U.S. patent application number 14/179310 was filed with the patent office on 2014-12-18 for hydantoins that modulate bace-mediated app processing.
This patent application is currently assigned to Buck Institute for Research on Aging. The applicant listed for this patent is Buck Institute for Research on Aging. Invention is credited to Dale E. BREDESEN, VARGHESE JOHN.
Application Number | 20140371283 14/179310 |
Document ID | / |
Family ID | 51354522 |
Filed Date | 2014-12-18 |
United States Patent
Application |
20140371283 |
Kind Code |
A1 |
JOHN; VARGHESE ; et
al. |
December 18, 2014 |
HYDANTOINS THAT MODULATE BACE-MEDIATED APP PROCESSING
Abstract
In certain embodiments hydantoin compounds are provided herein
that are effective to inhibit BACE activity against APP. Without
being bound to a particular theory, it is believed the activity of
the hydantoins identified herein appears to be associated with
binding to BACE and/or to APP particularly when these moieties form
a BACE/APP complex. Accordingly, it is believed the compounds
described herein represent a new class of compounds designated
herein as APP-Binding-BACE Inhibitors (ABBIs) and provide a new
mechanism to modulate APP processing. The hydantoins described
herein appear to show improved brain permeability and functional
BACE inhibition.
Inventors: |
JOHN; VARGHESE; (San
Francisco, CA) ; BREDESEN; Dale E.; (Novato,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Buck Institute for Research on Aging |
Novato |
CA |
US |
|
|
Assignee: |
Buck Institute for Research on
Aging
Novato
CA
|
Family ID: |
51354522 |
Appl. No.: |
14/179310 |
Filed: |
February 12, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61763830 |
Feb 12, 2013 |
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Current U.S.
Class: |
514/391 ;
514/386; 548/320.5; 548/321.5 |
Current CPC
Class: |
A61K 31/506 20130101;
C07D 233/88 20130101; A61P 25/02 20180101; C07D 401/10 20130101;
A61P 25/28 20180101; A61P 25/00 20180101; A61P 27/02 20180101; A61K
31/4439 20130101; A61P 9/00 20180101; A61K 45/06 20130101; C07D
233/76 20130101; A61K 31/4168 20130101; A61K 31/4166 20130101; A61P
43/00 20180101; C07D 403/10 20130101; C07D 233/70 20130101; C07D
401/04 20130101; A61K 31/46 20130101; A61K 31/4166 20130101; A61K
2300/00 20130101; A61K 31/4439 20130101; A61K 2300/00 20130101;
A61K 31/506 20130101; A61K 2300/00 20130101; A61K 31/46 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
514/391 ;
548/320.5; 548/321.5; 514/386 |
International
Class: |
C07D 233/88 20060101
C07D233/88; C07D 233/76 20060101 C07D233/76 |
Claims
1. A compound of Formula I: ##STR00142## wherein: R.sup.7 is
selected from the group consisting of C.dbd.O, C.dbd.S,
C--NH.sub.2, and C.dbd.NH, and the bond represented by the wavy
line is a single bond when R.sup.7 is C.dbd.O, C.dbd.S, or
C.dbd.NH, and a double bond when R.sup.7 is C--NH.sub.2; R.sup.8
and R.sup.9 are independently selected from the group consisting of
H, alkyl, cycloalkyl, and aryl, provided that when the bond
represented by the wavy line is a double bond, then R.sup.9 is
absent; R.sup.0 is selected from the group consisting of aryl,
substituted aryl, disubstituted aryl, heteroaryl, substituted
heteroaryl, disubstituted heteroaryl, alkyl, haloalkyl, cycloalkyl,
alkenyl, and alkynyl; X.sup.1 is selected from the group consisting
of CH, and N; R.sup.5 and R.sup.6 are independently selected from
halogen; R.sup.3 and R.sup.4 are independently absent or selected
from the group consisting of alkyl, cycloalkyl, alkoxy, thioalkyl;
and when X.sup.1 is C, then R.sup.0 is not phenyl monosubstituted
at the para position with --OCHF.sub.2, or a pharmaceutically
acceptable salt thereof, a tautomer thereof, a pharmaceutically
acceptable salt of a tautomer thereof, an enantiomer thereof, or a
pharmaceutically acceptable salt of an enantiomer thereof.
2. The compound of claim 1, wherein R.sup.7 is C.dbd.NH or
C.dbd.O.
3. (canceled)
4. The compound of claim 2, wherein said compound is a compound of
the formula: ##STR00143##
5. (canceled)
6. The compound of claim 2, wherein R.sup.7 is C--NH.sub.2 and said
compound is a compound of Formula II: ##STR00144##
7. The compound of claim 6, wherein said compound is a compound of
Formula III: ##STR00145## wherein: R.sup.1 and R.sup.2 are
independently absent or selected from the group consisting of
alkyl, haloalkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, thioalkyl,
aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and
X.sup.2, Y, and Z are independently CH or N.
8-10. (canceled)
11. The compound of claim 7, wherein said compound is a compound of
Formula IV: ##STR00146##
12-13. (canceled)
14. The compound of claim 1, wherein said compound is a compound
selected from the group consisting of ##STR00147##
15-17. (canceled)
18. A compound of Formula VI: ##STR00148## or a pharmaceutically
acceptable salt thereof, a tautomer thereof, a pharmaceutically
acceptable salt of a tautomer thereof, an enantiomer thereof, or a
pharmaceutically acceptable salt of an enantiomer thereof.
19-20. (canceled)
21. The compound of claim 1, wherein said compound binds to APP
and/or to the enzyme BACE and/or to an APP/BACE complex.
22. The compound of claim 1, wherein said compound binds to APP and
inhibits the enzyme BACE.
23. A pharmaceutical formulation comprising a pharmaceutically
acceptable carrier and a compound of claim 1.
24-27. (canceled)
28. A method of preventing or delaying the onset of a
pre-Alzheimer's condition and/or cognitive dysfunction, and/or
ameliorating one or more symptoms of a pre-Alzheimer's condition
and/or cognitive dysfunction, or preventing or delaying the
progression of a pre-Alzheimer's condition or cognitive dysfunction
to Alzheimer's disease, said method comprising: administering to a
subject in need thereof a compound of claim 1 in an amount
sufficient to prevent or delay the onset of a pre-Alzheimer's
cognitive dysfunction, and/or to ameliorate one or more symptoms of
a pre-Alzheimer's cognitive dysfunction, and/or to prevent or delay
the progression of a pre-Alzheimer's cognitive dysfunction to
Alzheimer's disease.
29. The method of claim 28, wherein said method is a method of:
preventing or delaying the transition from a cognitively
asymptomatic pre-Alzheimer's condition to a pre-Alzheimer's
cognitive dysfunction; and/or preventing or delaying the onset of a
pre-Alzheimer's cognitive dysfunction; and/or preventing or
delaying the progression of a pre-Alzheimer's cognitive dysfunction
to Alzheimer's disease.
30-50. (canceled)
51. The method of claim 28, wherein: said administration produces a
reduction in the CSF of levels of one or more components selected
from the group consisting of A.beta.42, sAPP.beta., total-Tau
(tTau), phospho-Tau (pTau), APPneo, soluble A.beta.40,
pTau/A.beta.42 ratio and tTau/A.beta.42 ratio, and/or an increase
in the CSF of levels of one or more components selected from the
group consisting of A.beta.42/A.beta.40 ratio, A.beta.42/A.beta.38
ratio, sAPP.alpha., sAPP.alpha./sAPP.beta. ratio,
sAPP.alpha./A.beta.40 ratio, and sAPP.alpha./A.beta.42 ratio;
and/or said administration produces a reduction of the plaque load
in the brain of the subject; and/or said administration produces a
reduction in the rate of plaque formation in the brain of the
subject; and/or said administration produces an improvement in the
cognitive abilities of the subject; and/or said administration
produces an improvement in, a stabilization of, or a reduction in
the rate of decline of the clinical dementia rating (CDR) of the
subject.
52-60. (canceled)
61. A method of ameliorating one or more symptoms of Alzheimer's
disease, and/or reversing Alzheimer's disease, and/or reducing the
rate of progression of Alzheimer's disease, said method comprising:
administering to a subject in need thereof a compound of claim 1 in
an amount sufficient to ameliorate one or more symptoms of
Alzheimer's disease, and/or to reverse Alzheimer's disease, and/or
to reduce the rate of progression of Alzheimer's disease.
62-63. (canceled)
64. The method of claim 61, wherein: said subject is diagnosed with
early stage Alzheimer's disease; or said subject is diagnosed with
mid-stage Alzheimer's disease; or said subject is diagnosed with
late-stage Alzheimer's disease.
65-67. (canceled)
68. The method of claim 61, wherein: said administering ameliorates
one or more symptoms of Alzheimer's disease; and/or said
administering reduces the rate of progression of Alzheimer's
disease; and/or said administering results in a reduction in the
CSF of levels of one or more components selected from the group
consisting of A.beta.42, sAPP.beta., total-Tau (tTau), phospho-Tau
(pTau), APPneo, soluble A.beta.40, pTau/A.beta.42 ratio and
tTau/A.beta.42 ratio, and/or an increase in the CSF of levels of
one or more components selected from the group consisting of
A.beta.42/A.beta.40 ratio, A.beta.42/A.beta.38 ratio, sAPP.alpha.,
sAPP.alpha./sAPP.beta. ratio, sAPP.alpha./A.beta.40 ratio, and
sAPP.alpha./A.beta.42 ratio; and/or said administration produces a
reduction of the plaque load in the brain of the subject; and/or
said administration produces a reduction in the rate of plaque
formation in the brain of the subject; and/or said administration
produces an improvement in the cognitive abilities of the subject;
and/or said administration produces an improvement in, a
stabilization of, or a reduction in the rate of decline of the
clinical dementia rating (CDR) of the subject; and/or said
administration produces a perceived improvement in quality of life
by the subject; and/or said administering results in reduced
cerebral amyloidosis and/or downstream neurodegeneration.
69-78. (canceled)
79. The method of claim 61, wherein: said subject shows a clinical
dementia rating indicative of Alzheimer's disease; and/or said
subject has a familial risk for having Alzheimer's disease; and/or
said subject has a familial Alzheimer's disease (FAD) mutation.
80-93. (canceled)
94. A method of slowing the progression, stopping, or reversing
age-related macular degeneration (AMD) in a mammal, said method
comprising administering to said mammal a compound of claim 1 in an
amount sufficient to slow the progression, stop, or reverse
age-related macular degeneration in said mammal.
95. A method for the treatment of a disease or disorder associated
with BACE activity in a subject in need thereof, wherein said
method comprises providing to said subject a therapeutically
effective amount of a compound of claim 1.
96-97. (canceled)
98. A kit comprising one or more containers containing a compound
of claim 1.
99-101. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of and priority to U.S. Ser.
No. 61/763,830, filed on Feb. 12, 2013, which is incorporated
herein by reference for all purposes.
STATEMENT OF GOVERNMENTAL SUPPORT
[0002] [Not Applicable]
BACKGROUND
[0003] Amyloid beta peptide (A.beta.) is a primary component of
beta amyloid fibrils and plaques, which are regarded as having a
role in an increasing number of pathologies. Examples of such
pathologies include, but are not limited to, Alzheimer's disease,
Down's syndrome, Parkinson's disease, memory loss (including memory
loss associated with Alzheimer's disease and Parkinson's disease),
attention deficit symptoms (including attention deficit symptoms
associated with Alzheimer's disease, Parkinson's disease, and
Down's syndrome), dementia (including pre-senile dementia, senile
dementia, dementia associated with Alzheimer's disease, Parkinson's
disease, and Down's syndrome), progressive supranuclear palsy,
cortical basal degeneration, neurodegeneration, olfactory
impairment (including olfactory impairment associated with
Alzheimer's disease, Parkinson's disease, and Down's syndrome),
.beta.-amyloid angiopathy (including cerebral amyloid angiopathy),
hereditary cerebral hemorrhage, mild cognitive impairment ("MCI"),
glaucoma, amyloidosis, type II diabetes, hemodialysis (.beta.2
microglobulins and complications arising therefrom),
neurodegenerative diseases such as scrapie, bovine spongiform
encephalitis, Creutzfeld Jakob disease, traumatic brain injury and
the like.
[0004] A.beta. peptides are short peptides that are produced by
proteolysis of the transmembrane protein called amyloid precursor
protein ("APP"). A.beta. peptides are made from the cleavage of APP
by .beta.-secretase activity at a position near the N-terminus of
A.beta., and by gamma secretase activity at a position near the
C-terminus of A.beta.. (APP is also cleaved by .alpha.-secretase
activity, resulting in the secreted, non-amyloidogenic fragment
known as soluble APP.alpha.). Beta site APP Cleaving Enzyme
("BACE-1") is regarded as the primary aspartyl protease responsible
for the production of A.beta. by .beta.-secretase activity. The
inhibition of BACE-1 has been shown to inhibit the production of
A.beta..
[0005] Alzheimer's disease (AD) is estimated to afflict more than
20 million people worldwide and is believed to be the most common
cause of dementia. As the World population ages, the number of
people with Alzheimer's disease (AD, currently approximately 5.4
million in the United States, will continue to rise. Alzheimer's is
a neurodegenerative disease associated with progressive dementia
and memory loss. Two key characteristics of AD are the accumulation
of extracellular deposits containing aggregated A.beta. peptide and
neuronal synaptic loss in the AD in specific brain regions.
Although AD pathogenesis is complex, compelling genetic and
biochemical evidence suggest that overproduction of A.beta., or
failure to clear this peptide is the earliest event in the amyloid
cascade that lead to AD primarily through amyloid deposition, which
is presumed to be involved in neurofibrillary tangle formation,
neuronal dysfunction and microglia activation, that characterize
AD-affected brain tissues.
[0006] The accumulation of A.beta. is considered to be the earliest
event in a complex cascade that leads to neurodegeneration, as
discerned from compelling genetic and biochemical evidence. The
amyloid cascade hypothesis (Hardy and Allsop (1991) Trends
Pharmacol. Sci., 12: 383-388; Selkoe (1996) J. Biol. Chem., 271:
18295-18298; Hardy (1997) Trends Neurosci., 20: 154-159; Hardy and
Selkoe (2002) Science, 297: 353-356) states that overproduction of
A.beta., or failure to clear this peptide, leads to AD, primarily
through amyloid deposition, which is presumed to be involved in
neurofibrillary tangle formation, neuronal dysfunction, and
microglia activation, that are hallmarks of AD-affected brain
tissues (Busciglio et al. (1995) Neuron, 14: 879-888; Gotz et al.
(1995) EMBO J., 14: 1304-1313; Lewis et al., (2001) Science, 293:
1487-1491; Hardy et al. (1985) Nat Neurosci., 1: 355-358).
[0007] Considering the causative role of A.beta. in AD etiology,
novel therapeutic strategies that lower A.beta. levels or prevent
the formation of the neurotoxic A.beta. species have been suggested
as a method to prevent or slow the progression of the disease.
Indeed, the major focus over the last decade has been to inhibit
brain A.beta. production and aggregation, to increase parenchymal
A.beta. clearance, and to interfere with A.beta.-induced cell
death.
[0008] The sequential cleavage of APP by membrane-bound proteases
.beta.-secretase and .gamma.-secretase results in the formation of
A.beta.. A competing proteolytic pathway to the .beta.-secretase
pathway, the .alpha.-secretase pathway, results in cleavage of APP
within the A.beta. domain, thereby precluding the generation of
A.beta. (Selkoe (2001) Physiol. Rev., 81: 741-766; Hussain et al.
(1999) Mol. Cell. Neurosci., 14: 419-427; Sinha et al. (1999)
Nature, 402: 537-540; Vassar et al. (1999) Science, 286: 735-741).
The .beta.-Site APP cleavage enzyme-1 (BACE1) was identified as the
major .beta.-secretase activity that mediates the first cleavage of
APP in the .beta.-amyloidogenic pathway (Id.).
[0009] BACE1 is a 501 amino acid protein that bears homology to
eukaryotic aspartic proteases, especially from the pepsin family
(Yan et al. (1999) Nature, 402: 533-537). Similar to other aspartic
proteases, BACE1 is synthesized as a zymogen with a pro-domain that
is cleaved by furin to release the mature protein. BACE1 is a
type-I transmembrane protein with a luminal active site that
cleaves APP to release an ectodomain (sAPP.beta.) into the
extracellular space. The remaining C-terminal fragment (CTF)
undergoes further cleavage by .gamma.-secretase, leading to the
release of A.beta. and the APP intracellular C-terminal domain
(AICD).
[0010] The presenilins have been proposed to be the major enzymatic
component of .gamma.-secretase, whose imprecise cleavage of APP
produces a spectrum of A.beta. peptides varying in length by a few
amino acids at the C-terminus. The majority of A.beta. normally
ends at amino acid 40 (A.beta.40), but the 42-amino acid variant
(A.beta.42) has been shown to be more susceptible to aggregation,
and has been hypothesized to nucleate senile plaque formation. The
modulation of the .gamma.-secretase can also lead to increase in
the 38-amino acid variant (A.beta.38). The competing
.alpha.-secretase pathway is the result of sequential cleavages by
.alpha.- and .gamma.-secretase. Three metalloproteases of the
disintegrin and metalloprotease family (ADAM 9, 10, and 17) have
been proposed as candidates for the .alpha.-secretase activity,
which cleaves APP at position 16 within the A.beta. sequence. Using
overexpression experiments, ADAM-10 has been shown to be the likely
.alpha.-secretase for cleavage of APP (Vassar (2002) Adv. Drug
Deliv. Rev., 54: 1589-1602; Buxbaum et al. (1998) J. Biol. Chem.,
273: 27765-27767; Koike et al. (1999) Biochem. J., 343(Pt 2):
371-375). This cleavage also releases an ectodomain (sAPP.alpha.),
which displays neuroprotective functions (Lammich et al. (1999)
Proc. Natl. Acad. Sci. USA, 96: 3922-3927). Subsequent cleavage of
the 83-amino acid CTF (C83) releases p3, which is
non-amyloidogenic, and AICD (Furukawa et al. (1996) J. Neurochem.,
67: 1882-1896). The functions of these fragments are not fully
elucidated, although AICD is hypothesized to mediate intracellular
signaling.
[0011] Research clarifying the metabolic pathways that regulate the
production of A.beta. from the Amyloid Precursor Protein (APP)
indicates that the secretases that produce A.beta. are good
therapeutic targets, since inhibition of either .beta.- or
.gamma.-secretase limits A.beta. production. The fact that
.beta.-secretase initiates APP processing, and thus serves as the
rate limiting step in production of A.beta., its inhibition has
attracted efforts by many research groups. Examples from the patent
literature are growing and include, for example, WO2006009653,
WO2007005404, WO2007005366, WO2007038271, WO2007016012,
US2005/0282826, US2007072925, WO2007149033, WO2007145568,
WO2007145569, WO2007145570, WO2007145571, WO2007114771,
US20070299087, WO2005/016876, WO2005/014540, WO2005/058311,
WO2006/065277, WO2006/014762, WO2006/014944, WO2006/138195,
WO2006/138264, WO2006/138192, WO2006/138217, WO2007/050721,
WO2007/053506, WO2007/146225, WO2006/138230, WO2006/138265,
WO2006/138266, WO2007/053506, WO2007/146225, WO2008/073365,
WO2008/073370, WO2008/103351, US2009/041201, US2009/041202, and
WO2010/047372.
[0012] One limitation of protease inhibitory strategies is the
inhibition of cleavage of all substrates of a given targeted
protease, such as BACE or the .gamma.-secretase complex. In the
case of .gamma.-secretase, substrates other than APP, such as
Notch, raise concerns for potential side effects of
.gamma.-secretase inhibition, and the recent failure of the
.gamma.-secretase inhibitor. Problems associated with the use of
semagacestat, serve to reinforce such concerns.
[0013] BACE is a key enzyme involved in processing of APP leading
to the production of A.beta.42 and the Alzheimer's disease (AD)
pathology. BACE-1 (also called BACE) has become a popular research
area since its discovery, and has perhaps surpassed
.gamma.-secretase as the most promising target for pharmaceutical
research. A problem with .gamma.-secretase as a target is its known
cleavage of Notch, which serves important functions in neuronal
development. Presenilin knockout mice demonstrated abnormal
somitogenesis and axial skeletal development with shortened body
length, as well as cerebral hemorrhages (Shen et al. (1997) Cell,
89: 629-639; Wong et al. (1997) Nature, 387: 288-292). In contrast,
several groups reported that BACE1 knockout mice are healthy and
show no signs of adverse effect (Luo et al. (2001) Nat. Neurosci.,
4: 231-232; Roberds et al. (2001) Hum. Mol. Genet., 10: 1317-1324),
while one group noticed subtle neurochemical deficits and
behavioral changes in otherwise viable and fertile mice (Harrison
et al. (2003) Mol. Cell Neurosci., 24: 646-655). Although recent
studies have shown that BACE1 knockout mice exhibit hypomyelination
of peripheral nerves (Willem et al. (2006) Science, 314: 664-666),
the consequences of BACE1 inhibition in adult animals, where
myelination has already taken place, are unclear. Recently BACE1
has been reported to cleave multiple substrates, including ST6Gal
I, PSGL-1, subunits of voltage-gated sodium channels, APP-like
proteins (APLPs), LDL receptor related protein (LRP) and, most
recently, type III neuregulin 1 (NRG1) (Willem et al. (2006)
Science, 314: 664-666; Hu et al. (2006) Nat. Neurosci., 9:
1520-1525). The consequences of inhibiting BACE1 directly are
therefore not yet fully understood.
[0014] Molecular modeling (Sauder et al. (2000) J. Mol. Biol., 300:
241-248) and subsequent X-ray crystallography (Hong et al. (2000)
Science, 290: 150-153; Maillard et al. (2007) J. Med. Chem., 50:
776-781) of the BACE-1 active site complexed with a
transition-state inhibitor provided crucial information about
BACE-1-substrate interactions. Structurally, the BACE-1 active site
is more open and less hydrophobic than other aspartyl proteases,
making development of effective in vivo BACE inhibitor candidates
difficult. While a there is a large drug discovery effort focused
on development of direct BACE inhibitors, none so far have advanced
significantly in clinical testing.
[0015] A few BACE inhibitors such as LY2811376 and CTS21166 entered
clinical testing, but did not go forward beyond Phase-1 due to
safety reasons. The discovery of other physiological substrates of
BACE raises a major concern in the clinical development of BACE
inhibitors or BACE modulators and could be a significant roadblock
in advancement of these inhibitors as a therapy for the
disease.
SUMMARY
[0016] In certain embodiments hydantoin compounds are provided
herein that are effective to inhibit BACE activity against APP.
Without being bound to a particular theory, it is believed the
activity of the hydantoins identified herein appears to be
associated with binding to BACE and/or to APP particularly when
these moieties form a BACE/APP complex. Accordingly, it is believed
the compounds described herein represent a new class of compounds
designated herein as APP-Binding-BACE Inhibitors (ABBIs) and
provide a new mechanism to modulate APP processing. The hydantoins
described herein appear to show improved brain permeability and
functional BACE inhibition.
[0017] In various aspects, the invention(s) contemplated herein may
include, but need not be limited to, any one or more of the
following embodiments:
Embodiment 1
[0018] A compound according to the formula:
##STR00001##
where M is
##STR00002##
R.sup.7 is selected from the group consisting of C.dbd.O, C.dbd.S,
C--NH.sub.2, and C.dbd.NH, and the bond represented by the wavy
line is a single bond when R.sup.7 is C.dbd.O, C.dbd.S, or
C.dbd.NH, and a double bond when R.sup.7 is C--NH.sub.2; R.sup.8
and R.sup.9 are independently selected from the group consisting of
H, alkyl, cycloalkyl, and aryl, provided that when the bond
represented by the wavy line is a double bond, then R.sup.9 is
absent; R.sup.0 is selected from the group consisting of aryl,
substituted aryl, disubstituted aryl, heteroaryl, substituted
heteroaryl, disubstituted heteroaryl, alkyl, haloalkyl, cycloalkyl,
alkenyl, and alkynyl; X.sup.1 is selected from the group consisting
of C-halogen (e.g., Cl or F), CH, and N; A is methyl or H; R.sup.5
and R.sup.6 are independently selected from halogen, H, alkyl,
aryl, trichloromethyl, and trifluoromethyl; R.sup.3 and R.sup.4 are
independently absent or selected from the group consisting of
alkyl, cycloalkyl, alkoxy, thioalky; and when X.sup.1 is C, then
R.sup.0 is not phenyl monosubstituted at the para position with
--OCHF.sub.2, or a pharmaceutically acceptable salt thereof, a
tautomer thereof, a pharmaceutically acceptable salt of a tautomer
thereof, an enantiomer thereof, or a pharmaceutically acceptable
salt of an enantiomer thereof.
Embodiment 2
[0019] The compound of embodiment 1, wherein said compound is a
compound according to the formula:
##STR00003##
Embodiment 3
[0020] The compound of embodiment 1, wherein said compound is a
compound according to the Formula:
##STR00004##
[0021] Embodiment 4
[0022] The compound according to any one of embodiments 1-3,
wherein R.sup.5 and R.sup.6 are independently selected from
halogen, H, alkyl, trichloromethyl, and trifluoromethyl.
Embodiment 5
[0023] The compound according to any one of embodiments 1-4,
wherein: X.sup.1 is selected from the group consisting of CH, and
N; and R.sup.5 and R.sup.6 are independently selected halogen.
Embodiment 6
[0024] The compound according to any one of embodiments 1-5,
wherein R.sup.7 is C.dbd.NH.
Embodiment 7
[0025] The compound according to any one of embodiments 1-5,
wherein R.sup.7 is C.dbd.O.
Embodiment 8
[0026] The compound of embodiment 6, wherein said compound is a
compound having the formula:
##STR00005##
Embodiment 9
[0027] The compound of embodiment 8, wherein R.sup.5 and R.sup.6
are independently selected halogens.
Embodiment 10
[0028] The compound of embodiment 9, wherein R.sup.5 and R.sup.6
are the same halogen.
Embodiment 11
[0029] The compound of embodiment 9, wherein R.sup.5 and R.sup.6
are both F.
Embodiment 12
[0030] The compound of embodiment 7, wherein said compound is a
compound of having the formula:
##STR00006##
Embodiment 13
[0031] The compound of embodiment 7, wherein said compound is a
compound of the formula:
##STR00007##
Embodiment 14
[0032] The compound of embodiment 7, wherein said compound is a
compound of the formula:
##STR00008##
Embodiment 15
[0033] The compound of embodiment 3, wherein R.sup.7 is
C.dbd.S.
Embodiment 16
[0034] The compound according to any one of embodiments 1-5,
wherein R.sup.7 is C--NH.sub.2 and said compound is a compound
having the formula:
##STR00009##
Embodiment 17
[0035] The compound of embodiment 16, wherein said compound is a
compound of Formula:
##STR00010##
where R.sup.1 and R.sup.2 are independently absent or selected from
the group consisting of alkyl, haloalkyl, cycloalkyl, alkenyl,
alkynyl, alkoxy, thioalkyl, aryl, substituted aryl, heteroaryl, and
substituted heteroaryl; and X.sup.2, Y, and Z are independently CH
or N.
Embodiment 18
[0036] The compound according to any one of embodiments 1-17,
wherein R.sup.5 and R.sup.6 are different halogens.
Embodiment 19
[0037] The compound according to any one of embodiments 1-17,
wherein R.sup.5 and R.sup.6 are the same halogen.
Embodiment 20
[0038] The compound according to any one of embodiments 1-19,
wherein R.sup.5 and R.sup.6 are independently Cl or F.
Embodiment 21
[0039] The compound of embodiment 17, wherein said compound is a
compound having the formula:
##STR00011##
Embodiment 22
[0040] The compound according to any one of embodiments 1-7 and
15-21, wherein X.sup.1 is CH.
Embodiment 23
[0041] The compound according to any one of embodiments 1-7 and
15-22, wherein R.sup.8 is H or CH.sub.3.
Embodiment 24
[0042] The compound of embodiment 3, wherein said compound is a
compound having the Formula:
##STR00012##
Embodiment 25
[0043] The compound of embodiment 3, wherein said compound is a
compound having the Formula:
##STR00013##
Embodiment 26
[0044] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00014##
Embodiment 27
[0045] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00015##
Embodiment 28
[0046] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00016##
Embodiment 29
[0047] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00017##
Embodiment 30
[0048] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00018##
Embodiment 31
[0049] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00019##
Embodiment 32
[0050] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00020##
Embodiment 33
[0051] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00021##
Embodiment 34
[0052] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00022##
Embodiment 35
[0053] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00023##
Embodiment 36
[0054] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00024##
Embodiment 37
[0055] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00025##
Embodiment 38
[0056] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00026##
Embodiment 39
[0057] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00027##
Embodiment 40
[0058] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00028##
Embodiment 41
[0059] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00029##
Embodiment 42
[0060] The compound of embodiment 3, wherein said compound is a
compound having the formula:
##STR00030##
Embodiment 43
[0061] A compound of Formula:
##STR00031##
or a pharmaceutically acceptable salt thereof, a tautomer thereof,
a pharmaceutically acceptable salt of a tautomer thereof, an
enantiomer thereof, or a pharmaceutically acceptable salt of an
enantiomer thereof.
Embodiment 44
[0062] The compound according to any one of embodiments 1-43,
wherein said compound is a substantially pure S enantiomer.
Embodiment 45
[0063] The compound according to any one of embodiments 1-43,
wherein said compound is a substantially pure R enantiomer.
Embodiment 46
[0064] The compound according to any one of embodiments 1-45,
wherein said compound binds to APP and/or to the enzyme BACE and/or
to an APP/BACE complex.
Embodiment 47
[0065] The compound according to any one of embodiments 1-45,
wherein said compound binds to APP and inhibits the enzyme
BACE.
Embodiment 48
[0066] A pharmaceutical formulation including a pharmaceutically
acceptable carrier and a compound according to any one of
embodiments 1-47.
Embodiment 49
[0067] The formulation of embodiment 48, wherein said formulation
is compounded for administration via a route selected from the
group consisting of oral delivery, isophoretic delivery,
transdermal delivery, parenteral delivery, aerosol administration,
administration via inhalation, intravenous administration, and
rectal administration.
Embodiment 50
[0068] The formulation of embodiment 48, wherein said formulation
is compounded for oral administration.
Embodiment 51
[0069] The formulation of embodiment 48, wherein said formulation
is sterile.
Embodiment 52
[0070] The formulation according to any one of embodiments 48-51,
wherein said formulation is a unit dosage formulation.
Embodiment 53
[0071] A method of preventing or delaying the onset of a
pre-Alzheimer's condition and/or cognitive dysfunction, and/or
ameliorating one or more symptoms of a pre-Alzheimer's condition
and/or cognitive dysfunction, or preventing or delaying the
progression of a pre-Alzheimer's condition or cognitive dysfunction
to Alzheimer's disease, said method including: administering to a
subject in need thereof a compound according to any one of
embodiments 1-47, or formulation according to any one of
embodiments 48-52 in an amount sufficient to prevent or delay the
onset of a pre-Alzheimer's cognitive dysfunction, and/or to
ameliorate one or more symptoms of a pre-Alzheimer's cognitive
dysfunction, and/or to prevent or delay the progression of a
pre-Alzheimer's cognitive dysfunction to Alzheimer's disease.
Embodiment 54
[0072] The method of embodiment 53, wherein said method is a method
of preventing or delaying the transition from a cognitively
asymptomatic pre-Alzheimer's condition to a pre-Alzheimer's
cognitive dysfunction.
Embodiment 55
[0073] The method of embodiment 53, wherein said method is a method
of preventing or delaying the onset of a pre-Alzheimer's cognitive
dysfunction.
Embodiment 56
[0074] The method of embodiment 53, wherein said method includes
ameliorating one or more symptoms of a pre-Alzheimer's cognitive
dysfunction.
Embodiment 57
[0075] The method of embodiment 53, wherein said method includes
preventing or delaying the progression of a pre-Alzheimer's
cognitive dysfunction to Alzheimer's disease.
Embodiment 58
[0076] The method according to any one of embodiments 53-57,
wherein said subject is a human.
Embodiment 59
[0077] The method according to any one of embodiments 53-58,
wherein said subject exhibits biomarker positivity of A.beta. in a
clinically normal human subject age 50 or older.
Embodiment 60
[0078] The method according to any one of embodiments 53-58,
wherein said subject exhibits asymptomatic cerebral
amyloidosis.
Embodiment 61
[0079] The method according to any one of embodiments 53-58,
wherein said subject exhibits cerebral amyloidosis in combination
with downstream neurodegeneration.
Embodiment 62
[0080] The method according to any one of embodiments 53-58,
wherein said subject exhibits cerebral amyloidosis in combination
with downstream neurodegeneration and subtle cognitive/behavioral
decline.
Embodiment 63
[0081] The method according to any one of embodiments 61-62,
wherein said downstream neurodegeneration is determined by one or
more elevated markers of neuronal injury selected from the group
consisting of tau, and FDG uptake.
Embodiment 64
[0082] The method according to any one of embodiments 60-63,
wherein said cerebral amyloidosis is determined by PET, or CSF
analysis, and structural MRI (sMRI).
Embodiment 65
[0083] The method according to any one of embodiments 53-64,
wherein said subject is a subject diagnosed with mild cognitive
impairment.
Embodiment 66
[0084] The method according to any one of embodiments 53-65,
wherein said subject shows a clinical dementia rating above zero
and below about 1.5.
Embodiment 67
[0085] The method according to any one of embodiments 53-66,
wherein the subject is human.
Embodiment 68
[0086] The method according to any one of embodiments 53-67,
wherein the subject is at risk of developing Alzheimer's
disease.
Embodiment 69
[0087] The method according to any one of embodiments 53-68,
wherein the subject has a familial risk for having Alzheimer's
disease.
Embodiment 70
[0088] The method according to any one of embodiments 53-68,
wherein the subject has a familial Alzheimer's disease (FAD)
mutation.
Embodiment 71
[0089] The method according to any one of embodiments 53-68,
wherein the subject has the APOE .epsilon.4 allele.
Embodiment 72
[0090] The method according to any one of embodiments 53-71,
wherein administration of said compound delays or prevents the
progression of MCI to Alzheimer's disease.
Embodiment 73
[0091] The method according to any one of embodiments 53-72,
wherein the subject is free of and does not have genetic risk
factors of Parkinson's disease or schizophrenia.
Embodiment 74
[0092] The method according to any one of embodiments 53-72,
wherein the subject is not diagnosed as having or at risk for
Parkinson's disease or schizophrenia.
Embodiment 75
[0093] The method according to any one of embodiments 53-72,
wherein the subject is not diagnosed as at risk for a neurological
disease or disorder other than Alzheimer's disease.
Embodiment 76
[0094] The method according to any one of embodiments 53-75,
wherein said administration produces a reduction in the CSF of
levels of one or more components selected from the group consisting
of A.beta.42, sAPP.beta., total-Tau (tTau), phospho-Tau (pTau),
APPneo, soluble A.beta.40, pTau/A.beta.42 ratio and tTau/A.beta.42
ratio, and/or an increase in the CSF of levels of one or more
components selected from the group consisting of
A.beta.42/A.beta.40 ratio, A.beta.42/A.beta.38 ratio, sAPP.alpha.,
sAPP.alpha./sAPP.beta. ratio, sAPP.alpha./A.beta.40 ratio, and
sAPP.alpha./A.beta.42 ratio.
Embodiment 77
[0095] The method according to any one of embodiments 53-76,
wherein said administration produces a reduction of the plaque load
in the brain of the subject.
Embodiment 78
[0096] The method according to any one of embodiments 53-76,
wherein said administration produces a reduction in the rate of
plaque formation in the brain of the subject.
Embodiment 79
[0097] The method according to any one of embodiments 53-76,
wherein said administration produces an improvement in the
cognitive abilities of the subject.
Embodiment 80
[0098] The method according to any one of embodiments 53-76,
wherein said administration produces an improvement in, a
stabilization of, or a reduction in the rate of decline of the
clinical dementia rating (CDR) of the subject.
Embodiment 81
[0099] The method according to any one of embodiments 53-76,
wherein the subject is a human and said administration produces a
perceived improvement in quality of life by the human.
Embodiment 82
[0100] The method according to any one of embodiments 53-81,
wherein the compound or formulation is administered via a route
selected from the group consisting of oral delivery, isophoretic
delivery, transdermal delivery, parenteral delivery, aerosol
administration, administration via inhalation, intravenous
administration, and rectal administration.
Embodiment 83
[0101] The method according to any one of embodiments 53-81,
wherein the compound or formulation is administered orally.
Embodiment 84
[0102] The method according to any one of embodiments 53-83,
wherein the administering is over a period of at least three
weeks.
Embodiment 85
[0103] The method according to any one of embodiments 53-83,
wherein the administering is over a period of at least 6
months.
Embodiment 86
[0104] A method of ameliorating one or more symptoms of Alzheimer's
disease, and/or reversing Alzheimer's disease, and/or reducing the
rate of progression of Alzheimer's disease, said method including:
administering to a subject in need thereof a compound according to
any one of embodiments 1-47, or formulation according to any one of
embodiments 48-52 in an amount sufficient to ameliorate one or more
symptoms of Alzheimer's disease, and/or to reverse Alzheimer's
disease, and/or to reduce the rate of progression of Alzheimer's
disease.
Embodiment 87
[0105] The method of embodiment 86, wherein said subject is a
human.
Embodiment 88
[0106] The method of embodiment 87, wherein said subject is a human
at least 50 years old.
Embodiment 89
[0107] The method according to any one of embodiments 86-88,
wherein said subject is diagnosed with early stage Alzheimer's
disease.
Embodiment 90
[0108] The method according to any one of embodiments 86-88,
wherein said subject is diagnosed with mid-stage Alzheimer's
disease.
Embodiment 91
[0109] The method according to any one of embodiments 86-88,
wherein said subject is diagnosed with late-stage Alzheimer's
disease.
Embodiment 92
[0110] The method according to any one of embodiments 86-91,
wherein said administering reduces the severity of Alzheimer's
disease.
Embodiment 93
[0111] The method according to any one of embodiments 86-91,
wherein said administering ameliorates one or more symptoms of
Alzheimer's disease.
Embodiment 94
[0112] The method according to any one of embodiments 86-91,
wherein said administering reduces the rate of progression of
Alzheimer's disease.
