NZ626662B2

NZ626662B2 - 6-difluoromethyl-5,6-dihydro-2h-[1,4]oxazin-3-amine derivatives

Info

Publication number: NZ626662B2
Application number: NZ626662A
Authority: NZ
Inventors: Henricus Jacobus Maria Gijsen; Hana Prokopcova; Michel Surkyn; Suarez Andres Avelino Trabanco
Original assignee: Janssen Pharmaceutica Nv
Priority date: 2011-12-05
Filing date: 2012-12-04
Publication date: 2015-09-01

Abstract

The disclosure relates to 6-difluoromethyl-5,6-dihydro-2H-[1,4]oxazin-3-amine derivatives of formula (I). These work as inhibitors of beta-secretase, also known as beta-site amyloid cleaving enzyme, BACE, BACE1, Asp2, or memapsin2. The disclosure also relates to pharmaceutical compositions comprising such compounds, to processes for preparing such compounds and compositions, and to the use of such compounds and compositions for the prevention and treatment of disorders in which beta-secretase is involved, such as Alzheimer's disease (AD), mild cognitive impairment, senility, dementia, dementia with Lewy bodies, cerebral amyloid angiopathy, multi-infarct dementia, Down's syndrome, dementia associated with stroke, dementia associated with Parkinson's disease and dementia associated with beta-amyloid. g such compounds, to processes for preparing such compounds and compositions, and to the use of such compounds and compositions for the prevention and treatment of disorders in which beta-secretase is involved, such as Alzheimer's disease (AD), mild cognitive impairment, senility, dementia, dementia with Lewy bodies, cerebral amyloid angiopathy, multi-infarct dementia, Down's syndrome, dementia associated with stroke, dementia associated with Parkinson's disease and dementia associated with beta-amyloid.

Description

-DIFLUOROMETHYL—5,6-DIHYDRO-2H-[1,4]OXAZINAMINE DERIVATIVES FIELD OF THE INVENTION The present invention relates to novel 6-diﬂuoromethyl—5,6—dihydro-2H- [1 ,4]oxazin—3-amine derivatives as inhibitors of beta—secretase, also known as beta—site amyloid cleaving enzyme, BACE, BACEl, Asp2, or memapsinZ. The invention is also directed to pharmaceutical compositions comprising such nds, to processes for preparing such compounds and itions, and to the use of such compounds and itions for the prevention and ent of disorders in which beta—secretase is involved, such as mer‘s disease (AD), mild cognitive impairment, senility, dementia, dementia with Lewy bodies, Down's syndrome, dementia associated with stroke, dementia associated with Parkinson's disease and dementia associated with beta- amyloid.

BACKGROUND OF THE INVENTION Alzheimer's Disease (AD) is a neurodegenerative e associated with aging.

AD patients suffer from cognition deﬁcits and memory loss as well as behavioral problems such as y. Over 90% ofthose afﬂicted with AD have a sporadic form of the disorder while less than 10% of the cases are familial or hereditary. In the United States, about 1 in 10 people at age 65 have AD while at age 85, 1 out of every two individuals are affected with AD. The average life ancy from the initial diagnosis is 7-10 years, and AD patients e extensive care either in an assisted living facility which is very costly or by family members. With the increasing number of elderly in the population, AD is a growing medical concern. Currently available therapies for AD merely treat the symptoms of the disease and include acetylcholinesterase tors to improve cognitive properties as well as anxiolytics and ychotics to control the oral problems associated with this ailment.

The hallmark ogical features in the brain ofAD patients are neuroﬁbillary tangles which are generated by hyperphosphorylation of tau protein and amyloid plaques which form by aggregation of beta-amyloid 1—42 (Abeta 1-42) peptide. Abeta 1-42 forms oligomers and then ﬁbrils, and ultimately amyloid plaques. The oligomers and ﬁbrils are believed to be especially neurotoxic and may cause most of the neurological damage associated with AD. Agents that prevent the formation of Abeta 1-42 have the potential to be disease—modifying agents for the treatment of AD. Abeta W0 2013(083557 2012/074351 1-42 is generated from the amyloid precursor protein (APP), comprised of 770 amino acids. The N-terminus of Abeta 1-42 is cleaved by beta-secretase (BACE), and then gamma-secretase cleaves the C-terminal end. In addition to Abeta 1-42, gamma- secretase also liberates Abeta 1-40 which is the predominant cleavage product as well as Abeta 1-38 and Abeta 1-43. These Abeta forms can also aggregate to form oligomers and ﬁbrils. Thus, inhibitors ofBACE would be expected to prevent the formation of Abeta 1—42 as well as Abeta 1-40, Abeta 1—38 and Abeta 1-43 and would be potential therapeutic agents in the treatment of AD.

WO-2011/009943 (Novartis) discloses unsubstituted and 2-substituted oxazine derivatives and their use as BACE inhibitors for the treatment of neurological disorders. WO—2011/020806 ann—LaRoche) discloses 2,6-unsubstituted 3—amino—5-phenyl-5,6-dihydro-2H—[l,4]oxazine derivatives having BACEl and /or BACE2 inhibitory properties.

SUMMARY OF THE INVENTION The present invention is directed to 5,6-dihydro-2H-[1,4]oxazin—3-amine derivatives of Formula (I) o CHF2 L R1 HZN N L\AI and the tautomers and the stereoisomeric forms thereof, wherein R1 is C1_3alkyl; R2 is hydrogen or ﬂuoro; L is a bond or —NHCO-; Ar is ed from the group consisting of pyridinyl, pyrimidinyl and pyrazinyl, each ally susbstituted with halo or C1_3a]koxy; and the ceutically acceptable addition salts thereof. rative of the invention is a ceutical composition sing a pharmaceutically acceptable carrier and any ofthe compounds described above. An illustration ofthe invention is a pharmaceutical composition made by mixing any of the compounds described above and a pharmaceutically acceptable carrier. Illustrating the invention is a process for making a pharmaceutical composition comprising mixing any ofthe compounds described above and a pharmaceutically acceptable carrier.

Exemplifying the invention are methods of treating a disorder mediated by the beta-secretase , comprising administering to a subject in need thereof a therapeutically effective amount of any ofthe compounds or pharmaceutical compositions described herein. r exemplifying the invention are s of inhibiting the ecretase enzyme, comprising administering to a t in need thereof a therapeutically effective amount of any ofthe nds or pharmaceutical itions described herein.

An example of the invention is a method of treating a disorder ed from the group consisting ofAlzheimer‘s disease, mild cognitive impairment, senility, dementia, dementia with Lewy bodies, cerebral amyloid angiopathy, multi-infarct dementia, Down's syndrome, dementia associated with stroke, ia associated with Parkinson's disease and dementia associated with beta-amyloid, preferably mer's disease, comprising administering to a subject in need thereof, a therapeutically effective amount of any of the compounds or pharmaceutical compositions described herein.

Another example of the ion is any of the nds described above for use in treating: (a) Alzheimer's Disease, (b) mild cognitive impairment, (0) senility, (d) dementia, (e) dementia with Lewy , (f) Down's syndrome, (g) dementia associated with stroke, (h) dementia associated with Parkinson's disease and (i) dementia associated with beta-amyloid, in a subject in need thereof.

DETAILED DESCRIPTION OF THE ION The present invention is directed to nds of Formula (I) as deﬁned hereinbefore and pharmaceutically acceptable salts and solvates thereof. The compounds ofFormula (I) are inhibitors of the beta—secretase enzyme (also known as beta—site cleaving enzyme, BACE, BACEl or memapsin 2), and are useful in the , Asp2 treatment of mer's disease, mild cognitive impairment, senility, dementia, dementia associated with stroke, dementia with Lewy bodies, Down's syndrome, dementia associated with Parkinson's disease and dementia associated with beta- amyloid, preferably Alzheimer's disease, mild cognitive impairment or dementia, more preferably mer's disease.

WO 2013083557 In an embodiment ofthe invention, R1 is methyl or ethyl.

In an embodiment of the invention Ar is ed from 5-methoxy-pyridinyl, -pyrimidiny1 and S-ﬂuoropyrazinyl.

In another embodiment of the ion, R2 is hydrogen or ﬂuoro.

In another embodiment, the quaternary carbon atom tuted with R1 has the R-conﬁguration.

DEFINITIONS "Halo" shall denote ﬂuoro, chloro and bromo; "C1_3alkyloxy" shall denote an ether radical wherein C1_3a]kyl is a straight or branched saturated alkyl group having 1, 2 or 3 carbon atoms, e.g. , ethyl, yl and 2-propy1.

The term "subj ect" as used herein, refers to an animal, preferably a mammal, most preferably a human, who is or has been the obj ect of treatment, observation or experiment.

The term peutically ive amoun " as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal se in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes ation of the symptoms of the disease or disorder being treated.

As used herein, the term "composition" is intended to encompass a product comprising the speciﬁed ingredients in the speciﬁed amounts, as well as any product which results, ly or indirectly, from combinations of the speciﬁed ingredients in the speciﬁed amounts.

Hereinbefore and hereinafter, the term "compound of formula (1)" is meant to include the addition salts, the solvates and the stereoisomers thereof.

The terms "stereoisomers" or "stereochemically isomeric forms" hereinbefore or hereinafter are used interchangeably.

The invention includes all stereoisomers of the compound of Formula (I) either as a pure stereoisomer or as a mixture of two or more stereoisomers.

Enantiomers are stereoisomers that are perimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemate or racemic mixture.

Diastereomers (or diastereoisomers) are stereoisomers that are not enantiomers, i.e. they are not related as mirror images. Therefore, the invention includes enantiomers, diastereomers, racemates.

The absolute conﬁguration is speciﬁed according to the Cahn-Ingold-Prelog system.

The conﬁguration at an asymmetric atom is speciﬁed by either R or S. Resolved compounds whose te conﬁguration is not known can be designated by (+) or (—) ing on the direction in which they rotate plane polarized light.

When a speciﬁc isomer is ﬁed, this means that said stereoisomer is substantially free, i.e. associated with less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, in particular less than 2% and most preferably less than 1%, of the other s. Thus, when a compound of formula (I) is for ce ed as (R), this means that the compound is substantially free of the (S) isomer.

