OA18265A - 2-amino-6-(difluoromethyl)-5,5-difluoro-6phenyl-3,4,5,6-tetrahydropyridines as bacel inhibitors. - Google Patents

Abstract

The present invention is directed to compounds according to formula (I)

Description

The présent invention is directed to compounds according to formula (I)

Separate aspects of the invention are directed to pharmaceutical compositions comprising said compounds and uses of the compounds to treat disorders for which the réduction of Αβ deposits is bénéficiai such as Alzheimer's disease.

Which compounds are inhibitors of the BACE1 enzyme.

Ο.Α.Ρ.Ι. - B.P. 887, YAOUNDE (Cameroun) - Tel. (237) 222 20 57 00-Site web: http:/www.oapi.int - Email: oapi@oapi.int

2-Amlno-6-(difluoromethyl)- S.S-difluoro-e-phenyl-O^.S.G-tetrahydropyridines as BACE1 Inhibitors

FIELD OF THE INVENTION

The présent Invention provides compounds which act as BACE1 inhibitors. Separate aspects of the invention are directed to pharmaceutical compositions comprising said compounds and uses of the compounds to treat neurodegenerative or cognitive disorders.

BACKGROUND ART

Dementia ls a clinical syndrome characterized by déficits In multiple areas of cognition that cannot be explained by normal aging, a noticeable décliné in function, and an absence of delirium. In addition, neuropsychiatrie symptoms and focal neurological findings are usually présent. Dementia is further classified based on etiology. Alzheimer's disease (AD) is the most common cause of dementia, followed by mixed AD and vascular dementia, Lewy body dementia (DLB), and fronto-temporal dementia. β-Amyloid deposits and neurofibrillary tangles are considered to be major pathologie characterizations associated with AD which is characterized by the loss of memory, cognition, reasonlng, judgment, and orientation. Also affected, as the disease progresses, are motor, sensory and linguistic abîlities until global Impairment of multiple cognitive functions occurs. β-Amylold deposits are predominantly an aggregate of Αβ peptide, which in tum ls a product of the proteolysis of amyloid precursor protein (APP) as part of the βamyloidogenic pathway. Αβ peptide results from the cleavage of APP at the C-terminal by one or more γ-secretases and at the N-terminal by β-secretase 1 (BACE1) also known as aspartyl protease 2. BACE1 activity ls correlated directly to the génération of Αβ peptide from APP.

Studies Indicate that the Inhibition of BACE1 impedes the production of Αβ peptide. Further, BACE1 co4ocalizes with its substrate APP in Golgl and endocytic compartments (Willem M, et al. Semin.Cell Dev. Biol, 2009,20,175-182). Knock-out studies in mice hâve demonstrated the absence of amyloid peptide formation white the animais are healthy and fertile (Ohno M, et al. Neurobiol. Dis., 2007,26,134-145). Genetic ablation of BACE1 în APP-overexpressing mice has demonstrated absence of plaque formation, and the reverse of cognitive déficits (Ohno M, et al. Neuroir, 2004,41,27-33). BACE1 levels are elevated In the brains of sporadic AD patients (Hampel and Shen, Scand. J. Clin. Lab. Invest. 2009,69,8-12).

These convergent findings indicate that the inhibition of BACE1 may be a therapeutic target for the treatment of AD as well as disorders for which the réduction of Αβ deposits ls bénéficiai.

AstraZeneca announced the discovery of AZD3839, a potent BACE1 Inhibitor clinical candidate forthe treatment of AD (Jeppsson, F., étal. J. Biol. Chem._t 2012,287,41245-41257) in October 2012. The effort which led to the discovery of AZD3839 was further described In Ginman, T., et al. J. Med. Chem., 2013, 56,4181-4205. The Ginman publication describes the Issues which were overcome in connection with the discovery and Identification of AZD3839. These Issues related to poor blood brain barrier pénétration and P-glycoprotein mediated efflux of the compounds resulting in lack of brain exposure.

The Ginman manuscript hypothesized that the différences In brain exposure would large! y be due to the core structures and Structure Activity Relationship data was provided wherein the in vitro properties on the reported compounds were given into four tables according to core subtypes. In table 4, a sériés of amidine containing compounds are described that were considered interesting from an activity perspective. However, the data suggests that the amidine containing core did not exhibit a favourable blood brain barrier permeability profile.

Researchers from Hoffmann-La Roche and Siena Biotech also reported the discovery of amidine containing compounds (Woltering, T. J., et al. Bioorg. Med. Chem. Lett. 2013,23, 42394243). These compounds (compounds 17 and 18 in the paper) were found not to hâve any in vivo effect (lack of Αβ40 réduction in brain In wild type mice).

Contrary to the teachings of Ginman, et al. and Woltering, T. J., et al., the Inventors hâve discovered a séries of amidine compounds which are brain pénétrant. Accordingly, the présent Invention relates to novel compounds having BACE1 Inhibitory activity, to their préparation, to their medical use and to médicaments comprising them.

SUMMARY OF THE INVENTION

An objective of the présent Invention Is to provide compounds that inhibit BACE1. Accordingly, the présent invention relates to compounds of Formula I.

NH₂

O W’* R¹

Formula I wherein Ar is selected from the group consisting of phenyl, pyridyl, pyrimldyl, pyrazinyl, Imidazolyl, pyrazolyl, thiazolyl, oxazolyl, Isoxazolyl, and where the Ar Is optionally substituted with one or more substituent selected from halogen, CN, CpCe alkyl, C^Ce alkenyl, QrCe alkynyl, C_rCe fluoroalkyl or C_rCe alkoxy; and

R1 is hydrogen, halogen, C1-C3 fluoroalkyl or CrQj alkyl;

or a pharmaceutically acceptable sait thereof.

In one embodiment the présent Invention provides compounds of Formula 1 or a pharmaceutically acceptable sait thereof for use in therapy.

The présent invention further provides a pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable sait thereof and a pharmaceutically acceptable carrier.

In one embodiment the invention provides the use of a compound of Formula I or a pharmaceutically acceptable sait thereof In the manufacture of a médicament for the treatment of neurodegenerative or cognitive disorder.

In one embodiment, the invention provides a compound of Formula I or a pharmaceutically acceptable sait thereof for use in a method for the treatment of a neurodegenerative or cognitive disorder.

In one embodiment the présent Invention provides a method of treating a neurodegenerative or cognitive disorder comprising administering a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable sait thereof to a patient In need thereof.

Further embodiments of the invention are provided immediately below: In one embodiment, the compound is of formula la

NH₂

An

R¹ Formula la;

or a pharmaceutically acceptable sait thereof.

In one embodiment, R¹ is F or H, particularly F.

In one embodiment, Ar Is optionally substituted with one or more F, Cl, Br, CN, CpCs alkyl, C1-C3 fluoroalkyl or CpCj alkoxy.

In one embodiment, Ar is optionally substituted phenyl.

In one embodiment, Ar Is optionally substituted pyridyl.

ln one embodiment, Ar Is optionally substituted pyrimidyl.

ln one embodiment, Ar Is optionally substituted pyrazinyl.

In one embodiment, Ar is optionally substituted imidazolyl.

ln one embodiment, Ar is optionally substituted pyrazolyl.

In one embodiment, Ar Is optionally substituted thiazolyl.

In one embodiment, Ar Is optionally substituted oxazolyl.

ln one embodiment, Ar is optionally substituted isoxazolyl.

In one embodiment, the compound Is selected from the group consisting of:

(S)-M(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2₁3,4₁5-tetrahydropyridin-2-yl)-4-fluorophenyl)-

5-chloropicolinamide (S)-/\43’{6’amino-2-(difluoiOmethyl)-3,3-difluoro-2,3₁4₁5-tetrahydropyridin-2-yl)-4-fluorophenyl)-

5-fluoropicolinamide (S)-W-(3-(6-amino-2-(difluoromethyf)-3,3-difluoro-2,3_l4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-

5-methoxypyrazine-2-ca rboxa mide (S)-//-(3-(6-amino-2-(difluoromethy1)-3,3-difluoro-2₁3₁4₁5-tetrahydropyridin-2-yl)-4-fluorophenyl)2-methyloxazole-4-carboxamide (S)-/7-{3-(6-amino-2-(difluoromethy1)-3₁3-clifluoro-2,3_>4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-

5-methoxypicolinamide (S)-AA(3-(6-amÎno-2-(difluoromethyl)-3,3-difluoro-2₁3₁4₁5-tetrahydropyridin-2-yl)-4-fluorophenyl)-

5-(difluorom ethyl )pyrazi ne-2-ca rboxamlde (S)-/\A(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3₁4₁5-tetrahydropyridin-2-yl)-4-fluorophenyl)-

5-cyanopicolinamide (S)-M-(3-(6-amino-2-{difluoromethyl)-3,3-difIuoro-2,3₁4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-

4- methylthiazole-2-carboxamide (S>N-(3-(6-amino-2-(difluoromethyl )-3,3-0^00(^2,3,4,5-tetrahydropyridin-2-yl)-4-fluorDphenyl)-

5- methoxypyrimidine-2-carboxamide (S)-N-(3-(6-amino-2-(difluoromethy1)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-

5-methoxy-3-methylpyrazine-2-carboxamide (S)-M(3-(6-amlno-2-(difluoiOmethyl)-3,3-difluoro-2,3₁4₁5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5cyano-3-methylpicolinamide (S)-M<3-(6-amino-2-{difluoromethyl)-3,3-difluoro-2₁3,4,5-tetrahydropyridin-2-yl)-4-fluonophenyl)-5bromopicolinamide (S)-N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2_I3_I4₁5-tetrahydropyridÎn-2-yl)-4-fluorophenyl)-5(methoxy-d3)picolinamide (S)-N-(3-(6-amino-2-{difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5(methoxy-d3)pyrazine-2-carboxamide or a pharmaceutically acceptable sait thereof.

A separate embodiment is directed to a pharmaceutical composition comprising a compound from the above list and a pharmaceutically acceptable carrier.

Another embodiment is directed to a method of treating a neurodegenerative or cognitive disorder comprising administering a therapeutically effective amount of a compound from the above list.

Yet another embodiment is directed to a use of a compound from the above list for the manufacture of a médicament for treating a neurodegenerative or cognitive disorder.

