U.S. patent application number 13/445644 was filed with the patent office on 2012-08-09 for compounds and compositions as itpkb inhibitors.
This patent application is currently assigned to IRM LLC. Invention is credited to Badry Bursulaya, Dai Cheng, Jiqing Jiang, Donald S. Karanewsky, Yi Liu, Shifeng Pan, Yongqin Wan, Xia Wang, Yun Feng Xie, Yang Yang.
Application Number | 20120202782 13/445644 |
Document ID | / |
Family ID | 39673403 |
Filed Date | 2012-08-09 |
United States Patent
Application |
20120202782 |
Kind Code |
A1 |
Bursulaya; Badry ; et
al. |
August 9, 2012 |
COMPOUNDS AND COMPOSITIONS AS ITPKB INHIBITORS
Abstract
The invention provides a novel class of compounds,
pharmaceutical compositions comprising such compounds and methods
of using such compounds to treat or prevent diseases or disorders
associated with abnormal or dysregulated B cell activities,
particularly diseases or disorders that involve aberrant activation
of inositol 1,4,5-trisphosphate 3-kinase B (ITPKb).
Inventors: |
Bursulaya; Badry; (San
Diego, CA) ; Cheng; Dai; (San Diego, CA) ;
Jiang; Jiqing; (San Diego, CA) ; Karanewsky; Donald
S.; (Escondido, CA) ; Liu; Yi; (San Diego,
CA) ; Pan; Shifeng; (San Diego, CA) ; Wan;
Yongqin; (Irvine, CA) ; Wang; Xia; (San Diego,
CA) ; Xie; Yun Feng; (San Diego, CA) ; Yang;
Yang; (San Diego, CA) |
Assignee: |
IRM LLC
Hamilton
BM
|
Family ID: |
39673403 |
Appl. No.: |
13/445644 |
Filed: |
April 12, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12664651 |
Feb 5, 2010 |
8178526 |
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PCT/US2008/066664 |
Jun 12, 2008 |
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13445644 |
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60944354 |
Jun 15, 2007 |
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Current U.S.
Class: |
514/210.16 ;
514/218; 514/230.5; 514/252.17; 514/253.05; 514/253.1; 514/254.02;
540/575; 544/105; 544/284; 544/363; 544/364; 544/368; 544/70;
544/92; 544/94 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 11/02 20180101; A61P 17/06 20180101; A61P 1/16 20180101; A61P
7/06 20180101; A61P 37/02 20180101; A61P 37/06 20180101; A61P 37/08
20180101; A61P 11/06 20180101; A61P 17/00 20180101; C07D 413/14
20130101; A61P 29/00 20180101; A61P 7/00 20180101; A61P 37/00
20180101; A61P 19/02 20180101; C07D 413/02 20130101; A61P 35/00
20180101 |
Class at
Publication: |
514/210.16 ;
540/575; 514/218; 544/92; 514/230.5; 544/364; 514/253.1; 544/94;
544/105; 544/368; 514/254.02; 544/363; 514/253.05; 544/284;
514/252.17; 544/70 |
International
Class: |
A61K 31/536 20060101
A61K031/536; A61K 31/551 20060101 A61K031/551; A61K 31/496 20060101
A61K031/496; A61K 31/4985 20060101 A61K031/4985; C07D 487/08
20060101 C07D487/08; A61K 31/538 20060101 A61K031/538; C07D 401/14
20060101 C07D401/14; A61K 31/517 20060101 A61K031/517; A61P 37/06
20060101 A61P037/06; A61P 29/00 20060101 A61P029/00; A61P 19/02
20060101 A61P019/02; A61P 17/06 20060101 A61P017/06; A61P 7/06
20060101 A61P007/06; A61P 35/00 20060101 A61P035/00; C07D 413/14
20060101 C07D413/14 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 12, 2008 |
US |
PCT/US2008/066664 |
Claims
1. A compound of Formula I: ##STR00056## wherein: n is selected
from 0, 1, 2 and 3; m is selected from 0, 1, 2 and 3; up to 3
groups of Ring A selected from --CR.sub.1.dbd., --CR.sub.2.dbd. and
--CR.sub.5.dbd. are optionally replaced with --N.dbd.; R.sub.1,
R.sub.2 and R.sub.5 are independently selected from hydrogen,
hydroxy, halo, cyano, C.sub.1-6alkyl,
halo-substituted-C.sub.1-6alkyl, hydroxy-substituted-C.sub.1-6alkyl
and cyano-substituted-C.sub.1-6alkyl; R.sub.3 and R.sub.4, together
with the carbon atoms to which R.sub.3 and R.sub.4 are attached,
form a 5 to 6 member heterocycle fused to ring A containing up to 4
radicals selected from O, C(O), S(O).sub.2, CR.sub.11R.sub.12 and
NH; wherein each R.sub.11 and R.sub.12 are independently selected
from hydrogen, C.sub.1-3alkyl, and halo-substituted-C.sub.1-3alkyl;
or R.sub.11 and R.sub.12, together with the carbon to which they
are both attached, forms C.sub.3-7cycloalkyl; R.sub.6 and R.sub.7
are independently selected from hydrogen, C.sub.1-3alkyl and
halo-substituted-C.sub.1-3alkyl; or R.sub.6 and R.sub.7, together
with the carbon to which they are both attached, forms
C.sub.3-7cycloalkyl; R.sub.8 is selected from C.sub.1-6alkyl,
C.sub.2-6alkenyl, halo-substituted-C.sub.1-6alkyl and
hydroxy-substituted-C.sub.1-6alkyl; or two R.sub.8 groups can
combine to form an alkyl bridge; or when two R.sub.8 groups are
attached to the same carbon atom, they, together with the carbon to
which they are both attached, form C.sub.3-7cycloalkyl; R.sub.9 is
selected from L.sub.1-C.sub.6-10aryl, L.sub.1-C.sub.1-10heteroaryl,
C.sub.1-6alkyl, L.sub.1-C.sub.3-12cycloalkyl and
L.sub.1-C.sub.3-8heterocycloalkyl; wherein said aryl, heteroaryl,
cycloalkyl and heterocycloalkyl of R.sub.9 can be optionally
substituted with 1 to 3 radicals independently selected from halo,
cyano, hydroxy, C.sub.1-3alkyl, halo-substituted-C.sub.1-3alkyl,
cyano-substituted-C.sub.1-3alkyl,
hydroxy-substituted-C.sub.1-3alkyl, --C(O)R.sub.13,
--C(O)NR.sub.13R.sub.14; wherein each R.sub.13 and R.sub.14 are
independently selected from hydrogen and C.sub.1-6alkyl; L.sub.1 is
a bond, C.sub.1-3 alkyl or halo-substituted-C.sub.1-3 alkyl; Y is N
or CR.sub.10; R.sub.10 is selected from hydrogen, C.sub.1-6alkyl,
--NR.sub.15R.sub.16, --NR.sub.15C(O)R.sub.16 and
--C(O)NR.sub.15R.sub.16; wherein each R.sub.15 and R.sub.16 are
independently selected from hydrogen, C.sub.1-6alkyl,
C.sub.6-10aryl, C.sub.1-10heteroaryl, C.sub.3-12cycloalkyl and
C.sub.3-8heterocycloalkyl; wherein said aryl, heteroaryl,
cycloalkyl and heterocycloalkyl can be optionally substituted with
1 to 3 radicals independently selected from halo, hydroxy, cyano,
C.sub.1-6alkyl, halo-substituted-C.sub.1-6alkyl, C.sub.1-6 alkoxy
and halo-substituted-C.sub.1-6alkoxy; and the pharmaceutically
acceptable salts thereof.
