U.S. patent application number 13/682151 was filed with the patent office on 2013-07-25 for compositions and methods for the treatment of disease associated with trp-p8 expression.
This patent application is currently assigned to Dendreon Corporation. The applicant listed for this patent is Dendreon Corporation. Invention is credited to Feng Chen, David F. Duncan, Thomas J. Graddis, Reiner Laus, Ofir Moreno, Sateesh K. Natarajan.
Application Number | 20130190296 13/682151 |
Document ID | / |
Family ID | 34272560 |
Filed Date | 2013-07-25 |
United States Patent
Application |
20130190296 |
Kind Code |
A1 |
Natarajan; Sateesh K. ; et
al. |
July 25, 2013 |
COMPOSITIONS AND METHODS FOR THE TREATMENT OF DISEASE ASSOCIATED
WITH TRP-P8 EXPRESSION
Abstract
Provided are small-molecule Trp-p8 modulators, including Trp-p8
agonists and Tip-p8 antagonists, and compositions comprising
small-molecule Trp-p8 agonists as well as methods for identifying
and characterizing novel small-molecule Trp-p8 modulators and
methods for decreasing viability and/or inhibiting growth of Trp-p8
expressing cells, methods for activating Trp-p8-mediated cation
influx, methods for stimulating apoptosis and/or necrosis, and
related methods for the treatment of diseases, including cancers
such as lung, breast, colon, and/or prostate cancers as well as
other diseases, such as benign prostatic hyperplasia, that are
associated with Trp-p8 expression.
Inventors: |
Natarajan; Sateesh K.;
(Bellevue, WA) ; Moreno; Ofir; (Seattle, WA)
; Graddis; Thomas J.; (Seattle, WA) ; Duncan;
David F.; (San Diego, CA) ; Laus; Reiner;
(Bellevue, WA) ; Chen; Feng; (Seattle,
WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dendreon Corporation; |
Seattle |
WA |
US |
|
|
Assignee: |
Dendreon Corporation
Seattle
WA
|
Family ID: |
34272560 |
Appl. No.: |
13/682151 |
Filed: |
November 20, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13115893 |
May 25, 2011 |
8389730 |
|
|
13682151 |
|
|
|
|
10923413 |
Aug 20, 2004 |
8362264 |
|
|
13115893 |
|
|
|
|
10923413 |
Aug 20, 2004 |
8362264 |
|
|
10923413 |
|
|
|
|
60497384 |
Aug 22, 2003 |
|
|
|
60497384 |
Aug 22, 2003 |
|
|
|
Current U.S.
Class: |
514/217.05 ;
435/29; 435/375; 514/212.01; 514/239.5; 514/249; 514/613; 514/614;
540/598; 540/604; 544/160; 544/166; 544/356; 564/123; 564/148 |
Current CPC
Class: |
A61P 11/00 20180101;
C07D 231/56 20130101; C07D 279/12 20130101; C07D 295/135 20130101;
C07D 471/04 20130101; C07C 243/38 20130101; C07D 403/14 20130101;
C07D 241/18 20130101; C07D 211/06 20130101; C07D 263/34 20130101;
C07C 311/20 20130101; C07C 2602/42 20170501; A61K 31/517 20130101;
C07D 231/14 20130101; C07D 317/66 20130101; C07D 513/04 20130101;
G01N 33/6872 20130101; C07D 215/04 20130101; C07D 213/40 20130101;
A61P 1/04 20180101; C07D 241/44 20130101; C07D 209/20 20130101;
C07D 295/096 20130101; C07D 307/52 20130101; A61K 31/498 20130101;
C07D 207/325 20130101; C07D 215/08 20130101; C07D 235/26 20130101;
A61P 35/00 20180101; C07D 401/04 20130101; C07D 333/34 20130101;
C07D 209/16 20130101; C07D 209/08 20130101; C07D 413/04 20130101;
C07D 207/09 20130101; C07D 487/04 20130101; A61K 31/517 20130101;
C07D 215/46 20130101; C07C 233/60 20130101; C07D 209/14 20130101;
C07D 405/12 20130101; C07C 323/44 20130101; A61P 15/00 20180101;
C07D 217/06 20130101; G01N 33/5011 20130101; G01N 2500/10 20130101;
C07C 311/37 20130101; C07C 2601/14 20170501; C07D 223/04 20130101;
C07D 493/08 20130101; C07D 207/06 20130101; C07D 209/48 20130101;
C07D 235/18 20130101; C07D 413/12 20130101; A61K 31/498 20130101;
C07C 233/58 20130101; C07C 323/40 20130101; C07D 231/12 20130101;
C07C 2601/18 20170501; C07D 239/42 20130101; C07D 263/57 20130101;
C07D 295/155 20130101; A61P 13/08 20180101; C07D 241/54 20130101;
A61P 43/00 20180101; C07D 495/04 20130101; C07D 251/54 20130101;
C07D 311/16 20130101; C07C 243/36 20130101; C07D 417/04 20130101;
A61K 45/06 20130101; C07D 295/26 20130101; A61K 2300/00 20130101;
C07C 333/06 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/217.05 ;
564/123; 514/613; 564/148; 514/614; 544/356; 514/249; 544/166;
514/239.5; 544/160; 540/604; 514/212.01; 540/598; 435/375;
435/29 |
International
Class: |
C07D 403/14 20060101
C07D403/14; C07C 243/36 20060101 C07C243/36; C07D 223/04 20060101
C07D223/04; C07D 295/155 20060101 C07D295/155; C07D 295/26 20060101
C07D295/26; C07C 233/60 20060101 C07C233/60; C07D 241/54 20060101
C07D241/54 |
Claims
1. A compound of Formula VIII ##STR00293## wherein R.sup.22 is a
linker moiety, which may be selected from the group consisting of
oxyacetamide, urea, carbamate, thiourea, sulfonamide, amine;
R.sup.23 is selected from the group consisting of H, an aliphatic
group of up to 25 carbons, and an aryl group of up to 10 carbons
selected from the group consisting of substituted phenyl,
phenalkyl, substituted phenalkyl, naphthyl, substituted naphthyl,
and pyridyl; and R.sup.24 is selected from the group consisting of
H, OH, and an aliphatic group containing up to 25 carbon atoms; and
R.sup.25 is selected from the group consisting of H.
2.-4. (canceled)
5. A composition comprising a compound of claim 1 and a
pharmaceutically acceptable excipient or diluent.
6.-7. (canceled)
8. A method for decreasing the viability of a Trp-p8 expressing
cell, said method comprising the step of contacting said cell with
a small-molecule Trp-p8 modulator in a concentration and for a time
required to decrease the viability of said cell.
9. The method of claim 8 wherein said small-molecule Trp-p8
modulator is selected from the group consisting of a compound of
Formula I, a compound of Formula II, a compound of Formula III, a
compound of Formula IV, a compound of Formula V, a compound of
Formula VI, a compound of Formula VII, and a compound of Formula
VIII.
10. The method of claim 9 wherein said small-molecule Trp-p8
modulator is a compound of Formula I ##STR00294## wherein R.sup.1
is selected from the group consisting of H, OH, CH.sub.3,
CH.sub.3--CH--CH.sub.3 (isopropyl), and CH.sub.3--C.dbd.CH.sub.2
(isopropenyl); R.sup.2 is selected from the group consisting of H;
R.sup.3 is selected from the group consisting of O, OH, acetate,
lactate, carboxamide, butanamide, sulphanamide, and propanetriol;
and R.sup.4 is selected from the group consisting of
CH.sub.3--CH--CH.sub.3 (isopropyl), isopropane-2-ol, and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl).
11. The method of claim 9 wherein said small-molecule Trp-p8
agonist is a compound of Formula II ##STR00295## wherein R.sup.5 is
selected from the group consisting of H, OH, CH.sub.3,
CH.sub.3--CH--CH.sub.3 (isopropyl), and CH.sub.3--C.dbd.CH.sub.2
(isopropenyl); R.sup.6 is selected from the group consisting of N;
R.sup.7 is selected from the group consisting of O and N; R.sup.8
is selected from the group consisting of NH, O, and S; and R.sup.9
is selected from the group consisting of NO.sub.2.
12. The method of claim 9 wherein said small-molecule Trp-p8
modulator is a compound of Formula III ##STR00296## wherein
R.sup.10 is selected from the group consisting of H and a
C.sub.1-C.sub.5 alkyl including, but not limited to, CH.sub.3,
C.sub.2H.sub.5, CH.sub.3--CH--CH.sub.3 (isopropyl) and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl); R.sup.11 is selected from
the group consisting of OH, carboxamide, butanamide, propanetriol,
and CONR'R'', wherein R' is selected from the group consisting of
H, CH.sub.3, C.sub.2H.sub.5, C.sub.4H.sub.8 (cyclobutyl), and
C.sub.4H.sub.8O, and wherein R'' is selected from the group
consisting of C.sub.2H.sub.5O00H.sub.2, C.sub.21-1.sub.5,
CH.sub.3--CH--CH.sub.3 (isopropyl), HOCH.sub.2C(CH.sub.3).sub.2,
HOCH.sub.2CH.sub.2, C.sub.4H.sub.9 (tertbutyl), and
C.sub.41-1.sub.9 (secbutyl); R.sup.12 is selected from the group
consisting of H and a C1-C5 alkyl including, but not limited to,
CH.sub.3, CH.sub.3--CH--CH.sub.3 (isopropyl),
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl), C.sub.4H.sub.9 (secbutyl),
C.sub.4H.sub.9 (isobutyl), C.sub.4H.sub.9 (n-butyl), and
C.sub.5H.sub.11 (isohexyl); and R.sup.13 is selected from the group
consisting of H and a C.sub.1-C.sub.5 alkyl including, but not
limited to, CH.sub.3, C.sub.2H.sub.5, CH.sub.3--CH--CH.sub.3
(isopropyl), CH.sub.3--C.dbd.CH.sub.2 (isopropenyl), C.sub.4H.sub.9
(secbutyl), and C.sub.4H.sub.9 (isobutyl).
13. The method of claim 9 wherein said small-molecule Trp-p8
modulator is a compound of Formula IV ##STR00297## wherein R.sup.14
is selected from the group consisting of H, an aliphatic group of
up to 25 carbons, and an aryl group of up to 10 carbons and
R.sup.15 is selected from the group consisting of H, OH, and an
aliphatic group containing up to 25 carbon atoms.
14-17. (canceled)
18. The method of claim 9 wherein said small-molecule Trp-p8
modulator is a compound of Formula V ##STR00298## wherein R.sup.16
is selected from the group consisting of a C.sub.2-C.sub.6 alkylene
group having at least one, but not more than three, hydroxyl
groups; and R.sup.17 and R.sup.18, independently of one another,
are selected from the group consisting of C.sub.1-C.sub.10-alkyl
groups, C.sub.5-C.sub.7-cycloalkyl, C.sub.6-C.sub.12-aryl, wherein
the total of the C atoms of R.sup.17 and R.sup.18 is not less than
3, or R.sup.17 and R.sup.18 together represent an alkylene group
that, together with the carbon atom that carries the groups
R.sup.17 and R.sup.18, forms a 5-7-membered ring.
19. (canceled)
20. The method of claim 9 wherein said small-molecule Trp-p8
modulator is a compound of Formula VII ##STR00299## wherein
R.sup.17 is selected from the group consisting of 2-pyridyl,
2-nitro-4-trifluoromethylphenyl, 2-nitro-4-chlorophenyl,
2-methoxyphenyl, 2-chlorophenyl, phenyl, 2-methyl-quinolin-3-yl,
4-methoxyphenyl, 4-fluorophenyl,
3-azepan1-yl-5-(4-trifluoromethoxy)phenylamino[1,3,5]triazyl,
cyclohexyl, diphenylmethyl, 2-phenylethyl, 4-hydroxy-cyclohexyl,
cycloheptyl, cyclopentyl, C-benzo[1,3]dioxol-5-yl-methyl,
2-pyridyl, and 4-chlorobenzyl; R.sup.18 is selected from the group
consisting of 1-benzyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,
3-benzylamino-2-nitrophenyl, 5-nitro-quinolin-8-yl,
1-yl-3-(2-isopropyl-5-methylcyclohexyloxy)-propan-2-ol,
1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,
benzyl-2-methylquinazolin-4-yl,
3-methyl-5-morpholin-4-yl-2-nitro-phenyl,
2-nitro-5-piperazin-1-yl-ethanol,
1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol,
4-(2,5-dimethyl-pyrrol-1-yl)-2-nitro-phenyl,
2-nitro-3-trifluoromethanesulfonyl-phenyl,
1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,
2-(2-Fluoro-phenoxymethyl)-2-cyano oxazolyl, adamantly,
5-(benzo[1,3]dioxol-5-ylamino)-10b,10c-dihydro-anthra[1,9-cd]isoxazol-6-o-
ne-yl, 2-methylthiazolo[3,2-b][1,2,4]triazol-6-01 4-methylphenyl
methyl, 3-benzyl-3H-quinazolin-4-one-2-yl, cyclopentyl,
tetrahydronapthyl, cyclooctyl, cyclohexyl,
C-[3-(4-chloro-phenyl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidin-7-yl]-methyl-
, C-(2-benzyl-5,6,7,8-tetrahydro
benzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-methyl, and
1-yl-3-(2-isopropyl-5-methyl cyclohexyloxy)-propan-2-ol; R.sup.19
and R.sup.20 are each independently selected from the group
consisting of H and O; and R.sup.21 is selected from the group
consisting of 4-methylphenyl, 2-chloro-4-fluorophenyl, and
4-chlorophenyl.
21. The method of claim 9 wherein said small-molecule Trp-p8
modulator is a compound of Formula VIII ##STR00300## wherein
R.sup.22 is a linker moiety, which may be selected from the group
consisting of oxyacetamide, urea, carbamate, thiourea, sulfonamide,
amine, amide; R.sup.23 is selected from the group consisting of H,
tetrahydro isoquinolinyl, tetrahydro quinolinyl, 3-methyl
indolinyl, indolinyl, 2-(N-methyl, N-phenylethyl)amino ethyl,
3-methyl indolinyl, 1-phenyl ethyl, 2-chloro benzyl,
2-methoxybenzyl, 2-methoxyphenyl, 2-cyclohex-1-enyl ethyl,
(1-phenyl-cyclophenyl)-methyl, 2-(tetrahydroquinolinyl)-ethyl,
1-propyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, cycloheptyl,
3-cyclohexylsulfanylpropyl, 2-cyclohex-1-enyl ethyl,
2-(N-isopropyl, N-phenylethyl)amino ethyl,
1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine,
2-cyclopentylethyl, 2-phenylcyclopropyl, 1-phenoxyethyl,
4-butyloxyphenyl, (2-nitrophenoxy)methyl,
4,7,7-trimethyl-2-oxa-bicyclo[2.2.1]heptan-3-one,
C-(1-phenyl-5-propyl-4H-pyrazol-4-yl)-methyl, benzyl,
2-chlorobenzyl,
1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-met-
hoxy-phenyl]-2,3,4,9-tetrahydro-1H-b-carboline,
C-[3-(4-butoxy-phenyl)-1H-pyrazol-4-yl]-methyl,
4-(azepane-1-sulfonyl)-phenyl, and
5-(7-chloro-quinolin-4-ylsulfanyl)-[1,3,4]thiadiazol-2-yl; R.sup.24
is selected from the group consisting of H, tetrahydro
isoquinolinyl, tetrahydro quinolinyl, 3-methyl indolinyl,
indolinyl, 3-methyl indolinyl,
1-propyl-1,2,3,4-pyrrolo[1,2-a]pyrazine,
1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, and
1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-met-
hoxy-phenyl]-2,3,4,9 tetrahydro-1H-b-carboline; and R.sup.25 is
selected from the group consisting of H.
22. A method for inducing apoptosis and/or necrosis in a cell
expressing Trp-p8 or for treating a disease associate with Trp-p8
expression, said method comprising the step of administering to
said cell a small-molecule Trp-p8 modulator selected from the group
consisting of a compound of Formula I, a compound of Formula II, a
compound of Formula III, a compound of Formula IV, a compound of
Formula V, a compound of Formula VI, a compound of Formula VII, and
a compound of Formula VIII.
23. A method for treating a disease associated with Trp-p8
expression of claim 22, said method comprising the steps of
administering to a mammal an efficacious amount of a composition
comprising a compound having the formula: ##STR00301## in
combination with a pharmaceutically acceptable carrier or diluent,
wherein R.sup.1 is selected from the group consisting of H, OH,
CH.sub.3, CH.sub.3--CH--CH.sub.3 (isopropyl), and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl); R.sup.2 is selected from
the group consisting of OH, carboxamide, butanamide, and
propanetriol; and R.sup.3 is selected from the group consisting of
CH.sub.3--CH--CH.sub.3 (isopropyl), isopropane-2-ol, and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl).
24. A method for treating a disease associated with Trp-p8
expression of claim 22, said method comprising the step of
administering to a mammal an efficacious amount of a composition
comprising a compound having the formula: ##STR00302## in
combination with a pharmaceutically acceptable carrier or diluent,
wherein R.sup.5 is selected from the group consisting of H, OH,
CH.sub.3, CH.sub.3--CH--CH.sub.3 (isopropyl), and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl); R.sup.6 is selected from
the group consisting of N; R.sup.7 is selected from the group
consisting of O, CH.sub.2, and CH.sub.3--C--CH.sub.3 (isopropyl);
R.sup.8 is selected from the group consisting of NH; and R.sup.9 is
selected from the group consisting of N02.
25. A method for treating a disease associated with Trp-p8
expression of claim 22, said method comprising the step of
administering to a mammal an efficacious amount of a composition
comprising a compound having the formula: ##STR00303## in
combination with a pharmaceutically acceptable carrier or diluent,
wherein R.sup.10 is selected from the group consisting of H,
CH.sub.3, CH.sub.3--CH--CH.sub.3 (isopropyl), and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl); R.sup.11 is selected from
the group consisting of OH, carboxamide, butanamide, and
propanetriol; R.sup.12 is selected from the group consisting of H,
CH.sub.3, CH.sub.3--CH--CH.sub.3 (isopropyl), and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl); and R.sup.13 is selected
from the group consisting of H, CH.sub.3, CH.sub.3--CH--CH.sub.3
(isopropyl), and CH.sub.3--C.dbd.CH.sub.2 (isopropenyl); and a
pharmaceutically acceptable carrier or diluent.
26. A method for treating a disease associated with Trp-p8
expression of claim 22, said method comprising the step of
administering to a mammal an efficacious amount of a composition
comprising a compound having the formula: ##STR00304## in
combination with a pharmaceutically acceptable carrier or diluent,
wherein R.sup.14 is selected from the group consisting of H, an
aliphatic group of up to 25 carbons, and an aryl group of up to 10
carbons and R.sup.15 is selected from the group consisting of H,
OH, and an aliphatic group containing up to 25 carbon atoms.
27.-30. (canceled)
31. A method for treating a disease associated with Trp-p8
expression of claim 22, said method comprising the step of
administering to a mammal an efficacious amount of a composition
comprising a compound having the formula: ##STR00305## in
combination with a pharmaceutically acceptable carrier or diluent,
wherein R.sup.16 is selected from the group consisting of a
C.sub.2-C.sub.6 alkylene group having at least one, but not more
than three, hydroxyl groups; and R.sup.17 and R.sup.18,
independently of one another, are selected from the group
consisting of C.sub.1-C.sub.10-alkyl groups,
C.sub.5-C.sub.7-cycloalkyl, C.sub.6-C.sub.12-aryl, wherein the
total of the C atoms of R.sup.17 and R.sup.18 is not less than 3,
or R.sup.17 and R.sup.18 together represent an alkylene group that,
together with the carbon atom that carries the groups R.sup.17 and
R.sup.18, forms a 5-7-membered ring.
32. (canceled)
33. A method for treating a disease associated with Trp-p8
expression of claim 22, said method comprising the step of
administering to a mammal an efficacious amount of a composition
comprising a compound having the formula: ##STR00306## in
combination with a pharmaceutically acceptable carrier or
diluent.
34. A method for treating a disease associated with Trp-p8
expression of claim 22, said method comprising the step of
administering to a mammal an efficacious amount of a composition
comprising a compound having the formula: ##STR00307## in
combination with a pharmaceutically acceptable carrier or diluent,
wherein R.sup.17 is selected from the group consisting of
2-pyridyl, 2-nitro-4-trifluoromethylphenyl, 2-nitro-4-chlorophenyl,
2-methoxyphenyl, 2-chlorophenyl, phenyl, 2-methyl-quinolin-3-yl,
4-methoxyphenyl, 4-fluorophenyl,
3-azepan1-yl-5-(4-trifluoromethoxy)phenylamino[1,3,5]triazyl,
cyclohexyl, diphenylmethyl, 2-phenylethyl, 4-hydroxy-cyclohexyl,
cycloheptyl, cyclopentyl, C-benzo[1,3]dioxol-5-yl-methyl,
2-pyridyl, and 4-chlorobenzyl; R.sup.18 is selected from the group
consisting of 1-benzyl-1-pyrazolo[3,4-d]pyrimidin-4-yl,
3-benzylamino-2-nitrophenyl, 5-nitro-quinolin-8-yl,
1-yl-3-(2-isopropyl-5-methylcyclohexyloxy)-propan-2-ol,
1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,
benzyl-2-methylquinazolin-4-yl,
3-methyl-5-morpholin-4-yl-2-nitro-phenyl,
2-nitro-5-piperazin-1-yl-ethanol,
1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol,
4-(2,5-dimethyl-pyrrol-1-yl)-2-nitro-phenyl,
2-nitro-3-trifluoromethanesulfonyl-phenyl,
1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,
2-(2-Fluoro-phenoxymethyl)-2-cyano oxazolyl, adamantly,
5-(benzo[1,3]dioxol-5-ylamino)-10b,10c-dihydro-anthra[1,9-cd]isoxazol-6-o-
ne-yl, 2-methylthiazolo[3,2-b][1,2,4]triazol-6-01 4-methyl-phenyl
methyl, 3-benzyl-3H-quinazolin-4-one-2-yl, cyclopentyl,
tetrahydronapthyl, cyclooctyl, cyclohexyl,
C-[3-(4-chloro-phenyl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidin-7-yl]-methyl-
, C-(2-benzyl-5,6,7,8-tetrahydro
benzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-methyl, and
1-yl-3-(2-isopropyl-5-methyl cyclohexyloxy)-propan-2-ol; R.sup.19
and R.sup.20 are each independently selected from the group
consisting of H and O; and R.sup.21 is selected from the group
consisting of 4-methylphenyl, 2-chloro-4-fluorophenyl, and
4-chlorophenyl.
35. A method for treating a disease associated with Trp-p8
expression of claim 22, said method comprising the step of
administering to a mammal an efficacious amount of a composition
comprising a compound having the formula: ##STR00308## in
combination with a pharmaceutically acceptable carrier or diluent,
wherein R.sup.22 is a linker moiety, which may be selected from the
group consisting of oxyacetamide, urea, carbamate, thiourea,
sulfonamide, amine, amide; R.sup.23 is selected from the group
consisting of H, tetrahydro isoquinolinyl, tetrahydro quinolinyl,
3-methyl indolinyl, indolinyl, 2-(N-methyl, N-phenylethyl)amino
ethyl, 3-methyl indolinyl, 1-phenyl ethyl, 2-chloro benzyl,
2-methoxybenzyl, 2-methoxyphenyl, 2-cyclohex-1-enyl ethyl,
(1-phenyl-cyclophenyl)-methyl, 2-(tetrahydroquinolinyl)-ethyl,
1-propyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, cycloheptyl,
3-cyclohexylsulfanylpropyl, 2-cyclohex-1-enyl ethyl,
2-(N-isopropyl, N-phenylethyl)amino ethyl,
1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine,
2-cyclopentylethyl, 2-phenylcyclopropyl, 1-phenoxyethyl,
4-butyloxyphenyl, (2-nitrophenoxy)methyl,
4,7,7-trimethyl-2-oxa-bicyclo[2.2.1]heptan-3-one,
C-(1-phenyl-5-propyl-1H-pyrazol-4-yl)-methyl, benzyl,
2-chlorobenzyl,
1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-met-
hoxy-phenyl]-2,3,4,9 tetrahydro-1H-b-carboline,
C-[3-(4-butoxy-phenyl)-1H-pyrazol-4-yl]-methyl,
4-(azepane-1-sulfonyl)-phenyl, and
5-(7-chloro-quinolin-4-ylsulfanyl)-[1,3,4]thiadiazol-2-yl; R.sup.24
is selected from the group consisting of H, tetrahydro
isoquinolinyl, tetrahydro quinolinyl, 3-methyl indolinyl,
indolinyl, 3-methyl indolinyl,
1-propyl-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine,
1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, and
1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-met-
hoxy-phenyl]-2,3,4,9 tetrahydro-1H-b-carboline; and R.sup.25 is
selected from the group consisting of H.
