U.S. patent application number 11/651320 was filed with the patent office on 2007-06-14 for substituted 7-aza-quinazoline compounds useful as p38 kinase inhibitors.
Invention is credited to Nolan James Dewdney, David Michael Goldstein.
Application Number | 20070135459 11/651320 |
Document ID | / |
Family ID | 33300055 |
Filed Date | 2007-06-14 |
United States Patent
Application |
20070135459 |
Kind Code |
A1 |
Dewdney; Nolan James ; et
al. |
June 14, 2007 |
Substituted 7-aza-quinazoline compounds useful as p38 kinase
inhibitors
Abstract
Compounds having the formula (I) or (II), ##STR1## are useful as
p38 kinase inhibitors, wherein R is an optionally substituted
alkyl, cycloalkyl, or aryl; R.sup.6 is hydrogen or lower alkyl;
R.sup.7 is hydrogen or a non-interfering substituent, and Q is a
non-aromatic moiety as defined in the specification.
Inventors: |
Dewdney; Nolan James; (San
Jose, CA) ; Goldstein; David Michael; (San Jose,
CA) |
Correspondence
Address: |
ROCHE PALO ALTO LLC;PATENT LAW DEPT. M/S A2-250
3431 HILLVIEW AVENUE
PALO ALTO
CA
94304
US
|
Family ID: |
33300055 |
Appl. No.: |
11/651320 |
Filed: |
January 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10824980 |
Apr 15, 2004 |
7189731 |
|
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11651320 |
Jan 9, 2007 |
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60463229 |
Apr 16, 2003 |
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Current U.S.
Class: |
514/264.11 ;
544/279 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 11/06 20180101; A61P 19/00 20180101; A61P 9/10 20180101; C07D
471/04 20130101; A61P 19/02 20180101; A61P 1/04 20180101; A61P
25/28 20180101; A61P 9/00 20180101; A61P 29/00 20180101; A61P 11/00
20180101; A61P 7/00 20180101 |
Class at
Publication: |
514/264.11 ;
544/279 |
International
Class: |
A61K 31/519 20060101
A61K031/519; C07D 487/02 20060101 C07D487/02 |
Claims
1-17. (canceled)
18. A method for treating a p38-mediated disorder in a patient
comprising administering to the patient in need of such treatment,
an effective amount of a compound of formula I or II: ##STR23## or
a pharmaceutically-acceptable salt thereof, wherein: R is selected
from: (a) alkyl optionally-substituted with one to three of
R.sup.17; (b) cycloalkyl optionally substituted with one, two or
three groups selected from R.sup.18; and (c) optionally-substituted
aryl; Q is selected from alkyl, cycloalkyl, substituted cycloalkyl,
heterocyclyl, substituted heterocyclyl, and alkyl substituted with
one, two or three of halogen, cyano, --OR.sup.8, --SR.sup.8,
--C(.dbd.O)R.sup.8, --C(O).sub.2R.sup.8,
--C(.dbd.O)NR.sup.8R.sup.9, --S(O).sub.pR.sup.10, --C(O).sub.p
NR.sup.8R.sup.9, --S(O).sub.2NR.sup.8R.sup.9, --NR.sup.8R.sup.9,
cycloalkyl, substituted cycloalkyl, heterocyclyl, and/or
substituted heterocyclyl; R.sup.6 is hydrogen or lower alkyl;
R.sup.7 is selected from hydrogen, alkyl, substituted alkyl,
halogen, cyano, nitro, hydroxy, alkoxy, haloalkoxy, amino,
alkylamino, and optionally-substituted cycloalkyl, heterocyclyl,
aryl, or heteroaryl: R.sup.8 and R.sup.9 are (i) independently
selected from hydrogen, alkyl, haloalkyl, hydroxyalkyl,
alkoxyalkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and
substituted heterocyclyl; or (ii) when R.sup.8 and R.sup.9 are
attached to the same nitrogen atom (as in
--C(O).sub.2NR.sup.8R.sup.9, --S(O).sub.2NR.sup.8R.sup.9, and
--NR.sup.8R.sup.9), R.sup.8 and R.sup.9 may be taken together to
form an optionally-substituted heterocyclyl tins; R.sup.10 is
alkyl, hydroxyalkyl, alkoxyalkyl, cycloalkyl, substituted
cycloalkyl, heterocyclyl, or substituted heterocyclyl; R.sup.17 is
at each occurrence independently selected from halogen, haloalkoxy,
haloalkyl, alkoxy, or optionally-substituted phenyl, benzyl,
phenyloxy, benzyloxy, or cycloalkyl: R.sup.18 is at each occurrence
independently selected from alkyl, substituted alkyl, halogen,
haloalkyl, haloalkoxy, cyano, alkoxy, acyl, alkoxycarbonyl,
alkylsulfonyl, or optionally-substituted phenyl, phenyloxy,
benzyloxy, cycloalkyl, heterocyclyl, or heteroaryl: and p is 1 or2
or a pharmaceutically acceptable salt thereof.
19. The method of claim 18, wherein the p38-mediated disorder is
selected from the group consisting of arthritis, Crohn's disease,
Alzheimer's disease, adult respiratory distress syndrome, chronic
obstructive pulmonary disease, asthma, stroke, sepsis, myocardial
infarction, and spondylitis.
20. A method for inhibiting p38 kinase in a mammal comprising
administering to said mammal a compound of formula I or II:
##STR24## or a pharmaceutically-acceptable salt thereof, wherein: R
is selected from: (d) alkyl optionally-substituted with one to
three of R.sup.17; (e) cycloalkyl optionally substituted with one,
two or three groups selected from R.sup.18l and (f)
optionally-substituted aryl; Q is selected from alkyl, cycloalkyl,
substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, and
alkyl substituted with one, two or three of halogen, cyano,
--OR.sup.8, --SR.sup.8, --C(.dbd.O)R.sup.8, --C(O)R.sup.8,
--C(.dbd.O)NR.sup.8R.sup.9, --S(O).sub.pR.sup.10,
--C(O)NR.sup.8R.sup.9, --S(O).sub.2NR.sup.8R.sup.9,
--NR.sup.8R.sup.9, cycloalkyl, substituted cycloalkyl.
heterocyclyl, and/or substituted heterocyclyl; R.sup.6is hydrogen
or lower alkyl: R.sup.7 is selected from hydrogen, alkyl,
substituted alkyl, halogen, cyano, nitro, hydroxy, alkoxy,
haloalkoxy, amino, alkylamino, and optionally-substituted
cycloalkyl, heterocyclyl, aryl, or heteroaryl; R.sup.8 and R.sup.9
are (i) independently selected from hydrogen, alkyl, haloalkyl,
hydroxyalkyl, alkoxyalkyl, cycloalkyl, substituted cycloalkyl,
heterocyclyl, and substituted heterocyclyl: or (ii) when R.sup.8
and R.sup.9 are attached to the same nitrogen atom (as in
--C(O).sub.2NR.sup.8R.sup.9, --S(O).sub.2NR.sup.8R.sup.9, and
--NR.sup.8R.sup.9), R.sup.8 and R.sup.9 may be taken together to
form an optionally-substituted heterocyclyl ring; R.sup.10 is
alkyl, hydroxyalkyl, alkoxyalkyl, cycloalkyl, substituted
cycloalkyl, heterocyclyl, or substituted heterocyclyl; R.sup.17 is
at each occurrence independently selected from halogen, haloalkoxy,
haloalkyl, alkoxy, or optionally-substituted phenyl, benzyl,
phenyloxy, benzyloxy, or cycloalkyl; R.sup.18 is at each occurrence
independently selected from alkyl, substituted alkyl, halogen,
haloalkyl, haloalkoxy, cyano, alkoxy, acyl, alkoxycarbonyl,
alkylsulfonyl, or optionally-substituted phenyl, phenyloxy,
benzyloxy, cycloalkyl, heterocyclyl, or heteroaryl; and p is 1 or
2.
21-22. (canceled)
23. The method of claim 20, wherein: Q is selected from an alkyl or
substituted alkyl having the formula --C(R.sup.1R.sup.2R.sup.3);
R.sup.1, R.sup.2 and R.sup.3 are selected from hydrogen, alkyl,
hydroxyalkyl, alkoxyalkyl,
--(C.sub.1-4alkylene)-S(O).sub.pR.sup.10,
-(C.sub.1-4alkylene)-C(O).sub.2R.sup.8, cycloalkyl,
cycloalkylalkyl, heterocyclyl, or heterocycloalkyl, wherein said
cycloalkyl and heterocyclyl groups are, in turn, optionally
substituted with up to one of R.sup.12 and up to one of R.sup.14;
and R.sup.12and R.sup.14 are independently selected where valence
allows from C.sub.1-4alkyl, hydroxy, oxo (.dbd.O),
--O(C.sub.1-4alkyl), --C(.dbd.O)H, --C(.dbd.O)(C.sub.1-4alkyl),
--C(O).sub.2H, --C(O).sub.2(C.sub.1-4alkyl), and
--S(O).sub.2(C.sub.1-4alkyl), or a pharmaceutically acceptable salt
thereof.
24. The method of claim 20, wherein said compound has the formula
##STR25## or a pharmaceutically acceptable salt thereof.
25. The method of claim 24, wherein said compound has the formula:
##STR26## wherein: X is --O--, --C(.dbd.O)--, --N(R.sup.12a)--, or
--CH(R.sup.12b)--; R.sup.12a is selected from hydrogen,
C.sub.1-4alkyl, --C(.dbd.O)R.sup.15, --C(O).sub.2R.sup.15, and
--S(O).sub.2(C.sub.1-4alkyl); R.sup.12b is selected from hydrogen,
C.sub.1-4alkyl, --OR.sup.15, --C(.dbd.O)R.sup.15,
--C(O).sub.2R.sup.15, and --S(O).sub.2(C.sub.1-4alkyl); R.sup.14 is
selected from C.sub.1-4alkyl, oxo (.dbd.O), --OR.sup.15,
--C(.dbd.O)R.sup.15, --C(O).sub.2R.sup.15, and
--S(O).sub.2(C.sub.1-4alkyl); R.sup.15 is selected from hydrogen
and C.sub.14alkyl; q is 0 or 1; and r is 0, 1 or 2, or a
pharmaceutically acceptable salt thereof.
