Compounds and methods to treat alzheimer's disease

Abstract

The present disclosure describes compounds and methods for disrupting the amyloid cascade.

Description

COMPOUNDS AND METHODS TO TREAT ALZHEIMER’S DISEASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of prior-filed United States provisional application number 63 / 611,294 filed on December 18, 2023, which is incorporated by reference in its entirety herein.GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under AG090241 awarded by National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The present disclosure describes novel compounds and methods for treating a patient suspected of having or diagnosed with Alzheimer’s disease. In particular, the disclosed compounds and methods may be used to reduce or eliminate amyloid plaque in the subject’s brain.BACKGROUND

[0004] The amyloid cascade of production and deposition of amyloid [3 (AJ3) peptides from amyloid precursor protein (APP) plays a role in the pathogenesis of Alzheimer’s disease (AD). Disease-modifying therapies developed in the past decades are designed to disrupt this amyloid cascade by inhibiting A(3 production with APP secretase inhibitors and clearance of accumulated A[3 peptides by A -targeted monoclonal antibodies. However, these two therapeutic approaches remain unsatisfactory as effective therapies for AD.SUMMARY OF THE INVENTION

[0005] The disclosure provides compounds and methods for treating a patient suspected of having or diagnosed with Alzheimer’s disease.

[0006] An aspect of the present disclosure relates to compounds, such as compounds of formula 1.

[0007] For example, one embodiment is a compound of formula 1 or a pharmaceutically acceptable salt thereof:Formula 1 wherein Rings A+B is selected fromorwherein R3 is selected from. Cl, CH3, NH2, or H; wherein R2 is selected from NO2, F, or H; and wherein R1 is selected fromCl, or H.

[0008] In some embodiments, the compound is Compound 1 of formulaor a pharmaceutically acceptable salt thereof.

[0009] In some embodiments, the compound is Compound 2 of formulaor a pharmaceutically acceptable salt thereof.

[0010] In some embodiments, a pharmaceutical composition comprises any one or combination of the aforementioned compounds and pharmaceutically acceptable carrier.

[0011] In some embodiments, a method of disrupting the amyloid cascade in a cell comprises contacting Cytoplasmic activation / proliferation-associate protein 1 (CAPRIN1) and / or APP, A[340, or A(342 with any one of the aforementioned compounds or pharmaceutically acceptable salt thereof. The result of this contacting reduces the concentration of at least one of APP, A(340, or A|342 compared to a second cell of the same type that has not been contacted with one of the aforementioned compounds or pharmaceutically acceptable salt thereof.

[0012] In some embodiments, a method of treating Alzheimer’s disease comprises administering to a mammal in need thereof a therapeutically effective amount of any one of the aforementioned compounds or pharmaceutical compositions.

[0013] In some embodiments, a method for treating a mammal suffering or suspected of suffering from Alzheimer’s disease comprises co-administering to a mammal in need thereof a therapeutically effective amount of any one of the aforementioned compounds and a therapeutically effective amount of at least one of a known APP secretase inhibitor and / or at least one of a known A0-targeted monoclonal antibody.

[0014] The methods disclosed herein are based on the discovery that the compounds have a high binding affinity between CAPRIN1 and at least one of APP, amyloid-beta peptide having a length of 40 residues (A(34O), or amyloid-beta peptide having a length of 42 residues (A042). When APP is cleaved, the Ap peptides are formed (e.g., A 40 or A 42). In part, the accumulation of the A peptides results in amyloid plaque formations.

[0015] Based upon the discoveries disclosed herein, and without being limited by theory, it is envisioned that the compounds disclosed are capable of binding first to either APP or CAPRINE Once first bound to either protein, the compound acts as a glue and binds the other protein (i.e., APP or CAPRIN1). The result is a targeted interaction between the two proteins facilitated by the compound leading to targeted degradation. With modifications, the compound can be tuned to form a targeted bond between distinct species of proteins (e.g., proteins containing a specific mutation or preferring a protein having 42 amino acids over a protein having 40 amino acids). The compounds disclosed are designed to bind at the interface of two proteins, such as APP and CAPRINE or CAPRIN1 and A[342, to enhance the protein-protein interaction and result in targeted protein degradation. By degrading the APP, A 4O, or Ap42. amyloid f> plaque formations are reduced or inhibited.

[0016] These and other embodiments and features of the disclosure will become more apparent through reference to the following description, the accompanying figures, and the claims. Furthermore, it is to be understood that the features of the various embodiments described herein are not mutually exclusive and can exist in various combinations and permutations.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 shows a table of several compounds tested.

[0018] FIG. 2 shows a graph of The A[342 levels in culture media of HEK-APP695WTtreated with various doses of 0043 (means ± SD, n=3).

[0019] FIG. 3 shows a western blot of the levels of the proteins as indicated (left) in HEK- APP695WTtreated with 0043 (10 pM) for 3 days.

[0020] FIG. 4 shows a western blot of the levels of the indicated proteins in HEK-APP695"Ttreated with various doses of 0043 for 3 days.

[0021] FIG. 5 shows a graph of results from an ELISA of A[342 in culture media of sAD2.3-Ns treated with each compound (10 pM) for 4 days.

[0022] FIG. 6 shows 8 graphs representing the results of ELISAs of A(342 levels in culture media of each of the panel of eight iPSC-Ns treated with indicated compounds (10 pM) for 4 days (means + SD; ****p<0.0001; n=4).

[0023] FIG. 7 shows a graph representing the results of an ELISA of A|342 levels in the cultures of APPSWEmutant and APPWTAG27606-Ns (means ± SD; ****p<0.0001; n=4).

[0024] FIG. 8 shows a graph representing the results of an ELISA of A042 levels in the cultures of APPSV EAG27606-Ns treated with 0043 (10 pM) for 4 days (means ± SD; ****p<0.0001; n=4)

[0025] FIG. 9 shows two graphs representing the results of an ELISA of A(342 in the cultures of sAD2.3-Ns treated with a series of doses of 0043 (top) for 5 days or one dose of 0043 (10 pM) for indicated days (bottom,' means ± SD; *p<0.05; ***p<0.001; n=3).

[0026] FIG. 10 shows two graphs representing results of an ELISA of A(342 in the cultures of sAD2.3-Ns and AG27606-Ns treated with a series of dilutions of 0043 for 5 days with the IC50 values indicated.

[0027] FIG. 11 show's tw o spectral fingerprints showing that Compound 2 (0043) binds directly to CAPRIN1 and APP, wherein NMR spectrum of 200 pM 0043 in green (top) and ID-WaterLOGSY spectra recorded of 200 pM 0043 where red and black spectrum corresponds to 0043 in the presence and absence of 20 pM CAPRIN1, respectively.

[0028] FIG. 12 shows graphical representations of backbone amide peaks in the ^-^N-HSQC spectrum of CAPRIN1 alone (blue) and in the presence of 0043 (red). The direction of peak shifts is shown by the black arrow s.

[0029] FIG. 13 shows a graph representation of the results of a ThermoFluor™ shift assay of rhCAPRINlEu11in the presence of indicated doses of 0043.

[0030] FIG. 14 shows a graph representation of the results of a ThermoFluor™ shift assay of rhAPPFu11in the presence of indicated doses of 0043.

[0031] FIG. 15 shows two western blots showing the presence of various proteins’ levels of indicated proteins at a temperature point relative to that 37°C (top) and in HEK-APP695WTin the absence and presence of 0043 (10 pM) with increased temperature (bottom).

[0032] FIG. 16 shows four graphs representing the results of western blots of CETS A in AG27606- Ns in the absence and presence of 0043 (10 pM) with increased temperature.

[0033] FIG. 17 shows a graph representing results of an ELISA of A[342 levels in culture media of sgCAPRINl and sgControl HEK-APP695WTclones after treated with 0043 (10 pM) for 3 days (means ± SD; ****P < 0.0001, ns: non-significance, n=3).

[0034] FIG. 18 shows a graph representing the results of a fractional distribution of individual and mixed rhCAPRINl and rhAPP695 across glycerol gradient showing that Compound 2 (0043) binding to C APRIN 1 and APP enhances protein-protein interaction.

[0035] FIG. 19 shows a western blot of anti-MBP antibody of the immunoprecipitation or by CAPRIN1 antibody of selected fractions as indicated with black arrows in the glycerol gradient.

[0036] FIG. 20 shows several western blots showing APP-biotin-Streptavidin pull-down of HEK- APP695WTclones (left) and AG27606-Ns (right) after treated with 0043 (10 pM) for APP interaction with CAPRIN1 or G3BP1. Whole cell lysate (WCL) was used as the loading control

[0037] FIG. 21 shows a western blot of avidin-APP-biotin antibodies pull-downs of rhAPPFu11and rhCAPRINlFu11in the presence of 0043 at indicated concentrations.

[0038] FIG. 22 show s a w estern blot of anti-C APRIN 1 antibod / agarose G bead pull-downs of rhC APRIN lFu11and rhAPPFull-MBP in the presence or absence of 0043.

[0039] FIG. 23 shows two graphs representing the results of a direct ELISA of C APRIN 1 interacted with rhAPPFu11or rhAPPTruncatedin the absence or presence of 0043.

[0040] FIG. 24 shows a graph representing a PK assessment of 0043 and 0152 by analyzing mouse plasma at different time points after oral administration of the compounds (n= 3), and a table showing the PK values of 0043 and 0152 determined by analyzing the compounds in mouse plasma samples after administrated orally in indicated doses and solution formulations.

[0041] FIG. 25 shows an NMR spectrum of 0152 in green (top) and ID-WaterLOGSY spectra recorded of 0152 where red and black spectrum corresponds to 0152 in the presence or absence of 20 pM CAPRINE

[0042] FIG. 26 shows a western blot of biotin-0152 / streptavidin-coated bead-pull downs of AG27606-Ns lysates in the presence of various doses of 0152 and HB007.

[0043] FIG. 27 show s tw o graphs representing the results of an ELISA of Ap42 in culture media of AG27606-Ns (I) and UKBiOl lA-Ns (g) after treated with 0043 and 0152 (10 pM) for 4 days (means ± SD; **p<0.01, ****p<0.0001; n=4).

[0044] FIG. 28 shows three graphs representing the results of an ELISA of A(342 in culture media of AG27606-Ns (left) and UKBiOl lA-Ns (middle) after treated with various doses of 0043 and0152 for 5 days with the IC50 values calculated, and ELISA of A|)42 in culture media of sAD2.3- Ns after treatment with 0043 and 0152 (10 pM) for 4 days (means ± SD; **p<0.01, n n> / =3) (right).

[0045] FIG. 29 shows a western blot of APP in AG27606-Ns after treated with 0043 (10 pM) and 0152 (10 pM) for 5 days.

[0046] FIG. 30 shows a table representing the results of an ELISA of A[>42 in culture media of sgControl and sgCAPRINl HEK-APP695WTclones after treated with 0043 and 0152 (10 pM) (means ± SD; ****p<0.0001).

[0047] FIG. 31 shows a graph representing the quantification of APP levels from a western blot.

[0048] FIG. 32 shows three western blots of AG27606-Ns after pretreated with MG132 (5 pM) for 30 minutes and then with 0043 (10 pM) for 24 hours (left), of sAD2.3-Ns (middle) and HEK- APP695WT(right) after treated with MG132 (5 pM) for 30 min and 0043 for 1 day.

[0049] FIG. 33 shows three western blots of of AG27606-Ns after pretreated with Bafilomycin Al (20 nM) for 30 minutes and then with 0043 (10 pM) for 24 hours (left), and sAD2.3-Ns (middle) and HEK-APP695WT(right) after treated with Baf Al (20 nM) for 30 min and 0043 for 1 day.

