XBERFOR

|ΘΘΟί] This- application claims priority to our US pim sioaal application having serial number 62/210945, which was filed 27-Aug-I5 and which is incorporated by reference herein,

^' Field of The Invention

[0002] The field of the invention is enzyme inhibitors and methods therefor, particularly as it relates to APP (amyloid precursor protein)-specific BACE (beta-secretase) inhibitors.

Background of the Invention

[0003] The background description includes information that may be useful in understanding the presen invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior ait.

[0004] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply, [0005] BACE inhibitors have gained significant attention due to the discovery that a mutation in tiie APP appears to protect against Alzheimer's Disease and oilier age-related cognitive decline. More specifically, the mutation is at the P2' residue of the BACE cleavage site and results in a significantly reduced Αβ production. Unfortunately, the substrate specificity of BACE is not exclusively limited to APP, and complete inhibition of BACE would thus have adverse effects.

[0006] More recently, certain B ACE inhibitors with at least some selectivity towards BACE were reported as is disclosed in WO 2014/127042, US 2016/0159746, and US■ 2014/0371 83. While such inhibitors had desirable selectivity against BACE wi th respect to APP, relati vel high concentrations were required for inhibition. Moreover., many of the co poun s ^' failed to penetrate the blood brain barrier. |0007] Thus , despite the relatively detailed knowledge of BACE activity and its siibsirate API* various drawbacks still remain for know BACE itrhibifors,, especially gh IC 50 and/or lack of penetrability across the blood brain barrier. Therefore, there is still a need to provide improved BACE inhibitors and methods. mmary of tlte laTeati&n

10008] Hie present iuventive subject wattes is drawn to various compounds, eonspositioas, and methods of BACE inhibition, and particularly to compounds and compositions that internet with both. BACE and APP to so increase selectivity of the inhibitor. Moreover, compounds presented herein exhibit desirably low IC50 values and penetrability across the blood brain barr ier.

[0009] in one aspect of the inventive subject matter, the inventors contemplate a compound that, has a structure according to Formula I

Formula I

wherein ¾ is phenyl substituted with (i) an alkoxyalkyi, (ii) an N-alkyialkyl, or (iii) a optionally halogenated heteroaryl (e.g. , pvridinyl. pvrimidmyl, or oxazoiyl), or wherein R _{ is a halogenated heteroaryl. _∑ is preferably an optionally halogenated lower alkoxy grou (e.g., difluoromeihoxy group) or an optionally halogenated N-aikylanuno group, and R ₃ is most preferably H. halogen, or optionally halogenated lower alkyl. Therefore, suitable compomids may have a structure according to Formula II or III

Formula H Formula III wherein R ₂ and R ₃ are as defined above, and wherein 4 is an optionally halogenated pyridiiryl. pyrhnidkiyi, or oxazolyl, and wherein 4 is halogen (e.g., F). In especially preferred aspects, the compounds will have a structure according to any one of Formulae IV- XII

Formula VI Formula VII

Formula XH

[ΘΘ10] Consequently, the inventors also contemplate a pharmaceutical composition comprising a coropoimd contemplated herein in combination wife a pharmaceutically acceptable earner. Most typically, the compound will be present in an amount effective to inhibit BACE in a patient when administered (e.g., orally or via injection) to the patient. As will t>e readily appreciated, as ueli formulations have BACE inhibitory activity, they will be -soitabie (alone or in combination with another pharmaceutical agent) for treatment of a neurological condition, and particularly for tr at-Beat a? Alzheimer's Disease. oll] Viewed from another perspective, the inventors also contemplate the use of contemplated compounds in. the mamifeettH e of a medicajiieni especially where the medicamen t is used ^' in the treatment of a neurodegenerative disease, and/or where the medicament is for the reduction of selective B ACE activity in a patient. Consequently, BACE inhibitors and uses thereof are also specifically contemplated herein and particularly include treatment of neurological conditions (e.g., Alzheimer's Disease). Tims, the inventors also contemplate treating a patient diagnosed with Akheimer's Disease or at risk for disease progression of inild cognitive decline.

|0012] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompan\½g drawing figures in which like numerals represent like components.

Brief Description of The Drawing

[0013] Figure 1 A depicts exemplary compounds according to the inventive subject matter, and Figures IB- ID depict exemplary test results for selected compounds.

[0014] Figure 2A depicts an exemplary compound according to the inventive subject matter, and Figures 2B-2C depict exemplary test results for selected compounds.

[0015] Figure 3A depicts exemplary compounds according to the inventive subject matter, and Figures 3B-3E depict exemplary test results for selected compounds.

[0016] Figure 4 depicts exemplary phaiiiiacokinetic test results for selected compounds.

J0017J Figure 5 A depicts an exemplary compound according to the inventive subject matter, and Figures 5B-5C depict exemplary test results for the compound of Figure 5 A.

