U.S. patent application number 12/368302 was filed with the patent office on 2009-11-19 for substituted quinolin-4-ylamine analogues.
This patent application is currently assigned to Neurogen Corporation. Invention is credited to Rajagopal Bakthavatchalam, Timothy M. Caldwell, Bertrand L. Chenard, Stephane De Lombaert, Kevin J. Hodgetts.
Application Number | 20090286767 12/368302 |
Document ID | / |
Family ID | 34079320 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090286767 |
Kind Code |
A1 |
Bakthavatchalam; Rajagopal ;
et al. |
November 19, 2009 |
SUBSTITUTED QUINOLIN-4-YLAMINE ANALOGUES
Abstract
Substituted quinolin-4-ylamine analogues are provided. Such
compounds are ligands that may be used to modulate specific
receptor activity in vivo or in vitro, and are particularly useful
in the treatment of conditions associated with pathological
receptor activation in humans, domesticated companion animals and
livestock animals. Pharmaceutical compositions and methods for
using them to treat such disorders are provided, as are methods for
using such ligands for receptor localization studies.
Inventors: |
Bakthavatchalam; Rajagopal;
(Madison, CT) ; Caldwell; Timothy M.; (Guilford,
CT) ; Chenard; Bertrand L.; (Waterford, CT) ;
De Lombaert; Stephane; (Madison, CT) ; Hodgetts;
Kevin J.; (Killingworth, CT) |
Correspondence
Address: |
EDWARD ANGELL PALMER & DODGE, LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Neurogen Corporation
Branford
CT
|
Family ID: |
34079320 |
Appl. No.: |
12/368302 |
Filed: |
February 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10891832 |
Jul 14, 2004 |
7488740 |
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12368302 |
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60486948 |
Jul 14, 2003 |
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Current U.S.
Class: |
514/210.21 ;
435/375; 514/234.5; 514/235.2; 514/252.04; 514/255.05; 514/275;
514/293; 514/300; 514/313; 544/127; 544/128; 544/238; 544/331;
544/405; 546/122; 546/160; 546/83 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 7/12 20180101; A61P 11/06 20180101; A61P 19/02 20180101; C07D
491/14 20130101; A61P 17/04 20180101; A61P 1/04 20180101; A61P
29/00 20180101; A61P 13/10 20180101; C07D 215/44 20130101; A61P
25/06 20180101; A61P 17/02 20180101; A61P 11/00 20180101; A61P
39/02 20180101; A61P 1/02 20180101; C07D 401/04 20130101; C07D
471/04 20130101; A61P 35/00 20180101; A61P 9/10 20180101; A61P
11/14 20180101; A61P 3/04 20180101; A61P 25/04 20180101; A61P 13/00
20180101; A61P 25/02 20180101 |
Class at
Publication: |
514/210.21 ;
435/375; 514/234.5; 514/235.2; 514/252.04; 514/255.05; 514/275;
514/293; 514/300; 514/313; 544/127; 544/128; 544/238; 544/331;
544/405; 546/83; 546/122; 546/160 |
International
Class: |
A61K 31/397 20060101
A61K031/397; C12N 5/00 20060101 C12N005/00; A61K 31/5377 20060101
A61K031/5377; A61K 31/501 20060101 A61K031/501; A61K 31/497
20060101 A61K031/497; A61K 31/506 20060101 A61K031/506; A61K
31/4375 20060101 A61K031/4375; A61K 31/4709 20060101 A61K031/4709;
C07D 413/14 20060101 C07D413/14; C07D 405/14 20060101 C07D405/14;
C07D 401/14 20060101 C07D401/14; C07D 401/10 20060101 C07D401/10;
A61P 3/04 20060101 A61P003/04; A61P 29/00 20060101 A61P029/00 |
Claims
1. A compound of the formula: ##STR00250## or a pharmaceutically
acceptable salt thereof, wherein: at least one of Y and Z is N; and
the other of Y and Z is N or CR.sub.1; R.sub.1 is hydrogen,
halogen, cyano, amino, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4haloalkyl, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.4haloalkoxy or mono- or
di-(C.sub.1-C.sub.4alkyl)amino; R.sub.2 is: (i) hydrogen, halogen
or cyano; (ii) a group of the formula --R.sub.c-M-A-R.sub.y,
wherein: R.sub.c is C.sub.0-C.sub.3alkyl or is joined to R.sub.y or
R.sub.z, to form a 4- to 10-membered carbocycle or heterocycle that
is substituted with from 0 to 2 substituents independently chosen
from R.sub.b; M is a single covalent bond, O, S, SO.sub.2,
C(.dbd.O), OC(.dbd.O), C(.dbd.O)O, O--C(.dbd.O)O,
C(.dbd.O)N(R.sub.z), OC(.dbd.O)N(R.sub.z), N(R.sub.z)C(.dbd.O),
N(R.sub.z)SO.sub.2, SO.sub.2N(R.sub.z) or N(R.sub.z); A is a single
covalent bond or C.sub.1-C.sub.8alkyl substituted with from 0 to 3
substituents independently chosen from R.sub.b; and R.sub.y and
R.sub.z, if present, are: (a) independently hydrogen,
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkyl ether,
C.sub.2-C.sub.8alkenyl, a 4-to 10-membered carbocycle or
heterocycle, or joined to R.sub.c to form a 4- to 10-membered
carbocycle or heterocycle, wherein each non-hydrogen R.sub.y and
R.sub.z is substituted with from 0 to 6 substituents independently
chosen from R.sub.b; or (b) joined to form a 4- to 10-membered
carbocycle or heterocycle that is substituted with from 0 to 6
substituents independently chosen from R.sub.b; such that R.sub.2
is not --NH.sub.2; or (iii) taken together with R.sub.7 to form a
fused 5- to 7-membered ring that is substituted with from 0 to 3
substituents independently chosen from oxo and
C.sub.1-C.sub.4alkyl; R.sub.7 is hydrogen, COOH,
C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.4alkoxycarbonyl or taken together with R.sub.2 to
form a fused, optionally substituted ring; Ar.sub.1 is phenyl or
6-membered heteroaryl, each of which is unsubstituted or
substituted ortho to the point of attachment with 1 or 2
substituents independently chosen from groups of the formula
LR.sub.a; Ar.sub.2 is 6- to 10-membered aryl or 5- to 10-membered
heteroaryl, each of which is substituted with from 0 to 6
substituents independently chosen from oxo and groups of the
formula LR.sub.a; L is independently selected at each occurrence
from a single covalent bond, O, C(.dbd.O), OC(.dbd.O), C(.dbd.O)O,
OC(.dbd.O)O, S(O).sub.m, N(R.sub.x), C(.dbd.O)N(R.sub.x),
N(R.sub.x)C(.dbd.O), N(R.sub.x)S(O).sub.m, S(O).sub.mN(R.sub.x) and
N[S(O).sub.mR.sub.w]S(O).sub.m; wherein m is independently selected
at each occurrence from 0, 1 and 2; R.sub.x is independently
selected at each occurrence from hydrogen, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkanoyl and C.sub.1-C.sub.6alkylsulfonyl; and
R.sub.w is hydrogen or C.sub.1-C.sub.6alkyl; R.sub.a is
independently selected at each occurrence from: (i) hydrogen,
halogen, cyano and nitro; and (ii) C.sub.1-C.sub.8alkyl,
C.sub.2-C.sub.8alkenyl, C.sub.2-C.sub.8alkynyl,
C.sub.1-C.sub.8haloalkyl, C.sub.2-C.sub.8alkyl ether, mono- and
di-(C.sub.1-C.sub.8alkyl)amino and (3- to 10-membered
heterocycle)C.sub.0-C.sub.6alkyl, each of which is substituted with
from 0 to 6 substituents independently selected from R.sub.b; and
R.sub.b is independently chosen at each occurrence from hydroxy,
halogen, amino, aminocarbonyl, cyano, nitro, oxo, COOH,
C.sub.1-C.sub.8alkyl, C.sub.1-C.sub.8alkoxy,
C.sub.1-C.sub.8alkylthio, C.sub.1-C.sub.8alkanoyl,
C.sub.1-C.sub.8alkanoyloxy, C.sub.1-C.sub.8alkoxycarbonyl,
C.sub.1-C.sub.8alkyl ether, C.sub.1-C.sub.8hydroxyalkyl,
C.sub.1-C.sub.8haloalkyl, phenylC.sub.0-C.sub.8alkyl, mono- and
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.8alkylsulfonyl and (4- to 7-membered
heterocycle)C.sub.0-C.sub.8alkyl.
2. A compound or salt according to claim 1, wherein Z is N.
3. A compound or salt according to claim 1, wherein Y is N.
4. A compound or salt according to claim 2, wherein Y is CH.
5. A compound or salt according to claim 1, wherein Y and Z are
N.
6. A compound or salt according to claim 1, wherein Ar.sub.2 is
phenyl or a 6-membered heteroaryl, each of which is substituted
with from 0 to 3 substituents independently selected from (a)
groups of the formula LR.sub.a and (b) groups that are taken
together to form a fused, 5- to 7-membered heterocyclic ring that
is substituted with from 0 to 3 substituents independently selected
from R.sub.b.
7. (canceled)
8. (canceled)
9. A compound or salt according to claim 6, wherein Ar.sub.1 is
phenyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl, each of
which is unsubstituted or substituted with halogen, cyano,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6alkoxy or C.sub.1-C.sub.6haloalkoxy.
10. A compound or salt according to claim 9, wherein: Ar.sub.1 is
phenyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl substituted
with halogen, cyano, C.sub.1-C.sub.4alkyl or
C.sub.1-C.sub.4haloalkyl; and Ar.sub.2 is phenyl, pyridyl,
pyrimidinyl, pyrazinyl or pyridazinyl, each of which is substituted
with halogen, cyano, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4hydroxyalkyl, C.sub.1-C.sub.4alkanoyl,
C.sub.1-C.sub.4haloalkyl, C.sub.1-C.sub.4alkylsulfonyl or
C.sub.1-C.sub.4haloalkylsulfonyl.
11. A compound or salt according to claim 1, wherein R.sub.2 is:
(i) hydrogen, hydroxy or halogen; or (ii) C.sub.1-C.sub.6alkyl,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6-aminoalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.2-C.sub.6alkyl ether, mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl or (4- to
7-membered heterocycloalkyl)C.sub.0-C.sub.4alkyl, each of which is
substituted with from 0 to 4 substituents independently chosen from
halogen, cyano, hydroxy, amino, oxo, mono- and
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkyl
C.sub.1-C.sub.6alkoxy and C.sub.1-C.sub.6haloalkyl.
12.-16. (canceled)
17. A compound of the formula: ##STR00251## or a pharmaceutically
acceptable salt thereof, wherein: Y and Z are each independently N
or CR.sub.1; R.sub.1 is independently selected at each occurrence
from hydrogen, halogen, cyano, amino, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6haloalkoxy and mono- and
di-(C.sub.1-C.sub.6alkyl)amino; R.sub.2 is: (i) hydrogen, halogen
or cyano; (ii) a group of the formula --R.sub.c-M-A-R.sub.Y,
wherein: R.sub.c is C.sub.0-C.sub.3alkyl or is joined to R.sub.y or
R.sub.z, to form a 4- to 10-membered carbocycle or heterocycle that
is substituted with from 0 to 2 substituents independently chosen
from R.sub.b; M is a single covalent bond, O, S, SO.sub.2,
C(.dbd.O), OC(.dbd.O), C(.dbd.O)O, O--C(.dbd.O)O,
C(.dbd.O)N(R.sub.z), OC(.dbd.O)N(R.sub.z), N(R.sub.z)C(.dbd.O),
N(R.sub.z)SO.sub.2, SO.sub.2N(R.sub.z) or N(R.sub.z); A is a single
covalent bond or C.sub.1-C.sub.8alkyl substituted with from 0 to 3
substituents independently chosen from R.sub.b; and R.sub.y and
R.sub.z, if present, are: (a) independently hydrogen,
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkyl ether,
C.sub.2-C.sub.8alkenyl, a 4-to 10-membered carbocycle or
heterocycle, or joined to R.sub.c to form a 4- to 10-membered
carbocycle or heterocycle, wherein each non-hydrogen R.sub.y and
R.sub.z is substituted with from 0 to 6 substituents independently
chosen from R.sub.b; or (b) joined to form a 4- to 10-membered
carbocycle or heterocycle that is substituted with from 0 to 6
substituents independently chosen from R.sub.b; or (iii) taken
together with R.sub.7 to form a fused 5- to 7-membered ring that is
substituted with from 0 to 3 substituents independently chosen from
oxo and C.sub.1-C.sub.4alkyl; R.sub.7 is hydrogen, COOH,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6alkoxycarbonyl or taken together with R.sub.2 to
form a fused, optionally substituted ring; Ar.sub.1 is phenyl or
6-membered heteroaryl, each of which is unsubstituted or
substituted ortho to the point of attachment with 1 or 2
substituents independently chosen from groups of the formula
LR.sub.a; Ar.sub.2 is 5- to 10-membered heteroaryl that is
substituted with from 0 to 6 substituents independently chosen from
oxo and groups of the formula LR.sub.a; L is independently selected
at each occurrence from a single covalent bond, O, C(.dbd.O),
OC(.dbd.O), C(.dbd.O)O, OC(.dbd.O)O, S(O).sub.m, N(R.sub.x),
C(.dbd.O)N(R.sub.x), N(R.sub.x)C(.dbd.O), N(R.sub.x)S(O).sub.m,
S(O).sub.mN(R.sub.x) and N[S(O).sub.mR.sub.w]S(O).sub.m; wherein m
is independently selected at each occurrence from 0, 1 and 2;
R.sub.x is independently selected at each occurrence from hydrogen,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkanoyl and
C.sub.1-C.sub.6alkylsulfonyl; and R.sub.w is hydrogen or
C.sub.1-C.sub.6alkyl; R.sub.a is independently selected at each
occurrence from: (i) hydrogen, halogen, cyano and nitro; and (ii)
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, C.sub.1-C.sub.8haloalkyl,
C.sub.2-C.sub.8alkyl ether, mono- and
di-(C.sub.1-C.sub.8alkyl)amino and (3- to 10-membered
heterocycle)C.sub.0-C.sub.6alkyl, each of which is substituted with
from 0 to 6 substituents independently selected from R.sub.b; and
R.sub.b is independently chosen at each occurrence from hydroxy,
halogen, amino, aminocarbonyl, cyano, nitro, oxo, COOH,
C.sub.1-C.sub.8alkyl, C.sub.1-C.sub.8alkoxy,
C.sub.1-C.sub.8alkylthio, C.sub.1-C.sub.8alkanoyl,
C.sub.1-C.sub.8alkanoyloxy, C.sub.1-C.sub.8alkoxycarbonyl,
C.sub.1-C.sub.8alkyl ether, C.sub.1-C.sub.8hydroxyalkyl,
C.sub.1-C.sub.8haloalkyl, phenylC.sub.0-C.sub.8alkyl, mono- and
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.8alkylsulfonyl and (4- to 7-membered
heterocycle)C.sub.0-C.sub.8alkyl.
18.-26. (canceled)
27. A compound of the formula: ##STR00252## or a pharmaceutically
acceptable salt thereof, wherein: Y and Z are each independently N
or CR.sub.1; R.sub.1 is independently selected at each occurrence
from hydrogen, halogen, cyano, amino, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6haloalkoxy and mono- and
di-(C.sub.1-C.sub.6alkyl)amino; R.sub.2 is: (i) halogen or cyano;
(ii) a group of the formula --R.sub.c-M-A-R.sub.y, wherein: R.sub.c
is C.sub.0-C.sub.3alkyl or is joined to R.sub.y or R.sub.z, to form
a 4- to 10-membered carbocycle or heterocycle that is substituted
with from 0 to 2 substituents independently chosen from R.sub.b; M
is a single covalent bond, O, S, SO.sub.2, C(.dbd.O), OC(.dbd.O),
C(.dbd.O)O, O--C(.dbd.O)O, C(.dbd.O)N(R.sub.z),
OC(.dbd.O)N(R.sub.z), N(R.sub.z)C(.dbd.O), N(R.sub.z)SO.sub.2,
SO.sub.2N(R.sub.z) or N(R.sub.z); A is a single covalent bond or
C.sub.1-C.sub.8alkyl substituted with from 0 to 3 substituents
independently chosen from R.sub.b; and R.sub.y and R.sub.z, if
present, are: (a) independently hydrogen, C.sub.1-C.sub.8alkyl,
C.sub.2-C.sub.8alkyl ether, C.sub.2-C.sub.8alkenyl, a 4-to
10-membered carbocycle or heterocycle, or joined to R.sub.c to form
a 4- to 10-membered carbocycle or heterocycle, wherein each
non-hydrogen R.sub.y and R.sub.z is substituted with from 0 to 6
substituents independently chosen from R.sub.b; or (b) joined to
form a 4- to 10-membered carbocycle or heterocycle that is
substituted with from 0 to 6 substituents independently chosen from
R.sub.b; such that R.sub.2 is not --NH.sub.2; or (iii) taken
together with R.sub.7 to form a fused 5- to 7-membered ring that is
substituted with from 0 to 3 substituents independently chosen from
oxo and C.sub.1-C.sub.4alkyl; R.sub.7 is hydrogen, COOH,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6alkoxycarbonyl or taken together with R.sub.2 to
form a fused, optionally substituted ring; Ar.sub.1 is phenyl or
6-membered heteroaryl, each of which is unsubstituted or
substituted ortho to the point of attachment with 1 or 2
substituents independently chosen from groups of the formula
LR.sub.a; Ar.sub.2 is 6- to 10-membered aryl or 5- to 10-membered
heteroaryl, each of which is substituted with from 0 to 6
substituents independently chosen from oxo and groups of the
formula LR.sub.a; L is independently selected at each occurrence
from a single covalent bond, O, C(.dbd.O), OC(.dbd.O), C(.dbd.O)O,
OC(.dbd.O)O, S(O).sub.m, N(R.sub.x), C(.dbd.O)N(R.sub.x),
N(R.sub.x)C(.dbd.O), N(R.sub.x)S(O).sub.m, S(O).sub.mN(R.sub.x) and
N[S(O).sub.mR.sub.w]S(O).sub.m; wherein m is independently selected
at each occurrence from 0, 1 and 2; R.sub.x is independently
selected at each occurrence from hydrogen, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkanoyl and C.sub.1-C.sub.6alkylsulfonyl; and
R.sub.w is hydrogen or C.sub.1-C.sub.6alkyl; R.sub.a is
independently selected at each occurrence from: (i) hydrogen,
halogen, cyano and nitro; and (ii) C.sub.1-C.sub.8alkyl,
C.sub.2-C.sub.8alkenyl, C.sub.2-C.sub.8alkynyl,
C.sub.1-C.sub.8haloalkyl, C.sub.2-C.sub.8alkyl ether, mono- and
di-(C.sub.1-C.sub.8alkyl)amino and (3- to 10-membered
heterocycle)C.sub.0-C.sub.6alkyl, each of which is substituted with
from 0 to 6 substituents independently selected from R.sub.b; and
R.sub.b is independently chosen at each occurrence from hydroxy,
halogen, amino, aminocarbonyl, cyano, nitro, oxo, COOH,
C.sub.1-C.sub.8alkyl, C.sub.1-C.sub.8alkoxy,
C.sub.1-C.sub.8alkylthio, C.sub.1-C.sub.8alkanoyl,
C.sub.1-C.sub.8alkanoyloxy, C.sub.1-C.sub.8alkoxycarbonyl,
C.sub.1-C.sub.8alkyl ether, C.sub.1-C.sub.8hydroxyalkyl,
C.sub.1-C.sub.8haloalkyl, phenylC.sub.0-C.sub.8alkyl, mono- and
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.8alkylsulfonyl and (4- to 7-membered
heterocycle)C.sub.0-C.sub.8alkyl.
28. A compound or salt according to claim 27, wherein at least one
of Y and Z is N.
29. A compound or salt according to claim 27, wherein Y and Z are
both CH.
30. A compound or salt according to claim 27, wherein Ar.sub.2 is
phenyl or a 6-membered heteroaryl, each of which is substituted
with from 0 to 3 substituents independently selected from (a)
groups of the formula LR.sub.a and (b) groups that are taken
together to form a fused, 5- to 7-membered heterocyclic ring that
is substituted with from 0 to 3 substituents independently selected
from R.sub.b.
31. (canceled)
32. (canceled)
33. A compound or salt according to claim 27, wherein Ar.sub.1 is
phenyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl, each of
which is unsubstituted or substituted with halogen, cyano,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6alkoxy or C.sub.1-C.sub.6haloalkoxy.
34.-46. (canceled)
47. A compound or salt according to claim 27, wherein the compound
has the formula: ##STR00253## wherein: Ar.sub.1 is phenyl, pyridyl,
pyrimidinyl, pyrazinyl or pyridazinyl, each of which is
unsubstituted or substituted with halogen, cyano,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6alkoxy or C.sub.1-C.sub.6haloalkoxy; Ar.sub.2 is
phenyl or a 6-membered heteroaryl, each of which is substituted
with from 0 to 3 substituents independently selected from (a)
groups of the formula LR.sub.a and (b) groups that are taken
together to form a fused, 5- to 7-membered heterocyclic ring that
is substituted with from 0 to 3 substituents independently selected
from R.sub.b; R.sub.3 is selected from: (i) hydrogen; and (ii)
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, C.sub.6-C.sub.10arylC.sub.0-C.sub.8alkyl,
and 5- to 10-membered heterocycleC.sub.0-C.sub.8alkyl, each of
which is substituted with from 0 to 6 substituents independently
selected from R.sub.b; R.sub.5 and R.sub.6 are, independently at
each occurrence: (i) each independently selected from hydrogen,
hydroxy and C.sub.1-C.sub.6alkyl; or (ii) taken together to form a
keto group; and n is 1, 2 or 3.
48. A compound or salt according to claim 47, wherein Ar.sub.2 is
phenyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl, each of
which is substituted with 0, 1 or 2 substituents independently
selected from halogen, cyano, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6alkyl ether, C.sub.1-C.sub.6alkanoyl,
C.sub.1-C.sub.6alkylsulfonyl, C.sub.1-C.sub.6haloalkylsulfonyl,
amino, mono- and di-(C.sub.1-C.sub.6alkyl)amino.
49.-59. (canceled)
60. A pharmaceutical composition, comprising at least one compound
or salt according to claim 1 in combination with a physiologically
acceptable carrier or excipient.
61. A method for reducing calcium conductance of a cellular
capsaicin receptor, comprising contacting a cell expressing a
capsaicin receptor with at least one compound or salt according to
claim 1, and thereby reducing calcium conductance of the capsaicin
receptor.
62.-70. (canceled)
71. A method for inhibiting binding of vanilloid ligand to a
capsaicin receptor in vitro, the method comprising contacting
capsaicin receptor with at least one compound or salt according to
claim 1, in an amount sufficient to detectably inhibit vanilloid
ligand binding to capsaicin receptor.
72. A method for inhibiting binding of vanilloid ligand to a
capsaicin receptor in a patient, the method comprising contacting
cells expressing capsaicin receptor with at least one compound or
salt according to claim 1, in an amount sufficient to detectably
inhibit vanilloid ligand binding to cells expressing a cloned
capsaicin receptor in vitro, and thereby inhibiting binding of
vanilloid ligand to the capsaicin receptor in the patient.
73. (canceled)
74. A method for treating a condition responsive to capsaicin
receptor modulation in a patient, comprising administering to the
patient a capsaicin receptor modulatory amount of a compound or
salt according to claim 1, and thereby alleviating the condition in
the patient.
75. (canceled)
76. (canceled)
77. A method for treating pain in a patient, comprising
administering to a patient suffering from pain a capsaicin receptor
modulatory amount of at least one compound or salt according to
claim 1, and thereby alleviating pain in the patient.
78.-80. (canceled)
81. A method according to claim 77, wherein the patient is
suffering from neuropathic pain.
82. A method according to claim 77, wherein the patient is
afflicted with a condition selected from: postmastectomy pain
syndrome, stump pain, phantom limb pain, oral neuropathic pain,
toothache, postherpetic neuralgia, diabetic neuropathy, reflex
sympathetic dystrophy, trigeminal neuralgia, osteoarthritis,
rheumatoid arthritis, fibromyalgia, Guillain-Barre syndrome,
meralgia paresthetica, burning-mouth syndrome, bilateral peripheral
neuropathy, causalgia, neuritis, neuronitis, neuralgia,
AIDS-related neuropathy, MS-related neuropathy, spinal cord
injury-related pain, surgery-related pain, musculoskeletal pain,
back pain, headache, migraine, angina, labor, hemorrhoids,
dyspepsia, Charcot's pains, intestinal gas, menstruation, cancer,
venom exposure, irritable bowel syndrome, inflammatory bowel
disease and trauma.
83. (canceled)
84. A method for treating a condition selected from the group
consisting of itch, cough, hiccup, urinary incontinence or
overactive bladder in a patient, comprising administering to a
patient a capsaicin receptor modulatory amount of a compound or
salt according to claim 1, and thereby alleviating the condition in
the patient.
85. (canceled)
86. (canceled)
87. A method promoting weight loss in an obese patient, comprising
administering to a patient a capsaicin receptor modulatory amount
of a compound or salt according to claim 1, and thereby promoting
weight loss in the patient.
88.-91. (canceled)
92. A packaged pharmaceutical preparation, comprising: (a) a
pharmaceutical composition according to claim 60 in a container;
and (b) instructions for using the composition to treat pain.
93.-97. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/891,832, now allowed, filed Jul. 14, 2004,
which claims the benefit of U.S. Provisional Patent Application No.
60/486,948, filed Jul. 14, 2003. The contents of each of these
applications are incorporated herein in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates generally to substituted
quinolin-4-ylamine analogues that have useful pharmacological
properties. The invention further relates to the use of such
compounds for treating conditions related to capsaicin receptor
activation, for identifying other agents that bind to capsaicin
receptor, and as probes for the detection and localization of
capsaicin receptors.
BACKGROUND OF THE INVENTION
[0003] Pain perception, or nociception, is mediated by the
peripheral terminals of a group of specialized sensory neurons,
termed "nociceptors." A wide variety of physical and chemical
stimuli induce activation of such neurons in mammals, leading to
recognition of a potentially harmful stimulus. Inappropriate or
excessive activation of nociceptors, however, can result in
debilitating acute or chronic pain.
[0004] Neuropathic pain involves pain signal transmission in the
absence of stimulus, and typically results from damage to the
nervous system. In most instances, such pain is thought to occur
because of sensitization in the peripheral and central nervous
systems following initial damage to the peripheral system (e.g.,
via direct injury or systemic disease). Neuropathic pain is
typically burning, shooting and unrelenting in its intensity and
can sometimes be more debilitating that the initial injury or
disease process that induced it.
[0005] Existing treatments for neuropathic pain are largely
ineffective. Opiates, such as morphine, are potent analgesics, but
their usefulness is limited because of adverse side effects, such
as physical addictiveness and withdrawal properties, as well as
respiratory depression, mood changes, and decreased intestinal
motility with concomitant constipation, nausea, vomiting, and
alterations in the endocrine and autonomic nervous systems. In
addition, neuropathic pain is frequently non-responsive or only
partially responsive to conventional opioid analgesic regimens.
Treatments employing the N-methyl-D-aspartate antagonist ketamine
or the alpha(2)-adrenergic agonist clonidine can reduce acute or
chronic pain, and permit a reduction in opioid consumption, but
these agents are often poorly tolerated due to side effects.
[0006] Topical treatment with capsaicin has been used to treat
chronic and acute pain, including neuropathic pain. Capsaicin is a
pungent substance derived from the plants of the Solanaceae family
(which includes hot chili peppers) and appears to act selectively
on the small diameter afferent nerve fibers (A-delta and C fibers)
that are believed to mediate pain. The response to capsaicin is
characterized by persistent activation of nociceptors in peripheral
tissues, followed by eventual desensitization of peripheral
nociceptors to one or more stimuli. From studies in animals,
capsaicin appears to trigger C fiber membrane depolarization by
opening cation selective channels for calcium and sodium.
[0007] Similar responses are also evoked by structural analogues of
capsaicin that share a common vanilloid moiety. One such analogue
is resiniferatoxin (RTX), a natural product of Euphorbia plants.
The term vanilloid receptor (VR) was coined to describe the
neuronal membrane recognition site for capsaicin and such related
irritant compounds. The capsaicin response is competitively
inhibited (and thereby antagonized) by another capsaicin analog,
capsazepine, and is also inhibited by the non-selective cation
channel blocker ruthenium red. These antagonists bind to VR with no
more than moderate affinity (typically with K.sub.i values of no
lower than 140 .mu.M).
[0008] Rat and human vanilloid receptors have been cloned from
dorsal root ganglion cells. The first type of vanilloid receptor to
be identified is known as vanilloid receptor type 1 (VR1), and the
terms "VR1" and "capsaicin receptor" are used interchangeably
herein to refer to rat and/or human receptors of this type, as well
as mammalian homologues. The role of VR1 in pain sensation has been
confirmed using mice lacking this receptor, which exhibit no
vanilloid-evoked pain behavior, and impaired responses to heat and
inflammation. VR1 is a nonselective cation channel with a threshold
for opening that is lowered in response to elevated temperatures,
low pH, and capsaicin receptor agonists. For example, the channel
usually opens at temperatures higher than about 45.degree. C.
Opening of the capsaicin receptor channel is generally followed by
the release of inflammatory peptides from neurons expressing the
receptor and other nearby neurons, increasing the pain response.
After initial activation by capsaicin, the capsaicin receptor
undergoes a rapid desensitization via phosphorylation by
cAMP-dependent protein kinase.
[0009] Because of their ability to desensitize nociceptors in
peripheral tissues, VR1 agonist vanilloid compounds have been used
as topical anesthetics. However, agonist application may itself
cause burning pain, which limits this therapeutic use. Recently, it
has been reported that VR1 antagonists, including nonvanilloid
compounds, are also useful for the treatment of pain (see PCT
International Application Publication Number WO 02/08221, which
published Jan. 31, 2002).
[0010] Thus, compounds that interact with VR1, but do not elicit
the initial painful sensation of VR1 agonist vanilloid compounds,
are desirable for the treatment of chronic and acute pain,
including neuropathic pain. Antagonists of this receptor are
particularly desirable for the treatment of pain, as well as
conditions such as tear gas exposure, itch and urinary tract
conditions such as urinary incontinence and overactive bladder. The
present invention fulfills this need, and provides further related
advantages.
SUMMARY OF THE INVENTION
[0011] The present invention provides substituted
quinolin-4-ylamine analogues characterized by the formula:
##STR00001##
as well as pharmaceutically acceptable salts of such compounds.
Within Formula I: [0012] Y and Z are each independently N or
CR.sub.1. In certain embodiments, Y and Z are independently N or
CH; in further embodiments, at least one of Y and Z is N (i.e., Y
is N, Z is N or both Y and Z are N). R.sub.1 is independently
selected at each occurrence from hydrogen, halogen, cyano, amino,
C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4haloalkyl,
C.sub.1-C.sub.4alkoxy, C.sub.1-C.sub.4haloalkoxy and mono- and
di-(C.sub.1-C.sub.4alkyl)amino. [0013] R.sub.2 is: (i) hydrogen,
halogen or cyano; [0014] (ii) a group of the formula
--R.sub.c-M-A-R.sub.y, wherein: [0015] R.sub.c is
C.sub.0-C.sub.3alkyl or is joined to R.sub.y or R.sub.z, to form a
4- to 10-membered carbocycle or heterocycle that is substituted
with from 0 to 2 substituents independently chosen from R.sub.b;
[0016] M is a single covalent bond, O, S, SO.sub.2, C(.dbd.O),
OC(.dbd.O), C(.dbd.O)O, O--C(.dbd.O)O, C(.dbd.O)N(R.sub.z),
OC(.dbd.O)N(R.sub.z), N(R.sub.z)C(.dbd.O), N(R.sub.z)SO.sub.2,
SO.sub.2N(R.sub.z) or N(R.sub.z); [0017] A is a single covalent
bond or C.sub.1-C.sub.8alkyl substituted with from 0 to 3
substituents independently chosen from R.sub.b; and [0018] R.sub.y
and R.sub.z, if present, are: [0019] (a) independently hydrogen,
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkyl ether,
C.sub.2-C.sub.8alkenyl, a 4-to 10-membered carbocycle or
heterocycle, or joined to R.sub.c, to form a 4- to 10-membered
carbocycle or heterocycle, wherein each non-hydrogen R.sub.y and
R.sub.z is substituted with from 0 to 6 substituents independently
chosen from R.sub.b; or [0020] (b) joined to form a 4- to
10-membered carbocycle or heterocycle that is substituted with from
0 to 6 substituents independently chosen from R.sub.b; or [0021]
(iii) taken together with R.sub.7 to form a fused 5- to 7-membered
ring that is substituted with from 0 to 3 substituents
independently chosen from oxo and C.sub.1-C.sub.4alkyl. In certain
embodiments, R.sub.2 is not --NH.sub.2. [0022] R.sub.7 is hydrogen,
COOH, C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.4alkoxycarbonyl or taken together with R.sub.2 to
form a fused, optionally substituted ring. [0023] Ar.sub.1 is
phenyl or 6-membered heteroaryl, each of which is unsubstituted or
substituted ortho to the point of attachment with 1 or 2
substituents independently chosen from groups of the formula
LR.sub.a. [0024] Ar.sub.2 is 6- to 10-membered aryl or 5- to
10-membered heteroaryl, each of which is substituted with from 0 to
6 substituents independently chosen from oxo and groups of the
formula LR.sub.a. Within certain compounds of Formula I, Ar.sub.2
is a 5- to 10-membered aromatic heterocycle that is optionally
substituted as described above. Within further such compounds,
Ar.sub.2 of Formula I is phenyl or a 6-membered aromatic
heterocycle, optionally substituted as described above. [0025] L is
independently selected at each occurrence from a single covalent
bond, O, C(.dbd.O), OC(.dbd.O), C(.dbd.O)O, OC(.dbd.O)O,
S(O).sub.m, N(R.sub.x), C(.dbd.O)N(R.sub.x), N(R.sub.x)C(.dbd.O),
N(R.sub.x)S(O).sub.m, S(O).sub.mN(R.sub.x) and
N[S(O).sub.mR.sub.w]S(O).sub.m; wherein m is independently selected
at each occurrence from 0, 1 and 2; R.sub.x is independently
selected at each occurrence from hydrogen, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkanoyl and C.sub.1-C.sub.6alkylsulfonyl; and
R.sub.w is hydrogen or C.sub.1-C.sub.6alkyl. [0026] R.sub.a is
independently selected at each occurrence from: [0027] (i)
hydrogen, halogen, cyano and nitro; and [0028] (ii)
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, C.sub.1-C.sub.8haloalkyl,
C.sub.2-C.sub.8alkyl ether, mono- and
di-(C.sub.1-C.sub.8alkyl)amino and (3- to 10-membered
heterocycle)C.sub.0-C.sub.6alkyl, each of which is substituted with
from 0 to 6 substituents independently selected from R.sub.b.
