EP1105116A1

EP1105116A1 - Hydroxamic acid derivatives as inhibitors of beta-amyloid production

Info

Publication number: EP1105116A1
Application number: EP99938450A
Authority: EP
Inventors: Mandy British Biotech Pharmac. Ltd. JOHNSTONE; Andrew P. British Biotech Pharmac. Ltd. AYSCOUGH
Original assignee: British Biotech Pharmaceuticals Ltd
Current assignee: Vernalis R&D Ltd
Priority date: 1998-08-11
Filing date: 1999-08-10
Publication date: 2001-06-13
Also published as: JP2002522489A; GB9817348D0; AU5295899A; WO2000009119A1

Abstract

A method for treatment of mammals suffering diseases responsive to inhibition of β-amyloid peptide (Aβ) 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 Aβ production wherein R1, R2, R3 are as defined in the specification and R4 is an ester or thioester group.

Description

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 β-amyioid peptide (Aβ).

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 β-amyloid peptide (Aβ) [Roher et al. (1986) PNAS USA. 83:2662-2666]. Aβ 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. Cell Biol. 10: 373-403] with the shorter 695 amino acid form being predominantly expressed in neurons. Cleavage occurs at the N- and C-termini of Aβ by as yet uncharacterised enzymes, termed β- secretase and γ-secretase, respectively.

Aβ 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 Aβ is present in cerebrospinal fluid of healthy individuals [Haass et al. (1992) Nature. 359:322-325; Seubert er 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 Aβ 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 invariably develop AD neuropathology in their forties or fifties) or by missense mutations that increase the amyloidogenic cleavages of APP at either the β-secretase site (leading to excessive production of both Aβ_1j(0 and Aβι-₄₂) or the γ-secretase site (resulting in selective increased production of Aβ_1^l2).

APP is anchored to internal membranes e.g. eπdoplasmic 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 Aβ domain by a protease designated 'α-secretase'. This results in the release of a large soluble ectodomain fragment (sAPPα) which is non- amyloidogenic. The regulated cleavage of APP (i.e. as enhanced by phorbol ester activation) at the α-secretase site is believed to be carried out by a isintegrin and metalloprotease (ADAM), specifically ADAM-17, also known as tumour-necrosis factor-α (TNF-α) converting enzyme (TACE), since it is capable of shedding the ectodomain of TNF-α [Bauxbaum et al. (1998) J. Biol. Chem. 273: 27765-27767]. Recently it has been shown that both the constitutive and regulated α-secretase cleavage of APP can be performed by ADAM-10 [Lammich et al. (1999) J. Biol. Chem. 96:3922-3927]. It would therefore seem feasible that stimulation of α- secretase cleavage may be a valid thereapeutic option, thereby precluding Aβ formation. However, since in most cells only a minority of APP molecules undergo α-secretory cleavage, any increase in proteolysis at this site would still potentially leave many APP molecules which could be subject to β- and γ-secretase cleavage. The most likely therapeutic option therefore, to reduce Aβ formation, would be to inhibit the β- and/or γ-secretase activities.

Although the β-secretase activity remains elusive recent reports have shown an intriguing connection between γ-secretase activity and the presenilin (PS) proteins [Wolfe et al. (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 β-amyloid derivative Aβ ₂ [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 γ-secretase activity and suggests that PS-1 is either a unique diaspartyl cofactor for γ-secretase activity or indeed is itself γ-secretase, an autoactivated intramembranous aspartyl protease [Wolfe et al. (1999) Nature. 398:513-517].

Because of its involvement in such neurological disorders as AD and cerebral amyloid neuropathy, inhibition of the production of Aβ 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 Aβ 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 β-secretase and/or γ-secretase enzymes, or of enzymes involved in the pathway leading to production of the putative β-secretase and/or γ-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 Aβ 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 Aβ production, but that activity is not suggested by, or predictable from, those publications.