36. A method for identifying a Trp-p8 agonist, said method
comprising the step of contacting a Trp-p8 expressing cell and a
non-Trp-p8 expressing cell with a candidate Trp-p8 agonist for a
time and in an amount sufficient to decrease the viability of said
Trp-p8 expressing cell but not said non-Trp-p8 expressing cell.
37. A method for identifying a Trp-p8 antagonist, said method
comprising the step of contacting a Trp-p8 expressing cell with a
Trp-p8 agonist and with a candidate Trp-p8 antagonist for a time
and in an amount sufficient for said agonist to decrease the
viability of said T rp-p8 expressing cell, wherein a Trp-p8
antagonist is detected by an increase in the viability of said
Trp-p8 expressing cell.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority under 35 U.S.C. 119(e) to
U.S. Provisional Patent Application No. 60/497,384 filed Aug. 22,
2003.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates generally to the fields of
cell biology, biochemistry, and organic chemistry. More
specifically, the present invention provides small-molecule
modulators of Trp-p8 activity, which include Trp-p8 agonists and
Trp-p8 antagonists, as well as compositions comprising
small-molecule Trp-p8 modulators. Also provided are methods for
identifying and characterizing novel small-molecule Trp-p8
modulators as well as methods for modulating Trp-p8-mediated cation
influx and/or apoptosis in a cell and related methods for the
treatment of diseases associated with Trp-p8 expression,
activation, and/or signaling. Exemplary diseases suitably treated
by the compositions and methods of the present invention include
cancers, such as lung, breast, colon, and/or prostate cancers.
BACKGROUND OF THE INVENTION
[0003] Prostate carcinoma is the most common cancer diagnosed in
men in the United States and has the second highest cancer death
rate yielding only to lung adenocarcinoma. Parker et al., CA Cancer
J. Clin. 46:5-27 (1996). Although it is possible to effectively
treat organ-confined prostate cancer, there are very limited
treatment options for metastatic disease. Thus, it is of great
importance to find novel ways to diagnose early stage disease and
to closely monitor both progression and treatment of the disease,
as well as to develop new therapeutic approaches. To achieve this,
it is important to understand the molecular mechanisms of prostate
cancer development and to identify new biochemical markers for
disease diagnosis and progression.
[0004] To date there are very few prostate-specific markers
available. The best-known and well-characterized markers of proven
prostate cancer diagnostic value are the proteins prostatic acid
phosphatase (PAP), prostate specific antigen (PSA), and
prostate-specific membrane antigen (PSMA). Each of these proteins
has also become the target for novel immunotherapy approaches to
the treatment of disease. Horoszewicz et al., Anticancer Res.
7:927-935 (1987); Barren et al., Prostate 30:65-68 (1997); Murphy
et al., Prostate 33:281-285 (1997); Murphy et al., Prostate
26:164-168 (1995); Rochon et al., Prostate 25:219-223 (1995);
Correale et al., J. Immunol. 161:3186-3194 (1998); and Murphy et
al., Prostate 38:73-78 (1999).
[0005] It has been reported that a cation channel protein,
variously designated Trp-p8 (transient receptor potential-p8),
TRPM8, and CMR1 (cold and menthol receptor 1), is preferentially
expressed in prostate. Cloning of the full-length human trp-p8 cDNA
revealed a transcript corresponding to an 1104 amino acid
polypeptide sharing homology with the trp family of calcium
channels. Clapham et al., Nature Reviews 2:387-396 (2001) and
Clapham et al., IUPHAR Compendium, TRP Channels (2002). Trp-p8
shows particularly high homology with the human TRPC7 gene--a
putative Ca.sup.2+ channel protein of the trp family that is highly
expressed in brain tissue. Nagamine et al., Genomics 54:124-131
(1998). Trp-p8 also shows significant homology to human melastatin,
another Trp family-related protein expressed in melanocytes and
believed to be a tumor suppressor gene. Duncan et al., Cancer Res.
58:1515-1520 (1998); and Hunter et al., Genomics 54:116-123 (1998).
Perhaps of greatest interest is the observation that the trp-p8
gene appears to be expressed in a large spectrum of nonprostatic in
addition to prostatic neoplastic lesions. Tsavaler et al., Cancer
Res. 61(9):3760-9 (2001).
[0006] The Trp superfamily comprises more than 20 related cation
channel proteins that have been implicated in processes including
sensory physiology to vasorelaxation and male fertility. Defects in
Trp channels have been associated with changes in growth control
and tumor suppression. While all Trp proteins are calcium channels,
they vary significantly in their selectivity and mode of
activation. Members of the Trp superfamily share significant
sequence homology and predicted structural similarities, such as
size of predicted transmembrane segments.
[0007] Trp-p8 is over-expressed in a range of cancers including
prostate, breast, lung and colon, while within normal tissues, it
is predominantly expressed in human prostate [Tsavaler et al.,
supra] and dorsal root ganglia (DRG, Dendreon, unpublished
observation). Fuessel et al. reported that Trp-p8 is a highly
prostate-specific and prostate carcinoma-associated gene thus
qualifying it as a potential target for specific therapies.
International J. of Oncology 23:221-228 (2003). Among other
species, Trp-p8 orthologues are reportedly expressed in a subset of
DRG and trigerminal ganglia (TG) neurons in rat [McKemy et al.,
Nature 416(6876):52-8 (2002)] and mouse [Peier et al., Cell
108(5):705-15 (2002)] as well. Thus, Trp-p8 is a pantumor-expressed
marker with significant potential use in disease diagnosis and
monitoring of disease progression during treatment as well as a
viable target for cancer therapy.
[0008] In two articles published concurrently, it was reported for
the first time that Trp-p8 orthologues, in response to cold and
certain cooling compounds, initiate an influx of cations, such as
calcium, from the extracellular space. McKemy et al., supra; and
Peier et al., supra. Two of the best known modulators of Trp-p8
activity are the Trp-p8 agonists menthol and Icilin. Menthol is
effective in inducing calcium influx at .sup..about.10-100 .mu.M
while Icilin is more potent with an effective concentration range
of 0.1-1 .mu.M.
[0009] The higher temperature threshold reported for Trp-p8
activation by the most widely studied agonist, menthol
(2-isopropyl-5-methyl-cyclohexanol), is about 30-32.degree. C. in a
variety of cells (cold-sensitive neurons, Trp-p8 heterologously
expressed in Xenopus oocytes, HEK293 and CHO cells). McKemy et al.,
Nature, supra; Peier et al., Cell, supra; Nealen et al., J
Neurophysiol. 90(1):515-520 (2003); and Reid et al., J Physiol.
545(Pt 2):595-614 (2002).
[0010] Although certain agonist compounds have been shown to
activate Trp-p8 expressing cells up to 32.degree. C., there has
been no report disclosing growth manipulation in cells at
physiological temperature (i.e. 37.degree. C.), the temperature at
which a compound must be active in order to be an efficacious in
vivo therapeutic.
[0011] Association of Trp-p8 with prostate, lung, breast, and colon
cancers and the important role various ion channels play in vital
cell functions suggest that Trp-p8 channel may have a significant
function in cancer cell signaling and/or proliferation. Modulation
of Trp-p8 activity, either by activating via an agonist or
inhibiting via an antagonist, at a physiological temperature can be
valuable as a therapeutic to manipulate the Trp-p8 expressing cells
in a specific manner.
[0012] Accordingly, there remains a need in the art for
small-molecule modulators of Trp-p8 activity, compositions
comprising one or more small-molecule Trp-p8 modulators, and
methods for the identification and use of small-molecules for
modulating the activity of Trp-p8 in a cell and for the treatment
of disease associated with the aberrant expression of Trp-p8.
SUMMARY OF THE INVENTION
[0013] The present invention fulfills these and other related needs
by providing small molecule modulators of Trp-p8 activity,
including Trp-p8 agonists and Trp-p8 antagonists, as well as
compositions comprising such Trp-p8 modulators, and methods for
identifying and using Trp-p8 modulators. Within certain
embodiments, compounds of the present invention bind to and
activate Trp-p8 and/or stimulate cation influx, including but not
limited to calcium influx, in a cell wherein cation influx is
correlative of Trp-p8 modulator induced toxicity. Thus, within
these and other embodiments, Trp-p8 agonists of the present
invention are effective in inhibiting growth of and/or inducing
apoptosis and/or necrosis in a cell expressing Trp-p8. Within
alternative embodiments are provided Trp-p8 antagonists that are
effective in reducing the basal activity of Trp-p8 in a cell
thereby reducing the viability of Trp-p8 expressing cells.
Advantageously, therefore, agonists and antagonists of the present
invention can be used to treat diseases including, but not limited
to, cancers of the breast, lung, colon, and/or prostate, that are
associated with Trp-p8 expression.
[0014] One or more Trp-p8 modulator can be formulated in
compositions, including pharmaceutical compositions, comprising one
or more pharmaceutically acceptable carrier or excipient and/or one
or more additional therapeutic compound. Such compositions will
find utility in methods for the treatment of one or more disease
associated with Trp-p8 expression.
[0015] Thus, in one embodiment, the present invention provides the
following Trp-p8 modulators and derivatives thereof:
[0016] A compound of Formula I
##STR00001##
wherein
[0017] R.sup.1 is selected from the group consisting of H, OH,
CH.sub.3, CH.sub.3--CH--CH.sub.3 (isopropyl), and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl);
[0018] R.sup.2 is selected from the group consisting of H;
[0019] R.sup.3 is selected from the group consisting of O, OH,
acetate, lactate, carboxamide, butanamide, sulphanamide, and
propanetriol; and
[0020] R.sup.4 is selected from the group consisting of
CH.sub.3--CH--CH.sub.3 (isopropyl), isopropane-2-ol, and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl).
[0021] Exemplary compounds of Formula I include the following:
##STR00002##
[0022] These exemplary Formula I Trp-p8 modulators have the
following trade and chemical names: Menthol
(2-isopropyl-5-methyl-cyclohexanol) (Sigma-Aldrich, Inc.; St.
Louis, Mo.); Frescolat ML (Harris & Ford, LLC; Indianapolis,
Ind.; Menthyl lactate); L-Menthyl Acetate (Millennium Chemicals;
Olympia Fields, Ill.;
Cyclohexanol-5-methyl-2-(1-methylethyl)-acetate-[1R-(1
alpha,2beta,5alpha)]-); Cooling Agent 10 (Takasago International
Corp.; Rockleigh, N.J.; (1)-Menthoxypropane-1,2-diol); Coolact
P.RTM. (Takasago International Corp.; (-)-Isopulegol); and Coolact
38D.RTM. (Takasago International Corp.).
[0023] In another embodiment, the present invention further
provides the following small-molecule Trp-p8 modulators and
derivatives thereof:
[0024] A compound of Formula II
##STR00003##
wherein
[0025] R.sup.5 is selected from the group consisting of H, OH,
CH.sub.3, CH.sub.3--CH--CH.sub.3 (isopropyl), and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl);
[0026] R.sup.6 is selected from the group consisting of N;
[0027] R.sup.7 is selected from the group consisting of O and
N;
[0028] R.sup.8 is selected from the group consisting of NH, O, and
S; and
[0029] R.sup.9 is selected from the group consisting of
NO.sub.2.
[0030] Compounds of Formula II are exemplified herein by Icilin
(1-(2-hydroxyphenyl)-4-(3-nitrophenyl)-1,2,3,6-tetrahydropyrimidine-2-one
aka
3,4-dihydro-3-(2-hydroxyphenyl)-6-(3-nitrophenyl)-(1H)-pyrimidin-2-on-
e).
##STR00004##
[0031] In another embodiment, the present invention further
provides the following acyclic carboxamide Trp-p8 agonists and
derivatives thereof as presented in U.S. Pat. No. 4,153,679,
incorporated herein by reference:
[0032] A compound of Formula III
##STR00005##
wherein
[0033] R.sup.10 is selected from the group consisting of H and a
C.sub.1-C.sub.5 alkyl including, but not limited to, CH.sub.3,
C.sub.2H.sub.5, CH.sub.3--CH--CH.sub.3 (isopropyl) and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl);
[0034] R.sup.11 is selected from the group consisting of OH,
carboxamide, butanamide, propanetriol, and CONR'R'', wherein R' is
selected from the group consisting of H, CH.sub.3,
[0035] C.sub.2H.sub.5, C.sub.4H.sub.8 (cyclobutyl), and
C.sub.4H.sub.8O, and wherein R'' is selected from the group
consisting of C.sub.2H.sub.5OOCH.sub.2, C.sub.2H.sub.5,
CH.sub.3--CH--CH.sub.3 (isopropyl), HOCH.sub.2C(CH.sub.3).sub.2,
HOCH.sub.2CH.sub.2, C.sub.4H.sub.9 (tertbutyl), and C.sub.4H.sub.9
(secbutyl);
[0036] R.sup.12 is selected from the group consisting of H and a
C.sub.1-C.sub.5 alkyl including, but not limited to, CH.sub.3,
CH.sub.3--CH--CH.sub.3 (isopropyl), CH.sub.3--C.dbd.CH.sub.2
(isopropenyl), C.sub.4H.sub.9 (secbutyl), C.sub.4H.sub.9
(isobutyl), C.sub.4H.sub.9 (n-butyl), and C.sub.5H.sub.11
(isohexyl); and
[0037] R.sup.13 is selected from the group consisting of H and a
C.sub.1-C.sub.5 alkyl including, but not limited to, CH.sub.3,
C.sub.2H.sub.5, CH.sub.3--CH--CH.sub.3 (isopropyl),
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl), C.sub.4H.sub.9 (secbutyl),
and C.sub.4H.sub.9 (isobutyl).
[0038] Compounds of Formula III are exemplified herein by WS-23
(2-Isopropyl-N,2,3-trimethylbutyramide aka
N,2,3-trimethyl-2-isopropyl butamide).
##STR00006##
[0039] In another embodiment, the present invention further
provides the following 3-substituted-p-menthane Trp-p8 modulators
and derivatives thereof exemplified by those presented in U.S. Pat.
No. 4,150,052, incorporated herein by reference:
[0040] A compound of Formula IV
##STR00007##
wherein
[0041] R.sup.14 is selected from the group consisting of H and an
aliphatic group containing up to 25 carbon atoms;
[0042] R.sup.15 is selected from the group consisting of H, OH and
an aliphatic group containing up to 25 carbon atoms, with the
proviso that when R.sup.15 is H, R.sup.14 may also be an aryl group
of up to 10 carbon atoms and selected from the group consisting of
substituted phenyl, phenalkyl, substituted phenalkyl, naphthyl,
substituted naphthyl, and pyridyl; and
[0043] R.sup.14 and R.sup.15, when taken together with the nitrogen
atom to which they are attached, may form a cyclic or heterocyclic
group of up to 25 carbon atoms, e.g., a piperidino or a morpholino
group. Exemplary such cyclical groups may be selected from the
group consisting of 3-phenyl-piperidin-1-yl,
3-phenyl-pyrrolidin-1-yl,
6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-yl, and
4-pyrimidin-2-yl-piperazin-1-yl.
[0044] Typical values for R.sup.14 and R.sup.15 when aliphatic are
methyl, ethyl, propyl, butyl, isobutyl, n-decyl, cyclopropyl,
cyclohexyl, cyclopentyl, cycloheptylmethyl, 2-hydroxyethyl,
3-hydroxy-n-propyl, 6-hydroxy-n-hexyl, 2-aminoethyl,
2-acetoxyethyl, 2-ethylcarboxyethyl, 4-hydroxybut-2-ynyl, and
carboxymethyl.
[0045] When R.sup.14 is aryl, typical values are benzyl, naphthyl,
4-methoxyphenyl, 2-methoxy-4-methoxyphenyl,
3-methoxy-5-methoxyphenyl, 4-methyl-5-chlorophenyl,
4-hydroxyphenyl, 4-methylphenyl, 3-methyl-4-methylphenyl,
3-hydroxy-4-methylphenyl, 4-fluorophenyl, 4-chlorophenyl,
4-bromophenyl, 4-ethylphenyl, 2-fluoro-4-fluorophenyl,
4-nitrophenyl, 2-hydroxynaphthyl, pyridyl,
[1-carbamoyl-2-(1H-indol-3-yl)-ethyl,
1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl,
1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl,
1-carbamoyl-2-(4-hydroxyphenyl)ethyl, 1-carbamoyl-2-phenylethyl,
1-hydroxymethyl-2-(1H-indol-3-yl)ethyl,
1-hydroxymethyl-2-(4-chlorophenyl)ethyl,
1-hydroxymethyl-2-(4-hydroxyphenyl)ethyl,
1-hydroxymethyl-2-hydroxy-2-phenylethyl, 1-hydroxymethyl-2-phenyl
ethyl, 1-methoxymethyl-2-phenylethyl,
1-methyl-2-(4-chlorophenyl)-ethyl,
1-methyl-2-(5-fluoro-1H-indol-3-yl)-ethyl,
1-methyl-2-hydroxy-2-phenylethyl, 1-oxo-2-phenylethyl,
1-phenyl-cyclopentylmethyl,
2-(1-cyclopentyl-pyrrolidin-3-yl)-ethyl, 2-(1H-indol-3-yl)ethyl,
dimethoxyphenyl)ethyl, 2-(2,4-dichlorophenyl)ethyl,
2-(2,4-dimethylphenyl)ethyl, 2-(2,5-dimethoxyphenyl)ethyl,
2-(2,5-dimethylphenyl)-ethyl, 2-(2,6-dimethylphenyl)ethyl,
2-(2-chloro-6-fluorophenyl)ethyl, 2-(2-chlorophenyl)ethyl,
2-(2-fluorophenyl)ethyl, 2-(2-furyl)ethyl,
2-(2-methoxy-5-bromophenyl)ethyl, 2-(2-methoxyphenyl)-ethyl,
2-(2-methylphenyl)ethyl, 2-(3,4-dichlorophenyl)ethyl,
2-(3,4-dimethoxyphenyl)ethyl, 2-(3,5-dimethoxyphenyl)ethyl,
2-(3-bromo-4-methoxyphenyl)ethyl, 2-(3-chlorophenyl)ethyl,
2-(3-ethoxyphenyl)ethyl, 2-(3-fluorophenyl)ethyl,
2-(3-hydroxy-4-methoxyphenyl)-ethyl, 2-(3-hydroxyphenyl)ethyl,
2-(3-methoxy-4-ethoxyphenyl)ethyl,
2-(3-methoxy-4-hydroxyphenyl)ethyl, 2-(3-methoxyphenyl)ethyl,
2-(3-methylphenyl)ethyl, 2-(3-trifluoromethylphenyl)ethyl,
2-(4-bromophenyl)-ethyl, 2-(4-chlorophenyl)ethyl,
2-(4-ethylphenyl)ethyl, 2-(4-fluorophenyl)ethyl,
2-(4-hydroxyphenyl)ethyl, 2-(4-methoxy-phenyl)-2-oxo-ethyl,
2-(4-methoxyphenyl)ethyl, 2-(4-methylphenyl)ethyl,
2-(4-methylphenyl)ethyl, 2-(4-methylsulfanylphenyl)ethyl,
2-(4-nitrophenyl)ethyl, 2-(4-sulfamoyl-phenyl)-ethyl,
2-(5-methoxy-1H-indol-3-yl)-ethyl,
2-(6-fluoro-1H-indol-3-yl)-ethyl,
2-(6-methoxy-1H-indol-3-yl)-ethyl,
2-(7-methyl-1H-indol-3-yl)-ethyl, 2-(N,N-dipropylamino)ethyl,
2-(pyridin-2-yl)-ethyl, 2-(pyridin-3-yl)-ethyl,
2-(pyridin-4-yl)-ethyl, 2,2-diphenylethyl, 2,3-difluorobenzyl,
2,3-dimethoxyphenyl, 2,4-dichlorophenyl, 2,4-difluorophenyl,
2,4-dimethoxyphenyl, 2,4-dimethylphenyl, 2-bromo-4-methylphenyl,
2-chloro-4-cyanophenyl, 2-chloro-4-fluorophenyl,
2-chloro-4-iodophenyl, 2-chloro-4-nitrophenyl,
2-chloro-5-nitrophenyl, 2-chlorophenyl, 2-cyclohex-1-enyl-ethyl,
2-fluoro-4-chlorophenyl, 2-fluoro-5-nitrophenyl,
2-hydroxy-2-(3-hydroxyphenyl)ethyl,
2-hydroxy-2-(4-hydroxyphenyl)ethyl, 2-hydroxy-2-phenylethyl,
2-iodophenyl, 2-methyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl,
2-methyl-2-phenylethyl, 2-methyl-4-bromophenyl,
2-methyl-5-nitrophenyl, 2-methylphenyl, 2-nitro-4-fluorophenyl,
2-nitrophenyl, 2-oxo-2,3-dihydro-1H-benzoimidazol-5-yl,
2-oxo-2-phenylethyl, 2-phenyl-1H-benzoimidazol-5-yl,
2-phenyl-2-(4-chlorophenyl)ethyl,
2-phenyl-2-(4-fluorophenyl)-ethyl,
2-phenyl-2-(4-methoxyphenyl)ethyl, 2-phenylethenyl, 2-phenylethyl,
2-pyridin-2-yl-benzooxazol-5-yl,
2-pyridin-3-yl-1H-benzoimidazol-5-yl, 2-thiophen-2-yl-ethyl),
2-trifluoromethyl-1H-benzoimidazol-5-yl, 3,4,5-trifluorophenyl,
3,4,5-trimethoxyphenyl, 3,4-cyclopentanephenyl, 3,4-dichlorophenyl,
3,4-dimethylphenyl, 3,5-dimethoxyphenyl, 3-acetamidophenyl,
3-bromo-4-methylphenyl, 3-carboxamidophenyl,
3-chloro-4-methoxyphenyl, 3-chloro-4-methylphenyl,
3-chloro-4-morpholin-4-yl-phenyl, 3-hydroxymethylphenyl,
3-nitrophenyl, 3-oxo-indan-5-yl, 3-phenylpropyl, 3-yl-acetophenone,
4-(1,1-dioxo-1l6-thiomorpholin-4-ylmethyl)-phenyl,
4-(1-hydroxyethyl)phenyl, 4-(2-hydroxyethyl)phenyl,
4-(4-ethyl-piperazin-1-yl)-phenyl,
4-(4-methyl-1H-benzoimidazol-2-yl)-phenyl,
4-(4-methyl-piperazin-1-ylmethyl)-phenyl,
4-(morpholine-4-sulfonyl)-phenyl,
4-[1,3-dioxo-2-(2-trifluoromethyl-phenyl)-2,3-dihydro-1H-isoindol-5-yloxy-
]-phenyl,
4-[2-(2-methoxy-phenyl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-ylox-
y]-phenyl,
4-{2-[2-(3,4-dimethoxy-phenyl)-ethyl]-1,3-dioxo-2,3-dihydro-1H--
isoindol-5-yloxy}-phenyl, 4-acetylphenyl,
4-azepan-1-ylmethyl-phenyl, 4-benzooxazol-2-yl-phenyl, 4-Biphenyl,
4-bromophenyl, 4-carboxamidophenyl, 4-chlorophenyl, 4-cyanophenyl,
4-ethoxyphenyl, 4-ethylphenyl, 4-fluorophenyl,
4-hydroxymethylphenyl, 4-methoxyphenyl,
4-methyl-2-oxo-2H-chromen-7-yl, 4-methylcyclohexyl, 4-methylphenyl,
4-methylsulfanylphenyl, 4-nitrobenzyl,
4-pyrrolidin-1-ylmethyl-phenyl, 4-trifluoromethylphenyl,
benzo[1,3]dioxol-5-yl, benzoylamino, benzyloxy,
bicyclo[2.2.1]hept-2-yl, C-1H-indazol-5-yl, cycloheptyl,
indan-2-yl, N-(2-diethylamino-ethyl)-benzamide-4-yl,
N'-quinoxalin-2-yl-amino, and phenylcyclopropyl.
