GB2512975A

GB2512975A - Novel compounds

Info

Publication number: GB2512975A
Application number: GB1401185.2A
Authority: GB
Inventors: Jose Luis Castro Pineiro; Carine Guerot
Original assignee: Eisai R&D Management Co Ltd
Current assignee: Eisai R&D Management Co Ltd
Priority date: 2013-01-25
Filing date: 2014-01-24
Publication date: 2014-10-15
Also published as: GB201301316D0; GB201401185D0

Abstract

4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3]thiazin-2-amine derivatives of formula (I) may be useful for the treatment of Alzheimer-type dementia and Down's syndrome. These compounds may have an amyloid-Î² (AÎ²) protein production inhibitory effect or a beta-site amyloid-Î² precursor protein cleavage enzyme 1 (BACE1 or beta-secretase) inhibitory effect. Ring A is an optionally substituted C6-14 aryl or a 5- to 10-membered heterocycle; Ring B is an optionally substituted C6-14 aryl or a 5- to 10-membered heterocycle; L is a bond, -O-, -NR8C(O)-, -C(O)NR8-, -NR8- or -C1-4 alkylene-; R8 is hydrogen or C1-6 alkyl; R1-R4 are H or a substituent. The compound may be (4aR,7aR)-7a-(3-(pyrimidin-5-yl)phenyl)-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3]thiazin-2-amine or N-(3-((4aR,7aR)-2-amino-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3]thiazin-7a-yl)phenyl)-5-methoxypyrazine-2-carboxamide.

Description

NOVEL COMPOUNDS

The present invention relates to a 4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3lthiazin-2-amine derivative and phannaceutical use thereof More particularly, the present invention relates to a 4a,6,7, 7a-tetrahydro-4H-furo[3,2-d] [1,3]thiazin-2-amine derivative which has an amyloid-J3 (hereinafter referred to as AJ3) protein production inhibitory effect or a beta-site amyloid-f3 precursor protein cleavage enzyme 1 (hereinafter referred to as BACE1 or beta-secretase) inhibitory effect and which therefore may be effective for treating a neurodegenerative disease caused by A13 protein, in particular, Alzheimer-type dementia, Down's syndrome or the like, and to a pharmaceutical composition comprising the 4a,6,7,7a-tetrahydro-4H-furo[3,2-dI [1,3]thiazin-2-amine derivative as an active ingredient.

Alzheimer's disease is characterized by degeneration and loss of neurons as well as formation of senile plaques and neurofibrillary tangles. Currently, only the symptoms of Alzheimer's disease are treated using a symptom-improving agent typified by an acetylcholinesterase inhibitor, and a fundamental remedy to inhibit progression of the disease has not yet been developed. It is necessary to develop a method for controlling the causative pathology in order to create a fundamental remedy for Alzheimer's disease.

It is believed that AJ3-proteins, as breakdown products of amyloid precursor proteins (hereinafter referred to as APP) are critical to the degeneration and loss of neurons and onset of symptoms of dementia. Af3-proteins have, as main components, Af340 consisting of 40 amino acids and A42 consisting of 42 amino acids wherein the two additional amino acids are present at the C-terminal, The Af340 md Af342 proteins are known to be highly prone to aggregation and to be the main components of the senile plaques linked to AD (Tanzi & Bertram, Cell 2005, 120: 545-555; Haass & Selkoe, Nat Rev Mol Cell Biol 2007, 8: 101-112).

Multiple mutations in the APP gene have been associated with early and late onset Alzheimer' s disease. The homozygous expression of one such mutation, A673V at the BACE1 cleavage site, has been linked to early onset AD (EOAD) (Di Fede et al, Science 2009, 323, t473-t477; Giaccone et al ActaNeuropathol. 2010, 120, 803-8 12). This mutation has, in cellular assays, been shown to significantly increase A1340 and Af342 production providing further support that genetic mutations in APP which increase its processing by BACEI result in increased amyloid production and AD, Similarly, aK67ON/MÔ7IL double mutant (Swedish mutation), which has previously been shown to be linked to early onset AD (Mullan et al Nat Genet. 1992 1(5), Aug, 345-347) produced even more pronounced increases in Af3 production than the A693V mutant.

A recent study of 1795 Icelanders (J'onsson et al, Nature 2012, 488, 2 August, 96-99) identified an APP mutation (A693T) which protects against Alzheimer's disease md cognitive decline, This mutation is proximal to the BACE cleavage site (position two of Af3) and was found to result in approximately 40% reduction of AJ3 peptide production in an iii vitro cellular assay which was attributed to the peptide being processed 50% less efficiently with 50% decrease in the rate of cleavage by BACE] This (heterozygous) mutation was previously identified in a single individual (Peacock et al, Neurology, 1993, 43, 1254-1256) who lived to 65 years of age and showed no signs of dementia and no amyloid pathology upon autopsy. The implication of these findings is that reducing the production of amyloid-f3 could prevent or delay the onset of AD. Furthermore, analysis of non-AD populations with and without the A693T mutant revealed that A693T carriers showed better conservation of cognitive function (Jonsson et a], Nature 2012,488,2 August, 96-99). Three homozygotes carrying the A673T mutation were found within the set and none were found to have suffered from dementia, including one who had died at the age of 88. Thus, there is strong genetic evidence linking APP mutations to AD pathology, both as protective and pathogenic.

A study on the rate of clearance of A13 from the CNS (Mawuenyeg et al Science 2010, 330, 24 December, 1774), reported that individuals with late onset AD (LOAD) had a 30% reduction versus controls. This data suggests that increasing the clearance of Af3 from the CNS or decreasing the formation of Af3 in the CNS by 30%, for example by inhibiting BACEI, may prevent or delay the onset of AD.

Accordingly, a compound that reduces production of Aj340 and A42 is predicted to be a disease progression inhibitor or prophylactic agent for Alzheimer's-type dementia (AD).

In addition to Alzheimer's-type dementia (AD), there are other amyloidogenic conditions such as Down's syndrome for which a compound that reduces production of A40 and Af342 may be beneficial (Mann, Neurobiol, Aging, t989, 10: 397-399; Mann et al,, J. Neurol Sci. 1989, 92: 247-260). Early onset dementia and Alzheimer's type dementia (AD) are common outcomes in individuals with Down's syndrome and evidence suggests A deposition may be a causative factor in cognitive impaimient associated with Down's syndrome. Another common co-morbidity in Down's syndrome is diabetes, being seven times more likely in individuals with Down's syndrome than the general population, and this increased frequency may be attributed to a gene product of chromosome 21, the triplication of all or some of which is characteristic of Down's syndrome (trisomy 21). As the gene encoding BACE2 is on chromosome 21 (Webb and Murphy, Curr. Gerontol, Geratr, Res, 2012) and BACE2 and one of its substrates, Tmem27, have been linked to diabetes, BACE2 inhibitors may be useful for the treatment of diabetes (Esterhazy et al, Cell Metab,, 2011, N: 365-377). Accordingly, a dual BACE1/2 inhibitor may provide an effective treatment for amyloid pathology and diabetes in the general or Down's syndrome populations.

Examples of other neurodegenerative diseases that might be treatable or preventable with a compound that reduces progression of Af340 and Af342 include cerebrovascular amyloid angiopathy (CAA), mild cognitive impairment (MCI), memory loss, presenile dementia, senile dementia, hereditary cerebral hemorrhage with amyloidosis, and other degenerative dementias such as dementias of mixed vascular and degenerative origin, dementia associated with supranuclear palsy, dementia associated with cortical basal degeneration, dementia associated with Parkinson's Disease (PD), and dementia associated with diffuse Lewy Body type of AD. Further conditions that might be treatable or preventable with a compound that reduces progression of AJ340 and AJ342 include Creutzfield-Jakob Disease (CJD), peripheral nerve injury, peripheral neuropathy, progressive supra-nuclear palsy, stroke, amyotrophic lateral sclerosis (ALS), autoimmune diseases, inflammation, arterial thrombosis, anxiety disorders, psychotic disorders, epilepsy, seizures, convulsions, stress disorders, vascular amyloi dosi s, pain, Gerstmann-Straeussl er-Scheinker syndrome, scrapie, encephal opathy, spino cerebel I ar ataxi a, Wilson's Disease, Graves Disease, Huntington's Disease, Whipple's Disease, Kostmann Disease, glaucoma, hereditary cerebral hemorrhage with amyloidosis, cerebral hemorrhage with amyloidosis, vascular amyloidosis, brain inflammation, fragile X syndrome, stroke, Tourette's syndrome, inclusion body myositis, stress disorders, depression, bipolar disorder and obsessive compulsive disorder.

Farah c/at (J. Neurosci, 13 April 2011, 31(15):5744-5754) have described how BACE1 inhibitors may also be useful in a therapeutic approach to accelerate regeneration and recovery after peripheral nerve damage.

A13 is produced by the cleavage of APP by beta-secretase (BACE]) and subsequently by gamma-secretase. For this reason, attempts have been made to create compounds which inhibit Aj3 production. Examples of compounds having an A13 or BACE I inhibitory effect are described for example in W020t 1009897, W020t t063233, W020t t063272, W0201 1090911, W0201 1002409, W0201 1005738, W0200913 1975, W0200902296t, W020090910]6, W020090974W and W02009097278, Several A13 inhibitors have been investigated in clinical studies as potential treatments for AD. For example, the compound MK-893 has been reported to reduce CSF A1340, A1342 and sAPPI3 in AD patients (Forman et al., International Conference on Alzheimer's & Parkinson's Diseases, Florence, Italy, 7th March 2013). Other examples include LY28 11376 (J. Neurosci 2011, Vol. 31(46):16507-16516) and LY2886721 (May etal., 1111'International Conference on Alzheimer's & Parkinson's Diseases, Florence, Italy, 7th March 2013).

An object of the present invention is to provide further compounds that have an Af3 production inhibitory effect or a BACE1 inhibitory effect and which may be useful as prophylactic or therapeutic agents for a neurodegenerative disease caused by Af3 and typified by Alzheimer-type dementia or Down's syndrome.

The invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof S2 NH2 S3 (I) wherein R' is hydrogen, halogen, C16 alicyl or C16 haloalicyl; R2 is hydrogen, halogen, C16 alicyl, C16 haloalkyl or a Ring C; R3 and R4 are each independently hydrogen, C16 alkyl or C16 haloalkyl; Ring A is a C614ary1 or a 5-to lO-membered heterocyclic group, either of which is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, cyano, nitro, -NIR'R', -C(O)NR5R5, -C(o)0R5, -C(O)R5, -NHS(O)2R', -NHC(O)R5, C26alkenyl, C26alkynyl, C3gcycloalkyl, C3gcycloalkoxy, Ci6alkoxy or 6alkyl; which C26alkenyl, C26alkynyl, C33cycloalkyl, C33cycloalkoxy, Ci5alkoxy or 6alkyl are each optionally substituted by one or more substituents independently selected from halogen, hydroxyl, cyano, C16 alkoxy, -NH2, -NH(Ci6alkyl) and -N(Ci6alky2; each R' independently is hydrogen or C16 allcyl; Ring B is a C6 14aryl or a 5-to lO-membered heterocyclic group, either of which is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, cyano, nitro, -NR6R6, -C(O)NR6R6, -C(O)0R6, -C(O)R6, -NHS(O)2R6, -NIHC(O)R6, C26alkenyl, C26alkynyl, C3gcycloalkyl, C38cycloalkoxy, Ci6alkoxy or C1.

6alkyl; which C26alkenyl, C26alkynyl, C33cycloalkyl, C33cycloalkoxy, Ci6alkoxy or C1 6alkyl are each optionally substituted by one or more substituents independently selected from halogen, hydroxyl, cyano, C16alkoxy, -NI-I2, -NH(Ci6alkyl) and -N(Ci6alky2; each R6 independently is hydrogen or C16 alkyl; Ring C is phenyl or a 5-to 6-membered heteroaryl group, either of which is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, cyano, nitro, -NIR7R', -C(O)NR7R7, -C(O)OR', -C(o)R7, -NHS(o)7R', -NHC(O)R', C2 6alkenyl, C26alkynyl, C3gcycloalkyl, C3gcycloalkoxy, Ci6alkoxy or Ci6alkyl; which C2 6alkenyl, C26allcynyl, C3gcycl oalkyl, C3gcycl oallcoxy, Ci6alkoxy or Ci6alkyl are each optionally substituted by one or more substituents independently selected from halogen, hydroxyl, cyano, C16 alkoxy, -NH2, -NH(Ci6alkyl) and -N(Ci6alkyl)2; each R' independently is hydrogen or C1(, alky; and L is a bond, -0-, -INIR8C(O)-, -C(O)NIR8-, -NR8-or -Ci4alkylene-; R8 is hydrogen or Ci6alkyl.

Certain compounds of formula (I) are capable of existing in stereoisomeric forms. It will be understood that the invention encompasses all geometric and structural isomers of the compounds of formula (I) and mixtures thereof including racemates, Tautomers and mixtures thereof also form an aspect of the present invention.

It will be understood that the compounds of the present invention and pharmaceutically acceptable salts thereof may exist in solvated, for example, hydrated, as well as unsolvated forms. It is to be understood that the compound of the present invention encompasses all such solvated forms.

In an embodiment of the present invention, Ring A is phenyl optionally substituted by one or more substituents independently selected from halogen, C14 alkyl and C1.4 haloakyl.

In an embodiment of the invention, Ring A is phenyl optionally substituted with one In an embodiment of the present invention, Ring B is a 6-membered heteroarvl group containing at east one nitrogen atom optionally substituted by one or more substituents independently selected from halogen, cyano, Ci4 haloakyl, Ci4 alkoxy, Ci4 haloalkoxy and C14 alkyl which C14 alkvl is optionally substituted with C14 alkoxy.

In an embodiment of the invention Ring B is pyridinyl, pyrimidinyl or pyrazinyl, wherein Ring B is optionally substituted by one or more substituents independently selected from halogen, cyano, C1.4 haloakyl, C1.4 alkoxy, C14 haloalkoxy and C14 alIcyl which C1.4 alkyl is optionally substituted with C1.4 allcoxy.

In an embodiment of the present invention, Lisa bond or -NHC(O)-.

In an embodiment of the present invention, Lisa bond.

In an embodiment of the present invention, L is -NHC(O)-.

