FUSED AMINODIHYDROTHIAZINE DERIVATIVE SALTS

AND USES THEREOF FIELD OF THE INVENTION

The present invention relates to new salts of a fused aminodihydrothiazine derivative and pharmaceutical uses thereof.

BACKGROUND

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 thought that Αβ -proteins as breakdown products of amyloid precursor proteins (hereinafter referred to as APP) are highly involved in degeneration and loss of neurons and onset of symptoms of dementia. Αβ-proteins have, as main components, Αβ40 consisting of 40 amino acids and Αβ42 with two amino acids added at the C- terminal. The Αβ40 and Αβ42 proteins are known to be highly prone to aggregation and to be the main components of senile plaques. Furthermore, it is known that Αβ40 and Αβ42 are increased by mutations in APP and presenilin genes associated with familial Alzheimer's disease (Jonsson et al, Nature 201 , 488, 2 August, 96-99).

Accordingly, a compound that reduces production of Αβ40 and Αβ42 is predicted to inhibit disease progression or be a 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 Αβ40 and Αβ42 may be beneficial. Early onset dementia and Alzheimer's type dementia (AD) are common outcomes in individuals with Down's syndrome and evidence suggests Αβ deposition may be a causative factor in cognitive impairment 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, and BACE2 and one of its substrates, Tmem27, have been linked to diabetes, BACE2 inhibitors may be useful for the treatment of diabetes. Accordingly, a dual BACEl/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 Αβ40 and Αβ42 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 Αβ40 and Αβ42 include type 2 diabetes, 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 amyloidosis, pain, Gerstmann-Straeussler-Scheinker syndrome, scrapie, encephalopathy, spino cerebellar ataxia, 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.

Αβ is produced by the cleavage of APP by beta-secretase (BACE1) and subsequently by gamma-secretase. For this reason, attempts have been made to create gamma-secretase and beta-secretase inhibitors in order to inhibit Αβ production. For example, WO2009/09101 describes a series of fused aminodihydrothiazine derivatives having an Αβ production inhibitory effect. International patent application

PCT/EP2012/050833 (WO2012/098213), incorporated by reference herein in its entirety, describes novel fused aminodihydrothiazine compounds that have high potency and attractive pharmacological properties. One such compound described in

PCT/EP2012/050833 is N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl) -5-(fluoromethyl)pyrazine- 2-carboxamide free base.

In the manufacture of pharmaceutical products it is important that the active compound is in a form that can be conveniently manipulated and processed in order to obtain a commercially- viable manufacturing process. In this regard, the chemical stability and physical stability of the active compound are important factors. The active compound and pharmaceutical compositions containing it must be capable of being effectively stored over long periods of time without exhibiting any significant change in physico-chemical characteristics (e.g. density, hygroscopicity and solubility).

The preparation of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl) -5-(fluoromethyl)pyrazine- 2-carboxamide free base described in PCT/EP2012/050833 yields a crystalline material. However, on further investigation it has been observed that the free base compound prepared according to the methods of PCT/EP2012/050833 can exist in more than one physical (polymorphic) form of similar melting points. Accordingly, there is a need for an alternative form of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl) -5-(fluoromethyl)pyrazine- 2-carboxamide suitable for pharmaceutical use and incorporation in a pharmaceutical composition.

SUMMARY

Provided herein is a salt of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)- 4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-flu orophenyl)-5- (fluoromethyl)pyrazine-2-carboxamide, which is selected from

i) N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H-furo[3,4- d] [ 1 ,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2 -carboxamide malonate, and

ii) N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H-furo[3,4- d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazi ne-2-carboxamide hippurate.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : X-Ray Powder Diffraction Pattern of N-(3-((4aS,5S,7aS)-2-amino-5- (trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]th iazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide free base Form B Polymorph.

Figure 2: X-Ray Powder Diffraction Pattern of N-(3-((4aS,5S,7aS)-2-amino-5- (trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-fui [3,4-d][l,3]thiazin-7a-yl)-4- fluoi phenyl)-5-(fluoiOmethyl)pyrazine-2-carboxamide free base Form A Polymorph.

Figure 3 : X-Ray Powder Diffraction Pattern of N-(3-((4aS,5S,7aS)-2-amino-5-

(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3 ]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide free base hydrate.

Figure 4: X-Ray Powder Diffraction Pattern of N-(3-((4aS,5S,7aS)-2-amino-5- (trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]th iazin-7a-yl)-4- fluoiOphenyl)-5-(fluoromethyl)pyrazine-2-carboxamide mono-malonate anhydrate.

Figure 5: DVS of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl) -5-(fluoromethyl)pyrazine- 2-carboxamide mono-malonate anhydrate. Figure 6: X-Ray Powder Diffraction Pattern of N-(3-((4aS,5S,7aS)-2-amino-5- (trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]th iazin-7a-yl)-4- fluorophenyl)-5 -(fluoromethyl)pyrazine-2-carboxamide mono-hippurate anhydrate.

Figure 7: DVS of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl) -5-(fluoromethyl)pyrazine- 2-carboxamide mono-hippurate anhydrate.

Figure 8: Single Crystal X-ray Structure of N-(3-((4aS,5S,7aS)-2-amino-5- (trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]th iazin-7a-yl)-4- fluorophenyl)-5 -(fluoromethyl)pyrazine-2-carboxamide mono-malonate anhydrate.

Figure 9: Single Crystal X-ray Structure of N-(3-((4aS,5S,7aS)-2-amino-5- (trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]th iazin-7a-yl)-4- fiuorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide mono-hippurate anhydrate.

DETAILED DESCRIPTION OF INVENTION

The name N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro- 4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorom ethyl)pyrazine-2- carboxamide was generated using ChemBioDraw Ultra 11.0 or 12.0, and denotes the compound depicted in Figure A.

Figure A

In a first aspect, the present invention provides a salt which is N-(3-

((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetra hydro-4H-furo[3,4- d] [ 1 ,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2 -carboxamide malonate. As known in the art, the term "malonate" denotes a salt of malonic acid (CH ₂ (COOH) ₂ ). In the present specification this salt may be referred to as the "malonate salt". The malonate salt of the present invention has good physico-chemical properties and is suitable for pharmaceutical use.

In an embodiment of the invention, the stoichiometric ratio of malonic acid and N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H-furo[3,4- d] [1 ,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2 -carboxamide in the malonate salt is approximately 1 :1, i.e. in the range 1 :0.9 to 1 : 1.1. In the present specification, the salt of this embodiment may be referred to as the 'mono-malonate salt'.

The present invention encompasses solvates (e.g. a hydrate) of the malonate salt. However, in one embodiment of the invention, the malonate salt is an anhydrate (i.e. a crystalline phase that does not contain water).

In the context of the present specification, a salt is an anhydrate and is non- hygroscopic if it has a water uptake value of less than 2% as measured by the increase in mass determined by Dynamic Vapor Sorption (DVS) from 5% to 95% relative humidity at 25 °C.

In one embodiment, the present invention provides a salt which is N-(3-

((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetra hydro-4H-furo[3,4- d] [1 ,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2 -carboxamide mono- malonate anhydrate. In a further aspect of this embodiment, there is provided a mono- malonate salt which is an anhydrate and has a water uptake value of less than 1.5 % as measured by the increase in mass determined by DVS from 5%> to 95% relative humidity at 25 °C.

The malonate salt of the invention has crystalline properties. In an embodiment of the invention, the malonate salt is at least 50 % crystalline. In a further embodiment the malonate salt is at least 60%> crystalline; in a still further embodiment at least 70% crystalline; and in a yet further embodiment at least 80% crystalline. In another embodiment, the malonate salt is from 50%, 60%, 70%, 80% or 90% to 95% or 100% crystalline. Crystallinity can be estimated by conventional X-ray diffractometry techniques or differential scanning calorimetry.

Generally, a diffraction angle (20) in X-ray powder diffractometry may have an error margin of +/- 0.2°. Therefore, in embodiments of the present invention where diffraction angles are quoted, the margin of error for X-ray powder diffraction peaks (expressed in degrees is 2Θ) is +/- 0.2°. Unless otherwise stated, X-ray powder diffraction peaks quoted in the present specification were measured using X rays generated from a Cu target.

In one embodiment, the present invention provides a N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide mono-malonate anhydrate salt, which exhibits at least the following characteristic X-ray powder diffraction peaks (expressed in degrees 2Θ +/- 0.2°): 16.2 and 18.5.

In another embodiment, the present invention provides a N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide mono-malonate anhydrate salt, which exhibits at least the following characteristic X-ray powder diffraction peaks (expressed in degrees 2Θ +/- 0.2°): 9.2 and 18.5.

In another embodiment, the present invention provides a N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide mono-malonate anhydrate salt, which exhibits at least the following characteristic X-ray powder diffraction peaks (expressed in degrees 2Θ +/- 0.2°): 9.2 and 16.2.

In another embodiment, the present invention provides a N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoiOmethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide mono-malonate anhydrate salt, which exhibits at least the following characteristic X-ray powder diffraction peaks (expressed in degrees 2Θ +/- 0.2°): 9.2, 16.2 and 18.5.

In another embodiment, the present invention provides a N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide mono-malonate anhydrate salt, which exhibits at least the following characteristic X-ray powder diffraction peaks (expressed in degrees 2Θ +/- 0.2°):

(1) : 9.2, 16.2, 18.5 and 25.6; or

(2) : 9.2, 16.2, 18.0, 18.5 and 25.6; or

(3): 9.2, 16.2, 18.0, 18.5, 25.6 and 26.4; or

(4) : 9.2, 16.2, 18.0, 18.5, 25.6, 26.4 and 29.8; or

(5) : 9.2, 16.2, 18.0, 18.5, 25.6, 26.4, 29.8 and 31.3.

The mono-malonate anhydrate salt of the present invention displays good manufacturability of a standard suitable for use in pharmaceutical development.

The malonate salt of the present invention may be prepared by dissolving or suspending N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H- furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorometh yl)pyrazine-2-carboxamide free base in a suitable solvent with heating as necessary, adding malonic acid and stirring the mixture, optionally with cooling or sonification, until crystallisation occurs, and then recovering the malonate salt crystals by conventional methods. Specific details of preparations of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl) -5-(fluoromethyl)pyrazine- 2-carboxamide malonate are described herein below in the examples.

Suitable solvents that may be used in the preparation of the malonate salt include ethanol, 1-propanol, 2-propanol 1-butanol, 2-butanol, acetone, THF, acetonitrile, ethyl acetate, isopropyl acetate, toluene or heptane (e.g. when mixed with polar solvents), water (e.g. when mixed with a suitable alcohol such as 2-propanol), methyl tert-butyl ether, and mixtures thereof. In one embodiment, the solvent used is 2-propanol. In one embodiment, the present invention provides a process of preparing N-(3- ((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahyd ro-4H-furo[3,4- d] [1 ,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2 -carboxamide mono- malonate anhydrate, comprising

(i) preparing a composition of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)- 4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l ,3]thiazin-7a-yl)-4-fluorophenyl)-5- (fluoromethyl)pyrazine-2-carboxamide free base in a suitable solvent,

(ii) adding malonic acid to the composition,

(iii) optionally after step (i) or (ii), heating the composition to a temperature in the range of 25 °C to solvent reflux temperature,

(iv) allowing crystals of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydro-4H-fuiO[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl) -5-(fluoromethyl)pyrazine- 2-carboxamide mono-malonate anhydrate to form.

The composition of (i) may be a suspension or solution of N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide free base.

The crystals of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydro-4H-furo [3 ,4-d] [ 1 ,3]thiazin-7a-yl)-4-fluorophenyl)-5 -(fluoromethyl)pyrazine- 2-carboxamide mono-malonate anhydrate prepared by this process may be recovered using conventional techniques, e.g. by filtration with optional washing and / or drying.

In one aspect of this embodiment, the solvent is 2-propanol. In step (ii), malonic acid may for example be added as a solid, as a solution in a suitable solvent, or as a suspension. In a further aspect of this embodiment, in step (ii) malonic acid is added as a suspension in 2-propanol. In a further aspect of this embodiment, the molar ratio of malonic acid to free base in the composition prepared in step (ii) is in the range of 1.2:1 to 1 : 1. In a further aspect of this embodiment, the composition prepared in step (i) is heated to a temperature in the range of 70 to 82°C prior to the addition of the malonic acid in step (ii). In a further aspect of this embodiment, the suspension prepared in step (ii) is cooled to a temperature in the range of -10 to 25°C to allow crystals of N-(3- ((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahyd ro-4H-furo[3,4- d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazi ne-2-carboxamide mono- malonate anhydrate to form.

In a second aspect the present invention provides a salt which is N-(3- ((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahyd ro-4H-furo[3,4- d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoiOmethyl)pyrazi ne-2-carboxamide hippurate. As known in the art, the term "hippurate" denotes a salt of hippuric acid (benzoylaminoethanoic acid). In the present specification this salt may be referred to as the "hippurate salt". The hippurate salt of the present invention has good physico- chemical properties and is suitable for pharmaceutical use.

The structure of hippuric acid is depicted in Figure B.

Figure B

In an embodiment of the invention, the stoichiometric ratio of hippuric acid and N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H-furo[3,4- d] [ 1 ,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2 -carboxamide in the hippurate salt is approximately 1 :1, i.e. in the range of 1 : 0.9 to 1 : 1.1. In the present specification, the salt of this embodiment may be referred to as the 'mono-hippurate salt'.

The present invention encompasses solvates (e.g. a hydrate) of the hippurate salt. However, in one embodiment of the invention, the hippurate salt is an anhydrate (i.e. a crystalline phase that does not contain water).

In the context of the present specification a salt is an anhydrate and is non- hygroscopic if it has a water uptake value of less than 2% as measured by the increase in mass determined by DVS from 5% to 95% relative humidity at 25 °C.

In one embodiment, the present invention provides a salt which is N-(3- ((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahyd ro-4H-furo[3,4- d] [1 ,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2 -carboxamide mono- hippurate anhydrate. In a further aspect of this embodiment, there is provided a mono- hippurate salt which is an anhydrate and has a water uptake value of less than 2 % as measured by the increase in mass determined by DVS from 5% to 95% relative humidity at 25 °C.

