U.S. patent application number 13/967818 was filed with the patent office on 2014-02-20 for selective glycosidase inhibitors and uses thereof.
This patent application is currently assigned to Simon Fraser University. The applicant listed for this patent is Simon Fraser University. Invention is credited to Ernest John McEachern, Keith Stubbs, David Jaro Vocadlo.
Application Number | 20140051719 13/967818 |
Document ID | / |
Family ID | 41609896 |
Filed Date | 2014-02-20 |
United States Patent
Application |
20140051719 |
Kind Code |
A1 |
Vocadlo; David Jaro ; et
al. |
February 20, 2014 |
Selective Glycosidase Inhibitors and Uses Thereof
Abstract
The invention provides compounds of Formula (I) for selectively
inhibiting glycosidases, prodrugs of the compounds, and
pharmaceutical compositions including the compounds or prodrugs of
the compounds. The invention also provides methods of treating
diseases and disorders related to deficiency or overexpression of
O-GlcNAcase, accumulation or deficiency of O-GlcNAc.
##STR00001##
Inventors: |
Vocadlo; David Jaro;
(Burnaby, CA) ; McEachern; Ernest John; (Burnaby,
CA) ; Stubbs; Keith; (Burnaby, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Simon Fraser University |
Burnaby |
|
CA |
|
|
Assignee: |
Simon Fraser University
Burnaby
CA
|
Family ID: |
41609896 |
Appl. No.: |
13/967818 |
Filed: |
August 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13057062 |
Jun 2, 2011 |
8541441 |
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PCT/CA09/01087 |
Jul 31, 2009 |
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13967818 |
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61085527 |
Aug 1, 2008 |
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Current U.S.
Class: |
514/299 ;
435/193; 546/183 |
Current CPC
Class: |
A61P 21/00 20180101;
A61P 13/12 20180101; A61P 37/06 20180101; A61P 27/14 20180101; A61P
25/28 20180101; A61P 37/02 20180101; A61P 1/04 20180101; A61P 1/00
20180101; A61P 35/00 20180101; A61P 37/08 20180101; C07D 471/04
20130101; C12Q 1/34 20130101; A61P 25/08 20180101; A61P 17/00
20180101; G01N 2500/00 20130101; A61P 17/06 20180101; A61P 17/04
20180101; A61P 25/00 20180101; A61P 9/10 20180101; A61P 11/00
20180101; A61P 11/02 20180101; A61P 11/06 20180101; A61P 9/00
20180101; A61P 19/02 20180101; A61P 29/00 20180101; A61P 25/16
20180101 |
Class at
Publication: |
514/299 ;
546/183; 435/193 |
International
Class: |
C07D 471/04 20060101
C07D471/04 |
Claims
1. A compound of Formula (I) or a pharmaceutically acceptable salt
thereof: ##STR00075## wherein each R.sup.1 is independently a
non-interfering substituent; and R.sup.2 is NR.sup.3.sub.2, wherein
each R.sup.3 is optionally independently a non-interfering
substituent, with the proviso that when each R.sup.1 is H, R.sup.2
excludes NH(COCH.sub.3), NH(CO(CH.sub.2).sub.2CH.sub.3),
NH(benzoyl), NH.sub.2, NH(CH.sub.3), NH(CH.sub.2CH.dbd.CH.sub.2),
NH((CH.sub.2).sub.3CH.sub.3), NH(CH.sub.2CH(CH.sub.3).sub.2),
NH(CH(CH.sub.3)(CH.sub.2CH.sub.3)),
NH(CH(CH.sub.3)(CH.sub.2CH.sub.2CH.sub.3), NH(benzyl),
NH(CH.sub.2CH.sub.2OCH.sub.3), N(CH.sub.2CH.sub.3).sub.2,
N(CH.sub.2CH.sub.3)((CH.sub.2).sub.3CH.sub.3),
NH(CH.sub.2).sub.7CH.sub.3, N(COCH.sub.3)(benzyl), N.sub.3, and
N(CH.sub.2CH.sub.2OH).sub.2; or when each R.sup.1 is C(O)CH.sub.3,
R.sup.2 excludes NH(COCH.sub.3), NH.sub.2, N.sub.3, and
N(CH.sub.2CH.sub.3).sub.2.
2. The compound of claim 1 wherein each R.sup.1 may be connected to
another R.sup.1 to form an additional ring structure.
3. The compound of claim 1 wherein R.sup.1 is H or
C(O)CH.sub.3.
4. The compound of any claim 1 wherein said non-interfering
substituent is selected from one or more of alkyl, branched alkyl,
alkenyl, branched alkenyl, alkynyl, branched alkynyl, aryl,
heteroaryl, arylalkyl, heteroarylalkyl, arylalkenyl,
heteroarylalkenyl, arylalkynyl, and heteroarylalkylnyl, each of
which may be optionally substituted with one or more heteroatoms or
additional non-interfering substituents.
5. The compound of claim 4 wherein said non-interfering substituent
comprises one or more heteroatoms selected from P, O, S, N, F, Cl,
Br, I, or B.
6. (canceled)
7. The compound of claim 1 wherein R.sup.2 is selected from the
group consisting of: N(CH.sub.3)(C(O)CH.sub.3),
N(CH.sub.2CH.sub.3)(C(O)CH.sub.3),
N(CH.sub.3)(C(O)CH.sub.2CH.sub.3), NH(C(O)NH.sub.2),
NH(C(O)NHCH.sub.3), NH(C(O)NHCH.sub.2CH.sub.3),
NH(C(O)NH(CH.sub.2).sub.2CH.sub.3),
NH(C(O)NH(CH.sub.2).sub.3CH.sub.3),
NH(C(O)NH(CH.sub.2CH.dbd.CH.sub.2)), NH(C(O)N(CH.sub.3).sub.2),
NH(C(O)N(CH.sub.3)(CH.sub.2CH.sub.3)),
NH(CO)(tetrahydropyrrol-1-yl), N(CH.sub.3)(C(O)NHCH.sub.3),
N(CH.sub.3)(C(O)N(CH.sub.3).sub.2), NH(CH.sub.2CH.sub.3),
NH(CH.sub.2).sub.2CH.sub.3, NH(CH(CH.sub.3).sub.2),
N(CH.sub.3).sub.2, N(CH.sub.3)(CH.sub.2CH.sub.3), N(CH.sub.3)
((CH.sub.2).sub.2CH.sub.3), N(CH.sub.3)((CH.sub.2).sub.3CH.sub.3),
N(CH.sub.3)(CH.sub.2CH.dbd.CH.sub.2), and tetrahydropyrrol 1
yl.
8. The compound of claim 1 wherein the compound is a prodrug.
9. The compound of claim 1 wherein the compound selectively
inhibits an O-glycoprotein
2-acetamido-2-deoxy-.beta.-D-glucopyranosidase (O-GlcNAcase).
10. (canceled)
11. The compound of claim 1 wherein the compound selectively
inhibits the cleavage of
2-acetamido-2-deoxy-.beta.-D-glucopyranoside (O-GlcNAc).
12. The compound of claim 9 wherein the O-GlcNAcase is a mammalian
O-GlcNAcase.
13. (canceled)
14. A pharmaceutical composition comprising the compound of claim 1
in combination with a pharmaceutically acceptable carrier.
15. A method of selectively inhibiting an O-GlcNAcase in a subject
in need thereof, the method comprising administering to the subject
an effective amount of a compound of Formula (I) or a
pharmaceutically acceptable salt thereof: ##STR00076## wherein each
R.sup.1 is independently a non-interfering substituent; and R.sup.2
is NR.sup.3.sub.2, wherein each R.sup.3 is optionally independently
a non-interfering substituent.
16. A method of elevating the level of O-GlcNAc in a subject in
need thereof, the method comprising administering to the subject an
effective amount of a compound of Formula (I) or a pharmaceutically
acceptable salt thereof: ##STR00077## wherein each R.sup.1 is
independently a non-interfering substituent; and R.sup.2 is
NR.sup.3.sub.2, wherein each R.sup.3 is optionally independently a
non-interfering substituent.
17. A method of treating a condition that is modulated by an
O-GlcNAcase, in a subject in need thereof, the method comprising
administering to the subject an effective amount of a compound of
Formula (I) or a pharmaceutically acceptable salt thereof:
##STR00078## wherein each R.sup.1 is independently a
non-interfering substituent; and R.sup.2 is NR.sup.3.sub.2, wherein
each R.sup.3 is optionally independently a non-interfering
substituent.
18. The method of claim 17 wherein the condition is selected from
one or more of the group consisting of an inflammatory disease, an
allergy, asthma, allergic rhinitis, hypersensitivity lung diseases,
hypersensitivity pneumonitis, eosinophilic pneumonias, delayed-type
hypersensitivity, atherosclerosis, interstitial lung disease (ILD),
idiopathic pulmonary fibrosis, ILD associated with rheumatoid
arthritis, systemic lupus erythematosus, ankylosing spondylitis,
systemic sclerosis, Sjogren's syndrome, polymyositis or
dermatomyositis, systemic anaphylaxis or hypersensitivity response,
drug allergy, insect sting allergy, autoimmune disease, rheumatoid
arthritis, psoriatic arthritis, multiple sclerosis, systemic lupus
erythematosus, myastenia gravis, glomerulonephritis, autoimmune
thyroiditis, graft rejection, allograft rejection,
graft-versus-host disease, inflammatory bowel disease, Crohn's
disease, ulcerative colitis, spondyloarthropathy, scleroderma,
psoriasis, T-cell mediated psoriasis, inflammatory dermatosis,
dermatitis, eczema, atopic dermatitis, allergic contact dermatitis,
urticaria, vasculitis, necrotizing, cutaneous, and hypersensitivity
vasculitis, eosinphilic myotis, eosiniphilic fasciitis, solid organ
transplant rejection, heart transplant rejection, lung transplant
rejection, liver transplant rejection, kidney transplant rejection,
pancreas transplant rejection, kidney allograft, lung allograft,
epilepsy, pain, stroke, neuroprotection.
19. A method of treating a condition selected from the group
consisting of a neurodegenerative disease, a tauopathy, cancer and
stress, in a subject in need thereof, the method comprising
administering to the subject an effective amount of a compound of
Formula (I) or a pharmaceutically acceptable salt thereof:
##STR00079## wherein each R.sup.1 is independently a
non-interfering substituent; and R.sup.2 is NR.sup.3.sub.2, wherein
each R.sup.3 is optionally independently a non-interfering
substituent.
20. The method of claim 19 wherein the condition is selected from
one or more of the group consisting of Alzheimer's disease,
Amyotrophic lateral sclerosis (ALS), Amyotrophic lateral sclerosis
with cognitive impairment (ALSci), Argyrophilic grain dementia,
Bluit disease, Corticobasal degeneration (CBD), Dementia
pugilistica, Diffuse neurofibrillary tangles with calcification,
Down's syndrome, Familial British dementia, Familial Danish
dementia, Frontotemporal dementia with parkinsonism linked to
chromosome 17 (FTDP-17), Gerstmann-Straussler-Scheinker disease,
Guadeloupean parkinsonism, Hallevorden-Spatz disease
(neurodegeneration with brain iron accumulation type 1), Multiple
system atrophy, Myotonic dystrophy, Niemann-Pick disease (type C),
Pallido-ponto-nigral degeneration, Parkinsonism-dementia complex of
Guam, Pick's disease (PiD), Post-encephalitic parkinsonism (PEP),
Prion diseases (including Creutzfeldt-Jakob Disease (CJD), Variant
Creutzfeldt-Jakob Disease (vCJD), Fatal Familial Insomnia, and
Kuru), Progressive supercortical gliosis, Progressive supranuclear
palsy (PSP), Richardson's syndrome, Subacute sclerosing
panencephalitis, Tangle-only dementia, Huntington's disease, and
Parkinson's disease.
21. The method of claim 19 wherein the stress is a cardiac
disorder.
22. The method of claim 21 wherein the cardiac disorder is selected
from one or more of the group consisting of ischemia; hemorrhage;
hypovolemic shock; myocardial infarction; an interventional
cardiology procedure; cardiac bypass surgery; fibrinolytic therapy;
angioplasty; and stent placement.
23-29. (canceled)
30. A method for screening for a selective inhibitor of an
O-GlcNAcase, the method comprising: a) contacting a first sample
with a test compound; b) contacting a second sample with a compound
of Formula (I) ##STR00080## wherein each R.sup.1 is independently a
non-interfering substituent; and R.sup.2 is NR.sup.3.sub.2, wherein
each R.sup.3 is optionally independently a non-interfering
substituent; c) determining the level of inhibition of the
O-GlcNAcase in the first and second samples, wherein the test
compound is a selective inhibitor of a O-GlcNAcase if the test
compound exhibits the same or greater inhibition of the O-GlcNAcase
when compared to the compound of Formula (I).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 13/057,062, filed on Jun. 2, 2011, which is a
national phase filing under 35 U.S.C. .sctn.371 of International
Application No. PCT/CA2009/001087, filed on Jul. 31, 2009, which
claims the benefit of and priority to U.S. Provisional Application
No. 61/085,527 filed Aug. 1, 2008, the disclosures of each of which
are hereby incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] This application relates to compounds which selectively
inhibit glycosidases and uses thereof.
BACKGROUND OF THE INVENTION
[0003] A wide range of cellular proteins, both nuclear and
cytoplasmic, are post-translationally modified by the addition of
the monosaccharide 2-acetamido-2-deoxy-.beta.-D-glucopyranoside
(.beta.-N-acetylglucosamine) which is attached via an O-glycosidic
linkage..sup.1 This modification is generally referred to as
O-linked N-acetylglucosamine or O-GlcNAc. The enzyme responsible
for post-translationally linking .beta.-N-acetylglucosamine
(GlcNAc) to specific serine and threonine residues of numerous
nucleocytoplasmic proteins is O-GlcNAc transferase (OGT)..sup.2-5 A
second enzyme, known as O-GlcNAcase.sup.6,7 removes this
post-translational modification to liberate proteins making the
O-GlcNAc-modification a dynamic cycle occurring several times
during the lifetime of a protein..sup.8
[0004] O-GlcNAc-modified proteins regulate a wide range of vital
cellular functions including, for example, transcription,.sup.9-12
proteasomal degradation,.sup.13 and cellular signaling..sup.14
O-GlcNAc is also found on many structural proteins..sup.15-17 For
example, it has been found on a number of cytoskeletal proteins,
including neurofilament proteins.sup.18,19 synapsins,.sup.6,20
synapsin-specific clathrin assembly protein AP-3,.sup.7 and
ankyrinG..sup.14 O-GlcNAc modification has been found to be
abundant in the brain..sup.21,22 It has also been found on proteins
clearly implicated in the etiology of several diseases including
Alzheimer's disease (AD) and cancer.
