HYDROXAMIC ACID DERIVATIVES AS INHIBITORS OF BETA-AMYLOID PRODUCTION The present invention relates to the use of certain esters and thioesters for the treatment of diseases responsive to inhibition of production of ß-amyloid peptide Background to the Invention Alzheimer's disease (AD) is the commonest form of adult onset dementia and is characterised neuropathologically by the accumulation of neuritic plaques in the cortical grey matter predominantly composed of the 4 kDa p-amy ! oid peptide (Aß) [Roher et al. (1986) PNAS USA. 83 : 2662-2666]. Ap is a 39-43 amino acid peptide [Glenner et al. (1984) Biochem. Biophys. Res. Comm. 120 : 8885-8908], derived from the proteolysis of Amyloid Precursor Protein (APP). APP is a ubiquitously expressed glycosylated transmembrane cell surface receptor-like protein which was first cloned, sequenced and mapped to chromosome 21 over a decade ago [Kang et al. (1987) Nature. 325 : 733-736]. As a group APP comprises four different polypeptides whose heterogeneity arises from alternative splicing and post- translational processing [Selkoe (1994) Ann. Rev. Ce ! t Biot. 10 : 373-403] with the shorter 695 amino acid form being predominantly expressed in neurons. Cleavage occurs at the N- and C-termini of Ap by as yet uncharacterised enzymes, termed ß- secretase and y-secretase, respectively.

Ap was once considered the result of abnormal processing of APP [Sisodia et al.

(1992) PNAS USA. 89 : 6075-6079] but in light of the fact that Ap is present in cerebrospinal fluid of healthy individuals [Haass et al. (1992) Nature. 359 : 322-325 ; Seubert et a/ (1992) Nature. 359 : 325-327 ; Shoji et al. (1992) Science. 258 : 126- 129] and that diffuse plaques are also present with no apparent ill effects in aged humans and primates [Sisodia & Price (1995) FASEB J. 9 : 366-370], this view has been reappraised. Rather it is now believed that it may be the elevated levels of Aß in affected individuals rather than simply its presence which causes the onset of AD. AD can result either by overexpression i. e. a gene dosage effect in trisomy 21 (Down's syndrome patients invariab ! y devetop AD neuropathology in their forties or fifties) or by missense mutations that increase the amyloidogenic cleavages of APP at either the p-secretase site (leading to excessive production of both Aß,4o and Aß142) or the y-secretase site (resulting in selective increased production of Aß,42).

APP is anchored to internal membranes e. g. endoplasmic reticulum (ER), Golgi, trans-Golgi network (TGN) and endosomes and in the plasmalemma by a 23 amino acid hydrophobic stretch near it's C-terminus. Both during and after APP is transported through the secretory pathway, on route to the cell surface, a proportion of APP molecules undergo endoproteolytic cleavage between residues 16 and 17 of the Ap domain by a protease designated'a-secretase'. This results in the release of a large soluble ectodomain fragment (sAPPa) which is non- amyloidogenic. The regulated cleavage of APP (i. e. as enhanced by phorbol ester activation) at the a-secretase site is believed to be carried out by a disintegrin and metalloprotease (ADAM), specifically ADAM-17, also known as tumour-necrosis factor-a (TNF-a) converting enzyme (TACE), since it is capable of shedding the ectodomain of TNF-a [Bauxbaum et al. (1998) J. Biol. Chem. 273 : 27765-27767].

Recently it has been shown that both the constitutive and regulated a-secretase cleavage of APP can be performed by ADAM-10 [Lammich et a/. (1999) J. Biol.

Chem. 96 : 3922-3927]. It would therefore seem feasible that stimulation of a- secretase cleavage may be a valid thereapeutic option, thereby precluding Aß formation. However, since in most cells only a minority of APP molecules undergo a-secretory cleavage, any increase in proteolysis at this site would still potentially leave many APP molecules which could be subject to ß- and y-secretase cleavage.

The most likely therapeutic option therefore, to reduce Ap formation, wouid be to inhibit the p- and/or y-secretase activities.

Although the p-secretase activity remains elusive recent reports have shown an intriguing connection between y-secretase activity and the presenilin (PS) proteins [Wolfe et a/. (1999) Nature. 398 : 513-517]. It has been known for some time that mutations in PS lead to early-onset AD and studies in transgenic mice and in transfected cells have shown that these PS mutations alter APP processing and caused increased levels of the 42-amino-acid p-amyioid derivative AP, -,, [Selkoe (1998) TICB. 8 : 447-453]. Mutagenesis studies have shown that two transmembrane (TM) aspartate residues within the PS-1 protein (within TM6 and TM7) are essential for y-secretase activity and suggests that PS-1 is either a unique diaspartyl cofactor for y-secretase activity or indeed is itself y-secretase, an autoactivated intramembranous aspartyl protease [Wolfe et a/. (1999) Nature.

398 : 513-517].

Because of its involvement in such neurological disorders as AD and cerebral amyloid neuropathy, inhibition of the production of Aß is currently being pursued as the most promising disease modifying mechanism, and there is a need in the art for agents which achieve such inhibition.

Brief Description of the invention This invention is based on the finding that certain esters and thioesters have the property of reducing Ap production by cells. Whilst the invention is not dependent on any particular theory of the mechanism by which such inhibition is achieved, it is presently believed that the compounds are inhibitors of the putative p-secretase and/or y-secretase enzymes, or of enzymes involved in the pathway leading to production of the putative p-secretase and/or y-secretase enzymes.

Our earlier international patent application WO 98/11063 discloses the use of the same class of esters and thioesters as inhibitors of the proliferation of rapidly dividing cells, and thus as agents for the treatment, inter alia, of cancer. However, the present utility as inhibitors of Aß production is unrelated to and not predictable from the teaching of that application.

A few patent publications (WO 92/09563, US 5183900, US 5270326, EP-A- 0489577, EP-A-0489579, WO 93/09097, WO 93/24449, WO 94/25434, WO 94/25435, WO 95/04033, WO 95/19965, and WO 95/22966) include within their generic disclosure carboxylate ester compounds having matrix metalloproteinase inhibitory activity. In accordance with the present invention, such compounds are now recognised to have activity as inhibitors of AR production, but that activity is not suggested by, or predictable from, those publications.