[0018] Figure 6A depicts an exemplary compound according to the inventive subject matter, and Figure 6B depicts exemplary test results for the compound of Figure 6 A.

[0019] Figure 7A depicts an exemplary compound according to the inventive subject matter, and Figure 7B depicts exemplary test, results for the compound of Figure 7 A. |ΘΘ20] Figure 8 depicts an exem lar compound according to the inventive subject matter and an exemplary test results tor the exemplary ccmspound.

Detailed . ^' Description

| 021] Tlie inventors have discovered that certain compounds as farther, described in mor detail belo had improved parameters with respect, to BACE mhib tion . Most notably, several of these ecanpoiinds also exhibited penetrance across the blood brain harrier and preferentially inhibited BACE with respect to APP as substrate.

[0022] The improved BACE inhibitors as contemplated herein will generall have a structure according to Formula I

Formula ί

wherein ¾ is typically a substituted aryl or heteroaryl group, and most preferably a phenyl substituted with (i) an alkoxyalkyl, (ii) an N-alkylalkyL or (iii) an optionally halogenaied heteroaiyi,, or R _} is a halogenaied heteroaryl. In further preferred aspects, R ₂ is an optionally halogeiiated lower alkoxy grou (typically having 1 -3 carbon atoms), an optionally

halogenaied N-alkyiarnino group (typically having 1-3 carbon atoms), an OH group, a C group, etc. In still further preferred aspects. R ₃ will be H, a halogen, or an optionally

halogenaied lower alkyl (typically having 1-3 carbon atoms). f ΘΘ23] For example, wherein Ri is a phenyl, it is preferred that the phenyl is substituted with a radical that includes a heteroatom, and most preferably an oxygen or nitrogen that, is separated from the phenyl by one carbon atom. Among oilier suitable radicals it is therefore contemplated that the phenyl may be substituted with an alkoxyalkyl (e.g., mefhoxyrnethyl) group or a N-alkylalkyl (N-niethyl methyl) group. Alternatively, the phenyl in R _{ may also be substituted with an optionally halogeiiated heteroaiyi. While numerous heterarul groups are deemed suitable, especially preferred heteroaryl groups will be five- or six-meinbered ring systems with one ox two eteioaioHis (e.g., aad½r 0). Consequently, suitable heteroary! groups include pyjidinyl, pjTimidmyl, aad oxazolyl /groups.

|ΘΘ24] In fiiiilier contemplaied aspects, RI niay also be a halogenated heteioaryl. Most typically, the heteroaryl will, be a five- or six-niembered ring aad include, one, two, or three heteroatoms. For example, suitable heteroaryl rings iiiclade imidazole, pyridine, pyiiaiidiae, etc. With respect to the halogen sithsiitaeat it is generally preferred tlxat the halogen is f!uoiiiie or chlorine, and most typically fluorine.

[0025] R2 in most preferred compounds will be a halogenated alkoxy group, and most typically will have 1-3 carbon atoms. For example, especially preferred R2 groups will be a halogenated methoxy group, and especially a fluoronieifaoxy, difluoromethoxy, or

trifliioroniethoxy group. Alternatively, R2 may also he a relatively small substiiuent, including halogens, a methyl group, a CN group, or hydrogen, likewise. R3 is preferably a relatively small substiiuent and may be hydrogen, lower alkyl (between 1-3 carbon atoms), CN, or a halogen. [0026] Therefore, in further contemplated aspects of the inventive subject matter, contemplaied compounds may also have a structure according to Formula II

Formula II

wherein R ₂ is an optionally halogenated lower alkoxy group as described above or an optionally halogenated N-aikylamino group as described above. For example, an especially preferred R ₂ group includes a halogenated methoxy group, particularly a difluoromethoxy group. Similarly, it is preferred that R ₃ may be a hydrogen, a halogen (and especially fluorine or chlorine), or an optionally halogenated lower alkyl as described above. In further preferred compounds, Rg is an optionally halogenated pyridinyl, pyrimidinyl, or oxazoiyt. Where R$ is halogenated, it is typically preferred that the halogen radical is a fluorine or chlorine radical. Moreover, it should be noted that while a single halogen is generaBy preferred, two, three, or more halogens are also expressly contemplated.

|0O27] ^" Where R¾ of Formula I is a heteroaryi it is generally prefeiTed that the heteroaryi will comprise one or two heieroaiorns {preferably nitrogen .and/or oxygen), and that the heteroai yi is a five- or six-niembered ring. Particularly preferred compounds will therefore have a structure according to Formula HI.

Formula HI

in which R ₂ is an optionally halogenated lower alkoxy group (e.g., difluoroniethoxy group) or an optionally halogenated N-aikylamino group as already described above. With respect to ¾ it is generally contemplated that tins R ₃ is H. halogen, or an optionally halogenated lower aikyi (e.g., optionally fluorinated methyl). R ₅ is a halogen, and most typically a fluorine or chlorine radical. Moreover, it is noted that the heteroaryl may be further halogenated with one or more additional halogen radicals, hi the compomid of Formula HI, X and Q are typically independently CH or N, and X and Q are not the same. However, one or more other heter atoms (including S and Se) are also expressly contemplated.