[0029] R.sub.b is independently chosen at each occurrence from
hydroxy, halogen, amino, aminocarbonyl, cyano, nitro, oxo, COOH,
C.sub.1-C.sub.8alkyl, C.sub.1-C.sub.8alkoxy,
C.sub.1-C.sub.8alkylthio, C.sub.1-C.sub.8alkanoyl,
C.sub.1-C.sub.8alkanoyloxy, C.sub.1-C.sub.8alkoxycarbonyl,
C.sub.1-C.sub.8alkyl ether, C.sub.1-C.sub.8hydroxyalkyl,
C.sub.1-C.sub.8haloalkyl, phenylC.sub.0-C.sub.8alkyl, mono- and
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.8alkylsulfonyl and (4- to 7-membered
heterocycle)C.sub.0-C.sub.8alkyl.
[0030] Within certain aspects, compounds of Formula I are VR1
modulators and exhibit a K.sub.i of no greater than 1 micromolar,
100 nanomolar, 50 nanomolar, 10 nanomolar or 1 nanomolar in a
capsaicin receptor binding assay and/or have an EC.sub.50 or
IC.sub.50 value of no greater than 1 micromolar, 100 nanomolar, 50
nanomolar, 10 nanomolar or 1 nanomolar in an assay for
determination of capsaicin receptor agonist or antagonist
activity.
[0031] In certain embodiments, VR1 modulators as described herein
are VR1 antagonists and exhibit no detectable agonist activity in
an in vitro assay of capsaicin receptor activation.
[0032] Within certain aspects, compounds as described herein are
labeled with a detectable marker (e.g., radiolabeled or fluorescein
conjugated).
[0033] The present invention further provides, within other
aspects, pharmaceutical compositions comprising at least one
compound as described herein (i.e., a compound as provided herein
or a pharmaceutically acceptable salt thereof) in combination with
a physiologically acceptable carrier or excipient.
[0034] Within further aspects, methods are provided for reducing
calcium conductance of a cellular capsaicin receptor, comprising
contacting a cell (e.g., neuronal) expressing a capsaicin receptor
with a capsaicin receptor modulatory amount of at least one VR1
modulator as described herein. Such contact may occur in vivo or in
vitro.
[0035] Methods are further provided for inhibiting binding of
vanilloid ligand to a capsaicin receptor. Within certain such
aspects, the inhibition takes place in vitro. Such methods comprise
contacting a capsaicin receptor with at least one VR1 modulator as
described herein, under conditions and in an amount sufficient to
detectably inhibit vanilloid ligand binding to the capsaicin
receptor. Within other such aspects, the capsaicin receptor is in a
patient. Such methods comprise contacting cells expressing a
capsaicin receptor in a patient with at least one VR1 modulator as
described herein in an amount sufficient to detectably inhibit
vanilloid ligand binding to cells expressing a cloned capsaicin
receptor in vitro, and thereby inhibiting binding of vanilloid
ligand to the capsaicin receptor in the patient.
[0036] The present invention further provides methods for treating
a condition responsive to capsaicin receptor modulation in a
patient, comprising administering to the patient a capsaicin
receptor modulatory amount of at least one VR1 modulator as
described herein.
[0037] Within other aspects, methods are provided for treating pain
in a patient, comprising administering to a patient suffering from
pain a capsaicin receptor modulatory amount of at least one VR1
modulator as described herein.
[0038] Methods are further provided for treating itch, urinary
incontinence, overactive bladder, cough and/or hiccup in a patient,
comprising administering to a patient suffering from one or more of
the foregoing conditions a capsaicin receptor modulatory amount of
at least one VR1 modulator as described herein.
[0039] The present invention further provides methods for promoting
weight loss in an obese patient, comprising administering to an
obese patient a capsaicin receptor modulatory amount of at least
one VR1 modulator as described herein.
[0040] Methods are further provided for identifying an agent that
binds to capsaicin receptor, comprising: (a) contacting capsaicin
receptor with a labeled VR1 modulator as described herein under
conditions that permit binding of the VR1 modulator to capsaicin
receptor, thereby generating bound, labeled VR1 modulator; (b)
detecting a signal that corresponds to the amount of bound, labeled
VR1 modulator in the absence of test agent; (c) contacting the
bound, labeled VR1 modulator with a test agent; (d) detecting a
signal that corresponds to the amount of bound labeled VR1
modulator in the presence of test agent; and (e) detecting a
decrease in signal detected in step (d), as compared to the signal
detected in step (b), and therefrom identifying an agent that binds
to capsaicin receptor.
[0041] Within further aspects, the present invention provides
methods for determining the presence or absence of capsaicin
receptor in a sample, comprising: (a) contacting a sample with a
VR1 modulator as described herein under conditions that permit
binding of the VR1 modulator to capsaicin receptor; and (b)
detecting a level of the VR1 modulator bound to capsaicin
receptor.
[0042] The present invention also provides packaged pharmaceutical
preparations, comprising: (a) a pharmaceutical composition as
described herein in a container; and (b) instructions for using the
composition to treat one or more conditions responsive to capsaicin
receptor modulation, such as pain, itch, urinary incontinence,
overactive bladder, cough, hiccup and/or obesity.
[0043] In yet another aspect, the present invention provides
methods of preparing the compounds disclosed herein, including the
intermediates.
[0044] These and other aspects of the invention will become
apparent upon reference to the following detailed description.
DETAILED DESCRIPTION
[0045] As noted above, the present invention provides substituted
quinolin-4-ylamine analogues. Such compounds may be used in vitro
or in vivo, to modulate (preferably inhibit) capsaicin receptor
activity in a variety of contexts.
Terminology
[0046] Compounds are generally described herein using standard
nomenclature. For compounds having asymmetric centers, it should be
understood that (unless otherwise specified) all of the optical
isomers and mixtures thereof are encompassed. In addition,
compounds with carbon-carbon double bonds may occur in Z- and
E-forms, with all isomeric forms of the compounds being included in
the present invention unless otherwise specified. Where a compound
exists in various tautomeric forms, a recited compound is not
limited to any one specific tautomer, but rather is intended to
encompass all tautomeric forms. Certain compounds are described
herein using a general formula that includes variables (e.g.,
R.sub.3, A.sub.1, X). Unless otherwise specified, each variable
within such a formula is defined independently of any other
variable, and any variable that occurs more than one time in a
formula is defined independently at each occurrence.
[0047] The term "quinolin-4-ylamine analogue," as used herein,
encompasses all compounds of Formula I as well as pharmaceutically
acceptable salts of such compounds. Such compounds include
analogues in which the quinoline core is modified by the addition
of ring nitrogen atoms, as well as analogues in which varied
substituents, as described in more detail below, are attached to
such a core structure. In other words, compounds that are
quinolin-4-ylamines, [1,8]naphthyridin-4-ylamines,
[1,5]naphthyridin-4-ylamines and pyrido[2,3-b]pyrazin-8-ylamines
are within the scope of quinolin-4-ylamine analogues.
[0048] A "pharmaceutically acceptable salt" of a compound recited
herein is an acid or base salt that is generally considered in the
art to be suitable for use in contact with the tissues of human
beings or animals without excessive toxicity, irritation, allergic
response, or other problem or complication. Such salts include
mineral and organic acid salts of basic residues such as amines, as
well as alkali or organic salts of acidic residues such as
carboxylic acids. Specific pharmaceutical salts include, but are
not limited to, salts of acids such as hydrochloric, phosphoric,
hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic,
sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene
sulfonic, ethane disulfonic, 2-hydroxyethylsulfonic, nitric,
benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic,
salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic,
propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such
as acetic, HOOC--(CH.sub.2).sub.n--COOH where n is 0-4, and the
like. Similarly, pharmaceutically acceptable cations include, but
are not limited to sodium, potassium, calcium, aluminum, lithium
and ammonium. Those of ordinary skill in the art will recognize
further pharmaceutically acceptable salts for the compounds
provided herein, including those listed by Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton,
Pa., p. 1418 (1985). In general, a pharmaceutically acceptable acid
or base salt can be synthesized from a parent compound that
contains a basic or acidic moiety by any conventional chemical
method. Briefly, such salts can be prepared by reacting the free
acid or base forms of these compounds with a stoichiometric amount
of the appropriate base or acid in water or in an organic solvent,
or in a mixture of the two; generally, the use of nonaqueous media,
such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile,
is preferred.
[0049] It will be apparent that each compound of Formula I may, but
need not, be formulated as a hydrate, solvate or non-covalent
complex. In addition, the various crystal forms and polymorphs are
within the scope of the present invention. Also provided herein are
prodrugs of the compounds of Formula I. A "prodrug" is a compound
that may not fully satisfy the structural requirements of the
compounds provided herein, but is modified in vivo, following
administration to a patient, to produce a compound of Formula I, or
other formula provided herein. For example, a prodrug may be an
acylated derivative of a compound as provided herein. Prodrugs
include compounds wherein hydroxy, amine or sulfhydryl groups are
bonded to any group that, when administered to a mammalian subject,
cleaves to form a free hydroxy, amino, or sulfhydryl group,
respectively. Examples of prodrugs include, but are not limited to,
acetate, formate and benzoate derivatives of alcohol and amine
functional groups within the compounds provided herein. Prodrugs of
the compounds provided herein may be prepared by modifying
functional groups present in the compounds in such a way that the
modifications are cleaved to the parent compounds.
[0050] As used herein, the term "alkyl" refers to a straight or
branched chain or cyclic saturated aliphatic hydrocarbon. Alkyl
groups include groups having from 1 to 8 carbon atoms
(C.sub.1-C.sub.8alkyl), from 1 to 6 carbon atoms
(C.sub.1-C.sub.6alkyl) and from 1 to 4 carbon atoms
(C.sub.1-C.sub.4alkyl), such as methyl, ethyl, propyl, isopropyl,
n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl,
neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, cyclopropyl,
cyclopropylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl,
cycloheptyl and norbornyl. "C.sub.0-C.sub.4alkyl" refers to a
single covalent bond (C.sub.0) or an alkyl group having 1, 2, 3 or
4 carbon atoms; "C.sub.0-C.sub.6alkyl" refers to a single covalent
bond or a C.sub.1-C.sub.6alkyl group; "C.sub.0-C.sub.8alkyl" refers
to a single covalent bond or a C.sub.1-C.sub.8alkyl group. In
certain embodiments, preferred alkyl groups are straight or
branched chain. In some instances herein, a substituent of an alkyl
group is specifically indicated. For example,
"C.sub.1-C.sub.6Cyanoalkyl" refers to a C.sub.1-C.sub.6alkyl group
that has at least one CN substituent. One representative branched
cyanoalkyl group is --C(CH.sub.3).sub.2CN. Similarly,
"C.sub.1-C.sub.6hydroxyalkyl" refers to a C.sub.1-C.sub.6alkyl
group that has at least one --OH substituent.
[0051] Similarly, "alkenyl" refers to straight or branched chain or
cyclic alkene groups, in which at least one unsaturated
carbon-carbon double bond is present. Alkenyl groups include
C.sub.2-C.sub.8alkenyl, C.sub.2-C.sub.6alkenyl and
C.sub.2-C.sub.4alkenyl groups, which have from 2 to 8, 2 to 6 or 2
to 4 carbon atoms, respectively, such as ethenyl, allyl or
isopropenyl. "Alkynyl" refers to straight or branched chain or
cyclic alkyne groups, which have one or more unsaturated
carbon-carbon bonds, at least one of which is a triple bond.
Alkynyl groups include C.sub.2-C.sub.8alkynyl,
C.sub.2-C.sub.6alkynyl and C.sub.2-C.sub.4alkynyl groups, which
have from 2 to 8, 2 to 6 or 2 to 4 carbon atoms, respectively. In
certain embodiments, preferred alkenyl and alkynyl groups are
straight or branched chain.
[0052] By "alkoxy," as used herein, is meant an alkyl group as
described above attached via an oxygen bridge. Alkoxy groups
include C.sub.1-C.sub.6alkoxy and C.sub.1-C.sub.4alkoxy groups,
which have from 1 to 6 or 1 to 4 carbon atoms, respectively.
Methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy,
tert-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy,
neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy are
specific alkoxy groups.
[0053] Similarly, "alkylthio" refers to an alkyl, alkenyl or
alkynyl group as described above attached via a sulfur bridge.
Preferred alkoxy and alkylthio groups are those in which an alkyl
group is attached via the heteroatom bridge.
[0054] The term "oxo," as used herein, refers to a keto (C.dbd.O)
group. An oxo group that is a substituent of a nonaromatic carbon
atom results in a conversion of --CH.sub.2-- to --C(.dbd.O)--.
[0055] The term "alkanoyl" refers to an acyl group in a linear or
branched arrangement (e.g., --(C.dbd.O)-alkyl), where attachment is
through the carbon of the keto group. Alkanoyl groups include
C.sub.2-C.sub.8alkanoyl, C.sub.2-C.sub.6alkanoyl and
C.sub.2-C.sub.4alkanoyl groups, which have from 2 to 8, 2 to 6 or 2
to 4 carbon atoms, respectively. "C.sub.1alkanoyl" refers to
--(C.dbd.O)--H, which (along with C.sub.2-C.sub.8alkanoyl) is
encompassed by the term "C.sub.1-C.sub.8alkanoyl." Ethanoyl is
C.sub.2alkanoyl.
[0056] An "alkanone" is a ketone group in which carbon atoms are in
a linear or branched alkyl arrangement. "C.sub.3-C.sub.8alkanone,"
"C.sub.3-C.sub.6alkanone" and "C.sub.3-C.sub.4alkanone" refer to an
alkanone having from 3 to 8, 6 or 4 carbon atoms, respectively. By
way of example, a C.sub.3 alkanone group has the structure
--CH.sub.2--(C.dbd.O)--CH.sub.3.
[0057] Similarly, "alkyl ether" refers to a linear or branched
ether substituent. Alkyl ether groups include C.sub.2-C.sub.8alkyl
ether, C.sub.2-C.sub.6alkyl ether and C.sub.2-C.sub.4alkyl ether
groups, which have 2 to 8, 6 or 4 carbon atoms, respectively. By
way of example, a C.sub.2 alkyl ether group has the structure
--CH.sub.2--O--CH.sub.3.
[0058] The term "alkoxycarbonyl" refers to an alkoxy group linked
via a carbonyl (i.e., a group having the general structure
--C(.dbd.O)--O-alkyl). Alkoxycarbonyl groups include
C.sub.2-C.sub.8, C.sub.2-C.sub.6 and C.sub.2-C.sub.4alkoxycarbonyl
groups, which have from 2 to 8, 6 or 4 carbon atoms, respectively.
"C.sub.1alkoxycarbonyl" refers to --C(.dbd.O)--OH, which is
encompassed by the term "C.sub.1-C.sub.8alkoxycarbonyl."
"Methoxycarbonyl" refers to C(.dbd.O)--OCH.sub.3.
[0059] "Alkanoyloxy," as used herein, refers to an alkanoyl group
linked via an oxygen bridge (i.e., a group having the general
structure --O--C(.dbd.O)-alkyl). Alkanoyloxy groups include
C.sub.2-C.sub.8, C.sub.2-C.sub.6 and C.sub.2-C.sub.4alkanoyloxy
groups, which have from 2 to 8, 6 or 4 carbon atoms,
respectively.
[0060] "Alkylsulfonyl" refers to groups of the formula
--(SO.sub.2)-alkyl, in which the sulfur atom is the point of
attachment. Alkylsulfonyl groups include
C.sub.1-C.sub.6alkylsulfonyl and C.sub.1-C.sub.4alkylsulfonyl
groups, which have from 1 to 6 or 1 to 4 carbon atoms,
respectively. Methylsulfonyl is one representative alkylsulfonyl
group.
[0061] "Alkylamino" refers to a secondary or tertiary amine having
the general structure --NH-alkyl or --N(alkyl)(alkyl), wherein each
alkyl may be the same or different. Such groups include, for
example, mono- and di-(C.sub.1-C.sub.8alkyl)amino groups, in which
each alkyl may be the same or different and may contain from 1 to 8
carbon atoms, as well as mono- and di-(C.sub.1-C.sub.6alkyl)amino
groups and mono- and di-(C.sub.1-C.sub.4alkyl)amino groups.
[0062] "Alkylaminoalkyl" refers to an alkylamino group linked via
an alkyl group (i.e., a group having the general structure
-alkyl-NH-alkyl or -alkyl-N(alkyl)(alkyl)) in which each alkyl is
selected independently. Such groups include, for example, mono- and
di-(C.sub.1-C.sub.8alkyl)aminoC.sub.1-C.sub.8alkyl, mono- and
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.1-C.sub.6alkyl and mono- and
di-(C.sub.1-C.sub.4alkyl)aminoC.sub.1-C.sub.4alkyl, in which each
alkyl may be the same or different. "Mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.6alkyl" refers to a
mono- or di-(C.sub.1-C.sub.6alkyl)amino group linked via a direct
bond or a C.sub.1-C.sub.6alkyl group. The following are
representative alkylaminoalkyl groups:
##STR00002##
[0063] The term "aminocarbonyl" refers to an amide group (i.e.,
--(C.dbd.O)NH.sub.2). "Mono- or
di-(C.sub.1-C.sub.8alkyl)aminocarbonyl" is an aminocarbonyl group
in which one or both of the hydrogen atoms is replaced with
C.sub.1-C.sub.8alkyl. If both hydrogen atoms are so replaced, the
C.sub.1-C.sub.8alkyl groups may be the same or different.
[0064] The term "halogen" refers to fluorine, chlorine, bromine or
iodine.
[0065] A "haloalkyl" is a branched, straight-chain or cyclic alkyl
group, substituted with 1 or more halogen atoms (e.g.,
"C.sub.1-C.sub.8haloalkyl" groups have from 1 to 8 carbon atoms;
"C.sub.1-C.sub.6haloalkyl" groups have from 1 to 6 carbon atoms).
Examples of haloalkyl groups include, but are not limited to,
mono-, di- or tri-fluoromethyl; mono-, di- or tri-chloromethyl;
mono-, di-, tri-, tetra- or penta-fluoroethyl; mono-, di-, tri-,
tetra- or penta-chloroethyl; and
1,2,2,2-tetrafluoro-1-trifluoromethyl-ethyl. Typical haloalkyl
groups are trifluoromethyl and difluoromethyl. The term
"haloalkoxy" refers to a haloalkyl group as defined above attached
via an oxygen bridge. "C.sub.1-C.sub.8haloalkoxy" groups have from
1 to 8 carbon atoms. "Haloalkylsulfonyl" refers to a haloalkyl
group attached via a --SO.sub.2-- bridge.
"C.sub.1-C.sub.6haloalkylsulfonyl" groups have from 1 to 6 carbon
atoms.
[0066] A dash ("-") that is not between two letters or symbols is
used to indicate a point of attachment for a substituent. For
example, --CONH.sub.2 is attached through the carbon atom.
[0067] A "heteroatom," as used herein, is oxygen, sulfur or
nitrogen.
[0068] A "carbocycle" or "carbocyclic group" comprises at least one
ring formed entirely by carbon-carbon bonds (referred to herein as
a carbocyclic ring), and does not contain a heterocyclic ring.
Unless otherwise specified, each carbocyclic ring within a
carbocycle may be saturated, partially saturated or aromatic. A
carbocycle generally has from 1 to 3 fused, pendant or spiro rings;
carbocycles within certain embodiments have one ring or two fused
rings. Typically, each ring contains from 3 to 8 ring members
(i.e., C.sub.3-C.sub.8); C.sub.5-C.sub.7 rings are recited in
certain embodiments. Carbocycles comprising fused, pendant or spiro
rings typically contain from 9 to 14 ring members. Certain
representative carbocycles are cycloalkyl (i.e., groups that
comprise saturated and/or partially saturated rings, such as
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl, adamantyl, decahydro-naphthalenyl, octahydro-indenyl,
and partially saturated variants of any of the foregoing, such as
cyclohexenyl). Other carbocycles are aryl (i.e., contain at least
one aromatic carbocyclic ring, with or without additional fused,
pendant or spiro cyclolkyl rings). Such carbocycles include, for
example, phenyl, naphthyl, fluorenyl, indanyl and
1,2,3,4-tetrahydro-naphthyl.
[0069] Certain carbocycles recited herein are
C.sub.6-C.sub.10arylC.sub.0-C.sub.8alkyl groups (i.e., groups in
which a carbocyclic group comprising at least one aromatic ring is
linked via a direct bond or a C.sub.1-C.sub.8alkyl group). Such
groups include, for example, phenyl and indanyl, as well as groups
in which either of the foregoing is linked via
C.sub.1-C.sub.8alkyl, preferably via C.sub.1-C.sub.4alkyl. Phenyl
groups linked via a direct bond or alkyl group may be designated
phenylC.sub.0-C.sub.8alkyl (e.g., benzyl, 1-phenyl-ethyl,
1-phenyl-propyl and 2-phenyl-ethyl). A phenylC.sub.0-C.sub.8alkoxy
group is a phenyl ring linked via an oxygen bridge or an alkoxy
group having from 1 to 8 carbon atoms (e.g., phenoxy or
benzoxy).
[0070] A "heterocycle" or "heterocyclic group" has from 1 to 3
fused, pendant or spiro rings, at least one of which is a
heterocyclic ring (i.e., one or more ring atoms is a heteroatom,
with the remaining ring atoms being carbon). Typically, a
heterocyclic ring comprises 1, 2, 3 or 4 heteroatoms; within
certain embodiments each heterocyclic ring has 1 or 2 heteroatoms
per ring. Each heterocyclic ring generally contains from 3 to 8
ring members (rings having from 4 or 5 to 7 ring members are
recited in certain embodiments) and heterocycles comprising fused,
pendant or spiro rings typically contain from 9 to 14 ring members.
Certain heterocycles comprise a sulfur atom as a ring member; in
certain embodiments, the sulfur atom is oxidized to SO or SO.sub.2.
Heterocycles may be optionally substituted with a variety of
substituents, as indicated. Unless otherwise specified, a
heterocycle may be a heterocycloalkyl group (i.e., each ring is
saturated or partially saturated) or a heteroaryl group (i.e., at
least one ring within the group is aromatic). A heterocyclic group
may generally be linked via any ring or substituent atom, provided
that a stable compound results. N-linked heterocyclic groups are
linked via a component nitrogen atom.
[0071] Heterocyclic groups include, for example, azepanyl,
azocinyl, benzimidazolyl, benzimidazolinyl, benzisothiazolyl,
benzisoxazolyl, benzofuranyl, benzothiofuranyl, benzoxazolyl,
benzothiazolyl, benztetrazolyl, chromanyl, chromenyl, cinnolinyl,
decahydroquinolinyl, dihydrofuro[2,3-b]tetrahydrofuranyl,
dihydroisoquinolinyl, dihydrotetrahydrofuranyl,
1,4-dioxa-8-aza-spiro[4.5]decyl, dithiazinyl, furanyl, furazanyl,
imidazolinyl, imidazolidinyl, imidazolyl, indazolyl, indolenyl,
indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl,
isoindazolyl, isoindolinyl, isoindolyl, isothiazolyl, isoxazolyl,
isoquinolinyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl,
oxadiazolyl, oxazolidinyl, oxazolyl, phthalazinyl, piperazinyl,
piperidinyl, piperidinyl, piperidonyl, pteridinyl, purinyl,
pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,
pyridazinyl, pyridoimidazolyl, pyridooxazolyl, pyridothiazolyl,
pyridyl, pyrimidyl, pyrrolidinyl, pyrrolidonyl, pyrrolinyl,
pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, quinuclidinyl,
tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl,
thiadiazinyl, thiadiazolyl, thiazolyl, thienothiazolyl,
thienooxazolyl, thienoimidazolyl, thienyl, thiophenyl,
thiomorpholinyl and variants thereof in which the sulfur atom is
oxidized, triazinyl, and any of the foregoing that are substituted
with from 1 to 4 substituents as described above.
[0072] Certain heterocyclic groups are 4- to 10-membered, 5- to
10-membered, 3- to 7-membered, 4- to 7-membered or 5- to 7-membered
groups that contain 1 heterocyclic ring or 2 fused or spiro rings,
optionally substituted. 4- to 10-membered heterocycloalkyl groups
include, for example, piperidinyl, piperazinyl, pyrrolidinyl,
azepanyl, 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, morpholino,
thiomorpholino and 1,1-dioxo-thiomorpholin-4-yl. Such groups may be
substituted as indicated. Representative aromatic heterocycles are
azocinyl, pyridyl, pyrimidyl, imidazolyl, tetrazolyl and
3,4-dihydro-1H-isoquinolin-2-yl.
(C.sub.3-C.sub.10)heterocycloalkyls include, for example,
piperidinyl, piperazinyl, pyrrolidinyl, azepanyl,
1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, morpholino, thiomorpholino, and
1,1-dioxo-thiomorpholin-4-yl, as well as groups in which each of
the foregoing is substituted. Representative aromatic heterocycles
are azocinyl, pyridyl, pyrimidyl, imidazolyl, tetrazolyl and
3,4-dihydro-1H-isoquinolin-2-yl.
[0073] Additional heterocyclic groups include, for example,
acridinyl, azepanyl, azocinyl, benzimidazolyl, benzimidazolinyl,
benzisothiazolyl, benzisoxazolyl, benzofuranyl, benzothiofuranyl,
benzothiophenyl, benzoxazolyl, benzothiazolyl,
benzotriazolylcarbazolyl, benztetrazolyl, NH-carbazolyl,
carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,
dihydrofuro[2,3-b]tetrahydrofuran, dihydroisoquinolinyl,
dihydrotetrahydrofuranyl, 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl,
dithiazinyl, furanyl, furazanyl, imidazolinyl, imidazolidinyl,
imidazolyl, indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,
isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl,
isoindolyl, isothiazolyl, isoxazolyl, isoquinolinyl, morpholinyl,
naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, oxazolidinyl,
oxazolyl, phenanthridinyl, phenanthrolinyl, phenazinyl,
phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl,
piperazinyl, piperidinyl, piperidinyl, piperidonyl, pteridinyl,
purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,
pyridazinyl, pyridoimidazolyl, pyridooxazolyl, pyridothiazolyl,
pyridyl, pyrimidyl, pyrrolidinyl, pyrrolidonyl, pyrrolinyl,
pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, quinuclidinyl,
tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl,
thiadiazinyl, thiadiazolyl, thianthrenyl, thiazolyl,
thienothiazolyl, thienooxazolyl, thienoimidazolyl, thienyl,
thiophenyl, thiomorpholinyl and variants thereof in which the
sulfur atom is oxidized, triazinyl, xanthenyl and any of the
foregoing that are substituted with from 1 to 4 substituents as
described above.
[0074] A "heterocycleC.sub.0-C.sub.8alkyl" is a heterocyclic group
linked via a single covalent bond or C.sub.1-C.sub.8alkyl group. A
(3- to 10-membered heterocycle)C.sub.0-C.sub.6alkyl is a
heterocyclic group having from 3 to 10 ring members linked via a
direct bond or a C.sub.1-C.sub.6alkyl group. A (5- to 7-membered
heterocycle)C.sub.0-C.sub.8alkyl is a 5- to 7-membered heterocyclic
ring linked via a single covalent bond or a C.sub.1-C.sub.8alkyl
group; a (4- to 7-membered heterocycloalkyl)C.sub.0-C.sub.4alkyl is
a 4-to 7-membered heterocycloalkyl ring linked via a single
covalent bond or a C.sub.1-C.sub.4alkyl group. I
[0075] A "substituent," as used herein, refers to a molecular
moiety that is covalently bonded to an atom within a molecule of
interest. For example, a "ring substituent" may be a moiety such as
a halogen, alkyl group, haloalkyl group or other group discussed
herein that is covalently bonded to an atom (preferably a carbon or
nitrogen atom) that is a ring member. The term "substitution"
refers to replacing a hydrogen atom in a molecular structure with a
substituent as described above, such that the valence on the
designated atom is not exceeded, and such that a chemically stable
compound (i.e., a compound that can be isolated, characterized, and
tested for biological activity) results from the substitution.
[0076] Groups that are "optionally substituted" are unsubstituted
or are substituted by other than hydrogen at one or more available
positions, typically 1, 2, 3, 4 or 5 positions, by one or more
suitable groups (which may be the same or different). Such optional
substituents include, for example, hydroxy, halogen, cyano, nitro,
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, C.sub.1-C.sub.8alkoxy, C.sub.2-C.sub.8alkyl
ether, C.sub.3-C.sub.8alkanone, C.sub.1-C.sub.8alkylthio, amino,
mono- or di-(C.sub.1-C.sub.8alkyl)amino, C.sub.1-C.sub.8haloalkyl,
C.sub.1-C.sub.8haloalkoxy, C.sub.1-C.sub.8alkanoyl,
C.sub.2-C.sub.8alkanoyloxy, C.sub.1-C.sub.8alkoxycarbonyl,
--COOH, --CONH.sub.2, mono- or
di-(C.sub.1-C.sub.8alkyl)aminocarbonyl, --SO.sub.2NH.sub.2, and/or
mono or di(C.sub.1-C.sub.8alkyl)sulfonamido, as well as carbocyclic
and heterocyclic groups. Optional substitution is also indicated by
the phrase "substituted with from 0 to X substituents," where X is
the maximum number of possible substituents. Certain optionally
substituted groups are substituted with from 0 to 2, 3 or 4
independently selected substituents (i.e., are unsubstituted or
substituted with up to the recited maximum number of
substitutents).
[0077] The terms "VR1" and "capsaicin receptor" are used
interchangeably herein to refer to a type 1 vanilloid receptor.
Unless otherwise specified, these terms encompass both rat and
human VR1 receptors (e.g., GenBank Accession Numbers AF327067,
AJ277028 and NM.sub.--018727; sequences of certain human VR1 cDNAs
are provided in SEQ ID NOs:1-3, and the encoded amino acid
sequences shown in SEQ ID NOs:4 and 5, of U.S. Pat. No. 6,482,611),
as well as homologues thereof found in other species.
[0078] A "VR1 modulator," also referred to herein as a "modulator,"
is a compound that modulates VR1 activation and/or VR1-mediated
signal transduction. VR1 modulators specifically provided herein
are compounds of Formula I and pharmaceutically acceptable salts of
compounds of Formula I. A VR1 modulator may be a VR1 agonist or
antagonist. A modulator binds with "high affinity" if the K.sub.i
at VR1 is less than 1 micromolar, preferably less than 100
nanomolar, 10 nanomolar or 1 nanomolar. A representative assay for
determining K.sub.i at VR1 is provided in Example 5, herein.
[0079] A modulator is considered an "antagonist" if it detectably
inhibits vanilloid ligand binding to VR1 and/or VR1-mediated signal
transduction (using, for example, the representative assay provided
in Example 6); in general, such an antagonist inhibits VR1
activation with a IC.sub.50 value of less than 1 micromolar,
preferably less than 100 nanomolar, and more preferably less than
10 nanomolar or 1 nanomolar within the assay provided in Example 6.
VR1 antagonists include neutral antagonists and inverse agonists.
In certain embodiments, capsaicin receptor antagonists provided
herein are not vanilloids.
[0080] An "inverse agonist" of VR1 is a compound that reduces the
activity of VR1 below its basal activity level in the absence of
added vanilloid ligand. Inverse agonists of VR1 may also inhibit
the activity of vanilloid ligand at VR1, and/or may also inhibit
binding of vanilloid ligand to VR1. The ability of a compound to
inhibit the binding of vanilloid ligand to VR1 may be measured by a
binding assay, such as the binding assay given in Example 5. The
basal activity of VR1, as well as the reduction in VR1 activity due
to the presence of VR1 antagonist, may be determined from a calcium
mobilization assay, such as the assay of Example 6.
[0081] A "neutral antagonist" of VR1 is a compound that inhibits
the activity of vanilloid ligand at VR1, but does not significantly
change the basal activity of the receptor (i.e., within a calcium
mobilization assay as described in Example 6 performed in the
absence of vanilloid ligand, VR1 activity is reduced by no more
than 10%, more preferably by no more than 5%, and even more
preferably by no more than 2%; most preferably, there is no
detectable reduction in activity). Neutral antagonists of VR1 may
inhibit the binding of vanilloid ligand to VR1.
[0082] As used herein a "capsaicin receptor agonist" or "VR1
agonist" is a compound that elevates the activity of the receptor
above the basal activity level of the receptor (i.e., enhances VR1
activation and/or VR1-mediated signal transduction). Capsaicin
receptor agonist activity may be identified using the
representative assay provided in Example 6. In general, such an
agonist has an EC.sub.50 value of less than 1 micromolar,
preferably less than 100 nanomolar, and more preferably less than
10 nanomolar within the assay provided in Example 6. In certain
embodiments, capsaicin receptor agonists provided herein are not
vanilloids.
[0083] A "vanilloid" is capsaicin or any capsaicin analogue that
comprises a phenyl ring with two oxygen atoms bound to adjacent
ring carbon atoms (one of which carbon atom is located para to the
point of attachment of a third moiety that is bound to the phenyl
ring). A vanilloid is a "vanilloid ligand" if it binds to VR1 with
a K.sub.i (determined as described herein) that is no greater than
10 .mu.M. Vanilloid ligand agonists include capsaicin, olvanil,
N-arachidonoyl-dopamine and resiniferatoxin (RTX). Vanilloid ligand
antagonists include capsazepine and iodo-resiniferatoxin.
[0084] A "capsaicin receptor modulatory amount" is an amount that,
upon administration to a patient, achieves a concentration of VR1
modulator at a capsaicin receptor within the patient that is
sufficient to alter the binding of vanilloid ligand to VR1 in vitro
(using the assay provided in Example 5) and/or VR1-mediated signal
transduction (using an assay provided in Example 6). The capsaicin
receptor may be present, or example, in a body fluid such as blood,
plasma, serum, CSF, synovial fluid, lymph, cellular interstitial
fluid, tears or urine.
[0085] A "therapeutically effective amount" is an amount that, upon
administration, is sufficient to provide detectable patient relief
from a condition being treated. Such relief may be detected using
any appropriate criteria, including alleviation of one or more
symptoms such as pain.
[0086] A "patient" is any individual treated with a compound (e.g.,
a VR1 modulator) as provided herein. Patients include humans, as
well as other animals such as companion animals (e.g., dogs and
cats) and livestock. Patients may be experiencing one or more
symptoms of a condition responsive to capsaicin receptor modulation
(e.g., pain, exposure to vanilloid ligand, itch, urinary
incontinence, overactive bladder, respiratory disorders, cough
and/or hiccup), or may be free of such symptom(s) (i.e., treatment
may be prophylactic).
