U.S. patent application number 12/201183 was filed with the patent office on 2009-03-05 for substituted n-aryl benzamides and related compounds for treatment of amyloid diseases and synucleinopathies.
Invention is credited to Gerardo Castillo, Thomas Lake, Lesley Larsen, Beth Nguyen, Alan Snow, Manfred Weigele.
Application Number | 20090060838 12/201183 |
Document ID | / |
Family ID | 34969702 |
Filed Date | 2009-03-05 |
United States Patent
Application |
20090060838 |
Kind Code |
A1 |
Snow; Alan ; et al. |
March 5, 2009 |
SUBSTITUTED N-ARYL BENZAMIDES AND RELATED COMPOUNDS FOR TREATMENT
OF AMYLOID DISEASES AND SYNUCLEINOPATHIES
Abstract
Substituted n-aryl benzamides, related compounds and their
pharmaceutically acceptable salts, their synthesis and labeling,
pharmaceutical compositions containing them, and their use in the
treatment of amyloid diseases, including A.beta. amyloidosis, such
as observed in Alzheimer's disease, IAPP amyloidosis, such as
observed in type 2 diabetes, and synucleinopathies, such as
observed in Parkinson's disease, and the manufacture of medicaments
for such treatment are provided. Also provided is the use of the
disclosed compounds as imaging agents and methods for in vivo
imaging and diagnosis of amyloid and synuclein diseases.
Inventors: |
Snow; Alan; (Lynnwood,
WA) ; Weigele; Manfred; (Cambridge, MA) ;
Larsen; Lesley; (Dunedin, NZ) ; Nguyen; Beth;
(Gurnee, IL) ; Lake; Thomas; (Snohomish, WA)
; Castillo; Gerardo; (Bothell, WA) |
Correspondence
Address: |
PROTEOTECH, INC.
12040 115TH AVE NE
KIRKLAND
WA
98034-6931
US
|
Family ID: |
34969702 |
Appl. No.: |
12/201183 |
Filed: |
August 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11328748 |
Jan 9, 2006 |
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12201183 |
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11129771 |
May 12, 2005 |
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11328748 |
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60570669 |
May 12, 2004 |
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60629525 |
Nov 18, 2004 |
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Current U.S.
Class: |
424/1.89 ;
514/617; 564/184 |
Current CPC
Class: |
A61P 21/00 20180101;
A61P 35/00 20180101; C07C 311/08 20130101; A61P 25/16 20180101;
A61P 25/28 20180101; A61P 43/00 20180101; A61P 25/00 20180101; A61P
3/10 20180101 |
Class at
Publication: |
424/1.89 ;
564/184; 514/617 |
International
Class: |
A61K 51/04 20060101
A61K051/04; C07C 233/88 20060101 C07C233/88; A61K 101/02 20060101
A61K101/02; A61K 31/167 20060101 A61K031/167 |
Claims
1. A compound having a formula: ##STR00081## or a pharmaceutically
acceptable salt thereof, where A.sub.1 and B.sub.1 are each
independently selected from halogen, pseudohalo, nitro,
.sup.+NH.sub.3, SO.sub.3H, carboxy and haloalkyl; and t and v are
each independently 1 to 3.
2. The compound of claim 1, wherein the compound has a formula
selected from: ##STR00082##
3. The compound of claim 1, wherein the compound has a formula
selected from:
N-(3,4-dihydroxyphenyl)-2-fluoro-3,4-dihydroxybenzamide,
N-(3,4-dihydroxyphenyl)-2-fluoro-4,5-dihydroxybenzamide,
N-(3,4-dihydroxyphenyl)-3-fluoro-4,5-dihydroxybenzamide,
N-(3-fluoro-4,5-dihydroxyphenyl)-3,4-dihydroxybenzamide,
N-(2-fluoro-4,5-dihydroxyphenyl)-3,4-dihydroxybenzamide, and
N-(2-fluoro-3,4-dihydroxyphenyl)-3,4-dihydroxybenzamide.
4. The compound of claim 1, wherein A.sub.1 and B.sub.1 are each
independently selected from F, Cl, Br, I, cyanide, cyanate,
thiocyanate, selenocyanate, trifluoromethoxy, and azide,
5. A pharmaceutical composition comprising the compound of claim 1
or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable excipient.
6. 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 amyloid
protein, and a label that indicates that the compound or
pharmaceutically acceptable salt thereof is used for treating the
formation, deposition, accumulation, or persistence of amyloid
protein.
7. A method of treating the formation, deposition, accumulation, or
persistence of amyloid protein, comprising administering an
effective amount of the compound of claim 1.
8. The method of claim 7, where the amyloid protein is
A.beta.amyloid fibrils.
9. The method of claim 7, where the amyloid protein is IAPP amyloid
fibrils.
10. The method of claim 7, where the compound administered is in an
amount between 0.1 mg/Kg/day and 1000 mg/Kg/day.
11. The method of claim 7, where the compound is administered in an
amount between 1 mg/Kg/day and 100 mg/Kg/day.
12. The method of claim 7, where amount of compound administered is
in an amount between 10 mg/Kg/day and 100 mg/Kg/day.
13. 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
.alpha.-synuclein protein, and a label that indicates that the
compound or pharmaceutically acceptable salt thereof is used for
treating the formation, deposition, accumulation, or persistence of
.alpha.-synuclein protein.
14. A method of treating the formation, deposition, accumulation,
or persistence of .alpha.-synuclein protein, comprising
administering an effective amount of the compound of claim 1.
15. The method of claim 14, where the .alpha.-synuclein proteins
are .alpha.-synuclein fibrils.
16. The method of claim 14, where the compound administered is in
an amount between 0.1 mg/Kg/day and 1000 mg/Kg/day.
17. The method of claim 14, where the compound is administered in
an amount between 1 mg/Kg/day and 100 mg/Kg/day.
18. The method of claim 14, where amount of compound administered
is in an amount between 10 mg/Kg/day and 100 mg/Kg/day.
19. The compound of claim 1 where A.sub.1 or B.sub.1 is
.sup.18F.
20. An amyloid imaging agent comprising the compound of claim 1
where A.sub.1 or B.sub.1 is .sup.18F.
21. A method of in vivo imaging of amyloid deposits, comprising
introducing into a mammal a detectable quantity of the compound of
claim 1 which has been radiolabeled; allowing sufficient time for
the labeled compound to become associated with amyloid deposits and
detecting the labeled compound associated with one or more amyloid
deposits.
22. The method of claim 21 wherein the step of detecting the
labeled compound is by positron emission tomography or single
photon emission computed tomography.
23. An amyloid diagnostic agent comprising the compound of claim 1
wherein A.sup.1 or B.sub.1 is .sup.18F.
24. A method of in vivo diagnosis of Alzheimer's disease,
comprising introducing into a mammal a detectable quantity of the
compound of claim 1 which has been radiolabeled, allowing
sufficient time for the labeled compound to become associated with
amyloid deposits and detecting the labeled compound associated with
one or more amyloid deposits.
25. The method of claim 24 wherein the step of detecting the
labeled compound is by positron emission tomography or single
photon emission computed tomography.
26. An .alpha.-synuclein imaging agent comprising the compound of
claim 1 wherein A.sub.1 or B.sub.1 is .sup.18F.
27. A method of in vivo imaging of .alpha.-synuclein deposits,
comprising introducing into a mammal a detectable quantity of the
compound of claim 1 which has been radiolabeled; allowing
sufficient time for the labeled compound to become associated with
.alpha.-synuclein deposits; and detecting the labeled compound
associated with one or more .alpha.-synuclein deposits.
28. The method of claim 27 wherein the step of detecting the
labeled compound is by positron emission tomography or single
photon emission computed tomography.
29. An a synuclein diagnostic agent comprising the compound of
claim 1 wherein A.sub.1 or B.sub.1 is .sup.18F.
30. A method of in vivo diagnosis of Parkinson's disease,
comprising introducing into a mammal a detectable quantity of the
compound of claim 1 which has been radiolabeled, allowing
sufficient time for the labeled compound to become associated with
Lewy body deposits and detecting the labeled compound associated
with one or more Lewy body deposits.
31. The method of claim 30 wherein the step of detecting the
labeled compound is by single photon emission computed tomography
or single photon emission computed tomography.
Description
RELATED APPLICATIONS
[0001] This application is a continuation in part of U.S.
application Ser. No. 11/328,748 which is a continuation of U.S.
application Ser. No. 11/129,771, filed May 12, 2005, entitled
"Substituted N-Aryl Benzamides and Related Compounds for Treatment
of Amyloid Diseases and Synucleinopathies." U.S. application Ser.
No. 11/129,771 claims priority under 35 U.S.C. .sctn. 119(e) to
U.S. provisional application Ser. Nos. 60/570,669, entitled
"Substituted N-Aryl Benzamides and Related Compounds for Treatment
of Amyloid Diseases and Synucleinopathies" to Snow et al., filed
May 12, 2004 and 60/629,525, entitled "Substituted N-Aryl
Benzamides and Related Compounds for Treatment of Amyloid Diseases
and Synucleinopathies" to Snow et al., filed Nov. 18, 2004. The
contents of the above-referenced applications are incorporated by
reference herein.
TECHNICAL FIELD
[0002] Provided herein are substituted N-aryl benzamides and
related compounds, pharmaceutical compositions and methods for
treatment of amyloid diseases, including beta-amyloid protein
(A.beta.), such as observed in Alzheimer's disease and Down's
syndrome, islet amyloid polypeptide (IAPP), such as observed in
type 2 diabetes, and alpha-synuclein, such as observed in
Parkinson's disease.
BACKGROUND
[0003] Alzheimer's disease is characterized by the accumulation of
a 39-43 amino acid peptide termed the .beta.-amyloid protein or
A.beta., in a fibrillar form, existing as extracellular amyloid
plaques and as amyloid within the walls of cerebral blood vessels.
Fibrillar A.beta. amyloid deposition in Alzheimer's disease is
believed to be detrimental to the patient and eventually leads to
toxicity and neuronal cell death, characteristic hallmarks of
Alzheimer's disease. Accumulating evidence implicates amyloid, and
more specifically, the formation, deposition, accumulation and/or
persistence of A.beta. fibrils, as a major causative factor of
Alzheimer's disease pathogenesis. In addition, besides Alzheimer's
disease, a number of other amyloid diseases involve formation,
deposition, accumulation and persistence of A.beta. fibrils,
including Down's syndrome, disorders involving congophilic
angiopathy, such as but not limited to, hereditary cerebral
hemorrhage of the Dutch type, inclusion body myositosis, dementia
pugilistica, cerebral .beta.-amyloid angiopathy, dementia
associated with progressive supranuclear palsy, dementia associated
with cortical basal degeneration and mild cognitive impairment.
[0004] Parkinson's disease is another human disorder characterized
by the formation, deposition, accumulation and/or persistence of
abnormal fibrillar protein deposits that demonstrate many of the
characteristics of amyloid. In Parkinson's disease, an accumulation
of cytoplasmic Lewy bodies consisting of filaments of
.alpha.-synuclein/NAC (non-A.beta. component) are believed
important in the pathogenesis and as therapeutic targets. New
agents or compounds able to inhibit .alpha.-synuclein and/or NAC
formation, deposition, accumulation and/or persistence, or disrupt
pre-formed .alpha.-synuclein/NAC fibrils (or portions thereof) are
regarded as potential therapeutics for the treatment of Parkinson's
and related synucleinopathies. NAC is a 35 amino acid fragment of
.alpha.-synuclein that has the ability to form amyloid-like fibrils
either in vitro or as observed in the brains of patients with
Parkinson's disease. The NAC fragment of .alpha.-synuclein is a
relative 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.
[0005] A variety of other human diseases also demonstrate amyloid
deposition and usually involve systemic organs (i.e. organs or
tissues lying outside the central nervous system), with the amyloid
accumulation leading to organ dysfunction or failure. These amyloid
diseases (discussed below) leading to marked amyloid accumulation
in a number of different organs and tissues, are known as systemic
amyloidoses. In other amyloid diseases, single organs may be
affected such as the pancreas in 90% of patients with type 2
diabetes. In this type of amyloid disease, the beta-cells in the
islets of Langerhans in pancreas are believed to be destroyed by
the accumulation of fibrillar amyloid deposits consisting primarily
of a protein known as islet amyloid polypeptide (IAPP). Inhibiting
or reducing such IAPP amyloid fibril formation, deposition,
accumulation and persistence is believed to lead to new effective
treatments for type 2 diabetes. In Alzheimer's disease, Parkinson's
and "systemic" amyloid diseases, there is currently no cure or
effective treatment, and the patient usually dies within 3 to 10
years from disease onset.
[0006] The amyloid diseases (amyloidoses) are classified according
to the type of amyloid protein present as well as the underlying
disease. Amyloid diseases have a number of common characteristics
including each amyloid consisting of a unique type of amyloid
protein. The amyloid diseases include, but are not limited to, the
amyloid associated with Alzheimer's disease, Down's syndrome,
hereditary cerebral hemorrhage with amyloidosis of the Dutch type,
dementia pugilistica, inclusion body myositosis (Askanas et al,
Ann. Neurol. 43:521-560, 1993) and mild cognitive impairment (where
the specific amyloid is referred to as beta-amyloid protein or
A.beta.), the amyloid associated with chronic inflammation, various
forms of malignancy and Familial Mediterranean Fever (where the
specific amyloid is referred to as AA amyloid or
inflammation-associated amyloidosis), the amyloid associated with
multiple myeloma and other B-cell dyscrasias (where the specific
amyloid is referred to as AL amyloid), the amyloid associated with
type 2 diabetes (where the specific amyloid protein is referred to
as amylin or islet amyloid polypeptide or IAPP), the amyloid
associated with the prion diseases including Creutzfeldt-Jakob
disease, Gerstmann-Straussler syndrome, kuru and animal scrapie
(where the specific amyloid is referred to as PrP amyloid), the
amyloid associated with long-term hemodialysis and carpal tunnel
syndrome (where the specific amyloid is referred to as
.alpha..sub.2-microglobulin amyloid), the amyloid associated with
senile cardiac amyloidosis and Familial Amyloidotic Polyneuropathy
(where the specific amyloid is referred to as transthyretin or
prealbumin), and the amyloid associated with endocrine tumors such
as medullary carcinoma of the thyroid (where the specific amyloid
is referred to as variants of procalcitonin). In addition, the
.alpha.-synuclein protein which forms amyloid-like fibrils, and is
Congo red and Thioflavin S positive (specific stains used to detect
amyloid fibrillar deposits), is found as part of Lewy bodies in the
brains of patients with Parkinson's disease, Lewy body disease
(Lewy in Handbuch der Neurologie, M. Lewandowski, ed., Springer,
Berlin pp. 920-933, 1912; Pollanen et al, J. Neuropath. Exp.
Neurol. 52:183-191, 1993; Spillantini et al, Proc. Natl. Acad. Sci.
USA 95:6469-6473, 1998; Arai et al, Neurosci. Lett. 259:83-86,
1999), multiple system atrophy (Wakabayashi et al, Acta Neuropath.
96:445-452, 1998), dementia with Lewy bodies, and the Lewy body
variant of Alzheimer's disease. For purposes of this disclosure,
Parkinson's disease, due to the fact that fibrils develop in the
brains of patients with this disease (which are Congo red and
Thioflavin S positive, and which contain predominant beta-pleated
sheet secondary structure), is now regarded as a disease that also
displays the characteristics of an amyloid-like disease.
[0007] Systemic amyloidoses which include the amyloid associated
with chronic inflammation, various forms of malignancy and familial
Mediterranean fever (i.e. AA amyloid or inflammation-associated
amyloidosis) (Benson and Cohen, Arth. Rheum. 22:36-42, 1979; Kamei
et al, Acta Path. Jpn. 32:123-133, 1982; McAdam et al., Lancet
2:572-573, 1975; Metaxas, Kidney Int. 20:676-685, 1981), and the
amyloid associated with multiple myeloma and other B-cell
dyscrasias (i.e. AL amyloid) (Harada et al., J. Histochem.
Cytochem. 19:1-15, 1971), as examples, are known to involve amyloid
deposition in a variety of different organs and tissues generally
lying outside the central nervous system. Amyloid deposition in
these diseases may occur, for example, in liver, heart, spleen,
gastrointestinal tract, kidney, skin, and/or lungs (Johnson et al,
N. Engl. J. Med. 321:513-518, 1989). For most of these amyloidoses,
there is no apparent cure or effective treatment and the
consequences of amyloid deposition can be detrimental to the
patient. For example, amyloid deposition in the kidney may lead to
renal failure, whereas amyloid deposition in the heart may lead to
heart failure. For these patients, amyloid accumulation in systemic
organs leads to eventual death generally within 3-5 years. Other
amyloidoses may affect a single organ or tissue such as observed
with the A.beta. amyloid deposits found in the brains of patients
with Alzheimer's disease and Down's syndrome: the PrP amyloid
deposits found in the brains of patients with Creutzfeldt-Jakob
disease, Gerstmann-Straussler syndrome, and kuru; the islet amyloid
(IAPP) deposits found in the islets of Langerhans in the pancreas
of 90% of patients with type 2 diabetes (Johnson et al, N. Engl. J.
Med. 321:513-518, 1989; Lab. Invest. 66:522 535, 1992); the
.alpha..sub.2-microglobulin amyloid deposits in the medial nerve
leading to carpal tunnel syndrome as observed in patients
undergoing long-term hemodialysis (Geyjo et al, Biochem. Biophys.
Res. Comm. 129:701-706, 1985; Kidney Int. 30:385-390, 1986); the
prealbumin/transthyretin amyloid observed in the hearts of patients
with senile cardiac amyloid; and the prealbumin/transthyretin
amyloid observed in peripheral nerves of patients who have familial
amyloidotic polyneuropathy (Skinner and Cohen, Biochem. Biophys.
Res. Comm. 99:1326-1332, 1981; Saraiva et al, J. Lab. Clin. Med.
102:590-603, 1983; J. Clin. Invest. 74:104-119, 1984; Tawara et al,
J. Lab. Clin. Med. 98:811-822, 1989).
[0008] Alzheimer's disease also puts a heavy economic burden on
society. A recent study estimated that the cost of caring for one
Alzheimer's disease patient with severe cognitive impairments at
home or in a nursing home, is more than $47,000 per year (A Guide
to Understanding Alzheimer's Disease and Related Disorders). For a
disease that can span from 2 to 20 years, the overall cost of
Alzheimer's disease to families and to society is staggering. The
annual economic toll of Alzheimer's disease in the United States in
terms of health care expenses and lost wages of both patients and
their caregivers is estimated at $80 to $100 billion (2003 Progress
Report on Alzheimer's Disease).
[0009] Amyloid as a Therapeutic Target for Alzheimer's Disease
[0010] Alzheimer's disease is characterized by the deposition and
accumulation of a 39-43 amino acid peptide termed the beta-amyloid
protein, A.beta. or .beta./A4 (Glenner and Wong, Biochem. Biophys.
Res. Comm. 120:885-890, 1984; Masters et al., Proc. Natl. Acad.
Sci. USA 82:4245-4249, 1985; Husby et al., Bull. WHO 71:105-108,
1993). A.beta. is derived by protease cleavage from larger
precursor proteins termed .beta.-amyloid precursor proteins (APPs)
of which there are several alternatively spliced variants. The most
abundant forms of the APPs include proteins consisting of 695, 751
and 770 amino acids (Tanzi et al., Nature 31:528-530, 1988).
[0011] The small A.beta. peptide is a major component that makes up
the amyloid deposits of "plaques" in the brains of patients with
Alzheimer's disease. In addition, Alzheimer's disease is
characterized by the presence of numerous neurofibrillary
"tangles", consisting of paired helical filaments which abnormally
accumulate in the neuronal cytoplasm (Grundke-Iqbal et al., Proc.
Natl. Acad. Sci. USA 83:4913-4917, 1986; Kosik et al., Proc. Natl.
Acad. Sci. USA 83:4044-4048, 1986; Lee et al., Science 251:675-678,
1991). The pathological hallmark of Alzheimer's disease is
therefore the presence of "plaques" and "tangles", with amyloid
being deposited in the central core of the plaques. The other major
type of lesion found in the Alzheimer's disease brain is the
accumulation of amyloid in the walls of blood vessels, both within
the brain parenchyma and in the walls of meningeal vessels that lie
outside the brain. The amyloid deposits localized to the walls of
blood vessels are referred to as cerebrovascular amyloid or
congophilic angiopathy (Mandybur, J. Neuropath. Exp. Neurol.
45:79-90, 1986; Pardridge et al., J. Neurochem. 49:1394-1401,
1987)
[0012] For many years there has been an ongoing scientific debate
as to the importance of "amyloid" in Alzheimer's disease, and
whether the "plaques" and "tangles" characteristic of this disease
were a cause or merely a consequence of the disease. Within the
last few years, studies now indicate that amyloid is indeed a
causative factor for Alzheimer's disease and should not be regarded
as merely an innocent bystander. The Alzheimer's A.beta. protein in
cell culture has been shown to cause degeneration of nerve cells
within short periods of time (Pike et al., Br. Res. 563:311-314,
1991; J. Neurochem. 64:253-265, 1995). Studies suggest that it is
the fibrillar structure (consisting of a predominant .beta.-pleated
sheet secondary structure), characteristic of all amyloids, that is
responsible for the neurotoxic effects. A.beta. has also been found
to be neurotoxic in slice cultures of hippocampus (Harrigan et al.,
Neurobiol. Aging 16:779-789, 1995) and induces nerve cell death in
transgenic mice (Games et al., Nature 373:523-527, 1995; Hsiao et
al., Science 274:99-102, 1996). Injection of the Alzheimer's
A.beta. into rat brain also causes memory impairment and neuronal
dysfunction (Flood et al., Proc. Natl. Acad. Sci. USA 88:3363-3366,
1991; Br. Res. 663:271-276, 1994).
[0013] Probably, the most convincing evidence that A.beta. amyloid
is directly involved in the pathogenesis of Alzheimer's disease
comes from genetic studies. It was discovered that the production
of A.beta. can result from mutations in the gene encoding, its
precursor, .beta.-amyloid precursor protein (Van Broeckhoven et
al., Science 248:1120-1122, 1990; Murrell et al., Science
254:97-99, 1991; Haass et al., Nature Med. 1: 1291-1296, 1995). The
identification of mutations in the beta-amyloid precursor protein
gene that cause early onset familial Alzheimer's disease is the
strongest argument that amyloid is central to the pathogenetic
process underlying this disease. Four reported disease-causing
mutations have been discovered which demonstrate the importance of
A.beta. in causing familial Alzheimer's disease (reviewed in Hardy,
Nature Genet. 1:233-234, 1992). All of these studies suggest that
providing a drug to reduce, eliminate or prevent fibrillar A.beta.
formation, deposition, accumulation and/or persistence in the
brains of human patients will serve as an effective
therapeutic.
[0014] Parkinson's Disease and Synucleinopathies
[0015] 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), an 140-amino acid protein (Ueda
et al., Proc. Natl. Acad. Sci. USA 90:11282-11286, 1993). Two
dominant mutations in .alpha.-synuclein causing 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). 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). Most importantly, both Parkinson's
disease-linked .alpha.-synuclein mutations accelerate this
aggregation process, demonstrating that such in vitro studies may
have relevance for Parkinson's disease pathogenesis.
Alpha-synuclein aggregation and fibril formation fulfills of the
criteria of a nucleation-dependent polymerization process (Wood et
al., J. Biol. Chem. 274:19509-19512, 1999). In this regard
.alpha.-synuclein fibril formation resembles that of Alzheimer's
.beta.-amyloid protein (A.beta.) fibrils. 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 when incubated at 37.degree.
C., and are positive with amyloid 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).
[0016] 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 and/or amyloid
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 amyloid-like fibrils 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 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".
[0017] Parkinson's disease .alpha.-synuclein fibrils, like the
A.beta. fibrils of Alzheimer's disease, also consist of a
predominantly .beta.-pleated sheet structure. Therefore, compounds
found to inhibit Alzheimer's disease A.beta. amyloid fibril
formation are also anticipated to be effective in the inhibition of
.alpha.-synuclein/NAC fibril formation, as shown from Examples
provided herein. These compounds would therefore also serve as
therapeutics for Parkinson's disease and other synucleinopathies,
in addition to having efficacy as a therapeutic for Alzheimer's
disease, type 2 diabetes, and other amyloid disorders.
[0018] Discovery and identification of new compounds or agents as
potential therapeutics to arrest amyloid formation, deposition,
accumulation and/or persistence that occurs in Alzheimer's disease,
Parkinson's disease, type II diabetes, and other amyloidoses are
desperately sought.
SUMMARY
[0019] Provided herein are compounds and pharmaceutical
compositions containing compounds having formula:
##STR00001##
or a pharmaceutically acceptable derivative thereof, where R is
selected from a 1) CONR' and 2) C.sub.1-C.sub.10 alkylene group, in
which: (a) when the number of carbon atoms is at least 2, there are
optionally 1 or 2 double bonds; (b) 1 to 3 non-adjacent methylene
groups are optionally replaced by NR', O, or S; (c) 1 or 2
methylene groups are optionally replaced by a carbonyl or
hydroxymethylene group; and (d) 1 or 2 methylene groups are
optionally replaced by a cycloalkyl or heterocyclyl group that is
optionally substituted with one or more substituents selected from
lower alkyl, NR', O, or S;
[0020] R' is H, alkyl, or acyl;
[0021] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently
selected as follows: [0022] i) R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are each independently selected from OH,
--NR.sup.5C(.dbd.O)R.sup.6, and --NR.sup.7S(O.sub.2)R.sup.5,
wherein R.sup.5 and R.sup.7 are each independently hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
aryl, substituted or unsubstituted aralkyl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroaralkyl, substituted or unsubstituted heterocyclyl or
substituted or unsubstituted heterocyclylalkyl; and R.sup.6 and
R.sup.8 are each independently substituted or unsubstituted alkoxy,
substituted or unsubstituted aralkoxy, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocyclyl, substituted or unsubstituted aryl or
--NR.sup.9R.sup.10 where R.sup.9 and R.sup.10 are each
independently hydrogen, alkyl, aralkyl, aryl, heteroaryl,
heteroaralkyl or heterocyclyl, with the proviso that at least one
of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 is not OH; [0023] ii)
R.sup.1 and R.sup.2 and/or R.sup.3 and R.sup.4 together are
--NH--C(.dbd.O)--NH--, --NH--S(O.sub.2)--NH--,
--CH.sub.2--C(.dbd.O)--NH-- or --CH.sub.2--S(O.sub.2)--NH and
together with the carbon atoms on which they are substituted form a
5 membered heterocyclic ring and the others of R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are each independently selected as in i); or
[0024] iii) at least one of R.sup.1, R.sup.2, R.sup.3 and R.sup.4
is --NH--CR.sup.a.dbd.CR.sup.b--, or
--NH--S(O.sub.2)CR.sup.cR.sup.d-- and together with two adjacent
carbon atoms of the phenyl ring forms a substituted or
unsubstituted heterocyclic or substituted or unsubstituted
heteroaromatic ring, and the others of R.sup.1, R.sup.2, R.sup.3
and R.sup.4 are each independently selected as in i) or ii);
and
[0025] wherein the rings A and B are substituted with one or more
substitutents selected from electron withdrawing groups including,
but not limited to halo, pseudohalo, nitro, .sup.+NH.sub.3,
SO.sub.3H, carboxy and haloalkyl.
[0026] In one embodiment, R.sup.1 to R.sup.10, R.sup.a, R.sup.b,
R.sup.c and R.sup.d are appropriately selected to optimize
physicochemical and/or biological properties such as,
bioavailability, pharmacokinetics, blood-brain barrier penetration,
optimized metabolism, and enhanced efficacy for treatment of
amyloid diseases and synucleinopathies.
[0027] Also provided are any pharmaceutically-acceptable
derivatives, 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, 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'-ylmethylbenzimidazole,
diethylamine and other alkylamines, piperazine,
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, salts of organic acids, such as but not limited to
acetates, lactates, malates, tartrates, citrates, ascorbates,
succinates, butyrates, valerates and fumarates.
[0028] 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 amyloid diseases, are also
provided.
[0029] 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.
[0030] Methods using such compounds and compositions for
disrupting, disaggregating and causing removal, reduction or
clearance of amyloid or synuclein fibrils are provided thereby
providing new treatments for amyloid diseases and
synucleinopathies.
[0031] Also provided are methods for treatment, prevention or
amelioration of one or more symptoms of amyloid diseases or
amyloidoses, including but not limited to diseases associated with
the formation, deposition, accumulation, or persistence of amyloid
fibrils, for example, the fibrils of an amyloid protein selected
from A.beta. amyloid, AA amyloid, AL amyloid, IAPP amyloid, PrP
amyloid, .alpha..sub.2-microglobulin amyloid, transthyretin,
prealbumin, and procalcitonin.
[0032] Methods for treatment of amyloid diseases, include, but are
not limited to Alzheimer's disease, Down's syndrome, dementia
pugilistica, multiple system atrophy, inclusion body myositosis,
hereditary cerebral hemorrhage with amyloidosis of the Dutch type,
Nieman-Pick disease type C, cerebral .beta.-amyloid angiopathy,
dementia associated with cortical basal degeneration, the
amyloidosis of type 2 diabetes, the amyloidosis of chronic
inflammation, the amyloidosis of malignancy and Familial
Mediterranean Fever, the amyloidosis of multiple myeloma and B-cell
dyscrasias, the amyloidosis of the prion diseases,
Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru,
scrapie, the amyloidosis associated with carpal tunnel syndrome,
senile cardiac amyloidosis, familial amyloidotic polyneuropathy,
and the amyloidosis associated with endocrine tumors.
[0033] Also 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, 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. Synuclein diseases include, but are not limited
to Parkinson's disease, 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.
DETAILED DESCRIPTION
A. Definitions
[0034] 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 are a plurality of definitions for a term
herein, those in this section prevail unless stated otherwise.
[0035] As used herein "Amyloid diseases" or "amyloidoses" are
diseases associated with the formation, deposition, accumulation,
or persistence of amyloid fibrils, including but not limited to the
fibrils of an amyloid protein selected from A.beta. amyloid, AA
amyloid, AL amyloid, IAPP amyloid, PrP amyloid,
.alpha..sub.2-microglobulin amyloid, transthyretin, prealbumin, and
procalcitonin. Such diseases include, but are not limited to
Alzheimer's disease, Down's syndrome, dementia pugilistica,
multiple system atrophy, inclusion body myositosis, hereditary
cerebral hemorrhage with amyloidosis of the Dutch type, Nieman-Pick
disease type C, cerebral .beta.-amyloid angiopathy, dementia
associated with cortical basal degeneration, the amyloidosis of
type 2 diabetes, the amyloidosis of chronic inflammation, the
amyloidosis of malignancy and Familial Mediterranean Fever, the
amyloidosis of multiple myeloma and B-cell dyscrasias, the
amyloidosis of the prion diseases, Creutzfeldt-Jakob disease,
Gerstmann-Straussler syndrome, kuru, scrapie, the amyloidosis
associated with carpal tunnel syndrome, senile cardiac amyloidosis,
familial amyloidotic polyneuropathy, and the amyloidosis associated
with endocrine tumors.
[0036] 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, 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.
