CN1838950A - Methods for treating protein aggregation disorders - Google Patents

Abstract

The present invention is based, at least in part on the discovery of therapeutic agents capable of preventing, inhibiting or modulating abnormal processing, misfolding or aggregation of protein. The therapeutic agents of the invention may prevent, inhibit or modulate the formation of inclusions. The therapeutic agents of the invention may also be capable of facilitating clearance and/or blocking the cellular toxicity of inclusions to treat or ameliorate disorders characterized by protein aggregation. Compounds which bind to structural motifs commonly found in protein aggregates, such as ss-sheets, would represent strong candidates for such compounds and are therefore desirable.

Description

Methods of treating protein aggregation diseases

Related applications

This application claims the benefits of U.S. Provisional Patent Application 60/480,918, filed June 23, 2003, U.S. Provisional Application 60/512,017, filed October 17, 2003, and U.S. Application 10/____,____, filed June 18, 2004 Priority, the titles of the above three applications are "Methods for Treating Protein Aggregation Disorders".

This application is related to U.S. Provisional Patent Application 60/480,984, filed June 23, 2003, U.S. Provisional Patent Application 60/512,116, filed October 17, 2003, and U.S. Application 10/____,____, filed June 18, 2004 (Docket No. NBI-152), entitled "Pharmaceutical Formulations of Amyloid-Inhibiting Compounds."

This application is related to U.S. Provisional Application 60/482,214, filed June 23, 2003, and U.S. Provisional Application 60/436,379, filed December 24, 2002, entitled "Combination Therapy for Alzheimer's Disease" the Treatment of Alzheimer's Disease), U.S. Utility Model Application 10/746,138, filed December 24, 2003, International Patent Application PCT/CA2003/002011 (designated NBI-154PC), and U.S. Application 10, filed June 18, 2004 /___, ___, both titled "Therapeutic Formulations for the Treatment of Beta-Amyloid Related Diseases".

This application is related to U.S. Provisional Patent Application 60/482,058, filed June 23, 2003, and U.S. Provisional Patent Application 60/512,135, filed October 17, 2003, both titled "Preparation of Compounds for the Treatment of Amyloidosis" "Synthetic Process for Preparing Compounds for Treating Anzyloidosis", and U.S. Application 10/___, ___ (file number NBI-156) filed on June 18, 2004, entitled "Improved drug candidates and methods for their preparation" (Improved Pharmaceutical Drug Candidates and Method for Preparation Thereof).

This application is also related to U.S. Provisional Patent Application 60/480,906, filed June 23, 2003, U.S. Provisional Patent Application 60/512,047, filed October 17, 2003, U.S. Application 10/____, filed June 18, 2004, ___ (Docket No. NBI-162A), U.S. Application 10/___, ___, filed June 18, 2004 (Docket No. NBI-162B), all titled "Methods and Compositions for Treating Amyloid-Related Diseases" "(Methods and Compositions for Treating Amyloid-Related Diseases).

This application is also related to U.S. Provisional Patent Application Serial No. 60/512,018, filed October 19, 2003, U.S. Provisional Patent Application Serial No. 60/480,928, and U.S. Patent Application 10/___,___(filed on June 18, 2004 No. NBI-163), all of the above application titles are "Methods and Compositions for Treating Amyloid-and Epileptogenesis-Associated Diseases".

This application is also related to US Patent Application 08/463,548 (now US Patent No. 5,972,328) "Method for Treating Amyloidosis".

The entire contents of each of the above patent applications are hereby expressly incorporated by reference, including without limitation the specification, claims and abstract and any drawings, tables or figures thereof.

Background technique

The biological function of a protein depends on its three-dimensional structure, which is largely determined by the protein's amino acid sequence, but is also influenced by the environment. In fact, the conformation of a protein governs its interactions with other factors that can participate in regulating protein function. The inability of peptides to adopt and maintain their correct structure through correct protein folding is a major threat to cell function and survival. Consequently, complex and precise systems have evolved to protect cells from the deleterious effects of misfolded proteins.

The first line of defense against misfolded proteins are chaperones, which bind to nascent polypeptides coming out of the ribosome, promoting proper folding of the polypeptide and preventing deleterious interactions. Chaperones also assist in the refolding of proteins damaged by stress and cellular damage (Netzer and Hartl. 1998. Trends Biochem Sci 23:68). Nonetheless, a large fraction of just-translated proteins fail to fold correctly, yielding large numbers of defective polypeptides (Schubert et al., 2000. Nature 404:770). These defective proteins are primarily degraded by the ubiquitin-proteasome system, a multicomponent system that recognizes and degrades excess proteins. In some cases, misfolded proteins escape this quality control system. When these misfolded proteins accumulate in sufficient quantities, they have a tendency to aggregate and are resistant to proteolysis. Insoluble protein aggregates (or precipitated proteins) can be deposited in microscopic inclusion bodies (also called inclusion bodies) or protein plaques, which are often characteristic of disease and contain disease-specific proteins.

When proteolysis is impaired, proteasomes and ubiquitinated proteins accumulate, forming organized aggregates or aggregates. Once formed, protein oligomers and larger aggregates have toxic properties by directly impairing vital cellular functions (Wojcik and DeMartino. 2003. Int J Biochem Cell Biol 35:579; Bence et al., 2001. Science 292: 1552). Furthermore, their accumulation in aggregates or inclusion bodies impairs the ubiquitin-proteasome system normally responsible for clearing such harmful misfolded proteins (see Garcia-Mata et al., 2002. Traffic 3:388). Since chaperone and ubiquitin-dependent proteolytic systems are critical for the regulation of fundamental cellular events such as cell division and apoptosis, disruption of this system can exacerbate cytotoxicity due to the accumulation of protein aggregates. However, data suggest that the presence of inclusion bodies, aggregates or plaques is not essential for the toxic response seen in proteopathy (Klement et al., 1998. Cell 95:41). Nonetheless, the presence of inclusion bodies, aggregates, or plaques is an excellent cellular marker to characterize cellular models of reverted human pathology and in vivo mouse models. This marker can be used for screening purposes as an indicator of unwanted protein aggregation activity leading to the formation of said inclusion bodies.

Thus, abnormal protein folding, oligomerization, aggregation or deposition can play an important role in the pathophysiology of various classes of chronic progressive degenerative diseases.

For example, many degenerative diseases are characterized by the presence of inclusion bodies and plaques. Non-limiting examples of such diseases include the following: Parkinson's disease (PD), diffuse Lewy body dementia (DLBD), multiple system atrophy (MSA), atrophic myotonia, dentate rubrum pallidal Lewy DRPLA, Friedrich's ataxia, Fragile X syndrome, Fragile XE mental retardation, Ma-Joe disease, Spinobulbar muscular atrophy (also known as Kennedy's disease), Spinocerebellar ataxia, Huntington's disease (HD), familial encephalopathy with neurofilin inclusions (FENIZB), Pick's disease, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis / Parkinson's Syndrome - Dementia Syndrome, Amyotrophic Lateral Sclerosis (ALS), Down's Syndrome, Age-Related Macular Degeneration, Cataracts and Wilson's Disease. In many cases, the genetic mutational basis of familial forms of these diseases has been identified. However, in most cases, the primary event that promotes or initiates the conformational transition of the target protein is not known, but it does eventually lead to abnormal processing, misfolding, oligomerization or aggregation of the protein, causing cytotoxicity . Abnormally folded proteins often accumulate in various types of cellular inclusions or aggregates as part of cellular detoxification, defense strategies or as an attempt to degrade abnormally folded proteins. Therefore, these inclusion bodies or aggregates have become one of the pathological hallmarks of said various classes of degenerative diseases. So far, there are no therapeutic drugs and fully effective treatments for these diseases.

Summary of Invention

It is desirable to have therapeutic compounds that prevent, inhibit or modulate abnormal processing, misfolding or aggregation of proteins, thereby preventing cell damage and death. These therapeutic compounds are useful in the treatment of the diseases mentioned in the background section above, and also in the treatment of those diseases described below. For example, compounds that directly bind target proteins and act on early protein oligomerization cascades can inhibit the formation of aggregates, aggregates or inclusion bodies. Compounds that bind aggregates and block the cytotoxicity associated with these aggregates are also effective in treating or ameliorating diseases caused by protein aggregation. Compounds that bind structural features commonly found in proteins leading to the formation of such aggregates, such as β-sheets, fibril-like structures or hydrophobic domains, represent strong candidates for such therapeutic applications and are therefore desirable. Such compounds are expected to facilitate the clearance and/or inhibit the toxicity of such abnormal proteins and established protein aggregates by preventing the de novo formation of aggregates.

The present invention is based at least in part on the discovery of therapeutic agents capable of preventing, inhibiting or modulating aberrant processing, misfolding or aggregation of proteins. The therapeutic drug of the present invention can prevent, inhibit or regulate the formation of inclusion bodies. The therapeutic agent of the present invention can promote the clearance of protein aggregates. The therapeutic drug of the present invention can also block the cytotoxicity of the inclusion body, thereby treating or improving diseases characterized by protein aggregation. The therapeutic drug of the present invention can also be used to prevent or treat diseases of protein conformation or protein aggregation.

In one embodiment of the present invention, there is provided a compound capable of binding a target protein with a propensity to form a β-sheet structure, thereby preventing, inhibiting or modulating misfolding, conformational transition, abnormal processing or aggregation of said protein. In another embodiment, the compound binds to structural motifs commonly found in protein aggregates, such as β-sheets.

Another aspect of the present invention provides a method for screening compounds for inhibiting protein aggregation or treating protein aggregation diseases, the method comprising screening for compounds that bind to proteins whose aggregation is indicative of a disease (eg synuclein in PD).

In another embodiment, compounds are provided that prevent self-association, oligomerization or aggregation of said protein, and the cytotoxicity associated with these activities.

In another embodiment, a compound that binds a target protein of interest prevents the conformational transition of the protein to a β-sheet and prevents the formation of naturally occurring oligomers, aggregates or fibrils following said transition.

In another embodiment, compounds are provided which prevent, inhibit or modulate the formation of aggregates or inclusion bodies indicative of toxic protein oligomers, aggregates or Assembly of fibrils.

In another embodiment, there is provided a method for screening compounds for the treatment or prevention of protein aggregation diseases, the method comprising administering the compound to transgenic mice undergoing progressive degenerative changes mimicking human disease, and screening for the ability to prevent some Or all of the compounds that are normally associated with the degenerative changes associated with this condition. In other embodiments, the method may comprise determining the effectiveness of the test compound to promote the clearance of unwanted protein aggregates, or determining the effectiveness of the test compound to promote the degradation of unwanted protein aggregates.

In one embodiment of the invention there is provided a method of treating or preventing a protein aggregation disorder comprising administering a compound of the invention to an individual suffering from or susceptible to such a disorder. In one embodiment, the protein aggregation disease is not Amyloid Proteopathy.

In another embodiment of the present invention, a compound-containing pharmaceutical composition is provided, which comprises an effective amount of the compound for treating protein aggregation diseases and a pharmaceutically acceptable carrier. In one embodiment, the protein aggregation disease is not an amyloid disease.

In one embodiment, a packaged composition for treating a protein aggregation disorder is provided, the composition comprising a compound having targeted therapeutic activity and instructions for using the composition to treat a protein aggregation disorder.

In another embodiment, a method for regulating harmful protein aggregation is provided, the method comprising contacting harmful protein aggregates or proteins with a tendency to form a β-sheet structure with an effective amount of the compound of the present invention, so that harmful protein aggregation is regulated , wherein the protein aggregation disease is not an amyloid disease.

In another embodiment, a method for regulating harmful protein aggregation is provided, the method comprising contacting harmful protein aggregates or proteins with a tendency to form a β-sheet structure with an effective amount of the compound of the present invention, so that the removal of harmful protein aggregation is modulated, thereby modulating unwanted protein aggregation, wherein the protein aggregation disease is not an amyloid disease.

In another embodiment, a method for regulating harmful protein aggregation is provided, the method comprising contacting harmful protein aggregates or proteins with a tendency to form a β-sheet structure with an effective amount of the compound of the present invention, causing the cells where the harmful protein aggregates Toxicity is modulated, thereby modulating unwanted protein aggregation, wherein the protein aggregation disease is not an amyloid disease.

In one embodiment, there is provided a method of treating or preventing tau-associated neurofibrillary tangles in a subject, the method comprising administering an effective amount of a compound of the invention such that the tau-associated neurofibrillary tangles are obtained treatment or prevention.

In one embodiment, there is provided a method of modulating tau-associated neurofibrillary tangles in a subject, the method comprising administering an effective amount of a compound of the invention such that tau-associated neurofibrillary tangles are modulated.

In one embodiment, there is provided a method of treating or preventing inclusion bodies containing α-synuclein NAC fragments in a subject, the method comprising administering an effective amount of a compound of the present invention such that α-synuclein NAC fragment-containing inclusion bodies are treated or prevented.

In one embodiment, there is provided a method of modulating inclusion bodies containing an NAC fragment of alpha-synuclein protein in a subject, the method comprising administering an effective amount of a compound of the invention to induce inclusion of an NAC fragment containing alpha-synuclein protein body is adjusted.

Brief description of the attached drawings

Figure 1 - This figure (computer generated) shows the assembly of NAC peptides into β-sheets at different concentrations using the Thioflavin (thioflavin) T assay described in the Examples below.

Figure 2 - Circular dichroism analysis (computer generated) of the conformation of the NAC peptide described in the Examples.

Figure 3 - Electron micrograph (computer generated) showing the appearance of NAC fibers described in the Examples below.

Figure 4 - Electron micrographs (computer generated) showing the effect of heparin on NAC fibril formation as described in the Examples below.

Figures 5-68 - Schematic representation of the compounds of the invention described herein. The compounds shown and their pharmaceutically acceptable salts, prodrugs (including their esters and amides), their pharmaceutical compositions and their use in the methods described hereinafter are included as part of the present invention.

Detailed description of the invention

The present invention provides methods and compounds useful for preventing, treating or modulating protein aggregation disorders. For convenience, certain definitions of terms referred to herein are set forth below.

The biological function of a protein depends on its three-dimensional structure, which is largely determined by the protein's amino acid sequence. When a protein emerges from the ribosome and begins the folding process to form the correct three-dimensional structure, its hydrophobic domains are exposed, which can lead to unsuccessful binding and unwanted protein aggregation (Wetzel.1994.Trends Biotechnol 12:193) . As used herein, "misfolded protein" refers to a protein or peptide whose conformation has not yet conformed to its correct three-dimensional structure, often resulting in aggregation, oligomerization or fibrillization of the abnormal protein by itself or with other proteins or peptides. As used herein, "conformational change" refers to the act of subjecting a normal protein to changes that result in changes in structural properties. Protein misfolding or conformational changes can occur during or after translation. This activity can be due to, for example, specific mutations (familial or idiopathic), extended polyglutamine repeats, DNA mutations or RNA modifications, amino acid misincorporation, or unequal synthesis of subunits in multisubunit proteins or other Occurs as a result of protein modification (Wetzel. 1994. Trends Biotechnol 12: 193; Bonifacino et al., 1989. J Cell Biol 109: 173; Hurle et al., 1994. Proc Natl Acad Sci USA, 91: 5446; see table below). Specifically, the absence (or reduction) of natural binding partners in multisubunit complexes or the absence (or reduction) of specific chaperone molecules (see below) results in interactions (direct or indirect) normally with these proteins or factors protein misfolding. These changes can occur during post-translational modifications of proteins, such as aberrant proteolytic processing, phosphorylation, methylation, acetylation, glycosylation, or nitrosylation of target proteins. Such modifications may be the result of specific mutations or the activation of specific cellular biochemical pathways.

The following table lists the various types of protein post-translational modifications: serial number protein post-translational modification 1 pPoly(ADP-ribosyl)ylation (Burkle, 2000) 2 Isoprenoids (Maltese, 1990) 3 Enzymatic glycosylation (Guevara et al., 1998) (Berninsone & Hirschberg, 1998) 4 Acetylation (Ogryzko, 2001) 5 Methylation (Person et al., 2003) 6 S-nitrosylation of protein Cys residues (Lane et al., 2001; Gu et al., 2002)) 7 Phosphorylation (Berninsone & Hirschberg, 1998) (Verkman & Mitra, 2000) 8 Mono-ADP-ribosylation (Okazaki & Moss, 1999) 9 Palmitoylation (Beers & Fisher, 1992) 10 Hydroxylation of prolyl residues (Rucker & Dubick, 1984) 11 Oxidative deamination of lysyl residues (Rucker & Dubick, 1984) 12 Crosslinks that covalently bond polypeptide chains (Rucker & Dubick, 1984) 13 Tyrosine Sulfation (Huttner, 1988) 14 Sulfation of glycans (Berninsone & Hirschberg, 1998) 15 Phosphorylation of glycans (Moses et al., 1997) 16 Carboxymethylation (Eggo et al., 1983) 17 Iodination (Eggo et al., 1983) 18 Tyrosination (Nath et al., 1981) 19 Oxidation of amino acid side chains, especially those of cysteine, prolyl, arginyl, lysyl, and histidyl residues) (Nath et al., 1981) 20 Deamination of asparaginyl and glutaminyl residues (Nath et al., 1981) twenty one Addition of metal ions (Waggoner et al., 2000) twenty two S-Glutathionylation of Cys (Dalle-Donne et al., 2003) twenty three Proteolysis (Nakayama et al., 2001) twenty four Non-enzymatic reactions of carbohydrates with protein amino groups (Munch et al., 1998) 25 Carboxylation of glutamine residues (Ware et al., 1989) 26 Hydroxylation of Proline and Lysine (Uzawa et al., 1998) 27 Carboxy-terminal amidation (Evans & Shine, 1991) 28 Citrullination (van Venrooij & Pruijn, 2000) 29 Carbamylation (Hasuike et al., 2002) 30 Sumoization (Freiman and Tjian 2003)

Beers, M.F. & Fisher, A.B. (1992). Am J Physiol 263, L151-60.

Berninsone, P. & Hirschberg, C.B.(1998)..Ann N Y Acad Sci 842, 91-9.

Burkle, A. (2000). Ann NY Acad Sci 908, 126-32.

Dalle-Donne, I., Giustarini, D., Rossi, R., Colombo, R. & Milzani, A. (2003). FreeRadic Biol Med 34, 23-32.

Eggo, M.C., Drucker, D., Cheifetz, R. & Burrow, G.N. (1983). Can J Biochem Cell Biol 61, 662-9.

Evans, H F. & Shine, J. (1991). Endocrinology 129, 1682-4.

Freiman RN, Tjian R. (2003). Cell 112: 11-7

Gu, Z., Kaul, M., Yan, B., Kridel, S.J., Cui, J., Strongin, A., Smith, J.W., Liddington, R.C. & Lipton, S.A. (2002). Science 297, 1186-90 .

Guevara, J., Espinosa, B., Zenteno, E., Vazguez, L., Luna, J., Perry, G. & Mena, R. (1998). J Neuropathol Exp Neurol 57, 905-14.

Hasuike, Y., Nakanishi, T., Maeda, K., Tanaka, T., Inoue, T. & Takamitsu, Y. (2002). Nephron 91, 228-34.

Huttner, W.B. (1988). Annu Rev Physiol 50, 363-76.

Lane, P., Hao, G. & Gross, S.S.(2001). Sci STKE 2001, RE1.

Maltes, W.A. (1990)..Faseb J 4, 3319-28.

Moses, J., Oldberg, A., Cheng, F. & Fransson, L.A. (1997). Eur J Biochem 248, 521-6.

Munch, G., Schinzel, R., Loske, C., Wong, A., Durany, N., Li, J.J., Vlassara, H., Smith, M.A., Perry, G. & Riederer, P. (1998) .J Neural Transm 105, 439-61.

Nakayama, K.I., Hatakeyama, S. & Nakayama, K. (2001). Biochem Biophys ResCommun 282, 853-60.

Nath, J., Flavin, M. & Schiffmann, E. (1981). J Cell Biol 91, 232-9.

Ogryzko, V.V.(2001). Cell Mol Life Sci 58, 683-92.

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Person, M.D., Monks, T.J. & Lau, S.S. (2003). Chem Res Toxicol 16, 598-608.

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Uzawa, K., Marshall, M.K., Katz, E.P., Tanzawa, H., Yeowell, H.N. & Yamauchi, M. (1998).. Biochem Biophys Res Commun 249, 652-5.

van Venrooij, W.J. & Pruijn, G.J. (2000). Arthritis Res 2, 249-51.

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Changes in post-translational modifications may be the result of specific mutations or the activation of specific cellular biochemical pathways. Misfolding, oligomerization or aggregation may also result from changes in the cellular environment, such as pH, temperature, ionic strength, redox environment or other stressors imposed on a particular biological environment. For example, changes in pH and temperature lead to partial unfolding of proteins and trigger a stress response that lessens cellular damage. Since a certain amount of misfolding is unavoidable, cells have evolved several systems to minimize this misfolding and to dispose of misfolded proteins prior to aggregation (Wickner et al., 1999. Science 286:1888) .

The first line of defense against misfolded proteins are molecular chaperones. As used herein, "molecular chaperone" or "chaperone molecule" refers to a molecule that binds to a nascent polypeptide exiting a ribosome, promoting proper folding and preventing deleterious interactions. Chaperones also assist in the refolding of proteins damaged by stress and cellular damage (Netzer and Hartl. 1998. Trends Biochem Sci 23:68). Chaperone molecules bind to and stabilize exposed hydrophobic amino acid residues, allowing proteins to adopt and maintain a correctly folded state by preventing incorrect intramolecular and intermolecular interactions of partially folded or unfolded polypeptides. Indeed, many proteins require chaperones for proper folding (Hartl. 1996. Nature 381:571). Despite this, a large fraction of newly translated proteins fail to fold correctly, resulting in a pool of defective polypeptides (Schubert et al., 2000. Nature 404:770). Transient or chronic absence of specific chaperones can also lead to accumulation of misfolded proteins.

These defective proteins are mainly degraded by the ubiquitin-proteasome system. As used herein, "ubiquitin-proteasome system" refers to a multicomponent system that recognizes and degrades excess proteins. As used herein, "proteasome" refers to a multi-subunit complex that occurs in the nucleus and cytoplasm. The proteasome mediates the degradation of cytoplasmic, nuclear (Hershko and Ciechanover. 1998. Ann Rev Biochem 67:425), secreted and transmembrane proteins (Hirsch and Ploegh. 2000. Trends Cell Biol 10:268). The ubiquitin-proteasome system contributes to the regulation of numerous cellular events by selectively degrading short-lived normal proteins in addition to clearing defective proteins. In some cases, misfolded proteins may escape the ubiquitin-proteasome surveillance system designed to promote correct folding and elimination of defective proteins. When these misfolded proteins accumulate in sufficient quantities, they tend to aggregate and become resistant to proteolysis. As used herein, "aggregates", "inclusion bodies", "inclusion bodies", "fibrils" and "plaques" refer to abnormal association and accumulation of abnormal proteins that are resistant to proteolysis and can either May not bind to molecules of the proteasome system. In many cases, these aggregates may colocalize with specific markers, such as the cytoskeletal microtubule markers vimentin, β-tubulin, and γ-tubulin, and may be extracellular, intracellular, or nuclear.

Proteasomes and ubiquitinated proteins often accumulate when proteolysis is impaired and can form organized clusters within inclusion bodies or plaques as part of cellular detoxification or defense strategies. When these clusters are specifically transported to inclusion bodies by dynein-dependent retrograde transport on microtubules and form in the pericentrosome region, these aggregates (as used herein) are referred to as aggregates. The aggregate pathway provides a mechanism by which aggregated proteins can form granular (approximately 200 nm) small aggregates that are transported to MTs on microtubules (MTs) by a process mediated by the minus-terminus dynein-dynein Organization Center (MTOC). Once at the MTOC, the individual particles are packed into individual, generally spherical aggregates (1-3 microns) surrounding the MTOC. Aggregates are motile: they recruit a variety of chaperones and the proteasome, possibly to assist in the processing of aggregated proteins, and serve as a cytoprotective mechanism against cytotoxicity (Taylor et al., 2003. Hum Mol Genet 12:749). Furthermore, the formation of aggregates likely activates autophagic clearance mechanisms that end in lysosomal degradation. Thus, the aggregate pathway may provide a new system to transport aggregated proteins from the cytosol to the lysosome for degradation. Once formed, accumulating aggregates (Wojcik and DeMartino. 2003. Int J Biochem Cell Biol 35:579) and protein oligomers or aggregates elsewhere in the cell (Bence et al., 2001. Science 292: 1552) can weaken ubiquitin- Function of the proteasome system and generation of toxicity. (For a review, see Garcia-Mata et al., 2002 Traffic 3: 388). Therefore, abnormalities in protein aggregation or deposition can play an important role in the pathophysiology of various classes of chronic progressive degenerative diseases.

In fact, it has been shown that many degenerative diseases are associated with oligomerization, aggregation or fibrosis of various proteins (see Kakizuka. 1998. TIG 14: 396 for a review). For example, many neurodegenerative diseases are caused by extended CAG repeats encoding polyglutamine stretches. Proteins containing extended polyglutamine repeats appear to aggregate themselves, resulting in neuronal cell death or degeneration. Non-limiting examples of these diseases are: spinal bulbar muscular atrophy, or Kennedy's disease, caused by extended polyglutamine repeats in the gene encoding the androgen receptor (AR); Huntington's disease (HD), caused by the huntingtin gene caused by an expanded polyglutamine repeat in the ataxia-1 gene; spinocerebellar ataxia type 1 (SCA1), caused by an increased polyglutamine repeat in the ataxin-1 gene; serpinopathy, caused by silk Caused by mutations in the ataxin (serine protease inhibitor) gene; spinocerebellar ataxia type 2 (SCA2), caused by increased polyglutamine repeats in the ataxia-3 gene; equine-joke disease (MJD or SCA3), caused by increased polyglutamine repeats in the ataxin-3 gene; spinocerebellar ataxia type 6 (SCA6), caused by increased polyglutamine repeats in the ataxin-6 gene Repeated sequences; spinocerebellar ataxia type 7 (SCA7), caused by increased polyglutamine repeats in the ataxia-7 gene; spinocerebellar ataxia type 17 (SCA17), caused by ataxia Caused by increased polyglutamine repeats in the dysregulin-17 gene; dentate rubin-pallidal Lewy body atrophy (DRPLA); and serpinopathy, caused by mutations in the serpin gene. While each of these diseases may be caused by mutations in different proteins, they all share a common feature, namely aggregation through self-association. Polyglutamine proteins have been shown to undergo aggregation facilitated by elongated polyglutamine segments, and this aggregation induces or increases cell death. Studies with transgenic mice have also shown that polyglutamine fragments are toxic, suggesting that polyglutamine accumulation is the root cause of neurodegeneration (Schilling et al., 1999. Hum Mol Genet 8:397; Reddy et al., 1998. Nat Gen20: 198; DiFiglia et al., 1997. Science 277:1990; Yamamoto et al., 2000. Cell 101:57).

Similarly, the predominant pathology in frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP17) is the assembly of tau proteins into fibrils (termed paired helical fibrils or PHFs) that form neurofibrillary tangles knot (NFT). This neuropathological feature is the characteristic change that defines the family of disorders known as tauopathies, which include Alzheimer's disease, dementia pugilistica, Down syndrome, prion diseases, muscular dystrophy Lateral sclerosis/parkinsonism-dementia syndrome, argyophilic graindementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcifications, frontotemporal dementia associated with chromosome 17/Park Kinson's syndrome, Hastings disease, multiple system atrophy (MSA), Nee-dermatosis type C, Pick's disease, progressive supranuclear palsy, subacute sclerosing panencephalitis, and tangle-predominant ) Alzheimer's disease (AD).

