U.S. patent application number 12/823039 was filed with the patent office on 2010-12-09 for c5ar antagonists.
This patent application is currently assigned to ChemoCentryx, Inc.. Invention is credited to Pingchen Fan, Kevin Lloyd Greenman, Manmohan Reddy Leleti, Yandong Li, Jay P. Powers, Hiroko Tanaka, Ju Yang, Yibin Zeng.
Application Number | 20100311753 12/823039 |
Document ID | / |
Family ID | 42267017 |
Filed Date | 2010-12-09 |
United States Patent
Application |
20100311753 |
Kind Code |
A1 |
Fan; Pingchen ; et
al. |
December 9, 2010 |
C5aR ANTAGONISTS
Abstract
Compounds are provided that are modulators of the C5a receptor.
The compounds are substituted piperidines and are useful in
pharmaceutical compositions, methods for the treatment of diseases
and disorders involving the pathologic activation of C5a
receptors.
Inventors: |
Fan; Pingchen; (Fremont,
CA) ; Greenman; Kevin Lloyd; (Sunnyvale, CA) ;
Leleti; Manmohan Reddy; (Sunnyvale, CA) ; Li;
Yandong; (San Jose, CA) ; Powers; Jay P.;
(Pacifica, CA) ; Tanaka; Hiroko; (Foster City,
CA) ; Yang; Ju; (Cupertino, CA) ; Zeng;
Yibin; (Foster City, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
ChemoCentryx, Inc.
Mountain View
CA
|
Family ID: |
42267017 |
Appl. No.: |
12/823039 |
Filed: |
June 24, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12643229 |
Dec 21, 2009 |
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12823039 |
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PCT/US2009/068941 |
Dec 21, 2009 |
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12643229 |
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61139919 |
Dec 22, 2008 |
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61139919 |
Dec 22, 2008 |
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Current U.S.
Class: |
514/237.2 ;
514/318; 514/326; 514/330; 544/130; 546/194; 546/208; 546/226 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
25/28 20180101; A61P 37/08 20180101; C07D 211/60 20130101; A61P
7/02 20180101; A61K 31/445 20130101; A61P 9/00 20180101; A61P 19/00
20180101; A61P 31/04 20180101; A61K 31/5377 20130101; A61P 37/00
20180101; A61P 1/04 20180101; C07D 401/10 20130101; A61P 13/12
20180101; A61P 43/00 20180101; C07D 413/14 20130101; A61K 31/454
20130101; A61P 11/00 20180101; A61P 21/04 20180101; A61P 37/06
20180101; A61K 31/451 20130101; A61P 11/06 20180101; C07D 401/06
20130101; C07D 401/14 20130101; A61P 17/00 20180101; A61P 37/02
20180101; C07D 401/12 20130101; C07D 405/10 20130101; C07D 413/12
20130101; A61P 35/00 20180101; A61P 1/18 20180101; A61P 7/04
20180101; A61P 19/02 20180101; A61P 29/00 20180101; A61P 17/04
20180101; A61P 17/02 20180101; A61P 3/10 20180101; A61P 25/00
20180101; A61K 31/4545 20130101; A61P 17/06 20180101 |
Class at
Publication: |
514/237.2 ;
546/226; 546/194; 546/208; 544/130; 514/330; 514/318; 514/326 |
International
Class: |
A61K 31/5377 20060101
A61K031/5377; C07D 211/32 20060101 C07D211/32; C07D 401/06 20060101
C07D401/06; C07D 401/12 20060101 C07D401/12; C07D 413/14 20060101
C07D413/14; A61K 31/451 20060101 A61K031/451; A61K 31/4545 20060101
A61K031/4545; A61K 31/454 20060101 A61K031/454; A61P 9/00 20060101
A61P009/00; A61P 9/10 20060101 A61P009/10; A61P 7/02 20060101
A61P007/02; A61P 25/00 20060101 A61P025/00; A61P 37/00 20060101
A61P037/00; A61P 29/00 20060101 A61P029/00; A61P 3/10 20060101
A61P003/10; A61P 25/28 20060101 A61P025/28 |
Claims
1. A compound having the formula ##STR00039## and pharmaceutically
acceptable salts, hydrates and rotomers thereof; wherein C.sup.1 is
selected from the group consisting of aryl and heteroaryl, wherein
the heteroaryl group has from 1-3 heteroatoms as ring members
selected from N, O and S; and wherein said aryl and heteroaryl
groups are optionally substituted with from 1 to 3 R.sup.1
substituents; C.sup.2 is selected from the group consisting of aryl
and heteroaryl, wherein the heteroaryl group has from 1-3
heteroatoms as ring members selected from N, O and S; and wherein
said aryl and heteroaryl groups are optionally substituted with
from 1 to 3 R.sup.2 substituents; C.sup.3 is selected from the
group consisting of C.sub.1-8 alkyl or heteroalkyl, C.sub.3-8
cycloalkyl, C.sub.3-8 cycloalkyl-C.sub.1-4 alkyl, aryl,
aryl-C.sub.1-4 alkyl, heteroaryl, heteroaryl-C.sub.1-4 alkyl,
heterocycloalkyl or heterocycloalkyl-C.sub.1-4 alkyl, wherein the
heterocycloalkyl group or portion has from 1-3 heteroatoms selected
from N, O and S, and wherein the heteroaryl group has from 1-3
heteroatoms as ring members selected from N, O and S, and each
C.sup.3 is optionally substituted with from 1-3 R.sup.3
substituents; each R.sup.1 is independently selected from the group
consisting of halogen, --CN, --R.sup.c, --CO.sub.2R.sup.a,
--CONR.sup.aR.sup.b, --C(O)R.sup.a, --OC(O)NR.sup.aR.sup.b,
--NR.sup.bC(O)R.sup.a, --NR.sup.bC(O).sub.2R.sup.c,
--NR.sup.a--C(O)NR.sup.aR.sup.b, --NR.sup.aC(O)NR.sup.aR.sup.b,
--NR.sup.aR.sup.b, --OR.sup.a, and --S(O).sub.2NR.sup.aR.sup.b;
wherein each R.sup.a and R.sup.b is independently selected from
hydrogen, C.sub.1-8 alkyl, and C.sub.1-8 haloalkyl, or when
attached to the same nitrogen atom can be combined with the
nitrogen atom to form a five or six-membered ring having from 0 to
2 additional heteroatoms as ring members selected from N, O or S,
and is optionally substituted with one or two oxo; each R.sup.c is
independently selected from the group consisting of C.sub.1-8 alkyl
or heteroalkyl, C.sub.1-8 haloalkyl, C.sub.3-6 cycloalkyl,
heterocycloalkyl, aryl and heteroaryl, and wherein the aliphatic
and cyclic portions of R.sup.a, R.sup.b and R.sup.c are optionally
further substituted with from one to three halogen, hydroxy,
methyl, amino, alkylamino and dialkylamino groups; and optionally
when two R.sup.1 substituents are on adjacent atoms, are combined
to form a fused five or six-membered carbocyclic or heterocyclic
ring; each R.sup.2 is independently selected from the group
consisting of halogen, --CN, --NO.sub.2, --R.sup.f,
--CO.sub.2R.sup.d, --CONR.sup.dR.sup.e, --C(O)R.sup.d,
--OC(O)NR.sup.dR.sup.e, --NR.sup.eC(O)R.sup.d,
--NR.sup.eC(O).sub.2R.sup.f, --NR.sup.dC(O)NR.sup.dR.sup.e,
--NR.sup.dC(O)NR.sup.dR.sup.e, --NR.sup.dR.sup.e, --OR.sup.d, and
--S(O).sub.2NR.sup.dR.sup.e; wherein each R.sup.d and R.sup.e is
independently selected from hydrogen, C.sub.1-8 alkyl, and
C.sub.1-8 haloalkyl, or when attached to the same nitrogen atom can
be combined with the nitrogen atom to form a five or six-membered
ring having from 0 to 2 additional heteroatoms as ring members
selected from N, O or S, and is optionally substituted with one or
two oxo; each R.sup.f is independently selected from the group
consisting of C.sub.1-8 alkyl or heteroalkyl, C.sub.1-8 haloalkyl,
C.sub.3-6 cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and
wherein the aliphatic and cyclic portions of R.sup.d, R.sup.e and
R.sup.f are optionally further substituted with from one to three
halogen, hydroxy, methyl, amino, alkylamino and dialkylamino
groups, and optionally when two R.sup.2 groups are on adjacent
atoms, they are combined to form a five- or six-membered ring; each
R.sup.3 is independently selected from the group consisting of
halogen, --CN, --R.sup.i, --CO.sub.2R.sup.g, --CONR.sup.gR.sup.h,
--C(O)R.sup.g, --C(O)R.sup.i, --OC(O)NR.sup.gR.sup.h,
--NR.sup.hC(O)R.sup.g, --NR.sup.hCO.sub.2R.sup.i,
--NR.sup.gC(O)NR.sup.gR.sup.h, --NR.sup.gR.sup.h, --OR.sup.g,
--OR.sup.j, --S(O).sub.2NR.sup.gR.sup.h, --X.sup.4--R.sup.j,
--NH--X.sup.4--R.sup.j, --O--X.sup.4--R.sup.j,
--X.sup.4--NR.sup.gR.sup.h, --X.sup.4--NHR.sup.j,
--X.sup.4--CONR.sup.gR.sup.h, --X.sup.4--NR.sup.hC(O)R.sup.g,
--X.sup.4--CO.sub.2--R.sup.g, --O--X.sup.4--CO.sub.2R.sup.g,
--NH--X.sup.4--CO.sub.2R.sup.g, --X.sup.4--NR.sup.hCO.sub.2R.sup.i,
--O--X.sup.4--NR.sup.hCO.sub.2R.sup.i,
--O--X.sup.4--NR.sup.hCO.sub.2R.sup.i, --NHR.sup.j and
--NHCH.sub.2R.sup.j, wherein X.sup.4 is a C.sub.1-4 alkylene; each
R.sup.g and R.sup.h is independently selected from hydrogen,
C.sub.1-8 alkyl or heteroalkyl, C.sub.3-6 cycloalkyl and C.sub.1-8
haloalkyl, or when attached to the same nitrogen atom can be
combined with the nitrogen atom to form a four-, five- or
six-membered ring having from 0 to 2 additional heteroatoms as ring
members selected from N, O or S and is optionally substituted with
one or two oxo; each R.sup.i is independently selected from the
group consisting of C.sub.1-8 alkyl or heteroalkyl, C.sub.1-8
haloalkyl, C.sub.3-6 cycloalkyl, heterocycloalkyl, aryl and
heteroaryl; and each R.sup.j is selected from the group consisting
of C.sub.3-6 cycloalkyl, imidazolyl, pyrimidinyl, pyrrolinyl,
piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, and
S,S-dioxo-tetrahydrothiopyranyl, and wherein the aliphatic and
cyclic portions of R.sup.g, R.sup.h, R.sup.i and R.sup.j are
optionally further substituted with from one to three halogen,
methyl, CF.sub.3, hydroxy, C.sub.1-4 alkoxy, C.sub.1-4
alkoxy-C.sub.1-4 alkyl, --C(O)O--C.sub.1-8 alkyl, amino, alkylamino
and dialkylamino groups, and optionally when two R.sup.3 groups are
on adjacent atoms, they are combined to form a five- or
six-membered ring; and X is hydrogen or CH.sub.3.
2. The compound of claim 1, wherein X is hydrogen.
3. The compound of claim 1, having the formula: ##STR00040##
4. The compound of claim 1, having the formula: ##STR00041##
5. The compound of claim 1, having the formula: ##STR00042##
wherein X.sup.1 is selected from the group consisting of N, CH and
CR.sup.1; the subscript n is an integer of from 0 to 2; X.sup.2 is
selected from the group consisting of N, CH and CR.sup.2; and the
subscript m is an integer of from 0 to 2.
6. The compound of claim 1, having the formula: ##STR00043##
wherein X.sup.1 is selected from the group consisting of N, CH and
CR.sup.1; the subscript n is an integer of from 0 to 2; X.sup.2 is
selected from the group consisting of N, CH and CR.sup.2; and the
subscript m is an integer of from 0 to 2.
7. The compound of claim 1, having the formula: ##STR00044##
wherein the subscript p is an integer of from 0 to 3; X.sup.1 is
selected from the group consisting of N, CH and CR.sup.1; the
subscript n is an integer of from 0 to 2; X.sup.2 is selected from
the group consisting of N, CH and CR.sup.2; and the subscript m is
an integer of from 0 to 2.
8. The compound of claim 1, having the formula: ##STR00045##
9. The compound of claim 1, having the formula: ##STR00046##
10. The compound of claim 1, having the formula: ##STR00047##
11. The compound of claim 1, having the formula: ##STR00048##
12. The compound of claim 1, having the formula: ##STR00049##
13. The compound of claim 1, having the formula: ##STR00050##
14. The compound of claim 1, having the formula: ##STR00051##
wherein R.sup.3 is a member selected from the group consisting of
--NR.sup.gR.sup.h, --NHR.sup.j and --NHCH.sub.2R.sup.j.
15. The compound of claim 1, having the formula: ##STR00052##
wherein R.sup.3 is a member selected from the group consisting of
--X.sup.4--NR.sup.gR.sup.h, --X.sup.4--R.sup.j and
--X.sup.4--NR.sup.hCOR.sup.g.
16. The compound of claim 1, wherein C.sup.1 is selected from the
group consisting of phenyl, pyridyl, indolyl and thiazolyl, each of
which is optionally substituted with from 1 to 3 R.sup.1
substituents.
17. The compound of claim 1, wherein C.sup.2 is selected from the
group consisting of phenyl, naphthyl, pyridyl and indolyl, each of
which is optionally substituted with from 1 to 3 R.sup.2
substituents.
18. The compound of claim 1, wherein C.sup.3 is selected from the
group consisting of C.sub.3-6 alkyl, C.sub.3-6 cycloalkyl,
C.sub.3-6 cycloalkyl C.sub.1-2alkyl, phenyl, pyridinyl, pyrazolyl,
piperidinyl, pyrrolidinyl, piperidinylmethyl and
pyrrolidinylmethyl, each of which is optionally substituted with
from 1 to 3 R.sup.3 substituents.
19. The compound of claim 16, wherein each R.sup.1 is independently
selected from the group consisting of halogen, --CN, --R.sup.c,
--NR.sup.aR.sup.b and --OR.sup.a, and wherein each R.sup.a and
R.sup.b is independently selected from hydrogen, C.sub.1-8 alkyl,
and C.sub.1-8 haloalkyl, or when attached to the same nitrogen atom
can be combined with the nitrogen atom to form a pyrrolidine ring;
each R.sup.c is independently selected from the group consisting of
C.sub.1-8 alkyl, C.sub.1-8 haloalkyl and C.sub.3-6 cycloalkyl, and
wherein the aliphatic and cyclic portions of R.sup.a, R.sup.b and
R.sup.c are optionally further substituted with from one to three
hydroxy, methyl, amino, alkylamino and dialkylamino groups; and
optionally when two R.sup.1 substituents are on adjacent atoms, are
combined to form a fused five or six-membered carbocyclic ring.
20. The compound of claim 17, wherein each R.sup.2 is independently
selected from the group consisting of halogen, --R.sup.f and
--OR.sup.d; wherein each R.sup.d is independently selected from
hydrogen, C.sub.1-8 alkyl, and C.sub.1-8 haloalkyl; each R.sup.f is
independently selected from the group consisting of C.sub.1-8
alkyl, C.sub.1-8 haloalkyl, C.sub.3-6 cycloalkyl, heterocycloalkyl
and heteroaryl, and wherein the aliphatic and cyclic portions of
R.sup.d and R.sup.f are optionally further substituted with from
one to three halogen, hydroxy, methyl, amino, alkylamino and
dialkylamino groups.
21. The compound of claim 18, wherein each R.sup.3 is independently
selected from the group consisting of halogen, --R.sup.i,
--CO.sub.2R.sup.g, --CONR.sup.gR.sup.h, --NR.sup.hC(O)R.sup.g,
--NR.sup.hC(O).sub.2R.sup.i, --NR.sup.gR.sup.h, --OR.sup.g,
--X.sup.4--R.sup.j, --X.sup.4--NR.sup.gR.sup.h,
--X.sup.4--CONR.sup.gR.sup.h, --X.sup.4--NR.sup.hC(O)R.sup.g,
--NHR.sup.j and --NHCH.sub.2R.sup.j, wherein X.sup.4 is a C.sub.1-3
alkylene; each R.sup.g and R.sup.h is independently selected from
hydrogen, C.sub.1-8 alkyl, C.sub.3-6 cycloalkyl and C.sub.1-8
haloalkyl, or when attached to the same nitrogen atom can be
combined with the nitrogen atom to form a five or six-membered ring
having from 0 to 1 additional heteroatoms as ring members selected
from N, O or S and is optionally substituted with one or two oxo;
each R.sup.i is independently selected from the group consisting of
C.sub.1-8 alkyl, C.sub.1-8 haloalkyl, C.sub.3-6 cycloalkyl,
heterocycloalkyl, aryl and heteroaryl; and each R.sup.j is selected
from the group consisting of C.sub.3-6 cycloalkyl, pyrrolinyl,
piperidinyl, morpholinyl, tetrahydrofuranyl, and tetrahydropyranyl,
and wherein the aliphatic and cyclic portions of R.sup.g, R.sup.h,
R.sup.i and R.sup.j are optionally further substituted with from
one to three halogen, methyl, CF.sub.3, hydroxy, amino, alkylamino
and dialkylamino groups
22. The compound of claim 20, wherein C.sup.2 is selected from the
group consisting of: ##STR00053##
23. The compound of claim 19, wherein C.sup.1 is selected from the
group consisting of: ##STR00054## ##STR00055##
24. The compound of claim 1, wherein C.sup.3 is selected from the
group consisting of: ##STR00056## ##STR00057##
25. The compound of claim 1, wherein C.sup.3 is selected from the
group consisting of: ##STR00058## ##STR00059##
26. The compound of claim 1, wherein said compound is selected from
the group in Table 1.
27. The compound of 1, having the formula: ##STR00060## and
pharmaceutically acceptable salts thereof.
28. The compound of 1, having the formula: ##STR00061## and
pharmaceutically acceptable salts thereof.
29. The compound of 1, having the formula: ##STR00062## and
pharmaceutically acceptable salts thereof.
