NZ623046B2 - Caffeinated compounds and compositions for treatment of amyloid diseases and synucleinopathies - Google Patents

Abstract

Compounds comprising a caffeine core (1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione) molecule substituted with two phenyl groups which in turn are substituted with two hydroxyl groups each and their pharmaceutically acceptable salts for treatment of ?-amyloid diseases, such as observed in Alzheimer's disease and synucleinopathies, such as Parkinson's disease. Alternative the compound can be described as xanthine core (3,7-dihydro-purine-2,6-dione) substituted with benzyl groups which in turn are substituted with two hydroxyl groups each. isease and synucleinopathies, such as Parkinson's disease. Alternative the compound can be described as xanthine core (3,7-dihydro-purine-2,6-dione) substituted with benzyl groups which in turn are substituted with two hydroxyl groups each.

Description

CAFFEINATED COMPOUNDS AND COMPOSITIONS FOR ENT OF AMYLOID DISEASES AND SYNUCLEINOPATHIES TECHNICAL FIELD This invention relates to compounds of the invention and pharmaceutically acceptable salts, their synthesis, ceutical compositions containing them, and their use in the treatment of b amyloid diseases, such as observed in Alzheimer's disease, and eopathies, such as Parkinson's disease, and in the manufacture of medicaments for such treatment.

BACKGROUND OF THE INVENTION Alzheimer's disease is characterized by the accumulation of a 39-43 amino acid peptide termed the b-amyloid protein or Ab, in a lar form, existing as extracellular amyloid plaques and as amyloid within the walls of cerebral blood vessels. Fibrillar Ab amyloid deposition in Alzheimer's disease is believed to be detrimental to the patient and eventually leads to toxicity and neuronal cell death, teristic hallmarks of Alzheimer's disease. Accumulating evidence ates amyloid, and more specifically, the formation, deposition, accumulation and/or persistence of A b fibrils, as a major causative factor of Alzheimer's e enesis. In addition, besides Alzheimer's disease, a number of other amyloid diseases involve formation, deposition, accumulation and tence of Ab fibrils, including Down's syndrome, disorders involving hilic angiopathy, such as but not limited to, tary cerebral hemorrhage of the Dutch type, and cerebral b-amyloid angiopathy.

Parkinson's disease is another human disorder characterized by the formation, deposition, accumulation and/or persistence of abnormal fibrillar protein deposits that demonstrate many of the characteristics of amyloid. In son's disease, an lation of asmic Lewy bodies consisting of filaments of a-synuclein are believed important in the pathogenesis and as therapeutic targets. New agents or compounds able to inhibit a-synuclein formation, deposition, accumulation and/or persistence, or disrupt pre-formed a-synuclein fibrils (or ns thereof) are regarded as potential therapeutics for the treatment of Parkinson's and related synucleopathies. A 35 amino acid fragment of a-synuclein that has the ability to form amyloid-like fibrils either in vitro or as observed in the brains of patients with Parkinson's disease. The fragment of a-synuclein is a relative important therapeutic target as this portion of a-synuclein is believed crucial for formation of Lewy bodies as observed in all patients with son's disease, synucleopathies and related disorders. In on, the a-synuclein protein which forms fibrils, and is Congo red and Thioflavin S positive (specific stains used to detect amyloid fibrillar deposits), is found as part of Lewy bodies in the brains of patients with Parkinson's disease, Lewy body disease (Lewy in ch der Neurologie, M. Lewandowski, ed., Springer, Berlin pp. 920-933, 1912; Pollanen et al, J. Neuropath.

Exp. Neurol. 52:183-191, 1993; Spillantini et al, Proc. Natl. Acad. Sci. USA 95:6469- 6473, 1998; Arai et al, Neurosci. Lett. 259:83-86, 1999), multiple system atrophy (Wakabayashi et al, Acta ath. 96:445-452, 1998), dementia with Lewy bodies, and the Lewy body variant of Alzheimer's disease. In son's disease, fibrils develop in the brains of patients with this disease which are Congo red and Thioflavin S positive, and which contain predominant beta-pleated sheet secondary structure.

Amyloid as a therapeutic target for Alzheimer's disease Alzheimer's disease also puts a heavy economic burden on society. A recent study estimated that the cost of caring for one Alzheimer's disease patient with severe cognitive impairments at home or in a nursing home, is more than $47,000 per year (A Guide to Understanding mer's Disease and Related Disorders). For a disease that can span from 2 to 20 years, the overall cost of Alzheimer's disease to families and to society is staggering. The annual economic toll of Alzheimer's disease in the United States in terms of health care expenses and lost wages of both patients and their caregivers is ted at $80 to $100 billion (2003 Progress Report on Alzheimer's Disease).

Tacrine hydrochloride ("Cognex"), the first FDA approved drug for mer's disease, is a acetylcholinesterase inhibitor (Cutler and Sramek, N. Engl. J. Med. 328:808 810, 1993). However, this drug has showed limited s in producing cognitive ement in Alzheimer's disease patients and initially had major side effects such as liver ty. The second FDA approved drug, donepezil ("Aricept"), which is also an acetylcholinesterase inhibitor, is more effective than tacrine, by demonstrating slight ive improvement in Alzheimer's e ts (Barner and Gray, Ann. cotherapy 32:70-77, 1998; Rogers and Friedhoff, Eur. Neuropsych. 8:67-75, 1998), but is not believed to be a cure. Therefore, it is clear that there is a need for more effective ents for Alzheimer's disease patients.

Alzheimer's disease is characterized by the deposition and lation of a 39- 43 amino acid peptide termed the beta-amyloid protein, Ab or b/A4 (Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890, 1984; Masters et al., Proc. Natl. Acad. Sci.

USA 5-4249, 1985; Husby et al., Bull. WHO 71:105-108, 1993). A b is derived by protease cleavage from larger precursor proteins termed b-amyloid precursor proteins (APPs) of which there are several alternatively spliced variants. The most abundant forms of the APPs include proteins consisting of 695, 751 and 770 amino acids (Tanzi et al., Nature -530, 1988).

The small Ab peptide is a major component that makes up the amyloid deposits of "plaques" in the brains of ts with Alzheimer's disease. In addition, Alzheimer's disease is characterized by the presence of numerous neurofibrillary "tangles", consisting of paired l filaments which abnormally accumulate in the neuronal cytoplasm (Grundke-Iqbal et al., Proc. Natl. Acad. Sci. USA 83:4913-4917, 1986; Kosik et al., Proc.

Natl. Acad. Sci. USA 83:4044-4048, 1986; Lee et al., Science 251:675-678, 1991). The pathological hallmark of mer's disease is therefore the presence of "plaques" and "tangles", with b-amyloid being deposited in the central core of the plaques. The other major type of lesion found in the Alzheimer's disease brain is the accumulation of b- d in the walls of blood vessels, both within the brain parenchyma and in the walls of meningeal vessels that lie outside the brain. The b-amyloid deposits localized to the walls of blood vessels are referred to as cerebrovascular amyloid or congophilic angiopathy (Mandybur, J. ath. Exp. . 45:79-90, 1986; Pardridge et al., J.

Neurochem. 49:1394-1401, 1987) For many years there has been an ongoing scientific debate as to the importance of "b-amyloid" in Alzheimer's disease, and whether the "plaques" and "tangles" characteristic of this disease were a cause or merely a consequence of the e. Within the last few years, studies now indicate that b-amyloid is indeed a causative factor for mer's disease and should not be regarded as merely an innocent bystander. The Alzheimer's A b protein in cell culture has been shown to cause degeneration of nerve cells within short periods of time (Pike et al., Br. Res. 563:311-314, 1991; J. Neurochem. 64:253-265, 1995). Studies suggest that it is the fibrillar structure (consisting of a predominant b-pleated sheet secondary structure), which is responsible for the neurotoxic effects. Ab has also been found to be neurotoxic in slice es of hippocampus (Harrigan et al., Neurobiol. Aging 16:779-789, 1995) and induces nerve cell death in transgenic mice (Games et al., Nature 373:523-527, 1995; Hsiao et al., e 274:99- 102, 1996). Injection of the Alzheimer's Ab into rat brain also causes memory impairment and neuronal dysfunction (Flood et al., Proc. Natl. Acad. Sci. USA 88:3363- 3366, 1991; Br. Res. 663:271-276, 1994).

Probably, the most convincing evidence that Ab amyloid is directly involved in the pathogenesis of Alzheimer's disease comes from c studies. It was discovered that the tion of Ab can result from mutations in the gene encoding, its precursor, b- d precursor protein (Van Broeckhoven et al., Science 248:1120-1122, 1990; Murrell et al., Science 254:97-99, 1991; Haass et al., Nature Med. 1:1291-1296, 1995).

The identification of mutations in the beta-amyloid precursor protein gene that cause early onset al Alzheimer's disease is the strongest argument that amyloid is central to the pathogenetic process underlying this disease. Four reported disease-causing mutations have been discovered which demonstrate the importance of Ab in causing familial Alzheimer's disease (reviewed in Hardy, Nature Genet. 234, 1992). All of these studies suggest that ing a drug to reduce, eliminate or prevent fibrillar Ab formation, deposition, lation and/or persistence in the brains of human patients will serve as an effective therapeutic.

A y of other human es also demonstrate amyloid deposition and usually involve systemic organs (i.e. organs or tissues lying outside the central nervous system), with the amyloid accumulation leading to organ dysfunction or failure. These amyloid diseases ssed below) leading to marked amyloid accumulation in a number of different organs and tissues, are known as systemic amyloidoses. In other amyloid diseases, single organs may be affected such as the pancreas in 90% of patients with type 2 diabetes. In this type of amyloid disease, the beta-cells in the islets of Langerhans in pancreas are believed to be yed by the accumulation of fibrillar amyloid deposits consisting primarily of a protein known as islet amyloid polypeptide (IAPP). Inhibiting or reducing such IAPP amyloid fibril formation, deposition, accumulation and persistence is believed to lead to new effective treatments for type 2 diabetes. In Alzheimer's disease, Parkinson's and "systemic" d diseases, there is currently no cure or effective treatment, and the patient usually dies within 3 to 10 years from disease onset.

The amyloid diseases (amyloidoses) are classified ing to the type of amyloid n present as well as the underlying e. Amyloid es have a number of common characteristics including each amyloid ting of a unique type of amyloid protein. The amyloid diseases include, but are not d to, the amyloid associated with mer's disease, Down's syndrome, hereditary cerebral hemorrhage with dosis of the Dutch type, dementia pugilistica, inclusion body myositosis (Askanas et al, Ann. Neurol. 43:521-560, 1993) and mild cognitive impairment (where the specific amyloid is referred to as myloid protein or Ab), the amyloid associated with chronic inflammation, various forms of malignancy and al Mediterranean Fever (where the specific amyloid is referred to as AA amyloid or inflammationassociated amyloidosis) (also known as systemic AA amyloidosis), the amyloid associated with le myeloma and other B-cell dyscrasias (where the specific amyloid is referred to as AL amyloid), the amyloid associated with type 2 diabetes (where the specific amyloid protein is referred to as amylin or islet amyloid polypeptide or IAPP), the amyloid associated with the prion diseases including Creutzfeldt- Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie (where the specific amyloid is referred to as PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (where the ic amyloid is referred to as a 2-microglobulin amyloid), the amyloid associated with senile cardiac amyloidosis and Familial dotic Polyneuropathy (where the specific amyloid is referred to as transthyretin or prealbumin), and the amyloid ated with endocrine tumors such as medullary carcinoma of the thyroid (where the specific amyloid is referred to as variants of citonin). In addition, the a-synuclein protein which forms amyloid-like fibrils, and is Congo red and Thioflavin S positive (specific stains used to detect d fibrillar deposits), is found as part of Lewy bodies in the brains of patients with Parkinson's disease, Lewy body disease (Lewy in Handbuch der Neuwlogie, M. Lewandowski, ed., Springer, Berlin pp. 920-933, 1912; Pollanen et al, J. Neuropath. Exp. Neurol. 52:183- 191, 1993; Spillantini et al, Proc. Natl. Acad. Sci. ¾4_95:6469-6473, 1998; Arai et al, Neurosci. Lett. 259:83-86, 1999), multiple system y (Wakabayashi et al, Acta ath. 96:445-452, 1998), dementia with Lewy bodies, and the Lewy body variant of Alzheimer's disease. For purposes of this disclosure, Parkinson's disease, due to the fact that fibrils develop in the brains of patients with this e (which are Congo red and Thioflavin S positive, and which contain predominant beta-pleated sheet secondary structure), is now regarded as a disease that also displays the characteristics of an amyloid-like disease.