The compounds ofFormula (I) co—exist in a dynamic equilibrium with the tautomers of Formula (I-a). o cm:2 L 1 ___._ HzN N L\Ar " R2 (I) (I-a) Furthermore, some ofthe crystalline forms for the compounds of the present invention may exist as polymorphs and as such are intended to be included in the present invention. In addition, some of the compounds of the present invention may form solvates with water (i.e., hydrates) or common organic solvents, and such so lvates are also intended to be encompassed within the scope of this invention.

For use in medicine, the salts of the compounds of this invention refer to non- toxic "pharmaceutically acceptable salts". Other salts may, however, be useful in the preparation of compounds according to this invention or of their pharmaceutically acceptable salts. le pharmaceutically acceptable salts of the compounds e acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, ﬁJmaric acid, maleic acid, ic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compounds of the invention carry an acidic moiety, le pharmaceutically acceptable salts thereofmay include alkali metal salts, e.g., sodium or potassium salts; WO 83557 2012/074351 alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.

Representative acids which may be used in the preparation of pharmaceutically able salts include, but are not limited to, the ing: acetic acid, 2,2—dichloro— acetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L—aspartic acid, benzenesulfonic acid, benzoic acid, 4- acetamidobenzoic acid, (+)—camphoric acid, camphorsulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2—hydroxy— ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-giuconic acid, D—glucoronic acid, L~g1utamic acid, beta-0x0- glutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, (+)—L—lactic acid, (i)-DL—1actic acid, lactobionic acid, maleic acid, (—)-L-malic acid, malonic acid, (dz)-DL-mandelic acid, methanesulfonic acid, alenesulfonic acid, naphthalene-1,5- disulfonic acid, 1—hydroxynaphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palrnitic acid, pamoic acid, phosphoric acid, L— pyroglutamic acid, salicylic acid, 4-amino-sa1icylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, anic acid, p-toluenesulfonic acid, triﬂuoromethylsulfonic acid, and undecylenic acid.

Representative bases which may be used in the preparation of pharmaceutically acceptable salts include, but are not limited to, the following: a, L—arginine, benethamine, benzathine, calcium hydroxide, choline, ylethanolamine, diethanolamine, diethylamine, 2-(diethylamino)—ethano1, ethanolamine, ethylene— diamine, N—methyl-glucamine, amine, 1H—imidazole, L—lysine, magnesium hydroxide, 4-(2—hydroxyethyl)—morpho line, zine, potassium hydroxide, 1—(2-hydroxyethyl)—pyrrolidine, secondary amine, sodium hydroxide, triethanolamine, tromethamine and zinc hydroxide.

The names of the compounds ofthe present invention were generated according to the nomenclature rules agreed upon by the Chemical cts Service (CAS) using Advanced Chemical Development, Inc., software (ACD/Name product version 10.01; Build 15494, 1 Dec 2006) or according to the nomenclature rules agreed upon by the International Union of Pure and Applied try (IUPAC) using Advanced Chemical Development, Inc., software (ACD/Name product version 4105, October 2006). In case oftautomeric forms, the name of the depicted tautomeric form ofthe structure was generated. The other non-depicted tautomeric form is also included within the scope of the present invention.

Preparation of the compounds Experimental procedure 1 The ﬁnal compounds according to Formula (I) can be prepared by catalytic hydrogenation of an intermediate compound of Formula (II-a) according to reaction scheme (1). Said conversion may be conducted by treatment of the intermediate compound of Formula (II—a) with hydrogen in the presence of a suitable catalyst such as, for example, palladium on carbon, a suitable catalyst poison, such as, for example, thiophene, in a suitable on-inert solvent, such as, for example, ethyl acetate or methanol. The mixture is stirred under hydrogen atmosphere, at a le temperature, lly room temperature, at a suitable pressure, such as, for example, heric pressure, for example for 16 hours. In reaction scheme (1), all les are deﬁned as in Formula (I).

L F ‘reduction" H2N N HZN/E: (II-a) R2 (I) R2 on Scheme 1 Ex erimental rocedure 2 The intermediate compounds of Formula (II-b) can generally be prepared by reacting an intermediate compound of Formula (111) with a compound of Formula (IV) according to reaction scheme (2), a reaction that is performed in a suitable reaction- inert solvent, such as, for example, dichloromethane or methanol, in the presence of a suitable base, such as, for example, triethylamine, in the presence of a condensation agent such as for e 0—(7azabenzotriazol—1-yl)-N,N,N’,N’-tetramethyluronium hexaﬂuorophosphate [HATU, CAS 1488931] or 4-(4,6-dimethoxy—1 ,3 azin- 4—methylmorpholinium chloride , CAS 3945—69-5] under thermal conditions such as, for example, heating the reaction mixture at 25 °C, for the required time to achieve tion of the reaction, for example 1-16 hours. In reaction scheme (2), all variables are deﬁned as in Formula (I). _ 8 ..

Howl/AI O F (IV) ,F \ H AI HZN N N (II-b) R2 Reaction Scheme 2 Ex erimental rocedure 3 Intermediate compounds of Formula (II-c) can generally be prepared by the on of intermediate compounds ofFormula (V1) with an appropriate oronate or aryl boronic acid in a Suzuki type reaction. Thus intermediate compounds of Formula (VI) can react with an aryl—boronate or aryl boronic acid in a suitable reaction- inert solvent, such as, for example, 1,4-dioxane, ethanol or mixtures of inert solvents such as, for example, 1,2-dimethoxyethane/water/ethanol, in the presence of a suitable base, such as, for example, aqueous K3PO4, Na2C03 or CSzCO3, a Pd—complex catalyst such as, for example, [1,1’-bis(diphenylphosphino)ferrocene]dichloropalladium(II) [CAS 722874] or trans-bisdicyclohexylamine)palladium ate [DAPCy, CAS 6283398] or tetrakis(triphenylphosphine) palladium (0) [CASl42213] under thermal conditions such as, for example, heating the reaction mixture at 80 °C, for example for a period of time between 2-20 hours or for example the reaction mixture at 130 °C, for e for 10 minutes under ave irradiation. In reaction scheme (3), all les are deﬁned as in Formula (I) and W is halo. R3 and R4 may be hydrogen or alkyl, or may be taken together to form for example a bivalent l of formula —CH2CH2—, ~CH2CH2CH2-, 01' -C(CH3)2C(CH3)2-. 9-19 Ar—‘B\O/R4 (II-c) Reaction Scheme 3 Experimental procedure 4 The intermediate compounds of Formula (111) can generally be prepared following the reaction steps shown in the reaction scheme (4) below.

W0 20131083557 _ 9- O / 0 IF B R _> E W HZN N R R_ (VII) (V1) I F HzN N NH2 (11]) R2 Reaction Scheme 4 A: Bromo—to-amine conversion B: thioamide-to—amidine sion C: amide-to—thioamide conversion (thionation) Intermediate compounds of Formula (111) in the above on scheme (4) can be prepared from the corresponding intermediate compounds of Formula (VI) following art—known copper catalyzed type coupling procedure (reaction step A). Said coupling may be conducted by treatment of said intermediate compounds of Formula (VI) with sodium azide in a suitable reaction-inert solvent, such as, for example, DMSO, in the presence of a mixture of suitable bases, such as, for example, dimethyl— ethylenediamine and , and a copper st such as, Cul, under thermal conditions such as, for example, heating the reaction mixture at 110 °C, until completion ofthe reaction, for example 1 hour.

Intermediate compounds of Formula (VI) in the above reaction scheme (4) can be prepared from the corresponding intermediate compounds of Formula (VII) following art-known thioamide-to-amidine conversion ures (reaction step B).

Said conversion may conveniently be conducted by treatment of intermediate compounds ofFormula (VII) with an ammonia source such as, for example, um chloride or aqueous ammonia, in a suitable on-inert solvent such as, for example, water or methanol and the like, under thermal conditions such as, for e, heating the reaction e at 60 °C, for example for 6 hours.

WO 2013083557 Intermediate compounds of Formula (VII) in the above reaction scheme (4) can be prepared from the corresponding intermediate compounds of Formula (VIII) following art-known thionation procedures (reaction step C). Said conversion may conveniently be ted by treatment of intermediate compounds of Formula (VIII) with a thionation agent such as, for example, phosphorous pentasulﬁde or 2,4-bis— (4-methoxyphenyl)~l,3—dithia-2,4—diphosphetane 2,4-disulﬁde [Lawesson’s reagent, CAS 19172-47—5], in a reaction inert solvent such as, for example, tetrahydrofuran or 1,4-dioxane and the like, under thermal ions such as, for example, heating the reaction mixture at 50 0C, for example for 50 s.

Experimental procedure 5 The intermediate compounds of Formula (VIII) and (IX) can generally be prepared from ediate compounds of Formula (X) following art-known reductive dehalogenation procedures (reaction step D). Said conversion may be conducted by treatment of the ediate of Formula (X) with a suitable zinc t, such as, for example, zinc dust or zinc copper couple in a suitable t, such as acetic acid, at a suitable temperature, typically from room temperature to 80 °C, for the required time to achieve completion ofthe reaction, for example 1-16 hours. This conversion affords a mixture ofthe intermediate compounds of a (VIII) and (IX) in different ratio depending on the reaction conditions and the reactants. 0 CF3 0 0 CF J; 3 R1 D R1 + ' /[ 2: O N W 0 N O E W H H (X) (IX) R2 (VIII) R2 R2 Reaction Scheme 5 Ex erimental rocedure 6 The intermediate compounds of a (X) can generally be prepared ing the reaction steps shown in the reaction scheme (6) below. _ 11 _ O OH O O R1 G R1 F O CF3 waoN O H we; ,1 O N w R2 H halo halo XIV x11 (XI) R2 1 C133 O N W (X) R2 Reaction Scheme 6 E: chlorination F: triﬂuoromethylation G: cyclization Intermediate compounds of Formula (X) in the above reaction scheme (6) can be prepared from intermediate compounds of Formula (XI) ing art-known chlorination procedures (reaction step E). Said sion may be conducted by treatment of the intermediate compound of Formula (XI) with a suitable chlorinating agent such as, for example, thionyl chloride, in the presence of a base such as, for example, pyridine in a reaction-inert t, such as, for example, dichloromethane.

