AU2002227222A1 - Cysteine protease inhibitors - Google Patents

Description

Cysteine Protease Inhibitors

Field of the invention.

This invention relates to inhibitors of cysteine proteases, especially those ofthe papain superfamily. The invention provides novel compounds useful in the prophylaxis or treatment of disorders stemming from misbalance of physiological proteases such as cathepsin F or S, or pathogenic proteases such as malarial falcipain.

Description of the related art.

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

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 ofthe human cathepsin L and H and plant cysteine proteases papain respectively, although only 31% homologous with Cathepsin B. It is found mainly in lymph nodes, spleen, 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 II molecules to bind antigenic peptides, and for transport ofthe 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 II molecules competent for binding peptides. Therefore, the inhibition of this enzyme may be useful in modulating class II-restricting immune response (WO 97/40066). Other disorders in which cathepsin S is implicated are chronic obstructive pulmonary disease and endometriosis.

WO 98/50533 describes the use of compounds according to the formula (I).

(I)

It is suggested the compounds of this formula, are useful as inhibitors to proteases, in particular the papain superfamily; specifically those ofthe Cathepsin family; and particularly Cathepsin K. The ketone bearing ring structure in these compounds has a tendency to spontaneously racemise, limiting their clinical utility. Other SKB applications describing ketone cathepsin K inhibitors include WO 98/46582, WO99/ 64399, WO00/29408, WO00/38687 and WO00/49011. However none of these applications disclose an α- ring substituent adjacent to the linkage to the peptidomimetic chain.

Shenai et al., J. Biol. Chem. 275 3729000-29010 describe the isolation of a major cysteine protease, denoted falcipain 2 from trophozoites of Plasmodium falciparium. The enzyme appears inter alia to hydrolyse erythrocyte haemoglobin in acidic food vacuoles. This publication also describes the isolation ofthe corresponding gene using an N-terminus tag, which is autocatalytically removed during folding.

SmithKline Beecham's WO 99/53039 describes the cysteine protease inhibitory activity of a diverse range of peptidomimetics on a trophozoite preparation from Plasmodium falciparium. No guidance is provided as to which cysteine protease is being inhibited. Although most ofthe peptidomimetics are linear structures, one compound (R, S)-3 - [N-(3 -benzyloxybenzoyl)-L-leucinylamino]tefr- ydrofuran-4-one belongs to the furanones of formula I depicted above. As would be expected of such chirally unstable structures, the ketone bearing ring is racemic. '

Summary ofthe invention

A first aspect ofthe invention provides a compound according to formula IN

where

Rl is R'-C(=O)- or R'-S(=O)2-

R' is

X, = O, S, ΝH,

W, Y, Z = CH, Ν;

R" = single or multiple ring substitution combinations taken from:

H, Cl-7-alkyl, C3-6-cycloalkyl, OH, SH, amine, halogen; R3 = Cl-7-alkyl, C2-C7 alkenyl, C2-C7 alkenyl, C3-7-cycloalkyl, Ar, Ar-Cl-7alkyl; R4 = H, Cl-7-alkyl, C3-7-cycloalkyl; C2-7alkenyl, Ar, Ar-Cl-C7-alkyl; R5 = Cl-7-alkyl, hydroxy- or halo-substituted Cl-C7alkyl, halogen, Ar-Cl-7-alkyl, C0-3-alkyl-CONR3R4 or R^iv;

R¹ IV _ where n = 1-3, m = 1-3;

R^v, R^vi = H, Cl -7-alkyl;

A = N, CH;

B =N, O, S, CH;

R^vii = absent when B = O„S; or R^vii = H, Cl-7-alkyl when B = N, CH;

R^viii = O, Cl -7-alkyl; R6 = H, Cl-7-alkyl, Ar-Cl -7-alkyl, Cl-3-alkyl-SO2-R^ix, Cl-3-alkyl-C(O)-NHR^ix or CH₂XAr; R^ix is CI -7-alkyl, Ar-Cl -7-alkyl or C3-6-cycloalkyl.

'CI -7-alkyl' as applied herein is meant to include straight and branched chain aliphatic carbon chains such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, heptyl and any simple isomers thereof. Additionally, any Cl- 7-alkyl may optionally be substituted by one or two halogens and/or a heteroatom S, O, NH. If the heteroatom is located at a chain terminus then it is appropriately substituted with one or 2 hydrogen atoms, for example as hydroxymethyl. An S heteroatom may be oxidised to the sulphone, especially in the case of R3 Cl-7 alkyl or ArCl-7alkyl.

'Cl-3-alkyT as applied herein includes methyl, ethyl, propyl, isopropyl, cyclopropyl, any of which may be optionally substituted as described in the paragraph above.

'Amine' includes NH2, NHCl-3-alkyl or N(Cl-3-alkyl)2.

'Halogen' as applied herein is meant to include F, CI, Br, I, particularly chloro and preferably fluoro. 'C3-6-cycloalkyT (or C3-C7 cycloalkyl) as applied herein is meant to include any variation of 'CI -7-alkyl' which additionally contains a C3-6 (or C3-7) carbocyclic ring such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. Alternatively the C3-6 or C3- 7 cyclopropyl may be spiro bound to the adjacent carbon without an intervening C1-C7 alkyl.

'Ar- CI -7-alkyl' as applied herein is meant to include a phenyl, pyrazolyl, pyridyl, imidazolyl, oxazolyl, isoxazolyl, thiazinolyl, isothiazinolyl, thiazolyl, oxadiazolyl, 1,2,3-triazolyl, 1 ,2,4-triazolyl, furanyl or thienyl aromatic ring (Ar) attached through a 'CI -7-alkyl' (defined above) to the dihydro-(3H)-furanone ring system or in the case of R2, R3 or R4 linked directly to the molecule backbone. Optionally, the aromatic ring Ar may be substituted with halogen, Cl-3-alkyl, OH, OCl-3-alkyl, SH, SCl-3-alkyl, amine and the like.

'Cl-3-alkyl-CONR'", R^lv' as applied herein is meant to include straight or branched carbon chain substituted with a 1°, 2° or 3° carboxamide wherein R'", R^IV includes H and Me.

'Cl-3-alkyl-SO₂-R^lx, as applied herein is meant to include straight or branched carbon chain substituted with a sulphone wherein R^1X includes 'CI -7-alkyl', 'Ar- CI -7-alkyl', 'C3-6-cycloalkyl'.

'Cl-3-alkyl-C(O)-NHR^lx, as applied herein is meant to include straight or branched carbon chain substituted with a secondary carboxamide wherein R^1X includes 'Cl-7- alkyl', 'Ar- CI -7-alkyl', *C3-6-cycloal yl'.

If a chiral centre is present, all isomeric forms are intended to be covered. Both (R) and (S) stereochemistries at the position corresponding to the pyranone 5-position (ie R5 adjacent the linkage to the peptidomimetic chain) are encompassed by the invention with (S) being preferred in some cases, for instance with cathepsin S inhibitors. Other cysteine proteases appear to favour the R stereoisomer at this position, such as cathepsin K and falcipain, but can accept the S. The compounds ofthe invention are cysteine protease inhibitors, notably against cathepsins or cathepsin-like proteases ofthe papain superfamily. Ideally the compound displays selective inhibition of a single protease in the complex mixture of proteolytic enzymes characterising the physiological environment, for example a greater than 10 fold selectivity, preferably greater than 100. Most preferably inhibitory specificity is exhibited over other members ofthe same enzyme class or family, such as the Cathepsin family, which have a high degree of homology, as incorrect regulation of proteolytic activity can lead to unwanted pathological conditions such as hypertension, blood clotting or worse. This is especially desirable for disorders such as autoimmune disorders where administration ofthe drug is likely to be protracted.

However, compounds can be useful notwithstanding .that they exhibit a degree of promiscuity in relation to inhibition of physiological proteases. For example the physiological functions of many cathepsins are redundant, that is inhibition of a particular cysteine protease can be compensated by the presence or upregulation of other non-inhibited proteases or alternative metabolic routes. Alternatively, treatments of short duration can result only in transient toxicity or other side effects.

The cross-specificity of cysteine proteases for a given putative inhibitor (ie the selectivity if the inhibitor) is readily ascertained with conventional enzyme and cell culture assays, for instance as depicted in the examples in relation to cathepsins S, K and L.

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

Examples of such diseases or conditions include those enumerated in WO 97/40066, such as autoimmune diseases, allergies, multiple sclerosis, rheumatoid arthritis and the like. The invention further provides the use ofthe 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 preferred embodiment, cathepsin S inhibitors have Rl = R'C(O)

Where R'

R4 and R6 = H;

R3 = n-butyl, t-butyl, 3-(2,2-dimethylpropyl), 4-(2-methylbutyl), 4-(3,3- dimethylbutyl), 4-(3,3-dimethyl-2-methylbutyl), 4-(3-methyl-2-methylbutyl), 5-

(2-methyl-3-methylpentyl, cyclohexylmethyl, cyclopentylmethyl);

R5 = CH₃, C₂H₅, CH₂OH, CH₂Ar, CH₂CONH₂, (CH₂)₂CONH₂,

or permutations thereof.

A favoured group of cathepsin S inhibitors comprises compounds otherwise as defined in the immediately preceding paragraph, wherein R5 is methyl, ethyl, propyl or hydroxymethyl. A further group of cathepsin inhibitors comprises compounds as defined in the paragraph above, but wherein R' as phenyl bears multiple substitutions, such as Cl-C7alkyl, hydroxy, halo and the like, typically at the 3 and 4 positions. Additional preferred definitions for R3 in formula IV include sulphone substituted Cl- 7 alkyl and especially sulphone substituted ArCl-7alkyl, such as benzenesulphonylmethyl, phenylsulphonylmethyl and phenylethylsulphonylmethyl. These R3 groups are conveniently combined wth the preferred variables in the preceding two paragraphs.

A further aspect ofthe invention provides methods for the treatment or prophylaxis of a parasitic infection, such as a protozoal or bacterial infection, comprising the administration of a compound of formula IV, to a mammal in need thereof. A still further aspect provides a method for the control of protozoal parasites comprising the administration of a compound of formula IV, to an invertebrate vector and/or to a locus prone to infestation of such a vector.

Conveniently the protozoal or bacterial parasite is a Plasmodium, Leishmania, Schistosoma, Giardia, Entamoeba, Trypansoma, Crithidia, Pneumocystis or Porphyromonas species.

Suitably, the treatment or prophylaxis of Plasmodium falciparium comprises inhibition of a falcipain II enzyme.

Preferred R3 groups for parasite treatment and prophylaxis include 2-methylpropen-l- yl, isobutyl and benzyl, especially the enantiomers defining the side chain of L-leucine or L-phenylalanine.

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

It will be appreciated that the invention extends to prodrugs, solvates, complexes and other forms releasing a compound of formula IV in vivo.

While it is possible for the active agent to be administered alone, it is preferable to present it as part of a pharmaceutical formulation. Such a formulation will comprise the above defined active agent together with one or more acceptable carriers/excipients and optionally other therapeutic ingredients. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients ofthe 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 ofthe 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 ofthe 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 ofthe active agent. Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.

The appropriate dosage for the compounds or formulations ofthe invention will depend upon the indication and the patient and is readily determined by conventional animal trials. Dosages providing intracellular (for inhibition of physiological proteases ofthe papain superamily) concentrations ofthe order 0.01-100 uM, more preferably .01-10 uM, such as 0.1-5uM are typically desirable and achievable. Ex vivo or topical administration against parasites will typically involve higher concentrations.

The term QSI -protecting group^Qor CN-protectedQand the like as used herein refers to those groups intended to protect the N-terminus of an amino acid or peptide or to protect an amino group against undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, ⁽^Protective Groups in Organic Synthesis^Q(John Wiley & Sons, New York, 1981), which is hereby incorporated by reference. N-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoracetyl, trichloroacetyl. phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4- chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl, and the like, carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3 ,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1 -(p-biphenylyl)- 1 -methylethoxycarbonyl, α, -dimethyl-3 , 5 - dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2 -trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like; alkyl gropus such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. Favoured N-protecting groups include formyl, acetyl, allyl, Fmoc, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, t-butoxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).

Hydroxy and/or carboxy protecting groups are also extensively reviewed in Greene ibid and include ethers such as methyl, substituted methyl ethers such as methoxymethyl, methylthiomethyl, benzyloxymethyl, t-butoxymethyl, 2-methoxyethoxymethyl and the like, silyl ethers such as trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS) tribenzylsilyl, triphenylsilyl, t-butyldiphenylsilyl (TBDPS), triisopropyl silyl and the like, substituted ethyl ethers such as 1-ethoxymethyl, 1 -methyl- 1-methoxyethyl, t-butyl, allyl, benzyl, p-methoxybenzyl, dipehenylmethyl, triphenylmethyl and the like, aralkyl groups such as trityl, and pixyl (9-hydroxy-9-phenylxanthene derivatives, especially the chloride). Ester hydroxy protecting groups include esters such as formate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate, pivaloate, adamantoate, mesitoate, benzoate and the like. Carbonate hydroxy protecting groups include methyl vinyl, allyl, cinnamyl, benzyl and the like.

Compounds are synthesised by a combination of chemistries, performed either in solution or on the solid phase.

The R5 substituent confers many beneficial qualities to molecules of general formula IV including improvements in potency and offers the potential to append inhibitor molecules with a basic functionality to improve solubility and pharmacokinetic properties. Additionally, molecules of formula IN where R5 is alkyl or other substituent and not simply hydrogen show good chiral stability at the pyranone alpha carbon (unless the context otherwise requires referred to as ring position 4 or C4 herein). By chirally stable is meant that the compounds ofthe invention exist as a predominant stereoisomer rather than an equal mixture of stereoisomers differing in stereochemistry at C4. Preferably the compounds ofthe invention are greater than 90% diastereomically pure after a protracted time period.. Note particularly the presence ofthe R5 substituent in the compounds ofthe invention in comparison with the absence of any substituent in the same position in formula (I) according to WO 98/50533, WO 98/46582, WO99/64399, WOOO/29408, WOOO/38687 and WOOO/49011.

Many active inhibitors contain commercially available amino acid residues such as L- leucine, L-norleucine etc . Alternatively, active inhibitors contain new and novel hydrophobic amino acids, which are prepared following the chemistry detailed in scheme 7. The synthesis detailed in Scheme 7 was adapted from Dexter, C. S. and Jackson, R. F. W. Chem.Commun. 75-76, 1998, and allows ready access to analogues embraced by R3 in formula IN. The side chains of some ofthe novel, multiply branched alpha-amino acid building blocks exemplified herein can be thought of as hybrids ofthe properties of combinations of other amino acid side chains, such as those of norleucine and t-butylalanine. This synthesis methodology is also described in Medivir UK's copending PCT application no PCT/GBO 1/02162 claiming priority from GB00025386.4 entitled Branched Amino Acids filed in the UK patent office on 17 October 2000, the contents of which are specifically incorporated herein.

