WO2009050227A1 - Dérivés de pyridazine pour inhiber la production de peptide béta amyloïde - Google Patents

Dérivés de pyridazine pour inhiber la production de peptide béta amyloïde Download PDF

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WO2009050227A1
WO2009050227A1 PCT/EP2008/063950 EP2008063950W WO2009050227A1 WO 2009050227 A1 WO2009050227 A1 WO 2009050227A1 EP 2008063950 W EP2008063950 W EP 2008063950W WO 2009050227 A1 WO2009050227 A1 WO 2009050227A1
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methyl
phenyl
methyloxy
mmol
imidazol
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PCT/EP2008/063950
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English (en)
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Andrew John Eatherton
Gerard Martin Paul Giblin
Adrian Hall
Matthew Russell Johnson
Joelle Le
William Leonard Mitchell
James Myatt
David Norton
Helen Susanne Price
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Glaxo Group Limited
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Publication of WO2009050227A1 publication Critical patent/WO2009050227A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings

Definitions

  • the present invention relates to novel compounds that inhibit the production of ⁇ - amyloid peptide (1-42), processes for their preparation, to compositions containing them and to their use in the treatment of diseases characterised by elevated ⁇ -amyloid levels or ⁇ -amyloid deposits, particularly Alzheimer's disease.
  • Alzheimer's disease is the most common cause of age-related cognitive decline, affecting greater than 12 million individuals worldwide (Citron M, (2002), Nat. Neurosci, 5, Suppl 1055-1057). The earliest stages of the disease are characterized by a progressive loss of memory with associated cognitive decline and language and behavioural deficits. In the later stages of the disease, patients develop global amnesia and have greatly reduced motor function. Death typically occurs 9 years following diagnosis and is often associated with other conditions, typically pneumonia (Davis K.L. and Samuels S. C. (1998) in Pharmacological Management of Neurological and
  • ⁇ -amyloid peptide is key to the progression of Alzheimer's disease and extracellular deposition of ⁇ -amyloid peptide in the form of senile plaques represents a key pathological hallmark (Selkoe, D. J. (2001 ) Physiological Reviews 81 : 741-766). The presence of senile plaques is accompanied by a prominent inflammatory response and neuronal loss.
  • the ⁇ -amyloid peptide exists in soluble and insoluble, fibrillar forms and a specific fibrillar form has been identified as the predominant neurotoxic species (Vassar, R. and Citron, M. (2000) Neuron 27: 419-422).
  • the ⁇ amyloid peptide is known to be produced by sequential cleavage of the membrane-bound beta amyloid precursor protein (also known as APP) in a process known as regulated intermembrane proteolysis (RIP). Firstly, APP is cleaved by an aspartyl protease enzyme known as Asp2 (also known as ⁇ -secretase, BACE1 or Memapsin-2), creating the N-terminus of the ⁇ -amyloid peptide (De Strooper, B. and Konig, G. (1999) Nature 402: 471-472).
  • Asp2 also known as ⁇ -secretase, BACE1 or Memapsin-2
  • the ⁇ -amyloid peptide is then released extracellularly by cleavage by gamma secretase, which also releases the APP intracellular domain into the cell (Haas, 2004).
  • Gamma secretase is a complex of four proteins (presenilin, Net, Aph-1 and PEN-2). It is able to cleave APP in several places, creating ⁇ amyloid peptides of different lengths. In normal individuals, the most abundant form of amyloid peptide is the 1-40 species (A ⁇ 1-40) forming approximately 90% of the detectable amyloid, with the more fibrillogenic 1-42 species (A ⁇ 1-42) forming approximately 10%.
  • a ⁇ (1-40) In Alzheimer's disease patients, however, the levels of A ⁇ (1-40) drop to approximately 50% whereas the levels of A ⁇ (1-42) rise to approximately 50%, whilst, concurrently, the total CNS amyloid load increases dramatically.
  • the A ⁇ (1-42) peptide is thought to play a critical role in Alzheimer's disease (McGowan et al (2005) Neuron 47:191-199). It has been shown to fibrilise more readily than A ⁇ (1-40), can induce oligomerisation of other ⁇ amyloid peptide species, is more neurotoxic than A ⁇ (1- 40) (Butterfield et al (2004) Brain Pathol. 14:426-432) and is the most abundant species within Alzheimer's disease plaques.
  • AD amyloid precursor protein
  • APP amyloid precursor protein
  • presenilin genes are associated with the familial forms of Alzheimer's disease (AD) characterised by early onset and accelerated pathology and result in increased levels of A ⁇ (1-42) (Selkoe DJ. , Podlisny M. B. (2002) Annu. Rev. Genomics Hum. Gemet. 3:67-99).
  • amyloid hypothesis centres around the theory that by reducing the amyloid accumulation in the brain, subsequent events would be prevented or reduced and that this would then have a beneficial effect on cognitive decline (Selkoe DJ. (2001 ) Physiological Reviews 81 : 741-766).
  • One mechanism by which this may be achieved is by inhibiting gamma secretase activity such that the levels of ⁇ amyloid are reduced.
  • Animal models of amyloid deposition have been generated by overexpression of mutant human transgenes in mice.
  • gamma secretase inhibitors reduce ⁇ amyloid levels and plaque load and improve cognition in these models and in wild type rats (El Mouedin M. et al (2006) Curr. Pharm. Des. 12:671-676; Comery TA et al (2005) J. Neurosci. 25:8898-8902).
  • Gamma secretase cleaves a number of other substrates in addition to APP (Medina M and Dotti CG (2003) Cell Signalling 15:829-841 ). Inhibition of gamma secretase activity has been associated with dramatic side effects in the gut and immune system which have been directly linked to inhibition of cleavage of Notch, another gamma secretase substrate (Wong GT. et al (2004) Journal of Biological Chemistry 279:12876-12882). It has recently been reported that it is possible to lower brain amyloid without affecting Notch (Eriksen J. L. (2003) J. Clin. Invest. 1 12:440-449).
