Compounds, compositions, and method of use to inhibit TAU protein and alpha-synuclein aggregation

Abstract

Compounds comprising an amide-linked coumarin scaffold, compositions comprising same, and method of using such compounds and compositions to inhibit tubulin-associated unit (tau) protein aggregation or alpha-synuclein ( α-syn) protein aggregation in a subject having, or at risk for, tau protein aggregation or α-syn protein aggregation, respectively.

Description

70835-02 COMPOUNDS, COMPOSITIONS, AND METHOD OF USE TO INHIBIT TAU PROTEIN AND ALPHA-SYNUCLEIN AGGREGATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from U.S. Appl. No. 63 / 685,312, filed August 21, 2025, which is incorporated by reference as if fully set forth herein. STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under AG070447 and AG071985 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0003] This disclosure relates to compounds comprising an amide-linked coumarin scaffold, compositions comprising same, and the use of such compounds and compositions to inhibittubulin-associated unit (tau) protein and alpha- -syn) protein aggregation, including,but not limited to, neurofibrillary tangles (NFTs), associated with tauopathies (e.g., Alzheimer’s disease, Downs syndrome, progressive supranuclear palsy, and traumatic brain injury) and Lewy bodies, associated with synucleinopathies (e.g., Parkinson’s disease, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA)). BACKGROUND

[0004] Protein fibrillization forms the basis of neurodegenerative disorders like Alzheimer’s disease (AD) and Parkinson’s disease (PD).1Fibril formation is a pathological event in which normally soluble proteins misfold and subsequently aggregate into insoluble fibrillar structures. These fibrils accumulate within neuronal tissues, disrupting cellular functions and progressively damaging nerve cells.2In Alzheimer's disease, the two primary pathological hallmarks widelyrecognized are the accumulation of toxic amyloid- 3 which form extracellularplaques, and the development of neurofibrillary tangles (NFT) derived from hyperphosphorylated tau proteins.4Both amyloid plaques and neurofibrillary tangles (NFTs) can aggregate between neurons, impairing cell signaling and contributing to the degeneration of synaptic neurites, whichleads to a failure in information transmission.5, 6 These conditions are considered central to the70835-02 plaques appear before NFTs in AD patients, epidemiological studies indicate that the severity of cognitive deficits is closely associated with the regional distribution of NFTs.7, 8

[0005] NFTs are formed by the aggregation of tau protein, which generally associates with and stabilizes microtubules in neurons.9When tau proteins undergo hyperphosphorylation, they detach from the microtubules to which they were originally bound. This detachment increases their propensity to interact with other tau monomers. As these individual tau proteins bind together, they form small oligomeric structures.10-sheet structure and form granular-like insoluble aggregates that merge to form larger, more complex aggregates (i.e., tau fibrils), which eventually develop into NFTs.11NFTs initially manifest in the trans- entorhinal region of the temporal lobe. Over time, they progressively spread to limbic regions, including the hippocampus, and ultimately extend their reach to encompass extensive areas of the neocortex, further driving the progression of AD.12

[0006] Parkinson's disease is distinguished by the pathological process wherein alpha-synuclein -syn) aggregates to form characteristic Lewy bodies (LB), contributing to the disease's progression.13-Syn is typically found in its unfolded, monomeric state and is abundantly expressed in various regions of the brain.14Through various post-translational modifications such as phosphorylation, acetylation, glycation, and ubiquitination, -syn undergoes a transformational shift in its shape, adopting a folded structure characterized by alpha-helices. This altered form tends to assemble into dimers and oligomers, marking a critical step in the aggregation process associated with neurodegenerative diseases.14-syn oligomers aggregate into immunoreactive structures within neurons, ultimately leading to the formation of LB.15The resulting Lewy bodies obstruct neurotransmitter transmission and activate pathways that lead to neuronal dysfunction.16, 17

[0007] Existing treatments for Alzheimer's disease (AD) and Parkinson's disease (PD) mainly target symptom alleviation without addressing the root causes of these neurodegenerative disorders. As a result, while they may provide temporary relief, they fail to deliver long-term improvements in overall health and quality of life. An antibody, which apparently stimulates the clearance of amyloid plaques has been approved by the Food and Drug Administration for the treatment of AD. However, it is the formation of the neurofibrillary tangles and the spatiotemporal distribution of the tangles that correlate with the loss of cognition. In view of the foregoing, there70835-02 is an unmet need for a small molecule that can inhibit the formation of oligomers, i.e., early-stage aggregation. Accordingly, it is an object of the present disclosure to provide such small molecules and related compositions, which can be used to inhibit tau protein aggregation and alpha-synuclein( -syn) protein aggregation (e.g., Lewy bodies and Lewy neurites). This and other objects, as wellas inventive features, will be apparent from the detailed description provided herein. SUMMARY

[0008] Provided is a compound of the formula:or a pharmaceutically acceptable salt or hydrate thereof, wherein R1 is (a) phenyl, which can be mono-, di- or tri-substituted with one or more substituents, which can be the same or different, selected from the group consisting of a C1-C6 alkyl, a C1-C6 alkyloxy, a C1-C6 alkylsulfanyl, a hydroxyl, and a halo or (b) a substituent selected from the group consisting of:70835-02R2 is hydrogen or a C1-C6 alkyl. In an embodiment, R1 is a C1-C6 alkyl, it is methyl. In an embodiment, when R1 is a C1-C6 alkyloxy, it is methoxy. In an embodiment, when R1 is a C1-C6 alkylsulfanyl, it is methylsulfanyl. In an embodiment, when R1 is a halo, it is fluoro, bromo, chloro, or iodo. In an embodiment, when R2is a C1-C6alkyl, it is methyl. In an embodiment, R1isand R2is hydrogen. In an embodiment, R1isand R2is hydrogen. In an embodiment, R1 isand R2 is hydrogen. In an embodiment, R1 isand R2 is hydrogen. In an embodiment, R1and R2is hydrogen.

[0009] Also provided is a pharmaceutical composition. The composition comprises an above- described compound, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

[0010] Further provided is a method of inhibiting alpha- -syn) protein aggregation-syn protein aggregation. The method comprises administering to the subject the above-described - syn protein aggregation. In an embodiment, the subject has, or is at risk for, Parkinson’s disease or dementia with Lewy bodies (DLB). BRIEF DESCRIPTION OF THE FIGURES

[0011] Fig. 1. -syn fibril formation. The ThT fibrillation -syn were measured under two conditions: in the absence of additives (0.25%70835-02 from three replicates conducted within the same experiment.

[0012] Figs. 2A-2B. Compounds 13 and 17 dose dependently reduced the -syn fibril formation. The Log(agonist) vs. normalized response (variable slope) correlation analysis conducted with Prism provided a LogEC50 of 1.36 ± 0.58 for compound 13 (Fig. 2A) and 1.30 ± 0.39 for compound 17 (Fig.2B). The curves display a dose-dependent response at concentrations --

[0013] Fig.3. Compound 13 diminished best the aggregation of tau isoform 2N4R, compared with compound 17. ThS fluorescence curves illustrate the aggregation kinetics of tau isoform 2N4R at mM chelex beads. The control sample consisted of 0.25% DMSO, while compounds were tested at 100 Each curve represents the average data obtained from three replicates.

[0014] Fig. 4A. Compounds 13, 15, and 17 successfully -syn oligomers triggered by Tris(bipyridyl)ruthenium(II)chloride (Ru(bpy)3) and ammonium persulfate -Syn, at a concentration of 60 - -syn oligomers of higher molecular weight were visible on Coomassie blue-stained polyacrylamide gels. Additional control samples, which were not exposed to light or lacked the cross-linking agent (Ru(bpy)3), showed no detectable cross-linked products.

[0015] Fig.4B. Compound 13 -syn oligomers. Compound9 (non- -syn fibrils) and compound 19 -syn fibrilformation) reduced partially the formation of oligomers. The oligomers were generated by exposing the -syn monomer with Tris(bipyridyl)ruthenium(II)chloride [Ru(bpy)3]3+ (RTD) and ammonium persulfate under brief light exposure (1 s) in the PICUP crosslinking assay. No incubation was necessary. The protein was used at a concentration of 30 control consisted -syn monomer are located at molecular mass of 15 kDa. The oligomers of higher molecular weight are visible between 35 and 40 kDa as well as between 55 and 180 kDa on Coomassie blue-stained 16% polyacrylamide gels. Additional control samples consisted of the protein not exposed to the light or the cross-linking70835-02 agent (RTD), and resulted in no detectable cross-linked products. Experiments were repeated three times. -syn anti-oligomerization, the compounds were assessed at a initial screening were subsequently tested to determine their dose-response effects on the -syn oligomer formation. Compounds 9 (4-hydroxybenzene), 13 (4,5- dihydroxybenzene), and 19 (3,5-dihydroxybenzene) demonstrated the most significant inhibition -syn oligomerization compared to the control, which contained 0.25% DMSO. In contrast, the other tested compounds (5, 8, 12, 16-18) showed very weak to no inhibitory effect. Compound 17, a 5,6-dihydroxybenzene derivative with strong anti-fibrillar activity, was among the inefficacious compounds. The dose-response studies of the promising compound 13 indicated they all have astrong, concentration- -syn oligomerization (see Figure 5C). The-syn anti-oligomer effect of compound 13 0N4R 13 and 17 as well as their nondemethylated counterparts (compounds 12 and 16) to assess the tau anti-oligomer activity. However, these coumarin-amide based derivatives did not halt the formation of tau 0N4R oligomers. Experiments were repeated twice with tau isoform 0N4R and one time with the tau isoform 2N4R (data not shown).

[0016] Figs.5A-5B. Gel electrophoresis, demonstrating the concentration-dependent inhibitory effect of compounds 9 (Fig.5A), 13 (Fig.5A), and 17 (Fig.5B). All three compounds reduced the -syn oligomers in a dose-dependent manner, as evidenced by the decreased intensity -syn (6 µM) was incubated with the compounds, and the PICUP assay was used to induce oligomer formation. DMSO (0.125%) served as the control.

[0017] Fig. 5C. Compound 13, a coumarin-amide-4,5- -synoligomer formation in a concentration-dependent manner, as evidenced by the decreased intensity of bands between the 35 and 40 kDa markers. The anti-oligomer effect disappeared with the 13 -syn (6put in the presence of different concentration of compound, and the PICUP assay was performed immediately to induce oligomer formation. DMSO (0.125%) served as the control. Experiments were repeated twice.70835-02

[0018] Fig. 6A. The dose-dependent inhibitory effect of compound 17 oligomerization was examined using the PICUP assay. No higher molecular weight oligomers were observed in the control samples, which did not include Ru(bpy)3and were not subjected to light exposure.

[0019] Fig. 6B. Compounds 12, 13, 16, and 17 oligomerization as assessed by the PICUP assay. Higher molecular weight oligomers (at 180 kDa) were generated with the control (0.25% DMSO) and remained with the compound treatments. The monomeric band present at 55 kDa was reduced for all conditions except for the control no light and no RTD (crosslinking reagent: Tris(bipyridyl)ruthenium(II)chloride [Ru(bpy)3]3+). Compounds 12 and 16 (the methoxy counterparts of compounds 13 and 17) were used as negative -syn oligomer and fibril formation. All compounds were tested at light exposure for 15 seconds. Experiments were repeated twice. Similar results were obtained with tau isoform 2N4R (data not shown).

[0020] Fig.7. Treatment with compounds 13 and 17 led to a decrease in the quantity and length -syn fibrils, as observed via transmission electron microscopy (TEM). After completing the ThT fibril kinetic formation assay, three replicate samples containing 6 µM alpha-synuclein and 100 µM compounds were retrieved from the 96-well plate. Subsequently, these samples were applied onto copper grids and examined using transmission electron microscopy (TEM). Scale bars = 200 nm.

[0021] Fig. 8. Transmission electron microscopy (TEM) images showing the anti-aggregation effect of compounds 12, 13, and 17 on tau 2N4R fibrils. DMSO (0.25%; 'CTRL') and the compounds (tested at 100 µM) were incubated with 12 µM tau for 5 days at 37oC in the presence acid. Compound 12 was used as the negative control. TEM images (scale bar: 200 nm) showed fewer tau fibrils in the samples treated with compounds 13 and 17 compared to the controls.

[0022] Figs. 9A-9B. Compounds 13 and 17 prevent S inclusion formation. M17D cells expressing the inclusion-prone S-3K::YFP fusion protein (dox-inducible) were treated with 0.1% DMSO (vehicle; “0 M”) as well as 5, 10, 20, and 40 M of compounds 13, 15 and 17 at t = 24 hours after plating. Cells were induced with doxycycline at t = 48 hours. Fig.9A: Incucyte-based analysis of punctate YFP signals relative to 0.1% DMSO was done at t = 96 hours (N = 370835-02 independent experiments, n = 6-18 individual wells total (0 M, n = 18; 10 M, n = 6; all other concentrations, n = 12). Fig.9B: Same as Fig.9A, but confluence fold changes relative to DMSO vehicle (0 M) were plotted.

[0023] Fig.9C. Representative IncuCyte -rich YFP-positive -changes relative to DMSO control + / - standard deviation. One-way ANOVA, Dunnett’s post-hoc test; , p < 0.05; , p < 0.01; , p < 0.001; , p < 0.0001; ns = non-significant.

[0024] Fig. 10. Compound 13 reduced the tau aggregation intracellularly, using a cell-based assay. Quantification of GFP-positive cell populations in the tau aggregation assay. ExpiCHO cells were transfected with split GFP-Tau fusion constructs (pmGFP11C-Tau and pmGFP10CTau) and analysis was performed to assess GFP fluorescence intensity, indicative of Tau aggregation, and cell viability was confirmed by ethidium homodimer-1 exclusion. Statistical analysis was conducted using GraphPad Prism. Data represent the mean ± standard deviation (SD) from three independent replicates. Significant differences between groups are indicated (****, p < 0.0001). DETAILED DESCRIPTION

[0025] The present disclosure is based on the investigation of the potential of amide-linked coumarin compounds as multitarget inhibitors against the aggregation of alpha-synuclein and tau proteins. Coumarin exists naturally in plants as an aromatic lactone compound.18It has been noted for its diverse range of biological activities depending on its chemical composition. Certain coumarin derivatives are recognized for their antioxidant19, antibacterial20, anti-proliferative21, and anti-cancer properties.22

[0026] Various aromatic moieties including aminoindoles, methoxy-substituted phenyl, and polyhydroxy aromatic functionalities were linked to the amide linker of the coumarin scaffold. Biophysical methods such as thioflavin T fluorescence assays (ThT), photo-induced cross-linking of unmodified proteins (PICUP), survival assays, and electron microscopic observations were used to evaluate the effect of analogs on inhibition of -syn and tau aggregation. The results showed that the dihydroxy and aminoindoles have promising effects on the inhibition of -syn and tau protein aggregation. In fibril formation assays using ThT and TEM, two compounds (13 and 17)70835-02 showed the best activity, meeting the cut-off criterion of 5% fibril reduction or less. Both compounds reduced the formation of -syn oligomers and in the inclusion formation assay using the M17D neuroblastoma cells expressing , compound 17 was the best in reducing - syn oligomers at low micromolar concentration.