[ΘΘ28] Consequently, especially preferred compounds according to the inventive subject matter will include those shown below having a structure according to Formulae XV-XIIX

Formula IV Formula V

Formula YJR Formula FX

Formula X Formula XI

Formula XII Formula XIII

[0029] In further contemplated aspects, it is noted the compounds according to Formulae I- XIII may include aiyl or heteroaryl groups other than those specified above, and suitable alternative aryl or heteroaryl groups include aromatic monocyclic or polycyclic gr oups, typically comprising between 5 and 18 carbo ring members, which may be unsubstituted or substituted by one or more suitable substituents, and which may be further fused one or more cycloalkyl groups, heterocycioalkyl groups, or heteroaiyl groups (which themselves may be imsiibsrituted or substituted by one or more suitable substituents). Examples for aryl groups include phenyl, biphenyl, 1,2,3,4-tetahydronaphtliyl, naphthyl anihryl, and phenanthryi. Suitable heteroaiyl groups will typically include aromatic monocyclic or polycyclic groups comprising generally between 4 and 18 ring members, including 1-5 heteroatoms selected from nitrogen, oxygen, and sulfur, which may be imsubstituted or substituted by one or more suitable substitoents as defined below, and to which may be fused one or more cycloalkyl groups, heterocycioalkyl groups, or aryl groups, which themselves may be unsubstituted or substituted by one or more suitable siibstituents. Examplary heteroaiyl gropits include iliienyl, furanyl, thiazolyl, triazolyl, imidazolyl, isoxazolyl, oxadiazolyl, tetrazolyl, pyridyl, pyrrolyi, thiadiazolyl, oxadiazolyl, oxafhiadiazolyl, tiiiatiiazolyl, pyrimidinyl, isoquinolinyl, quinolinyl, iiapthyridmyl, phmalimidyL benzimidazolyi, and benzoxazolyl. f ΘΘ30] hi general, the various moieties or functional groups for variables in the formulae may be substituted by one or more suitable "snbstitueiits". lire term "substituent" or is intended to mean any suitable substitue t that may be recognized or selected, by those skilled in the art. Illustrative examples of useful substitoents are those found in the exemplary compounds that follow, as well as halogen (chloro, iodo, bromo, or fhioro); Ci.g-alkyl; Ci^-alkeny!; C s~ alkynyl; hydroxy!: alkoxyl; aniiae; iniro; thiol; tMoetber; iisine; eyano; arniifo;

sulfhnamine; .'Sulfonamide; ketone; aldehyde; ester, oxygen .(=0); haloalkyl (eg.,

trifliroromethyl); earbocyclie cyeloalkyi which may be monocyclic or fused or non-fused polycyelic (eg., cyclopropy!. cyclobutyl eyciopeniyl or eyelohexyl), or a heierocy oalk l which may be monocyclic or fused or non-fused polyeyelie (e.g., pyiToiidinyl, piperidhry!, piperaziiiyL moipboliiiyi or t aziiiyl); earbocyclie or heterocyclic, monocyclic or fused or non-fused polycyclic aryl (e.g. , phenyl, naphthyl, pyrrolyi, indolyl, feranyl, thiophenyL knidazoiy!, oxazolyl, isoxazolyl, miazoiyi, triazolyL tetrazolyl, pyrazo!yl, pyridinyl, qumoliiiyl, isoquinoliiiyl, acridinyl, pyrazmyl pyridazinyl, pyrimidinyl, benzimidazolyl, beiizoiliiopiienyi, or beuzofijranyi); amino (primary, secondary, or tertiary); niiro; thiol; mioether, G-lower alky!; O-aryl, aryl; aryi-iower alkyi; C0 ₂ CH ₃ ; CON¾; OCH ₂ CONH ₂ : N¾; SO ₂ H ₂ ; OCH¾ CF ₃ ; OCF ₃ ; and the like. Such moieties may also be optionally substituted by a fused-ring structure or bridge, for example QC¾-Q, [00311 All of these substituents may optionally be fiuiiier substituted with a substituent selected from groups such as hydroxy! groups, halogens, oxo groups, alkyl groups, acyl groups, siilfonyl groups, mercapto groups, alkylthio groups, alkyloxyl groups, cyeloalkyi groups, beterocyeloalkyl groups, aryl groups, beteroaryl groups, carboxyl groups, amino groups, aikylammo groups, dialkylamino groups, carbamoyl groups, aryloxyl groups, heteiOaryioxyl groups, arylthio groups, beteroarylthio groups, and the like. The term

"optionally substituted" is intended to expressly indicate that the specified group is

misubstituted or substituted by one or more suitable substituents. unless the optional substitoents are expressly specified, in which case the term indicates that the group is unsubstitiited or substituted with the specified substituents. As defined above, various groups may be unsubstiruted or substituted (i.e., they are optionally substituted) unless indicated otherwise herein (e.g... by indicating that the specified group is unsubstiruted).

