COMPOUNDS

The present invention relates to novel heterocyclic compounds which are useful in the prevention and treatment of neurodegenerative disorders, such as Alzheimer's, Parkinson's and Huntington's diseases as well as type II diabetes.

A number of incurable, ageing-related or degenerative diseases have been linked to a generic and fundamental pathogenic process of protein or peptide misfolding and aggregation called "amyloidosis". These include Alzheimer's, Parkinson's and Huntington's diseases and type II diabetes. The amyloid deposits present in these diseases consist of particular peptides that are characteristic for each of these diseases but regardless of their sequence the amyloid fibrils have a characteristic β-sheet structure. In each disease, a specific protein or peptide misfolds, adopts β-sheet structure and oligomerizes to form soluble aggregation intermediates en route to fibril formation ultimately forming insoluble amyloid fibres, plaques or inclusions. These insoluble forms of the aggregated protein or peptide form by the intermolecular association of β-strands into β-sheets. Recent evidence suggests that the soluble amyloid oligomers may be the principal cause of neurotoxicity.

The amyloidoses are defined as diseases in which normally soluble proteins accumulate in various tissues as insoluble deposits of fibrils that are rich in β-sheet structure and have characteristic dye-binding properties (Glenner, 1980a, 1980b). Although the specific polypeptides that comprise the deposits are different for each amyloidosis, the disorders have several key features in common. The most prominent of these is the ability of proteins that are highly soluble in biological fluids to be gradually converted into insoluble filamentous polymers enriched in β-pleated sheet conformation.

Furthermore, they tend to form by a similar molecular mechanism (by the intermolecular association of β-strands into extended β-sheets), so they tend to share a similar molecular structure and a common ability to bind certain dyes such as Congo Red and Thioflavin T (Selkoe 2003; Stefani 2004).

These diseases and disorders, which are collectively referred to herein as "amyloid- related diseases", fall into two main categories: those which affect the brain and other parts of the central nervous system and those which affect other organs or tissues around the body, outside of the brain.

Examples of amyloid-related diseases which fall under these two categories are listed below in the following two sections, however many other examples of rare hereditary amyloid-related diseases are known which are not included here and more forms of amyloid-related disease are likely to be discovered in the future.

Neurodegenerative diseases associated with amyloidosis

Many different neurodegenerative diseases are associated with the misfolding and aggregation of a specific protein or peptide in a particular part of the brain, or elsewhere in the central nervous system, depending on the specific disease (Le Vine 2004; Caughey and Lansbury 2003; Dev et al. 2003; Taylor et al. 2002; Wood et al. 2003; Masino 2004; Ross and Poirier 2004; Soto and Castilla 2004; Forman et al. 2004). For example:

Various forms of Alzheimer's disease (AD/FAD) as well as Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (HCHWA, Dutch type), cerebral amyloid angiopathy, and possibly also mild cognitive impairment and other forms of dementia are associated with the aggregation of a 40/42-residue peptide called β- amyloid, Aβ(l-40) or Aβ(l-42), which forms insoluble amyloid fibres and plaques in

the cerebral cortex, hippocampus or elsewhere in the brain, depending on the specific disease;

Alzheimer's disease is also associated with the formation of neurofibrillary tangles by aggregation of a hyperphosphorylated protein called tau, which also occurs in frontotemporal dementia (Pick's disease);

Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) are associated with the aggregation of a protein called α-synuclein, which results in the formation of insoluble inclusions called "Lewy bodies";

Huntington's disease (HD), spinal and bulbar muscular atrophy (SBMA, also known as Kennedy's disease), dentatorubral pallidoluysian atrophy (DRPLA), different forms of spinocerebellar ataxia (SCA, types 1, 2, 3, 6 and 7), and possibly several other inheritable neurodegenerative diseases are associated with the aggregation of various proteins and peptides that contain abnormally expanded glutamine repeats (extended tracts of polyglutamine); Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE) in cows, scrapie in sheep, kuru, Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia, and possibly all other forms of transmissible encephalopathy are associated with the self-propagating misfolding and aggregation of prion proteins;

Amyotrophic lateral sclerosis (ALS), and possibly also some other forms of motor neuron disease (MND) are associated with the aggregation of a protein called superoxide dismutase;

Familial British dementia (FBD) and familial Danish dementia (FDD) are respectively associated with aggregation of the ABri and ADan peptide sequences derived from the BRI protein; and Hereditary cerebral hemorrhage with amyloidosis (HCHWA, Icelandic type) is associated with the aggregation of a protein called cystatin C.

Systemic diseases associated with amyloidosis

In addition to the neurodegenerative diseases listed above, a wide variety of systemic ageing-related or degenerative diseases are associated with the misfolding and aggregation of a particular protein or peptide in various other tissues around the body, outside of the brain (Gejyo et al. 1985; Jaikaran and Clark 2001; Buxbaum 2004). For example:

Type π diabetes (also known as adult-onset diabetes, or non-insulin dependent diabetes mellitus) is associated with the aggregation of a 37-residue peptide called the islet amyloid polypeptide (IAPP, or "amylin"), which forms insoluble deposits that are associated with the progressive destruction of insulin-producing β cells in the islets of Langerhans within the pancreas;

Dialysis-related amyloidosis (DRA) and prostatic amyloid are associated with the aggregation of a protein called β ₂ -microglobulin, either in bones, joints and tendons in DRA, which develops during prolonged periods of haemodialysis, or within the prostate in the case of prostatic amyloid;

Primary systemic amyloidosis, systemic AL amyloidosis and myeloma-associated amyloidosis are associated with the aggregation of immunoglobulin light chain (or in some cases immunoglobulin heavy chain) into insoluble amyloid deposits, which gradually accumulate in various major organs such as the liver, kidneys, heart and gastrointestinal (GI) tract;

Reactive systemic AA amyloidosis, secondary systemic amyloidosis, familial Mediterranean fever and chronic inflammatory disease are associated with the aggregation of serum amyloid A protein, which forms insoluble amyloid deposits that accumulate in major organs such as the liver, kidneys and spleen; Senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC) are associated with the misfolding and aggregation of different mutants of transthyretin protein (TTR), which form insoluble

inclusions in various organs and tissues such as the heart (especially in FAC), peripheral nerves (especially in FAP) and gastrointestinal (GI) tract

Another form of familial amyloid polyneuropathy (FAP, type II) is associated with the aggregation of apolipoprotein AI in the peripheral nerves; Familial visceral amyloidosis and hereditary non-neuropathic systemic amyloidosis are associated with misfolding and aggregation of various mutants of lysozyme, which form insoluble deposits in major organs such as the liver, kidneys and spleen;

Finnish hereditary systemic amyloidosis is associated with aggregation of a protein called gelsolin in the eyes (particularly in the cornea); Fibrinogen α-chain amyloidosis is associated with aggregation of the fibrinogen A α- chain, which forms insoluble amyloid deposits in various organs such as the liver and kidneys;

Insulin-related amyloidosis occurs by the aggregation of insulin at the site of injection in diabetics; Medullary carcinoma of the thyroid is associated with the aggregation of calcitonin in surrounding tissues;

Isolated atrial amyloidosis is associated with the aggregation of atrial natriuretic peptide (ANP) in the heart; and

Various forms of cataract are associated with the aggregation of γ-crystallin proteins in the lens of the eyes.

Pathogenic mechanism of amyloid-related diseases

While all these amyloid-related diseases share a common association with the pathogenic process of amyloidosis, the precise molecular mechanism by which this generic process of protein/peptide misfolding and aggregation is linked to the progressive degeneration of affected tissues is unclear. In some cases, including many

of the systemic amyloid-related diseases, it is thought that the sheer mass of insoluble protein or peptide simply overwhelms the affected tissues, ultimately leading to acute organ failure. In other cases, including most of the neurodegenerative diseases listed above, however, the symptoms of disease develop with the appearance of only very small aggregates and it was suggested that these insoluble deposits are inherently toxic and might cause the progressive destruction of cells in some way, for example by causing inflammation and oxidative stress, or by directly interfering with cell membranes or other cellular components or processes.

More recently, however, it has been established that the specific proteins and peptides involved in at least some of these amyloid-related diseases form various soluble oligomeric species during their aggregation, which range in size from dimers and trimers, to much larger species comprising tens or even hundreds or thousands of protein or peptide monomers. Moreover, the oligomers are inherently toxic to cells in vitro in the absence of insoluble aggregates, and they appear to share a common structural feature as they can all be recognised by the same antibody despite the fact that they may be formed by proteins or peptides with very different amino acid sequences (Kayed et al. 2003; Glabe 2004; Walsh et al. 2002; Walsh and Selkoe 2004).

