U.S. patent application number 14/223967 was filed with the patent office on 2014-09-18 for compounds that inhibit nfkb and bace1 activity.
This patent application is currently assigned to The Trustees of Columbia University in the City of New York. The applicant listed for this patent is The Trustees of Columbia University in the City of New York. Invention is credited to Shi-Xian Deng, Gangli Gong, Jeremy C. Hwang, Tae-Wan Kim, Donald W. Landry, Yidong Liu, Kirsten Alison Rinderspacher, Yuli Xie.
Application Number | 20140275165 14/223967 |
Document ID | / |
Family ID | 42129180 |
Filed Date | 2014-09-18 |
United States Patent
Application |
20140275165 |
Kind Code |
A1 |
Landry; Donald W. ; et
al. |
September 18, 2014 |
COMPOUNDS THAT INHIBIT NFkB AND BACE1 ACTIVITY
Abstract
The present invention relates to compounds with activity as
BACE1 and NF.kappa.B modulators, and methods for treating,
preventing, or ameliorating neurodegenerative diseases, such as
Alzheimer's disease. The present invention is also directed to the
treatment of diseases related to dysfunction of cell proliferation,
the immune system and/or inflammation using such compounds or
pharmaceutical compositions containing such candidate
compounds.
Inventors: |
Landry; Donald W.; (New
York, NY) ; Kim; Tae-Wan; (East Brunswick, NJ)
; Deng; Shi-Xian; (White Plains, NY) ; Gong;
Gangli; (Little Neck, NY) ; Hwang; Jeremy C.;
(Great Neck, NY) ; Xie; Yuli; (New York, NY)
; Liu; Yidong; (New York, NY) ; Rinderspacher;
Kirsten Alison; (Bronx, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees of Columbia University in the City of New
York |
New York |
NY |
US |
|
|
Assignee: |
The Trustees of Columbia University
in the City of New York
New York
NY
|
Family ID: |
42129180 |
Appl. No.: |
14/223967 |
Filed: |
March 24, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13094504 |
Apr 26, 2011 |
8685963 |
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14223967 |
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PCT/US2009/043011 |
May 6, 2009 |
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13094504 |
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61109891 |
Oct 30, 2008 |
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61143404 |
Jan 8, 2009 |
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61143532 |
Jan 9, 2009 |
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Current U.S.
Class: |
514/314 ;
435/184; 514/311; 546/171 |
Current CPC
Class: |
A61K 31/47 20130101;
A61P 29/00 20180101; C07D 409/12 20130101; A61P 37/02 20180101;
C07D 215/40 20130101; C07D 401/12 20130101; A61P 25/28 20180101;
A61P 35/00 20180101 |
Class at
Publication: |
514/314 ;
546/171; 514/311; 435/184 |
International
Class: |
C07D 215/40 20060101
C07D215/40; C07D 401/12 20060101 C07D401/12; C07D 409/12 20060101
C07D409/12 |
Goverment Interests
GRANT INFORMATION
[0002] This invention was made with government support under grants
P50 AG08702, and 5RO1 AT001643 awarded by the National Institutes
of Health, the Molecular Libraries Initiative of the National
Institutes of Health Roadmap for Medical Research and the
Intramural Research Program of the National Human Genome Research
Institute, National Institutes of Health. The government has
certain rights in the invention.
Claims
1. A composition comprising a compound of Formula I: ##STR00018##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are
independently selected for each occurrence from the group
consisting of hydrogen, halogen, alkyl, aryl, alkoxy, aryloxy,
alkylthiol, arylthiol, CN, and NO.sub.2; and wherein R.sup.10 is
selected from the group consisting of substituted or unsubstituted
alkyl, cycloalkyl, aryl, heteroaryl and alkenyl.
2. The composition of claim 1, wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4, and R.sup.5 are independently selected for each occurrence
from the group consisting of: hydrogen, methyl, Cl, OCH.sub.3,
CF.sub.3 and F.
3. The composition of claim 1, wherein R.sup.10 is selected from
the group consisting of phenyl, 2-Nitrophenyl, 3-Nitrophenyl,
4-Nitrophenyl, 4-Methyl-2-nitrophenyl, 2-Methyl-5-nitrophenyl,
2-Nitro-4-(trifluoromethyl)phenyl, 4-Methoxy-2-nitrophenyl,
2-Methyl-5-nitrophenyl, 4-Methyl-2-nitrophenyl, 4-Methylphenyl,
2-Aminophenyl, 2-Amino-4-methyl phenyl, Thiophen-2-yl,
5-Chlorothiophen-2-yl, 5-Bromothiophen-2-yl.
4-9. (canceled)
10. A method for inhibiting the activity of a .beta.-site APP
cleavage enzyme 1 (BACE1) in a cell which comprises contacting the
cell with a compound of claim 1 in an amount effective to inhibit
.beta.-site APP cleavage enzyme 1 activity.
11. The method of claim 10, wherein the inhibition of .beta.-site
APP cleavage enzyme 1 activity reduces the metabolism of an amyloid
precursor protein (APP).
12. The method of claim 10, wherein the cell is a mammalian
cell.
13. The method of claim 10, wherein the cell is contacted in
vitro.
14. A method for treating Alzheimer's disease in an individual,
which method comprises administering to the individual an effective
amount of a compound according to claim 1.
15. A method for inhibiting the activity of a Nuclear
factor-.kappa. B (NF.kappa.B) in a cell which comprises contacting
the cell with a compound of claim 1 in an amount effective to
inhibit Nuclear factor-.kappa. B (NF.kappa.B) activity.
16. The method of claim 15, wherein the inhibition of Nuclear
factor-.kappa. B (NF.kappa.B) activity reduces nuclear
translocation of the Nuclear factor-.kappa. B (NF.kappa.B).
17. The method of claim 15, wherein the cell is a mammalian
cell.
18. The method of claim 15, wherein the cell is contacted in
vitro.
19. A method for treating a disease related to dysfunction of cell
proliferation, the immune system, and/or inflammation in an
individual, which method comprises administering to the individual
an effective amount of a compound according to claim 1.
20. The method of claim 19, wherein the disease is cancer.
21. A pharmaceutical composition comprising a therapeutically
effective amount of a compound of claim 1 and a pharmaceutically
acceptable carrier.
22. The composition of claim 1, wherein R.sup.10 is a substituted
phenyl, and wherein the substituents are selected from the group
consisting of hydrogen, halogen and combinations thereof.
23. The composition of claim 22, wherein the substituents are
selected from the group consisting of hydrogen, fluorine, chlorine
and combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of International
Application No. PCT/US2009043011, filed May 6, 2009 and published
in English as WO2010051064 on May 6, 2010, which claims the
priority benefits of U.S. Provisional Application No. 61/109,891,
filed Oct. 30, 2008; U.S. Provisional Application No. 61/143,404,
filed Jan. 8, 2009; and U.S. Provisional Application No.
61/143,532, filed Jan. 9, 2009, all four of which are hereby
incorporated by reference in their entireties.
I. INTRODUCTION
[0003] The present invention relates to compounds with activity as
NF.kappa.B inhibitors. The present invention also relates to
methods for treating, preventing, and/or ameliorating diseases
related to dysfunction of cell proliferation, the immune system,
inflammation and/or neurodegeneration using such compounds or
pharmaceutical compositions comprising such compounds.
2. BACKGROUND OF THE INVENTION
[0004] Nuclear factor-.kappa. B (NF.kappa.B) signaling is an
essential signal transduction pathway involved in inflammatory
responses, oncogenesis, viral infection, the regulation of cell
proliferation and apoptosis and, in particularly in the case of B
and T lymphocytes, in antigenic stimulation (Ghosh, 1998, Annu.
Rev. Immunol., 16, 225-260; Karin, 1999, J. Biol. Chem., 274,
27339-27342; Israel, 2000, Trends Cell. Biol., 10, 129-133;
Santoro, 2003, EMBO J., 22, 2552-2560). In mammalian cells, there
are five NF.kappa.B family members that dimerize: RelA, RelB,
c-Rel, NF.kappa.B 2/p100/p52 and NF.kappa.B 1/p105/p50. NF.kappa.B,
whose predominant form is a heterodimeric transcription factor
composed of p50 and RelA subunits, remains sequestered in the
cytoplasm through association with members of an inhibitory family
of proteins known as I.kappa.B. Upon stimulation by the cytokines
TNF-.alpha. and interleukin-1, endotoxin (LPS), microbial and viral
infections, pro-inflammatory signals converge on the canonical IkB
kinase complex (IKK), a protein complex that is composed of two
kinase subunits, IKK.alpha./IKK-1 and IKK.beta./IKK-2 and a
structural/regulatory subunit NEMO/IKK-.gamma.. Once activated IKK
complex phosphorylates IkB proteins, triggering their
ubiquitination and subsequent degradation by the proteasome. Free
NF.kappa.B can then move into nucleus to initiate or up-regulate
gene expression.
[0005] Although IKK.alpha. and IKK.beta. exhibit striking
structural similarity (52%), genetic studies have shown that they
are involved in two pathways for the activation of NF.kappa.B
(Pomerantz, 2002, Molecular Cell 2002 10: 693-695). IKK.beta. has
been identified as the pro-inflammatory kinase responsible of
activation of classical NF.kappa.B complexes, whereas IKK.alpha. in
association with NF.kappa.B inducing kinase (NIK) plays an
essential role in the non-canonical NF.kappa.B signaling pathway
(Senftleben, 2001, 293: 1495-1499).
[0006] NF.kappa.B plays an essential role in the development and
progression of cancer, including breast cancer. Animal studies
suggest the presence of constitutively active NF.kappa.B at an
early stage during neoplastic transformation of mammary cells
(Clarkson et al., 2000, J Bio Chem. 275(17):12737-42). NF.kappa.B
inhibits apoptosis in mouse mammary epithelia (Soy ak et al., 1999,
Cell Growth Differ. 10(8):537-44) and selective activation of
NF.kappa.B subunits have been found in human breast cancer cell
lines and patient samples (Sovak et at, 1997, J Clin Invest.
100(12):2952-60; Cogswell et al., 2000, Oncogene 19(9):1123-31). An
inverse correlation between the levels of NF.kappa.B activation and
estrogen receptor expression has been reported (Nakshatri et al.,
1997, Mol Cell Biol. 17(7):3629-39) and inhibition of NF.kappa.B in
breast cancer cells induces spontaneous apoptosis (Sovak et al.,
1999, Cell Growth Differ. 10(8):537-44; Cogswell et al., 2000,
Oncogene 19(9):1123-31). Paclitaxel-induced sensitivity of breast
cancer cell lines was enhanced by an NF.kappa.B inhibitor,
parthenolide (Patel et al., 2000, Oncogene 19(36):4159-69; Newton
et al., 1999, J Bio Chem. 274(26):18827-35). The Mullerian
inhibiting substance was also found to inhibit breast cancer growth
through NF.kappa.B mediated pathway (Segev et al., 2000, J Bio
Chem. 275(37):28371-9). Furthermore, the transactivation function
of NF.kappa.B is negatively regulated by IKI411 in breast cancer
cell lines (Newton et al., 1999, J Bio Chem. 274(26):18827-35).
Lastly, overexpression of HER2neu can activate NF.kappa.B through
the activation of Akt pathway and block apoptosis (Zhou et al.,
2000, J Bio Chem. 275(10:8027-31). All these reports together
suggest that NF.kappa.B plays an important role in cancer generally
and in breast cancer specifically.
[0007] In light of the foregoing, inhibition of NF.kappa.B
activation represents a target for development of new
anti-inflammatory and anti-cancer drugs (Poulaki, 2002, Am J
Pathol. 161: 2229-2240). Among many protein actors in NF.kappa.B
signaling pathway, IKK complex represents one of the most promising
molecular targets for discoveries of new specific NF.kappa.B
inhibitors. To minimize the potential toxicity effects in vivo,
therapeutic success will greatly depend on the abilities of the
NF.kappa.B inhibitors to block activating signals without modifying
the basal level of NF.kappa.B activity. For example, May et al.
described a cell-permeable peptidic inhibitor that specifically
blocks the pro-inflammatory NF.kappa.B activation by disrupting the
constitutive NEMO interaction with IKK kinases (May, 2000, Science
289, 1550-1554; May, 2002, J. Biol. Chem. 277, 45992-46000).
Accordingly, it would be desirable to identify additional small
molecules that can reduce NF.kappa.B activity by selectively
inhibiting NF.kappa.B and/or components of the NF.kappa.B signaling
pathway.
3. SUMMARY OF THE INVENTION
[0008] The present invention relates to compounds which inhibit
NF.kappa.B activity. The compounds of the invention may be used to
inhibit NF.kappa.B activity in a subject, or in a cell in
culture.
