preceding step (433 mg, 1.48 mmol) was dissolved in 4 ml 1 :1 TFA/H₂O and heated at 5O⁰C for 3 hours. The reaction was concentrated in vacuo and azeotroped with toluene. The black residue was dissolved in pyridine and 1 ml Ac₂O added. The reaction was stirred at room temperature overnight, concentrated in vacuo and the residue given an aqueous basic work-up to yield the corresponding diacetate. ¹H NMR of the dark brown residue was consistent with structure.

The diacetate crude product was dissolved in 15 ml DCM and cooled to O⁰C HBR/AcOH (33% wt, 1.4 ml) was added and the reaction stirred at O⁰C to RT overnight. The reaction was concentrated in vacuo, azeoptroped with toluene and purity confirmed with TLC. The crude material was dissolved in THF (10 ml) and cooled to O⁰C. LiAIH₄ (1 N in THF, 2eq, 2.96 ml) was added dropwise and the reaction stirred at O⁰C for 1.5 hours. The reaction was quenched with 60 μl H₂O, 60 μl NaOH (2N), 180 μl H₂O. The reaction was filtered and the filtrate extracted with EtOAc. ¹H NMR consistent with structure Yield 240 mg 62%.

The resultant P1 building block can be N-deprotected, coupled with the P2 and P3 building blocks and then oxidized to the ketone. Alternatively, the hydroxyl groups can be protected, for example as the dimethylketal for coupling, followed by regeneration of the ketone. A further alternative is to generate the ketone on the P1 building block prior to coupling with P2 and/or P3.

Example 6

An alternative P1 building block

OaS.ΘS.ΘaSVΘ-Chloro-S^-dimethoxy-hexahvdro-furofS^-άipyrrole^-carboxylic acid benzyl ester

Step a) (3aS,6R,6aS)-6-Hydroxy-3,3-dimethoxy-hexahydro-furo[3,2-/b]pyrrole-4- carboxylic acid benzyl ester

To a stirred solution of (3aS,6R,6aS)-6-Hydroxy-3,3-dimethoxy-hexahydro-furo[3,2-/b]pyrrole-4- carboxylic acid terf-butyl ester (3800 mgs, 13.13 mmol) in CH₂CI₂ (100 mis) was added 2M HCI in MeOH (50 mis). The resulting solution was stirred overnight and then concentrated and azeotroped with Toluene (3 x 100 mis). The crude was then dissolved in CH₂CI₂ (100 mis) cooled to O⁰C and pyridine (1071 uls, 13.13 mmol) added followed by dropwise addition of CBZCI (1875 uls, 13.13 mmol). The reaction was stirred at room temperature for 2 hours then washed with 2 M HCI (2 x 50 mis), saturated NaHCO₃ (2 x 50 mis), dried (MgSO₄) and concentrated. The residue was purified by flash column chromatography (5-100% isohexane: EtOAc) to obtain the title compound as a clear oil (2510 mgs, 59 %). MS M + H 324, Retention Time 3.4 mins 10-90 MeCN:0.05%TFA 6 min Gradient C12 Reverse Phase 50mm ^* 4.6mm i.d. column.

Step b) (3aS,6R,6aS)-6-Methanesulfonyloxy-3,3-dimethoxy-hexahydro-furo[3,2-

/b]pyrrole-4-carboxylic acid benzyl ester

To a stirred solution of (3aS,6R,6aS)-6-Hydroxy-3,3-dimethoxy-hexahydro-furo[3,2-/b]pyrrole-4- carboxylic acid benzyl ester (500 mgs, 1.55 mmol) in CH₂CI₂ (20 mis) was added triethyl amine (332 uls, 2.32 mmol) and mesyl chloride (266 mgs, 2.32 mmol). After stirring for 30 minutes the reaction was washed with saturated NaHCO₃ (1 x 20 mis), 2M HCI (1 x 20 mis), dried MgSO₄ and concentrated to give the title product (655 mgs, 99%) as a yellow oil. MS M + H 402, Retention Time 4.37 mins 10-97 MeCN:0.05%TFA 6 min Gradient C12 Reverse Phase 50mm ^* 4.6mm i.d. column. Step c) (3aS,6S,6aS)-6-Chloro-3,3-dimethoxy-hexahydro-furo[3,2-/b]pyrrole-4-carboxylic acid benzyl ester

To a stirred solution of (3aS,6R,6aS)-6-Methanesulfonyloxy-3,3-dimethoxy-hexahydro-furo[3,2- /b]pyrrole-4-carboxylic acid benzyl ester (550 mgs, 1.37 mmol) in DMF (30 mis) was added lithium chloride (721 mgs, 13.7 mmol). After stirring for 120 minutes at 12O⁰C the reaction was concentrated, the residue was diluted with CH₂Cb (50 mis) washed with water (1 x 20 mis), dried MgSO₄ and concentrated. The residue was purified by flash column chromatography (5- 66% isohexane: EtOAc) to give the title product (330 mgs, 72%) as a yellow oil. MS M + H 342, 344, Retention Time 4.29 mins 10-97 MeCN:0.05%TFA 6 min Gradient C12 Reverse Phase 50mm ^* 4.6mm i.d. column.

The building block is coupled to P2/P3 as described and above and the P1 ketone generated.

Example 7

An alternative P1 building block

Step a)

J4°y ^■,iio MsCl, Pyridine

Quant.

1

1 (See Chem. Ber., 101 (1968), 3802 & Tetrahedron, 43, 13 (1987), 3095-3108)(7.0 g, 28.5 mmol) was dissolved in dry pyridine (50 ml.) and the solution cooled to O⁰C. Mesyl chloride was slowly added to the solution after approximately 10 minutes and the solution could adjust to room temperature. The reaction was stirred o.n (which is not necessary) and after 14 hrs MeOH (10 imL) was added followed by EtOAc (150 ml_). The solution was washed three times with 2 M H₂SO_{4 (aq)} (3^*100 ml.) and two times with NaHCO₃ sat. _(aq) (2^*100 mL) and thereafter the organic phase was dried with Na₂SO₄, filtered and the solvent was removed by rotary avaporation. The crude product was put on a high vacuum pump over night and gave product 2 as a slightly yellow oil in quantitative yield (1 1.5 g).

