U.S. patent application number 14/043861 was filed with the patent office on 2014-02-06 for heteroaryl amide analogues as p2x7 antagonists.
This patent application is currently assigned to H. Lundbeck A/S. The applicant listed for this patent is H. Lundbeck A/S. Invention is credited to Qin GUO, Kevin HODGETTS, David C. IHLE, Jun YUAN.
Application Number | 20140038955 14/043861 |
Document ID | / |
Family ID | 39545100 |
Filed Date | 2014-02-06 |
United States Patent
Application |
20140038955 |
Kind Code |
A1 |
IHLE; David C. ; et
al. |
February 6, 2014 |
HETEROARYL AMIDE ANALOGUES AS P2X7 ANTAGONISTS
Abstract
Heteroaryl amide analogues are provided, of the Formula:
##STR00001## wherein variables are as described herein. 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 such compounds to treat such disorders are provided, as are
methods for using such ligands for receptor localization
studies.
Inventors: |
IHLE; David C.; (Branford,
CT) ; GUO; Qin; (Waterford, CT) ; HODGETTS;
Kevin; (Killingworth, CT) ; YUAN; Jun;
(Guilford, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
H. Lundbeck A/S |
Valby |
|
DK |
|
|
Assignee: |
H. Lundbeck A/S
Valby
DK
|
Family ID: |
39545100 |
Appl. No.: |
14/043861 |
Filed: |
October 2, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12595394 |
Jun 28, 2010 |
8580812 |
|
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PCT/US2008/004563 |
Apr 9, 2008 |
|
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14043861 |
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60910864 |
Apr 10, 2007 |
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Current U.S.
Class: |
514/233.2 ;
514/249; 514/253.04; 514/255.05; 514/264.1; 514/278; 514/299;
514/300; 544/127; 544/279; 544/350; 544/362; 544/405; 546/112;
546/121; 546/16; 546/19 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
1/00 20180101; C07D 455/02 20130101; A61P 29/00 20180101; A61P
19/02 20180101; A61P 43/00 20180101; A61P 25/00 20180101; A61P
25/28 20180101; C07D 471/10 20130101; A61P 17/02 20180101; A61P
25/04 20180101; A61P 11/00 20180101; A61P 25/18 20180101; A61P
25/24 20180101; A61P 27/16 20180101; A61P 25/20 20180101; A61P
25/22 20180101; C07D 498/10 20130101; C07D 519/00 20130101; A61P
19/00 20180101; A61P 19/06 20180101; A61P 27/02 20180101; A61P
35/00 20180101; A61P 1/02 20180101; A61P 37/02 20180101; C07D
471/04 20130101; A61P 27/06 20180101; A61P 11/06 20180101 |
Class at
Publication: |
514/233.2 ;
514/249; 514/253.04; 514/255.05; 514/264.1; 514/278; 514/299;
514/300; 544/127; 544/279; 544/350; 544/362; 544/405; 546/16;
546/19; 546/112; 546/121 |
International
Class: |
C07D 471/04 20060101
C07D471/04; C07D 455/02 20060101 C07D455/02; C07D 471/10 20060101
C07D471/10; C07D 498/10 20060101 C07D498/10 |
Claims
1. A compound of the formula: ##STR00175## or a pharmaceutically
acceptable salt thereof, wherein: T, U and V are independently
chosen from CR.sub.3, CR.sub.A and N, such that (i) exactly one of
T, U and V is CR; and (ii) if X is CH.sub.2, Y is phenyl, and U and
V are both substituted carbon, then T is not N; W is
--C(.dbd.O)NR.sub.4--, --NR.sub.4C(.dbd.O)-- or
--NR.sub.4--NR.sub.4--C(.dbd.O)--; X is absent or
C.sub.1-C.sub.6alkylene that is substituted with from 0 to 4
substituents independently chosen from: (i) C.sub.1-C.sub.4alkyl,
(C.sub.3-C.sub.8cycloalkyl)C.sub.0-C.sub.2alkyl and
phenylC.sub.0-C.sub.2alkyl; (ii) substituents that are taken
together to form a 3- to 7-membered cycloalkyl or heterocycloalkyl
ring; and (iii) substituents that are taken together with R.sub.4
to form a 4- to 7-membered heterocycloalkyl; Y is
C.sub.3-C.sub.16cycloalkyl, 4- to 16-membered heterocycloalkyl, 6-
to 16-membered aryl or 5- to 16-membered heteroaryl, each of which
is substituted with from 0 to 6 substituents independently chosen
from hydroxy, halogen, cyano, amino, nitro, oxo, aminocarbonyl,
aminosulfonyl, COOH, C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6aminoalkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy,
C.sub.2-C.sub.6alkyl ether, C.sub.1-C.sub.6alkanoyl,
C.sub.1-C.sub.6alkylsulfonyl,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkanoylamino, mono-
or di-(C.sub.1-C.sub.6alkyl)aminocarbonyl, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl and
(C.sub.1-C.sub.6alkyl)sulfonylamino; Z.sub.1 and Z.sub.3 are
independently N or CR.sub.2; Z.sub.2 is N, CR.sub.2 or CR.sub.A;
Each R.sub.2 and each R.sub.3 is independently chosen from
hydrogen, halogen, cyano, amino, nitro, aminocarbonyl,
aminosulfonyl, COOH, C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6aminoalkyl
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy,
C.sub.1-C.sub.6alkanoyl, C.sub.2-C.sub.6alkyl ether,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminocarbonyl, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl and
(C.sub.1-C.sub.6alkyl)sulfonylamino; Each R.sub.4 is independently
hydrogen, C.sub.1-C.sub.6alkyl,
(C.sub.3-C.sub.8cycloalkyl)C.sub.0-C.sub.2alkyl or taken together
with a substituent of X to form a 4- to 7-membered
heterocycloalkyl; R.sub.A is a group of the formula -L-A-M,
wherein: L is absent or C.sub.1-C.sub.6alkylene that is optionally
modified by the replacement of a carbon-carbon single bond with a
double or triple carbon-carbon bond, which alkylene is optionally
substituted with oxo, --COOH, --SO.sub.2N, --PO.sub.3H.sub.2,
tetrazole or oxadizaolone; A is absent or CO, O, NR.sub.6, S. SO,
SO.sub.2, CONR.sub.6, NR.sub.6CO,
(C.sub.4-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkylene or (4- to
7-membered heterocycloalkyl)C.sub.0-C.sub.4alkylene; wherein
R.sub.6 is hydrogen or C.sub.1-C.sub.6alkyl; and M is: (i) hydroxy,
cyano, amino, aminocarbonyl, aminosulfonyl or COOH; or (ii)
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy, 5- to 10-membered
carbocycle, 4- to 10-membered heterocycle,
C.sub.1-C.sub.6alkanoyloxy, C.sub.1-C.sub.6alkanoylamino,
C.sub.1-C.sub.6alkylsulfonyl, C.sub.1-C.sub.6alkylsulfonylamino,
C.sub.1-C.sub.6alkylsulfonyloxy, mono- or
di-C.sub.1-C.sub.6alkylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl, or mono- or
di-(C.sub.1-C.sub.6alkyl)aminocarbonyl; each of which is optionally
substituted and each of which is preferably substituted with from 0
to 4 substituents independently chosen from oxo, amino, halogen,
hydroxy, cyano, aminocarbonyl, aminosulfonyl, COOH,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6haloalkoxy, C.sub.2-C.sub.6alkyl ether,
C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl, mono- or
di-(C.sub.1-C.sub.6alkylamino)carbonyl, and 4- to 7-membered
heterocycle.
2. A compound or salt thereof according to claim 1, wherein:
##STR00176##
3. A compound or sail thereof according to claim 2, wherein:
##STR00177##
4. A compound or salt thereof according to claim 1, wherein each
R.sub.3 is independently hydrogen or C.sub.1-C.sub.4alkyl.
5. A compound or salt thereof according to claim 1, wherein R.sub.A
is C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6cyanoalkyl,
C.sub.2-C.sub.6alkenyl, C.sub.2-C.sub.6alkyl ether,
phenylC.sub.0-C.sub.4alkyl, (4- to 10-membered
heterocycle)C.sub.0-C.sub.4alkyl,
(C.sub.1-C.sub.6alkylsulfonylamino)C.sub.0-C.sub.4alkyl,
(C.sub.1-C.sub.6alkanoyloxy)C.sub.0-C.sub.4alkyl,
(C.sub.1-C.sub.6alkylsulfonyloxy)C.sub.0-C.sub.4alkyl, (mono- or
di-C.sub.1-C.sub.6alkylamino)C.sub.0-C.sub.4alkyl, and (mono- or
di-C.sub.1-C.sub.6alkylaminocarbonyl)C.sub.0-C.sub.4alkyl; each of
which is substituted with from 0 to 4 substituents independently
chosen from: (i) oxo, halogen, amino, cyano, hydroxy,
aminocarbonyl, aminosulfonyl and COOH; and (ii)
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6alkoxy, C.sub.2-C.sub.6alkyl ether,
C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonylamino, mono- or
di-C.sub.1-C.sub.6alkylaminocarbonyl, mono- or
di-C.sub.1-C.sub.6alkylaminosulfonyl, phenyl and 4- to 7-membered
heterocycle; each of which is substituted with from 0 to 4
substituents independently chosen from halogen, hydroxy, amino,
oxo, aminocarbonyl, aminosulfonyl, COOH, C.sub.1-C.sub.4alkyl and
C.sub.1-C.sub.4haloalkyl.
6. A compound or salt thereof according to claim 5, wherein R.sub.A
is C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6cyanoalkyl,
C.sub.2-C.sub.6cyanoalkenyl, C.sub.2-C.sub.6alkyl ether, (mono- or
di-C.sub.1-C.sub.6alkylamino)C.sub.0-C.sub.4alkyl, (mono- or
di-C.sub.1-C.sub.6alkylaminocarbonyl)C.sub.0-C.sub.4alkyl, or (4-
to 7-membered heterocycle)C.sub.1-C.sub.4alkyl; each of which is
substituted with from 0 to 4 substituents independently chosen from
amino, hydroxy, oxo, halogen, aminocarbonyl, aminosulfonyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6alkoxy,
C.sub.2-C.sub.6alkyl ether, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, mono- or
di-C.sub.1-C.sub.6alkylaminocarbonyl, C.sub.1-C.sub.6alkylsulfonyl;
C.sub.1-C.sub.6alkylsulfonylamino, 4- to 7-membered
heterocycloalkyl, and 5- or 6-membered heteroaryl.
7. A compound or salt thereof according to claim 5, wherein R.sub.A
is a group of the formula: ##STR00178## wherein: L is absent or
C.sub.1-C.sub.6alkylene that is optionally substituted with oxo;
##STR00179## represents a 4- to 7-membered heterocycloalkyl that is
optionally fused to phenyl or to a 5- or 6-membered heteroaryl, and
R.sub.7 represents from 0 to 4 substituents independently chosen
from: (i) hydroxy, amino, oxo, aminocarbonyl, aminosulfonyl and
COOH; (ii) C.sub.1-C.sub.6alkyl, mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.6alkylsulfonylC.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.6alkylsulfonylaminoC.sub.0-C.sub.4alkyl, and 4- to
7-membered heterocycle; each of which is substituted with from 0 to
4 substituents independently chosen from halogen, hydroxy, amino,
oxo, aminocarbonyl, aminosulfonyl, COOK, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy, mono- or di-(C.sub.1-C.sub.6alkyl)amino, and
C.sub.1-C.sub.6alkylsulfonylamino; (iii) substituents that are
taken together to form a bridge, of the Formula
--(CH.sub.2).sub.q--P--(CH.sub.2).sub.r--, wherein q and r are
independently 0 or 1 and P is CH.sub.2, O, NH or 5; and (iv)
substituents that are taken together to form a spiro 4- to
7-membered heterocycloalkyl ring that is substituted with from 0 to
2 substituents independently chosen from oxo and
C.sub.1-C.sub.4alkyl.
8. A compound or salt thereof according to claim 7, wherein R.sub.A
is a group of the formula: ##STR00180## wherein: L is
C.sub.1-C.sub.2alkylene that is optionally substituted with oxo; G
is CHR.sub.8, NH or O; s and t are independently 0, 1, 2, 3 or 4,
such that the sum of s and t ranges from 2 to 5; and R.sub.8 is:
(i) hydrogen, amino, aminocarbonyl, aminosulfonyl or COOH; or
C.sub.1-C.sub.6alkyl, mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.6alkylsulfonylC.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.6alkylsulfonylaminoC.sub.0-C.sub.4alkyl, or 4- to
7-membered heterocycle; each of which is substituted with from 0 to
4 substituents independently chosen from halogen, hydroxy, amino,
oxo, aminocarbonyl, aminosulfonyl, COOH, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy, mono- or di-(C.sub.1-C.sub.6alkyl)amino, and
C.sub.1-C.sub.6alkylsulfonylamino.
9. A compound or salt thereof according to claim 7, wherein R.sub.A
is: ##STR00181## wherein: J is CH or N; B, D, E and F are
independently chosen from, CH.sub.2, NH and O; and R.sub.9
represents from 0 to 2 substituents independently chosen from: (i)
amino, aminocarbonyl and COOH; and (ii) C.sub.1-C.sub.6alkyl, mono-
or di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.2alkyl,
C.sub.1-C.sub.6alkylsulfonyl and C.sub.1-C.sub.6alkylsulfonylamino;
each of which is substituted with from 0 to 3 substituents
independently chosen from halogen, hydroxy, oxo and COOH.
10. A compound or salt thereof according to claim 5, wherein
R.sub.A is C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkyl ether, or mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl, each of which
is substituted with from 1 to 4 substituents independently chosen
hum halogen, hydroxy, cyano, amino, oxo, aminocarbonyl,
aminosulfonyl, COOH, C.sub.1-C.sub.6alkoxy, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkanoylamino,
C.sub.1-C.sub.6alkylsulfonyl, C.sub.1-C.sub.6alkylsulfonyloxy,
C.sub.1-C.sub.6alkylsulfonylamino, phenyl that is optionally
substituted with halogen or C.sub.1-C.sub.4alkyl, and 4- to
7-membered heterocycle that is optionally substituted with
C.sub.1-C.sub.4alkyl.
11. A compound or salt thereof according to claim 10, wherein
R.sub.A is mono-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.2alkyl or
C.sub.2-C.sub.6alkyl ether, each of which is substituted with from
1 to 4 substituents independently chosen from hydroxy, halogen,
oxo, COOH, C.sub.1-C.sub.4alkyl and C.sub.1-C.sub.4alkoxy.
12. A compound or salt thereof according to claim 1, wherein: L is
not absent; A is absent; and M is phenyl or a 5- or 6-membered
heteroaryl, each of which is substituted with from 0 to 4
substituents independently chosen from oxo, amino, halogen,
hydroxy, cyano, aminocarbonyl, aminosulfonyl, COOH,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6haloalkoxy, C.sub.2-C.sub.6alkyl ether,
C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
--C.sub.1-C.sub.6alkysulfonylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl, mono- or
di-(C.sub.1-C.sub.6alkylamino)carbonyl, and 4- to 7-membered
heterocycle.
13. A compound or salt thereof according to claim 1, wherein: L is
C.sub.0-C.sub.3alkylene that is optionally substituted with oxo or
COOH; A is absent; and M is phenyl that is substituted with amino,
cyano, aminocarbonyl, aminosulfonyl, COOH or
C.sub.1-C.sub.6alkyl.
14. A compound or salt thereof according to claim 1, wherein: L is
C.sub.1-C.sub.2alkylene that is optionally substituted with oxo; A
is absent; and M is mono- or di-(C.sub.1-C.sub.6alkyl)amino that is
substituted with a 5- or 6-membered heteroaryl, each of which
heteroaryl is substituted with from 0 to 4 substituents
independently chosen from oxo, amino, halogen, hydroxy, cyano,
aminocarbonyl, aminosulfonyl, COOH, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6hydroxyalkyl, --C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy,
C.sub.2-C.sub.6alkyl ether, C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl, mono- or
di-(C.sub.1-C.sub.6alkylamino)carbonyl, and 4- to 7-membered
heterocycle.
15. A compound or salt thereof according to claim 12 or claim 14,
wherein the 5- or 6-membered heteroaryl is: (i) pyridyl or
pyrimidinyl, each of which is substituted with from 0 to 4
substituents independently chosen from oxo, amino, halogen,
hydroxy, cyano, aminocarbonyl, aminosulfonyl, COOH,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6alkoxy, C.sub.2-C.sub.6alkyl ether,
C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl, mono- or
di-(C.sub.1-C.sub.6alkylamino)carbonyl, and 4- to 7-membered
heterocycle; or (ii) a heteroaryl chosen from: ##STR00182## each of
which is substituted with from 0 to 2 substituents independently
chosen from amino, halogen, hydroxy, cyano, aminocarbonyl,
aminosulfonyl, COOH, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6alkoxy,
C.sub.2-C.sub.6alkyl ether, C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl, mono- or
di-(C.sub.1-C.sub.6alkylamino)carbonyl, and 4- to 7-membered
heterocycle.
16. A compound or salt thereof according to claim 1, wherein each
R.sub.2 is hydrogen or C.sub.1-C.sub.6alkyl.
17. A pharmaceutical composition, comprising at least one compound
or salt thereof according to claim 1 in combination with a
physiologically acceptable carrier or excipient.
18. A method for treating a condition responsive to P2X.sub.7
receptor modulation in a patient, comprising administering to the
patient a therapeutically effective amount of at least one compound
or salt thereof according to claim 1, and thereby alleviating the
condition in the patient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application patent
application claiming benefit of U.S. Ser. No. 12/595,395, now
allowed, which is a .sctn.371 national stage filing of
PCT/US2008/004563, which claims the benefit of U.S. Provisional
Application No. 60/910,864, filed Apr. 10, 2007, which is
incorporated herein by reference for any matter therein that is
omitted herein.
FIELD OF THE INVENTION
[0002] This invention relates generally to heteroaryl amide
analogues that have useful pharmacological properties. The
invention further relates to the use of such compounds for treating
conditions related to P2X.sub.7 receptor activation, for
identifying other agents that bind to P2X.sub.7 receptor, and as
probes for the detection and localization of P2X.sub.7
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, which typically results from damage to the
nervous system, involves pain signal transmission in the absence of
stimulus, pain from a normally innocuous stimulus (allodynia) and
increased pain from a normally painful stimulus (hyperalgesia). In
most instances, neuropathic pain is thought to occur because of
sensitization in the peripheral and central nervous systems
following initial damage to the peripheral nervous 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 than the initial injury or
disease process that induced it.
[0005] Existing treatments for neuropathic pain are generally
suboptimal. 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, or
to treatment with other drugs, such as gabapentin. 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] Another common condition for which existing therapies are
insufficient or problematic is inflammation. Transient inflammation
is a beneficial mechanism that protects mammals from invading
pathogens. Uncontrolled inflammation, however, causes tissue damage
and pain and is the underlying cause of many illnesses, including
asthma, as well as other allergic, infectious, autoimmune,
degenerative, and idiopathic diseases. Existing treatments often
exhibit low, delayed or only temporary efficacy, undesirable
side-effects and/or a lack of selectivity. There is a continuing
need for new drugs that overcome one or more of the shortcomings of
drugs currently used for immunosuppression or in the treatment or
prevention of inflammatory disorders, including allergic disorders,
autoimmune disorders, fibrogenic disorders, and neurodegenerative
diseases, such as multiple sclerosis, amyotrophic lateral
sclerosis, Alzheimer's disease, and Huntington's disease.
[0007] P2X.sub.7 receptor ("P2X.sub.7") is a ligand-gated ion
channel that is activated by ATP and is present on a variety of
cell types, including microglia in the central nervous system and
cells involved in inflammation and immune system function. In
particular, P2X.sub.7 is involved in activation of lymphocytes and
monocyte/macrophages leading to the increased release of
pro-inflammatory cytokines (e.g., TNFalpha and IL-1 beta) from
these cells. Recent studies indicate that inhibiting P2X.sub.7
activation in situations of inflammation (e.g., rheumatoid
arthritis and other autoimmune diseases, osteoarthritis, uveitis,
asthma, chronic obstructive pulmonary disease and inflammatory
bowel disease) or interstitial fibrosis results in a therapeutic
effect. These and other studies indicate that P2X.sub.7 antagonists
may find use in the treatment and prophylaxis of pain, including
acute, chronic and neuropathic pain, as well as a variety of other
conditions including osteoarthritis, rheumatoid arthritis,
arthrosclerosis, inflammatory bowel disease, Alzheimer's disease,
traumatic brain injury, asthma, chronic obstructive pulmonary
disease, and fibrosis of internal organs (e.g., interstitial
fibrosis).
[0008] Small molecule P2X.sub.7 antagonists are desirable for such
therapies. The present invention fulfills this need, and provides
further related advantages.
SUMMARY OF THE INVENTION
[0009] The present invention provides heteroaryl amide analogues of
Formula I:
##STR00002##
as well as pharmaceutically acceptable salts of such compounds.