30. A pharmaceutical composition comprising a compound a
pharmaceutically acceptable carrier and a compound having formula
##STR00063## and pharmaceutically acceptable salts, hydrates and
rotomers thereof; wherein C.sup.1 is selected from the group
consisting of aryl and heteroaryl, wherein the heteroaryl group has
from 1-3 heteroatoms as ring members selected from N, O and S; and
wherein said aryl and heteroaryl groups are optionally substituted
with from 1 to 3 R.sup.1 substituents; C.sup.2 is selected from the
group consisting of aryl and heteroaryl, wherein the heteroaryl
group has from 1-3 heteroatoms as ring members selected from N, O
and S; and wherein said aryl and heteroaryl groups are optionally
substituted with from 1 to 3 R.sup.2 substituents; C.sup.3 is
selected from the group consisting of C.sub.1-8 alkyl or
heteroalkyl, C.sub.3-8 cycloalkyl, C.sub.3-8 cycloalkyl-C.sub.1-4
alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl, heteroaryl-C.sub.1-4
alkyl, heterocycloalkyl or heterocycloalkyl-C.sub.1-4 alkyl,
wherein the heterocycloalkyl group or portion has from 1-3
heteroatoms selected from N, O and S, and wherein the heteroaryl
group has from 1-3 heteroatoms as ring members selected from N, O
and S, and each C.sup.3 is optionally substituted with from 1-3
R.sup.3 substituents; each R.sup.1 is independently selected from
the group consisting of halogen, --CN, --R.sup.c,
--CO.sub.2R.sup.a, --CONR.sup.aR.sup.b, --C(O)R.sup.a,
--OC(O)NR.sup.aR.sup.b, --NR.sup.bC(O)R.sup.a,
--NR.sup.bC(O).sub.2R.sup.c, --NR.sup.a--C(O)NR.sup.aR.sup.b,
--NR.sup.aC(O)NR.sup.aR.sup.b, --NR.sup.aR.sup.b, --OR.sup.a, and
--S(O).sub.2NR.sup.aR.sup.b; wherein each R.sup.a and R.sup.b is
independently selected from hydrogen, C.sub.1-8 alkyl, and
C.sub.1-8 haloalkyl, or when attached to the same nitrogen atom can
be combined with the nitrogen atom to form a five or six-membered
ring having from 0 to 2 additional heteroatoms as ring members
selected from N, O or S, and is optionally substituted with one or
two oxo; each R.sup.c is independently selected from the group
consisting of C.sub.1-8 alkyl or heteroalkyl, C.sub.1-8 haloalkyl,
C.sub.3-6 cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and
wherein the aliphatic and cyclic portions of R.sup.a, R.sup.b and
R.sup.c are optionally further substituted with from one to three
halogen, hydroxy, methyl, amino, alkylamino and dialkylamino
groups; and optionally when two R.sup.1 substituents are on
adjacent atoms, are combined to form a fused five or six-membered
carbocyclic or heterocyclic ring; each R.sup.2 is independently
selected from the group consisting of halogen, --CN, --NO.sub.2,
--R.sup.f, --CO.sub.2R.sup.d, --CONR.sup.dR.sup.e, --C(O)R.sup.d,
--OC(O)NR.sup.dR.sup.e, --NR.sup.eC(O)R.sup.d,
--NR.sup.eC(O).sub.2R.sup.f, --NR.sup.dC(O)NR.sup.dR.sup.e,
--NR.sup.dC(O)NR.sup.dR.sup.e, --NR.sup.dR.sup.e, --OR.sup.d, and
--S(O).sub.2NR.sup.dR.sup.e; wherein each R.sup.d and R.sup.e is
independently selected from hydrogen, C.sub.1-8 alkyl, and
C.sub.1-8 haloalkyl, or when attached to the same nitrogen atom can
be combined with the nitrogen atom to form a five or six-membered
ring having from 0 to 2 additional heteroatoms as ring members
selected from N, O or S, and is optionally substituted with one or
two oxo; each R.sup.f is independently selected from the group
consisting of C.sub.1-8 alkyl or heteroalkyl, C.sub.1-8 haloalkyl,
C.sub.3-6 cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and
wherein the aliphatic and cyclic portions of R.sup.d, R.sup.e and
R.sup.f are optionally further substituted with from one to three
halogen, hydroxy, methyl, amino, alkylamino and dialkylamino
groups, and optionally when two R.sup.2 groups are on adjacent
atoms, they are combined to form a five- or six-membered ring; each
R.sup.3 is independently selected from the group consisting of
halogen, --CN, --R.sup.i, --CO.sub.2R.sup.g, --CONR.sup.gR.sup.h,
--C(O)R.sup.g, --C(O)R.sup.i, --OC(O)NR.sup.gR.sup.h,
--NR.sup.hC(O)R.sup.g, --NR.sup.hCO.sub.2R.sup.i,
--NR.sup.gC(O)NR.sup.gR.sup.h, --NR.sup.gR.sup.h, --OR.sup.g,
--OR.sup.j, --S(O).sub.2NR.sup.gR.sup.h, --X.sup.4--R.sup.j,
--NH--X.sup.4--R.sup.j, --O--X.sup.4--R.sup.j,
--X.sup.4--NR.sup.gR.sup.h, --X.sup.4--NHR.sup.j,
--X.sup.4--CONR.sup.gR.sup.h, --X.sup.4--NR.sup.hC(O)R.sup.g,
--X.sup.4--NHC(O)R.sup.g, --X.sup.4--CO.sub.2R.sup.g,
--O--X.sup.4--CO.sub.2R.sup.g, --NH--X.sup.4--CO.sub.2R.sup.g,
--X.sup.4--NR.sup.hCO.sub.2R.sup.i,
--O--X.sup.4--NR.sup.hCO.sub.2R.sup.i, --NHR.sup.j and
--NHCH.sub.2R.sup.j, wherein X.sup.4 is a C.sub.1-4 alkylene; each
R.sup.g and R.sup.h is independently selected from hydrogen,
C.sub.1-8 alkyl or heteroalkyl, C.sub.3-6 cycloalkyl and C.sub.1-8
haloalkyl, or when attached to the same nitrogen atom can be
combined with the nitrogen atom to form a four-, five- or
six-membered ring having from 0 to 2 additional heteroatoms as ring
members selected from N, O or S and is optionally substituted with
one or two oxo; each R.sup.i is independently selected from the
group consisting of C.sub.1-8 alkyl or heteroalkyl, C.sub.1-8
haloalkyl, C.sub.3-6 cycloalkyl, heterocycloalkyl, aryl and
heteroaryl; and each R.sup.i is selected from the group consisting
of C.sub.3-6 cycloalkyl, imidazolyl, pyrimidinyl, pyrrolinyl,
piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, and
S,S-dioxo-tetrahydrothiopyranyl and wherein the aliphatic and
cyclic portions of R.sup.g, R.sup.h, R.sup.i and R.sup.j are
optionally further substituted with from one to three halogen,
methyl, CF.sub.3, hydroxy, C.sub.1-4 alkoxy, C.sub.1-4
alkoxy-C.sub.1-4 alkyl, --C(O)O--C.sub.1-8 alkyl, amino, alkylamino
and dialkylamino groups, and optionally when two R.sup.3 groups are
on adjacent atoms, they are combined to form a five- or
six-membered ring; and X is hydrogen or CH.sub.3.
31. A method for treating a mammal suffering from or susceptible to
a disease or disorder involving pathologic activation of C5a
receptors, comprising administering to the mammal an effective
amount of a compound of claim 1.
32. A method of inhibiting C5a receptor-mediated cellular
chemotaxis comprising contacting mammalian white blood cells with a
C5a receptor modulatory amount of a compound of claim 1.
33. The method of claim 31, wherein the disease or disorder is an
inflammatory disease or disorder.
34. The method of claim 33, wherein the disease or disorder is
selected from the group consisting of neutropenia, sepsis, septic
shock, Alzheimer's disease, multiple sclerosis, stroke,
inflammatory bowel disease, age-related macular degeneration,
chronic obstructive pulmonary disorder, inflammation associated
with burns, lung injury, osteoarthritis, atopic dermatitis, chronic
urticaria, ischemia-reperfusion injury, acute respiratory distress
syndrome, systemic inflammatory response syndrome, multiple organ
dysfunction syndrome, tissue graft rejection, cancer and hyperacute
rejection of transplanted organs.
35. The method of claim 31, wherein the disease or disorder is a
cardiovascular or cerebrovascular disorder.
36. The method of claim 35, wherein the disease or disorder is
selected from the group consisting of myocardial infarction,
coronary thrombosis, vascular occlusion, post-surgical vascular
reocclusion, artherosclerosis, traumatic central nervous system
injury and ischemic heart disease.
37. The method of claim 31, wherein the disease or disorder is an
autoimmune disorder.
38. The method of claim 37, wherein the disease or disorder is
selected from the group consisting of rheumatoid arthritis,
systemic lupus erythematosus, Guillain-Barre syndrome,
pancreatitis, lupus nephritis, lupus glomerulonephritis, psoriasis,
Crohn's disease, vasculitis, irritable bowel syndrome,
dermatomyositis, multiple sclerosis, bronchial asthma, pemphigus,
pemphigoid, scleroderma, myasthenia gravis, autoimmune hemolytic
and thrombocytopenic states, Goodpasture's syndrome,
immunovasculitis, tissue graft rejection and hyperacute rejection
of transplanted organs.
39. The method of claim 31, wherein the disease or disorder is a
pathologic sequelae associated with the group consisting of
insulin-dependent diabetes, mellitus, lupus nephropathy, Heyman
nephritis, membranous nephritis, glomerulonephritis, contact
sensitivity responses, and inflammation resulting from contact of
blood with artificial surfaces.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
12/643,229 and PCT Application No. PCT/US2009/068941, filed Dec.
21, 2009, which claims the benefit of U.S. Ser. No. 61/139,919
filed Dec. 22, 2008; the entire content of which is incorporated
herein by reference.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] Not Applicable
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED ON A COMPACT DISK
[0003] Not Applicable
BACKGROUND OF THE INVENTION
[0004] The complement system plays a central role in the clearance
of immune complexes and in immune responses to infectious agents,
foreign antigens, virus infected cells and tumor cells.
Inappropriate or excessive activation of the complement system can
lead to harmful, and even potentially life-threatening consequences
due to severe inflammation and resulting tissue destruction. These
consequences are clinically manifested in various disorders
including septic shock; myocardial, as well as, intestinal
ischemia/reperfusion injury; graft rejection; organ failure;
nephritis; pathological inflammation; and autoimmune diseases.
[0005] The complement system is composed of a group of proteins
that are normally present in the serum in an inactive state.
Activation of the complement system encompasses mainly three
distinct pathways, i.e., the classical, the alternative, and the
lectin pathway (V. M. Holers, In Clinical Immunology: Principles
and Practice, ed. R. R. Rich, Mosby Press; 1996, 363-391): 1) The
classical pathway is a calcium/magnesium-dependent cascade, which
is normally activated by the formation of antigen-antibody
complexes. It can also be activated in an antibody-independent
manner by the binding of C-reactive protein, complexed with ligand,
and by many pathogens including gram-negative bacteria. 2) The
alternative pathway is a magnesium-dependent cascade which is
activated by deposition and activation of C3 on certain susceptible
surfaces (e.g. cell wall polysaccharides of yeast and bacteria, and
certain biopolymer materials). 3) The lectin pathway involves the
initial binding of mannose-binding lectin and the subsequent
activation of C2 and C4, which are common to the classical pathway
(Matsushita, M. et al., J. Exp. Med. 176: 1497-1502 (1992);
Suankratay, C. et al., J. Immunol. 160: 3006-3013 (1998)).
[0006] The activation of the complement pathway generates
biologically active fragments of complement proteins, e.g. C3a, C4a
and C5a anaphylatoxins and C5b-9 membrane attack complexes (MAC),
all which mediate inflammatory responses by affecting leukocyte
chemotaxis; activating macrophages, neutrophils, platelets, mast
cells and endothelial cells; and increasing vascular permeability,
cytolysis and tissue injury.
[0007] Complement C5a is one of the most potent proinflammatory
mediators of the complement system. (The anaphylactic C5a peptide
is 100 times more potent, on a molar basis, in eliciting
inflammatory responses than C3a.) C5a is the activated form of C5
(190 kD, molecular weight). C5a is present in human serum at
approximately 80 .mu.g/ml (Kohler, P. F. et al., J. Immunol. 99:
1211-1216 (1967)). It is composed of two polypeptide chains, a and
.beta., with approximate molecular weights of 115 kD and 75 kD,
respectively (Tack, B. F. et al., Biochemistry 18: 1490-1497
(1979)). Biosynthesized as a single-chain promolecule, C5 is
enzymatically cleaved into a two-chain structure during processing
and secretion. After cleavage, the two chains are held together by
at least one disulphide bond as well as noncovalent interactions
(Ooi, Y. M. et al., J. Immunol. 124: 2494-2498 (1980)).
[0008] C5 is cleaved into the C5a and C5b fragments during
activation of the complement pathways. The convertase enzymes
responsible for C5 activation are multi-subunit complexes of C4b,
C2a, and C3b for the classical pathway and of (C3b).sub.2, Bb, and
P for the alternative pathway (Goldlust, M. B. et al., J. Immunol.
113: 998-1007 (1974); Schreiber, R. D. et al, Proc. Natl. Acad.
Sci. 75: 3948-3952 (1978)). C5 is activated by cleavage at position
74-75 (Arg-Leu) in the .alpha.-chain. After activation, the 11.2
kD, 74 amino acid peptide C5a from the amino-terminus portion of
the .alpha.-chain is released. Both C5a and C3a are potent
stimulators of neutrophils and monocytes (Schindler, R. et al.,
Blood 76: 1631-1638 (1990); Haeffner-Cavaillon, N. et al., J.
Immunol. 138: 794-700 (1987); Cavaillon, J. M. et al., Eur. J.
Immunol. 20: 253-257 (1990)).
[0009] In addition to its anaphylatoxic properties, C5a induces
chemotactic migration of neutrophils (Ward, P. A. et al., J.
Immunol. 102: 93-99 (1969)), eosinophils (Kay, A. B. et al.,
Immunol. 24: 969-976 (1973)), basophils (Lett-Brown, M. A. et al.,
J. Immunol. 117: 246-252 1976)), and monocytes (Snyderman, R. et
al., Proc. Soc. Exp. Biol. Med. 138: 387-390 1971)). Both C5a and
C5b-9 activate endothelial cells to express adhesion molecules
essential for sequestration of activated leukocytes, which mediate
tissue inflammation and injury (Foreman, K. E. et al., J. Clin.
Invest. 94: 1147-1155 (1994); Foreman, K. E. et al., Inflammation
20: 1-9 (1996); Rollins, S. A. et al., Transplantation 69:
1959-1967 (2000)). C5a also mediates inflammatory reactions by
causing smooth muscle contraction, increasing vascular
permeability, inducing basophil and mast cell degranulation and
inducing release of lysosomal proteases and oxidative free radicals
(Gerard, C. et al., Ann. Rev. Immunol. 12: 775-808 (1994)).
Furthermore, C5a modulates the hepatic acute-phase gene expression
and augments the overall immune response by increasing the
production of TNF-.alpha., IL-1-13, IL-6, IL-8, prostaglandins and
leukotrienes (Lambris, J. D. et al., In: The Human Complement
System in Health and Disease, Volanakis, J. E. ed., Marcel Dekker,
New York, pp. 83-118).
[0010] The anaphylactic and chemotactic effects of C5a are believed
to be mediated through its interaction with the C5a receptor. The
human C5a receptor (C5aR) is a 52 kD membrane bound G
protein-coupled receptor, and is expressed on neutrophils,
monocytes, basophils, eosinophils, hepatocytes, lung smooth muscle
and endothelial cells, and renal glomerular tissues (Van-Epps, D.
E. et al., J. Immunol. 132: 2862-2867 (1984); Haviland, D. L. et
al, J. Immunol. 154:1861-1869 (1995); Wetsel, R. A., Immunol. Leff.
44: 183-187 (1995); Buchner, R. R. et al., J. Immunol. 155: 308-315
(1995); Chenoweth, D. E. et al., Proc. Natl. Acad. Sci. 75:
3943-3947 (1978); Zwirner, J. et al., Mol. Immunol. 36:877-884
(1999)). The ligand-binding site of C5aR is complex and consists of
at least two physically separable binding domains. One binds the
C5a amino terminus (amino acids 1-20) and disulfide-linked core
(amino acids 21-61), while the second binds the C5a
carboxy-terminal end (amino acids 62-74) (Wetsel, R. A., Curr.
Opin. Immunol. 7: 48-53 (1995)).
[0011] C5a plays important roles in inflammation and tissue injury.
In cardiopulmonary bypass and hemodialysis, C5a is formed as a
result of activation of the alternative complement pathway when
human blood makes contact with the artificial surface of the
heart-lung machine or kidney dialysis machine (Howard, R. J. et
al., Arch. Surg. 123: 1496-1501 (1988); Kirklin, J. K. et al., J.
Cardiovasc. Surg. 86: 845-857 (1983); Craddock, P. R. et al., N.
Engl. J. Med. 296: 769-774 (1977)). C5a causes increased capillary
permeability and edema, bronchoconstriction, pulmonary
vasoconstriction, leukocyte and platelet activation and
infiltration to tissues, in particular the lung (Czermak, B. J. et
al., J. Leukoc. Biol. 64: 40-48 (1998)). Administration of an
anti-C5a monoclonal antibody was shown to reduce cardiopulmonary
bypass and cardioplegia-induced coronary endothelial dysfunction
(Tofukuji, M. et al., J. Thorac. Cardiovasc. Surg. 116: 1060-1068
(1998)).
[0012] C5a is also involved in acute respiratory distress syndrome
(ARDS), Chronic Obstructive Pulmonary Disorder (COPD) and multiple
organ failure (MOF) (Hack, C. E. et al., Am. J. Med. 1989: 86:
20-26; Hammerschmidt D E et al. Lancet 1980; 1: 947-949; Heideman
M. et al. J. Trauma 1984; 4: 1038-1043; Marc, M M, et al., Am. J.
Respir. Cell and Mol. Biol., 2004: 31: 216-219). C5a augments
monocyte production of two important pro-inflammatory cytokines,
TNF-.alpha. and IL-1. C5a has also been shown to play an important
role in the development of tissue injury, and particularly
pulmonary injury, in animal models of septic shock (Smedegard G et
al. Am. J. Pathol. 1989; 135: 489-497; Markus, S., et al., FASEB
Journal (2001), 15: 568-570). In sepsis models using rats, pigs and
non-human primates, anti-C5a antibodies administered to the animals
before treatment with endotoxin or E. coli resulted in decreased
tissue injury, as well as decreased production of IL-6 (Smedegard,
G. et al., Am. J. Pathol. 135: 489-497 (1989); Hopken, U. et al.,
Eur. J. Immunol. 26: 1103-1109 (1996); Stevens, J. H. et al., J.
Clin. Invest. 77: 1812-1816 (1986)). More importantly, blockade or
C5a with anti-C5a polyclonal antibodies has been shown to
significantly improve survival rates in a caecal ligation/puncture
model of sepsis in rats (Czermak, B. J. et al., Nat. Med. 5:
788-792 (1999)). This model share many aspects of the clinical
manifestation of sepsis in humans. (Parker, S. J. et al., Br. J.
Surg. 88: 22-30 (2001)). In the same sepsis model, anti-C5a
antibodies were shown to inhibit apoptosis of thymocytes (Guo, R.
F. et al., J. Clin. Invest. 106: 1271-1280 (2000)) and prevent MOF
(Huber-Lang, M. et al., J. Immunol. 166: 1193-1199 (2001)).
Anti-C5a antibodies were also protective in a cobra venom factor
model of lung injury in rats, and in immune complex-induced lung
injury (Mulligan, M. S. et al. J. Clin. Invest. 98: 503-512
(1996)). The importance of C5a in immune complex-mediated lung
injury was later confirmed in mice (Bozic, C. R. et al., Science
26: 1103-1109 (1996)).
[0013] C5a is found to be a major mediator in myocardial
ischemia-reperfusion injury. Complement depletion reduced
myocardial infarct size in mice (Weisman, H. F. et al., Science
249: 146-151 (1990)), and treatment with anti-C5a antibodies
reduced injury in a rat model of hindlimb ischemia-reperfusion
(Bless, N. M. et al., Am. J. Physiol. 276: L57-L63 (1999)).
Reperfusion injury during myocardial infarction was also markedly
reduced in pigs that were retreated with a monoclonal anti-C5a IgG
(Amsterdam, E. A. et al., Am. J. Physiol. 268:H448-H457 (1995)). A
recombinant human C5aR antagonist reduces infarct size in a porcine
model of surgical revascularization (Riley, R. D. et al., J.
Thorac. Cardiovasc. Surg. 120: 350-358 (2000)).
[0014] C5a driven neutrophils also contribute to many bullous
diseases (e.g., bullous pemphigoid, pemphigus vulgaris and
pemphigus foliaceus). These are chronic and recurring inflammatory
disorders clinically characterized by sterile blisters that appear
in the sub-epidermal space of the skin and mucosa. While
autoantibodies to keratinocytes located at the cutaneous basement
membranes are believed to underlie the detachment of epidermal
basal keratinocytes from the underlying basement membrane, blisters
are also characterized by accumulation of neutrophils in both the
upper dermal layers and within the blister cavities. In
experimental models a reduction of neutrophils or absence of
complement (total or C5-selective) can inhibit formation of
sub-epidermal blisters, even in the presence of high auto-antibody
titers.
[0015] Complement levels are elevated in patients with rheumatoid
arthritis (Jose, P. J. et al., Ann. Rheum. Dis. 49: 747-752 (1990);
Grant, E. P., et al., J. of Exp. Med., 196(11): 1461-1471, (2002)),
lupus nephritis (Bao, L., et al., Eur. J. of Immunol., 35(8),
2496-2506, (2005)) and systemic lupus erythematosus (SLE) (Porcel,
J. M. et al., Clin. Immunol. Immunopathol. 74: 283-288 (1995)). C5a
levels correlate with the severity of the disease state.
Collagen-induced arthritis in mice and rats resembles the
rheumatoid arthritic disease in human. Mice deficient in the C5a
receptor demonstrated a complete protection from arthritis induced
by injection of monoclonal anti-collagen Abs (Banda, N. K., et al.,
J. of Immunol., 2003, 171: 2109-2115). Therefore, inhibition of C5a
and/or C5a receptor (C5aR) could be useful in treating these
chronic diseases.
[0016] The complement system is believed to be activated in
patients with inflammatory bowel disease (IBD) and is thought to
play a role in the disease pathogenesis. Activated complement
products were found at the luminal face of surface epithelial
cells, as well as in the muscularis mucosa and submucosal blood
vessels in IBD patients (Woodruff, T. M., et al., J of Immunol.,
2003, 171: 5514-5520).
[0017] C5aR expression is upregulated on reactive astrocytes,
microglia, and endothelial cells in an inflamed human central
nervous system (Gasque, P. et al., Am. J. Pathol. 150: 31-41
(1997)). C5a might be involved in neurodegenerative diseases, such
as Alzheimer disease (Mukherjee, P. et al., J. Neuroimmunol. 105:
124-130 (2000); O'Barr, S. et al., J. Neuroimmunol. (2000) 105:
87-94; Farkas, I., et al. J. Immunol. (2003) 170:5764-5771),
Parkinson's disease, Pick disease and transmissible spongiform
encephalopathies. Activation of neuronal C5aR may induce apoptosis
(Farkas I et al. J. Physiol. 1998; 507: 679-687). Therefore,
inhibition of C5a and/or C5aR could also be useful in treating
neurodegenerative diseases.