One embodiment is a compound from the above list for use in therapy.

Yet another embodiment Is directed to a compound form the above Isit for use in the treatment of a neurodegenerative or cognitive disorder.

DETAILED DESCRIPTION OF THE INVENTION

The présent Invention is based on the discovery of the compounds of Formula I are inhibitors of BACE1, and as such, are useful for the treatment of related disorders. Certain aspects of the invention are explained in greater detail below but this description is not intended to be a detailed catalog of ali the different ways In which the Invention may be implemented, or ali the features that may be added to the instant invention. Hence, the following spécification is Intended to illustrate some embodiments of the invention, and not to exhaustively specify ali permutations, combinations and variations thereof.

As used herein, the terni CrCe alkyl refers to a straight chained or branched saturated hydrocarbon having from one to six carbon atoms inclusive. Examples of C_rCe alkyl include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1 -butyl, 2-butyl, 2-methyl-2-propyl, 2-methyl-1propyl, n-pentyl and n-hexyl. Similarly, the term ‘CrCa alkyl refers to a straight chained or branched saturated hydrocarbon having from one to three carbon atoms inclusive. Examples of such substituents Indude, but are not limited to, methyl, ethyl and n-propyl.

Likewise, the term ‘CrCe alkoxy refers to a straight chained or branched saturated alkoxy group having from one to six carbon atoms indusive with the open valency on the oxygen.

Examples of C_rCe alkoxy indude, but are not limited to, methoxy, ethoxy, n-butoxy, t-butoxy and n-hexyloxy. The CrCealkoxy is optionally substituted with one or more fluorine atoms.

As used herein, the term ‘CrCe fluoroalkyl' refers to a straight chained or branched saturated hydrocarbon having from one to six carbon atoms Indusive substituted with one or more fluorine atoms. Examples of C_rCe fluoroalkyl Include, but are not limited to, trifluoromethyl, pentafluoroethyl, 1 fluoroethyl, monofluoromethyl, difluoromethyf, 1,2-difluoroethyl and 3,4 difluorohexyl. Similarly, the term C1-C3 fluoroalkyl refers to a straight chained or branched saturated hydrocarbon having from one to three carbon atoms inclusive substituted with one or more fluorine atoms per carbon atom.

The term halogen refers to fluorine, chlorine, bromine and iodine.

The term C2-C_e alkenyl refers to a branched or unbranched alkenyl group having from two to six carbon atoms and one double bond, Including but not limited to ethenyl, propenyl, and butenyl.

The term “CrCe alkynyl shall mean a branched or unbranched alkynyl group having from two to six carbon atoms and one triple bond, including but not limited to ethynyl, propynyl and butynyl.

As used herein, the phrase effective amount when applied to a compound ofthe Invention, Is intended to dénoté an amount suffident to cause an Intended biological effect. The phrase therapeutically effective amount when applied to a compound ofthe invention Is intended to dénoté an amount ofthe compound that Is suffident to ameliorate, palliate, stabilize, reverse, slow or delay the progression of a disorder or disease state, or of a symptom of the disorder or disease. In an embodiment, the method ofthe présent Invention provides for administration of combinations of compounds. In such instances, the ‘effective amount Is the amount of a compound of the présent invention In the combination suffident to cause the intended biological effect.

The term “treatment or treating as used herein means ameliorating or reverslng the progress or severity of a disease or disorder, or ameliorating or reversing one or more symptoms or side effects of such disease or disorder. “Treatment or “treating, as used herein, also means to inhibit or block, as in retard, arrest, restrain, impede or obstruct, the progress of a system, condition or state of a disease or disorder. For purposes of this Invention, “treatment or “treating further means an approach for obtaining benefldal or desired clinical results, where “benefidal or desired dinlcal results indude, without limitation, alleviation of a symptom, dîminishment of the extent of a disorder or disease, stabilized (i.e., not worsening) disease or disorder state, delay or slowing of a disease or disorder state, amelioration or palliation of a disease or disorder state, and rémission of a disease or disorder, whether partial or total.

The présent Invention Is based on the discovery that compounds of Formula I are

Inhibitors of BACE1, and as such, are useful for the treatment of disorders which pathological characteristics comprise β-amyloid deposits and neurofi britlary tangles, such as neurodegenerative or cognitive disorders.

The compounds ofthe présent Invention are, as discussed above, expected to be useful In the treatment of Alzheimer’s disease due to their effects on β-amyloid deposits and neurofibrillary tangles. This includes familial Alzheimer’s disease where patients carry mutations on spécifie genes Intimately involved in the production of Αβ peptide. It is, however, important to note that aggregates of Αβ peptide is not limited to familial Alzheimer's disease but is similarly an Important pathophysiological characterlstics ofthe more common sporadic Alzheimer’s disease [Mo/ Cell Neuroscl, 66.3-11,2015].

The compounds of the présent invention are also believed to be useful in the treatment of early-stage Alzheimer’s disease, I.e. disease stages where the biological and structural changes hâve started but the clinical manifestations of the disease hâve not yet become évident or are not yet well developed. Early-stage Alzheimer's disease may, in fact, start years before any clinical manifestation of the disease becomes manifest. Early-stage Alzheimer's disease includes prodromal Alzheimer's disease, preclinical Alzheimer’s disease and mild cognitive impairment. Although mild cognitive impairment may be unrelated to Alzheimer’s disease It Is often a transitional stage to Alzheimer's disease or due to Alzheimer's disease. Preclinical and prodromal Alzheimer's disease are asymptomatic stages, and they are typically diagnosed by the presence of Alzheimer's disease related blomarkers. In this context the compounds ofthe présent invention are believed to be useful In slowing down the progression of early-stage Alzheimer’s disease, such as mild cognitive Impairment to Alzheimer's disease. The compounds of the présent Invention are also believed to be useful In the treatment of memory loss, attention déficits and dementia associated with Alzheimer’s disease.

Other diseases, in addition to the continuum of Alzheimer’s disease, are characterized by β-amylold deposits and neurofibrillary tangles. This includes e.g. Trisomy 21 also known as Down’s syndrome. Patients suffering from Down’s syndrome hâve an extra chromosome 21 which chromosome contains the gene for the amylold precursor protein (APP). The extra chromosome 21 leadsto overexpression of APP, which leads to increased levels of Αβ peptide, which eventually causes the markedly Increased risk of developing Alzheimer’s disease seen in Down’s syndrome patients [A/zhe/mer's & Dementia, 11,700-709,201]. Cérébral amyloid angiopathy Is also characterized by β-amyloid deposits and neurofibrillary tangles In blood vessels of the central nervous system [Pharmacol Reports, 67,195-203,2015] and Is as such expected to be treatable with compounds ofthe présent invention.

In one embodiment, the présent invention provides a method of treating a disease selected from Alzheimer's disease (familial or sporadic), preclinical Alzheimer’s disease, prodromal Alzheimer’s disease, mild cognitive impairment, Down’s syndrome and cérébral amyloid anglopathy, the method comprising the administration of a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable sait thereof to a patient In need thereof.

The présent invention further provides a method of inhibiting BACE1 in a patient comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable sait thereof.

The présent invention also provides a method of inhibiting β-secretase mediated deavage of amyloid precursor protein comprising administering to a patient In need of such treatment a therapeutically effedive amount a compound of Formula l or a pharmaceutically acceptable sait thereof.

In further embodiments, the présent invention provides the use of a compound of Formula I or a pharmaceutically acceptable sait thereof for the manufadure of a médicament for the treatment of disease selected from Alzheimer’s disease (familial or sporadic), predinical Alzheimer’s disease, prodromal Alzheimer’s disease, mild cognitive impairment, Down’s syndrome or cérébral amyloid angiopathy.

The présent invention also provldes the use of a compound of Formula I or a pharmaceutically acceptable sait thereof for the manufacture of a médicament for the inhibition of BACE1. The présent invention further provides the use of a compound of Formula I or a pharmaceutically acceptable sait thereof for the manufacture of a médicament for the inhibition of production or accumulation of Αβ peptide.

In one embodiment, the présent invention provides a compound of Formula I or a pharmaceutically acceptable sait thereof for use In a method for the treatment of a disease selected form Alzheimer’s disease (familial or sporadic), predinical Alzheimer’s disease, prodromal Alzheimer’s disease, mild cognitive impairment, Down’s syndrome or cérébral amyloid angiopathy.

In one embodiment, the présent invention relates to a compound of Formula I or a pharmaceutically acceptable sait thereof for use In a method for inhibiting of BACE1 or In a method for inhibiting of production or accumulation of Αβ peptide.

In a further embodiment, the invention provides a pharmaceutical formulation adapted for any of the above treatments and uses.

In one embodiment, a mammal is a human.

In one embodiment, the patient is a human patient.

The présent Invention also comprises salts of the présent compounds, typically, pharmaceutically acceptable salts. Such salts Include pharmaceutically acceptable acid addition salts. Acid addition salts Include salts of inorganic acids as well as organic acids.

Représentative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, sulfamic, nitric acids and the like. Représentative examples of suitable organic acids include formlc, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, itaconic, lactic, methanesulfonlc, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methane sulfonic, ethanesulfonlc, tartane, ascorblc, pamolc, bismethylene salicylic, ethanedisulfonic, gluconlc, citraconlc, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzotc, glutamic, benzenesulfonic, p-toluenesulfonic acids, theophylline acetic acids, as well as the 8-halotheophyllïnes (for example, 8-bromotheophylline and the like). Further examples of pharmaceutically acceptable inorganic or organic acid addition salts Include the pharmaceutically acceptable salts listed In S. M. Berge, et al., J. Pharm. Sci.,

1977,66,2.

Furthermore, the compounds of this invention may exist in unsolvated as well as In solvated forms with pharmaceutically acceptable solvents such as water, éthanol and the like.

The compounds of the présent invention may hâve one or more asymmetric centres and it is intended that any optical isomers (i.e. enantiomers or diastereomers), as separated, pure or partially purified optical isomers and any mixtures thereof including racemic mixtures, I.e. a mixture of stereolsomeres, are included within the scope of the invention.

