U.S. patent application number 10/999128 was filed with the patent office on 2005-10-13 for protein kinase inhibitors and uses thereof.
Invention is credited to Choquette, Deb, Davies, Robert, Green, Jeremy, Ledeboer, Mark, Moon, Young Choon, Pierce, Albert.
Application Number | 20050228005 10/999128 |
Document ID | / |
Family ID | 23151418 |
Filed Date | 2005-10-13 |
United States Patent
Application |
20050228005 |
Kind Code |
A1 |
Moon, Young Choon ; et
al. |
October 13, 2005 |
Protein kinase inhibitors and uses thereof
Abstract
Described herein are benzisoxazole compounds of formula I: 1 or
a pharmaceutically acceptable derivative or prodrug thereof,
wherein A-B is N--O or O--N; Ar is an optionally substituted
C.sub.5-10 aryl group; R.sup.1 is hydrogen or an optionally
substituted group selected from C.sub.1-10 aliphatic, C.sub.5-10
aryl, C.sub.6-12 aralkyl, C.sub.3-10 heterocyclyl, or C.sub.4-12
heterocyclylalkyl; and T, n, R.sup.2 and R.sup.3 are as described
in the specification. These compounds are inhibitors of protein
kinases, particularly inhibitors of GSK-3 and JAK mammalian protein
kinases. The invention also provides pharmaceutically acceptable
compositions comprising the compounds of the invention and methods
of utilizing those compounds and compositions in the treatment of
various protein kinase mediated disorders.
Inventors: |
Moon, Young Choon;
(Lexington, MA) ; Green, Jeremy; (Burlington,
MA) ; Davies, Robert; (Arlington, MA) ;
Choquette, Deb; (Medford, MA) ; Pierce, Albert;
(Cambridge, MA) ; Ledeboer, Mark; (Acton,
MA) |
Correspondence
Address: |
CHOATE, HALL & STEWART LLP
TWO INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Family ID: |
23151418 |
Appl. No.: |
10/999128 |
Filed: |
November 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10999128 |
Nov 29, 2004 |
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10172888 |
Jun 14, 2002 |
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6825190 |
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60298646 |
Jun 15, 2001 |
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Current U.S.
Class: |
514/275 ;
544/331 |
Current CPC
Class: |
A61P 37/00 20180101;
A61P 11/06 20180101; A61P 35/02 20180101; A61P 25/02 20180101; A61P
37/02 20180101; A61P 43/00 20180101; A61P 35/00 20180101; A61P 1/16
20180101; A61P 25/28 20180101; A61P 37/08 20180101; A61P 19/02
20180101; A61P 25/18 20180101; A61P 9/10 20180101; A61P 9/04
20180101; A61P 9/00 20180101; A61P 3/10 20180101; A61P 25/16
20180101; A61P 37/06 20180101; C07D 413/04 20130101; A61P 21/00
20180101; A61P 25/14 20180101; A61P 29/00 20180101; C07D 413/14
20130101; A61P 25/00 20180101 |
Class at
Publication: |
514/275 ;
544/331 |
International
Class: |
A61K 031/506; C07D
043/02; C07D 043/14 |
Claims
1-10. (canceled)
11. A composition comprising a compound of formula I: 116or a
pharmaceutically acceptable salt thereof, wherein: A-B is N--O or
O--N; Ar is an optionally substituted C.sub.5-10 aryl group; T is a
C.sub.1-4 alkylidene chain wherein one or two methylene units of T
are optionally and independently replaced by O, NR, S, C(O),
C(O)NR, NRC(O)NR, SO.sub.2, SO.sub.2NR, NRSO.sub.2, NRSO.sub.2NR,
CO.sub.2, OC(O), NRCO.sub.2, or OC(O)NR; n is zero or one; R.sup.1
is hydrogen or an optionally substituted group selected from
C.sub.1-10 aliphatic. C.sub.5-10 aryl, C.sub.6-12 aralkyl,
C.sub.3-10 heterocyclyl, or C.sub.4-12 heterocyclylalkyl; each
