Therapeutic targeting of fibronectin 1 for mitigating alzheimer's disease pathology

Abstract

Methods and compositions for treating or reducing the development of neurodegenerative diseases.

Description

Dkt.93597 / 7434 92376-A-PCT THERAPEUTIC TARGETING OF FIBRONECTIN 1 FOR MITIGATING ALZHEIMER’S DISEASE PATHOLOGY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 705,622, filedOctober 10, 2024, the contents of which are hereby incorporated by reference. GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under AG056270, AG026395,AG067501, AG021886, AG041797, and AG066107 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING

[0003] This application incorporates-by-reference nucleotide and / or amino acid sequenceswhich are present in the file named “93597-7434_92376-A-PCT_Sequence_Listing_AWG.xml”, which is 35,083 bytes in size, and which was created on October 7, 2025 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the XML file filed October 9, 2025 as part of this application. BACKGROUND

[0004] The disclosures of all publications, patents, patent application publications and booksreferred to in this application are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

[0005] Alzheimer's disease (AD) is a complex neurodegenerative disorder significantlyassociated with APOE^4 and other genes. Our research targets the fibronectin 1 (FN1) gene, a key component of the extracellular matrix (ECM) that plays a crucial role in regulating blood-brain barrier (BBB) integrity and function[1-3]. BBB dysfunction occurs in AD, allowing the entry of neurotoxic substances into the brain, reducing clearance of toxic protein aggregates such as amyloid and tau, exacerbating inflammation, and accelerating neurodegeneration[4-8]. In APOE^4 4929-9458-4176v.2carriers with AD, FN1 protein levels are elevated, correlating with increased BBB permeability and subsequent AD pathology[9,10]. SUMMARY OF THE INVENTION

[0006] A method for treating, or for delaying or reducing the development of, aneurodegenerative disease, comprising the step of administering to a subject in need thereof an effective amount of a compound having the following structure: whereinatom Z is present or absent, and when present is a carbon bound to A, and when absent the two N atoms of the ring are directly bound to each other, wherein when Z is present: bonds ^^^^^^^^^^, and ^ are present, and bonds ^ and ^ are absent; A has the following structure: ,E is CH2, O, or S; and R9are R10are each, independently, H, optionally substituted or unsubstituted C1- C6 alkyl, C3-C12 cycloalkyl, aromatic, or alkylaromatic, 2 4929-9458-4176v.2wherein R9are R10are each independently optionally substituted or unsubstituted with halo, -OH, -SH, alkyl, alkyl-OH, alkyl-SH, alkyl-NH2, - O-alkyl, -S-alkyl, or -NHx(alkyl)y where x + y = 2; X is a C atom; Y is O or NR6; wherein when Y is O, R1is H, an optionally substituted or unsubstituted C1-C6alkyl, aromatic, alkyl carbonyl, alkyl carboxy, alkyl N-amido, or alkylaromatic group, wherein R1is optionally substituted or unsubstituted with halo, -OH, -SH, C1-C6alkyl, C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S- alkyl, -NHx(alkyl)ywhere x + y = 2, -C(O)NH2, or -SO2NH2; and wherein when Y is NR6, R1is H, alkyl, aromatic, alkyl carbonyl, alkyl carboxy, alkyl N-amido, or alkylaromatic; R6is H, alkyl, alkyl carbonyl, alkyl N-amido, alkylaromatic, or C(O)R16; or , wherein R16is C1-C6alkyl; R1and R6or R16may, together, form a 5- to 8-membered optionally substitututed or unsubstituted unsaturated heterocycle, wherein the heterocycle is optionally substituted with R7wherein R7is a C1-C6alkyl or an alkylamide and R2, R3, R4, and R5are, independently, H, halo, -OH, alkyl or -O-alkyl; wherein when Z is absent: bonds ^ and ^ are present, and bonds ^^^^^^^^^^^^and ^ are absent; X is a C or N atom; 3 4929-9458-4176v.2R2is H or a optionally substituted or unsubstituted benzoyl; R3, R4, and R5are H; Y is C(O)NHR8, and R8is H, C1-C6alkyl or alkyl aromatic; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0007] A method for treating a neurodegenerative disease, comprising the step ofadministering to a subject in need thereof an effective amount of a compound having the following structure: whereinatom Z is present or absent, and when present is a carbon bound to A, and when absent the two N atoms of the ring are directly bound to each other, wherein when Z is present: bonds ^^^^^^^^^^, and ^ are present, and bonds ^ and ^ are absent; A is a C1-C6 alkyl carboxamide linked through either -CH2-, -O-, or -S-; X is a C atom; Y is NR6or O; and when Y is O, R1is H, C1-C6 alkyl, alkyl carbonyl, alkyl N-amido, or alkylaromatic, and when Y is NR6, either R1and R6together form a 5- to 8-membered unsaturated heterocycle, or R6is C(O)R7, wherein R7is C1-C6alkyl and R1is H, C2-C6alkyl, C2-C6alkyl carbonyl, C2-C6alkyl N-amido, or alkylaromatic; 4 4929-9458-4176v.2R2, R3, R4, and R5are, independently, H, halo, -OH, C1-C6alkyl or C1-C6alkoxy; wherein when Z is absent: bonds ^ and ^ are present, and bonds ^^^^^^^^^^, and ^ are absent; X is an N atom; R2is H or benzoyl; R3, R4, and R5are H; Y is C(O)NHR8, and R8is H, C1-C6alkyl or alkyl aromatic; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0008] A method for preventing neurodegenerative disease, comprising the step ofadministering to a subject in need thereof an effective amount of a compound having the following structure: whereinatom Z is present or absent, and when present is a carbon bound to A, and when absent the two N atoms of the ring are directly bound to each other, wherein when Z is present: bonds ^^^^^^^^^^, and q are present, and bonds ^ and ^^are absent; A is a C1-C6alkyl carboxamide linked through either -CH2-, -O-, or -S-; X is a C atom; Y is NR6or O; and when Y is O, R1is H, C1-C6alkyl, alkyl carbonyl, alkyl N-amido, or alkylaromatic, 5 4929-9458-4176v.2and when Y is NR6, either R1and R6together form a 5- to 8-membered unsaturated heterocycle, or R6is C(O)R7, wherein R7is C1-C6alkyl and R1is H, C2-C6alkyl, C2-C6alkyl carbonyl, C2-C6alkyl N-amido, or alkylaromatic; R2, R3, R4, and R5are, independently, H, halo, -OH, C1-C6alkyl or C1-C6alkoxy; wherein when Z is absent: bonds ^ and ^ are present, and bonds ^^^^^^^^^^, and ^ are absent; X is an N atom; R2is H or benzoyl; R3, R4, and R5are H; Y is C(O)NHR8, and R8is H, C1-C6alkyl or alkyl aromatic; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0009] A method for treating, or for reducing the development of, a neurodegenerative disease,comprising the step of administering to a subject in need thereof an effective amount of a compound having the following structure:or unsubstituted mono or bicyclic aromatic; R17is an optionally substituted or unsubstituted mono or bicyclic aromatic; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0010] A method for treating, or for reducing the development of, a neurodegenerative disease,comprising the step of administering to a subject in need thereof an effective amount of a compound having the following structure: 6 4929-9458-4176v.2wherein R18and R19is C2-Cxalkyl; R18and R19may come together to form a 5-x-membered unsaturated optionally substituted or unsubstituted heterocycle; R20is a 5-6-membered optionally substituted heteroaromatic ring containing, selected independently, at least one of N, O, or S; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0011] A compound having the following structure:whereinatom Z is present or absent, and when present is a carbon bound to A, and when absent the two N atoms of the ring are directly bound to each other, wherein when Z is present: bonds ^^^^^^^^^^, and ^ are present, and bonds ^ and ^ are absent; A is a C1-C6 alkyl carboxamide linked through either -CH2-, or -O-, or -S-; X is a C atom; Y is NR6or O; 7 4929-9458-4176v.2and when Y is O, R1is H, C1-C6alkyl, alkyl carbonyl, alkyl N-amido, or alkylaromatic, and when Y is NR6, either R1and R6together form a 5- to 8-membered unsaturated heterocycle, or R6is C(O)R7, wherein R7is C1-C6alkyl and R1is H, C2-C6alkyl, C2-C6 alkyl carbonyl, C2-C6 alkyl N-amido, or alkylaromatic; wherein when A is linked through either -CH2-, or -O-: R2, R3, R4, and R5are, independently, H, -OH, -SH, C1-C4 alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, or -NHx(alkyl)y where x + y = 2; wherein when A is linked through -S-: at least one of R2, R3, R4, and R5are -H, -OH, -SH, C1-C4 alkyl, -C1-C4-OH, C1-C4-SH, C1- C4-NH2, -S-alkyl, or -NHx(alkyl)y where x + y = 2; and up to three of R2, R3, R4, and R5are -H wherein when Z is absent: bonds ^ and ^ are present, and bonds ^^^^^^^^^^, and ^ are absent; X is a C or N atom; R3, R4, and R5are H; Y is C(O)NHR8wherein when X is a C atom: R2is H or benzoyl; R8is H, a substituted or unsubstituted C1-C6 alkyl or alkyl aromatic; wherein when X is an N atom: R2is H, poly-substituted benzoyl, or optionally substituted or unsubstituted benzyl or naphthoyl 8 4929-9458-4176v.2R8is a substituted or unsubstituted C1-C6alkyl or alkyl aromatic; and the aromatic is substituted, or pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0012] A pharmaceutical composition comprising a compound described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGS. 1A-1F: Targeting FN1 in APOEe3 and APOEe4 humanized mouse astrocytes.(A) Schematic representation of the experimental design. Cortical astrocytes were isolated from humanized APOE3 (E3) and APOE4 (E4) mice and cultured in vitro. These astrocytes were treated with FN1-targeting compounds. Immunostaining for GFAP and FN1 was performed, and FN1 levels were quantified using immunohistochemistry and image analyses for protein intensity (intracellular and membrane bound FN1) and with ELISA (for FN1 in the supernatant). (B) Representative immunofluorescence images of FN1 (red), GFAP (green), and DAPI (blue) in E3 and E4 astrocytes under control conditions or following treatment with compound #3. Scale bars indicate 50 µm. (C-D) Quantification of normalized FN1 immunofluorescence intensity in E3 and E4 astrocytes. In E4 astrocytes (D) but not in E3 (C), treatment with compound #3 significantly reduced tissue / cellular FN1 intensity, that is more prone to insoluble deposition / accumulation. (E) Quantification of GFAP immunofluorescence intensity in E4 astrocytes shows a significant reduction following treatment with compound #3 (*p < 0.05), indicating reduced gliosis. (F) Relative FN1 levels measured by ELISA in E3 and E4 astrocyte cultures’ supernatant. Compound #3 does not affect secreted / soluble FN1 levels in E3 and E4 astrocytes, while compound #9 showed a significant decrease in soluble FN1 levels compared to controls in E4 astrocytes. Statistical significance was determined using one-way ANOVA, with ns indicating non-significance.

[0014] FIGS. 2A-2G: Figure 2: a, b, Double immunofluorescent staining (IFS) for CD31(green) and Fibronectin 1 (FN1, red) with DAPI nuclear counterstain (blue) in control (a, a’) and AD (b, b’) in humans. c, Mean intensity values for FN1 in control and AD. d, Comparative analysis of FN1 expression in APOEε3 and APOEε4 mouse. The left panel displays merged double immunofluorescence staining for CD31 (yellow) and Fibronectin 1 (FN1, cyan). Middle and right panels exhibit individual staining for CD31 and FN1, respectively. e, Quantifications of 9 4929-9458-4176v.2fluorescence intensity of FN1 across a number of observations indicated as n. f, Assessment of FN1 expression in control and APP knock-in mouse. g, The graph quantify the fluorescence intensity for FN1, providing statistical analysis with n representing the number of observations. Scale bars equal 100 µm.

[0015] FIGS. 3A-3D: Structure of inactive Fibronectin-GBD homodimer (PDB ID: 3M7P).

[0016] FIG. 4: Workflow for the discovery of FN-GBD modulators including exemplarycompounds. Of the compounds: 8003-2753 = Compound 1 K279-0860 = Compound 2 K284-6001 = Compound 3 K284-7630 = Compound 4 L574-0506 = Compound 7 P759-4281 = Compound 8 P759-4284 = Compound 9 P759-4513 = Compound 10.

[0017] FIG.5: Determination of safe doses for chemicals. Dotted line represents 80% viabilityin CTG assay. We determined the maximum dose for every compound that allows a viability of more than 80% in astrocyte cultures.

[0018] FIG. 6: Toxicity analyses of the compounds in APOE flox mice astrocytes in vitro.Initial ceiling safe doses for efficacy studies selected. For compounds: 2: 10 uM, 3: 5 uM, 9: 10 uM, 10: 10 uM.

[0019] FIG. 7: FN1 ELISA performed from supernatants of the compound-treated mouseastrocytes with APOE E3 and E4. #9 and #10 changing the secreted FN1 levels in APOE E4 genotyped astrocytes. Others are not. In APOE E3, no compound changes the supernatant levels.

[0020] FIG. 8: Detection of insoluble fraction of FN1 in mouse astrocytes with APOE E3 andAPOE E4. Compound 3 shows statistically significant reduction in insoluble FN1 levels as determined by IHC. Compounds 2, 9 and 10 also show a trend of reduction. Selected effective doses were used.

[0021] FIGS. 9A-9F: A. Experimental protocol. B. Detection of insoluble fraction of FN1 inmouse astrocytes with APOE E3 and APOE E4. Compound 3 effectively reduces insoluble FN1 deposition in human APOE E4 expressing mouse astrocytes (see C-F). 10 4929-9458-4176v.2

[0022] FIG. 10: Detection of GFAP as a measure of gliosis in mouse astrocytes with APOEE3 and APOE E4. Almost all compounds reduce GFAP expression, suggesting a reduction in gliosis.

[0023] FIG. 11: Validation in human iPSC-derived astrocytes with APOE 3 / 3 and 4 / 4(KOLF2.1J -.

[0024] FIG. 12: ELISA for cell-produced FN1 in the supernatant in human iPSC-derivedastrocytes. #2 and #3 reduce FN1 in APOE E4 cells at 10 and 1 uM, respectively. APOE E3 is not affected. There is a dose response.

[0025] FIG.13: IHC for cellular (insoluble) FN1 - in vitro efficacy study. Panel indicates fieldimages of APOE, GFAP and FN1 proteins in APOE3 and APOE4 astrocytes in vitro – control and compound treated.

[0026] FIG. 14: Quantification graphs. Every dot indicates one cell analyzed. Compound 3and 10 reduces cellular insoluble FN1 and APOE (potential pathogenic aggregation).

[0027] FIG 15: Colocalization of FN1 and APOE. APOE3 and FN1 co-localize (might indicateco-aggregation). Compounds 3, 9 and 10 reduce co-localization of APOE and FN1 in APOE E4 astrocytes in human in vitro. in E3 astrocytes, there is not much of this co-localization and compounds do not affect the co-localization.

[0028] FIG.16: Correlation of FN1 and APOE protein levels. n E3 astrocytes, FN1 and APOEexpression do not correlate. But in E4, FN1 expression is correlating with APOE E4, suggesting a potential causative pathogenicity of FN1 dependent on E4. The correlation coefficient is reduced in E4 astrocytes with all compounds. This suggests that compounds reduce the pathogenic insoluble aggregations of FN1 in APOE E4-genotype specific manner. In APOE E4 astrocytes, this is not the case.

[0029] FIG.17: In vitro efficacy. Compound 3consistently reduces FN1 levels in every APOE4 expression level. Compound 9 and 10 also reduces APOE4-dependent FN1 to a lesser extent but consistently. Compound 3, 9 and 10 are efficacious for reducing FN1 levels in APOE E4 genotype in vitro.

[0030] FIGS. 18A-18C: A. Locus of APOE gene in chromosome 19. B. Regions withintranslated APOE protein. C. Known genetic variants of APOE protein.

[0031] FIGS. 19A-19B: A. Geographic distribution of APOE alleles. B. Racial distribution ofAPOE alleles. 11 4929-9458-4176v.2

[0032] FIGS. 20A-20D.: A-D Likelihood and age-of-onset comparison for developing ADbased upon sex and age. stratified according to APOE genotypes.

[0033] FIG. 21: Representation of generalized increase in risk for AD based upon APOEe4allele count.

[0034] FIGS.22A-22F: Medicinal approaches to treating AD by targeting APOe4.A. blockingAPOE interactions; B. increasing APOE2 levels; C. decreasing APOE4 by immunotherapy; D. decreasing APOE4 by antisense oligos; E. increasing APOE lipidation; and F. correcting APOE4 conformation.

[0035] FIGS. 23A-23B: A. APOE relationship with blood-brain barrier and blood vesselfunction and clearance pathways of Aβ. B. APOE role in Aβ aggregation.

[0036] FIGS. 24A-24C: Aspects of the Blood-brain-barrier (BBB) in brain cells relative toblood flow. A. Cell types composing the BBB. B. Healthy BBB topology where astrocyte endfeet covers the vessels, enhancing the barrier function. C. BBB cells experiencing pathology. Astrocyte endfeet is remodeled to generate more space for clearance and extravasation of peripheral cells such as immune cells.

[0037] FIG.25: Whole genome sequencing of participants with two APOEε4 alleles identified510 rare coding variants enriched in extracellular matrix–related pathways. Variants in fibronectin- 1 (FN1) were prioritized, with a specific variant prioritized and demonstrating a protective effect in APOEε4 homozygotes. Meta-analysis across independent cohorts showed that carriers of this variant exhibit reduced Alzheimer’s disease risk (OR = 0.29; 95% CI 0.11–0.78, P = 0.014) and delayed age of onset by 3.37 years (95% CI 0.42–6.32, P = 0.025).

[0038] FIGS. 26A-26B: (A) Double immunofluorescent staining (IFS) for CD31 (green) andFibronectin-1 (FN1, red) with DAPI nuclear counterstain (blue) in brain tissue from mice homozygous for the APOEε3 allele (left), heterozygous for APOEε3 and APOEε4 (middle), and homozygous for APOEε4 (right). Top panels show merged double immunofluorescence staining, and bottom panels show individual FN1 staining. Scale bars equal 100 µm. (B) Quantification of FN1 fluorescence intensity across the indicated number of vessels (n), demonstrating significantly increased FN1 accumulation in APOEε4 / 4 compared to APOEε3 / 4 and APOEε3 / 3 genotypes (p < 0.0001), establishing a dose-dependent relationship between APOEε4 allele copy number and extracellular matrix remodeling at the blood–brain barrier. 12 4929-9458-4176v.2

[0039] FIGS. 27A-27C: Immunofluorescent staining of brain tissue demonstrates differentialfibronectin-1 (FN1, red) deposition at the blood–brain barrier in Alzheimer’s disease (AD) versus non-AD cases. (A) APOEε3 / 3 AD brain tissue exhibits limited FN1 deposition. (B) APOEε4 / 4 AD brain tissue shows extensive FN1 accumulation and reactive gliosis surrounding blood vessels. (C) In contrast, APOEε4 / 4 non-AD individuals exhibit markedly reduced FN1 deposition and attenuated gliosis despite APOEε4 homozygosity. Nuclei are counterstained with DAPI (blue). Scale bars equal 10 µm.

[0040] FIG. 28: Schematic illustration of extracellular matrix and blood-brain barrier (BBB)changes in relation to APOE genotype and fibronectin-1 (FN1) variants. APOE3 (left panel): Under physiological conditions, the extracellular matrix and basement membrane maintain normal BBB integrity. Blood vessels are surrounded by astrocytic endfeet, neurons, and microglia, with limited amyloid deposition and minimal gliosis. APOE4 (middle panel): Homozygosity for APOE4 is associated with pathological extracellular matrix remodeling, characterized by excessive FN1 deposition at the vascular basement membrane, increased amyloid accumulation, perivascular gliosis, and impaired microglial clearance. This leads to BBB dysfunction and exacerbation of Alzheimer's disease pathology. APOE4 with protective FN1 variant (right panel): Individuals exhibiting reduced fibronectin deposition despite APOE4 homozygosity are thereby protected. This attenuates extracellular matrix thickening, preserves BBB integrity, limits reactive gliosis, and enhances neuronal and microglial homeostasis, thereby mitigating Alzheimer's disease risk in APOE4 carriers. Fibronectin reduction is a therapeutically relevant protection and treatment strategy with disease-modifying potential.

[0041] FIG 29: FN1 domains and sites of protein interactions and signaling.

[0042] FIGS. 30A-30C: (A) Immunofluorescent staining of control brain tissue (Braak stageI) demonstrates minimal deposition of fibronectin isoforms FN1-EDA and FN1-EDB, with sparse microglial activation (IBA1, red). (B) In contrast, Alzheimer’s disease brain tissue (Braak stage VI) exhibits abundant FN1-EDA and FN1-EDB deposition (white), colocalizing with areas of extensive microgliosis (IBA1, red) and nuclei (DAPI, blue). (C) High-magnification image of AD brain reveals microglia (IBA1-positive, red) directly engulfing FN1 deposits (white), indicating an active role of microglia in the clearance of fibronectin aggregates, and fibronectin being an immune trigger. 13 4929-9458-4176v.2

[0043] FIGS. 31A-31B: Peripheral fibronectin is not associated with pathologies attached toAD. A. Minimal correlation of plasma FN1 with known AD pathologies and markers. B. Correlation of plasma FN1 with plasma VEGFA is very low.