In this context Is understood that when specifying the enantiomeric form, then the compound is In enantiomeric excess, e.g. essentially In a pure form. Accordingly, one embodiment of the Invention relates to a compound of the invention having an enantiomeric excess of at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 96%, preferably at least 98%.

Racemic forms may be resolved into the optical antipodes by known methods, for example, by séparation of diastereomeric salts thereof with an optically active acid, and liberating the optically active amine compound by treatment with a base. Séparation of such diastereomeric salts can be achieved, e.g. by fractional crystallization. The optically active acids suitable for this purpose may include, but are not limited to d- or I- tartane, mandelic or camphorsulfonic acids. Another method for resolving racemates into the optical antipodes is based upon chromatography on an optically active matrix. The compounds of the présent Invention may also be resolved by the formation and chromatographie séparation of diastereomeric dérivatives from chiral derivatizlng reagents, such as, chiral alkylating or acylating reagents, followed by cleavage of the chiral auxiliary. Any of the above methods may be applied either to résolve the optical antipodes of the compounds of the invention per se or to résolve the optical antipodes of synthetic intermediates, which can then be converted by methods described herein into the optically resolved final products which are the compounds ofthe Invention.

Additional methods for the resolution of optical isomers, known to those skilled in the art, may be used. Such methods include those discussed by J. Jaques, A. Collet and S. Wilen in Enantiomers, Racemates, and Resolutions, John Wiley and Sons, New York, 1981. Optically active compounds can also be prepared from optically active starting materials.

The présent invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable sait thereof and a pharmaceutically acceptable carrier. The présent invention also provides a pharmaceutical composition comprising a therapeutically effective amount of one of the spécifie compounds disclosed in the Experimental Section and a pharmaceutically acceptable carrier.

The compounds of the Invention may be administered alone or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses. The pharmaceutical compositions according to the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 22^m Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 2013.

Pharmaceutical compositions for oral administration Include solid dosage forms such as capsules, tablets, dragees, pills, lozenges, powders and granules. Where appropriate, the compositions may be prepared with coatings such as enteric coatings or they may be formulated so as to provide controlled release of the active ingrédient such as sustaîned or prolonged release according to methods well known in the art. Liquid dosage forms for oral administration Include solutions, émulsions, suspensions, syrups and élixirs. Pharmaceutical compositions for parentéral administration include stérile aqueous and nonaqueous injectable solutions, dispersions, suspensions or émulsions as well as stérile powders to be reconstituted in stérile injectable solutions or dispersions prior to use. Other suitable administration forms include, but are not limited to, suppositories, sprays, ointments, creams, gels, inhalants, dermal patches and Implants.

Typical oral dosages range from about 0.01 to about 100 mg/kg body weight per day.

The compounds of this Invention are generally utilized as the free base or as a pharmaceutically acceptable sait thereof. A pharmaceutically acceptable sait of a compound of

Formula I is prepared e.g. in a conventlonal manner by treating a solution or suspension of a free base of Formula I with a molar équivalent of a pharmaceutically acceptable acid. Représentative examples of suitable organic and Inorganic adds are described above.

Suitable pharmaceutical carriers include inert solid diluents or fillers, stérile aqueous solutions and various organic solvents. Examples of solid carriers Include lactose, terra alba, sucrose, cydodextrin, talc, gelatin, agar, pectin, acacia, magnésium stéarate, stearic acid and lower alkyl ethers of cellulose. Examples of liquid carriers include, but are not limited to, syrup, peanut oil, olive oil, phospholipids, fatty adds, fatty add amines, polyoxyethylene and water. Similarly, the carrier or diluent may indude any sustalned release material known in the art, such as glyceryl monostearate or glyceryi distearate, atone or mixed with a wax. The pharmaceutical compositions formed by combining the compounds of Formula I or a pharmaceutically acceptable sait thereof and a pharmaceutically acceptable carrier are readily administered in a variety of dosage forms suitable for the disdosed routes of administration. The formulations may convenlentiy be presented In unit dosage form by methods known In the art of pharmacy.

lf a solid carrier is used for oral administration, the préparation may be tabletted, placed in a hard gelatin capsule in powder or pellet form or lt may be in the form of a troche or lozenge. The amount of solid carrier will vary widely but will range from about 25 mg to about 1 g per dosage unit, lf a liquid carrier is used, the préparation may be tn the form of a syrup, émulsion, soft gelatin capsule or stérile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.

EXPERIMENTAL SECTION The compounds of the présent invention of the general formula I, wherein R’ and Ar are as defined above can be prepared by the methods outlined In the following reaction schemes 1-4 and in the examples. In the described methods, it Is possible to make use of variants or modifications, which are themselves known to chemists skilled In the art or could be apparent to the person of ordînary skill in this art. Furthermore, other methods for preparing compounds of the invention will be readily apparent to the person skilled In the art In light of the following reaction schemes and examples.

For example, Scheme 2 describe the use of sélective protecting groups during the synthesis of the compounds of the invention. One skilled In the art would be able to select the appropriate protecting group for a particular réaction. Moreover, lt may be necessary to incorporate protection and deprotection strategies for substituents such as amino, amido, keto and hydroxyl groups In the synthetic methods described below to syntheslze the compounds of

Formula I. Methods for protection and deprotection of such groups are well known in the art, and may be found In T. Green, et al., Protective Groups In Organic Synthesis, 1991,2™¹ Edition, John Wiley & Sons, New York.

For compounds, which can exist as a mixture or equilibrium between two or more tautomers, only one tautomer Is represented In the schemes, although It may not be the most stable tautomer. For compounds, which can exist in enantiomeric, stereoisomeric or géométrie Isomeric forms their géométrie configuration is specified; otherwise the structure represents a mixture of stereoisomers.

Analytical LC-MS data was obtained using the following methods.

Method A:

LC-MS was run on Waters Aquity UPLC-MS consisting of Waters Aquity Including column mamager, binary solvent manager, sample organizer, PDA detector (operating at 254 nM), ELS detector, and SQ-MS equîpped with APPI-source operating In positive ion mode.

LC-conditions: The column was Acquity UPLC BEH C181,7pm ; 2.1x150mm operating at 60’C with 0.6 ml/minutes of a binary gradient consisting of water + 0.05 % trifluoroacetic acid (A) and acetonitrile + 5% water + 0.03 % trifluoroacetic acid (B). Gradient: 0.00 min: 10% B; 3.00 min: 99.9% B; 3.01 min: 10% B; 3.60 min: 10% B. Total run time: 3.60 min.

Method B:

LC-MS was run on Waters Acquity UPLC-MS consisting of Waters Acquity including column manager, binary solvent manager, sample organizer, PDA detector (operating at 254 nm), ELS detector, and TQ-MS equîpped with APPI-source operating in positive ion mode.

LC-conditions: The column was Acquity UPLC BEH C18 1.7pm ; 2.1x50mm operating at 60*C with 1.2 ml/minutes of a binary gradient consisting of water + 0.05 % trifluoroacetic acid (A) and acetonitrile + 5% water + 0.05 % trifluoroacetic acid (B). Gradient: 0.00 min: 10% B; 1.00 min: 100% B; 1,01 min: 10% B; 1.15 min: 10% B. Total run time: 1.15 min.

Ή NMR spectra were recorded at 600 MHz on a Broker Avance AV-lll-600 instrument or at 400

MHz on a Broker Avance AV-lll-400 Instrument or a Varian 400 Instrument. Chemical shift values are expressed in ppm-values relative. The following abbreviations are used for multiplicity of NMR signais: s = singlet, d = doublet, t = triplet, q = quartet, dd = double doublet, ddd = double double doublet, dt = double triplet, br = broad, and m = multiplet

As an example and wherein R¹ îs fluorine In the ortho position of the phenyl ring, compounds of 5 the general formula IV may be prepared as shown In Scheme 1.

Scheme 1

Il IV where R¹ Is as defined under formula I and R is an alkyl group such as methyl or ethyl.

Compounds of the general formula IV (Scheme 1 ) may be prepared by reacting compounds of the general formula II with a halogen-metal exchange reagent such as butyllithium followed by addition to an ester of general formula lll.

As an example and wherein R¹ Is fluorine In the ortho position of the phenyl ring, compound of 15 the general formula XV| may be prepared as shown In Scheme 2.

Scheme 2

IV

VII

where R² and R³ are an alkyl group such as methyl or ethyl.

Compounds of the general formula Vil (Scheme 2) may be prepared by reacting compounds of the general formula IV with a sulfinamlde such as Vl In the presence of a Lewis acld/drylng agent such as titanium tetra ethoxide. Treatment of compounds ofthe general formula Vil with compounds of the general formula VIII such as ethyl bromodifluoroacetate In the presence of Zn powder or in the presence of diethyî zinc and trîs(triphenylphosphine)r hodium(l) chloride gives compounds of the general formula IX. Compounds of the general formula X are obtained from compounds of the general formula IX by treatment with a reducing agent such as dilsobutyfaluminium hydride. In some cases compound X might be In equilibrium with the hydrate form. Treatment of compounds of the general formula X with conditions such as methyl 2-(dimethoxyphosphoryl)-acetate in the presence of lithium chloride and a base such as N.N-diisopropylethylamine gives compounds of the general formula XI. Compounds ofthe general formula XII are obtained by hydrogénation of compounds of the general formula XI in the precense of a catalyst such as palladium on carbon. Compounds of the general formula XIII are obtained by treatment of compounds of the general formula XII with an acid such as hydrochloric acid in methanol followed by treatment with potassium carbonate in methanol or heating in a solvent such as toluene. Compounds of the general formula XIII can be nitrated using nltric acid to give compounds ofthe general formula XIV. Treatment of compounds of the general formula XIV with a reagent such as Lawesson’s reagent (2,4-bis(4-methoxyphenyl)-

1,3,2,4-dithladiphosphetane-2,4-disulfide) gives compounds ofthe general formula XV. Réduction of the nitro group of compounds of the general formula XV gives compounds of the general formula XVI.