2. The compound of claim 1, wherein: n is selected from 1 and 2; m
is selected from 0, 1 and 2; up to 3 groups of Ring A selected from
--CR.sub.1.dbd., --CR.sub.2.dbd. and --CR.sub.5.dbd. are optionally
replaced with --N.dbd. R.sub.1, R.sub.2 and R.sub.5 are hydrogen;
R.sub.6 and R.sub.7 are hydrogen; R.sub.8 is selected from
C.sub.1-6alkyl, halo-substituted-C.sub.1-6alkyl and
hydroxy-substituted-C.sub.1-6alkyl; or two R.sub.8 groups can
combine to form an alkyl bridge; or when two R.sub.8 groups are
attached to the same carbon, they, together with the carbon to
which they are both attached, form C.sub.3-7cycloalkyl; R.sub.9 is
selected from L.sub.1-C.sub.6-10aryl, L.sub.1-C.sub.1-10heteroaryl,
C.sub.1-6alkyl, L.sub.1-C.sub.3-12cycloalkyl and
L.sub.1-C.sub.3-8heterocycloalkyl; wherein said aryl, heteroaryl,
cycloalkyl and heterocycloalkyl of R.sub.9 can be optionally
substituted with 1 to 3 radicals independently selected from halo,
cyano, hydroxy, C.sub.1-3alkyl, halo-substituted-C.sub.1-3alkyl,
cyano-substituted-C.sub.1-3alkyl,
hydroxy-substituted-C.sub.1-3alkyl, --C(O)R.sub.13,
--C(O)NR.sub.13R.sub.14; wherein each R.sub.13 and R.sub.14 are
independently selected from hydrogen and C.sub.1-6alkyl; L.sub.1 is
a bond or C.sub.1-3alkyl; Y is CR.sub.10, and R.sub.10 is
hydrogen.
3. The compound of claim 2, wherein the 5 to 6 member heterocycle
fused to ring A formed from R.sub.3 and R.sub.4, together with the
carbon atoms to which R.sub.3 and R.sub.4 are attached, is selected
from: ##STR00057##
4. The compound of claim 3, wherein R.sub.8 is selected from
methyl, ethyl, trifluoromethyl, difluoromethyl, fluoromethyl and
hydroxy-methyl; or two R.sub.8 groups can combine to form an alkyl
bridge selected from methyl, ethyl and propyl; or two R.sub.8
groups are attached to the same carbon, they, together with the
carbon to which they are both attached, form cyclopropyl.
5. The compound of claim 4, wherein R.sub.9 is selected from
C.sub.3-7cycloalkyl, C.sub.4-7heterocycloalkyl, phenyl, pyridinyl,
pyrazinyl, pyrimidinyl and furo[3,2-c]pyridin-4-yl; wherein said
phenyl, pyridinyl, pyrazinyl, pyrimidinyl or
furo[3,2-c]pyridin-4-ylis optionally substituted with 1 to 3
radicals independently selected from trifluoromethyl, cyano, bromo,
chloro, hydroxy-methyl, methyl-carbonyl, methyl, amino-carbonyl,
nitro, iodo, fluoro, methoxy-carbonyl, hydroxy, amino, carboxy and
methoxy.
6. The compound of claim 1 selected from:
6-{4-[4-(5-trifluoromethyl-pyridin-2-yl)-[1,4]diazepan-1-ylmethyl]-1H-pyr-
azol-3-yl]-benzo[e][1,3]oxazine-2,4-dione;
6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)
methyl)-1H-pyrazol-3-yl)-3H-benzo[e][1,3]oxazine-2,4-dione;
6-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)
methyl)-1H-pyrazol-3-yl)-3H-benzo[e][1,3]oxazine-2,4-dione;
6-(4-(((S)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)
methyl)-1H-pyrazol-3-yl)-3H-benzo[e][1,3]oxazine-2,4-dione;
6-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one;
6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)-1,4-diazepan-1-yl)methyl)-1H-p-
yrazol-3-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one;
7-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-1,2-dihydroisoquinolin-3(4H)-one;
7-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)isoquinoline-1,3(2H,4H)-dione;
6-(4-44-(5-(trifluoromethyl)pyridin-2-yl)-1,4-diazepan-1-yl)methyl)-1H-py-
razol-3-yl)-3,4-dihydroquinazolin-2(1H)-one;
7-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)-1,4-diazepan-1-yl)methyl)-1H-p-
yrazol-3-yl)-1,2-dihydroisoquinolin-3(4H)-one; and
6-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-3,4-dihydroquinazolin-2(1H)-one;
(R)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2(3H)-one;
6-(4-43-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1--
yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyraz-
ol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-5-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2(3H)-one;
6-(4-((3-(5-(trifluoromethyl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptan--
6-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
6-(4-42-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1--
yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
6-(4-((3-(5-(trifluoromethyl)pyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-
-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
6-(4-((3-(5-(trifluoromethyl)pyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-
-yl)methyl)-1H-pyrazol-3-yl)-2H-benzo[e][1,3]oxazine-2,4(3H)-dione;
(S)-6-(4-43-(fluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-
-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
6-(4-((4-(2,3-dimethylphenyl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-2H-b-
enzo[e][1,3]oxazine-2,4(3H)-dione;
6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyraz-
ol-3-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one;
6-(4-((4-(2,3-dimethylphenyl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)benzo-
[d]oxazol-2(3H)-one;
(S)-6-(4-43-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazi-
n-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-((3-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperaz-
in-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(S)-6-(4-((3-(hydroxymethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-
-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-2-(2-methyl-4-((3-(2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H--
pyrazol-4-yl)methyl)piperazin-1-yl)isonicotinonitrile;
(R)-6-(4-((3-methyl-4-(4-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)m-
ethyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-((3-methyl-4-(5-methylpyridin-2-yl)piperazin-1-yl)methyl)-1H-pyr-
azol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(S)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-((4-cyclohexyl-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3-
,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(5-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)met-
hyl)-2H-1,2,3-triazol-4-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-4,4-dimethyl-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piper-
azin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-on-
e;
6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)-4,7-diazaspiro[2.5]octan-7-y-
l)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(2-methyl-4-((3-(2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H--
pyrazol-4-yl)methyl)piperazin-1-yl)nicotinonitrile;
(R)-6-(4-((4-(5-chloropyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyr-
azol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(S)-1-((3-(2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H-pyrazol-4-yl-
)methyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazine-2-carboxylic
acid;
(S)-6-(4-((2-(hydroxymethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-
-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(S)-6-(4-((2-(fluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin--
1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-((3-methyl-4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)methyl)-1-
H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-((4-(2-fluorobenzyl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-
-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-((4-(4-chlorophenyl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-
-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(S)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(S)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2(3H)-one, and
(R)-6-(4-((3-methyl-4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methyl)-1-
H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(S)-6-(4-((4-cyclohexyl-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3-
,4-dihydro-2H-benzo[e][1,3]oxazin-2-one.
7. A method for modulating T and B lymphocyte development and
function in a subject for the treatment of autoimmune diseases, the
method comprising administering to the subject a pharmaceutical
composition comprising an effective amount of an agent which
modulates the kinase activity or cellular level of an ITPKb
molecule; thereby modulating B lymphocyte differentiation and
function in a subject.
8. The method of claim 7 wherein the agent down-regulates the
cellular level of the ITPKb molecule.
9. The method of claim 8 wherein the agent is a compound of claim
1.
10. The method of claim 9 wherein the agent inhibits the kinase
activity of the ITPKb molecule.
11. The method of claim 10 wherein the subject is human and the
ITPKb molecule is human ITPK.beta..
12. The method of claim 11 in which the autoimmune disease is
selected from rheumatoid arthritis, systemic lupus erythematosus,
idiopathic thrombocytopenic purpura, hemolytic anemia, and
psoriasis.
13. The method of claim 11 wherein the subject suffers from B cell
lymphoma.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
application Ser. No. 60/944,345, filed Jun. 15, 2007, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention provides a novel class of compounds,
pharmaceutical compositions comprising such compounds and methods
of using such compounds to treat or prevent diseases or disorders
associated with abnormal or dysregulated B cell activities,
particularly diseases or disorders that involve aberrant activation
of inositol 1,4,5-trisphosphate 3-kinase B (ITPKb).
BACKGROUND
[0003] The protein kinases represent a large family of proteins,
which play a central role in the regulation of a wide variety of
cellular processes and maintaining control over cellular function.