In an embodiment of the present invention, one of R1 and R2 is hydrogen and the other of R and R is hydrogen, fluorine, methyl, monofluoromethyl, difluoromethyl or trifluoromethyl.

In an embodiment of the present invention, R' is hydrogen and R2 is hydrogen.

Those skilled in the art will recognise that the compounds of formula (I) comprise chiral centers located at the 7a and 4a positions as herein depicted below. In one aspect of the invention, the stereochemical configuration at these chiral centers is as depicted in Formula (IA) below.

RI

R2 NH2

S H 4

R3R (IA) For compounds of formula (IA), embodiments of the invention include those wherein each of A, B, L, R', R2, R3 and R4 are as defined herein above in embodiments of the invention concerning compounds of formula (I).

In one aspect, the present invention provides a compound of formula (IB) or a pharmaceutically acceptable salt thereof,

KNH (F) (IB)

N N H2

H

wherein nisO, t or2; A is CH or N; Y is fluorine, cyano, methyl, ethyl, monofluoromethyl, difluoromethyl, trifluoromethyl, difluoroethyl (-CH2CI-1F2), methoxy, ethoxy or methoxymethyl; one ofR' and R2 is hydrogen and the other ofR' and R2 is hydrogen, fluorine, methyl, monofluoromethyl, difluoromethyl or trifluoromethyl.

In one embodiment, the present invention provides a compound of formuTa (TB) or a pharmaceutically acceptable salt thereof wherein n is 0; A is N; Y is fluorine, monofluoromethyl, difluoromethyl or methoxy; H) is hydrogen; and ft2 is hydrogen.

In one aspect, the present invention provides a compound of formula (IC) or a pharmaceutically acceptable salt thereof,

JO

R2 NH2 H (IC) wherein n isO, t or 2; Ring B is a 6-membered heteroaryl group containing at least one nitrogen atom optionally substituted by one or more substituents independently selected from fluorine, cyano, methyl, ethyl, monofluoromethyl, difluoromethyl, trifluoromethyl, difluoroethyl (-CH2CHF2), methoxy, ethoxy or methoxymethyl; and one of R1 and R2 is hydrogen and the other of R1 and R2 is hydrogen, fluorine, methyl, monofluoromethyl, difluoromethyl or trifluoromethyl.

In one embodiment, the present invention provides a compound of formula (IC) or a pharmaceutically acceptable salt thereof wherein n is 0; Ring B is pyrimidinyl optionally substituted with I or 2 substituents independently selected from fluoine, cyano, methyl, ethyl, m onofluoromethyl, difluoromethyl, trifluoromethyl, difluoroethyl (-CH2CT-1F2), methoxy, ethoxy or methoxymethyl; R1 is hydrogen; and R2 is hydrogen.

For the avoidance of doubt, the compounds of formula (IA), (TB) and (IC) may be present as a mixture with one or more of the other possible stereoisomers, for example in a racemic mixture.

In one embodiment, the present invention provides the compound (4aR,7aR)-7a-(3-(pyrimidin-3-yl)phenyl)-4a,6,7,7a-tetrahydro-4H-furo[3, 2-d][l,3]thiazin-2-amine; )c N NH2 or a pharmaceutically acceptable salt thereof In one embodiment, the present invention provides the compound N-(3-((4alR,7aR)-2-amino-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3] thiazin-7a-yl)phenyl)-5-methoxypyrazine-2-carboxamide; N OMe 0tCNT

C-

Ifr Ny NH2

H

or a pharmaceutically acceptable salt thereof In one embodiment, the present invention provides the compound N-(3-((4aR,7aR)-2-amino-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3] thiazin-7a-yl)phenyl)-5- (difluoromethyl)pyrazine-2-carboxamide,

F

N F

ON

NH NyNH2

S

H

or a pharmaceutically acceptable salt thereof Further compounds according to formula (I) of the present invention include:-CH2F N CHF2 N OMe

NH NH NH F 2

NH F N1NH2

H H H

N CH2F

ONT F F

NH NH NH vv 17 H-

S S

<r-f N1NH2 NyNH2 F NtNH2

H H H

CN CN CN

NH NH NH

I, 17 y NH2 N1NH2 F N1NH2

H H H

FNH and FNH and pharmaceutically acceptable salts thereof As used herein, the term "C1 6allcyl" refers to an alkyl group having 1 to 6 carbon atoms. Examples of the group include linear and branched alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butvl, n-pentyl, isopentyl, neopentyl, n-hexyl, 1 -methylpropyl, I,2-dimethylpropyl, 1 -ethylpropyl, t -methyl-2-ethylpropyl, 1 -ethyl-2- methylpropyl, t, t,2-trimethylpropyl, t-methylbutyl, 2-methylbutyl, 1, t-dimethylbutyl, 2,2-dimethylbutyl, 2-ethylbutyl, 1,3 -dimethylbutyl, 2-methylpentyl and 3 -methylpentyl. The group is preferably methyl, ethyl or n-propyl.

As used herein, the term "Ci.4alkylene" refers to a divalent group derived by excluding any one hydrogen atom from a Ci.3alkyl group according to the definition of "C1.

6alkyl" above. Examples of a "Ci.4alkylene" group include methylene, 1,2-ethylene, 1,1-ethylene and 1,3-propylene.

As used herein, the term "C26alkenyl" refers to an alkenyl group having 2 to 6 carbon atoms. Examples of the group include linear and branched alkenyl groups such as vinyl, allyl, 1-propenyl, isopropenyl, ]-buten-]-yl, l-buten-2-yl, 1-buten-3-yl, 2-buten-]-yl and 2-buten-2-yl.

As used herein, the term "C2.6alkynyl" refers to an alkynyl group having 2 to 6 carbon atoms. Examples of the group include linear and branched alkynyl groups such as ethylnyl, 1-propynyl, 2-propynyl, butynyl, pentynyl and hexynyl.

As used herein, the term "C3scycloalkyl" refers to a cyclic alkyl group having a ring of 3 to 8 carbon atoms, Examples of the group include eyclopropyl, eyelobutyl, cyclopentyl, cyclohexyl, eyeloheptyl arid cyclooctyl.

As used herein, the term "C16alkoxy" refers to an alkyl group having 1 to 6 carbon atoms which is attached via an oxygen atom. Examples of the group include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, t-butoxy, n-pentoxy, isopentoxy, sec-pentoxy, t-pentoxy, n-hexyloxy, isohexyloxy, 1,2-dimethylpropoxy, 2-ethylpropoxy, 1 -methyl-2-ethylpropoxy, t -ethyl-2-methylpropoxy, 1,1,2-trimethylpropoxy, 1,1 -dimethylbutoxy, 2,2-dimethylbutoxy, 2-ethylbutoxy, 1,3 -dimethylbutoxy, 2-methylpentoxy, 3 -methylpentoxy and hexyloxy.

As used herein, the term "C3.8cycloalkoxy" refers to a cyclic alkyl group having a ring of 3 to 8 carbon atoms which is attached via an oxygen atom, Examples of the group include cyclopropoxy, cycl obutoxy, eye] opentoxy, cycl ohexyloxy, eye] oheptyloxy and cyclooctyloxy.

As used herein, the term "C6.14 aryl" refers to an aromatic hydrocarbon ring group having 6 to N carbon atoms. Examples of the group include phenyl, naphthyl and anthryl.

As used herein, the term "5-to 10-membered heterocyclic group" refers to a heteroatom-containing cyclic group having 5 to 10 ring atoms, including heteroaryl rings, non-aromatic rings such as heterocycloalkyl rings and partially saturated rings. Examples of the group include piperidinyl, pyrrolidinyl, azepinyl, azocanyl, piperazinyl, I,4-diazepanyl, morpholi nyl, thi omorpholinyl, pyrrolyl, imidazolyl, pyrazolyl, pyri dinyl, pyri dazinyl, pyrimidinyl, pyrazinyl, triazolyl, triazinyl, tetrazolyl, isoxazolyl, oxazolyl, oxadiazolyl, isothiazolyl, thiazolyl, thiadiazolyl, friryl, thienyl, quinolinyl, isoquinolinyl, benzofuryl, benzopyranyl, benzimidazolyl, benzotriazolyl, benzisothiazolyl, indolinyl, isoindolinyl, chromanyl, isochromanyl, 1,3 -dioxaindanyl and 1,4-dioxatetralinyl.

As used herein, the term 5-to 6-membered heteroaryl' denotes an aromatic monocyclic ring having 5 or 6 ring atoms wherein at least one ring atom is selected from nitrogen, oxygen and sulphur. Examples include furyl, thienyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, isooxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl, As used herein, the term C1.6 haloalkyl' denotes a C1.6 alkyl group substituted with one or more halogen atoms wherein each halogen is independently selected from fluorine, chlorine, bromine and iodine. Examples of C 1-6 haloalkyl groups include CF3 (trifluoromethyl), CHF2 (difluoromethyl), CH2F (monofluoromethyl), CH2CF3 and CH2CHF2.

As used herein, the term C.4ha1oalkoxy' denotes a C.4 alkoxy group substituted with one or more halogen atoms wherein each halogen is independently selected from fluorine, chlorine, bromine and iodine. Examples of C1.4 haloalkoxy groups include OCF3 (trifluoromethyloxy).

As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine.

In the context of the present specification, where it is stated that a group is optionally substituted by one or more substituents, the group may be unsubstituted or substituted, When substituted the group may for example be substituted with 1, 2 or 3 substituents, In the present invention, although crystal polymorphs of the compound of formula (I) may be present, the compound is not limited thereto and may be present as a single crystal form or a mixture of single crystal forms.

The present invention also includes isotopically-labelled compounds, which are identical to the compounds of formula (I), except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature, Examples of isotopes that can be incorporated into compounds of -10-the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, phosphorous, chlorine, technetium and iodine, such as 2H, 3H, c, "C, N, o, "t, 32P,9VIh1Tc, 23l and 1311.

Compounds of the present invention and pharmaceutically acceptable derivatives (e.g. salts) of said compounds that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present invention. Isotopically-labelled compounds of the present invention, for example those into which radioactive isotopes such as 11 and/or "C are incorporated, are useful in drug and/or substrate tissue distribution assays, and 14C are considered useful due to their ease of preparation and detectability. C, 150 and 8F isotopes are considered useful in PET (positron emission tomography), and 99''Tc, 123j and 131j isotopes are considered useful in SPECT (single photon emission computerized tomography), all useful in brain imaging. Substitution with heavier isotopes such as 211 can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased iii vivo half-life or reduced dosage requirements and, hence, are considered useful in some circumstances, Isotopically labelled compounds of formula (I) of this invention can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples below, by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.

The 4a,6,7,7a-tetrahydro-41-1-furo[3,2-d] [ ,3]thiazin-2-amine derivative of the formula (I) according to the present invention may be a pharmaceutically acceptable salt, Pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse, J, Pharm, Sci,, 1977, 66, 1-19. Specific examples of the pharmaceutically acceptable salt include inorganic acid salts (such as sulfates, nitrates, perchlorates, phosphates, carbonates, bicarbonates, hydrofluori des, hydrochl orides, hydrobromi des and hydroi odi des), organic carboxylates (such as acetates, oxalates, maleates, tartrates, fumarates, citrates, malonates, lactates and hippurates), organic sulfonates (such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, benzenesulfonates, toluenesulfonates and camphorsulfonates), amino acid salts (such as aspartates and glutamates), quaternary amine salts, alkali metal salts (such as sodium salts and potassium salts) and alkali earth metal salts (such as magnesium salts and calcium salts), The compound of the formula (I) according to the present invention may be converted to a pharmaceutically acceptable salt by a conventional method where necessary. The salt maybe prepared by a method in which methods typically used in the field of organic synthetic chemistry and the like are appropriately combined. Specific examples of the method include neutralization titration of a free solution of the compound of the present invention with an acid solution, The compound of formula (I) or a pharmaceutically acceptable salt thereof according to the present invention may be converted to a solvate by subjecting the compound to a solvate forming step known in the art,

-H -

The present invention further provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in therapy.

The 4a,6,7,7a-tetrahydro-4l-1-furo[3,2-d[ ,3]thiazin-2-amine derivative or pharmaceutically acceptable salt thereof according to the present invention has an AJ3 production inhibitory effect or BACE1 inhibitory effect and may be useful as a prophylactic or therapeutic agent for a neurodegenerative disease caused by A13 and typified by Alzheimer-type dementia. The compounds of the invention may reduce both A1340 and A1342.

Furthermore, the compounds of the present invention may have a BACE 2 inhibitory effect.

Thus, in another aspect, the present invention provides a compound of formula (1) as defined above, or a pharmaceutically acceptable salt thereof, for inhibiting production of amyloid-J3 protein.

In a further aspect, the present invention provides a compound of formula (I) as defined above, or a pharmaceutically acceptable sail thereof, for inhibiting beta-site amyloid- 3 precursor protein cleaving enzyme 1 IBACE I), In a further aspect, the present invention provides a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for treating or preventing a neurodegenerative disease.

In a further aspect, the present invention provides a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for treating or preventing Alzheimer-type dementia (AD).

In a further aspect, the present invention provides a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for treating Down's syndrome.

In another aspect, the invention provides the use of a compound of formula (1) as defined above, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment or prevention of a neurodegenerative disease.

In another aspect, the invention provides the use of a compound of fonnula (I) as defined above, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment or prevention of Alzheimer-type dementia (AD).

In another aspect, the invention provides the use of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of Down's syndrome.

In another aspect, the invention provides a method of inhibiting production of amyloid-J3 protein involving administering to a human subject in need thereof a therapeutically or prophylactically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. "Effective amount" means an amount sufficient to cause a benefit to the subject or at least to cause a change in the subject's condition.

In another aspect, the invention provides a method of treating or preventing Alzheimer-type dementia (AD) involving administering to a human subj ect in need thereof a -12-therapeutically or prophylactically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a method of treating Down's syndrome involving administering to a human subject in need thereof a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof In one aspect the present invention further provides a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for treating type 2 diabetes.

Tn a further aspect the present invention provides the use of a compound of formula (T) as defined above, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment or prevention of type 2 diabetes.