[0037] "Fibrillogenesis" refers to the formation, deposition,
accumulation and/or persistence of amyloid fibrils, filaments,
inclusions, deposits, as well as synuclein (usually involving
.alpha.-synuclein) and/or NAC fibrils, filaments, inclusions,
deposits or the like.
[0038] "Inhibition of fibrillogenesis" refers to the inhibition of
formation, deposition, accumulation and/or persistence of such
amyloid fibrils or synuclein fibril-like deposits.
[0039] "Disruption of fibrils or fibrillogenesis" refers to the
disruption of pre-formed amyloid or 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 amyloid or 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.
[0040] "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).
[0041] "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.
[0042] 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 amyloid or
synuclein fibril formation, deposition, accumulation and/or
persistence, or treats, prevents, or ameliorates one or more
symptoms of a disease associated with these conditions, such as an
amyloid disease or a synucleinopathy, in a measurable amount in one
embodiment, by at least 20%, in other embodiment, by at least 40%,
in other embodiment by at least 60%, and in still other embodiment
by at least 80%, relative to an untreated subject. Effective
amounts of a compound provided herein or composition 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.
[0043] 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.
[0044] 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
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.
[0045] 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.
[0046] 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. Treatment of a disease also
includes preventing the disease from occurring in a subject that
may be predisposed to the disease but does not yet experience or
exhibit symptoms of the disease (prophylactic treatment),
inhibiting the disease (slowing or arresting its development),
providing relief from the symptoms or side-effects of the disease
(including palliative treatment), and relieving the disease
(causing regression of the disease), such as by disruption of
pre-formed amyloid or synuclein fibrils. One such preventive
treatment may be use of the disclosed compounds for the treatment
of Mild Cognitive impairment (MCI).
[0047] 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.
[0048] 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 amyloid-like fibrils
when incubated at 37.degree. C., and is positive with amyloid
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.
[0049] 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).
[0050] 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. In the case of amino
acid residues, such residues may be of either the L- or D-form. The
configuration for naturally occurring amino acid residues is
generally L. When not specified the residue is the L form. As used
herein, the term "amino acid" refers to .alpha.-amino acids which
are racemic, or of either the D- or L-configuration. The
designation "d" preceding an amino acid designation (e.g., dAla,
dSer, dVal, etc.) refers to the D-isomer of the amino acid. The
designation "dl" preceding an amino acid designation (e.g., dlPip)
refers to a mixture of the L- and D-isomers of the amino acid. 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.
[0051] As used herein, substantially pure means sufficiently
homogeneous to appear free of readily detectable impurities as
determined by standard methods of analysis, such as thin layer
chromatography (TLC), gel electrophoresis, high performance liquid
chromatography (HPLC) and mass spectrometry (MS), used by those of
skill in the art to assess such purity, or sufficiently pure such
that further purification would not detectably alter the physical
and chemical properties, such as enzymatic and biological
activities, of the substance. Methods for purification of the
compounds to produce substantially chemically pure compounds are
known to those of skill in the art. A substantially chemically pure
compound may, however, be a mixture of stereoisomers. In such
instances, further purification might increase the specific
activity of the compound.
[0052] As used herein, alkyl, alkenyl and alkynyl carbon chains, if
not specified, contain from 1 to 20 carbons, or 1 or 2 to 16
carbons, and are straight or branched. Alkenyl carbon chains of
from 2 to 20 carbons, in certain embodiments, contain 1 to 8 double
bonds and alkenyl carbon chains of 2 to 16 carbons, in certain
embodiments, contain 1 to 5 double bonds. Alkynyl carbon chains of
from 2 to 20 carbons, in certain embodiments, contain 1 to 8 triple
bonds, and the alkynyl carbon chains of 2 to 16 carbons, in certain
embodiments, contain 1 to 5 triple bonds. Exemplary alkyl, alkenyl
and alkynyl groups herein include, but are not limited to, methyl,
ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl,
isopentyl, neopentyl, tert-pentyl, isohexyl, allyl (propenyl) and
propargyl (propynyl). As used herein, lower alkyl, lower alkenyl,
and lower alkynyl refer to carbon chains having from about 1 or
about 2 carbons up to about 6 carbons. As used herein,
"alk(en)(yn)yl" refers to an alkyl group containing at least one
double bond and at least one triple bond.
[0053] As used herein, "cycloalkyl" refers to a saturated mono- or
multi-cyclic ring system, in certain embodiments of 3 to 10 carbon
atoms, in other embodiments of 3 to 6 carbon atoms; cycloalkenyl
and cycloalkynyl refer to mono- or multicyclic ring systems that
respectively include at least one double bond and at least one
triple bond. Cycloalkenyl and cycloalkynyl groups may, in certain
embodiments, contain 3 to 10 carbon atoms, with cycloalkenyl
groups, in further embodiments, containing 4 to 7 carbon atoms and
cycloalkynyl groups, in further embodiments, containing 8 to 10
carbon atoms. The ring systems of the cycloalkyl, cycloalkenyl and
cycloalkynyl groups may be composed of one ring or two or more
rings which may be joined together in a fused, bridged or
spiro-connected fashion. "Cycloalk(en)(yn)yl" refers to a
cycloalkyl group containing at least one double bond and at least
one triple bond.
[0054] As used herein, "aryl" refers to aromatic monocyclic or
multicyclic groups containing from 6 to 19 carbon atoms. Aryl
groups include, but are not limited to groups such as unsubstituted
or substituted fluorenyl, unsubstituted or substituted phenyl, and
unsubstituted or substituted naphthyl.
[0055] As used herein, "heteroaryl" refers to a monocyclic or
multicyclic aromatic ring system, in certain embodiments, of about
5 to about 15 members where one or more, in one embodiment 1 to 3,
of the atoms in the ring system is a heteroatom, that is, an
element other than carbon, including but not limited to, nitrogen,
oxygen or sulfur. The heteroaryl group may be optionally fused to a
benzene ring. Heteroaryl groups include, but are not limited to,
furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl,
pyrrolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl,
quinolinyl and isoquinolinyl.
[0056] As used herein, "heterocyclyl" refers to a monocyclic or
multicyclic non-aromatic ring system, in one embodiment of 3 to 10
members, in another embodiment of 4 to 7 members, in a further
embodiment of 5 to 6 members, where one or more, in certain
embodiments, 1 to 3, of the atoms in the ring system is a
heteroatom, that is, an element other than carbon, including but
not limited to, nitrogen, oxygen or sulfur. In embodiments where
the heteroatom(s) is(are) nitrogen, the nitrogen is optionally
substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl,
aralkyl, heteroaralkyl, cycloalkyl, heterocyclyl, cycloalkylalkyl,
heterocyclylalkyl, acyl, guanidino, or the nitrogen may be
quaternized to form an ammonium group where the substituents are
selected as above.
[0057] As used herein, "aralkyl" refers to an alkyl group in which
one of the hydrogen atoms of the alkyl is replaced by an aryl
group.
[0058] As used herein, "heteroaralkyl" refers to an alkyl group in
which one of the hydrogen atoms of the alkyl is replaced by a
heteroaryl group.
[0059] As used herein, "halo", "halogen" or "halide" refers to F,
Cl, Br or I.
[0060] As used herein, pseudohalides or pseudohalo groups are
groups that behave substantially similar to halides. Such compounds
can be used in the same manner and treated in the same manner as
halides. Pseudohalides include, but are not limited to, cyanide,
cyanate, thiocyanate, selenocyanate, trifluoromethoxy, and
azide.
[0061] As used herein, "haloalkyl" refers to an alkyl group in
which one or more of the hydrogen atoms are replaced by halogen.
Such groups include, but are not limited to, chloromethyl,
trifluoromethyl andl-chloro-2-fluoroethyl.
[0062] As used herein, "haloalkoxy" refers to RO-- in which R is a
haloalkyl group.
[0063] As used herein, "sulfinyl" or "thionyl" refers to --S(O)--.
As used herein, "sulfonyl" or "sulfuryl" refers to --S(O).sub.2--.
As used herein, "sulfo" refers to --S(O).sub.2O--.
[0064] As used herein, "carboxy" refers to a divalent radical,
--C(O)O--.
[0065] As used herein, "aminocarbonyl" refers to
--C(O)NH.sub.2.
[0066] As used herein, "alkylaminocarbonyl" refers to --C(O)NHR in
which R is alkyl, including lower alkyl. As used herein,
"dialkylaminocarbonyl" refers to --C(O)NR'R in which R' and R are
each independently alkyl, including lower alkyl; "carboxamide"
refers to groups of formula --NR'COR in which R' and R are each
independently alkyl, including lower alkyl.
[0067] As used herein, "arylalkylaminocarbonyl" refers to
--C(O)NRR' in which one of R and R' is aryl, including lower aryl,
such as phenyl, and the other of R and R' is alkyl, including lower
alkyl.
[0068] As used herein, "arylaminocarbonyl" refers to --C(O)NHR in
which R is aryl, including lower aryl, such as phenyl.
[0069] As used herein, "hydroxycarbonyl" refers to --COOH.
[0070] As used herein, "alkoxycarbonyl" refers to --C(O)OR in which
R is alkyl, including lower alkyl.
[0071] As used herein, "aryloxycarbonyl" refers to --C(O)OR in
which R is aryl, including lower aryl, such as phenyl.
[0072] As used herein, "alkoxy" and "alkylthio" refer to RO-- and
RS--, in which R is alkyl, including lower alkyl.
[0073] As used herein, "aryloxy" and "arylthio" refer to RO-- and
RS--, in which R is aryl, including lower aryl, such as phenyl.
[0074] As used herein, "alkylene" refers to a straight, branched or
cyclic, in certain embodiments straight or branched, divalent
aliphatic hydrocarbon group, in one embodiment having from 1 to
about 20 carbon atoms, in another embodiment having from 1 to 12
carbons. In a further embodiment alkylene includes lower alkylene.
There may be optionally inserted along the alkylene group one or
more oxygen, sulfur, including S(.dbd.O) and S(.dbd.O).sub.2
groups, or substituted or unsubstituted nitrogen atoms, including
--NR-- and --N.sup.+RR-- groups, where the nitrogen substituent(s)
is(are) alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl or COR',
where R' is alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, --OY
or --NYY, where Y is hydrogen, alkyl, aryl, heteroaryl, cycloalkyl
or heterocyclyl. Alkylene groups include, but are not limited to,
methylene (--CH.sub.2--), ethylene (--CH.sub.2CH.sub.2--),
propylene (--(CH.sub.2).sub.3--), methylenedioxy
(--O--CH.sub.2--O--) and ethylenedioxy
(--O--(CH.sub.2).sub.2--O--). The term "lower alkylene" refers to
alkylene groups having 1 to 6 carbons. In certain embodiments,
alkylene groups are lower alkylene, including alkylene of 1 to 3
carbon atoms.
[0075] As used herein, "azaalkylene" refers to
--(CRR).sub.n--NR--(CRR).sub.m--, where n and m are each
independently an integer from 0 to 4. As used herein, "oxaalkylene"
refers to --(CRR).sub.n--O--(CRR).sub.m--, where n and m are each
independently an integer from 0 to 4. As used herein,
"thiaalkylene" refers to --(CRR).sub.n--S--(CRR).sub.m--,
--(CRR).sub.n--S(.dbd.O)--(CRR).sub.m--, and
--(CRR).sub.n--S(.dbd.O).sub.2--(CRR).sub.m--, where n and m are
each independently an integer from 0 to 4.
[0076] As used herein, "alkenylene" refers to a straight, branched
or cyclic, in one embodiment straight or branched, divalent
aliphatic hydrocarbon group, in certain embodiments having from 2
to about 20 carbon atoms and at least one double bond, in other
embodiments 1 to 12 carbons. In further embodiments, alkenylene
groups include lower alkenylene. There may be optionally inserted
along the alkenylene group one or more oxygen, sulfur or
substituted or unsubstituted nitrogen atoms, where the nitrogen
substituent is alkyl. Alkenylene groups include, but are not
limited to, --CH.dbd.CH--CH.dbd.CH-- and --CH.dbd.CH--CH.sub.2--.
The term "lower alkenylene" refers to alkenylene groups having 2 to
6 carbons. In certain embodiments, alkenylene groups are lower
alkenylene, including alkenylene of 3 to 4 carbon atoms.
[0077] As used herein, "alkynylene" refers to a straight, branched
or cyclic, in certain embodiments straight or branched, divalent
aliphatic hydrocarbon group, in one embodiment having from 2 to
about 20 carbon atoms and at least one triple bond, in another
embodiment 1 to 12 carbons. In a further embodiment, alkynylene
includes lower alkynylene. There may be optionally inserted along
the alkynylene group one or more oxygen, sulfur or substituted or
unsubstituted nitrogen atoms, where the nitrogen substituent is
alkyl. Alkynylene groups include, but are not limited to,
--C.ident.C--C.ident.C--, --C.ident.C-- and
--C.ident.C--CH.sub.2--. The term "lower alkynylene" refers to
alkynylene groups having 2 to 6 carbons. In certain embodiments,
alkynylene groups are lower alkynylene, including alkynylene of 3
to 4 carbon atoms.
[0078] As used herein, "alk(en)(yn)ylene" refers to a straight,
branched or cyclic, in certain embodiments straight or branched,
divalent aliphatic hydrocarbon group, in one embodiment having from
2 to about 20 carbon atoms and at least one triple bond, and at
least one double bond; in another embodiment 1 to 12 carbons. In
further embodiments, alk(en)(yn)ylene includes lower
alk(en)(yn)ylene. There may be optionally inserted along the
alkynylene group one or more oxygen, sulfur or substituted or
unsubstituted nitrogen atoms, where the nitrogen substituent is
alkyl. Alk(en)(yn)ylene groups include, but are not limited to,
--C.dbd.C--(CH.sub.2).sub.n--C.ident.C--, where n is 1 or 2. The
term "lower alk(en)(yn)ylene" refers to alk(en)(yn)ylene groups
having up to 6 carbons. In certain embodiments, alk(en)(yn)ylene
groups have about 4 carbon atoms.
[0079] As used herein, "cycloalkylene" refers to a divalent
saturated mono- or multicyclic ring system, in certain embodiments
of 3 to 10 carbon atoms, in other embodiments 3 to 6 carbon atoms;
cycloalkenylene and cycloalkynylene refer to divalent mono- or
multicyclic ring systems that respectively include at least one
double bond and at least one triple bond. Cycloalkenylene and
cycloalkynylene groups may, in certain embodiments, contain 3 to 10
carbon atoms, with cycloalkenylene groups in certain embodiments
containing 4 to 7 carbon atoms and cycloalkynylene groups in
certain embodiments containing 8 to 10 carbon atoms. The ring
systems of the cycloalkylene, cycloalkenylene and cycloalkynylene
groups may be composed of one ring or two or more rings which may
be joined together in a fused, bridged or spiro-connected
fashion.
[0080] "Cycloalk(en)(yn)ylene" refers to a cycloalkylene group
containing at least one double bond and at least one triple
bond.
[0081] As used herein, "arylene" refers to a monocyclic or
polycyclic, in certain embodiments monocyclic, divalent aromatic
group, in one embodiment having from 5 to about 20 carbon atoms and
at least one aromatic ring, in another embodiment 5 to 12 carbons.
In further embodiments, arylene includes lower arylene. Arylene
groups include, but are not limited to, 1,2-, 1,3- and
1,4-phenylene. The term "lower arylene" refers to arylene groups
having 6 carbons.
[0082] As used herein, "heteroarylene" refers to a divalent
monocyclic or multicyclic aromatic ring system, in one embodiment
of about 5 to about 15 atoms in the ring(s), where one or more, in
certain embodiments 1 to 3, of the atoms in the ring system is a
heteroatom, that is, an element other than carbon, including but
not limited to, nitrogen, oxygen or sulfur. The term "lower
heteroarylene" refers to heteroarylene groups having 5 or 6 atoms
in the ring.
[0083] As used herein, "heterocyclylene" refers to a divalent
monocyclic or multicyclic non-aromatic ring system, in certain
embodiments of 3 to 10 members, in one embodiment 4 to 7 members,
in another embodiment 5 to 6 members, where one or more, including
1 to 3, of the atoms in the ring system is a heteroatom, that is,
an element other than carbon, including but not limited to,
nitrogen, oxygen or sulfur.
[0084] As used herein, "substituted alkyl," "substituted alkenyl,"
"substituted alkynyl," "substituted cycloalkyl," "substituted
cycloalkenyl," "substituted cycloalkynyl," "substituted aryl,"
"substituted heteroaryl," "substituted heterocyclyl," "substituted
alkylene," "substituted alkenylene," "substituted alkynylene,"
"substituted cycloalkylene," "substituted cycloalkenylene,"
"substituted cycloalkynylene," "substituted arylene," "substituted
heteroarylene" and "substituted heterocyclylene" refer to alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heterocyclyl, alkylene, alkenylene, alkynylene,
cycloalkylene, cycloalkenylene, cycloalkynylene, arylene,
heteroarylene and heterocyclylene groups, respectively, that are
substituted with one or more substituents, in certain embodiments
one, two, three or four substituents, where the substituents are as
defined herein, in one embodiment selected from Q.sup.1.
[0085] As used herein, "alkylidene" refers to a divalent group,
such as .dbd.CR'R'', which is attached to one atom of another
group, forming a double bond. Alkylidene groups include, but are
not limited to, methylidene (.dbd.CH.sub.2) and ethylidene
(.dbd.CHCH.sub.3). As used herein, "arylalkylidene" refers to an
alkylidene group in which either R' or R'' is an aryl group.
"Cycloalkylidene" groups are those where R' and R'' are linked to
form a carbocyclic ring. "Heterocyclylidene" groups are those where
at least one of R' and R'' contain a heteroatom in the chain, and
R' and R'' are linked to form a heterocyclic ring.
[0086] As used herein, "amido" refers to the divalent group
--C(O)NH--. "Thioamido" refers to the divalent group --C(S)NH--.
"Oxyamido" refers to the divalent group --OC(O)NH--. "Thiaamido"
refers to the divalent group --SC(O)NH--. "Dithiaamido" refers to
the divalent group --SC(S)NH--. "Ureido" refers to the divalent
group --HNC(O)NH--. "Thioureido" refers to the divalent group
--HNC(S)NH--.
[0087] As used herein, "semicarbazide" refers to --NHC(O)NHNH--.
"Carbazate" refers to the divalent group --OC(O)NHNH--.
"Isothiocarbazate" refers to the divalent group --SC(O)NHNH--.
"Thiocarbazate" refers to the divalent group --OC(S)NHNH--.
"Sulfonylhydrazide" refers to the divalent group --SO.sub.2NHNH--.
"Hydrazide" refers to the divalent group --C(O)NHNH--. "Azo" refers
to the divalent group --N.dbd.N--. "Hydrazinyl" refers to the
divalent group --NH--NH--.
[0088] Where the number of any given substituent is not specified
(e.g., haloalkyl), there may be one or more substituents present.
For example, "haloalkyl" may include one or more of the same or
different halogens. As another example, "C.sub.1-3alkoxyphenyl" may
include one or more of the same or different alkoxy groups
containing one, two or three carbons.
[0089] 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
[0090] Provided herein are compounds and pharmaceutical
compositions containing compounds having formula:
##STR00002##
[0091] or a pharmaceutically acceptable derivative thereof, where R
is selected as follows: 1) R is CONR' or 2) R is C.sub.1-C.sub.10
alkylene group, in which: (a) when the number of carbon atoms is at
least 2, there are optionally 1 or 2 double bonds; (b) 1 to 3
non-adjacent methylene groups are optionally replaced by NR', O, or
S; (c) 1 or 2 methylene groups are optionally replaced by a
carbonyl or hydroxymethylene group; and (d) 1 or 2 methylene groups
are optionally replaced by a cycloalkyl or heterocyclyl group that
is optionally substituted with one or more substituents selected
from lower alkyl, NR', O, or S;
[0092] R' is H, alkyl, or acyl;
[0093] A.sub.1 and B.sub.1 are each independently selected from
halogen, pseudohalo, nitro, .sup.+NH.sub.3, SO.sub.3H, carboxy and
haloalkyl;
[0094] t and v are each independently 0 to 3;
[0095] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently
selected as follows: [0096] i) R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are each independently selected from OH,
--NR.sup.5C(.dbd.O)R.sup.6 and --NR.sup.7S(O.sub.2)R.sup.8, wherein
R.sup.5 and R.sup.7 are each independently hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted aryl, substituted
or unsubstituted aralkyl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted heteroaralkyl, substituted or
unsubstituted heterocyclyl or substituted or unsubstituted
heterocyclylalkyl; and R.sup.6 and R.sup.8 are each independently
substituted or unsubstituted alkoxy, substituted or unsubstituted
aralkoxy, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocyclyl, substituted or unsubstituted aryl or
--NR.sup.9R.sup.10 where R.sup.9 and R.sup.10 are each
independently hydrogen, alkyl, aralkyl, aryl, heteroaryl,
heteroaralkyl or heterocyclyl, with the proviso that at least one
of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 is not OH; [0097] ii)
R.sup.1 and R.sup.2 and/or R.sup.3 and R.sup.4 together are
--NH--C(.dbd.O)--NH--, --NH--S(O.sub.2)--NH--,
--CH.sub.2--C(.dbd.O)--NH-- or --CH.sub.2--S(O.sub.2)--NH and
together with the carbon atoms on which they are substituted form a
5 membered heterocyclic ring and the others of R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are each independently selected as in i); or
[0098] iii) at least one of R.sup.1, R.sup.2, R.sup.3 and R.sup.4
is --NH--CR.sup.a.dbd.CR.sup.b--, or
--NH--S(O.sub.2)CR.sup.cR.sup.d-- and together with two adjacent
carbon atoms of the phenyl ring forms a substituted or
unsubstituted heterocyclic or substituted or unsubstituted
heteroaromatic ring, and the others of R.sup.1, R.sup.2, R.sup.3
and R.sup.4 are each independently selected as in i) or ii); where
R.sup.a, R.sup.b, R.sup.c and R.sup.d are each independently
hydrogen or substituted or unsubstituted alkyl, wherein the
substituents when present are selected from one or more
substituents, in one embodiment one to three or four substituents,
each independently selected from Q.sup.1, where Q.sup.1 is halo,
pseudohalo, hydroxy, oxo, thia, nitrile, nitro, formyl, mercapto,
hydroxycarbonyl, hydroxycarbonylalkyl, alkyl, haloalkyl,
polyhaloalkyl, aminoalkyl, diaminoalkyl, alkenyl containing 1 to 2
double bonds, alkynyl containing 1 to 2 triple bonds, cycloalkyl,
cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, heteroaryl,
aralkyl, aralkenyl, aralkynyl, heteroarylalkyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkylidene,
arylalkylidene, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl,
alkoxycarbonyl, alkoxycarbonylalkyl, aryloxycarbonyl,
aryloxycarbonylalkyl, aralkoxycarbonyl, aralkoxycarbonylalkyl,
arylcarbonylalkyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, arylaminocarbonyl, diarylaminocarbonyl,
arylalkylaminocarbonyl, alkoxy, aryloxy, heteroaryloxy,
heteroaralkoxy, heterocyclyloxy, cycloalkoxy, perfluoroalkoxy,
alkenyloxy, alkynyloxy, aralkoxy, alkylcarbonyloxy,
arylcarbonyloxy, aralkylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, aralkoxycarbonyloxy, aminocarbonyloxy,
alkylaminocarbonyloxy, dialkylaminocarbonyloxy,
alkylarylaminocarbonyloxy, diarylaminocarbonyloxy, guanidino,
isothioureido, ureido, N-alkylureido, N-arylureido, N'-alkylureido,
N',N'-dialkylureido, N'-alkyl-N'-arylureido, N',N'-diarylureido,
N'-arylureido, N,N'-dialkylureido, N-alkyl-N'-arylureido,
N-aryl-N'-alkylureido, N,N'-diarylureido, N,N',N'-trialkylureido,
N,N'-dialkyl-N'-arylureido, N-alkyl-N',N'-diarylureido,
N-aryl-N',N'-dialkylureido, N,N'-diaryl-N'-alkylureido,
N,N',N'-triarylureido, amidino, alkylamidino, arylamidino, imino,
hydroxyimino, alkoxyimino, aryloxyimino, aralkoxyimino, alkylazo,
arylazo, aralkylazo, aminothiocarbonyl, alkylaminothiocarbonyl,
arylaminothiocarbonyl, amino, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, arylaminoalkyl, diarylaminoalkyl,
alkylarylaminoalkyl, alkylamino, dialkylamino, haloalkylamino,
arylamino, diarylamino, alkylarylamino, alkylcarbonylamino,
alkoxycarbonylamino, aralkoxycarbonylamino, arylcarbonylamino,
arylcarbonylaminoalkyl, aryloxycarbonylaminoalkyl,
aryloxyarylcarbonylamino, aryloxycarbonylamino, alkylsulfonylamino,
arylsulfonylamino, heteroarylsulfonylamino,
heterocyclylsulfonylamino, heteroarylthio, azido,
--N.sup.+R.sup.51R.sup.52R.sup.53, P(R.sup.50).sub.2,
P(.dbd.O)(R.sup.50).sub.2, OP(.dbd.O)(R.sup.50).sub.2,
--NR.sup.60C(.dbd.O)R.sup.63, dialkylphosphonyl,
alkylarylphosphonyl, diarylphosphonyl, hydroxyphosphonyl,
alkylthio, arylthio, perfluoroalkylthio, hydroxycarbonylalkylthio,
thiocyano, isothiocyano, alkylsulfinyloxy, alkylsulfonyloxy,
arylsulfinyloxy, arylsulfonyloxy, hydroxysulfonyloxy,
alkoxysulfonyloxy, aminosulfonyloxy, alkylaminosulfonyloxy,
dialkylaminosulfonyloxy, arylaminosulfonyloxy,
diarylaminosulfonyloxy, alkylarylaminosulfonyloxy, alkylsulfinyl,
alkylsulfonyl, arylsulfinyl, arylsulfonyl, hydroxysulfonyl,
alkoxysulfonyl, aminosulfonyl, alkylaminosulfonyl,
dialkylaminosulfonyl, arylaminosulfonyl, diarylaminosulfonyl or
alkylarylaminosulfonyl; or two Q.sup.1 groups, which substitute
atoms in a 1, 2 or 1,3 arrangement, together form alkylenedioxy
(i.e., --O--(CH.sub.2).sub.y--O--), thioalkylenoxy (i.e.,
--S--(CH.sub.2).sub.y--O--) or alkylenedithioxy (i.e.,
--S--(CH.sub.2).sub.y--S--) where y is 1 or 2; or two Q.sup.1
groups, which substitute the same atom, together form alkylene;
wherein
[0099] R.sup.50 is hydroxy, alkoxy, aralkoxy, alkyl, heteroaryl,
heterocyclyl, aryl or --NR.sup.70R.sup.71, where R.sup.70 and
R.sup.71 are each independently hydrogen, alkyl, aralkyl, aryl,
heteroaryl, heteroaralkyl or heterocyclyl, or R.sup.70 and R.sup.71
together form alkylene, azaalkylene, oxaalkylene or
thiaalkylene;
[0100] R.sup.51, R.sup.52 and R.sup.53 are each independently
hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl,
heterocyclyl or heterocyclylalkyl;
[0101] R.sup.60 is hydrogen, alkyl, aryl, aralkyl, heteroaryl,
heteroaralkyl, heterocyclyl or heterocyclylalkyl; and
[0102] R.sup.63 is alkoxy, aralkoxy, alkyl, heteroaryl,
heterocyclyl, aryl or --NR.sup.70R.sup.71.
[0103] In certain embodiments, the compounds provided herein have
formula:
##STR00003##
or a pharmaceutically acceptable salt thereof,
[0104] where R is selected from C.sub.1-C.sub.10 alkylene group, in
which, (a) when the number of carbon atoms is at least 2, there are
optionally 1 or 2 double bonds; (b) 1 to 3 non-adjacent methylene
groups are optionally replaced by NR' (where R' is H, alkyl, or
acyl), O, or S; (c) 1 or 2 methylene groups are optionally replaced
by a carbonyl or hydroxymethylene group; and (d) 1 or 2 methylene
groups are optionally replaced by a cycloalkyl or heterocyclyl
group that is optionally substituted with one or more substituents
selected from lower alkyl, NR' (where R' is H, alkyl, or acyl), O,
or S,
[0105] A.sub.1 and B.sub.1 are each independently selected from
halogen, pseudohalo, nitro, .sup.+NH.sub.3, SO.sub.3H, carboxy and
haloalkyl;
[0106] t and v are each independently 0 to 3.