WO 95/04033 discloses N-hydroxy-N'- (1- (S) -methoxycarbonyt-2, 2- dimethylpropyl) -2- (R) - (4-chlorophenylpropyl) succinamide as an intermediate for the preparation of the corresponding methylamide MMP inhibitor. In addition, Int. J.

Pept. Protein Res. (1996), 48 (2), 148-155 discloses the compound Ph-CH2CH (CO-Ile-OtBu) CH2CONHOH as an intermediate in the preparation of compounds which are inhibitors of neurotensin-degrading enzymes.

Detailed Description of the Invention In its broadest aspect, the present invention provides a method for treatment of mammals suffering diseases responsive to inhibition of Ap production, comprising administering to the mammal an amount of a compound of general formula (I) or a pharmaceutically acceptable salt hydrate or solvate thereof sufficient to inhibit Ap production : wherein R is hydrogen or (C,-C6)alkyl; R, is hydrogen ; (C,-C6)alkyl; (C2-C6)alkenyl; phenyl or substituted phenyl ; phenyl (C, -C6) alkyl or substituted phenyl(C,-C6)alkyl; phenyl(C2-C6)alkenyl or substituted phenyl(C2-C6)alkenyl heterocyclyl or substituted heterocyclyl ; heterocyclyi(C1 -C6)alkyl or substituted heterocyclyl (C, -C6) alkyl ; a group BSOnA- wherein n is 0,1 or 2 and B is hydrogen or a (C,-C6) alkyl, phenyl, substituted phenyl, heterocyclyl substituted heterocyclyl, (C,-C6)acyi, phenacyl or substituted phenacyl group, and A represents (C,-C6)alkylene; hydroxy or (C,-C6)alkoxy; amino, protected amino, acylamino, (C,-C6)alkylamino or di-(C,- C6)alkylamino; mercapto or (C,-C6)aikylthio; amino(C,-C6)alkyl, (C,-C6)alkylamino(C,-C6)alkyl, di(C,-C6)alkylamino(C,- C6)alkyl, hydroxy(C,-C6)alkyl, mercapto(C,-C6)aikyl or carboxy(C,-C6) alkyl wherein the amino-, hydroxy-, mercapto- or carboxyl-group are optionally protected or the carboxyl- group amidated ; lower alkyl substituted by carbamoyl, mono(lower alkyl)carbamoyl, di(lower alkyl) carbamoyl, di (lower alkyl) amino, or carboxy-lower alkanoylamino; or a cycloalkyl, cycloalkenyl or non-aromatic heterocydic ring containing up to 3 heteroatoms, any of which may be (i) substituted by one or more substituents selected from C1-C6 alkyl, C2-C6 alkenyl, halo, cyano (-CN), - CO2H, -CO2R, -CONH2, -CONHR, -CON(R)2, -OH, -OR, oxo-, -SH, -SR, - NHCOR, and -NHCO2R wherein R is C1-C6 alkyl or benzyl and/or (ii) fused to a cycloalkyl or heterocyclic ring ; R2 is a C1-C12 alkyl, C2-Cl2 alkenyl, C2-C,2 alkynyl, phenyl(C,-C6 alkyl)-, heteroaryl(C,-C6 alkyl)-, phenyl(C2-C6 alkenyl)-, heteroaryi(C2-C6 alkenyl)-, phenyl(C2-C6 alkynyl)-, <BR> <BR> <BR> <BR> heteroaryl (C2-C6 alkynyl) -, <BR> <BR> <BR> <BR> <BR> <BR> cycloalkyl(C,-C6 alkyl)-, <BR> <BR> <BR> <BR> <BR> <BR> cycloalkyl (C2-C6 alkenyl) -, cycloalkyl(C2-C6 alkynyl)-, cycloaikenyl(C1-C6 alkyl)-, cycloalkenyl(C2-C6 alkenyl)-, cycloalkenyl(C2-C6 alkynyl)-, phenyl(C,-C6 alkyl)O(C1-C6 alkyl)-, or heteroaryl(C,-C6 alkyl)O(C1-C6 alkyl)- group, any one of which may be optionally substituted by C, -C6 alkyl, C1-C6 alkoxy, halo, cyano (-CN), phenyl or heteroaryl, or phenyl or heteroaryl substituted by C,-C6 alkyl, <BR> Ci-Cgaikoxy, halo, or cyano (-CN) ; R3 is the characterising group of a natural or non-natural a amino acid in which any functional groups may be protected ; and R4 is an ester or thioester group, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

In another broad aspect of the invention, there is provided the use of a compound of formula (I) as defined in the immediately preceding paragraph, in the preparation of a pharmaceutical composition for the treatment of mammals suffering diseases responsive to inhibition ofAp production.

In another particular aspect of the invention, the compound used is one of general formula (I) above wherein : R, R, and R4 are as defined above with reference to formula (I) R2 is C,-C,2 alkyl, C2-C,2 alkenyl, C2-C,2 alkynyl, <BR> <BR> biphenyl(C,-C6 alkyl)-, phenylheteroaryl(C,-C6 alkyl)-, heteroarylphenyl(C,-C6 <BR> alkyl)-, biphenyl(C2-C6 alkenyl)-, phenylheteroaryl(C2-C6 alkenyl)-, heteroarylphenyl (CZ-C6 alkenyl) -, <BR> <BR> phenyl (C2-C6 alkynyl) -, heteroaryl (C2-C6 alkynyl) -, biphenyl(C2-C6 alkynyl)-, phenylheteroaryl(C2-C6 alkynyl)-, heteroarylphenyl(C2-C6 alkynyl)-, phenyl(C,-C6 alkyl)O(C,-C6 alkyl)-, or heteroaryl(C,-C6 alkyl)O(C,-C6 alkyl)-, any one of which may be optionally substituted on a ring carbon atom by C,-C6 aikyl, C,-C6 alkoxy, halo, or cyano (-CN) ; and R3 is C, -C6 alkyl, optionally substituted benzyl, optionally substituted phenyl, optionally substituted heteroaryl ; or the characterising group of a natural a amino acid, in which any functional group may be protected, any amino group may be acylated and any carboxy ! group present may be amidated ; or a heterocyclic(C,-C6)alkyi group, optionally substituted in the heterocyclic ring ; and pharmaceutically acceptable salts, hydrates or solvates thereof.

