U.S. patent application number 15/499216 was filed with the patent office on 2018-01-04 for compounds for the treatment of neurodegenerative diseases.
The applicant listed for this patent is PROTAMED, INC.. Invention is credited to JUDY CAM, JOEL CUMMINGS, LUKE A. ESPOSITO, QUBAI HU, F. MICHAEL HUDSON, THOMAS LAKE, ALAN D. SNOW, MARISA C. YADON.
Application Number | 20180002298 15/499216 |
Document ID | / |
Family ID | 46758284 |
Filed Date | 2018-01-04 |
United States Patent
Application |
20180002298 |
Kind Code |
A1 |
ESPOSITO; LUKE A. ; et
al. |
January 4, 2018 |
COMPOUNDS FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES
Abstract
Compounds and their pharmaceutically acceptable salts for
treatment of tauopathies, such as Alzheimer's disease, Pick's
disease, progressive supranuclear palsy, corticobasal degeneration,
familial frontotemporal dementia/Parkinsonism linked to chromosome
17, amyotrophic lateral sclerosis/Parkinsonism-dementia complex,
argyrophilic grain dementia, dementia pugilistic, diffuse
neurofibrillary tangles with calcification, progressive subcortical
gliosis and tangle only dementia.
Inventors: |
ESPOSITO; LUKE A.; (SEATTLE,
WA) ; YADON; MARISA C.; (DURBAN, ZA) ;
CUMMINGS; JOEL; (SEATTLE, WA) ; HUDSON; F.
MICHAEL; (OAKLAND, CA) ; LAKE; THOMAS;
(SNOHOMISH, WA) ; HU; QUBAI; (KIRKLAND, WA)
; CAM; JUDY; (BELLEVUE, WA) ; SNOW; ALAN D.;
(LYNNWOOD, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PROTAMED, INC. |
Woodinville |
WA |
US |
|
|
Family ID: |
46758284 |
Appl. No.: |
15/499216 |
Filed: |
April 27, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15151409 |
May 10, 2016 |
|
|
|
15499216 |
|
|
|
|
14740595 |
Jun 16, 2015 |
9334251 |
|
|
15151409 |
|
|
|
|
14486040 |
Sep 15, 2014 |
9085549 |
|
|
14740595 |
|
|
|
|
14002164 |
Aug 29, 2013 |
8865754 |
|
|
PCT/US12/27222 |
Mar 1, 2012 |
|
|
|
14486040 |
|
|
|
|
61448935 |
Mar 3, 2011 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07C 205/22 20130101;
C07D 271/107 20130101; C07D 277/24 20130101; C07D 263/56 20130101;
C07D 409/14 20130101; C07C 205/20 20130101; C07D 271/10 20130101;
C07D 333/16 20130101; C07C 255/53 20130101; C07D 263/57 20130101;
C07D 277/32 20130101; C07C 39/15 20130101; C07D 249/06 20130101;
C07D 285/08 20130101; A61J 1/00 20130101; C07D 285/12 20130101;
C07C 235/56 20130101; C07D 231/06 20130101; C07D 333/28 20130101;
C07D 263/32 20130101 |
International
Class: |
C07D 285/08 20060101
C07D285/08; A61J 1/00 20060101 A61J001/00; C07D 333/16 20060101
C07D333/16; C07D 285/12 20060101 C07D285/12; C07D 277/32 20060101
C07D277/32; C07D 277/24 20060101 C07D277/24; C07D 271/107 20060101
C07D271/107; C07D 271/10 20060101 C07D271/10; C07D 263/57 20060101
C07D263/57; C07D 263/56 20060101 C07D263/56; C07D 263/32 20060101
C07D263/32; C07D 249/06 20060101 C07D249/06; C07D 231/06 20060101
C07D231/06; C07C 255/53 20060101 C07C255/53; C07C 235/56 20060101
C07C235/56; C07C 205/22 20060101 C07C205/22; C07C 205/20 20060101
C07C205/20; C07C 39/15 20060101 C07C039/15; C07D 333/28 20060101
C07D333/28; C07D 409/14 20060101 C07D409/14 |
Claims
1. A compound selected from the group consisting of:
##STR00045##
2. A method of disrupting or inhibiting the formation, deposition,
accumulation, or persistence of tau fibrils and/or aggregates,
comprising administering a therapeutically effective amount of the
compounds of claim 1.
3. The method of claim 2, where the compound administered is in an
amount between 0.1 mg/Kg/day and 1000 mg/Kg/day.
4. The method of claim 2, where the compound is administered in an
amount between 1 mg/Kg/day and 100 mg/Kg/day.
5. The method of claim 2, where amount of compound administered is
in an amount between 10 mg/Kg/day and 100 mg/Kg/day.
6. A method resulting in neuroprotection from a tauopathy in a
mammal comprising the step of administrating a therapeutically
effective amount of a compound of claim 1.
7. The method of claim 6 where the tauopathy is one selected from;
Alzheimer's disease, Pick's disease, progressive supranuclear
palsy, corticobasal degeneration, familial frontotemporal
dementia/Parkinsonism linked to chromosome 17, amyotrophic lateral
sclerosis/Parkinsonism-dementia complex, argyrophilic grain
dementia, dementia pugilistic, diffuse neurofibrillary tangles with
calcification, progressive subcortical gliosis and tangle only
dementia.
8. An article of manufacture, comprising packaging material, the
compound of claim 1, or a pharmaceutically acceptable salt thereof,
contained within packaging material, which is used for treating the
formation, deposition, accumulation, or persistence of tau fibrils
and/or aggregates, and a label that indicates that the compound or
pharmaceutically acceptable salt thereof is used for treating the
formation, deposition, accumulation, or persistence of tau fibrils
and/or aggregates.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/151,409, filed May 10, 2016, which is a continuation of U.S.
application Ser. No. 14/740,595, filed Jun. 16, 2015, now U.S. Pat.
No. 9,334,251, which is a divisional of U.S. application Ser. No.
14/486,040 filed Sep. 15, 2014, now U.S. Pat. No. 9,085,549, which
is a divisional of U.S. application Ser. No. 14/002,164 filed Aug.
29, 2013, now U.S. Pat. No. 8,865,754, which was filed under 35
U.S.C. 371 and is a U.S. National Stage Application of
PCT/US2012/027222 filed Mar. 1, 2012, which claims priority to U.S.
Provisional Application No. 61/448,935, filed Mar. 3, 2011, each of
which are incorporated herein by reference.
TECHNICAL FIELD
[0002] Provided herein are compounds, pharmaceutical compositions,
and methods for the treatment of neurodegenerative diseases such as
Parkinson's disease and various tauopathies.
BACKGROUND OF INVENTION
[0003] Parkinson's disease (PD) is a neurodegenerative human
disorder characterized clinically by both motor (movement) and
non-motor behavioral dysfunction, and histopathologically by the
formation, deposition, accumulation and/or persistence of abnormal
fibrillar protein deposits and/or aggregates. This accumulation of
cytoplasmic Lewy bodies consisting of fibrils/aggregates of
.alpha.-synuclein/NAC (non-A.beta. component) is believed important
in the pathogenesis of PD. Lewy bodies occur mostly in the
substantia nigra and locus ceruleus sections of the brain stem and
the olfactory bulb, but also, to a lesser extent, in other
subcortical and cortical regions of the brain. Because of this
specific localization in the brain, Lewy bodies interfere with the
health and integrity of dopaminergic neuronal projections from the
substantia nigra to the striatum, thus adversely affecting the
ability to initiate, carry out and control voluntary movements.
Lewy bodies present in these brain regions may also impact the
production of acetylcholine and/or the balance between dopamine and
acetylcholine in the brain, thus causing disruption in perception,
thinking and behavior as well as other non-motor symptoms including
loss of smell and sleep disorders.
[0004] Dementia with Lewy Bodies (DLB) is a progressive
neurodegenerative disorder characterized by symptoms which display
various degrees of manifestation. Such symptoms include progressive
dementia, Parkinsonian movement difficulties, hallucinations, and
increased sensitivity to neuroleptic drugs. As with Alzheimer's
disease (AD), advanced age is considered to be a risk factor for
DLB, with average onset typically between the ages of 50-85. Twenty
percent of all dementia cases are caused by DLB and over 50% of PD
patients develop Parkinson's Disease Dementia (PDD), a type of DLB.
DLB may occur alone or in conjunction with other brain
abnormalities, including those involved in AD and PD, as mentioned
above. Currently, conclusive diagnosis of DLB is made during
postmortem autopsy.
[0005] New agents or compounds able to bind and/or inhibit
.alpha.-synuclein and/or NAC formation, deposition, accumulation
and/or persistence, or disrupt pre-formed .alpha.-synuclein/NAC
fibrils and/or aggregates (or portions thereof) are regarded as
potential therapeutics for the treatment of Parkinson's and related
synucleinopathies. Compounds which protect neurons from
degeneration and damage associated with Parkinson's and related
synucleinopathies could also prove useful as therapeutics.
[0006] Parkinson's Disease and Synucleinopathies
[0007] 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. Neuropath.
Exp. Neurol. 52:183-191, 1993), the major components of which are
filaments consisting of .alpha.-synuclein (Spillantini et al.,
Proc. Natl. Acad. Sci. USA 95:6469-6473, 1998; Arai et al.,
Neurosci. Lett. 259:83-86, 1999), a 140-amino acid protein (Ueda et
al., Proc. Natl. Acad. Sci. USA 90:11282-11286, 1993). Three
dominant mutations in .alpha.-synuclein causing increased tendency
to aggregate and resulting in familial early onset Parkinson's
disease have been described suggesting that Lewy bodies contribute
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;
Zarranz et al., Ann. Neurol. 55:164-173, 2004). Recently, in vitro
studies have demonstrated that recombinant .alpha.-synuclein can
indeed form Lewy body-like fibrils (Conway et al., Nature Med.
4:1318-1320, 1998; Hashimoto et al., Brain Res. 799:301-306, 1998;
Nahri et al., J. Biol. Chem. 274:9843-9846, 1999; Choi et al., FEBS
Lett. 576:363-368, 2004). Most importantly, both the A53T and the
E46K Parkinson's disease-linked .alpha.-synuclein mutations
accelerate this fibril-forming aggregation process, demonstrating
that such in vitro studies may have relevance for Parkinson's
disease pathogenesis. Alpha-synuclein aggregation and fibril
formation fulfills the criteria of a nucleation-dependent
polymerization process (Wood et al., J. Biol. Chem.
274:19509-19512, 1999). Alpha-synuclein recombinant protein, and
non-A.beta. component (known as NAC), which is a 35-amino acid
peptide fragment of .alpha.-synuclein, both have the ability to
form fibrils and/or aggregates when incubated at 37.degree. C., and
are positive with stains such as Congo red (demonstrating a
red/green birefringence when viewed under polarized light) and
Thioflavin S (demonstrating positive fluorescence) (Hashimoto et
al., Brain Res. 799:301-306, 1998; Ueda et al., Proc. Natl. Acad.
Sci. USA 90:11282-11286, 1993).
[0008] Synucleins are a family of small, presynaptic neuronal
proteins composed of .alpha.-, .beta.-, and .gamma.-synucleins, of
which only .alpha.-synuclein aggregates have been associated with
several neurological diseases (Ian et al., Clinical Neurosc. Res.
1:445-455, 2001; Trojanowski and Lee, Neurotoxicology 23:457-460,
2002). The role of synucleins (and in particular,
.alpha.-synuclein) in the etiology of a number of neurodegenerative
diseases has developed 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 amyloid plaques of a different neurological
disease, Alzheimer's disease. Biochemically, recombinant
.alpha.-synuclein was shown to form fibrils and/or aggregates that
recapitulated 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 .alpha.-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
.alpha.-synuclein in a spectrum of diseases such as Parkinson's
disease, dementia with Lewy bodies, multiple system atrophy and the
Lewy body variant of Alzheimer's disease has led to the
classification of these diseases under the umbrella term of
"synucleinopathies".
[0009] NAC is a 35 amino acid fragment of .alpha.-synuclein that
has the ability to form fibrils and/or aggregates either in vitro
or as observed in the brains of patients with Parkinson's disease.
The NAC fragment of .alpha.-synuclein is a relatively important
therapeutic target as this portion of .alpha.-synuclein is believed
crucial for formation of Lewy bodies as observed in all patients
with Parkinson's disease, synucleinopathies and related
disorders.
[0010] Currently available therapeutics such as carbidopa/levodopa
(Sinemet, Stalevo, Parcopa), dopamine agonists (Apokyn, Parlodel,
Neupro, Mirapex, Requip), anticholinergics (Cogentin, Artane),
MAO-B inhibitors (Eldepryi, Carbex, Zelapar, Azilect), COMT
inhibitors (Comtan, Tasmar), and other medications like Symmetrel
and Exelon aim to slow the loss of dopamine or improve just the
symptoms of the patient.
[0011] Discovery and identification of new compounds or agents as
potential therapeutics to arrest fibril and/or aggregate formation,
deposition, accumulation and/or persistence of .alpha.-synuclein in
Parkinson's disease or provide neuroprotection are desperately
sought.
[0012] Parkinson's disease .alpha.-synuclein fibrils and/or
aggregates consist of a predominantly .beta.-pleated sheet
structure. Compounds of this invention have been shown to be
effective in the inhibition of .alpha.-synuclein/NAC fibril
formation and/or aggregates as well as in the disruption of
pre-formed fibrils and/or aggregates, as shown from Examples
provided herein. These compounds could serve as therapeutics for
Parkinson's disease and other synucleinopathies.
[0013] Tau is a microtubule associated protein found primarily in
neuronal axons. Tau hyperphosphorylation is a common characteristic
of a number of dementing disorders collectively known as
tauopathies, some of which have distinct tau pathology combined
with other brain pathologies. Tauopathies include Alzheimer's
disease (AD), Pick's disease (PiD), progressive supranuclear palsy
(PSP), corticobasal degeneration (CBD) and familial frontotemporal
dementia/Parkinsonism linked to chromosome 17 (FTDP-17),
amyotrophic lateral sclerosis/Parkinsonism-dementia complex,
argyrophilic grain dementia, dementia pugilistic, diffuse
neurofibrillary tangles with calcification, progressive subcortical
gliosis and tangle only dementia. (Spillantini, M G and Goedert M,
1998 Trends Neurosci. October 21(10):428-33). In AD, tau pathology
is typically limited to the neurons while other tauopathies can
pathologically exhibit both neuronal and glial tau deposition
(Higuchi, M, et al., 2002. Neuropsychopharmacology: The Fifth
Generation of Progress, Chapter 94: Tau protein and tauopathy).
[0014] It has recently been postulated that tau protein may link
Parkinson's and Alzheimer's disease (Shulman, J. M. and DeJager, P.
L. 2009 Nature Genetics 41(12):1261-1262). This study examined
whether any genome wide association occurs between the two diseases
and found that three genes and two new loci were linked to
increased susceptibility.
[0015] Physiological phosphorylation of tau regulates the dynamics
of the association of tau with tubulin, and thereby microtubule
stability (Mazanetz. M. P. and Fischer, P. M. 2007. Nature Reviews
6:464-479). The stabilization of the microtubules in axons ensures
that maintain their function for axonal transport, growth and
branching (Bulic, B et al., 2009 Angew. Chem. Int. Ed. 48:2-15).
Hyperphosphorylation and misfolding of the tau protein is thought
to be the causative factor in abnormal intracellular aggregation
leading ultimately to neuronal dysfunction. Protein aggregates have
been found to be toxic to neurons.
[0016] Abnormal intraneuronal tau aggregation has three basic
pathological manifestations; neurofibrillary tangles (NFT's),
neuropil threads (NT's) and the argyrophilic dystrophic neurite
plaques (Braak, H and Braak, E, Neurobio. of Aging. 1997
18(4):351-357). Structurally, the NFT's are principally comprised
of paired helical filaments (PHF) comprised of two filamentous tau
proteins twisted around one another with a crossover repeat of 80
nm and a width of 8-20 nm (Li, D., et al., 2008. Computational
Biology 4(12) and Kidd, M 1963 Nature, 197:192). There are six
stages (Braak stages I-VI) of tau deposition in the brain, which
progress temporally at defined anatomical locations with the
initial stages characterized primarily by the deposition of NFT's
and NT's and the secondary stages further accompanied by NP (Braak,
1997). In Alzheimers Disease and other neuropathies, Braak's stages
correlate well with clinical disease progression as demonstrated by
increasing cognitive dysfunction. Severe cortical destruction which
occurs around stages III-IV coincides with the first manifestations
of the clinical onset of AD. Although no tau mutations have been
identified in AD there is a strong correlation between NFT density
and cognitive decline in AD (Brunden, K. R., Trojanowski, J. Q.,
and Lee, V. M. 2009 Nature Reviews 8:783-93).
[0017] New biomarkers and models of their temporal characteristics
are becoming even more useful for the diagnosis and
characterization of AD (Jack et al., 2010. Lancet 9:119-28).
Specifically, tau deposition is associated with neurodegeneration
in AD and an increase in CSF tau is an important indicator of tau
pathologic changes and correlates well with clinical disease
severity. A decrease in FDG-PET correlates well with increased CSF
tau and both are valid indicators of synaptic dysfunction (Jack et
al, ibid). This model of biomarker ordering, especially in mildly
cognitive impaired individuals, has important implications for
clinical trials. Potential therapeutics could be more accurately
assessed for efficacy is they are able to change the trajectory of
cognitive deterioration and individuals might be more selectively
chosen for trials (Jack et al, ibid).
[0018] It is presently not known if tau is a causative factor in
disease but it is likely that either a loss or gain for function
results in pathology. In FTLD17, a missense mutation affects the
alternative splicing of tau resulting in the disruption of the
ratio of the 4R to 3R tau isoform. More of the 4R isoform with an
extra repeat of the microtubule binding region may lead to
overstabilization of the microtubules resulting in disease. Other
post-translational events such as alterations in kinase activity
and glycosylation could also cause hyperphosphorylation and result
in disease or alternatively proteolytic cleavage could produce
truncated tau products more inclined to aggregate (Brunden,
ibid).
[0019] Recently tau toxicity has been re-emphasized as an important
therapeutic target in neurodegenerative tauopathies (Keystone
Symposium, March 2009). Routes for developing therapeutics are
either directed to inhibiting tau-phosphorylation kinases or
seeking compounds effective in the modulation of tau aggregation
and/or the dissolution or disruption of tau aggregates which may
prove equally useful or more specific for the alleviation of
tauopathies (Rafii, M and Aisen, P. 2009 BMC Medicine 7:7). A
recent paper surveyed the efficacy of several classes of compounds
for their ability to prevent tau aggregation and disaggregate
pre-formed tau fibrils (Bulic et al). Although there are general
concerns regarding the toxicity of disassembled fibrils, Bulic et
al., were able to show that reversing tau aggregation resulted in
increased cell viability.
SUMMARY OF INVENTION
[0020] In a first aspect, provided herein are compounds such as,
but not limited to:
##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005##
##STR00006## ##STR00007##
[0021] In a second aspect, this invention is a method of treating a
synucleinopathy in a mammal, especially a human, by administration
of a therapeutically effective amount of a compound of the first
aspect of this invention, for example as a pharmaceutical
composition. Methods using such compounds and compositions for
disrupting, disaggregating and causing removal, reduction or
clearance of .alpha.-synuclein fibrils and/or aggregates are
provided thereby providing new treatments for synucleinopathies.
The treatment of disease may also include the inhibiting the
formation of .alpha.-synuclein fibrils and/or aggregates or
providing neuroprotection for neurons at risk.