In frontotemporal dementia (FTD), aberrant assembly results from the post-translational modification, hyperphosphorylation, of the fully mature tau protein. Protein modifications can result from upstream events, such as those induced by overproduction of the Aβ peptide in AD, or by abnormal splicing activity of the tau gene resulting from mutations in the tau intron sequence, and have been associated with frontotemporal dementia (Spillantini et al., 1998. Proc Natl Acad Sci USA 95:7737). Expression of a mutant form of tau in transgenic mice is sufficient to induce the formation of neurofibrillary tangles similar to neurofibrillary tangles characteristic of human tauopathy, demonstrating that abnormal tau is sufficient to induce some forms of neurodegeneration. In transgenic mice expressing the amyloid precursor protein (APP) gene and mutant forms of tau, the mutant tau protein cooperates with mutant APP to produce a disease whose neuropathology is more similar to that observed in AD (for review, see Lee et al. et al., 2001. Science 293:1446; Gotz et al., 2001. Science 293:1491; Lewis et al., 2001. Science 293:1487).

A pathological feature of Parkinson's disease (PD) is the formation of cytoplasmic endosomes called Lewy bodies; these Lewy bodies are also seen in dementia with Lewy bodies (DLBD) and multiple system atrophy (MSA). The main component of Lewy bodies is α-synuclein. Thus, α-synuclein becomes another example of a protein whose aggregation is associated with neurodegeneration. Accumulation of α-synuclein in Lewy bodies has been shown to be associated with several autosomal dominant mutations in the α-synuclein gene. These substitution mutations clearly favor conformational transitions in the mature protein, leading to protein accumulation in the form of pathological oligomers and fibrils (Polymeropoulos et al., 1997. Science 276:2045; Kruger et al., 1998. Nat Genet 18:106; Conway et al., 2000. Proc Natl Acad Sci USA 97:571). PD is also associated with recessive mutations in the parkin gene (Kitada et al., 1998. Nature 392:605; Lucking et al., 2000. N Engl J Med 3421560; Ishikawa and Tsuji. 1996. Neurology 47160), parkin protein as E2 Ubiquitin-dependent protein ligase whose activity is important for the ubiquitination of proteins destined for degradation by the proteasome (Shimura et al., 2000. Nat Genet 25:302). Thus, mutations that reduce or eliminate parkin function can lead to accumulation or aggregation of protein substrates that are otherwise targeted for degradation. In the absence of parkin activity, parkin binding partners or substrates such as PaelR1, cdc-rel1, synphilin-1, α-synuclein, β- and γ-tubulin (some of which are critical for cellular Directly toxic) (Petrucelli et al., 2002.Neuron 36:1007; Imai et al., 2001.Cell 105:891; Ren et al., 2003.J Neurosci 23:316) accumulate and cause cell damage and death (reviewed in Cookson. 2003. Neuron 37: 7). Another recent report suggested that dysregulation (mutation) of another α-synuclein-interacting protein, synphilin-1, present in Lewy bodies, can lead to some sporadic cases of PD (Marx et al., 2003 . Hum Mol Genet 12:1223). Similarly, in amyotrophic lateral sclerosis (ALS), inclusion bodies called hyaloid inclusions, known to contain deposits of the mutated superoxide dismutase (SOD1) protein, are often observed. About 20% of familial ALS cases are also associated with mutations in the SOD1 gene (Rosen et al., 1993. Nature 362:59; Orrell, 2000. Neuromuscular Disord 10:63).

Altogether, these observations suggest that dysregulation of the proteasomal ubiquitin-mediated degradation pathway can lead to the accumulation of misfolded and aggregated proteins, forming inclusion bodies, which are responsible for cytotoxicity and degeneration. Electron microscopy analysis revealed that these proteins often contained granular, fibrillar, and fibril-like structures. These structures, while distinct, are similar to amyloid structures observed in, for example, Alzheimer's disease and prion diseases. Insoluble "amyloid" aggregates stained with Congo red exhibit characteristic red-green birefringence under polarized light. Although the aggregated proteins discussed above are not amyloid themselves, they can form similar structures, including β-sheet secondary structures. Hydrophobic regions are also characteristic of aggregated proteins. Thus, although many of the aggregated proteins found in protein aggregation diseases are not amyloid themselves, they share many of the structural features of amyloid, namely β-sheets, fibril-like structures and/or hydrophobic domains. Since all aggregates share common structural features, compounds that bind or inhibit amyloid formation by interacting with these structural motifs (e.g., β-sheets) are effective in preventing or inhibiting protein aggregation involved in protein aggregation diseases effect.

Therefore, screening for compounds that treat, modulate, prevent or inhibit unwanted protein aggregation represents a rational and general approach to treating or preventing diseases of protein aggregation.

Generally, a "protein aggregation disease or protein aggretinopathy" includes a disease, disorder or condition associated with unwanted protein aggregation in a subject. "Detrimental protein aggregation" is the unwanted and deleterious accumulation, oligomerization, fibrosis or aggregation of two or more heterologous or homologous proteins or peptides. Detrimental protein aggregates can be deposited in inclusion bodies, inclusion bodies, or plaques, which are often characteristic of disease and contain disease-specific proteins, such as in Parkinson's disease Contains α-synuclein Lewy bodies. Detrimental protein aggregates are three-dimensional structures that can contain, for example, misfolded proteins composed of β-sheets, fibril-like structures, and/or highly hydrophobic domains that tend to aggregate and are toxic to cells. Furthermore, although noxious protein aggregates do not contain amyloid deposits, and since it does not meet the strict definition of amyloid, it does not show the characteristic red-green or apple-green color when stained with Congo red under polarized light Birefringence, therefore, is also not considered to be associated with amyloidosis, but it can nevertheless be described as amyloid-like aggregates. As used herein, "non-amyloid" deleterious protein aggregates or "proteinopathies" refer to deleterious protein aggregates that do not contain amyloid deposits. Non-limiting classes of protein aggregation disorders or proteinopathies include Protein Conformational Disorders, Alpha-Synucleinopathies, Polyglutamine disorders, Serpinopathies, Taupathies, or others related diseases. Non-limiting examples of protein aggregation diseases include Parkinson's disease (PD), diffuse Lewy body dementia (DLBD), multiple system atrophy (MSA), atrophic myotonia, dentate rubrum pallidal Lewy body atrophy (DRPLA), Friedrich's ataxia, Fragile X syndrome, Fragile XE mental retardation, Horse-Johnson disease (MJD or SCA3), Spinobulbar muscular atrophy (also known as Kennedy disease), Spinocerebellar comorbidity Ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia Type 17 (SCA17), chronic liver disease, Huntington's disease (HD), familial encephalopathy with neurofilin inclusions (FENIZB), Pick's disease, corticobasal degeneration (CBD), progressive supranuclear palsy ( PSP), amyotrophic lateral sclerosis/parkinsonism-dementia syndrome, amyotrophic lateral sclerosis (ALS), cataract, serpinopathy, hemolytic anemia, cystic fibrosis, Wilson's disease, neurological Fibromatosis type 2, demyelinating peripheral neuropathy, retinitis pigmentosa, Marfan syndrome, emphysema, idiopathic pulmonary fibrosis, argyrophilic granular dementia, corticobasal degeneration, diffuse neurofibrillary Tangles with calcifications, frontotemporal dementia/parkinsonism associated with chromosome 17, Haas disease, Nee-dermatosis type C, or subacute sclerosing panencephalitis.

These disease categories and examples are discussed in further detail below. It is to be understood that the present invention includes embodiments of protein aggregation disorders associated with proteins having structural features that can be selectively targeted by compounds of the present invention. Examples of such structural features include β-sheets, fibril-like structures and/or hydrophobic domains. It should also be understood that the protein aggregation diseases described herein are not meant to include amyloid diseases or amyloidosis or methods of modulating or inhibiting amyloid deposition. See below for specific examples.

The effects of harmful protein aggregation are often cumulative, with progressive loss of cellular function culminating in cell death, which is responsible for a variety of pathological conditions.

Amyloidosis

Pathological deposition of amyloid is characteristic of a group of diseases known as amyloidoses, which include AA (reactive) amyloidosis, AL amyloidosis, senile systemic amyloidosis, cerebral amyloidosis ( including Alzheimer's disease and cerebral vascular amyloidosis), dialysis-associated amyloidosis, type II diabetes (caused by the islet amyloid polypeptide "IAPP"), and others, all of which are characterized by the presence of shared morphological properties amyloid fibrils that can be stained with specific dyes (such as Congo red) and have a characteristic red-green birefringent appearance under polarized light after staining. See for example WO 2003/017994A1. They also share common superstructural features and common X-ray diffraction phenomena and infrared spectra. The amyloidogenic proteins associated with each of these diseases differ in amino acid sequence but all have similar properties of self-association, forming oligomers and fibrils, and binding with various other components such as proteoglycans, Amyloid P and/or complement component binding. Moreover, each amyloidogenic protein, although different in amino acid sequence, displays similarities in functional domains capable of binding the glycosaminoglycan (GAG) portion of proteoglycans (known as the GAG-binding site) and promoting β - the area formed by the folds. Many amyloidogenic proteins are formed by proteolytic cleavage of precursor proteins such as serum amyloid A protein ("ApoSAA", yielding the AA peptide), transthyretin (sometimes called prealbumin ), β-amyloid precursor protein ("βAPP", yielding Aβ peptide), and peptides derived from the N-terminal region of monoclonal immunoglobulin κ or λ light chains. See eg 2001. Physiological Reviews Vol. 81.

Amyloid-related disease may be confined to one organ, or spread to several organs. The former is called "limited amyloidosis" and the latter is called "systemic amyloidosis".

Some amyloid diseases may be idiopathic, but most of these diseases occur as complications of pre-existing disease. For example, primary amyloidosis can occur without any other pathology, or it can follow plasma cell disease or multiple myeloma.

Secondary amyloidosis is often found to be associated with chronic infection (such as tuberculosis) or chronic inflammation (such as rheumatoid arthritis). Familial secondary amyloidosis is also seen in familial Mediterranean fever (FMF). As one of the other types of familial amyloidosis, this form of familial amyloidosis is hereditary and occurs in certain groups of people. In both primary and secondary amyloidoses, deposits are found in several organs, so they can be considered systemic amyloid diseases.

Another type of systemic amyloidosis is seen in long-term hemodialysis patients. Each of these cases had a different amyloidogenic protein involved in amyloid deposition.

A "localized amyloidosis" is an amyloidosis that tends to affect a single organ system. Different amyloidoses can also be characterized by the type of protein in the deposit. For example, neurodegenerative diseases such as scrapie, bovine spongiform encephalitis, Creutzfeldt-Jakob disease, and others can be diagnosed by the appearance and accumulation of a protease-resistant form of the prion protein (called PrP ^Sc or PrP27-30) in the central nervous system. characterization. Similarly, Alzheimer's disease, another neurodegenerative disorder, can be characterized by neuritic plaques and neurofibrillary tangles. In this case, plaques and vascular amyloid are formed by the deposition of fibrillar Aβ amyloid. Other diseases, such as adult onset diabetes (type II diabetes), can be characterized by localized accumulation of amyloid in the pancreas.

Unless otherwise specified, the term "β-amyloid" or "amyloid β" as used herein refers to amyloid β protein or peptide, amyloid β precursor protein or peptide, their intermediates, modifications and fragments. Specifically, "Aβ" refers to any peptide produced by the proteolytic processing of the amyloid precursor protein (APP) gene product, especially those involved in amyloid pathology, including Aβ _1-39 , Aβ _1-40 , Aβ _1-41 , Aβ _1-42 and Aβ _1-43 . The terms "β-amyloid", "amyloid β" and "Aβ" are used synonymously herein.

Unless otherwise stated, the term "amyloid" refers to an amyloidogenic protein, peptide or pieces of them.

In one embodiment of the invention protein aggregation diseases do not include amyloid diseases. The term "amyloidopathy" as used herein refers to the following disorders (the relevant amyloidogenic protein in parentheses after the disease): reactive or secondary amyloidosis (AA); idiopathic (primary) amyloidosis Degeneration; myeloma or macroglobulinemia-associated amyloidosis (amyloid kappa light chain or amyloid lambda light chain); familial amyloid polyneuropathy (Portuguese, Japanese, Swedish) (ATTR); Familial amyloid cardiomyopathy [Danish type] (ATTR); isolated cardiac amyloidosis (ATTR); systemic senile amyloidosis (ATTR); medullary carcinoma of the thyroid (calcitonin) ; isolated atrial amyloidosis (atrial natriuretic peptide); familial amyloidosis [Finnish type] (gelsolin); hereditary cerebral hemorrhage with amyloidosis [Icelandic type] (cysteine Protease Inhibitor C); Familial Amyloid Polyneuropathy [Iowa Type] (AApoA-I); Accelerated Aging in Mouse (AApoA-II); Fibrinogen-Associated Amyloidosis; Lysozyme-Associated Amyloidosis; Human Prion diseases; Transmissible spongiform encephalopathy; Scrapie (prion or PrP); Creutzfeldt-Jakob disease (PrP); GSS syndrome (PrP); Fatal familial insomnia (PrP); Bovine spongiform encephalitis ( PrP); Alzheimer's disease (Aβ); Cerebral amyloid angiopathy (Aβ); Hereditary cerebral hemorrhage (Aβ); Familial Mediterranean fever; Familial amyloid nephropathy with urticaria and deafness; Dialysis-associated amyloidosis (β2-microglobulin); Type II diabetes mellitus (IAPP); Down syndrome (Aβ); age-related macular degeneration (Aβ); and inclusion body myositis (Aβ). Amyloid diseases may be familial, or idiopathic, or sporadic, or infectious, and are intended to include all forms of the diseases listed above. It should be understood that the term "amyloid disease" is intended to include WO 96/28287 published on September 19, 1996, WO 00/64420 published on November 2, 2000 and WO 94/2000 published on October 13, 1994. Diseases described in 22437.

protein conformational disease

A group of diverse disorders has been grouped together under the name Protein Conformational Diseases (PCD) (Carrell and Lomas. 1997. Lancet 350: 134; Kelly. 1996. Curr Opin Struct Biol 6: 11; Thomas et al., 1995. Trends Biochem Sci 20: 456; Soto. 1999. JMol Med 77: 412; Carrell and Gooptu. 1998. Curr Opin Struct Biol 8: 799). Non-limiting examples of such diseases include serpinopathy, hemolytic anemia, Huntington's disease (HD), cystic fibrosis, amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). This category also includes amyloid-related diseases such as Alzheimer's disease (AD), transmissible spongiform encephalopathy (TSE), type II diabetes, dialysis-associated amyloidosis, secondary (AA) amyloidosis, Cerebral amyloid angiopathy, inclusion body myositis, Down syndrome, and age-related macular degeneration. PCD also includes spinobulbar muscular atrophy (SBMA) or Kennedy disease, Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1); spinocerebellar ataxia type 2 (SCA2), horse-young disease Ataxia disease (MJD or SCA3), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia type 17 (SCA17), dentate rubrum pallidum Atrophy with Lewy bodies (DRPLA), Myotonia atrophicus, Pick's disease, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis/parkinsonism-dementia syndrome , Friedrich's ataxia, fragile XE brain developmental delay, fragile X syndrome, Wilson's disease, chronic liver disease, and cataracts.

The defining feature of PCD is changes in the secondary or tertiary structure of normal proteins. Changes in protein conformation can contribute to disease by acquiring toxicity or by disabling the biological function of natively folded proteins (Thomas et al., 1995. Trends Biochem Sci20: 456; Carrell and Gooptu. 1998. Curr Opin Struct Biol 8: 799) . There is no apparent sequence or structural homology among the proteins involved in PCD, but these proteins are distinguished by their inherent ability to adopt at least two different stable conformations (Carrell and Gooptu. 1998. Curr Opin Struct Biol 8:799) . In most PCDs, misfolded proteins are enriched in β-sheet conformations (Soto. 1999. J Mol Med 77: 412; Carrell and Gooptu. 1998. Curr Opin Struct Biol 8: 799). β-sheets are one of the ubiquitous repeating secondary structures in folded proteins, consisting of alternating peptide fold chains connected by hydrogen bonds between NH groups and CO groups of peptide bonds. Hydrogen bonds in an α-helix occur between groups within the same strand, while hydrogen bonds in a β-sheet occur between one strand and another. Since the second β-strand originates from a different region of the same protein or from a different protein molecule, β-sheet formation is usually stabilized by oligomerization or aggregation of the protein. Indeed, in most PCDs, misfolded proteins self-associate and deposit in multiple organs in the form of various types of aggregates or inclusion bodies, causing tissue damage and organ dysfunction (Kelly. 1996 Curr Opin Struct Biol 6 :11). In one embodiment of the invention protein conformational diseases exclude amyloid diseases.

Alpha-synucleinopathies

In general, alpha-synucleinopathies include Parkinson's disease (PD) and other related disorders, including diffuse Lewy body dementia (DLBD; also called Lewy body disease), Heidelberg syndrome, neurological Orthostatic hypotension, Schmidt-Merde syndrome, Parkinson's plus syndrome, and multiple system atrophy (MSA; four brain degenerative diseases of Schmidt-Merde syndrome, neurogenic orthostatic hypotension and Parkinson's plus syndrome grouped together). A common feature of this group of diseases is abnormal deposition of α-synuclein in the cytoplasm of neurons or glial cells, forming inclusion bodies called Lewy bodies.

Alpha-synuclein is a major component of Lewy bodies and dystrophic axons in Parkinson's disease and diffuse Lewy body dementia; alpha-synuclein also accumulates in the cytoplasm of glial cells . In multiple system atrophy, α-synuclein forms cytoplasmic oligodendroglial and neuronal inclusions that are hallmarks of the disease.

Alpha-synuclein is a protein of 140 amino acids. Its function is unknown, but it has been shown to have chaperone activity (Souza et al., 2000. FEBS Lett 474:116) and has been proposed to play a role in regulating synaptic vesicle formation (Murphy et al., 2000. JNeurosci 29:3214) . In addition, α-synuclein has been shown to bind tau protein (Jensen et al., 1999. J Biol Chem 274:25481) and the microtubule-associated protein MAP-1B (Jensen et al., 2000. J Biol Chem 275:21500).

Accumulation of α-synuclein in α-synucleinopathies has a common fibrillar structure that produces insoluble and high-molecular-weight aggregates in vitro, but these There are differences in the binding of ubiquitin, with the exception of ubiquitin, whose binding to α-synuclein is common to all α-synuclein inclusion bodies (see Ferrer. 2001. Neurologia 16: 163 for a review). For example, Lewy bodies in MSA patients were found to contain the 14-3-3 protein, which mediates several types of signal transduction pathways (Kawamoto et al., 2002. Ann Neurol 52:722); Lewy bodies in PD patients contained -Synuclein-associated synphilin-1 (Wakabayashi et al., 2000.Ann Neurol47:521), while in the Lewy bodies of DLBD patients, cyclin-dependent kinase 5 (Cdk5) was found (Takahashi et al., 2000.Brain Res 862:253).

Transgenic mice expressing α-synuclein mutants A30P and A53T have been constructed. These mice showed an early-onset progressive decline in motor function. Neuropathologically, the axons of these animals showed typical α-synuclein immunoreactive Lewy body inclusions (Putten et al., 2000. J Neurosci 20:6021; Sommer et al., 2000. ExpGerontol 35:1389; Kahle et al., 2002. J Clin Invest 110: 1429; Kahle et al., 2001. Am J Pathol. 159: 2215; Gomez-Isla et al., 2003. Neurobiology Aging 24: 245-258; Lee et al., 2002. Proc Natl Acad Sci USA 99 :8968).

Parkinson's disease

Parkinson's disease (PD) is a slowly progressive late-onset neurodegenerative disorder. It is characterized by muscle stiffness, postural instability, and tremors.

Recent data imply several genetic factors in the etiology of PD. Studies of familial clusters suggest that late-onset PD has a significant genetic etiology (Payami et al., 2002. Arch Neurol 59:848). Hereditary forms of PD result from genetic mutations. To date, four genes (α-synuclein, parkin, COOH-terminal hydrolase L1, and DJ-1) and several additional loci have been shown to be associated with familial forms of PD (for review see Shastry. 2000. Neuroscientist 6: 234; Shasrty. 2001. Neurosci Res 41: 5; Cookson. 2003. Neuron 37: 7). Autosomal dominant PD is caused by mutations in the alpha-synuclein gene, while autosomal recessive PD is caused by mutations in the parkin gene.

Several lines of evidence suggest that noxious protein aggregation in substantia nigra dopaminergic neurons is a common mechanism of neurodegeneration in all known forms of PD. Three proteins whose mutations are associated with the development of PD (α-synuclein, parkin, and COOH-terminal hydrolase L1) are also present in sporadic PD (Mouradian. 2002. Neurology 58:179) and DLBL (Schlossmacher et al. , 2002.Am J Pathol 160:1655) in Lewy bodies. A pathological hallmark of Parkinson's disease is the formation of cytoplasmic inclusions known as Lewy bodies; these inclusions are also seen in DLBD and MSA. The main component of Lewy bodies is α-synuclein. Thus, α-synuclein is another example of a protein whose aggregation is associated with neurodegeneration. Aggregation of α-synuclein in Lewy bodies has been shown to be associated with certain autosomal dominant mutations within the α-synuclein gene. These substitution mutations apparently favor conformational transitions within the mature protein, resulting in the aggregation of the mature protein in the form of pathological oligomers and prefibrils (Polymeropoulos et al., 1997. Science 276:2045; Kruger et al., 1998. Nat Genet 18:106; Conway et al., 2000. Proc Natl Acad Sci USA 97:571). PD is also associated with recessive mutations in the parkin gene (Kitada et al., 1998. Nature 392: 605; Lucking et al., 2000. N Engl J Med 342: 1560; Ishikawa and Tsuji. 1996. Neurology 47: 160), the The activity of parkin as an E2-dependent ubiquitin protein ligase is important for the ubiquitination of proteins destined for degradation by the proteasome (Shimura et al., 2000. Nat Genet 25:302). Thus, loss of parkin function leads to accumulation or aggregation of protein substrates that would otherwise be targeted for degradation. In the absence of parkin activity, parkin binding partners or substrates such as PaelR1, cdc-rel1, synphilin-1, α-synuclein, α- and γ-tubulin (some of which are critical for cellular Directly toxic) (Petrucelli et al., 2002.Neuron 36:1007; Imai et al., 2001.Cell 105:891; Ren et al., 2003.J Neurosci 23:316) accumulate and cause cell damage and death (reviewed in Cookson. 2003. Neuron 37: 7). Another recent report suggested that dysregulation (mutation) of another α-synuclein-interacting protein, synphilin-1, present in Lewy bodies, can lead to some sporadic cases of PD (Marx et al., 2003 . Hum Mol Genet 12:1223). Altogether, these observations suggest that dysregulation of the proteasomal ubiquitin-mediated degradation pathway can lead to the accumulation of misfolded and aggregated proteins, forming inclusion bodies, which are responsible for cytotoxicity and degeneration.

Importantly, aggregation of α-synuclein in cultured human cells selectively denatures dopaminergic but not non-dopaminergic neurons in the presence of dopamine, suggesting α-synuclein aggregation Selective toxicity of (Xu et al., 2002.Nat Med, 8, 600). Recombinant α-synuclein incubated at 37°C for long periods of time forms aggregates and fibrils in vitro whose morphology is similar to that of fibrils isolated from Lewy bodies (E1-Agnaf et al., 1998. FEBS Lett 440:67 ; Conway et al., 1998. Nat Med 4:1318). Overexpression of parkin in the presence of proteasome inhibitors leads to the formation of aggregate-like inclusion bodies (Junn et al., 2002. J Biol Chem 6:47870). Furthermore, mice expressing known mutations in human α-synuclein showed adult-onset neurodegeneration and accumulation of α-synuclein in the brain (Lee et al., 2002. Proc Natl Acad Sci USA, 99, 8968; Kahle et al., 2001. Am J Pathol. 159:2215; Kahle et al., 2002. J Clin Invest 110:1429; Gomez-Isla et al., 2003. Neurobiology Aging 24:245).

Recent findings have shown that Lewy bodies are also formed in a large proportion of Alzheimer's disease (AD) patients, most in tonsils (Hamilton.2000.Brain Pathol, 10:378; Mukaetova-Ladinska et al., 2000.J Neuropathol Exp Neurol 59:408). Interestingly, the highly hydrophobic non-amyloid component (NAC) domain of α-synuclein has also been described as the second most abundant component of amyloid plaques in the brains of AD patients, after Aβ.

[alpha]-synuclein has been shown to form fibrils in vitro. Furthermore, it binds Aβ and promotes its aggregation (Yoshimoto et al., 1995. Proc Natl Acad Sci USA 92:9141). In fact, it was originally identified as a precursor of the non-amyloid beta (Aβ) component (NAD) of AD plaques (Ueda et al., 1993. Proc Natl Acad Sci USA 90: 11282; Iwai. 2000. Biochem Biophys Acta 1502:95; Masliah et al., 1996. Am J Pathol 148:201). NAC is a 35 amino acid long peptide with highly hydrophobic segments that can self-aggregate to form fibrils in vitro. Moreover, these fibrils efficiently elicit the formation of A[beta] fibrils in vitro (Han et al., 1995. Chem Biol. 2: 163-169; Iwai et al., 1995. Biochemistry 34: 10139). α-synuclein actually maintains its fibrillogenic properties through its NAC domain. Therefore, modulating the properties of NAC or directing the compounds of the present invention to NAC may be an effective therapeutic approach to inhibit the formation of protein aggregates and inclusion bodies associated with α-synucleinopathy, and inhibit the formation of protein aggregates and inclusion bodies in β-amyloid peptide and α -Aggregate formation between NACs of synuclein.

polyglutamine disease

Several often fatal adult-onset disorders with progressive neurodegenerative degeneration have been shown to result from expansions of the [CAG]n (polyglutamine or polyQ) segment in specific target proteins. To date, these disorders have included atrophic myotonia, dentate rubrum pallidal Lewy atrophy (DRPLA), Friedrich's ataxia, fragile X syndrome, fragile XE mental retardation, May-John disease , spinobulbar muscular atrophy (also known as Kennedy disease), spinocerebellar ataxia, and Huntington's disease (HD) (Kaneko et al., 1997. Proc NatlAcad Sci 94:10069; Zoghbi and Orr. 2000. Ann Rev Neurosci, 23 :217).

The prototypical polyglutamine disorder, Huntington's disease (HD), is an autosomal dominant neurodegenerative disorder characterized by involuntary movements, cognitive decline that progresses to dementia, and emotional dysregulation. The disease is characterized by a selective loss of striatal neurons, caused by an expansion of the polyglutamine segment in the huntingtin (HD) gene (Shastry.1994. Nasir et al., 1996; Tobin and Singer.2000. Trends Cell Biol, 10, 531). Mutant proteins (or polyglutamine-containing subfragments) formed ubiquitinated aggregates in neurons from patients or mouse models, and in most cases in neuronal nuclei.

Although the genes responsible for polyQ disease appear to be functionally unrelated, they all share the common feature of a CAG trinucleotide repeat in the coding region of each gene, which results in polyglutamine in the protein Amide segment. In the normal population, the length of polyQ is generally in the range of 10-36 consecutive glutamine residues. However, in each of these conditions, the polyQ segment expands beyond the normal range, resulting in adult-onset, slowly progressive neurodegeneration. The longer the extension, the earlier the onset and the severity of the disease.