WO 95/04033 discloses N⁴-hydroxy-N¹-(1-(S)-methoxycarbonyl-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-lle-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 Aβ 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 Aβ production:

wherein

R is hydrogen or (C₁-C₆)alkyl;

R₁ is hydrogen;

(C_rC₆)alkyl; (C₂-C₆)alkenyl;

phenyl or substituted phenyl;

phenyl (C_rC₆)alkyl or substituted phenyl(C₁-C₆)alkyl;

phenyl (C₂-C₆)alkenyl or substituted phenyl(C₂-C₆)alkenyl

heterocyclyl or substituted heterocyclyl;

heterocyclyl(C C₆)alkyl or substituted heterocyclyl(C,-C₆)alkyl;

a group BSO_nA- wherein n is 0, 1 or 2 and B is hydrogen or a (C C₆) alkyl, phenyl, substituted phenyl, heterocyclyl substituted heterocyclyl, (C C₆)acyl, phenacyl or substituted phenacyl group, and A represents (C_rC₆)alkylene;

hydroxy or (C_rC₆)alkoxy;

amino, protected amino, acylamino, (C_rC₆)alkylamino or di-(C_r C₆)alkylamino;

mercapto or (C_rC₆)alkylthio;

amino(C₁-C₆)alkyl, (C₁-C₆)alkylamino(C₁-C₆)alkyl, di(C₁-C₆)alkylamino(C₁- C₆)alkyl, hydroxy(C C₆)alkyl, mercapto(C_rC₆)alkyl or carboxy(C₁-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)carbamoyi, di(lower alkyl)amino, or carboxy-lower alkanoylamino; or a cycloalkyi, cycloalkenyl or non-aromatic heterocyclic ring containing up to 3 heteroatoms, any of which may be (i) substituted by one or more substituents selected from C_rC₆ alkyl, C₂-C₆ alkenyl, halo, cyano ( -CN), - C0₂H, -CO₂R, -CONH₂, -CONHR, -CON(R)₂, -OH, -OR, oxo-, -SH, -SR, - NHCOR, and -NHCO₂R wherein R is alkyl or benzyl and/or (ii) fused to a cycloalkyi or heterocyclic ring;

is a 0,-0,₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, phenyl(C_rC₆ alkyl)-, alkyl)-, phenyl(C₂-C₆ alkenyl)-, heteroaryl(C₂-C₆ alkenyl)-, phenyl(C₂-C₆ alkynyl)-, heteroaryl(C₂-C₆ alkynyl)-, cycloalkyl(C_rC₆ alkyl)-, cycloalkyl(C₂-C₆ alkenyl)-, cycloalkyl(C₂-C₆ alkynyl)-, cycloalkenyl(C C₆ alkyl)-, cycloalkenyl(C₂-C₆ alkenyl)-, cycloalkenyl(C₂-C₆ alkynyl)-, pheny Cj-Cg alkyl)O(C_rC₆ alkyl)-, or heteroaryl(C C₆ alkyl)O(C_rC₆ alkyl)- group, any one of which may be optionally substituted by

C_rC₆ alkyl,

C,-C₆ alkoxy, halo, cyano (-CN), phenyl or heteroaryl, or phenyl or heteroaryl substituted by C_rC₆ alkyl, C C₆ alkoxy, halo, or cyano (-CN);

R₃ is the characterising group of a natural or non-natural α 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 of Aβ production.

In another particular aspect of the invention, the compound used is one of general formula (I) above wherein:

R, R₁ and R₄ are as defined above with reference to formula (I)

R₂ is C,-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,

biphenyl(C,-C₆ alkyl)-, phenylheteroaryl(C,-C₆ alkyl)-, heteroarylphenyl(C C₆ alkyl)-,

biphenyl(C₂-C₆ alkenyl)-, phenylheteroaryl(C₂-C₆ alkenyl)-, heteroarylphenyl(C₂-C₆ alkenyl)-,

phenyl(C₂-C₆ alkynyl)-, heteroaryl(C₂-C₆ alkynyl)-, biphenyl(C₂-C₆ alkynyl)-, phenylheteroaryl(C₂-C₆ alkynyl)-, heteroarylphenyl(C₂-C₆ alkynyl)-,

phenyl(C₁-C₆ alkyl)0(C C₆ alkyl)-, or heteroaryl(C_rC₆ alkyl)O(C_rC₆ alkyl)-,

any one of which may be optionally substituted on a ring carbon atom by C,-C₆ alkyl, C_rC₆ 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 α amino acid, in which any functional group may be protected, any amino group may be acylated and any carboxyi group present may be amidated; or

a heterocyclic(C C₆)alkyl group, optionally substituted in the heterocyclic ring;

and pharmaceutically acceptable salts, hydrates or solvates thereof.