[0027] In view of the above, provided is a compound of the formula:or a pharmaceutically acceptable salt or hydrate thereof, wherein R1is (a) phenyl, which can be mono-, di- or tri-substituted with one or more substituents, which can be the same or different, selected from the group consisting of a C1-C6alkyl, a C1-C6 alkyloxy, a C1-C6 alkylsulfanyl, a hydroxyl, and a halo or (b) a substituent selected from the group consisting of:, wherein the wavy line represents the point of attachment to of R1 to the nitrogen atom; and70835-02 R2 is hydrogen or a C1-C6 alkyl. In an embodiment, R1 is mono-, di- or tri-substituted with a C1- C6 alkyl (e.g., wherein one, two or three R1is / are CH3). In an embodiment, R1 is mono-, di- or tri-substituted with a C1-C6 alkyloxy (e.g., wherein one, two or three R1is / are OCH3). In an embodiment, when R1is mono-, di- or tri-substituted with a C1-C6alkylsulfanyl (e.g., wherein one, two or three R1is SCH3). In an embodiment, R1is mono-, di- or tri-substituted with a halo (e.g., wherein one, two or three R1is / are F, Cl, Br or I or combinations thereof). In an embodiment, when R2 is a C1-C6 alkyl, it is methyl. In an embodiment, R1 is

[0028] Also provided are compounds of the formula (I):or a pharmaceutically acceptable salt or hydrate thereof, wherein: R3is 6- to 13-membered aryl substituted with (R4)nor 6- to 10-membered heteroaryl (wherein 1- 4 heteroaryl members are independently selected from N, O, and S) substituted with (R4)n; n is 0, 1, 2 or 3; R2 is hydrogen or a C1-C6 alkyl; and R4 in each instance is independently OH, halo, 6- to 10-membered heterocycloalkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), 6- to 10-membered heterocycloalkyl alkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), S(O)xR5(wherein R5is H, C1-C6alkyl or 6- to 10-membered heterocycloalkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S) and x is 0, 1 or 2), -SCN, -NCS, C1-C6-alkyl, C1-C6-alkoxy, C1-C6-haloalkyl, 6- to 10-membered aryl, 6- to70835-02 10-membered aryloxy, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), NHC(O)-6- to 10-membered heterocycloalkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), C(O)NH- 6- to 10-membered heterocycloalkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S) or two R4groups, together with carbon atoms to which they are attached, form a 5- to 6-membered heterocycloalkyl (wherein 1 or 2 heterocycloalkyl members are independently selected from N, O, and S).

[0029] In one example, the compound of the formula (I) is a compound wherein R3 is 6- to 13- membered aryl substituted with (R4)n and the compound is of the formula (II):or a pharmaceutically acceptable salt or hydrate thereof.

[0030] In the compounds of the formula (I) and / or (II), R4can be OH, halo, C1-C6-alkyl, C1-C6- alkoxy, C1-C6-haloalkyl, S(O)xR5(wherein R5is H, C1-C6alkyl or 6-membered heterocycloalkyl), 6-membered aryloxy, 6-membered heterocycloalkyl, 6-membered heterocycloalkyl alky or 5- membered heteroaryl. In examples, R4can be OH, CH3, CH3O, SCH3, Cl, F, Br, piperidinyl, morpholinyl, thiomorpholino, piperazinyl, SO2-6-membered heterocycloalkyl, phenoxy, SCN, thiophenyl, CF3, or two R4 groups, together with carbon atoms to which they are attached, form:.

[0031] In one example, the compound of the formula (I) is a compound wherein R3is 6- to 10- membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S) substituted with (R4)n and the compound is of the formula (III):such as:70835-02or a pharmaceutically acceptable salt or hydrate thereof.

[0032] Examples of compounds of the formula (I) include:which is independently substituted with (R4)n, such as:70835-02 570835-02hydrate thereof, each of which is independently substituted with (R4)n.

[0033] In the compounds of the formulae (I)-(III), R2 can be H. Alternatively, in the compounds of the formula (I)-(III), R2 can be CH3, CH2CH3 or CH(CH3)2. Alternatively, or in addition, n can be 0 or 1. In one example n is 0. In another example, n is 1. In another example, n is 2, wherein each R4can be the same or different. In yet another exmaple, n is 3, wherein each R4can be the same or different.

[0034] In one example, n is 1 and R4is C1-C6alkyl, such as CH3.

[0035] In another example, n is 1 and R4 is halo, such as F, Cl or Br.

[0036] In another example, n is 1 and R4 is OH.

[0037] In an example, n is 1 and R4 is C1-C6-alkoxy, such as OCH3.

[0038] In an example, n is 1 and R4 is C1-C6-haloalkyl, such as CF3. example, n is 1 and R4is 6- to 10-membered heterocycloalkyl, such as70835-02 wherein X1 is CR6 (wherein R6 is H or C1-C6 alkyl) or N; and X2 is (CR6)2 (wherein each R6 is independently H or C1-C6 alkyl), NR6, O or S. In an example, X1 is N and X2 is (CR6)2, wherein each R6 is H or X1 is CR6 and X2 is NR6, wherein each R6 is H. In an example, X1 is N and X2 is NR6, wherein R6is C1-C6-alkyl, such as CH3. In an example, X1is N and X2is S.

[0040] In another example, n is 1 and R4is 6- to 10-membered heterocycloalkyl alkyl-O-, suchwherein y is 1-6; X1is CR6(wherein R6is H or C1-C6alkyl) or N; and X2is (CR6)2(wherein each R6is independently H or C1-C6alkyl), NR6, O or S. In an example, y is 1, X1is CR6(wherein R6 is H), and X2 is NR6, wherein R6 is C1-C6 alkyl. In an example, y is 1, X1 is CR6 (wherein R6 is H), and X2 is NR6, wherein R6 is CH3. example, n is 1 and R4 is 5- to 10-membered heteroaryl, such aswherein X3 is O, S or NR6 (wherein R6 is H or C1-C6 alkyl). In an example, X3 is S.

[0042] In an example, n is 1 and R4 is NHC(O)-6- to 10-membered heterocycloalkyl, such as.

[0043] In an example, n is 1 and R4is C(O)NH-6- to 10-membered heterocycloalkyl, such as.

[0044] In one example, n is 2 and R4is C1-C6 alkyl, such as CH3.

[0045] In another example, n is 2 and R4 is halo, such as F, Cl or Br.

[0046] In another example, n is 2 and R4 is OH.

[0047] In an example, n is 2 and R4 is C1-C6-alkoxy, such as OCH3.

[0048] In an example, n is 2 and R4is C1-C6-haloalkyl, such as CF3.

[0049] In one example, n is 3 and R4is C1-C6alkyl, such as CH3.

[0050] In another example, n is 3 and R4is halo, such as F, Cl or Br.

[0051] In another example, n is 3 and R4is OH.70835-02

[0052] In an example, n is 3 and R4 is C1-C6-alkoxy, such as OCH3.

[0053] In an example, n is 3 and R4 is C1-C6-haloalkyl, such as CF3.

[0054] In one example, n is 1 and R4 is S(O)xR5. In one example, x is 0 and R5 is C1-C6 alkyl, such as CH3. In one example, x is 2 and R5is 6- to 10-membered heterocycloalkyl, such as, wherein X1 is CR6 (wherein R6 is H or C1-C6 alkyl) or N; and X2 is (CR6)2 (wherein each R6 is independently H or C1-C6 alkyl), NR6, O or S. In one example, X1 is N and X2 is NR6, wherein R6 is H or C1-C6 alkyl. In one example, R6 is C1-C6 alkyl, such as CH3.

[0055] The above compounds include isotopic variants and compounds in which one or more hydrogen atoms have been substituted with deuterium. The compounds may contain one or more chiral centers or may otherwise be capable of existing as multiple stereoisomers. In one embodiment, the compounds are not limited to any particular stereochemical requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be optically pure, or may be any of a variety of stereoisomeric mixtures, including racemic and other mixtures of enantiomers, other mixtures of diastereomers, and the like. Such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including mixtures of stereochemical configuration at one or more other chiral centers.

[0056] Similarly, the compounds may include geometric centers, such as cis, trans isomers, diastereomers, enantiomers, and E and Z double bonds. In another embodiment, the compounds are not limited to any particular geometric isomer requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be pure, or may be any of a variety of geometric isomer mixtures. Such mixtures of geometric isomers may include a single configuration at one or more double bonds and chiral carbons, while including mixtures of geometry at one or more other double bonds and chiral carbons.

[0057] The above compounds, and pharmaceutically acceptable salts and solvates, such as hydrates, thereof, can be synthesized in accordance with methods known in the art and exemplified herein. See, e.g., Example 1.

[0058] The compounds can be used to inhibit the aggregation of proteins prone to aggregate in a state of disease. The ability of such compounds to inhibit aggregation can include, but is not limited to, inhibition of oligomer formation, inhibition of fibril formation, and / or inhibition of70835-02 -syn inclusions. “Aggregation” and its derivatives are used herein to refer to all such inhibitory actions for ease of reference. Proteins prone to aggregate include, but are notlimited to, islet amyloid polypeptide, amyloid- - -syn), tubulin associated unit(tau), and transthyretin. The tau can be tau isoform 0N4R, 1N4R, 2N4R, 0N3R, 1N3R, and / or 2N3R with and without post-translational changes (such as phosphorylation). The compounds -syn. Diseases involving protein aggregation include, but are not limited to, AA amyloidosis, Alzheimer's disease, monoclonal immunoglobulin light-chain amyloidosis, Huntington's disease, Parkinson's disease, Creutzfeldt- Jacob disease, prion disorders, amyotrophic lateral sclerosis, type 2 diabetes, or transthyretin amyloidosis. The compounds described herein can be used to inhibit the aggregation of -syn in a subject having, or at risk for, Alzheimer’s disease, dementia with Lewy bodies (DLB), or multiple system atrophy (MSA). The compounds described herein also can be used to inhibit the -syn inclusions in a subject with a neuroblastoma.

[0059] The compounds can be used to inhibit tau protein aggregation in tauopathies. Tauopathies are a group of disorders that result from abnormal tau phosphorylation, abnormal levels of tau, abnormal tau splicing, and mutations in the tau gene, for example. Neurodegenerative diseases have been classified based on this protein accumulation. Tauopathies encompass more than 20 clinicopathological conditions, including Alzheimer’s disease (AD), which is the most common tauopathy. Other tauopathies include, but are not limited to, familial AD, primary age-related tauopathy (PART), Creutzfeldt-Jacob disease, dementia pugilistica, Gerstmann-Straussler- Scheinker disease (GSS), inclusion-body myositis, cortico-basal degeneration (CBD), Picks disease (PiD), progressive supranuclear palsy (also known as Steele, Richardson, and Olszewski disorder), Down syndrome, Parkinsonism with dementia, myotonic dystrophy, prion protein cerebral amyloid angiopathy, traumatic brain injury (TBI), amyotrophic lateral sclerosis (ALS), Parkinsonism-dementia complex of Guam, non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain disease, diffuse neurofibrillary tangles with calcification, frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), Haller-vorden-Spatz disease, multiple system atrophy (MSA), Niemann-Pick disease type C, pallido-ponto-nigral degeneration, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle predominant dementia, postencephalitic Parkinsonism, myotonic dystrophy, subacute sclerosis panencephalopathy, mutations in LRRK2,70835-02 chronic traumatic encephalopathy (CTE), familial British dementia, familial Danish dementia, other frontotemporal lobar degenerations, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, white matter tauopathy with globular glial inclusions, epilepsy, Lewy body dementia (LBD), mild cognitive impairment (MCI), multiple sclerosis, Parkinson’s disease, HIV-related dementia, adult onset diabetes, senile cardiac amyloidosis, glaucoma, ischemic stroke, psychosis in AD, Huntington’s disease, and prion diseases with tangles. The majority of neurodegenerative diseases are characterized by the deposition of insoluble protein in cells of the neuromuscular system.

[0060] The compounds can be formulated as pharmaceutical compositions comprising a pharmaceutically acceptable carrier using methods well-known in the art. “Carrier” is used generically herein to refer to pharmaceutically acceptable carriers, diluents, adjuvants, and excipients. See, e.g., Remington: The Science and Practice of Pharmacy, 23rdedition, October 30, 2020, Adeboye Adejare, ed. In view of the foregoing, also provided is a pharmaceutical composition. The composition comprises an above-described compound, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

[0061] Further in view of the above, provided is a method of inhibiting alpha- -syn)-syn protein aggregation. The method comprises administering to the subject the above-described pharmaceutical composition in an -syn protein aggregation. In an embodiment, the subject has, or is at risk for, Parkinson’s disease or dementia with Lewy bodies (DLB).

[0062] Any suitable route of administration can be used in the above methods. Examples include, but are not limited to, oral, parenteral, intravenous, intracranial, intracerebroventricular, and intracerebral. An effective amount can be determined by one of ordinary skill in the art using dosage range determining methods known in the art. Typically, a physician (or veterinarian for non-human subjects) will determine the actual dosage, which will be most suitable for an individual subject. The specific dose level and frequency of dosage for an individual may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, gender, diet, mode and time of administration, rate of excretion, other administered drugs, and the severity of the particular condition. The compound / compositions described herein can be administered with other biologically active compounds as appropriate.70835-02

[0063] The terms “substituted,” “substituent,” and “functional group” refer to a group that can be or is substituted onto a molecule or onto another group (e.g., on an aryl or an alkyl group). Examples of substituents include, but are not limited to, a halogen (e.g., F, Cl, Br, and I), OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, -(CH2)0-2P(O)(OR)2, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)0-2N(R)C(O)R, (CH2)0-2N(R)C(O)OR, (CH2)0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, or C(=NOR)R wherein each R can be, independently, hydrogen, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl, wherein any alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl or two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl, which can be mono- or independently multi-substituted.