[0050] FIG. 34 shows both Compound 2 (0043) and Compound 1 (0152).DETAILED DESCRIPTION

[0051] Definitions

[0052] The definitions of terms used herein are meant to incorporate the present state-of-the-art definitions recognized for each term in the chemical and pharmaceutical fields. Where appropriate, illustration is provided. The definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group. Unless defined to the contrary herein, a term will have its ordinary meaning understood by those having ordinary skill in the art.

[0053] Where stereochemistry is not specifically indicated, all stereoisomers of the inventive compounds are included within the scope of the disclosure, as pure compounds as well as mixtures thereof. Unless otherwise indicated, individual enantiomers, diastereomers, geometrical isomers, and combinations and mixtures thereof are all encompassed by the present disclosure. Polymorphic crystalline forms and solvates are also encompassed within the scope of this disclosure.

[0054] As used herein, the term “isolated” in connection with a compound of the present disclosure means the compound is not in a cell or organism and the compound is separated from some or all of the components that typically accompany it in nature.

[0055] As used herein, the term ’‘disrupting the amyloid cascade” refers to altering the expression level, such as reducing the amount of APP, A [MO. or A[J42 protein.

[0056] As used herein, the term “pure” in connection with an isolated sample of a compound of the present disclosure means the isolated sample includes at least 60% by weight of the compound. In certain embodiments, the isolated sample includes at least 70% by weight of the compound. In certain embodiments, the isolated sample includes at least 80% by weight of the compound. In certain embodiments, the isolated sample includes at least 90% by weight of the compound. In certain embodiments, the isolated sample includes at least 95% by weight of the compound. The purity of an isolated sample of a compound of the present disclosure may be assessed by a number of methods or a combination of them; e.g., thin-layer, preparative, or flash chromatography, mass spectrometry7, HPLC, NMR analysis, and the like.

[0057] The term “heteroatom” is art-recognized and refers to an atom of any element other than carbon or hydrogen. Illustrative heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur and selenium.

[0058] The term “alkyl” is art-recognized, and includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alky l groups. In certain embodiments, a straight chain or branched chain alkyl has about 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), and alternatively, about 20 or fewer.

[0059] Likewise, cycloalkyds have from about 3 to about 10 carbon atoms in their ring structure, and alternatively about 5, about 6, about 7, or about 8 carbons in the ring structure.

[0060] Unless the number of carbons is otherwise specified, “lower alkyl” refers to an alkyl group, as defined above, but having from one to about ten carbons, alternatively from one to about six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths.

[0061] The term “aralkyl” is art-recognized and refers to an alkyl group substituted with an ary l group (e.g., an aromatic or heteroaromatic group).

[0062] The terms “alkenyl” and “alkynyl” are art-recognized and refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that include at least one double or triple bond respectively.

[0063] The term “aryl” is art-recognized and refers to multi-membered single-ring or fused ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, naphthalene, anthracene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles” or “heteroaromatics. ”

[0064] The aromatic ring may be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, — CF3, — CN, or the like. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and / or heterocyclyls.

[0065] The terms ortho, meta and para are art-recognized and refer to 1,2-. 1,3- and 1,4- disubstituted benzenes, respectively. For example, the names 1,2-dimethylbenzene and orthodimethylbenzene are synonymous.

[0066] The terms “heterocyclyl”, “heteroaryl”, or “heterocyclic group” are art-recognized and refer to 3- to about 10-membered ring structures, alternatively 3- to about 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles may also be poly cycles. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline. cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, piperonyl, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring may be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, — CF3, — CN, or the like.

[0067] The term “optionally substituted” refers to a chemical group, such as alkyd, cycloalkyl aryl, and the like, wherein one or more hydrogen may be replaced with a substituent as described herein, including but not limited to halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, — CF3, — CN, or the like.

[0068] The terms ■■polycyclyl” or ‘"polycyclic group’" are art-recognized and refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and / or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are “fused rings”. Rings that are joined through non-adjacent atoms are termed “bridged” rings. Each of the rings of the poly cycle may be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyL cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, — CF3, — CN, or the like.

[0069] The term “carbocycle” is art-recognized and refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.

[0070] The term “nitro” is art-recognized and refers to — NO2.

[0071] The term “halogen” is art-recognized and refers to — F, — Cl, — Br or — I.

[0072] “Halide” designates the corresponding anion of the halogens, and “pseudohalide” designates polyatomic analogues of halogens whose chemical behavior mimics that of the halogens. Examples include, but are not limited to, cyanide, cyanate, thiocyanate, and azide.

[0073] The term “sulfhydryl” is art-recognized and refers to — SH.

[0074] The term “hydroxyl” means — OH.

[0075] The term “sulfonyl” is art-recognized and refers to — SO2 ".

[0076] The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that may be represented by the general formulas:wherein R50, R51 and R52 each independently represent a hydrogen, an alkyd, an alkenyl, — (CH2) m R61 , or R50 and R51 , taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R61 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In other embodiments, R50 and R51 (and optionally R52) each independently represent a hydrogen, an alkyl, an alkenyl, or — (CH2)m— R61. Thus, the term “alkylamine” includes an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R50 and R51 is an alkyl group.

[0077] The term “acylamino” is art-recognized and refers to a moiety7that may be represented by the general formula:wherein R50 is as defined above, and R54 represents a hydrogen, an alky l, an alkenyl or — (CH2)m— R61, where m and R61 are as defined above.

[0078] The term "amido” is art recognized as an amino-substituted carbonyl and includes a moiety that may be represented by the general formula:wherein R50 and R51 are as defined above. Certain embodiments of the amide in the present disclosure will not include imides which may be unstable.

[0079] The term “alkylthio” refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In certain embodiments, the “alkylthio” moiety is represented by one of — S- alkyl, — S-alkenyl, — S-alkynyl, and — S — (CH2)m— R61, wherein m and R61 are defined above. Representative alkydthio groups include methylthio, ethylthio, and the like.

[0080] The term “carboxyl” is art recognized and includes such moieties as may be represented by the general formulas:wherein X50 is a bond or represents an oxygen or a sulfur, and R55 and R56 represents a hydrogen, an alkyl, an alkenyl, — (CH2)m— R61 or a pharmaceutically acceptable salt, R56 represents a hydrogen, an alkyl, an alkenyl or — (CH2)m— R61, where m and R61 are defined above. Where X50 is an oxygen and R55 or R56 is not hydrogen, the formula represents an “ester”. Where X50 is an oxygen, and R55 is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when R55 is a hydrogen, the formula represents a “carboxylic acid”. Where X50 is an oxygen, and R56 is hydrogen, the formula represents a “formate”. In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a “thiolcarbonyl” group. Where X50 is a sulfur and R55 or R56 is not hydrogen, the formula represents a “thiolester.” Where X50 is a sulfur and R55 is hydrogen, the formula represents a “thiolcarboxylic acid.” Where X50 is a sulfur and R56 is hydrogen, the formula represents a “thiolformate.” On the other hand,where X50 is a bond, and R55 is not hydrogen, the above formula represents a “ketone” group.Where X50 is a bond, and R55 is hydrogen, the above formula represents an “aldehyde” group.

[0081] The term “carbamoyl” refers to — O(C=O)NRR', where R and R' are independently H, aliphatic groups, aryl groups or heteroaryl groups.

[0082] The term “oxo” refers to a carbonyl oxygen (=0).

[0083] The terms “oxime” and “oxime ether” are art-recognized and refer to moieties that may be represented by the general formula:wherein R75 is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralky l, or — (CH2)m— R61. The moiety is an “oxime’7when R is H; and it is an “oxime ether” when R is alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, or — (CH2)m— R61.

[0084] The terms “alkoxy!” or “alkoxy” are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of — O-alkyl, — O-alkenyl, — O-alkynyl, — O — (CH2)m— R61, where m and R61 are described above.

[0085] The term “sulfonate” is art recognized and refers to a moiety that may be represented by the general formula:in which R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

[0086] The term “sulfate” is art recognized and includes a moiety that may be represented by the general formula: in which R57 is as defined above.

[0087] The term “sulfonamido” is art recognized and includes a moiety that may be represented by the general formula:in which R50 and R56 are as defined above.

[0088] The term “sulfamoyl'’ is art-recognized and refers to a moiety that may be represented by the general formula:in which R50 and R51 are as defined above.

[0089] The term “sulfonyl” is art-recognized and refers to a moiety that may be represented by the general formula:in which R58 is one of the following: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.

[0090] The term “sulfoxido” is art-recognized and refers to a moiety that may be represented by the general formula:in which R58 is defined above.

[0091] The term “phosphoryl” is art-recognized and may in general be represented by the formula:wherein Q50 represents S or O, and R59 represents hydrogen, a lower alkyl or an aryl. When used to substitute, e.g., an alkyl, the phosphory l group of the phosphorylalkyl may be represented by the general formulas:wherein Q50 and R59, each independently, are defined above, and Q51 represents O, S or N. When Q50 is S, the phosphoryl moiety is a “phosphorothioate”.

[0092] The term “phosphoramidite” is art-recognized and may be represented in the general formulas:wherein Q51, R50, R51 and R59 are as defined above.

[0093] The term “phosphonamidite” is art-recognized and may be represented in the general formulas:wherein Q51, R50. R51 and R59 are as defined above, and R60 represents a lower alkyl or an aryl.

[0094] Analogous substitutions may be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls. iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.

[0095] The definition of each expression, e.g., alkyl, m, n, and the like, when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

[0096] The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively. The terms triflate. tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that include said groups, respectively.

[0097] The abbreviations Me. Et. Ph, Tf, Nf, Ts, and Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry, this list is typically presented in a table entitled "‘Standard List of Abbreviations.”

[0098] Certain compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. In addition, polymers of the present disclosure may also be optically active. The present disclosure contemplates all such compounds, including cis- and trans-isomers, E- and Z-isomers, R- and S -enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the disclosure. Additionalasymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this disclosure.

[0099] If, for instance, a particular enantiomer of compound of the present disclosure is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule includes a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art. and subsequent recovery of the pure enantiomers.

[0100] It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.

[0101] The term “substituted” is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds.

[0102] The phrase “protecting group” as used herein means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. Examples of nitrogen protecting groups include an amide ( — NRC(=O)R) or a urethane ( — NRC(=O)OR), for example, as: a methyl amide ( — NHC(=O)CH3): a benzyloxy amide ( — NHC4=O)OCH2C'6H5: — NHCbz); as at-butoxy amide ( — NHC(=O)OC(CH?)?, — NHBoc); a 2-biphenyl-2-propoxy amide ( —NHC(=O)OC(CHS)2C6H4C6H5), as a 9-fluorenylmethoxy amide ( — NHFmoc), as a 6- nitroveratryloxy amide ( — NHNvoc), as a 2-trimethylsilylethyloxy amide ( — NHTeoc), as a 2,2,2-tri chloroethyl oxy amide ( — NHTroc), as an allyloxy amide ( — NHAlloc), as a 2- (phenylsulfonyl)ethyloxy amide ( — NHPsec); or, in suitable cases (e.g., cyclic amines), as a nitroxide radical. The field of protecting group chemistry' has been reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis. 2nded.; Wiley: New York, 1991). Protected forms of the inventive compounds are included within the scope of this disclosure.

[0103] The term “pharmaceutically acceptable salt” or “salt” refers to a salt of one or more compounds. Suitable pharmaceutically acceptable salts of compounds include acid addition salts, such as those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and also those formed with organic acids such as maleic acid. For example, acids commonly employed to form pharmaceutically acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, as w ell as organic acids such as para-toluenesulfonic, salicylic, tartaric, bitartaric, ascorbic, maleic, besylic, fumaric, gluconic, glucuronic, formic, glutamic, methanesulfonic, ethanesulfonic, benzenesulfonic, lactic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric, benzoic and acetic acid, and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acry late, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne- 1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephathalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, [3-hydroxybutyrate. glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1 -sulfonate, naphthalene-2-sulfonate, mandelate and the like.