[0044] FIGS. 32A-32H: Analysis of post-mortem human brains demonstrates vascularfibronectin (FN1) deposition in Alzheimer's disease and cerebrovascular pathology. (A-D) Double immunofluorescent staining for CD31 (endothelial marker, green), FN1 (red), and DAPI (blue) shows elevated vascular FN1 deposition in AD (B), CVP (C), and combined CVP+AD (D) compared to controls (A). (A’-D’) Corresponding FN1-only channels highlight marked increases in fibronectin signal intensity in AD, CVP, and CVP+AD compared to controls. (E) High- magnification images confirm FN1 colocalization with vascular structures across disease states. (F) Morphometric analysis of 2,384 vessels from 23 individuals shows an average vessel diameter of 11.48 ± 4.12 micron, with quantification across multiple vascular profiles. (G) QQ plot validates model fit for statistical analysis of FN1 intensity data. (H) Quantification of normalized fibronectin intensity demonstrates significantly increased FN1 accumulation in AD, CVP, and CVP+AD compared to controls (two-way ANOVA, F(3,2384) = 231.59, p < 0.0001). These findings confirm that both Alzheimer’s disease and cerebrovascular pathology independently, and synergistically, drive fibronectin deposition at the human cerebrovasculature, linking extracellular matrix remodeling to vascular dysfunction in neurodegeneration. Therefore, therapeutic reduction of fibronectin deposition may mitigate vascular dysfunction and attenuates combined Alzheimer’s and cerebrovascular pathology.

[0045] FIGS. 33I-33P: (I) Schematic of experimental workflow: APOEε3 and APOEε4targeted replacement mouse models were subjected to histological sectioning, immunofluorescent staining, and quantitative image analysis. (J) Representative double immunofluorescence images of brain vasculature stained for CD31 (endothelial marker, yellow) and fibronectin (FN1, cyan) in APOEε3 / 3 versus APOEε4 / 4 mice. Merged panels show markedly increased fibronectin deposition in APOEε4 / 4 compared to APOEε3 / 3. (K) Quantification of mean fibronectin intensity across multiple brain regions demonstrates significantly higher FN1 deposition in APOEε4 / 4 compared with APOEε3 / 3 mice (t(10) = 11.68, F(10,1) = 136.48, p = 3.57 × 10⁻⁷). (L) Additional staining for fibronectin (green), fibrinogen (white), and DAPI (blue) shows perivascular accumulation of FN1 and colocalization with vascular fibrinogen in APOEε4 / 4 mice compared to APOEε3 / 3. Insets highlight vascular remodeling and FN1–fibrinogen overlap. (M) Lognormal QQ 14 4929-9458-4176v.2plot validates regression model fit for fibronectin intensity distribution across APOE genotypes. (N) Quantification confirms significantly higher fibrinogen intensity in APOEε4 / 4 compared with APOEε3 / 3 mice (p < 0.0001). (O) Scatter plot of fibrinogen leakage across animals demonstrates genotype-dependent segregation of values (χ²(1) = 28.59, F(10,1) = 8.172). (P) Bar graph of vascular fibronectin and fibrinogen correlation after automated image analyses normalized across experiments shows consistent increase of fibrinogen and fibronectin colocalization in APOEε4 / 4 relative to APOEε3 / 3. These results establish that APOEε4 homozygosity drives fibronectin over- deposition and vascular leakage in vivo, consistent with our other findings, and provides a mechanistic pathological link between APOEε4 and extracellular matrix–mediated BBB dysfunction and leakage. Therefore, therapeutic modulation of fibronectin deposition may reduce vascular leakage and preserves BBB integrity in APOEε4 carriers.

[0046] FIGS. 34A-34F: (A) Immunofluorescent staining of human post-mortem brain tissueshows fibronectin (FN1, red), aquaporin-4 (AQP4, white), and DAPI (blue). In control tissue, FN1 deposition is sparse and colocalizes with astrocytic endfeet (AQP4). In Alzheimer’s disease (AD), FN1 is markedly increased, with disrupted alignment relative to AQP4. (B) Quantification of FN1– AQP4 spatial distance demonstrates significant disorganization in AD compared to controls (nested t-test, t = 9.50, p < 1.0 × 10⁻¹⁵, n = 200). (C) Immunostaining for cadherin-5 (CDH5, endothelial marker) shows robust vascular signal in controls and reduced CDH5 intensity in AD. (D) Quantification reveals significantly decreased CDH5 intensity in AD versus controls (nested t-test, t = 9.49, p = 6.97 × 10⁻⁴, n = 602), consistent with endothelial dysfunction. (E) Double staining for FN1 (red) and CDH5 (white) highlights colocalization of fibronectin deposition with vascular endothelial disruption, particularly in AD. (F) 2D histogram analysis of FN1 versus CDH5 intensity shows reduced correlation in AD relative to controls (Pearson r = 0.36 in AD vs 0.82 in control; Manders coefficient = 0.613 vs 0.822), confirming impaired vascular integrity with excessive FN1 deposition. Conclusion: These findings establish that in AD brains, fibronectin deposition misaligns astrocytic endfeet, disrupts endothelial cadherin-5 integrity, and contributes to blood–brain barrier breakdown. Therefore, therapeutic reduction of fibronectin deposition may preserve astrocytic interaction and contact with vasculature and thereby the BBB function by maintaining endothelial integrity, and prevents BBB dysfunction.

[0047] FIG. 35: Immunofluorescent analysis of mouse brain vasculature comparing APOE3 / 3and APOE4 / 4 genotypes. Top panels: Triple staining with dextran-red (vascular tracer, red), 15 4929-9458-4176v.2fibronectin (green), CDH5 (endothelial marker, white), and DAPI (nuclei, blue). In APOE3 / 3 mice, dextran is largely contained within intact CDH5-positive vessels with minimal fibronectin deposition. In contrast, APOE4 / 4 mice show perivascular dextran leakage coinciding with increased fibronectin deposition and disrupted CDH5 signal, indicative of blood-brain barrier (BBB) dysfunction. Bottom panels: Dual staining of dextran-red with fibronectin (green) confirms colocalization of fibronectin accumulation with vascular leakage in APOE4 / 4 mice compared to APOE3 / 3 controls. These findings demonstrate that APOEε4 homozygosity promotes fibronectin- associated BBB breakdown. Therefore, therapeutic reduction of fibronectin deposition may preserve vascular integrity and mitigate APOEε4-driven neurovascular pathology.

[0048] FIGS. 36A-36B: (A) Single-nucleus sequencing (snSeq) of human brain tissuedemonstrates total FN1 expression across major cell types, with the highest expression observed in endothelial cells (EN, 23.87%) and astrocytic glia (AG, 21.29%), followed by pericytes (PE, 13.55%), excitatory neurons (ExcN, 13.55%), oligodendrocyte progenitor cells (OPC, 10.32%), inhibitory neurons (InhN, 9.03%), oligodendrocytes (OD, 5.16%), and microglia (MG, 3.23%). (B) Differential expression analysis of FN1 in Hispanic brain tissue reveals that APOEε4 carriers exhibit significant downregulation of FN1 across multiple non-vascular cell populations compared to non-carriers, consistent with cell-type–specific dysregulation in the setting of higher cerebrovascular pathology burden observed in this population. These findings establish that FN1 expression is enriched in vascular and astrocytic compartments and differentially regulated in APOEε4 Hispanic carriers. Therefore, therapeutic targeting of fibronectin pathways in high-risk populations or individuals with disproportionate vascular pathology burden may mitigate BBB dysfunction and reduce Alzheimer’s disease progression.

[0049] FIGS. 37A-37B: A. Immunofluorescent staining for FN1 (brown) with DAPI nuclearcounterstain (blue) (left) and zoom-in (right) in iPSC-derived human astrocytes homozygous for APOEe3 (top) and homozygous for APOEe4 (bottom), individual staining for FN1 (middle). B. Quantifications and statistical analysis of fluorescence intensity of FN1 in iPSC-derived human astrocytes homozygous for APOEe3 or APOEe4 (upper graph), and normalized Western blot levels (lower graph).

[0050] FIGS. 38A-38D: A. Double immunofluorescent staining for FN1 (brown) and APOE(purple) with DAPI nuclear counterstain (blue) in iPSC-derived human astrocytes homozygous for APOEe3 (top) and homozygous for APOEe4 (bottom). B Quantifications and statistical analysis 16 4929-9458-4176v.2of fluorescence intensity of FN1 in in iPSC-derived human astrocytes homozygous for APOEe3 (left) and homozygous for APOEe4 (right). Quantifications of fluorescence intensity of APOE in iPSC-derived human astrocytes homozygous for APOEe3 (left) and homozygous for APOEe4 (right). C. Comparison of intensities of FN1 and APOE in iPSC-derived human astrocytes homozygous for APOEe3 and homozygous for APOEe4 and regression curves for each. D. Histogram comparing intensities of FN1 and APOE in iPSC-derived human astrocytes homozygous for APOEe3 (left) and homozygous for APOEe4 (right). These findings establish that APOEε4 astrocytes drive coordinated upregulation of APOE and fibronectin, wherein therapeutic modulation of FN1 expression in astrocytes may reduce extracellular matrix accumulation, restores cellular homeostasis, and mitigates APOEε4-mediated vascular and neurodegenerative pathology.

[0051] FIGS. 39E-39H: E & F. (E) Immunofluorescent staining of post-mortem human brainsshows APOE (magenta), fibronectin (FN1, red), GFAP (astrocytic marker, green), and DAPI (blue) in APOEε3 / 3 versus APOEε4 / 4 individuals. APOEε4 / 4 brains demonstrate markedly increased FN1 deposition colocalizing with astrocytic GFAP signal. (F) High-magnification images reveal co-expression of APOE and FN1 in GFAP-positive astrocytic structures, with APOEε4 / 4 showing higher deposition than APOEε3 / 3. (G) Quantification demonstrates significantly elevated APOE intensity (t(459) = 4.11, p = 4.61 × 10⁻⁵) and FN1 intensity (t(498) = 5.28, p = 1.88 × 10⁻⁷) in APOEε4 / 4 compared with APOEε3 / 3 brains. (H) Correlation analysis of FN1 and APOE intensity shows a positive relationship, with APOEε4 / 4 individuals exhibiting stronger co-upregulation than APOEε3 / 3 controls. These findings establish that in human brains, APOEε4 carriers exhibit astrocytic co-upregulation of APOE and fibronectin deposition. Therapeutic reduction of FN1 expression in astrocytes may preserve blood–brain barrier homeostasis and mitigate APOEε4-driven gliovascular BBB pathology.

[0052] FIGS.40J-40L: J. Triple immunofluorescent staining for FN1 (cyan), APOE (purple),and AQP4 (grey) with DAPI nuclear counterstain (blue) in human brains homozygous for APOEe3 (left) and homozygous for APOEe4 (right). K. Pearson correlation between FN1 and AQP4 in human brains homozygous for APOEe3 (left) and homozygous for APOEe4 (right). L. Pearson correlation between APOE and AQP4 in human brains homozygous for APOEe3 (left) and homozygous for APOEe4 (right). These findings demonstrate that while global correlations between FN1, APOE, and astrocytic AQP4 are maintained, APOEε4 homozygosity drives 17 4929-9458-4176v.2abnormal FN1 and APOE deposition around astrocytic endfeet. Therapeutic reduction of FN1 accumulation may preserve astrocytic polarity, stabilize perivascular endfeet, and prevent APOEε4-mediated BBB dysfunction.

[0053] FIG.41: Zebrafish model demonstrating causal role of fibronectin (FN1) in blood-brainbarrier (BBB) leakage. Top row (Control + Dextran-Red): Dextran-Red vascular tracer remains contained within cerebral vessels, with minimal background FN1 signal, indicating intact BBB. Bottom row (her4.1:FN1-T2A-GFP + Dextran-Red): Zebrafish engineered to express human FN1 under the astrocytic her4.1 promoter exhibit strong FN1 signal and associated Dextran-Red extravasation, demonstrating BBB leakage. These findings establish that astrocytic expression of human FN1 is sufficient to drive BBB breakdown in vivo. Therapeutic inhibition of FN1 expression or activity may prevent fibronectin-induced vascular leakage and preserves BBB integrity.

[0054] FIG. 42: Schematic illustration of the pathological mechanism of fibronectin (FN1)accumulation at the blood-brain barrier (BBB). ApoE3 (left panel): Under normal conditions, endothelial and astrocytic crosstalk maintains BBB integrity. FN1 levels are low, with balanced signaling across VEGFR, EGFR, and IGF1R pathways. HB-EGF and VEGFA signaling support endothelial stability, while integrin-FAK signaling maintains controlled astrocytic-endothelial communication. ApoE4 (right panel): APOE4 increases FN1 expression and deposition in both endothelial and astrocytic compartments. Excess FN1 activates integrin-FAK signaling, disrupts VEGFR and EGFR crosstalk, and reduces HB-EGF and IGF1 activity. This pathological remodeling impairs endothelial barrier function, leading to BBB breakdown and neurovascular dysfunction. This model establishes that APOE4-driven fibronectin accumulation impairs astrocyte-endothelial communication and BBB integrity. Therapeutic reduction of FN1 expression or blockade of FN1-integrin-FAK signaling preserves endothelial function, restores growth factor signaling balance, and prevents BBB impairment.

[0055] FIG. 43: Human cerebrospinal fluid (CSF) analysis of co-expression betweenfibronectin (FN1) and vascular endothelial growth factor A (VEGFA), stratified by APOE4 carrier status and disease state. Control group: In cognitively normal controls, FN1 and VEGFA expression show weak or no positive correlation, regardless of APOE4 status. Alzheimer's disease (AD) group: In AD subjects, APOE4 carriers demonstrate a strong positive correlation between FN1 and VEGFA levels, whereas non-carriers show a flat or negative association. These findings 18 4929-9458-4176v.2establish that APOE4 carriers exhibit a pathological co-regulation of FN1 and VEGFA in the CSF during Alzheimer's disease. Therefore, inhibition of FN1 normalizes VEGFA signaling and provides a measurable CSF biomarker signature for treatment response and patient stratification.

[0056] FIG. 44: Fit interaction model in human brain.

[0057] FIGS. 45A-45B: (A) Control group: In untreated APOEε4 / 4 astrocytes, VEGFA (red)and IGF1 (green) are robustly expressed, indicating heightened astrocytic secretion of vascular growth factors. (B) Pyrintegrin-treated group: Upon exposure to 4 μM pyrintegrin, APOE4 / 4 astrocytes exhibit markedly reduced VEGFA (−25%) and IGF1 (−34.5%) expression, as quantified across >1,100 cells per condition. Immunofluorescence images confirm reduced growth factor intensity, consistent with suppression of fibronectin–integrin–mediated signaling These findings establish that pharmacological inhibition of fibronectin–integrin signaling attenuates maladaptive VEGFA and IGF1 production in APOEε4 astrocytes. Pyrintegrin or related integrin pathway inhibitors provide a therapeutic approach to normalize astrocytic signaling and mitigate blood– brain barrier dysfunction in APOEε4 carriers.

[0058] FIGS. 46A-46B: Fibronectin activation and accumulation. (A) Inactive fibronectinhomodimer is mechanically activated and changes its conformation to ECM fibrils. (B) APOE-ε4 increases fibronectin accumulation.

[0059] FIG. 47: Hit compound selection using in silico simulated docking followed bymolecular dynamics simulations.

[0060] FIGS. 48A-48B: (A) Control group: In wild-type zebrafish brains, immunostaining forSV2 (neuronal marker, green) and L-plastin (microglial marker, red) reveals normal distributions of neurons and microglia. fn1b knockout and compound treatment: In zebrafish lacking fibronectin (fn1b knockout), administration of candidate compounds #2 and #3 does not alter neuronal (SV2) or microglial (L-plastin) staining compared to untreated controls. (B) Quantification shows no significant differences across multiple brain sections in measures of SV2 density, L-plastin distribution, or microglial number, demonstrating preserved neuronal and immune cell architecture. These findings establish that fibronectin-targeting compounds #2 and #3 exhibit no detectable off-target neurotoxicity in fn1b-deficient zebrafish. Therefore, compound administration maintains neuronal and microglial integrity, confirming a favorable safety profile for therapeutic use in targeting fibronectin-driven blood–brain barrier dysfunction. Additionally, 19 4929-9458-4176v.2safety is validated in fibronectin-null models, ensuring compound activity is restricted to fibronectin-dependent pathways.

[0061] FIGS. 49A-49B: (A) Control group: In fn1b knockout zebrafish brains,immunostaining for ZO-1 (tight junction protein, red) and BLBP (astrocytic lineage marker, green) shows intact blood–brain barrier structure and normal astrocytic distribution. Compound treatment: Administration of candidate compounds #2 and #3 does not alter ZO-1 intensity or BLBP expression relative to untreated controls. (B) Quantitative analyses confirm no significant changes in either marker, demonstrating preserved BBB integrity and absence of reactive gliosis. These findings establish that fibronectin-targeting compounds #2 and #3 do not disrupt blood– brain barrier structure or induce astrocytic gliosis in fn1b-deficient zebrafish, wherein compound administration maintains tight junction integrity and astrocytic homeostasis, confirming safety and on-target selectivity for fibronectin-dependent pathways..

[0062] FIGS. 50A-50B: (A) Control (Aβ42-only): Treatment of Aβ42, which also enhancesfibronectin overexpression and deposition at the BBB, into zebrafish brains induces pathological changes, including reduced SV2+ neuronal density and increased L-plastin+ microglial infiltration, consistent with neuroinflammation and neuronal injury. Compound treatment: Co- treatment with candidate compounds #2 or #3 following Aβ42 exposure restores SV2+ neuronal staining and significantly reduces L-plastin+ microglial infiltration compared to Aβ42 alone. (B) Quantitative analyses confirm improved neuronal preservation and attenuated neuroinflammation, demonstrating protective effects of the compounds in vivo. These findings establish that fibronectin-targeting compounds #2 and #3 rescue Aβ42-induced neuronal loss and neuroinflammation in zebrafish, wherein compound administration restores neuronal integrity and reduces microglial activation, providing preclinical evidence of therapeutic efficacy against Alzheimer’s disease–associated vascular and inflammatory pathology.

[0063] FIGS. 51A-51B: (A) Aβ42 group: Injection of Aβ42 into zebrafish brains inducesfibronectin (FN1) deposition and astrocytic reactivity (BLBP), consistent with extracellular matrix remodeling and gliosis. Compound treatment: Co-treatment with candidate compounds #2 or #3 following Aβ42 exposure significantly reduces FN1 accumulation and BLBP intensity relative to Aβ42 alone. (B) Quantitative analyses confirm marked reductions in FN1 and BLBP, indicating suppression of pathological ECM deposition and reactive gliosis. These findings establish that fibronectin-targeting compounds #2 and #3 reduce Aβ42-induced fibronectin deposition and 20 4929-9458-4176v.2astrocytic gliosis in zebrafish, wherein compound administration mitigates extracellular matrix remodeling and neuroinflammatory responses, providing mechanistic evidence of therapeutic efficacy against Alzheimer’s disease–associated cerebrovascular pathology.

[0064] FIG. 52: Second protective FN1 variant rs116558455 indicating sites of mutationsrelative to wild-type FN1.

[0065] FIGS. 53A-53B: FN1 domains. A. Domains by type. B. Structural and functionalaspects of each FN1 domain.

[0066] FIGS.54A-54B: Cellular signaling pathways associated with FN1-integrin interactionsfor both A. outside-in signaling and B. inside-out signaling.

[0067] FIGS. 55A-55E: (A) (FAK inhibition): In iPSC-derived astrocytes, APOEε4 / 4 cellsexhibit suppressed VEGFA compared to APOEε3 / 3 controls. Pharmacological inhibition of focal adhesion kinase (FAK) restores VEGFA expression levels in APOEε4 astrocytes, indicating FAK as a critical downstream mediator of fibronectin–integrin signaling. (B) (αvβ3 / β5 integrin inhibition): Treatment with cilengitide, an αvβ3 / β5 integrin inhibitor, does not rescue VEGFA or IGF1 expression in APOEε4 astrocytes, and instead induces partial apoptosis, suggesting that αvβ3 blockade is not protective. (C and D) (β1 integrin inhibition with ATN-161): In contrast, inhibition of β1 integrin with ATN-161 significantly restores both VEGFA and IGF1 levels in APOEε4 astrocytes, normalizing protective growth factor secretion. (E) (schematic): Diagrammatic representation of fibronectin domains and integrin binding sites highlights that ATN-161 interacts with α5β1 non-RGD binding, thereby disrupting pathological fibronectin–integrin–FAK / MAPK signaling, while cilengitide engages RGD-binding αvβ3 integrins and induces apoptosis. These findings establish that fibronectin promotes pathological suppression of VEGFA and IGF1 through β1 integrin–FAK signaling in APOEε4 astrocytes, wherein inhibition of β1 integrin (e.g., ATN- 161) or FAK restores protective VEGFA / IGF1 secretion and provides a targeted therapeutic strategy for mitigating APOEε4-associated blood–brain barrier dysfunction.

[0068] FIG. 56: Schematic model of fibronectin-driven extracellular matrix (ECM)remodeling as an APOE4-mediated Alzheimer's disease (AD) pathology and its therapeutic targeting. Homeostatic state (left): Under normal APOE3 conditions, fibronectin (FN1) levels are low, astrocyte-endothelial interactions are intact, and VEGFA / IGF1 signaling supports blood- brain barrier (BBB) integrity. Pathological state (middle): In APOE4 carriers, FN1 deposition accumulates at the gliovascular interface, activating integrin-FAK signaling and pSMAD3, 21 4929-9458-4176v.2disrupting VEGFA / IGF1 signaling, impairing astrocyte-endothelial crosstalk, reducing amyloid clearance, and driving BBB dysfunction. Therapeutic intervention (right): FN1 modulation or integrin / FAK inhibition rebalances ECM signaling, restores VEGFA / IGF1 signaling, rescues astrocyte-endothelial interactions, prevents BBB dysfunction, and supports cognitive health. These findings establish that APOE4-mediated fibronectin remodeling constitutes a pathological ECM- signaling axis in AD, wherein therapeutic modulation of FN1-integrin-FAK signaling restores astrocyte-endothelial communication, reactivates VEGFA / IGF1 pathways, and prevents blood- brain barrier breakdown.