26. The method of claim 25, wherein R.sup.4 and R.sup.5 are both
fluoro.
27. The method of claim 19, wherein said disorder comprises
rheumatoid arthritis.
28. The method of claim 19, wherein said disorder comprises Crohn's
disease.
29. The method of claim 19, wherein said disorder comprises
Alzheimer's disease.
30. The method of claim 19, wherein said disorder comprises adult
respiratory distress syndrome.
31. The method of claim 19, wherein said disorder comprises chronic
obstructive pulmonary disease.
32. The method of claim 19, wherein said disorder comprises
asthma.
33. The method of claim 19, wherein said disorder comprises
stroke.
34. The method of claim 19, wherein said disorder comprises
sepsis.
35. The method of claim 19, wherein said disorder comprises
myocardial infarction.
36. The method of claim 19, wherein said disorder comprises
spondylitis.
Description
RELATED APPLICATIONS
[0001] This applications claims priority from U.S. provisional
patent application Ser. No. 60/463,229, filed Apr. 16, 2003,
incorporated herein by reference in full.
FIELD OF THE INVENTION
[0002] The present invention relates to certain quinazoline
compounds useful as p38 protein kinase inhibitors. In particular,
the present invention relates to 2-amino-6-phenyloxy substituted
7-azaquinazoline compounds, pharmaceutical preparations comprising
the same, and methods for using them.
BACKGROUND OF THE INVENTION
[0003] Mitogen-activated protein kinases (MAP) are a family of
proline-directed serine/threonine kinases that activate their
substrates by dual phosphorylation. The kinases are activated by a
variety of signals including nutritional and osmotic stress, UV
light, growth factors, endotoxin, and inflammatory cytokines. One
group of MAP kinases is the p38 kinase group which includes various
isoforms (e.g., p38.alpha., p39.beta., p38.gamma. and p38.delta.).
The p38 kinases are responsible for phosphorylating and activating
transcription factors (as well as other kinases), and are
themselves activated by physical and chemical stress,
pro-inflammatory cytokines, and bacterial lipopolysaccharide.
[0004] More importantly, the products of the p38 phosphorylation
have been shown to mediate the production of inflammatory
cytokines, including TNF, IL-1, IL-6, and cyclooxygenase-2 (COX-2).
Each of these cytokines has been implicated in numerous disease
states and conditions. For example, TNF-.alpha. is a cytokine
produced primarily by activated monocytes and macrophages.
Excessive or unregulated production of TNF-.alpha. has been
implicated as playing a causative role in the pathogenesis of
rheumatoid arthritis. More recently, inhibition of TNF production
has been shown to have broad application in the treatment of
inflammatory conditions, inflammatory bowel disease, Alzheimer's
disease, Crohn's disease, multiple sclerosis, and asthma.
[0005] Additionally, TNF has been implicated in viral infections,
such as HIV, influenza virus, and herpes virus including herpes
simplex virus type-1 (HSV-1), herpes simplex virus type-2 (HSV-2),
cytomegalovirus (CMV), varicella-zoster virus (VZV), Epstein-Barr
virus, human herpes virus-6 (HHV-6), human herpes virus-7 (HHV-7),
human herpes virus-8 (HHV-8), pseudorabies, and rhinotracheitis,
among others.
[0006] Similarly, IL-1 is produced by activated monocytes and
macrophages and plays a role in many pathophysiological responses
including rheumatoid arthritis, fever and reduction of bone
resorption.
[0007] The inhibition of these cytokines by inhibition of the p38
kinase would be beneficial in controlling, reducing and alleviating
many of these disease states. p38 MAP kinase inhibitors have
demonstrated efficacy in several disease models including arthritis
and other joint diseases, sepsis, stroke, myocardial injury,
respiratory inflammatory diseases such as chronic obstructive
pulmonary disease and asthma, and a wide range of inflammatory
conditions. The present invention provides certain
2-amino-6-phenoxy substituted quinazoline compounds useful in
inhibiting p38 kinase. U.S. patent application Ser. No. ______,
filed concomitantly herewith and assigned to the present assignee,
discloses 2-amino-6-phenoxy substituted quinazoline compounds
useful as p38 kinase inhibitors, and the entire contents of said
application is hereby incorporated herein by reference.
SUMMARY OF THE INVENTION
[0008] The present invention is directed to compounds having the
Formula (I) or (II): ##STR2##
[0009] and isomers, prodrugs, and pharmaceutically-acceptable salts
thereof, wherein: [0010] R is selected from: [0011] (a) alkyl
optionally-substituted with one to three of R.sup.17; [0012] (b)
cycloalkyl optionally substituted with one, two or three groups
selected from R.sup.18; and [0013] (c) optionally-substituted aryl;
[0014] Q is selected from alkyl, cycloalkyl, substituted
cycloalkyl, heterocyclyl, substituted heterocyclyl, and alkyl
substituted with one, two or three of halogen, cyano, --OR.sup.8,
--SR.sup.8, --C(.dbd.O)R.sup.8, --C(O).sub.2R.sup.8,
--C(.dbd.O)NR.sup.8R.sup.9, --C(O).sub.2NR.sup.8R.sup.9,
--S(O).sub.pR.sup.10, --S(O).sub.2NR.sup.8R.sup.9,
--NR.sup.8R.sup.9, cycloalkyl, substituted cycloalkyl,
heterocyclyl, and/or substituted heterocyclyl; [0015] R.sup.6 is
hydrogen or lower alkyl; [0016] R.sup.7 is selected from hydrogen,
alkyl, substituted alkyl, halogen, cyano, nitro, hydroxy, alkoxy,
haloalkoxy, amino, alkylamino, and optionally-substituted
cycloalkyl, heterocyclyl, aryl, or heteroaryl; [0017] R.sup.8 and
R.sup.9 are (i) independently selected from hydrogen, alkyl,
haloalkyl, hydroxyalkyl, alkoxyalkyl, cycloalkyl, substituted
cycloalkyl, heterocyclyl, and substituted heterocyclyl; or (ii)
when R.sup.8 and R.sup.9 are attached to the same nitrogen atom (as
in --C(.dbd.O)NR.sup.8R.sup.9, --S(O).sub.2NR.sup.8R.sup.9, and
--NR.sup.8R.sup.9), R.sup.8 and R.sup.9 may be taken together to
form an optionally-substituted heterocyclyl ring; [0018] R.sup.10
is alkyl, hydroxyalkyl, alkoxyalkyl, cycloalkyl, substituted
cycloalkyl, heterocyclyl, or substituted heterocyclyl; [0019]
R.sup.17 is at each occurrence independently selected from halogen,
haloalkoxy, haloalkyl, alkoxy, or optionally-substituted phenyl,
benzyl, phenyloxy, benzyloxy, or cycloalkyl; [0020] R.sup.18 is at
each occurrence independently selected from alkyl, substituted
alkyl, halogen, haloalkyl, haloalkoxy, cyano, alkoxy, acyl,
alkoxycarbonyl, alkylsulfonyl, or optionally-substituted phenyl,
phenyloxy, benzyloxy, cycloalkyl, heterocyclyl, or heteroaryl; and
[0021] p is 1 or 2.
[0022] Also provided are pharmaceutical compositions containing at
least one compound according to Formula (I) and/or (II), or a
pharmaceutically-acceptable salt thereof.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
[0023] The following definitions shall apply in the instant
specification and claims, unless otherwise specifically
indicated.
[0024] As used herein, the term "alkyl" means a linear or branched,
saturated monovalent hydrocarbon moiety of one to eight carbon
atoms (preferably one to six carbon atoms), e.g., methyl, ethyl,
n-propyl, 2-propyl, tert-butyl, pentyl, and the like. "Lower alkyl"
means an alkyl of one to four carbon atoms. When a subscript is
used herein following a carbon atom, the subscript refers to the
number of carbon atoms the named group may contain. Thus, for
example, C.sub.1-4alkyl means an alkyl of one to four carbon atoms
(i.e., lower alkyl) including methyl, ethyl, propyl, iso-propyl,
butyl, and tert-butyl.
[0025] "Alkylene" means a linear or branched, saturated divalent
hydrocarbon moiety of one to eight (preferably one to six) carbon
atoms, e.g., methylene, ethylene, propylene, and the like. When
reference is made to an alkylene linker group, as in
--Y--S(O).sub.2R, --Y--C(O).sub.2NRR, --Y--S(O).sub.2NRR, and so
forth, wherein Y is alkylene, it should be understood that the
alkylene may be a straight or branched-chain alkylene, and the
referenced substituent may be attached to any carbon atom of the
alkylene straight or branched chain. Thus, for example, the group
--Y--S(O).sub.2R, may include, without limitation,
--CH.sub.2--S(O).sub.2R, --CH.sub.2--CH[S(O).sub.2R]--CH.sub.3,
--CH.sub.2--CH{CH.sub.2CH[S(O).sub.2R]CH.sub.3}CH.sub.3, and so
forth.