Compounds of the general formula XIV may also be prepared as shown in Scheme 3. Starting from nitro substituted acetophenones of general formula IVb, compounds ofthe general formula Xlb may be prepared as described In Scheme 2 Compounds of the general formula Xllb are obtained by hydrogénation of compounds of the general formula Xlb in the presence of a catalyst such as palladium on carbon. Compounds ofthe general formula XIV may be prepared as described Scheme 2 for the préparation of compounds of the general formula XIII from compounds of the general formula XII. Protection of the aniline moiety of compounds of the general formula XIV gives compounds of the general formula XlVb. Treatment of compounds of the general formula XlVb with a reagent such as Lawesson’s reagent (2,4-bis(4-methoxyphenyl)-

1,3,2,4-dithladiphosphetane-2,4-disulfide) followed by deprotection of the aniline moiety gives compounds of the general formula XVI.

Scheme 3

where R¹ Is as defined under formula I and R³ Is an alkyl group such as methyl or ethyl

Compounds of the general formula I may be prepared as shown In Scheme 4.

Scheme 4

where R¹ and Ar are as defined under formula I.

Compounds of the general formula XIX may be prepared by reacting compounds of the general formula XVI with a carboxylic acid chloride of general formula XVII or by reaction with a carboxylic acid of general formula XVIII using procedures known to chemists skilled in the art. Treatment of compounds of the general formula XIX with ammonia gives compounds of the general formula I. In some cases, the addition of an oxidizing reagent such as tert-butyl hydroperoxide might be necessary to facilitate the reaction.

PREPARATION OF INTERMEDIATES

INTERMEDIATE: 2,2-difluoro-1-(2-fluorophenyl)ethan-1-one

Br

O

To a solution of 1-bromo-2-fluorobenzene (10.00 g, 57.14 mmol) In THF (200 mL) was added n-butyllithlum (2.5 M, 24.00 mL) drop-wise at -78°C over a period of 15 minutes under N₂. The mixture was stirred at -78^eC for 30 min. Ethyl 2,2-dîfluoroacetate (10.64 g, 85.71 mmol) was added dropwise at -78’C and stirred for 2 hours at -78*C. TLC showed no starting material remalned. Saturated aqueous NH4CI (15 mL) was added dropwise at -78’C. The reaction mixture was warmed to 25^eC, extracted with ethyl acetate (100 mL, three times). The combined organic phases were washed with brine (30 mL), dried over anhydrous Na2SO₄₁ filtered and concentrated In vacuo. The residue was purified by column chromatography on silica gel (petroleum ether. ethyl acetate - 95:5) to afford 2,2-difluoro-1-(2-fluorophenyl)ethan-1-one (5.60 g, 47.8% yield, 85% purity). ¹H NMR (CDCI₃,400MHz): δ 7.99-7.95 (m, 1H), 7.70 - 7.64 (m, 1H). 7.33 (t, J=7.6 Hz 2H), 7.24 (dd, J=10.8, 8.4 Hz. 1 H), 6.59-6.32 (m, 1H).

INTERMEDIATE: (R)-N-(2,2-difluoro-1-(2-fluorophenyl)ethylidene)-2-methy!propane-2sulfinamide

H₂N'^S,/tBu

To a solution of 2,2-difluoro-1-(2-fluorophenyl)ethan-1-one (5.60 g, 32.16 mmol) and (R)-2~ methylpropane-2-sulfinamlde (5.07 g, 41.81 mmol) In THF (110 mL), was added tetraethoxytitanium (14.67 g, 64.32 mmol) in one portion at 26*C. The yellow solution was stirred at 80’Cfor 2.5 hr. TLC (petroleum ether: ethyl acetate=3:1) showed no starting material remained. The mixture was cooled to 26’C. Water (10 mL) was added to the mixture and it was filtered and extracted with ethyl acetate (60 mL, three times). The organic layer was washed with brine, dried over Na₂SO4, filtered and concentrated and then purified by column chromatography on silica gel (petroleum ether: ethyl acetate=91:9) to afford (R)-A/-2,2-difluoro1-(2-fluorophenyl)ethylidene)-2-methylpropane-2-sulfinamide (5.60 g, 61.6% yield, 98.1% purity).

INTERMEDIATE: ethyl (S)-3-(((R)-tert-butylsulfinyl)amino)-2,2,4,4-tetrafluoro-3-(2-fluorophenyljbutanoate

To a solution of (R)-N-(2,2-dîfluoro-1-(2-fluorophenyl)ethylÎdene)-2-methylpropane-2-sulfinamide (4.60 g, 16.6 mmol), ethyl 2-bromo-2,2-difluoro-acetate (6.73 g, 33.18 mmol) and Rh(PPh₃)₃CI (469 mg, 498 pmol) in THF (90 mL) was added a solution of diethyl zinc (1 M in THF, 33 mL) dropwise at -78’C over a period of 20 minutes under Ar, during which the température was maintained below -65^eC. The reaction mixture was warmed to 0°C over a period of 10 minutes and stirred at 0^dC for 2 hours. TLC (petroleum ether/ethyl acetate=3:1) showed the starting material was consumed completely. The dark red solution was quenched by water (40 mL) and then filtered. The filtrate was extracted with ethyl acetate (100 mL, twice). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous Na₃SO<, filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate=4:1) to give ethyl (S)-3-(((R)-tert-butylsulfinyl)amino)-2,2,4,4tetrafluoro-3-(2-fluorophenyl)butanoate (4.26 g, 62.1% yield). ’H NMR (DMSO-d6,400MHz): δ 7.65 - 7.31 (m, 1H), 7.49 - 7.44 (m, 1H), 7.23 - 7.12 (m, 2H), 6.93 - 6.66 (m, 1H), 5.00 (s, 1H),

4.39 - 4.29 (m, 2H), 1.39 (s, 9H), 1.32 (t, J=8.0 Hz, 3H).

INTERMEDIATE: (R)-2-methyl-/V-((S)-1,1,3,3-tetrafluoro-2-(2-fluorophenyl)-4-oxobutan-2-yl)propane-2-sulfinamide

To a solution of ethyl (S)-3-(((R)-tert-butylsulfiny1)amino)-2,2,4,4-tetrafluoro-3-(2-fluoiOphenyl)butanoate (3.20 g, 7.97 mmol) in dry THF (35 mL) was added dropwise a solution of DIBAL-H (diisobutylaluminium hydride) in toluene (1.0 M, 16 mL, 16 mmol) at -78’C under N₂. The mixture was stirred at -78’C for 2 hours. The reaction was quenched carefully with methanol (3 mL) at -78’C. Then water (20 mL) and ethyl acetate (200 mL) were added and the mixture was warmed to 25’C. The mixture was aged for 30 minutes. The resulting mixture was filtered through a Celite pad. The organic layer was washed with brine and dried over Na₂SO₄. The organic layer was concentrated to give crude product (R)-2-methyl-N-((S)-1,1,3,3-tetrafluoro-2(2-fluorophenyi)-4-oxobutan-2-yl)propane-2-sulfinamide, which was used immediately in the next step without further purification.

INTERMEDIATE: ethyl (S)-5-(((R)-tert-butylsulfinyl)amino)-4,4,6,6-tetrafluoro-5-(2-fluorophenyl)hex-2-enoate

To a stirred suspension of UCI (405 mg, 9.56 mmol) in acetonitrile (30 mL) under N₂ were added ethyl 2-diethoxyphosphorylacetate (2.14 g, 9.56 mmol) and DIPEA (N.Ndiisopropylethylamine) (2.06 g, 15.94 mmol) at O’C. After 20 min, (R)-2-methyi-N-((S)-1,1,3,3tetrafluoro-2-(2-fluorophenyi)-4-oxobutan-2-yl)propane-2-sulfinamide (2.85 g, 7.97 mmol) in acetonitrile (10 mL) was added dropwise to the mixture at O’C and the mixture was stirred at 25’C for 17.5 hours. The reaction mixture was concentrated to remove acetonitrile, water (50 ml) was added and extracted with ethyl acetate (200 ml). The organic layer was dried and evaporated. The crude product was purified by column chromatography (petrolium ether: ethyl acetate = 5:1 to 4:1) to afford ethyl (S)-5-(((R)-tert-butylsulfinyl)amino)-4,4,6₁6-tetrafluoro-5-(2fluorophenyl)hex-2-enoate (1.77 g, 46.8% yield).

INTERMEDIATE: ethyl (S)-5-(((R)-tert-butylsulfinyl)amÎno)-4,4,6,6’tetrafluoro-5-(2-fluorophenyl)hexanoate

To a solution of ethyl (S)-5-(((R)-tert-butylsulfinyl)amino)-4,4,6,6-tetrafluoro-5-(2fluorophenyl}hex-2-enoate (1.77 g, 4.28 mmol) In ethyl acetate (100 mL) was added Pd/C (400 mg, 10%). The black suspension was stirred at 25*C for 18 hours under 45-50 psi H₂. It was filtrated and concentrated to give ethyl (S)-5-(((R)-tert-butylsulfinyl)amino)-4,4,6,6-tetrafluoro-5(2-fluorophenyl)hexanoate (1.70 g, 95.5% yield) which was used in the next step immediately without further purification.

INTERMEDIATE: (S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluorophenyl)piperidin-2-one

To a solution of ethyl (S)-5-(((R)-tert-butylsulfinyl)amino)-4-4.6.6-tetrafluoro-5-(2-fluorophenyl)hexanoate (1.70 g, 4.09 mmol) in dichloromethane (15 mL) was added HCI/MeOH (4 M, 17 mL). The colorless solution was stirred at 25’C for 1 hour. TLC analysis showed no starting material was left. The mixture was concentrated and the residue was dissolved In toluene. The resulting mixture was concentrated again to give 1.5 g of a colorless oil. This oil was dissolved in toluene (30 mL) and was stirred at 100*C for 18 hours. After the mixture was cooled to 25*0, it was concentrated to give the crude product which was purified by flash chromatography on siiica gel (petroleum ethenethyl acetate = 2:1) to give (S)-6-(difluoromethyl)-5₁5-difluoro-6-(2fluorophenyl)piperidin-2-one (880 mg, 3.15 mmol, 73% yield).