[0022] Also provided are any pharmaceutically-acceptable
derivatives of the compounds of the first aspect of this invention,
including salts, esters, enol ethers or esters, acetals, ketals,
orthoesters, hemiacetals, hemiketals, solvates, hydrates or
prodrugs of the compounds. Pharmaceutically-acceptable salts,
include, but are not limited to, amine salts, alkali metal salts,
such as but not limited to lithium, potassium and sodium, alkali
earth metal salts, such as but not limited to barium, calcium 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 mineral acids, such as but not limited to
hydrochlorides and sulfates, salts of organic acids, such as but
not limited to acetates, lactates, malates, tartrates, citrates,
ascorbates, succinates, butyrates, valerates and fumarates.
[0023] Pharmaceutical formulations for administration by an
appropriate route and means containing effective concentrations of
one or more of the compounds provided herein or pharmaceutically
acceptable derivatives, such as salts, esters, enol ethers or
esters, acetals, ketals, orthoesters, hemiacetals, hemiketals,
solvates, hydrates or prodrugs, of the compounds that deliver
amounts effective for the treatment of synucleinopathies, are also
provided.
[0024] The formulations are compositions suitable for
administration by any desired route and include solutions,
suspensions, emulsions, tablets, dispersible tablets, pills,
capsules, powders, dry powders for inhalation, sustained release
formulations, aerosols for nasal and respiratory delivery, patches
for transdermal delivery and any other suitable route. The
compositions should be suitable for oral administration, parenteral
administration by injection, including subcutaneously,
intramuscularly or intravenously as an injectable aqueous or oily
solution or emulsion, transdermal administration and other selected
routes.
[0025] Also provided are methods for treatment, prevention or
amelioration of one or more symptoms of synucleinopathies,
including but not limited to diseases associated with the
formation, deposition, accumulation, or persistence of
alpha-synuclein.
[0026] Provided are methods for treatment, prevention or
amelioration of one or more symptoms of synuclein diseases or
synucleinopathies. In one embodiment, the methods inhibit or
prevent .alpha.-synuclein/NAC fibril formation and/or aggregation,
inhibit or prevent .alpha.-synuclein/NAC fibril growth, and/or
cause disassembly, disruption, and/or disaggregation of preformed
.alpha.-synuclein/NAC fibrils and .alpha.-synuclein/NAC-associated
protein deposits and/or aggregates. Synuclein diseases include, but
are not limited to Parkinson's disease, PDD, familial Parkinson's
disease, Lewy body disease, the Lewy body variant of Alzheimer's
disease, dementia with Lewy bodies (DLB), multiple system atrophy,
and the Parkinsonism-dementia complex of Guam.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a graph illustrating that a compound of the
invention causes a dose-dependent inhibition of of
.alpha.-synuclein aggregation and fibril formation as assessed by
Thioflavin T fluorometry.
[0028] FIG. 2 is a graph illustrating that a compound of the
invention causes the dose-dependent inhibition of of
.alpha.-synuclein aggregation and fibril formation as assessed by
Congo Red binding.
[0029] FIG. 3 is a graph illustrating the rotenone dose-dependent
increase in Thioflavin S fluorescence in BE-M17 neuroblastoma cells
overexpressing A53T-mutant .alpha.-synuclein.
[0030] FIG. 4 is a graph illustrating that a compound of the
invention causes dose-dependent inhibition of rotenone-induced cell
death as assessed by the XTT cell viability assay.
[0031] FIG. 5 is a graph illustrating that a compound of the
invention causes the dose-dependent inhibition of .alpha.-synuclein
j-sheet formation as assessed by circular dichroism
spectroscopy.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
[0032] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which this invention belongs. All
patents, applications, published applications and other
publications are incorporated by reference in their entirety. In
the event that there is a plurality of definitions for a term
herein, those in this section prevail unless stated otherwise.
[0033] As used herein, "Synuclein diseases" or "synucleinopathies"
are diseases associated with the formation, deposition,
accumulation, or persistence of synuclein fibrils, including, but
not limited to .alpha.-synuclein fibrils. Such diseases include,
but are not limited to Parkinson's disease, Familial Parkinson's
disease, PDD, Lewy body disease, the Lewy body variant of
Alzheimer's disease, dementia with Lewy bodies, multiple system
atrophy, and the Parkinsonism-dementia complex of Guam.
[0034] Aggregation or Fibrillogenesis refers to the formation,
deposition, accumulation and/or persistence of synuclein fibrils,
filaments, inclusions, deposits, and/or NAC fibrils, filaments,
inclusions, deposits, and/or aggregates or the like.
[0035] Inhibition of aggregation or fibrillogenesis refers to the
inhibition of formation, deposition, accumulation and/or
persistence of such fibrils or fibril-like deposits.
[0036] Disruption of fibrils or fibrillogenesis refers to the
disruption of pre-formed .alpha.-synuclein fibrils, that usually
exist in a pre-dominant .beta.-pleated sheet secondary structure.
Such disruption by compounds provided herein may involve marked
reduction or disassembly of synuclein fibrils as assessed by
various methods such as Thioflavin T fluorometry, Congo red
binding, SDS-PAGE/Western blotting, as demonstrated by the Examples
presented in this application.
[0037] "Mammal" includes both humans and non-human mammals, such as
companion animals (cats, dogs, and the like), laboratory animals
(such as mice, rats, guinea pigs, and the like) and farm animals
(cattle, horses, sheep, goats, swine, and the like).
[0038] "Pharmaceutically acceptable excipient" means an excipient
that is conventionally 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 aerosol composition, gaseous.
[0039] 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 treatment, prevention or
symptom amelioration for the disease. A "therapeutically effective
amount" or a "therapeutically effective dosage" in certain
embodiments inhibits, reduces, disrupts, disassembles 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 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 thereof 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 100 mg/Kg/day. A broad range of disclosed
composition dosages are believed to be both safe and effective.
[0040] The term "sustained 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.
[0041] 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
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 physiologically
suitable 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.
[0042] As used herein, pharmaceutically acceptable derivatives of a
compound include salts, esters, enol ethers, enol esters, acetals,
ketals, orthoesters, 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 derivatization.
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-chlorobenzyl-2-pyrrolidin-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, calcium 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 mineral 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 include, but are not limited to, alkyl, alkenyl,
alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and
heterocyclyl esters of acidic groups, including, but not limited
to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic
acids, sulfinic acids and boronic acids. Pharmaceutically
acceptable enol ethers include, but are not limited to, derivatives
of formula C.dbd.C(OR) where R is hydrogen, alkyl, alkenyl,
alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or
heterocyclyl. Pharmaceutically acceptable enol esters include, but
are not limited to, derivatives of formula C.dbd.C(OC(O)R) where R
is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl,
heteroaralkyl, cycloalkyl or heterocyclyl. Pharmaceutically
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.
[0043] As used herein, treatment means any manner in which one or
more of the symptoms of a disease or disorder are ameliorated or
otherwise beneficially altered. For example, slowing or arresting
disease development, providing relief from the symptoms or
side-effects of the disease, and relieving the disease by causing
regression of the disease, such as by disruption of pre-formed
synuclein fibrils could all be considered as treatment.
[0044] As used herein, amelioration of the symptoms of a particular
disorder by administration of a particular compound or
pharmaceutical composition refers to any lessening, whether
permanent or temporary, lasting or transient that can be attributed
to or associated with administration of the composition.
[0045] As used herein, "NAC" (non-A.beta. component) is a 35-amino
acid peptide fragment of .alpha.-synuclein, which like
.alpha.-synuclein, has the ability to form fibrils when incubated
at 37.degree. C., and is positive with stains such as Congo red
(demonstrating a red/green birefringence when viewed under
polarized light) and Thioflavin S (demonstrating positive
fluorescence) (Hashimoto et al., Brain Res. 799:301-306, 1998; Ueda
et al., Proc. Natl. Acad. Sci. U.S.A. 90:11282-11286, 1993).
Inhibition of NAC fibril formation, deposition, accumulation,
aggregation, and/or persistence is believed to be effective
treatment for a number of diseases involving .alpha.-synuclein,
such as Parkinson's disease, Lewy body disease and multiple system
atrophy.
[0046] 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 compound is modified such that the
active compound will be regenerated by metabolic processes. The
prodrug may be designed to alter the metabolic stability 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 prodrugs of the compound (see, e.g., Nogrady
(1985) Medicinal Chemistry A Biochemical Approach, Oxford
University Press, New York, pages 388-392).
[0047] It is to be understood that the compounds provided herein
may contain chiral centers. Such chiral centers may be of either
the (R) or (S) configuration, or may be a mixture 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.
[0048] As used herein, the abbreviations for any protective 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. 11:942-944).
B. Compounds
[0049] Provided herein are compounds and pharmaceutical
compositions containing compounds, not limited to those shown above
in the summary section and in the following examples.
C. Preparation of the Compounds
[0050] The compounds provided herein can be prepared by standard
synthetic methods known in the art, and are shown in general
schemes provided herein. The examples that follow describe the
exemplary embodiments and are not purported to limit the scope of
the claimed subject matter. It is intended that the specification,
together with the following examples, be considered exemplary only,
with the scope and spirit of the claimed subject matter being
indicated by the claims that follow these examples. Other
embodiments within the scope of claims herein will be apparent to
one skilled in the art from consideration of the specification as
described herein.
[0051] The starting materials and reagents used in preparing these
compounds are either available from commercial suppliers such as
the Aldrich Chemical Company (Milwaukee, Wis.), Bachem (Torrance,
Calif.), Sigma (St. Louis, Mo.), or Lancaster Synthesis Inc.
(Windham, N.H.) or are prepared by methods well known to a person
of ordinary skill in the art, following procedures described in
such references as Fieser and Fieser's Reagents for Organic
Synthesis, vols. 1-17, John Wiley and Sons, New York, N.Y., 1991;
Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supps.,
Elsevier Science Publishers, 1989; Organic Reactions, vols. 1-40,
John Wiley and Sons, New York, N.Y., 1991; March J.: Advanced
Organic Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.;
and Larock: Comprehensive Organic Transformations, VCH Publishers,
New York, 1989.
[0052] 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 materials,
intermediates, and compounds provided herein may be isolated and
purified using conventional techniques, including precipitation,
filtration, distillation, crystallization, chromatography, and the
like. The compounds may be characterized using conventional
methods, including physical constants and spectroscopic
methods.
D. Pharmaceutical Compositions and Administration
[0053] The compounds provided herein can be used as such, be
administered in the form of pharmaceutically acceptable salts
derived from inorganic 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 purification 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, phosphate, glutamate,
and bicarbonate. Typical salts derived from organic or inorganic
bases include, but are not limited to lithium, sodium, potassium,
calcium, magnesium, ammonium, monoalkylammonium such as meglumine,
dialkylammonium, trialkylammonium, and tetralkylammonium. The mode
of administration of the pharmaceutical compositions can be oral,
rectal, intravenous, intramuscular, intracisternal, intravaginal,
intraperitoneal, bucal, subcutaneous, intrasternal, nasal, or
topical. The compositions can also be delivered at the target site
through a catheter, an intracoronary stent (a tubular device
composed of a fine wire mesh), a biodegradable polymer, or
biological carriers including, but are not limited to antibodies,
biotin-avidin complexes, and the like. Dosage forms for topical
administration of a compound provided herein include powders,
sprays, ointments and inhalants. The active compound is mixed under
sterile conditions with a pharmaceutically acceptable carrier and
any needed preservatives, buffers or propellants. Opthalmic
formulations, eye ointments, powders and solutions are also
provided herein.
[0054] 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 patient. The phrase "therapeutically
effective amount" of the compound provided herein means a
sufficient amount of the compound to treat disorders, at a
reasonable 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 judgment. The
total daily dose of the compounds provided herein may range from
about 0.0001 to about 1000 mg/kg/day. For purposes of oral
administration, doses can be in the range from about 0.001 to about
50 mg/kg/day. If desired, the effective daily dose can be divided
into multiple doses for purposes of administration; consequently,
single dose compositions 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, general health, sex and diet of the
patient, the time of administration, 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.
[0055] The compounds provided can be formulated 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 vehicle 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 crystal size and crystalline form.
Prolonged absorption of an injectable pharmaceutical form can be
achieved by the use of absorption delaying agents such as aluminum
monostearate or gelatin.
[0056] The compound provided herein can be administered enterally
or parenterally in solid or liquid forms. Compositions suitable for
parenteral injection may comprise physiologically acceptable,
isotonic sterile aqueous or nonaqueous solutions, dispersions,
suspensions, or emulsions, and sterile powders for reconstitution
into sterile injectable solutions or dispersions. Examples of
suitable aqueous and nonaqueous carriers, diluents, solvents or
vehicles include water, ethanol, polyols (propyleneglycol,
polyethyleneglycol, 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 contain
adjuvants such as preserving, wetting, emulsifying, and dispensing
agents. Suspensions, in addition to the active compounds, may
contain suspending agents such as ethoxylated isostearyl alcohols,
polyoxyethylene sorbitol and sorbitan esters, microcrystalline
cellulose, aluminum metahydroxide, bentonite, agar-agar and
tragacanth, or mixtures of these substances.
[0057] The compounds provided herein can also be administered by
injection or infusion, either subcutaneously or intravenously, or
intramuscularly, or intrasternally, or intranasally, or by infusion
techniques in the form of sterile injectable or oleaginous
suspension. The compound may be in the form of a sterile injectable
aqueous or oleaginous 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
injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose any bland fixed oils may be conventionally employed
including synthetic mono- or diglycerides. In addition fatty acids
such as oleic acid find use in the preparation of injectables.
Dosage regimens can be adjusted 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.
[0058] Injectable dosage 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 employed, 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 compatible with body tissues.
The injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved or dispersed in sterile water or other sterile
injectable medium just prior to use.
[0059] 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 extenders such
as starches, lactose, sucrose, glucose, mannitol and silicic acid;
(b) binders such as carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidone, sucrose and acacia; (c) humectants such as
glycerol; (d) disintegrating agents such as agar-agar, 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) absorbents such as kaolin and bentonite
clay and (i) lubricants such as talc, calcium stearate, magnesium
stearate, 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
hard-filled gelatin capsules using such excipients as lactose or
milk sugar as well as high molecular weight polyethylene glycols
and the like.
[0060] The solid dosage forms of tablets, dragees, capsules, pills
and granules can be prepared with coatings and shells such as
enteric coatings and other coatings well-known in the
pharmaceutical formulating 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, optionally, in a delayed
manner. Examples of embedding compositions which can be used
include polymeric substances and waxes. Tablets contain the
compound in admixture 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 example, 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 provide 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.
[0061] Liquid dosage forms for oral administration include
pharmaceutically 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, isopropyl 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
glycols and fatty acid esters of sorbitan and mixtures thereof.
Besides inert diluents, the oral compositions may also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring and perfuming agents.
[0062] Aqueous suspensions contain the compound in admixture with
excipients suitable for the manufacture of aqueous suspensions.
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 example 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 ethylene
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 coloring agents, one or more flavoring agents, or one
or more sweetening agents, such as sucrose or saccharin.
[0063] 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 antioxidant 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 exemplified by those
already described above. Additional excipients, for example
sweetening, flavoring and agents, may also be present.
[0064] 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 phosphatides, for example soy bean,
lecithin, and esters or partial esters derived from fatty acids and
hexitol anhydrides, for example sorbitan monooleate, and
condensation products of the said partial esters with ethylene
oxide, for example polyoxyethylene 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 formulations may also contain a
demulcent, preservative and flavoring and coloring agent.
[0065] 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
.alpha.-synuclein/NAC fibril formation such as Parkinson's disease.
Compositions for rectal or vaginal administration 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 suppository 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.
[0066] Compounds provided herein can also be administered in the
form of liposomes. Methods to form liposomes are known in the art
(Prescott, Ed., Method 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
crystals 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).
[0067] The compounds provided herein can also be administered in
the form of a `prodrug` wherein the active pharmaceutical
ingredients are released in vivo upon contact with hydrolytic
enzymes such as esterases and phosphatases in the body. The term
"pharmaceutically acceptable prodrugs" as used herein represents
those prodrugs of the compounds provided herein, 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, commensurate with a reasonable benefit/risk
ratio, and effective for their intended use. A thorough discussion
is provided in T. Higuchi and V. Stella (Higuchi, T. and Stella, V.
Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S. Symposium
Series; Edward B. Roche, Ed., Bioreversible Carriers in Drug Design
1987, American Pharmaceutical Association and Pergamon Press),
which is incorporated herein by reference.
[0068] The compounds provided herein, or pharmaceutically
acceptable derivatives thereof, may also be formulated to be
targeted to a particular 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 examples of targeting methods, see, e.g., U.S. Pat.
Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865,
6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975,
6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542
and 5,709,874.
[0069] In one embodiment, liposomal suspensions, including
tissue-targeted liposomes, such as tumor-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 formulations may be prepared as described in
U.S. Pat. No. 4,522,811. Briefly, liposomes such as multilamellar
vesicles (MLVs) 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 compound provided herein in
phosphate buffered saline lacking 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
[0070] Also provided are sustained release formulations to deliver
the compounds to the desired target (i.e. brain) at high
circulating levels (between 10.sup.-9 and 10.sup.-4 M). In a
certain embodiment for the treatment of Parkinson's disease, the
circulating levels of the compounds are maintained up to 10.sup.-7
M. The levels are either circulating in the patient systemically,
or in one embodiment, present in brain tissue, and in other
embodiments, localized to the .alpha.-synuclein fibril deposits in
brain.
[0071] It is understood that the compound levels are maintained
over a certain period of time as is desired and can be easily
determined by one skilled in the art. In one embodiment, the
administration of a sustained release formulation is effected so
that a constant level of therapeutic compound is maintained between
10.sup.-8 and 10.sup.-6 M between 48 to 96 hours in the sera.
[0072] 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
U.S. Pat. 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; 5,354,556 and 5,733,566, the disclosures of
which are each incorporated herein by reference. These
pharmaceutical 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 systems, multilayer 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 provided herein. 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 contemplated
herein.
[0073] In one embodiment, the sustained release formulation
contains active compound such as, but not limited to,
microcrystalline cellulose, maltodextrin, ethylcellulose, and
magnesium stearate. As described above, all known methods for
encapsulation which are compatible with properties of the disclosed
compounds are contemplated herein. The sustained release
formulation is encapsulated by coating particles or granules of the
pharmaceutical compositions provided herein with varying thickness
of slowly soluble polymers or by microencapsulation. In one
embodiment, the sustained release formulation is encapsulated with
a coating material of varying thickness (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.
[0074] In another embodiment, the sustained 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
embodiment, the coated particles have a size range between about
0.1 to about 300 microns, as disclosed in U.S. Pat. Nos. 4,710,384
and 5,354,556, which are incorporated herein by reference in their
entireties. Each of the particles is in the form of a micromatrix,
with the active ingredient uniformly distributed throughout the
polymer.
[0075] Sustained release formulations such as those described in
U.S. Pat. No. 4,710,384, which is incorporated herein by reference
in its entirety, having a relatively 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 depending 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 tablets formed. If the medicament is released too quickly, then
more plasticizer is used. Release characteristics are also a
function of the thickness of the coating. When substantial amounts
of plasticizer are used, the sustained release capacity of the
coating diminishes. 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, in one embodiment 20 to 25%, and the
amount of coating will be from 10 to 25% of the weight of the
active material, and in another embodiment, 15 to 20% of the weight
of active material. Any conventional pharmaceutically acceptable
plasticizer may be incorporated into the coating.
[0076] The compounds provided herein can be formulated as a
sustained and/or timed release formulation. All sustained release
pharmaceutical products have a common goal of improving drug
therapy over that achieved by their non-sustained counterparts.
Ideally, the use of an optimally designed sustained release
preparation in medical treatment is characterized by a minimum of
drug substance being employed 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) increased 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.
[0077] The sustained release formulations provided herein are
designed to initially release an amount of the therapeutic
composition that promptly produces the desired therapeutic effect,
and gradually and continually 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.
[0078] 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.
[0079] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound. In
one embodiment, the compounds are formulated as controlled release
powders of discrete microparticles that can be readily formulated
in liquid form. The sustained release powder comprises particles
containing an active ingredient and optionally, an excipient with
at least one non-toxic polymer.
