U.S. patent application number 11/375152 was filed with the patent office on 2006-07-20 for methods and compositions for treatment of central nervous system disorders.
Invention is credited to Merouane Bencherif, Mario B. Marrero.
Application Number | 20060160835 11/375152 |
Document ID | / |
Family ID | 26992172 |
Filed Date | 2006-07-20 |
United States Patent
Application |
20060160835 |
Kind Code |
A1 |
Bencherif; Merouane ; et
al. |
July 20, 2006 |
Methods and compositions for treatment of central nervous system
disorders
Abstract
The invention provides methods of screening for substances
having an effect on a nicotine receptor by contacting a cell having
a nicotine receptor with a test substance; and determining any
increase or decrease in phosphorylation of Janus-Activated Kinase 2
(JAK2). An increase in phosphorylation of JAK2 indicates that the
test substance stimulates the nicotine receptor, and wherein a
decrease in phosphorylation of JAK2 indicates that the test
substance inhibits the nicotine receptor. The invention also
provides screening methods for identification of substances that
affect nicotine receptor activity through activity mediated by the
AT2 receptor. Related pharmaceutical compositions and methods of
treatment are also provided.
Inventors: |
Bencherif; Merouane;
(Winston-Salem, NC) ; Marrero; Mario B.; (Evans,
GA) |
Correspondence
Address: |
WOMBLE CARLYLE SANDRIDGE & RICE, PLLC
ATTN: PATENT DOCKETING 32ND FLOOR
P.O. BOX 7037
ATLANTA
GA
30357-0037
US
|
Family ID: |
26992172 |
Appl. No.: |
11/375152 |
Filed: |
March 14, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10318842 |
Dec 13, 2002 |
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11375152 |
Mar 14, 2006 |
|
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60340582 |
Dec 14, 2001 |
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60369934 |
Apr 4, 2002 |
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Current U.S.
Class: |
514/278 |
Current CPC
Class: |
G01N 33/946 20130101;
A61P 25/28 20180101; G01N 33/9406 20130101; A61P 43/00 20180101;
A61P 25/00 20180101; A61K 31/44 20130101; A61K 31/505 20130101;
G01N 2500/00 20130101; C12Q 1/485 20130101 |
Class at
Publication: |
514/278 |
International
Class: |
A61K 31/4747 20060101
A61K031/4747 |
Claims
1. A composition comprising, a) a substance that binds .alpha.7
nicotinic acetylcholine receptors (.alpha.7-nAChR); and b) at least
one inhibitor of the AT2 receptor or at least one inhibitor of a
substance that stimulates the AT2 receptor; and c) a
pharmaceutically acceptable carrier.
2. The composition of claim 1, wherein the substance that binds
.alpha.7-nAChR is selected from the group consisting of
2-(3-pyridyl)azaadamantanes, diazabicyclic compounds,
pyridylazabicyclic compounds, cinnamamides of 3-aminoquinuclidine,
arylcarbamates of 3-quinuclidinol, aromatic amides of
3-aminoquinuclidine, spiroquinuclidines, and
benzylideneanabaseines.
3. The composition of claim 1, wherein the substance that inhibits
the AT2 receptor or that inhibits a substance that stimulates the
AT2 receptor is a substance that inhibits a substance that
stimulates AT2.
4. The composition of claim 1, wherein the substance that inhibits
the AT2 receptor or that inhibits a substance that stimulates the
AT2 receptor is a substance that inhibits AT2.
5. The composition of claim 1, wherein the substance that binds
.alpha.7 nAChR is a 2-(3-pyridyl)azaadamantane compound selected
from the group consisting of
1-aza-2-(5-bromo(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane;
1-aza-2-(5-amino-(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane;
1-aza-2-(5-ethoxy-(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane;
1-aza-2-(5-isopropoxy-(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane;
2-(3-pyridyl)-1-azatricyclo[3.3.1.1.sup.3,7]decane;
5-aza-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]decan-2-ol;
5-aza-1-(hydroxymethyl)-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]-decan-2-on-
e; 1-aza-2-(3-pyridyl)-1-azatricyclo[3.3.1.1.sup.3,7]decane;
5-aza-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]decan-2-ol;
5-aza-1-(hydroxymethyl)-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]-decan-2-on-
e; 5-aza-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]decan-2-one;
enantiomers thereof; and pharmaceutically acceptable salts
thereof.
6. The composition of claim 1, where the substance that binds
.alpha.7 nAChR is a diazabicyclic compound selected from the group
consisting of
(1S,4S)-2-(5-phenoxy-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane;
(1R,4R)-2-(5-phenoxy-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane;
(1S,4S)-2-(5-(3-methoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-
;
(1R,4R)-2-(5-(3-methoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]hepta-
ne;
(1S,4S)-2-(5-(4-methoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]hep-
tane;
(1R,4R)-2-(5-(4-methoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]h-
eptane;
(1S,4S)-2-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2-
.2.1]heptane;
(1R,4R)-2-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]hep-
tane;
(1S,4S)-2-(5-(4-fluorophenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]he-
ptane;
(1R,4R)-2-(5-(4-fluorophenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]h-
eptane;
(1S,4S)-2-(5-benzoyl-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane;
(1R,4R)-2-(5-benzoyl-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane;
(1S,4S)-2-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-2,5-diazabicyclo[2.2-
.1]heptane;
(1R,4R)-2-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-2,5-diazabicyclo[2.2-
.1]heptane;
(1S,4S)-2-(5-(4-(N-trifluoroacetylpiperidinyl)oxy)-3-pyridyl)-2,5-diazabi-
cyclo[2.2.1]heptane;
(1R,4R)-2-(5-(4-(N-trifluoroacetylpiperidinyl)oxy)-3-pyridyl)-2,5-diazabi-
cyclo[2.2.1]heptane;
6-methyl-3-(5-phenyl-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
6-methyl-3-(5-phenoxy-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
6-methyl-3-(5-(3-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane-
;
6-methyl-3-(5-(4-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]octa-
ne;
6-methyl-3-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.-
1]octane;
6-methyl-3-(5-(4-fluorophenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2-
.1]octane;
6-methyl-3-(5-benzoyl-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
6-methyl-3-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,6-diazabicyclo[3.-
2.1]octane;
3-methyl-6-(5-phenyl-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
3-methyl-6-(5-phenoxy-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
3-methyl-6-(5-(3-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane-
;
3-methyl-6-(5-(4-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]octa-
ne;
3-methyl-6-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.-
1]octane;
3-methyl-6-(5-(4-fluorophenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2-
.1]octane;
3-methyl-6-(5-benzoyl-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
3-methyl-6-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,6-diazabicyclo[3.-
2.1]octane; 6-(5-phenyl-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-phenoxy-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-(3-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-(4-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-(4-fluorophenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-benzoyl-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonan-
e; 3-(5-phenyl-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-phenoxy-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-(3-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-(4-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-(4-fluorophenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-benzoyl-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonan-
e; 3-(5-phenyl-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-phenoxy-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-(3-methoxyphenoxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-(4-methoxyphenoxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-(4-fluorophenoxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-benzoyl-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonan-
e; enantiomers thereof; and a pharmaceutically acceptable salt
thereof.
7. The composition of claim 1, wherein the substance that binds
.alpha.7 nAChR is a cinnamamide of 3-aminoquinuclidine selected
from the group consisting of
N-(1-Azabicyclo[2.2.2]oct-3-yl)(E-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)(3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-nitrophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-nitrophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-aminophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[Z-3-(2-methoxyphenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)-N-methyl-(E-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-2-phenylcyclopropane-1-carboxamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)(Z-2-fluoro-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-formamidophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-nitrophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)(E-3-(4-aminophenyl)propenamide;
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-formamidophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)(Z-3-methyl-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-N-methylanlinophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-N,N-dimethylaminophenyl)propenami-
de]; N-(1-Azabicyclo[2.2.2]oct-3-yl)(Z-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)(E-3-methyl-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)(E-2,3-diphenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-methoxyphenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)(E methyl-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-methylphenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-methoxyphenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-fluorophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-fluorophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-(2-chlorophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-chlorophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-chlorophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3,4-dichlorophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)-[E-3-(3-bromophenyl)propenamide]
N-(1-Azabicyclo[2.2.2]oct-3-yl)-[E-3-(4-bromophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-iodophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-iodophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-trifluoromethylphenyl)propenamide]-
;
N-(1-Azabicyclo[2.2.2]oct-3-yl)-[E-3-(3-trifluoromethylphenyl)propenami-
de]; N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-furyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-furyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-pyridyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-pyridyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-pyridyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-thienyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-thienyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(5-nitro-2-furyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(5-methoxy-3-pyridyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(5-hydroxy-3-pyridyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-imidazolyl)propenamide];
N-(endo-8-Aza-8-methylbicyclo[3.2.1]oct yl)(E-3-phenylpropenamide);
N-(exo-8-Aza-8-methylbicyclo[3.2.1]oct yl)(E-3-phenylpropenamide);
enantiomers thereof; and a pharmaceutically acceptable salt
thereof.
8. The composition of claim 1, wherein the substance that binds
.alpha.7 nAChR is an arylcarbamate of 3-quinuclidinol selected from
the group consisting of N-phenylcarbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; N-(4-bromophenyl)carbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; N-(4-methylphenyl)carbamic
acid 1-azabicyclo[2.2.2]octan-3-yl ester;
N-(4-methoxyphenyl)carbamic acid 1-azabicyclo[2.2.2]octan-3-yl
ester; N-(3,4-dichlorophenyl)carbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; N-(4-cyanophenyl)carbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; N-phenylcarbamic acid
1-azabicyclo[2.2.1]heptan-3-yl ester; N-(3-methoxyphenyl)carbamic
acid 1-azabicyclo[2.2.2]octan-3-yl ester; N-phenylthiocarbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; N-(2-pyridyl)carbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; N-(1-naphthyl)carbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; N-phenylcarbamic acid
(3R)-1-azabicyclo[2.2.2]octan-3-yl ester; N-phenylcarbamic acid
(3S)-1-azabicyclo[2.2.2]octan-3-yl ester; N-(4-pyridyl)carbamic
acid 1-azabicyclo[2.2.2]octan-3-yl ester; N-(m-biphenyl)carbamic
acid 1-azabicyclo[2.2.2]octan-3-yl ester; N-(3-quinolinyl)carbamic
acid 1-azabicyclo[2.2.2]octan-3-yl ester; enantiomers thereof; and
pharmaceutically acceptable salts thereof.
9. The composition of claim 1, wherein the substance that binds
.alpha.7 nAChR is an aromatic amide of 3-aminoquinuclidine selected
from the group consisting of
N-((3R)-1-azabicyclo[2.2.2]oct-3-yl)-5-phenylthiophene-2-carboxamide;
N-((3R)-1-azabicyclo[2.2.2]oct-3-yl)-5-phenyl-1,3,4-oxadiazole-2-carboxam-
ide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-y-1]-4-(4-hydroxyphenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(-4-acetamidophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-phenoxybenzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-benzylbenzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(phenylsulfanyl)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-3-phenoxybenzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-benzoylbenzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-fluorophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(2-fluorophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-fluorophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(2-chlorophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-chlorophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-chlorophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(2-methoxyphenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-methoxyphenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-methoxyphenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-chlorophenylsulfanyl)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-methoxyphenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-chlorophenylsulfanyl)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-chlorophenylsulfanyl)benzamide-
;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-methoxyphenylsulfanyl)-benzam-
ide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4(2-methoxyphenylsulfanyl)-benz-
amide; N-(2-methyl-1-azabicyclo[2.2.2]oct-3-yl)4-phenoxybenzamide;
enantiomers thereof; and pharmaceutically acceptable salts
thereof.
10. The composition of claim 1, wherein the substance that binds
.alpha.7 nAChR is a spiroquinuclidines selected from the group
consisting of
spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
5'-bromospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
5'-phenylspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
5'-nitrospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
1'-chlorospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]isoquinol-
ine];
5'-(phenylcarboxamido)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-fu-
ro[2,3-b]pyridine];
5'-(phenylaminocarbonylamino)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-f-
uro[2,3-b]pyridine];
5'-(phenylsulfonylamido)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2-
,3-b]pyridine];
5'-aminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
5'-N-methylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyr-
idine];
5'-N,N-dimethylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-fu-
ro[2,3-b]pyridine];
5'-N,N-diethylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]-
pyridine];
5'-N-ethylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyri-
dine];
5'-N-benzylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,-
3-b]pyridine];
5'-N-formamidospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyrid-
ine];
5'-N-acetamidospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b-
]pyridine];
spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]isoquinoline];
spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]quinoline];
5'-ethenylspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine]-
;
5'-(E)-(phenylethenyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2-
,3-b]pyridine];
5'-(4-morpholino)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]py-
ridine];
5'-(1-azetidinyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo-
[2,3-b]pyridine];
5'-(E)-(2-(4-pyridyl)ethenyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-f-
uro[2,3-b]pyridine;
5'-(E)-(2-(2-pyridyl)ethenyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-f-
uro[2,3-b]pyridine;
5'-(2-trimethylsilylethynyl(spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-fu-
ro[2,3-b]pyridine;
5'-ethynylspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine]-
;
5'-(2-furyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyrid-
ine];
5'-(3-pyridyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b-
]pyridine];
5'-methylspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
spiro[1-azabicyclo[2.2.2]octane-3,2'-3'H)-furo[2,3-b]pyridine-5'carbonit-
rile];
spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine-5'c-
arboxamide];
5'-N'-(3chlorophenyl)ureidoaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H-
)-furo[2,3-b]pyridine];
5'-N'-(2-nitrophenyl)ureidoaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H-
)-furo[2,3-b]pyridine];
4'chlorospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
4'-methoxyspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine]-
;
4'-phenylthiospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyri-
dine];
4'-(N-2-aminoethyl)aminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-
-furo[2,3-b]pyridine];
4'-phenylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyrid-
ine];
4'-methylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b-
]pyridine];
4'-(4-N-methylpiperazin-1-yl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-f-
uro[2,3-b]pyridine;
4'-chloro-spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[3,2-c]pyridine]-
; spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[3,2-c]pyridine];
6'-fluorospiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine];
spiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine-6'-carbonit-
rile];
6'-chlorospiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyri-
dine]; enantiomers thereof; and pharmaceutically acceptable salts
thereof.
11. The composition of claim 1, wherein the substance that binds
.alpha.7 nAChR is a benzylideneanabaseine selected from the group
consisting of 3-(2,4-dimethoxybenzylidene)anabaseine,
3-(4-hydroxybenzylidene)anabaseine;
3-(4-methoxybenzylidene)anabaseine,
3-(4-aminobenzylidene)anabaseine;
3-(4-hydroxy-2-methoxybenzylidene)anabaseine;
3-(2-hydroxy-4-methoxybenzylidene)anabaseine;
3-(4-isopropoxybenzylidene)anabaseine;
(7'-methyl-3-(2,4dimethoxybenzylidene))anabaseine;
3-(4-acetylaminocinnamylidene)anabaseine;
3-(4-hydroxycinnamylidene)anabaseine;
3-(4-methoxycinnamylidene)anabaseine;
3-(4-hydroxy-2-methoxycinnamylidene)anabaseine;
3-(2,4-dimethoxycinnamylidene)anabaseine; and
3-(4-acetoxycinnamylidene)anabaseine; enantiomers thereof; and
pharmaceutically acceptable salts thereof.
12. The composition of claim 4, wherein the substance that inhibits
AT2 is PD123177 or PD123319.
13. A composition for reducing apoptosis mediated by a
.beta.-amyloid polypeptide in mammalian neurons, comprising, a) a
substance that binds .alpha.7-nAChR; and b) at least one inhibitor
of the AT2 receptor or at least one inhibitor of a substance that
stimulates the AT2 receptor; and c) a pharmaceutically acceptable
carrier.
14. The composition of claim 13, wherein the substance that binds
.alpha.7-nAChR is selected from the group consisting of
2-(3-pyridyl)azaadamantanes, diazabicyclic compounds,
pyridylazabicyclic compounds, cinnamamides of 3-aminoquinuclidine,
arylcarbamates of 3-quinuclidinol, aromatic amides of
3-aminoquinuclidine, spiroquinuclidines, and
benzylideneanabaseines.
15. The composition of claim 13, wherein the substance that
inhibits the AT2 receptor or that inhibits a substance that
stimulates the AT2 receptor is a substance that inhibits a
substance that stimulates AT2.
16. The composition of claim 13, wherein the substance that
inhibits the AT2 receptor or that inhibits a substance that
stimulates the AT2 receptor is a substance that inhibits AT2.
17. The composition of claim 16, wherein the substance that
inhibits AT2 is PD123177 or PD123319.
18. The composition of claim 13, wherein the substance that binds
.alpha.7-nAChR is selected from the group consisting of:
1-aza-2-(5-bromo(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane;
1-aza-2-(5-amino-(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane;
1-aza-2-(5-ethoxy-(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane;
1-aza-2-(5-isopropoxy-(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane;
2-(3-pyridyl)-1-azatricyclo[3.3.1.1.sup.3,7]decane;
5-aza-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]decan-2-ol;
5-aza-1-(hydroxymethyl)-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]-decan-2-on-
e; 1-aza-2-(3-pyridyl)-1-azatricyclo[3.3.1.1.sup.3,7]decane;
5-aza-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]decan-2-ol;
5-aza-1-(hydroxymethyl)-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]-decan-2-on-
e; 5-aza-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]decan-2-one;
(1S,4S)-2-(5-phenoxy-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane;
(1R,4R)-2-(5-phenoxy-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane;
(1S,4S)-2-(5-(3-methoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-
;
(1R,4R)-2-(5-(3-methoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]hepta-
ne;
(1S,4S)-2-(5-(4-methoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]hep-
tane;
(1R,4R)-2-(5-(4-methoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]h-
eptane;
(1S,4S)-2-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2-
.2.1]heptane;
(1R,4R)-2-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]hep-
tane;
(1S,4S)-2-(5-(4-fluorophenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]he-
ptane;
(1R,4R)-2-(5-(4-fluorophenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]h-
eptane;
(1S,4S)-2-(5-benzoyl-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane;
(1R,4R)-2-(5-benzoyl-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane;
(1S,4S)-2-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-2,5-diazabicyclo[2.2-
.1]heptane;
(1R,4R)-2-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-2,5-diazabicyclo[2.2-
.1]heptane;
(1S,4S)-2-(5-(4-(N-trifluoroacetylpiperidinyl)oxy)-3-pyridyl)-2,5-diazabi-
cyclo[2.2.1]heptane;
(1R,4R)-2-(5-(4-(N-trifluoroacetylpiperidinyl)oxy)-3-pyridyl)-2,5-diazabi-
cyclo[2.2.1]heptane;
6-methyl-3-(5-phenyl-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
6-methyl-3-(5-phenoxy-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
6-methyl-3-(5-(3-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane-
;
6-methyl-3-(5-(4-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]octa-
ne;
6-methyl-3-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.-
1]octane;
6-methyl-3-(5-(4-fluorophenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2-
.1]octane;
6-methyl-3-(5-benzoyl-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
6-methyl-3-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,6-diazabicyclo[3.-
2.1]octane;
3-methyl-6-(5-phenyl-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
3-methyl-6-(5-phenoxy-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
3-methyl-6-(5-(3-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane-
;
3-methyl-6-(5-(4-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]octa-
ne;
3-methyl-6-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.-
1]octane;
3-methyl-6-(5-(4-fluorophenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2-
.1]octane;
3-methyl-6-(5-benzoyl-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
3-methyl-6-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,6-diazabicyclo[3.-
2.1]octane; 6-(5-phenyl-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-phenoxy-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-(3-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-(4-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-(4-fluorophenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-benzoyl-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
6-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonan-
e; 3-(5-phenyl-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-phenoxy-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-(3-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-(4-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-(4-fluorophenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-benzoyl-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
3-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonan-
e; 3-(5-phenyl-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-phenoxy-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-(3-methoxyphenoxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-(4-methoxyphenoxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-(4-fluorophenoxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-benzoyl-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
3-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonan-
e; N-(1-Azabicyclo[2.2.2]oct-3-yl)(E-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)(3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-nitrophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-nitrophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-aminophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[Z-3-(2-methoxyphenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)-N-methyl-(E-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-2-phenylcyclopropane-1-carboxamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)(Z-2-fluoro-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-formamidophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-nitrophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)(E-3-(4-aminophenyl)propenamide;
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-formamidophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)(Z-3-methyl-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-N-methylanlinophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-N,N-dimethylaminophenyl)propenami-
de]; N-(1-Azabicyclo[2.2.2]oct-3-yl)(Z-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)(E-3-methyl-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)(E-2,3-diphenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-methoxyphenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)(E methyl-3-phenylpropenamide);
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-methylphenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-methoxyphenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-fluorophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-fluorophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-(2-chlorophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-chlorophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-chlorophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3,4-dichlorophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)-[E-3-(3-bromophenyl)propenamide]
N-(1-Azabicyclo[2.2.2]oct-3-yl)-[E-3-(4-bromophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-iodophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-iodophenyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-trifluoromethylphenyl)propenamide]-
;
N-(1-Azabicyclo[2.2.2]oct-3-yl)-[E-3-(3-trifluoromethylphenyl)propenami-
de]; N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-furyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-furyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-pyridyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-pyridyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-pyridyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-thienyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-thienyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(5-nitro-2-furyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(5-methoxy-3-pyridyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(5-hydroxy-3-pyridyl)propenamide];
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-imidazolyl)propenamide];
N-(endo-8-Aza-8-methylbicyclo[3.2.1]oct yl)(E-3-phenylpropenamide);
N-(exo-8-Aza-8-methylbicyclo[3.2.1]oct yl)(E-3-phenylpropenamide);
N-phenylcarbamic acid 1-azabicyclo[2.2.2]octan-3-yl ester;
N-(4-bromophenyl)carbamic acid 1-azabicyclo[2.2.2]octan-3-yl ester;
N-(4-methylphenyl)carbamic acid 1-azabicyclo[2.2.2]octan-3-yl
ester; N-(4-methoxyphenyl)carbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; N-(3,4-dichlorophenyl)carbamic
acid 1-azabicyclo[2.2.2]octan-3-yl ester; N-(4-cyanophenyl)carbamic
acid 1-azabicyclo[2.2.2]octan-3-yl ester; N-phenylcarbamic acid
1-azabicyclo[2.2.1]heptan-3-yl ester; N-(3-methoxyphenyl)carbamic
acid 1-azabicyclo[2.2.2]octan-3-yl ester; N-phenylthiocarbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; N-(2-pyridyl)carbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; N-(1-naphthyl)carbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; N-phenylcarbamic acid
(3R)-1-azabicyclo[2.2.2]octan-3-yl ester; N-phenylcarbamic acid
(3S)-1-azabicyclo[2.2.2]octan-3-yl ester; N-(4-pyridyl)carbamic
acid 1-azabicyclo[2.2.2]octan-3-yl ester; N-(m-biphenyl)carbamic
acid 1-azabicyclo[2.2.2]octan-3-yl ester; N-(3-quinolinyl)carbamic
acid 1-azabicyclo[2.2.2]octan-3-yl ester;
N-((3R)-1-azabicyclo[2.2.2]oct-3-yl)-5-phenylthiophene-2-carboxamide;
N-((3R)-1-azabicyclo[2.2.2]oct-3-yl)-5-phenyl-1,3,4-oxadiazole-2-carboxam-
ide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-y-1]-4-(4-hydroxyphenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(-4-acetamidophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-phenoxybenzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-benzylbenzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(phenylsulfanyl)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-3-phenoxybenzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-benzoylbenzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-fluorophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(2-fluorophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-fluorophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(2-chlorophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-chlorophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-chlorophenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(2-methoxyphenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-methoxyphenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-methoxyphenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-chlorophenylsulfanyl)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-methoxyphenoxy)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-chlorophenylsulfanyl)benzamide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-chlorophenylsulfanyl)benzamide-
;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-methoxyphenylsulfanyl)-benzam-
ide;
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4(2-methoxyphenylsulfanyl)-benz-
amide; N-(2-methyl-1-azabicyclo[2.2.2]oct-3-yl)4-phenoxybenzamide;
spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
5'-bromospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
5'-phenylspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
5'-nitrospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
1'-chlorospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]isoquinol-
ine];
5'-(phenylcarboxamido)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-fu-
ro[2,3-b]pyridine];
5'-(phenylaminocarbonylamino)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-f-
uro[2,3-b]pyridine];
5'-(phenylsulfonylamido)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2-
,3-b]pyridine];
5'-aminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
5'-N-methylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyr-
idine];
5'-N,N-dimethylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-fu-
ro[2,3-b]pyridine];
5'-N,N-diethylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]-
pyridine];
5'-N-ethylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyri-
dine];
5'-N-benzylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,-
3-b]pyridine];
5'-N-formamidospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyrid-
ine];
5'-N-acetamidospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b-
]pyridine];
spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]isoquinoline];
spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]quinoline];
5'-ethenylspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine]-
;
5'-(E)-(phenylethenyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2-
,3-b]pyridine];
5'-(4-morpholino)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]py-
ridine];
5'-(1-azetidinyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo-
[2,3-b]pyridine];
5'-(E)-(2-(4-pyridyl)ethenyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-f-
uro[2,3-b]pyridine;
5'-(E)-(2-(2-pyridyl)ethenyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-f-
uro[2,3-b]pyridine;
5'-(2-trimethylsilylethynyl(spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-fu-
ro[2,3-b]pyridine;
5'-ethynylspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine]-
;
5'-(2-furyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyrid-
ine]
5'-(3-pyridyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]p-
yridine];
5'-methylspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]-
pyridine];
spiro[1-azabicyclo[2.2.2]octane-3,2'-3'H)-furo[2,3-b]pyridine-5'carbonitr-
ile];
spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine-5'ca-
rboxamide];
5'-N'-(3chlorophenyl)ureidoaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H-
)-furo[2,3-b]pyridine];
5'-N'-(2-nitrophenyl)ureidoaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H-
)-furo[2,3-b]pyridine];
4'chlorospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
4'-methoxyspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine]-
;
4'-phenylthiospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyri-
dine];
4'-(N-2-aminoethyl)aminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-
-furo[2,3-b]pyridine];
4'-phenylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyrid-
ine];
4'-methylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b-
]pyridine];
4'-(4-N-methylpiperazin-1-yl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-f-
uro[2,3-b]pyridine;
4'-chloro-spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[3,2-c]pyridine]-
; spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[3,2-c]pyridine];
6'-fluorospiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine];
spiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine-6'-carbonit-
rile];
6'-chlorospiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyri-
dine]; 3-(2,4-dimethoxybenzylidene)anabaseine,
3-(4-hydroxybenzylidene)anabaseine;
3-(4-methoxybenzylidene)anabaseine,
3-(4-aminobenzylidene)anabaseine;
3-(4-hydroxy-2-methoxybenzylidene)anabaseine;
3-(2-hydroxy-4-methoxybenzylidene)anabaseine;
3-(4-isopropoxybenzylidene)anabaseine;
(7'-methyl-3-(2,4dimethoxybenzylidene))anabaseine;
3-(4-acetylaminocinnamylidene)anabaseine;
3-(4-hydroxycinnamylidene)anabaseine;
3-(4-methoxycinnamylidene)anabaseine;
3-(4-hydroxy-2-methoxycinnamylidene)anabaseine;
3-(2,4-dimethoxycinnamylidene)anabaseine;
3-(4-acetoxycinnamylidene)anabaseine; enantiomers thereof; and
pharmaceutically acceptable salts thereof.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/318,842, fully incorporated herein by reference, itself
claiming benefit of U.S. Provisional Patent Application No.