[0018] There is some evidence that C5a production worsens
inflammation associated with atopic dermatitis (Neuber, K., et al.,
Immunology 73:83-87, (1991)), and chronic urticaria (Kaplan, A. P.,
J. Allergy Clin. Immunol. 114; 465-474, (2004).
[0019] Psoriasis is now known to be a T cell-mediated disease
(Gottlieb, E. L. et al., Nat. Med. 1: 442-447 (1995)). However,
neutrophils and mast cells may also be involved in the pathogenesis
of the disease (Terui, T. et al., Exp. Dermatol. 9: 1-10; 2000);
Werfel, T. et al., Arch. Dermatol. Res. 289: 83-86 (1997)).
Neutrophil accumulation under the stratum corneum is observed in
the highly inflamed areas of psoriatic plaques, and psoriatic
lesion (scale) extracts contain highly elevated levels of C5a and
exhibit potent chemotactic activity towards neutrophils, an effect
that can be inhibited by addition of a C5a antibody. T cells and
neutrophils are chemo-attracted by C5a (Nataf, S. et al., J.
Immunol. 162: 4018-4023 (1999); Tsuji, R. F. et al., J. Immunol.
165: 1588-1598 (2000); Cavaillon, J. M. et al., Eur. J. Immunol.
20: 253-257 (1990)). Additionally expression of C5aR has been
demonstrated in plasmacytoid dendritic cells (pDC) isolated from
lesions of cutaneous lupus erythematosus and these cells were shown
to display chemotactic behavior towards C5a, suggesting that
blockade of C5aR on pDC might be efficacious in reducing pDC
infiltration into inflamed skin in both SLE and psoriasis.
Therefore C5a could be an important therapeutic target for
treatment of psoriasis.
[0020] Immunoglobulin G-containing immune complexes (IC) contribute
to the pathophysiology in a number of autoimmune diseases, such as
systemic lupus erythematosus, rheumatoid arthritis, Sjogren's
disease, Goodpasture's syndrome, and hypersensitivity pneumonitis
(Madaio, M. P., Semin. Nephrol. 19: 48-56 (1999); Korganow, A. S.
et al., Immunity 10: 451-459 (1999); Bolten, W. K., Kidney Int. 50:
1754-1760 (1996); Ando, M. et al., Curr. Opin. Pulm. Med. 3:
391-399 (1997)). These diseases are highly heterogeneous and
generally affect one or more of the following organs: skin, blood
vessels, joints, kidneys, heart, lungs, nervous system and liver
(including cirrhosis and liver fibrosis). The classical animal
model for the inflammatory response in these IC diseases is the
Arthus reaction, which features the infiltration of
polymorphonuclear cells, hemorrhage, and plasma exudation (Arthus,
M., C. R. Soc. Biol. 55: 817-824 (1903)). Recent studies show that
C5aR deficient mice are protected from tissue injury induced by IC
(Kohl, J. et al., Mol. Immunol. 36: 893-903 (1999); Baumann, U. et
al., J. Immunol. 164: 1065-1070 (2000)). The results are consistent
with the observation that a small peptidic anti-C5aR antagonist
inhibits the inflammatory response caused by IC deposition
(Strachan, A. J. et al., J. Immunol. 164: 6560-6565 (2000)).
Together with its receptor, C5a plays an important role in the
pathogenesis of IC diseases. Inhibitors of C5a and C5aR could be
useful to treat these diseases.
DESCRIPTION OF RELATED ART
[0021] Only recently have non-peptide based C5a receptor
antagonists been described in the literature (e.g., Sumichika, H.,
et al., J. Biol. Chem. (2002), 277, 49403-49407). Non-peptide based
C5a receptor antagonist have been reported as being effective for
treating endotoxic shock in rats (Stracham, A. J., et al., J. of
Immunol. (2000), 164(12): 6560-6565); and for treating IBD in a rat
model (Woodruff, T. M., et al., J of Immunol., 2003, 171:
5514-5520). Non-peptide based C5a receptor modulators also have
been described in the patent literature by Neurogen Corporation,
(e.g., WO2004/043925, WO2004/018460, WO2005/007087, WO03/082826,
WO03/08828, WO02/49993, WO03/084524); Dompe S.P.A. (WO02/029187);
and The University of Queenland (WO2004/100975).
[0022] There is considerable experimental evidence in the
literature that implicates increased levels of C5a with a number of
diseases and disorders, in particular in autoimmune and
inflammatory diseases and disorders. Thus, there remains a need in
the art for new small organic molecule modulators, e.g., agonists,
preferably antagonists, partial agonists, of the C5a receptor
(C5aR) that are useful for inhibiting pathogenic events, e.g.,
chemotaxis, associated with increased levels anaphylatoxin
activity. The present invention fulfills this and other needs.
BRIEF SUMMARY OF THE INVENTION
[0023] In one aspect, the present invention provides compounds
having the formula:
##STR00001##
and pharmaceutically acceptable salts, hydrates and rotomers
thereof; wherein [0024] C.sup.1 is selected from the group
consisting of aryl and heteroaryl, wherein the heteroaryl group has
from 1-3 heteroatoms as ring members selected from N, O and S; and
wherein said aryl and heteroaryl groups are optionally substituted
with from 1 to 3 R.sup.1 substituents; [0025] C.sup.2 is selected
from the group consisting of aryl and heteroaryl, wherein the
heteroaryl group has from 1-3 heteroatoms as ring members selected
from N, O and S; and wherein said aryl and heteroaryl groups are
optionally substituted with from 1 to 3 R.sup.2 substituents;
[0026] C.sup.3 is selected from the group consisting of C.sub.1-8
alkyl or heteroalkyl, C.sub.3-8 cycloalkyl, C.sub.3-8
cycloalkyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl,
heteroaryl-C.sub.1-4 alkyl, heterocycloalkyl or
heterocycloalkyl-C.sub.1-4 alkyl, wherein the heterocycloalkyl
group or portion has from 1-3 heteroatoms selected from N, O and S,
and wherein the heteroaryl group has from 1-3 heteroatoms as ring
members selected from N, O and S, and each C.sup.3 is optionally
substituted with from 1-3 R.sup.3 substituents; [0027] each R.sup.1
is independently selected from the group consisting of halogen,
--CN, --R.sup.c, --CO.sub.2R.sup.a, --CONR.sup.aR.sup.b,
--C(O)R.sup.a, --OC(O)NR.sup.aR.sup.b, --NR.sup.bC(O)R.sup.a,
--NR.sup.bC(O).sub.2R.sup.c, --NR.sup.a--C(O)NR.sup.aR.sup.b,
--NR.sup.aC(O)NR.sup.aR.sup.b, --NR.sup.aR.sup.b, --OR.sup.a, and
--S(O).sub.2NR.sup.aR.sup.b; wherein each R.sup.a and R.sup.b is
independently selected from hydrogen, C.sub.1-8 alkyl, and
C.sub.1-8 haloalkyl, or when attached to the same nitrogen atom can
be combined with the nitrogen atom to form a five or six-membered
ring having from 0 to 2 additional heteroatoms as ring members
selected from N, O or S, and is optionally substituted with one or
two oxo; each R.sup.c is independently selected from the group
consisting of C.sub.1-8 alkyl or heteroalkyl, C.sub.1-8 haloalkyl,
C.sub.3-6 cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and
wherein the aliphatic and cyclic portions of R.sup.a, R.sup.b and
R.sup.c are optionally further substituted with from one to three
halogen, hydroxy, methyl, amino, alkylamino and dialkylamino
groups; and optionally when two R.sup.1 substituents are on
adjacent atoms, are combined to form a fused five or six-membered
carbocyclic or heterocyclic ring; [0028] each R.sup.2 is
independently selected from the group consisting of halogen, --CN,
--NO.sub.2, --R.sup.f, --CO.sub.2R.sup.d, --CONR.sup.dR.sup.e,
--C(O)R.sup.d, --OC(O)NR.sup.dR.sup.e, --NR.sup.eC(O)R.sup.d,
--NR.sup.eC(O).sub.2R.sup.f, --NR.sup.dC(O)NR.sup.dR.sup.e,
--NR.sup.dC(O)NR.sup.dR.sup.e, --NR.sup.dR.sup.e, --OR.sup.d, and
--S(O).sub.2NR.sup.dR.sup.e; wherein each R.sup.d and R.sup.e is
independently selected from hydrogen, C.sub.1-8 alkyl, and
C.sub.1-8 haloalkyl, or when attached to the same nitrogen atom can
be combined with the nitrogen atom to form a five or six-membered
ring having from 0 to 2 additional heteroatoms as ring members
selected from N, O or S, and is optionally substituted with one or
two oxo; each R.sup.f is independently selected from the group
consisting of C.sub.1-8 alkyl or heteroalkyl, C.sub.1-8 haloalkyl,
C.sub.3-6 cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and
wherein the aliphatic and cyclic portions of R.sup.d, R.sup.e and
R.sup.f are optionally further substituted with from one to three
halogen, hydroxy, methyl, amino, alkylamino and dialkylamino
groups, and optionally when two R.sup.2 groups are on adjacent
atoms, they are combined to form a five- or six-membered ring;
[0029] each R.sup.3 is independently selected from the group
consisting of halogen, --CN, --R.sup.i, --CO.sub.2R.sup.g,
--CONR.sup.gR.sup.h, --C(O)R.sup.g, --C(O)R.sup.i,
--OC(O)NR.sup.gR.sup.h, --NR.sup.hC(O)R.sup.g,
--NR.sup.hCO.sub.2R.sup.i, --NR.sup.gC(O)NR.sup.gR.sup.h,
--NR.sup.gR.sup.h, --OR.sup.g, --OR.sup.j,
--S(O).sub.2NR.sup.gR.sup.h, --X.sup.4--R.sup.j,
--NH--X.sup.4--R.sup.j, --O--X.sup.4--R.sup.j,
--X.sup.4--NR.sup.gR.sup.h, --X.sup.4--NHR.sup.j,
--X.sup.4--CONR.sup.gR.sup.h, --X.sup.4--NR.sup.hC(O)R.sup.g,
--X.sup.4--CO.sub.2--R.sup.g, --O--X.sup.4--CO.sub.2R.sup.g,
--NH--X.sup.4--CO.sub.2R.sup.g, --X.sup.4--NR.sup.hCO.sub.2R.sup.i,
--O--X.sup.4--NR.sup.hCO.sub.2R.sup.i, --NHR.sup.j and
--NHCH.sub.2R.sup.j, wherein X.sup.4 is a C.sub.1-4 alkylene; each
R.sup.g and R.sup.h is independently selected from hydrogen,
C.sub.1-8 alkyl or heteroalkyl, C.sub.3-6 cycloalkyl and C.sub.1-8
haloalkyl, or when attached to the same nitrogen atom can be
combined with the nitrogen atom to form a four-, five- or
six-membered ring having from 0 to 2 additional heteroatoms as ring
members selected from N, O or S and is optionally substituted with
one or two oxo; each R.sup.i is independently selected from the
group consisting of C.sub.1-8 alkyl or heteroalkyl, C.sub.1-8
haloalkyl, C.sub.3-6 cycloalkyl, heterocycloalkyl, aryl and
heteroaryl; and each R.sup.j is selected from the group consisting
of C.sub.3-6 cycloalkyl, imidazolyl, pyrimidinyl, pyrrolinyl,
piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, and
S,S-dioxo-tetrahydrothiopyranyl, and wherein the aliphatic and
cyclic portions of R.sup.g, R.sup.h, R.sup.i and R.sup.j are
optionally further substituted with from one to three halogen,
methyl, CF.sub.3, hydroxy, C.sub.1-4 alkoxy, C.sub.1-4
alkoxy-C.sub.1-4 alkyl, --C(O)O--C.sub.1-8 alkyl, amino, alkylamino
and dialkylamino groups, and optionally when two R.sup.3 groups are
on adjacent atoms, they are combined to form a five- or
six-membered ring; and [0030] X is hydrogen or CH.sub.3.
[0031] In addition to the compounds provided herein, the present
invention further provides pharmaceutical compositions containing
one or more of these compounds, as well as methods for the use of
these compounds in therapeutic methods, primarily to treat diseases
associated with C5a signalling activity.
[0032] In yet another aspect, the present invention provides
methods of diagnosing disease in an individual. In these methods,
the compounds provided herein are administered in labeled form to a
subject, followed by diagnostic imaging to determine the presence
or absence of C5aR. In a related aspect, a method of diagnosing
disease is carried out by contacting a tissue or blood sample with
a labeled compound as provided herein and determining the presence,
absence, or amount of C5aR in the sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 provides structures and activity for representative
compounds of the present invention. The compounds were prepared
using methods as described generally below, as well as methods
provided in the Examples.
DETAILED DESCRIPTION OF THE INVENTION
I. Abbreviation and Definitions
[0034] The term "alkyl", by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain hydrocarbon radical, having the number of carbon atoms
designated (i.e. C.sub.1-8 means one to eight carbons). Examples of
alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl,
t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,
and the like. The term "alkyl" in its broadest sense is also meant
to include those unsaturated groups such as alkenyl and alkynyl
groups. The teem "alkenyl" refers to an unsaturated alkyl group
having one or more double bonds. Similarly, the term "alkynyl"
refers to an unsaturated alkyl group having one or more triple
bonds. Examples of such unsaturated alkyl groups include vinyl,
2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,
3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the
higher homologs and isomers. The term "cycloalkyl" refers to
hydrocarbon rings having the indicated number of ring atoms (e.g.,
C.sub.3-6cycloalkyl) and being fully saturated or having no more
than one double bond between ring vertices. "Cycloalkyl" is also
meant to refer to bicyclic and polycyclic hydrocarbon rings such
as, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc.
The term "heterocycloalkyl" refers to a cycloalkyl group that
contain from one to five heteroatoms selected from N, O, and S,
wherein the nitrogen and sulfur atoms are optionally oxidized, and
the nitrogen atom(s) are optionally quaternized. The
heterocycloalkyl may be a monocyclic, a bicyclic or a polycyclic
ring system. Non limiting examples of heterocycloalkyl groups
include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam,
valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide,
piperidine, 1,4-dioxane, morpholine, thiomorpholine,
thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine,
pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran,
tetrahydrothiophene, quinuclidine, and the like. A heterocycloalkyl
group can be attached to the remainder of the molecule through a
ring carbon or a heteroatom.
[0035] The term "alkylene" by itself or as part of another
substituent means a divalent radical derived from an alkane, as
exemplified by --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. Typically, an
alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with
those groups having 10 or fewer carbon atoms being preferred in the
present invention. A "lower alkyl" or "lower alkylene" is a shorter
chain alkyl or alkylene group, generally having four or fewer
carbon atoms. Similarly, "alkenylene" and "alkynylene" refer to the
unsaturated forms of "alkylene" having double or triple bonds,
respectively.
[0036] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon radical, or combinations
thereof, consisting of the stated number of carbon atoms and from
one to three heteroatoms selected from the group consisting of O,
N, Si and S, and wherein the nitrogen and sulfur atoms may
optionally be oxidized and the nitrogen heteroatom may optionally
be quaternized. The heteroatom(s) O, N and S may be placed at any
interior position of the heteroalkyl group. The heteroatom Si may
be placed at any position of the heteroalkyl group, including the
position at which the alkyl group is attached to the remainder of
the molecule. Examples include --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.3. Similarly, the terms
"heteroalkenyl" and "heteroalkynyl" by itself or in combination
with another term, means, unless otherwise stated, an alkenyl group
or alkynyl group, respectively, that contains the stated number of
carbons and having from one to three heteroatoms selected from the
group consisting of O, N, Si and S, and wherein the nitrogen and
sulfur atoms may optionally be oxidized and the nitrogen heteroatom
may optionally be quaternized. The heteroatom(s) O, N and S may be
placed at any interior position of the heteroalkyl group.
[0037] The term "heteroalkylene" by itself or as part of another
substituent means a divalent radical, saturated or unsaturated or
polyunsaturated, derived from heteroalkyl, as exemplified by
--CH.sub.2--CH.sub.2--S--CH.sub.2CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--,
--O--CH.sub.2--CH.dbd.CH--,
--CH.sub.2--CH.dbd.C(H)CH.sub.2--O--CH.sub.2-- and
--S--CH.sub.2--C.ident.C--. For heteroalkylene groups, heteroatoms
can also occupy either or both of the chain termini (e.g.,
alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the
like).
[0038] The terms "alkoxy," "alkylamino" and "alkylthio" (or
thioalkoxy) are used in their conventional sense, and refer to
those alkyl groups attached to the remainder of the molecule via an
oxygen atom, an amino group, or a sulfur atom, respectively.
Additionally, for dialkylamino groups, the alkyl portions can be
the same or different and can also be combined to form a 3-7
membered ring with the nitrogen atom to which each is attached.
Accordingly, a group represented as --NR.sup.aR.sup.b is meant to
include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl and the
like.
[0039] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "C.sub.1-4 haloalkyl" is mean to include
trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,
3-bromopropyl, and the like.
[0040] The term "aryl" means, unless otherwise stated, a
polyunsaturated, typically aromatic, hydrocarbon group which can be
a single ring or multiple rings (up to three rings) which are fused
together or linked covalently. The term "heteroaryl" refers to aryl
groups (or rings) that contain from one to five heteroatoms
selected from N, O, and S, wherein the nitrogen and sulfur atoms
are optionally oxidized, and the nitrogen atom(s) are optionally
quaternized. A heteroaryl group can be attached to the remainder of
the molecule through a heteroatom. Non-limiting examples of aryl
groups include phenyl, naphthyl and biphenyl, while non-limiting
examples of heteroaryl groups include pyridyl, pyridazinyl,
pyrazinyl, pyrimindinyl, triazinyl, quinolinyl, quinoxalinyl,
quinazolinyl, cinnolinyl, phthalaziniyl, benzotriazinyl, purinyl,
benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl,
isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl,
thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl,
imidazopyridines, benzothiaxolyl, benzofuranyl, benzothienyl,
indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl,
pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl,
isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl and
the like. Substituents for each of the above noted aryl and
heteroaryl ring systems are selected from the group of acceptable
substituents described below.
[0041] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl and the like).
[0042] The above terms (e.g., "alkyl," "aryl" and "heteroaryl"), in
some embodiments, will include both substituted and unsubstituted
forms of the indicated radical. Preferred substituents for each
type of radical are provided below. For brevity, the terms aryl and
heteroaryl will refer to substituted or unsubstituted versions as
provided below, while the term "alkyl" and related aliphatic
radicals is meant to refer to unsubstituted version, unless
indicated to be substituted.
[0043] Substituents for the alkyl radicals (including those groups
often referred to as alkylene, alkenyl, alkynyl and cycloalkyl) can
be a variety of groups selected from: -halogen, --OR', --NR'R'',
--SR', --SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R',
--CONR'R'', --OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --NR'S(O).sub.2R'', --CN and
--NO.sub.2 in a number ranging from zero to (2m'+1), where m' is
the total number of carbon atoms in such radical. R', R'' and R'''
each independently refer to hydrogen, unsubstituted C.sub.1-8
alkyl, unsubstituted heteroalkyl, unsubstituted aryl, aryl
substituted with 1-3 halogens, unsubstituted C.sub.1-8 alkyl,
C.sub.1-8 alkoxy or C.sub.1-8 thioalkoxy groups, or unsubstituted
aryl-C.sub.1-4 alkyl groups. When R' and R'' are attached to the
same nitrogen atom, they can be combined with the nitrogen atom to
form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, --NR'R'' is
meant to include 1-pyrrolidinyl and 4-morpholinyl. The term "acyl"
as used by itself or as part of another group refers to an alkyl
radical wherein two substitutents on the carbon that is closest to
the point of attachment for the radical is replaced with the
substitutent .dbd.O (e.g., --C(O)CH.sub.3,
--C(O)CH.sub.2CH.sub.2OR' and the like).
[0044] Similarly, substituents for the aryl and heteroaryl groups
are varied and are generally selected from: -halogen, --OR',
--OC(O)R', --NR'R'', --SR', --R', --CN, --NO.sub.2, --CO.sub.2R',
--CONR'R'', --C(O)R', --OC(O)NR' R'', --NR''C(O)R',
--NR''C(O).sub.2R', --NR'--C(O)NR''R''', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --NR'S(O).sub.2R'', --N.sub.3,
perfluoro(C.sub.1-C.sub.4)alkoxy, and
perfluoro(C.sub.1-C.sub.4)alkyl, in a number ranging from zero to
the total number of open valences on the aromatic ring system; and
where R', R'' and R''' are independently selected from hydrogen,
C.sub.1-8 alkyl, C.sub.3-6 cycloalkyl, C.sub.2-8 alkenyl, C.sub.2-8
alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted
aryl)-C.sub.1-4 alkyl, and unsubstituted aryloxy-C.sub.1-4 alkyl.
Other suitable substituents include each of the above aryl
substituents attached to a ring atom by an alkylene tether of from
1-4 carbon atoms.