INTERMEDIATE: (S)-6-(difluoromethyl)-5₁5-difiuoro-6-(2-fluoro-5-nitrophenyl)piperidin-2-one

(S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluorophenyl)piperidin-2-one (880 mg, 3.15 mmol) was suspended In trifluoroacetic acid (2.55 mL). The mixture was cooled to O’C and concentrated H2SO4 (2.46 g, 24.3 mmol) was added. Then, HNO₃ (661.61 mg, 6.30 mmol) was added dropwise. After 2 hours of stirring at 25 ’C, the reaction mixture was poured onto 100 g ice and baslfied to pH > 11 using 5 M NaOH (aq). The suspension was extracted with ethyl acetate (150 mL). The phases were separated and the aqueous layer was extracted with ethyl acetate (2 x 70 mL). The combined organic phases were washed with a solution of saturated aqueous NH4CI (30 mL) and water (30 mL), dried over MgSO₄, filtered, and concentrated under reduced pressure to afford (S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluoro-5-nitrophenyl)piperidin-2-one (1.00 g, crude).

INTERMEDIATE: (S)-6-(dÎfluoromethyf)-5,5-difluoro-6-(2-fluoro-5’nitrophenyl)plperidine-2-thione

To a solution of (S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluoro-5-nitrophenyl)piperidin-2-one (1.00 g, 3.08 mmol) in toluene (5 mL) was added Lawesson’s reagent (2,4-bis(4-methoxyphenyi)-

1,3,2,4-dithiadiphosphetane-2,4-disulfide) (686 mg, 1.70 mmol). The mixture was stirred at 100*C for 2 hours. TLC analysis showed no starting material remained. The mixture was concentrated and the crude product was purified by flash chromatography on silica gel (petroleum ethenethyl acetate = 5:1) to give (S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluoro-5’ nitrophenyl)piperidine-2-thione (1.00 g, 2.94 mmol, 95.4% yield).

INTERMEDIATE: (S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5-difluoroplperidine-2thlone s, o₂n

s,

To a suspension of (S)-6-(difluoromethyl)-5,5-difluoro-6-(2-fluoro-5-nitrophenyl)piperidine-2thîone (1.00 g, 2.94 mmol) In éthanol (15 mL) and water (4 mL) was added Iron powder (821 mg, 145 mmol) and NhLCI (786 mg, 14.7 mmol, 5.0 Eq). The black mixture was stirred at 60*C for 18 hours. After the reaction mixture was cooled to 25 *C, the crude product was filtered and the residue was washed with éthanol (100 mL). The combined filtrâtes were concentrated and the resulting solid was dispersed in ethyl acetate (100 mL). The mixture was filtered and the filtrate was washed with water (30 mL), brine (20 mL) and concentrated. The crude product was purified by flash chromatography on silica (petroleum ethenethyl acetate=3:1~2:1) to give (S)-6(5-amino-2-fiuorophenyl)-e-(difluoromethyl)-5,5-difluoropiperidine-2-thîone (819 mg, 2.51 mmol, 85.3% yield). ¹H NMR (DMSO-d6,400MHz): δ 10.97 (s, 1H), 7.03 - 6.90 (m, 2H), 6.64 - 6.55 (m, 2H), 5.19 (s, 2H), 3.19 - 3.15 (m, 1H), 3.03 - 2.94 (m, 1H), 2.35 - 2.24 (m, 2H).

INTERMEDIATE: methyl-cf₃-5-(methoxy-d₃)pyrazine-2-carboxylate

O

Sodium (0.094 g, 4.10 mmol) was added In small portions methanol-d, (2.94 ml) and the reaction mixture was stirred until ail sodium has reacted. The soultion was the added to another soultion of methyl-5-chloropyrazine-2-carboxy!ate (0.6 g, 3.48 mmol) in methanol-d₄ (0.98 ml). The reaction mixture was stirred for 1.5 hours at room température. The reaction mixture was concentrated In vacuo. 2 ml of water was added. The mixture was extracted with ethyl acetate. The organic phase was washed with brine, dried over MgSO< and concentrated In vacuo to give methyl-d₃-5-(methoxy-d₃)pyrazine-2-carboxylate.

INTERMEDIATE: methyl 5-(methoxy-d₃)picoIinate

O

O

Methyl 5-hydroxypicolinate (2.88 g, 18.8 mmol) was dissolved In dimethylformamide (108 ml) under argon. Potassium carbonate (7.20 g, 52.1 mmol) was added and the orange suspension was stirred for 45 minutes at room température. Iodomethane-d₃ (1.41 ml, 22.6 mmol) was added. The reaction mixture was stirred for 2 hours. Water was added. The mixture was extracted with ethyl acetate. The organic phase was washed with brine, dried over MgSO₄ and concentrated In vacuo and purified by column chromatography on silica gel (heptane: ethyl acetate) to give methyl 5-(methoxy-d₃)picolinate.

PREPARATION OFTHE COMPOUNDS OF THE INVENTION

Example 1 (S)-/V-(3-(6-amÎno-2-(difluoromethyl)-3₁3-clifluoro-2₁3₁4,5-tetrahydropyriclin-2-yl)4-fluorophenyl)-5-chloropicolinamide (compound 1)

CL

5-chloropicolinic acid (19 mg, 0.12 mmol) and HATU (1-[bis(dimethylamino)methylene]1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) (67.4 mg, 0.177 mmol) was dissolved DMF (1 mL). The reaction mixture was stirred for 5 minutes. Then (S)-6-(5-amino-2fluorophenyO-e-idifluoromethyO-S.S-difluoropiperidine^-thione (25mg, 0.061 mmol) and DIPEA (N,W-di-lsopropyl ethylamine) (52 mg, 0.07 ml, 0.4 mmol) were added. The réaction mixture was stirred for 1 hour at room température. Satureted aqueous NH₄CI was added and the mixture was extracted with ethyl acetate. The combined organic phases were washed with brine, dried over MgSO₄, filtered, and concentrated under reduced pressure. Ammonia in methanol (7M, 2mL) was added and the reaction mixture was stirred at 55‘C In a sealed vial overnight. The reaction mixture was concentrated under reduced pressure. The crude product was purified by flash chromatography on silica (heptane:ethyl acetate). The product was dissolved In ethyl acetate and washed 5 times with satureted aqueous NaHCO3/water to remove thiourea byproducts. The combined organic phases were washed with brine, dried over MgSO₄, filtered, and concentrated under reduced pressure to give (S)-W-(3-{6-amino-2-(difluoromethyl)-3,3difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-chloropicolinamide. ’H NMR (DMSO- d6,600MHz): δ 10.78 (s, 1H), 8.79 (dt, J = 2.4,1.1 Hz, 1H), 8.20 (dd, J = 8.4,2.4 Hz, 1 H), 8.16 (dd, J = 8.4, 0.7 Hz, 1 H), 7.96 - 7.90 (m, 1H), 7.88 (dd, J = 6.8,2.7 Hz, 1H), 7.20 (dd, J = 11.6,

8.8 Hz, 1H), 6.74 (t, J = 55.2 Hz, 1H), 6.38 (s, 2H), 2.51 (dt, J = 3.7,1.8 Hz, 2H), 2.19 -1.98 (m,

2H).

LC-MS (m/z) 433 (MH*): tu = 0.53 minutes (Method A)

Example 2 (S)-N-(3-(6-amino-2-(difluoromethyl)-3₁3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)4-fluoropheny!)-5-fluoropicolinamIde (compound 2)

Prepared as In example 1 from (S)-6-(5-amino-2-fluorophenyl)-6-(dÎfluoromethyl)-5,5difluoropiperidine-2-thlone and 5-fluoroplcolinlc acid.

’H NMR (600 MHz, DMSO) δ 10.72 (s, 1H), 8.74 (d, J- 2.8 Hz, 1H), 8.24 (dd, 8.7,4.6 Hz, 1 H), 7.99 (td, J = 8.7,2.8 Hz, 1 H), 7.95 - 7.91 (m, 1 H), 7.88 (dd, J - 6.8,2.7 Hz, 1 H), 7.20 (dd, J = 11.6, 8.8 Hz, 1 H), 6.74 (t, J = 55.2 Hz, 1 H), 6.38 (s, 2H), 2.56 - 2.50 (m, 2H), 2.22 - 1.98 (m, 2H).

LC-MS (m/z) 417.1 (MH*); fe = 0.49 minutes (Method A)

Example 3 (S)-N-(3-(6-amino-2-(difluoromethyl)-3,3-dÎfluoro-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl)-5-methoxypyrazine-2-carboxamide (compound 3)

Prepared as In example 1 from (S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5difluoroplperidine-2-thione and 5-methoxypyrazine-2-carboxylic acid.

’H NMR (600 MHz, DMSO) δ 10.61 (s, 1 H), 8.89 (d, J = 1.3 Hz, 1 H), 8.42 (d, J = 1.3 Hz, 1 H),

7.90 (m, 2H), 7.20 (dd, J - 11.6,8.9 Hz, 1H), 6.86 - 6.62 (m, 1 H), 6.37 (s, 2H), 4.02 (s, 3H), 2.56 - 2.50 (m, 2H), 2.19 -1.96 (m, 2H).

LC-MS (m/z) 430.1 (MH*); U = 0.48 minutes (Method A)

Example 4 (S)-N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-y1)4-fluorophenyî)-2-methyloxazole-4-carboxamlde (compound 4)

Prepared as In exampîe 1 from (S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5difluoropfperidine-2-thione and 2-methyioxazole-4-carboxylic acid.

’H NMR (600 MHz, DMSO) δ 10.25 (s, 1H), 8.64 (s, 1H), 7.82 (m, 2H), 7.16 (dd, J-11.6,8.7 Hz, 1H), 6.73 (t, J= 55.2 Hz, 1H), 6.36 (s, 2H), 3.01 (s. 1H), 2.59-2.41 (m, 5H), 2.19-1.96 (m, 2H).

LC-MS (m/z) 403 (MH*); fe = 0.42 minutes (Method A)

Example 5 (S)-N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl)-5-methoxypicolinamide (compound 5)

Prepared as In example 1 from (S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyi)-5,5dlfluoropiperidine-2-thione and 5-methoxypicolinic acid.

Ή NMR (600 MHz, DMSO) δ 10.55 (s, 1H). 8.40 (d, J-2.9 Hz, 1H), 8.13 (d, J= 8.7 Hz, 1H),

7.94 - 7.90 (m, 1H), 7.84 (dd, J = 6.7,2.7 Hz, 1 H), 7.62 (dd, J= 8.7,2.9 Hz, 1H), 7.18 (dd, J =

11.6, 8.8 Hz, 1H), 6.85-6.61 (m, 1H), 6.36 (s, 2H), 3.93 (s, 3H), 2.55-2.47 (m, 2H), 2.172.00 (m, 2H).