In a yet furher aspect the present invention further provides a method of treating or preventing type 2 diabetes involving administering to a human subject in need thereof a therapeutically or prophylactically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof A further aspect of the invention provides a pharmaceutical composition comprising a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, as active ingredient in association with a pharmaceutically acceptable carrier, The composition may be in any suitable form, depending on the intended method of administration. It may for example be in the form of a tablet, capsule or liquid for oral administration, or of a solution or suspension for administration parenterally.

The compound of formula (I) or pharmaceutically acceptable salt thereof according to the present invention may be formulated by a conventional method, Preferable examples of the dosage form include tablets, coated tablets such as film tablets and sugar-coated tablets, fine granules, granules, powders, capsules, syrups, troches, inhal ants, suppositories, injections, ointments, eye drops, nasal drops, ear drops, cataplasms and lotions, These solid preparations such as tablets, capsules, granules and powders may contain generally 0.01 to 100 wt%, and preferably 0.1 to 100 wt% of the compound of formula (I) or pharmaceutically acceptable salt thereof according to the present invention as an active ingredient.

The active ingredient is formulated by blending ingredients generally used as materials for a pharmaceutical preparation and adding an excipient, a disintegrant, a binder, a lubricant, a colorant and a corrective typically used, and adding a stabilizer, an emulsifier, an absorbefacient, a surfactant, a p11 adjuster, a preservative and an antioxidant where necessary, for example, using a conventional method. Examples of such ingredients include animal and vegetable oils such as soybean oil, beef tallow and synthetic glyceride; hydrocarbons such as liquid paraffin, squahme and solid paraffin; ester oils such as octyldodecyl myristate arid isopropyl myristate; higher alcohols such as cetostearyl alcohol and behenyl alcohol; a silicone resin; silicone oil; surfactants such as polyoxyethylene fatty acid ester, sorbitan fatty in -I.) -acid ester, glycerol fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil and a polyoxyethylene-polyoxypropylene block copolymer; water-soluble polymers such as hydroxyethylcellulose, polyacrylic acid, a carboxyvinyl polymer, polyethylene glycol, polyvinylpyrrolidone and methylcellulose; lower alcohols such as ethanol and isopropanoL polyhydric alcohols such as glycerol, propy'ene glycol, dipropylene glycol and sorbitol; sugars such as glucose and sucrose; inorganic powders such as silicic anhydride, magnesium aluminum silicate and aluminum silicate; and purified water. Examples of the excipient used include lactose, corn starch, saccharose, glucose, mannitol, sorbitol, crystalline cellulose and silicon dioxide. Examples of the binder used include polyvinyl alcohol, polyvinyl ether, methylcellulose, ethylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, a polypropylene glycol-polyoxyethylene block copolymer and meglumine. Examples of the disintegrant used include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin and carboxymethylcellulose calcium. Examples of the lubricant used include magnesium stearate, talc, polyethylene glycol, silica and hydrogenated vegetable oil. Examples of the colorant used include those permitted to be added to pharmaceuticals. Examples of the corrective used include cocoa powder, menthol, empasm, mentha oil, borneol and cinnamon powder. Obviously, the ingredients are not limited to the above additive ingredients.

For example, an oral preparation is prepared by adding the compound of formula (I) or pharmaceutically acceptable salt thereof according to the present invention as an active ingredient, an excipient and, where necessary, a binder, a disintegrant, a lubricant, a colorant, a corrective and the like, and then forming the mixture into powder, tine granules, granules, tablets, coated tablets, capsules or the like by a conventional method. Obviously, tablets or granules may be appropriately coated, for example, sugar coated, where necessary.

For example, a symp or an injection preparation is prepared by adding a pH adjuster, a solubilizer, an isotonizing agent and the like, and a solubilizing agent, a stabilizer and the like where necessary by a conventional method. The injection may be a previously prepared solution, or may be powder itself or powder containing a suitable additive, which is dissolved before use. The injection may contain usually 0.01 to 100 wt%, and preferably 0.1 to 100 wt% of the active ingredient. Further, a liquid preparation for oral administration such as a suspension or a symp may contain usually 0.01 to 100 wt%, and preferably 0.1 to 00 wt% of the active ingredient.

For example, an external preparation may be prepared by any conventional method without specific limitations. As a base material, any of various materials usually used for a pharmaceutical, a quasi drug, a cosmetic or the like can be used. Examples of the base material include materials such as animal and vegetable oils, mineral oils, ester oils, waxes, higher alcohols, fatty acids, silicone oils, surfactants, phospholipids, alcohols, polyhydric -14-alcohols, water-soluble polymers, clay minerals and purified water. A pH adjuster, an antioxidant, a chelator, a preservative and fungicide, a colorant, a flavor or the like can be added where necessary. Further, ingredients such as an ingredient having a differentiation inducing effect, a blood flow enhancer, a bactericide, an antiphlogistic, a cell activator, vitamin, amino acid, a humectant and a keratolytic agent can be blended where necessary.

The dose of the compound of formula (I) or pharmaceutically acceptable salt thereof according to the present invention may vary according to the degree of symptoms, age, sex, body weight, mode of administration, type of salt and specific type of disease, for example.

Typically, the active ingredient is orally administered to an adult at about 30 j.tg to 10 g, preferably 100 jig to 5 g, and more preferably 100 jig to I g per day, or is administered to an adult by injection at about 30 jig to 1 g, preferably 100 jig to 500 mg, and more preferably jig to 300 mg per day, in one or several doses, respectively.

Compounds of formula U) may be used in combination with other therapeutic agents, for example medicaments claimed to be useful as either disease modifying or symptomatic treatments of a neurodegenerative disease such as Alzheimer' s disease. Thus, in a further aspect, the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is a compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one further active ingredient useful in treating a neurodegenerative disease. In one embodiment of the invention, the neurodegenerative disease is Alzheimer-type dementia (AD). Suitable examples of such further active ingredients may be symptomatic agents, for example those known to modify cholinergic transmission such as Ml and M3 muscarinic receptor agonists or allosteric modulators, M2 muscarinic antagonists, M4 agonists or positive allosteric modulators (PAM5), acetylcholinesterase inhibitors (such as tetrahydroami noacridine, donepezil hydrochloride and rivastigmine), nicotinic receptor agonists or allosteric modulators (such as cz7 agonists or allosteric modulators or a4J32 agonists or allosteric modulators), PPAR agonists (such as PPAR7 agonists), 5-HT4 receptor agonists or partial agonists, histamine H3 antagonists, 5-HT5 receptor antagonists or SHT1A receptor ligands and NIIVIDA receptor antagonists or modulators (such as memantine), 511T2A antagonists, 5-HT7 antagonists, D t agonists or PAMs, D4 agonists or PAMs, D5 agonists or PAMs, GABA-A aS inverse agonists or negative allosteric modulators (NAIVIs), GABA-A a2/3 agonists or PAMs, mGluR2 modulators (PAM5 or NAMs), mGluR3 PAMs, mGluRS PAMs, PDE I inhibitors, PDE 2 inhibitors, PDE 4 inhibitors, PDE 5 inhibitors, PDE 9 inhibitors, PDE 10 inhibitors, G1yT1 inhibitors, DAAO inhibitors, ASC1 inhibitors, AIVIIPA modulators, SIRT1 activators or inhibitors, AT4 antagonists, GaIRl antagonists, GaIR3 ligands, adenosine Al antagonists, adenosine A2a antagonists, a2A antagonists or agonists, selective and unselective norepinephrine reuptake inhibitors (SNRI5), or potential disease modifying agents such as gamma secretase inhibitors or modulators, alpha secretase activators or modulators, amyloid -15 -aggregation inhibitors, amyloid antibodies, tau aggregation inhibitors or tau phosphoryl ati on/kinase inhibitors, tau dephosphoryl ati on / phosphatase activators, mitogen-activated protein kinase kinase 4 (MKK4/MEK4/MAP2K4) inhibitors, c-Jun N-terminal kinase (INK) inhibitors, casein kinase inhibitors, MK2 (mitogen activated protein kinase-activated protein kinase 2) inhibitors, MARK (microtubule affinity regulating kinase) inhibitors, CDKS (cyclin dependent kinase 5) inhibitors, GSK-3 (glycogen synthase kinase-3) inhibitors and tau-tubulin kinase-t (TTBKt) inhibitors. Further examples of such other therapeutic agents may be calcium channel blockers, HMG-CoA (3-hydroxy-3-methyl-glutaryl-CoA) reductase inhibitors (statins) and lipid lowering agents, NGF (nerve growth factor) mimics, antioxidants, GPR3 ligands, plasmin activators, neprilysin (NEP) activators, IDE (insulin degrading enzyme) activators, melatonin MT1 and/or MT2 agonists, TLX/NR2E1 (tailless X receptor) ligands, G1uR1 ligands, RAGE (receptor for advanced glycation end-products) antagonists, EGFR (epidermal growth factor receptor) inhibitors, FPRL-i (formyl peptide-like receptor-i) ligands, GABA antagonists, and MIICAL (molecule interacting with casL) inhibitors, e.g. oxoreductase inhibitors, CBi antagonists/inverse agonists, non-steroidal anti-inflammatory drugs (NSAIDs), anti-inflammatory agents (for example agents that could be used to treat neuroinflammation either by enhancing or reducing neuroinflammation), amyloid precursor protein (APP) ligands, anti-amyloid vaccines and / or antibodies, agents that promote or enhance amyloid efflux and / or clearance, histone deacetylase (FIDAC) inhbitors, EP2 antagonists, il-beta HSDt (hydroxysteroid dehydrogenase) inhibitors, liver X receptor (LXR) agonists or PAMs, lipoprotein receptor-related protein (LRP) mimics and / or ligands and/or enhancers and/or inhibitors, butyryl cholinesterase inhibitors, kynurinic acid antagonists and / or inhibitors of kynurenine aminotransferease (KAT), orphanin FQ / nociceptin (NOP) / opioid-like receptor I (ORLI) antagonists, excitatory amino acid transporter (EAAT) ligands (activators or inhibitors), and plasminogen activator inhibitori (PAl-I) inhibitors, niacin and /or GPR 109 agonists or PAMs in combination with cholesterol lowering agents and / or HMGCoA reductase inhibitors (statins), dimebolin or similar agents, antihistamines, metal binding / chelating agents, antibiotics, growth hormone secretagogues, cholesterol lowering agents, vitamin E, cholesterol absorption inhibitors, cholesterol efflux promoters and / or activators, and insulin upregulating agents.

In one embodiment, the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is a compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one further active ingredient selected from:-cholinesterase inhibitors, e.g. donepezil, galantamine, rivastigamine, tetrahydroaminoacridine and pharmaceutically acceptable salts thereof -16- * NMDA receptor antagonists e.g. memantine and pharmaceutically acceptable salts thereof, and any other compounds which elicit their effects by a similar mechanism of action, * 5-I-TT6 antagonists, e.g. SB-742457 and pharmaceutically acceptable salts thereof, * HIvIGCoA reductase inhibitors e.g. lovastatin, rosuvastatin, atorvastatin, simvastatin, fluvastatin, pitavastatin, pravastatin and pharmaceutically acceptable salts thereof The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.

Consequently, the pharmaceutical product may, for example be a pharmaceutical composition comprising the first and further active ingredients in admixture, Alternatively, the pharmaceutical product may for example comprise the first and further active ingredients in separate pharmaceutical preparations suitable for simultaneous, sequential or separate administration to a patient in need thereof The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the invention, When a compound of formula (I) or a pharmaceutically acceptable salt thereof is used in combination with a second therapeutic agent active, the dose of each compound may differ from that when the compound is used alone, Appropriate doses will be readily appreciated by those skilled in the art, Thus, an additional aspect of the invention provides a method of preparing a pharmaceutical composition, involving admixing at least one compound of formula (I) as defined above, or a pharmaceutically acceptable salt therof, with one or more pharmaceutically acceptable adjuvants, diluents or carriers and/or with one or more other therapeutically or prophylactically active agents.

General Preparation Methods Compounds of formula (I) may be prepared by the General Preparation Methods described herein below.

It will be appreciated by those skilled in the art that when preparing certain compounds of formula (I) it may be appropriate to modify the general preparation methods by alternating the sequence of reaction steps and/or incorporating additional steps to vary substituent groups on intermediate compounds. Moreover, it will also be recognised that compounds of formula (I) prepared according to the general preparation methods may subsequently be converted to other compounds of formula (I) using known chemistry.

It will also be appreciated by those skilled in the art that in some instances certain functional groups such as hydroxyl, carboxyl or amino groups in starting reagents or intermediate compounds may need to be protected by protecting groups. Thus the following Preparation -17-Methods may involve at certain stages the incorporation of one or more protecting groups.

The protection and deprotection of frmnctional groups is, for example, described in Protective Groups in Organic Synthesis, 3rd edition, T.W Greene and PG.M. Wuts, Wiley-lnterscience (1999) and Protecting Groups', P1. Kocienski, Georg Thieme Verlag (1994). The choice of solvent used in the steps described in the general preparation methods may vary according to the specific reagents used. Unless otherwise stated the choice of solvent is not particularly limited insofar as it does not inhibit the reaction, allows the reagents to be dissolved therein to a certain extent, and is inert during the reaction.

In the reaction schemes accompanying the General Preparation Methods the groups equivalent to R' R2, R3 and R4 in formula (I) are, for simplicity and clarity, depicted as hydrogen atoms. It will be understood by those skilled in the art that analogous chemistry 1 4 may be used to prepare alternate compounds of formula (I) wherein any of R, it, R and R are groups other than hydrogen.

General Preparation Method I -Intermediates (th and (1) General Preparation Method 1 (Scheme 1) is a method of preparing Intermediates (th) and (ig) from compound (la). In the formula of Scheme t, Ring A is as defined in formula (1), LV is a suitable leaving group e.g. a halogen atom (such as Cl, Br or I) or a sulfonyloxy group (such as OTf, OMs, OT5), P is a suitable protecting group (Bn, PMB, etc.), P2 is a suitable protecting group (SOtBu, Boc, OBn etc.) and alkyl i is an alkyl group, such as Me, Et, Pr, Bu and the like. The compound (ta) may be prepared as described in J. Am. Chem, Soc., 2004,126, 13600-13601.

Step 1,1: This is a step of obtaining a compound (Ib) by the oxidation of the alcohol moiety in compound (Ia) to a ketone when Pi is defined as above.

Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions.