[0005] For example, it is well established that AD and a number of
related tauopathies including Downs' syndrome, Pick's disease,
Niemann-Pick Type C disease, and amyotrophic lateral sclerosis
(ALS) are characterized, in part, by the development of
neurofibrillary tangles (NFTs). These NFTs are aggregates of paired
helical filaments (PHFs) and are composed of an abnormal form of
the cytoskeletal protein "tau". Normally tau stabilizes a key
cellular network of microtubules that is essential for distributing
proteins and nutrients within neurons. In AD patients, however, tau
becomes hyperphosphorylated, disrupting its normal functions,
forming PHFs and ultimately aggregating to form NFTs. Six isoforms
of tau are found in the human brain. In AD patients, all six
isoforms of tau are found in NFTs, and all are markedly
hyperphosphorylated..sup.23,24 Tau in healthy brain tissue bears
only 2 or 3 phosphate groups, whereas those found in the brains of
AD patients bear, on average, 8 phosphate groups..sup.25,26 A clear
parallel between NFT levels in the brains of AD patients and the
severity of dementia strongly supports a key role for tau
dysfunction in AD..sup.27,28 The precise causes of this
hyperphosphorylation of tau remain elusive. Accordingly,
considerable effort has been dedicated toward: a) elucidating the
molecular physiological basis of tau hyperphosphorylation;.sup.29
and b) identifying strategies that could limit tau
hyperphosphorylation in the hope that these might halt, or even
reverse, the progression of Alzheimer's disease.sup.30-33 Thus far,
several lines of evidence suggest that up-regulation of a number of
kinases may be involved in hyperphosphorylation of
tau,.sup.21,34,35 although very recently, an alternative basis for
this hyperphosphorylation has been advanced..sup.21
[0006] In particular, it has recently emerged that phosphate levels
of tau are regulated by the levels of O-GlcNAc on tau. The presence
of O-GlcNAc on tau has stimulated studies that correlate O-GlcNAc
levels with tau phosphorylation levels. The recent interest in this
field stems from the observation that O-GlcNAc modification has
been found to occur on many proteins at amino acid residues that
are also known to be phosphorylated..sup.36-38 Consistent with this
observation, it has been found that increases in phosphorylation
levels result in decreased O-GlcNAc levels and conversely,
increased O-GlcNAc levels correlate with decreased phosphorylation
levels..sup.39 This reciprocal relationship between O-GlcNAc and
phosphorylation has been termed the "Yin-Yang hypothesis".sup.40
and has gained strong biochemical support by the recent discovery
that the enzyme OGT.sup.4 forms a functional complex with
phosphatases that act to remove phosphate groups from
proteins..sup.41 Like phosphorylation, O-GlcNAc is a dynamic
modification that can be removed and reinstalled several times
during the lifespan of a protein. Suggestively, the gene encoding
O-GlcNAcase has been mapped to a chromosomal locus that is linked
to AD..sup.7,42 Hyperphosphorylated tau in human AD brains has
markedly lower levels of O-GlcNAc than are found in healthy human
brains..sup.21 Very recently, it has been shown that O-GlcNAc
levels of soluble tau protein from human brains affected with AD
are markedly lower than those from healthy brain..sup.21
Furthermore, PHF from diseased brain was suggested to lack
completely any O-GlcNAc modification whatsoever..sup.21 The
molecular basis of this hypoglycosylation of tau is not known,
although it may stem from increased activity of kinases and/or
dysfunction of one of the enzymes involved in processing O-GlcNAc.
Supporting this latter view, in both PC-12 neuronal cells and in
brain tissue sections from mice, a nonselective
N-acetylglucosamindase inhibitor was used to increase tau O-GlcNAc
levels, whereupon it was observed that phosphorylation levels
decreased..sup.21 The implication of these collective results is
that by maintaining healthy O-GlcNAc levels in AD patients, such as
by inhibiting the action of O-GlcNAcase, one should be able to
block hyperphosphorylation of tau and all of the associated effects
of tau hyperphosphorylation, including the formation of NFTs and
downstream effects. However, because the proper functioning of the
.beta.-hexosaminidases is critical, any potential therapeutic
intervention for the treatment of AD that blocks the action of
O-GlcNAcase would have to avoid the concomitant inhibition of both
hexosaminidases A and B.
[0007] Neurons do not store glucose and therefore the brain relies
on glucose supplied by blood to maintain its essential metabolic
functions. Notably, it has been shown that within brain, glucose
uptake and metabolism decreases with aging..sup.43 Within the
brains of AD patients marked decreases in glucose utilization occur
and are thought to be a potential cause of
neurodegeneration..sup.44 The basis for this decreased glucose
supply in AD brain.sup.45-47 is thought to stem from any of
decreased glucose transport,.sup.48,49 impaired insulin
signaling,.sup.50,51 and decreased blood flow..sup.52
[0008] In light of this impaired glucose metabolism, it is worth
noting that of all glucose entering into cells, 2-5% is shunted
into the hexosamine biosynthetic pathway, thereby regulating
cellular concentrations of the end product of this pathway, uridine
diphosphate-N-acetylglucosamine (UDP-GlcNAc)..sup.53 UDP-GlcNAc is
a substrate of the nucleocytoplasmic enzyme O-GlcNAc transferase
(OGT),.sup.2-5 which acts to post-translationally add GlcNAc to
specific serine and threonine residues of numerous
nucleocytoplasmic proteins. OGT recognizes many of its
substrates.sup.54,55 and binding partners.sup.41,56 through its
tetratricopeptide repeat (TPR) domains..sup.57,58 As described
above, O-GlcNAcase.sup.6,7 removes this post-translational
modification to liberate proteins making the O-GlcNAc-modification
a dynamic cycle occurring several times during the lifetime of a
protein..sup.8 O-GlcNAc has been found in several proteins on known
phosphorylation sites,.sup.10,37,38,59 including tau and
neurofilaments..sup.60 Additionally, OGT shows unusual kinetic
behaviour making it exquisitely sensitive to intracellular
UDP-GlcNAc substrate concentrations and therefore glucose
supply..sup.41
[0009] Consistent with the known properties of the hexosamine
biosynthetic pathway, the enzymatic properties of OGT, and the
reciprocal relationship between O-GlcNAc and phosphorylation, it
has been shown that decreased glucose availability in brain leads
to tau hyperphosphorylation..sup.44 Therefore the gradual
impairment of glucose transport and metabolism, whatever its
causes, leads to decreased O-GlcNAc and hyperphosphorylation of tau
(and other proteins). Accordingly, the inhibition of O-GlcNAcase
should compensate for the age related impairment of glucose
metabolism within the brains of health individuals as well as
patients suffering from AD or related neurodegenerative
diseases.
[0010] These results suggest that a malfunction in the mechanisms
regulating tau O-GlcNAc levels may be vitally important in the
formation of NFTs and associated neurodegeneration. Good support
for blocking tau hyperphosphorylation as a therapeutically useful
intervention.sup.61 comes from recent studies showing that when
transgenic mice harbouring human tau are treated with kinase
inhibitors, they do not develop typical motor defects.sup.33 and,
in another case,.sup.32 show decreased levels of insoluble tau.
These studies provide a clear link between lowering tau
phosphorylation levels and alleviating AD-like behavioural symptoms
in a murine model of this disease. Indeed, pharmacological
modulation of tau hyperphosphorylation is widely recognized as a
valid therapeutic strategy for treating AD and other
neurodegenerative disorders..sup.62
[0011] Recent studies.sup.63 support the therapeutic potential of
small-molecule O-GlcNAcase inhibitors to limit tau
hyperphosphorylation for treatment of AD and related tauopathies.
Specifically, the O-GlcNAcase inhibitor thiamet-G has been
implicated in the reduction of tau phosphorylation in cultured
PC-12 cells at pathologically relevant sites..sup.63 Moreover, oral
administration of thiamet-G to healthy Sprague-Dawley rats has been
implicated in reduced phosphorylation of tau at Thr231, Ser396 and
Ser422 in both rat cortex and hippocampus..sup.63
[0012] There is also a large body of evidence indicating that
increased levels of O-GlcNAc protein modification provides
protection against pathogenic effects of stress in cardiac tissue,
including stress caused by ischemia, hemorrhage, hypervolemic
shock, and calcium paradox. For example, activation of the
hexosamine biosynthetic pathway (HBP) by administration of
glucosamine has been demonstrated to exert a protective effect in
animals models of ischemia/reperfusion,.sup.64-70 trauma
hemorrhage,.sup.71-73 hypervolemic shock,.sup.74 and calcium
paradox..sup.64,75 Moreover, strong evidence indicates that these
cardioprotective effects are mediated by elevated levels of protein
O-GlcNAc modification..sup.64,65,67,70,72,75-78 There is also
evidence that the O-GlcNAc modification plays a role in a variety
of neurodegenerative diseases, including Parkinson's disease and
Huntington's disease..sup.79
[0013] Humans have three genes encoding enzymes that cleave
terminal .beta.-N-acetyl-glucosamine residues from glycoconjugates.
The first of these encodes the enzyme O-glycoprotein
2-acetamido-2-deoxy-.beta.-D-glucopyranosidase (O-GlcNAcase).
O-GlcNAcase is a member of family 84 of glycoside hydrolases that
includes enzymes from organisms as diverse as prokaryotic pathogens
to humans (for the family classification of glycoside hydrolases
see Coutinho, P. M. & Henrissat, B. (1999) Carbohydrate-Active
Enzymes server at URL: http://afmb.cnrs-mrs.fr/CAZY/..sup.27,28
O-GlcNAcase acts to hydrolyse O-GlcNAc off of serine and threonine
residues of post-translationally modified proteins..sup.1,6,7,80,81
Consistent with the presence of O-GlcNAc on many intracellular
proteins, the enzyme O-GlcNAcase appears to have a role in the
etiology of several diseases including type II diabetes,.sup.14,82
AD,.sup.16,21,83 and cancer..sup.22,84 Although O-GlcNAcase was
likely isolated earlier on,.sup.18,19 about 20 years elapsed before
its biochemical role in acting to cleave O-GlcNAc from serine and
threonine residues of proteins was understood..sup.6 More recently
O-GlcNAcase has been cloned,.sup.7 partially characterized,.sup.20
and suggested to have additional activity as a histone
acetyltransferase..sup.20 However, little was known about the
catalytic mechanism of this enzyme.
[0014] The other two genes, HEXA and HEXB, encode enzymes
catalyzing the hydrolytic cleavage of terminal
.beta.-N-acetylglucosamine residues from glycoconjugates. The gene
products of HEXA and HEXB predominantly yield two dimeric isozymes,
hexosaminidase A and hexosaminidase B, respectively. Hexosaminidase
A (.alpha..beta.), a heterodimeric isozyme, is composed of an
.alpha.- and a .beta.-subunit. Hexosaminidase B (.beta..beta.), a
homodimeric isozyme, is composed of two .beta.-subunits. The two
subunits, .alpha.- and .beta.-, bear a high level of sequence
identity. Both of these enzymes are classified as members of family
20 of glycoside hydrolases and are normally localized within
lysosomes. The proper functioning of these lysosomal
.beta.-hexosaminidases is critical for human development, a fact
that is underscored by the tragic genetic illnesses, Tay-Sach's and
Sandhoff diseases which stem from a dysfunction in, respectively,
hexosaminidase A and hexosaminidase B..sup.85 These enzymatic
deficiencies cause an accumulation of glycolipids and
glycoconjugates in the lysosomes resulting in neurological
impairment and deformation. The deleterious effects of accumulation
of gangliosides at the organismal level are still being
uncovered..sup.86
[0015] As a result of the biological importance of these
.beta.-N-acetyl-glucosaminidases, small molecule inhibitors of
glycosidases.sup.87-90 have received a great deal of
attention,.sup.91 both as tools for elucidating the role of these
enzymes in biological processes and in developing potential
therapeutic applications. The control of glycosidase function using
small molecules offers several advantages over genetic knockout
studies including the ability to rapidly vary doses or to entirely
withdraw treatment.
[0016] However, a major challenge in developing inhibitors for
blocking the function of mammalian glycosidases, including
O-GlcNAcase, is the large number of functionally related enzymes
present in tissues of higher eukaryotes. Accordingly, the use of
non-selective inhibitors in studying the cellular and organismal
physiological role of one particular enzyme is complicated because
complex phenotypes arise from the concomitant inhibition of such
functionally related enzymes. In the case of
.beta.-N-acetylglucosaminidases, existing compounds that act to
block O-GlcNAcase function are non-specific and act potently to
inhibit the lysosomal .beta.-hexosaminidases.
[0017] A few of the better characterized inhibitors of
.beta.-N-acetyl-glucosaminidases which have been used in studies of
O-GlcNAc post-translational modification within both cells and
tissues are streptozotocin (STZ),
2'-methyl-.alpha.-D-glucopyrano-[2,1-d]-.DELTA.2'-thiazoline
(NAG-thiazoline) and
O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino
N-phenylcarbamate (PUGNAc)..sup.14,92-95
[0018] STZ has long been used as a diabetogenic compound because it
has a particularly detrimental effect on .beta.-islet cells..sup.96
STZ exerts its cytotoxic effects through both the alkylation of
cellular DNA.sup.96,97 as well as the generation of radical species
including nitric oxide..sup.98 The resulting DNA strand breakage
promotes the activation of poly(ADP-ribose) polymerase
(PARP).sup.99 with the net effect of depleting cellular NAD+ levels
and, ultimately, leading to cell death..sup.100,101 Other
investigators have proposed instead that STZ toxicity is a
consequence of the irreversible inhibition of O-GlcNAcase, which is
highly expressed within .beta.-islet cells..sup.92,102 This
hypothesis has, however, been brought into question by two
independent research groups..sup.103,104 Because cellular O-GlcNAc
levels on proteins increase in response to many forms of cellular
stress.sup.105 it seems possible that STZ results in increased
O-GlcNAc-modification levels on proteins by inducing cellular
stress rather than through any specific and direct action on
O-GlcNAcase. Indeed, Hanover and coworkers have shown that STZ
functions as a poor and somewhat selective inhibitor of
O-GlcNAcase.sup.106 and although it has been proposed by others
that STZ acts to irreversibly inhibit O-GlcNAcase,.sup.107 there
has been no clear demonstration of this mode of action. Recently,
it has been shown that STZ does not irreversibly inhibit
O-GlcNAcase..sup.108
[0019] NAG-thiazoline has been found to be a potent inhibitor of
family 20 hexosaminidases,.sup.90,109 and more recently, the family
84 O-GlcNAcases..sup.108 Despite its potency, a downside to using
NAG-thiazoline in a complex biological context is that it lacks
selectivity and therefore perturbs multiple cellular processes.