The hippurate salt of the invention has crystalline properties. In an embodiment of the invention, the hippurate salt is at least 50 % crystalline. In a further embodiment the hippurate salt is at least 60% crystalline; in a still further embodiment at least 70% crystalline; and in a yet further embodiment at least 80% crystalline. In another embodiment, the hippurate salt is from 50%, 60%, 70%, 80% or 90% to 95% or 100% crystalline. Crystallinity can be estimated by conventional X-ray diffractometry techniques or differential scanning calorimetry.

Generally, a diffraction angle (2Θ) in X-ray powder diffractometry may have an error margin of +/- 0.2°. Therefore, in embodiments of the present invention where diffraction angles are quoted, the margin of error for X-ray powder diffraction peaks (expressed in degrees is 2Θ) is +/- 0.2°. Unless otherwise stated, X-ray powder diffraction peaks quoted in the present specification were measured using X rays generated from a Cu target.

In one embodiment, the present invention provides a N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide mono-hippurate anydrate salt, which exhibits at least the following characteristic X-ray powder diffraction peaks (expressed in degrees 2Θ +/- 0.2°): 14.0 and 18.2.

In another embodiment, the present invention provides a N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide mono-hippurate anydrate salt, which exhibits at least the following characteristic X-ray powder diffraction peaks (expressed in degrees 2Θ +/- 0.2°): 14.0 and 22.6.

In another embodiment, the present invention provides a N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide mono-hippurate anydrate salt, which exhibits at least the following characteristic X-ray powder diffraction peaks (expressed in degrees 2Θ +/- 0.2°): 18.2 and 22.6.

In another embodiment, the present invention provides a N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide mono-hippurate anydrate salt, which exhibits at least the following characteristic X-ray powder diffraction peaks (expressed in degrees 2Θ +/- 0.2°): 14.0, 18.2 and 22.6.

In another embodiment, the present invention provides a N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydiO-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide mono-hippurate anydrate salt, which exhibits at least the following characteristic X-ray powder diffraction peaks (expressed in degrees 2Θ +/- 0.2°):

(1) : 11.2, 14.0, 18.2 and 22.6 or

(2) : 6.3, 11.2, 14.0, 18.2 and 22.6 or

(3): 6.3, 11.2, 14.0, 18.2, 19.1 and 22.6 or

(4) : 6.3, 11.2, 14.0, 18.2, 19.1, 22.6 and 24.4 or

(5) : 6.3, 11.2, 14.0, 18.2, 19.1, 22.6, 24.4 and 25.2.

The mono-hippurate anydrate salt of the present invention displays good manufacturability of a standard suitable for use in pharmaceutical development.

The hippurate salt of the present invention may be prepared by dissolving or suspending N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H- furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorometh yl)pyrazine-2-carboxamide free base in a suitable solvent with heating as necessary, adding hippuric acid and stirring the mixture, optionally with cooling or sonification, until crystallisation occurs, and then recovering the hippurate salt crystals by conventional methods. Specific details of a preparation of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl) -5-(fluoromethyl)pyrazine- 2-carboxamide hippurate are described herein below in the examples.

Suitable solvents that may be used in the preparation of the hippurate salt (e.g. the mono-hippurate anhydrate salt) include ethanol, 1-propanol, 2-propanol 1-butanol, 2-butanol, acetone, THF, acetonitrile, ethyl acetate, isopropyl acetate, toluene or heptane (e.g. when mixed with polar solvents), water (e.g. when mixed with a suitable alcohol such as 2-propanol), methyl tert-butyl ether, and mixtures thereof. In one embodiment, the solvent used is 2-propanol.

In one embodiment, the present invention provides a process of preparing N-(3- ((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahyd ro-4H-furo[3,4- d] [1 ,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2 -carboxamide mono- hippurate anhydrate, comprising

(i) preparing a composition comprising N-(3-((4aS,5S,7aS)-2-amino-5- (trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]th iazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide free base and hippuric acid in a suitable solvent,

(ii) optionally heating the composition to a temperature in the range of 25 °C to solvent reflux temperature, and

(iii) allowing crystals of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydi -4H-furo[3,4-d][l,3]tMazin-7a-yl)-4-fluorophenyl)-5-(fluorom ethyl)pyrazine- 2-carboxamide mono-hippurate anhydrate to form.

The crystals of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl) -5-(fluoromethyl)pyrazine- 2-carboxamide mono-hippurate anhydrate prepared by this process may be recovered using conventional techniques, e.g. by filtration with optional washing and / or drying.

In one aspect of this embodiment, the solvent is 2-propanol. In a further aspect of this embodiment, the molar ratio of hippuric acid to free base in the composition of

(i) is in the range of 1.25: 1 to 1 : 1. In a further aspect of this embodiment, the composition of (i) is heated to a temperature in the range of 65 to 82°C. In a further aspect of this embodiment, the composition is cooled to a temperature in the range of -10 to 25°C to allow crystals of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)- 4a,5,7,7a-tetrahydi -4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5- (fluoromethyl)pyrazine-2-carboxamide mono-hippurate anhydrate to form.

The composition of (i) may be for example be prepared by (a) preparing a composition of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro- 4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorom ethyl)pyrazine-2- carboxamide free base in a suitable solvent; (b) adding hippuric acid to the composition; and (c) optionally after step (a) or (b), heating the composition to a temperature in the range of 25 °C to solvent reflux temperature.

Further salts of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydro-4H-furo [3 ,4-d] [ 1 ,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine- 2-carboxamide have been prepared. These include an L-malate salt, an L-tartrate salt and a succinate salt. Procedures for preparing these salts are described herein below in the experimental section.

N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H- furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorometh yl)pyrazine-2-carboxamide free base may be prepared according to the procedures described in

PCT/EP2012/050833 (WO2012/098213), for example by the procedure described on page 90 of WO2012/098213. Analysis of different batches of free base material prepared by this procedure revealed that at least two different polymorphs of the free base could be formed and difficulties were encountered in selectively preparing a single polymorph using this preparation. Moreover, two of the polymorphic forms identified (Form A and Form B) were found to have very similar melting points and batch to batch variation in each polymorph's melting point was recorded. Batch variability was also observed in hygroscopicity measurements for Form A. Further research subsequently identified that a single polymorph (Form A) could consistently be prepared using an alternative preparation wherein the reaction for preparing (4aS,5S,7aS)-7a-(5-amino-2- fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-fu ro[3,4-d][l,3]thiazin-2- amine and 5-(fluoromethyl)pyrazine-2-carboxylic acid is quenched with aqueous sodium hydroxide in place of aqueous ammonium hydroxide. A description of this alternate preparation is provided herein below in the experimental section.

N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H- fuiO[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorometh yl)pyrazine-2-carboxamide contains three chiral centers located on the tetrahydrofuro-thiazinyl ring. The stereochemical configuration at each of these chiral centers is preferably S, i.e. they are (4aS,5S,7aS) stereoisomers. For the avoidance of doubt, in the present invention the (4aS,5S,7aS) stereoisomers may be present as a mixture with one or more of the other possible stereoisomers, for example in a racemic or diastereomeric mixture.

In one embodiment, the present invention provides N-(3-((4aS,5S,7aS)-2-amino- 5-(trifluoi methyl)-4a,5,7,7a-tetrahydro-4H-fuiO[3,4-d][l,3]thiazin-7a-y l)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide salt which is stereochemically pure at the (4aS,5S,7aS) chiral centers. In the context of the present specification, the term stereochemically pure denotes a compound which has 80 % or greater by weight of the (4aS,5S,7aS) stereoisomer and 20% or less by weight of other stereoisomers (weight based on free base). In a further embodiment, the salt has 90 % or greater by weight of the (4aS,5S,7aS) stereoisomer and 10% or less by weight of other stereoisomers. In a yet further embodiment, the salt has 95 % or greater by weight of the (4aS,5S,7aS) stereoisomer and 5% or less by weight of other stereoisomers. In a still further embodiment, the salt has 97 % or greater by weight of the (4aS,5S,7aS) stereoisomer and 3% or less by weight of other stereoisomers.

The present invention also includes isotopically-labelled versions of the salts wherein 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 the salt of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, phosphorous, chlorine, technetium and iodine, such as H, ³ H, ⁿ C, ¹⁴ C, ¹³ N, ¹⁵ 0, ¹⁸ F, ³² P, ^99m Tc, ¹²³ I and ¹³¹ I. Such isotopically-labelled salts are for example useful in drug and/or substrate tissue distribution assays. ³ H and ¹⁴ C are considered useful due to their ease of preparation and detectability. ⁿ C, ¹⁵ 0 and ¹⁸ F isotopes are considered useful in PET (positron emission tomography), and ^99m Tc, ¹²³ I and ^{1 1} I isotopes are considered useful in SPECT (single photon emission computerized tomography), all useful in brain imaging.

Substitution with heavier isotopes such as ² H can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, are considered useful in some circumstances. Isotopically labelled salts of this invention can generally be prepared by carrying out the procedures described herein below, by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.

The present invention further provides a N-(3-((4aS,5S,7aS)-2-amino-5-

(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3 ]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide salt according to the present invention for use in therapy.

The salt of the present invention may be useful as a prophylactic or therapeutic agent for a neurodegenerative disease caused by Αβ and typified by Alzheimer-type dementia (AD) or Down's syndrome. It may be used to reduce both Αβ40 and Αβ42. Furthermore, it may have a BACE 1 and / or a BACE 2 inhibitory effect.

Thus, in another aspect, the present invention provides a N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide salt according to the present invention for inhibiting production of amyloid-β protein.

In a further aspect, the present invention provides a N-(3-((4aS,5S,7aS)-2-amino- 5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3] thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide salt according to the present invention for inhibiting beta-site amyloid-β precursor protein cleaving enzyme 1 (BACE 1)·

In a further aspect, the present invention provides a N-(3-((4aS,5S,7aS)-2-amino- 5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3] thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide salt according to the present invention, for use in treating or preventing Alzheimer-type dementia (AD).

In a further aspect, the present invention provides a N-(3-((4aS,5S,7aS)-2-amino- 5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3] thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide salt according to the present invention, for use in treating Down's syndrome.

In another aspect, the invention provides the use of a N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide salt according to the present invention 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 N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide salt according to the present invention for the manufacture of a medicament for the treatment of Down' syndrome.

In another aspect, the invention provides a method of treating or preventing Alzheimer-type dementia (AD) involving administering to a human subject in need thereof a therapeutically or prophylactically effective amount of a N-(3-((4aS,5S,7aS)- 2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4 -d][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide salt according to the present invention.

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 N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H- furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorometh yl)pyrazine-2-carboxamide salt according to the present invention.

In the present specification, the term "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 one aspect the present invention further provides a N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydi -4H-furo[3,4-d][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide salt according to the present invention for use in treating type 2 diabetes. In a further aspect the present invention further provides the use of a N-(3- ((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahyd ro-4H-furo[3,4- d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazi ne-2-carboxamide salt according to the present invention for the manufacture of a medicament for the treatment or prevention of type 2 diabetes.

In a yet further aspect the present invention 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 N-(3-((4aS,5S,7aS)-2-amino-5- (trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]th iazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide salt according to the present invention.

A further aspect of the invention provides a pharmaceutical composition comprising a N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H- fui [3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)p yrazine-2-carboxamide salt according to the present invention 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 N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H- furo [3 ,4-d] [ 1 ,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2 -carboxamide salt according to the present invention can 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, inhalants, suppositories, injections, ointments, eye drops, nasal drops, ear drops, cataplasms and lotions. These solid preparations such as tablets, capsules, granules and powders can contain generally 0.01 to 100 wt%, and preferably 0.1 to 100 wt% of the malonate or hippurate salt as the 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 pH 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, squalane and solid paraffin; ester oils such as octyldodecyl myristate and 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 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, propylene 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. The ingredients are not limited to the above additive ingredients.

For example, an oral preparation may be prepared by adding the malonate or hippurate salt according to the present invention, 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, fine granules, granules, tablets, coated tablets, capsules or the like by a conventional method. Tablets or granules may be appropriately coated, for example, sugar coated, where necessary.

For example, a syrup 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 can 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 syrup can contain usually 0.01 to 100 wt%, and preferably 0.1 to 100 wt% of the active ingredient.

For example, an external preparation can 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 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 N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl) -5-(fluoromethyl)pyrazine- 2-carboxamide salt according to the present invention may be varied according to the degree of symptoms, age, sex, body weight, mode of administration, and specific type of disease. Typically, the active ingredient is orally administered to an adult at about 30 μg to 10 g, preferably 100 μg to 5 g, and more preferably 100 μg to 1 g per day, or is administered to an adult by injection at about 30 μg to 1 g, preferably 100 μg to 500 mg, and more preferably 100 μg to 300 mg per day, in one or several doses, respectively.