60/340,582 filed Dec. 14, 2001, and U.S. Provisional Patent
Application No. 60/369,934 filed Apr. 4, 2002.
FIELD OF THE INVENTION
[0002] The present invention relates to compounds having
pharmaceutical properties, and in particular, to compounds useful
for prophylaxis and/or treatment of central nervous system (CNS)
disorders, including disease states associated with Alzheimer's
disease.
[0003] The present invention relates to methods for prophylaxis
and/or treatment of patients suffering from or susceptible to such
disorders, and in particular, to a method for prophylaxis and/or
treatment of patients suffering from those disorders which are
associated with neurodegeneration of brain neurons. The present
invention also relates to compositions of matter useful as
pharmaceutical compositions in the prophylaxis and/or treatment of
CNS disorders that have been attributed to neurodegenerative
diseases.
BACKGROUND OF THE INVENTION
[0004] Nicotinic receptors. Nicotinic acetylcholine receptors
(nAChRs) are composed of various combinations of .alpha.-subunits
(.alpha.2-.alpha.9) and .beta.-subunits (.beta.2-.beta.4), and are
classified into two classes according to their affinity for
nicotine or .alpha.-bungarotoxin (.alpha.BTX) (Vijayaraghavan, S.,
et al., Neuron 8:353-362 (1992)). Of the known .alpha.BTX-binding
subtypes .alpha.7-.alpha.9, only .alpha.7 receptors are expressed
throughout the mammalian brain. Alpha7 receptors form functional
homomeric ion channels that promote Ca.sup.2+ influx, which are
rapidly desensitized (Breese, C. R., et al., J. Com. Neurol.
387:385-398 (1997); Vijayaraghavan, S., et al., Neuron 8:353-362
(1992)) and are thus assumed to be involved in synaptic
transmission (McGehee, D. S., et al., Science 269:1692-1696
(1995)). Nicotinic agonists selective for the .alpha.7 receptor
have demonstrated efficacy in improving cognitive functions in
rats, primates and AD patients. Due to the multifaceted deficits
observed in AD and the limited pharmacopae for management of AD
patients, there is an urgent need for new therapies and approaches
to optimize existing and emerging therapies.
[0005] Nicotine has also been found to inhibit death of PC12 cells
cultured in serum-free medium (Yamashita, H. and S. Nakamura,
Neurosci. Lett. 213:145-147 (1996)). In addition, a selective
.alpha.7 receptor agonist, anabaseine-derived
3-(4)-dimethylaminocinamylidine (DMAC) (de Fiebre, C. M., et al.,
Mol. Pharmacol. 47:164-171 (1995)), and an activator of nAChR,
including the .alpha.7 subtype, ABT-418 (Donnelly-Roberts, D. L.,
et al., Brain Res. 719:36-44 (1996)) have also been reported to
exert cytoprotective effects.
[0006] Nicotine-induced protection in neuronal cells is suppressed
by .alpha.BTX, a phosphatidylinositol 3-kinase (PI3K) inhibitor
(LY294002 and wortmannin), and a Src inhibitor (PP2). In addition,
the levels of phosphorylated Akt, an effector of PI3K, are
increased by nicotine ((Kihara, T., S. et al., J. Biol. Chem.
276:13541-13546 (2001)). These findings suggest that the .alpha.7
nicotinic receptor transduces signals to PI3K in a cascade, which
ultimately contributes to a neuroprotective effect.
[0007] Ang II signaling pathways. The actions of Angiotensin II
(Ang II) are mediated through two types of cell surface receptors,
(AT1 and AT2). Most of the physiological responses to Ang II in
glomerular mesangial cells (GMC) occur via the AT1 receptor subtype
(Bernstein, K. E. and M. B. Marrero, Trends. Cardiovasc. Med.
6:179-187 (1996); Marrero, M. B., et al., Cell. Signal. 8:21-26
(1996)). For AT1 receptors, activation by Ang II results in G
protein mediated signaling, including phospholipase C-dependent
activation of protein kinase C and release of calcium from
intracellular stores (Bernstein, K. E. and M. B. Marrero, Trends.
Cardiovasc. Med. 6:179-187 (1996)). AT1 receptors also activate
signaling pathways traditionally associated with growth factor and
cytokine receptors that induce the production of early growth
response genes. The signaling cascades whereby Ang II induces early
growth response genes, such as c-fos and c-jun proto-oncogenes,
does not in general require new protein synthesis and appear to be
regulated by post-translational modifications of pre-existing
transcription factors (Sadoshima, J. and S. Izumo, Circ. Res.
73:413-423 (1993); Okuda, M., Y. Kawahara, and M. Yokoyama, Am. J.
Physiol. 271: H595-H601, (1996); Sadoshima, J. and S. Izumo, Circ.
Res. 73:413-423 (1993); Sadoshima, J. and S. Izumo, Circ. Res.
73:424-438 (1993); Taubman, M. B., et al., J. Biol. Chem.
264:526-530 (1989)). Therefore, the Ang II-induced expression of
these early growth response genes is under the direct regulation of
intracellular signal transduction pathways. Three intracellular
signaling pathways have recently been implicated in the activation
of proto-oncogenes: the JAK/STAT, p21ras/Raf-1/MAP kinase, and the
PLC-.gamma.1 cascades (Bernstein, K. E. and M. B. Marrero, Trends.
Cardiovasc. Med. 6:179-187 (1996); Marrero, M. B., et al., Cell.
Signal. 8:21-26 (1996); Sayeski, P. P., et al. Regulatory Peptides
78:19-29 (1998)). From multiple studies focusing on AT1 receptor
signal transduction pathways, it has become apparent that the
temporal arrangement of agonist-stimulated signaling varies from
seconds (i.e., the activation of PLC-.gamma.1 and generation of
inositol phosphates) to minutes (e.g., MAP kinase activation) to
hours (e.g., JAK/STAT pathway) (Bernstein, K. E. and M. B. Marrero,
Trends. Cardiovasc. Med. 6:179-187 (1996); Sayeski, P. P., et al.,
Regulatory Peptides 78:19-29 (1998)). The exact mechanism(s) by
which the AT1 receptor is able to differentially couple to
disparate signal transduction pathways is not clear, but presumably
involves a complex series of steps that selectively recruits,
activates and then inactivates each signaling system in a
time-dependent manner.
[0008] Role of the JAK/STAT pathway in Ang II signaling. The JAK
family of cytosolic tyrosine kinases, traditionally thought to be
coupled to cytokine receptors such as those for the interleukins
and interferons, have four members (JAK1, JAK2, JAK3 and TYK2)
(Darnell, J. E., Jr., et al., Science 264:1415-1421 (1994);
Taubman, M. B., et al., J. Biol. Chem. 264:526-530 (1989)). In
response to ligand binding, these JAK tyrosine kinases associate
with, tyrosine-phosphorylate, and activate the cytokine receptor
itself. Once activated, JAKs tyrosine-phosphorylate and activate
other signaling molecules including the STAT family of nuclear
transcription factors after binding of the STATs to the receptor
(Darnell, J. E., Jr., et al., Science 264:1415-1421 (1994);
Taubman, M. B., et al., J. Biol. Chem. 264:526-530 (1989)). Thus,
the JAK/STAT pathway is an important link between cell surface
receptors and nuclear transcriptional events leading to cell
growth. Recently, Baker and colleagues have shown that STAT1,
STAT3, and STAT5 are tyrosine-phosphorylated in response to Ang II
in cardiac fibroblasts and AT1 receptor-transfected CHO cells
(Bhat, G. J., et al., J. Biol. Chem. 269:31443-31449 (1994); Bhat,
G. J., et al., J. Biol. Chem. 270:19059-19065 (1995); McWhinney, C.
D., et al., J. Mol. Cell. Cardiol. 30:751-761 (1998)). These
investigators also found that Ang II exposure stimulated the
phosphorylated monomeric STAT proteins to form homo- (STAT1.sub.2,
STAT3.sub.2 or STAT5.sub.2) or hetero- (STAT1:STAT3) dimer
complexes referred to as SIF (sis-inducing factors). These SIF
complexes subsequently translocate to the nucleus and interact with
specific DNA motifs called SIE (sis-inducing elements) or PIE
(prolactin-inducing element)-like elements within the c-fos
promoter, culminating in the activation of this early growth
response gene (Bhat, G. J., et al., J. Biol. Chem. 269:31443-31449
(1994); Darnell, J. E., Jr., et al., Science 264:1415-1421 (1994);
McWhinney, C. D., et al., J. Mol. Cell. Cardiol. 30:751-761 (1998);
Schindler, C. and J. E. Darnell, Jr., Annu. Rev. Biochem.
64:621-651 (1995)). The JAK/STAT cascade can be activated by Ang II
resulting in tyrosine phosphorylation of JAK2, STAT1 and STAT3, and
the translocation of STAT1 and STAT3 to the nucleus (Bhat, G. J.,
et al., J. Biol. Chem. 270:19059-19065 (1995); Marrero, M. B., et
al. Clin. Exp. Pharmacol. Physiol. 23:83-88 1996; Marrero, M. B.,
et al., Nature 375:247-250 (1995)). Furthermore, the
carboxyl-terminal tail of the AT1 receptor binds to JAK2 in an Ang
II-dependent manner (Ali, M. S., et al., J. Biol. Chem.
272:23382-23388 (1997)). In addition, inhibition of JAK2 tyrosine
phosphorylation with the pharmacologic JAK2 inhibitor, AG490, or
electroporation of blocking antibodies against STAT1 or STAT3
inhibits Ang II-induced vascular smooth muscle cell (VSMC)
proliferation and DNA synthesis (Marrero, M. B., et al., J. Biol.
Chem. 272:24684-24690 (1997)). These results indicate that
G-protein-coupled receptors, in particular the AT1 receptor, can
operate via the same intracellular tyrosine phosphorylation
pathways previously linked to mitogenic cytokine and growth factor
receptors. Finally, the tyrosine phosphatases, SHP-1 and SHP-2,
have opposite roles in Ang II-induced JAK2 phosphorylation. SHP-1
appears responsible for JAK2 dephosphorylation and termination of
the Ang II-induced JAK/STAT cascade, whereas SHP-2 appears to have
an essential role in JAK2 phosphorylation and initiation of the Ang
II-induced JAK/STAT cascade leading to cell proliferation (See
Jiao, H., et al., Direct association with and dephosphorylation of
Jak2 kinase by the SH2-domain-containing protein tyrosine
phosphatase SHP-1. Mol Cell Biol 16(12):6985-92(1996)).
[0009] The motif in the AT1 receptor that is required for
association with JAK2 is also required for association with SHP-2
(Marrero, M. B., et al., Am. J. Physiol. 275:C1216-C1223 (1998)).
Furthermore, SHP-2 is also required for JAK2-Ang II AT1 receptor
association (Marrero, M. B., et al., Am. J. Physiol.
275:C1216-C1223 (1998)). SHP-2 may thus play a role as an adaptor
protein for JAK2 association with the receptor, thereby
facilitating JAK2 phosphorylation and activation (Marrero, M. B.,
et al., Am. J. Physiol. 275: C1216-C1223, 1998).
[0010] Nicotinic Acetylcholine Receptors and .beta.-Amyloid
Toxicity. The cholinergic deficit in Alzheimer's Disease has been
clearly established and is the basis for the current symptomatic
strategy. There is an early and significant depletion of high
affinity nicotinic receptors in Alzheimer's patient's brains
(Court, J., et al. Biol. Psychiatry 49: 175-184 (2001)), and a
number of studies have shown cognitive improvement in rodent,
primates including humans following administration of ligands
targeting nAChRs (Newhouse P A, et al., Biol Psychiatry
49(3):268-78 (2001)). In addition to their known symptomatic
effects, neuronal nicotinic ligands have shown neuroprotective
activity in vitro (Donnelly-Roberts, D. L., et al. Brain Res. 719:
36-44 (1996)) and in vivo (Ryan R E, et al., 132(8):1650-6 (2001))
suggesting an additional potential for disease modification.
[0011] The .alpha.7 receptor forms functional homomeric
ligand-gated ion channels that promote rapidly desensitizing
Ca.sup.2+ influx, is widely expressed throughout the mammalian
brain, and has been implicated in sensory gating, cognition, and
neuroprotection (Seo, J., et al., Biol Psychiatry 49(3):240-7
(2001). Nicotine-induced neuroprotection against
.beta.-Amyloid-induced toxicity is suppressed by .alpha.-Bgt and a
selective .alpha.7-nAChR agonist, anabaseine-derived
3-(4)-dimethylaminocinamylidine (DMAC) exerts cytoprotective
effects (De Fiebre C. M., et al., Mol. Pharmacol. 47:164-171
(1995); Kem W R., Behav Brain Res. 113(1-2):169-81 (2000)). The
level of phosphorylated Akt, an effector of PI-3-K, is increased by
nicotine and cytoprotective effects are suppressed by
phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and
wortmannin), and Src inhibitor (PP2) (Kihara,T., et al., J. Biol.
Chem. 276:13541-13546 (2001)). The .alpha.7-nAChR transduces
signals to PI3K in a cascade, which ultimately contributes to a
neuroprotective effect against A.beta..
[0012] In contrast to the decrease in .alpha.7-nAChR, the
angiotensin converting enzyme (ACE--the enzyme that converts
Angiotensin I to Angiotensin II) density is increased in the
temporal cortex from patients with Alzheimer's disease (Barnes, N.
M., et al., Eur. J. Pharmacol. 200:289-292 (1991)), and the ACE
genotype is associated with AD in some populations (Narain Y et
al., J Med Genet., 37(9):695-7 (2000)). AT2 receptors exert growth
inhibitory effects or apoptosis both in cultured cells and in vivo
(Horiuchi, M., et al., Endocr. Res. 24:307-314 (1998)), are
expressed in PC12 cells, and have been shown to inhibit the
JAK/STAT signaling cascade (Horiuchi, M., et al., Circ. Res.
84:876-882 (1999)).
[0013] It would be desirable to further understand any
relationships between .alpha.7 nAChR-mediated beneficial pathways
and the apoptotic effects mediated by AT2, in order to maximize
cell survival by modulation of nAChR and/or AT2 activity.
[0014] It would also be desirable to further understand any
relationship between A.beta.-mediated toxicity and signaling
pathways affected by nicotinic receptors. For example, further
elucidation of these relationships can provide for discovery of
therapeutic compositions useful in mitigating the effects of
Alzheimer's Disease.
SUMMARY OF THE INVENTION
[0015] The present invention provides a method of determining
substances that stimulate or inhibit nicotine receptors. The
invention also provides for enhancement of effects of substances
that stimulate nicotine receptor activity mediated by
phosphorylation of the Janus-Activated Kinase 2 (JAK2).
[0016] The present inventors have shown that antagonists of the
angiotensin II, type 2 (AT2) receptor (or inhibitors of substances
that stimulate the AT2 receptor) enhance the effects of nicotinic
stimulatory substances by reducing or eliminating AT2-mediated
interference with nicotine receptor-induced phosphorylation of
JAK2. Further, the inventors have shown that nicotinic protection
against the effects of .beta.-amyloid-mediated toxicity operates
via JAK2 phosphorylation. Methods and compositions for prophylaxis
and/or treatment, and screening assays, including assays adapted
for high-throughput screening (HTS), are provided.
[0017] Accordingly, in one aspect, the invention relates to a
method of screening for a substance or for substances having an
effect on a nicotine receptor. The method comprises contacting a
cell having a nicotine receptor with a test compound; and
determining any increase or decrease in phosphorylation of
JAK2.
[0018] In another aspect, the invention relates to a method of
screening for a substance that increases or decreases an effect of
a substance that stimulates a nicotine receptor. The method
comprises contacting a cell having a nicotine receptor with the
substance that stimulates a nicotine receptor; contacting the cell
with a test substance; and determining any increase or decrease in
phosphorylation of JAK2 in the presence of the test substance
relative to a level of JAK2 phosphorylation measured when the cell
is in contact with a substance that stimulates a nicotine receptor
in the absence of the test substance.
[0019] In yet another aspect, the invention relates to a method of
screening for a substance that inhibits or stimulates an AT2
receptor and/or impairs or enhances the effect of a substance that
mediates an effect on the AT2 receptor. The method comprises
contacting a cell having a nicotine receptor and an AT2 receptor
with a substance that stimulates the nicotine receptor; contacting
the cell with a test substance; and determining any increase or
decrease in phosphorylation of JAK2 in the presence of the test
substance relative to a level of JAK2 phosphorylation measured when
the cell is in contact with the substance that stimulates the
nicotine receptor in the absence of the test substance. An increase
in JAK2 phosphorylation indicates that the test substance inhibits
the AT2 receptor, impairs the effect of a substance that stimulates
the AT2 receptor, or enhances the effect of a substance that
inhibits the AT2 receptor. A decrease in JAK2 phosphorylation
indicates that the test substance stimulates the AT2 receptor,
enhances the effect of a substance that stimulates the AT2
receptor, or impairs the effect of a substance that inhibits the
AT2 receptor.
[0020] In yet another aspect, the invention relates to a method of
decreasing apoptosis in cells comprising a nicotine receptor and an
AT2 receptor. The method comprises contacting the cells with a
substance that stimulates a nicotine receptor and either or both of
an inhibitor of the AT2 receptor or an inhibitor of a substance
that stimulates the AT2 receptor.
[0021] In yet another aspect, the invention relates to a method of
decreasing apoptosis in cells comprising a nicotine receptor and an
AT2 receptor. The method comprises contacting the cells with a
substance that stimulates the nicotine receptor; and contacting the
cell with either or both of an inhibitor of the AT2 receptor or an
inhibitor of a substance that stimulates the AT2 receptor.
[0022] In yet another aspect, the invention relates to a method of
treatment and/or prophylaxis for subjects suffering from a central
nervous system disorder mediated by a nicotine receptor. The method
comprises administering an effective amount of a pharmaceutical
composition including either or both of at least one inhibitor of
the AT2 receptor or at least one inhibitor of a substance that
stimulates the AT2 receptor; a substance that stimulates a nicotine
receptor; and a pharmaceutically acceptable carrier. The amount of
the pharmaceutical composition is effective to stimulate the
nicotine receptor.
[0023] In still another aspect, the invention relates to a
pharmaceutical composition for treatment and/or prophylaxis of a
central nervous disorder for administration to a subject suffering
from the disorder. The composition comprises either or both of at
least one inhibitor of the AT2 receptor or at least one inhibitor
of a substance that stimulates the AT2 receptor; a substance that
stimulates a nicotine receptor; and a pharmaceutically acceptable
carrier.
[0024] The invention also provides methods and compositions related
to the interaction of .beta.-amyloid and nicotinic receptors.
Accordingly, in one aspect, the invention relates to a method of
screening for substances that have an effect on
.beta.-amyloid-associated neurotoxicity mediated by binding of a
.beta.-amyloid peptide, polypeptide or protein to a nicotinic
receptor. The method comprises contacting a cell having a nicotine
receptor with a .beta.-amyloid polypeptide and determining a level
of Janus-Activated Kinase 2 (JAK2) phosphorylation; and contacting
the cell with a test substance and determining any increase or
decrease in phosphorylation of JAK2.
[0025] In another aspect, the invention also relates to a method of
screening for substances that decrease the neurotoxicity of
.beta.-amyloid polypeptides mediated by binding of a .beta.-amyloid
polypeptide to a nicotinic receptor. The method comprises
contacting a cell having a nicotine receptor with a .beta.-amyloid
polypeptide and determining a level of Janus-Activated Kinase 2
(JAK2) phosphorylation; and contacting the cell with a test
substance and determining any increase or decrease in
phosphorylation of JAK2. Any increase in JAK2 phosphorylation
indicates that the test substance decreases the neurotoxicity of
.beta.-amyloid peptides.
[0026] In another aspect, the invention relates to a method of
preventing or decreasing apoptosis in cells having nicotinic
receptors comprising contacting the cells with a substance that
increases phosphorylation of JAK2. The apoptosis can be that
associated with .beta.-amyloid-mediated toxicity.
[0027] In another aspect, the invention relates to a method of
prophylaxis and/or treatment of neurodegeneration associated with
Alzheimer's disease comprising administering a therapeutically
effective amount of a substance that increases phosphorylation of
JAK2.
[0028] In yet another aspect, the invention relates to a
composition for prophylaxis and/or treatment of neurodegeneration
associated with Alzheimer's disease comprising a therapeutically
effective amount of a substance that increases phosphorylation of
JAK2.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1 shows the effects of the JAK2 inhibitor AG-490 on the
nicotine-induced tyrosine phosphorylation of JAK2 and PI-3 kinase
plus serine phosphorylation of Akt in PC12 cells. PC12 cells,
pre-incubated for 1 hour in the presence or absence of the JAK2
inhibitor AG-490 at 10 .mu.M, were stimulated with nicotine (10
.mu.M) for various times (0, 5, 10, 30, 60, and 120 min). Cells
were either lysed and immunoblotted with phospho-specific and
nonphosphospecific anti-JAK2 and anti-Akt antibodies or lysed and
immunoprecipitated with anti-PI-3 kinase antibody. The PI-3 kinase
immunoprecipitated proteins were then immunoblotted with
anti-phosphotyrosine and anti-PI-3 kinase antibodies. Results shown
are representative of three experiments.
[0030] FIG. 2 shows Western blot analysis illustrating the effect
of nicotine on the JAK2 complex formation with the .alpha.7
receptor in PC12 cells. PC12 cells were stimulated with nicotine
(10 .mu.M) for various times (0, 1, 5, 10, 30, and 60 min). Cells
were lysed, and JAK2 was immunoprecipitated from lysates (1 mg of
protein) with an anti-JAK2 antibody. Immunoprecipitates were then
immunoblotted with an anti-.alpha.7 antibody. Similar results were
obtained in three experiments.
[0031] FIG. 3 shows effects of Ang II pretreatment with or without
Ang II receptor antagonists on the nicotine-induced activation of
JAK2 in PC12 cells. PC12 cells, pre-incubated for 8 hours in the
presence or absence of Ang II at 100 nM with or without 100 nM AT1
antagonist (candesartan), or 100 nM AT2 antagonist (PD 123177),
were stimulated with nicotine (10 .mu.M) for various times (0, 10,
and 30 min). Cells were either lysed and immunoblotted with
phospho-specific and nonphosphospecific anti-JAK2. Results shown
are representative of three experiments.
[0032] FIG. 4 is a graphic representation of the effects of the
JAK2 inhibitor AG-490 on nicotine protection against A.beta.-and
Ang II-induced cell death in PC12 cells. PC12 cells were treated
for 8 hours with either A.beta.(1-42) peptide at 100 nM or Ang II
at 100 nM in the presence or absence of nicotine and/or AG-490.
PC12 cells cultures were processed as described in the Examples,
and cell number was counted at the end of respective incubations.
Results represent the mean.+-.SEM of four independent cultures.
A.beta. induced a significant decrease in cell number ( *
P<0.01) which was significantly inhibited by co-incubation with
nicotine ( ** P<0.01). Nicotine, on the other hand, had no
effect in the presence of AG-490. Ang II also significantly reduced
PC12 cell number (+P<0.01), a result not significantly affected
by nicotine ( # P>0.05).