[0046] Trp-p8 modulators of Formula IV are exemplified herein by
the Trp-p8 agonists WS-3 (Millennium Chemicals;
N-Ethyl-p-menthane-3-carboxamide aka cyclohexanecarboxamide,
N-ethyl-5-methyl-2(1-methylethyl)) and by WS-12
(N-(4-methoxyphenyl)-p-menthan-3-carboxamide aka
cyclohexanecarboxamide,
N-(4-methoxyphenyl)-5-methyl-2(1-methylethyl)), and the compounds
presented in Table 1.
##STR00008##
[0047] In a further embodiment, the present invention provides the
following Trp-p8 modulators and derivatives thereof that comprise
at least one ketal moiety, including, but not limited to the Trp-p8
agonists 1-menthone glycerol ketal and 3,3,5-trimethylcyclohexaone
glycerol ketal presented in U.S. Pat. No. 5,266,592, incorporated
herein by reference:
[0048] A compound comprising at least one ketal of Formula V
##STR00009##
wherein
[0049] R.sup.16 is selected from the group consisting of a
C.sub.2-C.sub.6 alkylene group having at least one, but not more
than three, hydroxyl group(s), preferably one hydroxyl group;
and
[0050] either R.sup.17 and R.sup.18, independently of one another,
represent C.sub.1-C.sub.10-alkyl which is optionally substituted by
1 to 3 groups selected from the group consisting of hydroxyl,
amino, thio, and halogen (e.g., fluorine, chlorine, bromine, or
iodine), C.sub.5-C.sub.7-cycloalkyl, preferably cyclohexyl,
C.sub.6-C.sub.12-aryl, preferably phenyl, with the proviso that the
total of the C atoms of R.sup.17 and R.sup.18 is not less than 3;
or
[0051] R.sup.17 and R.sup.18 together represent an alkylene group
that, together with the carbon atom that carries the groups
R.sup.17 and R.sup.18, forms a 5-7-membered ring, it being possible
for this alkylene group, in turn, to be substituted by
C.sub.1-C.sub.6-alkyl groups.
[0052] Preferred groups R.sup.17 and R.sup.18 comprise methyl,
isopropyl, and tert-butyl.
[0053] Trp-p8 modulators comprising a ketal of Formula V include
the following compounds wherein R.sup.16 is as defined above:
##STR00010##
[0054] Compounds comprising a ketal of Formula V are exemplified
herein by the Trp-p8 agonist Frescolat MGA (Harris & Ford, LLC;
Menthone Glycerin Acetal),
##STR00011##
[0055] In yet a further embodiment, the present invention provides
Formula VI small-molecule Trp-p8 agonists and derivatives thereof
exemplified herein by L-Carvone (Millennium Chemicals;
(R)-5-Isopropenyl-2-methyl-2-cyclohexenone
p-Mentha-6,8-dien-2-one),
##STR00012##
[0056] Other embodiments of the present invention provide Trp-p8
modulators of Formula VII. A compound of Formula VII
##STR00013##
wherein
[0057] R.sup.17 is selected from the group consisting of 2-pyridyl,
2-nitro-4-trifluoromethylphenyl, 2-nitro-4-chlorophenyl,
2-methoxyphenyl, 2-chlorophenyl, phenyl, 2-methyl-quinolin-3-yl,
4-methoxyphenyl, 4-fluorophenyl,
3-azepan1-yl-5-(4-trifluoromethoxy)phenylamino[1,3,5]triazyl,
cyclohexyl, diphenylmethyl, 2-phenylethyl, 4-hydroxy-cyclohexyl,
cycloheptyl, cyclopentyl, C-benzo[1,3]dioxol-5-yl-methyl,
2-pyridyl, and 4-chlorobenzyl;
[0058] R.sup.18 is selected from the group consisting of
1-benzyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,
3-benzylamino-2-nitrophenyl, 5-nitro-quinolin-8-yl,
1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol,
1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,
benzyl-2-methyl-quinazolin-4-yl,
3-methyl-5-morpholin-4-yl-2-nitro-phenyl,
2-nitro-5-piperazin-1-yl-ethanol,
1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol,
4-(2,5-dimethyl-pyrrol-1-yl)-2-nitro-phenyl,
2-nitro-3-trifluoromethanesulfonyl-phenyl,
1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,
2-(2-Fluoro-phenoxymethyl)-2-cyano oxazolyl, adamantly,
5-(benzo[1,3]dioxol-5-ylamino)-10b,10c-dihydro-anthra[1,9-cd]isoxazol-6-o-
ne-yl, 2-methyl-thiazolo[3,2-b][1,2,4]triazol-6-ol 4-methylphenyl
methyl, 3-benzyl-3H-quinazolin-4-one-2-yl, cyclopentyl,
tetrahydronapthyl, cyclooctyl, cyclohexyl,
C-[3-(4-chloro-phenyl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidin-7-yl]-methyl-
,
C-(2-benzyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-me-
thyl, and
1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol;
[0059] R.sup.19 and R.sup.20 are each independently selected from
the group consisting of H and O; and
[0060] R.sup.21 is selected from the group consisting of
4-methylphenyl, 2-chloro-4-fluorophenyl, and 4-chlorophenyl.
[0061] In other embodiments, the present invention provides Formula
VIII small-molecule Trp-p8 modulators. A compound of Formula
VIII
##STR00014##
wherein
[0062] R.sup.22 is a linker moiety, which may be selected from the
group consisting of oxyacetamide, urea, carbamate, thiourea,
sulfonamide, amine, amide. Formula VIII antagonists are represented
by the following sub-formulae (Formula VIII-A, Formula VIII-B,
Formula VIII-C, Formula VIII-D, Formula VIII-E, Formula VIII-F, and
Formula VIII-G):
##STR00015##
[0063] Irrespective of which of the seven R.sup.22 linker moieties
is employed, R.sup.23 is selected from the group consisting of H,
tetrahydro isoquinolinyl, tetrahydro quinolinyl, 3-methyl
indolinyl, indolinyl, 2-(N-methyl, N-phenylethyl) amino ethyl,
3-methyl indolinyl, 1-phenyl ethyl, 2-chloro benzyl,
2-methoxybenzyl, 2-methoxyphenyl, 2-cyclohex-1-enyl ethyl,
(1-phenyl-cyclophenyl)-methyl, 2-(tetrahydroquinolinyl)-ethyl,
1-propyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, cycloheptyl,
3-cyclohexylsulfanylpropyl, 2-cyclohex-1-enyl ethyl,
2-(N-isopropyl, N-phenylethyl)amino ethyl,
1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine,
2-cyclopentylethyl, 2-phenylcyclopropyl, 1-phenoxyethyl,
4-butyloxyphenyl, (2-nitrophenoxy)methyl,
4,7,7-trimethyl-2-oxa-bicyclo[2.2.1]heptan-3-one,
C-(1-phenyl-5-propyl-1H-pyrazol-4-yl)-methyl, benzyl,
2-chlorobenzyl,
1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-met-
hoxy-phenyl]-2,3,4,9-tetrahydro-1H-b-carboline,
C-[3-(4-butoxy-phenyl)-1H-pyrazol-4-yl]-methyl,
4-(azepane-1-sulfonyl)-phenyl, and
5-(7-chloro-quinolin-4-ylsulfanyl)-[1,3,4]thiadiazol-2-yl;
[0064] R.sup.24 is selected from the group consisting of H,
tetrahydro isoquinolinyl, tetrahydro quinolinyl, 3-methyl
indolinyl, indolinyl, 3-methyl indolinyl,
1-propyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine,
1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, and
1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-met-
hoxy-phenyl]-2,3,4,9-tetrahydro-1H-b-carboline; and
[0065] R.sup.25 is selected from the group consisting of H.
[0066] Other aspects of the present invention provide compositions,
including pharmaceutical compositions, comprising one or more
small-molecule Trp-p8 modulators of Formula I, Formula II, Formula
III, Formula IV, Formula V, Formula VI, Formula VII, and Formula
VIII in combination with a pharmaceutically acceptable excipient,
carrier and/or diluent. Exemplified herein within the Examples are
specific Trp-p8 agonists of Formula I, Formula II, Formula III,
Formula IV, Formula V, and Formula VI and Trp-p8 antagonists of
Formula VII and Formula VIII. Also provided are Trp-p8 antagonists
of Formula I, Formula II, Formula III, Formula IV, Formula V, and
Formula VI and Trp-p8 agonists of Formula VII and Formula VIII.
[0067] Within still further aspects, compositions of the present
invention comprise one or more compound of Formula I, Formula II,
Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/or
Formula VIII formulated together with one or more cancer
therapeutic agent. Alternatively, compositions of the present
invention comprise a compound of Formula I, Formula II, Formula
III, Formula IV, Formula V, Formula VI, Formula VII, and/or Formula
VIII independently formulated with one or more cancer therapeutic
agent. That is, one or more compound of Formula I, Formula II,
Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/or
Formula VIII and the cancer therapeutic agent are separately
formulated.
[0068] Suitable cancer therapeutic agents include, but are not
limited to, antimitotic agents including, but not limited to,
paclitaxel, vincristine, and etoposide; alkylating agents
including, but not limited to, mechlorethamine, cyclophosphamide,
and carmustine; antimetabolites including, but not limited to,
methotrexate, gemcitabine, lometrexol, 5-fluorouracil, and
6-mercaptopurine; cytotoxic antibiotics including, but not limited
to, doxorubicin, daunorubicin, bleomycin, mitomycin C, and
streptozocin; platinum agents including, but not limited to,
cisplatin and carboplatin; hormonal agents including, but not
limited to, anti-estrogens such as tamoxifen and diethylstilbestrol
as well as anti-androgens such as flutamide; antiangiogenesis
agents; and farnesyl transferase inhibitors.
[0069] In certain aspects, compounds of Formula I, Formula II,
Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/or
Formula VIII are administered in combination with cancer
therapeutic agents that are themselves ineffective for modulating
Trp-p8 activity in a cell expressing Trp-p8. Surprisingly, these
types of combination therapy result in enhanced efficacy relative
to the use of a single compound of Formula I, Formula II, Formula
III, Formula IV, Formula V, Formula VI, Formula VII, and/or Formula
VIII alone.
[0070] In other aspects, compounds of Formula I, Formula II,
Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/or
Formula VIII are administered in combination with one or more
additional Trp-p8 modulator including, but not limited to, a
compound of Formula I, Formula II, Formula III, Formula IV, Formula
V, Formula VI, Formula VII, and/or Formula VIII.
[0071] Within certain of these embodiments are provided
small-molecule antagonists of the small-molecule Trp-p8 agonists
presented herein. Thus, within certain embodiments are provided
small-molecule Trp-p8 antagonists of Formula I, Formula II, Formula
III, Formula IV, Formula V, Formula VI, Formula VII, and/or Formula
VIII, and derivatives thereof, of one or more Formula I, Formula
II, Formula III, Formula IV, Formula V, Formula VI, Formula VII,
and/or Formula VIII Trp-p8 agonist.
[0072] Further embodiments of the present invention provide methods
for decreasing cell viability and/or inhibiting cell growth,
methods for stimulating cation influx, and methods for inducing
apoptosis and/or necrosis in a cell expressing Trp-p8. Exemplary
such methods comprise the step of contacting a cell with a compound
of Formula I, Formula II, Formula III, Formula IV, Formula V,
Formula VI, Formula VII, and/or Formula VIII in a concentration and
for a time required to decrease cell viability and/or inhibit cell
growth, to raise intracellular calcium, and/or to induce apoptosis
and/or necrosis of the cell.
[0073] In still further embodiments, the present invention provides
methods for treating a disease in a mammal, most typically a human,
by administering one or more compound and/or composition of the
present invention. In certain aspects, the methods include the
administration of a composition comprising a combination of a
compound of Formula I, Formula II, Formula III, Formula IV, Formula
V, Formula VI, Formula VII, and/or Formula VIE with one or more
cancer therapeutic agent delivered in a simultaneous manner, such
as in a single formulation. In certain other aspects, the methods
of the present invention include combination therapy wherein the
compound of Formula I, Formula II, Formula III, Formula IV, Formula
V, Formula VI, Formula VII, and/or Formula VIII is administered
first in one formulation, followed by the cancer therapeutic agent
in a separate formulation. The methods also include a cancer
therapeutic agent being delivered first in one formulation,
followed by a compound of Formula I, Formula II, Formula III,
Formula IV, Formula V, Formula VI, Formula VII, and/or Formula VIII
in a separate formulation.
[0074] Therapeutic methods of the present invention are
particularly effective in the treatment of cancers associated with
the expression of Trp-p8 including, but not limited to, certain
colon, lung, breast, and prostate cancers.
[0075] The above-mentioned and additional features of the present
invention and the manner of obtaining them will become apparent,
and the invention will be best understood by reference to the
following more detailed description, read in conjunction with the
accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
[0076] FIG. 1 is a graph depicting an exemplary ATP viability
assay. Trp-p8 agonists were tested at 10 .mu.M and agonist-specific
killing of Trp-p8 expressing CHO cells (CHO/Trp-p8) measured at
37.degree. C.
[0077] FIGS. 2A-2C are graphs depicting Trp-p8 agonist-induced
increases in intracellular calcium as determined by a calcium flux
assay performed at 37.degree. C. FIG. 2A is a positive control
demonstrating that CHO and CHO/Trp-p8 cells respond similarly to 1
.mu.M lonomycin at 37.degree. C. in the calcium flux assay. FIG. 2B
is a negative control demonstrating that parental CHO cells that do
not express endogenous or exogenous Trp-p8 do not respond to Trp-p8
agonists even at a concentration of 10 .mu.M. FIG. 2C demonstrates
that the Trp-p8 agonist, designated herein as compound 1603,
induced a specific, concentration-dependent response in CHO/Trp-p8
cells at 37.degree. C.
[0078] FIG. 3 are plots of flow cytometry data demonstrating that a
Trp-p8 agonist is capable of specifically inducing apoptosis in
Trp-p8 expressing CHO cells at 37.degree. C.
[0079] FIG. 4 is a graph depicting exemplary results from a primary
screen for Trp-p8 antagonists using the ATP viability assay,
described herein, with CHO/Trp-p8 cells at 37.degree. C. CHO/Trp-p8
cells were exposed to compounds, at different concentrations, in 1%
DMSO or 1% DMSO in combination with a toxic concentration of the
Trp-p8 agonist 1607. The viability of cells was measured after
24-26 hours at 37.degree. C. using the ATP assay. The compounds
D-2258 and D-2212 protected the cells from the toxic effect of the
Trp-p8 agonist D-1607 and, consequently, are classified as Trp-p8
antagonists. D-2250 had no protective effect and is shown here for
the purpose of illustration of the assay.
[0080] FIG. 5 is a graph depicting the screening and
characterization of Trp-p8 antagonists by the calcium flux assay
performed at 37.degree. C. CHO/Trp-p8 cells were loaded with the
calcium indicator dye, Fura-2, and the increase in intracellular
calcium in response to compounds was determined by the increase in
fluorescence. Fura-2 dye loaded CHO/Trp-p8 cells were exposed to 1%
DMSO or D-2258, at different concentrations, in 1% DMSO at
37.degree. C. Three minutes later, D-1607 was added to the c ells.
When c ells w ere exposed to effective concentrations of the
antagonist, D-2258, their ability to respond to the agonist D-1607
was significantly reduced or eliminated altogether.
[0081] SEQ ID NO: 1 is the nucleotide sequence of a human Trp-p8
cDNA (GenBank Accession No. AY090109).
[0082] SEQ ID NO: 2 is the amino acid sequence encoded by the
nucleotide sequence of SEQ ID NO: 1 (GenBank Accession No.
NP.sub.--076985).
DETAILED DESCRIPTION OF THE INVENTION
[0083] The present invention is based upon the discovery that
certain small-molecule agonists of Trp-p8 activity are capable of
inhibiting the growth of and/or inducing apoptosis and/or necrosis
in cells that express Trp-p8. Without wishing to be limited to any
specific mode of action, it is believed that Trp-p8
agonist-mediated activation of the Trp-p8 receptor substantially
increases cation influx, which is correlative of cellular toxicity.
I t is further believed that Trp-p8 antagonists can inhibit the
basal level and/or native ligand-induced activity of endogenous
Trp-p8 activation which, consequently, leads t or educed growth or
death of cells expressing this cation channel protein.
[0084] Thus, the present invention provides small-molecule Trp-p8
modulators, including agonists and antagonists of Trp-p8 activity,
as well as compositions, including pharmaceutical compositions,
comprising one or more small-molecule Trp-p8 modulator in
combination with one or more pharmaceutically acceptable carrier
and/or excipient. The present invention also provides combination
compositions comprising one or more Trp-p8 modulator and one or
more additional therapeutic compound such as, for example, a cancer
therapeutic agent. Trp-p8 modulators and compositions comprising
Trp-p8 modulators will find utility in methods for activating
Trp-p8-mediated cation influx in a cell, methods for inducing
apoptosis and/or necrosis in a cell, as well as methods for the
treatment of diseases associated with Trp-p8 expression including,
but not limited to, cancers, such as breast, colon, lung, and
prostate cancers.
DEFINITIONS
[0085] The term "Trp-p8 modulators" refers collectively to
small-molecule agonists and antagonists that bind to and either
increase or decrease, respectively, the activity of Trp-p8 in a
cell. Trp-p8 agonists include compounds of Formula I, Formula II,
Formula III, Formula IV, Formula V, Formula VI, Formula VII, and
Formula VIII and are exemplified herein by various compounds of
Formulas I-VI, and chemical derivatives thereof. Trp-p8 antagonists
include compounds of Formula I, Formula II, Formula III, Formula
IV, Formula V, Formula VI, Formula VII, and Formula VIII and are
exemplified herein by various compounds of Formulas VII-VIII, and
chemical derivatives thereof. Additional Trp-p8 agonists or
antagonists of Formula I, Formula II, Formula III, Formula IV,
Formula V, Formula VI, Formula VII, and Formula VIII, not
specifically exemplified herein, may be readily synthesized and
characterized by a skilled artisan by employing the methodology
expressly provided herein and/or as is readily available in the
art.
[0086] The phrase "activate Trp-p8" means agonist-mediated
activation of Trp-p8 expressed on the surface of a cell. For
example, within certain embodiments, agonists of the present
invention, when contacted with a cell and/or administered in vivo
to a mammalian subject, activate Trp-p8 thereby facilitating the
influx of cations, such as calcium ions, to such an intracellular
level and/or for such a duration that is sufficient to cause
toxicity to the cell as evidenced by a diminution in cell growth
and/or an onset of necrotic and/or apoptotic cell death.
[0087] The term "aliphatic" is intended to include any
straight-chained, branched-chained, or cyclic group free of
aromatic unsaturation, and thus embraces alkyl, cycloalkyl,
alkenyl, cycloalkenyl, alkynyl, hydroxyalkyl, acyloxyalkyl, alkoxy,
alkoxyalkyl, aminoalkyl, acylaminoalkyl, carboxyalkyl, and similar
combinations.
[0088] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon group, or combination thereof, which
may be fully saturated, mono or polyunsaturated and can include di
and multivalent groups, having the number of carbon atoms
designated (i.e. C.sub.1-C.sub.10 means one to ten carbons).
Examples of saturated hydrocarbon groups include groups such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,
sec-butyl, cyclohexyl, (cyclohexyl)ethyl, cyclopropylmethyl,
homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl,
n-octyl, and the like. An unsaturated alkyl group is one having one
or more double bonds or triple bonds. Examples of unsaturated alkyl
groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl,
2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
[0089] The term "alkenyl" denotes branched or unbranched
hydrocarbon chains containing one or more carbon-carbon double
bonds.
[0090] The term "alkynyl" refers to branched or unbranched
hydrocarbon chains containing one or more carbon-carbon triple
bonds.
[0091] The term "alkylene" by itself or as part of another
substituent means a divalent group derived from an alkane, as
exemplified by --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. Typically, an
alkylene group will have from 1 to 24 carbon atoms, with those
groups having 10 or fewer carbon atoms being preferred in the
present invention. A "lower alkyl" or "lower alkylene" is a shorter
chain alkyl or alkylene group, generally having eight or fewer
carbon atoms.
[0092] The term "cycloalkylene" by itself or as part of another
substituent means a divalent group derived from a cycloalkane, as
exemplified by cyclohexylene. Typically, a cycloalkylene group will
have from 5-8 carbon atoms, with those groups having 6 carbon atoms
being preferred in the present invention.
[0093] The term "alkenylene" by itself or as part of another
substituent means a divalent group derived from an alkenyl, as
exemplified by --CH.dbd.CHCH.sub.2CH.sub.2--. Typically, alkenylene
groups will have from 2 to 24 carbon atoms, with those groups
having 10 or fewer carbon atoms being preferred in the present
invention.
[0094] The terms "alkoxy," "alkylamino" and "alkylthio" refer to
those groups having an alkyl group attached to the remainder of the
molecule through an oxygen, nitrogen or sulfur atom, respectively.
Similarly, the term "dialkylamino" is used in a conventional sense
to refer to --NR'R'' wherein the R groups can be the same or
different alkyl groups.
[0095] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon, or combinations thereof,
fully saturated or containing from 1 to 3 degrees of unsaturation,
consisting of the stated number of carbon atoms and from one to
three heteroatoms selected from the group consisting of O, N, Si
and S, and wherein the nitrogen and sulfur atoms may optionally be
oxidized and the nitrogen heteroatom may optionally be quaternized.
The heteroatom(s) O, N and S may be placed at any interior position
of the heteroalkyl group. The heteroatom Si may be placed at any
position of the heteroalkyl group, including the position at which
the alkyl group is attached to the remainder of the molecule.
Examples include --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3--)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3,
--CH.sub.2--CH.sub.2--S(O)--CH.sub.3,
--CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.3. Also included in the term
"heteroalkyl" are those groups described in more detail below as
"heterocycloalkyl." The term "heteroalkylene" by itself or as part
of another substituent means a divalent group derived from
heteroalkyl, as exemplified by
--CH.sub.2--CH.sub.2--S--CH.sub.2CH.sub.2-- and
--CH.sub.2--S--CH.sub.2CH.sub.2--NH--CH.sub.2--. For heteroalkylene
groups, heteroatoms can also occupy either or both of the chain
termini. Still further, for alkylene and heteroalkylene linking
groups, no orientation of the linking group is implied.
[0096] The term "acyl" refers to those groups derived from an
organic acid by removal of the hydroxy portion of the acid.
Accordingly, acyl is meant to include, for example, acetyl,
propionyl, butyryl, decanoyl, pivaloyl, benzoyl and the like.
[0097] An "activated carbonyl" group is a carbonyl group whose
electrophilicity is enhanced as a result of the groups attached to
either side of the carbonyl. Examples of such activated carbonyl
groups are (polyfluoroalkyl)ketones, (polyfluoroalkyl)aldehydes,
alpha-keto esters, alpha-keto acids, alpha-keto amides,
1,2-diketones, 2-acylthiazoles, 2-acylimidazoles, and the like.
[0098] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include cyclopentyl,
cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the
like. Examples of heterocycloalkyl include
1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,
tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,
1-piperazinyl, 2-piperazinyl, and the like.
[0099] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"fluoroalkyl," are meant to include monofluoroalkyl and
polyfluoroalkyl.
[0100] The term "aryl," employed alone or in combination with other
terms (e.g., aryloxy, arylthioxy, arylalkyl) means, unless
otherwise stated, an aromatic substituent which can be a single
ring or multiple rings (up to three rings) which are fused together
or linked covalently. The term "heteroaryl" is meant to include
those aryl rings which contain from zero to four heteroatoms
selected from N, O, and S, wherein the nitrogen and sulfur atoms
are optionally oxidized, and the nitrogen atom(s) are optionally
quaternized. The "heteroaryl" groups can be attached to the
remainder of the molecule through a heteroatom. Non-limiting
examples of aryl and heteroaryl groups include phenyl, 1-naphthyl,
2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,
3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,
4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,
4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,
2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl,
4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl ring systems are
selected from the group of acceptable substituents described below.
The term "arylalkyl" is meant to include those groups in which an
aryl or heteroaryl group is attached to an alkyl group (e.g.,
benzyl, phenethyl, pyridylmethyl and the like) or a heteroalkyl
group (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0101] Each of the above terms (e.g., "alkyl," "heteroalkyl" and
"aryl") are meant to include both substituted and unsubstituted
forms of the indicated group. Preferred substituents for each type
of group are provided below.