[0026] When the term "alkyl" is used as a suffix following another
term, as in "phenylalkyl," or "hydroxyalkyl," this is intended to
refer to an alkyl group, as defined above, being substituted with
one or more (preferably one) substituent selected from the other,
specifically-named group. Thus, "phenylalkyl" includes benzyl,
phenylethyl, 2-phenylbutyl, and so forth. "Hydroxyalkyl" includes
2-hydroxyethyl, 1-(hydroxymethyl)-2-methylpropyl,
3,4-dihydroxybutyl, and so forth. Accordingly, as used herein, the
term "hydroxyalkyl" is used to define a subset of heteroalkyl
groups defined below. In the case of a "substituted
cycloalkylalkyl," this is intended to refer to an alkyl group, as
defined above, being substituted with one to two substituted
cycloalkyl groups, as defined below, and likewise, a "substituted
heterocycloalkylalkyl" refers to an alkyl group, as defined above,
being substituted with one to two substituted heterocyclyl groups,
as defined below.
[0027] The term "substituted alkyl" refers to an alkyl group as
defined above having one, two, three, or four substituents
(preferably one to two), independently selected from the group
consisting of halo, haloalkoxy, trifluoromethyl, cyano, nitro,
--OR.sup.a, --SR.sup.a, --S(O)R.sup.c, --S(O).sub.2R.sup.c,
--C(.dbd.O)R.sup.a, --C(.dbd.O)NR.sup.aR.sup.b,
--C(O).sub.2R.sup.a, --C(O).sub.2NR.sup.aR.sup.b,
--S(O).sub.2NR.sup.aR.sup.b, --NR.sup.aR.sup.b, aryl, substituted
aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted
cycloalkyl, heterocyclyl, and/or substituted heterocyclyl, wherein
R.sup.a and R.sup.b are independently selected from hydrogen,
C.sub.1-6alkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl, and
R.sup.c is selected from C.sub.1-6alkyl, aryl, heteroaryl,
cycloalkyl, and heterocyclyl, and each of R.sup.a, R.sup.b, and
R.sup.c in turn is optionally substituted with one, two, or three
of alkyl, halo, haloalkyl, hydroxy, alkoxy, haloalkoxy, cyano,
amino, alkylamino, SO.sub.2(alkyl), CO.sub.2H, CO.sub.2(alkyl),
C(.dbd.O)H, and/or C(.dbd.O)alkyl, and/or a C.sub.1-4 alkyl
substituted with one to two of halo, hydroxy, alkoxy, haloalkoxy,
cyano, amino, alkylamino, --SO.sub.2(alkyl), CO.sub.2H,
CO.sub.2(alkyl), C(.dbd.O)H, and/or C(.dbd.O)alkyl.
[0028] "Acyl" refers to the group --C(.dbd.O)R', wherein R' is
alkyl, substituted alkyl, aryl, or arylalkyl.
[0029] "Alkoxy" refers to the group OR', wherein R' is alkyl or
substituted alkyl. A "lower alkoxy" is a group --OR' wherein R' is
C.sub.1-4alkyl.
[0030] "Alkoxycarbonyl" refers to the group COOR', wherein R' is
alkyl or substituted alkyl as defined above.
[0031] "Alkylsulfonyl" refers to the group --S(O).sub.2R', wherein
R' is alkyl or substituted alkyl as defined above.
[0032] When reference is made herein to a carboxmide group
--CO.sub.2NRR (e.g., as in --C(O).sub.2NR.sup.8R.sup.9), it should
be understood this is intended to refer to the group
--O--C(.dbd.O)--NRR.
[0033] When the term "oxy" is used as a suffix following another
specifically-named group, as in "aryloxy", "heteroaryloxy," or
"arylalkyloxy", this means that an oxygen atom is present as a
linker to the other, specifically-named group. Thus, for example,
"aryloxy" refers to the group --O--R, wherein R is aryl;
"heteroaryloxy" refers to the group --O--R', wherein R' is
heteroaryl; and "arylalkyloxy" refers to the group --O--R'',
wherein R'' is arylalkyl such as benzyl. Similarly, a "substituted
aryloxy" means the group --O--R, wherein R is substituted aryl, and
a "substituted heteroaryloxy" means the group --O--R', wherein R'
is substituted heteroaryl.
[0034] "Amino" refers to the group NH.sub.2. Thus, an aminoalkyl
refers to an alkyl group having an amino substituent, e.g.,
--CH.sub.2--NH.sub.2, --CH.sub.2--CH.sub.2--NH.sub.2,
--CH.sub.2--CH(NH.sub.2)--CH.sub.3, and so forth. An alkylamino
refers to monoalkylamino groups having the formula --NHR, as well
as dialkylamino groups having the formula --NRR, wherein each R is
independently alkyl, substituted alkyl, heterocyclyl, substituted
heterocyclyl, cycloalkyl, or substituted cycloalkyl. Accordingly,
an alkylaminoalkyl refers to an alkyl group substituted by one to
two of --NHR and/or --NRR. A "lower aminoalkyl" refers to a group
--NHR' or --NR'R', wherein each R' is C.sub.1-4alkyl.
[0035] The term "aryl" refers to a monovalent, monocyclic or
bicyclic moiety in which at least one of the rings is an aromatic,
carbocyclic moiety. Thus, the term "aryl" includes phenyl,
1-napthyl, and 2-napthyl. The term "aryl" also includes phenyl
rings having fused thereto a second non-aromatic carbocyclic ring,
or a heteroaryl or heterocyclic ring such as benzothienyl,
benzo-pyrazolyl, benzopiperadinyl, benzocyclohexyl, and the like,
with the understanding, however, that the point of attachment will
be to the phenyl ring.
[0036] A "substituted aryl" is an aryl group as defined above
having one or more (preferably one, two, or three) substituents
independently selected from the group consisting of halo,
haloalkyl, haloalkoxy, cyano, nitro, --Y--R.sup.p, --Y-aryl,
--Y-heteroaryl, --Y-cycloalkyl, --Y-hetero-cyclyl, --Y--OR.sup.p,
--Y--NR.sup.pR.sup.q, --Y--C(.dbd.O)R.sup.p,
--Y--C(O).sub.2R.sup.p, --Y--C(.dbd.O)NR.sup.pR.sup.q,
--Y--C(O).sub.2NR.sup.pR.sup.q, --Y--S(O).sub.0-2R.sup.p,
--Y--NRS(O).sub.2R.sup.q, --Y--S(O).sub.2NR.sup.pR.sup.q, and/or
--Y--NRC(.dbd.O)NR.sup.pR.sup.q, where Y is absent or a
C.sub.1-4alkylene group, R is hydrogen, lower alkyl, or
hydroxyC.sub.1-4alkyl, and R.sup.p and R.sup.q are independently
selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, and
heterocyclyl, except when said substituent is
--YS(O).sub.1-2R.sup.p or --Y--NRS(O).sub.2R.sup.p, then R.sup.P in
these instances is not hydrogen. In each instance, each of R.sup.p
and/or R.sup.q in turn is optionally substituted with one to two of
alkyl, halo, cyano, hydroxy, alkoxy, amino, alkylamino, halo alkyl,
halo alkoxy, hydroxyalkyl, alkoxyalkyl, aminoalkyl,
alkylaminoalkyl, --SO.sub.2(alkyl), CO.sub.2H, CO.sub.2(alkyl),
C(.dbd.O)H, and/or C(.dbd.O)alkyl. A preferred group of aryl
substituents are those selected from alkyl, haloalkyl, halo,
hydroxy, amino, alkylamino, haloalkoxy and alkoxy. Within this
group, especially preferred aryl substituents are halo, alkyl, and
alkoxy. More specifically, the term "substituted aryl" includes,
but is not limited to, fluorophenyl, difluorophenyl, chlorophenyl,
methoxyphenyl, and the like.
[0037] The term "carbocyclic" means a cyclic moiety in which all
ring atoms are carbon atoms, including saturated, partially
unsaturated, and unsaturated rings.
[0038] The term "cycloalkyl" as used herein refers to saturated or
partially unsaturated, mono-valent, monocyclic carbocyclic moieties
of three to seven ring carbon atoms and further includes such rings
having a carbon-carbon bridge of one, two, or three bridgehead
carbon atoms, and/or having a second ring fused thereto, with the
understanding that said second fused ring may be a non-aromatic
carbocyclic or heterocyclic ring in which case the point of
attachment will be to the non-aromatic carbocyclic ring moiety.
Thus, the term "cycloalkyl" includes such rings as cyclo-propyl,
cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and the like.
Additionally, one or two carbon atoms of a cycloalkyl group may
optionally contain a carbonyl oxygen group, e.g., one or two atoms
in the ring may be a moiety of the formula --C(.dbd.O)--.
[0039] A "substituted cycloalkyl" is a cycloalkyl group as defined
above having one, two, or three substituents independently selected
from the group consisting of halo, haloalkyl, halo-alkoxy, cyano,
nitro, --Y--R.sup.s, --Y-cycloalkyl, --Y-heterocyclyl,
--Y--OR.sup.s, --Y--NR.sup.sR.sup.t, --Y--C(.dbd.O)R.sup.s,
--Y--C(O).sub.2R.sup.s, --Y--C(.dbd.O)NR.sup.sR.sup.t,
--Y--C(O).sub.2NR.sup.sR.sup.t, --Y--S(O).sub.0-2R.sup.s,
--Y--NRS(O).sub.2R.sup.s, --Y--S(O).sub.2NR.sup.sR.sup.t, and/or
--Y--NRC(.dbd.O)NR.sup.sR.sup.t, wherein Y is absent or a
C.sub.1-4alkylene group, R is hydrogen, lower alkyl, or
hydroxyC.sub.1-4alkyl, and R.sup.s and R.sup.t are independently
selected from hydrogen, alkyl, cycloalkyl, and heterocyclyl, except
when said substituent is --YS(O).sub.1-2R.sup.s or
--Y--NRS(O).sub.2R.sup.s, then R.sup.s in these instances is not
hydrogen. In each instance, each of R.sup.s and/or R.sup.t in turn
is optionally substituted with one to two of lower alkyl, halo,
cyano, hydroxy, alkoxy, amino, alkylamino, haloalkyl, haloalkoxy,
hydroxyalkyl, alkoxyalkyl, aminoalkyl, alkylaminoalkyl,
--SO.sub.2(alkyl), CO.sub.2H, CO.sub.2(alkyl), C(.dbd.O)H, and/or
C(.dbd.O)alkyl. Preferred substituents for substituted cycloalkyl
groups include -(alkylene).sub.n-hydroxy, -(alkylene).sub.n-lower
alkoxy, -(alkylene).sub.n-S(O).sub.2(lower alkyl), and
-(alkylene).sub.n-CO.sub.2(lower alkyl), where n is 0, 1, or 2.