[0107] In certain embodiments, the compounds provided herein have
formula:
##STR00004##
or a pharmaceutically acceptable derivative thereof; where R is
selected from C.sub.1-C.sub.10 alkylene group, in which, (a) when
the number of carbon atoms is at least 2, there are optionally 1 or
2 double bonds; (b) 1 to 3 non-adjacent methylene groups are
optionally replaced by NR' (where R' is H, alkyl, or acyl), O, or
S; (c) 1 or 2 methylene groups are optionally replaced by a
carbonyl or hydroxymethylene group; and (d) 1 or 2 methylene groups
are optionally replaced by a cycloalkyl or heterocyclyl group that
is optionally substituted with one or more substituents selected
from lower alkyl, NR' (where R' is H, alkyl, or acyl), O, or S,
[0108] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently
selected as follows: [0109] i) R.sup.1, R.sup.2, and R.sup.4 are
each independently selected from OH, --NR.sup.5C(.dbd.O)R.sup.6 and
--NR.sup.7S(O.sub.2)R.sup.8, wherein R.sup.5 and R.sup.7 are each
independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
aralkyl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted heteroaralkyl, substituted or unsubstituted
heterocyclyl or substituted or unsubstituted heterocyclylalkyl; and
R.sup.6 and R.sup.8 are each independently substituted or
unsubstituted alkoxy, substituted or unsubstituted aralkoxy,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocyclyl, substituted
or unsubstituted aryl or --NR.sup.9R.sup.10 where R.sup.9 and
R.sup.10 are each independently hydrogen, alkyl, aralkyl, aryl,
heteroaryl, heteroaralkyl or heterocyclyl, with the proviso that at
least one of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 is not OH;
[0110] ii) R.sup.1 and R.sup.2 and/or R.sup.3 and R.sup.4 together
are --NH--C(.dbd.O)--NH--, --NH--S(O.sub.2)--NH--,
--CH.sub.2--C(.dbd.O)--NH-- or --CH.sub.2--S(O.sub.2)--NH and
together with the carbon atoms on which they are substituted form a
5 membered heterocyclic ring and the others of R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are each independently selected as in i); or
[0111] iii) at least one of R.sup.1, R.sup.2, R.sup.3 and R.sup.4
is --NH--CR.sup.a.dbd.CR.sup.b--, or
--NH--S(O.sub.2)CR.sup.cR.sup.d-- and together with two adjacent
carbon atoms of the phenyl ring forms a substituted or
unsubstituted heterocyclic or substituted or unsubstituted
heteroaromatic ring, and the others of R.sup.1, R.sup.2, R.sup.3
and R.sup.4 are each independently selected as in i) or ii); where
R.sup.a, R.sup.b, R.sup.c and R.sup.d are each independently
hydrogen or substituted or unsubstituted alkyl,
[0112] wherein the substituents when present are selected from one
or more substituents, in one embodiment one to three or four
substituents, each independently selected from Q.sup.1, where
Q.sup.1 is halo, pseudohalo, hydroxy, oxo, thia, nitrile, nitro,
formyl, mercapto, hydroxycarbonyl, hydroxycarbonylalkyl, alkyl,
haloalkyl, polyhaloalkyl, aminoalkyl, diaminoalkyl, alkenyl
containing 1 to 2 double bonds, alkynyl containing 1 to 2 triple
bonds, cycloalkyl, cycloalkylalkyl, heterocyclyl,
heterocyclylalkyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl,
heteroarylalkyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl,
triarylsilyl, alkylidene, arylalkylidene, alkylcarbonyl,
arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl,
alkoxycarbonylalkyl, aryloxycarbonyl, aryloxycarbonylalkyl,
aralkoxycarbonyl, aralkoxycarbonylalkyl, arylcarbonylalkyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
arylaminocarbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl,
alkoxy, aryloxy, heteroaryloxy, heteroaralkoxy, heterocyclyloxy,
cycloalkoxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, aralkoxy,
alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, aralkoxycarbonyloxy,
aminocarbonyloxy, alkylaminocarbonyloxy, dialkylaminocarbonyloxy,
alkylarylaminocarbonyloxy, diarylaminocarbonyloxy, guanidino,
isothioureido, ureido, N-alkylureido, N-arylureido, N'-alkylureido,
N',N'-dialkylureido, N'-alkyl-N'-arylureido, N',N'-diarylureido,
N'-arylureido, N,N'-dialkylureido, N-alkyl-N'-arylureido,
N-aryl-N'-alkylureido, N,N'-diarylureido, N,N',N'-trialkylureido,
N,N'-dialkyl-N'-arylureido, N-alkyl-N',N'-diarylureido,
N-aryl-N',N'-dialkylureido, N,N'-diaryl-N'-alkylureido,
N,N',N'-triarylureido, amidino, alkylamidino, arylamidino, imino,
hydroxyimino, alkoxyimino, aryloxyimino, aralkoxyimino, alkylazo,
arylazo, aralkylazo, aminothiocarbonyl, alkylaminothiocarbonyl,
arylaminothiocarbonyl, amino, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, arylaminoalkyl, diarylaminoalkyl,
alkylarylaminoalkyl, alkylamino, dialkylamino, haloalkylamino,
arylamino, diarylamino, alkylarylamino, alkylcarbonylamino,
alkoxycarbonylamino, aralkoxycarbonylamino, arylcarbonylamino,
arylcarbonylaminoalkyl, aryloxycarbonylaminoalkyl,
aryloxyarylcarbonylamino, aryloxycarbonylamino, alkylsulfonylamino,
arylsulfonylamino, heteroarylsulfonylamino,
heterocyclylsulfonylamino, heteroarylthio, azido,
--N+R.sup.51R.sup.52R.sup.53, P(R.sup.50).sub.2,
P(.dbd.O)(R.sup.50).sub.2, OP(.dbd.O)(R.sup.50).sub.2,
--NR.sup.60C(.dbd.O)R.sup.63, dialkylphosphonyl,
alkylarylphosphonyl, diarylphosphonyl, hydroxyphosphonyl,
alkylthio, arylthio, perfluoroalkylthio, hydroxycarbonylalkylthio,
thiocyano, isothiocyano, alkylsulfinyloxy, alkylsulfonyloxy,
arylsulfinyloxy, arylsulfonyloxy, hydroxysulfonyloxy,
alkoxysulfonyloxy, aminosulfonyloxy, alkylaminosulfonyloxy,
dialkylaminosulfonyloxy, arylaminosulfonyloxy,
diarylaminosulfonyloxy, alkylarylaminosulfonyloxy, alkylsulfinyl,
alkylsulfonyl, arylsulfinyl, arylsulfonyl, hydroxysulfonyl,
alkoxysulfonyl, aminosulfonyl, alkylaminosulfonyl,
dialkylaminosulfonyl, arylaminosulfonyl, diarylaminosulfonyl or
alkylarylaminosulfonyl; or two Q.sup.1 groups, which substitute
atoms in a 1, 2 or 1,3 arrangement, together form alkylenedioxy
(i.e., --O--(CH.sub.2).sub.y--O--), thioalkylenoxy (i.e.,
--S--(CH.sub.2).sub.y--O--) or alkylenedithioxy (i.e.,
--S--(CH.sub.2).sub.y--S--) where y is 1 or 2; or two Q.sup.1
groups, which substitute the same atom, together form alkylene;
wherein
[0113] R.sup.50 is hydroxy, alkoxy, aralkoxy, alkyl, heteroaryl,
heterocyclyl, aryl or --NR.sup.70R.sup.71, where R.sup.70 and
R.sup.71 are each independently hydrogen, alkyl, aralkyl, aryl,
heteroaryl, heteroaralkyl or heterocyclyl, or R.sup.70 and R.sup.71
together form alkylene, azaalkylene, oxaalkylene or
thiaalkylene;
[0114] R.sup.51, R.sup.52 and R.sup.53 are each independently
hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl,
heterocyclyl or heterocyclylalkyl;
[0115] R.sup.60 is hydrogen, alkyl, aryl, aralkyl, heteroaryl,
heteroaralkyl, heterocyclyl or heterocyclylalkyl; and
[0116] R.sup.63 is alkoxy, aralkoxy, alkyl, heteroaryl,
heterocyclyl, aryl or --NR.sup.70R.sup.71.
[0117] In certain embodiments, Q.sup.1 is oxo, alkyl, haloalkyl,
polyhaloalkyl, aminoalkyl, diaminoalkyl, alkenyl containing 1 to 2
double bonds, alkynyl containing 1 to 2 triple bonds, cycloalkyl,
cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, heteroaryl,
aralkyl, aralkenyl, aralkynyl or heteroarylalkyl.
[0118] In certain embodiments, Q.sup.1 is oxo or alkyl. In certain
embodiments, Q.sup.1 is oxo. In certain embodiments, Q.sup.1 is
alkyl. In certain embodiments, Q.sup.1 is lower alkyl. In certain
embodiments, Q.sup.1 is methyl.
[0119] In certain embodiments, R' is H or alkyl. In other
embodiments, R' is H.
[0120] In certain embodiments, t is 0, 1 or 2. In certain
embodiments, t is 0 or 1. In certain embodiments, t is 1. In
certain embodiments, v is 0, 1 or 2. In certain embodiments, v is 0
or 1. In certain embodiments, v is 1.
[0121] In one embodiment, R is
--(CH.sub.2).sub.mC(O)(CH.sub.2).sub.sNH(CH.sub.2).sub.r,
--(CH.sub.2)-- or --(CH.sub.2).sub.sY(CH.sub.2).sub.r--, in which Y
is a cycloalkyl or heterocyclyl group that is optionally
substituted with one or more substituents selected from alkyl, NR',
O, or S; p is 1 to 10; and m, s and r are each independently 0 to
6.
[0122] In one embodiment, R is --C(O)NH, CH.sub.2CH.sub.2--, or
--(CH.sub.2)Y(CH.sub.2)--. In one embodiment, R is --C(O)NH--. In
one embodiment, R is --CH.sub.2CH.sub.2--. In one embodiment, R is
--(CH.sub.2)Y(CH.sub.2#.
[0123] In one embodiment, Y is heterocyclyl, optionally substituted
with one or more substituents selected from alkyl and oxo. In one
embodiment, Y is bridged heterocyclyl, optionally substituted with
one or more substituents selected from alkyl and oxo. In one
embodiment, Y is bicycloheterocyclyl substituted with methyl and
oxo. In one embodiment, Y is bicycloheterocyclyl where the
heteroatom is N. In another embodiment, Y is
##STR00005##
[0124] where Q.sup.2 is alkyl.
[0125] In certain embodiments, Y is
##STR00006##
In certain embodiments, R is substituted with alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,
heterocyclylalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl.
[0126] In certain embodiments, R is substituted with alkyl. In
other embodiments, R substituted with lower alkyl. In certain
embodiments, R is substituted with methyl.
[0127] In another embodiment, the compounds for use in the
compositions and methods provided herein have formula:
##STR00007##
[0128] In another embodiment, the compounds for use in the
compositions and methods provided herein have formula
##STR00008##
[0129] In certain embodiments, R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are each independently selected from [0130] i) OH,
formylamide, alkylamide, alkylarylamide, aralkylamide, arylamide,
N-alkyl-N-alkylsulfonamide, alkylsulfonamide, alkylarylsulfonamide,
arylsulfonamide or aralkylsulfonamide, with the proviso that at
least one of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 is not OH;
[0131] ii) R.sup.1 and R.sup.2 and/or R.sup.3 and R.sup.4 together
are --NH--C(.dbd.O)--NH--, --NH--S(O.sub.2)--NH--,
--CH.sub.2--C(.dbd.O)--NH-- or --CH.sub.2--S(O.sub.2)--NH and
together with the carbon atoms on which they are substituted form a
5 membered heterocyclic ring and the others of R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are each independently selected as in i); or
[0132] iii) at least one of R.sup.1, R.sup.2, R.sup.3 and R.sup.4
is --NH--CR.sup.a.dbd.CR.sup.b--, or
--NH--S(O.sub.2)CR.sup.cR.sup.d-- and together with two adjacent
carbon atoms of the phenyl ring forms a substituted or
unsubstituted heterocyclic or substituted or unsubstituted
heteroaromatic ring, and the others of R.sup.1, R.sup.2, R.sup.3
and R.sup.4 are each independently selected as in i) or ii); where
R.sup.a, R.sup.b, R.sup.c and R.sup.d are each independently
hydrogen or alkyl.
[0133] In one embodiment, R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are
each independently OH, formylamide, alkylamide, alkylarylamide,
aralkylamide, arylamide, alkylsulfonamide,
N-alkyl-N-alkylsulfonamide, alkylarylsulfonamide, arylsulfonamide
or aralkylsulfonamide, with the proviso that at least one of
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 is not OH.
[0134] In another embodiment, R.sup.1, R.sup.2, R.sup.3 and R.sup.4
are each independently OH, formylamide or alkylsulfonamide, with
the proviso that at least one of R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 is not OH.
[0135] In another embodiment, R.sup.1, R.sup.2, R.sup.3 and R.sup.4
are each independently OH, formylamide or methylsulfonamide, with
the proviso that at least one of R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 is not OH.
[0136] In another embodiment, R.sup.1 and R.sup.2 and/or R.sup.3
and R.sup.4 together are --NH--C(.dbd.O)--NH-- and the others of
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently OH,
formylamide, alkylsulfonamide.
[0137] In another embodiment, R.sup.1 and R.sup.2 together are
--NH--C(.dbd.O)--NH-- and R.sup.3 and R.sup.4 are each
independently OH, formylamide or methylsulfonamide.
[0138] In another embodiment, R.sup.3 and R.sup.4 together are
--NH--C(.dbd.O)--NH-- and R.sup.1 and R.sup.2 are each
independently OH, formylamide or methylsulfonamide.
[0139] In another embodiment, at least one of R.sup.1, R.sup.2,
R.sup.3 or R.sup.4 is --NH--CR.sup.a.dbd.CR.sup.b--, or
--NH--S(O.sub.2)CR.sup.cR.sup.d-- and together with two adjacent
carbon atoms of the phenyl ring forms a substituted or
unsubstituted heterocyclic or substituted or unsubstituted
heteroaromatic ring, and the others of R.sup.1, R.sup.2, R.sup.3
and R.sup.4 are each independently selected as in i) or ii); where
R.sup.a, R.sup.b, R.sup.c and R.sup.d are each independently
hydrogen or alkyl, and the others of R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are each independently selected from OH, formylamide,
alkylamide, alkylarylamide, aralkylamide, arylamide,
alkylsulfonamide, alkylarylsulfonamide, arylsulfonamide and
aralkylsulfonamide.
[0140] In another embodiment, at least one of R.sup.1, R.sup.2,
R.sup.3 or R.sup.4 is --NH--CH.dbd.CH--, and together with two
adjacent carbon atoms of the phenyl ring forms an indole ring, and
the others of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each
independently selected from OH, formylamide, alkylamide,
alkylarylamide, aralkylamide, arylamide, alkylsulfonamide,
alkylarylsulfonamide, arylsulfonamide and aralkylsulfonamide.
[0141] In another embodiment, at least one of R.sup.1, R.sup.2,
R.sup.3 or R.sup.4 is --NH--CH.dbd.CH-- and together with two
adjacent carbon atoms of the phenyl ring forms an indole ring, and
the others of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each
independently selected from OH, formylamide, and
methylsulfonamide.
[0142] In another embodiment, at least one of R.sup.1, R.sup.2,
R.sup.3 or R.sup.4 is --NH--S(O.sub.2)CH.sub.2-- and together with
two adjacent carbon atoms of the phenyl ring forms a
benzisothiazole-1,1-dioxide and the others of R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are each independently selected from OH,
formylamide, and methylsulfonamide.
[0143] In certain embodiments, the compounds have formula selected
from:
##STR00009##
wherein i) when M is C(O), R.sup.x is hydrogen or alkyl and ii)
when M is S(O).sub.2, R.sup.x is alkyl. In one embodiment, M is
C(O) and R.sup.x is alkyl.
[0144] In one embodiment, M is C(O) and R.sup.5 is isopropyl. In
one embodiment, M is S(O).sub.2 and R.sup.x is methyl.
[0145] In certain embodiments, the compounds have formula selected
from:
##STR00010##
[0146] In certain embodiments, the compounds have formula selected
from:
##STR00011##
[0147] In other embodiments, the compounds are selected from
formula:
##STR00012##
[0148] In certain embodiments, the compound has formula:
##STR00013##
wherein A.sub.2, A.sub.3, B.sub.2 and B.sub.3 are each
independently selected from halogen, cyanide, cyanate, thiocyanate,
selenocyanate, trifluoromethoxy, azide, nitro and trifluoromethyl;
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are selected as follows:
[0149] i) R.sup.1 and R.sup.2 are OH and R.sup.3 and R.sup.4 are
each independently selected as described elsewhere herein or
[0150] ii) R.sup.3 and R.sup.4 are OH and R.sup.1 and R.sup.2 are
each independently selected as described elsewhere herein and the
other variables are as described herein.
[0151] In certain embodiments, the compound has formula:
##STR00014##
wherein A.sub.2, A.sub.3, B.sub.2, and B.sub.3 are each
independently selected from halogen, pseudohalo, nitro,
.sup.+NH.sub.3, SO.sub.3H, carboxy and haloalkyl; and the other
variables are as described elsewhere herein.
[0152] In certain embodiments, the compound has formula:
##STR00015##
wherein the variables are as described elsewhere herein.
[0153] In certain embodiments, the compound has formula:
##STR00016##
[0154] wherein the variables are as described elsewhere herein.
[0155] In other embodiments, the compound has the formula:
##STR00017##
[0156] In one embodiment, the compound provided herein is selected
from
2-Oxo-N-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-2,3-dihydro-1H-benzo-
[d]imidazole-5-carboxide;
N-(3,4-dihydroxyphenyl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5-carboxam-
ide and
3,4-dihydroxy-N-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)benzam-
ide.
[0157] In one embodiment, the compound is selected from a group of
3,4,3',4'-tetrahydroxybenzoin; 3,4,3',4'-tetrahydroxydesoxybenzoin;
3,4,3',4'-tetrahydroxydiphenylmethane;
1,2-bis(3,4-dihydroxyphenyl)ethane;
1,3-bis(3,4-dihydroxyphenyl)propane;
3,4,3',4'-tetrahydroxychalcone;
3,5-bis(3,4-dihydroxyphenyl)-1-methyl-2-pyrazoline;
4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine;
1,4-bis(3,4-dihydroxybenzyl)piperazine;
N,N'-bis(3,4-dihydroxybenzyl)-N,N'-dimethyl-ethylenediamine;
2,5-bis(3,4-dihydroxybenzyl)-2,5-diaza[2.2.1]bicycloheptane;
N,N'-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane;
N,N'-bis(3,4-dihydroxybenzyl)-trans-1,4-diaminocyclohexane;
N,N'-bis(3,4-dihydroxybenzyl)-cis-1,3-bis(aminomethyl)cyclohexane;
N-(3,4-dihydroxybenzyl)proline 3,4-dihydroxybenzylamide;
2-(3,4-dihydroxybenzyl)isoquinoline-3-carboxylic acid
3,4-dihydroxyphenethylamide;
2,6-bis(3,4-dihydroxybenzyl)cyclohexanone;
3,5-bis(3,4-dihydroxybenzyl)-1-methyl-4-piperidinone;
2,4-bis(3,4-dihydroxybenzyl)-3-tropinone;
tris(3,4-dihydroxybenzyl)methane;
.alpha.-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic acid
3,4-dihydroxybenzyl amide;
4-(3,4-dihydroxybenzylaminomethylene)-2-(3,4-dihydroxyphenyl)oxazolin-5-o-
ne; 1,4-bis(3,4-dihydroxybenzoyl)piperazine;
N,N'-bis(3,4-dihydroxybenzoyl)-N,N'-dimethylethylenediamine;
2,5-bis(3,4-dihydroxybenzoyl)-2,5-diaza[2.2.1]bicycloheptane;
N,N'-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane;
N,N'-bis(3,4-dihydroxybenzoyl)-cis-1,3-bis(aminomethyl)cyclohexane;
3,6-bis(3,4-dihydroxybenzyl)-2,5-diketopiperazine;
3,6-bis(3,4-dihydroxybenzylidene)-1,4-dimethyl-2,5-diketopiperazine;
N-(3,4-dihydroxyphenylacetyl)proline-3,4-dihydroxyanilide;
2,3-bis(3,4-dihydroxyphenyl)butane;
1,3-bis(3,4-dihydroxybenzyl)benzene;
1,4-bis(3,4-dihydroxybenzyl)benzene;
2,6-bis(3,4-dihydroxybenzyl)pyridine;
2,5-bis(3,4-dihydroxybenzyl)thiophene;
2,3-bis(3,4-dihydroxybenzyl)thiophene;
1,2-bis(3,4-dihydroxyphenyl)cyclohexane;
1,4-bis(3,4-dihydroxyphenyl)cyclohexane;
3,7-bis(3,4-dihydroxyphenyl)bicyclo[3.3.0]octane;
2,3-bis(3,4-dihydroxyphenyl)-1,7,7-trimethyl-bicyclo[2.2.1]heptane;
1,2-bis(3,4-dihydroxyphenoxy)ethane;
1,3-bis(3,4-dihydroxyphenoxy)propane;
trans-1,2-bis(3,4-dihydroxyphenoxy)cyclopentane;
N-(3,4-dihydroxybenzyl)-3-(3,4-dihydroxyphenoxy)-2-hydroxypropylamine;
3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyanilide;
3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxybenzylamide;
3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyphenethylamide;
3,4-dihydroxybenzoic acid p-(3,4-dihydroxyphenoxy)anilide;
3,4-dihydroxybenzoic acid o-(3,4-dihydroxyphenoxy)anilide;
2,6-bis(3,4-dihydroxyphenoxy)pyridine; 3,4-dihydroxybenzoic acid
3,4-dihydroxyanilide; 3,4-dihydroxybenzoic acid
3,4-dihydroxybenzylamide; 3,4-dihydroxybenzoic acid
3,4-dihydroxyphenethylamide; 3,4-dihydroxyphenyl acetic acid
3,4-dihydroxyanilide; 3,4-dihydroxyphenylacetic acid
3,4-dihydroxybenzylamide; 3,4-dihydroxyphenylacetic acid
3,4-dihydroxyphenethylamide; 3-(3,4-dihydroxyphenyl)propionic acid
3,4-dihydroxyanilide; 3-(3,4-dihydroxyphenyl) propionic acid
3,4-dihydroxybenzylamide; 3-(3,4-dihydroxyphenyl)propionic acid
3,4-dihydroxyphenethylamide; 3,4-dihydroxycinnamic acid
3,4-dihydroxyanilide; 3,4-dihydroxycinnamic acid
3,4-dihydroxybenzylamide; 3,4-dihydroxycinnamic acid
3,4-dihydroxyphenethylamide; oxalic acid bis(3,4-dihydroxyanilide);
oxalic acid bis(3,4-dihydroxybenzylamide); oxalic acid
bis(3,4-dihydroxyphenethylamide); succinic acid
bis(3,4-dihydroxyanilide); succinic acid
bis(3,4-dihydroxybenzylamide); succinic acid
bis(3,4-dihydroxyphenethylamide); maleic acid
bis(3,4-dihydroxyanilide); maleic acid
bis(3,4-dihydroxybenzylamide); fumaric acid
bis(3,4-dihydroxyanilide); fumaric acid
bis(3,4-dihydroxybenzylamide); bis(3,4-dihydroxybenzyl)amine;
N-(3,4-dihydroxybenzyl)-3,4-dihydroxyphenethylamine;
tris(3,4-dihydroxybenzyl)amine; 1,3-bis(3,4-dihydroxyphenyl)urea;
1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxybenzyl)urea;
1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea;
3-deoxy-3-(3,4-dihydroxybenzyl)aminoepicatechin;
3-deoxy-3-(3,4-dihydroxyphenethyl)-aminoepicatechin;
2,3,6,7-tetrahydroxy-9,10-epoxy-9,10-dihydroacridine;
10-aminoanthracene-1,2,7,8-tetraol; acridine-1,2,6,7-tetraol;
phenoxazine-2,3,7,8,10-pentaol;
dibenzo[c,f][2,7]napthyridine-2,3,10,11-tetraol; and
6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-2,10,11-triol,
wherein at least one of the phenolic hydroxy groups of the compound
is replaced by:
[0158] i) --NR.sup.5C(.dbd.O)R.sup.6, --NR.sup.7S(O.sub.2)R.sup.8,
wherein R.sup.5 and R.sup.7 are each independently hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
aryl, substituted or unsubstituted aralkyl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroaralkyl, substituted or unsubstituted heterocyclyl or
substituted or unsubstituted heterocyclylalkyl; and R.sup.6 and
R.sup.8 are each independently substituted or unsubstituted alkoxy,
substituted or unsubstituted aralkoxy, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocyclyl, substituted or unsubstituted aryl or
--NR.sup.9R.sup.10 where R.sup.9 and R.sup.10 are each
independently hydrogen, alkyl, aralkyl, aryl, heteroaryl,
heteroaralkyl or heterocyclyl;
[0159] ii) R.sup.1 and R.sup.2 and/or R.sup.3 and R.sup.4 together
form a benzimidazolinone, benzothiadiazolidine-S,S-dioxide or
benzoxazolinone; or
[0160] iii) at least one of R.sup.1, R.sup.2, R.sup.3 and R.sup.4
together with an adjacent carbon atom forms a substituted or
unsubstituted heterocyclic or substituted or unsubstituted
heteroaromatic ring; wherein the substituents when present are
selected from one or more substituents, in one embodiment one to
three or four substituents, each independently selected from
Q.sup.1, where Q.sup.1 is halo, pseudohalo, hydroxy, oxo, thia,
nitrile, nitro, formyl, mercapto, hydroxycarbonyl,
hydroxycarbonylalkyl, alkyl, haloalkyl, polyhaloalkyl, aminoalkyl,
diaminoalkyl, alkenyl containing 1 to 2 double bonds, alkynyl
containing 1 to 2 triple bonds, cycloalkyl, cycloalkylalkyl,
heterocyclyl, heterocyclylalkyl, aryl, heteroaryl, aralkyl,
aralkenyl, aralkynyl, heteroarylalkyl, trialkylsilyl,
dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkylidene,
arylalkylidene, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl,
alkoxycarbonyl, alkoxycarbonylalkyl, aryloxycarbonyl,
aryloxycarbonylalkyl, aralkoxycarbonyl, aralkoxycarbonylalkyl,
arylcarbonylalkyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, arylaminocarbonyl, diarylaminocarbonyl,
arylalkylaminocarbonyl, alkoxy, aryloxy, heteroaryloxy,
heteroaralkoxy, heterocyclyloxy, cycloalkoxy, perfluoroalkoxy,
alkenyloxy, alkynyloxy, aralkoxy, alkylcarbonyloxy,
arylcarbonyloxy, aralkylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, aralkoxycarbonyloxy, aminocarbonyloxy,
alkylaminocarbonyloxy, dialkylaminocarbonyloxy,
alkylarylaminocarbonyloxy, diarylaminocarbonyloxy, guanidino,
isothioureido, ureido, N-alkylureido, N-arylureido, N'-alkylureido,
N',N'-dialkylureido, N'-alkyl-N'-arylureido, N',N'-diarylureido,
N'-arylureido, N,N'-dialkylureido, N-alkyl-N'-arylureido,
N-aryl-N'-alkylureido, N,N'-diarylureido, N,N',N'-trialkylureido,
N,N'-dialkyl-N'-arylureido, N-alkyl-N',N'-diarylureido,
N-aryl-N',N'-dialkylureido, N,N'-diaryl-N'-alkylureido,
N,N',N'-triarylureido, amidino, alkylamidino, arylamidino, imino,
hydroxyimino, alkoxyimino, aryloxyimino, aralkoxyimino, alkylazo,
arylazo, aralkylazo, aminothiocarbonyl, alkylaminothiocarbonyl,
arylaminothiocarbonyl, amino, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, arylaminoalkyl, diarylaminoalkyl,
alkylarylaminoalkyl, alkylamino, dialkylamino, haloalkylamino,
arylamino, diarylamino, alkylarylamino, alkylcarbonylamino,
alkoxycarbonylamino, aralkoxycarbonylamino, arylcarbonylamino,
arylcarbonylaminoalkyl, aryloxycarbonylaminoalkyl,
aryloxyarylcarbonylamino, aryloxycarbonylamino, alkylsulfonylamino,
arylsulfonylamino, heteroarylsulfonylamino,
heterocyclylsulfonylamino, heteroarylthio, azido,
--N.sup.+R.sup.51R.sup.52R.sup.53, P(R.sup.50).sub.2,
P(.dbd.O)(R.sup.50).sub.2, OP(.dbd.O)(R.sup.5).sub.2,
--NR.sup.60C(.dbd.O)R.sup.63, dialkylphosphonyl,
alkylarylphosphonyl, diarylphosphonyl, hydroxyphosphonyl,
alkylthio, arylthio, perfluoroalkylthio, hydroxycarbonylalkylthio,
thiocyano, isothiocyano, alkylsulfinyloxy, alkylsulfonyloxy,
arylsulfinyloxy, arylsulfonyloxy, hydroxysulfonyloxy,
alkoxysulfonyloxy, aminosulfonyloxy, alkylaminosulfonyloxy,
dialkylaminosulfonyloxy, arylaminosulfonyloxy,
diarylaminosulfonyloxy, alkylarylaminosulfonyloxy, alkylsulfinyl,
alkylsulfonyl, arylsulfinyl, arylsulfonyl, hydroxysulfonyl,
alkoxysulfonyl, aminosulfonyl, alkylaminosulfonyl,
dialkylaminosulfonyl, arylaminosulfonyl, diarylaminosulfonyl or
alkylarylaminosulfonyl; or two Q.sup.1 groups, which substitute
atoms in a 1, 2 or 1,3 arrangement, together form alkylenedioxy
(i.e., --O--(CH.sub.2).sub.y--O--), thioalkylenoxy (i.e.,
--S--(CH.sub.2).sub.y--O--) or alkylenedithioxy (i.e.,
--S--(CH.sub.2).sub.y--S--) where y is 1 or 2; or two Q.sup.1
groups, which substitute the same atom, together form alkylene;
wherein
[0161] R.sup.50 is hydroxy, alkoxy, aralkoxy, alkyl, heteroaryl,
heterocyclyl, aryl or --NR.sup.70R.sup.71, where R.sup.70 and
R.sup.71 are each independently hydrogen, alkyl, aralkyl, aryl,
heteroaryl, heteroaralkyl or heterocyclyl, or R.sup.70 and R.sup.71
together form alkylene, azaalkylene, oxaalkylene or
thiaalkylene;
[0162] R.sup.51, R.sup.52 and R.sup.53 are each independently
hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl,
heterocyclyl or heterocyclylalkyl;
[0163] R.sup.60 is hydrogen, alkyl, aryl, aralkyl, heteroaryl,
heteroaralkyl, heterocyclyl or heterocyclylalkyl; and
[0164] R.sup.63 is alkoxy, aralkoxy, alkyl, heteroaryl,
heterocyclyl, aryl or --NR.sup.70R.sup.71.
[0165] In one embodiment, the compound provided herein is selected
from; N-(3,4-dihydroxyphenyl)-2-fluoro-3,4-dihydroxybenzamide,
N-(3,4-dihydroxyphenyl)-2-fluoro-4,5-dihydroxybenzamide,
N-(3,4-dihydroxyphenyl)-3-fluoro-4,5-dihydroxybenzamide,
N-(3-fluoro-4,5-dihydroxyphenyl)-3,4-dihydroxybenzamide,
N-(2-fluoro-4,5-dihydroxyphenyl)-3,4-dihydroxybenzamide, and
N-(2-fluoro-3,4-dihydroxyphenyl)-3,4-dihydroxybenzamide.
[0166] In certain embodiments, the compound is selected from
##STR00018## ##STR00019##
[0167] In other embodiments, the compound is selected from
##STR00020##
[0168] In other embodiments, the compound is selected from
##STR00021##
[0169] In certain embodiments, the compound is selected from
##STR00022##
[0170] In certain embodiments, the compound is selected from
##STR00023##
[0171] In certain embodiments, the compound is
##STR00024##
[0172] In other embodiments, the compound is selected from
##STR00025##
[0173] In other embodiments, the compound is selected from
##STR00026##
[0174] In certain embodiments, the compound is selected from
##STR00027##
[0175] In certain embodiments, the compounds provided herein have
formula:
##STR00028##
[0176] wherein A.sub.1 and B.sub.1 are each independently selected
from halogen, pseudohalo, nitro, .sup.+NH.sub.3, SO.sub.3H, carboxy
and haloalkyl; and t.sub.1 and v.sub.1 are each independently 1 to
3; and the other variables are as described elsewhere herein.
[0177] In certain embodiments, t.sub.1 and v.sub.1 are each
independently 1 or 2. In certain embodiments, t.sub.1 is 1. In
certain embodiments, v.sub.1 is 1.
[0178] In certain embodiments, the compound is selected from
##STR00029##
[0179] wherein variables are as described elsewhere herein.
In certain embodiments, the compound is selected from
##STR00030## ##STR00031##
[0180] wherein A.sub.2, A.sub.3, B.sub.2 and B.sub.3 are each
independently selected from Cl, F, cyanide, cyanate, thiocyanate,
selenocyanate, trifluoromethoxy, azide, nitro and
trifluoromethyl.
C. Preparation of the Compounds
[0181] 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.
[0182] 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.
[0183] 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.
[0184] Provided herein are general reaction schemes for the
preparation of exemplary compounds.
[0185] i) Achesom et al. J. Med. Chem. (1981) 24, 1300-1304,
describe use of thionyl chloride for preparing benxthiadiazolidine
S,S-dioxide as follows:
##STR00032##
[0186] ii) Preparation of nitro benzothiadiazolidine S,S-dioxide is
described by Burke et al. in JCS Perkin Transactions (1984) 11,
1851-4, as follows:
##STR00033##
[0187] Further compounds provided herein can be prepared by
reactions described in the literature as follows:
##STR00034##
[0188] See, Roberts et al., J. O. Chen. (1997) 62, 568-577
##STR00035##
[0189] See, Hughes et al., J. Med. Chem. (1957) 18, 1077-1088.