Embodiment 95
[0113] The method according to any one of embodiments 86-94,
wherein said administering results in a reduction in the CSF of
levels of one or more components selected from the group consisting
of A.beta.42, sAPP.beta., total-Tau (tTau), phospho-Tau (pTau),
APPneo, soluble A.beta.40, pTau/A.beta.42 ratio and tTau/A.beta.42
ratio, and/or an increase in the CSF of levels of one or more
components selected from the group consisting of
A.beta.42/A.beta.40 ratio, A.beta.42/A.beta.38 ratio, sAPP.alpha.,
sAPP.alpha./sAPP.beta. ratio, sAPP.alpha./A.beta.40 ratio, and
sAPP.alpha./A.beta.42 ratio, is a method of preventing or delaying
the transition from a cognitively asymptomatic pre-Alzheimer's
condition to a pre-Alzheimer's cognitive dysfunction.
Embodiment 96
[0114] The method according to any one of embodiments 86-95,
wherein said administration produces a reduction of the plaque load
in the brain of the subject.
Embodiment 97
[0115] The method according to any one of embodiments 86-95,
wherein said administration produces a reduction in the rate of
plaque formation in the brain of the subject.
Embodiment 98
[0116] The method according to any one of embodiments 86-95,
wherein said administration produces an improvement in the
cognitive abilities of the subject.
Embodiment 99
[0117] The method according to any one of embodiments 86-95,
wherein said administration produces an improvement in, a
stabilization of, or a reduction in the rate of decline of the
clinical dementia rating (CDR) of the subject.
Embodiment 100
[0118] The method according to any one of embodiments 86-95,
wherein the subject is a human and said administration produces a
perceived improvement in quality of life by the human.
Embodiment 101
[0119] The method according to any one of embodiments 86-95,
wherein said administering results in reduced cerebral amyloidosis
and/or downstream neurodegeneration.
Embodiment 102
[0120] The method of embodiment 101, wherein said downstream
neurodegeneration is determined by one or more markers of neuronal
injury selected from the group consisting of tau, FDG uptake,
decrease in sAPPalpha, increase in sAPPbeta, and Abeta.
Embodiment 103
[0121] The method according to any one of embodiments 101-102,
wherein said cerebral amyloidosis is determined by PET using
amyloid/tau binding agents, CSF analysis, and structural MRI
(sMRI).
Embodiment 104
[0122] The method according to any one of embodiments 86-103,
wherein said subject shows a clinical dementia rating indicative of
Alzheimer's disease.
Embodiment 105
[0123] The method according to any one of embodiments 86-104,
wherein the subject has a familial risk for having Alzheimer's
disease.
Embodiment 106
[0124] The method according to any one of embodiments 86-105,
wherein the subject has a familial Alzheimer's disease (FAD)
mutation.
Embodiment 107
[0125] The method according to any one of embodiments 86-105,
wherein the subject has the APOE .epsilon.4 allele.
Embodiment 108
[0126] The method according to any one of embodiments 86-107,
wherein the subject is free of and does not have genetic risk
factors of Parkinson's disease or schizophrenia.
Embodiment 109
[0127] The method according to any one of embodiments 86-107,
wherein the subject is not diagnosed as having or at risk for
Parkinson's disease or schizophrenia.
Embodiment 110
[0128] The method according to any one of embodiments 86-109,
wherein the subject does not have a neurological disease or
disorder other than Alzheimer's disease.
Embodiment 111
[0129] The method according to any one of embodiments 86-110,
wherein the subject is not diagnosed as having or at risk for a
neurological disease or disorder other than Alzheimer's
disease.
Embodiment 112
[0130] The method according to any one of embodiments 86-111,
wherein the compound is administered via a route selected from the
group consisting of oral delivery, isophoretic delivery,
transdermal delivery, parenteral delivery, aerosol administration,
administration via inhalation, intravenous administration, and
rectal administration.
Embodiment 113
[0131] The method according to any one of embodiments 86-112,
wherein the compound is formulated for administration via a route
selected from the group consisting of oral delivery, isophoretic
delivery, transdermal delivery, parenteral delivery, aerosol
administration, administration via inhalation, intravenous
administration, and rectal administration.
Embodiment 114
[0132] The method according to any one of embodiments 86-113,
wherein the compound is administered orally.
Embodiment 115
[0133] The method according to any one of embodiments 86-114,
wherein the administering is over a period of at least three
weeks.
Embodiment 116
[0134] The method according to any one of embodiments 86-114,
wherein the administering is over a period of at least 6
months.
Embodiment 117
[0135] The method according to any one of embodiments 53-116,
wherein said compound is administered in combination with one or
more agents selected from the group consisting of disulfuram and/or
analogues thereof, honokiol and/or analogues thereof, tropisetron
and/or analogues thereof, nimetazepam and/or analogues thereof,
tropinol-esters and/or related esters and/or analogues thereof,
TrkA kinase inhibitors (e.g., ADDN-1351) and/or analogues thereof,
D2 receptor agonists, alpha1-adrenergic receptor antagonists, and
APP-specific BACE Inhibitors including, but not limited to
galangin, a galangin prodrug, rutin, a rutin prodrug, and other
flavonoids and flavonoid prodrugs.
Embodiment 118
[0136] The method of embodiment 117, wherein said compound is
administered in combination with tropisetron.
Embodiment 119
[0137] A method of slowing the progression, stopping, or reversing
age-related macular degeneration (AMD) in a mammal, said method
including administering to said mammal a compound according to any
one of embodiments 1-47, or formulation according to any one of
embodiments 48-52 in an amount sufficient to slow the progression,
stop, or reverse age-related macular degeneration in said
mammal.
Embodiment 120
[0138] A method for the treatment of a disease or disorder
associated with BACE activity in a subject in need thereof, wherein
said method includes providing to said subject a therapeutically
effective amount of a compound according to any one of embodiments
1-47, or formulation according to any one of embodiments 48-52.
Embodiment 121
[0139] The method of embodiment 120, wherein said disease or
disorder is selected from the group consisting of Alzheimer's
disease; cognitive impairment, Down's Syndrome, HCHWA-D, cognitive
decline, senile dementia, cerebral amyloid angiopathy, and a
neurodegenerative disorder.
Embodiment 122
[0140] The method of embodiment 121, wherein said disease or
disorder is characterized by the production of amyloid deposits
and/or neurofibrillary tangles.
Embodiment 123
[0141] A kit including one or more containers containing a compound
according to any one of embodiments 1-47, or formulation according
to any one of embodiments 48-52.
Embodiment 124
[0142] The kit of embodiment 123, wherein said kit further includes
a second agent selected from the group consisting of disulfuram
and/or analogues thereof, honokiol and/or analogues thereof,
tropisetron and/or analogues thereof, nimetazepam and/or analogues
thereof, tropinol-esters and/or related esters and/or analogues
thereof, TrkA kinase inhibitors (e.g., ADDN-1351) and/or analogues
thereof, D2 receptor agonists, alpha1-adrenergic receptor
antagonists, and APP-specific BACE Inhibitors including, but not
limited to galangin, a galangin prodrug, rutin, a rutin prodrug,
and other flavonoids and flavonoid prodrugs.
Embodiment 125
[0143] The kit of embodiment 124, wherein said second agent is
tropisetron.
Embodiment 126
[0144] The kit according to any one of embodiments 123-125, further
including instructional materials teaching dosages and treatment
regimen for the active agents contained in the kit.
Embodiment 127
[0145] The compounds, methods, or kits according to any one of
embodiments 1-126, wherein said embodiments expressly exclude
FAH-2.
Embodiment 128
[0146] The compounds, methods, or kits according to any one of
embodiments 1-127, wherein said embodiments expressly exclude
FAH-3.
Embodiment 129
[0147] The compounds, methods, or kits according to any one of
embodiments 1-128, wherein said embodiments expressly exclude
FAH-1.
Embodiment 130
[0148] The compounds, methods, or kits according to any one of
embodiments 1-129, wherein said embodiments expressly exclude
FAH-4.
Embodiment 131
[0149] The compounds, methods, or kits according to any one of
embodiments 1-130, wherein said embodiments expressly exclude
FAH-5.
Embodiment 132
[0150] The compounds, methods, or kits according to any one of
embodiments 1-131, wherein said embodiments expressly exclude
FAH-6.
Embodiment 133
[0151] The compounds, methods, or kits according to any one of
embodiments 1-132, wherein said embodiments expressly exclude
FAH-7.
Embodiment 134
[0152] The compounds, methods, or kits according to any one of
embodiments 1-133, wherein said embodiments expressly exclude
FAH-8.
Embodiment 135
[0153] The compounds, methods, or kits according to any one of
embodiments 1-134, wherein said embodiments expressly exclude
FAH-10
Embodiment 136
[0154] The compounds, methods, or kits according to any one of
embodiments 1-135, wherein said embodiments expressly exclude
FAH-11.
Embodiment 137
[0155] The compounds, methods, or kits according to any one of
embodiments 1-136, wherein said embodiments expressly exclude
FAH-12.
Embodiment 138
[0156] The compounds, methods, or kits according to any one of
embodiments 1-137, wherein said embodiments expressly exclude
FAH-13.
Embodiment 139
[0157] The compounds, methods, or kits according to any one of
embodiments 1-138, wherein said embodiments expressly exclude
FAH-14.
Embodiment 140
[0158] The compounds, methods, or kits according to any one of
embodiments 1-139, wherein said embodiments expressly exclude
FAH-15.
Embodiment 141
[0159] The compounds, methods, or kits according to any one of
embodiments 1-140, wherein said embodiments expressly exclude
FAH-17.
Embodiment 142
[0160] The compounds, methods, or kits according to any one of
embodiments 1-141, wherein said embodiments expressly exclude
FAH-19.
Embodiment 143
[0161] The compounds, methods, or kits according to any one of
embodiments 1-142, wherein said embodiments expressly exclude
FAH-22.
Embodiment 144
[0162] The compounds, methods, or kits according to any one of
embodiments 1-143, wherein said embodiments expressly exclude
FAH-23.
Embodiment 145
[0163] The compounds, methods, or kits according to any one of
embodiments 1-144, wherein said embodiments expressly exclude
FAH-25.
Embodiment 146
[0164] The compounds, methods, or kits according to any one of
embodiments 1-145, wherein said embodiments expressly exclude
FAH-27.
Embodiment 147
[0165] The compounds, methods, or kits according to any one of
embodiments 1-146, wherein said embodiments expressly exclude
FAH-28.
Embodiment 148
[0166] The compounds, methods, or kits according to any one of
embodiments 1-147, wherein the compound(s) described herein (or a
tautomer or stereoisomer thereof; or pharmaceutically acceptable
salt or solvate of said compound, said stereoisomer, or said
tautomer) are administered to a subject not diagnosed with or under
treatment for convulsions and/or epilepsy.
Embodiment 149
[0167] The compounds, methods, or kits according to any one of
embodiments 1-148 where the compound(s) described herein (or a
tautomer or stereoisomer thereof; or pharmaceutically acceptable
salt or solvate of said compound, said stereoisomer, or said
tautomer) are administered to a subject not subject to, and/or
diagnosed with, and/or under treatment for one or more of the
following: arrhythmia, epilepsy, neurosurgery, peripheral
neuropathy, rheumatoid arthritis, seizure prevention, seizures,
status epilepticus, and/or trigeminal neuralgia.
DEFINITIONS
[0168] Unless otherwise indicated, reference to a compound (e.g.,
to a hydantoins as described herein) should be construed broadly to
include pharmaceutically acceptable salts, prodrugs, tautomers,
alternate solid forms, non-covalent complexes, and combinations
thereof, of a chemical entity of the depicted structure or chemical
name.
[0169] Generally, reference to a certain element such as hydrogen
or H is meant to include all isotopes of that element. For example,
if an R group is defined to include hydrogen or H, it also includes
deuterium and tritium. Accordingly, isotopically labeled compounds
are within the scope of this invention.
[0170] A pharmaceutically acceptable salt is any salt of the parent
compound that is suitable for administration to an animal or human.
A pharmaceutically acceptable salt also refers to any salt which
may form in vivo as a result of administration of an acid, another
salt, or a prodrug which is converted into an acid or salt. A salt
comprises one or more ionic forms of the compound, such as a
conjugate acid or base, associated with one or more corresponding
counterions. Salts can form from or incorporate one or more
deprotonated acidic groups (e.g. carboxylic acids), one or more
protonated basic groups (e.g. amines), or both (e.g.
zwitterions).
[0171] A prodrug is a compound that is converted to a
therapeutically active compound after administration. For example,
conversion may occur by hydrolysis of an ester group, such as a
C.sub.1-C.sub.6 alkyl ester of the carboxylic acid group of the
present compounds, or some other biologically labile group. Prodrug
preparation is well known in the art. For example, "Prodrugs and
Drug Delivery Systems," which is a chapter in Richard B. Silverman,
Organic Chemistry of Drug Design and Drug Action, 2d Ed., Elsevier
Academic Press: Amsterdam, 2004, pp. 496-557, provides further
detail on the subject.
[0172] Tautomers are isomers that are in equilibrium with one
another. For example, tautomers may be related by transfer of a
proton, hydrogen atom, or hydride ion.
[0173] Unless stereochemistry is explicitly depicted, a structure
is intended to include every possible stereoisomer, both pure or in
any possible mixture.
[0174] Alternate solid forms are different solid forms than those
that may result from practicing the procedures described herein.
For example, alternate solid forms may be polymorphs, different
kinds of amorphous solid forms, glasses, and the like. In various
embodiments alternate solid forms of any of the compounds described
herein are contemplated.
[0175] In general, "substituted" refers to an organic group as
defined below (e.g., an alkyl group) in which one or more bonds to
a hydrogen atom contained therein are replaced by a bond to
non-hydrogen or non-carbon atoms. Substituted groups also include
groups in which one or more bonds to a carbon(s) or hydrogen(s)
atom are replaced by one or more bonds, including double or triple
bonds, to a heteroatom. Thus, a substituted group will be
substituted with one or more substituents, unless otherwise
specified. In some embodiments, a substituted group is substituted
with 1, 2, 3, 4, 5, or 6 substituents. Examples of substituent
groups include: halogens (i.e., F, Cl, Br, and I); hydroxyls;
alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy,
and heterocyclylalkoxy groups; carbonyls (oxo); carboxyls; esters;
urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines;
thiols; sulfides; sulfoxides; sulfones; sulfonyls; sulfonamides;
amines; N-oxides; hydrazines; hydrazides; hydrazones; azides;
amides; ureas; amidines; guanidines; enamines; imides; isocyanates;
isothiocyanates; cyanates; thiocyanates; imines; nitro groups;
nitriles (i.e., CN), and the like.
[0176] The term "alkyl" refers to and covers any and all groups
that are known as normal alkyl, branched-chain alkyl, cycloalkyl
and also cycloalkyl-alkyl. Illustrative alkyl groups include, but
are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, sec-butyl, t-butyl, octyl, and decyl. The term
"cycloalkyl" refers to cyclic, including polycyclic, saturated
hydrocarbyl groups. Examples include, but are not limited to
cyclopentyl, cyclohexyl, dicyclopentyl, norbornyl,
octahydronapthyl, and spiro[3.4]octyl. In certain embodiments,
alkyl groups contain 1-12 carbon atoms (C1-12 alkyl), or 1-9 carbon
atoms (C.sub.1-9 alkyl), or 1-6 carbon atoms (C.sub.1-6 alkyl), or
1-5 carbon atoms (C.sub.1-5 alkyl), or carbon atoms (C.sub.1-4
alkyl), or 1-3 carbon atoms (C.sub.1-3 alkyl), or 1-2 carbon atoms
(C.sub.1-2 alkyl).
[0177] By way of example, the term "C.sub.1-6 alkyl group" refers
to a straight chain or branched chain alkyl group having 1 to 6
carbon atoms, and may be exemplified by a methyl group, an ethyl
group, an n-propyl group, an isopropyl group, an n-butyl group, an
isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl
group, a tert-amyl group, a 3-methylbutyl group, a neopentyl group,
and an n-hexyl group.
[0178] The term "alkoxy" as used herein means an alkyl group bound
through a single, terminal oxygen atom. An "alkoxy" group may be
represented as --O-alkyl where alkyl is as defined above. The term
"aryloxy" is used in a similar fashion, and may be represented as
--O-aryl, with aryl as defined below. The term "hydroxy" refers to
--OH.
[0179] Similarly, the term "alkylthio" as used herein means an
alkyl group bound through a single, terminal sulfur atom. An
"alkylthio" group may be represented as --S-alkyl where alkyl is as
defined above. The term "arylthio" is used similarly, and may be
represented as --S-aryl, with aryl as defined below. The term
"mercapto" refers to --SH.
[0180] Aryl groups are cyclic aromatic hydrocarbons that do not
contain heteroatoms. Aryl groups include monocyclic, bicyclic and
polycyclic ring systems. Thus, aryl groups include, but are not
limited to, phenyl, azulenyl, heptalenyl, biphenylenyl, indacenyl,
fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl,
chrysenyl, biphenyl, anthracenyl, indenyl, indanyl, pentalenyl, and
naphthyl groups. In some embodiments, aryl groups contain 6-14
carbons, and in others from 6 to 12 or even 6-10 carbon atoms in
the ring portions of the groups. Although the phrase "aryl groups"
includes groups containing fused rings, such as fused
aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl,
and the like), it does not include aryl groups that have other
groups, such as alkyl or halo groups, bonded to one of the ring
members. Rather, groups such as tolyl are referred to as
substituted aryl groups. Representative substituted aryl groups may
be mono-substituted or substituted more than once. For example,
monosubstituted aryl groups include, but are not limited to, 2-,
3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may
be substituted with substituents such as those listed above.
[0181] The term "heteroaryl group" refers to a monocyclic or
condensed-ring aromatic heterocyclic group containing one or more
hetero-atoms selected from O, S and N. If the aromatic heterocyclic
group has a condensed ring, it can include a partially hydrogenated
monocyclic group. Examples of such a heteroaryl group include a
pyrazolyl group, a thiazolyl group, an isothiazolyl group, a
thiadiazolyl group, an imidazolyl group, a furyl group, a thienyl
group, an oxazolyl group, an isoxazolyl group, a pyrrolyl group, an
imidazolyl group, a (1,2,3)- and (1,2,4)-triazolyl group, a
tetrazolyl group, a pyranyl group, a pyridyl group, a pyrimidinyl
group, a pyrazinyl group, a pyridazinyl group, a quinolyl group, an
isoquinolyl group, a benzofuranyl group, an isobenzofuranyl group,
an indolyl group, an isoindolyl group, an indazolyl group, a
benzoimidazolyl group, a benzotriazolyl group, a benzoxazolyl
group, a benzothiazolyl group, a benzo[b]thiophenyl group, a
thieno[2,3-b]thiophenyl group, a (1,2)- and (1,3)-benzoxathiol
group, a chromenyl group, a 2-oxochromenyl group, a
benzothiadiazolyl group, a quinolizinyl group, a phthalazinyl
group, a naphthyridinyl group, a quinoxalinyl group, a quinazolinyl
group, a cinnolinyl group, and a carbazolyl group.
[0182] A "derivative" of a compound means a chemically modified
compound wherein the chemical modification takes place at one or
more functional groups of the compound. The derivative however, is
expected to retain, or enhance, the pharmacological activity of the
compound from which it is derived.
[0183] As used herein, "administering" refers to local and systemic
administration, e.g., including enteral, parenteral, pulmonary, and
topical/transdermal administration. Routes of administration for
agents (e.g., hydantoins described herein, or a tautomer(s) or
stereoisomer(s) thereof, or pharmaceutically acceptable salts or
solvates of said hydantoin(s), said stereoisomer(s), or said
tautomer(s), or analogues, derivatives, or prodrugs thereof) that
find use in the methods described herein include, e.g., oral (per
os (p.o.)) administration, nasal or inhalation administration,
administration as a suppository, topical contact, transdermal
delivery (e.g., via a transdermal patch), intrathecal (IT)
administration, intravenous ("iv") administration, intraperitoneal
("ip") administration, intramuscular ("im") administration,
intralesional administration, or subcutaneous ("sc")
administration, or the implantation of a slow-release device e.g.,
a mini-osmotic pump, a depot formulation, etc., to a subject.
Administration can be by any route including parenteral and
transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal).
Parenteral administration includes, e.g., intravenous,
intramuscular, intra-arterial, intradermal, subcutaneous,
intraperitoneal, intraventricular, ionophoretic and intracranial.
Other modes of delivery include, but are not limited to, the use of
liposomal formulations, intravenous infusion, transdermal patches,
etc.
[0184] The terms "systemic administration" and "systemically
administered" refer to a method of administering the agent(s)
described herein or composition to a mammal so that the agent(s) or
composition is delivered to sites in the body, including the
targeted site of pharmaceutical action, via the circulatory system.
Systemic administration includes, but is not limited to, oral,
intranasal, rectal and parenteral (e.g., other than through the
alimentary tract, such as intramuscular, intravenous,
intra-arterial, transdermal and subcutaneous) administration.
[0185] The term "co-administering" or "concurrent administration"
or "administering in conjunction with" when used, for example with
respect to the active agent(s) described herein e.g., hydantoins
described herein, or a tautomer(s) or stereoisomer(s) thereof, or
pharmaceutically acceptable salts or solvates of said hydantoin(s),
said stereoisomer(s), or said tautomer(s), or analogues,
derivatives, or prodrugs thereof and a second active agent (e.g., a
cognition enhancer), refers to administration of the agent(s)
and/the second active agent such that both can simultaneously
achieve a physiological effect. The two agents, however, need not
be administered together. In certain embodiments, administration of
one agent can precede administration of the other. Simultaneous
physiological effect need not necessarily require presence of both
agents in the circulation at the same time. However, in certain
embodiments, co-administering typically results in both agents
being simultaneously present in the body (e.g., in the plasma) at a
significant fraction (e.g., 20% or greater, preferably 30% or 40%
or greater, more preferably 50% or 60% or greater, most preferably
70% or 80% or 90% or greater) of their maximum serum concentration
for any given dose.
[0186] The term "effective amount" or "pharmaceutically effective
amount" refer to the amount and/or dosage, and/or dosage regime of
one or more agent(s) necessary to bring about the desired result
e.g., an amount sufficient to mitigating in a mammal one or more
symptoms associated with mild cognitive impairment (MCI), or an
amount sufficient to lessen the severity or delay the progression
of a disease characterized by amyloid deposits in the brain in a
mammal (e.g., therapeutically effective amounts), an amount
sufficient to reduce the risk or delaying the onset, and/or reduce
the ultimate severity of a disease characterized by amyloid
deposits in the brain in a mammal (e.g., prophylactically effective
amounts).
[0187] The phrase "cause to be administered" refers to the actions
taken by a medical professional (e.g., a physician), or a person
controlling medical care of a subject, that control and/or permit
the administration of the agent(s) at issue to the subject. Causing
to be administered can involve diagnosis and/or determination of an
appropriate therapeutic or prophylactic regimen, and/or prescribing
particular agent(s) for a subject. Such prescribing can include,
for example, drafting a prescription form, annotating a medical
record, and the like.
[0188] As used herein, the terms "treating" and "treatment" refer
to delaying the onset of, retarding or reversing the progress of,
reducing the severity of, or alleviating or preventing either the
disease or condition to which the term applies, or one or more
symptoms of such disease or condition.
[0189] The term "mitigating" refers to reduction or elimination of
one or more symptoms of that pathology or disease, and/or a
reduction in the rate or delay of onset or severity of one or more
symptoms of that pathology or disease, and/or the prevention of
that pathology or disease. In certain embodiments, the reduction or
elimination of one or more symptoms of pathology or disease can
include, but is not limited to, reduction or elimination of one or
more markers that are characteristic of the pathology or disease
(e.g., of total-Tau (tTau), phospho-Tau (pTau), APPneo, soluble
A.beta.40, pTau/A.beta.42 ratio and tTau/A.beta.42 ratio, and/or an
increase in the CSF of levels of one or more components selected
from the group consisting of A.beta.42/A.beta.40 ratio,
A.beta.42/A.beta.38 ratio, sAPP.alpha., sAPP.alpha./sAPP.beta.
ratio, sAPP.alpha./A.beta.40 ratio, sAPP.alpha./A.beta.42 ratio,
etc.) and/or reduction, stabilization or reversal of one or more
diagnostic criteria (e.g., clinical dementia rating (CDR)).
[0190] As used herein, the phrase "consisting essentially of"
refers to the genera or species of active pharmaceutical agents
recited in a method or composition, and further can include other
agents that, on their own do not substantial activity for the
recited indication or purpose. In some embodiments, the phrase
"consisting essentially of" expressly excludes the inclusion of one
or more additional agents that have neuropharmacological activity
other than the recited agent(s) (e.g., other than ASBIs such as
galangin, rutin, and analogues, derivatives, or prodrugs thereof).
In some embodiments, the phrase "consisting essentially of"
expressly excludes the inclusion of one or more additional active
agents other than the active agent(s) described herein (e.g., other
than ASBIs such as galangin, rutin, and analogues, derivatives, or
prodrugs thereof). In some embodiments, the phrase "consisting
essentially of" expressly excludes the inclusion of one or more
acetylcholinesterase inhibitors.
[0191] The terms "subject", "individual", and "patient"
interchangeably refer to a mammal, preferably a human or a
non-human primate, but also domesticated mammals (e.g., canine or
feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster,
guinea pig) and agricultural mammals (e.g., equine, bovine,
porcine, ovine). In various embodiments, the subject can be a human
(e.g., adult male, adult female, adolescent male, adolescent
female, male child, female child) under the care of a physician or
other health worker in a hospital, psychiatric care facility, as an
outpatient, or other clinical context. In certain embodiments the
subject may not be under the care or prescription of a physician or
other health worker.
[0192] The term "formulation" or "drug formulation" or "dosage
form" or "pharmaceutical formulation" as used herein refers to a
composition containing at least one therapeutic agent or medication
for delivery to a subject. In certain embodiments the dosage form
comprises a given "formulation" or "drug formulation" and may be
administered to a patient in the form of a lozenge, pill, tablet,
capsule, suppository, membrane, strip, liquid, patch, film, gel,
spray or other form.
[0193] The term "mucosal membrane" refers generally to any of the
mucus-coated biological membranes in the body. In certain
embodiments active agent(s) described herein can be administered
herein via any mucous membrane found in the body, including, but
not limited to buccal, perlingual, nasal, sublingual, pulmonary,
rectal, and vaginal mucosa. Absorption through the mucosal
membranes of the oral cavity and those of the gut are of interest.
Thus, peroral, buccal, sublingual, gingival and palatal absorption
are contemplated herein.
[0194] The term "transmucosal" delivery of a drug and the like is
meant to encompass all forms of delivery across or through a
mucosal membrane.
[0195] The term "bioadhesion" as used herein refers to the process
of adhesion of the dosage form(s) to a biological surface, e.g.,
mucosal membranes.
[0196] "Controlled drug delivery" refers to release or
administration of a drug from a given dosage form in a controlled
fashion in order to achieve the desired pharmacokinetic profile in
vivo. An aspect of "controlled" drug delivery is the ability to
manipulate the formulation and/or dosage form in order to establish
the desired kinetics of drug release.
[0197] "Sustained drug delivery" refers to release or
administration of a drug from a source (e.g., a drug formulation)
in a sustained fashion over a protracted yet specific period of
time, that may extend from several minutes to a few hours, days,
weeks or months. In various embodiments the term "sustained" will
be used to refer to delivery of consistent and/oe substantially
constant levels of drug over a time period ranging from a few
minutes to a day, with a profile characterized by the absence of an
immediate release phase, such as the one obtained from IV
administration.
[0198] The term "T." as used herein means the time point of maximum
observed plasma concentration.
[0199] The term "C.sub.max" as used herein means the maximum
observed plasma concentration.
[0200] The term "plasma t.sub.1/2" as used herein means the
observed "plasma half-life" and represents the time required for
the drug plasma concentration to reach the 50% of its maximal value
(C.sub.max). This facilitates determination of the mean duration of
pharmacological effects. In addition, it facilitates direct and
meaningful comparisons of the duration of different test articles
after delivery via the same or different routes.
[0201] The term "Optimal Therapeutic Targeting Ratio" or "OTTR"
represents the average time that the drug is present at therapeutic
levels, defined as time within which the drug plasma concentration
is maintained above 50% of C.sub.max normalized by the drug's
elimination half-life multiplied by the ratio of the C.sub.max
obtained in the dosage form of interest over the C.sub.max
following IV administration of equivalent doses and it is
calculated by the formula:
OTTR=(C.sup.IV.sub.max/C.sub.max).times.(Dose/Dose.sup.IV)(Time
above 50% of C.sub.max)/(Terminal.sup.IV elimination half-life of
the drug).
[0202] The term "substantial pure" means sufficiently homogeneous
to appear free of readily detectable impurities as determined by
standard methods of analysis, such as thin layer chromatography
(TLC), gel electrophoresis and high performance liquid
chromatography (HPLC), used by those of skill in the art to assess
such purity, or sufficiently pure such that further purification
would not detectably alter the physical or chemical properties, of
the compound. Methods for purification of the compounds to produce
substantially chemically pure compounds are known to those of skill
in the art. A substantially chemically pure compound may, however,
be a mixture of stereoisomers or isomers. In such instances,
further purification might increase the specific activity of the
compound.
[0203] The term "substantially pure" when used with respect to
enantiomers indicates that one particular enantiomer (e.g. an S
enantiomer or an R enantiomer) is substantially free of its
stereoisomer. In various embodiments substantially pure indicates
that a particular enantiomer is at least 70%, or at least 80%, or
at least 90%, or at least 95%, or at least 98%, or at least 99% of
the purified compound. Methods of producing substantially pure
enantiomers are well known to those of skill in the art. For
example, a single stereoisomer, e.g., an enantiomer, substantially
free of its stereoisomer may be obtained by resolution of the
racemic mixture using a method such as formation of diastereomers
using optically active resolving agents (Stereochemistry of Carbon
Compounds, (1962) by E. L. Eliel, McGraw Hill; Lochmuller (1975) J.
Chromatogr., 113(3): 283-302). Racemic mixtures of chiral compounds
of the can be separated and isolated by any suitable method,
including, but not limited to: (1) formation of ionic,
diastereomeric salts with chiral compounds and separation by
fractional crystallization or other methods, (2) formation of
diastereomeric compounds with chiral derivatizing reagents,
separation of the diastereomers, and conversion to the pure
stereoisomers, and (3) separation of the substantially pure or
enriched stereoisomers directly under chiral conditions. Another
approach for separation of the enantiomers is to use a Diacel
chiral column and elution using an organic mobile phase such as
done by Chiral Technologies (www.chiraltech.com) on a fee for
service basis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0204] FIG. 1 illustrates various hydantoins.
[0205] FIG. 2 illustrates various hydantoins.
[0206] FIG. 3 models of proposed interaction of the hydantoin with
the FLAP region of BACE1. The lower panel illustrates interaction
of the B-ring 3,4-substituent with the FLAP, Trp76 disrupts
Trp-76-.fwdarw.Tyr-71 H-bonding causing Tyr-71 to flip to the left
and interact with the difluoro containing A ring.
[0207] FIG. 4 illustrates APP Binding BACE Inhibitor (ABBI) FAH-3
binding to eAPP.sub.575-624 as measured by surface plasmon
resonance (SPR) screening. The binding affinity of the compounds
for the ectodomain of APP was determined using SPR. We have
developed a technique for measuring the affinity of compounds to
fragments of the ectodomain of APP. For the compound 3 binding
experiments a TRX-eAPP575-624 substrate was used. The eAPP was
crosslinked linked to the CM5 Biacore chips (GE Healthcare).
Compound 3 at various concentrations were used in the flow through
over the chip and the plasmon resonance signal was determined using
a Biacore T100.
[0208] FIG. 5 illustrates inhibition of A.beta. production by
FAH-3.
[0209] FIG. 6A illustrates selectivity of ABBI for inhibition of
the APP-BACE cleavage as compared to the PSGL-BACE cleavage shown
in FIG. 6B.
DETAILED DESCRIPTION
[0210] In various embodiments, hydantoins are identified that
appear to inhibit .beta.-secretase mediated APP processing by a
novel mechanism. In particular, without being bound to a particular
theory, it is believed that these molecules interact with BACE
and/or with APP and/or with a BACE/APP complex and thereby inhibit
the BACE cleavage of the MBP-C125 APP substrate, resulting in the
inhibition of the production of C99 and the .beta.-site peptide
substrate (P5-P5'). In addition, the various hydantoins identified
herein inhibit A.beta.42 in neuroblastoma SHSY5Y cells. Further we
demonstrate the activity of the hydantoins identified herein
appears to be associated with binding to BACE and/or to APP
particularly when these moieties form a BACE/APP complex.
Accordingly, it is believed the compounds described herein
represent a new class of compounds designated herein as
APP-Binding-BACE Inhibitors (ABBIs) and provide a new mechanism to
modulate APP processing. The hydantoins described herein appear to
show improved brain permeability and functional BACE
inhibition.
[0211] The ABBIs are specific for the APP and/or BACE and/or the
APP/BACE complex and are believed to show fewer undesired
side-effects because the ABBIs are typically not active on other
substrates for the enzyme or other enzyme complexes. With respect
to inhibitors of .gamma.-secretase, substrates other than APP, such
as Notch, raise concerns for potential side effects of
.gamma.-secretase inhibition, and the recent failure of the
.gamma.-secretase inhibitor, Semagacestat, serves to reinforce such
concerns. Similarly in the case of BACE, for example, inhibition of
non-APP substrates such as PSGL1 or LRP could produce adverse
side-effects. Therefore, a desirable BACE inhibitor would be one
that would bind/interact not with BACE but rather to APP, or to the
APP/BACE complex leading to APP-specific BACE complex inhibition
(ABBI).
[0212] Such ABBIs would potentially interact with the APP-BACE
complex, e.g., at the membrane and prevent its transition to the
"active" complex in early endosomes, where at pH<5 BACE is fully
active. Some .beta.-site binding antibodies have been shown to
block the cleavage of APP by BACE and also work in animal models of
AD, however for effective pharmaceutical development small organic
molecules are typically preferred to relatively large biomolecules
such as antibodies.
[0213] The data we report herein on the identification of the first
ABBIs demonstrates that such an approach is feasible. Without being
bound to a particular theory, ABBIs appear to inhibit BACE activity
by interacting with APP, particular when in an APP/BACE complex
thereby inhibiting the BACE cleavage of the Amyloid Precursor
Protein (APP) but not the proteolytic cleavage of other substrates.
Such therapeutics are believed to represent a new class of
Alzheimer's disease (or other amyloidogenic disease)
therapeutics.
[0214] The active site of BACE1 is covered by flaps. A single flap
of 14 residues in length forms an .alpha.-hairpin structure that is
perpendicular to a cleft that houses the active site and covers the
central part of that active site. During the catalytic cycle, the
flaps open to allow entrance of substrate (APP) into the catalytic
cleft and also to release hydrolytic products. Initially,
hydantoins described herein were produced by introducing a dihalo
(e.g., difluoro) ring into the amino hydantoin of Compound 0 (shown
in FIG. 1) to produce compound 1 (also shown in FIG. 1). Without
being bound to a particular theory, it is believed the dihalo-ring
introduces a FLAP interaction (e.g. an interaction with FLAP
residues Tyr-71 (through pi-stacking) and Trp-76 (through
interaction with OCF2)) and restricts FLAP movement limiting APP
entry into the active site and/or the exit of cleavage products
(see, e.g., FIG. 3). This provides a new family of small
(MW<400) brain penetrant BACE inhibitors (ABCIs).
[0215] Compound 2 and other hydantoins were produced (see, e.g.,
compounds 1-5 pharmacokinetic evaluation of these hydantoins was
determined in brain uptake assays using NTg mice (see, e.g., Table
1). It was also determined that Compound 1 lowered A.beta.42 in the
same animals at 5 mpk, while compound 3 lowered A.beta. at 1
mpk.
TABLE-US-00001 TABLE 1 Biological properties of illustrative
hydantoins as compared to BACE IV (.beta.-sectretase inhibitor IV
from Calbiochem (cat #565788). BACE APP binding Brain/Plasma
Compound IC.sub.50 (.mu.M) Kd (uM) Ratio at Cmax MW FAH-1 3 5
(moderate ~3:1 301.3 binding) FAH-2 2 8 (moderate ~1:1 367.3
binding) FAH-3 0.52 0.3 (strong ~1:2 381.3 binding) FAH-4 2.1 ~3:1
367.3 FAH-5 5.0 329.3 FAR-17 0.15 <1 uM (strong 0.5:1 363.3
binding) *BACE 0.05 >50 (essentially <0.1:1.sup. 578
inhibitor IV no binding) *.beta.-sectretase inhibitor IV from
Calbiochem (cat #565788)
[0216] It was also demonstrated that the compounds interacted with
eAPP (see, e.g., FIG. 4) using a BiaCore assay. The hydantoins
contemplated herein thus show desirable pharmacokinetic profiles
and have the desired activity as evidenced by interaction with APP
and/or BACE/APP complexes and lowering of A.beta.42.
[0217] The sequential cleavage of APP by membrane-bound proteases
.beta.-secretase and .gamma.-secretase results in the formation of
A.beta.. The .beta.-Site APP cleavage enzyme-1 (BACE1) was
identified as the major .beta.-secretase activity that mediates the
first cleavage of APP in the .beta.-amyloidogenic pathway. In view
of the ability of the ABBI compounds described herein to
specifically block BACE1 activity at APP, it is believed (and the
data presented herein show) that these ABBI compounds can lower
A.beta. levels or prevent the formation of the neurotoxic A.beta.
species. Accordingly, these compounds are believed to prevent or
slow the progression of the disease and/or to prevent or slow the
progression of pre-clinical manifestations of the amyloidogenic
disease pathway.
[0218] Accordingly it is believed that these agents) (e.g.,
hydantoins described herein, or a tautomer(s) or stereoisomer(s)
thereof, or pharmaceutically acceptable salts or solvates of said
hydantoin(s), said stereoisomer(s), or said tautomer(s), or
analogues, derivatives, or prodrugs thereof) can be used to prevent
or delay the onset of a pre-Alzheimer's cognitive dysfunction,
and/or to ameliorate one or more symptoms of a pre-Alzheimer's
cognitive dysfunction, and/or to prevent or delay the progression
of a pre-Alzheimer's condition or cognitive dysfunction to
Alzheimer's disease, and/or to promote the processing of amyloid
precursor protein (APP) by the non-amyloidogenic pathway. In
certain embodiments these agents can be used in the treatment of
Alzheimer's disease (e.g., to lessen the severity of the disease,
and/or to ameliorate one or more symptoms of the disease, and/or to
slow the progression of the disease).
Therapeutic and Prophylactic Methods.