  • the present invention provides, in a first aspect, a compound of formula (I) or a pharmaceutically acceptable salt thereof
  • R1 is selected from the group consisting of C1-6 alkoxy, C1-4 alkyl, and hydrogen;
  • R2 represents hydrogen, methyl or cyano, or -CO.R5, where R5 is -OH or -NR6R7 in which R6 and R7 are both H or alkyl; or R6 is H and R7 is alkyl, haloalkyl, hydroxyalkyl, or cycloalkyl; or R6 and R7 together with N form a 5 or 6 membered heterocylic ring optionally containing an additional heteroatom, which is optionally substituted; and
  • R3 and R4 together with the nitrogen atom to which they are attached, represent a five or six-membered saturated ring which optionally contains an additional heteroatom ,and which is optionally substituted or fused to a phenyl or heteroaryl group; or
  • R3 and R4 is hydrogen and the other of R3 and R4 is cycloalkyl or unsaturated heterocyclyl (each of which is optionally substituted and optionally fused to a phenyl group); or C4-6 alkyl which is alpha-branched and optionally substituted; or a C1-3 alkyl group which is alpha-substituted by a moiety selected from the group consisting of; optionally substituted phenyl, cycloalkyl which is optionally fused to a phenyl group, and optionally substituted saturated or unsaturated heterocyclyl, which C1-3 alkyl group is optionally additionally substituted elsewhere.
  • alkyl' refers to a linear or branched saturated hydrocarbon group.
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert butyl, n-pentyl, isopentyl (3-methylbutyl), neopentyl (2,2- dimethylpropyl), hexyl, 4-methylpentyl, 3,3-dimethylbutyl, 1 ,2,2 trimethy propyl and the like.
  • Alkyl groups may optionally be substituted by for example halogen or hydroxy, such as hydroxypropyl and hydroxyethyl.
  • 'cycloalkyl' refers to a saturated monocyclic hydrocarbon ring.
  • cycloalkyl groups include cyclopentyl and cyclohexyl.
  • Optionally substituted phenyl may be substituted by up to three groups which may be the same or different and may be independently selected from halogen, trifluoromethyl, cyano, phenyl, methyl, methoxy, ethyl, morpholino and pyrazolyl.
  • 'halogen' refers to a fluorine, chlorine, bromine or iodine atom.
  • 'heteroatom means oxygen, nitrogen or sulphur.
  • R1 may be C1-4 alkoxy, such as methoxy, or C1-4 alkyl, such as methyl.
  • R2 represents -CO.R5
  • typical values include carboxyl, carboxamide, pyrrolidinylcarbonyl, morpholino-carbonyl, cyclopropyl-carboxamide, ethyl-carboxamide, hydroxyethyl-carboxamide, dimethyl-carboxamide,
  • R3 and R4 together with the nitrogen atom to which they are attached, may represent a five or six-membered saturated ring such as piperidinyl and pyrrolidinyl. This group may be substituted as in methylpiperidinyl, hydroxyethylpiperidinyl, hydroxypropylpiperidinyl, methoxypiperidinyl,and methylpyrrolidinyl.
  • Such rings which are fused to a phenyl group include dihydroisoindolyl.
  • Suitable groups which optionally contain an additional heteroatom include morpholinyl and phenylmorpholinyl, piperazinyl and N-methylpiperazinyl, N-phenylpiperazinyl, N- (methylphenyl)piperazinyl and phenyl-N-methylpiperazinyl.
  • R3 and R4 When one of R3 and R4 is hydrogen and the other of R3 and R4 is cycloalkyl or unsaturated heterocyclyl suitable groups include cyclopentyl, cyclohexyl, and tetrahydropyranyl,
  • Such groups fused to a phenyl group include dihydroindenyl, dihydrobenzofuranyl, dihydrochromenyl.
  • Alpha-branched, optionally substituted C4-6 alkyl groups include trimethylpropyl, hydroxy-methylbutyl and hydroxy-dimethylbutyl.
  • R3/R4 is an alpha-substituted C1-3 alkyl group
  • suitable values include fluorophenyl-ethyl, biphenylyl-methyl, fluorophenyl-methyl, chlorophenyl-ethyl, methyloxy-phenyl, cyanophenyl-methyl, morpholinyl-phenyl-methyl, phenyl-methyl, cyclopentyl-methyl, tetrahydropyranyl-methyl, phenyl-furanyl-methyl, pyrazolyl-phenyl- methyl, ethylphenyl-methyl, fluorophenyl-propyl, dimethyl-isoxazolyl-methyl, methyl- pyrazinyl-piperidinyl-methyl, difluoropyridinyl-methyl, trifluoromethyl-phenyl-ethyl.
  • difluorophenyl-methyl trifluorophenyl-methyl, phenyl-hydroxyethyl, morpholinyl-phenyl- ethyl, phenyl-(pyrrolidinyl)ethyl, phenyl-methoxyethyl, and (dimethylethyloxycarbony ⁇ piperidinyl-methyl.
  • R1 is selected from the group consisting of methoxy, methyl, and hydrogen;
  • R2 represents methyl or hydrogen; and R3 and R4, together with the nitrogen atom to which they are attached, represent a five or six-membered saturated ring which is optionally substituted (for example by methyl) or fused to a phenyl group; or one of R3 and R4 is hydrogen and the other of R3 and R4 is cycloalkyl or a C1-3 alkyl group which is substituted at the 1- or 2-position by a moiety selected from the group consisting of; optionally substituted phenyl, cycloalkyl which is optionally fused to a phenyl group, and 1 -(1 , 1 -dimethylethyloxycarbonyl)piperidin-4-yl.
  • a particular embodiment of the invention provides compounds of formula (IA):
  • R1 is methoxy
  • R2 is hydrogen or methyl
  • R5 is optionally substituted phenyl, especially fluoro-substituted phenyl such as 2-fluorophenyl or 4-fluorophenyl.
  • Compounds of the invention include those of the Examples below, and pharmaceutically acceptable salts thereof.
  • Pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse, J. Pharm. Sci., 1977, 66, 1-19.
  • pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminium, ammonium, bismuth, calcium, copper, ferric, ferrous, lithium, magnesium, potassium, sodium, zinc and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, and cyclic amines.