[0009] The present invention also provides a method for the
treatment of a dysfunction of cell proliferation, the immune system
and/or inflammation in an individual, wherein the dysfunction is
associated with NF.kappa.B activity, by administering to an
individual in need of such treatment a pharmaceutical composition
comprising at least one compound of Formulas I-III in an amount
effective to treat the dysfunction.
[0010] In particular non-limiting embodiments, the present
invention relates to a compound of Formula I:
##STR00001##
and to salts, esters and prodrugs of the compounds of Formula I.
Additionally, the present invention describes methods of
synthesizing and using compounds of Formula I.
[0011] In other non-limiting embodiments, the present invention
relates to a compound of Formula II:
##STR00002##
and to salts, esters and prodrugs of the compounds of Formula II.
Additionally, the present invention describes methods of
synthesizing and using compounds of Formula II.
[0012] In further non-limiting embodiments, the present invention
relates to a compound of Formula III:
##STR00003##
and to salts, esters and prodrugs of the compounds of Formula III.
Additionally, the present invention describes methods of
synthesizing and using compounds of Formula III.
[0013] The present invention also provides a method of inhibiting
the activity of NF.kappa.B, by contacting the NF.kappa.B, an
NF.kappa.B associated protein, or a cell expressing NF.kappa.B,
with at least one compound of Formulas I-III (meaning Formula I,
Formula II, or Formula III) in an amount effective to inhibit the
activity of NF.kappa.B.
[0014] In one non-limiting embodiment, the NF.kappa.B is expressed
by a cell, for example, a mammalian cell, and the cell is contacted
with an effective amount of at least one compound of Formulas
I-III.
[0015] In another embodiment, the NF.kappa.B, or cell expressing
NF.kappa.B, is contacted with an effective amount of at least one
compound of Formulas I-III in vitro.
[0016] In other non-limiting embodiments, the pharmaceutical
composition may optionally be used in conjunction with one or more
additional compound for the treatment of a dysfunction of cell
proliferation, the immune system and/or inflammation.
[0017] It has further been discovered that some of these compounds
inhibit BACE1, an enzyme that participates in the production of
A.beta.. As A.beta. has been linked to neurodegeneration and to
Alzheimer's Disease in particular, such compounds may be used in
the treatment of neurodegenerative conditions and in particular in
Alzheimer's Disease and related conditions.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1A-B depicts general structure of
N-(quinolin-8-yl)benzenesulfonamides. A) Open chain and B) cyclized
structures.
[0019] FIG. 2 depicts a method for synthesizing a particular
compound of Formula II (the particular compound identified as
compound 9) and a particular compound of formula III (the
particular compound identified as compound I).
[0020] FIG. 3A-B depicts (A) a one-pot synthesis reaction mechanism
synthesizing compound 9a of Formula II and compound I a of Formula
III. (B) depicts the synthesis of particular compounds of Formula
II (the particular compounds identified as 9i, 9j, and 9k).
[0021] FIG. 4 depicts a one-pot synthesis reaction mechanism for
converting particular compounds of Formula II into particular
compounds of Formula III. The particular R groups of the starting
compounds (9a-p) and the corresponding synthesized compounds (1a-p)
are identical in each synthesis, and are illustrated in the table
along with the individual yields of compounds 1a-p.
[0022] FIG. 5A-B depicts (A) The two-color dual luciferase based
assay. I.kappa.B.alpha. is fused to a luciferase emitting
greenlight (CBG68), while a red light-emitting luciferase (CBR) is
present in its native state. Both luciferases are regulated by
binding of the tet repressor to the tet operator (TO) binding site
(TO), allowing simultaneous induction of the luciferases by the CMV
promoter upon addition of doxycycline and test compounds. The CBR
protein serves to normalize for non-specific effects, while the
levels of the I.kappa.B.alpha.-CBG68 protein increase uniquely with
inhibition of the NF.kappa.B pathway leading to I.kappa.B.alpha.
degradation. (B) The nuclear translocation assay. TNF.alpha.
stimulation results in nuclear translocation of NF.kappa.B, which
is detected by immunofluorescence using an anti-p65 antibody and a
secondary antibody labeled with an Alexa 647 fluorophore
(A647).
[0023] FIG. 6 depicts several compounds identified by the NFkB
inhibition screens illustrated in FIG. 4, including Structures of
MG-132 (1), BAY11-7082 (2), a quinazoline based inhibitor of AP1
and NF.kappa.B mediated transcription (3),
N-(quinolin-8-yl)benzenesulfonamide; PubChem CID: 161167 (4), and
C7-locked N-(quinolin-8-yebenzenesulfonamide (5); PubChem CID:
659101.
[0024] FIG. 7A-D depicts the NF.kappa.B inhibitory effect achieved
by exemplary compounds of Formulas I and III in three cell-based
NF.kappa.B activity assays. FIGS. 7A and C show the inhibitory
effect of the compounds as measured by an I.kappa.B.alpha.
stabilization assay, NF.kappa.B nuclear-translocation assay, and
NF.kappa.B-bla expression assay. In the I.kappa.B.alpha.
stabilization assay, a higher efficacy of I.kappa.B.alpha.
stabilization compared to stabilization achieved by MG-132 (an
I.kappa.B.alpha. stabilizing agent) indicates greater NF.kappa.B
inhibition. I.kappa.B.alpha. stabilization was measured as an
increase in luminescence of a green luciferase I.kappa.B.alpha.
construct. Stabilization at a lower EC.sub.50 concentration further
indicates more potent NF.kappa.B inhibition. A red luciferase under
the same regulatory control as the I.kappa.B.alpha. construct was
used as a control to monitor cell uniformity and non-specific
effects. Compounds that stabilized I.kappa.B.alpha. were those that
increased green luminescence with minimal effects on red
luminescence. (.sup.aEC.sub.50 values from the I.kappa.B.alpha.
stabilization assay shown for the green luminescence reporter along
with the % efficacy. .sup.bEC.sub.50 values from the ratio of the
green and red luminescent values for the I.kappa.B.alpha.
stabilization. Data are averages from two to three experiments
where each experiment consisted of concentration-titration for each
compound performed in duplicate and fitting concentration-response
curves to the response after bioassay. NA, not applicable; the
compound only showed a strong inhibitory response in the original
qHTS (IC.sub.50=2.5 .mu.M, 95% inhibition) in the non-specific (red
luminescence) dataset.)
[0025] In the nuclear-translocation and NF.kappa.B-bla expression
assays, inhibition of NF.kappa.B nuclear-translocation or
NF.kappa.B-bla expression at a lower IC.sub.50 concentration
indicates a stronger NF.kappa.B inhibitory effect. Cytotoxicity for
each compound is also shown. (.sup.cIC.sub.50 values are derived
from curve-fitting to data from a single experiment performed in
triplicate. ND, not determined. All compounds showed >90%
efficacy in the translocation assay except compounds 15 (78%) and
27 (75%). The cytotoxicity assay was performed in OCI-Ly3 cells
using a 4 h endpoint.) FIG. 7B shows the synthetic scheme used to
generate compounds 6-18 and 23-28 of Formula I. FIG. 7D shows the
synthetic scheme used to generate compounds 19-22 of Formula I and
compounds 28-40 of Formula III.
[0026] FIG. 8 depicts a sAPP.beta. assay utilized to identify
inhibitors of BACE1. The assay utilizes SY5Y cells stably
overexpressing BACE-GFP and SEAP-APPwt. Cells are incubated with a
candidate compound for 6 hours prior to harvesting media.
BACE1-mediated cleavage of APP results in the release of sAPP.beta.
into the media. .alpha.-secretase, a competing enzyme that is
non-amyloidogenic, also cleaves APP to release sAPP.beta.. Using an
sAPP.beta.-specific antibody (s.beta.wt), SEAP-sAPP.beta. is
specifically captured in a modified ELISA assay. Detection is
achieved by addition of the fluorescent alkaline phosphatase
substrate, 4-methylumbelliferyl phosphate (4-MUP).
[0027] FIG. 9A-B depicts a targeted screen of
N-(quinolin-8-yl)benzenesulfonamides, a small-scale screen using
176 structurally-related compounds based on the scaffold of
N-(quinolin-8-yl)benzenesulfonamides. A) Primary screen was
conducted in 96-well format at 2 .mu.M concentration in duplicate.
Data points represent mean of two determinations. The Z' factors
for the four 96-well plates used in the screen were all above 0.7,
B) Statistics from the primary screen. The threshold value for hit
selection was set at 3 standard deviations, or 59.76%, yielding 6
hit compounds and a hit rate of 3.41%.
[0028] FIG. 10A-B depicts SAR studies of
N-(quinolin-8-yl)benzenesulfonamides. 28 full and partial hits,
shown in (A) for compounds 1-26 and (B) for compounds 27 and 28,
from the targeted screen of N-(quinolin-8-yl)benzeriesulfonamides
were characterized in SY5Y-BACEGFP-SEAPAPPwt cells using the
cell-based BACE1 assay. All compounds were tested at 8
concentrations (30, 10, 3, 1, 0.3, 0.1, 0.03, and 0.01 .mu.M), and
data points were analyzed with Origin software and fitted using a
logistic model for IC.sub.50 determination.
[0029] FIG. 11A-B depicts Lentiviral-mediated transduction of APPsw
in primary neurons. Primary cortical neurons were harvested from
wild-type P0 mice and cultured according to established protocols.
Lentivirus harboring human Swedish mutant APP (Lenti-APPsw) was
packaged using ViraPower Lentiviral Packaging mix (Invitrogen)
according to the manufacturer's protocol. DIV-14 neurons were
incubated for 24 hours with primary culture media containing the
indicated volume (in .mu.l) of virus (LV-1 and LV-2 denote 2
separate batches of virus, 0 denotes no virus was used). After
infection, neurons were incubated for 72 hours with primary culture
media. Media was collected for A.beta.40 ELISA (BioSource), and
cell lysates were probed with APPCT antibody to visualize
transduced full-length APP. (A) Bar graph depicting results; (B)
Assay results.
[0030] FIG. 12 depicts immunocytochemistry of primary cortical
neurons. DIV-14 primary cortical neurons from wild-type mice were
fixed, permeabilized, and stained with TUT I to visualize neuronal
.beta.-tubulin (green). Cell nuclei (blue) were visualized with a
DAPI co-stain. Cells were imaged with the Nikon C1 digital confocal
system at 60.times. magnification.
[0031] FIG. 13A-B depicts the effect of CU-264 on A.beta.
expressing primary cortical neuron cultures from wild-type mice
transformed with lentivirus harboring APPsw. CU-264, which
exhibited sub-micromolar potency for sAPP.beta. reduction in
SY5Y-BACEGFP-SEAPAPPwt cells, does not reduce A.beta..sub.40 levels
in primary neurons when the cells are incubated with 2 .mu.M
CU-264. At 10 .mu.M, CU-264 increased A.beta. levels. (A) Bar graph
depicting results; (B) Assay results.
5. DETAILED DESCRIPTION
[0032] For clarity and not by way of limitation, this detailed
description is divided into the following sub-portions:
[0033] (i) definitions;
[0034] (ii) synthesis schemes;
[0035] (iii) methods of treatment; and
[0036] (iv) pharmaceutical compositions.
5.1 Definitions
[0037] The terms used in this specification generally have their
ordinary meanings in the art, within the context of this invention
and in the specific context where each term is used. Certain terms
are discussed below, or elsewhere in the specification, to provide
additional guidance to the practitioner in describing the
compositions and methods of the invention and how to make and use
them.
[0038] In one non-limiting embodiment, the NF.kappa.B is a human
NF.kappa.B. The NF.kappa.B is preferably, but not by way of
limitation, encoded by the Homo sapiens nuclear factor of kappa
light polypeptide gene enhancer in B-cells 1 (NF.kappa.B1) (GenBank
accession number NM.sub.--003998), Homo sapiens nuclear factor of
kappa light polypeptide gene enhancer in B-cells 2 (p49/p100)
(NF.kappa.B2) (GenBank accession numbers NM.sub.--001077493,
NM.sub.--001077494, or NM.sub.--002502), Homo sapiens v-rel
reticuloendotheliosis viral oncogene homolog A (avian) (RELA)
(GenBank accession number NM.sub.--021975), Homo sapiens v-rel
reticuloendotheliosis viral oncogene homolog B (RELB) (GenBank
accession number NM.sub.--006509), or Homo sapiens v-rel
reticuloendotheliosis viral oncogene homolog (avian) (REL) (GenBank
accession number NM.sub.--002908), or any nucleic acid that encodes
a human NF.kappa.B polypeptide. Alternatively, NF.kappa.B can be
encoded by any nucleic acid molecule exhibiting at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%,
or up to 100% homology to any one of the NF.kappa.B genes (as
determined by standard software, e.g. BLAST or FASTA), and any
sequences which hybridize under stringent conditions to these
sequences, which retain NF.kappa.B activity.