[0064] The term “alkyl” as used herein refers to substituted or unsubstituted straight chain and branched mono- or divalent alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms (C1-C40), 1 to about 20 carbon atoms (C1-C20), 1 to 12 carbons (C1-C12), 1 to 8 carbon atoms (C1-C8), or, in some embodiments, from 1 to 6 carbon atoms (C1-C6). Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and ante-isoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0065] The term “alkenyl” as used herein refers to substituted or unsubstituted straight chain and branched mono- or divalent alkenyl groups and cycloalkenyl groups having at least one double bond and having from 1 to 40 carbon atoms (C1-C40), 1 to about 20 carbon atoms (C1-C20), 1 to 12 carbons (C1-C12), 1 to 8 carbon atoms (C1-C8), or, in some embodiments, from 1 to 6 carbon atoms (C1-C6). Examples of straight chain alkenyl groups include those with from 1 to 8 carbonatoms such as -CH=CH-, -CH=CHCH3, and -CH2CH=CHCH2- groups, wherein the double70835-02 bonds can have an E- or Z-configuration. And when there are multiple bonds, each double bond can, independently, have an E- or a Z-configuration. Examples of branched alkenyl groups include, but are not limited to, -CH=C(CH3)- and CH2C=CH(CH3) groups. Representative substituted alkenyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0066] The term “cycloalkyl” as used herein refers to substituted or unsubstituted cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups can have any number of carbon atoms, e.g., 3 to 8 carbon atoms (C3-C8), 3 to 6 carbon atoms (C3-C6), and 4 to 8 carbon atoms (C4-C8). Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like.

[0067] The term “cycloalkylalkyl” as used herein refers to substituted or unsubstituted alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a cycloalkyl group as defined herein. Representative cycloalkylalkyl groups include, but are not limited to, cyclopentylalkyl.

[0068] The term “alkylcycloalkyl” as used herein refers to substituted or unsubstituted cycloalkyl groups as defined herein in which a hydrogen of a cycloalkyl group as defined herein is replaced with a bond to an alkyl group as defined herein. Representative alkylcycloalkyl groups include, but are not limited to, alkylcyclopropyl.

[0069] The term “acyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which can be part of a substituted or unsubstituted alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. In the special case wherein the carbonyl carbon atom is bonded to a hydrogen, the group is a “formyl” group, an acyl group as the term is defined herein. An acyl group can include 0 to about 12-40, 6-10, 1-5 or 2-5 additional carbon atoms bonded to the carbonyl group. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning here. A nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group70835-02 within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a “haloacyl” group. An example is a trifluoroacetyl group.

[0070] The term “aryl” as used herein refers to substituted or unsubstituted cyclic aromatic hydrocarbons that do not contain heteroatoms in the ring. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons (C6-C14) or from 6 to 10 carbon atoms (C6-C10) in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. “Aryl” and the phrase “aryl group” includes fused ring species including those that include fused aromatic and non-aromatic groups. Accordingly, “aryl” and the phrase “aryl group” include groups of the formula:each of which can be substituted or unsubstituted, such as hydroxy substituted.

[0071] Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or 2-8 substituted naphthyl groups, which can be substituted with carbon or non-carbon groups such as those listed herein.

[0072] The terms “aralkyl” and “arylalkyl” refer to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.

[0073] “Heterocycloalkyl” is a saturated or partially unsaturated non-aromatic monocyclic, bicyclic, tricyclic or polycyclic ring system that has from 3 to 14 members, such as 6 to 10 and 3 to 6 members, in which 1 to 3 carbon atoms in the ring are replaced by heteroatoms of O, S or N. The ring heteroatoms can also include oxidized S or N, such as sulfinyl, sulfonyl, and N-oxides70835-02 of a tertiary ring nitrogen. A heterocycloalkyl can be fused to another ring system, such as with an aryl or heteroaryl of 5-6 ring members. The point of attachment of the heterocycloalkyl ring is at a carbon or heteroatom such that a stable ring is retained. Examples of heterocycloalkyl groups include without limitation morpholino, tetrahydrofuranyl, dihydropyridinyl, piperidinyl, pyrrolidinyl, piperazinyl, dihydrobenzofuryl, and dihydroindolyl. A heterocycloalkyl group can be unsubstituted or optionally substituted with one or more substituents as described herein.

[0074] “Heteroaryl,” alone or in combination with any other moiety described herein, is a monocyclic aromatic ring structure containing 6 to 10, such as 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing one or more, such as 1-4, 1-3, or 1-2, heteroatoms independently selected from the group consisting of O, S, and N. Heteroaryl is also intended to include oxidized S or N, such as sulfinyl, sulfonyl and N-oxide of a tertiary ring nitrogen. A carbon or heteroatom is the point of attachment of the heteroaryl ring structure such that a stable compound is produced. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrazinyl, quinaoxalyl, indolizinyl, benzo[b]thienyl, quinazolinyl, purinyl, indolyl, quinolinyl, pyrimidinyl, pyrrolyl, pyrazolyl, oxazolyl, thiazolyl, thienyl, isoxazolyl, oxathiadiazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazolyl, furanyl, benzofuryl, and indolyl. A heteroaryl group can be unsubstituted or optionally substituted with one or more substituents as described herein.

[0075] The term “alkoxy” refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include one to about 12-20 or about 12-40 carbon atoms bonded to the oxygen atom, can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group is an alkoxy group within the meaning herein. A methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.

[0076] The terms “amine,” “amine group,” “amino,” and “amino group” refer to a substituent of the form -NH2, -NHR, -NR2, or -NR3+, wherein each R is defined herein, and70835-02 protonated forms of each, except for -NR3+, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group.

[0077] An “alkylamino” group includes a monoalkylamino, dialkylamino, and trialkylamino group. An example of a “alkylamino” is -NH-alkyl and -N(alkyl)2.

[0078] An example of a “cycloalkylamino” group is -NH-cycloalkyl and -N(cycloalkyl)2.

[0079] An example of a “cycloalkyl heterocycloamino” group is -NH-(heterocyclo cycloalkyl), wherein the heterocyclo group is attached to the nitrogen and the cycloalkyl group is attached to the heterocyclo group.

[0080] An example of a “heterocyclo cycloamino” group is -NH-(cycloalkyl heterocycle), wherein the cycloalkyl group is attached to the nitrogen and the heterocyclo group is attached to the cycloalkyl group.

[0081] The term “amido” refers to a group of the formula -C(O)NR2, wherein R is defined herein.

[0082] The terms “halo,” “halogen,” and “halide” group, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0083] The term “haloalkyl” group includes mono-halo alkyl groups, poly-halo alkyl groups, wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, -CF(CH3)2and the like.

[0084] The terms “treat,” “treating,” “treated,” or “treatment” (with respect to a disease or condition) is an approach for obtaining beneficial or desired results including and preferably clinical results and includes, but is not limited to, one or more of the following: improving a condition associated with a disease, curing a disease, lessening severity of a disease, delaying progression of a disease, alleviating one or more symptoms associated with a disease, increasing the quality of life of one suffering from a disease, prolonging survival and / or prophylactic or preventative treatment.

[0085] An “effective amount” refers to any amount that is sufficient to achieve a desired biological effect. Combined with the teachings provided herein, by choosing among the various70835-02 active conjugates or compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial unwanted toxicity and yet is effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular compound being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound and / or other therapeutic agent without necessitating undue experimentation. A maximum dose can be used, that is, the highest safe dose according to some medical judgment. Multiple doses per day can be used to achieve appropriate systemic levels of compounds. Appropriate systemic levels can be determined by, for example, measurement of the patient’s peak or sustained plasma level of the drug. “Dose” and “dosage” are used interchangeably herein.

[0086] Generally, daily oral doses of a compound are, for human subjects, from about 0.01 milligrams / kg per day to 1,000 milligrams / kg per day. Oral doses in the range of 0.5 to 50 milligrams / kg, in one or more administrations per day, can yield therapeutic results. Dosage can be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. For example, intravenous administration can vary from one order to several orders of magnitude lower dose per day. If the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) can be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of the compound.

[0087] A “therapeutically effective amount” (or “effective amount”) of a compound with respect to use in treatment, refers to an amount of the compound in a preparation which, when administered as part of a desired dosage regimen (to a mammal, such as a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0088] For any compound a therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data for compounds which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. Higher doses may be70835-02 required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.

[0089] The formulations can be administered in pharmaceutically acceptable solutions, which can routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. For use in therapy, an effective amount of the compound can be administered to a subject by any mode that delivers the compound to the desired surface. Administering a pharmaceutical composition can be accomplished by any means known to the skilled artisan. Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (urinary bladder), oral, subcutaneous, direct injection (for example, into a tumor or abscess), mucosal (e.g., topical to eye), inhalation, and topical.

[0090] For intravenous and other parenteral routes of administration, a compound can be formulated as a lyophilized preparation, as a lyophilized preparation of liposome-intercalated or -encapsulated active compound, as a lipid complex in aqueous suspension, or as a salt complex. Lyophilized formulations are generally reconstituted in suitable aqueous solution, e.g., in sterile water or saline, shortly prior to administration.

[0091] For oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well-known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. A pharmaceutical preparation for oral use can be obtained as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinyl pyrrolidone (PVP). If desired, disintegrating agents can be added, such as the cross-linked PVP, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations70835-02 can also be formulated in saline or buffers, e.g., EDTA for neutralizing internal acid conditions, or can be administered without any carriers.

[0092] Also contemplated are oral dosage forms of the compounds. The compounds can be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the compound itself, where said moiety permits (a) inhibition of acid hydrolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compounds and increase in circulation time in the body. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, PVP and polyproline. Abuchowski and Davis, “Soluble Polymer-Enzyme Adducts,” In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley- Interscience, New York, N.Y., pp. 367-383 (1981); Newmark et al., J Appl Biochem 4:185-189 (1982). Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. For pharmaceutical usage, as indicated above, polyethylene glycol moieties are suitable.

[0093] The location of release of a compound hereof can be the stomach, the small intestine (e.g., the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations, which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. The release can avoid the deleterious effects of the stomach environment, either by protection of the compound or by release of the compound beyond the stomach environment, such as in the intestine.

[0094] To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings can be used as mixed films.

[0095] A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules can consist of a hard shell (such as gelatin) for delivery of dry therapeutic (e.g., powder); for liquid forms, a soft gelatin shell can be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.70835-02

[0096] The compound can be included in the formulation as fine multi-particulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. Therapeutic agent could be prepared by compression.

[0097] Colorants and flavoring agents may all be included. For example, the compound can be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

[0098] One may dilute or increase the volume of the compound with an inert material. These diluents can include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts also can be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

[0099] Disintegrants can be included in the formulation of therapeutic agent into a solid dosage form. Materials used as disintegrates include, but are not limited to, starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrant is the insoluble cationic exchange resin. Powdered gums can be used as disintegrants and as binders, and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

[0100] Binders can be used to hold the compound together to form a hard tablet and can include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl celluloseethyl cellulose (EC) and carboxymethyl cellulose (CMC). PVP and hydroxypropylmethyl cellulose (HPMC) can both be used in alcoholic solutions to granulate therapeutic agent.

[0101] An anti-frictional agent can be included in the formulation of therapeutic to prevent sticking during the formulation process. Lubricants can be used as a layer between therapeutic agent and the die wall, and these can include, but are not limited to, stearic acid, including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and70835-02 waxes. Soluble lubricants can also be used, such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

[0102] Glidants, which can improve the flow properties of the drug during formulation and aid rearrangement during compression, can be added. The glidants can include starch, talc, pyrogenic silica and hydrated silicoaluminate.

[0103] To aid dissolution of therapeutic agent into the aqueous environment a surfactant can be added as a wetting agent. Surfactants can include anionic detergents, such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents, which can be used, include benzalkonium chloride and benzethonium chloride. Potential non-ionic detergents that can be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the compound or derivative thereof either alone or as a mixture in different ratios.

[0104] Pharmaceutical preparations, which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. Microspheres formulated for oral administration can also be used. Such microspheres have been well-defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

[0105] For buccal administration, the compositions can take the form of tablets or lozenges formulated in conventional manner.

[0106] For topical administration, the compound can be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.70835-02

[0107] For administration by inhalation, compounds can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator, can be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0108] Also contemplated is pulmonary delivery of the compounds (or salts thereof). The compound is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Other reports of inhaled molecules include Adjei et al., Pharm Res 7:565-569 (1990); Adjei et al., Int J Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al., J Cardiovasc Pharmacol 13(suppl. 5):143-146 (1989) (endothelin-1); Hubbard et al., Annal Int Med 3:206-212 (1989) (a1-antitrypsin); Smith et al., 1989, J Clin Invest 84:1145-1146 (a-1-proteinase); Oswein et al., 1990, "Aerosolization of Proteins," Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant hepatocyte growth hormone); Debs et al., 1988, J Immunol 140:3482-3488 (interferon-gamma and tumor necrosis factor alpha) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor; incorporated herein by reference). A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Pat. No. 5,451,569 (specifically incorporated herein by reference for its disclosure regarding same), issued Sep.19, 1995, to Wong et al.

[0109] Contemplated for use are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

[0110] Nasal delivery of a pharmaceutical composition is also contemplated. Nasal delivery allows the passage of a pharmaceutical composition to the blood stream directly after administering therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran.

[0111] The compounds, when it is desirable to deliver them systemically, can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in70835-02 multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0112] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0113] Alternatively, the active compounds can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0114] The compounds can also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0115] In addition to the formulations described above, a compound can also be formulated as a depot preparation. Such long-acting formulations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0116] The pharmaceutical compositions also can comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0117] Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosolized, pelleted for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and / or auxiliaries such as70835-02 disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R, Science 249:1527-1533 (1990).

[0118] The compound and optionally one or more other therapeutic agents can be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

[0119] Suitable buffering agents include acetic acid and a salt (1-2% w / v); citric acid and a salt (1-3% w / v); boric acid and a salt (0.5-2.5% w / v); and phosphoric acid and a salt (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); parabens (0.01-0.25% w / v); and thimerosal (0.004-0.02% w / v).

[0120] Pharmaceutical compositions contain an effective amount of a compound as described herein and optionally one or more other therapeutic agents included in a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” means one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a human or other vertebrate animal. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also can be commingled with the compounds, and with each other, in a manner such that there is no interaction, which would substantially impair the desired pharmaceutical efficiency.

