WO2000009119A1 - Derives d'acide hydroxamique en tant qu'inhibiteurs de la production du peptide beta-amyloide - Google Patents

Derives d'acide hydroxamique en tant qu'inhibiteurs de la production du peptide beta-amyloide Download PDF

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WO2000009119A1
WO2000009119A1 PCT/GB1999/002626 GB9902626W WO0009119A1 WO 2000009119 A1 WO2000009119 A1 WO 2000009119A1 GB 9902626 W GB9902626 W GB 9902626W WO 0009119 A1 WO0009119 A1 WO 0009119A1
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alkyl
phenyl
group
methyl
substituted
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PCT/GB1999/002626
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English (en)
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Mandy Johnstone
Andrew Paul Ayscough
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British Biotech Pharmaceuticals Limited
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Priority to JP2000564622A priority Critical patent/JP2002522489A/ja
Priority to EP99938450A priority patent/EP1105116A1/fr
Priority to AU52958/99A priority patent/AU5295899A/en
Publication of WO2000009119A1 publication Critical patent/WO2000009119A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to the use of certain esters and thioesters for the treatment of diseases responsive to inhibition of production of ⁇ -amyioid peptide (A ⁇ ).
  • AD Alzheimer's disease
  • a ⁇ 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 A ⁇ by as yet uncharacterised enzymes, termed ⁇ - secretase and ⁇ -secretase, respectively.
  • a ⁇ 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 A ⁇ 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.
  • 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 ⁇ -secretase site (leading to excessive production of both A ⁇ 1j(0 and A ⁇ - 42 ) or the ⁇ -secretase site (resulting in selective increased production of A ⁇ 1 ⁇ l2 ).
  • 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 ⁇ -secretase site (leading to excessive production of both A ⁇ 1j(0 and A ⁇ - 42 ) or the ⁇ -secretase site (resulting in selective increased production of A ⁇ 1 ⁇ l2 ).
  • APP is anchored to internal membranes e.g. e ⁇ doplasmic 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.
  • ER e ⁇ doplasmic reticulum
  • Golgi Golgi
  • TGN trans-Golgi network
  • endosomes in the plasmalemma by a 23 amino acid hydrophobic stretch near it's C-terminus.
  • ER e ⁇ doplasmic reticulum
  • TGN trans-Golgi network
  • endosomes in the plasmalemma by a 23 amino acid hydrophobic stretch near it's C-terminus.
  • ADAM-17 also known as tumour-necrosis factor- ⁇ (TNF- ⁇ ) converting enzyme (TACE)
  • TNF- ⁇ tumour-necrosis factor- ⁇
  • ADAM-10 both the constitutive and regulated ⁇ -secretase cleavage of APP can be performed by ADAM-10 [Lammich et al. (1999) J. Biol. Chem. 96:3922-3927].
  • This invention is based on the finding that certain esters and thioesters have the property of reducing A ⁇ 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 ⁇ -secretase and/or ⁇ -secretase enzymes, or of enzymes involved in the pathway leading to production of the putative ⁇ -secretase and/or ⁇ -secretase enzymes.
  • WO 95/04033 discloses N 4 -hydroxy-N 1 -(1-(S)-methoxycarbonyl-2,2- dimethylpropyl)-2-(R)-(4-chlorophenylpropyl)succinamide as an intermediate for the preparation of the corresponding methylamide MMP inhibitor.
  • Int. J. Pept. Protein Res. (1996), 48(2), 148-155 discloses the compound
  • Ph-CH 2 CH(CO-lle-OtBu)CH 2 CONHOH as an intermediate in the preparation of compounds which are inhibitors of neurotensin-degrading enzymes.
  • the present invention provides 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:
  • R is hydrogen or (C 1 -C 6 )alkyl
  • R 1 is hydrogen
  • heterocyclyl or substituted heterocyclyl
  • n 0, 1 or 2 and B is hydrogen or a (C C 6 ) alkyl, phenyl, substituted phenyl, heterocyclyl substituted heterocyclyl, (C C 6 )acyl, phenacyl or substituted phenacyl group, and A represents (C r C 6 )alkylene;
  • R 3 is the characterising group of a natural or non-natural ⁇ amino acid in which any functional groups may be protected.
  • R 4 is an ester or thioester group
  • the compound used is one of general formula (I) above wherein:
  • R, R 1 and R 4 are as defined above with reference to formula (I)
  • R 2 is C,-C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl,