[0020] PUGNAc is another compound that suffers from the same
problem of lack of selectivity, yet has enjoyed use as an inhibitor
of both human O-GlcNAcase.sup.6,110 and the family 20 human
.beta.-hexosaminidases..sup.111 This molecule, developed by Vasella
and coworkers, was found to be a potent competitive inhibitor of
the .beta.-N-acetyl-glucosaminidases from Canavalia ensiformis,
Mucor rouxii, and the .beta.-hexosaminidase from bovine
kidney..sup.88 It has been demonstrated that administration of
PUGNAc in a rat model of trauma hemorrhage decreases circulating
levels of the pro-inflammatory cytokines TNF-.alpha. and
IL-6..sup.112 It has also been shown that administration of PUGNAc
in a cell-based model of lymphocyte activation decreases production
of the cytokine IL-2..sup.113 Recent studies have indicated that
PUGNAc can be used in an animal model to reduce myocardial infarct
size after left coronary artery occlusions..sup.114 Of particular
significance is the fact that elevation of O-GlcNAc levels by
administration of PUGNAc, an inhibitor of O-GlcNAcase, in a rat
model of trauma hemorrhage improves cardiac function..sup.112,115
In addition, elevation of O-GlcNAc levels by treatment with PUGNAc
in a cellular model of ischemia/reperfusion injury using neonatal
rat ventricular myocytes improved cell viability and reduced
necrosis and apoptosis compared to untreated cells..sup.116
[0021] More recently, it has been suggested that the selective
O-GlcNAcase inhibitor NButGT exhibits protective activity in
cell-based models of ischemia/reperfusion and cellular stresses,
including oxidative stress..sup.117 This study suggests the use of
O-GlcNAcase inhibitors to elevate protein O-GlcNAc levels and
thereby prevent the pathogenic effects of stress in cardiac
tissue.
[0022] International patent applications PCT/CA2006/000300, filed 1
Mar. 2006, published under No. W0 2006/092049 on 8 Sep. 2006, and
PCT/CA2007/001554, filed 31 Aug. 2007, published under No. WO
2008/025170 on 6 Mar. 2008, which are hereby incorporated by
reference, describe selective inhibitors of O-GlcNAcase.
SUMMARY OF THE INVENTION
[0023] The invention provides, in part, compounds for selectively
inhibiting glycosidases, prodrugs of the compounds, uses of the
compounds and the prodrugs, pharmaceutical compositions including
the compounds or prodrugs of the compounds, and methods of treating
diseases and disorders related to deficiency or overexpression of
O-GlcNAcase, and/or accumulation or deficiency of O-GlcNAc.
[0024] In one aspect, the invention provides a compound of Formula
(I) or a pharmaceutically acceptable salt thereof:
##STR00002##
where each R.sup.1 may be independently a non-interfering
substituent; and R.sup.2 may be NR.sup.3.sub.2, where each R.sup.3
may be optionally independently a non-interfering substituent,
[0025] with the proviso that when each R.sup.1 is H, R.sup.2
excludes NH(COCH.sub.3), NH(CO(CH.sub.2).sub.2CH.sub.3),
NH(benzoyl), NH.sub.2, NH(CH.sub.3), NH(CH.sub.2CH.dbd.CH.sub.2),
NH((CH.sub.2).sub.3CH.sub.3), NH(CH.sub.2CH(CH.sub.3).sub.2),
NH(CH(CH.sub.3)(CH.sub.2CH.sub.3)),
NH(CH(CH.sub.3)(CH.sub.2CH.sub.2CH.sub.3), NH(benzyl),
NH(CH.sub.2CH.sub.2OCH.sub.3), N(CH.sub.2CH.sub.3).sub.2,
N(CH.sub.2CH.sub.3)((CH.sub.2).sub.3CH.sub.3),
NH(CH.sub.2).sub.7CH.sub.3, N(COCH.sub.3)(benzyl), N.sub.3, and
N(CH.sub.2CH.sub.2OH).sub.2; or when each R.sup.1 is C(O)CH.sub.3,
R.sup.2 excludes NH(COCH.sub.3), NH.sub.2, N.sub.3, and
N(CH.sub.2CH.sub.3).sub.2.
[0026] In alternative embodiments, each R.sup.1 may be connected to
another R.sup.1 to form an additional ring structure.
[0027] In alternative embodiments, the non-interfering substituent
may be alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, or
arylalkynyl, or may include one or more heteroatoms selected from
P, O, S, N, F, Cl, Br, I, or B. The non-interfering substituent may
be optionally substituted.
[0028] In alternative embodiments, the compound may be a prodrug;
the compound may selectively inhibit an O-glycoprotein
2-acetamido-2-deoxy-.beta.-D-glucopyranosidase (O-GlcNAcase); the
compound may selectively bind an O-GlcNAcase (e.g., a mammalian
O-GlcNAcase); the compound may selectively inhibit the cleavage of
a 2-acetamido-2-deoxy-.beta.-D-glucopyranoside (O-GlcNAc); the
compound may not substantially inhibit a mammalian
.beta.-hexosaminidase.
[0029] In alternative aspects, the invention provides a
pharmaceutical composition including a compound according to the
invention, in combination with a pharmaceutically acceptable
carrier.
[0030] In alternative aspects, the invention provides methods of
selectively inhibiting an O-GlcNAcase, or of inhibiting an
O-GlcNAcase in a subject in need thereof, or of increasing the
level of O-GlcNAc, or of treating a neurodegenerative disease, a
tauopathy, cancer or stress, in a subject in need thereof, by
administering to the subject an effective amount of a compound of
Formula (I) or a pharmaceutically acceptable salt thereof:
##STR00003##
where each R.sup.1 may be independently a non-interfering
substituent; and R.sup.2 may be NR.sup.3.sub.2, where each R.sup.3
may be optionally independently a non-interfering substituent.
[0031] The condition may be Alzheimer's disease, Amyotrophic
lateral sclerosis (ALS), Amyotrophic lateral sclerosis with
cognitive impairment (ALSci), Argyrophilic grain dementia, Bluit
disease, Corticobasal degeneration (CBD), Dementia pugilistica,
Diffuse neurofibrillary tangles with calcification, Down's
syndrome, Familial British dementia, Familial Danish dementia,
Frontotemporal dementia with parkinsonism linked to chromosome 17
(FTDP-17), Gerstmann-Straussler-Scheinker disease, Guadeloupean
parkinsonism, Hallevorden-Spatz disease (neurodegeneration with
brain iron accumulation type 1), Multiple system atrophy, Myotonic
dystrophy, Niemann-Pick disease (type C), Pallido-ponto-nigral
degeneration, Parkinsonism-dementia complex of Guam, Pick's disease
(PiD), Post-encephalitic parkinsonism (PEP), Prion diseases
(including Creutzfeldt-Jakob Disease (CJD), Variant
Creutzfeldt-Jakob Disease (vCJD), Fatal Familial Insomnia, and
Kuru), Progressive supercortical gliosis, Progressive supranuclear
palsy (PSP), Richardson's syndrome, Subacute sclerosing
panencephalitis, Tangle-only dementia, Huntington's disease, or
Parkinson's disease. The stress may be a cardiac disorder, e.g.,
ischemia; hemorrhage; hypovolemic shock; myocardial infarction; an
interventional cardiology procedure; cardiac bypass surgery;
fibrinolytic therapy; angioplasty; or stent placement.
[0032] In alternative aspects, the invention provides a method of
treating an O-GlcNAcase-mediated condition that excludes a
neurodegenerative disease, a tauopathy, cancer or stress, in a
subject in need thereof, by administering to the subject an
effective amount of a compound of Formula (I) or a pharmaceutically
acceptable salt thereof:
##STR00004##
[0033] wherein each R.sup.1 may be independently a non-interfering
substituent; R.sup.2 may be NR.sup.3.sub.2, where each R.sup.3 may
be optionally independently a non-interfering substituent. In some
embodiments, the condition may be inflammatory or allergic diseases
such as asthma, allergic rhinitis, hypersensitivity lung diseases,
hypersensitivity pneumonitis, eosinophilic pneumonias, delayed-type
hypersensitivity, atherosclerosis, interstitial lung disease (ILD)
(e.g., idiopathic pulmonary fibrosis, or ILD associated with
rheumatoid arthritis, systemic lupus erythematosus, ankylosing
spondylitis, systemic sclerosis, Sjogren's syndrome, polymyositis
or dermatomyositis); systemic anaphylaxis or hypersensitivity
responses, drug allergies, insect sting allergies; autoimmune
diseases, such as rheumatoid arthritis, psoriatic arthritis,
multiple sclerosis, systemic lupus erythematosus, myastenia gravis,
glomerulonephritis, autoimmune thyroiditis, graft rejection,
including allograft rejection or graft-versus-host disease;
inflammatory bowel diseases, such as Crohn's disease and ulcerative
colitis; spondyloarthropathies; scleroderma; psoriasis (including
T-cell mediated psoriasis) and inflammatory dermatoses such as
dermatitis, eczema, atopic dermatitis, allergic contact dermatitis,
urticaria; vasculitis (e.g., necrotizing, cutaneous, and
hypersensitivity vasculitis); eosinphilic myotis, and eosiniphilic
fasciitis; graft rejection, in particular but not limited to solid
organ transplants, such as heart, lung, liver, kidney, and pancreas
transplants (e.g. kidney and lung allografts); epilepsy; pain;
stroke, e.g., neuroprotection following a stroke.
[0034] In alternative embodiments, R.sup.1 may be H or
C(O)CH.sub.3. The administering may increase the level of O-GlcNAc
in the subject. The subject may be a human.
[0035] In alternative aspects, the invention provides use of a
compound of an effective amount of a compound of Formula (I) or a
pharmaceutically acceptable salt thereof:
##STR00005##
[0036] where each R.sup.1 may be independently a non-interfering
substituent; R.sup.2 may be NR.sup.3.sub.2, where each R.sup.3 may
be optionally independently a non-interfering substituent, in the
preparation of a medicament. The medicament may be for selectively
inhibiting an O-GlcNAcase, for increasing the level of O-GlcNAc,
for treating a condition modulated by an O-GlcNAcase, for treating
a neurodegenerative disease, a tauopathy, a cancer, or stress.
[0037] In alternative aspects, the invention provides a method for
screening for a selective inhibitor of an O-GlcNAcase, by a)
contacting a first sample with a test compound; b) contacting a
second sample with a compound of Formula (I)
##STR00006##
[0038] where each R.sup.1 may be independently a non-interfering
substituent; R.sup.2 may be NR.sup.3.sub.2, where each R.sup.3 may
be optionally independently a non-interfering substituent, c)
determining the level of inhibition of the O-GlcNAcase in the first
and second samples, where the test compound is a selective
inhibitor of a O-GlcNAcase if the test compound exhibits the same
or greater inhibition of the O-GlcNAcase when compared to the
compound of Formula (I).
[0039] This summary of the invention does not necessarily describe
all features of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] These and other features of the invention will become more
apparent from the following description in which reference is made
to the appended drawings wherein:
[0041] FIGS. 1A-B show Western blots of proteins from 3T3-L1 cells
cultured for 16 hours in the presence or absence of 100 .mu.M of
Compound 1 (Table 1). Incubation of 3T3-L1 cells with Compound 1
causes an increase in cellular levels of O-GlcNAc-modified
proteins; plus signs ("+") indicate presence of Compound 1, while
minus signs ("-") indicate absence of Compound 1. FIG. 1A: Western
blot analysis of cellular levels of O-GlcNAc-modified proteins
using anti-O-GlcNAc MAb CTD 110.6 followed by an anti-mouse IgG-HRP
conjugate. FIG. 1B: Western blot of samples loaded in FIG. 1A
treated with anti-.beta.-actin mAb Clone AC-40 followed by an
anti-mouse IgG-HRP conjugate reveals equivalent .beta.-actin levels
in each sample.
DETAILED DESCRIPTION
[0042] The invention provides, in part, novel compounds that are
capable of inhibiting an O-glycoprotein
2-acetamido-2-deoxy-.beta.-D-glucopyranosidase (O-GlcNAcase). In
some embodiments, the O-GlcNAcase is a mammalian O-GlcNAcase, such
as a rat, mouse or human O-GlcNAcase. In some embodiments, the
.beta.-hexosaminidase is a mammalian .beta.-hexosaminidase, such as
a rat, mouse or human .beta.-hexosaminidase.
[0043] In some embodiments, compounds according to the invention
exhibit a surprising and unexpected selectivity in inhibiting an
O-GlcNAcase. In some embodiments, the compounds according to the
invention are surprisingly more selective for an O-GlcNAcase over a
.beta.-hexosaminidase. In some embodiments, the compounds
selectively inhibit the activity of a mammalian O-GlcNAcase over a
mammalian .beta.-hexosaminidase. In some embodiments, a selective
inhibitor of an O-GlcNAcase does not substantially inhibit a
.beta.-hexosaminidase. A compound that "selectively" inhibits an
O-GlcNAcase is a compound that inhibits the activity or biological
function of an O-GlcNAcase, but does not substantially inhibit the
activity or biological function of a .beta.-hexosaminidase. For
example, in some embodiments, a selective inhibitor of an
O-GlcNAcase selectively inhibits the cleavage of
2-acetamido-2-deoxy-.beta.-D-glucopyranoside (O-GlcNAc) from
polypeptides. In some embodiments, a selective inhibitor of an
O-GlcNAcase selectively binds to an O-GlcNAcase. In some
embodiments, a selective inhibitor of an O-GlcNAcase inhibits
hyperphosphorylation of a tau protein and/or inhibits formations of
NFTs. By "inhibits," "inhibition" or "inhibiting" means a decrease
by any value between 10% and 90%, or of any integer value between
30% and 60%, or over 100%, or a decrease by 1-fold, 2-fold, 5-fold,
10-fold or more. It is to be understood that the inhibiting does
not require full inhibition. In some embodiments, a selective
inhibitor of an O-GlcNAcase elevates or enhances O-GlcNAc levels
e.g., O-GlcNAc-modified polypeptide or protein levels, in cells,
tissues, or organs (e.g., in brain, muscle, or heart (cardiac)
tissue) and in animals. By "elevating" or "enhancing" is meant an
increase by any value between 10% and 90%, or of any integer value
between 30% and 60%, or over 100%, or an increase by 1-fold,
2-fold, 5-fold, 10-fold, 15-fold, 25-fold, 50-fold, 100-fold or
more. In some embodiments, a selective inhibitor of an O-GlcNAcase
exhibits a selectivity ratio, as described herein, in the range 100
to 100000, or in the range 1000 to 100000, or at least 100, 200,
500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000,
7000, 10,000, 25,000, 50,000, 75,000, or any value within or about
the described range.