[0033] FIG. 5 is a graphic representation of the effects of JAK2
inhibitor AG-490 and nicotine on the A.beta.(1-42) amyloid-induced
activation of caspase 3 in PC12 cells. PC12 cells were incubated
for 0, 2, 4, 8, 12, and 24 hours by A.beta.(1-42) peptide at 100 nM
in the presence or absence of nicotine at 10 .mu.M and nicotine
co-incubated with AG-490 (10 .mu.M). Caspase 3 activities were
determined as described in the Examples. Results represent the
mean.+-.SEM of three independent cultures. A.beta. induced a
significant increase in caspase 3 activity at 4, 8, 12 and 24 hours
( * P<0.01) which was significantly inhibited by co-incubation
with nicotine ( ** P<0.01). Nicotine, on the other hand, had no
effect in the presence of AG-490.
[0034] FIG. 6 shows Western blot analysis illustrating the effects
of JAK2 inhibitor (AG-490) on nicotine protection against
A.beta.-induced apoptosis in PC12 cells. Poly-(ADP-ribose)
polymerase (PARP) is marker of cells undergoing apoptosis. PARP
expression was determined by Western analysis of PC12 cells nuclear
extract treated for 8 hours by A.beta. in the presence or absence
of nicotine and/or AG-490.
[0035] FIG. 7 shows Western blot analysis illustrating the effects
of the JAK2 inhibitor (AG-490) on nicotine protection against
A.beta.-and Ang II-induced apoptosis in PC12 cells. PARP is marker
of cells undergoing apoptosis. PARP expression was determined by
Western analysis of PC12 cells lysate treated for 8 hours by
A.beta.(1-42) peptide at 100 nM or Ang II at 100 nM in the presence
or absence of nicotine and/or AG-490.
[0036] FIG. 8 shows Western blot analysis illustrating the
co-immunoprecipitation of .alpha.7nAChR with A.beta.(1-42) amyloid.
Equal amounts of PC12 cells membrane proteins prepared from PC12
cells treated with A.beta.(1-42) peptide at 10 .mu.M for 5 minutes
were immunoprecipitated with anti-A.beta.(1-42) and subjected to
Western analysis with anti-.alpha.7nAChR. Lane 1 are cells treated
with A.beta.(1-42) peptide alone, and lane 2 are cells treated with
A.beta.(1-42) peptide in the presence of AG-490 (10 .mu.M). PC12
cells treated with A.beta.(1-42) peptide at 10 .mu.M in the
presence of 10 .mu.M nicotine with or with out AG-490 are shown in
lanes 3 and 4 respectively.
[0037] FIG. 9 shows Western blot analysis illustrating the effects
of AG-490 on the A.beta.(1-42) amyloid-induced phosphorylation of
JAK2. PC12 cells, pre-incubated for 1 hour in the presence or
absence of the JAK2 inhibitor AG-490 at 10 .mu.M were stimulated
with A.beta.(1-42) amyloid peptide at 100 nM (A) or 1 .mu.M (B) for
various times (0, 5, 10, 30, 60, and 120 min). Cells were either
lysed and immunoblotted with phospho-specific and
nonphosphospecific anti-JAK2 antibodies. Results shown are
representative of three experiments.
[0038] FIG. 10 is a schematic of the nicotine receptor mediated
survival pathway, illustrating the relationship of this pathway to
AT2- and A.beta.-mediated apoptotic pathways.
[0039] FIG. 11 shows Western blot analyses performed essential as
indicated for FIG. 1, using compound TC-1698 and showing activation
of JAK2, Akt, and PI-3 Kinase in PC12 cells, with suppression of
activation in the presence of the JAK2 inhibitor AG-490.
[0040] FIG. 12 illustrates angiotensin II-induced phosphorylation
of SHP-1 in PC12 cells.
[0041] FIG. 13 illustrates the effect of the SHP-1 inhibitor
vanadate on the angiotensin II-induced activation of SHP-1 in PC12
cells. (See Jiao, H., et al., Mol Cell Biol 16(12):6985-921996),
incorporated fully herein by reference, for unit definition of
SHP-1 activity).
[0042] FIG. 14 illustrates the effects of the JAK2 inhibitor
AG-490, and the SHP-1 inhibitor vanadate, on TC-1698-induced
neuroprotection against A.beta. (1-42) and angiotensin II-induced
apoptosis (PARP is a marker for apoptosis--absence is indicative of
a neuoprotective effect, as indicated above and discussed
herein).
[0043] FIG. 15 shows the TC-1698-induced activation of JAK2 in PC12
cells, in the presence and absence of the SHP-1 inhibitor
vanadate.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention provides screening assays, including
high throughput methods, for detecting substances that stimulate or
inhibit (directly or indirectly) nicotine receptors. Such factors
can be detected and characterized for .alpha.7 nicotine receptors.
Measuring tyrosine phosphorylation of the Janus Activated Kinase 2
(JAK2) allows detection of stimulation or inhibition of the
nicotine receptor. Further, screening assays are provided that
allow detection and characterization of substances that stimulate
or inhibit (directly or indirectly) the AT2 receptor. Methods are
also provided for decreasing apoptosis and increasing the effects
of substances that stimulate nicotine receptors.
[0045] In some instances, it may be desirable to perform either
preliminary or subsequent screening analysis to determine whether a
test substance is exerting an inhibitory effect on an apoptotic
pathway (e.g., the AT2 or A.beta. pathways) or a stimulatory effect
on a survival pathway (e.g., the nicotinic pathway). For example,
individual ligands may be tested to rule out either pathway through
competitive binding assays using selective agonists or antagonists.
The screening assays of the invention do not necessarily requires
such additional determinations, however, and desired evaluation of
a test substance can be accomplished without further screening.
[0046] The present invention also demonstrates a direct interaction
of the .beta.-amyloid peptide with the .alpha.7 nicotinic
acetylcholine receptor (.alpha.7-nAChR). Further, the molecular
mechanisms of nAChR-mediated neuroprotection are demonstrated to
involve a JAK2 phosphorylation signal cascade. Nicotine-stimulation
of .alpha.7-nAChR results in an initial increase in levels of
phosphorylated tyrosine kinase Janus-Activated Kinase-2 (JAK2) and
subsequent phosphorylation of PI-3 kinase, and Akt. These effects
are blocked by preincubation with the JAK2 specific inhibitor
AG-490 and by .alpha.-Bgt. The .alpha.7-nAChR co-precipitates with
phosphorylated JAK2 and this effect and the neuroprotective effect
of nicotine on A.beta. toxicity are reversed by AG-490. In the
absence of nicotine, exposure to .beta.-amyloid results in
uncoupling to JAK2, induction of caspase-3, and induction of the
DNA-repairing enzyme poly-(ADP-ribose) polymerase (PARP). This
cascade is inhibited by nicotine through JAK2 phosphorylation.
These findings suggest that the .alpha.7-nAChR transduces signals
to PI3K and Akt via JAK2 in a cascade that results in
neuroprotection. A negative feed back regulation between the
.alpha.7-nAChR and .beta.-amyloid-induced cell death is mediated
through JAK2 phosphorylation. Nicotine-stimulated JAK2 and its
neuroprotective effects can be prevented through activation of AT2
receptors. Accordingly, the present inventors have identified novel
mechanisms of receptor interactions relevant to neuronal viability.
Such mechanisms provide novel therapeutic strategies for optimizing
neuroprotection.
[0047] The methods and compositions of the invention can be
selective for the .alpha.7nAChR. This aspect of the invention can
provide for selective enhancement of .alpha.7-mediated activity,
thereby allowing the beneficial effects as described herein to be
achieved with a low incidence of side effects, e.g. those side
effects mediated primarily by non-.alpha.7 receptor subtypes. In
order to achieve the benefits of this aspect of the invention, the
selectively of binding to .alpha.7 receptors can be from about 10-
to about 100-fold greater than to another receptor subtype.
Further, the selectivity can be from about 100- to about 1000-fold
greater for .alpha.7-nAChR. As will be recognized, selectivity can
also be established by evaluating functional stimulation or
inhibition, e.g. by measuring JAK2 phosphorylation according to the
invention.
[0048] Methods for the prophylaxis and/or treatment of central
nervous system disorders are also provided, as are pharmaceutical
compositions useful in such methods.
[0049] As used herein the following terms have the meanings
indicated:
[0050] An "agonist" is a substance that stimulates its binding
partner, typically a receptor. Stimulation is defined in the
context of the particular assay, or may be apparent in the
literature from a discussion herein that makes a comparison to a
factor or substance that is accepted as an "agonist" or "partial
agonist" of the particular binding partner by those of skill in the
art. Stimulation may be defined with respect to an increase in a
particular effect or function that is induced by interaction of the
agonist or partial agonist with a binding partner and can include
allosteric effects.
[0051] An "antagonist" is a substance that inhibits its binding
partner, typically a receptor. Inhibition is defined in the context
of the particular assay, or may be apparent in the literature from
a discussion herein that makes a comparison to a factor or
substance that is accepted as an "antagonist" of the particular
binding partner by those of skill in the art. Inhibition may be
defined with respect to an decrease in a particular effect or
function that is induced by interaction of the agonist with a
binding partner, and can include allosteric effects.
[0052] Herein, the terms A.beta., A.beta. peptide(s),
.beta.-amyloid, amyloid .beta., and amyloid refer to any species of
peptide, polypeptide, or protein associated with the amyloid
characteristic of Alzheimer's Disease. The term "A.beta. (1-42)"
contemplates the 42 amino acid .beta.-amyloid protein, but also
includes fragments thereof that can bind to or mediate an effect
through an nicotinic receptor.
[0053] The term ".alpha.7 nAChR" refers to homopentameric nicotinic
acetylcholine receptors reported to be involved in cognition and
neuroprotection both in animals and human. These effects are shared
by the heteropentamer .alpha.4.beta.2 which is also widely
expressed in human brain (reviewed in Bencheri, M. and J. D.
Schmitt, Current Drug Targets Volume 1, number 4, August 2002; pp
349-357--also see entire issue, which is dedicated to nicotinic
receptor distribution and effects (e.g., .alpha.4.beta.2 and
.alpha.7)). Accordingly, the terms "nAChR" and "nicotine receptor,"
as used herein, encompass such receptors comprising these
subunits.
[0054] As used herein, "PC12" refers to the rat adrenal chromaffin
tumor cell line, PC12. This cell line, originally established by
Greene and Tischler (Greene, L. A. and A. S. Tischler,
Establishment of a noradrenergic clonal line of rat adrenal
pheochromocytoma cells which respond to nerve growth factor. Proc
Natl Acad Sci USA, 73(7):2424-8(1976), has been utilized frequently
in the study of neuronal acetylcholine receptors, including
.alpha.7 nAChR (See, e.g., Patrick, J. and W. B. Stallcup,
Immunological distinction between acetylcholine receptor and the
alpha-bungarotoxin-binding component on sympathetic neurons. Proc
Natl Acad Sci USA., 74(10):4689-92(1977); Whiting, et al.,
Functional acetylcholine receptor in PC12 cells. Nature 327:515-518
(1987); Cooper, S. T. and N. S. Millar, Host cell-specific folding
and assembly of the neuronal nicotinic acetylcholine receptor
alpha7 subunit. J Neurochem. 68(5):2140-51 (1997), all fully
incorporated herein by reference). The angiotensin receptor subtype
II (AT2) has also been expressed in PC12 cells (see, e.g., Wolf,
G., et al., Angiotensin II's antiproliferative effects mediated
through AT2-receptors depend on down-regulation of SM-20. Lab
Invest. 82(10):1305-17 (2002); and Lehtonen, J. Y., et al.,
Analysis of functional domains of angiotensin II type 2 receptor
involved in apoptosis. Mol Endocrinol. 13(7):1051-60(1999), all
fully incorporated herein by reference). The PC12 cell line is very
well-known and has been deposited, for example, with the American
Type Culture Collection under ATCC Number: CRL-1721.
[0055] Various nucleic acid sequences encoding nAChRs are
available, including for .alpha.7 (See, e.g., Chini, B., et al.,
Molecular cloning and chromosomal localization of the human
alpha7-nicotinic receptor subunit gene (CHRNA7), Genomics 19(2):
379-381 (1994); ACCESSION NM.sub.--000746, version
NM.sub.--000746.2 GI:21536283, fully incorporated herein by
reference).
[0056] Nucleic acid sequences encoding AT2 receptors are also
known, and guidance regarding expression cloning is available in
the literature (see, e.g., Mukoyama, M., et al., Expression cloning
of type 2 angiotensin II receptor reveals a unique class of
seven-transmembrane receptors. J Biol Chem. 268(33):24539-42
(1993); Nakajima, M., et al., Cloning of cDNA and analysis of the
gene for mouse angiotensin II type 2 receptor. Biochem Biophys Res
Commun. 197(2):393-9(1993); and Nakajima, M., et al., The
angiotensin II type 2 (AT2) receptor antagonizes the growth effects
of the AT1 receptor: gain-of-function study using gene transfer.
Proc Natl Acad Sci USA. 92(23):10663-7(1995); Homo sapiens
angiotensin receptor 2 (AGTR2), mRNA, ACCESSION XM.sub.--030897,
version XM.sub.--030897.2 GI:22058388, all fully incorporated
herein by reference).
[0057] Of course, other cells lines exist and may be prepared that
are suitable for the methods of the present invention. For example,
the human neuroblastoma cell line SH-SY5Y are also
".alpha.7-permissive" and may be used with .alpha.7-specific
methods according to the present invention (see, e.g., Cooper, S.
T. and N. S. Millar, J Neurochem. 68(5):2140-51 (1997)). Other
cells lines may be appropriate, depending on the subtype of nAChR
being evaluated.
[0058] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, cell biology and recombinant DNA, which are within
the skill of the art. See, e.g., Sambrook, Fritsch, and Maniatis,
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, (F. M. Ausubel et al. eds., 1987);
the series METHODS IN ENZYMOLOGY (Academic Press, INC.), PCR 2: A
PRACTICAL APPROACH (M. J. McPherson, B. D. Hames and (G. R. Taylor
eds., 1995); ANIMAL CELL CULTURE (R. I. Freshney. Ed., 1987); and
ANTIBODIES: A LABORATORY MANUAL (Harlow et al. eds., 1987), all
fully incorporated herein by reference.
[0059] Accordingly, in one aspect, the present invention relates to
a method of screening for substances having an effect on a nicotine
receptor by contacting a cell having a nicotine receptor with a
test substance; and determining any increase or decrease in
phosphorylation of Janus-Activated Kinase 2 (JAK2). An increase in
phosphorylation of JAK2 indicates that the test substance
stimulates the nicotine receptor, and a decrease in phosphorylation
of JAK2 indicates that the test substance inhibits the nicotine
receptor.
[0060] The test substance can be a member of a library of test
substances, and the library can be a combinatorial chemical
library, a peptide, polypeptide, nonpeptidal peptidominetic, an
antibody, or small molecule organic compound library. The library
can also be a random combination of compounds. The test substances
can be screened by high throughput screening.
[0061] In another aspect, the invention relates to a method of
screening for a substance that increases or decreases an effect of
a substance that stimulates a nicotine receptor by contacting a
cell having a nicotine receptor with the substance that stimulates
a nicotine receptor; contacting the cell with a test substance; and
determining any increase or decrease in phosphorylation of JAK2 in
the presence of the test substance relative to a level of JAK2
phosphorylation measured when the cell is in contact with a
substance that stimulates a nicotine receptor in the absence of the
test substance. An increase in JAK2 phosphorylation indicates that
the test substance increases the effect of the substance that
stimulates a nicotine receptor on the nicotine receptor; and a
decrease in JAK2 phosphorylation indicates that the test substance
decreases the effect of the substance that stimulates a nicotine
receptor on the nicotine receptor.
[0062] In another aspect, the invention relates to a method of
screening for substance that inhibits or stimulates an AT2
receptor, by contacting a cell having a nicotine receptor and an
AT2 receptor with a substance that stimulates the nicotine
receptor; contacting the cell with a test substance; and
determining any increase or decrease in phosphorylation of JAK2 in
the presence of the test substance relative to a level of JAK2
phosphorylation measured when the cell is in contact with the
substance that stimulates the nicotine receptor in the absence of
the test substance. An increase in JAK2 phosphorylation indicates
that the test substance inhibits the AT2 receptor; and a decrease
in JAK2 phosphorylation indicates that the test substance
stimulates the AT2 receptor. Any effect of the test substance on
the nicotine receptor in the absence of the AT2 receptor is
predetermined and any increase or decrease in JAK2 phosphorylation
is determined relative to any JAK2 phosphorylation related to such
effects. The test substance can be a member of a library of test
substances, and the library is a combinatorial chemical library, a
peptide, polypeptide, nonpeptidal peptidominetic, an antibody, or
small molecule organic compound library. The library can also be a
random combination of compounds.
[0063] The test substance can be screened by high throughput
screening.
[0064] In another aspect, the invention relates to a method of
screening for a substance that impairs or enhances the effect of a
substance that stimulates the AT2 receptor by contacting a cell
having a nicotine receptor and an AT2 receptor with a substance
that stimulates the nicotine receptor and a substance that
stimulates the AT2 receptor; contacting the cell with a test
substance; and determining any increase or decrease in
phosphorylation of JAK2 in the presence of the test substance
relative to a level of JAK2 phosphorylation measured when the cell
is in contact with the substance that stimulates the nicotine
receptor and the substance that stimulates the AT2 receptor in the
absence of the test substance. An increase in JAK2 phosphorylation
indicates that the test substance impairs the effect of the
substance that stimulates the AT2 receptor, or enhances the effect
of the substance that inhibits the AT2 receptor; and a decrease in
JAK2 phosphorylation indicates that the test substance enhances the
effect of the substance that stimulates the AT2 receptor, or
impairs the effect of the substance that inhibits the AT2
receptor.
[0065] In another aspect, the invention relates to a method of
screening for substances that have an effect on .beta.
amyloid-associated neurotoxicity mediated by a nicotinic receptor
by contacting a cell having a nicotine receptor with a .beta.
amyloid peptide and determining a level of Janus-Activated Kinase 2
(JAK2) phosphorylation; and contacting the cell with a test
substance and determining any increase or decrease in
phosphorylation of JAK2. An increase in JAK2 phosphorylation
indicates that the test substance is a candidate substance for
further evaluation as a substance capable of decreasing .beta.
amyloid-associated neurotoxicity.
[0066] In another aspect, the invention relates to a method of
screening for substances that decrease the neurotoxicity of .beta.
amyloid peptides mediated by a nicotinic receptor by contacting a
cell having a nicotine receptor with a .beta. amyloid peptide and
determining a level of Janus-Activated Kinase 2 (JAK2)
phosphorylation; and contacting the cell with a test substance and
determining any increase or decrease in phosphorylation of JAK2.
Any increase in JAK2 phosphorylation indicates that the test
substance decreases the neurotoxicity of .beta. amyloid
peptides.
[0067] In another aspect, the invention relates to a method of
increasing an effect of a substance that stimulates a nicotine
receptor in cells comprising a nicotine receptor and an AT2
receptor by contacting the cells with a substance that stimulates
the nicotine receptor; and contacting the cell with a substance
selected from the group consisting of an inhibitor of the AT2
receptor and an inhibitor of a substance that stimulates the AT2
receptor.
[0068] In another aspect, the invention relates to a method of
decreasing apoptosis in cells comprising a nicotine receptor and an
AT2 receptor by contacting the cells with a substance that
stimulates a nicotine receptor; and contacting the cells with a
substance selected from the group consisting of an inhibitor of the
AT2 receptor and an inhibitor of a substance that stimulates the
AT2 receptor. Increased cell survival can indicate a decrease in
apoptosis. A decrease in apoptosis can also be indicated by an
observation of decreased poly-(ADP-ribose) polymerase (PARP)
activity, decreased caspase 3 activity, or induction of Bcl2.
[0069] In another aspect, the invention relates to a method of
treatment or prophylaxis for subject suffering from a central
nervous system disorder mediated by a nicotine receptor by
administering an amount of a pharmaceutical composition comprising
a substance that stimulates a nicotine receptor; either or both of
at least one inhibitor of the AT2 receptor or at least one
inhibitor of a substance that stimulates the AT2 receptor; and a
pharmaceutically acceptable carrier. The amount of the
pharmaceutical composition is effective to stimulate the
receptor.
[0070] In another aspect, the invention relates to a method of
treating neurodegeneration associated with Alzheimer's disease by
administering an therapeutically effective amount of a substance
that increases tyrosine phosphorylation of JAK2.
[0071] In another aspect, the invention relates to a method of
treatment or prophylaxis of neurodegeneration associated with
Alzheimer's disease by administering an amount of a pharmaceutical
composition comprising a substance that stimulates a nicotine
receptor; either or both of at least one inhibitor of the AT2
receptor or at least one inhibitor of a substance that stimulates
the AT2 receptor; and a pharmaceutically acceptable carrier. The
amount of the pharmaceutical composition is effective to stimulate
the receptor.
[0072] In another aspect, the invention relates to a pharmaceutical
composition for treatment or prophylaxis of a central nervous
disorder for administration to a subject suffering from the
disorder, comprising a substance that stimulates a nicotine
receptor; and either or both of at least one inhibitor of the AT2
receptor or at least one inhibitor of a substance that stimulates
the AT2 receptor; and a pharmaceutically acceptable carrier. The
substance that stimulates a nicotine receptor can be a cholinergic
ligand, nicotinic agonist, or an acetylcholinesterase inhibitor.
The substance that stimulates a nicotine receptor can also be
selective for .alpha.7-nAChR. The substance selective for
.alpha.7-nAChR can be a substituted quinuclidine compound. The
substance can also be represented by a formula selected from the
group consisting of I, II, and III: ##STR1## The substance that
inhibits the AT2 receptor or that inhibits of a substance that
stimulates the AT2 receptor can be a substance that inhibits a
substance that stimulates AT2, including, for example, an
angiotensin II converting enzyme (ACE) inhibitor.
[0073] In another aspect, the invention relates to a pharmaceutical
composition for treatment or prophylaxis of a neurodegenerative
disorder for administration to a subject suffering from the
disorder, comprising a substance that stimulates a nicotine
receptor; and either or both of at least one inhibitor of the AT2
receptor or at least one inhibitor of a substance that stimulates
the AT2 receptor; and a pharmaceutically acceptable carrier. The
substance that stimulates a nicotine receptor can be a cholinergic
ligand, nicotinic agonist, or an acetylcholinesterase inhibitor.
The substance that stimulates a nicotine receptor can be selective
for .alpha.7-nAChR. The substance selective for .alpha.7-nAChR can
be a substituted quinuclidine compound. The substance that
stimulates a nicotine receptor can be represented by a formula
selected from the group consisting of I, II, and III: ##STR2## The
substance that inhibits the AT2 receptor or that inhibits a
substance that stimulates the AT2 receptor can be a substance that
inhibits a substance that stimulates AT2, for example, an
angiotensin II converting enzyme (ACE) inhibitor. The substance
that inhibits the AT2 receptor or that inhibits a substance that
stimulates the AT2 receptor is a substance that stimulates AT2. The
substance that stimulates AT2 can be PD123177 and/or PD123319.
[0074] Nicotine and the JAK/STAT pathway in neuronal signaling.
Nicotine activates the growth promoting enzyme Janus-Activated
Kinase 2 (JAK2) in PC12 cells. Pre-incubation of these cells with
the JAK2 specific inhibitor AG490 (see Kumano, K., A. et al.,
Biochem. Biophys. Res. Commun. 270: 209-214 (2000)) blocks the
nicotine-induced activation of PI3K and Akt (FIG. 1). Moreover,
nicotine also induces a complex between JAK2 and the .alpha.7
receptor (FIG. 2). These results provide direct evidence for
linkages between JAK2 and the nicotine-induced activation of the
PI3K cascade in PC12 cells.
[0075] Effects of the JAK2 inhibitor AG-490 on the nicotine-induced
tyrosine phosphorylation of JAK2 and PI-3 kinase and serine
phosphorylation of Akt in PC12 cells. JAK2 is tyrosine
phosphorylated in response to nicotine within 5 to 10 min and this
activation remains above basal levels even after longer exposure
(120 min) to nicotine (FIG. 1). The JAK2 inhibitor AG-490 inhibits
the basal and nicotine-stimulated JAK2 tyrosine phosphorylation,
the tyrosine phosphorylation of PI-3K and the serine
phosphorylation of Akt (FIG. 1). Similar results are observed in
the human cell line SH-SY5Y. These results suggest that JAK2
activation by nicotine precedes the activation of PI-3K and its
effector Akt. JAK2 activation is completely prevented by
pre-incubation of .alpha.-bungarotoxin, indicating a
receptor-mediated effect (FIG. 3).
[0076] Effects of Nicotine on the JAK2 complex formation with the
.alpha.7 receptor. To test the hypothesis that JAK2 interacts
directly with .alpha.7-nAChR, immuno-precipitation studies were
conducted using a rabbit polyclonal anti-JAK2 antibody. Cultured
PC12 cells were stimulated with nicotine (10 .mu.M) for various
times, lysed, and JAK2 was immunoprecipitated with anti-JAK2
antibody. Immunoprecipitated proteins were separated by gel
electrophoresis, transferred to nitrocellulose, and immunoblotted
with anti-.alpha.7 antibodies. As shown in FIG. 2, nicotine induced
a rapid association of JAK2 with the .alpha.7 receptor within 5
min. This time course of .alpha.7-receptor association with JAK2 is
similar to that of the nicotine-induced activation of JAK2 (FIG.
1). Identical results were also obtained when the experiments were
repeated using anti-.alpha.7 receptor antibody to immunoprecipitate
the receptor and probing the Western blot with the anti-JAK2
antibody.