Within Formula I:
[0010] T, U and V are independently chosen from CR.sub.3, CR.sub.A
and N; in certain embodiments, exactly one of T, U and V is
CR.sub.A; [0011] W is C(.dbd.O)NR.sub.4--, NR.sub.4C(.dbd.O)-- or
NR.sub.4--NR.sub.4--C(.dbd.O)--; [0012] X is absent or
C.sub.1-C.sub.6alkylene that is substituted with from 0 to 4
substituents independently chosen from: (i) C.sub.1-C.sub.4alkyl,
(C.sub.3-C.sub.8cycloalkyl)C.sub.0-C.sub.2alkyl and
phenylC.sub.0-C.sub.2alkyl; (ii) substituents that are taken
together to form a 3- to 7-membered cycloalkyl or heterocycloalkyl
ring; and (iii) substituents that are taken together with R.sub.4
to form a 4- to 7-membered heterocycloalkyl; [0013] Y is
C.sub.1-C.sub.8alkyl, C.sub.3-C.sub.16cycloalkyl, 4- to 16-membered
heterocycloalkyl, 6- to 16-membered aryl or 5- to 16-membered
heteroaryl, each of which is optionally substituted and each of
which is preferably substituted with from 0 to 6 substituents
independently chosen from hydroxy, halogen, cyano, amino, nitro,
oxo, aminocarbonyl, aminosulfonyl, COOH, C.sub.1-C.sub.6alkyl,
C.sub.2-C.sub.6alkenyl, C.sub.2-C.sub.6alkynyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6aminoalkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6haloalkoxy, C.sub.2-C.sub.6alkyl ether,
C.sub.1-C.sub.6alkanoyl, C.sub.1-C.sub.6alkylsulfonyl,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkanoylamino, mono-
or di-(C.sub.1-C.sub.6alkyl)aminocarbonyl, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl and
(C.sub.1-C.sub.6alkyl)sulfonylamino; [0014] Z.sub.1 and Z.sub.3 are
independently N, CH or substituted carbon (e.g., CR.sub.2); [0015]
Z.sub.2 is N, CH or substituted carbon (e.g., CR.sub.A or
CR.sub.2); [0016] Each R.sub.2 and each R.sub.3 is independently
chosen from hydrogen, halogen, cyano, amino, nitro, aminocarbonyl,
aminosulfonyl, COOH, C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6aminoalkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy,
C.sub.1-C.sub.6alkanoyl, C.sub.2-C.sub.6alkyl ether,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminocarbonyl, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl and
(C.sub.1-C.sub.6alkyl)sulfonylamino; [0017] Each R.sub.4 is
independently hydrogen, C.sub.1-C.sub.6alkyl,
(C.sub.3-C.sub.8cycloalkyl)C.sub.0-C.sub.2alkyl or taken together
with a substituent of X to form a 4- to 7-membered
heterocycloalkyl; and [0018] R.sub.A is a group of the formula
-L-A-M, wherein: [0019] L is absent or C.sub.1-C.sub.6alkylene that
is optionally modified by the replacement of a carbon-carbon single
bond with a double or triple carbon-carbon bond, which alkylene is
optionally substituted with oxo or an acidic moiety such as --COOH,
--SO.sub.3H, --SO.sub.2NH.sub.2, --PO.sub.3H.sub.2, tetrazole or
oxadizaolone; [0020] A is absent or CO, O, NR.sub.6, S, SO,
SO.sub.2, CONR.sub.6, NR.sub.6CO,
(C.sub.4-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkylene or (4- to
7-membered heterocycloalkyl)C.sub.0-C.sub.4alkylene; wherein
R.sub.6 is hydrogen or C.sub.1-C.sub.6alkyl; and [0021] M is:
[0022] (i) hydroxy, cyano, amino, aminocarbonyl, aminosulfonyl or
COOH; or [0023] (ii) C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6alkoxy, 5- to 10-membered carbocycle, 4- to
10-membered heterocycle, C.sub.1-C.sub.6alkanoyloxy,
C.sub.1-C.sub.6alkanoylamino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonylamino, C.sub.1-C.sub.6alkylsulfonyloxy,
mono- or di-C.sub.1-C.sub.6alkylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl, or mono- or
di-(C.sub.1-C.sub.6alkyl)aminocarbonyl; each of which is optionally
substituted and each of which is preferably substituted with from 0
to 4 substituents independently chosen from oxo, amino, halogen,
hydroxy, cyano, aminocarbonyl, aminosulfonyl, COOH,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6haloalkoxy, C.sub.2-C.sub.6alkyl ether,
C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl, mono- or
di-(C.sub.1-C.sub.6alkylamino)carbonyl, and 4- to 7-membered
heterocycle.
[0024] Within certain aspects, Y is C.sub.3-C.sub.16cycloalkyl, 4-
to 16-membered heterocycloalkyl, 6- to 16-membered aryl or 5- to
16-membered heteroaryl, each of which is substituted with from 0 to
6 substituents independently chosen from hydroxy, halogen, cyano,
amino, nitro, oxo, aminocarbonyl, aminosulfonyl, COOH,
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6aminoalkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy,
C.sub.2-C.sub.6alkyl ether, C.sub.1-C.sub.6alkanoyl,
C.sub.1-C.sub.6alkylsulfonyl,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkanoylamino, mono-
or di-(C.sub.1-C.sub.6alkyl)aminocarbonyl, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl and
(C.sub.1-C.sub.6alkyl)sulfonylamino;
[0025] Within certain aspects, heteroaryl amide analogues of
Formula I are P2X.sub.7 antagonists with an IC.sub.50 value no
greater than 20 micromolar, 10 micromolar, 5 micromolar, 1
micromolar, 500 nanomolar or 100 nanomolar in an in vitro assay for
determination of P2X.sub.7 antagonist activity. In certain
embodiments, such P2X.sub.7 antagonists exhibit no detectable
agonist activity in an in vitro assay of P2X.sub.7 activity (i.e.,
in an assay provided in Example 4, herein) at a concentration equal
to the IC.sub.50, 10 times the IC.sub.50 or 100 times the IC.sub.50
and/or at a concentration of 2,500 nM.
[0026] Within certain aspects, heteroaryl amide analogues provided
herein are labeled with a detectable marker (e.g., radiolabeled or
fluorescein conjugated).
[0027] The present invention further provides, within other
aspects, pharmaceutical compositions comprising at least one
heteroaryl amide analogue provided herein in combination with a
physiologically acceptable carrier or excipient.
[0028] Within further aspects, methods are provided for modulating
(e.g., reducing) cellular P2X.sub.7 activation or activity,
comprising contacting a cell (e.g., microglia, astrocyte or
peripheral macrophage or monocyte) that expresses a P2X.sub.7 with
at least one P2X.sub.7 modulator as described herein. Such contact
may occur in vivo or in vitro and is generally performed using a
concentration of P2X.sub.7 modulator that is sufficient to
detectably alter P2X.sub.7 activity in vitro (as determined using
an assay provided in Example 4).
[0029] The present invention further provides methods for treating
a condition responsive to P2X.sub.7 modulation in a patient,
comprising administering to the patient a therapeutically effective
amount of at least one P2X.sub.7 antagonist as described
herein.
[0030] Within other aspects, methods are provided for treating pain
in a patient, comprising administering to a patient suffering from
(or at risk for) pain a therapeutically effective amount of at
least one P2X.sub.7 antagonist as described herein.
[0031] Within other aspects, methods are provided for treating
inflammation in a patient, comprising administering to a patient
suffering from (or at risk for) inflammation a therapeutically
effective amount of at least one P2X.sub.7 antagonist as described
herein.
[0032] Methods are further provided for treating osteoarthritis,
rheumatoid arthritis, arthrosclerosis, inflammatory bowel disease,
Alzheimer's disease, traumatic brain injury, asthma, chronic
obstructive pulmonary disease, ocular conditions (e.g., glaucoma),
cirrhosis, lupus, scleroderma, or fibrosis of internal organs
(e.g., interstitial fibrosis) in a patient, comprising
administering to a patient suffering from (or at risk for) one or
more of the foregoing conditions a therapeutically effective amount
of at least one P2X.sub.7 antagonist as described herein.
[0033] Within still further aspects, the present invention provides
methods for inhibiting death of retinal ganglion cells in a
patient, comprising administering to the patient a therapeutically
effective amount of at least one P2X.sub.7 antagonist as described
herein.
[0034] Methods are further provided for identifying an agent that
binds to P2X.sub.7, comprising: (a) contacting P2X.sub.7 with a
labeled compound that is a heteroaryl amide analogue as described
herein under conditions that permit binding of the compound to
P2X.sub.7, thereby generating bound, labeled compound; (b)
detecting a signal that corresponds to the amount of bound, labeled
compound in the absence of test agent; (c) contacting the bound,
labeled compound with a test agent; (d) detecting a signal that
corresponds to the amount of bound labeled compound 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).
[0035] Within further aspects, the present invention provides
methods for determining the presence or absence of P2X.sub.7 in a
sample, comprising: (a) contacting a sample with a compound as
described herein under conditions that permit modulation by the
compound of P2X.sub.7 activity; and (b) detecting a signal
indicative of a level of the compound modulating P2X.sub.7
activity.
[0036] 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 (i) treat one or more conditions responsive to
P2X.sub.7 modulation, such as pain, osteoarthritis, rheumatoid
arthritis, arthrosclerosis, inflammatory bowel disease, Alzheimer's
disease, traumatic brain injury, asthma, chronic obstructive
pulmonary disease, ocular conditions (e.g., glaucoma), cirrhosis,
lupus, scleroderma, and/or fibrosis of internal organs (e.g.,
interstitial fibrosis) or (ii) provide retinal neuroprotection
(e.g., inhibit death of retinal ganglion cells).
[0037] In yet another aspect, the present invention provides
methods for preparing the compounds disclosed herein, including the
intermediates.
[0038] These and other aspects of the invention will become
apparent upon reference to the following detailed description.
DETAILED DESCRIPTION
[0039] As noted above, the present invention provides heteroaryl
amide analogues. Such compounds may be used in vitro or in vivo, to
modulate P2X.sub.7 activity in a variety of contexts.
Terminology
[0040] 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.1, A, 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.
[0041] The phrase "heteroaryl amide analogue," as used herein,
encompasses all compounds of Formula I, as well as compounds of
other Formulas provided herein (including any enantiomers,
racemates and stereoisomers) and pharmaceutically acceptable salts
thereof. In certain embodiments, substituted pyrimidinones provided
herein are isolated so as to be substantially free of residual
organic solvent (i.e., any such solvent in the preparation is
present in an amount that is at or below the limit set for that
solvent by the International Council on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human Use
(ICH)).
[0042] A "pharmaceutically acceptable salt" of a compound recited
herein is an acid or base salt that is suitable for use in contact
with the tissues of human beings or animals without excessive
toxicity or carcinogenicity, and preferably without 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 pharmaceutically acceptable anions
for use in salt formation include, but are not limited to, acetate,
2-acetoxybenzoate, ascorbate, benzoate, bicarbonate, bromide,
calcium edetate, carbonate, chloride, citrate, dihydrochloride,
diphosphate, ditartrate, edetate, estolate (ethylsuccinate),
formate, fumarate, gluceptate, gluconate, glutamate, glycolate,
glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,
hydrochloride, hydroiodide, hydroxymaleate, hydroxynaphthoate,
iodide, isethionate, lactate, lactobionate, malate, maleate,
mandelate, methylbromide, methylnitrate, methylsulfate, mucate,
napsylate, nitrate, pamoate, pantothenate, phenylacetate,
phosphate, polygalacturonate, propionate, salicylate, stearate,
subacetate, succinate, sulfamate, sulfanilate, sulfate, sulfonates
including besylate (benzenesulfonate), camsylate
(camphorsulfonate), edisylate (ethane-1,2-disulfonate), esylate
(ethanesulfonate) 2-hydroxyethylsulfonate,
mesylate(methanesulfonate), triflate (trifluoromethanesulfonate)
and tosylate (p-toluenesulfonate), tannate, tartrate, teoclate and
triethiodide. Similarly, pharmaceutically acceptable cations for
use in salt formation include, but are not limited to ammonium,
benzathine, chloroprocaine, choline, diethanolamine,
ethylenediamine, meglumine, procaine, and metals such as aluminum,
calcium, lithium, magnesium, potassium, sodium and zinc. Those of
ordinary skill in the art will recognize further pharmaceutically
acceptable salts for the compounds provided herein. 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, methanol, isopropanol or acetonitrile, is
preferred.
[0043] It will be apparent that compounds and salts thereof
provided herein may, but need not, be formulated as a hydrate, and
that such hydrates are encompassed by the formulas, names and
structures recited herein. In addition, the various non-hydrate
solvates, non-covalent complexes, crystal forms and polymorphs of
the compounds provided herein are within the scope of the present
invention. Also provided herein are prodrugs of the compounds of
the recited Formulas. 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 a formula provided herein. For
example, a prodrug may be an acylated derivative of such a
compound. 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, benzoate and peptide
derivatives of alcohol and amine functional groups within a
compound provided herein. Prodrugs may generally be prepared by
modifying functional groups present in the compounds in such a way
that the modifications are cleaved in vivo to yield the parent
compounds.
[0044] As used herein, the term "alkyl" refers to a straight or
branched chain 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 and 3-methylpentyl.
"C.sub.0-C.sub.nalkyl" refers to a single covalent bond (C.sub.0)
or an alkyl group having from 1 to n carbon atoms; for example
"C.sub.0-C.sub.4alkyl" refers to a single covalent bond or a
C.sub.1-C.sub.4alkyl group. In some instances, a substituent of an
alkyl group is specifically indicated. For example,
"C.sub.1-C.sub.6hydroxyalkyl" is a C.sub.1-C.sub.6alkyl group
substituted with at least one --OH; "C.sub.1-C.sub.6aminoalkyl" is
a C.sub.1-C.sub.6alkyl group substituted with at least one
NH.sub.2; C.sub.1-C.sub.6cyanoalkyl is a C.sub.1-C.sub.6alkyl group
substituted with at least one --CN.
[0045] "Alkenyl" refers to straight or branched chain alkene
groups, which comprise at least one unsaturated carbon-carbon
double bond. 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. "C.sub.2-C.sub.6cyanoalkenyl" is
a C.sub.2-C.sub.6alkenyl group substituted with at least one
--CN.
[0046] "Alkynyl" refers to straight or branched chain 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.
[0047] "Alkylene" refers to a divalent alkyl group, as defined
above. C.sub.1-C.sub.2alkylene is methylene or ethylene;
C.sub.0-C.sub.4alkylene is a single covalent bond or an alkylene
group having 1, 2, 3 or carbon atoms; C.sub.0-C.sub.2alkylene is a
single covalent bond, methylene or ethylene. A
"C.sub.1-C.sub.6alkylene that is optionally modified by the
replacement of a carbon-carbon single bond with a double or triple
carbon-carbon bond" is a C.sub.1-C.sub.6alkylene group as described
above, or a divalent C.sub.2-C.sub.6alkene or
C.sub.2-C.sub.6alkyne.
[0048] A "cycloalkyl" is a group that comprises one or more
saturated and/or partially saturated rings in which all ring
members are carbon, such as cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, myrtanyl and
partially saturated variants of the foregoing, such as
cyclohexenyl. Cycloalkyl groups do not comprise an aromatic ring or
a heterocyclic ring. Certain cycloalkyl groups are
C.sub.3-C.sub.7cycloalkyl, in which the cycloalkyl group contains a
single ring having from 3 to 7 ring members, all of which are
carbon. A "(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl" is a
C.sub.3-C.sub.7cycloalkyl group linked via a single covalent bond
or a C.sub.1-C.sub.4alkylene group.
[0049] A "(C.sub.4-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkylene" is a
divalent (C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl group that
is linked via two single covalent bonds to two specified moieties.
In general, one single covalent bond is located on the cyclic
portion and the other is located on the alkylene portion, if
present; alternatively, if no alkylene group is present, both
single covalent bonds are on different ring members. For example,
with respect to the group R.sub.A, if A is
(C.sub.6cycloalkyl)C.sub.2alkylene and M is COOH, one R.sub.A
moiety so formed is:
##STR00003##
[0050] 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 from 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
representative alkoxy groups.
[0051] The term "oxo" is used herein to refer to an oxygen
substituent of a carbon atom that results in the formation of a
carbonyl group (C.dbd.O). An oxo group that is a substituent of a
nonaromatic carbon atom results in a conversion of --CH.sub.2-- to
--C(.dbd.O)--. An oxo group that is a substituent of an aromatic
carbon atom results in a conversion of --CH-- to --C(.dbd.O)-- and
may result in a loss of aromaticity.
[0052] The term "alkanoyl" refers to an acyl group (e.g.,
--(C.dbd.O)-alkyl), in which carbon atoms are in a linear or
branched alkyl arrangement and where attachment is through the
carbon of the keto group. Alkanoyl groups have the indicated number
of carbon atoms, with the carbon of the keto group being included
in the numbered carbon atoms. For example a C.sub.2alkanoyl group
is --(C.dbd.O)CH.sub.3. Alkanoyl groups include, for example,
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, from 2 to 6
or from 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."
[0053] "Alkyl ether" refers to a linear or branched ether
substituent (i.e., an alkyl group that is substituted with an
alkoxy group). 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. A
C.sub.2 alkyl ether substituent is --CH.sub.2--O--CH.sub.3.
[0054] The term "alkoxycarbonyl" refers to an alkoxy group attached
through a keto (--(C.dbd.O)--) bridge (i.e., a group having the
general structure --C(.dbd.O)--O-alkyl). Alkoxycarbonyl groups
include C.sub.1-C.sub.8, C.sub.1-C.sub.6 and
C.sub.1-C.sub.4alkoxycarbonyl groups, which have from 1 to 8, 6 or
4 carbon atoms, respectively, in the alkyl portion of the group
(i.e., the carbon of the keto bridge is not included in the
indicated number of carbon atoms). "C.sub.1alkoxycarbonyl" refers
to --C(.dbd.O)--O--CH.sub.3; C.sub.3alkoxycarbonyl indicates
C(.dbd.O)--O--(CH.sub.2).sub.2CH.sub.3 or
--C(.dbd.O)--O--(CH)(CH.sub.3).sub.2.
[0055] "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.1-C.sub.8, C.sub.1-C.sub.6 and C.sub.1-C.sub.4alkanoyloxy
groups, which have from 1 to 8, 6 or 4 carbon atoms, respectively,
in the alkyl portion of the group. For example,
"C.sub.1alkanoyloxy" refers to O--C(.dbd.O)--CH.sub.3.
[0056] Similarly, "alkanoylamino," as used herein, refers to an
alkanoyl group linked via a nitrogen bridge (i.e., a group having
the general structure --N(R)--C(.dbd.O)-alkyl), in which R is
hydrogen or C.sub.1-C.sub.6alkyl. Alkanoylamino groups include
C.sub.1-C.sub.8, C.sub.1-C.sub.6 and C.sub.1-C.sub.4alkanoylamino
groups, which have from 1 to 8, 6 or 4 carbon atoms within the
alkanoyl group, respectively, in the alkyl portion of the
group.
[0057] "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 from 1 to 4 carbon atoms,
respectively. Methylsulfonyl is one representative alkylsulfonyl
group. "C.sub.1-C.sub.4haloalkylsulfonyl" is an alkylsulfonyl group
that has from 1 to 4 carbon atoms and is substituted with at least
one halogen (e.g., trifluoromethylsulfonyl).
[0058] "Alkylsulfonylamino" refers to groups of the formula
N(R)--(SO.sub.2)-alkyl, in which R is hydrogen or
C.sub.1-C.sub.6alkyl and the nitrogen atom is the point of
attachment. Alkylsulfonylamino groups include
C.sub.1-C.sub.6alkylsulfonylamino and
C.sub.1-C.sub.4alkylsulfonylamino groups, which have from 1 to 6 or
1 to 4 carbon atoms, respectively. Methylsulfonylamino is a
representative alkylsulfonylamino group.
"C.sub.1-C.sub.6haloalkylsulfonylamino" is an alkylsulfonylamino
group that has from 1 to 6 carbon atoms and is substituted with at
least one halogen (e.g., trifluoromethylsulfonylamino).
[0059] "Aminosulfonyl" refers to groups of the formula
--(SO.sub.2)--NH.sub.2, in which the sulfur atom is the point of
attachment. The term "mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl" refers to groups that
satisfy the formula --(SO.sub.2)--NR.sub.2, in which the sulfur
atom is the point of attachment, and in which one R is
C.sub.1-C.sub.6alkyl and the other R is hydrogen or an
independently chosen C.sub.1-C.sub.6alkyl.
[0060] "Alkylaminoalkyl" refers to an alkylamino group linked via
an alkylene group (i.e., a group having the general structure
alkyleneNHalkyl or alkylene-N(alkyl)(alkyl)) in which each alkyl is
selected independently from alkyl, cycloalkyl and (cycloalkyl)alkyl
groups. Alkylaminoalkyl 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.6alkyl)aminoC.sub.1-C.sub.4alkyl. "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 single
covalent bond or a C.sub.1-C.sub.6alkylene group. The following are
representative alkylaminoalkyl groups:
##STR00004##
It will be apparent that the definition of "alkyl" as used in the
terms "alkylamino" and "alkylaminoalkyl" differs from the
definition of "alkyl" used for all other alkyl-containing groups,
in the inclusion of cycloalkyl and (cycloalkyl)alkyl groups (e.g.,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.6alkyl).
[0061] The term "aminocarbonyl" refers to an amide group (i.e.,
--(C.dbd.O)NH.sub.2). "Mono- or
di-(C.sub.1-C.sub.6alkyl)aminocarbonyl" refers to groups of the
formula --(C.dbd.O)--N(R).sub.2, in which the carbonyl is the point
of attachment, one R is C.sub.1-C.sub.6alkyl and the other R is
hydrogen or an independently chosen C.sub.1-C.sub.6alkyl.
[0062] "Mono- or
di-(C.sub.1-C.sub.6alkyl)aminocarbonylC.sub.0-C.sub.4alkyl" is an
aminocarbonyl group in which one or both of the hydrogen atoms is
replaced with C.sub.1-C.sub.6alkyl, and which is linked via a
single covalent bond (i.e., mono- or
di-(C.sub.1-C.sub.6alkyl)aminocarbonyl) or a
C.sub.1-C.sub.4alkylene group (i.e.,
--(C.sub.0-C.sub.4alkyl)---(C.dbd.O)--N(C.sub.1-C.sub.6alkyl).sub.2).
If both hydrogen atoms are so replaced, the C.sub.1-C.sub.6alkyl
groups may be the same or different.
[0063] The term "aminosulfonyl" refers to a sulfonamide group
(i.e., --(SO.sub.2)NH.sub.2). "Mono- or
di-(C.sub.1-C.sub.8alkyl)aminosulfonyl" refers to groups of the
formula --(SO.sub.2)--N(R).sub.2, in which the sulfur atom is the
point of attachment, one R is C.sub.1-C.sub.8alkyl and the other R
is hydrogen or an independently chosen C.sub.1-C.sub.8alkyl.
[0064] "Mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonylC.sub.0-C.sub.4alkyl" is an
aminosulfonyl group in which one or both of the hydrogen atoms is
replaced with C.sub.1-C.sub.6alkyl, and which is linked via a
single covalent bond (i.e., mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl) or a
C.sub.1-C.sub.4alkylene group (i.e.,
--(C.sub.1-C.sub.4alkyl)-(SO.sub.2)N(C.sub.1-C.sub.6alkyl).sub.2).
If both hydrogen atoms are so replaced, the C.sub.1-C.sub.6alkyl
groups may be the same or different.
[0065] The term "halogen" refers to fluorine, chlorine, bromine or
iodine.
[0066] A "haloalkyl" is an alkyl group that is substituted with 1
or more independently chosen halogens (e.g.,
"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 that is
linked via an oxygen bridge.
[0067] 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.
[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 heterocycle. Unless
otherwise specified, each ring within a carbocycle may be
independently saturated, partially saturated or aromatic, and is
optionally substituted as indicated. A carbocycle generally has
from 1 to 3 fused, pendant or spiro rings and optionally further
contains one or more alkylene bridges; 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 16 ring members. Certain representative
carbocycles are cycloalkyl as described above (e.g., cyclohexyl,
cycloheptyl or adamantly). Other carbocycles are aryl (i.e.,
contain at least one aromatic carbocyclic ring, with or without one
or more additional aromatic and/or cycloalkyl rings). Such aryl
carbocycles include, for example, phenyl, naphthyl (e.g.,
1-naphthyl and 2-naphthyl), fluorenyl, indanyl and
1,2,3,4-tetrahydronaphthyl.
[0069] Certain carbocycles recited herein are
C.sub.6-C.sub.10arylC.sub.0-C.sub.8alkyl groups (i.e., groups in
which a 6- to 10-membered carbocyclic group comprising at least one
aromatic ring is linked via a single covalent bond or a
C.sub.1-C.sub.8alkylene group). Phenyl groups linked via a single
covalent bond or C.sub.1-C.sub.2alkylene group are designated
phenylC.sub.0-C.sub.2alkyl (e.g., benzyl, 1-phenyl-ethyl and
2-phenyl-ethyl).