  • Particular pharmaceutically acceptable organic bases include arginine, caffeine, choline, N, N'- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tris(hydroxymethyl)aminomethane (TRIS, trometamol) and the like.
  • Salts may also be formed from basic ion exchange resins, for example, polyamine resins. Salts may also be prepared from pharmaceutically acceptable acids including inorganic and organic acids. Such acids include acetic, L-ascorbic acid (vitamin C), L-aspartic acid, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanedisulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, malonic, mandelic, methanesulfonic, mucic, nicotinic, phosphoric, succinic, sulphuric, tartaric, p-toluenesulfonic, perchloric, fluoboric, and the like.
  • acids include acetic, L-ascorbic acid (vitamin C), L-aspartic acid, benzenesulfonic, benzoic, camphorsulf
  • the invention includes within its scope all possible stoichiometric and non-stoichiometric forms of the salts of the compounds of formula (I).
  • the invention also includes within its scope solvates (including hydrates) of the compounds of formula (I) and their pharmaceutically acceptable salts.
  • Certain compounds of formula (I) are capable of existing in stereoisomeric forms. It will be understood that the invention encompasses all geometric and optical isomers of these compounds and the mixtures thereof including racemates.
  • the different stereoisomeric forms may be separated one from the other by methods known in the art (e.g. separation by chiral HPLC), or any given isomer may be obtained by stereospecific or asymmetric synthesis.
  • the invention also extends to any tautomeric forms and mixtures thereof.
  • the present invention also provides a process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof, which process comprises reacting a compound of formula (II) with a compound of formula (III):
  • L1 and L2 are a leaving group such as halogen (eg bromine or iodine) and the other of L1 and L2 is a metal or metalloid residue such as B(OH) 2 , using conditions suitable for cross coupling reactions, typically involving use of a transition metal such as palladium in the presence of a suitable ligand such as triphenylphosphine, a base such as sodium carbonate and an appropriate solvent such as 1 ,2-dimethoxyethane, either at ambient temperature or elevated temperature (e.g.
  • the invention also provides a process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof, which process comprises treating a compound of formula (IV):
  • diseases characterised by elevated ⁇ -amyloid levels or ⁇ - amyloid deposits include Alzheimer's disease, mild cognitive impairment, Down's syndrome, hereditary cerebral haemorrhage with ⁇ -amyloidosis of the Dutch type, cerebral ⁇ -amyloid angiopathy and various types of degenerative dementias, such as those associated with Parkinson's disease, progressive supranuclear palsy, cortical basal degeneration, diffuse Lewis body type of Alzheimer's disease, multiple sclerosis and stroke.
  • Certain compounds of formula (I) may spare Notch signalling pathways.
  • the invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, for use as a therapeutic substance in the treatment or prophylaxis of the above disorders, in particular Alzheimer's disease.
  • the invention further provides a method of treatment or prophylaxis of the above disorders, in mammals including humans, which comprises administering to the sufferer a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • the invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of the above disorders.
  • the compounds of formula (I) are usually formulated in a pharmaceutical composition.
  • Such compositions can be prepared using standard procedures.
  • the present invention further provides a pharmaceutical composition for use in the treatment of the above disorders which comprises the compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • the present invention further provides a pharmaceutical composition which comprises the compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • Compounds of formula (I) may be used in combination with other therapeutic agents, for example medicaments claimed to be useful as either disease modifying or symptomatic treatments of Alzheimer's disease.
  • Suitable examples of such other therapeutic agents may be symptomatic agents, for example those known to modify cholinergic transmission such as M1 and M3 muscarinic receptor agonists or allosteric modulators, M2 muscarinic antagonists, acetylcholinesterase inhibitors (such as tetrahydroaminoacridine, donepezil hydrochloride and rivastigmine), nicotinic receptor agonists or allosteric modulators (such as ⁇ 7 agonists or allosteric modulators or ⁇ 4 ⁇ 2 agonists or allosteric modulators), PPAR agonists (such as PPAR ⁇ agonists), 5-HT 4 receptor agonists or partial agonists, histamine H3 antagonists, 5-HT 6 receptor antagonists or 5HT1 A receptor ligands and
  • the invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof together with a further therapeutic agent or agents.
  • Therapeutic agents which have been suggested for use in the treatment or prophylaxis of diseases characterised by elevated ⁇ -amyloid levels or ⁇ -amyloid deposits include, in addition to those set out above, immunotherapeutic agents such as antibodies, including antibodies against A ⁇ , alpha secretase activators, A ⁇ aggregation inhibitors, antioxidants, NGF (nerve growth factor) mimics, HMG-CoA (3-hydroxy-3-methyl-glutaryl- CoA) reductase inhibitors (statins) and other lipid-lowering drugs, AMPA ligands, calcium channel blockers, GABA receptor antagonists, GSK-3 (Glycogen Synthase Kinase-3) inhibitors, phosphodiesterase inhibitors, 5HT antagonists, melatonin (MT1 and/or MT2) agonists, insulin degrading enzyme (IDE) activators, neprilysin (NEP) activators, plasmin activators, MARK (microtubule affinity regulating kinase) inhibitors,
  • compositions comprising a combination as defined above together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the invention.
  • the individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.
  • each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.
  • a pharmaceutical composition of the invention which may be prepared by admixture, suitably at ambient temperature and atmospheric pressure, is usually adapted for oral, parenteral or rectal administration and, as such, may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable or infusible solutions or suspensions or suppositories. Orally administrable compositions are generally preferred.
  • Tablets and capsules for oral administration may be in unit dose form, and may contain conventional excipients, such as binding agents, fillers, tabletting lubricants, disintegrants and acceptable wetting agents.
  • 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 suspension, solutions, emulsions, syrups or elixirs, or may be in the form of a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), preservatives, and, if desired, conventional flavourings or colorants.
  • fluid unit dosage forms are prepared utilising a compound of the invention or pharmaceutically acceptable salt thereof and a sterile vehicle.
  • the compound depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle.
  • the compound can be dissolved for injection and filter sterilised before filling into a suitable vial or ampoule and sealing.
  • adjuvants such as a local anaesthetic, preservatives and buffering agents are 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 sterilisation cannot be accomplished by filtration.