[0039] In other non-limiting embodiments, an NF.kappa.B of the
invention may be characterized as having an amino acid sequence
described by GenBank accession numbers: NP.sub.--003989,
NP.sub.--001070961, NP.sub.--001070962, NP.sub.--002493,
NP.sub.--068810, NP.sub.--006500, and NP.sub.--002899, or any other
amino acid sequence at least 90% homologous thereto which retains
NF.kappa.B activity.
[0040] In another embodiment, NF.kappa.B comprises a family of
proteins which includes, but is not limited to, the proteins
NF.kappa.B1 (p50), NF.kappa.B2 (p52), RelA (p65), RelB and
c-Rel.
[0041] The BACE1, NF.kappa.B or APP may be a recombinant BACE1,
NF.kappa.B or APP polypeptide encoded by a recombinant nucleic
acid, for example, a recombinant DNA molecule, or may be of natural
origin.
[0042] The term "dysfunction of the immune system" refers to any
irregular activation of the innate and adaptive immune response
associated with an increase in NF.kappa.B activity, for example,
aberrant T-cell or B-cell development, maturation, and/or
proliferation.
[0043] The term "inflammation" encompasses both acute responses
(i.e., responses in which the inflammatory processes are active) as
well as chronic responses (i.e., responses marked by slow
progression and formation of new connective tissue). In certain
non-limiting embodiments, a disease associated with inflammation
that is to be treated by a compound of the instant invention is, by
way of example, but not by way of limitation, type I
hypersensitivity, atopy, anaphylaxis, asthma, osteoarthritis,
rheumatoid arthritis, septic arthritis, gout, juvenile idiopathic
arthritis, still's disease, ankylosing spondylitis, inflammatory
bowel disease, Crohn's disease or inflammation associated with
vertebral disc herniation.
[0044] According to the invention, a "subject" or "patient" is a
human or non-human animal. Although the animal subject is
preferably a human, the compounds and compositions of the invention
have application in veterinary medicine as well, e.g., for the
treatment of domesticated species such as canine, feline, and
various other pets; farm animal species such as bovine, equine,
ovine, caprine, porcine, etc.; wild animals, e.g., in the wild or
in a zoological garden; and avian species, such as chickens,
turkeys, quail, songbirds, etc.
[0045] The term `alkyl` refers to a straight or branched
C.sub.1-C.sub.20, preferably C.sub.1-C.sub.5, hydrocarbon group
consisting solely of carbon and hydrogen atoms, containing no
unsaturation, and which is attached to the rest of the molecule by
a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl
(isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl).
[0046] The term "alkenyl" refers to a C.sub.2-C.sub.20, preferably
C.sub.1-C.sub.5, aliphatic hydrocarbon group containing at least
one carbon-carbon double bond and which may be a straight or
branched chain, e.g., ethenyl, 1-propenyl, 2-propenyl (allyl),
iso-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl
[0047] The term "cycloalkyl" denotes an unsaturated, non-aromatic
mono- or multicyclic hydrocarbon ring system such as cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl. Examples of multicyclic
cycloalkyl groups include perhydronapththyl, adamantyl and
norbornyl groups bridged cyclic group or sprirobicyclic groups,
e.g., Spiro (4,4) non-2-yl.
[0048] The term "aryl" refers to aromatic radicals having in the
range of about 6 to about 14 carbon atoms such as phenyl, naphthyl,
tetrahydronapthyl, indanyl, biphenyl.
[0049] The term "heterocyclic" refers to a stable 3- to 15-membered
ring radical which consists of carbon atoms and one or more, for
example, from one to five, heteroatoms selected from the group
consisting of nitrogen, oxygen and sulfur. For purposes of this
invention, the heterocyclic ring radical may be a monocyclic or
bicyclic ring system, which may include fused or bridged ring
systems, and the nitrogen, carbon, oxygen or sulfur atoms in the
heterocyclic ring radical may be optionally oxidized to various
oxidation states. In addition, a nitrogen atom, where present, may
be optionally quaternized; and the ring radical may be partially or
fully saturated (i.e., heteroaromatic or heteroaryl aromatic).
[0050] The heterocyclic ring radical may be attached to the main
structure at any heteroatom or carbon atom that results in the
creation of a stable structure.
[0051] The term "heteroaryl" refers to a heterocyclic ring wherein
the ring is aromatic.
[0052] The substituents in the `substituted alkyl`, `substituted
alkenyl`, `substituted cycloalkyl`, `substituted aryl,`
`substituted heteroaryl` `substituted alkoxy,` `substituted
aryloxy,` `substituted alkylthiol,` and `substituted arylthiol` may
be the same or different, with one or more selected from the groups
hydrogen, halogen, acetyl, nitro, oxo (.dbd.O), CF.sub.3, NH.sub.2,
OCH.sub.3, or optionally substituted groups selected from alkyl,
alkoxy and aryl.
[0053] The term "halogen" refers to radicals of fluorine, chlorine,
bromine and iodine.
[0054] The term "BACE 1" refers to a polypeptide which mediates the
cleavage of APP in the .beta.-amyloidgenic pathway, producing an
sAPP.beta. ectodomain APP metabolite, which is released into the
extracellular space, and an intracellular C-terminal fragment
(CTF). In one non-limiting embodiment, the BACE1 is a human BACE1.
The BACE1 is preferably encoded by the Homo sapiens beta-site
APP-cleaving enzyme 1 (BACE1) gene (GenBank accession numbers
NM.sub.--012104, NM.sub.--138972, NM.sub.--138971, or
NM.sub.--138973), or any nucleic acid which encodes a human BACE1
polypeptide. Alternatively, BACE1 can be encoded by any nucleic
acid molecule exhibiting at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, or up to 100% homology to
a BACE1 gene (as determined by standard software, e.g. BLAST or
FASTA), and any sequences which hybridize under stringent
conditions to these sequences which retain BACE1 activity, where
stringent conditions are as described in U.S. Published Patent
Application US20030082140, which is hereby incorporated by
reference in its entirety and for all purposes.
[0055] In other non-limiting embodiments, a BACE1 of the invention
may be characterized as having an amino acid sequence described by
GenBank accession numbers: NP.sub.--036236, NP.sub.--620428,
NP.sub.--620427 and NP.sub.--620429, or any other amino acid
sequence at least 90%, or at least 95% homologous thereto, which
retains BACE1 activity.
[0056] The terms "APP" or "amyloid precursor protein" refers to a
substrate of BACE1 which may be metabolized into an ectodomain
sAPP.beta. fragment and a C-terminal fragment (CTF). In one
embodiment, APP is an integral membrane protein expressed in many
tissues and concentrated in, for example, the synapses of neurons.
In one non-limiting embodiment, APP is a human APP, for example,
Homo sapiens amyloid beta (A4) precursor protein (APP) encoded by
an APP gene (e.g., GenBank Accession numbers: NM.sub.--201414,
NM.sub.--201413, or NM.sub.--000484), or any nucleic acid that
encodes a human APP polypeptide. Alternatively, APP can be encoded
by any nucleic acid molecule exhibiting at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, at least 95%, or up to
100% homology to any one of the APP genes (as determined by
standard software, e.g. BLAST or FASTA), and any sequences which
hybridize under stringent conditions to these sequences.
[0057] In other non-limiting embodiments, APP may be characterized
as comprising an amino acid sequence described by GenBank accession
numbers: NP.sub.--958817, NP.sub.--958816, or NP.sub.--000475, or
any other amino acid sequence at least 90% or at least 95%
homologous thereto and is cleavable by a human BACE1 protein. In
non-limiting embodiments APP may be comprised in a fusion
protein.
5.2 Synthesis Schemes
[0058] The present invention provides for compounds that inhibit
the activity of NFkB and/or BACE1.
[0059] In certain non-limiting embodiments, the invention provides
for compounds of the following Formula
##STR00004##
[0060] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 are
independently selected for each occurrence from the group
consisting of hydrogen, halogen, alkyl, aryl, alkoxy, aryloxy,
alkylthiol, arylthiol, CN, and NO.sub.2; and
[0061] wherein R.sup.10 is selected from the group consisting of
substituted or unsubstituted alkyl, substituted or unsubstituted
cycloalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl and substituted or unsubstituted
alkenyl.
[0062] In non-limiting embodiments, R.sup.1, R.sup.2, R.sup.3,
R.sup.4 and R.sup.5 are independently selected for each occurrence
from the group consisting of hydrogen, methyl, Cl, OCH.sub.3,
CF.sub.3, Br, and F. In a specific non-limiting embodiment, R.sup.4
and R.sup.5 are hydrogen, and R.sup.1, R.sup.2, and R.sup.3 are
independently hydrogen, halogen, alkyl, aryl, CN, alkoxy, aryloxy,
NO.sub.2, alkylthio, and arylthio.
[0063] In other preferred embodiments, R.sup.10 is selected from
the group consisting of phenyl, naphthyl, 2-Nitrophenyl,
3-Nitrophenyl, 4-Nitrophenyl, 4-Methyl-2-nitrophenyl,
2-Methyl-5-nitrophenyl, 2-Nitro-4-(trifluoromethyl)phenyl,
4-Methoxy-2-nitrophenyl, 2-Methyl-5-nitrophenyl,
4-Methyl-2-nitrophenyl, 4-Methylphenyl, 2-Aminophenyl,
2-Amino-4-methyl phenyl, Thiophen-2-yl, 5-Chlorothiophen-2-yl,
5-Bromothiophen-2-yl.
[0064] In one non-limiting embodiment, compounds of Formula I may
be synthesized according to the following scheme:
##STR00005##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.10
are defined as above for Formula I.
[0065] In other non-limiting embodiments, compounds of Formula I
may be synthesized by any means known in the art.
[0066] In another non-limiting embodiment, the compound defined by
Foiinula I is:
##STR00006##
[0067] In other non-limiting embodiments, the compound defined by
Formula I is:
TABLE-US-00001 A ##STR00007## Compound R1 R2 R3 IC.sub.50
(sAPP.beta.) (.mu.M) CU-131 1-naphthalene H H 6.46 CU-138
1-naphthalene Br H 9.51 CU-150 2-thiophene H H 8.03 CU-151
4-methoxyphenyl H H 5.92 CU-152 4-nitrophenyl H H 4.63 CU-153
2-nitrophenyl H H 1.58 CU-155 2-naphthalene H H 2.20 CU-156
8-quinoline H H 4.23 CU-163 2-naphthalene Br H 0.79 CU-166 phenyl
Br H 13.45 CU-167 4-methylphenyl Br H 1.09 CU-168 8-quinoline Br H
17.17 CU-225 4-methylphenyl H Me 3.79 CU-242 2-aminophenyl H H 3.82
CU-261 4-methoxy-2-nitrophenyl H H 1.92 CU-264
4-trifluoromethyl-2-aminophenyl H H 0.53 CU-265
4-trifluoromethyl-2-nitrophenyl H H 2.95 CU-268 2-nitrophenyl OMe H
8.11 CU-271 4-methoxy-2-aminophenyl H H 2.74 CU-273 2-nitrophenyl H
Me 12.43 CU-274 2-aminophenyl H Me 11.11 CU-278
4-methoxy-2-aminophenyl H Me 11.48 CU-280
4-trifluoromethyl-2-nitrophenyl H Me 5.51 CU-281
4-trifluoromethyl-2-aminophenyl H Me 3.28 CU-294 2-naphthalene OMe
H 3.75 CU-295 1-naphthalene OMe H 3.63 B Compound Structure
IC.sub.50 (sAPP.beta.) (.mu.M) CU-262 ##STR00008## 0.82 CU-282
##STR00009## 1.11
[0068] In other non-limiting embodiments, the invention provides
for compounds of the following Formula II:
##STR00010##
[0069] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are independently selected
for each occurrence from the group consisting of hydrogen, halogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
aryl, substituted or unsubstituted alkoxy, substituted or
unsubstituted aryloxy, substituted or unsubstituted alkylthiol,
substituted or unsubstituted arylthiol, CN, and NO.sub.2. In a
specific non-limiting embodiment, R.sup.4 and R.sup.5 are hydrogen,
and R.sup.1, R.sup.2, and R.sup.3 are independently hydrogen,
halogen, alkyl, aryl, CN, alkoxy, aryloxy, NO.sub.2, alkylthio, and
arylthio.
[0070] In a non-limiting embodiment, R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are
independently selected for each occurrence from the group
consisting of hydrogen, methyl, Cl, OCH.sub.3, CF.sub.3 and F.