The reaction mixture is stirred at suitable temperature, for example 0 °C for the required time to e completion of the reaction, for e 30—60 minutes.

Intermediate compounds ula (XI) of the above reaction scheme (6) can be prepared from intermediate compounds ofFormula (XII) following art-known triﬂuoromethylation procedures ion step F). Said conversion may be conducted by treatment of the intermediate compound of Formula (XII) in the presence of tetrabutyl ammonium ﬂuoride (TBAF) or tetrabutyl ammonium triphenyldiﬂuoro- silicate (TBAT), with a triﬂuoromethylating agent such as, for example, (triﬂuoromethyl)trimethyl , in a suitable reaction-inert solvent, such as, for example, tetrahydrofuran. The reaction mixture is stirred at suitable temperature, for example room ature for the required time to achieve completion ofthe reaction, for e two hours.

Intermediate compounds of Formula (XII) in the above reaction scheme (6) can be prepared from intermediate compounds of Formula (XIV) following art-known two— step cyclization procedures (reaction step G). Said conversion may be ted by ﬁrst, treatment ofthe intermediate compounds of Formula (XIV) with an intermediate W0 2013/‘083557 compound ofFormula , such as, for example, chloroacetylchloride in the presence of a base such as, for example, NaOH or DIPEA, in a suitable reaction-inert solvent, such as, for example, dichloromethane, or mixtures of inert solvents such as, for example, water and 1,4-dioxane or water and THF. The pH ofthe reaction e may be adjusted to a suitable pH value, for example, 10-11, by on of a suitable base such as, for example, NaOH. The reaction mixture is stirred at a suitable temperature, for example, 0 °C to 25 °C for the required time to achieve completion of the reaction, for example 1—4 hours. The obtained crude residue can uently be cyclised to provide the intermediate (XII) by the on of a suitable base such as, for example, K2CO3, CszCO3, MN—diisopropylethylamine or NaHC03, in a suitable reaction-inert solvent, such as for example, acetonitrile or DMF. The reaction mixture is stirred under thermal conditions such as, for example, heating the reaction mixture at °C to 80°C for 2-24 hours or for e, heating the reaction mixture at 140 °C for -30 minutes under microwave ation. This conversion can also be performed in the absence of a base in a suitable reaction-inert solvent, such as for example, acetonitrile or DMF, at a suitable temperature, typically 40 °C to 110 °C, for a period of, for example, 24-48 hours.

PHARMACOLOGY The compounds of the present invention and the ceutically acceptable compositions f inhibit BACE and therefore may be useful in the ent or prevention of Alzheimer’s Disease (AD), mild cognitive impairment (MCI), ty, dementia, dementia with Lewy bodies, al amyloid angiopathy, multi—infarct dementia, Down’s me, dementia associated with Parkinson’s disease and dementia associated with beta—amyloid.

The invention relates to a compound according to the general a (I), a stereoisomeric form f or a pharmaceutically acceptable acid or base addition salt thereof, for use as a medicament.

The invention also relates to a compound according to the l Formula (I), a stereoisomeric form thereof or a the pharmaceutically acceptable acid or base addition salt thereof, for use in the treatment or prevention of diseases or conditions selected from the group consisting ofAD, MCI, senility, dementia, dementia with Lewy bodies, cerebral amyloid angiopathy, multi-infarct dementia, Down’s syndrome, dementia associated with Parkinson‘s disease and dementia associated with beta— amyloid.

W0 2013f083557 The invention also relates to the use of a compound according to the general a (I), a stereoisomeric form thereof or a pharmaceutically acceptable acid or base addition salt thereof, for the cture of a medicament for the treatment or prevention of any one ofthe e conditions mentioned hereinbefore.

In View of the utility of the compound of Formula (I), there is ed a method of treating subjects such as warm-blooded animals, including humans, suffering from or a method of preventing subjects such as warm—blooded s, including humans, to suffer from any one of the diseases mentioned before.

Said methods comprise the administration, i.e. the systemic or l administration, preferably oral administration, of an ive amount of a compound of Formula (I), a stereoisomeric form thereof, a pharmaceutically acceptable addition salt or solvate thereof, to a subject such as a warm-blooded animal, including a human.

A method oftreatment may also include administering the active ingredient on a regimen ofbetween one and four intakes per day. In these methods of treatment the compounds according to the invention are preferably formulated prior to administration. As described herein below, suitable pharmaceutical formulations are prepared by known procedures using well known and readily available ingredients.

The compounds of the present invention, that can be suitable to treat or prevent Alzheimer’s disease or the symptoms thereof, may be stered alone or in combination with one or more additional therapeutic agents. Combination therapy includes administration of a single pharmaceutical dosage formulation which ns a compound of Formula (I) and one or more additional therapeutic agents, as well as administration of the compound of Formula (I) and each additional therapeutic agents in its own separate pharmaceutical dosage formulation. For example, a compound of a (I) and a therapeutic agent may be stered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent may be administered in separate oral dosage formulations.

CEUTICAL COMPOSITIONS The present invention also provides itions for preventing or ng diseases in which inhibition of beta-secretase is beneﬁcial, such as Alzheimer's disease (AD), mild cognitive ment, senility, dementia, dementia with Lewy bodies, Down's syndrome, dementia associated with stroke, dementia associated with Parkinson's disease and dementia associated with beta-amyloid. Said compositions W0 20131083557 comprising a therapeutically effective amount of a compound according to formula (I) and a pharmaceutically acceptable carrier or diluent.

While it is le for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical composition. Accordingly, the t invention r provides a pharmaceutical composition comprising a compound according to the present invention, er with a pharmaceutically acceptable carrier or diluent. The r or diluent must be "acceptable" in the sense g compatible with the other ients of the composition and not deleterious to the recipients thereof.

The pharmaceutical compositions of this invention may be ed by any methods well known in the art of pharmacy. A therapeutically effective amount of the particular compound, in base form or addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which may take a wide variety of forms depending on the form ofpreparation desired for administration. These pharmaceutical compositions are desirably in unitary dosage form suitable, preferably, for systemic administration such as oral, percutaneous or parenteral administration; or topical administration such as Via inhalation, a nose spray, eye drops or via a cream, gel, shampoo or the like. For example, in ing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions: or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case ers, pills, es and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be ed.

Injectable solutions, for e, may be prepared in which the carrier comprises saline solution, glucose solution or a e of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wettable agent, optionally combined with suitable additives of any nature in minor tions, which additives do not cause any signiﬁcant deleterious effects on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired itions. These compositions may be or as an administered in various ways, e.g., as a transdermal patch, as a spot-on ointment. the aforementioned pharmaceutical It is especially ageous to formulate and uniformity of dosage. compositions in dosage unit form for ease of administration and claims herein refers to ally Dosage unit form as used in the speciﬁcation unit containing a predetermined quantity discrete units suitable as y dosages, each desired therapeutic effect in association of active ingredient calculated to produce the of such dosage unit forms are with the required pharmaceutical carrier. Examples wafers, tablets (including scored or coated tablets), capsules, pills, powder packets, and the like, and injectable ons or suspensions, teaspoonfuls, tablespoonfuls segregated multiples thereof. on the particular The exact dosage and frequency of administration depends treated, the severity of the compound of formula (I) used, the particular condition being of disorder and general physical condition being treated, the age, weight, sex, extent medication the individual may be condition of the particular t as well as other Furthermore, it is evident that said taking, as is well known to those skilled in the art. on the response of the effective daily amount may be lowered or increased depending of the physician prescribing the treated t and/or depending on the evaluation compounds of the instant ion. composition will Depending on the mode of administration, the ceutical from 0.1 to 70 % by weight, more comprise from 0.05 to 99 % by weight, preferably and, from 1 to 99.95 % preferably from 0.1 to 50 % by weight ofthe active ingredient, preferably from 50 to 99.9 % by , ably from 30 to 99.9 % by weight, more all percentages being based on the by weight of a pharmaceutically acceptable carrier, total weight of the composition. administration such as oral, The present compounds can be used for systemic administration such as via percutaneous or parenteral administration; or topical or the like. The inhalation, a nose spray, eye drops or via a cream, gel, shampoo and frequency of compounds are ably orally administered. The exact dosage to formula (I) used, the administration s on the particular nd according the condition being treated, the age, particular condition being treated, the severity of condition of the particular patient weight, sex, extent of disorder and general physical individual may be taking, as is well known to those as well as other medication the amount may be skilled in the art. Furthermore, it is evident that said effective daily 2012/074351 ~16- of the treated subject and/or depending lowered or increased depending on the se the compounds of the instant invention. on the evaluation of the physician prescribing be combined with a carrier The amount of a compound of Formula (I) that can the disease treated, al to produce a single dosage form will vary depending upon of administration. However, as a the mammalian species, and the particular mode of the present invention can, for general guide, suitable unit doses for the compounds 1000 mg ofthe active compound. example, preferably contain between 0.1 mg to about 500 mg. A more preferred unit dose is A preferred unit dose is between 1 mg to about unit dose is between 1 mg to about between 1 mg to about 300mg. Even more preferred than once a day, for example, 2, 3, 100 mg. Such unit doses can be administered more for a 4, 5 or 6 times a day, but ably 1 or 2 times per day, so that the total dosage mg per kg weight of t per 70 kg adult is in the range of 0.001 to about of subject per administration. A preferred dosage is 0.01 to about 1.5 mg per kg weight ofweeks or months, and in administration, and such therapy can extend for a number however, that the c dose level for any some cases, years. It will be understood, the activity of the particular patient will depend on a variety of factors ing health, sex and diet of the c compound employed; the age, body weight, general the rate of excretion; individual being treated; the time and route of administration; and the severity of the particular other drugs that have previously been administered; those of skill in the area. disease undergoing therapy, as is well understood by 1 mg to about 100 mg tablet or 1 mg to about 300 mg A typical dosage can be one time—release capsule or tablet taken taken once a day, or, le times per day, or one content of active ingredient. The once a day and containing a proportionally higher materials that dissolve at different pH time—release effect can be ed by capsule or by any other known values, by capsules that release slowly by osmotic pressure, means of controlled release. these ranges in some cases as will be It can be necessary to use dosages outside it is noted that the clinician or treating apparent to those skilled in the art. Further, terminate therapy in physician will know how and when to start, interrupt, adjust, or conjunction with individual patient response. one of skill in the art will For the compositions and methods ed above, those compounds that are understand that red compounds for use in each are for the compositions and noted as preferred above. Still further red compounds below. methods are those compounds provided in the examples -17..