  • the compound can be sterilised by exposure to ethylene oxide before suspension in a sterile vehicle.
  • a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.
  • the composition may contain from 0.1 % to 99% by weight, preferably from 10 to 60% by weight, of the active material, depending on the method of administration.
  • the dose of the compound used in the treatment of the aforementioned disorders will vary in the usual way with the seriousness of the disorders, the weight of the sufferer, and other similar factors.
  • the following Descriptions and Examples illustrate the preparation of compounds of the invention. In the procedures that follow, after each starting material, reference to a Description is typically provided. This is provided merely for assistance to the skilled chemist. The starting material may not necessarily have been prepared from the batch referred to.
  • references in the Examples below relating to the drying of organic layers or phases may refer to drying the solution over magnesium sulfate or sodium sulfate and filtering off the drying agent in accordance with conventional techniques. Products may generally be obtained by removing the solvent by evaporation under reduced pressure.
  • Chromatographic methods are known to the skilled person and include e.g. column chromatography, flash chromatography, HPLC (high performance liquid chromatography), and MDAP (mass directed autopreparation, also referred to as mass directed LCMS purification).
  • MDAP is described in e.g. W. Goetzinger et al, Int. J. Mass Spectrom., 2004, 238, 153-162.
  • the column used is a Waters Atlantis, the dimensions of which are 4.6mm x 50mm.
  • the stationary phase particle size is 3um.
  • the generic method used has a 5 minute runtime.
  • the above method has a flow rate of 3ml/min.
  • the injection volume for the generic method is 5ul
  • the column temperature is 30 degrees
  • the UV detection range is from 220 to 330nm
  • the column used is a Waters Acquity BEH UPLC C18, the dimensions of which are 2.1 mm x 50mm.
  • the stationary phase particle size is 1.7m m.
  • Aqueous solvent Water + 0.05% Formic Acid
  • the eneric method used has a 2 minute runtime.
  • the above method has a flow rate of 1 ml/min.
  • the UV detection range is from 220 to 330nm
  • the columns used are Waters Atlantis, the dimensions of which are 19mm x 100mm (small scale) and 30mm x 100mm (large scale).
  • the stationary phase particle size is 5m m.
  • Trifluoroacetic anhydride (3.45 ml) was added dropwise to a pyridine (10 ml) solution of 4-bromo-2-(methyloxy)aniline (3.34 g) under ice cooling and the reaction mixture was stirred at 0 0 C for one hour. Iced water (100 ml) was added and the mixture extracted with ethyl acetate (2 x 50 ml). The combined ethyl acetate was washed with water, dried (MgSO 4 ), evaporated and the residue loaded onto three Biotage Si 40+M columns. Purification was carried out on the Biotage SP4 (t.l.c. method based on a t.l.c.
  • the solid obtained was suspended in methanol (40 ml) and 2N aqueous sodium hydroxide solution (6 ml) and the mixture stirred for one hour to give a solution. After a total of ninety minutes, iced water (200 ml) was added and the aqueous extracted with ethyl acetate. The organic layer was dried (MgSO 4 ) and evaporated and the residue was dissolved in dichloromethane and loaded onto a Biotage Si 40+M column. Purification was carried out on the Biotage SP4 (t.l.c. method based on a t.l.c. run in 50 % dichloromethane / hexane) to afford the title compound (1.83 g) as a yellow oil.
  • Biotage SP4 t.l.c. method based on a t.l.c. run in 50 % dichloromethane / hexane
  • the reaction mixture was poured into a saturated aqueous solution of NaHCO 3 solution (100 ml) and extracted with EtOAc (3 x 100 ml). The combined organic solutions were washed with brine, dried over MgSO 4 and evaporated to dryness. The crude residue on a 12+M Biotage silica cartridge, eluting with a 0 to 10 % mixture of MeOH in Et 2 O to give the title compound (46 mg) as a light brown solid.
  • N-(3-Biphenylylmethyl)-6-chloro-3-pyridazinamine (2.22 g, 7.49 mmol) was dissolved in 1 ,2-dimethoxyethane (37 ml), degassed by purging with argon, treated with 4-methyl-1- [2-(methyloxy)-4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)phenyl]-1 /-/-imidazole (2.35 g, 7.49 mmol), PdCI 2 (dppf)-CH 2 CI 2 adduct (0.183 g, 0.225 mmol) and a degassed 2 M aqueous solution of sodium carbonate (15.0 ml, 30.0 mmol) then heated to 100 0 C for 3 hours.
  • the reaction mixture was then cooled to RT, poured into a saturated aqueous solution of NaHCO 3 (100 ml) and extracted with EtOAc (100 ml X 3). The combined organic solutions were washed with brine (50 ml), dried over MgSO 4 , filtered and evaporated to dryness to give the crude product (5.65 g) as a dark brown oil.
  • the crude product was purified by flash column chromatography on silica, eluting with a 0 to 5 % mixture of MeOH in DCM. The product was freeze-dried from 1 ,4-dioxane/water, dissolved in 1 ,4-dioxane, treated with 1.5 eq.
  • reaction time was 5 mins.
  • the crude product was purified by MDAP, extracted from aqueous K 2 CO 3 with EtOAc (x 3) and the combined organic solutions dried over MgSO 4 , filtered and evaporated.
  • the product residue was treated with 1 M ethereal HCI and dried to give the title compound (1 1 mg) as an off-white solid.
  • the crude product was purified by flash column chromatography on silica, eluting with a 0 to 10 % mixture of MeOH in DCM.
  • the product was further purified using a C18 reversed phase column, eluting with a 5 to 100% mixture of MeCN in water and then by MDAP.
  • the product was dissolved in DCM, treated with 2 equivalents of a 1 M solution of HCI in Et 2 O. This precipitated the title compound (37 mg) as a light yellow solid.
  • the reaction mixture was diluted with sodium bicarbonate (30ml) and ethyl acetate (30ml). The mixture was then washed with water (2 times 30ml) and saturated sodium chloride (30ml) and the organic layer extracted and dried with magnesium sulphate. This was then filtered and the solvent removed in vacuo to leave a brown oil (248mg). This was purified via Biotage SP4 (0 to 50% ethyl acetate /hexane, 25+S column, 15CVs). The appropriate fractions were combined and the solvent removed in vacuo to give the title compound as a yellow solid (159mg).