A partial, non-limiting, list of these kinases include: non-protein
substrate kinases such as IPTKb; receptor tyrosine kinases such as
platelet-derived growth factor receptor kinase (PDGF-R), the nerve
growth factor receptor, trkB, Met, and the fibroblast growth factor
receptor, FGFR3; non-receptor tyrosine kinases such Abl and the
fusion kinase BCR-Abl, Lck, Csk, Fes, Bmx and c-src; and
serine/threonine kinases such as b-RAF, c-RAF, sgk, MAP kinases
(e.g., MKK4, MKK6, etc.) and SAPK2a, SAPK2.beta. and SAPK3.
Aberrant kinase activity has been observed in many disease states
including benign and malignant proliferative disorders as well as
diseases resulting from inappropriate activation of the immune and
nervous systems.
[0004] The novel compounds of this invention inhibit the activity
of ITPKb and are, therefore, expected to be useful in the treatment
of ITPKb-associated diseases.
SUMMARY OF THE INVENTION
[0005] In one aspect, the present invention provides compounds of
Formula I:
##STR00001##
[0006] in which: [0007] n is selected from 0, 1, 2 and 3; [0008] m
is selected from 0, 1, 2 and 3; [0009] A can have up to 3 groups
selected from --CR.sub.1.dbd., --CR.sub.2.dbd. and --CR.sub.5.dbd.
replaced with --N.dbd.; [0010] R.sub.1, R.sub.2 and R.sub.5 are
independently selected from hydrogen, hydroxy, halo, cyano,
C.sub.1-6alkyl, halo-substituted-C.sub.1-6alkyl,
hydroxy-substituted-C.sub.1-6alkyl and
cyano-substituted-C.sub.1-6alkyl; [0011] R.sub.3 and R.sub.4,
together with the carbon atoms to which R.sub.3 and R.sub.4 are
attached, form a 5 to 6 member heterocycle fused to ring A
containing up to 4 radicals selected from O, C(O), S(O).sub.2,
CR.sub.11R.sub.12 and NH; wherein each R.sub.11 and R.sub.12 are
independently selected from hydrogen, C.sub.1-3alkyl, and
halo-substituted-C.sub.1-3alkyl; or R.sub.11 and R.sub.12, together
with the carbon to which they are both attached, forms
C.sub.3-7cycloalkyl; [0012] R.sub.6 and R.sub.7 are independently
selected from hydrogen, C.sub.1-3alkyl and
halo-substituted-C.sub.1-3alkyl; or R.sub.6 and R.sub.7, together
with the carbon to which they are both attached, forms
C.sub.3-7cycloalkyl; [0013] R.sub.8 is selected from
C.sub.1-6alkyl, C.sub.2-6alkenyl, halo-substituted-C.sub.1-6alkyl
and hydroxy-substituted-C.sub.1-6alkyl; or two R.sub.8 groups can
combine to form an alkyl bridge; or when two R.sub.8 groups are
attached to the same carbon atom, they, together with the carbon to
which they are both attached, form C.sub.3-7cycloalkyl; [0014]
R.sub.9 is selected from L.sub.1-C.sub.6-10aryl,
L.sub.1-C.sub.1-10heteroaryl, C.sub.1-6alkyl,
L.sub.1-C.sub.3-12cycloalkyl and L.sub.1-C.sub.3-8heterocycloalkyl;
wherein said aryl, heteroaryl, cycloalkyl and heterocycloalkyl of
R.sub.9 can be optionally substituted with 1 to 3 radicals
independently selected from halo, cyano, hydroxy, C.sub.1-3alkyl,
halo-substituted-C.sub.1-3alkyl, cyano-substituted-C.sub.1-3alkyl,
hydroxy-substituted-C.sub.1-3alkyl, --C(O)R.sub.13,
--C(O)NR.sub.13R.sub.14; wherein each R.sub.13 and R.sub.14 are
independently selected from hydrogen and C.sub.1-6alkyl; [0015]
L.sub.1 is a bond, C.sub.1-3alkyl or
halo-substituted-C.sub.1-3alkyl; [0016] Y is N or CR.sub.10; [0017]
R.sub.10 is selected from hydrogen, C.sub.1-6alkyl,
--NR.sub.15R.sub.16, --NR.sub.15C(O)R.sub.16 and
--C(O)NR.sub.15R.sub.16; wherein each R.sub.15 and R.sub.16 are
independently selected from hydrogen, C.sub.1-6alkyl,
C.sub.6-10aryl, C.sub.1-10heteroaryl, C.sub.3-12cycloalkyl and
C.sub.3-8heterocycloalkyl; wherein said aryl, heteroaryl,
cycloalkyl and heterocycloalkyl can be optionally substituted with
1 to 3 radicals independently selected from halo, hydroxy, cyano,
C.sub.1-6alkyl, halo-substituted-C.sub.1-6alkyl, C.sub.1-6alkoxy
and halo-substituted-C.sub.1-6alkoxy; and the N-oxide derivatives,
prodrug derivatives, protected derivatives, individual isomers and
mixture of isomers thereof; [0018] and the pharmaceutically
acceptable salts and solvates (e.g. hydrates) of such
compounds.
[0019] In a second aspect, the present invention provides a
pharmaceutical composition which contains a compound of Formula I
or a N-oxide derivative, individual isomers and mixture of isomers
thereof; or a pharmaceutically acceptable salt thereof, in
admixture with one or more suitable excipients.
[0020] In a third aspect, the present invention provides a method
of treating a disease in an animal in which inhibition of kinase
activity, particularly ITPKb activity, can prevent, inhibit or
ameliorate the pathology and/or symptomology of the diseases, which
method comprises administering to the animal a therapeutically
effective amount of a compound of Formula I or a N-oxide
derivative, individual isomers and mixture of isomers thereof, or a
pharmaceutically acceptable salt thereof.
[0021] In a fourth aspect, the present invention provides the use
of a compound of Formula I in the manufacture of a medicament for
treating a disease in an animal in which kinase activity,
particularly ITPKb activity, contributes to the pathology and/or
symptomology of the disease.
[0022] In a fifth aspect, the present invention provides a process
for preparing compounds of Formula I and the N-oxide derivatives,
prodrug derivatives, protected derivatives, individual isomers and
mixture of isomers thereof, and the pharmaceutically acceptable
salts thereof.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0023] "Alkyl" as a group and as a structural element of other
groups, for example halo-substituted-alkyl and alkoxy, can be
either straight-chained or branched. C.sub.1-4-alkoxy includes,
methoxy, ethoxy, and the like. Halo-substituted alkyl includes
trifluoromethyl, pentafluoroethyl, and the like.
Hydroxy-substituted alkyl includes hydroxy-methyl, hydroxy-ethyl
and the like.
[0024] "Aryl" means a monocyclic or fused bicyclic aromatic ring
assembly containing six to ten ring carbon atoms. For example, aryl
may be phenyl or naphthyl, preferably phenyl. "Arylene" means a
divalent radical derived from an aryl group.
[0025] "Heteroaryl" is as defined for aryl above where one or more
of the ring members can be a heteroatom selected from --O--,
--N.dbd., --NR--, --C(O)--, --S--, --S(O)-- or --S(O).sub.2--,
wherein R is hydrogen, C.sub.1-4alkyl or a nitrogen protecting
group. For example C.sub.1-10heteroaryl includes pyridyl, indolyl,
indazolyl, quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl,
benzothiopyranyl, benzo[1,3]dioxole, imidazolyl, benzo-imidazolyl,
pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl,
pyrazolyl, thienyl, etc.
[0026] "Cycloalkyl" means a saturated or partially unsaturated,
monocyclic, fused bicyclic or bridged polycyclic ring assembly
containing the number of ring atoms indicated. For example,
C.sub.3-10cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, etc.
[0027] "Heterocycloalkyl" means cycloalkyl, as defined in this
application, provided that one or more of the ring carbons
indicated, are replaced by a moiety selected from --O--, --N.dbd.,
--NR--, --C(O)--, --S--, --S(O)-- or --S(O).sub.2--, wherein R is
hydrogen, C.sub.1-4alkyl or a nitrogen protecting group. For
example, C.sub.3-8heterocycloalkyl as used in this application to
describe compounds of the invention includes morpholino,
pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl, piperidinyl,
piperidinylone, 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, etc.
[0028] "Halogen" (or halo) preferably represents chloro or fluoro,
but may also be bromo or iodo.