[0077] Effects of Ang II pretreatment with or without Ang II
receptor antagonists on nicotine-induced activation of JAK2. Ang II
exerts its biological effects via the activation of two different
receptors known as AT1 and AT2 receptors both belonging to the G
protein-coupled receptor family (Horiuchi, M., W. et al., Circ.
Res. 84: 876-882 (1999)). The AT1 receptor stimulates
proliferation, whereas the AT2 receptor exerts growth inhibitory
effects both in cultured cells, among others PC12 cells, as well as
in vivo (Gallinat, S., S., et al., FEBS Lett. 443: 75-79 (1999)).
In addition, it has also been reported that the angiotensin
converting enzyme density is increased in the temporal cortex from
patients with Alzheimer's disease (Barnes, N. M., et al., Eur. J.
Pharmacol. 200:289-292 (1991)). Pre-incubation of PC12 cells with
angiotensin II (Ang II) blocks the nicotine-induced activation of
JAK2 via the AT2 receptor (FIG. 3).
[0078] Preincubation of PC12 cells with Ang II blocks the
nicotine-induced tyrosine phosphorylation of JAK2 via the AT2
receptor (FIG. 3). This inhibition is completely prevented by
pre-incubation with an AT2 antagonist (PD 123177 at 100 nM), but
not by an AT1 antagonist (candesartan at 100 nM), consistent with
the receptor phenotype expressed in PC12 cells. This inhibition of
nicotine-induced JAK2 phosphorylation was accompanied by a complete
reversal of nicotine-induced neuroprotection as shown by cell
viability and by a nicotine-insensitive PARP induction.
[0079] Co-immunoprecipitation of .alpha.7-nAChR with A.beta. (1-42)
amyloid and JAK2 phosphorylation. Recent studies have shown that
A.beta.(1-42) bind with high affinity to .alpha.7-nAChR and that
this interaction can be inhibited by .alpha.7-nAChR antagonist. The
studies leading to the present invention confirm the molecular
association between A.beta.(1-42) and .alpha.7-nAChR in cells
treated with A.beta.(1-42) (10 .mu.M for 5 minutes) and
immunoprecipitated with anti-A.beta.(1-42) antibodies. Western
analyses using anti-.alpha.7-nAChR antibodies identifies a 57 kDa
protein reactive to anti-.alpha.7-nAChR which co-immunoprecipitates
with endogenous A.beta.(1-42) (FIG. 8). This effect is prevented by
AG-490 pre-treatment of cells (FIG. 8). However, when cells were
co-incubated with 10 .mu.M nicotine, the complex formation between
A.beta.(1-42) and .alpha.7-nAChR was blocked even in the presence
of AG-490 (FIG. 8). While not wishing to be bound by any particular
theory, these results appear to suggest that the interaction
between A.beta.(1-42) and .alpha.7-nAChR can be inhibited by
nicotine independently of JAK2. Pretreatment with A.beta.(1-42)
(0.1 .mu.M to 1 .mu.M) does not result in activation of JAK2 (FIG.
9A and 9B) even at very high concentrations (e.g., at 10 .mu.M and
100 .mu.M, not shown in FIGS. 9A and 9B).
[0080] Effects of nicotine on the A.beta.(1-42)-induced apoptosis
and the role of JAK2. Caspase 3 is expressed in PC12 cells and is
known to be involved in apoptosis. Caspase 3 activity was examined
following A.beta.(1-42)-induced apoptosis. The fluorescent peptide
substrate Ac-DEVD-7AMC was used to measure caspase 3-like activity
in cell lysates. As shown in FIG. 5, the caspase 3 activity that
resulted in the cleavage of the peptide substrate Ac-DEVD-7AMC is
evident after 4 hours of A.beta.(1-42) treatment and increased over
time until it reached a peak after 8 hours of treatment. The
A.beta.(1-42)-induced activation of caspase 3 is blocked by
nicotine (P<0.01), and this inhibition is prevented by AG-490
(FIG. 5).
[0081] The activation of caspase 3 following A.beta.(1-42)
treatment was further explored by measuring the cleavage of the
DNA-repairing enzyme poly-(ADP-ribose) polymerase (PARP) using
Western blot assay. PARP is an endogenous substrate for caspase 3
which is cleaved to a typical 85-kDa fragment during various forms
of apoptosis. As shown in FIG. 7, PARP (116-kDa) was cleaved to its
85-kDa fragment following A.beta.(1-42) treatment. This PARP
cleavage further indicates that caspase 3 or caspase 3-like
proteases are activated in A.beta.(1-42)-induced cell death.
[0082] The involvement of JAK2 in nicotine-induced neuroprotection
in the presence or absence of A.beta.(1-42) was tested. The
decrease in PC12 cell number was measured using a COULTER counter
(Model ZM, Coulter, Hialeah, Fla.) following A.beta.(1-42) and Ang
II treatments in the presence or absence of nicotine and AG-490. As
shown in FIG. 4, cell death induced by A.beta.(1-42) treatment is
significantly reduced in the presence of nicotine (P<0.01).
Nicotine had no effect on A.beta.(1-42)-induced cell death when
co-incubated with AG-490 (FIG. 4). These results demonstrate that
JAK2 plays a role in the nicotine-induced neuroprotection against
A.beta.(1-42)-induced cell death. In contrast, Ang II-induced
apoptosis was not affected by nicotine (FIG. 4).
[0083] .alpha.7-nAChR/JAK2 Neuroprotective Cascade. Direct linkages
between .alpha.7-nAChR and the tyrosine-phosphorylated enzyme JAK2
result in subsequent activation of PI-3-K, Akt, and induction of
Bcl-2. This complex formation and downstream neuroprotective
cascade is prevented when A.beta.(1-42) interacts with
.alpha.7-nAChR. This is evidenced by the stimulation of
pro-apoptotic events including induction of caspase 3, PARP
induction, and decreased cell viability. Whereas nicotine
interaction with .alpha.7-nAChR is "dominant" over A.beta.(1-42)
toxicity through JAK2 activation, nicotine neuroprotective effect
can be neutralized through activation of AT2 receptor as evidenced
by the reversal of JAK2 phosphorylation and inhibition of
nicotine-induced neuroprotection.
[0084] Nicotinic neurotransmission is compromised in the brains of
AD patients and selective loss of nAChR predominates in brain
regions with .beta.-amyloid deposition (Court, J., et al. Biol.
Psychiatry 49: 175-184 (2001)). Accumulating evidence suggests that
neuronal nicotinic receptor (NNR)-selective ligands can also offer
neuroprotective effects in a number of cellular and animal models
including neuronal death resulting from .beta.-amyloid toxicity. A
direct interaction of the .beta.-amyloid peptide with the
.alpha.7-nAChR is suggested by recent findings. .beta.-amyloid
peptide interacts with high affinity to the .alpha.7-nAChR and
results in functional non-competitive blockade .alpha.7-nAChR in
hippocampal neurons (Wang, H. Y., et al. J. Biol. Chem. 275:
5626-5632 (2000); Liu, Q., et al. Proc Natl Acad Sci USA
98(8):4734-9 (2001)). Neuroprotective mechanisms mediated by
nicotine in clonal cells have implicated tyrosine phosphorylation
of PI3P kinase, an enzyme involved in phosphoinositide metabolism
and linked to cell survival and apoptosis. Anti-apoptotic signals
transduced via JAK2 have been reported from several studies. In
hematopoietic cells, the kinase domain of JAK2 mediates the
induction of Bcl-2 and inhibits cell death (Sakai, I. and A. S.
Kraft, J. Biol. Chem. 272:12350-12358 (1997)). Treatment with the
JAK2 inhibitor AG-490 reduced the phosphorylation of PI-3-K
(Kumano, K., et al., Biochem. Biophys. Res. Commun. 270:209-214
(2000)), and that of STAT3 resulting in an increase in caspase-3
activity and Bax protein in acute myocardial infarction (Negoro,
S., et al., Cardiovasc. Res. 47:797-805 (2000)). Activation of
neuronal EPO receptors (EPORs) prevents apoptosis induced by NMDA
(N-methyl-D-aspartate) or NO by triggering crosstalk between the
signaling pathways of JAK2 and nuclear factor-kappaB (NF-kappaB)
(Digicaylioglu, M. and S. A. Lipton. Nature 412:641-647
(2001)).
[0085] The present inventors have shown that .alpha.7-nAChR
activation induces JAK2 phosphorylation and this initial event is
followed by PI-3 kinase phosphorylation and Akt phosphorylation, as
indicated by the inhibitory effect of AG-490 on the phosphorylation
of both proteins. The JAK2 phosphorylation in the presence of
nicotine is completely inhibited by .alpha.-bungarotoxin, an
antagonist to .alpha.7-nAChR. Nicotine-stimulated .alpha.7-nAChR
results in the formation of a complex between the .alpha.7 receptor
protein and the tyrosine phosphorylated JAK2.
[0086] Because interaction between .alpha.7-nAChR and A.beta.(1-42)
has been reported based on ligand-binding and functional studies,
the possibility that .beta.-amyloid could also induce an
.alpha.7-nAChR/JAK2 complex was tested. The association of
.beta.-amyloid and .alpha.7-nAChR was confirmed but no detectable
levels of tyrosine-phosphorylated JAK2 were indicated in response
to binding. In the presence of nicotine, no A.beta.
immunoreactivity was be detected in cell lysates, indicating that
nicotine has "displaced" A.beta. from .alpha.7-nAChR. This effect
is independent of JAK2 phosphorylation, as shown by the lack of any
reversal of this effect in the presence of AG-490.
[0087] The mechanism by which nicotine inhibits A.beta. toxicity is
unclear. The present inventions demonstrates a central role for
tyrosine phosphorylation of JAK2 in .alpha.7-nAChR activation of
key cellular enzymes involved in cell survival and in inhibition of
pro-apoptotic pathways. Nicotine inhibits .beta.-amyloid
cytotoxicity and this effect is completely prevented by inhibition
of tyrosine phosphorylation of JAK2. These effects can be shown by
measuring markers of cytotoxicity (e.g., PARP), induction of
pro-apoptotic enzymes (e.g., caspase 3), cell number, or induction
of Bcl2.
[0088] AT2 receptors are expressed in PC12 and have been shown to
inhibit the JAK/STAT signaling cascade (Kunioku, H., et al.,
Neurosci. Lett. 309: 13-16 (2001). In contrast to nicotine-induced
neuroprotection against A.beta.(1-42), pre-treatment of cells with
Ang II blocks the nicotine-induced activation of JAK2 via the AT2
receptor and completely prevents .alpha.7-nAChR-mediated
neuroprotective effects further suggesting a pivotal role for JAK2
phosphorylation. The present invention demonstrates opposite roles
on cell viability between .alpha.7-nAChR and AT2 receptor activity
(activation of the latter overriding the potential benefit through
the former). These results and the convergence of these pathways on
phosphorylated JAK2 indicate that recruitment of
.alpha.7-nAChR-mediated neuroprotection against A.beta.(1-42) may
be optimized under conditions where AT2-mediated inhibition of JAK2
phosphorylation is minimized. These findings identify novel
molecular mechanisms that are fully consistent with the role
attributed to AT2 and .beta.-amyloid on the pathophysiology
observed in the brain of Alzheimer's Disease patients.
[0089] High-throughput Screening (HTS). The screening methods of
the invention may be readily adapted to facilitate high-throughput
analysis. Assessment of the tyrosine phosphorylation state provides
information regarding candidate substances that either stimulate or
inhibit nicotine receptors such as the .alpha.7 receptor, or that
either stimulate or inhibit the AT2 receptor.
[0090] The substance (compounds) can be present in combinatorial or
other compound libraries, for example, lead generation and/or lead
optimization libraries. For purposes of this invention, lead
generation libraries are relatively large libraries that contain
potential lead compounds, and lead optimization libraries are
developed around compounds identified as potential leads by
assaying lead generation libraries. Such libraries typically
include a large number of compounds, include at least two
compounds, and can include upwards of tens of thousands of
compounds.
[0091] Logically arranged collections of potentially active
compounds can be evaluated using the high throughput bioassays
described herein, such that structure-reactivity relationships
(SARs) can be obtained. Methods for arranging compounds to be
assayed in logical arrangements are known to those of skill in the
art, and described, for example, in U.S. Pat. No. 5,962,736 to
Zambias et al., the contents of which are hereby incorporated by
reference. In one embodiment, the compounds are added to multi-well
plates in the form of an "array," which is defined herein as a
logical positional ordering of compounds in Cartesian coordinates,
where the array includes compounds with a similar core structure
and varying substitutions. Additional guidance regarding HTS assays
methods can be found in U.S. Pat. No. 6,468,736 to Brooker; and in
U.S. Appln. Publication No. 2002/0039749A1 in the name of Wu.
[0092] By placing the compounds in a logical array in multi-tube
arrays or multi-well plates, the effect of individual compounds can
be evaluated, and compared to that of structurally similar
compounds to generate SAR data.
[0093] In one embodiment, the identity and activity of the
compounds are stored on a relational database. By evaluating the
SAR data, lead compounds can be identified, and lead optimization
libraries designed. The logically arranged arrays can be evaluated
in a manner which automatically generates complete relational
structural information such that a positive result provides: (1)
information on a compound within any given spatial address on the
multi-well plates and (2) the ability to extract relational
structural information from negative results in the presence of
positive results.
[0094] Any chemical compound can be used as a test substance
(compound) in the assays of the invention. The assays can be
designed to screen large chemical libraries by automating the assay
steps and providing compounds from any convenient source to assays,
which are typically run in parallel (e.g., in microtiter formats on
microtiter plates in robotic assays). It will be appreciated that
there are many suppliers of chemical compounds, including Sigma
(St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St.
Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs,
Switzerland) and the like.
[0095] In one embodiment, high throughput screening methods involve
providing a combinatorial library containing a large number of test
compounds. Such "combinatorial chemical libraries" are then
screened in one or more assays, as described herein, to identify
those library members (particular chemical species or subclasses)
that display a desired characteristic activity. The compounds thus
identified can serve as conventional "lead compounds" or can
themselves be used as potential or actual therapeutics.
[0096] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0097] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int.
J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (PCT Publication No. WO 91/19735),
encoded peptides (PCT Publication WO 93/20242), random
bio-oligomers (PCT Publication No. WO 92/00091), benzodiazepines
(U.S. Pat. No. 5,288,514), diversomers such as hydantoins,
benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci.
USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al.,
J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics
with .beta.-D-glucose scaffolding (Hirschmann et al., J. Amer.
Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of
small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661
(1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)),
and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658
(1994)), nucleic acid libraries (see, Ausubel, Berger and Sambrook,
all supra), peptide nucleic acid libraries (see, e.g. U.S. Pat. No.
5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature
Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287),
carbohydrate libraries (see, e.g. Liang et al., Science,
274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organic
molecule libraries (see, e.g., benzodiazepines, Baum C&EN,
January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;
thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;
pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino
compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.
5,288,514, and the like).
[0098] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar,
Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek
Biosciences, Columbia, Md., etc.).
[0099] As noted, the invention provides in vitro assays for test
substances that affect nicotine receptor activity as detected by
JAK2 tyrosine phosphorylation in a high throughput format. Control
reactions that measure the level of JAK2 phosphorylation in a
reaction that does not include any test substance are optional, as
the assays are highly uniform. Such optional control reactions are
appropriate and increase the reliability of the assay. Accordingly,
in one embodiment, the methods of the invention include such a
control reaction. For each of the assay formats described, control
reactions which do not include a test substance can provide a
background level of JAK2 phosphorylation.
[0100] In some assays it will be desirable to use positive controls
to ensure that the components of the assays are working properly.
At least two types of positive controls are appropriate. First, a
known stimulator of nAChR which increases JAK2 phosphorylation can
be incubated with one sample of the assay, and the resulting
increase in signal can be determined according to the methods
herein. Second, a known inhibitor of the nAChR can be added, and
the resulting decrease in activity similarly detected. It will be
appreciated that test substances can also be combined with
stimulatory substances or inhibitors to find test substances which
inhibit activation or repression that is otherwise caused by the
presence of the known stimulatory substance or inhibitor. Because
the level of JAK2 phosphorylation resulting from stimulation of the
nAChR is shown herein to be decreased by substances that stimulate
the AT2 receptor, it will be understood that a coordinate protocol
for testing substances mediating an effect through the AT2 receptor
can be arranged for testing substances for effects mediated through
AT2 or, indirectly, through effects on substances that are
themselves modulators of AT2 activity (e.g., the effects of ACE
inhibitors, preventing the formation of Ang II).
[0101] In the high throughput assays of the invention, it is
possible to screen up to several thousand different test substances
in a single day. In particular, each well of a microtiter plate can
be used to run a separate assay against a selected test substance,
or, if concentration or incubation time effects are to be observed,
every 5-10 wells can test a single test substance. Thus, a single
standard microtiter plate can assay about 100 (96) substances. If
1536 well plates are used, then a single plate can easily assay
from about 100- about 1500 different compounds. It is possible to
assay many different plates per day; assay screens for up to about
6,000-20,000, and even up to about 100,000-1,000,000 different
compounds is possible using the integrated systems of the
invention.
[0102] Animal Model Testing. Regarding compositions and methods of
the invention directed toward treatment and/or prophylaxis of
neurodegerative disorders, animal models based on the effects
induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are
relevant (Behmand, R. A. and S. I. Harik. Nicotine enhances
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity. J.
Neurochem., 58:776-9 (1992); Ferger, B., et al., Effects of
nicotine on hydroxyl free radical formation in vitro and on
MPTP-induced neurotoxicity in vivo. Naunyn Schmiedebergs Arch
Pharmacol, 358:351-9 (1998); Fung, Y. K., et al., Chronic
administration of nicotine fails to alter the MPTP-induced
neurotoxicity in mice. Gen Pharmacol 22(4):669-72 (1991); Maggio,
R., et al., Nicotine prevents experimental parkinsonism in rodents
and induces striatal increase of neurotrophic factors. J Neurochem,
71:2439-46(1998); and Parain, K, et al., Nicotine, but not
cotinine, partially protects dopaminergic neurons against
MPTP-induced degeneration in mice. Brain Res. 890:347-350(2001),
all incorporated fully herein by reference).
[0103] Models based on MPTP-induced effects include chronic
hemi-Parkinsonian monkeys (Domino, E. F., et al., Nicotine alone
and in combination with L-DOPA methyl ester or the D(2) agonist
N-0923 in MPTP-induced chronic hemiparkinsonian monkeys. Exp
Neurol, 158:414-21(1999), incorporated fully herein by reference),
degeneration of nigrostriatal dopamine neurons in mice (Janson, A.
M., et al., Differential effects of acute and chronic nicotine
treatment on MPTP-(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)
induced degeneration of nigrostriatal dopamine neurons in the black
mouse. Clin Investig, 70:232-8 (1992), incorporated fully herein by
reference), evaluations of cognitive function in MPTP-treated
animals (Schneider, J. S., et al., Nicotinic acetylcholine receptor
agonist SIB-1508Y improves cognitive functioning in chronic
low-dose MPTP-treated monkeys. J Pharmacol Exp Ther, 290:731-9
(1999), incorporated fully herein by reference), and measurement of
striatal levels of 1-methyl-4-phenylpyridinium (MPP+) (Quik, M. and
D. A. Di Monte, Nicotine administration reduces striatal MPP+
levels in mice. Brain Res, 917:219-24 (2001), incorporated fully
herein by reference).
[0104] Other relevant animal models include kainic acid-induced
effects (Borlongan, C. V., et al., (-)-nicotine protects against
systemic kainic acid-induced excitotoxic effects. Exp Neurol,
136:261-5(1995), incorporated fully herein by reference),
6-hydroxydopamine (6-OHDA) lesion in rat (Costa, G., et al.,
Nicotine prevents striatal dopamine loss produced by
6-hydroxydopamine lesion in the substantia nigra. Brain Res,
888:336-342(2001); Ryan, R. E., et al., Dose-related
neuroprotective effects of chronic nicotine in 6-hydroxydopamine
treated rats, and loss of neuroprotection in .alpha.4 nicotinic
receptor subunit knockout mice. Br J Pharmacol. 132:1650-6(2001);
and Soto-Otero, R. et al., Effects of (-)-nicotine and (-)-cotinine
on 6-hydroxydopamine-induced oxidative stress and neurotoxicity:
relevance for Parkinson's disease. Biochem Pharmacol,
64(1):125-35(2002), all incorporated fully herein by reference);
quinolinic acid-induced hippocampal neurodegeneration (O'Neill, A.
B., et al., Histological and behavioral protection by (-)-nicotine
against quinolinic acid-induced neurodegeneration in the
hippocampus. Neurobiol Learn Mem, 69:46-64 (1998), incorporated
fully herein by reference); murine models of neonatal excitotoxic
brain injury (Laudenbach, V., et al., Selective activation of
central subtypes of nicotinic acetylcholine receptor has opposite
effects on neonatal excitotoxic brain injuries. FASEB J
16:423-425(2002), incorporated fully herein by reference); and
reserpine-induced striatal dopamine deficiency (Oishi, R., et al.,
Possible explanations for the antagonism by nicotine against
reserpine-induced depletion of monoamines in mouse brain. Naunyn
Schmiedebergs Arch Pharmacol. 348:154-7(1993)).
[0105] Effects on the age-associated loss of nigrostriatal
dopaminergic neurons may also be evaluated to determine the
potential for preventing or alleviating neurodegenerative disease
(See, e.g., Prasad, C., et al., Chronic nicotine intake decelerates
aging of nigrostriatal dopaminergic neurons. Life Sci, 54:1169-84
(1994); and see, generally, Picciotto, M. R. and M. Zoli, Nicotinic
receptors in aging and dementia. J Neurobiol. 53:641-55(2002), all
incorporated fully herein by reference).
[0106] Compositions. The methods of prophylaxis and/or treatment,
as well as the pharmaceutical compositions, can include substances
that stimulate nicotine receptors and substances that inhibit an
AT2 receptor, either directly or indirectly.
[0107] Substances that stimulate nicotine receptors, either
directly or indirectly, include .alpha.7 agonists, cholinergic
ligands, nicotinic agonists, and/or acetylcholinesterase
inhibitors. Nicotine receptor agonists of the invention can include
those discussed in, e.g., U.S. Pat. No. 5,977,144; U.S. Pat. No.
6,218,383; U.S. Pat. No. 6,310,102; U.S. Pat. No. 6,232,316;
Miller, et al., published international patent application No.
WO0190109A1; Bencherif, et al., published international patent
application No. WO0182978A2; Dull et al., published international
patent application No. WO0071520A2; Bencherif, published
international patent application No. WO0007600A1; and Caldwell, et
al., published international patent application No. WO9965876A1
(all fully incorporated herein by reference). Substances that can
be used according to the methods of the present invention include
those compounds as represented by the following formulae I-III:
##STR3## Substances that can be used also include compounds
disclosed in U.S. Pat. Nos. 5,952,339; 5,986,100; 6,057,446; and
6,211,372 (all fully incorporated herein by reference). Substances
that selectively stimulate particular receptors, e.g.,
.alpha.7-nAChR, can be used. Schmitt and Bencherif provide guidance
regarding the selection of compounds that selectively interact with
particular receptors, including the .alpha.7 subtype. (See Schmitt,
J. and M. Bencherif, Chapter 5, "Nicotinic Acetylcholine
Receptors," in Ann. Rep. Med. Chem. 35:41-51 (2000); and Schmitt,
J., Curr. Med. Chem., 7(8):749-800 (2000), both fully incorporated
herein by reference).
[0108] Various compounds have been reported to interact with alpha
7 nicotinic receptors and have been proposed as therapies on that
basis. See, for instance, PCT WO 99/62505, PCT WO 99/03859, PCT WO
97/30998, PCT WO 01/36417, PCT WO 02/15662, PCT WO 02/16355, PCT WO
02/16356, PCT WO 02/16357, PCT WO 02/16358, PCT WO 02/17358,
Stevens, et al., Psychopharm. 136:320 (1998), Dolle, et al., J.
Labelled Comp. Radiopharm. 44: 785-795 (2001) and Macor, et al.,
Bioorg. Med. Chem. Lett. 11:319-321 (2001) and references therein.
Among these compounds, a common structural theme is that of the
substituted tertiary bicylic amine (e.g., quinuclidine). Similar
substituted quinuclidine compounds have also been reported to bind
at muscarinic receptors. See, for instance, U.S. Pat. No. 5,712,270
to Sabb and PCTs WO 02/00652 and WO 02/051841.
[0109] Compounds of useful according to the present invention
include azaadamantane compounds, e.g., as taught in U.S. Pat. Nos.