For example compound (ta) can be oxidised to (Ib) with Swern oxidation (Synthesis 1981, 165-185; Org. React. 39: 297-572; Synthesis 1990: 857-870) in a suitable solvent, for example DCM, at an appropriate reaction temperature, for example be in the range -78°C to room temperature. Those skilled in the art will also appreciate that it may be possible to conduct this reaction with a range of alternative conditions, for example using Dess-Martin periodinane(J. Org. Chem. 1983, 48, 455; Org. Synth. CoIl. Vol. 10, 696), TPAP (Synthesis 1994, 639), Parikh-Doering oxidation (J. Am. Chem. Soc., 1967, 89: 5505-5507), Corey-Kim oxidation (J. Am. Chem. Soc 1972, 94, 7586), Pfitzner-Moffatt (J. Am. Chem. Soc. 1963,85, 3027) or using pyridinium chlorochromate (PCC) (Tetrahedron 1990, 46: 44 17-4420; Tetrahedron Lett,, 1975, 16, 2647-2650).

Scheme I H::r g Im:fflion 2! Ia H I H lb c Id Step 1.4 deprotection

LV LV LV LV

A OBU, aIkyI1OH A 1120 A H BzNCS A NyNH2 or K2C03, aIkyI1OH NNHBZ DCM/pyr NJ DCM NH2 H Step'1.7 H Stepl.6 H Stepl.5 H

OH OH

Iii Ig If le Step 1,2: This is a step of obtaining a compound (ic) by conversion of the ketone group of the compound (Ib) to an imine or imine derivatives (sulfinimines, sulfonamides, N-Boc-imines, oxime ethers) when P1 and P2 are defined as above. Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions.

For example compound (ib) can be converted to N-terv-butanesulfinyl ketimine (Ic) when P2 is SotBu with tBuSONH2 (racemic or enantiopure) in presence of a Lewis acid such as Ti(OEt)4, Ti(O'Pr)4, (J. Am. Chem. Soc. 1999, 121, 268-269; Chem, Rev, 2010, 110, 3600-3740) in a suitable organic solvent, (for example THF, Et20, CH2CI2) at an appropriate reaction temperature, between room temperature to solvent reflux, For example compound (Ib) can be converted to N-Boc imine (Ic) when P2 is Boc with N-Boc imino-(triphenylphosphorane) in a suitable organic solvent, (for example THF, toluene) at an appropriate reaction temperature, between room temperature and solvent reflux.

For example compound (tb) can be converted to O-benzyl ketoxime (Ic) when P2 is OBn with HC1.H2NOCH2Ph in presence of a base such as pyridine or sodium acetate, in a suitable organic solvent, (for example MeOH) at an appropriate reaction temperature, between room temperature and solvent reflux.

Step 1.3: This step is a step of obtaining a compound (Id) by addition of an organometalic reagent (LV-A-M) to the imine group of the compound (ic) when Pi and P2 are defined as above, Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions. -19-

For example, compound (Ic) can be treated with a suitable organometallic reagent (LV-A-M) such as organolithium and Grignard reagents in presence or absence of a Lewis acid such as BF3OEt2, AIMe3, (J. Am, Chem, Soc. 1999, 121, 268-269; Chem, Rev, 998, 98, 1407-1438) in a suitable organic solvent, (for example THF, Et,O, toluene, CH2CI2) at an appropriate reaction temperature, between -78°C to room temperature, preferably less than 0 °C.

Step 1.4: This is a step of obtaining a compound (Ic) by deprotection of the amino and alcohol groups of the compound (Id) when Pi and P2 are defined as above. Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions and can refer to a document such as T. W. Green and P.G.M Wuts, "Protective Groups in Organic Chemistry, Third Edition", John Wiley& Sons.

For example compound (td) can be treated with concentrated HC1 when P2 is SOtBu or Boc at an appropriate reaction temperature, between room temperature and solvent reflux, For example compound (td) can be transformed into compound (te) when P2 is OBn by hydrogenolysis using a Pd-catalyst, in particular Pd on carbon under an hydrogen atmosphere in a suitable organic solvent, (for example MeOH or EtOI-l) at an appropriate reaction temperature, between room temperature to reflux.

Step 1,5: This is a step of obtaining a compound (if) by conversion of the amino group of the compound (te) to a thiourea derivative, Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions.

For example compound (Ic) can be transformed into the thiourea compound (If) when reacting th benzoylisothiocyanate in a suitable organic solvent, (for example dichloromethane or toluene) at an appropriate reaction temperature, between -78°C to room temperature. This reaction can be performed under the same conditions as those described in J. Med. Chem. 1990, 33, 2393-2407, for example.

Step 1.6: This is a step of obtaining a compound (ig)by cyclisation of the compound (If).

For example compound (If) can be cyclised to isothiourea (Ig) th triflic anyhdride in the presence of a base such as pyridine, in a suitable organic solvent, (for example CH2CI2) at an appropriate reaction temperature, between -78°C to room temperature, typically -20°C to 0°C.

Step 1,7: This is a step of obtaining compounds (lh) from compound (ig) by utilizing a deprotection reaction of the benzamide group when Ring A is defined as above, -20 -Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions, For example compound (Ig) can be transformed to (lh) by using a base, such as DBU in a suitable solvent, such as an alkyl alcohol, such as methanol (alkyl i = Me). This reaction can be performed under the same conditions as those described in Synth. Commun. 2002, 32 (2), 265-272, for example. The reaction can be performed using I equivalent to a large excess of base. The reaction temperature is usually room temperature to solvent retlux temperature.

Alternatively the same transformation may be effected by treating (Ig) with a base such a K2C03 in an alcoholic solvent, such as methanol (alkyl i = Me), ethanol (alkyl i = Pt) or I -propanol (alkyl i = Pr). The reaction can be performed using I equivalent to a large excess of base. The reaction temperature may for example be in the range 50°C to 100°C.

General Preparation Method 2 General Preparation Method 2 (Scheme 2) is a method for preparing compounds of the present invention (formula (I)) wherein L is a bond from Intermediate (ig). In the formula of Scheme 2, Ring A and Ring B are as defined in formula (I), LV may be a halogen atom (Cl, Br or I) or a trifluoromethanesulfonyloxy group (OTf), arid Xis as defined below in Step 2,1, Intermediate (ig) may be prepared as described in General Preparation Method 1.

Scheme 2 A LV x A B DBU, aIkyI1OH NNHBz base, "Pd' NNHBz øí KCO3, aIkyI1OH o S Step2.1 a S Step22 0

H H H

Ig 2a 2b Step 2.1: This is a step of obtaining compounds (2a) from compound (lg)by utilizing a transition metal-mediated coupling reaction with the coupling partner (B-X) when Ring A, Ring B arid LV are defined as above, Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions, For example compound (Ig) can be transformed to (2a) by using a transition metal catalyst, for example a palladium catalyst such as dich1orobis(triphenyphosphine)pa1ladium or palladium dichloride with triphenylphosphine in a 1:2 ratio. Alternatively, a wide variety of related palladium catalysts may also be suitable for this transformation, for example tetrakis(triphenylphosphine)palladium and the like, Those skilled in the art will understand that many such catalysts are known and that many of such catalysts are capable of effecting this transformation and that the substrate (ig) or the coupling partner may dictate which catalyst can or cannot be used.

The aforementioned transition metal mediated coupling reactions require a suitably functionalized reaction partner (B-X), examples include B-X being boronic acids/esters (e.g.

--

Suzuki-Miyaura reaction; Pure AppI. Chem. 991, 63, 419-422; Organometallic Chem. 1999, 576, 147-68; Chem. Rev., 1979, 95 (7): 2457-2483; J. Org. Chem. 2007, 72, 7207-7213; J. Am. Chem. Soc. 2000, 122, 4020-4028 and J. Org. Chem. 2007, 72, 5960-5967), B-X being stannanes(eg Stille reaction; J. Am. Chem. Soc. 1978, 00, 3636; Org. Synth., 1998, CoIl.

Vol. 9, 553 & 1993, Vol. 71; . Angew. Chem. Tnt. Ed. EngI. 1986, 25, 508-524; Org. React.

1998, 50, 1-652 and J. Am. Chem. Soc. 1990, 112, 3093-3100), B-X being zinc reagents (e.g. Negishi reaction; J. Chem. Soc., Chem. Commun.,1977, 683; J. Org. Chem., 2008, 73, 7380- 7382; J. Am. Chem, Soc., 2003, 125, 12527-12530) and even B-X being Grignard reagents (catalysed by palladium or nickel, eg Kumada coupling; J. Am, Chem. Soc. 1972, 94 (12), 4374-4376), Those skilled in the art will appreciate the intricacies of these reagents and which ones it is most appropriate to use. Moreover, those skilled in the art will also appreciate that in some instances the position of the coupling groups may be reversed with the leaving group LV located on Ring B and X (e.g. a boronic acid) located on Ring A. In addition to the aforementioned catalyst and reaction partner, these transition-metal mediated reactions require a solvent and often a base is present. Suitable solvents include mixtures of water and DIvIE or toluene and ethanol or toluene and water or toluene and DIVIIE or the like. The reaction may be conducted at various temperatures, for example room temperature to 120°C, or for example 100°C.

Step 2,2: This is a step of obtaining compounds (2b) from compound (2a) by utilizing a deprotection reaction of the benzamide group.

Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions and can refer to Step 1.7 in General Preparation Method 1.

General Preparation Method 3 General Preparation Method 3 (Scheme 3) is a method for preparing compounds of the present invention (formula (1)) wherein L is -NHC(O)-from Intermediate (ig). In the formula of Scheme 3, Ring A and Ring B are as defined in formula (I) and LV may be a halogen atom (Cl, Br or I). Intermediate (ig) may be prepared as described in General Preparation Method 1.

Scheme 3 Oy® -.NH2 (Th rNH A A -co2H A DBU, aIkyI1OH A NNHBz NNHBz (Zb) NNHBZ or K2C03, aIkyI1OH NNH2 o Stepal S Step3 o Step3.3 0

H H H H

Ig 3a Sc ad -22 -Step 3,1: This is a step of obtaining compound (3a) from Intermediate compound (Ig) by conversion of the halogen atom (LV) into the corresponding amino group. Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions.

For example compound (3a) can be prepared by a copper coupling reaction between the appropriate aryl (or heteroaryl) halide (2a) and ammonia or an ammonia surrogate, (such as 2,2,2-trifluoroacetamide or acetamidine hydrochloride) with a source of copper catalyst (for example Cu20, Gui), in the presence or absence of a base such as K2G03 or Gs2GO3 and a ligand (such as trans-N,N'-dimethyl l,2-cyclohexanediamine or L-proline), Suitable solvents include mixtures of water and NMP or dioxane or DMF or the like, The reaction may be conducted at various temperatures, for example room temperature to 120°G, or for example 100°G. In the case where the trifluoroacetamide intermediate is formed, a one-pot hydrolysis may be performed by the addition of water and MeOH to give the primary aryl (or heteroaryl) amine (3a), This reaction can be performed under the same conditions as those described in Tet, Lett, 2008, 49, 70-75, Ghem, Gomm, 2009, 3035-3037, J, Org. Ghem, 2008, 73, 6864-6866 for example, Alternatively, an azido-dehalogenation is performed on the appropriate arylhalide (2a) in the presence of an azide source such as sodium azide, Reduction of the resulting intermediate azide to the amine (3a) can be effected by using a number of reducing agents, such as LiA1H4, NaBH4 or via hydrogenation conditions, Step 3,2: This is a step of obtaining compounds (3c) from compound (3a) by a coupling reaction of the amine group with the appropriate carboxylic acid, The condensation reaction of the compound (3a) with the compound (314 using a condensing agent can be perfonned under the same conditions as those usually used and described in the following documents. Examples of the known method include those in Rosowsky, A,; Forsch, R. A.; Moran, R. G.; Freisheim, J. H.; J. Med. Chem., 34 (1), 227-234 (1991), Brzostwska, lvi.; Brossi, A,; Flippen-Anderson, J. L.; Heterocycles, 32 (10), 1968- 1972 (1991), and Romero, D. L.; Morge, R. A.; Biles, C.; Berrios-Pena, N.; May, P. D.; Palmer, J, R,; Johnson, P. D.; Smith, H. W.; Busso, lvi.; Tan, C-K,; Voorman. R, L.; Reusser, F,; Althaus, I, W,; Downey, K. lvi.; So, A. G,; Resnick, L,; Tarpley, W, G,, Aristoff, P, A.; J, Med. Chem,, 37(7), 998-1014 (1994), The solvent in this reaction is not particularly limited insofar as it does not inhibit the reaction. Examples of the solvent include tetrahydrofuran, 1,4-dioxane, ethyl acetate, methyl acetate, dichloromethane, chloroform, N,N-dimethylformamide, acetonitrile, toluene and xylene. Examples of the condensing agent include CDI (N,N'-carbonyldiimidazole), Bop (IH-1,2,3 -benzotriazol-1 -yloxy(tri(dimethylamino))phosphonium hexafluorophosphate), DCC (N,N-dicyclohexylcarbodiimide), diethylphosphoryl cyanide, PyBOP (benzotriazol-t -fin -L.3 - yloxytris(pyrrolidino)phosphonium hexafluorophosphate) and EDC*HCI (I -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride). One equivalent to a large excess of the compound (3b) is used with respect to the compound (3a). One equivalent to a large excess of an organic base such as triethylamine may be added where necessary. The reaction time is not particularly limited and is usually 0.5 to 48 hours, and preferably 0.5 to 24 hours. The reaction temperature varies according to the raw material used, the solvent and the like and is not particularly limited. Ice-cold temperature to solvent reflux temperature is preferable.

Alternatively, the compound (3c) can be obtained by converting the desired carboxylic acid (3b) to the coresponding acid chloride and then reacting the acid chloride with the compound (3a). The acid chloride can be synthesized by a means known to a person skilled in the art. For example the desired carboxylic acid may converted to the corresponding acid chloride by reaction with thionyl chloride in the presence or absence of a solvent, for example dichioromethane, N,N' -dimethylimidazoline-2-one, NIMP or DV1IF. One to two equivalents or a large excess of thionyl chloride may be used with respect to the desired carboxylic acid.