  • reaction mixture was filtered and diluted with dichloromethane (40ml). The solvents were then removed in vacuo to give a brown oil.
  • D15 6-chloro- ⁇ /-[(1 S)-1-(4-fluorophenyl)propyl]-3-pyridazinamine (199 mg, 0.749 mmol) and 2M aqueous sodium carbonate (1.497 ml, 2.99 mmol) in 1 ,2- dimethoxyethane (4.5ml).
  • the crude product was purified on a silica column (25M) by flash chromatography (Biotage SP4, methanol/dichloromethane, 0-5% (12 CV), 5% (5 CV)) to give a yellow oil (394 mg).
  • the product was re-purified by mass- directed auto-preparation to give the formate of the title compound as a yellow gum (230 mg).
  • the product was dissolved in methanol and converted to the hydrochloride salt by the addition of 1 M hydrochloric acid in diethyl ether (1.1eq), then dried to give the title compound as a yellow solid (222mg).
  • 3,6-Dichloropyridazine (1.5 g, 10.07 mmol) was dissolved in ⁇ /-methyl-2-pyrrolidone (NMP) (7.5 ml) in a 20ml microwave vessel. To this was added (1 S)-1-(4-fluorophenyl)- ⁇ /-methylethanamine (1.542 g, 10.07 mmol) and di-isopropylethylamine (1.759 ml, 10.07 mmol). The solution was irradiated at 200 0 C in a sealed vessel using microwaves for 1 hour.
  • NMP ⁇ /-methyl-2-pyrrolidone
  • (2S)-3,3-dimethyl-2-butanamine (40mg) was added and the reaction continued as above for a further 30 minutes.
  • the reaction mixture was transferred in methanol and then concentrated in vacuo to give a brown oil (218mg).
  • This was purified by column chromatography on Biotage SP4 25S silica eluting in 0% 2M ammonia in methanol/dichloromethane (5CV), 0-5% 2M ammonia in methanol/dichloromethane (8CV), 5% 2M ammonia in methanol/dichloromethane (5CV).
  • the appropriate fractions were combined and concentrated in vacuo to yield crude product (119mg). This was now purified by mass- directed auto-preparation.
  • (2S)-2- amino-3,3-dimethyl-1-butanol (30mg) was added and the reaction irradiated at 18O 0 C for a further 15mins.
  • the reaction was transferred in methanol and concentrated in vacuo to yield a dark oil (509mg).
  • This was purified by column chromatography on Biotage SP4 4OM silica, eluting in 0% 2M ammonia in methanol/dichloromethane (5CV), 0-5% 2M ammonia in methanol/dichloromethane (8CV), 5% 2M ammonia in methanol/dichloromethane (6CV). The appropriate fractions were combined and concentrated in vacuo to yield crude product (61 mg).
  • Tetrakis(triphenylphosphine)palladium(0) (33.3 mg, 0.029 mmol) and 2M aqueous sodium carbonate (3.84 ml, 7.69 mmol) were added.
  • the reaction mixture was heated at reflux under argon for 4.5 hours.
  • the reaction mixture was diluted with ethyl acetate and water and washed thoroughly with saturated aqueous sodium carbonate solution.
  • the organics were separated and the aqueous extracted with 3 times ethyl acetate.
  • the combined organics were then washed with brine and dried over magnesium sulphate, filtered and concentrated in vacuo to yield a yellow oil (508mg).
  • Phosphorus oxychloride (459 ⁇ l, 4.93 mmol) was added to 3- ⁇ [(1 S)-1-(4- fluorophenyl)ethyl]amino ⁇ -6-[4-(4-methyl-1 H-imidazol-1-yl)-3-(methyloxy)phenyl]-4- pyridazinecarboxamide (110 mg, 0.246 mmol) and the mixture heated to 100 0 C for 1 h. Further phosphorus oxychloride (459 ⁇ l, 4.93 mmol) was added and the reaction heated for a further 3 h. The reaction was allowed to stand at room temperature overnight.
  • the POCI 3 was removed by rotary evaporator and the residue partitioned between ethyl acetate (20 ml.) and a mixture of water (10 ml.) and aqueous sodium bicarbonate (10 ml.) - a small amount of methanol was added to aid dissolution of some solid.
  • the organic phase was washed with further water (10 ml.) and brine (10 ml.) before it was dried (MgSO 4 ), filtered and the solvent removed in vacuo.
  • the compound of E38 was used as the free acid in E39 et seq, and was converted to its hydrochloride salt (for testing in biological assays - see hereinbelow) using the following method:
  • the reaction mixture was partitioned between ethyl acetate (20 ml) and aqueous sodium bicarbonate (20 ml) before the organic phase was washed with further sodium bicarbonate (20 ml_), water (10 ml.) and brine (10ml), dried (MgSO 4 ), filtered and the solvent removed in vacuo.
  • the residue was purified by silica chromatography, eluting 0-5% MeOH in DCM.
  • the resulting oil was dissolved in DCM and 1 M HCI in ether (0.12ml, 2 eq.) was added. Due to an impurity present, the material was loaded onto an SCX cartridge, washed with methanol and eluted with 2M ammonia in methanol.
  • the solid product was then dissolved in DCM and 1 M HCI in ether (0,07 ml.) added before concentrating to give the title compound (12 mg, 0.021 mmol) as a yellow solid.
  • Ethylamine hydrochloride was used for this reaction instead of the free base and four equivalents of DIPEA were added.
  • the organic layer was dried through a hydrophobic frit and the mixture was evaporated to an orange oil.
  • the crude product was purified using chromatography (40-M Si) eluting with 1 CV 12% EtOAc/hexane, 10CV 12-100% EtOAc/hexane and 1 CV 100% EtOAc/hexane.
  • the desired fractions were combined and evaporated to a yellow oily solid (1.638g) This was re-purified using chromatography (40-M Si) eluting with 1 CV 12% EtOAc/hexane, 10CV 12-100% EtOAc/hexane and 1 CV 100% EtOAc/hexane.