¹H NMR (CDCI₃, 400 MHz) 1.34 (s, 3H), 1.51 (s, 3H), 3.07 (s, 3H), 3.16 (s, 3H), 4.18 (d, 1 H, J = 3.1 ), 4.36 (dd, 1 H, J = 8.6, 3.2), 4.42 (dd, 1 H, J = 12.0, 5.0), 4.67 (dd, 1 H, J = 1 1.9, 2.3), 4.74 (d, 1 H₁ J = 3.7), 5.11 (ddd, 1 H₁ J = 8,6, 5.0, 2.3), 5.89 (d, 1 H₁ J = 3.6).

Step b)

Compound 2 (1 1.5 g, 28.5 mmol) was dissolved in DMF (50 mL). NaOAc (23.4 g, 285 mmol) and Ac₂O (48.6 mL, 0.514 mol) was added to the solution, which was heated to 125 ⁰C for 86 hrs. Some of the solvent was removed by rotary evaporation before addition of 500 mL EtOAc. The very dark solution was filtered through celite, with little or no effect. The wash with H₂O (3^* 350 mL) was tedious and took a long time. (Diethyl ether and brine was also added to increase the separation between the phases.) The organic phase was dried with Na₂SO₄, filtered and the solvent was removed by rotary evaporation. The crude products were purified with flash chromatography (heptane: ethyl acetate 7:3 -> 2:1 ) giving compound 3 in 61 % yield (5.70 g) and compound 4 in 22% yield (2.34 g).

Compound 3: ¹H NMR (CDCI₃, 400 MHz) 1.34 (s, 3H), 1.53 (s, 3H), 2.09 (s, 3H), 2.1 1 (s, 3H), 3.94 (d, 1 H, J = 3.4), 4.19 (dd, 1 H₁ J = 12.2, 5.0), 4.32 (dd, 1 H, J = 8.0, 3.3), 4.37 (dd, 1 H₁ J = 12.3, 3.5), 4.73 (d, 1 H, J = 3.6), 5.32-5.37 (m, 1 H), 5.94 (d, 1 H₁ J = 3.8).

Compound 4: ¹H NMR (CDCI₃, 400 MHz) 1.34 (s, 3H), 1.51 (s, 3H), 2.10 (s, 3H), 3.10 (s, 3H), 4.11 (d, 1 H, J = 3.6), 4.21 (dd, 1 H, J = 12.8, 6.2), 4.32 (dd, 1 H, J = 8.3, 3.2), 4.65 (dd, 1 H, J = 12.7, 2.2), 4.73 (d, 1 H, J = 3.5), 5.09 (ddd, 1 H, J = 8,3, 6.1 , 2.3), 5.89 (d, 1 H₁ J = 3.5). Step c)

3 days, 41%

Triethyl silane (27.6 ml_, 173 mmol) was added to compound 3 (5.70 g, 17.3 mmol) dissolved in dry DCM (40 ml_). The round bottomed flask was placed under inert atmosphere (N₂) in an ice bath, and allowed to cool, before slow addition of BF₃ ^*Et₂0 (23.1 ml_, 173 mmol). The reaction went slowly and was stirred for 3 days. There was still starting material but also a byproduct at work-up. Slow addition of NaHCO_{3 (sa}t _aq) (70 ml.) was followed by spoonwise addition of solid NaHCO₃ until the gas evolution ceased. The aqueous phase was extracted with DCM (150 ml.) and washed with NaHCO_{3 (sa}t _aq) (70 ml_), NH₄CI _(sat _aq> (70 ml_). The organic phase was dried with Na₂SO₄, filtered and evaporated. The crude product was purified by flash chromatography (Heptane: Ethyl acetate (2:1 ) to give a yield of 41 % (1.95 g). 0.69 g of starting material was also isolated.

¹H NMR (CDCI₃, 400 MHz) 2.08 (s, 3H), 2.1 1 (s, 3H), 3.72 (dd, 1 H, J = 10.0, 2.6), 3.95 (dd, 1 H, J = 4.6, 2.1 ), 4.17- 4.27 (m, 3H), 4.37 (dd, 1 H₁ J = 12.1 , 3.7), 4.52- 4.57 (m, 1 H), 5.26- 5.31 (m, 1 H).

Step d)

Imidazole (1.46g, 21.4 mmol) was added to a solution of compound 5 (1.95g, 7.14 mmol) in DMF (50 ml_). TBDPSCI was added after a couple of minutes and reaction was stirred at room temperature over night. Ethyl acetate (200 ml.) was added to the reaction and the solution was washed with 10% citric acid_(aq) (3^* 50 ml.) and NaHCO_{3 (sa}t _aq> (50 ml_). The organic phase was dried with Na₂SO₄, filtered and evaporated. The crude product was purified by flash chromatography (Heptane: Ethyl acetate (4:1 ) to give a yield of 92% (3.39g). ¹H NMR (CDCI₃, 400 MHz) 1.09 (s, 9H), 2.04 (s, 3H), 2.05 (s, 3H), 3.71 (dd, 1 H, J = 4.3, 2.1 ), 3.78 (dd, 1 H, J = 9.5, 2.2), 3.99 (dd, 1 H₁ J = 9.6, 4.6), 4.12 (dd, 1 H, J = 12.2, 5.1 ), 4.28- 4.33 (m, 2H), 4.36- 4.40 (m, 1 H), 5.17- 5.22 (m, 1 H), 7.37- 7.51 (m, 6H), 7.58- 7.74 (m, 4H). Step e)

NaOMe, MeOH ^Quant

NaOMe (10 ml_, 0.5 M in MeOH) was added to a solution of 6 (3.39 g, 6.63 mmol) dissolved in MeOH (60 ml_). The reaction was stirred at room temperature for 2 hrs before the solution was neutralized by adding Dowex 50 WX8 (H⁺ form) until a neutral pH was reached. The beads were filtered off and the solvent was removed by rotary evaporation followed by high vacuum. The product was obtained in quantitative yield (2.66 g).