[0040] The term "halo," "halide" or "halogen," when referring to a
substituent means fluoro, chloro, bromo, or iodo (preferably fluoro
or chloro).
[0041] The term "haloalkyl" means alkyl substituted with one or
more same or different halo atoms, e.g., --CH.sub.2Cl, --CF.sub.3,
--CH.sub.2CF.sub.3, --CH.sub.2CCl.sub.3, and the like, and further
includes those alkyl groups such as perfluoroalkyl in which all
alkyl hydrogen atoms are replaced by fluorine atoms.
[0042] The term "haloalkoxy" means a haloalkyl group as defined
above linked through an oxygen atom, e.g., it includes
--O--CH.sub.2Cl, --O--CF.sub.3, --O--CH.sub.2CF.sub.3,
--O--CH.sub.2CCl.sub.3, and the like.
[0043] The term "heteroalkyl" as used herein means an alkyl moiety
defined above, wherein one, two or three hydrogen atoms have been
replaced with a substituent independently selected from the group
consisting of --OR.sup.d, --NR.sup.dR.sup.e, and S(O).sub.nR.sup.d
(where n is an integer from 0 to 2), with the understanding that
the point of attachment of the heteroalkyl moiety is through a
carbon atom, wherein R.sup.d and R.sup.e are selected from
hydrogen, alkyl, substituted alkyl (but not including arylalkyl or
heteroarylalkyl), cycloalkyl, substituted cycloalkyl, heterocyclyl,
and substituted heterocyclyl, except when R.sup.d is attached to
S(O).sub.nR.sup.d and n is 1 or 2, then R.sup.d is not hydrogen.
Additionally, when R.sup.d and R.sup.e are attached to the same
nitrogen atom, they may be taken together to form an
optionally-substituted heterocyclyl or heteroaryl ring.
Representative examples include, but are not limited to,
2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxymethylethyl,
2,3-dihydroxypropyl, 1-hydroxymethylethyl, 3-hydroxybutyl,
2,3-dihydroxybutyl, 2-hydroxy-1-methylpropyl, 2-aminoethyl,
3-aminopropyl, 2-methylsulfonylethyl, aminosulfonylmethyl,
aminosulfonylethyl, methylaminosulfonylmethyl,
methylaminosulfonylethyl, methylaminosulfonylpropyl,
2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxymethylethyl,
2,3-dihydroxypropyl, and so forth.
[0044] "Heterocyclo," "heterocyclyl," or "heterocyclic" refers to a
saturated or partially-unsaturated non-aromatic monocyclic or
bicyclic moiety in which one or two ring atoms are heteroatoms
selected from N, O, or S(O).sub.n (where n is an integer from 0 to
2), the remaining ring atoms being carbon atoms, and additionally,
one or two carbon atoms may optionally contain a carbonyl oxygen
group, e.g., one or two atoms in the ring may be a moiety of the
formula --C(.dbd.O)--. Thus, the term heterocyclyl includes rings
such as tetrahydropyranyl, tetrahydrofuryl, piperidinyl,
piperazinyl, morpholinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl,
imidazolinyl, and the like. In the case of a bicyclic heterocyclyl,
one of the two rings may be a non-aromatic carbocyclic ring with
the point of attachment, however, being to the heterocyclic
ring.
[0045] A "substituted heterocyclyl" or "substituted heterocycle"
refers to a heterocyclyl group as defined above having one, two, or
three substituents (preferably one to two substituents) selected
from the group consisting of halo, haloalkyl, haloalkoxy, cyano,
nitro, --Y--R.sup.s, --Y-cycloalkyl, --Y-heterocyclyl,
--Y--OR.sup.s, --Y--NR.sup.sR.sup.t, --Y--C(.dbd.O)R.sup.s,
--Y--C(O).sub.2R.sup.s, --Y--C(.dbd.O)NR.sup.sR.sup.t,
--Y--C(O).sub.2NR.sup.sR.sup.t, --Y--S(O).sub.0-2R.sup.s,
--Y--NRS(O).sub.2R.sup.s, --Y--S(O).sub.2NR.sup.sR.sup.t, and/or
--Y--NRC(.dbd.O)NR.sup.sR.sup.t, wherein Y, R, R.sup.s and R.sup.t
are as defined above for substituted cycloalkyl groups, such that
R.sup.s and R.sup.t are, in turn, at each instance independently
optionally substituted with one to two further groups as recited
above in the definition for substituted cycloalkyl. Preferred
substituents for substituted heterocyclyl groups include
-(alkylene).sub.n-hydroxy, -(alkylene).sub.n-lower alkoxy,
-(alkylene).sub.n-S(O).sub.2(lower alkyl), and
-(alkylene).sub.n-CO.sub.2(lower alkyl), where n is 0, 1, or 2.
[0046] "Heteroaryl" means a monovalent, monocyclic aromatic moiety
of 5 to 6 ring atoms containing one, two, three, or four ring
heteroatoms, each independently selected from N, O, or S, the
remaining ring atoms being carbon, and it also includes such rings
having a second ring fused thereto of five to six ring atoms,
wherein the second fused ring may be aromatic or non-aromatic and
may be carbocyclic, heterocyclic, or a heteroaryl ring, with the
understanding, however, that in such cases the point of attachment
will be to an aromatic ring containing at least one heteroatom.
Thus, the term heteroaryl includes, but is not limited to, pyridyl,
furyl, thio-phenyl, thiazolyl, isothiazolyl, triazolyl, imidazolyl,
isoxazolyl, pyrrolyl, pyrazolyl, pyrimidinyl, benzofuryl,
isobenzofuryl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl,
indolyl, isoindolyl, benzoxazolyl, quinolyl, isoquinolyl,
benzimidazolyl, benzisoxazolyl, benzothiophenyl, dibenzo-furan, and
benzodiazepin-2-one-5-yl, and derivatives thereof.
[0047] A "substituted heteroaryl" is a heteroaryl ring as defined
above having one, two or three (preferably one or two) substituents
selected from the group consisting of halo, haloalkyl, haloalkoxy,
cyano, nitro, --Y--R.sup.P, --Y-aryl, --Y-heteroaryl,
--Y-cycloalkyl, --Y-heterocyclyl, --Y--OR.sup.p,
--Y--NR.sup.pR.sup.q, --Y--C(.dbd.O)R.sup.p,
--Y--C(O).sub.2R.sup.p, --Y--C(.dbd.O)NR.sup.pR.sup.q,
--Y--C(O).sub.2NR.sup.pR.sup.q, --Y--S(O).sub.0-2R.sup.P,
--Y--NRS(O).sub.2R.sup.q, --Y--S(O).sub.2NR.sup.pR.sup.q, and/or
--Y--NRC(.dbd.O)NR.sup.pR.sup.q, wherein Y, R, R.sup.p and R.sup.q
are as defined above for substituted aryl groups, such that R.sup.p
and R.sup.q are, in turn, at each instance independently optionally
substituted with one to two further substituents as recited above
in the definition for substituted aryl. Preferred substituents for
substituted heteroaryl groups include alkyl, haloalkyl,
heterocyclyl, halo, nitro, cyano, and -(alkylene).sub.n-CO.sub.2R
(where n is 0 or 1 and R is hydrogen or alkyl).
[0048] "Leaving group" has the meaning conventionally associated
with it in synthetic organic chemistry, i.e., an atom or a group
capable of being displaced by a nucleophile and includes halo (such
as chloro, bromo, and iodo), alkanesulfonyloxy, arenesulfonyloxy,
alkylcarbonyloxy (e.g., acetoxy), arylcarbonyloxy, mesyloxy,
tosyloxy, trifluoromethanesulfonyloxy, aryloxy (e.g.,
2,4-dinitrophenoxy), methoxy, N,O-dimethylhydroxylamino, and the
like.
[0049] "Optional" or "optionally" means that the subsequently
described event may but need not occur, and it includes instances
where the event occurs and instances in which it does not. For
example, "optionally-substituted cycloalkyl" refers to both
cycloalkyl groups and substituted cycloalkyl groups, as defined
above. When the term "optionally-substituted" precedes a number of
different types of rings in one line or string, e.g., as in
"optionally-substituted cycloalkyl or heterocyclyl", or
"optionally-substituted carbocyclic or heterocyclic ring," or
"optionally-substituted aryl, heteroaryl, cycloalkyl, or
heterocyclyl," it is intended that the term
"optionally-substituted" modifies each of the rings identified in
the line or string.
[0050] When the term "optionally-substituted" is used with respect
to a particularly-named cyclic group, such as
"optionally-substituted cyclohexyl," or "optionally-substituted
piperidinyl," it should be understood that the optional
substituents for such particularly-named rings may be selected from
the group of substituents recited above with respect to which the
genus of which the particularly-named group is a member. Thus, for
example, an "optionally-substituted cyclohexyl" may be an
unsubstituted cyclohexyl or a cyclohexyl group having one, two, or
three substituents selected from those recited above for
substituted cycloalkyl.