[0069] FIGS. 57A-57B: Longitudinal trajectories of plasma P-tau217 and cognitive decline(PACC-4) stratified by APOE genotype in the WRAP and ADRC cohorts. (A) (Biomarker trajectory): Plasma P-tau217 levels rise earlier and more steeply in APOE4 carriers compared to non-carriers, with E4 / 4 individuals crossing the 95% specificity threshold for Abeta42 positivity at age ~72 and the 95% sensitivity threshold at age ~80, whereas non-carriers reach these cut-offs substantially later. (B) (Cognitive trajectory): Cognitive performance (PACC-4) declines earlier and faster in APOE4 carriers, with thresholds for MCI and AD discrimination crossed approximately 5-10 years before non-carriers, highlighting accelerated disease progression. These findings establish that APOE4 carriers exhibit accelerated biomarker and cognitive trajectories, wherein P-tau217 and PACC-4 thresholds are reached nearly a decade earlier compared to non- carriers, providing a genotype-stratified framework for predicting disease onset and timing therapeutic intervention.

[0070] FIG. 58: Comparison of time from reaching the plasma P-tau217 specificity cut point(0.62 pg / ml) to onset of dementia symptoms, stratified by APOE genotype across multiple longitudinal cohorts (Wisconsin, ADNI, EFIGA, WHICAP, and combined meta-analysis). Methodological context: ROC-AUC analyses determined that 0.62 pg / ml was the optimal cut point for diagnostic specificity, minimizing false positives. Individuals were followed longitudinally after reaching this biomarker threshold, and survival-type curves plotted the time to symptom onset. Findings across cohorts: APOE4 / 4 carriers consistently demonstrated earlier onset of dementia symptoms after crossing the specificity cut point compared to non-carriers and APOE3 / 3 individuals. The average window from biomarker positivity to clinical symptom emergence was ~2-3 years, with APOE4 carriers showing the shortest lag time. Cross-cohort robustness: These patterns held across all four independent cohorts (Wisconsin, ADNI, EFIGA, WHICAP), as well 22 4929-9458-4176v.2as in the combined analysis, underscoring the reproducibility of the biomarker-to-symptom transition across diverse populations. These findings establish that plasma P-tau217 positivity predicts symptom onset within a narrow 2-3 year window, wherein APOE4 carriers show significantly accelerated progression, providing a critical biomarker-defined opportunity for therapeutic intervention prior to overt dementia.

[0071] FIG. 59:

[0264] Integrated evidence that APOE4 reduces VEGFA signaling throughfibronectin over-deposition across human brains, CSF, and iPSC-derived astrocytes. Astrocytic VEGFA production in AD brains: Immunostaining of post-mortem human brains reveals significantly lower VEGFA intensity in astrocytes from AD compared to controls, indicating disease-associated suppression of pro-angiogenic signaling. CSF transcriptomics and co- expression: In human CSF, co-expression analyses demonstrate that APOE4 carriers fail to upregulate VEGFA in response to FN1, whereas non-carriers show a protective coupling between VEGFA and FN1 expression during AD. APOE4-driven fibronectin accumulation: iPSC-derived astrocytes carrying APOE4 show robust over-deposition of FN1 compared to APOE3 cells, consistent with extracellular matrix remodeling linked to vascular pathology. Direct suppression of VEGFA by fibronectin: Experimental FN1 coating of astrocytes is sufficient to reduce VEGFA expression, demonstrating causality between excess fibronectin and impaired VEGFA signaling. Functional readouts in human astrocytes: APOE4 / 4 astrocytes show markedly reduced VEGFA levels compared to APOE3 / 3 cells, establishing a direct APOE genotype effect on VEGFA bioavailability. These findings establish that APOE4 promotes fibronectin over-deposition in astrocytes, leading to a maladaptive suppression of VEGFA signaling across human brain tissue, CSF, and iPSC models, wherein excessive fibronectin is sufficient to blunt astrocytic VEGFA output and drive vascular vulnerability in Alzheimer's disease.

[0072] FIG.60: Fibronectin-integrin signaling through focal adhesion kinase (FAK) is a majorpathway driving VEGFA / IGF1 suppression in APOE4 astrocytes and can be targeted pharmacologically. FAK inhibition rescues VEGFA levels: In iPSC-derived APOE4 / 4 astrocytes, pharmacological blockade of FAK restores VEGFA expression to levels comparable with APOE3 / 3, demonstrating that downstream signaling from fibronectin-integrin engagement suppresses growth factor expression. Integrin β1 blockade restores VEGFA and IGF1: Treatment with ATN-161, a non-RGD-binding α5β1 integrin antagonist, successfully rescues both VEGFA and IGF1 expression in APOEε4 astrocytes, supporting a causal role for β1-mediated signaling in 23 4929-9458-4176v.2fibronectin-driven pathology. Integrin αvβ3 blockade fails to restore signaling: By contrast, cilengitide-mediated αvβ3 inhibition does not rescue VEGFA or IGF1, indicating pathway specificity. Mechanistic schema: Fibronectin’s extracellular matrix domains (FNIII9, FNIII10, RGD) bind integrins, triggering FAK / MAPK activation. ATN-161 selectively disrupts α5β1- mediated non-RGD signaling, restoring pro-vascular growth factor production, while cilengitide blocks adhesion but induces endothelial apoptosis, underscoring divergent therapeutic implications. These findings establish that fibronectin suppresses VEGFA and IGF1 in APOE4 astrocytes through integrin α5β1-FAK signaling, wherein targeted inhibition with ATN-161, but not αvβ3 blockade, restores pro-vascular growth factor output and highlights a selective therapeutic axis for BBB protection in Alzheimer's disease. DETAILED DESCRIPTION

[0073] A method for treating, or for reducing the development of, a neurodegenerative disease,comprising the step of administering to a subject in need thereof an effective amount of a compound having the following structure: whereinatom Z is present or absent, and when present is a carbon bound to A, and when absent the two N atoms of the ring are directly bound to each other, wherein when Z is present: bonds ^^^^^^^^^^, and ^ are present, and bonds ^ and ^ are absent; A is a C1-C6 alkyl carboxamide linked through either -CH2-, -O-, or -S-; X is a C atom; Y is NR6or O; 24 4929-9458-4176v.2and when Y is O, R1is H, alkyl, alkyl carbonyl, alkyl N-amido, or alkylaromatic, and when Y is NR6, either R1and R6together form a 5- to 8-membered unsaturated heterocycle, or R6is C(O)R7, wherein R7is alkyl and R1is H, alkyl, C2-C6 alkyl carbonyl, C2-C6alkyl N-amido, or alkylaromatic; R2, R3, R4, and R5are, independently, H, halo, -OH, C1-C6 alkyl or C1-C6 alkoxy; wherein when Z is absent: bonds ^ and ^ are present, and bonds ^^^^^^^^^^^^and ^ are absent; X is an N atom; R2is H or benzoyl; R3, R4, and R5are H; Y is C(O)NHR8, and R8is H, C1-C6 alkyl or alkyl aromatic; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0074] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Z is present and has the following structure: .

[0075] In embodiments in theacceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, A has the following structure: 25 4929-9458-4176v.2wherein E is CH2, O, or S; and R9are R10are each, independently, H, optionally substituted or unsubstituted C1-C6 alkyl, C4-C8cycloalkyl, benzyl, aromatic, or alkylaromatic.

[0076] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R9is H or C1-C6 alkyl.

[0077] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is an optionally substituted or unsubstituted C4-C8 cycloalkyl, benzyl, or alkylaromatic.

[0078] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the cycloalkyl or alkylaromatic is optionally substituted with halo, -OH, -SH, C1-C4 alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, or -NHx(alkyl)y where x + y = 2.

[0079] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the aromatic is a carbocyclic or heteroaromatic ring containing, selected independently, at least one of N, O, or S.

[0080] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the cycloalkyl or aromatic is a monocycle or bicycle.

[0081] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Y is O.

[0082] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1is an optionally substituted or unsubstituted alkylaromatic.

[0083] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the alkylaromatic has the following structure: 26 4929-9458-4176v.2wherein n = 1-4; and wherein R11, R12, R13, R14, R15are each, independently H, -C(O)NH2, or -SO2NH2.

[0084] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R9is ethyl.

[0085] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is a benzyl, and wherein the benzyl is substituted with halo, -OH, -SH, C1-C4alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O- alkyl, -S-alkyl, or -NHx(alkyl)y where x + y = 2.

[0086] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is a p-methyl benzyl.

[0087] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2and R3are C1-C6 alkoxy, and wherein R2and R3are methoxy.

[0088] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

[0089] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R9is H. 27 4929-9458-4176v.2

[0090] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is a an optionally substituted or unsubstituted C4-C8 cycloalkyl, and wherein R10is a cyclopentane.

[0091] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2and R3are H.

[0092] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound may exist as a dimer linked by R1C1-C6 alkyl, and wherein the alkyl linker is propyl.

[0093] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

[0094] In embodimentsacceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Y is NR6, and wherein R6is C(O)R7.

[0095] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1and R6together form a 5- to 8-membered unsaturated heterocycle.

[0096] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R7is C1-C6alkyl or an alkylamide.

[0097] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the alkylamide has the following structure: 28 4929-9458-4176v.2wherein n = 1-4; and R16is an optionally substituted or unsubstituted alkylaromatic.

[0098] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R16is an optionally substituted or unsubstituted ethyl benzene.

[0099] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R9is H.

[0100] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is an optionally substituted or unsubstituted aromatic, and wherein the aromatic is a monocycle or bicycle.

[0101] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the aromatic is optionally substituted with halo, -OH, -SH, C1-C4 alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S- alkyl, or -NHx(alkyl)y where x + y = 2.

[0102] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the aromatic is a carbocyclic or heteroaromatic ring containing, selected independently, at least one of N, O, or S.

[0103] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is a substituted benzene, and wherein, R10is a 2,5-dimethoxy phenyl.

[0104] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: 29 4929-9458-4176v.2

[0105] In embodiments acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Z is absent and which has the following structure: .

[0106] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2is benzoyl, and wherein the benzoyl is substituted with halo, -OH, -SH, C1-C4alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, - O-alkyl, -S-alkyl, or -NHx(alkyl)y where x + y = 2.

[0107] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

[0108] In embodiments inacceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: 30 4929-9458-4176v.2.

[0109] In embodiments in utically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

[0110] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, compound has the following structure:wherein R16is an optionally or mono or bicyclic aromatic; R17is an optionally substituted or unsubstituted mono or bicyclic aromatic; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0111] In embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: 31 4929-9458-4176v.2wherein R18and R19is C2-Cx R18and R19may come together to form a 5-x-membered unsaturated optionally substituted or unsubstituted heterocycle; R20is a 5-6-membered optionally substituted heteroaromatic ring containing, selected independently, at least one of N, O, or S; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0112] A method for treating, or for reducing the development of, a neurodegenerative disease,comprising the step of administering to a subject in need thereof an effective amount of a compound having the following structure:substituted or unsubstituted mono or bicyclic aromatic; R17is an optionally substituted or unsubstituted mono or bicyclic aromatic; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0113] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .4929-9458-4176v.2

[0114] A method for treating, or for reducing the development of, a neurodegenerative disease,comprising the step of administering to a subject in need thereof an effective amount of a compound having the following structure: is C2-Cx alkyl;may come together to form a 5-x-membered unsaturated optionally substituted or unsubstituted heterocycle; R20is a 5-6-membered optionally substituted heteroaromatic ring containing, selected independently, at least one of N, O, or S; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0115] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .disease comprises improving one or more cognitiveperformance measures.

[0117] In embodiments, the neurodegenerative disease is Alzheimer’s disease.

[0118] A compound having the following structure:wherein33 4929-9458-4176v.2atom Z is present or absent, and when present is a carbon bound to A, and when absent the two N atoms of the ring are directly bound to each other, wherein when Z is present: bonds ^^^^^^^^^^, and ^ are present, and bonds ^ and ^ are absent; A is a C1-C6 alkyl carboxamide linked through either -CH2-, or -O-, or -S-; X is a C atom; Y is NR6or O; and when Y is O, R1is H, C1-C6alkyl, alkyl carbonyl, alkyl N-amido, or alkylaromatic, and when Y is NR6, either R1and R6together form a 5- to 8-membered unsaturated heterocycle, or R6is C(O)R7, wherein R7is C1-C6alkyl and R1is H, C2-C6alkyl, C2-C6 alkyl carbonyl, C2-C6 alkyl N-amido, or alkylaromatic; wherein when A is linked through either -CH2-, or -O-: R2, R3, R4, and R5are, independently, H, -OH, -SH, C1-C4 alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, or -NHx(alkyl)y where x + y = 2; wherein when A is linked through -S-: at least one of R2, R3, R4, and R5are -H, -OH, -SH, C1-C4 alkyl, -C1-C4-OH, C1-C4-SH, C1- C4-NH2, -S-alkyl, or -NHx(alkyl)ywhere x + y = 2; and up to three of R2, R3, R4, and R5are -H wherein when Z is absent: bonds ^ and ^ are present, and bonds ^^^^^^^^^^, and ^ are absent; 34 4929-9458-4176v.2X is a C or N atom; R3, R4, and R5are H; Y is C(O)NHR8wherein when X is a C atom: R2is H or benzoyl; R8is H, a substituted or unsubstituted C1-C6 alkyl or alkyl aromatic; wherein when X is an N atom: R2is H, poly-substituted benzoyl, or optionally substituted or unsubstituted benzyl or naphthoyl R8is a substituted or unsubstituted C1-C6alkyl or alkyl aromatic; and the aromatic is substituted, or pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0119] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Z is present and which has the following structure: .in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, A has the following structure:35 4929-9458-4176v.2wherein E is CH2, O, or S; and R9are R10are each, independently, H, optionally substituted or unsubstituted C1-C6 alkyl, C4-C8 cycloalkyl, benzyl, aromatic, or alkylaromatic.

[0121] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R9is H or C1-C6 alkyl.

[0122] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is an optionally substituted or unsubstituted C4-C8 cycloalkyl, aromatic, or alkylaromatic.

[0123] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the cycloalkyl, aromatic, or alkylaromatic is optionally substituted with halo, -OH, -SH, C1-C4alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, or -NHx(alkyl)y where x + y = 2.

[0124] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the aromatic is a carbocyclic or heteroaromatic ring containing, selected independently, at least one of N, O, or S.

[0125] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the cycloalkyl or aromatic is a monocycle or bicycle.

[0126] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1is an optionally substituted or unsubstituted C1-C11alkyl, alkylaromatic, aromatic, or alkylcarboxamide, or alkylcarboxalate.

[0127] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the alkylaromatic has the following structure:4; and wherein R11, R12, R13, R14, R15are each, independently H, -C(O)NH2, or -SO2NH2. 36 4929-9458-4176v.2

[0128] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, wherein the compound may exist as a dimer linked by R1C1-C6 alkyl.

[0129] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Y is NR6, and wherein R6is C(O)R7.

[0130] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1and R6together form a 5- to 8-membered unsaturated heterocycle.

[0131] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R7is C1-C6alkyl or an alkylcarboxamide.

[0132] In embodiments, in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the alkylcarboxyamide has the following structure: wherein n = 1-4; and R16is an optionally substituted or unsubstituted C1-C11 alkyl or alkylaromatic.

[0133] A method of treating or preventing blood–brain barrier (BBB) dysfunction in a subjectcarrying at least one APOEε4 allele, the method comprising administering an agent that reduces fibronectin-1 (FN1) expression, activity, or deposition. In some embodiments, reduction of FN1 expression preserves astrocyte–endothelial crosstalk, stabilizes vascular integrity, and / or mitigates neurodegenerative pathology.

[0134] In some embodiments, the agent inhibits FN1–integrin–FAK signaling to restoreendothelial growth factor signaling balance.

[0135] In some embodiments, the agent comprises an antisense oligonucleotide, siRNA, orshRNA targeting FN1 expression. In some embodiments, the siRNA comprises a sequence of one or more of the following FN1-siRNA:

[0136] Sense CCAUUUCACCUUCAGACAATT (SEQ ID NO:1)

[0137] Antisense UUGUCUGAAGGUGAAAUGGTT (SEQ ID NO:2)37 4929-9458-4176v.2

[0138] Sense GCAAGCAGCAAGCCAAUUUTT (SEQ ID NO:3)

[0139] Antisense AAAUUGGCUUGCUGCUUGCTT (SEQ ID NO:4)

[0140] FN1-siRNA are available commercially, e.g., FN1-siRNA were commercially obtainedfrom APExBio company (Shanghai, China). In some embodiments, the antisense compound directed against FN1 is set forth in WO2013181666A2, which is hereby incorporated by reference in its entirely, including the sequences set forth therein.

[0141] In some embodiments, the agent is a monoclonal antibody or small molecule thatblocks FN1–integrin binding. In some embodiments, the monoclonal antibody is an α5β1 anti- integrin antibody. In some embodiments, the agent is Volociximab.

[0142] In some embodiments of any of the methods disclosed herein, the subject ishomozygous for APOEε4 (APOEε4 / ε4). In some embodiments, the subject does not have cancer, or has not been diagnosed with a cancer or has not been treated for cancer. In some embodiments, the method further comprises identifying, or having identified, the subject as homozygous for APOEε4 (APOEε4 / ε4) prior to treatment.

[0143] In some embodiments, the treatment reduces perivascular fibronectin deposition, asmeasured by immunohistochemistry in endothelial and astrocytic compartments.

[0144] In some embodiments, the agent enhances microglial clearance of FN1 deposits.

[0145] In some embodiments, the subject is selected from a population with elevated vascularpathology burden, e.g., a Hispanic group.

[0146] In some embodiments, treatment delays the onset or progression of Alzheimer’s diseasein APOEε4 carriers.

[0147] A method of identifying impaired vascular protective signaling in an individual withAlzheimer’s disease, wherein the method comprises: providing or obtaining a cerebrospinal fluid (CSF) sample from the individual; measuring fibronectin (FN1) expression and vascular endothelial growth factor A (VEGFA) expression in the sample; determining the co-expression relationship between FN1 and VEGFA; identifying the individual as an APOEε4 carrier with impaired vascular protection when VEGFA expression does not increase with FN1 expression.

[0148] In some embodiments, the individual is an APOEε3 carriers, in whom VEGFAexpression increases with FN1 expression. 38 4929-9458-4176v.2

[0149] In some embodiments, VEGFA upregulation in APOEε3 carriers serves as acompensatory protective response to FN1 deposition, but this protective response is absent in APOEε4 carriers.

[0150] In some embodiments, impaired VEGFA co-expression in APOEε4 carriers indicateshigher susceptibility to blood–brain barrier dysfunction and vascular leakage.

[0151] In some embodiments, therapeutic administration of VEGFA, VEGFR agonists, orVEGFA-inducing agents is used to restore vascular protective signaling in APOEε4 carriers.

[0152] In some embodiments, FN1 and / or VEGFA measurement is performed byimmunoassay, mass spectrometry, or RNA-sequencing–based biomarker analysis.

[0153] A method of treating blood–brain barrier (BBB) dysfunction in a subject carryingAPOEε4, the method comprising administering to the subject (a) an inhibitor of β1 integrin and / or a focal adhesion kinase (FAK) inhibitor, and (b) a VEGFA agonist or IGF1 mimetic, so as to reduce BBB dysfunction.,

[0154] In some embodiments, (a) restores growth factor signaling in APOEε4 astrocytes. Insome embodiments, (b) enhances neurovascular protection. In some embodiments, (a) and (b) synergistically reverses APOEε4-driven extracellular matrix–mediated BBB breakdown.

[0155] In some embodiments, the inhibitor of β1 integrin is a small molecule. In someembodiments, the inhibitor of beta1 integrin comprises ATN-161, CLT 38643, or JSM-6427. In some embodiments, the inhibitor of β1 integrin is an inhibitor of alpha5beta1 integrin.

[0156] In some embodiments, the FAK inhibitor is a small molecule. In some embodiments,the FAK inhibitor comprises PF573228, Defactinib VS-6063 / PF-04554878, GSK2256098, CEP- 37440, BI 853520 or VS-4718 (PND-1186).

[0157] In embodiments, the combination is administered into the CNS. In some embodiments,the combination is administered in a manner such that the combination enters the CNS or the vasculature of the CNS. In some embodiments, (a) and (b) are administered simultaneously, e.g., as part of a single pharmaceutical composition. In some embodiments, (a) and (b) are administered separately.

[0158] A method for treating, or for delaying or reducing the development of, aneurodegenerative disease, comprising the step of administering to a subject in need thereof an effective amount of a compound having the following structure: 39 4929-9458-4176v.2wherein atom Z is present or absent, and when present is a carbon bound to A, and when absent the two N atoms of the ring are directly bound to each other, wherein when Z is present: bonds ^^^^^^^^^^, and ^ are present, and bonds ^ and ^ are absent; A has the following structure: ,E is CH2, O, or S; and R9are R10are each, independently, H, optionally substituted or unsubstituted C1- C6alkyl, C3-C12cycloalkyl, aromatic, or alkylaromatic, wherein R9are R10are each independently optionally substituted or unsubstituted with halo, -OH, -SH, alkyl, alkyl-OH, alkyl-SH, alkyl-NH2, - O-alkyl, -S-alkyl, or -NHx(alkyl)ywhere x + y = 2; X is a C atom; Y is O or NR6; wherein when Y is O, R1is H, an optionally substituted or unsubstituted C1-C6 alkyl, aromatic, alkyl carbonyl, alkyl carboxy, alkyl N-amido, or alkylaromatic group, 40 4929-9458-4176v.2wherein R1is optionally substituted or unsubstituted with halo, -OH, -SH, C1-C6 alkyl, C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S- alkyl, -NHx(alkyl)y where x + y = 2, -C(O)NH2, or -SO2NH2; and wherein when Y is NR6, R1is H, alkyl, aromatic, alkyl carbonyl, alkyl carboxy, alkyl N-amido, or alkylaromatic; R6is H, alkyl, alkyl carbonyl, alkyl N-amido, alkylaromatic, or C(O)R16; or , wherein R16is C1-C6 alkyl; R1and R6or R16may, together, form a 5- to 8-membered optionally substitututed or unsubstituted unsaturated heterocycle, wherein the heterocycle is optionally substituted with R7wherein R7is a C1-C6 alkyl or an alkylamide and R2, R3, R4, and R5are, independently, H, halo, -OH, alkyl or -O-alkyl; wherein when Z is absent: bonds ^ and ^ are present, and bonds ^^^^^^^^^^^^and ^ are absent; X is a C or N atom; R2is H or a optionally substituted or unsubstituted benzoyl; R3, R4, and R5are H; Y is C(O)NHR8, and R8is H, C1-C6 alkyl or alkyl aromatic; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof. 41 4929-9458-4176v.2

[0159] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Z is present.