[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
independently chosen from O, S and N, with the remaining ring atoms
being carbon). Additional rings, if present, may be heterocyclic or
carbocyclic. 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. Unless otherwise specified, a heterocycle may be
a heterocycloalkyl group (i.e., each ring is saturated or partially
saturated), such as a 4- to 7-membered heterocycloalkyl, which
generally comprises 1, 2, 3 or 4 ring atoms that are independently
chosen from C, O, N and S; or a heteroaryl group (i.e., at least
one ring within the group is aromatic), such as a 5- to 10-membered
heteroaryl (which may be monocyclic or bicyclic) or a 6-membered
heteroaryl (e.g., pyridyl or pyrimidyl). N-linked heterocyclic
groups are linked via a component nitrogen atom.
[0071] A "heterocycleC.sub.0-C.sub.4alkyl" is a heterocyclic group
linked via a single covalent bond or C.sub.1-C.sub.4alkylene group.
A "(4- to 7-membered heterocycloalkyl)C.sub.1-C.sub.4alkyl" is a
heterocycloalkyl ring with from 4 to 7 ring members that is linked
via a C.sub.1-C.sub.4alkylene group.
[0072] A "(4- to 7-membered
heterocycloalkyl)C.sub.0-C.sub.4alkylene" is a divalent (4- to
7-membered heterocycloalkyl)C.sub.0-C.sub.4alkyl group that is
linked via two single covalent bonds to two specified moieties. In
general, one such single covalent bond is located on the cyclic
portion and the other is located on the alkylene portion, if
present; alternatively, if no alkylene group is present, both such
single covalent bonds are located on different ring members. For
example, with respect to the group R.sub.A, if A is a
(piperidinyl)C.sub.2alkylene and M is COOH, one R.sub.A moiety so
formed is:
##STR00005##
[0073] 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 that is
covalently bonded to an atom (preferably a carbon or nitrogen atom)
that is a ring member. Substituents of aromatic groups are
generally covalently bonded to a ring carbon atom. The term
"substitution" refers to replacing a hydrogen atom in a molecular
structure with a substituent, 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.
[0074] 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). 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 substituents). Other optionally substituted groups are
substituted with at least one substituent (e.g., substituted with
from 1 to 2, 3 or 4 independently selected substituents).
[0075] The term "P2X.sub.7" hereinbelow refers to any P2X.sub.7
receptor, preferably a mammalian receptor such as the human or rat
P2X.sub.7 receptors disclosed in U.S. Pat. No. 6,133,434, as well
as homologues thereof found in other species.
[0076] A "P2X.sub.7 modulator," also referred to herein as a
"modulator," is a compound that modulates P2X.sub.7 activation
and/or P2X.sub.7-mediated activity (e.g., signal transduction).
P2X.sub.7 modulators specifically provided herein are compounds of
Formula I and pharmaceutically acceptable salts thereof. A
modulator may be a P2X.sub.7 agonist or antagonist.
[0077] A modulator is considered an "antagonist" if it detectably
inhibits P2X.sub.7-mediated signal transduction (using, for
example, a representative assay provided in Example 4); in general,
such an antagonist inhibits P2X.sub.7 activation with a IC.sub.50
value of less than 20 micromolar, preferably less than 10
micromolar, more preferably less than 5 micromolar, more preferably
less than 1 micromolar, still more preferably less than 500
nanomolar, and most preferably less than 100 nanomolar within an
assay provided in Example 4. P2X.sub.7 antagonists include neutral
antagonists and inverse agonists.
[0078] An "inverse agonist" of P2X.sub.7 is a compound that reduces
the activity of P2X.sub.7 below its basal activity level in the
absence of added ligand. Inverse agonists of P2X.sub.7 may also
inhibit the activity of ligand at P2X.sub.7 and/or binding of
ligand to P2X.sub.7. The basal activity of P2X.sub.7, as well as a
reduction in P2X.sub.7 activity due to the presence of P2X.sub.7
antagonist, may be determined from a calcium mobilization assay
(e.g., the assay of Example 4).
[0079] A "neutral antagonist" of P2X.sub.7 is a compound that
inhibits the activity of ligand at P2X.sub.7, but does not
significantly change the basal activity of P2X.sub.7 (i.e., within
a calcium mobilization assay as described in Example 4 performed in
the absence of ligand, P2X.sub.7 activity is reduced by no more
than 10%, preferably by no more than 5%, and more preferably by no
more than 2%; most preferably, there is no detectable reduction in
activity). Neutral antagonists of P2X.sub.7 may inhibit the binding
of ligand to P2X.sub.7.
[0080] As used herein a "P2X.sub.7 agonist" is a compound that
elevates the activity of the P2X.sub.7 above the basal activity
level of P2X.sub.7 (i.e., enhances P2X.sub.7 activation and/or
P2X.sub.7-mediated activity, such as signal transduction).
P2X.sub.7 agonist activity may be detected using the representative
assay provided in Example 4. P2X.sub.7 agonists include ATP and
2'(3')-O-(4-benzoyl-benzoyl)adenosine 5'-triphosephate (BzATP).
[0081] A "therapeutically effective amount" (or dose) is an amount
that, upon administration to a patient, results in a discernible
patient benefit (e.g., provides detectable relief from at least one
condition being treated). Such relief may be detected using any
appropriate criteria, including alleviation of one or more symptoms
such as pain. A therapeutically effective amount or dose generally
results in a concentration of compound in a body fluid (such as
blood, plasma, serum, CSF, synovial fluid, lymph, cellular
interstitial fluid, tears or urine) that is sufficient to alter
P2X.sub.7-mediated signal transduction (using an assay provided in
Example 4). It will be apparent that the discernible patient
benefit may be apparent after administration of a single dose, or
may become apparent following repeated administration of the
therapeutically effective dose according to a predetermined
regimen, depending upon the indication for which the compound is
administered.
[0082] By "statistically significant," as used herein, is meant
results varying from control at the p<0.1 level of significance
as measured using a standard parametric assay of statistical
significance such as a student's T test.
[0083] A "patient" is any individual treated with a compound
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 P2X.sub.7 modulation or may be free of such
symptom(s) (i.e., treatment may be prophylactic in a patient
considered at risk for the development of such symptoms).
Heteroaryl Amide Analogues
[0084] As noted above, the present invention provides heteroaryl
amide analogues of Formula I. Within certain aspects, such
compounds are modulators that may be used in a variety of contexts,
including in the treatment of conditions responsive to P2X.sub.7
modulation, such as pain. Such modulators are also useful as probes
for detection and localization of P2X.sub.7 and as standards in
P2X.sub.7-mediated signal transduction assays.
[0085] Within Formula I, the heteroaryl core:
##STR00006##
comprises at least one nitrogen atom, as indicated, and optionally
comprises additional nitrogen atom(s) at one or more of T, U, V,
Z.sub.1, Z.sub.2 and/or Z.sub.3. In certain embodiments, Z.sub.3 is
CR.sub.2; in further embodiments, Z.sub.3 is CH. Within other
embodiments, Z.sub.1, Z.sub.2 and Z.sub.3 are each CR.sub.2. The
5-membered ring portion of the core:
##STR00007##
within certain embodiments,
##STR00008##
[0086] Where present, each R.sub.3 is generally as described above;
in certain compounds each R.sub.3 is independently hydrogen or
C.sub.1-C.sub.4alkyl.
[0087] The variable R.sub.A is a ring substituent as described
above. In certain compounds, exactly one of T, U and V is CR.sub.A
(i.e., one and only one of T, U and V is a carbon atom that is
substituted with R.sub.A). Representative R.sub.A groups include,
for example, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6cyanoalkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkyl ether, phenylC.sub.0-C.sub.4alkyl (e.g.,
phenylC.sub.1-C.sub.4alkyl), (4- to 7-membered
heterocycle)C.sub.0-C.sub.4alkyl,
(C.sub.1-C.sub.6alkylsulfonylamino)C.sub.0-C.sub.4alkyl,
(C.sub.1-C.sub.6alkanoyloxy)C.sub.0-C.sub.4alkyl,
(C.sub.1-C.sub.6alkylsulfonyloxy)C.sub.0-C.sub.4alkyl, (mono- or
di-C.sub.1-C.sub.6alkylamino)C.sub.0-C.sub.4alkyl, and (mono- or
di-C.sub.1-C.sub.6alkylaminocarbonyl)C.sub.0-C.sub.4alkyl; each of
which is substituted with from 0 to 4 substituents independently
chosen from: (i) oxo, halogen, amino, cyano, hydroxy,
aminocarbonyl, aminosulfonyl and COOH; and (ii)
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6alkoxy, C.sub.2-C.sub.6alkyl ether,
C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonylamino, mono- or
di-C.sub.1-C.sub.6alkylaminocarbonyl, mono- or
di-C.sub.1-C.sub.6alkylaminosulfonyl, phenyl and 4- to 7-membered
heterocycle; each of which is substituted with from 0 to 4
substituents independently chosen from halogen, hydroxy, amino,
oxo, aminocarbonyl, aminosulfonyl, COOH, C.sub.1-C.sub.4alkyl and
C.sub.1-C.sub.4haloalkyl.
[0088] Within certain embodiments, R.sub.A is
C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6cyanoalkyl,
C.sub.2-C.sub.6cyanoalkenyl, C.sub.2-C.sub.6alkyl ether, (mono- or
di-C.sub.1-C.sub.6alkylamino)C.sub.0-C.sub.4alkyl, (mono- or
di-C.sub.1-C.sub.6alkylaminocarbonyl)C.sub.0-C.sub.4alkyl, or (4-
to 7-membered heterocycle)C.sub.1-C.sub.4alkyl; each of which is
substituted with from 0 to 4 substituents independently chosen from
amino, hydroxy, oxo, halogen, aminocarbonyl, aminosulfonyl,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6alkoxy, C.sub.2-C.sub.6alkyl ether, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, mono- or
di-C.sub.1-C.sub.6alkylaminocarbonyl, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonylamino, 4- to 7-membered
heterocycloalkyl, and 5- or 6-membered heteroaryl.
[0089] Within further embodiments, the "M" portion of R.sub.A is a
N-linked heterocycloalkyl. Certain such R.sub.A groups satisfy the
Formula:
##STR00009##
wherein: L is absent or C.sub.1-C.sub.6alkylene that is optionally
substituted with oxo;
##STR00010##
represents a 4- to 7-membered heterocycloalkyl that is optionally
fused to phenyl or to a 5- or 6-membered heteroaryl; and R.sub.7
represents from 0 to 4 substituents independently chosen from: (i)
hydroxy, amino, oxo, aminocarbonyl, aminosulfonyl and COOH; (ii)
C.sub.1-C.sub.6alkyl, mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.6alkylsulfonylC.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.6alkylsulfonylaminoC.sub.0-C.sub.4alkyl, and 4- to
7-membered heterocycle; each of which is substituted with from 0 to
4 substituents independently chosen from halogen, hydroxy, amino,
oxo, aminocarbonyl, aminosulfonyl, COOH, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy, mono- or di-(C.sub.1-C.sub.6alkyl)amino, and
C.sub.1-C.sub.6alkylsulfonylamino; (iii) substituents that are
taken together to form a bridge of the Formula
--(CH.sub.2).sub.q--P--(CH.sub.2), --, wherein q and r are
independently 0 or 1 and P is CH.sub.2, O, NH or S; and (iv)
substituents that are taken together to form a spiro 4- to
7-membered heterocycloalkyl ring that is substituted with from 0 to
2 substituents independently chosen from oxo and
C.sub.1-C.sub.4alkyl.
[0090] Certain such R.sub.A moieties further satisfy the
Formula:
##STR00011##
wherein: L is C.sub.1-C.sub.2alkylene that is optionally
substituted with oxo; G is CHR.sub.8, NH or O; s and t are
independently 0, 1, 2, 3 or 4, such that the sum of s and t ranges
from 2 to 5; and R.sub.8 is: (i) hydrogen, amino, aminocarbonyl,
aminosulfonyl or COOH; or (ii) C.sub.1-C.sub.6alkyl, mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.6alkylsulfonylC.sub.0-C.sub.4alkyl,
C.sub.1-C.sub.6alkylsulfonylaminoC.sub.0-C.sub.4alkyl, or 4- to
7-membered heterocycle; each of which is substituted with from 0 to
4 substituents independently chosen from halogen, hydroxy, amino,
oxo, aminocarbonyl, aminosulfonyl, COOH, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy, mono- or di-(C.sub.1-C.sub.6alkyl)amino, and
C.sub.1-C.sub.6alkylsulfonylamino.
[0091] Other such R.sub.A moieties further satisfy one of the
following Formulas:
##STR00012##
wherein J is CH or N; B, D, E and F are independently chosen from
CH.sub.2, NH and O; and R.sub.9 represents from 0 to 2 substituents
independently chosen from: (i) amino, aminocarbonyl and COOH; and
(ii) C.sub.1-C.sub.6alkyl, mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.2alkyl,
C.sub.1-C.sub.6alkylsulfonyl and C.sub.1-C.sub.6alkylsulfonylamino;
each of which is substituted with from 0 to 3 substituents
independently chosen from halogen, hydroxy, oxo and COOH.
[0092] Within other embodiments, R.sub.A is C.sub.1-C.sub.6alkyl,
C.sub.2-C.sub.6alkenyl, C.sub.2-C.sub.6alkyl ether, or mono- or
di-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.4alkyl, each of which
is substituted with from 1 to 4 substituents independently chosen
from halogen, hydroxy, cyano, amino, oxo, aminocarbonyl,
aminosulfonyl, COOH, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy,
mono- or di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkanoyl
amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonyloxy, C.sub.1-C.sub.6alkylsulfonylamino,
phenyl that is optionally substituted with halogen or
C.sub.1-C.sub.4alkyl, and 4- to 7-membered heterocycle that is
optionally substituted with C.sub.1-C.sub.4alkyl. Representative
such R.sub.A groups include, for example,
mono-(C.sub.1-C.sub.6alkyl)aminoC.sub.0-C.sub.2alkyl and
C.sub.2-C.sub.6alkyl ether, each of which is substituted with from
1 to 4 substituents independently chosen from halogen, hydroxy,
oxo, COOH, C.sub.1-C.sub.4alkyl and C.sub.1-C.sub.4alkoxy.
[0093] Within still further embodiments, R.sub.A is a group of the
Formula L-A-M as described above, wherein L is not absent; A is
absent; and M is phenyl or a 5- or 6-membered heteroaryl, each of
which is substituted with from 0 to 4 substituents independently
chosen from oxo, amino, halogen, hydroxy, cyano, aminocarbonyl,
aminosulfonyl, COOH, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy,
C.sub.2-C.sub.6alkyl ether, C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl, mono- or
di-(C.sub.1-C.sub.6alkylamino)carbonyl, and 4- to 7-membered
heterocycle. Within certain such compounds, M is a 5- or 6-membered
heteroaryl, each of which is optionally substituted. Certain such
5- or 6-membered heteroaryl moieties include, for example: [0094]
(i) imidazolyl, triazolyl, tetrazolyl, pyridyl, pyridazinyl or
pyrimidinyl, each of which is substituted with from 0 to 4
substituents independently chosen from oxo, amino, halogen,
hydroxy, cyano, aminocarbonyl, aminosulfonyl, COOH,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6haloalkoxy, C.sub.2-C.sub.6alkyl ether,
C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl, mono- or
di-(C.sub.1-C.sub.6alkylamino)carbonyl, and 4- to 7-membered
heterocycle; and
##STR00013##
[0094] each of which is substituted with from 0 to 2 substituents
independently chosen from amino, halogen, hydroxy, cyano,
aminocarbonyl, aminosulfonyl, COOH, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy,
C.sub.2-C.sub.6alkyl ether, C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl, mono- or
di-(C.sub.1-C.sub.6alkylamino)carbonyl, and 4- to 7-membered
heterocycle.
[0095] Within other embodiments, R.sub.A is a group of the Formula
L-A-M, wherein L is C.sub.0-C.sub.3alkylene that is optionally
substituted with oxo or COOH; A is absent; and M is phenyl that is
substituted with amino, cyano, aminocarbonyl, aminosulfonyl, COOH
or C.sub.1-C.sub.6alkyl.
[0096] Within other embodiments, R.sub.A is a group of the Formula
L-A-M, wherein L is C.sub.1-C.sub.2alkylene that is optionally
substituted with oxo; A is absent; and M is mono- or
di-(C.sub.1-C.sub.6alkyl)amino that is substituted with a 5- or
6-membered heteroaryl, each of which heteroaryl is substituted with
from 0 to 4 substituents independently chosen from oxo, amino,
halogen, hydroxy, cyano, aminocarbonyl, aminosulfonyl, COOH,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6haloalkoxy, C.sub.2-C.sub.6alkyl ether,
C.sub.1-C.sub.6alkanoylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)amino, C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonylamino, mono- or
di-(C.sub.1-C.sub.6alkyl)aminosulfonyl, mono- or
di-(C.sub.1-C.sub.6alkylamino)carbonyl, and 4- to 7-membered
heterocycle. Representative 5- or 6-membered heteroaryls are as
illustrated above.
[0097] As noted above, each of the variables Z.sub.1, Z.sub.2 and
Z.sub.3 in Formula A or Formula I is generally N, CH or substituted
carbon. Within certain embodiments, Z.sub.1, Z.sub.2 and Z.sub.3
are each CR.sub.2; Z.sub.1 is N and Z.sub.2 and Z.sub.3 are each
CR.sub.2; Z.sub.2 is N and Z.sub.1 and Z.sub.3 are each CR.sub.2;
Z.sub.3 is N and Z.sub.1 and Z.sub.3 are each CR.sub.2; or Z.sub.1
and Z.sub.3 are N and Z.sub.2 is CR.sub.2. Each R.sub.2, within
certain such compounds, is hydrogen or C.sub.1-C.sub.6alkyl.
[0098] Certain representative heteroaryl cores:
##STR00014##
include, for example:
##STR00015##
[0099] The variable "W," as noted above is generally W is
--C(.dbd.O)NR.sub.4--, --NR.sub.4C(.dbd.O)-- or
--NR.sub.4--NR.sub.4--C(.dbd.O)--. It will be apparent that the
orientation of these groups is intended to be retained; for
example, in a compound in which W is C(.dbd.O)NR.sub.4--, the
carbonyl of W is directly linked to the 6-membered ring of the
bicyclic core and the nitrogen of W is directly linked to X.
R.sub.4 is generally as described above; in certain embodiments,
R.sub.4 is hydrogen or methyl.
[0100] The variable "X" is generally as described above; in certain
embodiments, X is C.sub.1-C.sub.4alkylene (e.g., methylene or
ethylene), each of which is substituted with from 0 to 4
substituents independently chosen from C.sub.1-C.sub.4alkyl,
(C.sub.3-C.sub.8cycloalkyl)C.sub.0-C.sub.2alkyl, phenyl and
substituents that are taken together to form a 3- to 7-membered
cycloalkyl or heterocycloalkyl ring.
[0101] The variable "Y" is generally a cyclic moiety, optionally
substituted. In certain compounds, Y is a cycloalkyl or
heterocycloalkyl group, such as cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, piperidinyl, piperazinyl,
morpholinyl, or adamantyl, each of which is optionally substituted
as described above; in certain such compounds, each Y moiety is
substituted with from 0 to 4 substituents independently chosen from
halogen, hydroxy, cyano, amino, C.sub.1-C.sub.6alkyl,
C.sub.2-C.sub.6alkenyl, C.sub.2-C.sub.6alkynyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6alkoxy, and mono- or
di-(C.sub.1-C.sub.6alkyl)amino.
[0102] Within certain heteroaryl amide analogues provided herein,
W--X--Y is:
##STR00016##
wherein: n is 0, 1 or 2; R.sub.1 represents from 0 to 2
substituents independently chosen from halogen, hydroxy, cyano,
amino, nitro, aminocarbonyl, aminosulfonyl, C.sub.1-C.sub.6alkyl,
C.sub.2-C.sub.6alkenyl, C.sub.2-C.sub.6alkynyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl, and mono- or
di-(C.sub.1-C.sub.6alkyl)amino; or two substituents represented by
R.sub.1 are taken together to form a C.sub.1-C.sub.3alkylene bridge
or a fused or spiro 3- to 7-membered carbocyclic or heterocyclic
ring; and each R.sub.5 is independently hydrogen,
C.sub.1-C.sub.6alkyl, C.sub.3-C.sub.7cycloalkyl or phenyl; or two
R.sub.5 are taken together to form a C.sub.3-C.sub.8cycloalkyl.
Certain such compounds further satisfy the formula:
##STR00017## ##STR00018##
[0103] Within other compounds, Y is an aromatic moiety, such as:
(i) phenyl or a 5- or 6-membered heteroaryl, each of which is
optionally fused to a 5- to 7-membered carbocyclic or heterocyclic
ring; or
##STR00019##
each of which Y is substituted with from 0 to 4 substituents
independently chosen from halogen, hydroxy, cyano, amino,
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl,
C.sub.2-C.sub.6alkynyl, C.sub.1-C.sub.6haloalkyl,
C.sub.1-C.sub.6hydroxyalkyl, C.sub.1-C.sub.6alkoxy and mono- or
di-(C.sub.1-C.sub.6alkyl)amino. Within certain compounds, W--X--Y
is:
##STR00020## ##STR00021##
wherein:
##STR00022##
is a 5- to 7-membered carbocyclic or heterocyclic ring;
##STR00023##
is a 5- or 6-membered heteroaryl; R.sub.1 represents from 0 to 2
substituents independently chosen from halogen, hydroxy, cyano,
amino, nitro, aminocarbonyl, aminosulfonyl, C.sub.1-C.sub.6alkyl,
C.sub.2-C.sub.6alkenyl, C.sub.2-C.sub.6alkynyl,
C.sub.1-C.sub.6haloalkyl, C.sub.1-C.sub.6hydroxyalkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6haloalkoxy,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl, and mono- or
di-(C.sub.1-C.sub.6alkyl)amino; or two substituents represented by
R.sub.1 are taken together to form a fused or spiro 3- to
7-membered carbocyclic or heterocyclic ring; each R.sub.5 is
independently hydrogen, C.sub.1-C.sub.6alkyl,
C.sub.3-C.sub.7cycloalkyl or phenyl; or two R.sub.5 are taken
together to form a C.sub.3-C.sub.8cycloalkyl; Q is CH.sub.2, CO, O,
NH, S, SO or SO.sub.2; and m is 0 or 1. Certain such compounds
further satisfy the formula:
##STR00024## ##STR00025##
wherein K is CH or N. It will be apparent that when K is CH, the
carbon atom at the K position may, but need not, be substituted
with a substituent represented by R.sub.1.
[0104] Representative heteroaryl amide analogues 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. In
addition, other forms such as hydrates and prodrugs of such
compounds are specifically contemplated by the present
invention.
[0105] Within certain aspects of the present invention, heteroaryl
amide analogues provided herein detectably alter (modulate)
P2X.sub.7 activity, as determined using an assay such as an assay
recited in Example 4, herein. Additional assays that may be used
for this purpose include assays that measure IL-1.beta. release;
assays that measure uptake of a membrane-impermeant fluorescent dye
such as YO-PRO1; assays that measure lucifer yellow uptake; assays
that measure ethidium bromide uptake; and assays that use calcium
imaging to detect P2X.sub.7 activity; all of which assays are well
known in the art. Certain modulators provided herein detectably
modulate P2X.sub.7 activity at micromolar concentrations, at
nanomolar concentrations, or at subnanomolar concentrations.