[0219] In various embodiments therapeutic and/or prophylactic
methods are provided that utilize the active agent(s) (e.g.,
hydantoins described herein, or a tautomer(s) or stereoisomer(s)
thereof, or pharmaceutically acceptable salts or solvates of said
hydantoin(s), said stereoisomer(s), or said tautomer(s), or
analogues, derivatives, or prodrugs thereof) are provided.
Typically the methods involve administering one or more active
agent(s) to a subject (e.g., to a human in need thereof) in an
amount sufficient to realize the desired therapeutic or
prophylactic result.
[0220] Prophylaxis
[0221] In certain embodiments active agent(s) (e.g., hydantoins
described herein, or a tautomer(s) or stereoisomer(s) thereof, or
pharmaceutically acceptable salts or solvates of said hydantoin(s),
said stereoisomer(s), or said tautomer(s), or analogues,
derivatives, or prodrugs thereof) are utilized in various
prophylactic contexts. Thus, for example, in certain embodiments,
the active agent(s) can be used to prevent or delay the onset of a
pre-Alzheimer's cognitive dysfunction, and/or to ameliorate one
more symptoms of a pre-Alzheimer's condition and/or cognitive
dysfunction, and/or to prevent or delay the progression of a
pre-Alzheimer's condition and/or cognitive dysfunction to
Alzheimer's disease.
[0222] Accordingly in certain embodiments, the prophylactic methods
described herein are contemplated for subjects identified as "at
risk" and/or as having evidence of early Alzheimer's Disease (AD)
pathological changes, but who do not meet clinical criteria for MCI
or dementia. Without being bound to a particular theory, it is
believed that even this "preclinical" stage of the disease
represents a continuum from completely asymptomatic individuals
with biomarker evidence suggestive of AD-pathophysiological
process(es) (abbreviated as AD-P, see, e.g., Sperling et al. (2011)
Alzheimer's & Dementia, 1-13) at risk for progression to AD
dementia to biomarker-positive individuals who are already
demonstrating very subtle decline but not yet meeting standardized
criteria for MCI(see, e.g., Albert et al. (2011) Alzheimer's and
Dementia, 1-10 (doi:10.1016/j.jalz.2011.03.008).
[0223] This latter group of individuals might be classified as "not
normal, not MCI" but would be can be designated "pre-symptomatic"
or "pre-clinical or "asymptomatic" or "premanifest"). In various
embodiments this continuum of pre-symptomatic AD can also
encompass, but is not necessarily limited to, (1) individuals who
carry one or more apolipoprotein E (APOE) .epsilon.4 alleles who
are known or believed to have an increased risk of developing AD
dementia, at the point they are AD-P biomarker-positive, and (2)
carriers of autosomal dominant mutations, who are in the
presymptomatic biomarker-positive stage of their illness, and who
will almost certainly manifest clinical symptoms and progress to
dementia.
[0224] A biomarker model has been proposed in which the most widely
validated biomarkers of AD-P become abnormal and likewise reach a
ceiling in an ordered manner (see, e.g., Jack et al. (2010) Lancet
Neurol., 9: 119-128.). This biomarker model parallels proposed
pathophysiological sequence of (pre-AD/AD), and is relevant to
tracking the preclinical (asymptomatic) stages of AD (see, e.g.,
FIG. 3 in Sperling et al. (2011) Alzheimer's & Dementia, 1-13).
Biomarkers of brain amyloidosis include, but are not limited to
reductions in CSF A.beta..sub.42 and increased amyloid tracer
retention on positron emission tomography (PET) imaging. Elevated
CSF tau is not specific to AD and is thought to be a biomarker of
neuronal injury. Decreased fluorodeoxyglucose 18F (FDG) uptake on
PET with a temporoparietal pattern of hypometabolism is a biomarker
of AD-related synaptic dysfunction. Brain atrophy on structural
magnetic resonance imaging (MRI) in a characteristic pattern
involving the medial temporal lobes, paralimbic and temporoparietal
cortices is a biomarker of AD-related neurodegeneration. Other
markers include, but are not limited to volumetric MRI, FDG-PET, or
plasma biomarkers (see, e.g., Vemuri et al. (2009) Neurology, 73:
294-301; Yaffe et al. (2011) JAMA 305: 261-266).
[0225] In certain embodiments the subjects suitable for the
prophylactic methods contemplated herein include, but are not
limited to, subjects characterized as having asymptomatic cerebral
amyloidosis. In various embodiments these individuals have
biomarker evidence of A.beta. accumulation with elevated tracer
retention on PET amyloid imaging and/or low A.beta.42 in CSF assay,
but typically no detectable evidence of additional brain
alterations suggestive of neurodegeneration or subtle cognitive
and/or behavioral symptomatology.
[0226] It is noted that currently available CSF and PET imaging
biomarkers of A.beta. primarily provide evidence of amyloid
accumulation and deposition of fibrillar forms of amyloid. Data
suggest that soluble or oligomeric forms of A.beta. are likely in
equilibrium with plaques, which may serve as reservoirs. In certain
embodiments it is contemplated that there is an identifiable
preplaque stage in which only soluble forms of A.beta. are present.
In certain embodiments it is contemplated that oligomeric forms of
amyloid may be critical in the pathological cascade, and provide
useful markers. In addition, early synaptic changes may be present
before evidence of amyloid accumulation.
[0227] In certain embodiments the subjects suitable for the
prophylactic methods contemplated herein include, but are not
limited to, subjects characterized as amyloid positive with
evidence of synaptic dysfunction and/or early neurodegeneration. In
various embodiments these subjects have evidence of amyloid
positivity and presence of one or more markers of "downstream"
AD-related neuronal injury. Illustrative, but non-limiting markers
of neuronal injury include, but are not limited to (1) elevated CSF
tau or phospho-tau, (2) hypometabolism in an AD-like pattern (i.e.,
posterior cingulate, precuneus, and/or temporoparietal cortices) on
FDG-PET, and (3) cortical thinning/gray matter loss in a specific
anatomic distribution (i.e., lateral and medial parietal, posterior
cingulate, and lateral temporal cortices) and/or hippocampal
atrophy on volumetric MRI. Other markers include, but are not
limited to fMRI measures of default network connectivity. In
certain embodiments early synaptic dysfunction, as assessed by
functional imaging techniques such as FDG-PET and fMRI, can be
detectable before volumetric loss. Without being bound to a
particular theory, it is believed that amyloid-positive individuals
with evidence of early neurodegeneration may be farther down the
trajectory (i.e., in later stages of preclinical (asymptomatic)
AD).
[0228] In certain embodiments the subjects suitable for the
prophylactic methods contemplated herein include, but are not
limited to, subjects characterized as amyloid positive with
evidence of neurodegeneration and subtle cognitive decline. Without
being bound to a particular theory, it is believed that those
individuals with biomarker evidence of amyloid accumulation, early
neurodegeneration, and evidence of subtle cognitive decline are in
the last stage of preclinical (asymptomatic) AD, and are
approaching the border zone with clinical criteria for mild
cognitive impairment (MCI). These individuals may demonstrate
evidence of decline from their own baseline (particularly if
proxies of cognitive reserve are taken into consideration), even if
they still perform within the "normal" range on standard cognitive
measures. Without being bound to a particular theory, it is
believed that more sensitive cognitive measures, particularly with
challenging episodic memory measures, may detect very subtle
cognitive impairment in amyloid-positive individuals. In certain
embodiments criteria include, but are not limited to,
self-complaint of memory decline or other subtle neurobehavioral
changes.
[0229] As indicated above, subjects/patients amenable to
prophylactic methods described herein include individuals at risk
of disease (e.g., a pathology characterized by amyloid plaque
formation such as MCI) but not showing symptoms, as well as
subjects presently showing certain symptoms or markers. It is known
that the risk of MCI and later Alzheimer's disease generally
increases with age. Accordingly, in asymptomatic subjects with no
other known risk factors, in certain embodiments, prophylactic
application is contemplated for subjects over 50 years of age, or
subjects over 55 years of age, or subjects over 60 years of age, or
subjects over 65 years of age, or subjects over 70 years of age, or
subjects over 75 years of age, or subjects over 80 years of age, in
particular to prevent or slow the onset or ultimate severity of
mild cognitive impairment (MCI), and/or to slow or prevent the
progression from MCI to early stage Alzheimer's disease (AD).
[0230] In certain embodiments, the methods described herein are
especially useful for individuals who do have a known genetic risk
of Alzheimer's disease (or other amyloidogenic pathologies),
whether they are asymptomatic or showing symptoms of disease. Such
individuals include those having relatives who have experienced MCI
or AD (e.g., a parent, a grandparent, a sibling), and those whose
risk is determined by analysis of genetic or biochemical markers.
Genetic markers of risk toward Alzheimer's disease include, for
example, mutations in the APP gene, particularly mutations at
position 717 and positions 670 and 671 referred to as the Hardy and
Swedish mutations respectively (see Hardy (1997) Trends. Neurosci.,
20: 154-159). Other markers of risk include mutations in the
presenilin genes (PS1 and PS2), family history of AD, having the
familial Alzheimer's disease (FAD) mutation, the APOE .epsilon.4
allele, hypercholesterolemia or atherosclerosis. Further
susceptibility genes for the development of Alzheimer's disease are
reviewed, e.g., in Sleegers, et al. (2010) Trends Genet. 26(2):
84-93.
[0231] In some embodiments, the subject is asymptomatic but has
familial and/or genetic risk factors for developing MCI or
Alzheimer's disease. In asymptomatic patients, treatment can begin
at any age (e.g., at about 20, about 30, about 40, about 50 years
of age). Usually, however, it is not necessary to begin treatment
until a patient reaches at least about 40, or at least about 50, or
at least about 55, or at least about 60, or at least about 65, or
at least about 70 years of age.
[0232] In some embodiments, the subject exhibits symptoms, for
example, of mild cognitive impairment (MCI) or Alzheimer's disease
(AD). Individuals presently suffering from Alzheimer's disease can
be recognized from characteristic dementia, as well as the presence
of risk factors described above. In addition, a number of
diagnostic tests are available for identifying individuals who have
AD. These include measurement of CSF Tau, phospho-tau (pTau),
A.beta.42 levels and C-terminally cleaved APP fragment (APPneo).
Elevated total-Tau (tTau), phospho-Tau (pTau), APPneo, soluble
A.beta.40, pTau/A.beta.42 ratio and tTau/A.beta.42 ratio, and
decreased A.beta.42 levels, A.beta.42/A.beta.40 ratio,
A.beta.42/A.beta.38 ratio, sAPP.alpha. levels,
sAPP.alpha./sAPP.beta. ratio, sAPP.alpha./A.beta.40 ratio, and
sAPP.alpha./A.beta.42 ratio signify the presence of AD. In some
embodiments, the subject or patient is diagnosed as having MCI.
Increased levels of neural thread protein (NTP) in urine and/or
increased levels of .alpha.2-macroglobulin (.alpha.2M) and/or
complement factor H(CFH) in plasma are also biomarkers of MCI
and/or AD (see, e.g., Anoop et al. (2010) Int. J. Alzheimer's Dis.
2010:606802).
[0233] In certain embodiments, subjects amenable to treatment may
have age-associated memory impairment (AAMI), or mild cognitive
impairment (MCI). The methods described herein are particularly
well-suited to the prophylaxis and/or treatment of MCI. In such
instances, the methods can delay or prevent the onset of MCI, and
or reduce one or more symptoms characteristic of MCI and/or delay
or prevent the progression from MCI to early-, mid- or late-stage
Alzheimer's disease or reduce the ultimate severity of the
disease.
[0234] Mild Cognitive Impairment (MCI)
[0235] Mild cognitive impairment (MCI, also known as incipient
dementia, or isolated memory impairment) is a diagnosis given to
individuals who have cognitive impairments beyond that expected for
their age and education, but that typically do not interfere
significantly with their daily activities (see, e.g., Petersen et
al. (1999) Arch. Neurol. 56(3): 303-308). It is considered in many
instances to be a boundary or transitional stage between normal
aging and dementia. Although MCI can present with a variety of
symptoms, when memory loss is the predominant symptom it is termed
"amnestic MCI" and is frequently seen as a risk factor for
Alzheimer's disease (see, e.g., Grundman et al. (2004) Arch.
Neurol. 61(1): 59-66; and on the internet at
en.wikipedia.org/wiki/Mild_cognitive_impairment-cite_note-Grundman-1).
When individuals have impairments in domains other than memory it
is often classified as non-amnestic single- or multiple-domain MCI
and these individuals are believed to be more likely to convert to
other dementias (e.g., dementia with Lewy bodies). There is
evidence suggesting that while amnestic MCI patients may not meet
neuropathologic criteria for Alzheimer's disease, patients may be
in a transitional stage of evolving Alzheimer's disease; patients
in this hypothesized transitional stage demonstrated diffuse
amyloid in the neocortex and frequent neurofibrillary tangles in
the medial temporal lobe (see, e.g., Petersen et al. (2006) Arch.
Neurol. 63(5): 665-72).
[0236] The diagnosis of MCI typically involves a comprehensive
clinical assessment including clinical observation, neuroimaging,
blood tests and neuropsychological testing. In certain embodiments
diagnostic criteria for MIC include, but are not limited to those
described by Albert et al. (2011) Alzheimer's & Dementia. 1-10.
As described therein, diagnostic criteria include (1) core clinical
criteria that could be used by healthcare providers without access
to advanced imaging techniques or cerebrospinal fluid analysis, and
(2) research criteria that could be used in clinical research
settings, including clinical trials. The second set of criteria
incorporate the use of biomarkers based on imaging and
cerebrospinal fluid measures. The final set of criteria for mild
cognitive impairment due to AD has four levels of certainty,
depending on the presence and nature of the biomarker findings.
[0237] In certain embodiments clinical evaluation/diagnosis of MCI
involves: (1) Concern reflecting a change in cognition reported by
patient or informant or clinician (i.e., historical or observed
evidence of decline over time); (2) Objective evidence of
Impairment in one or more cognitive domains, typically including
memory (i.e., formal or bedside testing to establish level of
cognitive function in multiple domains); (3) Preservation of
independence in functional abilities; (4) Not demented; and in
certain embodiments, (5) An etiology of MCI consistent with AD
pathophysiological processes. Typically vascular, traumatic, and
medical causes of cognitive decline, are ruled out where possible.
In certain embodiments, when feasible, evidence of longitudinal
decline in cognition is identified. Diagnosis is reinforced by a
history consistent with AD genetic factors, where relevant.
[0238] With respect to impairment in cognitive domain(s), there
should be evidence of concern about a change in cognition, in
comparison with the person's previous level. There should be
evidence of lower performance in one or more cognitive domains that
is greater than would be expected for the patient's age and
educational background. If repeated assessments are available, then
a decline in performance should be evident over time. This change
can occur in a variety of cognitive domains, including memory,
executive function, attention, language, and visuospatial skills.
An impairment in episodic memory (i.e., the ability to learn and
retain new information) is seen most commonly in MCI patients who
subsequently progress to a diagnosis of AD dementia.
[0239] With respect to preservation of independence in functional
abilities, it is noted that persons with MCI commonly have mild
problems performing complex functional tasks which they used to
perform shopping. They may take more time, be less efficient, and
make more errors at performing such activities than in the past.
Nevertheless, they generally maintain their independence of
function in daily life, with minimal aids or assistance.
[0240] With respect to dementia, the cognitive changes should be
sufficiently mild that there is no evidence of a significant
impairment in social or occupational functioning.
[0241] If an individual has only been evaluated once, change will
be inferred from the history and/or evidence that cognitive
performance is impaired beyond what would have been expected for
that individual.
[0242] Cognitive testing is optimal for objectively assessing the
degree of cognitive impairment for an individual. Scores on
cognitive tests for individuals with MCI are typically 1 to 1.5
standard deviations below the mean for their age and education
matched peers on culturally appropriate normative data (i.e., for
the impaired domain(s), when available).
[0243] Episodic memory (i.e., the ability to learn and retain new
information) is most commonly seen in MCI patients who subsequently
progress to a diagnosis of AD dementia. There are a variety of
episodic memory tests that are useful for identifying those MCI
patients who have a high likelihood of progressing to AD dementia
within a few years. These tests typically assess both immediate and
delayed recall, so that it is possible to determine retention over
a delay. Many, although not all, of the tests that have proven
useful in this regard are wordlist learning tests with multiple
trials. Such tests reveal the rate of learning over time, as well
as the maximum amount acquired over the course of the learning
trials. They are also useful for demonstrating that the individual
is, in fact, paying attention to the task on immediate recall,
which then can be used as a baseline to assess the relative amount
of material retained on delayed recall. Examples of such tests
include (but are not limited to: the Free and Cued Selective
Reminding Test, the Rey Auditory Verbal Learning Test, and the
California Verbal Learning Test. Other episodic memory measures
include, but are not limited to: immediate and delayed recall of a
paragraph such as the Logical Memory I and II of the Wechsler
Memory Scale Revised (or other versions) and immediate and delayed
recall of nonverbal materials, such as the Visual Reproduction
subtests of the Wechsler Memory Scale-Revised I and II.
[0244] Because other cognitive domains can be impaired among
individuals with MCI, it is desirable to examine domains in
addition to memory. These include, but are not limited to executive
functions (e.g., set-shifting, reasoning, problem-solving,
planning), language (e.g., naming, fluency, expressive speech, and
comprehension), visuospatial skills, and attentional control (e.g.,
simple and divided attention). Many clinical neuropsychological
measures are available to assess these cognitive domains, including
(but not limited to the Trail Making Test (executive function), the
Boston Naming Test, letter and category fluency (language), figure
copying (spatial skills), and digit span forward (attention).
[0245] As indicated above, genetic factors can be incorporated into
the diagnosis of MCI. If an autosomal dominant form of AD is known
to be present (i.e., mutation in APP, PS1, PS2), then the
development of MCI is most likely the precursor to AD dementia. The
large majority of these cases develop early onset AD (i.e., onset
below 65 years of age).
[0246] In addition, there are genetic influences on the development
of late onset AD dementia. For example, the presence of one or two
.epsilon.4 alleles in the apolipoprotein E (APOE) gene is a genetic
variant broadly accepted as increasing risk for late-onset AD
dementia. Evidence suggests that an individual who meets the
clinical, cognitive, and etiologic criteria for MCI, and is also
APOE .epsilon.4 positive, is more likely to progress to AD dementia
within a few years than an individual without this genetic
characteristic. It is believed that additional genes play an
important, but smaller role than APOE and also confer changes in
risk for progression to AD dementia (see, e.g., Bertram et al.
(2010) Neuron, 21: 270-281).
[0247] In certain embodiments subjects suitable for the
prophylactic methods described herein include, but need not be
limited to, subjects identified having one or more of the core
clinical criteria described above and/or subjects identified with
one or more "research criteria" for MCI, e.g., as described
below.
[0248] "Research criteria" for the identification/prognosis of MCI
include, but are not limited to biomarkers that increase the
likelihood that MCI syndrome is due to the pathophysiological
processes of AD. Without being bound to a particular theory, it is
believed that the conjoint application of clinical criteria and
biomarkers can result in various levels of certainty that the MCI
syndrome is due to AD pathophysiological processes. In certain
embodiments, two categories of biomarkers have been the most
studied and applied to clinical outcomes are contemplated. These
include "A13" (which includes CSF A.beta..sub.42 and/or PET amyloid
imaging) and "biomarkers of neuronal injury" (which include, but
are not limited to CSF tau/p-tau, hippocampal, or medial temporal
lobe atrophy on MRI, and temporoparietal/precuneus hypometabolism
or hypoperfusion on PET or SPECT).
[0249] Without being bound to a particular theory, it is believed
that evidence of both A.beta., and neuronal injury (either an
increase in tau/p-tau or imaging biomarkers in a topographical
pattern characteristic of AD), together confers the highest
probability that the AD pathophysiological process is present.
Conversely, if these biomarkers are negative, this may provide
information concerning the likelihood of an alternate diagnosis. It
is recognized that biomarker findings may be contradictory and
accordingly any biomarker combination is indicative (an indicator)
used on the context of a differential diagnosis and not itself
dispositive. It is recognized that varying severities of an
abnormality may confer different likelihoods or prognoses, that are
difficult to quantify accurately for broad application.
[0250] For those potential MCI subjects whose clinical and
cognitive MCI syndrome is consistent with AD as the etiology, the
addition of biomarker analysis effects levels of certainty in the
diagnosis. In the most typical example in which the clinical and
cognitive syndrome of MCI has been established, including evidence
of an episodic memory disorder and a presumed degenerative
etiology, the most likely cause is the neurodegenerative process of
AD. However, the eventual outcome still has variable degrees of
certainty. The likelihood of progression to AD dementia will vary
with the severity of the cognitive decline and the nature of the
evidence suggesting that AD pathophysiology is the underlying
cause. Without being bound to a particular theory it is believed
that positive biomarkers reflecting neuronal injury increase the
likelihood that progression to dementia will occur within a few
years and that positive findings reflecting both A.beta.
accumulation and neuronal injury together confer the highest
likelihood that the diagnosis is MCI due to AD.
[0251] A positive A.beta. biomarker and a positive biomarker of
neuronal injury provide an indication that the MCI syndrome is due
to AD processes and the subject is well suited for the methods
described herein.
[0252] A positive A.beta. biomarker in a situation in which
neuronal injury biomarkers have not been or cannot be tested or a
positive biomarker of neuronal injury in a situation in which
A.beta. biomarkers have not been or cannot be tested indicate an
intermediate likelihood that the MCI syndrome is due to AD. Such
subjects are believed to be is well suited for the methods
described herein
[0253] Negative biomarkers for both A.beta. and neuronal injury
suggest that the MCI syndrome is not due to AD. In such instances
the subjects may not be well suited for the methods described
herein.
[0254] There is evidence that magnetic resonance imaging can
observe deterioration, including progressive loss of gray matter in
the brain, from mild cognitive impairment to full-blown Alzheimer
disease (see, e.g., Whitwell et al. (2008) Neurology 70(7):
512-520). A technique known as PiB PET imaging is used to clearly
show the sites and shapes of beta amyloid deposits in living
subjects using a C11 tracer that binds selectively to such deposits
(see, e.g., Jack et al. (2008) Brain 131(Pt 3): 665-680).
[0255] In certain embodiments, MCI is typically diagnosed when
there is 1) Evidence of memory impairment; 2) Preservation of
general cognitive and functional abilities; and 3) Absence of
diagnosed dementia.
[0256] In certain embodiments MCI and stages of Alzheimer's disease
can be identified/categorized, in part by Clinical Dementia Rating
(CDR) scores. The CDR is a five point scale used to characterize
six domains of cognitive and functional performance applicable to
Alzheimer disease and related dementias: Memory, Orientation,
Judgment & Problem Solving, Community Affairs, Home &
Hobbies, and Personal Care. The information to make each rating can
be obtained through a semi-structured interview of the patient and
a reliable informant or collateral source (e.g., family
member).
[0257] The CDR table provides descriptive anchors that guide the
clinician in making appropriate ratings based on interview data and
clinical judgment. In addition to ratings for each domain, an
overall CDR score may be calculated through the use of an
algorithm. This score is useful for characterizing and tracking a
patient's level of impairment/dementia: 0=Normal; 0.5=Very Mild
Dementia; 1=Mild Dementia; 2=Moderate Dementia; and 3=Severe
Dementia. An illustrative CDR table is shown in Table 2.
TABLE-US-00002 TABLE 2 Illustrative clinical dementia rating (CDR)
table. Impairment: None Questionable Mild Moderate Severe CDR: 0
0.5 1 2 3 Memory No memory Consistent Moderate Severe Severe loss
or slight memory memory memory slight forgetfulness; loss; more
loss; only loss; only inconsistent partial marked for highly
fragments forgetfulness recollection recent learned remain of
events' events; material "benign" defect retained; forgetfulness
interferes new with material everyday rapidly lost activities
Orientation Fully Fully Moderate Severe Oriented to oriented
oriented difficulty difficulty person only except for with time
with time slight relationships; relationships; difficulty oriented
for usually with time place at disoriented relationships
examination; to time, may have often to geographic place.
disorientation elsewhere Judgment & Solves Slight Moderate
Severely Unable to Problem everyday impairment difficulty in
impaired in make Solving problems & in solving handling
handling judgments handles problems, problems, problems, or solve
business & similarities, similarities similarities problems
financial and and and affairs well; differences differences;
differences; judgment social social good in judgment judgment
relation to usually usually past maintained impaired performance
Community Independent Slight Unable to No pretense of Affairs
function at impairment function independent function usual level in
these independently outside of home in job, activities at these
Appears Appears shopping, activities well enough too ill volunteer,
although to be taken to be taken and social may still be to
functions to functions groups engaged in outside a outside a some;
family family appears home home. normal to casual inspection Home
and Life at Life at Mild bit Only simple No Hobbies home, home,
definite chores significant hobbies, and hobbies, and impairment
preserved; function in intellectual intellectual of function very
home interests interests at home; restricted well slightly more
interests, maintained impaired difficult poorly chores maintained
abandoned; more complicated hobbies and interests abandoned
Personal Fully capable of self-care Needs Requires Requires Care
prompting assistance much help in dressing, with hygiene, personal
keeping of care; personal frequent effects incontinence
[0258] A CDR rating of .about.0.5 or .about.0.5 to 1.0 is often
considered clinically relevant MCI. Higher CDR ratings can be
indicative of progression into Alzheimer's disease.
[0259] In certain embodiments administration of one or more agents
described herein (e.g., hydantoins described herein, or a
tautomer(s) or stereoisomer(s) thereof, or pharmaceutically
acceptable salts or solvates of said hydantoin(s), said
stereoisomer(s), or said tautomer(s), or analogues, derivatives, or
prodrugs thereof) is deemed effective when there is a reduction in
the CSF of levels of one or more components selected from the group
consisting of Tau, phospho-Tau (pTau), APPneo, soluble A.beta.40,
soluble A.beta.42, and/or A.beta.42/A.beta.40 ratio, and/or when
there is a reduction of the plaque load in the brain of the
subject, and/or when there is a reduction in the rate of plaque
formation in the brain of the subject, and/or when there is an
improvement in the cognitive abilities of the subject, and/or when
there is a perceived improvement in quality of life by the subject,
and/or when there is a significant reduction in clinical dementia
rating (CDR), and/or when the rate of increase in clinical dementia
rating is slowed or stopped and/or when the progression from MCI to
early stage AD is slowed or stopped.
[0260] In some embodiments, a diagnosis of MCI can be determined by
considering the results of several clinical tests. For example,
Grundman, et al. (2004) Arch Neurol 61: 59-66, report that a
diagnosis of MCI can be established with clinical efficiency using
a simple memory test (paragraph recall) to establish an objective
memory deficit, a measure of general cognition (Mini-Mental State
Exam (MMSE), discussed in greater detail below) to exclude a
broader cognitive decline beyond memory, and a structured clinical
interview (CDR) with patients and caregivers to verify the
patient's memory complaint and memory loss and to ensure that the
patient was not demented. Patients with MCI perform, on average,
less than 1 standard deviation (SD) below normal on
nonmemorycognitive measures included in the battery. Tests of
learning, attention, perceptual speed, category fluency, and
executive function may be impaired in patients with MCI, but these
are far less prominent than the memory deficit.
[0261] Alzheimer's Disease (AD).
[0262] In certain embodiments the active agent(s(e.g., hydantoins
described herein, or a tautomer(s) or stereoisomer(s) thereof, or
pharmaceutically acceptable salts or solvates of said hydantoin(s),
said stereoisomer(s), or said tautomer(s), or analogues,
derivatives, or prodrugs thereof) are contemplated for the
treatment of Alzheimer's disease. In such instances the methods
described herein are useful in preventing or slowing the onset of
Alzheimer's disease (AD), in reducing the severity of AD when the
subject has transitioned to clinical AD diagnosis, and/or in
mitigating one or more symptoms of Alzheimer's disease.
[0263] In particular, where the Alzheimer's disease is early stage,
the methods can reduce or eliminate one or more symptoms
characteristic of AD and/or delay or prevent the progression from
MCI to early or later stage Alzheimer's disease.
[0264] Individuals presently suffering from Alzheimer's disease can
be recognized from characteristic dementia, as well as the presence
of risk factors described above. In addition, a number of
diagnostic tests are available for identifying individuals who have
AD. Individuals presently suffering from Alzheimer's disease can be
recognized from characteristic dementia, as well as the presence of
risk factors described above. In addition, a number of diagnostic
tests are available for identifying individuals who have AD. These
include measurement of CSF Tau, phospho-tau (pTau), sAPP.alpha.,
sAPP.beta., A.beta.40, A.beta.42 levels and/or C terminally cleaved
APP fragment (APPneo). Elevated Tau, pTau, sAPP.beta. and/or
APPneo, and/or decreased sAPP.alpha., soluble A.beta.40 and/or
soluble A.beta.42 levels, particularly in the context of a
differential diagnosis, can signify the presence of AD.
[0265] In certain embodiments subjects amenable to treatment may
have Alzheimer's disease. Individuals suffering from Alzheimer's
disease can also be diagnosed by Alzheimer's disease and Related
Disorders Association (ADRDA) criteria. The NINCDS-ADRDA
Alzheimer's Criteria were proposed in 1984 by the National
Institute of Neurological and Communicative Disorders and Stroke
and the Alzheimer's Disease and Related Disorders Association (now
known as the Alzheimer's Association) and are among the most used
in the diagnosis of Alzheimer's disease (AD). McKhann, et al.
(1984) Neurology 34(7): 939-44. According to these criteria, the
presence of cognitive impairment and a suspected dementia syndrome
should be confirmed by neuropsychological testing for a clinical
diagnosis of possible or probable AD. However, histopathologic
confirmation (microscopic examination of brain tissue) is generally
used for a dispositive diagnosis. The NINCDS-ADRDA Alzheimer's
Criteria specify eight cognitive domains that may be impaired in
AD: memory, language, perceptual skills, attention, constructive
abilities, orientation, problem solving and functional abilities).
These criteria have shown good reliability and validity.
[0266] Baseline evaluations of patient function can made using
classic psychometric measures, such as the Mini-Mental State Exam
(MMSE) (Folstein et al. (1975) J. Psychiatric Research 12 (3):
189-198), and the Alzheimer's Disease Assessment Scale (ADAS),
which is a comprehensive scale for evaluating patients with
Alzheimer's Disease status and function (see, e.g., Rosen, et al.
(1984) Am. J. Psychiatr., 141: 1356-1364). These psychometric
scales provide a measure of progression of the Alzheimer's
condition. Suitable qualitative life scales can also be used to
monitor treatment. The extent of disease progression can be
determined using a Mini-Mental State Exam (MMSE) (see, e.g.,
Folstein, et al. supra). Any score greater than or equal to 25
points (out of 30) is effectively normal (intact). Below this,
scores can indicate severe (<9 points), moderate (10-20 points)
or mild (21-24 points) Alzheimer's disease.
[0267] Alzheimer's disease can be broken down into various stages
including: 1) Moderate cognitive decline (Mild or early-stage
Alzheimer's disease), 2) Moderately severe cognitive decline
(Moderate or mid-stage Alzheimer's disease), 3) Severe cognitive
decline (Moderately severe or mid-stage Alzheimer's disease), and
4) Very severe cognitive decline (Severe or late-stage Alzheimer's
disease) as shown in Table 3.
TABLE-US-00003 TABLE 3 Illustrative stages of Alzheimer's disease.
Moderate Cognitive Decline (Mild or early stage AD) At this stage,
a careful medical interview detects clear-cut deficiencies in the
following areas: Decreased knowledge of recent events. Impaired
ability to perform challenging mental arithmetic. For example, to
count backward from 100 by 7s. Decreased capacity to perform
complex tasks, such as marketing, planning dinner for guests, or
paying bills and managing finances. Reduced memory of personal
history. The affected individual may seem subdued and withdrawn,
especially in socially or mentally challenging situations.
Moderately severe cognitive decline (Moderate or mid-stage
Alzheimer's disease) Major gaps in memory and deficits in cognitive
function emerge. Some assistance with day-to-day activities becomes
essential. At this stage, individuals may: Be unable during a
medical interview to recall such important details as their current
address, their telephone number, or the name of the college or high
school from which they graduated. Become confused about where they
are or about the date, day of the week or season. Have trouble with
less challenging mental arithmetic; for example, counting backward
from 40 by 4s or from 20 by 2s. Need help choosing proper clothing
for the season or the occasion. Usually retain substantial
knowledge about themselves and know their own name and the names of
their spouse or children. Usually require no assistance with eating
or using the toilet. Severe cognitive decline (Moderately severe or
mid-stage Alzheimer's disease) Memory difficulties continue to
worsen, significant personality changes may emerge, and affected
individuals need extensive help with daily activities. At this
stage, individuals may: Lose most awareness of recent experiences
and events as well as of their surroundings. Recollect their
personal history imperfectly, although they generally recall their
own name. Occasionally forget the name of their spouse or primary
caregiver but generally can distinguish familiar from unfamiliar
faces. Need help getting dressed properly; without supervision, may
make such errors as putting pajamas over daytime clothes or shoes
on wrong feet. Experience disruption of their normal sleep/waking
cycle. Need help with handling details of toileting (flushing
toilet, wiping and disposing of tissue properly). Have increasing
episodes of urinary or fecal incontinence. Experience significant
personality changes and behavioral symptoms, including
suspiciousness and delusions (for example, believing that their
caregiver is an impostor); hallucinations (seeing or hearing things
that are not really there); or compulsive, repetitive behaviors
such as hand-wringing or tissue shredding. Tend to wander and
become lost. Very severe cognitive decline (Severe or late-stage
Alzheimer's disease) This is the final stage of the disease when
individuals lose the ability to respond to their environment, the
ability to speak, and, ultimately, the ability to control movement.
Frequently individuals lose their capacity for recognizable speech,
although words or phrases may occasionally be uttered. Individuals
need help with eating and toileting and there is general
incontinence. Individuals lose the ability to walk without
assistance, then the ability to sit without support, the ability to
smile, and the ability to hold their head up. Reflexes become
abnormal and muscles grow rigid. Swallowing is impaired.
[0268] In various embodiments administration of one or more agents
described herein to subjects diagnosed with Alzheimer's disease is
deemed effective when the there is a reduction in the CSF of levels
of one or more components selected from the group consisting of
Tau, phospho-Tau (pTau), APPneo, soluble A.beta.40, soluble
A.beta.42, and/or and A.beta.42/A.beta.40 ratio, and/or when there
is a reduction of the plaque load in the brain of the subject,
and/or when there is a reduction in the rate of plaque formation in
the brain of the subject, and/or when there is an improvement in
the cognitive abilities of the subject, and/or when there is a
perceived improvement in quality of life by the subject, and/or
when there is a significant reduction in clinical dementia rating
(CDR) of the subject, and/or when the rate of increase in clinical
dementia rating is slowed or stopped and/or when the progression of
AD is slowed or stopped (e.g., when the transition from one stage
to another as listed in Table 3 is slowed or stopped).
[0269] In certain embodiments subjects amenable to the present
methods generally are free of a neurological disease or disorder
other than Alzheimer's disease. For example, in certain
embodiments, the subject does not have and is not at risk of
developing a neurological disease or disorder such as Parkinson's
disease, and/or schizophrenia, and/or psychosis.
Active Agent(s).
[0270] The methods described herein are based, in part, on the
discovery that administration of one or more active agents (e.g.,
hydantoins described herein, or a tautomer(s) or stereoisomer(s)
thereof, or pharmaceutically acceptable salts or solvates of said
hydantoin(s), said stereoisomer(s), or said tautomer(s), or
analogues, derivatives, or prodrugs thereof) find use in the
treatment and/or prophylaxis of diseases characterized by amyloid
deposits in the brain, for example, mild cognitive impairment,
Alzheimer's disease, macular degeneration, and the like.
[0271] In certain embodiments the active agent is a compound (e.g.,
a hydantoin) according to Formula I:
##STR00032##
where
M is
##STR00033##
[0272] and R.sup.7 is selected from the group consisting of
C.dbd.O, C.dbd.S, C--NH2, and C.dbd.NH, and the bond represented by
the wavy line is a single bond when R.sup.7 is C.dbd.O, C.dbd.S, or
C.dbd.NH, and a double bond when R.sup.7 is C--NH.sub.2; R.sup.8
and R.sup.9 are independently selected from the group consisting of
H, alkyl, cycloalkyl, and aryl, provided that when the bond
represented by the wavy line is a double bond, then R.sup.9 is
absent; R.sup.0 is selected from the group consisting of aryl,
substituted aryl, disubstituted aryl, heteroaryl, substituted
heteroaryl, disubstituted heteroaryl, alkyl, haloalkyl, cycloalkyl,
alkenyl, and alkynyl; X.sup.1 is selected from the group consisting
of C-halogen, CH, and N; A is methyl or H; R.sup.5 and R.sup.6 are
independently selected from halogen, H, alkyl, trichloromethyl, and
trifluoromethyl; R.sup.3 and R.sup.4 are independently absent or
selected from the group consisting of alkyl, cycloalkyl, alkoxy,
thioalky; and when X.sup.1 is C, then R.sup.0 is not phenyl
monosubstituted at the para position with --OCHF.sub.2. Also
contemplated are pharmaceutically acceptable salts thereof,
tautomer thereofs, pharmaceutically acceptable salts of a tautomer
thereof, an enantiomer thereof, a pharmaceutically acceptable salt
of an enantiomer thereof, and the like.
[0273] In certain embodiments, the compound is a compound according
to Formula IV:
##STR00034##
or a compound according to Formula V:
##STR00035##
[0274] In certain embodiments, of any of the foregoing compounds,
X.sup.1 is selected from the group consisting of C-halogen, CH, and
N or from the group consisting of CH, and N; and R.sup.5 and
R.sup.6 are independently selected halogen. In certain embodiments,
of any of the foregoing compounds, R.sup.7 is C.dbd.NH or R.sup.7
is C.dbd.O.
[0275] In certain embodiments the compound is a compound according
to Formula VI:
##STR00036##
In certain embodiments of the compound of Formula VI R.sup.5 and
R.sup.6 are independently selected halogens. In certain embodiments
of the compound of Formula VI R.sup.5 and R.sup.6 are the same
halogen (e.g., both F, both Cl, etc.).
[0276] In certain embodiments the compound is a compound according
to Formula V, where said compound is a compound of Formula VII:
##STR00037##
[0277] In certain embodiments the compound is a compound according
to Formula VIII:
##STR00038##
[0278] In certain embodiments the compound is a compound according
to Formula IX:
##STR00039##
[0279] In certain embodiments the compound is a compound of Formula
V where R.sup.7 is C.dbd.S.