R.sup.2 is independently selected from R, halo, CN, OR, N(R).sub.2,
SR, C(.dbd.O)R, CO.sub.2R, CONR.sub.2, NRC(.dbd.O)R,
NRCO.sub.2(C.sub.1-6 aliphatic), OC(.dbd.O)R, SO.sub.2R,
S(.dbd.O)R, SO.sub.2NR.sub.2, or NRSO.sub.2(C.sub.1-6 aliphatic);
each R.sup.3 is independently selected from R, halo, CN, OR,
N(R).sub.2, SR, C(.dbd.O)R, CO.sub.2R, CONR.sub.2, NRC(.dbd.O)R,
NRCO.sub.2(C.sub.1-6 aliphatic), OC(.dbd.O)R, SO.sub.2R,
S(.dbd.O)R, SO.sub.2NR.sub.2, or NRSO.sub.2(C.sub.1-6 aliphatic);
and each R is independently selected from hydrogen, a C.sub.1-8
aliphatic group, or two R on the same nitrogen are taken together
with the nitrogen to form a 4-8 membered heterocyclic ring having
1-3 heteroatoms selected from nitrogen, oxygen or sulfur, and a
pharmaceutically acceptable carrier, adjuvant or vehicle,
additionally comprising an additional therapeutic agent selected
from an a chemotherapeutic or anti-proliferative agent, a treatment
for Alzheimer's Disease, a treatment for Parkinson's Disease, an
agent for treating Multiple Sclerosis (MS), a treatment for asthma,
an anti-inflammatory agent, an immunomodulatory or
immunosuppressive agent, a neurotrophic factor, an agent for
treating cardiovascular disease, an agent for treating liver
disease, an agent for treating a blood disorder, or an agent for
treating an immunodeficiency disorder.
12. (canceled)
13. A method of treating or lessening the severity of a GSK-3- or
JAK-mediated disease or condition in a patient, comprising the step
of administering to said patient: a compound of formula I: 117or a
pharmaceutically acceptable salt thereof, wherein: A-B is N-0 or
O--N; Ar is an optionally substituted C.sub.5-10 aryl group; T is a
C.sub.1-4 alkylidene chain wherein one or two methylene units of T
are optionally and independently replaced by O, NR, S, C(O),
C(O)NR, NRC(O)NR, SO.sub.2, SO.sub.2NR, NRSO.sub.2, NRSO.sub.2NR,
CO.sub.2, OC(O), NRCO.sub.2, or OC(O)NR; n is zero or one; R.sup.1
is hydrogen or an optionally substituted group selected from
C.sub.1-10 aliphatic, C.sub.5-10 aryl, C.sub.6-12 aralkyl, C
.sub.3-10 heterocyclyl, or C.sub.4-12 heterocyclylalkyl; each
R.sup.2 is independently selected from R, halo, CN, OR, N(R).sub.2,
SR, C(.dbd.O)R, CO.sub.2R, CONR.sub.2, NRC(.dbd.O)R,
NRCO.sub.2(C.sub.1-6aliphatic), OC(.dbd.O)R, SO.sub.2R, S(.dbd.O)R,
SO.sub.2NR.sub.2, or NRSO.sub.2(C.sub.1-6 aliphatic); each R.sup.3
is independently selected from R, halo, CN, OR, N(R).sub.2, SR,
C(.dbd.O)R, CO.sub.2R, CONR.sub.2, NRC(.dbd.O)R,
NRCO.sub.2(C.sub.1-6aliphatic), OC(.dbd.O)R, SO.sub.2R, S(.dbd.O)R,
SO.sub.2NR.sub.2, or NRSO.sub.2(C.sub.1-6 aliphatic), and each R is
independently selected from hydrogen, a C.sub.1-8 aliphatic group,
or two R on the same nitrogen are taken together with the nitrogen
to form a 4-8 membered heterocyclic ring having 1-3 heteroatoms
selected from nitrogen, oxygen or sulfur.
14. The method according to claim 13, wherein said GSK-3-mediated
disease is selected from diabetes, Alzheimer's disease,
Huntington's disease, Parkinson's, AIDS-associated dementia,
amyotrophic lateral sclerosis (AML), multiple sclerosis (MS),
schizophrenia, cardiomycete hypertrophy, ischemia/reperfusion and
baldness.
15. The method according to claim 13, wherein said JAK-mediated
disease is selected from an immune response, an autoimmune disease,
a neurodegenerative disease, or a solid or hematologic
malignancy.