LC-MS (m/z) 429.1 (MH*); fe = 0.5 minutes (Method A)

Example 6 (S)-N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3₁4₁5-tetrahydropyridin-2-yl )4-fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamlde (compound 6)

F

Prepared as in example 1 from (SHJ-tS-amino^-fluorophenylJ-e-idÎfluoromethylJ-S.Sdifluoropiperidine-2-thlone and 5-difluoromethyl)pyrazine-2-carboxylÎc acid.

Ή NMR (600 MHz. DMSO) δ 11.02 (s, 1H), 9.40 (d, 1.3 Hz. 1H). 9.10 (s, 1H). 7.96-7.91 (m. 2H), 7.38 - 7.17 (m, 2H), 6.75 (t, J = 55.1 Hz, 1 H), 6.38 (s, 2H). 2.56-2.50 (m, 2H). 2.20 1.98 (m, 2H).

LC-MS (mfz) 450.1 (MH*); = 0.48 minutes (Method A)

Example 7 (S)-N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2₁3,4,5-tetrahydropyridÎn-2-yl)4-fluorophenyi)-5-cyanoplcolinamide (compound 7)

Prepared as in example 1 from (S)-6-(5-amino-2-fluorophenyl}-6-(difluoromethyl)-5,5dinuoropiperidine-2-thione and 5-cyanopicolinic acid.

Ή NMR (600 MHz, DMSO) δ 10.94 (s, 1H). 9.21 (d, J- 1.3 Hz. 1H), 8.59 (dd. J~ 8.2,1.9 Hz, 1 H), 8.29 (d, J = 8.1 Hz. 1 H), 7.96 - 7.88 (m, 2H). 7.22 (dd. J - 11.5.8.8 Hz, 1 H), 6.74 (t, J = 55.0 Hz, 1 H), 6.37 (s, 2H), 2.57 - 2.48 (m. 2H ), 2.07 (m, 2H).

LC-MS (m/z) 424.5 (MH⁺); = 0.45 minutes (Method B)

Example 8 (S)-/'A(3-(6-amino-2-(difluoromethyl)-3_I3-dinuoro-2,3,4₁5-tetrahydropyridin-2-yl)4-fluorophenyl)-4-methylthiazole-2-carboxamide (compound 8)

Prepared as In example 1 from (S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5difluoropiperidine-2-thlone and 4-methylthiazole-2-carboxytic acid.

Ή NMR (600 MHz, DMSO) δ 10.84 (s, 1 H), 7.90 (dd, J = 6.7, 2.5 Hz, 1 H), 7.88 - 7.83 (m, 1 H), 7.70(s, 1H), 7.19 (dd, J= 11.5, 8.9Hz, 1H), 6.74 (t. J= 55.1 Hz, 1H), 6.38 (s, 2H), 2.59-2.46 (m, 5H), 2.18-1.95 (m, 2H).

LC-MS (m/z) 419.4 (MH*); t« = 0.47 minutes (Method B)

Example 9 (S)-N-(3-(6-amlno-2-(difluoromethyl}-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)4-fluoropheny1)-5-methoxypyrimidine-2-carboxamlde (compound 9)

Prepared as in example 1 from (S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5difluoropiperidine-2-thione and 5-methoxypyrimldine-2-carboxylic acid.

’H NMR (600 MHz, DMSO) δ 10.73 (s, 1H), 8.73 (s, 2H), 7.95-7.91 (m, 1H), 7.82 (dd, J= 6.7,

2.5 Hz, 1H). 7.22-7.18 (m, 1H), 6.74 (t, J= 55.2 Hz. 1H). 6.37 (s, 2H), 4.03 (s, 3H), 2.582.50 (m, 2H), 2.18-1.99 (m, 2H).

LC-MS (m/z) 430.5 (MH*); U = 0.39 minutes (Method B)

Example 10 (S)-N-(3-(6-amino-2-(dinuoromethyl)-3,3-difluoro-2,3₁4,5-tetrahydropyridin-2-yl)4-fiuorophenyl)-5-methoxy-3-methylpyrazine-2-carboxamide (compound 10)

Prepared as In example 1 from (S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5difluoropiperidine-2-thione and 5-methoxy-3-methylpyrazine-2-carboxylic acid.

¹H NMR (600 MHz, DMSO) δ 10.52 (s, 1H), 8.24 (d, J- 0.6 Hz, 1H), 7.91 (ddd, J = 8.8, 4.1,2.8 Hz, 1H), 7.74 (dd, J= 6.8, 2.7 Hz, 1H), 7.18 (dd, J~ 11.7, 8.8 Hz, 1H), 6.85-6.62 (m, 1H), 6.38 (s, 2H), 3.99 (s, 3H), 2.76 (d, J- 0.5 Hz, 3H), 2.55 - 2.49 (m. 2H), 2.18-1.99 (m, 2H).

LC-MS (m/z) 444.5 (MH*); Îr = 0.52 minutes (Method B)

Example 11 (S)-N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-cyano-3-methylpicolinamide (compound 11)

Prepared as In example 1 from (S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5difluoroplperidine-2-thlone and 5-cyano-3-methylplcolinic acid.

Ή NMR (600 MHz, DMSO) δ 10.80 (s, J = 27.6 Hz, 1 H), 8.99 (d, J- 1.2 Hz, 1 H), 8.40 (s, 1H), 7.99 - 7.87 (m, 1H), 7.82 - 7.69 (m, 1H), 7.36 - 7.13 (m, 1H), 6.73 (t, J= 55.0 Hz, 1H), 6.36 (s, 2H), 2.56 - 2.47 (m, 2H), 2.21-1.97 (m, 2H).

LC-MS (m/z) 438.1 (MH*); îr = 0.49 minutes (Method B)

Example 12 (S)-N-(3-(6-amino-2-(difluoromethyî)-3,3-difluoro-2₁3₁4,5-tetrahydropyridin-2-y1)-4fluorophenyl)-5-bromoplcolinamlde (compound 12)

Prepared as in example 1 from (S)-6-(5-amino-2-fluorophenyl)-6-(dÎfluoromethyl)-5,5difluoropiperidine-2-thione and 5-bromoplcolinic acid.

Ή NMR (600 MHz, DMSO) δ 10.78 (s. 1H), 8.87 (dd, J = 2.3, 0.7 Hz, 1H), 8.33 (dd, J= 8.4,2.3 Hz, 1 H), 8.09 (dd, J = 8.4, 0.6 Hz, 1H), 7.94 (ddd, J = 8.8, 4.1,2.8 Hz, 1H), 7.89 (dd, 6.8,2.7 Hz, 1 H), 7.21 (dd, J = 11.6,8.8 Hz, 1 H), 6.88 - 6.62 (m, 1 H), 6.39 (s, 2H), 2.60 - 2.49 (m, 2H). 2.19-1.96 (m, 2H).

LC-MS (m/z) 479.1 (MH*); fe = 0.53 minutes (Method B)

Example 13 (S)-N-(3-(6-amino-2-(difluoromethyl)-3_I3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyi)-5-(methoxy-d3)picolinamlde (compound 13)

(S)-6-(5-amino-2-fluoropheny1)-6-(difluoromethyl)-5,5-difluoroplperidine-2-thione (25 mg, 0.081 mmol) In was dissolved in dichloromethane (1 mL) under an atmosphère of argon. Trimethylalumlnum (52 pl, 0.105 mmol, 2 molar, toluene) was added slowly, then methyl(methoxy-d3)pico!inate (18 mg, 0.11 mmol) In 0.5mL dichloromethane was added. The réaction mixture was stirred at room température for 2 hours. The réaction mixture was poured into cooled 4N HCl (aq). The mixture was extracted with ethyl acetate. The organic phase was washed with brine, dried over magnésium sulphate and concentrated In vacuo. 7M ammonia in methanol (4mL) was added and the reaction mixture was stined In a sealed vial at 50^eC overnight. The reaction mixture was concentrated In vacuo and was purified by flash chromatography on silica gel (heptane/ethyl acetate) followd by purification by preperatlve HPLC to obtain the title compound as the trifluroacetic acid sait.

Ή NMR (600 MHz, DMSO) δ 11.13 (s, 1H), 10.75 (s, 1H), 9.98 (s, 1H), 9.10 (s. 1H), 8.40 (dd. J = 2.9,0.5 Hz, 1H), 8.21 (ddd, J = 8.9,4.1, 2.6 Hz, 1H), 8.15 (dd, J =8.7,0.5 Hz, 1H), 8.09 (dd, J = 6.8,2.6 Hz, 1H), 7.64 (dd, J = 8.7, 2.9 Hz, 1H), 7.40 (dd, J = 11.9,9.0 Hz, 1H), 7.20 (t, J = 53.0 Hz, 1H), 3.17 - 3.01 (m, 2H), 2.48-2.38 (m, 2H).

LC-MS (m/z) 432 (MH*); L = 0.49 minutes (Method A)

Example 14 (S)-W-(3-(6-amlno-2-(difluoromethyl)-3,3-difIuoro-2,3₁4,5-tetrahydropyridin-2-y1)-4fluorophenyl)-5-(methoxy-d3)pyrazlne-2-carboxamide (compound 14)

Prepared as In example 13 from (S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5difluoroplperidine-2-thione and methyl 5-(methoxy-d₃)picolînate.

¹H NMR (600 MHz, DMSO) δ 10.61 (s, 1 H), 8.89 (d, J = 1.2 Hz, 1H), 8.42 (d. J = 1.2 Hz, 1H),

7.91 -7,86 (m, 2H), 7.19 (dd, J = 11.5, 8.9 Hz, 1H), 6.84-6.61 (m, 1H), 6.36 (s, 2H), 2.532.49 (m, 2H), 2.16 -1.96 (m, 2H).