[0104] Where the compounds carry one or more acidic moieties, pharmaceutically acceptable salts may be formed by treatment of a solution of the compound with a solution of a pharmaceutically acceptable base. Suitable bases for forming pharmaceutically acceptable salts with acidic functional groups include, but are not limited to, hydroxides and carbonates of alkali metals such as sodium, potassium, and lithium; alkaline earth metal such as calcium and magnesium; and other metals, such as aluminum and zinc. Suitable bases also include ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkyl amines), such as mono-, bis-, or tris-(2- hydroxyethyl)amine, 2-hydroxy-tert-butylamine. or tris-(hydroxymethyl)methylamine, N,N-dialkyl-N-(hydroxy alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine, or tri-(2- hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.

[0105] Certain compounds of the disclosure and their salts may exist in more than one crystalline form (i.e., polymorph); the present disclosure includes each of the crystal forms and mixtures thereof.

[0106] Certain compounds of the disclosure and their salts may also exist in the form of solvates, for example hydrates, and the present disclosure includes each solvate and mixtures thereof.

[0107] Certain compounds of the disclosure may include one or more chiral centers, and exist in different optically active forms. When compounds of the disclosure have one chiral center, the compounds exist in two enantiomeric forms and the present disclosure includes both enantiomers and mixtures of enantiomers, such as racemic mixtures thereof. The enantiomers may be resolved by methods known to those skilled in the art; for example, enantiomers may be resolved by formation of diastereoisomeric salts which may be separated, for example, by crystallization; formation of diastereoisomeric derivatives or complexes which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example, via enzymatic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example, on a chiral support; suitable include chiral supports (e.g., silica with a bound chiral ligand) or in the presence of a chiral solvent. Where the desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step may be used to liberate the desired purified enantiomer. Alternatively, specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.

[0108] When a compound of the disclosure includes more than one chiral center, it may exist in diastereoisomeric forms. The diastereoisomeric compounds may be separated by methods known to those skilled in the art (for example, chromatography or crystallization) and the individual enantiomers may be separated as described above. The present disclosure includes the various diastereoisomers of compounds of the disclosure, and mixtures thereof. Compounds of the disclosure may exist in different tautomeric forms or as different geometric isomers, and the present disclosure includes each tautomer and / or geometric isomer of compounds of the disclosure, and mixtures thereof. For example, any olefins present in the compounds may exist as either theE- or Z-geometric isomers or a mixture thereof unless stated otherwise. Compounds of the disclosure may exist in zwitterionic form. The present disclosure includes each zwitterionic form of compounds of the disclosure, and mixtures thereof.

[0109] As used herein the term “pro-drug” refers to an agent, which is converted into the parent drug in vivo by some physiological chemical process (e.g., a prodrug on being brought to the physiological pH is converted to the desired drug form). Pro-drugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmacological compositions over the parent drug. An example, without limitation, of a pro-drug would be a compound of the present disclosure wherein it is administered as an ester (the “pro-drug”) to facilitate transmittal across a cell membrane where water solubility is not beneficial, but then it is metabolically hydroly zed to the carboxylic acid once inside the cell where water solubility is beneficial. Pro-drugs have many useful properties. For example, a prodrug may be more water soluble than the ultimate drug, thereby facilitating intravenous administration of the drug. A pro-drug may also have a higher level of oral bioavailability' than the ultimate drug. After administration, the prodrug is enzymatically or chemically cleaved to deliver the ultimate drug in the blood or tissue.

[0110] Exemplary pro-drugs release an amine of a compound of the disclosure wherein the free hydrogen of an amine or alcohol is replaced by — CH2OP(=O)(OH)2, — CH2O(P=O)(ORs)2,(C=O)OCHR8O(C=O)CH3, or — (C=O)OCH2O(P=O)(OH)2(Ci-C6)alkanoyloxymethyl, 1- ((Ci-C6)alkanoyloxy)ethyl, l-methyl-l-((Ci-C6)alkanoyloxy)ethyl, (Ci-Ce)alkoxy carbonyloxymethyl. N — (Ci-C6)alkoxycarbonylamino-methyl, succinoyl, (Ci-Cs)alkanoyl, a-amino(Ci- C4)alkanoyl, arylactyl and a-aminoacyl, or a-aminoacyl-a-aminoacyl wherein said a-aminoacyl moieties are independently any of the naturally occurring L-amino acids found in proteins, — P(O)(OH)2, — P(O)(O(Ci-C6)alkyl)2 or glycosyl (the radical resulting from detachment of the hydroxyl of the hemiacetal of a carbohydrate).

[0111] Other exemplary pro-drugs upon cleavage release a corresponding free acid, and such hydrolyzable ester-forming residues of the compounds of this disclosure include but are not limited to carboxylic acid substituents (e.g., — (CH2)C(O)OH or a moiety that includes a carboxylic acid) wherein the free hydrogen is replaced by (Ci-C4)alkyl, (C2-Ci2)alkanoyloxymethyl, 1-((C4- C9)alkanoyloxy)ethyl, 1 -methyl- l-(alkanoyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1 -(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-l -(alkoxy carbonyloxy)ethyl having from 5 to 8 carbon atoms,N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1 -(N- (alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N — (Ci-C2)alk lamino(C2-C3)alkyl (such as 0- dimethylaminoethyl), carbamoyl-(Ci-C2)alkyl, N,N-di(Ci-C2)-alkylcarbamoyl-(Ci-C2)alkyl and piperidino-, pyrrolidino- or morph olino(C2-C3)alkyl.

[0112] The term ’‘subject” as used herein, refers to an animal, typically a mammal or a human, that will be or has been the object of treatment, observation, and / or experiment. When the term is used in conjunction with administration of a compound or drug, then the subject has been the object of treatment, observation, and / or administration of the compound or drug.

[0113] The terms “co-administration” and “co-administering” refer to both concurrent administration (administration of two or more therapeutic agents at the same time) and time varied administration (administration of one or more therapeutic agents at a time different from that of the administration of an additional therapeutic agent or agents), as long as the therapeutic agents are present in the patient to some extent at the same time.

[0114] The term “therapeutically effective amount” as used herein, means that amount of active compound or pharmaceutical agent that elicits a biological or medicinal response in a cell culture, tissue system, animal, or human that is being sought by a researcher, veterinarian, clinician, or physician, which includes alleviation of the symptoms of the disease, condition, or disorder being treated.

[0115] The term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.

[0116] The term “pharmaceutically acceptable carrier” refers to a medium that is used to prepare a desired dosage form of a compound. A pharmaceutically acceptable carrier can include one or more solvents, diluents, or other liquid vehicles; dispersion or suspension aids; surface active agents; isotonic agents; thickening or emulsifying agents; preservatives; solid binders; lubricants; and the like. Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa, 1975) and Flandbook of Pharmaceutical Excipients, Third Edition, A. FI. Kibbe ed. (American Pharmaceutical Assoc. 2000), disclose various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

[0117] Various quantities, such as amounts, sizes, dimensions, proportions, and the like, are presented in a range format throughout this disclosure. It should be understood that the description of a quantity in range format is merely for convenience and brevity' and should not beconstrued as an inflexible limitation on the scope of any embodiment. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as all individual numerical values within that range unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 4.62, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

[0118] The terminology used herein is to describe particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a.” “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “includes”, “comprises”, “including” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. Additionally, it should be appreciated that items included in a list in the form of “at least one of A, B, and C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A. B, and C). Similarly, items listed in the form of “at least one of A. B, or C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C).

[0119] Unless expressly stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0120] In any of the embodiments disclosed herein, the terms “treating” or “to treat” includes restraining, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder.

[0121] In any of the embodiments disclosed herein, the term “patient” refers to a human.

[0122] Exemplary Embodiments

[0123] An aspect of the disclosure relates to a compound of Formula 1 or a pharmaceutically acceptable salt thereof:Formula 1 wherein the polycyclic group represented as the Rings A+B is selected from benzthiazole, a fused thiazole, cyanobenzthiazole, or more specifically,wherein R3 is selected from a sulfonyl, an acylamino, an amide, a morpholine amide, a ketone, an ethyl ester, a methyl ester, a nitrile, an ether, a t-butyl ester, a dimethylamine, or more specifically, , a halogen such as Cl, an alkyl such as CH3. an amine such asNH2, or H; wherein R2is selected from NO2, a halogen, such as F, or H; and wherein Ri is selected from a nitrile, a morpholine, an imidazolidinone, a cyclohexadiene, a pyrzoline, an ether, or more specifically, , , , , , a halogen such as Cl, or H.

[0124] In some embodiments, the compound is Compound 1 of formula

[0125] In some embodiments, the compound is Compound 2 of formulaor a pharmaceutically acceptable salt thereof.

[0126] Any one of the aforementioned compounds may exist as the E-geometric isomer, the Z-geometric isomer, or mixtures thereof.

[0127] In one embodiment, any one of the aforementioned compounds is a degrader of APP, A(340, or A(342, or a combination thereof.

[0128] In one embodiment, the compound has an IC50 of less than about 5000 nM for APP, A(340, or A(342, or a combination thereof.

[0129] In one embodiment, the compound has an IC50 of less than about 1000 nM for APP, A(340, or A 42, or a combination thereof.

[0130] In one embodiment, the compound has an IC50 of less than about 500 nM for APP,A(340, or A(342, or a combination thereof.

[0131] In addition, it may be convenient or desirable to prepare, purify, and / or handle the active compound in a chemically protected form. The term “chemically protected form,’’ as used herein, pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions (i.e., they have been modified with a protecting group).By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed without affecting the protected group; the protecting group may be removed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, Protective Groups in Organic Synthesis (T. Green and P. Wuts, Wiley, 1991), and Protective Groups in Organic Synthesis (T. Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999).

[0132] For example, a hydroxy group may be protected as an ether ( — OR) or an ester ( — OC(=O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl)ether; a trimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester ( — OC(=O)CH3, — OAC).

[0133] For example, an aldehyde or ketone group may be protected as an acetal or ketal, respectively, in which the carbonyl group (C(=O)) is converted to a diether (( / (ORfy), by reaction with, for example, a primary7alcohol. The aldehyde or ketone group is readily regenerated by hydrolysis using a large excess of water in the presence of acid.

[0134] For example, an amine group may be protected, for example, as an amide ( — NRC(=0)R) or a urethane ( — NRC(=O)OR), for example, as: a methyl amide ( — NHC(=O)CHs); a benzyloxy amide ( — NHC^OjOCEECgEE; — NHCbz); as a t-butoxy amide ( — NHC(=O)OC(CH?)3, — NHBoc); a 2-biphenyl-2-propoxy amide ( —NHC(=O)OC(CH3)2C6H4CeH5), as a 9-fluorenylmethoxy amide ( — NHFmoc), as a 6- nitroveratryloxy amide ( — NHNvoc), as a 2-trimethylsilylethyloxy amide ( — NHTeoc), as a 2,2,2- trichloroethyloxy amide ( — NHTroc), as an allyloxy amide ( — NHAlloc), as a 2- (phenylsulfonyl)ethyloxy amide ( — NHPsec); or, in suitable cases (e.g., cyclic amines), as a nitroxide radical.

[0135] For example, a carboxylic acid group may be protected as an ester or an amide, for example, as: a benzy l ester; a t-butyl ester; a methyl ester; or a methyl amide.

[0136] For example, a thiol group may be protected as a thioether ( — SR), for example, as: a benzyl thioether; or an acetamidomethyl ether ( — SCH2NHC(=O)CH3).

[0137] Exemplary Pharmaceutical Compositions

[0138] One or more compounds of this disclosure can be administered to a mammal by itself or by themselves or in pharmaceutical compositions where it is or they are mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a disease or condition as described herein. Mixtures of these compounds can also be administered to the patient as a simple mixture or in suitable formulated pharmaceutical compositions. For example, one aspect of the disclosure relates to pharmaceutical composition comprising a therapeutically effective dose of a compound of formula 1, or a pharmaceutically acceptable salt, solvate, enantiomer or stereoisomer thereof; and a pharmaceutically acceptable diluent or carrier.