[0102] Substituents for the alkyl and heteroalkyl groups (including
those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a
variety of groups selected from: --OR', .dbd.O, .dbd.NR',
.dbd.N--OR', --NR'R'', --SR', -halogen, --SiR'R''R''', --OC(O)R',
--C(O)R', --CO.sub.2R', CONR'R'', --OC(O)NR'R'', --NR''C(O)R',
--NR'--C(O)NR''R''', --NR''C(O).sub.2R', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
S(O).sub.2R', --S(O).sub.2NR'R'', --CN and --NO.sub.2 in a number
ranging from zero to (2N+1), where N is the total number of carbon
atoms in such group. R', R'' and R''' each independently refer to
hydrogen, unsubstituted(C.sub.1-C.sub.8)alkyl and heteroalkyl,
unsubstituted aryl, aryl substituted with 1-3 halogens,
unsubstituted alkyl, alkoxy or thioalkoxy groups, or
aryl-(C.sub.1-C.sub.4)alkyl groups. When R' and R'' are attached to
the same nitrogen atom, they can be combined with the nitrogen atom
to form a 5-, 6-, or 7-membered ring. For example, --NR'R'' is
meant to include 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0103] Similarly, substituents for the aryl groups are varied and
are selected from: -halogen, --OR', --OC(O)R', --NR'R'', --SR',
--R', --CN, --NO.sub.2, --CO.sub.2R', --CONR'R'', --C(O)R',
--OC(O)NR'R'', --NR''C(O)R', --NR''C(O).sub.2R',
--NR'--C(O)NR''R''', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --NR''--S(O).sub.2--R',
--N.sub.3, --CH(Ph).sub.2, perfluoro(C.sub.1-C.sub.4)alkoxy, and
perfluoro(C.sub.1-C.sub.4)alkyl, in a number ranging from zero to
the total number of open valences on the aromatic ring system; and
where R', R'' and R''' are independently selected from hydrogen,
(C.sub.1-C.sub.8)alkyl and heteroalkyl, unsubstituted aryl,
(unsubstituted aryl)-(C.sub.1-C.sub.4)alkyl, and (unsubstituted
aryl)oxy-(C.sub.1-C.sub.4)alkyl.
[0104] Two of the substituents on adjacent atoms of the aryl ring
may optionally be replaced with a substituent of the formula
-T--C(O)--(CH.sub.2).sub.q--U--, wherein T and U are independently
--NH--, --CH.sub.2-- or a single bond, and the subscript q is an
integer of from 0 to 2. Alternatively, two of the substituents on
adjacent atoms of the aryl ring may optionally be replaced with a
substituent of the formula -A-(CH.sub.2).sub.r--B--, wherein A and
B are independently --CH.sub.2--, --NH--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2NR'-- or a single bond, and r is an integer of from 1
to 3. One of the single bonds of the new ring so formed may
optionally b e replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of the aryl ring may optionally
be replaced with a substituent of the formula
--(CH.sub.2).sub.s--X--(CH.sub.2).sub.t--, where s and t are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The substituent R'
in --NR'-- and --S(O).sub.2NR'-- is selected from hydrogen or
unsubstituted (C.sub.1-C.sub.6)alkyl.
[0105] As used herein, the term "heteroatom" is meant to include
oxygen (O), nitrogen (N), and sulfur (S).
[0106] The term "pharmaceutically acceptable salts" is meant to
include salts of the active compounds of Formula I, Formula II,
Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/or
Formula VIII that are prepared with relatively nontoxic acids or
bases, depending on the particular substituents found on the
compounds described herein. Examples of pharmaceutically acceptable
base addition salts include, but are not limited to, sodium,
potassium, calcium, ammonium, organic amino, magnesium salt, or
other similar salt. Examples of pharmaceutically acceptable acid
addition salts include, but are not limited to, those derived from
inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,
monohydrogencarbonic, phosphoric, monohydrogenphosphoric,
dihydrophosphoric, sulfuric, monohydrogensulfuric, hydriodic, or
phosphorous acids and the like, as well as the salts derived from
relatively nontoxic organic acids like acetic, propionic,
isobutyric, oxalic, maleic, malonic, benzoic, succinic, suberic,
fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic,
citric, tartaric, methanesulfonic, and the like.
Small-Molecule Modulators of Trp-p8 Activity
[0107] Small-molecule Trp-p8 modulators that are suitably employed
in the compositions and methods of the present invention are
exemplified herein by the following Trop-p8 agonist compounds:
Menthol (Sigma-Aldrich, Inc.; St. Louis, Mo.;
(2-isopropyl-5-methyl-cyclohexanol)); Frescolat ML (Harris &
Ford, LLC; Indianapolis, Ind.; Menthyl lactate); Frescolat MGA
(Harris & Ford, LLC; Menthone Glycerin Acetal); L-Menthyl
Acetate (Millennium Chemicals; Olympia Fields, Ill.;
Cyclohexanol-5-methyl-2-(1-methylethyl)-acetate-[1R-(1alpha,2beta,5
alpha)]-); L-Carvone (Millennium Chemicals;
(R)-5-Isopropenyl-2-methyl-2-cyclohexenone
p-Mentha-6,8-dien-2-one); WS-3 (Millennium Chemicals;
N-Ethyl-p-menthane-3-carboxamide aka cyclohexanecarboxamide,
N-ethyl-5-methyl-2(1-methylethyl)); Cooling Agent 10 (Takasago
International Corp.; Rockleigh, N.J.;
(1)-Menthoxypropane-1,2-diol); Coolact P.RTM. (Takasago
International Corp.; (-)-Isopulegol); Coolact 38D.RTM. (Takasago
International Corp.); Icilin
(1-(2-hydroxyphenyl)-4-(3-nitrophenyl)-1,2,3,6-tetrahydropyrimidin-
e-2-one aka
3,4-dihydro-3-(2-hydroxyphenyl)-6-(3-nitrophenyl)-(1H)-pyrimidin-2-one);
WS-23 (2-Isopropyl-N,2,3-trimethylbutyramide aka
N,2,3-trimethyl-2-isopropyl butamide), and WS-12
(N-(4-methoxyphenyl)-p-menthan-3-carboxamide aka
cyclohexanecarboxamide,
N-(4-methoxyphenyl)-5-methyl-2(1-methylethyl)).
[0108] The present invention further contemplates that additional
Trp-p8 agonists, including derivatives of the compounds of Formulas
I, II, III, IV, V, VI, VII, and/or VIII disclosed herein, may also
be suitably employed in the compositions and methods of the present
invention.
[0109] Thus, in one embodiment, the present invention provides the
following Trp-p8 modulators and derivatives thereof:
[0110] A compound of Formula I
##STR00016##
wherein
[0111] R.sup.1 is selected from the group consisting of H, OH,
CH.sub.3, CH.sub.3--CH--CH.sub.3 (isopropyl), and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl);
[0112] R.sup.2 is selected from the group consisting of H;
[0113] R.sup.3 is selected from the group consisting of O, OH,
acetate, lactate, carboxamide, butanamide, sulphanamide, and
propanetriol; and
[0114] R.sup.4 is selected from the group consisting of
CH.sub.3--CH--CH.sub.3 (isopropyl), isopropane-2-ol, and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl).
[0115] Exemplary compounds of Formula I include the following
Trp-p8 agonists:
##STR00017##
[0116] These exemplary Formula I Trp-p8 agonists have the following
trade and chemical names: Menthol
(2-isopropyl-5-methyl-cyclohexanol) (Sigma-Aldrich, Inc.; St.
Louis, Mo.); Frescolat ML (Harris & Ford, LLC; Indianapolis,
Ind.; Menthyl lactate); L-Menthyl Acetate (Millennium Chemicals;
Olympia Fields, Ill.;
Cyclohexanol-5-methyl-2-(1-methylethyl)-acetate-[1R-(1
alpha,2beta,5alpha)]-); Cooling Agent 10 (Takasago International
Corp.; Rockleigh, N.J.; (1)-Menthoxypropane-1,2-diol); Coolact
P.RTM. (Takasago International Corp.; (-)-Isopulegol); and Coolact
38D.RTM. (Takasago International Corp.).
[0117] In another embodiment, the present invention further
provides the following small-molecule Trp-p8 modulators and
derivatives thereof:
[0118] A compound of Formula II
##STR00018##
wherein
[0119] R.sup.5 is selected from the group consisting of H, OH,
CH.sub.3, CH.sub.3--CH--CH.sub.3 (isopropyl), and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl);
[0120] R.sup.6 is selected from the group consisting of N;
[0121] R.sup.7 is selected from the group consisting of O and
N;
[0122] R.sup.8 is selected from the group consisting of NH, O, and
S; and
[0123] R.sup.9 is selected from the group consisting of
NO.sub.2.
[0124] Compounds of Formula II are exemplified herein by the Trp-p8
agonist Icilin
(1-(2-hydroxyphenyl)-4-(3-nitrophenyl)-1,2,3,6-tetrahydropyrimidine-2-one
aka
3,4-dihydro-3-(2-hydroxyphenyl)-6-(3-nitrophenyl)-(1H)-pyrimidin-2-on-
e).
##STR00019##
[0125] In another embodiment, the present invention further
provides the following acyclic carboxamide Trp-p8 modulators and
derivatives thereof as presented in U.S. Pat. No. 4,153,679,
incorporated herein by reference:
[0126] A compound of Formula III
##STR00020##
wherein
[0127] R.sup.10 is selected from the group consisting of H and a
C.sub.1-C.sub.5 alkyl including, but not limited to, CH.sub.3,
C.sub.2H.sub.5, CH.sub.3--CH--CH.sub.3 (isopropyl), and
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl);
[0128] R.sup.11 is selected from the group consisting of OH,
carboxamide, butanamide, propanetriol, and CONR'R'', wherein R' is
selected from the group consisting of H, CH.sub.3, C.sub.2H.sub.5,
C.sub.4H.sub.8 (cyclobutyl), and C.sub.4H.sub.8O, and wherein R''
is selected from the group consisting of C.sub.2H.sub.5OOCH.sub.2,
C.sub.2H.sub.5, CH.sub.3--CH--CH.sub.3 (isopropyl),
HOCH.sub.2C(CH.sub.3).sub.2, HOCH.sub.2CH.sub.2, C.sub.4H.sub.9
(tertbutyl), C.sub.4H.sub.9 (secbutyl);
[0129] R.sup.12 is selected from the group consisting of H and a
C.sub.1-C.sub.5 alkyl including, but not limited to, CH.sub.3,
CH.sub.3--CH--CH.sub.3 (isopropyl), and CH.sub.3--C.dbd.CH.sub.2
(isopropenyl), C.sub.4H.sub.9 (secbutyl), C.sub.4H.sub.9
(isobutyl), C.sub.4H.sub.9 (n-butyl), C.sub.5H.sub.11 (isohexyl);
and
[0130] R.sup.13 is selected from the group consisting of H and a
C.sub.1-C.sub.5 alkyl including, but not limited to, CH.sub.3,
C.sub.2H.sub.5, CH.sub.3--CH--CH.sub.3 (isopropyl),
CH.sub.3--C.dbd.CH.sub.2 (isopropenyl), C.sub.4H.sub.9 (secbutyl),
C.sub.4H.sub.9 (isobutyl).
[0131] Compounds of Formula III are exemplified herein by the
Trp-p8 agonist WS-23 (2-Isopropyl-N,2,3-trimethylbutyramide aka
N,2,3-trimethyl-2-isopropyl butamide).
##STR00021##
[0132] In another embodiment, the present invention further
provides the following 3-substituted-p-menthane Trp-p8 modulators
and derivatives thereof as presented in U.S. Pat. No. 4,150,052,
incorporated herein by reference:
[0133] A compound of Formula IV
##STR00022##
wherein
[0134] R.sup.14 is selected from the group consisting of H and an
aliphatic group containing up to 25 carbon atoms;
[0135] R.sup.15 is selected from the group consisting of H, OH and
an aliphatic group containing up to 25 carbon atoms, with the
proviso that when R.sup.15 is H, R.sup.14 may also be an aryl group
of up to 10 carbon atoms and selected from the group consisting of
substituted phenyl, phenalkyl, substituted phenalkyl, naphthyl,
substituted naphthyl, and pyridyl; and
[0136] R.sup.14 and R.sup.15, when taken together with the nitrogen
atom to which they are attached, may form a cyclic or heterocyclic
group of up to 25 carbon atoms, e.g., a piperidino or a morpholino
group. Exemplary such cyclical groups may be selected from the
group consisting of 3-phenyl-piperidin-1-yl,
3-phenyl-pyrrolidin-1-yl,
6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-yl, and
4-pyrimidin-2-yl-piperazin-1-yl.
[0137] Typical values for R.sup.14 and R.sup.15 when aliphatic are
methyl, ethyl, propyl, butyl, isobutyl, n-decyl, cyclopropyl,
cyclohexyl, cyclopentyl, cycloheptylmethyl, 2-hydroxyethyl,
3-hydroxy-n-propyl, 6-hydroxy-n-hexyl, 2-aminoethyl,
2-acetoxyethyl, 2-ethylcarboxyethyl, 4-hydroxybut-2-ynyl, and
carboxymethyl.
[0138] When R.sup.14 is aryl, typical values are benzyl, naphthyl,
4-methoxyphenyl, 2-methoxy-4-methoxyphenyl,
3-methoxy-5-methoxyphenyl, 4-methyl-5-chlorophenyl,
4-hydroxyphenyl, 4-methylphenyl, 3-methyl-4-methylphenyl,
3-hydroxy-4-methylphenyl, 4-fluorophenyl, 4-chlorophenyl,
4-bromophenyl, 4-ethylphenyl, 2-fluoro-4-fluorophenyl,
4-nitrophenyl, 2-hydroxynaphthyl, pyridyl,
[1-carbamoyl-2-(1H-indol-3-yl)-ethyl,
1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl,
1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl,
1-carbamoyl-2-(4-hydroxyphenyl)ethyl, 1-carbamoyl-2-phenylethyl,
1-hydroxymethyl-2-(1H-indol-3-yl)ethyl,
1-hydroxymethyl-2-(4-chlorophenyl)ethyl,
1-hydroxymethyl-2-(4-hydroxyphenyl)ethyl,
1-hydroxymethyl-2-hydroxy-2-phenylethyl, 1-hydroxymethyl-2-phenyl
ethyl, 1-methoxymethyl-2-phenylethyl,
1-methyl-2-(4-chlorophenyl)-ethyl,
1-methyl-2-(5-fluoro-1H-indol-3-yl)-ethyl,
1-methyl-2-hydroxy-2-phenylethyl, 1-oxo-2-phenylethyl,
1-phenyl-cyclopentylmethyl,
2-(1-cyclopentyl-pyrrolidin-3-yl)-ethyl, 2-(1H-indol-3-yl)ethyl,
2-(2,3-dimethoxyphenyl)ethyl, 2-(2,4-dichlorophenyl)ethyl,
2-(2,4-dimethylphenyl)ethyl, dimethoxyphenyl)ethyl,
2-(2,5-dimethylphenyl)-ethyl, 2-(2,6-dimethylphenyl)ethyl,
2-(2-chloro-6-fluorophenyl)ethyl, 2-(2-chlorophenyl)ethyl,
2-(2-fluorophenyl)ethyl, 2-(2-furyl)ethyl,
2-(2-methoxy-5-bromophenyl)ethyl, 2-(2-methoxyphenyl)-ethyl,
2-(2-methylphenyl)ethyl, 2-(3,4-dichlorophenyl)ethyl,
2-(3,4-dimethoxyphenyl)ethyl, dimethoxyphenyl)ethyl,
2-(3-bromo-4-methoxyphenyl)ethyl, 2-(3-chlorophenyl)ethyl,
2-(3-ethoxyphenyl)ethyl, 2-(3-fluorophenyl)ethyl,
2-(3-hydroxy-4-methoxyphenyl)-ethyl, 2-(3-hydroxyphenyl)ethyl,
2-(3-methoxy-4-ethoxyphenyl)ethyl,
2-(3-methoxy-4-hydroxyphenyl)ethyl, 2-(3-methoxyphenyl)ethyl,
2-(3-methylphenyl)ethyl, 2-(3-trifluoromethylphenyl)ethyl,
2-(4-bromophenyl)-ethyl, 2-(4-chlorophenyl)ethyl,
2-(4-ethylphenyl)ethyl, 2-(4-fluorophenyl)ethyl,
2-(4-hydroxyphenyl)ethyl, 2-(4-methoxy-phenyl)-2-oxo-ethyl,
2-(4-methoxyphenyl)ethyl, 2-(4-methylphenyl)ethyl,
2-(4-methylphenyl)ethyl, 2-(4-methylsulfanylphenyl)ethyl,
2-(4-nitrophenyl)ethyl, 2-(4-sulfamoyl-phenyl)-ethyl,
2-(5-methoxy-1H-indol-3-yl)-ethyl,
2-(6-fluoro-1H-indol-3-yl)-ethyl,
2-(6-methoxy-1H-indol-3-yl)-ethyl,
2-(7-methyl-1H-indol-3-yl)-ethyl, 2-(N,N-dipropylamino)ethyl,
2-(pyridin-2-yl)-ethyl, 2-(pyridin-3-yl)-ethyl,
2-(pyridin-4-yl)-ethyl, 2,2-diphenylethyl, 2,3-difluorobenzyl,
2,3-dimethoxyphenyl, 2,4-dichlorophenyl, 2,4-difluorophenyl,
2,4-dimethoxyphenyl, 2,4-dimethylphenyl, 2-bromo-4-methylphenyl,
2-chloro-4-cyanophenyl, 2-chloro-4-fluorophenyl,
2-chloro-4-iodophenyl, 2-chloro-4-nitrophenyl,
2-chloro-5-nitrophenyl, 2-chlorophenyl, 2-cyclohex-1-enyl-ethyl,
2-fluoro-4-chlorophenyl, 2-fluoro-5-nitrophenyl,
2-hydroxy-2-(3-hydroxyphenyl)ethyl,
2-hydroxy-2-(4-hydroxyphenyl)ethyl, 2-hydroxy-2-phenylethyl,
2-iodophenyl, 2-methyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl,
2-methyl-2-phenylethyl, 2-methyl-4-bromophenyl,
2-methyl-5-nitrophenyl, 2-methylphenyl, 2-nitro-4-fluorophenyl,
2-nitrophenyl, 2-oxo-2,3-dihydro-1H-benzoimidazol-5-yl,
2-oxo-2-phenylethyl, 2-phenyl-1H-benzoimidazol-5-yl,
2-phenyl-2-(4-chlorophenyl)ethyl,
2-phenyl-2-(4-fluorophenyl)-ethyl,
2-phenyl-2-(4-methoxyphenyl)ethyl, 2-phenylethenyl, 2-phenylethyl,
2-pyridin-2-yl-benzooxazol-5-yl,
2-pyridin-3-yl-1H-benzoimidazol-5-yl, 2-thiophen-2-yl-ethyl),
2-trifluoromethyl-1H-benzoimidazol-5-yl, 3,4,5-trifluorophenyl,
3,4,5-trimethoxyphenyl, 3,4-cyclopentanephenyl, 3,4-dichlorophenyl,
3,4-dimethylphenyl, 3,5-dimethoxyphenyl, 3-acetamidophenyl,
3-bromo-4-methylphenyl, 3-carboxamidophenyl,
3-chloro-4-methoxyphenyl, 3-chloro-4-methylphenyl,
3-chloro-4-morpholin-4-yl-phenyl, 3-hydroxymethylphenyl,
3-nitrophenyl, 3-oxo-indan-5-yl, 3-phenylpropyl, 3-yl-acetophenone,
4-(1,1-dioxo-1l6-thiomorpholin-4-ylmethyl)-phenyl,
4-(1-hydroxyethyl)phenyl, 4-(2-hydroxyethyl)phenyl,
4-(4-ethyl-piperazin-1-yl)-phenyl,
4-(4-methyl-1H-benzoimidazol-2-yl)-phenyl,
4-(4-methyl-piperazin-1-ylmethyl)-phenyl,
4-(morpholine-4-sulfonyl)-phenyl,
4-[1,3-dioxo-2-(2-trifluoromethyl-phenyl)-2,3-dihydro-1H-isoindol-5-yloxy-
]-phenyl,
4-[2-(2-methoxy-phenyl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-ylox-
y]-phenyl,
4-{2-[2-(3,4-dimethoxy-phenyl)-ethyl]-1,3-dioxo-2,3-dihydro-1H--
isoindol-5-yloxy}-phenyl, 4-acetylphenyl,
4-azepan-1-ylmethyl-phenyl, 4-benzooxazol-2-yl-phenyl, 4-Biphenyl,
4-bromophenyl, 4-carboxamidophenyl, 4-chlorophenyl, 4-cyanophenyl,
4-ethoxyphenyl, 4-ethylphenyl, 4-fluorophenyl,
4-hydroxymethylphenyl, 4-methoxyphenyl,
4-methyl-2-oxo-2H-chromen-7-yl, 4-methylcyclohexyl, 4-methylphenyl,
4-methylsulfanylphenyl, 4-nitrobenzyl,
4-pyrrolidin-1-ylmethyl-phenyl, 4-trifluoromethylphenyl,
benzo[1,3]dioxol-5-yl, benzoylamino, benzyloxy,
bicyclo[2.2.1]hept-2-yl, C-1H-indazol-5-yl, cycloheptyl,
indan-2-yl, N-(2-diethylamino-ethyl)-benzamide-4-yl,
N'-quinoxalin-2-yl-amino, and phenylcyclopropyl.
[0139] Trp-p8 modulators of Formula IV are exemplified herein by
the Trp-p8 agonists WS-3 (Millennium Chemicals;
N-Ethyl-p-menthane-3-carboxamide aka cyclohexanecarboxamide,
N-ethyl-5-methyl-2(1-methylethyl)) and by WS-12
(N-(4-methoxyphenyl)-p-menthan-3-carboxamide aka
cyclohexanecarboxamide,
N-(4-methoxyphenyl)-5-methyl-2(1-methylethyl)), and the compounds
presented in Table 1.
##STR00023##
[0140] In a further embodiment, the present invention provides the
following Trp-p8 modulators and derivatives thereof that comprise
at least one ketal moiety, including, but not limited to the Trp-p8
agonists 1-menthone glycerol ketal and 3,3,5-trimethylcyclohexaone
glycerol ketal presented in U.S. Pat. No. 5,266,592, incorporated
herein by reference:
[0141] A compound comprising at least one ketal of Formula V
##STR00024##
wherein
[0142] R.sup.16 is selected from the group consisting of a
C.sub.2-C.sub.6 alkylene group having at least one, but not more
than 3, hydroxyl group(s), preferably 1 hydroxyl group, and
[0143] Either R.sup.17 and R.sup.18 independently of one another
represent C.sub.1-C.sub.10-alkyl which is optionally substituted by
1 to 3 groups selected from the group consisting of hydroxyl,
amino, thio, and halogen (e.g., fluorine, chlorine, bromine, or
iodine), C.sub.5-C.sub.7-cycloalkyl, preferably cyclohexyl,
C.sub.6-C.sub.12-aryl, preferably phenyl, with the proviso that the
total of the C atoms of R.sup.17 and R.sup.18 is not less than 3,
or
[0144] R.sup.17 and R.sup.18 together represent an alkylene group
that, together with the carbon atom that carries the groups
R.sup.17 and R.sup.18, forms a 5-7-membered ring, it being possible
for this alkylene group, in turn, to be substituted by
C.sub.1-C.sub.6-alkyl groups.
[0145] Preferred groups R.sup.17 and R.sup.18 comprise methyl,
isopropyl, and tert-butyl.
[0146] Compounds comprising a ketal of Formula V include the
following Trp-p8 modulator compounds wherein R.sup.16 is as defined
above:
##STR00025##
[0147] Compounds comprising a ketal of Formula V are exemplified
herein by the Trp-p8 agonist Frescolat MGA (Harris & Ford, LLC;
Menthone Glycerin Acetal),
##STR00026##
[0148] In yet a further embodiment, the present invention provides
Formula VI small-molecule Trp-p8 modulators and derivatives thereof
exemplified herein by L-Carvone (Millennium Chemicals;
(R)-5-Isopropenyl-2-methyl-2-cyclohexenone
p-Mentha-6,8-dien-2-one),
##STR00027##
[0149] Still further embodiments of the present invention provide
small-molecule antagonists of the small-molecule Trp-p8 agonists
presented herein. Thus, within certain embodiments are provided
Formula I, Formula II, Formula III, Formula IV, Formula V, Formula
VI, Formula VII, and/or Formula VIII small-molecule antagonists,
and derivatives thereof, of the Formula I, Formula II, Formula III,
Formula IV, Formula V, Formula VI, Formula VII, and/or Formula VIII
Trp-p8 agonists disclosed herein above.