Substituted Quinolin-4-Ylamine Analogues
[0087] As noted above, the present invention provides substituted
quinolin-4-ylamine analogues that may be used in a variety of
contexts, including in the treatment of pain (e.g., neuropathic or
peripheral nerve-mediated pain); exposure to capsaicin; exposure to
acid, heat, light, tear gas air pollutants, pepper spray or related
agents; respiratory conditions such as asthma or chronic
obstructive pulmonary disease; itch; urinary incontinence or
overactive bladder; cough or hiccup; and/or obesity. Such compounds
may also be used within in vitro assays (e.g., assays for receptor
activity), as probes for detection and localization of VR1 and as
standards in ligand binding and VR1-mediated signal transduction
assays.
[0088] Certain compounds provided herein detectably modulate the
binding of capsaicin to VR1 at nanomolar (i.e., submicromolar)
concentrations, preferably at subnanomolar concentrations, more
preferably at concentrations below 100 picomolar, 20 picomolar, 10
picomolar or 5 picomolar. Such modulators are preferably not
vanilloids. Certain preferred modulators are VR1 antagonists and
have no detectable agonist activity in the assay described in
Example 6. Preferred VR1 modulators further bind with high affinity
to VR1, and do not substantially inhibit activity of human EGF
receptor tyrosine kinase.
[0089] Certain compounds further satisfy Formula II:
##STR00003##
or are a pharmaceutically acceptable salt of such a compound,
wherein: [0090] At least one of Y and Z is N; and the other of Y
and Z is N or CR.sub.1. In certain embodiments, Z is N (e.g., Z is
N and Y is CH, or both Y and Z are N). In further embodiments, Y is
N. R.sub.1 is hydrogen, halogen, cyano, amino,
C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4haloalkyl,
C.sub.1-C.sub.4alkoxy, C.sub.1-C.sub.4haloalkoxy or mono- or
di-(C.sub.1-C.sub.4alkyl)amino; in certain embodiments, R.sub.1 is
hydrogen, C.sub.1-C.sub.4alkyl or halo C.sub.1-C.sub.4alkyl, with
hydrogen preferred. [0091] R.sub.2 is: (i) hydrogen, halogen or
cyano; [0092] (ii) a group of the formula --R.sub.c-M-A-R.sub.y,
wherein: [0093] R.sub.c is C.sub.0-C.sub.3alkyl or is joined to
R.sub.y or R.sub.z, to form a 4- to 10-membered carbocycle or
heterocycle that is substituted with from 0 to 2 substituents
independently chosen from R.sub.b; [0094] M is a single covalent
bond, O, S, SO.sub.2,
[0094] ##STR00004## [0095] A is a single covalent bond or
C.sub.1-C.sub.8alkyl substituted with from 0 to 3 substituents
independently chosen from R.sub.b; and [0096] R.sub.y and R.sub.z,
if present, are: [0097] (a) independently hydrogen,
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkyl ether,
C.sub.2-C.sub.8alkenyl, a 4-to 10-membered carbocycle or
heterocycle, or joined to R.sub.c to form a 4- to 10-membered
carbocycle or heterocycle, wherein each non-hydrogen R.sub.y and
R.sub.z is substituted with from 0 to 6 substituents independently
chosen from R.sub.b; or [0098] (b) joined to form a 4- to
10-membered carbocycle or heterocycle that is substituted with from
0 to 6 substituents independently chosen from R.sub.b; [0099] such
that R.sub.2 is not --NH.sub.2; or [0100] (iii) taken together with
R.sub.7 to form a fused 5- to 7-membered ring that is substituted
with from 0 to 3 substituents independently chosen from oxo and
C.sub.1-C.sub.4alkyl.
[0101] It will be apparent that, within groups of the formula
R.sub.c-M-A-R.sub.y, if two adjacent variables are bonds, then the
two variables are taken together to form a single bond. For
example, if R.sub.c is C.sub.0alkyl and M and A are both single
covalent bonds, then R.sub.2 is --R.sub.y. [0102] In certain
compounds, R.sub.2 is: (i) hydrogen, hydroxy or halogen; or (ii)
C.sub.1-C.sub.6alkyl,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6-aminoalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.2-C.sub.6alkyl ether, mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl or (4- to
7-membered heterocycloalkyl)C.sub.0-C.sub.4alkyl, each of which is
substituted with from 0 to 4 substituents independently chosen from
halogen, cyano, hydroxy, amino, oxo, mono- and
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkyl
C.sub.1-C.sub.6alkoxy and C.sub.1-C.sub.6haloalkyl. Representative
R.sub.2 groups include hydrogen, C.sub.1-C.sub.6alkyl,
C.sub.2-C.sub.6alkyl ether, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, morpholinylC.sub.0-C.sub.2alkyl,
piperazinylC.sub.0-C.sub.2alkyl, piperidinylC.sub.0-C.sub.2alkyl,
phenylC.sub.0-C.sub.2alkyl and pyridylC.sub.0-C.sub.2alkyl, each of
which is substituted with from 0 to 4 substituents independently
chosen from halogen, cyano, hydroxy, amino, oxo, mono- and
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkyl and
C.sub.1-C.sub.6haloalkyl. [0103] R.sub.7 is hydrogen, COOH,
C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.4alkoxycarbonyl or taken together with R.sub.2 to
form a fused, optionally substituted ring. In certain compounds,
R.sub.7 is hydrogen. [0104] Ar.sub.1 is phenyl or 6-membered
heteroaryl, each of which is unsubstituted or substituted ortho to
the point of attachment with 1 or 2 substituents independently
chosen from groups of the formula LR.sub.a. In other words, if
Ar.sub.1 is mono-substituted phenyl, the substitution is at the
2-position; and if Ar.sub.1 is di-substituted phenyl, the
substitutions are located at the 2- and 6-postions. Similarly, if
Ar.sub.1 is substituted pyridin-2-yl, then the substitution is at
the 3-position. Preferred Ar.sub.1 groups are phenyl, pyridyl,
pyrimidinyl, pyrazinyl and pyridazinyl, each of which is
unsubstituted or monosubstituted at the ortho position with
halogen, cyano, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6alkoxy or C.sub.1-C.sub.6haloalkoxy. Particularly
preferred groups are phenyl and pyridyl, optionally substituted as
described above. [0105] Ar.sub.2 is 6- to 10-membered aryl or 5- to
10-membered heteroaryl, each of which is substituted with from 0 to
6 substituents independently chosen from oxo and groups of the
formula LR.sub.a. In certain embodiments, Ar.sub.2 is phenyl or a
5- or 6-membered heteroaryl (e.g., a 6-membered heteroaryl), each
of which is substituted with from 0 to 3 substituents independently
selected from (a) groups of the formula LR.sub.a and (b) groups
that are taken together to form a fused, 5- to 7-membered
heterocyclic ring that is substituted with from 0 to 3 substituents
independently selected from R.sub.b. Representative Ar.sub.2 groups
include phenyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl,
each of which is substituted with 0, 1 or 2 substituents
independently chosen from halogen, cyano, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6alkyl ether, C.sub.1-C.sub.6alkanoyl,
C.sub.1-C.sub.6alkylsulfonyl, C.sub.1-C.sub.6haloalkylsulfonyl,
amino, and mono- and di-(C.sub.1-C.sub.6alkyl)amino. Preferably,
Ar.sub.2 is phenyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl
that is unsubstituted or substituted with halogen, cyano,
C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4hydroxyalkyl,
C.sub.1-C.sub.4alkanoyl, C.sub.1-C.sub.4haloalkyl,
C.sub.1-C.sub.4alkylsulfonyl or C.sub.1-C.sub.4haloalkylsulfonyl.
In certain such compounds, Ar.sub.1 is pyridyl, pyrimidinyl,
pyrazinyl or pyridazinyl, each of which is substituted with
halogen, cyano, C.sub.1-C.sub.4alkyl or C.sub.1-C.sub.4haloalkyl;
and Ar.sub.2 is pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl,
each of which is substituted with halogen, cyano,
C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4hydroxyalkyl,
C.sub.1-C.sub.4alkanoyl, C.sub.1-C.sub.4haloalkyl,
C.sub.1-C.sub.4alkylsulfonyl or C.sub.1-C.sub.4haloalkylsulfonyl.
[0106] L is independently selected at each occurrence from a single
covalent bond, O, C(.dbd.O),
##STR00005##
[0106] wherein m is independently selected at each occurrence from
0, 1 and 2; R.sub.x is independently selected at each occurrence
from hydrogen, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkanoyl and
C.sub.1-C.sub.6alkylsulfonyl; and R.sub.w is hydrogen or
C.sub.1-C.sub.6alkyl; [0107] R.sub.a is independently selected at
each occurrence from: [0108] (i) hydrogen, halogen, cyano and
nitro; and [0109] (ii) C.sub.1-C.sub.8alkyl,
C.sub.2-C.sub.8alkenyl, C.sub.2-C.sub.8alkynyl,
C.sub.1-C.sub.8haloalkyl, C.sub.2-C.sub.8alkyl ether, mono- and
di-(C.sub.1-C.sub.8alkyl)amino and (3- to 10-membered
heterocycle)C.sub.0-C.sub.6alkyl, each of which is substituted with
from 0 to 6 substituents independently selected from R.sub.b; and
[0110] R.sub.b is independently chosen at each occurrence from
hydroxy, halogen, amino, aminocarbonyl, cyano, nitro, oxo, COOH,
C.sub.1-C.sub.8alkyl, C.sub.1-C.sub.8alkoxy,
C.sub.1-C.sub.8alkylthio, C.sub.1-C.sub.8alkanoyl,
C.sub.1-C.sub.8alkanoyloxy, C.sub.1-C.sub.8alkoxycarbonyl,
C.sub.1-C.sub.8alkyl ether, C.sub.1-C.sub.8hydroxyalkyl,
C.sub.1-C.sub.8haloalkyl, phenylC.sub.0-C.sub.8alkyl, mono- and
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.8alkylsulfonyl and (4- to 7-membered
heterocycle)C.sub.0-C.sub.8alkyl.
[0111] Within certain compounds of Formula II: [0112] Ar.sub.1 is
pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl, each of which is
substituted with halogen, cyano, C.sub.1-C.sub.4alkyl or
C.sub.1-C.sub.4haloalkyl; [0113] Ar.sub.2 is phenyl, pyridyl,
pyrimidinyl, pyrazinyl or pyridazinyl, each of which is substituted
with halogen, cyano, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4hydroxyalkyl, C.sub.1-C.sub.4alkanoyl,
C.sub.1-C.sub.4haloalkyl, C.sub.1-C.sub.4alkylsulfonyl or
C.sub.1-C.sub.4haloalkylsulfonyl; [0114] R.sub.2 is: [0115] (i)
hydrogen, hydroxy or halogen; or [0116] (ii) C.sub.1-C.sub.6alkyl,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6-aminoalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.2-C.sub.6alkyl ether, mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl or (4- to
7-membered heterocycloalkyl)C.sub.0-C.sub.4alkyl, each of which is
substituted with from 0 to 4 substituents independently chosen from
halogen, cyano, hydroxy, amino, oxo, mono- and
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkyl
C.sub.1-C.sub.6alkoxy and C.sub.1-C.sub.6haloalkyl; and [0117]
R.sub.7 is hydrogen.
[0118] Certain compounds of Formula II further satisfy Formula
IIa
##STR00006##
wherein: [0119] Ar.sub.2 is phenyl or a 6-membered aromatic
heterocycle, each of which is substituted with from 0 to 3
substituents independently selected from groups of the formula
LR.sub.a; [0120] R.sub.2a is hydrogen, halogen or
C.sub.1-C.sub.4alkyl; and the remaining variables are as described
for Formula II.
[0121] Within Formula IIa, representative Ar.sub.2 groups include
phenyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl, each of
which is substituted with 0, 1 or 2 substituents independently
selected from halogen, cyano, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6alkyl ether, C.sub.1-C.sub.6alkanoyl,
C.sub.1-C.sub.6alkylsulfonyl, C.sub.1-C.sub.6haloalkylsulfonyl,
amino, mono- and di-(C.sub.1-C.sub.6alkyl)amino. Within certain
embodiments, Ar.sub.2 is pyridyl, pyrimidinyl, pyrazinyl or
pyridazinyl, each of which is unsubstituted or substituted with
halogen, cyano, C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4hydroxyalkyl,
C.sub.1-C.sub.4alkanoyl, C.sub.1-C.sub.4haloalkyl,
C.sub.1-C.sub.4alkylsulfonyl or C.sub.1-C.sub.4haloalkylsulfonyl.
Representative Ar.sub.1 groups are phenyl, pyridyl, pyrimidinyl,
pyrazinyl and pyridazinyl, each of which is unsubstituted or
substituted with halogen, cyano, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy or
C.sub.1-C.sub.6haloalkoxy. In certain such compounds, Ar.sub.1 is
pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl, each of which is
substituted with halogen, cyano, C.sub.1-C.sub.4alkyl or
C.sub.1-C.sub.4haloalkyl; and Ar.sub.2 is phenyl, pyridyl,
pyrimidinyl, pyrazinyl or pyridazinyl, each of which is substituted
with halogen, cyano, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4hydroxyalkyl, C.sub.1-C.sub.4alkanoyl,
C.sub.1-C.sub.4haloalkyl, C.sub.1-C.sub.4alkylsulfonyl or
C.sub.1-C.sub.4haloalkylsulfonyl.
[0122] Certain compounds of Formula I further satisfy Formula
III
##STR00007##
[0123] Within Formula III, Ar.sub.1, Y, Z, R.sub.2 and R.sub.7 are
as described for Formula I; and Ar.sub.2 is 5-to 10-membered
heteroaryl that substituted with from 0 to 6 substituents
independently chosen from oxo and groups of the formula LR.sub.a,
as described above for Formula I.
[0124] In certain compounds of Formula III, each R.sub.1 is
hydrogen.
[0125] Within further compounds of Formula III, R.sub.2 is: (i)
hydrogen, hydroxy or halogen; or (ii) C.sub.1-C.sub.6alkyl,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6-aminoalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.2-C.sub.6alkyl ether, mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl or (4- to
7-membered heterocycloalkyl)C.sub.0-C.sub.4alkyl, each of which is
substituted with from 0 to 4 substituents independently chosen from
halogen, cyano, hydroxy, amino, oxo, mono- and
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkyl
C.sub.1-C.sub.6alkoxy and C.sub.1-C.sub.6haloalkyl. Representative
R.sub.2 groups include C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkyl
ether, mono- or di-(C.sub.1-C.sub.6alkyl)amino,
morpholinylC.sub.0-C.sub.2alkyl, piperazinylC.sub.0-C.sub.2alkyl,
piperidinylC.sub.0-C.sub.2alkyl, phenylC.sub.0-C.sub.2alkyl and
pyridylC.sub.0-C.sub.2alkyl, each of which is substituted with from
0 to 4 substituents independently chosen from halogen, cyano,
hydroxy, amino, oxo, mono- and di-(C.sub.1-C.sub.6alkyl)amino,
C.sub.1-C.sub.6alkyl and C.sub.1-C.sub.6haloalkyl.
[0126] Within certain compounds of Formula III, Ar.sub.2 is a 5- or
6-membered heteroaryl (e.g., a 6-membered heteroaryl) that is
substituted with from 0 to 3 substituents independently selected
from (a) groups of the formula LR.sub.a and (b) groups that are
taken together to form a fused, 5- to 7-membered heterocyclic ring
that is substituted with from 0 to 3 substituents independently
selected from R.sub.b. Representative Ar.sub.2 groups include
pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl, each of which is
substituted with 0, 1 or 2 substituents independently selected from
halogen, cyano, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6alkyl ether,
C.sub.1-C.sub.6alkanoyl, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6haloalkylsulfonyl, amino, mono- and
di-(C.sub.1-C.sub.6alkyl)amino. Preferably, Ar.sub.2 is pyridyl,
pyrimidinyl, pyrazinyl or pyridazinyl, each of which is
unsubstituted or substituted with halogen, cyano,
C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4hydroxyalkyl,
C.sub.1-C.sub.4alkanoyl, C.sub.1-C.sub.4haloalkyl,
C.sub.1-C.sub.4alkylsulfonyl or C.sub.1-C.sub.4haloalkylsulfonyl.
Representative Ar.sub.1 groups include phenyl, pyridyl,
pyrimidinyl, pyrazinyl and pyridazinyl, each of which is
unsubstituted or substituted with halogen, cyano,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6alkoxy or C.sub.1-C.sub.6haloalkoxy. In certain such
compounds, Ar.sub.1 is pyridyl, pyrimidinyl, pyrazinyl or
pyridazinyl, each of which is substituted with halogen, cyano,
C.sub.1-C.sub.4alkyl or C.sub.1-C.sub.4haloalkyl; and Ar.sub.2 is
pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl, each of which is
substituted with halogen, cyano, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4hydroxyalkyl, C.sub.1-C.sub.4alkanoyl,
C.sub.1-C.sub.4haloalkyl, C.sub.1-C.sub.4alkylsulfonyl or
C.sub.1-C.sub.4haloalkylsulfonyl.
[0127] Within further compounds of Formula III: [0128] Ar.sub.1 is
pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl, each of which is
substituted with halogen, cyano, C.sub.1-C.sub.4alkyl or
C.sub.1-C.sub.4haloalkyl; [0129] Ar.sub.2 is pyridyl, pyrimidinyl,
pyrazinyl or pyridazinyl, each of which is substituted with
halogen, cyano, C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4hydroxyalkyl,
C.sub.1-C.sub.4alkanoyl, C.sub.1-C.sub.4haloalkyl,
C.sub.1-C.sub.4alkylsulfonyl or C.sub.1-C.sub.4haloalkylsulfonyl;
[0130] Y and Z are independently N or CH; [0131] R.sub.2 is: [0132]
(i) hydrogen, hydroxy or halogen; or [0133] (ii)
C.sub.1-C.sub.6alkyl,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6-aminoalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.2-C.sub.6alkyl ether, mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl or (4- to
7-membered heterocycloalkyl)C.sub.0-C.sub.4alkyl, each of which is
substituted with from 0 to 4 substituents independently chosen from
halogen, cyano, hydroxy, amino, oxo, mono- and
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkyl
C.sub.1-C.sub.6alkoxy and C.sub.1-C.sub.6haloalkyl; and [0134]
R.sub.7 is hydrogen.
[0135] Certain compounds of Formula I further satisfy Formula
IV
##STR00008##
Within Formula IV:
[0136] R.sub.7, Y, Z, Ar.sub.1 and Ar.sub.2 are as described above
for Formula I; and R.sub.2 is: (i) halogen or cyano; [0137] (ii) a
group of the formula --R.sub.c-M-A-R.sub.y, wherein: [0138] R.sub.1
is C.sub.0-C.sub.3alkyl or is joined to R.sub.y or R.sub.z, to form
a 4- to 10-membered carbocycle or heterocycle that is substituted
with from 0 to 2 substituents independently chosen from R.sub.b;
[0139] M is a single covalent bond, O, S, SO.sub.2, C(.dbd.O),
OC(.dbd.O), C(.dbd.O)O, O--C(.dbd.O)O, C(.dbd.O)N(R.sub.z),
OC(.dbd.O)N(R.sub.z), N(R.sub.z)C(.dbd.O), N(R.sub.z)SO.sub.2,
SO.sub.2N(R.sub.z) or N(R.sub.z); [0140] A is a single covalent
bond or C.sub.1-C.sub.8alkyl substituted with from 0 to 3
substituents independently chosen from R.sub.b; and [0141] R.sub.y
and R.sub.z, if present, are: [0142] (a) independently hydrogen,
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkyl ether,
C.sub.2-C.sub.8alkenyl, a 4-to 10-membered carbocycle or
heterocycle, or joined to R.sub.c to form a 4- to 10-membered
carbocycle or heterocycle, wherein each non-hydrogen R.sub.y and
R.sub.z is substituted with from 0 to 6 substituents independently
chosen from R.sub.b; or [0143] (b) joined to form a 4- to
10-membered carbocycle or heterocycle that is substituted with from
0 to 6 substituents independently chosen from R.sub.b; [0144] such
that R.sub.2 is not --NH.sub.2; or [0145] (iii) taken together with
R.sub.7 to form a fused 5- to 7-membered ring that is substituted
with from 0 to 3 substituents independently chosen from oxo and
C.sub.1-C.sub.4alkyl.
[0146] Within certain compounds of Formula IV, R.sub.2 is: (i)
hydroxy or halogen; or (ii) C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6aminoalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.2-C.sub.6alkyl ether, mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl, or (4- to
7-membered heterocycloalkyl)C.sub.0-C.sub.4alkyl, each of which is
substituted with from 0 to 4 substituents independently chosen from
halogen, cyano, hydroxy, amino, oxo, mono- and
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkyl
C.sub.1-C.sub.6alkoxy and C.sub.1-C.sub.6haloalkyl. Representative
such R.sub.2 groups include C.sub.1-C.sub.6alkyl,
C.sub.2-C.sub.6alkyl ether, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, morpholinylC.sub.0-C.sub.2alkyl,
piperazinylC.sub.0-C.sub.2alkyl, piperidinylC.sub.0-C.sub.2alkyl,
phenylC.sub.0-C.sub.2alkyl and pyridylC.sub.0-C.sub.2alkyl, each of
which is substituted with from 0 to 4 substituents independently
chosen from halogen, cyano, hydroxy, amino, oxo, mono- and
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkyl and
C.sub.1-C.sub.6haloalkyl.
[0147] Within certain compounds of Formula IV: [0148] Ar.sub.1 is
pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl, each of which is
substituted with halogen, cyano, C.sub.1-C.sub.4alkyl or
C.sub.1-C.sub.4haloalkyl; [0149] Ar.sub.2 is pyridyl, pyrimidinyl,
pyrazinyl or pyridazinyl, each of which is substituted with
halogen, cyano, C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4hydroxyalkyl,
C.sub.1-C.sub.4alkanoyl, C.sub.1-C.sub.4haloalkyl,
C.sub.1-C.sub.4alkylsulfonyl or C.sub.1-C.sub.4haloalkylsulfonyl;
[0150] Y and Z are independently N or CH; [0151] R.sub.2 is: [0152]
(i) hydroxy or halogen; or [0153] (ii) C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6aminoalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.2-C.sub.6alkyl ether, mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl or (4- to
7-membered heterocycloalkyl)C.sub.0-C.sub.4alkyl, each of which is
substituted with from 0 to 4 substituents independently chosen from
halogen, cyano, hydroxy, amino, oxo, mono- and
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkyl
C.sub.1-C.sub.6alkoxy and C.sub.1-C.sub.6haloalkyl; and [0154]
R.sub.7 is hydrogen.
[0155] Certain compounds of Formula IV further satisfy Formula
IVa
##STR00009##
Within Formula IVa:
[0156] Ar.sub.1, Y and Z are as described for Formula IV; [0157]
Ar.sub.2 is phenyl or a 6-membered heteroaryl, each of which is
substituted with from 0 to 3 substituents independently selected
from (a) groups of the formula LR.sub.a and (b) groups that are
taken together to form a fused, 5- to 7-membered heterocyclic ring
that is substituted with from 0 to 3 substituents independently
selected from R.sub.b; [0158] R.sub.3 and R.sub.4 are: [0159] (i)
each independently selected from: [0160] (a) hydrogen; and [0161]
(b) C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, C.sub.1-C.sub.8alkoxy,
C.sub.3-C.sub.8alkanone, C.sub.2-C.sub.8alkanoyl,
C.sub.2-C.sub.8alkyl ether,
C.sub.6-C.sub.10arylC.sub.0-C.sub.8alkyl, (5- to 10-membered
heterocycle)C.sub.0-C.sub.8alkyl and C.sub.1-C.sub.8alkylsulfonyl,
each of which is substituted with from 0 to 6 substituents
independently selected from R.sub.b; or [0162] (ii) joined to form,
with the N to which they are bound, a 4- to 10-membered
heterocyclic group that is substituted with from 0 to 6
substituents independently selected from R.sub.b; [0163] R.sub.5
and R.sub.6 are, independently at each occurrence: [0164] (i) each
independently selected from hydrogen, hydroxy and
C.sub.1-C.sub.6alkyl; or [0165] (ii) taken together to form a keto
group; and [0166] n is 1, 2 or 3.
[0167] In certain compounds of Formula IVa, R.sub.3 and R.sub.4 are
each independently: (i) hydrogen; or (ii) C.sub.1-C.sub.8alkyl,
C.sub.2-C.sub.8alkenyl or C.sub.1-C.sub.8alkylsulfonyl, each of
which is substituted with from 0 to 4 substituents independently
selected from hydroxy, halogen, amino, oxo, COOH,
C.sub.1-C.sub.6alkyl, haloC.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy and haloC.sub.1-C.sub.6alkoxy. In other
compounds of Formula IVa, R.sub.3 and R.sub.4 are joined to form
azetidine, pyrrolidine, morpholine, piperidine or piperazine, each
of which is substituted with from 0 to 4 substituents independently
selected from hydroxy, halogen, amino, oxo, COOH,
C.sub.1-C.sub.6alkyl, haloC.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy and haloC.sub.1-C.sub.6alkoxy.
[0168] Within certain embodiments, R.sub.3 and R.sub.4 of Formula
IVa are each independently selected from (i) hydrogen or (ii)
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, C.sub.3-C.sub.8alkanone,
C.sub.1-C.sub.8alkanoyl, C.sub.2-C.sub.8alkyl ether,
C.sub.6-C.sub.10arylC.sub.0-C.sub.8alkyl, 5- to 10-membered
heterocycleC.sub.0-C.sub.8alkyl and
--(SO.sub.2)C.sub.1-C.sub.8alkyl, each of which is optionally
substituted. Within other embodiments, R.sub.3 and R.sub.4 are each
independently selected from (i) hydrogen and (ii)
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
phenylC.sub.0-C.sub.4alkyl, indanylC.sub.0-C.sub.4alkyl, 5- to
6-membered heteroarylC.sub.0-C.sub.4alkyl and 4- to 7-membered
heterocycloalkylC.sub.0-C.sub.4alkyl, each of which is optionally
substituted with from 1 to 4 substituents independently selected
from hydroxy, halogen, amino, C.sub.1-C.sub.6alkyl,
haloC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy and
haloC.sub.1-C.sub.6alkoxy. Representative such R.sub.3 and R.sub.4
groups include C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl, 5- to
7-membered heterocycloC.sub.0-C.sub.4alkyl, C.sub.2-C.sub.6alkyl
ether, indanyl, benzyl, 1-phenyl-ethyl, 1-phenyl-propyl and
2-phenyl-ethyl, each of which is substituted with from 0 to 3
substituents independently selected from hydroxy, halogen and
C.sub.1-C.sub.4alkyl. For example, at least one of R.sub.3 and
R.sub.4 may be pyridylC.sub.0-C.sub.4alkyl,
pyrimidylC.sub.0-C.sub.4alkyl, imidazolylC.sub.0-C.sub.4alkyl or
tetrazolylC.sub.0-C.sub.4alkyl, each of which is substituted with
0, 1 or 2 substituents. Alternatively, R.sub.3 and/or R.sub.4 may
be joined to an R.sub.5 or R.sub.6 group (along with the N to which
R.sub.3 and R.sub.4 are bound and any carbon atoms between the N
and R.sub.5 or R.sub.6) to form an optionally substituted
heterocycle, such as a 5- to 10-membered mono- or bi-cyclic
group.
[0169] Within other embodiments, R.sub.3 and/or R.sub.4 of Formula
II may form an optionally substituted heterocycle. For example,
R.sub.3 and R.sub.4 may be joined to form, with the N to which they
are bound, an optionally substituted heterocycle; or R.sub.3 or
R.sub.4 may be joined to an R.sub.5 or R.sub.6 moiety to from an
optionally substituted heterocycle. In either case, the resulting
heterocycle may be, for example, a 4- or 5- to 10-membered, mono-
or bi-cyclic group substituted with from 0 to 4 substituents (e.g.,
from 1 to 4 substituents or 0, 1 or 2 substituents). In certain
embodiments, each substituent is independently selected from
hydroxy, halogen, C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4alkoxy, haloC.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.4alkanoyl, C.sub.1-C.sub.4alkoxycarbonyl,
aminocarbonyl, heterocycleC.sub.0-C.sub.8alkyl and
heterocycleC.sub.1-C.sub.8alkoxycarbonyl. In certain embodiments,
such substituents are lower alkyl groups such as methyl and/or
ethyl.
[0170] A heterocyclic group that comprises R.sub.3 and/or R.sub.4
may be a heteroaryl group, which comprises an aromatic ring (e.g.,
optionally substituted acridinyl, benzimidazolinyl, benzimidazolyl,
benzotriazolyl, carbazolyl, cinnolinyl, indazolyl, indolinyl,
indolyl, isoquinolinyl, quinoxalinyl, naphthyridinyl,
phenanthridinyl, phenazinyl, phenothiazinyl, phenoxazinyl,
phthalazinyl, pteridinyl, purinyl, quinolinyl, quinoxalinyl,
quinazolinyl, tetrahydroisoquinolinyl or tetrahydroquinolinyl). One
such heteroaryl is 3,4-dihydro-1H-isoquinolin-2-yl. Alternatively,
the heterocycle may be an optionally substituted heterocycloalkyl
group, such as azepanyl, azocinyl, decahydroquinolinyl,
1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, imidazolidinyl, imidazolinyl,
morpholino, piperadinyl, piperazinyl, pyridazinyl, pyrazolidinyl,
pyrazolinyl, pyrrolidinyl, pyrrolinyl, thiomorpholino or
1,1-dioxo-thiomorpholin-4-yl. Representative heterocycles that may
be formed from R.sub.3 and R.sub.4 include, but are not limited to,
optionally substituted azepane, azocane, dihydroisoquinoline,
imidazole, morpholine, octahydroquinoline, piperazine, piperidine
and pyrrolidine. Representative heterocycles that may be formed
from R.sub.3 or R.sub.4, in combination with an R.sub.5 or R.sub.6,
include (but are not limited to) optionally substituted piperadine
and pyrrolidine
[0171] R.sub.5 and R.sub.6 of Formula IVa, within certain
embodiments, are independently (at each occurrence) hydrogen or
optionally substituted C.sub.1-C.sub.6alkyl; in addition, or
alternatively, any R.sub.5 or R.sub.6 may be joined with any other
R.sub.5 or R.sub.6 to form an optionally substituted 5- to
7-membered cycloalkyl, or (as discussed above) joined with R.sub.3
or R.sub.4 to form an optionally substituted heterocycle. In
certain embodiments, each R.sub.5 and R.sub.6 is
C.sub.1-C.sub.2alkyl or hydrogen. n may be 1, 2 or 3, with 1
preferred in certain embodiments.
[0172] Certain compounds of Formula IV further satisfy Formula
IVb
##STR00010##
Within Formula IVb:
[0173] Ar.sub.1, Ar.sub.2, Y and Z are as described for Formula IV;
R.sub.3 is selected from: [0174] (i) hydrogen; and [0175] (ii)
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, C.sub.6-C.sub.10arylC.sub.0-C.sub.8alkyl,
and 5- to 10-membered heterocycleC.sub.0-C.sub.8alkyl, each of
which is substituted with from 0 to 6 substituents independently
selected from R.sub.b; R.sub.5 and R.sub.6 are, independently at
each occurrence:
[0176] (i) each independently selected from hydrogen, hydroxy and
C.sub.1-C.sub.6alkyl; or
[0177] (ii) taken together to form a keto group; and
n is 1, 2 or 3.
[0178] In certain compounds of Formula IVb, R.sub.3 is: (i)
hydrogen; or (ii) C.sub.1-C.sub.8alkyl substituted with from 0 to 4
substituents independently selected from hydroxy, halogen, amino,
oxo, C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6haloalkoxy and mono- and
di-(C.sub.1-C.sub.6alkyl)amino. Within certain such compounds:
[0179] Y and Z are independently N or CH; [0180] Ar.sub.1 is
pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl, each of which is
substituted with halogen, cyano, C.sub.1-C.sub.4alkyl or
C.sub.1-C.sub.4haloalkyl; [0181] Ar.sub.2 is pyridyl, pyrimidinyl,
pyrazinyl or pyridazinyl, each of which is substituted with
halogen, cyano, C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4hydroxyalkyl,
C.sub.1-C.sub.4alkanoyl, C.sub.1-C.sub.4haloalkyl,
C.sub.1-C.sub.4alkylsulfonyl or C.sub.1-C.sub.4haloalkylsulfonyl;
[0182] Each R.sub.5 and R.sub.6 is independently selected from
hydrogen and C.sub.1-C.sub.2alkyl; and [0183] n is 1.
[0184] Within certain embodiments of Formula IVb, R.sub.3 is: (i)
hydrogen or (ii) C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, C.sub.3-C.sub.8alkanone,
C.sub.2-C.sub.8alkyl ether,
C.sub.6-C.sub.10arylC.sub.0-C.sub.8alkyl, or 5- to 10-membered
heterocycleC.sub.0-C.sub.8alkyl, each of which is optionally
substituted. Within other embodiments, R.sub.3 of Formula IV is (i)
hydrogen or (ii) C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkyl ether,
phenylC.sub.0-C.sub.4alkyl, 5- to 6-membered
heteroarylC.sub.0-C.sub.4alkyl, or 4- to 7-membered
heterocycloalkylC.sub.0-C.sub.4alkyl, each of which is optionally
substituted with from 1 to 4 substituents independently selected
from hydroxy, halogen, amino, C.sub.1-C.sub.6alkyl,
haloC.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy and
haloC.sub.1-C.sub.6alkoxy. Representative R.sub.3 groups include
hydrogen, C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkyl ether and
benzyl, each of which is unsubstituted or substituted with from 1
to 3 substituents independently selected from hydroxy, halogen and
C.sub.1-C.sub.4alkyl. Alternatively, R.sub.3 may be joined to an
R.sub.5 or R.sub.6 group (along with the 0 to which R.sub.3 is
bound and any carbon atoms between the O and R.sub.5 or R.sub.6) to
form an optionally substituted heterocycle, such as a 5- to
10-membered mono- or bi-cyclic group. The resulting heterocycle
may, for example, be substituted with from 0 to 4 (e.g., 0, 1 or 2)
substituents independently chosen from hydroxy, halogen,
C.sub.1-C.sub.4alkyl, haloC.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4alkoxy, haloC.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.4alkanoyl, C.sub.1-C.sub.4alkoxycarbonyl,
aminocarbonyl, heterocycleC.sub.0-C.sub.8alkyl and
heterocycleC.sub.1-C.sub.8alkoxycarbonyl.
[0185] R.sub.5 and R.sub.6, within certain embodiments of Formula
III, are independently (at each occurrence) hydrogen or optionally
substituted C.sub.1-C.sub.6alkyl; in addition, or alternatively,
any R.sub.5 or R.sub.6 may be joined with any other R.sub.5 or
R.sub.6 to form an optionally substituted 5- to 7-membered
cycloalkyl, or (as discussed above) joined with R.sub.3 to form an
optionally substituted heterocycle. In certain embodiments, each
R.sub.5 and R.sub.6 is C.sub.1-C.sub.2alkyl or hydrogen. n may be
1, 2 or 3, with 1 preferred in certain embodiments.