[0071] In another non-limiting embodiment, the compound defined by
Formula II is:
##STR00011##
[0072] In another non-limiting embodiment, compounds of Formula II
may be synthesized according to the following scheme:
##STR00012##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are defined as above for Formula
II.
[0073] In other non-limiting embodiments, compounds of Formula II
may be synthesized by any means known in the art.
[0074] In other non-limiting embodiments, the compounds of Formula
II may be synthesized according to the following scheme:
##STR00013##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are defined as above for Formula
II.
[0075] In other non-limiting embodiments, the invention provides
for compounds of the following Formula III:
##STR00014##
[0076] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are independently selected
for each occurrence from the group consisting of hydrogen, halogen,
alkyl, aryl, alkoxy, aryloxy, alkylthiol, arylthiol, CN, and
NO.sub.2.
[0077] In non-limiting embodiments, R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are
independently selected for each occurrence from the group
consisting of hydrogen, methyl, Cl, OCH.sub.3, CF.sub.3, OH, and F.
In a specific non-limiting embodiment, R.sup.4 and R.sup.5 are
hydrogen, and R.sup.1, R.sup.2, and R.sup.3 are independently
hydrogen, halogen, alkyl, aryl, CN, alkoxy, aryloxy, NO.sub.2,
alkylthio, and arylthio.
[0078] In non-limiting embodiments, compounds of Formula III may be
synthesized according to the following scheme:
##STR00015##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are defined as above for Formula
III.
[0079] In other non-limiting embodiments, compounds of Formula III
may be synthesized by any means known in the art.
[0080] In other non-limiting embodiments, the compounds of Formula
III may be synthesized according to the following scheme:
##STR00016## ##STR00017##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8 and R.sup.9 are defined as above for Formula
III.
5.3 Methods of Treatment
[0081] In accordance with the invention, there are provided methods
of using the compounds of Formulas I-III. The compounds of the
instant disclosure can inhibit NFkB activity to exert beneficial
effects. A compound of Formula I, II or III that inhibits
NF.kappa.B activity may be used, in an effective amount, for the
treatment of conditions including, but not limited to, cancer,
tumorogenesis, and inflammatory conditions including, but not
limited to, type I hypersensitivity, atopy, anaphylaxis, asthma,
osteoarthritis, rheumatoid arthritis, septic arthritis, gout,
juvenile idiopathic arthritis, still's disease, ankylosing
spondylitis, inflammatory bowel disease, Crohn's disease or
inflammation associated with vertebral disc herniation. A compound
of Formula I, II or III that inhibits BACE 1 activity may be used,
in an effective amount, in the treatment of neurodegenerative
diseases, such as Alzheimer's disease. In addition, the present
invention is directed to the treatment of diseases related to
dysfunction of cell proliferation, the immune system and/or
inflammation.
5.3.1 Treatment of Disease Related to Dysfunction of Cell
Proliferation, the Immune System and/or Inflammation
[0082] In non-limiting embodiments, the present invention provides
for methods of treating diseases related to dysfunction of cell
proliferation, the immune system and/or inflammation in a subject
in need of such treatment by administration of a therapeutic
formulation which comprises at least one compound of Formulas
I-III.
[0083] In particular embodiments, the formulation may be
administered to a subject in need of such treatment in an amount
effective to inhibit NFkB activity. Where the formulation is to be
administered to a subject in vivo, the formulation may be
administered systemically (e.g. by intravenous injection, oral
administration, inhalation, subcutaneous, intramuscular, etc.),
intraventricularly, intrathecally, or by any other means known in
the art. The amount of the formulation to be administered may be
determined using methods known in the art, for example, by
performing dose response studies in one or more model system,
followed by approved clinical testing in humans.
[0084] In one embodiment, the subject or patient has been diagnosed
with, or has been identified as having an increased risk of
developing a disease associated with dysfunction of cell
proliferation, the immune system and/or inflammation.
[0085] In other non-limiting embodiments, the present invention
provides for methods of reducing in a subject, the risk of
inflammatory damage comprising administering to the subject, an
effective amount of a composition according to the invention. An
effective amount may be a local concentration or, in a
pharmaceutical composition, an amount that, when administered to a
subject, results in a therapeutic benefit.
[0086] According to the invention, an effective amount is an amount
of at least one compound of Formulas I-III which reduces one or
more clinical symptom of one or more of the aforementioned
diseases. In one example, an effective amount is an amount of at
least one compound of Formulas I-III that reduces the activity of
NF.kappa.B, for example, by stabilizing I.kappa.B.alpha. or by
inhibiting nuclear translocation of NF.kappa.B.
[0087] In one non-limiting embodiment, the effective amount of at
least one compound of Formulas I-III may be determined, for
example, via an in vitro assay wherein the effective amount of a
compound of Formulas I-III may be correlated with the compound's
ability to stabilize I.kappa.B.alpha.. By way of example, and not
of limitation, such an assay may comprise a cell-based assay
designed to identify compounds that stabilize I.kappa.B.alpha., and
thus reduce nuclear translocation of NFkB. The cell-based in vitro
IkB.alpha. stabilization assay may utilize a dual reporter system,
such as a luciferase reporter system in the cell line OCILy3, a
model for primary tumors of the activated B-cell subtype (ABC) of
diffuse large B-cell lymphoma (DLBCL) (FIG. 5). The cell line used
in the assay may exhibit a high NF.kappa.B activity level, for
example, constitutive NFkB activity levels due to high levels of
IKK.beta. activity leading to elevated expression of NFkB targeted
genes. IKK and proteasome activity may produce high levels of
I.kappa.B.alpha. degradation, liberating NF.kappa.B heterodimers to
translocate to the nucleus.
[0088] According to the assay, modulators of IkB.alpha. stability
may be identified by measuring changes in the level of expression
of an IkB.alpha. reporter, for example, an exogenous
IkB.alpha.-luciferase fusion reporter construct expressed by an
NFkB-insensitive promoter. IkB.alpha. reporter expression may be
measured in the presence or absence of a compound of the invention,
wherein a greater increase in IkB.alpha. reporter expression by a
cell contacted with the compound compared to the IkB.alpha.
reporter expression in a cell not contacted with the compound is
correlative with the therapeutic efficacy of the compound.
[0089] In one example, a dual luciferase IkB.alpha. stabilization
screen in OCI-Ly3 may be used for high throughput screening (HTS).
For example, but not by way of limitation, IkB.alpha. fused to a
green light-emitting beetle luciferase and a red light-emitting
beetle luciferase expressed in a native form can be used to monitor
cell uniformity and non-specific effects. Fold-responsiveness may
be further increased by having both reporters under the control of
inducible promoters, for example, promoters regulated by
doxycycline. Upon doxycycline induction of both luciferase
reporters, compounds that increased green luminescence with minimal
effects on the red luminescence signal may be identified as
IkB.alpha. stabilizers, and thus an inhibitor of NF.kappa.B
activity.
[0090] In one non-limiting embodiment, the cell based in vitro
IkB.alpha. stabilization assay may be the assay depicted in FIG.
5a.
[0091] In one embodiment, an effective amount of a compound of
Formulas I-III may be that amount which stabilizes IkB.alpha., and
a greater level of IkB.alpha. stabilization at a lower
concentration when compared to a control level of IkB.alpha.
stabilization, for example, as exhibited by a known IkB.alpha.
stabilizing agent, such as MG-132 (FIG. 6) is correlative with the
therapeutic efficacy of the compound.
[0092] In one embodiment, the compound may stabilize IkB.alpha.
with an efficacy of at least about 10-20%, at least about 20-50%,
at least about 50-100%, at least about 100-200%, at least about
200-400%, or at least about 400-800% when compared to the
IkB.alpha. stabilization achieved by a known IkB.alpha. stabilizing
agent, such as MG-132.
[0093] In one embodiment, an effective amount of a compound of
Formulas may be that amount which stabilizes IkB.alpha. with an
EC.sub.50 at a concentration ranging from about 200 .mu.M to about
0.01 .mu.M, preferably from about 100 .mu.M to about 0.01 .mu.M,
more preferably from about 50 .mu.M to about 0.01 .mu.M, and more
preferably from about 20 .mu.M to about 0.01 .mu.M in the in vitro
assay, wherein an EC.sub.50 at a lower concentration in the in
vitro assay is correlative with the compound's therapeutic
efficacy.
[0094] In another non-limiting embodiment, the effective amount of
at least one compound of Formulas I-III may be determined, for
example, via an in vitro assay wherein the effective amount of a
compound of Formulas I-III may be correlated with the compound's
ability to reduce the nuclear translocation of NF.kappa.B. By way
of example, and not of limitation, such an assay may comprise a
cell-based assay that utilizes an agent, for example, a cytokine
such as TNF.alpha., to stimulate nuclear translocation of
endogenous NFkB. Stimulation by the agent may result in proteasome
degradation of IkB.alpha. and subsequent translocation of NFkB from
the cytoplasm to the nucleus, while in the absence of such a
stimulatory agent, NFkB is sequestered in the cytoplasm due to its
binding to IkB.alpha..
[0095] In the nuclear-translocation in vitro assay, nuclear
translocation of NFkB, for example, the endogenous p65 RelA
subunit, may be detected and/or measured following stimulation with
the agent through the use of, for example, but not limited to,
fluorescent antibody detection and an automated imaging platform.
Compounds of the invention may be contacted with cells of the in
vitro assay, wherein a reduction in NFkB nuclear transport compared
to a cell not contacted with the compound is indicative of the
compound's ability to inhibit NF.kappa.B activity. According to the
invention, a reduction in nuclear translocation of NF.kappa.B may
be correlative with the compound's therapeutic efficacy.
[0096] In one non-limiting embodiment, the cell based
nuclear-translocation in vitro assay may be the assay depicted in
FIG. 5b.
[0097] In one embodiment, an effective amount of a compound of
Formulas I-III may be correlated with the compound's ability to
inhibit NF.kappa.B nuclear-translocation, wherein a greater level
of inhibition at a lower concentration when compared to a control
level of inhibition, for example, as exhibited by a known NFiB
nuclear-translocation inhibitor, such as BAY 11-7082 (FIG. 6), is
indicative of greater therapeutic efficacy of the compound.
[0098] In one embodiment, an effective amount of a compound of
Formulas I-III may be that amount which inhibits NF.kappa.B
nuclear-translocation with an efficacy of at least about 10-20%, at
least about 20-50%, at least about 50-80%, or at least about
80-100% or more when compared to the NF.kappa.B
nuclear-translocation inhibition achieved by a known inhibitor,
such as BAY 11-7082.
[0099] In one non-limiting embodiment, an effective amount of a
compound of Formulas I-III may be that amount which inhibits
NF.kappa.B nuclear-translocation by at least 50% when the compound
is administered at a concentration ranging from about 200 .mu.M to
about 0.01 .mu.M, preferably from about 100 .mu.M to about 0.01
.mu.M, more preferably from about 50 .mu.M to about 0.01 .mu.M, and
more preferably from about 10 .mu.M to about 0.01 .mu.M in the in
vitro assay, wherein inhibition of NF.kappa.B nuclear-translocation
at a lower concentration in the in vitro assay is correlative with
the compound's therapeutic efficacy.
[0100] In another non-limiting embodiment, the effective amount of
at least one compound of Formulas I-III may be determined, for
example, via an in vitro assay wherein the effective amount of a
compound of Formulas I-III may be correlated with the compound's
ability to reduce expression of an NF.kappa.B-dependent reporter
construct, for example, a .beta.-lactamase reporter
(NF.kappa.B-bla). By way of example, and not of limitation, such an
assay may comprise contacting a cell expressing the
NF.kappa.B-dependent reporter construct, and monitoring the level
of .beta.-lactamase expression, wherein a decrease in expression
compared to a cell not contacted with the compound indicates a
reduction in NF.kappa.B activity. According to the invention, the
reduction in expression of the NF.kappa.B-dependent reporter may be
correlative with the compound's therapeutic efficacy.
[0101] In one non-limiting embodiment, an effective amount of a
compound of Formulas I-III may be that amount which reduces
expression of an NF.kappa.B-bla construct by at least 50% when the
compound is administered at a concentration ranging from about 200
.mu.M to about 0.01 .mu.M, preferably from about 100 .mu.M to about
0.01 .mu.M, more preferably from about 50 .mu.M to about 0.01
.mu.M, and more preferably from about 10 .mu.M to about 0.01 .mu.M
in the in vitro assay, wherein a reduction of NF.kappa.B-bla
expression at a lower concentration in the in vitro assay is
correlative with the compound's therapeutic efficacy.