As used herein the term" (C, -C6) alkyl" or"lower alkyl"means a straight or branched chain alkyl moiety having from 1 to 6 carbon atoms, including for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl and n-hexyl.

The term" (C2-C6) alkenyl" means a straight or branched chain alkenyl moiety having from 2 to 6 carbon atoms having at least one double bond of either E or Z stereochemistry where applicable. This term would include, for example, vinyl, allyl, 1- and 2-butenyl and 2-methyl-2-propenyl.

The term "C2-C6 alkynyl" refers to straight chain or branched chain hydrocarbon groups having from two to six carbon atoms and having in addition one triple bond.

This term would include for example, ethynyl,1-propynyl,1- and 2-butynyl, 2- methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4- hexynyl and 5-hexynyl.

The term"cycloalkyl"means a saturated alicyclic moiety having from 3-8 carbon atoms and includes, for example, cyclohexyl, cyclooctyl, cycloheptyl, cyclopentyl, cyclobutyl and cyclopropyl.

The term"cycloalkenyl"means an unsaturated alicyclic moiety having from 4-8 carbon atoms and inclues, for example, cyclohexenyl, cyclooctenyl, cycloheptenyl, cyclopentenyl, and cyclobutenyl. In the case of cycloalkenyl rings of from 5-8 carbon atoms, the ring may contain more than one double bond.

The term"aryl"means an unsaturated aromatic carbocyclic group which is monocyclic (eg phenyl) or polycyclic (eg naphthyl).

The unqualified term"heterocyclyl"or"heterocyclic"means (i) a 5-7 membered heterocyclic ring containing one or more heteroatoms selected from S, N and O, and optionally fused to a benzene ring, including for example, pyrrolyl, furyl, thienyl, piperidinyl, imidazolyl, oxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, <BR> pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, benzimidazotyl, maleimido, succinimido, phthalimido, 1, 2-dimethyl-3, 5-dioxo-1, 2, 4-triazolidin-4-yl, 3,4,4-trimethyl-2,5-d ioxo- 1 -imidazol id inyl, 2-methyl-3,5-d ioxo- 1,2,4-oxad iazol-4-yl, <BR> 3-methyl-2, 4, 5-trioxo-1-imidazolidinyl, 2, 5-dioxo-3-phenyl-1-imidazolidinyl, 2-oxo-1- pyrrolidinyl,2,5-dioxo-1-pyrrolidinyl or 2, 6-dioxopiperidinyl, or (ii) a naphththatimido (ie 1, 3-dihydro-1, 3-dioxo-2H-benz [f] isoindot-2-y !), 1, 3-dihydro-l- oxo-2H-benz [flisoindol-2-yl, 1, 3-dihydro-1, 3-dioxo-2H-pyrrolo [3, 4-b] quinolin-2-yl, or 2, 3-dihydro-1, 3-dioxo-1H-benz [d, e] isoquinolin-2-yl group.

The term"heteroaryl"means a 5-7 membered substituted or unsubstituted aromatic heterocycle containing one or more heteroatoms. Illustrative of such rings are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, pyrazolyl, isoxazolyl, isothiazolyi, trizolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl.

The term"ester"or"esterified carboxyl group"means a group RgO (C=O) - in which Rg is the group characterising the ester, notionally derived from the alcohol RgOH.

The term"thioester"means a group R, S (C=O) - or RgS(C=S)- or R90 (C=S) -in which Rg is the group characterising the thioester, notionally derived from the alcohol RgOH or the thioalcohol RgSH.

Unless otherwise specified in the context in which it occurs, the term"substituted" as applied to any moiety herein means substituted with up to four substituents, each of which independently may be (C,-C6)alkyl, (C,-C6)alkoxy, hydroxy, mercapto, (C1-C6)alkylthio, amino, halo (including fluoro, chloro, bromo and iodo), nitro, trifluoromethyl, -COOH, -CONH2, -CN, -COORA, -CONH RA or -CON H RARA wherein RA is a (C, -C6) alkyl group or the residue of a natural alpha-amino acid.

The term"side chain of a natural or non-natural alpha-amino acid"means the group R'in a natural or non-natural amino acid of formula NH2-CH (R') -COOH.

Examples of side chains of natural alpha amino acids include those of alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, histidine, 5- hydroxyiysine, 4-hydroxyproline, isoleucine, leucine, Iysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, a- aminoadipic acid, a-amino-n-butyric acid, 3, 4-dihydroxyphenylalanine, homoserine, a-methylserine, ornithine, pipecolic acid, and thyroxine.

Natural alpha-amino acids which contain functional substituents, for example amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, or indolyl groups in their characteristic side chains include arginine, lysine, glutamic acid, aspartic acid, tryptophan, histidine, serine, threonine, tyrosine, and cysteine. When R3 in the compounds of the invention is one of those side chains, the functional substituent may optionally be protected.

The term"protected"when used in relation to a functional substituent in a side chain of a natural alpha-amino acid means a derivative of such a substituent which is substantially non-functional. For example, carboxyl groups may be esterified (for example as a C1-C6 alkyl ester), amino groups may be converted to amides (for example as a NHCOC1-C6 alkyl amide) or carbamates (for example as an NHC(=O)OC1-C6 alkyl or NHC (=O) OCH2Ph carbamate), hydroxyl groups may be converted to ethers (for example an OC,-C6 alkyl or a O(C,-C6 alkyl)phenyl ether) or esters (for example a OC(=O)C,-C6 alkyl ester) and thiol groups may be converted to thioethers (for example a tert-butyl or benzyl thioether) or thioesters (for example a SC(=O)C1-C6 alkyl thioester).

Examples of side chains of non-natural alpha amino acids include those referred to below in the discussion of suitable R3 groups for use in compounds of the present invention.