[0044] The compounds of the present invention elevate O-GlcNAc
levels on O-GlcNAc-modified polypeptides or proteins in vivo
specifically via interaction with an O-GlcNAcase enzyme, and are
effective in treating conditions which require or respond to
inhibition of O-GlcNAcase activity.
[0045] In some embodiments, the compounds of the present invention
are useful as agents that produce a decrease in tau phosphorylation
and NFT formation. In some embodiments, the compounds are therefore
useful to treat Alzheimer's disease and related tauopathies. In
some embodiments, the compounds are thus capable of treating
Alzheimer's disease and related tauopathies by lowering tau
phosphorylation and reducing NFT formation as a result of
increasing tau O-GlcNAc levels. In some embodiments, the compounds
produce an increase in levels of O-GlcNAc modification on
O-GlcNAc-modified polypeptides or proteins, and are therefore
useful for treatment of disorders responsive to such increases in
O-GlcNAc modification; these disorders include without limitation
neurodegenerative, inflammatory, cardiovascular, and
immunoregulatory diseases. In some embodiments, the compounds are
also useful as a result of other biological activities related to
their ability to inhibit the activity of glycosidase enzymes. In
alternative embodiments, the compounds of the invention are
valuable tools in studying the physiological role of O-GlcNAc at
the cellular and organismal level.
[0046] In alternative embodiments, the invention provides methods
of enhancing or elevating levels of protein O-GlcNAc modification
in animal subjects, such as, veterinary and human subjects. In
alternative embodiments, the invention provides methods of
selectively inhibiting an O-GlcNAcase enzyme in animal subjects,
such as, veterinary and human subjects. In alternative embodiments,
the invention provides methods of inhibiting phosphorylation of tau
polypeptides, or inhibiting formation of NFTs, in animal subjects,
such as, veterinary and human subjects.
[0047] In specific embodiments, the invention provides compounds
described generally by Formula (I) and the salts, prodrugs, and
stereoisomeric forms thereof:
##STR00007##
[0048] As set forth in Formula (I): each R.sup.1 can be
independently a non-interfering substituent; R.sup.2 can be
NR.sup.3.sub.2, where each R.sup.3 may be optionally independently
a non-interfering substituent. In some embodiments, each R.sup.1
may be connected to another R.sup.1 to form an additional ring
structure.
[0049] In the above Formula (I), each optionally substituted moiety
may be substituted with one or more non-interfering substituents.
For example, each optionally substituted moiety may be substituted
with one or more inorganic substituents; phosphoryl; halo; .dbd.O;
.dbd.NR.sup.4; OR; C.sub.1-10 alkyl or C.sub.2-10 alkenyl
optionally containing one or more P, N, O, S, N, F, Cl, Br, I, or B
and optionally substituted with halo; CN; optionally substituted
carbonyl; NR.sup.4.sub.2; C.dbd.NR.sup.4; an optionally substituted
carbocyclic or heterocyclic ring; or an optionally substituted aryl
or heteroaryl. R.sup.4 may be alkyl, branched alkyl, cycloalkyl,
aryl, or heteroaryl.
[0050] In some embodiments, R.sup.1 as set forth in Formula (I),
may be either hydrogen or a substituent that includes 1-20 atoms
that are other than hydrogen. In some embodiments, R.sup.1 may be
H, alkyl, or C(O)R.sup.4, where R.sup.4 may be alkyl, branched
alkyl, cycloalkyl, aryl, or heteroaryl. In some embodiments,
R.sup.1 may be H or C(O)CH.sub.3.
[0051] In some embodiments, R.sup.2 as set forth in Formula (I),
may be optionally substituted NR.sup.5.sub.2, where R.sup.5 may be
H, alkyl, branched alkyl, cycloalkyl, aryl, or heteroaryl.
[0052] In specific embodiments of the invention, compounds
according to Formula (I) include the compounds described in Table
1.
TABLE-US-00001 TABLE 1 Compound Name Structure 1
(N-((1S,6S,7R,8R,8aR)-1,7,8- trihydroxy-octahydroindolizin-6-
yl)acetamide ##STR00008## 2 N-((1S,6S,7R,8R,8aR)-1,7,8-
trihydroxy-octahydroindolizin-6- yl)butyramide ##STR00009## 3
N-((1S,6S,7R,8R,8aR)-1,7,8- trihydroxy-octahydroindolizin-6-
yl)benzamide ##STR00010## 4 (1S,6S,7R,8R,8aR)-6-amino-
octahydroindolizine-1,7,8-triol ##STR00011## 5
(1S,6S,7R,8R,8aR)-6-(methylamino)- octahydroindolizine-1,7,8-triol
##STR00012## 6 (1S,6S,7R,8R,8aR)-6-(allylamino)-
octahydroindolizine-1,7,8-triol ##STR00013## 7
(1S,6S,7R,8R,8aR)-6-(butylamino)- octahydroindolizine-1,7,8-triol
##STR00014## 8 (1S,6S,7R,8R,8aR)-6-
(isobutylamino)-octahydroindolizine- 1,7,8-triol ##STR00015## 9
(1S,6S,7R,8R,8aR)-6-(sec- butylamino)-octahydroindolizine-
1,7,8-triol ##STR00016## 10 (1S,6S,7R,8R,8aR)-6-(pentan-2-
ylamino)-octahydroindolizine-1,7,8- triol ##STR00017## 11
(1S,6S,7R,8R,8aR)-6-(benzylamino)- octahydroindolizine-1,7,8-triol
##STR00018## 12 (1S,6S,7R,8R,8aR)-6-(2- methoxyethylamino)-
octahydroindolizine-1,7,8-triol ##STR00019## 13
(1S,6S,7R,8R,8aR)-6-(diethylamino)- octahydroindolizine-1,7,8-triol
##STR00020## 14 (1S,6S,7R,8R,8aR)-6- (butyl(ethyl)amino)-
octahydroindolizine-1,7,8-triol ##STR00021## 15
(1S,6S,7R,8R,8aR)-6-(bis(2- hydroxyethyl)amino)-
octahydroindolizine-1,7,8-triol ##STR00022## 16
(1S,6S,7R,8R,8aR)-6-(octylamino)- octahydroindolizine-1,7,8-triol
##STR00023## 17 N-benzyl-N-((1S,6S,7R,8R,8aR)-
1,7,8-trihydroxy-octahydroindolizin- 6-yl)acetamide ##STR00024## 18
(1S,6S,7R,8R,8aR)-6-azido- octahydroindolizine-1,7,8-triol
##STR00025## 19 (1S,6S,7R,8R,8aR)-6-acetamido-
octahydroindolizine-1,7,8-triyl triacetate ##STR00026## 20
(1S,6S,7R,8R,8aR)-6-amino- octahydroindolizine-1,7,8-triyl
triacetate ##STR00027## 21 (1S,6S,7R,8R,8aR)-6-azido-
octahydroindolizine-1,7,8-triyl triacetate ##STR00028## 22
(1S,6S,7R,8R,8aR)-6-(diethylamino)- octahydroindolizine-1,7,8-triyl
triacetate ##STR00029##
[0053] In alternative embodiments of the invention, compounds
according to Formula (I) include one or more of the compounds
described in Table 2.
TABLE-US-00002 TABLE 2 Compound Name Structure 23
N-((1S,6S,7R,8R,8aR)-1,7,8- trihydroxy-octahydroindolizin-
6-yl)formamide ##STR00030## 24 N-((1S,6S,7R,8R,8aR)-1,7,8-
trihydroxy-octahydroindolizin- 6-yl)propionamide ##STR00031## 25
N-((1S,6S,7R,8R,8aR)-1,7,8- trihydroxy-octahydroindolizin-
6-yl)pentanamide ##STR00032## 26 N-((1S,6S,7R,8R,8aR)-1,7,8-
trihydroxy-octahydroindolizin- 6-yl)hexanamide ##STR00033## 27
N-((1S,6S,7R,8R,8aR)-1,7,8- trihydroxy-octahydroindolizin-
6-yl)isobutyramide ##STR00034## 28 3-methyl-N-
((1S,6S,7R,8R,8aR)-1,7,8- trihydroxy-octahydroindolizin-
6-yl)butanamide ##STR00035## 29 N-methyl-N-
((1S,6S,7R,8R,8aR)-1,7,8- trihydroxy-octahydroindolizin-
6-yl)acetamide ##STR00036## 30 N-ethyl-N- ((1S,6S,7R,8R,8aR)-1,7,8-
trihydroxy-octahydroindolizin- 6-yl)acetamide ##STR00037## 31
N-methyl-N- ((1S,6S,7R,8R,8aR)-1,7,8-
trihydroxy-octahydroindolizin- 6-yl)propionamide ##STR00038## 32
1-((1S,6S,7R,8R,8aR)-1,7,8- trihydroxy-octahydroindolizin-
6-yl)urea ##STR00039## 33 1-methyl-3- ((1S,6S,7R,8R,8aR)-1,7,8-
trihydroxy-octahydroindolizin- 6-yl)urea ##STR00040## 34
1-ethyl-3-((1S,6S,7R,8R,8aR)- 1,7,8-trihydroxy-
octahydroindolizin-6-yl)urea ##STR00041## 35 1-propyl-3-
((1S,6S,7R,8R,8aR)-1,7,8- trihydroxy-octahydroindolizin- 6-yl)urea
##STR00042## 36 1-butyl-3-((1S,6S,7R,8R,8aR)- 1,7,8-trihydroxy-
octahydroindolizin-6-yl)urea ##STR00043## 37
1-allyl-3-((1S,6S,7R,8R,8aR)- 1,7,8-trihydroxy-
octahydroindolizin-6-yl)urea ##STR00044## 38 1,1-dimethyl-3-
((1S,6S,7R,8R,8aR)-1,7,8- trihydroxy-octahydroindolizin- 6-yl)urea
##STR00045## 39 1-ethyl-1-methyl-3- ((1S,6S,7R,8R,8aR)-1,7,8-
trihydroxy-octahydroindolizin- 6-yl)urea ##STR00046## 40
N-((1S,6S,7R,8R,8aR)-1,7,8- trihydroxy-octahydroindolizin-
6-yl)pyrrolidine-1- carboxamide ##STR00047## 41 1,3-dimethyl-1-
((1S,6S,7R,8R,8aR)-1,7,8- trihydroxy-octahydroindolizin- 6-yl)urea
##STR00048## 42 1,1,3-trimethyl-3- ((1S,6S,7R,8R,8aR)-1,7,8-
trihydroxy-octahydroindolizin- 6-yl)urea ##STR00049## 43
(1S,6S,7R,8R,8aR)-6- (ethylamino)- octahydroindolizine-1,7,8-triol
##STR00050## 44 (1S,6S,7R,8R,8aR)-6- (propylamino)-
octahydroindolizine-1,7,8-triol ##STR00051## 45
(1S,6S,7R,8R,8aR)-6- (isopropylamino)-
octahydroindolizine-1,7,8-triol ##STR00052## 46
(1S,6S,7R,8R,8aR)-6- (dimethylamino)-
octahydroindolizine-1,7,8-triol ##STR00053## 47
(1S,6S,7R,8R,8aR)-6- (ethyl(methyl)amino)-
octahydroindolizine-1,7,8-triol ##STR00054## 48
(1S,6S,7R,8R,8aR)-6- (methyl(propyl)amino)-
octahydroindolizine-1,7,8-triol ##STR00055## 49
(1S,6S,7R,8R,8aR)-6- (butyl(methyl)amino)-
octahydroindolizine-1,7,8-triol ##STR00056## 50
(1S,6S,7R,8R,8aR)-6- (allyl(methyl)amino)-
octahydroindolizine-1,7,8-triol ##STR00057## 51
(1S,6S,7R,8R,8aR)-6- (pyrrolidin-1-yl)-
octahydroindolizine-1,7,8-triol ##STR00058##
[0054] In alternative embodiments of the invention, one or more of
the compounds described in Table 1 are specifically excluded from
the compounds described in Formula (I). In alternative embodiments
of the invention, specific stereoisomers or enantiomers of one or
more of the compounds described in Table 1 are specifically
excluded from the compounds described in Formula (I). In
alternative embodiments of the invention, specific precursors of
one or more of the compounds described in Table 1 are specifically
excluded from the compounds described in Formula (I).
[0055] In alternative embodiments, when each R.sup.1 is H, R.sup.2
excludes NH(COCH.sub.3), NH(CO(CH.sub.2).sub.2CH.sub.3),
NH(benzoyl), NH.sub.2, NH(CH.sub.3), NH(CH.sub.2CH.dbd.CH.sub.2),
NH((CH.sub.2).sub.3CH.sub.3), NH(CH.sub.2CH(CH.sub.3).sub.2),
NH(CH(CH.sub.3)(CH.sub.2CH.sub.3)),
NH(CH(CH.sub.3)(CH.sub.2CH.sub.2CH.sub.3), NH(benzyl),
NH(CH.sub.2CH.sub.2OCH.sub.3), N(CH.sub.2CH.sub.3).sub.2,
N(CH.sub.2CH.sub.3)((CH.sub.2).sub.3CH.sub.3),
NH(CH.sub.2).sub.7CH.sub.3, N(COCH.sub.3)(benzyl), N.sub.3, and
N(CH.sub.2CH.sub.2OH).sub.2; and when each R.sup.1 is C(O)CH.sub.3,
R.sup.2 excludes NH(COCH.sub.3), NH.sub.2, N.sub.3, and
N(CH.sub.2CH.sub.3).sub.2.