Access to sulphonyl bearing Cl-C7alkyl or ArCl-C7alkyl R3 groups, for instance arylalkylC0-2sulphonylmethyl functionalities can come from the suitably protected amino acid cysteine. Mitsunobu coupling ofthe cysteinyl thiol with aryl alcohols such as phenol yield the protected amino acid containing the phenylthiomethyl R3 sidechain that is readily oxidised using m-chloroperbenzoic acid to provide the R3 sidechain phenylsulphonylrnethyl. The benzylsulphonylrnethyl and phenethylsulphonylrnethyl R3 sidechain containing amino acids can be prepared by nucleophilic substitution of the cysteinyl thiol with benzyl bromide and phenethyl bromide respectively. Oxidation ofthe resulting sulphides with m-chloroperbenzoic acid provides the suitably protected amino acids with the benzylsulphonylrnethyl and phenethylsulphonylrnethyl R3 sidechain.

The pyranone or Ν-protected aminopyranone building blocks (synthesis exemplified in Schemes 1-5 A) are utilised in a solid phase synthesis of inhibitor molecules (typically 5-25mg product) detailed in Scheme 8. Alternatively, for larger scale syntheses, full preparation of inhibitors can be achieved by solution phase chemistry.

Compounds ofthe invention can be accessed as illustrated below with reference to Schemes 1-4 Scheme 1

8

a) OsO₄, NMM; b) TBDPSCl, imidazole, DMF/CH₂C1₂; c) allyl bromide, TBAF, Bu₂SnO; d) LiOH in THF/H₂O; e) 'BuOCOCl, NMM; f) diazomethane in Et₂O; g) LiCl (lOeq) in 80 % acetic acid; h) (Ph₃P)₄Pd, CHC1₃, AcOH, NMM; i) (MeO)₃CH_{5 j}p- toluenesulphonic acid, MeOH; j) TsCl, pyridine; k) Me₂CuCNLi₂; 1) 10 % Pd on carbon, H₂; m) Boc-Leu-Opfp, HOBt, NMM, DMF; n) 4M HC1 in dioxane; o) R¹ capping group eg benzoic acid, HBTU, HOBt, NMM, DMF; p) TFA, NaHCO₃ Compounds of the general formula IN, are prepared by methods shown in Scheme 1. Treatment of the known Cbz-ethyl ester 1 -Scheme- 1 with osmium tetroxide and 4- methylmorpholine provides the diol 2-Scheme-l. Protection of the primary alcohol may be effected with tert-butyldiphenylsilylchloride and imidazole to provide 3^ Scheme- 1. Protection ofthe secondary alcohol 3 -Scheme- 1 may be achieved with allyl bromide and subsequent base hydrolysis of the ethyl ester provides 4-Scheme-l. Activation ofthe acid 4-Scheme-l may be achieved with isobutyl chloroformate and 4- methylmorpholine to provide 5-Scheme-l. Subsequent treatment of 5-Scheme-l with diazomethane provides the diazoketone 6-Scheme-l. Cyclization of diazoketone (X Scheme- 1 can be effected by lithium chloride/aqueous acetic acid to give the 3- pyranone 7-Scheme-l. The allyl protection may be removed from 7-Scheme-l., by treatment with palladium(O) and acid, to provide alcohol 8-Scheme-l. Ketal formation from ketone 8-Scheme-l may be effected by treatment with trimethylorthoformate and _/D-toluenesulphonic acid to provide 9-Scheme-l. Conversion ofthe alcohol 9-Scheme-l to the methyl derivative 10-Scheme 1 can be achieved utilising methods that are known in the art, such as tosylation with tosylchloride and pyridine, with subsequent reaction with the higher order cuprate prepared from methyl lithium. Removal of the Cbz protecting group from 10-Scheme 1 may be achieved with 10% Pd on carbon in the presence of hydrogen to provide 11 -Scheme- 1. The amine 11 -Scheme- 1 can be coupled with a carboxylic acid by methods that are known in the art, such as coupling with a pentafluorophenol derivative in the presence of HOBT and ΝMM, to provide the amide 12-Scheme-l. The tert-butoxycarbonyl group may be removed by treatment with an acid, such as hydrogen chloride in dioxane and the amine salt subsequently coupled with a carboxylic acid by methods that are known in the art, such as coupling with an acid in the presence of HBTU and HOBT, to provide the amide 13-Scheme-l. Removal of the ketal functionality from 13 -Scheme- 1 may be achieved with trifluoroacetic acid in the presence of sodium hydrogen carbonate to provide 14- Scheme-1. Building blocks toward compounds of general formula IV are additionally conveniently prepared by Schemes 2-4:

Scheme 2

10 11 12

13 14 a) pyridine, acetic anhydride; b) triethylsilane, trimethylsilyl triflate; c) sodium methoxide, methanol; d) cyclohexanone diethylacetal; e) Swern oxidation; f) PPh₃CHCH3, THF; g) H₂, palladium on carbon, sodium bicarbonate; h) 80% aqueous acetic acid; i) sodium hydride, benzyl bromide; j) mesyl chloride, pyridine; k) sodium azide, DMF; I) H₂, palladium on carbon, di-(fetf- butyloxy)carbonyl; m) Dess-Martin periodinane Lyxose 1-scheme-2 can be peracetylated to give 2-Scheme-2 with acetic anhydride in pyridine at room temperature overnight. Reduction at the anomeric centre to afford 3-Scheme-2 may be achieved using triethylsilane in the presence of trimethylsilyl triflate. Hydrolysis of the triacetate 3-Scheme-2 affords 4-Scheme-2 whereupon the vicinal diol can be protected as the cyclohexanone acetal 5-Scheme-2. Swern oxidation of the unprotected alcohol functionality gives 6-Scheme-2, a key intermediate for the introduction of the required C5 pyranone substitution. Ethyl substitution is achieved here by treatment with ethyl triphenylphosphonium bromide with potassium tert- butoxide in THF at 0°C to produce 7-Scheme-2. Hydrogenation of 7-Scheme- 2 in ethyl acetate with sodium bicarbonate gives the ethyl derivative 8- Scheme-2 with the stereochemistry shown. Deprotection of the cyclohexanone acetal 8-Scheme-2 can be achieved with aqueous acetic acid overnight to afford the diol 9-Scheme-2. Selective benzylation of the equatorial hydroxyl group gives 10-Scheme-2, which can then be mesylated using mesyl chloride in pyridine at 50°C to produce 11-Scheme-2. Azide displacement of mesylate anion using sodium azide in DMF at 80°C affords 12-Scheme-14, from which the pyranol 13-Scheme-2 can be obtained by hydrogenolysis in the presence of BOC-anhydride. Oxidation to the pyranone 14-Scheme-2 is achieved using the Dess-Martin periodinane.

In Scheme 2, the C5 substitution is introduced using Wittig chemistry followed by hydrogenation, and hence compound 6-Scheme-2 could be converted to the C5 ethyl derivative 8-Scheme-2. Alternative C5 substitution can be achieved using this route. For example, alternative Wittig or Horner-Emmons chemistry will lead to different alkyl substituents. In an analogous manner, the C5 hydroxymethyl group can be prepared and this itself can be further derivatised to other groups such as halogen, amino and other basic groups and sulfhydryi.

A general methodology starting from L-lyxose has been established for the preparation of various 5-substituted 4-amino 3-hydroxy pyranols with all four possible combinations of configuration at position 4 and 5 i.e. 4S.5S; 4S,5R; 4R.5S and 4R,5R. This methodology is exemplified in Scheme 2A. The pyranols can then N-extended and capped as described herein and subsequently oxidised to the keto compounds, for example by Dess-Martin periodination.

L-Lyxose 1 R=acyl 2 R=acyl 3 R=H

Scheme 2A

6 l=ketal 5 R=ketal 4 R=ketal

7 R2=alkyl

8 Rl,R2=ketal, R3=alkyl, R4=H 13 R1=H, R2=N₃, R3=alkyl, R4=H

9 R1=R2=H, R3=alkyl, R4=H 14 R1=H, R2=N₃, R3=H, R4=alkyl 10 R1=R2=H, R3=H, R4=alkyl

11 Rl=Bn, R2=H, R3=alkyl, R4=H 15 R1=N₃, R2=H, R3=alkyl, R4=H

12 Rl=Bn, R2=H, R3=H, R4=alkyl 16 R1=N₃, R2=H, R3=H, R4=alkyl

L-lyxose can be acylated with a suitable acylating agent such as acid anhydride, acyl halide in an organic solvent like pyridine or other mixed organic solvents, to give the peracylated compound l-scheme-2A. This compound can then be subjected to anomeric reduction with a trialkyl silane together with a Lewis acid such as triethyl silane and trimethylsilyl frifluormethanesulphonate. Transforming the compound into the corresponding halo-, sulpho- or thiocarbo-glycoside followed by a radical reduction, using known methodology, can also bring about the anomeric reduction. Deacylation under basic condition provides the triol 3-scheme-2A- which can be selectively protected on the 2,3-hydroxylgroups forming a ketal 4-scheme 2A by using standard protecting group methodology. Oxidation ofthe 4-OH group into the keto function 5-scheme-2A can be performed with the Swern procedure, Dess-Martin or any other suitable oxidation method. Various 4-substituted alkenes 6-scheme-2A can be achieved by using appropriate Wittig reagents for example triphenylalkylphosphonium halide or triphenylalkylarylphosphonium halide together with a base. Catalytic hydrogenation ofthe Wittig product in the presence of a buffer provides predominantly compound 8-scheme-2A. Alternatively, the compound with the other configuration at this position 10-scheme-2A can be obtained by removal ofthe ketal protecting group prior to the hydrogenation. The alkene compound can also be subjected to hydroboration, which will introduce a hydroxyl group, suitable for further modifications.

Another possibility to achieve the 4-alkyl compounds is to transform the 4-OH group into a leaving group for example a sulphonate followed by displacement by a cuprous or Grignard reagent ofthe desired alkylgroup.

The ketal protecting group can be removed under acidic conditions such as 1M HC1/THF 1 :1 at room temperature or heating to 80 °C in aqueous acetic acid which will give the diol 8-scheme-2A. Selective protection ofthe 2-OH group with an alkylating agent such as benzyl halide or any other similar reagent in the presence of a base can give exclusively or predominantly the 2-O-protected compound 11,12- scheme-2A. The 3 -OH can be converted to a suitable leaving group such as a sulphonate, which subsequently can be displaced by an azide 13,14-scheme-2A. Alternatively, a Mitsunobu reaction can be used to produce the azide-substituted compound. Hydrogenation ofthe azide-compound in the presence of a carbamoylating agent like di-tert-butyl dicarbonate provides the desired l,5-anhydro-3-[(tert- butoxycarbonyl)amino]-3,4-dideoxy-4-ethyl-D-xylitol and 1 ,5-anhydro-3-[(tert- butoxycarbonyl)amino] -2,3 -dideoxy-2-ethyl-L-arabinitol.

The series of compounds with the other configuration at carbon 3 can be prepared by inversion ofthe configuration ofthe 3 -OH in compound l l,12-scheme-2A by methods that are known in the art, followed by the above procedure i.e. putting on a leaving group and azide displacement. They can also be prepared by the following sequence. Oxidation ofthe 3 -OH into a ketone, using the oxidation reagents previously described, transformation ofthe ketone into an oxime, utilising reagents such as benzyloxyamine halide and finally reduction ofthe oxime into the amino function. This will provide a mixture ofthe compounds with the two different configurations, which can be separated using known methodology. Boc-protection ofthe amino group and reductive removal ofthe benzyl protecting group provides the compounds with the remaining two configurations 4R,5S and 4R,5R. Scheme 3 Bno₂σ^^^cθ2Bn

NH,

8

10 a) TFA; b) Me₃Al, HCl.HNMe(OMe), DCM; c) CCk NH^Bu, BF₃.Et₂O, DCM, cyclohexane; d) LAH in Et₂O; e) TiuOH, 2-methyl-2-butene, NaClO₂, NaH₂PO₄, H₂O; f) *BuOK, Et₂O, H₂O; g) ^jBuOCOCl, NMM, THF; h) diazomethane in Et₂O; i) LiCl (lOeq) in 80 % acetic acid.

Compounds of the general formula IV are alternatively prepared by methods shown in Scheme 3. Alcohol 2-Scheme-3 can be prepared following the literature procedure reported by J. E. Baldwin et al. (Tetrahedron, 1995, 51 (43), 11581). Removal of the ester functionality from 2-Scheme-3 can be achieved with trifluoroacetic acid to provide the lactone 3-Scheme-3. Lactone 3-Scheme-3 can be ring opened by MeONHMe in the presence of Me₃Al to provide the alcohol 4-Scheme-3. The tert- butoxycarbonyl group may be introduced onto alcohol 4-Scheme-3 to provide 5^ Scheme-3. The Weinreb amide 5-Scheme-3 can then be treated with lithium aluminum hydride to provide the aldehyde 6-Scheme-3. Oxidation of the aldehyde 6-Scheme-3 can be effected by sodium chlorite to provide the acid 7-Scheme-3. Alternatively, the Weinreb amide 5-Scheme-3 can then be treated with potassium-tert-butoxide to provide the acid 7-Scheme-3. Activation of the acid 7-Scheme-3 with isobutyl chloroformate and 4-methylmorpholine provides 8-Scheme-3. Subsequent treatment of 8-Scheme-3 with diazomethane provides the diazoketone 9-Scheme-3. Cyclization of diazoketone 9-Scheme-3 can be effected by lithium chloride/aqueous acetic acid to give the dihydro-3(2H)-furanone 10-Scheme-3.

Scheme 4

10 a) (CF₃SO₂)₂O, pyridine, DCM; b) (nBu)₄NN₃, toluene; c) H₂, 10% Pd/C, pTsOH, MeOH; d) Boc₂O, NEt₃, THF; e) 1M LiOH, THF; f) TBDMSC1, NEt₃, cat DMAP, DCM; g) BuOCOCl, NMM, THF; h) diazomethane in Et₂O; i) LiCl (lOeq) in 80 % acetic acid.

Compounds of the general formula (IV) can be prepared analogously to the model compound depicted in scheme 4. Pantolactone l-Scheme-4 is commercially available and is first converted to the triflate 2-Scheme-4. The triflate 2-Scheme-4 may be displaced with tetrabutylammonium azide to provide the corresponding azide 3-Scheme-4. Azide 3-Scheme-4 may be reduced to provide the amine salt 4-Scheme-4. Protection ofthe amine salt 4-Scheme-4 provides 5-Scheme-4. Ring opening ofthe lactone 5-Scheme-4 with lithπim hydroxide provides the acid 6-Scheme-4. Protection of the primary alcohol 6-Scheme-4 with tetrabutyldimethylsilyl chloride in the presence of base provides acid 7-Scheme-4. Activation of the acid 7-Scheme-4 with isobutyl chloroformate and 4- methylmoφholine provides 8-Scheme-4. Subsequent treatment of 8-Scheme-4 with diazomethane provides the diazoketone 9-Scheme-4. Cyclization of diazoketone 9z Scheme-4 can be effected by lithium chloride/aqueous acetic acid to give the model dihydro-3(2H)-pyranone 10-Scheme-4. Corresponding ring closure can be perfomed on mono-R5 variants ofthe invention.