The reaction temperature is -30°C to reflux, and preferably -10°C to room temperature. An additive, such as carbonyl diimida.zole may also be used. The acid chloride may also be formed by treating the acid with oxalyl chloride in a solvent such as dichloromethane in the presence of DMF. The reaction temperature is -30°C to room temperature, and preferably - 1 0°C to room temperature Alternatively the compound (3c) can be obtained by converting the desired carboxylic acid (3b) to a mixed acid anhydride and then reacting the mixed acid anhydride with the compound (3a). The mixed acid anhydride can be synthesized by a means known to a person skilled in the art, The synthesis is performed by reacting the desired carboxylic acid with a chloroformate such as ethyl chloroformate in the presence of a base such as triethylamine, for example. One to two equivalents of the chloroformate and the base are used with respect to the desired carboxylic acid. The reaction temperature is -30°C to room temperature, and preferably -20°C to room temperature. The step of condensing the mixed acid anhydride with the compound (3a) is performed by reacting the mixed acid anhydride with the compound (3a) in a solvent such as dichloromethane, tetrahydrofuran or N,N-dimethylformamide, for example, One equivalent to a large excess of the desired acid anhydride is used with respect to the compound (3a), The reaction time is not particularly limited and is usually 0,5 to 48 hours, and preferably 0.5 to 12 hours, The reaction temperature is -20°C to 50°C, and preferably -20°C to room temperature.

Alternatively the compound (3c) can be obtained by converting the desired carboxylic acid (3b) to an active ester and then reacting the active ester with the compound (3a). The step of obtaining the active ester is performed by reacting the desired carboxylic acid with an active ester synthesis reagent in a solvent such as t,4-dioxane, tetrahydrofuran or N,N-dimethylformamide in the presence of a condensing agent such as DCC, for example, -24 -Examples of the active ester synthesis reagent include N-hydroxysuccinimide One to 1.5 equivalents of the active ester synthesis reagent and the condensing agent are used with respect to the compound (3a). The reaction time is not particularly limited and is usually 0,5 to 48 hours, and preferably 0.5 to 24 hours. The reaction temperature is -20°C to 50°C, and preferably -20°C to room temperature. The step of condensing the active ester with the compound (3a) is performed by reacting the active ester with the compound (3a) in a solvent such as dichloromethane, tetrahydrofuran or N.N-dimethylformamide, for example. One equivalent to a large excess of the active ester is used with respect to the compound (3a).

The reaction time is not particularly limited and is usually 0.5 to 48 hours, and preferably 0.5 to 24 hours. The reaction temperature is -20°C to 50°C, and preferably -20°C to room temperature.

Step 3.3: This is a step of obtaining compounds (3d) from compound (3c) by utilizing a deprotection reaction of the benzamide group, Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions and can refer to Step 1,7 in General Preparation Method 1, First Alternate Preparation of Intermediate Compound (Ic) Intermediate compound (Ic) described in General Preparation Method I may alternatively be prepared, as outlined in Scheme 4, from intermediate (I b), In the formula of Scheme 4, Ring A is as defined in formula (I), and LV and P1 are as defined for Scheme t.

Intermediate (ib) may be prepared as described in General Preparation Method 1, Scheme 4 A LV A LV reduction! A LV M OH NaN3, TFA N3 deprotection NH2 Step4.1 0 Step4.2 0 Step4.3 0 H OF1 H H H OH lb 4a 4b 10 Step 4.1: This step is a step of obtaining a compound (4a) by addition of an organometalic reagent (LV-A-M) to the ketone group of the compound (ib) when P1 is defined as above.

Those skilled in the art will appreciate that this transformation may be accomplished by a range of conditions.

For example compound (Ib) may be treated with a suitable organometallic reagent (LV-A-M) such as organolithium and Grignard reagents in a suitable organic solvent, (for example THE, Et20, toluene) at an appropriate reaction temperature, between -78°C to room temperature, preferaNy less than 0°C.

Step 4,2: -25 -This step is a step of obtaining a compound (4b) by converting the hydroxyl group of the compound (4a) to an azide group when h is defined as above. Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions.

For example compound (4a) could be treated with sodium azide in presence of an acid (such as trifluoroacetic acid or sulfuric acid) in a suitable organic solvent, (for example chloroform or dichloromethane) at an appropriate reaction temperature, between 0°C to room temperature.

Step 4,3: This step is a step of obtaining a compound (Ic) by concomitant reduction of the azide group and deprotection of the alcohol group of the compound (4b) when Pi is defined as above. Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions.

For example compound (4b) may be transformed into compound (le) by hydrogenolysis using a Pd-catalyst, in particular Pd on carbon or Pearlman's catalyst under an hydrogen atmosphere in a suitable organic solvent, (for example MeOH or EtOH) at an appropriate reaction temperature, between room temperature to reflux.

Second Alternate Preparations of Intermediate Compound (1c Intermediate compound (ic) described in General Preparation Method I may alternatively be prepared, as outlined in Scheme 5, from intermediate (4a). In the formula of Scheme 5, Ring A is as defined in formula (I) and LV and P1 are as defined for Scheme t.

Intermediate (4a) may be prepared as described in General Preparation Method 4. Those skilled in the art will appreciate that the steps and conditions of Scheme 5 are generally analogous to those shown in Scheme 4, the principal difference being the route designed between compound 4a and Ic, Scheme 5 0LV 6LV cxLV (JOH MeCN, H2SO4 NaOH deprotection (4NH2 0 Step 5.1 ot 0 Step 5.2 0 Step 5.3 0 H oi'i H o1 H H OH 4a 5a Sb 10 Step 5.1: This step is a step of obtaining a compound (5a) by use of a Ritter reaction to convert the alcohol group of compound (4a) into the acetamide derivative 5a when P1 is defined as above, Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions.

For example compound (4a) may be treated with acetonitrile in presence of sulfuric acid at an appropriate reaction temperature, between 0°C to room temperature.

-26 -Those skilled in the art will also appreciate that this reaction may be conducted with functionalised acetonitrile derivatives. Examples of functionalised acetonitrile derivatives include, but are not limited to chloroacetonitrile, Suitable acids include sulfuric acid or mixtures of sulfuric and acetic acid. Suitable reaction temperatures include 0°C to room temperature.

Step 5.2: This step is a step of obtaining a compound (Sb) by conversion of the acetamide group of the compound (Sa) to the amino group. Those skilled in the art will appreciate that this transfonuation can be accomplished by a range of conditions.

For example compound (Sn) may be treated with a base (such as NaOH, KOI-T or K2C03) in a suitable solvent, (for example MeOH, ethylene glycol) at an appropriate reaction temperature, between 0°C to reflux.

Those skilled in the art will also appreciate that this reaction may be conducted with milder conditions when a functionalised acetonitrile derivative, such chloroacetonitrile, is used in the previous step. If chloroacetonitrile was used in the previous step then resultant chloroamide may be deprotected by treating with thiourea in in the presence of a mild acid, such as acetic acid, in a suitable solvent, such as an alcoholic solvent, for example ethanol, at an appropriate reaction temperature, for example solvent reflux.

Step 5,3: This step is a step of obtaining a compound (te) by deprotection of the alcohol group of the compound (Sb) when Pi is defined as above, Those skilled in the art will appreciate that this transformation can be accomplished by a range of conditions.

For example compound (Sb) may be transformed into compound (Ic) by hydrogenolysis using a Pd-catalyst, in particular Pd on carbon or Pearlman's catalyst under an hydrogen atmosphere in a suitable organic solvent, (for example MeOH or EtOH) at an appropriate reaction temperature, between room temperature to reflux.

The present invention will be described more specifically below with reference to Examples, However, the present invention is not limited thereto. The abbreviations used in the Examples are conventional abbreviations known to a person skilled in the art, Some abbreviations are shown below: Abbreviations BOC & Boc: tert-butoxycarbonyl; br: broad; Bn: benzyl; Bu: butyl; BuLi: n-butyl lithium; Bz: benzoyl; d: doublet; DBU: 1,8-Diazabicyclo[5.4.0]undec-7-ene, DCM: dichloromethane; dd: doublet of doublets; DIBAL: disobutylaluminium hydride; DMF (N,N-dimethylformamide); DMAP (4-N,N-dimethylaminopyridine; DMSO dimethylsulfoxide; EDC & EDAC: (N-3 (-dimethylaminopropyl)N' ethylcarbodiimide hydrochloride); Et: ethyl; Et20: diethyl ether; EtOAc: ethyl acetate; EtOH: ethanol; h, hr, hrs: hours; HC1: hydrochloric -27 -acid; HPLC: high performance liquid chromatography; LCMS, LC/MS & LC-MS: liquid chromatography / mass spectrometry; m: multiplet; Me: methyl; MeCN: acetonitrile; MeOH: methanol; MS: mass spectrometry; MDAP: mass directed auto purification; mm & mins: minutes; NaOH: sodium hydroxide; NMP: N-methylpyrolidinone or I -methyl-2-pyrrolidinone; NMR: nuclear magnetic resonance; Ph: phenyl; PhCH3 & PhMe: toluene; PMB: p-methoxybenzyl; Pr: propyl; Rt: retention time; RT, rt & r.t.: room temperature; 5: singlet; SCX: strong cation exchange:-Isolute Flash SCX-2, Biotage; t: triplet; TBAF: tetrabutylammonium fluoride; TBAT: tetrabutylammonium iodide; TEA: triethylamine; THF: tetrahydrofuran; TFA: Trifluoroacetic acid; tic: thin layer chromatography; UV (ultraviolet).

H NMR spectra were recorded on a Bruker AM series spectrometer operating at a (reported) frequency of 400 IVIHz. Chemical shifts in proton nuclear magnetic resonance spectra are recorded in 8 units (ppm) relative to tetramethylsilane and coupling constants (J) are recorded in Hertz (Hz).

Chemical names were generated from chemical structures using ChemBioDraw Ultra 11.0 and 12,0 or ELN, both from Cambridgesoft.

Purification: Method D Gilson large scale reverse phase preparatory HPLC MANUAL2.GCT method: reverse phase 1-IPLC (Phenomenex Luna C18, 250 x 50mm, lOjim, 8OmL per mm, gradient 2O?b to 95% (over 25mm) then)5% (10mm) MeCN in H20 [0. t% acetic acidj).

Intermediate 1: (Z-4-(benzyloxybut-2-en-1-01 HOO Bn To a suspension of NaH (4.39 g of a 60% dispersion in mineral oil, 109. 9 mmol, washed with dry hexanes) in 500 mL of 4:1 mixture of dry THF/DMSO was added a solution of(Z)-but-2-ene-1,4-diol (8.21 mL, 99.9 mmol) in THF (250 mL) at rt. After being stirred for mm, a solution of benzyl bromide (t3.1 mL, t09.9 mmol) in THF (250 mL)was added dropwise, followed immediately by TBAI (t8.45 g, 49.9 mmol) in one portion. The mixture was heated at 60°C overnight. The reaction mixture was diluted with H20 (1 L) and was extracted with Et20 (3 x 200 mL). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The residue was then purified by silica gel column chromatography (gradient from 30% to 50% EtOAc in Hexanes) to give the title product as a yellow oil (12.31 g, 69%).

111 NIMR (400 MHz, CDC13) 6 ppm 4.1 t (dd, 1=5.93, 0.79 Hz, 2 H), 4.16-4.25 (m, 2 H), 4.54 (s, 2 H), 5.69-5.92 (m, 2 H), 7.29-7.44 (m, S H).

-28 -Intermediate 2: ((2R,3 S)-3-ftbenzyloxymethyDoxiran-2-yl)methano1 B nO To a suspension of activated Molecular sieves 4A (dried overnight at 130°C under high vacuum, ig) in DCM (40 mL, 621.67 mmol) under N2 at -23°C was added Ti(O'Pr4) (0.740 ml, 2.52 mmol), followed by (-)-diethyltartrate (0.48 mL, 2.80 mmol). After 30 mm at -23°C, tBuOOH (3.06 mE, 16.83 mmol, 5.5 NI solution in nonane) was added. After 30 mm at -23°C, the allylic alcohol (Z)-4-(benzyloxy)but-2-en-l -01 ( g, 5.61 mmol) in CH2CI2 (13 mL) was added dropwise. The reaction mixture was then stirred at -23°C for I Sb, then warmed to -12°C for lb. Sodium hydroxide (30% in sat, NaCI, SmL) was added and the reaction mixture stirred and allowed to warm tort for 1 h. The Molecular sieves were filtered on a celite pad. The aqueous layer extracted with CH2CI2, and the combined organic phases were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The residue was then purified by silica gel column chromatography (50% EtOAc in Hexanes) to give the title product as a colorless oil (996 mg, 91%, >95% cc determined via NMR analysis of its corresponding Mosher's ester).

1H NMR (400 MHz, CDC13) S ppm 1.99 (br. s., 1 H), 3.20-3.27 (m, 1 H), 3.31 (dt, J=5,99, 4.77 Hz, I H), 3.67 (dd,J=I 1.00, 5.01 Hz, I H), 3.71-3.80(m, 3 H), 4.55 (d,J= 1.74 Hz, I I-I), 4.63 (d, J=l 1.74 Hz, I H), 7.29-7.41 (m, S H).

Intermediate 3: (2S,3 S'-2-Ubenzyloxy'methyl)tetrahydrofuran-3 ol1 0H OBn To a suspension of NaH (10.88 g of a 60% dispersion in mineral oil, 0.27 mol, washed with dw hexanes) in DMSO (250 mL) in a flame dried flask was added portionwise over 10 mm trimethylsulfoxonium iodide (60 g, 0.27 mol, previously dried overnight at 30°C under high vacuum). The reaction mixture was then stirred for 30 mm until bubbling stopped, then ((2R,3S)-3-((benzyloxy)methyl)oxiran-2-yl)methanol (5.28 g, 27.2 mmol) in DMSO (2 mL) was added dropwise and the reaction stirred at 80°C fo 72h. It was then cooled and diluted with H,O (500 mL) and a saturated solution of NIHCl (10 mL). The reaction was extracted with Et20 (x3), the combined organic extracts were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The residue was then purified by silica gel column chromatography (45 to 65% EtOAc in Hexanes) to give the title product as a colorless oil (4.03g, 71%).

1HNMR(400 MHz, CDC13) S ppm 1.93-2.01 (m, 1 H), 2.16 (dtd, 1=13.37, 8.23, 8.23, 5.62 Hz, 1 H), 3.79-3.87 (m, 3 H), 3.88-3.94 (m, I H), 4.02-4.13 (m, 1 H), 4.48 (ddd, 1=5.72, 3.88, 2.26 Hz, 1 H), 4.60 (d, 1=0.98 Hz, 2 H), 7.28-7.39 (m, 5 H).