[0106] As noted above, compounds that are P2X.sub.7 antagonists are
preferred within certain embodiments. IC.sub.50 values for such
compounds may be determined using a standard in vitro
P2X.sub.7-mediated calcium mobilization assay, as provided in
Example 4. Briefly, cells expressing P2X.sub.7 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, Fluo-4 or Fura-2 (Invitrogen, Carlsbad, Calif.),
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
P2X.sub.7 agonist (e.g., ATP or
2'(3')-O-(4-benzoyl-benzoyl)adenosine 5'-triphosephate at, for
example, a concentration equal to the agonist's EC.sub.50), and a
fluorescence response is measured. When agonist-contacted cells are
contacted with a compound that is a P2X.sub.7 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. IC.sub.50 stands for 50% Inhibitory
Concentration, i.e., the concentration of compound that inhibits
receptor (e.g., P2X.sub.7) activity by 50% in an assay. Note that a
lower IC.sub.50 value for a compound at a particular receptor
corresponds to the compound exhibiting more potency at that
receptor, and higher IC.sub.50 values correspond to less potency at
that receptor. In certain embodiments, P2X.sub.7 antagonists
provided herein exhibit no detectable agonist activity an in vitro
assay of P2X.sub.7 agonism at a concentration of compound equal to
the IC.sub.50. Certain such antagonists exhibit no detectable
agonist activity an in vitro assay of P2X.sub.7 agonism at a
concentration of compound that is 100-fold higher than the
IC.sub.50.
[0107] P2X.sub.7 modulating activity may also, or alternatively, be
assessed using an in vivo pain relief assay as provided in Example
5. Modulators provided herein preferably have a statistically
significant specific effect on P2X.sub.7 activity within such a
functional assay.
[0108] In certain embodiments, preferred modulators are
non-sedating. In other words, a dose of modulator 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 5,
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 modulator 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).
[0109] 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 compound is nontoxic when a
therapeutically effective amount is administered to a subject),
side effects (a preferred compound 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 compound exhibits an in vivo
half-life allowing for no more than Q.I.D. dosing, preferably
T.I.D. dosing, more preferably B.I.D. dosing, and most preferably
once-a-day dosing). In addition, it may be desirable to select
compounds with differential penetration of the blood brain barrier.
For modulators used to treat pain or neurodegenerative disease by
modulating CNS P2X.sub.7 activity, high blood brain barrier
penetration is preferred, such that total daily oral doses as
described above provide such modulation in the CNS to a
therapeutically effective extent, while compounds exhibiting low
penetration of the blood brain barrier resulting in low brain
levels of modulators may be preferred to treat peripheral nerve
mediated pain or certain peripheral inflammatory diseases (e.g.
rheumatoid arthritis). Preferably such low blood brain barrier
penetrant compounds at such doses do not provide brain (e.g., CSF)
levels of the compound sufficient to modulate P2X.sub.7 activity to
a clinically or therapeutically effective extent. 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,
for example, within Example 7 of U.S. Patent Application
Publication Number 2005/0070547.
[0110] As noted above, preferred compounds provided herein are
nontoxic. In general, the term "nontoxic" 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, or (4) does not cause substantial release of liver
enzymes.
[0111] 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 of U.S. Patent Application
Publication Number 2005/0070547. In other words, cells treated as
described therein 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 cells.
[0112] A 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 a dose that yields a serum concentration equal to
the EC.sub.50 or IC.sub.50 for the compound. 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.
[0113] A compound does not cause substantial liver enlargement if
daily treatment of laboratory rodents (e.g., mice or rats) for 5-10
days with a dose that yields a serum concentration equal to the
EC.sub.50 or IC.sub.50 for the compound 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.
[0114] Similarly, a compound does not promote substantial release
of liver enzymes if administration of twice the minimum dose that
yields a serum concentration equal to the EC.sub.50 or IC.sub.50 at
P2X.sub.7 for the compound does not elevate serum levels of ALT,
LDH or AST in laboratory animals (e.g., 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) that are equal to the EC.sub.50 or IC.sub.50 for 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
EC.sub.50 or IC.sub.50 for the compound.
[0115] 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 EC.sub.50 or IC.sub.50 at P2X.sub.7 for
the compound.
[0116] 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 the EC.sub.50 or IC.sub.50 for
the compound. In other embodiments, certain preferred compounds do
not induce sister chromatid exchange (e.g., in Chinese hamster
ovary cells) at such concentrations.
[0117] For detection purposes, as discussed in more detail below,
modulators provided herein may be isotopically-labeled or
radiolabeled. Substitution with heavy isotopes such as deuterium
(i.e., .sup.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 Heteroaryl Amide Analogues
[0118] Heteroaryl amide 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. In some cases, protecting groups may be required
during preparation. Such protecting groups can be removed by
methods well known to those of ordinary skill in the art, such as
methods described in Greene and Wuts, "Protective Groups in Organic
Synthesis" (2nd Edition, John Wiley & Sons, 1991). In some
cases, further organic transformations may be performed using
methods well known to those of ordinary skill in the art, such as
methods described in Richard C. Larock, "Comprehensive Organic
Transformation," (VCH Publisher, Inc. 1989). Each variable in the
following Schemes refers to any group consistent with the
description of the compounds provided herein. Representative
reaction conditions for use within the following schemes are
provided in the Examples.
[0119] Certain abbreviations used in the following Schemes and
elsewhere herein include: [0120] Ac acetyl [0121] aq. aqueous
[0122] ACN acetonitrile [0123] BOP
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate [0124] Bu butyl [0125] chemical shift [0126]
DCM dichloromethane [0127] DIBAL-H diisobutylaluminium hydride
[0128] DMF dimethylformamide [0129] DMSO dimethylsulfoxide [0130]
Et ethyl [0131] EtOAc ethyl acetate [0132] EtOH ethanol [0133] h
hour(s) [0134] .sup.1H NMR proton nuclear magnetic resonance [0135]
Hz hertz [0136] iPr isopropyl [0137] MeOH methanol [0138] min
minute(s) [0139] Ms methanesulfonyl [0140] (M+1) mass+1 [0141]
Ph.sub.3P triphenylphosphine [0142] PTLC preparative thin layer
chromatography [0143] rt room temperature [0144] TEA triethylamine
[0145] TFA trifluoroacetic acid [0146] THF tetrahydrofuran
##STR00026##
[0147] In Scheme I, dicarboxylic acid dimethyl ester 1 is prepared
by reaction of the 3-pyridylcarbinol analogue with
O-mesitylenesulfonylhydroxylamine in the presence of acetylene
dicarboxylic acid dimethyl ester. Reaction of 1 with aqueous
H.sub.2SO.sub.4 affords the
4-hydroxymethylpyrazolo[1,5-a]pyridine-2-carboxylic acid analogue,
which is converted to the ethyl ester 2 by reaction with acetyl
chloride in absolute ethanol. Oxidation of 2 with Jones reagent
yields carboxylic acid 3, which is converted to amide 4 by reaction
with an appropriate amine in the presence of BOP.
##STR00027##
[0148] In Scheme II, ester 4 is hydrolyzed to yield the carboxylic
acid 5. Amide 6 is prepared by reaction with an appropriate amine
in the presence of BOP.
##STR00028##
[0149] In Scheme III, ester 4 is reduced to the alcohol 7, which is
converted to mesylate 8. Mesylate 8 is then used to generate amine
10 by reaction with an appropriate amine in the presence of
potassium carbonate, or the cyanomethyl derivative 9 by reaction
with sodium cyanide.
##STR00029##
[0150] Scheme IV illustrates the conversion of alcohol 7 to any of
a variety of ether moieties 11, via reaction with the appropriate
brominated reagent.
##STR00030##
[0151] In Scheme V, dicarboxylic acid dimethyl ester 12 is prepared
by reaction of the 3-aminopyridine analogue with
O-mesitylenesulfonylhydroxylamine. Reaction of 12 with aqueous
H.sub.2SO.sub.4 affords 4-aminopyrazolo[1,5-a]pyridine-2-carboxylic
acid analogue 13, which is converted to the amide 14 by reaction
with an appropriate carboxylic acid.
##STR00031##
[0152] Scheme VI illustrates the synthesis of certain
representative compounds of Formula I from ester 4, discussed
above. The formyl derivative is generated by reaction with DIBAL,
and is converted to the cyanovinyl derivative (a mixture of Z and E
isomers) using Ph.sub.3P.dbd.CHCN. Isomers can be separated using
standard techniques. Reduction of the cyanovinyl compound (e.g.,
with Pd/C and 1 atm H.sub.2) yields the cyanoethyl derivative,
which can be converted to a variety of compounds, including the
carboxylic acid and the tetrazolylethyl compounds illustrated.
##STR00032##
[0153] In Scheme VII, carboxylic acid methyl ester 16 is prepared
by reaction of the 3-pyridylcarbinol analogue 15 with
O-mesitylenesulfonylhydroxylamine in the presence of a propargyllic
ester derivative, wherein R is hydrogen or any suitable group such
as alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl each of
which is optionally substituted. Reaction of 16 with aqueous
H.sub.2SO.sub.4 affords the 4-hydroxymethylpyrazolo[1,5-a]pyridine
17. Oxidation of 17 with Jones reagent yields carboxylic acid 18,
which is converted to amide 19 by reaction with an appropriate
amine in the presence of BOP.
##STR00033##
[0154] In Scheme VIII, carboxylic acid methyl ester 21 is prepared
by reaction of the 3-aminopyridine analogue 20 with
O-mesitylenesulfonylhydroxylamine in the presence of a methyl
parpargellate derivative. Reaction of 21 with aqueous
H.sub.2SO.sub.4 affords 4-aminopyrazolo[1,5-a]pyridine-2-carboxylic
acid analogue 23, which is converted to the amide 24 by reaction
with an appropriate carboxylic acid. Alternatively 21 can be
directly reacted with an appropriate carboxylic acid to give amide
22.
##STR00034##
[0155] In Scheme IX, a nitrogen-containing heterocyclic halide 25
is converted to the aminoheterocycle 26 using any suitable method,
such as ammonia in EtOH at rt or elevated temperature. Amino
heterocycle 26 is reacted with an alpha halocarbonyl compound to
give the substituted imidazoheterocycle 28. The alpha haloaldehyde
29 is reacted with aminoheterocycle 26 to give the unsubstituted
imidazo heterocycle 30, which can be subsequently converted to
substituted imidazoheterocycle 31 through electrophilic
substitution of the imidazo functionality (e.g., via bromination to
give the bromide), which can be converted to the desired
imidazoheterocycle 31.
##STR00035##
[0156] In Scheme X, a nitrogen-containing heterocyclic halide 25 is
converted to the hydrazinoheterocycle 32 using any suitable method,
such as, for example, reacting hydrazine in EtOH at RT or elevated
temperature. Intermediate 32 is converted to triazoloheterocycle 34
by reacting with acid 33 without solvent or with solvent at rt or
elevated temperature.
##STR00036##
[0157] In Scheme XI, 35 is reacted with isothiocyanate 36 to form
37. The primary amine in compound 37 can be mono or di alylated or
acylated to give compound 38. Hydrolysis of 38 (when Q is CN or an
ester) under acidic conditions gives 39. Alternatively, if Q is a
halogen, treatment of 38 with ammonium gives 40. 39 and 40 are
readily converted to compounds of Formula I as illustrated
above.
##STR00037##
[0158] In Scheme XII, 35 is reacted with, for example,
O-mesitylenesulfonyhydroxylamine at rt. Aldehyde 41 and aqueous KOH
are then added to give intermediate 42. Hydrolysis of 42 (when Q is
CN or an ester) under acidic conditions gives 43. Alternatively, if
Q is a halogen, treatment of 42 with ammonium gives 44. 43 and 44
are readily converted to compounds of Formula I as illustrated
above.
##STR00038##
[0159] In Scheme XIII, a methyl substituted nitrogen heterocycle
carboxylate ester 45 is reacted with an alpha-halocarbonyl compound
and the intermediate slat is treated with ammonium hydroxide to
give the ester 46. This is converted to the target compound 47
through standard conditions.
##STR00039##
[0160] In Scheme XIV, a halogenated a nitrogen containing
heterocyclic halide 46 is reacted with potassium cyanide and the
resulting nitrile subjected to hydrogenation to give 47. 47 is
condensed with acid 48 to give the heterocycle 49, which is
converted to 50 by methods described herein.
[0161] 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.
[0162] 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
[0163] The present invention also provides pharmaceutical
compositions comprising one or more compounds provided herein,
together with at least one physiologically acceptable carrier or
excipient. Pharmaceutical compositions may comprise, for example,
one or more of water, buffers (e.g., sodium bicarbonate, neutral
buffered saline or phosphate buffered saline), ethanol, mineral
oil, vegetable oil, dimethylsulfoxide, carbohydrates (e.g.,
glucose, mannose, sucrose, starch, mannitol or dextrans), 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.
[0164] Pharmaceutical compositions may be formulated for any
appropriate manner of administration, including, for example,
topical, oral, nasal, rectal, vaginal, 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, the compositions may be
prepared as suppositories. In other 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, pharmaceutical
compositions 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.
[0165] 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). Tablets may be formed using standard techniques, including
dry granulation, direct compression and wet granulation. The
tablets may be uncoated or they may be coated by known
techniques.
[0166] 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).
[0167] Aqueous suspensions contain the active material(s) in
admixture with suitable excipients, such as 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/or one or
more sweetening agents, such as sucrose or saccharin.
[0168] 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.
[0169] 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, a
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.
[0170] 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.
[0171] 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.
[0172] 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 Remington: The Science and
Practice of Pharmacy, 21.sup.st ed., Lippincott Williams &
Wilkins, Philadelphia, Pa. (2005). Formulations may comprise
microcapsules, such as hydroxymethylcellulose or
gelatin-microcapsules, liposomes, albumin microspheres,
microemulsions, nanoparticles or nanocapsules.
[0173] A topical formulation may be prepared in any of a variety of
physical forms including, for example, solids, pastes, ointments,
creams, foams, lotions, gels, powders, aqueous liquids and
emulsions. The physical appearance and viscosity of such
pharmaceutically acceptable 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.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] A pharmaceutical composition may be prepared as a sterile
injectible aqueous or oleaginous suspension. The compound(s)
provided herein, 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.
[0178] Pharmaceutical compositions 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. Compositions for inhalation typically can
be provided in the form of a solution, suspension or emulsion that
can be administered as a dry powder or in the form of an aerosol
using a conventional propellant (e.g., dichlorodifluoromethane or
trichlorofluoromethane).
[0179] Pharmaceutical compositions may be formulated for release at
a predetermined rate. Instantaneous release may be achieved, for
example, via sublingual administration (i.e., administration by
mouth in such a way that the active ingredient(s) are rapidly
absorbed via the blood vessels under the tongue rather than via the
digestive tract). Controlled release formulations (i.e.,
formulations such as a capsule, tablet or coated tablet that slows
and/or delays release of active ingredient(s) following
administration) may be administered by, for example, oral, rectal
or subcutaneous implantation, or by implantation at a target site.
In general, a controlled release formulation comprises a matrix
and/or coating that delays disintegration and absorption in the
gastrointestinal tract (or implantation site) and thereby provides
a delayed action or a sustained action over a longer period. One
type of controlled-release formulation is a sustained-release
formulation, in which at least one active ingredient is
continuously released over a period of time at a constant rate.
Preferably, the therapeutic agent is released at such a rate that
blood (e.g., plasma) concentrations are maintained within the
therapeutic range, but below toxic levels, over a period of time
that is at least 4 hours, preferably at least 8 hours, and more
preferably at least 12 hours. 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.
[0180] Controlled release may be achieved by combining the active
ingredient(s) with a matrix material that itself alters release
rate and/or through the use of a controlled-release coating. The
release rate can be varied using methods well known in the art,
including (a) varying the thickness or composition of coating, (b)
altering the amount or manner of addition of plasticizer in a
coating, (c) including additional ingredients, such as
release-modifying agents, (d) altering the composition, particle
size or particle shape of the matrix, and (e) providing one or more
passageways through the coating. The amount of modulator contained
within a sustained release formulation depends upon, for example,
the method of administration (e.g., the site of implantation), the
rate and expected duration of release and the nature of the
condition to be treated or prevented.
[0181] The matrix material, which itself may or may not serve a
controlled-release function, is generally any material that
supports the active ingredient(s). For example, a time delay
material such as glyceryl monosterate or glyceryl distearate may be
employed. Active ingredient(s) may be combined with matrix material
prior to formation of the dosage form (e.g., a tablet, a capsule, a
troche, or a sprinkle). Alternatively, or in addition, active
ingredient(s) may be coated on the surface of a particle, granule,
sphere, microsphere, bead or pellet that comprises the matrix
material. Such coating may be achieved by conventional means, such
as by dissolving the active ingredient(s) in water or other
suitable solvent and spraying. Optionally, additional ingredients
are added prior to coating (e.g., to assist binding of the active
ingredient(s) to the matrix material or to color the solution). The
matrix may then be coated with a barrier agent prior to application
of controlled-release coating. Multiple coated matrix units may, if
desired, be encapsulated to generate the final dosage form.
[0182] In certain embodiments, a controlled release is achieved
through the use of a controlled release coating (i.e., a coating
that permits release of active ingredient(s) at a controlled rate
in aqueous medium). The controlled release coating should be a
strong, continuous film that is smooth, capable of supporting
pigments and other additives, non-toxic, inert and tack-free.
Coatings that regulate release of the modulator include
pH-independent coatings, pH-dependent coatings (which may be used
to release modulator in the stomach) and enteric coatings (which
allow the formulation to pass intact through the stomach and into
the small intestine, where the coating dissolves and the contents
are absorbed by the body). It will be apparent that multiple
coatings may be employed (e.g., to allow release of a portion of
the dose in the stomach and a portion further along the
gastrointestinal tract). For example, a portion of active
ingredient(s) may be coated over an enteric coating, and thereby
released in the stomach, while the remainder of active
ingredient(s) in the matrix core is protected by the enteric
coating and released further down the GI tract. pH dependent
coatings include, for example, shellac, cellulose acetate
phthalate, polyvinyl acetate phthalate,
hydroxypropylmethylcellulose phthalate, methacrylic acid ester
copolymers and zein.
[0183] In certain embodiments, the coating is a hydrophobic
material, preferably used in an amount effective to slow the
hydration of the gelling agent following administration. Suitable
hydrophobic materials include alkyl celluloses (e.g.,
ethylcellulose or carboxymethylcellulose), cellulose ethers,
cellulose esters, acrylic polymers (e.g., poly(acrylic acid),
poly(methacrylic acid), acrylic acid and methacrylic acid
copolymers, methyl methacrylate copolymers, ethoxy ethyl
methacrylates, cyanoethyl methacrylate, methacrylic acid alkamide
copolymer, poly(methyl methacrylate), polyacrylamide, ammonio
methacrylate copolymers, aminoalkyl methacrylate copolymer,
poly(methacrylic acid anhydride) and glycidyl methacrylate
copolymers) and mixtures of the foregoing. Representative aqueous
dispersions of ethylcellulose include, for example, AQUACOAT.RTM.
(FMC Corp., Philadelphia, Pa.) and SURELEASE.RTM. (Colorcon, Inc.,
West Point, Pa.), both of which can be applied to the substrate
according to the manufacturer's instructions. Representative
acrylic polymers include, for example, the various EUDRAGIT.RTM.
(Rohm America, Piscataway, N.J.) polymers, which may be used singly
or in combination depending on the desired release profile,
according to the manufacturer's instructions.
[0184] The physical properties of coatings that comprise an aqueous
dispersion of a hydrophobic material may be improved by the
addition or one or more plasticizers. Suitable plasticizers for
alkyl celluloses include, for example, dibutyl sebacate, diethyl
phthalate, triethyl citrate, tributyl citrate and triacetin.
Suitable plasticizers for acrylic polymers include, for example,
citric acid esters such as triethyl citrate and tributyl citrate,
dibutyl phthalate, polyethylene glycols, propylene glycol, diethyl
phthalate, castor oil and triacetin.
[0185] Controlled-release coatings are generally applied using
conventional techniques, such as by spraying in the form of an
aqueous dispersion. If desired, the coating may comprise pores or
channels or to facilitate release of active ingredient. Pores and
channels may be generated by well known methods, including the
addition of organic or inorganic material that is dissolved,
extracted or leached from the coating in the environment of use.
Certain such pore-forming materials include hydrophilic polymers,
such as hydroxyalkylcelluloses (e.g.,
hydroxypropylmethylcellulose), cellulose ethers, synthetic
water-soluble polymers (e.g., polyvinylpyrrolidone, cross-linked
polyvinylpyrrolidone and polyethylene oxide), water-soluble
polydextrose, saccharides and polysaccharides and alkali metal
salts. Alternatively, or in addition, a controlled release coating
may include one or more orifices, which may be formed my methods
such as those described in U.S. Pat. Nos. 3,845,770; 4,034,758;
4,077,407; 4,088,864; 4,783,337 and 5,071,607. Controlled-release
may also be achieved through the use of transdermal patches, using
conventional technology (see, e.g., U.S. Pat. No. 4,668,232).
[0186] Further examples of controlled release formulations, and
components thereof, may be found, for example, in U.S. Pat. Nos.
4,572,833; 4,587,117; 4,606,909; 4,610,870; 4,684,516; 4,777,049;
4,994,276; 4,996,058; 5,128,143; 5,202,128; 5,376,384; 5,384,133;
5,445,829; 5,510,119; 5,618,560; 5,643,604; 5,891,474; 5,958,456;
6,039,980; 6,143,353; 6,126,969; 6,156,342; 6,197,347; 6,387,394;
6,399,096; 6,437,000; 6,447,796; 6,475,493; 6,491,950; 6,524,615;
6,838,094; 6,905,709; 6,923,984; 6,923,988; and 6,911,217; each of
which is hereby incorporated by reference for its teaching of the
preparation of controlled release dosage forms.
[0187] In addition to or together with the above modes of
administration, a compound provided herein 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.
[0188] Compounds are generally administered in 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).
[0189] 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, the
particular mode of administration and any other co-administered
drugs. Dosage units generally contain between from about 10 .mu.g
to about 500 mg of active ingredient. Optimal dosages may be
established using routine testing, and procedures that are well
known in the art.
[0190] Pharmaceutical compositions may be packaged for treating
conditions responsive to P2X.sub.7 modulation (e.g., pain,
inflammation, neurodegeneration or other condition described
herein). Packaged pharmaceutical compositions generally include (i)
a container holding a pharmaceutical composition that comprises at
least one modulator as described herein and (ii) instructions
(e.g., labeling or a package insert) indicating that the contained
composition is to be used for treating a condition responsive to
P2X.sub.7 modulation in the patient.
