WO1997043250A1 - Composes a base d'acide hydroxamique, inhibiteurs de la formation de cd23 et de facteur de necrose des tumeurs - Google Patents

Composes a base d'acide hydroxamique, inhibiteurs de la formation de cd23 et de facteur de necrose des tumeurs Download PDF

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WO1997043250A1
WO1997043250A1 PCT/EP1997/002500 EP9702500W WO9743250A1 WO 1997043250 A1 WO1997043250 A1 WO 1997043250A1 EP 9702500 W EP9702500 W EP 9702500W WO 9743250 A1 WO9743250 A1 WO 9743250A1
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compound
formula
compound according
alkyl
prophylaxis
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PCT/EP1997/002500
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David Glynn Smith
Stuart Bailey
Andrew Faller
Derek Richard Buckle
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Smithkline Beecham Plc
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Priority to JP09540534A priority Critical patent/JP2000510138A/ja
Priority to EP97923088A priority patent/EP0901464A1/fr
Publication of WO1997043250A1 publication Critical patent/WO1997043250A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/60—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton with the carbon atom of at least one of the carboxyl groups bound to nitrogen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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

  • This invention relates to novel inhibitors ofthe formation of soluble human CD23 and their use in the treatment of conditions associated with excess production of soluble CD23 (s- CD23) such as autoimmune disease and allergy.
  • the compounds of the invention are also inhibitors ofthe release of tumour necrosis factor (TNF).
  • CD23 (the low affinity IgE receptor FceRII, Blast 2), is a 45 kDa type II integral protein expressed on the surface of a variety of mature cells, including B and T lymphocytes, macrophages, natural killer cells, Langerhans cells, monocytes and platelets (Delespesse et al, Adv Immunol, 42 [1991] 149-191). There is also a CD23-like molecule on eosinophils (Grangette et al, J Immunol, 14_3_ [1989] 3580-3588). CD23 has been implicated in the regulation of the immune response (Delespesse et al, Immunol Rev, 125 . [1992] 77-97).
  • Human CD23 exists as two differentially regulated isoforms, a and b, which differ only in the amino acids at the intracellular N-terminus (Yokota et al, Cell, 15 . [1988] 611-618). In man the constitutive a isoform is found only on B-lymphocytes, whereas type b, inducible by IL4, is found on all cells capable of expressing CD23.
  • i-CD23 cell bound CD23
  • s-CD23 well-defined soluble fragments
  • S-CD23 has been implicated in the overproduction of IgE, the hallmark of allergic diseases such as extrinsic asthma, rhinitis, allergic conjuctivitis, eczema, atopic dermatitis and anaphylaxis (Sutton and Gould, Nature, 3J>6_, [1993] 421-428).
  • Other biological activities attributed to S-CD23 include the stimulation of B cell growth and the induction of the release of mediators from monocytes.
  • S-CD23 has been observed in the serum of patients having B-chronic lymphocytic leukaemia (Sarfati et al, Blood, 7_L [1988] 94-98) and in the synovial fluids of patients with rheumatoid arthritis (Chomarat et al, Arthritis and Rheumatism, 26 [1993] 234-242). That there is a role for CD23 in inflammation is suggested by a number of sources. First, sCD23 has been reported to bind to extracellular receptors which when activated are involved in cell-mediated events of inflammation.
  • sCD23 is reported to directly activate monocyte TNF, IL-1, and IL-6 release (Armant et al, vol 180, J.Exp. Med., 1005-1011 (1994)).
  • CD23 has been reported to interact with the B2-integrin adhesion molecules, CDl lb and CDl lc on monocyte/macrophage (S. Lecoanet-Henchoz et al, Immunity, vol 3; 119-125 (1995)) which trigger NO2" , hydrogen peroxide and cytokine ( IL-1, IL-6, and TNF) release.
  • IL-4 or IFN induce the expression of CD23 and its release as sCD23 by human monocytes.
  • compounds which inhibit the formation of S-CD23 should have twofold actions of a) enhancing negative feedback inhibition of IgE synthesis by maintaining levels of i-CD23 on the surface of B cells, and b) inhibiting the immunostimulatory cytokine activities of higher molecular weight soluble fragments (Mr 37, 33 and 29 kDa) of S-CD23.
  • inhibition of CD23 cleavage should mitigate sCD23- induced monocyte activation and mediator formation, thereby reducing the inflammatory response.
  • TNF ⁇ is a pro-inflammatory cytokine which is released from stimulated cells by specific cleavage of a 76-amino acid signal sequence in the inactive precursor to generate the mature form.
  • the cleavage of TNF ⁇ has been reported to be carried out by a metalloprotease (Gearing, A.J.H. et al, (1994) Nature 370, 555-557; McGeehan, G.M. et al, (1994) Nature 370, 558-561; Mohler, K.M. et al, (1994) Nature 370, 218-220).