The N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoiOmethyl)-4a,5,7,7a-tet rahydro-4H- furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorometh yl)pyrazine-2-carboxamide salt according to the present invention 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 or Down's syndrome. Thus, in a further aspect, the present invention provides a pharmaceutical product comprising, in combination, a N-(3- ((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahyd ro-4H-furo[3,4- d] [1 ,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2 -carboxamide salt according to the present invention, 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) or Down's syndrome. 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 (PAMs), acetylcholinesterase inhibitors (such as tetrahydroaminoacridine, donepezil hydrochloride and rivastigmine), nicotinic receptor agonists or allosteric modulators (such as a7 agonists or allosteric modulators or α4β2 agonists or allosteric modulators), PPAR agonists (such as PPARy agonists), 5- HT ₄ receptor agonists or partial agonists, histamine H3 antagonists, 5-HT ₆ receptor antagonists or 5HTi _A receptor ligands and NMDA receptor antagonists or modulators such as memantine, 5-HT ₂ A antagonists, 5-HT ₇ antagonists, Dl agonists or PAMs, D4 agonists or PAMs, D5 agonists or PAMs, GABA-A a5 inverse agonists or negative allosteric modulators (NAMs), GABA-A ct2/3 agonists or PAMs, mGluR2 modulators (PAMs or NAMs), mGluR3 PAMs, mGluR5 PAMs, PDE 1 inhibitors, PDE 2 inhibitors, PDE 4 inhibitors, PDE 5 inhibitors, PDE 9 inhibitors, PDE 10 inhibitors, GlyTl inhibitors, DAAO inhibitors, ASCI inhibitors, AMPA modulators, SIRT1 activators or inhibitors, AT4 antagonists, GalRl antagonists, GalR3 ligands, adenosine Al

antagonists, adenosine A2a antagonists, oc2A antagonists or agonists, selective and unselective norepinephrine reuptake inhibitors (SNRIs), or potential disease modifying agents such as gamma secretase inhibitors or modulators, alpha secretase activators or modulators, amyloid aggregation inhibitors, amyloid antibodies, tau aggregation inhibitors or tau phosphorylation/kinase inhibitors, tau dephosphorylation / phosphatase activators, mitogen-activated protein kinase kinase 4 (MKK4/MEK4/MAP2K4) inhibitors, c-Jun N-terminal kinase (JNK) inhibitors, casein kinase inhibitors, MK2 (mitogen activated protein kinase-activated protein kinase 2) inhibitors, MARK

(microtubule affinity regulating kinase) inhibitors, CDK5 (cyclin dependent kinase 5) inhibitors, GSK-3 (glycogen synthase kinase-3) inhibitors and tau-tubulin kinase- 1 (TTBK1) 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, GluRl ligands, RAGE (receptor for advanced glycation end-products) antagonists, EGFR (epidermal growth factor receptor) inhibitors, FPRL-1 (formyl peptide-like receptor- 1) ligands, GABA antagonists, and MICAL (molecule interacting with casL) inhibitors, e.g. oxoreductase inhibitors, CB1 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 (HDAC) inhbitors, EP2 antagonists, 11 -beta HSD1 (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 1 (ORL1) antagonists, excitatory amino acid transporter (EAAT) ligands (activators or inhibitors), and plasminogen activator inhibitor- 1 (PAI-1) inhibitors, niacin and /or GPR109 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 N-(3-((4aS,5S,7aS)-2- amino-5-(trifluoiOmethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d ][l,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide salt according to the present invention, and at least one further active ingredient selected from:

• cholinesterase inhibitors, e.g. donepezil, galantamine, rivastigamine,

tetrahydroaminoacridine and pharmaceutically acceptable salts thereof,

· 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-HT ₆ antagonists, e.g. SB-742457 and pharmaceutically acceptable salts thereof,

• HMGCoA 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 N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro- 4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorom ethyl)pyrazine-2- carboxamide salt according to the present invention 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.

The present invention is illustrated by the following non-limiting Examples. The abbreviations used in the Examples are conventional abbreviations known to a person skilled in the art. Some abbreviations are shown below:

LCMS, LC/MS & LC-MS (liquid chromatography/mass spectrometry); MS (mass spectrometry); MDAP (mass directed auto purification); NMR (nuclear magnetic resonance); s, d, t, dd, m, br (singlet, doublet, triplet, doublet of doublets, multiplet, broad); Ph, Me, Et, Pr, Bu, Bn (phenyl, methyl, ethyl, propyl, butyl, benzyl); THF (tetrahydrofuran); DCM (dichloromethane); DMF (Ν,Ν-dimethylformamide); h, hr, hrs (hours); EDC & EDAC (N-3-(dimethylaminopropyl)-N'ethylcarbodiimide

hydrochloride); DMAP (4-N,N-dimethylaminopyridine); DMSO (dimethylsulfoxide); UV (ultraviolet); RT & rt (room temperature); Rt (retention time); min & mins (minutes); EtOAc (ethyl acetate); Et ₂ 0 (diethyl ether); MeCN (acetonitrile); EtOH (ethanol); MeOH (methanol); PhCH ₃ & PhMe (toluene); tic (thin layer

chromatography); TFA (trifluoroactic acid); NaOH (sodium hydroxide); HC1

(hydrochloric acid); NMP (N-methylpyrrolidinone or l-methyl-2-pyrrolidinone); HPLC (high performance liquid chromatography); TBAF (tetrabutylammonium fluoride); BuLi (n-butyl lithium); PyBOP: benzotriazol-l-yloxytris(pyrrolidino)phosphonium hexafluorophosphate; Pd ₂ dba ₃ : tris(dibenzylideneacetone)dipalladium; Pd(t-Bu P) ₂ : bis(tri-t-butylphosphine)palladium; TFA: trifluoroacetic acid; pTLC: preparative thin- layer chromatography; HRMS (high resolution mass spectrometry); Tr or Trt (trityl or triphenylmethyl); Bz: benzyl.

Synthesis of N-(3-((4aS,5SJaSV2-amino-5-(triflttoromethylV4a,5,7,7a-tetra hvdro- 4H-furo[3,4-dHl,31thiazin-7a-yl)-4-fluorophenylV5-(fluoromet hyl)pyrazine-2- carboxamide Free Base - Preparation 1 - Scheme 1

General Conditions: 1H NMR spectra were recorded on a Bruker AM series spectrometer operating at a (reported) frequency of 400 MHz. Chemical shifts in proton nuclear magnetic resonance spectra are recorded in δ units (ppm) relative to tetramethylsilane and coupling constants (J) are recorded in Hertz (Hz). Patterns are designated as s: singlet, d: doublet, t: triplet, br: broad. The term "room temperature" typically refers to about 10°C to about 35°C. "%" indicates wt% unless otherwise specified. Chemical names were generated from chemical structures using

ChemBioDraw Ultra 11.0 and 12.0.

1 -(2) fe -butyl ( IT2S)-1 , 1.1 -trifluorobut-3-en-2-ylloxy)acetate

To a suspension of trimethylsulfonium iodide (110 g) in THF (500 mL) at -30°C was added lithium hexamethyldisilazide (530 mL, IN in THF) portionwise over 45 mins. After stirring at -20°C for 20 mins, (S)-2-trifluoromethyloxirane (37.97 g) was added at the same temperature over 15 mins, and the mixture was allowed to warm to RT and stirred for 3 h. The slurry was then added portionwise to an ice-cold solution of tert-butyl bromoacetate (105.68 g) in NMP (200 mL). The resulting mixture was allowed to warm to RT and stir for 2 days, before dilution with EtOAc (1 L). The organic layer was washed with sodium bicarbonate (sat,, aq., 4 x 400 mL), dried over MgS0 ₄ and evaporated. The residue was purified by silica gel column chromatography (5% EtOAc in hexanes) to obtain the title compound (70.1 g) which was used in the subsequent step without purification. 1H-NMR (400 MHz, CDC1 ₃ ) δ (ppm): 1.30 (s, 9 H) 3.83 - 3.96 (m, 2 H) 4.14 - 4.21 (m, 1 H) 5.34 - 5.48 (m, 2 H) 5.56 - 5.71 (m, 1 H)

Scheme 1.

1 -(3) (S)-N-methoxy-N-methyl-2-(( 1 , 1 , 1 -trifluorobut-3 -en-2-yl oxy)acetamide

tert-Butyl {[(2S)-l,l,l-trifluorobut-3-en-2-yl]oxy}acetate (70.1 g, crude) was dissolved in ice-cold formic acid (200 mL). The mixture was allowed to warm to RT and stir overnight. The reaction mixture was then concentrated under reduced pressure, toluene (200 mL) was added, the mixture concentrated, before a second addition of toluene (200 mL) and concentration to an oil. The residue was dissolved in DCM (600 mL), cooled in an ice-bath, and Ν,Ν'-carbonyl diimidazole (35 g) was added portionwise over 20 mins. After stirring for 45 mins, N, O-dimethyl hydroxylamine hydrochloride (22 g) was added, and the reaction mixture was allowed to warm to RT and stirred overnight. Saturated NaHC0 ₃ (500 mL) and brine (250 mL) were then added, and the mixture extracted with EtOAc (3 x 750 mL). The combined organic portions were dried over MgS0 ₄ and evaporated. The residue was purified by silica gel column chromatography (1% to 30% EtOAc in hexanes) to obtain the title compound (25.17 g). 1H-NMR (400 MHz, CDC1 ₃ ) δ (ppm) 3.21 (s, 3 H), 3.71 (m, 3 H), 4.36 - 4.51 (m, 3 H), 5.54 - 5.69 (m, 2 H), 5.84 (ddd, J=17.7, 10.4, 7.3 Hz, 1 H) 1 -(4) (S 1 - (2-fluorophenylV2-( (1.1.1 -trifluorobut-3 -en-2-yl oxy)ethanone

A solution of n-butyllithium in hexane (2.50 M; 90 mL) was added dropwise over 25 mins to a solution of 2-bromofluorobenzene (40.35 g) in THF (250 mL) under a N ₂ atmosphere at -78°C. The reaction solution was allowed to warm to -60°C and stir for 60 min. (S)-N-Methoxy-N-methyl-2-((l,l,l-trifluorobut-3-en-2-yl)oxy) acetamide (40 g) in THF (25 mL) was added dropwise to the reaction solution, and after stirring at -60°C for 2 h, aqueous NH ₄ C1 (100 mL) was added to the reaction solution, followed by warming to RT. Brine (200 mL) was added to the reaction solution, and the mixture was extracted with EtOAc (3 x 400 mL). The combined organic portions were dried over MgS0 ₄ , evaporated, and the residue was purified by silica gel column

chromatography (1% to 10% EtOAc in hexanes) to obtain the title compound (33.59 g).

Ή-NMR (400 MHz, CDC1 ₃ ) δ (ppm): 4.40 (pentet, j 6.3 Hz, 1 H) 4.81 - 4.87 (m, 2 H), 5.54 - 5.69 (m, 2 H), 5.86 (ddd, J 17.4, 10.4, 7.3 Hz, 1 H) 7.12 - 7.22 (m, 1 H) 7.24 - 7.34 (m, 1 H) 7.54 - 7.63 (m, 1 H) 7.94 - 8.02 (m, 1 H).

l-(5) (SV 1 -(2-fluorophenyl -2-(Yl .1.1 -trifluorobut-3 -en-2-yl)oxy)efhanone oxime

(S)-l-(2-Fluorophenyl)-2-((l,l,l-trifluorobut-3-en-2-yl)oxy) ethanone (41.22 g) was dissolved in anhydrous methanol (400 mL) and hydroxylamine hydrochloride (14.0 g) and sodium acetate (19.0 g) were added. The reaction mixture was heated to 50°C for 90 min, then cooled to RT, concentrated in vacuo and the residue purified by silica gel chromatography (2% to 15% EtOAc in hexanes) to afford the title compound as a mixture of geometric isomers (40.54 g). 1H-NMR (400 MHz, CDC1 ₃ ) δ (ppm): 4.04 -

4.15 (m, 0.8 H), 4.18 - 4.26 (m, 0.2 H), 4.44 - 4.57 (m, 0.4 H) 4.79 - 4.90 (m, 1.6 H) 5.37 - 5.56 (m, 2 H) 5.64-5.78 (m, 1 H) 7.03 - 7.26 (m, 2 H) 7.33 - 7.54 (m, 2 H), 7.90 (br s, 0.2 H), 8.51 (br s, 0.8 H).

1-C6 (3aR.4S.6aS)-6a-(2-fluorophenyl)-4-(trifluoromethyl hexahvdrofuror3.4- c]isoxazole

(S)- 1 -(2-fiuorophenyl)-2-(( 1,1,1 -trifluorobut-3 -en-2-yl)oxy)ethanone oxime (40.54 g) was dissolved in xylenes (400 mL) and hydroquinone (4.0 g) was added. The reaction mixture was heated to reflux (heating block temperature 140°C) for 22 h, then cooled and evaporated. The residue was purified by silica gel column

chromatography (1% to 30% EtOAc in hexanes) to obtain the title compound (28.76 g). 1H-NMR (400 MHz, CDC1 ₃ ) δ (ppm): 3.71-3.81 (m, 1 H), 4.04 - 4.35 (m, 4 H), 4.51- 4.62 (m, 1 H), 5.38-5.54 (m, 1 H), 7.07 - 7.26 (m, 2 H), 7.32 - 7.42 (m, 1 H), 7.54-7.67 (m, 1 H).

l-(7 (( ^' 2S,3R,4S -4-amino-4-(2-fluorophenyl -2-(trifluoromethyl)tetrahvdrofuran-3- yOmethanol

(3aR,4S,6aS)-6a-(2-fluorophenyl)-4-(trifluoromethyl)hexahydr ofuro[3,4- cjisoxazole (28.76 g) was dissolved in acetic acid (200 n L) and the solution was cooled to 0°C. Zinc (50 g) was added, and the reaction mixture was allowed to warm and stir at RT for 16h. The reaction mixture was then diluted with EtOAc (500 mL) and filtered through celite, washing with a further 500 mL of EtOAc. The combined organic portions were evaporated, dissolved in chloroform (200 mL), and ammonia (28% aq., 250 mL) was added slowly. The layers were separated, and the aqueous portion was further extracted with chloroform (2 x 250 mL). The combined organic extracts were dried over anhydrous MgS0 ₄ and evaporated to afford the title compound (31.12 g) which was used in the subsequent step without further purification. ¹ H-NMR (400 MHz, CDC1 ₃ ) δ (ppm): 2.93 (ddd, J=7.7, 4.9, 2.5 Hz, 1 H), 3.84 (dd, J=12.4, 4.8 Hz, 1 H), 4.05 (dd, J=9.2, 3.2 Hz, 1 H), 4.17 (dd, J=12.4, 2.3 Hz, 1 H), 4.31 (d, J=9.3 Hz, 1 H), 4.72 (quin, J=7.3 Hz, 1 H), 7.13 (ddd, 7=13.1, 8.8, 1.3 Hz, 1 H), 7.22 (td, J=7.6, 1.3 Hz, 1 H), 7.31 - 7.40 (m, 1 H), 7.51 (td, J=8.0, 1.6 Hz, 1 H)

1-C8 N-(i(3S.4R.5S)-3-f2-fluorophenylV4-ihvdroxymethvn-5- (trifiuoromethyl tetrahydrofuran-3-vncarbamothioyl benzamide