Systemic amyloidoses which include the amyloid associated with chronic inflammation, various forms of malignancy and familial Mediterranean fever (i.e. AA amyloid or inflammation-associated amyloidosis) (Benson and Cohen, Arth. Rheum. 22:36-42, 1979; Kamei et al, Acta Path. Jpn. -133, 1982; McAdam et al., Lancet 573, 1975; Metaxas, Kidney Int. -685, 1981), and the amyloid associated with multiple myeloma and other B-cell dyscrasias (i.e. AL amyloid) a et al., J.

Histochem. em. 5, 1971), as examples, are known to involve amyloid deposition in a variety of different organs and tissues generally lying outside the central s system. Amyloid deposition in these diseases may occur, for example, in liver, heart, spleen, gastrointestinal tract, kidney, skin, and/or lungs (Johnson et al, N . Engl. J.

Med. 321:513-518,1989). For most of these amyloidoses, there is no apparent cure or effective treatment and the consequences of amyloid deposition can be detrimental to the patient. For example, amyloid deposition in the kidney may lead to renal failure, whereas amyloid deposition in the heart may lead to heart failure. For these patients, amyloid accumulation in systemic organs leads to eventual death generally within 3-5 years. Other amyloidoses may affect a single organ or tissue such as observed with the A b amyloid deposits found in the brains of patients with Alzheimer's disease and Down's syndrome: the PrP d deposits found in the brains of patients with Creutzfeldt-Jakob disease, ann-Straussler syndrome, and kuru; the islet amyloid (IAPP) deposits found in the islets of Langerhans in the pancreas of 90% of patients with type 2 diabetes (Johnson et al, N . Engl. J. Med. 321:513-518, 1989; Lab. Invest. 66:522 535, 1992); the a oglobulin amyloid deposits in the medial nerve leading to carpal tunnel me as observed in patients undergoing long-term hemodialysis (Geyjo et al, Biochem.

Biophys. Res. Comm. 129:701-706, 1985; Kidney Int. 30:385-390, 1986); the prealbumin/ transthyretin amyloid observed in the hearts of patients with senile cardiac amyloid; and the prealbumin/transthyretin amyloid observed in peripheral nerves of ts who have familial amyloidotic polyneuropathy (Skinner and Cohen, m. Biophys. Res. Comm. 99:1326-1332, 1981; Saraiva et al, J. Lab. Clin. Med. 102:590-603, 1983; J. Clin. Invest. 74:104-119, 1984; Tawara et al, J. Lab. Clin. Med. 98:811-822, 1989).

Parkinson's Disease and Synucleopathies Parkinson's disease is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies (Lewy in Handbuch der Neurologie, M. Lewandowski, ed., Springer, Berlin, pp. 920-933, 1912; Pollanen et al., J. ath. Exp. Neurol. 52:183-191, 1993), the major components of which are filaments consisting of a-synuclein (Spillantini et al., Proc. Natl. Acad. Sci. USA 9-6473, 1998; Arai et al., Neurosci. Lett. 259:83-86, 1999), an 140-amino acid protein (Ueda et al., Proc. Natl. Acad. Sci. USA 90:11282-11286, 1993). Two dominant mutations in a-synuclein causing familial early onset Parkinson's disease have been described suggesting that Lewy bodies bute mechanistically to the degeneration of neurons in Parkinson's disease and related disorders (Polymeropoulos et al., Science 276:2045-2047, 1997; Kruger et al., Nature Genet. 18:106-108, 1998). Recently, in vitro studies have demonstrated that recombinant a-synuclein can indeed form Lewy body-like fibrils (Conway et al., Nature Med. 4:1318-1320, 1998; oto et al., Brain Res. 799:301-306, 1998; Nahri et al., J. Biol. Chem. 274:9843-9846, 1999). Most importantly, both Parkinson's disease-linked clein mutations accelerate this aggregation process, demonstrating that such in vitro studies may have relevance for son's e pathogenesis. Alpha-synuclein aggregation and fibril formation fulfills the ia of a nucleation-dependent polymerization process (Wood et al., J. Biol. Chem. 274:19509-19512, 1999). In this regard a-synuclein fibril formation resembles that of Alzheimer's b-amyloid protein (A b) s. Alpha-synuclein recombinant protein, and hoh-A b component (known as NAC), which is a 35-amino acid peptide fragment of asynuclein , both have the ability to form fibrils when incubated at 37°C, and are positive with amyloid stains such as Congo red (demonstrating a red/green birefringence when viewed under polarized light) and avin S strating positive fluorescence) (Hashimoto et al., Brain Res. 799:301-306, 1998; Ueda et al., Proc. Natl. Acad. Sci. USA 90:11282-11286, 1993).

Synucleins are a family of small, presynaptic neuronal proteins composed of a-, b-, and g-synucleins, of which only clein aggregates have been associated with several neurological diseases (Ian et al., Clinical c. Res. 1:445-455, 2001; Trojanowski and Lee, Neurotoxicology 23 60, 2002). The role of synucleins (and in particular, alpha-synuclein) in the etiology of a number of neurodegenerative diseases has ped from several observations. Pathologically, synuclein was identified as a major component of Lewy bodies, the hallmark inclusions of Parkinson's disease, and a fragment thereof was isolated from d plaques of a different neurological disease, Alzheimer's disease. Biochemically, recombinant a-synuclein was shown to form fibrils that tulated the ultrastructural features of alpha-synuclein isolated from patients with dementia with Lewy bodies, Parkinson's disease and multiple system atrophy.

Additionally, the identification of mutations within the synuclein gene, albeit in rare cases of familial Parkinson's disease, demonstrated an unequivocal link between synuclein pathology and neurodegenerative diseases. The common involvement of asynuclein in a spectrum of diseases such as Parkinson's e, dementia with Lewy bodies, multiple system y and the Lewy body variant of Alzheimer's disease has led to the classification of these diseases under the umbrella term of "synucleopathies".

Parkinson's disease a-synuclein fibrils, and the A b fibrils of Alzheimer's disease, both consist of a predominantly b-pleated sheet structure. nds found to t mer's disease Ab amyloid fibril formation have also been shown to be effective in the inhibition of a-synuclein fibril ion, as illustrated in the Examples of the present invention. These compounds would therefore also serve as therapeutics for Parkinson's disease and other synucleopathies, in addition to having efficacy as a therapeutic for Alzheimer's disease.

Parkinson's disease and Alzheimer's e are characterized by the inappropriate lation of insoluble aggregates comprised primarily of misfolded proteins that are enriched in b-pleated sheet secondary structure (reviewed in Cohen et al., Nature 426 09, 2003; Chiti et al., Annu. Rev. Biochem., 75:333-366, 2006). In Parkinson's disease, -synuclein is the major constituent of these aggregates, as part of Lewy Bodies, and mutations in -synuclein that increase its propensity to misfold and ate are observed in familial Parkinson's disease (Polymeropoulos et al., Science 276 :1197-1199, 1997; Papadimitriou et al., Neurology 52:651-654, 1999).

Mitochondrial dysfunction, specifically as a result of impairment at complex I of the electron transport chain, is also a common feature of Parkinson's disease (Schapira et al., J. Neurochem., 54:823-827, 1990; reviewed in Greenamyre et al., IUBMB Life, 52:135- 141, 2001). Direct evidence for ondrial deficits in the etiology of Parkinson's disease came first from the observation that MPP+ (l-methylphenyl-2,3- dihydropyridinium), the active metabolite of the parkinsonism toxin N-methylphenyl- 1,2,3,6-tetrahydropyridine (MPTP), inhibits complex I (Nicklas et al., Life Sci., 36:2503- 2508, 1985). uently, rotenone, another complex I inhibitor, was shown to be an ed model for a-synuclein aggregation because it reproduces the above-mentioned a -synuclein-positive intracytoplasmic aggregates, in addition to the behavioral changes and loss of dopaminergic neurons seen in the MPTP model. ne ty of this type is seen in multiple model systems including rats (Betarbet et al., Nat. Neuwsci., 3:1301-1306, 2000; Panov et al., J. Biol. Chem., 280 :42026-42035, 2005), rat brain slices (Sherer et al., J. Neuwsci., 23:10756-10764, 2003; Testa et al., Mol. Brain Res., 134 :109-118, 2005), C. elegans (Ved et al., J. Biol. Chem., 280 :42655-42668, 2005) and cultured cells (Sherer et al., J. Neuwsci., 22:7006-7015, 2002) and has been shown to be a consequence of increased oxidative damage ing from complex I inhibition.

To better understand the relationship of ive damage to mutant a -synuclein pathogenesis, a neuroblastoma cell line (using BE-M17 cells) has been established in the art that overexpresses A53T a-synuclein. In these cells, A53T a -synuclein aggregates in response to a variety of oxidative stress-inducing agents and potentiates mitochondrial dysfunction and cell death (Ostrerova-Golts et al., J. Neuwsci., 20:6048-6054, 2000).

These cells are amenable to ne treatment as an oxidative stress inducer and hence, are particularly useful for testing agents that might t a -synuclein aggregation/fibrillogenesis .

Discovery and identification of new compounds or agents as potential therapeutics to arrest amyloid formation, deposition, accumulation and/or persistence that occurs in Alzheimer's disease, and Parkinson's disease, are desperately sought.

Y OF THE INVENTION This invention relates to the following compounds and other modification and derivates of these compounds and their use in the treatment of amyloid es and synucleopathies.

Compounds of this invention have the l a where: R are independently substituted with hydrogen, methyl and benzyl groups, and R4 is substituted with a hydrogen or phenyl group, wherein the phenyl or benzyl groups are independently tuted with up to 2 groups selected from H, OH, F, CI, Br, glucuronide, sulfate, cyano, methyl, NH2, SH, CH2OH, CN, CF3, NHSO2CH3, N(CH3)2, NHCH3, N(CN)2, NHCN, C(CN)3, NH(C=O)NH2, NH(C=O)CH3, NH2, (C=NOH)NH2, O(C=O)OCH3, and )H and pharmaceutically acceptable salts thereof.

Compounds of this invention have the general formula above where is substituted with a benzyl group, R2 or R3 are independently substituted with either a methyl or benzyl group and R is substituted with a hydrogen and wherein the benzyl groups are each substituted with two hydroxyl groups.

Compounds of the invention include, but are not limited to the ing: nd PD 150 Compound PD 151 Compound PD 152 Compound PD 153 Compound PD 154 In another aspect, this invention is pharmaceutical compositions comprising a compound of this invention and a pharmaceutically acceptable excipient; and ceutical compositions sing a pharmaceutically acceptable excipient and, as the sole active ingredient, a compound of the ion.

In another aspect, this invention is a method of treating an amyloid disease or eopathy in a , especially a human, by administration of a therapeutically effective amount of a compound of the invention, for example as a pharmaceutical composition.

In another aspect, this invention is the use of a nd of the invention in the manufacture of a medicament for the treatment of an amyloid e or synucleopathy.

In another aspect, this invention is a method of preparation of the compounds of the invention, i.e. the compounds of the formula or list above, and/or the formation of pharmaceutically acceptable salts thereof.