LC-MS (m/z) 433.1 (MH*); t« = 0.49 minutes (Method A)

Stereochemlstry

Crystals were obtained by recrystallization of (S)-5-bromo-N-(3-(2-(difluoromethyl)-3,3-difluoro-

6-thioxoplperidin-2-yl)-4-fluorophenyl)picolinamlde from a mixture of heptane and ethyl acetate. The structure of (S)-5-bromo-N-(3-(2-(difluoromethyl)-3,3-difluoro-6-thioxopiperidin-2-yl)-4fluorophenylJpicolÎnamide was elucidated by X-ray crystallography of said crystals. The structure shows the absolute and relative configuration of (S)-5-bromo-N-(3-(2-(difluoromethyl)-

3,3-difluoro-6-thioxopiperidin-2-yl)-4-fluorophenyl}picolinamide. (S)-5-bromo-A/-(3-(2(difluoromethyl)-3,3-difluoro-6-thloxopiperidin-2-y1)-4-fluorophenyl)picolinamide was prepared as described in example 1 starting from (S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)-5,5difluoroplperidine-2-thione and 5-bromopicolinic acid.

π

Figure 1 : X-ray structure of (S)-5-bromo-N-(3-(2-(difluoromethyl)-3,3-difluoro-6-thioxopiperidtn2-yl)-4-fluorophenyl)picolinaniide

The absolute configurations of the exemplified compounds of the présent invention can thus be rationalized. (S)-5-bromo-N-(3-(2-(difluoromethyl)-3,3-difluoro-6-thioxopiperidin-2-yl)-4fluoro phenyl Jpicolinamide was prepared from (S)-6-(5-amino-2-fluorophenyl)-6-(difluoromethyl)5,5-difluoropiperidine-2-thione which Is staring material for ail exemplified compounds of the présent Invention.

Pharmacologlcal Testlnq

BACE1 binding assay

The binding assay was performed as SPA-based assay using a biotinylated form of human BACE1 recombinantly expressed and subsequently purified from Freestyie HEK293 cells. The binding assay was run in a 50 mM sodium acetate buffer, pH 4.5 containing 50 mM NaCI and 0.03% Tween-20 in white clear bottom 384 plates (Coming #3653). 10 nM (final concentration) radioligand ([³H]-N-((1S,2R)-1-benzyl-3-cyclopropylamino-2-hydroxy-propyl)-5(methanesulfonyl-methyl-amino)-N-((R)-1-phenyl-ethyl)-lsophthalamide) (TRQ11569 purchased from GE Healthcare) was mixed with test compound at a given concentration, 6 nM (final concentration) human BACE1 and 25 pg Streptavidin coated PVT core SPA beads (RPNQ0007, GE Healthcare Life Sciences) in a total volume of 40 pi. Several concentrations of each test compound were tested in the assay for IC50 détermination. The plates were Incubated for one hour at room température and counted In a Wallac Trilux counter. Total and non-spedfic binding were determined using buffer and 1 pM (final concentration) of the high affinity BACE1 reference inhibitor (S)-6-[3-chloro-5-(5-prop-1 -ynyl-pyridin-3-yl)-thiophen-2-yl]-2-lmino-3,6dimethyl-tetrahydro-pyrimidÎn-4-one, respectively. For each test compound, a IC_M value (the concentration mediatîng 50% Inhibition of the spécifie binding of the radioligand) was determined from concentratron-response curve and used to calculate the K| from the équation K= ICso/fl+L/Kd), where L and Kj are the final concentration of the radioligand used in the assay and the dissociation constant of the radioligand, respectively. The Kd ofthe radioligand was 5 determined from saturation binding experiments.

Table 1: binding affinity of selected compounds

Compound No	BACE1 Kl (nM)
1	18
2	23
3	8.5
4	20
5	6.7
6	20
7	7.4
8	71
9	6.9
10	16
11	7.8
12	6.1
13	14
14	18

BACE1 efficacy assay

The efficacy assay was performed as a FRET-based assay using a commercially available BACE1 kit (Life Technologies, P2985). 2 pl test compound at 10 μΜ (final concentration) and 15 μΙ BACE1 enzyme from the kit (final concentration 3 nM) were preincubated for 15 minutes at room température before addition of 15 μΙ of substrate from the kit (250 nM final concentration) and Incubated for additional 90 minutes at room température. The assay plate was subsequently read in a Pherastar (Ex540/Em590). The enzyme activity observed in presence of test compound were normalized to the enzyme activity observed in presence of buffer and 10 μΜ (final concentration) of the high affinity BACE1 reference Inhibitor (S)-6-[3-Chloro-5-(5-prop1-yny1-pyridin-3-yi)-thiophen-2-yl]-2-imino-3,6-dimethy!-tetrahydropyrimidin-4-one, respectively.

The efficacy of the test compounds was evaluated at 10 μΜ (final concentration) and defined as the percent inhibition of the enzyme activity using the équation %inhibîtion - 100% - normalized enzyme activity In percent.

Table 2: BACE1 activity of selected compounds

CompoundNo	BACE1 Inhibition at 10μΜ (%)
1	106
2	100
3	103
4	104
5	101
6	103
8	102
9	103
10	106
13	103
14	108

Assessment of Αβ levels In rat brain and plasma following BACE1 Inhibition, Animais.

Ail rat care and experimental procedures were approved by Lundbeck Veterinary Staff, according to Danish législature. The rats were maintained In a barrier facility with a 12/12-h light/dark cycle and ad libitum food and water access.

Treatment of naïve Rats.

Young adult Male Sprague Dawley rats of approximately 250g weight were purchased from Charles River and received 0-30 mg/kg of vehicle (10% HP betaCD + 1M MeSO<, pH 2.5) or test compounds (dissolved In vehicle) only by oral gavage (p.o). The compounds are dosed at a volume of 5ml/kg. Cohorts of 5-10 animais were established for each treatment condition.

The animais undergoing treatment were dosely monitored by veterinary staff for any signs of toxicity. Monitoring parameters Included body weight, physical appearance, changes In coat appearance, occurrence of unprovoked behavior, and blunted or exaggerated responses to extemal stimuli.

Tissue collection,

At T =180 minutes after initial dosing the animais were stunned and decapitated with a guillotine. Trunk-blood was sampled în EDTA coated tubes after décapitation of the animal. The blood was centrifùged at 2200G at 4'C for 15 minutes and the plasma was collected and frozen at -80‘C. The blood was aliquoted for Αβ ELISA and DMPK analysis. Immediately following sacrifice, the brain was extracted and split Into 2 halves. The right hemibrains were snap frozen on dry Ice and stored at -80‘C. The left half was dissected; with the front forebrain taken for Αβ ELISA and the remainder used for DMPK analysis. These samples were also snap frozen on dry ice and stored at -80*C until use for analysis.

Tissue processing.

The cortex samples were thawed slightly on wet Ice before they were homogenized with a small volume dispersing instrument (T10 basic ULTRA-TURRAX®) which was set at speed 5 for approximately 5-7 sec. The tissue was processed In a 10 times volume of the weight, for example 100mg of tissue was homogenized in 1000pL of Homogenization buffer.

Homogenization buffer. 50ml Milli Q water + 50nM NaCI + 0.2% Diethylamin (DEA) + 1 tablet of Complété Protease Inhibitor cocktail + 1nM 4-(2-amlnoethyl) benzenesulfonyl fluoride hydrochloride irréversible serine protease inhibitor (AEBSF).

After homogenization 450 pL aliquots of the samples are collected into a 1.5ml Eppendorf tube and placed on wet Ice, 0.5% NP-40 (50ul) was added to ail samples and then they were incubated on Ice for 30 min. After which ail samples were sonlcated using an Ultrasonic homogenizer with 20 kHz homogeneous sound (SONOPLUS HD2070, Bandelîn Electronic) 10 puise set at 12-13 % power to extract ail the Ap species. The samples were then centrifùged (Ole Dich 157 MPRF Micro centrifuge) at 20000G for 20 minutes at 4‘C. After centrifugation 285pL of the supematant was plpetted Into 600pL microtubes tubes and neutralized with 15pL of1MTris-HCL buffer.

ELISA protocol.

WAKO 294-62501 Human/Rat Abeta amylold (40) kit was used for ail ELISA analyses. 30 pL plasma samples or 30 pL of the cortex supematants generated as described above were placed in 600 pL microtubes tubes on wet Ice. To this 30 pL of 8M Urea (AppliChem A1049, 9025) are added to generate a 2-fold dilution. Both plasma and cortex supematants are incubated on ice for 30 min. Standard rows were prepared from the standard peptide stock provided in the kit and standard diluent containing 1.6M Urea (200 pL 8M Urea + 800 pL of standard diluent) and 0.8M Urea (400pL 8M Urea + 3600pL Standard diluent). A serial 2-fold dilution of Αβ40 from 100 pmol/ml to 0 pmol/L was prepared for the assay.

After Incubation with urea, ail samples were further diluted by addition of 5 times standard diluent from the Kit. This was done by adding 240 pL Standard Diluent to 60 pL sample/urea mixture, which was then mixed well. 100 pL of each diluted sample was pipetted into designated wells of the ELISA plate In duplicates. The plate was then covered and incubated ovemight at 4’C. The following day, the ELISA kit was brought to room température before use. The incubated plate was washed 5 times with the 20x washing solution diluted in Milli Q water. 100 pL HRP-conjugate was applied to each well, and the plate was covered and incubâtes at 4'C for 1 hr. The wash was repeated again for 5 times. 100 pL 3₍3',5,5'-Tetramethylbenzidine (TMB) solution was applied to each well and the plate was covered and incubated In the dark at room température for 30 minutes. 100 pL STOP-solution was next applied to each well, and the plate was read at 450 nm wavelength In a spectrophotometer (Labsystems Muîtiscan Ascent) within 30 min of adding the STOP-solution to the wells.

Concentration of Αβ in the samples was determined based on a standard curve generated from standards containing known concentrations of synthetic Αβ40. Those skilled in the art will appreciate that diethylamine (DEA) and urea extractions will release soluble Αβ, and insoluble Αβ respectively. Since the ELISA kit Is validated and widely used, it is accepted that as long as the treatment conditions and assay conditions are the same for each compound tested, then the assay should yield consistent robust data for the compounds tested and produce minimal discrepancies.