These diseases likely share a common molecular pathogenesis caused by toxicity of the extended polyglutamine tract. It is now clear that expanding polyQ dramatically increases the function of disease proteins, which is toxic to neurons. Each polyQ disorder has a different characteristic pattern of neurodegeneration and, therefore, a different clinical presentation. The selective vulnerability of different neuronal populations in these diseases is poorly understood, but is likely to be related to the expression patterns of the various disease genes and to the normal function and interaction of disease gene products (Dragatsis et al., 2000. Nature Genet 26:300; Zuccato et al., 2001. Science 293:493).

Extended polyQ has been recognized to form neuronal intranuclear inclusions in animal models of polyQ disease and in the central nervous system of patients with the disease (Ross. 1997. Neuron 19: 1147). These inclusion bodies consist of insoluble aggregates of polyQ-containing proteins or aggregates of polyQ-containing fragments associated with other proteins. Proteins with long polyQ segments have been proposed to misfold and aggregate into antiparallel β-strands, termed "polar zippers" (Perutz. 1994. Proc Natl Acad Sci USA, 91:5355). The correlation between the threshold PolyQ length for aggregation to occur in experimental systems and the length of the CAG repeat that causes disease in humans supports the contention that self-association or aggregation of extended polyQ is responsible for its acquired toxic function. Although in some experimental systems the toxicity of extended polyQ was not associated with the formation of visible inclusion bodies, the formation of insoluble molecular aggregates appears to be a stable feature of cytotoxicity (Sisodia.1998.Cell 95:1; Klement et al., 1998.Cell 95:41; Saudou et al., 1998. Cell 95:55; Muchowski et al., 2002. Proc Natl Acad Sci USA99:727).

Transgenic mice expressing multiple sets of huntingtin alleles with extended polyglutamine segments exhibit an early clasping phenotype, impaired motor coordination, and hyperactivity. These phenotypes are associated with cortical inclusions, septal inclusions, hippocampal inclusions, reactive gliosis, cell loss, general brain atrophy, and neuropathological phenomena of cellular inclusions that are commonly seen in patients with Huntington's disease (Schilling et al., 1999. Hum Mol Genet 8: 397; Reddy et al., 1998. Nat Genet 20: 198; DiFiglia, et al., 1997. Science 277: 1990; Yamamoto et al., 2000. Cell 101: 57). Similar animal models have been developed for other polyglutamine disorders.

Serpinopathy

Serpinopathies include alpha(1)-antitrypsin (SERPINA1) deficiency and the newly characterized familial encephalopathy with neuroserpinin inclusions (FENIB) resulting from mutations in the neuroserpinin (SERPINI1) gene.

Serpins, or serine protease inhibitors, are a superfamily of proteins found in a wide range of species, including viruses, plants and humans. This family includes many different members such as α1-antichymotrypsin, C1 inhibitor, antithrombin and plasminogen activator inhibitor-1. In addition to its role in inflammation, complement, coagulation, and fibrinolysis, serpin is also involved in chromatin packaging, including the proteins MENT and neurospin. Its classification into the serpin superfamily is based on more than 30% amino acid homology to α1-trypsin, as well as on the basis of three β-sheets (A-C) and an exposed mobile reactive loop (which converts the peptide sequence as The pseudosubstrate presents the conserved tertiary structure of the target protease).

This conformation is required for the function of the proteases, but also makes them susceptible to conformational transitions that lead to disease. Point mutations can destabilize β-sheet A, allowing the incorporation of another loop of the serpin molecule. Sequential insertion of reactive loops produces polymer chains that are then retained in the cell, accumulating leading to tissue damage (Stein and Carrell. 1995. NatStruct Biol 2:96).

This common mechanism has been proposed to explain changes in disease phenotype associated with mutations in members of the serpinin superfamily, leading to the classification of these distinct diseases as serpinopathies (for review see Lomas and Carrell. 2002 . Nat Rev Genet 3:759).

tauopathy

Filamentous tau protein inclusions, similar to those observed in AD and prion patients, are central features of a wide variety of sporadic neuropathological disorders, an observation that has led researchers to propose that tau protein is the Causes of diseases of tauopathy. Such diseases include Alzheimer's disease, dementia pugilistica, Down syndrome, prion diseases, cerebral amyloid angiopathy, amyotrophic lateral sclerosis/parkinsonian-dementia syndrome, argyrophilic granular dementia , corticobasal degeneration, diffuse neurofibrillary tangles with calcifications, frontotemporal dementia/parkinsonism associated with chromosome 17, Haas disease, multiple system atrophy (MSA), Nye-dermatosis type C , Pick's disease, progressive supranuclear palsy, subacute sclerosing panencephalitis, and tangle-dominant Alzheimer's disease (AD) (Lee and Goedert. 2001. Ann Rev Neurosci 24:1121).

Tau protein is a low molecular weight microtubule-associated protein that is abundant in the central and peripheral nervous systems. The discovery that multiple mutations in the gene encoding tau are associated with frontotemporal dementia and Parkinsonism (FTDP-17) provides strong evidence that abnormal forms of tau contribute to these and other underlying neurodegenerative diseases (Reed et al., 2001. Neurobiol 22:89). In addition, polymorphisms associated with the tau gene appear to be risk factors for sporadic corticobasal degeneration, progressive supranuclear palsy, and Parkinson's disease (Martin et al., 2001. J Am MedAssoc 286:2245; Cole et al., 1999. Semin Neurol 19:407). AD-specific neurofibrillary tangles (NFTs) (see above) also consist of intracellular aggregates of hyperphosphorylated microtubule tau. Indeed, hyperphosphorylation appears to be a post-translational event that promotes the assembly of tau proteins into aggregates.

Although the regions of the brain affected by each of these disorders vary, evidence suggests that some common features appear to be present in all regions: impaired splicing of tau leading to aberrant hyperphosphorylation of tau, tau Fibrosis occurs and tau protein is eventually deposited as aggregates (for review see Avila. 2000. FEBS Lett 476: 89; Taylor et al., 2002. Science, 296: 1991).

Transgenic mice expressing a long isoform of tau protein carrying mutations seen in patients with frontotemporal dementia and Parkinsonism develop tauopathy characterized by the formation of congophilic hyperphosphorylated tau in forebrain neurons inclusion body. These inclusions appear as early as 18 months of age. In human cases, tau inclusion bodies consist of mutant and endogenous wild-type tau proteins and are associated with microtubule disruption and flame-shaped transformations of affected neurons. Behaviorally, aged transgenic mutant tau mice exhibit cognitive deficits, particularly impaired associative memory (Lewis et al., 2000. Nat Genet 25:402; Gotz et al., 2001. J Biol Chem276:529; Tatebayashi et al., 2002. Proc Natl Acad Sci USA 99: 13896; Gotz et al., 2001. Eur J Neurosci. 13: 2131; Tanemura et al., 2002. J Neurosci 22: 133; Ishihara et al., 1999. Neuron 24: 751; Spittaels et al., 1999. Am J Pathol 155:2153; Probst et al., 2000. Acta Neuropathol 99:469).

Other related protein aggregation disorders

Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of upper and lower motor neurons. About 10% of ALS cases are hereditary; the remainder are believed to be sporadic cases (Cole et al., 1999. Semm Neurol, 19:407), with neuropathology characterized by degeneration and damage of motor neurons and glial hyperplasia. Intracellular inclusions are found in degenerated neurons and glia (Rowland et al., 2001. N Eng J Med, 344:1688). Among inherited cases, about 20% of cases are caused by mutations in the gene encoding superoxide dismutase 1 (SOD1). More than 70 different pathogenic SOD1 mutations have been described. The neuropathological hallmark of familial ALS is neuronal Lewy body-like hyaloid inclusions and astrocytic hyaloid inclusions composed of mutant SOD1. Supporting the virogenicity of mutant SOD1 aggregates is the observation that murine models of mutant SOD1-mediated disease are characterized by prominent intracellular inclusions in motor neurons and, in some cases, motor neurons. There are prominent intracellular inclusions in astrocytes surrounding neurons (Cleveland and Liu. 2000. Nature Med 6:1320).

cataract

Mutations in alpha-crystallin can lead to cataracts. Alpha-crystallin is the major protein component of the lens of the mammalian eye. They are molecular chaperones involved in the folding of many proteins, and several families are known to exist in mammalian cells, including the small heat shock protein (sHSP) family. The molecular weight of sHSP is about 15-30 kDa. Alpha-crystallin is thought to play a key role in maintaining the clarity of the lens through its ability to inhibit stress-induced aggregation of unwanted proteins. α-crystallin prevents aggregation of other unfolded lens crystallins and proteins by 'trapping' them in high molecular weight complexes. However, during aging, the chaperone function of α-crystallin is gradually impaired, leading to the formation of light-scattering aggregates or inclusion bodies. In the central part of the lens, damaged proteins are not renewed, so post-translational modifications of α-crystallin accumulate, which further reduces the chaperone function of α-crystallin, leading to the formation of a cataract lens (for review see Horwitz.2003 .ExpEye Res. 76:145).

Wilson's disease

Wilson's disease is a genetic disorder characterized by the accumulation of copper in the body due to defective secretion of copper from liver cells. The intracellular localization of the Wilson's disease gene product ATP7B was recently identified as late endosomes. ATP7B is a membrane copper transporter. Multiple mutations have been documented in patients with Wilson's disease. The clinical presentation of patients varies widely. The green fluorescent protein-tagged common ATP7B mutant His1069Gln was expressed in Huh7 cells and HEK293 cells. This mutant protein is not localized to late endosomes and is degraded by the proteasome in the cytoplasm. Furthermore, His1069Gln forms aggregates consisting of degradants and intermediate filaments at the microtubule organizing center (Harada et al., 2001. Gastroenterology 121:1264). In addition, ubiquitin and the heat shock proteins hsp70 and hsp90 were shown to co-localize in aggregates in liver biopsies from patients with Wilson's disease (Riley. 2003. Exp Mol Pathol 74: 168). Based on the characteristics of aggregates in patients with Wilson's disease (localization in paranuclear regions, colocalization with proteasome subunits, transglutaminase, and heat shock proteins), it has been suggested that Wilson's disease can be attributed to the protein A member of conformational disease (French.2001.Gastroenterology 121:1264).

Compounds of the invention

The present invention relates to a method for treating or preventing a protein aggregation disease other than an amyloid disease (as defined herein) in a subject (preferably a mammal, more preferably a human), said method comprising administering to the subject an effective amount of any of the following formulae The compounds shown can treat or prevent protein aggregation diseases. In another embodiment, the present invention relates to a method of preventing, inhibiting, treating or modulating a protein aggregation disease other than an amyloid disease (as defined herein) in a subject, preferably a mammal, more preferably a human, said method It includes administering an effective amount of the compound of the present invention to a subject to prevent, inhibit, treat or regulate protein aggregation diseases.

A group of examples of compounds of the present invention are alkylsulfonic acids, which have the structure of formula Q-[-Y ^-- X ⁺ ] _n , wherein Q is a carrier molecule; Y is SO ₃ ^- X ⁺ , OSO ₃ ^- X ⁺ or SSO ₃ ⁻ X ⁺ ; X ⁺ is a cationic group, such as a positively charged atom or other moiety. Suitable carrier molecules include carbohydrates, polymers, peptides, peptide derivatives, aliphatic groups, cycloaliphatic groups, heterocyclic groups, aromatic groups, or combinations thereof. The carrier molecule may be substituted, for example by one or more amino, nitro, halogen, mercapto or hydroxyl groups. See WO 96/28187, WO 01/85093 and US Patent No. 5,840,294.

A specific group of compounds of the present invention have the following structures:

Wherein Y is an amino group (with the structure of -NR ^a R ^b ) or a sulfonic acid group (with the formula -SO ₃ ^- X ⁺ ), n is an integer of 1-5, and X is hydrogen or a cationic group (such as sodium). Some exemplary alkyl sulfonic acids include the following:

In some instances, the alkylsulfonic acids are "small molecules", that is, the compounds themselves are not products of gene transcription or translation (e.g., proteins, RNA, or DNA) and have low molecular weight (e.g., less than about 2500), and in other cases, compounds of the invention It can be a biological product, such as an antibody or an immunogenic peptide.

Alkylsulfonic acids can be prepared by the methods illustrated in the general reaction schemes, for example, in U.S. Patent Nos. Synthetic Process" ("SYNTHETIC PROCESS FOR PREPARING COMPOUNDS FOR TREATING AMYLOIDOSIS"), using readily available starting materials, reagents , prepared using conventional synthetic procedures. In these reactions it is also possible to use variations which are known per se but not mentioned here. Functional and structural equivalents of the compounds described herein having the same general properties, wherein one or more simple changes to the substituents do not adversely affect the basic properties or applications of the compounds, such equivalents can be obtained through the art Various methods are known to prepare.

The term "alkylsulfonic acid" as used herein includes substituted or unsubstituted alkylsulfonic acids, and substituted or unsubstituted lower alkylsulfonic acids. Amino-substituted compounds are of particular interest, so the invention relates to substituted or unsubstituted amino-substituted alkylsulfonic acids and substituted or unsubstituted amino-substituted lower alkylsulfonic acids, an example of which is 3-amino-1-propanesulfonic acid acid. Moreover, it should also be noted that the term "alkanesulfonic acid" as used herein should be understood as synonymous with the term "alkanesulfonic acid".

The present invention relates to substituted or unsubstituted alkylsulfonic acids, substituted or unsubstituted alkylsulfuric acids, substituted or unsubstituted alkylthiosulfonic acids, substituted or unsubstituted alkylthiosulfuric acids, or Their esters or amides, including their pharmaceutically acceptable salts. For example, the compounds of the present invention are substituted or unsubstituted alkylsulfonic acids, or their esters or amides, including their pharmaceutically acceptable salts. In another embodiment, the compound of the present invention is a substituted or unsubstituted lower alkylsulfonic acid, or its ester or amide, including its pharmaceutically acceptable salt. Similarly, the present invention includes compounds that are (substituted or unsubstituted amino) substituted alkylsulfonic acids, or esters or amides thereof, including pharmaceutically acceptable salts thereof. In yet another embodiment, the compound is a (substituted or unsubstituted amino) substituted lower alkylsulfonic acid, or an ester or amide thereof, including pharmaceutically acceptable salts thereof.

"Alkyl" as used herein includes saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl base, decyl, etc.), cyclic alkyl (or "cycloalkyl" or "alicyclic" or "carbocyclyl") (such as cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclo octyl, etc.), branched alkyl groups (isopropyl, tert-butyl, sec-butyl, isobutyl, etc.), and alkyl-substituted alkyl groups (such as alkyl-substituted cycloalkyl and cycloalkyl-substituted alkyl). The term "aliphatic" includes organic moieties typically having from 1 to 22 carbon atoms, characterized by straight or branched chains. In complex structures, the chains may be branched, bridged or cross-linked. Aliphatic groups include alkyl, alkenyl and alkynyl groups.

Accordingly, the present invention relates to substituted or unsubstituted alkylsulfonic acids, which are substituted or unsubstituted linear alkylsulfonic acids, substituted or unsubstituted cycloalkylsulfonic acids, and substituted or unsubstituted Branched chain alkyl sulfonic acid.

The structures of some of the compounds of the invention include chiral carbon atoms. It is to be understood that isomers arising from such asymmetries (eg, all enantiomers and diastereomers) are included within the scope of the invention unless otherwise indicated. That is, unless otherwise specified, any chiral carbon center can adopt either the (R)- or (S)-stereochemical configuration. Said isomers can be obtained in substantially pure form by classical separation techniques as well as by stereochemically controlled syntheses. Additionally, the compounds of the present invention may exist in unsolvated form, or form solvates with acceptable solvents such as water, THF, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention. The term "solvate" denotes a polymer comprising one or more molecules of a compound and one or more molecules of a pharmaceutical solvent such as water, ethanol, and the like.

In certain embodiments, the straight chain or branched chain alkyl group may have 30 or less carbon atoms in its main chain, such as C ₁ -C ₃₀ straight chain or C ₃ -C ₃₀ branched chain. In some embodiments, the main chain of the linear or branched alkyl group may have 20 or less than 20 carbon atoms, such as C ₁ -C ₂₀ straight chain or C ₃ -C ₂₀ branched chain, and may even have 18 or less carbon atoms. Likewise, exemplary cycloalkyls have 4-10 carbon atoms in the ring structure, or 4-7 carbon atoms in the ring structure.

The term "lower alkyl" refers to an alkyl group having 1-6 carbons in the chain, or a cycloalkyl group having 3-6 carbons in the ring structure. "Lower" in "lower alkyl" means that the moiety in question has at least one and less than about 8 carbon atoms, unless the number of carbons is specified otherwise. In some embodiments, the straight chain or branched lower alkyl group may have 6 or less than 6 carbon atoms in the main chain (such as C ₁ -C ₆ straight chain or C ₃ -C ₆ branched chain), for example Methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl. Likewise, a cycloalkyl group can have 3-8 carbon atoms in its ring structure, for example, it can have 5 or 6 carbon atoms in its ring structure. The term "C ₁ -C ₆ " in "C ₁ -C ₆ alkyl" refers to an alkyl group containing 1 to 6 carbon atoms.

Furthermore, unless otherwise specified, the term alkyl includes "unsubstituted alkyl" and "substituted alkyl", the latter of which refers to an alkyl group having substitutions for one or more hydrogens on one or more carbons of the hydrocarbon backbone base. Such substituents may include, for example, alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxy, aryloxycarbonyloxy, carboxy, alkane Alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphoric acid, phosphonato, phosphonous acid (phosphinato), cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, amino formyl and ureido), imino, mercapto, alkylthio, arylthio, thiocarboxyl, sulfate, alkylsulfinyl, sulfonato, sulfonamide, sulfonamido, nitro group, trifluoromethyl group, cyano group, azido group, heterocyclyl group, alkylaryl group or aromatic group (including heteroaryl group).

An "arylalkyl" group is an alkyl group substituted with an aryl group (eg, phenylmethyl (ie, benzyl)). An "alkylaryl" moiety is an aryl group substituted with an alkyl group (eg, p-methylphenyl (ie, p-tolyl)). The term "n-alkyl" refers to straight chain (ie, non-branched) unsubstituted alkyl groups. "Alkylene" is the divalent analog of the corresponding alkyl group. The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups similar to alkyl, but containing at least one carbon-carbon double bond or triple bond, respectively. Suitable alkenyl and alkynyl groups include groups having 2 to about 12 carbon atoms, preferably 2 to about 6 carbon atoms.

The term "aromatic group" or "aryl" includes unsaturated aromatic cyclic hydrocarbons containing one or more rings as well as unsaturated aromatic heterocycles. Aryl groups can also be fused or bridged with aliphatic rings or non-aromatic heterocycles to form polycyclic rings (eg, tetralin). "Arylene" is the divalent analog of the corresponding aryl group. Aryl groups can also be fused or bridged with non-aromatic aliphatic or heterocyclic rings to form polycyclic rings (eg, tetralin).

The term "heterocyclyl" includes closed ring structures similar to carbocyclyls in which one or more atoms in the ring are elements other than carbon, such as nitrogen, sulfur or oxygen. A heterocyclyl group can be saturated or unsaturated. In addition, heterocyclic groups such as pyrrolyl, pyridyl, isoquinolyl, quinolinyl, purinyl and furyl may be of aromatic nature, in which case they may be referred to as "heteroaryl" or "Heteroaromatic group".

Unless otherwise specified, aryl and heterocyclyl (including heteroaryl) groups may also be substituted on one or more of their constituent atoms. Examples of heteroaromatic and heteroaliphatic groups can have 1 to 3 separate or fused rings, each ring is 3 to about 8 membered, with one or more N, O or S hetero atom. In general, the term "heteroatom" includes any element other than carbon or hydrogen, examples of preferred heteroatoms include nitrogen, oxygen, sulfur and phosphorus. A heterocyclyl group can be saturated or unsaturated or aromatic.

Examples of heterocycles include, but are not limited to, acridine; aziocinyl; benzimidazolyl; benzofuryl; benzothiofuryl; benzothienyl; benzoxazolyl; Triazolyl; Benzotetrazolyl; Benziisoxazolyl; Benziisothiazolyl; Benzimidazolinyl; Carbazolyl; 4aH-carbazolyl; benzopyranyl; cinnolinyl; decahydroquinolinyl; 2H,6H-1,5,2-dithiazinyl; dihydrofuro[2,3-b]tetrahydrofuran; furyl; furan Zanyl; imidazolidinyl; imidazolinyl; imidazolyl; 1H-indazolyl; indolenyl; indolinyl; chromanyl; isoindazolyl; isoindolinyl; isoindolyl; isoquinolinyl; isothiazolyl; isoxazolyl; methylenedioxyphenyl; morpholinyl ; 1,5-Naphthyridine; Octahydroisoquinolyl; Oxadiazolyl; -oxadiazolyl; 1,3,4-oxadiazolyl; oxazolidinyl; oxazolyl; oxazolidinyl; pyrimidinyl; base; phenoxathiinyl; phenoxazinyl; 2,3-diazinyl; piperazinyl; piperidinyl; base; purinyl; pyranyl; pyrazinyl; pyrazolidinyl; pyrazolinyl; pyrazolyl; pyridazinyl; pyridoxazolyl; pyridimidazolyl; Pyridyl; pyrimidinyl; pyrrolidinyl; pyrrolinyl; 2H-pyrrolyl; pyrrolyl; quinazolinyl; quinolinyl; 4H-quinazinyl; Tetrahydroisoquinolyl; tetrahydroquinolyl; tetrazolyl; 6H-1,2,5-thiadiazinyl; 1,2,3-thiadiazolyl; 1,2,4-thiadiazolyl Base; 1,2,5-thiadiazolyl; 1,3,4-thiadiazolyl; thianthranyl; thiazolyl; thienyl; thienothiazolyl; thienooxazolyl; thienyl; triazinyl; 1,2,3-triazolyl; 1,2,4-triazolyl; 1,2,5-triazolyl; 1,3,4-triazolyl; and xanthene base. Preferred heterocycles include, but are not limited to, pyridyl; furyl; thienyl; pyrrolyl; pyrazolyl; pyrrolidinyl; imidazolyl; ; benzotriazolyl; benzisoxazolyl; oxindolyl; benzoxazolinyl; and isatinoyl. Also included are fused ring compounds and spiro compounds containing, for example, the aforementioned heterocycles.

A common aryl group is phenyl with one ring. Bicyclic hydrocarbon aryls include naphthyl, indenyl, benzocyclooctenyl, benzocycloheptenyl, pentalenyl and azulenyl, and their partially hydrogenated analogs such as indanyl and tetra hydronaphthyl. Exemplary tricyclic hydrocarbon aryl groups include acephthylenyl, fluorenyl, phenalenyl, phenanthrenyl, and anthracenyl.

Aryl also includes heteromonocyclic aryl, i.e. monocyclic heteroaryl, such as thienyl, furyl, pyranyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl and pyridazine and their oxidized analogues such as pyridinonyl, oxazolone, pyrazolone, isoxazolone and thiazolone. Corresponding hydrogenated (i.e. non-aromatic) heteromonocyclic groups include pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidinyl and piperidino, piperidino Zinc and morpholino and morpholino.

Aryl also includes fused bicyclic heteroaryl, such as indolyl, isoindolyl, indolizyl, indazolyl, quinolinyl, isoquinolyl, 2,3-naphthyridine, Quinoxalinyl, quinazolinyl, cinnolinyl, benzopyranyl, isobenzopyranyl, benzothienyl, benzimidazolyl, benzothiazolyl, purinyl, quinazinyl, isobenzopyranyl Quinolonyl, quinolonyl, 1,5-diazinyl and pteridinyl, and partially hydrogenated analogues such as chromanyl, isochromanyl, indolinyl, Isoindolinyl and tetrahydroindolyl. Aryl also includes fused tricyclic groups, such as phenothioxyl, carbazolyl, phenanthridinyl, acridine, perimidinyl, phenanthrolinyl, phenazinyl, phenanthrinyl, Thiazinyl, phenoxazinyl and dibenzofuranyl.

Some typical aryl groups include substituted or unsubstituted 5- and 6-membered monocyclic aryl groups. In another aspect, each Ar group may be selected from substituted or unsubstituted phenyl, pyrrolyl, furyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyryl Azolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyridazinyl and pyrimidinyl. Further examples include substituted or unsubstituted phenyl, 1-naphthyl, 2-naphthyl, biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2 -Imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazole Base, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridine Base, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolinyl, 5-isoquinolinyl, 2 -quinoxalinyl, 5-quinoxalinyl, 3-quinolinyl and 6-quinoxalinyl.

The term "amine" or "amino" as used herein refers to an unsubstituted or substituted moiety of the formula -NR ^a R ^b , wherein R ^a and R ^b are each independently hydrogen, alkyl, aryl or heterocyclyl, or R ^a and R ^b together with the nitrogen atom to which they are attached form a cyclic moiety having 3 to 8 atoms in the ring. Thus, the term amino includes cyclic amino moieties such as piperidinyl or pyrrolidinyl unless otherwise specified. Thus, the term "alkylamino" as used herein refers to an alkyl group to which is attached an amino group. Suitable alkylamino groups include groups having 1 to about 12 carbon atoms, for example having 1 to about 6 carbon atoms. The term amino includes compounds or moieties wherein a nitrogen atom is covalently bonded to at least one carbon atom or heteroatom. The term "dialkylamino" includes groups in which a nitrogen atom is bonded to at least two alkyl groups. The terms "arylamino" and "diarylamino" include groups in which the nitrogen is bonded to at least one or two aryl groups, respectively. The term "alkylarylamino" refers to an amino group bonded to at least one alkyl group and at least one aryl group. The term "alkaminoalkyl" refers to an alkyl, alkenyl or alkynyl group substituted with an alkylamino group. The term "amide" or "aminocarbonyl" includes compounds or moieties containing a nitrogen atom bonded to a carbon of a carbonyl or thiocarbonyl group.

The term "alkylthio" refers to an alkyl group having an attachment to a mercapto group. Suitable alkylthio groups include groups having 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms.

The term "alkylcarboxy" as used herein refers to an alkyl group having a carboxyl attachment.

The term "alkoxy" as used herein refers to an alkyl group having an oxygen atom attached to it. Representative alkoxy groups include alkoxy groups having 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms, such as methoxy, ethoxy, propoxy, tert-butoxy, and the like. Examples of alkoxy include methoxy, ethoxy, isopropoxy, propoxy, butoxy and pentyloxy. Alkoxy may be substituted by, for example, alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxy, alkoxy Alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonate, phosphonite, cyano , amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido) , imino group, mercapto group, alkylthio group, arylthio group, thiocarboxy group, sulfate group, alkylsulfinyl group, sulfonic acid group, sulfamoyl group, sulfonylamino group, nitro group, trifluoromethyl group, cyano group, Azido, heterocyclyl, alkylaryl or aromatic or heteroaromatic moiety. Examples of halogen-substituted alkoxy include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc., and perhaloalkoxy Oxygen.

The term "acylamino" includes moieties wherein the amino moiety is bonded to an acyl group. For example, acylamino groups include alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.

The terms "alkoxyalkyl", "alkylaminoalkyl" and "thioalkoxyalkyl" include such alkyl groups further comprising an oxygen, nitrogen or sulfur atom replacing one or more carbons in the hydrocarbon backbone .

The term "carbonyl" or "carboxy" includes compounds or moieties containing a carbon double bonded to an oxygen atom. Examples of carbonyl-containing moieties include aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, and the like.

The term "ether" or "etheric" includes compounds or moieties containing oxygen bonded to two carbon atoms. For example, an ether group or group of ethers includes "alkoxyalkyl," which refers to an alkyl, alkenyl, or alkynyl group substituted with an alkoxy group.