Methods of Use
[0191] P2X.sub.7 modulators provided herein may be used to alter
activity and/or activation of P2X.sub.7 in a variety of contexts,
both in vitro and in vivo. Within certain aspects, P2X.sub.7
antagonists may be used to inhibit the binding of ligand agonist to
P2X.sub.7 in vitro or in vivo. In general, such methods comprise
the step of contacting a P2X.sub.7 with one or more P2X.sub.7
modulators provided herein, in the presence of ligand in aqueous
solution and under conditions otherwise suitable for binding of the
ligand to P2X.sub.7. The modulator(s) are generally present at a
concentration that is sufficient to alter P2X.sub.7-mediated signal
transduction (using an assay provided in Example 4). The P2X.sub.7
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 P2X.sub.7 is expressed by a cell
that is present in a patient, and the aqueous solution is a body
fluid. Preferably, one or more modulators are administered to an
animal in an amount such that the modulator is present in at least
one body fluid of the animal at a therapeutically effective
concentration that is 20 micromolar or less, 10 micromolar or less,
5 micromolar or less, or 1 micromolar or less. For example, such
compounds may be administered at a therapeutically effective 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.
[0192] Also provided herein are methods for modulating, preferably
reducing, cellular P2X.sub.7 activation and/or activity, such as
signal-transducing activity (e.g., calcium conductance). Such
modulation may be achieved by contacting a P2X.sub.7 (either in
vitro or in vivo) with one or more modulators provided herein under
conditions suitable for binding of the modulator(s) to P2X.sub.7.
The modulator(s) are generally present at a concentration that is
sufficient to alter P2X.sub.7-mediated signal transduction as
described herein. P2X.sub.7 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 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 or inflammation) of a patient being
treated with one or more modulators provided herein.
[0193] P2X.sub.7 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 P2X.sub.7 signal-transducing activity.
[0194] The present invention further provides methods for treating
conditions responsive to P2X.sub.7 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 P2X.sub.7 modulation" if it is characterized by
inappropriate activity of a P2X.sub.7, regardless of the amount of
P2X.sub.7 agonist present locally, and/or if modulation of
P2X.sub.7 activity results in alleviation of the condition or a
symptom thereof. Such conditions include, for example, pain,
inflammation, cardiovascular disorders, ocular disorders,
neurodegenerative disorders and respiratory disorders (such as
cough, asthma, chronic obstructive pulmonary disease, chronic
bronchitis, cystic fibrosis and rhinitis, including allergic
rhinitis, such as seasonal an perennial rhinitis, and non-allergic
rhinitis), fibrosis as well as other conditions 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.
[0195] 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.
[0196] Pain that may be treated using the modulators provided
herein includes, for example, acute, chronic, inflammatory, and
neuropathic pain. Specific pain indications that may be treated as
described herein include, but are not limited to, pain associated
with osteoarthritis or rheumatoid arthritis; various neuropathic
pain syndromes (such as post-herpetic neuralgia, trigeminal
neuralgia, reflex sympathetic dystrophy, diabetic neuropathy,
Guillian Barre syndrome, fibromyalgia, oral neuropathic pain,
phantom limb pain, post-mastectomy pain, peripheral neuropathy,
myofascial pain syndromes, MS-related neuropathy, HIV or
AIDS-related neuropathy, and chemotherapy-induced and other
iatrogenic neuropathies); visceral pain, (such as that associated
with gastroesophageal reflux disease (GERD), irritable bowel
syndrome, inflammatory bowel disease, pancreatitis, intestinal gas,
gynecological disorders (e.g., menstrual pain, dysmenorrhoea, pain
associated with cystitis, labor pain, chronic pelvic pain, chronic
prostitis, endometriosis, heart pain and abdominal pain), and
urological disorders); dental pain (e.g., toothache, denture pain,
nerve root pain, pain resulting from periodontal disease, and pain
due to dental surgery including operative and post-operative pain);
headache (e.g., headaches involving peripheral nerve activity,
sinus headache, cluster headache (i.e., migranous neuralgia)
tension headache, migraine, temporomandibular pain and maxillary
sinus pain); stump pain; meralgia paresthetica; burning-mouth
syndrome; pain associated with nerve and root damage, including as
pain associated with peripheral nerve disorders (e.g., nerve
entrapment and brachial plexus avulsions, amputation, peripheral
neuropathies including bilateral peripheral neuropathy, tic
douloureux, atypical facial pain, nerve root damage, and
arachnoiditis), causalgia, 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;
central nervous system pain (e.g., pain due to brain stem damage,
sciatica, and ankylosing spondylitis); and spinal pain, including
spinal cord injury-related pain.
[0197] Further pain conditions that can be treated as described
herein include Charcot's pains, ear pain, muscle pain, eye pain,
orofacial pain (e.g., odontalgia), carpel tunnel syndrome, acute
and chronic back pain (e.g., lower back pain), gout, scar pain,
hemorrhoidal pain, dyspeptic pains, angina, nerve root pain,
"non-painful" neuropathies, complex regional pain syndrome,
homotopic pain and heterotopic pain--including pain associated with
carcinoma, often referred to as 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,
episiotomy pain, pain from cuts, musculoskeletal pain, bruises and
broken bones, and burn pain, especially primary hyperalgesia
associated therewith). Additional pain conditions that may be
treated as described herein include pain associated with autoimmune
diseases or immunodeficiency disorders, hot flashes, burns,
sunburn, and pain that results from exposure to heat, cold or
external chemical stimuli.
[0198] Conditions associated with inflammation and/or immune system
disorders that may be treated using the modulators provided herein
include, but are not limited to, arthritis (including
osteoarthritis, rheumatoid arthritis, psoriatic arthritis, Reiter's
syndrome, gout, traumatic arthritis, rubella arthritis, rheumatoid
spondylitis, gouty arthritis and juvenile arthritis); cystic
fibrosis; uveitis; systemic lupus erythematosus (and associated
glomerulonephritis); spondyloarthropathies; psoriasis; scleritis;
allergic conditions (including allergic reactions, allergic
rhinitis, allergic contact hypersensitivity, allergic dermatitis,
eczema and contact dermatitis), reperfusion injury (e.g., cardiac
and renal reperfusion injury), respiratory system disorders
(including hyper-responsiveness of the airway, cough, asthma (e.g.,
to prevent or decrease the severity of both acute early phase
asthma attack and the late phase reactions that follow such an
asthma attack; including bronchial, allergic, intrinsic, extrinsic,
exercise-induced, drug-induced (e.g., aspirin or NSAID-induced) and
dust-induced asthma), reactive airway disease, emphysema, acute
(adult) respiratory distress syndrome (ARDS), bronchitis (e.g.,
infectious and eosinophilic bronchitis), bronchiectasis, chronic
pulmonary obstructive disorder (COPD), chronic pulmonary
inflammatory disease, silicosis, pulmonary sarcoidosis, farmer's
lung, hypersensitivity pneumonitis and lung fibrosis), viral
infection, fungal infection, bacterial infection, Crohn's disease,
glomerulornephritis, HIV infection and AIDS, irritable bowel
syndrome, inflammatory bowel disease, dermatomyositis, multiple
sclerosis, pemphigus, pemphigoid, scleroderma, myasthenia gravis,
autoimmune hemolytic and thrombocytopenic states, Goodpasture's
syndrome (and associated glomerulonephritis and pulmonary
hemorrhage), tissue graft rejection, hyperacute rejection of
transplanted organs, allograft rejection, organ transplant
toxicity, neutropenia, sepsis, septic shock, endotoxic shock,
conjunctivitis shock, toxic shock syndrome, Alzheimer's disease,
inflammation associated with severe burns, lung injury, systemic
inflammatory response syndrome (SIRS), neonatal-onset multisystem
inflammatory disease (NOMID), Hashimoto's thyroiditis, Grave's
disease, Addison's disease, idiopathic thrombocytopaenic purprua,
eosinophilic fascitis, hyper-IgE syndrome, antiphospholipid
syndrome, leprosy, Sezary syndrome, paraneoplastic syndromes,
Muckle-Wells syndrome, lichen planus, familial cold
autoinflammatory syndrome (FCAS), colitis, ruptured abdominal
aortic aneurysm and multiple organ dysfunction syndrome (MODS).
Also included are pathologic sequellae associated with
insulin-dependent diabetes mellitus (including diabetic
retinopathy), lupus nephropathy, Heyman nephritis, membranous
nephritis and other forms of glomerulonephritis, macular
degeneration, contact sensitivity responses, and inflammation
resulting from contact of blood with artificial surfaces as occurs,
for example, during extracorporeal circulation of blood (e.g.,
during hemodialysis or via a heart-lung machine, for example, in
association with vascular surgery such as coronary artery bypass
grafting or heart valve replacement) such as extracorporeal
post-dialysis syndrome, or in association with contact with other
artificial vessel or container surfaces (e.g., ventricular assist
devices, artificial heart machines, transfusion tubing, blood
storage bags, plasmapheresis, plateletpheresis, and the like).
[0199] Still further conditions that may be treated using the
modulators provided herein include: [0200] Cardiovascular
disorders, such as cardiovascular disease, stroke, cerebral
ischemia, myocardial infarction, atherosclerosis, ischemic heart
disease, ischemia-reperfusion injury, aortic aneurysm, and
congestive heart failure; [0201] Ocular disorders such as glaucoma;
[0202] Neurological disorders (e.g., neurodegeneration), such as
neurodegenerative conditions associated with progressive CNS
disorders, including, but not limited to, Alzheimer's disease,
Parkinson's disease, amyotrophic lateral sclerosis, Huntington's
disease, Creutzfeldt-Jakob disease, dementia with Lewy bodies,
traumatic brain injury, spinal cord injury, neurotrauma, cerebral
amyloid angiopathy, and encephalitis; epilepsy and seizure
disorders; multiple sclerosis and other demyelinating syndromes;
cerebral atherosclerosis; vasculitis; temporal arteritis;
myasthenia gravis; neurosarcoidosis; and central and peripheral
nervous system complications of malignant, infectious or autoimmune
processes; the modulators provided herein may also be used to
promote neuroregeneration; [0203] Centrally-mediated
neuropsychiatric disorders, such as depression, depression mania,
bipolar disease, anxiety, schizophrenia, eating disorders, sleep
disorders and cognition disorders; and [0204] Other disorders, such
as cirrhosis, interstitial fibrosis, prostate, bladder and bowel
dysfunction (e.g., urinary incontinence, urinary hesitancy, rectal
hypersensitivity, fecal incontinence and benign prostatic
hypertrophy); itch/pruritus; obesity; lipid disorders; cancer;
hypertension; renal disorders; abnormal wound healing; myoblastic
leukemia; diabetes; meningitis; varicose veins; muscle
degeneration; cachexia; restenosis; thrombosis; cerebral malaria;
disorders of bones and joints (e.g., osteoporosis, bone resorption
disease, loosening of artificial joint implants, and others listed
above); epidermolysis bullosa; ocular angiogenesis; corneal injury;
corneal scarring; and tissue ulceration.
[0205] Modulators provided herein may also be used for
neuroprotection of the optic nerve (e.g., toinhibit the death of
retinal ganglion cells in a patient).
[0206] Within other aspects, modulators provided herein may be used
within combination therapy for the treatment of conditions
responsive to P2X.sub.7 modulation (e.g., conditions involving pain
and/or 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 cerebro-vascular disease and certain
infectious diseases.
[0207] Within such combination therapy, a modulator is administered
to a patient along with a second therapeutic agent (e.g., an
analgesic and/or anti-inflammatory agent). The modulator and second
therapeutic 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, leflunomide, cyclosporine A, IM
gold, minocycline, azathioprine, tumor necrosis factor (TNF)
receptor antagonists, soluble TNF alpha receptor (etanercept),
anti-TNF alpha antibodies (e.g., infliximab and adalimumab),
anti-CS antibodies, interleukin-1 (IL-1) receptor antagonists
(e.g., anakinra or IL-1 trap), IL-18 binding protein, CTLA4-Ig
(e.g., abatacept), anti-human IL-6 receptor monoclonal antibody
(e.g., tocilizumab), LFA-3-Ig fusion proteins (e.g., alefacept),
LFA-1 antagonists, anti-VLA4 monoantibody (e.g., natalizumab),
anti-CD11a monoclonal antibody, anti-CD20 monoclonal antibody
(e.g., rituximab), anti-IL-12 monoclonal antibody, anti-IL-15
monoclonal antibody, CDP 484, CDP 870, chemokine receptor
antagonists, selective iNOS inhibitors, p38 kinase inhibitors,
integrin antagonists, angiogenesis inhibitors, and TMI-1 dual
inhibitors. Further anti-inflammatory agents include meloxicam,
rofecoxib, celecoxib, etoricoxib, parecoxib, valdecoxib and
tilicoxib.
[0208] NSAIDs include, but are not limited to, ibuprofen,
flurbiprofen, naproxen or naproxen sodium, diclofenac, combinations
of diclofenac sodium and misoprostol, sulindac, oxaprozin,
diflunisal, piroxicam, indomethacin, etodolac, fenoprofen calcium,
ketoprofen, sodium nabumetone, sulfasalazine, tolmetin sodium, and
hydroxychloroquine. One class of NSAIDs consists of compounds that
inhibit cyclooxygenase (COX) enzymes; such compounds include
celecoxib and rofecoxib. NSAIDs further include salicylates such as
acetylsalicylic acid or aspirin, sodium salicylate, choline and
magnesium salicylates, and salsalate, as well as corticosteroids
such as cortisone, dexamethasone, methylprednisolone, prednisolone,
prednisolone sodium phosphate, and prednisone.
[0209] Suitable dosages for P2X.sub.7 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 modulator with an anti-inflammatory agent
results in a reduction of the dosage of the anti-inflammatory agent
required to produce a therapeutic effect (i.e., a decrease in the
minimum therapeutically effective amount). Thus, preferably, the
dosage of anti-inflammatory agent in a combination or combination
treatment method is less than the maximum dose advised by the
manufacturer for administration of the anti-inflammatory agent
without combination administration of a modulator. 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 modulator. It will be apparent that the dosage
amount of modulator component of the combination needed to achieve
the desired effect may similarly be reduced by the
co-administraction of the anti-inflammatory agent.
[0210] In certain preferred embodiments, the combination
administration of a modulator with an anti-inflammatory agent is
accomplished by packaging one or more 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 modulators 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 modulators and one or more anti-inflammatory agents are to
be taken together for the treatment of an inflammatory pain
condition.
[0211] Within further aspects, 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 analgesic
agents, including narcotic 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, alfentanil, alphaprodine, anileridine, bezitramide,
buprenorphine, butorphanol, codeine, diacetyldihydromorphine,
diacetylmorphine, dihydrocodeine, diphenoxylate, ethylmorphine,
fentanyl, heroin, hydrocodone, hydromorphone, isomethadone,
levomethorphan, levorphane, levorphanol, meperidine, metazocine,
methadone, methorphan, metopon, morphine, nalbuphine, opium
extracts, opium fluid extracts, powdered opium, granulated opium,
raw opium, tincture of opium, oxycodone, oxymorphone, paregoric,
pentazocine, pethidine, phenazocine, piminodine, propoxyphene,
racemethorphan, racemorphan, sulfentanyl, thebaine and
pharmaceutically acceptable salts and hydrates of the foregoing
agents.
[0212] Other examples of narcotic analgesic agents include
acetorphine, acetyldihydrocodeine, acetylmethadol, allylprodine,
alphracetylmethadol, alphameprodine, alphamethadol, benzethidine,
benzylmorphine, betacetylmethadol, betameprodine, betamethadol,
betaprodine, 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,
naltyhexone, nicocodeine, nicomorphine, noracymethadol,
norlevorphanol, normethadone, normorphine, norpipanone,
pentazocaine, phenadoxone, phenampromide, phenomorphan,
phenoperidine, piritramide, pholcodine, proheptazoine, properidine,
propiran, racemoramide, thebacon, trimeperidine and the
pharmaceutically acceptable salts and hydrates thereof.
[0213] Further specific representative analgesic agents include,
for example acetaminophen (paracetamol); aspirin and other NSAIDs
described above; NR2B antagonists; bradykinin antagonists;
anti-migraine agents; anticonvulsants such as oxcarbazepine and
carbamazepine; antidepressants (such as TCAs, SSRIs, SNRIs,
substance P antagonists, etc.); spinal blocks;
pentazocine/naloxone; meperidine; levorphanol; buprenorphine;
hydromorphone; fentanyl; sufentanyl; oxycodone;
oxycodone/acetaminophen, nalbuphine and oxymorphone. Still further
analgesic agents include CB2-receptor agonists, such as AM1241,
capsaicin receptor antagonists and compounds that bind to the 1126
subunit of voltage-gated calcium channels, such as gabapentin and
pregabalin.
[0214] Representative anti-migraine agents for use in combination
with a modulator provided herein include CGRP antagonists,
capsaicin receptor antagonists, ergotamines and 5-HT.sub.1
agonists, such as sumatripan, naratriptan, zolmatriptan and
rizatriptan.
[0215] Within still further aspects, modulators provided herein may
be used, for example, in the treatment of pulmonary disorders such
as asthma, in combination with one or more beta(2)-adrenergic
receptor agonists or leukotriene receptor antagonists (e.g., agents
that inhibits the cysteinyl leukotriene CysLT.sub.1 receptor).
CysLT.sub.1 antagonists include montelukast, zafirlukast, and
pranlukast.
[0216] For retinal neuroprotection and treatment of ocular
disorders, P2X.sub.7 modulators may be administered to the eye in
combination with, for example, one or more of an agent that
inhibits ATP release, an agent that enhances conversion of ATP to
adenosine and/or an agent that inhibits Ca.sup.+2 influx into
retinal ganglion cells. Such agents include, for example, adenosine
A.sub.3 receptor agonists, adenosine A.sub.1 receptor agonists,
ectonucleotidase agonists, Ca.sup.+2 chelating agents and NMDA
receptor antagonists.
[0217] Suitable dosages for P2X.sub.7 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 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).
[0218] For use in combination therapy, pharmaceutical compositions
as described above may further comprise one or more additional
medications as described above. In certain such compositions, the
additional medication is an analgesic. Also provided herein are
packaged pharmaceutical preparations comprising one or more
modulators and one or more additional medications (e.g.,
analgesics) in the same package. Such packaged pharmaceutical
preparations generally include (i) a container holding a
pharmaceutical composition that comprises at least one modulator as
described herein; (ii) a container holding a pharmaceutical
composition that comprises at least one additional medication (such
as a pain relief and/or anti-inflammatory medication) as described
above and (iii) instructions (e.g., labeling or a package insert)
indicating that the compositions are to be used simultaneously,
separately or sequentially for treating or preventing a condition
responsive to P2X.sub.7 modulation in the patient (such as a
condition in which pain and/or inflammation predominates).
[0219] Within separate aspects, the present invention provides a
variety of non-pharmaceutical in vitro and in vivo uses for the
modulator compounds provided herein. For example, such compounds
may be labeled and used as probes for the detection and
localization of P2X.sub.7 (in samples such as cell preparations or
tissue sections, preparations or fractions thereof). In addition,
modulators 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, modulators provided herein may be used as positive
controls in assays for receptor activity or as radiotracers (e.g.,
in receptor mapping procedures). For example, a modulator compound
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 used as a probe for receptor autoradiography
(receptor mapping) of P2X.sub.7 in cultured cells or tissue
samples, which 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, which sections are incorporated herein
by reference. Such receptor mapping procedures also include methods
that can be used to characterize P2X.sub.7 in living subjects, such
as positron emission tomography (PET) imaging or single photon
emission computerized tomography (SPECT).
[0220] 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
[0221] Mass spectroscopy data provided herein is Electrospray MS,
obtained in positive ion mode. Unless otherwise specified, such
data is obtained using a Micromass Time-of-Flight LCT (Waters
Corp.; Milford, Mass.), equipped with a Waters 600 pump (Waters
Corp.), Waters 996 photodiode array detector (Waters Corp.), and a
Gilson 215 autosampler (Gilson, Inc.; Middleton, Wis.).
MassLynx.TM. (Waters Corp.) version 4.0 software with OpenLynx
Global Server.TM., OpenLynx.TM. and AutoLynx.TM. processing is used
for data collection and analysis. MS conditions are as follows:
capillary voltage=3.5 kV; cone voltage=30 V, desolvation and source
temperature=350.degree. C. and 120.degree. C., respectively; mass
range=181-750 with a scan time of 0.22 seconds and an interscan
delay of 0.05 seconds.
[0222] For data marked with a ".sctn.," mass spectroscopy data is
obtained using a Waters ZMD II Mass Spectrometer (Waters Corp.),
equipped with a Waters 600 pump (Waters Corp.), Waters 996
photodiode array detector (Waters Corp.), and a Gilson 215
autosampler (Gilson, Inc.; Middleton, Wis.). MassLynx.TM. (Waters
Corp.) version 4.0 software with OpenLynx Global Server.TM.,
OpenLynx.TM. and AutoLynx.TM. processing is used for data
collection and analysis. MS conditions are as follows: capillary
voltage=3.5 kV; cone voltage=30 V, desolvation and source
temperature=250.degree. C. and 100.degree. C., respectively; mass
range=100-800 with a scan time of 0.5 seconds and an interscan
delay of 0.1 seconds.
[0223] For either method, sample volume of 1 microliter is injected
onto a 50.times.4.6 mm Chromolith SpeedROD RP-18e column (Merck
KGaA, Darmstadt, Germany), and eluted using a 2-phase linear
gradient at a flow rate of 6 ml/min. Sample is detected using total
absorbance count over the 220-340 nm UV range. The elution
conditions are: Mobile Phase A--95% water, 5% MeOH with 0.05% TFA;
Mobile Phase B--5% water, 95% MeOH with 0.025% TFA. The following
gradient is used: 0-0.5 min 10-100% B, hold at 100% B to 1.2 min,
return to 10% B at 1.21 min. Inject to inject cycle is 2.15
min.
[0224] Where indicated, retention times (R.sub.T) are provided in
minutes.
Example 1
Preparation of Representative Heteroaryl Amide Analogues
[0225] This Example illustrates the preparation of representative
heteroaryl amide analogues of Formula I, as well as certain
intermediates useful in the preparation of such compounds. The
P2X.sub.7 IC.sub.50, determined as described in Example 4A, is 2
micromolar or less for Compounds 1-15.
A.
4-[(Adamantan-1-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyridine-2-carboxylic
acid ethyl ester (Compound 1)
Step 1. 4-Hydroxymethylpyrazolo[1,5-a]pyridine-2,3-dicarboxylic
acid dimethyl ester
##STR00040##
[0227] A solution of O-mesitylenesulfonylhydroxylamine,
.about.30-35% water, (14.5 g, 52 mmol) and CH.sub.2Cl.sub.2 (100
mL) is added to a solution of 3-pyridylcarbinol (5.0 mL, 52 mmol)
and CH.sub.2Cl.sub.2 (100 mL) at 0.degree. C. dropwise over 40 min.
After 30 min at 0.degree. C., the ice bath is removed and the
solution is stirred at ambient temperature for 20 min. The
volatiles are removed under reduced pressure to afford
N-amino-3-hydroxymethylpyridinium mesityl sulfonate as a yellow
oil. DMF (100 mL) and K.sub.2CO.sub.3 (15 g, 110 mmol) are added.