[0029] "Treat", "treating" and "treatment" refer to a method of
alleviating or abating a disease and/or its attendant symptoms.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0030] The present invention provides compounds, compositions and
methods for the treatment of kinase related disease, particularly
IPTKb related diseases. For example, autoimmune diseases,
particularly B cell associated diseases, are related to IPTKb. For
example, rheumatoid arthritis, systemic lupus erythematosus (SLE),
immune thrombocytopenic purpura (ITP) and hemolytic anemia.
[0031] In one embodiment, with reference to compounds of Formula I,
n is selected from 1 and 2; m is selected from 0, 1 and 2; A can
have up to 3 groups selected from --CR.sub.1.dbd., --CR.sub.2.dbd.
and --CR.sub.5.dbd. replaced with --N.dbd.; R.sub.1, R.sub.2 and
R.sub.5 are hydrogen; R.sub.6 and R.sub.7 are hydrogen; R.sub.8 is
selected from C.sub.1-6alkyl, halo-substituted-C.sub.1-6alkyl and
hydroxy-substituted-C.sub.1-6alkyl; or two R.sub.8 groups can
combine to form an alkyl bridge; or when two R.sub.8 groups are
attached to the same carbon, they, together with the carbon to
which they are both attached, form C.sub.3-7cycloalkyl; R.sub.9 is
selected from L.sub.1-C.sub.6-10aryl, L.sub.1-C.sub.1-10heteroaryl,
C.sub.1-6alkyl, L.sub.1-C.sub.3-12cycloalkyl and
L.sub.1-C.sub.3-8heterocycloalkyl; wherein said aryl, heteroaryl,
cycloalkyl and heterocycloalkyl of R.sub.9 can be optionally
substituted with 1 to 3 radicals independently selected from halo,
cyano, hydroxy, C.sub.1-3alkyl, halo-substituted-C.sub.1-3alkyl,
cyano-substituted-C.sub.1-3alkyl,
hydroxy-substituted-C.sub.1-3alkyl, --C(O)R.sub.13,
--C(O)NR.sub.13R.sub.14; wherein each R.sub.13 and R.sub.14 are
independently selected from hydrogen and C.sub.1-6alkyl; L.sub.1 is
a bond or C.sub.1-3alkyl; Y is CR.sub.10, and R.sub.10 is
hydrogen.
[0032] In another embodiment, the 5 to 6 member heterocycle fused
to ring A formed from R.sub.3 and R.sub.4, together with the carbon
atoms to which R.sub.3 and R.sub.4 are attached, is selected
from:
##STR00002##
[0033] In another embodiment, R.sub.8 is selected from methyl,
ethyl, trifluoromethyl, difluoromethyl, fluoromethyl and
hydroxy-methyl; or two R.sub.8 groups can combine to form an alkyl
bridge selected from methyl, ethyl and propyl; or two R.sub.8
groups are attached to the same carbon, they, together with the
carbon to which they are both attached, form cyclopropyl.
[0034] In another embodiment, R.sub.9 is selected from
C.sub.3-7cycloalkyl, C.sub.4-7heterocycloalkyl, phenyl, pyridinyl,
pyrazinyl, pyrimidinyl and furo[3,2-c]pyridin-4-yl; wherein said
phenyl, pyridinyl, pyrazinyl, pyrimidinyl or
furo[3,2-c]pyridin-4-ylis optionally substituted with 1 to 3
radicals independently selected from trifluoromethyl, cyano, bromo,
chloro, hydroxy-methyl, methyl-carbonyl, methyl, amino-carbonyl,
nitro, iodo, fluoro, methoxy-carbonyl, hydroxy, amino, carboxy and
methoxy.
[0035] In another embodiment, are compounds selected from:
6-{4-[4-(5-Trifluoromethyl-pyridin-2-yl)-[1,4]diazepan-1-ylmethyl]-1H-pyr-
azol-3-yl}-benzo[e][1,3]oxazine-2,4-dione;
6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyraz-
ol-3-yl)-3H-benzo[e][1,3]oxazine-2,4-dione;
6-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-3H-benzo[e][1,3]oxazine-2,4-dione;
6-(4-(((S)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-3H-benzo[e][1,3]oxazine-2,4-dione;
6-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one;
6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)-1,4-diazepan-1-yl)methyl)-1H-p-
yrazol-3-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one;
7-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-1,2-dihydroisoquinolin-3(4H)-one;
7-(4-4(R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)meth-
yl)-1H-pyrazol-3-yl)isoquinoline-1,3 (2H,4H)-dione;
6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)-1,4-diazepan-1-yl)methyl)-1H-p-
yrazol-3-yl)-3,4-dihydroquinazolin-2(1H)-one;
7-(4-((4(5-(trifluoromethyl)pyridin-2-yl)-1,4-diazepan-1-yl)methyl)-1H-py-
razol-3-yl)-1,2-dihydroisoquinolin-3(4H)-one; and
6-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-3,4-dihydroquinazolin-2(1H)-one;
(R)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2 (3H)-one;
6-(4-((3-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-
-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyraz-
ol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-5-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2(3H)-one;
6-(4-((4(3-(5-(trifluoromethyl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]hepta-
n-6-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
6-(4-((2-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-
-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
6-(4-((3-(5-(trifluoromethyl)pyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-
-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
6-(4-((4(3-(5-(trifluoromethyl)pyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-
-8-yl)methyl)-1H-pyrazol-3-yl)-2H-benzo[e][1,3]oxazine-2,4(3H)-dione;
(S)-6-(4-((3-(fluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin--
1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
6-(4-((4-(2,3-dimethylphenyl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-2H-b-
enzo[e][1,3]oxazine-2,4(3H)-dione;
6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyraz-
ol-3-yl)-2H-benzo[1)][1,4]oxazin-3(4H)-one;
6-(4-((4-(2,3-dimethylphenyl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)benzo-
[d]oxazol-2(3H)-one;
(S)-6-(4-((3-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperaz-
in-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-((3-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperaz-
in-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(S)-6-(4-((3-(hydroxymethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-
-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-2-(2-methyl-4-((3-(2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H--
pyrazol-4-yl)methyl)piperazin-1-yl)isonicotinonitrile;
(R)-6-(4-((3-methyl-4-(4-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)m-
ethyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-((3-methyl-4-(5-methylpyridin-2-yl)piperazin-1-yl)methyl)-1H-pyr-
azol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(S)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-((4-cyclohexyl-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3-
,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(54(3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)meth-
yl)-2H-1,2,3-triazol-4-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-4,4-dimethyl-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piper-
azin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-on-
e;
6-(4-44-(5-(trifluoromethyl)pyridin-2-yl)-4,7-diazaspiro[2.5]octan-7-yl-
)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(2-methyl-4-43-(2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H-p-
yrazol-4-yl)methyl)piperazin-1-yl)nicotinonitrile;
(R)-6-(4-44-(5-chloropyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyra-
zol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(S)-1-((3-(2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H-pyrazol-4-yl-
)methyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazine-2-carboxylic
acid;
(S)-6-(4-((2-(hydroxymethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-
-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(S)-6-(4-((2-(fluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin--
1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-((3-methyl-4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)methyl)-1-
H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-44-(2-fluorobenzyl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3--
yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-((4-(4-chlorophenyl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-
-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(R)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(S)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;
(S)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)met-
hyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2(3H)-one,
(R)-6-(4-((3-methyl-4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methyl)-1-
H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one, and
(S)-6-(4-((4-cyclohexyl-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3-
,4-dihydro-2H-benzo[e][1,3]oxazin-2-one.
[0036] Further compounds of the invention are detailed in the
Examples and Table I, infra.
Pharmacology and Utility
[0037] Compounds of the invention modulate the activity of IPTKb
and, as such, are useful for treating diseases or disorders in
which aberrant activity of IPTKb, contributes to the pathology
and/or symptomology of diseases.