Furthermore it should be noted that the compounds contemplated herein may be prepar ed as prodrugs. The term "prodrug" as used herein refers to a modification of contemplated compounds, wherein the modified compound exhibits less pharmacological activity (as compared to tire modified compound) and wherein tire modified compound is converted within a target cell (eg., B-cell) or target organ anatomic structure (e.g., joint) back into the modified form. For example, conversion of contemplated compounds into prodrugs may be useful where the active drug is too toxic for safe systemic administration, or where the ccaitemplated compound is poorly absorbed by the digestive tract or other cornpartoent or cell,, or where the body breaks down the conterapiated compound before reacttng its tar get. Tb .i it should be recognized .thai &e compounds according to-i e inventive subject matter can be modified in numerous manners, and especiall preferred mo^fications include, those that improve one or more pharmacokinetic and/or phatiBacodynamic parameter. For example, erne or more substiftients may be added or re laced to achieve a higher AUC in serum,

[0032] On the other hand, arid especially where increased solubility is desired, hydrophilic groups may be added. Still further, where contemplated compounds contain one or more bonds that can be hydiolyzed (or otherwise cleaved), reaction products are also expressly contemplated. Exemplary suitable protocols for conversion of contemplated compounds into the corresponding prodrug form can be found in "Prodrugs (Drugs and he Pharmaceutical Sciences: a Series of Textbooks and Monographs)" by Kenneth B, Sloan (ISBN:

0824786297). and "Hydrolysis in Drag and Prodrug Metabolism: Chemistry, Biochemistry, and Enz mology" by Bernard Testa, Joachim M. Mayer (ISBN: 390639025X), both of which are incorporated by reference herein. Moreover, especially where contemplated compounds have a higher activity when the compound is metabolized (e.g. , hydiolyzed, hydroxylated, glucuronidated, etc. ), it should be appreciated that metabolites of contemplated compounds are also expressly contemplated herein.

[ΘΘ33] Of course, it should be appreciated that (where appropriate) contemplated compounds may have one or more asymmetric centers or groups that may give rise to isomeric, tautomeric, or other steric isoforms (e.g., R- and/or S-coiifigiiiation, E/Z configuration, tautomeric isoforms, enantiomers, diastereoiners, etc.), and each of such forms and mixtures thereof are expressly contemplated herein. f ΘΘ34] hi further contemplated aspects, the compounds may be formulated into a

pharmaceutical composition, typically in combination with a pharmaceutically acceptable carrier. Preferably, the compound will be present at a concentration effective to tr eat

Alzheimer's Disease or signs and or symptoms associated with Alzheimer's Disease. Viewed from another perspective, it is also contemplated that the compounds will be present in the pharmaceutical composition in an amount effective to reduce BACE activity in a patie t when the formulation is administered to the patient. Therefore, contemplated compounds and pharmaceutical compositions will also be advantageous in reducing Αβ level in neuronal tissue and associated plaque build-up. P 35] Depending on the structure of contemplated compounds, it .is. therefore contemplated -that the compounds according to the inventive subject matter are present in the composition ■trraa amount between. 1 microgram to 1000 milligram, more .typicall between 10 microgram to 500 milligram, arid most typically between 50 microgram to 500 muligram per. single dosage unit Thus, preferre concentrations of contemplated compounds in. vivo or in vitro. will generally be ^' between 0.1 nM and 500 imexoM, more t pically between 50 nM and 400 nacroM, and most typically betwee 100 nM and 200 microM. CoBsetpeiitly, in vi vo concentrations will generally be suitable to reduce BACE act vity in vivo with respect to APP cleavage by at least 10%, and more typically by at least 25%. [ΘΘ36] Furthermore, it should be recognized that all formulations are deemed suitable for use herein and especiall include oral and parenteral formulations. For example, for oral administration, contemplated compositions may be in the form of a tablet, capsule, suspension, or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredien Examples of such dosage units are tablets or capsules. The active ingredient may also be administered by injection as a composition wherein, for example, saline, dextrose or water may be used as a suitable carrier. Furthermore, where the compound is formulated for intrathecal adinnusfration, it is generally preferred that the compound is prepared as an injectable solution, suspension, or emulsion, hi still further contemplated formulations, contemplated compounds may be formulated for aerosol delivery (e.g., micropowderized, coated onto a dispersible carrier, dissolved hi atomizable solvent, etc.)