[0139] Techniques for formulation and administration of the compounds of the instant disclosure may be found in references well known to one of ordinary skill in the art, such as “Remington's Pharmaceutical Sciences,'’ Mack Publishing Co., Easton, Pa., latest edition.

[0140] Suitable routes of administration may. for example, include oral, eyedrop, rectal, transmucosal, topical, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0141] Alternatively, one may administer a compound in a local rather than a systemic manner, for example, via injection of the compound directly into an oedematous site, often in a depot or sustained release formulation.

[0142] Furthermore, one may administer a compound in a targeted drug delivery system, for example, in a liposome coated with endothelial-cell-specific antibody.

[0143] The pharmaceutical compositions of the present disclosure may be manufactured, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0144] Pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0145] For injection, the agents of the disclosure may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants are used in the formulation appropriate to the barrier to be permeated. Such penetrants are generally known in the art.

[0146] For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents maybe added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0147] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally include gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0148] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

[0149] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0150] For administration by inhalation, the compounds for use according to the present disclosure are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, tri chlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0151] The compounds can be formulated for parenteral administration by injection, e.g., bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0152] Pharmaceutical formulations for parenteral administration may include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil. or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may include substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also include suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0153] Alternatively, the active ingredient may be in powder form for reconstitution before use with a suitable vehicle, e.g., sterile pyrogen-free water.

[0154] The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., including conventional suppository bases such as cocoa butter or other glycerides.

[0155] In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly or by intramuscular injection). Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (for example, as a sparingly soluble salt).

[0156] Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethy sulfoxide also may be employed. Additionally, the compounds may be delivered using a sustained-release system, such as semi- permeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

[0157] The pharmaceutical compositions may also comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers, such as polyethylene glycols.

[0158] Exemplary Methods of Treatment

[0159] Also provided herein are methods of disrupting an amyloid cascade comprising contacting a CAPRIN1 with one of the aforementioned compounds or a pharmaceutically acceptable salt. In some embodiments, the method comprises contacting APP with one of the aforementioned compounds or a pharmaceutically acceptable salt. In some embodiments, the compound binds at the interface of CAPRIN1 and either APP, A|34O, or Ap42 to form a stable compound. Once the stable compound is formed, the APP, A(340, or A(342 is degraded. In some embodiments, a cell expressing APP, Ap40, or Ap42 is contacted with one or more of the disclosed compounds resulting in a decreased concentration of APP, AP40, or AP42 compared to a second cell of similar type that was not contacted by one or more of the disclosed compounds.

[0160] In some embodiments, a method of treating a subject diagnosed with or suspected of having Alzheimer’s disease comprises administering therapeutically effective amount of one or more of the disclosed compounds to the subject resulting in the inhibition or reduction of amyloid plaques. In some embodiments, a method of preventing or slowing the progression of Alzheimer’s disease comprises administering to a subject therapeutically effective amount of one or more of the disclosed compounds. In some embodiments, the method further comprises co-administering a therapeutically effective amount of at least one of a known APP secretase inhibitor and / or at least one of a known A0-targeted monoclonal antibody.

[0161] Exemplary Dosage

[0162] As used herein, a “therapeutically effective amount” or “therapeutically effective dose” is an amount of a compound of the disclosure or a combination of two or more such compounds, which inhibits, totally or partially, the progression of the condition or alleviates, at least partially, one or more symptoms of the condition. A therapeutically effective amount can also be an amount which is prophylactically effective. The amount which is therapeutically effective will depend upon the patient's size and gender, the condition to be treated, the severity of the condition and the result sought. For a given patient, a therapeutically effective amount may be determined by methods known to those of skill in the art.

[0163] A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the maximum tolerated dose (MTD) and the EDso (effective dose for 50% maximal response). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between MTD and ED50. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.

[0164] The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view7of the patient's condition. In the treatment of crises, the administration of an acute bolus or an infusion approaching the MTD may be required to obtain a rapid response.

[0165] Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the effect of disrupting the amyloid cascade, or minimal effective concentration (MEC). The MEC will vary7for each compound but can beestimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. HPLC assays or bioassays can be used to determine plasma concentrations.

[0166] Dosage intervals can also be determined using the MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for about 10-90% of the time, between about 30-90%, or between about 50-90% until the desired amelioration of symptoms is achieved. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0167] The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

[0168] Exemplary Kits

[0169] The compounds and compositions of the disclosure (e.g., compounds and compositions of formula 1) may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms including the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the disclosure formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition. Instructions for use may also be provided.

[0170] EXEMPLIFICATION

[0171] Further reference is made to the following experimental examples.

[0172] Table of Abbreviations

[0173] The compounds and methods now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the invention.

[0174] Example 1

[0175] Synthesis of Compound 1

[0176] Step 1. Methyl 2-amino-l,3-benzthiazole-6-carboxylate (2g, 9.6mmol) was suspended in THF (60mL) and reacted with l-methoxy-3-isocyanato-benzene (1.72g, 11.5mmol) at 25°C for 16hr. TLC was run in 1: 1 EtOAc / Hex to show completion. The reaction was concentrated then diluted with 40mL of EtOEI. After heating the reaction mixture to 70°C, 40mL of DCM was added and the reaction was stirred for 30mins. The solid was then cooled to 25°C, filtered and rinsed with EtOH to give 2.6g of Compound A as a white solid (76%).

[0177] Step 2. Compound A (2.6g, 7.3mmol) was suspended in a 2: 1 solution of THF / EtOH (80mL). An aqueous IN LiOH solution (58.1mmol) was added to the reaction and stirred at 25°C for 2 days. TLC in 1 : 1 EtOAc / Hex showed completion. The reaction was concentrated to remove the organic solvents and was diluted with 20mL of H2O and 80mL of IP A. The pH was adjusted to 5 using AcOH and after stirring for Ihr, the reaction was filtered and rinsed with IPA to obtain 2.42g (97%) of compound B as a white solid.Step 3. Compound B (2.42g, 7.3mmol) was suspended in DMF (90mL), then EDC (2.8g, 14.6mmol) and HOBT (1.19g, 8.78mmol) was added to the reaction. After stirring at 25°C for lOmins, morpholine (2.55g, 29.3mmol) and triethylamine (8.21mL, 58.5mmol) was added, then the reaction for stirred for 2 days at 25 °C. TLC in 1: 1 EtOAc / Hex showed completion, the reaction was poured into 200mL of H2O and washed three times with 200mL of EtOAc. The organic washes were combined and concentrated to ~100mL and lOOmL of H2O was added. The organic and aqueous were left alone for 8hrs until white solid crashed out of solution. The solid was then filtered and rinsed with EI2O to give 0152 (800mg, 27%) as a white solid of Compound 1.

[0178] Synthesis of Compound 2

[0179] Step 1. Methyl 2-amino-l,3-benzthiazole-6-carboxylate (2g, 9.6mmol) was suspended in THF (60mL) and reacted with 3-isocyanatobenzonitrile (1.65g. 11.5mmol) at 25°C for 16hr. TLC was run in 1 : 1 EtOAc / Hex to show completion. The reaction was concentrated then diluted with 40mL of EtOH. After heating the reaction mixture to 70°C, 40mL of CH2CI2 was added and the reaction was stirred for 30mins. The solid was then cooled to 25°C, filtered and rinsed with EtOH to give 2.8g of methyl 2-[(3-cyanophenyl)carbamoylamino]-L3-benzothiazole-6- carboxylate as a white solid (83%).

[0180] Step 2. Methyl 2-[(3-cyanophenyl)carbamoylamino]-l,3-benzothiazole-6- carboxylate (2.8g, 7.9 mmol) was suspended in a 2:1 solution of THF / EtOH (80mL). An aqueous IN LiOH solution (58. Immol) was added to the reaction and stirred at 25°C for 2 days. TLC in 1 : 1 EtOAc / Hex showed completion. The reaction was concentrated to remove the organic solvents and was diluted with 20mL of H2O and 80mL of Isopropyl alcohol. The pH was adjusted to 5 using AcOH and after stirring for Ihr, the reaction was filtered and rinsed with Isopropyl alcohol to obtain 2.3g (88%) of 2-[(3-cyanophenyl)carbamoylamino]-l,3-benzothiazole-6-carboxylic acid as a white solid.

[0181] Step 3. 2-[(3-cyanophenyl)carbamoylamino]-l,3-benzothiazole-6-carboxylic acid (2.3g, 6.8 mmol) was suspended in DMF (90mL), then EDCT (2.1g, 13.6 mmol) and HOBT (1 . 1g, 8.16 mmol) were added to the reaction. After stirring at 25°C for lOmins, morpholine (2.9g, 34 mmol) and triethylamine (9.4 mL, 68 mmol) was added, then the reaction for stirred for 2 days at 25°C. TLC in 1 : 1 EtOAc / Hex showed completion, the reaction was poured into 200mL of H2O and washed three times with 200mL of EtOAc. The organic washes were combined and concentrated to ~100mL and lOOmL of H2O was added. The organic and aqueous were left alone for 8hrs until white solid crashed out of solution. The solid was then filtered and rinsed with H2O to give the title compound HB0043 (1.2 g. 43%) as a white solid.

[0182] The next set of Examples provides the Materials and Methods for the Biological Assay Data.

[0183] Example 2

[0184] Cell lines and human iPSCs

[0185] HEK293 cells were purchased from the American Type Culture Collection(ATCC). The human iPSC SAD2.3 (UCSD234i-SAD2-3) were purchased from Wicell, UKBiOl 1 A from European Bank for induced pluripotent stem cells, Sigma, AG25367, AG25370, AG27606, AG27608, AG27609. and AG27602 from Coriell.

[0186] Example 3

[0187] iPSC-Ns differentiation

[0188] The human 1PSC SAD2.3 (UCSD234i-SAD2-3; Wicell), UKBiOl lA,AG25367, AG25370, AG27606, AG27608. AG27609. and AG27602 were maintained and expanded on Matrigel (Growth factor reduced Matrigel matrix, Coming) in mTeSRl complete medium (mTeSRl medium + 5X supplement, Stem Cell Technologies).

[0189] For iPSC-Ns transgene delivery, each iPSC lines was transduced with lentiviral particles for pLVX-UbC-rtTA-Ngn2:2A: Ascii (Addgene #127289) or pLVX-UbC- rtTA-Ngn2:2A: EGFP (Addgene #84777). followed by selection in the presence of puromycin (0.5 mg / ml, Gibco). To initiate conversion, the transgene delivered iPSCs were dissociated with StemPro™ Accutase™ (Gibco) and reseeded in Neuro culture media (Neurobasal media (Gibco), 2% B27 supplement (Gibco), IX Glutamax (Gibco)) with doxycycline (2 pg / ml, Sigma-Aldrich) for 5 days and without doxycycline for 3 days.

[0190] Example 4

[0191] Brain 3D organoid formation

[0192] For generating brain 3D organoids, the Lancaster’s protocol and Pa§ca's protocol were used with minor modifications. In brief, 9,000 cells / 100 pL of each well were seeded in Ultra-low attachment 96 well U-bottom plates (Sbio) supplemented with 10 pM of ROCK inhibitor. Next day, 100 pl of mTeSRl was added and the cells were cultured as embryonic bodies (EB) for 5 days at 37°C in 5% CO2 incubator. When the size of EB reached 400-600 pm, EBs were transferred into ultra-low binding 24 well (Coming) with 200 pl of neural differentiation medium (Neurobasal medium (Gibco), 2% B27, IX Glutamax) with 2 pg / ml of Doxycycline and incubated for 5 days at 37°C in 5% CO2 incubator until the size of EB was 700-800 pm. During this step, each EB was in its own well and culture medium was changed every day with fresh neural differentiation medium with Doxycycline. At day 10. the EB was embedded into 15 pl of 100% Matrigel (Coming) and the embedded EBs were cultured in neural differentiation medium without Doxycycline in 6 well ultra-low binding plates (Coming). In this step, each well of 6 well plates had 16 EBs and plates were incubated in stationary methods for 5 days at 37°C in 5% CO2 incubator to expand neuroepithelial buds, followed by placing the plates on the orbital shaker (Benchmark Scientific) at 75 rpm at 37°C in 5% CO2 incubator. Fresh medium was changed every 3 days.