Benzoyl isothiocyanate (19.0 mL) was added to a solution containing

((2S,3R,4S)-4-amino-4-(2-fluorophenyl)-2-(trifluoromethyl )tetrahydrofuran-3- yl)methanol (28.72 g) in DCM (150 mL), and the mixture was stirred at RT for 18 h. Sodium bicarbonate (sat., aq., 200 mL) was then added, the mixture extracted with EtOAc (3 x 300 mL), dried over MgS0 ₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (5% to 30% EtOAc in hexanes) to obtain the title compound (37.07 g). 1H-NMR (400 MHz, CDC1 ₃ ) δ

(ppm): 3.22 (dd, J=8.1, 4.5 Hz, 1 H), 3.31 (td, J=8.0, 3.0 Hz, 1 H), 3.94 - 4.07 (m, 1 H), 4.31 - 4.46 (m, 1 H), 4.53 (d, J=9.9 Hz, 1 H), 4.83 (d, J=9.9 Hz, 1 H), 6.97 - 7.14 (m, 1 H), 7.22 (td, J-7.7, 1.3 Hz, 1 H), 7.31 - 7.45 (m, 1 H), 7.49 - 7.61 (m, 2 H), 7.61 - 7.70 (m, 1 H), 7.75 (td, J=8.1, 1.5 Hz, 1 H), 7.79 - 7.93 (m, 2 H), 8.90 (s, 1 H), 11.85 (s, 1 H) l-(9) N-((4aS,5SJaS -7a-r2-fluoi phenvn-5-itrifluoiOmethyl -4a.5.7.7a-tetrahvdro-4H- furo[3 ,4-d 1 ,31thiazin-2-yl benzamide

N-(((3S,4R,5S)-3-(2-Fluorophenyl)-4-(hydroxymethyl)-5- (trifluoi methyl)tetrahydrofuran-3-yl)carbamothioyl)benzamide (31.1 g) was dissolved in pyridine (150 mL), and the mixture cooled to -20°C. Trifluoromethanesulfonic anhydride (14.0 mL) was added dropwise over 30 min and the reaction was allowed to warm to 0°C. After stirring for 2 h, the reaction was quenched by the addition of ammonium chloride (sat., aq., 400 mL) and extracted with EtOAc (3 x 500 mL). The combined organic extracts were dried over MgS0 ₄ , concentrated in vacuo and purified by silica gel column chromatography (2% to 30% EtO Ac/hex) to obtain the title compound (18.50 g). 1H NMR (400 MHz, CDC13) δ ppm 2.86 (dd, J=13.9, 3.5 Hz, 1H), 3.25 (d, J=13.6 Hz, 1 H), 3.61 (br. s., 1 H), 4.00 - 4.10 (m, 1 H), 4.66 (d, J=8.8 Hz, 1 H), 4.78 - 4.87 (m, 1 H), 7.12 - 7.60 (m, 6 H), 7.68 - 7.73 (m, 1 H), 7.99 - 8.16 (br. s., 2 H), 8.62 - 8.66 (m, 1 H)

1-ilO N-((4aS.5S.7aS -7a-(2-fluorophenyl -5-(trifluoromethvn-4a,5J.7a-tetrahvdro- 4H-furo Γ3.4-d1 Γ 1 ,31thiazin-2-vnbenzamide

N-((4aS,5S,7aS)-7a-(2-Fluorophenyl)-5-(trifluoromethyl)-4a,5 ,7,7a-tetrahydro- 4H-furo[3,4-d][l,3]thiazin-2-yl)benzamide (21.5 g) was dissolved in methanol (160 mL), l,8-diazabicyclo[5.4.0]undec-7-ene (16.29g) was added, and the solution was heated to reflux (heating block temperature 80°C). After 16 h, the reaction mixture was concentrated under reduced pressure, and the residue purified by silica gel column chromatography (10% to 60% EtOAc in hexanes) to afford the title compound (13.82 g). 1H NMR (400 MHz, CDC13) δ ppm 2.85 (dd, J=13.6, 3.8 Hz, 1 H), 3.14 (dd, J=13.5, 3.2 Hz, 1 H), 3.33 - 3.45 (m, 1 H), 3.92 (dd, J=8.1, 2.0 Hz, 1 H), 4.49 (br. s„ 2 H), 4.63

- 4.76 (m, 2 H), 7.08 (ddd, J=12.6, 8.1, 1.0 Hz, 1 H), 7.13 - 7.22 (m, 1 H), 7.25 - 7.36 (m, 1 H), 7.44 (td, J=8.0, 1.9 Hz, 1 H)

1-q 1) (4aS.5S.7aS)-7a-(2-fluoro-5-nitrophenyl -5-rtrifluoromethylV4a,5 ja- tetrahydro-4H-furo 3,4-dl | ^" L31thiazin-2-amine

N-((4aS,5S,7aS)-7a-(2-Fluorophenyl)-5-(trifluoromethyl)-4a,5 ,7,7a-tetrahydro- 4H-furo[3,4-d][l,3]thiazin-2-yl)benzamide (5.15 g) was dissolved in TFA (75 mL), and the solution was cooled to 0°C. Sulfuric acid (cone, 20 mL) was added, followed by fuming nitric acid (2 mL) dropwise over 20 mins. After stirring at 0°C for 90 mins, the reaction mixture was poured onto ice (200 g) and basified to pH 12 with 6N NaOH (aq.). After allowing the ice to melt, the mixture was extracted with EtOAc (3 x 500 mL), and the combined organic portions dried over MgS0 ₄ and evaporated to afford the title compound (22.1 g, purity approx. 71%) which was used in the subsequent step without purification. ^! H NMR (400 MHz, CDC1 ₃ ) δ ppm 2.89 (d, J=3.8 Hz, 1 H), 3.09 (br. s., 1 H), 3.28 - 3.54 (m, 1 H), 3.80 - 4.03 (m, 1 H), 4.50 - 4.70 (m, 3 H), 4.71 - 4.86 (m, 1 H), 7.21 - 7.30 (m, 1 H), 8.18 - 8.28 (m, 1 H), 8.45 (dd, J=6.8, 2.8 Hz, 1 H)

1-(T2 fe -butyl ((4aS,5SJaS -7a-( ^' 2-fluoi -5-nitrophenylV5-(trifluoromethyl)- 4a.5,7,7a-tetrahvdro-4H-furo Γ3 ,4-dl Γ 1.31thiazin-2-yl)carbamate (4aS,5S,7aS)-7a-(2-Fluoro-5-nitroprienyl)-5-(trifluoromethyl )-4a,5,7,7a- tetrahydro-4H-furo[3,4-d][l,3]thiazin-2-amine (20.6 g, crude) was dissolved in THF (300 mL), di-tert-butyl dicarbonate (12 g) was added portionwise over 20 mins and the reaction mixture was heated to 60°C. Further portions of di-tert-butyl dicarbonate (10 g) were added until starting material was consumed by TLC. The reaction mixture was cooled and sodium bicarbonate (sat., aq., 200 mL) and brine (200 mL) were added. The mixture was then extracted with EtOAc (3 x 500 mL) and the combined organic portions were dried over MgS0 ₄ and evaporated. The residue was purified by silica gel column chromatography (10% to 25% EtOAc in hexanes) to afford the title compound (16.62g). 1H NMR (400 MHz, CDC1 ₃ ) δ ppm 1.55 (s, 9 H), 2.73 - 2.84 (m, 1 H), 2.92 - 3.05 (m, 1 H), 3.43 - 3.55 (m, 1 H), 3.81 - 3.94 (m, 1 H), 4.57 (d, J=8.3 Hz, 1 H), 4.73 - 4.83 (m, 1 H), 7.19 - 7.39 (m, 2 H), 8.20 - 8.29 (m, 1 H), 8.32 (d, J=6.8 Hz, 1 H)

l-fl3) fert-butvKi4aS,5SJaS -7a-i5-amino-2-fluorophenvn-5-(trifluoromethvn- 4a,5 JJa-tetrahvdro-4H-furo[3,4-d][l,3]thiazin-2-yl carbamate

rt-Butyl ((4aS,5S,7aS)-7a-(2-fluoro-5-nitrophenyl)-5-(trifluoromethyl )- 4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]thiazin-2-yl)carbama te (16.61 g) was dissolved in ethanol (250 mL) and tin chloride dihydrate (25.0 g) was added. After stirring at RT for 18 h, the solution was poured onto NaOH (2N aq., 300 mL) and celite ^® (-50 g) was added. The resulting mixture was filtered through more celite ^® and extracted with EtOAc (2 x 500 mL). The combined organic portions were dried over MgS0 ₄ and evaporated to afford the title compound (15.52 g). This material could be used crude but a portion was purified by silica gel column chromatography (20% to 50% EtOAc in hexanes) to afford pure material (recovery 79%). ^! H NMR (400 MHz, CDC1 ₃ ) δ ppm 1.53 (s, 9H), 2.77 (d, J=14.4 Hz, 1H), 3.09 (br. s., 1H), 3.46 (br. s., 1H), 3.62 (br. s., 2H), 3.87 (br. s., 1H), 4.61 (d, J=8.6 Hz, 1H), 4.71 (br. s., 1H), 6.61 (br. s., 2H), 6.85 - 6.95 (m, 1H)

N-(3-((4aS,5SJaS -2-ammo-5-itrifluoromethvn-4a,5,7,7a-tetrahvdro-4H-furor3,4- d] [1 lthiazin-7a-yl)-4-fluoiOphenyl -5-(fluoromethyl)pyrazine-2-carboxamide free base

fert-Butyl ((4aS,5S,7aS)-7a-(5-amino-2-fluoiOphenyl)-5-(trifluoromethyl )- 4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]thiazin-2-yl)carbama te (500 mg) was dissolved in DCM (10 mL) and 5-fluoromethyl-pyrazine-2-carboxylic acid (223 mg), N,N- diisopropylethylamine (521 mg) and (lH-benzotriazol-l-yloxy)tripyrrolidin-l- yl)phosphonium hexafluorophophate (750 mg) were added. The reaction mixture was stirred at RT for 1 h, and sodium bicarbonate (sat., aq., 50 mL) was added. The mixture was extracted with EtOAc (2 x 75 mL), the combined organic portions were dried over MgSC , evaporated and purified by silica gel chromatography (0% to 30%

EtOAc/hexanes gradient) to afford the amide (613 mg) as a white solid. The amide was dissolved in DCM (2 mL) and TFA (1 mL) was added. After stirring at RT for 2 h, the reaction mixture was evaporated and sodium bicarbonate (sat., aq., 25 mL) was added. The mixture was extracted with EtOAc (3 x 25 mL), and the combined organic portions dried over MgS0 ₄ and evaporated to afford the title compound as a white solid

1H NMR (400 MHz, CDC1 ₃ ) δ ppm 2.89 (dd, J=13.8, 3.7 Hz, 1 H), 3.21 (dd, J=13.9, 2.5 Hz, 1 H), 3.40 - 3.51 (m, 1 H), 3.96 (d, J=7.3 Hz, 1 H), 4.42 - 4.85 (m, 4 H), 5.69 (d, J= 46.5 Hz, 2 H), 7.15 (dd, J=11.9, 8.8 Hz, 1 H), 7.64 (dd, J=7.1, 2.8 Hz, 1 H), 7.94 - 8.00 (m, 1 H), 8.77 (s, 1 H), 9.47 (s, 1 H), 9.68 (s, 1 H)

Synthesis of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyI)-4a,5,7,7a-tet rahvdro- 4H-furof3,4-dl [l,31thiazin-7a-vn-4-fluorophenyl)-5-(fluoromethyl)pyrazine- 2- carboxamide Free Base -Preparation 2

For Preparation 2, 1H NMR and ¹³ C NMR spectra were recorded on a Varian 400

MHz or 500 MHz instrument with vNMR 6.1C software.

1 -CI 4 ^s ) Synthesis of tert-Butyl 2-fl J,l-trifluorobut-3-en-2-yloxy)acetate

A reaction vessel was charged with toluene (3.2 L), THF (0.60 L) and acrolein

(0.40 L, 5.985 mol) at rt under nitrogen. (Trifluoromethyl)trimethylsilane (1.003 kg, 7.059 mol) was added at 17 °C. The reaction mixture was cooled to 2.5 °C and TBAF (0.01 M in THF, 0.400 L, 0.004 mol) was added over 2 h. During addition of TBAF, the temperature of the reaction mixture increased to 65 °C. The reaction mixture was cooled to 0 °C, and after 2h, tetra-n-butylammonium hydrogen sulfate (0.171 kg, 0.503 mol) was added, followed by tert-butyl bromoacetate (0.987 kg, 5.064 mol). Sodium hydroxide (50% wt in water, 4.2 kg, 52.6 mol) was added over 2h while maintaining the temperature under 10 °C. After 2h at 0-5 °C, to the reaction mixture was added water (2.9 L) and methyl tert-butyl ether (6.0 L). The aq. phase was extracted one more time with methyl tert-butyl ether (6.0 L). The organic phases were combined and washed with 14% aq. NaCl (3 x 1.6 L). The organics were concentrated under vacuum to afford the title compound as an oil (1.150 kg, 94.5%) which was used in the subsequent stage without additional purification. 1H NMR (500 MHz, CDC1 ₃ ) δ ppm 5.86 - 5.74 (m, 1H), 5.59 (d, J= 17.5 Hz, 1H), 5.56 (d, J= 10.9 Hz, 1H), 4.37 - 4.30 (m, 1H), 4.11 (d, J= 16.5 Hz, 1H), 4.06 (d, J- 16.4 Hz, 1H), 1.40 (s, 9H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ ppm 168.51, 128.49 (d, J= 1.7 Hz), 123.86, 123.71 (q, J= 281.8 Hz), 82.22, 78.67 (q, J= 31.5 Hz), 66.60, 28.02.