[0037] It should be appreciated thai the choice of the particular formulation and carrier will at least in part depend on the specific use and type of compound. There are numerous manners of drug formulation known in the art, and all of those are deemed suitable for use herein (see e.g.. Pharmaceutical Prefonnulation and Formulation: A Practical Guide from Candidate Drug Selection to Commercial Dosage Form b Mark Gibson; Informa HealthCare, ISBN: 1574911201 ; or Advanced Drug Formulation Design to Optimize Therapeutic Outcomes by Robert O. Williams, David R. Taft, and Jason T. MeConvilie; Informa HealthCare; ISBN: 1420043870).

|0038] The amount of therapeutically active compound that is administered and the dosage regimen for treating a dis ease condition with the compounds and/or compositions of this invention depends on a variety of factors, including the age, weight, sex and medical condition of the subject, the severity of the disease, the route an frequency of adniiiiistralioii, and the particular compound employed, and thus may vary widely. However, especially suitable quantities are provided above, and may therefore allow for a. daily dose of about 0.(8)1 (or even less) to 10 mg l g body weight, preferably between about 0.01 and about 50 n¾¾g body weight arid most preferably from about 0.1 to 20 nig¾g body weight. Typically, a daily dose can be adinuiistered in one. to four doses per day.

[0039] For therapeutic or prophylactic purposes , contemplated compounds are ordinarily combined with one or more excipients appropriate to the indicated route of adrinnistration. If administered per as, the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkaiioic acids, cellulose aikyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and or polyvinyl alcohol, and then tabieted or encapsulated for convenient admmistiation. Such capsules or tablets may contain a controlled-release formulation as may be provided in a dispersion of active compound in hydroxypropyi-methyl cellulose. Formulations for parenteral administration ma be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulations for oral administration . The compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, com oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. Oilier excipients and modes of administration are well and widely known in the pharmaceutical art. f ΘΘ40] Additionally, it is contemplated that contemplated compounds may be combined (in v 'o or in a pharmaceutical formulation or administration regimen) with another

pharmaceutically active ingredient, and especially contemplated other ingredients include various drugs targeting amyloid plaque, tau hyperphosphorylafion, various

nmunomodulatoiy drugs, and/or anti-inflammatoiy drags (e.g., steroids and NSAIDS), etc. Concentrations of second pharmaceutically active ingredients ar e typically at or preferably below those recommended for stand-alone administration, however, higher concentrations are also deemed suitable for use herein, f 0041] Preferably, contemplated compounds will have an IC ₅₀ (with respect to BACE inhibition and APP as substrate) of equal or less than 10 μΜ. more preferably of equal or less than 1 μΜ, and most preferably of equal or less, than 100 aM, and will have m apparent toxicity at the ICgo as rneasiu¾d above. Once candidate compounds fe icall having !¾ of e ual r less than 1.0 μΜ, aiore typically equal or less than 0.1 μΜ, and most typically equal or less than 0.0,1. μΜ,) are identified, such compounds can be further modified to ascertain S AR and to produce compounds with higher potency, reduced . toxicity, and/or increased bioavailability. Therefore, particularly preferred compounds not only inclnde those as shown in Fomrulae I-XXIi above, but also include those of Tables 1 _^ 4 below.

Experimental Data and Results

[0042] Synthetic Protocols: [0043] Contemplated compounds can be prepared using various methods known hi the ail and all of those are deemed suitable for use herein. Further particularly suitable methods will follow the generally synthetic protocol as described in US .2014/0371283 with slight modifications to accommodate various substituents,

[0044] Assay Systems to Evaluate APP Processing: Without being bound to a particular theory, it is believed that the active agent(s) described, herein promote processing of APP by the noiianiyloidogenic pathway and/or reduce or inhibit processing of APP by the amyloidogenic pathway. In the nonamyioidogeic pathway, APP is first cleaved by a-secretase within the Αβ sequence, releasing the APPsot eetodoniain ("sAPPa"). hi contrast, the amyloidogenic pathway is initiated when β-secretase cleaves APP at the amino tenrritius of the Αβ, thereby releasing the APPsp ectodomain ("sAPPp").

[0045] One method to evaluate the efficacy of the active agent(s) is to determine a reduction or elimina tion in the level of APP processing by the amyloidogenic pathway, e.g. , a reduction or elimina tion in the level of APP processing by β-secretase cleavage in response to the administration of the agent(s) of interest. Assays for determining the extent of APP cleavage at the β-seeretase cleavage site are well known in the art. Illustrative assays are described, for example, in U.S. Pat. Nos. 5,744,346 and 5,942,400. Kits for determining the presence and levels in a biological sample of sAPPa and sAPPp, as well as APPneo and Αβ commercially available, e.g., from PerkmElrner.