[0080] 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 alginic acid.
Representative synthetic polymers include those described, but not
limited to, those described in column 3, lines 33-45 of U.S. Pat.
No. 5,354,556, which is incorporated by reference in its entirety.
Particularly suitable polymers include those described, but not
limited to those described in column 3, line 46-column 4, line 8 of
U.S. Pat. No. 5,354,556 which is incorporated by reference in its
entirety.
[0081] The sustained release compositions provided herein may be
formulated for parenteral administration, e.g., by intramuscular
injections or implants for subcutaneous tissues and various body
cavities and transdermal devices. In one embodiment, intramuscular
injections 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.
[0082] A highly developed form of drug delivery that imparts
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.
Article of Manufacture
[0083] The compounds or pharmaceutically acceptable derivatives may
be packaged as articles of manufacture containing packaging
material, a compound or pharmaceutically acceptable derivative
thereof provided herein, which is effective for treatment,
prevention or amelioration of one or more symptoms of synuclein
diseases, within the packaging material, and a label that indicates
that the compound or composition, or pharmaceutically acceptable
derivative thereof; is used for treatment, prevention or
amelioration of one or more symptoms of synuclein diseases. The
articles of manufacture provided herein contain packaging
materials. Packaging materials for use in packaging pharmaceutical
products are well known to those of skill in the art. See, e.g.,
U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252. Examples of
pharmaceutical packaging materials include, but are not limited to,
blister packs, bottles, tubes, inhalers, pumps, bags, vials,
containers, syringes, bottles, and any packaging material suitable
for a selected formulation and intended mode of administration and
treatment. A wide array of formulations of the compounds and
compositions provided herein are contemplated as are a variety of
treatments for synuclein diseases.
E. Evaluation of the Activity of the Compounds
[0084] The biological activity of the compounds provided herein as
disruptors/inhibitors of Parkinson's disease .alpha.-synuclein
fibrils was assessed by determining the efficacy of the compounds
to cause a disassembly/disruption of pre-formed Parkinson's disease
.alpha.-synuclein fibrils. In one study, Thioflavin T fluorometry
was used to determine the effects of the compounds, and of a
negative control reference compound). In this assay Thioflavin T
binds specifically to fibrillar protein, and this binding produces
a fluorescence enhancement at 485 nm that is directly proportional
to the amount of fibrils present. The higher the fluorescence, the
greater the amount of fibrils present (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).
[0085] In the Congo red binding assay the ability of a given test
compound to alter .alpha.-synuclein fibril binding to Congo red was
quantified. In this assay, .alpha.-synuclein fibrils and test
compounds were incubated for 2 days and then vacuum filtered
through a 0.2 .mu.m filter. The amount of .alpha.-synuclein fibrils
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 compound) was indicative of the
test compound's ability to diminish/alter the amount of aggregated
and congophilic .alpha.-synuclein fibrils.
F. Combination Therapy
[0086] In another embodiment, the compounds may be administered in
combination, or sequentially, with another therapeutic agent. Such
other therapeutic agents include those known for treatment,
prevention, or amelioration of one or more symptoms of synuclein
diseases. Such therapeutic agents include, but are not limited to;
carbidopa/levodopa (Sinemet, Stalevo, Parcopa), dopamine agonists
(Apokyn, Parlodel, Neupro, Mirapex, Requip), anticholinergics
(Cogentin, Artane), MAO-B inhibitors (Eldepryl, Carbex, Zelapar,
Azilect), COMT inhibitors (Comtan, Tasmar), and other medications
like Symmetrel and Exelon.
G. Methods of Use of the Compounds and Compositions
[0087] The compounds and compositions provided herein are useful in
methods of treatment, prevention, or amelioration of one or more
symptoms of synucleopathies, including but not limited to diseases
associated with the formation, deposition, accumulation, or
persistence of synuclein fibrils. Also provided are methods to
inhibit or prevent .alpha.-synuclein/NAC fibril formation and/or
aggregation, methods to inhibit or prevent .alpha.-synuclein/NAC
fibril growth, and methods to cause disassembly, disruption, and/or
disaggregation of preformed .alpha.-synuclein/NAC fibrils and
.alpha.-synuclein/NAC-associated protein deposits.
[0088] In certain embodiments, the synuclein diseases or
synucleinopathies treated, prevented or whose symptoms are
ameliorated by the compounds and compositions provided herein
include, but are not limited to diseases associated with the
formation, deposition, accumulation, or persistence of synuclein
fibrils, including .alpha.-synuclein fibrils and/or aggregates. In
certain embodiments, such diseases include Parkinson's disease,
PDD, familial Parkinson's disease, Lewy body disease, the Lewy body
variant of Alzheimer's disease, dementia with Lewy bodies, multiple
system atrophy, and the Parkinsonism-dementia complex of Guam.
[0089] The following non-limiting Examples are given by way of
illustration only and are not considered a limitation of the
subject matter, many apparent variations of which are possible
without departing from the spirit or scope thereof.
EXAMPLES
Example 1--Synthesis of SA-52
##STR00008##
[0091] 3,4-dimethoxybenzoic acid (1) (1.00 g, 5.5 mmol) was
slurried in 5 mL of DCM. Oxalyl chloride (0.9 mL, 10.5 mmol) was
then added, and five drops of DMF were added to initiate the
reaction. The mixture was stirred overnight during which time it
became a yellow solution. The solution was concentrated and dried
in vacuo to remove the solvent and oxalyl chloride. The resultant
solid was redissolved in 7 mL of DCM, cooled to -78.degree. C., and
1.5 mL of pyridine was added. Then, 0.696 g of
2-bromo-4,5-dimethoxy aniline (3 mmol) was added in 5 mL of DCM. A
solid formed causing stirring to be difficult, and therefore, an
additional 6 mL portion of DCM was added. The mixture was warmed to
23.degree. C., and after the reaction was complete, quenched with
20 mL of water. The layers were separated, the organic was washed
with 20 mL of brine, dried with Na.sub.2SO.sub.4, and concentrated.
The crude product was purified by column chromatography and then
PTLC (0.5% MeOH in DCM as eluent) to give 1.05 g (88% yield) of
benzamide (2) as an off-white solid. .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta. 8.25 (bs, 2H, overlapping peak), 7.57 (d, J=2
Hz, 1H), 7.48 (dd, J=2 Hz, 8.4 Hz, 1H), 7.06 (s, 1H), 6.98 (d,
J=8.4 Hz, 1H), 4.09-3.99 (3 overlapping singlets, 9H), 3.90 (s,
3H).
[0092] To 0.049 g (0.14 mmol) of 2 in 3 mL of DCM was added 2.5 mL
1 M BBr.sub.3 in DCM. The solution was stirred 18 h. The mixture
was quenched with 10 mL MeOH and concentrated. The concentrate was
diluted again with 5 mL of MeOH, and 20 mL of MeOH was added. The
resultant solid was filtered, collected, and dried under vacuum
overnight to give 0.012 g (29% yield) of SA-52 as a brown solid.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. HRMS calculated for
C.sub.13H.sub.11BrNO.sub.5 (M+H).sup.+ 339.9821, found
339.9828.
Example 2--Synthesis of SA-53
##STR00009##
[0094] To 0.401 g (1.5 mmol) of 2-bromo-4,5-dimethoxybenzoic acid
(3) in 4 mL of DCM was added 0.4 mL (4.7 mmol) of oxalyl chloride,
and 1 drop of DMF. The mixture was stirred for 7 h, concentrated,
dried in vacuo in a similar manner to compound 2, and diluted with
5 mL of DCM. This solution was cooled to -78.degree. C., treated
with 0.8 mL pyridine and 0.137 (1 mmol) 3,4-methylenedioxy aniline.
The mixture was then brought to 23.degree. C., stirred 16 h,
quenched with 10 mL of water, and the resultant layers separated.
The organic layer was washed twice with 10 mL of water, dried with
Na.sub.2SO.sub.4, and concentrated. The crude product was purified
by PTLC using 10% EtOAc in DCM as the eluent to give 0.310 g (82%
yield) of benzamide 4 as a brown solid. .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta. 7.90 (bs, 1H), 7.40 (bs, 1H), 7.27 (d, J=2.6
Hz, 1H), 7.15 (s, 1H), 6.96 (d, J=8.6 Hz, 1H), 6.81 (d, J=8.6 Hz,
1H), 6.00 (s, 2H), 3.93 (s, 6H), HRMS calculated for
C.sub.16H.sub.15BrNO.sub.5 (M+H).sup.+ 380.0134, found
380.0145.
Example 3--Synthesis of SA-54
##STR00010##
[0096] To 0.230 g (0.58 mmol) of benzamide 2 was added 0.017 g of
CuI (0.09 mmol), 0.031 g (0.17 mmol) of 1,10 phenanthroline, and
0.560 g (1.7 mmol) of Cs.sub.2CO.sub.3. The mixture was suspended
in 5 mL of diglyme, and heated to 140.degree. C. for 20 h. The
mixture was diluted with 30 mL DCM, washed three times with 20 mL
of water, dried (Na.sub.2SO.sub.4), and concentrated to give an
orange oil as the crude product. This was heated under vacuum (1 mm
Hg) until a light orange solid formed to give 0.135 g (75% yield)
of benzoxazole 5, which was used without further purification.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 7.74 (dd, J=2.0 Hz, 8.4
Hz, 1H), 7.67 (d, J=2.2 Hz, 1H), 7.21 (s, 3H), 7.10 (s, 3H), 6.95
(d, J=8.4 Hz, 1H), 3.98-3.89 (4 overlapping singlets, 12H).
[0097] To 0.071 g (0.23 mmol) of 5 in 5 mL of DCM at 0.degree. C.
was added 2 mL 1M BBr.sub.3 in DCM. The dark brown mixture was
stirred 6 h, quenched with 5 mL MeOH, and concentrated. The MeOH
dilution-concentration procedure was repeated three more times to
give 0.081 g of crude product. This product was purified by PTLC
using 5% MeOH/DCM followed by 10% MeOH/DCM as the eluent and gave
0.023 g (39% yield) of SA-54 as an off-white solid. .sup.1H NMR
(200 MHz) .delta..quadrature.8.84 (bs, 2H), 8.34 (bs, 1H), 7.35
(bs, 1H), 7.47 (d, J=2 Hz, 1H), 7.40 (dd, J=2 Hz, 8.2 Hz, 1H), 7.05
(s, 2H), 7.01 (s, 2H). HRMS Calculated for C.sub.13H.sub.10NO.sub.5
(M+H).sup.+ 260.0586. found 260.0559.
Example 4--Synthesis of SA-55
##STR00011##
[0099] To 10.00 g (51 mmol) of methyl 3,4 dimethoxybenzoate (6) in
50 mL of AcOH at 0.degree. C. was added 8.90 g (56 mmol) of
Br.sub.2 in 50 mL of AcOH over 1.5 h. The ice bath was removed and
the mixture stirred 45 min. The reaction was quenched by pouring
into 700 mL of H.sub.2O, stirred 30 min, left quiescent for 1 h,
and filtered. The collected solid was washed with H.sub.2O and
washed with sat. aq. Na.sub.2S.sub.2O.sub.3. The solid was
partially dried, dissolved in 300 mL hot MeOH, and the resultant
solution was cooled. The cool methanolic solution of product was
treated with 200 mL of H.sub.2O and the white solid filtered to
give 8.92 g (64% yield) of methyl-2-bromo-4,5-dimethoxybenzoate (7)
as a white powder. The compound matched the physical and spectral
properties of the known compound.
[0100] A mixture of 0.960 g (3.48 mmol) of 7, 1.60 g (35 mmol) of
hydrazine hydrate (62% hydrazine), and 5 mL of EtOH was refluxed
for 15 h. The mixture was cooled to -20.degree. C., vacuum
filtered, washed with 50 mL of ice-cold 1:1 EtOH:H.sub.2O, and
dried to give 0.832 g (87% yield) of (2-bromo-4,5-dimethoxy
benzoyloxy) hydrazine (8) as a white, needle-like crystalline
solid. The above procedure was repeated on 2.79 g of the starting
ester 7 to result in 2.77 g (99% yield) of 8. .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta. 6.97 (s, 1H), 6.86 (s, 1H), 3.85 (s, 6H). HRMS
Calculated for C.sub.9H.sub.12O.sub.3N.sub.2Br 275.0031, found
275.0037.
[0101] To 2.15 g (11.8 mmol) of 3,4-dimethoxybenzoic acid (1) in 10
mL of DCM was added sequentially 2.5 mL (29.1 mmol) oxalyl chloride
and 0.2 mL of DMF. The mixture was stirred for 16 h during which
time it became a clear, light yellow solution. This solution was
concentrated and dried thoroughly to remove the excess oxalyl
chloride to generate the crude acid chloride as a light yellow
solid. This solid was taken up in 20 mL of DCM, the solution cooled
to 0.degree. C., and treated with 10 mL of pyridine and 0.25 g of
DMAP. The resultant solution was treated with 1.69 g (6.15 mmol) of
8 in 10 mL DCM and 10 mL of pyridine. The mixture was stirred 3 h
at 0.degree. C. and warmed to 23.degree. C. The reaction was
stirred an additional 16 h, concentrated, taken up in 50 mL of
EtOAc, and the layers separated. The aqueous was extracted once
more with 50 mL EtOAc. The combined organic layers were washed
three times with 100 mL H.sub.2O, dried (Na.sub.2SO.sub.4), and
concentrated. The concentrate was purified by Flash 40+M column
chromatography (Biotage) eluting first with 150 mL of 1:1 EtOAc/Hex
and then 2 L 5:1 EtOAc/Hex to give 1.27 g (47% yield) of hydrazide
9 as a yellow-brown powder. The reaction was repeated using 2.20 g
of 3,4 dihydroxybenzoic acid (1) and 2.77 g of hydrazine 8 to give
2.60 g (49% yield) of hydrazide 9. .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta. 9.91 (d, J=5.2 Hz, 1H), 9.53 (d, J=5.0 Hz, 1H),
7.46 (dd, J=2.0 Hz, 8.4 Hz, 1H), 7.41 (d, J=2.2 Hz, 1H), 7.23 (s,
1H), 6.98 (s, 1H), 6.80 (d, J=8.4 Hz, 1H), 3.96-3.89 (4 overlapping
singlets, 12H).
[0102] A solution of 0.352 g of intermediate 9 in 3 mL of
POCl.sub.3 was refluxed for 3 h. The reaction mixture is cooled,
poured into 125 mL of water, and sonicated for one minute. The
suspension was allowed to stand for 1 h, and the solid was
filtered, washed with excess water, collected, and air-dried to
give 0.302 g (90% yield) of brominated tetramethoxyoxadiazole (10)
as a white solid. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 7.71
(dd, J=2.0 Hz, 8.4 Hz, 1H), 7.67 (d, J=2.0 Hz, 1H), 7.57 (s, 1H),
7.17 (s, 1H), 6.98 (d, J=8.2 Hz, 1H), 3.96 (4 overlapping singlets,
12H). HRMS Calculated for C.sub.14H.sub.10BrN.sub.2O.sub.5
(M+H).sup.+ 421.0339, found 421.0334.
[0103] A mixture of 0.038 g of intermediate 10 in 3 mL of DCM was
cooled to -78.degree. C., and treated dropwise with a solution of
0.450 g of BBr.sub.3 in 5 mL of DCM. The mixture was stirred at
-78.degree. C. for 1 h, then at 23.degree. C. for 2.5 h. The
mixture was quenched by adding it carefully to 5 mL of MeOH in a
100 mL flask. The methanol solution was concentrated to 1 mL,
diluted with 5 mL water, and filtered to give 0.017 g (56% yield)
of SA-55 as a light yellow solid. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 10.5-9.5 (overlapping broad singlets, 4H), 7.56 (d, J=2 Hz,
1H), 7.52 (s, 1H), 7.48 (dd, J=2 Hz, 8.6 Hz, 1H), 7.25 (s, 1H),
7.04 (d, J=8.4 Hz, 1H)
Example 5--Synthesis of SA-57
##STR00012##
[0105] To a thick-walled 15 mL tube with a resealable Teflon
screw-cap was added 0.584 g (4.32 mmol) of 3,4-dihydroxbenzonitrile
(12), 5 mL of triethylene glycol, 1.172 g of NaSH.xH.sub.2O, and
0.25 mL of concentrated H.sub.2SO.sub.4. The tube was sealed with
the cap, and the mixture was warmed to 110.degree. C. and stirred
for 3 days at this temperature. The reaction was quenched by
pouring into 100 mL sat. aq. NH.sub.4.sup.+Cl, and extracted twice
with 50 mL of EtOAc. The combined organics were washed three times
with 15 mL water, dried with NaSO.sub.4, and concentrated to give
0.512 g (71% yield) of 13 as a golden colored solid. .sup.1H NMR
(200 MHz, CDCl.sub.3) .delta. 9.67 (s, 1H), 9.29 (s, 1H), 7.56 (dd,
J=1.8 Hz, 8.2 Hz), 7.49 (d, J=1.8 Hz), 6.94 (d, J=8.2 Hz), 6.09 (s,
2H). .sup.13C NMR (50 MHz, CDCl.sub.3) .delta. 198.9, 150.5, 147.4,
133.7, 123.3, 108.2, 107.8, 102.3.
[0106] A solution of 0.40 g (2.37 mmol) of 13 in 7 mL of DMSO was
treated with 12 drops of concentrated HCl, warmed to 38.degree. C.
for 18h, and poured into 25 mL of brine. The resultant solid was
filtered and washed with water to give 0.21 g (59% yield) of SA-57
as a yellow solid. .sup.1H-NMR (DMSO-d6) 9.85 (bs, 1H) 9.52 (bs,
1H), 9.43 (bs, 1H), 9.28 (bs, 1H), 7.69 (d, 1H, J=2 Hz), 7.59 (dd,
1H, J=2.2, 8.2 Hz), 7.46 (d, 1H, J=2 Hz), 7.38 (dd, 1H, J=2.2, 8.2
Hz), 6.90 (d, 1H, J=8.2 Hz), 6.86 (d, 1H, J=8.4 Hz). .sup.13C-NMR
(DMSO-d6) 187.8, 173.4, 150.2, 148.5, 146.4, 145.9, 124.6, 122.0,
120.4, 120.2, 116.8, 116.2, 115.7, 114.5. HRMS-ESI Calculated for
C.sub.14H.sub.11N.sub.2O.sub.4S (M+H).sup.+ 303.0440, found
303.0448.
Example 6--Synthesis of SA-58
##STR00013##
[0108] To a solution of 2,5-dibromothiophene (14) (242 mg, 1 mmol),
(3, 4-dimethoxy phenyl)boronic acid (15) (455 mg, 2.5 mmol), and
Pd(PPh.sub.3).sub.4 (58 mg, 0.05 mmol) in dioxane (10 mL) was added
Na.sub.2CO.sub.3 (12 mL, 2.0 M aqueous solution). The resultant
mixture was purged with nitrogen and stirred rapidly while heating
at 90.degree. C. overnight. The reaction mixture was cooled to
23.degree. C., acidified with 1M HCl and extracted with EtOAc. The
combined organic extracts were washed with H.sub.2O, dried over
MgSO.sub.4, filtered and concentrated under reduced pressure.
2,5-bis(3',4'-dimethoxyphenyl)thiophene (16) was obtained
quantitatively as a green-yellow solid after the purification by
column chromatography (10%-20% EtOAc in hexanes).
[0109] To a solution of 2,5-bis(3',4'-dimethoxyphenyl)thiophene 16
(110 mg, 0.3 mmol) in dry dichloromethane at -78.degree. C. was
added BBr.sub.3 (3 mL, 1M solution in DCM, 2.5 equiv per methoxy
function) dropwise. The reaction mixture was stirred at -78.degree.