[0186] In certain embodiments of the Formulas provided herein,
R.sub.2 is hydrogen, amino, hydroxy, halogen, or optionally
substituted --(CH.sub.2).sub.nNH.sub.2,
--(CH.sub.2).sub.nNH(C.sub.1-C.sub.8alkyl),
--(CH.sub.2).sub.nN(C.sub.1-C.sub.8alkyl).sub.2,
--(CH.sub.2).sub.n(5- to 8-membered heterocycloalkyl),
--(CH.sub.2).sub.nOH or --(CH.sub.2).sub.nO(C.sub.1-C.sub.8alkyl).
Optionally substituted groups include, for example, unsubstituted
groups and groups substituted with from 1 to 4 substituents
independently chosen from halogen, cyano, hydroxy, amino, mono- and
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkyl, and
haloC.sub.1-C.sub.6alkyl. Heterocycloalkyl groups include those in
which the heterocycloalkyl comprises a nitrogen or oxygen atom
directly linked to the --(CH.sub.2).sub.n.
[0187] In certain embodiments of the Formulas provided herein,
Ar.sub.1 is phenyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl,
each of which is unsubstituted or substituted with 1 or 2
substituents as described above; preferably such substituents, if
any, are independently selected from halogen, hydroxy, cyano,
amino, nitro, mono- and di-(C.sub.1-C.sub.6alkyl)amino,
C.sub.1-C.sub.6alkyl, haloC.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy and haloC.sub.1-C.sub.6alkoxy. For example,
Ar.sub.1 may have one substituent selected from halogen,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy,
haloC.sub.1-C.sub.6alkyl and haloC.sub.1-C.sub.6alkoxy. Ar.sub.1
groups include, but are not limited to, pyridin-2-yl,
3-methyl-pyridin-2-yl, 3-trifluoromethyl-pyridin-2-yl and
3-halo-pyridin-2-yl.
[0188] Ar.sub.2, within certain embodiments of the formulas
provided herein, is phenyl, pyridyl, pyrimidinyl, pyrazinyl or
pyridazinyl, each of which is unsubstituted or substituted with 1
or 2 substituents as described above. In certain embodiments, one
such substituent is located at the para position of a 6-membered
Ar.sub.2. Optional Ar.sub.2 substituents are as described above and
include, for example, halogen, hydroxy, cyano, amino,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6alkyl ether,
C.sub.2-C.sub.8alkenyl, C.sub.2-C.sub.8alkynyl,
C.sub.1-C.sub.6alkanoyl, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6haloalkylsulfonyl, C.sub.1-C.sub.6alkylsulfonamide,
C.sub.1-C.sub.6haloalkylsulfonamide, mono- and
di-(C.sub.1-C.sub.6alkyl)amino and 3- to 10-membered heterocycles.
Preferred Ar.sub.2 substituents include C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4halolkyl and groups of the formula
--(SO.sub.2)R.sub.a, wherein R.sub.a is C.sub.1-C.sub.4alkyl or
haloC.sub.1-C.sub.4alkyl. Ar.sub.2 groups include phenyl, pyridyl,
pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, imidazolyl,
pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl and
thiadiazolyl, each of which is optionally substituted with 1 or 2
substituents independently selected from halogen, cyano,
C.sub.1-C.sub.6alkyl, haloC.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy, haloC.sub.1-C.sub.6alkoxy,
--SO.sub.2--R.sub.a and --SO.sub.2NR.sub.x--R.sub.a. Ar.sub.2
groups include, but are not limited to, phenyl, 2-pyridyl and
3-pyridyl, each of which is substituted at the para-position with
halogen, cyano, methyl, ethyl, propyl, isopropyl, t-butyl,
trifluoromethyl, 2,2,2-trifluoroethyl,
2,2,2-trifluoro-1-methyl-ethyl, methanesulfonyl, ethanesulfonyl,
propanesulfonyl, propane-2-sulfonyl, trifluoromethanesulfonyl or
2,2,2-trifluoroethanesulfonyl.
[0189] Representative compounds provided herein include, but are
not limited to, those specifically described in Examples 1-3. It
will be apparent that the specific compounds recited herein are
representative only, and are not intended to limit the scope of the
present invention. Further, as noted above, all compounds of the
present invention may be present as a free acid or base or as a
pharmaceutically acceptable salt.
[0190] Within certain aspects of the present invention, substituted
quinolin-4-ylamine analogues provided herein detectably alter
(modulate) VR1 activity, as determined using an in vitro VR1
functional assay such as a calcium mobilization assay, dorsal root
ganglion assay or in vivo pain relief assay. As an initial screen
for such activity, a VR1 ligand binding assay may be used.
References herein to a "VR1 ligand binding assay" are intended to
refer to a standard in vitro receptor binding assay such as that
provided in Example 5, and a "calcium mobilization assay" (also
referred to herein as a "signal transduction assay") may be
performed as described in Example 6. Briefly, to assess binding to
VR1, a competition assay may be performed in which a VR1
preparation is incubated with labeled (e.g., .sup.125I or .sup.3H)
compound that binds to VR1 (e.g., a capsaicin receptor agonist such
as RTX) and unlabeled test compound. Within the assays provided
herein, the VR1 used is preferably mammalian VR1, more preferably
human or rat VR1. The receptor may be recombinantly expressed or
naturally expressed. The VR1 preparation may be, for example, a
membrane preparation from HEK293 or CHO cells that recombinantly
express human VR1. Incubation with a compound that detectably
modulates vanilloid ligand binding to VR1 results in a decrease or
increase in the amount of label bound to the VR1 preparation,
relative to the amount of label bound in the absence of the
compound. This decrease or increase may be used to determine the
K.sub.i at VR1 as described herein. In general, compounds that
decrease the amount of label bound to the VR1 preparation within
such an assay are preferred.
[0191] As noted above, compounds that are VR1 antagonists are
preferred within certain embodiments. IC.sub.50 values for such
compounds may be determined using a standard in vitro VR1-mediated
calcium mobilization assay, as provided in Example 6. Briefly,
cells expressing capsaicin receptor are contacted with a compound
of interest and with an indicator of intracellular calcium
concentration (e.g., a membrane permeable calcium sensitivity dye
such as Fluo-3 or Fura-2 (both of which are available, for example,
from Molecular Probes, Eugene, Oreg.), each of which produce a
fluorescent signal when bound to Ca.sup.++). Such contact is
preferably carried out by one or more incubations of the cells in
buffer or culture medium comprising either or both of the compound
and the indicator in solution. Contact is maintained for an amount
of time sufficient to allow the dye to enter the cells (e.g., 1-2
hours). Cells are washed or filtered to remove excess dye and are
then contacted with a vanilloid receptor agonist (e.g., capsaicin,
RTX or olvanil), typically at a concentration equal to the
EC.sub.50 concentration, and a fluorescence response is measured.
When agonist-contacted cells are contacted with a compound that is
a VR1 antagonist the fluorescence response is generally reduced by
at least 20%, preferably at least 50% and more preferably at least
80%, as compared to cells that are contacted with the agonist in
the absence of test compound. The IC.sub.50 for VR1 antagonists
provided herein is preferably less than 1 micromolar, less than 100
nM, less than 10 nM or less than 1 nM.
[0192] In other embodiments, compounds that are capsaicin receptor
agonists are preferred. Capsaicin receptor agonist activity may
generally be determined as described in Example 6. When cells are
contacted with 1 micromolar of a compound that is a VR1 agonist,
the fluorescence response is generally increased by an amount that
is at least 30% of the increase observed when cells are contacted
with 100 nM capsaicin. The EC.sub.50 for VR1 agonists provided
herein is preferably less than 1 micromolar, less than 100 nM or
less than 10 nM.
[0193] VR1 modulating activity may also, or alternatively, be
assessed using a cultured dorsal root ganglion assay as provided in
Example 9 and/or an in vivo pain relief assay as provided in
Example 10. Compounds provided herein preferably have a
statistically significant specific effect on VR1 activity within
one or more functional assays provided herein.
[0194] Within certain embodiments, VR1 modulators provided herein
do not substantially modulate ligand binding to other cell surface
receptors, such as EGF receptor tyrosine kinase or the nicotinic
acetylcholine receptor. In other words, such modulators do not
substantially inhibit activity of a cell surface receptor such as
the human epidermal growth factor (EGF) receptor tyrosine kinase or
the nicotinic acetylcholine receptor (e.g., the IC.sub.50 or
IC.sub.40 at such a receptor is preferably greater than 1
micromolar, and most preferably greater than 10 micromolar).
Preferably, a modulator does not detectably inhibit EGF receptor
activity or nicotinic acetylcholine receptor activity at a
concentration of 0.5 micromolar, 1 micromolar or more preferably 10
micromolar. Assays for determining cell surface receptor activity
are commercially available, and include the tyrosine kinase assay
kits available from Panvera (Madison, Wis.).
[0195] Preferred compounds provided herein are non-sedating. In
other words, a dose of compound that is twice the minimum dose
sufficient to provide analgesia in an animal model for determining
pain relief (such as a model provided in Example 10, herein) causes
only transient (i.e., lasting for no more than 1/2 the time that
pain relief lasts) or preferably no statistically significant
sedation in an animal model assay of sedation (using the method
described by Fitzgerald et al. (1988) Toxicology 49 (2-3):433-9).
Preferably, a dose that is five times the minimum dose sufficient
to provide analgesia does not produce statistically significant
sedation. More preferably, a compound provided herein does not
produce sedation at intravenous doses of less than 25 mg/kg
(preferably less than 10 mg/kg) or at oral doses of less than 140
mg/kg (preferably less than 50 mg/kg, more preferably less than 30
mg/kg).
[0196] If desired, compounds provided herein may be evaluated for
certain pharmacological properties including, but not limited to,
oral bioavailability (preferred compounds are orally bioavailable
to an extent allowing for therapeutically effective concentrations
of the compound to be achieved at oral doses of less than 140
mg/kg, preferably less than 50 mg/kg, more preferably less than 30
mg/kg, even more preferably less than 10 mg/kg, still more
preferably less than 1 mg/kg and most preferably less than 0.1
mg/kg), toxicity (a preferred VR1 modulator is nontoxic when a
capsaicin receptor modulatory amount is administered to a subject),
side effects (a preferred VR1 modulator produces side effects
comparable to placebo when a therapeutically effective amount of
the compound is administered to a subject), serum protein binding
and in vitro and in vivo half-life (a preferred VR1 modulator
exhibits an in vitro half-life that is equal to an in vivo
half-life allowing for Q.I.D. dosing, preferably T.I.D. dosing,
more preferably B.I.D. dosing, and most preferably once-a-day
dosing). In addition, differential penetration of the blood brain
barrier may be desirable for VR1 modulators used to treat pain by
modulating CNS VR1 activity such that total daily oral doses as
described above provide such modulation to a therapeutically
effective extent, while low brain levels of VR1 modulators used to
treat peripheral nerve mediated pain may be preferred (i.e., such
doses do not provide brain (e.g., CSF) levels of the compound
sufficient to significantly modulate VR1 activity). Routine assays
that are well known in the art may be used to assess these
properties, and identify superior compounds for a particular use.
For example, assays used to predict bioavailability include
transport across human intestinal cell monolayers, including Caco-2
cell monolayers. Penetration of the blood brain barrier of a
compound in humans may be predicted from the brain levels of the
compound in laboratory animals given the compound (e.g.,
intravenously). Serum protein binding may be predicted from albumin
binding assays. Compound half-life is inversely proportional to the
frequency of dosage of a compound. In vitro half-lives of compounds
may be predicted from assays of microsomal half-life as described
within Example 7, herein.
[0197] As noted above, preferred compounds provided herein are
nontoxic. In general, the term "nontoxic" as used herein shall be
understood in a relative sense and is intended to refer to any
substance that has been approved by the United States Food and Drug
Administration ("FDA") for administration to mammals (preferably
humans) or, in keeping with established criteria, is susceptible to
approval by the FDA for administration to mammals (preferably
humans). In addition, a highly preferred nontoxic compound
generally satisfies one or more of the following criteria: (1) does
not substantially inhibit cellular ATP production; (2) does not
significantly prolong heart QT intervals; (3) does not cause
substantial liver enlargement, and (4) does not cause substantial
release of liver enzymes.
[0198] As used herein, a compound that "does not substantially
inhibit cellular ATP production" is a compound that satisfies the
criteria set forth in Example 8, herein. In other words, cells
treated as described in Example 8 with 100 .mu.M of such a compound
exhibit ATP levels that are at least 50% of the ATP levels detected
in untreated cells. In more highly preferred embodiments, such
cells exhibit ATP levels that are at least 80% of the ATP levels
detected in untreated compound that "does not significantly prolong
heart QT intervals" is a compound that does not result in a
statistically significant prolongation of heart QT intervals (as
determined by electrocardiography) in guinea pigs, minipigs or dogs
upon administration of twice the minimum dose yielding a
therapeutically effective in vivo concentration. In certain
preferred embodiments, a dose of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 40
or 50 mg/kg administered parenterally or orally does not result in
a statistically significant prolongation of heart QT intervals. By
"statistically significant" is meant results varying from control
at the p<0.1 level or more preferably at the p<0.05 level of
significance as measured using a standard parametric assay of
statistical significance such as a student's T test.
[0199] A compound "does not cause substantial liver enlargement" if
daily treatment of laboratory rodents (e.g., mice or rats) for 5-10
days with twice the minimum dose that yields a therapeutically
effective in vivo concentration results in an increase in liver to
body weight ratio that is no more than 100% over matched controls.
In more highly preferred embodiments, such doses do not cause liver
enlargement of more than 75% or 50% over matched controls. If
non-rodent mammals (e.g., dogs) are used, such doses should not
result in an increase of liver to body weight ratio of more than
50%, preferably not more than 25%, and more preferably not more
than 10% over matched untreated controls. Preferred doses within
such assays include 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 40 or 50 mg/kg
administered parenterally or orally.
[0200] Similarly, a compound "does not promote substantial release
of liver enzymes" if administration of twice the minimum dose
yielding a therapeutically effective in vivo concentration does not
elevate serum levels of ALT, LDH or AST in laboratory rodents by
more than 100% over matched mock-treated controls. In more highly
preferred embodiments, such doses do not elevate such serum levels
by more than 75% or 50% over matched controls. Alternatively, a
compound "does not promote substantial release of liver enzymes"
if, in an in vitro hepatocyte assay, concentrations (in culture
media or other such solutions that are contacted and incubated with
hepatocytes in vitro) equivalent to two-fold the minimum in vivo
therapeutic concentration of the compound do not cause detectable
release of any of such liver enzymes into culture medium above
baseline levels seen in media from matched mock-treated control
cells. In more highly preferred embodiments, there is no detectable
release of any of such liver enzymes into culture medium above
baseline levels when such compound concentrations are five-fold,
and preferably ten-fold the minimum in vivo therapeutic
concentration of the compound.
[0201] In other embodiments, certain preferred compounds do not
inhibit or induce microsomal cytochrome P450 enzyme activities,
such as CYP1A2 activity, CYP2A6 activity, CYP2C9 activity, CYP2C19
activity, CYP2D6 activity, CYP2E1 activity or CYP3A4 activity at a
concentration equal to the minimum therapeutically effective in
vivo concentration.
[0202] Certain preferred compounds are not clastogenic (e.g., as
determined using a mouse erythrocyte precursor cell micronucleus
assay, an Ames micronucleus assay, a spiral micronucleus assay or
the like) at a concentration equal to the minimum therapeutically
effective in vivo concentration. In other embodiments, certain
preferred compounds do not induce sister chromatid exchange (e.g.,
in Chinese hamster ovary cells) at such concentrations.
[0203] For detection purposes, as discussed in more detail below,
VR1 modulators provided herein may be isotopically-labeled or
radiolabeled. For example, compounds recited in Formulas I-III may
have one or more atoms replaced by an atom of the same element
having an atomic mass or mass number different from the atomic mass
or mass number usually found in nature. Examples of isotopes that
can be present in the compounds provided herein include isotopes of
hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and
chlorine, such as .sup.2H, .sup.3H, .sup.11C, .sup.13C, .sup.14C,
.sup.15N, .sup.18O, .sup.17O, .sup.31P, .sup.32P, .sup.35S,
.sup.18F and .sup.36Cl. In addition, substitution with heavy
isotopes such as deuterium (i.e., 2H) can afford certain
therapeutic advantages resulting from greater metabolic stability,
for example increased in vivo half-life or reduced dosage
requirements and, hence, may be preferred in some
circumstances.
Preparation of Substituted Quinolin-4-Ylamine Analogues
[0204] Substituted quinolin-4-ylamine analogues may generally be
prepared using standard synthetic methods. Starting materials are
commercially available from suppliers such as Sigma-Aldrich Corp.
(St. Louis, Mo.), or may be synthesized from commercially available
precursors using established protocols. By way of example, a
synthetic route similar to that shown in any of the following
Schemes may be used, together with synthetic methods known in the
art of synthetic organic chemistry. Each variable in the following
schemes refers to any group consistent with the description of the
compounds provided herein.
[0205] Other definitions used in the following Schemes and in the
Examples are:
Ac.sub.2O acetic anhydride AcOH acetic acid CDCl.sub.3 deuterated
chloroform .delta. chemical shift DME ethylene glycol dimethyl
ether DMF dimethylformamide DPPF
1,1'-bis(diphenylphosphino)ferrocene EDCl
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Et
ethyl EtOH ethanol .sup.1H NMR proton nuclear magnetic resonance
HPLC high pressure liquid chromatography Hz hertz iPr isopropyl
iPrOH isopropanol LCMS liquid chromatography/mass spectrometry
KHMDS potassium bis(trimethylsilyl)amide MS mass spectrometry (M+1)
mass+1 KtBuO potassium tert-butoxide MeOH methanol THF
tetrahydrofuran Pd.sub.2(dba).sub.3
tris[dibenzylidineacetone]di-palladium Pd(PPh.sub.3).sub.4
tetrakis(triphenylphosphine) palladium (0) Xantphos
4,5-bis(diphenylphosphino)-9,9-dimethyl-xanthene
##STR00011##
##STR00012##
##STR00013##
##STR00014##
##STR00015##
##STR00016##
##STR00017##
##STR00018## ##STR00019##
##STR00020##
##STR00021## ##STR00022##
##STR00023##
[0206] In certain embodiments, a compound provided herein may
contain one or more asymmetric carbon atoms, so that the compound
can exist in different stereoisomeric forms. Such forms can be, for
example, racemates or optically active forms. As noted above, all
stereoisomers are encompassed by the present invention.
Nonetheless, it may be desirable to obtain single enantiomers
(i.e., optically active forms). Standard methods for preparing
single enantiomers include asymmetric synthesis and resolution of
the racemates. Resolution of the racemates can be accomplished, for
example, by conventional methods such as crystallization in the
presence of a resolving agent, or chromatography using, for example
a chiral HPLC column.
[0207] Compounds may be radiolabeled by carrying out their
synthesis using precursors comprising at least one atom that is a
radioisotope. Each radioisotope is preferably carbon (e.g.,
.sup.14C), hydrogen (e.g., .sup.3H), sulfur (e.g., .sup.35S), or
iodine (e.g., .sup.125I). Tritium labeled compounds may also be
prepared catalytically via platinum-catalyzed exchange in tritiated
acetic acid, acid-catalyzed exchange in tritiated trifluoroacetic
acid, or heterogeneous-catalyzed exchange with tritium gas using
the compound as substrate. In addition, certain precursors may be
subjected to tritium-halogen exchange with tritium gas, tritium gas
reduction of unsaturated bonds, or reduction using sodium
borotritide, as appropriate. Preparation of radiolabeled compounds
may be conveniently performed by a radioisotope supplier
specializing in custom synthesis of radiolabeled probe
compounds.
Pharmaceutical Compositions
[0208] The present invention also provides pharmaceutical
compositions comprising one or more substituted quinolin-4-ylamine
analogues, together with at least one physiologically acceptable
carrier or excipient. Pharmaceutical compositions may comprise, for
example, one or more of water, buffers (e.g., neutral buffered
saline or phosphate buffered saline), ethanol, mineral oil,
vegetable oil, dimethylsulfoxide, carbohydrates (e.g., glucose,
mannose, sucrose or dextrans), mannitol, proteins, adjuvants,
polypeptides or amino acids such as glycine, antioxidants,
chelating agents such as EDTA or glutathione and/or preservatives.
In addition, other active ingredients may (but need not) be
included in the pharmaceutical compositions provided herein.
[0209] Pharmaceutical compositions may be formulated for any
appropriate manner of administration, including, for example,
topical, oral, nasal, rectal or parenteral administration. The term
parenteral as used herein includes subcutaneous, intradermal,
intravascular (e.g., intravenous), intramuscular, spinal,
intracranial, intrathecal and intraperitoneal injection, as well as
any similar injection or infusion technique. In certain
embodiments, compositions suitable for oral use are preferred. Such
compositions include, for example, tablets, troches, lozenges,
aqueous or oily suspensions, dispersible powders or granules,
emulsion, hard or soft capsules, or syrups or elixirs. Within yet
other embodiments, compositions of the present invention may be
formulated as a lyophilizate. Formulation for topical
administration may be preferred for certain conditions (e.g., in
the treatment of skin conditions such as burns or itch).
Formulation for direct administration into the bladder
(intravesicular administration) may be preferred for treatment of
urinary incontinence and overactive bladder.
[0210] Compositions intended for oral use may further comprise one
or more components such as sweetening agents, flavoring agents,
coloring agents and/or preserving agents in order to provide
appealing and palatable preparations. Tablets contain the active
ingredient in admixture with physiologically acceptable excipients
that are suitable for the manufacture of tablets. Such excipients
include, for example, inert diluents (e.g., calcium carbonate,
sodium carbonate, lactose, calcium phosphate or sodium phosphate),
granulating and disintegrating agents (e.g., corn starch or alginic
acid), binding agents (e.g., starch, gelatin or acacia) and
lubricating agents (e.g., magnesium stearate, stearic acid or
talc). The tablets may be uncoated or they may be coated by known
techniques to delay disintegration and absorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time delay material such as
glyceryl monosterate or glyceryl distearate may be employed.
[0211] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent (e.g., calcium carbonate, calcium phosphate or
kaolin), or as soft gelatin capsules wherein the active ingredient
is mixed with water or an oil medium (e.g., peanut oil, liquid
paraffin or olive oil).
[0212] Aqueous suspensions contain the active material(s) in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients include suspending agents (e.g.,
sodium carboxymethylcellulose, methylcellulose,
hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia); and dispersing or wetting agents
(e.g., naturally-occurring phosphatides such as lecithin,
condensation products of an alkylene oxide with fatty acids such as
polyoxyethylene stearate, condensation products of ethylene oxide
with long chain aliphatic alcohols such as
heptadecaethyleneoxycetanol, condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides such as polyethylene sorbitan
monooleate). Aqueous suspensions may also comprise one or more
preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or
more coloring agents, one or more flavoring agents, and one or more
sweetening agents, such as sucrose or saccharin.
[0213] Oily suspensions may be formulated by suspending the active
ingredient(s) in a vegetable oil (e.g., arachis oil, olive oil,
sesame oil or coconut oil) or in a mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent such
as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such
as those set forth above, and/or flavoring agents may be added to
provide palatable oral preparations. Such suspensions may be
preserved by the addition of an anti-oxidant such as ascorbic
acid.
[0214] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, such as sweetening,
flavoring and coloring agents, may also be present.
[0215] Pharmaceutical compositions may also be formulated as
oil-in-water emulsions. The oily phase may be a vegetable oil
(e.g., olive oil or arachis oil), a mineral oil (e.g., liquid
paraffin) or a mixture thereof. Suitable emulsifying agents include
naturally-occurring gums (e.g., gum acacia or gum tragacanth),
naturally-occurring phosphatides (e.g., soy bean lecithin, and
esters or partial esters derived from fatty acids and hexitol),
anhydrides (e.g., sorbitan monoleate) and condensation products of
partial esters derived from fatty acids and hexitol with ethylene
oxide (e.g., polyoxyethylene sorbitan monoleate). An emulsion may
also comprise one or more sweetening and/or flavoring agents.
[0216] Syrups and elixirs may be formulated with sweetening agents,
such as glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also comprise one or more demulcents,
preservatives, flavoring agents and/or coloring agents.
[0217] Formulations for topical administration typically comprise a
topical vehicle combined with active agent(s), with or without
additional optional components. Suitable topical vehicles and
additional components are well known in the art, and it will be
apparent that the choice of a vehicle will depend on the particular
physical form and mode of delivery. Topical vehicles include water;
organic solvents such as alcohols (e.g., ethanol or isopropyl
alcohol) or glycerin; glycols (e.g., butylene, isoprene or
propylene glycol); aliphatic alcohols (e.g., lanolin); mixtures of
water and organic solvents and mixtures of organic solvents such as
alcohol and glycerin; lipid-based materials such as fatty acids,
acylglycerols (including oils, such as mineral oil, and fats of
natural or synthetic origin), phosphoglycerides, sphingolipids and
waxes; protein-based materials such as collagen and gelatin;
silicone-based materials (both non-volatile and volatile); and
hydrocarbon-based materials such as microsponges and polymer
matrices. A composition may further include one or more components
adapted to improve the stability or effectiveness of the applied
formulation, such as stabilizing agents, suspending agents,
emulsifying agents, viscosity adjusters, gelling agents,
preservatives, antioxidants, skin penetration enhancers,
moisturizers and sustained release materials. Examples of such
components are described in Martindale--The Extra Pharmacopoeia
(Pharmaceutical Press, London 1993) and Martin (ed.), Remington's
Pharmaceutical Sciences. Formulations may comprise microcapsules,
such as hydroxymethylcellulose or gelatin-microcapsules, liposomes,
albumin microspheres, microemulsions, nanoparticles or
nanocapsules.
[0218] A topical formulation may be prepared in a variety of
physical forms including, for example, solids, pastes, creams,
foams, lotions, gels, powders, aqueous liquids and emulsions. The
physical appearance and viscosity of such forms can be governed by
the presence and amount of emulsifier(s) and viscosity adjuster(s)
present in the formulation. Solids are generally firm and
non-pourable and commonly are formulated as bars or sticks, or in
particulate form; solids can be opaque or transparent, and
optionally can contain solvents, emulsifiers, moisturizers,
emollients, fragrances, dyes/colorants, preservatives and other
active ingredients that increase or enhance the efficacy of the
final product. Creams and lotions are often similar to one another,
differing mainly in their viscosity; both lotions and creams may be
opaque, translucent or clear and often contain emulsifiers,
solvents, and viscosity adjusting agents, as well as moisturizers,
emollients, fragrances, dyes/colorants, preservatives and other
active ingredients that increase or enhance the efficacy of the
final product. Gels can be prepared with a range of viscosities,
from thick or high viscosity to thin or low viscosity. These
formulations, like those of lotions and creams, may also contain
solvents, emulsifiers, moisturizers, emollients, fragrances,
dyes/colorants, preservatives and other active ingredients that
increase or enhance the efficacy of the final product. Liquids are
thinner than creams, lotions, or gels and often do not contain
emulsifiers. Liquid topical products often contain solvents,
emulsifiers, moisturizers, emollients, fragrances, dyes/colorants,
preservatives and other active ingredients that increase or enhance
the efficacy of the final product.
[0219] Suitable emulsifiers for use in topical formulations
include, but are not limited to, ionic emulsifiers, cetearyl
alcohol, non-ionic emulsifiers like polyoxyethylene oleyl ether,
PEG-40 stearate, ceteareth-12, ceteareth-20, ceteareth-30,
ceteareth alcohol, PEG-100 stearate and glyceryl stearate. Suitable
viscosity adjusting agents include, but are not limited to,
protective colloids or non-ionic gums such as
hydroxyethylcellulose, xanthan gum, magnesium aluminum silicate,
silica, microcrystalline wax, beeswax, paraffin, and cetyl
palmitate. A gel composition may be formed by the addition of a
gelling agent such as chitosan, methyl cellulose, ethyl cellulose,
polyvinyl alcohol, polyquaterniums, hydroxyethylcellulose,
hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer or
ammoniated glycyrrhizinate. Suitable surfactants include, but are
not limited to, nonionic, amphoteric, ionic and anionic
surfactants. For example, one or more of dimethicone copolyol,
polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80,
lauramide DEA, cocamide DEA, and cocamide MEA, oleyl betaine,
cocamidopropyl phosphatidyl PG-dimonium chloride, and ammonium
laureth sulfate may be used within topical formulations. Suitable
preservatives include, but are not limited to, antimicrobials such
as methylparaben, propylparaben, sorbic acid, benzoic acid, and
formaldehyde, as well as physical stabilizers and antioxidants such
as vitamin E, sodium ascorbate/ascorbic acid and propyl gallate.
Suitable moisturizers include, but are not limited to, lactic acid
and other hydroxy acids and their salts, glycerin, propylene
glycol, and butylene glycol. Suitable emollients include lanolin
alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum,
isostearyl neopentanoate and mineral oils. Suitable fragrances and
colors include, but are not limited to, FD&C Red No. 40 and
FD&C Yellow No. 5. Other suitable additional ingredients that
may be included a topical formulation include, but are not limited
to, abrasives, absorbents, anti-caking agents, anti-foaming agents,
anti-static agents, astringents (e.g., witch hazel, alcohol and
herbal extracts such as chamomile extract), binders/excipients,
buffering agents, chelating agents, film forming agents,
conditioning agents, propellants, opacifying agents, pH adjusters
and protectants.
[0220] An example of a suitable topical vehicle for formulation of
a gel is: hydroxypropylcellulose (2.1%); 70/30 isopropyl
alcohol/water (90.9%); propylene glycol (5.1%); and Polysorbate 80
(1.9%). An example of a suitable topical vehicle for formulation as
a foam is: cetyl alcohol (1.1%); stearyl alcohol (0.5%; Quaternium
52 (1.0%); propylene glycol (2.0%); Ethanol 95 PGF3 (61.05%);
deionized water (30.05%); P75 hydrocarbon propellant (4.30%). All
percents are by weight.
[0221] Typical modes of delivery for topical compositions include
application using the fingers; application using a physical
applicator such as a cloth, tissue, swab, stick or brush; spraying
(including mist, aerosol or foam spraying); dropper application;
sprinkling; soaking; and rinsing. Controlled release vehicles can
also be used.
[0222] A pharmaceutical composition may be prepared as a sterile
injectible aqueous or oleaginous suspension. The modulator,
depending on the vehicle and concentration used, can either be
suspended or dissolved in the vehicle. Such a composition may be
formulated according to the known art using suitable dispersing,
wetting agents and/or suspending agents such as those mentioned
above. Among the acceptable vehicles and solvents that may be
employed are water, 1,3-butanediol, Ringer's solution and isotonic
sodium chloride solution. In addition, sterile, fixed oils may be
employed as a solvent or suspending medium. For this purpose any
bland fixed oil may be employed, including synthetic mono- or
diglycerides. In addition, fatty acids such as oleic acid find use
in the preparation of injectible compositions, and adjuvants such
as local anesthetics, preservatives and/or buffering agents can be
dissolved in the vehicle.
[0223] Compounds may also be formulated as suppositories (e.g., for
rectal administration). Such compositions can be prepared by mixing
the drug with a suitable non-irritating excipient that is solid at
ordinary temperatures but liquid at the rectal temperature and will
therefore melt in the rectum to release the drug. Suitable
excipients include, for example, cocoa butter and polyethylene
glycols.
[0224] Pharmaceutical compositions may be formulated as sustained
release formulations (i.e., a formulation such as a capsule that
effects a slow release of modulator following administration). Such
formulations may generally be prepared using well known technology
and administered by, for example, oral, rectal or subcutaneous
implantation, or by implantation at the desired target site.
Carriers for use within such formulations are biocompatible, and
may also be biodegradable; preferably the formulation provides a
relatively constant level of modulator release. The amount of
modulator contained within a sustained release formulation depends
upon, for example, the site of implantation, the rate and expected
duration of release and the nature of the condition to be treated
or prevented.
[0225] In addition to or together with the above modes of
administration, a modulator may be conveniently added to food or
drinking water (e.g., for administration to non-human animals
including companion animals (such as dogs and cats) and livestock).
Animal feed and drinking water compositions may be formulated so
that the animal takes in an appropriate quantity of the composition
along with its diet. It may also be convenient to present the
composition as a premix for addition to feed or drinking water.
[0226] VR1 modulators are generally administered in a capsaicin
receptor modulatory amount, and preferably a therapeutically
effective amount. Preferred systemic doses are no higher than 50 mg
per kilogram of body weight per day (e.g., ranging from about 0.001
mg to about 50 mg per kilogram of body weight per day), with oral
doses generally being about 5-20 fold higher than intravenous doses
(e.g., ranging from 0.01 to 40 mg per kilogram of body weight per
day).
[0227] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage unit will vary
depending, for example, upon the patient being treated and the
particular mode of administration. Dosage units will generally
contain between from about 10 .mu.g to about 500 mg of an active
ingredient. Optimal dosages may be established using routine
testing, and procedures that are well known in the art.
[0228] Pharmaceutical compositions may be packaged for treating
conditions responsive to VR1 modulation (e.g., treatment of
exposure to vanilloid ligand, pain, itch, obesity or urinary
incontinence). Packaged pharmaceutical compositions may include a
container holding a therapeutically effective amount of at least
one VR1 modulator as described herein and instructions (e.g.,
labeling) indicating that the contained composition is to be used
for treating a condition responsive to VR1 modulation in the
patient.
Methods of Use
[0229] Compounds provided herein may be used to alter activity
and/or activation of capsaicin receptors in a variety of contexts,
both in vitro and in vivo. Within certain aspects, VR1 antagonists
may be used to inhibit the binding of vanilloid ligand agonist
(such as capsaicin and/or RTX) to capsaicin receptor in vitro or in
vivo. In general, such methods comprise the step of contacting a
capsaicin receptor with a capsaicin receptor modulatory amount of
one or more VR1 modulators provided herein, in the presence of
vanilloid ligand in aqueous solution and under conditions otherwise
suitable for binding of the ligand to capsaicin receptor. The
capsaicin receptor may be present in solution or suspension (e.g.,
in an isolated membrane or cell preparation), or in a cultured or
isolated cell. Within certain embodiments, the capsaicin receptor
is expressed by a neuronal cell present in a patient, and the
aqueous solution is a body fluid. Preferably, one or more VR1
modulators are administered to an animal in an amount such that the
analogue is present in at least one body fluid of the animal at a
therapeutically effective concentration that is 1 micromolar or
less; preferably 500 nanomolar or less; more preferably 100
nanomolar or less, 50 nanomolar or less, 20 nanomolar or less, or
10 nanomolar or less. For example, such compounds may be
administered at a dose that is less than 20 mg/kg body weight,
preferably less than 5 mg/kg and, in some instances, less than 1
mg/kg.
[0230] Also provided herein are methods for modulating, preferably
reducing, the signal-transducing activity (i.e., the calcium
conductance) of a cellular capsaicin receptor. Such modulation may
be achieved by contacting a capsaicin receptor (either in vitro or
in vivo) with a capsaicin receptor modulatory amount of one or more
VR1 modulators provided herein under conditions suitable for
binding of the modulator(s) to the receptor. The receptor may be
present in solution or suspension, in a cultured or isolated cell
preparation or in a cell within a patient. For example, the cell
may be a neuronal cell that is contacted in vivo in an animal.