A "sulfonic acid" or "sulfonate" group is a _-SO3H or _-SO3 ^- X ⁺ group bonded to a carbon atom, where X ⁺ is a cationic counterion group. Similarly, "sulfonic acid" compounds have a _-SO3H or _-SO3 ^- X ⁺ group bonded to a carbon atom, where X ⁺ is a cationic group. "Sulphate" as used herein is an _-OSO3H or _-OSO3 ^- X ⁺ group bound to a carbon atom, and a "sulfuric acid" compound has an _-OSO3H or _-OSO3 ^- X ⁺ group bound to a carbon atom , where X ⁺ is a cationic group. According to the invention, suitable cationic groups may be hydrogen atoms. In some cases, the cationic group may actually be another group on the therapeutic compound that is positively charged at physiological pH, such as an amino group.

"Counter ions" are required to maintain electrical neutrality and are pharmaceutically acceptable in the compositions of the invention. Compounds containing cationic groups covalently bonded to anionic groups may be referred to as "inner salts". Examples of anionic counterions include halides, triflate, sulfate, nitrate, hydroxide, carbonate, bicarbonate, acetate, phosphate, oxalate, cyanide, alkyl carboxylate, N-Hydroxysuccinimide, N-Hydroxybenzotriazole, Alcohol Anion, Thioalcohol Anion, Alkylsulfonyloxy, Haloalkylsulfonyloxy, Arylsulfonyloxy, Bisulfate, Oxalic Acid valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, formazan Sulfonate, Glucoheptonate or Lactobionate. Compounds containing cationic groups covalently bonded to anionic groups may be referred to as "inner salts".

The term "nitro" refers to -NO ₂ ; the term "halogen" or "halo" refers to -F, -Cl, -Br or -I; the term "thio" or "mercapto" refers to SH; the term "hydroxy" refers to - Oh.

The term "acyl" refers to a carbonyl group attached to a hydrogen through its carbon atom (ie, formyl), a carbonyl group attached to an aliphatic group (eg, acetyl), a carbonyl group attached to an aromatic group (eg, benzoyl), and the like. The term "substituted acyl" includes acyl groups in which one or more hydrogen atoms on one or more carbon atoms are replaced by groups such as: alkyl, alkynyl, halo, hydroxy, alkylcarbonyloxy, arylcarbonyloxy radical, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl , alkoxy, phosphate, phosphonate, phosphonite, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), imino, mercapto, alkylthio, arylthio, thiocarboxy, sulfate, alkylsulfinyl, sulfonic acid, Sulfamoyl, sulfonylamino, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Unless otherwise specified, chemical moieties of the compounds of the invention, including the genes discussed above, may be "substituted or unsubstituted". In some embodiments, the term "substituted" means that the moiety has a non-hydrogen substituent (ie, in most cases, replacing a hydrogen) placed on the moiety that enables the molecule to perform its intended function. Examples of the substituent include moieties selected from the group consisting of straight or branched chain alkyl (eg C ₁ -C ₅ ), cycloalkyl (eg C ₃ -C ₈ ), amino (including -NH ₂ ), - _SO3H , _-OSO3H , -CN, _-NO2 , halogen (eg -F, -Cl, -Br or -I), _-CH2OCH3 , _{-OCH3 ,} -SH, _-SCH3 , - OH and _-CO2H . Examples of such substituents include moieties selected from the group consisting of linear or branched alkyl (preferably C ₁ -C ₅ ), cycloalkyl (preferably C ₃ -C ₈ ), alkoxy (preferably C ₁ -C 8 ₆ ), thioalkyl (preferably C ₁ -C ₆ ), alkenyl (preferably C ₂ -C ₆ ), alkynyl (preferably C ₂ -C ₆ ), heterocyclyl, carbocyclyl, aryl (eg phenyl), aryloxy (e.g. phenoxy), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxyalkyl), arylacetamidoyl, alkylaryl, Heteroaralkyl, alkylcarbonyl and arylcarbonyl or other such acyl, heteroarylcarbonyl and heteroaryl, and (CR'R") _0-3 NR'R" (eg -NH ₂ ), (CR 'R") _0-3 CN (eg -CN), -NO ₂ , halogen (eg -F, -Cl, -Br or -I), (CR'R") _0-3 (halogen) ₃ (eg - CF ₃ ), (CR'R") _0-3 CH(halogen) ₂ , (CR'R") _0-3 CH ₂ (halogen), (CR'R") _0-3 CONR'R", (CR 'R") _0-3 (CNH)NR'R", (CR'R") _0-3 S(O) _1-2 NR'R", (CR'R") _0-3 CHO, (CR'R") _0-3 O (CR'R") _0-3 H, (CR'R") _0-3 S(O) _0-3 R' (eg _-SO3H ), (CR'R") _0-3 O(CR'R") _0-3 H (eg -CH ₂ OCH ₃ and -OCH ₃ ), (CR'R") _0-3 S(CR'R") _0-3 H (eg - SH and -SCH ₃ ), (CR'R") _0-3 OH (eg -OH), (CR'R") _0-3 COR', (CR'R") _0-3 (substituted or unsubstituted phenyl), (CR'R") _{0- 3} (C ₃ -C ₈ cycloalkyl), (CR'R") _0-3 CO ₂ R' (eg -CO ₂ H) and (CR'R ”) _0-3 OR' groups, wherein R' and R" are each independently hydrogen, C ₁ -C ₅ alkyl, C ₂ -C ₅ alkenyl, C ₂ -C ₅ alkynyl, or aryl; or The side chain of any natural amino acid.

In another embodiment, the substituents may be selected from linear or branched alkyl (preferably C ₁ -C ₅ ), cycloalkyl (preferably C ₃ -C ₈ ), alkoxy (preferably C ₁ -C ₆ ), thioalkyl (preferably C ₁ -C ₆ ), alkenyl (preferably C ₂ -C ₆ ), alkynyl (preferably C ₂ -C ₆ ), heterocyclyl, carbocyclyl, aryl (eg benzene radical), aryloxy (e.g. phenoxy), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxyalkyl), arylacetamido, alkylaryl, heteroaralkyl , alkylcarbonyl and arylcarbonyl or other such acyl, heteroarylcarbonyl and heteroaryl, (CR'R") _0-10 NR'R" (eg -NH ₂ ), (CR'R") _{0 -10} CN (eg -CN), -NO ₂ , halogen (eg F, Cl, Br or I), (CR'R") _0-10 (halogen) ₃ (eg -CF ₃ ), (CR'R" ) _0-10 CH(halogen) ₂ , (CR'R") _0-10 CH ₂ (halogen), (CR'R") _0-10 CONR'R", (CR'R") _0-10 (CNH )NR'R", (CR'R") _0-10 S(O) _1-2 NR'R", (CR'R") _0-10 CHO, (CR'R") _0-10 O(CR 'R'') _0-10 H, (CR'R'') _0-10 S(O) _0-3 R' (eg _-SO3H ), (CR'R'') _0-10 O(CR'R'' ) _0-10 H (eg -CH ₂ OCH ₃ and -OCH ₃ ), (CR'R") _0-10 S (CR'R") _0-3 H (eg -SH and -SCH ₃ ), (CR 'R") _0-10 OH (eg -OH), (CR'R") _0-10 COR', (CR'R") _0-10 (substituted or unsubstituted phenyl), (CR'R ") _0-10 (C ₃ -C ₈ cycloalkyl), (CR'R") _0-10 CO ₂ R' (eg -CO ₂ H) or (CR'R") _0-10 OR' group , or the side chain of any natural amino acid; wherein R' and R" are each independently hydrogen, C ₁ -C ₅ alkyl, C ₂ -C ₅ alkenyl, C ₂ -C ₅ alkynyl or aryl, or R ' and R" together form a benzylidene or -(CH ₂ ) ₂ O(CH ₂ ) ₂ - group.

It should be understood that "substituted" or "substituted" implies the proviso that the substitution is made in accordance with the permissible valencies of the atom being substituted and the substituent and that the substitution results in a stable compound, e.g. Reactions such as rearrangements, cyclizations, eliminations, etc. transform spontaneously. As used herein, the term "substituted" is meant to include all permissible substituents of organic compounds. In a broad sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compound substituents. The permissible substituents may be one or more.

It should be understood that "substituted" or "substituted" implies the proviso that the substitution is made in accordance with the permissible valencies of the atom being substituted and the substituent and that the substitution results in a stable compound, e.g. Reactions such as rearrangements, cyclizations, eliminations, etc. transform spontaneously. As used herein, the term "substituted" is meant to include all permissible substituents of organic compounds. In a broad sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compound substituents. The permissible substituents may be one or more and may be the same or different for appropriate organic compounds.

In some embodiments, a "substituent" may be selected from, for example, halogen, trifluoromethyl, nitro, cyano, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl , C ₁ -C ₆ alkylcarbonyloxy, arylcarbonyloxy, C ₁ -C ₆ alkoxycarbonyloxy, aryloxycarbonyloxy, C ₁ -C ₆ alkylcarbonyl, C ₁ -C ₆ alkoxycarbonyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylthio, arylthio, heterocyclyl, aralkyl and aryl (including heteroaryl).

Further examples of compounds useful as compounds of the present invention are included in the publication entitled "METHODS and COMPOSITIONS FOR TREATING AMYLOID-RELA TED DISEASES" (Attorney Docket No. NBI-162- 1, submitted June 23, 2003) and "METHODSAND COMPOSITIONS FOR THE TREA TMENT OFAMYLOID-ANDEPILEPTOGENESIS-ASSOCIATED DISEASES" (Attorney Docket No. NBI-163-1 , filed June 23, 2003) in a US provisional patent application.

In one embodiment, the present invention relates at least in part to a composition comprising a compound of formula I-A or a pharmaceutically acceptable salt thereof:

in:

R ¹ is substituted or unsubstituted cycloalkyl, aryl, arylcycloalkyl, bicyclic or tricyclic, bicyclic or tricyclic condensed ring group, or substituted or unsubstituted C ₂ -C ₁₀ alkyl;

^R is selected from hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl and benzimidazolyl ;

Y is SO ₃ ^- X ⁺ , OSO ₃ ^- X ⁺ or SSO ₃ ^- X ⁺ ;

X ⁺ is hydrogen, a cationic group, or an ester-forming group (i.e., as in a prodrug, described elsewhere herein);

^L and ^L are each independently substituted or unsubstituted C ₁ -C ₅ alkyl or absent,

with the proviso that when ^R1 is alkyl, ^L1 is absent.

In another embodiment, the present invention relates at least in part to a composition comprising a compound represented by formula II-A or a pharmaceutically acceptable salt thereof:

in:

R ¹ is a substituted or unsubstituted cyclic, bicyclic, tricyclic or benzoheterocyclic group or a substituted or unsubstituted C ₂ -C ₁₀ alkyl group;

^R is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, benzimidazolyl, Or link with ^R1 to form a heterocycle;

Y is SO ₃ ^- X ⁺ , OSO ₃ ^- X ⁺ or SSO ₃ ^- X ⁺ ;

X ⁺ is hydrogen, a cationic group or an ester-forming moiety;

m is 0 or 1;

n is 1, 2, 3 or 4;

L is substituted or unsubstituted C ₁ -C ₃ alkyl or absent,

with the proviso that when ^R1 is alkyl, L is absent.

In yet another embodiment, the present invention relates at least in part to a composition comprising a compound represented by formula III-A or a pharmaceutically acceptable salt and ester thereof:

in:

A is nitrogen or oxygen;

R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together may be a natural or unnatural amino acid residue or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl;

x is 0, 1, 2, 3 or 4;

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

R ³ , R ^3a , R ⁴ , R ^4a , R ⁵ , R ^5a , R ⁶ , R ^6a , R ⁷ and R ^7a are each independently hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl , aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino, tetrazolyl, or two R groups on adjacent ring atoms form a double bond with the ring atom, provided that One of R ³ , R ^3a , R ⁴ , R ^4a , R ⁵ , R ^5a , R ⁶ , R ^6a , R ⁷ and R ^7a is a moiety shown in formula IIIa-A:

in:

m is 0, 1, 2, 3 or 4;

R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are independently selected from hydrogen, halogen, hydroxyl, alkyl, alkoxy, haloalkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, Cyano, thiazolyl, triazolyl, imidazolyl, tetrazolyl, benzothiazolyl and benzimidazolyl;

Provided that the compound is not 3-(4-phenyl-1,2,3,6-tetrahydro-1-pyridyl)-1-propanesulfonic acid.

In yet another embodiment, the present invention relates at least in part to compositions comprising compounds of formula IV-A and pharmaceutically acceptable salts and esters thereof:

in:

A is nitrogen or oxygen;

R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together may be a natural or unnatural amino acid residue or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl;

x is 0, 1, 2, 3 or 4;

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

R ⁴ , R ^4a , R ⁵ , R ^5a , R ⁶ , R ^6a , R ⁷ and R ^7a are each independently hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkyl Carbonyl, arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino, tetrazolyl, ^R4 and ^R5 form a double bond together with the ring atom to which they are attached, or ^R6 and ^R7 form a double bond with the ring atom to which they are attached together form a double bond;

m is 0, 1, 2, 3 or 4;

R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are independently selected from hydrogen, halogen, hydroxyl, alkyl, alkoxy, haloalkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, Cyano, thiazolyl, triazolyl, imidazolyl, tetrazolyl, benzothiazolyl and benzimidazolyl.

In another embodiment, the present invention includes compositions containing compounds shown in formula V-A and pharmaceutically acceptable salts and prodrugs thereof:

in:

A is nitrogen or oxygen;

R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together may be a natural or unnatural amino acid residue or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl;

x is 0, 1, 2, 3 or 4;

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

aa is a natural or unnatural amino acid residue;

m is 0, 1, 2 or 3;

R ¹⁴ is hydrogen or a protecting group;

R ¹⁵ is hydrogen, alkyl or aryl.

In another embodiment, the present invention includes a composition comprising a compound represented by formula VI-A or a pharmaceutically acceptable salt thereof:

in:

n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

A is oxygen or nitrogen;

R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together may be a natural or unnatural amino acid residue or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl;

x is 0, 1, 2, 3 or 4;

R ¹⁹ is hydrogen, alkyl or aryl;

^Y1 is oxygen, sulfur or nitrogen;

^Y2 is carbon, nitrogen or oxygen;

^R20 is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl or benzimidazolyl;

^R is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, benzene And imidazolyl, or if Y ² is oxygen, then R ²¹ is absent;

^R is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, benzene and imidazolyl; or if Y ¹ is nitrogen, then R ²² is hydrogen, hydroxyl, alkoxy or aryloxy; or if Y ¹ is oxygen or sulfur, then R ²² does not exist; or if Y ¹ is nitrogen, then R ²² and R ²¹ can be linked together to form a ring moiety.

In another embodiment, the present invention includes compositions containing compounds shown in formula VII-A and pharmaceutically acceptable salts thereof:

in:

n is 2, 3 or 4;

A is oxygen or nitrogen;

R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together may be a natural or unnatural amino acid residue or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl;

x is 0, 1, 2, 3 or 4;

G is a direct bond or is oxygen, nitrogen or sulfur;

z is 0, 1, 2, 3, 4 or 5;

m is 0 or 1;

^R is selected from hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, aroyl, alkylcarbonyl, aminoalkylcarbonyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl and benzimidazolyl;

Each ^R is independently selected from hydrogen, halogen, cyano, hydroxy, alkoxy, mercapto, amino, nitro, alkyl, aryl, carbocyclyl, or heterocyclyl.

Additional compounds include, for example, compounds of formula I-B and pharmaceutically acceptable salts, esters and prodrugs thereof:

in:

X is oxygen or nitrogen;

Z is C=O, S(O) ₂ or P(O)OR ⁷ ;

m and n are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

R and ^R are each independently hydrogen, metal ion, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, together with ^X form part of a natural or unnatural amino acid residue, or -(CH ₂ ) _p -Y;

Y is hydrogen or a heterocyclic moiety selected from thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl and benzimidazolyl;

p is 0, 1, 2, 3 or 4;

^R is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, or alkoxycarbonyl;

^R is hydrogen, amino, cyano, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, substituted or unsubstituted aryl, heteroaryl, thiazolyl, triazolyl , tetrazolyl, imidazolyl, benzothiazolyl or benzimidazolyl;

In yet another embodiment, m is 0, 1 or 2. In yet another embodiment, n is 0, 1 or 2. In yet another embodiment, ^R3 is aryl, such as heteroaryl or phenyl. In yet another embodiment, Z is S(O) ₂ .

In another embodiment, the compound of the present invention is a compound shown in formula II-B and pharmaceutically acceptable salts, esters and prodrugs thereof:

in:

Each R ^is independently selected from hydrogen, halogen, hydroxyl, mercapto, amino, cyano, nitro, alkyl, aryl, carbocyclyl, or heterocyclyl;

J is absent, oxygen, nitrogen, sulfur, or a divalent bonding moiety including, but not limited to, lower alkylene, alkyleneoxy, alkyleneamino, alkylenethio, alkyleneoxyalkyl , alkyleneaminoalkyl, alkylenethioalkyl, alkenyl, alkenyloxy, alkenylamino or alkenylthio;

q is 1, 2, 3, 4 or 5.

In still another embodiment, the compound of the present invention is a compound shown in formula III-B and pharmaceutically acceptable salts, esters and prodrugs thereof:

in:

X is oxygen or nitrogen;

m and n are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

q is 1, 2, 3, 4 or 5;

R ¹ is hydrogen, metal ion, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl or together with X forms part of a natural or unnatural amino acid residue or -(CH ₂ ) _p -Y ;

Y is hydrogen or a heterocyclic moiety selected from thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl and benzimidazolyl;

p is 0, 1, 2, 3 or 4;

^R is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, or alkoxycarbonyl;

^R is selected from hydrogen, halogen, amino, nitro, hydroxyl, carbonyl, mercapto, carboxyl, alkyl, alkoxy, alkoxycarbonyl, acyl, alkylamino, acylamino;

q is an integer selected from 1-5;

J is absent, oxygen, nitrogen, sulfur, or a divalent bonding moiety including, but not limited to, lower alkylene, alkyleneoxy, alkyleneamino, alkylenethio, alkyleneoxyalkyl , alkyleneaminoalkyl, alkylenethioalkyl, alkenyl, alkenyloxy, alkenylamino or alkenylthio.

In yet another embodiment, the compound of the invention is;

In yet another embodiment, m is zero.

In another embodiment, the present invention relates to the compound shown in formula V-B:

in:

^R6 is a substituted or unsubstituted heterocyclic moiety.

In yet another embodiment, m is 0 or 1 . In another embodiment, n is 0 or 1. In yet another embodiment, R is ^thiazolyl , oxazolyl, pyrazolyl, indolyl, pyridyl, thiazinyl, thienyl, benzothienyl, dihydroimidazolyl, dihydrothiazolyl , oxazolidinyl, thiazolidinyl, tetrahydropyrimidinyl, or oxazinyl. In yet another embodiment, Z is S(O) ₂ .

The present invention also relates to compounds shown in formula I-C (seeing WO 00/64,420):

Wherein R ¹ and R ² are each independently a hydrogen atom or a substituted or unsubstituted aliphatic group or an aryl group; Z and Q are each independently a carbonyl group (C=O), a thiocarbonyl group (C=S), a sulfonyl group ( _SO2 ) or sulfoxide (S=O) groups; k and m are 0 or 1, with the proviso that when k is 1, ^R1 is not a hydrogen atom, and when m is 1, ^R2 is not a hydrogen atom. In one embodiment, at least one of k or m must be equal to one. The variables p and s are each independently positive integers selected such that the biodistribution of the therapeutic compound to the intended target site is unhindered while maintaining the activity of the therapeutic compound. T is a linking group (such as an alkylene group), Y is a group represented by the formula SO ₃ ^- X ⁺ , OSO ₃ ^- X ⁺ or SSO ₃ ^- X ⁺ ; wherein X ⁺ is a cationic group; and the drug of the compound Salts and prodrugs are acceptable.

In another embodiment of the compound shown in formula IC, R ¹ is an alkyl group, an alkenyl group or a monocyclic aromatic group, wherein the alkyl group may be substituted by a hydroxyl group; R ² is an alkyl group, an alkenyl group, a hydroxyalkane group A group, a monocyclic aryl group or a hydrogen atom, or R ¹ and R ² form a heterocyclic group with a condensed ring structure together with their connected nitrogen; k and m are 0, p and s are 1; T is an alkylene group; Y is _SO3X , X is a cationic group; and pharmaceutically acceptable salts and prodrugs of the compound.

In yet another embodiment of the compound shown in formula IC, R ¹ is an alkyl group, an alkenyl group or an aryl group; R ² is a hydrogen atom, an alkyl group or an aryl group, or R ¹ and R ² form a condensed ring structure together Heterocyclyl; Z and Q are each independently a carbonyl (C=O), thiocarbonyl (C=S), sulfonyl (SO ₂ ) or sulfoxide (S=O) group; k is 1, m is 0 or 1, provided that when k is 1, ^R1 is not a hydrogen atom, and when m is 1, ^R2 is not a hydrogen atom; p and s are each 1; T is an alkylene group; Y is _SO3X , and X is a cationic group; and a pharmaceutically acceptable salt of the compound.

On the other hand, R ¹ and R ² of the compound shown in formula IC can be alkyl, alkenyl or monocyclic aryl, or R ¹ and R ² can be a heterocyclic group of condensed ring structure together with the nitrogen they are connected to ; k and m are 0, p and s are 1; T is an alkylene group; Y is SO ₃ X, X is a cationic group; and pharmaceutically acceptable salts and prodrugs of the compound.

In another embodiment of the compound shown in formula IC, R ¹ is alkyl, alkenyl or monocyclic aryl, wherein said alkyl can be substituted by hydroxyl; R ² is alkyl, alkenyl, monocyclic aryl or a hydrogen atom, wherein the alkyl group may be substituted by a hydroxyl group; or R ¹ and R ² together with the nitrogen to which they are attached form a heterocyclic group of a condensed ring structure; k and m are 0, p and s are 1; T is an alkylene group; Y is SO ₃ X, X is a cationic group; and pharmaceutically acceptable salts and prodrugs of the compound.

In yet another embodiment, the invention relates to compounds of formula I-D:

in:

R ¹ is substituted or unsubstituted cycloalkyl, heterocyclyl, aryl, arylcycloalkyl, bicyclic or tricyclic, bicyclic or tricyclic fused ring group or substituted or unsubstituted C ₂ -C ₁₀ alkyl;

^R is selected from hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl and benzimidazolyl ;

Y is SO ₃ ^- X ⁺ , OSO ₃ ^- X ⁺ or SSO ₃ ^- X ⁺ ;

X ⁺ is hydrogen, a cationic group or an ester-forming group;

^L1 and ^L2 are each substituted or unsubstituted _C1 - _C5 alkyl or absent, or a pharmaceutically acceptable salt of the compound, provided that when ^R1 is alkyl, ^L1 is absent.

In yet another embodiment, the invention relates to compounds of formula II-D:

in:

R ¹ is a substituted or unsubstituted cyclic, bicyclic, tricyclic or benzoheterocyclic group or a substituted or unsubstituted C ₂ -C ₁₀ alkyl;

^R is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, benzimidazolyl, or Linked with ^R1 to form a heterocycle;

Y is SO ₃ ^- X ⁺ , OSO ₃ ^- X ⁺ or SSO ₃ ^- X ⁺ ;

X ⁺ is hydrogen, a cationic group or an ester-forming moiety;

m is 0 or 1;

n is 1, 2, 3 or 4;

L is substituted or unsubstituted C ₁ -C ₃ alkyl or absent, or a pharmaceutically acceptable salt of the compound, with the proviso that when R ¹ is alkyl, L is absent.

In yet another embodiment, ^R2 is hydrogen. In yet another embodiment, R ¹ is straight chain alkyl, such as ethyl, n-pentyl, n-heptyl or n-octyl. In another embodiment, R ¹ is tert-butyl. In yet another optional embodiment, R ¹ is C ₇ -C ₁₀ bicycloalkyl or tricycloalkyl, such as tricyclo[3.3.1.0 ^3,7 ]decyl (or adamantyl), bicyclo [2.1.2] Heptyl or indolyl. In another embodiment, R ¹ is tetrahydronaphthyl.

In one embodiment, ^L2 is -( _CH2 ) _3- . In yet another embodiment, L ² is -(CH ₂ ) ₄ - or -(CH ₂ ) ₅ -. In yet another embodiment, ^L2 is -( _CH2 ) _2- . In yet another embodiment, ^L2 is substituted alkyl, eg _-CH2- (CHOH) _-CH2- .

In _another embodiment, ^L1 is _CH2CH2 or is absent.

In yet another embodiment, R ¹ is branched alkyl, such as tert-butyl. In another embodiment, R ¹ is adamantyl. In another embodiment, ^R is cyclic alkyl, such as cyclopropyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. Cycloalkyl moieties may be further substituted, for example, with additional alkyl or other groups that enable the molecule to perform its intended function. In another embodiment, ^R1 is alkyl substituted with a propargyl moiety (eg, HC≡C-). In another embodiment, R ¹ is cyclohexyl substituted with one or more methyl or propargyl groups.

In other embodiments, L ¹ is a C ₁ -C ₂ alkyl linking group (eg -CH(CH ₃ )- or -(CH ₂ ) ₂ -). In yet another embodiment, ^R1 is phenyl. In certain embodiments, R ¹ is substituted with methoxy. In other embodiments, L ¹ is C ₃ , eg -(CH ₂ ) ₃ - or C(CH ₃ ) ₂ -. In certain embodiments, ^L is substituted, for example, with alkoxy, carboxy (-COOH), benzyl, amido (-C=O-NH-), or ester (C=OCO). In certain embodiments, the ester group is methyl, ethyl, propyl, butyl, cyclohexyl, or benzyl. In other embodiments, the ester group may be acrylate. In other embodiments, ^L is substituted with carboxy. In yet another embodiment, ^R is substituted with a substituted amido, wherein said amido is substituted with an alkyl, eg methyl, ethyl, propyl, butyl, pentyl or hexyl. In another embodiment, alkyl ^R1 is substituted with -C=O-NH-OH, C=O- _NH2 or amido. In certain embodiments, the amido group is substituted with an alkyl group such as methyl, ethyl, propyl, butyl, pentyl, hexyl, cyclohexyl, benzyl, or aryl. In another embodiment, the amido group is substituted with a -CH( _CH2 ) ₂ group. R ¹ itself may be substituted by phenyl, or may be a branched or straight chain alkyl. In certain embodiments, R ¹ can also be substituted with a thioether moiety. Examples of thioethers include S-Me, S-Et, and the like. In certain embodiments, the alkyl R ^moiety is substituted with both an aryl or thioether moiety and an amido moiety. In other embodiments, the alkyl R ^moieties can be substituted with both thioether and carboxyl moieties. In other embodiments, the alkyl ^R1 group is substituted with hydroxy. R ¹ groups such as alkyl R ¹ groups may also be substituted by both thioether and hydroxyl. In other embodiments, R ¹ groups, such as alkyl R ¹ groups, are substituted with cyano. Examples of ^R1 groups containing a -CN moiety include -C( _CH3 ) _2CN , cyclohexyl substituted with one or more cyano groups, and the like.

In other embodiments, the alkyl ^R1 group is substituted with aryl. Aryl groups may for example be substituted phenyl groups. Substituted phenyl groups may be substituted with one or more substituents such as hydroxy, cyano and alkoxy. In other embodiments, the alkyl ^R1 group is substituted with tetrazolyl or substituted or unsubstituted benzyl.