D. Pharmaceutical Compositions and Administration
[0190] 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.
[0191] In certain embodiments, the compositions contain a compound
provided herein that is at least substantially pure. In general
"pure" means better than 95% pure, and "substantially pure" means a
compound synthesized such that the compound, as made as available
for consideration into a therapeutic dosage, has only those
impurities that can not readily nor reasonably be removed by
conventional purification processes.
[0192] The mode of administration of the pharmaceutical
compositions can be oral, rectal, intravenous, intramuscular,
intracistemal, 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.
[0193] 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
5 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.
[0194] 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.
[0195] 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.
[0196] 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.
[0197] Injectable depot forms are made by forming microencapsule
matrices of the drug in biodegradable polymers such as
polylactide-polyglycolide. Depending upon the ratio of drug to
polymer and the nature of the particular polymer 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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, a preservative and flavoring and coloring agents.
[0204] 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 an amyloid disease, for example an
amyloidosis such as Alzheimer's disease or 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.
[0205] Compounds provided herein can also be administered in the
form of liposomes. Methods to form liposomes are known in the art
(Prescott, Ed., Methods in Cell Biology 1976, Volume XIV, Academic
Press, New York, N.Y.) As is known in the art, liposomes are
generally derived from phospholipids or other lipid substances.
Liposomes are formed by mono- or multi-lamellar hydrated liquid
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).
[0206] The compounds provided herein can also be administered in
the form of a `prodrug` wherein the active pharmaceutical
ingredients, represented by Formulas 1-3, are released in vivo upon
contact with hydrolytic enzymes such as esterases and phophatases
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.
[0207] 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.
[0208] 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 (MLV's) may be formed by drying down egg phosphatidyl
choline and brain phosphatidyl serine (7:3 molar ratio) on the
inside of a flask. A solution of a 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.
[0209] Article of Manufacture
[0210] 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 amyloidosis
and 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
amyloidosis and synuclein diseases.
[0211] 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 amyloidosis and synuclein diseases.
[0212] Sustained Release Formulations
[0213] Also provided are sustained release formulations to deliver
the compounds to the desired target (i.e. brain or systemic organs)
at high circulating levels (between 10.sup.-9 and 10.sup.-4 M). In
a certain embodiment for the treatment of Alzheimer's or
Parkinson's disease, the circulating levels of the compounds is
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 a another embodiments, localized to the amyloid or
.alpha.-synuclein fibril deposits in brain or other tissues.
[0214] 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.
[0215] 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.
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] 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.
[0221] 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.
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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.
E. Evaluation of the Activity of the Compounds
[0226] The biological activity of the compounds provided herein as
disruptors/inhibitors of Alzheimer's disease .beta.-amyloid protein
(A.beta.) fibrils, type 2 diabetes IAPP fibrils and Parkinson's
disease NAC fibrils was assessed by determining the efficacy of the
compounds to cause a disassembly/disruption of pre-formed amyloid
fibrils of Alzheimer's disease (i.e. consisting of A.beta. 1-42
fibrils), IAPP fibrils and Parkinson's disease NAC fibrils. In one
study, Thioflavin T fluorometry was used to determine the effects
of the compounds, and of EDTA (as a negative control). In this
assay Thioflavin T binds specifically to fibrillar amyloid, and
this binding produces a fluorescence enhancement at 485 nm that is
directly proportional to the amount of 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).
The disruption of A.beta. 1-42, even in its monomeric form, was
confirmed by a study involving the use of SDS-PAGE and Western
blotting methods.
[0227] In the Congo red binding assay the ability of a given test
compound to alter amyloid (A.beta. 1-42 fibrils, IAPP fibrils or
NAC fibrils) binding to Congo red was quantified. In this assay,
A.beta. 1-42 fibrils, IAPP fibril or NAC fibrils and test compounds
were incubated for 3 days and then vacuum filtered through a 0.2
.mu.m filter. The amount of A.beta. 1-42 fibrils, IAPP fibrils or
NAC 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 amyloid 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 A.beta.
1-42 fibrils, IAPP fibrils or NAC fibrils.
F. Combination Therapy
[0228] 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 amyloidosis
and synuclein diseases. Such therapeutic agents include, but are
not limited to, donepezil hydrochloride (Aracept), rivastigmine
tartrate (Exelon), tacrine hydrochloride (Cognex) and galantamine
hydrobromide (Reminyl).
G. Methods of Use of the Compounds and Compositions
[0229] The compounds and compositions provided herein are useful in
methods of treatment, prevention, or amelioration of one or more
symptoms of amyloid diseases or disorders, including but not
limited to diseases associated with the formation, deposition,
accumulation, or persistence of amyloid fibrils diseases associated
with the formation, deposition, accumulation, or persistence of
amyloid fibrils. In one embodiment, the fibrils of an amyloid
protein are selected from the group of A.beta. amyloid, AA amyloid,
AL amyloid, IAPP amyloid, PrP amyloid, .alpha..sub.2-microglobulin
amyloid, transthyretin, prealbumin, and procalcitonin. In certain
embodiments, the fibrils of an amyloid protein are A.beta. amyloid
and IAPP amyloid. In certain embodiments, the compounds and
compositions provided herein are used for treatment, prevention, or
amelioration of one or more symptoms of diseases including, but not
limited to Alzheimer's disease, Down's syndrome, dementia
pugilistica, multiple system atrophy, inclusion body myositosis,
hereditary cerebral hemorrhage with amyloidosis of the Dutch type,
Nieman-Pick disease type C, cerebral .beta.-amyloid angiopathy,
dementia associated with cortical basal degeneration, the
amyloidosis of type 2 diabetes, the amyloidosis of chronic
inflammation, the amyloidosis of malignancy and Familial
Mediterranean Fever, the amyloidosis of multiple myeloma and B-cell
dyscrasias, the amyloidosis of the prion diseases,
Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru,
scrapie, the amyloidosis associated with carpal tunnel syndrome,
senile cardiac amyloidosis, familial amyloidotic polyneuropathy,
and the amyloidosis associated with endocrine tumors. In certain
embodiments, the diseases are Alzheimer's disease or type 2
diabetes.
[0230] Also provided are methods to inhibit or prevent
.alpha.-synuclein/NAC fibril formation, 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.
[0231] 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. In certain
embodiments, such diseases include Parkinson's disease, 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.
H. Methods of Use of the Compounds and Compositions for
Diagnosis
[0232] Methods for detecting the presence or absence of amyloid or
.alpha.-synuclein aggregates in a biological sample are provided.
These methods include contacting a biological sample with a
selected compound, wherein the compound is labeled with a
detectable substance, for example, with a radionucleotide,
phosphorescent compound, fluorescent compound, fluorescent protein,
paramagnetic compound, metal chelators, or enzyme, all of which are
readily detectable in various assays and diagnostics know to those
skilled in the art, and then detecting the detectable substance
bound to the amyloid or .alpha.-synuclein aggregates in the
biological sample.
[0233] Methods for imaging the presence or absence of amyloid or
.alpha.-synuclein aggregates in the body or biological tissues are
provided. These methods include contacting amyloid or
.alpha.-synuclein aggregates in the body with a compound, wherein
the compound is labeled with detectable substance, for example,
with a radionucleotide, phosphorescent compound, fluorescent
compound, fluorescent protein, paramagnetic compound, metal
chelator, or enzyme, and detecting the detectable substance bound
to the amyloid or .alpha.-synuclein aggregates in the body or
biological tissues.
Diagnostic Applications
[0234] Accordingly, the compounds can also be used as diagnostic
agents to detect the presence or absence of amyloid or
.alpha.-synuclein aggregates in a biological sample or in vivo in a
subject. Furthermore, detection of amyloid or .alpha.-synuclein
aggregates using the compounds can be used to diagnose amyloidosis
or synucleopathies in a subject.
[0235] Compounds which interact with A.beta. amyloid or
.alpha.-synuclein or derivatives thereof are also disclosed herein.
The compounds can be used for a number of important diagnostic
and/or therapeutic applications as described herein.
[0236] In another embodiment, a compound is used in vivo to detect,
and if desired, quantitate, A.beta. amyloid or .alpha.-synuclein
deposition in a subject, for example, to aid in the diagnosis of
A.beta. amyloidosis or synucleopathies in the subject. To aid in
detection, the compound can be modified with a detectable
substance, preferably .sup.99mTc or radioactive iodine or fluorine.
Methods for labeling peptide compounds with technetium are known in
the art. A modifying group can be chosen that provides a site at
which a chelation group for .sup.99mTc can be introduced, such as a
derivative of cholic acid, which has a free amino group. Also
provided are compounds labeled with radioactive fluorine. For
example the isotope of radioactive fluorine can be incorporated to
create a diagnostic agent. Preferably, .sup.18F for positron
emission tomography (PET) or single photon emission computed
tomography (SPECT) studies.
[0237] 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
General Experimental Procedures
[0238] All solvents were distilled before use and were removed by
rotary evaporation at temperatures up to 35.degree. C. Merck silica
gel 60, 200-400 mesh, 40-63 .mu.m, was used for silica gel flash
chromatography. TLC was carried out using Merck
DC.cndot.plastikfolien Kieselgel 60 F.sub.254, first visualised
with a UV lamp, and then by dipping in a vanillin solution (1%
vanillin, 1% H.sub.2SO.sub.4 in EtOH), and heating. Mass spectra
were recorded on a Kratos MS-80 instrument. NMR spectra, at
25.degree. C., were recorded at 500 or 300 MHz for .sup.1H and 125
or 75 MHz for .sup.13C on Varian INOVA-500 or VXR-300
spectrometers. Chemical shifts are given in ppm on the 6 scale
referenced to the solvent peaks CHCl.sub.3 at 7.25 and CDCl.sub.3
at 77.0 ppm or (CH.sub.3).sub.2CO at 2.15 and (CD.sub.3).sub.2CO at
30.5 ppm or CH.sub.3OD at 3.30 and CD.sub.3OD at 39.0 ppm.
HPLC Conditions
[0239] The analytical HPLC equipment consisted of a Waters 717
autosampler, 600 pump and controller, and a 2487 UV detector
controlled by Omega software for method 2, and a Waters 717
autosampler, 600 pump and controller, and a 490 UV detector
controlled by Millennium software for method 1. Samples were
analysed by using an RP-18 semi-preparative column (Phenomenex
Prodigy 5 mm C18 100A, 250.times.4.6 mm) with a guard column
(Phonomenex SecurityGuard cartridge containing a C18 ODS 4.times.3
mm, 5 mm column) fitted at 30.degree. C. Samples (5 mL) were
analysed using a mobile phase flow rate of 5.0 mL/min, with UV
detection at 280 nm.
Solvent A--CH.sub.3CN
[0240] Solvent B--H.sub.2O containing 0.1% TFA
TABLE-US-00001 Method 1 Time (minutes) solvent A solvent B 0 11 89
20 11 89 30 100 0 31 11 89 40 11 89
[0241] HPLC Method 2 (for Compounds DC-0051-B1 Through
DC-0051-B4)
[0242] The method 2 constitutes using a C18 column with
2.1.times.50 mm dimensions. The run time is set at 7 minutes. The
mobile phase included (A) acetonitrile with 0.05% TFA, and (B)
distilled water with 0.05% TFA. All runs with method 2 employed a
gradient elution from 10% to 90% of solvent A.
Example 1
Synthesis of 3-methanesulfonylamino-4-hydroxybenzoic acid
3,4-dihydroxyanilide (DC-0051-S1; also referred to as
DC-0051-CB)
##STR00036##
[0244] Formation of the methanesulfonylamine derivative of the
amide DC-0051 was carried out by initial formation of the known
3-nitro-4-methoxybenzoic acid, then formation of the anilide of
with 3,4-methylenedioxyaniline which gave 3-nitro-4-methoxy-amide.
Catalytic reduction followed by immediate mesylation gave the
mesylamine which was demethylated simply by reaction with
borontribromide to give 3-methanesulfonylamino-4-hydroxybenzoic
acid 3,4-dihydroxyanilide (DC-0051-S1; also referred to as
DC-0051-CB)).
A) 3-Nitro-4-methoxybenzoic acid
[0245] To a suspension of p-anisic acid (3 g) in acetic anhydride
(20 ml) at 0.degree. C. was added dropwise conc. nitric acid (6
ml). The resultant clear solution was allowed to come to room
temperature, then stood for 30 minutes. The mixture was poured onto
ice (100 ml) and the white solid formed filtered off, then washed
with more ice cold water to give the product (2.8 g, 72%).
[0246] H NMR ((CD.sub.3).sub.2CO) 8.44 (1H, d, J 2 Hz), 8.29 (1H,
dd, J 2, 8 Hz), 7.51 (1H, d, J 8 Hz) and 4.11 (3H, s).
B) 3-Nitro-4-methoxybenzoic acid 3,4-methylenedioxyanilide
[0247] A suspension of 3-nitro-4-methoxybenzoic acid (1.4 g) in
thionyl chloride (10 ml) was heated at reflux for one hour. The
solvents were removed in vacuo to give the acid chloride as a white
solid, which was redissolved in dry dichloromethane (20 ml) and a
mixture of pyridine (1 ml) and 3,4-methylenedioxyaniline (1 g) in
dichloromethane (5 ml) was added dropwise. The mixture was left at
room temperature for 24 hours, then more dichloromethane (50 ml)
and hydrochloric acid (1 M, 50 ml) added and the precipitate
filtered off and washed with water to give 3-nitro-4-methoxybenzoic
acid 3,4-methylenedioxyanilide (1.72 g, 72%).
[0248] .sup.1H NMR ((CD.sub.3).sub.2CO) 9.79 (1H, bs, NH), 8.58
(1H, d, J 2 Hz), 8.41 (1H, dd, J 2, 8 Hz), 7.63 (1H, d, J 2 Hz),
7.62 (1H, d, J 8 Hz), 7.35 (1H, dd, J 2, 8 Hz), 6.96 (1H, d, J 8
Hz), 6.15 (2H, s) and 4.22 (3H, s).
C) 3-methanesulfonylamino-4-methoxybenzoic acid
3,4-methylenedioxyanilide
[0249] A suspension of 3-Nitro-4-methoxybenzoic acid
3,4-methylenedioxyanilide (0.44 g) in methanol (20 ml) with formic
acid (1 ml) was stirred under hydrogen with palladium hydroxide on
carbon (10%, 200 mg) for 5 hours. The mixture was filtered through
cotton wool and the solvents removed In vacuo. Purification by
column chromatography over silica gel eluting with 20 to 100% ethyl
acetate in dichloromethane gave the pure amine (270 mg, 68%). This
was immediately dissolved in pyridine (5 ml) and methanesulfonyl
chloride (0.2 ml) added dropwise, then the mixture left at room
temperature overnight. Hydrochloric acid (1 M, 100 ml) and ethyl
acetate (100 ml) were added, then the organic layer dried and
evaporated in vacuo to give the crude product. Crystallisation from
dichloromethane gave 3-methanesulfonylamino-4-methoxybenzoic acid
3,4-methylenedioxyanilide as white crystals (155 mg, 47%).
[0250] .sup.1H NMR ((CD.sub.3).sub.2CO) 9.62 (1H, bs, NH), 8.07
(1H, d, J 2 Hz), 7.97 (1H, bs, NH), 7.87 (1H, dd, J 2, 8 Hz), 7.55
(1H, d, J 2 Hz), 7.22 (1H, dd. J 2, 8 Hz), 7.21 (1H, d, J 8 Hz),
6.83 (1H, d, J 8 Hz), 6.13 (2H, s), 4.14 (3H, s) and 3.16 (3H,
s).
D) 3-Methanesulfonylamino-4-hydroxybenzoic acid
3,4-dihydroxyanilide (DC0051-S1), see, J. van Alphen. Rec. trav.
Chim. 1929, 48, 1112-23.
[0251] To a stirred suspension of
3-methanesulfonylamino-4-methoxybenzoic acid
3,4-methylenedioxyanilide (100 mg) in dry CH.sub.2Cl.sub.2 (20 mL)
under nitrogen, was added boron tribromide (0.2 ml) then stirring
continued for a further 20 hours. Methanol (50 ml-) was added
carefully, then the solvent evaporated in vacuo to a volume of 1
ml, this was repeated 2 more times. Purification by crystallisation
from methanol gave 3-methanesulfanylamino-4-hydroxybenzoic
acid-3,4-dihydroxyanilide (DC0051-A1) (45 mg, 47%) as pale brown
crystals.
[0252] H NMR ((CD.sub.3).sub.2CO) 9.68 (1H, bs, NH), 927 (1H, bs,
NH), 8.03 (.sup.1H, d, J 2 Hz), 8.02 (1H, bs, OH), 7.91 (1H, bs,
OH), 7.76 (1H, dd, J 2, 8 Hz), 7.75 (1H, bs, OH), 7.49 (1H, d, J 2
Hz), 7.10 (1H, dd, J 2, 8 Hz), 7.09 (1H, d, J 8 Hz), 6.79 (1H, d, J
8 Hz) and 3.05 (3H, s).
[0253] M/z 337 ((M-H), 100%).
[0254] Hplc (method 1) 21.1 min.
Example 2
3-Hydroxy-4-methanesulfonylamino-N-(3,4-dihydroxyphenyl)benzamide,
(DC0051-S8; also referred to as DC-0051-DB)
##STR00037##
[0256] Formation of the anilide of 3-methoxy-4-nitrobenzoic acid
with 3,4-methylenedioxyaniline gave 3-nitro-4-methoxy-amide.
Reduction by catalytic hydrogenation followed by immediate
mesylation gave the mesylamine. This was demethylated by reaction
with borontribromide to give
3-Hydroxy-4-methanesulfonylamino-N-(3,4-dihydroxyphenyl)benzamide
(DC-0051-S8; also referred to as DC0051-BD).
A) 3-Methoxy-4-nitro-N-(3,4-methylenedioxyphenyl)benzamide
[0257] A suspension of 3-methoxy-4-nitrobenzoic acid (0.5 g) in
thionyl chloride (10 ml) was heated at reflux for one hour. The
solvents were removed in vacuo to give the acid chloride as a white
solid. The acid chloride was dissolved in dry dichloromethane (10
ml) and a mixture of pyridine (0.5 ml) and
3,4-methylenedioxyaniline (0.4 g) in dichloromethane (5 ml) was
added dropwise. The mixture was left at room temperature for 24
hours, then dichloromethane (50 ml) and hydrochloric acid (1 M, 50
ml) added and the precipitate filtered off and washed with water to
give 3-Methoxy-4-nitro-N-(3,4-methylenedioxyphenyl)benzamide (0.43
g, 54%).
[0258] .sup.1H NMR ((CD.sub.3).sub.2CO) 9.79 (1H, bs, NH), 8.03
(1H, d, J 8 Hz), 7.98 (1H, d, J 2 Hz), 7.78 (1H, dd, J 2, 8 Hz),
7.63 (1H, d, J 2 Hz), 7.32 (1H, dd, J 2, 5 Hz), 6.93 (1H, d, J 5
Hz), 6.11 (2H, s). and 4.17 (3H, s).
B)
3-Methoxy-4-methanesulfonylamino-N-(3,4-methylenedioxyphenyl)benzamide
[0259] A suspension of
3-Methoxy-4-nitro-N-(3,4-methylenedioxyphenyl)benzamide (100 mg) in
methanol (20 ml) was stirred under hydrogen with palladium on
carbon (10%, 50 mg) for 18 hours.
The solvents were removed in vacuo to give a brown gum. The residue
was dissolved in pyridine (0.5 ml) and cooled to 0.degree. C. when
methanesulfonyl chloride (0.1 ml) was added, the mixture was kept
at 0.degree. C. for a further 30 minutes then brought to room
temperature for 1 h. Dilute hydrochloric acid (10 ml, 1 M) and
dichloromethane were added, the organic layer separated, dried and
evaporated in vacuo to give the product as a brown gum.
Purification by column chromatography over silica gel eluting with
dichloromethane containing ethyl acetate (0-100%) gave
3-Methoxy-4-methanesulfonylamino-N-(3,4-methylenedioxyphenyl)benzami-
de (65 mg, 55%) as a white solid.
[0260] 'H NMR ((CD.sub.3).sub.2CO) 9.52 (1H, bs, NH), 8.13 (1H, bs,
NH), 7.74 (1H, d, J 2 Hz), 7.72 (1H, dd, J 2, 8 Hz), 7.64 (1H, d, J
8 Hz), 7.62 (1H, d, J 2 Hz), 7.26 (1H, dd, J 2, 8 Hz), 6.91 (1H, d,
J 8 Hz), 6.09 (2H, s), 4.07 (3H, s) and 3.16 (3H, s).
C)
3-Hydroxy-4-methanesulfonylamino-N-(3,4-dihydroxyphenyl)benzamide
(DC0051-S8)
[0261] To a stirred suspension of
3-Methoxy-4-methanesulfonylamino-N-(3,4-methylenedioxyphenyl)benzamide
(200 mg) in dry CH.sub.2Cl.sub.2 (20 ml) under nitrogen, was added
boron tribromide (0.3 ml) then stirring continued for a further 20
hours. Methanol (50 ml) was added carefully, then the solvent
evaporated in vacuo to a volume of 1 ml, this was repeated 2 more
times. Purification by column chromatography over silica gel
eluting with chloroform containing methanol (10-20%) gave
3-Hydroxy-4-methanesulfonylamino-N-(3,4-dihydroxyphenyl)benzamide
(DC51-DB) (65 mg, 34%) as pale brown crystals.
[0262] 'H NMR (CD.sub.3OD) 7.45 (.sup.1H, d, J 8 Hz), 7.40 (1H, d,
J 2 Hz), 7.36 (1H, dd, J 2, 8 Hz), 7.20 (.sup.1H, d, J 2 Hz), 6.88
(1H, dd, J 2, 8 Hz), 6.73 (1H, d, J 8 Hz) and 2.98 (3H, s). M/z 337
((M-H)--, 100%)
[0263] Hplc (method 1) 29.2 min.
Example 3
N-(3-methanesulfonylamino-4-hydroxyphenyl)-3,4-dihydroxybenzamide.
(DC0051-S6; also referred to as DC-0051-AE)
##STR00038##
[0265] Treatment of commercially available 2-methoxy-5-nitroaniline
with methanesulfonyl chloride gave the mesylamine. Catalytic
reduction of the nitro group then gave the required aniline to
condense with 3,4-methylenedioxybenzoyl chloride to give the
anilide. Removal of the methoxy and methylene dioxy groups with
borontribromide then gave
N-(3-methanesulfonylamino-4-hydroxyphenyl)-3,4-dihydroxybenzamide
(DC0051-S6; also referred to as DC0051-AE).
A) 2-Methoxy-5-nitro-methanesulfonylaminobenzene
[0266] To a solution of 2-methoxy-5-nitroaniline (5 g) in pyridine
(25 ml) at 0.degree. C. was added dropwise methanesulfonyl chloride
(3.5 ml) then pyridine (0.5 ml). The mixture was left at 0.degree.
C. for 1 hour, then brought to room temperature for 2 h. The
mixture was poured onto ice (100 g) and dilute hydrochloric acid
(3M, 100 ml), the solid formed was filtered then washed with water
to give 2-Methoxy-5-nitromethanesulfonylaminobenzene (5.29, 71%) as
an off-white crystalline solid.
[0267] 'H NMR (CDCl.sub.3 8.39 (1H, d, J 2 Hz), 8.05 (1H, dd, J 2,
8 Hz), 6.99 (1H, d, J 8 Hz) and 6.98 (1H, bs, NH),
B) 2-Methoxy-5-amino-methanesulfonylaminobenzene
[0268] A solution of 2-Methoxy-5-nitro-methanesulfonylaminobenzene
(1 g) in methanol (20 ml) containing palladium on carbon (10%, 100
mg) was stirred at room temperature under hydrogen for 48 h. The
mixture was filtered through celite then evaporated to give
2-methoxy-5-aminomethanesulfonylaminobenzene as a brown gum. This
was used without purification in the following reaction.
C)
N-(3-methanesulfonylamino-4-methoxyphenyl)3,4-methylenedioxybenzamide
[0269] A suspension of 3,4-methylenedioxybenzoic acid` (300 mg) in
thionyl chloride (10 ml) was heated at reflux for one hour. The
solvents were removed in vacuo to give the acid chloride as a white
solid. 2-Methoxy-5-aminomethanesulfonylaminobenzene (from the
previous reaction) was dissolved in pyridine (20 ml) and added
dropwise to the acid chloride. The mixture was left at room
temperature for 24 hours, then poured onto ice (50 g) and
hydrochloric acid (3M, 100 ml) and the precipitate filtered off and
washed with water to give
N-(3-methanesulfonylamino-4-methoxyphenyl)-3,4-methylenedioxybenzamide
(1.37 g, 93%).
[0270] 'H NMR ((CD.sub.3).sub.2CO) 9.52 (1H, bs, NH), 7.88 (1H, dd,
J 2, 8 Hz), 7.87 (1H, d, J 2 Hz), 7.71 (1H, dd, J 2, 8 Hz). 7.59
(1H, d, J 2 Hz), 7.14 (1H, d, J 8 Hz), 7.04 (1H, d, J 8 Hz), 6.20
(2H, s), 4.00 (3H, s) and 3.10 (3H, s).
D)
N-(3-methanesulfonylamino-4-hydroxyphenyl)-3,4-dihydroxybenzamide
[0271] To a stirred suspension of
N-(3-methanesulfonylamino-4-methoxyphenyl)-3,4
methylenedioxybenzamide (200 mg) in dry CH.sub.2Cl.sub.2 (20 ml)
under nitrogen, was added boron tribromide (0.3 ml) then stirring
continued for further 20 hours. Methanol (50 ml) was added
carefully, then the solvent evaporated in vacuo to a volume of 1
ml, this was repeated 2 more times. Purification by column
chromatography over silica gel eluting with chloroform containing
methanol (10-20%) gave
N-(3-methanasulfonylamino-4-hydroxyphenyl)-3,4-dihydroxybenzamide
(62 mg, 33%) as pale brown crystals.
[0272] .sup.1H NMR ((CD.sub.3).sub.2CO) 7.87 (1H, d, J 2 Hz), 7.70
(1H, dd, J 2, 8 Hz), 7.65 (1H, d, J 2 Hz), 7.05 (1H, d, J 8 Hz),
7.00 (1H, d, J 8 Hz) and 3.12 (3H, s).
[0273] M/z 337 ((M-H)--, 100%)
[0274] Hplc (method 1) 22.1 min,
Example 4
N-(3-hydroxy-4-methanesulfonylaminophenyl)-3,4-dihydroxybenzamide
(DC-0051-S7; also referred to as DC-0051-AF)
##STR00039##
[0276] Treatment of commercially available 2-methoxy-4-nitroaniline
with methanesulfonyl chloride gave the mesylamine. Reduction by
catalytic hydrogenation of the nitro group then gave the required
aniline to condense with 3,4-methylenedioxybenzoyl acid chloride to
give the anilide. Removal of the methoxy and methylene dioxy groups
with borontribromide then gave
N-(3-hydroxy-4-methanesulfonylaminophenyl)-3,4-dihydroxybenzamide.
(DC-0051-S7; also referred to as DC-0051-AF).
A) 2-Methoxy-4-nitro-methanesulfonylaminobenzene
[0277] To a solution of 2-methoxy-4-nitroaniline (5 g) in pyridine
(25 ml) at 0.degree. C. was added dropwise methanesulfonyl chloride
(3.5 ml) then pyridine (0.5 ml). The mixture was left at 0.degree.
C. for 1 hour, then brought to room temperature for 2 h. The
mixture was poured onto ice (100 g) and dilute hydrochloric acid
(3M, 100 ml), the solid formed was filtered, then washed with water
to give 2-Methoxy-4-nitromethanesulfonylaminobenzene (7.32 g, 98%)
as an off-white crystalline solid.
[0278] .sup.1H NMR (CDCl.sub.3) 7.92 (1H, dd, J 2, 8 Hz), 7.78 (1H,
d, J 2 Hz), 7.64 (1H, d, J 8 Hz) and 7.23 (1H, bs, NH).
B) 2-Methoxy-4-aminomethanesulfonylaminobenzene
[0279] A solution of 2-Methoxy-4-nitro-methanesulfonylaminobenzene
(1 g) in methanol (20 ml) containing palladium on carbon (10%, 100
mg) was stirred at room temperature under hydrogen for 48 h. The
mixture was filtered through celite then evaporated to give
2-methoxy-4-aminomethanesulfonylaminobenzene as a brown gum. This
was used without purification in the following reaction.
C)
N-(3-methoxy-4-methanesulfonylaminophenyl)-3,4-methylenedioxybenzamide
[0280] A suspension of 3,4-methylenedioxybenzoic acid (300 mg) in
thionyl chloride (10 ml) was heated at reflux for one hour. The
solvents were removed in vacuo to give the acid chloride as a white
solid. 2-Methoxy-4-amino-methanesulfonylaminobenzene (from the
previous reaction) was dissolved in pyridine (20 ml) and added
dropwise to the acid chloride. The mixture was left at room
temperature for 24 hours, then poured onto ice (50 g) and
hydrochloric acid (3M, 100 ml) and the precipitate filtered off and
washed with water to give
N-(3-methoxy-4-methanesulfonylaminophenyl)-3,4-methylenedioxybenzamide
(1.37 g, 93%).
[0281] 1H NMR (CDCl.sub.3) 7.81 (1H, d, J 2 Hz), 7.70 (1H, bs, NH),
7.47 (1H, d, J 8 Hz), 7.38 (.sup.1H dd, J 2, 8 Hz), 7.34 (1H, d, J
2 Hz), 6.88 (1H, d, J 8 Hz), 6.79 (1H, dd, J 2, 8 Hz), 6.63 (1H,
bs, NH), 6.06 (2H, s) 3.92 (3H, s) and 2.91 (3H, s).
D)
N-(3-hydroxy-4-methanesulfonylaminophenyl)-3,4-dihydroxybenzamide
[0282] To a stirred suspension of
N-(3-methoxy-4-methanesulfonylaminophenyl)-3,4-methylenedioxybenzamide
(200 mg) in dry CH.sub.3Cl.sub.2 (20 ml) under nitrogen, was added
boron tribromide (0.3 ml) then stirring continued for a further 20
hours. Methanol (50 ml) was added carefully, then the solvent
evaporated in vacuo to a volume of 1 ml, this was repeated 2 more
times. Purification by column chromatography over silica gel
eluting with chloroform containing methanol (10-20%) gave
N-(3-hydroxy-4-methanesulfonylaminophenyl)-3,4-dihydroxybenzamide
(62 mg, 33%) as pale brown crystals.
[0283] 1H NMR ((CD.sub.3).sub.2CO) 7.86 (1H, d, J 2 Hz), 7.60 (1H,
d, J 2 Hz), 7.51 (1H, dd, J 2, 8 Hz), 7.40 (1H, d, J 8 Hz), 7.30
(1H, dd, J 2, 8 Hz), 7.00 (1H, d. J 8 Hz) and 3.06 (3H, s).
[0284] M/z 337 ((M-H).sup.-, 100%)
[0285] Hplc (method 1) 29.5 min.