[0102] In one non-limiting embodiment, an effective amount of a
compound of Formulas I-III may be an amount which achieves a local
concentration at the therapeutic site of about 100 .mu.M to about
0.01 .mu.M, preferably from about 50 .mu.M to about 0.01 .mu.M,
more preferably from about 20 .mu.M to about 0.01 .mu.M, and more
preferably from about 10 .mu.M to about 0.01 .mu.M in the in vitro
assay.
5.3.2 Treatment of Neurodegenerative Diseases
[0103] The present invention provides for methods of treating a
neurodegenerative disease in a subject in need of such treatment
comprising administering, to the subject, a therapeutically
effective amount of at least one compound of Formulas I-III.
Non-limiting examples of neurodegenerative diseases include
Alzheimer's disease, lewy body dementia, inclusion body myositis,
and cerebral amyloid angiopathy.
[0104] In particular embodiments, the present invention provides
for methods of treating diseases related to metabolism of APP by
BACE1 in a subject in need of such treatment by administration of a
therapeutic formulation which comprises an effective amount of at
least one compound of Formulas I-III. In particular embodiments,
the formulation may be administered to a subject in need of such
treatment in an amount effective to inhibit BACE1 activity and/or
reduce the presence of sAPP.beta. and/or A.beta.. Where the
formulation is to be administered to a subject in viva, the
formulation may be administered systemically (e.g. by intravenous
injection, oral administration, inhalation, etc.),
intraventricularly, intrathecally, or by any other means known in
the art. The amount of the formulation to be administered may be
determined using methods known in the art, for example, by
performing dose response studies in one or more model system,
followed by approved clinical testing in humans.
[0105] In one embodiment, the subject or patient has been diagnosed
with, or has been identified as having an increased risk of
developing a neurodegenerative disease, such as Alzheimer's
Disease.
[0106] In other non-limiting embodiments, the present invention
provides for methods of reducing in a subject, the risk of neural
damage comprising administering, to the subject, an effective
amount of a composition according to the invention. An effective
amount may be a local concentration or, in a pharmaceutical
composition, an amount that, when administered to a subject,
results in a therapeutic benefit.
[0107] According to the invention, an effective amount is an amount
of at least one compound of Formulas I-III which reduces one or
more clinical symptom of one or more of the aforementioned
diseases. In one example, an effective amount is an amount of at
least one compound of Formulas I-III that reduces the production of
sAPP.beta. or A.beta. generated by the metabolism of APP by
BACE1.
[0108] In one non-limiting embodiment, the effective amount of at
least one compound of Formulas I-III may be determined via an in
vitro assay, for example, as described in International Patent
Application No. PCT/US2007015938 (Publication No. WO 2008008463),
which is incorporated in its entirety herein for all purposes,
wherein the effective amount may be correlated with the compound's
ability to reduce the level of sAPP.beta.. By way of example, and
not of limitation, such an assay may comprise a cell-based modified
ELISA assay for measuring sAPP.beta., the secreted ectodomain of
.beta.-amyloid precursor protein (APP) following .beta.-secretase
(BACE1) cleavage. Such an in vitro assay may be used to identify
compounds of Formulas I-III that interfere with the first step of
sAPP.beta. generation.
[0109] As described in the Examples below, and depicted in FIG. 8,
the sAPP.beta. ELISA assay may comprise cells, for example, SY5Y
cells, transfected with a BACE1 reporter construct, such as a
GFP-tagged BACE 1 (BACE-GFP), and a wild type APP reporter
construct, such as a secreted alkaline phosphatase (SEAP)-tagged
wild type APP (SEAP-APPwt). BACE 1 cleavage of the reporter-tagged
APP (e.g. SEAP-APPwt) may result in secretion into the media of
SEAP-tagged sAPP.beta., which may be collected and specifically
captured using an sAPP.beta. cleavage site-specific antibody (e.g.
s.beta.wt). Following washing, a substrate may be used, for
example, the fluorescent alkaline phosphatase substrate
4-methylumbelliferyl phosphate (4-MUP), to detect the captured
SEAP-sAPP.beta..
[0110] In one non-limiting example, an effective amount of a
compound of Formulas I-III may be correlated with the compound's
ability to reduce the level of sAPP.beta. detected in the in vitro
assay compared to a control cell culture that was not contacted
with the candidate compound, wherein a reduction of sAPP.beta.
compared to the control cell culture correlates with the compound's
therapeutic efficacy.
[0111] In another non-limiting example, an effective amount of a
compound of Formulas I-III may be that amount which reduces the
level of sAPP.beta. detected in the in vitro assay by at least 0.1,
or by at least 0.5, or by at least 1, or by at least 1.5, or by at
least 2, or by at least 2.5, or by at least 3, or by at least 3.5,
or by at least 4, or by at least 4.5, or by at least 5, or by at
least 5.5, or by at least 6 or more standard deviations above a
control level of sAPP.beta. reduction detected in the in vitro
assay when the compound is tested at a concentration of about 2
.mu.M, or about 10 .mu.M, wherein such a reduction of sAPP.beta.
correlates with a compound's therapeutic efficacy. In one
embodiment, the control level of sAPP.beta. reduction may be the
average sAPP.beta. level in control cell cultures that are not
contacted with the candidate compound. In other embodiments, the
control level may be the average level of sAPP.beta. reduction
achieved by a series of compounds tested in the in vitro assay.
[0112] In one preferred non-limiting embodiment, an effective
amount of a compound of Formulas I-III may be correlated with the
compound's ability to reduce sAPP.beta. levels by about 3 standard
deviations greater than a control level of sAPP.beta. reduction
when the compound is administered at a concentration of 2 .mu.M in
the in vitro assay.
[0113] In other preferred non-limiting embodiments, an effective
amount of a compound of Formulas I-III may be correlated with the
compound's ability to reduce the sAPP.beta. level by about 3
standard deviations greater than a control level of sAPP.beta.
reduction when the compound is administered at a concentration of
10 .mu.M in the in vitro assay.
[0114] In another example, an effective amount of a compound of
Formulas may be that amount which reduces the level of sAPP.beta.
by at least 5%, by at least 10%, by at least 20%, by at least 30%,
by at least 40%, by at least 50%, by at least 60%, by at least 70%,
by at least 80%, by at least 90%, or by 100% when compared to
sAPP.beta. level in a control cell culture that was not contacted
with the candidate compound, wherein such a reduction of sAPP.beta.
correlates with a compound's therapeutic efficacy.
[0115] In another example, an effective amount of a compound of
Formulas may be that amount which reduces the level of sAPP.beta.
by at least about 50% compared to a control cell culture that was
not contacted with the candidate compound, wherein the compound is
tested at a concentration ranging from about 200 .mu.M to about
0.01 .mu.M, preferably from about 100 .mu.M to about 0.01 .mu.M,
and more preferably from about 10 .mu.M to about 0.01 .mu.M in the
in vitro assay, wherein such a reduction of sAPP.beta. at the
above-described concentrations is correlative with the compound's
therapeutic efficacy.
[0116] In other preferred non-limiting embodiments, an effective
amount of a compound of Formulas I-III may be correlated with the
compound's ability to reduce the sAPP.beta. level by about at least
50% when the compound is administered at a concentration of about
0.5 .mu.M in the in vitro assay.
[0117] In other preferred non-limiting embodiments, an effective
amount of a compound of Formulas I-III may be correlated with the
compound's ability to reduce the sAPP.beta. level by about at least
50% when the compound is administered at a concentration of about 1
.mu.M in the in vitro assay.
[0118] In other preferred non-limiting embodiments, an effective
amount of a compound of Formulas I-III may be correlated with the
compound's ability to reduce the sAPP.beta. level by about at least
50% when the compound is administered at a concentration of about 2
.mu.M in the in vitro assay.
[0119] In other preferred non-limiting embodiments, an effective
amount of a compound of Formulas I-III may be correlated with the
compound's ability to reduce the sAPP.beta. level by about at least
50% when the compound is administered at a concentration of less
than 3 .mu.M in the in vitro assay.
[0120] In other preferred non-limiting embodiments, an effective
amount of a compound of Formulas I-III may be correlated with the
compound's ability to reduce the sAPP.beta. level by about at least
50% when the compound is administered at a concentration of about 5
.mu.M in the in vitro assay.
[0121] In other preferred non-limiting embodiments, an effective
amount of a compound of Formulas I-III may be correlated with the
compound's ability to reduce the sAPP.beta. level by about at least
50% when the compound is administered at a concentration of about 8
.mu.M in the in vitro assay.
[0122] In other preferred non-limiting embodiments, an effective
amount of a compound of Formulas I-III may be correlated with the
compound's ability to reduce the sAPP.beta. level by about at least
50% when the compound is administered at a concentration of about
10 .mu.M in the in vitro assay.
[0123] In other non-limiting embodiments, the effective amount of
at least one compound of Formulas I-III may be correlated with the
compound's ability to inhibit the enzymatic activity of a BACE1
enzyme. The compound's BACE1 inhibitory effect may be assayed, for
example, through use of a BACE1 FRET Assay kit (Invitrogen Corp.,
Carlsbad, Calif., U.S.A.), wherein the fluorescence resonance
energy transfer (FRET)-based assay measures the cleavage by
purified recombinant .beta.-secretase of a peptide substrate
corresponding to the BACE1 cleavage site of Swedish mutant APP. In
such an assay, a greater reduction in fluorescence in the reaction
mixture following incubation with a compound of the invention
compared to a control cell culture not contacted with the compound
is correlative with the compound's therapeutic efficacy.
[0124] In one example, an effective amount of a compound of
Formulas I-III may be that amount which inhibits BACE1 enzymatic
activity by at least about 50% compared to a control cell culture
that was not contacted with the candidate compound. Preferably the
compound is tested at a concentration ranging from about 200 .mu.M
to about 0.01 .mu.M, preferably from about 100 .mu.M to about 0.01
.mu.M, and more preferably from about 10 .mu.M to about 0.01 .mu.M
in the in vitro assay, wherein such an inhibition of BACE1
enzymatic activity at the above-described concentrations is
correlative with the compound's therapeutic efficacy.
[0125] In other non-limiting embodiments, the effective amount of
at least one compound of Formulas I-III may be correlated with the
compound's ability to reduce the level of A.beta..sub.40 in an in
vitro assay that measures the level of A.beta..sub.40 produced in a
cell line, for example, an SY5Y-BACEGFP-SEAPAPPwt cell line. In one
non-limiting example, the level of A.beta..sub.40 expressed by the
cell line may be measured through the use of an A.beta..sub.40
Elisa kit (BioSource). In one embodiment, the assay comprises
incubating the A.beta..sub.40 expressing cells with a compound of
Formulas I-III, followed by assaying the concentration of
A.beta..sub.40 in the cell culture medium. In such an assay, a
greater reduction of A.beta..sub.40 concentration in the cell
culture medium following incubation with a compound compared to a
control cell culture not contacted with the compound is correlative
with the compound's therapeutic efficacy.
[0126] In one example, an effective amount of a compound of
Formulas I-III may be that amount which reduces the level of
A.beta..sub.40 in an in vitro assay by at least about 50% compared
to a control cell culture that was not contacted with the candidate
compound. Preferably the compound is tested at a concentration
ranging from about 200 .mu.M to about 0.01 .mu.M, preferably from
about 100 .mu.M to about 0.01 .mu.M, and more preferably from about
10 .mu.M to about 0.01 .mu.M in the in vitro assay, wherein such a
reduction in the level of A.beta..sub.40 at the above-described
concentrations is correlative with the compound's therapeutic
efficacy.
[0127] In other non-limiting embodiments, the effective amount of
at least one compound of Formulas I-III may be correlated with the
compound's ability to reduce the level of A.beta..sub.40 in an in
vitro assay that measures the level of A.beta..sub.40 produced in a
cell culture, for example, a culture of primary cortical neurons
transduced with lentivirus carrying Swedish mutant APP (APPsw). In
one embodiment, the assay comprises incubating the A.beta..sub.40
expressing cells with a compound of Formulas I-III, followed by
assaying the concentration of A.beta..sub.40 in the cell culture
medium. In such an assay, a greater reduction of A.beta..sub.40
concentration in the cell culture medium following incubation with
a compound compared to a control cell culture not contacted with
the compound is correlative with the compound's therapeutic
efficacy.
[0128] In one non-limiting embodiment, an effective amount of a
compound of Formulas I-III may be that amount which reduces
A.beta..sub.40 by about 1-10%, more preferably from about 10-20%,
more preferably from about 20-30%, more preferably from about
30-40%, more preferably from about 40-50%, more preferably from
about 50-60%, more preferably from about 60-70%, more preferably
from about 70-80%, more preferably from about 80-90%, and more
preferably from about 90-100%, compared to A.beta..sub.40 levels in
the cell culture medium of a control cell culture that was not
incubated with the compound. Preferably the compound is incubated
at a concentration of about 200 .mu.M to about 0.01 .mu.M,
preferably from about 100 .mu.M to about 0.01 .mu.M, and more
preferably from about 10 .mu.M to about 0.01 .mu.M in the in vitro
assay, wherein a greater level of A.beta..sub.40 reduction at a
lower concentration in the in vitro assay is correlative with the
compound's therapeutic efficacy.