[0038] By inhibiting B cell activation and development, the ITPKb
inhibitors of the present invention are useful in various
therapeutic applications. Pharmacological inhibition of ITPKb
provides a means to inhibit B cell malfunction in pathological
settings. For example, B cells play a pathological role in chronic
transplant rejection, and the development of autoimmune diseases
(e.g. Rheumatoid Arthritis, SLE, lupus, and the like), Psoriasis,
Allergy (Asthma, Rhinitis, COPD, Dermatitis) and others, including
anaphylaxis and many complement mediated diseases. The
ITPKb-inhibiting compounds of the invention can be effective agents
to treat these diseases where ITPKb acts to promote
pathogenesis.
[0039] Other diseases and conditions that are amenable to treatment
include diseases associated with or mediated by abnormal B cell
proliferation, e.g., B cell lymphoma. They also encompass other
antibody-mediated disorders, e.g., allergies, psoriasis, systematic
lupus erythematosus (SLE), primary binary cirrhosis (PBC), and
idiopathic thrombocytopenic purpura (ITP). In addition to treating
these diseases or conditions, ITPKb inhibitors of the present
invention are also useful for preventing or modulating the
development of such diseases or disorders in a subject (including
human and animals such as other mammals) suspected of being, or
known to be, prone to such diseases or disorders. The B-cell
modulators that can be employed in the therapeutic applications of
the invention include the specific ITPKb-inhibitors described in
the Examples and tables, infra.
[0040] The invention thus provides a method for modulating B
lymphocyte development and function in a subject (human or other
mammal) for the treatment of autoimmune diseases, the method
comprising administering to the subject a compound of formula I or
a pharmaceutical composition thereof in an effective amount to
modulate the kinase activity or cellular level of ITPKb (such as
demonstrated by the in vitro assays described, infra); thereby
modulating B lymphocyte differentiation and function in a subject.
The compound can down-regulate the cellular level of the ITPKb
molecule by inhibiting the kinase activity of ITPKb.
[0041] In accordance with the foregoing, the present invention
further provides a method for preventing, treating and/or
ameliorating the condition of any of the diseases or disorders
described above in a subject in need of such treatment, which
method comprises administering to said subject a therapeutically
effective amount (See, "Administration and Pharmaceutical
Compositions", infra) of a compound of Formula I or a
pharmaceutically acceptable salt thereof. Compounds of Formula I
can down-regulate the cellular level of the ITPKb molecule by
inhibiting the kinase activity of ITPKb such as described by the in
vitro assays described, infra. For any of the above uses, the
required dosage will vary depending on the mode of administration,
the particular condition to be treated and the effect desired.
Administration and Pharmaceutical Compositions
[0042] In general, compounds of the invention will be administered
in therapeutically effective amounts via any of the usual and
acceptable modes known in the art, either singly or in combination
with one or more therapeutic agents. A therapeutically effective
amount may vary widely depending on the severity of the disease,
the age and relative health of the subject, the potency of the
compound used and other factors. In general, satisfactory results
are indicated to be obtained systemically at daily dosages of from
about 0.03 to 2.5 mg/kg per body weight. An indicated daily dosage
in the larger mammal, e.g. humans, is in the range from about 0.5
mg to about 100 mg, conveniently administered, e.g. in divided
doses up to four times a day or in retard form. Suitable unit
dosage forms for oral administration comprise from ca. 1 to 50 mg
active ingredient.
[0043] Compounds of the invention can be administered as
pharmaceutical compositions by any conventional route, in
particular enterally, e.g., orally, e.g., in the form of tablets or
capsules, or parenterally, e.g., in the form of injectable
solutions or suspensions, topically, e.g., in the form of lotions,
gels, ointments or creams, or in a nasal or suppository form.
Pharmaceutical compositions comprising a compound of the present
invention in free form or in a pharmaceutically acceptable salt
form in association with at least one pharmaceutically acceptable
carrier or diluent can be manufactured in a conventional manner by
mixing, granulating or coating methods. For example, oral
compositions can be tablets or gelatin capsules comprising the
active ingredient together with a) diluents, e.g., lactose,
dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b)
lubricants, e.g., silica, talcum, stearic acid, its magnesium or
calcium salt and/or polyethyleneglycol; for tablets also c)
binders, e.g., magnesium aluminum silicate, starch paste, gelatin,
tragacanth, methylcellulose, sodium carboxymethylcellulose and or
polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches,
agar, alginic acid or its sodium salt, or effervescent mixtures;
and/or e) absorbents, colorants, flavors and sweeteners. Injectable
compositions can be aqueous isotonic solutions or suspensions, and
suppositories can be prepared from fatty emulsions or suspensions.
The compositions may be sterilized and/or contain adjuvants, such
as preserving, stabilizing, wetting or emulsifying agents, solution
promoters, salts for regulating the osmotic pressure and/or
buffers. In addition, they may also contain other therapeutically
valuable substances. Suitable formulations for transdermal
applications include an effective amount of a compound of the
present invention with a carrier. A carrier can include absorbable
pharmacologically acceptable solvents to assist passage through the
skin of the host. For example, transdermal devices are in the form
of a bandage comprising a backing member, a reservoir containing
the compound optionally with carriers, optionally a rate
controlling barrier to deliver the compound to the skin of the host
at a controlled and predetermined rate over a prolonged period of
time, and means to secure the device to the skin. Matrix
transdermal formulations may also be used. Suitable formulations
for topical application, e.g., to the skin and eyes, are preferably
aqueous solutions, ointments, creams or gels well-known in the art.
Such may contain solubilizers, stabilizers, tonicity enhancing
agents, buffers and preservatives.
[0044] Compounds of the invention can be administered in
therapeutically effective amounts in combination with one or more
therapeutic agents (pharmaceutical combinations). For example,
synergistic effects can occur with other immunomodulatory or
anti-inflammatory substances, for example when used in combination
with cyclosporin, rapamycin, or ascomycin, or immunosuppressant
analogues thereof, for example cyclosporin A (CsA), cyclosporin G,
FK-506, rapamycin, or comparable compounds, corticosteroids,
cyclophosphamide, azathioprine, methotrexate, brequinar,
leflunomide, mizoribine, mycophenolic acid, mycophenolate mofetil,
15-deoxyspergualin, immunosuppressant antibodies, especially
monoclonal antibodies for leukocyte receptors, for example MHC,
CD2, CD3, CD4, CD7, CD25, CD28, B7, CD45, CD58 or their ligands, or
other immunomodulatory compounds, such as CTLA41 g. Where the
compounds of the invention are administered in conjunction with
other therapies, dosages of the co-administered compounds will of
course vary depending on the type of co-drug employed, on the
specific drug employed, on the condition being treated and so
forth.
[0045] The invention also provides for a pharmaceutical
combinations, e.g. a kit, comprising a) a first agent which is a
compound of the invention as disclosed herein, in free form or in
pharmaceutically acceptable salt form, and b) at least one
co-agent. The kit can comprise instructions for its
administration.
[0046] The terms "co-administration" or "combined administration"
or the like as utilized herein are meant to encompass
administration of the selected therapeutic agents to a single
patient, and are intended to include treatment regimens in which
the agents are not necessarily administered by the same route of
administration or at the same time.
[0047] The term "pharmaceutical combination" as used herein means a
product that results from the mixing or combining of more than one
active ingredient and includes both fixed and non-fixed
combinations of the active ingredients. The term "fixed
combination" means that the active ingredients, e.g. a compound of
Formula I and a co-agent, are both administered to a patient
simultaneously in the form of a single entity or dosage. The term
"non-fixed combination" means that the active ingredients, e.g. a
compound of Formula I and a co-agent, are both administered to a
patient as separate entities either simultaneously, concurrently or
sequentially with no specific time limits, wherein such
administration provides therapeutically effective levels of the 2
compounds in the body of the patient. The latter also applies to
cocktail therapy, e.g. the administration of 3 or more active
ingredients.
Processes for Making Compounds of the Invention
[0048] The present invention also includes processes for the
preparation of compounds of the invention. In the reactions
described, it can be necessary to protect reactive functional
groups, for example hydroxy, amino, imino, thio or carboxy groups,
where these are desired in the final product, to avoid their
unwanted participation in the reactions. Conventional protecting
groups can be used in accordance with standard practice, for
example, see T.W. Greene and P. G. M. Wuts in "Protective Groups in
Organic Chemistry", John Wiley and Sons, 1991.