[0051] When reference is made herein to the C5, C6, 7-aza, or C8
carbon atoms of the quinazoline ring, the numbering of the ring
atoms is intended to be as follows: ##STR3##
[0052] "Pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic and neither biologically nor otherwise
undesirable. The term includes excipients that are acceptable for
veterinary use as well as human pharmaceutical use. A
"pharmaceutically acceptable excipient" as used in the
specification and claims includes both one and more than one such
excipient.
[0053] "Pharmaceutically acceptable salt" of a compound means a
salt that is generally safe, non-toxic and neither biologically nor
otherwise undesirable and that possesses the desired
pharmacological activity of the parent compound. Such salts
include: (1) acid addition salts, formed with inorganic acids such
as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid, and the like; or formed with organic acids such as
acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic
acid, glycolic acid, pyruvic acid, lactic acid, malonic acid,
succinic acid, malic acid, maleic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid,
cinnamic acid, mandelic acid, methanesulfonic acid, ethane-sulfonic
acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid,
benzenesulfonic acid, 4-chlorobenzenesulfonic acid,
2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic
acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1carboxylic acid,
glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic
acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary
butyl-acetic acid, lauryl sulfuric acid, gluconic acid, glutamic
acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic
acid, and the like; or (2) salts formed when an acidic proton
present in the parent compound either is replaced by a metal ion,
e.g., an alkali metal ion, an alkaline earth ion, or an aluminum
ion; or coordinates with an organic base such as ethanolamine,
diethanolamine, triethanolamine, tromethamine, N-methylglucamine,
and the like.
[0054] The terms "pro-drug" and "prodrug" are used interchangeably
herein and refer to any compound which releases an active parent
drug according to Formula I in vivo when such prodrug is
administered to a mammalian subject. Prodrugs of a compound of
Formula I are prepared by modifying one or more functional group(s)
present in the compound of Formula I in such a way that the
modification(s) may be cleaved in vivo to release the parent
compound. Prodrugs include compounds of Formula I wherein a
hydroxy, amino, or sulfhydryl group in a compound of Formula I is
bonded to any group that may be cleaved in vivo to regenerate the
free hydroxyl, amino, or sulfhydryl group, respectively. Examples
of prodrugs include, but are not limited to, esters (e.g., acetate,
formate, and benzoate derivatives), carbamates (e.g.,
N,N-di-methylaminocarbonyl) of hydroxy functional groups in
compounds of Formula I, and the like.
[0055] "Protecting group" refers to an atom or group of atoms that
is attached to a reactive group in a molecule and masks, reduces,
or prevents the reactivity of the group to which it is attached.
Examples of protecting groups can be found in Green and Wuts,
Protective Groups in Organic Chemistry (Wiley, 2.sup.nd ed. 1991),
and Harrison and Harrison et al., Compendium of Synthetic Organic
Methods, Vols. 1-8 (John Wiley and Sons, 1971-1996). Representative
amino protecting groups include formyl, acetyl, trifluoroacetyl,
benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl (Boc),
trimethyl silyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES),
trityl and substituted trityl groups, allyloxycarbonyl,
9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl
(NVOC), and the like. Representative hydroxy protecting groups
include those where the hydroxy group is either acylated or
alkylated such as with benzyl or lower alkyl and trityl ethers as
well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl
ethers, and allyl ethers.
[0056] "Treating" or "treatment" of a disease includes: (1)
preventing the disease, i.e., causing the clinical symptoms of the
disease not to develop in a mammal that may be exposed to or
predisposed to the disease but does not yet experience or display
symptoms of the disease; (2) inhibiting the progression of the
disease, i.e., arresting or reducing the development of the disease
or its symptoms; and (3) relieving the disease, i.e., causing
regression of the disease or its symptoms.
[0057] "A therapeutically effective amount" means the amount of a
compound that, when administered to a mammal for treating a
disease, is sufficient to effect a treatment for the disease. The
"therapeutically effective amount" will vary depending on such
factors as the compound being administered, the type of disease
being treated, the progression or severity of the disease state,
and the age, weight, and general health of the mammal being
treated.
[0058] Compounds that have the same molecular formula but differ in
the nature or sequence of bonding of their atoms or the arrangement
of their atoms in space are termed "isomers." Isomers that differ
in the arrangement of their atoms in space are termed
"stereoisomers." Stereoisomers that are not mirror images of one
another are termed "diastereomers," and those that are
non-superimposable mirror images of each other are termed
"enantiomers." When a compound has an asymmetric center, for
example, if a carbon atom is bonded to four different groups, a
pair of enantiomers is possible. An enantiomer can be characterized
by the absolute configuration of its asymmetric center and is
described by the (R) and (S) sequencing rules of Cahn and Prelog,
or by the manner in which the molecule rotates the plane of
polarized light and designated as dextrorotatory or levorotatory
(i.e., as (+) or (-)-isomers respectively). A chiral compound can
exist as either an individual enantiomer or as a mixture thereof. A
mixture containing different enantiomers is called a "racemic
mixture."
[0059] The compounds of this invention may possess one or more
asymmetric centers; such compounds can therefore be produced as
individual (R)-- or (S)-stereoisomers or as mixtures thereof.
Unless indicated otherwise, the description or naming of a
particular compound in the specification and claims is intended to
include both individual enantiomers and mixtures (racemic or
otherwise) thereof. The methods for the determination of
stereochemistry and the separation of stereoisomers are well-known
in the art (see March, Advanced Organic Chemistry, Chap. 4, 4th
edition, John Wiley and Sons, New York [1992]).
Preferred Embodiments
[0060] While the definition of this invention is set forth in the
Summary of the Invention, certain compounds of Formula (I) are
preferred.
[0061] Preferred compounds according to the invention are those
having Formula (Ip), ##STR4##
[0062] and isomers, prodrugs, and pharmaceutically-acceptable salts
thereof, wherein: [0063] R is optionally-substituted aryl; [0064] Q
is alkyl substituted with one, two or three of --OR.sup.8,
--SR.sup.8, --C(.dbd.O)R.sup.8, --C(O).sub.2R,
--C(.dbd.O)NR.sup.8R.sup.9, --S(O).sub.pR.sup.10,
--C(O).sub.2NR.sup.8R.sup.9, --S(O).sub.2NR.sup.8R.sup.9,
--NR.sup.8R.sup.9, cycloalkyl, substituted cycloalkyl,
heterocyclyl, and/or substituted heterocyclyl; [0065] R.sup.8 and
R.sup.9 are independently selected from hydrogen, alkyl,
hydroxyalkyl, alkoxyalkyl, cycloalkyl, substituted cycloalkyl,
heterocyclyl, and substituted heterocyclyl; [0066] R.sup.10 is
alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or
substituted heterocyclyl; and [0067] p is 1 or2.
[0068] More preferred are compounds of Formula (Ip), as immediately
defined above, wherein R is ##STR5## and R.sup.4 and R.sup.5 are
both halogen.
[0069] According to one aspect of the invention, preferred
compounds are compounds of Formula (Ip), as immediately defined
above, wherein Q is selected from C.sub.1-6alkyl or C.sub.1-6alkyl
substituted with one to two of hydroxy, --O(C.sub.1-4alkyl),
--S(O).sub.2(C.sub.1-4alkyl), C.sub.3-7cycloalkyl, and/or a five to
six membered monocyclic heterocyclic ring, wherein each of said
rings is optionally-substituted with up to one of R.sup.12 and/or
up to one of R.sup.14; and R.sup.12 and R.sup.14 are independently
selected where valence allows from C.sub.1-4alkyl, hydroxy, oxo
(.dbd.O), --O(C.sub.1-4alkyl), --C(.dbd.O)H,
--C(.dbd.O)(C.sub.1-4alkyl), --C(O).sub.2H,
--C(O).sub.2(C.sub.1-4alkyl), and --S(O).sub.2(C.sub.1-4alkyl).
[0070] According to another aspect of the invention, preferred
compounds are compounds of Formulae (I) and (Ip), wherein Q is an
alkyl or substituted alkyl having the formula
--C(R.sup.1R.sup.2R.sup.3), wherein R.sup.1, R.sup.2 and R.sup.3
are selected from hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl,
--(C.sub.1-4-alkylene)--S(O).sub.pR.sup.10, and
--(C.sub.1-4alkylene)-C(O).sub.2R.sup.8 (wherein R.sup.8 and
R.sup.10 are lower alkyl).
[0071] According to another aspect of the invention, preferred
compounds are compounds of Formulae (I) and (Ip), where Q is an
optionally-substituted C.sub.3-7cycloalkyl,
C.sub.3-7cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl. More
preferred are compounds where Q is a C.sub.1-4alkyl substituted
with an optionally-substituted cyclohexyl, piperidin-4-yl, or
tetrahydropyran-4-yl. Preferably, said cycloalkyl and heterocyclic
groups are unsubstituted or in turn, are substituted with OH,
--O(C.sub.1-4alkyl), --C(O).sub.2(C.sub.1-4alkyl) and/or
--S(O).sub.2(C.sub.1-4alkyl), more preferably with one of
--C(O).sub.2(Et) or --S(O).sub.2(CH.sub.3).
[0072] In compounds of Formula (I) and (Ip), preferably R is
di-substituted phenyl, more preferably 2,4-disubstituted phenyl,
and even more preferably 2,4-dihalosubstituted phenyl. Most
preferred are compounds where R is 2,4-difluorophenyl.