The molecular structure of these toxic soluble oligomers is not known and the precise mechanism by which they kill cells is also unclear, but several theories have been proposed. According to just one theory called the "channel hypothesis", for example, the oligomers form heterogeneous pores or leaky ion channels, which allow ions to flow freely through cell membranes, thereby destroying their integrity which ultimately causes cell death (Kagan et al. 2002). Alternatively, or in addition, the oligomers may form protofibrils which kill cells by a similar or completely different mechanism.

Regardless of the precise pathogenic mechanism, however, an overwhelming amount of evidence has now been accumulated which suggests that the general process of protein/peptide aggregation is the primary cause of all these, and possibly other, different amyloid-related diseases.

The present invention relates to chemical compounds and compositions which are inhibitors of amyloid toxicity and as such have use in the treatment of amyloid-related diseases and disorders.

Thus, in a first aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt or prodrug thereof:

(D wherein

X is an oxygen or sulphur atom

A is CH(R ⁶ )-, -O- or NR ⁷ or when A is NR ⁷ , then R ¹ and NR ⁷ can together form a ring such that R ¹ and R ⁷ are (CH ₂ ) _n , where n is 2-4, preferably 2-3;

Y is a bond, CH(R ⁶ )-, -O- or NR ⁷ or A and Y together form a single bond between the aromatic group and the carbonyl group but when A is-O- then Y is not -O

R ¹ and R ² are independently hydrogen, halogen, OR ⁸ , NR ⁹ R ¹⁰ , NR ⁹ COR ¹¹ , NR ⁹ SO ₂ R ¹¹ or C _1-6 alkyl optionally substituted by fluorine, hydroxyl, C _1-6 alkoxy or NR ⁹ R ¹⁰ ;

R ³ is C _1-6 alkyl, C _3-8 cycloalkyl C _4-10 alkylcycloalkyl optionally substituted by hydroxyl, C _1-6 alkoxy or NR ⁹ R ¹⁰ ;

R ⁴ is hydrogen, halogen, CF ₃ , OR ⁹ , NR ⁹ R ¹⁰ , NR ⁹ COR ¹¹ , NR ⁹ SO ₂ R ¹¹ or C _1-6 alkyl optionally substituted by hydroxyl, C _1-6 alkoxy or NR ⁹ R ¹⁰ ;

R ⁵ is hydrogen, halogen, CF ₃ , OR ⁸ , COOR ⁹ , CONR ⁹ R ¹⁰ or SO ₂ R ¹¹ ;

R ⁶ is hydrogen, C _1-6 alkyl, C _3-8 cycloalkyl C _4-10 alkylcycloalkyl, C _1-6 alkyl optionally substituted by hydroxyl, C _1-6 alkoxy or NR ⁹ R ¹⁰ , C _1-6 alkoxy or NR ⁹ R ¹⁰ ;

R ⁷ is hydrogen, C _1-6 alkyl, optionally substituted by C _1-6 alkoxy or NR ⁹ R ¹⁰ , phenyl wherein said phenyl group is optionally substituted by one or more substituents selected from halogen, C _1-6 alkyl, CF ₃ , OCF ₃ or OR ⁹ ;

R ⁸ is hydrogen or C _1-6 alkyl optionally substituted by fluorine, C _1-6 alkoxy or NR ⁹ R ¹⁰ ;

R ⁹ is hydrogen, C _1-6 alkyl or C _1-3 alkylphenyl wherein said phenyl group is optionally substituted by one or more substituents selected from halogen, C _1-6 alkyl optionally substituted by fluorine, OR ⁸ , NR ⁹ R ¹⁰ ;

R ¹⁰ is hydrogen, C _1-6 alkyl, C _3-8 cycloalkyl C _4-10 alkylcycloalkyl, C _1-6 alkenyl, phenyl or C _1-3 alkylphenyl wherein said phenyl groups are optionally substituted by one or more substituents selected from halogen, C _1-6 alkyl optionally substituted by fluorine, OR ;

or the groups R ⁹ and R ¹⁰ when they are attached to a nitrogen atom may together form a 5- or 6-membered ring which optionally contains one further heteroatom selected from NR ⁹ , S and O; and

R ¹¹ is C _1-6 alkyl or a phenyl group optionally substituted by one or more substituents selected from halogen, C _1-6 alkyl, CF ₃ , OCF _{3 or} OR ⁸ ; with the provisos that when A is -O- or NR ⁷ and Y is CH(R ⁶ ) then R ⁶ is not C _1-6 alkoxy or NR ⁹ R ¹⁰ ; when R ² and R ⁵ are both hydrogen, A is -NH- and Y is a bond, R ¹ is Cl, F or methoxy and R ⁴ is Cl or hydrogen, then R ³ is not ethyl; when R ² is hydrogen or methoxy, R ⁵ is hydrogen, A is -CH ₂ - or -CH(CH ₂ CH ₃ )-, Y is a bond, R ¹ is hydrogen or methoxy and R ⁴ is hydrogen, methyl, t-butyl, Cl, F, methoxy or ethoxy, then R ³ is not C _1-6 alkyl or 2,2 methoxyethyl; when R ² is hydrogen or Cl, R ⁵ is hydrogen or methoxy, A is -O- and Y is -CH ₂ -, R ¹ is hydrogen, Cl, ethyl or methoxy and R ⁴ is Cl, F, methyl, t-butyl, methoxy or ethoxy, then R ³ is not C _1-6 alkyl or 2,2 methoxyethyl; and when R ² is hydrogen, methyl or methoxy, R ⁵ is hydrogen or methoxy, A and Y together form a single bond between the aromatic group and the carbonyl group, R ¹ is hydrogen, methyl, t-butyl, F, Cl, Br, methoxy or ethoxy and R ⁴ is hydrogen, methyl, t- butyl, Cl, F, methoxy or ethoxy, then R ³ is not C _1-6 alkyl or 2,2 methoxyethyl.

Preferred compounds include: N-Isobutyl-2-phenoxy-N-(5-phenyl-[l,3,4]thiadiazol-2-yl)acet amide N-isobutyl-2-phenoxy-N-(5-phenyl-l,3,4-thiadiazol-2-yl)aceta mide

N-isobutyl-2-phenoxy-N-[5-(4-ethoxyphenyl)]-l,3,4-thiadia zol-2-yl)acetamide N-isobutyl-2-phenoxy-N-[5-(4-methoxyphenyl)]-l,3,4-thiadiazo l-2-yl)acetamide N-isobutyl-2-phenoxy-N-[5-(4-fluorophenyl)]-l,3,4-thiadiazol -2-yl)acetamide N-isobutyl-2-phenoxy-iV-[5-(3-fluorophenyl)]-l,3,4-thiadiazo l-2-yl)acetamide iV-isobutyl-2-phenoxy-iV- [5-(3 ,4-difluoro phenyl)]- 1 ,3 ,4-thiadiazol-2-yl)acetamide N-propyl-2-phenoxy-N-[5-(4-chlorophenyl)]-l,3,4-thiadiazol-2 -yl)acetamide N-propyl-2-phenoxy-N-[5-(3,4-dichlorophenyl)]-l,3,4-thiadiaz ol-2-yl)acetamide N-propyl-2-phenoxy-N-[5-(3-trifluoromethylphenyl)]-l,3,4-thi adiazol-2-yl)acetamide iV-isobutyl-2-(4-fluorophenoxy)-N-[5-(4-ethoxyphenyl)]-l,3,4 -thiadiazol-2- yl)acetamide

iV-isobutyl-2-(4-fluorophenoxy)-iV-[5-(3-fluoro phenyl)]-l,3,4-thiadiazol-2~ yl)acetamide

N-isobutyl-2-(4-(trifluoromethoxy)phenoxy)-iV-[5-(3-fluor ophenyl)]-l,3,4thiadiazol-2- yl)acetamide iV-[5-(3-fluorophenyl)-l,3,4-thiadiazol-2-yl]-N- isobutylbenzamide

2-(4-fluorophenyl)-iV-[5-(3-fluorophenyl)-l,3,4-thiadiazo l-2-yl]-N-isobutylacetamide

(2E)-iV-[5-(3-fluorop]ienyl)-l,3,4-thiadiazol-2-yl]-N-iso butyl-3-phenylacrylainide