C. for 3 h, warmed to 23.degree. C., and stirred 16 h under
nitrogen atmosphere. Water (10 mL) was added to quench the
reaction, and the aqueous layer was extracted with EtOAc. The
combined organic layer was washed with brine, dried over
MgSO.sub.4, filtered, and concentrated under reduced pressure. The
product was purified by recrystallization in MeOH/DCM and SA-58 was
obtained quantitatively as a greenish solid.
Example 7--Synthesis of SA-59, SA-60 and SA-61
##STR00014## ##STR00015##
[0111] 3-bromo-2,5-bis(3',4'-dimethoxyphenyl)thiophene (18) was
prepared by the reaction of 2,3,5-tribromothiophene (17) (321 mg, 1
mmol) and (3, 4-dimethoxyphenyl)boronic acid (15) (419 mg, 2.3
mmol) according to the similar procedure for compound 16. The
reaction mixture was purified by column chromatography (10%-30%
EtOAc in hexanes) and afforded 18 (337 mg, 77% yield) as a yellow
solid. SA-60 was also isolated (97 mg, 20% yield) from the reaction
above as a dark yellow solid.
[0112] SA-59 was prepared by the reaction of
3-bromo-2,5-bis(3',4'-dimethoxyphenyl) thiophene (18) (258 mg, 0.59
mmol) and BBr.sub.3 (6 mL, 6 mmol) according to the similar
procedure for compound SA-58. SA-59 (157 mg, 70% yield) was
obtained after preparative thin layer chromatography (PTLC)
purification (10% MeOH in DCM) as a green solid.
[0113] SA-61 was prepared by the reaction of
2,3,5-tri(3',4'-dimethoxyphenyl) thiophene (SA-60) (68 mg, 0.14
mmol) and BBr.sub.3 (2 mL, 2 mmol) according to the similar
procedure for compound SA-58. SA-61 (34 mg, 60% yield) was obtained
after PTLC purification (10% MeOH in DCM) as a brown oil.
Example 8--Synthesis of SA-62
##STR00016##
[0115] Compound 19 was prepared by the reaction of
2,5-dibromothiophene (17) (1.14 g, 5.24 mmol) and
(3,4-dimethoxyphenyl) boronic acid (15) (910 mg, 5 mmol) according
to the similar procedure for compound 16. The reaction mixture was
purified by flash column chromatography (FCC) (5%-20% EtOAc in
hexanes) and afforded compound 19 (509 mg, 34% yield) as a
yellowish crystal. Compound 16 was also isolated (578 mg, 65%
yield) as a yellow solid.
[0116] SA-62 was prepared by the reaction of
2-bromo-5-(3,4-dimethoxyphenyl)thiophene (19) (60 mg, 0.2 mmol) and
BBr.sub.3 (1M in DCM, 1 mL, 1 mmol) according to the similar
procedure for compound SA-52 and isolated as a green solid in
quantitative yield.
Example 9--Synthesis of SA-63
##STR00017## ##STR00018##
[0118] Compound 21 was prepared by the reaction of
2-bromo-5-(3,4-dimethoxyphenyl) thiophene (19) (449 mg, 1.5 mmol)
and (3,4,5-trimethoxyphenyl)boronic acid (20) (382 mg, 1.8 mmol)
according to the similar procedure for compound 16 and purified by
column chromatography (10%/-20% EtOAc in hexanes), then
recrystallization (EtOAc) provided the desired compound as a yellow
solid (524 mg, 91% yield).
[0119] SA-63 was prepared by the reaction of
2-(3,4-dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)thiophene (21)
(47 mg, 0.12 mmol) and BBr.sub.3 (1M in DCM, 0.8 mL, 0.8 mmol)
according to the similar procedure for compound SA-52. SA-63 was
obtained quantitatively as dark blue solid.
Example 10--Synthesis of SA-64
##STR00019##
[0121] To a solution of dioxane/EtOH/H.sub.2O (6 mL, 1/1/1) in a
microwave reaction vial (Biotage) was added 2,5-dibromothiazole
(22) (122 mg, 0.5 mmol), (3,4-dimethoxyphenyl)boronic acid (15)
(218 mg, 1.2 mmol), Pd(PPh.sub.3).sub.4 (29 mg, 0.025 mmol) and
Cs.sub.2CO.sub.3 (0.72 g, 2.2 mmol). The mixture was purged with
nitrogen and heated in a microwave reactor (Biotage) to 160.degree.
C. for 1 h. The reaction mixture was cooled to 23.degree. C.,
acidified with 1M HCl until the pH was 1, and extracted with EtOAc.
The combined organic extracts were washed with H.sub.2O before
being dried over MgSO.sub.4, filtered, and concentrated under
reduced pressure. Compound 24 was purified by column chromatography
(5%-35% EtOAc in hexanes) as brown-yellow solid (28 mg, 16% yield).
5-bromo-2-(3,4-dimethoxyphenyl)thiazole (23) was also isolated from
the reaction above as brown-yellow crystalline solid (28 mg, 19%
yield).
[0122] SA-64 was prepared by the reaction of
2,5-bis(3,4-dimethoxyphenyl)thiazole (24) (28 mg, 0.078 mmol) and
BBr.sub.3 (1M in DCM, 0.75 mL, 0.75 mmol) according to the similar
procedure for compound SA-58, and was obtained as a yellow solid
(14 mg, 60% yield).
Example 11--Synthesis of SA-65
[0123] A mixture of 0.100 g (0.237 mmol) of 10, 0.600 g (1.03 mmol)
of hexabutylditin, 5 mL of PhMe, and 1 mL of TEA was degassed by
nitrogen purge. Then 0.050 g of Pd(PPh.sub.3).sub.4 was added. The
reaction mixture was refluxed for 16 h. At this time, the reaction
was incomplete, but a decomposition product was seen in addition to
the desired product. The reaction was at this point concentrated
and purified by PTLC to prevent further decomposition to give 0.090
g (60% yield) of SA-65 as an off-white solid. .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta. 7.68-7.65 (overlapping peak doublet and doublet
of doublets, 2H), 7.53 (s, 1H), 7.16 (s, 1H), 6.98 (d, J=9.0 Hz,
1H), 3.99-3.96 (4 overlapping singlets, 12H), 1.55 (m, 6H), 1.44
(m, 6H), 1.15 (m, 6H), 0.84 (t, J=7.2 Hz, 9H).
Example 12--Synthesis of SA-66
##STR00020##
[0125] A mixture of 0.174 g of Lawesson's reagent, 0.175 g of
compound 9, and 20 mL of PhMe was heated to 60.degree. C. for 3 h.
The mixture was concentrated, and applied directly to a PTLC plate
for purification. The brominated thiadiazole 11 was purified by
PTLC and the middle of the desired product spot (fluoresces blue
under UV light) was collected to give 0.112 g (65% yield) of
compound 11 as an off-white to brown solid.
[0126] The above experiment was repeated using 1.13 g of 9, 1.21 g
of Lawesson's reagent, and 200 mL of PhMe. The mixture was refluxed
3 h, and quenched with 150 mL of water. The layers were separated,
and the aqueous extracted twice with 30 mL of EtOAc. The organic
layers were combined and washed twice with 50 mL 1 N aq. HCl, twice
with 50 mL saturated aq. NaHCO.sub.3, once with 25 mL of water, and
once with 50 mL of brine. The organic was dried, concentrated, and
purified by PTLC to give 1.04 g of the desired 11 as a yellow solid
(97% yield). .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 7.88 (s,
1H), 7.68 (bs, 1H), 7.51 (d, J=7.8 Hz, 1H), 7.15 (s, 1H), 6.95 (d,
J=8.4 Hz, 1H), 4.00-3.95 (4 overlapping singlets, 12H).
[0127] A solution of 0.051 g of 11 in 5 mL DCM was cooled to
-78.degree. C., treated with 1.5 mL of 2 M BBr.sub.3 in DCM,
stirred at -78.degree. C. for 0.5 h, and stirred at 23.degree. C.
for 3 h. The mixture was quenched with water carefully, poured into
100 mL brine, and extracted twice with 75 mL of EtOAc. The combined
organic layers were dried and concentrated to yield 36 mg (81%
yield) of the crude title compound as a yellow solid. The mixture
was recrystallized from hot MeOH and precipitated with water to
give 0.021 g (50% yield) of SA-66 as a brown solid.
[0128] The above experiment was repeated on 0.077 g of starting
material using the same procedure with the exception of slightly
different stirring times (1 h at -78.degree. C., 4 h at 23.degree.
C.) to give 0.054 g (83% yield) of SA-66. .sup.1H NMR (200 MHz,
DMSO-d.sub.6) .delta. 10.10 (s, 1H), 9.78 (s, 1H), 9.68 (s, 1H),
9.47 (s, 1H), 7.55 (s, 1H), 7.43 (d, J=1.8 Hz, 1H), 7.29 (dd, J=1.8
Hz, 8.0 Hz, 1H), 7.13 (s, 1H), 6.87 (d, J=8.2 Hz, 1H).
Example 13--Synthesis of SA-67
##STR00021##
[0130] To 0.285 g (1.57 mmol) of 3,4 dimethoxybenzoic acid (1) in 5
mL of DCM was added 0.3 mL (3.5 mmol) of oxalyl chloride and one
drop of DMF. The mixture was stirred for 2 h, quenched with
hydrazine hydrate (3 mL), concentrated, and dried. The solid was
taken up in 25 mL water, sonicated five minutes, filtered, and the
resultant solid washed with 40 mL water. The solid was dried, taken
up in 20:1 DCM:DMF, and purified by PTLC (8:2 EtOAc:Hexanes) to
give 0.056 g (20% yield) of 25 as an off-white solid. .sup.1H NMR
(200 MHz, DMSO-d.sub.6) .delta. 10.30 (s, 2H), 7.58 (d, J=8.4 Hz,
1H), 7.51 (bs, 1H), 7.18 (d, J=8.4 Hz, 1H), 3.82 (bs, 12H).
[0131] To 0.050 g (0.14 mmol) of hydrazide 25 was added 3 mL of
POCl.sub.3. The mixture was refluxed 2 h, quenched with 50 g of
ice, allowed to warm to 23.degree. C., and extracted twice with 25
mL of EtOAc. The organic layers were washed once with 25 mL water,
twice with 25 mL of brine, dried with Na.sub.2SO.sub.4, and
concentrated to give 0.043 g (93% yield) of 26 as a white solid.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 7.72 (d, J=8.0 Hz, 2H),
7.60 (d, J=1.2 Hz, 1H), 7.07 (d, J=8.4 Hz, 1H), 3.89 (s, 6H), 3.87
(s, 6H).
[0132] To 0.038 g (0.11 mmol) of oxadiazole 26 was added 3 mL of
DCM. The solution was cooled to -78.degree., and 5 mL of DCM
containing 0.450 g (1.8 mmol) of BBr.sub.3 was added. The mixture
was stirred at -78.degree. C. for 1 h, at 23.degree. C. for 2.5 h,
quenched by pouring into 5 mL of MeOH, and concentrated to 1 mL.
The resultant oil was diluted with 5 mL water and the resultant
precipitate was filtered to give 0.017 g (56% yield) of SA-67 as a
yellow solid. .sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 9.74 (bs,
2H), 9.51 (bs, 2H), 7.43 (d, J=2.0 Hz, 1H), 7.37 (dd, J=2.0 Hz, 8.4
Hz, 1H), 6.93 (d, J=8.0 Hz, 1H).
Example 14--Synthesis of SA-68
##STR00022##
[0134] 4-azido-1,2-dimethoxybenzene (27) (62 mg, 0.31 mmol),
4-ethynyl-1,2-dimethoxy benzene (28) (52 mg, 0.31 mmol), sodium
ascorbate (0.25 mL of 1M solution, 0.25 mmol), CuSO.sub.4 (0.020 mL
of 1M solution, 0.020 mmol) and tBuOH/H.sub.2O (2 mL, 1/1) were
added to a vial. The reaction mixture was purged with nitrogen,
stirred at 23.degree. C. overnight, poured into water at 0.degree.
C., and the resultant brown solid was filtered. This solid was
washed with water (1 mL) and Et.sub.2O (1 mL). Compound 29 (99 mg,
93% yield) was used in the next step without further
purification.
[0135] SA-68 was prepared by the reaction of
1,4-bis(3,4-dimethoxyphenyl)-1H-1,2,3-triazole (29) (67 mg, 0.20
mmol) and BBr.sub.3 (1M in DCM, 0.79 mL, 0.79 mmol) according to
the similar procedure for compound SA-52. SA-68 was obtained as a
dark brown solid (56 mg, 98% yield).
Example 15--Synthesis of SA-69
##STR00023##
[0137] To a solution of dioxane/EtOH/H.sub.2O (6 mL, 1/1/1) were
added 2,5-dibromothiazole (22) (756 mg, 3.1 mmol), (3,
4-dimethoxyphenyl)boronic acid (15) (380 mg, 2.1 mmol),
Pd(PPh.sub.3).sub.4 (58 mg, 0.05 mmol) and Cs.sub.2CO.sub.3 (1.4 g,
4.4 mmol) in a 20 mL microwave reaction vial (Biotage). The
solution was purged with nitrogen and heated in a microwave reactor
(Biotage) to 160.degree. C. for 30 min. The reaction mixture was
cooled to 23.degree. C., acidified with 1M HCl until pH=1, and
extracted with EtOAc. The combined organic extracts were washed
with H.sub.2O before being dried over MgSO.sub.4, filtered, and
concentrated under reduced pressure. Compound 23 was purified by
flash column chromatography (8%-50% EtOAc in hexanes) as
brown-yellow solid (193 mg, 32% yield).
2,5-bis(3,4-dimethoxyphenyl)thiazole (24), was also isolated as a
brown-yellow solid (34 mg, 5% yield).
[0138] SA-69 was prepared by the reaction of
5-bromo-2-(3',4'-dimethoxyphenyl)thiazole (23) (78 mg, 0.26 mmol)
and BBr.sub.3 (1M in DCM, 0.65 mL, 0.65 mmol) according to the
similar procedure for compound SA-52, and was obtained as a
brown-yellow solid quantitatively.
Example 16--Synthesis of SA-70
##STR00024##
[0140] A mixture of 0.996 g (6 mmol) 3,4-dimethoxybenzaldehyde
(30), 1.25 g of TosMIC (6.4 mmol), and 0.923 g (6.6 mmol) of
K.sub.2CO.sub.3 was refluxed in 30 mL of MeOH for 3 h. The reaction
mixture was quenched by pouring into 200 mL of a 1:1 mixture of
brine and water, cooled to -20.degree. C. for 1 h, and the
resultant solid filtered to give 0.952 g (77% yield) of 31 as an
off-white solid. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 7.85 (s,
1H), 7.22 (s, 1H), 7.20 (overlapping peak, 1H), 7.15 (dd, J=2.0 Hz,
12.4 Hz, 1H), 6.88 (d, J=1H), 3.91 (s, 3H), 3.88 (s, 3H).
[0141] A suspension of 0.212 g of Na.sub.2CO.sub.3 (2 mmol), 0.262
g of PPh.sub.3 (1 mmol), 0.206 g (1 mmol) of 31 and 0.316 g of
4-iodoveratrole (1.2 mmol) was formed in 1 mL of DMF. Then, 0.190 g
(1 mmol) of CuI was added. The mixture was stirred at 160.degree.
C. for 3 h and quenched by pouring into 50 mL of water containing
5% NH.sub.4.sup.+OH.sup.-. The product was extracted with 50 mL
DCM, the organic layer dried and concentrated, and the crude
product purified by PTLC to give 0.174 g (51% yield) of 32 as a
yellow solid. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 7.60 (d,
J=8.4 Hz, 1H), 7.54, (bs, 1H), 7.24 (s, 1H), 7.19 bs, 1H), 7.10 (s,
1H), 6.90-6.83 (m, 2H), 3.91-3.86 (4 overlapping multiplets, 12H).
.sup.13C NMR (50 MHz, CDCl.sub.3) .delta. 160.7, 150.9, 149.3,
149.2, 132.1, 132.0, 122.0, 121.2, 120.4, 119.3, 117.1, 111.5,
111.1, 109.1, 107.5, 56.01, 55.92.
[0142] A mixture of 0.075 g (0.22 mmol) of 32 in 15 mL DCM was
treated with 0.500 g (2 mmol) of BBr.sub.3 at -78.degree. C. The
mixture was stirred at -78.degree. C. for 0.5 h, then 2 h at
23.degree. C. The reaction was then quenched with 5 mL MeOH,
concentrated to 1 mmol and diluted with 5 mL of water. The
resultant precipitate was filtered to give 0.021 g (33% yield) of
SA-70 as a yellow solid. .sup.1H NMR (200 MHz, DMSO-d.sub.6)
.delta. 7.40 (s, 2H), 7.31 (dd, J 2.0 Hz, 8.0 Hz, 1H), 7.13 (d,
J=2.2 Hz), 7.05 (dd, J 2.2 Hz, 8.2 Hz, 1H), 6.86 (d, J=7.8 Hz, 1H),
6.82 (d, J=7.8 Hz, 1H). HRMS Calculated for
C.sub.15H.sub.12NO.sub.5 (M+H).sup.+ 286.0715, found 286.0717.
Example 17--Synthesis of SA-72
##STR00025##
[0144] A solution of 0.115 g (0.32 mmol) of 25 in 30 mL of PhMe was
treated with 0.140 g of Lawesson's reagent (0.35 mmol) and stirred
3 h at 100.degree. C. The reaction mixture was poured into 75 mL
water, shaken vigorously, and extracted with 50 mL EtOAc. The
organic layers were combined, washed with 25 ml saturated aqueous
NaHCO.sub.3, washed with 50 mL brine, dried, and concentrated. The
crude product was purified by PTLC to give 0.088 g (74% yield) of
33 as a yellow solid. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.
7.60 (bs, 2H), 7.37 (d, J=8.2 Hz, 2H), 6.86 (d, J=8.4 Hz, 2H), 3.95
(s, 6H), 3.90 (s, 6H). .sup.13C NMR (200 MHz, CDCl.sub.3) 167.3,
151.6, 149.4, 123.2, 121.6, 111.2, 110.0, 56.1, 56.0. HRMS
Calculated for C.sub.16H.sub.19N.sub.2O.sub.4S (M+H).sup.+
359.1066, found 359.1061.
[0145] A solution of 0.077 g (0.22 mmol) of 33 and 3 mL DCM was
treated with 0.250 g BBr.sub.3 at -78.degree. C. The reaction
mixture was stirred 1 h, treated with 0.250 g BBr.sub.3, and
stirred an addition 15 min at -78.degree. C. The mixture was warmed
to 23.degree. C., stirred for 1 h, quenched with 10 mL MeOH, and
concentrated. The concentrate was taken up in 1 mL MeOH, warmed
until a solution, and precipitated with 10 mL water. The resultant
solid was filtered and dried to give 0.054 g of SA-72 (83% yield)
as an off-white solid. .sup.1H NMR (200 MHz, DMSO-d.sub.6) 10.0-9.0
(broad peak, 4H), 7.41 (d, J=2.2 Hz, 2H), 7.24 (dd, J=2.2 Hz, 8.2
Hz, 1H), 6.87 (d, J=8.2 Hz, 2H).
Example 18--Synthesis of SA-74
##STR00026##
[0147] To a mixture of 0.219 g (0.5 mmol) of 11 in 20 mL PhMe was
added 1.09 g (Bu.sub.3Sn).sub.2 and 2 mL of triethylamine. The
mixture was degassed via a nitrogen purge, treated with 0.150 g
(0.13 mmol) of Pd(PPh.sub.3).sub.4, and refluxed for 15 h. The
mixture was concentrated and purified directly by PTLC by eluting
the plates with 1% TEA in hexanes then 50% EtOAc in hexanes with 1%
TEA successively. This gave 0.224 g (69% yield) SA-74 as an
off-white solid.