1 -(15) Synthesis of N-Methoxy-jV-methyl-2-(l , 1 , 1 -trifluorobut-3 -en-2-yloxy)acetamide

stage 2

To a reactor containing tert- butyl 2-( 1 , 1 , 1 -trifluorobut-3 -en-2-yloxy)acetate

(1.150 kg, 4.788 mol) was added formic acid (6.2 kg) at rt. The reaction mixture was heated to 55-60 °C for 4-5 h. The formic acid was evaporated under vacuum (T = 40- 45 °C) and chased with toluene (2 x 3.0 L). To the residue was added CH ₂ C1 ₂ (2.0 L) and further concentrated under vacuum. To the resulting residue was added CH ₂ C1 ₂ (4.6 L) and the solution was cooled to 0 °C, followed by N,N-carbonyldiimidazole (1.05kg, 6.49 mol) in five portions. The mixture was stirred for 30 mins, and Ν,Ο- dimethylhydroxylamine hydrochloride (0.67 kg, 6.72 mol) was added in portions while maintaining temperature below 10 °C. The reaction mixture was warmed to rt and stirred over 14 h. The reaction mixture was cooled to 3.2 °C and imidazole (100.7 g, 1.48 mol) was charged in two portions. The reaction mixture was warmed to rt and water (1.4 kg) was added, followed by methyl fert-butyl ether (14.0 L). The organic phase was washed with 2.0 N aq. HC1 (1.0 L and 0.7 L), followed by sat. aq. NaHC0 ₃ (1.2 L) and sat. aq. NaCl (1.20 L). The organics were concentrated to afford the title compound as an oil (0.95kg, 87.2%). 1H NMR (500 MHz, CDC1 ₃ ) δ ppm 5.85 - 5.76 (m, 1H), 5.62 (d, J= 17.2 Hz, 1H), 5.56 (d, J= 10.4 Hz, 1H), 4.49 - 4.34 (m, 3H), 3.68 (s, 3H), 3.67 (s, 1H), 3.18 (s, 3H), 3.08 (s, 1H); ¹³ C NMR (126 MHz, cdcl ₃ ) δ ppm 169.9*, 163.4*, 128.61, 123.87 (d, J = 282.0 Hz), 123.82, 78.54 (q, J= 31.3 Hz), 66.12, 61.52, 60.56, 36.20, 32.24. Note: this compound is a 3:1 mixture of amide bond rotamers. *Carbonyl chemical shifts estimated indirectly through 1 H- 13 C HMBC (heteronuclear multiple-bond correlation).

HRMS Calculated for C ₈ H ₁₂ F ₃ N0 ₃ [M+H] ⁺ 228.0848; found 228.0836.

1-(16 Synthesis of l-(2-Fluorophenyl -2-(l ,1 ,1 -trifluorobut-3 -en-2-yloxy)ethanone

stage 3

To a solution l-bromo-2-fluorobenzene (0.967 kg, 5.527 mol) in THF (6.2 L) at - 75 °C, was added n-butyllithium (2.50 M in hexane, 2.09 L, 5.22 mol) while

maintaining temperature below -65 °C (ca. 100 min.). After 15 mins, a solution of N- methoxy-N-methyl-2-(l,l,l-trifluorobut-3-en-2-yloxy)acetamid e (0.949 kg, 4.178 mol) in THF (1.6 L) was added while maintaining temperature below -65 °C (ca. 70 min.). After 2.5 h at -78 °C, the reaction was quenched by addition of sat. aq. NH ₄ C1 (3.0 L) and methyl fert-butyl ether (9.0 L). The reaction mixture was warmed to rt, the aq. phase was extracted again with methyl fert-butyl ether (2.5 L). The organic phases were combined, washed with sat. aq. NaCl (2 x 0.3 L) and concentrated under vacuum to afford the title compound as an oil (1.007kg, 80.0%). 1H NMR (500 MHz, CDC1 ₃ ) δ ppm 7.96 (td, J= 7.6, 1.8 Hz, 1H), 7.62 - 7.54 (m, 1H), 7.33 - 7.25 (m, 1H), 7.20 - 7.12 (m, 1H), 5.86 (ddd, J= 17.5, 10.4, 7.3 Hz, 1H), 5.60 (dd, J= 20.5, 13.8 Hz, 2H), 4.91 - 4.76 (m, 2H), 4.39 (dq, J= 12.8, 6.4 Hz, 1H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ ppm 193.55, 162.14 (d, J _CF = 254.1 Hz), 135.36 (d, J _CF = 9.2 Hz), 130.62 (d, J _CF = 3.2 Hz), 128.49, 124.85 (d, J _CF = 3.3 Hz), 123.89, 122.93, 122.72 (d, J _CF - 24.5 Hz), 116.50 (d, J _CF = 23.7 Hz), 78.97 (q, J _CF = 31.4 Hz), 74.56 (d, J _CF = 12.4 Hz).

HRMS Calculated for C ₁₂ H ₁₀ F ₄ O ₂ [M+H] ⁺ 263.0695; found 263.0709.

1-07) Synthesis of l-(2-Fluorophenyl -2-( ^" U J-trifluorobut-3-en-2-yloxy ethanone oxime

To a reactor was added hydroxylamine hydrochloride (0.34 kg, 4.95 mol), sodium acetate (0.47 kg, 5.70 mol) and MeOH (2.68 L). To this suspension was charged a solution of l-(2-fluoiOphenyl)-2-(l,l,l-trifluorobut-3-en-2-yloxy)ethano ne (0.998 kg, 3.806 mol) in MeOH (1.8 L) and the reaction mixture was heated to 40-50 °C. Upon completion (ca. 2 h) the reaction mixture was cooled to rt, and filtered over Celite (0.5 wt) and rinsed with EtOAc (3.0 L). The filtrate was concentrated under vacuum and to the resulting residue was added methyl fert-butyl ether (6.3 L), water (0.94 L) and sat. aq. NaHC0 ₃ (2.5 L). The organic phase was washed once with water (1.6 L) and sat. aq. NaCl (0.1 L). The organic phase was concentrated under vacuum to afford the title compound as an oil (1.03 kg, 95.0%). 1H NMR (500 MHz, CDC1 ₃ ) δ ppm 7.49

- 7.35 (m, 2H), 7.24 - 7.06 (m, 2H), 5.78 - 5.65 (m, 1H), 5.54 - 5.40 (m, 2H), 4.89 - 4.81 (m, 1H), 4.53 (d, J= 12.6 Hz, 1H), 4.47 (d, J= 12.6 Hz, 0.5H), 4.27 - 4.18 (m, 1H), 4.13 - 4.05 (m, 0.5H).

HRMS Calculated for C ₁₂ HnF ₄ N0 ₂ [M+H] ⁺ 278.0804; found 278.0780.

Note: l-(2-Fluorophenyl)-2-(l,l,l-trifluorobut-3-en-2-yloxy)ethano ne oxime exists as an equilibrium of structural isomers, which accounts for the less-than- whole-number integral values.

1-(18 Svntheis of GaR*,4S*.6aS*V6a-(2-fluoiOphenylV4-

oxime (1.085 kg, 3.328 mol) in xylenes (6.9 L) was added hydroquinone (86.2g, 0.8 mol) at rt. The solution was heated to 128 °C (internal temperature) for 18 h. The solution was cooled to rt, and hexanes (7.0 L) was added, followed by 4.0 M aq. HCl (2.4 L). The reaction mixture was stirred for 1 h, and filtered. To the solid was added water (2.0 L), methyl teri-butyl ether (7.0 L) and 25% wt. aq. NaOH (0.4 L).

The aq. layer was extracted once with methyl tert-butyl ether (7.0 L), the organics were combined, washed with 27% aq. NaCl (2.0 L) and concentrated under vacuum to a black oil (512.0g, 56%). 1H NMR (500 MHz, CDC1 ₃ ) δ ppm 7.64 - 7.52 (m, 1H), 7.39 - 7.31 (m, 1H), 7.19 (td, J= 7.7, 1,2 Hz, 1H), 7.11 (ddd, J= 11.9, 8.2, 1.0 Hz, 1H), 4.54 (d, J- 10.1 Hz, 1H), 4.34 - 4.23 (m, 1H), 4.26 - 4.17 (m, 1H), 4.16 (d, J= 10.2 Hz,

1H), 4.10 (d, J= 8.5 Hz, 1H), 3.71 (d, J= 20.2 Hz, 1H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ ppm 160.59 (d, J _CF = 247.0 Hz), 130.50 (d, J _CF = 8.7 Hz), 128.72, 124.69 (d, J _CF = 3.3 Hz), 124.45 (q, J _CF = 281.8 Hz), 124.43 (d, J _CF = 11.9 Hz), 116.66 (d, J _CF = 22.7 Hz), 83.70 (q, J _CF = 32.1 Hz), 78.17 (d, J _CF = 3.1 Hz), 77.63. 54.53.

HRMS Calculated for C ₁₂ HnF ₄ N0 ₂ [M+H] ⁺ 278.0804; found 278.0802.

1-(T9 Synthesis of ((2S ^;i! .3R*.4S*V4-amino-4-( ^' 2-fluorophenyl -2-

racemic

Zinc (389.2 g, 5.95 mol) was placed in a reaction vessel, and water was added (893 mL). Acetic acid (135 mL, 2.38 mol) was added while maintaining the temperature below 10 °C. After 15 min, 6a-(2-fluorophenyi)-4- (trifluoromethyl)hexahydrofuro[3,4-c]isoxazole (550.0 g, 1.98 mol) was added as a solution in THF (665 mL). The reaction mixture was stirred over 16 h at rt.

Methylene chloride (1.89 L) was added, followed by 28% aq. NH ₄ OH (552 mL) while the temperature was kept below 30 °C. The mixture was stirred for 30 min, and then filtered over Celite (80 g) rinsing with methylene chloride (378 mL). The aq. layer was extracted with methylene chloride (1.89 L). The organics were combined, washed with sat. aq. NaCl (1.0 L) and concentrated under vacuum to afford an oil (502 g, 90.6%). The crude residue was used in the following step without additional purification.

HRMS Calculated for C ₁₂ H ₁₃ F ₄ N0 ₂ [M+H] ⁺ 280.0961; found 280.0972.

l-(20 Synthesis of (i ',ii?,^-4-Amino-4-(2-fluorophenyl)-2-

a solution of 4-amino-4-(2-fluorophenyl)-2-(trifluoromethyl)tetrahydrofura n-

3-yl)methanol (0.502 kg, 1.798 mol) in ethanol (4.865 L) was added dibenzoyl-D- tartaric acid (0.642 kg, 1.798 mol). The resulting suspension was heated to 67 °C. Water (94.0 mL, 5.2 mol) was added over 15 min while maintaining temperature >66 °C. The resulting solution was cooled to to 45 °C while precipitation occurred. The slurry was reheated to 60 °C, and then cooled to ambient temperature at 5 °C/hour. The slurry was filtered, and the solid was rinsed with premixed and cooled solution of ethanol (950 mL) and water (20 mL). The solid was dried until constant weight under vacuum (370 g, 97.6% ee). 1H NMR (500 MHz, CD ₃ OD) δ ppm 8.13 (d, J- 7.2 Hz, 4H), 7.66 - 7.58 (m, 3H), 7.54 - 7.45 (m, 5H), 7.36 - 7.20 (m, 2H), 5.92 (s, 2H), 4.79 - 4.66 (m, 1H), 4.40 - 4.28 (m, 1H), 4.04 (dd, J= 12.1, 3.4 Hz, 1H), 3.92 (dd, J= 12.1, 5.4 Hz, 1H), 3.30 - 3.24 (m, 1H); ¹³ C NMR (125 MHz, DMSO) δ ppm 169.61, 165.81, 160.23 (d, J= 246.1 Hz), 133.00, 131.34 (d, J= 9.1 Hz), 129.65, 129.55, 128.08, 127.97 (d, J= 3.5 Hz), 124.95 (d, J= 3.3 Hz), 116.56 (d, J = 23.5 Hz), 77.48 (q, J _CF = 31.0 Hz), 76.33, 73.20, 65.61 (d, J= 3.1 Hz), 57.11.

HRMS Calculated for d ₂ Hi ₃ F ₄ N0 ₂ [M+H] ⁺ 280.0961 ; found 280.0967 (for amino alcohol).

The absolute stereochemistry of the title compound was assigned by comparison with a sample prepared starting from enantioenriched (S)-2-(trifluoromethyl)oxirane. Chiral HPLC parameters:

Equipment, Reagents, and Mobile Phase:

Equipment:

Mobile Phase:

Add 70 mL 2-propanol and 930 mL heptane (measured separately with a 100 mL and 1000-mL graduated cylinders) and 1.0 mL triethylamine (measured with volumetric glass pipette) to an appropriate flask and mix. Degas in-line during use. Diluting Solution: 2-Propanol

HPLC Parameters:

Column Maximum Pressure: 35 Bar

Needle Wash: 2-propanol

*Flow rate may be adjusted ± 0.2 ml/min to obtain specified retention times.

1 -(21) Synthesis of N-((3&^.5S -3-(2-fluorophenvn-4-(hvdiOxymethyl -5-

To chiral salt ((2 ₁ S ^, ,5i?, 5 -4-Amino-4-(2-fluorophenyl)-2- (trifluoromethyl)tetrahydrofuran-3 -yl)methanol (2S, 3S)-2,3 -bis(benzoyloxy)succinate (0.361 kg, 0.556 mol) was added EtOAc (1.08 L) and the suspension was cooled to - 3 °C. 1.0 N aq. NaOH (1.30 L) was added over 20 mins while maintaining T < 5 °C. After 5 mins, benzoyl isothiocyanate (80.0 mL, 594 mmol) was added over 8 mins while maintaining T < 5 °C. After 1 h, EtOAc (722 mL) was charged. The aq. layer was removed, and the organics were washed with sat. aq. NaHC0 ₃ (361 mL) and sat. aq. NaCl (361 mL). The organics were filtered over celite (90 g) and rinsed with EtOAc (360 mL). The organics were concentrated under vacuum to afford a residue which was re-dissolved into CH ₂ C1 ₂ (1.1 L) and concentrated to afford the title compound as yellow foam (261 g, 99% _> yield accounting for residual solvents) which was used in the following step. 1H NMR (500 MHz, DMSO) δ ppm 12.04 (s, 2H), 11.20 (s, 2H), 7.95 (d, J= 7.4 Hz, 2H), 7.69 - 7.60 (m, 1H), 7.56 - 7.42 (m, 2H), 7.37 - 7.28 (m, 1H), 7.24 - 7.12 (m, 2H). 5.59 (t, J= 4.5 Hz, 1H), 5.03 (d, J= 9.7 Hz, 1H), 4.92 (d, J= 9.7 Hz, 1H), 4.75 - 4.63 (m, 1H), 3.92 - 3.74 (m, 2H), 2.77 - 2.66 (m, 1H); ¹³ C NMR (125 MHz, DMSO) δ ppm 179.98, 167.85, 159.75 (d, J _CF = 245.0 Hz), 133.44, 132.58, 129.88, 129.81, 129.04, 128.85, 126.31 (d, J _CF = 9.8 Hz), 124.36, 116.83 (d, J _C F = 23.4 Hz), 76.11 (q, J _CF = 31.0 Hz). 74.37 (d, J _CF = 6.1 Hz), 68.77 (d, J _CF = 3.4 Hz), 57.03, 52.23.