Alternatively, the cell may be an epithelial cell, such as a
urinary bladder epithelial cell (urothelial cell) or an airway
epithelial cell that is contacted in vivo in an animal. Modulation
of signal tranducing activity may be assessed by detecting an
effect on calcium ion conductance (also referred to as calcium
mobilization or flux). Modulation of signal transducing activity
may alternatively be assessed by detecting an alteration of a
symptom (e.g., pain, burning sensation, broncho-constriction,
inflammation, cough, hiccup, itch, urinary incontinence or
overactive bladder) of a patient being treated with one or more VR1
modulators provided herein.
[0231] VR1 modulator(s) provided herein are preferably administered
to a patient (e.g., a human) orally or topically, and are present
within at least one body fluid of the animal while modulating VR1
signal-transducing activity. Preferred VR1 modulators for use in
such methods modulate VR1 signal-transducing activity in vitro at a
concentration of 1 nanomolar or less, preferably 100 picomolar or
less, more preferably 20 picomolar or less, and in vivo at a
concentration of 1 micromolar or less, 500 nanomolar or less, or
100 nanomolar or less in a body fluid such as blood.
[0232] The present invention further provides methods for treating
conditions responsive to VR1 modulation. Within the context of the
present invention, the term "treatment" encompasses both
disease-modifying treatment and symptomatic treatment, either of
which may be prophylactic (i.e., before the onset of symptoms, in
order to prevent, delay or reduce the severity of symptoms) or
therapeutic (i.e., after the onset of symptoms, in order to reduce
the severity and/or duration of symptoms). A condition is
"responsive to VR1 modulation" if it is characterized by
inappropriate activity of a capsaicin receptor, regardless of the
amount of vanilloid ligand present locally, and/or if modulation of
capsaicin receptor activity results in alleviation of the condition
or a symptom thereof. Such conditions include, for example,
symptoms resulting from exposure to VR1-activating stimuli, pain,
respiratory disorders such as asthma and chronic obstructive
pulmonary disease, itch, urinary incontinence, overactive bladder,
cough, hiccup, and obesity, as described in more detail below. Such
conditions may be diagnosed and monitored using criteria that have
been established in the art. Patients may include humans,
domesticated companion animals and livestock, with dosages as
described above.
[0233] Treatment regimens may vary depending on the compound used
and the particular condition to be treated. However, for treatment
of most disorders, a frequency of administration of 4 times daily
or less is preferred. In general, a dosage regimen of 2 times daily
is more preferred, with once a day dosing particularly preferred.
For the treatment of acute pain, a single dose that rapidly reaches
effective concentrations is desirable. It will be understood,
however, that the specific dose level and treatment regimen for any
particular patient will depend upon a variety of factors including
the activity of the specific compound employed, the age, body
weight, general health, sex, diet, time of administration, route of
administration, and rate of excretion, drug combination and the
severity of the particular disease undergoing therapy. In general,
the use of the minimum dose sufficient to provide effective therapy
is preferred. Patients may generally be monitored for therapeutic
effectiveness using medical or veterinary criteria suitable for the
condition being treated or prevented.
[0234] Patients experiencing symptoms resulting from exposure to
capsaicin receptor-activating stimuli include individuals with
burns caused by heat, light, tear gas or acid and those whose
mucous membranes are exposed (e.g., via ingestion, inhalation or
eye contact) to capsaicin (e.g., from hot peppers or in pepper
spray) or a related irritant such as acid, tear gas or air
pollutants. The resulting symptoms (which may be treated using VR1
modulators, especially antagonists, provided herein) may include,
for example, pain, broncho-constriction and inflammation.
[0235] Pain that may be treated using the VR1 modulators provided
herein may be chronic or acute and includes, but is not limited to,
peripheral nerve-mediated pain (especially neuropathic pain).
Compounds provided herein may be used in the treatment of, for
example, postmastectomy pain syndrome, stump pain, phantom limb
pain, oral neuropathic pain, toothache (dental pain), denture pain,
postherpetic neuralgia, diabetic neuropathy, reflex sympathetic
dystrophy, trigeminal neuralgia, osteoarthritis, rheumatoid
arthritis, fibromyalgia, Guillain-Barre syndrome, meralgia
paresthetica, burning-mouth syndrome and/or bilateral peripheral
neuropathy. Additional neuropathic pain conditions include
causalgia (reflex sympathetic dystrophy --RSD, secondary to injury
of a peripheral nerve), neuritis (including, for example, sciatic
neuritis, peripheral neuritis, polyneuritis, optic neuritis,
postfebrile neuritis, migrating neuritis, segmental neuritis and
Gombault's neuritis), neuronitis, neuralgias (e.g., those mentioned
above, cervicobrachial neuralgia, cranial neuralgia, geniculate
neuralgia, glossopharyngial neuralgia, migranous neuralgia,
idiopathic neuralgia, intercostals neuralgia, mammary neuralgia,
mandibular joint neuralgia, Morton's neuralgia, nasociliary
neuralgia, occipital neuralgia, red neuralgia, Sluder's neuralgia,
splenopalatine neuralgia, supraorbital neuralgia and vidian
neuralgia), surgery-related pain, musculoskeletal pain,
AIDS-related neuropathy, MS-related neuropathy, and spinal cord
injury-related pain. Headache, including headaches involving
peripheral nerve activity, such as sinus, cluster (i.e., migranous
neuralgia) and some tension headaches and migraine, may also be
treated as described herein. For example, migraine headaches may be
prevented by administration of a compound provided herein as soon
as a pre-migrainous aura is experienced by the patient. Further
pain conditions that can be treated as described herein include
"burning mouth syndrome," labor pains, Charcot's pains, intestinal
gas pains, menstrual pain, acute and chronic back pain (e.g., lower
back pain), hemorrhoidal pain, dyspeptic pains, angina, nerve root
pain, homotopic pain and heterotopic pain--including cancer
associated pain (e.g., in patients with bone cancer), pain (and
inflammation) associated with venom exposure (e.g., due to snake
bite, spider bite, or insect sting) and trauma associated pain
(e.g., post-surgical pain, pain from cuts, bruises and broken
bones, and burn pain). Additional pain conditions that may be
treated as described herein include pain associated with
inflammatory bowel disease, irritable bowel syndrome and/or
inflammatory bowel disease.
[0236] Within certain aspects, VR1 modulators provided herein may
be used for the treatment of mechanical pain. As used herein, the
term "mechanical pain" refers to pain other than headache pain that
is not neuropathic or a result of exposure to heat, cold or
external chemical stimuli. Mechanical pain includes physical trauma
(other than thermal or chemical burns or other irritating and/or
painful exposures to noxious chemicals) such as post-surgical pain
and pain from cuts, bruises and broken bones; toothache, denture
pain; nerve root pain; osteoarthritis; rheumatoid arthritis;
fibromyalgia; meralgia paresthetica; back pain; cancer-associated
pain; angina; carpel tunnel syndrome; and pain resulting from bone
fracture, labor, hemorrhoids, intestinal gas, dyspepsia, and
menstruation.
[0237] Itching conditions that may be treated include psoriatic
pruritis, itch due to hemodialysis, aguagenic pruritus, and itching
associated with vulvar vestibulitis, contact dermatitis, insect
bites and skin allergies. Urinary tract conditions that may be
treated as described herein include urinary incontinence (including
overflow incontinence, urge incontinence and stress incontinence),
as well as overactive or unstable bladder conditions (including
detrusor hyperflexia of spinal origin and bladder
hypersensitivity). In certain such treatment methods, VR1 modulator
is administered via a catheter or similar device, resulting in
direct injection of VR1 modulator into the bladder. Compounds
provided herein may also be used as anti-tussive agents (to
prevent, relieve or suppress coughing) and for the treatment of
hiccup, and to promote weight loss in an obese patient.
[0238] Within other aspects, VR1 modulators provided herein may be
used within combination therapy for the treatment of conditions
involving inflammatory components. Such conditions include, for
example, autoimmune disorders and pathologic autoimmune responses
known to have an inflammatory component including, but not limited
to, arthritis (especially rheumatoid arthritis), psoriasis, Crohn's
disease, lupus erythematosus, irritable bowel syndrome, tissue
graft rejection, and hyperacute rejection of transplanted organs.
Other such conditions include trauma (e.g., injury to the head or
spinal cord), cardio- and cerebo-vascular disease and certain
infectious diseases.
[0239] Within such combination therapy, a VR1 modulator is
administered to a patient along with an anti-inflammatory agent.
The VR1 modulator and anti-inflammatory agent may be present in the
same pharmaceutical composition, or may be administered separately
in either order. Anti-inflammatory agents include, for example,
non-steroidal anti-inflammatory drugs (NSAIDs), non-specific and
cyclooxygenase-2 (COX-2) specific cyclooxygenase enzyme inhibitors,
gold compounds, corticosteroids, methotrexate, tumor necrosis
factor (TNF) receptor antagonists, anti-TNF alpha antibodies,
anti-C5 antibodies, and interleukin-1 (IL-1) receptor antagonists.
Examples of NSAIDs include, but are not limited to ibuprofen (e.g.,
ADVIL.TM., MOTRIN.TM.), flurbiprofen (ANSAID.TM.), naproxen or
naproxen sodium (e.g., NAPROSYN, ANAPROX, ALEVE.TM.), diclofenac
(e.g., CATAFLAM.TM., VOLTAREN.TM.), combinations of diclofenac
sodium and misoprostol (e.g., ARTHROTEC.TM.), sulindac
(CLINORIL.TM.), oxaprozin (DAYPRO.TM.), diflunisal (DOLOBID.TM.),
piroxicam (FELDENE.TM.), indomethacin (INDOCIN.TM.), etodolac
(LODINE.TM.), fenoprofen calcium (NALFON.TM.), ketoprofen (e.g.,
ORUDIS.TM., ORUVAIL.TM.), sodium nabumetone (RELAFEN.TM.),
sulfasalazine (AZULFIDINE.TM.), tolmetin sodium (TOLECTIN.TM.), and
hydroxychloroquine (PLAQUENIL.TM.). A particular class of NSAIDs
consists of compounds that inhibit cyclooxygenase (COX) enzymes,
such as celecoxib (CELEBREX.TM.) and rofecoxib (VIOXX.TM.). NSAIDs
further include salicylates such as acetylsalicylic acid or
aspirin, sodium salicylate, choline and magnesium salicylates
(TRILISATE.TM.), and salsalate (DISALCID.TM.), as well as
corticosteroids such as cortisone (CORTONE.TM. acetate),
dexamethasone (e.g., DECADRON.TM.), methylprednisolone (MEDROL.TM.)
prednisolone (PRELONE.TM.), prednisolone sodium phosphate
(PEDIAPRED.TM.), and prednisone (e.g., PREDNICEN-M.TM.,
DELTASONE.TM., STERAPRED.TM.).
[0240] Suitable dosages for VR1 modulator within such combination
therapy are generally as described above. Dosages and methods of
administration of anti-inflammatory agents can be found, for
example, in the manufacturer's instructions in the Physician's Desk
Reference. In certain embodiments, the combination administration
of a VR1 modulator with an anti-inflammatory agent results in a
reduction of the dosage of the anti-inflammatory agent required to
produce a therapeutic effect. Thus, preferably, the dosage of
anti-inflammatory agent in a combination or combination treatment
method of the invention is less than the maximum dose advised by
the manufacturer for administration of the anti-inflammatory agent
without combination administration of a VR1 antagonist. More
preferably this dosage is less than 3/4, even more preferably less
than 1/2, and highly preferably, less than 1/4 of the maximum dose,
while most preferably the dose is less than 10% of the maximum dose
advised by the manufacturer for administration of the
anti-inflammatory agent(s) when administered without combination
administration of a VR1 antagonist. It will be apparent that the
dosage amount of VR1 antagonist component of the combination needed
to achieve the desired effect may similarly be affected by the
dosage amount and potency of the anti-inflammatory agent component
of the combination.
[0241] In certain preferred embodiments, the combination
administration of a VR1 modulator with an anti-inflammatory agent
is accomplished by packaging one or more VR1 modulators and one or
more anti-inflammatory agents in the same package, either in
separate containers within the package or in the same contained as
a mixture of one or more VR1 antagonists and one or more
anti-inflammatory agents. Preferred mixtures are formulated for
oral administration (e.g., as pills, capsules, tablets or the
like). In certain embodiments, the package comprises a label
bearing indicia indicating that the one or more VR1 modulators and
one or more anti-inflammatory agents are to be taken together for
the treatment of an inflammatory pain condition. A highly preferred
combination is one in which the anti-inflammatory agent(s) include
at least one COX-2 specific cyclooxygenase enzyme inhibitor such as
valdecoxib (BEXTRA.RTM.), lumiracoxib (PREXIGE.TM.), etoricoxib
(ARCOXIA.RTM.), celecoxib (CELEBREX.RTM.) and/or rofecoxib
(VIOXX.RTM.).
[0242] Within further aspects, VR1 modulators provided herein may
be used in combination with one or more additional pain relief
medications. Certain such medications are also anti-inflammatory
agents, and are listed above. Other such medications are narcotic
analgesic agents, which typically act at one or more opioid
receptor subtypes (e.g., .mu., .kappa. and/or .delta.), preferably
as agonists or partial agonists. Such agents include opiates,
opiate derivatives and opioids, as well as pharmaceutically
acceptable salts and hydrates thereof. Specific examples of
narcotic analgesics include, within preferred embodiments,
alfentanyl, alphaprodine, anileridine, bezitramide, buprenorphine,
codeine, diacetyldihydromorphine, diacetylmorphine, dihydrocodeine,
diphenoxylate, ethylmorphine, fentanyl, heroin, hydrocodone,
hydromorphone, isomethadone, levomethorphan, levorphane,
levorphanol, meperidine, metazocine, methadone, methorphan,
metopon, morphine, opium extracts, opium fluid extracts, powdered
opium, granulated opium, raw opium, tincture of opium, oxycodone,
oxymorphone, paregoric, pentazocine, pethidine, phenazocine,
piminodine, propoxyphene, racemethorphan, racemorphan, thebaine and
pharmaceutically acceptable salts and hydrates of the foregoing
agents.
[0243] Other examples of narcotic analgesic agents include
acetorphine, acetyldihydrocodeine, acetylmethadol, allylprodine,
alphracetylmethadol, alphameprodine, alphamethadol, benzethidine,
benzylmorphine, betacetylmethadol, betameprodine, betamethadol,
betaprodine, butorphanol, clonitazene, codeine methylbromide,
codeine-N-oxide, cyprenorphine, desomorphine, dextromoramide,
diampromide, diethylthiambutene, dihydromorphine, dimenoxadol,
dimepheptanol, dimethylthiamubutene, dioxaphetyl butyrate,
dipipanone, drotebanol, ethanol, ethylmethylthiambutene,
etonitazene, etorphine, etoxeridine, furethidine, hydromorphinol,
hydroxypethidine, ketobemidone, levomoramide, levophenacylmorphan,
methyldesorphine, methyldihydromorphine, morpheridine, morphine
methylpromide, morphine methylsulfonate, morphine-N-oxide,
myrophin, naloxone, nalbuyphine, naltyhexone, nicocodeine,
nicomorphine, noracymethadol, norlevorphanol, normethadone,
normorphine, norpipanone, pentazocaine, phenadoxone, phenampromide,
phenomorphan, phenoperidine, piritramide, pholcodine,
proheptazoineii, properidine, propiran, racemoramide, thebacon,
trimeperidine and the pharmaceutically acceptable salts and
hydrates thereof.
[0244] Further specific representative analgesic agents include,
for example: TALWIN.RTM. Nx and DEMEROL.RTM. (both available from
Sanofi Winthrop Pharmaceuticals; New York, N.Y.);
LEVO-DROMORAN.RTM.; BUPRENEX.RTM. (Reckitt & Coleman
Pharmaceuticals, Inc.; Richmond, Va.); MSIR.RTM. (Purdue Pharma
L.P.; Norwalk, Conn.); DILAUDID.RTM. (Knoll Pharmaceutical Co.;
Mount Olive, N.J.); SUBLIMAZE.RTM.; SUFENTA.RTM. (Janssen
Pharmaceutica Inc.; Titusville, N.J.); PERCOCET.RTM., NUBAIN.RTM.
and NUMORPHAN.RTM. (all available from Endo Pharmaceuticals Inc.;
Chadds Ford, Pa.) HYDROSTAT.RTM. IR, MS/S and MS/L (all available
from Richwood Pharmaceutical Co. Inc; Florence, Ky.), ORAMORPH.RTM.
SR and ROXICODONE.RTM. (both available from Roxanne Laboratories;
Columbus Ohio) and STADOL.RTM. (Bristol-Myers Squibb; New York,
N.Y.). Still further analgesic agents include CB2-receptor
agonists, such as AM1241, and compounds that bind to the
.alpha.2.delta. subunit, such as Neurontin (Gabapentin) and
pregabalin.
[0245] Suitable dosages for VR1 modulator within such combination
therapy are generally as described above. Dosages and methods of
administration of other pain relief medications can be found, for
example, in the manufacturer's instructions in the Physician's Desk
Reference. In certain embodiments, the combination administration
of a VR1 modulator with one or more additional pain medications
results in a reduction of the dosage of each therapeutic agent
required to produce a therapeutic effect (e.g., the dosage or one
or both agent may less than 3/4, less than 1/2, less than 1/4 or
less than 10% of the maximum dose listed above or advised by the
manufacturer). In certain preferred embodiments, the combination
administration of a VR1 modulator with one or more additional pain
relief medications is accomplished by packaging one or more VR1
modulators and one or more additional pain relief medications in
the same package, as described above.
[0246] Modulators that are VR1 agonists may further be used, for
example, in crowd control (as a substitute for tear gas) or
personal protection (e.g., in a spray formulation) or as
pharmaceutical agents for the treatment of pain, itch, urinary
incontinence or overactive bladder via capsaicin receptor
desensitization. In general, compounds for use in crowd control or
personal protection are formulated and used according to
conventional tear gas or pepper spray technology.
[0247] Within separate aspects, the present invention provides a
variety of non-pharmaceutical in vitro and in vivo uses for the
compounds provided herein. For example, such compounds may be
labeled and used as probes for the detection and localization of
capsaicin receptor (in samples such as cell preparations or tissue
sections, preparations or fractions thereof). In addition,
compounds provided herein that comprise a suitable reactive group
(such as an aryl carbonyl, nitro or azide group) may be used in
photoaffinity labeling studies of receptor binding sites. In
addition, compounds provided herein may be used as positive
controls in assays for receptor activity, as standards for
determining the ability of a candidate agent to bind to capsaicin
receptor, or as radiotracers for positron emission tomography (PET)
imaging or for single photon emission computerized tomography
(SPECT). Such methods can be used to characterize capsaicin
receptors in living subjects. For example, a VR1 modulator may be
labeled using any of a variety of well known techniques (e.g.,
radiolabeled with a radionuclide such as tritium, as described
herein), and incubated with a sample for a suitable incubation time
(e.g., determined by first assaying a time course of binding).
Following incubation, unbound compound is removed (e.g., by
washing), and bound compound detected using any method suitable for
the label employed (e.g., autoradiography or scintillation counting
for radiolabeled compounds; spectroscopic methods may be used to
detect luminescent groups and fluorescent groups). As a control, a
matched sample containing labeled compound and a greater (e.g.,
10-fold greater) amount of unlabeled compound may be processed in
the same manner. A greater amount of detectable label remaining in
the test sample than in the control indicates the presence of
capsaicin receptor in the sample. Detection assays, including
receptor autoradiography (receptor mapping) of capsaicin receptor
in cultured cells or tissue samples may be performed as described
by Kuhar in sections 8.1.1 to 8.1.9 of Current Protocols in
Pharmacology (1998) John Wiley & Sons, New York.
[0248] Modulators provided herein may also be used within a variety
of well known cell separation methods. For example, modulators may
be linked to the interior surface of a tissue culture plate or
other support, for use as affinity ligands for immobilizing and
thereby isolating, capsaicin receptors (e.g., isolating
receptor-expressing cells) in vitro. Within one preferred
embodiment, a modulator linked to a fluorescent marker, such as
fluorescein, is contacted with the cells, which are then analyzed
(or isolated) by fluorescence activated cell sorting (FACS).
[0249] Modulators provided herein may further be used within assays
for the identification of other agents that bind to capsaicin
receptor. In general, such assays are standard competition binding
assays, in which bound, labeled VR1 modulator is displaced by a
test compound. Briefly, such assays are performed by: (a)
contacting capsaicin receptor with a radiolabeled VR1 modulator as
described herein, under conditions that permit binding of the VR1
modulator to capsaicin receptor, thereby generating bound, labeled
VR1 modulator; (b) detecting a signal that corresponds to the
amount of bound, labeled VR1 modulator in the absence of test
agent; (c) contacting the bound, labeled VR1 modulator with a test
agent; (d) detecting a signal that corresponds to the amount of
bound labeled VR1 modulator in the presence of test agent; and (e)
detecting a decrease in signal detected in step (d), as compared to
the signal detected in step (b), and therefrom identifying an agent
that binds to capsaicin receptor.
[0250] The following Examples are offered by way of illustration
and not by way of limitation. Unless otherwise specified all
reagents and solvent are of standard commercial grade and are used
without further purification. Using routine modifications, the
starting materials may be varied and additional steps employed to
produce other compounds provided herein.
EXAMPLES
[0251] In the following Examples, mass spectroscopy data is
Electrospray MS, obtained in positive ion mode with a 15V or 30V
cone voltage, using a Micromass Time-of-Flight LCT, equipped with a
Waters 600 pump, Waters 996 photodiode array detector, Gilson 215
autosampler, and a Gilson 841 microinjector. MassLynx (Advanced
Chemistry Development, Inc; Toronto, Canada) version 4.0 software
was used for data collection and analysis. Sample volume of 1
microliter was injected onto a 50.times.4.6 mm Chromolith SpeedROD
C18 column, and eluted using a 2-phase linear gradient at 6 ml/min
flow rate. Sample was detected using total absorbance count over
the 220-340 nm UV range. The elution conditions were: Mobile Phase
A--95/5/0.05 Water/Methanol/TFA; Mobile Phase B--5/95/0.025
Water/Methanol/TFA.
TABLE-US-00001 Gradient: Time(min) % B 0 10 0.5 100 1.2 100 1.21
10
[0252] The total run time was 2 minutes inject to inject.
[0253] For the compounds described in Examples 1 and 2, the
IC.sub.50 determined as described in Example 6, herein, is 1
micromolar or less.
Example 1
Preparation of Representative Substituted Quinolin-4-ylamine
Analogues
[0254] A. Using the procedure illustrated in Scheme
1,2-methoxymethyl-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]napthyridin-4-y-
l-(4-trifluoromethylphenyl)-amine is prepared by the following
steps:
1. 2-cyano-3-trifluoromethylpyridine
##STR00024##
[0256] DMF (3500 mL), H.sub.2O (35 mL),
2-chloro-3-trifluoromethyl-pyridine (250 g; 1.38 mol), Zn(CN).sub.2
(97 g; 0.83 mol), Pd.sub.2(dba).sub.3 (19.0 g) and DPPF (22.4 g)
are combined in a 22.0 liter flask. The reaction mixture is
degassed by bubbling N.sub.2 into the reaction mixture over a
period of 30 minutes. The reaction mixture is then heated to
120.degree. C. for 4.5 hours, at which time an additional 9.5 g of
Pd.sub.2(dba).sub.3 and 11.2 g of DPPF is added to the reaction
mixture. The reaction mixture is then heated at 120.degree. C. for
another 2 hours, and then allowed to cool to room temperature
overnight. The resulting dark brown solution is cooled in ice and
cold water. A mixture of saturated NH.sub.4Cl (1380 mL), 28%
NH.sub.4OH (345 mL and water (1380 mL) is then added and the
mixture is stirred in an ice bath for 1 hour. To this stirred
mixture is added 3.0 liters of EtOAc, and the mixture is stirred
for 15 minutes. The EtOAc layer is separated from the mixture by
suction the extraction is repeated another four times with EtOAc
(2.times.2 liters, 2.times.1.5 liters). The combined EtOAc extracts
are filtered through one inch of celite and dried with
Na.sub.2SO.sub.4 (500 g). The dried extract is filtered and
concentrated in vacuum at 40.degree. C. initially to a volume of
three liters. The mixture is concentrated at 80.degree. C. under
vacuum to afford a dark brown oil, which is distilled under vacuum
to give 2-cyano-3-trifluoromethylpyridine.
2. 2-acetyl-3-trifluoromethylpyridine
##STR00025##
[0258] 2-cyano-3-trifluoromethylpyridine (179 g; 1.04 mol) and THF
(1200 mL) are combined in a 5.0 liter flask and cooled with and ice
and salt mixture. 3.0 M MeMgI/Et2O (694 mL) is added drop wise over
a period of 90 minutes while maintaining the inside temperature of
the reaction mixture below 5.degree. C. After the addition, the
reaction mixture is stirred at 0.degree. C. for another 30 minutes,
and is then slowly poured over 3.0 kg of crushed ice in a 12 liter
vessel with stirring (6.degree. C.). The undissolved magnesium
salts from the original reaction vessel are quenched with ice (750
g) and transferred to the 12 liter vessel. The resulting mixture is
acidified with 6.0 N aq. HCl to pH 2.0 and stirred for 30 minutes
at <10.degree. C. The mixture is then extracted with EtOAc
(5.times.1 liters), and the combined extracts are washed with brine
(1.5 liters) and dried with Na.sub.2SO.sub.4 (500 g). The dried
extract is filtered and concentrated in vacuum at 40.degree. C. to
afford a dark brown oil. The crude product is distilled under
vacuum to give 2-acetyl-3-trifluoromethylpyridine as a clear pale
yellow liquid.
3. 3-dimethylamino-1-(3-trifluoromethyl-pyridin-2-yl)-propenone
##STR00026##
[0260] 2-acetyl-3-trifluoromethylpyridine (150 g; 0.79 mol) and
(Me).sub.2N--CH(OMe).sub.2 (236 g; 1.98 mol) are combined in a 1
liter flask. The mixture is heated with stirring at 105.degree. C.
for 5 hours. Excess (Me).sub.2N--CH(OMe).sub.2 is removed under
vacuum at 60.degree. C. and the mixture is dried under high vacuum
(0.1 torr) for 1 hour to yield
3-dimethylamino-1-(3-trifluoromethyl-pyridin-2-yl)-propenone as a
brown oil.
4. 3-amino-3-methoxyacrylonitrile hydrochloride
##STR00027##
[0262] Malononitrile (198 g; 3 mol), HCO.sub.2Me (1.0 L) and MeOH
(240 mL) are added to a 3 liter flask and cooled with an ice and
salt mixture. SOCl.sub.2 is added drop wise over a period of 60
minutes while maintaining the inside temperature of the reaction
mixture below 10.degree. C. After the addition, the reaction
mixture is stirred at 0-10.degree. C. for another 60 minutes to
yield a yellow solid suspension. The salt that separated from the
mixture is filtered, and the solid is washed with HCO.sub.2Me
(2.times.75 mL) and air dried for 15 minutes. The salt is dried
under vacuum at 25.degree. C. for 1 hour to yield
3-amino-3-methoxyacrylonitrile hydrochloride as a white solid.
5. 6-Amino-3'-trifluoromethyl-[2,2']bipyridinyl-5-carbonitrile
##STR00028##
[0264] 3-dimethylamino-1-(3-trifluoromethyl-pyridin-2-yl)-propenone
(191.7 g; 0.79 mol), EtOH (2000 mL), NH4OAc (302.6 g; 9.93 mol) and
3-amino-3-methoxyacrylonitrile hydrochloride (211.2 g; 1.57 mol)
are combined in a 5 liter flask. The reaction mixture is heated to
80.degree. C. for 7 hours. The mixture is then allowed to cool to
room temperature and the solvent is removed under reduced pressure.
The solid that separates at this stage is filtered and washed with
small amount of cold EtOH to afford 133 g of light brick solid. The
solid is treated with 750 mL of EtOAc and the mixture is washed
with saturated NaHCO.sub.3. The organic extract is dried with
Na.sub.2SO.sub.4 (100 g), filtered and concentrated in vacuum at
40.degree. C. to yield 6-amino-3'-trifluoromethyl-[2,2']
bipyridinyl-5-carbonitrile as a light brick colored solid.
6. 6-Amino-3'-trifluoromethyl-[2,2']bipyridinyl-5-carboxylic
acid
##STR00029##
[0266] 6-Amino-3'-trifluoromethyl-[2,2']bipyridinyl-5-carbonitrile
(2.33 g, 8.82 mmol) is dissolved in 12M HCl (50 mL) and heated at
110.degree. C. overnight. The aqueous acid is removed under reduced
pressure to yield the title compound as its hydrochloride salt.
7. 6-Amino-3'-trifluoromethyl-[2,2']bipyridinyl-5-carboxylic acid
2,5-dioxo-pyrrolidin-1-yl ester
##STR00030##
[0268] 6-Amino-3'-trifluoromethyl-[2,2']bipyridinyl-5-carboxylic
acid hydrochloride (11.33 g, 35.44 mmol), N-hydroxy-succinimide
(8.15 g, 70.9 mmol), and EDCl (10.19 g, 53.16 mmol) are dissolved
in a solution of dry THF (100 mL) and Hunig's base (16.12 g, 125
mmol). The reaction mixture is stirred overnight at room
temperature. Ethyl acetate (200 mL) is added and the organic phase
extracted with water (3.times.100 mL) and brine (100 mL). The
organic extract is dried over Na.sub.2SO.sub.4 and the solvent
removed under reduced pressure to yield the title compound as a
brown foam.
8.
4-Hydroxy-2-methoxymethyl-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]napth-
yridine-3-carboxylic acid methyl ester
##STR00031##
[0270] A solution of
6-amino-3'-trifluoromethyl-[2,2']bipyridinyl-5-carboxylic acid
2,5-dioxo-pyrrolidin-1-yl ester (10.4 g, 27.3 mmol) in 50 mL dry
THF is added in one portion to a mixture of potassium t-butoxide
(7.36 g, 65.6 mmol) and methyl 4-methoxy-acetoacetate (8.77 g, 60.7
mmol) in dry THF (100 mL). The reaction is stirred overnight at
room temperature. Water (30 mL) is added and the solution
concentrated (.about.30 mL). The resulting mixture is extracted
with ether (2.times.50 mL). The aqueous portion is acidified with
concentrated hydrochloric acid and extracted with CH.sub.2Cl.sub.2
(4.times.100 mL). The combined organic extracts are dried over
Na.sub.2SO.sub.4 and the solvent removed under reduced pressure to
yield the title compound as a light brown oil that solidifies upon
standing.
9.
2-Methoxymethyl-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]napthyridin-4-o-
l
##STR00032##
[0272]
4-Hydroxy-2-methoxymethyl-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]n-
apthyridine-3-carboxylic acid methyl ester (200 mg, 0.508 mmol) is
dissolved in 12 M HCl (20 mL) and heated at 110.degree. C. for 6
hours. The reaction mixture is poured onto ice (100 g) and
extracted with CH.sub.2Cl.sub.2 (4.times.150 mL). The combined
organic extracts are dried over Na.sub.2SO.sub.4 and the solvent
removed under reduced pressure. The crude product is purified by
silica gel preparatory TLC eluting with hexanes/acetone (3:1)
yielding the title compound as a white solid.
10.
4-Chloro-2-methoxymethyl-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]napth-
yridine
##STR00033##
[0274]
2-Methoxymethyl-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]napthyridin-
-4-ol (191 mg, 0.569 mmol) is dissolved in a solution of chloroform
(15 mL), POCl.sub.3 (0.212 mL, 2.28 mmol) and 2,6-lutidine (0.256
mL, 2.28 mmol). The reaction is heated at reflux overnight. The
mixture is concentrated under reduced pressure. The resulting
residue is dissolved in EtOAc (50 mL) and extracted with water (50
mL), saturated NaHCO.sub.3 (aq) (50 mL) and brine (50 mL). The
organic extract is dried over Na.sub.2SO.sub.4 and the solvent
removed under reduced pressure. The crude product is purified by
column chromatography on silica gel eluting with hexanes/EtOAc
(1:1) to yield the title compound as a white solid.
11.
2-Methoxymethyl-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]napthyridin-4--
yl]-(4-trifluoromethylphenyl)-amine
##STR00034##
[0276]
4-Chloro-2-methoxymethyl-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]na-
pthyridine (25 mg, 0.0708 mmol) is dissolved in a solution of
isopropyl alcohol (2 mL) and 4-trifluoromethyl-aniline (25 mg,
0.155 mmol). The mixture is heated overnight at 60.degree. C. The
solution is concentrated under reduced pressure. The title compound
is isolated as a light yellow solid after silica gel preparatory
TLC eluting with hexanes/acetone (2:1). Mass Spec 479 (M+1).
[0277] B. Using a similar procedure,
2-methoxymethyl-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]napthyridin-4-yl--
(4-t-butylphenyl)-amine (Mass spec 467.2) is prepared:
##STR00035##
[0278] C.
2-Methoxymethyl-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(triflu-
oromethyl)pyridin-2-yl]-[1,8]naphthyridin-4-amine is prepared
according to the procedure shown in Scheme 2, as follows:
##STR00036##
[0279] To a de-gassed mixture of
4-chloro-2-methoxymethyl-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]naphthyr-
idine (1 mmol), cesium carbonate (2 mmol), 2-amino-trifluoromethyl
pyridine (1 mmol) in dioxane (10 mL) under nitrogen, add Pd.sub.2
dba.sub.3 (0.05 mmol) and xantphos (0.05 mol). Stir the mixture at
90.degree. C. overnight, concentrate, and extract with EtOAc. Dry
over Na.sub.2SO.sub.4 and concentrate under vacuum. Purify by
column chromatography eluting with
dichloromethane/methanol/ammonium hydroxide mixture to give
2-methoxymethyl-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl-
)pyridin-2-yl]-[1,8]naphthyridin-4-amine. MS 480 (M+1). .sup.1H NMR
.delta. (CDCl.sub.3) 8.88 (1H, d), 8.62 (1H, s), 8.18 (1H, d), 8.07
(1H, s), 7.88 (1H, dd), 7.84 (1H, d), 7.56 (1H, d), 7.54 (1H, d),
7.30 (1H, d), 4.77 (2H, s), 3.52 (3H, s), 2.63 (1H, br s).
[0280] D.