  • phenyl(C 2 -C 6 alkynyl)- phenyl(C 2 -C 6 alkynyl)-, heteroaryl(C 2 -C 6 alkynyl)-, biphenyl(C 2 -C 6 alkynyl)-, phenylheteroaryl(C 2 -C 6 alkynyl)-, heteroarylphenyl(C 2 -C 6 alkynyl)-,
  • R 3 is C C 6 alkyl, optionally substituted benzyl, optionally substituted phenyl, optionally substituted heteroaryl ; or
  • heterocyclic(C C 6 )alkyl group optionally substituted in the heterocyclic ring
  • (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.
  • (C 2 -C 6 )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.
  • C 2 -C 6 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.
  • cycloalkyi means a saturated alicyclic moiety having from 3-8 carbon atoms and includes, for example, cyclohexyl, cyclooctyl, cycloheptyl, cyclopentyl, cyclobutyl and cyclopropyl.
  • 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.
  • aryl means an unsaturated aromatic carbocyclic group which is monocyclic (eg phenyl) or polycyclic (eg naphthyl).
  • 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-dio
  • 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.
  • substituted as applied to any moiety herein means substituted with up to four substituents, each of which independently may be (C 1 -C 6 )alkyl, (C ⁇ C ⁇ alkoxy, hydroxy, mercapto, (C,-C 6 )alkyIthio, amino, halo (including fluoro, chloro, bromo and iodo), nitro, trifluoromethyl, -COOH, -CONH 2 , -CN, -COOR A , -CONHR A or -CONHR A R A wherein R A is a (C,-C 6 )alkyl group or the residue of a natural alpha-amino acid.
  • side chain of a natural or non-natural alpha-amino acid means the group R 1 in a natural or non-natural amino acid of formula NH 2 -CH(R 1 )-COOH.
  • 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, ⁇ - aminoadipic acid, ⁇ -amino-n-butyric acid, 3,4-dihydroxyphenylalanine, homoserine, ⁇ -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.
  • R 3 in the compounds of the invention is one of those side chains, the functional substituent may optionally be protected.
  • side chains of non-natural alpha amino acids include those referred to below in the discussion of suitable R 3 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.
  • the C atom carrying the hydroxamic acid and R 1 groups may be in the R or S configuration
  • the C atom carrying the R 2 group may be predominantly in the R configuration
  • the C atom carrying the R 3 and R 4 groups may be in either the R or S configuration, with the predominantly S configuration presently preferred.
  • compounds of formula (I) above are useful in human or veterinary medicine since they inhibit A ⁇ 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 A ⁇ 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.
  • binding agents for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone
  • fillers for example
  • 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.
  • 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
  • almond oil fractionated coconut oil
  • oily esters such as glycerine, propylene
  • the active ingredient may also be administered parenterally, including injection into the cerebrao-spinal fluid, in a sterile medium.
  • the drug can either be suspended or dissolved in the vehicle.
  • 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.
  • 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,-C 4 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
  • R, groups include n-propyl, allyl, hydroxy, methoxy and thienylsulfanyl-methyl.
  • R 2 may for example be
  • 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
  • R 2 groups include isobutyl, n-hexyl, 3-(2-chlorophenyl)prop-2- yn-1-yl.
  • R 3 may for example be C,-C 6 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 r C 6 alkoxybenzyl, or benzyloxy(C C 6 alkyl)- group; or
  • Alk is a (C,-C 6 )alkyl or (C 2 -C 6 )alkenyl group optionally interrupted by one or more -O-, or -S- atoms or -N(R 7 )- groups [where R 7 is a hydrogen atom or a (C C 6 )alkyl group], n is 0 or 1 , and R 6 is an optionally substituted cycloalkyi or cycloalkenyl group; or
  • a heterocyclic(C r C 6 )alkyl group either being unsubstituted or mono- or di- substituted in the heterocyclic ring with halo, nitro, carboxy, cyano, (C C 6 )alkanoyl, trifluoromethyl (C 1 -C 6 )alkyl, hydroxy, formyl, amino, (C 1 -C 6 )alkylamino, di-(C 1 -C 6 )alkylamino, mercapto, (C 1 -C 6 )alkylthio, hydroxy(C,-C 6 )alkyl, mercapto(C 1 -C 6 )alkyl or (C 1 -C 6 )alkylphenylmethyl.
  • R 3 groups examples include benzyl, phenyl, cyclohexylmethyl, pyridin- 3-ylmethyl, tert-butoxymethyl, iso-butyl and sec-butyl.