The dark brown mixture is cooled in a water bath. Dimethyl
acetylenedicarboxylate (6.9 mL, 56 mmol) are added dropwise over 10
min. After 15 min, the water bath is removed and the mixture is
left to vigorously stir under air for 18 h. After filtering through
Celite (CH.sub.2Cl.sub.2 rinse), the volatiles are removed under
reduced pressure. The residue is slurried in EtOAc and the mixture
is filtered through Celite (EtOAc rinse). The volatiles are removed
under reduced pressure to afford a dark brown oil. Purification by
flash column chromatography (2:1 hexanes:EtOAc to 1:1
hexanes:EtOAc) affords the title compound as a light yellow solid.
LC-MS m/z (M+Na.sup.+): 287.08.sup..sctn..
Step 2. 4-Hydroxymethylpyrazolo[1,5-a]pyridine-2-carboxylic acid
ethyl ester
##STR00041##
[0229] 50% Aqueous H.sub.2SO.sub.4 (400 mL) is added to
4-hydroxymethylpyrazolo[1,5-a]pyridine-2,3-dicarboxylic acid
dimethyl ester (11.6 g, 43.9 mmol) under air. The mixture is warmed
to 80.degree. C. After 3.5 h, the solution is transferred to a 2 L
flask and cooled to 0.degree. C. The solution is neutralized with
10 N aq. NaOH. The resulting slurry is acidified to pH 3 with 1 M
aq. HCl. The volatiles are removed under reduced pressure. The
residue is washed with 30% EtOH in CH.sub.2Cl.sub.2. The volatiles
are removed under reduced pressure to afford the title compound as
a tan solid. LC-MS m/z (M+H.sup.+): 193.07.
[0230] Acetyl chloride (9.5 mL, 130 mmol) is added to absolute
ethanol (200 mL). After 30 min, the solution is poured into the
flask containing
4-hydroxymethylpyrazolo[1,5-a]pyridine-2-carboxylic acid. The
mixture is warmed to 50.degree. C. for 3.5 h. After cooling to rt,
the mixture is made basic with dropwise addition of sat. aq.
NaHCO.sub.3. The volatiles are removed under reduced pressure. The
aqueous solution is extracted with EtOAc. The organics are dried
over Na.sub.2SO.sub.4, filtered, and concentrated to afford the
title compound as a tan powder. LC-MS m/z (M+Na.sup.+):
242.98.sup..sctn..
Step 3. Pyrazolo[1,5-a]pyridine-2,4-dicarboxylic acid 2-ethyl
ester
##STR00042##
[0232] A 2.65 M solution of the Jones reagent (28 mL) is added
dropwise over 40 min to a solution of
4-hydroxymethylpyrazolo[1,5-a]pyridine-2-carboxylic acid ethyl
ester (5.30 g, 24.1 mmol) and acetone (240 mL) under air cooled by
a water bath. After 20 min, isopropanol (3 mL) is added dropwise.
After stirring for 30 min, the blue-green mixture is filtered
(acetone rinse). The organics are concentrated under reduced
pressure. The residue is dissolved in water. Et.sub.2O is added and
the mixture is stirred for 15 min. The resulting solid is collected
by filtration. The filtrate is dried to afford the title compound
as a tan solid. LC-MS m/z (M+Na.sup.+): 257.02.sup..sctn..
Step 4.
4-[(Adamantan-1-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyridine-2-carbo-
xylic acid ethyl ester
##STR00043##
[0234] BOP (11 g, 24 mmol) is added to a slurry of
pyrazolo[1,5-a]pyridine-2,4-dicarboxylic acid 2-ethyl ester (5.6 g,
24 mmol), adamantan-1-ylmethylamine (4.4 g, 27 mmol), iPr.sub.2NEt
(21 mL, 120 mmol), and DMF (240 mL) under N.sub.2. The reaction
vessel is sealed and the mixture is left to stir for 15 h. EtOAc
(200 mL) is added. The solution is washed with 50% sat. aq.
NH.sub.4Cl (2.times.500 mL). The aqueous phases are extracted with
EtOAc (3.times.200 mL). The combined organics are dried over
Na.sub.2SO.sub.4, filtered, and concentrated. Purification by flash
column chromatography (2:1 hexanes:EtOAc to 1:1 hexanes:EtOAc)
affords the title compound as a tan solid. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta.: 8.62 (d, 1H), 7.60 (d, 1H), 7.44 (s, 1H), 7.00
(t, 1H), 6.23 (bs, 1H), 4.48 (q, 2H), 3.23 (d, 2H), 1.98-2.07 (m,
3H), 1.57-1.78 (m, 12H), 1.45 (t, 3H). LC-MS m/z (M+H.sup.+):
382.2; R.sub.T=1.09 min.
B.
N-(adamantan-1-ylmethyl)-2-{[(3R)-3-aminopyrrolidin-1-yl]carbonyl}pyraz-
olo[1,5-a]pyridine-4-carboxamide (Compound 2)
Step 1.
4-[(Adamantan-1-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyridine-2-carbo-
xylic acid
##STR00044##
[0236] 3 M Aqueous KOH (2.6 mL) is added to a mixture of
4-[(adamantan-1-ylmethyl)carbamoyl]-pyrazolo[1,5-a]pyridine-2-carboxylic
acid ethyl ester (503 mg, 1.32 mmol) and EtOH (6.6 mL). After 4 h,
the volatiles are removed under reduced pressure. The aqueous
residue is diluted with water (2 mL) and then acidified with 1 M
aq. HCl to pH 2. The solids are collected by filtration and then
dried to afford the title compound as a light yellow powder.
.sup.1H NMR (400 MHz, ((CD.sub.3).sub.2SO) .delta.: 8.86 (d, 1H),
8.50 (t, 1H), 7.71 (d, 1H), 7.26 (s, 1H), 7.12 (t, 1H), 3.00 (d,
2H), 1.88-1.98 (m, 3H), 1.48-1.69 (m, 12H). LC-MS m/z (M+H.sup.+):
354.09.
Step 2.
N-(adamantan-1-ylmethyl)-2-{[(3R)-3-aminopyrrolidin-1-yl]carbonyl}-
pyrazolo[1,5-a]pyridine-4-carboxamide
##STR00045##
[0238] BOP (680 mg, 1.5 mmol) is added to a mixture of
4-[(adamantan-1-ylmethyl)carbamoyl]-pyrazolo[1,5-a]pyridine-2-carboxylic
acid (417 mg, 1.18 mmol), tert-butyl(R)-pyrrolidin-3-ylcarbamate
(242 mg, 1.30 mmol), iPr.sub.2Net (620 .mu.L, 3.6 mmol) and DMF (12
mL) under N.sub.2. The reaction vessel is sealed and the solution
is left to stir for 20 h. EtOAc (50 mL) is added. The solution is
washed with H.sub.2O (2.times.50 mL) and brine (50 mL). The
organics are dried over Na.sub.2SO.sub.4, filtered, and
concentrated. Purification by flash column chromatography (2% MeOH
in CH.sub.2Cl.sub.2) affords (R)--
(1-{4-[(adamantan-1-ylmethyl)carbamoyl]pyrazolo[1,5-a]-pyridine-2-carbony-
l}pyrrolidin-3-yl)carbamic acid tert-butyl ester as a tan solid.
MeOH (6.8 mL) and 4 M HCl in dioxane (1.7 mL) are added. After 20
h, the volatiles are removed under reduced pressure to afford a
light tan solid. 15% Aqueous K.sub.2CO.sub.3 (50 mL) is added. The
solution is extracted with EtOAc (5.times.50 mL). The combined
organics are dried over Na.sub.2SO.sub.4, filtered, and
concentrated. Purification by flash column chromatography (1%
NH.sub.4OH in 9:1 CH.sub.2Cl.sub.2:MeOH) affords the title compound
as a tan foam. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.: 8.48
(ddd, 1H), 7.62 (dd, 1H), 7.22 (dd, 1H), 6.87 (t, 1H), 6.50 (bt,
1H), 3.42-4.20 (m, 5H), 3.20 (d, 2H), 2.14 (septet, 1H), 1.96-2.04
(m, 3H), 1.55-1.88 (m, 12H). LC-MS m/z (M+H.sup.+): 422.3;
R.sub.T=1.2 min.
C.
N-(adamantan-1-ylmethyl)-2-(hydroxymethyl)pyrazolo[1,5-a]pyridine-4-car-
boxamide (Compound 3)
##STR00046##
[0240] A 2.0 M solution of LiBH.sub.4 in THF (5.8 mL) is added
dropwise to a solution of
4-[(adamantan-1-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyridine-2-carboxylic
acid ethyl ester (2.01 g, 5.27 mmol) and THF (50 mL) at 0.degree.
C. under N.sub.2. After 10 min, the ice bath is removed. After 3 h,
additional 2.0 M solution of LiBH.sub.4 in THF (2.0 mL) is added.
After 4 h, sat. aq. NH.sub.4Cl is added dropwise. The volatiles are
removed under reduced pressure. H.sub.2O (20 mL) is added and the
solids are collected by filtration. The solids are slurried in MeOH
(75 mL) and warmed to 50.degree. C. for 4 h. The volatiles are
removed under reduced pressure to afford the title compound as a
light tan solid. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.: 8.58
(ddd, 1H), 7.56 (dd, 1H), 6.92 (t, 1H), 6.90 (s, 1H), 4.80 (s, 2H),
3.12 (s, 2H), 1.96-2.04 (m, 3H), 1.62-1.84 (m, 12H). LC-MS m/z
(M+H.sup.+): 340.3; R.sub.T=1.27 min.
D.
N-(adamantan-1-ylmethyl)-2-{[(3R)-3-aminopyrrolidin-1-yl]methyl}pyrazol-
o[1,5-a]pyridine-4-carboxamide (Compound 4)
Step 1. Methanesulfonic acid
4-[(adamantly-1-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyridine-2-ylmethyl
ester
##STR00047##
[0242] Methanesulfonyl chloride (140 .mu.l, 1.2 mmol) is added to a
slurry of 2-hydroxymethyl-pyrazolo[1,5-a]pyridine-4-carboxylic acid
(adamantan-1-ylmethyl)amide (510 mg, 1.5 mmol) in THF (15 mL) and
triethylamine (310 .mu.L, 2.3 mmol) under N.sub.2. After 1 h, the
mixture is filtered (THF rinse). The volatiles are removed under
reduced pressure to afford the title compound as a pale yellow
solid. LC-MS m/z (M+Na.sup.+): 440.04.
Step 2.
N-(adamantan-1-ylmethyl)-2-{[(3R)-3-aminopyrrolidin-1-yl]methyl}py-
razolo[1,5-a]pyridine-4-carboxamide
##STR00048##
[0244] tert-Butyl(R)-pyrrolidin-3-ylcarbamate (420 mg, 2.3 mmol) is
added to a mixture of methanesulfonic acid
4-[(adamantly-1-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyridine-2-ylmethyl
ester (630 mg, 1.5 mmol), K.sub.2CO.sub.3 (530 mg, 3.8 mmol) and
DMF (15 mL) under N.sub.2. After 3 h, H.sub.2O (50 mL) is added.
The solution is extracted with EtOAc (50 mL). The organics are
washed with H.sub.2O (50 mL) and brine (50 mL) and then dried over
Na.sub.2SO.sub.4, filtered and concentrated. Purification by flash
column chromatography (1% NH.sub.4OH in 95:5 CH.sub.2Cl.sub.2:MeOH)
affords
(R)-(1-{4-[(adamantan-1-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyridin-2-ylmet-
hyl}pyrrolidin-3-yl)carbamic acid tert-butyl ester as a tan foam.
The foam is dissolved in MeOH (12 mL) and 4 M HCl in dioxane (3.0
mL). After 6 h, the volatiles are removed under reduced pressure to
afford a tan foam. 15% Sat. aq. K.sub.2CO.sub.3 (50 mL) is added
and the mixture is extracted with EtOAc (5.times.25 mL). The
combined organics are dried over Na.sub.2SO.sub.4, filtered and
concentrated. Purification by flash column chromatography (1%
NH.sub.4OH in 9:1 CH.sub.2Cl.sub.2:MeOH to 2% NH.sub.4OH in 4:1
CH.sub.2Cl.sub.2:MeOH) affords the title compound as a tan foam.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta.: 8.49 (d, 1H), 7.54 (dd,
1H), 6.77 (s, 1H, 6.75 (t, 1H), 6.30 (bt, 1H), 4.85 (dd, 2H),
3.48-3.58 (m, 1H), 3.20 (d, 2H), 2.77 2.88 (m, 2H), 2.52 (m, 1H),
2.43 (dd, 1H), 2.14-2.28 (m, 1H), 1.45-2.05 (m, 18H). LC-MS m/z
(M+Fr): 408.3; R.sub.T=1.15 min.
E.
N-(adamantan-1-ylmethyl)-2-(cyanomethyl)pyrazolo[1,5-a]pyridine-4-carbo-
xamide (Compound 5)
##STR00049##
[0246] Sodium cyanide (520 mg, 11 mmol) is added to a solution of
methanesulfonic acid
4-[(adamantly-1-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyridine-2-ylmethyl
ester (720 mg, 2.1 mmol) and DMF (20 mL) under N.sub.2. The mixture
is warmed to 60.degree. C. for 1 h. After cooling to rt, the
mixture is diluted with EtOAc (50 mL) and then washed with 50% sat.
aq. NaHCO.sub.3 (2.times.50 mL) and brine (50 mL). The organics are
dried over Na.sub.2SO.sub.4, filtered and concentrated.
Purification by flash column chromatography (2:1 EtOAc:hexanes)
affords the title compound as a tan foam. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta.: 8.48 (d, 1H), 7.54 (dd, 1H), 6.91 (s, 1H),
6.83 (t, 1H), 6.26 (bs, 1H), 3.97 (s, 2H), 3.20 (d, 2H), 1.97-2.05
(m, 3H), 1.55-1.79 (m, 12H). LC-MS m/z (M+H.sup.+): 349.3;
R.sub.T=1.28 min.
F.
{4-[(Adamantan-1-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyridin-2-yl}acetic
acid (Compound 6)
##STR00050##
[0248] A solution of conc. HCl (2.3 mL) and AcOH (4.6 mL) is added
to 2-cyanomethylpyrazolo[1,5-a]pyridine-4-carboxylic acid
(adamantan-1-ylmethyl)amide. The solution is warmed to 60.degree.
C. for 20 h. The volatiles are removed under reduced pressure, and
the residue is dissolved in 1 M aq. NaOH (10 mL). The solution is
washed with Et.sub.2O (20 mL). The aqueous layer is acidified to
.about.pH 3 with 1 M aq. HCl. The mixture is filtered and the
filtrate dried to afford the title compound as a light tan solid.
.sup.1H NMR (400 MHz, ((CD.sub.3).sub.2SO) .delta.: 8.71 (d, 1H),
8.40 (bt, 1H), 7.60 (d, 1H), 6.90 (t, 1H), 6.79 (s, 1H), 3.75 (s,
2H), 2.99 (d, 2H), 1.88-1.96 (m, 3H), 1.46-1.70 (m, 12H). LC-MS m/z
(M+Yr): 368.3; R.sub.T=1.27 min.
G.
N-(adamantan-1-ylmethyl)-2-{2-[(3R)-3-aminopiperidin-1-yl]-2-oxoethyl}p-
yrazolo[1,5-a]pyridine-4-carboxamide (Compound 7)
##STR00051##
[0250] BOP (250 mg, 570 mmol) is added to a mixture of
{4-[(adamantan-1-ylmethyl)carbamoyl]-pyrazolo[1,5-a]pyridin-2-yl}acetic
acid (160 mg, 435 .mu.mol), tert-butyl(R)-piperidin-3-ylcarbamate
(96 mg, 480 .mu.mol), iPr.sub.2NEt (230 .mu.L, 1.3 mmol), and DMF
(2.2 mL) under N.sub.2. The reaction vessel is sealed and the
solution is left to stir for 15 h. EtOAc (25 mL) is added. The
solution is washed with H.sub.2O (2.times.25 mL) and brine (25 mL).
The organics are dried over Na.sub.2SO.sub.4, filtered and
concentrated. Purification by flash column chromatography (95:5
CH.sub.2Cl.sub.2:MeOH) affords
(R)-(1-{4-[(adamantan-1-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyridin-2-yl}ac-
etyl)piperidin-3-yl]carbamic acid tert-butyl ester as an orange
foam. MeOH (4.2 mL) and 4 M HCl in dioxane (1.1 mL) are added.
After 16 h, the volatiles are removed under reduced pressure to
afford a light tan solid. 15% Aqueous K.sub.2CO.sub.3 (30 mL) is
added. The solution is extracted with EtOAc (3.times.30 mL). The
combined organics are dried over Na.sub.2SO.sub.4, filtered and
concentrated. Purification by flash column chromatography (1%
NH.sub.4OH in 9:1 CH.sub.2Cl.sub.2:MeOH) affords the title compound
as a tan foam. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 8.46 8.64
(m, 1H), 7.44 (bs, 1H), 6.68 6.80 (m, 2H), 3.86 4.17 (m, 3H), 3.38
3.61 (m, 3H), 3.12 3.25 (m, 3H), 1.40 2.04 (m, 19H). LC-MS m/z
(M+Fr): 450.3; R.sub.T=1.17.
H.
{4-[(adamantan-1-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyridin-2-ylmethoxy}-
acetic acid ethyl ester (Compound 8)
##STR00052##
[0252] A 1.0 M solution of tBuOK in THF (0.3 mL) is added to a
mixture of 2-hydroxymethyl-pyrazolo[1,5-a]pyridine-4-carboxylic
acid (adamantan-1-ylmethyl)amide (73 mg, 210 .mu.mol) and DMF (1
mL) at 0.degree. C. under N.sub.2. After 5 min, ethyl bromoacetate
(30 .mu.L, 280 .mu.mol) is added. The cold bath is removed and the
mixture is left to stir for 23 h. 50% Sat. aq. NaHCO.sub.3 is added
and the solution is extracted with EtOAc (20 mL). The organics are
dried over Na.sub.2SO.sub.4, filtered and concentrated.
Purification by PTLC (2:1 EtOAc:hexanes) affords the title compound
as a pale yellow film. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.:
8.50 (dd, 1H), 7.57 (d, 1H), 6.88 (s, 1H), 6.81 (dt, 1H), 6.27 (bs,
1H), 4.88 (s, 2H), 4.22 (q, 2H), 4.19 (s, 2H), 3.21 (d, 2H),
1.94-2.04 (m, 3H), 1.56-1.78 (m, 12H), 1.28 (t, 3H). LC-MS m/z
(M+H.sup.+): 426.3; R.sub.T=1.31.
I.
{4-[(adamantan-1-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyridin-2-ylmethoxy}-
acetic acid (Compound 9)
##STR00053##
[0254] 3 M Aq. KOH (50 .mu.L) is added to a solution of
{4-[(adamantan-1-ylmethyl)carbamoyl]-pyrazolo[1,5-a]pyridin-2-ylmethoxy}a-
cetic acid ethyl ester (11 mg, 25 .mu.mmol) and EtOH (250 4). After
18 h, the volatiles are removed under reduced pressure. The residue
is diluted with H.sub.2O (0.3 mL) and then acidified with 1 M aq.
HCl. The title compound is collected by filtration. .sup.1H NMR
(400 MHz, ((CD.sub.3).sub.2SO) .delta.: 8.74 (d, 1H), 8.42 (bs,
1H), 7.62 (d, 1H), 6.93 (t, 1H), 6.84 (s, 1H), 4.70 (s, 2H), 4.08
(s, 2H), 2.99 (d, 2H), 1.86-2.00 (m, 3H), 1.48-1.73 (m, 12H). LC-MS
m/z (M+H.sup.+): 398.3; R.sub.T=1.26.
J.
4-(2-Adamantan-1-ylacetylamino)pyrazolo[1,5-a]pyridine-2-carboxylic
acid ethyl ester (Compound 10)
Step 1. 4-Aminopyrazolo[1,5-a]pyridine-2,3-dicarboxylic acid
dimethyl ester
##STR00054##
[0256] A solution of O-mesitylenesulfonylhydroxylamine, 30-35%
water, (14.5 g, 52 mmol) and CH.sub.2Cl.sub.2 (100 mL) is added to
a solution of 3-aminopyridine (4.9 g, 52 mmol) and CH.sub.2Cl.sub.2
(100 mL) at 0.degree. C. dropwise over 30 min. After 30 min at
0.degree. C., the ice bath is removed and the solution is stirred
at rt for 30 min. The volatiles are removed under reduced pressure
to afford a yellow oil, N-amino-3-aminopyridinium mesityl
sulfonate. DMF (100 mL) and K.sub.2CO.sub.3 (15 g, 110 mmol) are
added. The dark brown mixture is cooled in a water bath. Dimethyl
acetylenedicarboxylate (6.9 mL, 56 mmol) are added dropwise over 10
min. After 15 min, the water bath is removed and the mixture is
left to vigorously stir under air for 66 h. The volatiles are
removed under reduced pressure. The residue is slurried in MeOH and
the mixture is filtered through Celite (MeOH rinse). The volatiles
are removed under reduced pressure to afford a dark brown oil. 33%
Sat. aq. NaHCO.sub.3 (150 mL) is added and the solution is
extracted with EtOAc (6.times.200 mL). The combined organics are
dried over Na.sub.2SO.sub.4, filtered, and concentrated.
Purification by flash column chromatography (CH.sub.2Cl.sub.2)
affords the title compound as an orange-red oil. LC-MS m/z
(M+Na.sup.+): 272.01.
Step 2. 4-Aminopyrazolo[1,5-a]pyridine-2-carboxylic acid ethyl
ester
##STR00055##
[0258] 50% Aqueous H.sub.2SO.sub.4 (60 mL) is added to
4-Aminopyrazolo[1,5-a]pyridine-2,3-dicarboxylic acid dimethyl ester
(1.47 g, 5.90 mmol) under air. The mixture is warmed to 80.degree.
C. After 6.5 h, the solution is cooled to 0.degree. C. and then
neutralized with 10 N aq. NaOH. The resulting slurry is acidified
to pH 3 with 1 M aq. HCl. The volatiles are removed under reduced
pressure. The residue is washed with 30% EtOH in CH.sub.2Cl.sub.2.
The volatiles are removed under reduced pressure to afford
4-aminopyrazolo[1,5-a]pyridine-2-carboxylic acid in
Na.sub.2SO.sub.4. LC-MS m/z (M+H.sup.+): 178.01. A solution of
conc. H.sub.2SO.sub.4 (6.0 mL) and ethanol (120 mL) is added to the
crude 4-aminopyrazolo[1,5-a]pyridine-2-carboxylic acid. The mixture
is warmed to 75.degree. C. for 3.5 h. After cooling to rt, the
mixture is made basic with dropwise addition of sat. aq.
NaHCO.sub.3. The volatiles are removed under reduced pressure. The
aqueous mixture is filtered (EtOAc rinse). The aqueous solution is
extracted with EtOAc (3.times.250 mL). The combined organics are
dried over Na.sub.2SO.sub.4, filtered, and concentrated to afford
the title compound as dark brown solid. LC-MS m/z (M+Na.sup.+):
228.03.
Step 3.