[0160] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R9is H or an optionally substituted or unsubstituted C1-C6alkyl.

[0161] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is H or an optionally substituted or unsubstituted C3-C12cycloalkyl, aromatic, or alkylaromatic group.

[0162] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the cycloalkyl, alkylaromatic, or aromatic group is a monocycle or bicycle.

[0163] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is an aromatic group.

[0164] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is an optionally substituted or unsubstituted phenyl or heteroaromatic ring.

[0165] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is a heteroaromatic group containing, selected independently, at least one of N, O, or S.

[0166] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: 42 4929-9458-4176v.2.

[0167] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Y is O.

[0168] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

[0169] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, E is S.

[0170] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1is an optionally substituted or unsubstituted mono- or bicyclic alkylaromatic group.

[0171] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1is an optionally substituted or unsubstituted monocyclic alkylaromatic group.

[0172] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1is an optionally 43 4929-9458-4176v.2substituted or unsubstituted alkylaromatic, wherein the alkylaromatic is a carbocycle or a heterocycle.

[0173] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1is an optionally substituted or unsubstituted alkylphenyl group.

[0174] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1 has the following structure: wherein n = 1-4; andwherein R11, R12, R13, R14, R15are each, independently H, -C(O)NH2, or -SO2NH2.

[0175] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, n is 2.

[0176] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R13is -SO2NH2.

[0177] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R9is C1-C4alkyl.

[0178] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R9is ethyl.

[0179] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is an optionally substituted or unsubstituted benzyl. 44 4929-9458-4176v.2

[0180] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is substituted with C1- C4alkyl.

[0181] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is a methyl benzyl.

[0182] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is a p-methyl benzyl.

[0183] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R4and R5are each H.

[0184] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2and R3are C1-C6 -O- alkyl.

[0185] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2and R3are methoxy.

[0186] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

[0187] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R9is H. 45 4929-9458-4176v.2

[0188] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is an optionally substituted or unsubstituted C4-C8cycloalkyl.

[0189] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is a cyclopentane.

[0190] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2, R3,R4and R5are H.

[0191] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound exists as a homodimer linked by their respective R1groups.

[0192] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound exists as a homodimer linked by their respective R1groups, wherein R1is C1-C6 alkyl.

[0193] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound exists as a homodimer linked by their respective R1groups, wherein R1is is n-propenyl.

[0194] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .46 4929-9458-4176v.2

[0195] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Y is NR6.

[0196] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1and R6together form a 5- to 8-membered heterocycle.

[0197] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1and R6together form a 5- to 8-membered unsaturated oxo- or thioxo-heterocycle.

[0198] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1and R6together form a 5- to 8-membered unsaturated oxo-heterocycle.

[0199] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R6is C(O)R16.

[0200] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1and R16together form a 5- to 8-membered unsaturated heterocycle.

[0201] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1and R16together form a 5-membered unsaturated oxo-heterocycle.

[0202] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: 47 4929-9458-4176v.2.

[0203] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R7is a C1-C6 alkyl or an alkylamide.

[0204] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R7is an alkylamide.

[0205] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R7has the following structure: .

[0206] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, wherein n = 1-4.

[0207] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R17is an optionally substituted or unsubstituted alkylaromatic.

[0208] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R17is an optionally substituted or unsubstituted ethyl benzene. 48 4929-9458-4176v.2

[0209] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, E is S.

[0210] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R9is H.

[0211] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is an optionally substituted or unsubstituted phenyl group.

[0212] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is a substituted phenyl group with one or more -O-alkyl groups.

[0213] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is a phenyl group substituted with one or more methoxy groups.

[0214] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is a 2,5-dimethoxy phenyl group.

[0215] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2, R3, R4and R5are H.

[0216] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: 49 4929-9458-4176v.2.

[0217] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Z is absent.

[0218] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, X is N.

[0219] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Y is C(O)NHR8.

[0220] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereofthe compound has the following structure: .

[0221] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R8is H, C1-C6 alkyl, or alkyl aromatic. 50 4929-9458-4176v.2

[0222] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R8is alkyl aromatic.

[0223] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R8is alkyl phenyl.

[0224] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R8is 3-phenyl propane.

[0225] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2is an optionally substituted or unsubstituted benzoyl.

[0226] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2is a benzoyl substituted with halo, -OH, -SH, C1-C4alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, or - NHx(alkyl)y where x + y = 2.

[0227] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2is a C1-C4alkyl- substituted benzoyl.

[0228] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2is a methyl-substituted benzoyl.

[0229] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2is 2-methyl- benzoyl group.

[0230] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: 51 4929-9458-4176v.2.

[0231] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2is a halo-substituted benzoyl.

[0232] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2is a flourine-substituted benzoyl.

[0233] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2is 2-fluoro- benzoyl.

[0234] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

[0235] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2is a chloro-substituted benzoyl.

[0236] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2is 3-chloro- benzoyl. 52 4929-9458-4176v.2

[0237] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

[0238] A method for treating, or for delaying or for reducing the development of, aneurodegenerative disease, comprising the step of administering to a subject in need thereof an effective amount of a compound having the following structure:R17is an optionally substituted or unsubstituted mono or bicyclic cycloalkyl or aromatic group; wherein R17is optionally substituted with halo, -OH, -SH, C1-C4alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, or -NHx(alkyl)y where x + y = 2. R18is an optionally substituted or unsubstituted mono or bicyclic cycloalkyl or aromatic group; wherein R18is optionally substituted with halo, -OH, -SH, C1-C4 alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, or -NHx(alkyl)y where x + y = 2. or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof. 53 4929-9458-4176v.2

[0239] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R17is an optionally substituted or unsubstituted mono or bicyclic carbocyclic aromatic or heteroaromatic.

[0240] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R17is an optionally substituted or unsubstituted bicyclic carbocyclic aromatic or heteroaromatic.

[0241] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R17is an optionally substituted or unsubstituted bicyclic carbocyclic aromatic.

[0242] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R17is an optionally substituted or unsubstituted naphthyl group.

[0243] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R17is an optionally substituted or unsubstituted 1-naphthyl group.

[0244] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R18is an optionally substituted or unsubstituted mono- or bicyclic carbocyclic aromatic or heteroaromatic.

[0245] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R18is an optionally substituted or unsubstituted monocyclic carbocyclic aromatic or heteroaromatic.

[0246] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R18is an optionally substituted or unsubstituted phenyl group. 54 4929-9458-4176v.2

[0247] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R18is a -OH substituted phenyl group.

[0248] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R18is 3-hydroxy phenyl.

[0249] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

[0250] A method for treating, or for delaying or for reducing the development of, aneurodegenerative disease, comprising the step of administering to a subject in need thereof an effective amount of a compound having the following structure:wherein R19and R20are, independently, H or C1-C12 alkyl;R19and R20may together form a 5-12-membered optionally substituted or unsubstituted saturated or unsaturated mono- or bicyclic heterocycle, wherein R19and R20are optionally independently substituted with halo, - OH, -SH, C1-C4alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S- alkyl, -NHx(alkyl)ywhere x + y = 2, or -C(O)-O-alkyl; 55 4929-9458-4176v.2R21is a 5- or 6-membered optionally substituted or unsubstituted aromatic group ; or wherein R21is optionally independently substituted with halo, -OH, -SH, C1-C4 alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, -NHx(alkyl)y where x + y = 2, or -C(O)-O-alkyl; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0251] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R19and R20together form a 5-12-membered optionally substituted or unsubstituted saturated or unsaturated mono- or bicyclic heterocycle.

[0252] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R19and R20together form a 5-12-membered optionally substituted or unsubstituted saturated or unsaturated bicyclic heterocycle.

[0253] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R19and R20together form a 5-12-membered optionally substituted or unsubstituted aliphatic bicyclic heterocycle.

[0254] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R19and R20together form a 5-12-membered unsaturated optionally substituted or unsubstituted bicyclic heterocycle comprising a piperidine.

[0255] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R19and R20together form a 5-12-membered unsaturated optionally substituted or unsubstituted bicyclic heterocycle comprising a dixolane. 56 4929-9458-4176v.2

[0256] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R19and R20together form a 5-12-membered unsaturated optionally substituted or unsubstituted spiro-bicyclic heterocycle.

[0257] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R19and R20together form an optionally substituted or unsubstituted spiro-piperidine-dioxolane.

[0258] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R19and R20together form an optionally substituted or unsubstituted spiro-piperidine-4,2’-dioxolane.

[0259] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R21is a 5- or 6-membered optionally substituted or unsubstituted aromatic or heteroaromatic group.

[0260] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R21is a 5- or 6-membered optionally substituted or unsubstituted heteroaromatic group.

[0261] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R21is a 5- or 6-membered optionally substituted or unsubstituted heteroaromatic group, containing, selected independently, at least one of N, O, or S.

[0262] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R21is a 5-membered optionally substituted or unsubstituted heteroaromatic group, containing, selected independently, at least one of N, O, or S.

[0263] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R21is a 5-membered 57 4929-9458-4176v.2optionally substituted or unsubstituted heteroaromatic group, containing, selected independently, at least one of N or O.

[0264] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R21is a 5-membered optionally substituted or unsubstituted heteroaromatic group, containing, selected independently, at least one of each N and O.

[0265] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R21is an optionally substituted or unsubstituted oxazole.

[0266] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R21is an optionally substituted or unsubstituted 5-oxazolyl group.

[0267] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R21is a carboxy-substituted 5-oxazolyl group.

[0268] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R21is an alkyl carboxy- substituted 5-oxazolyl group.

[0269] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R21is an alkyl-4- oxazolecarboxylate.

[0270] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R21is a methyl-5-oxazole- 4-carboxylate. 58 4929-9458-4176v.2

[0271] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

[0272] A method for treating, or for reducing the development of, a neurodegenerative disease,comprising the step of administering to a subject in need thereof an effective amount of a compound is a human integrin antagonist.

[0273] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

[0274] In some embodiments, human integrin antagonist is an inhibitor of β1 integrin. Insome embodiments, human integrin antagonist comprises ATN-161, CLT 38643, or JSM-6427. In some embodiments, the inhibitor of β1 integrin is an inhibitor of alpha5beta1 integrin.

[0275] In some embodiments, treating the disease comprises improving one or more cognitiveperformance measures. 59 4929-9458-4176v.2

[0276] In some embodiments, the method reduces vascular dysfunction, reduces inflammationin the brain, or increases amyloid plaque clearance in the brain.

[0277] In some embodiments, the neurodegenerative disease is Alzheimer’s disease.

[0278] In some embodiments, the neurodegenerative disease is an APOE^4 related pathology.

[0279] In some embodiments, the subject is an APOE^4 carrier.

[0280] In some embodiments, the methods further comprise identifying, or having identified,the subject as an APOE^4 carrier prior to treating.

[0281] In some embodiments, the method further comprises identifying, or having identified,the subject as having a blood level of P-tau271 of 0.39pg / ml or greater prior to treating. In some embodiments, further comprising identifying, or having identified, the subject as having a blood level of P-tau271 of 0.4pg / ml or greater prior to treating. In some embodiments, the method further comprises identifying, or having identified, the subject as having a blood level of P-tau271 of about 0.39pg / ml or greater prior to treating.

[0282] In some embodiments, the subject has not shown symptoms of AD and the method isfor delaying the development of the neurodegenerative disease.

[0283] In some embodiments, the treatment increases the pre-clinical phase length of theneurodegenerative disease.

[0284] In some embodiments, the neurodegenerative disease is Cerebral Amyloid Angiopathy(CAA), but evidence also suggests it increases the risk for other neurological conditions including Parkinson's disease, amyotrophic lateral sclerosis (ALS), or frontotemporal dementia (FTD).

[0285] A compound having the following structure:60 4929-9458-4176v.2atom Z is present or absent, and when present is a carbon bound to A, and when absent the two N atoms of the ring are directly bound to each other, wherein when Z is present: bonds ^^^^^^^^^^, and ^ are present, and bonds ^ and ^ are absent; A has the following structure: ,E is CH2, O, or S; and R9are R10are each, independently, H, optionally substituted or unsubstituted C1- C6alkyl, C3-C12cycloalkyl, aromatic, or alkylaromatic, wherein R9are R10are each independently optionally substituted or unsubstituted with halo, -OH, -SH, alkyl, alkyl-OH, alkyl-SH, alkyl-NH2, - O-alkyl, -S-alkyl, or -NHx(alkyl)ywhere x + y = 2;-; X is a C atom; Y is NR6or O; wherein when Y is O, R1is H, an optionally substituted or unsubstituted C1-C6 alkyl, aromatic, alkyl carbonyl, alkyl carboxy, alkyl N-amido, or alkylaromatic group, 61 4929-9458-4176v.2wherein R1is optionally substituted or unsubstituted with halo, -OH, -SH, C1-C6 alkyl, C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S- alkyl, -NHx(alkyl)y where x + y = 2, -C(O)NH2, or -SO2NH2; and wherein when Y is NR6, R1is H, alkyl, alkyl carbonyl, alkyl carboxy, alkyl N-amido, or alkylaromatic; R6is H, alkyl, alkyl carbonyl, alkyl N-amido, alkylaromatic or C(O)R16; or wherein R16is C1-C6alkyl; R1and R6or R16may, together, form an optionally substituted or unsubstituted unsaturated heterocycle, wherein the heterocycle is optionally substituted with R7, wherein R7is a C1-C6 alkyl or an alkylamide;; and R2, R3, R4, and R5are, independently, H, -OH, -SH, C1-C4alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, or -NHx(alkyl)y where x + y = 2; wherein when E is -S: at least one of R2, R3, R4, and R5are -OH, -SH, C1-C4alkyl, -C1-C4-OH, C1-C4- SH, C1-C4-NH2, -S-alkyl, or -NHx(alkyl)y where x + y = 2; and up to three of R2, R3, R4, and R5are -H wherein when Z is absent: bonds ^ and ^ are present, and bonds ^^^^^^^^^^, and ^ are absent; X is a C or N atom; R3, R4, and R5are H; Y is C(O)NHR8, wherein R8is H, C1-C6 alkyl or alkyl aromatic; 62 4929-9458-4176v.2wherein when X is a C atom: R2is H, or an optionally substituted or unsubstituted benzoyl, benzyl or naphthoyl; R8is H, or a substituted or unsubstituted C1-C6 alkyl, aromatic, or alkyl aromatic; wherein when X is an N atom: R2is H, poly-substituted benzoyl, or optionally substituted or unsubstituted benzyl or naphthoyl; R8is H, or a substituted or unsubstituted C1-C6alkyl, aromatic, or substituted alkyl aromatic; or pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

[0286] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Z is present.

[0287] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R9is H or an optionally substituted or unsubstituted C1-C6 alkyl.

[0288] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is H or an optionally substituted or unsubstituted C4-C8cycloalkyl, aromatic, or alkylaromatic.

[0289] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the cycloalkyl, aromatic, or alkylaromatic is optionally substituted with halo, -OH, -SH, C1-C4alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, or -NHx(alkyl)ywhere x + y = 2.

[0290] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the aromatic is a carbocyclic or heteroaromatic ring containing, selected independently, at least one of N, O, or S. 63 4929-9458-4176v.2

[0291] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the cycloalkyl or aromatic is a monocycle or bicycle.

[0292] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .some the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Y is O.

[0294] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

[0295] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1is an optionally substituted or unsubstituted C1-C11 alkyl, alkylaromatic, aromatic, or alkylcarboxamide, or alkylcarboxalate. 64 4929-9458-4176v.2

[0296] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the alkylaromatic has the following structure: 4; andwherein R13, R14, R15are each, independently H, -C(O)NH2, or -SO2NH2.

[0297] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound may exist as a dimer.

[0298] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound may exist as a dimer.linked by the respective R1groups.

[0299] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound may exist as a dimer linked by the respective R1, wherein the R1groups are C1-C6alkyl.

[0300] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Y is NR6.

[0301] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: 65 4929-9458-4176v.2.

[0302] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R6is H, alkyl, alkyl carbonyl, alkyl N-amido, alkylaromatic or C(O)R16.

[0303] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1and R6together form unsaturated heterocycle B.

[0304] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R7is C1-C6alkyl or an alkylcarboxamide.

[0306] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R7has the following structure: 66 4929-9458-4176v.2wherein n = 1-4; and R16is an optionally substituted or unsubstituted C1-C11alkyl or alkylaromatic.

[0307] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Z is absent.

[0308] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, X is C or N.

[0309] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Y is C(O)NHR8.

[0310] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: H N .

[0311] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, X is C.

[0312] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2is H or an optionally substituted or unsubstituted benzoyl, benzyl or naphthoyl. 67 4929-9458-4176v.2

[0313] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R8is H or a substituted or unsubstituted C1-C6 alkyl or substituted alkyl aromatic.

[0314] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, X is N.

[0315] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R2is H, poly-substituted benzoyl, or optionally substituted or unsubstituted benzyl or naphthoyl.

[0316] In some embodiments in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R8is H or a substituted or unsubstituted C1-C6alkyl or substituted alkyl aromatic.

[0317] A pharmaceutical composition comprising a compound described herein and apharmaceutical excipient.

[0318] A method for treating a neurodegenerative disease, comprising the step ofadministering to a subject in need thereof an effective amount of compound described herein or an effective amount of a pharmaceutical composition comprising a compound described herein.

[0319] In embodiments, treating the disease comprises improving one or more cognitiveperformance measures.

[0320] A method for preventing neurodegenerative disease, comprising the step ofadministering to a subject in need thereof an effective amount of compound described herein or an effective amount of a pharmaceutical composition comprising a compound described herein.

[0321] In embodiments of the methods, the neurodegenerative disease is Alzheimer’sdisease.

[0322] In some embodiments, a pharmaceutically acceptable salt of any of the abovecompounds is provided. In some embodiments, an ester of any of the above compounds is provided. In some embodiments, an enantiomer of any of the above compounds is provided. 68 4929-9458-4176v.2

[0323] In some embodiments, a package or composition comprising any of the abovecompounds is provided.

[0324] In some embodiments, a salt of the compound of the present invention is used in anyof the above methods, uses, packages or compositions. In some embodiments, a pharmaceutically acceptable salt of the compound of the present invention is used in any of the above methods, uses, packages or compositions. In some embodiments, an ester of the compound of the present invention is used in any of the above methods, uses, packages or compositions. Any of the above compounds may be used in any of the disclosed methods, uses, packages or pharmaceutical compositions.

[0325] Any of the compounds used in the disclosed methods, uses, packages or pharmaceuticalcompositions may be replaced with any other compound disclosed in the present invention. Any of the above generic compounds may be used in any of the disclosed methods, uses, packages or compositions.

[0326] Except where otherwise specified, where the structure of a compound of this inventionincludes an asymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, a scalemic mixture or isolated single enantiomer. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis, such as those described in "Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.

[0327] The subject invention is also intended to include all isotopes of atoms occurring on thecompounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

[0328] It will be noted that any notation of a carbon in structures throughout this application,when used without further notation, are intended to represent all isotopes of carbon, such as 12C, 69 4929-9458-4176v.213C, or 14C. Furthermore, any compounds containing 13C or 14C may specifically have the structure of any of the compounds disclosed herein.

[0329] It will also be noted that any notation of a hydrogen in structures throughout thisapplication, when used without further notation, are intended to represent all isotopes of hydrogen, such as 1H, 2H (D), or 3H (T). Furthermore, any compounds containing 2H or 3H may specifically have the structure of any of the compounds disclosed herein.

[0330] Isotopically-labeled compounds can generally be prepared by conventional techniquesknown to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.

[0331] Deuterium (2H or D) is a stable, non-radioactive isotope of hydrogen and has an atomicweight of 2.0144. Hydrogen atom in a compound naturally occurs as a mixture of the isotopes 1H (hydrogen or protium), D (2H or deuterium), and T (3H or tritium). The natural abundance of deuterium is 0.0156%. Thus, a compound with a level of deuterium at any site of hydrogen atom in the compound that has been enriched to be greater than its natural abundance of 0.0156%, is novel over its non-enriched counterpart.

[0332] In the compounds used in the method of the present invention, the substituents may besubstituted or unsubstituted, unless specifically defined otherwise.

[0333] In the compounds used in the method of the present invention, alkyl, heteroalkyl,monocycle, bicycle, aryl, heteroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

[0334] It is understood that substituents and substitution patterns on the compounds used inthe method of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

[0335] In choosing the compounds used in the method of the present invention, one of ordinaryskill in the art will recognize that the various substituents, e.g., R1, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity. 70 4929-9458-4176v.2

[0336] As used herein, "alkyl" is intended to include both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number of carbon atoms. Thus, C1- Cn as in “C1–Cn alkyl" is defined to include groups having 1, 2......, n-1 or n carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, isopropyl, isobutyl, sec-butyl and so on. An embodiment can be C1-C12 alkyl, C2-C12 alkyl, C3-C12 alkyl, C4-C12 alkyl and so on. An embodiment can be C1-C8 alkyl, C2-C8 alkyl, C3-C8 alkyl, C4-C8 alkyl and so on. “Alkoxy" represents an alkyl group as described above attached through an oxygen bridge.