16. (canceled)
17. The method according to claim 13, comprising the additional
step of administering to said patient an additional therapeutic
agent selected from a chemotherapeutic or anti-proliferatic agent,
a treatment for Alzheimer's Disease, a treatment for Parkinson's
Disease, an agent for treating Multiple Sclerosis (MS), a treatment
for asthma, an agent for treating schizophrenia, an
anti-inflammatory agent, an immunomodulatory or immunosuppressive
agent, a neurotrophic factor, an agent for treating cardiovascular
disease, an agent for treating liver disease, an agent for treating
a blood disorder, or an agent for treating an immunodeficiency
disorder, wherein: said additional therapeutic agent is appropriate
for the disease being treated; and said additional therapeutic
agent is administered together with said composition as a single
dosage form or separately from said composition as part of a
multiple dosage form.
18. A method of inhibiting the production of hyperphosphorylated
Tau protein in a patient in need thereof, which method comprises
administering to the patient a therapeutically effective amount of
a compound of formula I: 118or a pharmaceutically acceptable salt
thereof, wherein: A-B is N--O or O--N; Ar is an optionally
substituted C.sub.5-10 aryl group; T is a C.sub.1-4 alkylidene
chain wherein one or two methylene units of T are optionally and
independently replaced by O, NR, S, C(O), C(O)NR, NRC(O)NR,
SO.sub.2, SO.sub.2NR, NRSO.sub.2, NRSO.sub.2NR, CO.sub.2, OC(O),
NRCO.sub.2, or OC(O)NR; n is zero or one; R.sup.1 is hydrogen or an
optionally substituted group selected from C.sub.1-10 aliphatic,
C.sub.5-10 aryl, C.sub.6-12 aralkyl, C.sub.3-10 heterocyclyl, or
C.sub.4-12 heterocyclylalkyl; each R.sup.2 is independently
selected from R, halo, CN, OR, N(R).sub.2, SR, C(.dbd.O)R,
CO.sub.2R, CONR.sub.2, NRC(.dbd.O)R,
NRCO.sub.2(C.sub.1-6aliphatic), OC(.dbd.O)R, SO.sub.2R, S(.dbd.O)R,
SO.sub.2NR.sub.2, or NRSO.sub.2(C.sub.1-6 aliphatic); each R.sup.3
is independently selected from R, halo, CN, OR, N(R).sub.2, SR,
C(.dbd.O)R, CO.sub.2R, CONR.sub.2, NRC(.dbd.O)R,
NRCO.sub.2(C.sub.1-6 aliphatic), OC(.dbd.O)R, SO.sub.2R,
S(.dbd.O)R, SO.sub.2NR.sub.2, or NRSO.sub.2(C.sub.1-6 aliphatic);
and each R is independently selected from hydrogen, a C.sub.1-8
aliphatic group, or two R on the same nitrogen are taken together
with the nitrogen to form a 4-8 membered heterocyclic ring having
1-3 heteroatoms selected from nitrogen, oxygen or sulfur.
19. A method of inhibiting the phosphorylation of .beta.-catenin in
a patient in need thereof, which method comprises administering to
the patient a therapeutically effective amount of a compound of
formula I: 119or a pharmaceutically acceptable salt thereof,
wherein: A-B is N--O or O--N; Ar is an optionally substituted
C.sub.5-10 aryl group: T is a C.sub.1-4 alkylidene chain wherein
one or two methylene units of T are optionally and independently
replaced by O, NR, S, C(O), C(O)NR, NRC(O)NR, SO.sub.2, SO.sub.2NR,
NRSO.sub.2, NRSO.sub.2NR, CO.sub.2, OC(O), NRCO.sub.2, or OC(O)NR;
n is zero or one: R.sup.1 is hydrogen or an optionally substituted
group selected from C.sub.1-10 aliphatic, C.sub.5-10 aryl,
C.sub.6-12 aralkyl, C.sub.3-10heterocyclyl, or C.sub.4-12
heterocyclylalkyl; each R.sup.2 is independently selected from R,
halo, CN, OR, N(R).sub.2, SR, C(.dbd.O)R, CO.sub.2R, CONR.sub.2,
NRC(.dbd.O)R, NRCO.sub.2(C.sub.1-6aliphatic), OC(.dbd.O)R,
SO.sub.2R, S(.dbd.O)R, SO.sub.2NR.sub.2, or NRSO.sub.2(C.sub.1-6
aliphatic); each R.sup.3 is independently selected from R, halo,
CN, OR, N(R).sub.2, SR, C(.dbd.O)R, CO.sub.2R, CONR.sub.2,
NRC(.dbd.O)R, NRCO.sub.2(C.sub.1-6 aliphatic), OC(.dbd.O)R,
SO.sub.2R, S(.dbd.O)R, SO.sub.2NR.sub.2, or NRSO.sub.2(C.sub.1-6
aliphatic); and each R is independently selected from hydrogen, a
C.sub.1-8 aliphatic group, or two R on the same nitrogen are taken
together with the nitrogen to form a 4-8 membered heterocyclic ring
having 1-3 heteroatoms selected from nitrogen, oxygen or
sulfur.