[0129] In other non-limiting embodiments, the effective amount of
at least one compound of Formulas I-III may be correlated with the
compound's ability to reduce the level of sAPP.beta. in an in vitro
assay that measures the level of sAPP.beta. produced in a cell
culture, for example, primary cortical neurons transduced with
lentivirus carrying Swedish mutant APP (APPsw). In one embodiment,
the assay comprises incubating the cells with a compound of
Formulas I-III, followed by assaying the concentration of
sAPP.beta. in the cell culture medium. In such an assay, a greater
reduction of sAPP.beta. concentration in the cell culture medium
following incubation with a compound compared to a control cell
culture not contacted with the compound is correlative with the
compound's therapeutic efficacy.
[0130] In one non-limiting embodiment, an effective amount of a
compound of Formulas I-III may be that amount which reduces
sAPP.beta. by about 1-10%, more preferably from about 10-20%, more
preferably from about 20-30%, more preferably from about 30-40%,
more preferably from about 40-50%, more preferably from about
50-60%, more preferably from about 60-70%, more preferably from
about 70-80%, more preferably from about 80-90%, and more
preferably from about 90-100%, compared to sAPP.beta. levels in the
cell culture medium of a control cell culture that was not
incubated with the compound. Preferably the compound is incubated
at a concentration of about 200 .mu.M to about 0.01 .mu.M,
preferably from about 100 .mu.M to about 0.01 .mu.M, and more
preferably from about 10 .mu.M to about 0.01 .mu.M in the in vitro
assay, wherein a greater level of sAPP.beta. reduction at a lower
concentration in the in vitro assay is correlative with the
compound's therapeutic efficacy.
[0131] In other non-limiting embodiments, an effective amount of a
compound of Formulas I-III may be that amount which reduces
A.beta..sub.40 by about 50% in an in vitro assay compared to
A.beta..sub.40 levels in the cell culture medium of a control cell
culture that was not incubated with the compound. Preferably the
compound is incubated at a concentration of about 200 .mu.M to
about 0.01 .mu.M, preferably from about 100 .mu.M to about 0.01
.mu.M, and more preferably from about 10 .mu.M to 0.01 .mu.M in the
in vitro assay, wherein a reduction of A.beta..sub.40 at a lower
concentration in the in vitro assay is correlative with the
compound's therapeutic efficacy.
[0132] In other non-limiting embodiments, the effective amount of
at least one compound of Formulas I-III may be correlated with the
compound's ability to reduce the level of A.beta..sub.40 in an in
vitro assay that measures the level of A.beta..sub.40 produced in a
cell culture, for example, cultured primary cortical neurons
prepared from Tg2576 mice (i.e. mice carrying human APPsw transgene
under the control of the PrP promoter). In one embodiment, the
assay comprises incubating the A.beta..sub.40 expressing cells with
a compound of Formulas I-III, followed by assaying the
concentration of A.beta..sub.40 in the cell culture medium. In such
an assay, a greater reduction of A.beta..sub.40 concentration in
the cell culture medium following incubation with a compound
compared to a control cell culture not contacted with the compound
is correlative with the compound's therapeutic efficacy.
[0133] In one non-limiting embodiment, an effective amount of a
compound of Formulas I-III may be that amount which reduces
A.beta..sub.40 by about 1-10%, more preferably from about 10-20%,
more preferably from about 20-30%, more preferably from about
30-40%, more preferably from about 40-50%, more preferably from
about 50-60%, more preferably from about 60-70%, more preferably
from about 70-80%, more preferably from about 80-90%, and more
preferably from about 90-100%, compared to A.beta..sub.40 levels in
the cell culture medium of a control cell culture that was not
incubated with the compound. Preferably when the compound is
incubated at a concentration of about 200 .mu.M to about 0.01
.mu.M, preferably from about 100 .mu.M to about 0.01 .mu.M, and
more preferably from about 10 .mu.M to about 0.01 .mu.M in the in
vitro assay, wherein a greater level of A.beta..sub.40 reduction at
a lower concentration in the in vitro assay is correlative with the
compound's therapeutic efficacy.
[0134] In other non-limiting embodiments, the effective amount of
at least one compound of Formulas I-III may be correlated with the
compound's ability to reduce the level of A.beta..sub.40 and/or
sAPP.beta. in an ex vivo assay that measures the level of
A.beta..sub.40 and/or sAPP.beta. produced in cells, for example,
organotypic brain slices from Tg2576 mice (i.e. mice carrying human
APPsw transgene under the control of the PrP promoter). In one
embodiment, the assay comprises incubating the brain slices with a
compound of Formulas followed by assaying the concentration of
A.beta..sub.40 and/or sAPP.beta. in the brain slices. In such an
assay, a greater reduction of A.beta..sub.40 and/or sAPP.beta.
concentration in the brain slices following incubation with a
compound compared to a control brain slice not contacted with the
compound is correlative with the compound's therapeutic
efficacy.
[0135] In one non-limiting embodiment, an effective amount of a
compound of Formulas I-III may be that amount which reduces
A.beta..sub.40 and/or sAPP.beta. by about 1-10%, more preferably
from about 10-20%, more preferably from about 20-30%, more
preferably from about 30-40%, more preferably from about 40-50%,
more preferably from about 50-60%, more preferably from about
60-70%, more preferably from about 70-80%, more preferably from
about 80-90%, and more preferably from about 90-100%, compared to
A.beta..sub.40 and/or sAPP.beta. levels in control brain slices
that were not incubated with the compound. Preferably the compound
is incubated at a concentration of about 200 .mu.M to about 0.01
.mu.M, preferably from about 100 .mu.M to about 0.01 .mu.M, and
more preferably from about 10 .mu.M to about 0.01 .mu.M in the ex
vivo assay, wherein a greater level of A.beta..sub.40 and/or
sAPP.beta. reduction at a lower concentration in the ex vivo assay
is correlative with the compound's therapeutic efficacy.
[0136] In other non-limiting embodiments, the effective amount of
at least one compound of Formulas I-III may be correlated with the
compound's ability to reduce the level of A.beta..sub.40 and/or
sAPP.beta. in an in vivo assay that measures the level of
A.beta..sub.40 and/or sAPP.beta. produced in a test subject, for
example, a Tg2576 mouse (i.e. mice carrying human APPsw transgene
under the control of the PrP promoter). In one embodiment, the
assay comprises administering at least one compound of Formulas
I-Ill to the test subject, for example, via interstitial fluid
(ISF) compound administration, intraperitoneal (IP) compound
injection, or through the use of a microdialysis apparatus for
infusion of the compound at multiple concentrations (for example,
in the hippocampus of the test subject), followed by assaying the
concentration of A.beta..sub.40 and/or sAPP.beta. in the test
subject. In such an assay, a greater reduction of A.beta..sub.40
and/or sAPP.beta. concentration in the test subject following
administration of the compound compared to a control subject not
administered the compound is correlative with the compound's
therapeutic efficacy.
[0137] In one non-limiting embodiment, an effective amount of a
compound of Formulas I-III may be that amount which reduces
A.beta..sub.40 and/or sAPP.beta. by about 1-10%, more preferably
from about 10-20%, more preferably from about 20-30%, more
preferably from about 30-40%, more preferably from about 40-50%,
more preferably from about 50-60%, more preferably from about
60-70%, more preferably from about 70-80%, more preferably from
about 80-90%, and more preferably from about 90-100%, compared to
A.beta..sub.40 and/or sAPP.beta. levels in brain homogenates of
subjects that were not administered the compound. Preferably the
compound is administered at a concentration of about 0.5 mg/kg to
about 20 mg/kg, preferably from about 1 mg/kg to about 20 mg/kg,
more preferably from about 3 mg/kg to about 20 mg/kg, more
preferably from about 5 mg/kg to about 20 mg/kg, more preferably
from about 10 mg/kg to about 20 mg/kg in the in vivo assay, wherein
a greater level of A.beta..sub.40 and/or sAPP.beta. reduction at a
lower concentration in the in vivo assay is correlative with the
compound's therapeutic efficacy.
[0138] In non-limiting embodiments, an effective amount of a
compound of Formulas I-III may be an amount which achieves a local
concentration at the therapeutic site of about 100 .mu.M to about
0.01 .mu.M, preferably from about 50 .mu.M to about 0.01 .mu.M,
more preferably from about 20 .mu.M to about 0.01 .mu.M, and more
preferably from about 10 .mu.M to about 0.01 .mu.M in the in vitro
assay.
5.3.3 Administration of Treatments
[0139] According to the invention, the component or components of a
pharmaceutical composition of the invention may be administered to
a subject by means including but not limited to intravenous,
intra-arterial, intramuscular, intradermal, transdermal,
subcutaneous, oral, intraperitoneal, intraventricular, and
intrathecal administration.
[0140] In particular non-limiting embodiments, the therapeutic
compound can be delivered in a controlled or sustained release
system. For example, a compound or composition may be administered
using intravenous infusion, an implantable osmotic pump, a
transdermal patch, liposomes, or other modes of administration. In
one embodiment, a pump may be used (see Sefton, 1987, CRC Crit.
Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507;
Saudek et al., 1989, N. Engl. J. Med. 321:574). In another
embodiment, polymeric materials can be used (see Langer and Wise
eds., 1974, Medical Applications of Controlled Release, CRC Press:
Boca Raton, Fla.; Smolen and Ball eds., 1984, Controlled Drug
Bioavailability, Drug Product Design and Performance, Wiley, N.Y.;
Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem.,
23:61; Levy et al., 1985, Science 228:190; During et al., 1989,
Ann. Neural., 25:351; Howard et al., 9189, J. Neurosurg. 71:105).
In yet another embodiment, a controlled release system can be
placed in proximity of the therapeutic target, i.e., the heart or a
blood vessel, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, 1984, in Medical Applications of Controlled
Release, supra, Vol. 2, pp. 115-138). Other controlled release
systems known in the art may also be used.
5.4 Pharmaceutical Compositions
[0141] The compounds and compositions of the invention may be
formulated as pharmaceutical compositions by admixture with a
pharmaceutically acceptable carrier or excipient.
[0142] For example, the pharmaceutical composition may comprise an
effective amount of at least one compound of Formulas I-III and a
physiologically acceptable diluent or carrier. The pharmaceutical
composition may further comprise a second drug, for example, but
not by way of limitation, an anti-cancer drug, an anti-inflammatory
drug, for example, but not limited to, a steroid compound and/or a
non-steroidal anti-inflammatory drug, or compound for the treatment
of Alzheimer's disease, such as an acetylcholinesterase inhibitor
or an NMDA glutamate receptor antagonist (e.g. memantine).
[0143] The phrase "pharmaceutically acceptable" indicates that a
substance is physiologically tolerable when administered to a
subject. Preferably, but not by way of limitation, as used herein,
the term "pharmaceutically acceptable" means approved by a
regulatory agency of the federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans. The term
"carrier" refers to a diluent, adjuvant, excipient, or vehicle with
which the compound is administered. Such pharmaceutical carriers
can be sterile liquids, such as water and oils, or, for solid
dosage forms, may be standard tabletting excipients. Water or
aqueous solution saline solutions and aqueous dextrose and glycerol
solutions are preferably employed as carriers, particularly for
injectable solutions. Suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin,
18th Edition, or other editions.
[0144] In a specific embodiment, the therapeutic compound can be
delivered in a vesicle, in particular a liposome (see Langer, 1990,
Science 249:1527-1533; Treat et al., 1989, in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler eds., Liss: New York, pp. 353-365; Lopez-Berestein, ibid.,
pp. 317-327; see generally Lopez-Berestein, ibid.).
[0145] The present invention is not to be limited in scope by the
specific embodiments described herein and the Examples that follow.
Indeed, various modifications of the invention in addition to those
described herein will become apparent to those skilled in the art
from the foregoing description and the accompanying Examples and
Figures. Such modifications are intended to fall within the scope
of the appended claims.
[0146] Patents, patent applications, publications, product
descriptions, GenBank Accession Numbers, and protocols are cited
throughout this application, the disclosures of which are
incorporated herein by reference in their entireties for all
purpose.
EXAMPLES
Example 1
Synthesis of Compounds of Formulas I-III
[0147] The compounds of Formulas I and II consist of
N-(quinolin-8-yl)benzensulfonamides, and the compounds of Formula
III consist of a structurally constrained cyclic sulfonamide with
the sultam bridging quinolinyl and phenyl moieties. This particular
scaffold lacks a general synthetic method. Described here is an
efficient synthesis of this class of compounds that allows a
diverse substitution pattern.