[0193] Example 5

[0194] HEK-APP695WTcell line construction

[0195] Lentiviral plasmids for APP695WTHEK293 were generated in the 3rdgeneration plasmid system through Vectorbuilder Company (pLV-Hygro-CMV-hAPP). HEK293T cells were transfected with targeting plasmid and 3rdgeneration packing plasmid (pMD2.G, pMDLg / pRRE; Addgene #12251, and pRSV-Rev; Addgene #12253) together using Lipofectamine 2000.. HEK293 cells were infected with virus particles and selected with hygromycin B (200 pg / ml, Invitrogen). For single clone selection, the cells were reseeded in 96-well plates as single cells. Once expanded, clones were analyzed by ELISA for amyloid beta 42 (A(342) and by western blot for APP expression. For sgCAPRINl HEK293 cell lines, the guide RNA sequences of CAPR1N 1 were cloned into LentiCRISPRv2 plasmid (Addgene #52961) according to protocol. HEK293 cells were infected, selected with puromycin (1 pg / ml) for 2 days, single cell selection, and followed by western blot for CAPRIN1 knock-out verification.

[0196] Example 6

[0197] Generation PSC

[0198] CRISPR Cas9 mediated knock-in cell Clones of APP-Swedish mutation inAG27606 iPSCs were generated by Synthego Corporation (Redwood City, CA, USA). Briefly, Ribonucleoproteins containing the Cas9 protein and synthetic chemically modified sgRNA were electroporated into the cells along with a single-stranded oligodeoxynucleotide donor using Synthego's optimized protocol. Editing efficiency was assessed upon recovery using Sanger sequencing, 48 hours post electroporation. The resulting chromatograms were processed using Synthego Inference of CRISPR edits software (ice.synthego.com). Edited cell pools were seeded at 1 cell / well using a single-cell printer into 96 or 384 well plates to create monoclonal cell populations. All wells were imaged every 3 days to ensure expansion from a single-cell clone. Clonal populations were screened and identified using the PCR-Sanger-ICE genotyping strategy.

[0199] Example 7

[0200] Cellular Thermal Shift Assay (CETSA)

[0201] For CETSA, as described previously, iPSC-Ns or APP695'A T-HEK were treated in the presence or absence of 0043 (100 pM) for 1 hour, trypsinized, washed and resuspended in 100 pl of PBS with protease inhibitors (600,000 cells / tube). Cells were incubated at their designated temperatures for 3 minutes, followed by 25°C for 3 minutes. Then, 1% NP40 was added to lyse the cells, followed by immediate snap freezing with liquid nitrogen and thawing using thermal cycler at 25°C. This freezing and thawing cycle was repeated twice. The lysates were spun at 13,500 rpm for 20 minutes at 4°C and supernatant was harvested, for western blotting analysis.

[0202] Example 8

[0203] ThermoFluor™ Assay

[0204] For the ThermoFluor™ assay, briefly, all proteins were used at a final concentration of 5 LLM for this assay and GloMelt™ Thermal Shift Protein Stability’ Kit (Biotium) was used at final concentration of 0.5X. All experiments were earned out with QuantStudio 6 Flex (Applied Biosystems). SYBR was used as a reporter and ROX was used as a passive reference according to the manufacturer's directions. Melting curve data was exported, followed by analysis with Boltzmann's sigmoidal curve in Prism 9.

[0205] Example 9

[0206] Cytotoxicity and cell viability’ assay

[0207] Cytotoxicity test was conducted using Neutral Red Uptake assay. In brief, iPSC-Ns seeded in 96 well plates (1 x 105cells / well) were treated dose-dependently for 5 days. Cells were washed with PBS containing CaCh and MgCh followed by the addition of Neutral red solution (40 ug / ml in PBS, Sigma-Aldrich) and incubation at 37°C for 3 hours. Cells were then washed with PBS containing CaCE and MgCh two times and de-stained using with Neutral red de-staining solution on plate shaker for 30 minutes, followed by reading at 540 nm with Microplate reader. Cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) in accordance with manufacturer instructions. In brief, HEK cells were plated at 5 x 104cells / well, iPSC-Ns at 1 x 105cells / well in 96 well plates and treated with drugs at 10 pM for 48 hours.

[0208] Example 10

[0209] Antibodies

[0210] The following antibodies w ere used for western blot, anti-beta- Amyloid, 1-16 (6E10, BioLegend, 803001), anti-beta-Amyloid, 1-42 (12F4, BioLegend, 805501), (B-Amyloid (Cell Signaling Technology', 8243), TUBB3 (Proteintech, 66375-1 -Ig), Biotin-anti-beta- Amyloid, 17-24 (4G8, BioLegend. 800704). Anti-Amyloid Precursor Protein, C-Terminal (Sigma- Aldrich, A8717), Anti-Human sAPPbeta (Tecan (IBL), JP18957), Presenilin 1 (Cell Signaling Technology, 5643), Presenilin 2 (Cell Signaling Technology, 9979), BACE1 (Abeam, ab2077), ADAM10 (Cell Signaling Technology, 14194), Nicastrin (Cell Signaling Technology7, 5665), GGA1 (Proteintech, 25674-1-AP), SORLA (Proteintech, 22592-1-AP), CDIO / Nepnlysin (Cell Signaling Technology, 65534), CD107a (LAMP-1) (BioLegend, 328601; Fisher Scientific, PA1-654A), SOX2 (Proteintech, 11064-1 -AP), His»Tag® (Millipore, 70796-3), anti-APP C-Terminal Fragment (BioLegend, 802803).

[0211] Example 1 1

[0212] Western blot analysis and immunoprecipitation

[0213] Cells were lysed in 1% triton X-100 buffer (50mM Tris (pH 7.4), 150mMNaCl, 10% glycerol, 1% Triton xlOO, 1 mM EDTA) or RIPA buffer (50 mM Tris (pH7.5) 150 mM Nad, 0.5% NP40, 0.5% deoxycholate, 0.1% SDS) supplemented with 1 mM PMSF, protease and phosphatase inhibitors. For immunoprecipitation, cell lysates were incubated with the primary antibodies (1-2 pg) overnight at 4°C and mixed with protein G beads for 4 hours. The immunoprecipitants were washed with 1 % triton X-100 buffer 3 times and eluted with 2X Laemmli buffer, followed by western blot.

[0214] Example 12

[0215] Real-time PCR

[0216] Total RNA was isolated using the RNeasy kit (Qiagen), followed by synthesis for cDNA with a reverse transcription reaction (Quantitect® Reverse Transcription kit). Quantitative PCR was performed on QuantStudio 6 Flex (Applied Biosystems). The expression levels of APP were normalized to the GAPDH mRNA levels and represented relative to the expression in control cells. The results were analyzed with the delta-delta-Ct methods.

[0217] Example 13

[0218] Enzyme-linked immunosorbent assay (ELISA)

[0219] For 2D culture, iPSC-Ns or HEK-APP695WTwere treated with drugs.Secreted A(342 peptides were quantified using the Human Amyloid (31-42 human ELISA kit (Thermo Fisher) according to the manufacturer's protocol.

[0220] For 3D organoids ELISA, tissue processing was performed as described with minor modifications. In brief, organoids were lysed with RIPA buffer supplemented with Protease and Phosphatase inhibitors (Sigma- Aldrich). Then, samples were sonicated and incubated on ice for 30 minutes, followed by centrifugation at 13,000 rpm for 30 minutes at 4°C. The supernatants (soluble fractions) were collected, and the pellets (insoluble fractions) were lysed with UREA buffer (8M Urea, 50 mM Tris (pH7.5), 100 mM NaCl, supplemented with protease and phosphatase inhibitors). Total protein in the soluble fraction was determined using BCA assay (Thermo Scientific). The A(342 in both RIPA and UREA fractions were measured using the Human Amyloid (31-42 human ELISA kit. The results were standardized with the amounts of proteins by BCA assay results.

[0221] Example 14

[0222] Immunofluorescence staining

[0223] Cells were grown on glass cover slips coated with 1 % Matrigel and treated with drugs. Cells were washed, fixed, permeabilized, and blocked and incubated in primary antibodies diluted in blocking solution (1% v / v BSA solution / 5% Goat serum in PBS containing 0. 1% Triton-X 100) overnight at 4°C (polyclonal Rabbit anti-CAPRINl, 15112-1 -AP, Proteintech; monoclonal mouse anti-APP (22C11), MAB348, Millipore sigma; monoclonal mouse anti-MAP2 (API 8), MA5- 12826, Thermo Fisher Scientific; polyclonal rabbit anti-NANOG, PAI -097; polyclonal rabbit anti-Calnexin, 10427-2-AP, Proteintech; polyclonal rabbit anti-ERGIC-53, 13364-1-AP, Proteintech; polyclonal rabbit anti-SEC31A, 17913-1-AP. Proteintech; polyclonal rabbit anti-TMED9, 21620-1-AP. Proteintech; monoclonal rabbit anti-GM130 (D6B1). 12480S, Cell Signaling Technology; polyclonal rabbit anti-TGN46, 13573-1-AP, Proteintech; monoclonal rabbit anti-EEAl (C45B10), 3288S, Cell Signaling Technology7; monoclonal rabbit anti-Rab7 (D95F2), 9367S, Cell Signaling Technology; monoclonal rabbit anti-LAMPl (D2D11), 9091S, Cell Signaling Technology). The cells were washed three times with PBS and incubated in secondary antibodies in blocking solution for 1 hour at room temperature (Alexa Fluor 488® Goat anti-Mouse IgG; Alexa Fluor 488® Goat anti-Rabbit IgG; Alexa Fluor 555® Goat anti-Mouse IgG; Alexa Fluor 555® Goat anti-Rabbit IgG), then washed with PBS, followed by HOECHST staining (Invitrogen) for 5 minutes and washed with PBS. The glass coverslips were put on a slide in mounting medium (VECTASHIELD) and the edges were sealed with coverslip sealant (BIOTIUM). Image data was captured and processed with a confocal microscope system (Olympus Fluoview FV1000) and ImageJ program.

[0224] For 3D organoids staining, organoids were fixed in 10% formalin overnight at room temperature followed by incubation in 30% sucrose solution overnight at room temperature, embedded in optimal cutting temperature compound (OCT, FisherScientific) and kept at -80°C overnight. Frozen samples were sectioned at 10 pm using a cry ostat (Leica) and mounted on microscope slides (Fisherbrand™ superfrost plus, FisherScientific). Prior to immunolabeling, sections were rinsed in PBS for 5 minutes, permeabilized for 30 minutes in PBS containing 0.3% Triton-X 100, blocked for 1 hour in PBS containing 5% BSA and 0.1% Triton-X 100, and incubated with primary' antibodies diluted in the same blocking solution. Then, secondary7staining, mounting, and imaging were followed.

[0225] Example 15

[0226] Immunohistochemistry of 3D organoids and human AD patient brain samples

[0227] Immunohistochemistry was performed in the Department of Pathology andLaboratory' Medicine. Autopsy samples of brain tissues of Alzheimer’s disease patients were provided by Indiana Pathology Institute (Indianapolis, Indiana, USA) in accordance with protocols approved by the institute. Paraffin-embedded 3D organoids and human AD patient brain samples subjected to Hematoxylin and Eosin (H&E), synapses, GFAP, CAPRIN1 (polyclonal Rabbit anti- CAPRIN1, 15112-1-AP, Proteintech), APP (monoclonal mouse anti-APP / Af> (6E10), 803001, Biolegend; MAB348, Millipore sigma) in a Dako automated instrument using the Dako Flex detection system. The primary’ antibodies and secondary antibodies were used in colocalization experiments with human AD brain biopsy samples, then imaged using a confocal microscope system.