4-[(4-Trifluoromethylphenyl)amino]-7-(3-trifluoromethylpyridin-2-
-yl)-2-methoxymethyl-[1,8]naphthyridine-3-carboxylic acid is
prepared according to the procedure shown in Scheme 3, by the
following steps:
1. Methyl
4-chloro-2-methoxymethyl-7-[3-(trifluoromethyl)pyridin-2-yl]-[1,-
8]naphthyridine-3-carboxylate
##STR00037##
[0282] Dissolve
4-hydroxy-2-methoxymethyl-7-(3-trifluoromethylpyridin-2-yl)-[1,8]naphthyr-
idine-3-carboxylic acid methyl ester (500 mg, 1.27 mmol) in a
solution of POCl.sub.3 (0.4 mL), 2,6-lutidine (0.43 mL) and
CHCl.sub.3 (25 mL). Heat the mixture at reflux overnight. Remove
the solvent under reduced pressure. Partition the residue between
CH.sub.2Cl.sub.2 and saturated NaHCO.sub.3 (aq). Extract the
aqueous layer twice with CH.sub.2Cl.sub.2 (2.times.100 mL). Dry the
combined organic extracts over Na.sub.2SO.sub.4. Remove the solvent
under reduced pressure. Purify the crude product by preparatory TLC
eluting with EtOAc\Hexanes (1:1) to yield the title compound.
2. Methyl
4-[(4-trifluoromethylphenyl)amino]-7-(3-trifluoromethylpyridin-2-
-yl)-2-methoxymethyl-[1,8]naphthyridine-3-carboxylate
##STR00038##
[0284] Dissolve methyl
4-chloro-2-methoxymethyl-7-[3-(trifluoromethyl)pyridin-2-yl]-[1,8]naphthy-
ridine-3-carboxylate (212 mg, 0.52 mmol) in acetonitrile (5 mL) and
add 4 drops 2M HCl (Et.sub.2O). Stir the mixture for 3 hours at
room temperature. Filter off the resulting yellow precipitate to
yield the title compound as its hydrochloride salt. MS 537.20
(M+1). .sup.1H NMR .delta. (Free base, CDCl.sub.3) 9.91 (1H, s),
8.88 (1H, m), 8.16 (2H, m), 7.67-7.55 (4H, m), 7.12 (1H, d), 6.99
(1H, d), 4.42 (2H, s), 4.02 (3H, s), 3.95 (3H, s).
3.
4-[(4-Trifluoromethylphenyl)amino]-7-(3-trifluoromethylpyridin-2-yl)-2--
methoxymethyl-[1,8]naphthyridine-3-carboxylic acid
##STR00039##
[0286] Stir a solution of methyl
4-[(4-trifluoromethylphenyl)amino]-7-(3-trifluoromethylpyridin-2-yl)-2-me-
thoxymethyl-[1,8]naphthyridine-3-carboxylate (114 mg, 0.212 mmol),
LiOH.H.sub.2O (45.0 mg, 1.06 mmol), THF (2 mL) and water (0.1 mL)
for 18 hours at room temperature. Add water (20 mL) and acidify
with acetic acid. Extract the solution with EtOAc (3.times.25 mL).
Wash the combined organic extracts with brine and dry over
Na.sub.2SO.sub.4. Remove the solvent under reduced pressure to
yield the title compound. MS 523.18 (M+1). .sup.1H NMR .delta.
(CDCl.sub.3) 11.50 (1H, br s), 8.89 (1H, s), 8.19 (2H, d), 7.88
(1H, dd), 7.67 (2H, m), 7.59 (1H, dd), 7.51 (2H, d), 7.22 (1H, br
s), 5.30 (2H, s), 3.65 (3H, s).
[0287] E.
4-(tert-Butyl-phenyl)-[2-methoxy-7-(3-trifluoromethyl-pyridin-2--
yl)-[1,8]naphthyridin-4-yl]-amine is prepared according to the
procedure shown in Scheme 4, as follows:
1. 6-Amino-3'-trifluoromethyl-[2,2']bipyridinyl-5-carboxylic
acid
##STR00040##
[0289] Heat a mixture of
6-amino-3'-trifluoromethyl-[2,2']bipyridinyl-5-carbonitrile (5 g)
in concentrated hydrochloric acid at 100.degree. C. for 12 hours.
Cool the mixture and evaporate to dryness to give the hydrochloride
salt of the title compound.
2. 6-Amino-3'-trifluoromethyl-[2,2']bipyridinyl-5-carboxylic acid
methyl ester
##STR00041##
[0291] Saturate a solution of
6-amino-3'-trifluoromethyl-[2,2']bipyridinyl-5-carboxylic acid (5
g) in methanol (100 ml) with hydrogen chloride gas. Heat the
mixture at reflux for 4 days and evaporate to dryness. Partition
the mixture between ethyl acetate and saturated sodium bicarbonate
solution. Separate the layers and extract the aqueous layer with
further ethyl acetate. Wash the combined organic extracts with
brine, dry (MgSO.sub.4) and evaporate to give the title
compound.
3.
4-Hydroxy-7-(3-trifluoromethyl-pyridin-2-yl)-1H-[1,8]naphthyridin-2-one
##STR00042##
[0293] Heat a solution of
6-amino-3'-trifluoromethyl-[2,2']bipyridinyl-5-carboxylic acid
methyl ester (1.0 mmol) acetic anhydride (2 mL) in pyridine (2 mL)
at 90.degree. C. for 8 hours. Cool the mixture and evaporate to
dryness. Add saturated aqueous sodium bicarbonate (30 mL) and
extract with ethyl acetate. Wash the combined organic extracts with
brine, dry and evaporate. Dissolve the solid in THF (4 mL) and add
drop wise to a solution of potassium bis(trimethylsilyl)amide (600
mg, 3.0 mmol) in toluene (6 mL) at -78.degree. C. Allow the
reaction to return to room temperature overnight. Add water (10 mL)
and extract with ethyl acetate. Acidify the aqueous layer with
hydrochloric acid and collect the precipitate by filtration.
Air-dry to give the title compound.
4.
2,4-Dichloro-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]naphthyridine
##STR00043##
[0295] Reflux
4-hydroxy-7-(3-trifluoromethyl-pyridin-2-yl)-1H-[1,8]naphthyridin-2-one
(1.2 g) for 18 hours in POCl.sub.3 (5 mL). Evaporate the solvent,
carefully neutralize with saturated NaHCO.sub.3 and extract with
EtOAc. Dry over Na.sub.2SO.sub.4 and concentrate under vacuum to
obtain the title compound.
5.
4-Chloro-2-methoxy-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]naphthyridin-
e
##STR00044##
[0297] Add sodium methoxide (4M, 1.0 mL, 4.0 mmol) to a solution of
2,4-dichloro-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]naphthyridine
(1.2 g, 3.5 mmol) in THF (30 mL). Stir at room temperature
overnight, add water (25 mL) and extract with ethyl acetate. Wash
the combined organic extracts with brine, dry (MgSO.sub.4) and
evaporate. Purify the residue by flash chromatography (elute with
1:2 hexane:ether) to give the title compound.
6.
4-tert-Butyl-phenyl)-[2-methoxy-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8-
]naphthyridin-4-yl]-amine
##STR00045##
[0299] Heat a mixture of
4-chloro-2-methoxy-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]naphthyridine
(500 mg, 1.47 mmol), 4-tert-butylaniline (240 mg, 1.6 mmol),
palladium acetate (33 mg, 0.15 mmol),
2-(dicyclohexylphosphino)biphenyl (53 mg, 0.15 mmol) and potassium
phosphate (0.5 M, 9 mL, 4.5 mmol) in dioxane (20 mL) at 100.degree.
C. for 16 hours. Cool the mixture and partition between ethyl
acetate and water. Separate the layers and extract the aqueous
layer with further ethyl acetate. Wash the combined organic
extracts with brine, dry (MgSO.sub.4) and evaporate. Triturate the
residue with ether to give the title compound as the hydrochloride
salt. MS 453 (M+1). .sup.1H NMR .delta. (DMSO) 9.07 (1H, s), 8.95
(1H, d), 8.86 (1H, d), 8.38 (1H, d), 7.77 (1H, m), 7.66 (1H, d),
7.44 (2H, d), 7.29 (2H, d), 6.27 (1H, s), 3.88 (3H, s), 1.30 (9H,
s).
[0300] F.
(4-Trifluoromethyl-phenyl)-[7-(3-trifluoromethyl-pyridin-2-yl)-[-
1,8]naphthyridin-4-yl-amine is prepared according to the procedure
shown in Scheme 5, as follows:
1.
4-(4-Trifluoromethyl-phenylamino)-7-(3-trifluoromethyl-pyridin-2-yl)-[1-
,8]naphthyridin-2-ol
##STR00046##
[0302] Heat
4-chloro-2-methoxy-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]naphthyridine
(600 mg, 1.8 mmol), 4-trifluoromethylaniline (322 mg, 2.0 mmol) and
2M hydrochloric acid in ether (1 mL, 2.0 mmol) in isopropanol (15
mL) at 80.degree. C. for 16 hours. Cool the mixture and collect the
precipitate by filtration. Partition the solid between ethyl
acetate and saturated sodium bicarbonate solution. Separate the
layers and extract the aqueous layer with further ethyl acetate.
Wash the combined organic extracts with brine, dry (MgSO.sub.4) and
evaporate to give the title compound.
2.
2-Chloro-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]naphthyridin-4-yl]-(4--
trifluoromethyl-phenyl)-amine
##STR00047##
[0304] Reflux
4-(4-trifluoromethyl-phenylamino)-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8-
]naphthyridin-2-ol (233 mg) for 2 hours in POCl.sub.3 (2 mL).
Evaporate the solvent, carefully neutralize with saturated
NaHCO.sub.3, and extract with EtOAc. Dry over Na.sub.2SO.sub.4,
concentrate under vacuum to obtain the title compound.
3.
(4-Trifluoromethyl-phenyl)-[7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]nap-
hthyridin-4-yl-amine
##STR00048##
[0306] Hydrogenate, at 50 psi, a mixture of 10% Pd--C (10 mg) and
2-chloro-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]naphthyridin-4-yl]-(4-tr-
ifluoromethyl-phenyl)-amine (40 mg) in 95% EtOH (10 mL). Filter
through a celite pad and concentrate the filtrate. Purify the
residue by preparative TLC (elute with 2:1 ethyl acetate:hexane) to
give the title compound. MS 435 (M+1). .sup.1H NMR .delta.
(CDCl.sub.3) 8.98 (1H, d), 8.74 (1H, s), 8.62 (1H, m), 8.18 (1H,
d), 7.78 (1H, d), 7.62 (2H, d), 7.50 (3H, m), 6.80 (1H, m).
[0307] G.
7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyridi-
n-2-yl]-1,8-naphthyridin-4-amine is prepared according to the
procedure shown in Scheme 5, as follows:
1.
2-Methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyr-
idin-2-yl]-1,8-naphthyridin-4-amine
##STR00049##
[0309] To a de-gassed mixture of
4-chloro-2-methoxy-7-(3-trifluoromethyl-pyridin-2-yl)-[1,8]naphthyridine
(1 mmol), cesium carbonate (2 mmol), 2-aminotrifluoromethylpyridine
(1 mmol) in dioxane (10 mL) under nitrogen, add Pd.sub.2 dba.sub.3
(0.05 mmol) and xantphos (0.05 mol). Stir the mixture at 90.degree.
C. overnight, concentrate, and extract with EtOAc. Dry over
Na.sub.2SO.sub.4 and concentrate under vacuum. Purify by column
chromatography eluting with dichloromethane/methanol/ammonium
hydroxide mixture to give the title compound.
2.
7-[3-(Trifluoromethyl)pyridin-2-yl]-4-{[5-(trifluoromethyl)pyridin-2-yl-
]amino}-1,8-naphthyridin-2-ol
##STR00050##
[0311] Heat
2-methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyrid-
in-2-yl]-[1,8]naphthyridin-4-amine (128 mg), 1M hydrochloric acid
in ether (0.25 mL) and isopropanol at 80.degree. C. overnight. Cool
and collect the precipitate by filtration to give the hydrochloride
salt of the title compound.
3.
2-Chloro-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyri-
din-2-yl]-1,8-naphthyridin-4-amine
##STR00051##
[0313] Heat
7-[3-(trifluoromethyl)pyridin-2-yl]-4-{[5-(trifluoromethyl)pyridin-2-yl]a-
mino}-[1,8]naphthyridin-2-ol (112 mg) and phosphorus oxychloride (1
mL) at reflux for 1 hour. Evaporate to dryness, partition between
ethyl acetate and saturated aqueous NaHCO.sub.3 and extract with
EtOAc. Dry the combined organics over Na.sub.2SO.sub.4, concentrate
under vacuum to obtain the title compound.
4.
7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyridin-2-yl]-
-1,8-naphthyridin-4-amine
##STR00052##
[0314] Stir a mixture of
2-chloro-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyridi-
n-2-yl]-[1,8]naphthyridin-4-amine (40 mg), ammonium formate (31
mg), 10% palladium on carbon (10 mg) in methanol (2 mL) at
50.degree. C. for 2 hours. Cool, filter through Celite and
evaporate to dryness. Purify by preparative thin layer
chromatography, eluting with dichloromethane/methanol/ammonium
hydroxide mixture to give the title compound. MS 436 (M+1). .sup.1H
NMR .delta. (CDCl.sub.3) 8.99 (1H, s), 8.84 (1H, d), 8.63 (1H, d),
8.61 (1H, s), 8.32 (1H, brs), 8.14 (1H, d), 8.05 (1H, brs),
7.79-7.85 (2H, m), 7.51 (1H, dd), 7.22 (1H, d).
[0315] H.
4-[(4-Trifluoromethylphenyl)amino]-7-(3-trifluoromethylpyridin-2-
-yl)-2-(methoxymethyl)-1,8-naphthyridine-3-carboxylic acid is
prepared according to the procedure shown in Scheme 6, as
follows:
1.
7-[3-(Trifluoromethyl)pyridin-2-yl]-4-{[5-(trifluoromethyl)pyridin-2-yl-
]amino}-1,8-naphthyridine-2-carbonitrile
##STR00053##
[0317] In a sealed tube, bubble argon through a solution of
2-chloro-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyridi-
n-2-yl]-1,8-naphthyridin-4-amine (360 mg, 0.766 mmol), ZnCN.sub.2
(63 mg, 0.536 mmol), DMF (10 mL) and water (0.1 mL). Add Pd.sub.2
dba.sub.3 (21 mg, 0.023 mmol) and DPPF (25 mg, 0.046 mmol) to the
degassed solution and heat the mixture at 120.degree. C. for one
hour. Cool the mixture and add EtOAc (100 mL). Extract the mixture
with 1N NaOH (3.times.100 mL). Dry the organic layer over
Na.sub.2SO.sub.4 and remove the solvent under reduced pressure.
Triturate the resulting solid with Et.sub.2O to yield the title
compound as an orange solid. MS 461.02 (M+1). .sup.1H NMR .delta.
(CDCl.sub.3) 8.90 (1H, d), 8.76 (1H, s), 8.73 (1H, s), 8.59 (1H,
d), 8.20 (1H, dd), 8.04 (1H, d), 7.96 (1H, dd), 7.91 (1H, m), 7.59
(1H, dd), 7.22 (1H, d).
2.
7-[3-(Trifluoromethyl)pyridin-2-yl]-4-{[5-(trifluoromethyl)pyridin-2-yl-
]amino}-1,8-naphthyridine-2-carboxamide
##STR00054##
[0319] Dissolve
7-[3-(trifluoromethyl)pyridin-2-yl]-4-{[5-(trifluoromethyl)pyridin-2-yl]a-
mino}-1,8-naphthyridine-2-carbonitrile (25 g, 0.054 mmol) in
concentrated H.sub.2SO.sub.4 (2 mL) and stir the mixture overnight
at ambient temperature. Pour the mixture onto ice and adjust the pH
to about 7-8. Filter off the resulting precipitate to yield the
title compound as an off-white solid. MS 479.02 (M+1). .sup.1H NMR
.delta. (CD.sub.3OD) 9.32 (1H, s), 9.10 (1H, d), 8.97 (1H, d), 8.71
(1H, s), 8.41 (1H, d), 8.05 (1H, d), 7.95 (1H, d), 7.82 (1H, m),
8.90 (1H, s), 7.44.
3.
7-[3-(Trifluoromethyl)pyridin-2-yl]-4-{[5-(trifluoromethyl)pyridin-2-yl-
]amino}-1,8-naphthyridine-2-carboxylic acid
##STR00055##
[0321] Dissolve
7-[3-(trifluoromethyl)pyridin-2-yl]-4-{[5-(trifluoromethyl)pyridin-2-yl]a-
mino}-1,8-naphthyridine-2-carboxamide (25 mg, 0.054 mmol) in 12M
HCl (3 mL) and heat at 85.degree. C. overnight. Pour onto ice and
adjust the pH to about 4. Extract with EtOAc (3.times.100 mL). Dry
the combined organic extracts over Na.sub.2SO.sub.4 and remove the
solvent under reduced pressure. Purify the crude residue by
preparatory TLC eluting with CH.sub.2Cl.sub.2/MeOH/AcOH (90:10:1)
to yield the title compound. MS 480.00 (M+1). .sup.1H NMR .delta.
(CD.sub.3OD) 9.58 (1H, s), 9.20 (1H, m), 8.95 (1H, m), 8.80 (1H,
s), 8.38 (1H, m), 8.13 (1H, d), 8.00 (1H, m), 7.75 (1H, m), 7.55
(1H, d).
Example 2
Preparation of Additional Representative Substituted
Quinolin-4-ylamine Analogues
[0322] A.
(4-tert-Butyl-phenyl)-[7-(2-trifluoromethyl-phenyl)-quinolin-4-y-
l]-amine is prepared by the following steps:
1. 7-(2-Trifluoromethyl-phenyl)-quinolin-4-ol
##STR00056##
[0324] Combine 7-chloroquinolin-4-ol (1000 mg, 5.55 mmol,)
2-(trifluoromethyl)phenylboronic acid (1583 mg, 8.33 mmol) and
toluene (50 mL), and bubble nitrogen into the solution for 10
minutes. Add palladium acetate (25 mg, 0.11 mmol),
2-(dicyclohexylphosphino)biphenyl (78 mg, 0.22 mmol), and
K.sub.3PO.sub.4 (2353 mg, 11.1 mmol) and heat at 90.degree. C. for
16 hours. Let cool, add water (25 mL) and EtOAc (50 mL), and remove
any insoluble material by filtration. Separate the EtOAc layer, and
extract the aqueous layer twice with EtOAc (25 mL each). Combine
the EtOAc extracts, dry (Na.sub.2SO.sub.4), and evaporate. Purify
by silica gel chromatography (94% CH.sub.2Cl.sub.2/5% MeOH/1%
NH.sub.4OH) to provide 7-(2-trifluoromethyl-phenyl)-quinolin-4-ol
as a white solid.
2. 4-Chloro-7-(2-trifluoromethyl-phenyl)-quinoline
##STR00057##
[0326] Heat a mixture of 7-(2-trifluoromethyl-phenyl)-quinolin-4-ol
(50 mg, 0.17 mmol) in POCl.sub.3 (10 mL) at 90.degree. C. for 16
hours. Evaporate the POCl.sub.3, and add ice (100 g) followed by
careful addition of saturated NaHCO.sub.3. Extract with EtOAc, dry
(Na.sub.2SO.sub.4), and evaporate to provide
4-chloro-7-(2-trifluoromethyl-phenyl)-quinoline as a tan solid.
3.
(4-tert-Butyl-phenyl)-[7-(2-trifluoromethyl-phenyl)-quinolin-4-yl]-amin-
e
##STR00058##
[0328] Heat a mixture of
4-chloro-7-(2-trifluoromethyl-phenyl)-quinoline (42 mg, 0.14 mmol)
and 4-(tert-butyl)aniline (41 mg, 0.29 mmol) in 2-propanol (10 mL)
at reflux for 3 hours. Evaporate the mixture, add 1M NaOH (10 mL),
extract twice with EtOAc (10 mL each), dry (Na.sub.2SO.sub.4), and
evaporate to provide the crude product. Purify by silica gel
chromatography, eluting with 75% hexane-EtOAc to provide
(4-tert-butyl-phenyl)-[7-(2-trifluoromethyl-phenyl)-quinolin-4-yl]-amine
as a white solid. Mass spec. 420.2.
[0329] B.
6-Methoxymethyl-3-(3-trifluoromethyl-pyridin-2-yl)-pyrido-[2,3-b-
]pyrazin-8-yl]-(4-trilfluoromethyl-phenyl)-amine is prepared by the
following steps:
1. 2-(1,2-Dimethoxy-ethylidene)-3-oxo-pentanedioic acid dimethyl
ester
##STR00059##
[0331] Dimethyl 1,3-acetonedicarboxylate (20.0 g, 115 mmol) is
dissolved in a solution of 1,1,1,2-tetramethoxy-ethane (23.8 g, 158
mmol) and acetic anhydride (420 mL) and the solution is heated to
reflux for 4 hours. The mixture is concentrated under reduced
pressure. Toluene (200 mL) is added and the solvent removed under
reduced pressure to yield the title compound.
2. 4,6-Dihydroxy-2-methoxymethyl-nicotinic acid methyl ester
##STR00060##
[0333] 2-(1,2-Dimethoxy-ethylidene)-3-oxo-pentanedioic acid
dimethyl ester (29.8 g, 115 mmol) is dissolved in a solution of
EtOH (250 mL), water (250 mL) and concentrated NH.sub.4OH(aq) (30
mL). The mixture is heated to 60.degree. C. for 5 hours. The
reaction mixture is cooled to room temperature and the EtOH removed
under reduced pressure. The remaining aqueous solution is cooled in
an ice bath and acidified with concentrated hydrochloric acid, at
which time a white precipitate appeared. The precipitate is
collected and dried in a vacuum oven to yield the title
compound.
3. 4,6-Dihydroxy-2-methoxymethyl-5-nitro-nicotinic acid methyl
ester
##STR00061##
[0335] 4,6-Dihydroxy-2-methoxymethyl-nicotinic acid methyl ester
(15.9 g, 74.75 mmol) is dissolved in AcOH (60 mL) and cooled to
0.degree. C. in an ice bath. Nitric acid (70%, 4.73 mL) is added
dropwise. The resulting solution is stirred at room temperature
overnight. Water (200 mL) is added to the mixture and a white
precipitate forms. The precipitate is collected and dried in a
vacuum oven to afford the title compound as a white solid.
4. 6-Methoxymethyl-3-nitro-pyridine-2,4-diol
##STR00062##
[0337] 4,6-Dihydroxy-2-methoxymethyl-5-nitro-nicotinic acid methyl
ester (2.00 g, 7.75 mmol) is placed in concentrated hydrochloric
acid (100 mL) and heated in a bomb at 120.degree. C. overnight. The
reaction mixture is cooled in an ice bath and poured onto ice (200
g). A white precipitate formed. The precipitate is collected and
dried in a vacuum oven to afford the title compound.
5. 2,4-Dichloro-6-methoxymethyl-3-nitro-pyridine
##STR00063##
[0339] 6-Methoxymethyl-3-nitro-pyridine-2,4-diol (725 mg, 3.63
mmol) is added to a solution of POCl.sub.3 (15 mL) at 0.degree. C.
The mixture is warmed to room temperature followed by heating at
reflux for 5 hours. Excess POCl.sub.3 is removed under reduced
pressure to yield a light brown oil. The crude oil is dissolved in
CH.sub.2Cl.sub.2 (100 mL) and extracted with water (100 mL),
NaHCO.sub.3 (100 mL), and brine (100 mL). The organic extract is
dried over Na.sub.2SO.sub.4 and the solvent removed under reduced
pressure. The crude product is chromatographed on silica gel
eluting with hexanes/EtOAc (4:1) to yield the title compound as a
light yellow oil.
6. 6-Methoxymethyl-3-nitro-pyridine-2,4-diamine
##STR00064##
[0341] A solution of saturated ammonia in methanol (20 mL) is added
to 2,4-dichloro-6-methoxymethyl-3-nitro-pyridine (620 mg, 2.61
mmol). The mixture is stirred for 1 hour at room temperature. A
white precipitate forms and is collected. The precipitate is dried
in a vacuum oven to afford the title compound as a white solid.
7. 6-Methoxymethyl-2,3,4-triamino-pyridine
##STR00065##
[0343] 6-Methoxymethyl-3-nitro-pyridine-2,4-diamine (455 mg, 2.29
mmol) is dissolved in EtOH (50 mL) and 10% Pd/C (50 mg) is added.
The mixture is hydrogenated at 50 psi for 2 hours. The reaction
mixture is filtered through Celite and the Celite bed washed with
EtOH (25 mL). The solvent is removed under reduced pressure to
yield the title compound as an off-white solid.
8.
6-Methoxymethyl-3-(3-trifluoromethyl-pyridin-2-yl)-pyrido-[2,3-b]pyrazi-
n-8-ylamine
##STR00066##
[0345] 6-Methoxymethyl-2,3,4-triamino-pyridine (353 mg, 2.10 mmol),
2-bromo-1-(3-trifluoromethyl-pyridin-2-yl)-ethanone hydrobromide
(771 mg, 2.21 mmol; synthesis described in Example 2E, below), and
NaHCO.sub.3 (554 mg, 6.59 mmol) are dissolved into a solution of
dioxane (20 mL) and water (20 mL). The reaction mixture is stirred
1 hour at room temperature and 3 hours at 100.degree. C. The
mixture is cooled and filtered through Celite. The Celite bed is
washed with EtOAc (20 mL). The aqueous mixture is extracted with
EtOAc (4.times.100 mL). The combined organic extracts are washed
with brine and dried over Na.sub.2SO.sub.4. The solvent is removed
under reduced pressure and the crude product purified by column
chromatography on silica gel eluting with acetone/hexanes (1:1) to
yield the title compound as a white solid.
9.
6-Methoxymethyl-3-(3-trifluoromethyl-pyridin-2-yl)-pyrido-[2,3-b]pyrazi-
n-8-ol
##STR00067##
[0347]
6-Methoxymethyl-3-(3-trifluoromethyl-pyridin-2-yl)-pyrido-[2,3-b]py-
razin-8-ylamine (252 mg, 0.751 mmol) is dissolved in a solution of
acetic acid (2 mL) and water (5 mL). The mixture is heated to
50.degree. C. and sodium nitrite (362 mg, 5.26 mmol) is added in
portions over 1 hour. The solution is warmed to 70.degree. C. and
stirred overnight. Water (20 mL) is added and the aqueous mixture
is extracted with EtOAc (4.times.50 mL). The combined organic
extracts are washed with brine and dried over Na.sub.2SO.sub.4. The
solvent is removed under reduced pressure. The crude product is
chromatographed on silica gel eluting with hexanes/acetone (1:1) to
afford the title compound as a white solid.
10.
8-Chloro-6-Methoxymethyl-3-(3-trifluoromethyl-pyridin-2-yl)-pyrido-[2,-
3-b]pyrazine
##STR00068##
[0349]
6-Methoxymethyl-3-(3-trifluoromethyl-pyridin-2-yl)-pyrido-[2,3-b]py-
razin-8-ol (146 mg, 0.436 mmol) is dissolved in a solution of
CHCl.sub.3 (20 mL), POCl.sub.3 (0.12 ml, 1.31 mmol) and
2,6-lutidine (0.2 mL, 1.31 mmol). The mixture is heated at reflux
overnight. The reaction is concentrated under reduced pressure.
EtOAc (30 mL) is added and the mixture is extracted with
NaHCO.sub.3 (aq) (30 mL) and brine (30 mL). The organic extract is
dried over Na.sub.2SO.sub.4 and the solvent removed under reduced
pressure. The crude product is purified by silica gel preparatory
TLC eluting with hexanes/EtOAc (1:1) to yield the title compound as
a light yellow oil that solidifies upon standing.
11.
6-Methoxymethyl-3-(3-trifluoromethyl-pyridin-2-yl)-pyrido-[2,3-b]pyraz-
in-8-yl]-(4-trilfluoromethyl-phenyl)-amine
##STR00069##
[0351] To a solution of acetonitrile (2.5 mL) and
4-trifluoromethylaniline (42 mg, 0.263 mmol) is added
8-chloro-(6-methoxymethyl-3-(3-trifluoromethyl-pyridin-2-yl)-pyrido-[2,3--
b]pyrazine (62 mg, 0.175 mmol) in acetonitrile (1 mL). The mixture
is heated at 80.degree. C. overnight. The solvent is removed under
reduced pressure and the crude reaction mixture purified by silica
gel preparatory TLC eluting with hexanes/acetone (2:1) to afford
the title compound as a light yellow solid.
[0352] C.
7-[3-(Trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyridi-
n-2-yl]-1,5-naphthyridin-4-amine is prepared according to the
procedure shown in Scheme 7, as follows:
1. 5-Amino-3-trifluoromethyl-[2,3']bipyridinyl-6'-carbonitrile
##STR00070##
[0354] Heat a solution of
6'-chloro-3-trifluoromethyl-[2,3']bipyridinyl-5'-ylamine (25 g,
0.091 mol; prepared essentially as described in PCT International
Application Publication Number WO 03/062209, published on Jul. 31,
2003), zinc cyanide (6.75 g, 0.058 mol), pd.sub.2(dba).sub.3 (2.63
g, 2.86 mmol), DPPF (3.16 g, 5.72 mmol) in DMF (250 mL) and water
(2.5 mL), under a nitrogen atmosphere, at 120.degree. C. for 1
hour. Cool the reaction to 0.degree. C. and add a solution of
saturated ammonium chloride (200 ml), water (200 mL) and
concentrated ammonium hydroxide (50 mL). After stirring at
0.degree. C. for 1 hour, filter the yellow precipitate, and wash
with water (200 mL) and a 1:1 mixture of ether-hexane (200 mL). Dry
the solid in air and then in a vacuum oven to give the title
compound.
2. 5-Amino-3-trifluoromethyl-[2,3']bipyridinyl-6'-carboxylic
acid
##STR00071##
[0356] Dissolve
5-amino-3-trifluoromethyl-[2,3']bipyridinyl-6'-carbonitrile (5 g,
18.9 mmol) in 12M HCl (100 mL) and heat at 100.degree. C.
overnight. Remove the aqueous acid under reduced pressure to yield
the title compound as its hydrochloride salt.
3. Ethyl
5'-amino-3-trifluoromethyl-[2,3']bipyridine-6'-carboxylate
##STR00072##
[0358] Saturate a solution of
5-amino-3-trifluoromethyl-[2,3']bipyridinyl-6'-carboxylic acid (5
g) in ethanol (100 ml) with hydrogen chloride gas. Heat the mixture
at reflux for 4 days and evaporate to dryness. Partition the
mixture between ethyl acetate and saturated sodium bicarbonate
solution. Separate the layers and extract the aqueous layer with
further ethyl acetate. Wash the combined organic extracts with
brine, dry (MgSO.sub.4) and evaporate to give the title
compound.
4.
7-[3-(Trifluoromethyl)pyridin-2-yl]-[1,5]naphthyridine-2,4-diol
##STR00073##
[0360] Heat a solution of ethyl
5'-amino-3-trifluoromethyl-[2,3']bipyridine-6'-carboxylate (10
mmol) and acetic anhydride (15 mL) in pyridine (15 mL) at
90.degree. C. for 8 hours. Cool the mixture and evaporate to
dryness. Add saturated aqueous sodium bicarbonate (30 mL) and
extract with ethyl acetate. Wash the combined organic extracts with
brine, dry and evaporate. Dissolve the solid in THF (30 mL) and add
drop wise to a solution of potassium bis(trimethylsilyl)amide (6 g,
30 mmol) in toluene (60 mL) at -78.degree. C. Allow the reaction to
return to room temperature overnight. Add water (100 mL) and
extract with ethyl acetate. Acidify the aqueous layer with
hydrochloric acid and collect the precipitate by filtration.
Air-dry to give the title compound.
5.
2,4-Dichloro-7-[3-(trifluoromethyl)pyridin-2-yl]-[1,5]naphthyridine
##STR00074##
[0362] Reflux
7-[3-(trifluoromethyl)pyridin-2-yl]-[1,5]naphthyridine-2,4-diol (1
g) for 18 hours in POCl.sub.3 (5 mL). Evaporate the solvent, then
carefully neutralize with saturated NaHCO.sub.3 and extract with
EtOAc. Dry over Na.sub.2SO.sub.4, concentrate under vacuum to
obtain the title compound.
6.
4-Chloro-2-methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]-[1,5]naphthyridi-
ne
##STR00075##
[0364] Add sodium methoxide (4M, 0.45 mL, 1.8 mmol) to a solution
of
2,4-dichloro-7-[3-(trifluoromethyl)pyridin-2-yl]-1,5-naphthyridine
(575 mg, 1.6 mmol) in THF (10 mL). Stir at room temperature for 1
hour, add water (15 mL) and extract with ethyl acetate. Wash the
combined organic extracts with brine, dry (MgSO.sub.4) and
evaporate. Purify the residue by flash chromatography (elute with
1:2 hexane:ether) to give the title compound.
7.
2-Methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyr-
idin-2-yl]-[1,5]naphthyridin-4-amine
##STR00076##
[0366] To a de-gassed mixture of
4-chloro-2-methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]-[1,5]naphthyridine
(1 mmol), cesium carbonate (2 mmol),
2-amino-trifluoromethylpyridine (1 mmol) in dioxane (10 mL) under
nitrogen, add Pd.sub.2 dba.sub.3 (46 mg) and xantphos (29 mg). Stir
the mixture at 100.degree. C. for 3 hours, cool, add water (10 mL)
and extract with EtOAc. Dry the combined extracts over
Na.sub.2SO.sub.4, concentrate under vacuum. Purify by
chromatography eluting with dichloromethane/methanol/ammonium
hydroxide mixture to give the title compound. MS 435.98 (M+1).
.sup.1H NMR .delta. (CDCl.sub.3) 8.95 (1H, d), 8.90 (1H, s), 8.58
(1H, s), 8.38 (1H, d), 8.30 (1H, s), 8.19 (1H, d), 8.06 (1H, s),
7.88 (1H, d), 7.55 (1H, m), 7.05 (1H, s), 4.16 (3H, s).
8.
7-[3-(Trifluoromethyl)pyridin-2-yl]-4-{[5-(trifluoromethyl)pyridin-2-yl-
]amino}-[1,5]naphthyridin-2-ol
##STR00077##
[0368] Heat a solution of
2-methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyrid-
in-2-yl]-[1,5]naphthyridin-4-amine (300 mg) in 33% hydrogen bromide
in acetic acid (10 mL) at 100.degree. C. for 18 hours. Evaporate to
dryness, add saturated aqueous sodium bicarbonate (10 mL) and
extract with EtOAc. Dry over Na.sub.2SO.sub.4, and concentrate
under vacuum.
9.