Salts of the compounds used in the invention include physiologically acceptable acid addition salts for example hydrochlorides, hydrobromides, sulphates, methane sulphonates, p-toluenesulphonates, phosphates, acetates, citrates, succinates, lactates, tartrates, fumarates and maleates. Salts may also be formed with bases, for example sodium, potassium, magnesium, and calcium salts.

There are several chiral centres in the compounds used according to the invention because of the presence of asymmetric carbon atoms. The presence of several asymmetric carbon atoms gives rise to a number of diastereomers with R or S stereochemistry at each chiral centre. For example, in the compounds used in the invention, the C atom carrying the hydroxamic acid and R, groups may be in the R or S configuration, the C atom carrying the R2 group may be predominantly in the R configuration, and the C atom carrying the R3 and R4 groups may be in either the R or S configuration, with the predominantly S configuration presently preferred.

As mentioned above, compounds of formula (I) above, and those of formula (I) excluded by the provisos in the definition of formula (I) above, are useful in human or veterinary medicine since they inhibit Ap production. They are therefore useful for the treatment AD and cerebral amyloid angiopathy in particular, as well as senile dementia of Alzheimer's type, neurodegenerative disorder associated with Down's syndrome, cerebral amyloid angiopathy-associated stroke, and hereditary cerebral hemorrhage with amyloidosis.

The compounds with which the invention is concerned may be prepared for administration by any route consistent with their pharmacokinetic properties and the requirement that they must contact the cells responsible for the disease- creating Ap production.

Orally administrable compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions. Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine ; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica ; disintegrants for example potato starch, or acceptab ! e wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, mulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats ; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia ; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol ; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.

The active ingredient may atso be administered parenterally, including injection into the cerebrao-spinal fluid, in a sterile medium. Depending on the vehicle and concentration used, the drug can either be suspended or dissolve in the vehicle.

Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.

Clinically safe and effective dosages for the compounds with which the invention is concerned will be determined by clinical trials, as is required by the regulatory authorities in the art. It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

In the compounds used in the invention, examples of substituents R, to R4 are given below : The groupR R, may be, for example, hydrogen, methyl, ethyl, n-propyl, n-butyl, isobutyl, hydroxy, methoxy, allyl, phenylpropyl, phenylprop-2-enyl, thienylsulphanylmethyl, thienylsulphinylmethyl, or thienylsulphonylmethyl ; or C,-C4 alkyl,eg methyl, ethyl n-propyl or n-butyl, substituted by a phthalimido, 1, 2-dimethyl-3, 5-dioxo-1, 2, 4-triazolidin-4-yl, 3-methyl-2, 5-dioxo-1- <BR> imidazolidinyl, 3, 4, 4-trimethyl-2, 5-dioxo-1-imidazolidinyl, 2-methyl-3, 5-dioxo- <BR> 1, 2, 4-oxadiazol-4-yl, 3-methyl-2, 4, 5-trioxo-1-imidazolidinyl, 2, 5-dioxo-3- phenyl-1-imidazolidinyl, 2-oxo-1-pyrrolidinyl, 2, 5-dioxo-1-pyrrolidinyl or 2, 6- dioxopiperidinyl, 5, 5-dimethyl-2, 4-dioxo-3-oxazolidinyl, hexahydro-1, 3- dioxopyrazolo [1, 2, a] [1, 2, 4] -triazol-2-yl, or a naphththalimido (ie 1, 3-dihydro- 1, 3-dioxo-2H-benz [f] isoindol-2-yl), 1, 3-dihydro-1-oxo-2H-benz [f] isoindol-2-yl, 1, 3-dihydro-l, 3-dioxo-2H-pyrroto [3, 4-b] quinotin-2-yf, or 2, 3-dihydro-1, 3- dioxo-1 H-benz [d, e] isoquinolin-2-yl group ; or cyclohexyl, cyclooctyl, cycloheptyl, cyclopentyl, cyclobutyl, cyclopropyl, tetrahydropyranyl or morpholinyl.

Presently preferred R, groups include n-propyl, allyl, hydroxy, methoxy and thienylsulfanyl-methyl.

The aroup B2 R2 may for example be C,-C,2 alkyl, C3-C6 alkenyl or C3-C6 alkynyl; phenyl (Cl-C6 alkyl) -, phenyl (C3-C6alkenyl) - or phenyl(C3-C6 alkynyl)- optionally substituted in the phenyl ring ; heteroaryl (C, -C6 alkyl) -, heteroaryl (C3-C6 alkenyl) - or heteroaryl (C3-C6 alkynyl) - optionally substituted in the heteroaryl ring ; 4-phenylphenyl(C,-C6 alkyl)-, 4-phenylphenyl(C3-C6 alkenyl)-, 4- phenylphenyl(C3-C6 alkynyl)-, 4-heteroarylphenyl(C1-C6 alkyl)-, 4- heteroaryiphenyi(C3-C6 alkenyl)-, 4-heteroarylphenyl(C3-C6 alkynyl)-, optionally substituted in the terminal phenyl or heteroaryl ring ; phenoxy(C,-C6 alkyl)- or heteroaryloxy(C,-C6 alkyl)- optionally substituted in the phenyl or heteroaryl ring ; Specific examples of such groups include methyl, ethyl, n- and iso-propyl, n-, iso- and tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-nonyl, n-decyl, prop-2-yn-1-yl, 3- phenylprop-2-yn-1-yl, 3-(2-chlorophenyl)prop-2-yn-1-yl, phenylpropyl, 4- chlorophenylpropyl, 4-methylphenylpropyl, 4-methoxyphenylpropyl, phenoxybutyl, <BR> 3- (4-pyridylphenyl) propyl-, 3- (4- (4-pyridyl) phenyl) prop-2-yn-1-yl, 3- (4- phenylphenyl) propyl-, 3- (4-phenyl) phenyl) prop-2-yn-1-yl and 3- [ (4- chlorophenyl) phenyl] propyl-.

Presently preferred R2 groups include isobutyl, n-hexyl, 3- (2-chlorophenyl) prop-2- yn-1-yl.