¹H NMR (CDCI₃, 400 MHz) 1.08 (s, 9H), 3.64 (dd, 1 H, J = 11.5, 5.5), 3.71 (dd, 1 H, J = 11.4, 3.9), 3.73- 3.77 (m, 2H), 3.85- 3.90 (m, 1 H), 3.95 (dd, 1 H, J = 9.6, 4.7), 4.15 (dd, 1 H, J = 6.1 , 4.2), 4.39- 4.43 (m, 1 H), 7.37- 7.51 (m, 6H), 7.58- 7.74 (m, 4H).

Step f)

Compound 7 (1.4 g, 3.27 mmol), dissolved in chloroform (10 ml.) and pyridine (4.77 ml_, 58.9 mmol) was cooled in a dry ice-, acetone bath. SO₂CI₂ (1.56 ml_, 19.6 mmol) was added and the bath was thereafter removed. The reaction mixture was stirred o.n and became more yellow/brown by time. After 16 hrs the reaction mixture was diluted with DCM (15 ml.) and washed with 10% citric acid (_aq) (15 ml.) and NaHCO_{3 (sa}t aq) (15 ml_). The organic phase was dried with Na₂SO₄, filtered and evaporated. The brown oil was dissolved in MeOH (10 ml.) and approx. 0.5 ml. of NaI (0.8% in MeOH: H₂O (1 :1 )) was added to the solution that was stirred for

15 minutes. The solvent was then evaporated and the crude product was purified by flash chromatography (Heptane: Ethyl acetate (4:1 ) to give a yield of 68%.

¹H NMR (CDCI₃, 400 MHz) 1.10 (s, 9H), 3.80- 3.85 (m, 2H), 3.89 (dd, 1 H, J = 12.1 , 5.8), 3.96 (dd, 1 H, J = 9.7, 3.8), 4.00 (dd, 1 H, J = 12.3, 2.6), 4.15 (ddd, 1 H, J = 9.7, 5.9, 2.6), 4.32- 4.36 (m, 2H), 7.35- 7.52 (m, 6H), 7.58- 7.75 (m, 4H) Step g)

PPh₃ (882 mg, 3.36 mmol) was added to a solution of compound 8 (1.04 g, 2.24 mmol) dissolved in MeOH (50 ml.) and H₂O (5 ml_). The reaction was stirred at room temperature over night. LC-MS showed no starting material but very little cyclized product. TEA (9.38 ml_, 67.2 mmol) and H₂O (5 ml.) was added to the solution which was heated to 50 ⁰C. After 4 hrs LC-MS showed no non-cyclized amine. The solvent was evaporated and the crude product purified by flash chromatography (heptane: ethyl acetate (3:2)) to give the product in 54% yield (0.49 g). LRMS (M+H) 402.

¹H NMR (CDCI₃, 400 MHz) 1.06 (s, 9H), 2.71 (dd, 1 H, J = 11.1 , 10.4), 3.18 (dd, 1 H, J = 11.2, 7.0), 3.73 (d, 1 H₁ J = 4.7), 3.78 (dd, 1 H₁ J = 9.8, 3.5), 3.84 (dd, 1 H₁ J = 9.8, 2.0), 3.95 (ddd, 1 H, J = 10.2, 7.1 , 4.1 ), 4.16- 4.19 (m, 1 H), 4.66 (dd, 1 H, J = 4.4, 4.4), 7.35- 7.46 (m, 6H), 7.61- 7.67 (m, 4H).

Step h)

BOC anhydride (0.52 g, 2.40 mmol) was added to a solution of compound 9 (0.48 g, 1.20 mmol) dissolved in 50 imL of MeOH: TEA (9:1 ). The reaction was stirred o.n. and thereafter the solvent was removed by rotary evaporation. The crude product was purified by flash chromatography (heptane: ethyl acetate (4:1-> 2:1 )) to give the product in quantitative yield (0.60 g). ¹H NMR (CDCI₃, 400 MHz) 1.07 (s, 9H), 1.24- 1.46 (m, 9H)^*, 3.05 (dd, 1 H, J = 10.4, 10.4), 3.56 (d, 1 H, J = 9.7), 3.70- 3.89 (m, 1 H)^*, 3.90- 4.15 (m, 2H)^*, 4.24- 4.89 (m, 3H)^*, 7.34- 7.47 (m, 6H), 7.59- 7.78 (m, 4H). ^* Denotes rotamers.

Step i)

Tetrabutylammonium flouride (1.79 ml_, 1.79 mmol) was added to a solution of compound 10 (0.60 g, 1.19 mmol) dissolved in THF (12 ml_). The reaction was stirred at room temperature for 3 hrs before the solvent was removed by rotary evaporation. The crude product was purified by flash chromatography (hetane: ethyl acetate (1 :1 -> 0:1 ) and obtained in 94% yield (0.29 g). ¹H NMR (CDCI₃, 400 MHz) 1.45- 1.52 (m, 9H)^*, 3.16- 3.32 (m, 1 H)^*, 3.83- 4.22 (m, 5H)^*, 4.41- 4.54 (m, 1 H)^*, 4.66- 4.71 (m, 1 H)^*. ^* Denotes rotamers.

Step j)

Dess- Martin periodinane (0.60 g, 1.42 mmol) was added to a solution of compound 1 1 (0.34 g, 1.29 mmol) dissolved in dry DCM. The reaction was stirred under N₂ for 2 hrs when the reaction was completed. The solution was washed 3 times (3^*20 ml.) with a 1 :1 mixture of 10 % Na₂S₂O_{3 (aq)} and NaHCO_{3 (sa}t aq>- The organic phase was dried with Na₂SO₄, filtered and evaporated. The crude product was purified by flash chromatography (Heptane: Ethyl acetate (3:1 ) to give a yield of 84% (284 mg).

¹H NMR (CDCI₃, 400 MHz) 1.48 (s, 9H), 3.45 (dd, 1 H, J = 1 1.3, 9.0), 4.01- 4.17 (m, 2H), 4.19- 4.41 (m, 3H), 4.68- 4.87 (m, 1 H).