  • the desired fractions were combined and evaporated to give the title compounds as a yellow solid (0.9146g, 2.83mmol).
  • Pd(Ph3P)4 (18.69 mg, 0.016 mmol), 2M aqueous sodium carbonate solution (0.647 ml, 1.294 mmol) and 6-chloro- ⁇ /-[(3,5-difluoro-2-pyridinyl)methyl]-3-pyridazinamine (83 mg, 0.323 mmol) were added to a solution of 4-methyl-1-[2-(methyloxy)-4-(4, 4,5,5- tetramethyl-1 ,3,2-dioxaborolan-2-yl)phenyl]-1 H-imidazole (102 mg, 0.323 mmol) in 1 ,2- dimethoxyethane (5 ml).
  • reaction mixture was diluted with ethyl acetate (60ml) and washed with water (3 x
  • reaction mixture was loaded onto a Biotage Si 40+M column and purified using the Biotage SP4 eluting with 0 to 5% methanol/dichloromethane over 10CV to afford the title compound (204mg) as a brown solid
  • reaction mixture was diluted with ethyl acetate (50ml) and washed with water (2 x 25ml) then evaporated.
  • the reaction mixture was loaded onto a Biotage Si 25+M column and purified using the Biotage SP4 eluting with 0 to 10% methanol/dichloromethane over 10CV to afford the title compound (406mg) as a brown solid.
  • reaction mixture was loaded onto a Biotage Si 40+M column and purified using the Biotage SP4 eluting with 0 to 2% methanol/dichloromethane over 10CV, followed by trituration with diethyl ether. Transferred to a glass vial and dried at 5O 0 C under high vacuum to afford the title compound (695mg) as a white solid.
  • reaction mixture was loaded onto a Biotage Si 25+M column and purified using the Biotage SP4 eluting with 0 to 5% methanol/dichloromethane over 20CV to afford the title compound (196mg) as a white solid.
  • the reaction mixture was diluted with ethyl acetate (25ml) and washed with water (3 x 25ml) then evaporated.
  • the residue was dissolved in dichloromethane and applied to a Biotage Si 25+M column and purified on the Biotage SP4 eluting with 0-5% methanol: dichloromethane over 20CV followed by MDAP.
  • the residue was dissolved in dichloromethane and treated with 1 N hydrochloric acid in diethyl ether (0.25ml). The mixture was evaporated and dried at 5O 0 C under high vacuum to afford the title compound (33mg) as a white solid.
  • the reaction mixture was diluted with ethyl acetate (25ml) and washed with water (3 x 25ml) then evaporated.
  • the residue was dissolved in dichloromethane and applied to a Biotage Si 12+M column and purified on the Biotage SP4 eluting with 0-5% methanol: dichloromethane over 20CV.
  • the residue was dissolved in dichloromethane and treated with 1 N hydrochloric acid in diethyl ether (0.25ml).
  • the mixture was evaporated to a pale yellow solid which was triturated with diethyl ether (decanted off diethyl ether and repeated) evaporated and transferred to a glass vial and the title compound (43mg) dried at 5O 0 C under high vacuum.
  • the enantiomers were separated using a Chiralcel OD (20mm x 250mm, 1OuM) column using heptane/ethanol (70:30) as eluant.
  • the faster running enantiomer was dissolved in dichloromethane and treated with 1 N hydrochloric acid in diethyl ether (0.1 ml). The mixture was evaporated to a yellow solid which was transferred to a glass vial and the title compound (19mg) dried at 5O 0 C under high vacuum.
  • reaction mixture was loaded onto a Biotage Si 25+M column and purified using the
  • reaction mixture was partitioned between ethyl acetate (50ml) and water (25ml) and washed with water (2 x 25ml) then evaporated and purified using MDAP.
  • reaction mixture was diluted with ethyl acetate (25ml) and washed with water (3 x 25ml) then evaporated and purified by MDAP
  • the residue was dissolved in dichloromethane and treated with 1 N hydrochloric acid in diethyl ether (0.6ml).
  • the mixture was evaporated to a pale yellow solid which was triturated with diethyl ether evaporated and transferred to a glass vial and the title compound (131 mg) dried at 5O 0 C under high vacuum.
  • reaction mixture was diluted with ethyl acetate (50ml) and washed with water (3 x 25ml) then evaporated to give the crude free base of the title compound as a brown foam (930mg).
  • a sample (190mg) was purified by MDAP to afford the title compound (117mg) as a pink oil.
  • the remaining unpurified material from example E64 (740mg) was purified by MDAP and combined with the pure material from example E64 to provide 6-[4-(4-Methyl-1 H- imidazol-1-yl)-3-(methyloxy)phenyl]- ⁇ /-[1-phenyl-2-(1-pyrrolidinyl)ethyl]-3-pyridazinamine formate (440mg).
  • the enantiomers were separated using a Chiralpak AD (50mm x 250mm, 2OuM) column using heptane/ethanol (50:50) as eluant.
  • the slower running enantiomer was dissolved in dichloromethane and treated with 1 N hydrochloric acid in diethyl ether (0.1 ml). The mixture was evaporated to a white solid which was transferred to a glass vial and the title compound (144mg) dried at 5O 0 C under high vacuum.
  • the residue was loaded onto a Biotage Si 25+M column and purified using the Biotage SP4 eluting with 0 to 5% methanol/dichloromethane over 10CV followed by MDAP.
  • the residue was dissolved in dichloromethane and treated with 1 N hydrochloric acid in diethyl ether (2ml). The mixture was evaporated to a white solid which was triturated with diethyl ether and filtered off washing thoroughly with diethyl ether. The solid was transferred to a glass vial and the title compound (379mg) dried at 5O 0 C under high vacuum.
  • reaction mixture was diluted with ethyl acetate (25ml) and washed with water (3 x 25ml) then evaporated.
  • reaction mixture was diluted with ethyl acetate (25ml) and washed with water (3 x 25ml) then evaporated and purified by MDAP.