[0056] As will be appreciated by a person skilled in the art,
Formula (I) above may also be represented alternatively as
follows:
##STR00059##
[0057] As used herein the singular forms "a", "and", and "the"
include plural referents unless the context clearly dictates
otherwise. For example, "a compound" refers to one or more of such
compounds, while "the enzyme" includes a particular enzyme as well
as other family members and equivalents thereof as known to those
skilled in the art.
[0058] Throughout this application, it is contemplated that the
term "compound" or "compounds" refers to the compounds discussed
herein and includes precursors and derivatives of the compounds,
including acyl-protected derivatives, and pharmaceutically
acceptable salts of the compounds, precursors, and derivatives. The
invention also includes prodrugs of the compounds, pharmaceutical
compositions including the compounds and a pharmaceutically
acceptable carrier, and pharmaceutical compositions including
prodrugs of the compounds and a pharmaceutically acceptable
carrier.
[0059] In some embodiments, all of the compounds of the invention
contain at least one chiral center. In some embodiments, the
formulations, preparation, and compositions including compounds
according to the invention include mixtures of stereoisomers,
individual stereoisomers, and enantiomeric mixtures, and mixtures
of multiple stereoisomers. In general, the compound may be supplied
in any desired degree of chiral purity.
[0060] In general, a "non-interfering substituent" is a substituent
whose presence does not destroy the ability of the compound of
Formula (I) to modulate the activity of the O-GlcNAcase enzyme.
Specifically, the presence of the substituent does not destroy the
effectiveness of the compound as a modulator of the activity of the
O-GlcNAcase enzyme.
[0061] Suitable non-interfering substituents include: H, alkyl
(C.sub.1-10), alkenyl (C.sub.2-10), alkynyl (C.sub.2-10), aryl
(5-12 members), arylalkyl, arylalkenyl, or arylalkynyl, each of
which may optionally contain one or more heteroatoms selected from
O, S, P, N, F, Cl, Br, I, or B and each of which may be further
substituted, for example, by .dbd.O; or optionally substituted
forms of acyl, arylacyl, alkyl- alkenyl-, alkynyl- or arylsulfonyl
and forms thereof which contain heteroatoms in the alkyl, alkenyl,
alkynyl or aryl moieties. Other noninterfering substituents include
.dbd.O, .dbd.NR, halo, CN, CF.sub.3, CHF.sub.2, NO.sub.2, OR, SR,
NR.sub.2, N.sub.3, COOR, and CONR.sub.2, where R is H or alkyl,
cycloalkyl, alkenyl, alkynyl, aryl, or heteroaryl. Where the
substituted atom is C, the substituents may include, in addition to
the substituents listed above, halo, OOCR, NROCR, where R is H or a
substituent set forth above.
[0062] "Alkyl" refers to a straight or branched hydrocarbon chain
group consisting solely of carbon and hydrogen atoms, containing no
unsaturation and including, for example, from one to ten carbon
atoms, and which is attached to the rest of the molecule by a
single bond. Unless stated otherwise specifically in the
specification, the alkyl group may be optionally substituted by one
or more substituents as described herein. Unless stated otherwise
specifically herein, it is understood that the substitution can
occur on any carbon of the alkyl group.
[0063] "Alkenyl" refers to a straight or branched hydrocarbon chain
group consisting solely of carbon and hydrogen atoms, containing at
least one double bond and including, for example, from two to ten
carbon atoms, and which is attached to the rest of the molecule by
a single bond or a double bond. Unless stated otherwise
specifically in the specification, the alkenyl group may be
optionally substituted by one or more substituents as described
herein. Unless stated otherwise specifically herein, it is
understood that the substitution can occur on any carbon of the
alkenyl group.
[0064] "Alkynyl" refers to a straight or branched hydrocarbon chain
group consisting solely of carbon and hydrogen atoms, containing at
least one triple bond and including, for example, from two to ten
carbon atoms. Unless stated otherwise specifically in the
specification, the alkenyl group may be optionally substituted by
one or more substituents as described herein.
[0065] "Aryl" refers to a phenyl or naphthyl group, including for
example, 5-12 members. Unless stated otherwise specifically herein,
the term "aryl" is meant to include aryl groups optionally
substituted by one or more substituents as described herein.
[0066] "Arylalkyl" refers to a group of the formula
--R.sub.aR.sub.b where R.sub.a is an alkyl group as described
herein and R.sub.b is one or more aryl moieties as described
herein. The aryl group(s) may be optionally substituted as
described herein.
[0067] "Arylalkenyl" refers to a group of the formula
--R.sub.cR.sub.b where R.sub.c is an alkenyl moiety as described
herein and R.sub.b is one or more aryl groups as described herein.
The aryl group(s) and the alkenyl group may be optionally
substituted as described herein.
[0068] "Acyl" refers to a group of the formula --C(O)R.sub.a, where
R.sub.a is an alkyl group as described herein. The alkyl group(s)
may be optionally substituted as described herein.
[0069] "Arylacyl" refers to a group of the formula --C(O)R.sub.b,
where R.sub.b is an aryl group as described herein. The aryl
group(s) may be optionally substituted as described herein.
[0070] "Cycloalkyl" refers to a stable monovalent monocyclic,
bicyclic or tricyclic hydrocarbon group consisting solely of carbon
and hydrogen atoms, having for example from 3 to 15 carbon atoms,
and which is saturated and attached to the rest of the molecule by
a single bond. Unless otherwise stated specifically herein, the
term "cycloalkyl" is meant to include cycloalkyl groups which are
optionally substituted as described herein.
[0071] By a "ring structure" is meant a cycloalkyl, aryl,
heteroaryl, or any cyclic structure that may be optionally
substituted.
[0072] "Optional" or "optionally" means that the subsequently
described event of circumstances may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances in which it does not. For example, "optionally
substituted alkyl" means that the alkyl group may or may not be
substituted and that the description includes both substituted
alkyl groups and alkyl groups having no substitution. Examples of
optionally substituted alkyl groups include, without limitation,
methyl, ethyl, propyl, etc. and including cycloalkyls such as
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
etc.; examples of optionally substituted alkenyl groups include
allyl, crotyl, 2-pentenyl, 3-hexenyl, 2-cyclopentenyl,
2-cyclohexenyl, 2-cyclopentenylmethyl, 2-cyclohexenylmethyl, etc.
In some embodiments, optionally substituted alkyl and alkenyl
groups include C.sub.1-6 alkyls or alkenyls.
[0073] "Halo" refers to bromo, chloro, fluoro, iodo, etc. In some
embodiments, suitable halogens include fluorine or chlorine.
[0074] An amino group may also be substituted once or twice (to
form a secondary or tertiary amine) with a group such as an
optionally substituted alkyl group including C.sub.1-10alkyl (e.g.,
methyl, ethyl propyl etc.); an optionally substituted alkenyl group
such as allyl, crotyl, 2-pentenyl, 3-hexenyl, etc., or an
optionally substituted cycloalkyl group such as cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. In these
cases, C.sub.1-6 alkyl, alkenyl and cycloalkyl are preferred. The
amine group may also be optionally substituted with an aromatic or
heterocyclic group, aralkyl (e.g., phenylC.sub.1-4alkyl) or
heteroalkyl for example, phenyl, pyridine, phenylmethyl (benzyl),
phenethyl, pyridinylmethyl, pyridinylethyl, etc. The heterocyclic
group may be a 5 or 6 membered ring containing 1-4 heteroatoms.
[0075] An amino group may be substituted with an optionally
substituted C.sub.2-4 alkanoyl, e.g., acetyl, propionyl, butyryl,
isobutyryl etc., or a C.sub.1-4alkylsulfonyl (e.g.,
methanesulfonyl, ethanesulfonyl, etc.) or a carbonyl or sulfonyl
substituted aromatic or heterocyclic ring, e.g., benzenesulfonyl,
benzoyl, pyridinesulfonyl, pyridinecarbonyl etc. The heterocycles
are as described herein.
Examples of optionally substituted carbonyl groups, or sulfonyl
groups include optionally substituted forms of such groups formed
from various hydrocarbyls such as alkyl, alkenyl and 5- to
6-membered monocyclic aromatic group (e.g., phenyl, pyridyl, etc.),
as described herein.
Therapeutic Indications
[0076] The invention provides methods of treating conditions that
are modulated, directly or indirectly, by an O-GlcNAcase enzyme or
by O-GlcNAc-modified protein levels, for example, a condition that
is benefited by inhibition of an O-GlcNAcase enzyme or by an
elevation of O-GlcNAc-modified protein levels Such conditions
include, without limitation, Glaucoma, Schizophrenia, tauopathies,
such as Alzheimer's disease, neurodegenerative diseases,
cardiovascular diseases, diseases associated with inflammation,
diseases associated with immunosuppression and cancers. The
compounds of the invention are also useful in the treatment of
diseases or disorders related to deficiency or over-expression of
O-GlcNAcase or accumulation or depletion of O-GlcNAc, or any
disease or disorder responsive to glycosidase inhibition therapy.
Such diseases and disorders include, but are not limited to,
Glaucoma, Schizophrenia, neurodegenerative disorders, such as
Alzheimer's disease (AD), or cancer. Such diseases and disorders
may also include diseases or disorders related to the accumulation
or deficiency in the enzyme OGT. Also included is a method of
protecting or treating target cells expressing proteins that are
modified by O-GlcNAc residues, the dysregulation of which
modification results in disease or pathology. The term "treating"
as used herein includes treatment, prevention, and
amelioration.
[0077] In alternative embodiments, the invention provides methods
of enhancing or elevating levels of protein O-GlcNAc modification
in animal subjects, such as, veterinary and human subjects. This
elevation of O-GlcNAc levels can be useful for the prevention or
treatment of Alzheimer's disease; prevention or treatment of other
neurodegenerative diseases (e.g. Parkinson's disease, Huntington's
disease); providing neuroprotective effects; preventing damage to
cardiac tissue; and treating diseases associated with inflammation
or immunosuppression.
[0078] In alternative embodiments, the invention provides methods
of selectively inhibiting an O-GlcNAcase enzyme in animal subjects,
such as veterinary and human subjects.
[0079] In alternative embodiments, the invention provides methods
of inhibiting phosphorylation of tau polypeptides, or inhibiting
formation of NFTs, in animal subjects, such as, veterinary and
human subjects. Accordingly, the compounds of the invention may be
used to study and treat AD and other tauopathies.
[0080] In general, the methods of the invention are effected by
administering a compound according to the invention to a subject in
need thereof, or by contacting a cell or a sample with a compound
according to the invention, for example, a pharmaceutical
composition comprising a therapeutically effective amount of the
compound according to Formula (I). More particularly, they are
useful in the treatment of a disorder in which the regulation of
O-GlcNAc protein modification is implicated, or any condition as
described herein. Disease states of interest include Alzheimer's
disease (AD) and related neurodegenerative tauopathies, in which
abnormal hyperphosphorylation of the microtubule-associated protein
tau is involved in disease pathogenesis. In some embodiments, the
compounds may be used to block hyperphosphorylation of tau by
maintaining elevated levels of O-GlcNAc on tau, thereby providing
therapeutic benefit.
[0081] Tauopathies that may be treated with the compounds of the
invention include: Alzheimer's disease, Amyotrophic lateral
sclerosis (ALS), Amyotrophic lateral sclerosis with cognitive
impairment (ALSci), Argyrophilic grain dementia, Bluit disease,
Corticobasal degeneration (CBD), Dementia pugilistica, Diffuse
neurofibrillary tangles with calcification, Down's syndrome,
Familial British dementia, Familial Danish dementia, Frontotemporal
dementia with parkinsonism linked to chromosome 17 (FTDP-17),
Gerstmann-Straussler-Scheinker disease, Guadeloupean parkinsonism,
Hallevorden-Spatz disease (neurodegeneration with brain iron
accumulation type 1), Multiple system atrophy, Myotonic dystrophy,
Niemann-Pick disease (type C), Pallido-ponto-nigral degeneration,
Parkinsonism-dementia complex of Guam, Pick's disease (PiD),
Post-encephalitic parkinsonism (PEP), Prion diseases (including
Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease
(vCJD), Fatal Familial Insomnia, and Kuru), Progressive
supercortical gliosis, Progressive supranuclear palsy (PSP),
Richardson's syndrome, Subacute sclerosing panencephalitis, and
Tangle-only dementia.
[0082] The compounds of this invention are also useful in the
treatment of conditions associate with tissue damage or stress,
stimulating cells, or promoting differentiation of cells.
Accordingly, in some embodiments, the compounds of this invention
may be used to provide therapeutic benefit in a variety of
conditions or medical procedures involving stress in cardiac
tissue, including but not limited to: ischemia; hemorrhage;
hypovolemic shock; myocardial infarction; an interventional
cardiology procedure; cardiac bypass surgery; fibrinolytic therapy;
angioplasty; and stent placement.
[0083] Compounds that selectively inhibit O-GlcNAcase activity may
be used for the treatment of diseases that are associated with
inflammation, including but not limited to, inflammatory or
allergic diseases such as asthma, allergic rhinitis,
hypersensitivity lung diseases, hypersensitivity pneumonitis,
eosinophilic pneumonias, delayed-type hypersensitivity,
atherosclerosis, interstitial lung disease (ILD) (e.g., idiopathic
pulmonary fibrosis, or ILD associated with rheumatoid arthritis,
systemic lupus erythematosus, ankylosing spondylitis, systemic
sclerosis, Sjogren's syndrome, polymyositis or dermatomyositis);
systemic anaphylaxis or hypersensitivity responses, drug allergies,
insect sting allergies; autoimmune diseases, such as rheumatoid
arthritis, psoriatic arthritis, multiple sclerosis, systemic lupus
erythematosus, myastenia gravis, glomerulonephritis, autoimmune
thyroiditis, graft rejection, including allograft rejection or
graft-versus-host disease; inflammatory bowel diseases, such as
Crohn's disease and ulcerative colitis; spondyloarthropathies;
scleroderma; psoriasis (including T-cell mediated psoriasis) and
inflammatory dermatoses such as dermatitis, eczema, atopic
dermatitis, allergic contact dermatitis, urticaria; vasculitis
(e.g., necrotizing, cutaneous, and hypersensitivity vasculitis);
eosinphilic myotis, eosiniphilic fasciitis; and cancers.