Example 5
3,4-Dimethanesulfonylamino-N-(3,4-dimethanesulfonylaminophenyl)benzamide,
(Referred to as DC0051-GH)
##STR00040##
[0287] Acid catalysed formation of the methyl ester of
3,4-diaminobenzoic acid followed by mesylation gave the
dimesylaminobenzoate. Basic hydrolysis of the ester then gave the
required 3,4-dimethanesulfonylaminobenzoic acid. Mesylation of
4-nitro-1,2-phenylenediamine gave the dimesylamino product which
after catalytic hydrogenation gave the required aniline.
Condensation of the acid with the amine in the presence of DCC then
gave the tetramesylamino-amide.
A) Methyl-3,4-diaminobenzoate
[0288] To dry methanol (20 ml) was carefully added thionyl chloride
(1 ml) dropwise with stirring. The 3,4-diaminobenzoic acid (1 g)
was added in portions at R.T. with stirring then the mixture heated
at reflux for 3 h. Saturated sodium bicarbonate was added until the
mixture was basic, then the mixture extracted into chloroform
containing 25% methanol. The extract was dried and evaporated in
vacuo to give the product (0.88 g, 81%) as a brown crystalline
solid.
[0289] .sup.1H NMR (CDCl.sub.3) 7.46 (1H, dd, J 2, 8 Hz), 7.40 (1H,
d, J 2 Hz), 6.67 (1H, d, J 8 Hz) and 3.84 (3H, s).
B) Methyl-3,4-dimethanesulfonylaminobenzoate
[0290] A solution of the diamine (0.88 g) in pyridine (10 ml) at
0.degree. C. was treated with methanesulfonyl chloride (2 ml). The
mixture was left at RT for 12 hours, then poured onto ice and
hydrochloric acid (3M, 50 ml) and the mixture filtered to give the
product as a white crystalline solid (0.54 g, 32%).
[0291] .sup.1H NMR ((CD.sub.3).sub.2SO) 9.39 (2H, bs), 8.11 (1H, d,
J 2 Hz), 7.95 (1H, dd, J 2, 8 Hz), 7.75 (1H, d, J 8 Hz), 3.97 (3H,
s), 3.28 (3H, s) and 3.17 (3H, s).
C) 3,4-Dimethanesulfonylaminobenzoic acid
[0292] A suspension of the ester (0.5 g) in acetone (25 ml) was
treated with sodium hydroxide solution (3M, 5 ml) and the resultant
orange solution left at R.T. for 2 hours. Aqueous hydrochloric acid
(3M) was added until the solution was acidic then extraction into
ethyl acetate containing 25% methanol gave the acid as a brown
solid (0.36 g, 75%).
[0293] .sup.1H NMR ((CD.sub.3).sub.2SO) 9.30 (2H, bs), 8.10 (1H, d,
J 2 Hz), 7.93 (1H, dd, J 2, 8 Hz), 7.72 (1H, d, J 8 Hz), 3.27 (3H,
s) and 3.17 (3H, s).
D) 3,4-Dimethanesulfonylamino-nitrobenzene
[0294] A solution of the diamine (2 g) in pyridine (10 ml) at
0.degree. C. was treated with methanesulfonyl chloride (3 ml). The
mixture was left at RT for 12 hours, then poured onto ice and
hydrochloric acid (3M, 50 ml) and the mixture filtered to give the
product as a white crystalline solid (1.21 g, 30%).
[0295] .sup.1H NMR ((CD.sub.3).sub.2SO) 8.37 (1H, d, J 2 Hz), 8.24
(1H, dd, J 2, 8 Hz), 7.86 (1H, d, J 8 Hz), 3.34 (3H, s) and 3.24
(3H, s).
E) 3,4-Dimethanesulfonylamino-aniline
[0296] A suspension of 3,4-Dimethanesulfonylamino-nitrobenzene (1.2
g) in methanol (50 ml) and ethyl acetate (50 ml) was stirred under
a hydrogen atmosphere with palladium on carbon (10%, 10 mg) for 18
hours. The catalyst was removed by filtration through celite and
the solvent removed in vacuo to give the amine (1.0 g) as a brown
gum. This was used without further purification.
F)
N-(3,4-dimethanesulfonylaminophenyl)-3,4-dimethanesulfonylaminobenzamid-
e (Referred to as DC0051-GH)
[0297] A suspension of the acid (1.5 g) and the amine (1.5 g) with
DCC (1.5 g) in dry THF (100 ml) were stirred together for 12 hours,
then the solvent removed in vacuo. Methanol (50 ml) was added to
the residue and the white solid filtered. Suspension of the residue
in more methanol (50 ml) followed by filtration gave the crude
product as the residue as an off-white solid. Suspension of the
solid in acetone (4.times.50 ml), filtration and removal of the
solvent in vacuo gave the pure product in the filtrate as a white
solid.
[0298] .sup.1H NMR ((CD.sub.3).sub.2CO) 10.03 (1H, bs), 8.45 (2H,
bs), 8.28 (1H, d, J 2 Hz), 8.12 (1H, d, J 2 Hz), 8.09 (1H, dd, J 2,
8 Hz), 7.93 (1H, dd, J 2, 8 Hz), 7.85 (1H, d, J 8 Hz), 7.63 (1H, d,
J 8 Hz), 3.24 (3H, s), 3.23 (3H, s), 3.19 (3H, s) and 3.17 (3H,
s).
[0299] hplc 30.3 minutes.
Example 6
4-Hydroxy-3-methanesulfonylamino-N-(3-hydroxy-4-methanesulfonylaminophenyl-
)benzamide, (Referred to as DC0051-CF)
##STR00041##
[0301] Treatment of commercially available 2-methoxy-4-nitroaniline
with methanesulfonyl chloride gave the mesylamine. Catalytic
reduction of the nitro group then gave the required aniline to
condense with 4-methoxy-3-nitrobenzoyl chloride to give the
anilide. Reduction by catalytic hydrogenation followed by immediate
mesylation gave the mesylamine. This was demethylated by reaction
with borontribromide to give
4-Hydroxy-3-methanesulfonylamino-N-(3-hydroxy-4-methanesulfonylamino-
phenyl)benzamide (DC0051-CF).
A) 2-Methoxy-4-nitro-methanesulfonylaminobenzene
[0302] To a solution of 2-methoxy-4-nitroaniline (5 g) in pyridine
(25 ml) at 0.degree. C. was added dropwise methanesulfonyl chloride
(3.5 ml). The mixture was left at 0.degree. C. for 1 hour, then
brought to RT for 2 h. The mixture was poured onto ice (100 g) and
dilute hydrochloric acid (3M, 100 ml), the solid formed was
filtered then washed with water and dried to give
2-methoxy-4-nitro-methanesulfonylaminobenzene (7.32 g, 98%) as an
off-white crystalline solid.
[0303] .sup.1H NMR (CDCl.sub.3) 7.93 (1H, dd, J 2, 8 Hz), 7.78 (1H,
d, J 2 Hz), 7.65 (1H, d, J 8 Hz), 7.23 (1H, bs), 4.00 (3H, s) and
3.09 (3H, s).
B) 2-Methoxy-4-amino-methanesulfonylaminobenzene
[0304] A solution of 2-methoxy-4-nitro-methanesulfonylaminobenzene
(1 g) in methanol (20 ml) containing palladium on carbon (10%, 100
mg) was stirred at RT under hydrogen for 48 h. The mixture was
filtered through celite then evaporated to give
2-methoxy-4-amino-methanesulfonylaminobenzene as a brown gum. This
was used without purification in the following reaction.
C)
4-Methoxy-3-nitro-N-(4-methanesulfonylamino-3-methoxyphenyl)benzamide
[0305] A suspension of 4-methoxy-3-nitrobenzoic acid (1 g) in
thionyl chloride (20 ml) was heated at reflux for two hours. Excess
thionyl chloride removed in vacuo to give the acid chloride as a
white solid. The acid chloride was dissolved in dry dichloromethane
(25 ml) and added to a mixture of pyridine (1 ml) and
2-methoxy-4-amino-methanesulfonylaminobenzene (0.4 g) in
dichloromethane (5 ml) dropwise. The mixture was left at room
temperature for 24 hours, then dichloromethane (50 ml) and
hydrochloric acid (1M, 50 ml) added and the precipitate filtered
off and washed with water to give
4-methoxy-3-nitro-N-(4-methanesulfonylamino-3-methoxyphenyl)benzamide
(0.43 g, 54%).
[0306] .sup.1H NMR ((CD.sub.3).sub.2CO) 8.57 (1H, d, J 2 Hz), 8.41
(1H, dd, J 2, 8 Hz), 7.89 (1H, d, J 2 Hz), 7.61 (1H, d, J 8 Hz),
7.47 (1H, d, J 8 Hz), 7.40 (1H, dd, J 2, 8 Hz), 4.18 (3H, s), 4.02
(3H, s) and 3.03 (3H, s).
D)
4-Methoxy-3-methanesulfonylamino-N-(3-methoxy-4-methanesulfonylaminophe-
nyl)-benzamide
[0307] A suspension of
4-methoxy-3-nitro-N-(4-methanesulfonylamino-3-methoxyphenyl)benzamide
(100 mg) in methanol (20 ml) was stirred under hydrogen with
palladium on carbon (10%, 50 mg) for 18 hours. The solvents were
removed in vacuo to give a brown gum. The residue was dissolved in
pyridine (0.5 ml) and cooled to 0.degree. C. when methanesulfonyl
chloride (0.1 ml) was added, the mixture was kept at 0.degree. C.
for a further 30 minutes then brought to R.T. for 1 h. Dilute
hydrochloric acid (10 ml, 1M) and dichloromethane were added, the
organic layer separated, dried and evaporated in vacuo to give the
product as a brown gum. Purification by column chromatography over
silica gel eluting with dichloromethane containing ethyl acetate
(0-100%) gave
3-methanesulfonylamino-4-methoxy-N-(3-methanesulfonylamino-4-methoxypheny-
l)benzamide (65 mg, 56%) as a white solid.
[0308] .sup.1H NMR ((CD.sub.3).sub.2CO) 9.61 (1H, bs, NH), 8.11
(1H, d, J 2 Hz), 7.96 (1H, bs, NH), 7.90 (1H, dd, J 2, 8 Hz), 7.85
(1H, d, J 2 Hz), 7.67 (1H, bs, NH), 7.39 (1H, d, J 8 Hz), 7.34 (1H,
dd, J 2, 8 Hz), 7.23 (1H, d, J 8 Hz), 4.02 (3H, s), 3.94 (3H, s),
3.04 (3H, s) and 2.95 (3H, s).
E)
4-Hydroxy-3-methanesulfonylamino-N-(3-hydroxy-4-methanesulfonylaminophe-
nyl)-benzamide
[0309] To a stirred suspension of
3-methanesulfonylamino-4-methoxy-N-(3-methanesulfonylamino-4-methoxypheny-
l)benzamide (200 mg) in dry CH.sub.2Cl.sub.2 (20 ml) under
nitrogen, was added boron tribromide (0.3 ml) then stirring
continued for a further 20 hours. Methanol (50 ml) was added
carefully, then the solvent evaporated in vacuo to a volume of 1
ml, this was repeated 2 more times. Purification by column
chromatography over silica gel eluting with chloroform containing
methanol (10-20%) gave
4-hydroxy-3-methanesulfonylamino-N-(3-hydroxy-4-methanesulfonylaminopheny-
l)-benzamide (62 mg, 33%) as pale brown crystals.
[0310] .sup.1H NMR (CD.sub.3OD) 7.92 (1H, d, J 2 Hz), 7.68 (1H, dd,
J 2, 8 Hz), 7.51 (1H, d, J 2 Hz), 7.26 (1H, d, J 8 Hz), 6.99 (1H,
dd, J 2, 8 Hz), 6.98 (1H, d, J 8 Hz), 2.99 (3H, s) and 2.92 (3H,
s).
[0311] Hplc (method 1) 29.0 min.
Example 7
3-Hydroxy-4-methanesulfonylamino-N-(4-hydroxy-3-methanesulfonylaminophenyl-
)benzamide, (Referred to as DC0051-DE)
##STR00042##
[0313] Treatment of commercially available 2-methoxy-5-nitroaniline
with methanesulfonyl chloride gave the mesylamine. Catalytic
reduction of the nitro group then gave the required aniline to
condense with 3-methoxy-4-nitrobenzoyl chloride to give the
anilide. Reduction by catalytic hydrogenation followed by immediate
mesylation gave the mesylamine. This was demethylated by reaction
with borontribromide to give a low yield of
3-Hydroxy-4-methanesulfonylamino-N-(4-hydroxy-3-methanesulfonylaminopheny-
l)benzamide (DC0051-DE), with a large amount of a stable borate
complex.
A) 2-Methoxy-5-nitro-methanesulfonylaminobenzene
[0314] To a solution of 2-methoxy-5-nitroaniline (5 g) in pyridine
(25 ml) at 0.degree. C. was added dropwise methanesulfonyl chloride
(3.5 ml) then pyridine (0.5 ml). The mixture was left at 0.degree.
C. for 1 hour, then brought to RT for 2 h. The mixture was poured
onto ice (100 g) and dilute hydrochloric acid (3M, 100 ml), the
solid formed was filtered then washed with water to give
2-Methoxy-5-nitro-methanesulfonylaminobenzene (5.2 g, 71%) as an
off-white crystalline solid.
[0315] .sup.1H NMR (CDCl.sub.3) 8.39 (1H, d, J 2 Hz), 8.05 (1H, dd,
J 2, 8 Hz), 6.99 (1H, d, J 8 Hz), 6.97 (1H, bs, NH), 4.01 (3H, s)
and 3.07 (3H, s).
B) 2-Methoxy-5-amino-methanesulfonylaminobenzene
[0316] A solution of 2-Methoxy-5-nitro-methanesulfonylaminobenzene
(1 g) in methanol (20 ml) containing palladium on carbon (10%, 100
mg) was stirred at RT under hydrogen for 48 h. The mixture was
filtered through celite then evaporated to give
2-methoxy-5-amino-methanesulfonylaminobenzene as a brown gum. This
was used without purification in the following reaction.
C)
3-Methoxy-4-nitro-N-(3-methanesulfonylamino-4-methoxyphenyl)benzamide
[0317] A suspension of 3-methoxy-4-nitrobenzoic acid (1.5 g) in
thionyl chloride (25 ml) was heated at reflux for two hours. Excess
thionyl chloride removed in vacuo to give the acid chloride as a
white solid. The acid chloride was dissolved in dry dichloromethane
(50 ml) then added to a mixture of pyridine (1.5 ml) and
4-methoxy-3-methanesulfonylamino-aniline (1.8 g) in dichloromethane
(50 ml) dropwise. The mixture was left at room temperature for 24
hours, then dichloromethane (100 ml) and hydrochloric acid (1M, 100
ml) added and the precipitate filtered off and washed with water to
give
3-methoxy-4-nitro-N-(3-methanesulfonylamino-4-methoxyphenyl)benzamide
(2.41 g, 80%).
[0318] .sup.1H NMR ((CD.sub.3).sub.2CO) 9.88 (1H, bs, NH), 8.03
(1H, d, J 8 Hz), 7.99 (1H, d, J 2 Hz), 7.87 (1H, dd, J 2, 8 Hz),
7.86 (1H, d, J 2 Hz), 7.81 (1H, dd, J 2, 8 Hz), 7.19 (1H, d, J 8
Hz), 4.17 (3H, s), 4.02 (3H, s) and 3.11 (3H, s).
D)
3-Methoxy-4-methanesulfonylamino-N-(3-methanesulfonylamino-4-methoxyphe-
nyl)benzamide
[0319] A suspension of
3-methoxy-4-nitro-N-(3-methanesulfonylamino-4-methoxyphenyl)benzamide
(1.4 g) in methanol (20 ml) was stirred under hydrogen with
palladium on carbon (10%, 50 mg) for 18 hours. The solvents were
removed in vacuo to give a brown gum. The residue was dissolved in
pyridine (5 ml) and cooled to 0.degree. C. when methanesulfonyl
chloride (0.5 ml) was added, the mixture was kept at 0.degree. C.
for a further 2 hours then brought to R.T. for 1 h. The mixture was
poured onto ice (50 g) and hydrochloric acid (3M, 50 g), the
resultant brown solid filtered and washed with water to give
4-methanesulfonylamino-3-methoxy-N-(3-methanesulfonylamino-4-meth-
oxyphenyl)benzamide (1.26 g, 86%) as a brown solid.
[0320] .sup.1H NMR ((CD.sub.3).sub.2CO) 9.66 (1H, bs, NH), 8.11
(1H, bs, NH), 7.88 (1H, dd, J 2, 8 Hz), 7.87 (1H, d, J 2 Hz), 7.84
(1H, bs, NH), 7.79 (1H, d, J 2 Hz), 7.76 (1H, dd, J 2, 8 Hz), 7.65
(1H, d, J 8 Hz), 7.17 (1H, d, J 8 Hz), 4.08 (3H, s), 4.01 (3H, s),
3.16 (3H, s) and 3.11 (3H, s).
E)
3-Hydroxy-4-methanesulfonylamino-N-(3-methanesulfonylamino-4-hydroxyphe-
nyl)benzamide
[0321] To a stirred suspension of
4-methanesulfonylamino-3-methoxy-N-(3-methanesulfonylamino-4-methoxypheny-
l)benzamide (1.25 g) in dry CH.sub.2Cl.sub.2 (50 ml) under
nitrogen, was added boron tribromide (1.5 ml) then stirring
continued for a further 20 hours. Methanol (50 ml) was added
carefully, then the solvent evaporated in vacuo to a volume of 1
ml, this was repeated 2 more times. Purification by column
chromatography over silica gel eluting with chloroform containing
methanol (10-20%) gave
3-hydroxy-4-methanesulfonylamino-N-(3-methanesulfonylamino-4-hydroxypheny-
l)benzamide (143 mg, 15%) as an off-white solid.
[0322] .sup.1H NMR ((CD.sub.3).sub.2CO) 9.55 (1H, bs), 8.82 (1H,
bs), 7.87 (1H, d, J 2 Hz), 7.73 (1H, dd, J 2, 8 Hz), 7.69 (1H, d, J
2 Hz), 7.64 (1H, dd, J 2, 8 Hz), 7.59 (1H, d, J 8 Hz), 7.04 (1H, d,
J 8 Hz), 3.16 (3H, s) and 3.12 (3H, s). Hplc (method 1) 29.5
min.
Example 8
3-Hydroxy-4-methanesulfonylamino-N-(3-hydroxy-4-methanesulfonylaminophenyl-
)benzamide, (Referred to as DC0051-DF)
##STR00043##
[0324] Treatment of commercially available 2-methoxy-4-nitroaniline
with methanesulfonyl chloride gave the mesylamine. Catalytic
reduction of the nitro group then gave the required aniline to
condense with 3-methoxy-4-nitrobenzoyl chloride to give the
anilide. Reduction by catalytic hydrogenation followed by immediate
mesylation gave the mesylamine. This was demethylated by reaction
with borontribromide to give
3-Hydroxy-4-methanesulfonylamino-N-(3-hydroxy-4-methanesulfonylamino-
phenyl)benzamide (DC0051-DF).
A) 2-Methoxy-4-nitro-methanesulfonylaminobenzene
[0325] To a solution of 2-methoxy-4-nitroaniline (5 g) in pyridine
(25 ml) at 0.degree. C. was added dropwise methanesulfonyl chloride
(3.5 ml). The mixture was left at 0.degree. C. for 1 hour, then
brought to RT for 2 h. The mixture was poured onto ice (100 g) and
dilute hydrochloric acid (3M, 100 ml), the solid formed was
filtered then washed with water and dried to give
2-methoxy-4-nitro-methanesulfonylaminobenzene (7.32 g, 98%) as an
off-white crystalline solid.
[0326] .sup.1H NMR (CDCl.sub.3) 7.93 (1H, dd, J 2, 8 Hz), 7.78 (1H,
d, J 2 Hz), 7.65 (1H, d, J 8 Hz), 7.23 (1H, bs), 4.00 (3H, s) and
3.09 (3H, s).
B) 2-Methoxy-4-amino-methanesulfonylaminobenzene
[0327] A solution of 2-methoxy-4-nitro-methanesulfonylaminobenzene
(1 g) in methanol (20 ml) containing palladium on carbon (10%, 100
mg) was stirred at RT under hydrogen for 48 h. The mixture was
filtered through celite then evaporated to give
2-methoxy-4-amino-methanesulfonylaminobenzene as a brown gum. This
was used without purification in the following reaction.
C)
3-Methoxy-4-nitro-N-(4-methanesulfonylamino-3-methoxyphenyl)benzamide
[0328] A suspension of 3-methoxy-4-nitrobenzoic acid (1.5 g) in
thionyl chloride (20 ml) was heated at reflux for two hours. Excess
thionyl chloride removed in vacuo to give the acid chloride as a
white solid. A solution of the acid chloride (1.64 g) in
dichloromethane (50 ml) was added to a suspension of
4-mesylamino-3-methoxyaniline (1.75 g) in dichloromethane (50 ml)
and then pyridine (1.5 ml) was added. The mixture was refluxed
together for 2 hours, then left at RT overnight. The resultant
mixture was added to dichloromethane (100 ml) and hydrochloric acid
(3M, 50 ml), the resultant precipitate was filtered off, washed
with water (100 ml) then dried to give
3-Methoxy-4-nitro-N-(4-methanesulfonylamino-3-methoxyphenyl)benzamide
(2.03 g, 67%).
[0329] .sup.1H NMR ((CD.sub.3).sub.2CO) 9.91 (1H, bs, NH), 8.05
(1H, d, J 8 Hz), 7.98 (1H, d, J 2 Hz), 7.90 (1H, d, J 2 Hz), 7.81
(1H, bs, NH), 7.80 (1H, dd, J 2, 8 Hz), 7.50 (1H, d, J 8 Hz), 7.39
(1H, dd, J 2, 8 Hz), 4.18 (3H, s), 4.02 (3H, s) and 3.04 (3H,
s).
D)
4-Methanesulfonylamino-3-methoxy-N-(4-methanesulfonylamino-3-methoxyphe-
nyl)benzamide
[0330] A suspension of
3-methoxy-4-nitro-N-(4-methanesulfonylamino-3-methoxyphenyl)benzamide
(2.03 g) in methanol (50 ml) and ethyl acetate (50 ml) was stirred
under hydrogen with palladium on carbon (10%, 50 mg) for 18 hours.
The solvents were removed in vacuo to give a brown gum. The residue
was dissolved in pyridine (5 ml) and cooled to 0.degree. C. when
methanesulfonyl chloride (1 ml) was added, the mixture was kept at
0.degree. C. for a further 2 hours then brought to R.T. for 1 h.
The mixture was poured onto ice (50 g) and hydrochloric acid (3M,
50 g), the resultant brown solid filtered and washed with water to
give
4-methanesulfonylamino-3-methoxy-N-(4-methanesulfonylamino-3-methoxypheny-
l)benzamide (2.1 g, 100%) as a pale brown solid.
[0331] .sup.1H NMR ((CD.sub.3).sub.2CO) 9.69 (1H, bs, NH), 8.15
(1H, bs, NH), 7.93 (1H, d, J 2 Hz), 7.78 (1H, d, J 2 Hz), 7.76 (1H,
bs, NH), 7.74 (1H, dd, J 2, 8 Hz), 7.66 (1H, d, J 8 Hz), 7.47 (1H,
d, J 8 Hz), 7.40 (1H, dd, J 2, 8 Hz), 4.09 (3H, s), 4.02 (3H, s),
3.17 (3H, s) and 3.03 (3H, s).
E)
4-Methanesulfonylamino-3-hydroxy-N-(4-methanesulfonylamino-3-hydroxyphe-
nyl)benzamide
[0332] To a stirred suspension of
4-methanesulfonylamino-3-methoxy-N-(3-methanesulfonylamino-4-methoxypheny-
l)benzamide (2 g) in dry CH.sub.2Cl.sub.2 (50 ml) under nitrogen,
was added boron tribromide (2 ml) and the resultant orange
suspension left for 3 hours. Methanol (50 ml) was added carefully
and the solution stood overnight. The solvent was evaporated in
vacuo to a volume of 1 ml, then methanol (50 ml) added, this was
repeated 2 more times. Purification by column chromatography over
silica gel eluting with chloroform containing methanol (10-20%)
gave
4-methanesulfonylamino-3-hydroxy-N-(4-methanesulfonylamino-3-hydroxypheny-
l)benzamide (DC0051-DF) (0.74 g, 40%) as a pale brown gum.
[0333] .sup.1H NMR ((CD.sub.3).sub.2SO) 10.37 (1H, bs, NH), 10.21
(1H, bs, NH), 10.01 (1H, bs, NH), 9.05 (1H, bs, OH), 8.76 (1H, bs,
OH), 7.68 (1H, bs), 7.53 (1H, bs), 7.51 (1H, dd, J 2, 8 Hz), 7.45
(1H, d, J 8 Hz), 7.21 (2H, bs), 3.14 (3H, s) and 3.03 (3H, s). Hplc
(method 1) 29.4 min.
Example 9
2-Oxo-N-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-2,3-dihydro-1H-benzo[-
d]imidazole-5-carboxide (Referred to as DC-0051-B1)
##STR00044##
[0335] The amide was synthesized by reacting
2-oxo-2,3-dihydro-1H-benzoimidazolyl-5-carboxylic acid with
5-amino-2,3-dihydro-1H-benzoimidazol-5-one in the presence of
1,3-N,N-diisopropylcarbodiimide and 1-hydroxybenzotriazole.
[0336] 1,3-N,N-Diisopropylcarbodiimde (0.504 g; 4 mmol) was added
to a solution of 2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic
acid (0.448 g; 2.5 mmol),
5-amino-2-oxo-2,3-dihydro-1H-benzoimidazole and
1-hydroxybenzotriazole (0.34 g; 2.5 mmol) in anhydrous
N,N-dimethylformamide (10 ml). The reaction mixture was stirred at
40.degree. C. for 12 hours. The precipitated product was isolated
by filtration of the reaction mixture followed by washing three
more times with N,N-dimethylformamide (3 ml). The product was
dissolved in dimethylsulfoxide (5 ml) and precipitated by diluting
the solution with acetonitrile (60 ml). Filtration and drying under
vacuum gave
2-Oxo-N-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-2,3-dihydro-1H-benzo-
[d]imidazole-5-carboxide
[0337] (also referred to as DC-0051-B1) (0.22 g; 28%).
[0338] .sup.1H NMR ((CD3).sub.2SO 10.62 (1H, s, NH), 10.53 (1H, s,
NH), 9.98 (1H, s, NH) 7.65 (1H, d, J 8 Hz) 7.55 (2H, bs) 7.23 (1H,
d, J 8 Hz) 7.05 (1H, d, J 8 Hz) 7.85 (1H, d, J 8 Hz).
Example 10
N-(3,4-dihydroxyphenyl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5-carboxami-
de (Referred to as DC-0051-B2)
##STR00045##
[0340] 3,4-Dihydroxy-1-nitrobenzene was benzylated by refluxing
with benzyl bromide with potassium carbonate as a base in acetone
which on reduction with sodium dithionite gave 3,4-dibenzyloxy
aniline. This was coupled to
2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid using
N,N-1,3-diisopropylcarbodiimide in the presence of
1-hydroxybenzotriazole to provide the amide. The amide was then
debenzylated by hydrogenation in presence of palladium on
carbon.
A) 3,4-Dibenzyloxy-1-nitro benzene
[0341] Potassium carbonate (4.14 g; 30 mmol) was added to a
solution of 3,4-dihydroxy-1-nitrobenzene (1.55 g; 10 mmol) and
benzyl bromide (3.42 g; 20 mmol) in acetone (100 ml). The reaction
mixture was refluxed for 12 hours. After the removal of the solvent
under reduced pressure, the residue was partitioned between ethyl
acetate (150 ml) and water (50 ml). The ethyl acetate layer was
washed with water (100 ml) and dried over anhydrous magnesium
sulfate. Removal of the solvent under reduced pressure provided
2.37 g of 3,4-Dibenzyloxy-1-nitro benzene. (Yield=70%)
[0342] .sup.1H NMR CDCl.sub.3 7.85 (1H, d, J 8 Hz) 7.8 (1H, s)
7.28-7.50 (m, 10H) 6.95 (1H, d, J 8 Hz) 5.24 (s, 2H) 5.21 (s,
2H)
B) 3,4-Dibenzyloxy aniline
[0343] Sodium dithionite (2 g) was added to a solution of
3,4-Dibenzyloxy-1-nitro benzene (2.37 gm) in a mixture of methanol
(30 ml)/aqueous ammonia (5 ml). After stirring for 12 hrs at room
temperature, the solvent was removed under reduced pressure. The
residue was partitioned between ethyl acetate (75 ml) and water (75
ml). The ethyl acetate layer was washed with water (25 ml), brine
solution (25 ml), dried over anhydrous magnesium sulfate and
concentrated under reduced pressure. Purification by flash
chromatography over silica gel eluting with ethyl acetate/hexane
(1:1) provided 1.0 g of 1-Benzyloxy-2-methoxy-5-aminobenzene
(Yield=47%).
[0344] .sup.1H NMR CDCl.sub.3 7.27-7.47 (10H, m) 6.8 (1H, d, J 8
Hz) 6.37 (1H, s) 6.22 (1H, d, J 8 Hz) 5.13 (2H, s) 5.06 (2H, s)
3.49 (2H, bs, NH.sub.2)
C) 2-Oxo-2,3-dihydro-1H-benzoimidazolyl-5-carboxyl
(1-N-3,4-dibenzyloxy phenyl)amide
[0345] 1,3-N,N-Diisopropylcarbodiimde (0.412 g; 3.27 mmol) was
added to a solution of
2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid (0.584 g;
3.27 mmol) 3,4-dibenzyloxy aniline (1.0 g, 3.27 mmol) and
1-hydroxybenzotriazole (0.442 g, 3.27 mmol) in anhydrous
N,N-dimethylformamide (15 ml). After stirring for 16 hrs at room
temperature the reaction mixture was poured in water (150 ml). The
pH of the mixture was adjusted to 2 with 1N hydrochloric acid and
stirred for 30 minutes. Filtration and washing the product with
ethyl acetate (3.times.10 ml) provided 1.12 grams of
2-Oxo-2,3-dihydro-1H-benzoimidazolyl-5-carboxyl
(1-N-3,4-dibenzyloxy phenyl)amide.
[0346] Yield=73.6%.
[0347] .sup.1H NMR (CD.sub.3).sub.2SO 10.5 (1H, s, NH) 7.65 (1H, d,
J 8 Hz) 7.6 (1H, s) 7.2-7.6 (m, 12H) 7.0 (2H, d, J 8 Hz) 5.15 (4H,
s).
D)
N-(3,4-dihydroxyphenyl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5-carbox-
amide
[0348] A solution of
2-Oxo-2,3-dihydro-1H-benzoimidazolyl-5-carboxyl
(1-N-3,4-dibenzyloxy phenyl)amide. (1.10 g) in a mixture of acetic
acid (100 ml) and N,N-dimethylformamide (25 ml) was hydrogenated at
40 Psi in presence of 10% palladium on carbon for 12 hrs at room
temperature. After removal of the catalyst by filtration, the
solvent was removed under reduced pressure. The residue was
dissolved in N,N-dimethylformamide (15 ml) and the product was
precipitated by diluting with a mixture of hexane/ethyl acetate
(1:1) (100 ml). Filtration provided 0.550 g of
N-(3,4-dihydroxyphenyl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5-carboxam-
ide. Yield=81%.