  • reaction mixture was loaded onto a Biotage 12+M Si column and eluted using 0 to
  • the column used is a Agilent SB C18, the dimensions of which are 4.6mm x30mm.
  • the stationary phase particle size is 1.8um Solvents
  • aqueous solvent Water+0.05% Trifluoroacetic acid
  • Organic solvent Acetonitrile+0.05% Trifluoroacetic acid
  • the above method has a flow rate of 1.5ml/min
  • the injection volume for generic method is 5ul
  • the column temperature is 50deg
  • the UV detection range is from 195to 400nm
  • the columns used are Waters Sunfire C18 and XBridge C18, the dimension of which are 19mmX50mm (small scale ) and 30mm x150mm (Large scale).
  • the station phase particle size is 5um.
  • Aqueous solvent water+0.05% Trifluoroacetic acid (TFA)
  • Aqueous solvent Water+0.05% Ammonia Solution (NH 4 OH)
  • Needle rinse solvent methanol Flow rate: All of purification methods have a flow rate of 30ml/min
  • Example 56 (resynthesis): ⁇ /-[(1 S)-1 -(4-fluorophenyl)ethyl]-4-methyl-6-[4-(4-methyl- 1H-imidazol-1-yl)-3- (methyloxy)phenyl]-3-pyridazinamine hydrochloride
  • Example 73 ⁇ /-[(1 S)-1-(4-fluorophenyl)propyl]-4-methyl-6-[4-(4-methyl-1H- imidazol-1-yl)-3-(methyloxy)phenyl]-3-pyridazinamine
  • Example 74 ⁇ /-[(3-chlorophenyl)methyl]-4-methyl-6-[4-(4-methyl-1 H-imidazol-1 -yl)- 3-(methyloxy)phenyl]-3-pyridazinamine
  • Example 75 4-methyl-6-[4-(4-methyl-1 H-imidazol-1 -yl)-3-(methyloxy)phenyl]- ⁇ /-[(4- methylphenyl)methyl]-3-pyridazinamine
  • Example 77 4-methyl-6-[4-(4-methyl-1 H-imidazol-1 -yl)-3-(methyloxy)phenyl]- ⁇ /- ⁇ [3- (methyloxy)phenyl]methyl ⁇ -3-pyridazinamine
  • Example 78 4-methyl-6-[4-(4-methyl-1 H-imidazol-1 -yl)-3-(methyloxy)phenyl]- ⁇ /- ⁇ [4- (methyloxy)phenyl]methyl ⁇ -3-pyridazinamine
  • Compounds of the invention may be tested for in vitro biological activity in accordance with the following assays.
  • DNA encoding the Swedish mutation variant amyloid precursor protein isoform 695 was cloned into a pcDNA3.1 vector (Invitrogen) using Hind 111 and Xbal sites and transfected into wild type SHSY5Y neuroblastoma cells. Stable cell clones were subsequently selected by growth in presence of geneticin (400ug/ml).
  • Assay ready frozen SHSY5Y Swedish APP cells were generated by growing the cells to confluence in culture media (Dulbecco's modified Eagle media F12 HAM containing 10% Heat inactivated Bovine Calf Serum, 0.1 mM Glutamine and 250Dg/ml Geneticin) over 4- 5 days at 37°C / 5% CO 2 . The medium was removed and the cells washed the cells in PBS. The cells were treated with Accutase (Innovative cell Technologies Inc, USA) until cells detached from surface of flask then neutralised by the further addition of warm culture medium. The cells were centrifuged for 4 minutes at 1000 rpm and the media discarded. Cells were resuspended in freezing medium (90% Dialysed Bovine Calf Serum, 10% DMSO) to give ⁇ 5xe7 cells/ml and control frozen in 1 ml aliquots.
  • culture media Dulbecco's modified Eagle media F12 HAM containing 10% Heat inactivated Bovine Calf Serum,
  • CB6/Balb c f1 mice were immunised with a conjugate of KLH (keyhole limpet hemocyanin) and a peptide comprising amino acids 37-42 of the ⁇ -amyloid peptide by the intraperitoneal route. Based on the results of an antisera ELISA for binding to peptide comprising amino acids 37-42 of the ⁇ -amyloid peptide, a mouse was selected for hybridoma fusion. This mouse was boosted twice intravenously with peptide conjugate prior to fusion. Hybridomas were formed using polyethylene glycol (PEG) to cause the membranes of B-cells and myeloma cells to fuse.
  • PEG polyethylene glycol
  • Som. Cell Genet. 1 397-400. Briefly, spleen cells were isolated from the mouse spleen and mixed with myeloma cells at a ratio of 5 Spleen cells : 1 myeloma. These were centrifuged to pellet the cells and the supernatant aspirated. PEG solution was added to the cell pellet and mixed with the cells to give cell fusion. The cell mixture was then diluted out with excess cell culture media (final volume 400 ml) and this was then plated out over 20 x 96 well plates.
  • Hybridomas were selected by the use of Hypoxantine Aminopterin and Thymidine (HAT) added to the media which kills off unfused myeloma cells. Unfused B-cells die naturally over the next 7-10 days. Hybridomas were screened for selective binding to peptide comprising amino acids 37-42 of the ⁇ -amyloid peptide. Based on the results of this, the monoclonal cell line 22545G5(1 )H14 was selected.
  • the cell line is cultured in JRH610 serum free media (commercially available from JRH Biosciences) until nutrients are depleted.
  • Antibody 5G5 is purified from the culture medium using standard protein purification methods.
  • mice were immunised with a conjugate of PPD (Purified protein derivative) and a peptide comprising amino acids 35-40 of the ⁇ -amyloid peptide by the intraperitoneal route. Based on the results of an antisera ELISA for binding to ⁇ -amyloid peptide (1-40), a mouse was selected for hybridoma fusion. This mouse was boosted twice intraperitonealy with peptide conjugate prior to fusion.