[0084] In addition, compounds that affects levels of protein
O-GlcNAc modification may be used for the treatment of diseases
associated with immunosuppression, such as in individuals
undergoing chemotherapy, radiation therapy, enhanced wound healing
and burn treatment, therapy for autoimmune disease or other drug
therapy (e.g., corticosteroid therapy) or combination of
conventional drugs used in the treatment of autoimmune diseases and
graft/transplantation rejection, which causes immunosuppression; or
immunosuppression due to congenital deficiency in receptor function
or other causes.
[0085] The compounds of the invention may be useful for treatment
of neurodegenerative diseases, including Parkinson's disease and
Huntington's disease. Other conditions that may be treated are
those triggered, affected, or in any other way correlated with
levels of O-GlcNAc post-translational protein modification. It is
expected that the compounds of this invention may be useful for the
treatment of such conditions and in particular, but not limited to,
the following for which a association with O-GlcNAc levels on
proteins has been established: graft rejection, in particular but
not limited to solid organ transplants, such as heart, lung, liver,
kidney, and pancreas transplants (e.g. kidney and lung allografts);
cancer, in particular but not limited to cancer of the breast,
lung, prostate, pancreas, colon, rectum, bladder, kidney, ovary; as
well as non-Hodgkin's lymphoma and melanoma; epilepsy, pain, or
stroke, e.g., for neuroprotection following a stroke.
Pharmaceutical & Veterinary Compositions, Dosages, and
Administration
[0086] Pharmaceutical compositions including compounds according to
the invention, or for use according to the invention, are
contemplated as being within the scope of the invention. In some
embodiments, pharmaceutical compositions including an effective
amount of a compound of Formula (I) are provided.
[0087] The compounds of formula (I) and their pharmaceutically
acceptable salts, stereoisomers, solvates, and derivatives are
useful because they have pharmacological activity in animals,
including humans. In some embodiments, the compounds according to
the invention are stable in plasma, when administered to a
subject.
[0088] In some embodiments, compounds according to the invention,
or for use according to the invention, may be provided in
combination with any other active agents or pharmaceutical
compositions where such combined therapy is useful to modulate
O-GlcNAcase activity, for example, to treat neurodegenerative,
inflammatory, cardiovascular, or immunoregulatory diseases, or any
condition described herein. In some embodiments, compounds
according to the invention, or for use according to the invention,
may be provided in combination with one or more agents useful in
the prevention or treatment of Alzheimer's disease. Examples of
such agents include, without limitation, [0089] acetylcholine
esterase inhibitors (AChEIs) such as Aricept.RTM. (Donepezil),
Exelon.RTM.(Rivastigmine), Razadyne.RTM. (Razadyne ER.RTM.,
Reminyl.RTM., Nivalin.RTM., Galantamine), Cognex.RTM. (Tacrine),
Dimebon, Huperzine A, Phenserine, Debio-9902 SR (ZT-1 SR),
Zanapezil (TAK0147), ganstigmine, NP7557, etc.; [0090] NMDA
receptor antagonists such as Namenda.RTM. (Axura.RTM.,
Akatinol.RTM., Ebixa.RTM., Memantine), Dimebon, SGS-742,
Neramexane, Debio-9902 SR (ZT-1 SR), etc.; [0091] gamma-secretase
inhibitors and/or modulators such as Flurizan.TM. (Tarenflurbil,
MPC-7869, R-flurbiprofen), LY450139, MK 0752, E2101, BMS-289948,
BMS-299897, BMS-433796, LY-411575, GSI-136, etc.; [0092]
beta-secretase inhibitors such as ATG-Z1, CTS-21166, etc.; [0093]
alpha-secretase activators, such as NGX267, etc; [0094]
amyloid-.beta. aggregation and/or fibrillization inhibitors such as
Alzhemed.TM. (3APS, Tramiprosate, 3-amino-1-propanesulfonic acid),
AL-108, AL-208, AZD-103, PBT2, Cereact, ONO-2506PO, PPI-558, etc.;
[0095] tau aggregation inhibitors such as methylene blue, etc.;
[0096] microtubule stabilizers such as AL-108, AL-208, paclitaxel,
etc.; [0097] RAGE inhibitors, such as TTP488, etc.; [0098] 5-HT1a
receptor antagonists, such as Xaliproden, Lecozotan, etc.; [0099]
5-HT4 receptor antagonists, such as PRX-03410, etc.; [0100] kinase
inhibitors such as SRN-003-556, amfurindamide, LiCl, AZD1080,
NP031112, SAR-502250, etc. [0101] humanized monoclonal anti-A.beta.
antibodies such as Bapineuzumab (AAB-001), LY2062430, RN1219,
ACU-5A5, etc.; [0102] amyloid vaccines such as AN-1792, ACC-001
[0103] neuroprotective agents such as Cerebrolysin, AL-108, AL-208,
Huperzine A, etc.; [0104] L-type calcium channel antagonists such
as MEM-1003, etc.; [0105] nicotinic receptor antagonists, such as
AZD3480, GTS-21, etc.; [0106] nicotinic receptor agonists, such as
MEM 3454, Nefiracetam, etc.; [0107] peroxisome
proliferator-activated receptor (PPAR) gamma agonists such as
Avandia.RTM. (Rosglitazone), etc.; [0108] phosphodiesterase IV
(PDE4) inhibitors, such as MK-0952, etc.; [0109] hormone
replacement therapy such as estrogen (Premarin), etc.; [0110]
monoamine oxidase (MAO) inhibitors such as NS2330, Rasagiline
(Azilect.RTM.), TVP-1012, etc.; [0111] AMPA receptor modulators
such as Ampalex (CX 516), etc.; [0112] nerve growth factors or NGF
potentiators, such as CERE-110 (AAV-NGF), T-588, T-817MA, etc.;
[0113] agents that prevent the release of luteinizing hormone (LH)
by the pituitary gland, such as leuoprolide (VP-4896), etc.; [0114]
GABA receptor modulators such as AC-3933, NGD 97-1, CP-457920,
etc.; [0115] benzodiazepine receptor inverse agonists such as
SB-737552 (S-8510), AC-3933, etc.; [0116] noradrenaline-releasing
agents such as T-588, T-817MA, etc.
[0117] It is to be understood that combination of compounds
according to the invention, or for use according to the invention,
with Alzheimer's agents is not limited to the examples described
herein, but includes combination with any agent useful for the
treatment of Alzheimer's disease. Combination of compounds
according to the invention, or for use according to the invention,
and other Alzheimer's agents may be administered separately or in
conjunction. The administration of one agent may be prior to,
concurrent to, or subsequent to the administration of other
agent(s).
[0118] In alternative embodiments, the compounds may be supplied as
"prodrugs" or protected forms, which release the compound after
administration to a subject. For example, the compound may carry a
protective group which is split off by hydrolysis in body fluids,
e.g., in the bloodstream, thus releasing the active compound or is
oxidized or reduced in body fluids to release the compound.
Accordingly, a "prodrug" is meant to indicate a compound that may
be converted under physiological conditions or by solvolysis to a
biologically active compound of the invention. Thus, the term
"prodrug" refers to a metabolic precursor of a compound of the
invention that is pharmaceutically acceptable. A prodrug may be
inactive when administered to a subject in need thereof, but is
converted in vivo to an active compound of the invention. Prodrugs
are typically rapidly transformed in vivo to yield the parent
compound of the invention, for example, by hydrolysis in blood. The
prodrug compound often offers advantages of solubility, tissue
compatibility or delayed release in a subject.
[0119] The term "prodrug" is also meant to include any covalently
bonded carriers which release the active compound of the invention
in vivo when such prodrug is administered to a subject. Prodrugs of
a compound of the invention may be prepared by modifying functional
groups present in the compound of the invention in such a way that
the modifications are cleaved, either in routine manipulation or in
vivo, to the parent compound of the invention. Prodrugs include
compounds of the invention wherein a hydroxy, amino or mercapto
group is bonded to any group that, when the prodrug of the compound
of the invention is administered to a mammalian subject, cleaves to
form a free hydroxy, free amino or free mercapto group,
respectively. Examples of prodrugs include, but are not limited to,
acetate, formate and benzoate derivatives of alcohol and acetamide,
formamide, and benzamide derivatives of amine functional groups in
the compounds of the invention and the like.
[0120] Additional examples of prodrugs for the compounds of the
invention include acetonide derivatives (also known as
isopropylidine derivatives) in which two OR.sup.1 groups in Formula
(I) may be linked in a ring, for example, as in Formulae (II) and
(III) shown below. Such acetonide groups may be cleaved in vivo to
liberate the parent compound of the invention, making these
acetonide derivatives prodrugs.
##STR00060##
[0121] A discussion of prodrugs may be found in "Smith and
Williams' Introduction to the Principles of Drug Design," H. J.
Smith, Wright, Second Edition, London (1988); Bundgard, H., Design
of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam); The
Practice of Medicinal Chemistry, Camille G. Wermuth et al., Ch 31,
(Academic Press, 1996); A Textbook of Drug Design and Development,
P. Krogsgaard-Larson and H. Bundgaard, eds. Ch 5, pgs 113 191
(Harwood Academic Publishers, 1991); Higuchi, T., et al.,
"Pro-drugs as Novel Delivery Systems," A.C.S. Symposium Series,
Vol. 14; or in Bioreversible Carriers in Drug Design, ed. Edward B.
Roche, American Pharmaceutical Association and Pergamon Press,
1987, all of which are incorporated in full by reference
herein.
[0122] Suitable prodrug forms of the compounds of the invention
include embodiments in which R.sup.1 is C(O)R, where R is
optionally substituted alkyl, alkenyl, alkynyl, aryl, or
heteroaryl. In these cases the ester groups may be hydrolyzed in
vivo (e.g. in bodily fluids), releasing the active compounds in
which R.sup.1 is H. Preferred prodrug embodiments of the invention
are the compounds of Formula (I) where R.sup.1 is C(O)CH.sub.3.
[0123] Compounds according to the invention, or for use according
to the invention, can be provided alone or in combination with
other compounds in the presence of a liposome, an adjuvant, or any
pharmaceutically acceptable carrier, diluent or excipient, in a
form suitable for administration to a subject such as a mammal, for
example, humans, cattle, sheep, etc. If desired, treatment with a
compound according to the invention may be combined with more
traditional and existing therapies for the therapeutic indications
described herein. Compounds according to the invention may be
provided chronically or intermittently. "Chronic" administration
refers to administration of the compound(s) in a continuous mode as
opposed to an acute mode, so as to maintain the initial therapeutic
effect (activity) for an extended period of time. "Intermittent"
administration is treatment that is not consecutively done without
interruption, but rather is cyclic in nature. The terms
"administration," "administrable," or "administering" as used
herein should be understood to mean providing a compound of the
invention to the subject in need of treatment.
[0124] "Pharmaceutically acceptable carrier, diluent or excipient"
includes without limitation any adjuvant, carrier, excipient,
glidant, sweetening agent, diluent, preservative, dye/colorant,
flavor enhancer, surfactant, wetting agent, dispersing agent,
suspending agent, stabilizer, isotonic agent, solvent, or
emulsifier that has been approved, for example, by the United
States Food and Drug Administration or other governmental agency as
being acceptable for use in humans or domestic animals.
[0125] The compounds of the present invention may be administered
in the form of pharmaceutically acceptable salts. In such cases,
pharmaceutical compositions in accordance with this invention may
comprise a salt of such a compound, preferably a physiologically
acceptable salt, which are known in the art. In some embodiments,
the term "pharmaceutically acceptable salt" as used herein means an
active ingredient comprising compounds of Formula I used in the
form of a salt thereof, particularly where the salt form confers on
the active ingredient improved pharmacokinetic properties as
compared to the free form of the active ingredient or other
previously disclosed salt form.
[0126] A "pharmaceutically acceptable salt" includes both acid and
base addition salts. A "pharmaceutically acceptable acid addition
salt" refers to those salts which retain the biological
effectiveness and properties of the free bases, which are not
biologically or otherwise undesirable, and which are formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid and the like, and
organic acids such as acetic acid, trifluoroacetic acid, propionic
acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,
malonic acid, succinic acid, fumaric acid, tartaric acid, citric
acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic
acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid,
and the like.
[0127] A "pharmaceutically acceptable base addition salt" refers to
those salts which retain the biological effectiveness and
properties of the free acids, which are not biologically or
otherwise undesirable. These salts are prepared from addition of an
inorganic base or an organic base to the free acid. Salts derived
from inorganic bases include, but are not limited to, the sodium,
potassium, lithium, ammonium, calcium, magnesium, iron, zinc,
copper, manganese, aluminum salts and the like. Preferred inorganic
salts are the ammonium, sodium, potassium, calcium, and magnesium
salts. Salts derived from organic bases include, but are not
limited to, salts of primary, secondary, and tertiary amines,
substituted amines including naturally occurring substituted
amines, cyclic amines and basic ion exchange resins, such as
isopropylamine, trimethylamine, diethylamine, triethylamine,
tripropylamine, ethanolamine, 2-dimethylaminoethanol,
2-diethylaminoethanol, dicyclohexylamine, lysine, arginine,
histidine, caffeine, procaine, hydrabamine, choline, betaine,
ethylenediamine, glucosamine, methylglucamine, theobromine,
purines, piperazine, piperidine, N-ethylpiperidine, polyamine
resins and the like. Particularly preferred organic bases are
isopropylamine, diethylamine, ethanolamine, trimethylamine,
dicyclohexylamine, choline and caffeine.
[0128] Thus, the term "pharmaceutically acceptable salt"
encompasses all acceptable salts including but not limited to
acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate,
bicarbonate, maleate, bisulfate, mandelate, bitartarate, mesylate,
borate, methylbromide, bromide, methylnitrite, calcium edetate,
methylsulfate, camsylate, mucate, carbonate, napsylate, chloride,
nitrate, clavulanate, N-methylglucamine, citrate, ammonium salt,
dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate
(embonate), estolate, palmitate, esylate, pantothenate, fumarate,
phosphate/diphosphate, gluceptate, polygalacturonate, gluconate,
salicylate, glutame, stearate, glycollylarsanilate, sulfate,
hexylresorcinate, subacetate, hydradamine, succinate, hydrobromide,
tannate, hydrochloride, tartrate, hydroxynaphthoate, teoclate,
iodide, tosylate, isothionate, triethiodide, lactate, panoate,
valerate, and the like.