[0150] Presented herein in the Examples are specific Trp-p8
antagonists that are exemplary of the Trp-p8 modulators of Formula
VII
##STR00028##
wherein
[0151] R.sup.17 is selected from the group consisting of 2-pyridyl,
2-nitro-4-trifluoromethylphenyl, 2-nitro-4-chlorophenyl,
2-methoxyphenyl, 2-chlorophenyl, phenyl, 2-methyl-quinolin-3-yl,
4-methoxyphenyl, 4-fluorophenyl,
3-azepan1-yl-5-(4-trifluoromethoxy)phenylamino[1,3,5]triazyl,
cyclohexyl, diphenylmethyl, 2-phenylethyl, 4-hydroxy-cyclohexyl,
cycloheptyl, cyclopentyl, C-benzo[1,3]dioxol-5-yl-methyl,
2-pyridyl, and 4-chlorobenzyl;
[0152] R.sup.18 is selected from the group consisting of
1-benzyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,
3-benzylamino-2-nitrophenyl, 5-nitro-quinolin-8-yl,
1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol,
1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,
benzyl-2-methyl-quinazolin-4-yl,
3-methyl-5-morpholin-4-yl-2-nitro-phenyl,
2-nitro-5-piperazin-1-yl-ethanol,
1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol,
4-(2,5-dimethyl-pyrrol-1-yl)-2-nitro-phenyl,
2-nitro-3-trifluoromethanesulfonyl-phenyl,
1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl,
2-(2-Fluoro-phenoxymethyl)-2-cyano oxazolyl, adamantly,
5-(benzo[1,3]dioxol-5-ylamino)-10b,10c-dihydro-anthra[1,9-cd]isoxazol-6-o-
ne-yl, 2-methyl-thiazolo[3,2-b][1,2,4]triazol-6-ol 4-methylphenyl
methyl, 3-benzyl-3H-quinazolin-4-one-2-yl, cyclopentyl,
tetrahydronapthyl, cyclooctyl, cyclohexyl,
C-[3-(4-chloro-phenyl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidin-7-yl]-methyl-
,
C-(2-benzyl-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-me-
thyl, and
1-yl-3-(2-isopropyl-5-methyl-cyclohexyloxy)-propan-2-ol;
[0153] R.sup.19 and R.sup.20 are each independently selected from
the group consisting of H and O; and
[0154] R.sup.21 is selected from the group consisting of
4-methylphenyl, 2-chloro-4-fluorophenyl, and 4-chlorophenyl.
[0155] In other embodiments, the present invention provides Formula
VIII small-molecule modulators, and derivatives thereof, including
Formula VIII antagonists of the Formula IV Trp-p8 agonists
disclosed herein above and in the Examples.
[0156] A compound of Formula VIII
##STR00029##
wherein
[0157] R.sup.22 is a linker moiety, which may be selected from the
group consisting of oxyacetamide, urea, carbamate, thiourea,
sulfonamide, amine, amide. Formula VIII antagonists are represented
by the following sub-formulae (Formula VIII-A, Formula VIII-B,
Formula VIII-C, Formula VIII-D, Formula VIII-E, Formula VIII-F, and
Formula VIII-G):
##STR00030##
[0158] Irrespective of which of the seven R.sup.22 linker moieties
is employed, R.sup.23 is selected from the group consisting of H,
tetrahydro isoquinolinyl, tetrahydro quinolinyl, 3-methyl
indolinyl, indolinyl, 2-(N-methyl, N-phenylethyl)amino ethyl,
3-methyl indolinyl, 1-phenyl ethyl, 2-chloro benzyl,
2-methoxybenzyl, 2-methoxyphenyl, 2-cyclohex-1-enyl ethyl,
(1-phenyl-cyclophenyl)-methyl, 2-(tetrahydroquinolinyl)-ethyl,
1-propyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, cycloheptyl,
3-cyclohexylsulfanylpropyl, 2-cyclohex-1-enyl ethyl,
2-(N-isopropyl,N-phenylethyl)amino ethyl,
1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine,
2-cyclopentylethyl, 2-phenylcyclopropyl, 1-phenoxyethyl,
4-butyloxyphenyl, (2-nitrophenoxy)methyl,
4,7,7-trimethyl-2-oxa-bicyclo[2.2.1]heptan-3-one,
C-(1-phenyl-5-propyl-1H-pyrazol-4-yl)-methyl, benzyl,
2-chlorobenzyl,
1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-met-
hoxy-phenyl]-2,3,4,9-tetrahydro-1H-b-carboline,
C-[3-(4-butoxy-phenyl)-1H-pyrazol-4-yl]-methyl,
4-(azepane-1-sulfonyl)-phenyl, and
547-chloro-quinolin-4-ylsulfanyl)-[1,3,4]thiadiazol-2-yl;
[0159] R.sup.24 is selected from the group consisting of H,
tetrahydro isoquinolinyl, tetrahydro quinolinyl, 3-methyl
indolinyl, indolinyl, 3-methyl indolinyl,
1-propyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine,
1-methyl-1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine, and
1-[3-(6,7-dimethoxy-1-methyl-3,4-dihydro-1H-isoquinolin-2-ylmethyl)-4-met-
hoxy-phenyl]-2,3,4,9-tetrahydro-1H-b-carboline; and
[0160] R.sup.25 is selected from the group consisting of H.
Synthesis of Small-Molecule Trp-p8 Modulators
[0161] As noted above, compounds of the present invention include
compounds of Formula I, Formula II, Formula III, Formula IV,
Formula V, Formula VI, Formula VII, and Formula VIII. Within
certain aspects, compounds of the present invention can be obtained
from commercial sources and/or can be made using commercially
available starting materials by employing synthetic methodology
readily available in the art. Compounds of Formulae I-VIII may be
isolated using typical isolation and purification techniques known
in the art, including, for example, chromatographic and
recrystallization methods.
[0162] Exemplary compositions and methodology for their synthesis
are disclosed in the following patents, each of which is
incorporated herein by reference: U.S. Pat. No. 4,150,052 discloses
N-substituted p-menthane 3-carboxyamide compounds having a
physiological cooling action on the skin; U.S. Pat. No. 4,153,679
discloses compositions comprising acyclic tertiary and secondary
carboxamides that have a physiological cooling action on the skin;
U.S. Pat. No. 4,020,153 discloses cyclic sulphonamides and
sulphinamides having a physiological cooling action on the skin;
European Patent Application No. 1 157 617 discloses alpha-keto
enamine derivatives in a variety of food, cosmetic, pharmaceutical,
and perfume compositions; U.S. Pat. No. 4,296,093 discloses alkyl
substituted cyclohexanamides having a physiological cooling effect
on the skin; U.S. Pat. No. 5,756,857 discloses cyclohexanol
derivatives having a cool feeling; U.S. Pat. No. 4,248,859
discloses alicyclic amides having a physiological cooling effect;
U.S. Pat. No. 5,266,592 discloses ketals, such as glycerol ketals,
for example 1-menthone glycerol ketal or
3,3,5-trimethylcyclohexanone glycerol ketal, that have a
physiological cooling effect; U.S. Pat. No. 6,328,982 discloses
compositions comprising a cooling compound such as 1-menthol
(2-isopropyl-5-methyl-cyclohexanol), 1-isopulegol,
3-(1-methoxy)propane-1,2-diol and p-menthane-3,8-diol; and U.S.
Pat. No. 4,459,425 discloses 3-1-menthoxypropane-1,2-diol and its
associated cooling activity.
[0163] Those of skill in the art will readily recognize that
compounds suitably included in the compositions and methods of the
present invention can exist in a number of cis and trans isomers,
E/Z forms, diastereomers, as well as optical isomers. Thus,
compounds used in the compositions and methods of the present
invention include all such combinations and variations.
[0164] In compounds of Formula I, Formula II, Formula III, Formula
IV, Formula V, and Formula VI carbon atoms to which four
non-identical substituents are bonded are asymmetric. Accordingly,
compounds of Formula I, Formula II, Formula III, Formula IV,
Formula V, or Formula VI may exist as enantiomers, diastereomers or
a mixture thereof. The enantiomers and diastereomers may be
separated by chromatographic or crystallization methods, or by
other methods known in the art. The asymmetric carbon atom may be
in one of two configurations, R or S, both of which are within the
scope of the present invention. The presence of small amounts of
the opposing enantiomer or diastereomer in the final purified
product does not affect the therapeutic application of such
compounds.
[0165] Compounds of Formulae I-VI may be further treated to form
pharmaceutically acceptable salts. Treatment of a compound of the
invention with an acid or base may form, respectively, a
pharmaceutically acceptable acid addition salt and a
pharmaceutically acceptable base addition salt, each as defined
above. Various inorganic and organic acids and bases known in the
art, including those described herein above, may be used to effect
the conversion to the salt.
[0166] The present invention also relates to pharmaceutically
acceptable isomers, hydrates, and solvates of compounds of Formula
I, Formula II, Formula III, Formula N, Formula V, and Formula VI.
Compounds of these formulae may also exist in various isomeric and
tautomeric forms including pharmaceutically acceptable salts,
hydrates and solvates of such isomers and tautomers.
[0167] This invention also encompasses prodrug derivatives of the
compounds of Formula I, Formula II, Formula III, Formula N, Formula
V, and Formula VI. The term "prodrug" refers to a pharmacologically
inactive derivative of a parent drug molecule that requires
biotransformation, either spontaneous or enzymatic, within the
organism to release the active drug. Prodrugs are variations or
derivatives of the compounds of Formulae I-VI of the present
invention that have groups cleavable under metabolic conditions.
Prodrugs become the compounds of the invention which are
pharmaceutically active in vivo when they undergo solvolysis under
physiological conditions or undergo enzymatic degradation. An
exemplary prodrug technology that may be suitably employed with the
compounds of the present invention is the protease activated cancer
therapy (PACT) technology described in detail within U.S. patent
application Ser. No. 10/156,214 and PCT Application Publication No.
WO 02/095007, both of which are incorporated herein by
reference.
[0168] Synthesis of compounds of Formula I, may be achieved, as
described below in reference to compounds of Formula IV, by
reacting an acid chloride, obtained by reacting
p-menthane-3-carboxylic acid with thionyl chloride, with the
appropriate amine. As noted below, typically, the reaction is
carried out in solution at room temperature in the presence of a
hydrogen chloride receptor (e.g., sodium hydroxide).
[0169] Synthesis of (1)-Menthoxypropane-1,2-diol (Cooling Agent 10)
from 1-menthol (2-isopropyl-5-methyl-cyclohexanol) is described in
U.S. Pat. No. 4,459,425, incorporated herein by reference. Briefly,
1-menthol and metallic sodium or sodium hydride are introduced into
a solvent (e.g., toluene or xylene) and heated. When the
temperature reaches 100.degree. C., or higher, the reaction starts
and generation of hydrogen gas occurs. After confirming that the
generation of hydrogen has stopped, the mixture is further heated
at the reflux temperature of the solvent to complete the
reaction.
[0170] Allyl halide (e.g., allyl chloride or allyl bromide) is then
added to the reaction mixture in small portions. As the reaction
proceeds, sodium halide deposits and the reaction solution becomes
slurry-like. After the reaction is completed, the reaction solution
is cooled, and after addition of water, the resulting mixture is
stirred. Then, a solvent, (e.g., benzene, toluene, ether, hexane,
or petroleum ether) is added. The organic layer is separated and
washed with saturated saline water. After the solvent is recovered,
the residue is distilled under reduced pressure to obtain
3-1-menthoxypropate-1-ene.
[0171] The 3-1-menthoxypropane-1-ne is oxidized into the
corresponding oxide by use of an organic peracid. The oxide is
hydrolyzed to form the desired 3-1-methoxypropane-1,2-diol. That
is, an organic acid, (e.g., formic acid or acetic acid) and aqueous
hydrogen peroxide are mixed with 3-1-menthoxypropane-1-ene and
gradually heated carefully while stirring. The organic acid and the
hydrogen peroxide react, forming an organic peracid that
participates in the oxidation reaction. The reaction is exothermic,
and rapid heating should be avoided. When the temperature of the
reaction solution reaches near 50.degree. C., the heating is
stopped. It is, thereafter, necessary for the temperature of the
reaction solution to be maintained at about 70.degree. C. by
external cooling to prevent a further temperature increase caused
by the heat of reaction. If the temperature of the reaction
solution is excessively high, the organic peracid decomposes before
it participates in the oxidation reaction, resulting in a reduction
in yield.
[0172] After the reaction is completed, a solvent (e.g., benzene,
toluene, xylene, or petroleum ether) is added to perform the
extraction. The extracted liquid is washed with water. Upon
recovery of the solvent by distillation, a crude oxide in the form
of an organic acid ester is produced. The crude oxide thus formed
is mixed with an about 20% aqueous solution or caustic soda, for
example, and is hydrolyzed by boiling for about 1 hour to produce
the desired 3-1-menthoxypropane-1,2-diol.
[0173] Synthesis of compounds of Formula II, exemplified herein by
1-(2-hydroxyphenyl)-4-(3-nitrophenyl)-1,2,3,6-tetrahydropyrimidine-2-one
(Icilin aka AG-3-5), is disclosed in U.S. Pat. No. 3,821,221,
incorporated herein by reference. Briefly,
.beta.-diethlyamino-m-nitropropiophenone hydrochloride is added to
50% aqueous ethanol and the mixture refluxed with stirring until
the hydrochloride is dissolved. O-aminophenol is added and the
solution refluxed for 30 minutes, then set aside to cool. The
reaction product is crystallized out of solution to yield
(3-(o-hydroxyanilino)-m-nitrophropiophenone (mp of 107.degree. C.
to 109.degree. C.). .beta.-(o-hydroxyanilino)-m-nitrophropiophenone
is dissolved in ethanol and concentrated HCl is added to acidify
the solution. The solvent is evaporated in vacuo and
.beta.-(o-hydroxyanilino-m-nitropropiophenone)-HCl (mp of
172.degree. C. to 173.degree. C.) is crystallized from
methanol-acetone.
.beta.-(o-hydroxyanilino-m-nitropropiophenone)-HCl is dissolved in
acetic acid at 60.degree. C. Potassium cyanate is added and the
reaction mixture cooled to room temperature. Water is added and the
crystalline
1-(2-hydroxyphenyl)-4-(3-nitrophenyl)-1,2,3,6-tetrahydropyrimidine-2-one
(mp of 228.degree. C. to 230.degree. C.) is recovered by
filtration.
[0174] Synthesis of acyclic carboxamide compounds of Formula III,
as exemplified herein by 2-isopropyl-N,2,3-trimethylbutyramide aka
N,2,3-trimethyl-2-isopropyl butamide (WS-23), is disclosed in U.S.
Pat. No. 4,153,679, incorporated herein by reference. Briefly,
Formula III amides may be prepared by conventional techniques known
to those of skill in the art, for example, by reacting an acid
chloride of the formula R.sub.10R.sub.12R.sub.13COCl with an amine
(R.sub.11), as indicated above, in the presence of hydrogen
chloride acceptor.
[0175] N,2,3-trimethyl-2-isopropyl butamide may be prepared, for
example, by heating 2,3-Dimethyl-2-isopropylbutanic acid under
reflux with thionyl chloride for 60 minutes. The excess of thionyl
chloride may be removed under reduced pressure and the
2,3-dimethyl-2-isopropylbutanoyl chloride distilled, bp. 73.degree.
C.-75.degree. C./15 mm.
[0176] The acid chloride in ether may be added dropwise to a
stirred solution of methylamine (70% soln. in water) in ether with
stirring. The ether layer may then be washed with water, dilute HCl
and water. The dried (MgSO.sub.4) ether solution was concentrated,
and the residue distilled to give N,2,3-trimethyl-2-isopropyl
butamide (mp 58.degree. C.-61.degree. C., bp. 83.degree.
C.-85.degree. C./0.35 mm.).
[0177] Synthesis of exemplary 3-substituted-p-menthane compounds
that may be suitably employed as Trp-p8 modulators in the
compositions and methods of the present invention is described in
U.S. Pat. No. 4,150,052, incorporated herein by reference in its
entirety. For example, the corresponding acid chloride (obtained by
reacting p-menthane-3-carboxylic acid with thionyl chloride) may be
reacted with the appropriate amine. The reaction will usually be
carried out at room temperature in solution in the presence of a
hydrogen chloride receptor, e.g., sodium hydroxide.
[0178] The basic p-menthane structure is a chair-shaped molecule
that can exist in cis or trans form. Substitution of the carboxyl
or amide group into the 3-position gives rise to four
configurational or geometric isomers depending upon whether the
substitution is axially or equatorially into the cis or trans
isomer, the four isomers are related as menthol is to neomenthol,
isomenthol, and neoisomenthol.
[0179] In an exemplary reaction protocol, p-Menthane-3-carboxylic
acid is heated under reflux with thionyl chloride. Excess thionyl
chloride is distilled off in vacuo. The crude p-menth-3-oyl
chloride is dissolved in diethyl ether and the ethereal solution
added with stirring and cooling to a solution of ethylamine and
sodium hydroxide in water. The mixture is stirred and the ethereal
layer separated. The aqueous layer is washed with ether and the
combined ethereal solution washed with dilute hydrochloric acid and
water. The ether solution is dried with MgSO.sub.4 and evaporated
to give a white crystalline solid. The solid is recrystallised from
acetone:water (9:1) by dissolving the crystals at room temperature
and then cooling to produce N-ethyl-p-menthane-3-carbozamide as a
white crystalline solid, mp. 82.5.degree. C.-84.5.degree. C.
Substitution of the amide group in the 3-position of the p-menthane
structure gives rise to optical and geometric isomerism.
[0180] When either R.sup.13 or R.sup.14 is aliphatic, the preferred
values are C.sub.1-C.sub.9 straight or branched chain alkyl,
C.sub.1-C.sub.9 straight or branched chain hydroxyalkyl or
aminoalkyl and C.sub.1-C.sub.4 acylated derivatives thereof, and
--C.sub.nH.sub.2nCOR.sup.15 or --C.sub.nH.sub.2nCOOR.sup.15, where
--C.sub.nH.sub.2n is a straight or branched chain alkylene in which
n is an integer of from 1-6 and R.sup.15 is H or a C.sub.1-C.sub.8
alkyl or hydroxyalkyl group, preferably a C.sub.1-C.sub.4 straight
chain alkyl group.
[0181] When R.sup.13 is H and R.sup.14 is OH or substituted phenyl,
e.g., alkylphenyl, hydroxyphenyl, alkoxyphenyl, halophenyl of up to
10 carbon atoms, phenalkyl or substituted phenalkyl, e.g., benzyl,
naphthyl, or substituted naphthyl, and compounds where R.sup.13 and
R.sup.14 are joined to form a cyclic group. When so joined,
R.sup.13 and R.sup.14 preferably represent an alkylene chain,
optionally interrupted by oxygen, which together with the nitrogen
atom to which R.sup.13 and R.sup.14 are attached forms a 5- or
6-membered heterocyclic ring.
[0182] Synthesis of exemplary compounds comprising one or more
ketal, including for example menthone glycerine ketals, that may be
suitably employed as Trp-p8 modulators in the compositions and
methods of the present invention is described in U.S. Pat. No.
5,266,592, incorporated herein by reference in its entirety.
[0183] For example, ketals of Formula V may be prepared by an
acid-catalysed reaction of a ketone on which the ketal of Formula V
is based and not less than the equivalent amount of aliphatic
C3-C6-alcohol having not less than 3 and not more than 5,
preferably 3, hydroxyl groups. The ketone on which the ketal of
Formula V is based and an excess amount of the C3-C6 alcohol having
3 to 5 hydroxyl groups will be employed.
[0184] Exemplary acid catalysts that can be used are
p-toluenesulphonic acid, phosphoric acid, or potassium hydrogen
sulphate in catalytically effective amounts. The reaction will
generally be carried out either in an organic solvent that together
with water forms an azeotrope, so that the water, which is
liberated during formation of the ketal, can be eliminated by
azeotropic entrainment or water-consuming co-reagents such as, for
example, trialkyl ortho esters are used. Exemplary organic solvents
include benzene, toluene, xylene, chloroform, methylene chloride
and trichloroethylene. The reaction is complete when water no
longer separates out or when the ester/alcohol mixture is no longer
separated out. The products may be washed subsequently with dilute
alkali and with water, to separate and dry the organic phase, to
strip off the solvent and, if appropriate, to purify the residue,
for example by distillation.
[0185] An exemplary compound comprising a ketal of Formula V is
1-menthone glycerol ketal (Frescolat MGA, aka menthone glycerin
acetal; Harris & Ford, LLC), which is synthesized as follows. 2
mol of 1-menthone, 3 mol of glycerol, and 5 g of potassium hydrogen
sulphate are mixed in a 2 liter three-neck flask in the presence of
toluene. This mixture is refluxed in a water separator. After 7
hours, water separates and the mixture is neutralized and
distilled.
[0186] Synthesis of the exemplary Formula VI terpene compound
L-Carvone (2-cyclohexen-1-one, 2-methyl-5-(1-methylethenyl)-(R);
Millennium Chemicals) from D-limonene is described in Ikan, Natural
Products--A Laboratory Guide pp. 151-155 (Academic Press, 1969),
incorporated herein by reference. Briefly, a solution of D-limonene
in isopropanol is cooled to below 10.degree. C. Solutions of HCl in
isopropanol and concentrated aqueous sodium nitrite are added
dropwise to the D-limonene solution to generate limonene
nitrosochloride. The limonene nitrosochloride and dimethylformamide
is refluxed with isopropanol. After cooling, crystallization is
induced and the precipitate filtered and washed with water. The
resulting L-carvoxime is refluxed with 0.5 M oxalic acid and the
mixture is steam distilled. The distillate is extracted with ether,
dried over anhydrous magnesium sulfate, and the excess ether
evaporated to leave L-carvone.
[0187] Additional synthetic methods for the preparation of Trp-p8
modulators of the present invention are presented herein in
Examples 1-5.
Compositions Comprising a Small-Molecule Trp-p8 Modulators
[0188] As discussed above, the present invention is directed to
small-molecule Trp-p8 modulators, including Trp-p8 agonists and
Trp-p8 antagonists that bind to and alter the activity of Trp-p8.
Within certain embodiments, Trp-p8 modulators are agonists that
are, in certain instances, capable of stimulating cation influx in,
and toxicity of, a cell expressing the Trp-p8 channel protein.
Within alternative embodiments, Trp-p8 modulators are antagonists
of Trp-p8 activity that are capable of reducing the activity of
Trp-p8 expressed in a cell. Thus, Trp-p8 modulators of the present
invention will find utility in compositions, including
pharmaceutical compositions, which are useful in the treatment of
diseases associated with Trp-p8 expression. Suitable compositions,
according to the present invention, comprise one or more Trp-p8
agonist of Formula I, Formula II, Formula III, Formula IV, Formula
V, Formula VI, Formula VII, and/or Formula VIII and/or one or more
Trp-p8 antagonist of Formula I, Formula II, Formula III, Formula
IV, Formula V, Formula VI, Formula VII, and/or Formula VIII, as
described above, in combination with one or more pharmaceutically
acceptable carrier or excipient.
[0189] In one embodiment, the present invention provides
small-molecule Trp-p8 modulators in combination with a
pharmaceutically acceptable excipient such as sterile saline or
other medium, water, gelatin, oil, etc., to form pharmaceutically
acceptable compositions. The compositions and/or agonists may be
administered alone or in combination with any convenient carrier,
diluent, etc. and such administration may be provided in single or
multiple dosages. Useful carriers include, but are not limited to,
solid, semi-solid, or liquid medium including water and non-toxic
organic solvents.
[0190] Pharmaceutical compositions of the present invention may be
prepared by mixing one or more Trp-p8 agonist of Formula I, Formula
II, Formula III, Formula IV, Formula V, Formula VI, Formula VII,
and/or Formula VIII with a pharmaceutically acceptable carrier or
agent. Alternatively, pharmaceutical compositions may be prepared
by mixing one or more Trp-p8 antagonist of Formula I, Formula II,
Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/or
Formula VIII with a pharmaceutically acceptable carrier or agent.