20. A composition for coating an implantable device comprising a
compound of formula I: 120or a pharmaceutically acceptable salt
thereof, wherein: A-B is N--O or O--N; Ar is an optionally
substituted C.sub.5-10 aryl group; T is a C.sub.1-4 alkylidene
chain wherein one or two methylene units of T are optionally and
independently replaced by O, NR, S, C(O), C(O)NR, NRC(O)NR,
SO.sub.2, SO.sub.2NR, NRSO.sub.2, NRSO.sub.2NR, CO.sub.2, OC(O),
NRCO.sub.2, or OC(O)NR; n is zero or one; R.sup.1 is hydrogen or an
optionally substituted group selected from C.sub.1-10 aliphatic,
C.sub.5-10 aryl, C.sub.6-12 aralkyl, C.sub.3-10 heterocyclyl, or
C.sub.4-12 heterocyclylalkyl; each R.sup.2 is independently
selected from R, halo, CN, OR, N(R).sub.2, SR, C(.dbd.O)R,
CO.sub.2R, CONR.sub.2, NRC(.dbd.O)R,
NRCO.sub.2(C.sub.1-6aliphatic), OC(.dbd.O)R, SO.sub.2R, S(.dbd.O)R,
SO.sub.2NR.sub.2, or NRSO.sub.2(C.sub.1-6 aliphatic); each R.sup.3
is independently selected from R, halo, CN, OR, N(R).sub.2, SR,
C(.dbd.O)R, CO.sub.2R, CONR.sub.2, NRC(.dbd.O)R,
NRCO.sub.2(C.sub.1-6alip- hatic), OC(.dbd.O)R, SO.sub.2R,
S(.dbd.O)R, SO.sub.2NR.sub.2, or NRSO.sub.2(C.sub.1-6 aliphatic);
and each R is independently selected from hydrogen, a C.sub.1-8
aliphatic group, or two R on the same nitrogen are taken together
with the nitrogen to form a 4-8 membered heterocyclic ring having
1-3 heteroatoms selected from nitrogen, oxygen or sulfur, and a
carrier suitable for coating said implantable device.
21. An implantable device coated with a composition according to
claim 20.
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of medicinal chemistry
and relates to compounds that are protein kinase inhibitors,
compositions comprising such compounds and methods of use. More
particularly, the compounds are inhibitors of GSK-3 and JAK and are
useful for treating disease states, such as diabetes and
Alzheimer's disease, that are alleviated by GSK-3 inhibitors, and
allergic disorders, autoimmune diseases, and conditions associated
with organ transplantation that are alleviated by JAK
inhibitors.
BACKGROUND OF THE INVENTION
[0002] The search for new therapeutic agents has been greatly aided
in recent years by a better understanding of the structure of
enzymes and other biomolecules associated with target diseases. One
important class of enzymes that has been the subject of extensive
study is the protein kinases.
[0003] Protein kinases mediate intracellular signal transduction.
They do this by effecting a phosphoryl transfer from a nucleoside
triphosphate to a protein acceptor that is involved in a signaling
pathway. There are a number of kinases and pathways through which
extracellular and other stimuli cause a variety of cellular
responses to occur inside the cell. Examples of such stimuli
include environmental and chemical stress signals (e.g. osmotic
shock; heat shock, ultraviolet radiation, bacterial endotoxin,
H.sub.2O.sub.2), cytokines (e.g. interleukin-1 (IL-1) and tumor
necrosis factor .alpha. (TNF-.alpha.)), and growth factors (e.g.
granulocyte macrophage-colony-stimulating factor (GM-CSF), and
fibroblast growth factor (FGF)). An extracellular stimulus may
effect one or more cellular responses related to cell growth,
migration, differentiation, secretion of hormones, activation of
transcription factors, muscle contraction, glucose metabolism,
control of protein synthesis and regulation of cell cycle.