As used herein the term "(C^C^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 "cycloalkyi" 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 includes, 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, thiazolyi, thiadiazolyi, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morphoiinyl, piperazinyl, indolyl, benzimidazolyl, maleimido, succinimido, phthalimido, 1 ,2-dimethyl-3,5-dioxo-1 ,2,4-triazolidin-4-yl, 3,4,4-trimethyl-2,5-dioxo-1-imidazolidinyl, 2-methyl-3,5-dioxo-1 ,2,4-oxadiazol-4-yl, 3-methyl-2,4,5-thoxo-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 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-1 ,3-dioxo-2H-pyrrolo[3,4-b]quinolin-2-yl, or 2,3-dihydro-1 ,3-dioxo-1 H-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, isothiazolyl, trizolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl.

The term "ester" or "esterified carboxyl group" means a group R_gO(C=O)- in which R₉ is the group characterising the ester, notionally derived from the alcohol R_gOH.

The term "thioester" means a group R₉S(C=O)- or R₉S(C=S)- or R₉O(C=S)-in which R₉ is the group characterising the thioester, notionally derived from the alcohol R₉OH or the thioalcohol R₉SH.

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 (C₁-C₆)alkyl, (C^C^alkoxy, hydroxy, mercapto, (C,-C₆)alkyIthio, amino, halo (including fluoro, chloro, bromo and iodo), nitro, trifluoromethyl, -COOH, -CONH₂, -CN, -COOR^A , -CONHR^Aor -CONHR^AR^A wherein R^A 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 R¹ in a natural or non-natural amino acid of formula NH₂-CH(R¹)-COOH.

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

Natural alpha-amino acids which contain functional substituents, for example amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, or indoiyl 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 C C₆ alkyl ester), amino groups may be converted to amides (for example as a NHCOC 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_rC₆ 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)C 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 R₁ 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 Aβ 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 Aβ 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 acceptable 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, emulsions, 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 also 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 dissolved 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 group R₁

R_T may be, for example, hydrogen, methyl, ethyl, n-propyl, n-butyl, isobutyl, hydroxy, methoxy, allyl, phenyipropyl, 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- imidazolidinyl, 3,4,4-trimethyl-2,5-dioxo-1 -imidazolidinyl, 2-methyl-3,5-dioxo- 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-1 ,3-dioxo-2H-pyrrolo[3,4-b]quinolin-2-yl, 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 group R,

R₂ may for example be

C C₁₂ alkyl, C₃-C₆ alkenyl or C₃-C₆ alkynyl;

phenyl(C_rC₆ alkyl)-, phenyl(C₃-C₆ alkenyl)- or phenyl(C₃-C₆ alkynyl)- optionally substituted in the phenyl ring;

heteroaryi(C₁-C₆ alkyl)-, heteroaryl(C₃-C₆ alkenyl)- or heteroaryl(C₃-C₆ alkynyl)- optionally substituted in the heteroaryl ring;

4-phenylphenyl(C_rC₆ alkyl)-, 4-phenylphenyi(C₃-C₆ alkenyl)-, 4- phenylphenyl(C₃-C₆ alkynyl)- , 4-heteroarylphenyl(C₁-C₆ alkyl)-, 4- heteroarylphenyl(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, 3-(4-pyridylphenyl)propyl-, 3-(4-(4-pyridyl)phenyl)prop-2-yn-1 -yl, 3-(4- phenylphenyl)propyi-, 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 R₃

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_rC₆ alkoxybenzyl, or benzyloxy(C C₆alkyl)- group; or

the characterising group of a natural α 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]_πR₆ 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 cycloalkyi 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_r C₆)alkoxy, di((C,-C₆)alkyl)amino, phenyl(C_r 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, α or β 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_rC₆)alkyl group, either being unsubstituted or mono- or di- substituted in the heterocyclic ring with halo, nitro, carboxy, cyano, (C C₆)alkanoyl, trifluoromethyl (C₁-C₆)alkyl, hydroxy, formyl, amino, (C₁-C₆)alkylamino, di-(C₁-C₆)alkylamino, mercapto, (C₁-C₆)alkylthio, hydroxy(C,-C₆)alkyl, mercapto(C₁-C₆)alkyl or (C₁-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 isobutyl.