[0121] Therapeutic agent(s), including specifically, but not limited to, a compound, can be provided in particles. “Particles” means nanoparticles or microparticles (or in some instances larger particles) that can consist in whole or in part of the compound or the other therapeutic agent(s). The particles can contain therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating. Therapeutic agent(s) also can be dispersed70835-02 throughout the particles. Therapeutic agent(s) also can be adsorbed into the particles. The particles can be of any order release kinetics, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle can include, in addition to therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof. The particles can be microcapsules which contain the compound in a solution or in a semi-solid state. The particles can be of virtually any shape.

[0122] Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering therapeutic agent(s). Such polymers can be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include bioerodible hydrogels described in Sawhney et al., Macromolecules 26:5823-2787 (1993), the teachings of which are specifically incorporated by reference herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[0123] Therapeutic agent(s) can be contained in controlled-release systems. The term “controlled release” refers to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non- immediate release formulations, with non-immediate release formulations including, but not limited to, sustained release and delayed release formulations. The term “sustained release” (also referred to as “extended release”) refers to a drug formulation that provides for gradual release of a drug over an extended period of time, and that can result in substantially constant blood levels of a drug over an extended time period. The term “delayed release” refers to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug therefrom. “Delayed release” may or may not involve gradual release of drug over an extended period of time, and thus may or may not be “sustained release.”

[0124] Use of a long-term, sustained-release implant can be particularly suitable for treatment of chronic conditions. “Long-term” release means that the implant is constructed and70835-02 arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and up to 30-60 days. Long-term sustained-release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.

[0125] The term “salts” and “pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic, and the like.

[0126] Pharmaceutically acceptable salts can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. In some instances, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa., 1990, the disclosure of which is hereby incorporated by reference.

[0127] The term “solvate” means a compound, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate.

[0128] The term “prodrug” means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide an active compound, particularly a compound of the invention. Examples of prodrugs include, but are not limited to, derivatives and metabolites of a compound of the invention that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates,70835-02 biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Specific prodrugs of compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid. The carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule. Prodrugs can typically be prepared using well- known methods, such as those described by Burger’s Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers GmbH).

[0129] Further, in each of the foregoing and following embodiments, it is to be understood that the formulae include and represent not only all pharmaceutically acceptable salts of the compounds, but also include any and all hydrates and / or solvates of the compound formulae or salts thereof. It is to be appreciated that certain functional groups, such as the hydroxy, amino, and like groups form complexes and / or coordination compounds with water and / or various solvents, in the various physical forms of the compounds. Accordingly, the above formulae are to be understood to include and represent those various hydrates and / or solvates. In each of the foregoing and following embodiments, it is also to be understood that the formulae include and represent each possible isomer, such as stereoisomers and geometric isomers, both individually and in any and all possible mixtures. In each of the foregoing and following embodiments, it is also to be understood that the formulae include and represent any and all crystalline forms, partially crystalline forms, and non-crystalline and / or amorphous forms of the compounds.

[0130] The term "pharmaceutically acceptable carrier" is art-recognized and refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof. Each carrier must be "acceptable" in the sense of being compatible with the subject composition and its components and not injurious to the patient. Some examples of materials, which may serve as pharmaceutically acceptable carriers, include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and70835-02 ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0131] The term “administering” includes all means of introducing the compounds and compositions described herein to the patient, including, but are not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, and the like. The compounds and compositions may be administered in unit dosage forms and / or formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

[0132] Illustrative formats for oral administration include tablets, capsules, elixirs, syrups, and the like. Illustrative routes for parenteral administration include intravenous, intraarterial, intraperitoneal, epidural, intraurethral, intrasternal, intramuscular and subcutaneous, as well as any other art recognized route of parenteral administration.

[0133] Illustrative means of parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques, as well as any other means of parenteral administration recognized in the art. Parenteral formulations are typically aqueous solutions, which may contain excipients such as salts, carbohydrates and buffering agents (preferably at a pH in the range from about 3 to about 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well-known to those skilled in the art. Parenteral administration of a compound is illustratively performed in the form of saline solutions or with the compound incorporated into liposomes. In cases where the compound, itself, is not sufficiently soluble to be dissolved, a solubilizer such as ethanol can be applied.

[0134] The dosage of each compound of the claimed combinations depends on several factors, including: the administration method, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the70835-02 pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect the dosage regimen used.

[0135] In the methods the individual components of a co-administration, or combination, can be administered by any suitable means, contemporaneously, simultaneously, sequentially in either order, separately or in a single pharmaceutical formulation. Where the co-administered compounds or compositions are administered in separate dosage forms, the number of dosages administered per day for each compound may be the same or different. The compounds or compositions may be administered via the same or different routes of administration. The compounds or compositions may be administered according to simultaneous or alternating regimens, at the same or different times during the course of the therapy, concurrently in divided or single forms.

[0136] The term “therapeutically effective amount” refers to that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated. In one aspect, the therapeutically effective amount is that which may treat or alleviate the disease or symptoms of the disease at a reasonable benefit / risk ratio applicable to any medical treatment. However, it is to be understood that the total daily usage of the compounds and compositions described herein may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidentally with the specific compound employed; and like factors well-known to the researcher, veterinarian, medical doctor or other clinician of ordinary skill.

[0137] Depending upon the route of administration, a wide range of permissible dosages dosages may be single or divided and may administered according to a wide variety of protocols, including q.d. (once a day), b.i.d. (twice a day), t.i.d. (three times a day), or even every other day,70835-02 once a week, once a month, once a quarter, and the like. In each of these cases the described therapeutically effective amounts correspond to the instance of administration, or alternatively to the total daily, weekly, month, or quarterly dose, as determined by the dosing protocol.

[0138] An effective amount of any one or a mixture of the compounds can be determined by the attending diagnostician or physician by the use of known techniques and / or by observing results obtained under analogous circumstances. In determining the effective amount or dose, a number of factors are considered by the attending diagnostician or physician, including, but not limited to, the species of mammal, including human, its size, age, and general health, the specific disease or disorder involved, the degree of or involvement or the severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances.

[0139] The term “patient” includes human and non-human animals such as companion animals (dogs and cats and the like) and livestock animals. Livestock animals are animals raised for food production. The patient to be treated is preferably a mammal, in particular a human.

[0140] The following enumerated Embodiments are also part of the disclosure and are listed in no particular order of importance:

[0141] Embodiment A1 relates to a compound of the formula:or a pharmaceutically acceptable salt or hydrate thereof, wherein R1 is (a) phenyl, which can be mono-, di- or tri-substituted with one or more substituents, which can be the same or different, selected from the group consisting of a C1-C6alkyl, a C1-C6alkyloxy, a C1-C6alkylsulfanyl, a hydroxyl, and a halo or (b) a substituent selected from the group consisting of:70835-02, wherein the wavy line represents the point of attachment to of R1 to the nitrogen atom; and R2 is hydrogen or a C1-C6 alkyl.

[0142] Embodiment A2 relates to the compound of Embodiment A1, wherein R1 is mono-, di- or tri-substituted with a C1-C6 alkyl.

[0143] Embodiment A3 relates to the compound of Embodiment A2, wherein one, two or three R1is / are CH3.

[0144] Embodiment A4 relates to the compound of Embodiment A1, wherein R1is mono-, di- or tri-substituted with a C1-C6 alkyloxy.

[0145] Embodiment A5 relates to the compound of Embodiment A4, wherein one, two or three R1is / are OCH3.

[0146] Embodiment A6 relates to the compound of any one of Embodiments A1-A3, wherein R1 is mono-, di- or tri-substituted with a C1-C6 alkylsulfanyl.

[0147] Embodiment A7 relates to the compound of Embodiment A6, wherein one, two or three R1is SCH3.70835-02

[0148] Embodiment A8 relates to the compound of Embodiment A1, wherein R1 is mono-, di- or tri-substituted with a halo.

[0149] Embodiment A9 relates to the compound of Embodiment A8, wherein one, two or three R1is / are F, Cl, Br or I.

[0150] Embodiment A10 relates to the compound of any one of Embodiments A1-A9, wherein R2is C1-C6alkyl.

[0151] Embodiment A11 relates to the compound of Embodiment A10, wherein R2 is CH3, CH2CH3 or CH(CH3)2.

[0152] Embodiment A12 relates to the compound of compound of Embodiment A1, wherein R1 isand R2is hydrogen.

[0153] Embodiment A13 relates to the compound of compound of Embodiment A1, wherein R1isand R2is hydrogen.

[0154] Embodiment A14 relates to the compound of compound of Embodiment A1, wherein R1isand R2 is hydrogen.

[0155] Embodiment A15 relates to the compound of compound of Embodiment A1, wherein R1 isand R2is hydrogen.70835-02

[0156] Embodiment A16 relates to the compound of compound of Embodiment A1, wherein R1 isand R2 is hydrogen.

[0157] Embodiment A17 relates to a compound of compound of the formula:or a pharmaceutically acceptable salt or hydrate thereof, wherein: R3is 6- to 13-membered aryl substituted with (R4)nor 6- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S) substituted with (R4)n; n is 0, 1, 2 or 3; R2 is hydrogen or a C1-C6 alkyl; and R4 in each instance is independently OH, halo, 6- to 10-membered heterocycloalkyl (wherein 1- 4 heterocycloalkyl members are independently selected from N, O, and S), 6- to 10-membered heterocycloalkyl alkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), 6- to 10-membered heterocycloalkyl alkyl-O- (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), S(O)xR5(wherein R5is H, C1-C6alkyl or 6- to 10-membered heterocycloalkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S) and x is 0, 1 or 2), -SCN, -NCS, C1-C6-alkyl, C1-C6- alkoxy, C1-C6-haloalkyl, 6- to 10-membered aryl, 6- to 10-membered aryloxy, 5- to 10- membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), NHC(O)-6- to 10-membered heterocycloalkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), C(O)NH-6- to 10-membered heterocycloalkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), NHC(O)-6- to 10-membered heterocycloalkyl alkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), C(O)NH-6- to 10-membered heterocycloalkyl alkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S) or two R470835-02 groups, together with carbon atoms to which they are attached, form a 5- to 6-membered heterocycloalkyl (wherein 1 or 2 heterocycloalkyl members are independently selected from N, O, and S).

[0158] Embodiment A18 relates to the compound of compound of Embodiment A17, or a pharmaceutically acceptable salt or hydrate thereof, wherein R3is 6- to 13-membered aryl substituted with (R4)nand the compound is of the formula:.

[0159] Embodiment A19 relates to the compound of Embodiment A18, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4is OH, halo, C1-C6-alkyl, C1-C6- alkoxy, C1-C6-haloalkyl, S(O)xR5(wherein R5is H, C1-C6alkyl or 6-membered heterocycloalkyl), 6-membered aryloxy, 6-membered heterocycloalkyl, 6-membered heterocycloalkyl alky or 5-membered heteroaryl.

[0160] Embodiment A20 relates to the compound of compound of Embodiment A18, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4 is OH, CH3, CH3O, SCH3, Cl, F, Br, piperidinyl, morpholinyl, thiomorpholino, piperazinyl, SO2-6-membered heterocycloalkyl, phenoxy, SCN, thiophenyl, CF3, or two R4 groups, together with carbon atoms to which they are attached, form:.

[0161] Embodiment A21 relates to the compound of compound of Embodiment A17, or a pharmaceutically acceptable salt or hydrate thereof, wherein R3is 6- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S) substituted with (R4)nand the compound is of the formula:.70835-02

[0162] Embodiment A22 relates to the compound of compound of Embodiment A17, or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is of the formula:

[0163] Embodiment A23 relates to the compound of compound of Embodiment A17, or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is of the formula:70835-02 570835-02 ,each of which is independently substituted with

[0164] Embodiment A24 relates to the compound of compound of Embodiments 17-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein R2 is H.

[0165] Embodiment A25 relates to the compound of compound of any of Embodiments A17-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 0 or 1.

[0166] Embodiment A26 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 0.

[0167] Embodiment A27 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4 is CH3.

[0168] Embodiment A28 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4is halo.

[0169] Embodiment A29 relates to the compound of compound of Embodiment A28, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4is F, Cl or Br.70835-02

[0170] Embodiment A30 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4 is OH.

[0171] Embodiment A31 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4is OCH3.

[0172] Embodiment A32 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4 is C1-C6-haloalkyl.

[0173] Embodiment A33 relates to the compound of compound of Embodiment A32, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4 is CF3.

[0174] Embodiment A34 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4is 6- to 10-membered heterocycloalkyl.

[0175] Embodiment A35 relates to the compound of compound of Embodiment A34, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4 is:, wherein X1is CR6(wherein R6is H or C1-C6alkyl) or N; and X2is (CR6)2(wherein each R6is independently H or C1-C6alkyl), NR6, O or S.

[0176] Embodiment A36 relates to the compound of compound of Embodiment A35, or a pharmaceutically acceptable salt or hydrate thereof, wherein X1 is N and X2 is (CR6)2, wherein each R6 is H or X1 is CR6 and X2 is NR6, wherein each R6 is H.

[0177] Embodiment A37 relates to the compound of compound of Embodiment A35, or a pharmaceutically acceptable salt or hydrate thereof, wherein X1 is N and X2 is NR6, wherein R6 is C1-C6-alkyl.

[0178] Embodiment A38 relates to the compound of compound of Embodiment A37, or a pharmaceutically acceptable salt or hydrate thereof, wherein R6is CH3.

[0179] Embodiment A39 relates to the compound of compound of Embodiment A35, or a pharmaceutically acceptable salt or hydrate thereof, wherein X1is N and X2is S.70835-02

[0180] Embodiment A40 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4 is 6- to 10-membered heterocycloalkyl alkyl-O-.

[0181] Embodiment A41 relates to the compound of compound of Embodiment A40, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4is:, wherein y is 1-6; X1is CR6(wherein R6is H or C1-C6alkyl) or N; and X2is (CR6)2(wherein each R6is independently H or C1-C6alkyl), NR6, O or S.

[0182] Embodiment A42 relates to the compound of compound of Embodiment A41, or a pharmaceutically acceptable salt or hydrate thereof, wherein y is 1, X1 is CR6 (wherein R6 is H), and X2 is NR6, wherein R6 is C1-C6 alkyl.

[0183] Embodiment A43 relates to the compound of compound of Embodiment A41, or a pharmaceutically acceptable salt or hydrate thereof, wherein y is 1, X1 is CR6 (wherein R6 is H), and X2is NR6, wherein R6is CH3.