5,986,100 and 5,952,339, having the general formula I: ##STR4##
[0110] wherein each of X and X' are individually nitrogen or carbon
bonded to a substituent species characterized as having a sigma m
value greater than 0, often greater than 0.1, and generally greater
than 0.2, and even greater than 0.3; less than 0, generally less
than -0.1; or 0 (i.e., is hydrogen); as determined in accordance
with Hansch, et al., Chem. Rev. 91:165 (1991); Z' is a substituent
other than hydrogen (e.g., alkyl, aryl, aralkyl, halo, hydroxyl,
alkoxyl, alkylhydroxy, cyano and mercapto); j is an integer from 0
to 5, preferably 0 or 1, and most preferably 0; and the wavy line
in the structure indicates that certain compounds can exist in the
form of enantiomers or diasteromers depending upon the placement of
substituent groups on the 1-aza-tricyclo[3.3.1.1.sup.3,7]decane
portion of the compound. The identity of A, A' and A'' can vary,
and individually represent those species described as substituent
species to the aromatic carbon atom previously described for X and
X'; and each of those substituent species often has a sigma m value
between about -0.3 and about 0.75, frequently between about -0.25
and about 0.6. More specifically, individual examples of the
substituent species to X and X' (when X and X' are carbon atoms),
Z', A, A' and A'' include F, Cl, Br, I, R', NR'R'', CF.sub.3, OH,
CN, NO.sub.2, C.sub.2R', SH, SCH.sub.3, N.sub.3, SO.sub.2,
CH.sub.3, OR', SR', C(.dbd.O)NR'R'', NR'C(.dbd.O)R', C(.dbd.O)R',
C(.dbd.O)OR', (CH.sub.2).sub.q OR', OC(.dbd.O)R', OC(.dbd.O)NR'R'',
and NR'C(.dbd.O)OR', where R' and R'' are individually hydrogen or
lower alkyl (e.g., C.sub.1-C.sub.10 alkyl, preferably
C.sub.1-C.sub.6 alkyl, and more preferably cyclohexyl, methyl,
ethyl, isopropyl or isobutyl), an aromatic group-containing
species, and q is an integer from 1 to 6. In certain circumstances,
it is preferred that when X' is carbon, the sigma m value of the
substituent bonded to that carbon is not equal to 0. However, for
certain compounds, the sigma m value of A'' is equal to 0; that is,
A'' is H. For certain preferred compounds, X' is carbon bonded to a
non-hydrogen substituent (i.e., such compounds are
5-substituted-3-pyridyl compounds). In addition, it is highly
preferred that A is hydrogen, it is preferred that A' is hydrogen,
and normally A'' is hydrogen. Generally, A and A' both are
hydrogen; sometimes A and A' are hydrogen, and A'' is halo, OR',
OH, NR'R'', SH or SR'; and often A, A' and A'' are all hydrogen. R'
and R'' can be straight chain or branched alkyl, or R' and R'' can
form a cycloalkyl functionality (e.g., cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, quinuclidinyl).
Representative aromatic group-containing species include pyridinyl,
quinolinyl, pyrimidinyl, phenyl, benzyl (where any of the foregoing
can be suitably substituted with at least one substitutent group,
such as alkyl, halo, or amino substituents). Representative
aromatic ring systems are set forth in Gibson, et al., J. Med.
Chem. 39:4065 (1996). For NR'R'', the nitrogen and R' and R'' can
form a ring structure, such as aziridinyl, azetidinyl,
pyrrolidinyl, piperidinyl, piperazinyl or morpholinyl. Z'' includes
hydrogen or Z' (where Z' is as previously defined), preferably
hydrogen. Preferably, Z' is attached to either of the carbon atoms
alpha to Y. Y includes C.dbd.O, C(OH)R', or C-A (where A is as
previously defined), but preferably Y is CH.sub.2. The compounds
represented in general formula I are optically active; and can be
provided and used in the form of racemates and enantiomers.
[0111] In a particular embodiment, X' is nitrogen characterized as
having a sigma m value greater than 0, less than 0 or 0; X is
nitrogen or carbon bonded to a substituent species characterized as
having a sigma m value equal to 0; A, A' and A'' are individually
substituent species characterized as having a sigma m value greater
than 0, less than 0 or 0; Z' is a substituent other than hydrogen;
j is an integer from 0 to 5; and the wavy line in the structure
indicates that the compound can exist in the form of an enantiomer
or a diasteromer; Z'' is hydrogen or a substituent other than
hydrogen; Y is C.dbd.O, C(OH)R' or C-A, where R' is hydrogen or
lower alkyl.
[0112] A representative compound is
5-aza-1-(hydroxymethyl)-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]-decan-2-on-
e, where A, A' and A'' each are hydrogen, X is CH, X' is nitrogen,
Y is C.dbd.O, Z'' is CH.sub.2OH and j is 0. Another representative
compound is
5-aza-6-(3-pyridyl)tricyclo[3.3.1.1..sup.3,7]decan-2-one, where A,
A' and A'' each are hydrogen, X is CH, X' is nitrogen, j is 0, Z''
is H and Y is C.dbd.O. Another representative compound is
5-aza-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]decan-2-ol, where A, A'
and A'' each are hydrogen, X is CH, X' is nitrogen, Y is
CH.sub.2OH, j is 0 and Z'' is H. These compounds are particularly
useful as intermediates for the preparation of other compounds of
the present invention.
[0113] A representative compound of the present invention is
1-aza-2-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]decane, where A, A' and
A'' each are hydrogen, X is CH, X' is nitrogen, Y is CH.sub.2, j is
0, Z'' is H and X is CH. Another representative compound of the
present invention is
1-aza-2-(5-bromo(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane, where
A, A' and A'' each are hydrogen, X is CBr, X' is nitrogen, Y is
CH.sub.2, j is 0 and Z'' is H. Another representative compound of
the present invention is
1-aza-2-(5-amino-(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane, where
A, A' and A'' each are hydrogen, X is CNH.sub.2, X' is nitrogen, Y
is CH.sub.2, j is 0 and Z'' is H. Another representative compound
of the present invention is
1-aza-2-(5-ethoxy-(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane,
where A, A' and A'' each are hydrogen, Y is CH.sub.2, j is 0, Z''
is H, X is COCH.sub.2, CH.sub.3, and X' is nitrogen. Another
representative compound of the present invention is
1-aza-2-(5-isopropoxy-(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane,
where A, A' and A'' each are hydrogen, Y is CH.sub.2, j is 0, Z''
is H, X is COC.sub.3H.sub.7, and X' is nitrogen. Another
representative compound of the present invention is
5-aza-6-(5-bromo-(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decan-2-ol,
where A, A' and A'' each are hydrogen, X is CBr, X' is nitrogen, Y
is CH.sub.2OH, j is 0 and Z'' is H.
[0114] Particular embodiments according to the foregoing general
formula include: [0115]
1-aza-2-(5-bromo(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane; [0116]
1-aza-2-(5-amino-(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane;
[0117]
1-aza-2-(5-ethoxy-(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane;
[0118]
1-aza-2-(5-isopropoxy-(3-pyridyl))tricyclo[3.3.1.1.sup.3,7]decane;
[0119] 2-(3-pyridyl)-1-azatricyclo[3.3.1.1.sup.3,7]decane; [0120]
5-aza-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]decan-2-ol; [0121]
5-aza-1-(hydroxymethyl)-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]-decan-2-on-
e; [0122] 1-aza-2-(3-pyridyl)-1-azatricyclo[3.3.1.1.sup.3,7]decane;
[0123] 5-aza-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]decan-2-ol;
[0124]
5-aza-1-(hydroxymethyl)-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]-decan-2-on-
e; [0125] 5-aza-6-(3-pyridyl)tricyclo[3.3.1.1.sup.3,7]decan-2-one;
and [0126] enantiomers and pharmaceutically acceptable salts
thereof.
[0127] Compounds of useful according to the present invention also
include diazabicyclic compounds, e.g., as taught in published
international patent application WO 01/90109, having the following
general formula: ##STR5## In the structure, Q is (CH.sub.2).sub.u,
Q.sup.i is (CH.sub.2).sub.v, Q.sup.ii is (CH.sub.2).sub.w,
Q.sup.iii is (CH.sub.2).sub.x, and Q.sup.iv is (CH.sub.2).sub.y,
where u, v, w and x are individually 0, 1, 2, 3 or 4, preferably 0
or 1, and y is 1 or 2. R is hydrogen or lower alkyl, preferably
hydrogen. In addition, the values of u, v, w, x and y are selected
such that the resulting diazabicyclic ring contains 7, 8 or 9
members, preferably 7 members. Z represents a suitable non-hydrogen
substituent species; exemplary species are set forth hereinafter.
In addition, j is an integer from 0 to 10, preferably 0, 1 or
2.
[0128] In the structure, preferably Cy represents a suitably
substituted 6-membered aromatic ring, as represented by the
formula: ##STR6## where each of X, X' and X'' are individually
nitrogen, nitrogen bonded to oxygen (e.g., an N-oxide or other N--O
functionality) or carbon bonded to a substituent species (i.e.,
hydrogen or a non-hydrogen species); A is O (oxygen) or C.dbd.O; D
is a suitable non-hydrogen substituent species, as set forth
hereinafter; k is either 0, 1 or 2, preferably 0 or 1; and Cx is
selected from a group consisting of aryl, substituted aryl,
heteroaryl, substituted heteroaryl, non-aromatic heterocyclyl,
substituted non-aromatic heterocyclyl, non-aromatic
heterocyclylalkyl and substituted non-aromatic heterocyclylalkyl. A
can also be a covalent bond, with the proviso that when A is so
defined, the diazabicyclic ring is not
2,5-diazabicyclo[2.2.1]heptane and/or Cx is not phenyl or
substituted phenyl. When A is a covalent bond, it is preferred that
Cx is aryl or heteroaryl.
[0129] Non-hydrogen substituent species Z and D, as well as those
substituent species attached to the various Cx groups, typically
have a sigma m value between about -0.3 and about 0.75 and include
alkyl, substituted alkyl, alkenyl, substituted alkenyl,
non-aromatic heterocyclyl, substituted non-aromatic heterocyclyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
alkylaryl, substituted alkylaryl, arylalkyl, substituted arylalkyl,
--F, --Cl, --Br, --I, --OR', --NR'R'', --CF.sub.3, --CN, --N.sub.3,
--NO.sub.2, --C.sub.2R', --SR', --SOR', --SO.sub.2CH.sub.3,
--SO.sub.2NR'R'', --C(.dbd.O)NR'R'', --NR'C(.dbd.O)R'',
--NR'SO.sub.2R'', --C(.dbd.O)R', --C(.dbd.O)OR',
--(CH.sub.2).sub.qOR', --OC(.dbd.O)R',
--(CR'R'').sub.qOCH.sub.2C.sub.2R', --(CR'R'').sub.qC(.dbd.O)R',
--O(CR'R'').sub.qC(.dbd.O)R', --C.sub.2(CR'R'').sub.qOR',
--(CR'R'').sub.qNR'R'', --OC(.dbd.O)NR'R'' and --NR'C(.dbd.O)OR'
where R' and R'' are individually hydrogen or lower alkyl (e.g.,
straight chain or branched alkyl including C.sub.1-C.sub.8,
preferably C.sub.1-C.sub.5, such as methyl, ethyl, or isopropyl),
an aromatic group-containing species or a substituted aromatic
group-containing species, and q is an integer from 1 to 6. R' and
R'' can form a cycloalkyl functionality. Representative aromatic
groups include carbocyclic (phenyl, biphenyl, naphthyl, etc.) and
heterocyclic (pyridinyl, pyrimidinyl, quinolinyl, indolyl, etc.)
rings.
[0130] As used herein in reference to the diazabicyclic compounds
useful according to the invention, "alkyl" refers to straight chain
or branched alkyl radicals including C.sub.1-C.sub.8, preferably
C.sub.1-C.sub.5, such as methyl, ethyl, or isopropyl, and cyclic
alkyl radicals up to 8 carbons; "substituted alkyl" refers to alkyl
radicals further bearing one or more substituent species such as
hydroxy, alkoxy, mercapto, aryl, heterocyclo, halo, amino,
carboxyl, carbamyl, cyano, and the like; "alkenyl" refers to
straight chain or branched hydrocarbon radicals including
C.sub.1-C.sub.8, preferably C.sub.1-C.sub.5 and having at least one
carbon-carbon double bond; "substituted alkenyl" refers to alkenyl
radicals further bearing one or more substituent species as defined
above; "aryl" refers to aromatic radicals having six to ten carbon
atoms; "substituted aryl" refers to aryl radicals further bearing
one or more substituent species as defined above; "alkylaryl"
refers to alkyl-substituted aryl radicals; "substituted alkylaryl"
refers to alkylaryl radicals further bearing one or more
substituent species as defined above; "arylalkyl" refers to
aryl-substituted alkyl radicals; "substituted arylalkyl" refers to
arylalkyl radicals further bearing one or more substituent species
as defined above; "heterocyclyl" refers to saturated or unsaturated
cyclic radicals containing one or more heteroatoms (e.g., O, N, S)
as part of the ring structure and having two to seven carbon atoms
in the ring; "substituted heterocyclyl" refers to heterocyclyl
radicals further bearing one or more substituent species as defined
above.
[0131] As used herein in reference to the diazabicyclic compounds
useful according to the invention, the term "heteroaryl" refers to
heterocyclic aromatic radicals, such as pyridinyl, pyrimidinyl,
pyrazinyl, pyridazinyl, quinolinyl, furanyl, thienyl, pyrrolyl,
indolyl, benzoxazolyl, etc.; "non-aromatic heterocyclyl" refers to
heterocyclic radicals, saturated or unsaturated, which are not
aromatic, such as tetrahydrofuranyl, tetrahydropyranyl,
tetrahydrothienyl, tetrahydrothiopyranyl, pyrrolidinyl,
piperidinyl, etc.; "non-aromatic heterocyclylalkyl" refers to
non-aromatic heterocyclyl radicals attached through an alkylene
chain of up to four carbon atoms; in each case, "substituted"
refers to the replacement of one or more of the hydrogens in the
group with a non-hydrogen substituent species, as described
above.
[0132] The preferred embodiments of the invention are those in
which one or two of X, X' and X'' are nitrogen, the most preferred
being the case in which only X'' is nitrogen. When only X'' is
nitrogen, it is preferred that A is attached at the C-5 position of
the pyridine ring and that k is 0 or 1.
[0133] Cx is preferably: ##STR7## wherein Y, Y', Y'' and Y''' are
individually nitrogen, nitrogen bonded to oxygen, or carbon bonded
to hydrogen or a substituent species, G; E is oxygen, sulfur or
nitrogen bonded to hydrogen or a substituent species, G; E', E''
and E''' are individually nitrogen or carbon bonded to hydrogen or
a substituent species, G; m is 0, 1, 2, 3 or 4; p is 0, 1, 2 or 3;
n is 0, 1, 2, 3 or 4; and G is selected from the group consisting
of alkyl, substituted alkyl, alkenyl, substituted alkenyl,
non-aromatic heterocyclyl, substituted non-aromatic heterocyclyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
alkylaryl, substituted alkylaryl, arylalkyl, substituted arylalkyl,
--F, --Cl, --Br, --I, --OR', --NR'R'', --CF.sub.3, --CN, --N.sub.3,
--NO.sub.2, --C.sub.2R', --SR', --SOR', --SO.sub.2CH.sub.3,
--SO.sub.2NR'R'', --C(.dbd.O)NR'R'', --NR'C(.dbd.O)R'',
--NR'SO.sub.2R'', --C(.dbd.O)R', --C(.dbd.O)OR',
--(CH.sub.2).sub.qOR', --OC(.dbd.O)R',
--(CR'R'').sub.qOCH.sub.2C.sub.2R', --(CR'R'').sub.qC(.dbd.O)R',
O(CR'R'').sub.qC(.dbd.O)R', --C.sub.2(CR'R'').sub.qOR',
--(CR'R'').sub.qNR'R'', --OC(.dbd.O)NR'R'' and --NR'C(.dbd.O)OR'
where R' and R'' are individually hydrogen, lower alkyl, an
aromatic group-containing species or a substituted aromatic
group-containing species, and q is an integer from 1 to 6.
Preferably Y, Y', Y'', Y''', E', E'' and E''' are all carbon bonded
to a substituent species. Alternatively one or two of Y, Y', Y'',
Y''', E', E'' and E''' are nitrogen and the remaining are carbon
bonded to a substituent species. R' and R'' can form a cycloalkyl
functionality. Representative aromatic groups include carbocyclic
(phenyl, biphenyl, naphthyl, etc.) and heterocyclic (pyridinyl,
pyrimidinyl, quinolinyl, indolyl, etc. ) rings. Adjacent
non-hydrogen substituent species, G, may combine to form one or
more saturated or unsaturated, substituted or unsubstituted,
carbocyclic or heterocyclic rings containing, but not limited to,
ether, acetal, ketal, amine, ketone, lactone, lactam, carbamate and
urea functionalities.
[0134] Representative diazabicyclic compounds useful according to
the present invention include the following: [0135]
(1S,4S)-2-(5-phenoxy-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane;
[0136]
(1R,4R)-2-(5-phenoxy-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane;
[0137]
(1S,4S)-2-(5-(3-methoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-
; [0138]
(1R,4R)-2-(5-(3-methoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2-
.1]heptane; [0139]
(1S,4S)-2-(5-(4-methoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-
; [0140]
(1R,4R)-2-(5-(4-methoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2-
.1]heptane; [0141]
(1S,4S)-2-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]hep-
tane; [0142]
(1R,4R)-2-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]hep-
tane; [0143]
(1S,4S)-2-(5-(4-fluorophenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane;
[0144]
(1R,4R)-2-(5-(4-fluorophenoxy)-3-pyridyl)-2,5-diazabicyclo[2.2.1-
]heptane; [0145]
(1S,4S)-2-(5-benzoyl-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane;
[0146]
(1R,4R)-2-(5-benzoyl-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane;
[0147]
(1S,4S)-2-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-2,5-diazabicyclo[2.2-
.1]heptane; [0148]
(1R,4R)-2-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-2,5-diazabicyclo[2.2-
.1]heptane; [0149]
(1S,4S)-2-(5-(4-(N-trifluoroacetylpiperidinyl)oxy)-3-pyridyl)-2,5-diazabi-
cyclo[2.2.1]heptane; [0150]
(1R,4R)-2-(5-(4-(N-trifluoroacetylpiperidinyl)oxy)-3-pyridyl)-2,5-diazabi-
cyclo[2.2.1]heptane; [0151]
6-methyl-3-(5-phenyl-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
[0152]
6-methyl-3-(5-phenoxy-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
[0153]
6-methyl-3-(5-(3-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane-
; [0154]
6-methyl-3-(5-(4-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.-
2.1]octane; [0155]
6-methyl-3-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]oc-
tane; [0156]
6-methyl-3-(5-(4-fluorophenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
[0157]
6-methyl-3-(5-benzoyl-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
[0158]
6-methyl-3-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,6-diazabi-
cyclo[3.2.1]octane; [0159]
3-methyl-6-(5-phenyl-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
[0160]
3-methyl-6-(5-phenoxy-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
[0161]
3-methyl-6-(5-(3-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane-
; [0162]
3-methyl-6-(5-(4-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.-
2.1]octane; [0163]
3-methyl-6-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]oc-
tane; [0164]
3-methyl-6-(5-(4-fluorophenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
[0165]
3-methyl-6-(5-benzoyl-3-pyridyl)-3,6-diazabicyclo[3.2.1]octane;
[0166]
3-methyl-6-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,6-diazabi-
cyclo[3.2.1]octane; [0167]
6-(5-phenyl-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane; [0168]
6-(5-phenoxy-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane; [0169]
6-(5-(3-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
[0170]
6-(5-(4-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
[0171]
6-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]non-
ane; [0172]
6-(5-(4-fluorophenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
[0173] 6-(5-benzoyl-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
[0174]
6-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonan-
e; [0175] 3-(5-phenyl-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
[0176] 3-(5-phenoxy-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
[0177]
3-(5-(3-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
[0178]
3-(5-(4-methoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
[0179]
3-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]non-
ane; [0180]
3-(5-(4-fluorophenoxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
[0181] 3-(5-benzoyl-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonane;
[0182]
3-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,6-diazabicyclo[3.2.2]nonan-
e; [0183] 3-(5-phenyl-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
[0184] 3-(5-phenoxy-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
[0185]
3-(5-(3-methoxyphenoxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
[0186]
3-(5-(4-methoxyphenoxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
[0187]
3-(5-(3,4-dimethoxyphenoxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]non-
ane; [0188]
3-(5-(4-fluorophenoxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane;
[0189] 3-(5-benzoyl-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonane; and
[0190]
3-(5-(4-(N-phenylpiperidinyl)oxy)-3-pyridyl)-3,7-diazabicyclo[3.3.1]nonan-
e, [0191] or an enantiomer, or a pharmaceutically acceptable salt
thereof.
[0192] Compounds useful according to the present invention also
include cinnamamides of 3-aminoquinuclidine, e.g., as taught in
published international patent application WO 01/36417, of the
general formula: ##STR8## wherein Arom represents a 5- or
6-membered aromatic or heteroaromatic ring containing zero to three
nitrogen atoms, zero to one oxygen atoms, and zero to one sulfur
atoms, or an 8-, 9- or 10-membered fused aromatic or heteroaromatic
ring system containing zero to four nitrogen atoms, zero to one
oxygen atom, and zero to one sulfur atoms, which may be optionally
substituted with one or more substituents selected from the
following: hydrogen, halogen, C.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4 alkynyl, aryl, heteroaryl,
--CO.sub.2R.sup.1, --CN, --NO.sub.2, --NR.sup.2R.sup.3, --CF.sub.3
or --OR.sup.4; [0193] R.sup.2, R.sup.3, and R.sup.4 are
independently hydrogen, C.sub.1-C.sub.4 alkyl, aryl, heteroaryl,
--C(O)R.sup.5, --C(O)NHR.sup.6, --C(O)R.sup.7 or --SO.sub.2R.sup.8,
or R.sup.2 and R.sup.3 may together be
(CH.sub.2).sub.jQ(CH.sub.2).sub.k where Q is O, S, NR.sup.9, or a
bond; [0194] j is 2 to 4; [0195] k is 0 to 2; and [0196] R.sup.1,
R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8 and R.sup.9, are
independently C.sub.1-C.sub.4 alkyl, aryl, or heteroaryl.
[0197] The compounds according to the foregoing general formula can
include: [0198]
N-(1-Azabicyclo[2.2.2]oct-3-yl)(E-3-phenylpropenamide); [0199]
N-(1-Azabicyclo[2.2.2]oct-3-yl)(3-phenylpropenamide); [0200]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-nitrophenyl)propenamide];
[0201]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-nitrophenyl)propenamide];
[0202]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-aminophenyl)propenamide];
[0203]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[Z-3-(2-methoxyphenyl)propenamide];
[0204]
N-(1-Azabicyclo[2.2.2]oct-3-yl)-N-methyl-(E-3-phenylpropenamide);
[0205]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-2-phenylcyclopropane-1-carboxa-
mide); [0206]
N-(1-Azabicyclo[2.2.2]oct-3-yl)(Z-2-fluoro-3-phenylpropenamide);
[0207]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-formamidophenyl)propenamide];
[0208]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-nitrophenyl)propenamide];
[0209]
N-(1-Azabicyclo[2.2.2]oct-3-yl)(E-3-(4-aminophenyl)propenamide;
[0210]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-formamidophenyl)propenamid-
e]; [0211]
N-(1-Azabicyclo[2.2.2]oct-3-yl)(Z-3-methyl-3-phenylpropenamide);
[0212]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-N-methylanlinophenyl)propenamide];
[0213]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-N,N-dimethylaminophenyl)p-
ropenamide]; [0214]
N-(1-Azabicyclo[2.2.2]oct-3-yl)(Z-3-phenylpropenamide); [0215]
N-(1-Azabicyclo[2.2.2]oct-3-yl)(E-3-methyl-3-phenylpropenamide);
[0216] N-(1-Azabicyclo[2.2.2]oct-3-yl)(E-2,3-diphenylpropenamide);
[0217]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-methoxyphenyl)propenamide];
[0218] N-(1-Azabicyclo[2.2.2]oct-3-yl)(E
methyl-3-phenylpropenamide); [0219]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-methylphenyl)propenamide];
[0220]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-methoxyphenyl)propenamide-
]; [0221]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-fluorophenyl)propenamide];
[0222]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-fluorophenyl)propenamide];
[0223]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-(2-chlorophenyl)propenamide];
[0224]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-chlorophenyl)propenamide];
[0225]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-chlorophenyl)propenamide]- ;
[0226]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3,4-dichlorophenyl)propena-
mide]; [0227]
N-(1-Azabicyclo[2.2.2]oct-3-yl)-[E-3-(3-bromophenyl)propenamide]
[0228]
N-(1-Azabicyclo[2.2.2]oct-3-yl)-[E-3-(4-bromophenyl)propenamide];
[0229]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-iodophenyl)propenamide];
[0230]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-iodophenyl)propenamide];
[0231]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-trifluoromethylphenyl)propenamide]-
; [0232]
N-(1-Azabicyclo[2.2.2]oct-3-yl)-[E-3-(3-trifluoromethylphenyl)p-
ropenamide]; [0233]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-furyl)propenamide]; [0234]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-furyl)propenamide]; [0235]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-pyridyl)propenamide]; [0236]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-pyridyl)propenamide]; [0237]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-pyridyl)propenamide]; [0238]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(2-thienyl)propenamide]; [0239]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(3-thienyl)propenamide]; [0240]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(5-nitro-2-furyl)propenamide];
[0241]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(5-methoxy-3-pyridyl)propenam-
ide]; [0242]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(5-hydroxy-3-pyridyl)propenamide];
[0243]
N-(1-Azabicyclo[2.2.2]oct-3-yl)[E-3-(4-imidazolyl)propenamide];
[0244] N-(endo-8-Aza-8-methylbicyclo[3.2.1]oct
yl)(E-3-phenylpropenamide); [0245]
N-(exo-8-Aza-8-methylbicyclo[3.2.1]oct yl)(E-3-phenylpropenamide);
[0246] or an enantiomer thereof, or a pharmaceutically acceptable
salt thereof.