Although schemes 1-4 have been illustrated by reference to particular R1-R4 values, it will be appreciated that the methodology is more generally applicable to precursors bearing the other claimed values in these positions, where necessary in conjunction with conventional protection of functionalities on R1-R4. Similarly other values for R5 and R6 can be accessed analogously.

Unless otherwise specified, where a chiral centre is present in a molecule but not assigned, both R and S isomers are intended. Preferably the stereochemistry at R3 is that ofthe corresponding L-amino acid. Most preferably the stereochemistry at R5 is S, especially when the adjacent linkage from the ring to the amine ofthe backbone, (ie C4) is also S. Alternatively the compounds ofthe invention are R,R at the latter stereo centres.

Currently preferred compounds ofthe present invention include, but are not limited to, the following examples:

Furan-3 -carboxylic acid (lS)-[3,3-dimethyl-l-(3-methyl-5-oxo-tetrahydro-pyran-4- ylcarbamoyl)-butyl] -amide Furan-3-carboxylic acid (lS)-[2-cyclohexyl-l-(3-methyl-5-oxo-tetrahydro-pyran-4- ylcarbamoyl)-ethyl] -amide

(15)-N-[3,3-Di-memyl-l-(3-methyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-butyl]- benzamide

( 1 S)-N- [2-Cyclohexyl- 1 -(3 -methyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl] - benzamide

( 1 S)-N- [3 ,3 -Dimethyl- 1 -(3 -methyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-butyl] -4- hydroxy-3 -methyl-benzamide

(15)-N-[2-Cyclohexyl-l-(3-methyl-5-oxo-tefrahydro-pyran-4-ylcarbamoyl)-ethyl]-4- hydroxy-3 -methyl-benzamide

Furan-3-carboxylic acid (l-S)-[2-cyclopenryl-l-(3-methyl-5-oxo-tetrahydro-pyran-4- ylcarbamoyl)-ethyl] -amide

( 1 S)-N- [2-Cyclopentyl- 1 -(3 -methyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl] - benzamide

(lS)-N-[2-Cyclopentyl-l-(3-methyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl]-4- hydroxy-3 -methyl-benzamide

Furan-3 -carboxylic acid (IS)- [3 ,3 -dimethyl- 1 -(3 -ethyl-5-oxo-tetrahydro-pyran-4- ylcarbamoyl)-butyl] -amide

Furan-3-carboxylic acid (lS)-[2-cyclohexyl-l-(3-ethyl-5-oxo-tetrahydro-pyran-4- ylcarbamoyl)-ethyl] -amide

( 1 S)-N- [3 ,3 -Dimethyl- 1 -(3 -ethyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-butyl] - benzamide

( 1 S)-N- [2-Cyclohexyl- 1 -(3 -ethyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl] - benzamide

( 1 S)-N- [3 ,3 -Dimethyl- 1 -(3 -ethyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-butyl] -4- hydroxy-3 -methyl-benzamide

( 1S)-N- [2-Cyclohexyl- 1 -(3 -ethyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl] -4- hydroxy-3-methyl-benzamide

Furan-3 -carboxylic acid (lS)-[2-cyclopentyl-l -(3-ethyl-5-oxo-tetrahydro-pyran-4- ylcarbamoyl)-ethyl] -amide ( 1 S)-N- [2-Cyclopentyl- 1 -(3 -ethyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl] - benzamide

( 1 S)-N- [2-Cyclopentyl- 1 -(3 -ethyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl] -4- hydroxy-3 -methyl-benzamide

Furan-3-carboxylic acid (lS)-[3,3-dimemyl-l-(3-propyl-5-oxo-tefr- ydro-pyran-4- ylcarbamoyl)-butyl] -amide

Furan-3 -carboxylic acid (15)-[2-cyclohexyl- 1 -(3 -propyl-5 -oxo-tetralιydro-pyran-4- ylcarbamoyl)-ethyl] -amide

( 1 S)-N- [3 ,3 -Dimethyl- 1 -(3 -propyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-butyl] - benzamide

(lS)-N-[2-Cyclohexyl-l-(3-propyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl]- benzamide

( 1 S)-N- [3 ,3 -Dimethyl- 1 -(3 -propyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-butyl] -4- hydroxy-3 -methyl-benzamide

( 1 S)-N- [2-Cyclohexyl- 1 -(3 -propyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl] -4- hydroxy-3-methyl-benzamide .

Furan-3 -carboxylic acid (IS)- [2-cyclopentyl- 1 -(3 -propyl-5-oxo-tetrahydro-pyran-4- ylcarbamoyl)-ethyl] -amide

(lS)-N-[2-Cyclopentyl-l-(3-propyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl]- benzamide

( 1 S)-N- [2-Cyclopentyl- 1 -(3 -propyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl] -4- hydroxy-3 -methyl-benzamide, particularly the respective 3R,4R enantiomers, most preferably the respective 3S, 4S enantiomers; and pharmaceutically acceptable salts thereof

Detailed disclosure ofthe embodiments Experimental Section

Solution Phase Chemistry Example 1. Following general chemistry schemes 5 and 5A

(a) General method for the synthesis of N-Boc protected 4-aminopyranol, exemplified by 1 ,5 -anhydro-3 - [(tert-butoxycarbonyl)amino] -3 ,4-dideoxy-4-ethyl-D-xylitol

4S, 5S

L-Lyxose

Scheme 5A l,2,3,4-Tetra-O-acetyI-L-lyxopyroanose (l).

L-Lyxopyroanose (25.0 g, 166 mmol) was dissolved in pyridine (150 ml) and cooled on an icebath, acetic anhydride (75 ml) was added and the solution was stirred at room temperature. After 2 hours tic (pentane: ethyl acetate 1:1) indicated complete conversion ofthe starting material into a higher migrating spot. The solution was concentrated and co-evaporated three times with toluene which gave a pale yellow syrup.

NMR data 400 MHz (CDC1₃): 1H, δ 2.06 (s, 3H), 2.08 (s, 3H), 2.14 (s, 3H), 2.16 (s,

3H), 3.71 (dd, 1H), 4.01 (dd, J=5.0, 11.7 Hz, 1H), 5.17-5.26 (m, 2H), 5.37 (dd, J=3.5,

8.8 Hz, 1H), 6.0 (d, J=3.2 Hz, 1H).

¹³C, δ 20.9, 20.9, 21.0, 21.0, 62.2, 66.7, 68.4, 68.4, 90.8, 168.8, 169.9, 170.0, 170.1.

2,3,4-Tri-(?-acetyl-l,5-anhydro- -arabinitol (2).

Trimethylsilyl frifluormethanesulphonate (60 ml, 333 mmol) was added to a solution of crude 1,2,3,4-tetra-O-acetyl-L-lyxopyroanose constituting the yield from the step above in acetonitrile (200 ml), the solution was cooled on an ice bath and triethylsilane (80 ml, 500 mmol) was added dropwise. The solution was stirred at room temperature and reaction was monitored by GC. When the reaction was complete (after 3 hours), the solution was neutralised with sodium hydrogen carbonate (s), diluted with dichloromethane and washed with water. The organic phase was dried with magnesium sulphate, filtered and concentrated. The obtained oil was purified by silica gel flash column chromatography (pentane: ethyl acetate 5:1, 4:1, 3:1) which gave 32 g, 74 % (from free lyxose) ofthe reduced compound.

NMR data 400 MHz (CDC1₃): 1H, δ 2.06 (s, 3H), 2.07 (s, 3H), 2.11 (s, 3H), 3.36-3.41 (m, 1H), 3.64 (dd, J=2.4, 12.2 Hz, 1H), 3.87 (m, 1H), 4.03 (m, 1H), 5.10-5.15 (m, 2H), 5.28-5.31 (m, 1H).

l,5-Anhydro-3,4-O-cyclohexylidene-L-arabinitol (3).

A solution of l-deoxy-2,3,4-tri-O-acetyl-L-lyxopyroanose (20.8 g, 80 mmol) in methanol (125 ml) was treated with a catalytic amount of 1M niethanolic sodium methoxide. After stirring for 1 hour at room temperature tic (ethyl acetate methanol 3:1) indicated complete conversion into a lower migrating spot. The solution was neutralised with Dowex -ET^1", filtered and concentrated, which gave a colourless oil. The oil was suspended in dichloromethane (70 ml) and cyclohexanone diethyl ketal (41 g, 240 mmol) was added followed by^»-toluenesulphonic acid until acidic pH. After a few minutes the suspension became a clear solution, which was stirred at room temperature. After 18 hours, when tic (pentane: ethyl acetate 1:2) indicated complete conversion into a higher migrating spot, the solution was neutralised with triethyl amine, concentrated and the residue was purified by silica gel flash column chromatography (toluene: ethyl acetate 3:2, 1:1) which gave 9.6 g, 56% ofthe title compound as white crystals.

NMR data 400 MHz (CDC1₃): 1H, δ 1.38-1.43 ( , 2H), 1.56-1.75 (m, 8H), 2.43 (d,

J=4.9 Hz, IH), 3.28 (m, IH), 3.75 (dd, J=3.9, 12.7 Hz, IH), 3.82-3.94 (m, 3H), 4.05 (t,

J=5.4 Hz, IH), 4.22 (m, IH).

¹³C, δ 23.9, 24.3, 25.2, 35.7, 38.3, 67.8, 68.7, 69.1, 71.9, 77.5, 110.5.

l,5-Anhydro-3,4-0-cyclohexylidene-L-ribulose (4).

A solution of dimethyl sulphoxide (2.65 ml, 37.3 mmol) in dichloromethane (30 ml) was added dropwise at -60 °C under nitrogen to a stirred solution of oxalyl chloride (1.79 ml, 20.5 mmol) in dichloromethane (30 ml) during a period of 15 min. To this solution a solution of 2,3-O-cyclohexylidene-l-deoxy-L-lyxopyroanose (4 g, 18.7 mmol) in dichloromethane (20 ml) was added dropwise during a period of 5 min. A white suspension was obtained and additional dichloromethane was added twice (10+30 ml). The temperature was allowed to rise to -25 °C when the suspension became a colourless solution. The temperature was again lowered to -45 °C and a solution of triethyl amine (12.9 ml, 93.3 mmol) in dichloromethane (20 ml) was added. After 10 min, when tic (toluene:ethyl acetate 1:1) indicated complete conversion ofthe alcohol into the ketone, the reaction mixture was poured into water (100 ml), the water layer was extracted once with dichloromethane (50 ml), the combined organic phases were dried with sodium sulphate, filtered and concentrated. Flash column chromatography on silica gel (eluent pentane :diethyl ether 1 : 1) of the residue gave a colourless solid 3.4 g, 86%.

The oxidation was also performed by the Dess-Martin procedure: A suspension of 2,3-O-cyclohexylidene-l-deoxy-L-lyxopyroanose (0.5 g, 2.33 mmol) and Dess-Martin periodinane (1.39 g, 3.29 mmol) in dichloromethane (5 ml) was stirred for 10 min then "wet dichloromethane^Q(46 μl water in 10 ml dichloromethane) was added dropwise during 15 min. After lh tic (toluene: ethyl acetate 1:1) indicated complete conversion ofthe starting material into a higher migrating spot. The reaction mixture was diluted with diethyl ether (100 ml) and washed with an aqueous solution of sodium hydrogen carbonate/sodium thiosulphate 1:1 (50 ml), dried with sodium sulphate, filtered and concentrated. Purification ofthe residue by flash column chromatography on silica gel (eluent pentane: diethyl ether 1:1) gave the title compound, 0.42 g, 84%, as a crystalline solid.

NMR data 400 MHz (CDC1₃): 1H, δ 1.39-1.43 (m, 2H), 1.56-1.72 (m, 8H), 3.92-4.07 (m, 3H), 4.18-4.23 (m, IH), 4.45 (d, J=6.8 Hz, IH), 4.64-4.67 (m, IH). ¹³C, δ 23.9, 24.1, 25.1, 35.3, 36.8, 68.5, 74.1, 75.1, 76.3, 112.4, 205.0.

l,5-Anhydro-4-deo-^-4-ethylidene-2^-0-cyclohe^lidene-D-e- '-' --^Ho-pentitol (5).

Potassium-t-butoxide (3.41 g, 30.4 mmol) was added in one portion to a stirred suspension of ethyltriphenylphosphonium bromide (11.9 g, 32.0 mmol) in THF (60 ml) at -10 °C under nitrogen. The obtained orange-red mixture was allowed to reach room temperature, then cooled again to -10 °C and a solution of l,5-anhydro-3,4-O- cyclohexylidene-L-ribulose (3.4 g, 16.0 mmol) in THF (40 ml) was added dropwise. The mixture was allowed to attain room temperature. Twenty minutes after the final addition, when tic (toluene. -ethyl acetate 1:1) indicated complete conversion ofthe starting material into a higher migrating spot, the reaction mixture was partitioned between diethyl ether (400 ml) and water (200 ml). The organic layer was washed with water (1x200 ml) and brine (1x200 ml), dried with sodium sulphate, filtered and concentrated to a 10 ml residue. The residue was purified by flash column chromatography on silica gel (eluent pentane:ethyl acetate 95:5, 9:1) and appropriate fractions were carefully concentrated (bath temperature 25 °C) to 10 g that was used directly in the next step.

l,5-Anhydro-4-deoxy-4-ethyl-2,3-O-cycϊohexylidene-D-ribitol (6).

The above solution was diluted with ethyl acetate (30 ml), Pd/C ( 10%, 0.2 g) was added and the mixture was hydrogenated at atmospheric pressure. Additional Pd/C was added (0.16 g + 0.20 g) after 40 and 90 minutes. After 100 minutes tic indicated almost complete consumption ofthe starting material. The reaction mixture was filtered through celite, concentrated into a liquid (5 ml) and purified by flash column chromatography on silica gel (eluent pentane:ethyl acetate 95:5, 9:1). Appropriate fractions were concentrated to 2.08 g and this solution was used directly in the next step.

NMR data 400 MHz (CDC1₃): 1H, δ 0.98 (t, 3H), 1.31-1.74 (m, 12H), 1.82-1.92 (m,

IH), 3.18-3.26 (m, 2H), 3.64-3.68 (m, IH), 3.84 (dd, J=6.4, 11.4 Hz, IH), 4.08-4.14

(m, IH), 4.27-4.29 (m, IH).