[0045] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CH.sub.2).sub.q--U--, wherein T and U are
independently --NH--, --O--, --CH.sub.2-- or a single bond, and q
is an integer of from 0 to 2. Alternatively, two of the
substituents on adjacent atoms of the aryl or heteroaryl ring may
optionally be replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CH.sub.2--, --O--, --NH--, --S--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2NR'-- or a single bond, and r is an integer of from 1
to 3. One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of the aryl or heteroaryl ring
may optionally be replaced with a substituent of the formula
--(CH.sub.2).sub.s--X--(CH.sub.2).sub.t--, where s and t are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituent R' in --NR'-- and --S(O).sub.2NR'-- is selected from
hydrogen or unsubstituted C.sub.1-6 alkyl.
[0046] As used herein, the term "heteroatom" is meant to include
oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
[0047] The term "ionic liquid" refers to any liquid that contains
mostly ions. Preferably, in the present invention, "ionic liquid"
refers to the salts whose melting point is relatively low (e.g.,
below 250.degree. C.). Examples of ionic liquids include but are
not limited to 1-butyl-3-methylimidazolium tetrafluoroborate,
1-hexyl-3-methylimidazolium tetrafluoroborate,
1-octyl-3-methylimidazolium tetrafluoroborate,
1-nonyl-3-methylimidazolium tetrafluoroborate,
1-decyl-3-methylimidazolium tetrafluoroborate,
1-hexyl-3-methylimidazolium hexafluorophosphate and
1-hexyl-3-methylimidazolium bromide, and the like.
[0048] The term "pharmaceutically acceptable salts" is meant to
include salts of the active compounds which are prepared with
relatively nontoxic acids or bases, depending on the particular
substituents found on the compounds described herein. When
compounds of the present invention contain relatively acidic
functionalities, base addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired base, either neat or in a suitable inert solvent. Examples
of salts derived from pharmaceutically-acceptable inorganic bases
include aluminum, ammonium, calcium, copper, ferric, ferrous,
lithium, magnesium, manganic, manganous, potassium, sodium, zinc
and the like. Salts derived from pharmaceutically-acceptable
organic bases include salts of primary, secondary and tertiary
amines, including substituted amines, cyclic amines,
naturally-occurring amines and the like, such as arginine, betaine,
caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine,
2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,
ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,
glucosamine, histidine, hydrabamine, isopropylamine, lysine,
methylglucamine, morpholine, piperazine, piperadine, polyamine
resins, procaine, purines, theobromine, triethylamine,
trimethylamine, tripropylamine, tromethamine and the like. When
compounds of the present invention contain relatively basic
functionalities, acid addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired acid, either neat or in a suitable inert solvent. Examples
of pharmaceutically acceptable acid addition salts include those
derived from inorganic acids like hydrochloric, hydrobromic,
nitric, carbonic, monohydrogencarbonic, phosphoric,
monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, malonic, benzoic, succinic,
suberic, fumaric, mandelic, phthalic, benzenesulfonic,
p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
Also included are salts of amino acids such as arginate and the
like, and salts of organic acids like glucuronic or galactunoric
acids and the like (see, for example, Berge, S. M., et al,
"Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977,
66, 1-19). Certain specific compounds of the present invention
contain both basic and acidic functionalities that allow the
compounds to be converted into either base or acid addition
salts.
[0049] The neutral forms of the compounds may be regenerated by
contacting the salt with a base or acid and isolating the parent
compound in the conventional manner. The parent form of the
compound differs from the various salt forms in certain physical
properties, such as solubility in polar solvents, but otherwise the
salts are equivalent to the parent form of the compound for the
purposes of the present invention.
[0050] In addition to salt forms, the present invention provides
compounds which are in a prodrug form. Prodrugs of the compounds
described herein are those compounds that readily undergo chemical
changes under physiological conditions to provide the compounds of
the present invention. Additionally, prodrugs can be converted to
the compounds of the present invention by chemical or biochemical
methods in an ex vivo environment. For example, prodrugs can be
slowly converted to the compounds of the present invention when
placed in a transdermal patch reservoir with a suitable enzyme or
chemical reagent.
[0051] Certain compounds of the present invention can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. In general, the solvated forms are equivalent to unsolvated
forms and are intended to be encompassed within the scope of the
present invention. Certain compounds of the present invention may
exist in multiple crystalline or amorphous forms. In general, all
physical forms are equivalent for the uses contemplated by the
present invention and are intended to be within the scope of the
present invention.
[0052] Certain compounds of the present invention possess
asymmetric carbon atoms (optical centers) or double bonds; the
racemates, diastereomers, geometric isomers, regioisomers and
individual isomers (e.g., separate enantiomers) are all intended to
be encompassed within the scope of the present invention. The
compounds of the present invention may also contain unnatural
proportions of atomic isotopes at one or more of the atoms that
constitute such compounds. For example, the compounds may be
radiolabeled with radioactive isotopes, such as for example tritium
(.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C). All
isotopic variations of the compounds of the present invention,
whether radioactive or not, are intended to be encompassed within
the scope of the present invention. For example, the compounds may
be prepared such that any number of hydrogen atoms are replaced
with a deuterium (.sup.2H) isotope.
II. Compounds
[0053] In one aspect, the present invention provides compounds
having the formula I:
##STR00002##
and pharmaceutically acceptable salts, hydrates and rotomers
thereof; wherein [0054] C.sup.1 is selected from the group
consisting of aryl and heteroaryl, wherein the heteroaryl group has
from 1-3 heteroatoms as ring members selected from N, O and S; and
wherein said aryl and heteroaryl groups are optionally substituted
with from 1 to 3 R.sup.1 substituents; [0055] C.sup.2 is selected
from the group consisting of aryl and heteroaryl, wherein the
heteroaryl group has from 1-3 heteroatoms as ring members selected
from N, O and S; and wherein said aryl and heteroaryl groups are
optionally substituted with from 1 to 3 R.sup.2 substituents;
[0056] C.sup.3 is selected from the group consisting of C.sub.1-8
alkyl or heteroalkyl, C.sub.3-8 cycloalkyl, C.sub.3-8
cycloalkyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl, heteroaryl,
heteroaryl-C.sub.1-4 alkyl, heterocycloalkyl or
heterocycloalkyl-C.sub.1-4 alkyl, wherein the heterocycloalkyl
group or portion has from 1-3 heteroatoms selected from N, O and S,
and wherein the heteroaryl group has from 1-3 heteroatoms as ring
members selected from N, O and S, and each C.sup.3 is optionally
substituted with from 1-3 R.sup.3 substituents; [0057] each R.sup.1
is independently selected from the group consisting of halogen,
--CN, --R.sup.c, --CO.sub.2R.sup.a, --CONR.sup.aR.sup.b,
--C(O)R.sup.a, --OC(O)NR.sup.aR.sup.b, --NR.sup.bC(O)R.sup.a,
--NR.sup.bC(O).sub.2R.sup.c, --NR.sup.a--C(O)NR.sup.aR.sup.b,
--NR.sup.aC(O)NR.sup.aR.sup.b, --NR.sup.aR.sup.b, --OR.sup.a, and
--S(O).sub.2NR.sup.aR.sup.b; wherein each R.sup.a and R.sup.b is
independently selected from hydrogen, C.sub.1-8 alkyl, and
C.sub.1-8 haloalkyl, or when attached to the same nitrogen atom can
be combined with the nitrogen atom to form a five or six-membered
ring having from 0 to 2 additional heteroatoms as ring members
selected from N, O or S, and is optionally substituted with one or
two oxo; each R.sup.c is independently selected from the group
consisting of C.sub.1-8 alkyl or heteroalkyl, C.sub.1-8 haloalkyl,
C.sub.3-6 cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and
wherein the aliphatic and cyclic portions of R.sup.a, R.sup.b and
R.sup.c are optionally further substituted with from one to three
halogen, hydroxy, methyl, amino, alkylamino and dialkylamino
groups; and optionally when two R.sup.1 substituents are on
adjacent atoms, are combined to form a fused five or six-membered
carbocyclic or heterocyclic ring; [0058] each R.sup.2 is
independently selected from the group consisting of halogen, --CN,
--NO.sub.2, --R.sup.f, --CO.sub.2R.sup.d, --CONR.sup.dR.sup.e,
--C(O)R.sup.d, --OC(O)NR.sup.dR.sup.e, --NR.sup.eC(O)R.sup.d,
--NR.sup.eC(O).sub.2R.sup.f, --NR.sup.dC(O)NR.sup.dR.sup.e,
--NR.sup.dC(O)NR.sup.dR.sup.e, --NR.sup.dR.sup.e, --OR.sup.d, and
--S(O).sub.2NR.sup.dR.sup.e; wherein each R.sup.d and R.sup.e is
independently selected from hydrogen, C.sub.1-8 alkyl, and
C.sub.1-8 haloalkyl, or when attached to the same nitrogen atom can
be combined with the nitrogen atom to form a five or six-membered
ring having from 0 to 2 additional heteroatoms as ring members
selected from N, O or S, and is optionally substituted with one or
two oxo; each R.sup.f is independently selected from the group
consisting of C.sub.1-8 alkyl or heteroalkyl, C.sub.1-8 haloalkyl,
C.sub.3-6 cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and
wherein the aliphatic and cyclic portions of R.sup.d, R.sup.e and
R.sup.f are optionally further substituted with from one to three
halogen, hydroxy, methyl, amino, alkylamino and dialkylamino
groups, and optionally when two R.sup.2 groups are on adjacent
atoms, they are combined to form a five- or six-membered ring;
[0059] each R.sup.3 is independently selected from the group
consisting of halogen, --CN, --R.sup.i, --CO.sub.2R.sup.g,
--CONR.sup.gR.sup.h, --C(O)R.sup.g, --C(O)R.sup.i,
--OC(O)NR.sup.gR.sup.h, --NR.sup.hC(O)R.sup.g,
--NR.sup.hCO.sub.2R.sup.i, --NR.sup.gC(O)NR.sup.gR.sup.h,
--NR.sup.gR.sup.h, --OR.sup.g, --OR.sup.j,
--S(O).sub.2NR.sup.gR.sup.h, --X.sup.4--R.sup.j,
--NH--X.sup.4--R.sup.j, --O--X.sup.4--R.sup.j,
--X.sup.4--NR.sup.gR.sup.h, --X.sup.4--NHR.sup.j,
--X.sup.4--CONR.sup.gR.sup.h, --X.sup.4--NR.sup.hC(O)R.sup.g,
--X.sup.4--CO.sub.2R.sup.g, --O--X.sup.4--CO.sub.2R.sup.g,
--NH--X.sup.4--CO.sub.2R.sup.g, --X.sup.4--NR.sup.hCO.sub.2R.sup.i,
--O--X.sup.4--NR.sup.hCO.sub.2R.sup.i, --NHR.sup.j and
--NHCH.sub.2R.sup.j, wherein X.sup.4 is a C.sub.1-4 alkylene; each
R.sup.g and R.sup.h is independently selected from hydrogen,
C.sub.1-8 alkyl or heteroalkyl, C.sub.3-6 cycloalkyl and C.sub.1-8
haloalkyl, or when attached to the same nitrogen atom can be
combined with the nitrogen atom to form a four-, five- or
six-membered ring having from 0 to 2 additional heteroatoms as ring
members selected from N, O or S and is optionally substituted with
one or two oxo; each R.sup.i is independently selected from the
group consisting of C.sub.1-8 alkyl or heteroalkyl, C.sub.1-8
haloalkyl, C.sub.3-6 cycloalkyl, heterocycloalkyl, aryl and
heteroaryl; and each R.sup.j is selected from the group consisting
of C.sub.3-6 cycloalkyl, imidazolyl, pyrimidinyl, pyrrolinyl,
piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, and
S,S-dioxo-tetrahydrothiopyranyl, and wherein the aliphatic and
cyclic portions of R.sup.g, R.sup.h, and R.sup.i are optionally
further substituted with from one to three halogen, methyl,
CF.sub.3, hydroxy, C.sub.1-4 alkoxy, C.sub.1-4 alkoxy-C.sub.1-4
alkyl, --C(O)O--C.sub.1-8 alkyl, amino, alkylamino and dialkylamino
groups, and optionally when two R.sup.3 groups are on adjacent
atoms, they are combined to form a five- or six-membered ring; and
[0060] X is hydrogen or CH.sub.3.
[0061] In formula I, the substituent C.sup.1 is, in one embodiment,
selected from the group consisting of phenyl, pyridyl, indolyl and
thiazolyl, each of which is optionally substituted with from 1 to 3
R.sup.1 substituents. Preferably, each R.sup.1 is independently
selected from the group consisting of halogen, --CN, --R.sup.c,
--NR.sup.aR.sup.b and --OR.sup.a, and wherein each R.sup.a and
R.sup.b is independently selected from hydrogen, C.sub.1-8 alkyl,
and C.sub.1-8 haloalkyl, or when attached to the same nitrogen atom
can be combined with the nitrogen atom to form a pyrrolidine ring;
each R.sup.c is independently selected from the group consisting of
C.sub.1-8 alkyl, C.sub.1-8 haloalkyl and C.sub.3-6 cycloalkyl, and
wherein the aliphatic and cyclic portions of R.sup.a, R.sup.b and
R.sup.c are optionally further substituted with from one to three
hydroxy, methyl, amino, alkylamino and dialkylamino groups; and
optionally when two R.sup.1 substituents are on adjacent atoms, are
combined to form a fused five or six-membered carbocyclic ring. In
selected embodiments of the invention, C.sup.1 is selected
from:
##STR00003## ##STR00004##
[0062] Returning to formula I, the substituents C.sup.2 is, in one
embodiment, selected from the group consisting of phenyl, naphthyl,
pyridyl and indolyl, each of which is optionally substituted with
from 1 to 3 R.sup.2 substituents. Preferably, each R.sup.2 is
independently selected from the group consisting of halogen,
--R.sup.f and --OR.sup.d; wherein each R.sup.d is independently
selected from hydrogen, C.sub.1-8 alkyl, and C.sub.1-8 haloalkyl;
each R.sup.f is independently selected from the group consisting of
C.sub.1-8 alkyl, C.sub.1-8 haloalkyl, C.sub.3-6 cycloalkyl,
heterocycloalkyl and heteroaryl, and wherein the aliphatic and
cyclic portions of R.sup.d and R.sup.f are optionally further
substituted with from one to three halogen, hydroxy, methyl, amino,
alkylamino and dialkylamino groups. In selected embodiments of the
invention, C.sup.2 is selected from the group consisting of:
##STR00005##
[0063] The substituent C.sup.3 is, in some embodiments, selected
from the group consisting of C.sub.3-6 alkyl, C.sub.3-6 cycloalkyl,
C.sub.3-6 cycloalkyl C.sub.1-2alkyl, phenyl, pyridinyl, pyrazolyl,
piperidinyl, pyrrolidinyl, piperidinylmethyl and
pyrrolidinylmethyl, each of which is optionally substituted with
from 1 to 3 R.sup.3 substituents. Preferably, each R.sup.3 is
independently selected from the group consisting of halogen,
--R.sup.i, --CO.sub.2R.sup.g, --CONR.sup.gR.sup.h,
--NR.sup.hC(O)R.sup.g, --NR.sup.hC(O).sub.2R.sup.i,
--NR.sup.gR.sup.h, --OR.sup.g, --X.sup.4--NR.sup.gR.sup.h,
--X.sup.4--CONR.sup.gR.sup.h, --X.sup.4--NR.sup.hC(O)R.sup.g,
--NHR.sup.j and --NHCH.sub.2R.sup.j, wherein X.sup.4 is a C.sub.1-3
alkylene; each R.sup.g and R.sup.h is independently selected from
hydrogen, C.sub.1-8 alkyl, C.sub.3-6 cycloalkyl and C.sub.1-8
haloalkyl, or when attached to the same nitrogen atom can be
combined with the nitrogen atom to form a five or six-membered ring
having from 0 to 1 additional heteroatoms as ring members selected
from N, O or S and is optionally substituted with one or two oxo;
each R.sup.i is independently selected from the group consisting of
C.sub.1-8 alkyl, C.sub.1-8 haloalkyl, C.sub.3-6 cycloalkyl,
heterocycloalkyl, aryl and heteroaryl; and each R.sup.j is selected
from the group consisting of C.sub.3-6 cycloalkyl, pyrrolinyl,
piperidinyl, morpholinyl, tetrahydrofuranyl, and tetrahydropyranyl,
and wherein the aliphatic and cyclic portions of R.sup.g, R.sup.h,
R.sup.i and R.sup.j are optionally further substituted with from
one to three halogen, methyl, CF.sub.3, hydroxy, amino, alkylamino
and dialkylamino groups. In selected embodiments of the invention,
C.sup.3 is selected from the group consisting of:
##STR00006## ##STR00007##
In other embodiments, C.sup.3 is selected from the group consisting
of:
##STR00008## ##STR00009##
[0064] Returning to formula I, X is preferably H.
Subformulae of Formula I:
[0065] In one embodiment of the invention, compounds of formula I
have subformula Ia:
##STR00010##
[0066] In a second embodiment of the invention, compounds of
formula I have subformula Ib:
##STR00011##
[0067] In a third embodiment of the invention, compounds of formula
I have subformula Ic:
##STR00012##
wherein X.sup.1 is selected from the group consisting of N, CH and
CR.sup.1; the subscript n is an integer of from 0 to 2; X.sup.2 is
selected from the group consisting of N, CH and CR.sup.2; and the
subscript m is an integer of from 0 to 2.
[0068] In a fourth embodiment of the invention, compounds of
formula I have subformula Id:
##STR00013##
wherein X.sup.1 is selected from the group consisting of N, CH and
CR.sup.1; the subscript n is an integer of from 0 to 2; X.sup.2 is
selected from the group consisting of N, CH and CR.sup.2; and the
subscript m is an integer of from 0 to 2.
[0069] In a fifth embodiment of the invention, compounds of formula
I have subformula Ie:
##STR00014##
wherein the subscript p is an integer of from 0 to 3; X.sup.1 is
selected from the group consisting of N, CH and CR.sup.1; the
subscript n is an integer of from 0 to 2; X.sup.2 is selected from
the group consisting of N, CH and CR.sup.2; and the subscript m is
an integer of from 0 to 2.
[0070] In other selected embodiments, the compounds of the
invention are represented by:
##STR00015##
wherein the substituents R.sup.1, R.sup.2 and R.sup.3, and the
subscript p all have the meanings provided with reference to
formula I.
[0071] In still other selected embodiments, the compounds of the
invention are represented by:
##STR00016##
wherein the substituents R.sup.1 and R.sup.3, and the subscript p
all have the meanings provided with reference to formula I.
[0072] In a particularly preferred group of embodiments, the
compounds of the invention are represented by formula (Ie.sup.5)
wherein R.sup.3 is a member selected from the group consisting of
--NR.sup.gR.sup.h, --NHR.sup.j and --NHCH.sub.2R.sup.j, and each
R.sup.g, R.sup.h and R.sup.j have the meanings provided with
reference to formula I.
[0073] In another particularly preferred group of embodiments, the
compounds of the invention are represented by formula (Ie.sup.5)
wherein R.sup.3 is a member selected from the group consisting of
--X.sup.4--NR.sup.gR.sup.h, --X.sup.4--R.sup.j and
--X.sup.4--NR.sup.hCOR.sup.g, and each of X.sup.4, R.sup.g, R.sup.h
and R.sup.j have the meanings provided with reference to formula
I.
[0074] Compounds of the invention having formula I can exist in
different diastereomeric forms, e.g., the substituents C.sup.1 and
C.sup.2 in subformulae Ia and Ic can be cis to each other or trans
to each other. As used herein, the terms cis or trans are used in
their conventional sense in the chemical arts, i.e., referring to
the position of the substituents to one another relative to a
reference plane, e.g., a double bond, or a ring system, such as a
decalin-type ring system or a hydroquinolone ring system: in the
cis isomer, the substituents are on the same side of the reference
plane, in the trans isomer the substituents are on opposite sides.
Additionally, different conformers are contemplated by the present
invention, as well as distinct rotamers. Conformers are
conformational isomers that can differ by rotations about one or
more .sigma. bonds. Rotamers are conformers that differ by rotation
about only a single .sigma. bond.
Preparation of Compounds
[0075] Those skilled in the art will recognize that there are a
variety of methods available to synthesize molecules represented in
the claims. In general, useful methods for synthesizing compounds
represented in the claims consist of four parts, which may be done
in any order: Formation of the piperidine ring, installation of two
amide bonds, and installation and/or modification of functional
groups on C.sup.1, C.sup.2, and C.sup.3.
[0076] Several methods for the preparation of claimed compounds are
illustrated below (eq. 1-6).
##STR00017##
[0077] Equations 1-4 demonstrate some methods of forming the
piperidine ring. Coupling at the 2-position of the pyridine ring
can be accomplished via transition metal mediated couplings as
shown in eq. 1-2, or metal catalyzed addition of an organometallic
species such as the zincate or magnesium salt (eq. 3). Subsequent
to coupling at the 2-position, transition metal mediated
hydrogenation of the pyridine ring yields the piperidine ring
system (eq. 1-3). Another method results in elaboration of a
n-amino acid to a piperidine ring as described in eq. 4. Those
skilled in the art will recognize that many synthetic methodologies
can yield substituted piperidines, including C--C or C--N
cyclization of acyclic precursors via alkylation or ring-closing
metathesis. Relative stereochemistry may be set by a variety of
methods, including facial selectivity during the hydrogenation
step. Absolute stereochemistry may also be set via a variety of
methods, via the use of chiral ligands or a chiral auxiliary,
separation of chiral diasteroisomers, use of chiral starting
materials, or classical resolution. Compounds with 2,3-trans
stereochemistry may have the relative stereochemistry set during
the piperidine formation, or may be derived via epimerization of a
2,3-cis piperidine as illustrated in eq. 5.