N-[5-(3-fluorophenyl)-l,3,4-thiadiazol-2-yl]-N-isobutyl-2 -phenoxypropanamide

N-[5-(3-fluorophenyl)-l,3,4-thiadiazol-2-yl]-iV-isobutyl- 2,3-dihydro-l,4- benzodioxine-2-carboxamide

N- [5-(3-fluorophenyl)- 1 ,3 ,4-thiadiazol-2~yl] -2-(4-hydroxyphenoxy)-JV- isobutylacetamide and

N- [5-(3-fluorophenyl)- 1 ,3 ,4-thiadiazol-2-yl] -3-(2-methoxyphenyl)-iV- propylpropanamide 3-(4-Fluorophenyl)-l-[5-(3-fluorophenyl)-(l,3,4-thiadiazol-2 -yl)-l-propylurea

3-(4-Fluorophenyl)-l-[5-(3-fluorophenyl)-(l,3,4-thiadiazo l-2-yl)-l-methyl-3- propylurea

5-Fluoro-2,3-dihydroindole-l-carboxylic acid, [5-(3-fluorophenyl)-l,3,4-thiadiazol-2- yl] -propylamide [5-(3-Fluoroρhenyl)-l,3,4-thiadiazol-2-yl]-isobutylcarbamic acid, 4-fluorophenyl ester

N-[5-(3-Fluorophenyl)-l,3,4-thiadiazol-2-yl]-4-methoxy-N- propyl-benzamide

N-[5-(3-Fluorophenyl)-l,3,4-oxadiazol-2-yl]-N-isobutyl-2- phenoxy-acetamide

2-(4-Fluorophenyl)-N-[5-(3-fluorophenyl)-l,3,4-oxadiazol- 2-yl]-N-isobutylacetamide

N-[5-(3-Fluorophenyl)-l,3,4-oxadiazol-2-yl]-3-(2-methoxyp henyl)-N-propyl- propionamide.

The term "alkyl" as used herein whether on its own or as part of a larger group e.g. "alkoxy" or "alkylphenyl" includes both straight and branched chain radicals, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl and

tert-butyl. The term alkyl also includes those radicals wherein one or more hydrogen atoms are replaced by fluorine, e.g. CF ₃ .

The term "alkenyl" and "alkynyl" as used herein includes both straight and branched chain radicals.

The term "halogen" as used herein includes fluorine, chlorine and bromine

The compounds of the first aspect may be provided as a salt, preferably as a pharmaceutically acceptable salt of compounds of formula (I). Examples of pharmaceutically acceptable salts of these compounds include those derived from organic acids such as acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid, mandelic acid, methanesulphonic acid, benzenesulphonic acid and p- toluenesulphonic acid, mineral acids such as hydrochloric and sulphuric acid and the like, giving methanesulphonate, benzenesulphonate, p-toluenesulphonate, hydrochloride and sulphate, and the like, respectively or those derived from bases such as organic and inorganic bases. Examples of suitable inorganic bases for the formation of salts of compounds for this invention include the hydroxides, carbonates, and bicarbonates of ammonia, lithium, sodium, calcium, potassium, aluminium, iron, magnesium, zinc and the like. Salts can also be formed with suitable organic bases. Such bases suitable for the formation of pharmaceutically acceptable base addition salts with compounds of the present invention include organic bases, which are nontoxic and strong enough to form salts. Such organic bases are already well known in the art and may include amino acids such as arginine and lysine, mono-, di-, or trihydroxyalkylamines such as mono-, di-, and triethanolamine, choline, mono-, di-, and trialkylamines, such as methylamine, dimethylamine, and trimethylamine, guanidine; N-methylglucosamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; tris(hydroxymethyl) aminomethane; and the like.

Salts may be prepared in a conventional manner using methods well known in the art. Acid addition salts of said basic compounds may be prepared by dissolving the free base compounds according to the first aspect of the invention in aqueous or aqueous alcohol solution or other suitable solvents containing the required acid. Where a compound of the invention contains an acidic function, a base salt of said compound may be prepared by reacting said compound with a suitable base. The acid or base salt may separate directly or can be obtained by concentrating the solution e.g. by evaporation. The pharmaceutically acceptable prodrugs of the compounds of formula (I) may be prepared by methods well known to those skilled in the art. A prodrug is commonly described as an inactive or protected derivative of an active ingredient or a drug, which is converted to the active ingredient or drug in the body. Examples of prodrugs include pharmaceutically acceptable esters, including C ₁ -C ₆ alkyl esters and pharmaceutically acceptable amides, including secondary C ₁ -C ₃ amides.

The compounds of the invention may exist in the form of optical isomers, e.g. diastereoisomers and mixtures of isomers in all ratios, e.g. racemic mixtures. The invention includes in particular the isomeric forms (R or S). The different isomeric forms may be separated or resolved one from the other by conventional methods, or any given isomer may be obtained by conventional synthetic methods or by stereospecific or asymmetric synthesis. Where a compound contains an alkene moiety, the alkene can be presented as a cis or trans isomer or a mixture thereof. When an isomeric form of a compound of the invention is provided substantially free of other isomers, it will preferably contain less than 5% w/w, more preferably less than 2% w/w and especially less than 1% w/w of the other isomers.

Since the compounds of the invention are intended for use in pharmaceutical compositions, it will readily be understood that they are each preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 98% pure (% are on a weight for

weight basis). Impure preparations of the compounds may be used for preparing the more pure forms used in the pharmaceutical compositions; these less pure preparations of the compounds should contain at least 1%, more suitably at least 5%, e.g. 10 to 59% of a compound of the formula (I).

A compound of formula (I), wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and X are as defined for formula (I), A = O and Y = CH(R ⁶ ), may be prepared from a compound of formula (II)

(II)

wherein R ³ , R ⁴ and R ⁵ are as defined in formula (I) by treatment with an appropriate aryloxyacetyl chloride of formula (HI) in the presence of a suitable inorganic base such as sodium bicarbonate or an organic base such as triethylamine in a suitable aprotic solvent such as dioxane with the application of heat, typically at the reflux temperature of the solvent.

(HI)

A compound of formula (II) wherein R ³ , R ⁴ and R ⁵ are as defined in formula (I) and X = sulfur may be prepared from a compound of formula (JY), wherein R ³ , R ⁴ and R ⁵ are as defined in formula (I), by treatment with concentrated sulphuric acid at 0 ⁰ C for an initial period of time, for example 1 hour, and then at room temperature for a period of time, for example up to 4 hours.

(IV)

A compound of formula (H) wherein R ³ , R ⁴ and R ⁵ are as defined in formula (I) and X = oxygen may be prepared via cyclodehydration of a semicarbazide of formula (V) by treatment with l^-dicyclohexylcarbodiimide in a suitable solvent such as DMF with the application of heat to, for example, 120°C. Alternatively, cyclodehydration may be effected by heating in phosphorus oxychloride. Alternatively, compounds of formula (II) wherein R ³ , R ⁴ and R ⁵ are as defined in formula (I) and X = oxygen may be prepared via cyclisation of a thiosemicarbazide of formula (IV) using tosyl chloride in the presence of a base such as pyridine in a suitable solvent such as THF at reflux.

(V)

A compound of formula (TV) wherein R ³ , R ⁴ and R ⁵ are as defined in formula (I) may be prepared by reaction of a hydrazide of formula (VI), wherein R ⁴ and R ⁵ are as defined in formula (I), with an isothiocyanate of formula (VII), wherein R ³ is as defined in formula (I), with the application of heat for a suitable period of time, for example 3 hours.

Compounds of formula (VI) and (VII) are either commercially available or can be synthesised by methods known in the art.

(VI)

R ^3'

(vπ)

A compound of formula (I), wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and X are as defined for formula (I), A = a single bond and Y = CH(R ⁶ ) or a single bond, may be prepared from

a compound of formula (II) wherein R , R and R are as defined in formula (I) by treatment with an appropriate arylacetyl chloride of formula (VIII) or an aroyl chloride of formula (IX) in the presence of a suitable inorganic base such as sodium bicarbonate or an organic base such as triethylamine in a suitable aprotic solvent such as dioxane with the application of heat, typically at the reflux temperature of the solvent.

(vπi) (IX)

A compound of formula (I), wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and X are as defined for formula (I), A = a single bond or CH(R ⁶ ) and Y = NR ⁷ , may be prepared from a compound of formula (II) by treatment with phosgene, in the presence of a base such as diisopropylethylamine, followed by treatment with an amine of formula (X) or (XI).