[0280] In certain embodiments the compound is a compound where
R.sup.7 is C--NH.sub.2 and the compound is a compound of Formula
X:
##STR00040##
and in certain embodiments of Formula VIII, the compound is a
compound according to Formula XI:
##STR00041##
where R.sup.1 and R.sup.2 are independently absent or selected from
the group consisting of alkyl, haloalkyl, cycloalkyl, alkenyl,
alkynyl, alkoxy, thioalkyl, aryl, substituted aryl, heteroaryl, and
substituted heteroaryl; and X.sup.2, Y, and Z are independently CH
or N. In certain embodiments of any of the foregoing Formulas
R.sup.5 and R.sup.6 are different halogens (e.g., R.sup.5.dbd.Cl
and R.sup.6.dbd.F, R.sup.5.dbd.F and R.sup.6.dbd.Cl, and the like).
In certain embodiments of any of the foregoing Formulas R.sup.5 and
R.sup.6 are the same halogen (e.g., both Cl, both F, etc).
[0281] In certain embodiments the compound is a compound of Formula
XII:
##STR00042##
[0282] In certain embodiments of any of the foregoing compounds,
X.sup.1 is CH. In certain embodiments of any of the foregoing
compounds R.sup.8 is H or CH.sub.3.
[0283] In certain embodiments the compound is a compound according
to Formula XIII:
##STR00043##
[0284] In certain embodiments the compound is a compound according
to Formula XIV:
##STR00044##
[0285] In certain embodiments the compound is a compound according
to Formula XV:
##STR00045##
[0286] In certain embodiments the compound is a compound according
to Formula XVI:
##STR00046##
[0287] In certain embodiments the compound is a compound according
to Formula XVII:
##STR00047##
[0288] In certain embodiments the compound is a compound according
to Formula XVIII:
##STR00048##
[0289] In certain embodiments the compound is a compound according
to Formula XIX:
##STR00049##
[0290] In certain embodiments the compound is a compound according
to Formula XX:
##STR00050##
[0291] In certain embodiments the compound is a compound according
to Formula XXI:
##STR00051##
[0292] In certain embodiments the compound is a compound according
to Formula XXII:
##STR00052##
[0293] In certain embodiments the compound is a compound according
to Formula XXIII:
##STR00053##
[0294] In certain embodiments the compound is a compound according
to Formula XXIV:
##STR00054##
[0295] In certain embodiments the compound is a compound according
to Formula XXV:
##STR00055##
[0296] In certain embodiments the compound is a compound according
to Formula XXVI:
##STR00056##
[0297] In certain embodiments the compound is a compound according
to Formula XXVII:
##STR00057##
[0298] In certain embodiments the compound is a compound according
to Formula XVIII:
##STR00058##
[0299] In certain embodiments the compound is a compound according
to Formula XXIX:
##STR00059##
[0300] In certain embodiments the compound is a compound according
to Formula XXX:
##STR00060##
[0301] In certain embodiments the compound is a compound according
to Formula XXXI:
##STR00061##
[0302] In certain embodiments any of the foregoing Formulas
expressly exclude FAH-2. In certain embodiments any of the
foregoing Formulas expressly exclude FAH-3. In certain embodiments
any of the foregoing Formulas expressly exclude FAH-2 and
FAH-3.
[0303] In certain embodiments the compound is a compound according
to Formula XXXII:
##STR00062##
or a pharmaceutically acceptable salt thereof, a tautomer thereof,
a pharmaceutically acceptable salt of a tautomer thereof, an
enantiomer thereof, or a pharmaceutically acceptable salt of an
enantiomer thereof.
[0304] In certain embodiments any of the foregoing compounds is a
substantially pure S enantiomer. In certain embodiments any of the
foregoing compounds is a substantially pure R enantiomer.
[0305] Various compounds contemplated herein include the compounds
shonw in Table 4.
TABLE-US-00004 TABLE 4 Illustrative, but non-limiting examples of
compounds contemplated herein. FAH # Structure MW FAH-3
##STR00063## 381.3 FAH-1 ##STR00064## 301.3 FAH-2 ##STR00065##
367.3 FAH-4 ##STR00066## 367.3 FAH-5 ##STR00067## 329.3 FAH-6
##STR00068## 345.3 FAH-8 ##STR00069## 345.3 FAH-9 ##STR00070##
359.3 FAH-10 ##STR00071## 333.3 FAH-11 ##STR00072## 332.31 FAH-12
##STR00073## 349.3 FAH-13 ##STR00074## 371.3 FAH-14 ##STR00075##
337.3 FAH-15 ##STR00076## 320.27 FAH-17 ##STR00077## 363.33 FAH-17
HCl ##STR00078## 399.8 FAH-19 ##STR00079## 399.32 FAH-22
##STR00080## 377.36 FAH-23 ##STR00081## 379.79 FAH-25 ##STR00082##
397.78 FAH-27 ##STR00083## 359.37 FAH-28 ##STR00084## 413.34
With respect to these compounds pharmaceutically acceptable salts,
tautomers, pharmaceutically acceptable salts of a tautomer,
enantiomers thereof, and pharmaceutically acceptable salts of an
enantiomer thereof are also contemplated. Additionally
substantially pure S enantiomers or substantially pure R
enantiomers of these compounds are contemplated.
[0306] Various illustrative, but non-limiting hydantoins are also
shown in FIGS. 1 and 2. In certain embodiments pharmaceutically
acceptable salts, tautomers, pharmaceutically acceptable salts of a
tautomer, enantiomers thereof, and pharmaceutically acceptable salt
of an enantiomer are contemplated.
[0307] With respect to certain molecules in FIG. 1, without being
bound to a particular theory, it is believed that the B-ring with
the methyl and the OCHF.sub.2 shows increased potency with a 3,4
substitution. It is believed that this type of a substitution
pattern interacts with the Trp-76 of the BACE flap disrupting the
interaction of the Tyr-71 of the flap with the Trp-76 and flipping
the Tyr-71 to the left allowing it to interact with the difluoro
groups of the A-ring (see also FIG. 3).
[0308] In certain embodiments the compound is a substantially pure
"S" enantiomer. In certain embodiments the compound is a
substantially pure "R" enantiomer. In certain embodiments the
compound binds to APP and/or to the enzyme BACE and/or to an
APP/BACE complex.
[0309] Methods of preparing hydantoins such as are described herein
are known to those of skill in the art. Generally, in one approach,
the relevant hydantoin (e.g., a diflouoro hydantoin) would be
prepared from 3,4 difluoro benzaldehyde transformed to dione and
condensed with urea to yield the hydantoin as described in Example
1.
[0310] Illustrative protocols for the synthesis of FAH-1, FAH-2,
FAH-3, FAH-4, FAH-5, FAH-17, FAH-17HCl salt, FAH-22, FAH-23,
FAH-27, and FAH-28 (see Table 4) are provided in Examples 1-11.
Synthesis of additional compounds described herein are
straightforward variations of the synthesis schemes provided
herein.
[0311] The various active agents and synthesis schemes are intended
to be illustrative and not limiting. Using the teachings provided
herein, numerous other (e.g., hydantoins or a tautomer(s) or
stereoisomer(s) thereof, or pharmaceutically acceptable salts or
solvates of said hydantoin(s), said stereoisomer(s), or said
tautomer(s), or analogues, derivatives, or prodrugs thereof can be
synthesized and identified by one of skill in the art.
[0312] Illustrative activity of certain APP selective BACE
inhibitors described above is shown in Table 5.
TABLE-US-00005 TABLE 5 Illustrative activity of certain APP
selective BACE inhibitors described herein. Primary Screen
Secondary Screen BACE1 NRG1- IC50 sAPP sAPP BACD1 APP Compound
cLogP (.mu.M) alpha beta sAPP(.alpha./.beta.) A.beta.1-42 IC50 Kd
FAH-3 3.34 0.53 .uparw. > 10% .dwnarw. < 20% .uparw.
.apprxeq. 10% >1 .mu.M NA .apprxeq.0.3 .mu.M FAH-17 3.2 0.15
.uparw. > 20% .dwnarw. < 50% .uparw. > 100% >1 .mu.M
>1 .mu.M .sup. <1 .mu.M FAH-22 0.71 .uparw. > 10% .dwnarw.
< 20% .uparw. .apprxeq. 50% NA NA FAH-23 0.32 .uparw. > 20%
.dwnarw. < 20% .uparw. > 50% NA NA FAH-27 0.13 .uparw. >
30% .dwnarw. < 30% .uparw. > 100% NA NA FAH-28 0.38 .uparw.
> 20% .dwnarw. < 20% .uparw. > 50% NA NA
Pharmaceutical Formulations.
[0313] In certain embodiments one or more active agents described
herein (e.g., hydantoins described herein, or a tautomer(s) or
stereoisomer(s) thereof, or pharmaceutically acceptable salts or
solvates of said hydantoin(s), said stereoisomer(s), or said
tautomer(s), or analogues, derivatives, or prodrugs thereof) are
administered to a mammal in need thereof, e.g., to a mammal at risk
for or suffering from a pathology characterized by abnormal
processing of amyloid precursor proteins, a mammal at risk for
progression of MCI to Alzheimer's disease, and so forth. In certain
embodiments the active agent(s) are administered to prevent or
delay the onset of a pre-Alzheimer's condition and/or cognitive
dysfunction, and/or to ameliorate one or more symptoms of a
pre-Alzheimer's cognitive dysfunction, and/or to prevent or delay
the progression of a pre-Alzheimer's condition or cognitive
dysfunction to Alzheimer's disease, and/or to promote the
processing of amyloid precursor protein (APP) by a
non-amyloidogenic pathway.
[0314] In certain embodiments one or more active agents described
herein (e.g., hydantoins described herein, or a tautomer(s) or
stereoisomer(s) thereof, or pharmaceutically acceptable salts or
solvates of said hydantoin(s), said stereoisomer(s), or said
tautomer(s), or analogues, derivatives, or prodrugs thereof) are
administered to a mammal in need thereof, e.g., to a mammal at risk
for or suffering from a pathology characterized by abnormal
processing of amyloid precursor proteins in conditions other than
Alzheimer's disease of MCI. Illustrative conditions, include, but
are not limited to AD-type symptoms of patients with Down's
syndrome, glaucoma, macular degeneration (e.g., age-related macular
degeneration (AMD), olfactory impairment. in the treatment of
type-II diabetes, including diabetes associated with
amyloidogenesis., neurodegenerative diseases such as scrapie,
bovine spongiform encaphalopathies (e.g., BSE), traumatic brain
injury ("TBI"), Creutzfeld-Jakob disease and the like, type II
diabetes. Other conditions characterized by characterized by
amyloid formation/deposition are contemplated. Such conditions
include, but are not limited to Huntington's Disease, medullary
carcinoma of the thyroid, cardiac arrhythmias, isolated atrial
amyloidosis, atherosclerosis, rheumatoid arthritis, aortic medial
amyloid, prolactinomas, familial amyloid polyneuropathy, hereditary
non-neuropathic systemic amyloidosis, dialysis related amyloidosis,
Finnish amyloidosis, Lattice corneal dystrophy, cerebral amyloid
angiopathy (e.g., Icelandic type), systemic AL amyloidosis,
sporadic inclusion body myositis, cerebrovascular dementia, and the
like.
[0315] The active agent(s) (e.g., hydantoins described herein) can
be administered in the "native" form or, if desired, in the form of
salts, esters, amides, prodrugs, derivatives, and the like,
provided the salt, ester, amide, prodrug or derivative is suitable
pharmacologically, i.e., effective in the present method(s). Salts,
esters, amides, prodrugs and other derivatives of the active agents
can be prepared using standard procedures known to those skilled in
the art of synthetic organic chemistry and described, for example,
by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms
and Structure, 4th Ed. N.Y. Wiley-Interscience, and as described
above.
[0316] For example, a pharmaceutically acceptable salt can be
prepared for any of the agent(s) described herein having a
functionality capable of forming a salt. A pharmaceutically
acceptable salt is any salt that retains the activity of the parent
compound and does not impart any deleterious or untoward effect on
the subject to which it is administered and in the context in which
it is administered.
[0317] In various embodiments pharmaceutically acceptable salts may
be derived from organic or inorganic bases. The salt may be a mono
or polyvalent ion. Of particular interest are the inorganic ions,
lithium, sodium, potassium, calcium, and magnesium. Organic salts
may be made with amines, particularly ammonium salts such as mono-,
di- and trialkyl amines or ethanol amines. Salts may also be formed
with caffeine, tromethamine and similar molecules.
[0318] Methods of formulating pharmaceutically active agents as
salts, esters, amide, prodrugs, and the like are well known to
those of skill in the art. For example, salts can be prepared from
the free base using conventional methodology that typically
involves reaction with a suitable acid. Generally, the base form of
the drug is dissolved in a polar organic solvent such as methanol
or ethanol and the acid is added thereto. The resulting salt either
precipitates or can be brought out of solution by addition of a
less polar solvent. Suitable acids for preparing acid addition
salts include, but are not limited to both organic acids, e.g.,
acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic
acid, malic acid, malonic acid, succinic acid, maleic 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, as well as
inorganic acids, e.g., hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid, and the like. An acid
addition salt can be reconverted to the free base by treatment with
a suitable base. Certain particularly preferred acid addition salts
of the active agents herein include halide salts, such as may be
prepared using hydrochloric or hydrobromic acids. Conversely,
preparation of basic salts of the active agents of this invention
are prepared in a similar manner using a pharmaceutically
acceptable base such as sodium hydroxide, potassium hydroxide,
ammonium hydroxide, calcium hydroxide, trimethylamine, or the like.
Particularly preferred basic salts include alkali metal salts,
e.g., the sodium salt, and copper salts.
[0319] For the preparation of salt forms of basic drugs, the pKa of
the counterion is preferably at least about 2 pH units lower than
the pKa of the drug. Similarly, for the preparation of salt forms
of acidic drugs, the pKa of the counterion is preferably at least
about 2 pH units higher than the pKa of the drug. This permits the
counterion to bring the solution's pH to a level lower than the
pH.sub.max to reach the salt plateau, at which the solubility of
salt prevails over the solubility of free acid or base. The
generalized rule of difference in pKa units of the ionizable group
in the active pharmaceutical ingredient (API) and in the acid or
base is meant to make the proton transfer energetically favorable.
When the pKa of the API and counterion are not significantly
different, a solid complex may form but may rapidly
disproportionate (i.e., break down into the individual entities of
drug and counterion) in an aqueous environment.
[0320] Preferably, the counterion is a pharmaceutically acceptable
counterion. Suitable anionic salt forms include, but are not
limited to acetate, benzoate, benzylate, bitartrate, bromide,
carbonate, chloride, citrate, edetate, edisylate, estolate,
fumarate, gluceptate, gluconate, hydrobromide, hydrochloride,
iodide, lactate, lactobionate, malate, maleate, mandelate,
mesylate, methyl bromide, methyl sulfate, mucate, napsylate,
nitrate, pamoate (embonate), phosphate and diphosphate, salicylate
and disalicylate, stearate, succinate, sulfate, tartrate, tosylate,
triethiodide, valerate, and the like, while suitable cationic salt
forms include, but are not limited to aluminum, benzathine,
calcium, ethylene diamine, lysine, magnesium, meglumine, potassium,
procaine, sodium, tromethamine, zinc, and the like.
[0321] Preparation of esters typically involves functionalization
of hydroxyl and/or carboxyl groups that are present within the
molecular structure of the active agent. In certain embodiments,
the esters are typically acyl-substituted derivatives of free
alcohol groups, i.e., moieties that are derived from carboxylic
acids of the formula RCOOH where R is alky, and preferably is lower
alkyl. Esters can be reconverted to the free acids, if desired, by
using conventional hydrogenolysis or hydrolysis procedures.
[0322] Amides can also be prepared using techniques known to those
skilled in the art or described in the pertinent literature. For
example, amides may be prepared from esters, using suitable amine
reactants, or they may be prepared from an anhydride or an acid
chloride by reaction with ammonia or a lower alkyl amine.
[0323] In various embodiments, the active agents identified herein
(e.g., hydantoins described herein, or a tautomer(s) or
stereoisomer(s) thereof, or pharmaceutically acceptable salts or
solvates of said hydantoin(s), said stereoisomer(s), or said
tautomer(s), or analogues, derivatives, or prodrugs thereof) are
useful for parenteral administration, topical administration, oral
administration, nasal administration (or otherwise inhaled), rectal
administration, or local administration, such as by aerosol or
transdermally, for prophylactic and/or therapeutic treatment of one
or more of the pathologies/indications described herein (e.g.,
pathologies characterized by excess amyloid plaque formation and/or
deposition or undesired amyloid or pre-amyloid processing).
[0324] In various embodiments the active agents described herein
can also be combined with a pharmaceutically acceptable carrier
(excipient) to form a pharmacological composition. Pharmaceutically
acceptable carriers can contain one or more physiologically
acceptable compound(s) that act, for example, to stabilize the
composition or to increase or decrease the absorption of the active
agent(s). Physiologically acceptable compounds can include, for
example, carbohydrates, such as glucose, sucrose, or dextrans,
antioxidants, such as ascorbic acid or glutathione, chelating
agents, low molecular weight proteins, protection and uptake
enhancers such as lipids, compositions that reduce the clearance or
hydrolysis of the active agents, or excipients or other stabilizers
and/or buffers.
[0325] Other physiologically acceptable compounds, particularly of
use in the preparation of tablets, capsules, gel caps, and the like
include, but are not limited to binders, diluent/fillers,
disintegrants, lubricants, suspending agents, and the like.
[0326] In certain embodiments, to manufacture an oral dosage form
(e.g., a tablet), an excipient (e.g., lactose, sucrose, starch,
mannitol, etc.), an optional disintegrator (e.g. calcium carbonate,
carboxymethylcellulose calcium, sodium starch glycollate,
crospovidone etc.), a binder (e.g. alpha-starch, gum arabic,
microcrystalline cellulose, carboxymethylcellulose,
polyvinylpyrrolidone, hydroxypropylcellulose, cyclodextrin, etc.),
and an optional lubricant (e.g., talc, magnesium stearate,
polyethylene glycol 6000, etc.), for instance, are added to the
active component or components (e.g., hydantoins described herein,
or a tautomer(s) or stereoisomer(s) thereof, or pharmaceutically
acceptable salts or solvates of said hydantoin(s), said
stereoisomer(s), or said tautomer(s), or analogues, derivatives, or
prodrugs thereof) and the resulting composition is compressed.
Where necessary the compressed product is coated, e.g., using known
methods for masking the taste or for enteric dissolution or
sustained release. Suitable coating materials include, but are not
limited to ethyl-cellulose, hydroxymethylcellulose, POLYOX.RTM.
ethylene glycol, cellulose acetate phthalate,
hydroxypropylmethylcellulose phthalate, and Eudragit (Rohm &
Haas, Germany; methacrylic-acrylic copolymer).
[0327] Other physiologically acceptable compounds include wetting
agents, emulsifying agents, dispersing agents or preservatives that
are particularly useful for preventing the growth or action of
microorganisms. Various preservatives are well known and include,
for example, phenol and ascorbic acid. One skilled in the art would
appreciate that the choice of pharmaceutically acceptable
carrier(s), including a physiologically acceptable compound
depends, for example, on the route of administration of the active
agent(s) and on the particular physiochemical characteristics of
the active agent(s).
[0328] In certain embodiments the excipients are sterile and
generally free of undesirable matter. These compositions can be
sterilized by conventional, well-known sterilization techniques.
For various oral dosage form excipients such as tablets and
capsules sterility is not required. The USP/NF standard is usually
sufficient.
[0329] The pharmaceutical compositions can be administered in a
variety of unit dosage forms depending upon the method of
administration. Suitable unit dosage forms, include, but are not
limited to powders, tablets, pills, capsules, lozenges,
suppositories, patches, nasal sprays, injectibles, implantable
sustained-release formulations, mucoadherent films, topical
varnishes, lipid complexes, etc.
[0330] Pharmaceutical compositions comprising the active agents
described herein (e.g., hydantoins described herein, or a
tautomer(s) or stereoisomer(s) thereof, or pharmaceutically
acceptable salts or solvates of said hydantoin(s), said
stereoisomer(s), or said tautomer(s), or analogues, derivatives, or
prodrugs thereof) can be manufactured by means of conventional
mixing, dissolving, granulating, dragee-making, levigating,
emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions can be formulated in a conventional
manner using one or more physiologically acceptable carriers,
diluents, excipients or auxiliaries that facilitate processing of
the active agent(s) into preparations that can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0331] In certain embodiments, the active agents described herein
are formulated for oral administration. For oral administration,
suitable formulations can be readily formulated by combining the
active agent(s) with pharmaceutically acceptable carriers suitable
for oral delivery well known in the art. Such carriers enable the
active agent(s) described herein to be formulated as tablets,
pills, dragees, caplets, lizenges, gelcaps, capsules, liquids,
gels, syrups, slurries, suspensions and the like, for oral
ingestion by a patient to be treated. For oral solid formulations
such as, for example, powders, capsules and tablets, suitable
excipients can include fillers such as sugars (e.g., lactose,
sucrose, mannitol and sorbitol), cellulose preparations (e.g.,
maize starch, wheat starch, rice starch, potato starch, gelatin,
gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium carboxymethylcellulose), synthetic polymers (e.g.,
polyvinylpyrrolidone (PVP)), granulating agents; and binding
agents. If desired, disintegrating agents may be added, such as the
cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. If desired, solid dosage forms may
be sugar-coated or enteric-coated using standard techniques. The
preparation of enteric-coated particles is disclosed for example in
U.S. Pat. Nos. 4,786,505 and 4,853,230.
[0332] For administration by inhalation, the active agent(s) are
conveniently delivered in the form of an aerosol spray from
pressurized packs or a nebulizer, with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit can be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0333] In various embodiments the active agent(s) can be formulated
in rectal or vaginal compositions such as suppositories or
retention enemas, e.g., containing conventional suppository bases
such as cocoa butter or other glycerides. Methods of formulating
active agents for rectal or vaginal delivery are well known to
those of skill in the art (see, e.g., Allen (2007) Suppositories,
Pharmaceutical Press) and typically involve combining the active
agents with a suitable base (e.g., hydrophilic (PEG), lipophilic
materials such as cocoa butter or Witepsol W45, amphiphilic
materials such as Suppocire AP and polyglycolized glyceride, and
the like). The base is selected and compounded for a desired
melting/delivery profile.
[0334] For topical administration the active agent(s) described
herein (e.g., hydantoins described herein, or a tautomer(s) or
stereoisomer(s) thereof, or pharmaceutically acceptable salts or
solvates of said hydantoin(s), said stereoisomer(s), or said
tautomer(s), or analogues, derivatives, or prodrugs thereof) can be
formulated as solutions, gels, ointments, creams, suspensions, and
the like as are well-known in the art.
[0335] In certain embodiments the active agents described herein
are formulated for systemic administration (e.g., as an injectable)
in accordance with standard methods well known to those of skill in
the art. Systemic formulations include, but are not limited to,
those designed for administration by injection, e.g. subcutaneous,
intravenous, intramuscular, intrathecal or intraperitoneal
injection, as well as those designed for transdermal, transmucosal
oral or pulmonary administration. For injection, the active agents
described herein can be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks solution,
Ringer's solution, or physiological saline buffer and/or in certain
emulsion formulations. The solution(s) can contain formulatory
agents such as suspending, stabilizing and/or dispersing agents. In
certain embodiments the active agent(s) can be provided in powder
form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use. For transmucosal administration,
and/or for blood/brain barrier passage, penetrants appropriate to
the barrier to be permeated can be used in the formulation. Such
penetrants are generally known in the art. Injectable formulations
and inhalable formulations are generally provided as a sterile or
substantially sterile formulation.
[0336] In addition to the formulations described previously, the
active agent(s) may also be formulated as a depot preparations.
Such long acting formulations can be administered by implantation
(for example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the active agent(s) may be formulated
with suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0337] In certain embodiments the active agent(s) described herein
can also be delivered through the skin using conventional
transdermal drug delivery systems, i.e., transdermal "patches"
wherein the active agent(s) are typically contained within a
laminated structure that serves as a drug delivery device to be
affixed to the skin. In such a structure, the drug composition is
typically contained in a layer, or "reservoir," underlying an upper
backing layer. It will be appreciated that the term "reservoir" in
this context refers to a quantity of "active ingredient(s)" that is
ultimately available for delivery to the surface of the skin. Thus,
for example, the "reservoir" may include the active ingredient(s)
in an adhesive on a backing layer of the patch, or in any of a
variety of different matrix formulations known to those of skill in
the art. The patch may contain a single reservoir, or it may
contain multiple reservoirs.
[0338] In one illustrative embodiment, the reservoir comprises a
polymeric matrix of a pharmaceutically acceptable contact adhesive
material that serves to affix the system to the skin during drug
delivery. Examples of suitable skin contact adhesive materials
include, but are not limited to, polyethylenes, polysiloxanes,
polyisobutylenes, polyacrylates, polyurethanes, and the like.
Alternatively, the drug-containing reservoir and skin contact
adhesive are present as separate and distinct layers, with the
adhesive underlying the reservoir which, in this case, may be
either a polymeric matrix as described above, or it may be a liquid
or hydrogel reservoir, or may take some other form. The backing
layer in these laminates, which serves as the upper surface of the
device, preferably functions as a primary structural element of the
"patch" and provides the device with much of its flexibility. The
material selected for the backing layer is preferably substantially
impermeable to the active agent(s) and any other materials that are
present.
[0339] Alternatively, other pharmaceutical delivery systems can be
employed. For example, liposomes, emulsions, and
microemulsions/nanoemulsions are well known examples of delivery
vehicles that may be used to protect and deliver pharmaceutically
active compounds. Certain organic solvents such as
dimethylsulfoxide also can be employed, although usually at the
cost of greater toxicity.
[0340] In certain embodiments the active agent(s) described herein
(e.g., hydantoins described herein, or a tautomer(s) or
stereoisomer(s) thereof, or pharmaceutically acceptable salts or
solvates of said hydantoin(s), said stereoisomer(s), or said
tautomer(s), or analogues, derivatives, or prodrugs thereof) are
formulated in a nanoemulsion. Nanoemulsions include, but are not
limited to oil in water (O/W) nanoemulsions, and water in oil (W/O)
nanoemulsions. Nanoemulsions can be defined as emulsions with mean
droplet diameters ranging from about 20 to about 1000 nm. Usually,
the average droplet size is between about 20 nm or 50 nm and about
500 nm. The terms sub-micron emulsion (SME) and mini-emulsion are
used as synonyms.
[0341] Illustrative oil in water (O/W) nanoemulsions include, but
are not limited to: Surfactant micelles--micelles composed of small
molecules surfactants or detergents (e.g., SDS/PBS/2-propanol);
Polymer micelles--micelles composed of polymer, copolymer, or block
copolymer surfactants (e.g., Pluronic L64/PBS/2-propanol); Blended
micelles--micelles in which there is more than one surfactant
component or in which one of the liquid phases (generally an
alcohol or fatty acid compound) participates in the formation of
the micelle (e.g., octanoic acid/PBS/EtOH); Integral
micelles--blended micelles in which the active agent(s) serve as an
auxiliary surfactant, forming an integral part of the micelle; and
Pickering (solid phase) emulsions--emulsions in which the active
agent(s) are associated with the exterior of a solid nanoparticle
(e.g., polystyrene nanoparticles/PBS/no oil phase).
[0342] Illustrative water in oil (W/O) nanoemulsions include, but
are not limited to: Surfactant micelles--micelles composed of small
molecules surfactants or detergents (e.g., dioctyl
sulfosuccinate/PBS/2-propanol, isopropylmyristate/PBS/2-propanol,
etc.); Polymer micelles--micelles composed of polymer, copolymer,
or block copolymer surfactants (e.g., PLURONIC.RTM.
L121/PBS/2-propanol); Blended micelles--micelles in which there is
more than one surfactant component or in which one of the liquid
phases (generally an alcohol or fatty acid compound) participates
in the formation of the micelle (e.g., capric/caprylic
diglyceride/PBS/EtOH); Integral micelles--blended micelles in which
the active agent(s) serve as an auxiliary surfactant, forming an
integral part of the micelle (e.g., active agent/PBS/polypropylene
glycol); and Pickering (solid phase) emulsions--emulsions in which
the active agent(s) are associated with the exterior of a solid
nanoparticle (e.g., chitosan nanoparticles/no aqueous phase/mineral
oil).
[0343] As indicated above, in certain embodiments the nanoemulsions
comprise one or more surfactants or detergents. In some embodiments
the surfactant is a non-anionic detergent (e.g., a polysorbate
surfactant, a polyoxyethylene ether, etc.). Surfactants that find
use in the present invention include, but are not limited to
surfactants such as the TWEEN.RTM., TRITON.RTM., and TYLOXAPOL.RTM.
families of compounds.
[0344] In certain embodiments the emulsions further comprise one or
more cationic halogen containing compounds, including but not
limited to, cetylpyridinium chloride. In still further embodiments,
the compositions further comprise one or more compounds that
increase the interaction ("interaction enhancers") of the
composition with microorganisms (e.g., chelating agents like
ethylenediaminetetraacetic acid, or
ethylenebis(oxyethylenenitrilo)tetraacetic acid in a buffer).
[0345] In some embodiments, the nanoemulsion further comprises an
emulsifying agent to aid in the formation of the emulsion.
Emulsifying agents include compounds that aggregate at the
oil/water interface to form a kind of continuous membrane that
prevents direct contact between two adjacent droplets. Certain
embodiments of the present invention feature oil-in-water emulsion
compositions that may readily be diluted with water to a desired
concentration without impairing their anti-pathogenic
properties.
[0346] In addition to discrete oil droplets dispersed in an aqueous
phase, certain oil-in-water emulsions can also contain other lipid
structures, such as small lipid vesicles (e.g., lipid spheres that
often consist of several substantially concentric lipid bilayers
separated from each other by layers of aqueous phase), micelles
(e.g., amphiphilic molecules in small clusters of 50-200 molecules
arranged so that the polar head groups face outward toward the
aqueous phase and the apolar tails are sequestered inward away from
the aqueous phase), or lamellar phases (lipid dispersions in which
each particle consists of parallel amphiphilic bilayers separated
by thin films of water).
[0347] These lipid structures are formed as a result of hydrophobic
forces that drive apolar residues (e.g., long hydrocarbon chains)
away from water. The above lipid preparations can generally be
described as surfactant lipid preparations (SLPs). SLPs are
minimally toxic to mucous membranes and are believed to be
metabolized within the small intestine (see e.g., Hamouda et al.,
(1998) J. Infect. Disease 180: 1939).
[0348] In certain embodiments the emulsion comprises a
discontinuous oil phase distributed in an aqueous phase, a first
component comprising an alcohol and/or glycerol, and a second
component comprising a surfactant or a halogen-containing compound.
The aqueous phase can comprise any type of aqueous phase including,
but not limited to, water (e.g., dionized water, distilled water,
tap water) and solutions (e.g., phosphate buffered saline solution
or other buffer systems). The oil phase can comprise any type of
oil including, but not limited to, plant oils (e.g., soybean oil,
avocado oil, flaxseed oil, coconut oil, cottonseed oil, squalene
oil, olive oil, canola oil, corn oil, rapeseed oil, safflower oil,
and sunflower oil), animal oils (e.g., fish oil), flavor oil, water
insoluble vitamins, mineral oil, and motor oil. In certain
embodiments, the oil phase comprises 30-90 vol % of the
oil-in-water emulsion (e.g., constitutes 30-90% of the total volume
of the final emulsion), more preferably 50-80%. The formulations
need not be limited to particular surfactants, however in certain
embodiments, the surfactant is a polysorbate surfactant (e.g.,
TWEEN 20.RTM., TWEEN 40.RTM., TWEEN 60.RTM., and TWEEN 80.RTM.), a
pheoxypolyethoxyethanol (e.g., TRITON.RTM. X-100, X-301, X-165,
X-102, and X-200, and TYLOXAPOL.RTM.), or sodium dodecyl sulfate,
and the like.
[0349] In certain embodiments a halogen-containing component is
present. the nature of the halogen-containing compound, in some
embodiments the halogen-containing compound comprises a chloride
salt (e.g., NaCl, KCl, etc.), a cetylpyridinium halide, a
cetyltrimethylammonium halide, a cetyldimethylethylammonium halide,
a cetyldimethylbenzylammonium halide, a cetyltributylphosphonium
halide, dodecyltrimethylammonium halides,
tetradecyltrimethylammonium halides, cetylpyridinium chloride,
cetyltrimethylammonium chloride, cetylbenzyldimethylammonium
chloride, cetylpyridinium bromide, cetyltrimethylammonium bromide,
cetyldimethylethylammonium bromide, cetyltributylphosphonium
bromide, dodecyltrimethylammonium bromide,
tetradecyltrimethylammonium bromide, and the like
[0350] In certain embodiments the emulsion comprises a quaternary
ammonium compound. Quaternary ammonium compounds include, but are
not limited to, N-alkyldimethyl benzyl ammonium saccharinate,
1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol; 1-Decanaminium,
N-decyl-N,N-dimethyl-, chloride (or) Didecyl dimethyl ammonium
chloride; 2-(2-(p-(Diisobuyl)cresosxy)ethoxy)ethyl dimethyl benzyl
ammonium chloride; 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl
dimethyl benzyl ammonium chloride; alkyl 1 or 3
benzyl-1-(2-hydroxethyl)-2-imidazolinium chloride; alkyl
bis(2-hydroxyethyl)benzyl ammonium chloride; alkyl dimethyl benzyl
ammonium chloride; alkyl dimethyl 3,4-dichlorobenzyl ammonium
chloride (100% C12); alkyl dimethyl 3,4-dichlorobenzyl ammonium
chloride (50% C14, 40% C12, 10% C16); alkyl dimethyl
3,4-dichlorobenzyl ammonium chloride (55% C14, 23% C12, 20% C16);
alkyl dimethyl benzyl ammonium chloride; alkyl dimethyl benzyl
ammonium chloride (100% C14); alkyl dimethyl benzyl ammonium
chloride (100% C16); alkyl dimethyl benzyl ammonium chloride (41%
C14, 28% C12); alkyl dimethyl benzyl ammonium chloride (47% C12,
18% C14); alkyl dimethyl benzyl ammonium chloride (55% C16, 20%
C14); alkyl dimethyl benzyl ammonium chloride (58% C14, 28% C16);
alkyl dimethyl benzyl ammonium chloride (60% C14, 25% C12); alkyl
dimethyl benzyl ammonium chloride (61% C11, 23% C14); alkyl
dimethyl benzyl ammonium chloride (61% C12, 23% C14); alkyl
dimethyl benzyl ammonium chloride (65% C12, 25% C14); alkyl
dimethyl benzyl ammonium chloride (67% C12, 24% C14); alkyl
dimethyl benzyl ammonium chloride (67% C12, 25% C14); alkyl
dimethyl benzyl ammonium chloride (90% C14, 5% C12); alkyl dimethyl
benzyl ammonium chloride (93% C14, 4% C12); alkyl dimethyl benzyl
ammonium chloride (95% C16, 5% C18); alkyl dimethyl benzyl ammonium
chloride (and) didecyl dimethyl ammonium chloride; alkyl dimethyl
benzyl ammonium chloride (as in fatty acids); alkyl dimethyl benzyl
ammonium chloride (C12-C16); alkyl dimethyl benzyl ammonium
chloride (C12-C18); alkyl dimethyl benzyl and dialkyl dimethyl
ammonium chloride; alkyl dimethyl dimethybenzyl ammonium chloride;
alkyl dimethyl ethyl ammonium bromide (90% C14, 5% C16, 5% C12);
alkyl dimethyl ethyl ammonium bromide (mixed alkyl and alkenyl
groups as in the fatty acids of soybean oil); alkyl dimethyl
ethylbenzyl ammonium chloride; alkyl dimethyl ethylbenzyl ammonium
chloride (60% C14); alkyl dimethyl isoproylbenzyl ammonium chloride
(50% C12, 30% C14, 17% C16, 3% C18); alkyl trimethyl ammonium
chloride (58% C18, 40% C16, 1% C14, 1% C12); alkyl trimethyl
ammonium chloride (90% C18, 10% C16);
alkyldimethyl(ethylbenzyl)ammonium chloride (C12-18);
Di-(C.sub.8-10)-alkyl dimethyl ammonium chlorides; dialkyl dimethyl
ammonium chloride; dialkyl dimethyl ammonium chloride; dialkyl
dimethyl ammonium chloride; dialkyl methyl benzyl ammonium
chloride; didecyl dimethyl ammonium chloride; diisodecyl dimethyl
ammonium chloride; dioctyl dimethyl ammonium chloride; dodecyl
bis(2-hydroxyethyl)octyl hydrogen ammonium chloride; dodecyl
dimethyl benzyl ammonium chloride; dodecylcarbamoyl methyl dimethyl
benzyl ammonium chloride; heptadecyl hydroxyethylimidazolinium
chloride; hexahydro-1,3,5-thris(2-hydroxyethyl)-s-triazine;
myristalkonium chloride (and) Quat RNIUM 14;
N,N-Dimethyl-2-hydroxypropylammonium chloride polymer; n-alkyl
dimethyl benzyl ammonium chloride; n-alkyl dimethyl ethylbenzyl
ammonium chloride; n-tetradecyl dimethyl benzyl ammonium chloride
monohydrate; octyl decyl dimethyl ammonium chloride; octyl dodecyl
dimethyl ammonium chloride; octyphenoxyethoxyethyl dimethyl benzyl
ammonium chloride; oxydiethylenebis (alkyl dimethyl ammonium
chloride); quaternary ammonium compounds, dicoco alkyldimethyl,
chloride; trimethoxysily propyl dimethyl octadecyl ammonium
chloride; trimethoxysilyl quats, trimethyl dodecylbenzyl ammonium
chloride; n-dodecyl dimethyl ethylbenzyl ammonium chloride;
n-hexadecyl dimethyl benzyl ammonium chloride; n-tetradecyl
dimethyl benzyl ammonium chloride; n-tetradecyl dimethyl
ethylbenzyl ammonium chloride; and n-octadecyl dimethyl benzyl
ammonium chloride.
[0351] Nanoemulsion formulations and methods of making such are
well known to those of skill in the art and described for example
in U.S. Pat. Nos. 7,476,393, 7,468,402, 7,314,624, 6,998,426,
6,902,737, 6,689,371, 6,541,018, 6,464,990, 6,461,625, 6,419,946,
6,413,527, 6,375,960, 6,335,022, 6,274,150, 6,120,778, 6,039,936,
5,925,341, 5,753,241, 5,698,219, and 5,152,923 and in Fanun et al.
(2009) Microemulsions: Properties and Applications (Surfactant
Science), CRC Press, Boca Ratan Fla.