In another aspect, this invention is a method of treatment of A b, IAPP, other amyloids, and a-synuclein or NAC fibrillogenesis, in an in vitro environment. The method includes the step of administering into the in vitro environment a therapeutically effective amount of a compound of this invention. Preferably the compound is selected from the groups described below with respect to their activity against Ab, IAPP, and NAC.

Also provided are any pharmaceutically-acceptable derivatives, including salts, esters, enol ethers or esters, s, ketals, orthoesters, hemiacetals, hemiketals, solvates, hydrates or prodrugs of the compounds.

Methods using such nds and itions for ting, disaggregating and causing removal, reduction or clearance of beta amyloid or a-synuclein fibrils or aggregates are provided thereby ing new treatments for synucleopathies.

Also provided are methods for treatment, prevention or amelioration of one or more symptoms of amyloid and synuclein diseases or synucleopathies. In one embodiment, the methods inhibit or prevent amyloid or a-synuclein fibril formation, inhibit or prevent d or a-synuclein fibril growth, and/or cause disassembly, disruption, and/or disaggregation of preformed amyloid or a-synuclein aggregates and amyloid or a-synuclein associated protein deposits. Amyloid diseases include, but are not limited to Alzheimer's disease, type II diabetes, ic AA and prion diseases.

Synuclein diseases include, but are not limited to Parkinson's disease, familial Parkinson's disease, Lewy body disease, dementia with Lewy bodies, multiple system atrophy, and the Parkinsonism-dementia complex of Guam.

DETAILED DESCRIPTION OF THE INVENTION Definitions In this application, the ing terms shall have the following meanings, without regard to whether the terms are used variantly elsewhere in the literature or otherwise in the known art.

As used herein "Amyloid diseases" or "amyloidoses" are diseases associated with the formation, deposition, accumulation, or persistence of Ab amyloid fibrils. Such diseases include, but are not limited to mer's e, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis of the Dutch type, and cerebral b- amyloid angiopathy. Other amyloid diseases such as systemic AA amyloidosis and IAPP amyloidosis of type II diabetes are also d diseases.

As used herein, "Synuclein diseases" or "synucleopathies" are diseases associated with the formation, deposition, accumulation, or persistence of a-synuclein fibrils. Such diseases include, but are not limited to Parkinson's disease, familial Parkinson's disease, Lewy body disease, dementia with Lewy bodies, multiple system y, and the Parkinsonism-dementia complex of Guam.

"Fibrillogenesis" refers to the formation, tion, accumulation and/or tence of b-amyloid fibrils, filaments, inclusions, deposits, as well as a-synuclein fibrils, filaments, inclusions, deposits or the like.

"Inhibition of fibrillogenesis" refers to the inhibition of formation, deposition, lation and/or persistence of such a b-amyloid fibrils or clein fibril-like deposits.

"Disruption of fibrils or fibrillogenesis" refers to the disruption of pre-formed b- amyloid or a-synuclein fibrils, that usually exist in a pre-dominant b-pleated sheet secondary structure. Such disruption by compounds provided herein may involve marked reduction or embly of amyloid or ein fibrils as assessed by s methods such as Thioflavin T fluorometry, Congo red binding, circular dichroism a, thioflavin S and cell based assays such as a-synuclein aggregation and XTT cytotoxicity assays and as demonstrated by the Examples presented in this application.

"Neuroprotection" or "neuroprotective" refers to the ability of a compound to protect, reduce, alleviate, rate, and/or attenuate damage to nerve cells (neurodegeneration) .

"Mammal" includes both humans and man mammals, such as companion animals (cats, dogs, and the like), laboratory animals (such as mice, rats, guinea pigs, and the like) and farm s (cattle, horses, sheep, goats, swine, and the like).

"Pharmaceutically acceptable excipient" means an excipient that is tionally useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use or for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an l composition, gaseous.

A "therapeutically effective amount" means the amount that, when administered to a subject or animal for treating a disease, is sufficient to affect the desired degree of ent, tion or symptom ration for the disease. A "therapeutically effective amount" or a "therapeutically effective dosage" in certain embodiments inhibits, s, disrupts, disassembles oid or a-synuclein fibril formation, deposition, accumulation and/or persistence, or treats, prevents, or ameliorates one or more symptoms of a disease associated with these conditions, such as an amyloid disease or a synucleinopathy, in a measurable amount in one embodiment, by at least 20%, in other embodiment, by at least 40%, in other embodiment by at least 60%, and in still other embodiment by at least 80%, relative to an untreated subject. Effective amounts of a compound provided herein or composition f for treatment of a mammalian subject are about 0.1 to about 1000 mg/Kg of body weight of the subject/day, such as from about 1 to about 100 mg/Kg/day, in other embodiment, from about 10 to about 500 mg/Kg/day.

A broad range of sed composition dosages are believed to be both safe and effective.

The term ined release component" is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, microspheres, or the like, or a combination thereof, that facilitates the sustained release of the active ingredient.

If the complex is water-soluble, it may be formulated in an appropriate buffer, for example, phosphate buffered saline, or other physiologically ible solutions.

Alternatively, if the resulting complex has poor solubility in aqueous solvents, then it may be formulated with a non-ionic surfactant such as Tween, or polyethylene glycol.

Thus, the compounds and their physiological solvents may be formulated for administration by tion or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, or rectal stration, as examples.

As used herein, pharmaceutically acceptable derivatives of a compound include salts, , enol ethers, enol esters, acetals, ketals, sters, hemiacetals, hemiketals, solvates, hydrates or prodrugs thereof. Such derivatives may be readily prepared by those of skill in this art using known methods for such tization. The compounds produced may be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs. Pharmaceutically acceptable salts include, but are not limited to, amine salts, such as but not limited to N ,N'- dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N- benzylphenethylamine, 1-para-chlorobenzylpyrrolidin- 1'-ylmethyl-benzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, m and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of l acids, such as but not limited to hydrochlorides and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates. Pharmaceutically acceptable esters e, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and cyclyl esters of acidic groups, ing, but not d to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids and c acids. Pharmaceutically acceptable enol ethers include, but are not limited to, derivatives of formula C=C(OR) where R is hydrogen, alkyl, alkenyl, l, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl. Pharmaceutically acceptable enol esters include, but are not limited to, derivatives of formula C=C(OC(0)R) where R is hydrogen, alkyl, alkenyl, l, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl. ceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.

As used herein, ent means any manner in which one or more of the symptoms of a disease or er are ameliorated or otherwise beneficially altered. ent of a disease also includes preventing the disease from occurring in a subject that may be predisposed to the disease but does not yet experience or exhibit ms of the disease (prophylactic treatment), ting the disease (slowing or arresting its pment), providing relief from the symptoms or side-effects of the disease (including palliative treatment), and relieving the disease (causing regression of the e), such as by disruption of pre-formed b-amyloid or a-synuclein fibrils. As used herein, amelioration of the symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any ing, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.

As used herein, inhibition of a-synuclein fibril formation, deposition, accumulation, aggregation, and/or persistence is ed to be effective treatment for a number of diseases involving a-synuclein, such as Parkinson's disease, Lewy body disease and multiple system atrophy.

As used herein, inhibition of amyloid fibril formation, deposition, accumulation, aggregation, and/or persistence is believed to be effective treatment for a number of diseases involving amyloid, such as Alzheimer' s disease, type II es and systemic AA amyloidosis.

As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized by one or more steps or processes or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. To produce a prodrug, the pharmaceutically active nd is ed such that the active compound will be regenerated by metabolic processes. The prodrug may be designed to alter the metabolic ity or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design gs of the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392).

Chemical structures for some of the compounds of this invention are shown. The names of the compounds are variously IUPAC names [names derived according to the accepted IUPAC (International Union of Pure and Applied Chemistry) system ished by the coalition of the Commission on Nomenclature of c Chemistry and the Commission on Physical Organic Chemistry, as can be found at http://www.chem.qmul.ac.uk/iupacL names derived from IUPAC names by addition or tution (for example, by the use of "3,4-methylenedioxyphenyl" derived from "phenyl" d of "benzo[l,3]dioxolyl"), and names derived from the names of reactants. However, the names used are explicitly equated to chemical structures, and are believed to be readily understood by a person of ry skill in the art.

"A pharmaceutical agent" or "pharmacological agent" or aceutical composition" refers to a nd or combination of compounds used for treatment, preferably in a pure or near pure form. In the specification, pharmaceutical or pharmacological agents include the compounds of this invention. The compounds are desirably purified to 80% homogeneity, and preferably to 90% homogeneity. Compounds and compositions purified to 99.9% homogeneity are believed to be advantageous. As a test or mation, a suitable neous compound on HPLC would yield, what those skilled in the art would identify as a single sharp-peak band.

It is to be understood that the compounds provided herein may contain chiral s. Such chiral centers may be of either the (R) or (S) configuration, or may be a e thereof. Thus, the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures. It is to be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of skill in the art will recognize that administration of a compound in its (R) form is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its (S) form.

As used herein, ntially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometry (MS), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not ably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to e ntially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the nd.

As used herein, the abbreviations for any tive groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. -944).

Compounds of the invention The compounds of this invention have the general formula where: R are independently substituted with hydrogen, methyl and benzyl groups, and R4 is tuted with a hydrogen, or phenyl group, wherein the phenyl or benzyl groups are independently substituted with up to 2 groups selected from H, OH, F, CI, Br, glucuronide, sulfate, cyano, methyl, NH2, SH, CH2OH, CN, CF3, NHS0 2CH3, N(CH3)2, NHCH3, N(CN)2, NHCN, C(CN)3, )NH 2, NH(C=0)CH 3, (C=NH)NH 2, (C=NOH)NH2, 0(C=0)OCH 3, and NH(C=0)H and pharmaceutically acceptable salts thereof.

The compounds of this invention are selected from the group consisting of: , and The compounds of this invention have the formula shown above where Ri is substituted with a benzyl group, R2 or R3 are ndently substituted with either a methyl or benzyl group and R4 is substituted with a hydrogen and wherein the benzyl groups are each substituted with two hydroxyl groups.

The compounds of this invention are incorporated into pharmaceutical compositions sing any of the compounds of this ion disclosed herein and a pharmaceutically acceptable excipient.

This invention also provides a method of treating the formation, deposition, accumulation, or persistence of amyloid or a-synuclein fibrils, comprising treating the fibrils with an effective amount of any of the compounds of this invention disclosed herein.

This invention also provides a method of treating an amyloid disease or a synucleinopathy in a mammal suffering rom, comprising administration of a therapeutically effective amount of any of the compounds of this invention disclosed herein.

This invention provides that the d disease is selected from the group of diseases consisting of Alzheimer's e, type II diabetes, ic AA amyloidosis, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis of the Dutch type, and cerebral b-amyloid angiopathy.

This invention provides that the amyloid disease is Alzheimer's disease.

This invention provides that the synucleinopathy is selected from the group consisting of Parkinson's disease, familial Parkinson's disease, and Lewy body disease, the Lewy body variant of Alzheimer's disease, ia with Lewy bodies, multiple system atrophy, and the Parkinsonism-dementia complex of Guam.

This invention es that the synucleinopathy is Parkinson's e.

This invention provides that in the methods of treating an amyloid disease or a synucleinopathy that the compounds of this invention are administered in an amount between 0.1 mg/Kg/day and 1000 day.

This invention provides that in the methods of treating an amyloid disease or a synucleinopathy that the compounds of this invention are stered in an amount between 1 mg/Kg/day and 100 mg/Kg/day.

This ion provides that in the methods of treating an amyloid disease or a synucleinopathy that the compounds of this invention are administered in an amount n 10 mg/Kg/day and 100 mg/Kg/day.