In yet another embodiment, L ¹ is -C(CH ₃ ) ₂ -(CH ₂ )-. In another embodiment, L ¹ is -(C(CH ₃ ) ₂ -CHOH-. In yet another embodiment, L ¹ is -(C(CH ₃ ) ₂ CH(OMe)-. In another embodiment In the scheme, ^R is a substituted or unsubstituted phenyl. In yet another embodiment, ^R is a para-substituted phenyl. Examples of substituents include, but are not limited to, fluorine, chlorine, bromine, iodine, methyl , tert-butyl, alkoxy, methoxy, etc. In other embodiments, ^R is substituted in the meta position. Examples of substituents include methoxy, chloro, methyl, tert-butyl, fluoro, alkyl , alkoxy, iodo, trifluoroalkyl, methoxy, etc. In another embodiment, ^L is phenyl substituted in the ortho position with a similar substituent. In another embodiment, ^L comprises the ring An alkyl moiety, such as cyclopentyl. In another embodiment, ^L comprises an alkylene group and an optionally substituted aryl group, the substituents being similar to those described above.

In certain embodiments, R ¹ is cyclopropyl or cyclohexyl. In certain embodiments, cyclopropyl or cyclohexyl is substituted with ether or alkyl. In certain other embodiments, the ether group is a benzyl ether group.

In another embodiment, wherein R ¹ is alkyl, it is substituted with a group such as phenyl or hydroxy.

In another embodiment, the present invention relates to compounds represented by formula III-D and pharmaceutically acceptable salts and esters thereof:

in:

A is nitrogen or oxygen;

R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together may be a natural or unnatural amino acid residue or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl;

x is 0, 1, 2, 3 or 4;

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

R ³ , R ^3a , R ⁴ , R ^4a , R ⁵ , R ^5a , R ⁶ , R ^6a , R ⁷ and R ^7a are each independently hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl , aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano, halo, amino, tetrazolyl, or two R groups on adjacent ring atoms form a double bond with the ring atom, provided that R ^3. One of R ^3a , R ⁴ , R ^4a , R ⁵ , R ^5a , R ⁶ , R ^6a , R ⁷ and R ^7a is the moiety shown in formula IIIa-D:

in:

m is 0, 1, 2, 3 or 4;

^RA , ^RB , ^RC , ^RD , and ^RE are independently selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkoxy, haloalkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, Cyano, thiazolyl, triazolyl, imidazolyl, tetrazolyl, benzothiazolyl and benzimidazolyl; provided that the compound is not 3-(4-phenyl-1,2,3,6-tetrahydro -1-pyridyl)-1-propanesulfonic acid. In yet another embodiment, n is 2, 3 or 4.

In another embodiment, R ¹¹ is a salt-forming cation. Examples of salt-forming cations include the pharmaceutically acceptable salts described herein as well as lithium, sodium, potassium, magnesium, calcium, barium, zinc, iron and ammonium. In another embodiment, R ¹¹ is an ester-forming group. Ester-forming groups include groups that, when combined, form esters. Examples of such groups include substituted or unsubstituted alkyl, aryl, alkenyl, alkynyl or cycloalkyl groups. In another embodiment, A is oxygen.

In another embodiment, ^R3 and ^R4 together with the carbon atom to which they are attached form a double bond. In another embodiment, ^RA , ^RB , ^RC , ^RD , and ^RE are each hydrogen. R ^A , R ^B , R ^D and ^RE are each hydrogen and R ^C is halogen such as fluorine, chlorine, iodine or bromine.

In another embodiment, R ³ or R ^5a is a moiety of formula IIIa-D.

In another embodiment, each of ^R4 , ^R5 , ^R6 and ^R7 is hydrogen. In yet another embodiment, each of R ^4a , R ^5a , R ^6a and R ^7a is hydrogen.

In another embodiment, R ^3a is hydroxy, cyano, acyl or hydroxy.

In yet another embodiment, R ¹¹ and A together are a natural or unnatural amino acid residue or a pharmaceutically acceptable salt or ester thereof. Examples of amino acid residues include esters and salts of phenylalanine and leucine.

In another embodiment, m is 0, 1 or 3.

In another embodiment, the present invention relates to compounds represented by formula IV-D and pharmaceutically acceptable salts and esters thereof:

in:

A is nitrogen or oxygen;

R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together may be a natural or unnatural amino acid residue or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl;

x is 0, 1, 2, 3 or 4;

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

R ⁴ , R ^4a , R ⁵ , R ^5a , R ⁶ , R ^6a , R ⁷ and R ^7a are each independently hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkyl Carbonyl, arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino, tetrazolyl, ^R4 and ^R5 form a double bond together with the ring atom to which they are attached, or ^R6 and ^R7 form a double bond with the ring atom to which they are attached together form a double bond;

m is 0, 1, 2, 3 or 4;

R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are independently selected from hydrogen, halogen, hydroxyl, alkyl, alkoxy, haloalkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, Cyano, thiazolyl, triazolyl, imidazolyl, tetrazolyl, benzothiazolyl and benzimidazolyl.

In another embodiment, R ¹¹ is a salt-forming cation. Examples of salt-forming cations include the pharmaceutically acceptable salts described herein as well as lithium, sodium, potassium, magnesium, calcium, barium, zinc, iron and ammonium. In another embodiment, R ¹¹ is an ester-forming group. Ester-forming groups include groups that, when combined, form esters. Examples of such groups include substituted or unsubstituted alkyl, aryl, alkenyl, alkynyl or cycloalkyl groups. In another embodiment, A is oxygen.

In another embodiment, m is 0 or 1. In yet another embodiment, n is 2, 3 or 4. In yet another embodiment, each of ^R4 , ^R5 , ^R6 and ^R7 is hydrogen. R ^4a , R ^5a , R ^6a and R ^7a may also be hydrogen. Examples of R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ and R ¹² include hydrogen. In other embodiments, each of R ⁸ , R ⁹ , R ¹¹ and R ¹² is hydrogen and R ¹⁰ is halogen (eg, fluorine, chlorine, bromine, or iodine), nitro, or alkyl (eg, methyl, ethyl, butyl, base).

In another embodiment, AR ¹¹ may be an amino acid residue, such as the residue of phenylalanine. In another embodiment, R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are each hydrogen and R ⁸ is other than hydrogen, for example halo, eg fluorine, chlorine, bromine or iodine.

In another embodiment, the present invention relates to compounds shown in formula V-D and pharmaceutically acceptable salts and prodrugs thereof:

in:

A is nitrogen or oxygen;

R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together may be a natural or unnatural amino acid residue or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl;

x is 0, 1, 2, 3 or 4;

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

aa is a natural or unnatural amino acid residue;

m is 0, 1, 2 or 3;

R ¹⁴ is hydrogen or a protecting group;

R ¹⁵ is hydrogen, alkyl or aryl.

In another embodiment, R ¹¹ is a salt-forming cation. Examples of salt-forming cations include the pharmaceutically acceptable salts described herein as well as lithium, sodium, potassium, magnesium, calcium, barium, zinc, iron and ammonium. In another embodiment, R ¹¹ is an ester-forming group. Ester-forming groups include groups that, when combined, form esters. Examples of such groups include substituted or unsubstituted alkyl, aryl, alkenyl, alkynyl or cycloalkyl groups. In another embodiment, A is oxygen.

In one embodiment, n is 2, 3 or 4. In certain embodiments, m is 0. In certain embodiments, AR ¹¹ is a natural amino acid residue or a salt or ester thereof. Examples of amino acid residues include, but are not limited to, leucine or phenylalanine residues and pharmaceutically acceptable salts and esters thereof. Examples of possible esters include methyl, ethyl and t-butyl.

In another embodiment, m is 1. Examples of aa include natural and unnatural amino acid residues such as phenylalanine, glycine and leucine.

In another embodiment, (aa) _m is a phe-phe residue; and a pharmaceutically acceptable salt or suitable protecting group.

In certain embodiments, R ¹⁵ is hydrogen or substituted alkyl, such as arylalkyl.

The term "unnatural amino acid" refers to any derivative of a natural amino acid, including the D form and α- and β-amino acid derivatives. Note that certain amino acids classified herein as unnatural amino acids, such as hydroxyproline, may be found in nature in certain organisms or in certain proteins. Amino acids with many different protecting groups suitable for direct use in solid phase synthesis of peptides are commercially available. In addition to the twenty most common natural amino acids, the following examples of unnatural amino acids and amino acid derivatives (common abbreviations are in parentheses) can be used in accordance with the invention: β-alanine (β-ALA), γ-aminobutyric acid (GABA), 2-aminobutyric acid (2-Abu), α, β-dihydro-2-aminobutyric acid (8-AU), 1-aminocyclopropane-1-carboxylic acid (ACPC), aminoisobutyric acid (Aib), 2-amino-thiazoline-4-carboxylic acid, 5-aminopentanoic acid (5-Ava), 6-aminocaproic acid (6-Ahx), 8-aminooctanoic acid (8-Aoc), 11-amino Undecanoic acid (11-Aun), 12-aminododecanoic acid (12-Ado), 2-aminobenzoic acid (2-Abz), 3-aminobenzoic acid (3-Abz), 4-aminobenzoic acid (4-Abz), 4-amino-3-hydroxy-6-methylheptanoic acid (Statine, Sta), aminooxyacetic acid (Aoa), 2-amino-tetralin-2-carboxylic acid (ATC), 4 -Amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA), p-aminophenylalanine (4-NH ₂ -Phe), biphenylalanine (Bip), p-bromophenylalanine (4-Br-Phe), o-chlorophenylalanine] (2-Cl-Phe), m-chlorophenylalanine (3-Cl-Phe), p-chlorophenylalanine (4-Cl -Phe), m-chlorotyrosine (3-Cl-Tyr), p-benzoylphenylalanine (Bpa), tert-butylglycine (TLG), cyclohexylalanine (Cha), cyclohexylglycine ( Chg), 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutyric acid (Dbu), 3,4-dichlorophenylalanine (3,4-Cl ₂ -Phe), 3 , 4-difluorophenylalanine (3,4-F ₂ -Phe), 3,5-diiodotyrosine (3,5-I ₂ -Tyr), o-fluorophenylalanine (2 -F-Phe), m-fluorophenylalanine (3-F-Phe), p-fluorophenylalanine (4-F-Phe), m-fluorotyrosine (3-F-Tyr), high Serine (Hse), Homophenylalanine (Hfe), Homotyrosine (Htyr), 5-Hydroxytryptophan (5-OH-Trp), Hydroxyproline (Hyp), p-Iodophenylalanine amino acid (4-I-Phe), 3-iodotyrosine (3-I-Tyr), indoline-2-carboxylic acid (Idc), isopiperidinecarboxylic acid (Inp), m-methyltyrosine (3-Me-Tyr), 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), p-nitrophenylalanine (4-NO ₂ -Phe) , 3-nitrotyrosine (3-NO ₂ -Tyr), norleucine (Nle), norvaline (Nva), ornithine (Orn), o-phosphotyrosine (H ₂ PO ₃ -Tyr), octahydroindole-2-carboxylic acid (Oic), penicillamine (Pen), pentafluorophenylalanine (F ₅ -Phe), phenylglycine (Phg), 2-piperidine acid ( Pip), propargylglycine (Pra), pyroglutamic acid (PGLU), sarcosine (Sar), tetrahydroisoquinoline-3-carboxylic acid (Tic) and thiazolidine-4-carboxylic acid (thioproline acid, Th). In addition, N-alkylated amino acids can also be used, as well as amino acids with amine-containing side chains (eg, Lys and Orn) where the amine has been acylated or alkylated.

In another embodiment, the present invention relates at least in part to a compound represented by formula VI-D or a pharmaceutically acceptable salt thereof:

in:

n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

A is oxygen or nitrogen;

R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together may be a natural or unnatural amino acid residue or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl;

x is 0, 1, 2, 3 or 4;

R ¹⁹ is hydrogen, alkyl or aryl;

^Y1 is oxygen, sulfur or nitrogen;

^Y2 is carbon, nitrogen or oxygen;

^R20 is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl or benzimidazolyl;

^R is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, benzene And imidazolyl, or if Y ² is oxygen, then R ²¹ is absent;

^R is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, benzene and imidazolyl; or if Y ¹ is nitrogen, R ²² is hydrogen, hydroxyl, alkoxy or aryloxy; or if Y ¹ is oxygen or sulfur, R ²² does not exist; or if Y ¹ is nitrogen, R ²² and R ²¹ can be linked together to form a ring moiety;

R ²³ is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl or benzimidazolyl, or if Y ² is nitrogen or oxygen, R ²³ is absent.

In another embodiment, R ¹¹ is a salt-forming cation. Examples of salt-forming cations include the pharmaceutically acceptable salts described herein as well as lithium, sodium, potassium, magnesium, calcium, barium, zinc, iron and ammonium. In yet another embodiment, the salt is the sodium salt. In yet another embodiment, A is oxygen.

In another embodiment, Y ¹ is oxygen or sulfur and R ²² is absent.

In another embodiment, Y ¹ is oxygen and R ²¹ is absent. Examples of ^R20 include benzyl, aryl (eg, phenyl), alkyl, cycloalkyl (eg, adamantyl), and the like. In other embodiments, Y ² is nitrogen and R ²¹ is hydrogen. In other embodiments, R ²¹ is benzyl. In yet another embodiment, R ²⁰ and R ²¹ are joined together to form a pyridine ring. In another embodiment, ^Y1 is sulfur.

In another embodiment, the present invention relates at least in part to compounds of formula VII-D and pharmaceutically acceptable salts thereof:

in:

n is 2, 3 or 4;

A is oxygen or nitrogen;

R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together may be a natural or unnatural amino acid residue or a salt or ester thereof;

Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl;

x is 0, 1, 2, 3 or 4;

G is a direct bond or is oxygen, nitrogen or sulfur;

z is 0, 1, 2, 3, 4 or 5;

m is 0 or 1;

^R is selected from hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, aroyl, alkylcarbonyl, aminoalkylcarbonyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl and benzimidazolyl;

Each ^R is independently selected from hydrogen, halogen, cyano, hydroxy, alkoxy, mercapto, amino, nitro, alkyl, aryl, carbocyclyl, or heterocyclyl.

In one embodiment, R ¹¹ is hydrogen. In another embodiment, A is oxygen. For example, n can be 3 and m can be 1. In other embodiments, ^R24 is hydrogen or benzyl.

In certain embodiments, z is 0, 2 or 3. In other embodiments, R ²⁵ is hydroxy or alkoxy, such as methoxy, ethoxy, and the like. In certain embodiments, two or more R ^substituents may be joined together to form a fused ring (eg, to form a methylenedioxyphenyl moiety).

The present invention relates to salt forms and acid/base forms of the compounds of the invention. For example, the invention is not only concerned with specific salt forms of the compounds referred to herein as salts, but also includes other pharmaceutically acceptable salts and acid and/or base forms of the compounds. The present invention also relates to the salt forms of the compounds shown herein.

Exemplary compounds of the invention are also shown in the figures herein. Exemplary compounds of any of the formulas listed herein (e.g., formulas I-D to VII-D) and specific groups and subsets thereof are part of the present invention, both titled "Methods and Compositions for Treating Amyloid" submitted on June 18, 2004 Related Diseases," U.S. Patent Application for "Methods and Compositions for the Treatment of Amyloid-Related Diseases," Attorney Docket Nos. NBI-162A and NBI-162B, and filed June 18, 2004, entitled "Methods and Compositions for the Treatment of Amyloid-and Epileptogenesis-Associated Diseases," "Methods and Compositions for Treating Amyloid and Epileptogenesis-Associated Diseases," Attorney Docket No. NBI-163, expressly incorporated by reference into In this article.

In one embodiment, the present invention does not relate to compounds described in WO 00/64420, WO 97/023458 and WO 96/28187. In one embodiment, the present invention does not relate to methods of using the compounds described in WO 00/64420, WO 97/023458 and WO 96/28187 to treat the diseases or conditions described in these three patents. In yet another embodiment, the present invention relates to the use of compounds described in WO 00/64420, WO 97/023458 and WO 96/28187 in the methods described in this application, the methods described in WO 00 /64420, WO 97/023458 and WO 96/28187 are not described. Furthermore, WO 00/64420, WO 97/023458 and WO 96/28187 are hereby incorporated by reference in their entirety.

In general, the compounds of the present invention can be prepared by the methods illustrated in the general reaction schemes, eg, in the reaction schemes described below, or by modifications of said methods, using readily available starting materials, reagents and conventional synthetic procedures. In these reactions it is also possible to use variations which are known per se but not mentioned here. Functional and structural equivalents of the compounds described herein having the same general properties, wherein one or more simple changes in the substituents do not adversely affect the essential properties or utility of the compound. Compounds of the invention can be readily prepared following the synthetic schemes and schemes described herein, which are illustrated in the specific procedures provided herein. However, those skilled in the art will recognize that other synthetic routes can be used to produce the compounds of the present invention, and the following is provided by way of example only and not as a limitation of the present invention. See, eg, "Comprehensive Organic Transformations" by R. Larock, VCH Publishers (1989). It will further be appreciated that various protection and deprotection strategies standard in the art can be employed (see, eg, "Protective Groups in Organic Synthesis" by Greene and Wuts). Those skilled in the relevant art will recognize that the choice of any particular protecting group (eg, amine and carboxyl protecting groups) will depend on the stability of the protected moiety under subsequent reaction conditions, and they will understand the consequences of appropriate selection. The following examples from the extensive chemical literature further illustrate the knowledge of those skilled in the art: "Chemistry of the amino Acids" by J.P. Greenstein and M. Winitz, John Wiley & Sons, Inc., New York (1961); by R. Larock "Comprehensive Organic Transformations", VCH Publishers (1989); T.D.Ocain et al., J.Med.Chem.31, 2193-99 (1988); E.M.Gordon et al., J.Med.Chem.31, 2199-10 (1988); "Practice of Peptide Synthesis" by M. Bodansky and A. Bodanszky, Springer-Verlag, New York (1984); "Protective Groups in Organic Synthesis" by T. Greene and P. Wuts (1991); by G.M. Coppola and H.F. Schuster "AsymmetricSynthesis: Construction of Chiral Molecules Using amino Acids", John Wiley & Sons, Inc., New York (1987); "The Chemical Synthesis of Peptides" by J. Jones, Oxford University Press, New York (1991); and P.D. "Introduction of Peptide Chemistry" by Bailey, John Wiley & Sons, Inc., New York (1992).

Chemical structures herein are drawn according to conventional standards well known in the art. Thus, when an atom, such as a carbon atom, is drawn as if to have an insufficient valence, it is assumed that the valence is satisfied by a hydrogen atom, although the hydrogen atom does not have to be explicitly drawn. The structures of some of the compounds of the invention contain chiral carbon atoms. It is to be understood that isomers arising from such asymmetries (eg, all enantiomers and diastereomers) are included within the scope of the invention unless otherwise indicated. That is, unless otherwise specified, any chiral carbon center can adopt either the (R)- or (S)-stereochemical configuration. Said isomers can be obtained in substantially pure form by classical separation techniques as well as by stereochemically controlled syntheses. Furthermore, alkenes may include E- or Z-geometry, where appropriate. Additionally, the compounds of the present invention can exist in unsolvated forms as well as solvated forms with acceptable solvents such as water, THF, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.

It is to be understood that the use of any of the compounds described herein or in the applications described in the "Related Applications" section falls within the scope of the present invention and is meant to be covered by the present invention, at least for those purposes expressly incorporated herein , and is expressly incorporated herein for all other purposes.

In one embodiment, the protein aggregation disease may be Pick's disease, corticobasal degeneration, progressive supranuclear palsy, amyotrophic lateral sclerosis/parkinsonism-dementia syndrome, Parkinson's disease (PD) , Huntington's disease (HD), atrophic myotonia, dentate rubrum pallidal Lewy body atrophy, Friedrich's ataxia, fragile X syndrome, fragile XE brain retardation, spinobulbar musculoskeletal Atrophy, Wilson's disease, and spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), Ma-Joe disease (MJD or SCA3), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia type 17 (SCA17), chronic liver disease, cataract, serpinopathy, hemolytic anemia, cystic fibrosis, neurofibroma Type 2 demyelinating peripheral neuropathy, retinitis pigmentosa, Marfan syndrome, emphysema, idiopathic pulmonary fibrosis, argyrophilic granular dementia, corticobasal degeneration, diffuse neurofibrillary tangles with Calcinosis, frontotemporal dementia/parkinsonism linked to chromosome 17, Hastings disease, Nye-dermatosis type C, or subacute sclerosing panencephalitis.

In one embodiment, the protein aggregation disorder is an alpha-synucleinopathy. In a preferred embodiment, the protein aggregation disorder is Parkinson's disease, Heidl-Herder syndrome, neurogenic orthostatic hypotension, Heider-Merder syndrome or Parkinson-plus syndrome.

In another embodiment, the protein aggregation disease is a tauopathy, with the proviso that the tauopathy is not Alzheimer's disease, prion disease, or cerebral amyloid angiopathy.

In a preferred embodiment, the tauopathy is amyotrophic lateral sclerosis/parkinsonism-dementia syndrome, argyrophilic granular dementia, DLBD, corticobasal degeneration, diffuse neurofibrillary tangles with calcifications, Chromosome 17-associated frontotemporal dementia/parkinsonism, Hastings disease, multiple system atrophy, Nee-dermatosis type C, Pick's disease, progressive supranuclear palsy, or subacute sclerosing panencephalitis .

The present invention relates to methods of treating, preventing or modulating protein aggregation diseases or proteinopathies other than amyloidopathies. One aspect of the invention relates to methods of treating, preventing or modulating protein aggregation diseases or protein diseases due to familial mutations in gene sequences, such as the following non-limiting examples of diseases: familial Parkinson's disease (wherein such as α-synaptic nucleoprotein, parkin, and COOH-terminal hydrolase L1 may form deleterious protein aggregates); myotonic dystrophy (eg, in which deleterious protein aggregates form myotonic myotonia protein kinase (DMPK); dentate nucleus Rhodopallidal Lewy body atrophy (DRPLA) (eg, in which aggregates of deleterious proteins of the DRPLA gene occur); Friedrich's ataxia (eg, in which aggregates of deleterious proteins of the gene for Fretschia's ataxia protein (FRDA) form) ; mutations in the androgen receptor (AR) where, for example, deleterious protein aggregates form in spinal bulbar muscular atrophy (also known as Kennedy disease); spinocerebellar ataxias, caused by, for example, mutations in the SCA1 gene Huntington's disease (HD), caused by, for example, mutations in the huntingtin protein or the IT15 gene that lead to the formation of harmful protein aggregates; Huntington's disease-like diseases, caused by, for example, connexin-3 (JPH3/HDL2) or mutations in the TBP gene caused by mutations; familial encephalopathy with neurofilin inclusion bodies (FENIB), caused by, for example, mutations in the neurofilin gene that lead to the formation of unwanted protein aggregates; or amyotrophic lateral sclerosis (ALS), which, for example, leads to the formation of Mutations in the SOD1 gene of deleterious protein aggregates; wherein the disease is not an amyloid disease.

In one embodiment of the invention, accumulation or oligomerization of deleterious protein aggregates may be associated with idiopathic mutations in the gene sequence, or occur sporadically, as in the following non-limiting disease examples: Amyotrophic side Cordon sclerosis/parkinsonism-dementia syndrome in which tau protein aggregates, forming harmful protein aggregates. One aspect of the invention relates to methods of treating, preventing or modulating idiopathic protein aggregation diseases or proteinopathies, such as the following: Parkinson's disease (PD), diffuse Lewy body dementia (DLBD), multiple Systemic Atrophy (MSA), Atrophic Myotonia, Dentatorrububal-Pallewy Lewy Atrophy (DRPLA), Friedrich's Ataxia, Fragile X Syndrome, Fragile XE Brain Retardation, Horse-John Spinobulbar muscular atrophy (also known as Kennedy disease), spinocerebellar ataxia, Huntington's disease (HD), familial encephalopathy with neurofilament inclusions (FENIB), Pick's disease, corticobasal Nuclear degeneration (CBD), progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis/parkinsonism-dementia syndrome, amyotrophic lateral sclerosis (ALS), cataract, or Wilson's disease, where the The disease is not amyloidosis.

The present invention also relates to a method of modulating deleterious protein aggregates, provided that said protein aggregates are not associated with an amyloid disease as defined herein, said method comprising contacting the deleterious protein aggregates with an effective amount of a compound of the invention so that the deleterious Protein aggregates are regulated.

In one embodiment of the invention, deleterious protein aggregates are associated with misfolding of mature proteins.

In another embodiment, the unwanted protein aggregates are extracellular aggregates.

In yet another embodiment, the unwanted protein aggregates are intracellular aggregates.

In another embodiment, the unwanted protein aggregates are cytoplasmic aggregates.

In one embodiment, the deleterious protein aggregates are nuclear aggregates.

In another embodiment, the deleterious protein aggregates are intramembrane aggregates.

In another embodiment, unwanted protein aggregates are associated with aggregates.

In yet another embodiment, deleterious protein aggregates are associated with improper degradation of proteins.

In another embodiment, the unwanted protein aggregates are in the endoplasmic reticulum.

In one embodiment, the deleterious protein aggregates are in the trans-Golgi network.

In one embodiment, unwanted protein aggregates are associated with misfolded proteins that evade the ubiquitin-proteasome system.

In another embodiment, unwanted protein aggregates are modulated by increasing their degradation.

In another embodiment, unwanted protein aggregates are inhibited.

In yet another embodiment, unwanted protein aggregates are modulated by increasing the rate of clearance of said protein aggregates.

In another embodiment, deleterious protein aggregates are associated with incorrect post-transcriptional modifications, incorrect post-translational modifications, misfolding of mature proteins due to, for example, lack of appropriate chaperones, incorrect degradation of proteins, The protein escapes the ubiquitin-proteasome system (which results in the formation of inclusion bodies, aggregates, aggregates, precipitated proteins, insoluble aggregates) or any combination of the above.

In another embodiment, unwanted protein aggregates are associated with fibrils, β-sheets, or hydrophobic domains.

As used herein, "treating" a subject includes applying or administering a therapeutic compound to a subject, or applying or administering a therapeutic compound to cells or tissues of a subject suffering from a disease or disorder, having symptoms of a disease or disorder or at risk of (or predisposed to) a disease or condition for the purpose of treating, curing, alleviating, relieving, altering, correcting, ameliorating, ameliorating, or affecting a disease or condition, a symptom of a disease or condition, or a disease or risk (or susceptibility) for a disorder.

The term "subject" includes living organisms in which protein aggregation disorders can occur. Examples of subjects include humans, monkeys, cows, sheep, goats, dogs, cats, mice, rats, and transgenic species thereof. Compositions of the present invention may be administered to a subject to be treated using known procedures at dosages and intervals effective to modulate unwanted protein aggregation in the subject as further described herein. The "effective amount" of a therapeutic compound required to achieve a therapeutic effect can vary depending on factors such as the amount of protein already deposited at the clinical site in the subject, the age, sex and weight of the subject, and the modulation of unwanted protein aggregation in the subject by the therapeutic compound Ability. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of treatment.

In certain embodiments of the invention, a subject is in need of treatment by the methods of the invention, and the treatment is selected based on such need. Subjects in need of treatment are well known in the art, and include those who have been identified as suffering from, have symptoms of, or are at risk of suffering from, a disease or disorder associated with a protein aggregation disorder, and are based on a diagnosis ( Such as a medical diagnosis) is a subject expected to benefit from treatment (eg, treat, cure, prevent, alleviate, relieve, alter, correct, ameliorate, ameliorate, or affect a disease or disorder, a symptom of a disease or disorder, or a risk of a disease or disorder).