¹³C, δ 11.3, 20.9, 24.0, 24.3, 25.3, 35.7, 38.3, 38.7, 67.7, 68.3, 70.8, 72.6, 109.5.

1 ,5- Anhy dro-4-deoxy-4-ethyl-D-ribitol (7).

The above l,5-anhydro-4-deoxy-4-ethyl-2,3-O-cyclohexylidene-D-ribitol was dissolved in aqueous acetic acid (80 %, 25 ml) and the solution was stirred at 70 °C.

After 18 hours, when tic (pentane:ethyl acetate 9:1 and 1 :1) indicated almost complete consumption ofthe starting material ( ~5% left), the solution was concentrated.

Purification ofthe residue by flash column chromatography on silica gel (eluent pentane: ethyl acetate 1:1, 2:3) gave 0.91 g 39 % (from the keto compound) of a colourless solid.

NMR data 400 MHz (CDCI3): 1H, δ 0.94 (t, 3H), 1.24-1.42 (m, 2H), 1.58-1.67 (m,

IH), 3.35 (t, IH), 3.43 (t, IH), 3.56 (dd, IH), 3.67-3.71 (m, 2H).

¹³C, δ 11.4, 20.1, 42.1, 66.1, 66.3, 68.3, 68.7.

l,5-Anhydro-2-0-benzyl-4-deoxy-4-ethyl-D-ribitol (8).

Sodium hydride (60%, 0.27 g, 6.84 mmol) was added in one portion, at room temperature, under nitrogen, to a stirred solution of l,5-anhydro-4-deoxy-4-ethyl-D- ribitol (0.5 g, 3.42 mmol) in dimethylformamide (7 ml). After 30 minutes benzyl bromide (0.53 ml, 4.45 mmol) was added dropwise during 30 minutes. After 20 minutes, when tic (petroleum ether:ethyl acetate 4:1) indicated complete conversion of the diol, methanol (1 ml) was added and the mixture was stirred for 20 minutes. The reaction mixture was diluted with ethyl acetate (100 ml), washed with water (3x50 ml), dried with sodium sulphate, filtered and concentrated. Purification of residue by flash column chromatography on silica gel (eluent pentane:ethyl acetate 9:1, 4:1) gave 0.52 g, 64% of a colourless solid. NMR data 400 MHz (CDC1₃): 1H, δ 0.94 (t, 3H), 1.25-1.36 (m, IH), 1.37-1.48 (m, IH), 1.54-1.62 (m, IH), 2.14 (s, IH), 3.40 (t, IH), 3.51-3.56 (m, 3H), 3.72-3.79 (m, IH), 4.13 (s, IH), 4.58 (d, J=l 1.7 Hz, IH), 4.63 (d, J=l 1.7 Hz, IH), 7.29-7.38 ( , 5H). ¹³C, δ 11.5, 20.1, 42.0, 64.1, 66.5, 66.6, 71.1, 75.6, 127.9, 128.2, 128.8, 138.1.

l,5-Anhydro-3-azido-2-0-benzyl-3,4-dideoxy-4-ethyl-D-xylitol (9).

Methanesulphonyl chloride (0.34 g, 2.96 mmol) was added to a stirred solution of 1,5- anhydro-2-O-benzyl-4-deoxy-4-ethyl-D-ribitol (0.28 g, 1.18 mmol) in pyridine (5 ml). The reaction mixture was warmed to 50 °C and stirred for one hour. Dichloromethane (100 ml) was added and the reaction mixture was washed successively with IM aqueous sulphuric acid (2x50 ml), IM aqueous sodium hydrogen carbonate, dried with sodium sulphate, filtered and concentrated. The residue was dissolved in dimethylformamide (10 ml) and sodium azide (0.31 g, 4.74 mmol) was added. The obtained mixture was stirred at 80 °C over night, diluted with ethyl acetate (100 ml), washed with water (3x50 ml), dried with sodium sulphate, filtered and concentrated. Purification of residue by flash column chromatography on silica gel (eluent toluene:ethyl acetate 95:5) gave 0.25 g, 81% of a colourless oil. NMR data 400 MHz (CDC1₃): 1H, δ 0,90 (t, 3H), 1.12-1.24 (m, IH), 1.44-1.54 (m, IH), 1.69-1.79 (m, IH), 3.01 (t, IH), 3.08-3.16 (m, 2H), 3.44-3.50 (m, IH), 3.92 (dd, J= .9, 11.7 Hz, IH), 4.04 (ddd, J=1.0, 4.9, 11.2 Hz, IH), 4.62 (d, J= 11.7 Hz, IH), 4.71 (d, J=l 1.2 Hz, IH) 7.29-7.37 (m, 5H). ¹³C, δ 11.3, 22.0, 42.4, 68.5, 69.2, 70.9, 73.1, 78.2, 128.2, 128.2, 128.7, 138.0.

l,5-Anhydro-3-[(tert-butoxycarbonyl)amino]-3,4-dideoxy-4-ethyl-D-syIitol (10).

Pd/C (10%, 30 mg) was added to solution of l,5-anhydro-3-azido-2-O-benzyl-3,4- dideoxy-4-ethyl-D-xylitol (88 mg, 0.34 mmol) and di-te/J-butyl dicarbonate (77 mg, 0.35 mmol) in ethyl acetate (4 ml) and the mixture was stirred under hydrogen. After 18 hours, when tic (pentane: ethyl acetate 9:1, ninhydride) indicated complete consumption ofthe starting material, the mixture was filtered through celite and concentrated. The residue was purified by flash column chromatography on silica gel (eluent toluene: ethyl acetate 4:1) which gave a colourless solid that still contained a benzyl group according to ¹Hnmr. The solid was dissolved in ethyl acetate.-ethanol 1:1 and hydrogenated over Pd/C (10% 20 mg). After 1 hour, when tic (toluene:ethyl acetate 1:1, ninhydride) indicated complete conversion ofthe starting material into a lower migrating spot, the mixture was filtered through celite and concentrated.

Purification of residue by flash column chromatography on silica gel (eluent toluene:ethyl acetate 1:1, 2:3) gave 59 mg, 71% ofthe desired monool as a colourless solid.

NMR data 400 MHz (CDC1₃): 1H, δ 0.90 (t, 3H), 1.12-1.24 (m, IH), 1.42-1.52 (m,

10H), 1.59-1.70 (m, 1H), 3.05-3.16 (m, 2H), 3.26-3.30 (m, IH), 3.43-3.48 (m, 2H),

3.96-4.05 (m, 2H).

¹³C, δ 11.5, 21.2, 28.5, 42.4, 59.2, 71.4, 71.8, 72J.

Alternative method for the preparation of 5-methyl pyranones as building blocks and intermediates towards 5-functionalised pyranones

2-Benzyloxycarbonylamino-4-hydroxy-3-methyl-butyric acid tert-butyl ester

2-Benzyloxycarbonylamino-4-hydroxy-3-methyl-butyric acid te/ -butyl ester was prepared following procedures reported by J.E. Baldwin et al (Tetrahedron 1995, 51(42), 11581).

(4-Methyl-2-oxo-tetrahydro-furan-3-yl)-carbamic acid benzyl ester

2-Benzyloxycarbonylamino-4-hydroxy-3-methyl-butyric acid fetf-butyl ester (1.00g, 3 mmol) was dissolved in TFA (30 mL). This solution was stirred for 45 minutes and then concentrated in vacuo. The residual TFA was removed azeotropically with toluene. This residue was purified by flash column chromatography to yield the title compound as a crystalline solid (750mg, 80%), MS (ES⁺) 250 (M+H). [ 3-Hydroxy-l- (methoxy-methyl-carbamoyl ) -2-methyl-propyl] ^■ carbamic acid benzyl ester 4

The lactone ring of (4-methyl-2-oxo-tetrahydro-furan-3-yl)-carbamic acid benzyl ester can be opened using Λ/,O-dimethylhydroxylamine hydrochloride in the presence of Me₃AI to give the title compound.

[3-te/ -Butoxy-1-(methoxy-methyl-carbamoyl)-2-methyl-propyl]-carbamic acid benzyl ester 5

The primary alcohol of [3-hydroxy-1-(methoxy-methyl-carbamoyl)-2-methyl- propyl]-carbamic acid benzyl ester can be protected using tert-butyl-2,2,2- trichloroacetimidate and boron trifluoride etherate to give the title compound.

(3-te/f-Butoxy-1-formyl-2-methyl-propyl)-carbamic acid benzyl ester 6

The Weinreb amide function of [3-fetf-butoxy-1-(methoxy-methyl-carbamoyl)- 2-methyl-propyl]-carbamic acid benzyl ester can be reduced using lithium aluminium hydride in ether to provide the title compound. 2-Benzyloxycarbonylamino-4-tert-butoxy-3-methyl-butyric acid 7

(3-tet -butoxy-1-formyl-2-methyl-propyl)-carbamic acid benzyl ester in tetf-butyl alcohol in the presence of 2-methyl-2-butene can be oxidised using a solution of sodium chlorite and monobasic sodium phosphate in water to give the title compound.

[3-tetf-Butoxy-1-(2-diazo-acetyl)-2-methyl-propyI]-carbamic acid benzyl ester 9

Activation of 2-benzyloxycarbonylamino-4-tetf-butoxy-3-methyl-butyric acid with isobutyl chloroformate and 4-methylmorpholine, and subsequent treatment of the activated acid with diazomethane allows for the preparation of the title compound.

(3-Methyl-5-oxo-tetrahydro-pyran-4-yl)-carbamic acid benzyl ester 10

Cyclisation of fe/f-butoxy-1-(2-diazo-acetyl)-2-methyl-propyl]-carbamic acid benzyl ester using lithium chloride in aqueous acetic acid gives the title compound. The CBz protecting group is readily replaced with Boc or Fmoc etc by conventional protecting group manipulation.

Pyranone or pyranol building blocks are N-terminal extended and capped as shown in Scheme 5A:

1. 4M HCl dioxan 2. R'COOPfp/HOBT 1. 4M HCl dioxan DMF, NMM 2. R'S0₂CI Et₃NH cat DMAP

Scheme 5A - N terminal extension & capping

(b) General method for N-terminal extension, exemplified by L-butylalanine The N-Boc-protected 4-aminopyranone from step a) is treated with a solution of 4.0M HCl in dioxan (25mL) at room temperature for lhr. The solvents are removed in vacuo and the residue azeotroped with 2 x toluene to give the hydrochloride salt. Boc-L-tert- butylalanine pentafluorophenyl ester ( 1.05eq) and 1-hydroxybenzotriazole hydrate (, 1.05eq) are dissolved in DMF (20mL) and after 5mins added to the above salt. The solution is then treated with N-methyliriorpholine (, 1.1 eq) and left at room temperature for 2hrs. The solvents are removed in vacuo and the crude product purified by flash chromatography over silica gel (50g) eluting with EtOAc / heptane (1 :3, v/v), then EtOAc / heptane (1 :2, v/v). Fractions are pooled and reduced in vacuo to give the title intermediate.

(c) General method for addition of capping group, exemplified by furylcarbonyl The N-extended aminopyranone from step b) is treated with a solution of 4.0M HCl in dioxan (25mL) at room temperature for lhr. The solvents were removed in vacuo and the residue azeotroped with 2 x toluene to give the hydrochloride salt.. Furan-3-carboxypentafluorophenyl ester (1.05eq) and 1-hydroxybenzotriazole hydrate (1.05eq) are dissolved in DMF (15mL) and after 5mins added to the above salt. The solution is then treated with N-methylmorpholine (l.leq) and left at room temperature for 2 hr. The solvents are removed in vacuo and the crude product purified by flash chromatography over silica gel (50g) eluting with EtOAc / heptane (3:2, v/v). Fractions are pooled and reduced in vacuo to give the title compound.

Example 2.

(a) General method for addition of sulphonyl capping group

The N-extended 4-aminopyranone from example 1 step b) is treated with a solution of 4.0M HCl in dioxan (5mL) at room temperature for lhr. The solvents were removed in vacuo and the residue azeotroped with 2 x toluene to give the hydrochloride salt. Hydrochloride salt was dissolved in dry DCM (2mL) and furan-3 -sulphonylchloride added followed by diisopropylethylamine (3 eq) and catalytic N,N- dimethylaminopyridine (2mg). After 2 hr at room temperature, the solution is diluted with DCM (15mL) and washed successively with 0.1N HCl (25mL), water (2 x 25mL) and brine (25mL), then dried over sodium sulphate. The solvent is removed in vacuo and the crude product purified by flash chromatography over silica gel (15g) eluting with EtOAc / heptane (1:1, v/v). Fractions are pooled and reduced in vacuo to give the title intermediate, lyophilised from 0.1 %aq TFA / acetonitrile.

Preparation of Building Block-Linker Constructs

General method for the synthesis of Pyranone - Linker Constructs (following Scheme

6).

Scheme 6 - Solid phase synthesis of compounds of formula IN

Ν-Fmoc-4-amino-5-ethylpyranone(1.0eq) is dissolved in a mixture of ethanol / water (7:1 v/v, lOmL per mmole compound) containing sodium acetate frihydrate (1.5eq). 4- [[(hydrazinocarbonyl)amino]methyl]cyclohexanecarboxylic acid trifluoro acetate (mw 329.3, l.Oeq) (see Murphy, A. M., et alJ. Am. Chem. Soc, 114, 3156-3157, 1992) is added and the mixture heated under reflux for 2hrs. The mixture is then cooled, poured into dichloromethane (lOO L per mmole compound) and water (lOOmL) added. The organic layer is separated, backwashed with saturated brine (lOOmL). The organic layer is dried (Na SO ), filtered and evaporated in vacuo to yield the title construct, which may be used without further purification

Chemistry Towards P2 Hybrid Aminoacids

The general chemistry depicted in Scheme 7 will shortly be published in full in the academic literature, by its inventors CS Dexter and RFW Jackson at the University of

Newcastle, England.

*{

Scheme 7 . Novel P2 hybrids by the CuCN catalysed cross coupling of Zn activated β- iodoalanine with allyl bromides

(a) General Procedure for the zinc coupling reactions

(b) Zinc activation

Zinc dust (150mg, 2.3mmol, 3.0eq, Aldrich) was weighed into a 25mL round bottom flask with a side arm and fitted with a three way tap. The zinc powder was heated with a heat gun under vacuum and the flask was flushed with nitrogen and evacuated and flushed a further three times. With the flask filled with nitrogen, dry DMF (lmL) was added. Trimethylsilylchloride (30μl, 0.23mmol, 0.3eq) was added and the zinc slurry was vigorously stirred for a further 30mins.

(c) Zinc insertion; N-(tert-Butoxycarbonyl)-3-iodozinc-L-alanine methyl ester (61) N-(tert-Butoxycarbonyl)-3-iodo-L-alanine methyl ester (247mg, 0.75mmol, l.Oeq) dissolved in dry DMF (0.5mL) was added dropwise, via cannula, to the activated zinc slurry at 0°C prepared as described above. The reaction mixture was then allowed to warm up to room temperature and stirred for lhr to give the organozinc reagent.