[0247] Compounds useful according to the present invention also
include arylcarbamates of 3-quinuclidinol, e.g., as taught in
published international patent application WO 97/30998, of the
general formula: ##STR9##
[0248] Particular embodiments of the foregoing general formula
include: [0249] N-phenylcarbamic acid 1-azabicyclo[2.2.2]octan-3-yl
ester; [0250] N-(4-bromophenyl)carbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; [0251]
N-(4-methylphenyl)carbamic acid 1-azabicyclo[2.2.2]octan-3-yl
ester; [0252] N-(4-methoxyphenyl)carbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; [0253]
N-(3,4-dichlorophenyl)carbamic acid 1-azabicyclo[2.2.2]octan-3-yl
ester; [0254] N-(4-cyanophenyl)carbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; [0255] N-phenylcarbamic acid
1-azabicyclo[2.2.1]heptan-3-yl ester; [0256]
N-(3-methoxyphenyl)carbamic acid 1-azabicyclo[2.2.2]octan-3-yl
ester; [0257] N-phenylthiocarbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; [0258] N-(2-pyridyl)carbamic
acid 1-azabicyclo[2.2.2]octan-3-yl ester; [0259]
N-(1-naphthyl)carbamic acid 1-azabicyclo[2.2.2]octan-3-yl ester;
[0260] N-phenylcarbamic acid (3R)-1-azabicyclo[2.2.2]octan-3-yl
ester; [0261] N-phenylcarbamic acid
(3S)-1-azabicyclo[2.2.2]octan-3-yl ester; [0262]
N-(4-pyridyl)carbamic acid 1-azabicyclo[2.2.2]octan-3-yl ester;
[0263] N-(m-biphenyl)carbamic acid 1-azabicyclo[2.2.2]octan-3-yl
ester; [0264] N-(3-quinolinyl)carbamic acid
1-azabicyclo[2.2.2]octan-3-yl ester; [0265] or an enantiomer
thereof, or a pharmaceutically acceptable salt thereof.
[0266] Compounds useful according to the present invention also
include heteroaromatic amides of 3-aminoquinuclidine, e.g., as
taught in published international patent application WO 02/15662,
of the general formula: ##STR10##
[0267] where Arom is a cyclic heteroaromatic moiety where the
heteroatoms can be from 1-3 atoms selected from oxygen, sulfur, or
nitrogen of the following structures: ##STR11##
[0268] wherein U is --O--, --S--, or --N(R.sub.1)--;
[0269] V and Y are independently selected from .dbd.N--, or
.dbd.C(R.sub.2)--;
[0270] Z is .dbd.N--, or .dbd.CH--, provided that when both V and Y
are .dbd.C(R.sub.2)-- and Z is .dbd.CH--, only one
.dbd.C(R.sub.2)-- can be .dbd.CH--, and further provided that when
U is --O--, Y is .dbd.C(R.sub.2)-- and Z is .dbd.C(R.sub.2)--, V
cannot be .dbd.N--;
[0271] R.sub.1 is --H, alkyl, cycloalkyl, heterocycloalkyl,
halogenated alkyl, halogenated cycloalkyl, halogenated
heterocycloalkyl, substituted alkyl, substituted cycloalkyl,
substituted heterocycloalkyl, or aryl, and provided that when W is
(b) and Z is .dbd.N-- and U is N(R.sub.1), R.sub.1 cannot be phenyl
or substituted phenyl;
[0272] Alkenyl is straight and branched-chain moieties having from
2-6 carbon atoms and having at least one carbon-carbon double
bond;
[0273] Halogenated alkenyl is an unsaturated alkenyl moiety having
from 2-6 carbon atoms and having 1 to (2n-1) substituent(s)
independently selected from --F, --Cl, --Br, or --I where n is the
maximum number of carbon atoms in the moiety;
[0274] Substituted alkenyl is an unsaturated alkenyl moiety having
from 2-6 carbon atoms and having 0-3 substituents independently
selected from --F, or --Cl, and further having 1 substituent
selected from --R.sub.3, --R.sub.5, --OR.sub.6, --SR.sub.6,
--NR.sub.6R.sub.6, --C(O)R.sub.6, --C(O)NR.sub.6R.sub.6, --CN,
--NR.sub.6C(O)R.sub.6, --S(O).sub.2NR.sub.6R.sub.6,
--NR.sub.6S(O).sub.2R.sub.6, phenyl, or substituted phenyl;
[0275] Alkynyl is straight and branched-chain moieties having from
2-6 carbon atoms and having at least one carbon-carbon triple
bond;
[0276] Halogenated alkynyl is an unsaturated alkynyl moiety having
from 3-6 carbon atoms and having 1 to (2n-3) substituent(s)
independently selected from --F, --Cl, --Br, or --I where n is the
maximum number of carbon atoms in the moiety;
[0277] Substituted alkynyl is an unsaturated alkynyl moiety having
from 3-6 carbon atoms and having 0-3 substituents independently
selected from --F, or --Cl, and further having 1 substituent
selected from --R.sub.3, --R.sub.5, --OR.sub.6, --SR.sub.6,
--NR.sub.6R.sub.6, --C(O)R.sub.6, --CN,
--C(O)NR.sub.6R.sub.6--NR.sub.6C(O)R.sub.6,
--S(O).sub.2NR.sub.6R.sub.6, --NR.sub.6S(O).sub.2R.sub.6, phenyl,
or substituted phenyl;
[0278] Halogenated cycloalkyl is a cyclic moiety having from 3-6
carbon atoms and having 1-4 substituents independently selected
from --F, or --Cl;
[0279] Substituted cycloalkyl is a cyclic moiety having from 3-6
carbon atoms and having 0-3 substituents independently selected
from --F, or --Cl, and further having 1 substituent selected from
--OR.sub.6, --SR.sub.6, --NR.sub.6R.sub.6, --C(O)R.sub.6,
--C(O)NR.sub.6R.sub.6, --CN,
--NR.sub.6C(O)R.sub.6--S(O).sub.2NR.sub.6R.sub.6,
--NR.sub.6S(O).sub.2R.sub.6, --NO.sub.2, phenyl, or substituted
phenyl;
[0280] Heterocycloalkyl is a cyclic moiety having 4-7 atoms with
1-2 atoms within the ring being --S--, --N(R.sub.1)--, or
--O--;
[0281] Halogenated heterocycloalkyl is a cyclic moiety having from
4-7 atoms with 12 atoms within the ring being --S--,
--N(R.sub.1)--, or --O--, and having 1-4 substituents independently
selected from --F, or --Cl;
[0282] Substituted heterocycloalkyl is a cyclic moiety having from
4-7 atoms with 1-2 atoms within the ring being --S--,
--N(R.sub.1)--, or --O-- and having 0-3 substituents independently
selected from --F, or --Cl, and further having 1 substituent
selected from --OR.sub.6, --SR.sub.6, --NR.sub.6R.sub.6,
--C(O)R.sub.6, --C(O)NR.sub.6R.sub.6, --CN, --NR.sub.6C(O)R.sub.6,
--NO.sub.2, --S(O).sub.2NR.sub.6R.sub.6,
--NR.sub.6S(O).sub.2R.sub.6, phenyl, or substituted phenyl;
[0283] R.sub.2 is independently selected from the group consisting
of --H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
halogenated alkyl, halogenated alkenyl, halogenated alkynyl,
halogenated cycloalkyl, halogenated heterocycloalkyl, substituted
alkyl, substituted alkenyl, substituted alkynyl, substituted
cycloalkyl, substituted heterocycloalkyl, aryl, --OR.sub.4,
--SR.sub.4, --F, --Cl, --Br, --I, --NR.sub.4R.sub.4, --C(O)R.sub.4,
--C(O)NR.sub.4R.sub.4, --CN, --NR.sub.4C(O)R.sub.7,
--S(O).sub.2NR.sub.4R.sub.4, --OS(O).sub.2R.sub.7,
--S(O).sub.2R.sub.4, --NR.sub.4S(O).sub.2R.sub.4,
--N(H)C(O)N(H)R.sub.4, --NO.sub.2, --R.sub.3, and --R.sub.5;
[0284] R.sub.3 is 5-membered heteroaromatic mono-cyclic moieties
containing within the ring 1-3 heteroatoms independently selected
from the group consisting of --O--, .dbd.N--, --N(R.sub.1)--, and
--S--, and having 0-1 substituent selected from --R.sub.8 and 0-3
substituents independently selected from --F, --Cl, --Br, or --I,
or R.sub.3 is a 9-membered fused-ring moiety having a 6-membered
ring fused to a 5-membered ring and having the formula ##STR12##
wherein E is O, S, or NR.sub.1, ##STR13## wherein E and G are
independently selected from CR.sub.10, O, S, or NR.sub.1, and A is
CR.sub.10 or N, or ##STR14## wherein E and G are independently
selected from CR.sub.10, O, S, or NR.sub.1, and A is CR.sub.10 or
N, each 9-membered fused-ring moiety having 0-1 substituent
selected from --R.sub.8 and 0-3 substituent(s) independently
selected from --F, --Cl, --Br, or --I, and having a bond directly
or indirectly attached to the core molecule where valency allows in
either the 6-membered or the 5-membered ring of the fused-ring
moiety;
[0285] Each R.sub.4 is independently selected from --H, alkyl,
halogenated alkyl, substituted alkyl, cycloalkyl, halogenated
cycloalkyl, substituted cycloalkyl, heterocycloalkyl, halogenated
heterocycloalkyl, substituted heterocycloalkyl, --R.sub.3,
--R.sub.5, phenyl, or substituted phenyl;
[0286] R.sub.5 is 6-membered heteroaromatic mono-cyclic moieties
containing within the ring 1-3 heteroatoms selected from .dbd.N--
and having 0-1 substituent selected from --R.sub.8 and 0-3
substituent(s) independently selected from --F, --Cl, --Br, or --I,
or 10-membered heteroaromatic bi-cyclic moieties containing within
one or both rings 1-3 heteroatoms selected from .dbd.N--, including
quinolinyl or isoquinolinyl, each 10-membered fused-ring moiety
having 0-1 substituent selected from --R.sub.8 and 0-3
substituent(s) independently selected from --F, --Cl, --Br, or --I
and having a bond directly or indirectly attached to the core
molecule where valency allows;
[0287] Each R.sub.6 is independently selected from --H, alkyl,
cycloalkyl, heterocycloalkyl, alkyl substituted with 1 substituent
selected from R.sub.9, cycloalkyl substituted with 1 substituent
selected from R.sub.9, heterocycloalkyl substituted with 1
substituent selected from R.sub.9, halogenated alkyl, halogenated
cycloalkyl, halogenated heterocycloalkyl, phenyl, substituted
phenyl, --R.sub.3, or --R.sub.5;
[0288] Each R.sub.7 is independently selected from --H, alkyl,
cycloalkyl, heterocycloalkyl, halogenated alkyl, halogenated
cycloalkyl, or halogenated heterocycloalkyl;
[0289] R.sub.8 is selected from --OR.sub.7, --SR.sub.7, alkyl,
cycloalkyl, heterocycloalkyl, halogenated alkyl, halogenated
cycloalkyl, halogenated heterocycloalkyl, substituted alkyl,
substituted cycloalkyl, substituted heterocycloalkyl,
--NR.sub.7R.sub.7, --C(O)R.sub.7, --NO.sub.2,
--C(O)NR.sub.7R.sub.7, --CN, --NR.sub.7C(O)R.sub.7,
--S(O).sub.2NR.sub.7R.sub.7, or --NR.sub.7S(O)2R.sub.7;
[0290] R.sub.9 is selected from --OR.sub.7, --SR.sub.7,
--NR.sub.7R.sub.7, --C(O)R.sub.7, --C(O)NR.sub.7R.sub.7,
--CN--NR.sub.7C(O)R.sub.7, --S(O).sub.2NR.sub.7R.sub.7,
--NR.sub.7S(O).sub.2R.sub.7, --CF.sub.3, or --NO.sub.2;
[0291] Each R.sub.10 is independently selected from --H, alkyl,
cycloalkyl, heterocycloalkyl, halogenated alkyl, halogenated
cycloalkyl, halogenated heterocycloalkyl, substituted alkyl,
substituted cycloalkyl, substituted heterocycloalkyl, --OR.sub.7,
--SR.sub.7, --NR.sub.7R.sub.7, --C(O)R.sub.7, --NO.sub.2,
--C(O)NR.sub.7R.sub.7, --CN, --NR.sub.7C(O)R.sub.7,
--S(O).sub.2NR.sub.7R.sub.7, or --NR.sub.7S(O).sub.2R.sub.7, --F,
--Cl, --Br, or --I, or a bond directly or indirectly attached to
the core molecule, provided that there is only one said bond to the
core molecule within the 9-membered fused-ring moiety, further
provided that the fused-ring moiety has 0-1 substituent selected
from alkyl, cycloalkyl, heterocycloalkyl, halogenated alkyl,
halogenated cycloalkyl, halogenated heterocycloalkyl, substituted
alkyl, substituted cycloalkyl, substituted heterocycloalkyl,
--OR.sub.7, --SR.sub.7, --NR.sub.7R.sub.7, --C(O)R.sub.7,
--NO.sub.2, --C(O)NR.sub.7R.sub.7, --CN, --NR.sub.7C(O)R.sub.7,
--S(O).sub.2NR.sub.7R.sub.7, or --NR.sub.7S(O).sub.2R.sub.7, and
further provided that the fused-ring moiety has 0-3 substituent(s)
selected from --F, --Cl, --Br, or --I.
[0292] Limited substituted alkyl is a substituted alkyl having from
1-6 carbon atoms and having 0-3 substituents independently selected
from --F, --Cl, --Br, or --I, and further having 1 substituent on
either only the .omega. carbon and selected from --OR.sub.7,
--SR.sub.7, --NR.sub.7R.sub.7, --C(O)R.sub.7, --NO.sub.2,
--C(O)NR.sub.7R.sub.7, --CN, --NR.sub.6C(O)R.sub.7,
--S(O).sub.2NR.sub.6R.sub.6, or --NR.sub.6S(O).sub.2R.sub.6, or on
any carbon with sufficient valency but not on the .omega. carbon
and selected from --R.sub.3, --R.sub.5, --OR.sub.6, --SR.sub.6,
--NR.sub.6R.sub.6, --C(O)R.sub.6, --NO.sub.2,
--C(O)NR.sub.6R.sub.6, --CN, --NR.sub.6C(O)R.sub.6,
--S(O).sub.2NR.sub.6R.sub.6, --NR.sub.6S(O).sub.2R.sub.6, phenyl,
or substituted phenyl;
[0293] Limited substituted alkenyl is a substituted alkenyl having
from 1-6 carbon atoms and having 0-3 substituents independently
selected from --F, --Cl, --Br, or --I, and further having 1
substituent on either only the .omega. carbon and selected from
--OR.sub.7, --SR.sub.7, --NR.sub.7R.sub.7, --C(O)R.sub.7,
--NO.sub.2, --C(O)NR.sub.7R.sub.7, --CN, --NR.sub.6C(O)R.sub.7,
--S(O).sub.2NR.sub.6R.sub.6, or --NR.sub.6S(O).sub.2R.sub.6, or on
any carbon with sufficient valency but not on the .omega. carbon
and selected from --R.sub.3, --R.sub.5, --OR.sub.6, --SR.sub.6,
--NR.sub.6R.sub.6, --C(O)R.sub.6, --NO.sub.2,
--C(O)NR.sub.6R.sub.6, --CN, --NR.sub.6C(O)R.sub.6,
--S(O).sub.2NR.sub.6NR.sub.6, --NR.sub.6S(O).sub.2R.sub.6, phenyl,
or substituted phenyl; and
[0294] Limited substituted alkynyl is a substituted alkynyl having
from 1-6 carbon atoms and having 0-3 substituents independently
selected from --F, --Cl, --Br, or --I, and further having 1
substituent on either only the .omega. carbon and selected from
--OR.sub.7, --SR.sub.7, --NR.sub.7R.sub.7, --C(O)R.sub.7,
--NO.sub.2, --C(O)NR.sub.7R.sub.7, --CN, --NR.sub.6C(O)R.sub.7,
--S(O).sub.2NR.sub.6R.sub.6, or --NR.sub.6S(O).sub.2R.sub.6, or on
any carbon with sufficient valency but not on the .omega. carbon
and selected from --R.sub.3, --R.sub.5, --OR.sub.6, --SR.sub.6,
--NR.sub.6R.sub.6, --C(O)R.sub.6, --NO.sub.2,
--C(O)NR.sub.6R.sub.6, --CN, --NR.sub.6C(O)R.sub.6,
--S(O).sub.2NR.sub.6R.sub.6, --NR.sub.6S(O).sub.2R.sub.6, phenyl,
or substituted phenyl.
[0295] Particular embodiments according to the foregoing general
formula include; [0296]
N-((3R)-1-azabicyclo[2.2.2]oct-3-yl)-5-phenylthiophene-2-carboxamide;
and [0297]
N-((3R)-1-azabicyclo[2.2.2]oct-3-yl)-5-phenyl-1,3,4-oxadiazole-2-
-carboxamide, or a pharmaceutically acceptable salt thereof.
[0298] Compounds useful according to the present invention also
include heteroaromatic amides of 3-aminoquinuclidine, e.g., as
taught in published international patent application WO 02/16356,
of the general formula I: ##STR15## or pharmaceutically acceptable
salts thereof, wherein X is O or S;
[0299] R.sub.1 is independently selected from the group consisting
of --H, alkyl, cycloalkyl, halogenated alkyl, and aryl;
[0300] Alkyl is both straight- and branched-chain moieties having
from 1-6 carbon atoms;
[0301] Halogenated alkyl is an alkyl moiety having from 1-6 carbon
atoms and having 1 to (2n+1) substituent(s) independently selected
from --F, --Cl, --Br, or --I where n is the maximum number of
carbon atoms in the moiety;
[0302] Cycloalkyl is a cyclic alkyl moiety having from 3-6 carbon
atoms;
[0303] Aryl is phenyl, substituted phenyl, naphthyl, or substituted
naphthyl;
[0304] Substituted phenyl is a phenyl either having 1-4
substituents independently selected from --F, --Cl, --Br, or --I,
or having 1 substituent selected from --R.sub.12 and 0-3
substituents independently selected from --F, --Cl, --Br, or
--I;
[0305] Substituted naphthyl is a naphthalene moiety either having
1-4 substituents independently selected from --F, --Cl, --Br, or
--I, or having 1 substituent selected from --R.sub.12 and 0-3
substituents independently selected from --F, --Cl, --Br, or --I,
where the substitution can be independently on either only one ring
or both rings of said naphthalene moiety;
[0306] R.sub.2 is --H, alkyl, halogenated alkyl, substituted alkyl,
cycloalkyl, benzyl, substituted benzyl, or aryl;
[0307] Substituted alkyl is an alkyl moiety from 1-6 carbon atoms
and having 0-3 substituents independently selected from --F, --Cl,
--Br, or --I and further having 1 substituent selected from
--OR.sub.10, --SR.sub.10, --NR.sub.10R.sub.10, --C(O)R.sub.10,
--C(O)NR.sub.10R.sub.10, --CN, --NR.sub.10C(O)R.sub.10,
--S(O).sub.2NR.sub.10R.sub.10, --NR.sub.10S(O).sub.2R.sub.10,
--NO.sub.2, --R.sub.7, --R.sub.9, phenyl, or substituted
phenyl;
[0308] Substituted benzyl is a benzyl either having 1-4
substituents independently selected from --F, --Cl, --Br, or --I,
or having 1 substituent selected from --R.sub.12 and 0-3
substituents independently selected from --F, --Cl, --Br, or --I,
provided that all substitution is on the phenyl ring of the
benzyl;
[0309] R.sub.3 is selected from the group consisting of --H, alkyl,
alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, halogenated alkyl,
halogenated alkenyl, halogenated alkynyl, halogenated cycloalkyl,
halogenated heterocycloalkyl, substituted alkyl, substituted
alkenyl, substituted alkynyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, --R.sub.7, --R.sub.9, --OR.sub.8,
--SR.sub.8, --F, --Cl, --Br, --I, --NR.sub.8R.sub.8, --C(O)R.sub.8,
--CN, --C(O)NR.sub.8R.sub.8, --NR.sub.8C(O)R.sub.8, --S(O)R.sub.8,
--OS(O).sub.2R.sub.8, --NR.sub.8S(O).sub.2R.sub.8, NO.sub.2, and
--N(H)C(O)N(H)R.sub.8;
[0310] Alkenyl is straight- and branched-chain moieties having from
2-6 carbon atoms and having at least one carbon-carbon double
bond;
[0311] Halogenated alkenyl is an unsaturated alkenyl moiety having
from 2-6 carbon atoms and having 1 to (2n-1) substituent(s)
independently selected from --F, --Cl, --Br, or --I where n is the
maximum number of carbon atoms in the moiety;
[0312] Substituted alkenyl is an unsaturated alkenyl moiety having
from 2-6 carbon atoms and having 0-3 substituents independently
selected from --F, or --Cl, and further having 1 substituent
selected from --R.sub.7, --R.sub.9, --OR.sub.10, --SR.sub.10,
--NR.sub.10R.sub.10, --C(O)R.sub.10, --C(O)NR.sub.10R.sub.10,
--NR.sub.10C(O)R.sub.10, --S(O).sub.2NR.sub.10R.sub.10,
--NR.sub.10S(O).sub.2R.sub.10, --CN, phenyl, or substituted
phenyl;
[0313] Alkynyl is straight- and branched-chained moieties having
from 2-6 carbon atoms and having at least one carbon-carbon triple
bond;
[0314] Halogenated alkynyl is an unsaturated alkynyl moiety having
from 3-6 carbon atoms and having 1 to (2n-3) substituent(s)
independently selected from --F, --Cl, --Br, or --I where n is the
maximum number of carbon atoms in the moiety;
[0315] Substituted alkynyl is an unsaturated alkynyl moiety having
from 3-6 carbon atoms and having 0-3 substituents independently
selected from --F, or --Cl, and further having 1 substituent
selected from --R.sub.7, --R.sub.9, --OR.sub.10, --SR.sub.10,
--NR.sub.10R.sub.10, --C(O)R.sub.10, --C(O)NR.sub.10R.sub.10,
--NR.sub.10C(O)R.sub.10, --S(O).sub.2NR.sub.10R.sub.10,
--NR.sub.10S(O).sub.2R.sub.10, --CN, phenyl, or substituted
phenyl;
[0316] Halogenated cycloalkyl is a cyclic moiety having from 3-6
carbon atoms and having 1-4 substituents independently selected
from --F, or --Cl;
[0317] Substituted cycloalkyl is a cyclic moiety having from 3-6
carbon atoms and having 0-3 substituents independently selected
from --F, or --Cl, and further having 1 substituent selected from
--OR.sub.10, --SR.sub.10, --NR.sub.10R.sub.10, --C(O)R.sub.10,
--CN, --C(O)NR.sub.10R.sub.10, --NR.sub.10C(O)R.sub.10,
--S(O).sub.2NR.sub.10R.sub.10, --NR.sub.10S(O).sub.2R.sub.10,
--NO.sub.2, phenyl, or substituted phenyl;
[0318] Heterocycloalkyl is a cyclic moiety having 4-7 atoms with
1-2 atoms within the ring being --S--, --N(R.sub.3)--, or
--O--;
[0319] Halogenated heterocycloalkyl is a cyclic moiety having from
4-7 atoms with 1-2 atoms within the ring being --S--,
--N(R.sub.3)--, or --O--, and having 1-4 substituents independently
selected from --F, or --Cl;
[0320] Substituted heterocycloalkyl is a cyclic moiety having from
4-7 atoms with 1-2 atoms within the ring being --S--,
--N(R.sub.3)--, or --O-- and having 0-3 substituents independently
selected from --F, or --Cl, and further having 1 substituent
selected from --OR.sub.10, --SR.sub.10, --NR.sub.10R.sub.10,
--C(O)R.sub.10, --C(O)NR.sub.10R.sub.10, --CN,
--NR.sub.10C(O)R.sub.10, --NO.sub.2, --S(O).sub.2NR.sub.10R.sub.10,
--NR.sub.10S(O).sub.2R.sub.10, phenyl, or substituted phenyl;
[0321] R.sub.4 is selected from the group consisting of
--O--R.sub.5, --S--R.sub.5, --S(O)--R.sub.5, --C(O)--R.sub.5, and
alkyl substituted on the .omega. carbon with R.sub.5 where said
.omega. carbon is determined by counting the longest carbon chain
of the alkyl moiety with the C-1 carbon being the carbon attached
to the phenyl ring of the core molecule and the .omega. carbon
being the carbon furthest from said C-1 carbon;
[0322] R.sub.5 is selected from aryl, R.sub.7, or R.sub.9;
[0323] R.sub.7 is 5-membered heteroaromatic mono-cyclic moieties
containing within the ring 1-3 heteroatoms independently selected
from the group consisting of --O--, .dbd.N--, --N(R.sub.14)--, and
--S--, and having 0-1 substituent selected from --R.sub.12 and 0-3
substituents independently selected from --F, --Cl, --Br, or --I,
or R.sub.7 is a 9-membered fused-ring moiety having a 6-membered
ring fused to a 5-membered ring and having the formula ##STR16##
wherein E is O, S, or NR.sub.14, ##STR17## wherein E and G are
independently selected from CR.sub.18, O, S, or NR.sub.14, and A is
CR.sub.18 or N, or ##STR18## wherein E and G are independently
selected from CR.sub.18, O, S, or NR.sub.14, and A is CR.sub.18 or
N, each 9-membered fused-ring moiety having 0-1 substituent
selected from --R.sub.12 and 0-3 substituent(s) independently
selected from --F, --Cl, --Br, or --I, and having a bond directly
or indirectly attached to the core molecule where valency allows in
either the 6-membered or the 5-membered ring of the fused-ring
moiety;
[0324] Each R.sub.8 is independently selected from --H, alkyl,
halogenated alkyl, substituted alkyl, cycloalkyl, halogenated
cycloalkyl, substituted cycloalkyl, heterocycloalkyl, halogenated
heterocycloalkyl, substituted heterocycloalkyl, R.sub.7, R.sub.9,
phenyl, or substituted phenyl;
[0325] R.sub.9 is 6-membered heteroaromatic mono-cyclic moieties
containing within the ring 1-3 heteroatoms selected from .dbd.N--
and having 0-1 substituent selected from --R.sub.12 and 0-3
substituent(s) independently selected from --F, --Cl, --Br, or --I,
or 10-membered heteroaromatic bi-cyclic moieties containing within
one or both rings 1-3 heteroatoms selected from .dbd.N--, including
quinolinyl or isoquinolinyl, each 10-membered fused-ring moiety
having 0-1 substituent selected from --R.sub.12 and 0-3
substituent(s) independently selected from --F, --Cl, --Br, or --I
and having a bond directly or indirectly attached to the core
molecule where valency allows;
[0326] Each R.sub.10 is independently selected from --H, alkyl,
cycloalkyl, heterocycloalkyl, alkyl substituted with 1 substituent
selected from R.sub.13, cycloalkyl substituted with 1 substituent
selected from R.sub.13, heterocycloalkyl substituted with 1
substituent selected from R.sub.13, halogenated alkyl, halogenated
cycloalkyl, halogenated heterocycloalkyl, phenyl, or substituted
phenyl;
[0327] Each R.sub.11 is independently selected from --H, alkyl,
cycloalkyl, heterocycloalkyl, halogenated alkyl, halogenated
cycloalkyl, or halogenated heterocycloalkyl;
[0328] R.sub.12 is selected from --OR.sub.11, --SR.sub.11, alkyl,
cycloalkyl, heterocycloalkyl, halogenated alkyl, halogenated
cycloalkyl, halogenated heterocycloalkyl, substituted alkyl,
substituted cycloalkyl, substituted heterocycloalkyl,
--NR.sub.11R.sub.11, --C(O)R.sub.11, --NO.sub.2,
--C(O)NR.sub.11R.sub.11, --CN, --NR.sub.11C(O)R.sub.11,
--S(O).sub.2NR.sub.11R.sub.11, or
--NR.sub.11S(O).sub.2R.sub.11;
[0329] R.sub.13 is selected from --OR.sub.11, --SR.sub.11,
--NR.sub.11R.sub.11, --C(O)R.sub.11, --C(O)NR.sub.11R.sub.11, --CN,
--CF.sub.3, --NR.sub.11C(O)R.sub.11, --S(O).sub.2NR.sub.11R.sub.11,
--NR.sub.11S(O).sub.2R.sub.11, or --NO.sub.2;
[0330] R.sub.14 is selected from --H, alkyl, halogenated alkyl,
substituted alkyl, cycloalkyl, halogenated cycloalkyl, substituted
cycloalkyl, heterocycloalkyl, halogenated heterocycloalkyl,
substituted heterocycloalkyl, phenyl, or substituted phenyl;
[0331] Each R.sub.15 is independently selected from alkyl,
halogenated alkyl, substituted alkyl, cycloalkyl, halogenated
cycloalkyl, substituted cycloalkyl, heterocycloalkyl, halogenated
heterocycloalkyl, substituted heterocycloalkyl, R.sub.7, R.sub.9,
phenyl, or substituted phenyl;
[0332] Each R.sub.16 is independently selected from cycloalkyl,
halogenated cycloalkyl, substituted cycloalkyl, heterocycloalkyl,
halogenated heterocycloalkyl, substituted heterocycloalkyl,
R.sub.7, R.sub.9, phenyl, or substituted phenyl;
[0333] R.sub.17 is selected from cycloalkyl, halogenated
cycloalkyl, substituted cycloalkyl, heterocycloalkyl, halogenated
heterocycloalkyl, or substituted heterocycloalkyl; and
[0334] Each R.sub.18 is independently selected from --H, alkyl,
cycloalkyl, heterocycloalkyl, halogenated alkyl, halogenated
cycloalkyl, halogenated heterocycloalkyl, substituted alkyl,
substituted cycloalkyl, substituted heterocycloalkyl, --OR.sub.11,
--SR.sub.11, --NR.sub.11R.sub.11, --C(O)R.sub.11, --NO.sub.2,
--C(O)NR.sub.11R.sub.11, --CN, --NR.sub.11C(O)R.sub.11,
--S(O).sub.2NR.sub.11R.sub.11, or --NR.sub.11S(O).sub.2R.sub.11,
--F, --Cl, --Br, or --I, or a bond directly or indirectly attached
to the core molecule, provided that there is only one said bond to
the core molecule within the 9-membered fused-ring moiety, further
provided that the fused-ring moiety has 0-1 substituent selected
from alkyl, cycloalkyl, heterocycloalkyl, halogenated alkyl,
halogenated cycloalkyl, halogenated heterocycloalkyl, substituted
alkyl, substituted cycloalkyl, substituted heterocycloalkyl,
--OR.sub.11, --SR.sub.11, --NR.sub.11R.sub.11, --C(O)R.sub.11,
--NO.sub.2, --C(O)NR.sub.11R.sub.11, --CN, --NR.sub.11C(O)R.sub.11,
--S(O).sub.2NR.sub.11R.sub.11, or --NR.sub.11S(O).sub.2R.sub.11,
and further provided that the fused-ring moiety has 0-3
substituent(s) selected from --F, --Cl, --Br, or --I.