[0004] Many disease states are associated with abnormal cellular
responses triggered by protein kinase-mediated events. These
diseases include autoimmune diseases, inflammatory diseases,
metabolic diseases, neurological and neurodegenerative diseases,
cancer, cardiovascular diseases, allergies and asthma, Alzheimer's
disease or hormone-related diseases. Accordingly, there has been a
substantial effort in medicinal chemistry to find protein kinase
inhibitors that are effective as therapeutic agents. A challenge
has been to find protein kinase inhibitors that act in a selective
manner. Since there are numerous protein kinases that are involved
in a variety of cellular responses, non-selective inhibitors may
lead to unwanted side effects.
[0005] Glycogen synthase kinase-3 (GSK-3) is a serine/threonine
protein kinase comprised of .alpha. and .beta. isforms that are
each encoded by distinct genes [Coghlan et al., Chemistry &
Biology, 7, 793-803 (2000); Kim and Kimmel, Curr. Opinion Genetics
Dev., 10, 508-514 (2000)]. GSK-3 has been implicated in various
diseases including diabetes, Alzheimer's disease, CNS disorders
such as manic depressive disorder and neurodegenerative diseases,
and cardiomyocete hypertrophy [WO 99/65897; WO 00/38675; and Haq et
al., J. Cell Biol. (2000) 151, 117]. These diseases may be caused
by, or result in, the abnormal operation of certain cell signaling
pathways in which GSK-3 plays a role. GSK-3 has been found to
phosphorylate and modulate the activity of a number of regulatory
proteins. These include glycogen synthase which is the rate
limiting enzyme necessary for glycogen synthesis, the microtubule
associated protein Tau, the gene transcription factor
.beta.-catenin, the translation initiation factor e1F2B, as well as
ATP citrate lyase, axin, heat shock factor-1, c-Jun, c-Myc, c-Myb,
CREB, and CEPB.alpha.. These diverse targets implicate GSK-3 in
many aspects of cellular metabolism, proliferation, differentiation
and development.
[0006] In a GSK-3 mediated pathway that is relevant for the
treatment of type II diabetes, insulin-induced signaling leads to
cellular glucose uptake and glycogen synthesis. Along this pathway,
GSK-3 is a negative regulator of the insulin-induced signal.
Normally, the presence of insulin causes inhibition of GSK-3
mediated phosphorylation and deactivation of glycogen synthase. The
inhibition of GSK-3 leads to increased glycogen synthesis and
glucose uptake [Klein et al., PNAS, 93, 8455-9 (1996); Cross et
al., Biochem. J., 303, 21-26 (1994); Cohen, Biochem. Soc. Trans.,
21, 555-567 (1993); Massillon et al., Biochem J. 299, 123-128
(1994)]. However, in a diabetic patient where the insulin response
is impaired, glycogen synthesis and glucose uptake fail to increase
despite the presence of relatively high blood levels of insulin.
This leads to abnormally high blood levels of glucose with acute
and chronic effects that may ultimately result in cardiovascular
disease, renal failure and blindness. In such patients, the normal
insulin-induced inhibition of GSK-3 fails to occur. It has also
been reported that in patients with type II diabetes, GSK-3 is
overexpressed [WO 00/38675]. Therapeutic inhibitors of GSK-3 are
therefore potentially useful for treating diabetic patients
suffering from an impaired response to insulin.
[0007] GSK-3 activity has also been associated with Alzheimer's
disease. This disease is characterized by the well-known
.beta.-amyloid peptide and the formation of intracellular
neurofibrillary tangles. The neurofibrillary tangles contain
hyperphosphorylated Tau protein where Tau is phosphorylated on
abnormal sites. GSK-3 has been shown to phosphorylate these
abnormal sites in cell and animal models. Furthermore, inhibition
of GSK-3 has been shown to prevent hyperphosphorylation of Tau in
cells [Lovestone et al., Current Biology 4, 1077-86 (1994);
Brownlees et al., Neuroreport 8, 3251-55 (1997)]. Therefore, it is
believed that GSK-3 activity may promote generation of the
neurofibrillary tangles and the progression of Alzheimer's
disease.
[0008] Another substrate of GSK-3 is .beta.-catenin which is
degradated after phosphorylation by GSK-3. Reduced levels of
.beta.-catenin have been reported in schizophrenic patients and
have also been associated with other diseases related to increase
in neuronal cell death [Zhong et al., Nature, 395, 698-702 (1998);
Takashima et al., PNAS, 90, 7789-93 (1993); Pei et al., J.