[0228] Example 16

[0229] Imaging Analysis

[0230] ImageJ (Fiji) program (NIH, Bethesda, MD, USA) was used to analyze images. Pearson’s correlation coefficients were calculated with JACop plugin from two channels. For trafficking analysis with organelles in iPSC-Ns, APP or CAPRIN1 or organelles signals were segmented using trainable weka segmentation plugin in Fiji. Colocalized area were calculated in image calculator tool (‘Or function) to get an overlay ed areas for the segmented signals, followed by using analyze particle tool in Fiji to determine by >1 pixel overlapping of segmented signal. For colocalization analysis, APP, CAPRIN1, or LAMP1 were segmented by using trainable weka segmentation plugin for iPSC-Ns, HEK293 cells, and 3D organoids. To determine the fluorescence intensity, the corrected total cell fluorescence (CTCF) was used using the formula: CTCF = Integrated Density - (Area of selected cell x Mean fluorescence of background readings).

[0231] Example 17

[0232] Recombinant proteins purification from E, coli

[0233] For full length CAPRIN1 purification, pET-6HIS-TEV-hCAPRINl(Vectorbuilder) were transformed into E.coli BL21(DE3)followed by culturing in Luria broth (LB) with 100 pg / mL of Ampicillin overnight at 37°C. 10 ml of primary culture was inoculated into fresh LB medium with Ampicillin, cultured until OD600 reached 0.8, expression of CAPRIN1 was induced with 0.5 mM isopropyl-P-D-thiogalactoside at 37°C for 4 hours. After centrifugation at 3,000 rpm for 20 minutes at 4°C, pellets were dissolved with prechilled lysis buffer (50 mM Na2HPO4, 300 mM NaCl, 10% Glycerol, 0.1% Triton X-100, 10 mM Imidazole at pH 8.0), followed by sonication for 16 minutes (10 seconds on and 30 seconds off for 24 cycles) on ice and centrifuged for 40 minutes (40,000 rpm at 4°C). The supernatant w as mixed with Ni-NTA agarosebeads (Qiagen) overnight at 4°C, then loaded on a PD-10 column (GE healthcare). The columns were washed with buffer (50 mM Na2HPO4, 300 mM NaCl, 10% Glycerol, 10 mM Imidazole at pH 8.0) and eluted with elution buffer (50 mM Na2HPC>4, 300 mM NaCl, 10% Glycerol, 250 mM Imidazole at pH 8.0), followed by dialysis (50 mM NaiHPCL. 250 mM NaCl. 10% Glycerol at pH 8.0) for 2 hours at 4°C. The samples were concentrated using Millipore Ami con filters (Millipore) and dialyzed with storing buffer (50 mM Na2HPC>4, 200 mM NaCl, 50% Glycerol at pH 8.0) for storing at -80°C.

[0234] For15N-CAPRIN1 samples for NMR, pCold-II-Halo-His8-HRV3C-CAPR1N 1112-260plasmid were gifted from Du. Plasmid transformed into E.coli BL21(DE3) cells and cultured overnight at 37°C. A single colony was selected and inoculated into 100 ml LB with 100 pg / mL Ampicillin overnight at 37°C for primary culture. The primary culture was then added to 1 L LB medium with Ampicillin (final OD600: 0.1) and was incubated at 37°C until OD600 0.8 at 37°C. Cells were then harvested and resuspended with 1 L15N-M9 minimal media (7.52 g of Na2HPO4-2H20, 3 g ofKH2PO4, 0.5 g of NaCl, 0.5 g ofNH4Cl, 50 mg of EDTA, 8.3 mg of FeCh- 6H20, 0.84 mg of ZnCl2, 0.13 mg of CUC12-2H20, 0.1 mg of COC12-2H20, 0.1 mg of H3BO3, 0.016 mg of MnC12-4H20, 1g of15NH4C1, 1 mM MgSCL, 1 mM CaCL, 1 mg of Thiamine-HCl, 1 mg of Biotin, and 100 pg / ml Ampicillin), followed by shaking incubation overnight at 10°C. Cells were harvested at 3,500 rpm for 20 min at 4°C and resuspended with lysis buffer (50 mM Tris, 500 mM NaCl, 20 mM Imidazole, and 2 mM P-mercaptoethanol at pH 8.0), sonicated for 20 minutes (5 s on and off cycles) on ice, and centrifuged at 40,000 rpm for 40 minutes at 4°C. Then, Ni-NTA beads were added into supernatant and rotated overnight at 4°C, loaded on a PD-10 column. Beads were washed with lysis buffer and eluted with elution buffer (50 mM Tris. 500 mM NaCl. 300 mM Imidazole, and 2 mM P-mercaptoethanol at pH 8.0), followed by dialysis with buffer (50 mM Tris, 100 mM NaCl, and 0.5 mM P-mercaptoethanol at pH 8.0). Then, HRV3C protease (TAKARA) was added overnight at 4°C and Ni-NTA was added to eliminate the cleaved portion overnight at 4°C. The flow-through was harvested and fractionated by NGC Chromatography Systems (BioRad) with buffer (50 mM Tris, 50 mM NaCl, and 0.5 mM P-mercaptoethanol at pH 8.0), followed by concentration and cutoff using Millipore Amicon fdters.

[0235] Example 18

[0236] In vitro binding assay

[0237] When CAP RIN 1 was used as a bait, recombinant CAPRIN1 (200 ng) was mixed with anti-CAPRINl antibodies (1 pg) in IP buffer (25 mM Tris, 150 mMNaCl, 5% Glycerol, and 1% Triton X-100 at pH 7.4) overnight at 4°C, followed by the addition of Protein-G agarosebeads for 4 hours at 4°C. CAPRIN1 conjugated beads were then washed with binding buffer (20 mM HEPES, 100 mM NaCl, 0.01% Triron X-100, and 5% Glycerol at pH 7.25) before the addition of recombinant human APP (prey) for 1 hour at 4°C. Beads were washed and eluted with 2X Laemmli buffer with boiling.

[0238] Example 19

[0239] Nuclear magnetic resonance INMR) spectroscopy

[0240] All NMR spectroscopy was performed at the Indiana University atIndianapolis on a Bruker Avance-III-600 NMR spectrometer operating at 600.2 MHz (1H), 61.081MHz (15N), and 150.9MHz (13C) and equipped with a TCI cryoprobe. All experiments were conducted at 298K in 5mm diameter NMR tubes with a sample volume of 500 pL. Solutions were buffered using 150 mM Potassium phosphate (pH 7.5) and 100 mM KC1 dissolved in 90% H2O and 10% D2O. For WaterLOGSY, 20 pM of unlabeled CAPRIN1112-260protein and 200 pM of compound were used, and the mixing time was set to 1.5 s. The data was collected with 256 scans, a sweep width of lOppm, an acquisition time of 1.58 s, and a relaxation delay 5 seconds. Prior to Fourier transformation the data were multiplied with an exponential function with a line broadening of 1 Hz. ForJH-15N HSQC experiments, 100 pM of15N-CAPRIN1112-260was used in free form as well in 0043 (200 pM) bound form. All spectra were processed using Bruker Topspin 4.1 software and analyzed using CARA (http: / / wiki.cara.nmr.ch / ). The amide CSPs between free and bound states were calculated as CSP = [(SHfree- 5Hbound)2+ ((SNfree- 5Nbound) / 5)2]1 / 2, where 5H and 5N are the chemical shift of the backbone amide proton and nitrogen, respectively

[0241] Example 20

[0242] In vitro binding assay

[0243] When APP was used as a bait, biotin conjugated APP antibody (1 pg; 4G8,Biolegend) was pre-mixed with recombinant APP (100 ng, Biolegend) in pulldown buffer (0.1% NP-40, 50 mM Tris, 150 mM NaCl, 0.2 mM EDTA, 100 mM KC1, 1 mM MgCl2, 0.2 mM CaCl2, and 10% glycerol) overnight at 4°C, followed by addition of 0.5 mg of Streptavidin magnetic beads (Pierce) for 1 hour at 4°C. After washing the beads, recombinant human CAPRIN1 (full length, 100 ng) was added together with dose dependent 0043 in the binding buffer for 1 hour at 4°C. Beads were w ashed and eluted w ith 2X Laemmli buffer with boiling.

[0244] Example 21

[0245] Recombinant CAPRIN1, APP695, and APP624 from insect cells

[0246] The DNA cassette (10xHis-3C site or 10xHis-MBP-3C site) was inserted immediately 5’ of the BnmHI site in the pFastBac vector (Invitrogen, 10360014) using the SLICmethod

[0036] , yielding constructs termed pSEP0(10His-3C) (pYT1353), or pSEP8 (lOHis-MBP- 3C) (pYT1566) vector respectively. The open reading frame (ORF) of human CAPRIN1 was codon-optimized, synthesized, and sub-cloned into the BamHI and Hindlll site of a pSEPO vector, yielding pSEPO-CAPRINl (10xHis-3C-CAPRINl) (pYT1837) vector. The open reading frame (ORF) of a full-length human APP (APP695) was codon-optimized, synthesized, and sub-cloned into the BamHI and Hindlll site of a pSEP8 vector, yielding pSEP8-APP695 (10xHis-MBP-3C- APP695) (pYT1842) vector. STOP codon was introduced into ORF of APP695 in pSEP8-APP695 construct, yielding pSEP8-APP624 vector (pYT1841) by which the C-terminal truncated mutant APP. APP624. was generated. Generation of baculovirus expressing 1 OxHis-C APRIN 1. lOxHis- MBP-APP695, or 10xHis-MBP-APP624 using pSEPO-CAPRINl, pSEP8-APP695, or pSEP8- APP624 vector in Sf9 cells (Expression Systems, Inc) was performed as described previously. Expression of recombinant CAPRIN1, or APP695, or APP624 was optimized using the TEQC method in which a 200 ml culture of Hi5 cells (Expression Systems, Inc, 94-002S) was infected at an estimated multiplicity of infection (eMOI) of 4. After a 96-hour incubation at 27°C, cells were harvested, frozen in liquid nitrogen and kept at -80°C until use.

[0247] For the purification of CAPRIN1, cell pellet was lysed in 50 mM Hepes-KOH pH 7.6. 400 mM potassium chloride (KC1, 10 % Glycerol. 5 mM [3-mercaptoethanol, 10 mM Imidazole, and 0.5 ml of 100X protease cocktail (6 mM Leupeptin, 0.2 mM Pepstatin A, 20 mM Benzamidine, and 10 mM PMSF). Cell lysate was stirred for 30 min followed by centrifugation at 100,000 g for 45 min. The supernatant was loaded onto Ni-resin (HIS-Select, Sigma- Aldrich) and washed with buffer 1 (50 mM Hepes-KOH pH7.6, 1 M KC1, 5% Glycerol, 10 mM Imidazole, and 5 mM [3-mercaptoethanol), and buffer 2 (50 mM Hepes-KOH pH7.6, 2 M KC1, 5% Glycerol, 10 mM Imidazole, and 5 mM |3-mercaptoethanol) followed by lysis buffer and the low salt buffer (50 mM Hepes-KOH pH7.6, 200 mM KC1, 5% Glycerol, 10 mM Imidazole, and 5 mM |3- mercaptoethanol). 1 OxHis-C APRIN 1 was eluted from Ni-resin with the elution buffer (50 mM Hepes-KOH pH7.6, 200 mM potassium acetate, 5% Glycerol, 300 mM Imidazole, and 5 mM [3- mercaptoethanol). Elution fractions containing 1 OxHis-C APRIN1 were pooled and dialyzed overnight against the low salt buffer. Dialysate was concentrated by spin column (Ami con 10k cut-off) to 4.0 mg / ml. Recombinant CAPRIN1 was further purified by 10-30% (v / v) glycerol gradient containing the buffer (50 mM Hepes-KOH pH7.6, 200 mM KC1, 5% glycerol and 5 mM [3-mercaptoethanol). After centrifugation at 35,000 rpm in a Beckman SW Ti-40 rotor for 19 hours at 4°C, the gradient was fractionated using a PGF Piston Gradient Fractionator (BioComp Instruments). Fractions containing 1 OxHis-C APRIN 1 were pooled and dialyzed overnight againstthe low salt buffer. Dialysate was concentrated by spin column (Amicon 10k cut-off) to 4 mg / ml. Concentration of purified protein was measured by Bradford method. When purifying APP695 and APP624, the same procedure as the CAPRIN1 purification was used. Buffer 1 (50 mM Hepes- KOH pH7.6. 1 M KC1, 5% Glycerol, 5 mM P-mercaptoethanol, and 0.1% DDM). wash buffer 2 (50 mM Hepes-KOH pH7.6, 2 M KCL 5% Glycerol, 5 mM P-mercaptoethanol, and 0.1% DDM), low salt buffer with 0.1% DDM, elution buffer (50 mM Hepes-KOH pH7.6, 200 mM potassium acetate, 5% Glycerol, 50 mM Maltose, and 5 mM P-mercaptoethanol), and Amylose-resin were used.