This invention also provides an article of manufacture, comprising packaging material, the compounds of this invention, or a pharmaceutically acceptable salts thereof, contained within ing material, which is used for treating the formation, deposition, accumulation, or tence of b-amyloid or a-synuclein fibrils and/or aggregates, and a label that indicates that the compound or pharmaceutically able salt thereof is used for treating the formation, deposition, accumulation, or persistence of oid or asynuclein fibrils and/or aggregates.

The compounds of this invention are compounds selected from but not limited to: Compound PD 150 Compound PD 151 Compound PD 152 Compound PD 153 Compound PD 154 Synthesis of the compounds of the invention The compounds of this invention may be prepared by methods generally known to the person of ordinary skill in the art, having regard to that knowledge and the disclosure of this application ing Example 1.

The starting materials and reagents used in ing these compounds are either available from commercial suppliers such as the Aldrich al Company (Milwaukee, WI), Bachem nce, CA), Sigma (St. Louis, MO), or Lancaster Synthesis Inc. (Windham, NH) or are prepared by methods well known to a person of ordinary skill in the art, following ures described in such references as Fieser and Fieser's Reagents for Organic sis, vols. 1-17, John Wiley and Sons, New York, NY, 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supps., Elsevier e Publishers, 1989; Organic Reactions, vols. 1-40, John Wiley and Sons, New York, NY, 1991; March J.: Advanced Organic Chemistry, 4th ed., John Wiley and Sons, New York, NY; and Larock: Comprehensive Organic Transformations, VCH Publishers, New York, 1989.

In most cases, protective groups for the hydroxy groups are introduced and finally removed. Suitable protective groups are described in Greene et al., Protective Groups in Organic Synthesis, Second Edition, John Wiley and Sons, New York, 1991. Other starting materials or early intermediates may be prepared by elaboration of the materials listed above, for example, by methods well known to a person of ordinary skill in the art.

The starting als, ediates, and compounds of this invention may be isolated and purified using conventional techniques, ing precipitation, filtration, distillation, crystallization, chromatography, and the like. The compounds may be characterized using conventional methods, including physical constants and spectroscopic methods.

Pharmacology and Utility The compounds provided herein can be used as such, be stered in the form of pharmaceutically acceptable salts derived from nic or organic acids, or used in combination with one or more pharmaceutically acceptable excipients. The phrase "pharmaceutically acceptable salt" means those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. The salts can be prepared either in situ during the final isolation and cation of the compounds provided herein or separately by reacting the acidic or basic drug substance with a suitable base or acid respectively. Typical salts derived from organic or inorganic acids salts include, but are not limited to hydrochloride, hydrobromide, hydroiodide, acetate, adipate, alginate, citrate, aspartate, benzoate, bisulfate, gluconate, fumarate, hydroiodide, lactate, maleate, oxalate, palmitoate, pectinate, succinate, tartrate, ate, glutamate, and onate. Typical salts derived from c or inorganic bases include, but are not d to lithium, sodium, potassium, calcium, magnesium, ammonium, monoalkylammonium such as meglumine, dialkylammonium, trialkylammonium, and tetralkylammonium.

Actual dosage levels of active ingredients and the mode of administration of the pharmaceutical compositions provided herein can be varied in order to achieve the effective therapeutic response for a particular t. The phrase peutically effective amount" of the compound provided herein means a sufficient amount of the compound to treat disorders, at a able benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the provided will be decided by the attending physician within the scope of sound medical nt. The total daily dose of the compounds provided herein may range from about 0.1 to about 1000 mg/kg/day. For purposes of oral administration, doses can be in the range from about 1 to about 500 mg/kg/day. If desired, the effective daily dose can be divided into multiple doses for purposes of stration; consequently, single dose itions may contain such amounts or submultiples thereof to make up the daily dose. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; medical history of the patient, activity of the specific compound employed; the specific composition employed, age, body weight, l health, sex and diet of the patient, the time of stration, route of administration, the duration of the treatment, rate of excretion of the specific compound employed, drugs used in combination or coincidental with the specific compound employed; and the like.

The compounds provided can be ated together with one or more non-toxic pharmaceutically acceptable diluents, carriers, adjuvants, and antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some cases, in order to prolong the effect of the drug, it is desirable to decrease the rate of absorption of the drug from subcutaneous or intramuscular injection.

This can be accomplished by suspending crystalline or amorphous drug substance in a e having poor water solubility such as oils. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon l size and lline form. Prolonged absorption of an able pharmaceutical form can be achieved by the use of absorption delaying agents such as aluminum monostearate or gelatin.

The compound provided herein can be stered enterally or parenterally in solid or liquid forms. Compositions suitable for parenteral ion may se physiologically acceptable, isotonic sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for titution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, ts, solvents or vehicles include water, ethanol, s (propylene glycol, polyethylene glycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, and suitable mixtures thereof. These compositions can also n adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Suspensions, in addition to the active compounds, may contain suspending agents such as lated isostearyl alcohols, yethylene sorbitol and sorbitan esters, microcrystalline ose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.

The compounds provided herein can also be administered by injection or infusion, either subcutaneously or enously, or intramuscularly, or intrasternally, or intranasally, or by infusion techniques in the form of sterile injectable or oleaginous sion. The compound may be in the form of a sterile injectable aqueous or nous suspensions. These suspensions may be formulated according to the known art using suitable dispersing of wetting agents and suspending agents that have been described above. The sterile injectable preparation may also be a sterile able solution or suspension in a non-toxic parenterally- acceptable diluent or solvent for example, as a solution in tanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride on. In addition, sterile, fixed oils are conventionally employed as a t or suspending medium. For this purpose any bland fixed oils may be conventionally employed including synthetic mono- or di-glycerides. In addition fatty acids such as oleic acid find use in the preparation of injectables. Dosage regimens can be ed to provide the optimum therapeutic response. For example, several divided dosages may be administered daily or the dosage may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer ed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are ible with body tissues. The injectable formulations can be ized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of e solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound may be mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or (a) fillers or ers such as starches, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as ymethylcellulose, alginates, n, polyvinylpyrrolidone, sucrose and acacia; (c) humectants such as glycerol; (d) disintegrating agents such as gar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (e) solution retarding agents such as paraffin; (f) absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glycerol monostearate; (h) ents such as kaolin and bentonite clay and (i) lubricants such as talc, m stearate, magnesium te, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hardfilled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The solid dosage forms of tablets, s, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings nown in the pharmaceutical ating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, ally, in a delayed manner.

Examples of embedding compositions which can be used include polymeric substances and waxes. Tablets n the compound in ure with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets.

These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch or alginic acid; binding agents, for e, maize starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate or stearic acid or tale. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby e a sustained action over a longer period. For example, a time delay material such as glycerol monostearate or glycerol distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the compound is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

Liquid dosage forms for oral administration include ceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, pyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene s and fatty acid esters of an and mixtures f. Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and ding agents, sweetening, flavoring and perfuming agents.

Other oral delivery systems such as self-microemulsifying drug ry systems (SMEDDS) in liquid and pellet forms that result in ed lity, dissolution, and in vivo oral absorption of the poorly water-soluble compounds can be ated such as those ped for curcumin. (European Journal of ceutics and Biopharmaceutics 76 (2010) 475-485) Aqueous suspensions contain the compound in admixture with excipients suitable for the manufacture of aqueous sions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be naturally occurring phosphatides, for e lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ne oxide with partial esters derived from fatty acids such as hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters from fatty acids and a hexitol anhydrides, for example, polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate, one or more ng agents, one or more flavoring agents, or one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the compound in a vegetable oil, for example arachis oil, olive oil, sesame oil, or coconut oil or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth below, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an idant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are ified by those already described above.

Additional ents, for example ning, flavoring and agents, may also be present.

The compounds provided herein may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oils, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally occurring phosphatides, for example soy bean, lecithin, and occurring atides, for example soy bean, in, and esters or l esters derived from fatty acids and hexitol ides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example yethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for example, glycerol, sorbitol or sucrose. Such ations may also contain a ent, a preservative and flavoring and coloring agents.

In one embodiment, the compounds are formulated 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 subjects to be treated; each containing a therapeutically effective quantity of the compound and at least one pharmaceutical excipient. A drug product will comprise a dosage unit form within a container that is labeled or accompanied by a label indicating the intended method of treatment, such as the treatment of a disease associated with a-synuclein fibril formation such as Parkinson's disease. Compositions for rectal or vaginal stration are preferably suppositories which can be prepared by mixing the compounds provided herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a itory wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Compounds provided herein can also be administered in the form of liposomes. s to form mes are known in the art ott, Ed., Methods in Cell Biology 1976, Volume XIV, Academic Press, New York, N.Y.) As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid ls which are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound provided herein, stabilizers, preservatives, excipients and the like. The preferred lipids are natural and synthetic phospholipids and phosphatidyl cholines (lecithins).

The compounds provided , or pharmaceutically acceptable derivatives thereof, may also be formulated to be targeted to a ular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. For non-limiting es of ing methods, see, e.g., U.S. Patent Nos. 652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 570, 6,120,751, 495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, ,900,252, 5,840,674, 542 and 5,709,874.

In one embodiment, liposomal suspensions, including tissue-targeted liposomes, such as targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art.

For example, liposome ations may be prepared as described in U.S. Patent No. 4,522,811. Briefly, liposomes such as multilamellar vesicles (MLV's) may be formed by drying down egg phosphatidyl choline and brain phosphatidyl serine (7:3 molar ratio) on the inside of a flask. A solution of a nd provided herein in phosphate buffered saline g divalent cations (PBS) is added and the flask shaken until the lipid film is dispersed. The resulting vesicles are washed to remove unencapsulated compound, pelleted by centrifugation, and then resuspended in PBS.

Sustained Release Formulations The ion also includes the use of ned release formulations to deliver the nds of the present invention to the desired target (i.e. brain or systemic organs) at high ating levels (between 10 9 and 10 4 M) are also disclosed. In a preferred ment for the treatment of Parkinson's disease, the circulating levels of the compounds is maintained up to 10 M. The levels are either circulating in the patient systemically, or in a preferred embodiment, present in brain tissue, and in a most red embodiments, localized to the a-synuclein fibril deposits in brain or other tissues.

It is tood that the nd levels are maintained over a certain period of time as is desired and can be easily determined by one skilled in the art using this disclosure and compounds of the invention. In a preferred embodiment, the invention includes a unique feature of administration comprising a sustained release formulation so that a constant level of therapeutic compound is maintained between 10 8 and 10 6 M between 48 to 96 hours in the sera.

Such sustained and/or timed release formulations may be made by sustained release means of delivery devices that are well known to those of ordinary skill in the art, such as those described in US Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3, 598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; ,354,556 and 5,733,566, the disclosures of which are each orated herein by reference. These ceutical compositions can be used to provide slow or sustained release of one or more of the active compounds using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic s, ayer coatings, microparticles, liposomes, microspheres, or the like. Suitable sustained release formulations known to those skilled in the art, including those described herein, may be readily selected for use with the pharmaceutical compositions of the invention. Thus, single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gelcaps, caplets, powders and the like, that are adapted for sustained release are encompassed by the present invention.

In a preferred embodiment, the sustained release formulation contains active compound such as, but not d to, rystalline cellulose, maltodextrin, ethylcellulose, and magnesium stearate. As described above, all known s for encapsulation which are compatible with properties of the disclosed compounds are encompassed by this invention. The sustained release formulation is encapsulated by g particles or granules of the pharmaceutical composition of the invention with varying ess of slowly soluble polymers or by microencapsulation. In a preferred embodiment, the sustained release formulation is encapsulated with a coating material of g ess (e.g. about 1 micron to 200 microns) that allow the dissolution of the pharmaceutical composition about 48 hours to about 72 hours after administration to a mammal. In another embodiment, the coating material is a food-approved additive.