[0148] The synthetic strategy was to generate N-8-quinolinyl
benzenesulfonamides, and then construct the sultam skeleton through
cyclization of the easily accessible N-8-quinolinyl
benzenesulfonamides. Diazotization-induced cyclization was employed
to synthesize the target compound (FIG. 2). Sulfonamide 8 was
prepared from 2-nitrobezenesufonyl chloride and 8-aminoquinoline 2
in pyridine. The nitro group was reduced with SnCl.sub.2 to provide
amine 9 and subsequent diazotization afforded triazine 10. A
procedure employing Cu (0) and NaOH (Ullman et al., 1911, Ber.
Dtsch. Chem. Ges. 43:2694), failed to yield the desired product,
but thermolysis in a variety of solvents (e.g. EtOH, H.sub.2O, DMSO
or HOAc) or neat afforded the product 1. The best result (41%
yield) was obtained by heating in glacial acetic acid at
120.degree. C. for ten minutes.
[0149] A second synthetic method involves a one-pot synthesis of
N-8-quinolinyl benzenesultams from
N-8-quinolinyl-2-aminobezenesulfonamides. In a typical reaction, as
shown in FIG. 3A, 2-aminobenzenesulfonamide 9a, prepared from
6-methoxy-2-methylquinolin-8-amine 2a (Qiu et al., 2007, Inorg.
Chim. Acta. 360:431), was dissolved in HOAc and treated with 1.5
equiv of t-BuONO at 10.degree. C. The reaction was allowed to warm
to room temperature over 10 minutes and afforded sultam 1a in (78%
yield) without isolation of the triazine intermediate.
[0150] A selection of N-8-quinolinyl-2-aminobezenesulfonamides
(9a-p) were prepared and subsequently cyclized in similar one-pot
reactions to give sultams (1a-p) (FIG. 4). In each case, the
product was isolated without intermediate purification (FIG. 4).
Aminobezenesulfonamides (9b-h and 9l-p) were synthesized in a
similar manner described in FIG. 3A. Intermediates 9i-k were
prepared from aniline 11 as outlined in FIG. 3B: Sulfonamidation of
aniline 11 with 2-nitrobezenesufonyl chloride provided sulfonamide
12. Skraup reaction under modified conditions (Matsugi et al.,
2000, Tetrahedron Lett. 41:8523) followed by reduction of nitro
group provided the N-quinolinyl sulfonamides (9i-k).
[0151] A typical synthesis of a compound of Formulas II and III
involves the following steps:
[0152] Compound 9a: To a solution of
6-methoxy-2-methylquinolin-8-amine 2a (0.19 g, 1.0 mmol) in
pyridine (5 ml) was added 4-methyl-2-nitrobenzenesulfonyl chloride
(0.24 g, 1.0 mmol). The mixture was stirred at room temperature
overnight and precipitated with H.sub.2O. The crude product was
filtered and recrystallized from EtOH to afford
nitrobenzenesulfonamide (0.31 g, 79%) as red crystal. .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 10.1 (br, 1H); 8.04 (d, 1H), 7.86 (d,
1H), 7.62 (m, 2H), 7.39 (d, 1H), 7.24 (d, 1H), 6.73 (s, 1H), 3.88
(s, 31-1), 2.66 (s, 3H), 2.41 (s, 3H); ESI-MS (M.sup..+-..+-.1):
388.0. To a suspension of above nitro compound (0.20 g, 0.56 mmol)
in EtOH (5 ml) was added SnCl.sub.2 (0.32 g, 1.7 mmol) slowly. The
mixture was refluxed for 2 h. After removal of EtOH, the residue
was treated with 1M NaOH. The aqueous solution was extracted with
CH.sub.2Cl.sub.2. The combined organic phases were washed by brine,
dried over Na.sub.2SO.sub.4, and concentrated to yield 9a (0.18 g,
92%) as white solid. .sup.1H NMR (300 MH.sub.z, CDCl.sub.3) .delta.
9.42 (br, 1H); 7.83 (d, 1H), 7.65 (d, I H), 7.30 (s, 1H), 7.23 (d,
1H), 6.64 (s, 1H), 6.48 (d, 1H), 6.42 (s, 111), 3.80 (s, 3H), 2.61
(s, 3H), 2.17 (s, 3H); ESI-MS (M.sup.++1): 358.1.
[0153] Compound 1a:
[0154] To a solution of 9a (0.1 g, 0.28 mmol) in HOAc (1 ml) at
10.degree. C. was added t-BuONO (0.05 mL, 0.42 mmol). The reaction
was slowly warmed to room temperature over 10 min and quenched with
H.sub.2O. The mixture was extracted with EtOAc. The combined
organic phases were washed with saturated NaHCO.sub.3, dried over
Na.sub.2SO.sub.4, and concentrated. Flash chromatography
(EtOAc/CH.sub.2Cl.sub.2 1:10 vv) gave 1a (74 mg, 78%) as yellow
solid. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.49 (s, 1H), 7.98
(d, 1H), 7.92 (d, 1H), 7.37 (d, 114), 7.25 (d, 1H), 6.87 (s, 1H),
4.00 (s, 3H), 2.69 (s, 3H), 2.52 (s, 3H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 156.2, 155.2, 142.2, 134.8, 134.5, 134.1,
132.2, 131.4, 130.1, 129.0, 126.8, 124.4, 122.0, 112.5, 100.0,
56.2, 25.2, 22.6; ESI-MS (M.sup.++1): 341.2.
[0155] In summary, a one-pot reaction for the conversion of
N-8-quinolinyl-2-aminobezenesulfonamides into their corresponding
sultams under mild conditions has been described. This procedure
offers access to a class of heterocyclic compounds that have shown
therapeutic potential as novel BACE1 and NF-.kappa.B inhibitors, as
described below.
Example 2
Inhibition of NFkB Activity
[0156] Small molecule regulation of signaling cascades associated
with NFkB may provide novel approaches to alleviate numerous
disease states, (D'Acquisto, et al., 2006 Curr. Opin. Pharmacol.
6:387; Gilmore, et al., 2006, Oncogene 25:6887). Several
high-throughput campaigns in search of novel inhibitors of the NFkB
signal transduction pathway have been undertaken. Two of these
include a cell-based assay designed to identify IkB.alpha.
stabilization, and a cell-based assay designed to identify
inhibitors of TNF.alpha.-induced translocation of NFkB. Despite the
differing assay design, cell type, and signal readout, a
N-(quinolin-8-yl)benzenesulfonamide core scaffold was identified in
both screens.
NFkB Inhibitory Assays
[0157] The first assay, an IkB.alpha. stabilization screen, was
performed using a dual luciferase reporter system in the cell line
OCILy3, an excellent model for primary tumors of the activated
B-cell subtype (ABC) of diffuse large B-cell lymphoma (DLBCL)
(Figure SA; PubChem AID: 445) (Davis, et al., 2007, Drug Dev.
Technol. 5:85; Davis, et al., 2001, J. Exp. Med. 184:1861; Okamoto,
et al., 2007, Curr. Pharm. Des. 13:447) Abnormally high
constitutive NFkB activity levels have been noted in ABC-DLBCL, as
in several types of cancer, due to high levels of IKK13 activity
leading to elevated expression of NFkB targeted genes. (Okamoto, et
al., 2007, Curr. Pharm. Des. 13:447) In this setting, IKK and
proteasome activity produce high degradation of IkB.alpha.,
liberating p50/65 and/or p50/c-Rel heterodimers to translocate to
the nucleus. Because lines of ABC-DLBCL and other cancer types are
dependent on constitutive NFkB activity, the NFkB pathway is a
therapeutic target. This is particularly true for those processes
governing IkB.alpha. degradation, and the therapeutic potential of
small molecules for this purpose has been shown by studies in
ABC-DLBCL lines using a specific IKK.beta. inhibitor. Furthermore,
the use of an ABC-DLBCL line for a small-molecule screen of
IkB.alpha. stabilization provides a context that is especially
close to that of the targeted disease, with the potential that
inhibitors may be found that affect specific upstream points in IKK
activation.
[0158] To identify modulators of IkB.alpha. stability in ABC-DLBCL
lines, such as small molecules inhibiting IKK or proteasome
activity, changes in the level of an exogenous
IkB.alpha.-luciferase fusion reporter expressed by an
NFkB-insensitive promoter were measured. (Lam et al., 2005, Clin.
Cancer res. 11:28; Ngo et al., 2006, Nature 441:106) The dual
luciferase IkB.alpha. stabilization screen in OCI-Ly3 employed was
designed to be suitable for HTS, by using IkB.alpha. fused to a
green light-emitting beetle luciferase, with a red light-emitting
beetle luciferase expressed in a native form to monitor cell
uniformity and non-specific effects. (Davis, et al., 2007, Drug
Dev. Technol. 5:85) Fold-responsiveness was further increased by
having both reporters under the control of inducible promoters
regulated by doxycycline. Upon doxycycline induction of both
luciferase reporters, compounds that increased green luminescence
with minimal effects on the red luminescence signal were scored as
IkB.alpha. stabilizers. (Davis, et al., 2007, Drug Dev. Technol.
5:85).
[0159] The second assay, a translocation-based assay, was a
high-content screen performed in human umbilical vein endothelial
cells (HUVEC) using TNF.alpha. to stimulate nuclear translocation
of endogenous NFkB (Figure SB; PubChem AID: 438). NFkB is
sequestered in the cytoplasm due to its binding to IkB.alpha.,
which blocks exposure of a nuclear localization sequence.
Activation by cytokines such as TNF.alpha. results in proteasome
degradation of IkB.alpha. and subsequent translocation of NFkB from
the cytoplasm to the nucleus. In the assay, nuclear translocation
of the endogenous p65 RelA subunit of NFkB, at 30 min
post-stimulation, was monitored using fluorescent antibody
detection and an automated imaging platform. (Mayer et al., 2006,
Methods Enzyme 414:266) NFkB inhibitors in this assay, such as
IkB.alpha. stabilizers, were detected as compounds that interfered
with p65 translocation to the nucleus.
[0160] The above-described assays were validated using known
inhibitors of the NFkB pathway (FIG. 6). The proteasome inhibitor
MG-132 (1) 16 served as a positive control for the IkB.alpha.
stabilization assay. The translocation assay used BAY 11-7082 (2),
an agent that inhibits the TNF.alpha.-induced phosphorylation of
IkB.alpha.. (Kamthong, 2001, Biochem 356:525; Izban et al., 2000,
Hum. Pathol. 31:1482; Pierce et al., 1997, J. Biol. Chem.
272:21096). Additionally, a substituted
2-(thiophen-2-yl)quinazoline 3 which acts as an inhibitor of NFkB
and AP1 mediated transcriptional activation (Palanki et al., 2003,
Bioorg. Med. Chem. Lett. 13:4077) was used as a positive control in
a secondary assay that used TNF.alpha. to stimulate NFkB-dependent
expression of a .beta.-lactamase reporter (NFkB-bla in FIG. 7).
This compound also served as a negative control for the dual
luciferase IkB.alpha. stabilization assay.
Results
[0161] N-(quinolin-8-yl)benzenesulfonamide (4) and the related
C7-locked N-(quinolin-8-yl)benzenesulfonamide (5) were identified
as two common structures in both screens (FIG. 6). To further
confirm that these agents interfered with the NFkB activation in a
genuine manner, a reporter assay obtained from Invitrogen where
induction of .beta.-lactamase occurred in an NFkB-dependent manner
was also performed on the active compounds. These data confirmed
the ability of these compounds to inhibit the NFkB pathway, and the
compounds were then advanced for further study (FIG. 7).
[0162] The synthesis of analogues of (4) was undertaken through a
one-step process involving the addition of unsubstituted or
2-substituted, 6-substituted or 2,6-disubstituted quinolin-8-amines
and a variety of sulfonyl chlorides (FIG. 7B). Amino-substituted
analogues (19-22) (Izban et al., 2000, Hum. Pathol. 31:1482) were
obtained by reduction of the corresponding nitro compounds with
stannous chloride in ethanol (FIG. 7D). Over 40 novel analogues
were prepared to establish preliminary structure activity
relationships.
Discussion
[0163] The synthesis of various analogues of (5) via a
diazotization-induced cyclization of accessible
N-8-quinolinyl-2-aminobenzenesulfonamides proved to be an efficient
way of synthesizing this type of C7-locked sulfonamide (FIG.
7D).
[0164] Analogues of (4) and (5) were analyzed within both screens.