[0248] Example 22

[0249] CAPRIN1-APP interaction by glycerol gradient

[0250] ~1 mg of 10xHis-MBP-APP695 or 10xHis-MBP-APP624, was mixed with~1 mg of lOxHis-CAPRINl and loaded onto 10-30% (v / v) glycerol gradient containing the buffer (50 mM Hepes-KOH pH7.6, 200 mM KC1. 5 mM P-mercaptoethanol, and 0.1% DDM). For controls, ~1 mg of 10xHis-MBP-APP695 (full-length) or 10xHis-MBP-APP624, alone was loaded onto 10-30% (v / v) glycerol gradient as well. After centrifugation at 35,000 rpm in a Beckman SW Ti-40 rotor for 19 hours at 4°C, the gradient was fractionated as described above. For fractions containing lOxHis-CAPRINl, a PGF Piston Gradient Fractionator was used. Fractions containing lOxHis-CAPRINl, 10xHis-MBP-APP695 or 10xHis-MBP-APP624 alone or mixture were subjected to 4-12% NuPAGE (Invitrogen) and stained with Coomassie blue.

[0251] The next set of Examples disclose biological data using the disclosed compounds.

[0252] Example 23

[0253] Identification of CAPRIN1 -targeted molecules that reduce AB42 and APP

[0254] To establish a cell model to screen for CAPRIN 1 -targeted molecules, we expressed wild type APP695 (APP695WT), a predominant form in human neurons in human embryonic kidney 293T (HEK) cells, to form the stable HEK-APP695WTclone #2. APP is cleaved by BACE1 and y-secretase to release Ap42 extracellularly); thus, ELISA was used to measure extracellular AP42 levels in culture medium with BACE1 (BI, P-Secretase Inhibitor IV) or y- secretase inhibitors (GSI, y-Secretase Inhibitor XXI, Compound E) used as controls. The clone #2 was treated with each molecule, (i.e., compound) of interest. The culture medium was subsequently tested by ELISA for Ap42. Cell viability’ was also evaluated to eliminate compounds toxic to the cells. The results identified seven active molecules that significantly reduced extracellular levels of Ap42.

[0255] These seven active compounds identified share three pharmacophore motifs: a bicyclic ring, a urea core, and a phenyl ring (Fig. 1). The urea core was consistent in all the compounds. For the bicyclic ring, the 6-5 ring system showed the desired activity with a benzothiazole ring such as 0043 being most active in this system. Semi -saturated rings such as hexahydro- 1,3-benzothiazole and hexahydrothiazolo [5,4-c]pyridine also preserve compound activity. For the phenyl ring moiety, carbonyl group in the meta-position of the ring improved the activity. Five out of seven active compounds contained a carbonyl moiety such as a tertiary amide or an ester on the phenyl ring. Of the seven compounds, 0043 was more potent in a dose-dependent manner (Fig. 2) and its activity was not affected by fetal bovine serum (FBS) levels in medium.

[0256] To evaluate the activity of the compounds on APP, western blots determined that 0043 treatment of HEK-APP695WTreduced APP but not BACE1, PSEN1, and presenilin enhancer 2 (PEN2) (Fig. 3). 0043 treatment reduced APP in dose-dependent manner (Fig. 4) but had no effects on the levels of CAPRIN1 and its binding partner Ras GTPase-activating proteinbinding protein 1 (G3BP1) (results not shown). The activity of 0043 on APP degradation was confirmed by western blots using the C-terminal antibody in HEK-APP695WTcells. In contrast, the mRNA levels of APP w ere not significantly changed by 0043. Neither did 0043 treatment have an effect on cancer cell growth nor SUMO1 protein levels in cancer cells. Interestingly, in this system, 0043 treatment had no effect on APP levels in cancer cells, due in part to a lack of APP expression in cancer cells. Collectively, the results suggest that compound 0043 reduces A042 through induction of APP degradation in the cells.

[0257] Example 24

[0258] Compound 2 (i.e., 0043) reduces extracellular AB42 and intracellular APP in AD-iPSC-Ns

[0259] To determine the activity of the compounds in AD, we differentiated human neurons from a panel of human iPSCs from sAD, fAD, and non-AD patients, using the Ascll / Mashl and Neurogenin 2 (Ngn2) protocol. iPSCs were infected with Ngn2 / Ascii and induced to differentiated neurons by doxycycline either in continuous or discontinuous treatment. ELISA detected higher A(342 levels in culture under discontinuous than continuous treatment. All iPSCs were successfully differentiated to mutual iPSC-Ns, as determined by the sternness marker NANOG and the neuronal marker MAP2, respectively.

[0260] The active compounds (i.e.. compounds of interest), as identified usingHEK-APP695UTcells in Example 23, were tested first using sAD-iPSC SAD2.3-differentiated neurons (SAD2.3-Ns). ELISA confirmed the activity of 0043, 0047, 0048 and 0062 on reductionof extracellular A|342 (Fig. 5), but 0062 was toxic to SAD2.3-Ns. The compound 0043, 0047 and 0062 were further tested in all iPSC differentiated neurons (iPSC-Ns). The results showed that 0043 treatment reduced the extracellular A|342 in all iPSC-Ns. and to a greater extent in AD-iPSC- Ns compared to non-AD iPSC-Ns (Fig. 6). The data showed that fAD-iPSC-Ns with PSEN1, PSEN2 mutation and APOE4 allele were responsive to the compounds. To evaluate how APP mutant neurons responded, the wild type PP gene (APPWT) in sAD-AG27606 iPSCs was edited by CRISPR-Cas9 to the Swedish APP mutant K595N / M596L. The APP-Swedish mutation (APPSWE) did not affect the neuronal differentiation of AG27606-iPSCs. nor the mRNA expression of APP, nor the protein expression of APP. C APR1N 1 and BACE1. The APP8'™, however, resulted in more than four times of extracellular release of A|342 (Fig. 7), consistent with the previous finding. Treatment with 0043 significantly reduced extracellular release of A[142 from the APPSWEneurons as compared with APPWTAG27606-iPSCs (Fig. 8).

[0261] The activity of 0043 in A(342 reduction was dose and time-dependent with the IC50 values of the compound activity approximately at 1.8 pM and 2.1 pM, respectively, in the treatment of SAD2.3-Ns and AG27606-Ns (Fig. 9 and Fig. 10). Western blots showed that 0043 treatment of SAD2.3-Ns reduced intraneuronal APP. but not other AD related proteins such as BACE1, ADAM10 (constitutive a-secretase), PSEN1, nicastrin, ADP ribosylation factor-binding protein 1 (GGA1, BACE1 binding protein), Soria (an intracellular sorting receptor to APP) and neprilysin in SAD2.3-iNs. The activity of 0043 in APP reduction in SAD2.3-Ns and AG27606-Ns was dose-dependent. In contrast, 0043 had no effects on the differentiation of SAD2.3 iPSCs in sAD2.3-iPSC-Ns or the mRNA transcriptional levels o APP gene in SAD2.3-Ns and AG27600- Ns. These studies have characterized the compound 0043 as an APP degrader that reduces extracellular A|342 in both sAD and fAD-iPSC-Ns.

[0262] Example 25

[0263] The compound 0043 acts as a molecular glue that directly binds CAPRIN1 and APP

[0264] To determine if the APP degrader 0043 binds CAPRIN1, we expressed and purified the recombinant human fragment of CAPRIN1 (rhCAPRINl112-260). The sensitive ligandprotein one-dimensional nuclear magnetic resonance (lD-NMR)-based water Ligand Observed via Gradient SpectroscopY (WaterLOGSY) showed the difference in peak intensities due to different tumbling rates of the compound 0043 in the presence or absence of rhCAPRINl112-260, indicating the binding of 0043 on rhCAPRINl112-260(Fig. 1 1). To test the binding further, ^-^N HSQC studies on isotopically15N-labelled rhCAPRINl112-260were conducted based on the protocolreported, and the changes were monitored in presence of 0043; the results indicated significant chemical shift perturbations (CSPs) for some amide cross-peaks of CAPRIN1 in the presence of 0043. which confirmed the binding of 0043 on CAPRIN1 (Fig. 12). Next, we conducted ThermoFluor™ assay and showed that the presence of 0043 stabilized CAPRIN1 upon binding and resulted in a thermal shift (Fig. 13). To determine if 0043 also binds and stabilizes APP, we repeated ThermoFluor™ experiments with rhAPP695and observed significant thermal shifts for rhAPP695in the presence of 0043 (Fig. 14). The data indicates that 0043 directly binds CAPRIN1 and APP and can stabilize the proteins, which suggests that 0043 may act as a molecular glue between CAPR1N1 and APP.

[0265] To determine whether 0043 binds to cellular CAPRIN1 and APP, we conducted a cellular thermal shift assay (CETSA), in which CAPRIN1 and APP proteins, unlike PSEN1, showed a clear thermal stabilization with increase in temperature in the presence of 0043 in AG27606-Ns and HEK-APP695Tcells (Fig. 15). The presence of 0043 shifted the heat denaturation curve of CAPRIN1 and APP to higher temperatures, but not PSEN1 in AG27606-Ns (Fig. 16) or HEK-APP695WT. These results indicate that CAPRIN1 and APP are the binding partners of the compound 0043 in neurons. To confirm the role of CAPRIN1 in 0043-mediated degradation of APP, we generated CAPRIN1 knockout (KO) clones from HEK-APP695WTcells using a lentiCRISPRv2 system. The sgCAPRINl-KO HEK-APP695WTclones were selected, as measured by western blots and immunofluorescence. The clone was treated with 0043, western blots and ELISA showed that CAP RIN 1 KO abolished the activity7of 0043 on APP degradation in the cells and the extracellular release of A(342 (Fig. 17). These results indicate 0043 binds on CAPRIN1 and APP and induces CAPRIN 1 -mediated APP degradation in cells.

[0266] Example 26

[0267] The compound 0043 binding of CAPRIN1 and APP enhances the proteinprotein interaction

[0268] To investigate whether CAPRIN 1 and APP interact with each other, we generated His-tagged rhCAPRINlFu11(rhCAPRINl) and His- and maltose-binding protein (MBP)- tagged rhAPP695. The extracellular domain of APP was also generated without the transmembrane domain (rhAPP624). To assess the stoichiometry of protein interaction, rhAPP624or rhAPP695, alone or mixed with rhCAPRINl, were sedimented on glycerol gradient; and fractions of each sedimentation were analyzed by SDS-PAGE. Mixing of rhCAPRINl and rhAPP resulted in the shift or spreading of the peak fractions of each protein towards higher molecular weights, suggesting one-to-one stoichiometric interaction of CAPRIN1 and APP (Fig. 18).Immunoprecipitation of each fraction by anti-CAPRINl antibodies and western blots of immunoprecipitated fractions using an anti-MBP antibody confirmed the complex formation of rhCAPRINl and rhAPP (Fig. 19).