Step k)

A pre-mixed solution of AcCI (42 uL, 0.601 mmol) and MeOH (5 ml.) was added to a solution of compound 12 (263 mg, 1.01 mmol). After stirring for 2 hrs additional AcCI (0.98 ml_, 14 mmol) was added and after stirring for 16 hrs additional AcCI (9.8 ml_, 140 mmol) was added. The reaction was completed shortly thereafter and evaporated in three aliquots to dryness and further dried by high vacuum to give 103 % crude yield (253 mg).

¹H NMR (CDCI₃, 400 MHz) 3.34 (s, 3H), 3.40 (s, 3H), 3.76 (d, 1 H, J = 10.6), 3.72- 3.90 (m, 1 H), 4.15 (d, 1 H₁ J = 10.4), 4.34 (d, 1 H, J = 4.6), 4.50- 4.60 (m, 1 H), 4.69- 4.75 (m, 1 H), 4.83 (s, 1 H).

The building block is elongated with P2 and P3 building blocks as shown above and the P1 ketone regenerated as described above.

Example 8

Morpholine-4-carboxylx acid [2-(6chloro-3-oxo-hexahydro-furo[3,2-b1pyrrol-4-yl)-1 -(1 -methyl- cvclopentylmethyl)-2-oxo-ethvπamide

Step a)

The P1 building block 8-1 (330 mg; 0.97 mmol) from Example 6 was dissolved in methanol (30 ml). 10% Palladium on carbon (30 mg) was added and the mixture stirred under a hydrogen atmosphere for 3h. The mixture was filtered and the filtrate concentrated under vacuum to afford compound 8-2 (182 mg; 0.87 mmol) as an orange oil. Step b)

Compound 8-3 (122 mg; 0.32 mmol) prepared as described in Example 2 of WO06/64286) was dissolved in DMF (3 ml) and HOAt (44 mg; 0.32 mmol) was added, followed by HATU (122 mg; 0.32 mmol), NMM (65 mg; 0.64 mmol) and compound 2 (60 mg; 0.29 mmol). The reaction mixture was stirred for 3h, concentrated under vacuum and the residue partitioned between EtOAc and sodium hydrogen carbonate solution. The aqueous layer was extracted with EtOAc and the combined organic layers were washed with sodium hydrogen carbonate solution, water, brine and dried over sodium sulphate. The solvent was removed, and the residue purified by flash column chromatography on silica (1 :1 EtOAc:isohexane) to afford compound 8-4 (1 19 mg; 0.25 mmol) as a clear oil.

ESI⁺, m/z: 474 and 476 (M⁺ +1 )

Step c)

Compound 8-4 (126 mg; 0.32 mmol) was dissolved in TFA (2 ml) and the reaction mixture was stirred for 4h. The mixture was concentrated under vacuum and the residue partitioned between EtOAc and sodium hydrogen carbonate solution. The aqueous layer was extracted with EtOAc and the combined organic layers were washed with water, brine and dried over sodium sulphate. The solvent was removed, and the residue purified by HPLC to afford the title compound as a white powder.

ESI⁺, m/z: 428 and 430 (M+1 ), 445 and 447 (M+H₂O) Example 9

Furan-3-carboxylic acid r2-(6-(R)-chloro-3-oxo-hexahvdro-furo-[3,2-b1pyrrol-4-yl)-1 -(1 -methyl- cvclopentylmethyl)-2-oxo-ethvHamide

Step a)

Compound 9-6 prepared as described in WO06/64286 (173 mg; 0.64 mmol) was dissolved in DMF (3 ml) and HOAt (95 mg; 0.70 mmol) was added, followed by HATU (266 mg; 0.70 mmol), NMM (176 mg; 1.74 mmol) and compound 8-2 (120 mg; 0.58 mmol). The reaction mixture was stirred overnight, concentrated under vacuum and the residue partitioned between EtOAc and sodium hydrogen carbonate solution. The aqueous layer was extracted with EtOAc and the combined organic layers were washed with sodium hydrogen carbonate solution, water, brine and dried over sodium sulphate. The solvent was removed, and the residue purified by flash column chromatography on silica (3:1 isohexane:MTBE) to afford compound 9-7 (228 mg; 0.50 mmol) as a clear oil.

ESI⁺, m/z: 461 and 463 (M+1 )

Acetyl chloride (2 ml) was added dropwise to ice-cooled anhydrous methanol (20 ml). The mixture was stirred for 10 min and a solution of compound 9-7 (228 mg; 0.50 mmol) in methanol (2 ml) was added and the reaction mixture stirred for 3h. The mixture was concentrated under vacuum to afford compound 9-8 (191 mg; 0.48 mmol) as a yellow solid. (Sample used directly in subsequent reaction).

Compound 9-8 (95 mg; 0.24 mmol) was dissolved in DMF (1 ml) and HATU (110 mg; 0.26 mmol) was added, followed by NMM (73 mg; 0.72 mmol) and compound 9-8 (95 mg; 0.24 mmol). The reaction mixture was stirred overnight, concentrated under vacuum and the residue partitioned between EtOAc and sodium hydrogen carbonate solution. The aqueous layer was extracted with EtOAc and the combined organic layers were washed with sodium hydrogen carbonate solution, water, brine and dried over sodium sulphate. The solvent was removed, and the residue purified by flash column chromatography on silica (3:1 isohexane: EtOAc) to afford compound 9-9 (67 mg; 0.15 mmol) as a clear oil.

Compound 9 (64 mg; 0.14 mmol) was dissolved in TFA (1 ml) and the reaction mixture was stirred for 4h. The mixture was concentrated under vacuum and the residue partitioned between EtOAc and sodium hydrogen carbonate solution. The aqueous layer was extracted with EtOAc and the combined organic layers were washed with water, brine and dried over sodium sulphate. The solvent was removed, and the residue purified by HPLC to afford the title compound as a white powder.