  • TNF ⁇ is induced in a variety of cell types in response to bacteria, endotoxin, various viruses and parasites, so that one physiological function ascribed to TNF ⁇ is a contribution to the inflammatory response to acute infection by bacteria, parasites, etc (Dinarello, CA. (1992) Immunol. 4, 133-145). Overproduction of TNF ⁇ has been implicated in disease states such as rheumatoid arthritis, septic shock, Crohn's disease and cachexia (Dinarello, 1992).
  • TNF ⁇ may also contribute to the destruction of tissue in autoimmune disease although it is not a initiating factor in these diseases.
  • TNF ⁇ antibodies have been shown to reduce the severity of disease in short term studies in rheumatoid arthritis models (Elliott, M.J., et al (1993) Arthrit. Rheum. 12, 1681-1690; Elliott et al (1994) Lancet 344, 1125-1127).
  • WO 96/02240 discloses that compounds which inhibit the action of matrix metalloproteases (eg collagenase, stromelysin and gelatinase) are effective inhibitors ofthe release of human soluble CD23 transfected into mammalian cell culture systems.
  • matrix metalloproteases eg collagenase, stromelysin and gelatinase
  • UK Patent Application No. 9601041.8 discloses that certain compounds of formula (I) are effective inhibitors ofthe release of human soluble CD23 transfected into mammalian cell culture systems:
  • R is (C2-6) a lkenylthiomethyl or (C2-6)alkynylthiomethyl
  • Rl is alkyl or alkenyl
  • R 2 is alkyl, alkenyl, aryl, cycloalkyl or cycloalkenyl
  • R3 is hydrogen, alkyl, alkenyl, alkynyl or aryl.
  • Alkyl, alkenyl and alkynyl groups referred to herein include straight and branched groups containing up to six carbon atoms and are optionally substituted by one or more groups selected from the group consisting of aryl, heterocyclyl, (C ⁇ _g)alkoxy, (C ⁇ _6)alkylthio, aryl(C ⁇ _6)alkoxy, aryl(C ⁇ _6)alkylthio, amino, mono- or di- (C ⁇ _6)alkylamino, cycloalkyl, cycloalkenyl, carboxy and esters thereof, hydroxy, and halogen.
  • Cycloalkyl and cycloalkenyl groups referred to herein include groups having between three and eight ring carbon atoms and are optionally substituted as described hereinabove for alkyl, alkenyl and alkynyl groups.
  • aryl means single and fused rings suitably containing from 4 to 7, preferably 5 or 6, ring atoms in each ring, which rings, may each be unsubstituted or substituted by, for example, up to three substituents.
  • a fused ring system may include aliphatic rings and need include only one aromatic ring.
  • Suitable aryl groups include phenyl and naphthyl such as 1 -naphthyl or 2-naphthyl.
  • any aryl group, including phenyl and naphthyl may be optionally substituted by up to five, preferably up to three substituents.
  • Suitable substituents include halogen,
  • heterocyclyl and “heterocyclic” suitably include, unless otherwise defined, aromatic and non-aromatic, single and fused, rings suitably containing up to four heteroatoms in each ring, each of which is selected from oxygen, nitrogen and sulphur, which rings, may be unsubstituted or substituted by, for example, up to three substituents.
  • Each heterocyclic ring suitably has from 4 to 7, preferably 5 or 6, ring atoms.
  • a fused heterocyclic ring system may include carbocyclic rings and need include only one heterocyclic ring.
  • a substituent for a heterocyclyl group is selected from halogen, (C i . 6)alkyl, aryl(C i _6)alky 1, (C i _6)alkoxy, (C i _6)alkoxy(C i -6)alkyl, halo(C ⁇ _ fj )alkyl, hydroxy, amino, mono- and di-N-(C ⁇ _6)alkyl-amino, acylamino, carboxy salts, carboxy esters, carbamoyl, mono- and di-N-(C]_6)alkylcarbonyl, aryloxycarbonyl, (C ⁇ 6)alkoxycarbonyl(C ⁇ _6)alkyl, aryl, oxy groups, ureido, guanidino, sulphonylamino, aminosulphonyl, (C ⁇ _6)alkylthio, (C ⁇ _6)alkylsulphinyl, (C ⁇ ⁇
  • R is methyl substituted by propargylthio, 2- butynylthio or allylthio; and/or R ⁇ is an isobutyl group; and/or R 2 is a benzyl group; and/or R3 is hydrogen or methyl.
  • each of R to R ⁇ is selected from the group consisting ofthe values ascribed to it in the Examples hereinbelow.
  • the compound of formula (I) ofthe invention is selected from the group consisting ofthe compounds described in the Examples hereinbelow.
  • the present invention provides the use of a compound of formula (I) for the production of a medicament for the treatment or prophylaxis of disorders such as allergy, inflammatory disorders and autoimmune disease in which the overproduction of S-CD23 is implicated.
  • the invention provides a method for the treatment or prophylaxis of disorders such as allergy, inflammatory disorders and autoimmune disease in which the overproduction of S-CD23 is implicated, which method comprises the administration of a compound of formula (I), to a human or non-human mammal in need thereof.