HRMS Calculated for C ₂₀ H ₁₈ F ₄ N ₂ O ₃ S [M+H] ⁺ 441.0896; found 441.0818.

22) Synthesis of N-((4aS, 5.9 a^-7a-(2-fluorophenylV5-(trifluorometfaylV4a,5J.7a-

A solution of N-((3S, 4R, 5S)-3 -(2-fluorophenyl)-4-(hydroxymethyl)-5- (trifluoromethyl)-tetrahydrofuran-3-ylcarbamothioyl)benzamid e (258.3 g, 583.8 mmol) in CH ₂ C1 ₂ (1.55 L) was cooled to -19.4 °C. Pyridine (118 mL, 1.46 mol) was added while maintaining temperature at -20 °C, and then the reaction mixture was cooled to - 24 °C. In another nitrogen purged vessel, CH ₂ C1 ₂ (258 mL) was added followed by trifluoromethanesulfonic anhydride (108.0 mL, 642.2 mmol). The resulting solution was added to the reaction mixture over 30 min, while maintaining temperature < - 19.7 °C. Upon completed addition, the reaction mixture was stirred for 30 min at - 20 °C to -15 °C, and then warmed to -11 °C over 20 min. Saturated aq. NH ₄ C1 (646 mL) and water (390 mL) was added. The mixture was warmed to ambient temperature and the aq. layer was removed. The organics were washed with premixed saturated aq. NH ₄ C1 (646 mL) and water (390 mL). The aq. layers were combined, and extracted once with CH ₂ C1 ₂ (520 mL). The organics were combined, and concentrated under vacuum to afford a light orange foam (250 g, 100%). The residue was used in the next stage without purification. 1H NMR (500 MHz, CDC1 ₃ ) δ ppm 8.03 (d, J= 6.7 Hz,

2H), 7.52 (t, J= 7.0 Hz, 1H), 7.48 - 7.31 (m, 4H), 7.20 (t, J= 7.4 Hz, 1H), 7.12 (dd, J= 12.0, 8.4 Hz, 1H), 4.82 - 4.73 (m, 1H), 4.60 (d, J= 8.9 Hz, 1H), 4.03 (d, J= 8.3 Hz, 1H), 3.57 (d, J= 2.7 Hz, 1H), 3.20 (d, J= 13.6 Hz, 1H), 2.81 (dd, J= 13.8, 2.5 Hz, 1H); ¹³ C NMR (125 MHz, CDC1 ₃ ) δ ppm 171.50, 159.57 (d, J _CF = 247.2 Hz), 134.62, 132.49, 130.65 (d, JCF J= 8.8 Hz), 129.77, 128.51, 128.45, 125.14 (q, J _CF = 281.8 Hz), 124.97 (d, JCF = 3.0 Hz), 124.66 (d, J _CF = 10.3 Hz), 117.05 (d, J _CF = 23.5 Hz), 66.81 (d, J _CF = 5.2 Hz), 38.90, 23.20.

HRMS Calculated for C ₂₀ Hi ₆ F ₄ N ₂ O ₂ S [M+H] ⁺ 425.0947; found 425.0945.

1-Γ23) Synthesis of (4ag.5^7a ₁ S ^f )-7a-i2-fluoiOphenyl -5-(trifluoromethyn-4a.5,7,7a- tetrahvdro-4H-furor3,4-d1 Π .31thiazin-2-amine

To a solution of N-((4aS, 5S,7a5 -7a-(2-fiuoiOphenyl)-5-(trifluoromethyl)- 4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]thiazin-2-yl)benzami de (250.2 g, 589.5 mmol) in methanol (1.25 L) was added K ₂ C0 ₃ (81.5 g, 590.0 mmol). The suspension was heated to 65 °C for 6 h. Upon cooling to ambient temperature, the solvent was evaporated under vacuum. To the resulting residue, was added 1.0 N aq NaOH (1.18 L) and THF (502 mL). The heterogeneous mixture was heated to 45 °C for 1 h. The mixture was cooled to ambient temperature, and EtOAc (1.38 L) was added. The aqueous layer was extracted with EtOAc (0.75 L). The organics were combined, washed with saturated aq. NaHC0 ₃ (500 mL) and saturated aq. NaCl (500 mL). The organics were concentrated under vacuum to afford the title compound as a brown oil (184.1 g, 91.6 % yield accounting for residual solvents). 1H NMR (500 MHz, DMSO) δ ppm 7.49 - 7.42 (m, 1H), 7.40 - 7.33 (m, 1H), 7.26 - 7.15 (m, 2H), 6.26 (s, 2H), 4.77 - 4.54 (m, 1H), 4.40 (d, J= 8.0 Hz, 1H). 3.80 (dd, J= 7.9, 2.3 Hz, 1H), 3.24 - 3.17 (m, 1H), 3.00 (dd, J= 13.9, 3.2 Hz, 1H), 2.85 (dd, J= 13.9, 3.9 Hz, 1H); ¹³ C NMR (125 MHz, DMSO) δ ppm 159.75 (d, J _CF = 245.1 Hz), 149.51, 131.31 (d, J _CF = 3.9 Hz), 130.13 (d, JCF = 8.8 Hz), 128.08 (d, J _CF = 10.4 Hz), 128.28 (q, J _CF = 282.1 Hz). 124.87 (d, JCF = 3.0 Hz), 116.80 (d, J= 23.8 Hz). 78.77, 76.80 (q, J _CF = 30.8 Hz), 66.31, 36.37, 23.27.

HRMS Calculated for C ₁₃ H ₁₂ F ₄ N ₂ OS [M+H] ⁺ 321.0685; found 321.0677.

1 -(24) Synthesis of i4ag.5^7a5 ^f )-7a-i2-fluoro-5-nitrophenyl)-5-(trifluoromethyl -

4a,5,7Ja-tetrahydro-4H-furo 3,4-dirL31thiazin-2-amine hydrochloride

To a cooled vessel containing (4aS',55',7a ₁ S)-7a-(2-fluorophenyl)-5- (trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]th iazin-2-amine (184.1 g, 574.8 mmol) was added trifluoroacetic acid (0.954 kg) in portions while the temperature was maintained below 20 °C. The mixture was cooled to 3.5 °C and sulfuric acid (146 mL, 2.73 mol) was added over 20 min while the temperature was maintained below 5 °C. Fuming nitric acid (39.8 mL, 0.948 mol) was added over 30 min, while the temperature was maintained below 10 °C. After 1.5 h at 0-10 °C, the reaction mixture was slowly quenched by transferring into an aq. solution of NaOH (575 g, 14.4 mol) in water (4.6 L) cooled to 5 °C. The resulting suspension was stirred for 1 h at 21 °C. The suspension was then filtered and the solid rinsed with cold water (920 mL). The solid was dried under vacuum until constant weight, and then dissolved into ethanol (1.05 L). The solution was heated to 35 °C, and cone. HCl (55.6 mL, 0.690 mol) was added while maintaining temperature below 40 °C. The suspension was then cooled to -5 °C, held for 1 hr and filtered. The solid was rinsed with cold ethanol (420 mL) and dried until constant weight to obtain the title compound (185.0 g, 87.3%). ^! H NMR (500 MHz, DMSO) δ ppm 11.80 (s, 2H), 8.45 - 8.36 (m, 1H), 8.31 (dd, J= 6.6, 2.5 Hz, 1H), 7.66 (dd, J= 11.1, 9.3 Hz, 1H), 4.96 - 4.72 (m, 1H), 4.58 (d, J= 10.0 Hz, 1H), 4.27 (d, J= 9.9 Hz, 1H), 3.76 - 3.66 (m, 1H), 3.39 (dd, J= 14.9, 3.6 Hz, 1H), 3.24 (dd, J= 14.3, 4.6 Hz, 1H); ¹³ C NMR (125 MHz, DMSO) δ ppm 168.34, 163.33 (d, J _CF = 257.8 Hz), 144.58, 127.61 (d, J _CF = 11.6 Hz), 125.84, 124.10, 1 19.28 (d, J _CF = 26.5 Hz), 77.38 (q, JCF = 31.5 Hz), 75.99, 65.88 (d, J _CF = 4.8 Hz), 40.36, 23.98.

HRMS Calculated for C ₁₃ HnF ₄ N ₃ 0 ₃ S [M+H] ⁺ 366.0536; found 366.0523.

\-(25) Synthesis of (4aiS',5 _l S',7a ₎ S ^r )-7a-(5-amino-2-fluorophenyl)-5-(trifluoromethyl)- 4a,5,7.7a-tetrahvdro-4H-furo[ ^" 3,4-dl[ ^" l,31thiazin-2-amine

Ethanol (0.975 L) was added to iron powder (62.5g, 1.12 mol) under nitrogen atmosphere. Concentrated HCl (9.03 mL) was added at ambient temperature and the suspension was heated to 65 °C for 1.5 h. The suspension was then cooled to 50 °C, and sat. aq. NH ₄ C1 (299 g) were added. The temperature of the reaction mixture was allowed to reach 50 °C, and (4aS,5S,7aS)-7a-(2-fluoro-5-nitrophenyl)-5- (trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]th iazin-2-amine

hydrochloride (75.0 g, 187.0 mol) was added in portions while maintaining

temperature below 68 °C. After 30 min, ethanol (0. 45 L) was added, and the reaction mixture was cooled to 20-25 °C over 1 h. The suspension was stirred for 2 h and filtered over Celite (75 g) rinsing with ethanol (0.972 L). The solution was

concentrated under vacuum to a brown solid. Water (0.9 L) was added followed by 3.0 N NaOH (0.187 L, 560 mmol) while maintaining temperature below 35 °C. The resulting suspension was stirred for 1 h at 20-25 °C. The suspension was filtered, and the solid was rinsed with cold water (0.38 L). The solid was dried under vacuum at 40-45 °C over 24 h to obtain the title compound (57.7 g, 95.5 %). 1H NMR (500 MHz,

DMSO) δ ppm 6.81 (dd, J= 12.5, 8.6 Hz, 1H), 6.62 (dd, J= 7.0, 2.9 Hz, 1H), 6.50 - 6.42 (m, 1H), 6.16 (s, 2H), 4.96 (s, 2H), 4.72 - 4.54 (m, 1H), 4.35 (d, J= 7.8 Hz, 1H), 3.74 (dd, J= 7.8, 2.5 Hz, 1H), 3.18 - 3.08 (m, 1H), 3.01 (dd, J= 13.9, 3.0 Hz, 1H). 2.84 (dd, J= 13.8, 3.8 Hz, 1H); ¹³ C NMR (125 MHz, DMSO) δ ppm 156.20 (d, J _CF = 243.0 Hz), 148.73, 145.49, 127.86 (d, J _CF = 11.0 Hz), 116.79 (d, J _CF = 24.8 Hz), 116.10 (d, J _CF = 3.3 Hz), 114.10 (d, J _CF = 8.0 Hz), 78.89, 76.57 (q, J _CF = 31.0 Hz), 66.35, 36.35, 23.11. HRMS Calculated for C ₁₃ H ₁₃ F4N ₃ OS [M+H] ⁺ 336.0794; found 336.0789.

N-( ^' 3-((4aS.5S.7aSV2-amino-5-rtrifluoromethvn-4a,5,7,7a-te trahvdro-4H-furo( ^" 3,4- dl Γ 1 ,31thiazin-7a-yl -4-fluoiOphenyl -5-(fluoromethyl ^' )pyrazine-2-carboxamide Free Base

(4aS,5S,7aS)-7a-(5-amino-2-fluoiOphenyl)-5-(trifluoromethyl) -4a,5,7,7a-tetrahydro-4H- furo[3,4-d][l,3]thiazin-2-amine (70.0 g, 1.0 equiv) were charged to a reactor and ethyl acetate (EtOAc, 630 mL) was added to the mixture to give a suspension. A solution of n-propane phosphonic acid anhydride (T3P, 146 g, 1.10 equiv, 50 wt% in EtOAc) was added at ambient temperature while controlling the internal temperature below 30 °C. The reaction mixture was stirred at 40-45 °C >3 hours and monitored by HPLC. The reaction mixture was cooled to 15-20 °C and water (140 mL) was charged. After 10-15 minutes charged 28% ammonium hydroxide (175 mL) while controlling the temperature below 30 °C. EtOAc (245 mLO was added and the reaction mixture was stirred for 30 minutes at ambient temperature. The aqueous phase was separated and back-extracted with EtOAc (490 mL). The organic phases were combined and washed with 15% aq. NaCl (140 mL) and water (140 mL). The organic layer was filtered over Celite (1.0 Wt) and rinsed with EtOAc (140 mL). The solution was concentrated under vacuum to obtain a beige solid (quantitative crude yield) which was recrystallized from 1-propanol to afford N-(3-((4aS,5S,7aS)-2-amino-5-(trifiuoromethyl)-4a,5,7,7a-tet rahydro-4H- furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorometh yl)pyrazine-2-carboxamide as a white solid (70.0 g).