[0352] In certain embodiments, one or more active agents described
herein can be provided as a "concentrate", e.g., in a storage
container (e.g., in a premeasured volume) ready for dilution, or in
a soluble capsule ready for addition to a volume of water, alcohol,
hydrogen peroxide, or other diluent.
[0353] Extended Release (Sustained Release) Formulations.
[0354] In certain embodiments "extended release" formulations of
the active agent(s) described herein (e.g., hydantoins described
herein, or a tautomer(s) or stereoisomer(s) thereof, or
pharmaceutically acceptable salts or solvates of said hydantoin(s),
said stereoisomer(s), or said tautomer(s), or analogues,
derivatives, or prodrugs thereof) are contemplated. In various
embodiments such extended release formulations are designed to
avoid the high peak plasma levels of intravenous and conventional
immediate release oral dosage forms.
[0355] Illustrative sustained-release formulations include, for
example, semipermeable matrices of solid polymers containing the
therapeutic agent. Various uses of sustained-release materials have
been established and are well known by those skilled in the art.
Sustained-release capsules may, depending on their chemical nature,
release the compounds for a few weeks up to over 100 days.
Depending on the chemical nature and the biological stability of
the therapeutic reagent, additional strategies for stabilization
can be employed.
[0356] In certain embodiments such "extended release" formulations
utilize the mucosa and can independently control tablet
disintegration (or erosion) and/or drug dissolution and release
from the tablet over time to provide a safer delivery profile. In
certain embodiments the oral formulations of active agent(s)
described herein (e.g., hydantoins described herein, or a
tautomer(s) or stereoisomer(s) thereof, or pharmaceutically
acceptable salts or solvates of said hydantoin(s), said
stereoisomer(s), or said tautomer(s), or analogues, derivatives, or
prodrugs thereof) provide individual, repetitive doses that include
a defined amount of the active agent that is delivered over a
defined amount of time.
[0357] One illustrative sustained release formulation is a
substantially homogeneous composition that comprises about 0.01% to
about 99% w/w, or about 0.1% to about 95%, or about 0.1%, or about
1%, or about 2%, or about 5%, or about 10%, or about 15%, or about
20% to about 80%, or to about 90%, or to about 95%, or to about
97%, or to about 98%, or to about 99%1 of the active ingredient(s)
(e.g., hydantoins described herein, or a tautomer(s) or
stereoisomer(s) thereof, or pharmaceutically acceptable salts or
solvates of said hydantoin(s), said stereoisomer(s), or said
tautomer(s), or analogues, derivatives, or prodrugs thereof) and
one or more mucoadhesives (also referred to herein as
"bioadhesives") that provide for adherence to the targeted mucosa
of the subject (patient) and that may further comprise one or more
of the following: one or more binders that provide binding of the
excipients in a single tablet; one or more hydrogel forming
excipients; one or more bulking agents; one or more lubricants; one
or more glidants; one or more solubilizers; one or more
surfactants; one or more flavors; one or more disintegrants; one or
more buffering excipients; one or more coatings; one or more
controlled release modifiers; and one or more other excipients and
factors that modify and control the drug's dissolution or
disintegration time and kinetics or protect the active drug from
degradation.
[0358] In various embodiments a sustained release pharmaceutical
dosage form for oral transmucosal delivery can be solid or
non-solid. In one illustrative embodiment, the dosage form is a
solid that turns into a hydrogel following contact with saliva.
[0359] Suitable excipients include, but are not limited to
substances added to the formulations that are required to produce a
commercial product and can include, but are not limited to: bulking
agents, binders, surfactants, bioadhesives, lubricants,
disintegrants, stabilizers, solubilizers, glidants, and additives
or factors that affect dissolution or disintegration time. Suitable
excipients are not limited to those above, and other suitable
nontoxic pharmaceutically acceptable carriers for use in oral
formulations can be found in Remington's Pharmaceutical Sciences,
17th Edition, 1985.
[0360] In certain embodiments extended release formulations of the
active agent(s) described herein for oral transmucosal drug
delivery include at least one bioadhesive (mucoadhesive) agent or a
mixture of several bioadhesives to promote adhesion to the oral
mucosa during drug delivery. In addition the bioadhesive agents may
also be effective in controlling the dosage form erosion time
and/or, the drug dissolution kinetics over time when the dosage
form is wetted. Such mucoadhesive drug delivery systems are very
beneficial, since they can prolong the residence time of the drug
at the site of absorption and increase drug bioavailability. The
mucoadhesive polymers forming hydrogels are typically hydrophilic
and swellable, containing numerous hydrogen bond-forming groups,
like hydroxyl, carboxyl or amine, which favor adhesion. When used
in a dry form, they attract water from the mucosal surface and
swell, leading to polymer/mucus interaction through hydrogen
bonding, electrostatic, hydrophobic or van der Waals
interaction.
[0361] Illustrative suitable mucoadhesive or bioadhesive materials,
include, but are not limited to natural, synthetic or biological
polymers, lipids, phospholipids, and the like. Examples of natural
and/or synthetic polymers include cellulosic derivatives (such as
methylcellulose, carboxymethyl cellulose, hydroxyethyl cellulose,
hydroxyethylmethyl cellulose, etc), natural gums (such as guar gum,
xanthan gum, locust bean gum, karaya gum, veegum etc.),
polyacrylates (such as CARBOPOL.RTM., polycarbophil, etc),
alginates, thiol-containing polymers, POLYOX.RTM. yethylenes,
polyethylene glycols (PEG) of all molecular weights (preferably
between 1000 and 40,000 Da, of any chemistry, linear or branched),
dextrans of all molecular weights (preferably between 1000 and
40,000 Da of any source), block copolymers, such as those prepared
by combinations of lactic and glycolic acid (PLA, PGA, PLGA of
various viscosities, molecular weights and lactic-to-glycolic acid
ratios) polyethylene glycol-polypropylene glycol block copolymers
of any number and combination of repeating units (such as
PLURONICS.RTM., TEKTRONIX.RTM. or GENAPOL.RTM. block copolymers),
combination of the above copolymers either physically or chemically
linked units (for example PEG-PLA or PEG-PLGA copolymers) mixtures.
Preferably the bioadhesive excipient is selected from the group of
polyethylene glycols, POLYOX.RTM. yethylenes, polyacrylic acid
polymers, such as CARBOPOL.RTM. (such as CARBOPOL.RTM. 71G, 934P,
971P, 974P, and the like) and polycarbophils (such as NOVEON.RTM.
AA-1, NOVEON.RTM. CA-1, NOVEON.RTM. CA-2, and the like), cellulose
and its derivatives and most preferably it is polyethylene glycol,
carbopol, and/or a cellulosic derivative or a combination
thereof.
[0362] In certain embodiments the mucoadhesive/bioadhesive
excipient is typically present at 1-50% w/w, preferably 1-40% w/w
or most preferably between 5-30% w/w. A particular formulation may
contain one or more different bioadhesives in any combination.
[0363] In certain embodiments the formulations for oral
transmucosal drug delivery also include a binder or mixture of two
or more binders which facilitate binding of the excipients into a
single dosage form. Illustrative binders include, binders selected
from the group consisting of cellulosic derivatives (such as
methylcellulose, carboxymethyl cellulose, hydroxyethyl cellulose,
hydroxyethylmethyl cellulose, etc.), polyacrylates (such as
CARBOPOL.RTM., polycarbophil, etc.), POVIDONE.RTM. (all grades),
POLYOX.RTM..RTM. of any molecular weight or grade, irradiated or
not, starch, polyvinylpyrrolidone (PVP), AVICEL.RTM., and the like.
In certain embodiments the binder is typically present at 0.5-60%
w/w, preferably 1-30% w/w and most preferably 1.5-15% w/w.
[0364] In certain embodiments the formulations also include at
least one hydrogel-forming excipient. Illustrative hydrogel forming
excipients include, but are not limited to those selected from the
group consisting of polyethylene glycols and other polymers having
an ethylene glycol backbone, whether homopolymers or cross linked
heteropolymers, block copolymers using ethylene glycol units, such
as POLYOX.RTM. yethylene homopolymers (such as POLYOX.RTM..RTM.
N10/MW=100,000 POLYOX.RTM.-80/MW=200,000; POLYOX.RTM.
1105/MW=900,000; POLYOX.RTM.-301/MW=4,000,000;
POLYOX.RTM.-303/MW=7,000,000, POLYOX.RTM. WSR-N-60K, all of which
are tradenames of Union Carbide), hydroxypropylmethylcellylose
(HPMC) of all molecular weights and grades (such as METOLOSE.RTM.
90SHS3000, METOLOSE.RTM. 90SH30000, all of which are tradenames of
Shin-Etsu Chemical company), Poloxamers (such as LUTROL.RTM. F-68,
LUTROL.RTM. F-127, F-105 etc., all tradenames of BASF Chemicals),
GENAPOL.RTM., polyethylene glycols (PEG, such as PEG-1500,
PEG-3500, PEG-4000, PEG-6000, PEG-8000, PEG-12000, PEG-20,000,
etc.), natural gums (xanthan gum, locust bean gum, etc.) and
cellulose derivatives (HC, HMC, HMPC, HPC, CP, CMC), polyacrylic
acid-based polymers either as free or cross-linked and combinations
thereof, biodegradable polymers such as poly lactic acids,
polyglycolic acids and any combination thereof, whether a physical
blend or cross-linked. In certain embodiments, the hydrogel
components may be cross-linked. The hydrogel forming excipient(s)
are typically present at 0.1-70% w/w, preferably 1-50% w/w or most
preferably 1-30% w/w.
[0365] In certain embodiments the formulations may also include at
least one controlled release modifier which is a substance that
upon hydration of the dosage form will preferentially adhere to the
drug molecules and thus reduce the rate of its diffusion from the
oral dosage form. Such excipients may also reduce the rate of water
uptake by the formulation and thus enable a more prolonged drug
dissolution and release from the tablet. In general the selected
excipient(s) are lipophilic and capable of naturally complexing to
the hydrophobic or lipophilic drugs. The degree of association of
the release modifier and the drug can be varied by altering the
modifier-to-drug ratio in the formulation. In addition, such
interaction may be appropriately enhanced by the appropriate
combination of the release modifier with the active drug in the
manufacturing process. Alternatively, the controlled release
modifier may be a charged polymer either synthetic or biopolymer
bearing a net charge, either positive or negative, and which is
capable of binding to the active via electrostatic interactions
thus modifying both its diffusion through the tablet and/or the
kinetics of its permeation through the mucosal surface. Similarly
to the other compounds mentioned above, such interaction is
reversible and does not involve permanent chemical bonds with the
active. In certain embodiments the controlled release modifier may
typically be present at 0-80% w/w, preferably 1-20% w/w, most
preferably 1-10% w/w.
[0366] In various embodiments the extended release formulations may
also include other conventional components required for the
development of oral dosage forms, which are known to those skilled
in the art. These components may include one or more bulking agents
(such as lactose USP, Starch 1500, mannitol, sorbitol, malitol or
other non-reducing sugars; microcrystalline cellulose (e.g.,
AVICEL.RTM.), dibasic calcium phosphate dehydrate, sucrose, and
mixtures thereof), at least one solubilizing agent(s) (such as
cyclodextrins, pH adjusters, salts and buffers, surfactants, fatty
acids, phospholipids, metals of fatty acids etc.), metal salts and
buffers organic (such as acetate, citrate, tartrate, etc.) or
inorganic (phosphate, carbonate, bicarbonate, borate, sulfate,
sulfite, bisulfite, metabisulfite, chloride, etc.), salts of metals
such as sodium, potassium, calcium, magnesium, etc.), at least one
lubricant (such as stearic acid and divalent cations of, such as
magnesium stearate, calcium stearate, etc., talc, glycerol
monostearate and the like), one or more glidants (such as colloidal
silicon dioxide, precipitated silicon dioxide, fumed silica
(CAB-O-SIL.RTM. M-5P, trademark of Cabot Corporation), stearowet
and sterotex, silicas (such as SILOID.RTM. and SILOX.RTM.
silicas--trademarks of Grace Davison Products, Aerosil--trademark
of Degussa Pharma), higher fatty acids, the metal salts thereof,
hydrogenated vegetable oils and the like), flavors or sweeteners
and colorants (such as aspartame, mannitol, lactose, sucrose, other
artificial sweeteners; ferric oxides and FD&C lakes), additives
to help stabilize the drug substance from chemical of physical
degradation (such as anti-oxidants, anti-hydrolytic agents,
aggregation-blockers etc. Anti-oxidants may include BHT, BHA,
vitamins, citric acid, EDTA, sodium bisulfate, sodium
metabisulfate, thiourea, methionine, surfactants, amino-acids, such
as arginine, glycine, histidine, methionine salts, pH adjusters,
chelating agents and buffers in the dry or solution form), one or
more excipients that may affect tablet disintegration kinetics and
drug release from the tablet, and thus pharmacokinetics
(disintegrants such as those known to those skilled in the art and
may be selected from a group consisting of starch,
carboxy-methycellulose type or crosslinked polyvinyl pyrrolidone
(such as cross-povidone, PVP-XL), alginates, cellulose-based
disintegrants (such as purified cellulose, methylcellulose,
crosslinked sodium carboxy methylcellulose (Ac-Di-Sol) and carboxy
methyl cellulose), low substituted hydroxypropyl ethers of
cellulose, microcrystalline cellulose (such as AVICEL.RTM.), ion
exchange resins (such as AMBRELITE.RTM. IPR 88), gums (such as
agar, locust bean, karaya, pectin and tragacanth), guar gums, gum
karaya, chitin and chitosan, smecta, gellan gum, isapghula husk,
polacrillin potassium (Tulsion.sup.339)' gas-evolving disintegrants
(such as citric acid and tartaric acid along with the sodium
bicarbonate, sodium carbonate, potassium bicarbonate or calcium
carbonate), sodium starch glycolate (such as EXPLOTAB.RTM. and
PRIMOGEL.RTM.), starch DC and the likes, at least one biodegradable
polymer of any type useful for extended drug release. Exemplary
polymer compositions include, but are not limited to,
polyanhydrides and co-polymers of lactic acid and glycolic acid,
poly(dl-lactide-co-glycolide) (PLGA), poly(lactic acid) (PLA),
poly(glycolic acid) (PGA), polyorthoesters, proteins, and
polysaccharides.
[0367] In certain embodiments, the active agent(s) can be
chemically modified to significantly modify the pharmacokinetics in
plasma. This may be accomplished for example by conjugation with
poly(ethylene glycol) (PEG), including site-specific PEGylation.
PEGylation, which may improve drug performance by optimizing
pharmacokinetics, decreasing immunogenicity and dosing
frequency.
[0368] Methods of making a formulation of the active agent(s)
described herein (e.g., hydantoins described herein, or a
tautomer(s) or stereoisomer(s) thereof, or pharmaceutically
acceptable salts or solvates of said hydantoin(s), said
stereoisomer(s), or said tautomer(s), or analogues, derivatives, or
prodrugs thereof) for GI or oral transmucosal delivery are also
provided. One method includes the steps of powder grinding, dry
powder mixing and tableting via direct compression. Alternatively,
a wet granulation process may be used. Such a method (such as high
shear granulation process) involves mixing the active ingredient
and possibly some excipients in a mixer. The binder may be one of
the excipients added in the dry mix state or dissolved in the fluid
used for granulating. The granulating solution or suspension is
added to the dry powders in the mixer and mixed until the desired
characteristics are achieved. This usually produces a granule that
will be of suitable characteristics for producing dosage forms with
adequate dissolution time, content uniformity, and other physical
characteristics. After the wet granulation step, the product is
most often dried and/or then milled after drying to get a major
percentage of the product within a desired size range. Sometimes,
the product is dried after being wet sized using a device such as
an oscillating granulator, or a mill. The dry granulation may then
processed to get an acceptable size range by first screening with a
sieving device, and then milling the oversized particles.
[0369] Additionally, the formulation may be manufactured by
alternative granulation processes, all known to those skilled in
the art, such as spray fluid bed granulation, extrusion and
spheronization or fluid bed rotor granulation.
[0370] Additionally, the tablet dosage form of the active agent(s)
described herein may be prepared by coating the primary tablet
manufactured as described above with suitable coatings known in the
art. Such coatings are meant to protect the active cores against
damage (abrasion, breakage, dust formation) against influences to
which the cores are exposed during transport and storage
(atmospheric humidity, temperature fluctuations), and naturally
these film coatings can also be colored. The sealing effect of film
coats against water vapor is expressed by the water vapor
permeability. Coating may be performed by one of the available
processes such as Wurster coating, dry coating, film coating, fluid
bed coating, pan coating, etc. Typical coating materials include
polyvinyl pyrrolidone (PVP), polyvinyl pyrrolidone vinyl acetate
copolymer (PVPVA), polyvinyl alcohol (PVA), polyvinyl
alcohol/polyethylene glycol copolymer (PVA/PEG), cellulose acetate
phthalate, ethyl cellulose, gellan gum, maltodextrin,
methacrylates, methyl cellulose, hydroxyl propyl methyl cellulose
(HPMC of all grades and molecular weights), carrageenan, shellac
and the like.
[0371] In certain embodiments the tablet core comprising the active
agent(s) described herein can be coated with a bioadhesive and/or
pH resistant material to enable material, such as those defined
above, to improve bioadhesion of the tablet in the sublingual
cavity.
[0372] In certain embodiments, the active agent(s) described herein
(e.g., hydantoins described herein, or a tautomer(s) or
stereoisomer(s) thereof, or pharmaceutically acceptable salts or
solvates of said hydantoin(s), said stereoisomer(s), or said
tautomer(s), or analogues, derivatives, or prodrugs thereof) are
formulated as inclusion complexes. While not limited to
cyclodextrin inclusion complexes, it is noted that cyclodextrin is
the agent most frequently used to form pharmaceutical inclusion
complexes. Cyclodextrins (CD) are cyclic oligomers of glucose, that
typically contain 6, 7, or 8 glucose monomers joined by .alpha.-1,4
linkages. These oligomers are commonly called .alpha.-CD,
.beta.-CD, and .gamma.-CD, respectively. Higher oligomers
containing up to 12 glucose monomers are known, and contemplated to
in the formulations described herein. Functionalized cyclodextrin
inclusion complexes are also contemplated. Illustrative, but
non-limiting functionalized cyclodextrins include, but are not
limited to sulfonates, sulfonates and sulfinates, or disulfonates
of hydroxybutenyl cyclodextrin; sulfonates, sulfonates and
sulfinates, or disulfonates of mixed ethers of cyclodextrins where
at least one of the ether substituents is hydroxybutenyl
cyclodextrin. Illustrative cyclodextrins include a polysaccharide
ether which comprises at least one 2-hydroxybutenyl substituent,
wherein the at least one hydroxybutenyl substituent is sulfonated
and sulfinated, or disulfonated, and an alkylpolyglycoside ether
which comprises at least one 2-hydroxybutenyl substituent, wherein
the at least one hydroxybutenyl substituent is sulfonated and
sulfinated, or disulfonated. In various embodiments inclusion
complexes formed between sulfonated hydroxybutenyl cyclodextrins
and one or more of the active agent(s) described herein are
contemplated. Methods of preparing cyclodextrins, and cyclodextrin
inclusion complexes are found for example in U.S. Patent
Publication No: 2004/0054164 and the references cited therein and
in U.S. Patent Publication No: 2011/0218173 and the references
cited therein.
[0373] Pharmacokinetics (PK) and Formulation Attributes
[0374] One advantage of the extended (controlled) release oral (GI
or transmucosal) formulations described herein is that they can
maintain the plasma drug concentration within a targeted
therapeutic window for a longer duration than with
immediate-release formulations, whether solid dosage forms or
liquid-based dosage forms. The high peak plasma levels typically
observed for such conventional immediate release formulations will
be blunted by the prolonged release of the drug over 1 to 12 hours
or longer. In addition, a rapid decline in plasma levels will be
avoided since the drug will continually be crossing from the oral
cavity into the bloodstream during the length of time of
dissolution of the tablet, thus providing plasma pharmacokinetics
with a more stable plateau. In addition, the dosage forms described
herein may improve treatment safety by minimizing the potentially
deleterious side effects due to the reduction of the peaks and
troughs in the plasma drug pharmacokinetics, which compromise
treatment safety.
[0375] In various embodiments the oral transmucosal formulations of
the active agent(s) described herein designed to avoid the high
peak plasma levels of intravenous and conventional immediate
release oral dosage forms by utilizing the mucosa and by
independently controlling both tablet disintegration (or erosion)
and drug dissolution and release from the tablet over time to
provide a safer delivery profile. The oral formulations described
herein provide individual, repetitive doses that include a defined
amount of the active agent.
[0376] An advantage of the bioadhesive oral transmucosal
formulations described herein is that they exhibit highly
consistent bioavailability and can maintain the plasma drug
concentration within a targeted therapeutic window with
significantly lower variability for a longer duration than
currently available dosage forms, whether solid dosage forms or IV
dosage forms. In addition, a rapid decline in plasma levels is
avoided since the drug is continually crossing from the oral cavity
or GI tract into the bloodstream during the length of time of
dissolution of the tablet or longer, thus providing plasma
pharmacokinetics with an extended plateau phase as compared to the
conventional immediate release oral dosage forms. Further, the
dosage forms described herein can improve treatment safety by
minimizing the potentially deleterious side effects due to the
relative reduction of the peaks and troughs in the plasma drug
pharmacokinetics, which compromise treatment safety and is typical
of currently available dosage forms.
[0377] In various embodiments bioadhesive formulations described
herein can be designed to manipulate and control the
pharmacokinetic profile of the active agent(s) described herein. As
such, the formulations can be adjusted to achieve `slow`
disintegration times (and erosion kinetic profiles) and slow drug
release and thus enable very prolonged pharmacokinetic profiles
that provide sustained drug action. Although such formulations may
be designed to still provide a fast onset, they are mostly intended
to enable the sustained drug PK and effect while maintaining the
other performance attributes of the tablet such as bioadhesion,
reproducibility of action, blunted C.sub.max, etc.
[0378] The performance and attributes of the bioadhesive
transmucosal formulations of this invention are independent of the
manufacturing process. A number of conventional, well-established
and known in the art processes can be used to manufacture the
formulations of the present invention (such as wet and dry
granulation, direct compression, etc.) without impacting the dosage
form physicochemical properties or in vivo performance.
[0379] An illustrative mathematical ratio that demonstrates the
prolonged plateau phase of the measured blood plasma levels of the
active agent(s) described herein, following administration of the
dosage forms of the invention is the term "Optimal Therapeutic
Targeting Ratio" or "OTTR", which represents the average time that
the drug is present at therapeutic levels, defined as time within
which the drug plasma concentration is maintained above 50% of
C.sub.max normalized by the drug's elimination half-life multiplied
by the ratio of the C.sub.max obtained in the dosage form of
interest over the normalized C.sub.max following IV administration
of equivalent doses. In certain embodiments the OTTR can be
calculated by the formula:
OTTR=(C.sup.IV/C.sub.max).times.(Dose/Dose.sup.IV)(Time above 50%
of C.sub.max)/(Terminal.sup.IV elimination half-life of the
drug).
[0380] In certain embodiments the OTTR is greater than about 15, or
greater than about 20, or greater than about 25, or greater than
about 30, or greater than about 40, or greater than about 50.
Administration
[0381] In certain embodiments one or more active agents described
herein (e.g., hydantoins described herein, or a tautomer(s) or
stereoisomer(s) thereof, or pharmaceutically acceptable salts or
solvates of said hydantoin(s), said stereoisomer(s), or said
tautomer(s), or analogues, derivatives, or prodrugs thereof) are
administered to a mammal in need thereof, e.g., to a mammal at risk
for or suffering from a pathology characterized by abnormal
processing of amyloid precursor proteins, a mammal at risk for
progression of MCI to Alzheimer's disease, and so forth. In certain
embodiments the active agent(s) are administered to prevent or
delay the onset of a pre-Alzheimer's cognitive dysfunction, and/or
to ameliorate one or more symptoms of a pre-Alzheimer's cognitive
dysfunction, and/or to prevent or delay the progression of a
pre-Alzheimer's condition or cognitive dysfunction to Alzheimer's
disease, and/or to promote the processing of amyloid precursor
protein (APP) by a non-amyloidogenic pathway. In certain
embodiments one or more active agent(s) are administered for the
treatment of early stage, mid stage, or late-stage Alzheimer's
disease, e.g., to reduce the severity of the disease, and/or to
ameliorate one or more symptoms of the disease, and/or to slow the
progression of the disease.
[0382] In various embodiments the active agent(s) described herein
(e.g., hydantoins described herein, or a tautomer(s) or
stereoisomer(s) thereof, or pharmaceutically acceptable salts or
solvates of said hydantoin(s), said stereoisomer(s), or said
tautomer(s), or analogues, derivatives, or prodrugs thereof) can be
administered by any of a number of routes. Thus, for example they
can be administered orally, parenterally, (intravenously (IV),
intramuscularly (IM), depo-IM, subcutaneously (SQ), and depo-SQ),
sublingually, intranasally (inhalation), intrathecally,
transdermally (e.g., via transdermal patch), topically,
ionophoretically or rectally. Typically the dosage form is selected
to facilitate delivery to the brain (e.g., passage through the
blood brain barrier). In this context it is noted that the
compounds described herein are readily delivered to the brain.
Dosage forms known to those of skill in the art are suitable for
delivery of the compound.
[0383] In various embodiments the active agent(s) are administered
in an amount/dosage regimen sufficient to exert a prophylactically
and/or therapeutically useful effect in the absence of undesirable
side effects on the subject treated (or with the presence of
acceptable levels and/or types of side effects). The specific
amount/dosage regimen will vary depending on the weight, gender,
age and health of the individual; the formulation, the biochemical
nature, bioactivity, bioavailability and the side effects of the
particular compound.
[0384] In certain embodiments the therapeutically or
prophylactically effective amount may be determined empirically by
testing the agent(s) in known in vitro and in vivo model systems
for the treated disorder. A therapeutically or prophylactically
effective dose can be determined by first administering a low dose,
and then incrementally increasing until a dose is reached that
achieves the desired effect with minimal or no undesired side
effects.
[0385] In certain embodiments, when administered orally, an
administered amount of the agent(s) described herein effective to
prevent or delay the onset of a pre-Alzheimer's cognitive
dysfunction, and/or to ameliorate one or more symptoms of a
pre-Alzheimer's cognitive dysfunction, and/or to prevent or delay
the progression of a pre-Alzheimer's condition or cognitive
dysfunction to Alzheimer's disease, and/or to promote the
processing of amyloid precursor protein (APP) by a
non-amyloidogenic pathway, and/or to treat or prevent AD ranges
from about 0.1 mg/day to about 500 mg/day or about 1,000 mg/day, or
from about 0.1 mg/day to about 200 mg/day, for example, from about
1 mg/day to about 100 mg/day, for example, from about 5 mg/day to
about 50 mg/day. In some embodiments, the subject is administered
the compound at a dose of about 0.05 to about 0.50 mg/kg, for
example, about 0.05 mg/kg, 0.10 mg/kg, 0.20 mg/kg, 0.33 mg/kg, 0.50
mg/kg. It is understood that while a patient may be started at one
dose, that dose may be varied (increased or decreased, as
appropriate) over time as the patient's condition changes.
[0386] Depending on outcome evaluations, higher doses may be used.
For example, in certain embodiments, up to as much as 1000 mg/day
can be administered, e.g., 5 mg/day, 10 mg/day, 25 mg/day, 50
mg/day, 100 mg/day, 200 mg/day, 300 mg/day, 400 mg/day, 500 mg/day,
600 mg/day, 700 mg/day, 800 mg/day, 900 mg/day or 1000 mg/day.
[0387] In various embodiments, active agent(s) described herein can
be administered parenterally, for example, by IV, IM, depo-1M, SC,
or depo-SC. In certain embodiments when administered parenterally,
a therapeutically effective amount of about 0.5 to about 100
mg/day, preferably from about 5 to about 50 mg daily can be
delivered. When a depot formulation is used for injection once a
month or once every two weeks, the dose in certain embodiments can
be about 0.5 mg/day to about 50 mg/day, or a monthly dose of from
about 15 mg to about 1,500 mg. In part because of the forgetfulness
of the patients with Alzheimer's disease, it is preferred that the
parenteral dosage form be a depo formulation.
[0388] In various embodiments, the active agent(s) described herein
can be administered sublingually. In some embodiments, when given
sublingually, the compounds and/or analogs thereof can be given one
to four times daily in the amounts described above for IM
administration.
[0389] In various embodiments, the active agent(s) described herein
can be administered intranasally. When given by this route, the
appropriate dosage forms are a nasal spray or dry powder, as is
known to those skilled in the art. In certain embodiments, the
dosage of compound and/or analog thereof for intranasal
administration is the amount described above for IM
administration.
[0390] In various embodiments, the active agent(s) described herein
can be administered intrathecally. When given by this route the
appropriate dosage form can be a parenteral dosage form as is known
to those skilled in the art. In certain embodiments, the dosage of
compound and/or analog thereof for intrathecal administration is
the amount described above for IM administration.
[0391] In certain embodiments, the active agent(s) described herein
can be administered topically. When given by this route, the
appropriate dosage form is a cream, ointment, or patch. When
administered topically, the dosage is from about 1.0 mg/day to
about 200 mg/day. Because the amount that can be delivered by a
patch is limited, two or more patches may be used. The number and
size of the patch is not important as long as a therapeutically
effective amount of compound be delivered as is known to those
skilled in the art. The compound can be administered rectally by
suppository as is known to those skilled in the art. In certain
embodiments, when administered by suppository, the therapeutically
effective amount is from about 1.0 mg to about 500 mg.
[0392] In various embodiments, the active agent(s) described herein
can be administered by implants as is known to those skilled in the
art. When administering the compound by implant, the
therapeutically effective amount is the amount described above for
depot administration.
[0393] In various embodiments, the active agent(s) described herein
thereof can be enclosed in multiple or single dose containers. The
enclosed agent(s) can be provided in kits, for example, including
component parts that can be assembled for use. For example, an
active agent in lyophilized form and a suitable diluent may be
provided as separated components for combination prior to use. A
kit may include an active agent and a second therapeutic agent for
co-administration. The active agent and second therapeutic agent
may be provided as separate component parts. A kit may include a
plurality of containers, each container holding one or more unit
dose of the compounds. The containers are preferably adapted for
the desired mode of administration, including, but not limited to
tablets, gel capsules, sustained-release capsules, and the like for
oral administration; depot products, pre-filled syringes, ampules,
vials, and the like for parenteral administration; and patches,
medipads, creams, and the like for topical administration, e.g., as
described herein.
[0394] In various embodiments the dosage forms can be administered
to the subject 1, 2, 3, or 4 times daily. In certain embodiments it
is preferred that the compound be administered either three or
fewer times, more preferably once or twice daily. In certain
embodiments, it is preferred that the agent(s) be administered in
oral dosage form.
[0395] It should be apparent to one skilled in the art that the
exact dosage and frequency of administration will depend on the
particular condition being treated, the severity of the condition
being treated, the age, weight, general physical condition of the
particular patient, and other medication the individual may be
taking as is well known to administering physicians who are skilled
in this art.
[0396] While the compositions and methods are described herein with
respect to use in humans, they are also suitable for animal, e.g.,
veterinary use. Thus certain organisms (subjects) contemplated
herein include, but are not limited to humans, non-human primates,
canines, equines, felines, porcines, ungulates, largomorphs, and
the like.
[0397] The foregoing formulations and administration methods are
intended to be illustrative and not limiting. It will be
appreciated that, using the teaching provided herein, other
suitable formulations and modes of administration can be readily
devised.
Combination Therapies
[0398] In certain embodiments, the active agent(s) described herein
(e.g., hydantoins described herein, or a tautomer(s) or
stereoisomer(s) thereof, or pharmaceutically acceptable salts or
solvates of said hydantoin(s), said stereoisomer(s), or said
tautomer(s), or analogues, derivatives, or prodrugs thereof) can be
used in combination with other therapeutic agents or approaches
used to treat or prevent diseases characterized by amyloid deposits
in the brain, including MCI and/or AD. Accordingly, in certain
embodiments, a pharmaceutical composition comprising at least
active agent described herein (e.g., a hydantoin described herein,
or a tautomer or stereoisomer thereof, or pharmaceutically
acceptable salts or solvate of said hydantoin, said stereoisomer,
or said tautomer, or an analogue, derivative, or prodrug thereof)
one together with at least one additional therapeutic agent, and a
pharmaceutically acceptable carrier or diluent is contemplated. In
certain embodiments a therapeutic or prophylactic method comprising
administering at least active agent described herein in conjunction
with at least one additional therapeutic agent is contemplated.
[0399] In certain embodiments non-limiting examples of additional
therapeutic agents include, but are not limited to disulfuram
and/or analogues thereof, honokiol and/or analogues thereof,
tropisetron and/or analogues thereof, nimetazepam and/or analogues
thereof (see, e.g., U.S. Ser. No. 13/213,960 (U.S. Patent
Publication No: US-2012-0071468-A1), and PCT/US2011/048472 (PCT
Publication No: WO 2012/024616) which are incorporated herein by
reference for the compounds described therein), tropinol-esters
and/or related esters and/or analogues thereof (see, e.g., U.S.
Ser. No. 61/514,381, which is incorporated herein by reference for
the compounds described herein), TrkA kinase inhibitors (e.g.,
ADDN-1351) and/or analogues thereof (see, e.g., U.S. Ser. No.
61/525,076, which is incorporated herein by reference for the
compounds described therein), D2 receptor agonists and
alpha1-adrenergic receptor antagonists, and APP-specific BACE
Inhibitors (ASBIs) as described and/or claimed in U.S. Ser. No.
61/728,688, filed on Nov. 20, 2012 which is incorporated herein by
reference for the active agents described herein including, but not
limited to galangin, a galangin prodrug, rutin, a rutin prodrug,
and other flavonoids and flavonoid prodrugs as described or claimed
therein.
[0400] Non-limiting examples of additional therapeutic agents
include drugs selected from the group consisting of: (a) drugs
useful for the treatment of Alzheimer's disease and/or drugs useful
for treating one or more symptoms of Alzheimer's disease, (b) drugs
useful for inhibiting the synthesis A.beta., and (c) drugs useful
for treating neurodegenerative diseases. Additional non-limiting
examples of additional therapeutic agents for use in combination
with the compounds (e.g., hydantoins) described herein include
drugs useful for the treatment, prevention, delay of onset,
amelioration of any pathology associated with A.beta. and/or a
symptom thereof. Non-limiting examples of pathologies associated
with A.beta. include: Alzheimer's disease, Down's syndrome,
Parkinson's disease, memory loss, memory loss associated with
Alzheimer's disease, memory loss associated with Parkinson's
disease, attention deficit symptoms, attention deficit symptoms
associated with Alzheimer's disease, Parkinson's disease, and/or
Down's syndrome, dementia, stroke, microgliosis and brain
inflammation, pre-senile dementia, senile dementia, dementia
associated with Alzheimer's disease, Parkinson's disease, and/or
Down's syndrome, progressive supranuclear palsy, cortical basal
degeneration, neurodegeneration, olfactory impairment, olfactory
impairment associated with Alzheimer's disease, Parkinson's
disease, and/or Down's syndrome, .beta.-amyloid angiopathy,
cerebral amyloid angiopathy, hereditary cerebral hemorrhage, mild
cognitive impairment ("MCI"), glaucoma, amyloidosis, type II
diabetes, hemodialysis complications (from .beta..sub.2
microglobulins and complications arising therefrom in hemodialysis
patients), scrapie, bovine spongiform encephalitis, traumatic brain
injury ("TBI"), and Creutzfeld-Jakob disease, comprising
administering to said patient at least one hydantoin compound
described herein, or a tautomer or isomer thereof; or
pharmaceutically acceptable salt or solvate of said compound or
said tautomer, in an amount effective to inhibit said pathology or
pathologies.
[0401] In certain embodiments such additional therapeutic agents
include, but are not limited to acetylcholinesterase inhibitors
(including without limitation, e.g., (-)-phenserine enantiomer,
tacrine, ipidacrine, galantamine, donepezil, icopezil, zanapezil,
rivastigmine, huperzine A, phenserine, physostigmine, neostigmine,
pyridostigmine, ambenonium, demarcarium, edrophonium, ladostigil
and ungeremine); NMDA receptor antagonist (including without
limitations e.g., Memantine); muscarinic receptor agonists
(including without limitation, e.g., Talsaclidine, AF-102B, AF-267B
(NGX-267)); nicotinic receptor agonists (including without
limitation, e.g., Ispronicline (AZD-3480)); beta-secretase
inhibitors (including without limitations e.g., thiazolidinediones,
including rosiglitazone and pioglitazone); gamma-secretase
inhibitors (including without limitation, e.g., semagacestat
(LY-450139), MK-0752, E-2012, BMS-708163, PF-3084014, begacestat
(GSI-953), and NIC.sub.5-15); inhibitors of A.beta. aggregation
(including without limitation, e.g., Clioquinol (PBT1), PBT2,
tramiprosate (homotaurine), Scyllo-inositol (a.k.a.,
scyllo-cyclohexanehexyl, AZD-103 and ELND-005), passive
immunotherapy with A.beta. fragments (including without limitations
e.g., Bapineuzemab) and Epigallocatechin-3-gallate (EGCg));
anti-inflammatory agents such as cyclooxygenase II inhibitors;
anti-oxidants such as Vitamin E and ginkolides; immunological
approaches, such as, for example, immunization with A.beta. peptide
or administration of anti-A.beta. peptide antibodies; statins; and
direct or indirect neurotrophic agents such as Cerebrolysin.TM.,
AIT-082 (Emilieu, 2000, Arch. Neurol. 57:454), Netrin (Luorenco
(2009) Cell Death Differ., 16: 655-663), Netrin mimetics, NGF, NGF
mimetics, BDNF and other neurotrophic agents of the future, agents
that promote neurogenesis e.g. stem cell therapy. Further
pharmacologic agents useful in the treatment or prevention diseases
characterized by amyloid deposits in the brain, including MCI
and/or AD, are described, e.g., in Mangialasche, et al. (2010)
Lancet Neurol., 9:702-716.