[0073] Another group of preferred compounds are those having the
formula, ##STR6## wherein Q is alkyl, cycloalkyl, substituted
cycloalkyl, heterocyclyl, substituted heterocyclyl, or
C.sub.1-6alkyl substituted with one, two or three of halogen,
cyano, haloalkoxy, --OR.sup.8, --SR.sup.8, --S(O).sub.pR.sup.10,
--C(O).sub.2R.sup.8, cycloalkyl, substituted cycloalkyl,
heterocyclyl and/or substituted heterocyclyl, wherein R.sup.8 is
selected from hydrogen, alkyl, and optionally-substituted
cycloalkyl or heterocyclyl, and R.sup.10 is alkyl or
optionally-substituted cycloalkyl or heterocyclyl.
[0074] Another group of preferred compounds are those having the
formula: ##STR7##
[0075] wherein: [0076] R.sup.4 and R.sup.5 are both halogen (more
preferably fluoro); [0077] X is --O--, --C(.dbd.O)--,
--N(R.sup.12a)--, or --CH(R.sup.12b)--; [0078] R.sup.12a is
selected from hydrogen, C.sub.1-4alkyl, --C(.dbd.O)R.sup.15,
--C(O).sub.2R.sup.15, and --S(O).sub.2(C.sub.1-4alkyl); [0079]
R.sup.12b is selected from hydrogen, C.sub.1-4alkyl, --OR.sup.15,
--C(.dbd.O)R.sup.15, --C(O).sub.2R.sup.15, and
--S(O).sub.2(C.sub.1-4alkyl); [0080] R.sup.14 is selected from
C.sub.1-4alkyl, oxo (.dbd.O), --OR.sup.15, --C(.dbd.O)R.sup.15,
--C(O).sub.2R.sup.15, and --S(O).sub.2(C.sub.1-4alkyl); [0081]
R.sup.15 is at each occurrence selected from hydrogen and
C.sub.1-4alkyl; [0082] q is 0 or 1; and [0083] r is 0, 1 or2.
[0084] Even more preferred are compounds as immediately defined
above, wherein X is --NR.sup.12a--, R.sup.12a is
S(O).sub.2(C.sub.1-4alkyl), and q is 1.
Utility
[0085] Compounds of Formula I are useful for the treatment of any
disorder or disease state in a human, or other mammal, which is
exacerbated or caused by excessive or unregulated proinflammatory
cytokines (i.e., TNF, IL-1, etc.), or p38 kinase activation by such
mammal. Compounds of Formula I inhibit p38 kinase in in vitro
assays and inhibit TNF-.alpha. or IL-1.beta. release in cell based
assays.
[0086] In view of their activity as inhibitors of p38 kinase, the
compounds of the invention are useful for treating inflammatory
diseases, autoimmune diseases, destructive bone disorders,
proliferative disorders, angiogenic disorders, infectious diseases,
neurodegenerative diseases, and viral diseases. Compounds of the
invention are useful in treating arthritis, including but not
limited to rheumatoid arthritis, spondylitis, gouty arthritis,
osteoarthritis, systemic lupus erythematosus (SLE), juvenile
arthritis, and other arthritic conditions. In addition, compounds
of the present invention are useful in treating pulmonary disorders
or lung inflammation, including adult respiratory distress
syndrome, pulmonary sarcoidosis, asthma, silicosis, and chronic
obstructive pulmonary disease. Furthermore, compounds of the
present invention are useful in treating viral and bacterial
infections, including sepsis, septic shock, gram negative sepsis,
malaria, meningitis, cachexia secondary to infection or malignancy,
cachexia secondary to acquired immune deficiency syndrome (AIDS),
AIDS, ARC (AIDS related complex), pneumonia, and herpes virus.
Moreover, compounds of the present invention are useful in the
treatment of bone resorption diseases, such as osteoporosis,
endotoxic shock, toxic shock syndrome, reperfusion injury,
autoimmune disease including graft vs. host reaction and allograft
rejections, cardiovascular diseases including myocardial
infarction, atherosclerosis, thrombosis, congestive heart failure,
cardiac reperfusion injury, renal reperfusion injury, liver disease
and nephritis, and myalgias due to infection.
[0087] The compounds of the present invention are also useful for
the treatment of influenza, diabetes, skin-related conditions such
as psoriasis, eczema, burns, dermatitis, keloid formation, and scar
tissue formation. Compounds of the present invention are also
useful in treating gastrointestinal conditions such as inflammatory
bowel disease, Crohn's disease, gastritis, irritable bowel
syndrome, and ulcerative colitis. The compounds of the present
invention can also be used in treating ophthalmic diseases, such as
retinitis, retinopathies, uveitis, ocular photophobia, and acute
injury to the eye tissue. Compounds of the invention also would be
useful for treatment of cancer and angiogenesis, including
neoplasia and metastasis; ophthalmological conditions such as
corneal graft rejection, ocular neovascularization, retinal
neovascularization including neovascularization following injury or
infection, diabetic retinopathy, retrolental fibroplasia and
neovascular glaucoma; ulcerative diseases such as gastric ulcer;
pathological, but non-malignant conditions such as hemangiomas,
including infantile hemangiomas, angiofibroma of the nasopharynx
and avascular necrosis of bone; diabetic nephropathy and
cardiomyopathy; and disorders of the female reproductive system
such as endometriosis.
[0088] The compounds of the present invention are also useful in
treating central nervous system disorders, such as Alzheimer's
disease, multiple sclerosis, and depression.
[0089] In addition, compounds of the present invention are also
useful for preventing the production of cyclooxygenase-2 and thus
are useful in treating those diseases responsive to inhibition of
COX-2, such as fever, edema, and pain, including headache,
neuromuscular pain, dental pain, arthritic pain and pain caused by
cancer.
[0090] Besides being useful for human treatment, compounds of the
present invention are also useful for veterinary treatment of
animals such as companion, exotic, and farm animals, including
mammals, rodents, and the like. More preferred animals include
horses, dogs, and cats.
[0091] Furthermore, compounds of the present invention can be used
in co-therapies, partially or completely, in place of other
conventional anti-inflammatory agents, such as together with
steroids, cyclooxygenase-2 inhibitors, NSAIDs, aspirin, DMARDS,
immunosuppressive agents, 5-lipoxygenase inhibitors, LTB.sub.4
antagonists and LTA.sub.4 hydrolase inhibitors.
[0092] As used herein, the term "TNF mediated disorder" refers to
any and all disorders and disease states in which TNF plays a role,
either by control of TNF itself, or by TNF causing another monokine
to be released, such as but not limited to IL-1, IL-6 or IL-8. A
disease state in which, for instance, IL-1 is a major component,
and whose production or action, is exacerbated or secreted in
response to TNF, would therefore be considered a disorder mediated
by TNF.
[0093] As used herein, the term "p38 mediated disorder" refers to
any and all disorders and disease states in which p38 plays a role,
either by control of p38 itself, or by p38 causing another factor
to be released, such as but not limited to IL-1, IL-6 or IL-8. A
disease state in which, for instance, IL-1 is a major component,
and whose production or action is exacerbated or secreted in
response to p38, would therefore be considered a disorder mediated
by p38.
[0094] As TNF-.beta. has close structural homology with TNF-.alpha.
(also known as cachectin), and since each induces similar biologic
responses and binds to the same cellular receptor, the synthesis of
both TNF-.alpha. and TNF-.beta. are inhibited by the compounds of
the present invention. When reference herein is made generally to
"TNF" inhibition, this is intended to encompass both TNF-.alpha.
and TNF-.beta. inhibition, unless specifically delineated
otherwise.
Abbreviations
[0095] For ease of reference, the following abbreviations are used
in the general synthetic schemes and Examples below: [0096]
EtOH=ethanol [0097] MeOH=methanol [0098] EtOAc=ethyl acetate [0099]
DCE=1,2-dichloroethane [0100] DCM=dichloromethane [0101]
DMF=dimethylformamide [0102] NaOH=sodium hydroxide [0103]
NMP=1-methyl-2-pyrrolidinone [0104] Pd/C=palladium on carbon [0105]
TEA or Et.sub.3N=triethylamine [0106] TFA=trifluoroacetic acid
[0107] THF=tetrahydrofuran [0108] TLC=thin layer chromotography
[0109] Mp=melting point [0110] MW=molecular weight [0111] h=hour(s)
[0112] rt.=room temperature General Synthetic Schemes
[0113] The compounds of the present invention can be prepared in a
number of ways known to one skilled in the art. Preferred methods
include, but are not limited to, the general synthetic procedures
described below.
[0114] The starting materials and reagents used are either
available from commercial suppliers such as Aldrich Chemical Co.
(Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA), Enika
Chemie or Sigma (St. Louis, Mo., USA), Maybridge (Dist: Ryan
Scientific, P.O. Box 6496, Columbia, S.C. 92960), etc.; or they can
be prepared by methods known to those skilled in the art following
procedures set forth in the literature. These schemes are merely
illustrative; various modifications to these schemes can be made
and will be apparent to one skilled in the art.
[0115] The starting materials and the intermediates of the reaction
may be isolated and purified if desired using conventional
techniques, including but not limited to filtration, distillation,
crystallization, chromatography, and the like. Such materials may
be characterized using conventional means, including physical
constants and spectral data. In the Schemes, the variables Q, R,
R.sup.6, R.sup.7 etc., are defined as set forth in the Summary of
Invention and claims. ##STR8##
[0116] Compound (2) can be prepared from
2-chloro-4-methyl-5-nitropyridine (1) by treatment with an oxidant
such as sodium dichromate dihydtrate in concentrated sulfuric acid,
followed by esterification with an appropriate agent such as
trimethylsilyldiazo methane. Compound (2) can be converted to the
desired 2-alkoxy or aryloxy 5-nitro-isonicotinic acid methyl ester
(3) by treatment with an appropriate alcohol ROH and base such as
sodium hydride. Treatment of compound (3) with hydrogen gas
provides compound (4), which when reacted with urea provides
pyridopyrimidine-2,4-dione (5). Compound (5) can be reacted with
phosphorus oxychloride and N,N-diethyl aniline to provide dichloro
compound (6), which upon treatment with ammonia in an appropriate
solvent such as MeOH provides compound (7), which in turn can be
converted to compound (8) upon treatment with tert-butylnitrite in
solvent such as THF. Coupling of compound (8) with an
appropriately-substituted amine NH.sub.2-Q in solvent such as NMP
provides compounds of formula (I). ##STR9##
[0117] Compounds of general formula (I) can be converted to the
corresponding N-oxides of formula (II) by treatment with an
appropriate oxidizing agent such as dimethyl dioxirane, urea
peroxide, or methyltrioxorhenium/H.sub.2O.sub.2.