2-Chloro-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyri-
din-2-yl]-[1,5]naphthyridin-4-amine
##STR00078##
[0370] Heat
7-[3-(trifluoromethyl)pyridin-2-yl]-4-{[5-(trifluoromethyl)pyridin-2-yl]a-
mino}-[1,5]naphthyridin-2-ol (190 mg) and phosphorus oxychloride (3
mL) at reflux for 30 minutes. Evaporate to dryness, partition
between ethyl acetate and saturated aqueous NaHCO.sub.3 and extract
with EtOAc. Wash the combined extracts with brine and dry over
Na.sub.2SO.sub.4, concentrate under vacuum to obtain the title
compound. MS 469.93 (M+1). .sup.1H NMR .delta. (CDCl.sub.3) 9.04
(1H, s), 8.85 (1H, d), 8.60 (1H, s), 8.3-8.36 (2H, m), 8.18 (1H,
d), 7.90-7.98 (2H, m), 7.58 (1H, m). 10.
7-[3-(Trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyridin-2-yl]-[-
1,5]naphthyridin-4-amine
##STR00079##
[0371] Stir a mixture of
2-chloro-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyridi-
n-2-yl]-[1,5]naphthyridin-4-amine (94 mg), ammonium formate (126
mg), 10% palladium on carbon (25 mg) in methanol (10 mL) at
50.degree. C. for 2 hours. Cool, filter through Celite and
evaporate to dryness. Purify by preparative thin layer
chromatography, eluting with dichloromethane/methanol/ammonium
hydroxide mixture to give the title compound. MS 436 (M+1). .sup.1H
NMR .delta. (CDCl.sub.3) 8.95-8.93 (2H, m), 8.63-8.57 (2H, m),
8.34-8.32 (2H, m), 8.26 (1H, s), 8.18 (1H, dd), 7.91 (1H, dd),
7.57-7.52 (2H, m).
[0372] D.
7-[3-(Trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyridi-
n-2-yl]quinolin-4-amine is prepared according to the procedure
shown in Scheme 8, as follows:
1. 2-p-Tolyl-3-trifluoromethyl-pyridine
##STR00080##
[0374] To a de-gassed mixture of
2-chloro-3-(trifluoromethyl)-pyridine (70.1 mmol), p-tolylboronic
acid (70.6 mmol), and 2M Na.sub.2CO.sub.3 (175.0 mmol), in DME (200
mL) under nitrogen, and add Pd(PPh.sub.3).sub.4 (2.8 mmol). Stir
the mixture at 80.degree. C. overnight, concentrate, extract with
EtOAc. Dry over Na.sub.2SO.sub.4, concentrate under vacuum, and
pass through a silica gel pad to give the title compound.
2. 2-(4-Methyl-3-nitro-phenyl)-3-(trifluoromethyl)-pyridine
##STR00081##
[0376] To a solution of 2-p-tolyl-3-trifluoromethyl-pyridine (8.4
mmol) in H.sub.2SO.sub.4 (6 mL) cautiously add fuming HNO.sub.3 (2
ml). Stir the mixture for 1 hour at room temperature. Pour the
mixture onto ice-water (30 mL), extract with EtOAc, wash with 1 N
NaOH, dry over Na.sub.2SO.sub.4, and concentrate under vacuum to
obtain the title compound.
3. 2-Nitro-4-(3-trifluoromethyl-pyridin-2-yl)-benzoic acid
##STR00082##
[0378] To a solution of
2-(4-methyl-3-nitro-phenyl)-3-(trifluoromethyl)-pyridine (7.1 mmol)
in a mixture of pyridine (10 mL) and water (5 ml) add KMnO.sub.4
(25.3 mmol) portion wise. Stir the mixture for 4 hours at
110.degree. C. then add another 25.3 mmol of KMnO.sub.4 and 10 ml
of water. Stir the mixture at 110.degree. C. overnight. Cool to
room temperature, filter through celite pad. Concentrate the
filtrate under vacuum, dilute with water, and wash the aqueous with
EtOAc. Neutralize the aqueous layer with 2 N HCl and collect the
precipitate to give the title compound.
4. 2-Nitro-4-(3-trifluoromethyl-pyridin-2-yl)-benzoic acid methyl
ester
##STR00083##
[0380] Saturate a solution of
2-nitro-4-(3-trifluoromethyl-pyridin-2-yl)-benzoic acid (5 g) in
methanol (100 ml) with hydrogen chloride gas. Heat the mixture at
reflux for 4 days and evaporate to dryness. Partition the mixture
between ethyl acetate and saturated sodium bicarbonate solution.
Separate the layers and extract the aqueous layer with further
ethyl acetate. Wash the combined organic extracts with brine, dry
(MgSO.sub.4) and evaporate to give the title compound.
5. 2-Amino-4-(3-trifluoromethyl-pyridin-2-yl)-benzoic acid methyl
ester
##STR00084##
[0382] Hydrogenate, at 50 psi, a mixture of 10% Pd--C (150 mg) and
2-nitro-4-(3-trifluoromethyl-pyridin-2-yl)-benzoic acid methyl
ester (2 g) in 95% EtOH (100 mL). Filter through a celite pad and
concentrate the filtrate to give the title compound.
6.
4-Hydroxy-7-(3-trifluoromethyl-pyridin-2-yl)-1H-quinolin-2-one
##STR00085##
[0384] Heat a solution of
2-amino-4-(3-trifluoromethyl-pyridin-2-yl)-benzoic acid methyl
ester (296 mg, 1.0 mmol) and acetic acid (1 mL) in dioxane (2 mL)
at 60.degree. C. for 3 hours. Cool the mixture, add water (1 mL)
and evaporate to dryness. Dissolve the solid in THF (4 mL) and add
drop wise to a solution of potassium bis(trimethylsilyl)amide (600
mg, 3.0 mmol) in toluene (6 mL) at -78.degree. C. Allow the
reaction to return to room temperature overnight. Add water (10 mL)
and extract with ethyl acetate. Acidify the aqueous layer with
hydrochloric acid and collect the precipitate by filtration.
Air-dry to give the title compound.
7. 2,4-Dichloro-7-(3-trifluoromethyl-pyridin-2-yl)-quinoline
##STR00086##
[0386] Reflux
4-hydroxy-7-(3-trifluoromethyl-pyridin-2-yl)-1H-quinolin-2-one (306
mg) for 18 hours in POCl.sub.3 (5 mL). Evaporate the solvent, then
carefully neutralize with saturated NaHCO.sub.3, and extract with
EtOAc. Dry over Na.sub.2SO.sub.4, concentrate under vacuum to
obtain the title compound.
8.
2-Chloro-4-methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]quinoline
##STR00087##
[0388] Add sodium methoxide (4M, 1.1 mmol) to a solution of
2,4-dichloro-7-(3-trifluoromethyl-pyridin-2-yl)-quinoline (1.0
mmol) in THF (10 mL). Stir at room temperature for 1 hour, add
water (15 mL) and extract with ethyl acetate. Wash the combined
organic extracts with brine, dry (MgSO.sub.4) and evaporate. Purify
the mixture of
2-chloro-4-methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]quinoline and
4-chloro-2-methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]quinoline by
flash chromatography (elute with 1:2 hexane:ether) to give the
title compound.
9. 4-Methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]quinoline
##STR00088##
[0390] Stir a mixture of
2-chloro-4-methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]quinoline
(111 mg), ammonium formate (190 mg), 10% palladium on carbon (30
mg) in methanol (10 mL) at room temperature for 2 hours. Cool,
filter through Celite and evaporate to give the title compound.
10. 7-[3-(Trifluoromethyl)pyridin-2-yl]quinolin-4-ol
##STR00089##
[0392] Heat a solution of
4-methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]quinoline (100 mg), in
33% hydrogen bromide in acetic acid (5 mL) at 100.degree. C. for 18
hours. Evaporate to dryness to give the hydrobromide salt of the
title compound.
11. 4-Chloro-7-[3-(trifluoromethyl)pyridin-2-yl]quinoline
##STR00090##
[0394] Reflux 7-[3-(trifluoromethyl)pyridin-2-yl]quinolin-4-ol (145
mg) for 2 hours in POCl.sub.3 (2 mL). Evaporate the solvent, then
carefully neutralize with saturated NaHCO.sub.3, and extract with
EtOAc. Dry over Na.sub.2SO.sub.4, concentrate under vacuum to
obtain the title compound.
12.
7-[3-(Trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyridin-2-yl-
]quinolin-4-amine
##STR00091##
[0396] To a de-gassed mixture of
4-chloro-7-[3-(trifluoromethyl)pyridin-2-yl]quinoline (0.5 mmol),
cesium carbonate (1 mmol), 2-amino-trifluoromethylpyridine (0.5
mmol) in dioxane (5 mL) under nitrogen add Pd.sub.2 dba.sub.3 (23
mg) and xantphos (15 mg). Stir the mixture at 100.degree. C. for 3
h, cool, add water (8 mL) and extract with EtOAc. Dry over
Na.sub.2SO.sub.4, concentrate under vacuum. Purify by
chromatography eluting with dichloromethane/methanol/ammonium
hydroxide mixture and triturate with ether/hexane to give the title
compound. MS 435 (M+1). .sup.1H NMR .delta. (CDCl.sub.3) 8.73 (1H,
d), 8.56 (1H, s), 8.51 (1H, s), 8.40 (2H, d), 8.11-8.09 (3H, m),
7.85 (1H, d), 7.67 (1H, d), 7.53 (1H, d), 7.47 (1H, dd).
[0397] E.
3-[3-(Trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyridi-
n-2-yl]pyrido[2,3-b]pyrazin-8-amine is prepared as shown in Scheme
9, as follows:
1. 2-Bromo-1-(3-trifluoromethyl-pyridin-2-yl)-ethanone
##STR00092##
[0399] Dissolve 1-(3-trifluoromethyl-pyridin-2-yl)-ethanone (2.10
g, 11.1 mmol) in HBr (30% by wt in AcOH) (14 mL). Cool the mixture
to 0.degree. C. and add bromine (0.62 mL) drop wise. Allow the
resulting solution to warm to room temperature and stir for 3
hours. Concentrate the reaction under reduced pressure to yield the
title compound as its HBr salt.
2.
3-[3-(trifluoromethyl)pyridin-2-yl]pyrido[2,3-b]pyrazin-8-amine
##STR00093##
[0401] Dissolve 2,3,4-triaminopyridine (2.5 mmol) in water (20 mL).
Add NaHCO.sub.3 (0.63 g, 7.5 mmol), dioxane (10 mL), and
2-bromo-1-(3-trifluoromethyl-pyridin-2-yl)-ethanone hydrobromide
(0.5 g) and stir at 100.degree. C. for 2 hours. Cool the mixture
and extract with EtOAc (4.times.10 mL). Wash the combined organic
extracts with brine and dry over Na.sub.2SO.sub.4. Purify the
residue by preparative HPLC to give the title compound.
3.
3-[3-(Trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyridin-2-yl]-
pyrido[2,3-b]pyrazin-8-amine
##STR00094##
[0403] To a de-gassed mixture of
3-[3-(trifluoromethyl)pyridin-2-yl]pyrido[2,3-b]pyrazin-8-amine (72
mg, 0.25 mmol), cesium carbonate (162 mg, 0.5 mmol),
2-amino-trifluoromethyl pyridine (45 mg, 0.25 mmol) in dioxane (5
mL) under nitrogen, add Pd.sub.2 dba.sub.3 (11 mg) and xantphos (7
mg). Stir the mixture at 100.degree. C. for 3 hours, cool, add
water (10 mL) and extract with EtOAc. Dry over Na.sub.2SO.sub.4,
concentrate under vacuum. Purify by chromatography eluting with
dichloromethane/methanol/ammonium hydroxide mixture to give the
title compound. MS 437 (M+1). .sup.1H NMR .delta. (CDCl.sub.3) 9.42
(1H, s), 9.28 (1H, s), 9.11 (1H, d), 8.95 (1H, d), 8.90 (1H, d),
8.72 (1H, s), 8.25 (1H, d), 7.89 (1H, d), 7.61 (1H, dd), 7.13 (1H,
d).
[0404] F.
7-(3-Chloropyridin-2-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)-1,8-
-naphthyridin-4-amine
1. tert-Butyl 4-chloropyridin-2-ylcarbamate
##STR00095##
[0406] Dissolve azido(4-chloropyridin-2-yl)methanone (1.5 g,
0.008216 moles, prepared essentially as described by Sundberg and
Jiang (1997) Org. Prep. Proced. Int. 29:117-122) in toluene (20.0
mL) and heat at 55.degree. C. for 2.0 hours. Add t-butanol (1.96
mL, 0.02054 moles) to the reaction mixture and continue heating at
80.degree. C. for 24 hours. Cool the mixture and concentrate under
reduced pressure to afford a residue. Dissolve the residue in
EtOAc/1.0 N aq. NaOH (50.0 mL each). Separate the organic layer,
extract the aqueous solution with EtOAc (3.times.20.0 mL), wash the
EtOAc with brine, dry (MgSO.sub.4) and concentrate under reduced
pressure to afford a red colored solid. Purify the crude product by
flash column chromatography using 5% EtOAc/hexane to afford the
title product as a white solid.
2. tert-Butyl 4-chloro-3-formylpyridin-2-ylcarbamate
##STR00096##
[0408] Dissolve tert-butyl 4-chloropyridin-2-ylcarbamate (1.95 g,
0.00855 moles) in dry THF (50 mL) and cool to -78.degree. C. under
nitrogen atmosphere. Add dropwise 1.6 M n-BuLi/hexane (12.8 mL,
0.02053 moles) over a period of 15 minutes while maintaining the
reaction temperature below -70.degree. C. Stir the resulting red
orange solution at -78.degree. C. for 2 hours. Add DMF (3.3 mL,
0.04275 moles) dropwise to the reaction mixture while maintaining
the reaction temperature below -70.degree. C. Stir further at
-78.degree. C. for 2 hours and then quench the reaction mixture
with saturated ammonium chloride (50 mL). Warm the reaction mixture
to room temperature, extract with EtOAc (3.times.50 mL) and dry
with MgSO.sub.4. Filter and concentrate under reduced pressure to
afford a yellow viscous oil. Purify the crude product by flash
column chromatography using 15-20% EtOAc/hexane to afford the title
product as a white solid.
3. 2-Amino-4-chloronicotinaldehyde
##STR00097##
[0410] Dissolve tert-butyl 4-chloro-3-formylpyridin-2-ylcarbamate
(1.6 g, 6.2 mmol) in anhydrous CH.sub.2Cl.sub.2 (50 mL) under
N.sub.2 atmosphere. Add dropwise trifluoroacetic acid (2.4 mL, 31.0
mmol) to the reaction mixture and stir at room temperature
overnight. Add saturated aq. sodium carbonate (50 mL) to the
reaction mixture, separate the organic layer, extract the aq. layer
with CH.sub.2Cl.sub.2 (2.times.20 mL) and dry with MgSO.sub.4.
Filter and concentrate under reduced pressure to afford the title
product as a yellow solid.
4. 5-Chloro-2-(3-chloropyridin-2-yl)-[1,8]naphthyridine
##STR00098##
[0412] Dissolve 2-amino-4-chloronicotinaldehyde (312 mg, 2.0 mmol)
and 2-acetyl-3-chloropyridine (310 mg, 2.0 mmol) in anhydrous THF
(5.0 mL) and cool it to -20.degree. C. under N.sub.2 atmosphere.
Add in portion t-BuOK (448 mg, 4.0 mmol) to the reaction mixture
and stir the mixture at 10.degree. C. for 2 hours. Concentrate the
reaction mixture under vacuum, dilute the residue with water (10
mL), filter the solid, wash the solid with water and dry under high
vacuum to afford the title product as a yellow solid.
5.
7-(3-Chloropyridin-2-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)-[1,8]napht-
hyridin-4-amine
##STR00099##
[0414] Heat a mixture of
5-chloro-2-(3-chloropyridin-2-yl)-[1,8]naphthyridine (82.5 mg, 0.3
mmol) and 2-amino-5-trifluoromethylpyridine (97.2 mg, 0.6 mmol) at
180.degree. C. for 2.0 hours. Cool the mixture, dilute with
EtOAc/1.0 N aq. NaOH (5.0 mL each), and separate the organic layer,
extract the aq. layer with EtOAc (2.times.5 mL) and dry the
combined organic layers with MgSO.sub.4. Filter the dried extract
and concentrate under vacuum to afford crude product. Purify by
column chromatography using EtOAc to 2% MeOH/EtOAc as eluents to
afford title compound as a yellow solid. .sup.1H NMR (400 MHZ,
DMSO-D.sub.6) .delta. 10.2 (s, 1H), 9.07 (d, 1H, J=1.9 Hz), 8.93
(d, 1H, J=1.2 Hz), 8.68 (m, 2H), 8.47 (s, 1H), 8.08 (m, 2H), 7.96
(d, 1H, J=2.2 Hz), 7.56 (dd, 1H), 7.46 (d, 1H, J=2.2 Hz). MS=402.22
(M+H).
[0415] G.
7-(3-Methylpyridin-2-yl)-N-(5-(trifluoromethyl)pyrimidin-2-yl)-[-
1,8]naphthyridin-4-amine
1. 5-Chloro-2-(3-methylpyridin-2-yl)-1,8-naphthyridine
##STR00100##
[0417] Dissolve 2-amino-4-chloronicotinaldehyde (156 mg, 1.0 mmol)
and 2-acetyl-3-methylpyridine (136 mg, 1.0 mmol) in anhydrous THF
(5.0 mL) and cool it to -20.degree. C. under N.sub.2 atmosphere.
Add in portion t-BuOK (224 mg, 2.0 mmol) to the reaction mixture
and stir the mixture at 10.degree. C. for 2 hours. Concentrate the
reaction mixture under vacuum, dilute the residue with water (10
mL), filter the solid, wash the solid with water and dry under high
vacuum to afford the title product as a yellow colored solid.
2.
7-(3-Methylpyridin-2-yl)-N-(5-(trifluoromethyl)pyrimidin-2-yl)-[1,8]nap-
hthyridin-4-amine
##STR00101##
[0419] Heat a mixture of
5-chloro-2-(3-methylpyridin-2-yl)-[1,8]naphthyridine (51 mg, 0.2
mmol), 2-amino-5-trifluoromethylpyrimidine (42.0 mg, 0.25 mmol),
xantphos (11.6 mg, 0.02 mmol), Pd.sub.2(dba).sub.3 (18.3 mg, 0.02
mmol) and Cs.sub.2CO.sub.3 (130 mg, 0.4 mmol) in dioxane (2.0 mL)
at 100.degree. C. for 20 hours. Cool the mixture, concentrate under
vacuum, dilute with EtOAc/water (5.0 mL each), filter through
celite, wash celite with EtOAc (2.times.5 mL) and dry the combined
organic layers with MgSO.sub.4. Filter the dried extract and
concentrate under vacuum to afford the crude product. Purify by
column chromatography using EtOAc as eluent to afford the title
compound as a yellow solid. .sup.1H NMR (400 MHZ, DMSO-D.sub.6)
.delta. 10.92 (s, 1H), 9.0 (m, 3H), 8.92 (d, 1H, J=1.8 Hz), 8.58
(d, 1H, J=1.0 Hz), 8.12 (m, 2H), 7.80 (d, 1H, J=2.1 Hz), 7.41 (dd,
1H), 2.65 (s, 3H). MS=383.3 (M+H).
[0420] H.
7-(3-Methylpyridin-2-yl)-N-(5-(trifluoromethyl)pyrazin-2-yl)-[1,-
8]naphthyridin-4-amine
##STR00102##
[0421] Heat a mixture of
5-chloro-2-(3-methylpyridin-2-yl)-[1,8]naphthyridine (51 mg, 0.2
mmol), 2-amino-5-trifluoromethylpyrazine (42.0 mg, 0.25 mmol),
xantphos (11.6 mg, 0.02 mmol), Pd.sub.2(dba).sub.3 (18.3 mg, 0.02
mmol) and Cs.sub.2CO.sub.3 (130 mg, 0.4 mmol) in dioxane (2.0 mL)
at 100.degree. C. for 20 hours. Cool the mixture, concentrate under
vacuum, dilute with EtOAc/water (5.0 mL each), filter through
celite, wash celite with EtOAc (2.times.5 mL) and dry the combined
organic layers with MgSO.sub.4. Filter the dried extract and
concentrate under vacuum to afford the crude product. Purify by
preparative TLC using 2% MeOH/EtOAc as eluent to afford the title
compound as a yellow solid. .sup.1H NMR (400 MHZ, DMSO-D.sub.6)
.delta. 10.6 (s, 1H), 9.04 (d, 1H, J=2.1 Hz), 9.0 (s, 1H), 8.77 (s,
2H), 8.59 (d, 1H, J=1.6 Hz), 8.4 (s, 1H), 8.20 (d, 1H, J=2.2 Hz),
7.81 (d, 1H, J=1.9 Hz), 7.42 (dd, 1H), 2.65 (s, 3H). MS=383.11
(M+H).
[0422] I.
7-(3-(Trifluoromethyl)pyridin-2-yl)-N-(5-(trifluoromethyl)pyridi-
n-2-yl)-1,8-naphthyridin-4-amine
1.
5-Chloro-2-(3-(trifluoromethyl)pyridin-2-yl)-[1,8]naphthyridine
##STR00103##
[0424] Dissolve 2-amino-4-chloronicotinaldehyde (78 mg, 0.5 mmol)
and 2-acetyl-3-trifluoromethylpyridine (95 mg, 0.5 mmol) in
anhydrous THF (2.0 mL) and cool it to -20.degree. C. under N.sub.2
atmosphere. Add in portion t-BuOK (112 mg, 1.0 mmol) to the
reaction mixture and stir the mixture at 10.degree. C. for 2 hours.
Concentrate the reaction mixture under vacuum, dilute the residue
with saturated aq. ammonium chloride (10 mL), filter the solid,
wash the solid with water and dry under high vacuum to afford
desired product as a cream colored solid.
2.
7-(3-(Trifluoromethyl)pyridin-2-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)-
-[1,8]naphthyridin-4-amine
##STR00104##
[0426] Heat a mixture of
5-chloro-2-(3-(trifluoromethyl)pyridin-2-yl)-[1,8]naphthyridine (62
mg, 0.2 mmol), 2-amino-5-trifluoromethylpyridine (32.4 mg, 0.2
mmol), xantphos (11.6 mg, 0.02 mmol), Pd.sub.2(dba).sub.3 (18.3 mg,
0.02 mmol) and Cs.sub.2CO.sub.3 (130 mg, 0.4 mmol) in dioxane (2.0
mL) at 100.degree. C. for 20 hours. Cool the mixture, concentrate
under vacuum, dilute with EtOAc/water (5.0 mL each), filter through
celite, wash celite with EtOAc (2.times.5 mL) and dry combined
organic layers with MgSO.sub.4. Filter the dried extract and
concentrate under vacuum to afford crude product. Purify by
preparative TLC using EtOAc as eluent to afford title compound as a
yellow solid. .sup.1H NMR (400 MHZ, DMSO-D.sub.6) .delta. 10.15 (s,
1H), 9.1 (d, 1H, J=2.2 Hz), 9.0 (d, 1H, J=1.1 Hz), 8.95 (d, 1H,
J=1.2 Hz), 8.68 (s, 1H), 8.50 (d, 1H, J=1.3 Hz), 8.43 (d, 1H, J=2.0
Hz), 8.12 (dd, 1H), 8.0 (d, 1H, J=2.2 Hz), 7.81 (m, 1H), 7.49 (d,
1H, J=2.2 Hz). MS=436.08 (M+H).
Example 3
Additional Representative Substituted Quinolin-4-ylamine
Analogues
[0427] Using routine modifications, the starting materials may be
varied and additional steps employed to produce other compounds
provided herein. Compounds listed in Table I were prepared using
such methods. In the column labeled "IC.sub.50" a * indicates that
the IC.sub.50 determined as described in Example 6 is 1 micromolar
or less (i.e., the concentration of such compounds that is required
to provide a 50% decrease in the fluorescence response of cells
exposed to one IC.sub.50 of capsaicin is 1 micromolar or less).
TABLE-US-00002 TABLE 1 Ret. MS Compound Name IC.sub.50 Time (M + 1)
1 ##STR00105## 2-methyl-N-[4- (trifluoromethyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2- yl]quinolin-4-amine * 1.19 448.15 2
##STR00106## 2-Methoxymethyl-7-(3- trifluoromethyl-pyridin-2-yl)-
[1,8]naphthyridin-4-yl]-(4- trifluoromethylphenyl)-amine * 1.17
479.16 3 ##STR00107## 2-Methoxymethyl-7-(3-
trifluoromethyl-pyridin-2-yl)- [1,8]naphthyridin-4-yl]-(4-t-
butylphenyl)-amine * 1.21 467.2 4 ##STR00108## methyl
2-(methoxymethyl)-4-{[4- (trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridine-3-carboxylate *
1.23 537.23 5 ##STR00109## {4-{[4-
(trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-2-yl}methanol *
1.14 465.2 6 ##STR00110## 2-(methoxymethyl)-4-{[4-
(trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridine-3-carboxylic acid
* 1.21 523.22 7 ##STR00111## 2-(isobutoxymethyl)-N-[4-
(trifluoromethyl)phenyl]-7-[3- (trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-amine * 1.24 521.25 8 ##STR00112##
N-(4-chlorophenyl)-2- (methoxymethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.16
445.19 9 ##STR00113## N-(4-fluorophenyl)-2- (methoxymethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.14
429.21 10 ##STR00114## N-(4-chloro-3-fluorophenyl)-2-
(methoxymethyl)-7-[3- (trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-amine * 1.17 463.19 11 ##STR00115##
4-({2-(methoxymethyl)-7-[3- (trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4- yl}amino)benzonitrile * 1.1 436.22 12 ##STR00116##
5-{[4- (trifluoromethyl)phenyl]amino}-2-[3-
(trifluoromethyl)pyridin-2- yl]furo[3,4-b]-1,8-naphthyridin-
6(8H)-one * 1.24 491.15 13 ##STR00117##
N-[4-(isopropylsulfonyl)phenyl]-2- (methoxymethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.11
517.29 14 ##STR00118## 2-(methoxymethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.18
480.25 15 ##STR00119## 2-(methoxymethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-{4-
[(trifluoromethyl)sulfonyl]phenyl}- 1,8-naphthyridin-4-amine * 1.17
543.24 16 ##STR00120## N-(4,4-dimethyl-1,2,3,4-
tetrahydroquinolin-7-yl)-2- (methoxymethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.18
494.37 17 ##STR00121## 2-(methoxymethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-N- (2,4,4-trimethyl-1,2,3,4-
tetrahydroisoquinolin-7-yl)-1,8- naphthyridin-4-amine * 1.06 508.39
18 ##STR00122## 2-isobutoxy-N-[4- (trifluoromethyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2- yl]quinolin-4-amine * 1.26 506.26 19
##STR00123## N-(4-tert-butylphenyl)-2-isobutoxy-
7-[3-(trifluoromethyl)pyridin-2- yl]quinolin-4-amine * 1.28 494.32
20 ##STR00124## 2-chloro-N-[4- (trifluoromethyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2- yl]quinolin-4-amine * 1.34 468.17 21
##STR00125## 4-[(4-tert-butylphenyl)amino]-7-[3-
(trifluoromethyl)pyridin-2- yl]quinolin-2-ol * 1.29 438.27 22
##STR00126## N-(4-tert-butylphenyl)-2-chloro-7-
[3-(trifluoromethyl)pyridin-2- yl]quinolin-4-amine * 1.36 456.23 23
##STR00127## N-(4-tert-butylphenyl)-2-morpholin-
4-yl-7-[3-(trifluoromethyl)pyridin-2- yl]quinolin-4-amine * 1.24
507.24 24 ##STR00128## 2-{[(2R,6S)-2,6-dimethylmorpholin-
4-yl]methyl}-N-[4- (trifluoromethyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.19
562.23 25 ##STR00129## 2-{[2- (dimethylamino)ethoxy]methyl}-N-
[4-(trifluoromethyl)phenyl]-7-{3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.1
536.22 26 ##STR00130## N-(4-tert-butylphenyl)-2-piperidin-1-
yl-7-[3-(trifluoromethyl)pyridin-2- yl]quinolin-4-amine * 1.28
505.25 27 ##STR00131## N~2~butyl-N~4~-(4-tert- butylphenyl)-7-[3-
(trifluoromethyl)pyridin-2- yl]quinoline-2,4-diamine * 1.29 493.28
28 ##STR00132## N-(4-tert-butylphenyl)-2-pyrrolidin-
1-yl-7-[3-(trifluoromethyl)pyridin-2- yl]quinolin-4-amine * 1.26
491.25 29 ##STR00133## N~4~-(4-tert-butylphenyl)-N~2~-
isopentyl-7-[3- (trifluoromethyl)pyridin-2-
yl]quinoline-2,4-diamine * 1.31 507.29 30 ##STR00134##
N-(4-tert-butylphenyl)-2-ethoxy-7- [3-(trifluoromethyl)pyridin-2-
yl]quinolin-4-amine * 1.24 466.31 31 ##STR00135## 4-{[4-
(trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-2-ol * 1.24 451.22
32 ##STR00136## N-(4-tert-butylphenyl)-2- (isopentyloxy)-7-[3-
(trifluoromethyl)pyridin-2- yl]quinolin-4-amine * 1.3 508.36 33
##STR00137## N-(4-tert-butylphenyl)-2-propoxy-7-
[3-(trifluoromethyl)pyridin-2- yl]quinolin-4-amine * 1.26 480.32 34
##STR00138## 2-chloro-N-[4- (trifluoromethyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.3
469.18 35 ##STR00139## N-[4-(trifluoromethyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 435.35
36 ##STR00140## 2-ethoxy-N-[4- (trifluoromethyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.28
479.22 37 ##STR00141## N-(4-tert-butylphenyl)-2-ethoxy-7-
[3-(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.26
467.29 38 ##STR00142## 2-ethoxy-N-(4-isopropylphenyl)-7-
[3-(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.24
453.27 39 ##STR00143## 2-methyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.17
450.21 40 ##STR00144## 2-(ethoxymethyl)-N-[4-
(trilfuoromethyl)phenyl]-7-[3- (trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-amine * 1.2 493.18 41 ##STR00145##
2-(isopropoxymethyl)-N-[4- (trifluoromethyl)phenyl]-7-[3-
(trilfuoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.22
507.19 42 ##STR00146## 2-[(isopentyloxy)methyl]-N-[4-
(trifluoromethyl)phenyl]-7-[3- (trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-amine * 1.26 535.23 43 ##STR00147##
2-(propoxymethyl)-N-[4- (trifluoromethyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.22
507.19 44 ##STR00148## 2-(isobutoxymethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.27
522.19 45 ##STR00149## 2-ethoxy-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.32
480.15 46 ##STR00150## 2-ethoxy-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-{4-
[(trifluoromethyl)sulfonyl]phenyl}- 1,8-naphthyridin-4-amine * 1.31
543.15 47 ##STR00151## N-(4-tert-butylphenyl)-2-methoxy-7-
[3-(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.25
453.2 48 ##STR00152## 2-methyl-N-[4- (trifluoromethyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.18
449.16 49 ##STR00153## 2-[(2-methylpyrrolidin-1-yl)methyl]-
N-[4-(trifluoromethyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.19
532.26 50 ##STR00154## 2-{[bis(2- methoxyethyl)amino]methyl}-N-[4-
(trifluoromethyl)phenyl]-7-[3- (trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-amine * 1.2 580.28 51 ##STR00155##
7-(3-chloropyridin-2-yl)-2- (methoxymethyl)-N-[4-
(trifluoromethyl)phenyl]-1,8- naphthyridin-4-amine * 1.17 445.17 52
##STR00156## 7-(3-chloropyridin-2-yl)-2- (isobutoxymethyl)-N-[4-
(trifluoromethyl)phenyl]-1,8- naphthyridin-4-amine * 1.25 487.23 53
##STR00157## 2-(4-{[7-(3-chloropyridin-2-yl)-2-
(methoxymethyl)-1,8-naphthyridin- 4-yl]amino}phenyl)propan-2-ol *
1.13 435.22 54 ##STR00158## N-(4-fluorophenyl)-2-
(isobutoxymethyl)-7-[3- (trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-amine * 1.22 471.25 55 ##STR00159##
2-(azetidin-1-ylmethyl)-N-[4- (trifluoromethyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.18
504.23 56 ##STR00160## 1-[4-({2-(isobutoxymethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-
yl}amino)phenyl]ethanone * 495.27 57 ##STR00161##
2-[4-({2-(isobutoxymethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-
yl}amino)phenyl]propan-2-ol * 1.21 511.3 58 ##STR00162##
2-[(isopropylamino)methyl]-N-[4- (trifluoromethyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.19
506.22 59 ##STR00163## 2-[(isobutylamino)methyl]-N-[4-
(trifluoromethyl)phenyl]-7-[3- (trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-amine * 1.19 520.24 60 ##STR00164##
2-{[isobutyl(methyl)amino]methyl}-
N-[4-(trifluoromethyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.21
534.26 61 ##STR00165## N-(5-bromopyridin-2-yl)-2-
(isobutoxymethyl)-7-[3- (trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-amine * 1.27 532.15 62 ##STR00166##
7-(3-fluoropyriidn-2-yl)-2- (methoxymethyl)-N-[4-
(trifluoromethyl)phenyl]-1,8- naphthyridin-4-amine * 1.16 429.16 63
##STR00167## 1-(4-{[7-(3-chloropyridin-2-yl)-2-
(isobutoxymethyl)-1,8-naphthyridin- 4-yl]amino}phenyl)ethanone *
1.2 461.21 64 ##STR00168## 2-(4-{[7-(3-chloropyridin-2-yl)-2-
(isobutoxymethyl)-1,8-naphthyridin- 4-yl]amino}phenyl)propan-2-ol *
1.21 477.25 65 ##STR00169## 2-(isobutoxymethyl)-N-[4-
(methylsulfonyl)phenyl]-7-[3- (trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-amine * 1.15 531.2 66 ##STR00170## 1-{[4-{[4-
(trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-2-yl}methoxy)propan- 2-ol * 1.18 523.19 67
##STR00171## 1-({4-{[4- (trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-2-yl}methoxy)acetone * 1.17 521.17 68 ##STR00172##
4-({2-(isobutoxymethyl)-7-[3- (trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-yl}amino)-N,N- dimethylbenzenesulfonamide * 1.19
560.25 69 ##STR00173## N-{[4-({2-(isobutoxymethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-
yl}amino)phenyl]sulfonyl}acetamide 1.16 574.28 70 ##STR00174##
N-methyl-N-({4-{[4- (trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-2-
yl}methyl)methanesulfonamide * 1.17 556.22 71 ##STR00175##
N-methyl-N-({4-{[4- (trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-2-
yl}methyl)ethanesulfonamide * 1.18 570.24 72 ##STR00176##
2-Iisobutoxymethyl)-N-[4- (isopropylsulfonyl)phenyl]-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.19
559.29 73 ##STR00177## 2-(isobutoxymethyl)-N-[4-
(propylsulfonyl)phenyl]-7-[3- (trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-amine * 1.19 559.3 74 ##STR00178##
2-(isobutoxymethyl)-7-[3- (trifluoromethyl)pyridin-2-yl]-N-{4-
[(trifluoromethyl)sulfonyl]phenyl}- 1,8-naphthyridin-4-amine * 1.24
585.24 75 ##STR00179## N,2-dimethyl-N-({4-{[4-
(trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-2-
yl}methyl)propanamide * 1.18 548.19 76 ##STR00180##
N-methyl-N-({4-{[4- (trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-2-yl}methyl)glycine * 1.17 495.15 77 ##STR00181##
3-[ethyl({4-{[4- (trifluoromethyl)phenyl]amino]-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-2-
yl}methyl)amino]propan-1-ol * 1.19 550.05 78 ##STR00182##
3-[isopropyl({4-{[4- (trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-2-
yl}methyl)amino]propan-1-ol * 1.19 564.06 79 ##STR00183##
3-[propyl({4-{[4- (trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-2-
yl}methyl)amino]propan-1-ol * 1.21 564.05 80 ##STR00184##
N-isobutyl-N-({4-{[4- (trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-2-
yl}methyl)methanesulfonamide * 1.22 597.99 81 ##STR00185##
N-isobutyl-N-({4-{[4- (trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-2-yl}methyl)glycine * 82 ##STR00186##
2-methyl-1-({4-{[4- (trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-2-yl}methoxy)propan- 2-ol * 1.19 537.17 83
##STR00187## 2-(isobutoxymethyl)-N-(5- methylpyridin-2-yl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.24
468.2 84 ##STR00188## 2-(isobutoxymethyl)-N-pyridin-2-yl-
7-[3-(tirlfuoromethyl)pyridin-2-yl]- 1,8-naphthyridin-4-amine *
1.21 454.19 85 ##STR00189## 2-[6-({2-(isobutoxymethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-yl}amino)pyridin-2- yl]propan-2-ol * 1.21 512.23 86
##STR00190## 2-methyl-3-({4-{[4-
(trifluoromethyl)phenyl]amino}-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-2-yl}methoxy)butan-2- ol * 1.21 551.2 87 ##STR00191##
6-({2-(isobutoxymethyl)-7-[3- (trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4- yl}amino)nicotinonitrile * 1.22 479.19 88
##STR00192## (7-[3-(trifluoromethyl)pyridin-2-yl]-
4-{[5-(trifluoromethyl)pyridin-2- yl]amino}-1,8-naphthyridin-2-
yl)methanol * 1.16 466.12 89 ##STR00193##
2-[(dimethylamino)methyl]-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.15
493.17 90 ##STR00194## rel-2-{[(2R,6S)-2,6-
dimethylmorpholin-4-yl]methyl}-7-
[3-(trifluoromethyl)pyridin-2-yl]-N-
[5-(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.2
563.22 91 ##STR00195## 2-(morpholin-4-ylmethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.17
535.19 92 ##STR00196## 2-(piperidin-1-ylmethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.17
533.21 93 ##STR00197## 2-[(2-methylpyrrolidin-1-yl)methyl]-
7-[3-(trifluoromethyl)pyridin-2-yl]-N-
[5-(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.18