(d) CuBr.SMe₂ preparation

Whilst the zinc insertion reaction was in progress, CuBr.SMe₂ (20mg, O.lmmol, 0.13eq) was weighed into a 25ml round bottom flask fitted with a three way tap and dried "gently^Qwith a heat gun under vacuum until CuBr.SMe₂ changed appearance from a brown powder to give a light green powder. Dry DMF (0.5mL) was then added followed by addition ofthe elecfrophile (either l-bromo-2-methylbut-2-ene, toluene-4- sulfonic acid-(E)-2-methyl-but-2-enyl ester or l-bromo-2,3-dimethylbut-2-ene) (l.Ommol, 1.3eq). The reaction mixture was then cooled to -15°C.

(e) Coupling Reaction

Stirring ofthe organozinc reagent solution was stopped to allow the zinc powder to settle and the supernatant was carefully removed via cannula (care taken to avoid transferring too much zinc powder) and added dropwise to the solution of elecfrophile and copper catalyst. The cooling bath was removed and the solution was stirred at room temperature overnight. Ethyl acetate (20mL) was added and stirring was continued for a further 15mins. The reaction mixture was transferred to a separating funnel and a further aliquot of EtOAc (30mL) was added. The organic phase was washed successively with IM Na₂S₂O₃ (20mL), water (2 x 20mL), brine (40mL), dried over sodium sulphate and filtered. The solvent was removed in vacuo and the crude product purified by flash chromatography on silica gel as described. (f) Hydrogenation of alkene

The alkene (l.Ommol) was dissolved in ethanol (lOmL), 10% palladium on carbon (80mg) added and hydrogen introduced. Once the reaction had been deemed to have reached completion, the hydrogen was removed, the reaction filtered through Celite and the catalyst washed with ethanol (30mL). The combined organic filtrate was concentrated in vacuo and the alkane used directly in the subsequent reaction.

(g) Saponification of methyl ester

The methyl ester (l.Ommol) was dissolved in THF (6mL) and whilst stirring, a solution of LiOH (1.2mmol, 1.2eq) in water (6mL) was added dropwise. Once the reaction was deemed to have reached completion, the THF was removed in vacuo and diethyl ether (lOmL) added to the residue. The reaction mixture was then acidified with 1.0M HCl until pH =3. The organic phase was then removed and the aqueous layer extracted with diethyl ether (2 x lOmL). The combined organic extracts were dried over magnesium sulphate, filtered and the solvent removed in vacuo to give the carboxylic acid used directly in the subsequent reaction.

(h) Removal of N-Boc protecting group

The N-Boc protected material (l.Ommol) was dissolved in DCM (2mL) and cooled to 0°C. Trifluoroacetic acid (2mL) was added dropwise and when the reaction was deemed to have reached completion, the solvents were removed in vacuo to yield the amine used directly in the subsequent reaction. Alternatively, the N-Boc protected material (l.Ommol) was cooled to 0°C and 4M HCl in dioxane (5mL) added dropwise and when the reaction was deemed to have reached completion, the solvents were removed in vacuo to yield the amine used directly in the subsequent reaction.

(i) Fmoc protection of amine

The amine (l.Ommol) in 1,4-dioxane (2mL) was cooled to 0°C and 10% sodium carbonate (2.2mmol, 2.2eq, 2mL) added. The biphasic reaction mixture was stirred vigorously and Fmoc-Cl (1.1 mmol, 1.1 eq) added. Once the reaction was deemed to have reached completion, diethyl ether (lOmL) added and the reaction mixture acidified to pH = 3 with IM HCl. The organic phase was removed and the aqueous layer extracted with diethyl ether (2 x lOmL). The combined organic extracts were dried over sodium sulphate, filtered, the solvent removed in vacuo and the residue purified by flash chromatography over silica gel.

Example Synthesis 1

Preparation of 2S-2-(9H-fluoren-9-ylmethoxycarbonylamino)-4,4-dimethylhexanoic acid (68)

The following scheme explains how optically pure (S)-2-tert-Butoxycarbonylamino- 4,4-dimethyl-hex-5-enoic acid methyl ester (62) was prepared and isolated.

Separated by column chromatography

(a) 2S-2-tert-Butoxycarbonylamino-4, 4-dimethyl-hex-5-enoic acid methyl ester (62), 2S-2-tert-butoxycarbonylamino-4-(2S-3,3-dimethyl-oxiranyl)-butyric acid methyl ester (63) and 2S-2-tert-butoxycarbonylamino-4-(2R-3,3-dimethyl-oxiranyl)- butyric acid methyl ester (64)

Following the general procedure for zinc coupling reactions, l-bromo-3-methylbut-2- ene (115μL, l.Ommol) was coupled to compound (61) (247mg, 0.75mmol) in the presence of CuBr.SMe₂ (20mg, 0.1 mmol) to give a residue which was purified by flash column chromatography over silica gel eluting with EtOAc / 40:60 petroleum ether (1 :9, v/v). Fractions were pooled and reduced in vacuo to give a mixture of regioisomers (2:1 formal SN2' vs SN2), inseparable by column chromatography, as a colourless oil, yield 190mg, 93%.

To a mixture of regioisomers (190mg, 0.7mmol) in chloroform (3mL) was added dropwise over 5mins, 3-chloroperbenzoic acid (156mg, 85% pure, O.Smmol, l.leq) in chloroform (2mL). The reaction mixture was stirred at room temperature for a further 2hr. The reaction mixture was then washed successively with IM Na₂S₂O₅ (5mL), saturated sodium bicarbonate solution (5mL) and brine (lOmL). The organic phase was dried over sodium sulfate, filtered, the solvent removed in vacuo and the residue was purified by flash chromatography over silica gel eluting with EtOAc / 40:60 petroleum ether (2:8, v/v). Three products were obtained; compound (62) was eluted first and further elution afforded an inseparable mixture of compound (63) and compound (64). Fractions ofthe initial component were pooled and reduced in vacuo to give 2S-2-tert- butoxycarbonylamino-4,4-dimethyl-hex-5-enoic acid methyl ester (62) as a clear oil, yield 93mg, 49%. Electrospray-MS m/z 272 (MH⁺). Analytical HPLC Rt = 21.45mins (95%), HRMS C₁₀H₁₇O₄N requires M, 215.1158, found: M⁺-C₄H₈215.1152 (δ - 2.8 ppm); IR (cap. fύmycm^"1 3369 (s), 3084 (m), 2965 (s), 1748 (s), 1715 (s), 1517 (s), 1167 (s), 1007 (s), 914 (s)

δ_H(500 MHz; CDC1₃) 1.06 (6H, s, CH₂=CHC(CH₃)₂), 1.42 (9H, s, QCH^) 1.55 (IH, dd, J 14, 9, CH₂=CHC(CH₃)₂CH2A), 1.82 (IH, dd, J 14, 3, CH₂=CHC(CH₃)₂CH_2B), 3.69 (3H, s, OCH₃), 4.30 (IH, m, NHCHCO₂CH₃), 4.83 (IH, br d, J 7, NH), 4.97 (2H, m, CHz-CH) and 5.78 (IH, dd, J_trans 17.5, J_cis 11, CH₂=CH) δ_c (125 MHz; CDCI3) 26.93 (CH₂=CHC(CH₃)₂), 28.34 (C(CH₃)₃), 36.33 (CH₂=CHC(CH₃)₂CH₂), 45.06 (CH₂=CHC(CH₃)₂), 51.25 (NHCHCO₂CH₃), 52.15 (OCH₃), 79.77 (C(CH₃)₃), 111.39 (CH₂=CH), 146.87 (CH₂=CH), 154.97 (NHCC Bu¹) and 174.04 (CO₂CH₃).

(b) 2S-2-tert-Butoxycarbonylamino-4,4-dimethyl-hexanoic acid methyl ester (65) Following the general procedure for alkene hydrogenation, compound (62) (93mg, 0.3mmol) yielded compound (65) as a colourless oil, yield 90mg, 96%) and used directly in the subsequent reaction. Electrospray-MS m/z 274 (MET^1"). Analytical HPLC Rt = 22.55mins (100%).

(c) 2S-2-tert-Butoxycarbonylamino-4,4-dimethyl-hexanoic acid (66) Following the general procedure for methyl ester saponification, compound (65) (90mg, 0.3mmol) gave compound (66) as crystals, yield 79mg, 92% and used directly in the subsequent reaction. Electrospray-MS m/z 260 (MH^1"). Analytical HPLC Rt = 20.90mins (100%).

(d) 2S-2-Amino-4,4-dimethyl-hexanoic acid trifluoroacetic acid salt (67) Following the general procedure of N-Boc removal using TFA, compound (66) (79mg, 0.3mmol) gave compound (67) as a solid, yield 80mg, 96% and used directly in the subsequent reaction. Electrospray-MS m/z 274 (MH⁺).

(e) 2S-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-4,4-dimethyl-hexanoic acid (68) Following the general procedure for Fmoc protection of an amine, compound (67) (80mg, 0.3mmol) gave on purification by flash chromatography over silica gel eluting with CΗC1₃ / CH₃OH (100:0 to 96:4, v/v) 2S-2-(9H-fluoren-9- ylmethoxycarbonylamino)-4,4-dimethyl-hexanoic acid (68) as a solid, yield 60mg, 54%. Electrospray-MS m/z 382(MΗ⁺). Analytical HPLC Rt = 23.63mins (100%); [α ]_D ¹⁷ -18.4 (c 0.25 in EtOH)

δ_H (500MHz, CDCI₃) 0.88 (3H, t, J7, CH₃CH₂), 0.95 (6H, s, CH₃CH₂C(CH₃)₂), 1.31 (2H, , CH3CH2), 1.46 (IH, dd, J 14.5, 10, 1.85 (IH, br d, J 14.5, CH₃CH₂C(CH₃)₂CH2B), 4.21_(1H, t, J6.5, CH-Fmoc), 4.41 (3H, m, NHCHCO₂H and CH₂O), 5.02 (IH, br d, J8, NH-Fmoc), 7.29 (2H, m, H-2' and H-7'), 7.38 (2H, m, H-3' and H-6'), 7.58 (2H, m, H-l ' and H-8') and 7.74 (2H, d, J7, H-4' and H-5').

Example Synthesis 2

Preparation of 2S,4RS-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-4,5-dimethyl- hexanoic acid (74)

Optically pure 2S,4S-2-tert-Butoxycarbonylamino-4,5-dimethyl-hex-5-enoic acid methyl ester (69) and 2S,4R-2-tert-Butoxy-carbonylamino-4,5-dimethyl-hex-5-enoic acid methyl ester (70) were obtained directly after zinc coupling reaction by flash chromatography.

(61) (69) (70)

(a) 2S,4S-2-te;J-Butoxycarbonylamino-4,5-dimethyl-hex-5-enoic acid methyl ester (69) and 2S,4R-2-tert-butoxy-carbonylamino-4,5-dimethyl-hex-5-enoic acid methyl ester (70) Following the general procedure for zinc coupling reactions, toluene-4-sulfonic acid (E)-2-methyl-but-2-enyl ester (1.45mL, l.Ommol) was coupled to compound (61) (247mg, 0.75mmol) in the presence of CuBr.SMe₂ (20mg, O.lOmmol) to give a residue which was purified by flash chromatography over silica gel eluting with EtOAc / 40:60 petroleum ether (1:9, v/v) to give two diastereoisomers. Analytical HPLC Rt = 22.49mins (60%) and Rt = 22.52mins (40%). Fractions ofthe first eluted component were pooled to give one ofthe diastereoisomers obtained as a colourless oil, yield 36mg, 18%). Next a mixture ofthe diastereomers as a colourless oil, yield 75mg, 37% was obtained. Pure fractions containing the later eluted component were pooled to give the other diastereoisomer as a colourless oil, yield 19mg, 9%. (The stereochemistry at the 4 position was not investigated). Spectral data obtained for the fast running diastereomer: Electrospray-MS m/z 272 (MH*); [α]_D ²⁰ +12.3 (c 1.06 in CHC1₃); IR (cap. filmycm^"1 3382 (s), 3070 (m), 2966 (s), 1746 (s), 1716 (s), 1616 (w), 1507 (s), 886 (m)

δ_H(500 MHz, CDCI3) 1.06 (3H, d, J7, CH₃CH), 1.45 (9H, s, C(CH₃)₃), 1-58 (IH, m, CH₃CH), 1.68 (3H, s, CH₃C=CH₂), 1.85 (IH, m, CH^CH), 1.97 (IH, m, CH^CH), 3.73 (3H, s, OCH₃), 4.29 (IH, m, NHCHCO₂CH₃), 4.72 (IH, s, CH^CH), 4.95 (IH, d, J1.5, CH2B=CH) and 5.04 (IH, d, J7, NH) δ_c (125 MHz, CDCI3) 18.61 (CH₃C=CH₂), 21.64 (CH₃CH), 28.32 (C(CH₃)₃), 30.79 (CH₃CHCH₂), 38.06 (CH₂CHNH), 52.00 (NHCHCO₂CH₃), 52.22 (OCH₃), 79.53 (C(CH₃)₃), 110.19 (CH₂=C(CH₃)), 144.62 (CH₂=C(CH₃)), 155.18 (OCONH) and 173.30 (CO₂CH₃). Spectral data obtained for the slow running diastereoismer: Electrospray-MS m/z 272 (MH⁺); [α]_D ²⁰ +16.0 (c 0.60 in CHC1₃); IR (cap. filmycm ¹ 3369 (s), 3073 (m), 2969 (s), 1747 (s), 1717 (s), 1617 (w), 1517 (s), 893 (m)

δ_H(500 MHz, CDC1₃) 1.04 (3H, d, J7, CH3CH), 1.44 (9H, s, C(CH₃)₃), 1.55 (IH, m, CH₃CH), 1.67 (3H, s, CH₃C=CH₂), 1.91 (IH, m, CH^_ACH), 2.37 (IH, m, CH^CH), 3.73 (3H, s, OCH3), 4.26 (IH, m, NHCHCO₂CH₃), 4J5 (IH, d, J1.5, CH^CH), 4.79 (IH, d, J1.5, CH_2B=CH) and 5.46 (IH, d, J6.1, NH) δ_c (125 MHz, CDCI₃) 18.51 (CH₃C=CH₂), 20.14 (CH₃CH), 28.31 (C(CH₃)₃), 30.55 (CH₃CHCH₂), 37.64 (CH₂CHNH), 52.17 (NHCHCO₂CH₃), 52.22 (OCH₃), 79.74 (C(CH₃)₃), 111.27 (CH₂=C(CH₃)), 147.94 (CH₂=C(CH₃)), 155.36 (OCONH) and 173.83 (CO₂CH₃).

These diastereoisomers were not separated routinely and used as a mixture in subsequent reactions.