ESI⁺, m/z: 409 and 41 1 (M+1 ), 427 and 429 (M+H₂O) Example 10

Furan-3-carboxylic acid [2-(6-chloro-3-oxo-hexahydro-furo-[3,2-b1pyrrol-4-yl)-1 -(1 -methyl- cvclopentylmethyl)-2-oxo-ethvHamide

Step a)

13 14

DIEA (320 uL, 1.93 mmol) and BOC-b-Methylcyclopentyl alanine-OH (144 mg, 0.532 mmol) was added to crude 13 from Example 7(1 18 mg, 0.483 mmol), dissolved in DMF (6 ml_). The reaction flask was cooled in an ice bath for 10 minutes before addition of HATU (202 mg, 0.532 mmol). The reaction was stirred for 3 hours at room temperature before 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 NaHCO_{3 (sa}t _aq) (10 ml_). The organic phase was dried with Na₂SO₄, filtered and evaporated. The crude product was purified by flash chromatography (Heptane: Ethyl acetate (2:1-> 1 :1 ) to give product 14 in a yield of 58% (130 mg).

Step b).

Acetyl chloride (0.4 ml.) was added dropwise to a solution of compound 14 (0.154 g, 0.334 mmol) in methanol (4 ml.) at 0°C. The reaction mixture was then stirred at rt over night, then concentrated. The residue was redissolved twice in dry DMF (5 ml) and concentrated to dryness, then again dissolved in DMF (6 ml_). 3-Furoic acid (45 mg, 0.401 mmol) and DIEA (221 L, 1.34 mmol) was added to the solution before it was cooled to 0°C and HATU (140 mg, 0.367 mmol) was added. The reaction was stirred for 3 hours at room temperature before 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 NaHCO_{3 (sa}t _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 -> 1 :2) to give the product as a transparent oil/solid in 87 % yield (132 mg).

LRMS (M+H) 455. NMR (CDCI₃, 400 MHz):1.05 (s, 3H), 1.35- 1.52 (m, 4H), 1.56- 1.72 (m, 5H), 1.89 (dd, 1 H, J = 14.6, 3.3), 3.24 (s, 3H), 3.42 (s, 3H), 3.47 (d, 1 H, J = 10.4), 3.70 (d, 1 H, J = 10.0), 3.91 (d, 1 H, J = 10.2), 4.07- 4.13 (m, 1 H), 4.50 (dd, 1 H, J = 10.2, 7.4), 4.58 (dd, 1 H, J = 5.4, 5.4), 4.71 (d, 1 H₁ J = 5.5), 4.98 (ddd, 1 H₁ J = 8.9, 8.9, 3.4), 6.47 (d, 1 H₁ J = 8.3), 6.58 (bs, 1 H), 7.40 (dd, 1 H, J = 1.7, 1.7), 7.89 (bs, 1 H)

Step c)

Compound 15 (125 mg, 0.275 mmol) was dissolved in 20 ml. of TFA: H₂O (97.5: 2.5) and stirred for 4 hours. The solvent was removed and the crude product evaporated on silica to be purified by flash chromatography (heptane: ethyl acetate (1 : 2)) to give the product as an off-white solid in 73 % yield (82 mg).

¹H NMR (CDCI₃, 400 MHz) for the major rotamer (3:1 mixture of rotamers):1.01 (s, 3H), 1.32- 1.49 (m, 4H), 1.55- 1.70 (m, 4H), 1.82 (d, 2H, J = 6.9), 3.69 (dd, 1 H, J = 10.4, 8.6), 4.1 1 (d, 1 H, J = 17.2), 4.28 (d, 1 H, J = 17.2), 4.39- 4.47 (m, 1 H), 4.65 (dd, 1 H, J = 10.5, 6.8), 4.79 (d, 1 H, J = 5.6), 4.84- 4.92 (m, 2H), 6.58 (bs, 1 H), 6.95 (d, 1 H₁ J = 8.3), 7.37 (dd, 1 H, J = 1.8, 1.8), 7.88 (bs, 1 H) Biological Example 1

Cathepsin S Ki determination

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

Substrate dilutions

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

Inhibitor dilutions.

100μl/well of assay buffer is added to columns 2-5 and 7-12 of 4 rows of a 96 well V bottom polypropylene plate. 200μl/well of assay buffer is added to columns 1 and 6.

The first test compound prepared in DMSO is added to column 1 of the top row, typically at a volume to provide between 10 and 30 times the initially determined rough K,. 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 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 K, up to a maximum of 100μM.

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

Using an 8-channel multistepping pipette set to 5 x 10μl, distribute 10μl/well of substrate to the 384 well assay plate. Distribute the first column of the substrate dilution plate to all columns of the assay plate starting at row A. The tip spacing of the multichannel pipette will correctly skip alternate rows. Distribute the second column to all columns starting at row B. Using a 12-channel multistepping pipette set to 4 x 10μl, distribute 10μl/well of inhibitor to the 384 well assay plate. Distribute the first row of the inhibitor dilution plate to alternate rows of the assay plate starting at A1. The tip spacing of the multichannel pipette will correctly skip alternate columns. Similarly, distribute the second, third and fourth rows to alternate rows and columns starting at A2, B1 and B2 respectively.

Mix 20ml assay buffer and 20μl 1 M DTT. Add sufficient cathepsin S to give 2nM final concentration.

Using the a distributor such as a Multidrop 384, add 30μl/well to all wells of the assay plate and read in fluorescent spectrophotometer 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.

Pre-incubation of the enzyme with a physiologically relevant concentration of the inhibitor can decrease Ki by a factor of 10 or more. This situation more accurately reflects the clinical situation where medication is taken for protracted periods. In general the inhibitor is incubated at a range of concentrations with 2nM cathepsin S in assay buffer for one hour on a sealed microtitre plate. 90μl are removed and added with mixing to 10μl 1 mM substrate on an assay plate and the plate read in a fluorescent plate reader as described above.

The compounds in table 1 have K₁ values (without pre-incubation) in the range 7.2-120 nM, indicating their utility in disorders mediated by aberrant cathepsin S activation or expression.

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 imM Na phosphate, 1 mM EDTA, 0.1 % PEG 4000, pH 6.5. The DMSO stock (see substrate dilutions) is diluted to 10% in assay buffer . 56 ul of substrate is added to row A and 2 x 256 ul of buffer is added to row A. The final cathepsin K concentration is 0.5 nM.

Compounds of the invention preferably have a Ki in this assay between 2 to in excess of 10 fold higher than the cath S Ki.

Biological Example 3

Cathepsin L Ki

The procedure of Biological Example 1 with the following amendments is used for the determination of Ki for cathepsin L.