Neuropathol. Exp, 56, 70-78 (1997); Smith et al., Bio-org. Med.
Chem. 11, 635-639 (2001)].
[0009] Small molecule inhibitors of GSK-3 have recently been
reported [WO 99/65897 (Chiron) and WO 00/38675 (SmithKline
Beecham)].
[0010] The Janus kinases (JAK) are a family of tyrosine kinases
consisting of JAK1, JAK2, JAK3 and TYK2. The JAKs play a critical
role in cytokine signaling. The down-stream substrates of the JAK
family of kinases include the signal transducer and activator of
transcription (STAT) proteins. JAK/STAT signaling has been
implicated in the mediation of many abnormal immune responses such
as allergies, asthma, autoimmune diseases such as transplant
rejection, rheumatoid arthritis, amyotrophic lateral sclerosis and
multiple sclerosis as well as in solid and hematologic malignancies
such as leukemias and lymphomas. The pharmaceutical intervention in
the JAK/STAT pathway has been reviewed [Frank Mol. Med. 5: 432-456
(1999) & Seidel, et al, Oncogene 19: 2645-2656 (2000)1.
[0011] JAK1, JAK2, and TYK2 are ubiquitously expressed, while JAK3
is predominantly expressed in hematopoietic cells. JAK3 binds
exclusively to the common cytokine receptor gamma chain
(.gamma..sub.c) and is activated by IL-2, IL-4, IL-7, IL-9, and
IL-15. The proliferation and survival of murine mast cells induced
by IL-4 and IL-9 have, in fact, been shown to be dependent on JAK3-
and .gamma..sub.c-signaling [Suzuki et al, Blood 96: 2172-2180
(2000)].
[0012] Cross-linking of the high-affinity immunoglobulin (Ig) E
receptors of sensitized mast cells leads to a release of
proinflammatory mediators, including a number of vasoactive
cytokines resulting in acute allergic, or immediate (type I)
hypersensitivity reactions [Gordon et al, Nature 346: 274-276
(1990) & Galli, N. Engl. J. Med., 328: 257-265 (1993)]. A
crucial role for JAK3 in IgE receptor-mediated mast cell responses
in vitro and in vivo has been established (Malaviya, et al,
Biochem. Biophys. Res. Commun. 257: 807-813 (1999)). In addition,
the prevention of type I hypersensitivity reactions, including
anaphylaxis, mediated by mast cell-activation through inhibition of
JAK3 has also been reported [Malaviya et al, J. Biol. Chem.
274:27028-27038 (1999)]. Targeting mast cells with JAK3 inhibitors
modulated mast cell degranulation in vitro and prevented IgE
receptor/antigen-mediated anaphylactic reactions in vivo.
[0013] A recent study described the successful targeting of JAK3
for immunosuppression and allograft acceptance. The study
demonstrated a dose-dependent survival of Buffalo heart allograft
in Wistar Furth recipients upon administration of inhibitors of
JAK3 indicating the possibility of regulating unwanted immune
responses in graft versus host disease [Kirken, transpl. proc. 33:
3268-3270 (2001)].
[0014] IL-4-mediated STAT-phosphorylation has been implicated as
the mechanism involved in early and late stages of rheumatoid
arthritis (RA). Up-regulation of proinflammatory cytokines in RA
synovium and synovial fluid is a characteristic of the disease. It
has been demostrated that IL-4-mediated activation of IL-4/STAT
pathway is mediated through the Janus Kinases (JAK 1 & 3) and
that IL-4-associated JAK kinases are expressed in the RA synovium
[Muller-Ladner, et al, J. Immunol. 164: 3894-3901 (2000)].
[0015] Familial amyoptrophic lateral sclerosis (FALS) is a fatal
neurodegenerative disorder affecting about 10% of ALS patients. The
survival rates of FALS mice were increased upon treatment with a
JAK3 specific inhibitor. This suggested that JAK3 plays a role in
FALS [Trieu, et al, Biochem. Biophys. Res. Commun. 267: 22-25
(2000)].
[0016] Signal transducer and activator of transcription (STAT)
proteins are activated by, among others, the JAK family kinases.