EXPERIMENTAL PART Hereinafter, the term "mp." means melting point, "aq." means aqueous, "rm." means reaction mixture, "rt." means room temperature, ’ means iso- propylethylamine, "DIPE" means diisopropylether, ‘THF’ means tetrahydroﬁlran, ‘DMF’ means dimethylformamide, ‘DCM’ means dichloromethane, "EtOH" means ethanol ‘EtOAc’ means ethylacetate, "AcOH" means acetic acid, " means isopropanol, "iPrNHz" means isopropylamine, "MeCN" means acetonitrile, "MeOH" means methanol, "Pd(OAc)2" means palladium(II)diacetate, "rac" means racemic, ‘sat.’ means ted, ‘SFC’ means supercritical ﬂuid chromatography, ‘SFC—MS’ means supercritical ﬂuid chromatography/mass spectrometry, "LC—MS" means liquid chromatography/mass spectrometry, "GCMS" means gas chromatography/mass ometry, "HPLC" means high—performance liquid chromatography, "RP" means ed phase, "UPLC" means ultra-performance liquid chromatography, "Rt" means retention time (in minutes), "[M+H]+" means the protonated mass of the free base of the compound, "DAST" means diethylaminosulfur triﬂuoride, "DMTMM" means 4— (4,6-dimethoxy—l ,3,5-triazin—2—yl)methylmorpholinium chloride, "HATU" means 0-(7-azabenzotriazo 1- 1 ,N,N’,N’-tetramethyluronium hexaﬂuorophosphate, "Xantphos" means. (9,9-dimethyl—9H—xanthene-4,5-diyl)bis[diphenylphosphine], "TBAT" means tetrabutyl ammonium triphenyldiﬂuorosilicate, "TFA" means trifuoroacetic acid, "Et20" means diethylether, "DMSO" means dimethylsulfoxide, "MeCN" means acetonitrile.

For key intermediates, as well as some ﬁnal compounds, the absolute conﬁguration of chiral centers (indicated as R and/or S) were established Via comparison with samples ofknown conﬁguration, or the use of ical techniques suitable for the determination of absolute conﬁguration, such as VCD (vibrational cicular dichroism) or X—ray crystallography. When the absolute ration at a chiral center is unknown, it is arbitrarily designated R*.

A. Preparation ofthe intermediates Example Al Preparation of ediate 1.

Br F Trimethylsilylcyanide (30.7 mL, 230 mmol) was added to a stirred solution of -bromo-2—ﬂuoroacetophenone (25 g, 115 mmol) and NH4C1(18.5 g, 345 mmol) in OH (150 mL). The mixture was stirred at room temperature for 3 days. Then the solvent was evaporated in vacuo and the residue was taken up in EtOAc (80 mL).

The solid was ﬁltered and the ﬁltrate was evaporated in vacuo to yield intermediate 1 (27.9 g, quant. yield) which was used in the next step without further puriﬁcation.

Example A2 Preparation of intermediate 2.

H2N 0 Br F Intermediate 1 (27 g, 111 mmol) was dissolved in HCl (37% in H20) (130 mL) and acetic acid (130 mL) and the mixture was reﬂuxed for 16 hours. After cooling to room ature, the mixture was concentrated in vacuo. Water was added and the aqueous layer was ted with EtOAc. The aqueous layer was basiﬁed with aq. NaOH solution (25%) to pH 7. The aqueous layer was partially concentrated in vacuo. The mixture was cooled down in an ice bath and the precipitate was ﬁltered off, washed with water and then EtZO and dried in vacuo to yield intermediate 2 (18 g, 62% yield) as a white solid.

Example A3 Preparation of intermediate 3.

HgN O 0...- Br F Intermediate 2 (15 g, 57.2 mmol) was dissolved in MeOH (300 mL). H2804 (330 mL) was added and the reaction mixture was reﬂuxed for 48 h. The r.m. was concentrated in. vacuo. Water was added and the solution was basiﬁed to pH 8 with sat. aq. NHC03 solution. The aqueous layer was then extracted with EtOAc. The c layer was separated, dried (MgSO4), ﬁltered and concentrated in vacuo to yield intermediate 3 (15 g, 95% yield). e A4 Preparation of intermediate 4.

H2N O Br F W0 2013/083557 2012/074351 Intermediate 3 (10 g) was separated into the corresponding enantiomers by preparative SFC on lpak® Daicel AD 30 x 250 mm). Mobile phase (CO2, MeOH with 0.2% iPrNH2) to yield intermediate 4 (4.2 g, 42% yield). 0 (365 an: —10.1 nm, 0 0.762 w/V %, MeOH, 20 °C).

Example A5 Preparation ofintermediate 5.

H2N o Br F THF (150 mL) was added to a solution of intermediate 4 (40 g, 145 mmol) in NaOH (1 M in H20, 360 mL). The mixture was stirred at It. for 4 hours. The mixture was concentrated in vacuo to afford intermediate 5 (42 g) as a white solid, which was used as such in the next reaction step.

Example A6 Preparation of intermediate 6.

NH O O R Br F To a cooled solution of intermediate 5 (41.3 g, 145 mmol) in H20 (150 mL), a solution of chloroacetyl chloride (24 mL, 304.5 mmol) in 1,4—dioxane (75 mL) was added dropwise. Simultaneously, NaOH (5M in H20, 29 mL) was added to adjust the pH at 10—11. The organic layer was separated, and the aqueous layer extracted with Et20. Then the s layer was ed with HCl (6 M, in H2O) until pH 2. The precipitated white solid was ted by ﬁltration, washed with H20 and dried to yield intermediate 6 (42 g, 86% yield).

Example A7 Pre aration ofintermediate 7.

Intermediate 6 (42 g, 124 mmol) and NaHCO; (20.8 g, 248 mmol) were dissolved in DMF (1000 mL), and the reaction mixture was stirred at 80 °C for 3 hours. The mixture was partially concentrated under reduced pressure, cooled to r.t. and then ﬁltered over W0 83557 diatomaceous earth . The ﬁltrate was concentrated in vacuo, and the residue was puriﬁed by ﬂash column chromatography (silica gel; eluent: MeOH/DCM 0/100 to /95). The desired fractions were collected and concentrated in vacuo to yield intermediate 7 (36 g, 96% yield).

Example A8 Pre aration ofintermediate 8.

HNJﬁ or?F3 To a on of intermediate 7 (11.6 g, 38.5 mmol) in THF (117 mL) was added TBAT (2.08 g, 3.85 mmol). Then, oromethyl)trimethyl silane (12.5 mL, 84.6 mmol) was added dropwise, and the r.m. was stirred at r.t. for 20 minutes. The mixture was quenched with s NaCl and extracted with EtOAc. The combined organic layers were dried (MgSO4), ﬁltered and concentrated in vacuo to yield intermediate 8 (14 g, 98% yield) as a mixture of cis and trans s, which was used as such in the next step. e A9 Pre n ofintermediate 9.

HN/Uﬁ Br R I:CI Intermediate 8 (14 g, 37.6 mmol) was dissolved in DCM (600 mL) and cooled down to 0 °C Then thionyl chloride (11.2 mL, 150 mmol) was added dropwise . The reaction mixture was stirred for 30 min at 0 OC and then pyridine (18.2 mL, 225.7 mmol) was added. After 30 minutes the reaction was hydrolyzed with an aqueous 1N HCl solution and then extracted with DCM. The organic layers were separated, dried (MgSO4), ﬁltered and evaporated in vacuo. The crude product was puriﬁed by ﬂash column chromatography (silica gel; eluent: 7 M solution of ammonia in methanol/DCM 0/100 to 2/98). The desired fractions were collected and concentrated in vacuo to yield intermediate 9 (6 g, 41% yield, mixture of diastereoisomers).

Example A10 Pre aration of intermediate 10.

F [SN/U} F F Intermediate 9 (7 g, 17.9 mmol) and zinc copper couple (8.55 g, 66.3 mmol) were stirred in acetic acid (420 mL) at r.t. for 16 hours. The reaction mixture was ﬁltered, washed with DCM and concentrated in vacuo. Ammonium hydroxide solution (28% in water) and DCM were added and the mixture was stirred at r.t. for 1 h. The organic layer was separated and the aqueous layer was extracted with DCM. The combined organic layers were dried (MgSO4), ﬁltered and evaporated in. vacuo to yield ediate 10 (6 g, 99% yield) as a white powder.

Example A11 Pre n ofintermediate 11.

F HNJS F F P2S5 (5.95 g, 26.8 mmol) was added to a solution of intermediate 10 (6 g, 17.9 mmol) in THF (145 mL) at room temperature. The reaction e was stirred at 70 °C for 90 minutes. Then the mixture was cooled down to r.t., ﬁltered off and the organic solvent evaporated in vacuo to yield intermediate 11 (5.9 g), which was used as such in the next step.

Example A12 Pre aration of intermediate 12.

F NJ} F F ediate 11 (5.9 g, 16.8 mmol) was dissolved in 7N ammonia in MeOH (390 mL) and the reaction mixture was stirred at 80 °C for 2 hours. The solvent was evaporated and the crude product puriﬁed by column chromatography a gel; eluent: 7 M solution of a in ol/DCM 0/100 to 5/95). The desired fractions were collected and concentrated in vacuo to yield intermediate 12 (4.04 g, 72% yield).

Example A13 Pre aration of intermediate 13.