Example 19--Synthesis of SA-75 and SA-76
##STR00027##
[0149] A solution of 0.051 g of SA-74 in 3 mL of DCM was treated
with a 1 M iodine in DCM solution until the orange/yellow color of
the iodine persists. The mixture was quenched with a 1 M solution
of KF in MeOH (2 mL) then sat. aq. Na.sub.2S.sub.2O.sub.3 (2 mL).
The mixture was extracted twice with 10 mL EtOAc. The organic
layers were combined, washed with 10 mL water, dried and
concentrated to give the crude title compound. The crude product
was purified by PTLC with another run of this reaction using 0.082
g of SA-74 and run in the same manner as described above. This gave
0.012 g (12% yield) of SA-75 as a yellow solid. .sup.1H NMR (200
MHz, CDCl.sub.3) .delta. 7.73 (d, J=2.2 Hz, 1H), 7.59 (s, 1H), 7.54
(dd, J=2.2 Hz, 8.4 Hz), 7.42 (s, 1H), 6.98 (d, J=8.4 Hz, 1H),
4.03-3.95 (4 overlapping singlets, 12H).
[0150] A solution of 0.008 g of SA-75 in 1 mL of DCM was cooled to
0.degree. C., treated with 0.5 mL of 1 M BBr.sub.3 in DCM, and
stirred for 1 h. The reaction was treated with an additional 1 mL
portion of 1 M BBr.sub.3 in DCM, stirred an additional 2 h at
0.degree. C., and warmed to 23.degree. C. The reaction mixture was
stirred an additional 0.5 h, quenched with 3 mL MeOH, and
concentrated to 0.5 mL. The product was precipitated with 5 mL
water, and the resultant yellow solid was filtered and dried to
give (0.002 g) of SA-76 as a yellow-green solid. .sup.1H NMR (200
MHz, DMSO-d.sub.6) .delta. 9.93-9.50 (overlapping broad singlets,
4H), 7.42-7.28 (overlapping irresolvable peaks, 4H), 6.98 (d, J=8.2
Hz, 1H).
Example 20--Synthesis of SA-77 and SA-78
##STR00028##
[0152] To a round-bottom flask charged with
5-(5-(3,4-dihydroxyphenyl)thiophen-2-yl)benzene-1,2,3-triol (SA-63)
(145 mg, 0.46 mmol), 4-methylbenzenesulfonic acid (p-TsOH.H.sub.2O)
(8.7 mg, 0.046 mmol), and 2,2-dimethoxypropane (0.45 mL, 3.7 mmol)
were added acetone (3 mL) and benzene (15 mL). A short column with
4 A molecular sieves and a condenser were installed on the flask.
The reaction was refluxed for 15 h. An additional portion of
2,2-dimethoxypropane (0.45 mL, 3.7 mmol) was added, and the
reaction was refluxed for another 24 h. The solution was
concentrated at reduced pressure and purified by PTLC, which
afforded 34 as a white solid (53 mg, 29%).
[0153] To a solution of
6-(5-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)thiophen-2-yl)-2,2-dimethylben-
zo [d][1,3]dioxol-4-ol (34) (53 mg, 0.13 mmol), triethylamine
(0.056 mL, 0.40 mmol) and DCM (2 mL) was added
trifluoromethanesulfonic anhydride (0.034 mL, 0.20 mmol) at
0.degree. C. The reaction was warmed to 23.degree. C. after 30 min
and extracted with DCM. The organic layer was washed with sat. aq.
NaHCO.sub.3, H.sub.2O and brine. The organic layers were dried over
MgSO.sub.4, concentrated, and 35 was obtained as yellowish oil (53
mg, 75% yield).
[0154] To
6-(5-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)thiophen-2-yl)-2,2-di-
methylbenzo[d][1,3]dioxol-4-yl trifluoromethanesulfonate (35) (32
mg, 0.061 mmol) was added Pd(PPh.sub.3).sub.4 (14 mg, 0.012 mmol),
LiCl (26 mg, 0.61 mmol), bistributylditin (0.153 mL, 0.305 mmol)
and dioxane (2 mL) (plus Triethythylamine??). The solution was
purged with nitrogen and refluxed for 3.5 h. The reaction was
concentrated, evaporated and subjected to PTLC (8% EtOAc in
hexanes). SA-77 was obtained as a yellowish oil (26 mg, 64%
yield).
##STR00029##
[0155] To the solution of
tributyl(6-(5-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)thiophen-2-yl)-2,2-di-
methylbenzo[d][1,3]dioxol-4-yl)stannane (SA-77) (26 mg, 0.039 mmol)
and THF (1 mL) was added the solution of 12 (20 mg, 0.078 mmol) in
THF (1 mL) dropwise. The reaction was stirred for 10 min and
concentrated, and 36 was obtained quantitatively as a yellow solid
after purification by PTLC (7% EtOAc in hexanes).
[0156] To
6-(5-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)thiophen-2-yl)-4-iodo-
-2,2-dimethylbenzo [d][1,3]dioxole (36) (15 mg, 0.029 mmol) were
added a few drops of H.sub.2O and trifluoroacetic acid (0.45 mL).
The reaction was stirred at 23.degree. C. for 1 h, and afforded
SA-78 quantitatively as a white crystal after concentration at
reduced pressure.
Example 21--Synthesis of SA-79 and 80
##STR00030##
[0158] To 4-(5-bromothiazol-2-yl)benzene-1,2-diol (SA-69) (90 mg,
0.32 mmol) was added 4-dimethylaminopyridine (DMAP) (117 mg) and
acetic anhydride (3 mL). The reaction was stirred at 23.degree. C.
for 1 h before it was extracted with EtOAc. The organic layer was
washed with sat. aq. NaHCO.sub.3, H.sub.2O and brine and dried over
MgSO.sub.4. SA-79 was obtained as yellow solid (74 mg, 65% yield)
after PTLC (20% EtOAc in hexanes).
[0159] To a solution of 4-(5-bromothiazol-2-yl)-1,2-phenylene
diacetate (SA-79) (36 mg, 0.10 mmol), Pd(PPh.sub.3).sub.4 and
dioxane (2 mL) was added bistributyltin (0.25 mL, 0.50 mmol). The
solution was purged with nitrogen and refluxed for 1.5 h. PTLC (25%
EtOAc in hexanes) provided SA-80 as yellow oil (22 mg, 39%
yield).
Example 22--Synthesis of SA-81
##STR00031##
[0161] To a solution of nBuLi (0.34 mL, 2.5 M in hexanes) and THF
(6 mL) was added 5-bromo-2-(3,4-dimethoxyphenyl)thiazole (23) (54
mg, 0.18 mmol) and additional THF (3 mL) was added dropwise at
-78.degree. C. under nitrogen atmosphere. SnBu.sub.3Cl (0.15 mL)
was added after the reaction was stirred at -78.degree. C. for 30
min. After another 30 min, saturated aqueous NaHCO.sub.3 was added
to quench the reaction. The mixture was extracted with EtOAc and
the organic layer was washed with water and brine. PTLC (25% EtOAc
in hexanes) provided SA-81 as a yellow oil (58 mg, 63% yield).
Example 23--Synthesis of SA-82
##STR00032##
[0163] A mixture of 0.26 g (1 mmol) of 2-bromo-4,5-dimethoxybenzoic
acid (3) in 5 mL of DCM was treated with 0.3 mL of (COCl).sub.2 and
two drops of DMF. The mixture was stirred for an additional hour
after a yellow solution had formed (about 2 h total). The mixture
was concentrated and dried in vacuo thoroughly, taken up in 15 mL
of DCM, cooled to 0.degree. C., and treated with 2 mL pyridine.
Then 0.79 g of known amine 37 was added. The mixture was warmed to
23.degree. C., stirred 3 days, and quenched with water. The product
was extracted with 10 mL of DCM, the combined organic was washed
with 10 mL water, dried, and concentrated to give the crude product
that was purified by PTLC (50% EtOAc in hexanes) to give 0.070 g
(16% yield) of compound 38 as a light brown solid. .sup.1H NMR (200
MHz, CDCl.sub.3) .delta. 7.67 (dd, J=2.0 Hz, 8.4 Hz, 1H), 7.56
(apparent broad singlet, overlapping peaks, 2H), 7.04 (s, 1H), 6.95
(d, J=6.95 Hz, 1H), 4.94 (d, J=4.2 Hz, 1H), 3.98 (s, 3H), 3.95 (s,
3H), 3.86 (bs, 6H). HRMS Calculated for C.sub.19H.sub.22O.sub.6NBr
(M+H).sup.+ 438.0552, found 438.0556.
[0164] A mixture of 0.103 g (0.24 mmol) of amide 38 in 10 mL of THF
was treated with 0.117 g (0.29 mmol) of Lawesson's reagent. The
mixture was refluxed for 2 h, cooled, and concentrated. The crude
product was purified directly by PLTC to give 0.048 g (45% yield)
of compound 39 as a light brown solid. .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta. 7.78 (apparent singlet, 1H), 7.55-7.50
(overlapping singlet, dd, 2H), 7.46 (s, 1H), 7.13 (s, 1H), 6.94 (d,
J=8.8 Hz, 1h), 3.97-3.89 (overlapping singlets, 12H).
[0165] A solution of 0.031 g 39 (0.07 mmol) in 4 mL of DCM was
cooled to -78.degree. C., and treated with 1.5 mL of 1 M BBr.sub.3
in DCM. The mixture was stirred for 1 h at -78.degree. C., 1 h at
23.degree. C., and quenched with 2 mL of MeOH. The mixture was
concentrated to 1 mL, diluted with 10 mL water, sonicated, and the
resultant solid filtered to give 0.009 g (35% yield) of SA-82 as an
off-white solid.
Example 24--Synthesis of SA-83
##STR00033##
[0167] To 0.012 g (0.03 mmol) of SA-76 in 0.5 mL pyridine was added
0.05 mL of acetic anhydride. The mixture was stirred at 140.degree.
C. for 3 h, cooled, and poured into 10 mL of water with 0.5 g
NH.sub.4.sup.+Cl.sup.-, and the product was extracted with 5 mL 10%
MeOH in DCM. The extract was concentrated, and purified by PTLC to
give 0.005 g (8.3*10.sup.-3 mmol) of SA-83 as a brown oil. .sup.1H
NMR (200 MHz, CDCl.sub.3) .delta. 7.94-7.88 (3 overlapping
irresolvable peaks, 3H), 7.82 (s, 1H), 7.36 (d, J=9.0 Hz),
2.34-2.32 (4 overlapping singlets, 12H).
Example 25--Synthesis of SA-84
##STR00034##
[0169] A mixture of 0.058 g (0.132 mmol) of amide 38 was refluxed
in 2 mL POCl.sub.3. The solution was added to 50 mL of water and
sonicated to precipitate a light yellow solid, which was filtered,
washed with cold water, and dried to give 0.041 g (74% yield) of 40
as a yellow solid. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 7.61
(s, 1H), 7.37 (d, J=2.0 Hz, 1H), 7.31 (dd, J=2.2 Hz, 8.4 Hz, 1H),
7.25 (s, 1H), 7.18 (s, 1H), 6.96 (d, J=8.4 Hz, 1H), 3.96
(overlapping singlets, 12H). HRMS Calculated for
C.sub.19H.sub.19BrNO.sub.5 (M+H).sup.+ 420.0448, found
420.0465.
[0170] To 0.100 g (0.24 mmol) of 40 in 20 mL of DCM at -78.degree.
C. was added 2.5 mL of 1 M BBr.sub.3 in DCM. The reaction was
warmed to 23.degree. C., stirred for 3 h, and quenched with 10 mL
of MeOH. The reaction mixture was concentrated, and water was added
to precipitate the product. This did not yield any solid, so the
crude product was reconcentrated and purified by PTLC (10% MeOH in
DCM) to give, after 2 weeks drying, 0.040 g (46% yield) of SA-84 as
a yellow-green solid. .sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta.
7.50 (s, 1H), 7.44 (s, 1H), 7.16 (d, J=2.0 Hz), 7.09 (s, 1H), 7.08
(dd, coupling constants not resolvable due to overlapping singlet,
1H), 6.83 (d, J=8.2 Hz, 1H).
Example 26--Synthesis of SA-86
##STR00035##
[0172] To a mixture of 1-bromo-2-iodo-4,5-dimethoxybenzene (41) (27
mg, 0.078 mmol), Pd(PPh.sub.3).sub.4 (4.1 mg, 5 mol %) and toluene
(1 mL) was added the solution of
2-(3,4-dimethoxyphenyl)-5-(tributylstannyl)thiazole (SA-81) (36 mg,
0.070 mmol) and toluene (1 mL). The reaction was purged with
nitrogen and refluxed 16 h. The concentrated reaction mixture was
purified by PTLC (35% EtOAc in hexanes) and afforded 42 (20 mg, 65%
yield).
[0173] SA-86 was prepared by the reaction of
5-(2-bromo-4,5-dimethoxyphenyl)-2-(3,4-dimethoxyphenyl) thiazole
(42) (20 mg, 0.046 mmol) and BBr.sub.3 (in DDM??) (0.46 mL, 0.46
mmol) according to the similar procedure for compound SA-58, and
obtained SA-86 as a yellow solid quantitatively.
Example 27--Synthesis of SA-87
##STR00036##
[0175] 2-Bromo-4,5-dimethoxybenzoic acid (3) (4.50 g, 17.6 mmol),
veratrole (2.44 g, 17.7 mmol), and 60 g of polyphosphoric acid
(PPA) were heated to 80.degree. C. for 45 min. The reaction mixture
turned a deep orange color during this time. The crude mixture was
treated with 400 mL of water, allowed stir overnight, and the
resultant crude product was filtered as a brownish solid. The solid
was recrystallized twice from ethanol to give 1.89 g (28% yield) of
benzophenone 43. .sup.1H NMR (CDCl.sub.3, .delta.) 7.54 (d, J=2.0
Hz, 1H), 7.26 (dd, J=2.0, 8.0 Hz), 7.07 (s, 1H), 6.90-6.83
(overlapping peaks, 3H), 3.95 (s, 6H), 3.88 (s, 3H), 3.84 (s,
3H).
Benzophenone 43 (1.31 g, 3.4 mmol) in 10 mL DCM was added to a
solution of 1.00 g NaBH.sub.4 (26 mmol) in 10 mL of TFA. Caution:
the reaction between NaBH.sub.4 and TFA is extremely exothermic
with the evolution of hydrogen gas. It is recommended that: (1) an
ice bath be used when the addition is occurring and (2) NaBH.sub.4
pellets are used as opposed to powder. The mixture was stirred 16
h, quenched with 15 mL of water, and diluted with 20 mL of DCM. The
aqueous layer was made basic with 10 N NaOH, the layers were
separated, the aqueous layer extracted with one portion of 10 mL
DCM, and the organic layer dried with Na.sub.2SO.sub.4 and
concentrated. Flash 40+M column chromatography (Biotage) gave 1.15
g (91% yield) of 44 as a thick, yellow oil.
[0176] .sup.1H NMR (CDCl.sub.3, .delta.) 7.04 (s, 1H), 6.80 (d,
J=8.2 Hz, 1H), 6.74-6.66 (overlapping peaks, 2H), 6.63 (s, 1H),
3.99 (s, 2H), 3.86 (s, 6H), 3.84 (s, 3H), 3.77 (s, 3H).
Bromide 44 (0.056 g, 0.172 mmol) in 2 mL of DCM was cooled to
0.degree. C. and treated with 1 mL of 2 M BBr.sub.3 in DCM. The
mixture was stirred 2h while allowing to warm to -23.degree. C.,
quenched with methanol, and concentrated. The crude, concentrated
product was treated with 10 mL of water, extracted twice with eight
mL of ethyl acetate, and the organic layers were concentrated to
give the SA-87 as an off-white solid.
[0177] .sup.1H NMR (CDCl.sub.3, .delta.) 6.93 (d, J=8.2 Hz, 1H),
6.62-6.57 (overlapping peaks, 2H), 6.51 (bs, 1H), 6.43-6.40 (m,
1H), 3.67 (s, 2H).
Example 28--Synthesis of SA-88 and SA-90
##STR00037##
[0179] 3,4-dimethoxybenzoic acid (1) (3.75 g, 20.6 mmol), veratrole
(2.86 g, 20.7 mmol), and 80 g of polyphosphoric acid (PPA) were
heated to 80.degree. C. with stirring via overhead stirrer for 1 h.
Water was added, and the mixture was stirred until a solid formed.
This solid was filtered, recrystallized twice from ethanol,
filtered, and dried to give 3.79 g (61% yield) of benzophenone
64.
[0180] .sup.1H NMR (CDCl, .delta.) 7.43 (d, J=2.0 Hz, 2H), 7.38
(dd, J=2.0, 8.2 Hz, 2H), 6.90 (d, J=8.2 Hz, 2H), 3.96 (s, 6H), 3.94
(s, 6H).
[0181] Benzophenone 64 (0.50 g, 1.66 mmol) was dissolved in 4 mL of
DCM and added to a solution made from NaBH.sub.4 (0.356 g, 9.4
mmol) and 3 mL TFA. The resultant mixture was stirred 18 h, diluted
with 15 mL of DCM and 10 mL of water, made basic with 10 N NaOH,
and the layers separated. The aqueous was extracted once more 10 mL
of DCM, and the combined organic layers were dried and
concentrated. The crude product was purified by PTLC to give 0.42 g
(88% yield) of 65.
The previous reaction was repeated using 1.12 g of benzophenone 64
with the other reagents scaled appropriately, and gave 0.86 g (81%
yield) of 65.
[0182] .sup.1H NMR (CDCl.sub.3, .delta.), 6.83-6.78 (m, 2H),
6.75-6.69 (m, 4H), 3.88-3.83 (multiple singlets, 14).
[0183] A solution of 65 (0.500 g, 1.7 mmol) in 3 mL of AcOH was
treated with 0.120 g of 69% aq. HNO.sub.3 (1.3 mmol) and stirred
for 1.5 h. The mixture was poured into 50 mL of water, and
extracted twice with 25 mL of ethyl acetate. The combined organic
layers were washed three times with 20 mL of water, dried, and
concentrated to give the crude product, which was purified by PTLC
to give 0.123 g of 65 (25% recovery), 0.137 g (24% yield) of 67,
and 0.041 g (6% yield) of 66.
[0184] .sup.1H NMR of 66 (CDCl.sub.3, .delta.), 7.65 (s, 2H), 6.54,
(s, 2H), 4.66 (s, 2H), 3.93 (s, 6H), 3.80 (s, 6H). [0185] .sup.1HMR
of 67 (CDCl.sub.3, .delta.), 7.60 (s, 1H), 6.79-6.63 (overlapping
peaks, 4H), 4.26 (s, 2H), 3.91-3.81 (m, 12H).
[0186] .sup.13C NMR of 67 (CDCl.sub.3, .delta.), 153.0, 149.1,
147.7, 147.3, 131.6, 131.3, 120.9, 113.6, 112.4, 111.3, 108.2,
56.42, 56.27, 55.9, 38.3.
[0187] A mixture of 66 (0.030 g, 0.08 mmol) in 3 mL of DCM was
treated with 1 mL of 2 M BBr.sub.3 and stirred 2.5 h. Methanol (10
mL) was added to quench the reaction, the mixture was concentrated,
and water was added. The resultant brown solid was filtered and not
purified further (due to lack of solubility of the product) to give
0.007 g (27% yield) of SA-88.
[0188] .sup.1H NMR (DMSO-d.sub.6, .delta.), 8.87 (bs, 2H), 8.71
(bs, 2H), 7.56 (s, 4H), 3.48 (s, 2H).
[0189] A mixture of 67 (0.030 g, 0.09 mmol) in 2 mL of DCM was
treated with 1 mL of 2 M BBr.sub.3 and stirred for 2.5 h. Methanol
(10 mL) was added to quench the reaction, the mixture concentrated,
and treated with 10 mL of water. This was extracted with 10 mL of
ethyl acetate, dried, and concentrated to give 8 mg (32% yield) of
SA-90 as a thick brown oil that slowly solidified.