(X) (XI)

Alternatively a compound of formula (I), wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and X are as defined for formula (I), A = a single bond or CH(R ⁶ ) and Y = NH, may be prepared from a compound of formula (II) by treatment with an isocyanate of formula (XII) or (XIII) in a suitable aprotic solvent such as dichloromethane or dioxane with, if necessary the application of heat. Compounds of formula (XH) and (XIII) are either commercially available or can be synthesised by methods known in the art.

(XE) (xiπ)

A compound of formula (I), wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and X are as defined for formula (I), A = NR ⁷ and Y = CH(R ⁶ ) may be prepared from a compound of formula (It) by treatment with an appropriate arylaminoacetyl chloride of formula (XIV) in the presence of a suitable inorganic base such as sodium bicarbonate or an organic base such as triethylamine in a suitable aprotic solvent such as dioxane with the application of heat, typically at the reflux temperature of the solvent.

(XIV)

A compound of formula (I), wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and X are as defined for formula (I), A = a single bond or CH(R ⁶ ) and Y = O may be prepared from a compound of formula (II) with lj'-carbonyldiimidazole followed by treatment with a phenol of formula (XV) or a benzyl alcohol of formula (XVI). Alternatively a compound of formula (I), wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and X are as defined for formula (I), A = a single bond or CH(R ⁶ ) and Y = O may be prepared from a compound of formula (II) by treatment with phosgene, in the presence of a base such as diisopropylethylamine, followed by treatment with a phenol of formula (XV) or a benzyl alcohol of formula (XVI) .

(XV) (XVI)

It will also be appreciated that the carboxylic acid chloride, phenol, amine and alcohol building blocks used in the synthesis of compounds of general formula (I) are either commercially available or can be synthesised by methods known in the art.

During the synthesis of the compounds of formula (I), labile functional groups in the intermediate compounds, e.g. hydroxy, carboxy and amino groups, may be protected. The protecting groups may be removed at any stage in the synthesis of the compounds of formula (I) or may be present on the final compound of formula (I). A comprehensive discussion of the ways in which various labile functional groups may be protected and methods for cleaving the resulting protected derivatives is given in for example Protective Groups in Organic Chemistry, T.W. Greene and P.G.M. Wuts (Wiley-Interscience, New York, 2 ^nd edition, 1991).

The pharmaceutically effective compounds of formula (I) may be administered in conventional dosage forms prepared by combining a compound of formula (I) ("active ingredient") with standard pharmaceutical carriers or excipients according to conventional procedures well known in the art. The procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.

Thus, in a second aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt or prodrug thereof, together with one or more pharmaceutically acceptable carriers or excipients.

The active ingredient or pharmaceutical composition can be administered simultaneously, separately or sequentially with another appropriate treatment for the amyloid-related disease being treated.

The active ingredient or pharmaceutical composition may be administered to a subject by any of the routes conventionally used for drug administration, for example they may be adapted for oral (including buccal, sublingual), topical (including transdermal), nasal (including inhalation), rectal, vaginal or parenteral (including

subcutaneous, intramuscular, intravenous or intradermal) administration to mammals including humans. The most suitable route for administration in any given case will depend upon the particular compound or pharmaceutical composition, the subject, and the nature and composition and severity of the disease and the physical condition of the subject. Such compositions may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

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 polyvinylpyrrolidone ; filler, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol;

preservatives, for example methyl or propyl p-hydoxybenzoate or sorbic acid, and, if desired, conventional flavouring or colouring agents.

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions powders, solutions, pastes, gels, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams. Such applications include those to the eye or other external tissues, for example the mouth and sin and the compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. The composition may also contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions.

Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epiderma of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6),318 (1986).

Pharmaceutical compositions adapted for controlled or sustained release may be administered by injection, for example by the subcutaneous route.

Pharmaceutical compositions adapted for nasal administration wherein the carrier is a solid include coarse powder having a particle size for example in the range of 20-500 microns which is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nose. Suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of an active ingredient.

Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurise aerosols, nebulizers or insufflators.

Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas. Suppositories will contain conventional suppository bases, e.g. cocoa-butter or other glyceride.

Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray compositions.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

Extemporaneous injection solution and suspensions may be prepared from sterile powders, granules and tablets.

For parenteral administration, fluid unit dosage forms are prepared utilising the active ingredient and a sterile vehicle, water being preferred. The active ingredient, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions the active ingredient can be dissolved in water for injection and filter sterilised before filling into a suitable vial or ampoule and sealing.

Advantageously, agents such as local anaesthetic, preservative and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use. Parenteral suspensions are prepared in substantially the same manner except that the active ingredient is suspended in the vehicle instead of being dissolved and sterilisation cannot be accomplished by filtration. The active ingredient can be sterilised by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active ingredient.

The pharmaceutical compositions according to the invention are preferably adapted for oral administration.

It should be understood that in addition to the ingredients particularly mentioned above, the compositions may also include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents. They may also contain therapeutically active agents in addition to the compounds of the present invention. Such carriers may

be present as from about 1% up to about 98% of the formulation. More usually they will form up to about 80% of the formulation.

The compositions may contain from 0.1% by weight, preferably from 10-60% by weight, of the active material, depending on the method of administration.

Pharmaceutical compositions may be presented in unit dose forms containing a predetermined amount of active ingredient per dose. Such a unit may contain for example 0.1mg/kg to 750mg/kg, more preferably O.lmg/kg to lOmg/kg depending on the condition being treated, the route of administration and the age, weight and condition of the patient. Preferred unit dosage compositions are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

It will be recognised by one of skill in the art that the optimal quantity and spacing of individual dosages of compounds in the first and second aspects of the invention will be determined by the nature and extent of the condition being treated the form, route and site of administration, and the particular subject being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment , i.e., the number of doses of the aforementioned compounds given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

Depending on the route of administration, the chemical compound or composition may be required to be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate it.

In order to administer the chemical compound or composition by other than parenteral administration, it may be coated by, or administered with, a material to prevent its

inactivation. For example, it may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.

Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes.

The active chemical compound or composition may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms .

The pharmaceutical compositions or formulations suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene gloycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.

The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol,

sorbic acid, thirnierosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active chemical compound or composition in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof.

When the chemical compound or composition is suitably protected as described above, it may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such

as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier.

Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.

As used herein "pharmaceutically acceptable carrier and/or diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such as active material for the treatment

of disease in living subjects having a diseased condition in which bodily health is impaired.

The principal active ingredients are compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

In other aspcts, the present invention provides:

1. The use of a compound as described herein in the manufacture of a medicament for the treatment of an amyloid-related disease. In particular, the medicament is for the treatment of: a) any form of Alzheimer's disease (AD or FAD); b) any form of mild cognitive impairment (MCI) or senile dementia; c) Down's syndrome; d) cerebral amyloid angiopathy, inclusion body myositis, hereditary cerebral hemorrhage with amyloidosis (HCHWA, Dutch type), or age-related macular degeneration (ARMD); e) fronto-temporal dementia; f) any form of Parkinson's disease (PD) or dementia with Lewy bodies; g) Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), spinocerebellar ataxia (SCA, types 1, 2, 3, 6 and 7), spinal and bulbar muscular atrophy (SBMA, Kennedy's disease), or any other polyglutamine disease; h) Creutzfeldt- Jakob disease (CJD), bovine spongiform encephalopathy

(BSE) in cows, scrapie in sheep, kuru, Gerstmann-Straussler-Scheinker disease (GSS),

fatal familial insomnia, or any other transmissible encephalopathy that is associated with the aggregation of prion proteins; i) amyotrophic lateral sclerosis (ALS) or any other form of motor neuron disease; j) familial British dementia (FBD) or familial Danish dementia (FDD); k) hereditary cerebral hemorrhage with amyloidosis (HCHWA, Icelandic type);

1) type II diabetes (adult onset diabetes, or non-insulin dependent diabetes mellitus, NIDDM); m) dialysis-related amyloidosis (DRA) or prostatic amyloid; n) primary systemic amyloidosis, systemic AL amyloidosis, or nodular AL amyloidosis; o) myeloma associated amyloidosis; p) systemic (reactive) AA amyloidosis, secondary systemic amyloidosis, chronic inflammatory disease, or familial Mediterranean fever; q) senile systemic amyloidosis, familial amyloid polyneuropathy, or familial cardiac amyloid; r) familial visceral amyloidosis, hereditary non-neuropathic systemic amyloidosis, or any other lysozyme-related amyloidosis; s) Finnish hereditary systemic amyloidosis; t) fibrinogen α-chain amyloidosis; u) insulin-related amyloidosis; v) medullary carcinoma of the thyroid; w) isolated atrial amyloidosis; x) any form of cataract; and

y) any other amyloid-related disease that is associated with the misfolding or aggregation of a specific target amyloid-forming protein or peptide into toxic soluble oligomers, protofibrils, ion channels, insoluble amyloid fibres, plaques or inclusions.