-29 - 1(2S,3S)-2-((benzyloxy)methyl)tetrahydroftiran-3-ol may be prepared as described in J, Am. Chem, Soc., 2004,126, 13600-13601, Intermediate 4: N-((R)-2-'benzyl oxy)methyl)dihydrofuran-3 (2H)-ylidene)-2-methylpropane- 2-sulfinamide cjNSOtBu OBn To a solution of oxalyl chloride (8.13 mL, 92.9 mmol) in CH2CI2 (160 mL) at -78°C was slowly added DM50 (15.38 mL, 216.7 mmol), the reaction mixture was allowed to stir for 15 mm. Then, a solution of(25,35)-2-((benzyloxy)methyl)tetrahydrofuran-3-ol (8.06 g, 38.7 mmol) in CH2C12 (32 mL) was slowly added. The mixture was stirred for 2 h at -78°C before adding Et3N (40 mL, 290 mmol). The reaction mixture was allowed to warm to r.t, for mm, and then diluted with CH2C12 (300 mL) and a saturated solution of NH4C1 (300 mL).

The aqueous layer was extracted with CH2C12 (200 mL) and the combined organic extracts were washed with brine (300 mL), dried over Na2SO4, then concentrated under reduced pressure. The resulting residue was triturated with diethyl ether and the filtrate concentrated under reduced pressure to give (S)-2-((benzyl oxy)methyl)dihydrofuran-3 (2W-one as a yellow oil (9.09 g), which was used directly in the next step without further purification.

To a solution of the previous ketone (9.09 g) and racemic 2-methylpropane-2-sulfinamide (5.88g, 48.5 mmol) in dry THE (58 mL) under N2 was added Ti(OEt)4 (18.45 mL, 88.2 mmol). The resulting yellow suspension was stirred at 70°C for 16h. The solution was cooled to r,t,, and then poured into brine (60 mL), filtered through celite and washed thoroughly with EtOAc, The aqueous phase was extracted with EtOAc, and the combined organic extracts were washed with brine, dried over Na2 SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (15 to 45% EtOAc in Hexanes) to give the title compound as a mixture of diastereoisomeric imine as yellow oil (9.6g, 80% over 2 steps).

1HNMR (400 MHz, CDC13) 6 ppm 1.23-1.26 (m, 9 H), 2.72-2.87 (m, 1 H), 3.27-3.39 (m, I H), 3.71-3.80 (m, I H), 3.80-3.87(m, 1 H), 3.96-4.10(m, 1 H), 4.27-4.41 (m, 2 H), 4.51-4.64 (m, 2 H), 7.27-7.39 (m, 5 H).

-30 - Intermediate 5: N-((2R,3R')-2-((benzyloxy')methyfl-3-(3-bromoyhenyfltetrahydrofuran-3-yfl- 2-methylpropane-2-sulfinamide Br ,NHSOtBu OBn To a solution of 1-bromo-3-iodobenzene (8.88 mL, 69.6 mmol) in dry toluene (28 mL) at 0°C was added 1BuLi (27.9 mL, 2.5M in hexanes, 69.6 mmol). The reaction was left to stir and warm up to reach 5°C over 2.Sh. To the previous yellow suspension at 0°C was added dropwise over 30 mm a solution of N-((R)-2-((benzyloxy)methyl)dihydrofuran-3(21-l)-ylidene) -2-methylpropane-2-sulfinamide (6.53g. 21.1 mmol) and AIMe3 (11.6 mL, 23.2 mmol) in dry toluene (13 mE) that was stirred under N2 at -78°C for 10 mm. The resulting solution was stirred and allowed to warm to r.t. over 20h, and was then quenched at 0°C with Na2 SO4. tOH2O and left to stir for 1.5h at r.t. The reaction mixture was diluted with EtOAc, Na2SO4 was then added, The reaction mixture was then filtered, rinsed thoroughly with EtOAc and concentrated under reduced pressure. The residue was then purified by silica gel column chromatography (S to 50% EtOAc in Hexanes) to give (S)-2- ((benzyloxy)methyl)dihydrofuran-3(2H)-one as a yellow oil (1.65 g), and the desired N- ((2R,3R)-2-((benzyloxy)methyl)-3-(3 -bromophenyl)tetrahydrofuran-3 -yl)-2-methylpropane- 2-sulfinamide as a yellow oil (3.63 g, 37%).

LCMS: Rt 1,69 mm, m/z = 466/468 M+Hf.

1HNIMR (400 MHz, CDC13) 5 ppm 1,18 (s, 9 H), 2.45 (dt, 1=13,02, 7,55 liz, 1 H), 2,77 (ddd, J12.90, 7,52, 5,26 liz, I H), 3,79 (dd, J=i0.88, 3,91 Hz, 1 H), 3,91-3,98 (m, 2 H),4.07-4.]S(m, I H),417(t,J=3.61 Hz, I H), 4.52(d,J11.86Hz, I H), 4.59(d,J=ll.62 Hz, I H), 5,07 (s, I H), 7,20-7,25 (m, I H), 7.29-7,38 (m, S H), 7.39-7.43 (m, I H), 7.50-7.54 (m, I H), 7.70 (t, J= .90 Hz, I H).

Intermediate 6: N-U(2R.3R-3 -(3 -bromophenyfl-2-(hvdroxymethyl)tetrahydrofuran-3 -yl)carbamothioyl)benzamide N-((2R,3 R)-2-((benzyl oxy)methyl)-3 -(3 -bromophenyl)tetrahydrofuran-3 -yI)-2-methylpropane-2-sulfinamide (3.928, 8.4 mmol) was treated with concentrated HCI (13.5 mL). The reaction mixture was heated at reflux for Ih. After cooling down, ice was added to

-

the reaction mixture, which was then basified with a 50% aq. NaOH solution. It was then extracted with EtOAc (x3), and the combined organic extracts were washed with brine, dried over Na2 SO4, and concentrated under reduced pressure to give the crude alcohol as a brown oil (3.08g), which was directly used in the next step without further purification.

LCMS: Rt 0.84 mm, m/z = 272/274 M+H]T Benzoyl isothiocyanate (1.25 mL, 9.3 mmol) was added dropwise to a solution of cmde ((2R,3R)-3 -amino-3 -(3-bromophenyl)tetrahydrofuran-2-yl)methanol (3.08g) in CH2C12 (60 mL). The reaction was stirred at r.t. for 40 mm, and was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (30 to 55% EtOAc in Hexanes) to afford the title compound as a colorless foam (2.93 g, 80% over 2 steps).

LCMS: Rt 1.50 mm, m/z = 435/437 M+H]T 1HNMR(600 MHz, CDCI3) 8 ppm 2.24 (br. s., 1 H), 2.74 (ddd, ,J13.3, 8.0, 6.6 Hz, 1 H), 3.28 (ddd, ,k13.2, 7.5, 6.3 Hz, 1 H), 3.87-3.93 (m, 1 H,) 3.94-3.99 (m, I H), 4.09 (td, 1=8.4, 6.0 Hz, 1 H), 4.1 t-4.16 (m, 1 H), 4.17-4.25 (m, t H), 7.24-7.27 (m, 1 H), 7.37 (dd, 1=7.9, 0.9 Hz, 1 H), 7.4t-7.45 (m, 1 H), 7.51-7.58 (m, 3 H), 7.61-7.67 (m, 1 H), 7.84-7.91 (m, 2 H), 8.91 (s, I H), 11.75 (s, 1 H).

Intermediate 7: N-((4aR,7aR)-7a-(3-bromophenyl)-4a,6,7,7a-tetrahydro-41-1-furo[3,2-d][l, 3lthiazin-2-yl)benzamide Br

H

NyNyPh

H

To a solution of N-(((2R,3R)-3 -ç -bromophenyl)-2-(hydroxymethyl)tetrahydrofuran- 3-yl)carbamothioyl)benzamide (2.0 g, 4.6 mmol) in CH2C12 (23 mL) was added pyridine (2.29 mL, 28.3 mmol). The solution was cooled to -20°C and trifluoromethanesulfonic anhydride (1,16 mL, 6.9 mmol) was added dropwise over 15 mm, The reaction mixture was left to warn to 0°C over I.Sh, and was quenched with a saturated soultion of NaI-1C03, The aqueous layer was extracted with CH2CI2 (x3). The combined organics were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (35 to 50% EtOAc in Hexanes) to afford the title product as a colorless foam (1.80 g, 94%).

LCMS: RI 1.Gomin,m/z=4l7/419[M+Hf'.

H NMR (400 Ml-lz, CDCI3) 8 ppm 2.33-2.44 (m, I H), 2.85 (dt, 1=13.0, 9.3 Hz, I I-I), 3.09 (d, 1=3.4 Hz, 2 H), 4.16-4.23 (m, I H), 4.27 (td, J8.7, 2.4 Hz, I H), 4.32 (t, 1=3.2 Hz, I H), 7.28-7.34 (m, 1 H), 7.37-7.55 (m, 5 H), 7.58 (t, 1=1.8 Hz, 1 H), 8.16 (d, 1=6.0 Hz, 2 H), 12.46 (br s., 1 H).

-32 - Intermediate 8: N-((4aR,7a10-7a-(3-aminophenyl)-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][I. 3lthiazin-2-vl)benzamide -.. NH çNNyPh

H

In a round bottomed flask, N-((4aR,7aR)-7a-(3 -bromophenyl)-4a,6, 7,7a-tetrahydro- 4H-furo[3,2-d][1,3jthiazin-2-yl)benzamide (976 mg, 234 mmol), trifluoroacetamide (952 mg, 842 mmol), Cul (151 mg, 0.79 mmol), Na! (596 mg, 3.9Smmol), K2C03 (614 mg, 4.44 mmol) were put under vacuum and filled back with nitrogen (x3), I,4-Dioxane (20 mL) (previously degased with bubbling N2) was then added followed by trans-I,2-bis(methylamino)cyclohexane (0.65 mL, 4.12 mmol). The mixture was then heated at 115°C for 1 6h. MeOll (1.5 mL) and water (1.5 mL) were then added to the reaction mixture which was then heated at 115°C for 2h. The reaction mixture was partially concentrated before water and EtOAc were added. The organic phase was separated and the aqueous layer extracted with EtOAc (x2), The combined organic phases were washed with brine, dried over Na2SO4 and solvent removed in vacuo, The residue was purified by silica gel column chromatography (40 to 80% EtOAc in Hexanes) to give the title compound as a colorless solid, (291 fig, 35%).

LCMS: Rt 1.12 mi m/z = 354 [M+H]t Example 1: (4aR,7aR)-7a-(3-(pyrimidin-5-yI)phenyl)-4a,6,7,7a-tetrahydro-4H-furo [3.2-dl [t..3lthiazin-2-amine

U NH2

To a solution of N-((4aR, 7aR)-7a-(3 -bromophenyl)-4a,6, 7,7a-tetrahydro-4H-furo[3,2-d][1,3]thiazin-2-yl)benzamide (Intermediate 7, 100 mg, 0.24 mmol) in DME (1.4 mL), EtOH (0.48 mL) and water (0.63 mL) was added pyrimidin-5-ylboronic acid (131 mg, .05 mmol), bis(triphenylphosphine)palladium(II) chloride (33.6 mg, 0.05 mmol) and Cs2CO3 (468 mg, 1.44 mmol). The reaction mixture was heated at 100°C for lh. After cooling down to r.t., it was partitioned between EtOAc and a saturated solution ofNaHCO3. The layers were separated, and the aqueous layer extacted with EtOAc (x 2). The combined organic extracts were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0 to 5% MeOH in CH2C12) to fin -.3.3 - give N-((4aR,7aR)-7a-(3-(pyrimidin-5-yl)phenyl)-4a,6,7,7a-tetrahydro-4H-furo[3, 2-d][l,3]thiazin-2-yl)benzamide (108 mg) as a brown oil.

It was then dissolved in MeOI-l (2 mL) and DBU (0.08 mL), the solution was stirred at refiux for 2.Sh. The solvents were then removed in vacun. The residue was partitioned between H20 and EtOAc. The aqueous phase was extracted with EtOAc, and the combined organic extracts were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The residue was purified first by SCX and by preparative HPLC Method D, (1 injection in 1 mL DMF). The residue was isolated by SCX to afford the title compound (40 mg) as a colorless solid.

LCMS:RtO,81 min,m/z=313[M+H]t 1HNMR(400 MHz, CDCI3) 6 ppm 2.32-2.40 (m, 1 H), 2.79-2.85 (m, 1 H), 3.04 (dd, *J13.5, 2.0 Hz, 1 H), 3.10 (dd, 1=13.5, 4.7 Hz, 1 H), 4.02-4.08 (m, I H), 4.20 (td, 1=8.4, 2.6 Hz, 1 H), 4.23-4.26 (m, 1 H), 7.48-7.52 (m, 1 H), 7.53-7.56 (m, 2 H), 7.63-7.65 (m, I H), 8.97 (s, 2 H), 9.23 (s, 1 H).

Examnle 2: N-(3-((4aR,7aR)-2-amino-4a,6,7,7a-tetrahydro-4H-furo [3.2-di F1.3lthiazin- 7a-ylffiheriyl)-5-methoxyrjyrazine-2-carboxamicle Step I: N-(3-((4aR,7aI-2-benzamido-4a,6,7,7a-tetrahydro-4H-furo13,2-d111, 3lthiazin-7a-yflphenyfl-5-methoxypyrazine-2-carboxami de N OMe N1NYPh

H

N,N'-Diisopropylethylamine (t42 1jL, 0,82 mmol) was added to a stirred mixture of N-((4aR,7aR)-7a-(3-aminophenyl)-4a,6,7,7a-tetrahydro-4H-furo[3,2-dj [I,3]thiazin-2-yl)benzamide (Intermediate 8, 145 mg, 0.41 mmol), 5-methoxypyrazine-2-carboxylic acid (95 mg, 0.62 mmol) and PyBOP (320 mg, 0.62 mmol) in dry CH2C12 (5.3 mL). The mixture was stirred at r.t. for 1.Sh, and then partitioned between CH2C12 aM a saturated aqueous solution of NaHCO1. The aqueous layer was extracted with CH2C12 (x2). The combined extracts were washed with biine, dried over Na2 SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (30 to 60% EtOAc in Hexanes) to give the title compound as a colorless foam (168 mg, 84%) LCMS: Rt.50 mm, m/z = 490 [M+H]t -34 - Step 2: N-(3-((4aR,7aRI2-amino-4a,6,7,7a-tetrahydro-4H-furol3,2-dlIl. 3lthiazin-7a-yflphenyl)-5-methoxypyrazine-2-carboxami de (Example 2) N OMe oi 1r1_fr N. NH2

H

N-(3-4aR,7aR)-2-benzamido-4a,6,7,7a-tetrahydro-4H-furo[3,2-d] [1,3]thiazin-7a-yl)phenyl)-5-methoxypyrazine-2-carboxamide (168 mg, 0.34 mmol) was dissolved in methao1 (2.9 mL), 1,8-diazabicyclo[5.4.0]undec-7-ene (113!IL, 0,75 mmol) was added, aM the solution was refluxed for 2h and then concentrated under reduced pressure. The residue was purified by preparative HPLC Method D, (1 injection in I mL DMF) and SCX to afford the title compound as a colorless solid, (68 mg).