Results for analogues of (4) are shown in FIG. 7A, while the
results for derivatives of (5) are shown in FIG. 7C. In general,
there was agreement in the relative potencies of analogues tested
in both screening assays, as well as the NFkB .beta.-lactamase
reporter assay (although exceptions do exist).
[0165] A more rigid structure was associated with compound 5,
because a bridge between the quinoline carbon 7 and the
ortho-position of the sulfonyl phenyl ring locks the core scaffold
in a rigid conformation. Two compounds (30 and 31) with the best
overall potency values were identified here. Substituents at the 9
position (para to the sulfonylamide moiety designated R''' in FIG.
7C) are generally well tolerated as are 6-methoxy and 2-methyl
substitutions (analogues 32 and 37-40, respectively).
Example 3
High- and Medium-Throughput Screening of Small Molecule Libraries
to Identify Small Molecule Modulators of BACE1
[0166] A cell-based modified ELISA assay for measuring sAPP.beta.,
the secreted ectodomain of .beta.-amyloid precursor protein (APP)
following .beta.-secretase (BACE 1) cleavage, was used to identify
a class of compounds that interfered with the first step of
sAPP.beta. generation. This assay has been described in
International Application PCT/US2007015938 (Published as
International Publication No. WO 08/008463), which is herein
incorporated in its entirety for all purposes.
[0167] BACE1-mediated cleavage of APP is a key and necessary event
in the generation of neurotoxic .beta.-amyloid (A.beta.), a widely
accepted contributor to the development of Alzheimer's disease
(AD). Studies in BACE 1 knockout mice showed that they are viable,
fertile, and do not produce A.beta., making BACE1 an attractive
target for AD therapeutic intervention.
[0168] The SY5Y-BACEGFP-SEAPAPPwt cell based assay was developed to
discover novel small molecule modulators of BACE1 activity. SY5Y
cells were stably transfected with GFP-tagged BACE1 (BACE-GFP) and
secreted alkaline phosphatase (SEAP)-tagged wildtype APP
(SEAP-APPwt). BACE1 cleavage of SEAP-APPwt results in secretion
into the media of SEAP-tagged sAPP.beta., which is collected and
specifically captured using an sAPP.beta. cleavage site-specific
antibody (s.beta.wt). After washing, the fluorescent alkaline
phosphatase substrate 4-methylumbelliferyl phosphate (4-MUP) is
used to detect the captured SEAP-sAPP.beta. (FIG. 8).
Targeted Screening Using N-(Quinolin-8-Yl)Benzenesulfonamides and
Related Compounds
[0169] N-(quinolin-8-yl)benzenesulfonamides (FIG. 2) were recently
reported as agents capable of down-regulating NF-.kappa.B activity
(Xie et al., 2008). NF-.kappa.B is a ubiquitous transcription
factor that is thought to play a role in many cellular processes,
including immune and inflammatory responses, developmental
processes, cellular growth, and apoptosis. Dysregulation of
NF-.kappa.B signaling has been associated with many disease states,
including asthma, arthritis, cancer, inflammatory conditions,
neurodegenerative diseases, and heart disease (reviewed in Kumar et
al., 2004). NF-.kappa.B inhibitors have also been reported to
inhibit Ap production (Paris et al., 2007). Furthermore,
N-(quinolin-8-yl)benzenesulfonamides bear structural similarity to
clioquinol and its second-generation compound PBT2. Both clioquinol
and PBT2 are believed to be strong metal ion chelators that may
interfere with oligomerization and aggregation of AP and promote
its clearance (Ritchie et al., 2003). PBT2, developed by Prana
Biotechnology, is currently in Phase II clinical trial for the
treatment of AD. Thus, a small library of
N-(quinolin-8-yl)benzenesulfonamides and related compounds was
screened for agents that reduce BACE1-mediated cleavage of APP.
[0170] 176 compounds were tested in the primary screen (FIG. 9).
Briefly, each compound (10 mM stock in DMSO) was diluted 1:25 in
DMSO and loaded onto 96-well plates to generate a 400 .mu.M stock
plate. 1 .mu.l of each compound was then diluted 1:200 with cell
culture media (0.5% DMSO concentration) in two separate 96-well
plates for a final screening concentration of 2 .mu.M. 150 .mu.l of
media containing compound was then transferred onto
SY5Y-BACEGFP-SEAPAPPwt cells in 96-well format, and the BACE1 assay
was performed as described previously. The primary screen was
conducted in duplicate. A parallel screen was conducted at 10 .mu.M
concentration, also in duplicate. This data is not shown. Percent
inhibition of the fluorescence signal was calculated as
100.times.[DMSO control compound]/DMSO control The majority of data
points were clustered between -25% and 25% inhibition of sAPP.beta.
fluorescence signal (FIG. 9A). The Z' factors for all 4 96-well
plates used in the primary screen were above 0.7 (data not shown).
The threshold for hit selection was set at 3 standard deviations,
or 59.76% inhibition, yielding 6 small molecule hits and a hit rate
of 3.41% (FIG. 9B).
[0171] Several partial hits were also identified in the targeted
screen of N-(quinolin-8-yl)benzenesulfonamides. Partial hits were
defined loosely as compounds that either exhibited activity just
below the threshold 59.76% inhibition in the 2 .mu.M screen or
those that displayed significant activity in the 10 .mu.M screen.
These compounds were characterized for 8-point dose-response and
structure-activity relationship studies.
[0172] In summary, a cell-based BACE1 assay was used to screen
N-(quinolin-8-yl)benzene-sulfonamides and related compounds, a
class of NF-.kappa.B inhibitors. Because of their potency,
availability, and the reported connection between NF-.kappa.B
inhibition and AP generation, structural analogs of
N-(quinolin-8-yl)benzenesulfonamides were also selected for
investigation in structure-activity relationship studies. Primary
and secondary screening using the cell-based BACE1 assay thus
yielded chemotypes for SAR studies and further characterization in
more physiological systems.
Example 4
Structure-Activity Relationship Studies and Characterization in
Physiological Systems
[0173] SAR studies were conducted for full and partial hits from
the targeted screen of N-(quinolin-8-yl)benzenesulfonamides.
Characterization in SY5 Y-BACEGFP-SEAPAPPwt cells identified CU-264
as the most potent analog. CU-264 was further characterized in a of
more physiological assay for its ability to reduce A.beta..sub.40
and sAPP.beta.. CU-264 had the effect of raising A.beta..sub.40 and
sAPP.beta. levels.
SAR Studies of N-(quinolin-8-yl)Benzenesulfonamides
[0174] Targeted screening of N-(quinolin-8-yl)benzenesulfonamides
and related compounds revealed several full and partial hits
capable of reducing the fluorescent signal from the cell-based
BACE1 assay. Based on the chemical structures of the 176 compounds
as well as the full and partial hits from the targeted screen, the
structural elements that were critical for activity were
identified. All full and partial hits contained the sulfonamide of
quinoline motif either in open or cyclized varieties, as shown in
FIG. 10. Substituting naphthalene for quinoline or removal of the
sulfonamide substituent resulted in loss of activity.
[0175] 28 full and partial hits were characterized in 8-point
dose-response experiments for IC.sub.50 determination in
SY5Y-BACEGFP-SEAPAPPwt cells (FIG. 10). The investigation revealed
3 compounds with sub-micromolar potencies for sAPP3
reduction--CU-163, CU-264, and CU-262. Analysis of the data
revealed a few structure-activity relationships. Addition of a
methyl group at R3 resulted in a reduction of potency (compare
CU-274, CU-278, CU-280, and CU-281 with CU-242, CU-271, CU-265, and
CU-264, respectively). The electronic properties of the R groups
did not affect compound activity based on the available data. The
addition of electron-withdrawing, electron-donating, and
electron-neutral groups did not have a clear and consistent effect
on activity. Although there are no direct comparisons, cyclization
of compounds (e.g. CU-262 and CU-282) did not significantly alter
the potency compared with non-cyclized compounds (e.g. CU-264,
CU-265, CU-280, and CU-281).
[0176] SAR studies of N-(quinolin-8-yl)benzenesulfonamides revealed
3 compounds with sub-micromolar potencies for sAPP.beta. reduction.
The most potent compound, CU-264, is further evaluated.
Evaluation of CU-264 in More Physiological Systems
[0177] The previously described hit was further analyzed in
physiologically relevant systems, e.g. primary neurons and
Alzheimer's model mice. Two complementary neuronal cell systems
were employed to test the effects of candidate compounds on various
aspects of APP processing: cultured mouse cortical neurons
(postnatal day 0) infected with recombinant lentivirus carrying
human APPsw (Lenti-APPsw; FIG. 11); and cultured cortical neurons
prepared from Tg2576 mice (carrying human APPsw transgene under the
control of the PrP promoter). Lenti-APPsw infected cortical neurons
can be prepared in a relatively large scale, but suffer from
variability in lentiviral infection efficiency, resulting in
variable levels of APP expression. In contrast, cortical neurons
derived from Tg2576 provide constant APP expression. However,
neuronal yield is generally much lower, since only half the pups
will contain the transgene when heterozygote crossbreeds were
conducted. Therefore, Initial characterization experiments were
performed using cortical neurons infected with Lenti-APPsw.
Compounds exhibiting inhibitory activity on sAPP.beta. and/or
A.beta. would then be re-evaluated in Tg2576 mice-derived
neurons.
[0178] Ex vivo and in vivo assays will be used to characterize
certain hit(s). Ex vivo systems, such as organotypic brain slices,
offer an alternative to in vivo assays, and can also be used to
test a large number of compounds. Further more, at least with
regard to the A.beta. release phenotype, brain slice data have been
shown to correlate well with the results obtained in vivo (reviewed
in Noraberg et al., 2005). Brain slices from p7 Tg2576 pups are
used herein for compound characterization.
[0179] For in vivo studies, interstitial fluid (ISF) compound
administration and A.beta. measurement, as well as intraperitoneal
(IP) compound injection, both utilizing Tg2576 mice, will be used
to characterize compounds. Positioning of a guide cannula to the
mouse hippocampus allows for insertion of a microdialysis
apparatus, which can be used to infuse compounds at multiple
concentrations sequentially in the awake mouse. A.beta.
measurements can be performed using the same apparatus, yielding
rapid dose-response determinations (Cirrito et al., 2003). This
method allows for rapid assessment of compound effect on AP on a
dynamic time scale. More conventionally, compounds can also be
administered via IP injection. For these experiments, 12-month old
Tg2576 mice will be used.
CU-264 does not Reduce A.beta.40 in Lenti-APPsw Infected Primary
Cortical Neurons
[0180] Lenti-APPsw infected primary cortical neurons were used for
the initial round of physiological experiments. Primary cortical
neurons were harvested from wild-type P0 pups using established
protocols. The majority of cells from the resulting culture exhibit
neuronal morphology on light microscopy and express neuronal
.beta.-tubulin as visualized by immunocytochemistry using the TUJ1
antibody (Covance, FIG. 12).
[0181] The Lenti-APPsw vector was co-transfected into HEK293 T
cells with ViraPower packaging mix (Invitrogen) to generate the
lentivirus. Lentiviral-mediated transduction of APPsw in primary
neurons was performed by adding neuron primary culture media
containing the lentiviral particles to wild-type DIV-14 primary
neurons (FIG. 11). After 24 hours, neurons were incubated with
media for 72 hours prior to collecting for A.beta..sub.40
measurement. Two batches of virus (LV-1 and LV-2) were tested,
showing that there is batch-to-batch variation in the viral
titer.
[0182] Using this experimental paradigm, CU-264 was tested at 10
and 2 .mu.M concentrations to examine the sAPP.beta.- and
A.beta.-lowering activity of the compound. The protocol was
modified slightly to allow for 24-hour compound treatment. DIV-14
wild-type primary neurons (FIG. 12) cultured on 6-well plates were
incubated for 24 hours with primary culture media containing
lentiviral particles. After infection, fresh media was mixed in a
1:1 ratio with conditioned media collected prior to lentiviral
infection, and applied to the cells for 48 hours to allow for APP
expression. At the end of this "pre-drug" incubation, media
containing compound was applied for 24-hour treatment. Control
experiments using DMSO alone showed that media collected after the
24-hour treatment period contained at least two times higher
A.beta..sub.40 than that collected just after the 48-hour pre-drug
period. CU-264, which exhibited sub-micromolar potency for
sAPP.beta. reduction in SY5Y-BACEGFP-SEAPAPPwt cells, did not
reduce A.beta..sub.40 in primary neurons (FIG. 13A). In contrast,
CU-264 was observed to increase AP at 10 .mu.M.
[0183] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0184] Patents, patent applications, publications, product
descriptions, GenBank Accession Numbers, and protocols are cited
throughout this application, the disclosures of which are
incorporated herein by reference in their entireties for all
purposes.
* * * * *