The term "modulate" is meant to encompass preventing or stopping the aggregation or accumulation of unwanted proteins, inhibiting or slowing down further aggregation of unwanted proteins in a subject who is developing a protein aggregation disorder (e.g., protein aggregates have developed), and reducing, reversing or promoting clearance Detrimental protein aggregates in a subject developing a protein aggregation disorder. Modulation of unwanted protein aggregation is determined by comparison to untreated subjects, or by comparison to treated subjects prior to treatment, or, for example, by a clinically measurable improvement, such as in patients with, for example, Parkinson's disease or synaptic In the case of patients with nucleopathies, the improvement is stabilization of cognitive function or prevention of further decline in cognitive function (ie preventing, slowing or stopping disease progression). The term "modulation" is meant to encompass both the inhibition (defined below) and enhancement of unwanted protein aggregation. Accordingly, the term "modulation" is meant to encompass 1) the prevention or cessation of aggregation or accumulation of unwanted proteins, the inhibition or slowing of further aggregation of unwanted proteins in a subject who is developing a protein aggregation disease (for example, protein aggregates have occurred), and developing Reduction or reversal of unwanted protein aggregation in a subject of a protein aggregation disease, and 2) enhancement of unwanted protein aggregation, such as increasing the rate or amount of unwanted protein aggregation in vivo or in vitro. Compounds that increase unwanted protein aggregation are useful in animal models of protein aggregation diseases, eg, can be used to enable unwanted protein aggregation to occur in an animal for a shorter period of time, or can be used to increase unwanted protein aggregation for a selected period of time. Compounds that enhance aggregation of unwanted proteins can be used in screening assays for compounds that inhibit aggregation of unwanted proteins in vivo, eg, in animal models of unwanted protein aggregation, cell assays, and in vitro assays. Such compounds can be used, for example, to provide faster or more sensitive screening assays for compounds. In some instances, compounds that promote unwanted protein aggregation may also be administered for therapeutic purposes, eg, to enhance deposition of unwanted protein aggregates. Modulation of unwanted protein aggregation is determined as compared to untreated subjects, or as compared to treated subjects prior to treatment.

"Inhibition" of unwanted protein aggregation includes prevention or cessation of aggregate formation, inhibition or slowing of further amyloid deposition in subjects with amyloidosis (e.g., protein aggregates have developed), and reduction or reversal of positively developing protein Protein aggregation disease or deposits in a subject of aggregation disease. Inhibition of unwanted protein aggregation is determined by comparison to untreated subjects, or by comparison to treated subjects prior to treatment, or, for example, by a clinically measurable improvement, such as in patients with, for example, Parkinson's disease or synaptic In the case of patients with nucleopathies, the improvement is stabilization of cognitive function or prevention of further decline in cognitive function (ie preventing, slowing or stopping disease progression).

The invention further provides a method of modulating cytotoxicity, preferably neurotoxicity, associated with a deleterious protein aggregate, said method comprising contacting the deleterious protein aggregate with an effective amount of a compound of the invention such that the cytotoxicity is modulated. In a preferred embodiment, the cytotoxicity is associated with inclusion bodies. In another preferred embodiment, cytotoxicity is modulated in neuronal cells or glial cells.

The present invention also provides a method for treating or preventing neurofibrillary tangles associated with tau protein in a subject (preferably a mammal, more preferably a human), the method comprising administering an effective amount of the compound of the present invention to the subject to induce neurofibrillary tangles associated with tau protein Fibrous tangles are treated or prevented. In another embodiment, the present invention is directed to a method of modulating tau-associated neurofibrillary tangles in a subject (preferably a mammal, more preferably a human), said method comprising administering to the subject an effective amount of a compound of the present invention that binds to tau Protein-associated neurofibrillary tangles are regulated.

The present invention provides a method for treating or preventing inclusion bodies containing α-synuclein NAC fragments in a subject (preferably a mammal, more preferably a human), the method comprising administering to the subject an effective amount of the compound of the present invention to make an inclusion body containing α-synuclein Inclusion bodies of the NAC fragment of synuclein are treated or prevented. In another embodiment, the present invention relates to a method for modulating inclusion bodies containing α-synuclein NAC fragment in a subject (preferably a mammal, more preferably a human), said method comprising administering to the subject an effective amount of a compound of the present invention such that Inclusion bodies containing the NAC fragment of α-synuclein are regulated.

In another embodiment, the deleterious protein aggregate is associated with one of the following proteins or fragments: alpha-synuclein, tau protein, NAC, huntingtin, DRPLA, meningiocytoma protein, cytokeratin, myelin 22. Rhodopsin, atrophin-1, fibrillin-1, ataxin-1, ataxin-2, ataxin-3, ataxin-6, ataxin- 17. Androgen receptor, surfactin-C or alpha 1 -antitrypsin.

The compounds of the invention may be administered therapeutically or prophylactically to treat diseases associated with the formation of unwanted protein aggregates, the development of unwanted protein aggregates into aggregates, or the deposition of unwanted protein aggregates into inclusion bodies such as Lewy bodies. The compounds of the invention may bind the aggregate-forming protein prior to its inclusion in the protein aggregate or when the protein becomes part of the aggregate, or may bind the aggregate itself. The compounds of the invention may also bind proteins in their native form, preventing their conformation from changing into a form that could form deleterious aggregates. The compounds of the present invention can use any of the following mechanisms (the mechanisms listed here are intended to be illustrative, not limiting) to act to improve the progress of protein aggregation diseases: slowing down the formation or deposition of harmful protein aggregates; The extent of deposition of unwanted protein aggregates; inhibition, reduction or prevention of formation of unwanted protein aggregate fibrils; inhibition of neurodegeneration or cytotoxicity caused by unwanted protein aggregates; inhibition of inflammation caused by unwanted protein aggregates; clearance of substances from the brain.

A compound of the invention may be used alone, or in combination with a second compound. The compound may be any compound or substance known in the art to be beneficial to the subject. The second compound can be any compound known in the art to treat, prevent or alleviate the symptoms of protein aggregation diseases such as Parkinson's disease. In addition, the second compound may be any compound that is beneficial to the subject when administered in combination with a compound of the present invention, eg, a neuroprotective compound. The phrase "administering" a second compound in combination includes the following meanings: co-administration of a compound of the invention with a second compound; administration of a compound of the invention first, followed by the second compound; and administration of the second compound first, followed by the compound of the invention .

After the compound of the present invention enters the brain (after penetrating the blood-brain barrier), or acts from the periphery, it can effectively control the deposition of harmful protein aggregates. When the compound acts peripherally, it can alter, for example, the balance of alpha-synuclein between the brain and plasma, thereby favoring the excretion of alpha-synuclein from the brain. Increased excretion of α-synuclein from the brain leads to a decrease in the concentration of α-synuclein in the brain, thus favoring less deposition of α-synuclein in aggregates or Lewy bodies. Alternatively, compounds that penetrate the brain could control deposition by acting directly on brain alpha-synuclein, for example by maintaining it in a non-fibrillar form or facilitating its clearance from the brain.

Subjects and Patient Populations

The term "subject" as used herein includes living organisms in which amyloidosis may occur or which are susceptible to protein aggregation diseases. Examples of subjects include humans, chickens, ducks, Peking ducks, geese, monkeys, cows, rabbits, sheep, goats, dogs, cats, mice, rats, and transgenic species thereof. Compositions of the present invention can be administered to a subject to be treated using known methods at dosages and intervals effective to modulate aggregation of unwanted proteins in the subject or the toxicity of the aggregates as further described herein. The effective amount of a therapeutic compound required to achieve a therapeutic effect may vary depending on the amount of deleterious protein aggregates already deposited at the clinical site in the subject, the subject's age, sex, and weight, and the degree to which the therapeutic compound modulates deleterious protein aggregates in the subject. ability. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of treatment.

In an exemplary aspect of the invention, the subject is a human. For example, the object is people over 30 years old, people over 40 years old, people over 50 years old, people over 60 years old, people over 70 years old, people over 80 years old, people over 85 years old, people over 90 years old people or people over the age of 95. The subject can be a woman, including a postmenopausal woman who may be receiving hormone (estrogen) replacement therapy. The subject can also be a man. In another embodiment, the subject is under 40 years old.

A subject may be a person at risk of developing a protein aggregation disorder, for example a person over the age of 40 or a person predisposed to developing a protein aggregation disorder. Predisposing factors for protein aggregation disorders identified or proposed in the scientific literature include: genotypes that predispose a subject to a protein aggregation disorder; environmental factors that predispose a subject to a protein aggregation disorder; predisposing a subject to a protein aggregation disorder A history of infection with viral or bacterial agents of the disease; and vascular factors that predispose the subject to protein aggregation diseases, etc. The subject may also have one or more risk factors for cardiovascular disease (coronary atherosclerosis, angina pectoris, and myocardial infarction) or cerebrovascular disease (intracranial or extracranial arteriosclerosis, stroke, syncope, and transient ischemic attack), Such as hypercholesterolemia, hypertension, diabetes, smoking, coronary artery disease, family history or past history of cerebrovascular disease and cardiovascular disease. Hypercholesterolemia is generally defined as a serum total cholesterol concentration greater than about 5.2 mmol/L (about 200 mg/dL).

The method of the present invention can be used in one or more of the following aspects: preventing protein aggregation diseases, treating protein aggregation diseases, improving symptoms of protein aggregation diseases or regulating the production of harmful protein aggregates. In one embodiment, the human has a family history of protein aggregation disease or dementia.

In another embodiment, the human is at least about 40 years old. In another embodiment, the human is at least about 60 years old. In another embodiment, the human is at least about 70 years old. In another embodiment, the human is at least about 80 years old. In another embodiment, the human is at least about 85 years old. In one embodiment, the human is between about 60 and about 100 years old.

In yet another embodiment, a subject is indicated to be at risk of a disease by diagnostic brain imaging techniques, such as techniques that measure brain activity, plaque deposition (eg, accumulation of harmful proteins), or brain atrophy.

In yet another embodiment, the subject is indicated to be at risk by a cognitive test, such as the Clinical Dementia Rating ("CDR") or the Mini-Mental Status Examination ("MMSE"). Subject scored below average on cognitive tests compared to previous controls of comparable age and educational background. Furthermore, the subject may exhibit a decrease in score compared to previous participation in the same or similar cognitive tests.

In determining CDR, subjects are typically assessed and rated against each of the following six cognitive and behavioral categories: memory, orientation, judgment and problem-solving, social interactions, family and hobbies, and personal care. The assessment may include past information provided by the subject or, preferably, a witness familiar with the subject. Evaluate and rate the object against each of the above to determine a general rating (0, 0.5, 1.0, 2.0, or 3.0). Grade 0 is considered normal. A grade of 1.0 may be considered equivalent to mild dementia. Subjects with a CDR of 0.5 are characterized by mild persistent amnesia, partial recall of past events, and "benign" amnesia. In one embodiment, the subject is assessed to have a CDR rating above 0, above about 0.5, above about 1.0, above about 1.5, above about 2.0, above about 2.5, or about Level 3.0.

Another test is the Mini-mental State Examination (MMSE) described by Folstein in "Mini-mental state. A practical method for grading the cognitive state of patients for the clinician." J. Psychiatr. Res. 12:189-198, 1975 . The MMSE assesses the presence of general mental decline. See also Folstein "Differential diagnosis of dementia. The clinical process." Psychiatr Clin North Am. 20:45-57, 1997. The MMSE is a means of assessing the onset of dementia and the presence of general mental decline. MMSE is scored on a scale of 0-30. The MMSE does not assess basic cognitive potential like, for example, so-called IQ tests. Instead, it tests intellectual skills. A person of "normal" intellectual ability scores "30" on an MMSE objective test (however, a person with an MMSE score of 30 can also score well below "normal" on an IQ test). See, eg, Kaufer, J. Neuropsychiatry Clin. Neurosci. 10:55-63, 1998; Becke, Alzheimer DisAssoc Disord. 12:54-57, 1998; Ellis, Arch. Neurol. 55:360-365, 1998; Magni, Int. . Psychogeriatr. 8: 127-134, 1996; Monsch, Acta Neurol. Scand. 92: 145-150, 1995. In one embodiment, the subject scores less than 30 on at least one of the MMSE tests. In another embodiment, the subject scores less than about 28, less than about 26, less than about 24, less than about 22, less than about 20, less than about 18, less than about 16 , less than about 14 points, less than about 12 points, less than about 10 points, less than about 8 points, less than about 6 points, less than about 4 points, less than about 2 points, or less than about 1 point.

In another embodiment, the subject does not exhibit symptoms of protein aggregation disease. In another embodiment, the subject is a human who is at least 40 years of age and does not exhibit symptoms of a protein aggregation disorder. In another embodiment, the subject is a human who is at least 40 years of age and exhibits one or more symptoms of a protein aggregation disorder.

The methods of the invention can be used as therapeutic methods in subjects with protein aggregation disorders, or as prophylactic methods in subjects predisposed to protein aggregation disorders or dementia (eg, subjects with genomic mutations).

It is to be understood that wherever any values and ranges are provided herein (eg, in terms of subject population age, dosage and blood levels), all values and ranges encompassed by such values and ranges are encompassed within the scope of the invention. Furthermore, all values between these values and ranges may also be upper or lower limits of the range.

pharmaceutical preparations

In another embodiment, the present invention is directed to pharmaceutical compositions comprising compounds of any of the formulas herein, and methods of preparing such pharmaceutical compositions for use in the treatment of protein aggregation disorders.

In general, compounds of the present invention can be synthesized by the methods illustrated in the general reaction schemes, such as those set forth in the patents and patent applications mentioned herein, or by modifications of said methods, using readily available starting materials, reagents, and prepared by conventional synthetic methods. In these reactions it is also possible to use variations which are known per se but not mentioned here. Also included are functional and structural equivalents of the compounds described herein having the same general properties, in which simple modification of one or more substituents does not adversely affect the essential properties or utility of the compounds.

The drug of the present invention may be provided as a solution in a suitable solvent or in a solvent-free form (eg lyophilized). In another aspect of the invention, the pharmaceuticals and buffers for carrying out the methods of the invention may be packaged as a kit, optionally including a container. Kits are available for commercial use according to the methods described herein and may include instructions for use in the methods of the invention. Kit supplementary components may include acids, bases, buffers, inorganic salts, solvents, antioxidants, preservatives or metal chelating agents. The kit supplement components are present as a pure composition, or in the form of an aqueous or organic solution into which one or more kit supplement components are incorporated. Any or all of the kit supplement components optionally further comprise a buffer.

The term "container" includes any receptacle for holding a therapeutic agent. For example, in one embodiment, the container is a package containing the formulation. In another embodiment, the container is not a packaging containing the formulation, that is, the container is a receptacle, such as a box or vial, containing a packaged formulation or an unpackaged formulation and instructions for use of the formulation. In addition, packaging techniques are well known in the art. It should be appreciated that the instructions for use of the therapeutic formulation may be on the packaging containing the therapeutic formulation such that the functional relationship of the instructions to the packaged product is enhanced.

Therapeutic compounds may also be administered by parenteral, intraperitoneal, intraspinal or intracerebral routes. Dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

To administer a therapeutic compound by a route other than parenteral administration, it may be necessary to coat or co-administer the compound with a substance to prevent inactivation of the compound. For example, therapeutic compounds can be administered to a subject in a suitable carrier such as liposomes or diluents. Pharmaceutically acceptable diluents include saline and aqueous buffered solutions. Liposomes include water-in-oil-in-water CGF emulsions and conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. The compositions must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size (in the case of dispersions) and by the use of surfactants. Prevention of the action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyalcohols, such as mannitol and sorbitol, can be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the compositions compounds which delay absorption, for example aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the therapeutic compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the therapeutic compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum-drying and freeze-drying of a previously sterile-filtered solution to yield the active ingredient (i.e., therapeutic compound) and any desired supplementary ingredients. powder.

Therapeutic compounds can be administered orally, eg, with an inert diluent or an absorbable edible carrier. Therapeutic compounds and other ingredients may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the patient's food. For oral therapy, the therapeutic compounds may be mixed with excipients and administered in the form of ingestible tablets, buccal tablets, tablets, capsules, elixirs, suspensions, syrups, wafers and the like. The percentage of therapeutic compound in the composition and formulation can of course vary. The therapeutic compound is present in such therapeutically useful compositions in such an amount that a suitable dosage will be obtained.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of a therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are limited by and directly dependent on (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the amyloid deposition in a subject skilled in the art using such a therapeutic compound inherent limitations.

Accordingly, the present invention includes pharmaceutical formulations comprising pharmaceutical agents described herein in a pharmaceutically acceptable carrier, including pharmaceutically acceptable salts thereof, for nebulized, oral and parenteral administration. Likewise, the invention includes medicaments and salts thereof which have been lyophilized and reconstituted into pharmaceutically acceptable formulations for administration, for example, by intravenous, intramuscular or subcutaneous injection. The administration method can also be intradermal administration or transdermal administration.

According to the present invention, the compounds of the various formulas herein and pharmaceutically acceptable salts thereof can be administered orally or inhaled in solid form, or administered intramuscularly or intravenously in the form of solution, suspension or emulsion. In addition, the drug or its salt can also be administered in the form of a liposomal suspension by inhalation, intravenously or intramuscularly.

Pharmaceutical formulations suitable for administration by inhalation as aerosols are also provided. Such formulations comprise the desired solution or suspension of a compound of any formula herein, or a salt thereof, or a plurality of solid particles of said compound or a salt thereof. The desired formulation can be filled into a small chamber for nebulization. Atomization can be achieved by compressed air or by ultrasonic energy to form a large number of liquid droplets or solid particles containing the drug or its salt. The particle size of the liquid droplets or solid particles should be in the range of about 0.5 to about 5 microns. Solid particles may be obtained by treating a solid compound of any formula described herein, or a salt thereof, by any suitable means known in the art, for example by micronization. Solid particles or droplets may be, for example, about 1 to about 2 microns in size. For this purpose, a commercial nebulizer can be used for this purpose.

Pharmaceutical formulations suitable for aerosol administration may be in liquid form, possibly comprising a water-soluble compound of any of the formulas described herein, or a salt thereof, in a carrier, including water. Surfactants can be present in the formulation, which, when the formulation is sprayed, lower the surface tension of the formulation sufficiently to result in the formation of droplets in the desired size range.

Oral compositions also include liquid solutions, emulsions, suspensions and the like. Pharmaceutically acceptable carriers suitable for use in the preparation of such compositions are well known in the art. Typical carrier ingredients for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For suspension formulations, typical suspending agents include methylcellulose, sodium carboxymethylcellulose, tragacanth, and sodium alginate; typical wetting agents include lecithin and polysorbate 80; typical preservatives include nitric acid Methyl Parkin and Sodium Benzoate. Oral liquid compositions may also contain one or more of the above disclosed ingredients such as sweetening, flavoring and coloring agents.

The pharmaceutical composition may also be coated in a conventional manner, usually with a pH- or time-dependent coating, so that the target compound is released adjacent to the desired site of topical application in the gastrointestinal tract, or at a different time to prolong the desired effect. Such dosage forms typically include, but are not limited to, one or more of cellulose acetate-phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, ethylcellulose, Wax and shellac.

Other compositions that may be used to achieve systemic delivery of the drug of interest include sublingual, buccal, and nasal dosage forms. Such compositions typically contain one or more soluble fillers, such as sucrose, sorbitol, and mannitol; and binders, such as gum arabic, microcrystalline cellulose, carboxymethylcellulose, and hydroxypropylmethyl cellulose. Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents disclosed above may also be included.

Compositions of the present invention may also be administered topically to a subject, for example, by directly applying or distributing the composition to the epidermis or epithelial tissue of a subject, or transdermally via a "patch". Such compositions include, for example, lotions, creams, solutions, gels and solids. These topical compositions may contain an effective amount of a compound of the present invention, usually at least about 0.1%, or even from about 1% to about 5%. Carriers suitable for topical administration generally remain on the skin in the form of a continuous film that resists removal by perspiration or water exposure. Typically, the carrier is organic in nature in which the therapeutic compound is dispersed or dissolved. The carrier may include pharmaceutically acceptable softeners, emulsifiers, thickeners, solvents and the like.

The active drug is administered in a therapeutically effective amount sufficient to inhibit aggregation of unwanted proteins in a subject. A "therapeutically effective" dose inhibits unwanted protein aggregation by, for example, at least about 20%, or at least about 40%, or even at least about 60%, or at least about 80%, relative to untreated subjects. In the case of, for example, patients with Parkinson's disease, a "therapeutically effective" dose stabilizes cognitive function or prevents further decline in cognitive function (ie, prevents, slows or stops the progression of the disease). The present invention thus provides therapeutic agents. "Therapeutic agent" or "drug" refers to a compound that has ameliorating or preventive beneficial effects on a particular disease or condition in a living human or non-human animal.

The toxicity and therapeutic efficacy of such drugs can be determined by standard pharmaceutical methods in cell culture or experimental animals, such as measuring LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio of LD50/ED50, and generally the greater the therapeutic index, the more effective it is. Although drugs with toxic side effects can be used, care should be taken to design delivery systems that target such drugs to damaged tissue sites to minimize potential damage to undamaged cells, thereby reducing side effects.

It is understood that the appropriate dosage will depend on many factors within the knowledge of the ordinary skilled physician, veterinarian or researcher. The dosage of the small molecule may vary depending on factors such as the nature, size and condition of the subject or sample being treated, and the route of administration of the composition (if applicable), and the effect of the small molecule on the nucleic acid or polypeptide of the invention desired by the practitioner. should have the effect. Exemplary doses include milligrams or micrograms of small molecules per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram. micrograms to approximately 50 micrograms per kilogram). It is also understood that appropriate dosages will depend on the potency of the expression or activity to be modulated. Such appropriate dosages can be determined using the assay methods described herein. When administering one or more of these small molecules to animals (such as humans) to regulate the expression or activity of the polypeptides or nucleic acids of the present invention, physicians, veterinarians or researchers can, for example, prescribe relatively low doses at the beginning, and then Increase dose until an appropriate response is obtained. In addition, it is to be understood that the particular dosage level administered to any particular animal subject may depend on a variety of factors, including the activity of the particular compound employed, the subject's age, weight, general health, sex and diet, time of administration, route of administration , excretion rate, any drug combination, degree of expression or activity to be modulated.

The ability of compounds to inhibit protein aggregation can be assessed in animal model systems, such as transgenic mice expressing human α-synuclein, which is predictive of inhibitory efficacy against protein aggregation in human disease, or where protein aggregation can be predicted Other relevant animal models, such as those described herein. Likewise, the ability of a compound to prevent or reduce cognitive impairment in model systems can be predictive of efficacy in humans. Alternatively, the ability of a compound can be assessed by testing its ability to inhibit protein aggregate formation in vitro, for example using a fibril formation assay as described herein, including ThT, CD or EM assays.

blood brain barrier

The medicaments of the invention can be formulated to ensure their proper distribution in the body. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic drugs. To ensure that more of the hydrophilic therapeutic agents of the invention cross the BBB, these agents can be formulated, for example, in liposomes. See, eg, US Patent Nos. 4,522,811, 5,374,548, and 5,399,331 for methods of preparing liposomes. Liposomes may contain one or more moieties that can be selectively transported into specific cells or organs ("targeting moieties"), thereby providing targeted drug delivery (see e.g. V.V. Ranade (1989) J. Clin. Pharmacol .29:685).

Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al), mannosides (Umezawa et al (1988) Biochem. (1995) FEBS Lett.357:140; M.Owais et al. (1995) Antimicrob.Agents Chemother.39:180), surfactant protein A receptor (Briscoe et al. (1995) Am.J Physio.1233:134), gp120 (Schreier et al. (1994) J Biol. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4:273. In one embodiment, the therapeutic agents of the invention are formulated in liposomes; which may include targeting moieties.

To ensure that the drug of the present invention crosses the BBB, the drug can be coupled to a BBB transporter (for a review on BBB transporters and mechanisms, see Bickel et al., Adv. Drug Delivery Reviews, Vol. 46, pp. 247-279, 2001). Exemplary transporters include cationized albumin or anti-transferrin receptor OX26 monoclonal antibody; these proteins cross the BBB by adsorption-mediated or receptor-mediated transcytosis, respectively.

Examples of other BBB transporters that target receptor-mediated transport systems into the brain include, for example, insulin, insulin-like growth factors (IGF-I, IGF-II), angiotensin II, atrial and brain natriuretic peptide (ANP , BNP), interleukin I (IL-1) and transferrin and other factors. Receptor-binding monoclonal antibodies to these factors can also be used as BBB transporters. BBB transporters targeting adsorption-mediated endocytosis mechanisms include cationic moieties such as cationized LDL, albumin conjugated to polylysine or horseradish peroxidase, cationized albumin, or cationized immune globulin. Small basic oligopeptides such as dynorphin analog E-2078 and ACTH analog ebiratide can also cross the brain through adsorption-mediated endocytosis, and they are potential transporters.

Other BBB transporter targeting systems direct nutrient transport systems to the brain. Examples of such BBB transporters include hexose moieties such as glucose, monocarboxylic acids such as lactic acid, neutral amino acids such as phenylalanine, amines such as choline, basic amino acids such as arginine, ), nucleosides (eg, adenosine), purine bases (eg, adenine), and thyroid hormones (eg, triiodothyridine). Antibodies to the extracellular domain of nutrient transporters can also be used as transport vehicles. Other possible carriers include angiotensin II and ANP, which may be involved in regulating BBB permeability.

In some cases, following transport into the brain, the bond linking the therapeutic compound to the transport vehicle can be cleaved to release the bioactive compound. Exemplary linkages include disulfide, ester, thioether, amide, acid labile, and Schiff base linkages. An avidin/biotin linker can also be used, where avidin is covalently coupled to the BBB drug delivery vehicle. Avidin itself can be a drug transporter.

Prodrug

The present invention also relates to prodrugs of the various drugs described herein. Prodrugs are drugs that can be converted in vivo into their active form (see for example R.B. Silverman, 1992, "The Organic Chemistry of Drug Design and Drug Action," Academic Press, Chapter 8). Prodrugs can be used to alter the biodistribution (eg, to allow entry of a drug that would not normally be able to access a protease reaction site) or pharmacokinetics of a particular compound. For example, carboxylic acid groups can be esterified, eg, with methyl or ethyl groups, to produce esters. When an ester is administered to a subject, the ester is cleaved by enzymatic or non-enzymatic, reduction, oxidation, or hydrolysis to reveal an anionic group. Anionic groups can be esterified with moieties (eg, acyloxymethyl esters) which upon cleavage exhibit intermediate compounds which subsequently decompose to yield the active compound. Prodrug moieties can also be metabolized to carboxylic acids in vivo by esterases or by other mechanisms.

Examples of prodrugs and their uses are well known in the art (see, eg, Berge et al. (1977) "Pharmaceutical Salts", J Pharm. Sci. 66:1-19). Prodrugs can be prepared in situ during the final isolation and purification of the drug, or by separately reacting the free acid form of the purified compound with a suitable derivatizing compound. Carboxylic acids can be converted to esters by treatment with ethanol in the presence of a catalyst.

Examples of cleavable carboxylic acid prodrug moieties include substituted or unsubstituted, branched or unbranched lower alkyl ester moieties (e.g. ethyl, propyl, butyl, pentyl, cyclopentyl, hexyl ester, cyclohexyl ester), lower alkenyl ester, di-lower alkylamino lower alkyl ester (such as dimethylaminoethyl ester), amido lower alkyl ester, acyloxy lower alkyl ester (such as pivaloyl oxymethyl ester), aryl ester (phenyl ester), aryl-lower alkyl ester (such as benzyl ester), substituted (such as methyl, halogen or methoxy substituted) aryl and aryl-lower Alkyl esters, amides, lower alkyl amides, di-lower alkyl amides and hydroxy amides.

pharmaceutically acceptable salt

Certain embodiments of the medicaments of the invention may contain basic functional groups, such as amino or alkylamino groups, and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salt" in this sense refers to the relatively non-toxic inorganic or organic acid addition salts of the medicaments of the invention. These salts can be prepared in situ during the final isolation and purification of the drug of the invention, or separately by reacting a purified compound of the invention in free base form with a suitable organic or inorganic acid and isolating the salt formed.