The enzyme is commercially available human cathepsin L (for example Calbiochem). The substrate is H-D-Val-Leu-Lys-AMC available from Bahcem. The assay buffer is 10OmM sodium acetate 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 1OmM inhbitor made up in 100% DMSO is dispensed into row A. 10ul of 50 uM substrate (=1/200 dilution of 1OmM stock in DMSO, diluted in assay buffer.) The majority of the compounds illustrated above provide selectivity over cathepsin L of at least 100 fold.

Representative compounds of the invention were tested in the above cathepisn S₁L and K assays. 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 2

"-" = not determined

The compounds of formula Il are thus potent inhibitors of cathepsin S and yet selective over the closely related cathepsin S and L.

Biological Example 4

Permeability

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

Apical to basolateral transport

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

Transport plate no 1 comprises 3 rows of 4 wells each. Row 1 is denoted Wash, row 2 "30 minutes" and row 3 "60 minutes". Transport plate no 2 comprises 3 rows of 4 wells, one denoted row 4 "90 minutes", row 5 "120 minutes and the remaining row unassigned. The culture medium from the apical wells is removed and the inserts are transferred to a wash row (No. 1 ) in a transport plate (plate no.1 ) out of 2 plates without inserts, which have already been prepared with 1.5 ml. transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5. In A→B screening the TB in basolateral well also contains 1 % Bovine Serum Albumin.

0.5 ml. transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts and the cell monolayers equilibrated in the transport buffer system for 30 minutes at 37 ⁰C in a polymix shaker. After being equilibrated to the buffer system the Transepithelial electrical resistance value (TEER) is measured in each well by an EVOM chop stick instrument. The TEER values are usually between 400 to 1000 Ω per well (depends on passage number used).

The transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to the 30 minutes row (No. 2) and fresh 425 μl_ TB (pH 6.5), including the test substance is added to the apical (donor) well. The plates are incubated in a polymix shaker at 37⁰C with a low shaking velocity of approximately 150 to 300 rpm.

After 30 minutes incubation in row 2 the inserts will be moved to new pre-warmed basolateral (receiver) wells every 30 minutes; row 3 (60 minutes), 4 (90 minutes) and 5 (120 minutes).

25 μl_ samples will be taken from the apical solution after ~2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.

300 μl_ will be taken from the basolateral (receiver) wells at each scheduled time point and the post value of TEER is measured at the end the experiment. To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at -2O⁰C until analysis by HPLC or LC-MS.

Basolateral to apical transport

Generally every compound will be tested in 2-4 wells. The basolateral and the apical wells will contain 1.55 mL and 0.4 mL TB, respectively, and the standard concentration of the tested substances is 10 μM. Furthermore all test solutions and buffers will contain 1 % DMSO. Prior to the experiment the transport plates are precoated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material.

After 21 to 28 days in culture on filter supports the cells are ready for permeability experiments. The culture medium from the apical wells are removed and the inserts are transferred to a wash row (No.1 ) in a new plate without inserts (Transport plate). The transport plate comprises 3 rows of 4 wells. Row 1 is denoted "wash" and row 3 is the "experimental row". The transport plate has previously been prepared with 1.5 ml. TB (pH 7.4) in wash row No. 1 and with 1.55 ml. TB (pH 7.4), including the test substance, in experimental row No. 3 (donor side).

0.5 ml. transport buffer (HBSS, 25 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 ⁰C in a polymix shaker. After being equilibrated to the buffer system the TEER value is measured in each well by an EVOM chop stick instrument.

The transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to row 3 and 400 μl_ fresh TB, pH 6.5 is added to the inserts. After 30 minutes 250 μl_ is withdrawn from the apical (receiver) well and replaced by fresh transport buffer. Thereafter 250 μl_ samples will be withdrawn and replaced by fresh transport buffer every 30 minutes until the end of the experiment at 120 minutes, and finally a post value of TEER is measured at the end of the experiment. A 25 μl_ samples will be taken from the basolateral (donor) compartment after ~2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.

To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at -2O⁰C until analysis by HPLC or LC-MS.

Calculation

Determination of the cumulative fraction absorbed, FA_cum, versus time. FA_cum is calculated from:

_F ^rA _Acum _ ^" L Yu ^C«/

C DI

Where CRJ is the receiver concentration at the end of the interval i and Crjj is the donor concentration at the beginning of interval i. A linear relationship should be obtained.

The determination of permeability coefficients (Papp. cm/s) are calculated from:

_ (k - V_R) ^PaPP ^" (A - 60) where k is the transport rate (mirr^ ) defined as the slope obtained by linear regression of cumulative fraction absorbed (FA_cum ) as a function of time (min), VR is the volume in the receiver chamber (ml_), and A is the area of the filter (cιτι2).

Reference compounds

Biological Example 5

Cellular cathepsin S K₁

This example describes procedures for assessing potency of cathepsin S inhibitors on inhibition of in vitro T cell activation by determining concentration of the compound required for reducing 50% of the IL-2 secretion in T cells stimulated with compound-treated antigen presenting cells in an antigen presentation assay using the 19.3 cells and the 9001 cells as the effector cells and the antigen presenting cells, respectively. 19.3 cells are murine T cell hybridomas recognizing type Il collagen (260-272) in the context of HLA-DR1 , and 9001 is an EBV-transformed human B cell line expressing homozygous DR1 molecule. The 9001 cells will be pre-treated with varying concentration of the compounds for 1 hour and then incubated with the T cells in the presence of collagen at a final concentration of 0.1 mg/ml. The cultures will be incubated overnight at 37°C with 5% CO₂ and amount of IL-2 in the supernatant determined with ELISA. The IC₅o-IL-2 values representing the concentration of compounds at which secretion of IL-2 from the T cells is reduced by 50% will be determined by regression analysis Major histocompatibility complex (MHC) class Il molecules bind peptides generated by degradation of endocytosed antigens and display them as MHC class ll-peptide complexes at the cell surface for recognition by CD4+ T cells. MHC class Il molecules are assembled with the assistance of invariant chain (Ii) in the endoplasmic reticulum (ER) and transported to an endocytic compartment where Ii undergoes rapid degradation by endosomal and lysosomal proteases. A peptide fragment of Ii, CLIP (class ll-associated Invariant chain Peptides) remains bound in the class Il peptide binding groove, until removed by the chaperone molecule H-2M in mouse or HLA-DM in humans. This allows peptides derived from proteolytic degradation of foreign and self proteins to bind class Il molecules and subsequently to be presented to T cells in the context of MHC molecules. In dendritic cells and B cells, cathepsin S is required for complete invariant chain processing and CLIP generation. Inactivating cathepsin S with inhibitors will impair MHC class Il peptide loading and formation of stable MHC/peptide complexes leading to reduced antigen presentation and T cell activation.