[0337] The term "alkenyl" refers to a non-aromatic hydrocarbon radical, straight or branched,containing at least 1 carbon to carbon double bond, and up to the maximum possible number of non-aromatic carbon-carbon double bonds may be present. Thus, C2-Cn alkenyl is defined to include groups having 1, 2...., n-1 or n carbons. For example, "C2-C6 alkenyl" means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and at least 1 carbon-carbon double bond, and up to, for example, 3 carbon-carbon double bonds in the case of a C6 alkenyl, respectively. Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl. As described above with respect to alkyl, the straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated. An embodiment can be C2-C12 alkenyl or C2-C8 alkenyl.

[0338] The term "alkynyl" refers to a hydrocarbon radical straight or branched, containing atleast 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present. Thus, C2-Cn alkynyl is defined to include groups having 1, 2...., n-1 or n carbons. For example, "C2-C6 alkynyl" means an alkynyl radical having 2 or 3 carbon atoms, and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms, and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms, and up to 3 carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl and butynyl. As described above with respect to alkyl, the straight or branched portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated. An embodiment can be a C2-Cn alkynyl. An embodiment can be C2-C12 alkynyl or C3-C8 alkynyl.

[0339] As used herein, “halo” or “halogen” refers to a substituent from the halogen group andincludes fluorine, chlorine, bromine, and iodine. Each of these can be referred to similarly as substituents as fluoride, chloride, bromide, and iodide when these each replace a hydrogen atom 71 4929-9458-4176v.2and are collectively referred to as “halides”. When used to describe a group they are attached to they may be referred to as fluoro, chloro, bromo, and iodo.

[0340] As used herein, "haloalkyl" includes alkyl groups as described above wherein one ormore bonds to hydrogen contained therein are replaced by a bond to a halogen. In some embodiments, C1-C12 haloalkyl or C1-C6 haloalkyl. C1-Cn as in “C1–Cn haloalkyl" is defined to include groups having 1, 2, ...., n-1 or n carbons in a linear or branched arrangement (e.g. C1– C2 haloalkyl, C1–C3 haloalkyl, C1–C4 haloalkyl, C1–C5 haloalkyl, or C1–C6 haloalkyl) For example, C1–C6, as in "C1–C6 haloalkyl" is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched alkyl arrangement wherein a hydrogen contained therein is replaced by a bond to a halogen.

[0341] As used herein, "hydroxyalkyl" includes alkyl groups as described above wherein oneor more bonds to hydrogen contained therein are replaced by a bond to an -OH group. In some embodiments, C1-C12 hydroxyalkyl or C1-C6 hydroxyalkyl. C1-Cn as in “C1–Cn hydroxy alkyl" is defined to include groups having 1, 2, ...., n-1 or n carbons in a linear or branched arrangement (e.g. C1–C2 hydroxyalkyl, C1–C3 hydroxyalkyl, C1–C4 hydroxyalkyl, C1–C5 hydroxyalkyl, or C1–C6 hydroxyalkyl) For example, C1–C6, as in "C1–C6 hydroxyalkyl" is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched alkyl arrangement wherein a hydrogen contained therein is replaced by a bond to an -OH group.

[0342] As used herein, "heteroalkyl" includes both branched and straight-chain saturatedaliphatic hydrocarbon groups having the specified number of carbon atoms and at least 1 heteroatom within the chain or branch.

[0343] As used herein, "monocycle" includes any stable polyatomic carbon ring of up to 10atoms and may be unsubstituted or substituted. Examples of such non-aromatic monocycle elements include but are not limited to: cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and piperidine. Examples of such aromatic monocycle elements include but are not limited to: phenyl and pyridine.

[0344] As used herein, "bicycle" includes any stable polyatomic carbon ring of up to 10 atomsthat is fused or directly bound to a polyatomic carbon ring of up to 10 atoms with each ring being independently unsubstituted or substituted. Examples of such non-aromatic bicycle elements include but are not limited to: decalin and decahydronaphthalene. Examples of such aromatic bicycle elements include but are not limited to: biphenyl and naphthalene. 72 4929-9458-4176v.2

[0345] As used herein, “spiro” or “spirocyclic” refers to any polycyclic groups which share asingle atom. This is in contrast to fused polycyclic groups which share at least two atoms.

[0346] As used herein, "aryl" or “aromatic” is intended to mean any stable monocyclic,bicyclic or polycyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic, and may be unsubstituted or substituted. Examples of such aryl elements include but are not limited to: phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, phenanthryl, anthryl or acenaphthyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.

[0347] The term "heteroaryl" or “heteroaromatic”, as used herein, represents a stablemonocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Bicyclic aromatic heteroaryl groups include phenyl, pyridine, pyrimidine or pyridazine rings that are (a) fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (d) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N or S. Heteroaryl groups within the scope of this definition include but are not limited to: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl, hexahydroazepinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, furanyl, thienyl, 73 4929-9458-4176v.2benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetra-hydroquinoline. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively. If the heteroaryl contains nitrogen atoms, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.

[0348] As used herein, “alkylaromatic” or “alkylaryl” includes alkyl groups as describedabove wherein one or more bonds to hydrogen contained therein are replaced by a bond to an aromatic group. In some embodiments, C1-C12 alkylaromatic or C1-C6 alkylaromatic. C1-Cn as in “C1–Cn alkylaromatic" is defined to include groups having 1, 2, ...., n-1 or n carbons in a linear or branched arrangement (e.g. C1–C2 alkylaromatic, C1–C3 alkylaromatic, C1–C4 alkylaromatic, C1–C5 alkylaromatic, or C1–C6 alkylaromatic) For example, C1–C6, as in "C1–C6 alkylaromatic" is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched alkyl arrangement wherein a hydrogen contained therein is replaced by a bond to an aromatic group.

[0349] The term "heterocycle", “heterocyclyl” or “heterocyclic” refers to a mono- or poly-cyclic ring system which can be saturated or contains one or more degrees of unsaturation and contains one or more heteroatoms. Preferred heteroatoms include N, O, and / or S, including N- oxides, sulfur oxides, and dioxides. Preferably the ring is three to ten-membered and is either saturated or has one or more degrees of unsaturation. The heterocycle may be unsubstituted or substituted, with multiple degrees of substitution being allowed. Such rings may be optionally fused to one or more of another "heterocyclic" ring(s), heteroaryl ring(s), aryl ring(s), or cycloalkyl ring(s). Examples of heterocycles include, but are not limited to, tetrahydrofuran, pyran, 1,4- dioxane, 1,3-dioxane, piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, 1,3-oxathiolane, and the like.

[0350] The term “ester” or “carboxy” is intended to a mean an organic compound containingthe R-O-CO-R’ group.

[0351] The term “amide” “amido” is intended to mean an organic compound containing theR-N-CO-R’ group.

[0352] As used herein, “alkylcarbonyl” or “carbonyl” includes alkyl groups as described abovewherein two bonds to hydrogen contained therein are replaced by two bonds to the same oxygen 74 4929-9458-4176v.2atom. In some embodiments, C1-C12 alkylcarbonyl or C1-C6 alkylcarbonyl C1-Cn as in “C1–Cn alkylcarbonyl" is defined to include groups having 1, 2, ...., n-1 or n carbons in a linear or branched arrangement (e.g. C1–C2 alkylcarbonyl, C1–C3 alkylcarbonyl, C1–C4 alkylcarbonyl, C1–C5 alkylcarbonyl, or C1–C6 alkylcarbonyl) For example, C1–C6, as in "C1–C6 alkylcarbonyl" is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched alkyl arrangement wherein two hydrogens contained therein are replaced by two bond to the same oxygen atom.

[0353]

[0354] The term “oxo” is intended to mean an organic compound containing a carbonyl, i.e. acarbon atom that has two bonds to an oxygen atom. Oxo groups are substituents wherein two bonds to hydrogens contained therein are replaced by two bonds to the same oxygen atom.

[0355] The term “thioxo” is intended to mean an organic compound containing a thionyl, i.e.a carbon atom that has two bonds to a sulfur atom. Thioxo groups are substituents wherein two bonds to hydrogens contained therein are replaced by two bonds to the same sulfur atom.

[0356] The term “substitution”, “substituted” and “substituent” refers to a functional group asdescribed above in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms, provided that normal valencies are maintained and that the substitution results in a stable compound. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Examples of substituent groups include the functional groups described above, and halogens (i.e., F, Cl, Br, and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl, n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, such as methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such as phenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) and p-trifluoromethylbenzyloxy (4- trifluoromethylphenylmethoxy); heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto; sulfanyl groups, such as methylsulfanyl, ethylsulfanyl and propylsulfanyl; cyano; amino groups, such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; and carboxyl. Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally. By independently substituted, it is meant that the (two or more) substituents can be the same or different. 75 4929-9458-4176v.2

[0357] The compounds used in the method of the present invention may be prepared bytechniques known in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.

[0358] Compounds used in the method of the present invention may be prepared by techniquesdescribed in Vogel’s Textbook of Practical Organic Chemistry, A.I. Vogel, A.R. Tatchell, B.S. Furnis, A.J. Hannaford, P.W.G. Smith, (Prentice Hall) 5th Edition (1996), March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5th Edition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.

[0359] The various R groups attached to the aromatic rings of the compounds disclosed hereinmay be added to the rings by standard procedures, for example those set forth in Advanced Organic Chemistry: Part B: Reactions and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), the content of which is hereby incorporated by reference.

[0360] Another aspect of the invention comprises a compound used in the method of thepresent invention as a pharmaceutical composition.

[0361] As used herein, the term “pharmaceutically active agent” means any substance orcompound suitable for administration to a subject and furnishes biological activity or other direct effect in the treatment, cure, mitigation, diagnosis, or prevention of disease, or affects the structure or any function of the subject. Pharmaceutically active agents include, but are not limited to, substances and compounds described in the Physicians’ Desk Reference (PDR Network, LLC; 64th edition; November 15, 2009) and “Approved Drug Products with Therapeutic Equivalence Evaluations” (U.S. Department Of Health And Human Services, 30th edition, 2010), which are hereby incorporated by reference. Pharmaceutically active agents which have pendant carboxylic acid groups may be modified in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Where a pharmaceutically active agent does not possess a carboxylic acid group, the ordinarily skilled artisan will be able to design and incorporate a carboxylic acid group into the pharmaceutically active agent where esterification may subsequently be carried out so 76 4929-9458-4176v.2long as the modification does not interfere with the pharmaceutically active agent’s biological activity or effect.

[0362] The compounds used in the method of the present invention may be in a salt form. Asused herein, a “salt” is a salt of the instant compounds which has been modified by making acid or base salts of the compounds. In the case of compounds used to treat an infection or disease caused by a pathogen, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols; alkali or organic salts of acidic residues such as carboxylic acids. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the alkali metal salts, sodium, potassium or lithium. The term "pharmaceutically acceptable salt" in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci.66:1-19).

[0363] As used herein, "treating" means inhibiting, slowing, halting, or reversing theprogression of a disease or infection. Treating may also mean improving one or more symptoms of a disease or infection.

[0364] The compounds used in the method of the present invention may be administered invarious forms, including those detailed herein. The treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the disease in conjunction with one or more of the instant compounds. This combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These can be 77 4929-9458-4176v.2administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.

[0365] The dosage of the compounds administered in treatment will vary depending uponfactors such as the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.

[0366] A dosage unit of the compounds used in the method of the present invention maycomprise a single compound or mixtures thereof with additional antibacterial agents. The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection, topical application, or other methods, into or onto a site of infection, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

[0367] The compounds used in the method of the present invention can be administered inadmixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for 78 4929-9458-4176v.2example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

[0368] Techniques and compositions for making dosage forms useful in the present inventionare described in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

[0369] Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents,flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

[0370] The compounds used in the method of the present invention may also be administeredin the form of liposome delivery systems, such as small unilamellar vesicles, large unilamallar 79 4929-9458-4176v.2vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue-targeted emulsions.

[0371] The compounds used in the method of the present invention may also be coupled tosoluble polymers as targetable drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.

[0372] Gelatin capsules may contain the active ingredient compounds and powdered carriers,such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

[0373] For oral administration in liquid dosage form, the oral drug components are combinedwith any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non- effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.

[0374] Liquid dosage forms for oral administration can contain coloring and flavoring toincrease patient acceptance. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain 80 4929-9458-4176v.2a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

[0375] The compounds used in the method of the present invention may also be administeredvia transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.

[0376] Parenteral and intravenous forms may also include minerals and other materials tomake them compatible with the type of injection or delivery system chosen.

[0377] Each embodiment disclosed herein is contemplated as being applicable to each of theother disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

[0378] “And / or” as used herein, for example, with option A and / or option B, encompasses theseparate embodiments of (i) option A, (ii) option B, and (iii) option A plus option B.

[0379] All combinations of the various elements described herein are within the scope of theinvention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0380] Definitions: The terms used in this specification generally have their ordinary meaningsin the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of 81 4929-9458-4176v.2the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments.

[0381] The term “subject” as used in this application means a mammal. Mammals includecanines, felines, rodents, bovine, equines, porcines, ovines, and primates including humans. Thus, the invention can be used in human medicine or also in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical applications. In a preferred embodiment the subject is a human.

[0382] The terms “therapeutically effective amount” or "amount effective to" encompasses,unless otherwise indicated, an amount sufficient to ameliorate or inhibit a symptom or sign of the medical condition. An effective amount for a particular subject may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects. An effective amount can be the maximal dose or dosing protocol that avoids significant side effects or toxic effects.

[0383] The term “about” or “approximately” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0384] Experiments

[0385] The inventors have identified a rare loss of function variant in FN1 gene thatsignificantly lowers the risk of AD in individuals carrying one or more APOE^4 alleles and is accompanied by a reduction of FN1 at the BBB[9]. Conversely, individuals who carry the APOE^4 allele but do not develop dementia exhibit lower FN1 protein levels that are comparable to those 82 4929-9458-4176v.2of control subjects[9]. These findings indicate that FN1 levels may be intricately linked to APOE^4 pathology and AD at the BBB, and that reduction of FN1 may be associated with reduced AD risk.

[0386] We performed functional studies in zebrafish amyloidosis models by comparing FN1knockout loss-of-function animals to controls, demonstrating that reduced FN1 increases amyloid clearance and synaptic integrity, and reduces inflammation, gliosis, and BBB dysfunction[9]. We observed that excessive FN1 deposition disrupted vascular endothelial growth factor (VEGF) signaling in the brain, leading to further compromise of the BBB integrity. We also found VEGFA downregulation in astrocytes and perturbed VEGFA target engagement in vascular cells in human postmortem and antemortem data, iPSCs, and mouse and zebrafish models of AD. Using humanized APOE^4-expressing mouse brains we saw more than a two-fold increase in FN1 deposition at the BBB compared to APOE^3-expressing animals. An independent study confirmed that the top upregulated protein in human iPSC-derived astrocytes expressing APOE^4 was FN1, when compared to APOE^3 astrocytes

[0011] . These observations underscores the critical interaction of FN1 with APOE^4 in mediating the disease pathology, particularly at the BBB.

[0387] The protective FN1 variant reduces aggregated FN1 levels, thereby preserving BBBfunction and preventing the accumulation of toxic amyloid-beta deposits in the brain by offsetting the pathological role of APOE^4. By targeting FN1 to chemically mimic the protective variant in individuals without the protective variant, a similar protective effect and mitigation of BBB dysfunction, restoration of critical cellular interactions between the brain and vasculature, and offset of the overall APOE^4 -mediated AD pathology are achievable.

[0388] Human genetic evidence: The discovery phase of the FN1 genetic variant is extensivelydetailed in our recent publication[9]. In this study we utilized whole-genome sequencing (WGS) data from the National Institute on Aging Alzheimer's Disease Family Based Study (NIA-AD FBS)25, Washington Heights / Inwood Columbia Aging Project (WHICAP)

[0026] , and Estudio Familiar de Influencia Genetica en Alzheimer (EFIGA) cohorts. These cohorts included over 700 non-Hispanic White and Caribbean Hispanic families affected by AD. We identified 510 rare coding variants segregating exclusively among unaffected APOEε4 carriers, with significant enrichment in extracellular matrix (ECM)-related processes. Notably, we found that the FN1 rs140926439 FN1 loss of function variant as protective of AD and it delayed the age at onset by 3.37 years. This variant showed a significant association with reduced AD risk in an independent cohort of 7,185 APOEε4 homozygous carriers. 83 4929-9458-4176v.2

[0389] Target expression in disease-relevant regions: We found that FN1 expression iselevated in the brains of APOEε4 carriers with AD, particularly at the BBB. We confirmed this through immunohistochemical analyses of human postmortem brain tissues, where FN1 levels were significantly higher in APOEε4 homozygous individuals compared to APOEε3 / 3 controls. Additionally, independent studies in human iPSC-derived astrocytes expressing APOEε4 demonstrated increased FN1 expression compared to those expressing APOEε3[10,11]. We also demonstrated in APOEε4 expressing humanized mouse models and APP knock in mouse, FN1 expression is elevated at the BBB compared to controls. Our results are further verified by independent studies where the most upregulated gene and protein in APOEε4+ iPSC-derived astrocytes compared to those expressing APOEε3 was indeed FN1

[0011] .

[0390] Changes in target expression or activity: Increased FN1 deposition at the BBB isassociated with APOEε4 dosage and AD pathology. In unaffected APOEε4 carriers, FN1 levels are comparable to levels among controls without AD, indicating a pathological involvement of FN1 in APOEe4-mediated AD pathology. In APOEε4 carriers without dementia, FN1 levels are 28.6% lower than APOEε4 carriers with AD[9]. Therefore, we will use 30% reduction in FN1 levels at the BBB as our therapeutic benchmark level of efficacy. Finally, we validated the pathological upregulation of FN1 at the BBB in mouse and zebrafish models as well as human iPSC-derived astrocytes.

[0391] Genetic or pharmacologic manipulation: Our functional studies in FN1 knockoutzebrafish models combined with amyloid toxicity demonstrated that reduced FN1 enhances amyloid clearance and synaptic integrity, reduces inflammation, hampers gliosis, and improves BBB dysfunction; indicating that reduced FN1 could serve as an improvement therapy for AD- related pathologies. In humanized APOEε4-expressing mouse models, we found that the FN1 deposition at the BBB was more than two-fold higher compared to APOEε3-expressing animals, solidifying the mechanistic link between APOEε4 and FN1. This evidence underscores the critical interaction of FN1 with APOEε4 in mediating disease pathology, particularly at the BBB. Our in vitro studies in mouse astrocytes expressing human APOEε variants (Figure 1) showed reduction in FN1 aggregation in APOEε4 genotype (Figure 1D, 39.8% reduction in insoluble FN1) but not in APOEε3 genotype (Figure 1C), indicating an exciting genotype specific effect. Additionally, compound #3 also reduced gliosis (as reduced GFAP expression, Figure 1E). Interestingly, compound #3 did not change the soluble FN1 levels but insoluble form, while another hit 84 4929-9458-4176v.2compound #9 did not change insoluble FN1 but reduced the levels of soluble FN1 (Figure 1F). Therefore, compound #3 fits to our initial assumptions and goals, and is our top lead compound.

[0392] Direct measures of target engagement: Measures of target engagement for FN1 includeassessing FN1 levels at the BBB and in the brain using immunohistochemistry and protein quantification techniques. We will employ functional assays in animal models, such as the zebrafish and mouse models, to evaluate the impact of FN1-targeting compounds on amyloid clearance, BBB integrity, and gliosis. Additionally, we will use VEGFA and SMAD3 as direct readouts. In our recent studies, we showed that (1) VEGFA is downregulated in astrocytes, (2) SMAD3 is upregulated vasculature in AD, and (3) FN1 upregulation reduces VEGFA and increase SMAD3, and FN1 loss-of-function reverts these measures. Therefore, by using FN1 aggregation as direct measure and VEGFA / SMAD3 as immediate consequences, we will measure target engagement and efficacy of our drug candidates.

[0393] Compelling nature of the target: FN1 is a uniquely compelling target for AD owing toits direct involvement in BBB integrity and interaction with APOEε4, a well-known genetic risk factor for AD. Unlike other targets, FN1 offers a novel mechanism to mitigate AD pathology by addressing ECM remodeling and vascular dysfunction, critical components of disease progression. One protective variant in FN1 in humans is in the FnII domain, which is not extensively studied and has a novel therapeutic relevance.

[0394] Strength of evidence for mode of action: The mode of action linking FN1 to AD issupported by strong genetic, biochemical, and functional evidence that we generated. Genetic: We identified the protective FN1 variant in a large cohort study involving over 7,000 APOEε4 carriers, including those with and without AD. We found that this variant is associated with a significant reduction in AD risk and delayed onset by more than three years in APOEε4 homozygous carriers. Biochemical: We observed elevated FN1 levels in the human brain, particularly at the BBB, in APOEε4 carriers with AD. Immunohistochemical analyses of human postmortem brain tissues revealed significantly higher FN1 levels in APOEε4 homozygous individuals compared to APOEε3 / 3 controls. We validated changes in the levels of FN1 in humans with ELISA assays. Additionally, studies in human iPSC-derived astrocytes expressing APOEε4 demonstrated increased FN1 expression compared to those expressing APOEε3[10,11]. This suggests that FN1 elevation is a critical factor in BBB dysfunction and AD pathology. Functional: We demonstrated that FN1 knockout zebrafish models combined with amyloid toxicity reduced FN1, and 85 4929-9458-4176v.2ameliorated amyloid clearance, inflammation, gliosis, BBB dysfunction, and synaptic integrity[9]. In humanized APOEε4-expressing mouse models, FN1 deposition at the BBB was more than two- fold higher compared to APOEε3-expressing animals. Mechanistic: The protective FN1 variant is a loss-of-function mutation that stabilizes the inactive form of FN1. FN1 usually exists as a dimer covalently linked via two disulfide bonds (Figure 3). Each dimer subunit contains nearly 2,500 amino acids and consists of a repetition of 56 modules of three types: type I, type II, and type III repeats. The protective mutation in the FNII domain alters the structural conformation, preventing excessive FN1 deposition and preserving BBB function. We validated this mechanism through structural analyses and thermal shift assays, showing that the mutation increases stability and reduces pathological aggregation of FN1. This comprehensive body of evidence highlights the strong rationale for targeting FN1 in AD, providing a robust foundation for our proposed therapeutic approach.