[0335] Particular embodiments according to the foregoing general
formula include; [0336]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-y-1]-4-(4-hydroxyphenoxy)benzamide;
[0337]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(-4-acetamidophenoxy)benza-
mide; [0338]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-phenoxybenzamide; [0339]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-benzylbenzamide; [0340]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(phenylsulfanyl)benzamide;
[0341] N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-3-phenoxybenzamide;
[0342] N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-benzoylbenzamide;
[0343]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-fluorophenoxy)benzamide;
[0344]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(2-fluorophenoxy)benzamide-
; [0345]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-fluorophenoxy)benzami-
de; [0346]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(2-chlorophenoxy)benzamide;
[0347]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-chlorophenoxy)benzamide-
; [0348]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-chlorophenoxy)benzami-
de; [0349]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(2-methoxyphenoxy)benzamide;
[0350]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-methoxyphenoxy)benzamid-
e; [0351]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-methoxyphenoxy)benzamide;
[0352]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-chlorophenylsulfanyl)be-
nzamide; [0353]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-methoxyphenoxy)benzamide;
[0354]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-chlorophenylsulfanyl)be-
nzamide; [0355]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(4-chlorophenylsulfanyl)benzamide;
[0356]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-(3-methoxyphenylsulfanyl)-
-benzamide; [0357]
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4(2-methoxyphenylsulfanyl)-benzamide-
; [0358]
N-(2-methyl-1-azabicyclo[2.2.2]oct-3-yl)4-phenoxybenzamide; and
[0359] pharmaceutically acceptable salts thereof.
[0360] Further,
N-((3R)-1-azabicyclo[2.2.2]oct-3-yl)-4-(pyridin-3-yloxy)benzamide
and pharmaceutically acceptable salts thereof are also useful.
[0361] Compounds useful according to the present invention also
include spiroquinuclidine compounds, e.g., as taught in published
international patent application WO 99/03859, of the general
formula: ##STR19## wherein n is 0 or 1; m is 0 or 1; p is 0 or 1; X
is oxygen or sulfur; Y is CH, N or NO; W is oxygen, H.sub.2 or
F.sub.2; A is N or C(R.sup.2); G is N or C(R.sup.3); D is N or
C(R.sup.4); [0362] with the proviso that no more than one of A, G,
and D is nitrogen but at least one of Y, A, G, and D is nitrogen or
NO; [0363] R.sup.1 is hydrogen or C.sub.1-C.sub.4 alkyl; [0364]
R.sup.2, R.sup.3, and R.sup.4 are independently hydrogen, halogen,
C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4
alkynyl, aryl, heteroaryl, OH, OC.sub.1--C.sub.4 alkyl,
CO.sub.2R.sup.1, --CN, --NO.sub.2, --NR.sup.5R.sup.6, --CF.sub.3,
--OSO.sub.2CF.sub.3, or R.sup.2 and R.sup.3, or R.sup.3 and
R.sup.4, respectively, may together form another six membered
aromatic or heteroaromatic ring sharing A and G, or G and D,
respectively, containing between zero and two nitrogen atoms, and
substituted with one to two of the following substituents:
independently hydrogen, halogen, C.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, C.sub.2-C.sub.4 alkynyl, aryl, heteroaryl,
OH, OC.sub.1--C.sub.4 alkyl, CO.sub.2R.sup.1, --CN, --NO.sub.2,
--NR.sup.5R.sup.6, --CF.sub.3, --OSO.sub.2CF.sub.3; [0365] R.sup.5
and R.sup.6 are independently hydrogen, C.sub.1-C.sub.4 alkyl,
C(O)R.sup.7, C(O)NHR.sup.8, C(O)OR.sup.9, SO.sub.2R.sup.10 or may
together be (CH.sub.2).sub.jQ(CH.sub.2).sub.k where [0366] Q is O,
S, NR.sup.11, or a bond; j is 2 to 7; k is 0 to 2; [0367] R.sup.7,
R.sup.8, R.sup.9, R.sup.10, and R.sup.11 are independently
C.sub.1-C.sub.4 alkyl, aryl, or heteroaryl, [0368] or an enantiomer
thereof, or a pharmaceutically acceptable salt thereof.
[0369] Particular embodiments according to the foregoing general
formula include: [0370]
spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
[0371]
5'-bromospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
[0372]
5'-phenylspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]py-
ridine]; [0373]
5'-nitrospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
[0374]
1'-chlorospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]is-
oquinoline]; [0375]
5'-(phenylcarboxamido)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-
-b]pyridine]; [0376]
5'-(phenylaminocarbonylamino)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-f-
uro[2,3-b]pyridine]; [0377]
5'-(phenylsulfonylamido)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2-
,3-b]pyridine]; [0378]
5'-aminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
[0379]
5'-N-methylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2-
,3-b]pyridine]; [0380]
5'-N,N-dimethylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b-
]pyridine]; [0381]
5'-N,N-diethylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]-
pyridine]; [0382]
5'-N-ethylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyri-
dine]; [0383]
5'-N-benzylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyr-
idine]; [0384]
5'-N-formamidospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyrid-
ine]; [0385]
5'-N-acetamidospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyrid-
ine]; [0386]
spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]isoquinoline];
[0387]
spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]quinoline];
[0388]
5'-ethenylspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]-
pyridine]; [0389]
5'-(E)-(phenylethenyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-
-b]pyridine]; [0390]
5'-(4-morpholino)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]py-
ridine]; [0391]
5'-(1-azetidinyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]py-
ridine]; [0392]
5'-(E)-(2-(4-pyridyl)ethenyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-f-
uro[2,3-b]pyridine]; [0393]
5'-(E)-(2-(2-pyridyl)ethenyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-f-
uro[2,3-b]pyridine]; [0394]
5'-(2-trimethylsilylethynyl(spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-fu-
ro[2,3-b]pyridine]; [0395]
5'-ethynylspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine]-
; [0396]
5'-(2-furyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-
-b]pyridine]; [0397]
5'-(3-pyridyl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyrid-
ine]; [0398]
5'-methylspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
[0399]
spiro[1-azabicyclo[2.2.2]octane-3,2'-3'H)-furo[2,3-b]pyridine-5'-
carbonitrile]; [0400]
spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine-5'carboxam-
ide]; [0401]
5'-N'-(3chlorophenyl)ureidoaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H-
)-furo[2,3-b]pyridine]; [0402]
5'-N'-(2-nitrophenyl)ureidoaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H-
)-furo[2,3-b]pyridine]; [0403]
4'chlorospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridine];
[0404]
4'-methoxyspiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]p-
yridine]; [0405]
4'-phenylthiospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyridi-
ne]; [0406]
4'-(N-2-aminoethyl)aminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2-
,3-b]pyridine]; [0407]
4'-Phenylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyrid-
ine]; [0408]
4'-methylaminospiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[2,3-b]pyrid-
ine]; [0409]
4'-(4-N-methylpiperazin-1-yl)spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-f-
uro[2,3-b]pyridine]; [0410]
4'-chloro-spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[3,2-c]pyridine]-
; [0411]
spiro[1-azabicyclo[2.2.2]octane-3,2'-(3'H)-furo[3,2-c]pyridine]- ;
[0412]
6'-fluorospiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]p-
yridine]; [0413]
spiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine-6'-carbonit-
rile]; [0414]
6'-chlorospiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine];
[0415] or an enantiomer, or pharmaceutically acceptable salt
thereof.
[0416] Compounds useful according to the present invention also
include anabaseine compounds, e.g., as taught in U.S. Pat. No.
5,977,144, of the general formula: ##STR20## or a salt thereof,
wherein R.sub.1, R.sub.6, and R.sub.7 are hydrogen or
C.sub.1-C.sub.4 alkyl; and R.sup.2 is .dbd.CHCH.dbd.CHX, wherein X
is ##STR21## wherein R.sup.3, R.sup.4, and R.sup.5 are selected
from the group consisting of hydrogen, C.sub.1-C.sub.4 alkyl
optionally substituted with N,N-dialkylamino having 1 to 4 carbons
in each of the alkyls, C.sub.1-C.sub.6 alkoxy optionally
substituted with N,N-dialkylamino having 1 to 4 carbons in each of
the alkyls, carboalkoxy having 1 to 4 carbons in the alkoxy, amino,
amino having 1 to 4 carbons in the acyl, cyano, N,N-dialkylamino
having 1 to 4 carbons in each of the alkyls, halo, hydroxyl, and
nitro.
[0417] Particular embodiments of compounds according to the
foregoing general formula include benzylidene- or
cinnamylidene-anabaseines including: [0418]
3-(2,4-dimethoxybenzylidene)anabaseine,
3-(4-hydroxybenzylidene)anabaseine; [0419]
3-(4-methoxybenzylidene)anabaseine,
3-(4-aminobenzylidene)anabaseine; [0420]
3-(4-hydroxy-2-methoxybenzylidene)anabaseine; [0421]
3-(2-hydroxy-4-methoxybenzylidene)anabaseine; [0422]
3-(4-isopropoxybenzylidene)anabaseine; [0423]
(7'-methyl-3-(2,4dimethoxybenzylidene))anabaseine; [0424]
3-(4-acetylaminocinnamylidene)anabaseine; [0425]
3-(4-hydroxycinnamylidene)anabaseine; [0426]
3-(4-methoxycinnamylidene)anabaseine; [0427]
3-(4-hydroxy-2-methoxycinnamylidene)anabaseine; [0428]
3-(2,4-dimethoxycinnamylidene)anabaseine; and [0429]
3-(4-acetoxycinnamylidene)anabaseine; [0430] and pharmaceutically
acceptable salts thereof.
[0431] Substances that inhibit an AT2 receptor, either directly or
indirectly, include ACE inhibitors that interfere with the action
of AT2 stimulator angiotensin II converting enzyme or ACE (see
Brown, N. J. and D. E. Vaughan, Circulation. 97:1411-1420 (1998),
incorporated fully herein by reference). Such substances also
include inhibitors of the AT2 receptor itself. (e.g. PD123177 and
PD123319; see Fischer, J. W., et al., Cardiovasc. Res. 51(4):784-91
(2001); and Horiuchi, et al. J. Clin. Invest. 103(1):63-71(1999),
both fully incorporated herein by reference).
[0432] ACE inhibitors useful according to the present invention
include those taught in Brown, N. J. and D. E. Vaughan,
Circulation, 97:1411-1420 (1998), e.g., captopril, enalapril,
lisinopril, benazepril, quinapril, ramapril, trandolapril,
moexipril, fosinopril, and zofenopril. Of course, this list is not
intended to be limiting, and other compounds known in the art as
ACE inhibitors can also be used.
[0433] Polypeptides, Antibodies and Related Methods. The compounds
can be, for example, antibodies, antibody fragments, enzymes,
proteins, peptides, nucleic acids such as oligonucleotides, or
small molecules.
[0434] For the protein or polypeptide compounds of the invention, a
mimic of one or more amino acids, otherwise known as a polypeptide
mimetic or peptidominetic, can also be used. As used herein, the
term "mimic" means an amino acid or an amino acid analog that has
the same or similar functional characteristic of an amino acid.
Thus, for example, a (D)arginine analog can be a mimic of
(D)arginine if the analog contains a side chain having a positive
charge at physiological pH, as is characteristic of the guinidinium
side chain reactive group of arginine. A polypeptide mimetic or
peptidomimetic is an organic molecule that retains similar
polypeptide chain pharmacophore groups as are present in the
corresponding polypeptide. The substitution of amino acids by
non-naturally occurring amino acids and peptidomimetics as
described above can enhance the overall activity or properties of
an individual polypeptide based on the modifications to the side
chain functionalities. For example, these types of alterations can
be employed along with the oligomer components of the present
invention to further enhance the polypeptide's resistance to
enzymatic breakdown and/or to improve biological activity.
[0435] The antibodies can be, for example, monoclonal, humanized
(chimeric) or polyclonal antibodies, and can be prepared using
conventional techniques.
[0436] Antibodies can be generated that bind to nicotinic
receptors, e.g., the .alpha.7-nAChR. Antibodies can also be
generated that bind to the AT2 receptor. These antibodies can be
selected such that they are effective in preventing activation of
the receptors when bound to their target. Antibodies can also be
produced that bind to substances that stimulate or inhibit
receptors, in a manner that interferes with the expected function
or activity of the substance. Antibodies can also be prepared that
bind and affect the activity of enzyme such as ACE, which generate
active ligands that stimulate or inhibit receptor activity. Herein,
receptors, ligands, related enzymes, and other effectors used to
prepare specific antibodies may be referred to simply as
"antigens." The term "ligand" can refer to a specific binding
partner of a receptor that is either stimulatory or inhibitory of a
particular activity.
[0437] Polyclonal antibodies can be used, provided their overall
effect is a desired effect (i.e., a stimulatory or inhibitory
effect, as desired). Monoclonal antibodies can be used, as well as
humanized (chimeric) antibodies. The antibodies may inhibit binding
between binding partners such as receptors and their ligands by
sterically interfering with and/or binding to all or part of the
actual binding site(s), either on the receptor or on the
ligand.
[0438] The term "antibody" refers to a polypeptide substantially
encoded by an immunoglobulin gene or immunoglobulin genes, or
fragments thereof, that specifically binds and recognizes an
analyte or antigen, e.g. a receptor and/or a ligand (stimulatory or
inhibitory substance) relevant to the methods of the invention).
Immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0439] An exemplary immunoglobulin (antibody) structural unit
includes a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain has a variable region of about 100 to 110 or more amino acids
primarily responsible for antigen recognition. The terms "variable
light chain" (or "VL") and "variable heavy chain" (or "VH") refer
to these light and heavy chains, respectively.
[0440] Antibodies exist, for example, as intact immunoglobulins or
as a number of well-characterized antigen-binding fragments
produced by digestion with various peptidases. For example, pepsin
digests an antibody below the disulfide linkages in the hinge
region to produce an F(ab').sub.2 fragment, a dimer of Fab which
itself is a light chain joined to VH-CH1 by a disulfide bond. The
F(ab').sub.2 fragment can be reduced under mild conditions to break
the disulfide linkage in the hinge region, thereby converting the
F(ab').sub.2 dimer into an Fab' monomer. The Fab' monomer is
essentially an Fab with part of the hinge region (see Fundamental
Immunology, Third Edition, W. E. Paul (ed.), Raven Press, N.Y.
(1993), the contents of which are hereby incorporated by
reference). While various antibody fragments are defined in terms
of the digestion of an intact antibody, one of ordinary skill in
the art will appreciate that such fragments can be synthesized de
novo either chemically or by using recombinant DNA methodology.
Thus, the term antibody, as used herein, also includes antibody
fragments, such as a single chain antibody, an antigen binding
F(ab')2 fragment, an antigen binding Fab' fragment, an antigen
binding Fab fragment, an antigen binding Fv fragment, a single
heavy chain or a chimeric (humanized) antibody. Such antibodies can
be produced by modifying whole antibodies or synthesized de novo
using recombinant DNA methodologies.
[0441] Receptors, ligands, or other species relevant to the methods
of the invention ("antigens"--including fragments, derivatives, and
analogs thereof) can be used as immunogens to generate antibodies
which immunospecifically bind such immunogens. Such antibodies
include, but are not limited to, polyclonal antibodies, monoclonal
antibodies, chimeric antibodies, single-chain antibodies,
antigen-binding antibody fragments (e.g., Fab, Fab', F(ab').sub.2,
Fv, or hypervariable regions), and mAb or Fab expression libraries.
In some embodiments, polyclonal and/or monoclonal antibodies to the
antigens are produced. In yet other embodiments, fragments of the
receptors or ligands that are identified as immunogenic are used as
immunogens for antibody production.
[0442] Various procedures known in the art can be used to produce
polyclonal antibodies. Various host animals (including, but not
limited to, rabbits, mice, rats, sheep, goats, camels, and the
like) can be immunized by injection with the antigen, fragment,
derivative or analog. Various adjuvants can be used to increase the
immunological response, depending on the host species. Such
adjuvants include, for example, Freund's adjuvant (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and other adjuvants, such as BCG (bacille
Calmette-Guerin) and Corynebacterium parvum.
[0443] Any technique that provides for the production of antibody
molecules by continuous cell lines in culture can be used to
prepare monoclonal antibodies directed toward the receptors or
ligands (stimulatory or inhibitory substances) according to the
methods of the invention. Such techniques include, for example, the
hybridoma technique originally developed by Kohler and Milstein
(see, e.g., Nature 256:495-97 (1975)), the trioma technique (see,
e.g., Hagiwara and Yuasa, Hum. Antibodies Hybridomas 4:15-19
(1993); Hering et al., Biomed. Biochim. Acta 47:211-16 (1988)), the
human B-cell hybridoma technique (see, e.g., Kozbor et al.,
Immunology Today 4:72 (1983)), and the EBV-hybridoma technique to
produce human monoclonal antibodies (see, e.g., Cole et al., In:
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96 (1985)). Human antibodies can be used and can be obtained by
using human hybridomas (see, e.g., Cote et al., Proc. Natl. Acad.
Sci. USA 80:2026-30 (1983)) or by transforming human B cells with
EBV virus in vitro (see, e.g., Cole et al., supra).
[0444] "Chimeric" or "humanized" antibodies (see, e.g., Morrison et
al., Proc. Natl. Acad. Sci. USA 81:6851-55 (1984); Neuberger et
al., Nature 312:604-08 (1984); Takeda et al., Nature 314:452-54
(1985)) can also be prepared. Methods for producing such "chimeric"
molecules are generally well known and described in, for example,
U.S. Pat. Nos. 4,816,567; 4,816,397; 5,693,762; and 5,712,120; PCT
Patent Publications WO 87/02671 and WO 90/00616; and European
Patent Publication EP 239 400 (the disclosures of which are
incorporated by reference herein). Alternatively, a human
monoclonal antibody or portions thereof can be identified by first
screening a cDNA library for nucleic acid molecules that encode
antibodies that specifically bind to the receptors or ligands of
the invention according to the method generally set forth by Huse
et al., Science 246:1275-81 (1989), the contents of which are
hereby incorporated by reference. The nucleic acid molecule can
then be cloned and amplified to obtain sequences that encode the
antibody (or antigen-binding domain) of the desired specificity.
Phage display technology offers another technique for selecting
antibodies that bind to the receptors, ligands, or enzymes relevant
to the methods of the invention, fragments, derivatives or analogs
thereof. (See, e.g., International Patent Publications WO 91/17271
and WO 92/01047; Huse et al., supra.)
[0445] Techniques for producing single chain antibodies (see, e.g.,
U.S. Pat. Nos. 4,946,778 and 5,969,108) can also be used. An
additional aspect of the invention utilizes the techniques
described for the construction of a Fab expression library (see,
e.g., Huse et al., supra) to allow rapid and easy identification of
monoclonal Fab fragments with the desired specificity for antigens,
fragments, derivatives, or analogs thereof.
[0446] Antibodies that contain the idiotype of the molecule can be
generated by known techniques. For example, such fragments include
but are not limited to, the F(ab').sub.2 fragment which can be
produced by pepsin digestion of the antibody molecule, the Fab'
fragments which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragment, the Fab fragments which can be
generated by treating the antibody molecule with papain and a
reducing agent, and Fv fragments. Recombinant Fv fragments can also
be produced in eukaryotic cells using, for example, the methods
described in U.S. Pat. No. 5,965,405 (the disclosure of which is
incorporated by reference herein).
[0447] Antibody screening can be accomplished by techniques known
in the art (e.g., ELISA (enzyme-linked immunosorbent assay)). In
one example, antibodies that recognize a specific domain of an
antigen can be used to assay generated hybridomas for a product
which binds to polypeptides containing that domain. Small amounts
of humanized antibody can be produced in a transient expression
system in CHO cells to establish that they bind to HUVEC cells
expressing antigens relevant to the methods of the invention.
Stable cell lines can then be isolated to produce larger quantities
of purified material. The binding affinity of murine and humanized
antibodies can be determined using the procedure described by
Krause et al., Behring Inst. Mitt., 87:56-67 (1990).