2. A method for the treatment of an amyloid-related disease, which comprises the step of administering to a subject an effective amount of a compound or pharmaceutical composition as described herein.

3. A compound of the invention for use in the treatment of an amyloid- related disease.

Examples The following examples are to be construed as being illustrative and not as a limitation on the scope of the invention in any way.

Example 1 Synthesis of N-isobutyl-2-phenoxy-iV-(5-phenyl-l,3,4-thiadiazol-2- yl)acetamide.

A solution of benzoic hydrazide (1.36g, O.Olmol) in 30ml of methanol (3OmL) was treated with isobutyl isothiocyanate (1.15ml O.Olmol) and heated at reflux for 3h. The reaction mixture was poured onto cracked ice. The white solid was collected by filtration and dried to give the 2-benzoyl-N-isobutylhydrazinecarbothioamide (2.2g, 90%). After checking by TLC (DCM: Methanol 9:1) this intermediate was of sufficient purity to use directly in the next step.

Concentrated sulfuric acid (15mL) was cooled to O ⁰ C and stirred while 2-benzoyl-iV- isobutylhydrazinecarbothioamide (1.Og, 3.9mmol) was added portion wise. The mixture was stirred for Ih at O ⁰ C and then allowed to warm to room temperature over

4 h. The solution was poured onto cracked ice and neutralized with dilute sodium hydroxide solution. The white solid was collected by filtration and dried to give the N- isobutyl-5-phenyl-l,3,4-thiadiazol-2-amine (0.67g, 72%). After checking by TLC (DCM: Methanol 9: 1) this intermediate was of sufficient purity to use directly in the next step.

A solution of phenoxyacetyl chloride (0.36mL, 2.145mmol) in dry dioxane (5.OmL) was added slowly to a stirred suspension of N-isobutyl-5-phenyl-l,3,4-thiadiazol-2- amine (0.5g, 2.145mmol) and sodium bicarbonate (4.0g) in dry dioxane (15ml). After the addition, the mixture was stirred for Ih at room temperature and then heated at reflux for 5h. The reaction mixture was then poured onto cracked ice. The resultant white solid was collected by filtration and washed with hexane to give the title compound (0.68g, 98%). δ _H (4-DMSO, 300 MHz): 0.97 (6H, d), 2.38 (IH, m), 4.09 (2H, m), 5.32 (2H, s), 6.98 (IH, t), 7.01 (2H, d), 7.31 (2H, t), 7.52 (3H, m), 7.93 (2H, m). m/z (ES+) 367.9 (M+H) ⁺ .

Example 2 Synthesis of N-isobutyl-2-phenoxy-N-[5-(4-ethoxyphenyl)]-l,3,4- thiadiazol-2-yI)acetamide

All the steps used to prepare the title compound are analogous to those used for Example 1 except that 4-ethoxybenzoic hydrazide was used instead of benzoic hydrazide. δ _H (4-DMSO, 300 MHz): 0.97 (6H, d), 1.34 (3H, t), 2.35 (IH, m), 4.08 (4H, m), 5.29 (2H, s), 6.98 (3H, m), 7.05 (2H, d), 7.28 (2H, t), 7.85 (2H, d).

m/z (ES+) 412.0 (M+H) ⁺ .

Example 3 Synthesis of N-isobutyl-2-phenoxy-N-[5-(4-methoxyphenyl)]-l,3,4- thiadiazol-2-yl)acetamide

All the steps used to prepare the title compound are analogous to those used for Example 1 except that 4-methoxybenzoic hydrazide was used instead of benzoic hydrazide. δ _H (J ₆ -DMSO, 300 MHz): 0.97 (6H, d), 2.35 (IH, m), 3.82 (3H, s), 4.06 (2H, m), 5.29 (2H, s), 6.97 (3H, m), 7.07 (2H, d), 7.30 (2H, t), 7.86 (2H, d). m/z (ES+) 397.9 (M+H) ⁺ .

Example 4 Synthesis of iV-isobutyl-2-phenoxy-iV-[5-(4-fluorophenyl)]-l,3,4- thiadiazol-2-yl)acetamide

All the steps used to prepare the title compound are analogous to those used for Example 1 except that 4-fluorobenzoic hydrazide was used instead of benzoic hydrazide. δ _H (J ₆ -DMSO, 300 MHz): 0.98 (6H, d), 2.37 (IH, m), 4.09 (2H, d), 5.32 (2H, s), 6.95-7.05 (3H, m), 7.38 (2H, t), 7.31 (2H, t), 8.00 (2H, m). m/z (ES+) 385.9 (M+H) ⁺ .

Example 5 Synthesis of iV-isobutyl-2-phenoxy-N-[5-(3-fluorophenyl)]-l,3,4- thiadiazol-2-yl)acetamide

All the steps used to prepare the title compound are analogous to those used for Example 1 except that 3-fluorobenzoic hydrazide was used instead of benzoic hydrazide. δ _H tø-DMSO, 300 MHz): 0.98 (6H, d), 2.38 (IH, m), 4.09 (2H, m), 5.33 (2H, s), 6.98 (IH, t), 7.02 (2H, d), 7.31 (2H, t), 7.39 (IH, m), 7.58 (IH, m) and 7.78 (2H, m). m/z (ES+) 386.1 (M+H) ⁺ .

Example 6 Synthesis of N-isobutyl-2-phenoxy-N-[5-(3,4-difluoro phenyl)]-l,3,4- thiadiazol-2-yl)acetamide

AU the steps used to prepare the title compound are analogous to those used for Example 1 except that 3,4-difluorobenzoic hydrazide was used instead of benzoic hydrazide. δ _H tø-DMSO, 300 MHz): 0.98 (6H, d), 2.37 (IH, m), 4.09 (2H, d), 5.32 (2H, s), 6.93- 7.05 (3H, m), 7.30 (IH, t), 7.31 (IH, t), 7.61 (IH, m), 7.81 (IH, m), 8.04 (IH, m). m/z (ES ⁺ ): 403.9 (M+H) ⁺ .

Example 7 Synthesis of N-propyl-2-phenoxy-iV-[5-(4-chlorophenyl)]-l,3,4- thiadiazol-2-yI)acetamide

A solution of 4-chlorobenzoic hydrazide (1.7Og, O.Olmol) in methanol (3OmL) was treated with propyl isothiocyanate (l.OlmL O.Olmol) and heated at reflux for 3h. The reaction mixture was poured onto cracked ice. The white solid was collected by filtration and dried to give 2-(4-chlorobenzoyl)-λf-propylhydrazinecarbofhioamide (2.5g, 95%). After checking the TLC (DCM: Methanol 9:1) this intermediate was of sufficient purity to use directly in the next step.

Concentrated sulfuric acid (25mL) was cooled to O ⁰ C and stirred while 2-(4- chlorobenzoyl)~N-propylhydrazinecarbothioamide (2.Og, 7.3mmol) was added portionwise. The mixture was stirred for Ih at O ⁰ C and then allowed to warm to room temperature over 4h. The solution was poured onto cracked ice and neutralized with dilute sodium hydroxide solution. The white solid was collected by filtration and dried to give 5-(4-chlorophenyl)-iV-propyl-l,3,4-thiadiazol-2-amine (1.3g, 70%). After checking the TLC (DCM: Methanol 9:1) this intermediate was of sufficient purity to use directly in the next step.

A solution of phenoxyacetyl chloride (0.33mL, 1.97mmol) in dry dioxane (5.OmL) was added slowly to a stirred suspension of 5-(4-chlorophenyl)-iV-propyl-l,3,4- thiadiazol-2-amine (0.5g, 1.97mmol) and sodium bicarbonate (5.0g) in dry dioxane (15mL). After the addition, the mixture was stirred for Ih at room temperature and then heated at reflux for 5h. The reaction mixture was then poured onto cracked ice. The resultant white solid was collected by filtration and washed with hexane to give the title compound (0.74g, 98%).