1H NMR (500 MHz, DMSO) δ 10.89 (s, 1H), 9.30 (s, 1H), 8.89 (s, 1H), 7.95 (dd, J= 7.3, 2.7 Hz, 1H), 7.94 - 7.89 (m, 1H), 7.21 (dd, J= 12.0, 8.8 Hz, 1H), 6.22 (s, 2H), 5.71

(d, J= 46.3 Hz, 2H), 4.77 - 4.61 (m, 1H), 4.37 (d, J= 8.1 Hz, 1H), 3.87 (dd, J= 8.0, 2.7 Hz, 1H), 3.20 (dt, J= 7.0, 3.5 Hz, 1H), 3.15 (dd, J= 13.9, 3.1 Hz, 1H), 2.91 (dd, J= 13.8, 3.8 Hz, 1H). ¹³ C NMR (126 MHz, DMSO) δ 161.32 (s), 155.82 (d, J= 243.4 Hz), 153.71 (d, J= 18.7 Hz), 148.77 (s), 144.71 (d, J= 1.9 Hz), 143.30 (s), 141.01 (d, J= 5.6 Hz), 134.36 (d, J= 2.0 Hz), 128.20 (d, J= 12.1 Hz), 125.57 (q, J= 283.0 Hz), 123.12 (d, J= 3.6 Hz), 121.64 (d, J= 8.6 Hz), 116.35 (d, J= 25.2 Hz), 82.55 (d, J= 165.8 Hz), 78.37 (s), 76.44 (q, J= 30.6 Hz), 65.89 (d, J= 5.3 Hz), 35.89 (s), 23.01 (s).

HRMS Calculated for Ci ₉ H ₁₇ F ₅ N ₅ 0 ₂ S [M+H] ⁺ 474.1023; found 474.1032.

Specific Optical Rotation: [a] _D ²⁰ = +102.4

Analysis of different batches of free base material recrystallized from 1-propanol revealed that two different polymorphs of the free base could be prepared.

Recrystallisation procedures that resulted in the preparation of i) free base Form B polymorph and ii) free base Form A polymorph, are described below. Physico-chemical properties of the Form A and Form B polymorphs, including XRPD and hygroscopicity measurements, are given in the analysis section.

i) Free base Form B polymorph: Crude free base (1.56 g) was suspended in 13.3 mL 1-propanol and heated to 90 °C. The solution was cooled to 0 °C and filtered. The solid was rinsed with 1-propanol (3.9 mL) and dried under vacuum to afford 1.23g of a solid.

ii) Free base Form A polymorph: Crude free base (10.4 g) was suspended in 27.5 mL of 1-propanol and heated to 90-95 °C. The mixture was cooled to ambient temperature and held for more than 15 hours. The solid was filtered and rinsed with 1- propanol (9.15 mL) and dried under vacuum to afford 4.1 g of a solid.

Alternative Procedure for Preparing N-(3-((4aS,5S,7aS)-2-amino-5- itrifluoroniethvl)-4a _< 5 _< 7,7a-tetrahvdro-4H-furo[3,4-dl [l,31thiazin-7a-vl)-4- fluorophenyI)-5-(fluoromethyl)pyrazine-2-carboxamide free base Form A

Polymorph.

(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(trifluoromethyl) -4a,5,7,7a- tetrahydro-4H-furo[3,4-d][l,3]thiazin-2-amine (1.35 kg, 4.026 mol) was azeotropically dried under rotary evaporation with EtOAc (2.7 L) and toluene (2.7 L) and then dissolved with EtOAc (4.05 L). The solution was transferred to another reactor with 5- (fluoromethyl)pyrazine-2-carboxylic acid (0.660 kg, 4.227 mol). The originating vessel and lines were rinsed with EtOAc (6.75 L). To the stirred suspension under nitrogen, was charged n-propane phosphonic acid anhydride (T3P, 2.82 kg, 4.429 mol, 50 wt % in EtOAc) with temperature below 30 °C. The mixture was heated to 45 °C and held for ca 1.5 hours. The mixture was cooled to 30 °C and water (2.7 L) was added. The mixture was further cooled to 0-10 °C. EtOAc (3.38L) and 3.0N aq NaOH (5.37 L, 16.104 mol) were added with temperature below 30 °C to achieve a pH ~10. The bottom aqueous phase was back-extracted with EtOAc (4.73 L). The organics were combined and concentrated under vacuum to afford crude N-(3-((4aS,5S,7aS)-2-amino- 5 -(trifluoromethyl)-4a,5 ,7,7a-tetrahydro-4H-furo [3 ,4-d] [ 1 ,3]thiazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide as a beige solid (1.95 kg,

100%). To the crude N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a- tetrahydiO-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl) -5-(fluoromethyl)pyrazine- 2-carboxamide (20.0 g, 42.24 mmol) was charged 1-propanol (160 mL). The suspension was heated to 88-92 °C to form a solution. The solution was then cooled to -10 to 0 °C at 10-15 °C and held for 1 hour. The suspension was filtered and the solids were rinsed with cold 1-propanol (40 mL). The solid was dried under vacuum and collected to afford crystalline N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro- 4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorom ethyl)pyrazine-2- carboxamide (15.8 g) as the Form A polymorph.

In the following preparations the choice of free base polymorph used as starting material is not critical as it will undergo dissolution in the reaction solvent. Synthesis of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethylV4a,5,7,7a-tetr ahydro- 4H-furo[3,4-dUl,31thiazin-7a-yl)-4-fluorophenyl)-5-(fluorome thvI)pyrazine-2- carboxamide free base hydrate

N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H- furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorometh yl)pyrazine-2-carboxamide free base (38.3 mg) was dispersed in 0.3 mL-volume of methanol/water (1 : 1, v/v) and then stirred with a magnetic stirrer for four days at room temperature. The solid of the title compound was collected by filtration through a membrane filter (0.2 um pore diameter).

1H-NMR (600 MHz, DMSO-</ ₆ ) δ (ppm): 2.90 (dd, J=14, 4 Hz, IH), 3.14 (dd, J=14, 3 Hz, IH), 3.19 (m, IH), 3.86 (dd, J-8, 2 Hz, IH), 4.36 (d, J=8 Hz, IH), 4.67 (dq, J=7, 7 Hz, IH), 5.71 (d, J=46 Hz, 2H), 6.22 (s, 2H), 7.21 (dd, J=12, 9 Hz, IH), 7.91 (m, IH), 7.95 (dd, J=7, 3 Hz, IH), 8.89 (s, IH), 9.29 (s, IH), 10.90 (s, IH).

Synthesis of N-(3-((4aS,5S,7aS -2-amino-5-rtrifluoromethylV4a,5,7,7a-tetrahvdro- 4H-furo|3,4-dl|l ,31thiazin-7a-vn-4-fluoronhenvl)-5-(fluoromethvl)i)vrazine-2 - carboxamide mono-malonate anhydrate - Preparation A

N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoiOmethyl)-4a,5,7,7a-tet rahydiO-4H- furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorometh yl)pyrazine-2-carboxamide free base (202.5 mg, Form B polymorph) and malonic acid (46.2 mg) were completely dissolved in ethanol (5mL) at room temperature. Crystallization was initiated when the solution was sonicated. The solution or suspension was stirred with a magnetic stirrer for three days to complete the crystallization. The white solid of the title compound was collected by filtration through a membrane filter (0.2 um pore diameter). 1H-NMR (600 MHz, OMSO-d ₆ ) δ (ppm): 3.00 (brd, J=14 Hz, IH), 3.09 (s, 2H), 3.21 (dd, J=14, 3 Hz, IH), 3.29 (m, IH), 3.96 (brd, J=8 Hz, IH), 4.40 (d, J=8 Hz, IH), 4.71 (dq, J=6, 6 Hz, IH), 5.71 (d, J=46 Hz, 2H), 6.90 (brs, 2H), 7.25 (dd, J-l 1, 9 Hz, IH), 7.98 (m, 2H), 8.89 (s, IH), 9.30 (s, IH), 10.93 (s, IH), 13.06 (brs, IH).

Synthesis of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethvI)-4a,5,7,7a-tet rahvdro- 4H-fui of3.4-dl[l,31thiaziii-7a-vl)-4-fluoi oT)licnvl)-5-(fluoromethvl)nvrazinc-2- carboxamide mono-malonate anhydrate - Preparation B

To a reaction vessel was charged N-(3-((4aS,5S,7aS)-2-amino-5- (trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]th iazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide free base (1.84 g, 3.89 mmol, (Form B polymorph) and 2-propanol (18.4 niL). The suspension was heated up to 75- 80 °C. A solution of malonic acid (0.45 g, 4.27 mmol) in 2-propanol (3.68 mL) was added. The resulting suspension was cooled to 20 °C. After 1 hour at 20 °C the suspension was filtered and the solid was rinsed with 2-propanol (3.68 mL). The solid was dried until constant weight to afford the title compound (2.10 g, 94%).

1H NMR (400 MHz, DMSO) δ 10.93 (s, IH), 9.26 (s, IH), 8.85 (s, IH), 7.99 - 7.94 (m, 2H), 7.51 (br s, IH), 7.23 (dd, J= 11.6, 8.4 Hz, IH), 5.67 (d, J= 46.4 Hz, 2H), 4.73 - 4.66 (m, IH), 4.39 (d, J= 8.8 Hz, IH), 3.97 (dd, J= 8.8, 1.6 Hz, IH), 3.31 (dt, J= 7.6, 3.8 Hz, IH), 3.19 (dd, J= 14.0, 3.2 Hz, IH), 3.02 (s, 2H), 3.01 (dd, J= 14.0, 4.0 Hz, IH). The 1H NMR were recorded on a Varian 400 Mhz instrument with vNMR 6.1C software. The malonic acid content was confirmed to be approximately 1 : 1 by ion chiOmatography (IC) with electrical conductivity detection, and by single crystal X-ray analysis.

Synthesis of N-(3-((4aS,5S,7aS)-2-amino-S-(trifluoromethyl)-4a,5,7,7a-tet rahydro- 4H-furof3,4-dl [l,31thiazin-7a-yl)-4-fluorophenyI)-5-(fluoromethyl ^' )pyrazine-2- carboxamide mono-hippurate anhydrate - Preparation A

N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydiO-4H- furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorometh yl)pyrazine-2-carboxamide free base (135 mg) and hippuric acid (52 mg) were completely dissolved in 2-propanol (4 mL) with heating to 70 °C for around an hour and then the solution or suspension was cooled down to room temperature and stirred overnight with a magnetic stirrer. The white solid was collected by filtration through a membrane filter (0.2 um pore diameter) and then dried at 60 °C for 2 hours. The recovery weight of the title compound was 178 mg.

1H-NMR (600 MHz, DMSO-<¾) δ (ppm): 2.90 (dd, J=14, 4 Hz, IH), 3.14 (dd, J=14, 3 Hz, IH), 3.19 (m, IH), 3.87 (dd, J=8, 2 Hz, IH), 3.91 (d, J=6 Hz, 2H), 4.36 (d, J=8 Hz, 1H), 4.67 (dq, J=7, 7 Hz, 1H), 5.71 (d, J=46 Hz, 2H), 6.25 (brs, 2H), 7.21 (dd, J=12, 9 Hz, 1H), 7.47 (dd, J=7, 7 Hz, 2H), 7.54 (t, J=7 Hz, 1H), 7.86 (d, J=7 Hz, 2H), 7.91 (m, 1H), 7.95 (dd, J=7, 3 Hz, 1H), 8.79 (t, J=6 Hz, 1H), 8.89 (s, 1H), 9.29 (s, 1H), 10.90 (s, 1H), 12.58 (brs, 1H).

Synthesis of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahvdro- 4H-furo[3,4-d1 [l,31thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine -2- carboxamide mono-hippurate anhydrate - Preparation B

To a reactor was charged N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)- 4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l ,3]thiazin-7a-yl)-4-fluorophenyl)-5-

(fluoromethyl)pyrazine-2-carboxamide (crude free base 118.8g, 1.0 equiv., Form A polymorph) and 2-propanol (1.4 L). The mixture was heated to 80-82 °C. Hippuric acid (0.416 Wt, 1.1 equiv.) was charged as a solid. The mixture was stirred at 80-82 °C for 30 minutes and then cooled to 17-22 °C. The slurry was filtered and the solid was rinsed with 2-propanol (120 mL). The solid was dried under vacuum at 45 °C to obtain the hippurate salt as a solid in 95% yield (156g).

The hippuric acid content was confirmed to be approximately 1 :1 by ion chromatography (IC) with electrical conductivity detection, and by single crystal X-ray analysis.

Synthesis of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahvdro- 4H-furo[3,4-dT [l,31thiazin-7a-yl)-4-fluorophenyl)-5-(flttoromethyl)pyrazin e-2- carboxamide L-malate anhydrate

N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H- furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorometh yl)pyrazine-2-carboxamide free base (105 mg) and L-malic acid (28 mg) were dissolved in the solvent mixture of ethanol (6 mL) and water (0.65 mL) with heating to 78 °C. Although a little bit of powder still remained, the mixture was cooled down to room temperature and stirred overnight with a magnetic stirrer. The white solid of the title compound (123 mg) was collected by filtration through a membrane filter (0.2 um pore diameter).

¹ H-NMR (600 MHz, DMSO-<¾) δ (ppm): 2.41 (dd, J=16, 7 Hz, 0.5H), 2.58 (dd, J=16, 6 Hz, 0.5H), 2.93 (dd, J=14, 4 Hz, 1H), 3.16 (dd, J=14, 3 Hz, 1H), 3.22 (m, 1H), 3.89 (dd, J=8, 2 Hz, 1H), 4.18 (dd, J=7, 6 Hz, 0.5H), 4.38 (d, J=8 Hz, 1H), 4.68 (dq, J=7, 7 Hz, 1H), 5.71 (d, J=46 Hz, 2H), 6.45 (brs, 2H), 7.22 (dd, J=12, 9 Hz, 1H), 7.93 (m, 1H), 7.96 (dd, J=7, 3 Hz, 1H), 8.89 (s, 1H), 9.29 (s, 1H), 10.91 (s, 1H).