[0184] Embodiment A44 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4is 5- to 10-membered heteroaryl.

[0185] Embodiment A45 relates to the compound of compound of Embodiment A44, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4 is:, wherein X3 is O, S or NR6 (wherein R6 is H or C1-C6 alkyl).

[0186] Embodiment A46 relates to the compound of compound of Embodiment A45, or a pharmaceutically acceptable salt or hydrate thereof, wherein X3is S.

[0187] Embodiment A47 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4 is NHC(O)-6- to 10-membered heterocycloalkyl.

[0188] Embodiment A48 relates to the compound of compound of Embodiment A47, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4 is:.70835-02

[0189] Embodiment A49 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4 is C(O)NH-6- to 10-membered heterocycloalkyl.

[0190] Embodiment A50 relates to the compound of compound of Embodiment A49, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4is:.

[0191] Embodiment A51 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 2 and R4 is OH.

[0192] Embodiment A52 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 2 and R4is OCH3.

[0193] Embodiment A53 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 3 and R4is OH.

[0194] Embodiment A54 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 3 and R4 is OCH3.

[0195] Embodiment A55 relates to the compound of compound of any of Embodiments A17, A18, or A21-A24, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4is S(O)xR5.

[0196] Embodiment A56 relates to the compound of compound of Embodiment A55, or a pharmaceutically acceptable salt or hydrate thereof, wherein x is 0 and R5is C1-C6alkyl.

[0197] Embodiment A57 relates to the compound of compound of Embodiment A56, or a pharmaceutically acceptable salt or hydrate thereof, wherein R5 is CH3.

[0198] Embodiment A58 relates to the compound of compound of Embodiment A55, or a pharmaceutically acceptable salt or hydrate thereof, wherein x is 2 and R5 is 6- to 10- membered heterocycloalkyl.70835-02

[0199] Embodiment A59 relates to the compound of compound of Embodiment A56, or a pharmaceutically acceptable salt or hydrate thereof, wherein R5 is:, wherein X1is CR6(wherein R6is H or C1-C6alkyl) or N; and X2is (CR6)2(wherein each R6 is independently H or C1-C6 alkyl), NR6, O or S.

[0200] Embodiment A60 relates to the compound of compound of Embodiment A59, or a pharmaceutically acceptable salt or hydrate thereof, wherein X1 is N and X2 is NR6, wherein R6 is H or C1-C6 alkyl.

[0201] Embodiment A61 relates to the compound of compound of Embodiment A60, or a pharmaceutically acceptable salt or hydrate thereof, wherein R6is C1-C6alkyl.

[0202] Embodiment A62 relates to the compound of compound of Embodiment A61, or a pharmaceutically acceptable salt or hydrate thereof, wherein R6is CH3.

[0203] Embodiment A63 relates to a pharmaceutical composition comprising the compound of any one of Embodiments A1-A62, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

[0204] Embodiment A64 relates to a method of inhibiting alpha- -syn)-syn protein aggregation, which method comprises administering to the subject the pharmaceutical composition of Embodiment A60 in --syn aggregation is-syn protein aggregation.

[0205] Embodiment A65 relates to the method of Embodiment A64, wherein the subject has, or is at risk for, Parkinson’s disease or dementia with Lewy bodies (DLB). EXAMPLES

[0206] The following examples serve to illustrate the present disclosure. The examples are not intended to limit the scope of the claimed invention in any way. Example 1 Synthesis70835-02Analogs: 1 - 6, 2110-,3142, 14, 15, 17, 19andScheme 1. Synthesis of coumarin-based-amide compounds (1-34) using primary aromatic amine (R1) via EDCI coupling to generate the final products presented in Table 1.Analog: 8 Amine =Scheme 2. Synthesis of coumarin-based-amides using secondary aromatic amine using (COCl)2.

[0207] General Characterizations.1H and13C{1H} NMR spectra were recorded using a 500 MHz Bruker instrument working at a frequency of 500 MHz for1H and at 126 MHz for13C.Chemical shifts are reported in ppm using residual solvent resonances as internal reference (2.50 and 39.51 for1H and13d6 respectively).1H NMR data are reported as follows: b = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. Coupling constants are given in hertz. The purity of all compounds and synthetic intermediates was judged to be 95% or better based on1H NMR. IR measurements were performed in a Nicolet FTIR as thin films in the Purdue Drug Discovery facility. High-resolution mass spectrometry analyses were conducted at the MSU Mass Spectrometry facility. General synthetic procedure

[0208] Synthesis of amides with primary aromatic amines: The coumarin 3-carboxylic acid (1.00 equiv.), respective primary aromatic amine (1.00 equiv.), and DMAP (0.10 equiv.) were dissolved in DCM at room temperature. The mixture was cooled to 0 °C in an ice bath. Then, EDCI (10 mL in DCM) was added dropwise at 0 °C for 10-20 min and gradually warmed to room temperature overnight. The mixture was stirred until all the starting material was70835-02 consumed. The reaction mixture was filtered and washed with HCl (10 %, 25 mL), water (25 mL), and DCM (10 mL). The precipitate was dried overnight to obtain the dry product.

[0209] Synthesis of amides with secondary aromatic amines: The coumarin 3-carboxylic acid (1.00 equiv.) and DMF (50 µL) were dissolved in DCM (8.0 mL). The mixture was cooled to 0 °C in an ice bath. Oxalyl chloride (1.25 equiv.) was added dropwise at 0 °C and gradually allowed to warm to room temperature overnight. The next day, the solvent was evaporated by vacuum, and crude acid chloride was redissolved in DCM (8.0 mL) and cooled to 0 °C. The secondary aromatic amine (1.00 equiv.) was added followed by Et3N (1.00 equiv.) and allowed to warm gradually to room temperature overnight. The next day, the reaction mixture was treated with aqueous HCl (10 %, 25 mL) and extracted with EtOAc. The organic layer was washed with brine (25 mL), dried with anhydrous MgSO4, and evaporated to obtain the crude. The crude was recrystallized with hexane and ethyl acetate to obtain a clean amide product.

[0210] Demethylation of coumarin-amides with BBr3(Analogs: 7, 9, 11, 13, 16, 18, 20): The methoxy amide substrate (1.00 equiv.) was dissolved in DCM (5.0 mL) at room temperature and cooled to 0 °C on an ice bath. Boron tribromide (5.00 equiv. per each methoxy functionality) was added dropwise at 0 °C, over 15 min. Then, the reaction mixture was stirred at 0 °C and allowed to warm to room temperature gradually overnight. The reaction progress was followed by TLC. Upon completion, the reaction was quenched with saturated NH4Cl, and the crude was purified by recrystallization with hexane and ethyl acetate (1:1).2-oxo-N-phenyl-2H-chromene-3-carboxamide (1)

[0211] Yellow color solid (376 mg, 71%).1H NMR 8.90 (s, 1H), 7.99 (d, J = 7.7 Hz, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.71 (d, J = 7.9 Hz, 2H), 7.53 (d, J = 8.4 Hz, 1H), 7.45 (t,= 7.5 Hz, 1H), 7.37 (t, J = 7.8 Hz, 2H), 7.13 (t, J = 7.4 Hz, 1H).13C NMR 124.8, 120.4, 119.0, 116.7.70835-02N-(4-methylphenyl)-2-oxo-2H-chromene-3-carboxamide (2)

[0212] Yellow color solid (365 mg, 65%).1H NMR 8.89 (s, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.83 – 7.68 (m, 1H), 7.59 (d, J = 7.5 Hz, 2H), 7.52 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.4 Hz, 1H), 7.16 (d, J = 7.6 Hz, 2H), 2.26 (s, 3H).13C NMR (126 MHz, 116.7, 21.0.2-oxo-N-(pyridin-4-yl)-2H-chromene-3-carboxamide (3)

[0213] Yellow color solid (257 mg, 43%).1H NMR 8.90 (s, 1H), 8.49 (d, J = 5.9 Hz, 2H), 7.99 (d, J = 7.7 Hz, 1H), 7.84 – 7.74 (m, 1H), 7.70 (d, J = 6.0 Hz, 2H), 7.54 (d, J = 8.3 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H).13C NMR 161.6, 160.5, 154.5, 151.1, 148.3, 145.1, 135.0, 130.9, 125.8, 118.8, 116.8, 114.4.N-(4-fluorophenyl)-2-oxo-2H-chromene-3-carboxamide (4)

[0214] Pale yellow color solid (474 mg, 83%).1H NMR 1H), 8.90 (s, 1H), 7.99 (d, J = 7.7 Hz, 1H), 7.76 (d, J = 8.0 Hz, 3H), 7.62 – 7.36 (m, 2H), 7.21 (s, 2H).13C NMR 120.4, 118.9, 116.7, 116.2, 116.0.70835-02N-(4-methoxyphenyl)-2-oxo-2H-chromene-3-carboxamide (6)

[0216] Yellow color solid (210 mg, 47%).1H NMR 8.89 (s, 1H), 7.98 (dd, J = 7.8, 1.6 Hz, 1H), 7.75 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.63 (d, J = 9.0 Hz, 2H), 7.52 (d, J = 8.4 Hz, 1H), 7.45 (td, J = 7.5, 1.1 Hz, 1H), 6.93 (d, J = 9.0 Hz, 2H), 3.73 (s, 3H).13C NMR 125.7, 121.9, 120.3, 119.0, 116.7, 114.6, 55.7.N-(4-hydroxyphenyl)-2-oxo-2H-chromene-3-carboxamide (7)

[0217] Yellow color solid (30 mg, 62%).1H NMR 9.36 (s, 1H), 8.88 (s, 1H), 7.98 (dd, J = 7.8, 1.6 Hz, 1H), 7.75 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.51 (t, J = 9.0 Hz, 3H), 7.44 (td, J = 7.6, 1.1 Hz, 1H), 6.75 (d, J = 8.8 Hz, 2H).13C NMR (126 MHz, 120.35, 119.02, 116.68, 115.84.70835-02N-(4-methoxyphenyl)-N-methyl-2-oxo-2H-chromene-3-carboxamide (8)

[0218] Dark yellow color solid (117 mg, 78%).1H NMR (500 MHz, CDCl31H), 7.51 – 7.43 (m, 1H), 7.40 (dd, J = 7.8, 1.6 Hz, 1H), 7.21 (dd, J = 21.2, 7.9 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 8.8 Hz, 2H), 3.70 (s, 3H), 3.42 (s, 3H).13C NMR (126 MHz, CDCl3) 55.3, 37.8.N-(4-hydroxyphenyl)-N-methyl-2-oxo-2H-chromene-3-carboxamide (9)

[0219] Pale yellow color solid (51 mg, 62%).1H NMR 1H), 7.90 (s, 1H), 7.61 (dd, J = 7.8, 1.6 Hz, 1H), 7.56 (ddd, J = 8.7, 7.3, 1.7 Hz, 1H), 7.36 – 7.26 (m, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.13 (d, J = 8.8 Hz, 2H), 6.72 (d, J = 8.8 Hz, 2H), 3.33 (s, 3H).13C NMR 128.128.65, 126.8, 124.6, 118.3, 116.2, 115.7, 115.6, 36.5. HRMS (ESI / Q-TOF) m / z: [M - H]+Calcd. for C17H13NO4295.2940; Found 295.0844.N-(2-methoxyphenyl)-2-oxo-2H-chromene-3-carboxamide (10)

[0220] Yellow color solid (739 mg, 83%).1H NMR 9.02 (s, 1H), 8.43 (d, J = 8.1 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.76 (t, J = 7.9 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.09 (d, J = 4.4 Hz, 2H), 7.04 – 6.89 (m, 1H), 3.89 (s,70835-02 3H).13C NMR 124.9, 121.1, 120.1, 119.1, 116.7, 111.5, 56.6.N-(2-hydroxyphenyl)-2-oxo-2H-chromene-3-carboxamide (11)

[0221] Yellow color solid (79 mg, 76 %).1H NMR 10.20 (s, 1H), 9.03 (s, 1H), 8.38 (dd, J = 8.0, 1.6 Hz, 1H), 8.03 (dd, J = 7.8, 1.6 Hz, 1H), 7.77 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.45 (td, J = 7.5, 1.1 Hz, 1H), 7.01 – 6.87 (m, 2H), 6.82 (ddd, J = 8.6, 7.1, 1.8 Hz, 1H).13C NMR 148.8, 147.1, 134.9, 131.0, 127.0, 125.7, 124.8, 120.4, 119.6, 119.2, 119.1, 116.7, 115.1.N-(3,4-dimethoxyphenyl)-2-oxo-2H-chromene-3-carboxamide (12)

[0222] Yellow color solid (479 mg, 73%).1H NMR 8.88 (s, 1H), 8.07 – 7.87 (m, 1H), 7.75 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.36 (d, J = 2.4 Hz, 1H), 7.26 (dd, J = 8.6, 2.4 Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H), 3.76 (s, 3H), 3.72 (s, 3H).13C NMR 147.8, 146.1, 134.7, 131.9, 130.7, 125.7, 120.2, 119.0, 116.7, 112.5, 112.4, 105.4, 56.1, 56.0.N-(3,4-dihydroxyphenyl)-2-oxo-2H-chromene-3-carboxamide (13)

[0223] Dark yellow color solid (57 mg, 40%).1H NMR 1H), 9.10 (s, 1H), 8.86 (s, 1H), 8.81 (s, 1H), 7.97 (dd, J = 7.8, 1.6 Hz, 1H), 7.74 (ddd, J = 8.7,70835-02 7.3, 1.6 Hz, 1H), 7.51 (d, J = 8.3 Hz, 1H), 7.43 (td, J = 7.5, 1.1 Hz, 1H), 7.29 (d, J = 2.5 Hz, 1H), 6.87 (dd, J = 8.5, 2.5 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H).13C NMR 159.4, 154.3, 147.5, 145.7, 142.7, 134.6, 130.7, 130.4, 125.7, 120.4, 119.0, 116.7, 116.0, 111.5, 108.8.2-oxo-N-(3,4,5-trimethoxyphenyl)-2H-chromene-3-carboxamide (14)

[0224] Yellow color solid (594 mg, 83%).1H NMR 8.89 (s, 1H), 7.97 (dd, J = 7.8, 1.6 Hz, 1H), 7.76 (ddd,= 8.7, 7.3, 1.6 Hz, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.45 (td, J = 7.5, 1.1 Hz, 1H), 7.10 (s, 2H), 3.77 (s, 6H), 3.63 (s, 3H).13C NMR (126 116.7, 98.2, 60.6, 56.3.2-oxo-N-(3,4,5-trihydroxyphenyl)-2H-chromene-3-carboxamide (15)