[0402] In certain embodiments, additional non-limiting examples of
additional therapeutic agents for use in combination with compounds
described herein include: muscarinic antagonists (e.g., m.sub.1
agonists (such as acetylcholine, oxotremorine, carbachol, or
McNa343), or m.sub.2 antagonists cholinesterase inhibitors (e.g.,
acetyl- and/or butyrylchlolinesterase inhibitors such as donepezil
(Aricept.RTM.), galantamine (Razadyne.RTM.), and rivastigimine
(Exelon.RTM.); N-methyl-D-aspartate receptor antagonists (e.g.,
NAMENDA.RTM. (memantine HCl); combinations of cholinesterase
inhibitors and N-methyl-D-aspartate receptor antagonists; gamma
secretase modulators; gamma secretase inhibitors; non-steroidal
anti-inflammatory agents; anti-inflammatory agents that can reduce
neuroinflammation; anti-amyloid antibodies (such as bapineuzemab,
Wyeth/Elan); vitamin E; nicotinic acetylcholine receptor agonists;
CB1 receptor inverse agonists or CB1 receptor antagonists;
antibiotics; growth hormone secretagogues; histamine H3
antagonists; AMPA agonists; PDE4 inhibitors; GABA.sub.A inverse
agonists; inhibitors of amyloid aggregation; glycogen synthase
kinase beta inhibitors; promoters of alpha secretase activity;
PDE-10 inhibitors; Tau kinase inhibitors (e.g., GSK3beta
inhibitors, cdk5 inhibitors, or ERK inhibitors); Tau aggregation
inhibitors (e.g., REMBER.RTM.; RAGE inhibitors (e.g., TTP 488
(PF-4494700)); anti-A.beta. vaccine; APP ligands; agents that
upregulate insulin, cholesterol lowering agents such as HMG-CoA
reductase inhibitors (for example, statins such as Atorvastatin,
Fluvastatin, Lovastatin, Mevastatin, Pitavastatin, Pravastatin,
Rosuvastatin, Simvastatin) and/or cholesterol absorption inhibitors
(such as Ezetimibe), or combinations of HMG-CoA reductase
inhibitors and cholesterol absorption inhibitors (such as, for
example, VYTORIN.RTM.); fibrates (such as, for example, clofibrate,
Clofibride, Etofibrate, and Aluminium Clofibrate); combinations of
fibrates and cholesterol lowering agents and/or cholesterol
absorption inhibitors; nicotinic receptor agonists; niacin;
combinations of niacin and cholesterol absorption inhibitors and/or
cholesterol lowering agents (e.g., SIMCOR.RTM. (niacin/simvastatin,
available from Abbott Laboratories, Inc.); LXR agonists; LRP
mimics; H3 receptor antagonists; histone deacetylase inhibitors;
hsp90 inhibitors; 5-HT4 agonists (e.g., PRX-03140 (Epix
Pharmaceuticals)); 5-HT6 receptor antagonists; mGluR1 receptor
modulators or antagonists; mGluR5 receptor modulators or
antagonists; mGluR2/3 antagonists; Prostaglandin EP2 receptor
antagonists; PAI-1 inhibitors; agents that can induce Abeta efflux
such as gelsolin; Metal-protein attenuating compound (e.g., PBT2);
and GPR3 modulators; and antihistamines such as Dimebolin (e.g.,
DIMEBON.RTM., Pfizer).
[0403] Accordingly certain embodiments provide a pharmaceutical
composition comprising an effective amount of one or more
hydantoins described herein and an additional therapeutic agent,
and/or a method of treatment or prophylaxis comprising
administration of one or more hydantoins described herein in
conjunction with an additional therapeutic agent where the
therapeutic agent in the formulation and/or method is disulfuram
and/or analogues thereof (see, e.g., U.S. Ser. No. 13/213,960 (U.S.
Patent Publication No: US-2012-0071468-A1), and PCT/US2011/048472
(PCT Publication No: WO 2012/024616)).
[0404] Certain embodiments provide a pharmaceutical composition
comprising an effective amount of one or more hydantoins described
herein and an additional therapeutic agent, and/or a method of
treatment or prophylaxis comprising administration of one or more
hydantoins described herein in conjunction with an additional
therapeutic agent where the therapeutic agent in the formulation
and/or method is honokiol and/or analogues thereof (see, e.g., U.S.
Ser. No. 13/213,960 (U.S. Patent Publication No:
US-2012-0071468-A1), and PCT/US2011/048472 (PCT Publication No: WO
2012/024616)).
[0405] Certain embodiments provide a pharmaceutical composition
comprising an effective amount of one or more hydantoins described
herein and an additional therapeutic agent, and/or a method of
treatment or prophylaxis comprising administration of one or more
hydantoins described herein in conjunction with an additional
therapeutic agent where the therapeutic agent in the formulation
and/or method is tropisetron and/or analogues thereof (see, e.g.,
U.S. Ser. No. 13/213,960 (U.S. Patent Publication No:
US-2012-0071468-A1), and PCT/US2011/048472 (PCT Publication No: WO
2012/024616)).
[0406] Certain embodiments provide a pharmaceutical composition
comprising an effective amount of one or more hydantoins described
herein and an additional therapeutic agent, and/or a method of
treatment or prophylaxis comprising administration of one or more
hydantoins described herein in conjunction with an additional
therapeutic agent where the therapeutic agent in the formulation
and/or method is tropisetron.
[0407] Certain embodiments provide a pharmaceutical composition
comprising an effective amount of one or more hydantoins described
herein and an additional therapeutic agent, and/or a method of
treatment or prophylaxis comprising administration of one or more
hydantoins described herein in conjunction with an additional
therapeutic agent where the therapeutic agent in the formulation
and/or method is nimetazepam and/or analogues thereof (see, e.g.,
U.S. Ser. No. 13/213,960 (U.S. Patent Publication No:
US-2012-0071468-A1), and PCT/US2011/048472 (PCT Publication No: WO
2012/024616)).
[0408] Certain embodiments provide a pharmaceutical composition
comprising an effective amount of one or more hydantoins described
herein and an additional therapeutic agent, and/or a method of
treatment or prophylaxis comprising administration of one or more
hydantoins described herein in conjunction with an additional
therapeutic agent where the therapeutic agent in the formulation
and/or method is a tropinol ester or related ester (see, e.g., U.S.
Ser. No. 61/514,381).
[0409] Certain embodiments provide a pharmaceutical composition
comprising an effective amount of one or more hydantoins described
herein and an additional therapeutic agent, and/or a method of
treatment or prophylaxis comprising administration of one or more
hydantoins described herein in conjunction with an additional
therapeutic agent where the therapeutic agent in the formulation
and/or method is a TrkA kinase inhibitor (e.g., ADDN-1351) and/or
analogues thereof (see, e.g., U.S. Ser. No. 61/525,076).
[0410] Certain embodiments provide a pharmaceutical composition
comprising an effective amount of one or more hydantoins described
herein and an additional therapeutic agent, and/or a method of
treatment or prophylaxis comprising administration of one or more
hydantoins described herein in conjunction with an additional
therapeutic agent where the therapeutic agent in the formulation
and/or method is a D2 receptor agonists and/or an alpha1-adrenergic
receptor antagonists.
[0411] Certain embodiments provide a pharmaceutical composition
comprising an effective amount of one or more hydantoins described
herein and an additional therapeutic agent, and/or a method of
treatment or prophylaxis comprising administration of one or more
hydantoins described herein in conjunction with an additional
therapeutic agent where the therapeutic agent in the formulation
and/or method is an ASBIs as described and/or claimed in U.S. Ser.
No. 61/728,688, filed on Nov. 20, 2012 which is incorporated herein
by reference for the active agents described herein including, but
not limited to galangin, a galangin prodrug, rutin, a, and other
flavonoids as described or claimed therein.
[0412] Certain embodiments provide a pharmaceutical composition
comprising an effective amount of one or more hydantoins described
herein and an additional therapeutic agent, and/or a method of
treatment or prophylaxis comprising administration of one or more
hydantoins described herein in conjunction with an additional
therapeutic agent where the therapeutic agent in the formulation
and/or method is one or more cholinesterase inhibitors (e.g.,
acetyl- and/or butyrylchlolinesterase inhibitors).
[0413] Certain embodiments provide a pharmaceutical composition
comprising an effective amount of one or more hydantoins described
herein and an additional therapeutic agent, and/or a method of
treatment or prophylaxis comprising administration of one or more
hydantoins described herein in conjunction with an additional
therapeutic agent where the therapeutic agent in the formulation
and/or method is one or more muscarinic antagonists (e.g., m.sub.1
agonists or m.sub.2 antagonists).
[0414] Certain embodiments provide a pharmaceutical composition
comprising an effective amount of one or more hydantoins described
herein and an additional therapeutic agent, and/or a method of
treatment or prophylaxis comprising administration of one or more
hydantoins described herein in conjunction with an additional
therapeutic agent where the therapeutic agent in the formulation
and/or method is one or more compounds selected from the group
consisting of cholinesterase inhibitors (such as, for example,
(.+-.)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidi-
nyl]methyl-1]-1H-inden-1-one hydrochloride, i.e, donepezil
hydrochloride, available as the ARICEPT.RTM. brand of donepezil
hydrochloride), N-methyl-D-aspartate receptor inhibitors (such as,
for example, Namenda.RTM. (memantine HCl)); anti-amyloid antibodies
(such as bapineuzumab, Wyeth/Elan), gamma secretase inhibitors,
gamma secretase modulators, and beta secretase inhibitors other
than the hydantoins described herein.
Additional Indications.
[0415] Use of Hydantoin in Age Related Macular Degeneration and
Glaucoma.
[0416] While in various embodiments, the use of APP-Binding-BACE
Inhibitors (ABBIs), e.g., the various hydantoins described herein,
are contemplated for the preventing or delaying the onset of a
pre-Alzheimer's condition and/or cognitive dysfunction, and/or
ameliorating one or more symptoms of a pre-Alzheimer's condition
and/or cognitive dysfunction, or preventing or delaying the
progression of a pre-Alzheimer's condition or cognitive dysfunction
to Alzheimer's disease, and/or for the treatment of Alzheimer's
disease, other uses of ABBIs are also contemplated. In particular,
in certain embodiments, the use of ABBIs is contemplated for the
treatment and/or prophylaxis of age-related macular degeneration
and/or glaucoma.
[0417] Without being bound to a particular theory, it is believed
that abnormal extracellular deposition of proteins may contribute
to age-related macular degeneration (AMD) pathogenesis and
progression, which is also the case in Alzheimer's disease and
atherosclerosis. In both conditions, the protein deposits contain
many shared constituents such as apoE, complement, and A.beta.
peptides. For instance, in human AMD, A.beta. peptide deposition is
associated with drusen, where it accumulates and colocalizes with
activated complement components (Anderson et al. (2004) Exp. Eye.
Res., 78:243-256; Dentchev et al. (2003) Mol. Vis., 9: 184-190;
Johnson et al. (2002) Proc Natl Acad Sci USA 99: 11830-11835.).
Luibl et al. (2006) J. Clin. Invest., 116: 378-385, showed the
presence of potentially toxic amyloid oligomers in drusen, sub-RPE
basal deposits, and RPE of human donor eyes using an antibody that
specifically recognizes the oligomeric form of A.beta.. These
A.beta. oligomers were not detected in control age-matched donor
eyes without drusen. Isas et al. (2010) Invest. Ophthalmol. Vis.
Sci., 51: 1304-1310, also detected soluble as well as mature
A.beta. fibrils in drusen. Collectively, these findings implicate
A.beta. in the pathogenesis of AMD. In addition, A.beta. peptide
has been detected in sub-RPE basal deposits and neovascular lesions
in a murine model of AMD (Ding et al. (2008) Vision Res., 48:
339-345; Malek et al. (2005) Proc Natl Acad Sci USA, 102:
11900-11905). In this model, aged human APOE4-targeted replacement
mice (APOE4 mice) fed a high-fat, cholesterol-enriched (HFC) diet
(APOE4-HFC mice) exhibit morphologic hallmarks observed in both dry
and wet AMD. These hallmarks include thick diffuse sub-RPE
deposits, lipid- and protein-containing focal drusen-like deposits,
thickening of Bruch's membrane, patchy regions of RPE atrophy
opposed to areas of photoreceptor degeneration, and CNV (Malek et
al. (2005) Proc Natl Acad Sci USA, 102: 11900-11905). It is
believed that, in the APOE4-HFC mouse model of AMD, A.beta.
accumulation provokes damage at the level of the RPE/choroid and
has previously been shown that systemic administration of
anti-A.beta.40-specific antibodies can partially attenuate the
decline in visual function exhibited in this model (Ding et al.
(2008) Vision Res., 48: 339-345). It has also been demonstrated
that anti-A.beta. immunotherapy simultaneously targeting both
A.beta.40 and A.beta.42 blocks histopathologic changes and
completely protects visual function in APOE4-HFC mice (Ding et al.
(2011) Proc. Nat'l. Acad. Sci. U.S.A., 108(28): E279-E287).
[0418] Without being bound by a particular theory, it is believed
that APP processing to A.beta. in the eye occurs by the activities
of BACE and .gamma.-secretase in the retina and retinal pigmented
epithelial (RPE) cell layers and that sAPP.alpha. and A.beta. are
secreted into the vitreous humor (see, e.g., (Prakasam et al.
(2008) J. Alzh. Dis., 20: 1243-1253). A.beta. is further
transported into the aqueous humor where it is readily
measured.
[0419] In view of these findings, it is believe that ABBIs, e.g.,
the hydantoins described herein, can find use in the treatment or
prophylaxis of age-related macular degeneration (AMD) and/or
glaucoma. Accordingly, it is believed that ABBIs can be
administered to a subject to slow or prevent the appearance of AMD
(and/or glaucoma), and/or to reduce one or more symptoms of AMD,
and/or to slow, stop, or reverse progression of the disease. In
various embodiments one or more ABBIs (e.g., any one or more of the
active agent(s) described herein) are administered to a subject
(e.g., a human, a non-human mammal) for these purposes. As
described above, in various embodiments, the ABBI is administered
via a route selected from the group consisting of oral delivery,
isophoretic delivery, transdermal delivery, parenteral delivery,
aerosol administration, administration via inhalation, intravenous
administration, and rectal administration.
[0420] In certain embodiments, the administration is directly to
the eye. Thus for example, in certain embodiments, the agent(s) can
be administered to the eye in the form of eye drops, via
intraocular injection, and the like.
[0421] Typically the ABBIs are administered in an effective amount
for the treatment and/or prophylaxis of AMD or glaucoma, where the
effective amount will vary by the modality of administration. In
certain embodiments effective amount is an amount sufficient to
mitigating in a mammal one or more symptoms associated with
age-related macular degeneration (AMD). In certain embodiments the
effective amount is an amount, an amount sufficient to reduce the
risk or delaying the onset, and/or reduce the ultimate severity of
a AMD disease (or glaucoma) characterized by reduction of A.beta.
in the vitreous and/or aqueous humor and/or the amyloid deposits on
the retina and/or the RPE cell layer.
Assay Systems to Evaluate APP Processing
[0422] Without being bound to a particular theory, it is believed
that the active agent(s) described herein (e.g., ABBIs such as the
hydantoins described herein) promote processing of APP by the
nonamyloidogenic pathway and/or reduce or inhibits processing of
APP by the amyloidogenic pathway. In the nonamyloidogeic pathway,
APP is first cleaved by .alpha.-secretase within the A.beta.
sequence, releasing the APPs.alpha. ectodomain ("sAPP.alpha."). In
contrast, the amyloidogenic pathway is initiated when
.beta.-secretase cleaves APP at the amino terminus of the A.beta.,
thereby releasing the APPs.beta. ectodomain ("sAPP.beta."). APP
processing by the nonamyloidogenic and amyloidogenic pathways is
known in the art and reviewed, e.g., by Xu (2009) J Alzheimers
Dis., 16(2): 211-224, and De Strooper, et al. (2010 Nat Rev Neurol
6(2): 99-107.
[0423] One method to evaluate the efficacy of the active agent(s)
is to determine a reduction or elimination in the level of APP
processing by the amyloidogenic pathway, e.g., a reduction or
elimination in the level of APP processing by .beta.-secretase
cleavage in response to the administration of the agent(s) of
interest. Assays for determining the extent of APP cleavage at the
.beta.-secretase cleavage site are well known in the art.
Illustrative assays are described, for example, in U.S. Pat. Nos.
5,744,346 and 5,942,400. Kits for determining the presence and
levels in a biological sample of sAPP.alpha. and sAPP.beta., as
well as APPneo and A.beta. commercially available, e.g., from
PerkinElmer.
[0424] ABBI Assay.
[0425] APP Binding BACE Inhibitor (ABBI) activity of any of the
compounds described herein can readily be verified using, for
example, assays described herein. Basically, in certain embodiments
a pair the assays are utilized to identify ABBI compounds that
inhibit BACE cleavage of the MBP-C125 APP substrate, resulting in
the inhibition of the production of C99 and the .beta.-site peptide
substrate (P5-P5') and also interacts with APP, e.g., as measured
by surface plasmon resonance (SPR) analysis.
[0426] In one illustrative embodiment, an MBP-C125 APP695 wt fusion
protein can be used as one of the substrates and the second
substrate can be the commercially available P5-P5' fluorescence
substrate. Each of these substrates is incubated with recombinant
BACE (R&D (cat#931-AS-050) in, for example, a 96 well plate
format. For the MBP-C125 substrate the C-99 product from the BACE
cleavage can be measured using an AlphaLisa assay as a readout. For
the P5-5' substrate the loss of fluorescence upon BACE cleavage can
be used as the readout. For the SPR assay the binding analysis of
the hydantoins to fragments of the ectodomain of APP (eAPP) that
are recombinantly prepared (Libeu et al. (2012) PLoS ONE 7(6):
e40027) would be done. An ABBI would inhibit the BACE cleavage of
the MBP-C125 and/or the fluorescence substrate and would also bind
to the ectodomain of APP such as the APP.sub.230-624 fragment.
[0427] Other Cell Free Assays
[0428] Illustrative assays that can be used to demonstrate the
inhibitory activity of the active agent(s) are described, for
example, in WO 2000/017369, WO 2000/0003819, and U.S. Pat. Nos.
5,942,400 and 5,744,346. Such assays can be performed in cell-free
incubations or in cellular incubations using cells expressing an
alpha-secretase and/or beta-secretase and an APP substrate having a
alpha-secretase and beta-secretase cleavage sites.
[0429] In one illustrative embodiment, the agent(s) of interest are
contacted with an APP substrate containing alpha-secretase and
beta-secretase cleavage sites of APP, for example, a complete APP
or variant, an APP fragment, or a recombinant or synthetic APP
substrate containing the amino acid sequence: KM-DA or NL-DA
(APP-SW), is incubated in the presence of an alpha-secretase and/or
beta-secretase enzyme, a fragment thereof, or a synthetic or
recombinant polypeptide variant having alpha-secretase or
beta-secretase activity and effective to cleave the alpha-secretase
or beta-secretase cleavage sites of APP, under incubation
conditions suitable for the cleavage activity of the enzyme.
Agent(s) having the desired activity reduce or prevent cleavage of
the APP substrate. Suitable substrates optionally include
derivatives that may be fusion proteins or peptides that contain
the substrate peptide and a modification useful to facilitate the
purification or detection of the peptide or its alpha-secretase
and/or beta-secretase cleavage products. Useful modifications
include the insertion of a known antigenic epitope for antibody
binding; the linking of a label or detectable moiety, the linking
of a binding substrate, and the like.
[0430] Suitable incubation conditions for a cell-free in vitro
assay include, for example: approximately 200 nanomolar to 10
micromolar substrate, approximately 10 to 200 picomolar enzyme, and
approximately 0.1 nanomolar to 10 micromolar of the agent(s), in
aqueous solution, at an approximate pH of 4-7, at approximately
37.degree. C., for a time period of approximately 10 minutes to 3
hours. These incubation conditions are illustrative only, and can
be varied as required for the particular assay components and/or
desired measurement system. Optimization of the incubation
conditions for the particular assay components should account for
the specific alpha-secretase and/or beta-secretase enzyme used and
its pH optimum, any additional enzymes and/or markers that might be
used in the assay, and the like. Such optimization is routine and
will not require undue experimentation.
[0431] Another illustrative assay utilizes a fusion peptide having
maltose binding protein (MBP) fused to the C-terminal 125 amino
acids of APP-SW. The MBP portion is captured on an assay substrate
by anti-MBP capture antibody. Incubation of the captured fusion
protein in the presence of alpha-secretase and/or beta-secretase
results in cleavage of the substrate at the alpha-secretase and/or
beta-secretase cleavage sites, respectively. This system can be
used to screen for the inhibitory activity of the agent(s) of
interest. Analysis of the cleavage activity can be, for example, by
immunoassay of cleavage products. One such immunoassay detects a
unique epitope exposed at the carboxy terminus of the cleaved
fusion protein, for example, using the antibody SW192. This assay
is described, for example, in U.S. Pat. No. 5,942,400.
[0432] Cellular Assays
[0433] Numerous cell-based assays can be used to evaluate the
activity of agent(s) of interest on relative alpha-secretase
activity to beta-secretase activity and/or processing of APP to
release amyloidogenic versus non-amyloidogenic A.beta. oligomers.
Contact of an APP substrate with an alpha-secretase and/or
beta-secretase enzyme within the cell and in the presence or
absence of the agent(s) can be used to demonstrate alpha-secretase
promoting and/or beta-secretase inhibitory activity of the
agent(s). Preferably, the assay in the presence of the agent(s)
provides at least about 30%, most preferably at least about 50%
inhibition of the enzymatic activity, as compared with a
non-inhibited control.
[0434] In one embodiment, cells that naturally express
alpha-secretase and/or beta-secretase are used. Alternatively,
cells are modified to express a recombinant alpha-secretase and/or
beta-secretase or synthetic variant enzymes, as discussed above.
The APP substrate may be added to the culture medium and is
preferably expressed in the cells. Cells that naturally express
APP, variant or mutant forms of APP, or cells transformed to
express an isoform of APP, mutant or variant APP, recombinant or
synthetic APP, APP fragment, or synthetic APP peptide or fusion
protein containing the alpha-secretase and/or beta-secretase APP
cleavage sites can be used, provided that the expressed APP is
permitted to contact the enzyme and enzymatic cleavage activity can
be analyzed.
[0435] Human cell lines that normally process A.beta. from APP
provide a useful means to assay inhibitory activities of the
agent(s). Production and release of A.beta. and/or other cleavage
products into the culture medium can be measured, for example by
immunoassay, such as Western blot or enzyme-linked immunoassay
(EIA) such as by ELISA.
[0436] Cells expressing an APP substrate and an active
alpha-secretase and/or beta-secretase can be incubated in the
presence of the agents to demonstrate relative enzymatic activity
of the alpha-secretase and/or beta-secretase as compared with a
control. Relative activity of the alpha-secretase to the
beta-secretase can be measured by analysis of one or more cleavage
products of the APP substrate. For example, inhibition of
beta-secretase activity against the substrate APP would be expected
to decrease release of specific beta-secretase induced APP cleavage
products such as A.beta. (e.g., A.beta.40 or A.beta.42), sAPP.beta.
and APPneo. Promotion or enhancement of alpha-secretase activity
against the substrate APP would be expected to increase release of
specific alpha-secretase induced APP cleavage products such as
sAPP.alpha. and p3 peptide.
[0437] Although both neural and non-neural cells process and
release A.beta., levels of endogenous beta-secretase activity are
low and often difficult to detect by EIA. The use of cell types
known to have enhanced beta-secretase activity, enhanced processing
of APP to A.beta., and/or enhanced production of A.beta. are
therefore preferred. For example, transfection of cells with the
Swedish Mutant form of APP (APP-SW); with the Indiana Mutant form
(APP-IN); or with APP-SW-IN provides cells having enhanced
beta-secretase activity and producing amounts of A.beta. that can
be readily measured.
[0438] In such assays, for example, the cells expressing APP,
alpha-secretase and/or beta-secretase are incubated in a culture
medium under conditions suitable for alpha-secretase and/or
beta-secretase enzymatic activity at its cleavage site on the APP
substrate. On exposure of the cells to the agent(s), the amount of
A.beta. released into the medium and/or the amount of CTF99
fragments of APP in the cell lysates is reduced as compared with
the control. The cleavage products of APP can be analyzed, for
example, by immune reactions with specific antibodies, as discussed
above.
[0439] In certain embodiments, preferred cells for analysis of
alpha-secretase and/or beta-secretase activity include primary
human neuronal cells, primary transgenic animal neuronal cells
where the transgene is APP, and other cells such as those of a
stable 293 cell line expressing APP, for example, APP-SW.
[0440] In Vivo Assays: Animal Models
[0441] Various animal models can be used to analyze the activity of
agent(s) of interest on relative alpha-secretase and/or
beta-secretase activity and/or processing of APP to release
A.beta.. For example, transgenic animals expressing APP substrate,
alpha-secretase and/or beta-secretase enzyme can be used to
demonstrate inhibitory activity of the agent(s). Certain transgenic
animal models have been described, for example, in U.S. Pat. Nos.
5,877,399; 5,612,486; 5,387,742; 5,720,936; 5,850,003; 5,877,015,
and 5,811,633, and in Ganes et al. (1995) Nature 373: 523.
Preferred are animals that exhibit characteristics associated with
the pathophysiology of AD. Administration of the agent(s) to the
transgenic mice described herein provides an alternative method for
demonstrating the inhibitory activity of the agent(s).
Administration of the agent(s) in a pharmaceutically effective
carrier and via an administrative route that reaches the target
tissue in an appropriate therapeutic amount is also preferred.
[0442] Inhibition of beta-secretase mediated cleavage of APP at the
beta-secretase cleavage site and of A.beta. release can be analyzed
in these animals by measure of cleavage fragments in the animal's
body fluids such as cerebral fluid or tissues. Likewise, promotion
or enhancement of alpha-secretase mediated cleavage of APP at the
alpha-secretase cleavage site and of release of sAPP.alpha. can be
analyzed in these animals by measure of cleavage fragments in the
animal's body fluids such as cerebral fluid or tissues. In certain
embodiments, analysis of brain tissues for A.beta. deposits or
plaques is preferred.
[0443] On contacting an APP substrate with an alpha-secretase
and/or beta-secretase enzyme in the presence of the agent(s) under
conditions sufficient to permit enzymatic mediated cleavage of APP
and/or release of A.beta. from the substrate, desirable agent(s)
are effective to reduce beta-secretase-mediated cleavage of APP at
the beta-secretase cleavage site and/or effective to reduce
released amounts of A.beta.. The agent(s) are also preferably
effective to enhance alpha-secretase-mediated cleavage of APP at
the alpha-secretase cleavage site and to increase released amounts
of sAPP.alpha.. Where such contacting is the administration of the
agent(s) to an animal model, for example, as described above, the
agent(s) is effective to reduce A.beta. deposition in brain tissues
of the animal, and to reduce the number and/or size of beta amyloid
plaques. Where such administration is to a human subject, the
agent(s) is effective to inhibit or slow the progression of disease
characterized by enhanced amounts of A.beta., to slow the
progression of AD in the, and/or to prevent onset or development of
AD in a patient at risk for the disease.
[0444] Methods of Monitoring Clinical Efficacy
[0445] In various embodiments, the effectiveness of treatment can
be determined by comparing a baseline measure of a parameter of
disease before administration of the agent(s) (e.g., hydantoins
described herein, or a tautomer(s) or stereoisomer(s) thereof, or
pharmaceutically acceptable salts or solvates of said hydantoin(s),
said stereoisomer(s), or said tautomer(s), or analogues,
derivatives, or prodrugs thereof) is commenced to the same
parameter one or more time points after the agent(s) or analog has
been administered. One illustrative parameter that can be measured
is a biomarker (e.g., a peptide oligomer) of APP processing. Such
biomarkers include, but are not limited to increased levels of
sAPP.alpha., p3 (A.beta.17-42 or A.beta.17-40), sAPP.beta., soluble
A.beta.40, and/or soluble A.beta.42 in the blood, plasma, serum,
urine, mucous or cerebrospinal fluid (CSF). Detection of increased
levels of sAPP.alpha. and/or p3, and decreased levels of sAPP.beta.
and/or APPneo is an indicator that the treatment is effective.
Conversely, detection of decreased levels of sAPP.alpha. and/or p3,
and/or increased levels of sAPP.beta., APPneo, Tau or phospho-Tau
(pTau) is an indicator that the treatment is not effective.
[0446] Another parameter to determine effectiveness of treatment is
the level of amyloid plaque deposits in the brain. Amyloid plaques
can be determined using any method known in the art, e.g., as
determined by CT, PET, PIB-PET and/or MRI. Administration of the
agent(s)) (e.g., hydantoins described herein, or a tautomer(s) or
stereoisomer(s) thereof, or pharmaceutically acceptable salts or
solvates of said hydantoin(s), said stereoisomer(s), or said
tautomer(s), or analogues, derivatives, or prodrugs thereof) can
result in a reduction in the rate of plaque formation, and even a
retraction or reduction of plaque deposits in the brain.
Effectiveness of treatment can also be determined by observing a
stabilization and/or improvement of cognitive abilities of the
subject. Cognitive abilities can be evaluated using any
art-accepted method, including for example, Clinical Dementia
Rating (CDR), the mini-mental state examination (MMSE) or Folstein
test, evaluative criteria listed in the DSM-IV (Diagnostic and
Statistical Manual of Mental Disorders, Fourth Edition) or DSM-V,
and the like.
[0447] Clinical efficacy can be monitored using any method known in
the art. Measurable biomarkers to monitor efficacy include, but are
not limited to, monitoring blood, plasma, serum, urine, mucous or
cerebrospinal fluid (CSF) levels of sAPP.alpha., sAPP.beta.,
A.beta.42, A.beta.40, APPneo and p3 (e.g., A.beta.17-42 or
A.beta.17-40). Detection of increased levels of sAPP.alpha. and/or
p3, and decreased levels of sAPP.beta. and/or APPneo are indicators
that the treatment or prevention regime is efficacious. Conversely,
detection of decreased levels of sAPP.alpha. and/or p3, and
increased levels of sAPP.beta. and/or APPneo are indicators that
the treatment or prevention regime is not efficacious. Other
biomarkers include Tau and phospho-Tau (pTau). Detection of
decreased levels of Tau and pTau are indicators that the treatment
or prevention regime is efficacious.
[0448] Efficacy can also be determined by measuring amyloid plaque
load in the brain. The treatment or prevention regime is considered
efficacious when the amyloid plaque load in the brain does not
increase or is reduced. Conversely, the treatment or prevention
regime is considered inefficacious when the amyloid plaque load in
the brain increases. Amyloid plaque load can be determined using
any method known in the art, e.g., including CT, PET, PIB-PET
and/or MRI.
[0449] Efficacy can also be determined by measuring the cognitive
abilities of the subject. Cognitive abilities can be measured using
any method known in the art. Illustrative tests include assigning a
Clinical Dementia Rating (CDR) score or applying the mini mental
state examination (MMSE) (Folstein, et al., Journal of Psychiatric
Research 12 (3): 189-98). Subjects who maintain the same score or
who achieve an improved score, e.g., when applying the CDR or MMSE,
indicate that the treatment or prevention regime is efficacious.
Conversely, subjects who receive a score indicating diminished
cognitive abilities, e.g., when applying the CDR or MMSE, indicate
that the treatment or prevention regime has not been
efficacious.
[0450] In certain embodiments, the monitoring methods can entail
determining a baseline value of a measurable biomarker or parameter
(e.g., amyloid plaque load or cognitive abilities) in a subject
before administering a dosage of the agent(s), and comparing this
with a value for the same measurable biomarker or parameter after
treatment.
[0451] In other methods, a control value (e.g., a mean and standard
deviation) of the measurable biomarker or parameter is determined
for a control population. In certain embodiments, the individuals
in the control population have not received prior treatment and do
not have AD, MCI, nor are at risk of developing AD or MCI. In such
cases, if the value of the measurable biomarker or clinical
parameter approaches the control value, then treatment is
considered efficacious. In other embodiments, the individuals in
the control population have not received prior treatment and have
been diagnosed with AD or MCI. In such cases, if the value of the
measurable biomarker or clinical parameter approaches the control
value, then treatment is considered inefficacious.
[0452] In other methods, a subject who is not presently receiving
treatment but has undergone a previous course of treatment is
monitored for one or more of the biomarkers or clinical parameters
to determine whether a resumption of treatment is required. The
measured value of one or more of the biomarkers or clinical
parameters in the subject can be compared with a value previously
achieved in the subject after a previous course of treatment.
Alternatively, the value measured in the subject can be compared
with a control value (mean plus standard deviation/ANOVA)
determined in population of subjects after undergoing a course of
treatment. Alternatively, the measured value in the subject can be
compared with a control value in populations of prophylactically
treated subjects who remain free of symptoms of disease, or
populations of therapeutically treated subjects who show
amelioration of disease characteristics. In such cases, if the
value of the measurable biomarker or clinical parameter approaches
the control value, then treatment is considered efficacious and
need not be resumed. In all of these cases, a significant
difference relative to the control level (e.g., more than a
standard deviation) is an indicator that treatment should be
resumed in the subject.
[0453] In certain embodiments the tissue sample for analysis is
typically blood, plasma, serum, urine, mucous or cerebrospinal
fluid from the subject.
Kits.
[0454] In various embodiments, the active agent(s) (e.g.,
hydantoins) described herein thereof can be enclosed in multiple or
single dose containers. The enclosed agent(s) can be provided in
kits, for example, including component parts that can be assembled
for use. For example, an active agent in lyophilized form and a
suitable diluent may be provided as separated components for
combination prior to use. A kit may include an active agent and a
second therapeutic agent for co-administration. The active agent
and second therapeutic agent may be provided as separate component
parts. A kit may include a plurality of containers, each container
holding one or more unit dose of the compounds. The containers are
preferably adapted for the desired mode of administration,
including, but not limited to tablets, gel capsules,
sustained-release capsules, and the like for oral administration;
depot products, pre-filled syringes, ampules, vials, and the like
for parenteral administration; and patches, medipads, creams, and
the like for topical administration, e.g., as described herein.
[0455] In certain embodiments, a kit is provided where the kit
comprises one or more hydantoin compounds described herein, or a
tautomer or stereoisomer thereof, or pharmaceutically acceptable
salt or solvate of said compound, said stereoisomer, or said
tautomer, preferably provided as a pharmaceutical composition and
in a suitable container or containers and/or with suitable
packaging; optionally one or more additional active agents, which
if present are preferably provided as a pharmaceutical composition
and in a suitable container or containers and/or with suitable
packaging; and optionally instructions for use, for example written
instructions on how to administer the compound or compositions.
[0456] In another embodiment, a kit is provided that comprises a
single container or multiple containers: (a) a pharmaceutically
acceptable composition comprising one or more compounds of claim 1
and/or any of compounds 1-10 shown in FIGS. 1 and 2, or a tautomer
or stereoisomer thereof, or pharmaceutically acceptable salt or
solvate of said compound, said stereoisomer, or said tautomer,
optionally a pharmaceutically acceptable composition comprising one
or more additional therapeutic agents; and optionally instructions
for use their use. The kit may optionally comprise labeling (e.g.,
instructional materials) appropriate to the intended use or
uses.
[0457] As with any pharmaceutical product, the packaging
material(s) and/or container(s) are designed to protect the
stability of the product during storage and shipment. In addition,
the kits can include instructions for use or other informational
material that can advise the user such as, for example, a
physician, technician or patient, regarding how to properly
administer the composition(s) as prophylactic, therapeutic, or
ameliorative treatment of the disease of concern. In some
embodiments, instructions can indicate or suggest a dosing regimen
that includes, but is not limited to, actual doses and monitoring
procedures.
[0458] In some embodiments, the instructions can include
informational material indicating that the administering of the
compositions can result in adverse reactions including but not
limited to allergic reactions such as, for example, anaphylaxis.
The informational material can indicate that allergic reactions may
exhibit only as mild pruritic rashes or may be severe and include
erythroderma, vasculitis, anaphylaxis, Steven-Johnson syndrome, and
the like. In certain embodiments the informational material(s) may
indicate that anaphylaxis can be fatal and may occur when any
foreign protein is introduced into the body. In certain embodiments
the informational material may indicate that these allergic
reactions can manifest themselves as urticaria or a rash and
develop into lethal systemic reactions and can occur soon after
exposure such as, for example, within 10 minutes. The informational
material can further indicate that an allergic reaction may cause a
subject to experience paresthesia, hypotension, laryngeal edema,
mental status changes, facial or pharyngeal angioedema, airway
obstruction, bronchospasm, urticaria and pruritus, serum sickness,
arthritis, allergic nephritis, glomerulonephritis, temporal
arthritis, eosinophilia, or a combination thereof.
[0459] While the instructional materials typically comprise written
or printed materials they are not limited to such. Any medium
capable of storing such instructions and communicating them to an
end user is contemplated herein. Such media include, but are not
limited to electronic storage media (e.g., magnetic discs, tapes,
cartridges, chips), optical media (e.g., CD ROM), and the like.
Such media may include addresses to internet sites that provide
such instructional materials.
[0460] In some embodiments, the kits can comprise one or more
packaging materials such as, for example, a box, bottle, tube,
vial, container, sprayer, insufflator, intravenous (I.V.) bag,
envelope, and the like; and at least one unit dosage form of an
agent comprising active agent(s) described herein and a packaging
material. In some embodiments, the kits also include instructions
for using the composition as prophylactic, therapeutic, or
ameliorative treatment for the disease of concern.
[0461] In some embodiments, the articles of manufacture can
comprise one or more packaging materials such as, for example, a
box, bottle, tube, vial, container, sprayer, insufflator,
intravenous (I.V.) bag, envelope, and the like; and a first
composition comprising at least one unit dosage form of an agent
comprising one or more hydantoins described herein, or a
tautomer(s) or stereoisomer(s) thereof, or pharmaceutically
acceptable salts or solvates of said hydantoin(s), said
stereoisomer(s), or said tautomer(s), or analogues, derivatives, or
prodrugs thereof within the packaging material, along with a second
composition comprising a second agent such as, for example, an
agent used in the treatment and/or prophylaxis of Alzheimer's
disease (e.g., as described herein), or any prodrugs, codrugs,
metabolites, analogs, homologues, congeners, derivatives, salts and
combinations thereof. In some embodiments, the articles of
manufacture may also include instructions for using the composition
as a prophylactic, therapeutic, or ameliorative treatment for the
disease of concern.
EXAMPLES
[0462] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
Synthesis of Compound 1
5-(3,5-difluorophenyl)-5-phenylimidazolidine-2,4-dione
(Hydantoin-1)
Step 1: Synthesis of
(3,5-difluorophenyl)(2-phenyl-1,3-dithian-2-yl)methanol
##STR00085##
[0464] 2-Phenyl-1,3-dithiane (1.791 g, 9.12 mmol) was dissolved in
20 ml of dry THF and cooled to 0.degree. C. BuLi (6.84 ml, 10.94
mmol) was added dropwise under nitrogen and the mixture was stirred
for 30 min at 0.degree. C. A solution of 3,5-difluorobenzaldehyde
(1.00 ml, 9.12 mmol) in THF (10 ml) was added and the mixture was
stirred for 30 minutes, then warmed to ambient temperature over 1
hour and quenched with saturated ammonium chloride solution. The
organic phase was washed with brine and dried with sodium sulfate.