F NAN F F Intermediate 12 (3.6 g, 10.7 mmol) was ed with NaN3 (1.75 g, 26.9 mmol), Cu] (2.56 g, 13.4 mrnol) and Na2C03 (2.28 g, 21.5 mmol) in DMSO (153 mL) and the reaction was degassed. After that, N,N’—dimethylethylenediamine (2 mL, 18.8 mmol) was added and the mixture was heated at 110 °C until completion of the reaction, about 3 hours. The reaction mixture was concentrated in vacuo. 7N ammonia in MeOH was added and the mixture was stirred overnight. The precipitate formed was ﬁltered off and the ﬁltrate was concentrated in vacuo. The crude product was puriﬁed by column chromatography (silica gel; eluent: 7 M solution of ammonia in methanol/DCM 0/100 to 30/70). The desired fractions were collected and concentrated in vacuo to yield intermediate 13 (1.52 g, 52% yield).

Example A14 Pre aration of ediate 14.

N/ O R \ Me / N H F \ N -Methoxypyrazinecarboxylic acid (0.218 g, 1.42 mmol) was dissolved in MeOH (30 mL) and DMTMM (0.456 g, 1.548 mmol) was added. After stirring the mixture for minutes, a solution of ediate 13 (0.35 g, 1.29 mmol) in MeOH (20 mL) was 2O added at 0 °C, and the mixture was stirred for 16 h at room temperature. The solvent was evaporated in vacuo. The crude material was d by ﬂash column tography (silica gel; eluent: 7 M solution of ammonia in methanol/DCM 0/100 to 5/95). The desired fractions were collected and concentrated in vacuo. The residue was suspended from DIPE/heptanes, ﬁltered and dried under high vacuum to yield intermediate 14 (0.266 g, 51% yield) as a white solid.

Example A15 Pre aration of intermediate 15.

H2]?/ m/[{1H \F Intermediate 15 was synthesized following the same approach described in the Example A14. Starting from intermediate 13 (0.35 g, 1.29 mmol) ediate 15 was obtained as white solid (0.362 g, 71% yield).

Example A16 Preparation of intermediate 16.

O F 1~(5-bromoﬂu0ro-phenyl)ethanone [(CAS 198477—89—3), 70 g, 322 mmol) and selenium oxide (71.6 g, 645 mmol) were ved in ne (520 mL). The reaction mixture was stirred at 100 °C for 2 hours. The solvent was evaporated and aqueous HCl 1N solution was added. The aqueous layer was extracted with EtOAc. The ed c layers were dried (Mg2804), ﬁltered and concentrated in vacuo to yield intermediate 16 (62 g, 78% yield), which was used as such in the next reaction.

Example A17 Preparation of intermediate 17.

O F Thionyl chloride (37 mL, 510 mmol) was added dropwise to a stirred on of intermediate 16 (42 g, 170 mmol) in MeOH (456 mL) at 0 °C. The mixture was reﬂuxed for 18 hours. The solvents were evaporated in vacuo and the residue was partitioned between saturated Na2C03 and DCM. The organic layer was separated, dried (MgZSO4), ﬁltered and concentrated in vacuo to yield intermediate 17 (30 g, 68% yield) as a yellow oil. e A18 Pre aration ofintermediate 18.

\\SS Yomw um(IV) isopropoxide (153 mL, 522 mmol) was added to a stirred mixture of intermediate 17 (68 g, 261 mmol) and (S)methylpropanesu1ﬁnamide (37.9 g, 313 mrnol) in ane (1000 mL). The mixture was d at 80 °C for 1.5 hours. The mixture was cooled down to r.t., and ice-water was added. The resulting mixture was ﬁltered over a diatomaceous earth pad and rinsed with n-heptane. The s layer was ted with EtOAc. The combined organic layers were dried (MgSO4), ﬁltered and concentrated in vacuo to yield intermediate 18 (87.9 g, 86% , which was used as such in the next reaction.

Example A19 Pre aration ofintermediate 19.

HN’S\\O YO Br Ethylmagnesium bromide (3 M, 86 mL, 259 mmol) was added dropwise to a stirred solution of intermediate 18 (72.6 g, 185 mmol) in DCM (1154 mL) at —78 °C under nitrogen. The mixture was stirred at this temperature for 30 min, and then the reaction was quenched by the addition of a sat. aq. NH4Cl solution, followed by water. The mixture was extracted with DCM and washed with water. The organic layer was separated, dried (MgSO4), ﬁltered and the solvents evaporated in vacuo. The crude product was puriﬁed by ﬂash column chromatography (silica gel; eluent: Heptanes/EtOAc 90/10 to 70/30). The desired fractions were collected and concentrated in vacuo to yield intermediate 19 (25.56 g, 33% yield, mixture of diastereomers) as a yellow oil. 2012/074351 Example A20 Pre aration ofintermediate 20.

HN’Se HO Br A 2M aq. NaOH solution (91 mL, 181.8 mmol) was added to a solution of crude intermediate 19 (25.6 g, 60.6 mrnol) in MeOH (68 mL). The resulting mixture was stirred at reﬂux for 5 hours. The e was cooled to r.t., and then partitioned n water and EtOAc. The aqueous layer was separated and neutralized by the addition of a 1M aq. HCl solution, and then extracted with DCM. The organic layer was separated, dried (MgSO4), ﬁltered and the solvents evaporated in vacuo. The residue was suspended from DIPE and the precipitate was ﬁltered off and dried in vacuo to yield intermediate 20 (17.5 g, 76% yield, mixture of diastereomers) as a white solid.

Exam le A21 Preparation ofintermediate 21.

HO Br 0 Hydrochloric acid salt Intermediate 20 (17.5 g, 46 mmol) was stirred in 4M HCl solution in dioxane (46 mL) at room temperature for 15 min. To the resulting suspension, DIPE was added, and the itate was d off and dried in vacuo to yield intermediate 21 (15.1 g, quant. yield, racemate) as a white solid.

Example A22 Preparation of intermediate 22.

C'\/U\ NH OH Br F To a cooled solution of intermediate 21 (15.1 g, 43.2 mmol) and DIPEA (35 mL, 203.7 mmol) in DCM (350 mL), chloroacetyl chloride (5.6 mL, 70.6 mmol) was added dropwise at 0 °C. After ng at 0 °C for 15 minutes, the reaction mixture was warmed to rt and acidiﬁed with HCl (2 M in H20, 10 mL). The mixture was extracted with EtOAc and washed with brine. The organic layer was separated, dried (MgSO4), ﬁltered and the solvents evaporated in vacuo. The crude product was triturated with DIPE and the precipitate was ﬁltered off and dried in vacuo to yield intermediate 22 (8.04 g, 53% yield) as a brown solid.

Example A23 Pre aration ofintermediate 23.

F HNJﬁ Intermediate 23 was synthesized following the same approach described in the e 7. ng from intermediate 22 (8 g, 22.69 mmol) intermediate 23 was obtained as a white solid (4.5 g, 63% yield).

Example A24 Pre aration ofintermediate 24.

F HN/Uﬁ Ol—PF3 Intermediate 24 was synthesized following the same approach described in the Example 8. Starting from intermediate 23 (2.34 g, 7.4 mmol) intermediate 24 was obtained as an oil (1.9 g, 66% . e A25 Pre aration of intermediate 25.

F HNJH Intermediate 25 was synthesized following the same approach described in the Example 9. Starting from ediate 24 (1.9 g, 4.92 mmol) intermediate 25 was obtained as a pale yellow solid (1.58 g, 79% yield). e A26 Pre aration of intermediate 26.

F HNJH 01:3 Intermediate 25 (1.4 g, 3.46 mmol) was stirred in acetic acid (42 mL) at 100 °C for minutes. Zinc (0.91 g, 13.8 mmol) was added and the mixture was stirred at 100 °C for l h. Extra zinc (0.452 g, 6.9 mmol) was added and the mixture was further stirred at 100 °C. After another hour, new zinc (0.226 g, 3.46 mmol) was added and the mixture was stirred at 100 °C for 2 h. Finally, extra zinc (0.91 g, 13.8 mmol) was added and the mixture was stirred at 100 °C for 1 h. After cooling, the reaction mixture was ﬁltered, washed with DCM and concentrated in vacuo. Ammonium hydroxide solution (28% in water), aq. sat. NaHC03 on and water were added. The aqueous layer was extracted with DCM. The combined organic layers were dried (MgSO4), ﬁltered and evaporated in vacuo to yield ediate 26 (1.26 g, 98% yield) as a white solid.

Example A27 Pre aration of intermediate 27.

F HNJS‘ Intermediate 27 was synthesized following the same approach bed in the Example 11. Starting from intermediate 26 (1.26 g, 3.4 mmol) intermediate 27 was obtained as a white solid (1.09 g, 83% yield).

Example A28 Pre aration of intermediate 28 and ediate 29.

"VS NH2 O Ngﬁ / CF3 I F F Br Intermediate 28 Br Intermediate 29 ~28~ Intermediate 27 (1 g, 2.59 mmol) was dissolved in 7N ammonia in MeOH (60 mL) and the reaction mixture was stirred at 130 °C for 15 minutes under microwave radiation. The reaction mixture was concentrated in vacuo and new 7N a in MeOH (30 mL) was added. The on mixture was stirred at 130 °C for another minutes under microwave radiation. The solvent was evaporated and the crude product puriﬁed by column chromatography a gel; eluent: 7 M solution of ammonia in methanol/DCM 0/100 to 2/98). The d fractions were collected and concentrated in vacuo to yield intermediate 28 (0.29 g, 30% yield, cis racemate) as a white solid and intermediate 29 (0.27 g, 30% yield) as an oil.

Example A29 Pre aration of intermediate 30.

FN2\ F F Intermediate 30 was synthesized ing the same ch described in the Example 13. Starting from intermediate 29 (0.189 g, 0.542 mmol) intermediate 30 was ed as an oil (0.16 g), which was used as such in the next reaction.

Example A30 Pre aration of intermediate 31. 011%"/ H2r>_\o Intermediate 31 was synthesized following the same approach described in the Example A14. Starting from intermediate 30 (0.09 g, 0.315 mmol) intermediate 31 was obtained as an off white solid (0.028 g, 21% yield).