[0225] Dark red color solid (90 mg, 69%).1H NMR 8.85 (s, 1H), 7.98 (d, J = 7.7 Hz, 1H), 7.74 (s, 1H), 7.61 – 7.31 (m, 2H), 6.71 (s, 2H).13C NMR 119.0, 116.7, 99.9.N-(2,3-dimethoxyphenyl)-2-oxo-2H-chromene-3-carboxamide (16)70835-02

[0226] Yellow color solid (458 mg, 90%).1H NMR 9.02 (s, 1H), 8.05 (dd, J = 19.9, 8.0 Hz, 2H), 7.86 – 7.69 (m, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.49 – 7.41 (m, 1H), 7.11 – 7.01 (m, 1H), 6.88 – 6.77 (m, 1H), 3.82 (s, 6H).13C NMR (126 MHz, 119.1, 116.7, 112.5, 109.0, 60.9, 56.3.N-(2,3-dihydroxyphenyl)-2-oxo-2H-chromene-3-carboxamide (17)

[0227] Yellow color solid (106 mg, 89%).1H NMR 9.46 (s, 1H), 9.10 (s, 1H), 9.02 (s, 1H), 8.02 (dd, J = 7.8, 1.6 Hz, 1H), 7.88 (dd, J = 8.1, 1.6 Hz, 1H), 7.76 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.45 (td, J = 7.5, 1.1 Hz, 1H), 6.68 – 6.53 (m, 2H).13C NMR 134.8, 130.9, 128.0, 125.7, 119.30, 119.27, 119.1, 116.7, 111.9, 111.6. HRMS (ESI / Q-TOF) m / z: [M + H]+Calcd. for C16H11NO5297.0637; 295.0479.N-(3,5-dimethoxyphenyl)-2-oxo-2H-chromene-3-carboxamide (18)

[0228] Yellow color solid (425 mg, 65%).1H NMR 8.88 (s, 1H), 7.98 (dd, J = 7.8, 1.6 Hz, 1H), 7.76 (ddd, J = 8.6, 7.4, 1.6 Hz, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.45 (td, J = 7.5, 1.0 Hz, 1H), 6.94 (d, J = 2.2 Hz, 2H), 6.28 (t, J = 2.3 Hz, 1H), 3.73 (s, 6H).13C NMR 125.8, 120.3, 119.0, 116.7, 98.7, 96.8, 55.7.70835-02N-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-oxo-2H-chromene-3-carboxamide (20)

[0230] 1H NMR J = 7.8 Hz, 1H), 7.75 (ddd, J = 8.7, 7.2, 1.6 Hz, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.49 – 7.42 (m, 1H), 7.39 (d, J = 2.5 Hz, 1H), 7.04 (dd, J = 8.7, 2.5 Hz, 1H), 6.83 (d, J = 8.7 Hz, 1H), 4.22 (td, J = 5.5, 3.8 Hz, 4H).13C NMR 125.7, 120.3, 119.0, 117.5, 116.7, 113.6, 109.5, 64.7, 64.5.N-(1H-indol-4-yl)-2-oxo-2H-chromene-3-carboxamide (21)

[0231] Yellow color solid (86 mg, 28%)NMR 9.77 (s, 1H), 9.52 (s, 1H), 7.62 (d, J = 8.7 Hz, 2H), 7.52 (s, 1H), 7.34 (dt, J = 5.9, 3.7 Hz, 4H), 7.14 – 6.93 (m, 1H), 6.41 (s, 1H).13C NMR 136.9, 134.9, 130.9, 130.2, 125.8, 125.7, 122.0, 119.9, 119.6, 119.2, 116.7, 109.7, 108.7, 97.5.70835-02N-(1H-indol-5-yl)-2-oxo-2H-chromene-3-carboxamide (22)

[0232] Yellow color solid (310 mg, quantitative).1H NMR 1H), 10.59 (s, 1H), 8.93 (s, 1H), 8.03 (d, J = 2.0 Hz, 1H), 8.00 (dd, J = 7.8, 1.6 Hz, 1H), 7.75 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.45 (td, J = 7.5, 1.1 Hz, 1H), 7.38 (d, J = 8.6 Hz, 1H), 7.34 (t, J = 2.7 Hz, 1H), 7.29 (dd, J = 8.7, 2.1 Hz, 1H), 6.42 (s, 1H).13C NMR 120.5, 119.1, 116.7, 115.5, 112.0, 111.8, 101.8. HRMS (ESI / Q-TOF) m / z: [M + H]+Calcd. for C18H12N2O3304.0848; Found 304.0856.N-(1H-indol-6-yl)-2-oxo-2H-chromene-3-carboxamide (23)

[0233] Yellow color solid (206 mg, 67%).1H NMR 10.68 (s, 1H), 8.92 (s, 1H), 8.14 (s, 1H), 8.00 (dd, J = 7.8, 1.6 Hz, 1H), 7.75 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.52 (dd, J = 16.6, 8.4 Hz, 2H), 7.45 (td, J = 7.5, 1.1 Hz, 1H), 7.31 (t, J = 2.7 Hz, 1H), 7.07 (dd, J = 8.4, 1.9 Hz, 1H), 6.39 (s, 1H).13C NMR 154.3, 147.5, 136.3, 134.6, 132.5, 130.7, 126.1, 125.7, 125.2, 120.7, 120.5, 119.1, 116.7, 113.1, 103.3, 101.6. HRMS (ESI / Q-TOF) m / z: [M + H]+Calcd. for C18H12N2O3304.0848; Found 304.0853.N-(1H-indol-7-yl)-2-oxo-2H-chromene-3-carboxamide (24)70835-022.36 (s, 3H).13C NMR 130.7, 130.3, 129.1, 125.7, 120.5, 119.1, 116.7, 114.3, 111.1, 110.8, 99.9, 13.9. HRMS (ESI / Q- TOF) m / z: [M + H]+Calcd. for C19H14N2O3318.1004; Found 318.1014.70835-022-oxo-N-(quinolin-6-yl)-2H-chromene-3-carboxamide (27)

[0237] Brownish yellow color solid (529 mg, 83%).1H NMR (s, 1H), 9.00 (dd, J = 4.8, 1.6 Hz, 1H), 8.90 (s, 1H), 8.78 (d, J = 8.3 Hz, 1H), 8.66 (d, J = 2.3 Hz, 1H), 8.23 (d, J = 9.1 Hz, 1H), 8.13 (dd, J = 9.2, 2.4 Hz, 1H), 7.99 (dd, J = 7.8, 1.6 Hz, 1H), 7.85 – 7.68 (m, 2H), 7.52 (d, J = 8.3 Hz, 1H), 7.44 (m, J = 7.5, 1.0 Hz, 1H).13C NMR (126 MHz, 125.8, 125.9, 122.7, 120.2, 118.8, 116.8, 116.6. HRMS (ESI / Q-TOF) m / z: [M + H]+Calcd. for C19H12N2O3316.0848; Found 316.0854.2-oxo-N-[2-(piperidin-1-yl)phenyl]-2H-chromene-3-carboxamide (28)

[0238] Yellow color solid (276 mg, 40%)9.04 (s, 1H), 8.48 (dd, J = 7.3, 1.6 Hz, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.84 – 7.67 (m, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.24 (dt, J = 7.6, 1.3 Hz, 1H), 7.17 – 6.99 (m, 2H), 2.90 – 2.66 (m, 4H), 1.84 – 1.67 (m, 4H), 1.53 (s, 2H).13C NMR 159.5, 154.5, 149.1, 144.2, 134.9, 134.0, 131.0, 125.7, 124.9, 124.8, 121.4, 120.1, 119.2, 119.1, 116.6, 53.9, 26.2, 24.2.N-[2-(morpholin-4-yl)phenyl]-2-oxo-2H-chromene-3-carboxamide (29)70835-02

[0239] Pale yellow color solid (529 mg, 75%) NMR 1H), 9.04 (s, 1H), 8.50 (dd, J = 8.1, 1.6 Hz, 1H), 8.11 – 7.94 (m, 1H), 7.77 (td, J = 7.9, 1.6 Hz, 1H), 7.52 (d, = 8.4 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.29 (dd, J = 7.7, 1.6 Hz, 1H), 7.13 (m, 2H), 3.83 (s, 4H), 2.83 (s, 4H).13C NMR 134.9, 134.0, 131.0, 125.7, 125.4, 124.9, 121.4, 120.2, 119.3, 119.1, 116.6, 66.8, 52.7.N-{4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}-2-oxo-2H-chromene-3-carboxamide (30)

[0240] White color solid (216 mg, 50%)NMR 8.90 (s, 1H), 8.00 (t, J = 8.9 Hz, 3H), 7.87 – 7.70 (m, 3H), 7.54 (d, J = 8.4 Hz, 1H), 7.50 – 7.41 (m, 1H), 3.32 (s, 8H), 2.63 (s, 3H).13C NMR 143.0, 135.0, 130.8, 129.5, 125.8, 120.6, 120.5, 118.8, 116.8, 52.2, 43.1, 42.8.2-oxo-N-(2-phenoxyphenyl)-2H-chromene-3-carboxamide (31)

[0241] Yellow color solid (314 mg, 88%).1H NMR 9.01 (s, 1H), 8.56 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.74 (tdd, J = 7.3, 1.8, 0.9 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 7.42 – 7.35 (m, 2H), 7.23 – 7.17 (m, 1H), 7.17 – 7.08 (m, 2H), 7.06 (d, J = 1.1 Hz, 2H), 7.00 – 6.93 (m, 1H).13C NMR (126 MHz, DMSO) 121.3, 119.3, 119.1, 118.9, 118.5, 116.7.70835-022-oxo-N-(3-phenoxyphenyl)-2H-chromene-3-carboxamide (32)

[0242] Pale yellow color solid (194 mg, 54%).1H NMR 1H), 8.85 (s, 1H), 7.96 (dd, J = 7.8, 1.6 Hz, 1H), 7.75 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.52 (d, J = 8.3 Hz, 2H), 7.48 – 7.30 (m, 5H), 7.16 (t, J = 7.4 Hz, 1H), 7.05 (d, J = 8.0 Hz, 2H), 6.78 (dt, J = 6.8, 2.5 Hz, 1H).13C NMR 134.8, 130.9, 130.7, 130.6, 125.8, 124.2, 120.5, 119.5, 118.9, 116.7, 115.2, 114.7, 110.2.N-[6-(cyanosulfanyl)-1,3-benzothiazol-2-yl]-2-oxo-2H-chromene-3-carboxamide (34)

[0244] Pale yellow color solid (251 mg, 66%).1H NMR 1H), 9.01 (s, 1H), 8.39 (d, J = 1.9 Hz, 1H), 7.99 (s, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.83 – 7.75 (m, 1H), 7.66 (dd, J = 8.6, 2.0 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H).13C NMR70835-02 , 130.0, 126.1, 126.0, 122.8, 119.0, 118.7, 117.8, 116.9, 112.5.

[0245] N-(4-chlorophenyl)-2-oxo-2H-chromene-3-carboxamide (35). Pale yellow color solid (436 mg, 95%).1H NMR (500 MHz, DMSO-d6J = 7.8 Hz, 1H), 7.78 (d, J = 8.86 Hz, 3H), 7.56 (d, J = 8.38 Hz, 1H), 7.50 – 7.43 (m, 3H).13C NMR (126 MHz, DMSO-d6122.0, 120.5, 118.9, 116.8. HRMS (ESI / Q-TOF) m / z: [M + H]+Calcd. for C16H11ClNO3 300.0427; Found 300.0425. 2-oxo-N-(4-(trifluoromethyl)phenyl)-2H-chromene-3-carboxamide (36).

[0246] White color solid (522 mg, 78%).1H NMR (500 MHz, CDCl3 9.07 (s, 1H), 7.90 (d, J = 7.9 Hz, 2H), 7.80 – 7.75 (m, 2H), 7.67 (d, J = 8.68 Hz, 2H), 7.51-7.45 (m, 2H).13C NMR (126 MHz, CDCl3126.3, 125.7, 125.2121.1, 118.6, 118.2, 116.8. HRMS (ESI / Q-TOF) m / z: [M + H]+Calcd. for C17H11F3NO3334.0691; Found 334.0685. 2-oxo-N-(4-(thiophen-2-yl)phenyl)-2H-chromene-3-carboxamide (37).

[0247] Yellow color solid (576 mg, 83%).1H NMR (500 MHz, DMSO-d6 1H), 8.94 (s, 1H), 8.03 (d, J = 7.44 Hz, 1H), 7.81 – 7.79 (m, 3H), 7.69 (d, J = 8.72 Hz, 2H), 7.57 (d, J = 8.15 Hz, 1H), 7.53 (d, J = 5.72 Hz, 1H), 7.50 – 7.47 (m, 2H), 7.14 (t, J = 4.58 Hz, 1H).13C NMR (126 MHz, DMSO-d6 130.3, 129.0, 126.5, 125.8, 123.8, 120.9, 120.5, 119.0, 116.8. HRMS (ESI / Q-TOF) m / z: [M+H]+Calcd. for C20H14NO3S 348.0694; Found 348.0693. N-(benzo[c][1,2,5]thiadiazol-4-yl)-2-oxo-2H-chromene-3-carboxamide (38).