  • the invention also provides a pharmaceutical composition for the treatment or prophylaxis of disorders such as allergy, inflammatory disorders and autoimmune disease in which the overproduction of S-CD23 is implicated which comprises a compound of formula (I) and optionally a pharmaceutically acceptable carrier therefor.
  • the present invention provides the use of a compound of formula (I) for the production of a medicament for the treatment or prophylaxis of conditions mediated by T ⁇ F, including, but not limited to, inflammation, fever, cardiovascular effects, haemorrhage, coagulation and acute phase response, cachexia and anorexia, acute infections, shock states, graft versus host reactions and autoimmune disease.
  • the invention provides a method for the treatment or prophylaxis of conditions mediated by TNF, which method comprises the administration of a compound of formula (I), to a human or non-human mammal in need thereof.
  • the invention also provides a pharmaceutical composition for the treatment or prophylaxis of conditions mediated by TNF, which comprises a compound of formula (I) and optionally a pharmaceutically acceptable carrier therefor.
  • Particular inflammatory disorders include CNS disorders such as Alzheimers disease, multiple sclerosis, and multi-infarct dementia, as well as the inflammation mediated sequelae of stroke and head trauma.
  • Salts of compounds of formula (I) include for example acid addition salts derived from inorganic or organic acids, such as hydrochlorides, hydrobromides, hydroiodides, p- toluenesulphonates, phosphates, sulphates, acetates, trifluoroacetates, propionates, citrates, maleates, fumarates, malonates, succinates, lactates, oxalates, tartarates and benzoates.
  • inorganic or organic acids such as hydrochlorides, hydrobromides, hydroiodides, p- toluenesulphonates, phosphates, sulphates, acetates, trifluoroacetates, propionates, citrates, maleates, fumarates, malonates, succinates, lactates, oxalates, tartarates and benzoates.
  • Salts may also be formed with bases.
  • Such salts include salts derived from inorganic or organic bases, for example alkali metal salts such as sodium or potassium salts, and organic amine salts such as morpholine, piperidine, dimethylamine or diethylamine salts.
  • the compounds of the present invention are potent inhibitors of CD23 processing and TNF release. Certain compounds ofthe invention exhibit reduced collagenase inhibitory activity in comparison with the above-mentioned compounds ofthe prior art.
  • the compounds ofthe invention may be prepared by use of any approriate conventional method, for example by analogy with the methods disclosed in patent publications WO 90/05716, WO 93/24475, WO 94/21625, WO 95/19956, WO 90/05719, WO 91/02716, WO 92/13831, WO 93/20047, EP-A-0214639, EP-A-0236872, EP-A- 0274453, EP-A-0489577, EP-A-0489579, EP-A-0497192, EP-A-0574758 and USP 4599361. Accordingly, a further aspect of the invention provides a process for preparing a compound of formula (I) as defined hereinabove, which process comprises: (a) deprotecting a compound of formula (II):
  • Rl to R ⁇ are as defined hereinabove, with a compound of formula R ⁇ .X, wherein R ⁇ is (C2-6) a lkenyl or (C2-6)alkynyl and X is a leaving group such as bromine or iodine; or
  • Compounds of formula (II) can be prepared from compounds of formula (III) by reaction with a protected hydroxylamme.
  • Compounds of formula (III) can be prepared by reacting a compound of formula (V):
  • Suitable protecting groups for a hydroxamic acid are well known in the art and include trimethylsilyl, t-butyl and t-butyldimethylsilyl.
  • R ⁇ is as defined hereinabove and R*> is a protecting group for carboxy such as t- butyl, with a compound of formula (IX):
  • the isomers, including stereoisomers, ofthe compounds ofthe present invention may be prepared as mixtures of such isomers or as individual isomers.
  • the individual isomers may be prepared by any appropriate method, for example individual stereoisomers may be prepared by stereospecific chemical synthesis starting from chiral substrates or by separating mixtures of diastereoisomers using known methods.
  • the invention provides compounds of formula (IA):
  • the compounds are isolated in substantially pure form.
  • an inhibitor ofthe formation of soluble human CD23 has useful medical properties.
  • the active compounds are administered as pharmaceutically acceptable compositions.
  • compositions are preferably adapted for oral administration. However, they may be adapted for other modes of administration, for example in the form of a spray, aerosol or other conventional method for inhalation, for treating respiratory tract disorders; or parenteral administration for patients suffering from heart failure. Other alternative modes of administration include sub lingual or transdermal administration.