[0269] In evaluation of the role of 0043 on CAPRIN1 and APP interaction, biotinylated anti-APP antibody was used in pull down of HEK-APP695WTcells and AG27606-Ns after treated with 0043. The results showed that 0043 treatment enhanced the interaction of CAPRIN1 and APP in HEK-APP695WTand AG27606-Ns (Fig. 20). To test this at protein levels, we used biotinylated anti-APP antibody as a bait and rhCAPRINl or 0043 as a prey, western blot showed that 0043 increased biotinylated anti-APP antibody pull down of CAPR1N1 (Fig. 21). In a reverse, anti-CAPRINl antibody was used to pull down rhAPP695and results showed that 0043 increased anti-CAPRINl antibody pull down of rhAPP (Fig. 22). Next, rhCAPRINl was coated on plates and incubated with rhAPP695or rhAPP624in the presence or absence of 0043. ELISA showed that 0043 increased the binding of both APP695and APP624to coated rhCAPRINl (Fig. 23). To determine the activity of 0043 in AD-iPSC-Ns, finally, AG27606-Ns were treated with 0043 and confocal fluorescent microscopy demonstrated that 0043 treatment increased the association of CAPRIN1 and APP in the neurons (not shown). Collectively, these results indicate that 0043 acts as a molecular glue that enhances CAPRIN1 and APP interaction and CAPRIN1- mediated APP degradation in neurons.

[0270] Example 27

[0271] Chemical modifications of 0043 identify' the more potent and soluble lead compound 0152 (i.e.. Compound 1)

[0272] To improve drug-like properties of 0043, we conducted structure-activity relationship (SAR) studies through chemical design, synthesis, and bioactivity analysis of 0043 derivatives. SAR results indicate that replacing the nitrile group with a methyl ether group significantly improved the drug-like properties of the derivative 0152 (i.e., Compound 1) as shown in Fig. 34. The mouse oral pharmacokinetics (PK) studies showed that the total drug exposure across time of the lead compound 0152, as determined by the area under the curve (AUC), was 6 times higher than that of the hit compound 0043 and the oral bioavailability of 0152 was markedly improved up to 30% from 5% of 0043 (Fig. 24). To analyze the interaction of the lead compound 0152 and rhCAPRINl, ID NMR WaterLOGSY was recorded for the compound in the presence of rhCAPRINl112'126and a change in peak intensities was observed, which confirmed the binding of 0152 on CAPRIN1 (Fig. 25). The compound 0152 was linked to biotin (0152-Biotin) and rhCAPRINl was incubated with 0152-Biotin; 0152-Biotin was subsequently pulled down bystreptavi din-coated beads. Western blots of the pull-down confirmed the binding of rhCAPRINl to 0152-Biotin and the binding was inhibited competitively by free 0152 (not shown). AG27606- Ns lysates were then included with 0152-Biotin and pull-down assay confirmed the binding of cellular CAPRIN1 and APP on 0152-Biotin while the binding was inhibited competitively by free 0152, but not free HB007 suggesting that 0152 and HB007 bind different sites of rhCAPRINl (Fig.26).

[0273] To evaluate the activity of 0152 in AD iPSC-Ns, an ELISA of A042 in culture medium showed the improved potency of 0152 in reduction of extracellular A042 release, as compared with the hit compound 0043 in sAD AG27606 and fAD UKBiOHA iPSC-Ns (Fig.27). AD-iPSC-Ns were then treated with a series of the dilutions of 0152; the ELISA showed the improved IC50 values ranging from 0.8-0.9 pM of the lead 0152 as compared with the IC50 of the hit 0043 at 2 pM in A042 reduction (Fig. 28). The potent activity of 0152 on reduction of extracellular A042 levels was also confirmed using sAD2.3-Ns as shown in Fig. 28.

[0274] Western blots further confirmed that the treatment of 0152 markedly reduced APP levels in AG27606-Ns as compared with 0043 (Fig. 29). Confocal microscopy revealed that 0152 treatment increased CAPRIN1 and APP interaction in AG27606-Ns and sAD2.3-Ns. Finally, CAPRIN1 KO sgCAPRINl-HEK-APP695WTclones were treated with 0043 and 0152; the ELISA showed 0152 was more potent than 0043 in reduction of extracellular A042; and western blots showed the significant reduction of APP levels (Fig. 30). In contrast, CAPRIN1 knockout drastically reduced the effects of 0043 and 0152 on both A042 and APP reduction (Fig. 31). Collectively, these results establish 0152 acts as a small-molecule degrader of APP with improved drug-like properties and potent activity in induction of APP degradation and decrease of Af>42 extracellular release.

[0275] Example 28

[0276] APP degraders induce CAPRIN1 -mediated APP lysosomal degradation in iPSC neurons

[0277] To reveal the mechanism of action, we determined whether the degraders induce APP degradation either through the proteasome or lysosome using the proteasome inhibitor MG132 and the lysosome inhibitor Bafilomycin Al (Baf Al). The presence of MG132 had no effects on 0043-induced degradation of APP in AG27606-Ns (Fig. 32), sAD2.3-Ns and HEK- APP695WT(Fig. 32). In contrast. Baf Al pretreatment abolished the activity of 0043 in APP degradation in AG27606-Ns (Fig. 33), sAD2.3-Ns and HEK-APP695WT(Fig. 33). The data suggest that the compound induces APP degradation through the lysosome. Next, we tracked APP throughcellular organelles using confocal co-localization of APP and CAPRTN1 with organelle-specific markers. APP was colocalized with the earlier endosome antigen 1 (EEA1), the late endosome Rab7 and the lysosomal associate membrane protein 1 (LAMP1) in AG27606-Ns; while CAPRIN1 was co-localized with Rab7 and LAMP1. as well as SEC31A, a subunit of the coat protein complex II (COPII) of the endoplasmic reticulum (ER) enriched with stress granules coated vesicle. The treatment of 0043 increased the co-localization of APP and CAPRIN1 with EEA1, Rad7 and LAMP 1. The 0043 -enhanced co-localization of LAMP 1 was further confirmed in a panel of AD-iPSC-Ns by confocal correlation coefficient analysis.

[0278] To further determine the role of CAPRIN1 on APP lysosomal degradation, we carried out immunoprecipitation of 0043-treated AG27606-Ns and UKBiOl lA-Ns by a CAPRIN1 antibody. Western blots of the immunoprecipitated showed that 0043 treatment increased CAPRIN1 pull-down of APP and LAMP1 in AD-iPSC-Ns. AG27606-Ns were then treated with 0043 and 0152 and confocal microscopy showed the enhanced potent activity of 0152 in enhancing of the colocalization of APP and LAMP1 in AD-iPSC-Ns. CAPRIN1 knockout (KO) in sgCAPRINl-HEK-APP695WTdrastically reduced the effects of 0043 and 0152 on the colocalization of APP and LAMP1. The results suggest that, through binding on CAPRIN1 and APP, the compounds 0043 and 0152 enhance CAPRIN 1 -mediated APP trafficking from the endosome to lysosome for APP degradation.

[0279] Example 29

[0280] APP degraders reduce AB42 levels in AD iPSC-derived brain organoids

[0281] To define the role of CAPRIN1 in AD, we first examined CAPRIN1 expression and localization in AD brains. Immunohistochemistry (IHC) of paraffin embedded, and formalin fixed sections of autopsy brains demonstrated that CAPRIN1 and APP were predominantly expressed in neurons in AD and non- AD brains, whereas A(342 was extracellular in AD brains. Confocal immunofluorescent stains of the section further showed the co-localization of CAPRIN1 and APP in neurons. Next, we developed 3 dimensional (3D) organoids from AD iPSCs as AD brain tissue models. Hematoxylin and eosin (H&E) histochemistry and immunohistochemistry of paraffin embedded, and formalin fixed sections of AG27606-3D organoids revealed the differentiation of neurons and astroglia as determined by neuronal synaptophysin and glial fibrillary acidic protein (GFAP). Confocal immunofluorescence stains of the sections of AG27606-3D organoids showed the expression of the neuronal marker MAP2 and TUJ1 and more accumulation of APP / Ap in a long-term culture than shorter or healthy organoids. Immunohistochemistry of AG27606-3D organoids demonstrated that CAPRIN1 and APP weredominantly expressed in organoids, as shown in human AD brains. Immunofluorescent stains with confocal microscopy showed that the compound increased colocalization of APP and CAPRIN1 in the organoids after 1-month and 3-months treatments; and the treatment of the compounds markedly reduced APP / A(3 in AD-3D-organoids. The compounds decreased A peptides was confirmed by the antibody that recognizes AP37-42 peptides in immunofluorescence microscopy and ELISA of Ap42. These results indicate that compounds 0043 and 0152 induce APP and CAPRIN1 co-localization and APP degradation, thus resulting in Ap reduction in AD iPSC derived organoids.

[0282] The above examples are provided for the purpose of illustrating various embodiments of the invention and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are provided only as examples, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.

[0283] INCORPORATION BY REFERENCE

[0284] All of the U.S. patents and U.S. published patent applications cited herein are hereby incorporated by reference.

[0285] EQUIVALENTS

[0286] While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and / or methoddescribed herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[0287] While embodiments of the present disclosure have been described herein, it is to be understood by those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS1. A compound of formula 1 or a pharmaceutically acceptable salt thereof:

2. The compound of claim 1, wherein polycyclic group represented as the Rings A+B is selected from benzthiazole, a fused thiazol, or cyanobenthiazole.

3. The compound of claim 2, wherein the polycyclic group represented as Rings A+B is selected from, orwherein Rs is selected from a sulfonyl, an acylamino, an amide, a morpholine amide, a ketone, an ethyl ester, a methyl ester, a nitrile, an ether, a t-but l ester, a dimethylamine.

4. The compound of claim 3, wherein Rs is selected from ®§,, a halogen, an alky l, an amine, or H.

5. The compound of claim 4, wherein the halogen is Cl.

6. The compound of claim 4, wherein the alkyl is CH3.

7. The compound of claim 4, wherein the amine is NH2.

8. The compound of claim 1, wherein R2 is selected from NO2, a halogen, or H.

9. The compound of claim 8, wherein the halogen is F.

10. The compound of claim 1. wherein Ri is selected from a nitrile, a morpholine, an imidazolidinone. a cyclohexadiene, a pyrzoline. an ether, a halogen, or H.i l. The compound of claim 10, wherein Ri is selected from, or12. The compound of claim 10, wherein the halogen is Cl.

13. The compound of claim 1, wherein the Rings A+B is , and Rs is selected from a sulfonyl, an acylamino, an amide, a morpholine amide, a ketone, an ethyl ester, a methyl ester, a nitrile, an ether, a t-butyl ester, a dimethylamine, a halogen, an alkyl, an amine, or H.

14. The compound of claim 13, wherein R2 is selected from NO2, a halogen, or H.

15. The compound of claim 13, wherein Ri is selected from a nitrile, a morpholine, an imidazolidinone, a cyclohexadiene, a pyrzoline, an ether, a halogen, or H.

16. The compound of claim 15, wherein Ri is selected from INor .

17. The compound of claim 1, wherein the compound has the formula19. A method of disrupting an amyloid cascade comprising contacting a CAPRIN1 and / or APP with a compound or salt thereof according to claim 1.

20. A pharmaceutical composition comprising the compound or a salt thereof of claim 1 and an excipient.

21. A method of treating a subject diagnosed with or suspected of having Alzheimer's disease comprising administering a therapeutically effective amount of a composition or a salt thereof according to claim 1.-so

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