δ _H (d ₆ -DMSO, 300 MHz): 0.99 (3H, t), 1.87 (2H, m), 4.16 (2H, m), 5.33 (2H, s), 6.98 (IH, t), 7.01 (2H, d), 7.31 (2H, t), 7.60 (2H, d), 7.96 (2H, d). m/z (ES+) 387.9 (M+H) ⁺ .

Example 8 Synthesis of N-propyl-2-pheπoxy-iV-[5-(3,4-dichlorophenyl)]-l,3,4- thiadiazol-2-yI)acetamide

All the steps used to prepare the title compound are analogous to those used for Example 7 except that 3,4-dichlorobenzoic hydrazide was used instead of 4- chlorobenzoic hydrazide. δ _H tøs-DMSO, 300 MHz): 1.00 (3H, t), 1.86 (2H, m), 4.17 (2H, m), 5.34 (2H, s), 6.98

(IH, t), 7.02 (2H, d), 7.31 (2H, t), 7.79 (IH, d), 7.92 (IH, dd), 8.17 (IH, dd). m/z (ES+) 321.9 (M+H) ⁺ . Example 9 Synthesis of N-propyl-2-phenoxy-N-[5-(3-trifluoromethyIphenyI)]-

1,3,4- thiadiazoI-2-yl)acetamide

All the steps used to prepare the title compound are analogous to those used for Example 7 except that 3-trifluoromethylbenzoic hydrazide was used instead of 4- chlorobenzoic hydrazide. δ _H (J ₆ -DMSO, 300 MHz): 0.91 (3H, t), 1.79 (2H, m), 4.09 (2H, m), 5.25 (2H, s), 6.88-

6.97 (3H, m), 7.22 (2H, t), 7.69 (IH, t), 7.81 (IH, d), 8.15 (2H, m). m/z (ES+) 421.9 (M+H) ⁺ .

Example 10 Synthesis of N-isobutyI-2-(4-fluorophenoxy)-N-[5-(4-ethoxyphenyl)]- l,3,4-thiadiazol-2-yl)acetamide.

AU the steps used to prepare the title compound are analogous to those used for Example 2 except that 4-fluorophenoxyacetyl chloride was used instead of phenoxyacetyl chloride. δ _H (J ₆ -DMSO, 300 MHz): 0.96 (6H, d), 1.34 (3H, t), 2.35 (IH, m), 4.08 (4H, m), 5.28 (2H, s), 7.04-7.15 (6H, m), 7.85 (2H, d). m/z (ES+) 430.0 (M+H) ⁺ .

Example 11 Synthesis of N-isobutyl-2-(4-fluorophenoxy)-N-[5-(3-fluoro phenyl)]- 1,3,4- thiadiazol-2-yl)acetamide

All the steps used to prepare the title compound are analogous to those used for Example 5 except that 4-fluorophenoxyacetyl chloride was used instead of phenoxyacetyl chloride. δ _H tø-DMSO, 300 MHz): 0.97 (6H, d), 2.35 (IH, m), 4.08 (2H, d), 5.31 (2H, s), 7.00- 7.05 (2H, m), 7.14 (2H, m), 7.38 (IH, m), 7.58 (IH, m), 7.77 (IH, d). m/z (ES ⁺ ): 403.9 (M+H) ⁺ .

Example 12 Synthesis of N-isobutyl-2-(4-(trifluoromethoxy)phenoxy)-N-[5-(3- fluoro phenyl)]-l,3,4thiadiazol-2-yl)acetamide

All the steps used to prepare the title compound are analogous to those used for

Example 5 except that 4-(trifluoromethoxy)phenoxyacetyl chloride was used instead of phenoxyacetyl chloride. δ _H fø-DMSO, 300 MHz): 0.98 (6H, d), 2.37 (IH, m), 4.09 (2H, d), 5.38 (2H, s), 7.11

(2H, d), 7.32 (2H, d), 7.38 (IH, m), 7.58 (IH, m), 7.77 (IH, d). m/z (ES ⁺ ): 470.0 (M+H) ⁺ .

Example 13 Synthesis of N-[5-(3-fluorophenyl)-l,3,4-thiadiazol-2-yl]-2V- isobutylbenzamide

AU the steps used to prepare the title compound are analogous to those used for Example 5 except that benzoyl chloride was used instead of phenoxyacetyl chloride. δ _H tø-DMSO, 300 MHz): 0.69 (6H, d), 2.35 (IH, m), 4.09 (2H, m), 7.59 (IH, m), 7.65-7.75 (6H, m), 7.83 (2H, m). m/z (ES+) 355.9 (M+H) ⁺ .

Example 14 Synthesis of 2-(4-fluorophenyl)-N-[5-(3-fluorophenyl)-l,3,4- thiadiazol-2-yl]-N-isobutylacetamide

All the steps used to prepare the title compound are analogous to those used for Example 5 except that 4-fluorophenylacetyl chloride was used instead of phenoxyacetyl chloride. δ _H fø-DMSO, 300 MHz): 0.97 (6H, d), 2.35 (IH, m), 4.20-4.25 (4H, m), 7.18 (2H, m), 7.35 (3H, m), 7.57 (IH, m), 7.75 (2H, m). m/z (ES+) 387.9 (M+H) ⁺ .

Example 15 Synthesis of (2£)-N-[5-(3-fluorophenyl)-l,3,4-thiadiazol-2-yl]-iV- isobutyl-3-phenylacrylamide

AU the steps used to prepare the title compound are analogous to those used for Example 5 except that cinnamoyl chloride was used instead of phenoxyacetyl chloride. δ _H (^ ₆ -DMSO, 300 MHz): 0.96 (6H, d), 1.90 (IH, m), 3.14 (2H, m), 6.83 (IH, d), 7.25-7.65 (7H, m), 7.75-8.00 (2H, m) and 8.10 (IH, m).

Example 16 Synthesis of N-[5-(3-fluorophenyl)-l,3,4-thiadiazol-2-yl]-N-isobutyl- 2-phenoxypropanamide

AU the steps used to prepare the title compound are analogous to those used for Example 5 except that 2-phenoxypropionyl chloride was used instead of phenoxyacetyl chloride. δ _H (J ₆ -DMSO, 300 MHz): 0.90 (3H, d), 0.87 (3H, d), 1.56 (3H, d), 2.32 (IH, m), 4.07 (IH, m), 4.19 (IH, m), 5.72 (IH, m), 6.98 (3H, m), 7.25-7.45 (3H, m), 7.58 (IH, m) and 7.77 (2H, m). m/z (ES+) 400.0 (M+H) ⁺ .

Example 17 Synthesis of iV-[5-(3-fluorophenyl)-l,3,4-thiadiazol-2-yl]-N-isobutyl- 2,3-dihydro-l,4-benzodioxine-2-carboxamide

AU the steps used to prepare the title compound are analogous to those used for Example 5 except that 2,3-dihydro-benzo[l,4]dioxane-2-carbonyl chloride was used instead of phenoxyacetyl chloride. δ _H (J ₆ -DMSO, 300 MHz): 0.94 (3H, d), 0.96 (3H, d), 2.31 (IH, m), 4.09 (IH, m), 4.36 (IH, m), 4.47 (2H, m), 5.75 (IH, m), 6.88 (3H, m), 6.96 (IH, m), 7.38 (IH, m), 7.58 (IH, m) and 7.78 (2H, m). m/z (ES+) 414.0 (M+H) ⁺ .

Example 18 Synthesis of iV-[5-(3-fluorophenyl)-l,3,4-thiadiazol-2-yl]-2-(4- hydroxyphenoxy)-iV-isobutylacetamide

AU the steps used to prepare the title compound are analogous to those used for Example 5 except that 4-hydroxyphenoxyacetyl chloride was used instead of phenoxyacetyl chloride.

Example 19 Synthesis of N-[5-(3-fluorophenyl)-l,3,4-thiadiazol-2-yl]-3-(2- methoxy-phenyO-N-propylpropanamide

A solution of 3-fluorobenzoic hydrazide (1.54g, O.Olmol) in methanol (3OmL) was treated with propyl isothiocyanate (l.OlmL, O.Olmol) and heated at reflux for 3h. The reaction mixture was then poured onto cracked ice. The resultant white solid was collected by filtration and dried to give 2-(3-fluorobenzoyl)-iV-propylhydrazine- carbothioamide (2.4g, 95%). After checking by TLC (DCM: Methanol 9:1) this intermediate was of sufficient purity to use directly in the next step.