The group R,,

Examples of particular ester and thioester groups R₄ groups include those of formula -(C=O)OR₉ , -(C=O)SR₉ , -(C=S)SR₉, and -(C=S)OR₉ wherein R₉ is (C_r C₆)alkyl, (C₂-C₆)alkenyl, cycloalkyi, cycloalkyl(C Cg)alkyl-, phenyl, heterocyclyl, phenyl(C_rC₆)alkyl-, heterocyclyl(C,-C₆)alkyl-, (C₁-C₆)alkoxy(C₁-C₆)alkyl-, (C_r C₆)alkoxy(C₁-C₆)alkoxy(C₁-Cg)alkyl-_l any of which may be substituted on a ring or non-ring carbon atom or on a ring heteroatom, if present. Examples of such R₉ 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- pyridylmethyl, 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)OR₉, wherein R₉ is benzyl or cyclopentyl.

The group R

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 following:

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 Aβ. In the Example the following abbreviations are used:

Aβ β-amyloid 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[β-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 class disclosed in WO 98/11063 to reduce Aβ production by APP transfected COS-1 cells was investigated. The test compound was 2S-(3S-hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3- phenylpropionic acid cyclopentyl ester (Example 20 of WO 98/1 1063)

i) Preparation of pGW1 HGAPP₆₉₅

The APP₆₉₅ gene [kindly provided in the prokaryote vector pUC1 19 by Professor Benno Mueller-Hill (Universtat Koln)] and its flanking segments were sequenced using the fmol® DNA Cycle Sequencing System (Promega, Southampton, UK). The APP gene was PCR-amplified and subcloned into the mammalian expression vector pGWI HG [Wells et al. (1996) Glia. 18:332-340] using standard molecular cloning techniques. Subcloning Efficiency DH5α cells (GibcoBRL, Paisley, UK) were transformed with the ligation products, according to manufacturer's instructions, and plated out on YT agar plates containing 100 μg/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 μl of miniprep DNA was digested with Hind\\\. Positive clones producing bands of 2,850 bp and 7,450 bp were subjected to further restriction enzyme (Smal, Xho\ and Sacl) 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 μg/ml penicillin/streptomycin mixture (Sigma)(DMEM/10% 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 pipetted into a 15 ml Falcon tube containing 64 μl Tris-EDTA (T.E.), followed by 6 μl of 100 mM chloroquine (Sigma) and 24 μl of 100 mg/ml DEAE- Dextran (M_r 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 ceils 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 μCi/ml ³⁵S-Met (activity>1000 Ci/mmol; Amersham Pharmacia Biotech, Bucks, UK)) containing test compound at a concentration of 10 μM. Control cells were radiolabelled in the absence of test compound. Cell homogenates were collected 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 μg/ml), Pepstatin (10 μg/ml), AEBSF (40 μg/ml) and Aprotinin (20 μg/ml)]. Homogenates were spun at 12,000gmax for 5 min to precipitate cell debris. Prior to immunoprecipitatioπ, conditioned media was processed by centrifuging at

10O.OOOgmax 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 μl per 200 μl of sample. Samples were pulse microcentrifuged to sediment the beads and any non-specific material bound to them. Aβ specific monoclonal antibodies (mAbs) 6E10 and 4G8 (Senetek, Maryland Heights, MO, USA; mAb 6E10 binds the first 17 residues of the β-amyloid region; mAb 4G8 recognises residues 17-28) were added at 2 μg/ml 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 μl of SDS/sample 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 μm polyvinylidene difluoride (PVDF) membranes (Novex) [Towbin et al. (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 Aβ formation by the COS-1 cells. The test compound was found to cause >95% inhibition of Aβ formation at 10μM. Example 2

The ability of representative compounds from the class disclosed in WO 98/11063 to reduce Aβ production by APP transfected CHO cells was investigated. The test compounds were:

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionic acid cyclopentyl ester (Example 20 of WO 98/11063)

2S-[2R-(1S-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-phenylpropionic acid isopropyl ester (Example 23 of WO 98/11063)

2S-[2R-(S-Hydroxy-hydroxycarbamoyl-methyl)-4-methyl-pentanoylamino]-3-phenyl' propionic acid cyclopentyl ester (Example 11 of WO 98/11063)

2S-{2R-[1 S-Hydroxycarbamoyl-2-(thiophen-2-ylsulphanyl)-ethyl]-4-methyl- pentanoylamino}-3-phenyl-propionic acid isopropyl ester (Example 17 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoyiamino)-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-pentanoyiamino]-3- phenyl-propionic acid cyclopentyl ester (Example 37 of WO 98/11063)

3-tert-Butoxy-2S-(3S-hydroxycarbamoyl-2R-isobutyl-hex-5-enoyiamino)-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 Aβ formation.