##STR00018##
[0078] Acylation of the piperidine ring is described in equation 6.
In the case of eq. 6, X may be chosen from an appropriate group
such as OH, Cl and F, or from any group capable of activating a
carbonyl group for addition of an amine (e.g, OSu, imidazole,
etc.). Such couplings may be assisted by the use of inorganic or
organic bases, activating agents such as HBTU, and also by
catalysts, in particular by those catalysts known in tha art which
assist in the formation of amide bonds, such as DMAP, HOBT, etc.
Suitable coupling partners include a carboxylic acid and a
piperidine, an acyl fluoride and an amine and so forth. Those
skilled in the art will recognize that there are other possible
combinations which will also result in the desired product.
##STR00019##
[0079] A variety of methods described above have been used to
prepare compounds of the invention, some of which are described in
the examples.
[0080] A family of specific compounds of particular interest having
formula I consists of compounds, pharmaceutically acceptable salts,
hydrates and rotomers thereof, as set forth in FIG. 1. For the
compounds of FIG. 1, any bond not illustrating at attached atom or
group is meant to be a methyl group. For example,
##STR00020##
is meant to illustrate
##STR00021##
III. Pharmaceutical Compositions
[0081] In addition to the compounds provided above, compositions
for modulating C5a activity in humans and animals will typically
contain a pharmaceutical carrier or diluent.
[0082] The term "composition" as used herein is intended to
encompass a product comprising the specified ingredients in the
specified amounts, as well as any product which results, directly
or indirectly, from combination of the specified ingredients in the
specified amounts. By "pharmaceutically acceptable" it is meant the
carrier, diluent or excipient must be compatible with the other
ingredients of the formulation and not deleterious to the recipient
thereof.
[0083] The pharmaceutical compositions for the administration of
the compounds of this invention may conveniently be presented in
unit dosage form and may be prepared by any of the methods well
known in the art of pharmacy and drug delivery. All methods include
the step of bringing the active ingredient into association with
the carrier which constitutes one or more accessory ingredients. In
general, the pharmaceutical compositions are prepared by uniformly
and intimately bringing the active ingredient into association with
a liquid carrier or a finely divided solid carrier or both, and
then, if necessary, shaping the product into the desired
formulation. In the pharmaceutical composition the active object
compound is included in an amount sufficient to produce the desired
effect upon the process or condition of diseases.
[0084] The pharmaceutical compositions containing the active
ingredient may be in a foam suitable for oral use, for example, as
tablets, troches, lozenges, aqueous or oily suspensions,
dispersible powders or granules, emulsions and self emulsifications
as described in U.S. Patent Application 2002-0012680, hard or soft
capsules, syrups, elixirs, solutions, buccal patch, oral gel,
chewing gum, chewable tablets, effervescent powder and effervescent
tablets. Compositions intended for oral use may be prepared
according to any method known to the art for the manufacture of
pharmaceutical compositions and such compositions may contain one
or more agents selected from the group consisting of sweetening
agents, flavoring agents, coloring agents, antioxidants and
preserving agents in order to provide pharmaceutically elegant and
palatable preparations. Tablets contain the active ingredient in
admixture with non-toxic pharmaceutically acceptable excipients
which are suitable for the manufacture of tablets. These excipients
may be for example, inert diluents, such as cellulose, silicon
dioxide, aluminum oxide, calcium carbonate, sodium carbonate,
glucose, mannitol, sorbitol, lactose, calcium phosphate or sodium
phosphate; granulating and disintegrating agents, for example, corn
starch, or alginic acid; binding agents, for example PVP,
cellulose, PEG, starch, gelatin or acacia, and lubricating agents,
for example magnesium stearate, stearic acid or talc. The tablets
may be uncoated or they may be coated, enterically or otherwise, by
known techniques to delay disintegration and absorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time delay material such as
glyceryl monostearate or glyceryl distearate may be employed. They
may also be coated by the techniques described in the U.S. Pat.
Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic
therapeutic tablets for control release.
[0085] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, polyethylene glycol (PEG) of various average
sizes (e.g., PEG400, PEG4000) and certain surfactants such as
cremophor or solutol, or as soft gelatin capsules wherein the
active ingredient is mixed with water or an oil medium, for example
peanut oil, liquid paraffin, or olive oil. Additionally, emulsions
can be prepared with a non-water miscible ingredient such as oils
and stabilized with surfactants such as mono- or di-glycerides, PEG
esters and the like.
[0086] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydroxy-propylmethylcellulose, sodium alginate,
polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents may be a naturally-occurring phosphatide, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids, for example polyoxy-ethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example heptadecaethyleneoxycetanol, or 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, for example polyethylene
sorbitan monooleate. The aqueous suspensions may also contain one
or more preservatives, for example ethyl, or n-propyl,
p-hydroxybenzoate, one or more coloring agents, one or more
flavoring agents, and one or more sweetening agents, such as
sucrose or saccharin.
[0087] Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil, for example 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, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set forth above, and flavoring agents may be added to
provide a palatable oral preparation. These compositions may be
preserved by the addition of an anti-oxidant such as ascorbic
acid.
[0088] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, may also be present.
[0089] The pharmaceutical compositions of the invention may also be
in the form of oil-in-water emulsions. The oily phase may be a
vegetable oil, for example olive oil or arachis oil, or a mineral
oil, for example liquid paraffin or mixtures of these. Suitable
emulsifying agents may be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions may also contain sweetening and flavoring
agents.
[0090] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, a preservative and
flavoring and coloring agents. Oral solutions can be prepared in
combination with, for example, cyclodextrin, PEG and
surfactants.
[0091] The pharmaceutical compositions may be in the form of a
sterile injectable aqueous or oleagenous suspension. This
suspension may be formulated according to the known art using those
suitable dispersing or wetting agents and suspending agents which
have been mentioned above. The sterile injectable preparation may
also be a sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butane diol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally 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 injectables.
[0092] The compounds of the present invention may also be
administered in the form of suppositories for rectal administration
of the drug. These compositions can be prepared by mixing the drug
with a suitable non-irritating excipient which is solid at ordinary
temperatures but liquid at the rectal temperature and will
therefore melt in the rectum to release the drug. Such materials
include cocoa butter and polyethylene glycols. Additionally, the
compounds can be administered via ocular delivery by means of
solutions or ointments. Still further, transdermal delivery of the
subject compounds can be accomplished by means of iontophoretic
patches and the like. For topical use, creams, ointments, jellies,
solutions or suspensions, etc., containing the compounds of the
present invention are employed. As used herein, topical application
is also meant to include the use of mouth washes and gargles.
[0093] The compounds of this invention may also be coupled a
carrier that is a suitable polymers as targetable drug carriers.
Such polymers can include polyvinylpyrrolidone, pyran copolymer,
polyhydroxy-propyl-methacrylamide-phenol,
polyhydroxyethyl-aspartamide-phenol, or
polyethyleneoxide-polylysine substituted with palmitoyl residues.
Furthermore, the compounds of the invention may be coupled to a
carrier that is a class of biodegradable polymers useful in
achieving controlled release of a drug, for example polylactic
acid, polyglycolic acid, copolymers of polylactic and polyglycolic
acid, polyepsilon caprolactone, polyhydroxy butyric acid,
polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates
and cross linked or amphipathic block copolymers of hydrogels.
Polymers and semipermeable polymer matrices may be formed into
shaped articles, such as valves, stents, tubing, prostheses and the
like. In one embodiment of the invention, the compound of the
invention is coupled to a polymer or semipermeable polymer matrix
that is formed as a stent or stent-graft device.
IV. Methods of Treating Diseases and Disorders Modulated by C5a
[0094] The compounds of the invention may be used as agonists,
(preferably) antagonists, partial agonists, inverse agonists, of
C5a receptors in a variety of contexts, both in vitro and in vivo.
In one embodiment, the compounds of the invention are C5aR
antagonist that can be used to inhibit the binding of C5a receptor
ligand (e.g., C5a) to C5a receptor in vitro or in vivo. In general,
such methods comprise the step of contacting a C5a receptor with a
sufficient amount of one or more C5a receptor modulators as
provided herein, in the presence of C5a receptor ligand in aqueous
solution and under conditions otherwise suitable for binding of the
ligand to C5a receptor. The C5a receptor may be present in
suspension (e.g., in an isolated membrane or cell preparation), in
a cultured or isolated cell, or in a tissue or organ.
[0095] Preferably, the amount of C5a receptor modulator contacted
with the receptor should be sufficient to inhibit C5a binding to
C5a receptor in vitro as measured, for example, using a radioligand
binding assay, calcium mobilization assay, or chemotaxis assay as
described herein.
[0096] In one embodiment of the invention, the C5a modulators of
the invention are used to modulate, preferably inhibit, the
signal-transducing activity of a C5a receptor, for example, by
contacting one or more compound(s) of the invention with a C5a
receptor (either in vitro or in vivo) under conditions suitable for
binding of the modulator(s) to the receptor. The receptor may be
present in solution or suspension, in a cultured or isolated cell
preparation or within a patient. Any modulation of the signal
transducing activity may be assessed by detecting an effect on
calcium ion calcium mobilization or by detecting an effect on C5a
receptor-mediated cellular chemotaxis. In general, an effective
amount of C5a modulator(s) is an amount sufficient to modulate C5a
receptor signal transducing activity in vitro within a calcium
mobilization assay or C5a receptor-mediated cellular chemotaxis
within a migration assay.
[0097] When compounds of the invention are used to inhibit C5a
receptor-mediated cellular chemotaxis, preferably leukocyte (e.g.,
neutrophil) chemotaxis, in an in vitro chemotaxis assay, such
methods comprise contacting white blood cells (particularly primate
white blood cells, especially human white blood cells) with one or
more compounds of the invention. Preferably the concentration is
sufficient to inhibit chemotaxis of white blood cells in an in
vitro chemotaxis assay, so that the levels of chemotaxis observed
in a control assay are significantly higher, as described above,
than the levels observed in an assay to which a compound of the
invention has been added.
[0098] In another embodiment, the compounds of the present
invention are useful for facilitating organ transplants. In this
embodiment, the compounds can be placed in a solution, with the
organ prior to transplant.
[0099] In another embodiment, the compounds of the present
invention further can be used for treating patients suffering from
conditions that are responsive to C5a receptor modulation. As used
herein, the term "treating" or "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). As used herein, a
condition is considered "responsive to C5a receptor modulation" if
modulation of C5a receptor activity results in the reduction of
inappropriate activity of a C5a receptor. As used herein, the term
"patients" include primates (especially humans), domesticated
companion animals (such as dogs, cats, horses, and the like) and
livestock (such as cattle, pigs, sheep, and the like), with dosages
as described herein.
Conditions that can be Treated by C5a Modulation:
[0100] Autoimmune disorders--e.g., Rheumatoid arthritis, systemic
lupus erythematosus, Guillain-Barre syndrome, pancreatitis, lupus
nephritis, lupus glomerulonephritis, psoriasis, Crohn's disease,
vasculitis, irritable bowel syndrome, dermatomyositis, multiple
sclerosis, bronchial asthma, pemphigus, pemphigoid, scleroderma,
myasthenia gravis, autoimmune hemolytic and thrombocytopenic
states, Goodpasture's syndrome (and associated glomerulonephritis
and pulmonary hemorrhage), immunovasculitis, tissue graft
rejection, hyperacute rejection of transplanted organs; and the
like.
[0101] Inflammatory disorders and related conditions--e.g.,
Neutropenia, sepsis, septic shock, Alzheimer's disease, multiple
sclerosis, stroke, inflammatory bowel disease (IBD), age-related
macular degeneration (AMD, both wet and dry forms), inflammation
associated with severe burns, lung injury, and ischemia-reperfusion
injury, osteoarthritis, as well as acute (adult) respiratory
distress syndrome (ARDS), chronic pulmonary obstructive disorder
(COPD), systemic inflammatory response syndrome (SIRS), atopic
dermatitis, psoriasis, chronic urticaria 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, contact
sensitivity responses, and inflammation resulting from contact of
blood with artificial surfaces that can cause complement
activation, 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), 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). Also included are diseases related
to ischemia/reperfusion injury, such as those resulting from
transplants, including solid organ transplant, and syndromes such
as ischemic reperfusion injury, ischemic colitis and cardiac
ischemia. Compounds of the instant invention may also be useful in
the treatment of age-related macular degeneration (Hageman et al,
P.N.A.S. 102: 7227-7232, 2005).
[0102] Cardiovascular and Cerebrovascular Disorders--e.g.,
myocardial infarction, coronary thrombosis, vascular occlusion,
post-surgical vascular reocclusion, atherosclerosis, traumatic
central nervous system injury, and ischemic heart disease. In one
embodiment, an effective amount of a compound of the invention may
be administered to a patient at risk for myocardial infarction or
thrombosis (i.e., a patient who has one or more recognized risk
factor for myocardial infarction or thrombosis, such as, but not
limited to, obesity, smoking, high blood pressure,
hypercholesterolemia, previous or genetic history of myocardial
infarction or thrombosis) in order reduce the risk of myocardial
infarction or thrombosis.
[0103] Diseases of Vasculitis--Vasculitic diseases are
characterized by inflammation of the vessels. Infiltration of
leukocytes leads to destruction of the vessel walls, and the
complement pathway is believed to play a major role in initiating
leukocyte migration as well as the resultant damage manifested at
the site of inflammation (Vasculitis, Second Edition, Edited by
Ball and Bridges, Oxford University Press, pp 47-53, 2008). The
compounds provided in the present invention can be used to treat
leukoclastic vasculitis, Wegener's granulomatosis, microscopic
polyangiitis, Churg-Strauss syndrome, Henoch-Schonlein purpura,
polyateritis nodosa, Rapidly Progressive Glomerulonephritis (RPGN),
cryoglobulinaemia, giant cell arteritis (GCA), Behcet's disease and
Takayasu's arteritis (TAK).
[0104] HIV infection and AIDS--C5a receptor modulators provided
herein may be used to inhibit HIV infection, delay AIDS progression
or decrease the severity of symptoms or HIV infection and AIDS.
[0105] Neurodegenerative disorders and related diseases--Within
further aspects, C5a antagonists provided herein may be used to
treat Alzheimer's disease, multiple sclerosis, and cognitive
function decline associated with cardiopulmonary bypass surgery and
related procedures.
[0106] Cancers--The C5a antagonists provided herein are also useful
for the treatment of cancers and precancerous conditions in a
subject. Specific cancers that can be treated include, but are not
limited to, sarcomas, carcinomas, and mixed tumors. Exemplary
conditions that may be treated according to the present invention
include fibrosarcomas, liposarcomas, chondrosarcomas, osteogenic
sarcomas, angiosarcomas, lymphangiosarcomas, synoviomas,
mesotheliomas, meningiomas, leukemias, lymphomas, leiomyosarcomas,
rhabdomyosarcomas, squamous cell carcinomas, basal cell carcinomas,
adenocarcinomas, papillary carcinomas, cystadenocarcinomas,
bronchogenic carcinomas, melanomas, renal cell carcinomas,
hepatocellular carcinomas, transitional cell carcinomas,
choriocarcinomas, seminomas, embryonal carcinomas, wilm's tumors,
pleomorphic adenomas, liver cell papillomas, renal tubular
adenomas, cystadenomas, papillomas, adenomas, leiomyomas,
rhabdomyomas, hemangiomas, lymphangiomas, osteomas, chondromas,
lipomas and fibromas.
[0107] In another embodiment, the compounds of the present
invention are useful in the treatment of cisplatin induced
nephrotoxicity. In this embodiment, compound treatment can
alleviate the nephrotoxicity induced by cisplatin chemotherapy of
malignancies (Hao Pan et al, Am J Physiol Renal Physiol, 296,
F496-504, 2009).
[0108] In one embodiment of the invention, the compounds of the
invention can be used for the treatment of diseases selected from
the group consisting of sepsis (and associated disorders), COPD,
rheumatoid arthritis, lupus nephritis and multiple sclerosis.
[0109] Treatment methods provided herein include, in general,
administration to a patient an effective amount of one or more
compounds provided herein. Suitable patients include those patients
suffering from or susceptible to (i.e., prophylactic treatment) a
disorder or disease identified herein. Typical patients for
treatment as described herein include mammals, particularly
primates, especially humans. Other suitable patients include
domesticated companion animals such as a dog, cat, horse, and the
like, or a livestock animal such as cattle, pig, sheep and the
like.
[0110] In general, treatment methods provided herein comprise
administering to a patient an effective amount of a compound one or
more compounds provided herein. In a preferred embodiment, the
compound(s) of the invention are preferably administered to a
patient (e.g., a human) orally or topically. The effective amount
may be an amount sufficient to modulate C5a receptor activity
and/or an amount sufficient to reduce or alleviate the symptoms
presented by the patient. Preferably, the amount administered is
sufficient to yield a plasma concentration of the compound (or its
active metabolite, if the compound is a pro-drug) high enough to
detectably inhibit white blood cell (e.g., neutrophil) chemotaxis
in vitro. Treatment regimens may vary depending on the compound
used and the particular condition to be treated; 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. 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, rate of excretion, drug
combination (i.e., other drugs being administered to the patient)
and the severity of the particular disease undergoing therapy, as
well as the judgment of the prescribing medical practitioner. 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.
[0111] Dosage levels of the order of from about 0.1 mg to about 140
mg per kilogram of body weight per day are useful in the treatment
or preventions of conditions involving pathogenic C5a activity
(about 0.5 mg to about 7 g per human patient per day). The amount
of active ingredient that may be combined with the carrier
materials to produce a single dosage form will vary depending upon
the host treated and the particular mode of administration. Dosage
unit forms will generally contain between from about 1 mg to about
500 mg of an active ingredient. For compounds administered orally,
transdermally, intravenously, or subcutaneously, it is preferred
that sufficient amount of the compound be administered to achieve a
serum concentration of 5 ng (nanograms)/mL-10 .mu.g (micrograms)/mL
serum, more preferably sufficient compound to achieve a serum
concentration of 20 ng-1 .mu.g/ml serum should be administered,
most preferably sufficient compound to achieve a serum
concentration of 50 ng/ml-200 ng/ml serum should be administered.
For direct injection into the synovium (for the treatment of
arthritis) sufficient compounds should be administered to achieve a
local concentration of approximately 1 micromolar.
[0112] Frequency of dosage may also vary depending on the compound
used and the particular disease treated. However, for treatment of
most disorders, a dosage regimen of 4 times daily, three times
daily, or less is preferred, with a dosage regimen of once daily or
2 times daily being particularly preferred. It will be understood,
however, that the specific dose level 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 (i.e., other drugs being
administered to the patient), the severity of the particular
disease undergoing therapy, and other factors, including the
judgment of the prescribing medical practitioner.
[0113] In another aspect of the invention, the compounds of the
invention can be used in a variety of non-pharmaceutical in vitro
and in vivo application. For example, the compounds of the
invention may be labeled and used as probes for the detection and
localization of C5a receptor (cell preparations or tissue sections
samples). The compounds of the invention may also be used as
positive controls in assays for C5a receptor activity, i.e., as
standards for determining the ability of a candidate agent to bind
to C5a receptor, or as radiotracers for positron emission
tomography (PET) imaging or for single photon emission computerized
tomography (SPECT). Such methods can be used to characterize C5a
receptors in living subjects. For example, a C5a receptor modulator
may be labeled using any of a variety of well known techniques
(e.g., radiolabeled with a radionuclide such as tritium), and
incubated with a sample for a suitable incubation time (e.g.,
determined by first assaying a time course of binding). Following
incubation, unbound compound is removed (e.g., by washing), and
bound compound detected using any method suitable for the label
employed (e.g., autoradiography or scintillation counting for
radiolabeled compounds; spectroscopic methods may be used to detect
luminescent groups and fluorescent groups). As a control, a matched
sample containing labeled compound and a greater (e.g., 10-fold
greater) amount of unlabeled compound may be processed in the same
manner. A greater amount of detectable label remaining in the test
sample than in the control indicates the presence of C5a receptor
in the sample. Detection assays, including receptor autoradiography
(receptor mapping) of C5a receptor in cultured cells or tissue
samples may be performed as described by Kuhar in sections 8.1.1 to
8.1.9 of Current Protocols in Pharmacology (1998) John Wiley &
Sons, New York.
[0114] The compounds provided herein may also be used within a
variety of well known cell separation methods. For example,
modulators may be linked to the interior surface of a tissue
culture plate or other support, for use as affinity ligands for
immobilizing and thereby isolating, C5a receptors (e.g., isolating
receptor-expressing cells) in vitro. In one preferred application,
a modulator linked to a fluorescent marker, such as fluorescein, is
contacted with the cells, which are then analyzed (or isolated) by
fluorescence activated cell sorting (FACS).