The group B3 R3 may for example be C,-C6 alkyl, phenyl, 2,- 3-, or 4-hydroxyphenyl, 2, - 3-, or 4-methoxyphenyl, 2, - 3-, or 4-pyridylmethyl, benzyl) 2, - 3-, or 4- hydroxybenzyl, 2, - 3-, or 4-benzyloxybenzyl, 2, - 3-, or 4-C,-C6 alkoxybenzyl, or benzyloxy(C,-C6alkyl)- group; or the characterising group of a natural a amino acid, in which any functional group may be protected, any amino group may be acylated and any carboxyl group present may be amidated ; or a group -[Alk]nR6 where Alk is a (C,-C6)alkyl or (C2-C6)alkenyl group optionally interrupted by one or more -O-, or -S- atoms or -N (R7) - groups [where R7 is a hydrogen atom or a (C,-C6)alkyl group], n is 0 or 1, and R6 is an optionally substituted cycloalkyl or cycloalkenyl group ; or a benzyl group substituted in the phenyl ring by a group of formula - OCH2COR8 where R8 is hydroxyl, amino, (C,-C6)alkoxy, phenyl(C,- C6)alkoxy, (C1-C6)alkylamino, di((C,-C6)alkyl)amino, phenyl(C,- C6) alkylamino, the residue of an amino acid or acid halide, ester or amide derivative thereof, said residue being linked via an amide bond, said amino acid being selected from glycine, a or p alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, histidine, arginine, glutamic acid, and aspartic acid ; or a heterocyclic(C,-C6)alkyl group, either being unsubstituted or mono- or di- substituted in the heterocyclic ring with halo, nitro, carboxy, (C,-C6)alkoxy, cyano, (C,-C6)alkanoyl, trifluoromethyl (C,-C6)alkyl, hydroxy, formyl, amino, (C,-C6)alkylamino, di-(C,-C6)alkylamino, mercapto, (C,-C6)alkylthio, hydroxy(C,-C6)alkyl, mercapto(C,-C6)alkyl or (C, -C6) alkylphenylmethyl.

Examples of particular R3 groups include benzyl, phenyl, cyclohexylmethyl, pyridin- 3-ylmethyl, tert-butoxymethyl, iso-butyl and sec-butyl.

Presently preferred R3 groups include phenyl, benzyl, tert-butoxymethyl and iso- butyl.

The group R1 Examples of particular ester and thioester groups R4 groups include those of formula -(C=O)ORg, -(C=O)SRg, -(C=S)SRg, and -(C=S)ORg wherein Rg is (C,- <BR> <BR> <BR> <BR> C6)alkyl, (C2-C6)alkenyl, cycloalkyl, cycloalkyl(C,-C6)alkyl-, phenyl, heterocyclyl, <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> phenyl(C,-C6)alkyl-, heterocyclyl(C,-C6)alkyl-, (C,-C6)alkoxy(C,-C6)alkyl-, (C,- C6)alkoxy(C,-C6)alkoxy(C,-C6)alkyl-, any of which may be substituted on a ring or non-ring carbon atom or on a ring heteroatom, if present. Examples of such Rg groups include methyl, ethyl, n-propyl, n-butyl, 1-ethyl-prop-1-yl, 1-methyl-prop-1-yl, 1-methyl-but-1-yl, cyclopentyl, cyclohexyl, allyl, phenyl, benzyl, 2-, 3- and 4- pyridyimethyl, N-methylpiperidin-4-yl, 1-methylcyclopent-1yl, adamantyl, tetrahydrofuran-3-yl and methoxyethyl. Presently preferred are compounds of formula (I) wherein R4 is a carboxylate ester of formula -(C=O)ORg, wherein Rg is benzyl or cyclopentyl.

ThegroupR Presently preferred R groups are hydrogen and methyl.

Compounds used according to the present invention may be prepared as described in WO 98/11063. Specific compounds for use in accordance with the invention include those disclosed in WO 98/11063, and the fo ! ! owing : 2 (R or S)-[2R-(S-Hydroxy-hydroxycarbamoyl-methyl)-4-methyl- pentanoylamine]-2-phenyl-ethanoic acid cyclopentyl ester, 2 (R or S) - (3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino) -2- phenylethanoic acid isopropyl ester, 2 (R or S) - [2R- (S-Hydroxycarbamoyl-methoxy-methyl) -4-methyl- pentanoylamino] -2-phenylethanoic acid cyclopentyl ester, 2 (R or S) - (3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino) -2- (4- methoxyphenyl) ethanoic acid cyclopentyl ester, 2 (R or S) - (3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino) -2- (thien-2- yl)ethanoic acid cyclopentyl ester, 2 (R or S) - (3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino) -2- (thien-3- yl) ethanoic acid cyclopentyl ester, and pharmaceutically or veterinarily acceptable salts, hydrates or solvates thereof.

The 2-S diastereomers of the above compounds are preferred.

The following Example illustrates the ability of a representative compound to reduce production of Ap. In the Example the following abbreviations are used : App-amyioid peptide AEBSF 4- (2-Aminoethyl) benzenesulphonyl fluoride APP Amyloid precursor protein CHAPS (3- [ (3'-Cholamidopropyl) dimethylammonio] -1-propanesulphonate CHO Chinese hamster ovary cells COS African green monkey kidney epithelial carcinoma cells DEAE Diethylaminoethyl DMEM Dulbecco's modified Eagle medium DMSO Dimethylsulphoxide EDTA Ethylenediamine tetra-acetic acid EGTA Ethylene glycol-bis [O-aminothyl ether] -N, N, N', N'-tetra-acetic acid ELISA Enzyme-linked immunosorbent assay FCS Foetal calf serum LB Lysis buffer Met Methionine PBS Phosphate buffered saline PCR Polymerase chain reaction PVDF Polyvinylidene difluoride sVCAM-1 Soluble vascular cell adhesion molecule-1 YT Yeast-tryptone Example 1 The ability of a representative compound from the clans disclosed in WO 98/11063 to reduce Ap production by APP transfected COS-1 cells was investigated. The test compound was 2S- (3S-hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino) -3- phenylpropionic acid cydopenty ! ester (Example 20 of WO 98/11063) i) Preparation of pGW1HGAPP695 The APP695 gene [kindly provided in the prokaryote vector pUC119 by Professor Benno Mueller-Hill (Universtat Koln)] and its flanking segments were sequenced using the fmol0 DNA Cycle Sequencing System (Promega, Southampton, UK).