Representative salts include hydrohalides (including hydrobromides and hydrochlorides), sulfates, bisulfates, phosphates, nitrates, acetates, valerates, oleates, palmitates , Stearate, Laurate, Benzoate, Lactate, Phosphate, Tosylate, Citrate, Maleate, Fumarate, Succinate, Tartrate, Naphthalene salt, methanesulfonate, glucoheptonate, lactobionate, 2-hydroxyethanesulfonate and laurylsulfonate, etc. (See eg Berge et al. (1977) "Pharmaceutical Salts", J Pharm. Sci. 66, 1-19).

In other cases, the agents of the invention may contain one or more acidic functional groups and thus are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" refers in these cases to the relatively non-toxic, inorganic and organic base addition salts of the medicaments of the invention.

These salts can also be prepared in situ during the final isolation and purification of the pharmaceuticals of the present invention, or separately by reacting the purified compound in free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation Salts, ammonia or pharmaceutically acceptable organic primary, secondary or tertiary amines. Representative alkali metal salts or alkaline earth metal salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, aluminum salts, and the like. Representative organic amines that can be used to form base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like.

Example

The present invention is further illustrated by the following examples, which should not be construed as further limiting the present invention.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific methods, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this invention and are covered by the appended claims of this invention. The contents of all references, issued patents, and published patent applications cited throughout the specification of this application are hereby incorporated by reference.

 Example 1: To detect protein aggregation disease-related

  Assays for harmful protein aggregates

Assay to detect deleterious protein aggregates associated with deleterious accumulation of NAC

Preparation of NAC peptide

As discussed herein, the acronym NAC refers to the non-amyloid component of amyloid plaques found in AD patients, specifically, residues 61-95 corresponding to α-synuclein of 35 amino acid peptides. NAC peptide was synthesized by Fmoc tert-butyl chemical method on the peptide synthesizer of Protein Technologies, Inc., with a purity of >98%. The peptide content was determined to be 71.6% by amino acid analysis.

Preparations of NAC may contain aggregated material. To remove these aggregates and monomerize the peptides, a lower deaggregation/filtration method was obtained adapted from the article by Walsh and Colleagues (J Biol Chem. 1997 Aug 29;272(35):22364-72). This method involves sonicating the peptide in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) to dissolve any structure that may be present. Any large aggregates that may remain in solution were then filtered through a 20 nm filter and stored at -80°C until use.

Compound preparation

A 2 mM solution was prepared in TBSA buffer (Tris 0.02M, NaCl 0.15M, _NaN3 0.005%, pH 7.4) and stored at 4°C. For the assay, the mixture was diluted to the desired concentration with TBSAE buffer (Tris 0.02M, NaCl 0.15M, NaN ₃ 0.005%, EDTA 100 μM, pH 7.4) and combined with NAC peptide.

fibril formation test

The ability of test compounds to inhibit NAC assembly into fibers was identified using a fibril formation assay. After removal of HFIP by evaporation under _N atmosphere, 20 μM NAC peptide was dissolved in assay buffer (0.02M Tris, 0.15M NaCl, 0.005% NaN ₃ , 100 μM EDTA in a Perkin-Elmer HTS 7000 plus microplate reader at 37°C ), shaking for 1 minute every 20 minutes. The presence of fibers can be monitored by three different methods.

The following assays were performed in clear polystyrene 96-well microplates as follows: 125 μL of 40 μM NAC peptide was combined with 125 μL of 200 μM test compound in 0.02M Tris, 0.15M NaCl, 0.005% _NaN3 , 100 μM pH 7.4. After the microplate was sealed with a plastic sheet, it was incubated in a Perkin-Elmer HTS 7000 microplate reader at 37°C with shaking for 1 minute every 20 minutes.

Thioflavin T analysis. For this assay, 5 [mu]M Thioflavin T was added to the NAC assembly conditions described above. During the experiment, fluorescence was measured every 20 minutes with a Perkin-Elmer HTS microplate reader at 430 excitation/485 emission. As can be seen in Figure 1, the presence of fibers can be detected by an increase in fluorescence. This assay allows the identification of compounds that inhibit the increase in fluorescence upon incubation.

Turbidity analysis. Alternatively, NAC peptides that have been incubated with test compounds under appropriate assembly conditions can be analyzed by reading absorbance at 40 nm to monitor the turbidity of the solution, which is also an indicator of aggregate formation.

Circular Dichroism (CD) Analysis. Circular dichroism detection analysis of the NAC peptide during its assembly indicated that NAC transitioned from a random coil conformation to a β-sheet/turn conformation after approximately 15-20 hours of incubation in the absence of the test compound. This assay allows the identification of compounds that prevent NAC from adopting a β-sheet/β-turn conformation upon incubation. At appropriate time points during the incubation, the test solution was transferred to a 0.1-cm optical path length quartz cuvette for CD spectroscopy at 37 °C with a Jasco J-715 spectropolarimeter between 190 and 260 nm , with a resolution of 0.1nm and a bandwidth of 1nm. NAC alone was found to adopt a β-sheet/turn conformation after overnight incubation. Some samples containing test compounds were observed to retain the random coil NAC, and some were undergoing a conformational transition but not yet to the β-sheet/β-turn conformation.

Electron microscope (EM) analysis. At the time points when CD scans were performed, 3 μl aliquots were removed from the samples, spotted onto Formvar grids, and negatively stained with 4% uranyl acetate. The grid was observed with a JOEL 2000 transmission electron microscope at 25,000X magnification to check for the presence of fibrils and amorphous aggregates. This assay can be used to confirm the absence of fibers in the presence of compounds identified by ThT and CD that inhibit NAC assembly. The final result is obtained by taking the average of 10 complete segments.

Example 2: Application of Thioflavin T Assay in Determining Formation of β-Sheets by Isolated NAC

Trials that have been performed have shown that Thioflavin T (ThT) can be used in high-throughput and validation work using NAC peptides. A fluorescent signal indicative of β-sheet formation started to appear at 10 hours of incubation (Fig. 1). The intensity of the fluorescent signal is directly related to the concentration of NAC in solution, reaching a maximum at 30 μM NAC. A similar ThT profile was observed (in 96-well plates) with a T _1/2 (time required to obtain a signal equal to half the maximum signal obtained) of ~15 hours.

Example 3: Circular dichroism analysis and electron microscope analysis of NAC peptide conformation

Circular dichroism analysis of the conformation of the NAC peptide after incubation for 10-72 h (Fig. 2) revealed a minimum at 227 nm, reminiscent of that observed in regions rich in α-helices . After incubation of NAC in the presence of heparin, its CD spectrum showed a distinct minimum at 218 nm, which is characteristic of the β-sheet conformation. Electron microscopic analysis (Fig. 3) detected the appearance of NAC fibers. The presence of heparin clearly promotes a conformational transition and favors a high β-sheet content that promotes aggregate/fibril formation (Figure 2). This result indicates that glycosaminoglycans are indeed involved in the oligomerization/fibril formation process of NAC, confirming the effectiveness of the approach of using GAG mimetic compounds as a means to prevent NAC oligomerization and formation of toxic aggregates. EM analysis supports this observation that NAC peptides appear to form longer and more tangled fibers in the presence of heparin (Figure 4).

The table below summarizes the CD test results for several compounds.

CD analysis results of test compounds

CD analysis observed that when NAC was incubated with the test compound, it displayed a different conformation: "random coil" indicates that NAC in the sample was observed to remain in its original random coil conformation, indicating that the compound was co-incubated with NAC Active; "β-sheet" or "β-sheet/β-turn" indicates that the compound cannot prevent the transition from random coil to β-sheet; "transition" indicates that NAC is observed to be in the transition from random coil to β-sheet In the process, it is shown that the compound is capable of slowing down the conformational change process and thus exhibits activity. Compounds tested multiple times were classified as active if they prevented the transition to β-sheet/β-turn in 50% or more of the tests.

Electron microscopy analysis: Fibril formation assays performed for CD analysis were also performed for EM analysis. Electron micrographs were visually inspected and scored as follows. Briefly, 10 representative frames from a 300 mesh grid were analyzed and compared with photomicrographs of NAC alone without the test compound (100% fibers) a score was given indicating the amount of fiber formed in the sample. According to the results of this analysis, score according to the classification method of 0%-100% fiber: (-) means that less than about 25% fiber is observed; (+) means about 25% fiber; (++) about 50% fiber Fiber; (+++) about 75% fiber; (++++) 100% fiber. (AA) indicates that amorphous aggregates were observed. The following table gives the EM analysis results of the samples.

EM analysis results

Example 4: Compounds that prevent protein aggregation diseases can be screened in cell models

Various cell culture models have been described to monitor aggregate formation associated with protein accumulation. Additionally, this protein oligomerizes and aggregates and induces toxicity in these cultured cells. For Parkinson's disease models, overexpression of various synuclein mutants induced aggregate formation in several cell lines: SH-SY5Y cells (Kanda et al., 2000. Neurosci 97:279), BE ( 2)-M17 neuroblastoma cells, HEK293 (Ko et al., 2000. J Neurochem 75: 2546), NT-2, SK-N-MC cell line (Lee et al., 2001. J Neurochem 76: 998) and BE-M17 Neuroblastoma cells (Ostrerova-Golts et al., 2000. J Neurosci 20:6048).

A similar model has been developed to study aggregate formation by other proteins such as huntingtin involved in the development of Huntington's disease; mutant huntingtin gene in PC-12 cell line (Wu et al., 2002. JBC 277:44208; Igarashi et al., 2003. Mol Neurosci 14:565) or expression in Cos-7 (Carmichael et al., 2002. Neurosci Lett 330:270-274; Yang et al., 2002. Hum Mol Gen 11:2905) results in the appearance of aggregates.

Target aggregated proteins (e.g., synuclein or huntingtin) can be immunocolocalized with various cytoskeletal proteins such as β-tubulin, γ-tubulin, or vimentin, allowing easy identification of aggregates or inclusion bodies detection. Exclusion of other markers (such as α-mannosidase II of the Golgi apparatus) can further determine the cellular distribution of inclusion bodies. As previously demonstrated for DPRLA and meningioma proteins (Shimohata et al., 2002; Gautreau et al., 2003; see table above), other markers such as ubiquitin can be used to characterize accumulation in the paranuclear domain of the microtubule organizing center (MTOC) Aggregates of aggregates. Other aggregates or inclusions can also be found in the cytoplasm (Lewy bodies, Mallory bodies) or the nucleus (huntingtin, ataxin).

Although these large aggregates can be detected visually by microscopic analysis, they may not themselves be toxic, and their presence may not be sufficient to induce toxicity. However, their presence indicates the accumulation of aberrant protein conformers and assembly intermediates, the latter two of which have been shown to induce cytotoxicity.

Recent evidence clearly demonstrates a toxic role for androgen-receptors containing extended polyglutamine segments that lead to spinal bulbar muscular atrophy. After transfection with an AR mutant with 112 glutamine repeats (AR-112Q), all cells showed cytoplasmic inclusions characteristic of aggregates and displayed marked cell death (Taylor et al., 2003. Hum Mol Genet 12: 749). Importantly, inhibition of aggregate formation with nocodazole resulted in a dose-dependent increase in cell death, highlighting the importance of aggregates in protecting cells from the toxic effects of oligomers/aggregates.

Cells with aggregate accumulation can be treated in vitro with proprietary compounds, and the formation of toxic aggregates can be monitored in a variety of ways. Following treatment of cells expressing the mutant protein, aggregates or inclusions can be quantified by microscopy using fluorescent labels, and cell viability can be monitored using the viability assay described above or by MTT or WST-1 staining. (Abee and Matsuke. 2000. Neurosc Res 38:3256; Berridge et al., 1996. Biochemica 4:11). The amount of cell death can be quantified using a FACS-based survival assay (Taylor et al., 2003. Hum Mol Genet 12:749).

Aggregates or aggregates are isolated by cell disruption and centrifugation for further characterization and quantification.

Example 5: Method of Isolating Aggregates Associated with Protein Aggregation Diseases

Starger's technique can be improved (Starger and Goldman, 1977.Proc NatlAcad Sci USA 74:2422) to optimize the separation of protein aggregates from aggregates. Briefly, cells were cultured to 85% confluence in 100-mm dishes and treated with 10 mg/ml ALLN for 12 hours before isolation. Cells were washed twice in PBS (6 mM sodium potassium phosphate buffer, 170 mM NaCl, 3 mM KCl), scraped and collected at 2,500 g for 3 min. Resuspend each 100-mm dish of washed cells in 1 ml PBS and pass through a 25-gauge needle three to four times until bright-field microscopy shows that most of the cells are broken. The material was washed by resuspending in PBS and sedimenting three times at 2,000 g. Examination of the resulting material by fluorescence microscopy confirmed the presence of isolated aggregates containing GFP. The resulting aggregate-rich cell fraction was collected a final time at 2,000 g and resuspended in 200 ml of PBS/1% BSA. This can be used as a starting material for immunoelectron microscopy. The formation of these protein aggregates can be further quantified by quantitative ELISA after protein denaturation, or by dot blot filter retention assay after centrifugation of aggregates, as described (Johnston et al., 1998. JCB 143:1883).

Example 6: Dot blot filtration retention test

Cells expressing the mutant protein were washed in ice-cold phosphate-buffered saline (PBS), scraped, and pelleted by centrifugation (2000 g, 10 min, 4°C). Lysis of cells in 500 μL containing protease inhibitors PMSF (2 mM), leupeptin (10 μg/ml), pepstatin (10 μg/ml), aprotinin (1 μg/ml) and antiprotease (50 μg/ml) Lyse on ice for 30 minutes in buffer [50 mM Tris-HCl (pH 8.8), 100 mM NaCl, 5 mM MgCl ₂ , 0.5% (w/v) Nonidet P-40 (NP-40), 1 mM EDTA]. Insoluble material was removed by centrifugation in a microcentrifuge at 14000 rpm for 5 minutes at 4°C. The pellet containing insoluble matter was resuspended in 100 μl DNase buffer [20 mM Tris-HCl (pH 8.0), 15 mM MgCl ₂ ], DNase I (Boehringer Mannheim) was added to a final concentration of 0.5 mg/ml, and then Incubate for 1 hour at 37°C. Protein concentration after DNase treatment was determined by dotMetric assay (Geno technology) using BSA as a standard. The incubation reaction was terminated by adjusting the resulting mixture with 20 mM EDTA, 2% (w/v) SDS and 50 mM DTT, followed by heating at 98°C for 5 minutes.

A filtration assay to detect polyglutamine-containing huntingtin aggregates has been described (Johnson et al., 1995. J Mol Recog 8:125). Denatured and reduced protein samples were prepared as described above by diluting an aliquot equivalent to 50-250 ng of fusion protein from transfected cells or 5-30 μg of extracted protein (precipitated fraction) into 200 μl of 2% SDS and infused in BRL. Cellulose acetate membranes (Sehleicher and Schuell, Keene, NH, pore size 0.2 [mu]m) that had been pre-equilibrated with 2% SDS were filtered in a dot blot filter unit. Filters were washed twice with 200 μl 0.1% SDS, then blocked with 3% nonfat dry milk in TBS (100 mM Tris-HCl, pH 7.4, 150 mM NaCl), followed by incubation with anti-HD1 antibody (1:1000). Filters were washed several times in TBS, then incubated with a secondary anti-rabbit antibody conjugated to horseradish peroxidase (Sigma, 1:5000), followed by ECL (enhanced chemiluminescence, Amersham) detection. Developed blots were exposed several times to Kodak (Rochester, NY) X-OMAT film or a Lumi-Imager (Boehringer Mannheim) to allow quantification of the immunoblots.

To detect and quantify polyglutamine-containing aggregates produced by protease-treated GST-x fusion proteins, a biotin/streptavidin-AP detection system was used. After filtration, cellulose acetate membranes were incubated with 1% (w/v) BSA in TBS for 1 hour at room temperature with gentle shaking on a reciprocating shaker. The filters were then incubated for 30 minutes with streptavidin-alkaline phosphatase (Promega, Madison, WI) diluted 1:1000 in TBS containing 1% BSA, with 0.1% (v/v ) Tween 20 TBS washed three times, and then washed three times with TBS, and finally together with fluorescent alkaline phosphatase substrate AttoPhos or chlorine-substituted 1,2-dioxetane chemiluminescence substrate CDPStar (Boehringer Mannheim) in 100mM Incubate for 3 min in Tris-HCl, pH 9.0, 100 mM NaCl and 1 mM MgCl ₂ . Fluorescent and chemiluminescent signals were imaged and quantified using the Boehringer Lumi-Imager F1 system and LumiAnalyst software (BoehringerMannheim).

Example 7: Filter trap test

The filter trap assay was used to detect the presence of aggregates in frozen brain samples in a simple assay. This method can be modified to detect various types of protein aggregates. Frozen mouse hemibrains and frozen human brain fragments were weighed and then treated with polytron in 10 volumes of phosphate-buffered saline (pH 7.4) containing 1× protease inhibitor cocktail (Cat.#P8340, Sigma, St.Louis, MO). ) homogenate. The homogenate was centrifuged in a microcentrifuge at 3,000 rpm for 5 minutes at 4°C. An aliquot of the supernatant was taken and the protein concentration was determined by the BCA method (Pierce Chemical Co., Rockford, IL). Aliquots were stored frozen at -70°C until use. Samples were thawed prior to filtration and then diluted with PBS to a final volume of 200 [mu]l with 1% SDS (with the exception of Figure 2, where the final SDS concentration varied between 0.1-5%). The resulting solution was then filtered through a 0.2-μm pore size cellulose acetate membrane (OE66, Schleicher & Schuell, Keene, NH) using a 96-well dot blot apparatus (Bio-Rad Laboratories, Hercules, CA). Prior to filtration, the filters were soaked in PBS containing 1% SDS. Filter blots were washed twice with 500 μL PBS (pH, 7.4). Filter-retained proteins were detected by immunostaining following the procedure used for immunoblotting (Xu et al., 2002 Alzheimer Dis Assoc Disord 16:191).

For analysis of filter-retained proteins, target blots were excised, boiled for 10 min in 40 μL 1x SDS loading buffer (Laemmli, 1970), and mixed vigorously (by vortex mixer). Samples (20 μL) were loaded onto SDS/polyacrylamide gels and blotted onto nitrocellulose membranes (BA-S85, Schleicher & Schuell, Keene, NH). Immobilized proteins were detected with mAb 6E10 and ECL (NEN Life Science Products, Inc., Boston, MA) as previously described (Jankowsky et al., 2001).

 Example 8: To detect protein aggregation disease-related

Multiple approaches to detrimental protein aggregates

The table below summarizes references detailing methods and techniques used to detect inclusion bodies or aggregates associated with various diseases. It should be noted that the techniques described herein can be employed by one of ordinary skill in the art to practice the methods described herein. protein under study Aggregate Detection Technology indication Inclusion body type and location references Parkin alpha-synuclein synphilin-1 1- Co-localization with Parkin/γ-Tubulin (MOTC-labeled) by immunofluorescence 2- Microtubule inhibitors/protease inhibitors Parkinson's disease Lewy body cytoplasm Junn et al., 2002. JBC 277, 47870-47877 DRPLA protein 1- Co-localization with β-tubulin/γ-tubulin/Vimentin by immunofluorescence 2- Microtubule inhibitors/protease inhibitors Atrophy of dentate nuclei rubrum pallidal Lewy bodies Aggregate paranuclear and nuclear inclusions Shimohata et al., 2002., Neurosci. Lett. 323, 215-218 Huntingtin Colocalization with γ-Tubulin/Vimentin by Immunofluorescence Huntington's disease Intranuclear inclusions and cytoplasmic aggregates of dystrophic axons or neuropilus Waelter et al., 2001. Mol. Biol Cell 12, 1393-1407 Cystic Fibrosis Transmembrane Regulator (CFTR) 1-Colocalization with Vimentin/γ-Tubulin by Immunofluorescence 2-Protease Inhibitors 3- Electron Microscopy 4- Subcellular Fractionation and Aggregate Separation cystic fibrosis aggregate cytoplasm Johnston et al, 1998, JCB143, 1883-1898 α-synuclein Co-localization with γ-tubulin by immunofluorescence Parkinson's disease Lewy body cytoplasm McNaught et al 2003. Eur. J. Neurosci. 16, 2136-2148 Meningioma protein 1-Colocalization with γ-tubulin by immunofluorescence 2-Nocodazole 3-Pulse chase/immunoprecipitation of EM4-components neurofibroma type 2 aggregate cytoplasm Gautreau et al. 2003. JBC 278, 6235-6242 Cytokeratin Colocalization with ubiquitin/proteasome subunit P25 by immunofluorescence Alcoholic hepatitis, nonalcoholic steatohepatitis, chronic biliary obstruction, copper poisoning, drug poisoning, and hepatocellular neoplasms Mallory bodies French et al, 2001. Exp. Mol. Pathol. 71, 241-246

Example 9: Screening of compounds in a transgenic mouse model of protein aggregation disease

The table below provides examples of transgenic mouse lines established to model human proteinopathies.

Constructs to express various mutant proteins have been designed and introduced into transgenic mice to mimic various protein aggregation diseases or proteinopathies. Expression of these genes results in a variety of neuropathological and behavioral changes consistent with human conditions associated with mutated genes.

For example, transgenic mice expressing modest levels of the long isoform of tau (carrying mutations seen in patients with frontotemporal dementia and Parkinson's disease) develop tauopathy, which is characterized by tropism in forebrain neurons. Congo red hyperphosphorylated tau inclusion bodies. These inclusions begin to appear at 18 months of age. In humans, tau inclusion bodies consist of both mutant tau and endogenous wild-type tau and are associated with microtubule disruption and flame-shaped transformation in affected neurons. Behaviorally, aged TgτR406W mice exhibited cognitive deficits, particularly impaired associative memory (see table below).

Another example of a successfully produced human disease model is a transgenic mouse model expressing the α-synuclein mutations A30P and A53T. These mice exhibited an early-onset progressive decline in motor function. Neuropathologically, these mice displayed typical α-synuclein immunoreactive Lewy body inclusions in axons (see table below).

Various huntingtin alleles have been introduced into mice. Transgenic mice expressing huntingtin alleles with multiple sets of extended polyglutamine segments exhibit an early clasp phenotype, impaired muscle movement coordination, and hyperactivity. The disease is associated with cortical inclusions, septal inclusions, hippocampal inclusions, reactive gliosis, cell loss, general brain atrophy, and neuropathological phenomena of cellular inclusions commonly seen in patients with Huntington's disease The changes are consistent (see table below).

Therefore, the application of this transgenic mouse model, which appears to mimic various human non-amyloid disease phenotypes, is to test the therapeutic efficacy of compounds that bind target proteins, prevent protein assembly, oligomerization, aggregation, or promote protein clearance. Animal models are provided.

A transgenic model of human tauopathy mutation/gene Transgene/Promoter genetic background behavioral phenotype neurological features citation τP301L Tau with 4 repeats, exon 10, but exons 2 and 3 deleted and P301L mutation/PrP promoter C57BL6DBA/2SW Early and severe motor and behavioral deficits Fibrillary gliosis in anterior horn, axonal degeneration, neuronal damage Lewis J et al 2000. Nat Genet 25: 402-5. τP301L Tau40 isoform with 4 repeats, exons 2 and 3 and P301L mutation/Thy12 promoter B6D2F1, C57BL/6 Signs of Wallerian degeneration, neurogenic muscle atrophy, muscle weakness. Numerous tau-responsive nerve cell bodies and dendrites; numerous pathologically enlarged axons with neurofilaments and tau-responsive spheroids Gotz J et al. 2001. J Biol Chem. 276: 529-34. τR406W τ/αCaMk-II promoter with R406W mutation B6SJL/F1C57BL/6J Impaired associative memory. Abnormal prepulse inhibition and the forced swim test. Accumulation of insoluble tau protein. Congophilic hyperphosphorylated tau inclusion bodies appear in forebrain neurons. TatebayashiY et al. 2002.Proc NatlAcad SciUSA.99:13896-901. τG272V Tau40/prion protein promoter with G272V mutation B6D2F1C57BL/6 no neurological deficit Fibrils appear in oligodendrocytes with phospho-tau phosphorylation. ThioS-positive fibril inclusions in spinal oligodendrocytes and motor neurons Gotz J et al 2001.Eur JNeurosci.13:2131-40 τV337M Tau/PDGF-b promoter with V337M, Thy12 promoter B6L Highly athletic. The results of the elevated + word maze test and the conditioned fear test are significantly different Irregularly shaped neurons in the hippocampus are immunoreactive to tau and contain paired helical filaments. Atrophic cell death. Tanemura K et al 2002 JNeurosci.22:133-41 τ3-repeat sequence The tau'/prion protein promoter with 3 repeats, B6D2/F1 Progressive muscle movement weakness, impaired coordination Insoluble tau accumulation, deep astrocytosis and axonal degeneration. Ishibara T et al 1999Neuron24:751-62 τ4-repeat sequence tau,/mouse thy-1 promoter with 4 repeats FVB/N Lack of muscle movement and sensation. Axonal degeneration, astrocytosis and ubiquitination of accumulated proteins Spittaels K et al 1999 AmJ Pathol.155.2153-65 τ4-repeat ALZ17 Tau/mouse Thy.-1.2 promoter with 4 repeats B6D2/F1xB6D2/F1C57BL/6 Lack of muscle movement and reflexes. Prominent somato-dendritic staining of hyperphosphorylated tau with marked axonopathy Probst A et al. 2000 ActaNeuropathol.99: 469-81

A transgenic model of human Huntington's disease mutation/gene Transgene/Promoter genetic background behavioral phenotype neurological features citation N171HD cDNA encoding the N-terminal fragment (171 amino acids) of huntingtin with 82, 44 or 18 glutamine/prion protein promoter C3HC57BL/6 Abnormal behavior only occurs in 82Q. Loss of coordination, tremor, hypokinesia, and abnormal gait are present. Early clasping and muscle-motor coordination are impaired. die early. Inclusion bodies were present in the striatum, cortex, hippocampus, amygdala, and cerebellum. Diffuse nuclear accumulation of htt protein. Striatal cell loss and global brain atrophy. Schilling G et al 1999. Human Molecular Genetics HD HD gene with 16, 48 or Q89 glutamine repeat sequence CMV promoter FVB/N A circling, hyperactive early clasping phenotype in 89Q and 48Q mice Inclusion bodies and gliosis appeared in the striatum, cerebral cortex, thalamus, and hippocampus. Striatal cell loss. Reddy PH et al. 1998. Nature Gen. 20: 198-202 HD HD gene exon 1-3 with 89Q repeat fragment FVB/N Prolonged hyperactivity and early clenching. Inclusion bodies are present throughout the brain. HD HD gene with 100Q, 48Q or 18Q repeat sequence/NSE promoter SJLC57BL/6 Hyperactive 100Q mice display an early clasp phenotype and impaired muscle motor coordination Dystrophic axons appear in neuronal intranuclear inclusions and in the cortex and striatum. Cell loss and brain shrinkage. DiFiglia et al. 1997Science277:1990-1993. conditional HD mutant Conditional expression of mutant huntingtin band 94Q (tet-off, CamKIIα-tTA) CBAC57BL/6 Late onset tremor and abnormal gait. Impaired early gripping and muscle-motor coordination Inclusion bodies appeared in the striatum, septum, cortex, and hippocampus. Reactive astrocytes. progressive global brain atrophy Yamamoto et al. 2002. Cell101: 57-66

A transgenic model of human Parkinson's disease mutation/gene Transgene/Promoter genetic background behavioral phenotype neurological features citation α-synuclein α-synuclein with A53T/Thy1 gene promoter C57BL/6 Early progressive impairment of motor function Astrocytic gliosis and microglial activation. Diffuse perinuclear α-synuclein staining. Intensely stained by Campbell-Switzer pyridinium silver, showing Lewy-like changes Putten H et al 2000. JNeurosci 20: 6021-9. Sommer B et al 2000 ExpGerontol. 35: 1389-403 α-synuclein A30P α-synuclein-with A30P/Thy1 promoter C57BL/6 No muscle movement abnormalities α-Syn positive axons display features of Lewy bodies, emanating from neuronal cell bodies Kahle PJ et al 2002 J Clin.Invest 110:1429-1439 α-synuclein wt α-synuclein C57BL/6 no record Abnormal Tgα-Syn positive axons, characteristic of Lewy body disease, Kahle et al 2001. Am JPathol.159:2215-25. Alpha-synuclein A30P or A53T α-synuclein A30P or A53T Tg5093 Progressive movement disorder with stiffness, dystonia, impaired gait, and tremor Discrete Lewy body-like α-synuclein inclusions could not be identified. There was no specific degeneration of the nigrostriatal dopaminergic system. Gomez-Isla et al. Neurobiology of Aging 24 245-258 α-synuclein Alpha-synuclein. A30P or A53T C3H/HeJxC57BL6/J Adult-onset neurodegenerative disease with progressive motor dysfunction Neuronal abnormalities in perinuclear bodies and axons, including pathological accumulation of α-Syn and ubiquitin Lee et al., 2002Proc NatlAcad SciUSA.99:8968-73 APP+α-synuclein Lineage D heterozygous α-SYN mice were crossed with lineage J9 heterozygous hAPP mice Masliah E et al 2001. ProcNat Acad SciUSA98: 12245-50

Example 11: Evaluation of compounds that bind NAC peptides by mass spectrometry

Compounds of the invention were evaluated for their ability to bind NAC peptides in aqueous solution. Binding capacity correlates with the intensity of the peptide-compound complex peaks observed by electrospray mass spectrometry. All aqueous solutions were prepared with Millipore distilled deionized water. The pH value was measured using a Beckman Φ36 pH meter equipped with a Corning Semi-Micro Combination pH electrode.