[0115] In FIG. 1, structures and activity are provided for
representative compounds described herein. Activity is provided as
follows for the binding assay as described herein: +, 500
nM.ltoreq.IC.sub.50<2000 nM; ++, 50 nM.ltoreq.IC.sub.50<500
nM; +++, 5 nM.ltoreq.IC.sub.50<50 nM; and ++++, IC.sub.50<5
nM.
V. Examples
[0116] The following examples are offered to illustrate, but not to
limit the claimed invention.
[0117] Reagents and solvents used below can be obtained from
commercial sources such as Aldrich Chemical Co. (Milwaukee, Wis.,
USA). .sup.1H-NMR spectra were recorded on a Varian Mercury 400 MHz
NMR spectrometer. Significant peaks are provided relative to TMS
and are tabulated in the order: multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet) and number of
protons. Mass spectrometry results are reported as the ratio of
mass over charge, followed by the relative abundance of each ion
(in parenthesis). In the examples, a single m/e value is reported
for the M+H (or, as noted, M-H) ion containing the most common
atomic isotopes. Isotope patterns correspond to the expected
formula in all cases. Electrospray ionization (ESI) mass
spectrometry analysis was conducted on a Hewlett-Packard MSD
electrospray mass spectrometer using the HP1100 HPLC for sample
delivery. Normally the analyte was dissolved in methanol at 0.1
mg/mL and 1 microlitre was infused with the delivery solvent into
the mass spectrometer, which scanned from 100 to 1500 daltons. All
compounds could be analyzed in the positive ESI mode, using
acetonitrile/water with 1% formic acid as the delivery solvent. The
compounds provided below could also be analyzed in the negative ESI
mode, using 2 mM NH.sub.4OAc in acetonitrile/water as delivery
system.
[0118] The following abbreviations are used in the Examples and
throughout the description of the invention:
EtOH: Ethanol
[0119] EtONa: Sodium ethoxide
THF: Tetrahydrofuran
[0120] TLC: Thin layer chromatography
MeOH: Methanol
[0121] Compounds within the scope of this invention can be
synthesized as described below, using a variety of reactions known
to the skilled artisan. One skilled in the art will also recognize
that alternative methods may be employed to synthesize the target
compounds of this invention, and that the approaches described
within the body of this document are not exhaustive, but do provide
broadly applicable and practical routes to compounds of
interest.
[0122] Certain molecules claimed in this patent can exist in
different enantiomeric and diastereomeric forms and all such
variants of these compounds are claimed.
[0123] The detailed description of the experimental procedures used
to synthesize key compounds in this text lead to molecules that are
described by the physical data identifying them as well as by the
structural depictions associated with them.
[0124] Those skilled in the art will also recognize that during
standard work up procedures in organic chemistry, acids and bases
are frequently used. Salts of the parent compounds are sometimes
produced, if they possess the necessary intrinsic acidity or
basicity, during the experimental procedures described within this
patent.
Example 1
Synthesis of
cis-1-(2-fluoro-6-methylbenzoyl)-2-phenylpiperidine-3-carboxylic
acid (3-trifluoromethylphenyl)amide
##STR00022##
[0126] a) Pd(PPh.sub.3).sub.4 (3.0 g, 2.6 mmol) was added to a
solution of 2-chloro-3-carboxyethylpyridine (25 g, 134.7 mmol),
phenylboronic acid (21.04 g, 172.6 mmol) and K.sub.2CO.sub.3 (55.1
g, 399 mmol) in 1,4-dioxane (200 mL) and water (200 mL). The
reaction mixture was heated at 100.degree. C. for 2 h. The solution
was then cooled to room temperature and the dioxane was removed
under reduced pressure. The resulting aqueous layer was extracted
with ethyl acetate, and the combined organic layers were dried
(Na.sub.2SO.sub.4), filtered through celite, and concentrated under
reduced pressure. The residue was purified by flash chromatography
(SiO.sub.2, 10-100% EtOAc/hexanes) to get the 2-phenylpyridine
derivative in 91% yield (27.98 g). LC-MS R.sub.t (retention time):
2.45 min, MS: (ES) m/z 228 (M+H.sup.+).
[0127] b) PtO.sub.2 (800 mg, 3.52 mmol) was added to a solution of
2-phenyl-nicotinic acid ethyl ester (20 g, 88 mmol, prepared in
step a above) in EtOH (60 mL) and concentrated HCl (15 mL). The
reaction mixture was hydrogenated using a Parr shaker at 40-45 psi,
for 1 h. The reaction mixture was then filtered through celite,
washed with EtOH, and the filtrate was concentrated under reduced
pressure. The residue was diluted with CH.sub.2Cl.sub.2 and washed
with saturated NaHCO.sub.3. Purification by flash chromatography
(SiO.sub.2, 0-20% MeOH/CH.sub.2Cl.sub.2) gave the desired product
in 85% yield (17.4 g). LC-MS R.sub.t (retention time): 1.73 min,
MS: (ES) m/z 234 (M+H.sup.+).
[0128] c) Oxalyl chloride (3.2 mL, 30.75 mmol) was added to the
solution of 2-fluoro-6-methylbenzoic acid (3.79 g, 24.6 mmol) in
CH.sub.2Cl.sub.2 (20 mL) in a reaction flask at room temperature,
followed by addition of a catalytic amount of DMF. The reaction was
kept stirring for 2 h at room temperature. Solvent and excess
oxalyl chloride were removed in vacuo and the residue was dried
under high vacuum for 20 min. The resulting acid chloride was
dissolved in dry CH.sub.2Cl.sub.2 (20 mL) and cooled to 0.degree.
C. followed by the addition of the piperidine made in step b (5.56
g, 20.5 mmol) and Et.sub.3N (8.6 mL, 61.5 mmol). The mixture was
then allowed to warm to room temperature and stirred overnight. The
reaction mixture was diluted with CH.sub.2Cl.sub.2 and water was
added. The layers were separated and the aqueous layer was
extracted with CH.sub.2Cl.sub.2. The combined organic layers were
dried (MgSO.sub.4) and concentrated under reduced pressure. The
residue was purified by flash chromatography (SiO.sub.2, 10-35%
EtOAc/hexanes) to give 7.47 g of the desired compound 99% yield).
LC-MS R.sub.t (retention time): 2.50 min and 2.58 min (two
rotamers), MS: (ES) m/z 370 (M+H.sup.+).
[0129] d) Lithium aluminum hydride solution (2.0 M in THF, 8.2 mL,
16.4 mmol) was added to a solution of the ester from step c (2.98
g, 8.06 mmol) in THF (100 ml) at 0.degree. C. The resulting
solution was kept stirring at 0.degree. C. for 2 h at which time
the reaction was completed. 15% Aqueous NaOH (625 .mu.L) was added
drop wise to quench the reaction followed by H.sub.2O (625 .mu.L).
To the cloudy colloidal mixture was added additional water (1.85
mL), and the mixture was kept stirring for 1 h at rt. The mixture
was then filtered through a celite plug, and the filtrate was
concentrated under reduced pressure. Purification by flash
chromatography (SiO.sub.2, 33-67% EtOAc/hexanes) gave 2.46 g of the
desired product (93% yield). LC-MS: R.sub.t (retention time):1.90
min and 2.09 min (two rotamers), MS: (ES) m/z 328 (M+H.sup.+).
[0130] e) A solution of the alcohol from step d (1.42 g, 4.33
mmol,) in acetic acid (65 ml) was added to a slurry of CrO.sub.3
(2.61 g, 26.1 mmol) in H.sub.2O (16 ml) at room temperature. The
resulting mixture was kept stirring at room temperature until the
reaction was completed (90 min). The mixture was filtered through a
Celite plug and the filtrate was concentrated under reduced
pressure. Purification by flash chromatography (SiO.sub.2, 3-10%
CH.sub.2Cl.sub.2:MeOH followed by 50-67% EtOAc/hexanes) gave 1.03 g
of the desired product (70% yield). LC-MS: R.sub.t (retention
time): 1.88 min and 2.12 min (two rotamers), MS: (ES) m/z 342
(M+H.sup.+).
[0131] f) 3-Trifluoromethylaniline (16.2 mg, 0.1 mmol, 1.0 eq) was
added to a solution of the acid prepared above (34.2 mg, 0.1 mmol)
and triethylamine (6 eq) in CH.sub.2Cl.sub.2 (1 mL). T3P (95.5 mg,
0.15 mmol) was then slowly added and the solution was allowed to
stir at room temperature for 1.5 h. The reaction mixture was
diluted with CH.sub.2Cl.sub.2 (1 mL), washed with 1 N aqueous HCl
followed by saturated aqueous NaHCO.sub.3. The organic layer was
separated, dried over anhydrous MgSO4, and concentrated under
reduced pressure Purification by flash chromatography (SiO.sub.2,
5-40% EtOAc/hexanes) gave 35 mg (73% yield) of the product as a
white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 1.22-2.45 (m, 8H),
2.93-3.32 (m, 3H), 6.77-7.82 (m, 12H), 9.10 (s, 0.38H), 9.30 (s,
0.62H). LC-MS: R.sub.t (retention time)=2.88 min, MS: (ES) m/z 485
(M+H.sup.+).
Example 2
Synthesis of
N-(3-tert-butylphenyl)-1-(5-chloro-3-methylpicolinoyl)-2-phenylpiperidine-
-3-carboxamide
##STR00023##
[0133] a) 2-Chloronicotinoyl chloride (1.05 eq) dissolved in
anhydrous dichloromethane (0.5 M) was added to a solution of
3-tert-butylaniline (1 eq) and 2 M aq K.sub.2CO.sub.3 (2.2 eq) in
anhydrous dichloromethane (0.5 M) at 0.degree. C. over a period of
30 min, and the reaction mixture was allowed to stir at room
temperature for an additional 1.5 h. The layers were separated and
the aqueous layer was extracted with dichloromethane. The combined
organic layer was washed with brine, dried (MgSO.sub.4), filtered
and concentrated to give the desired amide as a foamy solid which
was used as such in the next step without further purification. MS:
(ES) m/z 289.1 (M+H.sup.+).
[0134] b) Pd(PPh.sub.3).sub.4 (2-5 mol %) was added to a solution
of the above pyridine amide (1 eq), phenylboronic acid (1.4 eq) and
2 M aq K.sub.2CO.sub.3 (2.4 eq) in toluene (0.7 M) and the reaction
mixture was heated at 100.degree. C. over night (.about.12 h).
After cooling to room temperature, the reaction mixture was
filtered through celite and the celite plug was washed with EtOAc.
The filtrate was diluted with water and extracted with EtOAc, dried
(MgSO.sub.4), filtered and concentrated and concentrated under
reduced pressure. The residue was purified by automated flash
chromatography (SiO.sub.2, 10% to 100% gradient of EtOAc-hexanes)
and dried in vacuo to give the 2-phenyl-3-carboxyamidepyridine in
60-75% yield, MS: (ES) m/z 331.2 (M+H.sup.+).
[0135] c) PtO.sub.2 (10 mol %) was added to a solution of the
2-phenylpyridine derivative prepares above (1 eq) in EtOH and
concentrated HCl (excess, 4:1 ratio) and the reaction mixture was
hydrogenated using a Parr shaker at 40-45 psi, for 1.5 h. It was
filtered through celite, washed with EtOH, and the filtrate was
concentrated. The residue was diluted with CH.sub.2Cl.sub.2 and
washed with saturated aq NaHCO.sub.3. The residue was then purified
by automated flash chromatography (SiO.sub.2, 1% to 30% gradient of
CH.sub.2Cl.sub.2-MeOH) and dried in vacuo to give the title
compound in .about.85% yield as a foamy solid. MS: (ES) m/z 337.2
(M+H.sup.+).
[0136] d) 5-Chloro-3-methylpicolinic acid (30 mg, 0.16 mmol) and
N-(3-tert-butylphenyl)-2-phenylpiperidine-3-carboxamide (50 mg,
0.15 mmol, prepared in step c above) were dissolved in anhydrous
DMF (1 mL). N,N-Diisopropylethylamine (0.15 mL) was added at room
temperature followed by HCTU (67 mg, 0.16 mmol). After stirring 2 h
at ambient temperature, LC-MS and TLC indicated the completion of
the reaction. The reaction mixture was diluted with EtOAc (50 mL)
and washed with 1 N HCl (20 mL), saturated NaHCO.sub.3 (30 mL), and
brine (30 mL) and the resulting solution was concentrated under
reduced pressure. The residue was purified by preparative HPLC
(20.fwdarw.95% gradient of MeCN--H.sub.2O with 0.1% TFA) and the
pure fractions were lyophilized to afford the title compound (50
mg, 67% yield). HPLC retention time=2.88 minutes. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 8.42 (d, 1H, J=0.8 Hz), 7.97 (br, 1H),
7.59 (d, 1H, J=0.8 Hz), 7.56 (d, 1H, J=7.6 Hz), 7.34 (m, 3H), 7.20
(m, 3H), 7.10 (d, 1H, J=7.6 Hz), 6.61 (two sets of br, 1H), 3.12
(two sets of m, 2H), 2.94 (three sets of m, 1H), 2.36 (s, 3H), 2.20
(two sets of br, 2H), 1.74 (br complex, 2H), 1.29 (s, 9H). MS: (ES)
m/z 490.2 (M+H.sup.+).
Example 3
Synthesis of
cis-1-(2-methylbenzoyl)-2-(3-fluorophenyl)piperidine-3-carboxylic
acid (3-tert-butylphenyl)amide
##STR00024##
[0138] a) To a mixture of
N-(3-tert-butylphenyl)-2-chloronicotinamide (570.2 mg, 2 mmol),
3-fluorophenylboronic acid (401.2 mg, 2.8 mmol), 3 mL of toluene,
and 1 mL of 2 N potassium carbonate in water was added
tetrakis(triphenylphosphine)palladium(0) (234.5 mg, 0.2 mmol). The
mixture was then heated at 90.degree. C. for 3 hour under nitrogen,
before it was cooled down to room temperature. The reaction mixture
was then diluted with 30 mL of water and 150 mL of EtOAc. The
organic layer was separated, washed with brine, and dried
(Na.sub.2SO.sub.4). The organic solvent was removed under reduced
pressure and the residue was purified by silica gel column (40%
EtOAc in hexane) to give
N-(3-tert-butylphenyl)-2-(3-fluorophenyl)nicotinamide (691.4 mg,
99%). MS: (ES) m/z 394.5 (M+H.sup.+).
[0139] b) A mixture of
N-(3-tert-butylphenyl)-2-(3-fluorophenyl)nicotinamide (501.2 mg,
1.4 mmol), platinum oxide (51.9 mg, 0.21 mmol), and concentrated
HCl (400 .mu.L, 5.2 mmol) in 5 mL of ethanol was stirred vigorously
under hydrogen balloon overnight. The mixture was filtered, and the
solids washed with 25 mL of methanol three times. The combined
solution was dried under reduced pressure. To the residue was added
30 mL of saturated sodium bicarbonate and 150 mL of EtOAc. The
organic layer was separated, and dried over sodium sulfate.
Evaporation of solvent gave the crude
2-(3-fluorophenyl)piperidine-3-carboxylic acid
(3-tert-butylphenyl)amide as a brown solid, which was taken on
directly to the next step. MS: (ES) m/z 355.7 (M+H.sup.+).
[0140] c) To a solution of
2-(3-fluorophenyl)piperidine-3-carboxylic acid
(3-tert-butylphenyl)amide (prepared above, 177.3 mg, 0.5 mmol) in 2
mL of dichloromethane was added Et.sub.3N (100 .mu.L, excess), and
2-methylbenzoyl chloride (92.3 mg, 0.6 mmol) at room temperature.
The resulting solution was then stirred at this temperature until
completion of the reaction (10 min.). The reaction mixture was then
directly loaded onto a silica gel column, and was purified by using
ISCO (30% EtOAc in hexane) to give the final product
2-(3-fluorophenyl)-1-(2-methylbenzoyl)piperidine-3-carboxylic acid
(3-tert-butylphenyl)amide (151.2 mg, 64% yield). .sup.1H NMR (400
MHZ, CDCl.sub.3, mixture of rotomers): .delta. 7.91 (s, 0.6H), 7.85
(s, 0.4H), 7.18-7.46 (m, 9H), 7.11 (m, 1H), 6.95 (m, 1H), 6.67 (d,
J=1.2 Hz, 1H), 3.36 (d, J=1.6 Hz, 0.4H), 3.26 (d, J=1.6 Hz, 1H),
3.05 (m, 1H), 2.89 (t, J=1.2 Hz, 1H), 2.45 (s, 1H), 2.02-2.40 (m,
4H), 1.70-1.84 (m, 3H), 1.44-1.64 (s, 1H), 1.32 (s, 6H), 1.25 (s,
1H). MS: (ES) m/z 473.2 (M+H.sup.+).
Example 4
Synthesis of
cis-1-(2-methylbenzoyl)-2-(2,2-dimethylpropyl)piperidine-3-carboxylic
acid (3-tert-butylphenyl)amide
##STR00025##
[0142] a) To a stirred solution of 2-bromonicotinic acid (1.01 g, 5
mmol) dissolved in anhydrous dichloromethane (8 mL) were added EDCI
(1.34 g, 7 mmol) and 3-tert-butylaniline (0.74 g, 5 mmol) at room
temperature and the reaction mixture was stirred for 12 hours. The
mixture was then diluted with dichloromethane, followed by
saturated sodium bicarbonate and water wash. The dichloromethane
layer was dried over anhydrous magnesium sulfate, filtered and
concentrated under reduced pressure. The residue was purified by
flash chromatography to obtain
2-bromo-N-(3-tert-butylphenyl)nicotinamide in 59% yield (950 mg).
Rt: 2.44 min (20-100-5 method). MS: (ES) m/z 333, 335
(M+H.sup.+).
[0143] b) 2,2-Dimethylpropylmagnesium chloride (1 M-diethylether,
4.8 mL, 4.8 mmol) was added to a suspension of copper cyanide (215
mg, 2.40 mmol) in THF (6 mL) at -78.degree. C. After stirring at
the same temperature for 1 hour,
2-bromo-N-(3-tert-butylphenyl)nicotinamide (200 mg, 0.601 mmol) was
added all at once as a solid. The reaction mixture was gradually
warmed to room temperature and the reaction was allowed to stir
overnight. Saturated ammonium chloride solution and ethyl acetate
was added, and the reaction mixture was filtered through celite and
rinsed with ethyl acetate. The layers were separated and the
product was extracted once more with ethyl acetate. The combined
organic layers were washed with brine and dried over anhydrous
sodium sulfate. After removing the solvent under reduced pressure,
the crude material was purified using silica gel column
chromatography using a gradient of 20%-50% ethyl acetate in hexanes
to yield N-(3-tert-butylphenyl)-2-(2,2-dimethylpropyl)nicotinamide
(168 mg, 0.517 mmol, 86%). Rf=0.45 (toluene:ethyl acetate=2:1).
[0144] c) N-(3-tert-Butylphenyl)-2-(2,2-dimethylpropyl)nicotinamide
(168 mg, 0.517 mmol) was dissolved in ethanol (5 mL). Platinum
oxide (11.6 mg, 0.0511 mmol) was added followed by concentrated
hydrochloric acid (250 .mu.L). The reaction mixture was
hydrogenated using a Parr apparatus for 1.5 hours at 45 psi.
Analysis of the reaction mixture showed incomplete conversion, and
the sequence was repeated one more time. Platinum oxide was
filtered off and the solvents were removed under reduced pressure.
The crude material was neutralized using saturated sodium
bicarbonate solution and extracted with ethyl acetate. The organic
layer was then washed with brine and dried over anhydrous magnesium
sulfate. Removal of solvent under reduced pressure gave the crude
2,3-cis-2-(2,2-dimethylpropyl)piperidine-3-carboxylic
acid-(3-tert-butylphenyl)amide (153 mg) which was used in the next
step without further purification.
[0145] d) To a solution of
2,3-cis-2-(2,2-dimethylpropyl)piperidine-3-carboxylic
acid-(3-tert-butylphenyl)amide (84.8 mg, 0.257 mmol) in pyridine
(415 .mu.L, 5.13 mmol) at room temperature was added
2-methylbenzoyl chloride (81.6 mg, 0.528 mmol) in chloroform (415
.mu.L). A catalytic amount (not weighed) of dimethylaminopyridine
was added to enhance the reaction and the mixture was stirred for
three days. Ethyl acetate and water was then added to the reaction
mixture and the product was extracted with ethyl acetate three
times. The combined organic layers were dried over anhydrous
magnesium sulfate. After removal of the solvent under reduced
pressure the crude material was purified via silica gel
chromatography using 10%-20% ethyl acetate in hexanes to give
2,3-cis-2-(2,2-dimethylpropyl)-1-(2-methylbenzoyl)piperidine-3-carboxylic
acid-(3-tert-butylphenyl)amide (47.0 mg, 0.105 mmol, 41%). Rf=0.6
(hexanes:ethyl acetate=2:1). Rt=3.16 min., 3.26 min. (compound
exists as mixtures of several conformers. 20-100-5 method). .sup.1H
NMR (CDCl.sub.3) .delta. 9.68 (s, 1H), 9.43 (s, 1H), 8.33 (s, 1H),
8.28 (s, 1H)), 6.97-7.79 (m, 8H), 5.48 (br, 1H), 5.39 (dd, J=4, 10
Hz, 1H), 5.33 (dd, J=6, 6 Hz, 1H), 3.38 (ddd, J=4, 14, 14 Hz, 2H),
3.25 (dd, J=13, 13 Hz, 2H), 2.66 (dd, J=4, 8.4 Hz, 1H), 2.63 (ddd,
J=2.8, 2.8, 8 Hz, 1H), 2.50 (s, 9H), 2.40 (s, 9H), 2.25 (s, 9H),
2.13 (s, 9H), 1.79-1.99 (m, 2H), 1.23-1.56 (m, 2H), 1.32 (s, 9H),
1.07 (s, 9H), 1.06 (s, 9H), 0.97 (s, 9H), 0.95 (s, 9H). MS: (ES)
m/z 449 (M+H.sup.+).