[0129] Pharmaceutically acceptable salts of the compounds of the
present invention can be used as a dosage for modifying solubility
or hydrolysis characteristics, or can be used in sustained release
or prodrug formulations. Also, pharmaceutically acceptable salts of
the compounds of this invention may include those formed from
cations such as sodium, potassium, aluminum, calcium, lithium,
magnesium, zinc, and from bases such as ammonia, ethylenediamine,
N-methyl-glutamine, lysine, arginine, ornithine, choline,
N,N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine,
procaine, N-benzylphenethyl-amine, diethylamine, piperazine,
tris(hydroxymethyl)aminomethane, and tetramethylammonium
hydroxide.
[0130] Pharmaceutical formulations will typically include one or
more carriers acceptable for the mode of administration of the
preparation, be it by injection, inhalation, topical
administration, lavage, or other modes suitable for the selected
treatment. Suitable carriers are those known in the art for use in
such modes of administration.
[0131] Suitable pharmaceutical compositions may be formulated by
means known in the art and their mode of administration and dose
determined by the skilled practitioner. For parenteral
administration, a compound may be dissolved in sterile water or
saline or a pharmaceutically acceptable vehicle used for
administration of non-water soluble compounds such as those used
for vitamin K. For enteral administration, the compound may be
administered in a tablet, capsule or dissolved in liquid form. The
table or capsule may be enteric coated, or in a formulation for
sustained release. Many suitable formulations are known, including,
polymeric or protein microparticles encapsulating a compound to be
released, ointments, gels, hydrogels, or solutions which can be
used topically or locally to administer a compound. A sustained
release patch or implant may be employed to provide release over a
prolonged period of time. Many techniques known to skilled
practitioners are described in Remington: the Science &
Practice of Pharmacy by Alfonso Gennaro, 20.sup.th ed., Williams
& Wilkins, (2000). Formulations for parenteral administration
may, for example, contain excipients, polyalkylene glycols such as
polyethylene glycol, oils of vegetable origin, or hydrogenated
naphthalenes. Biocompatible, biodegradable lactide polymer,
lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene
copolymers may be used to control the release of the compounds.
Other potentially useful parenteral delivery systems for modulatory
compounds include ethylene-vinyl acetate copolymer particles,
osmotic pumps, implantable infusion systems, and liposomes.
Formulations for inhalation may contain excipients, for example,
lactose, or may be aqueous solutions containing, for example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or
may be oily solutions for administration in the form of nasal
drops, or as a gel.
[0132] The compounds or pharmaceutical compositions according to
the present invention may be administered by oral or non-oral,
e.g., intramuscular, intraperitoneal, intravenous, intracisternal
injection or infusion, subcutaneous injection, transdermal or
transmucosal routes. In some embodiments, compounds or
pharmaceutical compositions in accordance with this invention or
for use in this invention may be administered by means of a medical
device or appliance such as an implant, graft, prosthesis, stent,
etc. Implants may be devised which are intended to contain and
release such compounds or compositions. An example would be an
implant made of a polymeric material adapted to release the
compound over a period of time. The compounds may be administered
alone or as a mixture with a pharmaceutically acceptable carrier
e.g., as solid formulations such as tablets, capsules, granules,
powders, etc.; liquid formulations such as syrups, injections,
etc.; injections, drops, suppositories, pessaryies. In some
embodiments, compounds or pharmaceutical compositions in accordance
with this invention or for use in this invention may be
administered by inhalation spray, nasal, vaginal, rectal,
sublingual, or topical routes and may be formulated, alone or
together, in suitable dosage unit formulations containing
conventional non-toxic pharmaceutically acceptable carriers,
adjuvants and vehicles appropriate for each route of
administration.
[0133] The compounds of the invention may be used to treat animals,
including mice, rats, horses, cattle, sheep, dogs, cats, and
monkeys. However, compounds of the invention can also be used in
other organisms, such as avian species (e.g., chickens). The
compounds of the invention may also be effective for use in humans.
The term "subject" or alternatively referred to herein as "patient"
is intended to be referred to an animal, preferably a mammal, most
preferably a human, who has been the object of treatment,
observation or experiment. However, the compounds, methods and
pharmaceutical compositions of the present invention may be used in
the treatment of animals. Accordingly, as used herein, a "subject"
may be a human, non-human primate, rat, mouse, cow, horse, pig,
sheep, goat, dog, cat, etc. The subject may be suspected of having
or at risk for having a condition requiring modulation of
O-GlcNAcase activity.
[0134] An "effective amount" of a compound according to the
invention includes a therapeutically effective amount or a
prophylactically effective amount. A "therapeutically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired therapeutic result, such
as inhibition of an O-GlcNAcase, elevation of O-GlcNAc levels,
inhibition of tau phosphorylation, or any condition described
herein. A therapeutically effective amount of a compound may vary
according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the compound to elicit
a desired response in the individual. Dosage regimens may be
adjusted to provide the optimum therapeutic response. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the compound are outweighed by the
therapeutically beneficial effects. A "prophylactically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired prophylactic result, such
as inhibition of an O-GlcNAcase, elevation of O-GlcNAc levels,
inhibition of tau phosphorylation, or any condition described
herein. Typically, a prophylactic dose is used in subjects prior to
or at an earlier stage of disease, so that a prophylactically
effective amount may be less than a therapeutically effective
amount. A suitable range for therapeutically or prophylactically
effective amounts of a compound may be any integer from 0.1
nM-0.1M, 0.1 nM-0.05M, 0.05 nM-15 .mu.M or 0.01 nM-10 .mu.M.
[0135] In alternative embodiments, in the treatment or prevention
of conditions which require modulation of O-GlcNAcase activity, an
appropriate dosage level will generally be about 0.01 to 500 mg per
kg subject body weight per day, and can be administered in singe or
multiple doses. In some embodiments, the dosage level will be about
0.1 to about 250 mg/kg per day. It will be understood that the
specific dose level and frequency of dosage for any particular
patient may be varied and will depend upon a variety of factors
including the activity of the specific compound used, the metabolic
stability and length of action of that compound, the age, body
weight, general health, sex, diet, mode and time of administration,
rate of excretion, drug combination, the severity of the particular
condition, and the patient undergoing therapy.
[0136] It is to be noted that dosage values may vary with the
severity of the condition to be alleviated. For any particular
subject, specific dosage regimens may be adjusted over time
according to the individual need and the professional judgement of
the person administering or supervising the administration of the
compositions. Dosage ranges set forth herein are exemplary only and
do not limit the dosage ranges that may be selected by medical
practitioners. The amount of active compound(s) in the composition
may vary according to factors such as the disease state, age, sex,
and weight of the subject. Dosage regimens may be adjusted to
provide the optimum therapeutic response. For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It may be advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. In general, compounds of the invention should
be used without causing substantial toxicity, and as described
herein, the compounds exhibit a suitable safety profile for
therapeutic use. Toxicity of the compounds of the invention can be
determined using standard techniques, for example, by testing in
cell cultures or experimental animals and determining the
therapeutic index, i.e., the ratio between the LD50 (the dose
lethal to 50% of the population) and the LD100 (the dose lethal to
100% of the population). In some circumstances however, such as in
severe disease conditions, it may be necessary to administer
substantial excesses of the compositions.
Other Uses and Assays
[0137] A compound of Formula (I) may be used in screening assays
for compounds which modulate the activity of glycosidase enzymes,
preferably the O-GlcNAcase enzyme. The ability of a test compound
to inhibit O-GlcNAcase-dependent cleavage of O-GlcNAc from a model
substrate may be measured using any assays, as described herein or
known to one of ordinary skill in the art. For example, a
fluoresence or UV-based assay known in the art may be used. A "test
compound" is any naturally-occurring or artificially-derived
chemical compound. Test compounds may include, without limitation,
peptides, polypeptides, synthesised organic molecules, naturally
occurring organic molecules, and nucleic acid molecules. A test
compound can "compete" with a known compound such as a compound of
Formula (I) by, for example, interfering with inhibition of
O-GlcNAcase-dependent cleavage of O-GlcNAc or by interfering with
any biological response induced by a compound of Formula (I).
[0138] Generally, a test compound can exhibit any value between 10%
and 200%, or over 500%, modulation when compared to a compound of
Formula (I) or other reference compound. For example, a test
compound may exhibit at least any positive or negative integer from
10% to 200% modulation, or at least any positive or negative
integer from 30% to 150% modulation, or at least any positive or
negative integer from 60% to 100% modulation, or any positive or
negative integer over 100% modulation. A compound that is a
negative modulator will in general decrease modulation relative to
a known compound, while a compound that is a positive modulator
will in general increase modulation relative to a known
compound.
[0139] In general, test compounds are identified from large
libraries of both natural products or synthetic (or semi-synthetic)
extracts or chemical libraries according to methods known in the
art. Those skilled in the field of drug discovery and development
will understand that the precise source of test extracts or
compounds is not critical to the method(s) of the invention.
Accordingly, virtually any number of chemical extracts or compounds
can be screened using the exemplary methods described herein.
Examples of such extracts or compounds include, but are not limited
to, plant-, fungal-, prokaryotic- or animal-based extracts,
fermentation broths, and synthetic compounds, as well as
modification of existing compounds. Numerous methods are also
available for generating random or directed synthesis (e.g.,
semi-synthesis or total synthesis) of any number of chemical
compounds, including, but not limited to, saccharide-, lipid-,
peptide-, and nucleic acid-based compounds. Synthetic compound
libraries are commercially available. Alternatively, libraries of
natural compounds in the form of bacterial, fungal, plant, and
animal extracts are commercially available from a number of
sources, including Biotics (Sussex, UK), Xenova (Slough, UK),
Harbor Branch Oceanographic Institute (Ft. Pierce, Fla., USA), and
PharmaMar, MA, USA. In addition, natural and synthetically produced
libraries are produced, if desired, according to methods known in
the art, e.g., by standard extraction and fractionation methods.
Furthermore, if desired, any library or compound is readily
modified using standard chemical, physical, or biochemical
methods.
[0140] When a crude extract is found to modulate inhibition of
O-GlcNAcase-dependent cleavage of O-GlcNAc, or any biological
response induced by a compound of Formula (I), further
fractionation of the positive lead extract is necessary to isolate
chemical constituents responsible for the observed effect. Thus,
the goal of the extraction, fractionation, and purification process
is the careful characterization and identification of a chemical
entity within the crude extract having O-GlcNAcase-inhibitory
activities. The same assays described herein for the detection of
activities in mixtures of compounds can be used to purify the
active component and to test derivatives thereof. Methods of
fractionation and purification of such heterogeneous extracts are
known in the art. If desired, compounds shown to be useful agents
for treatment are chemically modified according to methods known in
the art. Compounds identified as being of therapeutic,
prophylactic, diagnostic, or other value may be subsequently
analyzed using a suitable animal model, as described herein or
known in the art.
[0141] In some embodiments, the compounds described herein (e.g.,
the compounds of Formula I) or test compounds may be analyzed using
established cellular.sup.118-120 and/or transgenic animal models of
disease.sup.32,33 and the ability of the compounds to, for example,
block the formation of toxic tau species determined. Such analyses
may be used for example to determine or confirm the efficacy of the
compounds in treating or preventing pathology associated with the
accumulation of toxic tau species (for example, Alzheimer's disease
and other tauopathies).
[0142] In some embodiments, the compounds described herein (e.g.,
the compounds of Formula I) or test compounds may be analyzed using
established cellular stress assays.sup.105,116,117 and/or animal
models of ischemia-reperfusion.sup.70,114 or
trauma-hemorrhage..sup.72,112,115 Such analyses may be used for
example to determine or confirm the efficacy of the compounds in
treating or preventing pathology associated with cellular stress
(including ischemia, hemorrhage, hypovolemic shock, myocardial
infarction, and other cardiovascular disorders) or in treating or
preventing tissue damage or promoting functional recovery.
[0143] In some embodiments, the compounds are useful in the
development of animal models for studying diseases or disorders
related to deficiencies in O-GlcNAcase, over-expression of
O-GlcNAcase, accumulation of O-GlcNAc, depletion of O-GlcNAc, and
for studying treatment of diseases and disorders related to
deficiency or over-expression of O-GlcNAcase, or accumulation or
depletion of O-GlcNAc. Such diseases and disorders include
neurodegenerative diseases, including Alzheimer's disease, and
cancer.
[0144] Various alternative embodiments and examples of the
invention are described herein. These embodiments and examples are
illustrative and should not be construed as limiting the scope of
the invention.
EXAMPLES
[0145] The following examples are intended to illustrate
embodiments of the invention and are not intended to be construed
in a limiting manner.
Example 1
[0146] Compounds of the invention having general structure D are
prepared according to the sequence described in Scheme 1. Thus,
hydrogenation of the known intermediate A.sup.121,122 in the
presence of catalytic Pd/C in MeOH provides the amine B. Coupling
of this material with the appropriate acid chloride followed by
deprotection with sodium methoxide in methanol provides the desired
products D. Alternatively, conversion of B to C is also effected by
reaction of B with the appropriate carboxylic acid in the presence
of EDCI, HOBt, and Hunig's base in dichloromethane.
##STR00061##
Example 2
[0147] Compounds of the invention having general structure G are
prepared according to the sequence described in Scheme 2. Thus,
reductive amination of intermediate B (Scheme 1) with the
appropriate aldehyde in the presence of NaBH(OAc).sub.3 provides
the secondary amine E. Coupling of this material with the
appropriate acid chloride followed by deprotection with sodium
methoxide in methanol provides the desired products G.
Alternatively, conversion of E to F is also effected by reaction of
B with the appropriate carboxylic acid in the presence of EDCI,
HOBt, and Hunig's base in dichloromethane.
##STR00062##
Example 3
[0148] Compounds of the invention having general structure I is
prepared according to the sequence described in Scheme 3. Thus,
reaction of either intermediate B (Scheme 1) or E (Scheme 2) with
the appropriate isocyanate provides the ureas H. Deprotection with
sodium methoxide in methanol then furnishes the desired products
I.
##STR00063##
Example 4
[0149] Compounds of the invention having general structure K is
prepared according to the sequence described in Scheme 4. Thus,
reaction of either intermediate B (Scheme 1) or E (Scheme 2) with
phosgene followed by the appropriate secondary amine provides the
ureas J. Deprotection with sodium methoxide in methanol then
furnishes the desired products K.