Example A31 Pre aration of intermediate 32.

Hzl‘i/ N 0 ("WPN\N FF Intermediate 12 (0.4 g, 1.194 mmol), 5-pyrimidinylboronic acid (0.296 g, 2.387 mmol) and tetrakis(triphenylphosphine) palladium(0) (0.207 g, 0.179 mo 1) were dissolved in a mixture of 1,4-dioxane (18 mL) and aqueous NaHC03 (sat. $01., 8.5 mL). The resulting mixture was flushed with N2 and then heated at 70 °C for 2 hours. The reaction mixture was then diluted with water and then extracted with DCM (3x). The combined organic layer was washed with brine, dried (NaZSO4), ﬁltered and the solvents evaporated in vacuo. The crude product was puriﬁed by ﬂash column chromatography (silica gel; 7 M solution of ammonia in methanol/DCM 0/100 to 5/95). The desired ons were collected and concentrated in vacuo to yield intermediate 32 (0.3 g, 75% yield) as a white foam.

Example A32 Pre aration of intermediate 33.

N 0 ng from 3—bromoacetophenone (CAS 21424), intermediate 33 was synthesized following the same reaction procedures as described for ediate 12 in Examples Al-A12.

Example A33 Pre aration ofintermediate 34. "2‘?/ N O (W R \F N\ F Intermediate 34 was synthesized ing the same approach described in the Example A31. Starting from ediate 33 (0.31 g, 0.978 mmol) intermediate 34 was obtained as a white solid (0.21 g, 68% yield).

Example A34 Pre n of intermediate 35.

F NJ} R"): F F WO 2013083557 _30_ Intermediate 13 (2.78 g, 10.25 mmol) was dissolved in EtOAc (70 mL) and palladium on carbon (10%) (1.09 g) and thiophene (0.4% solution in THF, 14 mL) were added.

The mixture was hydrogenated at rt and atmospheric pressure for 16 hours. The catalyst was ﬁltered off and the solvents ated in vacuo. The crude product was puriﬁed by ﬂash column chromatography (silica gel; eluent: 7 M solution of ammonia in methanol/DCM 0/100 to 10/90). The desired fractions were collected and concentrated in vacuo to yield intermediate 35 (0.478 g, 17% yield) B. Pre aration of the ﬁnal com ounds Example B1 Preparation of compound 1: N~f3;L(_2R*,3R)-5—Amino(diﬂuoromethyl1methyl- hydro-2H- 1 ,4-oxazin—3~yl]—4—ﬂuorophenyl} methoxypyrazinecarboxamide H211/ R R* MemNN H CHF2 N\ F ediate 14 (0.154 g, 0.378 mmol) was dissolved in EtOAc (5 ml.) and palladium on carbon (10%) (0.04 g, 0.038 mmol) and thiophene (04% solution in THF, 0.5 mL, 0.026 mmol) were added. The mixture was hydrogenated at rt and atmospheric re for 16 hours. The catalyst was ﬁltered off and the solvents evaporated in vacuo. The crude product was puriﬁed by ﬂash column chromatography (silica gel; eluent: 7 M solution of ammonia in methanol/DCM 0/100 to 2/98). The desired fractions were collected and concentrated in vacuo. The residue was suspended from DIPE, ﬁltered and dried under high vacuum to yield compound 1 (0.067 g, 43% yield).

Example B2 Preparation of compound 2: N- {3—[(2R*,3R)Amino~2—( diﬂuoromethyl)—3-methyl- 3,6-dihydro-2H—1,4-oxazinyl]—4-fluorophenyl} ﬂuoropypidinecarboxamide H2":/ R R" / R1 H CHF2 \ F Compound 2 was synthesized following the same approach described in the Example B1. ng from intermediate 15 (0.251 g, 0.637 mmol) compound 2 was obtained as white solid (0.114 g, 45% yield).

W0 20132083557 Example B3 Pre aration of com ound 3: ac-N- Intermediate 31 (0.028 g, 0.066 mmol) was dissolved in MeOH (1.3 mL) and enated in a H-cube reactor (1 mL/min, 10% Pd/C cartridge, full H2 mode), ﬁrst at 25 °C, then at 50 °C and ﬁnally at 80 °C. The solvent was evaporated in vacuo. The crude product was puriﬁed by preparative HPLC (C18 XBridge 19 x 100 5 um), mobile phase (gradient from 80% 0.1% NH4C03H/NH4OH pH 9 solution in Water, 20% CH3CN to 0% 0.1% NH4C03H/NH4OH pH 9 solution in Water, 100% CH3CN). The d fractions were ted and concentrated in vacuo to yield compound 3 (0.0032 g, 11% yield).

Example B4 Pre aration ofcom ound 4: 5R 6R* ( R R* \ CHF2 Compound 4 was synthesized following the same approach described in the Example B1. Starting from intermediate 32 (0.155 g, 0.464 mmol) compound 4 was obtained (0.018 g, 12% .

Example B5 Pre aration ofcom ound 5: 5R 6R* ~6— Difluorometh l meth 1 3- imidin—5~ ylphenyl)-5,6-dihydro-2H- 1 zinamine H2"?/ N O (/ R R* \ CHF2 Compound 5 was synthesized following the same approach described in the Example Bl. Starting from intermediate 34 (0.124 g, 0.392 mmol) compound 5 was obtained as a white solid (0.04 g, 32% yield).

WO 83557 Example B6 Pre aration ofcom ound 6: N- c1 / N H cm:2 -Chloropyridine—Z—carboxylic acid (63 mg, 0.4 mmol) was dissolved in MeOH (7 mL) and DMTMM (129 mg, 0.44 mmol) was added. After ng the mixture for minutes, a solution of intermediate 35 (100 mg, 0.366 mmol) in MeOH (8 mL) was added at 0 °C, and the mixture was stirred for 16 h at room temperature. The solvent was evaporated in vacuo. The crude material was puriﬁed by ﬂash column chromatography a gel; eluent: 7 M solution of ammonia in methanol/DCM 0/100 to 5/95). The desired fractions were collected and concentrated in vacuo. The residue was triturated with DIPE, ﬁltered and dried under high vacuum to yield compound 6 (0.116 g, 74% yield) as a white solid Example B7 Pre aration ofcom ound 7: N— 2R* 3R Amino diﬂuorometh l meth 1- hydro-2H—1,4-oxazinyl]ﬂuorophenyl} cyanopyridine-Z-carboxamide H",/ R R* NC / N H CHF2 \ N \ F Compound 7 was synthesized ing the same approach described in the Example B6. Starting from intermediate 35(100 mg, 0.4 mmol) compound 7 was obtained (110 mg, 75% yield).

Compounds 1 to 7 in tablel list the compounds that were prepared ing to one of the above Examples. ‘Ex. No.’ refers to the Example number according to which protocol the compound was synthesized. ‘Co. No.’ means compound number. C2(R*) means that the absolute conﬁguration at C2 is either R or S but unknown yet.

W0 2013.1083557 _ 33 - Table 1: C2(R*);C3(R) Single diastereoisomer Pure enantiomer C2(R*);C3(R) Single diastereoisomer Pure enantiomer C2(RS)SC3(RS) Single diastereoisomer (CiS) C2(R*);C3(R) Single diastereoisomer Pure enantiomer C2(R*);C3(R) Single diastereoisomer Pure omer C2(R*);C3(R) Single diastereoisomer Pure enantiomer C2(R*);C3(R) Single diastereoisomer Pure enantiomer C. Analﬁical Part LCMS For (LC)MS-characterization of the compounds ofthe present invention, the following methods were used.

The LC measurement was performed using an Acquity UPLC (Waters) system sing a binary pump, a sample organizer, a column heater (set at 55°C), a diode- array detector (DAD) and a column as ed in the respective methods below. Flow from the column was split to a MS ometer. The MS detector was conﬁgured with an electrospray tion source. Mass spectra were acquired by seaming from 100 to 1000 in 0.18 seconds using a dwell time of 0.02 seconds. The capillary needle voltage was 3.5 kV and the source temperature was maintained at 140°C. Nitrogen was used as the nebulizer gas. Data acquisition was performed with a Waters-Micromass MassLynx—Openlynx data system.

Reversed phase UPLC (Ultra Performance Liquid Chromatography) was carried out on a d ethylsiloxane/silica hybrid (BEH) C18 column (1.7 um, 2.1 x 50 mm; Waters Acquity) with a ﬂow rate of 0.8 ml/min. Two mobile phases (10 mM ammonium acetate in HzO/acetonitrile 95/5; mobile phase B: acetonitrile) were used to run a gradient condition from 95 % A and 5 % B to 5 % A and 95 % B in 1.3 minutes and hold for 0.7 minutes. An injection volume of 0.75 ul was used.

Cone voltage was 10 V for positive ionization mode and 20 V for negative ionization mode.

Alethod 2: The UPLC (Ultra Performance Liquid tography) measurement was performed using an Acquity UPLC (Waters) system comprising a sampler organizer, a binary pump with degasser, a four column’s oven, a diode-array detector (DAD) and a column as speciﬁed in the respective methods. The MS detector was conﬁgured with an ESCI dual ionization source (electrospray combined with atmospheric re al tion). Nitrogen was used as the nebulizer gas. The source temperature was maintained at 140 °C. Data acquisition was med with nx—Openlynx software.

Reversed phase UPLC (Ultra Performance Liquid Chromatography) was carried out on a RRHD Eclipse Plus-C18 (1.8 mm, 2.1 X 50 mm) from Agilent, with a ﬂow rate of 1.0 ml/min, at 50°C without split to the MS detector. The gradient conditions used are: 95 % A (0.5 g/l ammonium acetate solution + 5 % acetonitrile), 5 % B (acetonitrile), to 40 % A, 60 % B in 3.8 minutes, to 5 % A, 95 % B in 4.6 minutes, kept till 5.0 minutes.

Injection volume 2 ul. Low-resolution mass spectra (single quadrupole, SQD detector) were acquired by ng from 100 to 1000 in 0.1 seconds using an inter—channel delay of 0.08 second. The capillary needle voltage was 3 kV. The cone voltage was V for positive ionization mode and 30 V for negative ionization mode.