[0190] .sup.1H NMR (DMSO-d.sub.6, .delta.), 7.54, (s, 3H),
6.97-6.93 (overlapping peaks, 2H), 6.50 (d, 1H, overlapping with
next peak), 6.45 (d, J=2.0 Hz, 1H), 3.85 (s, 2H)
Example 29--Synthesis of SA-93
##STR00038##
[0192] 2-Iodo-4,5-dimethoxybenzoic acid (49) (2.76 g, 9.0 mmol),
veratrole (1.20 g, 8.6 mmol), and 50 g of polyphosphoric acid (PPA)
were heated to 90.degree. C. for 15 min. An additional 0.30 g (2.1
mmol) of veratrole was added. The reaction mixture turned a deep
orange color during the reaction. The crude mixture was treated
with 600 mL of ice water, sonicated, and the resultant crude
product was filtered as a brownish solid (1.55 g, 40% yield). This
product 50 was used without further purification.
[0193] Benzophenone 50 (1.55 g, 3.6 mmol) in 10 mL DCM was added to
a solution of 2.00 g NaBH.sub.4 (26 mmol) in 10 mL of TFA. Caution:
the reaction between NaBH.sub.4 and TFA is extremely exothermic
with the evolution of hydrogen gas. It is recommended that: (1) an
ice bath be used when the addition is occurring and (2) NaBH.sub.4
pellets are used as opposed to powder. The mixture was stirred 16
h, quenched with 50 mL of water, and diluted with 15 mL of DCM. The
aqueous layer was made basic with 10 N NaOH, the layers were
separated, the aqueous layer extracted once with 25 mL DCM, and the
organic layer dried with Na.sub.2SO.sub.4 and concentrated. The
crude product consisted of the iodo compound 51 and the des-iodo
46. The products practically co-elute when Flash 40+M column
chromatography is used, so the crude was purified using PTLC with
0.4% ethyl acetate as the eluent and eluting the PTLC plates
several times to give 0.30 g (20% yield) of 51 as a thick, yellow
oil.
[0194] .sup.1H NMR (CDCl.sub.3, .delta.) 6.80 (d, J=8.0 Hz, 1H),
6.74-6.64 (overlapping peaks, 4H), 3.98 (s, 2H), 3.85 (bs, 9H),
3.75 (s, 3H).
[0195] .sup.13C NMR (CDCl.sub.3, .delta.) 149.5, 149.0, 148.1,
147.6, 136.3, 132.6, 121.8, 120.8, 113.1, 112.3, 111.3, 88.8, 56.2,
55.9, 45.6.
[0196] Iodide 51 (0.150 g, 0.36 mmol) in 40 mL of DCM was cooled to
-78.degree. C. and treated with BBr.sub.3 (0.75 g, 3 mmol) neat.
The mixture was stirred 2 h, allowed to warm to -23.degree. C.,
stirred 3h more, quenched with water, extracted with 20 mL ethyl
acetate, and the organic dried and concentrated. The crude,
concentrated product was precipitated with DCM, and filtered to
give 0.022 g of SA-93 (17% yield).
[0197] .sup.1H NMR (DMSO-d.sub.6, .delta.) 6.62-6.52 (overlapping
peaks, 3H), 6.50 (d, J=1.9 Hz, 1H), 6.41 (dd, J=2.0, 8.2 Hz, 1H),
3.54 (s, 2H), 4.5-3.5 (broad peak, 4H, "--OH")
Example 30--Synthesis of SA-94 and SA-98
##STR00039##
[0199] A mixture of 3',4'-dimethoxyacetophenone (52) (0.54 g, 3
mmol) and 3,4-dimethoxybenzaldehyde (0.51 g, 3.06 mmol) (30) in 15
mL of absolute ethanol was treated with 1.75 g of NaOH, sonicated
for 10 min, then stirred overnight. The resultant mixture was
cooled to 0.degree. C., filtered, and washed with 4.degree. C.
ethanol to obtain a yellow solid that was dried to give 0.88 g of
53 (89% yield).
[0200] .sup.1H NMR (CDCl.sub.3, .delta.) 7.76 (d, J=15.4 Hz, 1H),
7.68 (dd, J=2.0, 8.4 Hz, 1H), 7.62 (d, J=2.0 Hz, 1H), 7.40 (d,
J=15.4 Hz, 1H), 7.19 (dd, J=2.0, 8.4 Hz, 1H), 6.93 (d, J=8.0 Hz,
1H), 6.90 (d, J=8.0 Hz, 1H).
[0201] A solution of 53 (0.14 g, 0.43 mmol) was dissolved in 40 mL
of DCM, cooled to -78.degree. C., and BBr.sub.3 (0.98 g, 3.9 mmol)
in 4 mL of DCM was added. The mixture was allowed to warm to
0.degree. C. over 2 h, and then immediately warmed to 23.degree. C.
The mixture was stirred 2 h, quenched with 20 mL of water, and
extracted with 100 mL of ethyl acetate. The organic layer was
dried, concentrated until .about.5 mL remained, and treated with 30
mL of DCM. A deep purple solid precipitated from the solution. This
solid was filtered, and dried to a yellow-green solid in the dark
to give 0.068 g (59% yield) of SA-94.
[0202] .sup.1H NMR (DMSO-d.sub.6) 10.5-8.0 (bs, 4H), 7.57-7.47 (m,
2H), 7.49 (s, 2H), 7.20 (bs, 1H), 7.12 (m, 1H), 6.84 (d, J=8.4 Hz,
1H), 6.79 (d, J=8.6 Hz, 1H).
[0203] A solution of 53 (0.082 g, 0.25 mmol) in 1.5 mL of acetic
acid was treated with 0.5 mL of hydrazine hydrate and heated to
135.degree. C. for 2 h and 140.degree. C. for 1 h. The yellow color
of the starting material gradually discharged throughout the course
of the reaction. The crude reaction mixture was poured into 20 mL
of water, extracted with 10 mL of ethyl acetate twice. The combined
organic layers were washed twice with 20 mL of water, dried, and
concentrated to give 0.058 g (61% yield) of 54 as a clear oil that
was not purified further.
[0204] A solution of 54 (0.056 g, 0.59 mmol) in 20 mL of DCM was
cooled to -78.degree. C., and treated with BBr.sub.3 (1.025 g, 4.1
mmol) in 5 mL of DCM. The mixture was stirred for 3 h at
-78.degree. C., stirred for 1 h at .about.0.degree. C., and
quenched with water. The resultant mixture was extracted with 100
mL of ethyl acetate, and the organic layer was dried and
concentrated to give the crude product which was taken up in 2 mL
of ethyl acetate, treated with 2 mL of hexanes, and treated with 50
mL of DCM. The precipitated product SA-98 (0.019 g, 40% yield) was
collected by filtration and dried to give an off-white powder.
[0205] .sup.1H NMR (DMSO-d.sub.6) 9.4 (bs, 1H), 9.2 (bs, 1H), 8.9
(bs, 1H), 8.8 (bs, 1H), 7.26 (d, J=2.2 Hz, 1H), 6.98 (dd, J=2.2,
8.4 Hz, 1H), 6.77 (d, J=8.2 Hz, 1H), 6.64 (d, J=8.4 Hz, 1H), 6.52
(d, J=2.2 Hz, 1H), 6.44 (dd, J=2.2, 8.0 Hz, 1H), 5.32 (dd, J=3.6,
11.0 Hz, 1H), 3.4 (m, 1H), 3.22 (m, 1H), 2.24 (s, 3H).
[0206] HRMS, Calculated for C.sub.17H.sub.17N.sub.2O.sub.5
(M+H).sup.+ 329.1137, found 329.1125.
Example 31--Synthesis of SA-95
##STR00040##
[0208] A mixture of 3',4'-dimethoxyacetophenone (52) (2 mmol, 0.36
g) and 6-bromoveratraldehyde (55) (2 mmol, 0.49 g) in 15 mL of
ethanol was treated with 1 g of NaOH and 0.015 g (2.78 mmol) of
NaOMe. The mixture was stirred at 23.degree. C. for 18 h, cooled to
4.degree. C., and the resultant yellow solid filtered to give the
bromochalcone 56 as a yellow solid (0.68 g, 83% yield).
[0209] .sup.1H NMR (CDCl.sub.3, .delta.), 8.07 (d, J=15.6 Hz, 1H),
7.75 (m, 1H), 7.67 (d, J=1.8 Hz, 1H), 7.38 (m, 1H), 7.22 (s, 1H),
7.12 (s, 1H), 6.95 (d, J=8.2 Hz, 1H), 3.98-3.94 (overlapping
singlets, 12H).
[0210] A mixture of bromochalcone 56 (0.20 g, 0.49 mmol) in 30 mL
of DCM was cooled to -78.degree. C. and treated with BBr.sub.3
(0.72 g, 2.88 mmol) in 4 mL of DCM. The mixture was stirred at
-78.degree. C. for 2.5 h, warmed gradually to 0.degree. C. over 1
h, warmed to 23.degree. C., and stirred an additional 1 h. The
reaction was quenched with 20 mL of water, and the mixture
extracted with 50 mL of ethyl acetate. The ethyl acetate layer was
washed once with 20 mL of brine, dried concentrated to 2 mL, and
treated with 50 mL of DCM. The product was filtered and dried to
get SA-95 as a reddish solid (0.056 g, 33% yield).
[0211] .sup.1H NMR (dmso-d.sub.6) 10.2 (bs, 1H), 9.8 (bs, 1H), 9.3
(bs, 2H), 7.81 (d, J=15.4 Hz, 1H), 7.61 (d, J=2.2 Hz, 1H),
7.56-7.49 (overlapping peaks, 3H), 7.05 (s, 1H), 6.86 (d, J=8.2 Hz,
1H).
Example 32--Synthesis of SA-96
##STR00041##
[0213] A mixture of 2'-bromo-4',5'-dimethoxyacetophenone (57)
(0.390 g, 1.5 mmol) and 3,4 dimethoxybenzaldehyde (30) (0.310 g,
1.9 mmol) in 10 mL of ethanol was treated with 0.75 g of NaOH. The
mixture was inverted ten times to thoroughly mix the starting
materials, sonicated 5 min, allowed to stand 2 h, and sonicated 5
min again. A yellow precipitate resulted, and this was filtered and
dried to give bromochalcone 58 as a light yellow solid (0.482 g,
79% yield).
[0214] .sup.1H NMR (CDCl.sub.3, .delta.) 7.41 (d, J=2.2 Hz, 1H),
7.39-7.34 (m, 3H), 7.25 (s, 1H), 7.17 (d, J=16.0 Hz, 1H), 7.10 (s,
1H), 7.00 (d, J=8.4 Hz, 1H), 3.86-3.81 (overlapping singlets,
12H).
[0215] .sup.13C NMR (CDCl.sub.3, .delta.) 194.0, 151.9, 151.1,
149.5, 148.5, 146.6, 133.4, 127.6, 124.6, 124.0, 116.5, 112.8,
112.1, 111.5, 110.2, 56.6, 56.4, 56.2, 56.1.
[0216] A mixture of bromochalcone 58 (0.27 g, 0.66 mmol) in 30 mL
of DCM was cooled to -78.degree. C., and treated with 1.25 g (5
mmol) of BBr.sub.3 in 5 mL of DCM. The mixture was stirred at
-78.degree. C. for 3 h, warmed immediately to 23.degree. C.,
stirred at 23.degree. C. for 1 h, quenched with 20 mL of water, and
extracted with 125 mL of ethyl acetate. The organic layer was dried
and concentrated to 1 mL. The concentrate was treated with an
excess of DCM to precipitate a purplish-red solid that was filtered
and dried to give SA-96 as a reddish-brown solid (0.036 g, 16%
yield).
[0217] .sup.1H NMR (DMSO-d.sub.6, .delta.) 7.26 (d, J=16.0 Hz, 1H),
7.09 (bs, 1H), 7.05-6.86 (overlapping peaks, 4H), 6.77 (d, J=8.2
Hz, 1H).
Example 33--Synthesis of SA-97
##STR00042##
[0219] A mixture of 2,4,5-trimethoxybenzoic acid (59) (1.06 g, 5
mmol), veratrole (0.69 g, 5 mmol), and 10 g of PPA was heated with
a heat gun and stirred with a stirring rod for 20 min. Water (100
mL) was added to the reaction mixture and the resultant mixture was
cooled to 4.degree. C. and stirred with a stirring rod for 20 min.
The water was decanted and the resultant grey, gummy solid was
taken up in a hot solution 20 mL of reagent alcohol and 10 mL of
water. The mixture was cooled and filtered to give 0.43 g of orange
crystals. Water (40 mL) was added to the filtrate and the resultant
orange solid was filtered to give 0.72 g of amorphous orange solid.
Both fractions were the desired 60 (1.15 g, 69% yield).
[0220] A mixture of benzophenone 60 (0.93 g, 2.9 mmol) in 20 mL of
DCM was added to 0.92 g of NaBH.sub.4 (24.3 mmol) in 10 mL of TFA.
Caution: the reaction between NaBH.sub.4 and TFA is extremely
exothermic with the evolution of hydrogen gas. It is recommended
that: (1) an ice bath be used when the addition is occurring and
(2) NaBH.sub.4 pellets are used as opposed to powder. The mixture
was stirred for 6 h, and an additional portion of NaBH.sub.4 (0.53
g, 14.0 mmol) was added. The mixture was stirred 20 h, diluted with
50 mL of DCM and 50 mL of water, and the aqueous layer made basic
with NaOH. The layers were separated, and the aqueous layer was
extracted once with 50 mL of DCM. The organic layers were combined,
dried, and concentrated to give a yellow oil that was purified by
Flash 40+M chromatography (Biotage) to give 61 as a pale yellow,
thick oil (0.63 g, 71% yield).
[0221] .sup.1H NMR (CDCl.sub.3, .delta.) 6.83-6.76 (overlapping
peaks, 3H), 6.66 (s, 1H), 6.57 (s, 1H), 3.91-3.86 (overlapping
singlets, 11H), 3.83 (s, 3H), 3.79 (s, 3H).
[0222] A mixture of pentamethoxy compound 61 (0.262 g, 0.82 mmol)
in 30 mL of DCM at -78.degree. C. was treated with BBr.sub.3 (2.00
g, 8 mmol) in 8 mL of DCM. The resultant mixture was stirred at
-78.degree. C. 1 h, warmed to 23.degree. C. immediately, stirred at
23.degree. C. for 4 h, quenched with 20 mL of water and extracted
with 100 mL of ethyl acetate. The ethyl acetate layer was
concentrated to dryness, taken up in approximately 0.5 mL of ethyl
acetate, precipitated with DCM, cooled to -20.degree. C., and
filtered to give SA-97 as a white solid (0.93 g, 46% yield).
[0223] .sup.1H NMR (DMSO-d.sub.6) 8.63-8.29 (bs, 4H), 6.58 (d,
J=7.8 Hz, 1H), 6.52 (d, J=2.0 Hz, 1H), 6.40 (dd, J=2.0, 8.0 Hz,
1H), 6.29 (s, 1H), 6.26 (s, 1H), 3.50 (s, 2H).
Example 34--Synthesis of SA-99
##STR00043##
[0225] A mixture of dibromide 62 (0.562 g, 2 mmol), boronic acid 15
(0.910 g, 5 mmol), 20 mL of dioxane, and 24 mL of 2 M
Na.sub.2CO.sub.3 (aqueous) was degassed by nitrogen purge. Then
Pd(PPh.sub.3).sub.4 (0.12 g, 0.10 mmol) was added. The resultant
mixture was refluxed 20 h, diluted with 50 mL of water, and
extracted three times with 50 mL of ethyl acetate. The combined
organic layers were washed once with 50 mL of water, dried, and
concentrated to give the crude product. This was purified by Flash
40+M chromatography (Biotage) using gradient ethyl acetate in
hexanes as the eluent to give 63 as a yellow solid (0.288 g, 36%
yield).
[0226] .sup.1H NMR (CDCl.sub.3, .delta.), 7.94 (d, J=1.8 Hz, 1H),
7.75 (dd, J=1.8, 8.0 Hz, 1H), 7.47 (d, J=8.0 Hz, 1H), 7.17 (dd,
J=2.2, 8.2 Hz, 1H), 7.10 (d, J=2.0 Hz, 1H), 6.96 (d, J=8.0 Hz, 1H),
6.90-6.85 (overlapping peaks, 3H), 3.95 (s, 3H), 3.92 (s, 3H), 3.90
(s, 3H), 3.87 (s, 3H).
[0227] .sup.13C NMR (CDCl.sub.3, .delta.) 149.9, 149.7, 149.6,
149.3, 149.1, 141.1, 133.9, 132.2, 131.2, 130.0, 129.5, 121.8,
120.5, 119.6, 111.7, 111.4, 111.2, 110.1, 56.11, 56.05, 56.0,
55.9.
[0228] A solution of 63 (0.062 g, 0.16 mmol) in 25 mL DCM was
cooled to -78.degree. C., treated with BBr.sub.3 (1.125 g, 4.5
mmol) in 6 mL of DCM, stirred 2.5 h at -78.degree. C., warmed to
23.degree. C., stirred at -23.degree. C. for 1.5 h, quenched with
20 mL water, and extracted once with 100 mL of ethyl acetate. The
ethyl acetate layer was dried and concentrated to 3 mL and
precipitated with excess DCM. The precipitate was filtered and
dried to give SA-99 as a yellow solid (0.037 g, 70% yield).
[0229] .sup.1H NMR (DMSO-d.sub.6) 9.17-9.11 (bs, 4H), 7.96 (d,
J=1.6 Hz, 1H), 7.83 (dd, J=2.2, 8.2 Hz, 1H), 7.49 (d, J=8.0 Hz,
1H), 7.14 (bs, 1H), 7.08 (d, J=8.4 Hz, 1H), 6.88-6.79 (overlapping
peaks, 2H), 6.72 (d, J=2.2 Hz, 1H), 6.63 (dd, J=2.0, 8.2 Hz,
1H),
[0230] HRMS Calculated for C.sub.18H.sub.13NO.sub.6Na (M+Na).sup.+
362.0641, Found 362.0645.
Example 35--Synthesis of SA-108
##STR00044##
[0232] To 1.00 g (4.07 mmol) of acid chloride 68 in 25 mL of DCM at
0.degree. C. was added 5 mL of pyridine. The mixture was stirred 3
min, and 0.76 g (3.87 mmol) of known hydrazide 69 was added at
once. The mixture was gradually allowed to warm to 21-23.degree. C.
and stirred at this temperature for 16 h. The mixture was
concentrated, treated with 10 mL of ethanol, warmed to reflux, and
diluted with 30 mL of water. After returning the mixture to reflux,
ethanol was added gradually until a solution formed. The solution
was allowed to cool, and the precipitated light yellow solid was
collected and dried to give 0.498 g (1.24 mmol, 32% yield) of
70.
[0233] .sup.1H NMR (CDCl.sub.3 and MeOD, .delta.) 7.54 (s, 1H),
7.45 (dd, J=2.2, 8.4 Hz, 1H), 7.39 (d, J=1.8 Hz), 7.16 (s, 1H),
6.82 (d, J=8.4 Hz, 1H), 3.98-3.73 (overlapping singlets+residual
H.sub.2O peak, 12H).
[0234] A mixture of 0.398 g (1 mmol) of 70 and 0.452 g (1.11 mmol)
of Lawesson's reagent in 50 mL of THF were refluxed for 16 h. The
mixture was concentrated and purified by PTLC using 10% EtOAc in
DCM as the eluent to give 0.255 g (0.62 mmol, 62% yield) 71 as a
bright yellow solid.
[0235] .sup.1H NMR (DMSO-d.sub.6) 7.77 (s, 1H), 7.60-7.58
(overlapping peaks, 2H), 7.40 (s, 1H), 7.15 (d, J=8.8 Hz, 1H), 3.97
(s, 3H), 3.95 (s, 3H), 3.89 (s, 3H), 3.87 (s, 3H).