Diastereoisomer A

¹H-NMR; δ (MeOD), 7.4-7.29 (5H, m), 5.43 (1 H, s), 5.2-5.14 (1 H, m), 4.02 (1 H, 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).

¹³C-NMR; δ (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

¹H-NMR; δ (MeOD), 7.33-7.19 (5H, m), 5.3 (1 H, s), 5.11-5.06 (1 H, m), 3.81 (1 H, d, J=7.3Hz), 2.83-2.74 (1 H, 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). ¹³C-NMR; δ (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 Aβ formation.

Diastereoisomer A

¹H-NMR; δ (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 (1 H, m), 3.53 (1 H, d, J=9.7Hz), 3.17 (3H, s), 2.89-2.79 (1 H, 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). ¹³C-NMR; δ (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; δ (MeOD), 7.39-7.30 (5H, m), 5.45 (1 H, s), 5.21-5.15 (1 H, m), 3.59 (1 H, 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 (1 H, m), 1.01 (1 H, ddd, J=3.7, 9.9, 13.3Hz), 0.83 (3H, d, J=6.5Hz) and 0.79 (3H, d, J=6.6Hz). ¹³C-NMR; δ (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 pGWI 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 Not\ which cuts within the ampicillin resistance gene. Forty micrograms of DNA was pipetted into 0.4 cm, gap 50, sterile cuvettes (Gene Puiser Cuvettes, Bio-Rad, Hemel Hempstead, Herts, UK) followed by 800 μl of 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 Puiser, Bio- Rad) set at 0.8 kV, 25 μFD and pulsed for 0.5-0.6 ms. The cell suspension was left on ice for a further 20 minutes and then 50 μl of cell suspension was pipetted into 50 ml of DMEM/10% FCS and mixed by gently inverting the tube. This diluted cell suspension was then aliquoted into 96-well plates (Falcon) by pipetting 100 μl per well. The cells were replaced 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 Aβ 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 μM) 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 Aβ secreted into the media was assessed using an ELISA specific for Aβ (Quality Controlled Biochemicals, Hopkinton, MA, USA).

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

Example 3

Proliferation study

To eliminate the possibility that the observed reduction in Aβ 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 μM over a 24 hour period, which represents the incubation period of the APP labelling and Aβ 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 Aβ 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.

Claims

1. A method for treatment of mammals suffering diseases responsive to inhibition of A╬▓ 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 A╬▓ production:

wherein

R is hydrogen or (C_rC₆)alkyl;

Ri is hydrogen;

(C_rC₆)alkyl;

(C₂-C₆)alkenyi;

phenyl or substituted phenyl;

phenyl (C C₆)alkyl or substituted phenyl(C₁-C₆)alkyl;

phenyl (C₂-C₆)alkenyl or substituted phenyl(C₂-C₆)alkenyl

heterocyclyl or substituted heterocyclyl;

heterocyclyl(C₁-C₆)alkyl or substituted heterocyclyl(C_rC₆)alkyl; a group BSO_RA- wherein n is 0, 1 or 2 and B is hydrogen or a (C_rC₆) alkyl, phenyl, substituted phenyl, heterocyclyl substituted heterocyclyl, (C₁-C₆)acyl, phenacyl or substituted phenacyl group, and A represents (C_rC₆)alkylene;

hydroxy or (C,-C₆)alkoxy;

amino, protected amino, acylamino, (C₁-C₆)alkylamino or di-(C_r C₆)alkylamino;

mercapto or (C C₆)alkylthio;

amino(C₁-C₆)alkyl, (C₁-C₆)alkylamino(C₁-C₆)alkyl, di(C₁-C₆)alkylamino(C₁- C₆)alkyl, hydroxy(C,-C₆)alkyl, mercapto(C₁-C₆)alkyl or carboxy(C₁-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 cycloalkyi, cycloalkenyl or non-aromatic heterocyclic ring containing up to 3 heteroatoms, any of which may be (i) substituted by one or more substituents selected from C,-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 C_rC₆ alkyl or benzyl and/or (ii) fused to a cycloalkyi or heterocyclic ring;