[0046] ABBI Assay: APP Binding BACE Inhibitor (ABBI) activity of any of the compounds described herein can readily be verified using, for example, assays described herein. Basically, ia certain embodiments a pair tiie assays are utilized to identify ABB! compounds that inhibit BACE cleavage of the MBP-C125 APP substrate, resulting in the inlnbition of the production of€99 and the β-site peptide substrate. (P5-P5*) .and 'also ^· interacts with APP, for example, as measured by surface plasmon resonance (SPR) analysis. [0047] In one ilhistmtive embodiment, an MBP-CI25 APP69S wt, fusion protein can be. used as one of the substrates and the second substr ate can be tire commercially available P5-P5' fluorescence substrate. Each of these substrates is incubated with recombinant BACE (R&D (cat# 3 l-AS-050) in, for example, a 96 well plate format. For the MBP-C125 substrate the C-99 product froni tiie BACE cleavage can be measured usin an AlphaLisa assay as a readout. For the P5-5' substrate, tiie loss of fluorescence upon BACE cleavage can be used as the readout. For the SPR assay the binding analysis of contemplated compounds to fragments of the ectodomain of APP (eAPP) that are recombinant]}' prepared (see e.g., Libeu et al. (2012) PLoS ONE 7(6): e40027) would be done. An ABBI would inhibit the BACE cleavage of the MBP-C125 and/or tiie fluorescence substrate and would also bind to the ectodomain of APP such as the APP230-624 fragment.

{0048] Other Cell Free Assays: Illustrative assays that can be used to demonstrate the inhibitory activity of the active agent(s) are described, for example, in WO 2000/017369, WO 2000/0003819, and US 5,942,400 and 5,744,346. Such assays can be performed in cell-free incubations or hi cellular incubations using ceils expressing an alpha-secretase and/or beta- secretase and an APP substrate having a alpha-secretase and beta-seeretase cleavage sites.

[0049] hi further aspects of the inventive subject matter, contemplated compounds are contacted with an APP substrate containing alpha -secretase and beta-seeretase cleavage sites of APP, for example, a complete APP or variant, an APP fragment, or a recombinant or synthetic APP substrate containing the amino acid sequence: KM-DA or NL-DA (APP-SWj, is incubated in the presence of an alpha-secretase and/or beta-seeretase enzyme, a fragment thereof, or a synthetic or recombinant polypeptide variant having alpha-secretase or beta- seeretase activity and effective to cleave the alpha-secretase or beta-seeretase cleavage sites of APP, under incubation conditions suitable for the cleavage activity of the enzyme.

Agent(s) having the desired activity reduce or prevent cleavage of the APP substrate. Suitable substrates optionally include derivatives that may be fusion proteins or peptides that contain the substrate peptide and a modification useful to facilitate the purification or detection of the peptide or its alpha-secretase and/or beta-seeretase cleavage products. Useful modifications include ike insertion of a known antigenic epitope for antibody binding; tile linking of a label or detectable moiety, the linking of a binding substrate, and the like. f ΘΘ59] Suitable incubation conditions for a cell-free in vitro assay include, for example: approximately 200 aM to 10 μ _. Μ substrate, approximately 1.0 to 200 pM enzyme, and approximately 0.1 »M to 10 μΜ of the agent(s), in aqueous solution, at an approximate pH of 4-7, at approximately 37° C, for a time period of approximately 10 minutes to 3 hoars. These incubation conditions ar e illustrative only, and can be varied as required for the particular assay components and or desir ed measurement system. Optimization of the incubation conditions for the particular assay components should account for the specific alpha-secretase and/or beta-secretase enzyme used and its pH optimum, any additional enzymes and or markers that might be used in the assay, and the like. Such optimization is routine and will not require undue experimentation.

[0051J Another exemplary assay utilizes a fusion peptide having maltose binding protein (MBP) fused to the C-tenninal 125 amino acids of APP-SW. The MBP portion is captured on an assay substrate by anti-MBP capture antibody, incubation of the captured fusion protein hi the presence of alpha-secretase and/or beta-secretase results in cleavage of the substrate at the alpha-secretase and/or beta-secretase cleavage sites, respectively. This system can be used to screen for the inhibitory activity of tiie agent(s) of interest. Analysis of tire cleavage activity can be, for example , by immunoassay of clea vage products. One such immunoassay detects a unique epitope exposed at the earboxy tennirms of the cleaved fusion protein, for example, using the antibody SW192. This assay is described, for example, in US 5,942,400.

[0052] Cellular- Assays: Numerous cell-based assays can be used to evaluate the activity of ageiit(s) of interest on relative alpha-secretase activity to beta-secretase activity and/or processing of APP to release amyloidogenic versus non-amyloidogenic Αβ oligomers.

Contact of an APP substra te with an alpha-secretase and/or beta-secretase enzyme within the cell and in the presence or absence of the agent(s) can be used to demonstrate alpha-secretase promoting and/or beta-secretase inhibitory activity of the ageiit(s). Preferably, the assay in the presence of the ageni(s) provides at least about 30%, most preferably at least about 50% inhibition of the enzymatic activity, as compared with a non-inhibited contr ol . [0053] in one embodiment, cells that naturally express alpha-secretase and/or beta-secretase are used. Alternatively, cells are modified to express a recombinant alpha-secretase and or beta-seeretase or synthetic variant enzymes, as discussed above. he-APP substrate may be added to fee culture medinm and is preferably expressed in the cells. Cells that naturally express APP, ^■■ variant or mutant forms of APP, or cells transformed to express an isoferm of APP, mutant or variant APP, recombinant or synthetic APP, APP fragment or synthetic APP peptide or fusion protein containing the alpha-seexetase and or befa-secxetase APP cleavage sites can be- ^' used, -provided that the expressed APP is permitted to contact the enzyrne and enzymatic cleavage activity can be analyzed.