  • R 3 groups include phenyl, benzyl, tert-butoxymethyl and isobutyl.
  • R 9 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 R groups are hydrogen and methyl.
  • PVDF Polyvinylidene difluoride sVCAM-1 Soluble vascular cell adhesion molecule-1
  • test compound was 2S-(3S-hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3- phenylpropionic acid cyclopentyl ester (Example 20 of WO 98/1 1063)
  • the APP 695 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 DH5 ⁇ cells (GibcoBRL, Paisley, UK) were transformed with the ligation products, according to manufacturer's instructions, and plated out on YT agar plates containing 100 ⁇ g/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 ⁇ l of miniprep DNA was digested with Hind ⁇ .
  • COS-1 cells European Collection of Cell Cultures, Salisbury, Wilts, UK
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS Foetal Calf Serum
  • FCS Foetal Calf Serum
  • FCS Foetal Calf Serum
  • DMEM/10% FCS penicillin/streptomycin mixture
  • 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.
  • PBS phosphate-buffered saline
  • 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 ⁇ Ci/ml 35 S-Met (activity>1000 Ci/mmol; Amersham Pharmacia Biotech, Bucks, UK)) containing test compound at a concentration of 10 ⁇ M. Control cells were radiolabelled in the absence of test compound.
  • labelling medium methionine-free DMEM containing 10% FCS, 5% L-Glu, 5% penicillin/streptomycin mix (Sigma) and 100 ⁇ Ci/ml 35 S-Met (activity>1000 Ci/mmol; Amersham Pharmacia Biotech, Bucks, UK)
  • 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 ⁇ l per 200 ⁇ l of sample. Samples were pulse microcentrifuged to sediment the beads and any non-specific material bound to them.
  • RT room temperature
  • a ⁇ specific monoclonal antibodies 6E10 and 4G8 (Senetek, Maryland Heights, MO, USA; mAb 6E10 binds the first 17 residues of the ⁇ -amyloid region; mAb 4G8 recognises residues 17-28) were added at 2 ⁇ g/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 ⁇ l 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 ⁇ m polyvinylidene difluoride (PVDF) membranes (Novex) [Towbin et al. (1979) PNAS USA. 76:4350-4354] at 150 V for 45 min.
  • PVDF polyvinylidene difluoride
  • 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.
  • test compound inhibited A ⁇ formation by the COS-1 cells.
  • the test compound was found to cause >95% inhibition of A ⁇ formation at 10 ⁇ M.
  • test compounds were:
  • Diastereoisomers A and B were each tested for their ability to inhibit A ⁇ formation.
  • the human APP 695 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 10 7 cells/ml.
  • 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 (Geneuterer Cuvettes, Bio-Rad, Hemel Hempstead, Herts, UK) followed by 800 ⁇ l of CHO cells resuspended in PBS (8 x 10 6 cells), mixed and left on ice for 10 minutes. The cuvette was then placed in the electroporator (Geneuterer, Bio- Rad) set at 0.8 kV, 25 ⁇ FD and pulsed for 0.5-0.6 ms.
  • the cell suspension was left on ice for a further 20 minutes and then 50 ⁇ l 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 ⁇ l 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].
  • XGPRT xanthine- guanine phosphoribosyltransferase
  • 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.
  • CHO cells (1 x 10 6 ) stably expressing APP 695 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 ⁇ M) 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% CO 2 for 24 hours. Conditioned media was centrifuged to pellet any remaining membrane fraction and the levels of A ⁇ secreted into the media was assessed using an ELISA specific for A ⁇ (Quality Controlled Biochemicals, Hopkinton, MA, USA).
  • test compounds inhibited A ⁇ formation by the CHO cells.
  • the test compounds were each found to cause from 10% to 100% inhibition of A ⁇ formation at 10 ⁇ M.
  • test compound 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 ⁇ M over a 24 hour period, which represents the incubation period of the APP labelling and A ⁇ ELISA experiments.
  • 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 APP 695 in the APP proteolysis studies) using standard molecular cloning techniques.
  • Cells (1 x 10 6 ) 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).
  • test compounds which were found to reduce A ⁇ production in the CHO cell line stably transfected with human APP 695 ) significantly reduced the levels of sVCAM-1 produced in the CHO cell line stably transfected with the human sVCAM-1 gene.