[0236] A mixture of 0.100 g (0.24 mmol) of 71 in 5 g of pyridine
hydrochloride was heated to 200.degree. C. for 30 min. The mixture
was cooled, treated with 30 mL of water, and filtered. The
resultant solid was recrystallized from aqueous methanol to yield
0.016 g of a brown solid as SA-108. The product is only sparingly
soluble in DMSO-d.sub.6, and insoluble in other deuterated solvents
or solvent mixtures.
[0237] .sup.1H NMR (DMSO-d.sub.6, .delta.), 8.79 (s, 1H), 7.80 (bs,
1H), 7.57 (s, 1H), 7.43 (m, 1H), 7.28 (m, 1H), 6.91 (s, 1H), 6.80
(d, J=8.0 Hz, 1H). Example 1--Compounds provided herein bind are
potent disruptors/inhibitors of Parkinson's disease
.alpha.-synuclein fibrils
Example 36--Compounds Provided Herein are Potent
Disruptors/Inhibitors of Parkinson's Disease .alpha.-synuclein
Fibrils
[0238] The compounds were found to be potent
disrupters/disaggregators of .alpha.-synuclein fibrils. In this set
of studies, the efficacy of certain compounds provided herein to
cause a disassembly/disruption/disaggregation of pre-formed fibrils
of Parkinson's disease (i.e. consisting of .alpha.-synuclein
fibrils) was analyzed. For the studies described below in Parts A
and B, 69 .mu.M of .alpha.-synuclein (rPeptide, Bogart, Calif.) was
first incubated at 37.degree. C. for 4 days in 20 mM sodium acetate
buffer at pH 4 with circular shaking (1,300 rpm) to cause
.alpha.-synuclein aggregation and fibril formation.
Part A: Thioflavin T Fluorometry Data
[0239] In one study, Thioflavin T fluorometry was used to determine
the effects of the compounds on .alpha.-synuclein fibrils. In
addition to test compounds, this experiment included a positive
control compound and a negative control compound for reference. 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 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).
[0240] Following initial .alpha.-synuclein fibrilization as
described above, 6.9 .mu.M .alpha.-synuclein was incubated at
37.degree. C. for 2 days with shaking (1,300 rpm), either alone, or
in the presence of one of the compounds (at test
compound:.alpha.-synuclein molar ratios of 50:1, 10:1, 5:1, 1:1,
0.5:1, 0.1:1, 0.05:1 and 0.01:1) in phosphate-buffered saline, pH
7.4+0.02% sodium azide. Following 2 days of co-incubation, 50 .mu.l
of each incubation mixture was transferred into a 96-well
microtiter plate containing 150 .mu.l of distilled water and 50
.mu.l of a Thioflavin T solution (i.e. 500 .mu.M Thioflavin T in
250 .mu.M phosphate buffer, pH 6.8). The final concentration of
Thioflavin T reagent is 100 .mu.M in 50 .mu.M phosphate buffer, pH
6.8. The fluorescence was read at 485 nm (444 nm excitation
wavelength) using an ELISA plate fluorometer after subtraction with
buffer alone or compound alone, as blank.
[0241] The results of the 2-day incubations are presented below.
For each compound, the % inhibition of Thioflavin T fluorescence
(i.e. the decrease compared to control reactions containing
.alpha.-synuclein alone) was plotted against the log of the
concentration of the test compound (expressed as mole ratio
relative to .alpha.-synuclein). Where possible, the effective
concentration of SA compound that yields 50% of maximal % decrease
of Thioflavin T fluorescence (EC.sub.50) was calculated from the
sigmoidal shaped dose response curve. The compounds (SA-52, SA-53,
SA-54, SA-55, SA-57, SA-58, SA-59, SA-61, SA-62, SA-63, SA-64,
SA-66, SA-67, SA-68, SA-69, SA-70, SA-72, SA-73, SA-93, SA-94,
SA-95, SA-96, SA-97, SA-98 and SA-99 and positive reference
compound #1) all caused a dose-dependent and extensive
disruption/disassembly of preformed .alpha.-synuclein fibrils
(Table 1). For example, compound SA-57 caused a significant
(p<0.01 relative to .alpha.-synuclein alone) nearly complete
inhibition (97-100%) when used at test compound:.alpha.-synuclein
molar ratios .gtoreq.5:1 and a significant 83% inhibition when used
at a test compound:.alpha.-synuclein molar ratio of 1:1 (FIG. 1),
whereas the negative reference compound showed no significant
inhibition of Thioflavin T fluorescence at any of the
concentrations tested (not shown). The EC.sub.50 of SA-57 for
inhibition of Thioflavin T fluorescence was determined to be 0.23
moles of test compound per mole of .alpha.-synuclein (Table 1). For
the compounds described here that caused a dose-dependent
disruption/disassembly of ca-synuclein fibrils, the maximum %
inhibition ranged from 76-100% and the EC.sub.50 values ranged from
0.08-4.3 moles of test compound:.alpha.-synuclein, with most
compounds showing highly potent effects (i.e. EC.sub.50<1 mole
of test compound:.alpha.-synuclein) in this assay.
[0242] This study indicated that the compounds provided herein are
potent disrupters/dissaggregators of Parkinson's disease
.alpha.-synuclein fibrils, and usually exert their effects in a
dose-dependent manner.
TABLE-US-00001 TABLE 1 SA compounds disrupt/disaggregate
.alpha.-synuclein aggregates as measured by Thioflavin T
fluorometry. SA # EC.sub.50 (mole ratio) Max % Decrease 52 0.28 99
53 0.17 100 54 0.74 98 55 0.08 92 57 0.23 100 58 0.19 100 59 0.58
96 61 0.10 100 62 1.09 95 63 0.86 100 64 0.39 100 66 0.69 100 67
4.3 76 68 0.77 100 69 0.73 88 70 0.17 100 72 0.12 100 73 0.52 100
93 0.16 100 94 0.15 100 95 0.16 100 96 0.14 100 97 0.16 99 98 0.65
100 99 0.65 100 Positive Reference #1 0.24 100 Negative Reference
not determined 0
Part B: Congo Red Binding Data
[0243] In the Congo red binding assay, the ability of a given test
compound to alter .alpha.-synuclein aggregate binding to Congo red
is quantified. In this assay Congo red binds specifically to
fibrillar amyloid, and this binding is directly proportional to the
amount of fibrils formed. Following initial .alpha.-synuclein
fibrilization as described above, .alpha.-synuclein aggregates and
test compounds were incubated for 2 days and then vacuum filtered
through a 0.2 .mu.m filter. The amount of .alpha.-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
amyloid protein in the absence of the test compound--i.e.
.alpha.-synuclein alone) was indicative of the test compound's
ability to diminish/alter the amount of aggregated and congophilic
.alpha.-synuclein and thus cause
disassembly/disruption/disaggregation of .alpha.-synuclein
fibrils.
[0244] In one study, the ability of .alpha.-synuclein fibrils to
bind Congo red in the absence or presence of increasing amounts of
the compounds provided herein, including a positive and a negative
reference compound (at test compound:.alpha.-synuclein molar ratios
of 50:1, 10:1, 5:1, 1:1, 0.5:1, 0.1:1, 0.05:1, 0.01:1) was
determined. The results of 2-day incubations are presented in Table
2 below. Whereas the negative reference compound caused no
significant inhibition of .alpha.-synuclein fibril binding to Congo
red at all concentrations tested (not shown), the test compounds
caused a dose-dependent inhibition of .alpha.-synuclein binding to
Congo red. For example, compound SA-57 caused a significant
(p<0.01) inhibition (66%) when used at a test
compound:.alpha.-synuclein molar ratio of 50:1 and a significant
49% inhibition when used at a test compound:.alpha.-synuclein molar
ratio of 5:1 (FIG. 2). The EC.sub.50 of SA-57 for inhibition of
Congo Red binding was determined to be 2.0 moles of test compound
per mole of .alpha.-synuclein (Table 2). For the compounds
described here that caused a dose-dependent disruption/disassembly
of .alpha.-synuclein fibrils, the maximum % inhibition ranged from
25-94% and the EC.sub.50 values ranged from 1.0-15.9 moles of test
compound per mole of .alpha.-synuclein. Taken together, the results
of this study indicated that compounds of this invention
disrupt/disaggregate/disassemble .alpha.-synuclein aggregates as
indicated by their ability to inhibit Parkinson's disease type
.alpha.-synuclein fibril binding to Congo red, and usually exert
their effects in a dose-dependent manner.
TABLE-US-00002 TABLE 2 SA compounds disrupt/disaggregate
.alpha.-synuclein fibrils/aggregates as measured by Congo Red
binding assay. SA # EC.sub.50 (mole ratio) Max % Decrease 52 10.7
68 53 6.8 75 54 15.9 57 55 4.5 38 57 2.0 66 58 1.9 94 59 7.6 85 61
2 84 63 9.5 72 64 2.3 85 66 2.2 48 67 6.8 25 68 6.4 60 69 14.2 39
70 3.4 88 72 2.9 29 73 1.1 35 93 1.9 62 94 4.5 65 95 4.5 64 96 5.1
72 97 1.0 40 98 10.1 44 99 14.2 54 Positive Reference #1 4.5 66
Negative Reference not determined 0
Example 37--Compounds of this Invention are Potent
Disruptors/Inhibitors of .alpha.-Synuclein Fibrils and/or
Aggregates Associated with Parkinson's Disease
[0245] Parkinson's Disease is characterized by the accumulation of
insoluble intraneuronal aggregates called Lewy Bodies, a major
component of which is .alpha.-synuclein (reviewed in Dauer et al.,
Neuron, 39:889-909, 2003). Since autosomal dominant mutations in
.alpha.-synuclein cause a subset of familial Parkinson's disease,
and since these mutations increase the likelihood of ca-synuclein
to aggregate and form Lewy Bodies, aggregated .alpha.-synuclein is
proposed to be directly involved in the etiology and disease
progression (Polymeropoulos et al., Science 276:1197-1199, 1997;
Papadimitriou et al., Neurology 52:651-654, 1999). Structural
studies have revealed that intracellular Lewy bodies contain a
large proportion of misfolded proteins with a high degree of
.beta.-pleated sheet secondary structure. Therefore, since many of
the compounds described herein cause
disassembly/disruption/disaggregation of .alpha.-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 inhibit or prevent .alpha.-synuclein aggregation
associated with Parkinson's disease.
[0246] To test the therapeutic potential of the compounds, two
cell-based assays were utilized. In both assays, rotenone is used
to induce mitochondrial oxidative stress and cause
.alpha.-synuclein aggregation. The first assay utilizes the binding
of the fluorescent dye Thioflavin S to structures with high .beta.
sheet content, including .alpha.-synuclein fibrils. Therefore,
quantitative assessment of the extent of Thioflavin S-positive
staining of fixed cells is used to test the ability of the test
compounds to inhibit/prevent or decrease the amount of
.alpha.-synuclein aggregates relative to cells that were treated
with rotenone only. In the second assay, cell viability is assessed
using the XTT cytotoxicity assay (Cell Proliferation Assay Kit II,
Roche, Mannheim, Germany), which is dependent on intact, functional
mitochondria in live cells. Thus, the XTT cytotoxicity assay is
used to test the ability of the compounds to ameliorate the
mitochondrial toxicity and resulting loss of viability associated
with the accumulation of .alpha.-synuclein aggregates. These
studies are presented in the following examples.
[0247] To carry out these studies, a cell culture model was used in
which human .alpha.-synuclein aggregation is experimentally
induced. BE-M17 human neuroblastoma cells stably transfected with
A53T-mutant human .alpha.-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 .mu.g/ml) and 500 .mu.g/ml G418 as previously
described (Ostrerova-Golts et al., J. Neurosci., 20:6048-6054,
2000).
[0248] Thioflavin S is commonly used to detect aggregated protein
structures in situ, including in brain tissue (Vallet et al., Acta
Neuropathol., 83:170-178, 1992), and cultured cells
(Ostrerova-Golts et al., J. Neurosci., 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
.beta.-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
.beta.-pleated structures that formed in response 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). Briefly, for these studies cells
were grown on Poly-D-Lysine coated glass slide chambers at
approximately 4.5-5.5.times.10.sup.4 cells/cm.sup.2. After 16-18
hours, cells were treated with 500 nM or 2 .mu.M rotenone (Sigma)
or vehicle (0.05% DMSO) as indicated. Within 15 minutes of rotenone
(or vehicle) addition, compounds were added at the indicated
concentration, or mock-treatment was performed in which cell
culture media only (no compound) 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 deionized water wash, the cells were incubated 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 mounted using an aqueous-based mountant designed to
protect against photobleaching. Aggregates that bind to Thioflavin
S were detected with a fluorescent microscope using a High Q FITC
filter set (480 to 535 nm bandwidth) and a 20.times. objective lens
unless otherwise 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 treatment
conditions. To assess the amount of Thioflavin 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 removed. 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
in Table 3 represent statistically significant (p<0.05)
reductions (reported as percent inhibition) in Thioflavin S
fluorescence in cells treated with test compound and rotenone
relative to cells treated with rotenone only.
[0249] To validate the ability of the assay to quantitatively
detect aggregates that bind Thioflavin S, staining of BE-M17 cells
overexpressing A53T .alpha.-synuclein was carried out and the
results revealed a rotenone dose-dependent increase in Thioflavin
S-positive aggregates relative to vehicle-treated control cells
(FIG. 3). Higher magnification images obtained with a 40.times.
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 .mu.M rotenone were sufficient to induce robust
aggregation (FIG. 3) and thus are effective doses to test the
ability of compounds to attenuate the formation of these
aggregates.
[0250] Using the protocol described above, several 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 .alpha.-synuclein. Examples of
results obtained from experiments using these compounds are
summarized in Table 4. Many of the compounds tested significantly
disrupted, prevented or inhibited .alpha.-synuclein aggregation and
fibril formation in the presence of rotenone as indicated by a
decrease in Thioflavin S-positive inclusions, relative to cells
treated with rotenone only. For example, cells treated with 500 nM
rotenone only exhibited a robust presence of Thioflavin S-positive
aggregates (not shown), whereas addition of 500 nM, 1 .mu.M or 5
.mu.M SA-72 markedly reduced the abundance of these
rotenone-induced aggregates by 63%, 65% and 83%, respectively,
relative to rotenone only-treated cells (Table 3). Similarly, in
cells treated with 2 .mu.M rotenone only, there was a robust
presence of Thioflavin S-positive aggregates (not shown), whereas
addition of 500 nM or 1 .mu.M SA-72 markedly reduced the abundance
of these rotenone-induced aggregates by 67% and 42%, respectively,
relative to rotenone only-treated cells (Table 3). Therefore, SA-72
reduced, inhibited, prevented and/or eliminated Thioflavin
S-positive aggregates in cells that express human A53T
.alpha.-synuclein.
[0251] In addition to SA-72, compounds SA-52, SA-53, SA-54, SA-58,
SA-59, SA-61, SA-62, SA-66, SA-67, SA-68, SA-93, SA-94, SA-95,
SA-96 and SA-98, at given concentrations, all showed significant
disruption/prevention/inhibition of rotenone-induced Thioflavin
S-positive inclusions when tested in a similar fashion. These
results are summarized in Table 3.
[0252] Taken together, we concluded that the tested compounds
SA-52, SA-53, SA-54, SA-58, SA-59, SA-61, SA-62, SA-66, SA-67,
SA-68, SA-72 SA-93, SA-94, SA-95, SA-96 and SA-98 effectively and
potently reduced, prevented and/or inhibited the formation,
deposition and/or accumulation of .alpha.-synuclein aggregates in
A53T .alpha.-synuclein-expressing BE-M17 cells.
TABLE-US-00003 TABLE 3 SA compounds prevent/inhibit
rotenone-induced Thioflavin S-positive .alpha.-synuclein aggregates
in cells. Concentration in .mu.M Efficacy SA # rotenone compound %
Inhibition 52 2 5 49 53 2 2 71 54 0.5 0.5 80 0.5 2 83 2 1 56 58 0.5
2 68 2 2 63 59 0.5 1 69 0.5 2 76 0.5 5 56 2 1 67 61 0.5 0.5 36 0.5
5 48 62 0.5 0.5 81 0.5 1 55 0.5 5 85 66 0.5 0.5 39 0.5 1 32 0.5 5
94 2 0.5 57 2 1 69 67 0.5 0.5 75 0.5 1 48 0.5 2 92 68 0.5 1 69 0.5
5 73 2 5 44 72 0.5 0.5 63 0.5 1 65 0.5 5 83 2 0.5 67 2 1 42 93 2 2
45 94 0.5 2 85 2 0.5 70 2 1 63 95 0.5 0.5 73 0.5 1 98 0.5 2 90 2
0.5 95 96 0.5 1 82 2 0.5 73 2 2 81 98 0.5 0.5 74 2 1 76 Positive
Ref. #1 0.5 0.5 47 0.5 1 65 0.5 2 70 2 0.5 49 2 1 56 2 2 60
Negative Ref. 0.5 5 none detected 2 5 none detected
Example 38--Compounds of this Invention are Neuroprotective Against
Rotenone-Induced Cytotoxicity
[0253] The XTT cytotoxicity assay (Cell Proliferation Assay Kit II)
was previously used to demonstrate that A53T .alpha.-synuclein
potentiates cell death in BE-M17 cells through an oxidative
stress-dependent mechanism (Ostrerova-Golts et al., J. Neurosci.,
20:6048-6054, 2000). Research has shown that the accumulation of
.alpha.-synuclein fibrils in Lewy bodies contributes
mechanistically to the degradation 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). Here, the XTT Cell Proliferation Assay Kit II (hereafter
referred to as the XTT assay) was used to measure the ability of
compounds to provide neuroprotection against rotenone-induced
cytotoxicity. The assay is based on the principle that conversion
of the yellow tetrazolium salt XTT to form an orange formazan dye
(that absorbs light around 490 nm) occurs only in metabolically
active, viable cells. Therefore, light absorbance at 490 nm is
proportional to cell viability. For this assay, cells were plated
in 96 well tissue culture dishes at 10.sup.4 cells per well. After
16-18 hours, cells were treated with 500 nM rotenone, or vehicle
(0.05% DMSO) as indicated. Approximately 15 minutes after rotenone
addition, compounds were added at the indicated concentration. As a
control, compounds were added without rotenone (in the presence of
0.05% DMSO vehicle) and resulted in no toxicity at the doses
presented. Mock-treatment consisted of cell culture media only (no
compound), in the presence or absence of rotenone. After 44-46
hours of treatment, conditioned media was removed and replaced with
100 .mu.l fresh media and 50 .mu.l XTT labeling reaction mixture
according to the manufacturer's recommendations. Five to six hours
later, the absorbance at 493 nm was measured and corrected for
absorbance at the 620 nm reference wavelength. Treatment with 500
nM rotenone decreased viability by 30-40%. Percent inhibition of
cell death was calculated as the proportion of the rotenone-induced
absorbance (viability) decrease that was eliminated by SA compound
treatment.
[0254] Using the protocol described above, several selected
compounds were tested for their ability to provide neuroprotection
against the rotenone-induced loss of cell viability (cell death) in
A53T .alpha.-synuclein-expressing BE-M17 cells. In this series of
experiments, there was a 30-40% loss of viability (cell death) in
500 nM rotenone-treated cells, relative to vehicle-treated cells,
as expected. However, treatment with the compounds SA-52, SA-58,
SA-59, SA-60, SA-61, SA-62, SA-64, SA-67, SA-68, SA-69, SA-70,
SA-72, SA-73, SA-93, SA-94, SA-95, SA-96, SA-97, SA-98, and both
positive reference compounds 2 and 3, resulted in a significant,
dose-dependent inhibition of rotenone-induced cell death. To define
the relative potency of each compound, the % inhibition of cell
death was plotted against the log of the dose (.mu.M), and, when
possible, the 50% inhibitory concentration (IC.sub.50) was
calculated from the dose response curve. For example, treatment
with 3.5 .mu.M SA-94 resulted in 35% inhibition of cell death and
treatment with 15 .mu.M SA-94 resulted in 58% inhibition of cell
death, with a calculated IC.sub.50 of 2.3 .mu.M (FIG. 4), whereas
treatment with the negative reference compound showed no inhibition
of rotenone-induced cell death in this dose range (not shown). The
positive results from this assay for the compounds described herein
are summarized in Table 4.