Example 5
Synthesis of
cis-2-cyclopentyl-1-(2-methylbenzoyl)piperidine-3-carboxylic acid
(3-tert-butylphenyl)amide
##STR00026##
[0147] a) Cyclopentylzinc bromide (0.5 M, 6.5 mL, 3.26 mmol) was
added to a room temperature stirred solution of the
2-chloronicotinic acid methyl ester (400 mg, 2.33 mmol), CuI (19
mg, 0.1 mmol) and Pd(dppf)Cl.sub.2 (42 mg, 0.06 mmol) in anhydrous
dimethylacetamide (1.7 mL) under nitrogen. The reaction mixture was
heated to 70.degree. C. for 3.5 hours, cooled to room temperature,
filtered through celite, and the cake was rinsed with ethyl
acetate. The filtrate was washed with water, brine, dried
(MgSO.sub.4), filtered and concentrated under reduced. The residue
was purified by flash chromatography (SiO2, 10-100% EtOAc/hexanes)
to get the desired compound in 83% yield (400 mg). LC-MS R.sub.t
(retention time): 1.87 min; MS: (ES) m/z 206 (M+H.sup.+).
[0148] b) n-BuLi (1.47 mL, 3.68 mmol) was added to the
3-tert-butylaniline (580 mg, 3.89 mmol) at -78.degree. C. in dry
THF (2 mL) under nitrogen and the solution was allowed to stir at
0.degree. C. for 10 minutes. The reaction mixture was re-cooled to
-78.degree. C. and 2-cyclopentyl-nicotinic acid methyl ester (400
mg, 1.94 mmol) dissolved in dry THF (2 mL) was added to it. The
reaction mixture was allowed to attain 0.degree. C. over a period
of 2 hours, quenched with saturated aqueous NH.sub.4Cl, and
extracted with ethyl acetate. The combined organic layers were
dried (MgSO.sub.4), filtered and concentrated under reduced
pressure. The residue was purified by flash chromatography (SiO2,
10-100% EtOAc/hexanes) to give the pure compound in 91% yield (572
mg). LC-MS R.sub.t (retention time): 2.61 min; MS: (ES) m/z 323
(M+H.sup.+).
[0149] c) To a solution of the
N-(3-tert-butylphenyl)-2-cyclopentylnicotinamide (570 mg, 1.77
mmol) in ethanol (10 mL) containing concentrated HCl (1 mL) was
added platinum oxide (40 mg, 0.17 mmol) and the solution was
hydrogenated using a Parr shaker at 40 psi for 1.5 hour. The
reaction mixture was filtered through Celite, and the cake was
rinsed with ethanol. The filtrate was concentrated, and the residue
was dried under high vacuum for 2 hours to get quantitative yield
of the desired piperidine as a HCl salt. LC-MS R.sub.t (retention
time): 1.97 min; MS: (ES) m/z 329 (M+H.sup.+).
[0150] d) To a solution of the
cis-2-cyclopentylpiperidine-3-carboxylic acid
(3-tert-butyl-phenyl)amide prepared above (123 mg, 0.34 mmol) in
dry CH.sub.2Cl.sub.2 (1 mL) containing Et.sub.3N (142 .mu.L, 1.02
mmol) was added 2-methylbenzoyl chloride (53 mg, 0.34 mmol) and the
mixture was stirred at room temperature for 2 hours. The reaction
mixture was then diluted with ethyl acetate (20 mL), washed with 1
N aqueous HCl, water, and brine. The organic layer was dried
(MgSO.sub.4), filtered and concentrated under reduced pressure. The
residue was purified by reverse phase preparative HPLC (20-95%
gradient of CH.sub.3CN--H.sub.2O) and dried (Lyophilizer) to give
the title compound in 65% yield (109 mg). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 1.22-1.48 (m, 11H), 1.56-1.80 (m, 5H),
1.84-2.06 (m, 4H), 2.10-2.23 (m, 1H), 2.30 (s, 1.6H), 2.39 (s,
1.4H), 2.41-2.50 (m, 1H), 2.71-2.76 (m, 1H), 3.02-3.09 (m, 1H),
3.25-3.39 (m, 1H), 5.11 (bs, 1H), 7.05-7.30 (m, 6H), 7.47-7.55 (m,
2H), 8.32 (bs, 1H). LC-MS R.sub.t (retention time): 3.16 min; MS:
(ES) m/z 447 (M+H).sup.+. LC-MS method: Agilent Zorbax SB-C18,
2.1.times.50 mm, 5.mu., 35.degree. C., 1 mL/min flow rate, a 2.5
min gradient of 20% to 100% B with a 1.0 min wash at 100% B; A=0.1%
formic acid/5% acetonitrile/94.9 water, B=0.1% formic acid/5%
water/94.9 acetonitrile.
Example 6
Synthesis of
(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-methylbenzoyl)piperidine-3-carb-
oxylic acid (3-chloro-4-methylphenyl)amide
##STR00027##
[0152] b)
cis-2-(4-tert-Butoxycarbonylaminophenyl)piperidine-3-carboxylic
acid ethyl ester was synthesized similarly as illustrated in
example 1.
[0153] c:1):
cis-2-(4-tert-Butoxycarbonylaminophenyl)piperidine-3-carboxylic
acid ethyl ester (61 g, 174.8 mmol) and di-p-toluoyl-L-tartaric
acid (62 g, 174.8 mmol) was dissolved in EtOH (500 ml). The clear
solution was concentrated and pumped to dry. The obtained white
salt was then dissolved into 250 ml of ethyl acetate to form a
clear solution. To this solution was added 500 ml of TBME slowly.
The obtained solution was left at rt undisturbed for 3 days. At
this time a lot of white crystals were formed. They were then
filtered and washed with 100 ml of TBME to obtain a white solid (60
g).
[0154] The above salt was re-dissolved in ethanol, concentrated and
pumped to dry. The obtained salt was dissolved into 500 ml of THF,
followed by adding TBME (500 ml). The obtained clear solution was
left at rt undisturbed for another 2.5 days. The obtained white
crystals were filtered to obtain 20.5 g (enrichment 64:1) of the
salt.
[0155] c:2) To a 0.degree. C. stirred suspension of the salt (16.7
g) in CH.sub.2Cl.sub.2 (150 mL) was added saturated aqueous
NaHCO.sub.3 solution (100 mL) and the reaction mixture was allowed
to stir at r.t over a period of 30 minutes. The layers were
separated and the aqueous layer was extracted with CH.sub.2Cl.sub.2
(50 mL). The combined organic layer was washed with saturated
aqueous NaHCO.sub.3 (2.times.100 mL), dried and concentrated to
give
(2R,3S)-2-(4-tert-Butoxycarbonylaminophenyl)piperidine-3-carboxylic
acid ethyl ester in 90% yield and .about. in 97% ee.
[0156] d) To a 0.degree. C. solution of the
(2R,3S)-2-(4-tert-Butoxycarbonylaminophenyl)-piperidine-3-carboxylic
acid ethyl ester prepared above (600 mg, 1.72 mmol) in dry
CH.sub.2Cl.sub.2 (5 mL) containing Et.sub.3N (480 .mu.L, 3.44 mmol)
was added 2-methylbenzoyl chloride (266 mg, 1.72 mmol) and the
mixture was stirred at room temperature for over night. The
reaction mixture was then diluted with CH.sub.2Cl.sub.2 (20 mL),
washed with 1 N aqueous HCl, water, and brine. The organic layer
was dried (MgSO.sub.4), filtered and concentrated under reduced
pressure to give
(2R,3S)-2-(4-tert-Butoxycarbonylaminophenyl)-1-(2-methylbenzoyl)piperidin-
e-3-carboxylic acid ethyl ester in quantitative yield and the crude
product was used as such in the next step.
[0157] e) 4N HCl in 1,4-dioxane (5 mL, 20 mmol) was slowly added to
a 0.degree. C. solution of the above crude product
(2R,3S)-2-(4-tert-Butoxycarbonylaminophenyl)-1-(2-methylbenzoyl)piperidin-
e-3-carboxylic acid ethyl ester (840 mg, 1.72 mmol) in dry
CH.sub.2Cl.sub.2 (4 mL). After the addition of the HCl, the
reaction mixture was allowed to attain r.t and stirred for 1 h. It
was diluted with CH.sub.2Cl.sub.2 (30 mL), cooled to 0.degree. C.
and neutralized with saturated aqueous NaHCO.sub.3 to get the
(2R,3S)-2-(4-aminophenyl)-1-(2-methylbenzoyl)piperidine-3-carboxylic
acid ethyl ester (612 mg) in 97% yield over two steps.
[0158] f) Na(OAC).sub.3BH (495 mg, 2.33 mmol) was added to a
solution of the
(2R,3S)-2-(4-aminophenyl)-1-(2-methylbenzoyl)piperidine-3-carboxylic
acid ethyl ester (612 mg, 1.67 mmol), cyclopentanone (140 mg, 1.67
mmol) and acetic acid (100 mg, 1.67 mmol) in dry dichloroethane at
r.t and the reaction mixture was heated to 50.degree. C. for 4 h,
cooled to r.t and stirred for 48 h. It was then diluted with
CH.sub.2Cl.sub.2 (30 mL), washed with saturated aqueous NaHCO.sub.3
solution, dried and concentrated in vacuo. The residue was purified
by ISCO flash column using ethyl acete and hexanes as mobile phase
(40 g column, 0-40% gradient) to afford
(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-methylbenzoyl)piperidine-3-carb-
oxylic acid ethyl ester (450 mg).
[0159] g) Me.sub.3Al (290 .mu.L, 0.57 mmol, 2M in toluene) was
added to a solution of the 3-Chloro-4-methylphenylamine (65 mg,
0.46 mmol) in dry dichloroethane (1 mL) at ambient temperature.
Stirred for 20 minutes, then
(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-methylbenzoyl)piperidine-3-
-carboxylic acid ethyl ester (100 mg, 0.23 mmol) dissolved in dry
dichloroethane (1 mL) was added to it. The reaction mixture was
then heated to 85.degree. C. for 3 h, cooled to r.t, diluted with
CH.sub.2Cl.sub.2 (20 mL), washed with saturated aqueous NaHCO.sub.3
solution. The aqueous layer was extracted with CH.sub.2Cl.sub.2 (20
mL) and the combined organic layer was dried (MgSO.sub.4) and
concentrated. The residue was purified by reverse phase preparative
HPLC (20-95% gradient of CH.sub.3CN--H.sub.2O with 0.1% TFA as
additive), the product containing fractions were pooled together
and concentrated. The residue was diluted with CH.sub.2Cl.sub.2 (30
mL), washed with saturated aqueous NaHCO.sub.3 solution. The
CH.sub.2Cl.sub.2 layer was dried (MgSO.sub.4) and concentrated to
get the pure
(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-methylbenzoyl)piperidine-3-carb-
oxylic acid (3-chloro-4-methylphenyl)amide in 50% yield.
[0160] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.4 (bs, 1H), 7.55 (s,
1H), 7.37-7.05 (m, 9H), 6.55-6.52 (m, 2H), 3.77-3.70 (m, 1H),
3.30-3.16 (m, 1H), 3.04-2.91 (m, 2H), 2.43-1.94 (m, 8H), 1.71-1.46
(m, 11H).
Example 7
[0161] The following are representative compounds prepared and
evaluated using methods similar to the examples herein.
Characterization data is provided for the compounds below.
Biological evaluation is shown in FIG. 1 for these compounds and
others prepared as described herein.
[0162]
(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-fluoro-6-methylbenzoyl)pi-
peridine-3-carboxylic acid
(4-methyl-3-trifluoromethylphenyl)amide
##STR00028##
[0163] .sup.1H NMR (400 MHz, TFA-d) .delta. 7.91 (d, J=8.6 Hz, 1H),
7.84 (d, J=8.6 Hz, 1H), 7.58-6.82 (m, 8H), 6.75 (t, J=8.6 Hz, 1H),
4.10-4.00 (m, 1H), 3.60-3.47 (m, 1H), 3.45-3.41 (m, 1H), 3.33-3.25
(m, 1H), 2.44-2.22 (m, 7H), 2.04-1.92 (m, 4H), 1.82-0.169 (m,
7H)
[0164]
(2R,3S)-1-(2-Chlorobenzoyl)-2-(4-cyclopentylaminophenyl)piperidine--
3-carboxylic acid (4-methyl-3-trifluoromethylphenyl)amide
##STR00029##
[0165] .sup.1H NMR (400 MHz, CDCl.sub.3) 9.41 (bs, 0.5H), 9.03 (bs,
0.5H), 7.55 (s, 1H), 7.49-7.39 (m, 3H), 7.31-7.27 (m, 2H),
7.18-7.04 (m, 2H), 6.83-6.74 (m, 3H), 3.76-3.64 (m, 1H), 3.22-2.90
(m, 5H), 2.39 (s, 3H), 2.32-2.20 (m, 1H), 2.16-2.04 (m, 1H),
2.0-1.86 (m, 2H) 1.80-1.72 (m, 3H), 1.56 (bs, 5H).
(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-fluoro-6-methylbenzoyl)piperidin-
e-3-carboxylic acid (3-chloro-4-methylphenyl)amide
##STR00030##
[0167] .sup.1H NMR (DMSO-d.sub.6) .delta. 10.22 (s, 1H), 7.67 (dd,
J=1.8 Hz, J=11.0 Hz, 1H), 7.04-7.33 (m, 9H), 6.30 (dd, J=5.8 Hz,
J=9.4 Hz, 1H), 5.52 (br, 1H), 3.56-3.64 (m, 1H), 3.00-3.17 (m, 2H),
2.90-2.98 (m, 1H), 2.23 (2.24) (s, 3H), 1.97 (2.33) (s, 3H),
1.32-2.22 (m, 12H)
(2R,3S)-1-(4-Chlorobenzoyl)-2-(4-Cyclopentylaminophenyl)piperidine-3-carbo-
xylic acid (4-methyl-3-trifluoromethylphenyl)amide
##STR00031##
[0169] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.79 (bs, 1H), 7.62 (s,
1H), 7.52-7.48 (m, 1H), 7.37-7.30 (m, 5H), 7.13 (d, J=8.4 Hz, 1H),
6.52-6.50 (m, 3H), 3.75-3.69 (m, 1H), 3.44 (bs, 1H), 3.09-2.97 (m,
2H), 2.39 (s, 3H), 2.37-2.30 (m, 1H), 2.13-2.08 (m, 1H), 2.10-1.93
(m, 2H), 1.80-1.59 (m, 7H), 1.48-1.42 (m, 2H)
(2R,3S)-2-(4-Cyclohexylaminophenyl)-1-(2-fluoro-6-methylbenzoyl)piperidine-
-3-carboxylic acid (3-.sup.t-butylphenyl)amide
##STR00032##
[0171] .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.24 (m, 1H),
7.40-6.85 (m, 8H), 6.65-6.40 (m, 3H), 3.57 (s, 1H), 3.30-2.90 (m,
4H), 2.50-1.85 (m, 9H), 1.80-1.50 (m, 5H), 1.40-1.00 (m, 13H)
(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-fluoro-6-methylbenzoyl)piperidin-
e-3-carboxylic acid (4-methyl-3-pyrrolidin-1-yl-phenyl)amide
##STR00033##
[0173] .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.98 (m, 1H),
7.40-7.18 (m, 3H), 7.10-6.80 (m, 4H), 6.64-6.40 (m, 3H), 3.80-3.50
(m, 2H), 3.30-2.90 (m, 6H), 2.50-2.10 (m, 7H), 2.10-1.80 (m, 8H),
1.80-1.20 (m, 9H)
(2R,3S)-2-[4-(Cyclopentyloxy)phenyl]-1-(2-fluoro-6-methylbenzoyl)piperidin-
e-3-carboxylic acid (3-chloro-4-methylphenyl)amide
##STR00034##
[0175] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.68 (bs, 0.6H), 8.58 (bs,
0.4H), 7.59-7.40 (m, 3H), 7.29-6.90 (m, 4H), 6.80 (m, 2H), 6.65 (m,
1H), 4.72 (m, 1H), 3.30-2.92 (m, 3H), 2.44 (s, 1H), 2.42-2.30 (m,
1H), 2.30 (s, 1H), 2.29 (s, 2H), 2.20 (s, 2H), 2.19-2.12 (m, 1H),
2.08-1.92 (m, 2H), 1.90-1.72 (m, 7H) 1.60 (m, 2H).
(.+-.)-(2R,3S)-2-(4-Cyclopentylaminophenyl)-1-(2-fluoro-6-methylbenzoyl)pi-
peridine-3-carboxylic acid (4-chloro-3-methylphenyl)amide
##STR00035##
[0177] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.25 (bs, 0.4H), 8.16 (bs,
0.6H), 7.44-7.20 (m, 6H), 7.06-6.84 (m, 2H), 6.59-6.50 (m, 2H),
3.75 (m, 1H), 3.66 (bs, 1H), 3.26-2.92 (m, 3H), 2.43 (s, 1H),
2.42-2.30 (m, 1H), 2.30 (s, 1H), 2.29 (s, 2H), 2.20 (s, 2H),
2.19-2.12 (m, 1H), 2.08-1.92 (m, 2H), 1.80-1.58 (m, 7H) 1.45 (m,
2H).
(2R,3S)-2-(4-Cyclobutylaminophenyl)-1-(2-fluoro-6-methylbenzoyl)piperidine-
-3-carboxylic acid (3-.sup.t-butylphenyl)amide
##STR00036##
[0179] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.20 (s, 0.6H),
8.39 (s, 0.4H), 7.44-6.88 (m, 10H), 6.25 (dd, J=12 Hz, J=6 Hz, 1H),
6.45 (t, J=8.4 Hz, 1H), 3.87 (m, 1H), 3.26-2.95 (m, 3H), 2.46-2.05
(m, 8H), 1.86-1.61 (m, 5H), 1.34-1.11 (m, 9H)
(2R,3S)-1-(2-fluoro-6-methylbenzoyl)-2-[4-(tetrahydropyran-4-ylamino)pheny-
l]piperidine-3-carboxylic acid (3-morpholin-4-yl-phenyl)amide
##STR00037##
[0181] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.61 (s, 1H),
7.34-6.92 (m, 10H), 6.78-6.65 (m, 1H), 6.62-6.53 (m, 1H), 3.98-3.85
(m, 4H), 3.83-3.70 (m, 1H), 3.55-3.30 (m, 3H), 3.27-2.98 (M, 4H),
2.42-1.92 (m, 8H), 1.81-1.45 (m, 7H)
(2R,3S)-1-(2-fluoro-6-methylbenzoyl)-2-[4-((R)-2-trifluoromethylpyrrolidin-
-1-ylmethyl)phenyl]piperidine-3-carboxylic acid
(3-.sup.t-butylphenyl)amide
##STR00038##
[0183] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.01 (bs, 0.5H), 7.96 (bs,
0.5H), 7.55-7.37 (m, 3H), 7.30-7.19 (m, 6H), 7.13-7.06 (m, 1H),
7.01-6.90 (m, 1H), 6.85-6.64 (m, 1H), 4.15-4.11 (m, 1H), 3.58-3.54
(m, 1H), 3.30-3.20 (m, 2H), 3.17-2.80 (m, 2H), 2.45-2.17 (m, 4H),
2.00-1.94 (m, 2H), 1.86-1.60 (m, 8H), 1.31-1.26 (m, 7H)
Example 8
Materials and Methods
[0184] A. Cells
[0185] 1. C5a Receptor Expressing Cells
[0186] a) U937 Cells
[0187] U937 cells are a monocytic cell line which express C5aR, and
are available from American Tissue Cell Collection (Virginia).
These cells were cultured as a suspension in RPMI-1640 medium
supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5
g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, and 10% FBS. Cells
were grown under 5% CO.sub.2/95% air, 100% humidity at 37.degree.
C. and subcultured twice weekly at 1:6 (cells were cultured at a
density range of 1.times.10.sup.5 to 2.times.10.sup.6 cells/mL) and
harvested at 1.times.10.sup.6 cells/mL. Prior to assay, cells are
treated overnight with 0.5 mM of cyclic AMP (Sigma, Ohio) and
washed once prior to use. cAMP treated U937 cells can be used in
C5aR ligand binding and functional assays.