The APP gene was PCR-amplified and subcloned into the mammalian expression vector pGW1 HG [Wells et a/. (1996) Glia. 18 : 332-340] using standard molecular cloning techniques. Subcloning Efficiency DH5a cells (GibcoBRL, Paisley, UK) were transformed with the ligation products, according to manufacturer's instructions, and plated out on YT agar plates containing 100 ,ug/ml carbenicillin.

Next day any colonies that had grown up were selected and used to inoculate 5 ml of 2xYT broth. Cultures were left to grow at 37°C for 6 hours. The plasmid DNA was then purified with a Wizard@ Miniprep Purification Kit (Promega).

Approximately 20 ui of miniprep DNA was digested with Hindlil. Positive clones producing bands of 2, 850 bp and 7, 450 bp were subjected to further restriction enzyme (Smal, Xhol and Saci) digests and also PCR tests (two sets of primers were used) ; clones producing the correct bands upon gel visualisation were then selected for further validation by sequencing. ii) Transfection and radiolabelling of COS-1 cells COS-1 cells (European Collection of Cell Cultures, Salisbury, Wilts, UK) were cultured in Dulbecco's Modified Eagle Medium (DMEM ; GibcoBRL) containing 10% Foetal Calf Serum (FCS ; GibcoBRL), 200 mM Glutamine (Sigma, Poole, Dorset, UK) and 100 ug/ml penicillin/streptomycin mixture (Sigma) (DMEM/1 0% FCS). The day before transfection the cells were trypsinised, counted, and plated into T-75 flasks (Falcon ; Becton Dickinson, Oxford, UK) at approximately 2. 4 x 106 cells per flask.

Cells were transiently transfected by the DEAE-Dextran method [Vaheri & Pagano (1965) Virology. 27 : 434-436]. Six micrograms of DNA (stock concentration of 1 mg/ml) was pipette into a 15 ml Falcon tube containing 64 ut Tris-EDTA (T. E.), followed by 6 i of 100 mM chloroquine (Sigma) and 24 pl of 100 mg/ml DEAE- Dextran (Mr 500, 000 ; Pharmacia Biotech, Uppsala, Sweden). The mixture was made up to 6 ml with serum-free media and added to the cells for 2 hours at 37°C.

Following removal of transfection media the cells were incubated with 10% DMSO in phosphate-buffered saline (PBS) for 2 min. Subsequently, the cells were washed twice with PBS and cultured in normal growth media for approximately 48 hours.

Forty-eight hours post-transfection the culture medium was removed and the cells were washed once with PBS (calcium and magnesium free ; Sigma). The cells were then incubated with 5 ml of methionine-free media (GibcoBRL) for 30 min to exhaust intracellular supplies of methionine. The cells were simultaneously contacted with the test compound and radiolabelled by incubating for 24 hours in 5 ml of labelling medium (methionine-free DMEM containing 10% FCS, 5% L-Glu, 5% penicillin/streptomycin mix (Sigma) and 100 pCi/ml 35S-Met (activity>1000 Ci/mmol; Amersham Pharmacia Biotech, Bucks, UK) ) containing test compound at a concentration of 10 IJM. Control cells were radiolabelled in the absence of test compound. Cell homogenates were collecte by scraping the adherent cells off the flasks into 1. 5 ml lysis buffer [LB ; 1 % CHAPS, 50 mM Tris-HCI, pH 7. 6, 150 mM NaCI, 10 mM EDTA, 5 mM EGTA containing protease inhibitors : Leupeptin (10 Jg/ml), Pepstatin (10 pg/ml), AEBSF (40 ,ug/ml) and Aprotinin (20 ug/ml)].

Homogenates were spun at 12, 000gmax for 5 min to precipitate cell debris. Prior to immunoprecipitation, conditioned media was processed by centrifuging at 100, 000gmax in a Beckman T-100 Ultramicrocentrifuge to pellet any remaining membrane fraction. iii) Immunoprecipitation and quantification of products Cell homogenates and conditioned media were first pre-cleared for 2 hours at room temperature (RT) with a 1 : 1 slurry of Protein G-Sepharose (Pharmacia Biotech) and dilution buffer [0.1% Triton X-100 (Sigma), 0. 1 % bovine albumin (Sigma) in TNE solution (50 mM Tris, pH 7. 6, 500 mM NaCI, 2 mM EDTA plus protease inhibitors]. The slurry was added at 10 ut per 200 pl of sample. Samples were pulse microcentrifuged to sediment the beads and any non-specific material bound to them. Ap specific monoclonal antibodies (mAbs) 6E10 and 4G8 (Senetek, Maryland Heights, MO, USA ; mAb 6E10 binds the first 17 residues of the ß-amyloid region ; mAb 4G8 recognises residues 17-28) were added at 2 ug/m) and tubes were placed on a rotating wheel for 4 hours at RT ; followed by incubation with the Sepharose beads for a further 2 hours at 4°C. The immunoprecipitates were washed four times, with 1 ml of the following wash solutions : dilution buffer (x2), TNE solution with 500 mM NaCI and TNE solution with 150 mM NaCI. The antigen was dissociated from the immune complexes by adding 40 u ! of SDS/sampfe buffer and heating at 95°C for 5 min. Samples were loaded onto either 16% Tris-Glycine gels (Novex, San Diego, CA, USA) or 10-20% Tris-Tricine gels (Novex) and run at 150 V for 90 min [Laemmli, (1970) Nature. 227:680-685]. Labelled proteins were transferred to 0.2 ,um poiyvinylidene difluoride (PVDF) membranes (Novex) [Towbin et a/. (1979) PNAS USA. 76 : 4350-4354] at 150 V for 45 min. Blots were left in an exposure cassette for 72 hours to transfer signal to a phosphor screen, which was scanned with a Phosphorlmager SF (Molecular Dynamics, Sunnyvale, CA, USA). Data was analysed with ImageQuant (Molecular Dynamics) software.

The above procedures established that test compound inhibited Aß formation by the COS-1 cells. The test compound was found to cause >95% inhibition ofAp formation at 101lu.