[ΘΘ54] Human cell lines that normally process Ap from APP provide a useful means to assay inhibitory activities of the ageiit(s). Production and release of A and or other cleavage products into the culture medium can be measured, for example by knaiiffioassay _; such as Western blot or enzyme-linked immunoassay (EIA) such as by EOS A.

[0055] Cells expressing an APP substrate and an active a pha-secretase and/or beta-seeretase can be incubated in the presence of the agents to demonstrate relative enzymatic activity of fee alpha-secretase and/or beta-seeretase as compared with a control. Relative activity of fee alpha-secretase to the beta-seeretase can be measured by analysis ^' of one or more cleavage products of the APP substrate. For example, inhibition, of beta-seeretase activity "against the substrate APP would be expected to decrease release of specific befa-secretase induced APP cleavage products such as Αβ {e.g., Αβ40 ox Αβ42), sAPPp* and APPneo. Promotion or enhancement of alpha-secretase activity against the substr ate APP would be expected to increase release of specific alpha-secretase induced APP cleavage products such as sAPPa and p3 peptide.

[0056] Although both neural and non-neural ceils process and release Αβ, levels of endogenous beta-seeretase activity are low and often difficult to detect by EIA. The use of cell types known to have enhanced beta-seeretase activity, enhanced processing of APP to Αβ, and/or enhanced production of Αβ are therefore preferred. For example, transfection of cells with the Swedish Mutant form of APP (APP-SW): with the Indiana Mutant form (APP- I ); or with APP-SW-IN provides cells having enhanced beta-seeretase activity and producing amounts of Αβ that can be readily measur ed.

[0057] In such assays, for example, the cells expressing APP, alpha-secretase and/or beta- seeretase are incubated in a culture medium under conditions suitable for alpha-secretase and or beta-seeretase enzymatic activit at its clea vage site on the APP substrate. On

IS exposure of ike cells to the ageni(s), the amount of Αβ released into the med um and/or tile anioimi of CTF99 fragnieiits of APP in the cell lysates is reduced as compared with the control. ¾e cleavage products of APP can t e analyzed, for example, by immune reactions wiiii specific, antibodies, as discussed above. [0058] In certain embodiments, preferred cells for analysis of alpha-secretase -and/or beta- secretase activity include primary human neuronal cefis, primary tramgenic animal neuronal cells where the traiisgene is APP, and other ceils such as those of a stable 293 cell line expressing APP, for example, APP-S .

J0059J In Vivo Assays - Animal Models: Various animal models can be used to analyze the activity of agerit(s) of interest on relative alpha-secretase and/or beta-secretase activity and/or processing of APP to release Αβ. For example, tr ansgenic animals expressing APP substrate, alpha-secretase and/or beta-secretase enzyme can be used to demonstrate inhibitory activity of the ageni(s). Certain transgenic animal models have been described, for example, in US 5,877,399; 5,612,486; 5,387,742; 5,720,936; 5,850,003; 5,877,015, and 5,811,633, and in Gaiies et a . (1995) Nature 373: 523. Preferred are animals that exhibit characteristics associated with the pathophysiology of AD. Administration of the agent(s) to the transgenic mice described herein provides an alternative method for demonstrating the inhibitory activity of the ageni(s). Adnrrmstration of the agerit{s) in a harmaceuti all effective carrier and via an administrative route that reaches the tar get tissue in an appropriate therapeutic amount is also preferred.

[0060] Inhibition of beta-secretase mediated clea vage of APP at the beta-secretase cleavage site and of Αβ reiease can be analyzed in these animals by measure of cleavage fr agments in the animal's body fluids such as cerebral fluid or tissues. Likewise, promotion or

enhancement of alpha-secretase mediated clea vage of APP at the alpha-secretase cleavage site and of release of sAPPot can be analyzed in these animals by measure of cleavage fragments in the animal's body fluids such as cerebral fluid or tissues, in certain

embodiments, analysis of brain tissues for Αβ deposits or plaques is preferred.