[0049] Compounds of Formula I, wherein R.sub.6 and R.sub.7 are both
hydrogen, can be prepared by proceeding as in the following
Reaction Scheme I:
##STR00003##
in which n, m, A, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.8, R.sub.9 and R.sub.10 are as defined in the Summary of the
Invention.
[0050] A compound of Formula I can be prepared by reacting of a
compound of formula 3 with a compound of formula 4 in the presence
of a suitable solvent (e.g., DCM) using an appropriate reducing
agents (e.g., NaCNBH.sub.3). A compound of formula 3 can be
prepared by reacting of a compound formula 2 with the complex of
POCl.sub.3 and DMF followed by the addition of a suitable base
(e.g., NaOH).
[0051] Detailed examples of the synthesis of a compound of Formula
I can be found in the Examples, infra.
Additional Processes for Making Compounds of the Invention
[0052] A compound of the invention can be prepared as a
pharmaceutically acceptable acid addition salt by reacting the free
base form of the compound with a pharmaceutically acceptable
inorganic or organic acid. Alternatively, a pharmaceutically
acceptable base addition salt of a compound of the invention can be
prepared by reacting the free acid form of the compound with a
pharmaceutically acceptable inorganic or organic base.
[0053] Alternatively, the salt forms of the compounds of the
invention can be prepared using salts of the starting materials or
intermediates.
[0054] The free acid or free base forms of the compounds of the
invention can be prepared from the corresponding base addition salt
or acid addition salt from, respectively. For example a compound of
the invention in an acid addition salt form can be converted to the
corresponding free base by treating with a suitable base (e.g.,
ammonium hydroxide solution, sodium hydroxide, and the like). A
compound of the invention in a base addition salt form can be
converted to the corresponding free acid by treating with a
suitable acid (e.g., hydrochloric acid, etc.).
[0055] Compounds of the invention in unoxidized form can be
prepared from N-oxides of compounds of the invention by treating
with a reducing agent (e.g., sulfur, sulfur dioxide, triphenyl
phosphine, lithium borohydride, sodium borohydride, phosphorus
trichloride, tribromide, or the like) in a suitable inert organic
solvent (e.g. acetonitrile, ethanol, aqueous dioxane, or the like)
at 0 to 80.degree. C.
[0056] Prodrug derivatives of the compounds of the invention can be
prepared by methods known to those of ordinary skill in the art
(e.g., for further details see Saulnier et al., (1994), Bioorganic
and Medicinal Chemistry Letters, Vol. 4, p. 1985). For example,
appropriate prodrugs can be prepared by reacting a non-derivatized
compound of the invention with a suitable carbamylating agent
(e.g., 1,1-acyloxyalkylcarbanochloridate, para-nitrophenyl
carbonate, or the like).
[0057] Protected derivatives of the compounds of the invention can
be made by means known to those of ordinary skill in the art. A
detailed description of techniques applicable to the creation of
protecting groups and their removal can be found in T. W. Greene,
"Protecting Groups in Organic Chemistry", 3.sup.rd edition, John
Wiley and Sons, Inc., 1999.
[0058] Compounds of the present invention can be conveniently
prepared, or formed during the process of the invention, as
solvates (e.g., hydrates). Hydrates of compounds of the present
invention can be conveniently prepared by recrystallization from an
aqueous/organic solvent mixture, using organic solvents such as
dioxin, tetrahydrofuran or methanol.
[0059] Compounds of the invention can be prepared as their
individual stereoisomers by reacting a racemic mixture of the
compound with an optically active resolving agent to form a pair of
diastereoisomeric compounds, separating the diastereomers and
recovering the optically pure enantiomers. While resolution of
enantiomers can be carried out using covalent diastereomeric
derivatives of the compounds of the invention, dissociable
complexes are preferred (e.g., crystalline diastereomeric salts).
Diastereomers have distinct physical properties (e.g., melting
points, boiling points, solubilities, reactivity, etc.) and can be
readily separated by taking advantage of these dissimilarities. The
diastereomers can be separated by chromatography, or preferably, by
separation/resolution techniques based upon differences in
solubility. The optically pure enantiomer is then recovered, along
with the resolving agent, by any practical means that would not
result in racemization. A more detailed description of the
techniques applicable to the resolution of stereoisomers of
compounds from their racemic mixture can be found in Jean Jacques,
Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and
Resolutions", John Wiley And Sons, Inc., 1981.
[0060] In summary, the compounds of Formula I can be made by a
process, which involves:
[0061] (a) that of reaction scheme I; and
[0062] (b) optionally converting a compound of the invention into a
pharmaceutically acceptable salt;
[0063] (c) optionally converting a salt form of a compound of the
invention to a non-salt form;
[0064] (d) optionally converting an unoxidized form of a compound
of the invention into a pharmaceutically acceptable N-oxide;
[0065] (e) optionally converting an N-oxide form of a compound of
the invention to its unoxidized form;
[0066] (f) optionally resolving an individual isomer of a compound
of the invention from a mixture of isomers;
[0067] (g) optionally converting a non-derivatized compound of the
invention into a pharmaceutically acceptable prodrug derivative;
and
[0068] (h) optionally converting a prodrug derivative of a compound
of the invention to its non-derivatized form.
[0069] The present invention also includes all suitable isotopic
variations of the compounds of the invention, or pharmaceutically
acceptable salts thereof. An isotopic variation of a compound of
the invention or a pharmaceutically acceptable salt thereof is
defined as one in which at least one atom is replaced by an atom
having the same atomic number but an atomic mass different from the
atomic mass usually found in nature. Examples of isotopes that may
be incorporated into the compounds of the invention and
pharmaceutically acceptable salts thereof include but are not
limited to isotopes of hydrogen, carbon, nitrogen and oxygen such
as as .sup.2H, .sup.3H, .sup.11C, .sup.13C, .sup.14C, .sup.15N,
.sup.17O, .sup.18O, .sup.35S, .sup.18O, .sup.35S, .sup.18O,
.sup.35S, .sup.18F, .sup.36Cl and .sup.123I. Certain isotopic
variations of the compounds of the invention and pharmaceutically
acceptable salts thereof, for example, those in which a radioactive
isotope such as .sup.3H or .sup.14C is incorporated, are useful in
drug and/or substrate tissue distribution studies. In particular
examples, .sup.3H and .sup.14C isotopes may be used for their ease
of preparation and detectability. In other examples, substitution
with isotopes such as may may afford certain therapeutic advantages
resulting from greater metabolic stability, such as increased in
vivo half-life or reduced dosage requirements. Isotopic variations
of the compounds of the invention or pharmaceutically acceptable
salts thereof can generally be prepared by conventional procedures
using appropriate isotopic variations of suitable reagents.
Isotopic variations of the compounds have the potential to change a
compound's metabolic fate and/or create small changes in physical
properties such as hydrophobicity, and the like. Isotopic variation
have the potential to enhance efficacy and safety, enhance
bioavailability and half-life, alter protein binding, change
biodistribution, increase the proportion of active metabolites
and/or decrease the formation of reactive or toxic metabolites.
[0070] Insofar as the production of the starting materials is not
particularly described, the compounds are known or can be prepared
analogously to methods known in the art or as disclosed in the
Examples hereinafter.
[0071] One of skill in the art will appreciate that the above
transformations are only representative of methods for preparation
of the compounds of the present invention, and that other well
known methods can similarly be used.
EXAMPLES
[0072] The present invention is further exemplified, but not
limited, by the following examples that illustrate the preparation
of compounds of Formula I according to the invention.
Example 1
6-{4-[4-(5-Trifluoromethyl-pyridin-2-yl)-[1,4]diazepan-1-ylmethyl]-1H-pyra-
zol-3-yl}-benzo[e][1,3]oxazine-2,4-dione
##STR00004## ##STR00005##
[0074] A mixture of 2-chloro-5-trifluoromethylpyridine (362 mg,
1.99 mmol) and [1,4]-diazepane (1000 mg, 9.98 mmol) in DMF (6.0 mL)
is stirred for 2 hours at room temperature followed by evaporation
of the solvent DMF under vacuo. The residue is distributed between
ethyl acetate (30 mL) and water (40 mL). The aqueous phase is
extracted once with ethyl acetate (20 m) and the combined organic
phases are dried with Na.sub.2SO.sub.4. Evaporation gives an oily
residue (crude 1-(5-trifluoromethyl-pyridin-2-yl)[1,4]-diazepane)
which is used directly for reductive amination in the last
step.