EXAMPLE 1
[6-(2,4-Difluoro-phenoxy)-pyrido[3,4-d]pyrimidin-2-yl]-(1-methanesulfonyl--
piperidin-4-yl)-amine
[0118] ##STR10##
1A. 2-Chloro-5-nitro-isonicotinic acid methyl ester
[0119] ##STR11##
[0120] To a solution of 2-chloro-4-methyl-5-nitropyridine (Aldrich
Chemical Co.) (5.0 g, 29.0 mmol) in concentrated sulfuric acid (60
mL) with stirring at 0.degree. C., was added a solution of sodium
dichromate dihydrate (14.3 g, 1.65 eq) in concentrated sulfuric
acid (100 mL) at a rate which kept the internal temperature less
than 10.degree. C. After addition was complete, the reaction
mixture was stirred from 0.degree. C. to room temperature over 6
hours. By TLC, the reaction was not complete, so additional sodium
dichromate dihydrate (8.4 g, 1 eq) was added directly to the
solution at 0.degree. C. The resulting solution was stirred from
0.degree. C. to room temperature overnight. The reaction was
complete by TLC the next morning, and it was poured into a mixture
of ice (1 L) and EtOAc (900 mL). The resulting mixture was stirred
for about 10 minutes and then the layers were separated. The
organic layer was washed with brine (2.times.600 mL), and the EtOAc
layer was dried over magnesium sulfate and filtered. With stirring
at room temperature, trimethylsilyl diazomethathane (2.0M in
hexanes, 15 mL) was added to the EtOAc filtrate. The resulting
mixture was stirred at room temperature for 30 min. By TLC, there
was no acid remaining, and then MeOH (150 mL) was added and the
solution stirred for 30 minutes. The solution was concentrated and
then EtOAc (700 mL) was added to the residue which was dried over
magnesium sulfate, filtered, concentrated and pumped to give the
title compound (1A) as an off-white powder (5.79 g, M.sup.+=216,
M.P.=60.5-64.1.degree. C.).
1B. 2-(2,4-Difluoro-phenoxy)-5-nitro-isonicotinic acid methyl
ester
[0121] ##STR12##
[0122] To 2,4 difluorophenol (Aldrich Chemical Co.) (2.68 mL, 1.1
eq) in THF (60 mL) at 0.degree. C. was added sodium hydride (60% in
oil) (1.123 g, 1.1 eq), and the resulting mixture was stirred from
0.degree. C. to room temperature over 1 hour. This solution was
then added to compound (1A) (5.79 g, 26.7 mmol), and the resulting
mixture was heated at reflux for 2 hours. TLC analysis confirmed
that the reaction was complete, and the mixture was cooled to room
temperature. EtOAc (300 mL) and water (100 mL) were added, the
resulting mixture was partitioned, and the layers were separated.
The organic layer was washed with water (3.times.100 mL) and brine
(1.times.100 mL). Then the EtOAc layer was dried over magnesium
sulfate, filtered, concentrated, and pumped to give the titled
compound (1B) as an off-white powder (7.99 g, M.sup.+=310,
Mp=90.2-91.4.degree. C.)
1C. 5-Amino-2-(2,4-difluoro-phenoxy)-isonicotinic acid methyl
ester
[0123] ##STR13##
[0124] Compound (1B) (7.99 g, 25.6 mmol) was taken up in EtOH (350
mL), and nitrogen gas was bubbled through the solution for five
minutes before 10% Pd/C (1.6 g) was carefully added. The resulting
mixture was put on the Parr Shaker under 60 psi hydrogen gas for 4
hours. By TLC, there was no starting material remaining. The
reaction mixture was filtered through a 3 cm bed of celite, and the
filtrate was concentrated to give 7.0 g of crude product.
Purification by column chromatography eluting on 130 g of silica
gel with a gradient of 5% EtOAc in hexanes through 10% EtOAc in
hexanes afforded the titled compound (IC) as an off-white powder
(4.223 g, (M+H).sup.+=281, Mp=102.0-103.9.degree. C.).
1D. 6-(2,4
-Difluoro-phenoxy)-1H-pyrido[3,4-d]pyrimidine-2,4-dione
[0125] ##STR14##
[0126] Compound (IC) (4.2 g, 15.0 mmol) and urea (9.0 g, 10 eq)
were heated to a melt at 160.degree. C. with stirring. After 2
hours, the melt solidified and by TLC the reaction was complete.
The reaction mixture was cooled to room temperature, water (250 mL)
was added, and the resulting mixture was left to stand for 1 hour.
Then the solids were broken up with a spatula and the mixture was
stirred vigorously for 1 hour. After suction filtration, a tan
powder was collected which was air-dried in the frit for two days.
Drying under high vacuum at 56.degree. C. gave the title compound
(1D) as a tan powder (4.307 g, (M-H).sup.-=290, M.P.>300.degree.
C.).
1E.
2,4-Dichloro-6-(2,4-difluoro-phenoxy)-pyrido[3,4-d]pyrimidine
[0127] ##STR15##
[0128] Compound (1D) (4.2 g, 14.4 mmol) was taken up in phosphorus
oxychloride (55 mL), and then N,N-diethyl aniline (1.34 mL, 0.6 eq)
was added. The resulting mixture was heated to reflux with stirring
for 2 hours. TLC analysis confirmed that the reaction was complete.
The reaction mixture was concentrated under reduced pressure at
55.degree. C. and then EtOAc (600 mL) was added to the residue. At
0.degree. C., sodium carbonate.sub.(aq) (pH=12) solution (300 mL)
was slowly added, and then the resulting mixture was stirred for 20
minutes. The layers were separated, then another 300 mL of sodium
carbonate.sub.(aq) (pH=12) solution was added, and the resulting
mixture was stirred for 15 minutes. The combined aqueous layers
were extracted with EtOAc (1.times.200 mL), and the combined
organic layers were finally washed with brine (1.times.300 mL). The
organic layer was dried over magnesium sulfate, filtered and
concentrated. The crude product was purified by column
chromatography eluting on 130 g of silica gel with 3% EtOAc in
hexanes to give the title compound (1E) as an off-white powder
(3.584 g, M.sup.+=327, Mp=144.3-146.2.degree. C.)
1F. 2-Chloro-6-(2,4-difluoro-phenoxy)-pyrido[3,
4-d]pyrimidine-4-ylamine
[0129] ##STR16##
[0130] Compound (1E) (1.75 g, 5.33 mmol) was placed in a
sealed-tube, and 7 N ammonia in MeOH (Aldrich Chemical Co.) (30 mL)
was added. The tube was tightly capped, and the resulting
homogeneous mixture was stirred at room temperature overnight. The
next morning, TLC confirmed that the reaction was complete. The
mixture was transferred to a round-bottom flask and concentrated
under reduced pressure at 55.degree. C. The residue was taken up in
EtOAc (700 mL) and water (250 mL). The layers were partitioned and
then separated. The organic layer was washed with water
(2.times.250 mL) and brine (1.times.250 mL). The organic layer was
dried over magnesium sulfate, filtered, concentrated, and pumped to
give the title compound (1F) as an off-white powder (1.859 g,
(M-H).sup.-=307, Mp>300.degree. C.).
1G. 2-Chloro-6-(2,4-difluoro-phenoxy)-pyrido[3,4-d]pyrimidine
[0131] ##STR17##
[0132] Compound (1F) (1.8 g, 5.83 mmol) was taken up in THF (100
mL) and tert-butylnitrite (Aldrich Chemical Co.) was added. The
resulting mixture was heated to reflux with stirring for 48 hours
and additional tert-butyl nitrite was added in portions (total 5
mL). By TLC , the reaction was complete. After cooling to room
temperature, the solvent was removed under reduced pressure at
55.degree. C. EtOAc (180 mL) and water (60 mL) were added to the
residue, and the layers were partitioned and separated. The organic
layer was washed with water (3.times.60 mL) and brine (1.times.60
mL). After drying over magnesium sulfate, the organic layer was
filtered, concentrated and pumped to give a crude oil (3.14 g).
Purification by column chromatography eluting on 68 g of silica gel
with 5% EtOAc in hexanes afforded the title compound (1G) as a
yellow powder (0.546 g, M.sup.+=293).
1H.
[6-(2,4-Difluoro-phenoxy)-pyrido[3,4-d]pyrimidin-2-yl-(1-methanesulfon-
yl-piperidin-4-yl)-amine (Example 1)
[0133] Compound (1G) (200 mg, 0.68 mmol) and
1-methanesulfonyl-piperidin-4-ylamine (365 mg, 3 eq) in NMP were
heated at 90.degree. C. with stirring for 1 hour. By TLC, the
reaction was complete and allowed to cool to room temperature.
EtOAc (80 mL) and water (40 mL) were added, and the layers were
partitioned and separated. The organic layer was washed with water
(3.times.40 mL) and brine (1.times.40 mL). The EtOAc layer was then
dried over magnesium sulfate, filtered, and concentrated.
Purification by preparative TLC eluting with 5% MeOH in DCM
afforded the above-titled compound (Example 1) as a yellow powder
(0.185 g, (M+H).sup.+=436, Mp=209.9-211.9.degree. C.).