In another embodiment, the ned release formulation is a matrix dissolution device that is prepared by compressing the drug with a slowly soluble polymer carrier into a tablet. In one preferred embodiment, the coated particles have a size range between about 0.1 to about 300 microns, as disclosed in U.S. Patent Nos. 4,710,384 and ,354,556, which are incorporated herein by reference in their entireties. Each of the particles is in the form of a micromatrix, with the active ient uniformly distributed throughout the polymer.

Sustained release formulations such as those described in U.S. Patent No. 4,710,384, which is incorporated herein by reference in its entirety, having a vely high percentage of plasticizer in the coating in order to permit sufficient flexibility to prevent substantial breakage during compression are disclosed. The specific amount of plasticizer varies ing on the nature of the coating and the particular plasticizer used. The amount may be readily determined empirically by testing the release characteristics of the s formed. If the medicament is released too quickly, then more plasticizer is used. Release characteristics are also a function of the thickness of the g. When substantial amounts of plasticizer are used, the sustained release capacity of the coating shes. Thus, the thickness of the coating may be increased slightly to make up for an increase in the amount of plasticizer. Generally, the plasticizer in such an embodiment will be present in an amount of about 15 to 30 of the sustained release material in the coating, preferably 20 to 25 %, and the amount of coating will be from 10 to 25% of the weight of the active material, ably 15 to 20 . Any conventional pharmaceutically acceptable cizer may be incorporated into the coating.

The compounds of the invention can be ated as a sustained and/or timed e formulation. All sustained release pharmaceutical products have a common goal of improving drug therapy over that achieved by their stained counterparts.

Ideally, the use of an optimally designed sustained release preparation in medical treatment is characterized by a minimum of drug substance being ed to cure or control the condition. Advantages of sustained release formulations may include: 1) extended activity of the composition, 2) reduced dosage frequency, and 3) sed patient compliance. In addition, sustained release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the composition, and thus can affect the occurrence of side effects.

The sustained release formulations of the invention are designed to initially release an amount of the therapeutic composition that promptly produces the d therapeutic effect, and gradually and ually release of other amounts of compositions to maintain this level of therapeutic effect over an extended period of time.

In order to maintain this constant level in the body, the therapeutic composition must be released from the dosage form at a rate that will replace the composition being metabolized and excreted from the body.

The sustained release of an active ingredient may be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds. The term "sustained release ent" in the context of the present invention is d herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, microspheres, or the like, or a combination thereof, that facilitates the sustained release of the active ingredient.

If the complex is water-soluble, it may be formulated in an appropriate buffer, for example, phosphate buffered saline, or other physiologically compatible solutions.

Alternatively, if the ing complex has poor solubility in aqueous solvents, then it may be formulated with a non-ionic surfactant such as Tween, or hylene .

Thus, the compounds and their physiologically solvents may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, or rectal administration, as examples.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound. In a preferred embodiment, the compounds of the present invention are formulated as controlled e powders of te microparticles that can be y ated in liquid form. The sustained release powder comprises particles containing an active ingredient and optionally, an excipient with at least one non-toxic polymer.

The powder can be dispersed or suspended in a liquid vehicle and will maintain its sustained release characteristics for a useful period of time. These dispersions or suspensions have both chemical stability and stability in terms of dissolution rate. The powder may contain an excipient comprising a polymer, which may be soluble, insoluble, permeable, impermeable, or biodegradable. The polymers may be polymers or copolymers. The polymer may be a natural or synthetic polymer. Natural polymers include polypeptides (e.g., zein), polysaccharides (e.g., cellulose), and c acid.

Representative synthetic polymers include those described, but not limited to, those described in column 3, lines 33-45 of U.S. Patent No. 5,354,556, which is orated by reference in its entirety. Particularly suitable polymers include those bed, but not limited to those described in column 3, line 46-column 4, line 8 of U.S. Patent No. ,354,556 which is incorporated by reference in its entirety.

The sustained release compounds of the ion may be formulated for parenteral administration, e.g., by intramuscular injections or implants for subcutaneous tissues and various body es and transdermal devices. In one embodiment, intramuscular ions are formulated as aqueous or oil suspensions. In an aqueous suspension, the sustained release effect is due to, in part, a reduction in solubility of the active compound upon complexation or a decrease in dissolution rate. A similar approach is taken with oil suspensions and solutions, wherein the release rate of an active compound is determined by partitioning of the active compound out of the oil into the surrounding aqueous medium. Only active compounds which are oil soluble and have the desired partition characteristics are suitable. Oils that may be used for intramuscular injection include, but are not limited to, sesame, olive, arachis, maize, almond, soybean, cottonseed and castor oil.

A highly developed form of drug delivery that s sustained release over periods of time ranging from days to years is to implant a drug-bearing polymeric device subcutaneously or in various body cavities. The polymer material used in an implant, which must be biocompatible and nontoxic, include but are not limited to hydrogels, silicones, polyethylenes, ethylene-vinyl acetate copolymers, or biodegradable polymers.

EXAMPLES Example 1: Synthesis of the compounds of the invention The compounds of this invention may be prepared by methods lly known to the person of ordinary skill in the art, having regard to that knowledge and the disclosure of this ation including the Examples presented below.

The starting als and reagents used in preparing these nds are either available from cial suppliers such as the Aldrich Chemical Company (Milwaukee, WI), Bachem (Torrance, CA), Sigma (St. Louis, MO), or Lancaster sis Inc. (Windham, NH) or are ed by methods well known to a person of ordinary skill in the art, following procedures bed in such references as Fieser and Fieser's Reagents for Organic Synthesis, vols. 1-17, John Wiley and Sons, New York, NY, 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supps., Elsevier e Publishers, 1989; Organic Reactions, vols. 1-40, John Wiley and Sons, New York, NY, 1991; March J.: Advanced Organic Chemistry, 4th ed., John Wiley and Sons, New York, NY; and Larock: Comprehensive Organic Transformations, VCH Publishers, New York, 1989. 3, 4-(bisbenzyloxy)benzoic acid benzyl ester: PRO35 Anhydrous potassium carbonate (10.9 g, 79.5 mmol) was added to a solution of 3, 4- Dihydroxybenzoic acid (3.5 g, 22.7 mmol) in anhydrous DMF (100 ml followed by benzyl chloride (8.8 g, 69.2 mmol). The resulting suspension was stirred under argon at 60 °C overnight. The reaction mixture was poured in water (150ml) and extracted with ethyl acetate (3x 50 ml). The combined c extract was washed with water, brine solution (50 ml each) dried over anhydrous magnesium sulfate. The solvent was d under reduced pressure. Yield = 9.4 g, 98% yield. PRO35. 3, benzyloxy)benzyl alcohol: LiAIH 4 / Ether PRO35 PRO36A A on of 3, 4-(bisbenzyloxy)benzoic acid benzyl ester (lot # PRO35, 9.0 g, 21.2 mmol) in 60 ml of anhydrous ether was added drop wise to a suspension of lithium aluminum hydride (0.89 g, 23.31 mmol) in ether (30 ml). The reaction mixture was quenched after three hours by slowly adding hydrated sodium sulfate . The reaction mixture was filtered after stirring for thirty minutes and the te was concentrated under reduced pressure to yield the alcohol as a white solid. PRO36A, 6.3 g, 93% yield. 3, 4-(bisbenzyloxy)benzyl chloride: PRO36A PRO36B 3, 4-(bisbenzyloxy)benzyl alcohol (Lot #PRO36A, 6.0 g, 18.7 mmol) was added to thionyl chloride (12 ml) and DMF (0.2 ml) at room temperature and the on mixture was heated to 60 °C for 2 hrs. Excess thionyl chloride was removed under reduced pressure. The yellow residue was dissolved in 20% ethyl acetate in hexane and was passed through a short bed of silica gel. The silica gel is flushed with with 20% ethyl acetate in hexane (200 ml). The combined washing was concentrated under reduced pressure to yield the desired chloride as a yellow solid, PRO36B, 5.1 g, 81% yield. 3-Meth l-l , 7-bis(3', 4'-dihydroxybenzyl)xanthine: PRO36B PD A solution of yl xanthine ( 200 mg, 1.2 mmol) in N,N-dimethylformamide (5 ml) was treated with s-benzyloxy-benzyl chloride (Lot #PRO36B, 1.02 g, 3 mmol) and NaH (96 mg, 4 mmol) and 0.5 equivalent tetrabutyl um iodide and was heated to 60 °C for 12 hrs. The on mixture was poured in water and extracted with ethyl acetate (3x30 ml). The ed organic extract was washed with water, brine solution (30 ml) each and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure and the product was purified by flash column chromatography over silica gel using 40% ethyl acetate/hexane to yield the desired product, 600 mg, 78% yield. This t was dissolved in ethyl acetate (25 ml) and methanol (10 ml) and acetic acid ( 1 ml) and hydrogenated in ce of 10% Pd-C at 55 PSI for 2 hr. Catalyst was filtered off and the solvent was removed under reduced pressure. The residue was purified on a silica gel column eluting with 50% ethyl acetate in hexane to 100% ethyl acetate. The desired product was isolated as an off white solid, Yield =400 mg PD 151. 7-Meth l-l , 3-bis(3', ydroxy)benzylxanthine: PD 150 7-Methyl-l, 3-bis(3', 4'-dihydroxy)benzylxanthine was sized from 7 -methyl xanthine ( 200 mg, 1.2 mmol) following the general procedure as described in the previous experiment. Yield 150 mg PD 150. 1-Meth l-l , 3-bis(3', 4'-dihydroxy)benzyl xanthine: PD 152 1-Methyl-l, 3-bis(3', ydroxy)benzyl xanthine was sized from 1-methyl xanthine ( 200 mg, 1.2 mmol) following the general procedure as described in the previous experiment. Yield 200 mg PD 152. 8-BromoMethyl(3', 4'-dibenzyloxy) benzyl xanthine: A solution of 3-methylbromo xanthine (294 mg, 1.2 mmol) in N, N- dimethylformamide (5 ml) was added to NaH (96 mg, 4 mmol) 3, 4-bis-benzyloxy- benzyl chloride (Lot #PRO36B, 1.02 g, 3 mmol) and tetrabutylammonium iodide (0.5 equivalent). The solution was heated to 60 °C for 12 hrs. The reaction mixture was poured in water and extracted with ethyl acetate (3x30 ml). The combined c extract was washed with water, brine on (30 ml) each and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure and the product was purified by flash column chromatography over silica gel using 40% ethyl acetate/hexane to yield the desired product, 511 mg, 78% yield. 8-Bromo-l, 3-dimethyl(3', 4'-dibenzyloxy) benzyl xanthine PRO42 A solution of 8-BromoMethyl(3', 4'-dibenzyloxy) benzyl xanthine (511 mg; 0.94 mmol) and methyl iodide (426 mg; 3mmol) in N, N-dimethylformamide (5 ml) was added to potassium carbonate (138 mg; l.Ommol) and was heated to 60 °C for 6 hrs. The reaction mixture was poured in water (25ml) and extracted with ethyl acetate (3 x 25ml).

The combined extract was dried over anhydrous Magnesium Sulfate and concentrated under reduced pressure. The product was purified by flash chromatography over silica gel eluting with 30% ethyl acetate/hexane to yield the d product. 535 mg, 95% PRO- 04-42 1, 3-dimethyl(3', 4'-dibenzyloxy) benzyl(3', 4'-dimethoxyphenyl) xanthine PRO43 A solution of 8-Bromo-l, 3-dimethyl(3', 4'-dibenzyloxy)benzyl xanthine ( 535 mg, 0.95 mmol) and methoxy phenylboronic acid (182 mg; 1 mmol) in 1,4-dioxane (5 ml) was treated is(triphenyl phosphine)palladium(O) (77 mg, 0.1 mmol) and potassium carbonate (277 mg, 2 mmol). The mixture was heated to 70 C for 12 hrs under argon atmosphere. The reaction mixture was concentrated under reduced pressure.