  • PPD Purified protein derivative
  • Hybridomas were formed using polyethylene glycol (PEG) to cause the membranes of B-cells and myeloma cells to fuse. This is standard methodology based on: Pontecorvo G: 1975, Production of somatic cell hybrids by means of polyethylene glycol treatment. Som. Cell Genet. 1 :397-400. Briefly, spleen cells were isolated from a mouse spleen and mixed with myeloma cells at a ratio of 5 Spleen cells : 1 myeloma. These were centrifuged to pellet the cells and the supernatant aspirated. PEG solution was added to the cell pellet and mixed with the cells to give cell fusion. The cell mixture was then diluted out with excess cell culture media (final volume 400 ml) and this was then plated out over 20 x 96 well plates. Hybridomas were selected by the use of Hypoxantine
  • HAT Thymidine
  • Aminopterin and Thymidine added to the media which kills off unfused myeloma cells. Unfused B-cells die naturally over the next 7-10 days. Hybridomas were screened for binding to ⁇ -amyloid peptide (1-40) and no binding to either ⁇ -amyloid peptide(1-38) or ⁇ -amyloid peptide (1-42) peptides. Based on the results of this, the monoclonal cell line S091 117E08(1 )A01 was selected. The cell line is cultured in JRH610 serum free media (commercially available from JRH Biosciences) until nutrients are depleted. Antibody 17E08 is purified from the culture medium using standard protein purification methods.
  • 5G5 antibody was incubated for 48 hours at 37 0 C with unconjugated AlphaLISA Acceptor Beads in a 10:1 Bead to antibody ratio in the presence of 1.25mg/ml Sodium Cyanoborohydride in a Conjugation Buffer of 0.1 M Sodium Phosphate pH 8.0 + 0.1% Tween 20.
  • 17E08 antibody was incubated for 48 hours at 37 0 C with unconjugated AlphaLISA Acceptor Beads in a 10:1 Bead to antibody ratio in the presence of 1.25mg/ml Sodium Cyanoborohydride in a Conjugation Buffer of 0.1 M Sodium Phosphate pH 8.0 + 0.1% Tween 20.
  • test compound was added in neat DMSO, then 50 ⁇ l/well of cell suspension. Plates were sealed and incubated at 37°C, 5% CO 2 . After 48 hours, 5 ⁇ l supernatant was transferred to each well of a white solid low volume 384 well microtitre plate. 2ul of 10ug/ml 17E08 conjugated AlphaLISA Acceptor Beads and 700ng/ml Biotinylated 6E10 antibody (Signet, USA) were added in Assay Buffer (Tris 3OmM pH 7.4, 0.02% Tween 20, 0.02% Casein).
  • Notch cells were produced by taking African Green Monkey CV-1 cells grown in Maintenance Medium (Advanced DMEM (Invitrogen), 5% Heat Inactivated Fetal Calf serum, 2mM Glutamax (Invitogen)) then passaging into Pre-Assay Cell Passaging media (Dulbeccos MEM with Hepes, 10% Charcoal stripped dextran treated fetal calf serum (Hyclone), 2mM Glutamax) for 2-3 days.
  • DMEM Invitrogen
  • 5% Heat Inactivated Fetal Calf serum 2mM Glutamax (Invitogen)
  • Pre-Assay Cell Passaging media Dulbeccos MEM with Hepes, 10% Charcoal stripped dextran treated fetal calf serum (Hyclone), 2mM Glutamax
  • the cells with confluency ⁇ 95% were harvested by washing them in PBS and dissociating from the flask with Trypsin/ EDTA and adding 7 mis of Assay media (Phenol Red Free DMEM (Gibco), 5% Charcoal stripped dextran treated fetal calf serum (Hyclone), 2mM Glutamax, 25mM HEPES buffer (Sigma)). The cells were centrifuged at IOOOrpm for 4 minutes, resuspended in 10 ml fresh Assay media and counted.
  • the transfected cells were washed in PBS, removed from the flasks using trypsin/EDTA. centrifuged at IOOOrpm taken up in Cell freezing medium (Sigma) to give 1-1.5e7cells/ml and control frozen to -14O 0 C for future use.
  • Examples 1 to 72 were tested (as free base or salt) in the above A ⁇ (1-42) assay. All exhibited a plC50 >4.8 in this assay. In addition, all exhibited a higher plC50 in this assay than in the Notch assay.
  • the rat primary cortical neurons were prepared from rat embryos at embryonic days 18. Briefly, pregnant rats were euthanized by an overdose of sodium pentobarbital. The uterus were then removed from the rat and placed on ice. The whole brains of pups were taken out with forceps. The cerebral cortex was dissected out from the whole brain by first peeling off the meninges. The cortical tissues are chopped thoroughly with a blade and 0.25% trypsin-EDTA solution (37 ° C pre-warmed) was added and treated for 10 min at 37 ° C .
  • the DMEM full medium (containing FBS and GluTAMax) were added to inhibit trypsin activity and then centrifuge at 60Og for 5 min at 4°C , the supernatants were carefully removed. The cell pellets were then re-suspended in DMEM full medium by proper blowing with a pipette until devoid of clumps. Strain the cell suspension first through a 100 micron then a 70 micron mesh. The cells were counted by 1 :5 dilution mixed with 0.4% trypan blue. The cells were then plated in 24 -well plate(poly-L-lysine pre-coated) at cell density of 4 x 10 5 cells/well in 300 ul of DMEM full medium. After incubation at 37 ° C for half an hour, the culture medium were carefully removed and 500 ul fresh DMEM full medium were then added into the wells. The medium were half- changed with culture medium containing NB+B27+GluTAMax.
  • test compounds To assay for inhibition of A ⁇ (1-40) and A ⁇ (1-42) production, 0.5 ul of test compounds in
  • % inhibition was plotted vs log concentration using a Grafit IC50 fit (Erithacus Software, UK) to determine the plC50 for each compound.

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Abstract

La présente invention concerne de nouveaux composés qui inhibent la production de peptide β-amyloïde (1-42), des procédés pour leur préparation, des compositions les contenant et leur utilisation dans le traitement de maladies caractérisées par des niveaux élevés de β-amyloïde ou des dépôts de β-amyloïde, en particulier la maladie d'Alzheimer.
PCT/EP2008/063950 2007-10-18 2008-10-16 Dérivés de pyridazine pour inhiber la production de peptide béta amyloïde WO2009050227A1 (fr)

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