[0349] .sup.1H NMR (CD.sub.3).sub.2SO 10.94 (1H, bs) 9.86 (1H, s)
8.85 (1H, bs) 7.61 (1H, d J 8 Hz) 7.59 (1H, s) 7.3 (1H, s) 7.0 (1H,
d, J 8 Hz) 6.96 (1H, d, J 8 Hz) 6.66 (1H, d, J 8 Hz)
[0350] M/z (286 (M+H.sup.+), 308 (M+Na.sup.+ 100%). HPLC (method 2)
3.256 min.
Example 11
3,4-dihydroxy-N-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)benzamide
(referred to as DC-0051-B3)
##STR00046##
[0352] 3,4-Dihydroxybenzoic acid was converted to its methyl ester
by refluxing in methanol in presence of acid. The dihydroxy group
was protected as its benzyl ether by treating with benzyl bromide
and potassium carbonate. Hydrolysis of the ester using sodium
hydroxide provided the acid which was coupled to
5-amino-2,3-dihydro-1H-benzoimidazol-5-one using
N,N-1,3-diisopropylcarbodiimide in presence of
1-hydroxybenzotriazole to provide the amide. The amide was
debenzylated by hydrogenation in presence of palladium on
carbon.
A) 3,4-Dihydroxy Benzoic Acid Methyl Ester
[0353] A solution of 3,4-dihydroxy benzoic acid (2.8 g) in methanol
(150 ml) was refluxed in presence of concentrated hydrochloric acid
(0.5 ml) for 12 hrs. After concentrating under reduced pressure,
the residue was dissolved in ethyl acetate (150 ml) and washed with
water (50 ml) 10% sodium bicarbonate solution (50 ml), brine
solution (50 ml) and dried over anhydrous magnesium sulfate.
Removal of the solvent under reduced pressure provided 2.64 g of
3,4-dihydroxybenzoic acid methyl ester. (Yield=86.5%).
[0354] .sup.1H NMR CDCl.sub.3 7.7 (1H, s) 7.63 (1H, d, J 8 Hz) 6.92
(1H, d, J 8 Hz) 5.7 (2H, bs) 3.92 (3H, s)
B) 3,4-Dibenzyloxy Benzoic Acid Methyl Ester
[0355] Potassium carbonate (6.5 g; 47 mmol) was added to a solution
of 3,4-dihydroxybenzoic acid methyl ester (2.6 g; 15.7 mmol) and
benzyl bromide (5.37 g; 31.4 mmol) in acetone (100 ml). The
reaction mixture was refluxed for 12 hrs. After the removal of the
solvent under reduced pressure, the residue was partitioned between
ethyl acetate (150 ml) and water (50 ml). The ethyl acetate layer
was washed with water (50 ml) and dried over anhydrous magnesium
sulfate. Removal of the solvent under reduced pressure provided
3.36 g of 3,4-Dibenzyloxy benzoic acid methyl ester
(Yield=86.6%)
[0356] .sup.1H NMR CDCl.sub.3 7.67 (1H, s) 7.65 (1H, d, J 8 Hz)
7.28-7.50 (m, 10H) 6.95 (1H, d, J 8 Hz) 5.24 (s, 2H) 5.21 (s, 2H)
3.89 (s, 3H)
C) 3,4-Dibenzyloxy Benzoic Acid
[0357] A solution of sodium hydroxide (1.2 g) in methanol (100 ml)
was added to a solution of
3,4-dibenzyloxy benzoic acid methyl ester (4.64 g) in methanol (50
ml) and refluxed for 4 hrs. After removal of methanol under reduced
pressure the residue was dissolved in water (100 ml) and washed
with ethyl acetate (2.times.50 ml). The aqueous layer was acidified
with 2N hydrochloric acid to pH 2. The precipitated product was
collected by filtration which on drying under vacuum provided 2.4 g
of 3,4-benzyloxy benzoic acid. (Yield=74%)
[0358] .sup.1H NMR CDCl.sub.3 7.7 (2H, b, s) 7.27-7.5 (10H, m) 6.98
(1H, d, J 8 Hz) 5.26 (2H, s) 5.22 (2H, s)
D)
3,4-Dibenzyloxy-(5-N-2-oxo-2,3-dihydro-1H-benzoimidazolyl)benzamide
[0359] 1,3-N,N-Diisopropylcarbodiimde (0.945 g; 7.5 mmol) was added
to a solution of 3,4-dibenzyloxy benzoic acid (1.67 g, 5 mmol),
5-amino-2,3-dihydro-1H-benzoimidazol-5-one (0.745 g, 5 mmol) and
1-hydroxybenzotriazole (0.675 g, 5 mmol) in anhydrous
N,N-dimethylformamide (20 ml). After stirring for 16 hrs at room
temperature the reaction mixture was poured in water (100 ml). The
pH of the mixture was adjusted to 2 with 1N hydrochloric acid and
stirred for 30 minutes. Filtration and washing the product with
ethyl acetate (3.times.10 ml) provided 1.06 grams of
3,4-dibenzyloxy-(5-N-2-oxo-2,3-dihydro-1H-benzoimidazolyl)benzamide.
(Yield=45.7%).
[0360] .sup.1H NMR (CD.sub.3).sub.2SO 9.94 (1H, s) 7.65-7.2 (14H,
m) 7.09 (2H, d, J 8 Hz) 5.1 (4H, s).
E)
3,4-dihydroxy-N-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)benzamide
[0361] A solution of
3,4-dibenzyloxy-(5-N-2-oxo-2,3-dihydro-1H-benzoimidazolyl)benzamide
(1.06 g; 2.28 mmol) in acetic acid (120 ml) was hydrogenated at 40
Psi in presence of 10% palladium on carbon for 12 hrs at room
temperature. After removal of the catalyst by filtration, the
solvent was removed under reduced pressure. The residue was
dissolved in N,N-dimethylformamide (15 ml) and the product was
precipitated by diluting with a mixture of hexane/ethyl acetate
(1:1) (100 ml). Filtration provided 0.334 g of
3,4-dihydroxy-N-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)benzamide.
Yield=50%.
[0362] .sup.1H NMR (CD.sub.3).sub.2SO 10.54 (1H, bs) 9.78 (1H, s)
9.41 (1H, bs) 7.54 (1H, s) 7.37 (1H, s) 7.32 (1H, d, J 8 Hz) 7.23
(1H, d, J 8 Hz) 6.85 (1H, d, J 8 Hz) 6.80 (1H, d, J 8 Hz). M/z (286
(M+H.sup.+) 100%, 308 (M+Na.sup.+). HPLC (method 2) 2.34 min.
Example 12
3-hydroxy-N-(3-hydroxy-4-methoxyphenyl)-4-methoxybenzamide
(referred to as DC-0051-B4)
##STR00047##
[0364] 3-Hydroxy-4-methoxy-1-nitrobenzene was benzylated by
refluxing with benzyl bromide with potassium carbonate as a base in
acetone which on reduction with sodium dithionite gave
3-benzyloxy-4-methoxy aniline. 4-Hydroxy-3-methoxy benzoic acid was
converted to its methyl ester by refluxing in methanol in presence
of an acid. The hydroxyl was benzylated using benzyl bromide and
potassium carbonate. The ester was hydrolyzed with sodium hydroxide
to provide the acid. The aniline and acid were coupled using
N,N-1,3-diisopropylcarbodiimide in presence of
1-hydroxybenzotriazole to provide the amide. Finally the benzyl
group was removed by hydrogenation in presence of palladium on
carbon.
A) 1-Benzyloxy-2-methoxy-5-nitrobenzene
[0365] Potassium carbonate (1.65 gm; 12 mmol) was added to a
solution of 2-methoxy-5-nitro phenol (1.69 g; 10 mmol) and benzyl
bromide (1.71 gm; 10 mmol) in acetone (60 ml). The reaction mixture
was refluxed for 12 hours. The solvent was removed under reduced
pressure and the residue was partitioned between ethyl acetate (150
ml) and water (50 ml). The ethyl acetate layer was separated,
washed with water (2.times.50 ml), dried over anhydrous magnesium
sulfate. Removal of the solvent under reduced pressure provided 2.5
g of 1-Benzyloxy-2-methoxy-5-nitrobenzene (Yield=96.5%)
[0366] .sup.1H NMR CDCl.sub.3 7.95 (1H, d, J 8 Hz) 7.81 (1H, s)
7.3-7.5 (5H, m) 6.92 (1H, d, J 8 Hz) 5.15 (2H, s) 3.95 (3H, s).
B) 1-Benzyloxy-2-methoxy-5-aminobenzene
[0367] Sodium dithionite (1.5 g) was added to a solution of
1-benzyloxy-2-methoxy-5-nitrobenzene (2.5 gm) in a mixture of
methanol (20 ml)/aqueous ammonia (4 ml). After stirring for 12 hrs
at room temperature, the solvent was removed under reduced
pressure. The residue was partitioned between ethyl acetate (75 ml)
and water (50 ml). The ethyl acetate layer was washed with water
(25 ml), brine solution (25 ml), dried over anhydrous magnesium
sulfate and concentrated under reduced pressure. Purification by
flash chromatography over silica gel eluting with ethyl
acetate/hexane (1:1) provided 0.771 g of
1-Benzyloxy-2-methoxy-5-aminobenzene (Yield=35%).
[0368] .sup.1H NMR CDCl.sub.3 7.25-7.5 (5H, m) 6.78 (1H, d, J 8 Hz)
6.35 (1H, s) 6.28 (1H, d, J 8 Hz) 5.1 (2H, s) 3.8 (3H, s)
C) 4-Hydroxy-3-methoxy benzoic acid methyl ester
[0369] A solution of 4-hydroxy-3-methoxy benzoic acid (7.2 g) in
methanol (150 ml) was refluxed in presence of concentrated
hydrochloric acid (0.5 ml) for 12 hrs. After concentrating under
reduced pressure, the residue was dissolved in ethyl acetate (200
ml) and washed with water (50 ml) 10% sodium bicarbonate solution
(2.times.50 ml), water (50 ml) and dried over anhydrous magnesium
sulfate. Removal of the solvent under reduced pressure provided
7.25 g of 4-hydroxy-3-methoxy benzoic acid methyl ester.
(Yield=91.5%).
[0370] .sup.1H NMR CDCl.sub.3 7.65 (1H, d, J 8 Hz) 7.55 (1H, s)
6.95 (1H, d, J 8 Hz) 6.15 (1H, bs, --OH) 3.95 (3H, s) 3.9 (3H,
s).
D) 4-Benzyloxy-3-methoxy benzoic acid methyl ester
[0371] Potassium carbonate (3.45 g; 25 mmol) was added to a
solution of 4-hydroxy-3-methoxy benzoic acid methyl ester (3.6 g;
20 mmol) and benzyl bromide (3.42 g; 20 mmol) in acetone (100 ml).
The reaction mixture was refluxed for 12 hrs. After the removal of
the solvent under reduced pressure, the residue was partitioned
between ethyl acetate (150 ml) and water (50 ml). The ethyl acetate
layer was washed with water (50 ml) and dried over anhydrous
magnesium sulfate. Removal of the solvent under reduced pressure
provided 4.64 g of 4-benzyloxy-3-methoxy benzoic acid methyl ester
(Yield=86.6%)
E) 4-Benzyloxy-3-methoxy benzoic acid
[0372] A solution of sodium hydroxide (2.0 g) in methanol (50 ml)
was added to a solution of
4-benzyloxy-3-methoxy benzoic acid methyl ester (4.64 g) in
methanol (50 ml) and refluxed for 4 hrs. After removal of methanol
under reduced pressure the residue was dissolved in water (150 ml)
and washed with ethyl acetate (2.times.50 ml). The aqueous layer
was acidified with 2N hydrochloric acid to pH 2. The precipitated
product was collected by filtration which on drying under vacuum
provided 4.17 g of 4-benzyloxy-3-methoxy benzoic acid.
(Yield=74%)
[0373] .sup.1H NMR CDCl.sub.3 7.7 (1H, d, J=8 Hz) 7.63 (1H, s)
7.3-7.5 (5H, m) 6.92 (1H, d, J 8 Hz) 5.25 (2H, s) 3.98 (3H, s)
F)
4-Benzyloxy-3-methoxy-N-(3-benzyloxy-4-methoxyphenyl)benzamide
[0374] N,N-1,3-Diisopropyl carbodiimide (0.40 g, 3.36 mmol) was
added to a solution of 1-benzyloxy-2-methoxy-5-aminobenzene (0.771
g, 3.36 mmol), 4-benzyloxy-3-methoxy benzoic acid (0.87 g, 3.36
mmol) and 1-hydroxybenzotriazole (0.454 g, 3.36 mmol) in
N,N-dimethylformamide (15 ml) and stirred for 12 hrs. The product
was precipitated by diluting with a mixture of ethyl acetate/hexane
(1:1) (120 ml). Filtration of the reaction mixture provided 1.12 g
of 4-Benzyloxy-3-methoxy-N-(3-benzyloxy-4-methoxyphenyl)benzamide.
Yield=69%.
[0375] .sup.1H NMR (CD.sub.3).sub.2SO 9.93 (1H, s) 7.29-7.59 (14H,
m) 7.16 (1H, d, J 8 Hz) 6.96 (1H, d, J 8 Hz) 5.18 (2H, s) 5.06 (2H,
s) 3.85 (3H, s) 3.76 (3H, s)
G) 3-hydroxy-N-(3-hydroxy-4-methoxyphenyl)-4-methoxybenzamide
[0376] A solution of the
4-Benzyloxy-3-methoxy-N-(3-benzyloxy-4-methoxyphenyl)benzamide
(1.05 g) in a mixture of N,N-dimethylformamide/methanol (1:5, 120
ml) was hydrogenated in presence of 10% palladium on carbon at 40
Psi at room temperature for 12 hrs. Removal of the catalyst by
filtration and purification by flash chromatography over silica gel
eluting with 65% ethyl acetate/hexane provided 0.26 g of
3-hydroxy-N-(3-hydroxy-4-methoxyphenyl)-4-methoxybenzamide.
Yield=41.6%
[0377] .sup.1H NMR (CD.sub.3).sub.2SO 9.73 (1H, s) 9.62 (1H, bs)
8.99 (1H, bs) 7.5 (1H, s) 7.45 (1H, d, J 8 Hz) 7.29 (1H, s) 7.09
(1H, d, J 8 Hz) 6.85 (1H, d, J 8 Hz) 3.84 (3H, s) 3.74 (3H, s) M/z
(290 (M+H.sup.+), 312 (M+Na.sup.+), 100%). HPLC (method 2) 3.86
min.
Example 13
[0378] The following compounds were prepared using procedures
similar those described herein:
[0379] i) DC-0051-A2 also referred as DC-0051-S2
##STR00048##
[0380] ii) DC-0051-A3 also referred as DC-0051-S3
##STR00049##
[0381] iii) DC-0051-A4 also referred as DC-0051-S4
##STR00050##
iv) DC-0051-A5 also referred as DC-0051-S5
##STR00051## ##STR00052##
Example 14
4-Hydroxy-3-methanesulfonylamino-N-(4-hydroxy-3-methane sulfonyl
amino phenyl)-benzamide borate complex (Referred to as DC0051-CE
borate complex)
[0382] Treatment of commercially available 2-methoxy-5-nitroaniline
with methanesulfonyl chloride gave the mesylamine. Catalytic
reduction of the nitro group then gave the required aniline to
condense with 4-methoxy-3-nitrobenzoyl chloride to give the
anilide. Reduction by catalytic hydrogenation followed by immediate
mesylation gave the mesylamine. Demethylation under usual
conditions gave a stable borate complex of the required
product.
Example 15
HPLC Method
[0383] For the following synthetic methods this HPLC method was
employed. Samples were analysed using an Agilent HP1100 instrument,
operated with EzChrom Elite software, and fitted with a C18 column
(Phenomenex Prodigy 5 .mu.m 100A, 250.times.4.6 mm) with a guard
column (Phenomenex ODS 4.times.3 mm, 5 .mu.m) held at 30.degree. C.
Peaks were detected at 280 nm. The mobile phase was acetonitrile in
water (with 0.1% TFA): t.sub.0=11%, t.sub.20=11%, t.sub.30=100%,
t.sub.31=11%, t.sub.40=11%. The flow rate was 1 mL/min and the
injection volume of 5 .mu.L.
Overview of Synthesis of
N-(2-fluoro-4,5-dihydroxyphenyl)-3,4-dihydroxy benzamide
(DC51-F5)
##STR00053##
[0385] Nitration (I. M. Takakis et al. J Heterocyclic Chem. 1991,
28, 625-634) of commercially available 4-fluoroveratrole gave, as
expected, just the 2-nitro isomer 1, as shown by NMR. Reduction
using tin II chloride (A. Kamal et al. Bioorg. Med. Chem. 2007, 15
(22), 6868-6875) of this gave the aniline 2, which was immediately
reacted with 3,4-methylenedioxybenzoyl chloride to give the anilide
3. Deprotection with boron tribromide under standard conditions
gave the free phenolic amide, DC51-F5
(N-(2-fluoro-4,5-dihydroxyphenyl)-3,4-dihydroxybenzamide) in
reasonable yield.
Step A: Preparation of 1-fluoro-4,5-dimethoxy-2-nitrobenzene
(1)
[0386] To ice cold nitric acid (10 ml) was added dropwise with
stirring 4-fluoroveratrole (1 g). The mixture was stirred at
0.degree. C. for 15 minutes, then brought to RT for 15 mins. The
orange solution was poured over ice and the resultant off white
solid was filtered, washed with water then dried to give
1-fluoro-4,5-dimethoxy-2-nitrobenzene (1) (1.25 g, 97%).
[0387] .sup.1H NMR (CDCl.sub.3, 500 MHz) 7.58 (1H, d, J 7 Hz, H3),
6.72 (1H, d, J 12 Hz, H6), 3.96 (3H, s) and 3.93 (3H, s).
##STR00054##
Step B: Preparation of 2-fluoro-4,5-dimethoxyaniline (2)
[0388] A solution of 1-fluoro-4,5-dimethoxy-2-nitrobenzene (1)
(0.28 g) in ethyl acetate/95% ethanol (7 ml/7 ml) and tin(II)
chloride (1.0 g) was heated to 70.degree. C. with stirring for 3 h,
when more tin(II) chloride (1.0 g) was added and the mixture heated
for a further 2 h, then left stirring at RT overnight. The solvents
were removed in vacuo, then methanol/DCM (1:10) and sodium
bicarbonate (sat.) were added. The mixture was filtered through
ceelite, then the ceelite washed with further methanol/DCM (1:10).
The organic phase was separated, dried, and evaporated in vacuo to
give the product as a dark brown solid. This was used directly in
the next step.
[0389] .sup.1H NMR (CDCl.sub.3, 300 MHz) 6.57 (1H, d, J 13 Hz, H3),
6.34 (1H, d, J 9 Hz, H6), 3.77 (3H, s) and 3.74 (3H, s).
##STR00055##
Step C: Preparation of 3,4-Methylenedioxy-N-(4,5-dimethoxy-2-fluoro
phenyl)benzamide (3)
[0390] A suspension of piperonylic acid (0.51 g, 3.1 mmol) in
thionyl chloride (5 ml) was refluxed in the absence of moisture for
2 hours when a clear solution had been formed. The excess thionyl
chloride was removed by evaporation in vacuo to leave the acid
chloride as an off-white solid.
[0391] A solution of the acid chloride in dichloromethane (20 ml)
was added to a solution of 2-fluoro-4,5-dimethoxyaniline (2) (0.40
g, 2.9 mmol) in dichloromethane (5 ml) and then pyridine (1 ml) was
added. The mixture was refluxed together for 3 hours, then left at
RT overnight. The resultant brown solution was diluted with
dichloromethane (100 ml) and methanol (15 ml), washed with
hydrochloric acid (1M, 50 ml) then dried and evaporated in vacuo to
give the product as a brown solid. Purification by column
chromatography over silica gel eluting with 10 to 20% ethyl acetate
in DCM gave 3,4-Methylenedioxy-N-(4,5-dimethoxy-2-fluoro
phenyl)benzamide (3) as a brown solid (480 mg, 57%).
[0392] M/z found 342.0767. C.sub.16H.sub.14FNNaO.sub.5 requires
342.0748.
[0393] .sup.1H NMR ((CD.sub.3).sub.2CO, 500 MHz) 9.01 (1H, bs, NH),
7.72 (1H, dd, J 2, 8 Hz, H6), 7.63 (1H, t, J 8 Hz, H6'), 7.59 (1H,
d, J 2 Hz, H2), 7.06 (1H, d, J 8 Hz, H5), 6.97 (1H, d, J 12 Hz,
H3'), 6.22 (2H, s), 3.93 (3H, s) and 3.90 (3H, s).
##STR00056##
Step D: Preparation of 3,4-Dihydroxy-N-(2-fluoro-4,5-dihydroxy
phenyl)benzamide (DC51-F5)
[0394] To a solution of amide (3) (400 mg, 1.25 mmol) in dry
dichloromethane (30 ml) at RT was added boron tribromide (1 ml). A
pale green solution was first formed which rapidly formed an
off-brown precipitate. This suspension was left at RT for 3 h, then
methanol (then 20 ml) was added carefully and the resultant brown
solution left at RT overnight, when a yellow solid was formed.
[0395] The solvents were removed in vacuo to leave about 1 ml, then
methanol (dropwise then 30 ml) was added carefully. This was
repeated 4 times, then solvents removed in vacuo to leave the
product as an off-white solid. The solid was dissolved in a minimum
amount of methanol, then dichloromethane added until just cloudy.
The mixture was left in the fridge overnight when the product was
formed as an off white crystalline solid (261 mg, 78%).
[0396] Hplc 18.12 minutes.
[0397] M]z found 278.0479. C.sub.13H.sub.9FNO.sub.5 requires
278.0470.
[0398] .sup.1H NMR (CD.sub.3OD, 300 MHz) 7.36 (1H, d, J 2 Hz, H2),
7.32 (1H, dd, J 2, 9 Hz, H6), 7.02 (1H, d, J 8 Hz, H6'), 6.83 (1H,
d, J 9 Hz, H5) and 6.60 (1H, d, J 12 Hz, H3').
##STR00057##
Example 16
Overview of Synthesis of Positional isomers
N-(3-fluoro-4,5-dihydroxyphenyl)-3,4-dihydroxy benzamide (DC51-F4)
and N-(2-fluoro-3,4-dihydroxyphenyl)-3,4-dihydroxy benzamide
(DC51-F6)
[0399] 3-Fluoroveratrole 4 was prepared by methylation.sup.4 of the
commercially available 3-fluorocatechol. Nitration of
3-fluoroveratrole gave a mixture of the two isomeric products 5 and
6 in a 2:1 ratio (structures and ratio determined by NMR
spectroscopy). Separation of these, followed by reduction with tin
II chloride.sup.3 gave the anilines 7 and 8. Reaction of the
anilines with 3,4-methylenedioxybenzoyl chloride gave the anilides
9 and 10. Deprotection with boron tribromide under standard
conditions gave the free phenolic anilides,
N-(3-fluoro-4,5-dihydroxyphenyl)-3,4-dihydroxybenzamide (DC51-F4)
and N-(2-fluoro-3,4-dihydroxyphenyl)-3,4-dihydroxybenzamide
(DC51-F6) in reasonable yield.
##STR00058##
Step A: Preparation of 1-fluoro-2,3-dimethoxy-5-nitrobenzene (5)
and 1-fluoro-2,3-dimethoxy-6-nitrobenzene (6)
[0400] To ice cold nitric acid (10 ml) was added dropwise with
stirring 3-fluoroveratrole (2 g). The mixture was stirred at
0.degree. C. for 15 minutes, then brought to RT for 15 mins. The
orange solution was poured over ice and the resultant off white
solid was filtered, washed with water then dried.
[0401] Column chromatography over silica eluting with pet ether/DCM
(1:1) gave the pure 1-fluoro-2,3-dimethoxy-5-nitrobenzene (5) (V M.
Cervera et al. Tetrahedron 1996, 52 (7), 2557-2564) (1.20 mg, 50%)
.sup.1H NMR (CDCl.sub.3, 500 MHz) 7.68 (1H, dd, J 2, 11 Hz, H6),
7.61 (1H, dd, J 1, 2 Hz, H4), 4.06 (3H, d, 2 Hz) and 3.96 (3H,
s).
[0402] This first fraction was followed by pure
1-fluoro-2,3-dimethoxy-6-nitrobenzene (6) (0.550 mg, 23%).
[0403] .sup.1H NMR (CDCl.sub.3, 500 MHz) 7.86 (1H, dd, J 9, 10 Hz,
H5), 6.75 (1H, dd, J 2, 9 Hz, H4), 3.96 (3H, s) and 3.97 (3H, d, 2
Hz).
##STR00059##
Step B1: Preparation of 3-fluoro-4,5-dimethoxyaniline (7)
[0404] A solution of 1-fluoro-2,3-dimethoxy-5-nitrobenzene (5)
(0.56 g) in ethyl acetate/95% ethanol (14 ml/14 ml) and tin(II)
chloride (2.0 g) was heated to 70.degree. C. with stirring for 3 h,
when more tin(II) chloride (1.0 g) was added and the mixture heated
for a further 2 h, then left stirring at RT overnight. The solvents
were removed in vacuo, then methanol/DCM (1:10) and sodium
bicarbonate (sat.) were added. The mixture was filtered through
ceelite, then the ceelite washed with further methanol/DCM (1:10).
The organic phase was separated, dried, and evaporated in vacuo to
give aniline (7) as a dark brown solid. This was used directly in
the following reaction.
Step B2: Preparation of -fluoro-3,4-dimethoxy-aniline (8)
[0405] A solution of 1-fluoro-2,3-dimethoxy-6-nitrobenzene (6)
(0.56 g) in ethyl acetate/95% ethanol (14 ml/14 ml) and tin(II)
chloride (2.0 g) was heated to 70.degree. C. with stirring for 3 h,
when more tin(II) chloride (1.0 g) was added and the mixture heated
for a further 2 h, then left stirring at RT overnight. The solvents
were removed in vacuo, then methanol/DCM (1:10) and sodium
bicarbonate (sat.) were added. The mixture was filtered through
ceelite, then the ceelite washed with further methanol/DCM (1:10).
The organic phase was separated, dried, and evaporated in vacuo to
give aniline (8) as a dark brown solid. This was used directly in
the following reaction.
Step C1: Preparation of
3,4-Methylenedioxy-N-(4,5-dimethoxy-3-fluoro phenyl)benzamide
(9)
[0406] A suspension of piperonylic acid (0.57 g, 3.45 mmol) in
thionyl chloride (5 ml) was refluxed in the absence of moisture for
2 hours when a clear solution had been formed. The excess thionyl
chloride was removed by evaporation in vacuo to leave the acid
chloride as an off-white solid.
[0407] A solution of the acid chloride in dichloromethane (20 ml)
was added to a solution of 3-fluoro-4,5-dimethoxy-aniline (7) (0.45
g, 3.3 mmol) in dichloromethane (5 ml) and then pyridine (1 ml) was
added. The mixture was refluxed together for 3 hours, then left at
RT overnight. The resultant brown solution was diluted with
dichloromethane (100 ml) and methanol (15 ml), washed with
hydrochloric acid (1M, 50 ml) then dried and evaporated in vacuo to
give the product as a brown solid. Purification by column
chromatography over silica gel eluting with 10 to 20% ethyl acetate
in DCM gave amide (9) as an off-white solid (400 mg, 43%).
[0408] M/z found 342.0751. C.sub.16H.sub.14FNNaO.sub.5 requires
342.0748.
[0409] .sup.1H NMR (CDCl.sub.3, 300 MHz) 7.58 (1H, bs, NH), 7.36
(1H, dd, J 2, 9 Hz, H6), 7.33 (1H, d, J 2 Hz, H2), 7.18 (1H, t, J 2
Hz, H6'), 6.93 (1H, dd, J 2, 13 Hz, H2'), 6.87 (1H, d, J 9 Hz, H5),
6.06 (2H, s), 3.90 (3H, s) and 3.89 (3H, s).
##STR00060##
Step C2: Preparation of
3,4-Methylenedioxy-N-(3,4-dimethoxy-2-fluorophenyl)benzamide
(10)
[0410] A suspension of piperonylic acid (0.57 g, 3.45 mmol) in
thionyl chloride (5 ml) was refluxed in the absence of moisture for
2 hours when a clear solution had been formed. The excess thionyl
chloride was removed by evaporation in vacuo to leave the acid
chloride as an off-white solid.
[0411] A solution of the acid chloride in dichloromethane (20 ml)
was added to a solution of 2-fluoro-3,4-dimethoxy-aniline (8) (0.45
g, 3.3 mmol) in dichloromethane (5 ml) and then pyridine (1 ml) was
added. The mixture was refluxed together for 3 hours, then left at
RT overnight. The resultant brown solution was diluted with
dichloromethane (100 ml) and methanol (15 ml), washed with
hydrochloric acid (1M, 50 ml) then dried and evaporated in vacuo to
give the product as a brown solid. Purification by column
chromatography over silica gel eluting with 10 to 20% ethyl acetate
in DCM gave amide (10) as an off-white solid (430 mg, 46%).
[0412] .sup.1H NMR (CDCl.sub.3, 300 MHz) 7.97 (1H, t, J 9 Hz, H6'),
7.71 (1H, bs, NH), 7.40 (1H, bd, J 9 Hz, H6), 7.36 (1H, bs, H2),
6.88 (1H, d, J 9 Hz, H5), 6.70 (1H, bd, J 10 Hz, H5'), 6.06 (2H,
s), 3.94 (3H, s) and 3.87 (3H, s).
##STR00061##
Step D1: Preparation of
3,4-Dihydroxy-N-(3-fluoro-4,5-dihydroxyphenyl)benzamide
(DC51-F4)
[0413] To a solution of amide (9) (400 mg, 1.25 mmol) in dry
dichloromethane (30 ml) at RT was added boron tribromide (1 ml). A
pale green solution was first formed with a light white
precipitate. This suspension was left at R. for 3 h, then methanol
(dropwise then 20 ml) was added carefully and the resultant brown
solution left at RT overnight, when a yellow solid was formed.
[0414] The solvents were removed in vacuo to leave about 1 ml, then
methanol (dropwise then 30 ml) was added carefully. This was
repeated 4 times, then solvents removed in vacuo to leave the
product as an off-white solid. The solid was dissolved in a minimum
amount of methanol, then dichloromethane added until just cloudy.
The mixture was left in the fridge overnight when (DC51-F4) was
formed as an off white crystalline solid (271 mg, 81%).
[0415] HPLC 22.44 minutes.
[0416] M/z found 278.0455. C.sub.13H.sub.9FNO.sub.5 requires
278.0470.
[0417] .sup.1H NMR (CD.sub.3OD, 300 MHz) 7.34 (1H, d, J 2 Hz, H2),
7.29 (1H, dd, J 2, 9 Hz, H6), 6.97 (1H, bs, H6'), 6.96 (1H, dd, J
2, 11 Hz, H2') and 6.82 (1H, d, J 9 Hz, H5).