Method 3: The LC measurement was performed using an Acquity UPLC (Waters) system comprising a binary pump, a sample organizer, a column heater (set at 55 °C), a diode— array or (DAD) and a column as speciﬁed in the respective methods below. Flow from the column was split to a MS spectrometer. The MS detector was conﬁgured with an ospray ionization source. Mass spectra were acquired by scanning from 100 to 1000 in 0.18 seconds using a dwell time of 0.02 s. The capillary needle e was 3.5 kV and the source temperature was maintained at 140 °C. Nitrogen was used as the nebulizer gas. Data acquisition was performed with a Waters-Micromass MassLynx-Openlynx data system.

Reversed phase UPLC (Ultra Performance Liquid Chromatography) was carried out on a bridged ethylsiloxane/silica hybrid (BEH) C18 column (1.7 um, 2.1 x 50 mm; Waters Acquity) with a ﬂow rate of 0.8 ml/min. Two mobile phases (10 mM ammonium acetate in HZO/acetonitrile 95/5; mobile phase B: acetonitrile) were used to run a nt condition from 95 % A and 5 % B to 5 % A and 95 % B in 1.3 minutes and hold for 0.3 minutes. An injection volume of 0.5 ul was used. Cone voltage was 10 V for positive ionization mode and 20 V for negative ionization mode.

Melting Points Values are either peak values or melt ranges, and are obtained with experimental uncertainties that are commonly associated with this analytical method.

DSC823e (indicated by DSC in Table 2) For a number of compounds, melting points were determined with a e (Mettler-Toledo). Melting points were ed with a temperature gradient of ° C/minute. Maximum temperature was 400°C.

Table 2: Analytical data — Rt means retention time (in minutes), [M+H]+ means the protonated mass of the compound, method refers to the method used for .

Melting Point 1 0.75 410 l 227.3°C 2 0.76 397 1 205.7°C 3 1.98 424 2 n.d. 4 0.59 337 l n.d. 0.55 319 l n.d. 6 3 203.9°C 7 3 230.9°C n.d. means not determined Optical Rotations Optical rotations were measured on a Perkin-Elmer 341 polarimeter with a sodium lamp and ed as follows: [01]),toc (c g/ 100 m1, t).

Table 3: Analytical data — Optical rotation values for omerically pure com ounds Wavelength Concentration Solvent For a number of compounds, 1H NMR spectra were recorded on a Bruker DPX—360, on a Bruker DPX—400 or on a Bruker Avance 600 spectrometer with standard pulse sequences, operating at 360 MHZ, 400 MHz and 600 MHz respectively, using CHLOROFORM-d (deuterated chloroform, CDClg) or DMSO-d6 (deuterated DMSO, dimethyl-d6 sulfoxide) as solvents. Chemical shifts (5) are reported in parts per million (ppm) relative to ethylsilane (TMS), which was used as internal standard.

Table 4: NMR result 1H NMR (360 MHz, DMSO-dg) 5 ppm 1.52 (s, 3 H), 3.93 — 4.01 (m, 1 H), 4.02 (s, 3 H), 4.10 (d, J=15.9 Hz, 1 H), 4.15 (d, J=15.9 Hz, 1 H), 5.64 (td, J=54.0, 4.4 Hz, 1 H), 5.80 (br. s, 2 H), 7.11 (dd, J=12.1, 8.8 Hz, 1 H), 7.80 (ddd, J=8.8, 4.1, 2.7 Hz, 1 H), 8.04 (dd, J=7.3, 2.8 Hz, 1 H), 8.42 (d, J=1.3 Hz, 1 H), 8.88 (d, J=l.3 Hz, 1 H), 10.44 (s, 1 H). 1H NMR (360 MHz, g) 8 ppm 1.52 (s, 3 H), 3.94 - 4.05 (m, 1 H), 4.10 (d, J=16.0 Hz, 1 H), 4.15 (d, J=16.0 Hz, 1 H), .65 (td, J=54.3, 4.3 Hz, 1 H), 5.81 (br. s, 2 H), 7.11 (dd, J=12.1, 8.8 Hz, 1 H), 7.82 (dt, J=8.5, 3.6 Hz, 1 H), 7.97 (dd, J=8.8, 2.9 Hz, 1 H), 8.03 (td, J=6.9, 2.7 Hz, 1 H), 8.22 (dd, J=8.8, 4.8 Hz, 1 H), 8.74 (d, J=2.9 Hz, 1 H), 10.55 (br. s, 1 H).

W0 20131083557 _ 37 _ NMR result H1 NMR (400 MHz, CHLOROFORM-a’) 5 ppm 0.77 (t, J=7.4 Hz, 3 H), 1.97 — 2.07 (m, 1 H), 2.13 _ 2.26 (m, 1 H), 4.07 (s, 3 H), 4.32 (br. s., 2 H), 4.18 (dd, J=15.7, 1.2 Hz, 1 H), 4.33 (dd, J=15.6, 1.5 Hz, 1 H), 4.36 (ddt, , 8.4, 2.2, 2.2 Hz, 1 H), 5.46 (td, J=54.0, 2.6 Hz, 1 H), 7.07 (dd, J=11.6, 8.8 Hz, 1 H), 7.83 (dd, J=6.7, 2.8 Hz, 1 H), 8.07 (ddd, J=8.8, 4.2, 2.9 Hz, 1 H), 8.16 (d, J=1.4 Hz, 1 H), 9.02 (d, J=1.2 Hz, 1 H), 9.56 (br. s, 1 H). 1H NMR (360 MHz, DMSO-ds) 5 ppm 1.56 (s, 3 H), 4.05 — 4.13 (m, 1 H), 4.11 _ 4.17 (m, 2 H), 5.80 (td, J=53.8, 4.0 Hz, 1 H), 4 5.90 (br. s., 2 H), 7.31 (dd, J=12.1, 8.4 Hz, 1 H), 7.76 (ddd, J=8.3, 4.5, 2.6 Hz, 1 H), 8.10 (dd, J=7.5, 2.4 Hz, 1 H), 9.08 (s, 2 H), 9.20 (s, 1 H). 1H NMR (360 MHz, DMSO—ds) 5 ppm 1.57 (s, 3 H), 3.85 (ddd, J=15.2, 7.5, 4.8 Hz, 2 H), 4.11 (d, J=15.7 Hz, 1 H), 4.26 (d, J=15.7 Hz, 1 H), 5.24 — 5.64 (m, 1 H), 5.78 (br. s., 2 H), 7.43 — 7.53 (m, 2 H), 7.64 — 7.74 (m, 1 H), 9.10 (s, 2 H), 9.20 (s, 1 H). 1H NMR (360 MHz, s) 5 ppm 1.53 (s, 3 H) 4.00 (ddd, J=14.2, 10.0, 4.0 Hz, 1 H) 4.07 — 4.22 (m, 2 H) 5.47 — 5.81 (m, 6 1 H) 5.82 (s, 2 H) 7.12 (dd, J=12.1, 8.8 Hz, 1 H) 7.78 — 7.88 (m, 1 H) 8.04 (dd, J=7.1, 2.7 Hz, 1 H) 8.11 - 8.18 (m, 1 H) 8.20 (dd, J=8.4, 2.6 Hz, 1 H) 8.79 (d, J=1.8 Hz, 1 H) 10.61 (s, 1 H) 1H NMR (360 MHz, DMSO-d6) 5 ppm 1.53 (s, 3 H) 3.90 — 4.06 (m, 1 H) 4.06 - 4.21 (m, 2 H) 5.47 — 5.81 (m, 1 H) 5.81 (br. s., 7 2 H) 7.13 (dd, J=12.1, 8.8 Hz, 1 H) 7.76 — 7.90 (m, 1 H) 8.07 (dd, J=7.3, 2.9 Hz, 1 H) 8.28 (d, J=8.4 Hz, 1 H) 8.58 (dd, J=8.2, 2.0 Hz, 1 H) 9.20 (d, J=1.8 Hz, 1 H) 10.78 (s, 1 H) D. Pharmacological examples The compounds provided in the t invention are inhibitors of the beta-site APP—cleaving enzyme 1 (BACEl). Inhibition ofBACEl, an aspartic protease, is believed to be nt for treatment of Alzheimer’s Disease (AD). The production and accumulation of beta-amyloid peptides (Abeta) from the beta-amyloid precursor protein -38..

(APP) is believed to play a key role in the onset and progression of AD. Abeta is produced from the d sor protein (APP) by sequential cleavage at the N— and C-termini of the Abeta domain by ecretase and gamma-secretase, respectively.

Compounds of Formula (I) are expected to have their effect substantially at BACE] by virtue oftheir ability to inhibit the enzymatic activity. The our of such inhibitors tested using a mical Fluorescence Resonance Energy Transfer (FRET) based assay and a cellular dLisa assay in SKNBE2 cells described below and which are suitable for the ﬁcation of such compounds, and more ularly the compounds according to Formula (I), are shown in Table 5 and Table 6.

Biochemical FRET based assay This assay is a Fluorescence Resonance Energy Transfer Assay (FRET) based assay. The substrate for this assay is an APP derived 13 amino acids peptide that contains the ‘Swedish’ Lys-Met/Asn-Leu mutation ofthe amyloid precursor protein (APP) ecretase cleavage site. This substrate also contains two ﬂuorophores: (7—methoxycoumarinyl) acetic acid (Mca) is a ﬂuorescent donor with excitation wavelength at 320 nm and emission at 405 nm and 2,4-Dinitrophenyl (an) is a proprietary quencher acceptor. The distance between those two groups has been selected so that upon light tion, the donor ﬂuorescence energy is signiﬁcantly quenched by the acceptor, h resonance energy transfer. Upon cleavage by BACEI the ﬂuorophore Mca is separated from the quenching group an, restoring the full fluorescence yield of the donor. The se in ﬂuorescence is linearly related to the rate of proteolysis.