The e was ed on a silica gel column g with 40% ethyl acetate in hexane to 70% ethyl acetate. The desired product was isolated as off white solid, Yield 500 mg, 81% PRO43 1, 3-dimethyl(3', 4'-dibenzyloxy) benzyl(3', 4'-dihydroxyphenyl) xanthine PD 154 Boron tribromide (1.62 g; 6.48 mmol) was added to a solution of 1, 3-dimethyl(3', 4'- dibenzyloxy) benzyl(3', 4'-dimethoxyphenyl) xanthine (500 mg, 0.81 mmol) in anhydrous dichloromethane (12 ml) at -70 °C. After 1 hr the reaction mixture was warmed to room temperature and d for 6 hr. Methanol (3 ml) was added and the reaction mixture was stirred overnight. The reaction mixture was concentrated under reduced pressure. The residue was purified on a silica gel column eluting with 70% ethyl acetate/hexane to 100% ethyl acetate. The desired product was ed as off white solid, Yield 110 mg; 26% PD 154. o-l, 3, 7-trimethyl- xanthine PRO45 A solution of 8-bromomethyl xanthine (300 mg; 1.2 mmol) and methyl iodide (1.42 g; .0 mmol) in N, N-dimethylformamide (5 ml) was added to potassium carbonate (662 mg; 4.8 mmol) and was heated to 60 °C for 6 hrs. The reaction mixture was poured in water (25ml) and extracted with ethyl acetate (3 x 25ml). The ed extract was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The product was purified by flash chromatography over silica gel eluting with ethyl acetate to yield the desired product. 315 mg; 92% PRO45 -trimethyl (3', 4'-dimethoxyphenyl) xanthine: A solution of 8-Bromo-l, 3, 7-trimethyl xanthine (315 mg, 1.15 mmol) and 3, 4- oxy phenylboronic acid (230 mg; 1.26 mmol) in 1, 4-dioxane (5 ml) was treated tetrakis (triphenyl phosphine) ium (0) (88 mg, 0.14 mmol) and potassium carbonate (277 mg, 2.0 mmol). The mixture was heated to 70 °C for 12 hrs under argon atmosphere. The reaction mixture was concentrated under reduced pressure. The residue was purified on a silica gel column eluting with 70% ethyl acetate in hexane to 100% ethyl acetate. The desired product was isolated as off white solid, Yield 297 mg, 78% PRO46 1, 3, ethyl (3', 4'-dihydroxyoxyphenyl) xanthine: Boron tribromide (1.62 g; 6.48 mmol) was added to a solution of 1, 3, 7-trimethyl , 4'-dimethoxyphenyl) xanthine (297mg, 0.90 mmol) in anhydrous dichloromethane (12 ml) at -70 °C. After 1 hr the reaction mixture was warmed to room temperature and stirred for 6 hr. Methanol (3 ml) was added and the reaction e was stirred overnight. The reaction mixture was concentrated under reduced pressure. The residue was purified on a silica gel column eluting with ethyl acetate. The desired product was isolated as off white solid, Yield 210 mg; 82% PD 153.

Example 2: Compounds disrupt/inhibit gregated Parkinson's disease o - synuclein fibrils The compounds were found to be disrupters/disaggregators of c -synuclein fibrils.

In this set of studies, the efficacy of n nds provided herein to cause a disassembly/disruption/disaggregation of pre-formed s of Parkinson's disease (i.e. consisting of cc-synuclein fibrils) was analyzed. For the studies described below in Parts A and B, 69 mM of cc-synuclein (rPeptide, Bogart, CA) was first ted at 37°C for 4 days in 20 mM sodium acetate buffer at pH 4 with ar shaking (1,300 rpm) to cause cc-synuclein aggregation and fibril formation.

Part A : Thioflavin T Fluorometry In one study, Thioflavin T fluorometry was used to determine the effects of the compounds on cc-synuclein fibrils. In addition to test compounds, this experiment included three control compounds unds 1, 2 and 3) for reference. In this assay Thioflavin T binds specifically to fibrillar protein, and this binding es a fluorescence enhancement at 485nm that is directly proportional to the amount of fibrils formed. The higher the fluorescence, the greater the amount of fibrils formed (Naki et al., Lab. Invest. 65:104-110, 1991; Levine III, Protein Sci. 2:404-410, 1993; Amyloid:Int. J .

Exp. Clin. Invest. 2:1-6, 1995).

Following l cc-synuclein fibrilization as described above, the cc-synuclein (6.9 mM) mixture was then incubated at 37 C for 2 days with shaking (200 rpm), either alone, or in the presence of one of the nds (at test compound: a-synuclein molar ratios of 10:1, 1:1, 0.1:1, and 0.01:1) in phosphate-buffered saline, pH 7.4 + 0.02% sodium azide. Following 2 days of co-incubation, 50m1(5 mg) of each incubation mixture was erred into a 96-well microtiter plate containing 150m1of distilled water and 50m1of a Thioflavin T solution (i.e. 500 mM Thioflavin T in 250 mM phosphate buffer, pH 6.8). The final concentration of Thioflavin T reagent is 100 mM in 50 mM phosphate buffer, pH 6.8. The fluorescence was read at 485nm (444nm excitation wavelength) using an ELISA plate fluorometer. Subtraction of the signal obtained from a diluted reaction (blank) containing buffer alone or nd alone at a concentration equivalent to that of its corresponding cc-synuclein-containing reaction was used to quantitate the amount of Thioflavin T fluorescence in each cc-synuclein-containing reaction that is proportional to the protein fibril content in that on.

The results of the 2-day incubations are presented below. For each compound, the % inhibition of Thioflavin T fluorescence is shown in Table 1. This study indicated that the nds provided herein disrupt/disaggregate pre-formed Parkinson's disease a-synuclein fibrils.

Table 1: Compounds disrupt/disaggregate a-synuclein aggregates as measured by Thioflavin T fluorometry.

Part B : Congo red binding data In the Congo red binding assay, the ability of a given test compound to alter a- synuclein aggregate binding to Congo red is quantified. In this assay Congo red binds specifically to fibrillar protein, and this binding is directly proportional to the amount of fibrils formed. Following initial -synuclein fibrilization as bed above, a-synuclein aggregates and test compounds were incubated for 2 days and then vacuum filtered through a 0.2 mih filter. The amount of a-synuclein retained in the filter was then quantitated following staining of the filter with Congo red. After appropriate washing of the filter, any lowering of the Congo red color on the filter in the presence of the test compound (compared to the Congo red staining of the protein in the absence of the test nd- i.e. clein alone) was indicative of the test compound's ability to diminish/alter the amount of aggregated and congophilic a-synuclein and thus cause disassembly/disruption/ disaggregation of a-synuclein fibrils.

In one study, the ability of a-synuclein fibrils to bind Congo red following a 2-day incubation of -synuclein in the absence or presence of increasing amounts of the compounds provided herein, including positive reference ol) compounds (at test nd: -synuclein molar ratios of 10:1, 1:1, 0.1:1, 0.01:1) was determined. The s of 2-day incubations are presented in Table 2 below. The results of this study indicate that compounds of this invention disrupt/ disaggregate/ emble pre-formed a-synuclein aggregates as indicated by their y to inhibit Parkinson's disease type a - synuclein fibril binding to Congo red.

Table 2 Compounds disrupt/disaggregate a-synuclein fibrils/aggregates as measured by a Congo red g assay.

Example 3: Compounds disrupt/inhibit freshly dissolved Parkinson's disease - synuclein protein from forming fibrils (i.e. b-sheet secondary structure) Thioflavin T Fluorometry To test whether the compounds can inhibit formation of a -synuclein b-sheet, the same assay as described in Example 2, was utilized but the a-synuclein was fresh and not pre-fibrillized. Fresh wild-type a-synuclein was dissolved in a buffer containing 9.5mM phosphate, 137mM sodium chloride and 2.7mM potassium chloride (phosphate-buffered ; PBS), and the pH was adjusted to pH 7.4. This solution was then lyophilized and dissolved in 1.0ml zed water at 0.5 mg/ml . As indicated above the test compounds ally at test compound: a-synuclein molar ratios of 10:1, 1:1, 0.1:1, and ) were then added to the -synuclein. Following 24-38 hours of co-incubation, the incubation mixtures were diluted 1:10 and 50m1of each diluted tion mixture was transferred into a 96-well microtiter plate ning 150m1of distilled water and 50m1of a Thioflavin T solution (i.e. 500 mM Thioflavin T in 250 mM phosphate buffer, pH 6.8).

The final concentration of a-synuclein was 0.7 mM and the concentration of Thioflavin T reagent was 100 mM in 50 mM ate buffer, pH 6.8. In some experiments, 200 mΐ of each diluted incubation mixture was combined in the 96-well microtiter plate with 50 mΐ of the 500 mM Thioflavin T solution to give 2.8 mM a-synuclein in the presence of 100 mM Thioflavin T reagent. The fluorescence was read at 485nm (444nm excitation wavelength) using an ELISA plate fluorometer after subtraction with buffer alone or nd alone, as blank. Positive control compound 1 performed nearly identically in inhibiting a-synuclein aggregation regardless of whether 0.7 mM or 2.8 mM a-synuclein was uently used in the Thioflavin T reaction.

The te results of this study presented in Table 3 indicated that compounds of this invention interfered with a-synuclein aggregation as indicated by their ability to prevent the formation of a-synuclein b-sheet secondary folding as assessed by Thioflavin T fluorometry.

Table 3 Compounds t formation of a-synuclein b-sheet-rich structures as measured by Thioflavin T Fluorometry Example 4- Compounds of this invention are potent disruptors/inhibitors of o - synuclein fibrils and/or aggregates associated with Parkinson's disease Parkinson's Disease is terized by the accumulation of insoluble intraneuronal aggregates called Lewy Bodies, a major ent of which is c - synuclein (reviewed in Dauer et al., Neuron, 39:889-909, 2003). Since autosomal dominant mutations in a -synuclein cause a subset of familial son's disease, and since these mutations increase the likelihood of a-synuclein to aggregate and form Lewy Bodies, aggregated a-synuclein is proposed to be directly involved in the etiology and disease progression (Polymeropoulos et al., e 276:1197-1199, 1997; Papadimitriou et al., Neurology 52:651-654, 1999). ural studies have ed that intracellular Lewy bodies contain a large proportion of ded proteins with a high degree of b- pleated sheet secondary structure. Therefore, since many of the compounds described herein cause disassembly/disruption/ disaggregation of a-synuclein aggregates in the in vitro assays (Thioflavin T fluorometry and Congo Red binding assays) described above, studies were also conducted in living cells to determine the efficacy of these compounds to t or prevent a-synuclein aggregation associated with Parkinson's disease.

To test the therapeutic potential of the compounds, a cell-based assay was utilized. In this assay, rotenone is used to induce ondrial oxidative stress and cause a-synuclein aggregation. The assay es the binding of the fluorescent dye Thioflavin S to structures with high b-sheet content, including a-synuclein fibrils.

Therefore, quantitative assessment of the extent of Thioflavin S-positive staining of fixed cells is used to test the y of the test compounds to inhibit/prevent or decrease the amount of a-synuclein aggregates ve to cells that were treated with rotenone only.

This study is presented in the following examples.

To carry out these studies, a cell culture model was used in which human a - ein aggregation is mentally induced. BE-M17 human neuroblastoma cells stably transfected with A53T-mutant human a-synuclein were obtained. Cell culture reagents were obtained from Gibco/Invitrogen, and cells were grown in OPTIMEM supplemented with 10% FBS, Penicillin (100 units/ml), Streptomycin (100 g/ml) and 500 / l G418 as previously described (Ostrerova-Golts et al., J. Neurosci., 20:6048- 6054, 2000).