4-(2-Adamantan-1-ylacetylamino)pyrazolo[1,5-a]pyridine-2-carboxyli-
c acid ethyl ester
##STR00056##
[0260] A 1.0 M solution of triethylamine in toluene (0.3 mL) is
added to a solution of 1-adamantaneacetic acid (14 mg, 80 mot) and
4-aminopyrazolo[1,5-a]pyridine-2-carboxylic acid ethyl ester (12
mg, 60 mol) in DMF (0.3 mL) under N.sub.2. A solution of
2-chloro-1,3-dimethylimidazolinium chloride (20 mg, 120 mop in ACN
(0.3 mL) is added. The reaction vessel is sealed and the mixture is
warmed to 50.degree. C. for 2 h. The mixture is cooled to rt. 50%
Sat. aq. NaHCO.sub.3 (2 mL) is added. The solution is extracted
with Et OAc (2.times.1 mL). The combined organics are concentrated.
Purification by preparative thin layer chromatography (2:1
hexanes:EtOAc) affords the title compound as a light tan solid.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 8.32 (d, 1H), 8.05 (d,
1H), 7.15 (s, 1H), 7.05 (s, 1H), 6.91 (t, 1H), 4.59 (q, 2H), 2.21
(s, 2H), 1.98-2.06 (m, 3H), 1.56-1.78 (m, 12H), 1.46 (t, 3H). LC-MS
m/z (M+H.sup.+): 382.10.
Example 2
Synthesis of Additional Representative Heteroaryl Amide
Analogues
[0261] This Example illustrates the synthesis of additional
representative heteroaryl amide analogues of Formula I, as well as
certain intermediates useful in the preparation of such
compounds.
A. 2-(2-Cyano-vinyl)-pyrazolo[1,5-a]pyridine-4-carboxylic acid
(adamantan-1-ylmethyl)-amide (Compound 11)
Step 1. 2-Formyl-pyrazolo[1,5-a]pyridine-4-carboxylic acid
(adaman-1-ylmethyl)-amide
##STR00057##
[0263] A solution of
4-[(adaman-1-ylmethyl)-carbomoy]-pyrazolo[1,5-a]pyridine-2-carboxylic
acid ethyl ester (1.2 g, 3.1 mmol) in THF (25 mL) is cooled to
-78.degree. C. A solution of DIBAL-H (25 mL, 1M in hexane) is added
dropwise. The mixture is stirred for 2 h at -78.degree. C. A
saturated Rochelle salt solution is added, and the mixture is
stirred for 4 h at rt. The mixture is extracted with DCM
(4.times.70 mL), and the organic layer is dried over
Na.sub.2SO.sub.4. Purification by a flash column (eluted with 2-3%
MeOH in DCM) gives the title compound.
Step 2. 2-(2-Cyano-vinyl)-pyrazolo[1,5-a]pyridine-4-carboxylic acid
(adamantan-1-ylmethyl)-amide
##STR00058##
[0265] Ph.sub.3P.dbd.CHCN (735 mg, 2.5 mmol) is added to a solution
of 2-formyl-pyrazolo[1,5-a]pyridine-4-carboxylic acid
(adaman-1-ylmethyl)-amide (700 mg, 2.1 mmol) in THF at rt. The
mixture is stirred for 2 h. The solvent is removed, and the
resulting mixture of Z and E isomers is separated by a flash column
(eluted with 20 to 30% EtOAc in hexane) into pure Z isomer, pure E
isomer and a mixture (Z/E).
b.
N-(adamantan-1-ylmethyl)-2-(2-cyanoethyl)pyrazolo[1,5-a]pyridine-4-carb-
oxamide (Compound 12)
##STR00059##
[0267] Pd/C (50 mg) is added to a solution of
2-(2-cyano-vinyl)-pyrazolo[1,5-a]pyridine-4-carboxylic acid
(adamantan-1-ylmethyl)-amide in MeOH. The mixture is stirred under
hydrogen balloon for 6 h. The mixture is filtered through celite
and the celite and solvent are removed to give the title compound.
LC-MS m/z (M+H.sup.+): 363.
C.
2-[2-(1H-Tetrazol-5yl)-ethyl]-pyrazolo[1,5-a]pyridine-4-carboxylic
acid (adamantan-1-ylmethyl)-amide (Compound 13)
##STR00060##
[0269] NaN.sub.3 (65 mg, 0.48 mmol) and NH.sub.4Cl (26 mg, 0.48
mmol) are added to a solution of
2-(2-cyano-ethyl)-pyrazolo[1,5-a]pyridine-4-carboxylic acid
(adamantan-1-ylmethyl)-amide (58 mg, 0.16 mmol) in DMSO at rt. The
mixture is heated to 130.degree. C. for 6 h. Additional NaN.sub.3
(65 mg, 0.48 mmol) is added, and the mixture is heated for 14 h.
The mixture is cooled to rt. H.sub.2O is added and pH is adjusted
to about 7. The aqueous layer is extracted with EtOAc (3.times.15
mL), dried over Na.sub.2SO.sub.4 and the solvent is removed to
dryness. The crude product is purified by PTLC (eluted with 2% MeOH
in DCM) to give the title compound. .sup.1H NMR (CDCl.sub.3):
8.49-8.47 (d, 1H), 7.52-7.50 (d, 1H), 6.79-6.69 (t, 1H), 6.69 (s,
1H), 6.40 (b, 1H), 3.44-3.41 (m, 2H), 3.31-3.29 (m, 2H), 3.20-3.19
(m, 2H), 2.00 (b, 3H), 1.74-1.60 (m, 12H). LC-MS m/z (M+H.sup.+):
406.07.
D.
N-(adamantan-1-ylmethyl)-3-(3-cyanobenzoyl)indolizine-8-carboxamide
(Compound 14)
Step 1. N-(Adamantan-1-ylmethyl)-2-methylnicotinamide
##STR00061##
[0271] To a mixture of 2-methylnicotinic acid (2.06 g, 15 mmol) in
40 mL of DMF is added sequentially diisopropylethylamine (5.2 mL,
30 mmol), 1-adamantan-1-ylmethanamine (2.5 g, 15 mmol), and
benzotriazol-1-yloxytris-(dimethylamino)-phosphonium
hexafluorophosphate (BOP reagent, 8 g, 18 mmol). The resulting
mixture is stirred at rt for 17 h. The mixture is then poured into
ice water (150 mL), and the precipitated solid is collected by
vacuum filtration and dried in vacuo to afford the title compound
as an off-white solid. Mass spec. (285.19, M+H).
Step 2.
3-[(Adamantan-1-ylmethyl)carbamoyl]-1-[2-(3-cyanophenyl)-2-oxoethy-
l]-2-methylpyridinium bromide
##STR00062##
[0273] A mixture of N-(adamantan-1-ylmethyl)-2-methylnicotinamide
(300 mg, 1.05 mmol) and 3-(bromoacetyl)benzonitrile (235 mg, 1.05
mmol) in 10 mL of acetone is stirred at reflux for 2.5 days. After
cooling to rt, most of the acetone is evaporated, and the remaining
mixture is filtered. The collected solid is dried in vacuo to give
the title compound as a white solid. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 8.93 (1H, m), 8.86 (1H, bs), 8.62 (1H, d),
8.58 (1H, s), 8.33 (1H, d), 8.26 (1H, d), 8.14 (1H, t), 7.87 (1H,
t), 6.60 (2H, s), 3.01 (2H, d), 2.67 (3H, s), 1.94 (3H, s), 1.63
(6H, m), 1.51 (6H, s).
Step 3.
N-(Adamantan-1-ylmethyl)-3-(3-cyanobenzoyl)indolizine-8-carboxamid-
e
##STR00063##
[0275] A mixture of
3-[(adamantan-1-ylmethyl)carbamoyl]-1-[2-(3-cyanophenyl)-2-oxoethyl]-2-me-
thylpyridinium bromide (400 mg, 0.81 mmol) and
N,N-dimethylformamide dimethyl acetal (0.22 mL, 1.62 mmol) in DMF
(2.0 mL) is stirred at 110.degree. C. for 3 h. The mixture is
concentrated in vacuo and purified by column chromatography
(gradient from CH.sub.2Cl.sub.2 to 10% EtOAc/CH.sub.2Cl.sub.2) to
give the title compound as a yellow solid. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 9.91 (1H, d), 8.53 (1H, t), 8.15 (1H, m),
8.05 (2H, m), 7.74 (1H, t), 7.66 (1H, dd), 7.44 (1H, d), 7.22 (1H,
t), 6.95 (1H, d), 3.01 (2H, d), 1.93 (3H, s), 1.63 (6H, m), 1.52
(6H, s). Mass spec. (438.19, M+H).
E.
N-(Adamantan-1-ylmethyl)-3-(3-carbamoylbenzoyl)indolizine-8-carboxamide
(Compound 15)
##STR00064##
[0277] To ice cold H.sub.2SO.sub.4 (0.5 mL) is added in one portion
N-(adamantan-1-ylmethyl)-3-(3-cyano-benzoyl)indolizine-8-carboxamide
(50 mg, 0.11 mmol). The mixture is stirred at 0.degree. C. until it
becomes homogeneous, at which time the ice bath is removed and the
reaction is warmed to rt. After stirring for 12 h, the mixture is
cooled back to 0.degree. C. and quenched with ice. The resulting
solution is neutralized to pH=7-8 with 5N NaOH and extracted with
EtOAc (20 mL). The EtOAc extract is dried (Na.sub.2SO.sub.4),
filtered, and evaporated to give the title compound as a yellow
solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 9.92 (1H, d),
8.50 (1H, t), 8.22 (1H, s), 8.09 (2H, m), 7.88 (1H, d), 7.62 (2H,
m), 7.48 (1H, s), 7.40 (1H, d), 7.19 (1H, t), 6.94 (1H, d), 3.00
(2H, s), 1.93 (3H, s), 1.62 (6H, m), 1.52 (6H, s). Mass spec.
(456.21, M+H).
Example 3
Synthesis of Additional Representative Heteroaryl Amide
Analogues
[0278] Using routine modifications, the starting materials may be
varied and additional steps employed to produce other compounds
provided herein. Compounds listed in Tables I and II are prepared
using such methods. In the column of Table I labeled "IC.sub.50," a
"*" indicates that the IC.sub.50 determined as described in Example
4A is 2 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 80 .mu.M of
(2'(3')-O-(4-benzoyl-benzoyl)adenosine 5'-triphosephate is 2
micromolar or less).
Mass spectroscopy data is provided in Table I as (M+1) in the
column headed "MS." The retention time, in minutes, is provided in
the column headed R.sub.T.
TABLE-US-00001 TABLE I Representative Heteroaryl Amide Analogues
Compound Name MS R.sub.T IC.sub.50 16 ##STR00065## N-(adamantan-1-
ylmethyl)-2-{[(3R)-3- (isopropylamino) pyrrolidin-1-
yl]carbonyl}pyrazolo[1, 5-a]pyridine-4- carboxamide 464.3 1.21 * 17
##STR00066## N-(adamantan-1- ylmethyl)-2-{[(3R)-3-
aminopiperidin-1- yl]carbonyl}pyrazolo[1, 5-a]pyridine-4-
carboxamide 436.3 1.18 * 18 ##STR00067## 4-N-(adamantan-1-
ylmethyl)-2-N-(2- methoxyethyl)-2-N- methylpyrazolo[1,5-
a]pyridine-2,4- dicarboxamide 425.3 1.3 * 19 ##STR00068##
4-N-(adamantan-1- ylmethyl)-2-N-methyl- 2-N-pyridin-2-
ylpyrazolo[1,5- a]pyridin-2,4- dicarboxamide 444.3 1.28 * 20
##STR00069## N-(adamantan-1- ylmethyl)-2-(5,6- dihydroimidazo[1,5-
a]pyrazin-7(8H)- ylcarbonyl)pyrazolo[1, 5-a]pyridine-4- carboxamide
459.3 1.2 * 21 ##STR00070## 4-N-(adamantan-1- ylmethyl)-2-N-methyl-
2-N-(pyridin-3- ylmethyl)pyrazolo[1,5- a]pyridine-2,4-
dicarboxamide 458.2 1.37 * 22 ##STR00071## N-(adamantan-1-
ylmethyl)-2- (morpholin-4- ylcarbonyl)pyrazolo[1,- 5-a]pyridine-4-
carboxamide 423.2 1.44 * 23 ##STR00072## ethyl N-({4-
[(adamantan-1- ylmethyl)carbamoyl] pyrazolo[1,5-a]pyridin-2-
yl}carbonyl)-N- methylglycine 453.3 1.3 * 24 ##STR00073##
N-(adamantan-1- ylmethyl)-2-(5,6- dihydro[1,2,4]triazolo[4,
3-a]pyrazin-7(8H)- ylcarbonyl)pyrazolo[1, 5-a]pyridine-4-
carboxamide 460.3 1.24 * 25 ##STR00074## N-(adamantan-1-
ylmethyl)-2-(2- morpholin-4-yl-2- oxoethyl)pyrazolo[1,5-
a]pyridine-4- carboxamide 437.2 1.39 * 26 ##STR00075##
4-N-(adamantan-1- ylmethyl)-2-N-methyl- 2-N-[(1-methyl-1H-
imidazol-5- yl)methyl]pyrazolo[1,5- a]pyridine-2,4- dicarboxamide
461.3 1.2 * 27 ##STR00076## N-(adamantan-1- ylmethyl)-2-{[(3R)-3-
(dimethylamino) pyrrolidin-1- yl]carbonyl}pyrazolo[1,
5-a]pyridine-4- carboxamide 450.3 1.17 * 28 ##STR00077##
4-N-(adamantan-1- ylmethyl)-2-N-methyl- 2-N-(tetrahydrofuran-
3-yl)pyrazolo[1,5- a]pyridine-2,4- dicarboxamide 437.3 1.28 * 29
##STR00078## ethyl N-({4- [(adamantan-1- ylmethyl)carbamoyl]
pyrazolo[1,5-a]pyridin-2- yl}methyl)-N- methylglycinate 439.3 1.22
* 30 ##STR00079## N-(adamantan-1- ylmethyl)-2-[(2-methyl-
4-oxo-3,5,7,8- tetrahydropyrido[4,3- d]pyrimidin-6(4H)-
yl)carbonyl]pyrazolo[1, 5-a]pyridine-4- carboxamide 501.3 1.26 * 31
##STR00080## N-(adamantan-1- ylmethyl)-2-{[(3R)-3- (diethylamino)
pyrrolidin-1- yl]carbonyl}pyrazolo[1, 5-a]pyridine-4- carboxamide *
32 ##STR00081## N-(adamantan-1- ylmethyl)-2-[(3- oxopiperazin-1-
yl)carbonyl]pyrazolo[1, 5-a]pyridine-4- carboxamide 436.2 1.26 * 33
##STR00082## 4-N-(adamantan-1- ylmethyl)-2-N-methyl-
2-N-(pyridin-4- ylmethyl)pyrazolo[1,5- a]pyridine-2,4-
dicarboxamide 458.2 1.31 * 34 ##STR00083## N-(adamantan-1-
ylmethyl)-2-[(1-methyl- 1,4,6,7-tetrahydro-5H-
pyrazolo[4,3-c]pyridin- 5- yl)carbonyl]pyrazolo[1, 5-a]pyridine-4-
carboxamide 473.3 1.29 * 35 ##STR00084## 4-N-(adamantan-1-
ylmethyl)-2-N- (tetrahydro-2H-pyran- 4-yl)pyrazolo[1,5-
a]pyridine-2,4- dicarboxamide 437.3 1.29 * 36 ##STR00085##
N-(adamantan-1- ylmethyl)-2-[(2-oxo-1- oxa-3,8-
diazaspiro[4.5]dec-8- yl)carbonyl]pyrazolo[1, 5-a]pyridine-4-
carboxamide 492.3 1.26 * 37 ##STR00086## 4-N-(adamantan-1-
ylmethyl)-2-N-methyl- 2-N-(tetrahydro-2H- pyran-4- yl)pyrazolo[1,5-
a]pyridine-2,4- dicarboxamide 451.3 1.3 * 38 ##STR00087##
4-N-(adamantan-1- ylmethyl)-2-N-(3- methoxypropyl)pyrazolo
[1,5-a]pyridine-2,4- dicarboxamide 425.3 1.3 * 39 ##STR00088##
4-N-(adamantan-1- ylmethyl)-2-N-methyl- 2-N-pyridin-4-
ylpyrazolo[1,5- a]pyridine-2,4- dicarboxamide 444.3 1.21 * 40
##STR00089## N-(adamantan-1- ylmethyl)-2-[(3-oxo-
2,8-diazaspiro[4.5]dec- 8- yl)carbonyl]pyrazolo[1,- 5-a]pyridine-4-
carboxamide 490.4 1.25 * 41 ##STR00090## ethyl N-({4-
[(adamantan-1- ylmethyl)carbamoyl] pyrazolo[1,5-a]pyridin-2-
yl}carbonyl)glycinate 439.3 1.3 * 42 ##STR00091## 4-N-(adamantan-1-
ylmethyl)-2-N-(2- methoxyethyl)pyrazolo [1,5-a]pyridine-2,4-
dicarboxamide 411.3 1.28 * 43 ##STR00092## 2-{[(3R)-3-
aminopiperidin-1- yl]carbonyl}-N-(4- methyl-2-
phenylpentyl)pyrazolo [1,5-a]pyridine-4- carboxamide 448.4 1.2 * 44
##STR00093## tert-butyl [1-({4-[(4- methyl-2-
phenylpentyl)carbamoyl] pyrazolo[1,5- a]pyridin-2-
yl}carbonyl)pyrrolidin- 3-yl]carbamate 478.3 1.33 45 ##STR00094##
2-{[(3R)-3- aminopyrrolidin-1- yl]carbonyl}-N-(4- methyl-2-
phenylpentyl)pyrazolo [1,5-a]pyridine-4- carboxamide 434.4 1.21 *
46 ##STR00095## N-(adamantan-1- ylmethyl)-2-{[(3R)-3-
(methylamino)pyrrolidin- 1- yl]carbonyl}pyrazolo[1, 5-a]pyridine-4-
carboxamide 436.4 1.2 * 47 ##STR00096## N-(adamantan-1-
ylmethyl)-2-{[(3R)-3- (ethylamino)pyrrolidin- 1-
yl]carbonyl}pyrazolo[1, 5-a]pyridine-4- carboxamide 450.4 1.22 * 48
##STR00097## 4-N-(adamantan-1- ylmethyl)-2-N-methyl-
2-N-(pyrazin-2- ylmethyl)pyrazolo[1,5- a]pyridine-2,4-
dicarboxamide 459.3 1.28 * 49 ##STR00098## 4-N-(adamantan-1-
ylmethyl)-2-N-methyl- 2-N-[(1-methyl-1H- pyrazol-4-
yl)methyl]pyrazolo[1,5- a]pyridine-2,4- dicarboxamide 461.3 1.28 *
50 ##STR00099## 4-N-(adamantan-1- ylmethyl)-2-N-methyl-
2-N-(pyridin-2- ylmethyl)pyrazolo[1,5- a]pyridine-2,4-
dicarboxamide 458.3 1.26 * 51 ##STR00100## 4-N-(adamantan-1-
ylmethyl)-2-N-(pyridin- 2- ylmethyl)pyrazolo[1,5- a]pyridine-2,4-
dicarboxamide 444.3 1.24 * 52 ##STR00101## N-(adamantan-1-
ylmethyl)-2-(5,6- dihydroimidazo[1,5- a]pyrazin-7(8H)-
ylmethyl)pyrazolo[1,5- a]pyridine-4- carboxamide 445.4 1.21 * 53
##STR00102## N-(adamantan-1- ylmethyl)-2- {[methyl(pyridin-2-
ylmethyl)amino]methyl} pyrazolo[1,5- a]pyridine-4- carboxamide
444.4 1.23 * 54 ##STR00103## N-(adamantan-1- ylmethyl)-2-
{[methyl(pyridin-3- ylmethyl)amino]methyl} pyrazolo[1,5-
a]pyridine-4- carboxamide 444.4 1.21 * 55 ##STR00104##
N-(adamantan-1- ylmethyl)-2- {[methyl(pyrazin-2-
ylmethyl)amino]methyl} pyrazolo[1,5- a]pyridine-4- carboxamide
445.4 1.21 * 56 ##STR00105## 2-{[(3R)-3- aminopyrrolidin-1-
yl]carbonyl}-N-(4- methyl-2-pyridin-3- ylpentyl)pyrazolo[1,5-
a]pyridine-4- carboxamide 435.4 1 * 57 ##STR00106## 2-{[(3R)-3-
aminopyrrolidin-1- yl]carbonyl}-N-[(1- pyridin-3-
ylcyclohexyl)methyl] pyrazolo[1,5-a]pyridine- 4-carboxamide 447.4
0.96 * 58 ##STR00107## 2-{[(3R)-3- aminopyrrolidin-1-
yl]carbonyl}-N-(2- cyclohexyl-4- methylpentyl)pyrazolo
[1,5-a]pyridine-4- carboxamide 440.4 1.28 59 ##STR00108##
N-(adamantan-1- ylmethyl)-2-{2-[(2- fluoroethyl)amino]-2-
oxoethyl}pyrazolo[1,5- a]pyridine-4- carboxamide 413.3 1.26 * 60
##STR00109## N-(adamantan-1- ylmethyl)-2-{2-[(2,2-
difluoroethyl)amino]-2- oxoethyl}pyrazolo[1,5- a]pyridine-4-
carboxamide 431.3 1.28 * 61 ##STR00110## N-(adamantan-1-
ylmethyl)-2-{2-oxo-2- [(2,2,2- trifluoroethyl)amino]
ethyl}pyrazolo[1,5- a]pyridine-4- carboxamide 449.3 1.29 * 62
##STR00111## 4-N-(adamantan-1- ylmethyl)-2-N-(2-
fluoroethyl)pyrazolo[1, 5-a]pyridine-2,4- dicarboxamide 399.3 1.28
* 63 ##STR00112## 4-N-(adamantan-1- ylmethyl)-2-N-(2,2-
difluoroethyl)pyrazolo [1,5-a]pyridine-2,4- dicarboxamide 417.3
1.29 * 64 ##STR00113## 4-N-(adamantan-1- ylmethyl)-2-N-(2,2,2-
trifluoroethyl)pyrazolo [1,5-a]pyridine-2,4- dicarboxamide 435.3
1.31 * 65 ##STR00114## 4-N-(adamantan-1- ylmethyl)-2-N-(2-
fluorobenzyl)-2-N- methylpyrazolo[1,5- a]pyridine-2,4-
dicarboxamide 475.3 1.36 * 66 ##STR00115## 4-N-(adamantan-1-
ylmethyl)-2-N-(3- fluorobenzyl)-2-N- methylpyrazolo[1,5-
a]pyridine-2,4- dicarboxamide 475.3 1.35 * 67 ##STR00116##
4-N-(adamantan-1- ylmethyl)-2-N-(4- fluorobenzyl)-2-N-
methylpyrazolo[1,5- a]pyridine-2,4- dicarboxamide 475.3 1.35 * 68
##STR00117## N-(adamantan-1- ylmethyl)-2-{[(2-
fluorobenzyl)(methyl) amino]methyl}pyrazolo [1,5-a]pyridine-4-
carboxamide 461.4 1.24 * 69 ##STR00118## N-(adamantan-1-
ylmethyl)-2-{[(3- fluorobenzyl)(methyl) amino]methyl}pyrazolo
[1,5-a]pyridine-4- carboxamide 461.4 1.24 * 70 ##STR00119##
N-(adamantan-1- ylmethyl)-2-{[(4- fluorobenzyl)(methyl)
amino]methyl}pyrazolo [1,5-a]pyridine-4- carboxamide 461.4 1.25 *
71 ##STR00120## 2-{[(3R)-3- aminopyrrolidin-1-
yl]carbonyl}-N-[2-(4- chlorophenyl)-4- methylpentyl]pyrazolo
[1,5-a]pyridine-4- carboxamide 468.3 1.23 * 72 ##STR00121##
2-{[(3R)-3- aminopyrrolidin-1- yl]carbonyl}-N-(3- cyclohexyl-2-
phenylpropyl)]pyrazolo [1,5-a]pyridine-4- carboxamide 474.4 1.27 *
73 ##STR00122## 2-{[(3R)-3- aminopyrrolidin-1- yl]carbonyl}-N-[4-
methyl-2-(4- methylphenyl)pentyl] pyrazolo[1,5-a]pyridine-
4-carboxamide 461.5 1.41 * 74 ##STR00123## 4-N-(adamantan-1-
ylmethyl)-2-N-(2- fluoroethyl)-2-N- methylpyrazolo[1,5-
a]pyridine-2,4- dicarboxamide 413.3 1.3 * 75 ##STR00124##
4-N-(adamantan-1- ylmethyl)-2-N-(2,2- difluoroethyl)-2-N-
methylpyrazolo[1,5- a]pyridine-2,4- dicarboxamide 431.3 1.31 * 76
##STR00125## 4-N-(adamantan-1- ylmethyl)-2-N-methyl- 2-N-(2,2,2-
trifluoroethyl)pyrazolo [1,5-a]pyridine-2,4- dicarboxamide 449.3