##STR00062##
Step D2: Preparation of 3,4-Dihydroxy-N-(2-fluoro-3,4-dihydroxy
phenyl)benzamide (DC51-F6)
[0418] To a solution of amide (10) (430 mg, 1.34 mmol) in dry
dichloromethane (30 ml) at RT was added boron tribromide (1 ml). A
pale orange solution was first formed with a light white ppt. This
suspension was left at RT for 3 h, then methanol (dropwise then 20
ml) was added carefully and the resultant brown solution left at RT
overnight, when a yellow solid was formed.
[0419] The solvents were removed in vacuo to leave about 1 ml, then
methanol (dropwise then 30 ml) was added carefully. This was
repeated 4 times, then solvents removed in vacuo to leave the
product as an off-white solid. The solid was dissolved in a minimum
amount of methanol, then dichloromethane added until just cloudy.
The mixture was left in the fridge overnight when the product was
formed as a brown crystalline solid (241 mg, 67%).
[0420] HPLC 12.13 minutes.
[0421] M/z found 278.0490. C.sub.13H.sub.9FNO.sub.5 requires
278.0470.
[0422] .sup.1H NMR (CD.sub.3OD, 300 MHz) 7.38 (1H, d, J 2.5 Hz,
H2), 7.33 (1H, dd, J 2.5, 9 Hz, H6), 6.83 (1H, d, J 9 Hz, H5), 6.81
(1H, t, J 9 Hz, H6') and 6.58 (1H, dd, J 2.5, 9 Hz, H5').
##STR00063##
Example 17
Overview of Synthesis of
N-(3,4-dihydroxyphenyl)-2-fluoro-4,5-dihydroxy benzamide
(DC51-F2)
[0423] Oxidation of commercially available 6-fluoroveratraldehyde
with `Jones reagent`.sup.6 gave the acid 11 in good yield.
Formation of the acid chloride from this and condensation with
3,4-methylenedioxyaniline then gave the anilide 12. Deprotection
with boron tribromide under standard conditions gave the free
phenolic anilide, DC51-F2 in good yield.
##STR00064##
Step A: Preparation of 2-fluoro-4,5-dimethoxybenzoic acid (11)
[0424] To a stirred solution of 2-fluoro-4,5-dimethoxybenzaldehyde
(0.46 g, 2.5 mmol) in acetone (20 ml) was added Jones reagent (6
ml) dropwise and the mixture stirred at RT for 4 h (P. B. Wakchaure
et al. Tetrahedron 2008, 64, 1786-1791.). The mixture was diluted
with water, extracted into ethyl acetate then dried and evaporated
in vacuo to give the crude acid. Recrystallisation from ethyl
acetate/pet ether (40-60) gave the pure acid (11) as a brown
crystalline solid (H. B. Stegmann et al. J.C.S. Perkin Trans 2,
1994, 547-555).
[0425] .sup.1H NMR (CDCl.sub.3, 300 MHz) 7.43 (1H, d, J 7 Hz, H6),
6.66 (1H, d, 13 Hz, H3), 3.93 (3H, s) and 3.90 (3H, s).
##STR00065##
Step B: Preparation of 4,5-Dimethoxy-2-fluoro-N-(3,4-methylene
dioxy phenyl)benzamide (12)
[0426] A suspension of 2-fluoro-4,5-dimethoxybenzoic acid (11)
(0.40 g, 2.0 mmol) in thionyl chloride (5 ml) was refluxed in the
absence of moisture for 1 hour when a clear solution had been
formed. The excess thionyl chloride was removed by evaporation in
vacuo to leave the acid chloride as an off-white solid.
[0427] A solution of the acid chloride in dichloromethane (15 ml)
was added to a solution of 3,4-methylenedioxyaniline (0.33 g, 2.4
mmol) in dichloromethane (5 ml) and then pyridine (0.5 ml) was
added. The mixture was refluxed together for 2 hours, then left at
RT overnight. The resultant brown solution was diluted with
DCM/MeOH (5:1) (50 ml) washed with hydrochloric acid (1M, 25 ml)
then dried and evaporated in vacuo to give the product as a brown
solid. The product was columned over silica gel when 10 to 20%
ethyl acetate in DCM gave amide (12) as an off-white solid (550 mg,
86%).
[0428] M/z found 342.0759. C.sub.16H.sub.14FNNaO.sub.5 requires
342.0748.
[0429] .sup.1H NMR ((CD.sub.3).sub.2CO, 300 MHz) 9.13 (1H, bs, NH),
7.62 (1H, bs, H2'), 7.48 (1H, d, J 8 Hz, H6), 7.24 (1H, bd, J 8 Hz,
H6'), 7.01 (1H, d, J 13 Hz, H3), 6.92 (1H, d, J 9 Hz, H5'), 6.10
(2H, s), 4.01 (3H, s) and 3.96 (3H, s).
##STR00066##
Step C: Preparation of 4,5-Dihydroxy-2-fluoro-N-(3,4-dihydroxy
phenyl)benzamide (DC51-F2)
[0430] To a solution of amide (12) (500 mg, 1.6 mmol) in dry
dichloromethane (30 ml) at RT was added boron tribromide (1 ml). A
pale orange solution was first formed with a light white ppt. This
suspension was left at RT for 3 h, then methanol (dropwise then 20
ml) was added carefully and the resultant brown solution left at RT
overnight, when a yellow solid was formed.
[0431] The solvents were removed in vacuo to leave about 1 ml, then
methanol (dropwise then 30 ml) was added carefully. This was
repeated 4 times, and then solvents removed in vacuo to leave the
product as an off-white solid. The solid was dissolved in a minimum
amount of methanol, and then dichloromethane added until just
cloudy. The mixture was left in the fridge overnight when (DC51-F2)
was formed as an off white crystalline solid (320 mg, 73%).
[0432] HPLC 25.71 minutes.
[0433] M/z found 278.0488. C.sub.13H.sub.9FNO.sub.5 requires
278.0470.
[0434] .sup.1H NMR (CD.sub.3OD, 300 MHz) 7.17-7.19 (2H, m, H6,
H2'), 6.84 (1H, dd, J 2.5, 9 Hz, H6'), 6.72 (1H, d, J 9 Hz, H5')
and 6.60 (1H, d, J 13 Hz, H3).
##STR00067##
Example 18
Overview of Synthesis of
N-(3,4-dihydroxyphenyl)-2-fluoro-3,4-dihydroxy benzamide
(DC51-F1)
[0435] Aldehyde 13 could be prepared, as described previously by
Ladd et al. using a reaction of hexamethylenetetramine and
trifluoroacetic acid (D. L. Ladd et al. J. Org. Chem. 1981, 46 (1),
203-206). Oxidation of aldehyde 13 gave the benzoic acid 14, then
formation of the acid chloride from this and condensation with
3,4-methylenedioxyaniline gave the anilide 15. Deprotection with
boron tribromide under standard conditions gave the free phenolic
anilide, DC51-F1 in good yield.
##STR00068##
Step A: Preparation of 1-Fluoro-2,3-dimethoxybenzene (4)
[0436] A suspension of 3-fluoro-catechol (3.7 g, 29 mmol) in
acetone (30 ml) with potassium carbonate (12 g, 87 mmol) and methyl
iodide (10 ml, 160 mmol) was stirred at RT for 24 h. The mixture
was diluted with ethyl acetate, filtered, washed with water, dried
and evaporated in vacuo to give the product as a pale yellow liquid
(3.42, 76%).
[0437] .sup.1H NMR (CDCl.sub.3, 300 MHz) 7.00-6.90 (1H, m),
6.76-6.64 (1H, m), 3.91 (3H, d, 1 Hz) and 3.86 (3H, s).
Step B: Preparation of 2-fluoro-3,4-dimethoxybenzaldehyde (13)
[0438] To a heated solution of hexamethylenetetraamine (2.2 g, 16
mmol) in TFA (10 ml) at 80.degree. C. was added dropwise
3-fluoroveratrole (1.20 g, 7.8 mmol) in TFA (10 ml). The mixture
was heated for an additional hour, then concentrated in vacuo.
[0439] Ice/water (30 ml) was added until the mixture turned cloudy,
the mixture was stirred for 15 mins then made basic with solid
sodium carbonate, stirred for 20 mins then extracted into ethyl
acetate, washed with water dried and evaporated in vacuo.
[0440] Purification by column chromatography over silica gel
eluting with ethyl acetate (0-50%) in dichloromethane gave aldehyde
(13) (D. L. Ladd et al. Syn. Comm, 1985, 15 (1), 61-69) as a
colourless gum (0.96 g, 68%).
[0441] .sup.1H NMR (CDCl.sub.3, 300 MHz) 10.19 (1H, s, CHO), 7.58
(1H, dd, J 7, 9 Hz, H6), 6.79 (1H, d, 9 Hz, H5) and 3.95 (6H,
s).
##STR00069##
Step C: Preparation of 2-Fluoro-3,4-dimethoxybenzoic acid (14)
[0442] To a stirred solution of 2-fluoro-3,4-dimethoxybenzaldehyde
(13) (0.46 g, 2.5 mmol) in acetone (20 ml) was added Jones reagent
(6 ml) dropwise and the mixture stirred at RT for 4 h..sup.6 The
mixture was diluted with water, extracted into ethyl acetate then
dried and evaporated in vacuo to give the crude acid.
Recrystallisation from ethyl acetate/pet ether (40-60) gave the
pure acid (14) as a brown crystalline solid (H. B. Stegmann et al.
J.C.S. Perkin Trans 2, 1994, 547-555).
[0443] .sup.1H NMR ((CD.sub.3).sub.2CO, 300 MHz) 7.80 (1H, dd, J 7,
8 Hz, H6), 7.07 (1H, d, 8 Hz, H5), 4.06 (3H, s) and 3.95 (3H,
s).
##STR00070##
Step D: Preparation of 3,4-Dimethoxy-2-fluoro-N-(3,4-methylene
dioxyphenyl)benzamide (15)
[0444] A suspension of 2-fluoro-3,4-dimethoxybenzoic acid (14)
(0.50 g, 2.5 mmol) in thionyl chloride (5 ml) was refluxed in the
absence of moisture for 1 hour when a clear solution had been
formed. The excess thionyl chloride was removed by evaporation in
vacuo to leave the acid chloride as an off-white solid.
[0445] A solution of the acid chloride in dichloromethane (15 ml)
was added to a solution of 3,4-methylenedioxyaniline (0.41 g, 3.0
mmol) in dichloromethane (5 ml) and then pyridine (0.5 ml) was
added. The mixture was refluxed together for 2 hours, then left at
RT overnight. The resultant brown solution was diluted with
DCM/MeOH (5:1) (50 ml) washed with hydrochloric acid (1M, 25 ml)
then dried and evaporated in vacuo to give the product as a brown
solid. The product was columned over silica gel when 10 to 20%
ethyl acetate in DCM gave amide (15) as an off-white solid (630 mg,
79%).
[0446] M/z found 342.0760. C.sub.16H.sub.14FNNaO.sub.5 requires
342.0748.
[0447] .sup.1H NMR ((CD.sub.3).sub.2CO, 300 MHz) 9.22 (1H, bs, NH),
7.66-7.60 (2H, m, H2', H6), 7.25 (1H, bd, J 8 Hz, H6'), 7.09 (1H,
bd, J 9 Hz, H5), 6.92 (1H, d, J 9 Hz, H5'), 6.09 (2H, s), 4.01 (3H,
s) and 3.96 (3H, s).
##STR00071##
Step E: Preparation of
3,4-Dihydroxy-2-fluoro-N-(3,4-dihydroxyphenyl)benzamide
(DC51-F1)
[0448] To a solution of amide (15) (500 mg, 1.6 mmol) in dry
dichloromethane (30 ml) at r.t. was added boron tribromide (1 ml).
A pale orange solution was first formed with a light white ppt.
This suspension was left at RT for 3 h, then methanol (dropwise
then 20 ml) was added carefully and the resultant brown solution
left at RT overnight, when a yellow solid was formed.
[0449] The solvents were removed in vacuo to leave about 1 ml, then
methanol (dropwise then 30 ml) was added carefully. This was
repeated 4 times, then solvents removed in vacuo to leave the
product as an off-white solid. The solid was dissolved in a minimum
amount of methanol, then dichloromethane added until just cloudy.
The mixture was left in the fridge overnight when (DC51-F1) was
formed as a brown crystalline solid (283 mg, 65%).
[0450] HPLC 15.10 minutes.
[0451] M/z found 278.0488. C.sub.13H.sub.9FNO.sub.5 requires
278.0470.
[0452] .sup.1H NMR (CD.sub.3OD, 300 MHz) 7.21 (1H, d, J 2.5, H2'),
7.09 (1H, t, J 9 Hz, H6), 6.85 (1H, dd, J 2.5, 9 Hz, H6'), 6.72
(1H, d, J 9 Hz, H5') and 6.66 (1H, dd, J 1, 9 Hz, H5).
##STR00072##
Example 19
Overview of Synthesis of
N-(3,4-dihydroxyphenyl)-3-fluoro-4,5-dihydroxy benzamide
(DC51-F3)
[0453] Work by Clark (M. T. Clark and D. D. Miller. J. Org. Chem.
1986, 51, 4072-4073) showed that the required formyl product can be
prepared in a three step process from 2-fluoro-6-methoxyphenol,
which in turn can be prepared from commercially available
3-fluoroanisole. Within the timeframe of the work, 3-fluoroanisole
was unavailable, so we looked for an alternative route to a
2-fluoro-6-alkoxyphenol.
[0454] Alkylation of 3-fluorocatechol with one equivalent of benzyl
bromide (benzyl chosen as a bulkier group than methyl which should
give a greater proportion of the monoalkylated products) gave a 2:1
mixture of mono and di-benzylated products. Separation of these
gave the two mono-benzylated products 16b and 16c as an inseparable
mixture. Treatment of this mixture under the formylation conditions
used for 2-fluoro-6-methoxyphenol (M. T. Clark and D. D. Miller. J.
Org. Chem. 1986, 51, 4072-4073) gave two readily separated
aldehydes 17b and 17c. Comparison of their NMR spectra with
previously formed compounds showed them to be the two expected
products. Methylation.sup.4 of the phenol gave the methoxide 18
which upon oxidation then gave the acid 19. Formation of the acid
chloride from this and condensation with 3,4-methylenedioxyaniline
gave the anilide 20. Deprotection with boron tribromide under
standard conditions gave the free phenolic anilide, DC51-F3
although in poor overall yield.
##STR00073##
Step A: Preparation of 2,3-Dibenzyloxy-1-fluorobenzene,
6-benzyloxy-2-fluorophenol and 2-benzyloxy-3-fluorophenol (16a)
(16b) and (16c)
[0455] A mixture of 3-fluorocatachol (2.0 g, 16 mmol), benzyl
bromide (2.67 g, 15.6 mmol) sodium iodide (1.2 g, 7.8 mmol) and
potassium carbonate (6 g, 47 mmol) in acetonitrile (100 ml) was
sonicated for 5 minutes, then stirred at RT overnight. Water (100
ml) was added, then the solution made acidic with 3M HCl and
extracted with ethyl acetate, washed with brine, dried and
evaporated in vacuo to give an orange gum. Separation by column
chromatography over silica gel eluting with DCM/Pet ether (1:1)
gave 2,3-Dibenzyloxy-1-fluorobenzene (16a) as a colourless oil (1.2
g, 25%) followed by a mixture of 6-benzyloxy-2-fluorophenol (16b)
and 2-benzyloxy-3-fluorophenol (16c) as a colourless oil (2.05 g,
60%).
[0456] .sup.1H NMR (CDCl.sub.3, 300 MHz) 7.42-7.35 (5H, m, Bn),
6.93-6.84 (1H, m), 6.73-6.60 (2H, m), 5.57 (1H, s, OH), 5.14 (1H,
s) and 5.13 (1H, s).
##STR00074##
Step B: Preparation of 6-Benzyloxy-2-fluorophenol (17b)
[0457] The mixture of phenols (16b) and (16c) (2.0 g, 9.2 mmol) was
added to a solution of 33% dimethylamine (2.5 g, 18 mmol) and 37%
formaldehyde (1.5 ml. 18 mmol) in absolute ethanol (15 ml). The
mixture was heated at reflux for 2 h, cooled and evaporated to give
a solid. Iodomethane (18 ml, 145 mmol) was added to a solution of
this solid in chloroform (60 ml) and the mixture stirred for 18 h
at RT. The white solid was filtered off then heated to 120.degree.
C. in acetic acid (6 ml) and water (6 ml), when HMTA (1.5 g, 11
mmol) was added to the mixture. The mixture was stirred at
120.degree. C. for 2 h then c.HCl (0.25 ml) was added and heating
continued for 5 mins. The cooled mixture was extracted into ether,
dried and evaporated in vacuo. Purification by column
chromatography over silica gel eluting with 0-100% DCM in pet ether
(40-60) gave aldehyde (17b) as a white solid (0.25 g, 11%).
[0458] .sup.1H NMR (CDCl.sub.3, 300 MHz) 9.76 (1H, d, J 1 Hz, CHO),
7.45-7.37 (5H, m, Bn), 7.33 (1H, t, J 2 Hz, H6), 7.29 (1H, dd, J 2,
11 Hz, H2) and 5.19 (2H, s).
##STR00075##
Step C: Preparation of
5-Benzyloxy-3-fluoro-4-methoxybenzaldehyde.sup.9 (18)
[0459] A suspension of phenol (17b) (250 mg, 29 mmol) in acetone
(10 ml) with potassium carbonate (1 g, 7 mmol) and methyl iodide (2
g, 14 mmol) was stirred at RT for 24 h. The mixture was diluted
with ethyl acetate, filtered, washed with water, dried and
evaporated in vacuo to give the product as a pale yellow liquid
(250 mg, 95%).
##STR00076##
Step D: Preparation of 5-Benzyloxy-3-fluoro-4-methoxybenzoic acid
(19)
[0460] To a stirred solution of aldehyde (18) (0.25 g, 1.02 mmol)
in acetone (20 ml) was added Jones reagent (6 ml) dropwise..sup.6
The mixture was stirred at RT for 4 h then diluted with water,
extracted into ethyl acetate then dried and evaporated in vacuo to
give the crude acid (250 mg, 94%).
##STR00077##
Step E: Preparation of
5-Benzyloxy-3-fluoro-4-methoxy-N-(3,4-methylene dioxy
phenyl)benzamide (20)
[0461] A suspension of 5-benzyloxy-3-fluoro-4-methoxybenzoic (19)
(0.25 g, 0.9 mmol) in thionyl chloride (5 ml) was refluxed in the
absence of moisture for 1 hour when a clear solution had been
formed. The excess thionyl chloride was removed by evaporation in
vacuo to leave the acid chloride as an off-white solid.
[0462] A solution of the acid chloride in dichloromethane (15 ml)
was added to a solution of 3,4-methylenedioxyaniline (0.15 g, 1.1
mmol) in dichloromethane (5 ml) and then pyridine (0.5 ml) was
added. The mixture was refluxed together for 2 hours, then left at
RT overnight. The resultant brown solution was diluted with
DCM/MeOH (5:1) (50 ml) washed with hydrochloric acid (1M, 25 ml)
then dried to give the product as a brown solid. The product was
columned over silica gel when 10 to 20% ethyl acetate in DCM gave
amide (20) as an off-white solid (320 mg, 89%).
[0463] .sup.1H NMR (CDCl.sub.3, 300 MHz) 7.48-7.33 (5H, m, Bn),
7.32-7.29 (2H, m, H6, H2'), 7.16 (1H, dd, J 2, 12 Hz, H2), 6.85
(1H, dd, J 2, 9 Hz, H6'), 6.77 (1H, d, J 9 Hz, H5'), 5.97 (2H, s),
5.19 (2H, s) and 4.00 (3H, s).
##STR00078##
Step F: Preparation of
4,5-Dihydroxy-3-fluoro-N-(3,4-dihydroxyphenyl)benzamide
(DC51-F3)
[0464] To a solution of amide (20) (340 mg, 0.86 mmol) in dry
dichloromethane (30 ml) at RT was added boron tribromide (1 ml). A
pale orange solution was first formed with a light white ppt. This
suspension was left at RT for 3 h, then methanol (dropwise then 20
ml) was added carefully and the resultant brown solution left at RT
overnight, when a yellow solid was formed.
[0465] The solvents were removed in vacuo to leave about 1 ml, then
methanol (dropwise then 30 ml) was added carefully. This was
repeated 4 times, then solvents removed in vacuo to leave the
product as a brown solid. The solid was freeze dried to give
(DC51-F3) as a brown solid (229 mg, 95%).
[0466] HPLC 20.58 minutes.
[0467] M/z found 280.0600. C.sub.13H.sub.11FNO.sub.5 requires
280.0616.
[0468] .sup.1H NMR (CD.sub.3OD, 300 MHz) 7.21 (1H, dd, J 2.5, 12
Hz, H2), 7.20 (1H, bs, H2'), 7.16 (1H, d, J 2.5 Hz, H6), 6.85 (1H,
dd, J 2.5, 9 Hz, H6') and 6.72 (1H, d, J 9 Hz, H5').
##STR00079##
[0469] Two alternative preparations of DC51-F3 could start either
from 3-fluoroanisole, to form 3-fluoro-4,5-dimethoxybenzaldehyde or
(as shown below) introduce the fluoride onto the benzene ring at a
later stage. One possibility would be the use of select fluor on
compound (21) where there is just one free ortho or para position.
After methylation the nitrile group could be hydrolysed to the
required acid.
##STR00080##
Example 20
Compounds Provided Herein are Potent Disrupters of Alzheimer's Ad
1-42 Fibrils
[0470] The compounds prepared in the preceding Examples were found
to be potent disrupters/inhibitors of Alzheimer's disease A.beta.
fibrils. In a set of studies, the efficacy of certain compounds
provided herein to cause a disassembly/disruption of pre-formed
amyloid fibrils of Alzheimer's disease (i.e. consisting of A.beta.
1-42 fibrils) was analyzed.
Part A--Thioflavin T Fluorometry Data
[0471] In one study, Thioflavin T fluorometry was used to determine
the effects of the compounds, and of EDTA (as a negative control).
In this assay Thioflavin T binds specifically to fibrillar amyloid,
and this binding produces a fluorescence enhancement at 485 nm that
is directly proportional to the amount of amyloid fibrils formed.
The higher the fluorescence, the greater the amount of amyloid
fibrils formed (Naki et al., Lab. Invest. 65:104-110, 1991; Levine
III, Protein Sci. 2:404-410, 1993; Amyloid:Int. J. Exp. Clin.
Invest. 2:1-6, 1995).
[0472] In this study, 25 .mu.M of pre-fibrillized A.beta. 1-42
(Bachem Inc) was incubated at 37.degree. C. for 3 days, either
alone, or in the presence of one of the compounds or EDTA (at
A.beta.:test compound weight ratios of 1:1, 1:0.1, 1:0.01 or
1:0.001). Following 3 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 mM Thioflavin T in 250 mM 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).
[0473] The results of the 3-day incubations are presented below.
For example, whereas EDTA caused no significant
inhibition/disruption of A.beta. 1-42 fibrils at all concentrations
tested, the compounds (DC-0051, DC-0051-S1, S3, S4, S5, S6, S7, S8
and S9) all caused a dose-dependent disruption/disassembly of
preformed A.beta. 1-42 fibrils to some extent (Table 1). For
example, compound DC-0051-S8 caused a significant (p<0.01)
87.9+/-0.78% inhibition when used at an A.beta.:test compound wt/wt
ratio of 1:0.1, and a significant 56.0+/-11.32% inhibition when
used at an A.beta.:test compound wt/wt ratio of 1:0.01 (Table 1).
Under the same conditions (i.e. A.beta.:test compound wt/wt ratio
of 1:0.01), compound DC-0051 caused an 89.5+/-3.26% disruption,
compound DC-0051-S5 caused an 80.0+/-0.63% disruption, and compound
DC-0051-S9 caused an 84.1+/-4.28% disruption. This study indicated
that the compound provided herein are disrupters/inhibitors of
Alzheimer's disease type A.beta. fibrils, and usually exert their
effects in a dose-dependent manner.
TABLE-US-00002 TABLE 1 Thioflavin T Fluorometry Data- Disruption of
A.beta. 1-42 Fibrils by Test Compounds Test Compound # 1:1 (wt/wt)
1:0.1 (wt/wt) 1:0.01 (wt/wt) 1:0.001 (wt/wt) EDTA (control) 0.0
.+-. 3.59% 0.0 .+-. 4.41% 0.2 .+-. 3.03% 0.0 .+-. 1.54% DC-0051
98.7 .+-. 0.07% 89.5 .+-. 3.26% 32.0 .+-. 4.31% 10.9 .+-. 2.24%
DC0051-S1 96.4 .+-. 0.58% 74.6 .+-. 3.71% 20.8 .+-. 4.63% 9.0 .+-.
3.53% DC0051-S3 92.5 .+-. 0.47% 59.9 .+-. 1.34% 16.1 .+-. 2.04%
14.6 .+-. 2.90% DC0051-S4 95.2 .+-. 0.42% 70.2 .+-. 7.01% 18.2 .+-.
3.68% 16.6 .+-. 4.14% DC0051-S5 99.0 .+-. 0.25% 80.0 .+-. 0.63%
28.4 .+-. 0.74% 20.3 .+-. 6.71% DC0051-S6 95.4 .+-. 0.72% 53.5 .+-.
14.88% 4.0 .+-. 4.33% 9.5 .+-. 1.64% DC0051-S7 92.8 .+-. 1.92% 50.2
.+-. 6.94% 10.1 .+-. 5.82% 13.4 .+-. 3.42% DC0051-S8 96.7 .+-.
0.73% 87.9 .+-. 0.78% 56.0 .+-. 11.32% 32.9 .+-. 2.70% DC0051-S9
98.8 .+-. 0.26% 84.1 .+-. 4.28% 60.7 .+-. 12.57% 13.6 .+-. 2.08% (%
inhibition of A.beta.; for A.beta.:test compound at given wt/wt
ratio)
Part B: SDS-PAGE/Western Blot Data
[0474] The disruption of A.beta. 1-42, even in its monomeric form,
was confirmed by a study involving the use of SDS-PAGE and Western
blotting methods (not shown). In this latter study, triplicate
samples of pre-fibrillized A.beta. 1-42 (25 .mu.M) was incubated at
37.degree. C. for 3 days, alone or in the presence of the compounds
or EDTA. Five micrograms of each sample was then filtered through a
0.2 .mu.m filter. Protein recovered from the filtrate was then
loaded, and ran on a 10-20% Tris-Tricine SDS-PAGE, blotted to
nitrocellulose and detected using an A.beta.-antibody (clone 6E10;
Senetek). In this study, A.beta. 1-42 was detected as a 4
kilodalton band (i.e. monomeric A.beta.) following incubation
alone, or in the presence of EDTA, at 3 days. For example, A.beta.
1-42 monmers were not detected following incubation of A.beta. 1-42
with compounds DC-0051, DC-0051-S1, DC-0051-S5, DC-0051-S8 and
DC-0051-S9, correlating nicely with the Thioflavin T fluorometry
data (described above) and suggesting that these compounds were
capable of causing a disappearance of monomeric A.beta. 1-42. This
study confirms that these compounds are also capable of causing a
disruption/removal of monomeric A.beta. 1-42.
Part C: Congo Red Binding Data
[0475] In the Congo red binding assay the ability of a test
compound to alter amyloid (in this case, A.beta.) binding to Congo
redc is quantified. In this assay, A.beta. 1-42 and test compounds
were incubated for 3 days and then vacuum filtered through a 0.2
.mu.m filter. The amount of A.beta. 1-42 retained in the filter was
then quantified 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) was indicative of the test compound's
ability to diminish/alter the amount of aggregated and congophilic
A.beta.. This particular assay appears to be more stringent in
character than Thioflavin T fluorometry, and it is more difficult
to remove Congo red binding to A.beta. 42 fibrils than assessed by
other assays, so the % inhibitions observed even with potent
compounds is usually lower than that observed as determined by
other assays such as Thioflavin T fluometry.
[0476] In one study, the ability of A.beta. fibrils to bind Congo
red in the absence or presence of increasing amounts of the
compounds or EDTA (at A.beta.:test compound weight ratios of
1:0.001, 1:0.01, 1:0.1 and 1:1) was determined. The results of the
3-day incubations are presented in Table 2 below. Wheras EDTA
caused no significant inhibition of A.beta. 1-42 fibril binding to
Congo red, the compounds (DC-0051, DC-0051-S1, S3, S4, S5, S6, S7,
S8 and S9) caused a dose-dependent inhibition of A.beta. binding to
Congo red (Table 2). For example, compound DC-0051-S5 caused a
significant 82.3+/-0.59% inhibition of Congo red binding to A.beta.
1-42 fibrils when used at an A.beta.:test compound wt/wt ratio of
1:1, and a 40.3+/-5.81% inhibition when used an A.beta.:test
compound wt/wt ratio of 1:0.1 (Table 2). Other good inhibitors as
compared to the A.beta.:test compound wt/wt ratio (of 1:0.1
appeared to be DC-0051-S1 (19.7+/-2.97% inhibition), DC-0051
(40.3+/-5.81% inhibition), DC-0051-S6 (17.1+/-4.94% inhibition),
DC-0051-S8 (19.8+/-2.43% inhibition) and DC-0051-S9 (17.4+/-6.11%
inhibition).
TABLE-US-00003 TABLE 2 Congo red Binding Data- Disruption of
A.beta. 1-42 Fibrils by Test Compounds Test Compound # 1:1 (wt/wt)
1:0.1 (wt/wt) 1:0.01 (wt/wt) 1:0.001 (wt/wt) EDTA (control) 3.9 +/-
1.37% 0.0 .+-. 0.76% 0.0 +/- 0.49% 0.0 .+-. 0.62% DC-0051 76.7 +/-
1.22% 40.3 +/- 5.81% 3.3 +/- 0.95% 1.7 +/- 0.10% DC0051-S1 48.6 +/-
2.01% 19.7 +/- 2.97% 2.4 +/- 0.92% 1.0 +/- 2.11% DC0051-S3 36.2 +/-
2.51% 16.6 +/- 1.87% 0.0 +/- 2.11% 0.0 +/- 2.12% DC0051-S4 48.8 +/-
2.29% 15.1 +/- 4.17% 0.0 +/- 2.13% 1.5 +/- 1.42% DC0051-S5 82.3 +/-
0.59% 17.5 +/- 1.23% 0.2 +/- 1.97% 0.0 +/- 1.37% DC0051-S6 48.5 +/-
3.58% 17.1 +/- 4.94% 2.1 +/- 1.14% 3.1 +/- 0.97% DC0051-S7 44.6 +/-
4.59% 8.8 +/- 1.70% 0.0 +/- 0.29% 2.4 +/- 2.23% DC0051-S8 41.2 +/-
6.83% 19.8 +/- 2.43% 3.9 +/- 0.54% 2.3 +/- 3.16% DC0051-S9 60.8 +/-
2.12% 17.4 +/- 6.11% 3.8 +/- 3.90% 0.0 +/- 1.27% (% inhibition of
A.beta.; for A.beta.:test compound at given wt/wt ratio)
Example 21
Additional Compounds Provided Herein are Potent Disrupters of
Alzheimer's A.beta. 1-42 Fibrils
[0477] The compounds prepared in the preceding Examples were found
to be potent disrupters/inhibitors of Alzheimer's disease A.beta.
fibrils. In another set of studies, the efficacy of certain
compounds provided herein (and referred to as DC-0051-B2,
DC-0051-B3 and DC-0051-B4) to cause a disassembly/disruption of
pre-formed amyloid fibrils of Alzheimer's disease (i.e. consisting
of A.beta. 1-42 fibrils) was analyzed.