Thioflavin S is commonly used to detect aggregated protein structures in situ, including in brain tissue (Vallet et al., Acta Neuwpathol., 83:170-178, 1992), and cultured cells (Ostrerova-Golts et al., J. ci., 20:6048-6054, 2000), whereas Thioflavin T is often used as an in vitro reagent to analyze the aggregation of soluble proteins into fibrils enriched in b-pleated sheet structures (LeVine III, Prot. Sci., 2:404- 410, 1993). Therefore, Thioflavin S histochemistry was used on cultured cells to detect aggregates containing a high degree of b-pleated structures that formed in se to oxidative stress-inducing agents (in this case rotenone) as previously described, with minor modifications (Ostrerova-Golts et al., J. Neurosci., 20:6048-6054, 2000). y, for these studies cells were grown on Poly-D- Lysine coated glass slide chambers at approximately 4.5-5.5 x 104 cells/cm 2 . After 16-18 hours, cells were treated with 500 nM or 2 mM rotenone (Sigma) or vehicle (0.05% DMSO) as indicated. Within 15 s of rotenone (or vehicle) addition, compounds were added at the indicated concentration, or mock-treatment was performed in which cell culture media only (no nd) was added. Identical treatments were repeated after 48 hours. After an additional 24 hours, cells were fixed for 25 minutes in 3% paraformaldehyde. After a PBS wash and a zed water wash, the cells were ted with 0.015% Thioflavin S in 50% ethanol for 25 minutes, washed twice for four minutes in 50% ethanol and twice for five minutes in deionized water and then d using an aqueous-based mountant designed to protect against photobleaching. ates that bind to Thioflavin S were detected with a scent microscope using a High Q FITC filter set (480 to 535 nm bandwidth) and a 20X objective lens unless ise indicated. Between 8 and 20 (usually 16-18) representative images per condition were selected and imaged using Q Capture software by an experimenter who was blinded to ent conditions. To assess the amount of avin S-positive aggregates, the total area per field covered by Thioflavin S-positive inclusions was determined by image analysis and quantitation. For this purpose, background fluorescence that failed to exceed pre- set size or pixel intensity threshold parameters was eliminated using Image Pro Plus software. Spurious, non-cell associated fluorescence was manually d. Unless indicated otherwise, comparisons between groups were made by comparing mean relative amounts of Thioflavin S-positive inclusions for a given treatment condition (i.e. cells treated with rotenone only versus cells treated with rotenone and test compound at a given concentration). Statistical analyses were performed with GraphPad Prism (GraphPad Inc). Differences between means (two samples) were assessed by the Student's t test. Differences among multiple means were assessed by one-factor ANOVA followed by Dunnett's post hoc test, compared to rotenone only treated cells. The data presented below represent tically significant (p<0.05) reductions ted as percent inhibition) in Thioflavin S scence in cells treated with test compound and rotenone ve to cells d with rotenone only.

To validate the ability of the assay to quantitatively detect aggregates that bind Thioflavin S, staining of BE-M17 cells overexpressing A53T -synuclein was carried out and the results ed a ne dose-dependent increase in avin S-positive aggregates relative to vehicle-treated control cells (not shown). Higher magnification images obtained with a 40X objective indicated that the Thioflavin S-positive aggregates were intracellular and cytoplasmic, analogous to the accumulation of intracytoplasmic Lewy bodies that are pathological hallmarks associated with Parkinson's disease (not shown). Quantitation of the area covered by Thioflavin-S-positive aggregates established that 500 nM and 2 mM ne were sufficient to induce robust aggregation (not shown) and thus are effective doses to test the ability of compounds to attenuate the formation of these aggregates.

Using the protocol described above, selected compounds were tested for their ability to reduce, inhibit, prevent or eliminate Thioflavin S-positive aggregates in rotenone-treated BE-M17 cells overexpressing A53T a-synuclein. Some of the nds tested significantly disrupted, prevented or inhibited a-synuclein ation and fibril formation in the presence of rotenone as indicated by a decrease in Thioflavin S-positive ions, relative to cells treated with rotenone only. For example, cells treated only with 500 nM rotenone exhibited a robust presence of Thioflavin S-positive aggregates, whereas addition of 500 nM or 2 mM PD-151 markedly reduced the abundance of these rotenone-induced aggregates by 52% and 84%, respectively, relative to rotenone only-treated cells. Similarly, in cells treated only with 2 mM rotenone, there was a robust presence of Thioflavin tive aggregates, whereas addition of 2 mM or 5 mM PD-151 ly reduced the abundance of these rotenone-induced aggregates by 58% and 60%, respectively, relative to rotenone only-treated cells. Therefore, PD-151 reduced, inhibited, prevented and/or eliminated Thioflavin S-positive aggregates in cells that express human A53T a -synuclein.

In addition, PD-152, at given trations, showed significant tion/prevention/inhibition of rotenone-induced Thioflavin S-positive inclusions when tested in a similar fashion. For example, cells treated only with 500 nM rotenone exhibited a robust presence of Thioflavin S-positive aggregates, whereas addition of 2 mM or 5 mM PD-152 markedly reduced the abundance of these rotenone-induced aggregates by 54% and 55%, respectively, ve to rotenone only-treated cells. rly, in cells treated only with 2 mM rotenone, there was a robust presence of avin S-positive aggregates, whereas addition of 500 nM or 2 mM PD-152 markedly reduced the abundance of these rotenone-induced aggregates by 78% and 79%, respectively, relative to rotenone only-treated cells. Therefore, PD-152 also d, inhibited, prevented and/or eliminated Thioflavin S-positive aggregates in cells that express human A53T a -synuclein.

Taken together, we concluded that the tested compounds PD-151 and PD-152 ively and ly reduced, prevented and/or inhibited the formation, deposition and/or accumulation of a -synuclein aggregates in A53T a -synuclein-expressing BE-M17 cells.

Example 5 : Compounds of this ion are potent disrupters/inhibitors of Alzheimer's Ab1-42 fibrils or aggregates The compounds prepared in the preceding Examples were found to be potent disruptors/inhibitors of Parkinson's disease a-synuclein protein fibrils or aggregates. In a set of studies, the efficacy of the compounds to cause a disassembly/disruption/ disaggregation of pre-formed amyloid fibrils of Alzheimer's disease (i.e. ting of Ab 1-42 fibrils) was analyzed.

Part A - Thioflavin T fluorometry In one study, Thioflavin T fluorometry was used to determine the effects of the compounds, and of caffeine (as a negative control). In this assay Thioflavin T binds specifically to fibrillar amyloid, and this binding produces a fluorescence enhancement at 485 nm that is directly proportional to the amount of amyloid fibrils formed. The higher the fluorescence, the greater the amount of amyloid fibrils formed (Naki et al., Lab.

Invest. 65:104-110, 1991; Levine III, Protein Sci. 410, 1993; Amyloid: Int. J. Exp.

Clin. Invest. 2:1-6, 1995).

In this study, 30 mΐ of a 1 mg/mL solution (in distilled water) of brillized human A b 1-42 (rPeptide) was incubated at 37°C for 2 days either alone, or in the presence of one of the compounds or caffeine (at test compound:Ap molar ratios of 10:1, :1, 1:1, 0.1:1 or 0.05:1). The final concentration of Ab in the reaction is 0.1 mg/mL (22 mM) in phosphate-buffered saline, pH 7.4 + 0.02% sodium azide in 300 final volume.

Following 2-days of co-incubation, 50 mΐ of each incubation mixture was transferred into a 96-well microtiter plate containing 150 mΐ of distilled water and 50 mΐ of a Thioflavin T solution (i.e. 500 mM Thioflavin T in 250 mM phosphate buffer, pH 6.8). The emission fluorescence was read at 485 nm (444 nm tion wavelength) using an ELISA plate fluorometer after subtraction with buffer alone or compound alone, as blank.

The results of the 2-day tions are presented in Table 4 . For example, whereas caffeine caused no significant inhibition of A b 1-42 fibrils at all concentrations tested, the compounds all caused a dose-dependent disruption/disassembly/disaggregation of preformed A b 1-42 fibrils. All of the nds tested were ive in disrupting pre-formed Ab 1-42 fibrils. These results are similar to the results obtained from a positive control nd (not shown) that demonstrated robust inhibition of Thioflavin T fluorescence. For example, all of the compounds in this invention caused at least 63% inhibition of Thioflavin T fluorescence when used at a test compound:A b: molar ratio of :1. At a test nd:A b molar ratio of 5:1 the levels of inhibition ranged from 17 to 87% and all compounds except PD-153 showed at least 74% inhibition at this 5:1 (test compound:AP) tration. Even at equimolar concentrations (test compound:AP molar ratio of 1:1) there was at least 54% inhibition of avin T fluorescence for all compounds except PD-153. Interestingly, PD-150 and PD-154 were ive against Ab fibrils/aggregates at substoichiometric concentrations (i.e. test compound: Ab molar ratios of 0.1:1 and 0.05:1) in this assay. This study indicated that the compounds of this ion are potent disruptors/inhibitors of Alzheimer's disease type A b fibrils, and usually exert their s in a dose-dependent manner.

Table 4 Compounds disrupt/disaggregate Ab fibrils/aggregates as measured by a Thioflavin T fluorometry assay.

Part B : Congo red In the Congo red binding assay the ability of a test compound to alter b-amyloid binding to Congo red is quantified. In this assay, Ab 1-42 (as prepared for the Thio T assay) and test compounds were incubated for 2 days and then vacuum filtered through a 0.2 mih filter. The amount of Ab 1-42 ed in the filter was then quantitated following staining of the filter with Congo red. After appropriate washing of the filter, any lowering of the Congo red color on the filter in the ce of the test compound (compared to the Congo red staining of the amyloid n in the e of the test compound) was indicative of the test compound's ability to diminish/alter the amount of aggregated and congophilic Ab.

In one study, the ability of Ab fibrils to bind Congo red in the absence or presence of increasing amounts of the compounds or caffeine (at test compound:A b molar ratios of :1, 5:1, 1:1, or 0.1:1) was determined. The s of 2-day incubations are presented in Table 5 . Whereas caffeine caused no significant inhibition of Ab 1-42 fibril binding to Congo red at all concentrations tested, the compounds caused a dose-dependent inhibition of Ab binding to Congo red. For e, PD-150, PD-151 and PD-152 each caused a significant inhibition (ranging from 59-63% inhibition) of Congo red g to Ab 1-42 fibrils when used at a test compound:A b molar ratio of 10:1, and a significant inhibition of Congo red binding when used at a test compound: Ab molar ratio of 5:1 (ranging from 46-48% inhibition). Similar to the results for the Thioflavin T fluorometry assay, this study also ted that compounds of this invention are potent disruptors/inhibitors of Ab fibrils as assessed by Ab fibril binding to Congo red, and usually exert their effects in a dose-dependent manner.

Table 5 Compounds disrupt/disaggregate Ab fibrils/aggregates as measured by a Congo Red binding assay.