Data analysis

To détermine the concentration of Αβ40 in the samples, the interpolated values of the samples loaded on plates are multiplied by 20 to account for the dilutions made when the volumes of

DEA, urea and neutralization solution were added up. Values are calculated as percentage change in Αβ40 compared to vehicle treated animais.

Bloanalysls of brain and plasma samples

TC was determined in plasma and brain homogenate using UltraPerformance LC® (UPLC®) chromatography followed by tandem-MS (MS/MS) détection.

Apparatus:

Tecan Genesis RSP 200; Biomek NXP, Beckman Coulter; Sigma 4K15 centrifuge; Acquity UPLC, Waters; Sciex API4000 TQ, Applied Biosystems; MS software: Analyst version 1.4.1 Chemicals

Acetonitrile, HPLC-grade, Fluka, No. 34967N; Methanol, HPLC-grade, Sigma-Aldrich, Lot 9003S; Formic acid, HPLC-grade, Riedel-de Haën, Lot 51660; Purified water, Millipore Synergy UV

Sample préparation

Brain homogenate was prepared by homogenizing the brain 1:4 (v/v) with water2propanokDMSO (50:30:20 v/v/v) followed by centrifugation and collection ofthe supematant. Calibration standards and QC samples were prepared using a Hamilton robot. 150 pL of ISTD In acetonitrile (1 ng/mL ISTD) was added to 25 pL of calibration standards, QC samples and test samples (plasma and brain homogenate) using a Biomek robot. After centrifugation (6200 g, 4 ^eC, 20 min) 100 pL supematant from each sample was transferred to a new plate and mixed with 100 pL water with 0.1 % formic acid using a Biomek robot (method file InVivo transfer). After a qulck centrifugation (6200 g, 4 °C, 5 mln) the samples were placed in the auto-sampler. UPLC-MS/MS analysis

MS/MS détection was done with an Applied Biosystems Sciex API 4000 instrument In positivelon electrospray ionisation mode. TC and ISTD were detected at a parent > daughter mass to charge ratio (m/z). Nitrogen was used for the nebulizer and collision gases. The peak area correlated linearly with the plasma and brain concentration of the analytes In the range of 1.00 — 1000 ng/mL plasma and 5.00 - 5000 ng/g brain (conected for dilution). If the plasma/brain sample drug concentration was above 1000 ng/mL or 5000 ng/g, the sample was diluted appropriately in blank plasma/blank brain homogenate before analysis.

Chromatographie system

Analytical columns:

Waters Acquity UPLC HSS C18 SB (pH 2-8) 1.8 pm, 2.1x30mm.

Mobile phase A: 0.1 % aq. formic acid or 0.1 % aq. ammonium hydroxide

Mobile phase B: Acetonitrile with 0.1 % aq. formic acid or 0.1 % aq. ammonium hydroxide.

Weak wash: Methanol

Strong wash: Acetonitrile/lsopropanol/formic acid (50/50/2 v/v/v)

Flow: 0.6 mL/mln Run time: 3 min.

To waste: 0-0.5 min

Température: 40*C

Gradient:

Time (min)	%A	%B
0	98	2
0.01	98	2
1.5	5	95
2	5	95
2.2	98	2
3	98	2

Compounds 3 and 5 were admistered at doses of 10 mg/kg p.o. and brain and plasma samples were collected at 3 hours post dose and the following exposures were measured as described above.

Table 3: Results for compound 3

-	Dose (mg/kg)	Exp (ng/g)	Brain/Plasma ratio	Αβ40 réduction (%)
Brain Rat Plasma Rat	10	511 1682	0.30	24 39
Brain Rat Plasma Rat	30	2284 7056	0.32	38 42

Table 4: Results for compound 5

	Dose (mg/kg)	Exp (ng/g)	Brain/Plasma ratio	Αβ40 réduction (%)
Brain Rat Plasma Rat	10	187 660	0.28	5 40
Brain Rat Plasma Rat	30	959 3348	0.29	36 49

As shown In tables 3 and 4, compounds of the présent Invention are able to penetrate the blood brain barrier and show efficacy in the CNS.

MDCK-MDR1 assay

The permeability of the test compounds was assessed In MDCK-MDR1 cells that were cultured to confluency (4-6 days) in a 96 transwell plate. Test compounds were diluted with the transport buffer (HBSS + 1% BSA) to a concentration of 0.5 μΜ and applied to the apical or basolateral side of the cell monolayer. Perméation of the test compounds from A to B direction or B to A direction was determined In triplicate over a 60-minute Incubation time at 37’C and 5% CO2 with a relative humidity of 95%. Test compounds were quantifiée! by LC-MS/MS analysis based on the peaks area ratios of analyte/IS In both the recelverand donor wells of the transwell plate. The apparent permeability coefficient Papp (cm/s) was calculated using the équation: Papp = (dCr/dt)xVr/(AxCO)

Where dCr/dt Is the cumulative concentration of compound In the receiver chamber as a function of time (μΜ/s); Vr is the solution volume in the receiver chamber (0.05 mL on the apical side; 0.25 mL on the basolateral side); A is the surface area for the transport, I.e. 0.0804 cm² for the area of the monolayer; C0 is the Initia! concentration In the donor chamber (μΜ). Compounds are dassified Pgp substrates when efflux ratio (Papp BA / Papp AB) Is 2 2.

Table 5: BACE1 activity of selected compounds

Compound	MDCK- MDR1 efflux ratio
1	1.16
2	1.75
3	1.22

4	4.01
5	0.99
6	1.36
7	2.43
8	0.92
9	10.99
10	0.98
11	2.68
12	1.31
13	0.83

As shown in tables 5, the majority of the exemplified compounds of the présent invention hâve MDCK- MDR1 efflux ratios below 2 and are thus likely to be able to cross the blood brain barrier (E Kems, L Di, Drug-like Properties: Concepts, Structure Design and Methods (2008) Elsevier).

I

- - t

Claims (14)

1.

A compound of formula I

NH₂

Formula I wherein Ar is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazinyl, imidazolyl, pyrazofyl, thiazolyl, oxazolyl, isoxazolyf, and where the Ar is optionally substituted with one or more substituent selected from halogen, CN, CrCe alkyl, CrCe alkenyl, CrCe alkynyl, C_rCe fluoroalkyl or Ci-Ce alkoxy; and

R¹ is hydrogen, halogen, C1-C3 fluoroalkyl or CpCa alkyl;

or a pharmaceutically acceptable sait thereof.

2. A compound according to claim 1, wherein the compound is of formula la

NH

R¹

Formula la;

or a pharmaceutically acceptable sait thereof.

3. The compound according to claim 1 or 2, wherein R¹ is F or H.

4. The compound according to claim 1 or 2, wherein Ar ls optionally substituted with one or more F, Cl, Br, CN, CrQj alkyl, CrCj fluoroalkyl or Cj-Ca alkoxy.

5. The compound according to anyone of claims 1-4, wherein Ar is optionally substituted pyridyl.

6. The compound according to anyone of claims 1-4, wherein Ar is optionally substituted pyrimidyl.

7. The compound according to anyone of daims 1-4, wherein Ar is optionally substituted pyrazinyl.

8. The compound according to anyone of daims 1 -4, wherein Ar is optionally substituted oxazolyl.

9. The compound according to anyone of daims 1-4, wherein Ar is optionally substituted thiazolyl.

10. The compound according to daim 1, wherein the compound is selected from the group consisting of: (S)-N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-€hloropicolinamide, (S)-N-(3-{6-amino-2-{difluo!Omethyl)-3,3-difluoro-2₁3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-fluOropicolinamtde, (S)-AH3-(6-amino-2-(dîfluoromethyi)-3₁3-difluoro-2,3,4,5-tetrahydropyridin-2-yiy4fluorophenyl)-5-methoxypyrazine-2-carboxamide₁ (S)-N^3-(6-amino-2-(dinuoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-2-methyloxazole-4-carboxamide₁ (S)-N-(3-(6-amino-2-(difluoiOmethy1)-3,3-difluoro-2,3,4,5-tetrahydrOpyridin-2-yl)-4fluorophenyl)-5-methoxypicolinamide, (S)-N-(3-(6-amino-2-{dinuoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-(difluorDmethyl)pyrazine-2-carboxamÎde, (S)-N-(3-(6-amino-2-(dÎfluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridÎn-2-yl)-4fluorophenyl)-5-cyanopicolÎnamide, (S)-N<3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2₁3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl>4-methylthiazole-2-carboxamide, (S)-N-(3-{6-amino-2-{difluoromethyl)-3₁3-difluoro-2,3₁4,5-tetrahydropyridÎr>-2-yl)-4fluorophenyl)-5-methoxypyrimidine-2-carboxamide, (S)-AA(3-(6-amino-2-(difluoromethyi)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluoropheny1)-5-methoxy-3-methylpyrazine-2-carboxamide, (S)-/V-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydiOpyridin-2-yl)-4fluorophenyl)-5-cyano-3-methyipicolinamide, (S)-N-(3-(6-amino-2-(difluoromethyl)-3₁3-difluoro-2₁3,4_>5-tetrahydropyridin-2-yl)-4fluorophenylJ-S-bromopicolinamide, (S)-/V-(3-(6-amino-2-(difluo ro m ethyl)-3,3-difIuoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-(methoxy-d3)picolinamide and (S)-N-(3-(6-amino-2-(difluorümethyiy3,3-difluorD-2,3,4,54etrahydropyridin-2-yl}4fluorophenyl)-5-(methoxy-d3)pyrazine-2-carboxamide;

or a pharmaceutically acceptable sait thereof.

11. A pharmaceutical composition comprising a compound according to anyone of claims 1-10 and a pharmaceutically acceptable carrier.

12. The use of a compound according to anyone of claims 1-10 for the manufacture of a médicament for treating a disease selected from Alzheimer’s disease (familial or sporadic), preclinical Alzheimer’s disease, prodromal Alzheimer’s disease, mild cognitive impairment, Down's syndrome and cérébral amyloid angiopathy.

13. A compound according to anyone of claims 1-10 for use In therapy.

14. A compound according to anyone of claims 1-10 for use in the treatment of a disease selected from Alzheimer’s disease (familial or sporadic), preclinical Alzheimer’s disease, prodromal Alzheimer's disease, mild cognitive impairment, Down's syndrome and cérébral amyloid angiopathy.