  • the compositions may be in the form of tablets, capsules, powders, granules, lozenges, suppositories, reconstitutable powders, or liquid preparations, such as oral or sterile parenteral solutions or suspensions.
  • composition ofthe invention is in the form of a unit dose.
  • Unit dose presentation forms for oral administration may be tablets and capsules and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycollate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulphate.
  • binding agents for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone
  • fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine
  • tabletting lubricants for example magnesium stearate
  • disintegrants for example star
  • the solid oral compositions may be prepared by conventional methods of blending, filling or tabletting. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. Such operations are of course conventional in the art.
  • the tablets may be coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.
  • Oral liquid preparations may be in the form of, for example, 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, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel, 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 esters of 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,
  • fluid unit dosage forms are prepared utilizing the compound and a sterile vehicle, and, depending on the concentration used, can be either suspended or dissolved in the vehicle. Jn preparing solutions the compound can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampoule and sealing.
  • adjuvants such as a local anaesthetic, a preservative and buffering agents can be dissolved in the vehicle.
  • the composition can be frozen after filling into the vial and the water removed under vacuum.
  • Parenteral suspensions are prepared in substantially the same manner, except that the compound is suspended in the vehicle instead of being dissolved, and sterilization cannot be accomplished by filtration.
  • the compound can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle.
  • a surfactant or wetting agent is included in the composition to facilitate uniform distribution ofthe compound.
  • compositions of this invention may also suitably be presented for administration to the respiratory tract as a snuff or an aerosol or solution for a nebulizer, or as a microf ⁇ ne powder for insufflation, alone or in combination with an inert carrier such as lactose.
  • the particles of active compound suitably have diameters of less than 50 microns, preferably less than 10 microns for example diameters in the range of 1-50 microns, 1-10 microns or 1-5 microns.
  • small amounts of other anti-asthmatics and bronchodilators for example sympathomimetic amines such as isoprenaline, isoetharine, salbutamol, phenylephrine and ephedrine; xanthine derivatives such as theophylline and aminophylline and corticosteroids such as prednisolone and adrenal stimulants such as ACTH may be included.
  • sympathomimetic amines such as isoprenaline, isoetharine, salbutamol, phenylephrine and ephedrine
  • xanthine derivatives such as theophylline and aminophylline and corticosteroids such as prednisolone and adrenal stimulants such as ACTH
  • ACTH adrenal stimulants
  • compositions may contain from 0.1% to 99% by weight, preferably from 10-60% by weight, ofthe active material, depending upon the method of administration.
  • a preferred range for inhaled administration is 10-99%, especially 60-99%, for example 90, 95 or 99%.
  • Microfine powder formulations may suitably be administered in an aerosol as a metered dose or by means of a suitable breath-activated device.
  • Suitable metered dose aerosol formulations comprise conventional propellants, cosolvents, such as ethanol, surfactants such as oleyl alcohol, lubricants such as oleyl alcohol, desiccants such as calcium sulphate and density modifiers such as sodium chloride.
  • Suitable solutions for a nebulizer are isotonic sterilised solutions, optionally buffered, at for example between pH 4-7, containing up to 20mg/ml of compound but more generally 0.1 to lOmg/ml, for use with standard nebulisation equipment.
  • a unit dose form of a composition ofthe invention may contain from 0.1 to lOOOmg of a compound ofthe invention (0.001 to lOmg via inhalation) and more usually from 1 to 500mg, for example 1 to 25 or 5 to 500mg.
  • Such compositions may be administered from 1 to 6 times a day, more usually from 2 to 4 times a day, in a manner such that the daily dose is from lmg to lg for a 70 kg human adult and more particularly from 5 to 500mg. That is in the range of about 1.4 x 10 ⁇ 2 mg/kg/day to 14 mg/kg/day and more particularly in the range of about 7 x 10"2 mg/kg/day to 7 mg/kg/day.
  • Procedure 1 The ability of test compounds to inhibit the release of soluble CD23 was investigated by use ofthe following procedure.