To assess the potency of the cathepsin S inhibitors, an antigen presentation assay uses a collagen specific, HLA-DR1 restricted mouse T cell hybridoma (19.3) as effector cells, human EBV-transformed B cells (9001 ) as antigen presenting cells (APC), and ιmlL-2 ELISA as the read-out system. Inhibition of Cathepsin S with specific inhibitors will impair the processing and presentation of collagen in APCs which in turn reduces the activation of the T cells. The extent of inhibition on T cells is measured by the degree of reduction in IL-2 secretion. IC₅₀-IL-2 represents the concentration of compounds at which secretion of IL-2 from the T cells is reduced by 50% in the antigen presentation assay.

MATERIALS

Cathepsin S inhibitors

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

Cells

All the cells will be cultured in DMEM medium (Invitrogen, cat #1 1995-065) supplemented with 10% fetal bovine serum (Hyclone, cat #SH30070.03), 100 U/ml penicillin, 100 ug/ml streptomycin and 2 mM L-glutamine (Invitrogen, cat #10378-016). T cell: 19.3, murine DR1 transgenic T cell hybridomas, DR1 restricted, Type Il collagen 260-272 specific

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

Antigen

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

EQUIPMENT

Tissue culture incubator (Forma Scientific, model. #3120)

Sorvall centrifuge (Sorvall RC-3B)

Plate washer

Plate-reader (Tecan, Spectra shell, cat. #20-074)

PROCEDURES

Antigen presentation assay

1. Two-fold serial dilutions of the compounds, starting at 40OuM in AIMV medium, will be transferred to a 96-well round-bottom microtiter plate at a volume of 50ul/well.

2. Antigen-presenting cells will be washed and resuspended in AIMV medium to a density of 0.8x10⁶/ml, and then added to the plates at a volume of 50ul/well, giving the number of cells per well as 40,000.

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

4. The T cells will be washed and resuspended in AIMV to a density of 0.8x10⁶/ml.

5. The antigen will be diluted to a 4X concentration in AIMV and mixed 1 to 1 with the T cells.

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

6. The plates will be incubated overnight at 37C with 5% CO₂. 7. Supernatant will be carefully removed from each well and measured for amount of IL-2 with ELISA.

IL-2 ELISA

Mouse IL-2 ELISA kits will be purchased from Pharmingen (Mouse IL-2 OptEIA set, #2614Kl). 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 degreesC.

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

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

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

5. After washing 4 times, the plate will be incubated for 1 hr at RT with a mixture of a biotinylated anti-mlL2 antibody and avidin-HRP prepared in blocking buffer.

6. Following 8 washes with wash buffer, the substrate (TMB) will be added to the plate and incubated at RT for 15-30 minutes until the color develops.

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

8. The plates will be measured at 450 nm with an ELISA plate reader (Spectra, Tecan).

9. A set of purified recombinant ιmlL-2 with known concentration will be prepared from the stock solution (provided in the kit) with the blocking buffer and assayed in each plate to provide a standard curve for quantification of IL-2.

DETERMINATION OF IC50-IL-2 OF CATHEPSIN S INHIBITORS

The potency of each compound will be measured by the IC₅₀ value derived from this assay. IC₅₀ represents the concentration of compound at which secretion of IL-2 from the T cells is reduced by 50%. The absorbance at 450 nm from each well will be converted into amount of IL-2 (pg/ml) using the Winselect software (Tecan) based on the standard curve generated from in-plate standards of purified recombinant mlL-2. Means and standard deviations will be calculated from triplicates with Excel.

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

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

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

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

Following controls are included and analyzed as appropriate:

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

T + APCs, with anti-CD3/CD28, with compounds, for toxicity associated with compounds.

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

Biological Example 6

Human Liver Micrososomes

Metabolic stability is determined by commercially available human liver microsome assays, such as XEN 042, assayed in accordace with manufacturer's recommendations. Biological example 7

Metabolic Stability

Conpounds 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 (Xenotech LLC Lenexa US, 1 mg/mL protein in 0.1 M phosphate buffer, pH 7.4) at 37° centigrade over a one hour period. The incubations are initiated by the addition of 1 mM NADPH co-factor. Timed sub- samples are taken at 0, 20, 40 and 60 minutes and "crash precipitated" by the addition of 3 volumes of ice-cold acetonitrile. The samples are centrifuged at reduced temperature and the supernatants are separated and analyzed by LC-MS-MS.

Alternatively, an analogous stability assay is carried out in human or monkey whole blood.

All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the following claims:

Claims

Claims

1. A compound of the formula I:

wherein:

one of R¹ and R² is halogen and the other is halogen or H;

E is -C(=O)-, -CH(CrC₃haloalkyl)-, -S(=O)_m-, -OC(=O)-, -NRaC(=O)-NRaS(=O)_m-; where m is 1 or 2;

R³ is a stable, optionally substituted, monocyclic or bicyclic, carbocyclic or heterocyclic ring system wherein the or each ring is saturated, partially saturated or aromatic and has 4, 5 or 6 ring atoms and 0 to 3 hetero atoms selected from S, O and N and wherein the optional substituents comprise 1 to 3 members selected from R⁴;

R⁴ is independently selected from halo, hydroxy, oxo, nitrile, amine, carboxy, carbamoyl, nitro, sulphonamide, CrC₄ alkyl (optionally substituted with one to three Rb), CrC₄ alkanoyl and XR⁵,

X is a bond or a 1-4 membered linkage comprising 0-4 carbon atoms and/or an amine, amide, sulphonamide, ester, ether, urea or carbamate function;

R⁵ is CrC₄ alkyl or a monocyclic ring selected from C₃-C₆ cycloalkyl, C₃-C₆ cycloalkenyl, phenyl, azepanyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, indolinyl, pyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, thiopyranyl, furanyl, tetrahydrofuranyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, tetrazolyl, pyrazolyl, indolyl, any of which alkyl or ring is substituted with one to three Rb:

Ra is independently H, CrC₄ alkyl or -C(=0)Cr₄ alkyl, either alkyl being optionally substituted with 1 to 3 members independently selected from Rb;

Rb is independently halo, hydroxyl, C_rC₄ alkyl, C_rC₄ haloalkyl, -O(C_rC₄ alkyl), -N(C₀-C₄ alkyl)₂, carbamoyl; -NHC(=O)C_rC₄ alkyl;

and pharmaceutically acceptable salts, solvates, hydrates and N-oxides thereof.