First, 20 μM NAC (MW 3260.6 Da) was analyzed at pH 7.40, and common sodium clusters were observed at +2, +3 and +4 at m/z 1335.5, 1116.7 and 843.4, respectively. The optimal cone voltage was determined to be 20V.

Mass spectrometry—Mass spectrometry was performed on a Waters ZQ4000 mass spectrometer equipped with Waters 2795 sample management software. Data processing and analysis were performed with MassLynx 4.0 (the previous version was MassLynx 3.5). Test compounds and depolymerized peptides were mixed in an aqueous medium (6.6% EtOH) in a ratio of 5:1 (20 μM NAC: 100 μM test compound or 40 μM NAC: 200 μM test compound). The pH of the resulting mixture was adjusted to 7.4 (±0.2) with 0.1% NaOH (3-5 μL). A 20 μM or 40 μM NAC peptide solution was regularly prepared in the same manner as a control. Mass spectra were acquired by introducing the solution into the electrospray source by direct infusion with a syringe pump at a flow rate of 25 μl/min and scanning in positive ion mode from 100-2100 Da. The scan time per scan was 0.9 s, the delay between scans was 0.1 s, and the run time per sample was 5 min. All mass spectra are the sum of 300 scans. The desolvation and electrospray source temperature was 70 °C, and the cone and capillary voltages were maintained at 20 V and 3.2 kV, respectively.

The quotient of the total area under the bound NAC-compound complex peak divided by the total area under the unbound NAC peak was determined for each test compound. The results are summarized in the table below.

NAC peptide binding data

^* +++ = strong; when total binding is 120% or higher

++ = Moderate; when total binding is between 120% and 70%

+ = weak; when total binding is between 70% and 30%

-=none; when total binding is between 30% and 0%

Claims (54)

1. A method of treating or preventing a protein aggregation disease in a subject, the method comprising Administering an effective amount of a compound to the subject suffering from a protein aggregation disease, so that the protein aggregation disease in the subject is treated or prevented, wherein the compound has one of the following formulas: Q-[-Y ^- -X ⁺ ] _n where Q is a carrier molecule; Y is SO ₃ ^- X ⁺ , OSO ₃ ^- X ⁺ or SSO ₃ ^- X ⁺ ; X ⁺ is a cationic group, such as a positively charged atom or other moiety; suitable carrier molecules include carbohydrates , polymers, peptides, peptide derivatives, aliphatic groups, alicyclic groups, heterocyclic groups, aromatic groups, or combinations thereof; or Wherein Y is an amino group or a sulfonic acid group, n is an integer of 1-5, and X is hydrogen or a cationic group; or Wherein R ¹ is substituted or unsubstituted cycloalkyl, aryl, arylcycloalkyl, bicyclic or tricyclic, bicyclic or tricyclic condensed ring group or substituted or unsubstituted C ₂ -C ₁₀ Alkyl; R ^is selected from hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl and benzene and imidazolyl; Y is SO ₃ ^- X ⁺ , OSO ₃ ^- X ⁺ or SSO ₃ ^- X ⁺ ; X ⁺ is hydrogen, a cationic group or an ester-forming group (ie, the ester-forming group in the prodrug, herein It is described there); L ^{and L} are each independently substituted or unsubstituted C ₁ -C ₅ alkyl or absent, or a pharmaceutically acceptable salt of the compound, provided that when ^R is an alkyl base time, L ¹ does not exist; or Wherein R ¹ is a substituted or unsubstituted ring, bicyclic, tricyclic or benzoheterocyclic group or a substituted or unsubstituted C ₂ -C ₁₀ alkyl; R ² is hydrogen, alkyl, mercaptoalkyl , alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, benzimidazolyl, or link with ^R to form a heterocycle; Y is SO ₃ ^- X ⁺ , OSO ₃ ^- X ⁺ or SSO ₃ ^- X ⁺ ; X ⁺ is hydrogen, a cationic group or an ester-forming moiety; m is 0 or 1; n is 1, 2, 3 or 4; L is a substitution or unsubstituted C ₁ -C ₃ alkyl or absent, with the proviso that when R ¹ is alkyl, L ¹ is absent; or

wherein A is nitrogen or oxygen; R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together may be a natural or unnatural amino acid residue or its salt or ester; Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl; x is 0, 1, 2, 3 or 4; n is 0, 1, 2 , 3, 4, 5, 6, 7, 8, 9 or 10; R ³ , R ^3a , R ⁴ , R ^4a , R ⁵ , R ^5a , R ⁶ , R ^6a , R ⁷ and R ^7a are each independently hydrogen , alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino, tetrazolyl, or on adjacent ring atoms The two R groups of R together form a double bond with a ring atom, provided that one of R ³ , R ^3a , R ⁴ , R ^4a , R ⁵ , R ^5a , R ⁶ , R ^6a , R ⁷ and R ^7a is of formula IIIa The part shown in -A: wherein m is 0, 1, 2, 3 or 4; R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are independently selected from hydrogen, halogen, hydroxyl, alkyl, alkoxy, haloalkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, cyano, thiazolyl, triazolyl, imidazolyl, tetrazolyl, benzothiazolyl and benzimidazolyl; and pharmaceutically acceptable salts of said compounds and Esters, with the proviso that the compound is not 3-(4-phenyl-1,2,3,6-tetrahydro-1-pyridyl)-1-propanesulfonic acid; or wherein A is nitrogen or oxygen; R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together may be a natural or unnatural amino acid residue or its salt or ester; Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl; x is 0, 1, 2, 3 or 4; n is 0, 1, 2 , 3, 4, 5, 6, 7, 8, 9 or 10; R ⁴ , R ^4a , R ⁵ , R ^5a , R ⁶ , R ^6a , R ⁷ and R ^7a are each independently hydrogen, alkyl, mercaptoalkane radical, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino, tetrazolyl, the ring atoms to which ^R4 and ^R5 are attached Form a double bond together, or R ⁶ and R ⁷ form a double bond together with the ring atoms to which they are attached; m is 0, 1, 2, 3 or 4; R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are independently selected from From hydrogen, halogen, hydroxyl, alkyl, alkoxy, haloalkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, cyano, thiazolyl, triazolyl, imidazolyl, tetrazolyl , benzothiazolyl and benzimidazolyl; or wherein A is nitrogen or oxygen; R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together may be a natural or unnatural amino acid residue or its salt or ester; Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl; x is 0, 1, 2, 3 or 4; n is 0, 1, 2 , 3, 4, 5, 6, 7, 8, 9 or 10; aa is a natural or unnatural amino acid residue; m is 0, 1, 2 or 3; R ¹⁴ is hydrogen or a protecting group; R ¹⁵ is hydrogen, alkyl or aryl; or

Wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; A is oxygen or nitrogen; R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x - Q, or when A is nitrogen, A and ^R together may be a natural or unnatural amino acid residue or a salt or ester thereof; Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzo imidazolyl; x is 0, 1, 2, 3 or 4; R ¹⁹ is hydrogen, alkyl or aryl; Y ¹ is oxygen, sulfur or nitrogen; Y ² is carbon, nitrogen or oxygen; R ²⁰ is hydrogen, alkane group, amino, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl or benzimidazolyl; ^R is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, benzene and imidazolyl, or if Y ² is oxygen, R ²¹ is absent; R ²² is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, Triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, benzimidazolyl; or if Y ¹ is nitrogen, then R ²² is hydrogen, hydroxyl, alkoxy, or aryloxy; or if Y ¹ is oxygen or sulfur, then R ²² is absent; or if Y ¹ is nitrogen, then R ²² and R ²¹ can be linked together to form a cyclic moiety; or

wherein n is 2, 3 or 4; A is oxygen or nitrogen; R ¹¹ is hydrogen, a salt-forming cation, an ester-forming group, -(CH ₂ ) _x -Q, or when A is nitrogen, A and R ¹¹ together Can be natural or unnatural amino acid residues or salts or esters thereof; Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl or benzimidazolyl; x is 0, 1, 2, 3 or 4 ; G is a direct bond or oxygen, nitrogen or sulfur; z is 0, 1, 2, 3, 4 or 5; m is 0 or 1; ^R is selected from hydrogen, alkyl, mercaptoalkyl, alkenyl, alkyne Aroyl, aroyl, alkylcarbonyl, aminoalkylcarbonyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl and benzimidazolyl; each R ²⁵ independently selected from hydrogen, halogen, cyano, hydroxy, alkoxy, mercapto, amino, nitro, alkyl, aryl, carbocyclyl or heterocyclyl; And wherein the protein aggregation disease is not an amyloid disease. 2. A method of treating or preventing a protein aggregation disease in a subject, the method comprising Administering an effective amount of a compound to the subject suffering from a protein aggregation disease, so that the protein aggregation disease in the subject is treated or prevented, wherein the compound has one of the following formulas:

Wherein X is oxygen or nitrogen; Z is C=O, S(O) ₂ or P(O)OR ⁷ ; m and n are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R ¹ and R ⁷ are each independently hydrogen, metal ion, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, and together with X form a part of a natural or unnatural amino acid residue , or -(CH ₂ ) _p -Y; Y is hydrogen or a heterocyclic moiety selected from thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl and benzimidazolyl; p is 0, 1 , 2, 3 or 4; R ² is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl or alkoxycarbonyl; R ³ is hydrogen, Amino, cyano, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, substituted or unsubstituted aryl, heteroaryl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl or benzimidazolyl; or wherein each R is ^{independently} selected from hydrogen, halogen, hydroxyl, mercapto, amino, cyano, nitro, alkyl, aryl, carbocyclyl or heterocyclyl; J is absent, oxygen, nitrogen, sulfur or di Valence bonding moieties, including but not limited to lower alkylene, alkyleneoxy, alkyleneamino, alkylenethio, alkyleneoxyalkyl, alkyleneaminoalkyl, alkylenethio q is 1, 2, 3, 4 or 5; or

or

wherein X is oxygen or nitrogen; m and n are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; q is 1, 2, 3, 4 or 5; ^R is hydrogen, metal ion, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, or together with X form part of a natural or unnatural amino acid residue, or -(CH ₂ ) _p -Y; Y is hydrogen or a heterocyclic moiety selected from thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl and benzimidazolyl; p is 0, 1, 2, 3 or 4; ^R is hydrogen , alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl or alkoxycarbonyl; ^R is selected from hydrogen, halogen, amino, nitro, hydroxyl, carbonyl , mercapto, carboxyl, alkyl, alkoxy, alkoxycarbonyl, acyl, alkylamino, acylamino; q is an integer selected from 1-5; J is absent, oxygen, nitrogen, sulfur or a divalent bond Combining moieties, including but not limited to lower alkylene, alkyleneoxy, alkyleneamino, alkylenethio, alkyleneoxyalkyl, alkyleneaminoalkyl, alkylenethioalkane group, alkenyl, alkenyloxy, alkenylamino or alkenylthio; or

wherein ^R is a substituted or unsubstituted heterocyclic moiety; wherein the remaining substituents are as defined above; and wherein said protein aggregation disease is not an amyloid disease. 3. A method of treating or preventing a protein aggregation disease in a subject, the method comprising Administering an effective amount of a compound to the subject suffering from a protein aggregation disease, so that the protein aggregation disease in the subject is treated or prevented, wherein the compound has one of the following formulas: Wherein R ¹ and R ² are each independently a hydrogen atom or a substituted or unsubstituted aliphatic group or an aryl group; Z and Q are each independently a carbonyl group (C=O), a thiocarbonyl group (C=S), a sulfonyl group (SO ₂ ) or sulfoxide (S=O) group; k and m are 0 or 1, provided that when k is 1, R ¹ is not a hydrogen atom, and when m is 1, R ² is not a hydrogen atom; T is a linking group (such as an alkylene group), Y is a gene of the formula SO ₃ ^- X ⁺ , OSO ₃ ^- X ⁺ or SSO ₃ ^- X ⁺ ; wherein X ⁺ is a cationic group; or Wherein R ¹ is an alkyl group, an alkenyl group or a single-ring aromatic group, wherein the alkyl group can be substituted by a hydroxyl group; R ² is an alkyl group, an alkenyl group, a hydroxyalkyl group, a single-ring aromatic group or a hydrogen atom, Or R ¹ and R ² form a heterocyclic group with a condensed ring structure together with their attached nitrogen; k and m are 0, p and s are 1; T is an alkylene group; Y is SO ₃ X, X is a cationic group group; or Wherein R ¹ is an alkyl group, an alkenyl group or an aromatic group; R ² is a hydrogen atom, an alkyl group or an aromatic group, or R ¹ and R ² together form a heterocyclic group of a condensed ring structure; Z and Q are each independently is a carbonyl (C=O), thiocarbonyl (C=S), sulfonyl (SO ₂ ) or sulfoxide (S=O) group; k is 1 and m is 0 or 1, provided that when k is When 1, ^R1 is not a hydrogen atom, when m is 1, ^R2 is not a hydrogen atom; p and s are each 1; T is an alkylene group; Y is _SO3X , X is a cationic group; or Wherein R ¹ and R ² are alkyl, alkenyl or monocyclic aromatic groups, or R ¹ and R ² form a heterocyclic group with a condensed ring structure together with their attached nitrogen; k and m are 0, p and s is 1; T is an alkylene group; Y is _SO3X , X is a cationic group; or Wherein R ¹ is an alkyl group, an alkenyl group or a single-ring aromatic group, wherein the alkyl group can be substituted by a hydroxyl group; R ² is an alkyl group, an alkenyl group, a single-ring aromatic group or a hydrogen atom, wherein the alkyl group The group can be substituted by hydroxyl; or R ¹ and R ² form a heterocyclic group with a condensed ring structure together with their attached nitrogen; k and m are 0, p and s are 1; T is an alkylene group; Y is SO ₃ X, X is a cationic group; and pharmaceutically acceptable salts and prodrugs of said compounds, wherein said protein aggregation disease is not an amyloid disease. 4. A method of modulating a protein aggregation disease in a subject, the method comprising Administration of an effective amount of a compound according to any one of claims 1-3 to said subject suffering from a protein aggregation disorder, wherein said protein aggregation disorder is not an amyloid disease, results in modulation of said protein aggregation disorder in said subject. 5. A method of regulating harmful protein aggregation, said method comprising The unwanted protein aggregation is modulated by contacting the unwanted protein aggregate with an effective amount of the compound of any one of claims 1-3, wherein the unwanted protein aggregate is not associated with an amyloid disease. 6. A method of modulating cytotoxicity, said method comprising Said cytotoxicity is modulated by contacting a cell presenting a deleterious protein aggregate not associated with an amyloid disease with an effective amount of a compound according to any one of claims 1-3. 7. The method of any one of claims 1-4, wherein the subject is a mammal. 8. The method of claim 7, wherein said mammal is a human. 9. The method of any one of claims 1-4, wherein the protein aggregation disorder is selected from the group consisting of Pick's disease, corticobasal degeneration, progressive supranuclear palsy, amyotrophic lateral sclerosis/parkinsonism-dementia syndrome, Parkinson's disease (PD), Huntington's disease (HD), atrophic myotonia, dentate rubrum pallidal Lewy body atrophy, Friedrich's ataxia, fragile X syndrome, Fragile XE brain retardation, spinobulbar muscular atrophy, Wilson's disease, and spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), Ma-Joe disease (MJD or SCA3 ), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia type 17 (SCA17), chronic liver disease, cataract, serpinopathy, hemolytic Anemia, cystic fibrosis, neurofibromatosis type 2, demyelinating peripheral neuropathy, retinitis pigmentosa, Marfan syndrome, emphysema, idiopathic pulmonary fibrosis, argyrophilic granular dementia, corticobasal Nuclear degeneration, diffuse neurofibrillary tangles with calcifications, frontotemporal dementia/parkinsonism associated with chromosome 17, Hastings disease, Nieder's disease type C, and subacute sclerosing panencephalitis. 10. The method of any one of claims 1-4, wherein the protein aggregation disorder is a familial disorder. 11. The method of any one of claims 1-4, wherein the protein aggregation disorder is an idiopathic disorder. 12. The method of any one of claims 1-4, wherein the protein aggregation disorder is an alpha-synucleinopathy. 13. The method of any one of claims 1-4, wherein the protein aggregation disease is a tauopathy, with the proviso that the tauopathy is not Alzheimer's disease, prion disease, or cerebral amyloid angiopathy. 14. The method of claim 13, wherein the tauopathy is selected from the group consisting of amyotrophic lateral sclerosis/parkinsonism-dementia syndrome, argyrophilic granular dementia, corticobasal degeneration, diffuse neurofibrillary tangles with Calcifications, frontotemporal dementia/parkinsonism associated with chromosome 17, Ha-S disease, multiple system atrophy, Nie-dermatosis type C, Pick's disease, progressive supranuclear palsy, and subacute sclerosing Panencephalitis. 15. The method of claim 12, wherein the alpha-synucleinopathies are Parkinson's disease, Hierde syndrome, neurogenic orthostatic hypotension, Hier-Merde syndrome, and Parkinson-plus syndrome . 16. The method of claim 6, wherein said cytotoxicity involves neurotoxicity. 17. The method of claim 6, wherein said cytotoxicity is associated with inclusion bodies. 18. A method of treating or preventing tau-associated neurofibrillary tangles in a subject, the method comprising administering to said subject having neurofibrillary tangles associated with tau protein an effective amount of a compound of any one of claims 1-3 such that said neurofibrillary tangles associated with tau protein in said subject is treated or prevention. 19. A method of modulating tau-associated neurofibrillary tangles in a subject, the method comprising administering to said subject suffering from tau protein-associated neurofibrillary tangles an effective amount of a compound of any one of claims 1-3, such that said tau protein-associated neurofibrillary tangles are modulated in said subject . 20. A method of treating or preventing inclusion bodies containing alpha-synuclein NAC fragments in a subject, said method comprising administering to the subject having inclusion bodies containing α-synuclein NAC fragments an effective amount of the compound of any one of claims 1-3, so that the inclusion of α-synuclein NAC fragments in the subject be treated or prevented. 21. A method of modulating inclusion bodies containing alpha-synuclein NAC fragments in a subject, said method comprising administering to the subject having inclusion bodies containing α-synuclein NAC fragments an effective amount of the compound of any one of claims 1-3, so that the inclusion of α-synuclein NAC fragments in the subject body is adjusted. 22. The method of any one of claims 5 or 52-56, wherein the effective amount is effective to inhibit unwanted protein aggregation. 23. The method of claim 6, wherein the effective amount is effective to inhibit cytotoxicity. 24. The method of any one of claims 1-6, 18-21, or 26-27, wherein the method further comprises administering the compound in combination with a pharmaceutically acceptable carrier. 25. A method for modulating unwanted protein aggregation, said method comprising contacting protein aggregates with a compound such that said unwanted protein aggregation is regulated, wherein said compound is a compound according to any one of claims 1-3. 26. The method of any one of claims 5, 6, or 50-54, wherein the unwanted protein aggregates are extracellular aggregates. 27. The method of any one of claims 5, 6, or 50-54, wherein the unwanted protein aggregates are intracellular aggregates. 28. The method of any one of claims 5, 6, or 50-54, wherein the unwanted protein aggregates are cytoplasmic aggregates. 29. The method of any one of claims 5, 6, or 50-54, wherein the unwanted protein aggregates are nuclear aggregates. 30. The method of any one of claims 5, 6, or 50-54, wherein the unwanted protein aggregates are intramembrane aggregates. 31. The method of any one of claims 5, 6, or 50-54, wherein the unwanted protein aggregates are in the endoplasmic reticulum. 32. The method of any one of claims 5, 6, or 50-54, wherein the unwanted protein aggregates are in the trans-Golgi network. 33. The method of any one of claims 5, 6, or 50-54, wherein the unwanted protein aggregates are associated with aggregates. 34. The method of any one of claims 5, 6, or 50-54, wherein the unwanted protein aggregates are associated with misfolding of mature proteins. 35. The method of any one of claims 5, 6, or 50-54, wherein the unwanted protein aggregates are associated with improper degradation of proteins. 36. The method of any one of claims 5, 6, or 50-54, wherein the unwanted protein aggregates are associated with misfolded proteins that evade the ubiquitin-proteasome system. 37. The method of any one of claims 5, 6, or 50-54, wherein aggregation of the unwanted protein is inhibited. 38. The method of any one of claims 5, 6, or 50-54, wherein said unwanted protein aggregation is modulated by increasing degradation of said protein aggregates. 39. The method of any one of claims 5, 6, or 50-54, wherein said unwanted protein aggregation is modulated by increasing the rate of clearance of said protein aggregates. 40. The method of any one of claims 5, 6, or 50-54, wherein the unwanted protein aggregation is associated with fibrils, beta-sheets, or hydrophobic domains. 41. A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1-3 and a pharmaceutically acceptable carrier, wherein the effective amount is effective for treating a protein aggregation disorder. 42. The pharmaceutical composition of claim 41, wherein the protein aggregation disease is selected from the group consisting of Pick's disease, corticobasal degeneration, progressive supranuclear palsy, amyotrophic lateral sclerosis/parkinsonism-dementia syndrome, Parkinson's disease (PD), Huntington's disease (HD), atrophic myotonia, dentate rubrum pallidal Lewy body atrophy, Friedrich's ataxia, fragile X syndrome, fragile XE brain Developmental delay, spinobulbar muscular atrophy, Wilson's disease, and genes for spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), Mae-Joe disease (MJD or SCA3), Spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia type 17 (SCA17), chronic liver disease, cataracts, serpinopathy, hemolytic anemia, and Cystic fibrosis, Pick's disease, corticobasal degeneration, progressive supranuclear palsy, amyotrophic lateral sclerosis/parkinsonism-dementia syndrome, Parkinson's disease (PD), Huntington's disease (HD ), atrophic myotonia, dentate rubrum pallidal Lewy body atrophy, Friedrich's ataxia, fragile X syndrome, fragile XE brain retardation, Ma-Joe disease, spinal bulbar muscular atrophy syndrome, Wilson's disease, spinocerebellar ataxia, and cataracts. 43. A packaging composition for treating protein aggregation diseases with the compound of the present invention having targeted therapeutic activity, said composition comprising the compound of the present invention having said targeted therapeutic activity and related to the use of said compound of the present invention Description of the compound for treating said protein aggregation disorder. 44. A method of identifying a candidate compound that can be used to treat or prevent protein aggregation diseases, the method comprising: a) administering the test compound to a mouse model of protein aggregation disease; b) determining the effectiveness of said test compound in preventing, modulating, reducing or inhibiting the development of progressive degenerative changes associated with said protein aggregation disease in said mouse model; c) determining that the selected compound is a candidate compound for treating or preventing a protein aggregation disease. 45. The method of any one of claims 5, 6, or 50-54, wherein said unwanted protein aggregates are associated with at least one protein selected from the group consisting of alpha-synuclein, tau protein, NAC, huntingtin, DRPLA, Meningioma protein, Cytokeratin, Myelin 22, Rhodopsin, Dystrophin-1, Fibrillin-1, Ataxin-1, Ataxin-2, Ataxin -3. Ataxin-6, ataxin-7, ataxin-17, androgen receptor, surfactin-C and α1-antitrypsin. 46. A method for identifying candidate compounds useful for preventing, modulating, reducing or inhibiting unwanted protein aggregates, the method comprising: a) Exposure to harmful protein aggregates in vitro; b) determining the effectiveness of the test compound in preventing, modulating, reducing or inhibiting unwanted protein aggregates; c) determining that the selected compound is a candidate compound for treating or preventing a protein aggregation disease. 47. The method of claim 6, wherein said cells are neuronal cells or glial cells. 48. The method of claim 44, further comprising determining the effectiveness of said test compound in promoting clearance of said unwanted protein aggregates. 49. The method of claim 44, further comprising determining the effectiveness of said test compound in promoting degradation of said unwanted protein aggregate. 50. A method of modulating unwanted protein aggregation, said method comprising contacting the harmful protein aggregates with an effective amount of the compound of any one of claims 1-3, so that the removal of the harmful protein aggregates is regulated, thereby regulating the harmful protein aggregates, wherein the harmful protein aggregates are associated with amyloid Illness has nothing to do with it. 51. A method of modulating unwanted protein aggregation, said method comprising Making harmful protein aggregates contact with an effective amount of the compound of any one of claims 1-3, so that the cytotoxicity of said harmful protein aggregation is adjusted, thereby regulating harmful protein aggregation, wherein said harmful protein aggregates are associated with amyloid Proteinopathies are irrelevant. 52. A method of modulating unwanted protein aggregation, said method comprising Contacting a protein with a propensity to form a beta-sheet structure with an effective amount of a compound according to any one of claims 1-3 results in regulation of unwanted protein aggregation, wherein the protein aggregation disease is not an amyloid disease. 53. A method of modulating unwanted protein aggregation, said method comprising contacting a protein with a tendency to form a β-sheet structure with an effective amount of the compound according to any one of claims 1-3, so that the removal of said harmful protein aggregation is regulated, thereby regulating harmful protein aggregation, wherein said protein aggregation disease Not amyloid disease. 54. A method of modulating unwanted protein aggregation, said method comprising The protein that has the tendency to form a β-sheet structure is contacted with an effective amount of the compound of any one of claims 1-3, so that the cytotoxicity of the aggregation of the harmful protein is adjusted, thereby regulating the aggregation of the harmful protein, wherein the aggregation of the protein The disease is not amyloidosis.

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