[0248] Beige color solid (226 mg, 70%).1H NMR (500 MHz, CDCl3 9.07 (s, 1H), 8.67 (d, J = 7.41 Hz, 1H), 7.78 – 7.64 (m, 3H), 7.66 (t, J = 8.05 Hz, 1H), 7.49 (d, J = 8.35 Hz, 1H), 7.43 (t, J = 7.46 Hz, 1H).13C NMR (126 MHz, CDCl3 154.9, 149.5, 148.5, 134.8, 131.0, 130.2, 130.2, 125.7, 118.8, 118.6, 117.1, 116.8, 116.3. HRMS (ESI / Q-TOF) m / z: [M + H]+Calcd. for C16H10N3O3S 324.0443; Found 324.0443. Example 2 -syn fibril formation Chemical and protein source70835-02

[0249] Heparin sodium salt was acquired from Millipore-Sigma; Thioflavin-T (ThT) for -syn ThT assays from Alfa Aesar (Ward Hill, MA); Recombinant -syn and tau 2N4R from rPeptide (Watkinsville, GA). The expression and purification of tau ON4R were carried out as previously described.28Dr. Benjamin Wolozin (Boston University) generously provided a bacterial expression plasmid that contains the vector pET30a with a cDNA sequence encoding the human Tau 0N4R isoform. Rosetta BL21 E. coli cells (CamR) containing the pET30a[0N4R tau wt] plasmid (KanR) were cultured in LB media with 50 µg / mL kanamycin and 50 µg / mL chloramphenicol. Protein expression was induced by adding 1 mM IPTG and incubating for about 18 h at 37°C. The cells were collected by centrifugation at 6,000 g for 15 min at 4°C. The cells were mixed with lysis buffer containing 10 mM Hepes (pH 7.4), 50 mM NaCl, 1 mM MgCl2, 1 mM PMSF, 1X protease inhibitor cocktail, and 0.5 mM DTT. Subsequently, the cell suspension underwent sonication in cycles of 30 seconds on and 1 minute off, operating at 30-45% power for a total duration of 5 min. Following sonication, the lysate underwent centrifugation at 10,000 rpm for 10 min at 4°C. The supernatant was then gently mixed with 7.8 ml of 3M NaCl and incubated in an 80°C water bath for 10 minutes, followed by cooling on ice for an additional 10 min. After centrifugation at 10,000 rpm for 10 min at 4°C, the resulting supernatant was carefully transferred into fresh tubes. The supernatant was subjected to overnight dialysis against a cation exchange buffer (50 mM MES, 1M NaCl, 1 mM DTT, pH 6.0). Following dialysis, the sample was applied onto a HiPrep SP HP column, where proteins were separated using a linear gradient of NaCl concentration ranging from 50 mM to 1 M. Fractions containing the tau isoform 0N4R were combined, and the resulting protein solution underwent dialysis against PBS (pH 7.4) before being stored at -80ºC. Thioflavin T (ThT) fluorescence assays

[0250] -synuclein fibrilization using the Thioflavin fluorescence assay, as previously described.29,30A 96-well microplate was utilized for the experiment. The compounds were added at a final concentration -Synuclein was ck solution of 277 –HCl (pH 7.4). The assay conditions also included 10 mM PBS buffer (pH 7.4), 300 mM NaCl, 0.5 mM SDS, and a 3 mm borosilicate bead. The 96-well plate was loaded into a Synergy HT multi-mode microplate reader (BioTek, Winooski,70835-02 VT) and incubated at 37°C. The ThT fluorescence intensity was monitored with excitation at 440 nm and emission at 485 nm. Measurements were taken every 20 minutes, with the plate shaken for 10 seconds before each reading. The kinetics were observed over a period of 27 h with each sample analyzed in triplicate. Table 1. The molecular structures of coumarin derivatives and their respective anti-fibrillary -syn were evaluated. The activity was expressed as the maximum thioflavin T (ThT) intensity percentage, with compounds tested at a final concentration of 100 -syn at 2 µM. The results, representing the average of three replicates, are presented with the standard error of the mean (SEM).70835-0270835-02Table 2. The molecular structures of coumarin derivatives and their respective anti-fibrillary -syn were evaluated. The activity was expressed as the maximum thioflavin T (ThT) -syn at 6 µM. The results, representing the average of three replicates, are presented with the standard error of the mean (SEM).70835-02 Table 3. The molecular structures of additional coumarin derivatives and their respective anti- -syn were evaluated. The activity was expressed as the maximum thioflavin - syn at 2 µM. The results, representing the average of three replicates, are presented with the standard error of the mean (SEM).70835-0270835-02Thioflavin S (ThS) fluorescence assay

[0251] To study the inhibitory effects of the compounds -syn fibrillation, we employed -sheet protein chains aligned parallel to the fibril axis,23restricting rotation around the C–C bond in the ThT molecule,24which leads to an increase in ThT fluorescence.

[0252] A black 384-well microplate with a flat bottom (ref 784076, medium binding; Brand) was used for the Thioflavin S (ThS) assay. PBS (pH 7.4) (Gibco, catalog number 10010- 023), treated with chelex beads (Biosciences, BTNM-0024), was dispensed into each well to a final volume of 10 µL. The peptide (2N4R) was added to each well at a final concentration of 12 µM, followed by the addition of DTT to a final concentration of 5 mM. The compound was then added at a final concentration of 100 µM, and heparin was added at a final concentration of 150 µM. The reaction mixture was supplemented with arachidonic acid to a final concentration of 0.092 µg / ml. (A0781, Fischer TCI). Thioflavin S (ThS) was added to each well at a final70835-02 concentration of 40 µM to monitor the kinetics of fibril formation. Fluorescence intensity was measured every 5 min with a 30 s agitation before each measurement.

[0253] -syn at ours. During this incubation period, the fluorescence of ThT was continuously measured at 20-min intervals to monitor the changes in fluorescent intensity. The addition of compounds 5, 8, 9, 12, 16, and 18 resulted in an increase in fluorescence intensity. In contrast, compounds 13, 17, and 19 decreased the fluorescence intensity compared -syn was incubated with DMSO (Fig.1), suggesting that these -syn fibril formation. The ThT fluorescence intensity for samples containing compounds 13, 17, and 19 was recorded as 0.81 ± 0.23, 10.71 ± 0.19, and 20.24 ± 1.76, respectively (Table 2). After conducting the initial ThT assay, dose-response curves were specifically generated for compounds 13 and 17 (Fig. 2). The analysis performed using Prism software showed a Log(agonist) versus normalized response correlation with a variable slope. This analysis provided LogEC50 values of 0.81 ± 0.23 for compound 13 and 10.71 ± 0.19 for compound 17. These values highlight the differential potency of the two compounds in their respective dose-response relationships. Only compound 17 was tested at approximately a 1:8 ratio (12 µM tau, 100 µM compound 17) for tau ThS (Fig. 3), demonstrating approximately a 25% reduction in tau fibril formation. Example 3 Compounds reduced the oligomer formation by photo-induced cross-linking of unmodified proteins (PICUP)

[0254] The aggregation of fibril-forming proteins into oligomers marks a critical step in the pathogenesis of protein-misfolding diseases. These oligomers, characterized by their cytotoxicity,25play a pivotal role in disease progression and are thus significant targets for therapeutic intervention. Photo-induced Cross-linking of Unmodified Proteins (PICUP) assay was used -syn and tau oligomers.26This approach allowed evaluation of the effectiveness of the compounds in inhibiting or reducing the formation of these oligomers.

[0255] a-Syn and tau (0N4R isoform) were diluted in PBS (pH 7.4) to achieve a final concentration of 6 µM for each protein in a PCR tube. 2 µL of tris(bipyridine)ruthenium(II) mM) were added initiate cross-linking. The tube was then placed in a homemade dark enclosure70835-02 (a small box). To assess the progressive impact of our compounds on suppressing protein oligomerization, we employed controls that were kept in the dark and lacked Ru(bpy) (i.e., 0.125% -Syn was exposed to light for 1 sec, while tau 0N4R was exposed for 60 sec. To stop the PICUP reaction, 15% 2-mercaptoethanol was added immediately following exposure to light. The reaction mixture was heated on a heating block at 95 °C for 10 minutes. A 16% SDS-PAGE gel was used to evaluate the samples with the aid of a protein marker (PageRuler Prestained Protein Ladder), which was used to examine the molecular weight of the samples. The electrophoresis was initially run at 60 mV for 15 min, after which the voltage was increased to a maximum of 80 mV and maintained until the dye migrated to the bottom of the gel. Gels were incubated in the fixing solution for 1 hour and then stained with Coomassie Blue for up to 48 hours to improve sensitivity. The gels were then destained with 10% acetic acid and incubated in deionized water for about 1 hour to remove the background color before scanning with the G:BOX F3 (Syngene).

[0256] The effects of the -syn and tau isoform 0N4R oligomerization wasinvestigated -syn anti-oligomerization, the compounds were assessedFig.4). The most promising compounds from this initial screening were subsequently tested to determine their dose-response effects on the -syn oligomer formation. Compounds 13, 15, and 17 demonstrated the most -synuclein oligomerization compared to the control, which contained 0.125% DMSO (Fig. 4). In contrast, the other tested compounds (10, 11, 12, 14, and 16) showed no inhibitory effect. The dose-response studies of the promising compounds (13, 15, and 17)indicated they all have a strong, concentration- -synucleinoligomerization (Figs. 5A-5Bdose- 17 was tested;however, it did not prevent the formation of tau 0N4R oligomers. Example 4 Confirmation of the anti-fibrillar activity by transmission electron microscopy (TEM)

[0257] - syn and tau 2N4R fibrils, we performed TEM analysis. TEM was performed following previously established methods.31,3270835-02 DMSO (0.25% as control), was incubated in a solution containing 10 mM PBS buffer (pH 7.4), -syn, the samples used for the ThT fibril kinetic formation assay were subsequently analyzed by TEM.10 µL of the samples were applied onto a 400-mesh Formvar-carbon-coated copper grid (Electron Microscopy Sciences, Hatfield, PA) and incubated for 1 minute before rinsing with water. After rinsing the grid with distilled water, it was air-dried, and a solution of 1% uranyl acetate was applied for 1 min and then dried using filter paper. The samples were examined using a JEOL 1400 Flash (Japan) electron microscope operating at 100 kV.

[0258] Figure 7 -syn fibrils extracted from the 96-well plate post-ThT assay. The images contrast fibrils formed under control conditions (without test compounds) and those formed in the presence of test compounds. The control sample, containing 1.5% DMSO, exhibited a molecular architecture molecular architecture marked by extensive and densely packed fibrils. In the sample containing compounds 13 and 17, there was a notable and significant reduction in both the length and density of the fibrils, with compound 13 being more effective, which is consistent with the ThT data. Compound 19 showed some effect, but the fibrils were more densely packed compared to the most effective compounds, 13 and 17. In contrast, compounds 8, 12, 16, and 18 showed no observable effect. In TEM images of tau 2N4R (Fig.8), it was observed that compounds 13 and 17 effectively inhibited the formation of protein fibrils. The compounds, including DMSO (0.25%; 'CTRL'), were co-incubated with tau 2N4R for 5 days before imaging. The reduction in fibrils observed in the samples treated with compounds 13 and 17 indicates that both compounds showed a stronger inhibitory effect on fibril formation compared to compound 12. Example 5 -syn inclusion in neuroblastoma cells

[0259] To investigate the effects of various compounds on inclusion formation and cell -synuclein- fusion protein in a doxycycline (dox)-inducible manner were used.33This model is particularly relevant for studying the pathological consequences of the familial Parkinson's disease-associated -synuclein E46K mutation. The pathology is further intensified by a triple mutation (E35K + hich leads to the formation of distinctive round cytoplasmic70835-02 inclusions in cultured cells, thereby providing a valuable system for examining disease mechanisms and testing potential therapeutic strategies.27

[0260] Cells were seeded at a density of 30,000 cells per well in 96-well plates. -3K::YFP transgene expression doxycycline to the culture media. Cells were cultured in the Incucyte Zoom 2000 platform (Essen Biosciences), where continuous imaging (green, bright field) was conducted. Analysis of inclusion formation or growth was performed at 48 h post-induction (96 hours post-plating). The Incucyte processing definition "Inclusions" was configured as follows: Parameters were set to Fixed Threshold with a Threshold (GCU) of 50; Edge Split was activated with Edge Sensitivity set to Cell confluence was assessed using the following criteria: 'Cells' processing definition with Parameters set at Segmentation Adjustment 0.7, Cleanup with all parameters at 0, and Filters applied with an Area

[0261] Cells were exposed to various concentrations of compounds 13, 15, and 17 starting 24 hours after plating. At 48 hours, doxycycline was added to induce transgene expression. By 96 hours, the dose-dependent effects of the compounds on the formation of inclusions were analyzed. Compound 13 -syn inclusion formation at concentrations of 10, 20, and 40 µM, with the most pronounced effect observed at 10 µM (Figure YA). Similarly, compound 17 -syn inclusions at these concentrations, with the greatest reduction at 40 µM. In contrast, compound 15 -syn inclusion formation. Treatment with all three compounds did not change cell confluence (Fig. 9). Example 6 Effect of Compounds on Tau Aggregation in ExpiCHO Cells.

[0262] To evaluate the impact of test compounds on rau aggregation, ExpiCHO cells were transfected with split GFP-Tau fusion constructs (pmGFP11C-Tau and pmGFP10C-Tau) and control (0.1% DMSO) for 72 hours. Flow cytometry analysis was used to quantify GFP-positive cell populations, reflecting tau aggregation, and ethidium homodimer-1 exclusion was used to confirm cell viability. Only viable GFP+ cells were used for analysis. Among the tested compounds, only compound 13 significantly reduced GFP fluorescence compared to the vehicle control, indicating its unique ability to inhibit tau aggregation70835-02 (Figure 10). None of the other compounds (5, 8, 9, 12, 17, 16, 18, 19) demonstrated a measurable effect on GFP-positive populations. These findings are consistent with the results observed in the TEM ThS analysis on 2N4R Tau, further supporting the inhibitory effect of compound 13 on tau aggregation. Example 7

[0263] Coumarin compounds anti-fibrillary activities can be applied to other diseases such as type 2 diabetes. Indeed, compounds 13, 15, 19 reduced the IAPP fibril formation at more than 90% (Table 5). Table 5. Coumarin compounds inhibit islet amyloid polypeptide (IAPP) fibril formation as tested with ThT fluorescence assays. Percentages of maximum ThT fluorescence were collected at the plateau phase of the kinetics of fibril formation. Compounds were tested at 100 µM with IAPP at 7 µM. The data represent the average of three replicates and the SEM. A lower percentage is indicative of reduction of fibril formation.70835-0270835-02Example 8. In vitro- absorption, distribution, metabolism, and excretion (ADME) Results

[0264] Compounds 12 and 14 can be used as pro-drug of compounds 13 and 15, respectively, based on the mouse liver microsome data (Table Y). The microsomal stability data70835-02 are higher for the methoxy coumarin compounds 12 and 14. However the demethylated counterparts (compounds 13 and 15) showed better anti-oligomer and anti-fibrillar activities.nointaoilaort nnamticioir)tosv to ni.aitap co aitR 7 9.8 9.9 9.1 9.2 7.0 6.3 9.mnreerctc i eta ertarE(00 0 0 0 0 0en Hd ocyttiliabdueltsos etci etereniw ksedn h uTo.ep m moosCor .ciS DM SreMvimLe 5.su0od nmanlidCanuNop M mm m 1 3oc0l0aco3 itmd p dnarthon a,%ecg 03ni7m.04.03.12.5.8.3 234 3.0ntid o n yn ni5 nc w a tilair iaddnaetC7 ba5ni5sne3u.3.6.6.0.9.4.lelcme1 m 4 1 1 81 76 86 1dn mto R a Scitauelh.5ni4.42.43.9.58.81.32.2opp2Mpeh m 2 1 2 5 8 8 5p musdeµo)ta1 d tetycts4.bcitil)0et7 ucanidedierb ulM - 56 06 82 01 63 34fHoptpµ((erset ,o S ytirle eeifcbfweauyerenare etaeH H O O M M H e OeeOeMsbh w STsel rc u HH H O tc1 - O O O MM M O O O OcilB.dn p u R O OoPMubourtH e Magnµ0pelaSteis0m Mu1-o. e1ccs,li- - - - - -6n,II )II )II )II )II )II )ofoyec3 5 7 nma1 1 1 2 41 61elbdetigliarDIpaV- G( V- 5 G( V- 1 G( V- 1V- 5 G(G( V- 7 G(8asanbaaereK1 K2 K3 K59 K1 1 T wartslcV S 1 S 1 S 1 S S 1 K S 1 570835-02

[0265] In conclusion, coumarin-based-amide compounds were designed and synthesized primarily to evaluate the anti-aggregation activity on two proteins involved in neurodegenerative -syn and tau. The coumarin-amide-dihydroxybenzene derivatives demonstrated -syn antiaggregation activities when compared to coumarin-amide-indole and methoxybenzene derivatives. Compound 13 (which bears a 4,5-dihydroxybenzene moiety) was -syn oligomer formation, tau fibril formation (cell free assay), and tau dimerization (cell-based assay) but failed -syn inclusions in neuroblastoma cells. Incontrast, compound 17, a 5,6- -syn inclusions, butfailed at inhibiting tau fibrils (cell free assay) and tau dimerization (cell-based assay). This study sets the stage to future exploration of dual inhibitors of aggregation and sEH to reduce neuroinflammation and improved neuronal health in transgenic animal models of synucleinopathies and tauopathies.