(b) 2S,4RS-2-tert-Butoxycarbonylamino-4,5-dimethyl-hexanoic acid methyl ester (71) Following the general procedure for alkene hydrogenation, compounds (69) and compound (70) (130mg, 0.48mmol) yielded a mixture of two diastereoisomers (71) which were not separated, obtained as a colourless oil, yield 128mg, 98%. Analytical HPLC Rt 22.49mins, electrospray-MS m/z 274 (MH⁺).

(c) 2S,4RS-2-tert-Butoxycarbonylamino-4,5-dimethyl-hexanoic acid (72) Following the general procedure for methyl ester saponification, compounds (71) (128mg, 0.47mmol) gave a inseparable mixture of compounds (72) as a colourless oil, yield 106mg, 87%. Electrospray-MS m/z 260 (MH⁺). Analytical HPLC Rt = 20.65mins (100%).

(d) 2S,4RS-2-Amino-4,5-dimethyl-hexanoic acid trifluoroacetic acid salt (73) Following the general procedure of N-Boc removal using TFA, compounds (72) (106mg, 0.41mmol) gave an inseparable mixture of compounds (73) as a solid, yield 107mg, 96%) and used directly in the subsequent reaction. Electrospray-MS m/z 160 (MH⁺). (e) 2S,4RS-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-4,5-dimethyl-hexanoic acid

(74) Following the general procedure for Fmoc protection of an amine, compounds (73) (107mg, 0.39mmol) gave on purification by flash chromatography over silica gel eluting with CHC1₃ / CH₃OH (100:0 to 95:5, v/v) 2S,4RS-2-(9H-fluoren-9- ylmethoxycarbonylamino)-4,5-dimethyl-hexanoic acid (74) as a solid, yield 60mg, 40% as a mixture of two diastereoisomers. Analytical HPLC Rt = 23.83mins (40%) and Rt = 24.06mins (60%). First eluted diastereomer: Electrospray-MS m z 382 (MH ). Later eluted diastereomer: Electrospray-MS m/z 382 (MH^1-).

Example Synthesis 3

Preparation of 2S,5RS-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-5,6-dimethyl- heptanoic acid (80) and 2S-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-4,4,5- trimethyl-hexanoic acid (84)

(S)-2-tert-butyloxycarbonylamino-5,6-dimethyl-hept-5-enoic methyl ester (75) and (S)- 2-tert-butyloxycarbonylamino-4,4,5-trimethyl-hex-5-enoic methyl ester (76) were obtained directly after zinc coupling reaction by flash chromatography.

(a) 2S-2-tert-Butyloxycarbonylamino-5,6-dimethyl-hept-5-enoic methyl ester (75) and 2S-2-tert-butyloxycarbonylamino-4,4,5-trimethyl-hex-5-enoic methyl ester (76) Following the general procedure for zinc coupling reactions, l-bromo-2,3-dimethylbut- 2-ene (163mg, l.Ommol) was coupled to compound (61) (247mg, 0J5mmol) in presence of CuBr.SMe (20mg, O.lOmmol) to give a residue which on purification by flash chromatography over silica gel eluting with EtOAc/ 40:60 petroleum ether (1 :9) gave two regioisomers. The first eluted component compound (75) as a colourless oil, yield 60mg, 28% and the second eluted component was compound (76) as a colourless oil, yield 51 mg, 24%.

Spectral data obtained for compound (75); Electrospray-MS m/z 285 (MH⁺). Analytical HPLC Rt = 22.85mins (100%); HRMS d₅H₂₇NO₄ requires M, 285.1940, found: M⁺ 285.1954 (δ - 4.9 ppm); [α]_D ²² +26.1 (c 1.01 in CH₂C1₂); elemental analysis C₁₅H₂₇NO₄ (req) %C 63.1, %H 9.5, %N 4.9, (fnd) %C 62.4, %H 9.6, %N 5.3; IR (cap. filmycm^"1 3366 (s), 3154 (m), 2978 (s), 1744 (s), 1718 (s), 1506 (s), 1366 (s), 1164 (s)

δ_H (500 MHz, CDC1₃) 1.45 (9H, s, C(CH₃)₃), 1.62 (9H, m, (CH₃)₂=C(CH₃)), 1.87 (IH, m, CH^_ACHZCH), 2.03 (IH, m, CH_2BCH₂CH), 2.09 (IH, dd, J6, 10.5, CH₂CH_2ACH), 2.12 (IH, dd, J6.5, 10.5, CH₂CH_2BCH), 3J4 (3H, s, OCH₃), 4.29 (IH, m, NHCHCO₂CH₃) and 5.02 (IH, d, J7, NH) δ_c (125 MHz, CDCI₃) 18.19 ((CH₃)₂C=C(CH₃)), 20.00 ((CH₃)_2cisC=C(CH₃)), 20.61 ((CH₃)_2transC=C(CH₃)), 28.33 (C(CH₃)₃), 30.07 (CH₂CH₂CH), 30.92 (CH₂CH₂CH), 52.20 (NHCHCO₂CH₃), 53.47 (OCH₃), 80.00 (C(CH₃)₃), 95.90 ((CH₃)₂C=C(CH₃)), 96.49 ((CH₃)₂C-C(CH₃), 155.33 (OCONH) and 173.42 (CO₂CH₃).

Spectral data obtained for compound (76); Electrospray-MS m/z 285 (MH ). Analytical HPLC Rt = 22.91mins (100%); HRMS C₁₁H₁ NO₄ requires 229.1314, found: '- Hs 229.1309 (δ - 2.2 ppm); [ ]_D ²³ +4.8 (c 1.01 in CH₂C1₂); elemental analysis Cι₅H₂₇NO₄ (req) %C 63.1, %H 9.5, %N 4.9, (fnd) %C 62.5, %H 9.5, %N; IR (cap. filmycm^"1 3368 (s), 3091 (m), 2934 (s), 1748 (s), 1717 (s), 1516 (s)

δ_H(500 MHz, CDCI₃) 1.10 (3H, s, (CH₃)_2AC), 1.12 (3H, s, (CH₃)_2BC), 1.43 (9H, s, C(CH₃)₃), 1.60 (IH, m, CH^CH), 1.74 (3H, s, CH₃C=CH₂), 1.92 (IH, dd, J 14.5, 4, CH^BCH), 3.70 (3H, s, OCH₃), 4.24 (IH, m, NHCHCO₂CH₃), 4.79 (IH, s, CH_2A ⁼C(CH₃)), 4.82 (IH, s, CH_2B=C(CH₃)) and 4.83 (IH, br d, Jl 1, NH) δ_c (125 MHz, CDCI₃) 19.38 (CH₃), 27.19 (CH₃), 27.61 (CH₃), 28.34 (C(CH₃)₃), 38.50 (CH₂CH), 38.95 ((CH₃)₂C), 51.34 (NHCHCO₂CH₃), 52.13 (OCH₃), 79.71 (C(CH₃)₃), 110.95 (CH₂=C(CH₃)), 150.62 (CH₂=C(CH₃)), 155.00 (OCONH) and 174.24 (CO₂CH₃). (b) 2S,5RS-2-tert-Butoxycarbonylamino-5,6-dimethyl-heptanoic acid methyl ester (77) Following the general procedure for alkene hydrogenation, 2S-2-tert- butyloxycarbonylamino-5,6-dimethyl-hept-5-enoic methyl ester (75) (60mg, 0.21mmol) yielded compound (77) as a colourless oil, yield 54mg, 89%). Electrospray- MS m z 288 (MH*). Analytical HPLC Rt = 24.06mins (100%).

(c) 2S,5RS-2-tert-Butoxycarbonylamino-5,6-dimethyl-heptanoic acid (78) Following the general procedure for methyl ester saponification, compounds (77) (54mg, 0.19mmol) gave compounds (78) as a colourless oil, yield 54mg, 100%. Electrospray-MS m/z 274 (MH^1"). Analytical HPLC Rt = 21.44mins (100%).

(d) 2S,5RS-2-Amino-5,6-dimethyl-heptanoic acid hydrochloride salt (79)

Following the general procedure of N-Boc removal using 4M HCl in dioxane, compounds (78) (54mg, 0.20mmol) gave compounds (79) as a solid, yield 40mg, 97%. Electrospray-MS m/z 174 (MH⁴).

(e) 2S,5RS-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-5,6-dimethyl-heptanoic acid

(80)

Following the general procedure for Fmoc protection of an amine, compounds (79) (40mg, 0.19mmol) gave on purification by flash chromatography over silica gel eluting with CHC1₃ / CH₃OH (100:0 to 95:5, v/v) 2S,5RS-2-(9H-fluoren-9- ylmethoxycarbonylamino)-5,6-dimethyl-heptanoic acid (80) as a solid, yield 27mg, 36%. Electrospray-MS m/z 395 (MH⁺). Analytical HPLC Rt = 24.52mins (100%), HRMS C₂₄H₂₉O₄NNa requires 418.1994, found: MNa⁺, 418.1993. (δ - 0.38 ppm)

δ_H (500 MHz; CDC1₃) 0.73 (6H, m, (CH₃)₂CH), 0.82 (3H, d, J6.5, (CH₃)₂CHCH(CH₃)), 1.23 (IH, m, (CH₃)₂CHCH(CH₃)CH2_A), 1.39 (IH, m, (CH₃)₂CHCH(CH₃)CH₂B)₅ 1.55 (2H, m, (CH₃)₂CHCH(CH₃) and (CH₃)₂CHCH(CH₃)CH₂CH_2A), 1-63 (IH, m, (CH₃)₂CHCH(CH₃)), 1.90 (IH, m, (CH₃)₂CHCH(CH₃)CH₂CH2B), 4.18 (IH, t, J6.5, CH-Fmoc), 4.40 (3H, m, NHCHCO₂H and CH₂O), 5.30 (IH, br d, J8, NH-Fmoc), 7.27 (2H, m, H-2' and H-7'), 7.37 (2H, m, H-3' and H-6'), 7.56(2H, m, H-l' and H-8') and 7J5 (2H, d, J7, H-4' and H-5')

δ_c (125 MHz; CDC1₃) 14.91 (CH₃)₂CHCH(CH₃)), 17.49 and 17.73 ((CH₃)_2ACH), 19.93 and 20.05 ((CH₃)_2BCH), 28.08 ((CH₃)₂CH), 29.26 and 29.44 ((CH₃)₂CHCH(CH₃)CH₂CH₂), 30.04 and 30.17 ((CH₃)₂CHCH(CH₃)CH₂CH₂), 31.38 and 31.68 ((CH₃)₂CHCH(CH₃)), 37.89 and 38.07 (NHCHCO₂H), 46.88 (CH-1'), 66.84 (CH₂O), 119.72 (CH-5' and CH-10'), 124.80 (CH-4' and CH-11'), 126.81 (CH-6' and CH-9'), 127.46 (CH-3' and CH-12'), 141.05 ( C-7' and C-8'), 143.47 (C-2' and C-13') and 155.89 (OCONH). The quaternary signal for the carboxylic acid was not observed.

(f) 2S-2-tert-Butoxycarbonylamino-4,4,5-trimethyl-hexanoic acid methyl ester (81) Following the general procedure for alkene hydrogenation, 2S-2-tert- butyloxycarbonylamino-4,4,5-frimethyl-hex-5-enoic methyl ester (76) (51mg, 0.18mmol) yielded compound (81) as a colourless oil, yield 46mg, 90%. Electrospray- MS m/z 288 (MH⁺). Analytical HPLC Rt = 22.91mins (100%).

(g) 2S-2-tert-Butoxycarbonylamino-4,4,5-trimethyl-hexanoic acid (82) Following the general procedure for methyl ester saponification, compound (81) (46mg, O.lόmmol) gave compound (82) as a colourless oil, yield 44mg, 100%. Electrospray-MS m/z 274 (MH⁺).

(h) 2S-2-Amino-4,4,5-trimethyl-hexanoic acid hydrochloride salt (83) Following the general procedure of N-Boc removal using 4M HCl in dioxane, compound (82) (44mg, 0.16mmol) gave compound (83) as a solid, yield 33mg, 99%. Electrospray-MS m/z 174 (MH ).

(i) 2S-2-(9H-Fluoren-9-ylmemoxycarbonylamino)-4,4,5-trimethyl-hexanoic acid (84) Following the general procedure for Fmoc protection of an amine, compound (83) (33mg, 0.16mmol) gave on purification by flash chromatography over silica gel eluting with CHCI3 / CH₃OH (100:0 to 95:5, v/v) 2S-2-(9H-fluoren-9- yhnethoxycarbonylamino)-4,4,5-trimethyl-hexanoic acid (84) as a solid, yield 20mg, 32%. Electrospray-MS m/z 396 (MH⁴). Analytical HPLC Rt = 24.28mins (100%), HRMS C₂₄H₂₉O₄NNa requires 418.1994, found: MNa⁺, 418.1993. (δ - 0.38 ppm)

δ_H (500 MHz; CDC1₃) 0.93 (9H, m, (CH3)₂CHC(CH₃)_2A), 0.98 (3H, s, (CH₃)₂CHC(CH3)_2B), 1.48 (lH, dd, J 14, 10, (CH₃)₂CHC(CH₃)₂CH2_A), 1.57 (lH, m, (CH₃)₂CH), 1.91 (IH, d, J 14, (CH₃)₂CHC(CH₃)₂CH₂B), 4.21 (IH, t, J6.5, CH-Fmoc), 4.40 (3H, m, NHCHCO₂H and CH₂O), 5.10 (IH, br d, J7.5, NH-Fmoc), 7.27 (2H, m, H-2' and H-7'), 7.36 (2H, m, H-3' and H-6'), 7.57 (2H, m, H-l' and H-8') and 7J4 (2H, d, J7, H-4' and H-5') δ_c (125 MHz; CDC1₃) 17.01 ((CH₃)_2ACH), 17.16 ((CH₃)_2BCH), 23.69 ((CH₃)₂CHC(CH₃)_2A), 24.27 ((CH₃)₂CHC(CH₃)_2B)₅ 35.27 ((CH₃)₂CHC(CH₃)₂), 35.73 ((CH₃)₂CH), 41.88 ((CH₃)₂CHC(CH₃)₂CH₂), 46.93 (CH-1'), 54.20 (NHCHCO₂H), 66.79 (CH₂O), 119.70 (CH-5' and CH-10'), 124.78 (CH-4' and CH-11'), 126.79 (CH- 6' and CH-9'), 127.44 (CH-3' and CH-12'), 141.05 ( C-7' and C-8'), 143.61 (C-2' and C-13') and 155.68 (OCONH). The quaternary signal for the carboxylic acid was not observed.

General Solid Phase procedures

Molecules are assembled using pyranone building blocks and novel protected aminoacids described earlier, by solid phase procedures on Chiron multipins following the protocols detailed below.