1.33 * 77 ##STR00126## N-(adamantan-1- ylmethyl)-2-{2-[(2-
fluoroethyl)(methyl) amino]-2- oxoethyl}pyrazolo[1,5- a]pyridine-4-
carboxamide 427.3 1.29 * 78 ##STR00127## N-(adamantan-1-
ylmethyl)-2-{2-[(2,2- difluoroethyl)(methyl) amino]-2-
oxoethyl}pyrazolo[1,5- a]pyridine-4- carboxamide 445.3 1.3 * 79
##STR00128## N-(adamantan-1- ylmethyl)-2-{2- [methyl(2,2,2-
trifluoroethyl)amino]-2- oxoethyl}pyrazolo[1,5- a]pyridine-4-
carboxamide 463.3 1.32 * 80 ##STR00129## 2-{[(3R)-3-
aminopyrrolidin-1- yl]carbonyl}-N-{4- methyl-2-[4-
(trifluoromethyl)phenyl] pentyl}pyrazolo[1,5- a]pyridine-4-
carboxamide 502.3 1.25 * 81 ##STR00130## 4-N-(adamantan-1-
ylmethyl)-2-N-methyl- 2-N-[2- (methylsulfonyl)ethyl]
pyrazolo[1,5-a]pyridine- 2,4-dicarboxamide 473.3 1.26 82
##STR00131## 4-N-(adamantan-1- ylmethyl)-2-N-(1,1-
dioxidotetrahydrothio- phen-3-yl)pyrazolo[1,5- a]pyridine-2,4-
dicarboxamide 471.3 1.28 * 83 ##STR00132## N-{[1-(4-
chlorophenyl)cyclohexyl] methyl}-2-(5,6- dihydroimidazo[1,5-
a]pyrazin-7(8H)- ylcarbonyl)pyrazolo[1,- 5-a]pyridine-4-
carboxamide 517.3 1.25 * 84 ##STR00133## N-{[1-(4-
chlorophenyl)cyclohexyl] methyl}-2- (morpholin-4-
ylcarbonyl)pyrazolo[1, 5-a]pyridine-4- carboxamide 481.3 1.33 *
85 ##STR00134## 4-N-{[1-(4- chlorophenyl)cyclohexyl]
methyl}-2-N-(2- fluoroethyl)-2-N- methylpyrazolo[1,5-
a]pyridine-2,4- dicarboxamide 471.3 1.33 * 86 ##STR00135##
2-N-(2-fluoroethyl)-2- N-methyl-4-N-{4- methyl-2-[4-
(trifluoromethyl)phenyl] pentyl}pyrazolo[1,5- a]pyridine-2,4-
dicarboxamide 493.3 1.34 * 87 ##STR00136## N-{4-methyl-2-[4-
(trifluoromethyl)phenyl] pentyl}-2-(morpholin-4-
tlcarbonyl)pyrazolo[1, 5-a]pyridine-4- carboxamide 503.3 1.33 * 88
##STR00137## 2-(5,6- dihydroimidazo[1,5- a]pyrazin-7(8H)-
ylcarbonyl)-N-{4- methyl-2-[4- (trifluoromethyl)phenyl]
pentyl}pyrazolo[1,5- a]pyridine-4- carboxamide 539.3 1.25 * 89
##STR00138## N-(adamantan-1- ylmethyl)-2-(4,5-
dihydro-1H-imidazol-2- ylmethyl)pyrazolo[1,5- a]pyridine-4-
carboxamide 405.5 1.31 * 90 ##STR00139## 2-{[(3R)-3-
aminopyrrolidin-1- yl]carbonyl}-N-{[1-(4- chlorophenyl)cyclo-
hexyl]methyl}pyrazolo[1,5- a]pyridine-4- carboxamide 480.3 1.25 *
91 ##STR00140## N-[2-(4-chlorophenyl)- 4-methylpentyl]-2-
(morpholin-4- ylcarbonyl)pyrazolo[1, 5-a]pyridine-4- carboxamide
468.98 1.32 * 92 ##STR00141## Pyrazolo[1,5- a]pyridine-2,4-
dicarboxylic acid 4-{[2- (4-chloro-phenyl)-4- methyl-pentyl]-amine}
2-[(2-fluoro-ethyl)- methyl-amide] 458.96 1.33 * 93 ##STR00142##
Pyrazolo[1,5- a]pyridine-2,4- dicarboxylic acid 2-[(2-
fluoro-ethyl)-methyl- amide]-4-{[1-(4- trifluoromethyl-phenyl)-
cyclohexylmethyl]- amide} 504.52 1.33 * 94 ##STR00143##
2-(morpholin-4- ylcarbonyl)-N-({1-[4- (trifluoromethyl)phenyl]
cyclohexyl}methyl) pyrazolo[1,5-a]pyridine-4- carboxamide 514.54
1.33 * 95 ##STR00144## N-(adamantan-1- ylmethyl)-2-[2-(4,5-
dihydro-1H-imidazol-2- yl)ethyl]pyrazolo[1,5- a]pyridine-4-
carboxamide 405.54 1.21 * 96 ##STR00145## 2-adamantan-1-yl-N-
[2-(morpholin-4- ylcarbonyl)pyrazolo[1, 5-a]pyridin-4- yl]acetamide
422.52 1.33 * 97 ##STR00146## 2-adamantan-1-yl-N- [2-(5,6-
dihydroimidazo[1,5- a]pyrazin-7(8H)- ylcarbonyl)pyrazolo[1,
5-a]pyridin-4- yl]acetamide 458.56 1.23 * 98 ##STR00147##
4-[(adamantan-1- ylacetyl)amino]-N-(2- fluoroethyl)-N-
methylpyrazolo[1,5- a]pyridine-2- carboxamide 412.50 1.33 * 99
##STR00148## 2-adamantan-1-yl-N- (2-{[(3R)-3- aminopyrrolidin-1-
yl]carbonyl}pyrazolo[1, 5-a]pyridin-4- yl)acetamide 421.54 1.22 *
100 ##STR00149## rel-2-{[(2R,6S)-2,6- dimethylmorpholin-4-
yl]carbonyl}-N-{4- methyl-2-[4- (trifluoromethyl)phenyl]
pentyl}pyrazolo[1,5- a]pyridine-4- carboxamide 530.58 1.36 * 101
##STR00150## 2-[(2,2- dimethylmorphlolin-4- yl)carbonyl]-N-{4-
methyl-2-[4- (trifluoromethyl)phenyl] pentyl}pyrazolo[1,5-
a]pyridine-4- carboxamide 530.58 1.36 * 102 ##STR00151##
rel-2-{[(3R,5S)-3,5- dimethylmorpholin-4- yl]carbonyl}-N-{4-
methyl-2-[4- (trifluoromethyl)phenyl] pentyl}pyrazolo[1,5-
a]pyridine-4- carboxamide 530.58 1.36 * 103 ##STR00152##
2-{4-[(adamantan-1- ylmethyl)carbamoyl] pyrazolo[1,5-a]pyridin-2-
yl}-3-phenylpropanoic acid 457.56 1.30 * 104 ##STR00153##
2-[4-({[1-(4- chlorophenyl)cyclohexyl] methyl}carbamoyl)
pyrazolo[1,5-a]pyridin-2- yl]-3-phenylpropanoic acid 516.03 1.38 *
105 ##STR00154## 3-{4-[(adamantan-1- ylmethyl)carbamoyl]
pyrazolo[1,5-a]pyridin-2- yl}butanoic acid 395.49 1.32 * 106
##STR00155## 3-{4-[(adamantan-1- ylmethyl)carbamoyl]
pyrazolo[1,5-a]pyridin-2- yl}-3-phenylpropanoic acid 457.56 1.36 *
107 ##STR00156## 3-{4-[(adamantan-1- ylmethyl)carbamoyl]
pyrazolo[1,5-a]pyridin-2- yl}-4-phenylbutanoic acid 471.59 1.38 *
108 ##STR00157## 4-{8-[(adamantan-1- ylmethyl)carbamoyl]
indolizin-2-yl}benzoic acid 428.11 1.58 * 109 ##STR00158##
N-(adamantan-1- ylmethyl)-3-(4- cyanobenzoyl)indolizine-
8-carboxamide 438.23 1.39 * 110 ##STR00159## N-(adamantan-1-
ylmethyl)-3-(4- carbamoylbenzoyl) indolizine-8-carboxamide 456.21
1.33 * 111 ##STR00160## 2-(4-cyanophenyl)-N- [(1-pyridin-3-
ylcyclohexyl)methyl] indolizine-8-carboxamide 435.20 1.17 112
##STR00161## N-(adamantan-1- ylmethyl)-2-(4- carbamoylphenyl)
indolizine-8-carboxamide 428.22 1.31 *
TABLE-US-00002 TABLE II Additional Representative Heteroaryl Amide
Analogues Compound Name 120 ##STR00162##
(R)-2-(3-aminopyrrolidine-1- carbonyl)-N-((1-(4-
(trifluoromethyl)phenyl)cyclo- hexyl)methyl)H-pyrazolo[1,5-
a]pyridine-4-carboxamide 121 ##STR00163## 2-(5,6,7,8-
tetrahydroimidazo[1,5- a]pyrazine-7-carbonyl)-N-((1-(4-
(trifluoromethyl)phenyl)cyclo- hexyl)methyl)H-pyrazolo[1,5-
a]pyridine-4-carboxamide 122 ##STR00164##
2-(morpholine-4-carbonyl)-N- ((1-(4- (trifluoromethyl)phenyl)cyclo-
hexyl)methyl)H-pyrazolo[1,5- a]pyridine-4-carboxamide 123
##STR00165## Pyrazolo[1,5-a]pyridine-2,4- dicarboxylic acid
2-[(2-fluoro- ethyl)-methyl-amide] 4-{[1-(4- trifluoromethyl-
phenyl)-cyclohexylmethyl]- amide} 124 ##STR00166##
N-(2-(4-chlorophenyl)-4- methylpentyl)-2-(5,6,7,8-
tetrahydroimidazo[1,5- a]pyrazine-7-carbonyl)H-
pyrazolo[1,5-a]pyridine-4- carboxamide 125 ##STR00167##
N-(2-(4-chlorophenyl)-4- methylpentyl)-2-(morpholine-4-
carbonyl)H-pyrazolo[1,5- a]pyridine-4-carboxamide 126 ##STR00168##
Pyrazolo[1,5-a]pyridine-2,4- dicarboxylic acid 2-[(2-fluoro-ethyl)-
methyl-amide] 4-{[4-methyl-2-(4- trifluoromethyl-phenyl)-pentyl]-
amide} 127 ##STR00169## N-(adamantan-1-ylmethyl)-2-
{[5-(2-hydroxyethyl)-2H- tetrazol-2-yl]methyl}pyrazolo[1,5-
a]pyridine-4-carboxamide 128 ##STR00170##
N-(adamantan-1-ylmethyl)-2- {[5-(2-hydroxyethyl)-1H-
tetrazol-1-yl]methyl}pyrazolo[1,5- a]pyridine-4-carboxamide 129
##STR00171## N-[2-(3-Amino-pyrrolidine-1- carbonyl)-pyrazolo[1,5-
a]pyridin-4-yl]-2-(3-ethyl- bicyclo[3.3.1]non-1-yl)- acetamide 130
##STR00172## 4-[2-(3-Ethyl-bicyclo[3.3.1]non-
1-yl)-acetylamino]-pyrazolo[1,5- a]pyridine-2-carboxylic acid
(2-fluoro-ethyl)-methyl-amide 131 ##STR00173##
2-(3-Ethyl-bicyclo[3.3.1]non-1- yl)-N-[2-(morpholine-4-carbonyl)-
pyrazolo[1,5-a]pyridin-4-yl]- acetamide 132 ##STR00174##
N-[2-(5,6-Dihydro-8H- imidazo[1,5-a] pyrazine-7-carbonyl)-
pyrazolo[1,5-a]pyridin- 4-yl]-2-(3-ethyl-bicyclo[3.3.1]non-
1-yl)-acetamide
Example 4
P2X.sub.7 Assays
[0279] This Example illustrates representative assays for use in
evaluating test compounds for agonist and antagonist activity.
A. High Throughput P2X.sub.7 Calcium Mobilization Assay
[0280] SH-SY5Y cells, ATCC Number CRL-2266, (American Type Culture
Collection, Manassas, Va.) are cultured under DMEM/High medium
supplemented with 10% FBS, and 10 mM HEPES (Invitrogen Corp.,
Carlsbad, Calif.) in 5% CO.sub.2 and at 37.degree. C. One day prior
to the experiment, cells are plated at a density of 100,000
cells/well in a 96 well black/clear TC plate (Corning.RTM.
Costar.RTM., Sigma-Aldrich Co., St. Louis, Mo.). At the beginning
of the experiment, the culture medium is removed and cells are
incubated with 50 .mu.L of 2.3 .mu.M Fluo-4 AM dye (Invitrogen
Corp.) in the assay solution (5 mM KCl, 9.6 mM
NaH.sub.2PO.sub.4H.sub.2O, 25 mM HEPES, 280 mM Sucrose, 5 mM
Glucose, and 0.5 mM CaCl.sub.2; pH is adjusted to 7.4 with NaOH)
for an hour at 37.degree. C. After one hour dye incubation, wells
are rinsed once with 50 .mu.L assay solution, and are then
incubated for an hour at room temperature with 100 .mu.L assay
solution containing the test compound. The final concentration of
test compound generally ranges from 1 to 2500 nM; for positive
control cells, no test compound is added. After the one hour
incubation, plates are transferred to a FLIPR.sup.TETRA instrument
(Molecular Devices, Sunnyvale, Calif.) for calcium mobilization
analysis.
[0281] For determination of antagonist activity, 50 .mu.L of
P2X.sub.7 agonist (2'(3')-O-(4-benzoyl-benzoyl)adenosine
5'-triphosephate (BzATP; Sigma-Aldrich) in the assay solution is
transferred using the FLIPR into the plate, such that the final
agonist concentration is 80 .mu.M (about EC.sub.50). In negative
control cells, 50 .mu.L of assay solution without agonist is added
at this stage. The peak fluorescence signal over a 2 minute period
is then measured.
[0282] The data is analyzed as follows. First, the average maximum
relative fluorescent unit (RFU) response from the negative control
wells (no agonist) is subtracted from the maximum response detected
for each of the other experimental wells. Second, average maximum
RFU response is calculated for the positive control wells (agonist
wells). Then, percent inhibition for each compound tested is
calculated using the equation:
Percent Inhibition=100-100.times.(Peak Signal in Test Cells/Peak
Signal in Control Cells)
The % inhibition data is plotted as a function of test compound
concentration and test compound IC.sub.50 is determined using, for
example, KALEIDAGRAPH software (Synergy Software, Reading, Pa.)
best fit of the data to the equation:
y=m.sub.1*(1/(1+(m.sub.2/m.sub.0).sup.m3))
where y is the percent inhibition, m.sub.0 is the concentration of
the agonist, m.sub.1 is the maximum RFU, m.sub.2 corresponds to the
test compound IC.sub.50 (the concentration required to provide a
50% decrease, relative to the response observed in the presence of
agonist and without antagonist) and m.sub.3 is the Hill
coefficient. Alternatively, test compound IC.sub.50 is determined
using a linear regression in which x is ln(concentration of test
compound) and y is ln(percent inhibition/(100-percent inhibition).
Data with a percent inhibition that is greater than 90% or less
than 15% are rejected and are not used in the regression. The
IC.sub.50 calculated in this fashion is e.sup.(-Intercept/slope).
For antagonists of the P2X.sub.7, the calculated IC.sub.50 is
preferably below 20 micromolar, more preferably below 10
micromolar, even more preferably below 5 micromolar and most
preferably below 1 micromolar.
[0283] Similar assays are performed in the absence of added agonist
for the determination of agonist activity of the test compounds.
Within such assays, the ability of a test compound to act as an
agonist of P2X.sub.7 is determined by measuring the fluorescence
response elicited by the test compound as a function of compound
concentration. P2X.sub.7 antagonists that exhibit no detectable
agonist activity elicit no detectable fluorescence response at a
concentration of 2,500 nM.
B. Electrophysiological P2X.sub.7 Assays
[0284] SH-SY5Y cells are cultured under DMEM/High medium
supplemented with 10% FBS, and 10 mM HEPES (Invitrogen Corp.,
Carlsbad, Calif.) in 5% CO.sub.2 and at 37.degree. C., and are
split onto 12 mm round Poly-D-Lysine (PDL) coated coverslips (BD
Biosciences, San Jose, Calif.) in a 35 mm dish with a density of
130K cells/dish a day prior to the experiment. Whole cell voltage
clamp recordings are made with the Axopatch-200B amplifier (Axon
Instruments, Foster City, Calif.). The recording electrodes are
pulled from borosilicate pipettes (World Precision Instruments,
Sarasota, Fla.) on a horizontal puller (Sutter Instrument Model
P-87) and have resistances ranging from 2 to 3 M.OMEGA. when
backfilled with internal solution. All voltage protocols are
generated using pClamp 8 (Axon Instruments) software. Data are
digitized at 1 or 5 kHz and recorded onto a PC for further
analysis. Data are analyzed using Clampfit (Axon Instruments),
Excel (Microsoft, Redmond, Wash.), and Origin software (MicroCal,
LLC; Northampton, Mass.). All whole-cell recordings are conducted
at room temperature. Internal solution contains (in mM): 100 KF, 40
KCl, 5 NaCl, 10 EGTA and 10 HEPES (pH=7.4 adjusted with KOH). The
external solution contains 70 mM NaCl, 0.3 mM CaCl.sub.2, 5 mM KCl,
20 mM HEPES, 10 mM glucose, and 134 mM sucrose (pH=7.4 adjusted
with NaOH). All chemicals are from Sigma, unless otherwise
stated.
[0285] P2X.sub.7 is activated by 200 .mu.M of P2X.sub.7 agonist,
BzATP. At a holding potential of -80 mV, the activated inward
current is recorded in the presence and absence of the test
compound. Then, percent inhibition for each compound tested is
calculated using the equation:
% Inhibition=100-100.times.(Current Amplitude in Compound/Current
Amplitude in Control).
To determine a test compound's IC.sub.50 for P2X.sub.7
electrophysiologically, several concentrations of the compound are
tested and their inhibitions on P2X.sub.7 currents are calculated
as above. This dose-response curve is best fitted using Origin
software (Microcal, MA) with the following equation:
Percent Inhibition=100/(1+(IC.sub.50/C).sup.N)
where C is the concentration of the antagonist, N is the Hill
coefficient, and IC.sub.50 represents the compound IC.sub.50 value
against P2X.sub.7.
Example 5
Carrageenan-Induced Mechanical Hyperalgesia (Paw Pressure) Assay
for Determining Pain Relief
[0286] This Example illustrates a representative method for
assessing the degree of pain relief provided by a test
compound.
[0287] Adult male Sprague Dawley rats (200-300 g; obtained from
Harlan Sprague Dawley, Inc., Indianapolis, Ind.) are housed under a
12 h light/dark cycle with access to food and water ad libitum. For
the assay, all animals are habituated once, baselined twice and
tested once, with each procedure being conducted on a separate day.
Prior to each day's procedure, animals are allowed to acclimate for
at least 1 hour in the testing room before the start of the
procedure. For habituation, each animal is gently restrained with
each hindpaw consecutively extended in front of the animal as is
necessary for testing. This procedure is performed by alternating
hindpaws and repeated three times for each hindpaw. Animals are
then subjected to the first baseline, second baseline and testing
on consecutive days. For each baseline, the animal is restrained as
in the habituation session and the paw tested using the paw
pressure testing apparatus (Digital Randall Selitto, IITC Inc.,
Woodland Hills, Calif.). Animals are baselined and tested in groups
of ten, each animal being tested once on the left and right
hindpaws, followed by the next consecutive animal. This procedure
is repeated three times for a total of three measurements on each
hindpaw. If any individual read is drastically different (varies by
more than about 100 g) from the other two on a given hindpaw, the
hindpaw is retested a 4.sup.th time, and the average of the three
most consistent scores is used. On test day, all animals are
injected with 0.1 mL intraplantar 0.5%-1.5% carrageenan (dissolved
in saline) 3 hours prior to testing. Test compounds or vehicle may
be administered by various routes at various timepoints prior to
testing, but for any particular assay, the routes and timepoints
are the same for animals in each treatment group administered test
compound (a different dosage of test compound may be administered
to each such group) and those in the treatment group administered
vehicle control. If a compound is orally administered, the animals
are food-deprived the evening before testing. As with the baseline,
each hindpaw is tested three times and the results recorded for
analysis.
[0288] Hypersensitivity of nociception values are calculated for
each treatment group as the mean of the left foot gram force scores
on test day (left foot only or LFO score). Statistical significance
between treatment groups is determined by running an ANOVA on LFO
scores followed with a least significant difference (LSD) post hoc
test. A p<0.05 is considered to be a statistically significant
difference.
[0289] Compounds are said to relieve pain in this model if they
result in a statistically significant reduction in hypersensitivity
of nociception values compared to vehicle controls, determined as
described above, 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.
* * * * *