  • Plasma membranes from RPMI 8866 cells, a human Epstein-Barr virus transformed B-cell line (Sarfati et al., Immunology 60 [1987] 539-547) expressing high levels of CD23 are purified using an aqueous extraction method.
  • Cells resuspended in homogenization buffer (20mM HEPES pH 7.4, 150 mM NaCI, 1.5 mM MgC12, 1 mM DTT) are broken by N2 cavitation in a Parr bomb and the plasma membrane fraction mixed with other membranes is recovered by centrifugation at 10,000Xg.
  • the light pellet is resuspended in 0.2 M potassium phosphate, pH 7.2 using 2 ml per 1-3 g wet cells and the nuclear pellet is discarded.
  • the membranes are further fractionated by partitioning between Dextran 500 (6.4% w/w) and polyethylene glycol (PEG) 5000 (6.4% w/w) (ref), at 0.25 M sucrose in a total of 16 g per 10-15 mg membrane proteins [Morre and Morre, BioTechniques 7, 946-957 (1989)].
  • the phases are separated by brief centrifugation at lOOOXg and the PEG (upper) phase is collected, diluted 3-5 fold with 20 mM potassium phosphate buffer pH 7.4, and centrifuged at 100,000Xg to recover membranes in that phase.
  • the pellet is resuspended in phosphate-buffered saline and consists of 3-4 fold enriched plasma membranes as well as some other cell membranes (e.g. lysosomes, Golgi).
  • the membranes are aliquoted and stored at -80°C Fractionation at 6.6 % Dextran/PEG yields plasma membranes enriched 10-fold.
  • fractionated membranes are incubated at 37°C for times up to 4 hrs to produce fragments of CD23 which are separated from the membrane by filtration in 0.2 micron
  • sCD23 released from the membrane is determined using the EIA kit from The Binding Site (Birmingham, UK) or a similar one utilizing MHM6 anti-CD23 mAb [Rowe et al., Int. J. Cancer, 29, 373-382 (1982)] or another anti-CD23 mAb as the capture antibody in a sandwich EIA..
  • the amount of soluble CD23 made by 0.5 ug membrane protein in a total volume of 50 ul phosphate-buffered saline is measured by EIA and compared to the amount made in the presence of various concentrations of inhibitors.
  • Inhibitors are prepared in solutions of water or dimethylsulfoxide (DMSO) and the final DMSO concentration is not more than 2 %. IC50's are determined by curve fitting as the concentration where 50 % inhibition of production of sCD23 is observed relative to the difference in sCD23 between controls incubated without inhibitor.
  • DMSO dimethylsulfoxide
  • Procedure 2 The ability of test compounds to inhibit collagenase was investigated using the following procedure.
  • the potency of compounds to act as inhibitors of collagenase was determined by the method of Cawston and Barrett (Anal. Biochem. 99, 340-345, 1979), hereby inco ⁇ orated by reference, whereby a 1 mM solution ofthe inhibitor being tested or dilutions thereof, was incubated at 37 °C for 18 h with collagen and human recombinant collagenase, from synovial fibroblasts cloned, expressed and purified from E. Coli, (buffered with 150 mM Tris, pH 7.6, containing 15 mM calcium chloride, 0.05% Brij 35, 200 mM sodium chloride and 0.02% sodium azide).
  • the collagen was acetylated 3 H type 1 bovine collagen prepared by the method of Cawston and Murphy (methods in Enzymology ⁇ Q, 711,1981)
  • the samples were centrifuged to sediment undigested collagen and an aliquot ofthe radioactive supernatant removed for assay on a scintillation counter as a measure of hydrolysis.
  • the collagenase activity in the presence of ImM inhibitor, or dilution thereof, was compared to activity in a control devoid of inhibitor and the results reported as that concentration effecting 50% ofthe collagenase (IC50).
  • Procedure 3 The ability of test compounds to inhibit TNF release was investigated using the following procedure.
  • Assay for inhibition of release of TNF ⁇ from human monocytes stimulated by lipopoiysaccharide (LPS) endotoxin Human moncytes, cultured in RPMI 1640 medium supplemented with 10 % fetal calf serum, are centrifuged at lOOOXg for 5 min and then resuspended in medium at 2 X 10 6 cells/ ml. The cell suspension is aliquoted in 24 well plates, 1 ml per well. Compounds to be tested are dissolved in neat dimethyl sulfoxide (DMSO) and added to culture with the final DMSO concentration at 0.1 %. Compounds are added to cells in triplicate wells.
  • DMSO dimethyl sulfoxide
  • TNF ⁇ release is stimulated by addition of LPS to the cells at a final concentration of 200 ng/ml. Appropriate control cultures are set up in triplicates also. The plates are incubated for 18-20 hrs at 37° C, 5% CO 2 , then centrifuged at 1000 Xg for 5 min. A specific ELISA for human TNF ⁇ (SmithKhne Beecham) is used to measure TNF levels in the cell-free culture supernatants.
  • the comparative example was Example 2 of WO 90/05719, the compound of formula:
  • R is CH2S-(2-thienyl) and R 1 is methyl