[0061] On contacting an APP substrate with an alpha-secretase and or beta-secretase enzyme in the presence of the agent(s) under conditions sufficient to permit enzymatic mediated cleavage of APP and/or release of Ap from the substrate, desirable agent(s) are effective to reduce beia-secretase-mediated cleavage of APP at the beta-secretase cleavage site and or effective -to reduce released amounts of Ap. Hie ageiii(s) are also preferably effective to enhance alpha-seeretase-mediated cleavage of APP at the alpha-seexetase cleavage site and to. increase released amounts of sAPPa. Where .such, contacting is the a¾iiiiistra?ioB of fee ageni(s) Jo an animal model.,, for example, as described above, the agent(s) is effective to reduce A deposition in brain tissues of the aniinal, and to reduce fee number and/or size of beta amyloid plaques.. ^" Where, su b aAihrhstration is to a buuian. subject, the agent(s) is effective to inhibit or slow fee progression of disease characterized by enhanced amounts of Αβ, to slow fee progression of AD in the, and/or to prevent onset or development of AD in a patient at risk for the disease.

[ΘΘ62] Using the experimental conditions and parameters as described above, the inventors investigated the effects of contemplated compounds on BACE activity wife respect to IC ₅₀ values, preference towards APP, and various p!iaiiiiaeokmetie parameters in various in vitro and in vivo experiments . Most notably, many of the compounds had submicroniolar IC _:¾ values, exhibited pronounced preference towards APP in BACE imiibition, and were able to cross the blood brain barrier in phaniiacologically meaningful quantities. Table 1 and Table 2 below list exemplariiy results for selected compounds presented herein.

Table 1

|0063] More particularly, Figure 1A shows the structures for FAH17 and FAH32, which were tested in an in vitro BACE inhibition assay along with two other compounds, FAH37 and FAH42 (for structures, see Figure 2A and Table 2. respectively). As can be readily seen from Figure IB, FAH32 had significantly reduced IC _¾ } in the BACE assay. When tested with CHO-7W ceils that were stably transfected with human APP (wildiype), ΑβΙ-42 peptide fomiation was substantially reduced using FAH32 versus control and FAH 17 as shown in Figure IC, On the other hand, FAH32 increased sAPPa fomiation as compared to FAH .17, indicating a higher selectivity of FAH32 towards APP, while sAPPp formation was reduced as compared to FAH17 as can be seen from Figure ID.

[0064] When compared with FAH37 as shown in Figure 2A. FAH37 was less effective in reducing BACE activity in vi.tro as shown in Figure 2B, and similar differences in inhibition were also evident from a P5-P5' fluorescence assay in vitro as is shown in Figure 2C.Further compounds were evaluated and selected compounds are shown in Figure 3A with test results for a BACE activity assay sho n in Figure 3B, Notably, even relatively moderate changes in structure resulted in substantial differences in observed BACE -activ ty. These -compounds were further tested for selectivity or preferenc towards APP in a _. cell based model, and the results are shown for sAPPp production in Figure 3C, for sAPPo, in Figure 3D, and for A l-42 prodiiction in Figure 3Έ.

Ι&Θ65] Table 3 exen^Iarily shews further physical and pharrnacokaietic data for selected compounds according to the inventive subject matter.

a e 3

|0066| Pharmacokinetic data for selected compounds of Table 3 are shown in Figure 4. As is readily apparent, not all compounds were equally able to cross the blood brain barrier, but most compounds did reach pliaimacologically meaningful ieveis given their low IC ₅₀ values. Figure 5A provides a farther exemplary compound FAH65) and Figure 5B depicts dose response d¾ta for Αβ1-42 production in a cell based system.. Further shown are effects observed with regard to Apl-42 production, sAPPp and sAPPa fomiation. As can be seen. FAH65 exhibits clear- preference towards APP in BACE inhibition, and strongly reduces ΑβΙ-42 production. Moreover, FAH65 has also desirable pharmacological properties as ca be seen irom Figure 5C. Here, data are presented for oral and subcutaneous administration (SQ at dosages of 10- and 30 mg/kg. Values are measured in plasma and brain, and the lower graph shows a magnified scale of the area in the box in the upper graph of Figure 5C. Given the low ICs _© value of the compotsrsd, it should be appreciated that pharmaceutically .meaningful concentrations can be achieved n the brain. Similar. results for the compounds of Figures 6A and 7A are presented in Figures 6B aud 7B. Once more, it should he noted tha these exemplary eorapotBids are preferential towards APP in BACE iiilnbition as can be taken from the data for AJ -42, sAPPp, and sAPPa formation. Similar data are expected for FAH74 as shown in Figure 8, which had a IC ₅₀ of 9 BM as determined by an in vitro BACE inhibition assay.

J0067] Additional compounds according to the inventive subject matter and associated data are shown in Table 4 below in which MW indicates molecular weight, cLogP is the partition coefficient {log TPSA is total polar surface area, BACE IC ₅₀ is in micromolar, and PAMPA is parallel artificial membrane permeability assay.

30

|ΘΘ68] It should be apparent to thorn skilled in the ait that many rnore modifications besides those already described are possible without departing froni the ii eative concepts herein. "The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in inteipretmg botf J the specification and the elaiirts, all terms should be in er re ed' hi the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C .... and N, the text should t>e interpreted as requiring only one element from the group, not A plus N, or B phis N, etc