X-ray powder diffraction peaks for the L-malate anhydrate salt (expressed in degrees 2Θ +/- 0.2°) include 15.2 and 19.1. Synthesis of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethylV4a,5,7,7a-tetr ahydro- 4H-furof3,4-d1[l,31thiazin-7a-yl)-4-fluorophenvn-5-(fluorome thyl)pyrazine-2- carboxamide L-tartrate

N-(3-((4aS,5S,7aS)-2-amino-5-(trifluorometliyl)-4a,5,7,7a-te traliydro-4H- furo[3,4-d] [1 ,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2 -carboxamide free base (50 mg) and L-tartaric acid (19 mg) were dissolved in ethanol (2 mL) with heating to 60 °C. Although a little bit of power still remained, the solution or suspension was cooled down to room temperature. Since gelation was observed during cooling, heptane (1 mL) was added, and then the mixture was stirred overnight with a magnetic stirrer. The white solid of the title compound was collected by filtration through a membrane filter (0.2 um pore diameter).

1H-NMR (600 MHz, DMSO-i/ ₆ ) δ (ppm): 2.92 (dd, J=14, 4 Hz, 1H), 3.15 (dd, J-14, 3 Hz, 1H), 3.21 (m, 1H), 3.89 (dd, J=8, 2 Hz, 1H), 4.23 (s, 1H), 4.37 (d, J=8 Hz, 1H), 4.68 (dq, J=7, 7 Hz, 1H), 5.71 (d, J=46 Hz, 2H), 6.40 (brs, 2H), 7.22 (dd, J=12, 9 Hz, 1H), 7.93 (m, 1H), 7.95 (dd, J=7, 3 Hz, 1H), 8.89 (s, 1H), 9.29 (s, 1H), 10.90 (s, 1H).

X-ray powder diffraction peaks for the L-tartrate salt (expressed in degrees 2Θ +/- 0.2°) include: 5.8, 10.4, 17.1, 20.8.

Synthesis of N-(3-((4aS,5S,7aSV2-amino-5-(trifluoromethvn-4a,5,7,7a-tetra hydro- 4H-furof3,4-d1 [l,31thiazin-7a-yl)-4-fluorophenyI)-5-(fluoromethyl)pyrazine -2- carboxamide succinate

N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro-4H- furo[3,4-d][l,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluorometh yl)pyrazine-2-carboxamide free base (107 mg) and succinic acid (29 mg) were dissolved in 2-propanol (4 mL) with heating to 70 °C. Although a little bit of power still remained, the mixture was cooled down to room temperature and stirred overnight with a magnetic stirrer. The white solid was collected by filtration through a membrane filter (0.2 um pore diameter) and then dried at 60 °C for 2 hours. The recovery weight of the title compound was 96 mg.

Ή-NMR (600 MHz, DMSO-< ₆ ) δ (ppm): 2.40 (s, 2H), 2.90 (dd, J=14, 4 Hz, 1H), 3.14 (dd, J=14, 3 Hz, 1H), 3.19 (m, 1H), 3.87 (dd, J=8, 2 Hz, 1H), 4.37 (d, J=8 Hz, 1H), 4.67 (dq, J-7, 7 Hz, 1H), 5.71 (d, J=46 Hz, 2H), 6.26 (brs, 2H), 7.21 (dd, J=12, 9 Hz, 1H), 7.91 (ddd, J=9, 3, 3 Hz, 1H), 7.95 (dd, J=7, 3 Hz, 1H), 8.89 (s, 1H), 9.29 (s, 1H), 10.90 (s, 1H), 12.21 (brs, 1H).

X-ray powder diffraction peaks for the succinate salt (expressed in degrees 20 +/- 0.2°) include: 8.8, 12.4, 17.7 and 22.3. Solid State and Physico-Chemical Analysis

Instrument Details and Methods for Solid State and Physico-Chemical Analysis X-Ray Powder Diffraction (XRPD) Measurement

Machine type: RINT-TTRIII (Rigaku, Japan)

Each sample is placed on the platform of an X ray system, and analyzed under the following conditions.

Target: Cu

Detector: scintillation counter

Tube voltage: 50 kV

Tube current: 300 mA

Slit: divergence slit 0.5 mm (Height limiting slit 2 mm), scatter slit open, receiving slit open

Scan speed: 5 min

Step/Sampling: 0.02°

Scan range: 5° to 35°

Sample holder: aluminum holder

Monochromator: not used

Differential scanning calorimetry (DSC)

Each sample is accurately weighed into an aluminium pan with a pinhole punched in the lid, and then heated under a nitrogen-purge atmosphere from 25 to 200 °C at 10 °C/min using an empty pan as reference. The temperature axis and the cell constant in both thermal analyses are calibrated with indium in advance. The followings are measurement conditions.

Machine type: DSC822 ⁶ (Mettler Toledo, Switzerland)

Atmosphere: N ₂ gas (40 niL/min)

Sample pan: aluminium

Reference: empty pan

Heating rate: 10 °C/min

Hygroscopicity Measurement (Dynamic Vapor Sorption - DVS)

To evaluate hygroscopicity of each sample, its vapor pressure isotherm is obtained using a dynamic vapor sorption analyzer. The test sample (7 to 13 mg) is humidified in an isothermal chamber where RH conditions are regulated from 5% to 95% at 25 °C by adjusting relative flow rates of dry (0% RH) and moist (100% RH) nitrogen. The sample weight is measured every 2 min with the microbalance. The weight stability criterion employed for the equilibrium is either of the folio wings.

1) The maximum weight change for each measurement is less than 0.02% (w/w).

2) The weight change per minute is less than 0.002% (w/w).

Analysis of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro- 4H-furo[3,4-dl [l,31thia¾in-7a-vn-4-fluorophciivl)-5-(fluoromcthvl)pyrazin e-2- carboxamide free base

Form B Polymorph

An XRPD spectrum of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-

4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4- fluorophenyl)-5- (fluoromethyl)pyrazine-2-carboxamide free base Form B Polymorph is presented in Figure 1. X-ray powder diffraction peaks for the free base Form B polymorph

(expressed in degrees 20 +/- 0.2°) include: 13.4, 14.4, 16.9, 17.8 and 20.1.

A water uptake value of less than 2% was recorded by DVS (relative humidity from 5% to 95% at 25 °C). The melting point of Form B displayed batch to batch differences but as measured by DSC (onset) was recorded to be in the range of 173 °C to 175 °C. Form A Polymorph

An XRPD spectrum of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)- 4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-flu orophenyl)-5- (fluoromethyl)pyrazine-2-carboxamide free base Form A Polymorph is presented in Figure 2. X-ray powder diffraction peaks for the free base Form A polymorph

(expressed in degrees 20 +/- 0.2°) include: 6.7, 11.5, 13.5, 14.8 and 19.0.

A water uptake value of greater than 10% was recorded by DVS (relative humidity from 5% to 95% at 25 °C). This was reversible. Moreover, batch variability was observed with subsequent batches of material giving water uptake values in the range of 4 to 5%. The melting point of Form A displayed batch to batch differences but as measured by DSC (onset) was recorded to be in the range of 165 to 172 °C.

Analysis of N-(3-((4aS,5S,7aS)-2-amino-5-(triflnoromethylV4a.5.7.7a-tetr ahydro- 4H-furo[3,4-diri,31thiazin-7a-yl)-4-flttorophenyl)-5-(fluoro methyl)pyrazine-2- carboxamide free base hydrate

An XRPD spectrum of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-

4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4- fluorophenyl)-5- (fluoromethyl)pyrazine-2-carboxamide free base hydrate is presented in Figure 3. X-ray powder diffraction peaks for the free base hydrate (expressed in degrees 2Θ +/- 0.2°) include: 14.8, 16.5, 16.8, 21.4 and 28.5.

Analysis of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5 ₁ 7,7a-tetrahvdro- 4H-furoi3,4-dl [l,31thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine -2- carboxamide mono-malonate anhydrate

Material obtained by Procedure A described herein above was analysed by XRPD and DVS.

An XRPD spectrum of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)- 4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-flu orophenyl)-5-

(fluoromethyl)pyrazine-2-carboxamide mono-malonate anhydrate is presented in Figure 4.

A DVS isotherm plot of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)- 4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-fiu orophenyl)-5- (fluoromethyl)pyrazine-2-carboxamide mono-malonate anhydrate is presented in Figure 5 ¹ . The water uptake is approximately 1.1% as measured by the increase in mass determined by DVS from 5% to 95% relative humidity at 25 °C.

¹ The DVS plot shown in Figure 5 was recorded on a batch of material prepared in an analogous manner to that obtained in Procedure A.

Analysis of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tet rahydro- 4H-furo[3,4-dl [l,3Uhiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl ^' )pyrazine-2- carboxamide mono-hippurate anhydrate

Material obtained by Procedure A described herein above was analysed by XRPD and DVS.

An XRPD spectrum of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)- 4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4-flu orophenyl)-5- (fluoromethyl)pyrazine-2-carboxamide mono-hippurate anhydrate is presented in Figure 6.

A DVS isotherm plot of N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-

4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]thiazin-7a-yl)-4- fluorophenyl)-5- (fluoromethyl)pyrazine-2-carboxamide mono-hippurate anhydrate is presented in Figure 7. The water uptake is less than 2 % as measured by the increase in mass determined by DVS from 5% to 95% relative humidity at 25 °C.

Single Crystal X-Rav Analysis: N-r3-((4aS,5SJaS)-2-amino-5-(trifluoromethyl)- 4a,5,7,7a-tetrahvdro-4H-furo[3,4-dl [l,3|thiaziii-7a-vn-4-fluorophenvl)-5- (fluoromethyl)pyrazine-2-carboxamide mono-malontae anhydrate Data were collected using a Bruker CCD (charge coupled device) diffractometer equipped with an Oxford Cyrostream low-temperature apparatus operating at 173K and a wavelength of 1.5418 A. The crystal size was 0.36 x 0.07 x 0.04 mm ³ . The crystallographic data and structural analysis results are given below. A three dimensional representation of the structure as determined by X-ray analysis is depicted in Figure 8.

Crystal System: triclinic

Space Group: PI

Unit Cell Dimensions a = 7.7712(14) A = 86.331 (2)°.

b = 11.430 (2) A β = 88.346 (2)°.

c = 15.878 (9) A γ = 89.1 15 (2)°.

Volume: 1225.8(4) A ³

Z value: 2

R indices (all data) Rl = 0.1304, wR2 0.1413

Single Crystal X-Rav Analysis; N-(3-f(4aS,5SJaSV2-amino-5-(triflttoromethyI - 4a,5,7,7a-tetrahydro-4H-furof3 _< 4-dl H,31thiazin-7a-vl)-4-fluoi oi)hcnyl)-5- (fluoromethyl)pyrazine-2-carboxamide mono-hippurate anhydrate

Data were collected using a Bruker CCD (charge coupled device) diffractometer equipped with an Oxford Cyrostream low-temperature apparatus operating at 173K and a wavelength of 1.5418 A. The crystal size was 0.25 x 0.11 x 0.04 mm ³ . The crystallographic data and structural analysis results are given below. A three dimensional representation of the structure as determined by X-ray analysis is depicted in Figure 9.

Crystal System: orthorhombic

Space Group: P2j2i2

Unit Cell Dimensions a = 28.0583(15) A a 90.000°.

b = 7.1741 (4) A P = 90.000°.

c = 14.7333(9) A γ = 90.000°.

Volume: 2965.7(3) A ³

Z value: 4

R indices (all data) Rl = 0.0829, wR2 = 0.1949

Stability Testing

The stability of the mono-hippurate salt was tested under stressed conditions including high temperature (25 °C, 60 °C, and 100 °C, 14 days), humidity (40°C/75% relative humidity, 14 days) and ultra-violet and visible light (7 days). No significant degredation was observed under any of the test conditions (see Table 1). Table 1 : Solid State Stabilit of Hippurate Salt

Area% refers to HPLC Area% analysis.

Wt/Wt refers to weight/weight HPLC analysis,

"nt" means "not tested at that time point"

a: stored in glass vial and Teflon lined cap

: as-is, mono-hippurate salt. Sample stored at -20 °C was used as standard

°: open vial

d: ultraviolet, 25 ±2 °C,>200watt-hours/M ²

e: visible light, 25 ±2 °C, >1.2 million lux-hours In vitro cellular assay:

Quantification of Αβ peptide in culture of neurons from rat fetus brain

(1) 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 under ether anesthesia. The brain was isolated from the embryo and immersed in HBSS (Sigma Aldrich #H9269) containing lOmM HEPES (Gibco #15630-056). 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 0.05% trypsin-EDTA solution (GIBCO, #25300) at 37°C for 20 minutes to disperse the cells. The cells were then washed twice and then gently resuspended in Neurobasal medium (Gibco #21103)

supplemented with 2% B27 supplement (GIBCO #17504-044), 0.5 mM L-glutamine (GIBCO #25030), lx N2 (GIBCO #17502-048), lOOug/ml Pen/Strep (GIBCO 15140- 122) and 5% heat inactivated FCS (PAA #A15-701). The cell dispersion was filtered through a 40-μηι 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 μΐ, /well at an initial cell density of 3.25 x 10 ⁵ 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% C0 ₂ -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 DMSO at a concentration of xlOOO that of the final assay concentration (FAC). Compound solutions were then prepared by adding 999ul of 'Assay Neurobasal media' (as described in above section) to lul of DMSO compound stock. The total amount of the medium was removed from each of the cell plate wells, and ΜΟμΙ,ΛνεΙΙ of 'Assay Neurobasal media' was added followed by 60ul of compound solution. The final DMSO concentration was 0.1%.

(3) Sampling

The cells were cultured for either 1 or 3 days after addition of the compound for ABx-40 and ABx-42 assays respectively. 150μ1 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, 50μ1 of 20% Alamar blue solution (Invitrogen #DAL1100) in assay Neurobasal media, was added to

50μ1 of remaining sample within each well. Cells were then incubated at 37°C in 5% C0 ₂ -95% air for lhr.

Measurement of fluorescence intensity for each well was the carried out at 540/590nm 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) Αβ ELISA

Human/Rat β Amyloid (42) ELISA Kit Wako (#290-62601) and Human/Rat β Amyloid (40) ELISA Kit Wako (#294-62501) from Wako Pure Chemical Industries, Ltd. were used for Αβ ELISA. Αβ 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 IC50 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, N-(3-((4aS,5S,7aS)-2-amino-5- (trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][l,3]th iazin-7a-yl)-4- fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide free base had an IC50 value (Αβ40) of 0.006 μΜ.