[0395] Therapeutic modality

[0396] Hit / lead molecule or series activity: Our peptidomimetic small molecule hit compoundswere selected through extensive in silico peptidomimetic library screens, molecular docking, and MD simulation studies. These compounds show strong affinity for the specific 3D structural pocket generated by inactive dimeric FN1, aiming to mimic the protective effects of the FN1 variant.

[0397] Solubility and scale-up feasibility: The selected compounds demonstrate adequatesolubility and scalability for further testing. We confirmed these properties through preliminary solubility testing.

[0398] Selectivity assessment: We will test the selectivity of our lead compounds amongrelated ECM proteins and unrelated family members to ensure targeted action on FN1 without off- target effects. We will achieve this through binding affinity assays and specificity testing in cell- based assays. One such method is surface plasmon resonance (SPR). SPR is a powerful technique used to measure the binding affinity and kinetics of interactions between the lead compounds and FN1. It provides real-time, label-free detection of binding events, allowing us to assess the specificity of the compounds by testing their interactions with a panel of related ECM proteins and unrelated family members. Another possible method is protein microarrays, where an array of related ECM protein peptides can be used to test the binding of the lead compounds. Protein 86 4929-9458-4176v.2microarrays allow for the simultaneous screening of multiple protein interactions, providing a comprehensive assessment of selectivity.

[0399] ADMET profiling: We identified FN1 variant only very recently and identified the hitcompounds after a comprehensive in silico screen and preliminary in vitro studies. Therefore, we did not have time to perform detailed in vitro or in vivo ADMET. However, we performed preliminary cytotoxicity studies in vitro (Figure 5) and determined a safe dose range to start iterative testing. In our proposal, we will first generate a lead library after SAR studies (for details, see Section 4 – Experimental design and methods). The lead library we will generate will be tested for extensive in vitro ADMET properties and PK data along with a funnel selection procedure including metabolic stability in liver microsomes, cytotoxicity, CYP enzyme activity modulation, target efficacy with direct and indirect measures, stability in solution, and penetration for GI tract and BBB using transwell cell assays and artificial membranes. We will generate a score matrix with all these parameters and the best 10 compounds, or their derivatives will be prioritized for zebrafish studies for preliminary in vivo testing. Here, we will assess the toxicity and dose- response relationship with established readouts on FN1 accumulation, BBB integrity, and inflammation[9]. We have used this approach successfully for funneling down hit and lead compounds before[27-29]. Preliminary in vivo testing will further prioritize 3 compounds or derivatives for in vivo ADMET in mouse. We will assess the DMPK characteristics in collaboration with a CRO that we will decide (representative quotes in supplementary). These measures will include two routes of administration (oral and intravenous), absorption and excretion, in vivo metabolite identification with LC / MS, tissue distribution with whole body quantitative radiolabeling or similar method (e.g., BBB penetration), plasma protein binding, hepatotoxicity (e.g., CYP enzyme activation or inhibition), and calculation of PK parameters (e.g., Cmax, AUC). The compound with the best profile (total score when every parameter is scored from 0-10), will be used for in vivo efficacy study in mice with two routes of administration and 3 doses.

[0400] Preclinical efficacy studies:

[0401] Blood-brain barrier penetration: One of the properties for us to select ourpeptidomimetic hits was BBB penetration prediction scores. These scores do not absolutely implicate BBB penetration yet distinguish and filter out non-BBB-penetrant molecules. In this 87 4929-9458-4176v.2proposal, we aim to generate BBB penetration data by in vivo tissue distribution studies in mouse models.

[0402] Dosing administration and regimen: We have initial dosing studies performed in vitro.We will justify the dosing regimen for our hit compounds by PK / PD data obtained from in vivo studies in zebrafish and mice. Dedicated goals for these studies are included in our proposal. The relevant aims will include in vivo dose-response analyses, dose-toxicity analyses, and dosing regiments for intravenous and oral administration to test biodistribution. We will refine the dosing regimen based on these results to inform efficacy studies.

[0403] Measures of target engagement: Direct and indirect measures of target engagement willinclude biochemical and immunohistochemical markers related to FN1 biology, such as FN1 deposition at the BBB (direct immunolabeling), VEGFA signaling (plasma VEGFA levels by ELISA measurements, brain VEGFA expression in astrocytes detected by immunolabeling and automated image quantification), gliosis markers (hypertrophic GFAP levels, elevated IL6 levels), BBB integrity marker analyses (CD31-ZO1 co-staining delineating the endothelial integrity), synaptic integrity (SV2, PSD95, Syn immunolabeling and quantification) and inflammatory markers (TNF^, IL1^, C3 levels in the plasma and in the brain tissue determined by ELISA and immunolabeling). We will prioritize these outcomes over behavioral outcomes to assess the pharmacodynamic responses related to FN1 targeting. We anticipate behavioral tests be performed after this proposal period, as future opportunities.

[0404] In some embodiments, preclinical efficacy studies may utilize well-characterizedmouse models expressing humanized APOEε4 and zebrafish models with fibronectin (FN1) knockout combined with amyloid toxicity. Such models are advantageous for assessing APOEε4- dependent pathology and Alzheimer’s disease–related amyloidosis[30,31]. They reproduce critical features of APOEε4-driven alterations, including amyloid deposition, blood–brain barrier (BBB) dysfunction, and gliosis.

[0405] The humanized APOEε4-expressing mouse model is useful for studying Alzheimer’spathology because it incorporates the genetic risk factor present in a substantial proportion of human patients. These mice exhibit robust amyloid-β plaque formation, BBB compromise with increased permeability, and associated neuroinflammation. Elevated levels of inflammatory cytokines such as IL1β and IL6 mirror gliosis observed in human AD brains[30–33]. This model enables quantitative assessment of FN1 deposition at the BBB and in brain parenchyma by 88 4929-9458-4176v.2immunohistochemistry or protein quantification. Interactions between FN1, VEGFA, and SMAD3 may also be evaluated as mechanistic readouts of BBB integrity.

[0406] The APP^NL-G-F / NL-G-F knock-in mouse model

[0034] provides a physiologicallyrelevant amyloid model. These mice carry humanized APP mutations that elevate amyloid-β42 production without overexpression artifacts. They develop progressive amyloid-β42 deposition from ~6 months of age, together with microgliosis, astrocytosis, and synaptic loss (e.g., reduced synaptophysin and PSD95). Cognitive deficits correlate with amyloid burden, paralleling human AD progression. Increased FN1 expression and aggregation at the BBB are also observed in this model (Figure 2).

[0407] In certain embodiments, additional animal models may be employed, including otherhumanized APOE knock-in systems or alternative genetic manipulations, provided they allow evaluation of fibronectin-associated pathology, VEGFA / SMAD3 signaling, and BBB function.

[0408] The zebrafish knockout model allows investigation of the effects of the FN1 loss-of-function combined with an amyloidosis system that has been using extensively for the last decade [4,9,27-29,35-50]. We expect that any compound that alleviates the pathological effects of FN1 should converge the phenotypes in amyloidosis zebrafish model to the FN1 knockout model, as the aim of the compounds is to mimic FN1 loss of function. The zebrafish model provides a complementary system for high-throughput screening and preliminary efficacy testing of FN1- targeting compounds, by offering several advantages. The zebrafish model (1) exhibit key features of AD pathology, including amyloid-beta accumulation and neuroinflammation, (2) the FN1 knockout model specifically allows us to study the role of FN1 in these processes, (3) facilitate the study of molecular pathways involved in AD with powered studies (these processes include amyloid clearance, synaptic integrity, immune system activity, inflammation, and BBB integrity), and (4) is cost-effective and allow for large-scale screening of compounds, accelerating the drug development process. The genetic and physiological responses in zebrafish are also relevant to human disease, making them a valuable tool for early-stage screening before moving to mammalian models. We have extensive data supporting this aspect molecularly and pathologically[4,9,27-29,35-37,43,44,46,49-51]. We also successfully used zebrafish to investigate the effects of human genetic variants in various genes and their pathological relevance to AD[9,35,38,41-43]. We will further validate the positive hits from zebrafish studies in mouse models, ensuring a comprehensive evaluation across different biological systems. By utilizing both 89 4929-9458-4176v.2mouse and zebrafish models, we ensure a robust and comprehensive preclinical evaluation of FN1- targeting compounds. Each model provides unique insights into different aspects of AD pathology and target engagement. Animal usage and power analyses are detailed in Animal Studies Questionnaire.

[0409] Target Validation Data

[0410] Rational biological connection: The biological connection of FN1 to AD is well-established through genetic studies, protein expression analyses, and functional assays in various models as documented above. The protective FN1 variant, identified in large genetic cohorts, reduces FN1 levels and mitigates AD pathology by preserving BBB integrity and preventing amyloid-beta accumulation, offering an unprecedented therapy option.

[0411] Lead / clinical molecule selection: Our lead compounds were selected through a rigorousscreening process, including in silico library screens, molecular docking, and MD simulations. These compounds were optimized for strong binding affinity to the specific 3D structural pocket of inactive dimeric FN1, aiming to mimic the protective effects of the FN1 variant. Subsequent in vitro tests in human APOE^3 and APOE^4-expressing mouse astrocytes confirmed their efficacy and proposed safety profiles (Figure 1). Our results are promising as we found a lead compound (#3) is reducing insoluble FN1 aggregates and GFAP in APOE^4 genotype-specific manner and does not alter the soluble FN1 levels (Figure 1). This positions our lead as a first-in-class molecule with high-potential for AD therapeutics.

[0412] Protein preparation: We obtained the X-ray structure biological assembly ofhomodimer Fibronectin-GBD (PDB ID:3M7P) from the RCSB Protein Data Bank

[0021] . We deleted the duplicate zinc atoms at the connection points of monomers and repositioned to their average coordinates (Figure 3A). For structural protein preparation, we used the Protein Preparation tool in the Maestro molecular modelling suite

[0052] . We used the Maestro’s Prime module[53,54] to fill the missing loops and side chains into the residues. To add Hydrogen atoms to the protein at physiological pH, we used PROPKA[55,56], ensuring accurate ionization states of amino acid residues. We performed structural minimization by using the OPLS3 force field

[0057] with a convergence criterion of 0.3 Å RMSD for heavy atoms.

[0413] Grid generation: We generated a 3D grid that represents the binding site of Fibronectin,which is essential for the evaluation of ligand binding energy during docking simulations. The Receptor Grid Generation tool was utilized to identify the binding site

[0058] . The grid was generated 90 4929-9458-4176v.2by specifying key residues, mutation site Gly357, and its interacting residues upon mutation to Glu357, Gln483 and Arg495 (Figure 3C). The coordinates for the centroid of the selected residues were determined as -80.49, -21.38, 6.27 in the x, y, z coordinate system, respectively.

[0414] Ligand preparation: We collected the screening database from ChemDiv library catalogset. Since the mutation site is on the surface of the protein (Figure 3B-D), we selected the protein- protein interaction library consisting of 214,102 compounds, peptidomimetics library consisting 36,711 compounds and non-peptide peptidomimetics library consisting 19,079 compounds. We prepared a total of 219,268 compounds using LigPrep module of the Maestro molecular modeling package (Schrodinger software). During the ligand preparation process, we calculated the protonation states of the compounds within a pH range of 7.0 ± 2.0 using the Epik module

[0059] . We retained the compounds with specified chiral centers and for other chiral centers, we generated up to five distinct stereoisomers. To ensure accurate representation of molecular characteristics, we applied the OPLS357 force field. Figure 4 gives a schematic overview of the selection procedure.

[0415] Molecular docking: We conducted the docking simulations by using the grid-baseGlide docking algorithm

[0060] , which systematically explores the lowest energy conformation of the docked compound at the binding site of the receptor. The ligands underwent hierarchical filtration to assess the complementarity of the ligand–receptor system. Ligands that pass this phase underwent energy minimization and we assigned them with scores. We employed Glide / SP docking protocol. We considered standard parameters, including ligand sampling with nitrogen inversions and ring conformations, bias sampling of torsion for amides, and post-docking minimization of 10 poses for each ligand. We ranked the docked poses based on docking scores. We considered only the top-scoring poses as the most active.

[0416] Molecular dynamics (MD): We performed all-atom MD simulations by usingDesmond

[0017] . We generated a solvation box using the TIP3P solvent model.18 We performed the simulations in the constant pressure, constant temperature (NPT) ensemble at 310 K with a pressure of 1.01325 bar. We maintained these parameters using a Nose–Hoover thermostat

[0019] and Martyina–Tobias–Klein barostat

[0020] . We balanced the system by adding 9 Cl– ions and 0.15 M NaCl solution to achieve a pH of 7.4 and neutralize the simulation medium. Prior to the simulations, we employed an energy minimization and relaxation of the structure in Desmond. We conducted each MD simulation for 10ns production run in triplicate seed numbers. We generated 91 4929-9458-4176v.21000 trajectory frames for all simulations. We collected the relevant MD simulations data individually and saved in trajectory files.

[0417] Molecular mechanics generalized Born surface area (MM-GBSA) calculations: Tocalculate average binding free energy of each compound at the Fibronectin-GBD in MD simulations, we performed MM-GBSA analysis by using Maestro’s Prime module[9,10]. We employed a systematic approach, where we performed MM-GBSA calculations using one frame out of every ten frames. We defined the dielectric constant by using the VSGB 2.0 implicit solvation model

[0021] . After the calculations for each complex, we computed the average MM- GBSA value and standard deviations for each compound. We set a threshold of -60 kcal / mol and selected eight compounds that display an average binding energy of above threshold in all triplicate simulations (Figure 4). We could not synthesize one compound, so we proceeded with our in vitro studies with 7 compounds. From the 7 compounds, we first performed IC50 determination and cell viability experiments using CTG assay. Here, we found specific concentrations that allow >80% of the astrocytes in cultures to be viable after treatment (Figure 5). We used these determined concentrations for our in vitro efficacy study as shown in Figure 1.

[0418] Additional findings and disclosures herein:- Association of FN1 with APOE4 genetic risk in developing AD;- Discovery of FN1 accumulation as an early pathogenesis in AD brains;- Identification that FN1 affects blood vessels and blood-brain barrier (BBB);- Uncovering the molecular mechanism how FN1 affects BBB;- Identification of FN1-related predictive biomarkers for AD;- Discovery two FN1 gene genetic variants that protect against AD;- Demonstration of both novel and repurposed compounds for improvement in predictivebiomarker for FN1 / BBB pathology in AD; and -Identification of treating AD by intervening in early vascular pathology via fibronectinmodulation.

[0419] Apolipoprotein E gene variant APOEε4 is the strongest genetic risk factor forAlzheimer's disease. APOEε4 frequency is 14% in general population, but, APOEε4 carriers constitute 40-60% of all AD patients. Therefore, therapies tailored to APOEε4 carriers could dramatically reduce AD prevalence and improve outcomes in the highest-risk group (Figure 21).

[0420] Pathophysiology of APOE in AD (Figures 22 and 23):92 4929-9458-4176v.2- Influences Aβ clearance and aggregation in an isoform-dependent manner- Can be targeted in several ways- Modifiers of APOE can be therapeutic targets- Modulating APOEε4’s downstream effects disclosed herein such as fibronectin depositionand BBB dysfunction, offers a novel, actionable therapeutic strategy.

[0421] BBB integrity depends on coordinated cellular interactions and its breakdown is linkedto AD (Figures 24 A-C). Targeting BBB dysfunction, especially via fibronectin, can restore vascular health and delay AD onset. No treatment is currently available for APOEε4 carriers. However, amyloid-related imaging abnormalities (ARIA) makes APOEε4 carriers poor candidates for anti-Aβ monoclonal antibody therapies. The vascular contribution to dementia is not well understood. Treatments are typically provided late and early interventions needed. However, early stage targets are limited. FN1 offers a novel, early-stage target that can overcome limitations of current AD treatments.

[0422] Two genetic variants in the fibronectin gene reduces AD risk by 71% and delays theonset by 3.4 years. Mimicking these protective variants pharmacologically could revolutionize AD prevention.

[0423] Whole-genome sequencing reveals protective mechanisms linked to FN1 (Figure 25).Understanding resilience mechanisms enables targeted drug development for high-risk individuals.

[0424] FN1 deposition increases with APOEε4 dosage, thus APOEε4 drives fibronectinaccumulation - a modifiable pathogenic process (Figures 26 A&B).

[0425] Resilient individuals show reduced FN1 accumulation. Lower FN1 levels may be abiomarker and therapeutic goal for vascular resilience in AD (Figures 27 A-C).

[0426] Reducing the fibronectin is a therapeutic opportunity as FN1 modulation is consideredto restore multiple systems disrupted in AD (Figure 28), providing a multi-benefit therapeutic approach to: -Lower fibronectin- Reduce vascular dysfunction and inflammation- Increase amyloid clearance, synaptic integrity, and cognitive capacity.93 4929-9458-4176v.2

[0427] FN1 is a modular protein with many domains required for protein binding and signaling(Figure 29). FN1’s modularity allows for precise drug targeting which enhances specificity and reducing off-target effects.

[0428] As shown in figures 30 A and B, tissue FN1 (with EDA / EDB domains) is upregulatedin AD and elicits immune responses. Tissue-specific FN1 targeting is feasible and therapeutically advantageous. However, peripheral FN1 in the blood is structurally different and is not associated with AD (Figure 31). This presents a unique opportunity for reducing off-target and systemic effects of therapeutics against FN1.

[0429] Figures 32 A-H show direct evidence of fibronectin accumulation in blood vessels ofhuman brains affected by AD or vascular pathology. FN1 levels are elevated in affected vasculature, reinforcing its role in early disease mechanisms. Fibronectin accumulation is not just a biomarker but a modifiable pathological feature, making it a prime therapeutic target in AD and related vascular neurodegenerative diseases.

[0430] FN1 is upregulated in APOE mouse models, and its expression correlates withfibrinogen leakage - a marker of blood-brain barrier breakdown (Figures 33 I-P). These findings confirm that FN1 is not only a pathological marker but a mechanistic driver of vascular dysfunction in APOE-related AD, reinforcing its value as a therapeutic target.

[0431] Data in figures 34 A-F links fibronectin to astrocyte dysfunction and vascularbreakdown in human AD brains. FN1 correlates with disrupted astrocyte endfeet and compromised vasculature. FN1 accumulation disrupts neurovascular unit integrity - a central mechanism in AD pathogenesis and a prime therapeutic target.

[0432] The leakiness in mouse brains shown in figure 35 validates human findings in animalmodels. FN1 deposition in mouse brain vessels correlates with vascular leakiness. FN1-driven vascular pathology is conserved across species, strengthening its candidacy for drug targeting.

[0433] FN1 is most expressed in astrocytes, endothelia, and pericytes (BBB cell types) withAPOEε4 driving upregulation in astrocytes (Figures 36 A&B). FN1’s cell-type specificity enables targeted intervention, especially in APOEε4-driven astrocytic pathology.

[0434] APOEε4 astrocytes show increased FN1 deposition in pathological forms, mirroringhuman brain findings (Figures 37 A&B). In vitro models confirm that FN1 pathology is APOEε4- dependent and reproducible, thereby enabling compound screening and therapeutic development. 94 4929-9458-4176v.2

[0435] APOEe4 and FN1 colocalize in vitro and in vivo in humans, suggesting a directpathological relationship (Figures 38 A-D; and figure 39 E-G). The physical proximity of APOE4 and FN1 in astrocytes supports a mechanistic link and targeting FN1 may disrupt this pathogenic interaction.

[0436] Thus, the APOE4–FN1 interaction is not just experimental, it is a real, disease-relevantphenomenon in humans, reinforcing FN1 as a therapeutic target, as demonstrated by figures 39 E- G.

[0437] FN1 expression in AQP4+ astrocyte endfeet, AQP4 depolarization and colocalizationwith APOE4 indicate impaired astrocyte–vascular interactions (Figures 40 J-L). FN1 pathology may drive astrocytic dysfunction, contributing to BBB breakdown making it a key therapeutic target. FN1 is inflammation-responsive, making it a dynamic and druggable node in AD pathology.

[0438] Further findings are that FN1 expression in astrocytes leads to BBB leakage inzebrafish, as shown in Figure 41. FN1 is sufficient to impair vascular integrity, reinforcing its pathogenic role and therapeutic relevance. Also, FN1 also disrupts integrin–FAK signalling, leading to vascular and astrocytic dysfunction through VEGFA, HBEGF and IGF1 signalling (Figure 42). Moreover, VEGFA and IGF1 signalling are altered in APOE4 carriers, consistent with FN1-driven pathology. These pathways also serve as clinical biomarkers and not just efficacy endpoints for FN1-targeting therapies (Figures 43 and 44).

[0439] Pyrintegrin, an activator of integrin signalling, reduces VEGFA and IGF1 in APOE4astrocytes (Figures 45 A&B). Active integrin signalling through FN1 is pathological and changes predictive FN1 related markers.

[0440] It is noted that two protective genetic variants reside in domains critical for FN1function. Protective variant 1 is in the FNII domain, as shown in Figure 29. Targeting the variant domains allows for rational drug design with high specificity.