The solvent was removed in vacuo to give crude
(3,5-difluorophenyl)(2-phenyl-1,3-dithian-2-yl)methanol (3.15 g,
9.31 mmol, 104% yield) as a thick yellow oil. The residue was
carried through to the next step.
Step 2: Synthesis of
2-(3,5-difluorophenyl)-2-hydroxy-1-phenylethanone
##STR00086##
[0466] (3,5-Difluorophenyl)(2-phenyl-1,3-dithian-2-yl)methanol
(3.15 g, 9.31 mmol) was dissolved in 15 ml of acetonitrile and 2.5
ml of water. Bis(trifluoroacetoxy)iodobenzene (5.00 g, 11.63 mmol)
in 10 ml of acetonitrile was slowly added at ambient temperature to
the vigorously stirred solution. After 30 minutes TLC (25%
EtOAC/hexane) analysis indicated a complete reaction. EtOAc (150
ml) was added and the mixture was rinsed with saturated sodium
bicarbonate solution (50 ml) and brine (50 ml). The organic
fractions were dried, and the solvent was removed in vacuo. The
crude product was purified by flash column chromatography (12.5%
EtOAc/hexane) to give
2-(3,5-difluorophenyl)-2-hydroxy-1-phenylethanone (1.10 g, 4.43
mmol, 48%) as a pale yellow solid. The proton NMR was consistent
with the proposed structure.
Step 3: Synthesis of
1-(3,5-difluorophenyl)-2-phenylethane-1,2-dione
##STR00087##
[0468] 2-(3,5-difluorophenyl)-2-hydroxy-1-phenylethanone (1.10 g,
4.43 mmol) was dissolved in 80% acetic acid together with
diacetoxycopper hydrate (44 mg, 0.22 mmol) and ammonium nitrate
(0.30 g, 3.75 mmol). The mixture was refluxed for 2.5 hours and
then cooled. The reaction mixture was poured into ethyl acetate (50
ml) and washed with brine (2.times.25 ml), dried over sodium
sulfate, filtered and evaporated. The crude material was purified
by column chromatography (5% EtOAc/hexane) to give
1-(3,5-difluorophenyl)-2-phenylethane-1,2-dione (1.10 g, 4.43 mmol,
quant.) as a bright yellow solid. The proton NMR was consistent
with the proposed structure.
Step 4: Synthesis of
5-(3,5-difluorophenyl)-5-phenylimidazolidine-2,4-dione
##STR00088##
[0470] To a solution of
1-(3,5-difluorophenyl)-2-phenylethane-1,2-dione (0.99 g, 4.02
mmol), urea (0.435 g, 7.24 mmol) in ethanol (20 ml) and water (5
ml) was added solid NaOH (0.29 g, 7.24 mmol). The reaction mixture
was refluxed until TLC (50% EtOAc/hexane) analysis indicated a
complete reaction. The reaction mixture was diluted with water (30
ml) and carefully acidified with 2M HCl to pH 5. The reaction
mixture was extracted with ethyl acetate (100 ml) and washed with
water (50 ml) and brine (50 ml). The organic extract was dried over
sodium sulfate, filtered and evaporated to give a residue that was
triturated with acetone and hexane mixtures to afford
5-(3,5-difluorophenyl)-5-phenylimidazolidine-2,4-dione (0.220 g) as
a solid that was highly hydrated with water as judged by NMR
spectroscopy. The solid, after heating (120.degree. C.) under
vacuum overnight afforded the desired product (0.20 g, 0.69 mmol,
17%) as a white powder. .sup.1H NMR (400 MHz, d.sub.6-DMSO) .delta.
ppm 11.31 (brs, 1H), 9.44 (s, 1H), 7.37 (m, 6H), 7.10 (d, J=6.77
Hz, 2H); .sup.13C NMR (100 MHz, d.sub.6-DMSO) .delta. ppm 173.89,
163.52, 163.39, 161.06, 160.93, 155.78, 143.79, 143.70, 143.61,
139.16, 128.82, 128.44, 126.26, 110.12, 110.04, 109.93, 109.85,
104.11, 103.86, 103.60, 69.43 (note: C-F coupling was observed in
several instances giving rise to doublet and triplet signals; LC
(220 nm): R.sub.t=4.09 min, LC purity: 95.8%, m/z (M-1): 300.3.
Example 2
Synthesis of FAH-2:
2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H--
imidazol-5(4H)-one
Step 1: Synthesis of 2-(3,5-difluorophenyl)-1,3-dithiane
##STR00089##
[0472] BF.sub.3.OMe.sub.2 (0.70 ml, 7.62 mmol) was added dropwise
to a solution of 1,3-propanedithiol (0.90 ml, 8.94 mmol) and
3,5-difluorobenzaldehyde (1.00 ml, 8.94 mmol) in DCM (50 ml) at
0.degree. C. The reaction was stirred at ambient temperature for 1
hour where TLC (5% EtOAc/hexane) indicated a complete reaction. The
reaction mixture was then diluted with DCM (50 ml), filtered
through Celite (and the Celite pad was washed with additional DCM
(3.times.20 ml) and the filtrate washed with brine (50 ml),
saturated NaHCO.sub.3 (3.times.50 ml), 10% KOH solution (50 ml),
water (50 ml) and brine (50 ml) and finally dried over sodium
sulfate. The organic extract was filtered and evaporated to afford
2-(3,5-difluorophenyl)-1,3-dithiane (2.14 g, 9.21 mmol, 103%) as
white crystalline needles. The proton NMR was consistent with the
proposed structure.
Step 2: Synthesis of
(4-(difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)metha-
nol
##STR00090##
[0474] 2-(3,5-Difluorophenyl)-1,3-dithiane (2.14 g, 9.21 mmol) was
dissolved in 20 ml of dry THF and cooled to 0.degree. C. BuLi (8.50
ml, 10.20 mmol) was added dropwise under nitrogen and the mixture
was stirred for 15 min at 0.degree. C. A solution of
4-(difluoromethoxy)benzaldehyde (1.30 ml, 9.33 mmol) in THF (10 ml)
was added and the mixture was stirred for 10 minutes, then warmed
to ambient temperature over 10 minutes and quenched with saturated
ammonium chloride solution. The organic phase was washed with brine
and dried with sodium sulfate. The solvent was removed in vacuo to
give a residue that was purified by flash column chromatography
(10% EtOAc/hexane) to afford
(4-(difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)metha-
nol (1.90 g, 4.70 mmol, 51%) as a thick yellow oil. The proton NMR
was consistent with the proposed structure.
Step 3: Synthesis of
2-(4-(difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone
##STR00091##
[0476]
(4-(Difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl-
)methanol (1.90 g, 4.70 mmol) was dissolved in 15 ml acetonitrile
and 2.5 ml of water. Bis(trifluoroacetoxy)iodobenzene (2.53 g, 5.87
mmol) in 10 ml of acetonitrile was slowly added at ambient
temperature to the vigorously stirred solution. After 30 minutes
TLC (25% EtOAC/hexane) analysis indicated a complete reaction.
EtOAc (150 ml) was added and the mixture was rinsed with saturated
sodium bicarbonate solution (50 ml) and brine (50 ml). The organic
fractions were dried, and the solvent was removed in vacuo. The
crude product was purified by flash column chromatography (12.5%
EtOAc/hexane) to give
2-(4-(difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone
(0.460 g, 1.46 mmol, 31%) as a pale yellow solid. The proton NMR
was consistent with the proposed structure.
Step 4: Synthesis of
1-(4-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione
##STR00092##
[0478]
2-(4-(Difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethan-
one (0.46 g, 1.46 mmol) was dissolved in 80% acetic acid together
with diacetoxycopper hydrate (26 mg, 0.13 mmol) and ammonium
nitrate (0.18 g, 2.25 mmol). The mixture was refluxed for 90
minutes and then cooled. The reaction mixture was poured into ethyl
acetate (50 ml) and washed with brine (2.times.25 ml), dried over
sodium sulfate, filtered and evaporated. The crude material was
passed through a silica-gel plug, evaporated and azeotroped with
toluene (3.times.20 ml) to remove excess acetic acid to give crude
1-(4-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione
(0.456 g, 1.46 mmol, 100%) as a bright yellow solid. The crude
solid was carried through to the next step.
Step 5: Synthesis of
2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H--
imidazol-5(4H)-one
##STR00093##
[0480]
1-(4-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dion-
e (0.456 g, 1.46 mmol) in ethanol (20 ml) and water (5 ml) was
added 1-methylguanidine hydrochloride (0.16 g, 1.46 mmol) and
potassium carbonate (0.61 g, 4.38 mmol). The mixture was allowed to
reflux for 3 hours and then cooled to ambient temperature. The
volatiles were removed in vacuo and the residue was taken up in
water and extracted into chloroform (50 ml). The organic fractions
were dried with sodium sulfate and the solvent was removed in
vacuo. The crude material was purified by column chromatography
(EtOAc) to afford
2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H--
imidazol-5(4H)-one (0.172 g, 0.47 mmol) as a glass that was heavily
contaminated with ethyl acetate as judged by NMR analysis. The
glass was taken into dry ethanol (3 ml) and layered with hexane (1
ml) to get a turbid solution. The solution was rotary evaporated to
give an oil that solidified on standing. This was dried overnight
and the weight obtained was 0.150 g. The solid contained ethanol
and hexane solvent residues as judged by NMR analysis. Therefore,
the solids were re-dissolved into iso-propanol, rotary evaporated
and dried under high vacuum at 90.degree. C. overnight to afford
2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H--
imidazol-5(4H)-one (0.130 g, 0.35 mmol, 24%) as an off-white solid.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 7.47 (d, J=8.79 Hz,
2H), 7.10-7.00 (m, 4H), 6.70 (tt, J=8.73, 2.32 Hz, 1H), 6.53 (t,
J.sub.H-F=73.76 Hz, 1H), 5.45 (brs, 1H), 3.11 (s, 3H); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta. ppm 178.619, 164.122, 163.996,
161.650, 161.524, 155.704, 150.742, 150.714, 150.687, 145.164,
145.081, 144.991, 137.735, 128.390, 119.444, 118.328, 115.743,
113.158, 110.329, 110.255, 110.138, 110.065, 103.421, 103.169,
102.917, 77.203, 25.966 (note: C-F coupling was observed in several
instances giving rise to doublet and triplet signals); LC (260 nm):
R.sub.t=3.899 min, LC Purity: 96.3%, m/z (M-1): 366.
Example 3
Synthesis of FAH-3:
2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H--
imidazol-5(4H)-one
Step 1: Synthesis of 2-(3,5-difluorophenyl)-1,3-dithiane
##STR00094##
[0482] BF.sub.3.OMe.sub.2 (1.40 ml, 15.25 mmol) was added dropwise
to a solution of 1,3-propanedithiol (1.81 ml, 17.87 mmol) and
3,5-difluorobenzaldehyde (2.00 ml, 17.87 mmol) in DCM (50 ml) at
0.degree. C. The reaction was stirred at ambient temperature for 1
hour where TLC (5% EtOAc/hexane) indicated a complete reaction. The
reaction mixture was then diluted with additional DCM (50 ml),
filtered through Celite, and the Celite pad was washed with
additional DCM (3.times.20 ml). The filtrate was washed with
saturated NaHCO3 (3.times.50 ml), 10% KOH solution (2.times.50 ml),
water (2.times.50 ml) and brine (50 ml) and finally dried over
sodium sulfate. The organic extract was filtered and evaporated to
afford 2-(3,5-difluorophenyl)-1,3-dithiane (4.12 g, 17.73 mmol,
99%) as a white solid. The proton NMR was consistent with the
proposed structure.
Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde
##STR00095##
[0484] Attempt 1:
[0485] A solution of sodium chlorodifluoroacetate (3.23 g, 21.15
mmol) and 4-hydroxy-3-methylbenzaldehyde (1.44 g, 10.58 mmol),
potassium carbonate (2.19 g, 15.87 mmol) in a mixture of DMF (8 ml)
and water (2 ml) was heated at 100.degree. C. for 2 hours. The
reaction mixture was cooled and conc. HCl (1.5 ml) followed by
water (2.1 ml). The reaction mixture was diluted with water (20 ml)
and extracted with ethyl acetate (3.times.25 ml). The organic
extract was washed with 10% (m/v) aqueous LiCl solution (3.times.25
ml), dried over sodium sulfate, filtered and evaporated to give a
residue that was flash chromatographed (15% EtOAc/hexane) to give
4-(difluoromethoxy)-3-methylbenzaldehyde (0.244 g, 1.31 mmol, 12%)
as a brown oil and recovered 4-hydroxy-3-methylbenzaldehyde (1.164
g, 8.55 mmol, 81%) as a brown solid.
[0486] The experiment was repeated again, except with the absence
of water. Briefly, the experimental is given below:
[0487] Attempt 2:
[0488] A solution of sodium chlorodifluoroacetate (2.60 g, 17.04
mmol) and 4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF
(15 ml) was added over 3 hours to a solution of DMF (15 ml)
containing potassium carbonate (1.77 g, 12.78 mmol) at 95.degree.
C. The reaction was allowed to age for an additional 15 minutes and
then cooled. The reaction mixture was diluted with water (50 ml)
and extracted with ethyl acetate (3.times.50 ml). The organic
extract was washed with 10% (m/v) aqueous LiCl solution (3.times.25
ml), dried over sodium sulfate, filtered and evaporated to give a
residue that was flash chromatographed (15% EtOAc/hexane) to give
4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053.sub.--1(2),
1.00 g, 5.37 mmol, 63%) as a yellow oil. This oil was combined with
that of the previous experiment and passed through a Pasteur
pipette column eluting with 10% EtOAC/hexane to give an oil that
solidified on standing (1.315 g, 7.06 mmol, 67% over the two
reactions). The proton NMR was consistent with the proposed
structure.
[0489] Finally, repeating the experiment using the conditions in
attempt 2, an additional 1.4 g of the desired product was
isolated.
Step 3: Synthesis of
(4-(difluoromethoxy)-3-methylphenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-
-yl)methanol
##STR00096##
[0491] 2-(3,5-Difluorophenyl)-1,3-dithiane (3.12 g, 13.43 mmol) was
dissolved in 30 ml of dry THF and cooled to -10.degree. C. BuLi
(1.6M, 12.0 ml, 19.20 mmol) was added dropwise under nitrogen and
the mixture was stirred for 15 min at -10.degree. C. to afford a
blood-red solution. A solution of
4-(difluoromethoxy)-3-methylbenzaldehyde (2.50 g, 13.43 mmol) in
THF (10 ml) was added dropwise and the mixture was stirred for 15
minutes, then warmed to ambient temperature over 10 minutes and
quenched with saturated ammonium chloride solution. The organic
phase was washed with brine and dried with sodium sulfate. The
solvent was removed and the residue flash chromatographed (10%
EtOAc/hexane) to give
(4-(difluoromethoxy)-3-methylphenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-
-yl)methanol (3.07 g, 7.22 mmol, 55%) as a thick oil that
solidified on standing. The NMR was consistent with the proposed
structure.
Step 4: Synthesis of
2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3,5-difluorophenyl)-2-hydroxyet-
hanone
##STR00097##
[0493]
(4-(Difluoromethoxy)-3-methylphenyl)(2-(3,5-difluorophenyl)-1,3-dit-
hian-2-yl)methanol (3.07 g, 7.34 mmol) was dissolved in
acetonitrile (15 ml) and water (2.5 ml).
Bis(trifluoroacetoxy)iodobenzene (3.94 g, 9.17 mmol) in
acetonitrile (10 ml) was slowly added at ambient temperature to the
vigorously stirred solution. After 20 minutes TLC (20%
EtOAc/hexane) analysis indicated a complete reaction. EtOAc (150
ml) was added and the mixture was rinsed with saturated sodium
bicarbonate solution (50 ml) and brine (50 ml). The organic
fractions were dried, and the solvent was removed in vacuo. The
crude product was purified twice by flash column chromatography
(10% EtOAc/hexane) to give
2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3,5-difluorophenyl)-2-hydroxyet-
hanone (0.853 g, 2.60 mmol, 35%) as a pale yellow oil. The proton
NMR was consistent with the proposed structure.
Step 5: Synthesis of
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3,5-difluorophenyl)ethane-1,2-d-
ione
##STR00098##
[0495]
2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3,5-difluorophenyl)-2-hyd-
roxyethanone (0.853 g, 2.60 mmol) was dissolved in 80% acetic acid
together with diacetoxycopper hydrate (52 mg, 0.26 mmol) and
ammonium nitrate (0.156 g, 1.95 mmol). The mixture was refluxed for
90 minutes and then cooled. The reaction mixture was poured into
ethyl acetate (50 ml) and washed with brine (2.times.25 ml), dried
over sodium sulfate, filtered and evaporated. The residue was
azeotroped with toluene to remove acetic acid and the residue
(0.737 g, 2.26 mmol, 87%) was used directly into the next
stage.
Step 6: Synthesis of
2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3,5-difluorophenyl)-1-m-
ethyl-1H-imidazol-5(4H)-one
##STR00099##
[0497] A mixture of
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3,5-difluorophenyl)ethane-1,2-d-
ione (737 mg, 2.26 mmol) in ethanol (15 ml) and dioxane (15 ml) was
added 1-methylguanidine hydrochloride (990 mg, 9.04 mmol) and
stirred at ambient temperature for 15 minutes. Sodium carbonate
(958 mg, 9.04 mmol) in water (5 ml) was added and the mixture
immersed into an oil bath at 85.degree. C. and stirred for 3 hours.
TLC (EtOAc) indicated a complete reaction. The reaction mixture was
cooled to ambient temperature and concentrated. Purification by
flash chromatography (EtOAc) afforded
2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3,5-difluorophenyl)-1-m-
ethyl-1H-imidazol-5(4H)-one (0.52 g, 1.36 mmol, 60%) as a yellow
solid. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 7.34-7.24 (m,
2H), 7.08-6.97 (m, 3H), 6.74-6.66 (m, 1H), 6.47 (t, J.sub.H-F=74.03
Hz, 1H), 5.45 (brs, 2H), 3.11 (s, 3H), 2.25 (s, 3H); LC (260 nm):
R=3.919 min, LC Purity: 96.1%, m/z (M+1): 382, LC (220 nm): R=3.922
min, LC Purity: 96.8%.
Example 4
Synthesis of FAH-5
2-amino-4-(3,5-difluorophenyl)-4-(3,5-dimethylphenyl)-1-methyl-1H-imidazol-
-5(4H)-one
Synthesis of 2-(3,5-difluorophenyl)-1,3-dithiane
##STR00100##
[0499] BF.sub.3.OMe.sub.2 (2.50 mL, 27.5 mmol) was added dropwise
to a solution of 1,3-propanedithiol (3.70 mL, 36.6 mmol) and
3,5-difluorobenzaldehyde (4.10 mL, 36.6 mmol) in DCM (75 mL) at
0.degree. C. The reaction was stirred at ambient temperature for 1
hour where TLC (5% EtOAc/hexane) indicated a complete reaction. The
reaction mixture was then diluted with additional DCM (50 mL),
filtered through Celite, and the Celite pad was washed with
additional DCM (3.times.50 mL). The filtrate was washed with
saturated NaHCO.sub.3 (3.times.100 mL), 10% KOH solution
(2.times.100 mL), water (100 mL) and brine (100 mL) and finally
dried over sodium sulfate. The organic extract was filtered through
a pad of silica and the silica pad washed with 10% Ethyl
acetate/hexane mixtures (3.times.20 mL). The organic extract was
evaporated to afford 2-(3,5-difluorophenyl)-1,3-dithiane (8.44 g,
36.3 mmol, 99%) as a crystalline white solid. The NMR was
consistent with the proposed structure.
Synthesis of
(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)(3,5-dimethylphenyl)methanol
##STR00101##
[0501] 2-(3,5-Difluorophenyl)-1,3-dithiane (8.00 g, 34.4 mmol) was
dissolved in 100 mL of dry THF and cooled to -10.degree. C. BuLi
(1.6M, 34 mL, 54.4 mmol) was added dropwise under nitrogen and the
mixture was stirred for 15 min at -10.degree. C. to afford a brown
solution. A solution of 3,5-dimethylbenzaldehyde (4.84 g, 36.1
mmol) in THF (10 mL) was added dropwise and the reaction mixture
was stirred for 15 minutes, then warmed to ambient temperature over
30 minutes and quenched with saturated ammonium chloride solution.
The organic phase was washed with brine and dried with sodium
sulfate. The solvent was removed and the residue purified by flash
chromatography (10% EtOAc/hexane) to give
(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)(3,5-dimethylphenyl)methanol
(6.60 g, 18.01 mmol, 52%) as a thick oil that solidified on
standing. The NMR was consistent with the proposed structure.
Synthesis of
1-(3,5-difluorophenyl)-2-(3,5-dimethylphenyl)-2-hydroxyethanone
##STR00102##
[0503]
(2-(3,5-Difluorophenyl)-1,3-dithian-2-yl)(3,5-dimethylphenyl)methan-
ol (6.60 g, 18.01 mmol) was dissolved in a solution of acetonitrile
(75 mL) and water (15 mL). Bis(trifluoroacetoxy)iodobenzene (9.68
g, 22.51 mmol) was added in several portions to the vigorously
stirred solution at ambient temperature. After 60 minutes, TLC (20%
EtOAC/hexane) analysis appeared to indicate a complete reaction.
Ethyl acetate (150 mL) was added and the mixture was rinsed with
saturated sodium bicarbonate solution (2.times.50 mL) and brine (50
mL). The organic fractions were dried over sodium sulfate, filtered
and evaporated. The residue was purified twice by flash column
chromatography (10% EtOAc/hexane) to give
1-(3,5-difluorophenyl)-2-(3,5-dimethylphenyl)-2-hydroxyethanone
(2.10 g, 7.60 mmol, 42%, ca. 90% purity by NMR) as a pale yellow
solid contaminated with starting material (ca. 10%); R.sub.f (10%
EtOAc/hexane): 0.20 was identical for both starting material and
product. However the NMR was consistent with the proposed structure
of the product which was the major component.
Synthesis of
1-(3,5-difluorophenyl)-2-(3,5-dimethylphenyl)ethane-1,2-dione
##STR00103##
[0505]
1-(3,5-Difluorophenyl)-2-(3,5-dimethylphenyl)-2-hydroxyethanone
(2.10 g, 7.60 mmol) was dissolved in 80% acetic acid (10 mL)
together with diacetoxycopper hydrate (0.15 g, 0.76 mmol) and
ammonium nitrate (0.46 g, 5.70 mmol). The mixture was refluxed for
90 minutes and then cooled. The green coloured reaction mixture was
poured into ethyl acetate (50 mL) and washed with brine (2.times.25
mL), dried over sodium sulfate, filtered and evaporated. The
residue was subjected to flash chromatography (20% EtOAc/hexane) to
afford
1-(3,5-difluorophenyl)-2-(3,5-dimethylphenyl)ethane-1,2-dione (1.13
g, 4.12 mmol, 54%) as a yellow solid. The NMR was consistent with
the proposed structure.
Synthesis of
2-amino-4-(3,5-difluorophenyl)-4-(3,5-dimethylphenyl)-1-methyl-1H-imidazo-
l-5(4H)-one (FAH5)
##STR00104##
[0507] A mixture of
1-(3,5-difluorophenyl)-2-(3,5-dimethylphenyl)ethane-1,2-dione (500
mg, 1.82 mmol) in ethanol (15 mL) and dioxane (15 mL) was added
1-methylguanidine hydrochloride (799 mg, 7.29 mmol) and stirred at
ambient temperature for 15 minutes. Sodium carbonate (773 mg, 7.29
mmol) in water (5 mL) was added and the mixture immersed into an
oil bath at 85.degree. C. and stirred for 4 hours. TLC (EtOAc)
indicated a complete reaction. The reaction mixture was cooled to
ambient temperature and concentrated. The residue was purified
twice by column chromatography (EtOAc, 50% EtOAc/hexane) and
finally by PTLC (Chloroform) to afford
2-amino-4-(3,5-difluorophenyl)-4-(3,5-dimethylphenyl)-1-methyl-1H-imidazo-
l-5(4H)-one (0.20 g, 0.61 mmol, 33%) as a white solid after drying
under high vacuum at 60.degree. C. for 36 hours. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. ppm 7.07 (d, J=6.83 Hz, 2H), 7.02 (brs,
2H), 6.91 (brs, 1H), 6.69 (t, J=8.48 Hz, 1H), 5.22 (s, 2H), 3.10
(s, 3H), 2.27 (s, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
ppm 164.1, 163.9, 161.6, 161.5, 155.3, 145.4, 140.4, 138.2, 129.7,
124.5, 110.5, 110.4, 110.3, 110.2, 103.0, 77.2, 25.9, 21.41,
(please note: due to presence of fluorine atoms,
J.sup.2.sub.C-F-J.sup.4.sub.C-F couplings giving rise to poorly
resolved triplets and doublets are noted); LC (230 nm) R.sub.t
(min)=3.97, LC purity=95.29%; m/z: found [M+H].sup.+=330.1,
expected [M+H].sup.+=330.1 (C.sub.18H.sub.18F.sub.2N.sub.3O).
Example 5
Synthesis of FAH-4 (ITH002329)
Synthesis of 2-(3,5-difluorophenyl)-1,3-dithiane
##STR00105##
[0509] BF3.OMe.sub.2 (1.40 ml, 15.25 mmol) was added dropwise to a
solution of 1,3-propanedithiol (1.81 ml, 17.87 mmol) and
3,5-difluorobenzaldehyde (2.00 ml, 17.87 mmol) in DCM (50 ml) at
0.degree. C. The reaction was stirred at ambient temperature for 1
hour where TLC (5% EtOAc/hexane) indicated a complete reaction. The
reaction mixture was then diluted with additional DCM (50 ml),
filtered through Celite, and the Celite pad was washed with
additional DCM (3.times.20 ml). The filtrate was washed with
saturated NaHCO3 (3.times.50 ml), 10% KOH solution (2.times.50 ml),
water (2.times.50 ml) and brine (50 ml) and finally dried over
sodium sulfate. The organic extract was filtered and evaporated to
afford 2-(3,5-difluorophenyl)-1,3-dithiane (4.12 g, 17.73 mmol,
99%) as a white solid.
Synthesis of 3-(difluoromethoxy)benzaldehyde
##STR00106##
[0511] A solution of sodium chlorodifluoroacetate (12.48 g, 82
mmol) and 3-hydroxybenzaldehyde (5.00 g, 40.9 mmol) in DMF (75 ml)
was added over 3 hours to a solution of DMF (25 ml) containing
potassium carbonate (8.49 g, 61.4 mmol) at 95.degree. C. The
reaction was allowed to age for an additional 2 hours and then
cooled. The reaction mixture was diluted with water (100 ml) and
extracted with ethyl acetate (4.times.50 ml). The organic extract
was washed with 10% (m/v) aqueous LiCl solution (3.times.25 ml),
dried over sodium sulfate, filtered and evaporated to give a
residue that was flash chromatographed (15% EtOAc/hexane) to give
3-(difluoromethoxy)benzaldehyde (2.50 g, 14.52 mmol, 36%) as a
yellow oil. The NMR was consistent with the proposed structure.
Synthesis of
(3-(difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)metha-
nol
##STR00107##
[0513] The 2-(3,5-difluorophenyl)-1,3-dithiane (3.37 g, 14.52 mmol)
was dissolved in 30 ml of dry THF and cooled to -10.degree. C. BuLi
(1.6M, 12.0 ml, 19.20 mmol) was added dropwise under nitrogen and
the mixture was stirred for 15 min at -10.degree. C. to afford a
blood-red solution. A solution of 3-(difluoromethoxy)benzaldehyde
(2.50 g, 14.52 mmol) in THF (10 ml) was added dropwise and the
mixture was stirred for 15 minutes, then warmed to ambient
temperature over 10 minutes and quenched with saturated ammonium
chloride solution. The organic phase was washed with brine and
dried with sodium sulfate. The solvent was removed and the residue
flash chromatographed (10% EtOAc/hexane) to give
(3-(difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)metha-
nol (3.33 g, 8.23 mmol, 57%) as a thick oil that solidified on
standing. The NMR was consistent with the proposed structure.
Synthesis of
2-(3-(difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone
##STR00108##
[0515]
(3-(Difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl-
)methanol (3.33 g, 8.23 mmol) was dissolved in 15 ml of
acetonitrile and 2.5 ml of water. Bis(trifluoroacetoxy)iodobenzene
(4.43 g, 10.29 mmol) in 10 ml of acetonitrile was slowly added to
the vigorously stirred solution at ambient temperature. After 30
minutes, TLC (20% EtOAC/hexane) analysis indicated a complete
reaction. EtOAc (150 ml) was added and the mixture was rinsed with
saturated sodium bicarbonate solution (50 ml) and brine (50 ml).
The organic fractions were dried, and the solvent was removed in
vacuo. The crude product was purified twice by flash column
chromatography (10% EtOAc/hexane) to give
2-(3-(difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone
(1.01 g, 3.21 mmol, 39%) as a pale yellow oil. The NMR was
consistent with the proposed structure.
Synthesis of
1-(3-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione
##STR00109##
[0517]
2-(3-(Difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethan-
one (1.00 g, 3.18 mmol) was dissolved in 80% acetic acid (10 ml)
together with diacetoxycopper hydrate (0.13 g, 0.64 mmol) and
ammonium nitrate (0.19 g, 2.39 mmol). The mixture was refluxed for
90 minutes and then cooled. The copper coloured reaction mixture
was poured into ethyl acetate (50 ml) and washed with brine
(2.times.25 ml), dried over sodium sulfate, filtered and
evaporated. The residue was subjected to flash chromatography (20%
EtOAc/hexane) to afford
1-(3-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione
(0.46 g, 1.48 mmol, 47%) as a yellow oil. Further elution of the
column afforded starting material (0.40 g, 1.27 mmol, 40% recovery)
as an oil. The desired product was used as received.
Synthesis of
2-amino-4-(3-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H--
imidazol-5(4H)-one
##STR00110##
[0519] A mixture of
1-(3-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione
(463 mg, 1.48 mmol) in ethanol (15 ml) and dioxane (15 ml) was
added 1-methylguanidine hydrochloride (650 mg, 5.93 mmol) and
stirred at ambient temperature for 15 minutes. Sodium carbonate
(629 mg, 5.93 mmol) in water (5 ml) was added and the mixture
immersed into an oil bath at 85.degree. C. and stirred for 3 hours.
TLC (EtOAc) indicated a complete reaction. The reaction mixture was
cooled to ambient temperature and concentrated. The residue was
purified twice by PTLC (EtOAc) to afford
2-amino-4-(3-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H--
imidazol-5(4H)-one (0.22 g, 0.60 mmol, 40%) as a yellow solid after
drying under high vacuum at 60.degree. C. for 48 hours. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. ppm 7.38-7.29 (m, 2H), 7.24 (broad m,
1H), 7.10-7.00 (m, 3H), 6.71 (m, 1H), 6.49 (t, J.sub.H-F=74.03 Hz,
1H), 5.61 (brs, 2H), 3.10 (s, 3H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. ppm 178.30, 164.13, 164.00, 161.66, 161.53,
156.03, 151.27, 151.24, 151.21, 145.00, 144.92, 144.83, 142.88,
129.90, 123.81, 118.73, 118.42, 118.17, 115.84, 113.25, 110.30,
110.22, 110.11, 110.03, 103.47, 103.22, 102.97, 74.92, 25.95
(please note: due to presence of fluorine atoms,
J.sup.2.sub.C-F-J.sup.4.sub.C-F couplings giving rise to triplets
and doublets are noted); LC (220 nm): R.sub.t=3.85 min, LC Purity:
95.6%, m/z [M].sup.+=367.9,
Example 6
FAH-17:
2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-fluorophenyl)-1-methyl--
1H-imidazol-5(4H)-one
Step 1: Synthesis of
2-(4-(difluoromethoxy)-3-methylphenyl)-1,3-dithiane
##STR00111##
[0521] BF.sub.3.OEt.sub.2 (4.30 ml, 34.8 mmol) was added dropwise
to a solution of 1,3-propanedithiol (4.07 ml, 40.3 mmol) and
3-fluorobenzaldehyde (5.00 g, 40.3 mmol) in DCM (201 ml) at
0.degree. C. The reaction was stirred at ambient temperature for 1
hour where TLC (5% EtOAc/hexane) indicated a complete reaction. The
reaction mixture was then diluted with DCM (50 ml), filtered
through Celite (and the Celite pad was washed with additional DCM
(3.times.50 ml)) and the filtrate washed with brine (100 ml),
saturated NaHCO.sub.3 (3.times.100 ml), 10% KOH solution (100 ml),
water (100 ml) and brine (100 ml) and finally dried over sodium
sulfate. The organic extract was filtered and evaporated. The
product was purified using 5% ethyl acetate:hexane to afford
2-(3-fluorophenyl)-1,3-dithiane (8.71 g, 39.0 mmol, 97%) as an
off-clear oil. The oil was used directly into the next step. The
NMR was consistent with the proposed structure.
Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde
##STR00112##
[0523] A solution of sodium chlorodifluoroacetate (2.60 g, 17.04
mmol) and 4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF
(15 ml) was added over 3 hours to a solution of DMF (15 ml)
containing potassium carbonate (1.77 g, 12.78 mmol) at 95.degree.
C. The reaction was allowed to age for an additional 15 minutes and
then cooled. The reaction mixture was diluted with water (50 ml)
and extracted with ethyl acetate (3.times.50 ml). The organic
extract was washed with 10% (m/v) aqueous LiCl solution (3.times.25
ml), dried over sodium sulfate, filtered and evaporated to give a
residue that was flash chromatographed (15% EtOAc/hexane) to give
4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053.sub.--1(2),
1.00 g, 5.37 mmol, 63%) as a yellow oil. This oil was combined with
that of the previous experiment and passed through a Pasteur
pipette column eluting with 10% EtOAC/hexane to give an oil that
solidified on standing (1.315 g, 7.06 mmol, 67% over the two
reactions). The proton NMR was consistent with the proposed
structure.
[0524] Finally, repeating the experiment using the conditions in
attempt 2, an additional 1.4 g of the desired product was
isolated.
Step 3: Synthesis of
(2-(4-(difluoromethoxy)-3-methylphenyl)-1,3-dithian-2-yl)(3-fluorophenyl)-
methanol
##STR00113##
[0526] 2-(3-fluorophenyl)-1,3-dithiane (4.00 g, 18.66 mmol) was
dissolved in dry THF (93.5 mL) and cooled to -10.degree. C. nBuLi
(1.6M, 14.00 ml, 22.40 mmol) was added dropwise under nitrogen and
the mixture was stirred for 30 min at -10.degree. C. to afford a
dark red solution. A solution of
4-(difluoromethoxy)-3-methylbenzaldehyde (3.47 g, 18.66 mmol) in
THF (93.5 ml) was added dropwise and the mixture at -10.degree. C.
and was stirred for 15 minutes, then warmed to ambient temperature
over 1 h and quenched with saturated ammonium chloride solution
(7.5 ml) followed by dilution with EtOAc (50 ml). The organic phase
was washed with water (2.times.20 ml), brine (1.times.20 ml) and
dried with sodium sulfate. After filtration and concentration the
crude product was purified by flash column chromatography (15%
EtOAc/Hex) to give
(4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluorophenyl)-1,3-dithian-2-yl)-
methanol (6.00 g, 14.96 mmol, 80%) as an oil.
Step 4: Synthesis of
2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluorophenyl)-2-hydroxyethano-
ne
##STR00114##
[0528]
(4-(Difluoromethoxy)-3-methylphenyl)(2-(3-difluorophenyl)-1,3-dithi-
an-2-yl)methanol (3.07 g, 7.34 mmol) was dissolved in acetonitrile
(15 ml) and water (2.5 ml). Bis(trifluoroacetoxy)iodobenzene (3.94
g, 9.17 mmol) in acetonitrile (10 ml) was slowly added at ambient
temperature to the vigorously stirred solution. After 20 minutes
TLC (20% EtOAc/hexane) analysis indicated a complete reaction.
EtOAc (150 ml) was added and the mixture was rinsed with saturated
sodium bicarbonate solution (50 ml) and brine (50 ml). The organic
fractions were dried, and the solvent was removed in vacuo. The
crude product was purified twice by flash column chromatography
(10% EtOAc/hexane) to give
2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluorophenyl)-2-hydroxyethano-
ne (0.853 g, 2.60 mmol, 35%) as a pale yellow oil. The proton NMR
was consistent with the proposed structure.
Step 5: Synthesis of
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)ethane-1,2-dione
##STR00115##
[0530]
1-(2,4-difluorophenyl)-2-(4-methoxy-3-fluorolphenyl)ethane-1,2-dion-
e was synthesized according to the representative procedure using
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)-2-hydroxyethano-
ne (0.500 g, 1.612 mmol) and gave
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)ethane-1,2-dione
(0.3881 g, 78%) as a yellow solid. The NMR was consistent with the
proposed structure.
Step 6: Synthesis of
2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-fluorophenyl)-1-methyl-1H-imid-
azol-5(4H)-one
##STR00116##
[0532] Potassium carbonate (0.516 g, 4.87 mmol) in water (4.6 mL)
was added into a mixture of
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)ethane-1,2-dione
(0.375 g, 1.217 mmol), 1-methylguanidine hydrochloride (0.533 g,
4.87 mmol), dioxane (19 mL), and ethyl alcohol (25 mL). The
reaction mixture was stirred at 85.degree. C. for 4 h. The
volatiles were removed in vacuo, and the residue was taken in
chloroform (100 ml) and washed with water (2.times.25 mL). The
organic extracts were dried over MgSO.sub.4. Evaporation and
purification by flash chromatography (60% EtOAc/Hex to 100% EtOAc)
followed by re-crystallization from CHCl.sub.3/hexanes gave
2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3-fluorophenyl)-1-methy-
l-1H-imidazol-5(4H)-one (216 mg, 47%) as a off-white solid. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.37-7.13 (m, 5H), 6.96 (m, 2H),
6.46 (t, J=74.1 Hz, 1H), 5.73 (s, 2H), 3.09 (s, 3H), 2.23 (s, 3H);
.sup.13C NMR (101 MHz, CDCl.sub.3) .delta. 178.87, 163.90, 161.46,
155.70, 149.21, 143.36, 138.01, 130.04, 130.02-129.79 (m), 125.73,
122.70 (d, J=2.9 Hz), 118.75, 116.17, 114.45 (dd, J=38.7, 22.0 Hz),
113.60, 75.07, 25.87, 16.35. (please note: due to presence of
fluorine atoms, J.sup.2.sub.C-F-J.sup.4.sub.C-F couplings giving
rise to poorly resolved triplets and doublets are noted); LC (260
nm) R.sub.t (min)=3.923, LC purity=96%; m/z: found
[M+H].sup.+=364.2, expected [M+H].sup.+=364.3
(C.sub.17H.sub.14F.sub.3N.sub.3O).