Preparation of Crown Assembly

The compounds are synthesised in parallel fashion using the appropriately loaded Fmoc-Building block-linker-DA MDA derivatised macrocrowns (see above) loaded at approximately 3.5 — 9.1 μmoles per crown. Prior to synthesis each crown is connected to its respective stem and slotted into the 8 x 12 stem holder. Coupling ofthe amino acids employs standard Fmoc amino acid chemistry as described in 'Solid Phase Peptide Synthesis', E. Atherton and R.C. Sheppard, IRL Press Ltd, Oxford, UK, 1989. Removal of Nα-Fmoc Protection

A 250 mL solvent resistant bath is charged with 200 mL of a 20% piperidine/DMF solution. The multipin assembly is added and deprotection allowed to proceed for 30 minutes. The assembly is then removed and excess solvent removed by brief shaking. The assembly is then washed consecutively with (200 mL each), DMF (5 minutes) and MeOH (5 minutes, 2 minutes, 2 minutes) and left to air dry for 15 minutes.

Quantitative UN Measurement of Fmoc Chromophore Release A lcm path length UN cell is charged with 1.2 mL of a 20% piperidine/DMF solution and used to zero the absorbance ofthe UN spectrometer at a wavelength of 290nm. A UN standard is then prepared consisting of 5.0 mg Fmoc-Asp(OBut)-Pepsyn KA (0.08 mmol/g) in 3.2 mL of a 20%) piperidine/DMF solution. This standard gives Abs₂ o = 0.55-0.65 (at room temperature). An aliquot ofthe multipin deprotection solution is then diluted as appropriate to give a theoretical Abs₂₉o = 0.6, and this value compared with the actual experimentally measured absorbance showing the efficiency of previous coupling reaction.

Standard Coupling Of Amino Acid Residues

Coupling reactions are performed by charging the appropriate wells of a polypropylene 96 well plate with the pattern of activated solutions required during a particular round of coupling. Macrocrown standard couplings were performed in DMF (500 μl).

Coupling of an Amino-acid Residue To Appropriate Well

Whilst the multipin assembly is drying, the appropriate Ν_α-Fmoc amino acid pfp esters (10 equivalents calculated from the loading of each crown) and HOBt (10 equivalents) required for the particular round of coupling are accurately weighed into suitable containers. Alternatively, the appropriate N_α-Fmoc amino acids (10 equivalents calculated from the loading of each crown), desired coupling agent e.g. HBTU (9.9 equivalents calculated from the loading of each crown) and activation e.g. HOBt (9.9 equivalents calculated from the loading of each crown), NMM (19.9 equivalents calculated from the loading of each crown) are accurately weighed into suitable containers. The protected and activated Fmoc amino acid derivatives are then dissolved in DMF (500 μl for each macrocrown e.g. for 20 macrocrowns, 20 x 10 eq. x 7 μmoles of derivative would be dissolved in 10 mL DMF). The appropriate derivatives are then dispensed to the appropriate wells ready for commencement ofthe 'coupling cycle'. As a standard, coupling reactions are allowed to proceed for 6 hours. The coupled assembly was then washed as detailed below.

Washing Following Coupling

If a 20%) piperidine/DMF deprotection is to immediately follow the coupling cycle, then the multipin assembly is briefly shaken to remove excess solvent washed consecutively with (200 mL each), MeOH (5 minutes) and DMF (5 minutes) and de- protected. If the multipin assembly is to be stored or reacted further, then a full washing cycle consisting brief shaking then consecutive washes with (200 mL each), DMF (5 minutes) and MeOH (5 minutes, 2 minutes, 2 minutes) is performed.

Addition of Capping Group

Whilst the multipin assembly is drying, the appropriate acid capping group (10 equivalents calculated from the loading of each crown), desired coupling agent e.g. HBTU (9.9 equivalents calculated from the loading of each crown) and activation e.g. HOBt (9.9 equivalents calculated from the loading of each crown), NMM (19.9 equivalents calculated from the loading of each crown) are accurately weighed into suitable containers. The acid derivatives / coupling agents are then dissolved in DMF (500 μl for each macrocrown e.g. for 20 macrocrowns, 20 x 10 eq. of derivative would be dissolved in 10 mL DMF) and left to activate for 5 minutes. The appropriate derivatives are then dispensed to the appropriate wells ready for commencement ofthe 'capping cycle'. As a standard, capping reactions are allowed to proceed for 18 hours overnight. The capped assembly was then washed as detailed above.

Acidolytic Mediated Cleavage of Molecule-Pin Assembly

Acid mediated cleavage protocols are strictly performed in a fume hood. A polystyrene

96 well plate (1 mL/well) is labelled and weighed to the nearest mg. Appropriate wells are then charged with a frifluoroacetic acid/water (95:5, v/v, 600 μl) cleavage solution, in a pattern corresponding to that ofthe multipin assembly to be cleaved.

The multipin assembly is added, the entire construct covered in tin foil and left for 2 hours. The multipin assembly in then added to another polystyrene 96 well plate (1 mL/well) containing frifluoroacetic acid/water (95:5, v/v, 600 μl) (as above) for 5 minutes.

Work up of Cleaved Molecules

The primary polystyrene cleavage plate (2 hour cleavage) and the secondary polystyrene plate (5 minute wash) are then placed in the Gene Vac evaporator and the solvents removed (minimum drying rate) for 90 minutes. The contents ofthe secondary polystyrene plate are transferred to their corresponding wells on the primary plate using an acetonitrile/water (50: 50 v/v/v) solution (3 x 150 μl) and the spent secondary plate discarded. Aliqouts (5-20μL) are taken for analysis. The plate was covered in tin foil, pin-pricked over wells containing compounds, placed into the freezer for lhr, then lyophilised.

Analysis and Purification of Molecules

The (5-20μL) aliquots are analysed by analytical HPLC and electrospray-MS. In virtually all cases, crude purities are >90% by HPLC with the desired m/z. Sample were purified by semi-preparative reverse phase HPLC, using Vydac .Appropriate fractions are combined and lyophilised in tared lOmL glass vials, then re- weighed. Molecules were prepared on a 15-90μmole scale, yielding 2.0-26.0mg of purified products. The purity of each product was confirmed by analytical HPLC at >95% (215nm UN detection) and gave the appropriate [MH]⁺ by electrospray mass specfrometry analysis.

Loading of Macrocrowns With Constructs

General method for the loading of multipins with Pyranone - Linker Constructs Amino functionalised DA/MDA macrocrowns (ex Chiron Mimotopes, Australia, 9.1μmole loading) or amino functionalised HEMA gears (ex Chiron Mimotopes, Australia, 1.3μmole loading) are used for all loadings and subsequent solid phase syntheses.

Pyranone - Linker Construct (3eq compared to total surface functionalisation of crowns

/ gears) is carboxyl activated with 2-(lH-benzofriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (3eq), 1-hydroxybenzotriazole (3eq) and N-methylmorpholine

(6eq) in dimethylformamide (5mL) for 5mins. This mixture is added to the crowns / gears, additional DMF added to cover the reaction surface and the mixture left overnight.

Standard washing and Fmoc deprotection readings (see procedures above) typically indicates virtually quantitative loading.

Kj determinations for cathepsins S, L, and K

Cathepsin S (Mammalian, murine and rat)

General

Assays were performed in 100 mM sodium phosphate, 100 mM NaCl, pH 6.5 (buffer) in white 384 well plates (Corning Costar). Eight inhibitors were assayed per plate.

Inhibitor dilutions

Inhibitor dilutions were performed on a 96 well N-bottomed polypropylene plate (Corning Costar). 100 μl of buffer was placed in wells 2-5 and 7-12 of rows A, B, C and D. Sufficient of each inhibitor at 10 mM in DMSO was placed into wells Al-Dl and A6-D6 to give the desired final concentration when the volume in the well was made up to 200 μl with buffer. Column 1 was made up to 200 μl with buffer, mixed by aspirating and dispensing 100 μl in the well, and 100 μl transferred to column 2. The pipette tips were changed and the mixing and fransferral repeated to column 5. This process was repeated for columns 6-10.

Substrate dilutions

Substrate dilutions were performed on a 96 deep well polypropylene plate (Beckman Coulter). 280 μl of buffer was placed in wells B-H of columns 1 and 2. 70 μl of 10 mM boc-Nal-Leu-Lys-AMC was placed in Al and A2. 2 x 250 μl of buffer was added to wells Al and A2, mixed by aspirating and dispensing 280 μl in the well, and 280 μl transferred to row B. The pipette tips were changed and the process repeated down the plate to row H.

Assay

Column 1 ofthe substrate dilution plate was distributed at 10 μl per well into alternate rows beginning at row A. Column 2 was distributed to alternate rows beginning at row B. Row A ofthe inhibitor dilution plate was distributed at 10 μl per well to alternate rows and columns starting at Al . Row B was distributed to alternate rows and columns starting at A2. Row C was distributed to alternate rows and columns starting at Bl and row D was distributed to alternate rows and columns starting at B2. The assay was started by the addition of 30 μl per well of 20 nM cathepsin S in buffer containing 10 mM 2-mercaptoethanol.

Data were saved as text files and imported into Excel. The initial rates were determined by linear regression and then fitted to the competitive inhibition equation using SigmaPlot.

Cathepsins L and K

Assays were performed essentially as above. For cathepsin L, the buffer used was 100 mM sodium acetate, 1 mM EDTA, pH 5.5 and the substrate was J>~Nal-Leu-Lys-AMC with a highest concentration of 100 μM. For cathepsin K, the buffer used was 100 mM MES/Tris, 1 mM EDTA, pH 7.0 and the substrate was E>-Ala-Leu-Lys-AMC with a highest concentration of 250 μM.

Determination of cathepsin K proteolytic catalytic activity

Convenient assays for cathepsin K arere carried out using human recombinant enzyme, as described above. Standard assay conditions for the determination of kinetic constants used a fluorogenic peptide substrate, typically H-£>-Ala-Leu-Lys-AMC, and were determined in either 100 mM Mes/Tris, pH 7.0 containing 1 mM EDTA and 10 mM 2-mercaptoethanol or 100 mM Na acetate, pH 5.5 containing 5 mM EDTA and 20 mM cysteine. The enzyme concentration used was 5 nM. The stock substrate solution was prepared at 10 mM in DMSO. Screens were carried out at a fixed substrate concentration of 60 μM and detailed kinetic studies with doubling dilutions of substrate from 250 μM. The total DMSO concentration in the assay was kept below 3%. All assays were conducted at ambient temperature. Product fluorescence (excitation at 390 nm, emission at 460 nm) was monitored with a Labsystems Fluoroskan Ascent fluorescent plate reader. Product progress curves were generated over 15 minutes following generation of AMC product.

Inhibition Studies

Potential inhibitors are screened using the above assay with variable concentrations of test compound. Reactions were initiated by addition of enzyme to buffered solutions of substrate and inhibitor. K; values were calculated according to equation 1

where vø is the velocity ofthe reaction, Vis the maximal velocity, Sis the concentration of substrate with Michaelis constant of K_M, and /is the concentration of inhibitor.

Determination of falcipain 2 proteolytic catalytic activity

Generation of Falcipain 2 Cloning

The deoxyoligonucleotide primers:

(SEQ ID NO.: 1) 5'CGCGGATCCGCCACCATGGAATTAAACAGATTTGCCGAT- 3' and (SEQ ID NO.:2)

5'CGCGTCGACTTAATGATGATGATGATGATGTTCAATTAATGGAATGAATG CATCAGT-3' were designed based on sequences deposited at the Sanger Centre, Cambridge, UK (httpJ/www.sanger.ac.uk/Proiects/P falciparurn/blast server, shlmf). These primers were designed to amplify a portion ofthe cDNA sequence ofthe cysteinyl proteinase now known as Falcipain 2 and to include relevant terminal cloning enzymes sites and a carboxy-terminal hexahistidine coding sequence immediately upsfream ofthe stop codon.

Polymerase chain reaction was performed with the above primers and Plasmodium falciparum phage library DNA as a template using the following conditions; 94°C for 2 minutes then 35 cycles of 94°C for 10 seconds, 50°C for 1 minute, and 60°C for 2 minutes, this was followed by a 60°C 5 minute incubation. The 880bp PCR amplicon was purified and phosphorylated using T4 polynucleotide kinase. This DNA was then ligated into EcoRV cleaved, dephosphorylated Bluescript II cloning vector and transformed into DH5 alpha E.coli. The DNA sequence of the plasmid inserts in isolated recombinant E.coli clones were determined using an Amersham Megabace sequencing instrument. To create an authentic ORF a three-way ligation was conducted bringing together the N-terminus of truncated falcipain-2 (Ncol/Ndel), the C-terminus of falcipain-2 (Ndel Ba Hl) and the vector ρQE-60 (NcoI/BamHI).

Nucleotide Sequence of TF2.10 (SEQ ID NO.: 3):

CCATGGAATTAAACAGATTTGCCGATTTAACTTATCATGAATTTAAAAACA

AATATCTTAGTTTAAGATCTTCAAAACCATTAAAGAATTCTAAATATTTATT

AGATCAAATGAATTATGAAGAAGTTATAAAAAAATATAGAGGAGAAGAAA

ATTTCGATCATGCAGCTTACGACTGGAGATTACACAGTGGTGTAACACCTG

TAAAGGATCAAAAAAATTGTGGATCTTGCTGGGCCTTTAGTAGTATAGGTT

CCGTAGAATCACAATATGCTATCAGAAAAAATAAATTAATAACCTTAAGTG

AACAAGAATTAGTAGATTGTTCATTTAAAAATTATGGTTGTAATGGAGGTC

TCATTAATAATGCCTTTGAGGATATGATTGAACTTGGAGGTATATGTCCAG

ATGGTGATTATCCATATGTGAGTGATGCTCCAAATTTATGTAACATAGATA

GATGTACTGAAAAATATGGAATCAAAAATTATTTATCCGTACCAGATAATA

AATTAAAAGAAGCACTTAGATTCTTGGGACCTATTAGTATTAGTGTAGCCG

TATCAGATGATTTTGCTTTTTACAAAGAAGGTATTTTCGATGGAGAATGTG

GTGATGAATTAAATCATGCCGTTATGCTTGTAGGTTTTGGTATGAAAGAAA

TTGTTAATCCATTAACCAAGAAAGGAGAAAAACATTATTATTATATAATTA

AGAACTCATGGGGACAACAATGGGGAGAAAGAGGTTTCATAAATATTGAA ACAGATGAATCAGGATTAATGAGAAAATGTGGATTAGGTACTGATGCATTC

ATTCCATTAATTGAACATCATCATCATCATCATTAAGTCGACGCGATCGAA

TTCCTGCAGCCCGGGGATCC

Coding for the Protein Sequence (SEQ ID NO.: 4):

MELNRFADLTYHEFKNKYLSLRSSKPLKNSKYLLDQMNYEEVIKKYRGEENFD

HAΛYDWRLHSGNTPVKDQKNCGSCWAFSSIGSVESQYAIRKNKLITLSEQELV

DCSFKNYGCNGGLINNAFEDMIELGGICPDGDYPYVSDAPNLCNIDRCTEKYGI

KNYLSVPDNKLKEALRFLGPISISVANSDDFAFYKEGIFDGECGDELNHANMLV

GFGMKEIVΝPLTKKGEKHYYYΠKΝSWGQQWGERGFIΝIETDESGLMRJKCGLG

TDAFIPLIEHHHHHH.