[0188] b) Isolated Human Neutrophils
[0189] Optionally, human or murine neutrophils can be used to assay
for compound activity. Neutrophils may be isolated from fresh human
blood using density separation and centrifugation. Briefly, whole
blood is incubated with equal parts 3% dextran and allowed to
separate for 45 minutes. After separation, the top layer is layered
on top of 15 mls of Ficoll (15 mls of Ficoll for every 30 mls of
blood suspension) and centrifuged for 30 minutes at 400.times.g
with no brake. The pellet at the bottom of the tube is then
isolated and resuspended into PharmLyse RBC Lysis Buffer (BD
Biosciences, San Jose, Calif.) after which the sample is again
centrifuged for 10 minutes at 400.times.g with brake. The remaining
cell pellet is resuspended as appropriate and consists of isolated
neutrophils.
[0190] B. Assays
[0191] 1. Inhibition of .sup.125I-C5a Binding to C5aR
[0192] cAMP treated U937 cells expressing C5aR were centrifuged and
resuspended in assay buffer (20 mM HEPES pH 7.1, 140 mM NaCl, 1 mM
CaCl.sub.2, 5 mM MgCl.sub.2, and with 0.1% bovine serum albumin) to
a concentration of 3.times.10.sup.6 cells/mL. Binding assays were
set up as follows. 0.1 mL of cells was added to the assay plates
containing 5 .mu.L of the compound, giving a final concentration of
.about.2-10 .mu.M each compound for screening (or part of a dose
response for compound IC.sub.50 determinations). Then 0.1 mL of
.sup.125I labeled C5a (obtained from Perkin Elmer Life Sciences,
Boston, Mass.) diluted in assay buffer to a final concentration of
.about.50 pM, yielding .about.30,000 cpm per well, was added, the
plates sealed and incubated for approximately 3 hours at 4.degree.
C. on a shaker platform. Reactions were aspirated onto GF/B glass
filters pre-soaked in 0.3% polyethyleneimine (PEI) solution, on a
vacuum cell harvester (Packard Instruments; Meriden, Conn.).
Scintillation fluid (40 .mu.l; Microscint 20, Packard Instruments)
was added to each well, the plates were sealed and radioactivity
measured in a Topcount scintillation counter (Packard Instruments).
Control wells containing either diluent only (for total counts) or
excess C5a (1 .mu.g/mL, for non-specific binding) were used to
calculate the percent of total inhibition for compound. The
computer program Prism from GraphPad, Inc. (San Diego, Calif.) was
used to calculate IC.sub.50 values. IC.sub.50 values are those
concentrations required to reduce the binding of radiolabeled C5a
to the receptor by 50%. (For further descriptions of ligand binding
and other functional assays, see Dairaghi, et al., J. Biol. Chem.
274:21569-21574 (1999), Penfold, et al., Proc. Natl. Acad. Sci.
USA. 96:9839-9844 (1999), and Dairaghi, et al., J. Biol. Chem.
272:28206-28209 (1997)).
[0193] 2. Calcium Mobilization
[0194] Optionally, compounds may be further assayed for their
ability to inhibit calcium flux in cells. To detect the release of
intracellular stores of calcium, cells (e.g., cAMP stimulated U937
or neutrophils) are incubated with 3 .mu.M of INDO-1AM dye
(Molecular Probes; Eugene, Oreg.) in cell media for 45 minutes at
room temperature and washed with phosphate buffered saline (PBS).
After INDO-1AM loading, the cells are resuspended in flux buffer
(Hank's balanced salt solution (HBSS) and 1% FBS). Calcium
mobilization is measured using a Photon Technology International
spectrophotometer (Photon Technology International; New Jersey)
with excitation at 350 nm and dual simultaneous recording of
fluorescence emission at 400 nm and 490 nm. Relative intracellular
calcium levels are expressed as the 400 nm/490 nm emission ratio.
Experiments are performed at 37.degree. C. with constant mixing in
cuvettes each containing 10.sup.6 cells in 2 mL of flux buffer. The
chemokine ligands may be used over a range from 1 to 100 nM. The
emission ratio is plotted over time (typically 2-3 minutes).
Candidate ligand blocking compounds (up to 10 .mu.M) are added at
10 seconds, followed by chemokines at 60 seconds (i.e., C5a;
R&D Systems; Minneapolis, Minn.) and control chemokine (i.e.,
SDF-1.alpha.; R&D Systems; Minneapolis, Minn.) at 150
seconds.
[0195] 3. Chemotaxis Assays
[0196] Optionally, compounds may be further assayed for their
ability to inhibit chemotaxis in cells. Chemotaxis assays are
performed using 5 .mu.m pore polycarbonate,
polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers
(Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's
balanced salt solution (HBSS) and 1% FBS). C5aR ligands (i.e., C5a,
R&D Systems; Minneapolis, Minn.) are use to evaluate compound
mediated inhibition of C5aR mediated migration. Other chemokines
(i.e., SDF-1.alpha.; R&D Systems; Minneapolis, Minn.) are used
as specificity controls. The lower chamber is loaded with 29 .mu.l
of chemokine (i.e., 0.03 nM C5a) and varying amounts of compound;
the top chamber contains 100,000 U937 or neutrophil cells in 20
.mu.l. The chambers are incubated 1.5 hours at 37.degree. C., and
the number of cells in the lower chamber quantified either by
direct cell counts in five high powered fields per well or by the
CyQuant assay (Molecular Probes), a fluorescent dye method that
measures nucleic acid content and microscopic observation.
[0197] C. Identification of Inhibitors of C5aR
[0198] 1. Assay
[0199] To evaluate small organic molecules that prevent the C5a
receptor from binding ligand, an assay was employed that detected
.sup.125I-C5a binding to cells expressing C5aR on the cell surface
(for example, cAMP stimulated U937 cells or isolated human
neutrophils). For compounds that inhibited binding, whether
competitive or not, fewer radioactive counts are observed when
compared to uninhibited controls.
[0200] Equal numbers of cells were added to each well in the plate.
The cells were then incubated with radiolabeled C5a. Unbound ligand
was removed by washing the cells, and bound ligand was determined
by quantifying radioactive counts. Cells that were incubated
without any organic compound gave total counts; non-specific
binding was determined by incubating the cells with unlabeled
ligand and labeled ligand. Percent inhibition was determined by the
equation:
% inhibition=(1-[(sample cpm)-(nonspecific cpm)]/[(total
cpm)-(nonspecific cpm)]).times.100.
[0201] 2. Dose Response Curves
[0202] To ascertain a candidate compound's affinity for C5aR as
well as confirm its ability to inhibit ligand binding, inhibitory
activity was titered over a 1.times.10.sup.-10 to 1.times.10.sup.-4
M range of compound concentrations. In the assay, the amount of
compound was varied; while cell number and ligand concentration
were held constant.
[0203] D. In Vivo Efficacy Models
[0204] The compounds of interest can be evaluated for potential
efficacy in treating a C5a mediated conditions by determining the
efficacy of the compound in an animal model. In addition to the
models described below, other suitable animal models for studying
the compound of interest can be found in Mizuno, M. et al., Expert
Opin. Investig. Drugs (2005), 14(7), 807-821, which is incorporated
herein by reference in its entirety.
[0205] 1. Models of C5a Induced Leukopenia
[0206] a) C5a Induced Leukopenia in a Human C5aR Knock-in Mouse
Model
[0207] To study the efficacy of compounds of the instant invention
in an animal model, a recombinant mouse can be created using
standard techniques, wherein the genetic sequence coding for the
mouse C5aR is replaced with sequence coding for the human C5aR, to
create a hC5aR-KI mouse. In this mouse, administration of hC5a
leads to upregulation of adhesion molecules on blood vessel walls
which bind blood leukocytes, sequestering them from the blood
stream. Animals are administered 20 ug/kg of hC5a and 1 minute
later leukocytes are quantified in peripheral blood by standard
techniques. Pretreatment of mice with varying doses of the present
compounds can almost completely block the hC5a induced
leukopenia.
[0208] b) C5a induced Leukopenia in a Cynomolgus Monkey Model
[0209] To study the efficacy of compounds of the instant invention
in a non-human primate model, C5a induced leukopenia is studied in
a cynomolgus model. In this model administration of hC5a leads to
upregulation of adhesion molecules on blood vessel walls which bind
blood leukocytes, hence sequestering them from the blood stream.
Animals are administered 10 ug/kg of hC5a and 1 minute later
leukocytes are quantified in peripheral blood.
[0210] c) Mouse Model of ANCA Induced Vasculitis
[0211] On day 0, hC5aR-KI mice are intravenously injected with 50
mg/kg purified antibody to myeloperoxidase (Xiao et al, J. Clin.
Invest. 110: 955-963 (2002)). Mice are further dosed with oral
daily doses of compounds of the invention or vehicle for seven
days, then mice are sacrificed and kidneys collected for
histological examination. Analysis of kidney sections can show
significantly reduced number and severity of crescentic and
necrotic lesions in the glomeruli when compared to vehicle treated
animals.
[0212] d) Mouse Model of Choroidal Neovascularization
[0213] To study the efficacy of compounds of the instant invention
in treatment of age related macular degeneration (AMD) the bruch
membrane in the eyes of hC5aR-KI mice are ruptured by laser
photocoagulation (Nozika et al, PNAS 103: 2328-2333 (2006). Mice
are treated with vehicle or a daily oral or appropriate
intra-vitreal dose of a compound of the invention for one to two
weeks. Repair of laser induced damage and neovascularization are
assessed by histology and angiography.
[0214] 2. Rheumatoid Arthritis Models
[0215] a) Rabbit Model of Destructive Joint Inflammation
[0216] To study the effects of candidate compounds on inhibiting
the inflammatory response of rabbits to an intra-articular
injection of the bacterial membrane component lipopolysaccharide
(LPS), a rabbit model of destructive joint inflammation is used.
This study design mimics the destructive joint inflammation seen in
arthritis. Intra-articular injection of LPS causes an acute
inflammatory response characterized by the release of cytokines and
chemokines, many of which have been identified in rheumatoid
arthritic joints. Marked increases in leukocytes occur in synovial
fluid and in synovium in response to elevation of these chemotactic
mediators. Selective antagonists of chemokine receptors have shown
efficacy in this model (see Podolin, et al., J. Immunol.
169(11):6435-6444 (2002)).
[0217] A rabbit LPS study is conducted essentially as described in
Podolin, et al. ibid., female New Zealand rabbits (approximately 2
kilograms) are treated intra-articularly in one knee with LPS (10
ng) together with either vehicle only (phosphate buffered saline
with 1% DMSO) or with addition of candidate compound (dose 1=50
.mu.M or dose 2=100 .mu.M) in a total volume of 1.0 mL. Sixteen
hours after the LPS injection, knees are lavaged and cells counts
are performed. Beneficial effects of treatment were determined by
histopathologic evaluation of synovial inflammation. Inflammation
scores are used for the histopathologic evaluation: 1--minimal,
2--mild, 3--moderate, 4--moderate-marked.
[0218] b) Evaluation of a Compound in a Rat Model of Collagen
Induced Arthritis
[0219] A 17 day developing type II collagen arthritis study is
conducted to evaluate the effects of a candidate compound on
arthritis induced clinical ankle swelling. Rat collagen arthritis
is an experimental model of polyarthritis that has been widely used
for preclinical testing of numerous anti-arthritic agents (see
Trentham, et al., J. Exp. Med. 146(3):857-868 (1977), Bendele, et
al., Toxicologic Pathol. 27:134-142 (1999), Bendele, et al.,
Arthritis Rheum. 42:498-506 (1999)). The hallmarks of this model
are reliable onset and progression of robust, easily measurable
polyarticular inflammation, marked cartilage destruction in
association with pannus formation and mild to moderate bone
resorption and periosteal bone proliferation.
[0220] Female Lewis rats (approximately 0.2 kilograms) are
anesthetized with isoflurane and injected with Freund's Incomplete
Adjuvant containing 2 mg/mL bovine type II collagen at the base of
the tail and two sites on the back on days 0 and 6 of this 17 day
study. A candidate compound is dosed daily in a sub-cutaneous
manner from day 0 till day 17 at a efficacious dose. Caliper
measurements of the ankle joint diameter were taken, and reducing
joint swelling is taken as a measure of efficacy.
[0221] 3. Rat Model of Sepsis
[0222] To study the effect of compounds of interest on inhibiting
the generalized inflammatory response that is associated with a
sepsis like disease, the Cecal Ligation and Puncture (CLP) rat
model of sepsis is used. A Rat CLP study is conducted essentially
as described in Fujimura N, et al. (American Journal Respiratory
Critical Care Medicine 2000; 161: 440-446). Briefly described here,
Wistar Albino Rats of both sexes weighing between 200-250 g are
fasted for twelve hours prior to experiments. Animals are kept on
normal 12 hour light and dark cycles and fed standard rat chow up
until 12 hours prior to experiment. Then animals are split into
four groups; (i) two sham operation groups and (ii) two CLP groups.
Each of these two groups (i.e., (i) and (ii)) is split into vehicle
control group and test compound group. Sepsis is induced by the CLP
method. Under brief anesthesia a midline laparotomy is made using
minimal dissection and the cecum is ligated just below the
ileocaecal valve with 3-0 silk, so the intestinal continuity is
maintained. The antimesinteric surface of the cecum is perforated
with an 18 gauge needle at two locations 1 cm apart and the cecum
is gently squeezed until fecal matter is extruded. The bowel is
then returned to the abdomen and the incision is closed. At the end
of the operation, all rats are resuscitated with saline, 3 ml/100 g
body weight, given subcutaneously. Postoperatively, the rats are
deprived of food, but have free access to water for the next 16
hours until they are sacrificed. The sham operated groups are given
a laparotomy and the cecum is manipulated but not ligated or
perforated. Beneficial effects of treatment are measured by
histopathological scoring of tissues and organs as well as
measurement of several key indicators of hepatic function, renal
function, and lipid peroxidation. To test for hepatic function
aspartate transaminase (AST) and alanine transaminase (ALT) are
measured. Blood urea nitrogen and creatinine concentrations are
studied to assess renal function. Pro-inflammatory cytokines such
as TNF-alpha and IL-1beta are also assayed by ELISA for serum
levels.
[0223] 4. Mouse SLE Model of Experimental Lupus Nephritis.
[0224] To study the effect of compounds of interest on a Systemic
Lupus Erythematosus (SLE), the MRL/lpr murine SLE model is used.
The MRL/Mp-Tmfrsf6.sup.lpr/lpr strain (MRL/lpr) is a commonly used
mouse model of human SLE. To test compounds efficacy in this model
male MRL/lpr mice are equally divided between control and C5aR
antagonists groups at 13 weeks of age. Then over the next 6 weeks
compound or vehicle is administered to the animals via osmotic
pumps to maintain coverage and minimize stress effects on the
animals. Serum and urine samples are collected bi-weekly during the
six weeks of disease onset and progression. In a minority of these
mice glomerulosclerosis develops leading to the death of the animal
from renal failure. Following mortality as an indicator of renal
failure is one of the measured criteria and successful treatment
will usually result in a delay in the onset of sudden death among
the test groups. In addition, the presence and magnitude of renal
disease may also be monitored continuously with blood urea nitrogen
(BUN) and albuminuria measurements. Tissues and organs were also
harvested at 19 weeks and subjected to histopathology and
immunohistochemistry and scored based on tissue damage and cellular
infiltration.
[0225] 5. Rat Model of COPD
[0226] Smoke induced airway inflammation in rodent models may be
used to assess efficacy of compounds in Chronic Obstructive
Pulmonary Disease (COPD). Selective antagonists of chemokines have
shown efficacy in this model (see, Stevenson, et al., Am. J.
Physiol Lung Cell Mol Physiol. 288 L514-L522, (2005)). An acute rat
model of COPD is conducted as described by Stevenson et al. A
compound of interest is administered either systemically via oral
or IV dosing; or locally with nebulized compound. Male
Sprague-Dawley rats (350-400 g) are placed in Perspex chambers and
exposed to cigarette smoke drawn in via a pump (50 mL every 30
seconds with fresh air in between). Rats are exposed for a total
period of 32 minutes. Rats are sacrificed up to 7 days after
initial exposure. Any beneficial effects of treatment are assessed
by a decrease inflammatory cell infiltrate, decreases in chemokine
and cytokine levels.
[0227] In a chronic model, mice or rats are exposed to daily
tobacco smoke exposures for up to 12 months. Compound is
administered systemically via once daily oral dosing, or
potentially locally via nebulized compound. In addition to the
inflammation observed with the acute model (Stevensen et al.),
animals may also exhibit other pathologies similar to that seen in
human COPD such as emphysema (as indicated by increased mean linear
intercept) as well as altered lung chemistry (see Martorana et al,
Am. J. Respir. Crit Care Med. 172(7): 848-53.
[0228] 6. Mouse EAE Model of Multiple Sclerosis
[0229] Experimental autoimmune encephalomyelitis (EAE) is a model
of human multiple sclerosis. Variations of the model have been
published, and are well known in the field. In a typical protocol,
C57BL/6 (Charles River Laboratories) mice are used for the EAE
model. Mice are immunized with 200 ug myelin oligodendrocyte
glycoprotein (MOG) 35-55 (Peptide International) emulsified in
Complete Freund's Adjuvant (CFA) containing 4 mg/ml Mycobacterium
tuberculosis (Sigma-Aldrich) s.c. on day 0. In addition, on day 0
and day 2 animals are given 200 ng of pertussis toxin (Calbiochem)
i.v. Clinical scoring is based on a scale of 0-5: 0, no signs of
disease; 1, flaccid tail; 2, hind limb weakness; 3, hind limb
paralysis; 4, forelimb weakness or paralysis; 5, moribund. Dosing
of the compounds of interest to be assessed can be initiated on day
0 (prophylactic) or day 7 (therapeutic, when histological evidence
of disease is present but few animals are presenting clinical
signs) and dosed once or more per day at concentrations appropriate
for their activity and pharmacokinetic properties, e.g. 100 mg/kg
s.c. Efficacy of compounds can be assessed by comparisons of
severity (maximum mean clinical score in presence of compound
compared to vehicle), or by measuring a decrease in the number of
macrophages (F4/80 positive) isolated from spinal cords. Spinal
cord mononuclear cells can be isolated via discontinuous
Percoll-gradient. Cells can be stained using rat anti-mouse
F4/80-PE or rat IgG2b-PE (Caltag Laboratories) and quantitated by
FACS analysis using 10 ul of Polybeads per sample
(Polysciences).
[0230] 7. Mouse Model of Kidney Transplantation
[0231] Transplantation models can be performed in mice, for
instance a model of allogenic kidney transplant from C57BL/6 to
BALB/c mice is described in Faikah Gueler et al, JASN Express, Aug.
27, 2008. Briefly, mice are anesthetized and the left donor kidney
attached to a cuff of the aorta and the renal vein with a small
caval cuff, and the ureters removed en block. After left
nephrectomy of the recipient, the vascular cuffs are anastomosed to
the recipient abdominal aorta and vena cava, respectively, below
the level of the native renal vessels. The ureter is directly
anastomosed into the bladder. Cold ischemia time is 60 min, and
warm ischemia time is 30 min. The right native kidney can be
removed at the time of allograft transplantation or at
posttransplantation day 4 for long-term survival studies. General
physical condition of the mice is monitored for evidence of
rejection. Compound treatment of animals can be started before
surgery or immediately after transplantation, eg by sub cut
injection once daily. Mice are studied for renal function and
survival. Serum creatinine levels are measured by an automated
method (Beckman Analyzer, Krefeld, Germany).
[0232] 8. Mouse Model of Ischemia/Reperfusion
[0233] A mouse model of ischemia/reperfusion injury can be
performed as described by Xiufen Zheng et al, Am. J. Pathol, Vol
173:4, October 2008. Briefly, CD1 mice aged 6-8 weeks are
anesthetized and placed on a heating pad to maintain warmth during
surgery. Following abdominal incisions, renal pedicles are bluntly
dissected and a microvascular clamp placed on the left renal
pedicle for 25-30 minutes. Following ischemia the clamps are
removed along with the right kidney, incisions sutured, and the
animals allowed to recover. Blood is collected for serum creatinine
and BUN analysis as an indicator of kidney health. Alternatively
animal survival is monitored over time. Compound can be
administered to animals before and/or after the surgery and the
effects on serum creatinine, BUN or animal survival used as
indicators of compound efficacy.
[0234] 9. Mouse Model of Tumor Growth
[0235] C57BL/6 mice 6-16 weeks of age are injected subcutaneously
with 1.times.105 TC-1 cells (ATCC, VA) in the right or left rear
flank. Beginning about 2 weeks after cell injection, tumors are
measured with calipers every 2-4 d until the tumor size required
the mice are killed. At the time of sacrifice animals are subjected
to a full necropsy and spleens and tumors removed. Excised tumors
are measured and weighed. Compounds may be administered before
and/or after tumor injections, and a delay or inhibition of tumor
growth used to assess compound efficacy.
* * * * *