[0266] Alzheimer's and Parkinson's diseases are major health concerns that impact millions of people around the globe, representing a substantial and growing crisis in global health. A significant challenge in treating AD and PD is the lack of effective therapeutics to prevent the aggregation of misfolded proteins (AD: tau and amyloid beta; PD: -syn) that are central to the onset and progression of these conditions. Most available drugs only address symptoms and do not offer a long-term cure. Most available drugs only address symptoms and do not offer a long-term cure. -syn and tau oligomerization and fibrilization. We designed and synthesized 35 amide coumarin compounds to evaluate their efficacy in blocking these processes. The results from the fibrilization kinetic assay (ThT) indicate that the dihydroxyphenylamide-coumarin derivatives (compounds 13, 17, and 19) -syn fibrilization. Notably, compounds 13 and 17 demonstrated the highest efficacy, as confirmed by analyzing the ultrastructural changes in fibril formation using TEM. Both compounds showed a dose-dependent effect in the prevention of the formation of - syn oligomers as observed from the PICUP experiment. For the tau anti-oligomerization assays, only compound 17 was tested, and no significant anti-oligomer activity was observed. However, in the TEM analysis assessing the anti-fibrilization effects of the two most promising compounds (13 and 17), both compounds effectively reduced the formation of tau 2N4R fibrils, suggesting their potential to inhibit tau aggregation.70835-02 model expressed in M17D neuroblastoma cells, the two most effective compounds (13 and 17) -synuclein inclusions, with compound 17 demonstrating the most pronounced effect at concentrations of 20 . 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[0267] All patents, patent application publications, journal articles, textbooks, and other publications mentioned in the specification are indicative of the level of skill of those in the art to which the disclosure pertains. All such publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.

[0268] The invention illustratively described herein may be suitably practiced in the absence of any element(s) or limitation(s), which is / are not specifically disclosed herein. Thus, for example, each instance herein of any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. Likewise, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods and / or steps of the type, which are described herein and / or which will become apparent to those ordinarily skilled in the art upon reading the disclosure.

[0269] The term “about,” when referring to a number or a numerical value or range (including, for example, whole numbers, fractions, and percentages), means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error) and thus the numerical value or range can vary between 1% and 15% of the stated number or numerical range (e.g., + / - 5 % to 15% of the recited value) provided that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). The term “substantially” can allow for a degree of variability in a value or range, for example, within 90%, within 95%, or within 99% of a stated value or of a stated limit of a range.

[0270] The terms and expressions, which have been employed, are used as terms of description and not of limitation. In this regard, where certain terms are defined under "Definitions" and are otherwise defined, described, or discussed elsewhere in the "Detailed70835-02 Description," all such definitions, descriptions, and discussions are intended to be attributed to such terms. There also is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. Furthermore, while subheadings, e.g., "Definitions," are used in the "Detailed Description," such use is solely for ease of reference and is not intended to limit any disclosure made in one section to that section only; rather, any disclosure made under one subheading is intended to constitute a disclosure under each and every other subheading.

[0271] It is recognized that various modifications are possible within the scope of the claimed invention. Thus, although the present invention has been specifically disclosed in the context of preferred embodiments and optional features, those skilled in the art may resort to modifications and variations of the concepts disclosed herein. Such modifications and variations are considered within the scope of the invention as claimed herein.

Claims

1. 70835-02 WHAT IS CLAIMED IS:

1. A compound of the formula:or a pharmaceutically acceptable salt or hydrate thereof, wherein R1 is (a) phenyl, which can be mono-, di- or tri-substituted with one or more substituents, which can be the same or different, selected from the group consisting of a C1-C6 alkyl, a C1-C6alkyloxy, a C1-C6alkylsulfanyl, a hydroxyl, and a halo or (b) a substituent selected from the group consisting of: ,, wherein the wavy line represents the point of attachment to of R1to the nitrogen atom; and R2is hydrogen or a C1-C6alkyl.70835-02 2. The compound of claim 1, wherein R1 is mono-, di- or tri-substituted with a C1-C6 alkyl.

3. The compound of claim 2, wherein one, two or three R1is / are CH3.

4. The compound of claim 1, wherein R1is mono-, di- or tri-substituted with a C1-C6alkyloxy.

5. The compound of claim 4, wherein one, two or three R1is / are OCH3.

6. The compound of any one of claims 1-3, wherein R1 is mono-, di- or tri-substituted with a C1- C6 alkylsulfanyl.

7. The compound of claim 6, wherein one, two or three R1is SCH3.

8. The compound of claim 1, wherein R1is mono-, di- or tri-substituted with a halo.

9. The compound of claim 8, wherein one, two or three R1 is / are F, Cl, Br or I.

10. The compound of any one of claims 1-9, wherein R2 is C1-C6 alkyl.

11. The compound of claim 10, wherein R2is CH3, CH2CH3or CH(CH3)2.

12. The compound of claim 1, wherein R1isand R2 is hydrogen.

13. The compound of claim 1, wherein R1isand R2is hydrogen.70835-02 14. The compound of claim 1, wherein R1 isand R2is hydrogen.

15. The compound of claim 1, wherein R1 isand R2is hydrogen.

16. The compound of claim 1, wherein R1 isand R2is hydrogen.

17. A compound of the formula:or a pharmaceutically acceptable salt or hydrate thereof, wherein: R3 is 6- to 13-membered aryl substituted with (R4)n or 6- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S) substituted with (R4)n; n is 0, 1, 2 or 3; R2is hydrogen or a C1-C6alkyl; and R4in each instance is independently OH, halo, 6- to 10-membered heterocycloalkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), 6- to 10-70835-02 membered heterocycloalkyl alkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), 6- to 10-membered heterocycloalkyl alkyl-O- (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), S(O)xR5 (wherein R5 is H, C1-C6alkyl or 6- to 10-membered heterocycloalkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S) and x is 0, 1 or 2), -SCN, -NCS, C1-C6-alkyl, C1- C6-alkoxy, C1-C6-haloalkyl, 6- to 10-membered aryl, 6- to 10-membered aryloxy, 5- to 10- membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), NHC(O)-6- to 10-membered heterocycloalkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), C(O)NH-6- to 10-membered heterocycloalkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), NHC(O)- 6- to 10-membered heterocycloalkyl alkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S), C(O)NH-6- to 10-membered heterocycloalkyl alkyl (wherein 1-4 heterocycloalkyl members are independently selected from N, O, and S) or two R4groups, together with carbon atoms to which they are attached, form a 5- to 6-membered heterocycloalkyl (wherein 1 or 2 heterocycloalkyl members are independently selected from N, O, and S).

18. The compound of claim 17, or a pharmaceutically acceptable salt or hydrate thereof, wherein R3 is 6- to 13-membered aryl substituted with (R4)n and the compound is of the formula:.

19. The compound of claim 18, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4is OH, halo, C1-C6-alkyl, C1-C6-alkoxy, C1-C6-haloalkyl, S(O)xR5(wherein R5is H, C1-C6alkyl or 6-membered heterocycloalkyl), 6-membered aryloxy, 6-membered heterocycloalkyl, 6-membered heterocycloalkyl alky or 5-membered heteroaryl.

20. The compound of claim 18, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4 is OH, CH3, CH3O, SCH3, Cl, F, Br, piperidinyl, morpholinyl, thiomorpholino, piperazinyl,70835-02 SO2-6-membered heterocycloalkyl, phenoxy, SCN, thiophenyl, CF3, or two R4 groups, together with carbon atoms to which they are attached, form:.

21. The compound of claim 17, or a pharmaceutically acceptable salt or hydrate thereof, wherein R3 is 6- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S) substituted with (R4)n and the compound is of the formula:.

22. The compound of claim 17, or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is of the formula:70835-02, each of which is independently substituted with (R4)n.

23. The compound of claim 17, or a pharmaceutically acceptable salt or hydrate thereof, wherein the compound is of the formula:, , ,70835-02 ,each of which is independently substituted with (R4)n.

24. The compound of claims 17-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein R2 is H.70835-02 25. The compound of any of claims 17-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 0 or 1.

26. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 0.

27. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4 is CH3.

28. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4is halo.

29. The compound of claim 28, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4 is F, Cl or Br.

30. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4 is OH.

31. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4is OCH3.

32. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4 is C1-C6-haloalkyl.

33. The compound of claim 32, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4 is CF3.

34. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4is 6- to 10-membered heterocycloalkyl.70835-02 35. The compound of claim 34, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4 is:, wherein X1is CR6(wherein R6is H or C1-C6alkyl) or N; and X2is (CR6)2(wherein each R6 is independently H or C1-C6 alkyl), NR6, O or S.

36. The compound of claim 35, or a pharmaceutically acceptable salt or hydrate thereof, wherein X1 is N and X2 is (CR6)2, wherein each R6 is H or X1 is CR6 and X2 is NR6, wherein each R6 is H.

37. The compound of claim 35, or a pharmaceutically acceptable salt or hydrate thereof, wherein X1is N and X2is NR6, wherein R6is C1-C6-alkyl.

38. The compound of claim 37, or a pharmaceutically acceptable salt or hydrate thereof, wherein R6 is CH3.

39. The compound of claim 35, or a pharmaceutically acceptable salt or hydrate thereof, wherein X1is N and X2is S.

40. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4 is 6- to 10-membered heterocycloalkyl alkyl-O-.

41. The compound of claim 40, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4 is:, wherein y is 1-6; X1 is CR6 (wherein R6 is H or C1-C6 alkyl) or N; and X2 is (CR6)2 (wherein each R6 is independently H or C1-C6 alkyl), NR6, O or S.

42. The compound of claim 41, or a pharmaceutically acceptable salt or hydrate thereof, wherein y is 1, X1is CR6(wherein R6is H), and X2is NR6, wherein R6is C1-C6alkyl.70835-02 43. The compound of claim 41, or a pharmaceutically acceptable salt or hydrate thereof, wherein y is 1, X1 is CR6 (wherein R6 is H), and X2 is NR6, wherein R6 is CH3.

44. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4is 5- to 10-membered heteroaryl.

45. The compound of claim 44, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4 is:, wherein X3 is O, S or NR6 (wherein R6 is H or C1-C6 alkyl).

46. The compound of claim 45, or a pharmaceutically acceptable salt or hydrate thereof, wherein X3is S.

47. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4 is NHC(O)-6- to 10-membered heterocycloalkyl.

48. The compound of claim 47, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4is:.

49. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4is C(O)NH-6- to 10-membered heterocycloalkyl.

50. The compound of claim 49, or a pharmaceutically acceptable salt or hydrate thereof, wherein R4is:.70835-02 51. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 2 and R4 is OH.

52. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 2 and R4is OCH3.

53. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 3 and R4 is OH.

54. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 3 and R4 is OCH3.

55. The compound of any of claims 17, 18, or 21-23, or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1 and R4is S(O)xR5.

56. The compound of claim 55, or a pharmaceutically acceptable salt or hydrate thereof, wherein x is 0 and R5 is C1-C6 alkyl.

57. The compound of claim 55, or a pharmaceutically acceptable salt or hydrate thereof, wherein R5is CH3.

58. The compound of claim 55, or a pharmaceutically acceptable salt or hydrate thereof, wherein x is 2 and R5 is 6- to 10-membered heterocycloalkyl.

59. The compound of claim 58, or a pharmaceutically acceptable salt or hydrate thereof, wherein R5 is:, wherein X1is CR6(wherein R6is H or C1-C6alkyl) or N; and X2is (CR6)2(wherein each R6is independently H or C1-C6alkyl), NR6, O or S.70835-02 60. The compound of claim 59, or a pharmaceutically acceptable salt or hydrate thereof, wherein X1 is N and X2 is NR6, wherein R6 is H or C1-C6 alkyl.

61. The compound of claim 60, or a pharmaceutically acceptable salt or hydrate thereof, wherein R6is C1-C6alkyl.

62. The compound of claim 61, or a pharmaceutically acceptable salt or hydrate thereof, wherein R6 is CH3.

63. A pharmaceutical composition comprising the compound of any one of claims 1-62, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

64. A method of inhibiting alpha- -syn) protein aggregation in a subject having, or at-syn protein aggregation, which method comprises administering to the subject the pharmaceutical composition of claim 63 -syn protein --synprotein aggregation.

65. The method of claim 64, wherein the subject has, or is at risk for, Parkinson’s disease or dementia with Lewy bodies (DLB).

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