Concentrated sulfuric acid (25mL) was cooled to 0 ⁰ C and stirred while 2-(3- fluorobenzoyl)-N-propylhydrazine-carbothioamide (2.Og, 7.8mmol) was added portion

wise. The reaction mixture was stirred for Ih at O ⁰ C and then allowed to warm to room temperature over 4h. The solution was poured onto cracked ice and neutralized with dilute sodium hydroxide solution. The resultant white solid was collected by filtration and dried to give 5-(3-fluorophenyl)~N-propyl-l,3,4~thiadiazol-2-amine (1.3g, 75%). After checking by TLC (DCM: Methanol 9:1) this intermediate was of sufficient purity to use directly in the next step.

A solution of 3-(2~methoxyphenyl)propanoyl chloride (0.4ImL, 2.1mmol) in dry dioxane (5.OmL) was added slowly to a stirred suspension of 5-(3-fluorophenyl)-iV- propyl-l,3,4-thiadiazol-2-amine (0.5g, 2.1mmol) and sodium bicarbonate (5.0g) in dry dioxane (15ml). After the addition , the mixture was stirred at room temperature for Ih and then heated at reflux for 5h. The reaction mixture was then poured onto cracked ice. The resultant white solid was collected by filtered and washed with hexane to give the title compound (0.79g, 95%). δ _H tø-DMSO, 300 MHz): 0.94 (3H, t), 1.74 (IH, m), 2.95 (2H, m), 3.02 (2H, m), 3.80 (3H, s), 4.17 (2H, m), 6.88 (IH, t), 6.97 (IH, d), 7.22 (2H, m), 7.38 (IH, m), 7.59 (IH, m), 7.78 (2H, m). m/z (ES+) 399.9 (M+H) ⁺ .

Example 20 3-(4-Fluorophenyl)-l-[5-(3-fluorophenyl)-(l,3,4-thiadiazol-2 -yl)-l- propylurea

A solution of 3-fluorobenzoic hydrazide (5g, 32mmol) in methanol (100ml) was treated with propyl isothiocyanate (5g, 49mmol) and heated at reflux for 3h. After this time the reaction mixture was poured onto cracked ice. The white solid was collected

by filtration and dried to give 2-(3-fluorobenzoyl)-λ ^L proρylhydrazinecarbothioamide. After checking the TLC (DCM: Methanol 9:1) this intermediate (7.5g) was of sufficient purity to use directly in the next step.

Concentrated sulphuric acid (12OmL) was cooled to 0 C and stirred while 2-(3- fluorobenzoyl)-N-propylhydrazinecarbothioamide (7.5g, 30mmol) was added portion- wise. The mixture was stirred for Ih in the cold and then allowed to warm to room temperature over 4h. The solution was poured into cracked ice and neutralized with dilute sodium hydroxide solution. The white solid was collected by filtration and dried to give the N-propyl-5-(3-fluorophenyl)-l,3,4-thiadiazol-2-amine. After checking the TLC (DCM: Methanol 9:1) this intermediate was of sufficient purity to use directly in the next step.

A solution of N-propyl-5-(3-fluorophenyl)-l,3,4-thiadiazol-2-amine (Ig, 4.2mmol), diisopropyl-ethylamine (1OmL) and THF (1OmL) was cooled to 0°C and stirred for 15 min. Triphosgene (0.66g) was added and the reaction mixture stirred at the same temperature for 15 min. 4-Fluoroaniline (0.5g, 4.5mmol) was added and the temperature raised to 10-12 ⁰ C and the reaction mixture stirred for 20 min. The reaction mixture was poured into water and the resulting product was extracted with ethyl acetate, dried over sodium sulphate and purified by column chromatography on silica gel, eluting with ethyl acetate:hexane (1:4), to give 500mg of the title compound. δ _H (CDCl ₃ , 300 MHz): 0.97 (3H, t), 1.70-1.85 (2H, m), 3.98 (2H, t), 6.94 (2H, t), 7.09 (IH, dt), 7.31-7.40 (IH, m), 7.40-7.48 (2H, m), 7.50-7.57 (2H, m). m/z (ES+) 374.9 (M+H) ⁺ .

Example 21 3-(4-Fluorophenyl)-l-[5-(3-fluorophenyl)-(l,3 _; ,4-thiadiazol-2-yl)-l- methyl-3-propyIurea

A mixture of N-propyl-5-(3-fluorophenyl)-l,3,4-thiadiazol-2-amine (Ig, 4.2mmol), diisopropyl-ethylamine (1OmL) and THF (1OmL) was cooled to 0 C and stirred for 15 min. Triphosgene (0.66g) was added and the reaction mixture stirred at same temperature for 15 min. 4-Fluoro-N-methylaniline (0.50g, 4.0mmol) was added and the temperature raised to 10-12°C and the reaction mixture stirred for 20 min. The reaction mixture was poured into water and the resulting product was extracted with ethyl acetate, dried over sodium sulphate and purified by column chromatography on silica gel, eluting with ethyl acetate:hexane (1:4), to give 400mg of the title compound. δ _H (CDCl ₃ , 300 MHz): 0.85 (3H, t), 1.50-1.65 (2H, m), 2.91 (2H, t), 3.39 (3H, s), 6.91 (2H, t), 7.05 (IH, m), 7.1-7.2 (4H, m), 7.2-7.3 (IH, m). m/z (ES+) 388.9 (M+H) ⁺ .

Example 22 5-Fluoro-2,3-dihydroindoIe-l-carboxylic acid, [5-(3-fluorophenyl)- l,3,4-thiadiazol-2-yl]-propylamide

A mixture of N-propyl-5-(3-fluorophenyl)-l,3,4-miadiazol-2-amine (Ig, 4.2mmol), diisopropyl-ethylamine (1OmL) and THF (1OmL) was cooled to 0°C and stirred for 15 min. Triphosgene (0.66g) was added and the reaction mixture stirred at same

temperature for 15 min. 4-Fluoroindoline (0.5Og, 3.65mmol) was added and the temperature raised to 10-12°C and the reaction mixture stirred for 20 min. The reaction mixture was poured into water and the resulting product was extracted with ethyl acetate, dried over sodium sulphate and purified by column chromatography on silica gel, eluting with ethyl acetate:hexane (1:4), to give 300mg of the title compound. δ _H (CDCl ₃ , 300 MHz): 0.98 (3H, t), 1.67-1.78 (2H, m), 3.22 (4H, m), 4.37 (2H, t), 6.9- 7.0 (2H, m), 7.18 (IH, m), 7.4-7.5 (3H, m), 8.0 (IH, m). m/z (ES+) 400.9 (M+H) ⁺ .

Example 23 [5-(3-Fluorophenyl)-l,3,4-thiadiazol-2-yl]-isobutylcarbamic acid, 4- fluorophenyl ester

λ ^r -isobutyl-5-(3-fluorophenyl)-l,3,4-thiadiazol-2-amine was prepared by the same method as for N-propyl-5-(3-fluorophenyl)-l,3,4-thiadiazol-2-amine except that isobutylisothiocyanate was used instead of propylisothiocyanate.

A mixture of iV-isobutyl-5-(3-fluorophenyl)-l,3,4-thiadiazol-2-amine (1.06g, 4.22mmol), diisopropylethylamine (1OmL) and THF (lOnϊL) was cooled to 0°C and stirred for 15 min. Triphosgene (0.66g) was added and the reaction mixture stirred at same temperature for 15 min. 4-Fluorophenol 0.50g, 4.46mmol) was added and the temperature raised to 10-12°C and the reaction mixture stirred for 20 min. The reaction mixture was poured into water and the resulting product was extracted with ethyl acetate, dried over sodium sulphate and purified by column chromatography on silica gel, eluting with ethyl acetate:hexane (1:4), to give lOOmg of the title compound.

δ _H (CDCl ₃ , 300 MHz): 1.00 (6H, d), 2.39 (IH, m), 4.30 (2H, d), 7.00-7.12 (4H, m), 7.28-7.38 (2H, m), 7.52-7.62 (2H, m). m/z (ES+) 390.1 (M+H) ⁺ .