Example 6: Compounds of this invention directly inhibit/disrupt the in vitro conversion of Ab to b-sheet containing fibril structures Part A : Thioflavin T Fluorometry To test whether the compounds can inhibit b-sheet formation of Ab, the same assay as described in Example 5 was utilized, but the Ab was prepared so that it is in a non-fibrillar state at the start of the assay. To e this brillar state, lyophilized human Ab 1-42 (rPeptide) was ved to 1 mg/mL (220 mM) using 2 mM NaOH and the pH was adjusted to 10.5 with small ( ) additions of 1M NaOH. The clear solution was then frozen, re-lyophilized, and dissolved in a buffer ning 9.5mM phosphate, 137mM sodium chloride and 2.7mM potassium chloride (phosphate-buffered saline; PBS) to a concentration of 2 mg/mL (440 mM) A b. In separate tubes, test compound stocks were prepared in PBS at s concentrations such that final reactions containing equal volumes of the test compound stocks and the Ab solution would result in a final Ab concentration of 1 mg/mL (220 mM) with test compound:A b molar ratios of 10:1, 5:1, 1:1, and 0.5:1. The reactions containing Ab + test compounds (or A b + PBS as a control for A b aggregation) were then incubated for 24 hours, the incubation mixtures were diluted 1:20 to 0.05 mg/mL A b and 50m of each diluted incubation mixture was transferred into a 96-well microtiter plate containing 150m of distilled water and 50m of a Thioflavin T solution (i.e. 500 mM Thioflavin T in 250 mM phosphate buffer, pH 6.8). The final concentration of Ab was 2.2 mM and the concentration of avin T reagent was 100 mM in 50 mM phosphate buffer, pH 6.8. The fluorescence was read at 485nm (444nm tion wavelength) using an ELISA plate fluorometer after subtraction with PBS buffer alone or compound alone, as blank.

The te results of this study presented in Table 6 indicated that nds of this invention interfered with Ab aggregation as indicated by their ability to prevent the formation of b-sheet secondary folding of Ab as assessed by Thioflavin T metry. For example, PD-150, PD-151 and PD-152 each caused a significant inhibition (ranging from 61-93% inhibition) of Thioflavin T fluorescence when used at a test compound:A b molar ratio of 10:1, and a significant inhibition of Thioflavin T fluorescence when used at a test compound:A b molar ratio of 5:1 (ranging from 23-87% inhibition). The positive control compound (Control 4) performed as expected and completely inhibited A b aggregation (by 100%) at test compound:A b molar ratios >1:1 whereas the negative control compound (caffeine) failed to inhibit Ab aggregation at any of the concentrations tested (50:1, 10:1 and 1:1). This study indicated that compounds of this invention are potent tors of t rich-Ab fibril formation as ed by Thioflavin T fluorometry, and the compounds usually exert their effects in a pendent manner.

Table 6 Compounds inhibit formation of b-sheet-rich structures of Ab as measured by Thioflavin T Fluorometry.

Part B : ar Dichroism (CD) Spectroscopy Since several compounds were shown to reduce the nce of Thioflavin T- positive aggregates (Table 6), we sought independent confirmation that the compounds directly inhibit the conversion of Ab to b-sheet ning structures by using circular dichroism (CD) spectroscopy. For this purpose, the Ab reactions that were used in the Thioflavin T fluorometry assay (Part A in this Example) were assessed at 24 hours of aggregation. Ab alone was also assessed by CD spectral analysis at t=0, prior to aggregation, (t=0, ed reference l). After 24 hours, reactions were diluted 20- fold in PBS and a CD spectrum for each reaction was acquired on a Jasco J-810 spectropolarimeter using a 0 .1 cm path length cell. All spectra were recorded with a step size of 0.1 nm, a bandwidth of 1 nm, and an Ab concentration of 0.05 mg/ml. The spectra were d at the shortest ngth that still provided a dynode e less than 600V. The trimmed spectra were then subjected to a data processing routine beginning with noise reduction by Fourier transform followed by subtraction of a blank spectrum (vehicle only without Ab). These blank corrected a were then zeroed at 260 nm and the units converted from millidegrees to specific icity.

Percent b-sheet was determined from processed spectra using the ellipticity minimum value at approximately 218 nm and referencing to a scale normalized to nearly fully folded and unfolded reference values, consistent with previous reports (Ramirez- Alvarado et al., J. Mol. Biol., 273:898-912, 1997; Andersen et al., J. Am. Chem. Soc, 121 :9879-9880, 1999). The fully folded reference value was found by performing the described calculation on the spectrum of Ab fibrillized for 24 hours (complete fibrillization), and assigning this difference the arbitrary value of 100% b-sheet. The unfolded reference was provided by the spectrum from the same sample at the initial time point (t=0) and ascribing the difference found here the ary value of 0% b-sheet.

These percent b-sheet values were then used to provide the respective relative % inhibition of t induced by the compounds at given molar ratio of test compound:A b.

First, in order to confirm that Ab 1-42 is indeed converted to a b-sheet-rich structure and to establish the timing of this conversion at 24 hours in our system, an aliquot of the Ab only incubation mixture (without compounds) was sampled and the CD um was collected. At 24 hours of incubation, CD analysis revealed a large abundance of a b-sheet-rich structure(s), indicated by the pronounced specific ellipticity minimum at 218 nm and maximum at 197 nm (not shown). However, when test compounds PD-150, PD-151, PD-152, PD-153 or the positive control compound (control 4) were included individually in the reaction mixture, at appropriate concentrations, at 24 hours of incubation the ude of the change of the minimum at 218 nm was reduced, relative to A b alone, and the spectra were more characteristic of random coil structure.

Thus, we conclude that some of the nds in this invention t, to varying degrees, the conversion of natively unfolded A b to a b-sheet-rich structure. On the other hand, the negative control compound, caffeine, had no effect on the magnitude of change in the ellipticity minima at 218 nm. These results are ized in Table 7 . As a ic example of a test compound that inhibits b-sheet formation in Ab, compound PD-150 resulted in at least 62% tion when used at test compound:Ab molar ratios >5:1. Taken together, these results indicate that some of the compounds in this invention show potent tion and prevention of Ab aggregation, a hallmark of the amyloid diseases such as Alzheimer's disease.

Table 7 Compounds inhibit formation of b-sheet-rich structures of Ab as ed by Circular Dichroism (CD) Spectroscopy Example 7- Compounds of this invention display therapeutically relevant levels in plasma and brain consistent with a drug intended for treatment of central nervous system disorders For select compounds in this invention, we have used wild type mice to determine the following plasma pharmacokinetic (PK) parameters: maximal concentration (C ), and area under the curve (AUC) as derived from a time versus concentration plot. We have also ined the maximal mouse brain levels, and overall brain re over time of select compounds in this invention, expressed as C -brain and AUC-brain, tively. In order to establish the method, we assessed brain and plasma compound levels over time utilizing 50 mg/kg intraperitoneal (i.p.) injections of 2 control compounds. These s indicated a rapid spike in plasma levels and brain uptake of these control compounds, followed by te clearance from blood by 6 hours post- dose (data not shown). In a typical experiment to assess the compounds in this invention, we used CD-I female mice with a sample size (n) equal to 4 mice per post-dose time- point (for example, 7, 15, 30, and 60 min post-dose). We chose early time points to assess initial exposure, when we expected plasma and brain exposure to be the high, though we may have missed even higher exposure at earlier time points (between 0 and 7 minutes) since brain and plasma levels were highest in our study at the earliest time point assessed (7 minutes).

For the purpose of these studies, each compound was formulated at 5 mg/mL in % polyethylene glycol (PEG)-400 in PBS + 0.1% ascorbic acid (w/v). The dose volume was 10 mL/kg of body weight. Mice were stered test compound via intraperitoneal injection, and at about 2-3 s before scheduled sacrifice, they were deeply anesthetized with 2.5% n. Once anesthetized, whole blood was removed by cardiac puncture, transferred to riate EDTA-containing tubes, and immediately chilled on ice. This was ed by te perfusion of each mouse with >15 ml cold 0.9% saline by cannulation of the left ventricle and clamping of the ding aorta.

Brains were harvested, frozen on dry ice and stored at -80°C for bioanalysis of the test compound. Plasma was extracted from whole blood by standard centrifugation techniques within 1 hour. Compounds were (liquid-liquid) extracted from plasma and brain homogenates using ethyl acetate, followed by HPLC/MS quantitation using methods (i.e. HPLC gradients and mass spectrometry parameters) developed for these novel nds. All methods established sufficient stability in relevant matrices and solvents, and used internal quantitation controls. Quantitation was achieved using a calibration curve ted with compounds spiked into the appropriate matrix (i.e. 20% 0/PBS + 0.1% ascorbic acid). We have established lower limit of quantitative sensitivities of 5-25 ng/ml (plasma) and 5-25 ng/g (brain), ient for these studies.

Following determination of the brain and plasma concentrations at the various post-dose time points, select plasma (C and AUC) and brain (C -brain and AUC-brain) PK max max parameters were determined with WinNonLin software (Pharsight Inc). We compared the values determined for the compounds in this ion to the values for the positive control compound that when administered at the same therapeutically relevant route and dose level (i.p. injection at 50 mg/kg) is known to be present in the plasma and the brain at levels sufficient for a biological effect (i.e. reduction of cc-synuclein brain levels and improved motor function; data not shown).

Using the ol described above, for example, we determined that PD-151 has a plasma C = 5,340 ng/mL and plasma AUC = 212,417 min*ng/mL. This plasma exposure compares favorably with the positive control compound that has a plasma C = 8,230 ng/mL and plasma AUC = 420,406. In brain, PD-151 has a C -brain = 59.2 ng/g and AUC-brain = 2,147 min*ng/g. This brain exposure es favorably with the positive control compound that has a C -brain = 94.6 ng/g and AUC-brain = 7,029 min*ng/g. PD-150 also showed acceptable plasma and brain exposure. For example, PD- 150 has a plasma C = 3,284 ng/mL, plasma AUC = 127,662 /mL, C -brain = max max 99.5 ng/g and AUC-brain = 3,048 min*ng/g.

Taken together, these results indicate that some of the nds in this invention are shown to have plasma and brain exposure that is consistent with a drug intended to treat a central nervous system disorder such as Alzheimer's or Parkinson's disease where the primary target is a brain protein. For example, the levels of the compounds in this ion have a brain and plasma exposure that is comparable to a l compound (i.e. no greater than ld different than the control compound) when that control compound is administered at a therapeutically effective amount in a disease-relevant animal model.

Claims (13)

We claim:

1. A compound selected from the group consisting of compounds of the formula where: R1-3 are independently hydrogen, methyl and benzyl groups, wherein at least two of R1-3 are benzyl substituted at any one time, and wherein the benzyl groups are substituted with two hydroxy groups, and pharmaceutically acceptable salts f.

2. The nd of claim 1 selected from the group consisting of: HO N N O HO O N HO N HO O N N , , and O N N 206524NZ_claims_20150715_PLH

3. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable excipient.

4. The use of a compound of claim 1 in the manufacture of a ment for the treatment of disorders characterized by the formation, deposition, accumulation, or persistence of amyloid or α-synuclein fibrils.

5. The use of a compound of claim 1 in the manufacture of a medicament for the treatment of an amyloid disease or a synucleinopathy.

6. The use of claim 5 where the amyloid disease is selected from the group of diseases consisting of Alzheimer’s e, type II diabetes, systemic AA amyloidosis, Down’s syndrome, hereditary al hemorrhage with dosis of the Dutch type, and cerebral β-amyloid angiopathy.

7. The used of claim 5 where the amyloid disease is mer’s disease.

8. The use of claim 5 where the synucleinopathy is selected from the group consisting of Parkinson’s disease, familial Parkinson’s disease, Lewy body disease, the Lewy body variant of Alzheimer’s disease, dementia with Lewy bodies, multiple system y, and the Parkinsonism-dementia complex of Guam.

9. The use of claim 5 where the synucleinopathy is Parkinson’s disease.

10. The use of claim 5, where the medicament is ated to be administered in an amount between 0.1 mg/Kg/day and 1000 mg/Kg/day.

11. The use of claim 5, where the medicament is formulated to be administered in an amount between 1 mg/Kg/day and 100 mg/Kg/day.

12. The use of claim 5, where the medicament is formulated to be administered in an amount between 10 mg/Kg/day and 100 mg/Kg/day. 206524NZ_claims_20150715_PLH

13. An article of manufacture, comprising packaging material, the compound of claim 1, or a pharmaceutically acceptable salt thereof, and a label that instructs the use of the article for the treatment of an d disease or a synucleinopathy. 206524NZ_claims_20150715_PLH