The TF2.10 insert was excised from the pQE-60 vector using the restriction enzymes Νcol and BamHI, ligated into ΝcoI/BamHI cut expression vector pET-l lD and transformed into DH5 alpha E.coli. The presence of a recombinant expression plasmid (pET-TF2.10) in an isolated E.coli colony was confirmed by restriction enzyme digest of plasmid DΝA. BL21(DE3) E.coli were transformed with pET-TF2.10 and used for expression ofthe recombinant cysteinyl proteinase.

Protein Expression

pET-TF2.10-Transformed BL21(DE3) E.coli (BLTF2.10) were grown up overnight at 200 rpm, 37°C in Luria broth containing 100 μg/ml ampicillin. Fresh medium was then inoculated and grown to an OD₆oo_nm of 0.8 before protein expression was induced using 1 mM IPTG. Induction was performed for 3 hours at 200 rpm, 37°C then the bacterial cells harvested by centrifugation and stored at -80°C until protein purification performed. Protein Purification and Refolding

An E.coli cell pellet equivalent to 250ml culture was lysed by resuspension in solubilisation buffer (6M guanidine hydrochloride, 20mM Tris-HCl, 250mM NaCl, 20mM imidazole, pH8.0) for 30 minutes at room temperature. After centrifugation at 12000g for 10 minutes at 4°C the cleared lysate was applied to 1 ml nickel-NTA agarose, and agitated for 1 hour at room temperature.

Protein Refolding Method 1

The protein bound to nickel-NTA was batch washed with 6M guanidine hydrochloride, 20mM Tris-HCl, pH 8.o, 250mM NaCl then 8M urea, Tris-HCl, pH 8.0, 500mM NaCl then 8M urea, Tris-HCl, pH 8.0 including 30 mM imidazole and protein elution performed using 8M urea, Tris-HCl, pH 8.0 with 1 M imidazole. The eluted protein was then diluted 100 fold in refolding buffer (lOOmM Tris-HCl, lmM EDTA, 20% glycerol, 250mM L-arginine, lmM reduced glutathione, O.lmM oxidised glutatione, pH8.0) and left stirring overnight at 4°C. The protein could then be concentrated either by filter centrifugation or repurification using a nickel-agarose column (after dialysis to remove the EDTA).

Protein Refolding Method 2

The protein bound to nickel-NTA was batch washed with 8M urea, Tris-HCl, 500mM NaCl, pH 8.0 then 8M urea, Tris-HCl, pH 8.0 including 20 mM imidazole, then 2M urea, Tris-HCl, pH 8.0. The protein was then refolded on the column by the addition of lOOmM Tris-HCl, pH8.0, 250mM L-arginine, lmM reduced glutathione, O.lmM oxidised glutatione with incubation at 4°C and protein elution performed using, lOOmM Tris-HCl, pH 8.0 with 0.5 M imidazole.

Immediately active (mature) proteinase was obtained using protein refolding method 1 and concentrating the dilute refolded enzyme by filter centrifugation. This method, however, did result in a large degree of enzyme loss due to autoproteolysis. Both concentrating the protein refolded using method 1 by nickel column purification and using refolding method 2 resulted in greater recovery of the enzyme in its stable inactive pro-form. The pro-form could also be used to generate mature active falcipain 2, after incubation at 37°C.

Convenient assays for falcipain 2 are carried out using the above recombinant enzyme. Alternatively, Sijali et al. Prot Exp Purif 22 128-134 2001 describes a useful assay. Standard assay conditions for the determination of kinetic constants used a fluorogenic peptide substrate, typically Boc-Nal-Leu-Lys-AMC, and were determined in either 100 mM Mes/Tris/acetate, pH 7.0 containing 1 M ΝaCl and 10 mM 2-mercaptoethanol or 100 mM Νa phosphate, pH 5.5 containing 1 M ΝaCl and 10 mM 2- mercaptoethanol. The enzyme concentration used was 2 nM. The stock substrate solution was prepared at 10 mM in DMSO. Screens were carried out at a fixed substrate concentration of 80 μM and detailed kinetic studies with doubling dilutions of substrate from 250 μM. The total DMSO concentration in the assay was kept below 3%>. All assays were conducted at ambient temperature. Product fluorescence (excitation at 390 nm, emission at 460 nm) was monitored with a Labsystems Fluoroskan Ascent fluorescent plate reader. Product progress curves were generated over 15 minutes following generation of AMC product.

Inhibition Studies

Potential inhibitors were screened using the above assay with variable concentrations of test compound. Reactions were initiated by addition of enzyme to buffered solutions of substrate and inhibitor. K; values were calculated according to equation 1

where vø is the velocity ofthe reaction, J^is the maximal velocity, S is the concentration of substrate with Michaelis constant of KM, and /is the concentration of inhibitor.

Claims (20)

Claims
1. A compound ofthe formula IN

   where

   Rl is R'-C(=O)- or R'-S(=O)2- R' is

   X, = O, S, ΝH,

   W, Y, Z = CH, Ν;

   R" = single or multiple ring substitution combinations taken from:

   H, CI -7-alkyl, C3-6-cycloalkyl, OH, SH, amine, halogen; R3 = Cl-7-alkyl, C2-C7 alkenyl, C3-7-cycloalkyl, Ar, Ar-Cl -7-alkyl; R4 = H, Cl-7-alkyl, C3-7-cycloalkyl; C2-7alkenyl, Ar, Ar-Cl -C7-alkyl; R5 = CI -7-alkyl, hydroxy- or halo-substituted Cl-C7alkylhalogen, Ar-Cl -7-alkyl, C0- 3-alkyl-COΝR3R4 orR^iv;

   R^p

   where n = 1-3, m = 1-3;

   R^v, R^vi = H, Cl -7-alkyl;

   A = N, CH;

   B = N, O, S, CH;

   R^vii = absent when B = O, S; or R^vii = H, Cl-7-aIkyl when B = N, CH;

   R^viii = O, Cl-7-alkyl; R6 = H, CI -7-alkyl, Ar-Cl -7-alkyl, Cl-3-alkyl-SO2-R^ix, Cl-3-alkyl-C(O)-NHR^ix or CH₂XAr;

   R^ix is CI -7-alkyl, Ar-Cl -7-alkyl, C3-C6-cycloalkyl and pharmaceutically acceptable salts thereof.
2. 2. A compound according to claim 1, wherein R4 is hydrogen.
3. 3. A compound according to claim 1, wherein Rl is R'C(O),

   Where R'
4. 4. A compound according to claim 1, wherein R' is fur-3-yl, thien-3-yl, or phenyl substituted with multiple substitutions.
5. 5. A compound according to claim 1, wherein R3 is n-butyl, t-butyl, 3 -(2,2- dimethylpropyl), 4-(2-methylbutyl), 4-(3,3-dimethylbutyl), 4-(3,3-dimethyl-2- methylbutyl), 4-(3-methyl-2-methylbutyl) or 5-(2-methyl-3-methylpentyl)
6. 6. A compound according to claim 5, wherein R3 is t-butyl, 3 -(2,2- dimethylpropyl), or 4-(3,3-dimethyl-2-methylbutyl).
7. 7. A compound according to claim 1, wherein R3 is C3-C6 cycloalkyl.
8. 8. A compound according to claim 7 wherein R3 is the side chain of L- cycohexylalanine or L-cyclopentylalanine.
9. 9. A compound according to claim 1, wherein R5 is CH₃, C₂H₅, CH₂OH, CH₂Ar, CH₂CONH₂, (CH₂)₂CONH₂,
10. 10. A compound according to claim 1, wherein R5 is CH₃, CH₂CH₃, CH₂CH₂CH₃ or CH₂OH.
11. 11. A compound according to claim 1, wherein R5 has (S) stereochemistry, preferably where the C4-bond also has the (S) stereochemistry
12. 12. A compound according to claim 1, wherein R5 has (R) stereochemistry, preferably where the C4-bond also has the (R) stereochemistry..
13. 13. A compound according to claim 1 , wherein

    Rl = R'C(O)

    Where R'

    R4 and R6 = H;

    R3 = n-butyl, t-butyl, 3-(2,2-dimethylpropyl), 4-(2-methylbutyl), 4-(3,3- dimethylbutyl), 4-(3,3-dimethyl-2-methylbutyl), 4-(3-methyl-2-methylbutyl), 5-

    (2-methyl-3- methylpentyl);

    R5 = CH₃, C₂H_S, CH₂Ar, CH₂CONH₂, (CH₂)₂CONH₂,
14. 14. A compound selected from the group consisting of

    Furan-3-carboxylic acid (lS)-[3,3-dimethyl-l-(3-methyl-5-oxo-tetrahydro-pyran-4- ylcarbamoyl)-butyl] -amide

    Furan-3-carboxylic acid (lS)-[2-cyclohexyl-l-(3-methyl-5-oxo-tefrahydro-pyran-4- ylcarbamoyl)-ethyl] -amide

    ( 1 S)-N- [3 ,3 -Dimethyl- 1 -(3 -methyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-butyl] - benzamide

    ( 1 S)-N- [2-Cyclohexyl- 1 -(3 -methyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl] - benzamide

    ( 1 S)-N- [3 ,3 -Dimethyl- 1 -(3 -methyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-butyl] -4- hydroxy-3 -methyl-benzamide

    (lS)-N-[2-Cyclohexyl-l-(3-methyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl]-4- hydroxy-3 -methyl-benzamide

    Furan-3 -carboxylic acid ( 1 S)- [2-cyclopentyl- 1 -(3 -methyl-5 -oxo-tetrahydro-pyran-4- ylcarbamoyl)-ethyl] -amide

    ( 1 S)-N- [2-Cyclopentyl- 1 -(3 -methyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl] - benzamide

    (1S)-N- [2-Cyclopentyl- 1 -(3 -methyl-5 -oxo-te trah.ydro-pyran-4-ylcarbamoyl)-ethyl] -4- hydroxy-3 -methyl-benzamide

    Furan-3-carboxylic acid (lS)-[3,3-dimethyl-l-(3-ethyl-5-oxo-tefrahydro-pyran-4- ylcarbamoyl)-butyl] -amide

    Furan-3 -carboxylic acid (lS)-[2-cyclohexyl-l -(3 -ethyl-5 -oxo-tetrahydro-pyran-4- ylcarbamoyl)-ethyl] -amide

    (lS)-N-[3,3-Dimethyl-l-(3-ethyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-butyl]- benzamide ( 1 S)-N- [2-Cyclohexyl- 1 -(3 -ethyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl] - benzamide

    (lS)-N-[3,3-Dimemyl-l-(3-ethyl-5-oxo-tefrahydro-pyran-4-ylcarbamoyl)-butyl]-4- hydroxy-3 -methyl-benzamide

    (l<S -N-[2-Cyclohexyl-l-(3-ethyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl]-4- hydroxy-3 -methyl-benzamide

    Furan-3 -carboxylic acid (IS)- [2-cyclopentyl- 1 -(3 -ethyl-5 -oxo-tetrahydro-pyran-4- ylcarbamoyl)-ethyl]-amide

    (lS)-N-[2-Cyclopentyl-l-(3-ethyl-5-oxo-tefrahydro-pyran-4-ylcarbamoyl)-ethyl]- benzamide

    (lS)-N-[2-Cyclopentyl-l-(3-ethyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl]-4- hydroxy-3 -methyl-benzamide

    Furan-3 -carboxylic acid (lS)-[3,3-dimethyl-l -(3 -propyl-5 -oxo-tetrahydro-pyran-4- ylcarbamoyl)-butyl] -amide

    Furan-3 -carboxylic acid (IS)- [2-cyclohexyl- 1 -(3 -propyl-5 -oxo-tetrahydro-pyran-4- ylcarbamoyl)-ethyl] -amide

    ( 1 S)-N- [3 ,3 -Dimethyl- 1 -(3 -propyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-butyl] - benzamide

    (lS)-N-[2-Cyclohexyl-l-(3-propyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl]- benzamide

    (lS)-N-[3,3-Dimethyl-l-(3-propyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-butyl]-4- hydroxy-3 -methyl-benzamide

    ( IS)-N- [2-Cyclohexyl- 1 -(3 -propyl-5 -oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl] -4- hydroxy-3 -methyl-benzamide

    Furan-3 -carboxylic acid (lS)-[2-cyclopentyl-l -(3-propyl-5-oxo-tetrahydro-pyran-4- ylcarbamoyl)-ethyl] -amide

    (lS)-N-[2-Cyclopentyl-l-(3-propyl-5-oxo-tetrahydro-pyran-4-ylcarbamoyl)-ethyl]- benzamide

    (lS)-N-[2-Cyclopenryl-l-(3-proρyl-5-oxo-tefrahydro-ρyran-4-ylcarbamoyl)-ethyl]-4- hydroxy-3 -methyl-benzamide and pharmaceutically acceptable salts thereof
15. 15. A pharmaceutical composition comprising a compound according to any preceding claim and a pharmaceutically acceptable carrier.
16. 16. A method of inhibiting the cysteine protease Cathepsin S which comprises administering to a patient in need thereof an effective amount of a compound according to any of claims 1 to 13.
17. 17. A method of inhibiting the cysteine protease Cathepsin S which comprises administering to a patient in need thereof an effective amount of a composition according to claim 15.
18. 18. Use of a compound according to any of claims 1 to 13 in the manufacture of a medicament for the treatment of a disease alleviated or moderated by inhibition of Cathepsin S cysteine protease activity.
19. 19. A method for the preparation of a compound as defined in claim 1 , comprising the steps of manipulating the protecting groups on a suitably protected carbohydrate derivative to effect deoxygenation at the anomeric postion, introducing the R5 substituent via a ketone functionality, for example by Wittig chemistry, introducing the 4-amino group by further manipulation ofthe C4 secondary alcohol functionality to provide a protected 4-amino-5-substituted pyranol, N-extending the amine function using peptide chemistry and adding the R'C(=^:O) or R'S(=O)₂ capping group, further including the step of oxidising the pyranol to pyranone before or after N-extension and/or capping.
20. 20. A method for the preparation of a compound as defined in claim 1 , comprising the steps of diazotising an O-protected, acyclic carboxylic derivative of a suitably derivatised 3-amino-4-substituted lactone, cyclising the diazomethylketone produced to afford a protected 4-amino-5-substituted pyranone, N-extending the amine function using peptide chemistry and adding the R'C(=O) or R'S(=O)₂ capping group.