2. A compound according to claim 1 , with the stereochemistry shown in the partial structure:

where R¹ is H and R² is fluoro or chloro or R² is H and R¹ is fluoro or chloro.

3. A compound according to claim 2, wherein R¹ is chloro.

4. A compound according to any preceding claim, wherein E is -C(=O)- or ('S_/)-CH(CF3)-.

5. A compound according to any preceding claim wherein R is optionally substituted furyl, thienyl, pyrazinyl, pyridyl, pyrrolyl or morpholinyl.

6. A compound according to claim 5, wherein R³ has a partial structure selected from the group:

wherein

R⁴' is H, halo, Od-C₄ alkyl, C(=O)NRkRI, NRkC(=O)C_rC₄ alkyl, NRkC(=O)NRkRI or - NRkC(=O)OCrC₄ alkyl, or NHC(=O)OMe,

Rk and Rl are independently H, CrC₄ alkyl or C(=O)Ci-C₄ alkyl or Rk, Rl and an adjacent N atom to which they are both attached defines a cyclic amine selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl or N-methylpiperazinyl.

7. A compound according to claim 6, wherein R^4' is fluoro, methoxy, dimethylcarbamoyl, NHC(=O)Me, -NHC(=O)NHCH₃, NHC(=O)N(CH3)₂ , NHC(=O)OMe or a cyclic amine NHC(=O)NRkRI

8. A compound according to any of claims 1-4, wherein R³ is optionally substituted phenyl.

9. A compound according to claim 8, wherein the phenyl is substituted with m-fluoro, p- fluoro, p-hydroxy, p-hydroxy-m-chloro, p-hydroxy-m-fluoro, p-hydroxy-m-methoxy, p-hydroxy-m- methyl, bis-p-chloro-p-hydroxy, m-cyano, p-acetamido or p-pyrimid-2-yl.

10. A compound according to claim 9, wherein R³ comprises the partial structure:

where Rm is-NRaSO_mR⁵ or -NHC(=O)NRkRI ;

Rk, Rl and the N atom to which they are both attached defines a cyclic amine selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl or N-methylpiperazinyl;

and R⁴" is H, CrC₄ alkyl, CrC₄ haloalkyl, halo, cyano, hydroxyl, or Ci-C₄alkoxy.

1 1. A compound according to claim 10, wherein R⁵ is CrC₄ alkyl, such as methyl, ethyl or propyl or t-butyl; halogenated CrC₄ alkyl such as trifluoromethyl; C3-C6 cycloalkyl, such as cyclopropyl or cyclohexyl; or phenyl or benzyl, any of which is optionally substituted with R⁶.

12. A compound according to claim 10, wherein R³ has the partial structure:

13. A compound according to claim 10, with the partial structure:

Ra is H or methyl and Rn is H or methyl, especially with the partial structure:

14. A compound according to any of claims 1-4, wherein R³ has the partial structure:

where R₄ is H or R⁴, and carbamates such as -NRaC(=0)0CrC₄ alkyl such as NHC(=O)OMe, Rz is CH, NH or O and the S atom is optionally oxidised to >S=O or preferably >S(=O)₂.

15. A compound according to any of claims 1 -4, wherein R³ has the partial structure:

where R⁴ is H, CrC₄ alkyl, NH₂, NHCi-C₄alkyl (such as methylamine), N(CrC₄alkyl)₂ such as dimethylamine), NHC(=O)CrC₄alkyl (such as acetamide); ring nitrogens are optionally substituted with CrC₄ alkyl (such as methyl, ethyl or t-butyl) , or C(=0)CrC₄ alkyl (such as acetyl); and R⁴ is an optional substituent R⁴ as defined in claim 1.

16. A compound according to any of claims 1 -4, wherein R³ has the partial structure:

where R⁴' is H, C_rC₄ alkyl, NH₂, NHC_rC₄alkyl (such as methylamide), N(C_rC₄alkyl)₂ such as dimethylamide), NHC(=O)CrC₄alkyl (such as acetamide); ring nitrogens are optionally substituted with CrC₄ alkyl (such as methyl, ethyl or t-butyl) , or C(=0)CrC₄ alkyl (such as acetyl);

R⁴ is an optional substituent as defined in claim 1. O' is absent (ie 2 hydrogen atoms) or keto.

17. A pharmaceutical composition comprising a compound as claimed in any of claims 1 -16 and a pharmaceutically acceptable carrier or vehicle therefor.

18. Use of a compound as claimed in claim 1 in the manufacture of a medicament for the treatment or prophylaxis of disorders caused by aberrant cathepsin S expression or activation.

19. Use according to claim 18, wherein the disorder is an autoimmune disorder such as MS, RA, juvenile diabetes or asthma.

20. Use according to claim 19, wherein the disorder is chronic pain.

21. A method for the treatment or prophylaxis of disorders caused by aberrant cathepsin S expression or activation comprising the administration of an effective amount of a compound as defined in claim 1 to an individual suffering from or threatened with the disorder.

22. A method according to claim 21 , wherein the disorder is an autoimmune disorder such as MS, RA, juvenile diabetes or asthma.

23. A method according to claim 21 , wherein the disorder is chronic pain or psoriasis.

24. A compound according to cany one of claims 1 to 16 for use as a medicament.