[0441] APOE4 promotes FN1 accumulation and potentially conformational change (Figures46 A&B). APOE4-driven FN1 activation is a modifiable step - ideal for therapeutic intervention.

[0442] Test doses for compounds include: 2: 10 uM, 3: 5 uM, 9: 10 uM, 10: 10 uM. Compound3 is especially significant in statistically significant reduction in insoluble FN1 levels as determined by IHC as shown in figures 1 A-F. Compounds 2, 9 and 10 also show a trend of reduction (Figure 8). Selected effective doses were used. Compound 3 effectively reduces insoluble FN1 deposition in human APOE E4 expressing mouse astrocytes. 95 4929-9458-4176v.2

[0443] As shown in figure 10, almost all evaluated compounds reduce GFAP expression,suggesting a reduction in gliosis. FN1-targeting compounds also reduce gliosis, thereby offering multi-modal therapeutic benefits.

[0444] Based on ELISA experiments, (Figure 12) compounds #2 and #3 reduce FN1 in thesupernatant of APOE E4 cells at 10 and 1 uM, respectively, while APOE E3 is not affected. FN1 modulation is genotype-specific.

[0445] The data (Figure 15) demonstrates APOE3 and FN1 colocalize. Compounds 3, 9 and10 reduce colocalization of APOE and FN1 in APOE E4 astrocytes in human in vitro. In E3 astrocytes, there is not much of this colocalization and compounds do not affect this.

[0446] In E3 astrocytes, FN1 and APOE expression do not correlate, as shown by the data infigure 16. But in E4, FN1 expression correlates with APOE E4, suggesting a synergistic pathogenicity of FN1 dependent on E4. The correlation coefficient is reduced in E4 astrocytes with all compounds. This suggests that compounds reduce the pathogenic insoluble aggregations / accumulations of FN1 in APOE E4-genotype specific manner. In APOE E4 astrocytes, this is not the case. Reducing pathogenic colocalization is a novel mechanism.

[0447] FN1-targeting compounds show no off-target effects in 10-fold excess in zebrafish.FN1-targeting compounds show no off-target effects in synapses or microglia even at 10× concentration in zebrafish without fn1 in their brains (Figures 48 A&B). High specificity and no off-target red flags supports safety and strengthens the patent’s novelty claims. No off-target effects were seen in BBB integrity and gliosis assays (Figures 49 A& B).

[0448] FN1-targeting compounds increase synaptic vesicles and reduce microglia, which aretwo beneficial responses (Figures 50 A&B). FN1 modulation improves synaptic health and reduces neuroinflammation, offering multi-system benefits. FN1 levels and gliosis are reduced with treatment by FN1-targeting compounds (Figures 51 A&B). These compounds reverse the core pathological features of AD, thus validating their therapeutic value.

[0449] FN1 & Integrin

[0450] A second protective variant of FN1, rs116558455, has also been discovered (Figures52 and 53 A&B). This variant stabilizes FN1 structure and may reduce pathological activation. Residue 1482 of human fibronectin falls within the tenth type III repeat (FNIII-10), which spans residues 1447–1536 and comprises the major cell-attachment (RGD) domain. R1482 lies in one of 96 4929-9458-4176v.2the β-strands forming the core of the FNIII β-sandwich. While it does not contact integrins directly, it helps stabilize the fold that presents the adjacent RGD loop for receptor engagement.

[0451] FN1–integrin interactions are altered in the diseased state. Targeting FN1–integrinsignalling is a path to restore vascular and astrocytic function (Figures 54 A&B). Fibronectin signals though integrin-mediated focal adhesion kinase (FAK). One of the protective FN1 variants herein disclosed is at the integrin binding site and potentially alters downstream FAK signaling. FAK blockage restores VEGFA levels in APOEe4 astrocytes (Figure 55 A). Blockage of integrein Beta 1 with ATN-161 restores VEGFA and IGF1 levels in iPSC-derived APOEe4 human astrocytes (Figures 55 C &D). Blockage of β3 integrin does not rescue VEGFA and IGF1 (Figure 55 B). Variant-linked changes are consistent across datasets. Multiple lines of evidence validate this variant’s protective role and alteration in FN1-dependent predictive markers VEGFA and IGF1.

[0452] Alzheimer’s Disease: When To Intervene?

[0453] Alzheimer’s disease is considered a complex genetic disorder. The most frequentlyencountered genetic variant is in apolipoprotein E gene, also known as APOE-e4. Nearly 25% of the population carries at least one APOE-e4 allele, and individuals with one copy of the APOE-e4 allele have a 3-fold increased risk while those with two copies of this allele have a 10-fold increased risk of developing Alzheimer’s disease. We have been interested in carriers of the APOE-e4 risk allele who have escaped Alzheimer’s disease. We hypothesized that APOE-ε4 carriers without dementia might have genetic variations that protect them from developing Alzheimer’s disease. To test this hypothesis, we leveraged whole-genome sequencing data from the National Institute on Aging Alzheimer's Disease Family Based Study (NIA-AD FBS), the Washington Heights / Inwood Columbia Aging Project (WHICAP), and The Estudio Familiar de Influencia Genética en Alzheimer and identified potentially protective variants segregating exclusively among APOE-ε4 allele carriers without dementia who were followed longitudinally. Rare loss of function genetic variants in fibronectin 1 (FN1) and collagen 6 (COL6) are constituents of the extracellular matrix forming the blood-brain-barrier and strongly associated with protection from Alzheimer’s disease. Subsequent work by our team has identified the role these genes in the extracellular matrix and the blood brain barrier. Moreover, with laboratory investigations we now understand how a loss of function in FN1 might delay or prevent the onset of Alzheimer’s disease among individuals with the APOE-e4 risk allele. We have also identified 97 4929-9458-4176v.2compounds that mimic the effects of these genetic variants and identified when to intervene. We investigated several AD cohorts with existing cognitive data and clinical diagnoses, APOE genotypes, and blood-based biomarkers (Ab40, Ab42, P-tau181, P-tau217). The goal was to determine the timing between an increase in P-tau217, a surrogate biomarker for Alzheimer’s disease pathology, and the onset of cognitive dysfunction, or a clinical diagnosis of mild cognitive impairment or Alzheimer’s disease. We then compared the intervals by APOE genotype. P-tau271 levels predict age of onset, and can be modifiable via FN1-targeting therapies (Figures 57 A&B). FN1 modulation may delay disease onset. APOE4 carriers show earlier symptom onset after biomarker threshold. FN1-targeting therapies in these patients is considered to extend the preclinical phase. Also, a clear window when to intervene can be identified for therapeutics to patients. We plotted the longitudinal trajectories of P-tau217 and cognitive decline for the entire Wisconsin Registry for Alzheimer’s Prevention (WRAP) and the Wisconsin ADRC cohorts, creating reference lines and the corresponding ages at which individuals crossed these thresholds. P-tau217 cut-off points were selected based on previously published literature from WRAP. Cognition cut-off points were derived using the Youden Index applied to each individual’s most recent cognitive score to determine thresholds that best discriminate MCI or AD from non- demented individuals. Figure 57 illustrates the overall longitudinal patterns in the full sample and is not stratified by diagnostic stage or disease status. For descriptive purposes, the trajectories can be interpreted as a visual approximation of when a 'typical' individual might cross biomarker or cognitive thresholds over time, representing a general pattern of progression from normal cognition to AD, stratified by APOE genotype. Similar observations were acquired when we investigated the same parameters in the Alzheimer’s Disease NeuroImaging Initiative (ADNI) Cohort, The Washington Heights Inwood Columbia Aging Project (WHICAP) and The Estudio Familiar de Influencia Genética en Alzheimer (EFIGA) cohorts.

[0454] Next, we plotted the trajectories base on the sensitivity and specificity of P-tau217diagnostically (Figure 58). We used established optimal cut points based on receiver operating curves and area under curve analyses (ROC-AUC). In plasma, the sensitivity was most robust for P-tau217 levels of 0.4 pg / ml, while the most robust specificity was a P-tau217 level 0.62 pg / ml. However, we chose to use the optimate cut point for specificity because this reduces the likelihood of false positives. In Figure 58, we compare the time between reaching the specificity cut point and the onset of clinical symptoms associated with onset of dementia. 98 4929-9458-4176v.2

[0455] A proposed model for the molecular pathology mechanism of APOE4-mediatedfibronectin at the BBB is shown in Fig. 59. The schematic at the top illustrates how APOE4, compared to the protective APOE3 isoform, promotes fibronectin overproduction by astrocytes. This excessive fibronectin engages integrin β1 on the astrocyte membrane, activating downstream focal adhesion kinase (FAK) and MAPK signaling pathways. These signaling events ultimately suppress the expression of key trophic factors, VEGFA and IGF1, which are critical for maintaining vascular health and BBB integrity. As a result, APOE4 leads to neurovascular dysfunction, a hallmark of AD progression. Supporting this model, post-mortem human brain samples reveal that VEGFA expression is significantly reduced in astrocytes from individuals with AD compared to age-matched controls. This is confirmed by immunofluorescence imaging and quantification, showing lower VEGFA levels in GFAP-positive astrocytes in AD brains. Additionally, cerebrospinal fluid (CSF) analyses and transcriptomic co-expression data demonstrate that APOE4 carriers have lower VEGFA levels and a disrupted positive correlation between FN1 and VEGFA expression, further implicating fibronectin in repressing VEGFA in AD. Human iPSC-derived astrocyte models offer mechanistic insight. Astrocytes expressing APOE4 (APOE4 / 4) produce significantly higher levels of fibronectin compared to APOE3 / 3 cells, as shown by immunostaining and intensity quantification. Correspondingly, VEGFA levels are markedly reduced in APOE4 astrocytes, highlighting a direct relationship between APOE4, fibronectin elevation, and VEGFA suppression. Importantly, simply plating astrocytes on fibronectin-coated dishes (in the absence of APOE4) is sufficient to repress VEGFA expression, indicating that fibronectin alone can drive this phenotype.

[0456] As shown in Fig. 60, FN1 signals through integrin-mediated focal adhesion kinase(FAK). One of the protective variants identified is at the integrin binding site and potentially alters downstream FAK signaling. Fibronectin regulation on VEGFA is mediated through integrin signaling. Inhibition of FAK, a key downstream effector of integrin activation (with general FAK inhibition drugs – Bhattarai et al., 2024), rescues VEGFA expression in APOE4 astrocytes, suggesting that FN1 signals through the integrin–FAK axis. More specifically, blocking the α5β1 integrin with ATN-161 (our discovery), a non-competitive antagonist that binds outside the RGD site, restores both VEGFA and IGF1 levels in APOE4 astrocytes. In contrast, blocking αvβ3 integrin using cilengitide, a competitive inhibitor that blocks adhesion and induces apoptosis, does not affect VEGFA or IGF1 levels (our discovery). These results demonstrate the specificity of 99 4929-9458-4176v.2α5β1-mediated signaling in the repression of growth factors and support the therapeutic relevance of ATN-161. A mechanistic diagram summarizes the distinct modes of action of ATN-161 and cilengitide. ATN-161 disrupts fibronectin–integrin β1 signaling without blocking adhesion, thereby preventing downstream FAK / MAPK activation and preserving VEGFA and IGF1 expression. Cilengitide, by contrast, targets αvβ3 integrins at the RGD-binding site, disrupting cell adhesion and inducing apoptosis, but fails to impact the fibronectin–β1–FAK pathway involved in VEGFA suppression. The final table contrasts the molecular features, mechanisms of action, and clinical status of ATN-161 and cilengitide. ATN-161 is a linear pentapeptide that acts as a non- competitive inhibitor of α5β1 integrin signaling, with established safety in early clinical trials and potential for repurposing in AD. Cilengitide, a cyclic RGD-mimetic peptide, is a competitive inhibitor of αvβ3 / β5 integrins and has undergone clinical testing in glioblastoma, though with limited efficacy. These findings show a novel APOE4-specific fibronectin–integrin–FAK signaling axis in astrocytes that suppresses VEGFA and IGF1, offering new therapeutic opportunities to restore BBB integrity and neurovascular health in Alzheimer’s disease.

[0457] The clinical trial endpoints in Table 1 below provide a mechanistically anchoredframework for testing therapies in APOEε4 carriers, wherein reductions in CSF fibronectin and downstream pFAK / pSMAD3 / pAkt signaling, alongside restoration of VEGFA and IGF1, directly report on target engagement, while standard AD biomarkers (P-tau217, Aβ42 / 40, YKL-40, sTREM2) and cognitive measures ensure translational relevance and capture disease modification.”

[0458] Table 1: Clinical endpoints100 4929-9458-4176v.2

[0459] 1. Cao, L., Nicosia, J., Larouche, J., Zhang, Y., Bachman, H., Brown, A.C.,Holmgren, L., and Barker, T.H. (2017). Detection of an Integrin-Binding Mechanoswitch within Fibronectin during Tissue Formation and Fibrosis. ACS Nano 11, 7110-7117. 10.1021 / acsnano.7b02755.

[0460] 2. Sottile, J., Shi, F., Rublyevska, I., Chiang, H.Y., Lust, J., and Chandler, J. (2007).Fibronectin-dependent collagen I deposition modulates the cell response to fibronectin. Am J Physiol Cell Physiol 293, C1934-1946.10.1152 / ajpcell.00130.2007.

[0461] 3. Guan, J.L. (1997). Role of focal adhesion kinase in integrin signaling. Int JBiochem Cell Biol 29, 1085-1096.10.1016 / s1357-2725(97)00051-4.

[0462] 4. Is, O., Wang, X., Reddy, J., Min, Y., Yilmaz, E., Bhattarai, P., Patel, T., Bergman,J., Quicksall, Z., Heckman, M., et al. (2024). Gliovascular transcriptional perturbations in Alzheimer's disease reveal molecular mechanisms of blood brain barrier dysfunction. Nat Commun 15, 4758.10.1038 / s41467-024-48926-6. 101 4929-9458-4176v.2

[0463] 5. Montagne, A., Nation, D.A., Sagare, A.P., Barisano, G., Sweeney, M.D.,Chakhoyan, A., Pachicano, M., Joe, E., Nelson, A.R., D'Orazio, L.M., et al. (2020). APOE4 leads to blood-brain barrier dysfunction predicting cognitive decline. Nature 581, 71-76. 10.1038 / s41586-020-2247-3.

[0464] 6. Thomsen, M.S., Routhe, L.J., and Moos, T. (2017). The vascular basementmembrane in the healthy and pathological brain. J Cereb Blood Flow Metab 37, 3300-3317. 10.1177 / 0271678X17722436.

[0465] 7. Tarasoff-Conway, J.M., Carare, R.O., Osorio, R.S., Glodzik, L., Butler, T.,Fieremans, E., Axel, L., Rusinek, H., Nicholson, C., Zlokovic, B.V., et al. (2015). Clearance systems in the brain-implications for Alzheimer disease. Nat Rev Neurol 11, 457-470. 10.1038 / nrneurol.2015.119.

[0466] 8. Ji, Y., Permanne, B., Sigurdsson, E.M., Holtzman, D.M., and Wisniewski, T.(2001). Amyloid beta40 / 42 clearance across the blood-brain barrier following intra-ventricular injections in wild-type, apoE knock-out and human apoE3 or E4 expressing transgenic mice. J Alzheimers Dis 3, 23-30.

[0467] 9. Bhattarai, P., Gunasekaran, T.I., Belloy, M.E., Reyes-Dumeyer, D., Julich, D.,Tayran, H., Yilmaz, E., Flaherty, D., Turgutalp, B., Sukumar, G., et al. (2024). Rare genetic variation in fibronectin 1 (FN1) protects against APOEepsilon4 in Alzheimer's disease. Acta neuropathologica 147, 70.10.1007 / s00401-024-02721-1.

[0468] 10. Keable, A., O'Neill, R., MacGregor Sharp, M., Gatherer, M., Yuen, H.M., Johnston,D.A., Weller, R.O., and Carare, R.O. (2020). ApoE4 Astrocytes Secrete Basement Membranes Rich in Fibronectin and Poor in Laminin Compared to ApoE3 Astrocytes. Int J Mol Sci 21. 10.3390 / ijms21124371.

[0469] 11. Feringa, F., and al., e. (2024). The Neurolipid Atlas: a lipidomics resource forneurodegenerative diseases uncovers cholesterol as a regulator of astrocyte reactivity impaired by ApoE4. bioRxiv.10.1101 / 2024.07.01.601474.

[0470] 12. van Schaik, P.E.M., Zuhorn, I.S., and Baron, W. (2022). Targeting Fibronectin toOvercome Remyelination Failure in Multiple Sclerosis: The Need for Brain- and Lesion-Targeted Drug Delivery. Int J Mol Sci 23.10.3390 / ijms23158418. 102 4929-9458-4176v.2

[0471] 13. Gong, Z., Chen, M., Ren, Q., Yue, X., and Dai, Z. (2020). Fibronectin-targeteddual-acting micelles for combination therapy of metastatic breast cancer. Signal Transduct Target Ther 5, 12.10.1038 / s41392-019-0104-3.

[0472] 14. Liu, J., Ren, Z., Sun, Y., Xu, L., Wei, D., Tan, W., and Ding, D. (2023).Investigation of the Relationship between Aptamers' Targeting Functions and Human Plasma Proteins. ACS Nano 17, 24329-24342.10.1021 / acsnano.3c10238.

[0473] 15. Martin-Otal, C., Lasarte-C1a, A., Serrano, D., Casares, N., Conde, E., Navarro, F.,Sanchez-Moreno, I., Gorraiz, M., Sarrion, P., Calvo, A., et al. (2022). Targeting the extra domain A of fibronectin for cancer therapy with CAR-T cells. J Immunother Cancer 10.10.1136 / jitc-2021- 004479.

[0474] 16. Wang, Y., Jiang, L., Zhang, Y., Lu, Y., Li, J., Wang, H., Yao, D., and Wang, D.(2020). Fibronectin-Targeting and Cathepsin B-Activatable Theranostic Nanoprobe for MR / Fluorescence Imaging and Enhanced Photodynamic Therapy for Triple Negative Breast Cancer. ACS Appl Mater Interfaces 12, 33564-33574.10.1021 / acsami.0c10397.

[0475] 17. Han, Z., and Lu, Z.R. (2017). Targeting Fibronectin for Cancer Imaging andTherapy. J Mater Chem B 5, 639-654.10.1039 / C6TB02008A.

[0476] 18. Schumacher, S., Dedden, D., Nunez, R.V., Matoba, K., Takagi, J., Biertumpfel, C.,and Mizuno, N. (2021). Structural insights into integrin alpha(5)beta(1) opening by fibronectin ligand. Sci Adv 7.10.1126 / sciadv.abe9716.

[0477] 19. Guillon, E., Das, D., Julich, D., Hassan, A.R., Geller, H., and Holley, S. (2020).Fibronectin is a smart adhesive that both influences and responds to the mechanics of early spinal column development. Elife 9.10.7554 / eLife.48964.

[0478] 20. Zou, L., Cao, S., Kang, N., Huebert, R.C., and Shah, V.H. (2012). Fibronectininduces endothelial cell migration through beta1 integrin and Src-dependent phosphorylation of fibroblast growth factor receptor-1 at tyrosines 653 / 654 and 766. J Biol Chem 287, 7190-7202. 10.1074 / jbc.M111.304972.

[0479] 21. Graille, M., Pagano, M., Rose, T., Ravaux, M.R., and van Tilbeurgh, H. (2010).Zinc induces structural reorganization of gelatin binding domain from human fibronectin and affects collagen binding. Structure 18, 710-718.10.1016 / j.str.2010.03.012. 103 4929-9458-4176v.2

[0480] 22. Obara, M., Kang, M.S., and Yamada, K.M. (1988). Site-directed mutagenesis ofthe cell-binding domain of human fibronectin: separable, synergistic sites mediate adhesive function. Cell 53, 649-657.10.1016 / 0092-8674(88)90580-6.

[0481] 23. Engvall, E., Ruoslahti, E., and Miller, E.J. (1978). Affinity of fibronectin tocollagens of different genetic types and to fibrinogen. J Exp Med 147, 1584-1595. 10.1084 / jem.147.6.1584.

[0482] 24. Yu, K.F., Zhang, W.Q., Luo, L.M., Song, P., Li, D., Du, R., Ren, W., Huang, D.,Lu, W.L., Zhang, X., and Zhang, Q. (2013). The antitumor activity of a doxorubicin loaded, iRGD- modified sterically-stabilized liposome on B16-F10 melanoma cells: in vitro and in vivo evaluation. Int J Nanomedicine 8, 2473-2485.10.2147 / IJN.S46962ijn-8-2473 [pii].

[0483] 25. Reyes-Dumeyer, D., Faber, K., Vardarajan, B., Goate, A., Renton, A., Chao, M.,Boeve, B., Cruchaga, C., Pericak-Vance, M., Haines, J.L., et al. (2022). The National Institute on Aging Late-Onset Alzheimer's Disease Family Based Study: A resource for genetic discovery. Alzheimers Dement 18, 1889-1897.10.1002 / alz.12514.

[0484] 26. Vardarajan, B.N., Faber, K.M., Bird, T.D., Bennett, D.A., Rosenberg, R., Boeve,B.F., Graff-Radford, N.R., Goate, A.M., Farlow, M., Sweet, R.A., et al. (2014). Age-specific incidence rates for dementia and Alzheimer disease in NIA-LOAD / NCRAD and EFIGA families: National Institute on Aging Genetics Initiative for Late-Onset Alzheimer Disease / National Cell Repository for Alzheimer Disease (NIA-LOAD / NCRAD) and Estudio Familiar de Influencia Genetica en Alzheimer (EFIGA). JAMA Neurol 71, 315-323.10.1001 / jamaneurol.2013.5570.