LCMS: Rt 0.99 mm, m/z = 386 [M+H]t 1HNMR(600 MHz, CDCI3) 5 ppm 2.23 (ddd, .1=12.3, 6.2, 2.3 Hz, 1 H), 2.76-2.87 (m, 1 H), 3.05 (d, J=3.2 Hz, 2 H), 3.98 (ddd, J9.9, 8.1, 6.3 Hz, 1 H), 4.08 (s, 3 H), 4.16(td, J=8.3, 2.5 Hz, 1 H), 4.19 (t, J=3.4 Hz, 1 H), 4.33-4.55 (m, 2 H), 7.22 (d, J=7.9 Hz, 1 H), 7.39 (t, J=7.9 Hz, I H), 7.73 (dd, J=8.2, 1.2 Hz, I H), 7.75-7.79 (m, I H), 8.18 (d, J.2 Hz, I H), 9.04 (d, J0.9 Hz, I H), 9.54 (s, I H), Example 3: N-(3-U4aR,7aR)-2-amino-4a,6,7,7a-tetrahydro-4H-furol3,2-dl II,3lthiazin- 7a-yI)phcnyl)-5-(clifluoromethyl)pyrazine-2-carboxamklc Step 1: N-(3-((4aR,7aR)-2-benzamido-4a,6,7,7a-tetrahydro-4H-furol3,2-dlIl, 3lthiazin-7a-yl)phenyl)-5-(difluoromethyl)pyrazine-2-carboxamide

F

H

N,N'-Diisopropylethylamine (142 RL, 0.82 mmol) was added to a stirred mixture of N-((4aR,7aR)-7a-(3-aminophenyl)-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3] thiazin-2- -35 -yl)benzamide (Intermediate 8, 145 mg, 0,41 mmol), 5-(difluoromethyl)pyrazine-2-carboxylic acid (107mg, 0,62 mmol) and PyBOP (320 mg, 0,62 mmol) in dry CH2CI2 (5.3 mL). The mixture was stirred at r.t, for 3h, and then partitioned between CH2CI2 and a saturated aqueous solution ofNal-TCO3. The aqueous layer was extracted with CH2CI2 (x2). The combined extracts were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (30 to 60% EtOAc in Hexanes) to give the title compound as a yellow oil (156 mg, 75%).

LCMS: Rt 1.50 mm, m/z= 510 [M+H]t Step 2: N-(3-U4aR,7aR)-2-amino-4a,6,7,7a-tetrahydro-4H-furol3,2-dlIl. 3lthiazin-7a-yl)phenyl)-5-(difluoromethyl)pyrazine-2-carboxamide (Example 3) TNH2 N-(3 -((4aR, 7aR)-2-benzamido-4a,6,7, 7a-tetrahydro-4H-furo[3,2-dI [1,3]thiazin-7a-yl)phenyl)-5-(difluoromethyl)pyrazine-2-carboxamide (t56 mg, 0.3t mmol) was dissolved in methanol (2.5 mL), 1,8-diazabicyclo[5.4.0]undec-7-ene (tOi 1jL, 0.67 mmol) was added, and the solution was refluxed for 3.Sh and then concentrated under reduced pressure.The residue was purified by preparative I-IPLC Method D, (1 injection in I mL DMF) and SCX to afford the title compound as a colorless solid, (68 mg).

LCMS: Rt.00 mi m/z = 406 [M+H]t HNMR (600 J\'IT-Tz, CDCI3)ö ppm 2.24 (ddd, J=2.3, 6.2, 2.6 Hz, H), 2.77-2.86 (m, I H), 3.01-3.11 (m, 2H), 3.99 (ddd, J=9.7, 8.2, 6.2 Hz, 1 H), 4.17 (td, .fr8.3, 2.5 Hz, 1 H), 4.20 (dd, ,J3.8, 2.9 Hz, 1 H), 4.45 (br. s, 2 H), 6.81 (t, ,h54.5 Hz, 1 H), 7.27-7.29 (m, 1 H), 7.43 (t, 1=7.8 Hz, 1 H), 7.74-7.81 (m, 2 H), 8.94 (s, I H), 9.55 (s, I H), 9.68 (br. s, 1 H).

Biolo2ical Data In vitro cellular assay: Ouantification of AS peptide in culture of neurons from rat fetus brain (I) Rat primary neuronal culture Primary neuronal cultures were prepared from the cerebral cortex of embryonic day 18 Wistar rats (Charles River, UK). Specifically, the embryos were aseptically removed from pregnant rats following CO2 overdose. The brain was isolated from the embryo and -36 -immersed in HBSS (Sigma Aldrich ilH9269) containing 10mM HEPES (Gibco Th 5630-056).

The cerebral cortex was collected from the isolated brain under a stereoscopic microscope.

The cerebral cortex fragments collected were enzymatically treated in an enzyme solution containing 005% trypsin-EDTA solution (GIECO, #25300) at 37°C for 20 minutes to disperse the celis. The cells were then washed twice and then gently resuspended in Neurobasal medium (Gibco #21103) supplemented with lx B27 supplement (GIBCO #17504-044), 2mM L-glutamine (GIBCO #25030), lxN2 (GIBCO #17502-048), 100U/ml Pen/Strep (GTBCO 15140-122) and 5% heat inactivated FCS (PAA #Al5-551). The cell dispersion was filtered through a 40-iim nylon mesh (BD Falcon #352340) to remove the remaining cell mass, and thus a neuronal cell suspension was obtained. The neuronal cell suspension was diluted with the medium above and then plated in a volume of 100 bit /well at an initial cell density of 3.25 x i05 cells/ml in poly-D-lysine coated 96-well culture plate (Greiner #655940). The plated cells were cultured in the culture plate at 37°C in 5% C02- 95% air for 24hrs. The total amount of the medium was replaced with assay Neurobasal medium' (as above excluding heat inactivated FCS), and then the cells were cultured for a further five days.

(2) Addition of compound The drug was added to the culture plate on Day 6 of culture as follows. 8 point compound serial dilutions were generated in DM50 at a concentration ofxl000 that of the final assay concentration (FAC). Compound solutions were then prepared by adding 3@0p1 of Assay Neurobasal media' (as described in above section) to lpl of DMSO compound stock.

The total amount of the medium was removed from each of the cell plate wells, and ]4OpL/well of fresh culture medium was added to cells, and subsequently a further 6Opl of compound solution was then added to the cells. The final DM50 concentration was 0.] %, (3) Sampling The cells were cultured for either I day after addition of the compound for ABx-40.

1 Sop1 of sample medium was collected and used as the ELISA sample.

(4) Evaluation of cell survival Cell survival was evaluated using an Alamar assay according to the following procedure. After collecting the sample to be used in the ELISA assay, SOpl of 20?/o Alamar blue solution (Invitrogen #DAL 1100) in assay Neurobasal media, was added to SOpl of remaining sample within each well. Cells were then incubated at 37°C in 5% C02-95% air for 45 minutes.

Measurement of fluorescence intensity for each well was carried out at 540/S9Onm using a Pherastar plus plate reader (BMG labtech). Upon measurement, wells having no cells plated and containing only the medium and Alamar solution were set as background (bkg).

(5) A13 ELISA -37 -Human/Rat JE Amyloid (42) ELISA Kit Wako (#290-6260 1) and Human/Rat 1 Amyloid (40) ELISA Kit Wako (#294-6250 1) from Wako Pure Chemical Industries, Ltd. were used for AI3 ELISA. A13 ELISA was carried out according to the protocols recommended by the manufacturers, described in the documents accompanying the kits. The results were shown as percentage of the control groups and ICSO values for each compound were determined using four parameter logistic fit model using the XLFIT5 software package (IDBS).

As measured by the above in vitro assay, compound Examples gave the IC50 values (A1340) recorded in Table 1,

Table 1

Example Cell 1C50 (pM) 1 0.110 2 0.010 3 0.008 In vivo analysis: Measurement of Compound Concentrations in Blood and Plasma Compound concentrations in blood or plasma and brain may be measured to calculate a brain:blood (Br:Bl) or brain:plasma (Br:Pl) ratio. This method has been used historically and has been widely accepted as a method of determining CNS penetration (Summerfield et al., S Pharmacol. Expt. Ther. 2007, 322, 205). Literature has been published to suggest that it is important to consider the free concentrations in i/va and that when no efflux occurs from the brain the free plasma concentration should be the same or equivalent to the free brain concentration (Kalvass and Maurer, Biopharmaceutics & Drug Disposition 2002, 23, 327; Mauer et al, Drug Metab. Disposition 2005, 33, 175; Trainor Expert Opin. Drug Discov.

2007, 2, Sfl, Thus, a compound that can freely penetrate the CNS and is not subjected to active efflux, for example by Pgp or another transporter, should demonstrate a free brain:free plasma (Br: Plfr) or an unbound brain:unbound plasma (Bra: Plo) of approximately 1:1.

Free or unbound concentrations can be calculated by multiplying the total brain or total plasma concentration by the fraction unbound in brain tissue or plasma, which can be measured by the assay described herein below, it has also been proposed that drug concentrations in cerebrospinal fluid (CSF) are equivalent to free brain concentrations for compounds which are not actively effluxed from the brain (He et al,, Xenobiotica 2009, 39, 687). Thus another method of determining CNS penetration would be to assess the CSF:free plasma (CSF: IPl) or CSF:unbound plasma (CSF: Pl). If the free drug in plasma is able to -38 -permeate into the CNS and is not actively inf'luxed or effluxed then the CSF:PIfr or CSF:Pl should be approximately 1:1.

Rat in vivo CNS penetration Male Sprague Dawley rats were acquired from Charles River UK Ltd. (Margate, UK) and housed according to UK Home Office guidelines. Drugs were made up to the appropriate concentrations in 0.5% methyl cellulose. Animals were dosed orally (2mL/kg) by gavage at the doses outlined below.

At the specified time point post-dosing, the animals were administered an i.p. injection of sodium pentobarbitone (approximately 330mg/kg for terminal anaesthesia).

Using a guillotine, the animals were decapitated and trunk blood collected into I Sml Falcon tubes containing 100 IU heparin. Blood was vortexed followed by centrifugation at 6000rpm, 4°C for 5 minutes. Plasma was collected for DTvIPK and ELISA assays and stored at -80°C until use. Brains were dissected out and divided along the midline, weighed and stored at -80°C until further use.

Method for Analysis of Plasma, Brain and CSF Samples Preparation of Acetonitrile Working Solutions Test compound was prepared as a I mg free base/mL solution in DMSO, vortexed and sonicated for 5 mm. The mg/mL DMSO solution was diluted to 30 and 00.tg/mL acetoniffile stocks, by adding 30 iL to 970 iL acetonitrile and 100 iL to 900 iL acetonitrile, respectively. The 30 and 100 g/mL acetonitrile stocks were then serially diluted 1:9 (v/v) (100 iL stock into 900 ML acetonitrile) to give the following solutions: 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 g/mL acetonitrile.

Preparation of Plasma Standards, Blanks and Samples Control male Sprague Dawley rat plasma and the study plasma samples were stored at -80°C until the day of analysis when they were thawed at room temperature. Control plasma and study samples were centrifuged (2,000 x g. 10 mm, 4°C) and the control plasma aliquoted (90 ML) into Micronics tubes for preparation of standards and blank samples. Study samples were aliquoted (100 ML) into Micronics tubes.

An aliquot (10 pL) of the appropriate acetonitrile stock was added to the control plasma (to give a final volume of 100 ML) to give the required calibration standards covering the range 1 -10,000 ng/mL. Double blank and blank samples were prepared by adding 10 ML of acetonitrile to 90 ML of blank plasma.

Preparation of Brain Standards. Blanks and Samples Control male Sprague Dawley rat brain and the study brain samples were weighed after collection and stored at -80°C until the day of analysis when they were thawed at room temperature. Once thawed brains were diluted with water (4 mL per gram of tissue) and homogenised using a mechanical homogeniser. An aliquot (100 ML) of each study sample -39 -was taken into Micronics tubes ready for analysis and sufficient aliquots (90 1iL) of control brain homogenate prepared for preparation of standards and blanks.

An aliquot (10 ML) of the appropriate acetonitrile stocks was added to the control brain homogenate (to give a final volume of 100 pL) to give the required calibration standards covering the range 1.5-15,000 ng/g. Double blank and blank samples were prepared by adding 10 1.tL of acetonitrile to 90 ML of blank brain homogenate.

Extraction of Plasma and Brain Samples. Standards and Blanks Each plasma and brain homogenate sample, standard and blank (100 ML) was extracted with an aliquot (300 ML) of acetonitrile (containing 0.1% formic acid and internal standard). Double blanks were extracted with an aliquot (300 ML) of acetonitrile containing 0.1% formic acid). All samples, standards and blanks were then vortex mixed and centrifuged (2000 x g, 15 mm, 4°C). An aliquot (50 ML) of the resulting supernatant was then taken into a 2 mL 96-deep well plate and diluted with acetonitrile:water (50:50 v/v) (150 ML) ready for analysis by a specific LC/MS/MS method.