##STR00064##
Example 5
[0150] Compounds of the invention having general structure L are
prepared according to the sequence described in Scheme 5. Thus,
deprotection of either intermediate B (Scheme 1) or E (Scheme 2)
with sodium methoxide in methanol provides the desired products
L.
##STR00065##
Example 6
[0151] Compounds of the invention having general structure N are
prepared according to the sequence described by Furneaux et
al..sup.123 and in Scheme 6. Following this route, deprotection of
intermediate M.sup.123 with sodium methoxide in methanol followed
by nucleophilic addition of the appropriate amine (to the resulting
intermediate epoxide) provides the desired products N.
##STR00066##
Example 7
Compound 1
(N-((1S,6S,7R,8R,8aR)-1,7,8-trihydroxy-octahydroindolizin-6-yl)acetamide
##STR00067##
[0153] Compound 1 was prepared using literature procedures.sup.123
and exhibited spectral characteristics consistent with those
reported..sup.123
Example 8
Compound 24
N-((1S,6S,7R,8R,8aR)-1,7,8-trihydroxy-octahydroindolizin-6-yl)propionamide
##STR00068##
[0155] Compound 24 was prepared following standard procedures (e.g.
Scheme 1). .sup.1H NMR (500 MHz, methanol-d.sub.4) .delta. 1.12 (t,
3H, J=7.6 Hz), 1.70-1.90 (m, 3H), 2.08 (q, 1H, J=8.8 Hz), 2.17-2.25
(m, 3H), 3.14-3.07 (m, 2H), 3.27 (dd, 1H, J=8.8, 10.2 Hz), 3.65
(dd, 1H, J=8.9, 8.9 Hz), 3.92 (dd, 1H, J=4.9, 9.4, 9.4 Hz),
4.28-4.32 (m, 1H); .sup.13C NMR (125 MHz, methanol-d.sub.4) .delta.
10.40, 30.24, 34.48, 52.70, 53.09, 55.84, 71.24, 71.54, 73.73,
78.25, 177.36; MS (HRMS): m/z 244.1408 (M+H).sup.+; Calcd. for
C.sub.11H.sub.20N.sub.2O.sub.4: 244.1423. Anal. Calcd for
C.sub.11H.sub.20N.sub.2O.sub.4: C, 54.08; H, 8.25; N, 11.47; Found:
C, 54.02; H, 8.28; N, 11.41.
Example 9
Compound 2
N-((1S,6S,7R,8R,8aR)-1,7,8-trihydroxy-octahydroindolizin-6-yl)butyramide
##STR00069##
[0157] Compound 2 was prepared using literature procedures.sup.123
and exhibited spectral characteristics consistent with those
reported..sup.123
Example 10
Compound 27
N-((1S,6S,7R,8R,8aR)-1,7,8-trihydroxy-octahydroindolizin-6-yl)isobutyramid-
e
##STR00070##
[0159] Compound 27 was prepared following standard procedures (e.g.
Scheme 1). .sup.1H NMR (500 MHz, methanol-d.sub.4) .delta. 1.09 (d,
3H, J=6.9 Hz), 1.12 (d, 3H, J=6.9 Hz), 1.70-1.87 (m, 3H), 2.09 (q,
1H, J=8.8 Hz), 2.19-2.25 (m, 1H), 2.45 (m, J=6.9 Hz), 3.07-3.14 (m,
2H), 3.28 (dd, 1H, J=8.8, 10.6 Hz), 3.65 (dd, 1H, J=8.9, 8.9 Hz),
3.90 (ddd, 1H, J=4.8, 10.6, 10.6 Hz), 4.27-4.31 (m, 1H); .sup.13C
NMR (125 MHz, methanol-d.sub.4) .delta. 19.77, 20.07, 34.48, 36.35,
52.59, 53.10, 55.84, 71.25, 71.61, 73.75, 78.20, 180.56; MS (HRMS):
m/z 258.1559 (M+H).sup.+; Calcd. for
C.sub.12H.sub.22N.sub.2O.sub.4: 258.1580. Anal. Calcd for
C.sub.12H.sub.22N.sub.2O.sub.4: C, 55.80; H, 8.58; N, 10.84; Found:
C, 55.92; H, 8.63; N, 10.79.
Example 11
Compound 25
N-((1S,6S,7R,8R,8aR)-1,7,8-trihydroxy-octahydroindolizin-6-yl)pentanamide
##STR00071##
[0161] Compound 25 was prepared following standard procedures (e.g.
Scheme 1). .sup.1H NMR (500 MHz, methanol-d.sub.4) .delta. 0.93 (t,
3H, J=7.3 Hz), 1.31-1.39 (m, 2H), 1.55-1.62 (m, 2H), 1.70-1.87 (m,
3H, H2, H5', H8a), 2.08 (q, J=8.7 Hz), 2.17-2.25 (m, 3H), 3.07-3.14
(m, 2H), 3.26 (dd, 1H, J=8.8, 10.7 Hz), 3.65 (dd, 1H, J=8.9, 8.9
Hz), 3.92 (dd, 1H, J=4.9, 10.7, 10.7 Hz, H6), 4.28-4.32 (m, 1H);
.sup.13C NMR (125 MHz, methanol-d.sub.4) .delta. 14.14, 23.36,
29.16, 34.48, 36.98, 52.70, 53.09, 55.86, 71.24, 71.57, 73.73,
78.23, 176.64; MS (HRMS): m/z 272.1759 (M+H).sup.+; Calcd. for
C.sub.13H.sub.24N.sub.2O.sub.4: 272.1736. Anal. Calcd for
C.sub.13H.sub.24N.sub.2O.sub.4: C, 57.33; H, 8.88; N, 10.29; Found:
C, 57.29; H, 8.83; N, 10.34.
Example 12
Compound 28
3-methyl-N-((1S,6S,7R,8R,8aR)-1,7,8-trihydroxy-octahydroindolizin-6-yl)but-
anamide
##STR00072##
[0163] Compound 28 was prepared following standard procedures (e.g.
Scheme 1). .sup.1H NMR (500 MHz, methanol-d.sub.4) .delta. 0.93 (d,
3H, J=6.2 Hz), 0.97 (d, 3H, J=6.2 Hz), 1.70-1.87 (m, 3H),
2.03-2.11, 2.18-2.24 (2m, 5H), 3.07-3.14 (m, 2H), 3.26 (dd, 1H),
3.64 (dd, 1H, J=9.3, 9.3 Hz), 3.93 (dd, 1H, J=4.7, 10.5, 10.5 Hz),
4.26-4.30 (m, 1H); .sup.13C NMR (125 MHz, methanol-d.sub.4) .delta.
22.62, 22.81, 27.42, 34.49, 46.49, 52.69, 53.09, 55.91, 71.26,
71.62, 73.74, 78.23, 175.91; MS (HRMS): m/z 272.1748 (M+H).sup.+;
Calcd. for C.sub.13H.sub.24N.sub.2O.sub.4: 272.1736. Anal. Calcd
for C.sub.13H.sub.24N.sub.2O.sub.4: C, 57.33; H, 8.88; N, 10.29;
Found: C, 57.29; H, 8.78; N, 10.21.
Example 13
Compound 26
N-((1S,6S,7R,8R,8aR)-1,7,8-trihydroxy-octahydroindolizin-6-yl)hexanamide
##STR00073##
[0165] Compound 26 was prepared following standard procedures (e.g.
Scheme 1). .sup.1H NMR (500 MHz, methanol-d.sub.4) .delta. 0.92 (t,
3H, J=6.9 Hz), 1.27-1.38 (m, 4H), 1.57-1.64 (m, 2H), 1.70-1.89 (m,
3H), 2.09 (q, 1H, J=8.9 Hz), 2.17-2.25 (m, 3H), 3.07-3.15 (m, 2H),
3.27 (dd, 1H), 3.65 (dd, 1H, J=9.1, 9.1 Hz), 3.93 (dd, 1H, J=4.8,
10.6 Hz), 4.28-4.32 (m, 1H); .sup.13C NMR (125 MHz,
methanol-d.sub.4) .delta. 14.28, 23.45, 26.69, 32.52, 34.46, 37.20,
52.67, 53.09, 55.82, 71.21, 71.54, 73.72, 78.19, 176.64; MS (HRMS):
m/z 286.1888 (M+H).sup.+; Calcd. for
C.sub.14H.sub.26N.sub.2O.sub.4: 286.1893. Anal. Calcd for
C.sub.14H.sub.26N.sub.2O.sub.4: C, 58.72; H, 9.15; N, 9.78; Found:
C, 58.88; H, 9.08; N, 9.75.
Example 14
Compound 43
(1S,6S,7R,8R,8aR)-6-(ethylamino)-octahydroindolizine-1,7,8-triol
##STR00074##
[0167] Compound 43 was prepared following standard procedures (e.g.
Schemes 1, 2, 5). .sup.1H NMR (500 MHz, methanol-d.sub.4) .delta.
0.92 (t, 3H, J=6.9 Hz), 1.27-1.38 (m, 4H), 1.57-1.64 (m, 2H),
1.70-1.89 (m, 3H), 2.09 (q, 1H, J=8.9 Hz), 2.17-2.25 (m, 3H),
3.07-3.15 (m, 2H), 3.27 (dd, 1H), 3.65 (dd, 1H, J=9.1, 9.1 Hz),
3.93 (dd, 1H, J=4.8, 10.6 Hz), 4.28-4.32 (m, 1H); .sup.13C NMR (125
MHz, methanol-d.sub.4) .delta. 14.28, 23.45, 26.69, 32.52, 34.46,
37.20, 52.67, 53.09, 55.82, 71.21, 71.54, 73.72, 78.19, 176.64; MS
(HRMS): m/z 286.1888 (M+H).sup.+; Calcd. for
C.sub.14H.sub.26N.sub.2O.sub.4: 286.1893. Anal. Calcd for
C.sub.14H.sub.26N.sub.2O.sub.4: C, 58.72; H, 9.15; N, 9.78; Found:
C, 58.88; H, 9.08; N, 9.75.
Example 15
Assay for Determination of K.sub.I Values for Inhibition of
O-GlcNAcase Activity
[0168] Experimental Procedure for Kinetic Analyses:
[0169] Enzymatic reactions were carried out in PBS buffer (pH 7.4)
using pNP-GlcNAc as a substrate (0.5 mM) and monitored continuously
at 37.degree. C. at 400 nm using a Cary 3E UV-VIS spectrophotometer
equipped with a Peltier temperature controller. Reactions were
pre-heated in a 500 .mu.L quartz cuvette for approximately 5
minutes followed by addition of 10 .mu.L enzyme via syringe (final
enzyme concentration 0.002 mg/mL). Reaction velocities were
determined by linear regression of the linear region of the
reaction progress curve between the first and third minutes. An
inhibitor concentration range of 1/5 to 5 times K.sub.1 was used in
each case.
[0170] When tested in this assay, compound 1 exhibited a K.sub.I
value for inhibition of O-GlcNAcase of 0.67 .mu.M.
[0171] When tested in this assay, many of the compounds described
herein exhibit K.sub.I values for inhibition of O-GlcNAcase in the
range 1 nM-50 .mu.M. All K.sub.I values are determined using linear
regression of Dixon plots.
Example 16
Assay for Determination of K.sub.I Values for Inhibition of
.beta.-Hexosaminidase Activity
[0172] Experimental Procedure for Kinetic Analyses:
[0173] All enzymatic assays are carried out in triplicate at
37.degree. C. using a stopped assay procedure by measuring the
amount of 4-nitrophenolate liberated as determined by absorption
measurements at 400 nm. Reactions (50 .mu.L) are initiated by the
addition, via syringe, of enzyme (3 .mu.L). Time-dependent assay of
.beta.-hexosaminidase has revealed that the enzyme is stable in the
buffer over the period of the assay: 50 mM citrate, 100 mM NaCl,
0.1% BSA, pH 4.25. .beta.-hexosaminidase is used at a concentration
of 0.036 mg/mL with pNP-GlcNAc as a substrate at a concentration of
0.5 mM. The inhibitor is tested at five concentrations ranging from
5 times to 1/5 K.sub.I. K.sub.I values are determined by linear
regression of data from Dixon plots.
[0174] When tested in this assay, many of the compounds described
herein exhibit K.sub.I values for inhibition of
.beta.-hexosaminidase in the range 1 .mu.M-10 mM.
[0175] The selectivity ratio for inhibition of O-GlcNAcase over
.beta.-hexosaminidase is defined here as:
K.sub.I(.beta.-hexosaminidase)/K.sub.I(O-GlcNAcase)
[0176] In general, the compounds described herein should exhibit a
selectivity ratio in the range of about 10 to 100000. Thus, many
compounds of the invention exhibit high selectivity for inhibition
of O-GlcNAcase over .beta.-hexosaminidase.
Example 17
[0177] Western Blot Analyses:
[0178] 3T3-L1 adipocytes were treated with Compound 1 at 100 .mu.M
for 16 hours. Cells were harvested by adding 400 .mu.L of 1% SDS
with 50 mM beta-ME, boiling this for 10 minutes, using these
lysates in subsequent Western blots. Briefly, samples were
separated by SDS-PAGE (10% gels), transferred to nitrocellulose
membrane (Bio-Rad), blocked for one hour at room temperature (RT)
with 1% bovine serum albumin (BSA) in PBS containing 0.1% Tween-20
(PBS-T) and then subsequently probed with the appropriate primary
antibody delivered in 1% BSA in PBS-T for overnight at 4.degree. C.
Membranes were then extensively washed with PBS-T, blocked again
for 30 minutes with 1% BSA in PBS-T at RT and then probed with the
appropriate HRP conjugated secondary antibody for one at RT
delivered in 1% BSA in PBS-T. Finally, the membranes were washed
extensively and then developed with SuperSignal West Pico
Chemiluminesence substrate.
[0179] Results from Cell Studies:
[0180] Cellular levels of O-GlcNAc-modified proteins within cells
cultured for 16 hours in the presence of Compound 1, or in its
absence was carried out using the O-GlcNAc directed monoclonal
antibody mAbCTD110.6. Marked increases in cellular levels of
O-GlcNAc-modified proteins within the cells were observed as
compared to the control (FIG. 1) indicating that this compound
readily gains access to the interior of the cell where it acts to
block O-GlcNAcase function.
[0181] The present invention has been described with regard to one
or more embodiments. However, it will be apparent to persons
skilled in the art that a number of variations and modifications
can be made without departing from the scope of the invention as
defined in the claims.
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* * * * *
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