Abstract

L'invention concerne un procédé de traitement d'un mammifère atteint d'une maladie réactive à l'inhibition de la production du peptide bêta-amyloïde (Aβ). Le procédé consiste à administrer au mammifère une quantité suffisante d'un composé de la formule générale (I) ou d'un sel hydraté ou solvate pharmaceutiquement acceptable dudit composé, pour inhiber la production d'Aβ. Dans ladite formule, R1, R2, R3 sont tels que définis dans le mémorandum descriptif, et R4 est un groupe ester ou thioester.
PCT/GB1999/002626 1998-08-11 1999-08-10 Derives d'acide hydroxamique en tant qu'inhibiteurs de la production du peptide beta-amyloide WO2000009119A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2000564622A JP2002522489A (ja) 1998-08-11 1999-08-10 ベータ−アミロイド産生の阻害剤としてのヒドロキサム酸誘導体
EP99938450A EP1105116A1 (fr) 1998-08-11 1999-08-10 Derives d'acide hydroxamique en tant qu'inhibiteurs de la production du peptide beta-amyloide
AU52958/99A AU5295899A (en) 1998-08-11 1999-08-10 Hydroxamic acid derivatives as inhibitors of beta-amyloid production

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9817348.7 1998-08-11
GBGB9817348.7A GB9817348D0 (en) 1998-08-11 1998-08-11 Pharmaceutical use of esters

Publications (1)

Publication Number Publication Date
WO2000009119A1 true WO2000009119A1 (fr) 2000-02-24

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JP (1) JP2002522489A (fr)
AU (1) AU5295899A (fr)
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WO (1) WO2000009119A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10043282A1 (de) * 2000-09-02 2002-03-28 Kurt Heininger Verwendung von Inhibitoren der Amyloid-beta-Protein-Bildung
WO2010107435A1 (fr) * 2009-03-19 2010-09-23 Bristol-Myers Squibb Company Nouveau composé alpha-(n-sulfonamido)acétamide en tant qu'inhibiteur de la production de peptide amyloïde bêta
EP3634410A4 (fr) * 2017-06-05 2021-03-03 The Methodist Hospital System Inhibiteurs de phosphorylation des protéines tau et méthodes de traitement ou de prévention de la maladie d'alzheimer

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011063A1 (fr) * 1996-09-10 1998-03-19 British Biotech Pharmaceuticals Limited Derives cytostatiques d'acide hydroxamique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011063A1 (fr) * 1996-09-10 1998-03-19 British Biotech Pharmaceuticals Limited Derives cytostatiques d'acide hydroxamique

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10043282A1 (de) * 2000-09-02 2002-03-28 Kurt Heininger Verwendung von Inhibitoren der Amyloid-beta-Protein-Bildung
WO2010107435A1 (fr) * 2009-03-19 2010-09-23 Bristol-Myers Squibb Company Nouveau composé alpha-(n-sulfonamido)acétamide en tant qu'inhibiteur de la production de peptide amyloïde bêta
EP3634410A4 (fr) * 2017-06-05 2021-03-03 The Methodist Hospital System Inhibiteurs de phosphorylation des protéines tau et méthodes de traitement ou de prévention de la maladie d'alzheimer

Also Published As

Publication number Publication date
EP1105116A1 (fr) 2001-06-13
GB9817348D0 (en) 1998-10-07
AU5295899A (en) 2000-03-06
JP2002522489A (ja) 2002-07-23

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