Example 7
FAH-17HCl Salt
##STR00117##
[0534]
2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3-fluorophenyl)-1-
-methyl-1H-imidazol-5(4H)-one (0.50 g, 1.38 mmol) was dissolved in
anhydrous DCM (66 ml) followed by addition of HCl (1M in diethyl
ether, 2.2 ml). The mixture was stirred at room temperature for 5
min and the solvent evaporated in vacuo to yield
2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3-fluorophenyl)-1-methy-
l-1H-imidazol-5(4H)-one hydrochloride (0.50 g, 1.20 mmol, 87%) as a
white solid. .sup.1H NMR (d.sub.6-DMSO): 11.78 (brs, 1H), 9.73
(brs, 1H), 7.53-7.06 (m, 8H), 3.19 (s, 3H), 2.23 (s, 3H); .sup.13C
NMR (d.sub.6-DMSO): 176.62, 176.99, 172.57, 163.63, 161.20, 157.95,
150.07, 150.04, 140.36, 140.30, 134.40, 129.86, 126.60, 123.70,
119.52, 119.00, 116.96, 116.47, 116.26, 114.64, 114.41, 70.30,
27.43, 16.4 0(please note: due to presence of fluorine atoms,
J.sup.2.sub.C-F-J.sup.4.sub.C-F couplings giving rise to poorly
resolved triplets and doublets are noted); LC (220 nm):
R.sub.t=3.84 min, purity 96.5%; MS: For
C.sub.18H.sub.16F.sub.3N.sub.3O.sub.2 expect [M+H].sup.+=364.3
obtained 364.1
Example 8
Synthesis of FAH-22
Synthesis of 2-(3-fluoro-5-methylphenyl)-1,3-dithiane
##STR00118##
[0536] BF.sub.3.OEt.sub.2 (2.61 ml, 21.16 mmol) was added dropwise
to a solution of 1,3-propanedithiol (2.48 ml, 24.47 mmol) and
3-fluoro-5-methylbenzaldehyde (3.38 g, 24.47 mmol) in DCM (122 ml)
at 0.degree. C. The reaction was stirred at ambient temperature for
1 hour where TLC (5% EtOAc/hexane) indicated a complete reaction.
The reaction mixture was then diluted with DCM (100 ml), filtered
through Celite (and the Celite pad was washed with additional DCM
(3.times.100 ml)) and the filtrate washed with brine (100 ml),
saturated NaHCO.sub.3 (3.times.100 ml), 10% KOH solution (100 ml),
water (100 ml) and brine (100 ml) and finally dried over sodium
sulfate. The organic extract was filtered and evaporated to afford
2-(3-fluoro-5-methylphenyl)-1,3-dithiane (4.69 g, 77%) as a light
pink solid. The product was used in the next step without further
purification. The NMR was consistent with the proposed
structure.
Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde
##STR00119##
[0538] A solution of sodium chlorodifluoroacetate (2.60 g, 17.04
mmol) and 4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF
(15 ml) was added over 3 hours to a solution of DMF (15 ml)
containing potassium carbonate (1.77 g, 12.78 mmol) at 95.degree.
C. The reaction was allowed to age for an additional 15 minutes and
then cooled. The reaction mixture was diluted with water (50 ml)
and extracted with ethyl acetate (3.times.50 ml). The organic
extract was washed with 10% (m/v) aqueous LiCl solution (3.times.25
ml), dried over sodium sulfate, filtered and evaporated to give a
residue that was flash chromatographed (15% EtOAc/hexane) to give
4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053.sub.--1(2),
1.00 g, 5.37 mmol, 63%) as a yellow oil. This oil was combined with
that of the previous experiment and passed through a Pasteur
pipette column eluting with 10% EtOAC/hexane to give an oil that
solidified on standing (1.315 g, 7.06 mmol, 67% over the two
reactions). The proton NMR was consistent with the proposed
structure. 1.4 g of the desired product was isolated.
Synthesis of
(4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,3-dith-
ian-2-yl)methanol
##STR00120##
[0540]
(4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,-
3-dithian-2-yl)methanol was prepared according to the
representative procedure using
2-(3-fluoro-5-methylphenyl)-1,3-dithiane (0.932 g, 4.08 mmol) and
4-(difluoromethoxy)-3-methylbenzaldehyde (0.760 g, 4.08 mmol) which
gave
(4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,3-dith-
ian-2-yl)methanol (0.413 g, 24%) as a yellow oil. The NMR was
consistent with the proposed structure.
Synthesis of
2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluoro-5-methylphenyl)-2-hydr-
oxyethanone
##STR00121##
[0542]
2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluoro-5-methylphenyl)--
2-hydroxyethanone was synthesized according to the representative
procedure using
(4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,3-dith-
ian-2-yl)methanol (0.400 g, 0.965 mmol) and gave
2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluoro-5-methylphenyl)-2-hydr-
oxyethanone (162 mg, 47%) as a yellow solid. The NMR was consistent
with the proposed structure. Note:
(4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,3-dith-
ian-2-yl)methanone (104 mg, 22%) was also recovered.
Synthesis of
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluoro-5-methylphenyl)ethane--
1,2-dione
##STR00122##
[0544]
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluoro-5-methylphenyl)e-
thane-1,2-dione was synthesized according to the representative
procedure using
2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluoro-5-methylphenyl)--
2-hydroxyethanone (0.150 g, 0.463 mmol) and gave
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluoro-5-methylphenyl)ethane--
1,2-dione (0.1134 g, 74%) as a yellow solid. The NMR was consistent
with the proposed structure.
Synthesis of 2-amino-4-(4-(difluoromethoxy)-3-methyl
phenyl)-4-(3-fluoro-5-methylphenyl)-1-methyl-1H-imidazol-5(4H)-one
##STR00123##
[0546] Potassium carbonate (0.149 g, 1.407 mmol) in water (2.3 mL)
was added into a mixture of
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)ethane-1,2-dione
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluoro-5-methylphenyl)ethane--
1,2-dione (0.1134 g, 0.352 mmol), 1-methylguanidine hydrochloride
(0.154 g, 1.407 mmol), dioxane (5.46 mL), and ethyl alcohol (7.10
mL). The reaction mixture was stirred at 85.degree. C. for 1.5 h.
The volatiles were removed in vacuo, and the residue was taken in
chloroform (50 ml) and washed with water (2.times.15 mL). The
organic extracts were dried over MgSO.sub.4. Evaporation and
purification five times by flash chromatography (1% methanol in
ethyl acetate) gave
2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3-fluoro-5-methylphenyl-
)-1-methyl-1H-imidazol-5(4H)-one (85 mg, 75%) as a off-white solid.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.30 (d, J=2.0 Hz, 1H),
7.28-7.20 (m, 1H), 7.02 (s, 1H), 6.95 (m, 2H), 6.77 (d, J=9.3 Hz,
1H), 6.45 (t, J=74.1 Hz, 1H), 5.26 (s, 2H), 3.07 (s, 3H), 2.28 (s,
3H), 2.23 (s, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) .delta.
178.52, 163.88, 161.44, 155.75, 149.22 (t, J=2.5 Hz), 141.88 (dd,
J=288.6, 7.8 Hz), 138.02, 130.05 (d, J=9.4 Hz), 125.75, 123.30 (d,
J=2.5 Hz), 118.75 (d, J=7.5 Hz), 116.21, 115.32 (d, J=21.0 Hz),
113.63, 111.33 (d, J=23.3 Hz), 74.72, 25.83, 21.46 (d, J=1.8 Hz),
16.33. (please note: due to presence of fluorine atoms,
J.sup.2.sub.C-F-J.sup.4.sub.C-F couplings giving rise to poorly
resolved triplets and doublets are noted); LC (220 nm) R.sub.t
(min)=4.007, LC purity=98%; m/z: found [M+H].sup.+=378.2, expected
[M+H].sup.+=378.4 (C.sub.19H.sub.18F.sub.3N.sub.3O.sub.2).
Example 9
Synthesis of FAH-23:
2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-chlorophenyl)-1-methyl-1H-imid-
azol-5(4H)-one
Step 1: Synthesis of 2-(3-chlorophenol)-1,3-dithiane
##STR00124##
[0548] BF.sub.3.OEt.sub.2 (2.28 ml, 18.46 mmol) was added dropwise
to a solution of 1,3-propanedithiol (2.16 ml, 21.34 mmol) and
3-chlorobenzaldehyde (3.00 g, 21.34 mmol) in DCM (107 ml) at
0.degree. C. The reaction was stirred at ambient temperature for 1
hour where TLC (5% EtOAc/hexane) indicated a complete reaction. The
reaction mixture was then diluted with DCM (50 ml), filtered
through Celite (and the Celite pad was washed with additional DCM
(3.times.10 ml)) and the filtrate washed with brine (100 ml),
saturated NaHCO.sub.3 (3.times.100 ml), 10% KOH solution (100 ml),
water (100 ml) and brine (100 ml) and finally dried over sodium
sulfate. The organic extract was filtered and evaporated to afford
2-(3-chlorophenyl)-1,3-dithiane (4.62 g, 92%) as a colourless
solid. The product was used in the next step without further
purification.
Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde
##STR00125##
[0550] A solution of sodium chlorodifluoroacetate (2.60 g, 17.04
mmol) and 4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF
(15 ml) was added over 3 hours to a solution of DMF (15 ml)
containing potassium carbonate (1.77 g, 12.78 mmol) at 95.degree.
C. The reaction was allowed to age for an additional 15 minutes and
then cooled. The reaction mixture was diluted with water (50 ml)
and extracted with ethyl acetate (3.times.50 ml). The organic
extract was washed with 10% (m/v) aqueous LiCl solution (3.times.25
ml), dried over sodium sulfate, filtered and evaporated to give a
residue that was flash chromatographed (15% EtOAc/hexane) to give
4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053.sub.--1(2),
1.00 g, 5.37 mmol, 63%) as a yellow oil. This oil was combined with
that of the previous experiment and passed through a Pasteur
pipette column eluting with 10% EtOAC/hexane to give an oil that
solidified on standing (1.315 g, 7.06 mmol, 67% over the two
reactions). The proton NMR was consistent with the proposed
structure. 1.4 g of the desired product was isolated.
Step 3: Synthesis of
(4-(difluoromethoxy)-3-methylphenyl)(2-(3-chlorophenyl)-1,3-dithian-2-yl)-
methanol
##STR00126##
[0552] 2-(3-chlorophenyl)-1,3-dithiane (0.92 g, 3.98 mmol) was
dissolved in dry THF (20 mL) and cooled to -29.degree. C. nBuLi
(1.6M, 2.99 ml, 4.78 mmol) was added dropwise under nitrogen and
the mixture was stirred for 30 min at -29.degree. C. to afford a
dark red solution. A solution of
4-(difluoromethoxy)-3-methylbenzaldehyde (0.74 g, 3.98 mmol) in THF
(19.8 ml) was added dropwise and the mixture at -29.degree. C. and
was stirred for 15 minutes, then warmed to ambient temperature over
1 h and quenched with saturated ammonium chloride solution (7.5 ml)
followed by dilution with EtOAc (50 ml). The organic phase was
washed with water (2.times.20 ml), brine (1.times.20 ml) and dried
with sodium sulfate. After filtration and concentration the crude
product was purified by flash column chromatography (15%
EtOAc/Hexane) to give
(2-(3-chlorophenyl)-1,3-dithian-2-yl)(4-(difluoromethoxy)-3-methylphenyl)-
methanol (0.81 g, 1.94 mmol, 49%) as an oil. The NMR was consistent
with the proposed structure.
Step 4: Synthesis of
2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-chlorophenyl)-2-hydroxyethano-
ne
##STR00127##
[0554]
(4-(Difluoromethoxy)-3-methylphenyl)(2-(3-chlorophenyl)-1,3-dithian-
-2-yl)methanol (3.07 g, 7.34 mmol) was dissolved in acetonitrile
(15 ml) and water (2.5 ml). Bis(trifluoroacetoxy)iodobenzene (3.94
g, 9.17 mmol) in acetonitrile (10 ml) was slowly added at ambient
temperature to the vigorously stirred solution. After 20 minutes
TLC (20% EtOAc/hexane) analysis indicated a complete reaction.
EtOAc (150 ml) was added and the mixture was rinsed with saturated
sodium bicarbonate solution (50 ml) and brine (50 ml). The organic
fractions were dried, and the solvent was removed in vacuo. The
crude product was purified twice by flash column chromatography
(10% EtOAc/hexane) to give
2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-chlorophenyl)-2-hydroxyethano-
ne (0.803 g, 2.4 mmol, 32%) as a pale yellow oil. The proton NMR
was consistent with the proposed structure.
Step 5: Synthesis of
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-chlorophenyl)ethane-1,2-dione
##STR00128##
[0556]
(2-(3-chlorophenyl)-1,3-dithian-2-yl)(4-(difluoromethoxy)-3-methylp-
henyl)methanol (0.80 g, 1.92 mmol) was dissolved in dichloromethane
(24.29 ml) and tert-butanol (5.14 ml, 53.7 mmol) under nitrogen
atmosphere. Dess-Martin Periodinane (2.04 g, 4.80 mmol) was added
and the reaction was stirred overnight at room temperature. Sodium
thiosulphate (5 ml, 1M) was added and the layers were separated.
The organic phase was washed with sodium hydrogen carbonate and the
solvent was evaporated. Purification on prep plate in 25% ethyl
acetate hexane gave
1-(3-chlorophenyl)-2-(4-(difluoromethoxy)-3-methylphenyl)ethane-1,2-dione
(0.41 g, 1.27 mmol, 66%) as a yellow solid was used directly into
the next stage.
Step 6: Synthesis of
2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-chlorophenyl)-1-methyl-1H-imid-
azol-5(4H)-one
##STR00129##
[0558] Potassium carbonate (0.522 g, 4.93 mmol) in water (7.85 mL)
was added into a mixture of
1-(3-chlorophenyl)-2-(4-(difluoromethoxy)-3-methylphenyl)ethane-1,2-dione
(0.40 g, 1.23 mmol), 1-methylguanidine hydrochloride (0.54 g, 4.93
mmol), dioxane (19.13 mL), and ethyl alcohol (24.87 mL). The
reaction mixture was stirred at 85.degree. C. for 1.5 h. The
volatiles were removed in vacuo, and the residue was taken in
chloroform (50 ml) and washed with water (2.times.15 mL). The
organic extracts were dried over MgSO.sub.4. Evaporation and
purified three times on PTLC (1% MeOH in EtOAc) and column
chromatography (50-90% ethyl acetate:hexane) to give
2-amino-4-(3-chlorophenyl)-4-(4-(difluoromethoxy)-3-methylphenyl)-1-methy-
l-1H-imidazol-5(4H)-one (0.20 g, 0.52 mmol, 43%) as an off-white
solid. .sup.1H NMR (CDCl.sub.3): 7.48 (s, 1H), 7.30-7.20 (m, 5H),
7.0 (s, 1H), 6.48 (t, 1H, J=74.1 Hz), 4.50 (brs, 2H), 3.12 (s, 3H),
2.26 (s, 3H); .sup.13C NMR (CDCl.sub.3): 178.45, 155.74, 149.25,
143.22, 137.90, 134.32, 130.05, 127.12, 125.74, 125.36, 118.78,
116.17, 113.60 74.73, 25.89, 16.35 (please note: due to presence of
fluorine atoms, J.sup.2.sub.C-F-J.sup.4.sub.C-F couplings giving
rise to poorly resolved triplets and doublets are noted); LC (220
nm): R.sub.t=3.95 min, purity 96.6%; MS: For
C.sub.18H.sub.16ClF.sub.2N.sub.3O.sub.2 expect [M+H].sup.+=380.8
obtained 380.1
Example 10
Synthesis of Compound FAH-27:
2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-methylphenyl)-1-methyl-1H-imid-
azol-5(4H)-one
Step 1: Synthesis of 2-(3-methylphenyl)-1,3-dithiane
##STR00130##
[0560] BF.sub.3.OEt.sub.2 (2.67 ml, 21.60 mmol) was added dropwise
to a solution of 1,3-propanedithiol (2.53 ml, 24.97 mmol) and
3-methylbenzaldehyde (3.00 g, 24.97 mmol) in DCM (125 ml) at
0.degree. C. The reaction was stirred at ambient temperature for 1
hour where TLC (5% EtOAc/hexane) indicated a complete reaction. The
reaction mixture was then diluted with DCM (50 ml), filtered
through Celite (and the Celite pad was washed with additional DCM
(3.times.10 ml)) and the filtrate washed with brine (100 ml),
saturated NaHCO.sub.3 (3.times.100 ml), 10% KOH solution (100 ml),
water (100 ml) and brine (100 ml) and finally dried over sodium
sulfate. The organic extract was filtered and evaporated to afford
2-(m-tolyl)-1,3-dithiane (4.66 g, 85%) as a light brown solid. The
product was used in the next step without further purification. The
proton NMR was consistent with the proposed structure.
Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde
##STR00131##
[0562] A solution of sodium chlorodifluoroacetate (2.60 g, 17.04
mmol) and 4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF
(15 ml) was added over 3 hours to a solution of DMF (15 ml)
containing potassium carbonate (1.77 g, 12.78 mmol) at 95.degree.
C. The reaction was allowed to age for an additional 15 minutes and
then cooled. The reaction mixture was diluted with water (50 ml)
and extracted with ethyl acetate (3.times.50 ml). The organic
extract was washed with 10% (m/v) aqueous LiCl solution (3.times.25
ml), dried over sodium sulfate, filtered and evaporated to give a
residue that was flash chromatographed (15% EtOAc/hexane) to give
4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053.sub.--1(2),
1.00 g, 5.37 mmol, 63%) as a yellow oil. This oil was combined with
that of the previous experiment and passed through a Pasteur
pipette column eluting with 10% EtOAC/hexane to give an oil that
solidified on standing (1.315 g, 7.06 mmol, 67% over the two
reactions). The proton NMR was consistent with the proposed
structure. 1.4 g of the desired product was isolated.
Step 3: Synthesis of
(4-(difluoromethoxy)-3-methylphenyl)(2-(3-methylphenyl)-1,3-dithian-2-yl)-
methanol
##STR00132##
[0564] 2-(m-Tolyl)-1,3-dithiane (2.00 g, 9.51 mmol) was dissolved
in dry THF (47.5 mL) and cooled to -29.degree. C. nBuLi (1.6M, 7.13
ml, 11.41 mmol) was added dropwise under nitrogen and the mixture
was stirred for 30 min at -10.degree. C. to afford a dark red
solution. A solution of 4-(difluoromethoxy)-3-methylbenzaldehyde
(1.77 g, 9.51 mmol) in THF (47.5 ml) was added dropwise and the
mixture at -29.degree. C. and was stirred for 15 minutes, then
warmed to ambient temperature over 1 h and quenched with saturated
ammonium chloride solution (7.5 ml) followed by dilution with EtOAc
(50 ml). The organic phase was washed with water (2.times.20 ml),
brine (1.times.20 ml) and dried with sodium sulfate. After
filtration and concentration the crude product was purified by
flash column chromatography (15% EtOAC/Hex) to give
(4-(difluoromethoxy)-3-methylphenyl)(2-(m-tolyl)-1,3-dithian-2-yl)methano-
l (2.73 g, 6.88 mmol, 72%) as an oil. The NMR was consistent with
the proposed structure.
Step 4: Synthesis of
2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-methylphenyl)-2-hydroxyethano-
ne
##STR00133##
[0566]
(4-(Difluoromethoxy)-3-methylphenyl)(2-(m-tolyl)-1,3-dithian-2-yl)m-
ethanol (2.70 g, 6.81 mmol) was dissolved in dichloromethane (86
ml) and tert-butanol (18.28 ml, 191 mmol) under nitrogen
atmosphere. Dess-Martin Periodinane (7.22 g, 17.02 mmol) was added
and the reaction was stirred overnight at room temperature. Sodium
thiosulphate (5 ml, 1M) was added and the layers were separated.
The organic phase was washed with sodium hydrogen carbonate and the
solvent was evaporated. Purification on column chromatography in 5%
ethyl acetate/hexane gave
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(m-tolyl)ethane-1,2-dione
(1.44 g, 4.75 mmol, 70%) as a yellow solid.
Step 5: Synthesis of
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-methylphenyl)ethane-1,2-dione
##STR00134##
[0568]
(4-(Difluoromethoxy)-3-methylphenyl)(2-(m-tolyl)-1,3-dithian-2-yl)m-
ethanol (2.70 g, 6.81 mmol) was dissolved in dichloromethane (86
ml) and tert-butanol (18.28 ml, 191 mmol) under nitrogen
atmosphere. Dess-Martin Periodinane (7.22 g, 17.02 mmol) was added
and the reaction was stirred overnight at room temperature. Sodium
thiosulphate (5 ml, 1M) was added and the layers were separated.
The organic phase was washed with sodium hydrogen carbonate and the
solvent was evaporated. Purification on column chromatography in 5%
ethyl acetate/hexane gave
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(m-tolyl)ethane-1,2-dione
(1.44 g, 4.75 mmol, 70%) as a yellow solid and was used directly
into the next stage.
Step 6: Synthesis of
2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-methylphenyl)-1-methyl-1H-imid-
azol-5(4H)-one
##STR00135##
[0570] Potassium carbonate (1.81 g, 17.09 mmol) in water (27.23 mL)
was added into a mixture of
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(m-tolyl)ethane-1,2-dione
(1.30 g, 4.27 mmol), 1-methylguanidine hydrochloride (1.87 g, 17.09
mmol), dioxane (66.3 mL), and ethyl alcohol (86 mL). The reaction
mixture was stirred at 85.degree. C. for 1.5 h. The volatiles were
removed in vacuo, and the residue was taken in chloroform (50 ml)
and washed with water (2.times.15 mL). The organic extracts were
dried over MgSO.sub.4. Evaporation and purified three times by PTLC
(1% MeOH in EtOAc) and once by column chromatography (50-90% ethyl
acetate:hexane) to give
2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-1-methyl-4-(m-tolyl)-1H-im-
idazol-5(4H)-one (0.39 g, 1.07 mmol, 25%) as an off-white solid.
.sup.1H NMR (CDCl.sub.3): 7.34 (s, 1H), 7.24-7.10 (m, 5H), 6.95 (d,
1H, J=8 Hz), 6.47 (t, 1H, J=74.1 Hz), 6.05 (brs, 2H), 3.04 (s, 3H),
2.30 (s, 3H), 2.23 (s, 3H); .sup.13C NMR (CDCl.sub.3): 178.55,
155.97, 149.14, 149.12, 149.09, 141.16, 138.14, 130.20, 128.35,
127.56, 125.94, 124.16, 118.83, 118.61, 116.25, 113.67, 74.74,
25.70, 21.52, 16.32 (please note: due to presence of fluorine
atoms, J.sup.2.sub.C-F-J.sup.4.sub.C-F couplings giving rise to
poorly resolved triplets and doublets are noted); LC (220 nm):
R.sub.t=3.85 min, purity 97.3%; MS: For
C.sub.19H.sub.19F.sub.2N.sub.3O.sub.2 expect [M+H].sup.+=360.4
obtained 360.2
Example 11
Synthesis of FAH-28:
2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-(trifluoromethyl)phenyl)-1-met-
hyl-1H-imidazol-5(4H)-one
Step 1: Synthesis of 2-(3-trifluoromethyl)phenyl)-1,3-dithiane
##STR00136##
[0572] BF.sub.3.OEt.sub.2 (1.84 ml, 14.90 mmol) was added dropwise
to a solution of 1,3-propanedithiol (1.74 ml, 17.23 mmol) and
3-(trifluoromethyl)benzaldehyde (3.00 g, 17.23 mmol) in DCM (86 ml)
at 0.degree. C. The reaction was stirred at ambient temperature for
1 hour where TLC (5% EtOAc/hexane) indicated a complete reaction.
The reaction mixture was then diluted with DCM (50 ml), filtered
through Celite (and the Celite pad was washed with additional DCM
(3.times.10 ml)) and the filtrate washed with brine (100 ml),
saturated NaHCO.sub.3 (3.times.100 ml), 10% KOH solution (100 ml),
water (100 ml) and brine (100 ml) and finally dried over sodium
sulfate. The organic extract was filtered and evaporated to afford
2-(3-(trifluoromethyl)phenyl)-1,3-dithiane (4.62 g, 17.30 mmol,
100%) as a colourless solid. The product was used in the next step
without further purification.
Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde
##STR00137##
[0574] A solution of sodium chlorodifluoroacetate (2.60 g, 17.04
mmol) and 4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF
(15 ml) was added over 3 hours to a solution of DMF (15 ml)
containing potassium carbonate (1.77 g, 12.78 mmol) at 95.degree.
C. The reaction was allowed to age for an additional 15 minutes and
then cooled. The reaction mixture was diluted with water (50 ml)
and extracted with ethyl acetate (3.times.50 ml). The organic
extract was washed with 10% (m/v) aqueous LiCl solution (3.times.25
ml), dried over sodium sulfate, filtered and evaporated to give a
residue that was flash chromatographed (15% EtOAc/hexane) to give
4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053.sub.--1(2),
1.00 g, 5.37 mmol, 63%) as a yellow oil. This oil was combined with
that of the previous experiment and passed through a Pasteur
pipette column eluting with 10% EtOAC/hexane to give an oil that
solidified on standing (1.315 g, 7.06 mmol, 67% over the two
reactions). The proton NMR was consistent with the proposed
structure. 1.4 g of the desired product was isolated.
Step 3: Synthesis of
(4-(difluoromethoxy)-3-methylphenyl)(2-(3-(trifluoromethyl)phenyl)-1,3-di-
thian-2-yl)methanol
##STR00138##
[0576] 2-(3-(trifluoromethyl)phenyl)-1,3-dithiane (2.00 g, 7.57
mmol) (021STM-080) was dissolved in dry THF (38 mL) and cooled to
-29.degree. C. nBuLi (1.6M, 5.67 ml, 9.08 mmol) was added dropwise
under nitrogen and the mixture was stirred for 30 min at
-29.degree. C. to afford a dark red solution. A solution of
4-(difluoromethoxy)-3-methylbenzaldehyde (1.41 g, 7.57 mmol) in THF
(38 ml) was added dropwise and the mixture at -29.degree. C. and
was stirred for 15 minutes, then warmed to ambient temperature over
1 h and quenched with saturated ammonium chloride solution (7.5 ml)
followed by dilution with EtOAc (50 ml). The organic phase was
washed with water (2.times.20 ml), brine (1.times.20 ml) and dried
with sodium sulfate. After filtration and concentration the crude
product was purified by flash column chromatography (5-15%
EtOAc/Hex) to give
(4-(difluoromethoxy)-3-methylphenyl)(2-(3-(trifluoromethyl)phenyl)-1-
,3-dithian-2-yl)methanol (2.29 g, 4.55 mmol, 60%) as a golden
oil.
Step 4: Synthesis of
2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-(trifluoromethyl)phenyl)-2-hy-
droxyethanone
##STR00139##
[0578]
(4-(Difluoromethoxy)-3-methylphenyl)(2-(3-trifluoromethylphenyl)-1,-
3-dithian-2-yl)methanol (3.07 g, 7.34 mmol) was dissolved in
acetonitrile (15 ml) and water (2.5 ml).
Bis(trifluoroacetoxy)iodobenzene (3.94 g, 9.17 mmol) in
acetonitrile (10 ml) was slowly added at ambient temperature to the
vigorously stirred solution. After 20 minutes TLC (20%
EtOAc/hexane) analysis indicated a complete reaction. EtOAc (150
ml) was added and the mixture was rinsed with saturated sodium
bicarbonate solution (50 ml) and brine (50 ml). The organic
fractions were dried, and the solvent was removed in vacuo. The
crude product was purified twice by flash column chromatography
(10% EtOAc/hexane) to give
2-(4-(difluoromethoxy)-3-trifluoromethylphenyl)-1-(3-methylphenyl)-2-hydr-
oxyethanone (0.803 g, 2.4 mmol, 32%) as a pale yellow oil. The
proton NMR was consistent with the proposed structure.
Step 5: Synthesis of
1-(4-(difluoromethoxy)-3-(trifluoromethyl)phenyl)-2-(3-(trifluoromethyl)p-
henyl)ethane-1,2-dione
##STR00140##
[0580]
(4-(difluoromethoxy)-3-methylphenyl)(2-(3-(trifluoromethyl)phenyl)--
1,3-dithian-2-yl)methanol (2.20 g, 4.88 mmol) was dissolved in
dichloromethane (61.8 ml) and tert-butanol (13.08 ml, 137 mmol)
under nitrogen atmosphere. Dess-Martin Periodinane (5.18 g, 12.21
mmol) was added and the reaction was stirred overnight at room
temperature. Sodium thiosulphate (5 ml, 1M) was added and the
layers were separated. The organic phase was washed with sodium
hydrogen carbonate and the solvent was evaporated. Purification on
column chromatography in 5% ethyl acetate/hexane gave
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-(trifluoromethyl)phenyl)ethan-
e-1,2-dione (1.20 g, 3.35 mmol, 69%) as a yellow solid was used
directly into the next stage.
Step 6: Synthesis of
2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-(trifluoromethyl)phenyl)-1-met-
hyl-1H-imidazol-5(4H)-one
##STR00141##
[0582] Potassium carbonate (1.36 g, 12.84 mmol) in water (20.46 mL)
was added into a mixture of
1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-(trifluoromethyl)phenyl)ethan-
e-1,2-dione (1.15 g, 3.21 mmol), 1-methylguanidine hydrochloride
(1.41 g, 12.84 mmol), dioxane (49.8 mL), and ethyl alcohol (64.8
mL). The reaction mixture was stirred at 85.degree. C. for 1.5 h.
The volatiles were removed in vacuo, and the residue was taken in
chloroform (50 ml) and washed with water (2.times.15 mL). The
organic extracts were dried over MgSO.sub.4. Evaporation and
purified three times by PTLC (1% MeOH in EtOAc) and once by column
chromatography (50-90% ethyl acetate:hexane) to give
2-amino-4-(3,5-difluorophenyl)-4-(6-methoxypyridin-3-yl)-1-methyl-1H-
-imidazol-5(4H)-one (0.18 g, 0.44 mmol, 14%) as an off-white solid.
.sup.1H NMR (CDCl.sub.3): 7.80 (s, 1H), 7.70 (d, 1H, J=8 Hz), 7.55
(d, 1H, J=8 Hz), 7.45-7.41 (m, 1H), 7.32 (s, 1H), 7.28-7.24 (m,
1H), 7.0 (d, 1H, J=8 Hz), 6.48 (t, 1H, J=74.1 Hz), 5.64 (brs, 2H),
3.10 (s, 3H), 2.26 (s, 3H); .sup.13C NMR (CDCl.sub.3): 178.78,
156.18, 156.09, 156.07, 149.23, 142.39, 138.11, 130.71, 130.13,
128.99, 125.72, 122.72, 118.73, 116.15, 113.57, 75.05, 25.84, 16.33
(please note: due to presence of fluorine atoms,
J.sup.2.sub.C-F-J.sup.4.sub.C-F couplings giving rise to poorly
resolved triplets and doublets are noted); LC (220 nm):
R.sub.t=4.04 min, purity 96.6%; MS: For
C.sub.19H.sub.16F.sub.5N.sub.3O.sub.2 expect [M+H].sup.+=414.3
obtained 414.1
Example 12
SPR Analysis
[0583] The surfaces of all two flow cell FC1 and FC2 of a
carboxymethylated-dextran (CM-5) chips were washed sequentially
with 50 mM NaOH, 1 mM HCl, 0.05% H.sub.3PO.sub.4 and 20 mM sodium
phosphate pH 7.4, 125 mM sodium chloride in parallel using a flow
rate of 30 .mu.l/min for 1 min using a Biacore T-100 (GE
Healthcare). The fusion protein was immobilized via amine coupling
using 20 mM phosphate, 125 mM sodium chloride pH 7.4 on to FC2.
This fusion protein TRX-eAPP.sub.575-624-contains a fusion of
thioredoxin (TRX) and residues 575-624 of the APP ectodomain
(20-kDa). The fusion protein is produced as described in Libeu et
al (JAD 2011). The protein was concentrated to 2 mg/ml in 20 mM
phosphate pH 6.5, 125 mM sodium chloride and then dissolved to a
concentration of 50 .mu.g per ml in 20 mM sodium acetate pH 5.0.
FC1 served as a reference cell following a mock immobilization with
buffer alone. For all cells, the flow rate was 10 .mu.l per min.
The chip was blocked with 1M ethanolamine (pH 8.5). The final RU
values were determined for BACE inhibitors binding to
TRX-eAPP.sub.575-624 by flowing varying concentrations of the
inhibitor in DMSO to 50 .mu.M. Compounds were diluted from 10 mM
solutions in DMSO to 50 .mu.M in 1% DMSO, 20 mM sodium phosphate pH
7.4, 125 mM sodium chloride, 0.05% Tween and then serially diluted
by 1.5 for 10 steps. Binding traces were recorded for each dilution
with a binding phase of 60 seconds and a dissociation phase of 240
seconds. Each cycle was performed at 20.degree. C. with a constant
flow rate of 20 .mu.l/min. An additional 240 seconds of buffer flow
at 60 .mu.l per min across the cells was applied as a regeneration
phase to facilitate complete dissociation of the compound from the
protein. The sensograms were obtained by subtraction of the
reference and buffer signals using the Biacore T100 Evaluation
software. The binding curves were modeled with the PRISM (Graphpad
Inc).
Example 13
Experimental Methods for Measurement A.beta.42 in SH-SY5Y Cells
In Vitro Abeta Testing Assay:
[0584] SH-SY5Y neuroblastoma cells were seeded at 50,000 cells/well
in a 96 wells plate for 24 h. Then their medium was changed for
fresh medium supplemented with desired concentration of the
hydantoin compound (e.g. compound 3). After 24 h, 20 .mu.l of the
medium was added to 2 .mu.l of the complete protease inhibitor with
1 .mu.M EDTA and kept at 4.degree. C. until analysis using the
ELISA assay below.
ELISA Assays:
[0585] ELISA kits from Invitrogen were also used to quantify
A.beta.1-42 (KHB3544) in duplicate. For the A.beta.1-42
ultrasensitive ELISA, samples were diluted 1:2 (50 .mu.l CSF plus
50 .mu.l kit-provided standard diluent buffer). Assays were
performed according to manufacturer's instructions. In short,
standards and samples were added to a plate pre-coated with a
monoclonal capture antibody specific for the amino terminus of Hu
A.beta.. The samples were co-incubated with a rabbit detection
antibody (Ab) specific for the carboxy terminus of the A.beta.
species being assayed for 3 hr at room temperature overnight at
4.degree. (A.beta.1-42) with gentle rocking. After washing, bound
rabbit Ab was detected using a horseradish peroxidase-labeled
anti-rabbit secondary Ab. After washing again, bound HRP-anti
rabbit Ab was detected colorimetrically (Spectramax 190, Molecular
Devices) by the addition of a substrate solution. 1 mM
4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF)
protease inhibitor (101500, Calbiochem) was added to standards and
samples.
Example 14
Brain Uptake Testing (PK)
[0586] In general, CNS exposure of the hydantoins were performed as
follows: Studies consisted of collection of heparinized plasma and
brains after treatment with the hydantoins, following subcutaneous
(sc) administration of the molecules at 10 mg/kg. Plasma and brain
levels of the compounds were determined by quantitative LC/MS/MS
methodology, conducted at Integrated Analytical Solutions (on the
internet at ianalytical.net). Plasma samples were precipitated with
acetonitrile:methanol (1:1) cocktail containing an internal
standard. The brain samples were homogenized directly in
ethylacetate or extracted from 5M guanidine homogenates using the
liquid-liquid method. The resulting supernatant were evaporated to
dryness and subjected to the LC/MS/MS analysis. For each compound 3
mice were used for analysis. The brain-to-plasma ratios and brain
levels were then be calculated.
Example 15
Selectivity of ABBI
Inhibition of APP-BACE Versus PSLG1-BACE or NRG1-BACE Cleavage
[0587] P5-P5' Assay:
[0588] In order to determine the APP-BACE1 IC50 Sigma BACE1
substrate (7-Methoxycumarin-4-acetyl-[Asn670, Lue671]-Amyloid b/A4
Precursor Protein 770 Fragment 667-676-(2,4 dinitrophenyl)
Lys-Arg-Arg amide trifluoroacetate salt) was used, manufacturer
protocol was followed. Briefly, 0.01 units of BACE1 were incubated
for 1 h at room temperature with a BACE inhibitor, then the
substrate was added to each well and the fluorescence was read
immediately and every 30 min for 2 h. Activity was determine by
dividing the fluorescence at a specific [BACE inhibitor] by the
fluorescence at [BACE inhibitor]=0 .mu.M, the % activity was
plotted vs log [BACE inhibitor] to determine the APP-BACE1 using
GraphPad Prism 5 (FIG. 6A)
[0589] PSGL1 and NRG1 Assays:
[0590] Briefly, in order to determine the PSGL-1-BACE1 IC50 IC50
HEK 293 cells were plated in 24 well plates and transiently
cotransfected with either PSGL1/lacZ or NRG1/lacZ constructs using
Lipofectamine 2000; manufacturer protocol was followed. Two hours
after adding the DNA-lipid complex to the cells a BACE inhibitor
was added to each well, then cells were incubated overnight at
37.degree. C. and 5% CO.sub.2. Cultured medium was collected to
determine NRG1 or PSGL1, and cells were lysed to measure lacZ
levels. Sigma SEAP kit standard protocol was conducted on the
cultured medium to detect levels of PSGL1 or NRG1. Promega kit
instructions were followed to determine lacZ concentration. The
ratio of PSGL1/lacz vs [BACE inhibitor] or were plotted to
determine the BACE1-IC50 on each of the substrate using GraphPad
Prism 5 (FIG. 6B). The ABBI FAH17 shows a >200 fold selectivity
for APP over PSGL1. Similar testing shows that FAH17 is >10 fold
selective for APP over NRG1.
[0591] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
* * * * *