[0485] 27. Turgutalp, B., and Kizil, C. (2024). Multi-target drugs for Alzheimer's disease.Trends Pharmacol Sci 45, 628-638.10.1016 / j.tips.2024.05.005.

[0486] 28. Turgutalp, B., Bhattarai, P., Ercetin, T., Luise, C., Reis, R., Gurdal, E., Isaak, A.,Biriken, D., Dinter, E., Sipahi, H., et al. (2022). Discovery of Potent Cholinesterase Inhibition- Based Multi-Target-Directed Lead Compounds for Synaptoprotection in Alzheimer’s Disease. J Med Chem.

[0487] 29. Bhattarai, P., Turgutalp, B., and Kizil, C. (2022). Zebrafish as an Experimental andPreclinical Model for Alzheimer's Disease. ACS Chem Neurosci 13, 2939-2941. 10.1021 / acschemneuro.2c00583.

[0488] 30. Miranda, A.M., Ashok, A., Chan, R.B., Zhou, B., Xu, Y., McIntire, L.B., Area-Gomez, E., Di Paolo, G., Duff, K.E., Oliveira, T.G., and Nuriel, T. (2022). Effects of APOE4 104 4929-9458-4176v.2allelic dosage on lipidomic signatures in the entorhinal cortex of aged mice. Transl Psychiatry 12, 129.10.1038 / s41398-022-01881-6.

[0489] 31. Nuriel, T., Angulo, S.L., Khan, U., Ashok, A., Chen, Q., Figueroa, H.Y., Emrani,S., Liu, L., Herman, M., Barrett, G., et al. (2017). Neuronal hyperactivity due to loss of inhibitory tone in APOE4 mice lacking Alzheimer's disease-like pathology. Nat Commun 8, 1464. 10.1038 / s41467-017-01444-0.

[0490] 32. Jackson, R.J., Hyman, B.T., and Serrano-Pozo, A. (2024). Multifaceted roles ofAPOE in Alzheimer disease. Nat Rev Neurol.10.1038 / s41582-024-00988-2.

[0491] 33. Sullivan, P.M., Mezdour, H., Aratani, Y., Knouff, C., Najib, J., Reddick, R.L.,Quarfordt, S.H., and Maeda, N. (1997). Targeted replacement of the mouse apolipoprotein E gene with the common human APOE3 allele enhances diet-induced hypercholesterolemia and atherosclerosis. J Biol Chem 272, 17972-17980.10.1074 / jbc.272.29.17972.

[0492] 34. Xia, D., Lianoglou, S., Sandmann, T., Calvert, M., Suh, J.H., Thomsen, E., Dugas,J., Pizzo, M.E., DeVos, S.L., Earr, T.K., et al. (2022). Novel App knock-in mouse model shows key features of amyloid pathology and reveals profound metabolic dysregulation of microglia. Mol Neurodegener 17, 41.10.1186 / s13024-022-00547-7.

[0493] 35. Tayran, H., Yilmaz, E., Bhattarai, P., Min, Y., Wang, X., Ma, Y., Nelson, N.,Kassara, N., Cosacak, M.I., Dogru, R.M., et al. (2024). ABCA7-dependent Neuropeptide-Y signalling is a resilience mechanism required for synaptic integrity in Alzheimer's disease. Cell Genomics in press.

[0494] 36. Haage, V., Tuddenham, J.F., Comandante-Lou, N., Bautista, A., Monzel, A., Chiu,R., Fujita, M., Garcia, F.G., Bhattarai, P., Patel, R., et al. (2024). A pharmacological toolkit for human microglia identifies Topoisomerase I inhibitors as immunomodulators for Alzheimer's disease. bioRxiv.10.1101 / 2024.02.06.579103.

[0495] 37. Siddiqui, T., Cosacak, M.I., Popova, S., Bhattarai, P., Yilmaz, E., Lee, A.J., Min,Y., Wang, X., Allen, M., Is, O., et al. (2023). Nerve growth factor receptor (Ngfr) induces neurogenic plasticity by suppressing reactive astroglial Lcn2 / Slc22a17 signaling in Alzheimer's disease. NPJ Regen Med 8, 33.10.1038 / s41536-023-00311-5.

[0496] 38. Ray, N., Kunkle BR, Hamilton-Nelson, K., Kurup, J., Rajabli, F., Cosacak, M.,Kizil, C., Jean-Francois, M., Cuccaro, M., Reyes-Dumeyer, D., et al. (2023). Extended genome- wide association study employing the African genome resources panel identifies novel 105 4929-9458-4176v.2susceptibility loci for Alzheimer's disease in individuals of African ancestry. Alzheimers Dement. 10.1002 / alz.13880.

[0497] 39. Lin, W., Gandhi, S., Bhattarai, P., Kasa, K., Kizil, C., and Zhang, Y. (2023).Combining high throughput array synthesis and growth algorithm to discover TNF-alpha binders with new structures and properties. Eur J Med Chem 248, 115078.10.1016 / j.ejmech.2022.115078.

[0498] 40. Kim, Y.A., Mellen, M., Kizil, C., and Santa-Maria, I. (2023). Mechanisms linkingcerebrovascular dysfunction and tauopathy: Adding a layer of epiregulatory complexity. Br J Pharmacol.10.1111 / bph.16280.

[0499] 41. Felsky, D., Santa-Maria, I., Cosacak, M.I., French, L., Schneider, J.A., Bennett,D.A., De Jager, P.L., Kizil, C., and Tosto, G. (2023). The Caribbean-Hispanic Alzheimer's disease brain transcriptome reveals ancestry-specific disease mechanisms. Neurobiol Dis 176, 105938. 10.1016 / j.nbd.2022.105938.

[0500] 42. Kizil, C., Sariya, S., Kim, Y.A., Rajabli, F., Martin, E., Reyes-Dumeyer, D.,Vardarajan, B., Maldonado, A., Haines, J.L., Mayeux, R., et al. (2022). Admixture Mapping of Alzheimer's disease in Caribbean Hispanics identifies a new locus on 22q13.1. Mol Psychiatry. 10.1038 / s41380-022-01526-6.

[0501] 43. Lee, A.J., Raghavan, N.S., Bhattarai, P., Siddiqui, T., Sariya, S., Reyes-Dumeyer,D., Flowers, X.E., Cardoso, S.A.L., De Jager, P.L., Bennett, D.A., et al. (2022). FMNL2 regulates gliovascular interactions and is associated with vascular risk factors and cerebrovascular pathology in Alzheimer’s disease. Acta neuropathologica.10.1007 / s00401-022-02431-6.

[0502] 44. Siddiqui, T., Bhattarai, P., Popova, S., Cosacak, M.I., Sariya, S., Zhang, Y.,Mayeux, R., Tosto, G., and Kizil, C. (2021). KYNA / Ahr Signaling Suppresses Neural Stem Cell Plasticity and Neurogenesis in Adult Zebrafish Model of Alzheimer's Disease. Cells 10. 10.3390 / cells10102748.

[0503] 45. Cosacak, M.I., Bhattarai, P., and Kizil, C. (2020). Alzheimer’s disease, neural stemcells and neurogenesis: cellular phase at single-cell level. Neural Reg Res 15, 824-827. 10.4103 / 1673-5374.268896.

[0504] 46. Bhattarai, P., Cosacak, M.I., Mashkaryan, V., Demir, S., Popova, S., Govindarajan,N., Brandt, K., Zhang, Y., Chang, W., Ampatzis, K., and Kizil, C. (2020). Neuron-glia interaction through Serotonin-BDNF-NGFR axis enables regenerative neurogenesis in Alzheimer’s model of adult zebrafish brain.. PLoS Biol 18, e3000585.10.1371 / journal.pbio.3000585. 106 4929-9458-4176v.2

[0505] 47. Cosacak, M.I., Bhattarai, P., Reinhardt, S., Petzold, A., Dahl, A., Zhang, Y., andKizil, C. (2019). Single-Cell Transcriptomics Analyses of Neural Stem Cell Heterogeneity and Contextual Plasticity in a Zebrafish Brain Model of Amyloid Toxicity. Cell Rep 27, 1307-1318 e1303.10.1016 / j.celrep.2019.03.090.

[0506] 48. Cosacak, M.I., Bhattarai, P., Bocova, L., Dzewas, T., Mashkaryan, V.,Papadimitriou, C., Brandt, K., Hollak, H., Antos, C.L., and Kizil, C. (2017). Human TAUP301L overexpression results in TAU hyperphosphorylation without neurofibrillary tangles in adult zebrafish brain Scientific Reports 7, 12959.10.1038 / s41598-017-13311-5.

[0507] 49. Bhattarai, P., Thomas, A.K., Cosacak, M.I., Papadimitriou, C., Mashkaryan, V.,Zhang, Y., and Kizil, C. (2017). Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain. Journal of Visualized Experiments 128.10.3791 / 56014.

[0508] 50. Bhattarai, P., Thomas, A.K., Papadimitriou, C., Cosacak, M.I., Mashkaryan, V.,Froc, C., Kurth, T., Dahl, A., Zhang, Y., and Kizil, C. (2016). IL4 / STAT6 signaling activates neural stem cell proliferation and neurogenesis upon Amyloid-β42 aggregation in adult zebrafish brain. Cell Reports 17, 941–948.10.1016 / j.celrep.2016.09.075.

[0509] 51. Papadimitriou, C., Celikkaya, H., Cosacak, M.I., Mashkaryan, V., Bray, L.,Bhattarai, P., Brandt, K., Hollak, H., Chen, X., He, S., et al. (2018). 3D Culture Method for Alzheimer's Disease Modeling Reveals Interleukin-4 Rescues Abeta42-Induced Loss of Human Neural Stem Cell Plasticity. Dev Cell 46, 85-101 e108.10.1016 / j.devcel.2018.06.005.

[0510] 52. Sastry, G.M., Adzhigirey, M., Day, T., Annabhimoju, R., and Sherman, W. (2013).Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J Comput Aided Mol Des 27, 221-234.10.1007 / s10822-013-9644-8.

[0511] 53. Jacobson, M.P., Pincus, D.L., Rapp, C.S., Day, T.J., Honig, B., Shaw, D.E., andFriesner, R.A. (2004). A hierarchical approach to all-atom protein loop prediction. Proteins 55, 351-367.10.1002 / prot.10613.

[0512] 54. Jacobson, M.P., Friesner, R.A., Xiang, Z., and Honig, B. (2002). On the role of thecrystal environment in determining protein side-chain conformations. J Mol Biol 320, 597-608. 10.1016 / s0022-2836(02)00470-9.

[0513] 55. Olsson, M.H., Sondergaard, C.R., Rostkowski, M., and Jensen, J.H. (2011).PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical pKa Predictions. J Chem Theory Comput 7, 525-537.10.1021 / ct100578z. 107 4929-9458-4176v.2

[0514] 56. Sondergaard, C.R., Olsson, M.H., Rostkowski, M., and Jensen, J.H. (2011).Improved Treatment of Ligands and Coupling Effects in Empirical Calculation and Rationalization of pKa Values. J Chem Theory Comput 7, 2284-2295.10.1021 / ct200133y.

[0515] 57. Lu, C., Wu, C., Ghoreishi, D., Chen, W., Wang, L., Damm, W., Ross, G.A.,Dahlgren, M.K., Russell, E., Von Bargen, C.D., et al. (2021). OPLS4: Improving Force Field Accuracy on Challenging Regimes of Chemical Space. J Chem Theory Comput 17, 4291-4300. 10.1021 / acs.jctc.1c00302.

[0516] 58. Friesner, R.A., Murphy, R.B., Repasky, M.P., Frye, L.L., Greenwood, J.R.,Halgren, T.A., Sanschagrin, P.C., and Mainz, D.T. (2006). Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med Chem 49, 6177-6196.10.1021 / jm051256o.

[0517] 59. Shelley, J.C., Cholleti, A., Frye, L.L., Greenwood, J.R., Timlin, M.R., andUchimaya, M. (2007). Epik: a software program for pK( a ) prediction and protonation state generation for drug-like molecules. J Comput Aided Mol Des 21, 681-691.10.1007 / s10822-007- 9133-z.

[0518] 60. Friesner, R.A., Banks, J.L., Murphy, R.B., Halgren, T.A., Klicic, J.J., Mainz, D.T.,Repasky, M.P., Knoll, E.H., Shelley, M., Perry, J.K., et al. (2004). Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem 47, 1739-1749.10.1021 / jm0306430.

[0519] 61. Sabrina Simoes, J.L.N., Gallen Triana-Baltzer, Setareh Moughadam, Emily I Chen,Milankumar Kothiya, Yasir H Qureshi, Vivek Patel, Lawrence S Honig, Hartmuth Kolb, Scott A Small (2020). Tau and other proteins found in Alzheimer's disease spinal fluid are linked to retromer-mediated endosomal traffic in mice and humans. Sci Transl Med 12, eaba6334. 10.1126 / scitranslmed.aba6334.

[0520] 62. Cosacak, M.I., Bhattarai, P., De Jager, P.L., Menon, V., Tosto, G., and Kizil, C.(2022). Single Cell / Nucleus Transcriptomics Comparison in Zebrafish and Humans Reveals Common and Distinct Molecular Responses to Alzheimer’s Disease. Cells 11, 1807. 10.3390 / cells11111807. 108 4929-9458-4176v.2

Claims

CLAIMS:

1. A method for treating, or for delaying or reducing the development of, a neurodegenerativedisease, comprising the step of administering to a subject in need thereof an effective amount of a compound having the following structure: whereinatom Z is present or absent, and when present is a carbon bound to A, and when absent the two N atoms of the ring are directly bound to each other, wherein when Z is present: bonds ^^^^^^^^^^, and ^ are present, and bonds ^ and ^ are absent; A has the following structure: ,E is CH2, O, or S; and R9are R10are each, independently, H, optionally substituted or unsubstituted C1- C6 alkyl, C3-C12 cycloalkyl, aromatic, or alkylaromatic, wherein R9are R10are each independently optionally substituted or unsubstituted with halo, -OH, -SH, alkyl, alkyl-OH, alkyl-SH, alkyl-NH2, - O-alkyl, -S-alkyl, or -NHx(alkyl)y where x + y = 2; X is a C atom; 109 4929-9458-4176v.2Y is O or NR6; wherein when Y is O, R1is H, an optionally substituted or unsubstituted C1-C6 alkyl, aromatic, alkyl carbonyl, alkyl carboxy, alkyl N-amido, or alkylaromatic group, wherein R1is optionally substituted or unsubstituted with halo, -OH, -SH, C1-C6 alkyl, C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S- alkyl, -NHx(alkyl)ywhere x + y = 2, -C(O)NH2, or -SO2NH2; and wherein when Y is NR6, R1is H, alkyl, aromatic, alkyl carbonyl, alkyl carboxy, alkyl N-amido, or alkylaromatic; R6is H, alkyl, alkyl carbonyl, alkyl N-amido, alkylaromatic, or C(O)R16; or , wherein R16is C1-C6 alkyl; R1and R6or R16may, together, form a 5- to 8-membered optionally substitututed or unsubstituted unsaturated heterocycle, wherein the heterocycle is optionally substituted with R7wherein R7is a C1-C6alkyl or an alkylamide and R2, R3, R4, and R5are, independently, H, halo, -OH, alkyl or -O-alkyl; wherein when Z is absent: bonds ^ and ^ are present, and bonds ^^^^^^^^^^^^and ^ are absent; X is a C or N atom; R2is H or a optionally substituted or unsubstituted benzoyl; R3, R4, and R5are H; Y is C(O)NHR8, and R8is H, C1-C6alkyl or alkyl aromatic; 110 4929-9458-4176v.2or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

2. The method of Claim 1, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Z is present.

3. The method of Claim 2, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R9is H or an optionally substituted or unsubstituted C1-C6alkyl.

4. The method of Claim 2, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is H or an optionally substituted or unsubstituted C3-C12 cycloalkyl, aromatic, or alkylaromatic group.

5. The method of Claim 4, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the cycloalkyl, alkylaromatic, or aromatic group is a monocycle or bicycle.

6. The method of Claim 5, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is an aromatic group.

7. The method of Claim 6, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is an optionally substituted or unsubstituted phenyl or heteroaromatic ring.

8. The method of Claim 7, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R10is a heteroaromatic group containing, selected independently, at least one of N, O, or S. 111 4929-9458-4176v.

29. The method of any of Claims 1-8, wherein in the compound or pharmaceuticallyacceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

10. The method of Claim 9, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, Y is O.

11. The method of Claim 10, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, the compound has the following structure: .

12. The method of Claim 11, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, E is S.

13. The method of Claim 12, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1is an optionally substituted or unsubstituted mono- or bicyclic alkylaromatic group. 112 4929-9458-4176v.

214. The method of Claim 13, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1is an optionally substituted or unsubstituted monocyclic alkylaromatic group.

15. The method of Claim 14, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1is an optionally substituted or unsubstituted alkylaromatic, wherein the alkylaromatic is a carbocycle or a heterocycle.

16. The method of Claim 15, wherein in the compound or pharmaceutically acceptable salt orpharmaceutically acceptable salt hydrate or deuterated analog thereof, R1is an optionally substituted or unsubstituted alkylphenyl group.

17. A method for treating, or for delaying or for reducing the development of, aneurodegenerative disease, comprising the step of administering to a subject in need thereof an effective amount of a compound having the following structure:R17is an optionally substituted or unsubstituted mono or bicyclic cycloalkyl or aromatic group; wherein R17is optionally substituted with halo, -OH, -SH, C1-C4 alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, or -NHx(alkyl)ywhere x + y = 2; R18is an optionally substituted or unsubstituted mono or bicyclic cycloalkyl or aromatic group; wherein R18is optionally substituted with halo, -OH, -SH, C1-C4 alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, or -NHx(alkyl)ywhere x + y = 2; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof. 113 4929-9458-4176v.

218. A method for treating, or for delaying or for reducing the development of, aneurodegenerative disease, comprising the step of administering to a subject in need thereof an effective amount of a compound having the following structure:wherein R19and R20are, independently, H or C1-C12 alkyl;R19and R20may together form a 5-12-membered optionally substituted or unsubstituted saturated or unsaturated mono- or bicyclic heterocycle, wherein R19and R20are optionally independently substituted with halo, - OH, -SH, C1-C4 alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S- alkyl, -NHx(alkyl)y where x + y = 2, or -C(O)-O-alkyl; R21is a 5- or 6-membered optionally substituted or unsubstituted aromatic group ; or wherein R21is optionally independently substituted with halo, -OH, -SH, C1-C4 alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, -NHx(alkyl)y where x + y = 2, or -C(O)-O-alkyl; or a pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof.

19. A method for treating, or for reducing the development of, a neurodegenerative disease,comprising the step of administering to a subject in need thereof an effective amount of a compound is a human integrin antagonist.

20. A compound having the following structure:114 4929-9458-4176v.2wherein atom Z is present or absent, and when present is a carbon bound to A, and when absent the two N atoms of the ring are directly bound to each other, wherein when Z is present: bonds ^^^^^^^^^^, and ^ are present, and bonds ^ and ^ are absent; A has the following structure: ,E is CH2, O, or S; and R9are R10are each, independently, H, optionally substituted or unsubstituted C1- C6alkyl, C3-C12cycloalkyl, aromatic, or alkylaromatic, wherein R9are R10are each independently optionally substituted or unsubstituted with halo, -OH, -SH, alkyl, alkyl-OH, alkyl-SH, alkyl-NH2, -O-alkyl, -S-alkyl, or -NHx(alkyl)ywhere x + y = 2; X is a C atom; Y is NR6or O; wherein when Y is O, R1is H, an optionally substituted or unsubstituted C1-C6 alkyl, aromatic, alkyl carbonyl, alkyl carboxy, alkyl N-amido, or alkylaromatic group, 115 4929-9458-4176v.2wherein R1is optionally substituted or unsubstituted with halo, -OH, -SH, C1-C6 alkyl, C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S- alkyl, -NHx(alkyl)y where x + y = 2, -C(O)NH2, or -SO2NH2; and wherein when Y is NR6, R1is H, alkyl, alkyl carbonyl, alkyl carboxy, alkyl N-amido, or alkylaromatic; R6is H, alkyl, alkyl carbonyl, alkyl N-amido, alkylaromatic or C(O)R16; or wherein R16is C1-C6alkyl; R1and R6or R16may, together, form an optionally substituted or unsubstituted unsaturated heterocycle, wherein the heterocycle is optionally substituted with R7, wherein R7is a C1-C6 alkyl or an alkylamide; and R2, R3, R4, and R5are, independently, H, -OH, -SH, C1-C4alkyl, -C1-C4-OH, C1-C4-SH, C1-C4-NH2, -O-alkyl, -S-alkyl, or -NHx(alkyl)y where x + y = 2; wherein when E is -S: at least one of R2, R3, R4, and R5are -OH, -SH, C1-C4alkyl, -C1-C4-OH, C1-C4- SH, C1-C4-NH2, -S-alkyl, or -NHx(alkyl)y where x + y = 2; and up to three of R2, R3, R4, and R5are -H wherein when Z is absent: bonds ^ and ^ are present, and bonds ^^^^^^^^^^, and ^ are absent; X is a C or N atom; R3, R4, and R5are H; Y is C(O)NHR8, wherein R8is H, C1-C6 alkyl or alkyl aromatic; 116 4929-9458-4176v.2wherein when X is a C atom: R2is H, or an optionally substituted or unsubstituted benzoyl, benzyl or naphthoyl; R8is H, or a substituted or unsubstituted C1-C6 alkyl, aromatic, or alkyl aromatic; wherein when X is an N atom: R2is H, poly-substituted benzoyl, or optionally substituted or unsubstituted benzyl or naphthoyl; R8is H, or a substituted or unsubstituted C1-C6alkyl, aromatic, or substituted alkyl aromatic; or pharmaceutically acceptable salt or pharmaceutically acceptable salt hydrate or deuterated analog thereof. 117 4929-9458-4176v.2

Images