EXAMPLES 2-5
[0134] ##STR18##
[0135] Compounds having the Formula (1b) above, wherein Q has the
values set forth in Table 1, were prepared following the same or
similar method as described above for Example 1, except the
appropriately-substituted amine was used in place of
1-(methylsulfonyl)piperidin-4-yl amine in step 1H. TABLE-US-00001
TABLE 1 Ex. No. Q Compound Name MW 2 ##STR19## [6-(2,4-Difluoro-
phenoxy)-pyrido[3,4- d]pyrimidin-2-yl]- isopropyl-amine 316.31 3
##STR20## 3-[6-(2,4-Difluoro- phenoxy)-pyrido[3,4-
d]pyrimidin-2-ylamino]- pentane-1,5-diol 376.36 4 ##STR21##
[6-(2,4-Difluoro- phenoxy)-pyrido[3,4- d]pyrimidin-2-yl]-
(tetrahydro-pyran-4-yl)- amine 358.34 5 ##STR22##
4-[6-(2,4-Difluoro- phenoxy)-pyrido[3,4- d]pyrimidin-2-ylamino]-
cyclohexanol 372.37
EXAMPLE 6
[0136] The following are representative pharmaceutical formulations
containing a compound of Formula (I) or (II).
Tablet Formulation
[0137] The following ingredients are mixed intimately and pressed
into single scored tablets. TABLE-US-00002 Ingredient tablet, mg
Quantity per compound of this invention 400 cornstarch 50
croscarmellose sodium 25 lactose 120 magnesium stearate 5
Capsule Formulation
[0138] The following ingredients are mixed intimately and loaded
into a hard-shell gelatin capsule. TABLE-US-00003 Ingredient
capsule, mg Quantity per compound of this invention 200 lactose,
spray-dried 148 magnesium stearate 2
Suspension Formulation
[0139] The following ingredients are mixed to form a suspension for
oral administration. TABLE-US-00004 Ingredient Amount compound of
this invention 1.0 g fumaric acid 0.5 g sodium chloride 2.0 g
methyl paraben 0.15 g propyl paraben 0.05 g granulated sugar 25.5 g
Sorbitol (70% solution) 12.85 g Veegum K (Vanderbilt Co.) 1.0 g
flavoring 0.035 ml colorings 0.5 mg distilled water q.s. to 100
ml
Injectable Formulation
[0140] The following ingredients are mixed to form an injectable
formulation. TABLE-US-00005 Ingredient Amount compound of this
invention 0.2 g sodium acetate buffer solution, 0.4 M 2.0 ml HCl
(1N) or NaOH (1N) q.s. to suitable pH water (distilled, sterile)
q.s. to 20 ml
[0141] All of the above ingredients, except water, are combined and
heated to 60-70.degree. C. with stirring. A sufficient quantity of
water at 60.degree. C. is then added with vigorous stirring to
emulsify the ingredients, and water then added q.s. to 100 g.
Suppository Formulation
[0142] A suppository of total weight 2.5 g is prepared by mixing
the compound of the invention with Witepsolg H-15 (triglycerides of
saturated vegetable fatty acid; Riches-Nelson, Inc., New York), and
has the following composition: TABLE-US-00006 compound of the
invention 500 mg Witepsol .RTM. H-15 balance
EXAMPLE 7
Inhibition of p38 (MAP) Kinase-In Vitro Assay
[0143] The p-38 MAP kinase inhibitory activity of compounds of this
invention in vitro was determined by measuring the transfer of the
.gamma.-phosphate from .gamma.-.sup.33P-ATP by p38 kinase to Myelin
Basic Protein (MBP), using the a minor modification of the method
described in Ahn et al., J. Biol. Chem., Vol. 266 (7), 4220-4227
(1991).
[0144] The phosphorylated form of the recombinant p38 MAP kinase
was expressed with SEK-1 and MEKK in E. Coli (see Khokhlatchev et
al., J Biol. Chem. Vol. 272(17), pp. 11057-11062 (1997)), and then
purified by affinity chromatography using a Nickel column.
[0145] The phosphorylated p38 MAP kinase was diluted in kinase
buffer (20 mM 3-(N-morpholino)propanesulfonic acid, pH 7.2, 25 mM
.beta.-glycerol phosphate, 5 mM ethylene glycol-bis(beta-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 1 mM sodium vanadate, 1 mM
dithio-threitol, 40 mM magnesium chloride). Test compound dissolved
in DMSO or only DMSO (control) was added, and the samples were
incubated for 10 min. at 30.degree. C. The kinase reaction was
initiated by the addition of a substrate cocktail containing MBP
and .gamma.-.sup.33P-ATP. After incubating for an additional 20
min. at 30.degree. C., the reaction was terminated by adding 0.75%
phosphoric acid. The phosphorylated MBP was then separated from the
residual .gamma.-.sup.33P-ATP using a phosphocellulose membrane
(Millipore, Bedfrod, Mass.) and quantitated using a scintillation
counter (Packard, Meriden, Conn.).
EXAMPLE 8
[0146] This example illustrates an in vitro assay to evaluate the
inhibition of LPS-induced TNF-.alpha. production in THP1 cells.
[0147] The ability of the compounds of this invention to inhibit
the TNF-.alpha. release was determined using a minor modification
of the methods described in Blifeld, et al. Transplantation,
51:498-503 (1991).
[0148] (a) Induction of TNF Biosynthesis:
[0149] THP-1 cells were suspended in culture medium [RPMI
(Gibco-BRL, Gailthersburg, Md.) containing 10% fetal bovine serum,
0.02 mM 2-mercaptoethanol], at a concentration of
2.5.times.10.sup.6 cells/mL and then plated in 96 well plate (0.2
mL aliquots in each well). Test compounds were dissolved in DMSO
and then diluted with the culture medium such that the final DMSO
concentration was 5%. Twenty five .mu.L aliquots of test solution
or only medium with DMSO (control) were added to each well. The
cells were incubated for 30 min., at 37.degree. C. LPS (Sigma, St.
Louis, Mo.) was added to the wells at a final concentration of 0.5
.mu.g/ml, and cells were incubated for an additional 2 h. At the
end of the incubation period, culture supernatants were collected
and the amount of TNF-.alpha. present was determined using an ELISA
assay as described below.
[0150] (b) ELISA Assay:
[0151] The amount of human TNF-.alpha. present was determined by a
specific trapping ELISA assay using two anti-TNF-.alpha. antibodies
(2TNF-H12 and 2TNF-H34) described in Reimund, J. M., et al. GUT.
Vol. 39(5), 684-689 (1996).
[0152] Polystyrene 96-well plates were coated with 50 .mu.l per
well of antibody 2TNF-H12 in PBS (5 .mu.g/mL) and incubated in a
humidified chamber at 4.degree. C. overnight. The plates were
washed with PBS and then blocked with 3% nonfat-dry milk in PBS for
1 hour at room temperature and washed with 0.1% BSA (bovine serum
albumin) in PBS.
[0153] TNF standards were prepared from a stock solution of human
recombinant TNF-.alpha. (R&D Systems, Minneapolis, Minn.). The
concentration of the standards in the assay began at 10 ng/mL
followed by 6 half log serial dilutions.
[0154] Twenty five .mu.L aliquots of the above culture supernatants
or TNF standards or only medium (control) were mixed with 25 .mu.L
aliquots of biotinylated monoclonal antibody 2TNF-H34 (1 .mu.g/mL
in PBS containing 0.1% BSA) and then added to each well. The
samples were incubated for 2 hr at room temperature with gentle
shaking and then washed 3 times with 0.1% BSA in PBS. 50 .mu.L of
peroxidase-streptavidin (Zymed, S. San Francisco, Calif.) solution
containing 0.416 .mu.g/mL of peroxidase-streptavidin and 0.1% BSA
in PBS was added to each well. The samples were incubated for an
additional 1 hr at room temperature and then washed 3 times with
0.1% BSA in PBS. Fifty .mu.L of O-phenylenediamine solution (1
.mu.g/mL O-phenylene-diamine and 0.03% hydrogen peroxide in 0.2M
citrate buffer pH 4.5) was added to each well and the samples were
incubated in the dark for 30 min., at room temperature. Optical
density of the sample and the reference were read at 450 nm and 650
nm, respectively. TNF-.alpha. levels were determined from a graph
relating the optical density at 450 nm to the concentration
used.
[0155] The IC.sub.50 value was defined as the concentration of the
test compound corresponding to half-maximal reduction in 450 nm
absorbance.
[0156] The compounds described in the Examples herein were tested
in the above assay and found to have a measurable level of p38
inhibitory activity. As an illustration, Table 2 below lists
approximate p38 inhibitory activities that were detected for
certain compounds according to the invention (expressed as
IC.sub.50, the concentration causing 50% inhibition of the p38
enzyme being analyzed). TABLE-US-00007 TABLE 2 Ex. No. IC.sub.50
.mu.M (P38 MAP KINASE E-03) 1 <.05 2 <.5 4 <.3
[0157] The foregoing discussion of the invention has been presented
for purposes of illustration and description. The foregoing is not
intended to limit the invention to the forms disclosed herein.
Although the description of the invention has included one or more
embodiments and certain variations and modifications, other
variations and modifications are within the scope of the invention,
e.g., as may be within the skill and knowledge of those in the art.
It is intended to obtain rights which include alternative
embodiments to the extent permitted, including alternate,
interchangeable and/or equivalent structures, functions, ranges or
steps to those claimed, whether or not such alternate,
interchangeable and/or equivalent structures, functions, ranges or
steps are disclosed herein, and without intending to publicly
dedicate any patentable subject matter. All publications, patents,
and patent applications cited herein are hereby incorporated by
reference in their entirety.
* * * * *