Example 2 The ability of representative compounds from the class disclosed in WO 98/11063 to reduce Ap production by APP transfected CHO cells was investigated. The test compounds were : <BR> <BR> 2S-(3S-Hyd roxyca rbamoyi-2 R-isobutyl-hex-5-enoylamino)-3-phenylpropionic acid cyclopentyl ester (Example 20 of WO 98/11063) 2S-[2R-(1 S-HydroxyCarbamoyl-ethyl)-4-methyl-pentanoylamino]-3-phenyl- propionic acid isopropyl ester (Example 9 of WO 98/11063) 2S- (2R-Hydroxycarbamoylmethyl-octanoylamino) -3-phenyl-propionic acid isopropyl ester. (Example 10 of WO 98/11063) 2R-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phen ylpropionic acid isopropyl ester (Example 23 of WO 98/11063) 2S- [2R- (S-Hydroxy-hydroxycarbamoyl-methyi) -4-methyl-pentanoylamino] -3-phenyl- propionic acid cyclopentyl ester (Example 11 of WO 98/11063) 2S- {2R- [1 S-Hydroxycarbamoyl-2- (thiophen-2-yisulphanyl) -ethyl] -4-methyl- pentanoylamino}-3-phenyl-propionic acid isopropyl ester (Example 17 of WO 98/11063) 2S- (3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino) -3S-methyl-pentanoic acid cyclopentyl ester (Example 12 of WO 98/11063) 2S- (3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino) -4-methyl-pentanoic acid cyclopentyl ester (Example 27 of WO 98/11063) 2S- (3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino) -3-phenylpropionic acid cyclohexyl ester (Example 32 of WO 98/11063) 2S-[2R-(1 S-Cyclopentyl-hydroxycarbamoyl-methyl)-4-methyl-pentanoyiami no]-3- phenyl-propionic acid cyclopentyl ester (Example 37 of WO 98/11063) 3-tert-Butoxy-2S-(3S-hydroxyCarbamoyl-2R-isobutyi-hex-5-enoy lamino)-propionic acid cyclopentyl ester (Example 40 of WO 98/11063) 2S- (3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino) -2-phenylethanoic acid cyclopentyl ester (Example 41 of WO 98/11063) 2- [2R- (S-Hydroxy-hydroxycarbamoyl-methyl) -4-methyl-pentanoylamine] -2-phenyl- ethanoic acid cyclopentyl ester Prepared using procedures similar to those described similar to those described in Example 8 of WO 98/11063, using phenylglycine cyclopentyl ester. Only diastereoisomer B was tested for its ability to inhibit A) 3 formation.

Diastereoisomer A 'H-NMR ; 8 (MeOD), 7. 4-7. 29 (5H, m), 5. 43 (lu, s), 5.2-5.14 (1H, m), 4.02 (1H, d, J=6. 9Hz), 2. 94-2. 85 (1 H, m), 1. 91-1. 34 (10H, bm), 1. 25-1. 14 (1 H, m) and 0. 86 (6H, dd, J=6. 5, 11. 5Hz).

'3C-NMR; 6 (MeOD), 175. 6, 171. 8, 171. 4, 137. 8, 129. 8, 129. 4, 128. 6, 80. 0, 73. 2, 58. 5, 49. 2, 39. 1, 33. 3, 33. 3, 26. 8, 24. 5, 24. 4, 23. 7 and 22. 1.

Diastereoisomer B 'H-NMR ; 8 (MeOD), 7. 33-7. 19 (5H, m), 5.3 (1H, s), 5.11-5.06 (1H, m), 3.81 (1H, d, J=7.3Hz), 2.83-2.74 (1H, m), 1.83-1.45 (1 OH, bm), 1. 12-1. 03 (1 H, m) and 0. 88-0. 81 (6H, dd, J=6.4, 12.3Hz). '3C-NMR; 6 (MeOD),175.8, 171.8, 171.5, 137.3, 129.8, 129.5, 128.8, 79.9, 73.3, 58.7, 48.9, 39.2, 33.3, 33.3, 26.7, 24.5, 24. 5, 24. 0 and 22. 2.

2- [2R- (S-Hydroxycarbamoyl-methoxy-methyl) -4-methyl-pentanoylamino] -3- phenylethanoic acid cyclopentyl ester.

Prepared using methods similar to those described similar to those described in Example 3 of WO 98/11063, using phenylglycine cyclopentyl ester.

Diastereoisomers A and B were each tested for their ability to inhibit Ap formation.

Diastereoisomer A 'H-NMR; 6 (MeOD), 8. 83 (1 H, d, J=6. 6Hz), 7. 48-7. 29 (5H, m), 5. 44-5. 42 (1 H, m), 5.20-5.16 (1H, m), 3.53 (1H, d, J=9.7Hz), 3. 17 (3H, s), 2.89-2.79 (1H, m), 1. 90-1. 54 (10H, bm), 1. 06-0. 99 (1 H, m), 0. 95 (3H, d, J=6. 5Hz) and 0. 90 (3H, d, J=6. 4Hz).'3C-NMR ; 8 (MeOD), 175.3, 171.6, 169.4, 137.5, 129.7, 129.4, 128.7, 83.1, 79.9, 58.7, 58.1, 48.5, 38.4, 33.4, 33.3, 26.7, 24.6, 24.5, 24.3 and 21. 8.