is a C_rC₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, pheny C -Cg alkyl)-, heteroaryl(C_rC₆ alkyl)-, phenyl(C₂-C₆ alkenyl)-, heteroaryl(C₂-C₆ alkenyl)-, phenyl(C₂-C₆ alkynyl)-, heteroaryl(C₂-C₆ alkynyl)-, cycloalkyl(C_rC₆ alkyl)-, cycloalkyl(C₂-C₆ alkenyl)-, cycloalkyl(C₂-C₆ alkynyl)-, cycloalkenyl(C,-C₆ alkyl)-, cycloalkenyl(C₂-C₆ alkenyl)-, cycloalkenyl(C₂-C₆ alkynyl)-, phenyl(C_rC₆ alkyl)O(C_rCg alkyl)-, or alkyl)O(C_rC₆ alkyl)- group, any one of which may be optionally substituted by

C_rC₆ alkyl,

C C₆ alkoxy, halo, cyano (-CN), phenyl or heteroaryl, or phenyl or heteroaryl substituted by C_rC₆ alkyl, C_rC₆ alkoxy, halo, or cyano (-CN);

R₃ is the characterising group of a natural or non-natural ╬▒ amino acid in which any functional groups may be protected; and

R₄ is an ester or thioester group.

2. A method as claimed in claim 1 wherein the stereochemical configuration of the carbon atom carrying the group R₂ is R, and that of the carbon atom carrying the groups R₃ and R₄ is S.

3. A method as claimed in claim 1 or claim 2 wherein R., is:

hydrogen, methyl, ethyl, n-propyl, n-butyl, isobutyl, hydroxy, methoxy, allyl, phenylpropyl, phenylprop-2-enyl, thienyisulphanylmethyl, thienylsulphinylmethyl, or thienylsulphonylmethyl; or

C_rC₄ 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- imidazolidinyl, 3,4,4-trimethyl-2,5-dioxo-1 -imidazolidinyl, 2-methyl-3,5-dioxo- 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-1 ,3-dioxo-2H-pyrrolo[3,4-b]quinolin-2-yl, or 2,3-dihydro-1 ,3- dioxo-1 H-benz[d,e]isoquinolin-2-yl group; or

4. A method as claimed in claim 1 or claim 2 wherein R, is n-propyl, allyl, hydroxy, methoxy or thienylsulfanyl-methyl.

5. A method as claimed in claim 1 or claim 2 wherein R₂ is:

C,-C₁₂ alkyl, C₃-C₆ alkenyl or C₃-C₆ alkynyl;

alkyl)-, phenyl(C₃-C₆ alkenyl)- or phenyl(C₃-C₆ alkynyl)- optionally substituted in the phenyl ring; heteroaryl(C_rC₆ alkyl)-, heteroaryl(C₃-C₆ alkenyl)- or heteroaryl(C₃-C₆ alkynyl)- optionally substituted in the heteroaryl ring;

4-phenylphenyi(C,-C₆ alkyl)-, 4-phenyiphenyl(C₃-C₆ alkenyl)-, 4- phenylphenyl(C₃-C₆ alkynyl)- , 4-heteroarylphenyl(C₁-C₆ alkyl)-, 4- heteroarylphenyl(C₃-C₆ alkenyl)-, 4-heteroarylphenyl(C₃-C₆ alkynyl)-, optionally substituted in the terminal phenyl or heteroaryl ring; or

phenoxy(C C₆ alkyl)- or heteroaryloxy(C₁-C₆ alkyl)- optionally substituted in the phenyl or heteroaryl ring.

6. A method as claimed in claim 1 or claim 2 wherein R₂ is: methyl, ethyl, n- or iso-propyl, n- , iso- or 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, 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 or 3-[(4- chlorophenyl)phenyl]propyl-.

7. A method as claimed in claim 1 or claim 2 wherein R₂ is isobutyl, n-hexyl, or 3-(2-chlorophenyl)prop-2-yn-1-yl.

8. A method as claimed in claim 1 or claim 2 wherein R₃ is 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_rC₆ alkoxybenzyl, or benzyloxy(C₁-C₆alkyl)-.