533.2 94 ##STR00198## 2-[(diethylamino)methyl]-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.19
521.2 95 ##STR00199## 2-(pyrrolidin-1-ylmethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.19
519.19 96 ##STR00200## 2-methoxy-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.31
466.12 97 ##STR00201## 7-[3-(trifluoromethyl)pyridin-2-yl]-4-
{[5-(trifluoromethyl)pyridin-2- yl]amino}-1,8-naphthyridin-2-ol *
1.26 452.08 98 ##STR00202## 2-cyclobutyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.25
490.14 99 ##STR00203## 2-cyclobutyl-N-(5-methylpyridin-2-
yl)-7-[3-(trifluoromethyl)pyridin-2- yl]-1,8-naphthyridin-4-amine *
1.2 436.18 100 ##STR00204## 6-({2-cyclobutyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-
yl}amino)nicotinonitrile * 1.19 447.16 101 ##STR00205##
N-(5-chloropyridin-2-yl)-2- cylcobutyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphtyridin-4-amine * 1.23
456.13 102 ##STR00206## 2-isopropyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.24
478.15 103 ##STR00207## 2-isopropyl-N-(5-methylpyridin-2-
yl)-7-[3-(trifluoromethyl)pyridin-2- yl]-1,8-naphthyridin-4-amine *
1.19 424.17 104 ##STR00208## 6-({2-isopropyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-
yl}amino)nicotinonitrile * 1.17 435.16 105 ##STR00209##
N-(5-chloropyriidn-2-yl)-2- isopropyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.22
444.12 106 ##STR00210## 7-[3-(trifluoromethyl)pyridin-2-yl]-N-
[5-(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.16
436.12 107 ##STR00211## 2-methoxy-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,5- naphthyriidn-4-amine 1.2 466.16
108 ##STR00212## 7-[3-(trifluoromethyl)pyridin-2-yl]-N-
[5-(trifluoromethyl)pyridin-2-yl]-1,5- naphthyridin-4-amine * 1.29
436.14 109 ##STR00213## 7-[3-(trifluoromethyl)pyridin-2-yl]-N-
[5-(trifluoromethyl)pyridin-2- yl]quinolin-4-amine * 1.19 435.15
110 ##STR00214## 2-ethyl-7-[3-(trifluoromethyl)pyridin-
2-yl]-N-[5-(trifluoromethyl)pyridin-2- yl]-1,8-naphthyridin-4-amine
* 1.2 464.16 111 ##STR00215## 1-[6-({2-ethyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-yl}amino)pyridin-3- yl]ethanone * 1.16 438.19 112
##STR00216## 2-[6-({2-ethyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-yl}amino)pyridin-3- yl]propan-2-ol * 1.16 454.25 113
##STR00217## 3-[3-(trifluoromethyl)pyridin-2-yl]-N-
[5-(trifluoromethyl)pyridin-2- yl]pyrido[2,3-b]pyrazin-8-amine *
1.23 437.12 114 ##STR00218## 2-propyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.25
478.16 115 ##STR00219## 1-[6-({2-propyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-yl}amino)pyridin-3- yl]ethanone * 1.19 452.19 116
##STR00220## 1-[6-({2-isopropyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-yl}amino)pyridin-3- yl]ethanone * 1.18 452.2 117
##STR00221## 2-[6-({2-propyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-yl}amino)pyridin-3- yl]propan-2-ol * 1.19 468.24 118
##STR00222## 2-[6-({2-isopropyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-yl}amino)pyridin-3- yl]propan-2-ol * 1.18 468.22 119
##STR00223## 2-(1-methoxyethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.23
494.17 120 ##STR00224## 1-[6-({2-(1-methoxyethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-yl}amino)pyridin-3- yl]ethanone * 1.17 468.2 121
##STR00225## 2-[6-({2-(1-methoxyethyl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8-
naphthyridin-4-yl}amino)pyridin-3- yl]propan-2-ol * 1.15 484.24 122
##STR00226## N-(5-chloropyridin-2-yl)-2-propyl-7-
[3-(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.24
444.13 123 ##STR00227## N-[5-(methylsulfonyl)pyridin-2-yl]-2-
propyl-7-[3-(trifluoromethyl)pyridin-
2-yl]-1,8-naphthyridin-4-amine * 1.16 488.15 124 ##STR00228##
2-(1-methylpiperidin-3-yl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 1.2
533.19 125 ##STR00229## 7-[3-(trifluoromethyl)pyridin-2-yl]-4-
{[5-(trifluoromethyl)pyridin-2- yl]amino}-1,8-naphthyridine-2-
carbonitrile * 1.31 461.11 126 ##STR00230##
7-[3-(trifluoromethyl)pyridin-2-yl]-4-
{[5-(trifluoromethyl)pyridin-2- yl]amino}-1,8-naphthyridine-2-
carboxamide * 127 ##STR00231##
7-[3-(trifluoromethyl)pyridin-2-yl]-4-
{[5-(trifluoromethyl)pyridin-2- yl]amino}-1,8-naphthyridine-2-
carboxylic acid 128 ##STR00232##
N-(5-ethylpyridin-2-yl)-2-propyl-7-
[3-(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 129
##STR00233## 2-morpholin-4-yl-7-[3-
(trifluoromethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 521.1
130 ##STR00234## N~2~-isobutyl-7-[3-
(trifluoromethyl)pyridin-2-yl]-N~4~-
[5-(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridine-2,4-diamine *
507.1 131 ##STR00235## N-[5-(trifluoromethyl)pyrazin-2-yl]-
7-[3-(trifluoromethyl)pyridin-2-yl]- 1,8-naphthyridin-4-amine *
437.04 132 ##STR00236## 7-[3-(trifluoromethyl)pyridin-2-yl]-N-
[5-(trifluoromethyl)pyrimidin-2-yl]- 1,8-naphthyridin-4-amine *
437.04 133 ##STR00237## 1-(7-[3-(trifluoromethyl)pyridin-2-
yl]-4-{[5-(trifluoromethyl)pyridin-2- yl]amino}-1,8-naphthyridin-2-
yl)ethanone * 134 ##STR00238## N-[6-(trifluoromethyl)pyridazin-3-
yl]-7-[3-(trifluoromethyl)pyridin-2- yl]-1,8-naphthyridin-4-amine
135 ##STR00239## 7-(3-methylpyridin-2-yl)-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 136
##STR00240## 7-(3-chloropyridin-2-yl)-N-[5-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 137
##STR00241## 7-(3-methylpyridin-2-yl)-N-[5-
(trifluoromethyl)pyrimidin-2-yl]-1,8- naphthyridin-4-amine *
138 ##STR00242## 7-(3-chloropyridin-2-yl)-N-[5-
(trifluoromethyl)pyrazin-2-yl]-1,8- naphthyridin-4-amine * 139
##STR00243## N-(5-chloropyridin-2-yl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 140
##STR00244## N-(5-methylpyridin-2-yl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 141
##STR00245## 1-[6-({7-[3-(trifluoromethyl)pyridin-
2-yl]-1,8-naphthyridin-4- yl]amino)pyridin-3-yl]ethanone * 142
##STR00246## 7-[3-(trifluoromethyl)pyridin-2-yl]-N-
[6-(trifluoromethyl)pyridin-3-yl]-1,8- naphthyridin-4-amine * 143
##STR00247## N-(5-ethylpyridin-2-yl)-7-[3-
(trifluoromethyl)pyridin-2-yl]-1,8- naphthyridin-4-amine * 144
##STR00248## 7-(3-methylpyridin-2-yl)-N-[5-
(trifluoromethyl)pyrazin-2-yl]-1,8- naphthyridin-4-amine * 145
##STR00249## 7-(3-chloropyridin-2-yl)-N-[5-
(trifluoromethyl)pyrimidin-2-yl]-1,8- naphthyridin-4-amine *
Example 4
VR1-Transfected Cells and Membrane Preparations
[0428] This Example illustrates the preparation of VR1-transfected
cells and membrane preparations for use in binding assays (Example
5) and functional assays (Example 6).
[0429] A cDNA encoding full length human capsaicin receptor (SEQ ID
NO:1, 2 or 3 of U.S. Pat. No. 6,482,611) was subcloned in the
plasmid pBK-CMV (Stratagene, La Jolla, Calif.) for recombinant
expression in mammalian cells.
[0430] Human embryonic kidney (HEK293) cells were transfected with
the pBK-CMV expression construct encoding the full length human
capsaicin receptor using standard methods. The transfected cells
were selected for two weeks in media containing G418 (400 .mu.g/ml)
to obtain a pool of stably transfected cells. Independent clones
were isolated from this pool by limiting dilution to obtain clonal
stable cell lines for use in subsequent experiments.
[0431] For radioligand binding experiments, cells were seeded in
T175 cell culture flasks in media without antibiotics and grown to
approximately 90% confluency. The flasks were then washed with PBS
and harvested in PBS containing 5 mM EDTA. The cells were pelleted
by gentle centrifugation and stored at -80.degree. C. until
assayed.
[0432] Previously frozen cells were disrupted with the aid of a
tissue homogenizer in ice-cold HEPES homogenization buffer (5 mM
KCl 5, 5.8 mM NaCl, 0.75 mM CaCl.sub.2, 2 mM MgCl.sub.2, 320 mM
sucrose, and 10 mM HEPES pH 7.4). Tissue homogenates were first
centrifuged for 10 minutes at 1000.times.g (4.degree. C.) to remove
the nuclear fraction and debris, and then the supernatant from the
first centrifugation is further centrifuged for 30 minutes at
35,000.times.g (4.degree. C.) to obtain a partially purified
membrane fraction. Membranes were resuspended in the HEPES
homogenization buffer prior to the assay. An aliquot of this
membrane homogenate is used to determine protein concentration via
the Bradford method (BIO-RAD Protein Assay Kit, #500-0001, BIO-RAD,
Hercules, Calif.).
Example 5
Capsaicin Receptor Binding Assay
[0433] This Example illustrates a representative assay of capsaicin
receptor binding that may be used to determine the binding affinity
of compounds for the capsaicin (VR1) receptor.
[0434] Binding studies with [.sup.3H] Resiniferatoxin (RTX) are
carried out essentially as described by Szallasi and Blumberg
(1992) J. Pharmacol. Exp. Ter. 262:883-888. In this protocol,
non-specific RTX binding is reduced by adding bovine alpha.sub.1
acid glycoprotein (100 g per tube) after the binding reaction has
been terminated.
[0435] [.sup.3H] RTX (37 Ci/mmol) is synthesized by and obtained
from the Chemical Synthesis and Analysis Laboratory, National
Cancer Institute-Frederick Cancer Research and Development Center,
Frederick, Md. [.sup.3H] RTX may also be obtained from commercial
vendors (e.g., Amersham Pharmacia Biotech, Inc.; Piscataway,
N.J.).
[0436] The membrane homogenate of Example 4 is centrifuged as
before and resuspended to a protein concentration of 333 .mu.g/ml
in homogenization buffer. Binding assay mixtures are set up on ice
and contain [.sup.3H]RTX (specific activity 2200 mCi/ml), 2 .mu.l
non-radioactive test compound, 0.25 mg/ml bovine serum albumin
(Cohn fraction V), and 5.times.10.sup.4-1.times.10.sup.5
VR1-transfected cells. The final volume is adjusted to 500 .mu.l
(for competition binding assays) or 1,000 .mu.l (for saturation
binding assays) with the ice-cold HEPES homogenization buffer
solution (pH 7.4) described above. Non-specific binding is defined
as that occurring in the presence of 1 .mu.M non-radioactive RTX
(Alexis Corp.; San Diego, Calif.). For saturation binding,
[.sup.3H]RTX is added in the concentration range of 7-1,000 pM,
using 1 to 2 dilutions. Typically 11 concentration points are
collected per saturation binding curve.
[0437] Competition binding assays are performed in the presence of
60 pM [.sup.3H]RTX and various concentrations of test compound. The
binding reactions are initiated by transferring the assay mixtures
into a 37.degree. C. water bath and are terminated following a 60
minute incubation period by cooling the tubes on ice.
Membrane-bound RTX is separated from free, as well as any
alpha.sub.1-acid glycoprotein-bound RTX, by filtration onto WALLAC
glass fiber filters (PERKIN-ELMER, Gaithersburg, Md.) which were
pre-soaked with 1.0% PEI (polyethyleneimine) for 2 hours prior to
use. Filters are allowed to dry overnight then counted in a WALLAC
1205 BETA PLATE counter after addition of WALLAC BETA SCINT
scintillation fluid.
[0438] Equilibrium binding parameters are determined by fitting the
allosteric Hill equation to the measured values with the aid of the
computer program FIT P (Biosoft, Ferguson, Mo.) as described by
Szallasi, et al. (1993) J. Pharmacol. Exp. Ther. 266:678-683.
Compounds provided herein generally exhibit K.sub.i values for
capsaicin receptor of less than 1 .mu.M, 100 nM, 50 nM, 25 nM, 10
nM, or 1 nM in this assay.
Example 6
Calcium Mobilization Assay
[0439] This Example illustrates a representative calcium
mobilization assay for use in monitoring the response of cells
expressing capsaicin receptor to capsaicin and other vanilloid
ligands of the capsaicin receptor, as well as for evaluating test
compounds for agonist and antagonist activity.
[0440] Cells transfected with expression plasmids (as described in
Example 4) and thereby expressing human capsaicin receptor are
seeded and grown to 70-90% confluency in FALCON black-walled,
clear-bottomed 96-well plates (#3904, BECTON-DICKINSON, Franklin
Lakes, N.J.). The culture medium is emptied from the 96 well plates
and FLUO-3 AM calcium sensitive dye (Molecular Probes, Eugene,
Oreg.) is added to each well (dye solution: 1 mg FLUO-3 AM, 440
.mu.L DMSO and 440 .mu.l 20% pluronic acid in DMSO, diluted 1:250
in Krebs-Ringer HEPES (KRH) buffer (25 mM HEPES, 5 mM KCl, 0.96 mM
NaH.sub.2PO.sub.4, 1 mM MgSO.sub.4, 2 mM CaCl.sub.2, 5 mM glucose,
1 mM probenecid, pH 7.4), 501 diluted solution per well). Plates
are covered with aluminum foil and incubated at 37.degree. C. for
1-2 hours in an environment containing 5% CO.sub.2. After the
incubation, the dye is emptied from the plates, and the cells are
washed once with KRH buffer, and resuspended in KRH buffer.
[0441] Agonist (e.g., olvanil, capsaicin, or RTX)-induced calcium
mobilization is monitored using either FLUOROSKAN ASCENT
(Labsystems, Franklin, Mass.) or FLIPR (fluorometric imaging plate
reader system, Molecular Devices, Sunnyvale, Calif.) instruments.
Varying concentrations of the antagonists ruthenium red or
capsazepine (RBI; Natick, Mass.) are added to cells concurrently
with agonist (e.g., 25-50 nM capsaicin). For agonist-induced
calcium responses, data obtained between 30 and 60 seconds after
agonist application are used to generate the IC.sub.50 values.
KALEIDAGRAPH software (Synergy Software, Reading, Pa.) is used to
fit the data to the equation:
y=a*(1/(1+(b/x).sup.c))
to determine the IC.sub.50 for the response. In this equation, y is
the maximum fluorescence signal, x is the concentration of the
agonist or antagonist, a is the E.sub.max, b corresponds to the
IC.sub.50 value and c is the Hill coefficient.
[0442] To measure the ability of a test compound to antagonize
(inhibit) the response of cells expressing capsaicin receptors to
capsaicin or other vanilloid agonist, the IC.sub.50 of capsaicin is
first determined. An additional 20 .mu.l of KRH buffer and 1 .mu.l
DMSO is added to each well of cells, prepared as described above.
100 .mu.l capsaicin in KRH buffer is automatically transferred by
the FLIPR instrument to each well. An 8-point concentration
response curve, with final capsaicin concentrations of 1 nM to 3
.mu.M, is used to determine capsaicin IC.sub.50.
[0443] Test compounds are dissolved in DMSO, diluted in 20 .mu.l
KRH buffer so that the final concentration of test compounds in the
assay well is between 1 .mu.M and 5 .mu.M, and added to cells
prepared as described above. The 96 well plates containing prepared
cells and test compounds are incubated in the dark, at room
temperature for 0.5 to 6 hours. It is important that the incubation
not continue beyond 6 hours. Just prior to determining the
fluorescence response, 100 .mu.l capsaicin in KRH buffer at twice
the IC.sub.50 concentration determined from the concentration
response curve is automatically added by the FLIPR instrument to
each well of the 96 well plate for a final sample volume of 200
.mu.l and a final capsaicin concentration equal to the IC.sub.50.
The final concentration of test compounds in the assay wells is
between 1 .mu.M and 5 .mu.M. Typically cells exposed to one
IC.sub.50 of capsaicin exhibit a fluorescence response of about
10,000 Relative Fluorescence Units. Antagonists of the capsaicin
receptor decrease this response by at least about 20%, preferably
by at least about 50%, and most preferably by at least 80% as
compared to matched control. The concentration of antagonist
required to provide a 50% decrease is the IC.sub.50 for the
antagonist, and is preferably below 1 micromolar, 100 nanomolar, 10
nanomolar or 1 nanomolar.
[0444] The ability of a compound to act as an agonist of the
capsaicin receptor is determined by measuring the fluorescence
response of cells expressing capsaicin receptors, using the methods
described above, in the absence of capsaicin, RTX, or other
capsaicin receptor agonists. Compounds that cause cells to exhibit
fluorescence above background are capsaicin receptor agonists.
Certain preferred compounds of the present invention are
antagonists that are essentially free of agonist activity as
demonstrated by the absence of detectable agonist activity in such
an assay at compound concentrations below 4 nM, more preferably at
concentrations below 10 .mu.M and most preferably at concentrations
less than or equal to 100 .mu.M.
Example 7
Microsomal In Vitro Half-Life
[0445] This Example illustrates the evaluation of compound
half-life values (t.sub.1/2 values) using a representative liver
microsomal half-life assay.
[0446] Pooled human liver microsomes are obtained from XenoTech
LLC, 3800 Cambridge St., Kansas City, Kans. 66103 (catalog #H0610).
Such liver microsomes may also be obtained from In Vitro
Technologies (Baltimore, Md.) or Tissue Transformation Technologies
(Edison, N.J.). Six test reactions are prepared, each containing 25
.mu.L microsomes, 5 .mu.L of a 100 .mu.M solution of test compound,
and 399 .mu.L 0.1 M phosphate buffer (19 mL 0.1 M
NaH.sub.2PO.sub.4, 81 mL 0.1 M Na.sub.2HPO.sub.4, adjusted to pH
7.4 with H.sub.3PO.sub.4). A seventh reaction is prepared as a
positive control containing 25 .mu.L microsomes, 399 .mu.L 0.1 M
phosphate buffer, and 5 .mu.L of a 100 .mu.M solution of a compound
with known metabolic properties (e.g., DIAZEPAM or CLOZAPINE).
Reactions are preincubated at 39.degree. C. for 10 minutes.
[0447] CoFactor Mixture is prepared by diluting 16.2 mg NADP and
45.4 mg Glucose-6-phosphate in 4 mL 100 mM MgCl.sub.2.
Glucose-6-phosphate dehydrogenase solution is prepared by diluting
214.3 .mu.L glucose-6-phosphate dehydrogenase suspension
(Boehringer-Manheim catalog no. 0737224, distributed by Roche
Molecular Biochemicals, Indianapolis, Ind.) into 1285.7 .mu.L
distilled water. 71 .mu.L Starting Reaction Mixture (3 mL CoFactor
Mixture; 1.2 mL Glucose-6-phosphate dehydrogenase solution) is
added to 5 of the 6 test reactions and to the positive control. 71
.mu.L 100 mM MgCl.sub.2 is added to the sixth test reaction, which
is used as a negative control. At each time point (0, 1, 3, 5, and
10 minutes), 75 .mu.L of each reaction mix is pipetted into a well
of a 96-well deep-well plate containing 75 .mu.L ice-cold
acetonitrile. Samples are vortexed and centrifuged 10 minutes at
3500 rpm (Sorval T 6000D centrifuge, H1000B rotor). 75 .mu.L of
supernatant from each reaction is transferred to a well of a
96-well plate containing 150 .mu.L of a 0.5 .mu.M solution of a
compound with a known LCMS profile (internal standard) per well.
LCMS analysis of each sample is carried out and the amount of
unmetabolized test compound is measured as AUC, compound
concentration vs. time is plotted, and the t.sub.1/2 value of the
test compound is extrapolated.
[0448] Preferred compounds of the present invention exhibit in
vitro t.sub.1/2 values of greater than 10 minutes and less than 4
hours, preferably between 30 minutes and 1 hour, in human liver
microsomes.
Example 8
MDCK Toxicity Assay
[0449] This Example illustrates the evaluation of compound toxicity
using a Madin Darby canine kidney (MDCK) cell cytotoxicity
assay.
[0450] 1 .mu.L of test compound is added to each well of a clear
bottom 96-well plate (PACKARD, Meriden, Conn.) to give final
concentration of compound in the assay of 10 micromolar, 100
micromolar or 200 micromolar. Solvent without test compound is
added to control wells.
[0451] MDCK cells, ATCC no. CCL-34 (American Type Culture
Collection, Manassas, Va.), are maintained in sterile conditions
following the instructions in the ATCC production information
sheet. Confluent MDCK cells are trypsinized, harvested, and diluted
to a concentration of 0.1.times.10.sup.6 cells/ml with warm
(37.degree. C.) medium (VITACELL Minimum Essential Medium Eagle,
ATCC catalog #30-2003). 100 .mu.L of diluted cells is added to each
well, except for five standard curve control wells that contain 100
.mu.L of warm medium without cells. The plate is then incubated at
37.degree. C. under 95% O.sub.2, 5% CO.sub.2 for 2 hours with
constant shaking. After incubation, 50 .mu.L of mammalian cell
lysis solution is added per well, the wells are covered with
PACKARD TOPSEAL stickers, and plates are shaken at approximately
700 rpm on a suitable shaker for 2 minutes.
[0452] Compounds causing toxicity will decrease ATP production,
relative to untreated cells. The PACKARD, (Meriden, Conn.)
ATP-LITE-M Luminescent ATP detection kit, product no. 6016941, is
generally used according to the manufacturer's instructions to
measure ATP production in treated and untreated MDCK cells. PACKARD
ATP LITE-M reagents are allowed to equilibrate to room temperature.
Once equilibrated, the lyophilized substrate solution is
reconstituted in 5.5 mls of substrate buffer solution (from kit).
Lyophilized ATP standard solution is reconstituted in deionized
water to give a 10 mM stock. For the five control wells, 10 .mu.L
of serially diluted PACKARD standard is added to each of the
standard curve control wells to yield a final concentration in each
subsequent well of 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM.
PACKARD substrate solution (50 .mu.L) is added to all wells, which
are then covered, and the plates are shaken at approximately 700
rpm on a suitable shaker for 2 minutes. A white PACKARD sticker is
attached to the bottom of each plate and samples are dark adapted
by wrapping plates in foil and placing in the dark for 10 minutes.
Luminescence is then measured at 22.degree. C. using a luminescence
counter (e.g., PACKARD TOPCOUNT Microplate Scintillation and
Luminescence Counter or TECAN SPECTRAFLUOR PLUS), and ATP levels
calculated from the standard curve. ATP levels in cells treated
with test compound(s) are compared to the levels determined for
untreated cells. Cells treated with 10 .mu.M of a preferred test
compound exhibit ATP levels that are at least 80%, preferably at
least 90%, of the untreated cells. When a 100 .mu.M concentration
of the test compound is used, cells treated with preferred test
compounds exhibit ATP levels that are at least 50%, preferably at
least 80%, of the ATP levels detected in untreated cells.
Example 9
Dorsal Root Ganglion Cell Assay
[0453] This Example illustrates a representative dorsal root
ganglian cell assay for evaluating VR1 antagonist activity of a
compound.
[0454] DRG are dissected from neonatal rats, dissociated and
cultured using standard methods (Aguayo and White (1992) Brain
Research 570:61-67). After 48 hour incubation, cells are washed
once and incubated for 30-60 minutes with the calcium sensitive dye
Fluo 4 AM (2.5-10 ug/ml; TefLabs, Austin, Tex.). Cells are then
washed once, and various concentrations of compound is added to the
cells. Addition of capsaicin to the cells results in a
VR1-dependent increase in intracellular calcium levels which is
monitored by a change in Fluo-4 fluorescence with a fluorometer.
Data are collected for 60-180 seconds to determine the maximum
fluorescent signal. Fluorescent signal is then plotted as a
function of compound concentration to identify the concentration
required to achieve a 50% inhibition of the capsaicin-activated
response, or IC.sub.50. Antagonists of the capsaicin receptor
preferably have an IC.sub.50 below 1 micromolar, 100 nanomolar, 10
nanomolar or 1 nanomolar.
Example 10
Animal Models for Determining Pain Relief
[0455] This Example illustrates representative methods for
assessing the degree of pain relief provided by a compound.
A. Pain Relief Testing
[0456] The following methods may be used to assess pain relief.
Mechanical Allodynia
[0457] Mechanical allodynia (an abnormal response to an innocuous
stimulus) is assessed essentially as described by Chaplan et al.
(1994) J. Neurosci. Methods 53:55-63 and Tal and Eliav (1998) Pain
64(3):511-518. A series of von Frey filaments of varying rigidity
(typically 8-14 filaments in a series) are applied to the plantar
surface of the hind paw with just enough force to bend the
filament. The filaments are held in this position for no more than
three seconds or until a positive allodynic response is displayed
by the rat. A positive allodynic response consists of lifting the
affected paw followed immediately by licking or shaking of the paw.
The order and frequency with which the individual filaments are
applied are determined by using Dixon up-down method. Testing is
initiated with the middle hair of the series with subsequent
filaments being applied in consecutive fashion, ascending or
descending, depending on whether a negative or positive response,
respectively, is obtained with the initial filament.
[0458] Compounds are effective in reversing or preventing
mechanical allodynia-like symptoms if rats treated with such
compounds require stimulation with a Von Frey filament of higher
rigidity strength to provoke a positive allodynic response as
compared to control untreated or vehicle treated rats.
Alternatively, or in addition, testing of an animal in chronic pain
may be done before and after compound administration. In such an
assay, an effective compound results in an increase in the rigidity
of the filament needed to induce a response after treatment, as
compared to the filament that induces a response before treatment
or in an animal that is also in chronic pain but is left untreated
or is treated with vehicle. Test compounds are administered before
or after onset of pain. When a test compound is administered after
pain onset, testing is performed 10 minutes to three hours after
administration.
Mechanical Hyperalgesia
[0459] Mechanical hyperalgesia (an exaggerated response to painful
stimulus) is tested essentially as described by Koch et al. (1996)
Analgesia 2(3):157-164. Rats are placed in individual compartments
of a cage with a warmed, perforated metal floor. Hind paw
withdrawal duration (i.e., the amount of time for which the animal
holds its paw up before placing it back on the floor) is measured
after a mild pinprick to the plantar surface of either hind
paw.
[0460] Compounds produce a reduction in mechanical hyperalgesia if
there is a statistically significant decrease in the duration of
hindpaw withdrawal. Test compound may be administered before or
after onset of pain. For compounds administered after pain onset,
testing is performed 10 minutes to three hours after
administration.
Thermal Hyperalgesia
[0461] Thermal hyperalgesia (an exaggerated response to noxious
thermal stimulus) is measured essentially as described by
Hargreaves et al. (1988) Pain. 32(1):77-88. Briefly, a constant
radiant heat source is applied the animals' plantar surface of
either hind paw. The time to withdrawal (i.e., the amount of time
that heat is applied before the animal moves its paw), otherwise
described as thermal threshold or latency, determines the animal's
hind paw sensitivity to heat.
[0462] Compounds produce a reduction in thermal hyperalgesia if
there is a statistically significant increase in the time to
hindpaw withdrawal (i.e., the thermal threshold to response or
latency is increased). Test compound may be administered before or
after onset of pain. For compounds administered after pain onset,
testing is performed 10 minutes to three hours after
administration.
B. Pain Models
[0463] Pain may be induced using any of the following methods, to
allow testing of analgesic efficacy of a compound. In general,
compounds provided herein result in a statistically significant
reduction in pain as determined by at least one of the previously
described testing methods, using male SD rats and at least one of
the following models.
Acute Inflammatory Pain Model
[0464] Acute inflammatory pain is induced using the carrageenan
model essentially as described by Field et al. (1997) Br. J.
Pharmacol. 121(8):1513-1522. 100-200 .mu.l of 1-2% carrageenan
solution is injected into the rats' hind paw. Three to four hours
following injection, the animals' sensitivity to thermal and
mechanical stimuli is tested using the methods described above. A
test compound (0.01 to 50 mg/kg) is administered to the animal,
prior to testing, or prior to injection of carrageenan. The
compound can be administered orally or through any parenteral
route, or topically on the paw. Compounds that relieve pain in this
model result in a statistically significant reduction in mechanical
allodynia and/or thermal hyperalgesia.
Chronic Inflammatory Pain Model
[0465] Chronic inflammatory pain is induced using one of the
following protocols: [0466] 1. Essentially as described by
Bertorelli et al. (1999) Br. J. Pharmacol. 128(6):1252-1258, and
Stein et al. (1998) Pharmacol. Biochem. Behav. 31(2):455-51, 200
.mu.L Complete Freund's Adjuvant (0.1 mg heat killed and dried M.
Tuberculosis) is injected to the rats' hind paw: 100 .mu.L into the
dorsal surface and 100 .mu.L into the plantar surface. [0467] 2.
Essentially as described by Abbadie et al. (1994) J. Neurosci.
14(10):5865-5871 rats are injected with 150 .mu.L of CFA (1.5 mg)
in the tibio-tarsal joint.
[0468] Prior to injection with CFA in either protocol, an
individual baseline sensitivity to mechanical and thermal
stimulation of the animals' hind paws is obtained for each
experimental animal.
[0469] Following injection of CFA, rats are tested for thermal
hyperalgesia, mechanical allodynia and mechanical hyperalgesia as
described above. To verify the development of symptoms, rats are
tested on days 5,6, and 7 following CFA injection. On day 7,
animals are treated with a test compound, morphine or vehicle. An
oral dose of morphine of 1-5 mg/kg is suitable as positive control.
Typically, a dose of 0.01-50 mg/kg of test compound is used.
Compounds can be administered as a single bolus prior to testing or
once or twice or three times daily, for several days prior to
testing. Drugs are administered orally or through any parenteral
route, or applied topically to the animal.
[0470] Results are expressed as Percent Maximum Potential Efficacy
(MPE). 0% MPE is defined as analgesic effect of vehicle, 100% MPE
is defined as an animal's return to pre-CFA baseline sensitivity.
Compounds that relieve pain in this model result in a MPE of at
least 30%.
Chronic Neuropathic Pain Model
[0471] Chronic neuropathic pain is induced using the chronic
constriction injury (CCI) to the rat's sciatic nerve essentially as
described by Bennett and Xie (1988) Pain 33:87-107. Rats are
anesthetized (e.g. with an intraperitoneal dose of 50-65 mg/kg
pentobarbital with additional doses administered as needed). The
lateral aspect of each hind limb is shaved and disinfected. Using
aseptic technique, an incision is made on the lateral aspect of the
hind limb at the mid thigh level. The biceps femoris is bluntly
dissected and the sciatic nerve is exposed. On one hind limb of
each animal, four loosely tied ligatures are made around the
sciatic nerve approximately 1-2 mm apart. On the other side the
sciatic nerve is not ligated and is not manipulated. The muscle is
closed with continuous pattern and the skin is closed with wound
clips or sutures. Rats are assessed for mechanical allodynia,
mechanical hyperalgesia and thermal hyperalgesia as described
above.
[0472] Compounds that relieve pain in this model result in a
statistically significant reduction in mechanical allodynia,
mechanical hyperalgesia and/or thermal hyperalgesia when
administered (0.01-50 mg/kg, orally, parenterally or topically)
immediately prior to testing as a single bolus, or for several
days: once or twice or three times daily prior to testing.
* * * * *