Example 24 N-[5-(3-Fluorophenyl)-l,3,4-thiadiazol-2-yl]-4-methoxy-N-pro pyl- benzamide

A mixture of N-propyl-5-(3-fluorophenyl)~l,3,4-thiadiazol-2-amine (0.5g, 2.1mmol), p-anisoylchloride (0.36g, 2.1mmol) and sodium carbonate (5g) in dry dioxan (2OmL) was heated at 60 C for 3h. The reaction mixture was poured into water and the resulting product was extracted with ethyl acetate, dried over sodium sulphate and purified it by column chromatography on silica gel, eluting with ethyl acetate:hexane (1:4), to give 500mg of the title compound. δ _H (CDCl ₃ , 300 MHz): 0.85 (3H, t), 1.82-1.93 (2H, m), 3.90 (3H, t), 4.30 (2H, t), 7.03 (2H, d), 7.18 (IH, br t), 7.47 (IH, m), 7.56 (2H, d), 7.72-7.79 (2H, m). m/z (ES+) 371.9 (M+H) ⁺ .

Example 25 N-[5-(3-Fluorophenyl)-l,3,4-oxadiazol-2-yl]-N-isobutyl-2-phe noxy- acetamide

A solution of 3-fluorobenzoic hydrazide (5g, 32.3mmol) in methanol (10OmL) was treated with isobutyl isothiocyanate (5g, 43mmol) and reflux for 3h. After this time the heating was stopped and the reaction mixture was poured onto cracked ice. The white solid was filtered and dried to give the 2-(3-fluorobenzoyl)-iV- isobutylhydrazinecarbothioamide. This material (7.5g) was of sufficient purity to use directly in the next step.

A mixture of 2-(3-fluorobenzoyl)-N-isobutylhydrazinecarbothioamide (3g, ll.δmol), tosyl chloride (2.54g, 13.3mmol), and pyridine(1.9g, 24.1mmol) in THF (3OmL) was stirred at 70°C for 2Oh and then cooled to room temperature. The solution was poured into cracked ice and neutralized with dilute hydrochloric acid. The white solid was collected by filtration, dried and purified by column chromatography on silica gel, eluting with ethyl acetaterhexane (1:2), to give iV-isobutyl-5-(3-fluorophenyl)-l,3,4- oxadiazol-2-amine (2g).

A mixture of N-isobutyl-5-(3-fluorophenyl)-l,3,4-oxadiazol-2-amine (l.Og, 4.26mmol), phenoxyacetyl chloride (0.789g, 4.6mmol) and sodium carbonate (7g) in dry dioxan (4OmL) was heated at reflux (105°C), with stirring, for 5h. The reaction mixture was poured into water and the resulting product was extracted by ethyl acetate, dried over sodium sulphate and purified by column chromatography on silica gel, eluting with ethyl acetate :hexane (1:9), to give the title compound (50mg). δ _H (CDCl ₃ , 300 MHz): 0.94 (6H, d), 1.98 (IH, m), 3.72 (2H, d), 5.02 (2H, s), 6.71 (2H, d), 6.85 (IH, t), 7.08-7.20 (3H, m), 7.35-7.44 (IH, m), 7.52 (IH, br d), 7.64 (IH, d). m/z (ES+) 370.0 (M+H) ⁺ .

Example 26 2-(4-Fluorophenyl)-N-[5-(3-fluorophenyl)-l,3,4-oxadiazol-2-y l]-N- isobutylacetamide

A mixture of iV-isobutyl-5-(3-fluorophenyl)-l,3,4-oxadiazol-2-amine (1.Og, 4.26mmol), 4-fluorophenylacetyl chloride (0.789g, 4.6mmol) and sodium carbonate (7g) in dry dioxan (4OmL) was heated at reflux (105°C), with stirring, for 5h. The reaction mixture was poured into water and the resulting product was extracted by ethyl acetate, dried over sodium sulphate and purified by column chromatography on silica gel, eluting with ethyl acetate:hexane (1:9), to give the title compound (35mg). δ _H (CDCl ₃ , 300 MHz): 0.84 (6H, d), 1.92 (IH, m), 3.65 (2H, d), 4.00 (2H, s), 6.85 (2H, t), 7.00-7.08 (2H, m), 7.14-7.21 (IH, m), 7.37-7.46 (IH, m), 7.53 (IH, td), 7.63 (IH, d). m/z (ES+) 372.0 (M+H) ⁺ .

Example 27 N-[5-(3-FluorophenyI)-l,3,4-oxadiazol-2-yl]-3-(2-methoxyphen yl)- N-propyl-propionamides

N-propyl-5-(3-fluorophenyl)-l,3,4-oxadiazol-2-amine was prepared by the same method as for N-isobutyl-5-(3-fluorophenyl)-l,3,4-oxadiazol-2-amine except that propyliso-thiocyanate was used instead of isobutylisothiocyanate.

A mixture of JV-propyl-5-(3-fluorophenyl)-l,3,4-oxadiazol-2-arnine (l.Og, 4.52mmol), 3-(2-methoxyphenyl)propionyl chloride (0.91gm, 4.6mmol) and sodium carbonate (7g) in dry dioxan (4OmL) was heated at reflux (105°C), with stirring, for 5h. The reaction mixture was poured into water and the resulting product was extracted by ethyl acetate, dried over sodium sulphate and purified by column chromatography on silica gel, eluting with ethyl acetate:hexane (1:9), to give the title compound (50mg). δ _H (CDCl ₃ , 300 MHz): 0.95 (3H, t), 1.5-1.7 (2H, m), 2.93 (4H, s), 3.65 (3H, s), 3.78 (2H, m), 6.67 (IH, d), 6.75 (IH, t), 7.0-7.1 (2H, m), 7.1-7.2 (IH, m), 7.40 (IH, m), 7.58 (IH, br d), 7.70 (IH, d). m/z (ES+) 385.2 (M+H) ⁺ .

Example 28 Preparation of stock solutions for biological assays

Aβ(l-42) preparation

Aβ(l-42) was prepared for amyloid aggregation and toxicity assays by dissolving Aβ(l-42) HCl salt in hexafluoroisopropanol (HFIP), with brief sonication and vortexing. This solution of the Aβ(l-42) peptide in HFIP was stored at 4°C @ 2mM. When required, an aliquot of this stock solution was freeze-dried and dissolved in DMSO to 200 times the required final assay concentration (e.g. 2mM for a final assay concentration of 10 μM). Compound preparation

A 2OmM stock solution of each test compound was prepared in DMSO, and aliquots of these solutions were used to prepare further stock solutions of each test compound in DMSO, ranging in concentration from 3μM up to 1OmM. These stock solutions were prepared for use as and when required and stored at -2O ⁰ C (maximum of 3 freeze-thaw cycles). The 2OmM parent stock solutions were stored frozen at -20 °C.

Example 29 Cell viability assay for amyloid toxicity using MTT reduction

The activity of compounds in protecting SH SY5Y cells from a toxic insult of lOμM Aβ(l-42) was assessed by using inhibition of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-tetrazolium bromide] reduction as a measure of cell viability. An aliquot (3μl) of test compound [various concentrations] in DMSO is added to 294 μl of Opti- Mem (containing 2% FBS, 1% Pen/Strep, 1% L-GIn) {daughter plate}. The well is mixed thoroughly. Then an aliquot (3 μl) of Aβ(l-42) [2mM] is added to the daughter plate wells and again mixed thoroughly. 50 μl is then aspirated and dispensed into wells containing 50 μl media + SH SY5Ycells (cells are also plated in Opti-Mem, at ~ 30,000 cells/well/50 μl). Final concentrations of compound on cells range from [50μM] to [~15nM] with a final concentration of Aβ(l-42) of [10μM].

Cell plates are incubated for 24 h and then the MTT assay (Shearman, 1999). is performed. Briefly, 15μl of MTT dye (from Promega) added to each well and the plates incubated in 5% CO ₂ at 37°C for 4 hours. 100 μl Stop/solubilsation solution (from Promega) was added to each well and the plates were left overnight in humidified box at room temperature. The plate was shaken and the absorbance was recorded at both 570 nm and 650 nm. δA values were calculated by subtracting absorbance at 650 nm from absorbance at 570 nm, to reduce non-specific background absorbance. δA values from equivalent experiments were averaged and % cell viability was determined as follows: % cell viability = FδACsample) - δAfdead cell control)! x 100%

[δA(live cell control) - δA(dead cell control)]

Live cell controls: 1% DMSO in Opti-Mem Dead cell controls: 0.1% Triton X-100 added to cells

Example 30Activity of compounds in protecting SH S Y5Y cells from a toxic insult of lOμM Aβ(l-42) using inhibition of MTT reduction as a measure of cell viability

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