In addition, pharmaceutical compositions may further include
excipients, stabilizers, diluents and the like and may be provided
in sustained release or timed release formulations. Acceptable
carriers, agents, excipients, stabilizers, diluents and the like
for therapeutic use are well known in the pharmaceutical field, and
are described, for example, in "Remington's Pharmaceutical
Sciences," (Mack Publishing Co., ed. A.R. Gennaro, 1985),
incorporated herein by reference. Such materials are nontoxic to
the recipients at the dosages and concentrations employed and
include buffers such a s phosphate, citrate, acetate, and other
organic acid salts, antioxidants such as ascorbic acid, low
molecular weight peptides such as polyarginine, proteins, such as
serum albumin, gelatin, or immunoglobulin, hydrophilic polymers
such as serum albumin, gelatin, or immunoglobulin, hydrophilic
polymers such as polyvinylpyrrolidinone, amino acids such as
glycine, glutamic acid, aspartic acid, or arginine,
monosaccharides, disaccharides, and other carbohydrates including
cellulose or its derivatives, glucose, mannose or dextrins,
chelating agents such as EDTA, sugar alcohols such as mannitol or
sorbitol, counterions such as sodium and/or nonionic surfactants
such as TWEEN, or polyethyleneglycol.
[0191] Within still further aspects, the compositions of the
present invention comprise a compound of Formula I, Formula II,
Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/or
Formula VIII formulated together with one or more cancer
therapeutic agent. Alternatively, the compositions of the present
invention comprise a compound of Formula I, Formula II, Formula
III, Formula IV, Formula V, Formula VI, Formula VII, and/or Formula
VIII independently formulated with one or more cancer therapeutic
agent. That is, the compound of Formula I, Formula II, Formula III,
Formula IV, Formula V, Formula VI, Formula VII, and/or Formula VIII
and the cancer therapeutic agent are separately formulated.
[0192] Suitable cancer therapeutic agents include, but are not
limited to, antimitotic agents including, but not limited to,
paclitaxel, vincristine, and etoposide; alkylating agents
including, but not limited to, mechlorethamine, cyclophosphamide,
and carmustine; antimetabolites including, but not limited to,
methotrexate, gemcitabine, lometrexol, 5-fluorouracil, and
6-mercaptopurine; cytotoxic antibiotics including, but not limited
to, doxorubicin, daunorubicin, bleomycin, mitomycin C, and
streptozocin; platinum agents including, but not limited to,
cisplatin and carboplatin; hormonal agents including, but not
limited to, anti-estrogens such as tamoxifen and diethylstilbestrol
as well as anti-androgens such as flutamide; antiangiogenesis
agents; and farnesyl transferase inhibitors.
[0193] In certain aspects, compounds of Formula I, Formula II,
Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/or
Formula VIII are administered in combination with a cancer
therapeutic agent that is ineffective in stimulating
Trp-p8-mediated cation influx.
[0194] In other aspects, compounds of Formula I, Formula II,
Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/or
Formula VIE are administered in combination with one or more
additional Trp-p8 modulator including, but not limited to a
compound of Formula I, Formula II, Formula III, Formula IV, Formula
V, Formula VI, Formula VII, and/or Formula VIII.
[0195] Depending upon the particular treatment regimen
contemplated, pharmaceutical compositions of the present invention
may be administered parenterally, topically, orally, or locally. In
certain aspects, the pharmaceutical compositions are administered
parenterally, e.g., intravenously, subcutaneously, intradermally,
or intramuscularly. In one embodiment, the present invention
provides compositions for parenteral administration that comprise a
compound of the present invention, dissolved or suspended in a
carrier such as an aqueous carrier.
[0196] For solid formulations, compounds may be admixed with
conventional nontoxic solid carriers such as, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharin, talcum, cellulose, glucose, sucrose,
magnesium carbonate, and the like.
[0197] For aerosol administration, compounds of the present
invention may be supplied in finely divided form along with a
nontoxic surfactant and propellant. Exemplary such agents are the
esters or partial esters of fatty acids containing from 6 to 22
carbon atoms, such as caproic, actanoic, lauric, palmitic, stearic,
linoleic, olesteric, and oleic acids.
[0198] Compositions of the present invention may be administered by
injection, i.e. intravenously, intramuscularly, intracutaneously,
subcutaneously, introaduodenally, or intraperitoneally.
Alternatively, compositions may be administered by inhalation, such
as intranasally, and may be administered transdermally, such as
through a patch or the like.
[0199] It will be understood that the actual preferred formulation
of compositions, including pharmaceutical compositions, will vary
according to the mode of administration as well as the particular
disease being treated. The optimal formulations and modes of
administration will be routinely determined on a disease by disease
and patient by patient basis by those of skill in the art.
Methods for Identifying and Characterizing the In Vitro and In Vivo
Efficacy of Small-Molecule Modulators of Trp-p8
[0200] As discussed above, the present invention is directed to
small-molecule Trp-p8 modulators, including agonists and
antagonists of Trp-p8 activity. Disclosed herein are Trp-p8
modulators exemplified by the compounds of Formula I, Formula II,
Formula III, Formula IV, Formula V, Formula VI, Formula VII, and/or
Formula VIII described herein above. The present invention further
contemplates that additional Trp-p8 modulators may also be suitably
employed in the compositions and methods of the present
invention.
[0201] Additional or alternative Trp-p8 agonists and antagonists
may be identified by the methodology disclosed in the accompanying
Examples. For instance, Trp-p8 agonists having efficacy in the
treatment of disease(s) associated with Trp-p8 expression include
small molecules that result in one or more of the following: (1)
inhibit the growth or decrease the viability of a cell expressing
Trp-p8; (2) stimulate calcium and/or other cation influx in a cell
expressing Trp-p8; (3) induction of a poptosis and/or necrosis in a
c ell expressing Trp-p8; and/or (4) efficacy in one or more animal
model systems of human disease. Trp-p8 antagonists having efficacy
in the treatment of disease(s) associated with Trp-p8 expression
include small molecules that that result in one or more of the
following: (1) protect Trp-p8 expressing cells from toxic effect of
agonists in in vitro model system (2) inhibit growth of and/or kill
cancer cell line with endogenous Trp-p8 expression (3) are
efficacious in one or more animal model systems of human
disease.
[0202] Thus, within certain embodiments, the present invention
provides methods for identifying Trp-p8 agonists comprising the
step of contacting a Trp-p8 expressing cell with a candidate Trp-p8
agonist for a time and in an amount sufficient to inhibit the
growth and/or decrease the viability of a Trp-p8 expressing cell,
wherein the inhibited growth and/or reduced viability indicate that
the candidate Trp-p8 agonist is capable of activating Trp-p8
expressed by the cell.
[0203] Other embodiments provide methods for identifying Trp-p8
agonists, comprising the step of contacting a Trp-p8 expressing
cell with a candidate Trp-p8 agonist for a time and in an amount
sufficient to induce influx of calcium and/or other cations into
the cell, wherein increased cation influx is correlative of
increased cellular toxicity.
[0204] Still further embodiments provide methods for identifying
Trp-p8 agonists comprising the step of administering a candidate
Trp-p8 agonist to an animal having one or more neoplastic cell that
expresses Trp-p8 for a time and in an amount sufficient to inhibit
the growth of and/or induce apoptosis and/or necrosis in the cell
thereby increasing the survival of the animal, wherein any one or
more of inhibition of cell growth, induction of apoptosis,
induction of necrosis, and/or increased survival of the animal
indicate efficacy of the Trp-p8 agonist.
[0205] The present invention provides methods for the
identification of Trp-p8 antagonists in addition to the Trp-p8
antagonists disclosed herein by the compounds of Formula VII and
Formula VIII. Such method include (1) in vitro assay systems for
detecting the protection of Trp-p8 expressing cells from toxicity
induced by Trp-p8 agonists; (2) in vitro and in vivo assay systems
of detecting the inhibition of growth of a cancer cell and/or
cancer cell line endogenously expressing Trp-p8; (3) in vivo animal
model systems whereby one or more candidate Trp-p8 antagonist is
administered to an animal having one or more neoplastic cell that
expresses Trp-p8 for a time and in an amount sufficient to inhibit
the growth of and/or induce apoptosis and/or necrosis in the cell
thereby increasing the survival of the animal.
Methods for Use of Trp-p8 Modulators
[0206] Small-molecule Trp-p8 modulators of the present invention
may be suitably employed in methods for modifying (i.e. activating
or reducing) Trp-p8-mediated calcium influx in a cell and
therapeutic methods for the treatment of one or more diseases
associated with expression of Trp-p8. For example, and as noted
above, it has been observed that abnormal Trp-p8 expression is
associated with a neoplastic phenotype in a variety of cancerous
tissues including breast, colon, lung, and prostate tissues.
Tsavaler et al., Cancer Research, supra.
[0207] Thus, within certain embodiments are provided methods for
activating Trp-p8-mediated calcium influx in a cell, such methods
comprising the step of contacting the Trp-p8 expressing cell with
an amount of a Trp-p8 agonist for a time sufficient to inhibit
growth of the cell and/or to induce necrosis and/or apoptosis in
the cell. Exemplary methods for activating Trp-p8 are provided
within the Examples presented herein.
[0208] Other embodiments of the present invention provide
therapeutic methods for the treatment of diseases associated with
expression of Trp-p8, such methods comprising the step of
administering to a mammal, typically a human, a therapeutically
effective amount of a composition comprising a Trp-p8 agonist for a
time sufficient to inhibit growth of the cell and/or to induce
necrosis and/or apoptosis in the cell. As used herein, the phrase
"therapeutically effective amount" refers to the amount of a
compound that, when administered to a mammal for treating a
disease, is sufficient to effect such treatment for the disease.
The "therapeutically effective amount" will vary depending upon the
compound, the disease, and its severity and the age, weight, etc.,
of the mammal to be treated.
[0209] As used herein, the terms "treat", "treating", and
"treatment" include: (1) preventing the disease, i.e. causing the
clinical symptoms of the disease not to develop in a mammal that
may be predisposed to the disease but does not yet experience any
symptoms of the disease; (2) inhibiting the disease, i.e. arresting
or reducing the development of the disease or its clinical
symptoms; or (3) relieving the disease, i.e. causing regression of
the disease or its clinical symptoms.
[0210] While the frequency and dosage of treatment regimens will
vary depending upon such factors as the disease and patient
treated, compositions comprising one or more compound of Formula I,
Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula
VII, and/or Formula VIII are typically administered in the range of
about 0.001 mg compound/kg body mass to about 100 mg/kg. Typically,
treatment is initiated with smaller dosages that are less than the
optimum dose of the compound. Thereafter, the dosage may be
increased until optimal effectiveness is achieved.
[0211] In most instances, administration of a composition(s) of the
present invention is achieved by any method that ensures systemic
exposure to the compound of Formula I, Formula II, Formula III,
Formula IV, Formula V, Formula VI, Formula VII, and/or Formula
VIII. Thus, compositions may be administered orally, parenterally,
intraduodenally, and intranasally. Typically, such compositions
comprise one or more such compound in combination with one or more
pharmaceutically acceptable carrier or diluent, as described in
further detail herein above.
[0212] Other embodiments of the present invention provide
combination therapies wherein one or more compound of Formula I,
Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula
VII, and/or Formula VIII is administered in conjunction with one or
more cancer therapeutic agent, as described in further detail
herein above, such as an antimitotic a gent, an alkylating agent,
an antimetabolite, a cytotoxic antibiotic, a platinum agent, a
hormonal agent, and/or an antiandrogen. Still further embodiments
of the present invention provide combination therapies wherein two
or more compounds of Formula I, Formula II, Formula III, Formula
IV, Formula V, Formula VI, Formula VII, and/or Formula VIII are
administered either simultaneously or sequentially to achieve the
desired therapeutic outcome.
[0213] Thus, as used herein, the term "combination" means that at
least two compounds can be delivered in a simultaneous manner, in
combination therapy wherein the first compound is administered
first, followed by the second compound, as well as wherein the
second compound is delivered first, followed by the first compound.
The desired result can be either a subjective relief of a
symptom(s) or an objectively identifiable improvement in the
recipient of the dosages.
[0214] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Synthesis of Menthane-3-carboxamide Compounds
[0215] This example discloses methodology for the synthesis of
menthane-3-carboxamide Trp-p8 modulators.
##STR00031##
[0216] Menthane-3-carboxylic acid (2).
[0217] Water (300 ml) was placed in a 2-L Erlenmeyer flask with a
large stir bar. Sulfuric acid (500 ml) was added carefully with
stirring. The solution was allowed to cool to 75.degree. C., and
N-ethyl-p-menthane-3-carboxamide (1, 62.5 g) was added. The
temperature was maintained at 75.degree. C. with a hot plate, and
sodium nitrite (31 g) was added carefully. Two more 31 gram
portions of NaNO.sub.2 were added at 1-hour intervals, and the
mixture was stirred overnight at 75.degree. C.
[0218] The mixture was cooled to room temperature, diluted with
.about.1 L of ice water, and extracted with .about.500 ml of ether.
The ether layer was separated, washed with water, and extracted
with 2.times.350 ml of 1M NaOH. The aqueous layer was made acidic
with concentrated HCl and extracted with ether. The ether layer was
dried with MgSO.sub.4 and evaporated to give menthane-3-carboxylic
acid (33.2 g, 61%) as a crystalline solid, [.quadrature.]=-50.3 deg
(c=1, CHCl.sub.3, 25.degree. C.).
[0219] Menthane-3-carbonyl chloride (3).
[0220] Menthane-3-carboxylic acid (54.35 g) was refluxed with 80 ml
of thionyl chloride for 3 hours. The SOCl.sub.2 was removed by
distillation, and the acid chloride was distilled at
114-115.degree. C. (8 Torr). (Lit. b.p. 84-85.degree. C. at 3.5
Torr). Yield: 50 g (84%).
[0221] General Procedure for Preparation of menthane-3-carboxamides
(5).
[0222] To a stirred solution of 0.2 mmol of the amine (4) in 1 ml
of acetonitrile or NMP and 0.4 mmol of DIPEA was added 0.022 ml of
menthane-3-carbonyl chloride (3). The reaction mixture was shaken
for 3 hours. For less reactive amines, the mixture was heated
(60.degree. C.) and shaken for 24 hours. The product was purified
from the crude reaction mixture by HPLC (40-95% gradient over 10
minutes using 0.05% TFA in CH.sub.3CN and 0.05% TFA in H.sub.2O)
and evaporated to dryness.
Example 2
Synthesis of Menthol Acetamide and Carbamate Compounds
[0223] This example discloses methodology for the synthesis of
Menthol Acetamide and Carbamate Trp-p8 modulators.
##STR00032##
[0224] General Procedures for the Preparation of Menthol Acetamides
(7) and Menthol Carbamates (9).
[0225] To a two-phase ether-water system containing 0.07 mmol of
amine (4) in 1 ml of ether and 1 ml of 0.1 M aqueous sodium
hydroxide was added 0.1 mmol of acid chloride (6) or chloroformate
(8). The reaction mixture was shaken for 2-3 hours. The upper layer
was removed and evaporated to dryness to afford the product.
Example 3
Synthesis of Menthyl-3-urea and Menthyl-3-thiourea Compounds
[0226] This example discloses methodology for the synthesis of
Menthyl-3-urea and Menthyl-3-thiourea Trp-p8 modulators.
##STR00033##
[0227] Menthyl-3-isocyanate (10).
[0228] To an ice-cooled solution of menthane-3-carboxylic acid (4
g, 22 mmol) and triethylamine (3.05 ml, 22 mmol) in dry DMF (45 ml)
was added diphenylphosphoryl azide (4.72 ml, 22 mmol). The mixture
was stirred at 0.degree. C. for 2 hours and at room temperature for
3 hours, then poured into a mixture of ether and ice water. The
ether layer was separated and washed with aqueous sodium
bicarbonate, dried with MgSO.sub.4, and evaporated. The residue was
distilled (85.degree. C., 10 Torr) to give menthyl-3-isocyanate
(2.9 g, 73%) as a colorless liquid.
[0229] General Procedure for the Preparation of menthyl-3-ureas
(12) and menthyl-3-thioureas (13).
[0230] To a stirred solution of 0.07 mmol of the amine (4) or thiol
(11) in 1 ml of dry ethyl acetate was added a solution of 0.07 mmol
of menthyl-3-isocyanate in 0.5 ml of ethyl acetate. The reaction
mixture was shaken overnight then evaporated to dryness to afford
the product.
Example 4
Synthesis of Menthane-3-amide, Menthane-3-sulfonamide and
Menthane-3-carbamate Compounds
[0231] This example discloses methodology for the synthesis of
Menthane-3-amide, Menthane-3-sulfonamide and Menthane-3-carbamate
Trp-p8 modulators.
##STR00034##
[0232] Menthyl-3-amine hydrochloride (14).
[0233] To a vigorously stirred mixture of diethyl ether (100 ml)
and 6N aqueous hydrochloric acid (100 ml) was added
menthyl-3-isocyanate (2.0 g, 11 mmol), and the mixture was stirred
overnight at room temperature. The aqueous phase was separated,
made basic with an excess of aqueous sodium hydroxide, and
extracted with ether. The ether phase was dried with MgSO.sub.4 and
filtered. An excess of anhydrous HCl (3M) in ethanol was added, and
a precipitate immediately formed. Filtration gave menthylamine
hydrochloride (1.9 g, 90%) as fine white crystals.
[0234] General Procedure for the Preparation of menthane-3-amides
(18), menthane-3-sulfonamides (19) and menthane-3-carbamates
(20).
[0235] A solution of menthyl-3-amine hydrochloride (0.078 mmol) in
0.7 ml of water was added to a solution of the acid chloride,
sulfonyl chloride, or chloroformate (0.078 mop in 1 ml Et.sub.2O,
followed by the addition 0.3 ml of 0.5 M NaOH. The mixture was
shaken at room temperature for 12-18 hours. The Et.sub.2O layer was
then separated and the solvent removed under reduced pressure to
afford the product.
Example 5
Synthesis of Menthyl-3-amine Compounds
[0236] This example discloses methodology for the synthesis of
Menthyl-3-amine Trp-p8 modulators.
##STR00035##
[0237] General Procedures for the Preparation of menthyl-3-amines
(23).
[0238] Method 1
[0239] A mixture of the amine (22, 0.1 mmol) and 15.4 mg (0.1 mmol)
of menthone (21) in 1 ml of 1,2-dichloroethane was treated with
sodium triacetoxyborohydride (32 mg, 0.15 mmol). The mixture was
stirred for 24 h (periodically monitored by LCMS). The reaction
mixture was quenched by adding 1N NaOH (1 ml) and the product was
extracted with ether. The extract was washed with brine, dried
(MgSO.sub.4) and evaporated to dryness to afforf the product.
[0240] Method 2
[0241] A mixture of menthyl-3-amine (0.1 mmol) and 15.4 mg (0.1
mmol) of aldehyde (21) in 1 ml of 1,2-dichloroethane was treated
with sodium triacetoxyborohydride (32 mg, 0.15 mmol). The mixture
was stirred for 24 h (periodically monitored by LCMS). The reaction
mixture was quenched by adding 1N NaOH (1 ml) and the product was
extracted with ether. The extract was washed with brine, dried
(MgSO.sub.4) and evaporated to dryness.
Example 6
Expression of Trp-p8 in CHO Cells
[0242] Human trp-p8 transfected CHO cells (referred to herein as
CHO/Trp-p8) were generated for use in experiments of the present
invention. Expression of Trp-p8 polypeptide in this transfectant
and the absence of any endogenous expression in the non-transfected
to CHO was confirmed by western blot and immunofluorescence using a
Trp-p8 specific antibody (GS2.20; disclosed within copending U.S.
patent application Ser. No. 10/______ incorporated herein by
reference in its entirety) as well as the calcium flux assay with
Icilin
(1-[2-hydroxyphenyl]-4-[3-nitrophenyl]-1,2,3,6-tetrahydropyrimidine-2-one-
) and menthol (2-isopropyl-5-methyl-cyclohexanol). Non-transfected
CHO cells were used to establish the specificity of the effects of
the compounds observed with CHO/Trp-p8.
Example 7
Trp-p8-Mediated Decrease in Cell Viability Following Exposure of
CHO/Trp-p8 Cells with Candidate Trp-P8 Agonist Compounds at
37.degree. C.
[0243] This Example discloses an ATP viability assay suitable for
screening for effective Trp-p8 agonists. The ATP viability assay
described herein employs CHO cells expressing an exogenous Trp-p8
cDNA. This example further establishes that Trp-p8 agonists of the
present invention are effective in decreasing the viability of
Trp-p8 expressing cells.
[0244] The concentration of intracellular ATP declines very rapidly
when metabolically active cells undergo necrosis and/or apoptosis.
The ATP concentration and consequently the relative cell viability
can be measured by established methods using commercially available
reagents. In the agonist screening methodology disclosed herein, a
compound that specifically decreases the viability of CHO/Trp-p8
cells is referred to as an agonist.
[0245] As a primary screen for efficacy and specificity for
agonists, both the non-transfected CHO and CHO/Trp-p8 cells were
exposed to 10 .mu.M of test compounds in 1% dimethylsulfoxide
(DMSO) or 1% DMSO (control) in a 96-well black walled,
black-bottomed, cell-culture treated plate. DMSO was the solvent
for all of the compounds tested. After 24-26 hours at 37.degree.
C., the cells were lysed and ATP concentration determined via a
chemiluminescence assay using a commercially available reagent
kit--Cell Titer-Glo (Promega; Madison, Wis.). Relative viability
(%), expressed as the ATP level in cells treated with compounds
expressed as a percentage of ATP levels in cells treated with the
DMSO alone, was a measure of the agonist activity of the candidate
compound--the lower the % viability, the more potent the Trp-p8
agonist. EC.sub.50 values were determined for the most active
candidate Trp-p8 agonists at 37.degree. C. by measuring viability
at 8-10 agonist concentrations. (EC.sub.50 is defined herein as the
agonist concentration at which there is a 50% reduction in cell
viability).
[0246] Exemplary Trp-p8 Agonists of Formula IV that were
efficacious in the ATP viability assay are presented herein in
Table 1. Table 1A presents Formula IV Trp-p8 agonists exhibiting an
EC.sub.50 value within the range of 0.05 to 0.20 .mu.M; Table 1B
presents Formula IV Trp-p8 agonists exhibiting an EC.sub.50 value
within the range of 0.20 to 0.50 .mu.M; Table 1C presents Formula
IV Trp-p8 agonists exhibiting an EC.sub.50 value within the range
of 0.50 to 1.00 .mu.M; and Table 1D presents Formula IV Trp-p8
agonists exhibiting an EC.sub.50 value within the range of 1.00 to
7.00 .mu.M.
[0247] Viability of CHO/Trp-p8 cells following treatment with
exemplary Trp-p8 agonists is presented in FIG. 1.