[0075] To the suspension of 5-acetylsalicylamide (900 mg, 5.02
mmol) in pyridine (3.5 mL) at 0.degree. C. under stirring is added
ethyl chloro-carbamate (600 mg, 5.53 mmol) dropwise and the
reaction is heated at 98.degree. C. overnight. Pyridine is
evaporated and the residue is distributed between ethyl acetate
(100 mL) and water (60 mL). The organic phase is washed with HCl
(2N, 50 mL) and water (60 mL) and dried with Na.sub.2SO.sub.4. As
the organic phase is evaporated, the product
6-acetyl-benzo[e][1,3]oxazine-2,4-dione starts to precipitate. The
precipitate is collected by filtration and then further
concentrated until the starting material (5-acetylsalicylamide)
begins to co-crystallize.
[0076] To a suspension of 6-acetyl-benzo[e][1,3]oxazine-2,4-dione
(626 mg, 3.05 mmol) obtained above in ethanol (5.0 mL), is added a
solution of NaOAc.3H.sub.2O (910 mg, 2.2 equiv.) and semicarbazide
hydrochloride (408 mg, 1.20 equiv.) in water (5.0 mL). The mixture
is heated at 92.degree. C. for 2 hours. After cooling to room
temperature, the product is collected by filtration, washed with
acetonitrile and dried under vacuum to give the corresponding
semicarbazone derivative.
[0077] To anhydrous DMF (2.50 mL) at 0.degree. C. under argon is
added POCl.sub.3 (0.60 mL, 1.01 g, 6.56 mmol) dropwise. After 10
minutes, the ice-bath is removed to allow the temperature to rise
to room temperature. The semicarbazone obtained above is added in
portions and the temperature is raised to and kept at 68.degree. C.
for 1 hour. The reaction is quenched into ice/water.
Na.sub.2CO.sub.3 is used to raise the pH to about 11.about.12
followed by neutralization with NH.sub.4Cl saturated aqueous
solution. The precipitate is collected by filtration and dried in
vacuum to give
3-(2,4-dioxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H-pyrazole-4-carba-
ldehyde.
[0078] The mixture of
3-(2,4-dioxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H-pyrazole-4-carba-
ldehyde (51.4 mg, 0.2 mmol) and
1-(5-trifluoromethyl-pyridin-2-yl)-[1,4]diazepane (24.5 mg, 0.1
mmol) in methylene chloride (1.0 mL), methanol (0.5 mL) and acetic
acid (15 .mu.L) is stirred for 30 minutes at room temperature. Then
Na(AcO).sub.3BH (84 mg, 4.0 equiv.) is added and the reaction is
stirred overnight at room temperature. After evaporation, the
residue is redissolved in DMF (1.0 mL) which is subject to
reverse-phase preparative LC-MS (acetonitrile/water/TFA gradient
10-90% CH.sub.3CN in 7.5 min, Ultro 120 5 uM C18Q, 75.times.30
mmID). The collected water/MeCN solution of the TFA salt of the
product is evaporated to remove the acetonitrile. A saturated
aqueous solution of NaHCO.sub.3 is added to raise the pH to about
8-9. Then ethyl acetate is used to extract the product and the
organic phase is dried with Na.sub.2SO.sub.4. Evaporation of the
solvent yields the free-based
6-{4-[4-(5-trifluoromethyl-pyridin-2-yl)-[1,4]diazepan-1-ylmethyl]-1H-pyr-
azol-3-yl}-benzo[e][1,3]oxazine-2,4-dione.
Example 2
6-(5-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl]meth-
yl)-2H-1,2,3-triazol-4-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one
##STR00006##
[0079] Example 2-1
Preparation of 2-(aminomethyl)-4-bromophenol
##STR00007##
[0081] 5-bromo-2-hydroxybenzamide (2.16 g, 10 mol) and NaBH.sub.4
(1.52 g, 40 mol) are dissolved in dry THF (150 ml) in a two-neck
septum capped round-bottom flask. Iodine (5.06 g, 20 mol)) in dry
THF is added under nitrogen atmosphere at 0.degree. C. over 2.5
hours. The reaction mixture is refluxed for 3 hours and then cooled
to 0.degree. C. The excess hydride is destroyed by careful addition
of 1N HCl. After removing most organic solvent by vacuum, the
acidic aqueous solution is diluted in 1N HCl (150 ml) and washed
three times with ether (30 ml each time). The pH of the aqueous
solution is adjusted to pH=6-7 by careful addition of sodium
bicarbonate solid. The product is collected by filtration and then
washed with water. The product is further dried in vacuum oven
overnight giving 2-(aminomethyl)-4-bromophenol which is used
directly in the next step without further purification.
Example 2-2
Preparation of 6-bromo-3,4-dihydrobenzo[e][1,3]oxazin-2-one
##STR00008##
[0083] A solution of 2-(aminomethyl)-4-bromophenol (I) (1.16 g, 5.7
mmol), triethylamthylamine (III) and carbonyldiimidazole (II) (1.86
g, 11.5 mmol) in anhydrous THF (100 ml) is refluxed for 4 hours.
The solvent is evaporated and the residue dissolved in
dichloromethane (DCM) (100 ml). The organic solution is washed with
1N HCl (20 ml.times.3) and then with brine (50 ml). The solvent is
then evaporated and the resulting residue is purified by
chromatography to give 6-bromo-3,4-dihydrobenzo[e][1,3]oxazin-2-one
as a white powder.
Example 2-3
Preparation of
3,4-dihydro-6-(3-hydroxyprop-1-ynyl)benzo[e][1,3]oxazin-2-one
##STR00009##
[0085] To a solution of
6-bromo-3,4-dihydrobenzo[e][1,3]oxazin-2-one (114 mg, 0.5 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (60 mg, 0.05 mmol), CuI (20 mg, 0.1
mmol), tetrabutylammonium iodide (74 mg, 0.2 mmol) in 1 mL of DMF
are added prop-2-yn-1-ol (150 mg, 2.5 mmol) and triethylamine (0.8
mL) via syringe under nitrogen atmosphere. The resultant mixture is
heated at 75.degree. C. until the aryl bromide is consumed, as
monitored by TLC. The cooled solution is partitioned between ethyl
acetate and water, and the organic layer is then washed with brine,
and dried over Na.sub.2SO.sub.4. Evaporation of the solution
followed by column chromatography afforded the coupling product:
3,4-dihydro-6-(3-hydroxyprop-1-ynyl)benzo[e][1,3]oxazin-2-one as
colorless oil. m/e: 204 (M+1)
Example 2-4
Preparation of
5-(3,4-dihydro-2-oxo-2H-benzo[e][1,3]oxazin-6-yl)-2H-1,2,3-triazole-4-car-
baldehyde
##STR00010##
[0087]
3,4-dihydro-6-(3-hydroxyprop-1-ynyl)benzo[e][1,3]oxazin-2-one (30
mg, 0.15 mmol) is dissolved in dimethyl formamide (DMF) (2 ml) and
is treated with Dess-Martin reagent (130 mg, 0.3 mmol) at room
temperature. After stirring for 1 hour, the reaction is quenched
with 1N HCl (10 ml) and then extracted with ethyl acetate (EtOAc)
(10 ml x5). The combined organic phase is washed with water, then
saturated aqueous ammonia chloride, and then brine. After drying
over sodium sulfate, the solvent is removed by vacuum to give crude
aldehyde which is used directly in the next step.
[0088] The crude aldehyde obtained above is dissolved in DMSO (2
ml) and then treated with sodium azide (98 mg, 1.5 mmol) at room
temperature. After stirring for 4 hours, the reaction is quenched
with aqueous sodium bicarbonate solution (10 ml) and then extracted
with EtOAc (10 ml x5). The combined organic phase is washed with
water, then saturated aqueous sodium bicarbonate, and then brine.