Diastereoisomer B 1H-NMR; 6 (MeOD), 7. 39-7. 30 (5H, m), 5.45 (1H, s), 5. 21-5. 15 (1H, m), 3.59 (1H, d, J=9. 4Hz), 3. 29 (3H, s), 2. 89-2. 79 (1 H, m), 1. 93-1. 49 (9H, bm), 1.42-1.21 (1H, m), 1.01 (1H, ddd, J=3. 7, 9. 9, 13. 3Hz), 0. 83 (3H, d, J=6.5Hz) and 0. 79 (3H, d, J=6. 6Hz).'3C-NMR ; 8 (MeOD), 175.1, 171.5, 169.5, 137.9, 129.7, 129.4, 128.7, 83.0, 79.8, 58.5, 58.3, 48.6, 38.5, 33.3, 27.8, 24.5, 24.4, 24.1 and 21. 7. i) Expression studies in CHO cells The human APP695 and sVCAM-1 genes were subcloned into the mammalian expression vector pGW1 HG and DNA was prepared for transfection studies in the Chinese hamster ovary (CHO) cell line (European Collection of Cell Cultures). Cells were stably transfected by electroporation [Neumann et al. (1982) EMBO J. 1 : 841- 845]. Briefly CHO cells grown in confluent monolayer cultures in DMEM/10% FCS were trypsinised (0. 25% trypsin/0. 02% EDTA ; Sigma), counted and resuspended in PBS at 107 cells/ml. To allow recombination of the plasmid DNA with the host CHO cell DNA the vector was linearised with Notl which cuts within the ampicillin resistance gene. Forty micrograms of DNA was pipette into 0. 4 cm, gap 50, sterile cuvettes (Gene Pulser Cuvettes, Bio-Rad, Hemel Hempstead, Herts, UK) followed by 800 pu ouf CHO cells resuspended in PBS (8 x 106 cells), mixed and left on ice for 10 minutes. The cuvette was then placed in the electroporator (Gene Pulser, Bio- Rad) set at 0. 8 kV, 25 jFD and pulsed for 0. 5-0. 6 ms. The cell suspension was left on ice for a further 20 minutes and then 50 ! of cell suspension was pipette into 50 ml of DMEM/10% FCS and mixed by gently inverting the tube. This diluted cell suspension was then aliquote into 96-well plates (Falcon) by pipetting 100 ti per well. The cells were replace in the 37°C incubator and left for 24 hours to recover from the electroporation. After this time the cultures were left to grow in xanthine- guanine phosphoribosyltransferase (XGPRT, gpt) selection media [Mulligan & Berg (1981) PNAS USA. 78 : 2072-2076]. Colonies of cells were screened for expression of the relevant gene (APP or sVCAM-1) by taking conditioned media from the respective wells and immunoblotting with mAb 6E10 or anti-soluble vascular cell adhesion molecule-1 (sVCAM-1) IgG (R & D Systems, Abingdon, UK), respectively.

Colonies of cells expressing the appropriate gene product were sub-cloned a further two times and then grown up. ii) Quantification of Aß in a CHO cell line stably transfected with APP695 CHO cells (1 x 106) stably expressing APP695 grown in GPT selection media were plated into T-75 flasks (Falcon) and left overnight to adhere to the bottom of the flasks. The following morning compounds were prepared at 100 mM in DMSO and diluted at various concentrations (0. 1, 1 and 10 s1M) in DMEM/5% FCS. Cultures were briefly washed once with PBS (calcium and magnesium free ; Sigma) and 4 ml of DMEM/5% FCS (containing compounds or DMSO vehicle) was added to each flask and incubated at 37°C in 6% CO2 for 24 hours. Conditioned media was centrifuged to pellet any remaining membrane fraction and the levels of Ap secreted into the media was assessed using an ELISA specific for Aß (Quality Controlled Biochemicals, Hopkinton, MA, USA).

The above procedures established that the test compounds inhibited Ap formation by the CHO cells. The test compounds were each found to cause from 10% to 100% inhibition of Aß formation at 10 pM.

Example 3 Proliferation study To eliminate the possibility that the observed reduction in Ap formation was due to inhibition of cell proliferation or protein synthesis by the test compounds, their effects on cell proliferation and the synthesis of an independent protein (sVCAM-1), cloned into the same mammalian expression vector, was investigated : The test compound (Example 20 of WO 98/11063) used in the labelling experiments was tested for its affect on the growth and proliferation of untransfected COS-1 cells. Cells (10, 000) were plated into each well of a 24-well plate (Falcon) and allowed to adhere for 4 h. The cells were incubated in various concentrations of compound diluted into DMEM/10%FCS at 37 °C for 24,48 and 72 hour intervals, after which they were fixed with 2 % formaldehyde in PBS and stained with 1 % crystal violet dye. The dye was then extracted with glacial acetic acid and the colorimetric measurement was done using an Anthos 2001 plate reader (AnthosLab Instruments, Salzburg, Austria) linked to Biolise software.

The test compound did not have a significant anti-proliferative effect at a concentration of 10 jJvt over a 24 hour period, which represents the incubation period of the APP labelling and Ap ELISA experiments.

The effects of the test compounds (2- [2R- (S-hydroxy-hydroxycarbamoyl-methyl) -4- methyl-pentanoylamine] -2-phenyl-ethanoic acid cyclopentyl ester, (diastereomer B), 2- [2R- (S-hydroxycarbamoyl-methoxy-methyl) -4-methyl-pentanoylamino] -3- phenylethanoic acid cyclopentyl ester (both diastereomers), and Examples 11,17, 27,37, 40 and 41 of WO 98/11063) on protein synthesis in healthy CHO cells stably transfected with human sVCAM-1 was also investigated. The human sVCAM-1 gene was PCR-amplified from a human cDNA library and subcloned into the vector pGW1 HG (used to stably transfect CHO cells with APP695 in the APP proteolysis studies) using standard molecular cloning techniques. Cells (1 x 106) were plated into T-75 flasks (Falcon) and allowed to adhere for 4 h. The cells were incubated in various concentrations of compound diluted into DMEM/5%FCS at 37°C for 24 hour intervals, after which the levels of sVCAM-1 released into the media was assessed using an ELISA specific for human sVCAM-1 (R & D Systems).

None of the test compounds (which were found to reduce Ap production in the CHO cell line stably transfected with human APP695) significantly reduced the levels of sVCAM-1 produced in the CHO cell line stably transfected with the human sVCAM-1 gene.

Example 4 Blood brain barrier penetration Following administration of the test compound of Example 20 of WO 98/11063 at 30mg/kg i. p. to five male Lewis rats, cerebrospinal fluid samples were taken at 1 hour post dosing. LCMS analysis showed a mean concentration of 750ng/ml of the carboxylic acid metabolite resulting from hydrolysis of the ester. This is evidence that the parent ester is able to cross the blood brain barrier in appreciable amounts, since the carboxylic acid metabolite, being a negatively charged species at physiological pH, is unlikely to achieve penetration.