TABLE-US-00001 TABLE 1A Exemplary Trp-p8 Agonists of Formula IV
Exhibiting an EC.sub.50 Value within the Range of 0.05 to 0.20
.mu.M Ref. No.: Chemical Structure R.sup.14/R.sup.15 R.sup.15 2905
##STR00036## 2-hydroxy-2-phenylethyl 3012 ##STR00037##
2-oxo-2-phenylethyl 2896 ##STR00038## 2-hydroxy-2-(3-
hydroxyphenyl)ethyl 3006 ##STR00039## 1-methyl-2-hydroxy-2-
phenylethyl 2926 ##STR00040## 2-phenylethenyl 3014 ##STR00041##
Benzoylamino 2963 ##STR00042## 4-acetylphenyl ##STR00043##
TABLE-US-00002 TABLE 1B Exemplary Trp-p8 Agonists of Formula IV
Exhibiting an EC.sub.50 Value within the Range of 0.20 to 0.50
.mu.M Ref. No.: Chemical Structure R.sup.14/R.sup.15 R.sup.15 3024
##STR00044## N'-quinoxalin-2-yl-amino 2913 ##STR00045##
2-(4-hydroxyphenyl)ethyl 2897 ##STR00046## 2-hydroxy-2-(4-
hydroxyphenyl)ethyl 2928 ##STR00047## 2-(3-hydroxyphenyl)ethyl 2901
##STR00048## Phenylcyclopropyl 1906 ##STR00049## 2-(2-furyl)ethyl
2920 ##STR00050## 2-(2-methylphenyl)ethyl 2952 ##STR00051##
2-(6-fluoro-1H-indol-3-yl)- ethyl 3013 ##STR00052##
2-(4-methoxy-phenyl)-2- oxo-ethyl 1603 ##STR00053## 2-phenylethyl
2264 ##STR00054## 2-(2-flourophenyl)ethyl 2261 ##STR00055##
2-hydroxy-2-phenylethyl 2904 ##STR00056## 2-hydroxy-2-phenylethyl
2932 ##STR00057## 2-(2,4-dichlorophenyl)ethyl 2931 ##STR00058##
2-(2-chloro-6- flourophenyl)ethyl 2942 ##STR00059##
2-(3-methylphenyl)ethyl 2930 ##STR00060## 2-(3-chlorophenyl)ethyl
1901 ##STR00061## 2-(2-methylphenyl)ethyl 2944 ##STR00062##
1-methyl-2-(5-fluoro-1H- indol-3-yl)-ethyl 3003 ##STR00063##
N-(2-diethylamino-ethyl)- benzamide-4-yl 2966 ##STR00064##
4-methylsulfanylphenyl 2973 ##STR00065## 2-chloro-4-cyanophenyl
2869 ##STR00066## 4-(2-hydroxyethyl)phenyl 2984 ##STR00067##
4-methyl-2-oxo-2H- chromen-7-yl 2832 ##STR00068##
4-(1-hydroxyethyl)phenyl 2836 ##STR00069## 3-oxo-indan-5-yl 2887
##STR00070## 4-[2-(2-methoxy-phenyl)- 1,3-dioxo-2,3-dihydro-1H-
isoindol-5-yloxyl-phenyl 2892 ##STR00071## 4-{2-[2-(3,4-dimethoxy-
phenyl)-ethyl]-1,3-dioxo- 2,3-dihydro-1H-isoindol-5- yloxy}-phenyl
2858 ##STR00072## 3-chloro-4-morpholin-4-yl- phenyl 2958
##STR00073## 4-hydroxymethylphenyl 2864 ##STR00074##
2-chloro-4-iodophenyl 2831 ##STR00075## 4-carboxamidophenyl 2983
##STR00076## 2-chloro-4-nitrophenyl 2961 ##STR00077##
3,4-cyclopentanephenyl 2828 ##STR00078## 4-cyanophenyl 2964
##STR00079## 4-ethoxyphenyl ##STR00080##
TABLE-US-00003 TABLE 1C Exemplary Trp-p8 Agonists of Formula IV
Exhibiting an EC.sub.50 Value within the Range of 0.50 to 1.00
.mu.M Ref. No.: Chemical Structure R.sup.14/R.sup.15 R.sup.15 3040
##STR00081## 2-(pyridin-3-yl)-ethyl 2903 ##STR00082##
2-methyl-2-phenylethyl 1903 ##STR00083## 2-thiophen-2-yl-ethyl)
2679 ##STR00084## 2-(1H-indol-3-yl)ethyl 2918 ##STR00085##
2-(3-methoxy-4- hydroxyphenyl)ethyl 2263 ##STR00086##
2-(4-flourophenyl)ethyl 3041 ##STR00087## 2-(pyridin-4-yl)-ethyl
3039 ##STR00088## 2-(pyridin-2-yl)-ethyl 1619 ##STR00089##
3-phenyl-piperidin-1-yl 2262 ##STR00090## 2-(3-flourophenyl)ethyl
2940 ##STR00091## 2-(2-methoxy-5- bromophenyl)ethyl 2270
##STR00092## 1-hydroxymethyl-2-phenyl ethyl 3009 ##STR00093##
2-methyl-2-phenylethyl 2939 ##STR00094## 2-(3-bromo-4-
methoxyphenyl) ethyl 2914 ##STR00095## 2-(4-methylphenyl) ethyl
3010 ##STR00096## 1-oxo-2-phenylethyl 2912 ##STR00097##
2-(4-bromophenyl)-ethyl 2922 ##STR00098## 2-phenyl-2-(4-
flourophenyl)-ethyl 2950 ##STR00099## 2-(6-methoxy-1H-indol-3-
yl)-ethyl 2868 ##STR00100## 4-methylphenyl 2891 ##STR00101##
4-[1,3-dioxo-2-(2- trifluoromethyl-phenyl)-
2,3-dihydro-1H-isoindol-5- yloxy]-phenyl 2998 ##STR00102##
4-(4-methyl-piperazin-1- ylmethyl)-phenyl 2960 ##STR00103##
C-1H-Indazol-5-yl 2970 ##STR00104## 2-flouro-4-chlorophenyl 2979
##STR00105## 4-triflouromethylphenyl 2993 ##STR00106##
2-methyl-4-broophenyl 2987 ##STR00107## 4-Pyrrolidin-1-ylmethyl-
phenyl 2853 ##STR00108## 2-phenyl-1H- benzoimidazol-5-yl 2875
##STR00109## 4-(morpholine-4-sulfonyl)- phenyl 2956 ##STR00110##
2,4-dimethylphenyl 2978 ##STR00111## 3-chloro-4-methoxyphenyl 2856
##STR00112## 2-pyridin-3-yl-1H- benzoimidazol-5-yl ##STR00113##
TABLE-US-00004 TABLE 1D Exemplary Trp-p8 Agonists of Formula IV
Exhibiting an EC.sub.50 Value within the Range of 1.00 to 7.00
.mu.M Ref. No.: Chemical Structure R.sup.14/R.sup.15 R.sup.15 2943
##STR00114## 2-(2,5-dimethylphenyl)- ethyl 2917 ##STR00115##
2-(3-hydroxy-4- methoxyphenyl)-ethyl 2269 ##STR00116##
2-(2-methoxyphenyl)-ethyl 3007 ##STR00117## 1-methoxymethyl-2-
phenylethyl 2898 ##STR00118## 1-methyl-2-(4- chlorophenyl)-ethyl
1627 ##STR00119## 3-phenyl-pyrrolidin-1-yl 2271 ##STR00120##
2-(2-chlorophenyl)ethyl 2933 ##STR00121## 2-(2,6-
dimethylphenyl)ethyl 2936 ##STR00122## 2-(3,4-dichlorophenyl)ethyl
2923 ##STR00123## 2-phenyl-2-(4- methoxyphenyl)ethyl 2919
##STR00124## 2-(2,4- dimethylphenyl)ethyl 2266 ##STR00125##
1-hydroxymethyl-2-phenyl ethyl 2929 ##STR00126## 2-(3-
triflouromethylphenyl)ethyl 2935 ##STR00127## 2-(2,5-
dimethoxyphenyl)ethyl 1591 ##STR00128## 2-cyclohex-1-enyl-ethyl
3035 ##STR00129## benzyloxy 1568 ##STR00130##
2-(4-chlorophenyl)ethyl 2894 ##STR00131## 1-hydroxymethyl-2-(4-
chlorophenyl)ethyl 2265 ##STR00132## 2-(4-methoxyphenyl)ethyl 2924
##STR00133## 2-phenyl-2-(4- chlorophenyl)ethyl 2677 ##STR00134##
3-phenylpropyl 1910 ##STR00135## 2-(4- methylsulfanylphenyl)ethyl
2273 ##STR00136## 1-hydroxymethyl-2- hydroxy-2-phenylethyl 2937
##STR00137## 2-(3,5- dimethoxyphenyl)ethyl 2949 ##STR00138##
2-(5-methoxy-1H-indol-3- yl)-ethyl 2941 ##STR00139##
2-(3-ethoxyphenyl)ethyl 2953 ##STR00140## 2-(7-methyl-1H-indol-3-
yl)-ethyl 2938 ##STR00141## 2-(4-ethylphenyl)ethyl 2934
##STR00142## 2-(2,3- dimethoxyphenyl)ethyl 2268 ##STR00143##
1-methyl-2-hydroxy-2- phenylethyl 2647 ##STR00144##
6,7-dimethoxy-1-methyl- 3,4-dihydro-1H- isoquinolin-2-yl 2915
##STR00145## 2-(4-sulfamoyl-phenyl)- ethyl 1912 ##STR00146##
2-(1-cyclopentyl- pyrrolidin-3-yl)-ethyl 3032 ##STR00147##
2-(3-methoxy-4- ethoxyphenyl)ethyl 2947 ##STR00148##
1-hydroxymethyl-2-(1H- indol-3-yl)ethyl 2945 ##STR00149##
[1-carbamoyl-2-(1H-indol- 3-yl)-ethyl 1599 ##STR00150## 1-phenyl-
cyclopentylmethyl 3008 ##STR00151## 1-hydroxymethyl-2-phenyl ethyl
2909 ##STR00152## 1-hydroxymethyl-2-(4- hydroxyphenyl)ethyl 2598
##STR00153## 4-nitrobenzyl 2593 ##STR00154## 2,3-diflourobenzyl
2899 ##STR00155## 1-carbamoyl-2-phenylethyl 2279 ##STR00156##
2,2-diphenylethyl 2267 ##STR00157## 2-(3-methoxyphenyl)ethyl 1611
##STR00158## 4-pyrimidin-2-yl- piperazin-1-yl 2277 ##STR00159##
2-(3,4- dimethoxyphenyl)ethyl 1566 ##STR00160## 4-methylcyclohexyl
2260 ##STR00161## indan-2-yl 2900 ##STR00162## 1-carbamoyl-2-(4-
hydroxyphenyl)ethyl 1637 ##STR00163## cycloheptyl 1629 ##STR00164##
bicyclo[2.2.1]hept-2-yl 1614 ##STR00165## 2-(N,N-
dipropylamino)ethyl 2272 ##STR00166## 2-(4-nitrophenyl)ethyl 2981
##STR00167## 4-Biphenyl 1617 ##STR00168## 3,4-dimethylphenyl 2862
##STR00169## 4-(1,1-dioxo-116- thiomorpholin-4-ylmethyl)- phenyl
2844 ##STR00170## 2-bromo-4-methylphenyl 2870 ##STR00171##
2-oxo-2,3-dihydro-1H- benzoimidazol-5-yl 3030 ##STR00172##
2-triflouromethyl-1H- benzoimidazol-5-yl 2974 ##STR00173##
2,3-dimethoxyphenyl 2849 ##STR00174## 4-Azepan-1-ylmethyl- phenyl
2850 ##STR00175## 4-(4-ethyl-piperazin-1-yl)- phenyl 1631
##STR00176## 4-chlorophenyl 2841 ##STR00177##
1,3-dioxo-2,3-dihydro-1H- isoindol-4-yl 2843 ##STR00178##
3-bromo-4-methylphenyl 1607 ##STR00179## 4-methoxyphenyl 2840
##STR00180## 2,4-dichlorophenyl 2962 ##STR00181## 3-yl-acetophenone
2872 ##STR00182## 2-flouro-5-nitrophenyl 2985 ##STR00183##
2-methyl-1,3-dioxo-2,3- dihydro-1H-isoindol-5-yl 1586 ##STR00184##
3-chloro-4-methylphenyl 2833 ##STR00185## 3-nitrophenyl 2957
##STR00186## 3-hydroxymethylphenyl 1639 ##STR00187## 4-bromophenyl
2835 ##STR00188## 2-chloro-4-flourophenyl 2982 ##STR00189##
2-chloro-5-nitrophenyl 2954 ##STR00190## 2-methylphenyl 2871
##STR00191## 3-acetamidophenyl 1575 ##STR00192## 4-ethylphenyl 2980
##STR00193## 3,4-dichlorophenyl 1642 ##STR00194##
benzo[1,3]dioxol-5-yl 2873 ##STR00195## 1,3-dioxo-2,3-dihydro-1H-
isoindol-5-yl 2965 ##STR00196## 2-nitrophenyl 2976 ##STR00197##
2-nitro-4-flourophenyl 1545 ##STR00198## 2,4-diflourophenyl 2990
##STR00199## 3,4,5-trimethoxyphenyl 2829 ##STR00200##
2-chlorophenyl 2837 ##STR00201## 2-methyl-5-nitrophenyl 2859
##STR00202## 2-iodophenyl 2972 ##STR00203## 3,4,5-triflourophenyl
3002 ##STR00204## 4-(4-methyl-1H- benzoimidazol-2-yl)-phenyl 2851
##STR00205## 4-benzooxazol-2-yl-phenyl 1616 ##STR00206##
4-flourophenyl 2855 ##STR00207## 2-pyridin-2-yl- benzooxazol-5-yl
2830 ##STR00208## 3-carboxamidophenyl 1577 ##STR00209##
2,4-dimethoxyphenyl 1585 ##STR00210## 3,5-dimethoxyphenyl
##STR00211##
Example 8
Screen and Characterization of Trp-p8 Agonist Compounds by
Measuring Calcium Influx in CHO/Trp-p8 Cells at 37.degree. C.
[0248] This example discloses a CHO/Trp-p8-based calcium influx
assay used to further assess the activity of candidate Trp-p8
agonists of the present invention.
[0249] Calcium influx was measured using a Flexstation Microplate
Fluorescence Plate Reader (Molecular Devices; Sunnyvale, Calif.). A
typical assay for calcium flux was performed as follows. Cells in
DMEM/Ham's F-12 based medium, typically at a density of 30,000
cells/well/100 .mu.l, were plated in a 96-well black-walled, clear
bottomed tissue culture plate (Greiner Bio-one) and incubated for
16-20 hours at 37.degree. C. Cells in each well were incubated for
one hour at 37.degree. C. with a Fura2-AM Fluorescent Dye/Pluronic
F-27 mixture (Molecular Probes; Eugene, Oreg.) and dissolved in the
medium containing Probenecid. Typical final concentrations were:
5-8 .mu.M of Fura2-AM, 0.01% Pluronic F-27, and 2.5 mM Probenecid
(an anion exchange inhibitor that reduces transport of the
hydrolyzed dye from inside the cell thereby minimizing loss of dye
during the experiment). After one hour, cells were washed in a
buffered solution (20 mM HEPES and Hanks Balanced Salt Solution
with 1.26 mM CaCl.sub.2), pH 7.4 containing Probenecid at a final
concentration of 2.5 mM and pre-incubated for at least 30 minutes
at the assay temperature of 37.degree. C.
[0250] Typically, the above described HEPES/HBSS-based buffer
containing either no additional calcium or with calcium to increase
the concentration to 2 mM and various concentrations of compounds
(at 5-times the final concentrations) were added to each well using
the robotic multi-channel pipettor. The compounds were preincubated
at 37.degree. C. for at least 30 minutes before performing the
assay (at 37.degree. C.). Signals were read with dual excitation
wavelengths of 340 and 380 nm and emission wavelength of 510 nm
with a cut-off filter at 495 nm. The signal was reported as the
ratio of emission when excited at 340 nm to the emission when
excited at 380 nm [Relative Fluorescence Units (RFU)]. Ionomycin
was routinely used as a positive control.
[0251] In the case of the agonist assay, the compounds at different
concentrations were added to the dye-loaded cells (as described
above). The increase in RFU was a measure of potency of the
compound as an agonist. Exemplary results are presented in FIG.
2.
Example 9
Increase in Apoptosis Following Exposure of CHO/Trp-p8 Cells with
Trp-P8 Agonist Compounds at 37.degree. C.
[0252] This example discloses the effectiveness of Trp-p8 agonist
compounds in inducing apoptosis in Trp-p8 expressing cells.
[0253] An Annexin V/Propidium Iodide (PI) flow cytometry assay was
used to provide additional insights into the mechanism of cell
death induced by Trp-p8 agonist compounds. Annexin V staining
detects translocation of Phosphatidylserine to the outer layer of
plasma membrane, an event characteristic of apoptosis, while PI
staining indicates dead cells with compromised membranes.
[0254] Cells were treated with compounds in 1% DMSO or with a 1%
DMSO (control) for 24-26 hours at 37.degree. C. The cells were
briefly trypsinized under controlled conditions and stained with an
Annexin V/PI reagent kit following the methodology provided by the
supplier (e.g., Southern Biotech; Birmingham, Ala.). Exemplary
results are presented in FIG. 3.
Example 10
In Vitro Screen Using a Cell Viability Assay for Trp-p8 Antagonist
Compounds Based Upon Protection of Trp-p8-Expressing Cells from
Toxic Agonist Compounds
[0255] This example discloses an assay system for identifying and
characterizing candidate Trp-p8 antagonist compounds.
[0256] Trp-p8 antagonists were identified by employing a cell
viability assay with CHO/Trp-p8 cells at 37.degree. C. (see Example
7) with the following modification. Within the context of the
present invention, compounds that protect CHO/Trp-p8 cells from the
toxic effect of a control agonist thereby maintaining the viability
of the CHO/Trp-p8 cell exposed to a Trp-p8 agonist is defined as
antagonist. As a primary screen for antagonists, CHO/Trp-p8 cells
were exposed to 10 .mu.M of test compounds in 1% dimethylsulfoxide
(DMSO) or 1% DMSO plus a toxic concentration of a control agonist,
D-1607. The relative viability at 10 .mu.M, determined as described
in Example 7, was a measure of the potential of the compound as a
Trp-p8 antagonist--the higher the viability, the more potent the
antagonist. Exemplary results are presented in FIG. 4.
TABLE-US-00005 TABLE 2 Exemplary Formula VIII Trp-p8 Antagonists of
Formula IV Agonist Compounds Relative Viability Ref. (%) at No.:
Chemical Structure R.sup.23/R.sup.24 R.sup.24 10 .mu.M 1457
##STR00212## Tetrahydro isoquinolinyl 112 1465 ##STR00213##
Tetrahydro quinolinyl 90 1475 ##STR00214## 3-methyl indolinyl 100
1504 ##STR00215## indolinyl 90 1582 ##STR00216## 2-(N-methyl, N-
Phenylethyl) amino ethyl 103 1588 ##STR00217## 3-methyl indolinyl
88 1664 ##STR00218## 1-phenyl ethyl 107 1669 ##STR00219## 2-chloro
benzyl 86 1673 ##STR00220## 2-methoxybenzyl 112 1688 ##STR00221##
Tetrahydro isoquinolinyl 91 1691 ##STR00222## 3-methyl indolinyl 92
1696 ##STR00223## Tetrahydro quinolinyl 82 1709 ##STR00224##
2-methoxyphenyl 95 1743 ##STR00225## 2-cyclohex-1-enyl ethyl 97
1745 ##STR00226## (1-Phenyl-cyclopentyl)- methyl 88 1781
##STR00227## 3-methyl indolinyl 91 1815 ##STR00228##
2-(tetrahydroquinolinyl)- ethyl 87 1819 ##STR00229## Tetrahydro
isoquinolinyl 86 1838 ##STR00230## 1-Propyl-1,2,3,4- tetrahydro-
pyrrolo[1,2-a] pyrazine 88 1876 ##STR00231## cyclohepttyl 86 1882
##STR00232## 3- Cyclohexylsulfanylpropyl 85 1883 ##STR00233##
2-cyclohex-1-enyl ethyl 84 1885 ##STR00234## 2-(N-isopropyl, N-
Phenylethyl) amino ethyl 97 1918 ##STR00235## 1-methyl-1,2,3,4-
tetrahydro- pyrrolo[1,2-a] pyrazine 94 1920 ##STR00236## 81 1923
##STR00237## 116 1925 ##STR00238## 94 1937 ##STR00239## 101 1940
##STR00240## 1-methyl-1,2,3,4- tetrahydro- pyrrolo[1,2-a] pyrazine
110 1941 ##STR00241## 1-Propyl-1,2,3,4- tetrahydro- pyrrolo[1,2-a]
pyrazine 98 1996 ##STR00242## 2-cyclopentylethyl 89 2013
##STR00243## 2-Phenylcyclopropyl 95 2018 ##STR00244##
1-phenoxyethyl 103 2044 ##STR00245## 4-butyloxyphenyl 94 2045
##STR00246## (2-nitrophenoxy)methyl 191 2046 ##STR00247##
4,7,7-trimethyl-2-oxa- bicyclo[2.2.1]heptan-3- one 89 2067
##STR00248## C-(1-Phenyl-5-propyl- 1H-pyrazol-4-yl)-methyl 105 2291
##STR00249## Benzyl 87 2306 ##STR00250## 2-chlorobenzyl 92 2639
##STR00251## benzyl; R2 = 2-(4- methyl)pyridyl 102 2676
##STR00252## 1-[3-(6,7-Dimethoxy- 1-methyl-3,4- dihydro-1H-
isoquinolin-2- ylmethyl)-4-methoxy- phenyl]-2,3,4,9-
tetrahydro-1H-b- carboline 83 2777 ##STR00253##
C-[3-(4-Butoxy-phenyl)- 1H-pyrazol-4-yl]-methyl 83 2865
##STR00254## 4-(Azepane-1-sulfonyl)- phenyl 91 3026 ##STR00255##
5-(7-Chloro-quinolin-4- ylsulfanyl)- [1,3,4]thiadiazol-2-yl 86 2131
##STR00256## 2,4,6-trichlorophenyl 59 2134 ##STR00257##
4,5-dibromothiophen-2-yl 63 2710 ##STR00258## 2-hydroxy-5-
methylphenyl 70 2745 ##STR00259## 3-phenyl-1H-pyrazol-4-yl 62 2752
##STR00260## 3-(4-flourophenyl)-1H- pyrazol-4-yl 65 2754
##STR00261## 3-(4-ethylphenyl)-1H- pyrazol-4-yl 74
TABLE-US-00006 TABLE 3 Exemplary Formula VII Trp-p8 Antagonists of
Formula II Agonist Compounds Rel. Viab Ref. R.sup.19/ (%) at No.
Chemical Structure R.sup.17 R.sup.18 R.sup.20 R.sup.21 10 .mu.M 13
##STR00262## 2-Pyridyl 1-Benzyl-1H- pyrazolo[3,4-d] pyrimidin-4-yl
H 91 27 ##STR00263## 2-Nitro-4- triflourome thylphenyl
3-benzylamino-2- nitrophenyl H 87 34 ##STR00264## 2-nitro-4-
chlorophenyl 5-Nitro-quinolin- 8-yl H 82 36 ##STR00265## 2-
methoxyphenyl 1-yl-3-(2- isopropyl-5- methyl- cyclohexyloxy)-
propan-2-ol H 87 51 ##STR00266## 2- chlorophenyl 1-Phenyl-1H-
pyrazolo[3,4-d] pyrimidin-4-yl H 100 67 ##STR00267## phenyl
Benzyl-2-methyl- quinazolin-4-yl H 102 69 ##STR00268## phenyl
3-Methyl-5- morpholin-4-yl-2- nitro-phenyl H 106 74 ##STR00269##
2-methyl- quinolin-3- yl 2-nitro-5- piperazin-1-yl- ethanol H 81 93
##STR00270## 4- methoxyphenyl 1-yl-3-(2- isopropyl-5- methyl-
cyclohexyloxy)- propan-2-ol H 111 103 ##STR00271## phenyl
4-(2,5-Dimethyl- pyrrol-1-yl)-2- nitro-phenyl H 99 107 ##STR00272##
phenyl 2-nitro-3- trifluoromethane- sulfonyl-phenyl H 98 159
##STR00273## 4- flourphenyl 1-Phenyl-1H- pyrazolo[3,4-d]
pyrimidin-4-yl H 95 711 ##STR00274## phenyl 2-(2-Fluoro-
phenoxymethyl)- 2-cyano oxazolyl H 94 809 ##STR00275## 3-azepan
1-yl-5-(4- triflouro- methoxy) phenylamino [1,3,5]triazyl adamantyl
H 89 812 ##STR00276## 3-azepan 1-yl-5-(4- triflouro- methoxy)
phenylamino [1,3,5]triazyl adamantyl H 99 881 ##STR00277##
cyclohexyl 5- (Benzo[1,3]dioxol- 5-ylamino)- 10b,10c-dihydro-
anthra[1,9-cd] isoxazol-6- one-yl H 82 882 ##STR00278## cyclohexyl
5- (Benzo[1,3]dioxol- 5-ylamino)- 10b,10c-dihydro- anthra[1,9-cd]
isoxazol-6- one-yl H 86 1019 ##STR00279## diphenylmethyl 2-Methyl-
thiazolo[3,2-b] [1,2,4]triazol-6- ol 4-methylphenyl methyl H 87
1021 ##STR00280## diphenylmethyl 2-Methyl- thiazolo[3,2-b]
[1,2,4]triazol-6- ol 4-methylphenyl methyl H 92 1026 ##STR00281##
diphenylmethyl 2-Methyl- thiazolo[3,2-b] [1,2,4]triazol-6- ol
4-methylphenyl methyl H 92 1027 ##STR00282## diphenylmethyl
2-Methyl- thiazolo[3,2-b] [1,2,4]triazol-6- ol 4-methylphenyl
methyl H 84 1028 ##STR00283## diphenylmethyl 2-Methyl-
thiazolo[3,2-b] [1,2,4]triazol-6- ol 4-methylphenyl methyl H 85
1039 ##STR00284## diphenylmethyl 2-Methyl- thiazolo[3,2-b]
[1,2,4]triazol-6- ol 4-methylphenyl methyl H 84 1069 ##STR00285##
2- phenylethyl 3-benzyl-3H- quinazolin-4-one- 2-yl O 86 1262
##STR00286## 4-hydroxy- cyclohexyl cyclopentyl O 4-methylphenyl 86
1280 ##STR00287## cycloheptyl tetrahydronapthyl O 2-chloro-4-
flourophenyl 87 1283 ##STR00288## cyclopentyl cyclooctyl O
4-chlorophenyl 100 1284 ##STR00289## cyclopentyl cyclohexyl O
4-chlorophenyl 95 1313 ##STR00290## C- Benzo[1,3] dioxol-5-
yl-methyl C-[3-(4-Chloro- phenyl)-2,5- dimethyl- pyrazolo[1,5-a]
pyrimidin-7-yl]- methyl H 92 1323 ##STR00291## 2-Pyridyl
C-(2-Benzyl- 5,6,7,8- tetrahydro- benzo[4,5]thieno
[2,3-d]pyrimidin-4- yl)-methyl H 88 2496 ##STR00292## 1-
chlorobenzyl 1-yl-3-(2- isopropyl-5- methyl- cyclohexyloxy)-
propan-2-ol H 82
Example 11
In Vitro Screen Using a Calcium Flux Assay for Trp-p8 Antagonist
Compounds Based Upon their Abilities to Suppress the Calcium Influx
Induced by Trp p8 Agonists in CHO/Trp-p8 Cells
[0257] This example discloses an in vitro assay system employed to
further screen and characterize candidate Trp-p8 antagonists.