[0448] Antibodies binding to antigens relevant to the methods of
the invention (including fragments, derivatives and analogs) can be
used for passive antibody treatment, according to methods known in
the art. The antibodies can be produced as described above and can
be polyclonal or monoclonal antibodies and administered
intravenously, enterally (e.g., as an enteric coated tablet form),
by aerosol, orally, transdermally, transmucosally, intrapleurally,
intrathecally, or by other suitable routes.
[0449] The foregoing methods relating to antibodies allow
production of antibodies which bind specifically to receptors, e.g.
AT2 or .alpha.7nAChR, or to substances that modulate activity of
receptors, e.g. substances which either stimulate or inhibit
activation of receptors such as angiotensin II, nicotine, or
.beta.-amyloid. Further, specific antibodies may be produced that
inhibit or affect the activity of enzymes, such as ACE, that
indirectly affect stimulation of a relevant receptor via activation
of a stimulatory substance.
[0450] Pharmaceutical Compositions. The present invention also
relates to compositions that can be used for prophylaxis of a
condition or disorder to a subject susceptible to such a condition
or disorder, and for providing treatment to a subject suffering
therefrom. For example, the compositions can be administered to a
patient an amount of a compound effective for providing some degree
of prevention of the progression of a CNS disorder (i.e., provide
protective effects), amelioration of the symptoms of a CNS
disorder, and amelioration of the reoccurrence of a CNS disorder.
The compositions can include an effective amount of a compound that
stimulates a nicotine receptor. The invention also involves the
administration of effective amounts of compounds that inhibit the
AT2 receptor, or that inhibit the effects of a stimulator of the
AT2 receptor. Compounds that prevent A.beta.-mediated interference
with JAK2 phosphorylation can also be administered as
pharmaceutical compositions according to the present invention.
[0451] The compounds can be employed in a free base form or in a
salt form (e.g., as pharmaceutically acceptable salts). Examples of
suitable pharmaceutically acceptable salts include inorganic acid
addition salts such as hydrochloride, hydrobromide, sulfate,
phosphate, and nitrate; organic acid addition salts such as
acetate, galactarate, hemigalactarate, propionate, succinate,
lactate, glycolate, malate, tartrate, citrate, maleate, fumarate,
methanesulfonate, p-toluenesulfonate, ditoluyl tartrate, and
ascorbate; salts with acidic amino acid such as aspartate and
glutamate; alkali metal salts such as sodium salt and potassium
salt; alkaline earth metal salts such as magnesium salt and calcium
salt; ammonium salt; organic basic salts such as trimethylamine
salt, triethylamine salt, pyridine salt, picoline salt,
dicyclohexylamine salt, and N,N'-dibenzylethylenediamine salt; and
salts with basic amino acid such as lysine salt and arginine salt.
The salts may be in some cases hydrates or ethanol solvates.
[0452] Compounds useful in the methods described herein can be
those described, for example, in, Williams et al. DN&P
7(4):205-227 (1994), Arneric et al., CNS Drug Rev. 1(1):1-26
(1995), Arneric et al., Exp. Opin. Invest. Drugs 5(1):79-100
(1996), Bencherif et al., JPET 279:1413 (1996), Lippiello et al.,
JPET 279:1422 (1996), Damaj et al., Neuroscience (1997), Holladay
et al., J. Med. Chem 40(28): 4169-4194 (1997), Bannon et al.,
Science 279: 77-80 (1998), PCT WO 94/08992, PCT WO 96/31475, and
U.S. Pat. No. 5,583,140 to Bencherif et al., U.S. Pat. No.
5,597,919 to Dull et al., and U.S. Pat. No. 5,604,231 to Smith et
al., the disclosures of which are incorporated herein by reference
in their entirety. Compounds of the present invention can be used
as analgesics, to prevent or treat a variety of neurodegenerative
diseases, and to treat convulsions such as those that are
symptomatic of epilepsy. CNS disorders which can be prevented or
treated in accordance with the present invention include presenile
dementia (early onset Alzheimer's disease), senile dementia
(dementia of the Alzheimer's type), HIV-dementia, multiple cerebral
infarcts, Parkinsonism including Parkinson's disease, Pick's
disease, Huntington's chorea, progressive supra nuclear palsy, Lewy
body dementia, and mild cognitive impairment. Compounds of the
present invention also can be used to prevent or treat conditions
such as syphilis and Creutzfeld-Jakob disease.
[0453] The pharmaceutical compositions also can include various
other components as additives or adjuncts. Exemplary
pharmaceutically acceptable components or adjuncts which are
employed in relevant circumstances include antioxidants, free
radical scavenging agents, peptides, growth factors, antibiotics,
bacteriostatic agents, immunosuppressives, anticoagulants,
buffering agents, anti-inflammatory agents, anti-pyretics, time
release binders, anesthetics, steroids and corticosteroids. Such
components can provide additional therapeutic benefit, act to
affect the therapeutic action of the pharmaceutical composition, or
act towards preventing any potential side effects which may be
posed as a result of administration of the pharmaceutical
composition.
[0454] The manner in which the compounds are administered can vary.
The compounds can be administered by inhalation (e.g., in the form
of an aerosol either nasally or using delivery articles of the type
set forth in U.S. Pat. No. 4,922,901 to Brooks et al., the
disclosure of which is incorporated herein in its entirety);
topically (e.g., in lotion form); orally (e.g., in liquid form
within a solvent such as an aqueous or non-aqueous liquid, or
within a solid carrier); intravenously (e.g., within a dextrose or
saline solution); as an infusion or injection (e.g., as a
suspension or as an emulsion in a pharmaceutically acceptable
liquid or mixture of liquids); intrathecally; intracerebro
ventricularly; or transdermally (e.g., using a transdermal patch).
Although it is possible to administer the compounds in the form of
a bulk active chemical, each compound can be presented in the form
of a pharmaceutical composition or formulation for efficient and
effective administration. Exemplary methods for administering such
compounds will be apparent to the skilled artisan. For example, the
compounds can be administered in the form of a tablet, a hard
gelatin capsule or as a time release capsule. As another example,
the compounds can be delivered transdermally using the types of
patch technologies available, for example, from Novartis and Alza
Corporation. The administration of the pharmaceutical compositions
of the present invention can be intermittent, or at a gradual,
continuous, constant or controlled rate to a warm-blooded animal,
(e.g., a mammal such as a mouse, rat, cat, rabbit, dog, pig, cow,
or monkey); but advantageously is administered to a human being. In
addition, the time of day and the number of times per day that the
pharmaceutical formulation is administered can vary. Administration
can be such that the active ingredients of the pharmaceutical
formulation interact with receptor sites within the body of the
subject that effect the functioning of the CNS. More specifically,
in prophylaxis and/or treatment of a CNS disorder administration
can be such as to optimize the effect upon those relevant receptor
subtypes that have an effect upon the functioning of the CNS, while
minimizing the effects upon muscle-type receptor subtypes. Other
suitable methods for administering the compounds of the present
invention are described in U.S. Pat. No. 5,604,231 to Smith et
al.
[0455] The appropriate dose of the compound is that amount
effective to prevent occurrence of the symptoms of the disorder or
to treat some symptoms of the disorder from which the patient
suffers. By "effective amount," "therapeutic amount," or "effective
dose" is meant that amount sufficient to elicit the desired
pharmacological or therapeutic effects, thus resulting in effective
prevention or treatment of the disorder. Thus, when treating a CNS
disorder, an effective amount of compound is an amount sufficient
to pass across the blood-brain barrier of the subject, to bind to
relevant receptor sites in the brain of the subject, and to
activate relevant nicotinic receptor subtypes (e.g., provide
neurotransmitter secretion, thus resulting in effective prevention
or treatment of the disorder). Prevention of the disorder is
manifested by delaying the onset of the symptoms of the disorder.
Treatment of the disorder is manifested by a decrease in the
symptoms associated with the disorder or an amelioration of the
reoccurrence of the symptoms of the disorder.
[0456] The effective dose can vary, depending upon factors such as
the condition of the patient, the severity of the symptoms of the
disorder, and the manner in which the pharmaceutical composition is
administered. For human patients, the effective dose of typical
compounds generally requires administering the compound in an
amount sufficient to activate relevant receptors to effect
neurotransmitter (e.g., dopamine) release, but the amount should be
insufficient to induce effects on skeletal muscles and ganglia to
any significant degree. The effective dose of compounds will of
course differ from patient to patient but in general includes
amounts starting where CNS effects or other desired therapeutic
effects occur, but below the amount where muscular effects are
observed.
[0457] Typically, the effective dose of compounds generally
requires administering the compound orally in an amount of from
about 1 .mu.g/kg to about 20 mg/kg of patient weight. Often, the
compounds of the present invention are administered in an amount
from about 10 .mu.g/kg to about 10 mg/kg of patient weight,
frequently from about 10 .mu.g to about 1 mg/kg of patient weight.
The foregoing effective doses typically represent that amount
administered as a single dose, or as one or more doses administered
over a 24 hour period.
[0458] For human patients, the effective dose of typical compounds
generally requires administering the compound in an amount of at
least about 1, often at least about 10, and frequently at least
about 1 to about 100 mg/24 hr/patient. For human patients, the
effective dose of typical compounds requires administering the
compound which generally does not exceed about 500, often does not
exceed about 400, and frequently does not exceed about 300 mg/24
hr/patient. In addition, administration of the effective dose is
such that the concentration of the compound within the plasma of
the patient normally does not exceed 10 .mu.g/ml, and frequently
does not exceed 1 .mu.g/ml.
[0459] The compounds according to the method of the present
invention can have the ability to pass across the blood-brain
barrier of the patient. Such compounds have the ability to enter
the central nervous system of the patient. The log P values of
typical compounds, which are useful in carrying out the present
invention are generally greater than about -0.5, often are greater
than about 0, and frequently are greater than about 0.5. The log P
values of such typical compounds generally are less than about 3,
often are less than about 2, and frequently are less than about 1.
Log P values provide a measure of the ability of a compound to pass
across a diffusion barrier, such as a biological membrane. See,
Hansch, et al., J. Med. Chem. 11:1 (1968).
[0460] The receptor binding constants of typical compounds useful
in carrying out the present invention generally exceed about 0.1
nM, often exceed about 1 nM, and frequently exceed about 10 nM. The
receptor binding constants of certain compounds are less than about
100 .mu.M, often are less than about 10 .mu.M and frequently are
less than about 5 .mu.M; and of other compounds generally are less
than about 1 .mu.M, often are less than about 100 nM, and
frequently are less than about 50 nM. Certain compounds can possess
receptor binding constants of less than 10 .mu.M, and even less
than 100 .mu.M. Receptor binding constants provide a measure of the
ability of the compound to bind to half of the relevant receptor
sites of certain brain cells of the patient. See, Cheng, et al.,
Biochem. Pharmacol. 22:3099 (1973).
[0461] Certain compounds useful according to the method of the
present invention can have the ability to demonstrate a nicotinic
function by effectively binding to a nicotinic acetylcholine
receptor. Such compounds have the ability to activate relevant
neurons to release or secrete acetylcholine, dopamine, and other
neurotransmitters. Generally, typical compounds useful in carrying
out the present invention provide for the activation of nicotinic
acetylcholine receptor in amounts of at least one third, typically
at least about 10 times less, frequently at least about 100 times
less, and sometimes at least about 1,000 times less, than those
required for activation of muscle-type nicotinic receptors. Certain
compounds of the present invention can provide secretion of
dopamine in an amount which is comparable to that elicited by an
equal molar amount of (S)-(-)-nicotine.
[0462] The compounds useful according to the methods of the present
invention, when employed in effective amounts in accordance with
the method of the present invention, are selective to certain
relevant nicotinic receptors, but do not cause significant
activation of receptors associated with undesirable side effects at
concentrations at least greater than those required for activation
of dopamine release. By this is meant that a particular dose of
compound resulting in prevention and/or treatment of a CNS
disorder, is essentially ineffective in eliciting activation of
certain muscle-type nicotinic receptors at concentration higher
than 5 times, higher than 100 times, and higher than 1,000 times,
than those required for activation of dopamine release. This
selectivity of certain compounds of the present invention against
those ganglia-type receptors responsible for cardiovascular side
effects is demonstrated by a lack of the ability of those compounds
to activate nicotinic function of adrenal chromaffin tissue at
concentrations greater than those required for activation of
dopamine release.
[0463] Certain compounds useful according to the methods of the
present invention, when employed in effective amounts in accordance
with the method of the present invention, can be effective towards
prevention of the progression of CNS disorders, amelioration of the
symptoms of CNS disorders, and amelioration of the reoccurrence of
CNS disorders. However, such effective amounts of those compounds
are not sufficient to elicit any appreciable side effects, as
demonstrated by increased effects relating to skeletal muscle. As
such, administration of certain compounds of the present invention
provides a therapeutic window in which treatment of certain CNS
disorders is provided and certain side effects are avoided. That
is, an effective dose of a compound of the present invention is
sufficient to provide the desired effects upon the CNS, but is
insufficient (i.e., is not at a high enough level) to provide
significant undesirable side effects.
[0464] The following detailed Examples are not limiting and are
only illustrative of the methods and compositions of the
invention.
EXAMPLES
[0465] The following examples utilized materials and methods as
indicated:
[0466] Chemicals. Molecular weight standards, acrylamide, sodium
dodecyl sulfate (SDS), N-N'-methylene-bisacrylamide,
N,N,N',N'-tetramethylenediamine, protein assay reagents and
nitrocellulose membranes were purchased from Bio-Rad Laboratories
(Hercules, Calif., USA). Protein A/G-agarose was obtained from
Santa-Cruz Biotechnology (Santa Cruz, Calif., USA) whereas
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum,
trypsin, and all medium additives were obtained from Mediatech Inc.
(Herndon, Va., USA). Monoclonal antibody to phosphotyrosine (PY20),
JAK2, Akt and PI3 Kinase were procured from Transduction
Laboratories (Lexington, Ky., USA). Anti-phospho Akt and PARP
antibodies were purchased from New England Biolabs (Beverly, Mass.,
USA). Anti-phosphotyrosine JAK2 antibody was obtained from
Biosource International (Camarillo, Calif., USA). The Pierce
SUPERSIGNAL substrate chemiluminescence detection kit was obtained
from PIERCE (Rockford, Ill., USA). Goat anti-mouse IgG and
anti-rabbit IgG were acquired from Amersham (Princeton, N.J., USA),
and TWEEN-20, nicotine, A.beta. (1-42) peptide, anti-A.beta.(1-42)
and anti-.alpha.7-nAChR and all other chemicals were purchased from
Sigma Chemical Corp. (St. Louis, Mo., USA).
[0467] Isolation and Culture of PC12 cells. PC12 (rat
pheochromocytoma) cells were maintained in proliferative growth
phase in DMEM (GIBCO/BRL, Gaithersburg, Md.) supplemented with 10%
horse serum, 5% fetal calf serum (Atlanta Biologicals, Norcross,
Ga.) and antibiotics (penicillin/streptomycin) according to routine
protocols (Liu Q., et al. Proc Natl Acad Sci USA 98(8):4734-9
(2001)). For particular examples, the media was changed to fresh
serum-free media containing either 10 .mu.M AG-490, 10 .mu.M
A.beta.(1-42) prior to nicotine stimulation.
[0468] Data analysis. All statistical comparisons were made using
Student's t test for paired data and analysis of variance (ANOVA).
Significance was P<0.05.
Example 1
Immunoprecipitation and Phosphorylation Analysis of Pathway
Proteins
[0469] PC12 cells were stimulated with nicotine for timed periods.
The immunoprecipitation and Western blotting was performed as
previously described (Liang, H., et al., J. Biol. Chem. 274:
19846-19851 (1999); Marrero, M. B., et al., Nature 375:247-250
(1995). Marrero, M. B., et al., Am. J. Physiol. 275:C1216-C1223
(1998)).
[0470] To immunoprecipitate proteins the following antibodies were
used: anti-.alpha.7 receptor, anti-PI-3 kinase (2 .mu.g/ml),
anti-JAK2 (2 .mu.g/ml), or anti-phosphotyrosine (PY20 clone, 10
.mu.g/ml). The recovered immunoprecipitated proteins was
transferred to nitrocellulose membrane and blotted with the
appropriate antibodies. For the phosphospecific JAK2 and Akt
proteins, the nitrocellulose membrane was incubated overnight at
4.degree. C. with affinity-purified anti-phosphospecific JAK2 and
Akt antibodies. Finally, proteins were visualized using a
horseradish-peroxidase conjugated to goat anti-mouse or donkey
anti-rabbit IgG and an enhanced chemiluminescence kit.
Example 2
Western Blotting Studies of JAK2 and Akt
[0471] The phosphorylation of JAK2 and Akt proteins was determined
in serum-starved PC12 cells stimulated with 10 .mu.M nicotine for
various times ranging from 0 min to 120 min in the presence or
absence of 10 .mu.M AG-490 (1 hour pre-incubation). At the end of
stimulation, cells were washed twice with ice-cold PBS-V
(phosphate-buffered saline with 1 mmol/L Na.sub.3VO.sub.4). Each
dish was then treated for 60 min with ice-cold lysis buffer (20
mmol/L Tris-HCl, pH 7.4, 2.5 mmol/L EDTA, 1% Triton X-100, 10%
glycerol, 1% deoxycholate, 0.1% SDS, 10 mmol/L
Na.sub.4P.sub.2O.sub.7, 50 mmol/L NaF, 1 mmol/L Na.sub.3VO.sub.4
and 1 mmol/L PMSF), and the supernatant fractions were obtained as
cell lysate by centrifugation at 58 000 g for 25 min at 4.degree.
C. Samples were resolved by 7.5% SDS-PAGE gel electrophoresis,
transferred to nitrocellulose membranes and blocked by 60 min
incubation at room temperature (22.degree. C.) in TTBS (TBS with
0.05% Tween-20, pH 7.4) plus 5% skimmed milk powder. The
nitrocellulose membranes were incubated overnight at 4.degree. C.
with affinity-purified anti-phospho specific JAK2 and Akt
antibodies. The nitrocellulose membranes were washed twice for 10
min each with TTBS and incubated for various times with goat
anti-rabbit IgG horseradish peroxidase conjugate. After extensive
washing, bound antibody was visualized on KODAK BIOMAX film, PIERCE
SUPERSIGNAL substrate chemiluminescence detection kit. Molecular
weight markers assessed specificity of the bands.
Example 3
Immunoprecipitation Studies of PI-3 Kinase
[0472] Serum-starved PC12 cells were stimulated with 10 .mu.M
nicotine for various times ranging from 0 min to 120 min, and
washed twice with ice-cold PBS-V (phosphate-buffered saline with 1
mmol/L Na.sub.3VO.sub.4). Each dish was treated for 60 min with
ice-cold lysis buffer (20 mmol/L Tris-HCl, pH 7.4, 2.5 mmol/L EDTA,
1% Triton X-100, 10% glycerol, 1% deoxycholate, 0.1% SDS, 10 mmol/L
Na.sub.4P.sub.2O.sub.7, 50 mmol/L NaF, 1 mmol/L Na.sub.3VO.sub.4
and 1 mmol/L PMSF), and the supernatant fraction obtained as cell
lysate by centrifugation at 58 000 g for 20 min at 4.degree. C. The
cell lysate was incubated with 10 .mu.g/ml of anti-PI3 kinase
monoclonal antibodies at 4.degree. C. for 2 hours and precipitated
by addition of 50 .mu.l of protein A/G agarose at 4.degree. C.
overnight. The immunoprecipitates were recovered by centrifugation
and washed three times with ice-cold wash buffer (TBS, 0.1% Triton
X-100, 1 mmol/L PMSF, and 1 mmol/L Na.sub.3VO.sub.4).
Immunoprecipitated proteins were dissolved in 100 ml of Laemmli
sample buffer and 80 ml of each sample was resolved by SDS-PAGE gel
electrophoresis. Samples were transferred to nitrocellulose
membranes and blocked by 60 min incubation at room temperature
(22.degree. C.) in TTBS (TBS with 0.05% Tween-20, pH 7.4) plus 5%
skimmed milk powder. Nitrocellulose membranes were then incubated
overnight at 4.degree. C. with 10 .mu.g/ml of affinity-purified,
anti-phosphotyrosine antibodies, and the bound antibodies were
visualized using a PIERCE SUPERSIGNAL chemiluminescence detection
kit.
Example 4
Assessment of PC12 Cell Apoptosis
[0473] Apoptosis was determined by assessing the cleavage of the
DNA-repairing enzyme poly-(ADP-ribose) polymerase (PARP) using a
Western blot assay. PARP (116-kDa) is an endogenous substrate for
caspase-3, which is cleaved to a typical 85-kDa fragment during
various forms of apoptosis. PC12 cells were treated with 100 nM
A.beta. for 8 hours in the presence or absence of nicotine and/or
AG-490. The cells were collected, washed with PBS, and lysed in 120
ul of SDS-PAGE sample buffer boiled for 10 min. Total cell lysates
(30 .mu.g of protein) were separated by SDS-PAGE and transferred to
nitrocellulose membranes. The membranes were blocked for 1 hr at
25.degree. C. with 5% nonfat dry milk in TBST (25 mM Tris-HCI, pH
7.5, 0.5 M NaCl, 0.05% Tween-20). Membranes were incubated with
primary PARP antibody specific for the 85-kDa fragment for 2-3 hr
at 25.degree. C., rinsed with TBST, and incubated with secondary
antibody for 1 hr at 25.degree. C. Immunodetection was performed
with appropriate antibody using an enhanced chemiluminescence (ECL)
system (Amersham).
[0474] Caspase 3 enzyme activity was determined with a fluorogenic
substrate for caspase-3 in crude PC12 cell extracts. The caspase-3
fluorogenic peptide Ac-DEVD-AMC (Promega, Madison, Wis.) contains
the specific caspase-3 cleavage sequence (DEVD) coupled at the
C-terminal to the fluorochrome 7-amino-4-methyl coumarin. The
substrate emits a blue fluorescence when excited at a wavelength of
360 nm. When cleaved from the peptide by the caspase 3 enzyme
activity in the cell lysate, free 7-amino-4-methyl coumarin is
released and can be detected by its yellow/green emission at 460
nm. Appropriate controls included a reversible aldehyde inhibitor
of caspase-3 to assess the specific contribution of the caspase 3
enzyme activity. Fluorescence units were normalized relative to
total protein concentration of the cell extract. The assays were
performed in triplicate, and the experiments were repeated three
times. In addition, the decrease in PC12 cell number was measured
using a COULTER counter (model ZM, Coulter, Hialeah, Fla.).
Example 5
Animal Models for Assessing Neuroprotective Effects
[0475] Animals models that are used to evaluate the neuroprotective
effects of compositions and methods according to the present
invention are provided below, along with an appropriate endpoint
(based on nicotine, as indicated) with citations which provide
guidance regarding use of each model (all incorporated fully herein
by reference):
[0476] 6-OHDA (Partial Lesion)--Rats
[0477] Nicotine (NIC) (1.0 mg/kg; s.c.) attenuates dopaminergic
neurotoxicity (i.e., loss of striatal dopamine loss) induced by
6-OHDA (6 .mu.g) into the substantia nigra (Costa, G., et al.,
Brain Res, 888:336-342(2001); Soto-Otero, R. et al., Biochem
Pharmacol, 64(1):125-35(2002)).
[0478] Reserpine-Induced Dopamine Depletion--Mice
[0479] NIC (3 mg/kg; s.c.) blocked the reserpine (0.5 mg/kg;
i.v.)-induced dopamine depletion and this effect of NIC was blocked
by mecamylamine but not by hexamethonium (Oishi, R., et al., Naunyn
Schmiedebergs Arch Pharmacol. 348:154-7(1993)).
[0480] Methamphetamine Toxicity--Mice and Rats
[0481] NIC (1.0 mg/kg) prevents methamphetamine (5 mg/kg)-toxicity
(brain levels) in mice similar to MK-801 (Maggio, R., et al., J
Neurochem, 71:2439-46(1998); and Parain, K, et al., Brain Res.
890:347-350(2001).
MPTP Effects
[0482] Mice
[0483] NIC (0.75 mg/kg) reduces striatal MPP+ levels induced by
MPTP (30 mg/kg; s.c.) similar to MK-801 (Quik, M. and D. A. Di
Monte, Brain Res, 917:219-24 (2001)).
[0484] Non-Human Primates
[0485] SIB-1508Y (1.8 mg/kg) was more effective than nicotine in
enhancing the effects of L-dopa on motor and cognitive function in
the primate MPTP model (Domino, E. F., et al., Exp Neurol,
158:414-21(1999); Schneider, J. S. et al., J Pharmacol Exp Ther.
290(2):731-739(1999)).
Example 6
TC-1698 Mediated Increase in JAK2 Phosphorylation
[0486] In PC12 cells, similarly to nicotine, TC-1698
(2-Pyridin-3-yl-1-aza-bicyclo[3.2.2]nonane) induces phosphorylation
of Jak-2 and potently activates this pathway. See FIGS. 11-15, and
the preceding descriptions thereof.
[0487] TC-1698 is a potent and selective .alpha.7 agonist.
Functional assays indicate that TC-1698 does not activate the
ganglionic subtype .alpha.3.beta.4, the muscle subtype
.alpha.1.beta.1.gamma..delta., the neuronal subtypes
.alpha.3.beta.2 and .alpha.4.beta.2. In contrast TC-1698 binds to
the .alpha.7 with high affinity (Ki=0.8 nM) and is 50-fold more
potent than the endogenous neurotransmitter acetylcholine in
activating .alpha.7 nAChR.
* * * * *