Abstract

L'invention concerne les composés présentant la formule (I), dans laquelle R est (C2-6)alcénylthiométhyle ou (C2-6)alcynylthiométhyle; R1 représente alkyle ou alcényle; R2 représente alkyle, alcényle, aryle, cycloalkyle ou cycloalcényle; et R3 représente hydrogène, alkyle, alcényle, alcynyle ou aryle. Ces composés sont utiles pour traiter les troubles provoqués par s-CD23.
PCT/EP1997/002500 1996-05-10 1997-05-07 Composes a base d'acide hydroxamique, inhibiteurs de la formation de cd23 et de facteur de necrose des tumeurs WO1997043250A1 (fr)

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JP09540534A JP2000510138A (ja) 1996-05-10 1997-05-07 ヒドロキサム酸をベースとするｃｄ２３およびｔｎｆ形成の阻害剤
EP97923088A EP0901464A1 (fr) 1996-05-10 1997-05-07 Composes a base d'acide hydroxamique, inhibiteurs de la formation de cd23 et de facteur de necrose des tumeurs

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GBGB9609795.1A GB9609795D0 (en) 1996-05-10 1996-05-10 Novel compounds
GB9609795.1 1996-05-10

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Cited By (5)

* Cited by examiner, † Cited by third party
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WO1998055449A1 (fr) * 1997-06-06 1998-12-10 The University Of Queensland Composes d'acide hydroxamique ayant des proprietes anticancereuses et antiparasitaires
WO2002006227A1 (fr) * 2000-07-18 2002-01-24 Chugai Seiyaku Kabushiki Kaisha Inhibiteurs de la metalloprotease matricielle
EP1992636A2 (fr) 1999-11-12 2008-11-19 Amgen Inc. Procédé pour la correction d'un mauvais repliement de bisulfure dans les molécules Fc
EP2087908A1 (fr) 2001-06-26 2009-08-12 Amgen, Inc. Anticorps opgl
WO2020070239A1 (fr) 2018-10-04 2020-04-09 INSERM (Institut National de la Santé et de la Recherche Médicale) Inhibiteurs de l'egfr pour traiter les kératodermies

Citations (3)

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EP1992636A2 (fr) 1999-11-12 2008-11-19 Amgen Inc. Procédé pour la correction d'un mauvais repliement de bisulfure dans les molécules Fc
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EP2087908A1 (fr) 2001-06-26 2009-08-12 Amgen, Inc. Anticorps opgl
EP3492100A1 (fr) 2001-06-26 2019-06-05 Amgen Inc. Anticorps pour opgl
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EP0901464A1 (fr) 1999-03-17
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