[0258] Trp-p8 antagonists were also screened and characterized
using a calcium flux assay at 37.degree. C. as described in Example
8 with the following two distinctions: (1) the compound was
pre-mixed with the control agonist or only the control agonist is
added to the cells and suppression of the response to the agonist
is a measure of the potency of the compound as an antagonist and
(2) the compound, at different concentrations, was added to the
cells followed by addition of the control agonist after 2-3 minutes
and the suppression of response induced by agonist was a measure of
potency of the compound as an antagonist. Exemplary results are
presented in FIG. 5.
Example 12
An Animal Model System for Assaying the In Vivo Efficacy of
Candidate Trp-p8 Agonists and Antagonists for the Treatment of
Cancer
[0259] This Example provides an animal model system suitable for
determining the in vivo efficacy of candidate Trp-p8
modulators--including both agonists and antagonists.
[0260] Human prostate cancer xenografts expressing Trp-P8 (from Dr.
Robert Vessella's lab in University of Washington--as assessed by
in situ hybridization and immunohistochemistry using a protein
specific rabbit polyclonal antibody, T-904) as well as CHO (Chinese
Hamster Ovary) and EL-4 (Mouse Thymoma) cell lines were engineered
to express Trp-P8 and used to establish tumor models in mice.
Trp-P8 expression in these transfectants was confirmed by western
blots and immunofluorescence using a Trp-p8 specific antibody (GS
2.20) as well as by response to known agonists in a calcium influx
functional assay. In addition, these transfected cell lines were
susceptible to killing by Trp-p8 agonists as evident from the ATP
viability and apoptosis assays (as described herein in Examples 7
and 8).
[0261] A tumor model in mice was established by subcutaneously
injecting CHO/Trp-P8 cells in SCID mice. Trp-p8 expression in
tumors excised from these mice was confirmed by RT-PCR and western
blot analysis. Further tumor model development is carried out using
the human prostate cancer xenografts described above in athymic
nude or SCID mice and using an EL4/Trp-p8 transfectant in normal
mice. Prostate xenografts from other sources and other cell lines
that may be engineered to express Trp-p8 are also potential
candidates for building more model systems.
[0262] Based on results from in vitro and in vivo evaluations, a
set of trp-p8 agonists will be chosen to determine efficacy in
mice. The in vitro evaluations would include potency in cell
killing assay, aqueous solubility, plasma binding study and
metabolic stability (potential for a compound to be metabolized by
liver as determined by using hepatocytes and/or mouse microsomes).
The in vivo evaluations would include pharmacokinetics and toxicity
studies. The chosen compounds will be administered to mice with
Trp-p8 expressing tumors by different routes [oral, intravenous,
intraperitoneal, subcutaneous, intramuscular]. Tumor reduction and
survival of these mice will be evaluated at different dosages of
these compounds. The compound most effective in fighting tumor will
be chosen for further investigations
[0263] Although the present invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, changes and modifications can be carried out
without departing from the scope of the invention which is intended
to be limited only by the scope of the appended claims.
Sequence CWU 1
1
215674DNAHuman 1aagaaaatcc tgcttgacaa aaaccgtcac ttaggaaaag
atgtcctttc gggcagccag 60gctcagcatg aggaacagaa ggaatgacac tctggacagc
acccggaccc tgtactccag 120cgcgtctcgg agcacagact tgtcttacag
tgaaagcgac ttggtgaatt ttattcaagc 180aaattttaag aaacgagaat
gtgtcttctt tatcaaagat tccaaggcca cggagaatgt 240gtgcaagtgt
ggctatgccc agagccagca catggaaggc acccagatca accaaagtga
300gaaatggaac tacaagaaac acaccaagga atttcctacc gacgcctttg
gggatattca 360gtttgagaca ctggggaaga aagggaagta tatacgtctg
tcctgcgaca cggacgcgga 420aatcctttac gagctgctga cccagcactg
gcacctgaaa acacccaacc tggtcatttc 480tgtgaccggg ggcgccaaga
acttcgccct gaagccgcgc atgcgcaaga tcttcagccg 540gctcatctac
atcgcgcagt ccaaaggtgc ttggattctc acgggaggca cccattatgg
600cctgatgaag tacatcgggg aggtggtgag agataacacc atcagcagga
gttcagagga 660gaatattgtg gccattggca tagcagcttg gggcatggtc
tccaaccggg acaccctcat 720caggaattgc gatgctgagg gctatttttt
agcccagtac cttatggatg acttcacaag 780agatccactg tatatcctgg
acaacaacca cacacatttg ctgctcgtgg acaatggctg 840tcatggacat
cccactgtcg aagcaaagct ccggaatcag ctagagaagt atatctctga
900gcgcactatt caagattcca actatggtgg caagatcccc attgtgtgtt
ttgcccaagg 960aggtggaaaa gagactttga aagccatcaa tacctccatc
aaaaataaaa ttccttgtgt 1020ggtggtggaa ggctcgggcc agatcgctga
tgtgatcgct agcctggtgg aggtggagga 1080tgccctgaca tcttctgccg
tcaaggagaa gctggtgcgc tttttacccc gcacggtgtc 1140ccggctgcct
gaggaggaga ctgagagttg gatcaaatgg ctcaaagaaa ttctcgaatg
1200ttctcaccta ttaacagtta ttaaaatgga agaagctggg gatgaaattg
tgagcaatgc 1260catctcctac gctctataca aagccttcag caccagtgag
caagacaagg ataactggaa 1320tgggcagctg aagcttctgc tggagtggaa
ccagctggac ttagccaatg atgagatttt 1380caccaatgac cgccgatggg
agtctgctga ccttcaagaa gtcatgttta cggctctcat 1440aaaggacaga
cccaagtttg tccgcctctt tctggagaat ggcttgaacc tacggaagtt
1500tctcacccat gatgtcctca ctgaactctt ctccaaccac ttcagcacgc
ttgtgtaccg 1560gaatctgcag atcgccaaga attcctataa tgatgccctc
ctcacgtttg tctggaaact 1620ggttgcgaac ttccgaagag gcttccggaa
ggaagacaga aatggccggg acgagatgga 1680catagaactc cacgacgtgt
ctcctattac tcggcacccc ctgcaagctc tcttcatctg 1740ggccattctt
cagaataaga aggaactctc caaagtcatt tgggagcaga ccaggggctg
1800cactctggca gccctgggag ccagcaagct tctgaagact ctggccaaag
tgaagaacga 1860catcaatgct gctggggagt ccgaggagct ggctaatgag
tacgagaccc gggctgttga 1920gctgttcact gagtgttaca gcagcgatga
agacttggca gaacagctgc tggtctattc 1980ctgtgaagct tggggtggaa
gcaactgtct ggagctggcg gtggaggcca cagaccagca 2040tttcatcgcc
cagcctgggg tccagaattt tctttctaag caatggtatg gagagatttc
2100ccgagacacc aagaactgga agattatcct gtgtctgttt attataccct
tggtgggctg 2160tggctttgta tcatttagga agaaacctgt cgacaagcac
aagaagctgc tttggtacta 2220tgtggcgttc ttcacctccc ccttcgtggt
cttctcctgg aatgtggtct tctacatcgc 2280cttcctcctg ctgtttgcct
acgtgctgct catggatttc cattcggtgc cacacccccc 2340cgagctggtc
ctgtactcgc tggtctttgt cctcttctgt gatgaagtga gacagtggta
2400cgtaaatggg gtgaattatt ttactgacct gtggaatgtg atggacacgc
tggggctttt 2460ttacttcata gcaggaattg tatttcggct ccactcttct
aataaaagct ctttgtattc 2520tggacgagtc attttctgtc tggactacat
tattttcact ctaagattga tccacatttt 2580tactgtaagc agaaacttag
gacccaagat tataatgctg cagaggatgc tgatcgatgt 2640gttcttcttc
ctgttcctct ttgcggtgtg gatggtggcc tttggcgtgg ccaggcaagg
2700gatccttagg cagaatgagc agcgctggag gtggatattc cgttcggtca
tctacgagcc 2760ctacctggcc atgttcggcc aggtgcccag tgacgtggat
ggtaccacgt atgactttgc 2820ccactgcacc ttcactggga atgagtccaa
gccactgtgt gtggagctgg atgagcacaa 2880cctgccccgg ttccccgagt
ggatcaccat ccccctggtg tgcatctaca tgttatccac 2940caacatcctg
ctggtcaacc tgctggtcgc catgtttggc tacacggtgg gcaccgtcca
3000ggagaacaat gaccaggtct ggaagttcca gaggtacttc ctggtgcagg
agtactgcag 3060ccgcctcaat atccccttcc ccttcatcgt cttcgcttac
ttctacatgg tggtgaagaa 3120gtgcttcaag tgttgctgca aggagaaaaa
catggagtct tctgtctgct gtttcaaaaa 3180tgaagacaat gagactctgg
catgggaggg tgtcatgaag gaaaactacc ttgtcaagat 3240caacacaaaa
gccaacgaca cctcagagga aatgaggcat cgatttagac aactggatac
3300aaagcttaat gatctcaagg gtcttctgaa agagattgct aataaaatca
aataaaatca 3360aataaaactg tatgaaactc taatggagaa aaatctaatt
atagcaagat catattaagg 3420aatgctgatg aacaattttg ctatcgacta
ctaaatgaga gattttcaga cccctgggta 3480catggtggat gattttaaat
caccctagtg tgctgagacc ttgagaataa agtgtgtgat 3540tggtttcata
cttgaagacg gatataaagg aagaatattt cctttatgtg tttctccaga
3600atggtgcctg tttctctctg tgtctcaatg cctgggactg gaggttgata
gtttaagtgt 3660gttcttaccg cctccttttt cctttaatct tatttttgat
gaacacatat ataggagaac 3720atctatccta tgaataagaa cctggtcatg
ctttactcct gtattgttat tttgttcatt 3780tccaattgat tctctacttt
tccctttttt gtattatgtg actaattagt tggcatattg 3840ttaaaagtct
ctcaaattag gccagattct aaaacatgct gcagcaagag gaccccgctc
3900tcttcaggaa aagtgttttc atttctcagg atgcttctta cctgtcagag
gaggtgacaa 3960ggcagtctct tgctctcttg gactcaccag gctcctattg
aaggaaccac ccccattcct 4020aaatatgtga aaagtcgccc aaaatgcaac
cttgaaaggc actactgact ttgttcttat 4080tggatactcc tcttatttat
tatttttcca ttaaaaataa tagctggcta ttatagaaaa 4140tttagaccat
acagagatgt agaaagaaca taaattgtcc ccattacctt aaggtaatca
4200ctgctaacaa tttctggatg gtttttcaag tctatttttt ttctatgtat
gtctcaattc 4260tctttcaaaa ttttacagaa tgttatcata ctacatatat
actttttatg taagcttttt 4320cacttagtat tttatcaaat atgtttttat
tatattcata gccttcttaa acattatatc 4380aataattgca taataggcaa
cctctagcga ttaccataat tttgctcatt gaaggctatc 4440tccagttgat
cattgggatg agcatctttg tgcatgaatc ctattgctgt atttgggaaa
4500attttccaag gttagattcc aataaatatc tatttattat taaatattaa
aatatcgatt 4560tattattaaa accatttata aggctttttc ataaatgtat
agcaaatagg aattattaac 4620ttgagcataa gatatgagat acatgaacct
gaactattaa aataaaatat tatatttaac 4680cctagtttaa gaagaagtca
atatgcttat ttaaatatta tggatggtgg gcagatcact 4740tgaggtcagg
agttcgagac cagcctggcc aacatggcaa aaccacatct ctactaaaaa
4800taaaaaaatt agctgggtgt ggtggtgcac tcctgtaatc ccagctactc
agaaggctga 4860ggtacaagaa ttgctggaac ctgggaggcg gaggttgcag
tgaaccaaga ttgcaccact 4920gcactccagc cggggtgaca gagtgagact
ccgactgaaa ataaataaat aaataaataa 4980ataaataaat aaataaatat
tatggatggt gaagggaatg gtatagaatt ggagagatta 5040tcttactgaa
cacctgtagt cccagctttc tctggaagtg gtggtatttg agcaggatgt
5100gcacaaggca attgaaatgc ccataattag tttctcagct ttgaatacac
tataaactca 5160gtggctgaag gaggaaattt tagaaggaag ctactaaaag
atctaatttg aaaaactaca 5220aaagcattaa ctaaaaaagt ttattttcct
tttgtctggg cagtagtgaa aataactact 5280cacaacattc actatgtttg
caaggaatta acacaaataa aagatgcctt tttacttaaa 5340cgccaagaca
gaaaacttgc ccaatactga gaagcaactt gcattagaga gggaactgtt
5400aaatgttttc aacccagttc atctggtgga tgtttttgca ggttactctg
agaattttgc 5460ttatgaaaaa tcattatttt tagtgtagtt cacaataatg
tattgaacat acttctaatc 5520aaaggtgcta tgtccttgtg tatggtacta
aatgtgtcct gtgtactttt gcacaactga 5580gaatcctgcg gcttggttta
atgagtgtgt tcatgaaata aataatggag gaattgtcaa 5640aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaa 567421104PRTHuman 2Met Ser Phe Arg Ala
Ala Arg Leu Ser Met Arg Asn Arg Arg Asn Asp1 5 10 15Thr Leu Asp Ser
Thr Arg Thr Leu Tyr Ser Ser Ala Ser Arg Ser Thr 20 25 30Asp Leu Ser
Tyr Ser Glu Ser Asp Leu Val Asn Phe Ile Gln Ala Asn 35 40 45Phe Lys
Lys Arg Glu Cys Val Phe Phe Ile Lys Asp Ser Lys Ala Thr 50 55 60Glu
Asn Val Cys Lys Cys Gly Tyr Ala Gln Ser Gln His Met Glu Gly65 70 75
80Thr Gln Ile Asn Gln Ser Glu Lys Trp Asn Tyr Lys Lys His Thr Lys
85 90 95Glu Phe Pro Thr Asp Ala Phe Gly Asp Ile Gln Phe Glu Thr Leu
Gly 100 105 110Lys Lys Gly Lys Tyr Ile Arg Leu Ser Cys Asp Thr Asp
Ala Glu Ile 115 120 125Leu Tyr Glu Leu Leu Thr Gln His Trp His Leu
Lys Thr Pro Asn Leu 130 135 140Val Ile Ser Val Thr Gly Gly Ala Lys
Asn Phe Ala Leu Lys Pro Arg145 150 155 160Met Arg Lys Ile Phe Ser
Arg Leu Ile Tyr Ile Ala Gln Ser Lys Gly 165 170 175Ala Trp Ile Leu
Thr Gly Gly Thr His Tyr Gly Leu Met Lys Tyr Ile 180 185 190Gly Glu
Val Val Arg Asp Asn Thr Ile Ser Arg Ser Ser Glu Glu Asn 195 200
205Ile Val Ala Ile Gly Ile Ala Ala Trp Gly Met Val Ser Asn Arg Asp
210 215 220Thr Leu Ile Arg Asn Cys Asp Ala Glu Gly Tyr Phe Leu Ala
Gln Tyr225 230 235 240Leu Met Asp Asp Phe Thr Arg Asp Pro Leu Tyr
Ile Leu Asp Asn Asn 245 250 255His Thr His Leu Leu Leu Val Asp Asn
Gly Cys His Gly His Pro Thr 260 265 270Val Glu Ala Lys Leu Arg Asn
Gln Leu Glu Lys Tyr Ile Ser Glu Arg 275 280 285Thr Ile Gln Asp Ser
Asn Tyr Gly Gly Lys Ile Pro Ile Val Cys Phe 290 295 300Ala Gln Gly
Gly Gly Lys Glu Thr Leu Lys Ala Ile Asn Thr Ser Ile305 310 315
320Lys Asn Lys Ile Pro Cys Val Val Val Glu Gly Ser Gly Gln Ile Ala
325 330 335Asp Val Ile Ala Ser Leu Val Glu Val Glu Asp Ala Leu Thr
Ser Ser 340 345 350Ala Val Lys Glu Lys Leu Val Arg Phe Leu Pro Arg
Thr Val Ser Arg 355 360 365Leu Pro Glu Glu Glu Thr Glu Ser Trp Ile
Lys Trp Leu Lys Glu Ile 370 375 380Leu Glu Cys Ser His Leu Leu Thr
Val Ile Lys Met Glu Glu Ala Gly385 390 395 400Asp Glu Ile Val Ser
Asn Ala Ile Ser Tyr Ala Leu Tyr Lys Ala Phe 405 410 415Ser Thr Ser
Glu Gln Asp Lys Asp Asn Trp Asn Gly Gln Leu Lys Leu 420 425 430Leu
Leu Glu Trp Asn Gln Leu Asp Leu Ala Asn Asp Glu Ile Phe Thr 435 440
445Asn Asp Arg Arg Trp Glu Ser Ala Asp Leu Gln Glu Val Met Phe Thr
450 455 460Ala Leu Ile Lys Asp Arg Pro Lys Phe Val Arg Leu Phe Leu
Glu Asn465 470 475 480Gly Leu Asn Leu Arg Lys Phe Leu Thr His Asp
Val Leu Thr Glu Leu 485 490 495Phe Ser Asn His Phe Ser Thr Leu Val
Tyr Arg Asn Leu Gln Ile Ala 500 505 510Lys Asn Ser Tyr Asn Asp Ala
Leu Leu Thr Phe Val Trp Lys Leu Val 515 520 525Ala Asn Phe Arg Arg
Gly Phe Arg Lys Glu Asp Arg Asn Gly Arg Asp 530 535 540Glu Met Asp
Ile Glu Leu His Asp Val Ser Pro Ile Thr Arg His Pro545 550 555
560Leu Gln Ala Leu Phe Ile Trp Ala Ile Leu Gln Asn Lys Lys Glu Leu
565 570 575Ser Lys Val Ile Trp Glu Gln Thr Arg Gly Cys Thr Leu Ala
Ala Leu 580 585 590Gly Ala Ser Lys Leu Leu Lys Thr Leu Ala Lys Val
Lys Asn Asp Ile 595 600 605Asn Ala Ala Gly Glu Ser Glu Glu Leu Ala
Asn Glu Tyr Glu Thr Arg 610 615 620Ala Val Glu Leu Phe Thr Glu Cys
Tyr Ser Ser Asp Glu Asp Leu Ala625 630 635 640Glu Gln Leu Leu Val
Tyr Ser Cys Glu Ala Trp Gly Gly Ser Asn Cys 645 650 655Leu Glu Leu
Ala Val Glu Ala Thr Asp Gln His Phe Ile Ala Gln Pro 660 665 670Gly
Val Gln Asn Phe Leu Ser Lys Gln Trp Tyr Gly Glu Ile Ser Arg 675 680
685Asp Thr Lys Asn Trp Lys Ile Ile Leu Cys Leu Phe Ile Ile Pro Leu
690 695 700Val Gly Cys Gly Phe Val Ser Phe Arg Lys Lys Pro Val Asp
Lys His705 710 715 720Lys Lys Leu Leu Trp Tyr Tyr Val Ala Phe Phe
Thr Ser Pro Phe Val 725 730 735Val Phe Ser Trp Asn Val Val Phe Tyr
Ile Ala Phe Leu Leu Leu Phe 740 745 750Ala Tyr Val Leu Leu Met Asp
Phe His Ser Val Pro His Pro Pro Glu 755 760 765Leu Val Leu Tyr Ser
Leu Val Phe Val Leu Phe Cys Asp Glu Val Arg 770 775 780Gln Trp Tyr
Val Asn Gly Val Asn Tyr Phe Thr Asp Leu Trp Asn Val785 790 795
800Met Asp Thr Leu Gly Leu Phe Tyr Phe Ile Ala Gly Ile Val Phe Arg
805 810 815Leu His Ser Ser Asn Lys Ser Ser Leu Tyr Ser Gly Arg Val
Ile Phe 820 825 830Cys Leu Asp Tyr Ile Ile Phe Thr Leu Arg Leu Ile
His Ile Phe Thr 835 840 845Val Ser Arg Asn Leu Gly Pro Lys Ile Ile
Met Leu Gln Arg Met Leu 850 855 860Ile Asp Val Phe Phe Phe Leu Phe
Leu Phe Ala Val Trp Met Val Ala865 870 875 880Phe Gly Val Ala Arg
Gln Gly Ile Leu Arg Gln Asn Glu Gln Arg Trp 885 890 895Arg Trp Ile
Phe Arg Ser Val Ile Tyr Glu Pro Tyr Leu Ala Met Phe 900 905 910Gly
Gln Val Pro Ser Asp Val Asp Gly Thr Thr Tyr Asp Phe Ala His 915 920
925Cys Thr Phe Thr Gly Asn Glu Ser Lys Pro Leu Cys Val Glu Leu Asp
930 935 940Glu His Asn Leu Pro Arg Phe Pro Glu Trp Ile Thr Ile Pro
Leu Val945 950 955 960Cys Ile Tyr Met Leu Ser Thr Asn Ile Leu Leu
Val Asn Leu Leu Val 965 970 975Ala Met Phe Gly Tyr Thr Val Gly Thr
Val Gln Glu Asn Asn Asp Gln 980 985 990Val Trp Lys Phe Gln Arg Tyr
Phe Leu Val Gln Glu Tyr Cys Ser Arg 995 1000 1005Leu Asn Ile Pro
Phe Pro Phe Ile Val Phe Ala Tyr Phe Tyr Met Val 1010 1015 1020Val
Lys Lys Cys Phe Lys Cys Cys Cys Lys Glu Lys Asn Met Glu Ser1025
1030 1035 1040Ser Val Cys Cys Phe Lys Asn Glu Asp Asn Glu Thr Leu
Ala Trp Glu 1045 1050 1055Gly Val Met Lys Glu Asn Tyr Leu Val Lys
Ile Asn Thr Lys Ala Asn 1060 1065 1070Asp Thr Ser Glu Glu Met Arg
His Arg Phe Arg Gln Leu Asp Thr Lys 1075 1080 1085Leu Asn Asp Leu
Lys Gly Leu Leu Lys Glu Ile Ala Asn Lys Ile Lys 1090 1095 1100
* * * * *