Preparation of CSF Samples, Standards and Blanks Control male Sprague Dawley rat CSF and the study CSF samples were stored at - 80°C until the day of analysis when they were thawed at room temperature. An aliquot (50 ML) of each study sample was taken into Micronics tubes ready for analysis and sufficient aliquots (45 ML) of control CSF prepared for preparation of standards and blanks.

An aliquot (5 ML) of the appropriate acetonitrile stocks was added to the control CSF (to give a final volume of 50 ML) to give the required calibration standards covering the range 1 -3,000 ng/mL. Double blank and blank samples were prepared by adding 5 ML of acetonitrile to 45 ML of blank CSF.

Extraction of CSF Samples, Standards and Blanks Each CSF sample, standard and blank (SO ML) was extracted with an aliquot (150 ML) of acetonitrile (containing 0.1% formic acid and internal standard). Double blanks were extracted with an aliquot (150 ML) of acetonitrile containing 0.1% formic acid. All samples were then vortex mixed and an aliquot (50 ML) of each was then further diluted in 150 ML of acetonitrile:water (50/50 v/v) in a 2 mL 96-deep well block ready for LC-MS/MS analysis.

All samples were then analysed using a Waters Acquity UPLC coupled to a Waters Xevo TQ mass spectrometer.

LC Conditions: Column: Acquity UPLC BEH C18, 1.7 urn, 2.1 x 50mm, maintained at4O'C Mobile Phase: A = Water containing 0.1% formic acid B = Acetonitrile containing 0.1% formic acid Gradient: -40 -Time (mm) B (%) 0 5 1.2 95 1.5 95 1.7 5 2.0 5 Flow rate: 0.6 mL/min; injection volume 5 pL; autosampler temperature 6°C LC flow was diverted to waste for the first 0.3 mm of each in] ection MS/MS transitions were optimised automatically by Waters QuanOptimise software.

Data Analysis Compound to internal standard peak area ratios were calculated by TargetLynx v4. I software and the data for calibration curves fitted appropriately. Concentrations of test compound were then quantified automatically in TargetLynx v4. 1 and exported to Microsoft Excel 2003.

Amyloid detection DEAl NaC1 Extraction of A13 peptides from rat brain: lOOmI of chilled 0,2?.' diethyl amine (DEA)in 50mM NaCI (pH 10) was freshly prepared and lml/2Smg brain tissue was added to each hemisphere (i.e. 40x brain volume).

The brains were immediately homogenized using a Polytron PT 1200 for 1.5 minutes and samples left to incubate on ice for one hour after homogenisation. 3m1 of the homogenate was transferred to a polyallomer tube (Beckman #362333) and spun at 133000 x g (55,000rpm) for 45 mm at 4° C. The supernatant was then neutralised to pH 8-8.3 by adding 1/10 volume 0.5M Tris/HCI, pH 6.8. The samples can be used fresh or snap frozen on dry-ice and stored at -80°C until required for analysis.

Human/ Rat Amvloid (40) ELISA (Wako Kifl The Wako Af340 ELISA kit (Code No. 294-62501) uses the monoclonal antibody BNT77, raised against epitope A13(1 1-28) and the monoclonal antibody BA27, which specifically detects the C-terminal portion of Af340. This kit is used for the quantitative determination of human or rat Aj3(l-40) and also N-terminally truncated Af340 species (A13(x- 40)) in biological matrices such as tissue culture medium, tissue homogenate, CSF and plasma.

For analysis, plasma and brain samples are diluted 1:1 with the standard diluent contained in the kit and CSF samples are diluted 1:8 with the standard diluent contained in the kit. The assay is carried out according to the manufacturers instructions and samples are analysed in duplicate. Data is analysed using Microsoft Excel 2003 and statistical analysis is carried out using Genstat 9' Edition.

-

Results Compounds were administered at a dose of 30mg/kg p.o. and plasma and brain samples were collected at 6 hours post-dose and the concentrations were measured. The results, including percentage A1340 reductions (%,j,) as measured in Plasma (°/4 P1), Brain (%J. Br) and CSF (%. CSF) are shown in Table 2.

Table 2: _______ _____ _______ _____ _____ _____ _____ ____ _____ _____ Ex. [P1] [P1j [Br] Br11] ICSFI Brtht: Bra: CSF: %J,, %.. %.j.

______ nM nM nM nM nM Pl01 Pl PU P1 Br CSF Ex.2 803 405 1158 95 120 1.4 0.2 0.3 46 60 77 Ex,3 859 514 791 108 101 1.0 0.2 0.2 45 56 84 1. Calculated by multiplying the IP1I by P1 Eu value recorded in Table 3.

2. Calculated by multiplying the [Br] by Br Fn value recorded in Table 3 below.

The results of this assay demonstrate that compound examples 2 and 3 can reduce brain and CSF Af3 after oral administration.

Method For Determination of Plasma Protein Binding (PPB) and Brain Tissue Binding (BTB) Compounds were grouped into cassettes of up to n=4 compounds based on molecular weights differing by a minimum of 5-Da for the purpose of cassetting compounds for incubation. Compounds were dissolved singly in DMSO to give 10 mM stock solutions before further dilution 100-fold in acetonitrile to give combined solutions containing 100 1.tM of each compound.

Naive male Sprague Dawley rat plasma and brains stored at -80 °C were thawed at room temperature. Plasma was centrifuged (2000 >< g, 10 minutes, 4 °C) to remove any precipitate. Brains were weighed and diluted with 2 mL of Phosphate Buffered Saline (PBS) (p1-I 7.4) per gram of tissue and homogenised using a Polytron homogeniser. An aliquot of each 100 iM combined compound solution was then added to plasma and brain homogenate and vortex mixed to give a final compound concentration of H''1 per compound in matrix, A Rapid Equilibrium Dialysis (RED) base plate was filled with the appropriate number of 8-kDa molecular weight cut-off disposable inserts (u3 per cassette) (Thermo Scientific). Matrix containing compounds was then added into the matrix chamber of the inserts (200 pL) and an aliquot (350 iL) of PBS added to the buffer chambers, The plate was covered with a gas permeable seal (Thermo Scientific) and incubated at 37 °C (with 5% CO2 and 95% humidity) for 6 hours with 200 rpm agitation. At the end of the incubation, the seal was removed and an equal aliquot taken from the matrix and PBS chambers and dispensed into Micronics tubes. Plasma and brain was then matrix matched with an equal volume of -42 -drug-free PBS and the PBS samples with an equal volume of the corresponding drug-free matrix, to give equal final compositions and volumes.

Samples are vortex mixed and extracted by protein precipitation with a 1:3 aliquot of acetonitrile containing 0.1% fonriic acid and internal standard. Samples are then vortex mixed and centrifuged (2000 < g, 15 minutes, 4 °C) and an aliquot of the supernatant removed into a 96-deep well plate and diluted with an equal volume of water ready for analysis by LC/MS/MS.

LC/J'vJS/MS analysis was conducted on a Waters Acquity UPLC and Xevo TQ MS (Massachusetts, US). Compound and internal standard were analyzed using specific MRM transitions. Peak area response ratios of parent compound to internal standard were determined using Waters TargetLynx v4. 1 (Tvlassachusetts, US).

Plasma protein binding was calculated using the foflowing equations: Plasma fu = PBS response ratio / Plasma response ratio % Binding = (1 -Plasma fu) x 100 Brain tissue binding was calculated using the following equations: Apparent fu = PBS response ratio / Brain response ratio Brain fu = (/D) / [((1 / Apparent fu) -1) + (lID)] %Binding=( -Brain fu)x 100 D = Brain homogenate dilution (i.e. 3' for a 2mL: ig dilution): (Kalvass JC. and Maurer TS.

(2002) Influence of nonspecific brain and plasma binding on CNS exposure: Implications for rational drug discovery. Biopharm. Drug Dispos. 23, 327), Results are presented in Table 3.

Table 3 _____________ _____________ _____________ ______________ Compound Rat PPB (%) Rat Plasma fu Rat BTB (%) Rat Brain fu _____________ _____________ (P1 Fu) _____________ (Br Fu) Ex,2 49.6 0.504 91.8 0.082 Ex.3 40.1 0.599 86.3 0.137 1')

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Claims

CLAIMS: 1 A compound of formula (I) or a pharmaceutically acceptable salt thereof R2Y NH2 R3 R4 (1) wherein R' is hydrogen, halogen, C16 alicyl or Ci6 haloalicyl; R2 is hydrogen, halogen, C16 alkyl, C16 haloalkyl or a Ring C; R3 and R4 are each independently hydrogen, C16 alkyl or C16 haloalkyl; Ring A is a C6 i4aiyl or a 5-to lO-membered heterocyclic group, either of which is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, cyano, nitro, -NIR'R5, -C(O)NR5R5, -C(O)0R5, -C(O)R5, -NHS(O)2R', -NI-IC(O)R', C26alkenyl, C26alkynyl, C3scycl oallcyl, C3 gcycl oalkoxy, C i6alkoxy or C1.6alkyl; which C26alkenyl, C26alkynyl, C38cycloalkyl, C38cycloalkoxy, C16alkoxy or C1 6alkyl are each optionally substituted by one or more substituents independently selected from halogen, hydroxyl, cyano, C6 alkoxy, -NH2, -NH(Ch6alkyl) and -N(C6alkyl)2; each R5 independently is hydrogen or C -6 alkyl; Ring B is a C614arv1 or a 5-to lO-membered heterocyclic group, either of which is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, cyano, nitro, -NR6R6, -C(O)NR6R6, -C(O)0R6, -C(O)R6, -NHS(O)2R6, -NHC(O)R6, C26alkenyl, C26alkynyl, C3gcycloalkyl, C38cycloalkoxy, C i6alkoxy or C1.6alkyl; which C26alkenyl, C26allq'nyl, C3gcycloalkyl, C3gcycloalkoxy, Ci6alkoxy or C1 6alkyl are each optionally substituted by one or more substituents independently selected from halogen, hydroxyl, cyano, C1, alkoxy, -NH2, -NH(C16a1ky1) and -N(C16a1ky1)2; each R6 independently is hydrogen or C 1-6 alkyl; Ring C is phenyl or a 5-to 6-membered heteroaryl group, either of which is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, cyano, nitro, -NRW, -C(O)NR7R7, -C(O)OR', -C(O)R7, -NHS(O)9R', -NHC(O)R7, C2.6alkenyl, C26allcynyl, C38cyc1 oalkyl, C38cyc1 oallcoxy, C16alkoxy or C16a1ky1; which C2 6alkenyl, C26alkynyl, C38cycloalkyl, C38cycloalkoxy, C16alkoxy or C16alkyl are each optionally substituted by one or more substituents independently selected from halogen, -44 -hydroxyl, cyano, C1.6 alkoxy, -NH2, -NH(Ci.6alkyl) and -N(Ci.6alkyl)2; each R' independently is hydrogen or C14, alkyl; and L is a bond, -0-, -NR8C(0)-, -C(0)NR5-, -NR8-or -Ci.4alkylene-; R is hydrogen or C1.6 alkyl.
2. A compound as claimed in claim 1, or a pharmaceuticafly acceptable salt thereof, wherein Ring A is phenyl optionally substituted by one or more substituents independently selected from halogen, C4alkyl and C4haloakyl.
3. A compound as claimed in claim I or claim 2, or a pharmaceutically acceptable salt thereof, wherein Ring B is a 6-membered heteroaryl group containing at least one nitrogen atom optionally substituted by one or more substituents independently selected from halogen, cyano, Cp4 haloakyl, C14 alkoxy, C14 haloalkoxy and C14 alkyl which C14 alkyl is optionally 4, A compound as claimed in any one of claims Ito 3, or a pharmaceutically acceptable salt thereof, wherein L is a bond or -NH-C(0)-.5. A compound as claimed in any one of claims I to 4, or a pharmaceutically acceptable salt thereof wherein one of R1 and R2 is hydrogen and the other of R1 and R2 is hydrogen, fluorine, methyl, monofluoromethyl, difluoromethyl or trifluoromethyl.6. A compound as claimed in claim I, or a pharmaceutically acceptable salt thereof, of formula (IB) _ oy( YNH (F) IR2 NH2 H (IB) wherein nisO, I or2; A is CH or N; Y is fluorine, cyano, methyl, ethyl, monofluoromethyl, difluoromethyl.trifluoromethyl, difluoroethyl, methoxy, ethoxy or methoxymethyl; -45 -one of R' and R2 is hydrogen and the other of R1 and R2 is hydrogen, fluorine, methyl, monofluoromethyl, difluoromethyl or trifluoromethyl 7. A compound as claimed in claim 6, or a pharmaceutically acceptable salt thereof, wherein n is 0; A is N; Y is fluorine, monofluoromethyl, difluoromethyl or methoxy; R' is hydrogen; and R2 is hydrogen.8. A compound as claimed in claim I, or a pharmaceutically acceptable salt thereof, of formula (IC) R2 NH2 H (IC) wherein n isO, 1 or 2; Ring B is a 6-membered heteroaryl group containing at least one nitrogen atom optionally substituted by one or more substituents independently selected from fluorine, cyano, methyl, ethyl, monofluoromethyl, difluoromethyl, trifluoromethyl, difluoroethyl, methoxy, ethoxy or methoxymethyl; and one of R' and R2 is hydrogen and the other of R1 and R2 is hydrogen, fluorine, methyl, monofluoromethyl, difluoromethyl or trifluoromethyl.9. A compound as claimed in any one of claims ito 8, or a pharmaceutically acceptable salt thereof, for use in therapy.10. A compound as claimed in any one of claims I to 8, or a pharmaceutically acceptable salt thereof, for treating or preventing Alzheimer-type dementia (AD).11. A compound as claimed in any one of claims I to 8, or a pharmaceutically acceptable salt thereof, for treating Down's syndrome.12. Use of a compound as claimed in any one of claims I to8, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment or prevention of Alzheimer-type dementia (AD).-46 - 13. Use of a compound as claimed in any one of claims I to 8, or a pharmaceutically acceptable salt thereoZ for the manufacture of a medicament for the treatment of Down's syndrome.14. A phannaceutical composition comprising the compound as claimed in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof; as an active ingredient in association with a pharmaceutically acceptable carrier.15. A pharmaceutical product comprising, in combination, a first active ingredient which isa compound as claimed in any one of claims Ito 8 or a pharmaceutically acceptable salt thereof; and at least one further active ingredient useful in treating a neurodegenerative disease.