[0255] Taken together, we concluded that the tested compounds that
were efficacious in inhibiting rotenone-induced cytotoxicity
demonstrate neuroprotective activity against .alpha.-synuclein
toxicity in this system.
TABLE-US-00004 TABLE 4 SA compounds prevent/inhibit
rotenone-induced cell death in A53T-mutant .alpha.-synuclein
neuroblastoma cells SA # IC.sub.50 (.mu.M) Max % Inhibition 52 23.8
64 58 3.5 100 59 3.1 63 60 12.5 33 61 3.6 93 62 3.6 31 64 2.6 100
67 8.5 44 68 24 61 69 35-75 30 70 5.6 78 72 10.3 39 73 35-75 77 93
19.8 100 94 2.3 58 95 3.8 63 96 4.3 61 97 39 42 98 48 56 Positive
Ref. #2 4.2 72 Positive Ref. #3 10.6 82 Negative Ref. not
determined 0
Example 39--Compounds of this Invention Directly Inhibit the In
Vitro Conversion of .alpha.-synuclein to .beta.-Sheet Containing
Structures
[0256] As described above, Thioflavin S histochemistry in
.alpha.-synuclein expressing cells was used to detect aggregates
containing a high degree of .beta.-pleated sheet structures that
formed in response to rotenone treatment. Since several compounds
were shown to reduce the abundance of Thioflavin S-positive
aggregates (Example 3), we sought independent confirmation that the
compounds directly inhibit the conversion of .alpha.-synuclein to
.beta.-sheet containing structures by using circular dichroism (CD)
spectroscopy. For this purpose, .alpha.-synuclein was obtained from
rPeptide as a lyophilized salt in 1 mg aliquots. Buffer components
and other solvents were obtained from Sigma as A.C.S. Reagent grade
or higher. Wild-type .alpha.-synuclein was dissolved in a buffer
containing 9.5 mM phosphate, 137 mM sodium chloride and 2.7 mM
potassium chloride (phosphate-buffered saline; PBS), and the pH was
adjusted to pH 7.4. This solution was then re-lyophilized and
dissolved in 0.5 mL deionized water at 2 mg/mL (138 .mu.M), and an
aliquot taken and diluted to 0.05 mg/mL in PBS for CD spectral
analysis (t=0, unfolded reference control). In order to induce
aggregation, 1 mg/ml .alpha.-synuclein (69 .mu.M) was incubated at
37'C for 24 hours with shaking (1,300 rpm), either alone, or in the
presence of one of the test compounds (at test
compound:.alpha.-synuclein molar ratios of 5:1, 1:1, 0.5:1, 0.1:1,
0.05:1, 0.01:1). After 24 hours, reactions were diluted 20-fold in
PBS and CD spectra for each reaction were 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 .alpha.-synuclein concentration of 0.05 mg/ml. The spectra were
trimmed at the shortest wavelength that still provided a dynode
voltage 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 .alpha.-synuclein). These blank corrected spectra were then
zeroed at 260 nm and the units converted from millidegrees to
specific ellipticity.
[0257] Percent .beta.-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 .alpha.-synuclein fibrillized for 24
hours (complete fibrilization), and assigning this difference the
arbitrary value of 100% .beta.-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 arbitrary
value of 0% .beta.-sheet. These percent .beta.-sheet values were
then used to provide the respective relative % inhibition of
.beta.-sheet induced by the compounds at given molar ratio of test
compound:.alpha.-synuclein. For each compound, the % inhibition of
.beta.-sheet formation was plotted against the log of the
concentration (mole ratio) of the test compound and, where
possible, the 50% inhibitory concentration (IC.sub.50) was
calculated from the dose response curve.
[0258] First, in order to confirm that .alpha.-synuclein is indeed
converted to a .beta.-sheet-rich structure and to establish the
timing of this conversion at 24 hours in our system, an aliquot of
the .alpha.-synuclein only incubation mixture (without compounds)
was sampled at various time points and CD spectra collected. At 24
hours of incubation, CD analysis revealed a large abundance of a
.beta.-sheet-rich structure, indicated by the pronounced specific
ellipticity minimum at 218 nm and maximum at 197 nm (not shown).
However, when test compounds SA-54, SA-55, SA-57, SA-58, SA-59,
SA-61, SA-62, SA-64, SA-67, SA-68, SA-70, SA-72, SA-93, SA-94,
SA-95, SA-96, SA-97, SA-98, SA-99 or positive reference compound #1
were included individually in the reaction mixture, at appropriate
concentrations, and the incubation mixture sampled 24 hours later,
there was an absence of the minimum at 218 nm. Instead, a spectrum
characteristic of random coil was exhibited (not shown). We
conclude that these compounds prevent the conversion of natively
unfolded .alpha.-synuclein to a .beta.-sheet-rich structure. These
results are summarized in Table 5. As a specific example, compound
SA-57 resulted in nearly complete inhibition when used at test
compound:.alpha.-synuclein molar ratios .gtoreq.0.05:1, with a
calculated IC.sub.50 of 0.026 moles of compound per mole of
.alpha.-synuclein (FIG. 5), whereas the negative reference compound
did not significantly inhibit .beta.-sheet formation at any of the
tested molar ratios (not shown). Nearly all of the compounds that
inhibited .alpha.-synuclein .beta.-sheet formation did so at less
than equimolar ratios relative to .alpha.-synuclein (i.e. IC.sub.50
molar ratios <1), although some compounds (for example, SA-99;
IC.sub.50=2.08) required higher concentrations in order to markedly
inhibit .alpha.-synuclein .beta.-sheet formation (Table 5). Taken
together, these results indicate that these SA compounds show
potent inhibition and prevention of .alpha.-synuclein aggregation a
hallmark of the synucleinopathies such as Parkinson's disease.
TABLE-US-00005 TABLE 5 SA compounds prevent/inhibit .beta.-sheet
containing .alpha.-synuclein aggregates as assessed by circular
dichroism spectroscopy. SA # IC.sub.50 (mole ratio) Max %
Inhibition 54 0.55 100 55 0.055 100 57 0.026 100 58 0.36 94 59 0.75
99 61 0.78 95 62 0.54 88 64 0.56 100 67 0.41 100 68 0.6 100 70 0.25
92 72 0.27 100 93 0.38 91 94 0.22 95 95 0.68 97 96 0.13 100 97 not
determined 100 98 0.08 100 99 2.08 98 Positive Ref. #1 0.04 100
Negative Ref. not determined none detected
Example 40--Compounds Provided Herein Bind with High Affinity to
Parkinson's Disease .alpha.-synuclein Fibrils
[0259] The compounds prepared in the preceding examples were found
to bind with high affinity to .alpha.-synuclein aggregates/fibrils
that are found in the hallmark Lewy Bodies of Parkinson's disease.
In order to assess relative binding affinities of the test
compounds for aggregated .alpha.-synuclein, competition assays were
set up with a radiolabeled molecule already known to bind to
.alpha.-synuclein fibrils and non-radiolabeled test compounds. In
order to induce its aggregation, .alpha.-synuclein was incubated in
phosphate buffered saline (PBS, pH 7.4) at 37.degree. C. for three
days with shaking (1,400 rpm). Competitive binding assays were
carried out in 12.times.75 mm borosilicate glass tubes. The
reaction mixture contained 100 .mu.L of .alpha.-synuclein
aggregates (0.5-1 .mu.g), [.sup.3H] positive reference compound #1
(100-200 nM diluted in PBS) and 50 .mu.L of competing compounds
(10.sup.-5-10.sup.-9 M, diluted serially in PBS containing 0.1%
bovine serum albumin) in a final volume of 0.25 ml. Non-specific
binding was defined in the presence of cold positive reference
compound #1 (50 .mu.M) in the same assay tubes. The mixture was
incubated for 120 min at 37.degree. C., and the bound and the free
radioactivity were separated by vacuum filtration through Whatman
GF/B filters using a Brandel M-24R cell harvester, followed by
washing with PBS buffer three times. Filters containing the bound
[.sup.3H] positive reference compound #1 were assayed for
radioactivity in a liquid scintillation counter (Beckman LS6500).
IC.sub.50 values were determined by a non-linear, least squares
regression analysis. Inhibition constants (Ki) values were
calculated using the equation of Cheng and Prusoff (Cheng et al.,
Biochemical Pharmacology 22:3099-3108, 1973) using the observed
IC.sub.50 of the tested compound, the concentration of radioligand
employed in the assay, and the value for the Kd of the ligand (600
nM).
[0260] The results from these experiments are reported in Table 6.
As an example, SA-64 binds with high affinity to .alpha.-synuclein
fibrils (Ki=89 nM) but does not show significant binding affinity
for the amyloid-.beta. peptide of Alzheimer's disease (not shown).
Similarly, SA-58 and SA-57 bind with high affinity to
.alpha.-synuclein aggregates, with binding constants (Ki) of 105 nM
and 124 nM, respectively. The increased binding affinity (by
4-5-fold) of SA-57 and SA-58, relative to positive reference
molecule #1 represents a significant improvement in binding to
.alpha.-synuclein aggregates for these new molecules. Taken
together, these results indicate that SA compounds bind to varying
degrees to the .alpha.-synuclein aggregates, a hallmark of
synucleinopathies such as Parkinson's disease.
TABLE-US-00006 TABLE 6 SA compounds bind to .alpha.-synuclein
aggregates as measured by an in vitro competition binding assay. SA
# K.sub.i (nM) 52 1260 53 813 54 1640 55 370 57 124 58 105 59 697
61 3100 62 506 63 283 64 89 66 135 67 466 68 697 69 765 70 659 72
270 76 219 78 330 79 537 82 177 83 330 84 639 86 224 87 2000 88 970
89 1200 90 1700 94 1100 95 890 96 1350 Positive reference #1 532
negative reference #1 no binding negative reference #2
>10000
Example 41--Use of Recombinant Tau Repeat Domain for In Vitro
Screening of Tau Aggregation Inhibitors
[0261] During in vitro screening for identification of tau
aggregation inhibitors, we found that under the same experimental
conditions, formation of paired helical filaments (PHFs) from
commercially-purchased full-length tau protein (e.g. Tau441;
rPeptide) was much slower (>11 days) than that from the tau
repeat domain (TauRD; containing Q244-E372 of Tau441) (.gtoreq.24
hours). Because of the remarkably short turn-around time and common
aggregation properties, we used TauRD for in vitro drug screening
to identify tau aggregation inhibitors [Barghorn S, Biernat J, and
Mandelkow E, Purification of recombinant tau protein and
preparation of Alzheimer-paired helical filaments in vitro. Methods
Mol Biol, 2005. 299: p. 35-51]. Since the TauRD protein is not
commercially available, we produced our own protein for this study.
A cDNA fragment coding for the human TauRD (Q244-E372 of Tau441)
was cloned into a bacterial expression vector and the construct was
then expressed in E. Coli. The recombinant TauRD protein was then
purified by heat-stability treatment and cation exchange
chromatography as described [Barghorn, et al.,] with minor
modifications. Using this method, we achieved a protein yield of 10
mg per liter of bacterial culture, with >95% purity. Aggregation
and PHF formation of purified TauRD were evaluated and validated by
independent assays including Thio S fluorometry, CD spectroscopy
and electron microscopy (Data not shown). The results consistently
demonstrate that TauRD (10 .mu.M) is able to form Thio S-positive,
.beta.-sheet-containing PHFs when incubated with an equal
concentration of heparin, at 37.degree. C. (with shaking at
800-1000 rpm for .gtoreq.1 day).
Example 42--Identification of Novel Tau Aggregation Inhibitors by
Thioflavin S Fluorometry Screening
[0262] The Thio S fluorometry assay as a primary screening method
to identify tau protein aggregation inhibitors from our small
molecule library. Aggregated tau fibrils were prepared in the
presence of equimolar ratios of TauRD and heparin (10 .mu.M each)
in 20 mM Na-phosphate buffer, pH7.4. The reaction mixture was
incubated at 37.degree. C. with shaking (800-1000 rpm) for 22-24 hr
(or for 3 days). In the Thio S inhibition assays, test compounds at
0, 0.1, 1, 10 and 100 .mu.M were added at time 0 into the reaction
containing TauRD and heparin. The same reaction mixture
(+/-increasing concentrations of compounds) but without TauRD were
also set up in parallel to serve as background controls. For all
test compounds background fluorescence readings were very low,
usually <5% of those of the TauRD-containing wells. For each
compound, the IC.sub.50 was calculated using Prism version 5
software (GraphPad Software) by nonlinear regression [(Log
[inhibitor] vs. normalized response; variable slope)]. In initial
screening, 20 test compounds demonstrated a broad range of
activities for inhibiting tau protein fibril formation: IC.sub.50
values ranged from .about.5 .mu.M to infinity (i.e. no activity at
all). The results suggested that the inhibitory activities were
structure specific. The Thio S screening results are summarized in
Table 7 (in which the reactions were incubated for 22 hours).
TABLE-US-00007 TABLE 7 SA compounds inhibit tau protein fibril
formation as measured by Thioflavin S fluorometry. Compounds ThioS
(IC.sub.50, .mu.M) SA-97 5.20 SA-54 7.34 SA-95 9.02 .+-. 4.66 (n =
2) SA-63 10.08 .+-. 0.56 (n = 2) SA-57 10.24 SA-61 12.54 .+-. 1.24
(n = 2) SA-64 17.61 SA-96 21.21 SA-94 21.44 SA-99 24.70 SA-52 32.83
SA-68 33.96 SA-98 78.51 SA-70 117.00 SA-59 211.10 SA-72 290.20
SA-67 3941.00 SA-62 no inhibition SA-55 no inhibition SA-60 no
inhibition
Example 43--Select Compounds Also Inhibit Tau Protein Formation of
.beta.-Sheet Secondary Structures Characteristic of Neurofibrillary
Tangles as Determined by Circular Dichroism Spectroscopy
[0263] CD spectroscopy was also performed to determine each
compound's potency in inhibiting .beta.-sheet secondary structure
in TauRD under aggregation-prone conditions. The CD spectroscopy
and Thio S assays were typically analyzed in parallel from the same
sample preparation in order to correlate the results from two
independent assays. CD spectra were taken from the samples
containing +/-TauRD with increasing concentrations of compounds,
and collected at 25.degree. C. on a JASCO Model J-810
Spectropolarimeter. To determine compound inhibitory potency, we
established a semi-quantitative scoring system to illustrate TauRD
conformational changes on CD spectra explained below. Since CD
spectra reflect a total population of secondary structures
(including random coil, .beta.-sheet, and various intermediate
conformers) of TauRD proteins under a given condition, the CD
scores were established based on (1) the CD spectra derived from
TauRD mixtures with different ratios of random coil/.beta.-sheet;
(2) time-dependent conformational changes of TauRD. CD analysis
revealed that non-aggregated TauRD proteins in solution (at time 0
in the presence of heparin, or at various times of incubation in
the absence of heparin) showed spectra with ellipticity minima near
195 nm, characteristic of largely random coil structures (not
shown). In contrast, aggregated and fibrillar TauRD showed spectra
with minima near 218 nm, characteristic of .beta.-sheet secondary
structure (not shown). Our CD analysis studies confirmed some of
our compounds that could inhibit formation of tau protein
.beta.-sheet structure.
TABLE-US-00008 TABLE 8 SA compounds prevent/inhibit .beta.-sheet
containing tau protein as assessed by circular dichroism
spectroscopy. Compounds CD SA-97 ++ SA-54 - SA-95 + SA-63 + SA-57 -
SA-61 + SA-64 - SA-96 - SA-94 - SA-99 + SA-52 + SA-68 No data SA-98
No data SA-70 No data SA-59 - SA-72 - SA-67 n/t SA-62 n/t SA-55 -
SA-60 n/t
[0264] Table 8 summarizes data from aggregated TauRD proteins in
the presence of various SA-compounds where the CD score of `-`
indicates that the CD spectrum is similar to that of no compound
controls with ellipticity minima at 218 nm (3-sheet) and `+`
indicates that the Minima remained at 218 nm but with a reduced
magnitude (intermediate conformers) and `++` indicates that the
minima shifted to between 195-218 nm (intermediate conformers)
Example 44--Compositions of Compounds Provided Herein
[0265] The compounds provided herein, as mentioned previously, are
desirably administered in the form of pharmaceutical compositions.
Suitable pharmaceutical compositions, and the method of preparing
them, are well-known to persons of ordinary skill in the art and
are described in such treatises as Remington: The Science and
Practice of Pharmacy, A. Gennaro, ed., 20th edition, Lippincott,
Williams & Wilkins, Philadelphia, Pa. Representative
compositions are as follows.
Oral Tablet Formulation
TABLE-US-00009 [0266] % w/w Compound provided herein 10.0 Magnesium
stearate 0.5 Starch 2.0 Hydroxypropylmethylcellulose 1.0
Microcrystalline cellulose 86.5
[0267] The ingredients are mixed to homogeneity, then granulated
with the aid of water, and the granulates dried. The granulate is
then compressed into tablets sized to give a suitable dose of the
compound. The tablet is optionally coated by applying a suspension
of a film forming agent (e.g. hydroxypropylmethylcellulose),
pigment (e.g. titanium dioxide), and plasticizer (e.g. diethyl
phthalate), and drying the film by evaporation of the solvent. The
film coat may comprise, for example, 2-6% of the tablet weight.
Oral Capsule Formulation
[0268] The granulate from the previous section of this Example is
filled into hard gelatin capsules of a size suitable to the
intended dose. The capsule is banded for sealing, if desired.
Softgel Formulation
[0269] A softgel formulation is prepared as follows:
TABLE-US-00010 % w/w Compound provided herein 20.0 Polyethylene
glycol 400 80.0
[0270] The compound is dissolved or dispersed in the polyethylene
glycol, and a thickening agent added if required. A quantity of the
formulation sufficient to provide the desired dose of the compound
is then filled into softgels.
Parenteral Formulation
[0271] A parenteral formulation is prepared as follows:
TABLE-US-00011 % w/w Compound provided herein 1.0 Normal saline
99.0
[0272] The compound is dissolved in the saline, and the resulting
solution is sterilized and filled into vials, ampoules, and
prefilled syringes, as appropriate.
Controlled-Release Oral Formulation
[0273] A sustained release formulation may be prepared by the
method of U.S. Pat. No. 4,710,384, as follows:
[0274] One kilogram of a compound provided herein is coated in a
modified Uni-Glatt powder coater with Dow Type 10 ethyl cellulose.
The spraying solution is an 8% solution of the ethyl cellulose in
90% acetone to 10% ethanol. Castor oil is added as plasticizer in
an amount equal to 20% of the ethyl cellulose present. The spraying
conditions are as follows: 1) speed, 1 liter/hour; 2) flap, 10-15%;
3) inlet temperature, 50.degree. C., 4) outlet temperature,
30.degree. C., 5) percent of coating, 17%. The coated compound is
sieved to particle sizes between 74 and 210 microns. Attention is
paid to ensure a good mix of particles of different sizes within
that range. Four hundred mg of the coated particles are mixed with
100 mg of starch and the mixture is compressed in a hand press to
1.5 tons to produce a 500 mg controlled release tablet.
[0275] The claimed subject matter is not limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the specific embodiments in addition to those
described will become apparent to those skilled in the art from the
foregoing descriptions. Such modifications are intended to fall
within the scope of the appended claims. Various publications are
cited herein, the disclosures of which are incorporated by
reference in their entireties.
* * * * *