After drying over sodium sulfate, the solvent is removed by vacuum
to give the crude product which is then purified by flash
chromatography yeilding
5-(3,4-dihydro-2-oxo-2H-benzo[e][1,3]oxazin-6-yl)-2H-1,2,3-triazole-4-car-
baldehyde as a colorless oil. m/e: 245 (M+1).
Example 2-5
Preparation of
6-(54(R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methy-
l)-2H-1,2,3-triazol-4-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one
##STR00011##
[0090]
5-(3,4-dihydro-2-oxo-2H-benzo[e][1,3]oxazin-6-yl)-2H-1,2,3-triazole-
-4-carbaldehyde (15 mg, 0.06 mmol) and
(R)-1-(5-(trifluoromethyl)pyridin-2-yl)-2-methylpiperazine (20 mg,
0.08 mmol) are mixed in DMF (1 ml) followed by addition of acetic
acid (AcOH) (30u1). The mizture is stirred at room temperature for
30 minute and then sodium triacetate boron hydride (42 mg, 0.2
mmol) is added. The resulting suspension is then stirred at room
temperature overnight. The reaction is quenched using an aqueous
saturated solution of sodium bicarbonate and then extracted with
ethyl acetate (10 ml.times.4). The combined organic phase is
concentrated and the resulting crude product is purified by HPLC
giving
6-(5-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-
-yl)
methyl)-2H-1,2,3-triazol-4-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one
as a white powder. m/e:474 (M+1).
[0091] By repeating the procedures described in the above example,
using appropriate starting materials, the following compounds of
Formula I, as identified in Table 1, are obtained.
TABLE-US-00001 TABLE 1 Physical Data Compound MS (m/z): Number
Structure (M + 1) 2 ##STR00012## 473.2 3 ##STR00013## 487.2 4
##STR00014## 487.2 5 ##STR00015## 473.2 6 ##STR00016## 459.2 7
##STR00017## 527.2 8 ##STR00018## 459.2 9 ##STR00019## 459.2 10
##STR00020## 471.2 11 ##STR00021## 527.2 12 ##STR00022## 485.2 13
##STR00023## 499.2 14 ##STR00024## 491.2 15 ##STR00025## 432.2 16
##STR00026## 459.2 17 ##STR00027## 404.2 18 ##STR00028## 527.2 19
##STR00029## 527.2 20 ##STR00030## 489.2 21 ##STR00031## 22
##STR00032## 23 ##STR00033## 24 ##STR00034## 25 ##STR00035## 26
##STR00036## 27 ##STR00037## 430.2 28 ##STR00038## 474.2 29
##STR00039## 419.2 30 ##STR00040## 473.2 31 ##STR00041## 410.3 32
##STR00042## 474.2 33 ##STR00043## 501.2 34 ##STR00044## 485.2 35
##STR00045## 430.2 36 ##STR00046## 439.2 37 ##STR00047## 503.2 38
##STR00048## 489.2 39 ##STR00049## 491.2 40 ##STR00050## 472.2 41
##STR00051## 436.2 42 ##STR00052## 438.2 43 ##STR00053## 472.2 44
##STR00054## 473.2 45 ##STR00055## 459.2
Assays
[0092] Compounds of the present invention are assayed to measure
their capacity to inhibit ITPKb according to the following
assays:
[0093] Purification of ITPKb: The DNA sequence encoding murine
ITPKb residues 640-942 is amplified from a full-length construct in
mammalian expression vector pKDNZ by PCR. The 3'-primer
incorporates a stop codon and an overhanging Pad site. The product
is digested with Pad before being ligated into the MH4 plasmid
which has been prepared by digestion with PmlI and PacI. Cloning
into the MH4 plasmid adds the sequence MGSDKIHHHHHH to the
N-terminus of the translated region. Mutant enzymes are made by
site-directed mutagenesis using the Stratagene Quikchange kit.
[0094] ITPKb is expressed in the HK100 strain of Escherichia coli.
Typically, a 4 L batch of cells is grown in LB with 0.1 .mu.g/mL
ampicillin to 0.5A.sub.600 at 30 degrees C., before induction with
0.02% L-arabinose for 6 hours. Cells are harvested by
centrifugation, and pellets are resuspended in 50 mL of 50 mM Tris
(pH 8), 100 mM NaCl, 1 mM TCEP, and 0.1 mg/mL lysozyme, with 1
Complete protease inhibitor tablet (Roche). Cells are disrupted by
sonication, and debris is removed by centrifugation for 40 minutes
at 35000 g.
[0095] Initial purification is performed using three
nickel-Sepharose Hi-Trap HP 1 mL columns (Amersham) connected in
series. After application of the pellet supernatants, the bound
material is washed with 20 mM Tris (pH 8.0), 20 mM imidazole, 10%
glycerol (v/v), and 1 mM TCEP before elution with an imidazole
gradient up to 200 mM.
[0096] Fractions containing ITPKb are identified by SDS-PAGE, and
the pure fractions ae concentrated and buffer exchanged using
centriprep 20 15 kDa columns into 20 mM Tris (pH 8), 200 mM KCl, 5
mM MgCl.sub.2, 0.5 mM DTT, 10% glycerol, 1 .mu.M IP.sub.3, and 20
.mu.M ATP to a final protein concentration of 7 mg/mL.
[0097] Biochemical Measurement of ITPKb Activity: ITPKb activity is
determined using the Kinase-Glo (Promega) ATP depletion assay. The
assay reaction buffer consists of 50 mM Tris (pH 8.0), 100 mM NaCl,
1 mM DTT, 10% glycerol, 5 mM MgCl.sub.2, 1 .mu.M ATP, and 10 .mu.M
IP.sub.3 (Alexis Biochemicals). 50 nl of inhibitor is then added to
each 40 .mu.L reaction followed by a 10 .mu.L addition of purified
ITPKb (final concentration of 60 nM). The reaction mixture is
incubated for 60 minutes at room temperature and stopped by the
addition of an equal volume of kinase-glo reagent (Promega).
Luminescence is measured using a Molecular Devices Acquest
instrument.
[0098] Compounds of Formula I preferably have an IC.sub.50 of less
than 500 nM, preferably less than 250 nM, more preferably less than
100 nM at inhibiting the phosphorylation of IP3.
[0099] Measuring Intracellular IP3, IP4, and IP5 levels by HPLC:
Jurkat cells are obtained from ATCC (clone E6-1) (www.ATCC.org
Cat#TIB-152). 10.sup.7 cells in 1 ml of inositol free RPMI-1640 w/o
serum, are pulse labeled at 37.degree. C. for 6 hours with 15 uCi
of 3H myo-inositol in inositol. Cells are then diluted to 4 ml of
RPMI-1640 with 10% FBS and incubated overnight at 37.degree. C.
Cells are then concentrated and resuspended in 1 ml of RPMI-1640
w/10% FBS. 1 .mu.l of inhibitor in DMSO is then added. 50 .mu.g of
OKT3 and 10 .mu.g of anti-human CD28 (BD Pharmingen clone CD28.2)
is added followed by a 5 minute incubation at 37.degree. C. Cells
are then concentrated and the reaction quenched with the
resuspension of the cell pellet in 100 .mu.L of PBS w/350 mM HCl.
Extracts are then spun to remove proteins and cellular debris.
Labeled inositol polyphosphates in the extracts are then resolved
by HPLC on a Partisphere SAX column (15 cm.times.4.6 mm). Samples
are eluted as follows with gradients generated by mixing buffer A
(10 mM (NH.sub.4)H.sub.2PO.sub.4, pH 3.35, with H.sub.3PO.sub.4)
with buffer B (1.7 M (NH.sub.4)H.sub.2PO.sub.4, pH 3.35, with
H.sub.3PO.sub.4). 0-12.5 minutes 0-100% Buffer B; 12-5-25 minutes
100% Buffer B; 25-30 minutes 0-100% buffer A; 30-45 minutes 100%
buffer A. Radioactivity is detected with an online .beta.-Ram
detector from IN/US systems.
[0100] Compounds of Formula I preferably have an IC.sub.50 of less
than 1 .mu.M, more preferably less than 500 nM in inhibiting the
conversion of IP3 to IP4.
[0101] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference for all purposes.
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