Thioflavin T Fluorometry Data
[0478] In one study, Thioflavin T fluorometry was used to determine
the effects of the compounds, and of EDTA (as a negative control).
In this assay Thioflavin T binds specifically to fibrillar amyloid,
and this binding produces a fluorescence enhancement at 485 nm that
is directly proportional to the amount of amyloid fibrils formed.
The higher the fluorescence, the greater the amount of amyloid
fibrils formed (Naki et al., Lab. Invest. 65:104-110, 1991; Levine
III, Protein Sci. 2:404-410, 1993; Amyloid:Int. J. Exp. Clin.
Invest. 2:1-6, 1995).
[0479] In this study, 25 .mu.M of pre-fibrillized A.beta. 1-42
(Bachem Inc) was incubated at 37.degree. C. for 3 days, either
alone, or in the presence of one of the compounds (DC-0051-B2,
DC-0051-B3 or DC-0051-B4). Following 3 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 mM Thioflavin T in 250
mM 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).
[0480] The results of the 3-day incubations are presented below.
For example, whereas EDTA caused no significant
inhibition/disruption of A.beta. 1-42 fibrils at all concentrations
tested, the compounds (DC-0051-B2, DC-0051-B3, and DC-0051-B4) all
caused a dose-dependent disruption/disassembly of preformed A.beta.
1-42 fibrils to some extent (Table 3). The most efficacious
compounds to disrupt pre-formed A.beta. 1-42 fibrils as assessed by
the Thioflavin T fluometry assay appeared to be DC-0051-B2. For
example, compound DC-0051-B2 caused a significant (p<0.01)
65.8+/-2.01% inhibition when used at an A.beta.:test compound wt/wt
ratio of 1:0.1, and a significant 85.5+/-1.27% inhibition when used
at an A.beta.:test compound wt/wt ratio of 1:1 (Table 3). This
study indicated that the additional compounds provided herein are
disrupters/inhibitors of Alzheimer's disease type A.beta. fibrils,
and usually exert their effects in a dose-dependent manner.
TABLE-US-00004 TABLE 3 Thioflavin T Fluorometry Data- Disruption of
A.beta. 1-42 Fibrils by Test Compounds Test Compound # 1:1 (wt/wt)
1:0.1 (wt/wt) 1:0.01 (wt/wt) 1:0.001 (wt/wt) EDTA (control) 5.0 +/-
11.39% 0.0 +/- 1.18% 0.0 +/- 2.26% 10.3 +/- 10.81% DC-0051 98.5 +/-
0.56% 88.8 +/- 0.76% 41.1 +/- 2.52% 18.6 +/- 8.89% DC0051-B2 85.5
+/- 1.27% 65.8 +/- 2.01% 19.2 +/- 6.18% 10.2 +/- 9.49% DC0051-B3
17.9 +/- 16.85% 22.2 +/- 2.63% 1.0 +/- 1.62% 15.7 +/- 7.34%
DC0051-B4 28.1 +/- 3.06% 21.1 +/- 4.00% 3.6 +/- 4.96% 17.2 +/-
4.32% (% inhibition of A.beta.; for A.beta.:test compound at given
wt/wt ratio)
Example 22
Compounds Provided Herein are Potent Disrupters of Type 2 Diabetes
IAPP Fibrils
[0481] The compounds prepared in the preceding Examples were also
found to be potent disrupters/inhibitors of type 2 diabetes IAPP
fibrils. In a set of studies, the efficacy of certain compounds
provided herein to cause a disassembly/disruption of pre-formed
amyloid fibrils of type 2 diabetes (i.e. consisting of IAPP
fibrils) was analyzed.
Part A--Thioflavin T Fluorometry Data
[0482] In one study, Thioflavin T fluorometry was used to determine
the effects of the compounds, and of EDTA (as a negative control).
In this assay Thioflavin T binds specifically to fibrillar amyloid,
and this binding produces a fluorescence enhancement at 485 nm that
is directly proportional to the amount of amyloid fibrils formed.
The higher the fluorescence, the greater the amount of amyloid
fibrils formed (Naki et al., Lab. Invest. 65:104-110, 1991; Levine
III, Protein Sci. 2:404-410, 1993; Amyloid:Int. J. Exp. Clin.
Invest. 2:1-6, 1995).
[0483] In this study, 25 .mu.M of IAPP (Bachem Inc) was incubated
at 37.degree. C. for 3 days, either alone, or in the presence of
one of the compounds or EDTA (at IAPP:test compound weight ratios
of 1:1, 1:0.1, 1:0.01 or 1:0.001). Following 3 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 mM
Thioflavin T in 250 mM 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).
[0484] The results of the 3-day incubations are presented below.
For example, whereas EDTA caused no significant
inhibition/disruption of IAPP fibrils at all concentrations tested,
the compounds (DC-0051, DC-0051-S1, S3, S4, S5, S6, S7, S8 and S9)
all caused a dose-dependent disruption/disassembly of preformed
IAPP fibrils to some extent (Table 4). For example, compound
DC-0051-S8 caused a significant (p<0.01) 91.4+/-1.06% inhibition
when used at an IAPP:test compound wt/wt ratio of 1:0.1, and a
significant 52.2+/-0.45% inhibition when used at an IAPP:test
compound wt/wt ratio of 1:0.011 (Table 4). Under the same
conditions (i.e. IAPP:test compound wt/wt ratio of 1:0.01),
compound DC-0051 caused a 63.9+/-0.56% disruption, compound
DC-0051-S1 caused a 47.2+/-5.48% disruption, and compound
DC-0051-S3 caused a 49.3+/-0.65% disruption. This study indicated
that the compound provided herein are also potent
disrupters/inhibitors of type 2 diabetes IAPP fibrils, and usually
exert their effects in a dose-dependent manner.
TABLE-US-00005 TABLE 4 Thioflavin T Fluorometry Data- Disruption of
IAPP Fibrils by Test Compounds Test Compound # 1:1 (wt/wt) 1:0.1
(wt/wt) 1:0.01 (wt/wt) 1:0.001 (wt/wt) EDTA (control) 0.0 .+-.
1.31% 1.6 +/- 5.86% 4. +/- 3.152% 0.0 +/- 0.56% DC-0051 99.6 +/-
0.12% 95.6 +/- 0.31% 63.9 +/- 0.56% 32.5 +/- 1.51% DC0051-S1 98.5
+/- 0.12% 86.2 +/- 1.95% 47.2 +/- 5.48% 6.7 +/- 0.64% DC0051-S3
98.7 +/- 0.24% 87.2 +/- 1.48% 49.3 +/- 0.65% 19.0 +/- 2.70%
DC0051-S4 97.5 +/- 0.11% 80.2 +/- 1.59% 36.7 +/- 0.74% 14.3 +/-
1.57% DC0051-S5 99.3 +/- 0.21% 87.1 +/- 1.46% 36.1 +/- 1.29% 15.0
+/- 2.38% DC0051-S6 98.7 +/- 0.52% 74.4 +/- 12.17% 19.7 +/- 1.64%
0.0 +/- 1.68% DC0051-S7 98.6 +/- 0.18% 77.7 +/- 2.68% 30.3 +/-
6.06% 7.7 +/- 2.60% DC0051-S8 99.5 +/- 0.32% 91.4 +/- 1.06% 52.2
+/- 0.45% 8.8 +/- 0.55% DC0051-S9 99.5 +/- 0.15% 82.8 +/- 4.28%
34.8 +/- 1.07% 7.0 +/- 2.49% (% inhibition of IAPP; for IAPP:test
compound at given wt/wt ratio)
Part B: Congo Red Binding Data
[0485] In the Congo red binding assay the ability of a test
compound to alter amyloid (in this case, IAPP) binding to Congo red
is quantified. In this assay, IAPP and test compounds were
incubated for 3 days and then vacuum filtered through a 0.2 .mu.m
filter. The amount of IAPP retained in the filter was then
quantified 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) was indicative of the test compound's ability to
diminish/alter the amount of aggregated and congophilic IAPP. This
particular assay appears to be more stringent in character than
Thioflavin T fluorometry, and it is more difficult to remove Congo
red binding to IAPP fibrils than assessed by other assays, so the %
inhibitions observed even with potent compounds is usually lower
than that observed as determined by other assays such as Thioflavin
T fluometry.
[0486] In one study, the ability of IAPP fibrils to bind Congo red
in the absence or presence of increasing amounts of the compounds
or EDTA (at IAPP:test compound weight ratios of 1:0.001, 1:0.01,
1:0.1 and 1:1) was determined. The results of the 3-day incubations
are presented in Table 5 below. Wheras EDTA caused no significant
inhibition of IAPP fibril binding to Congo red, the compounds
(DC-0051, DC-0051-S1, S3, S4, S5, S6, S7, S8 and S9) caused a
dose-dependent inhibition of IAPP binding to Congo red (Table 5).
For example, compound DC-0051-S8 caused a significant 41.0+/-4.15%
inhibition of Congo red binding to IAPP fibrils when used at an
IAPP:test compound wt/wt ratio of 1:1, and a 26.7+/-0.82%
inhibition when used an an IAPP:test compound wt/wt ratio of 1:0.1
(Table 5). Other good inhibitors as compared to the IAPP:test
compound wt/wt ratio of 1:0.1 appeared to be DC-0051 (51+/-0.63%
inhibition), DC-0051-S1 (24.1+/-1.99% inhibition), DC-0051-S4
(22.0+/-0.26% inhibition), and DC-0051-S9 (21.2+/-2.70%
inhibition)
TABLE-US-00006 TABLE 5 Congo red Binding Data- Disruption of IAPP
Fibrils by Test Compounds Test Compound # 1:1 (wt/wt) 1:0.1 (wt/wt)
1:0.01 (wt/wt) 1:0.001 (wt/wt) EDTA (control) 13.9 +/- 4.71% 0.0
+/- 2.40% 0.0 +/- 1.65% 0.0 .+-. 1.82% DC-0051 73.6 +/- 2.15% 51.0
+/- 0.63% 8.7 +/- 2.60% 0.0 +/- 4.07% DC0051-S1 44.1 +/- 1.03% 24.1
+/- 1.99% 0.0 +/- 2.55% 0.0 +/- 3.60% DC0051-S3 52.5 +/- 1.84% 17.4
+/- 2.21% 3.4 +/- 3.63% 0.0 +/- 1.94% DC0051-S4 30.0 +/- 1.38% 22.0
+/- 0.26% 2.4 +/- 3.24% 0.0 +/- 2.89% DC0051-S5 59.3 +/- 0.93% 11.0
+/- 3.94% 0.0 +/- 1.50% 0.0 +/- 2.26% DC0051-S6 46.0 +/- 0.65% 7.7
+/- 5.15% 0.0 +/- 4.57% 0.0 +/- 1.41% DC0051-S7 42.7 +/- 2.82% 3.6
+/- 1.15% 3.0 +/- 3.54% 1.8 +/- 3.25% DC0051-S8 41.0 +/- 4.15% 26.7
+/- 0.82% 0.0 +/- 5.19% 0.3 +/- 2.37% DC0051-S9 52.3 +/- 1.00% 21.2
+/- 2.70% 1.1 +/- 5.40% 2.5 +/- 5.02% (% inhibition of IAPP; for
IAPP:test compound at given wt/wt ratio)
Example 23
Compounds Provided Herein are Potent Disrupters of Alpha-Synuclein
Fibrils
[0487] The compounds prepared in the preceding Examples were also
found to be potent disrupters/inhibitors of alpha-synuclein
fibrils. In a set of studies, the efficacy of certain compounds
provided herein to cause a disassembly/disruption of pre-formed
amyloid-like fibrils of Parkinson's disease (i.e. consisting of
alpha-synuclein fibrils) was analyzed.
Thioflavin T Fluorometry Data
[0488] In one study, Thioflavin T fluorometry was used to determine
the effects of the compounds, and of EDTA (as a negative control).
In this assay Thioflavin T binds specifically to fibrillar amyloid,
and this binding produces a fluorescence enhancement at 485 nm that
is directly proportional to the amount of amyloid fibrils formed.
The higher the fluorescence, the greater the amount of amyloid
fibrils formed (Naki et al., Lab. Invest. 65:104-110, 1991; Levine
III, Protein Sci. 2:404-410, 1993; Amyloid:Int. J. Exp. Clin.
Invest. 2:1-6, 1995).
[0489] In this study, 25 .mu.M of alpha-synuclein (Recombinant
Peptide) was first incubated at 55.degree. C. for 2 days with
heparin (Sigma) to cause alpha-synuclein aggregation and fibril
formation. Heparin, is a highly sulfated glycosaminoglycan known to
cause aggregation of amyloid proteins. Following initial
alpha-synulcein fibrillization, alpha-synuclein+heparin was
incubated at 37.degree. C. for 3 days, either alone, or in the
presence of one of the compounds or EDTA (at .alpha.-synuclein:test
compound weight ratios of 1:1, 1:0.1, 1:0.01 or 1:0.001). Following
3 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
mM Thioflavin T in 250 mM 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).
[0490] The results of the 3-day incubations are presented below.
The compounds (DC-0051, DC-0051-S1, S3, S4, S5, S6, S7, S8 and S9)
all caused a dose-dependent disruption/disassembly of preformed
alpha-synuclein fibrils to some extent (Table 6). For example,
compound DC-005'-S1 caused a significant (p<0.01) 94.5+/-2.11%
inhibition when used at an .alpha.-synuclein:test compound wt/wt
ratio of 1:0.1, and a significant 99.1+/-0.12% inhibition when used
at an .alpha.-synuclein:test compound wt/wt ratio of 1:1 (Table 6).
On the other hand, compounds DC-0051-S8 caused a significant
(p<0.01) 84.6+/-0.47% inhibition when used at an
.alpha.-synuclein:test compound wt/wt ratio of 1:0.1, and a
significant 96.1+/-1.14% inhibition when used at an
.alpha.-synuclein:test compound wt/wt ratio of 1:1 (Table 6). This
study indicated that the compounds provided herein are also potent
disrupters/inhibitors of Parkinson's disease alpha-synuclein
fibrils, and usually exert their effects in a dose-dependent
manner.
TABLE-US-00007 TABLE 6 Thioflavin T Fluorometry Data- Disruption of
Apha-Synuclein Fibrils by Test Compounds Test Compound # 1:1
(wt/wt) 1:0.1 (wt/wt) 1:0.01 (wt/wt) 1:0.001 (wt/wt) DC-0051 99.7
+/- 0.09% 98.6 +/- 0.26% 82.7 +/- 2.40% 64.5 +/- 1.64% DC0051-S1
99.1 +/- 0.12% 94.5 +/- 2.11% 55.9+/13.31% 52.9 +/- 1.34% DC0051-S3
97.0 +/- 0.85% 87.6 +/- 4.07% 43.6 +/- 11.73% 37.6 +/- 5.18%
DC0051-S4 96.0 -+/- 0.50% 86.8 +/- 1.55% 53.7 +/- 10.98% 41.1 +/-
6.53% DC0051-S5 98.5 +/- 0.09% 91.6 +/- 0.37% 65.0 +/- 4.42% 49.1
+/- 2.61% DC0051-S6 96.0 +/- 1.65% 66.6 +/- 5.77% 46.9 +/- 5.52%
53.3 +/- 1.70% DC0051-S7 96.0 +/- 0.78% 82.1 +/- 8.94% 33.4 +/-
4.77% 47.9 +/- 6.32% DC0051-S8 96.1 +/- 1.14% 84.6 +/- 0.47% 44.1
+/- 2.19% 38.7 +/- 4.76% DC0051-S9 99.6 +/- 0.19% 96.1 +/- 0.65%
64.5 +/- 5.56% 50.9 +/- 1.86% (% inhibition of .alpha.-synuclein;
for .alpha.-synuclein:test compound at given wt/wt ratio)
Example 24
Compositions of Compounds Provided Herein
[0491] 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.
[0492] Representative compositions are as follows:
Oral Tablet Formulation
[0493] An oral tablet formulation of a compound provided herein is
prepared as follows:
TABLE-US-00008 % w/w Compound provided herein 10.0 Magnesium
stearate 0.5 Starch 2.0 Hydroxypropylmethylcellulose 1.0
Microcrystalline cellulose 86.5
[0494] 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
[0495] 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
[0496] A softgel formulation is prepared as follows:
TABLE-US-00009 % w/w Compound provided herein 20.0 Polyethylene
glycol 400 80.0
[0497] 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
[0498] A parenteral formulation is prepared as follows:
TABLE-US-00010 % w/w Compound provided herein 1.0 Normal saline
99.0
[0499] 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
[0500] A sustained release formulation may be prepared by the
method of U.S. Pat. No. 4,710,384, as follows:
[0501] One Kg 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.
Example 25
Compounds of this Invention are Potent Disrupters of Alzheimer's
A.beta. 1-42 Fibrils
[0502] In a set of studies, the efficacy of the compounds to cause
a disassembly/disruption of pre-formed amyloid fibrils of
Alzheimer's disease (i.e. consisting of A.beta. 1-42 fibrils) was
analyzed.
Part A--Thioflavin T fluorometry Data
[0503] In one study, Thioflavin T fluorometry was used to determine
the effects of the compounds, and of EDTA (as a negative control).
In this assay Thioflavin T binds specifically to fibrillar amyloid,
and this binding produces a fluorescence enhancement at 485 nm that
is directly proportional to the amount of amyloid fibrils formed.
The higher the fluorescence, the greater the amount of amyloid
fibrils formed (Naki et al., Lab. Invest. 65:104-110, 1991; Levine
III, Protein Sci. 2:404-410, 1993; Amyloid: Int. J. Exp. Clin.
Invest. 2:1-6, 1995).
[0504] In this study, 30 .mu.g of pre-fibrillized A.beta. 1-42
(rPeptide) was incubated at 37.degree. C. for 3 days either alone,
or in the presence of one of the compounds or EDTA (at A.beta.:test
compound weight ratios of 1:1, 1:0.1, 1:0.01 or 1:0.001). Following
3-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
mM Thioflavin T in 250 mM 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.
[0505] The results of the 3-day incubations are presented in Table
7 below. For example, whereas EDTA caused no significant inhibition
of A.beta. 1-42 fibrils at all concentrations tested, the compounds
all caused a dose-dependent disruption/disassembly of preformed
A.beta. 1-42 fibrils to some extent. The most efficacious compounds
to disrupt pre-formed A.beta. 1-42 fibrils appeared to be compounds
DC-0051-F2 and DC-0051-F5. For example, compound F2 caused a
significant (p<0.01) 95.9.+-.0.88% inhibition when used at an
A.beta.:test compound wt/wt ratio of 1:0.1, and a 53.7.+-.6.29%
disruption when used at an A.beta.:compound wt/wt ratio of 1:0.01.
Under the same conditions (i.e. A.beta.:test compound wt/wt ratio
of 1:0.1), compound F5 caused a 91.8.+-.0.22% disruption, and a
43.9.+-.2.01% disruption when uses at an A.beta.:compound wt/wt
ratio of 1:0.01. This study indicated that the compounds of this
invention are potent disruptors/inhibitors of Alzheimer's disease
type A.beta. fibrils, and usually exert their effects in a
dose-dependent manner.
TABLE-US-00011 TABLE 7 Thioflavin T fluorometry data - disruption
of A.beta. 1-42 Alzheimer's fibrils % Inhibition A.beta. (result
.+-. S.D.) at A.beta.:compound wt/wt ratio given Test Compound #
1:1 1:0.1 1:0.01 1:0.001 EDTA (control) 0.0 .+-. 3.46 0.0 .+-. 6.02
2.4 .+-. 2.86 0.0 .+-. 1.70 DC-0051-F1 98.7 .+-. 0.38 91.3 .+-.
1.39 37.1 .+-. 1.44 15.6 .+-. 2.01 DC-0051-F2 99.6 .+-. 0.55 95.9
.+-. 0.88 53.7 .+-. 6.29 17.1 .+-. 4.32 DC-0051-F3 99.0 .+-. 0.77
92.3 .+-. 0.19 34.6 .+-. 2.21 15.1 .+-. 3.81 DC-0051-F4 99.7 .+-.
0.58 79.8 .+-. 0.77 33.4 .+-. 2.09 16.0 .+-. 6.64 DC-0051-F5 99.8
.+-. 0.40 91.8 .+-. 0.22 43.9 .+-. 2.01 19.5 .+-. 1.49 DC-0051-F6
99.4 .+-. 0.86 88.0 .+-. 1.09 36.8 .+-. 3.43 9.6 .+-. 0.96
Part B: Congo Red Binding Data
[0506] In the Congo red binding assay, the ability of a test
compound to alter amyloid (in this case, A.beta.) binding to Congo
red is quantified. In this assay, A.beta. 1-42 and test compounds
were incubated for 3 days and then vacuum filtered through a 0.2
.mu.m filter. The amount of A.beta. 1-42 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) was indicative of the test compound's
ability to diminish/alter the amount of aggregated and congophilic
A.beta..
[0507] In one study, the ability of A.beta. fibrils to bind Congo
red in the absence or presence of increasing amounts of the
compounds or EDTA (at A.beta.:test compound weight ratios of 1:1,
1:0.1, 1:0.01 or 1:0.001) was determined. The results of 3-day
incubations are presented in Table 8 below. Whereas EDTA caused no
significant inhibition of A.beta. 1-42 fibril binding to Congo red
at all concentrations tested, the compounds caused a dose-dependent
inhibition of A.beta. binding to Congo red. For example, compound
DC-0051-F2 caused a significant (p<0.01) 80.1.+-.3.79%
inhibition of Congo red binding to A.beta. 1-42 fibrils when used
at an A.beta.:test compound wt/wt ratio of 1:1, and a significant
(p<0.01) 49.9.+-.9.13% inhibition of Congo red binding when used
at an A.beta.:test compound wt/wt ratio of 1:0.1. In another
example, compound DC-0051-F1 caused a significant (p<0.01)
80.1.+-.2.61% inhibition of Congo red binding to A.beta. 1-42
fibrils when used at an A.beta.:test compound wt/wt ratio of 1:1,
and a significant (p<0.01) 27.5.+-.2.39% inhibition of Congo red
binding when used at an A.beta.:test compound wt/wt ratio of 1:0.1.
This study also indicated that compounds of this invention are
potent inhibitors of A.beta. fibril binding to Congo red, and
usually exert their effects in a dose-dependent manner.
TABLE-US-00012 TABLE 8 Congo red binding data % Inhibition A.beta.
(result .+-. S.D.) at A.beta.:test compound wt/wt ratio given Test
Compound # 1:1 1:0.1 1:0.01 1:0.001 EDTA (control) 8.4 .+-. 2.74
0.0 .+-. 6.83 8.4 .+-. 4.00 1.2 .+-. 7.95 DC-0051-F1 80.1 .+-. 2.61
27.5 .+-. 2.39 2.2 .+-. 7.70 3.7 .+-. 4.36 DC-0051-F2 80.1 .+-.
3.79 49.9 .+-. 9.13 0.5 .+-. 5.24 4.9 .+-. 7.88 DC-0051-F3 74.5
.+-. 2.69 23.3 .+-. 5.01 0.0 .+-. 3.25 0.0 .+-. 2.64 DC-0051-F4
71.0 .+-. 2.39 10.9 .+-. 5.05 8.8 .+-. 5.07 4.3 .+-. 11.81
DC-0051-F5 69.9 .+-. 2.06 25.7 .+-. 5.69 9.8 .+-. 2.54 0.0 .+-.
2.50 DC-0051-F6 68.0 .+-. 2.11 18.3 .+-. 5.32 6.8 .+-. 1.51 3.7
.+-. 3.24
Example 27
Compounds of this Invention are Potent Disrupters of Parkinson's
Disease Alpha (.alpha.) Synuclein Fibrils
[0508] The tested compounds of this invention were found also to be
potent disruptors/inhibitors of Parkinson's disease
.alpha.-synuclein fibrils. .alpha.-synuclein has been demonstrated
to form amyloid-like fibrils when incubated at 37.degree. C. for a
few days. It is postulated to play an important role in the
pathogenesis of Parkinson's disease and other synucleinopathies. In
a set of studies, the efficacy of the compounds to cause a
disassembly/disruption of pre-formed .alpha.-synuclein fibrils of
Parkinson's disease was analyzed.
Part A--Thioflavin T Fluorometry Data
[0509] In one study, Thioflavin T fluorometry was used to determine
the effects of compounds DC-0051-F1, F2, F3, F4, F5, F6 and EDTA
(as a negative control). In this assay, Thioflavin T binds
specifically to .alpha.-synuclein fibrils, and this binding
produces a fluorescence enhancement at 485 nm that is directly
proportional to the amount of .alpha.-synuclein fibrils present.
The higher the fluorescence, the greater the amount of
.alpha.-synuclein 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).
[0510] In this study, 30 .mu.g of pre-fibrillized .alpha.-synuclein
(rpeptide) was incubated at 37.degree. C. for 3 days either alone
or in the presence of compounds DC-0051-F1, F2, F3, F4, F5, F6 or
EDTA (at .alpha.-synuclein:test compound weight ratios of 1:1,
1:0.1, 1:0.01 or 1:0.001). Following 3-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 mM Thioflavin T in 250
mM 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.
[0511] The results of the 3-day incubations are presented below in
Table 9. For example, whereas EDTA caused no significant inhibition
of .alpha.-synuclein fibrils at all concentrations tested,
compounds DC-0051-F1, F2, F3, F4, F5 and F6 all caused a
dose-dependent disruption/disassembly of pre-formed
.alpha.-synuclein fibrils to various extents. For example, compound
F2 caused a significant (p<0.01) 94.7.+-.2.57% inhibition when
used at an .alpha.-synuclein:test compound ratio of 1:0.1, and a
61.1.+-.1.37% disruption when used at a .alpha.-synuclein:compound
wt/wt ratio of 1:0.01. Under the same conditions (i.e.
.alpha.-synuclein:test compound wt/wt ratio of 1:0.1), compound F3
caused a 92.5.+-.2.35% disruption, and a 59.6.+-.6.39% disruption
when uses at an .alpha.-synuclein:compound wt/wt ratio of 1:0.01.
This study indicated that compounds of this invention are potent
disruptors/inhibitors of Parkinson's disease .alpha.-synuclein
fibrils, and usually exert their effects in a dose-dependent
manner.
TABLE-US-00013 TABLE 9 Thioflavin T fluorometry data - disruption
of Parkinson's disease .alpha.-synuclein fibrils % Inhibition
.alpha.-synuclein (result .+-. S.D.) at .alpha.-synuclein:test
compound wt/wt ratio given Test Compound # 1:1 1:0.1 1:0.01 1:0.001
EDTA (control) 0.0 .+-. 2.03 0.0 .+-. 6.97 0.0 .+-. 17.06 0.0 .+-.
4.11 DC-0051-F1 100.0 .+-. 3.14 90.4 .+-. 2.07 58.4 .+-. 5.06 13.7
.+-. 6.06 DC-0051-F2 100.0 .+-. 1.49 94.7 .+-. 2.57 61.1 .+-. 1.37
15.9 .+-. 7.20 DC-0051-F3 96.8 .+-. 2.82 92.5 .+-. 2.35 59.6 .+-.
6.39 13.8 .+-. 5.12 DC-0051-F4 100.0 .+-. 2.84 94.3 .+-. 1.22 40.6
.+-. 1.49 6.1 .+-. 9.44 DC-0051-F5 98.9 .+-. 0.65 94.7 .+-. 1.70
59.0 .+-. 1.18 5.9 .+-. 10.41 DC-0051-F6 100.0 .+-. 1.98 94.7 .+-.
0.68 56.2 .+-. 1.49 4.2 .+-. 5.02
Part B: Congo Red Binding Data
[0512] In the Congo red binding assay, the ability of a given test
compound to alter amyloid (in this case, .alpha.-synuclein) binding
to Congo red is quantified. In this assay, .alpha.-synuclein and
test compounds were incubated for 3 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) was indicative
of the test compound's ability to diminish/alter the amount of
aggregated and congophilic .alpha.-synuclein.
[0513] In one study, the ability of .alpha.-synuclein fibrils to
bind Congo red in the absence or presence of increasing amounts of
compounds DC-0051-F1, F2, F3, F4, F5, F6 and EDTA (at
.alpha.-synuclein:test compound weight ratios of 1:1, 1:0.1, 1:0.01
or 1:0.001) was determined. The results of 3-day incubations are
presented in Table 10 below. Whereas EDTA caused no significant
inhibition of .alpha.-synuclein fibril binding to Congo red at all
concentrations tested, the compounds caused a dose-dependent
inhibition of .alpha.-synuclein binding to Congo red. For example,
compound F2 caused a significant (p<0.01) 67.8.+-.3.52%
inhibition of Congo red binding to .alpha.-synuclein fibrils when
used at a .alpha.-synuclein:test compound wt/wt ratio of 1:1, and a
significant (p<0.01) 36.5.+-.5.34% inhibition of Congo red
binding when used at a .alpha.-synuclein:test compound wt/wt ratio
of 1:0.1. In comparison, compound F1 caused a 69.8.+-.2.67%
inhibition of Congo red binding to .alpha.-synuclein fibrils when
used at a .alpha.-synuclein:test compound wt/wt ratio of 1:1, and a
32.9.+-.2.97% inhibition of Congo red binding when used at a
.alpha.-synuclein:test compound wt/wt ratio of 1:0.1. This study
also indicated that compounds of this invention are also potent
inhibitors of Parkinson's disease type .alpha.-synuclein fibril
binding to Congo red, and usually exert their effects in a
dose-dependent manner.
TABLE-US-00014 TABLE 10 Congo red binding data - disruption of
Parkinson's disease .alpha.-synuclein fibrils % Inhibition
.alpha.-synuclein (result .+-. S.D.) at .alpha.-synuclein:test
compound wt/wt ratio given Test Compound # 1:1 1:0.1 1:0.01 1:0.001
EDTA (control) 0.0 .+-. 5.50 2.2 .+-. 5.16 2.9 .+-. 5.0 3.3 .+-.
1.85 DC-0051-F1 69.8 .+-. 2.67 32.9 .+-. 2.97 3.8 .+-. 5.82 0.0
.+-. 3.42 DC-0051-F2 67.8 .+-. 3.52 36.5 .+-. 5.34 3.7 .+-. 2.83
0.0 .+-. 6.71 DC-0051-F3 55.9 .+-. 2.65 23.3 .+-. 2.24 2.6 .+-.
4.90 0.0 .+-. 3.93 DC-0051-F4 64.8 .+-. 5.08 18.0 .+-. 0.58 0.0
.+-. 3.44 6.1 .+-. 8.92 DC-0051-F5 69.4 .+-. 0.13 32.7 .+-. 3.96
5.0 .+-. 5.47 0.0 .+-. 1.01 DC-0051-F6 59.8 .+-. 3.62 29.9 .+-.
2.41 1.4 .+-. 5.69 0.0 .+-. 4.20
[0514] 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.
* * * * *