Brieﬂy in a 384-well format recombinant BACEl n in a ﬁnal concentration of 1 ug/ml is incubated for 120 minutes at room temperature with 10 um substrate in incubation buffer (40 mM Citrate buffer pH 5.0, 0.04 % PEG, 4 % DMSO) in the absence or presence of compound. Next the amount of proteolysis is directly measured by ﬂuorescence measurement at T=0 and T=120 (excitation at 320 nm and emission at 405 nm). Results are expressed in RFU (Relative Fluorescence Units), as difference between T120 and TO.

A best-ﬁt curve is ﬁtted by a minimum sum of squares method to the plot of %Controlmin versus compound concentration. From this an IC50 value (inhibitory concentration causing 50% inhibition of activity) can be obtained.

LC = Median of the low control values = Low control: Reaction without enzyme HC = Median ofthe High control values = High Control: Reaction with enzyme %Effect = 100-[(sample-LC) / (HC-LC) *100] %Control = (sample /HC)* 100 %Controlmin = (sample—LC) / (HC-LC) * 100 The following exempliﬁed compounds were tested ially as described above and exhibited the following the activity: Table 5: Biochemical FRET based Cellular OLLisa assay in SKNBEZ cells In two aLisa assays the levels of Abeta total and Abeta 1-42 produced and secreted into the medium n neuroblastoma SKNBEZ cells are quantiﬁed. The assay is based on the human neuroblastoma SKNBEZ expressing the wild type Amyloid sor n 95). The compounds are diluted and added to these cells, incubated for 18 hours and then measurements ofAbeta 1—42 and Abeta total are taken. Abeta total and Abeta 1-42 are measured by sandwich (xLisa. aLisa is a sandwich assay using biotinylated dy AbN/25 attached to streptavidin coated beads and antibody Ab4G8 or cAb42/26 conjugated acceptor beads for the detection of Abeta total and Abeta 1—42 respectively. In the presence of Abeta total or Abeta 1-42, the beads come into close proximity. The excitation ofthe donor beads provokes the release of t oxygen molecules that trigger a cascade of energy transfer in the acceptor beads, resulting in light emission. Light emission is measured aﬁer 1 hour incubation (excitation at 650 nm and emission at 615 nm). _ 40 _ A t curve is ﬁtted by a minimum sum of squares method to the plot of %Controlmin versus nd concentration. From this an ICso value (inhibitory tration causing 50 % inhibition of activity) can be obtained.

LC = Median ofthe low control values = Low control: cells preincubated without compound, without biotinylated Ab in the ctLisa HC = Median ofthe High control values = High Control: cells preincubated without compound %Effect = lOO—[(sample~LC) / (HC~LC) * 100] %Control = (sample /HC)* 100 %C0ntrolmin = e—LC) / (HC-LC) * 100 The following exempliﬁed compounds were tested ially as described above and exhibited the ing the activity: Table 6: Cellular aLisa assay in Cellular aLisa assay in SKNBE2 cells SKNBE2 cells Abetatotal Demonstration ofin viva eﬂz‘cacy AB peptide lowering agents of the invention can be used to treat AD in mammals such as humans or alternatively demonstrating efﬁcacy in animal models such as, but not limited to, the mouse, rat, or guinea pig. The mammal may not be diagnosed with AD, or may not have a genetic predisposition for AD, but may be transgenic such that it overproduces and eventually deposits AB in a manner similar to that seen in humans afﬂicted with AD.

W0 2013l083557 AB peptide lowering agents can be administered in any standard form using any standard method. For e, but not limited to, AB e lowering agents can be in the form of liquid, tablets or capsules that are taken orally or by injection. AB peptide lowering agents can be administered at any dose that is sufﬁcient to signiﬁcantly reduce levels ofAB peptides in the blood, blood , serum, cerebrospinal fluid (CSF), or brain.

To determine whether acute administration of an AB42 peptide lowering agent would reduce AB e levels in vivo, ansgenic rodents, e.g. mice or rats were used. Animals treated with the AB peptide lowering agent were examined and compared to those untreated or treated with vehicle and brain levels of soluble AB42 and total AB were quantitated by standard techniques, for example, using ELISA.

Treatment periods varied from hours (h) to days and were adjusted based on the results ofthe AB42 lowering once a time course of onset of effect could be established.

A typical protocol for measuring AB42 ng in vivo is shown but it is only one ofmany variations that could be used to optimize the levels of detectable AB. For example, AB peptide lowering compounds were formulated in 20 % hydroxypropyl B cyclodextrin. The AB peptide lowering agents were administered as a single oral dose (p.o.) or a single subcutaneous dose (3.0.) to overnight fasted animals. After a certain time, usually 2 or 4 h (as indicated in Table 7), the animals were sacriﬁced and AB42 levels were analysed.

Blood was collected by decapitation and exsanguinations in EDTA-treated collection tubes. Blood was fuged at 1900 g for 10 minutes (min) at 4 °C and the plasma recovered and ﬂash frozen for later analysis. The brain was removed from the m and hindbrain. The cerebellum was removed and the left and right hemisphere were separated. The left hemisphere was stored at —18 °C for quantitative analysis of test compound levels. The right hemisphere was rinsed with phosphate—buffered saline (PBS) buffer and immediately frozen on dry ice and stored at -80 °C until homogenization for biochemical assays.

Mouse brains from non-transgenic animals were resuspended in 8 volumes of 0.4 % DEA (diethylamine) /50 mM NaCl containing protease tors (Roche- 80001 or 04693159001) per gram oftissue, e.g. for 0.158 g brain, add 1.264 ml of 0.4 % DEA. All samples were homogenized in the FastPrep-24 system (MP Biomedicals) using lysing matrix D (MPBio #6913-100) at 6m/s for 20 seconds. nates were centrifuged at 0 x g for 50 min. The resulting high speed supernatants were then erred to fresh eppendorf tubes. Nine parts of supernatant were neutralized with 1 part 0.5 M Tris-HCl pH 6.8 and used to quantify ABtotal and A1342.

To quantify the amount ofABtotal and A1342 in the soluble fraction of the brain homogenates, Enzyme-Linked—Immunosorbent—Assays were used . Brieﬂy, the rds (a dilution of synthetic ABl-40 and ABl-42, ) were prepared in 1.5 ml Eppendorftube in Ultraculture, with ﬁnal concentrations ranging from 10000 to 0.3 pg/ml. The samples and standards were co-incubated with HRPO-labelled N—terminal antibody for A1542 detection and with the biotinylated mid-domain antibody 4G8 for ABtotal detection. 50 ul of conjugate/sample or conjugate/standards es were then added to the antibody-coated plate (the capture antibodies selectively ize the C—terminal end of A1342, antibody B42/26, for A1342 detection and the N—terminus of A13, antibody JRF/rAB/2, for A13total detection). The plate was allowed to incubate ght at 4 °C in order to allow formation of the antibody- amyloid complex. Following this incubation and subsequent wash steps the ELISA for A1342 quantiﬁcation was ﬁnished by addition of Quanta Blu fluorogenic peroxidase substrate according to the manufacturer’s ctions (Pierce Corp, Rockford, 11). A reading was performed after 10 to 15 min ation 320 nm /emission 420 nm).

For ABtotal detection, a Streptavidine-Peroxidase-Conjugate was added, ed 60 min later by an addional wash step and addition of Quanta Blu ﬂuorogenic peroxidase substrate according to the manufacturer’s instructions (Pierce Corp., Rockford, II). A reading was performed after 10 to 15 min (excitation 320 nm /emission 420 nm).

In this model at least 20 % A1342 lowering compared to untreated animals would be advantageous.

The following exempliﬁed compounds were tested ially as described above and exhibited the following the ty: Table 7: A1342 Aﬁtotal Dose Route of Time after (%Ctrl)_Mea (%Ctrl)_Mea administration administration 2012/074351 _ 43 _ M342 ABtotal Dose Route of Time after (%Ctrl)_Mea (%Ctrl)_Mea administration administration n n 7 53 l 55 l 30 mpk p.0. 4 h j n.t. means not tested; s.c. means subcutaneous ; p.o. means oral

Claims

1.A compound of Formula (I) o CHF2 L R1 HZN N L\Ar 5 R2 or a tautomer or a stereoisomeric form thereof, n R1 is C;_3alkyl; R2 is hydrogen or ﬂuoro; L is a bond or —NHCO-; 10 Ar is selected from the group consisting of pyridinyl, dinyl and pyrazinyl, each optionally susbstituted with halo or koxy; or a pharmaceutically acceptable addition salt thereof.

2. The compound of claim 1 wherein R1 is methyl or ethyl.

3. The compound of claim 2 n Ar is selected from 5—methoxy—pyridinyl, 5-pyrimidinyl and S—ﬂuoropyrazinyl.

4. The compound of claim 1 wherein R2 is hydrogen or ﬂuoro.

5. The compound of claim 1 wherein the quaternary carbon atom substituted with R1 has the R-conﬁguration.

6. A pharmaceutical composition comprising a therapeutically effective amount of 25 a compound as deﬁned in any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. A process for preparing a pharmaceutical composition as deﬁned in claim 6, wherein a pharmaceutically acceptable carrier is intimately mixed with a 3O therapeutically effective amount of a compound as deﬁned in any one of claims 1 to 5.

8. A compound as deﬁned in any one of claims 1 to 5 for use in the ent or prevention of Alzheimer's disease (AD), mild cognitive ment, senility, dementia, dementia with Lewy , cerebral d angiopathy, multi- infarct dementia, Down’s me, dementia associated with , dementia associated with Parkinson's disease or dementia associated with beta-amyloid, 5

9. The use of a compound as deﬁned in any one of claims 1 to 5 or a pharmaceutical composition as deﬁned in claim 6 in the manufacture of a medicament for treating a disorder selected from the group consisting of Alzheimer‘s disease, mild cognitive impairment, senility, dementia, dementia with Lewy bodies, cerebral amyloid angiopathy, multi-infarct dementia, Down's 10 syndrome, dementia associated with stroke, dementia associated with son's disease and dementia associated with beta-amyloid.

10. A nd according to claim 1, substantially as herein described with reference to any one of the accompanying examples.

11. The use ing to claim 9, substantially as herein described with reference to any one of the accompanying examples.

12. A process according to claim 7, substantially as herein described with reference 20 to any one of the accompanying examples.