9. A method as claimed in claim 1 or claim 2 wherein R₃ is the characterising group of a natural ╬▒ amino acid, in which any functional group may be protected, any amino group may be acylated and any carboxyl group present may be amidated.

10. A method as claimed in claim 1 or claim 2 wherein R₃ is 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 cycloalkyi or cycloalkenyl group.

11. A method as claimed in claim 1 or claim 2 wherein R₃ is a benzyl group substituted in the phenyl ring by a group of formula -OCH₂COR₈ where R₈ is hydroxyl, amino, phenyl(C_rC₆)alkoxy, (C_rC₆)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, ╬▒ or ╬▓ alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, histidine, arginine, glutamic acid, and aspartic acid.

12. A method as claimed in claim 1 or claim 2 wherein R₃ is a heterocyclk^C,- C₆)alkyl group, either being unsubstituted or mono- or di-substituted in the heterocyclic ring with halo, nitro, carboxy, (C₁-C₆)alkoxy, cyano, (C,-C₆)alkanoyl, trifluoromethyl hydroxy, formyl, amino, (C₁-C₆)alkylamino, di-(C_r C₆)alkylamino, mercapto, (C_rC₆)alkylthio, hydroxy(C₁-C₆)alkyl, mercapto(C₁- C₆)alkyl or (C_rC₆)alkylphenylmethyl.

13. A method as claimed in claim 1 or claim 2 wherein R₃ is phenyl, benzyl, tert- butoxymethyl or iso-butyl.

14. A method as claimed in claim 1 or claim 2 wherein R₄ is a group of formula - (C=O)OR₉ , -(C=O)SR₉ , -(C=S)SR₉₎ and -(C=S)OR₉ wherein R₉ is (C_rC₆)alkyl, (C₂-C₆)alkenyl, cycloalkyi, cycloalkyl(C C₆)alkyl-, phenyl, heterocyclyl, phenyl(C_r C₆)alkyl-, heterocyclyl(C,-C₆)alkyl-₎ (C₁-C₆)alkoxy(C₁-Cg)alkyl-₎ or (C₁-C₆)alkoxy(C₁- 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.

15. A method as claimed in claim 1 or claim 2 wherein R₄ is a group of formula - (C=O)OR₉ wherein R₉ is 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-pyridylmethyl, N-methylpiperidin-4-yl, 1-methylcyclopent-1yl, adamantyl, tetrahydrofuran-3-yl or methoxyethyl.

16. A method as claimed in claim 1 or claim 2 wherein R₄ is a group of formula - (C=O)OR₉ wherein R₉ is benzyl or cyclopentyl.

17. A method as claimed in claim 1 or claim 2 wherein R is hydrogen or methyl.

18. A method as claimed in claim 1 or claim 2 wherein R, is n-propyl, allyl, methoxy or thienylsulfanyl-methyl, R₂ is isobutyl, n-hexyl, or 3-(2-chlorophenyl)prop- 2-yn-1-yl, R₃ is phenyl, benzyl, tert-butoxymethyl or iso-butyl, R₄ is a group of formula -(C=O)OR₉ wherein R₉ is benzyl or cyclopentyl and R is hydrogen or methyl.

19. A method as claimed in claim 1 wherein the compound of formula (I) is selected from

2(R or S)-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-2- phenyiethanoic acid isopropyl ester,

2(R or S)-[2R-(S-Hydroxycarbamoyl-methoxy-methyl)-4-methyl- pentanoyiamino]-2-phenylethanoic acid cyclopentyl ester, 2(R or S)-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-2-(4- methoxyphenyl)ethanoic acid cyclopentyl ester,

and pharmaceutically or veterinarily acceptable salts, hydrates or soivates thereof.

20. A method as claimed in claim 19 wherein the 2S diastereomer of the compound is used.

21. The use of a compound of formula (I) as defined in any of claims 1 to 20, in the preparation of a pharmaceutical composition for the treatment of mammals suffering diseases responsive to inhibition of A╬▓ production.

22. A method as claimed in any of claims 1 to 20, or a use as claimed in claim 21 , wherein the disease to be treated is Altzheimer's disease, senile dementia of Alzheimer's type, neurodegenerative disorder associated with Down's syndrome, neurodegeneration secondary to traumatic injury to the brain, cerebral amyloid angiopathy, cerebral amyloid angiopathy-associated stroke, or hereditary cerebral hemorrhage with amyloidosis.