Results from a recent study suggested the possibility of
intervention in the JAK/STAT signaling pathway by targeting JAK
family kinases with specific inhibitors for the treatment of
leukemia [Sudbeck, et al, Clin. Cancer Res. 5: 1569-1582 (1999)].
JAK3 specific compounds were shown to inhibit the clonogenic growth
of JAK3-expressing cell lines DAUDI, RAMOS, LCl; 19, NALM-6, MOLT-3
and HL-60.
[0017] In animal models, TEL/JAK2 fusion proteins have induced
myeloproliferative disorders and in hematopoietic cell lines,
introduction of TEL/JAK2 resulted in activation of STAT1, STAT3,
STAT5, and cytokine-independent growth [Schwaller, et al, EMBO J.
17: 5321-5333 (1998)].
[0018] Inhibition of JAK3 and TYK2 abrogated tyrosine
phosphorylation of STAT3, and inhibited cell growth of mycosis
fungoides, a form of cutaneous T cell lymphoma. These results
implicated JAK family kinases in the constitutively activated
JAK/STAT pathway that is present in mycosis fungoides [Nielsen, et
al, Proc. Nat. Acad. Sci. U.S.A. 94: 6764-6769 (1997)]. Similarly,
STAT3, STAT5, JAK1 and JAK2 were demonstrated to be constitutively
activated in mouse T cell lymphoma characterized initially by LCK
over-expression, thus further implicating the JAK/STAT pathway in
abnormal cell growth [Yu, et al, J. Immunol. 159: 5206-5210
(1997)]. In addition, IL-6-mediated STAT3 activation was blocked by
an inhibitor of JAK, leading to sensitization of myeloma cells to
apoptosis [Catlett-Falcone, et al, Immunity 10:105-115 (1999)].
[0019] There is a continued need to find new therapeutic agents to
treat human diseases. Accordingly, there is a great need to develop
inhibitors of GSK-3 and JAK protein kinases that are useful in
treating various diseases or conditions associated with GSK-3 and
JAK activation, particularly given the inadequate treatments
currently available for the majority of these disorders.
DESCRIPTION OF THE INVENTION
[0020] It has now been found that compounds of this invention and
pharmaceutical compositions thereof are effective as protein kinase
inhibitors, particularly as inhibitors of GSK-3 and JAK. These
compounds have the general formula I: 2
[0021] or a pharmaceutically acceptable derivative or prodrug
thereof, wherein:
[0022] A-B is N--O or O--N;
[0023] Ar is an optionally substituted C.sub.5-10 aryl group;
[0024] T is a C.sub.1-4 alkylidene chain wherein one or two
methylene units of T are optionally and independently replaced by
O, NR, S, C(O), C(O)NR, NRC(O)NR, SO.sub.2, SO.sub.2NR, NRSO.sub.2,
NRSO.sub.2NR, CO.sub.2, OC(O), NRCO.sub.2, or OC(O)NR;
[0025] n is zero or one;
[0026] R.sup.1 is hydrogen or an optionally substituted group
selected from C.sub.1-10 aliphatic, C.sub.5-10 aryl, C.sub.6-12
aralkyl, C.sub.3-10 heterocyclyl, or C.sub.4-12
heterocyclylalkyl;
[0027] each R.sup.2 is independently selectea from R, halo, CN, OR,
N(R).sub.2, SR, C(.dbd.O)R, CO.sub.2R, CONR.sub.2, NRC(.dbd.O)R,
NRCO.sub.2(C.sub.1-6 aliphatic), OC(.dbd.O)R, SO.sub.2R,
S(.dbd.O)R, SO.sub.2NR.sub.2, or NRSO.sub.2(C.sub.16
aliphatic);
[0028] each R.sup.3 is independently selected from R, halo, CN, OR,
N(R).sub.2, SR, C(.dbd.O)R, CO.sub.2R, CONR.sub.2, NRC(.dbd.O)R,
NRCO.sub.2(C.sub.1-6 aliphatic), OC(.dbd.O)R, SO.sub.2R,
S(.dbd.O)R, SO.sub.2NR.sub.2, or NRSO.sub.2(C.sub.1-6 aliphatic);
and
[0029] each R is independently selected from hydrogen, a C.sub.1-8
aliphatic group, or two R on the same nitrogen are taken together
with the nitrogen to form a 4-8 membered heterocyclic ring having
1-3 heteroatoms selected from nitrogen, oxygen or sulfur.
[0030] As used herein, the following definitions shall apply unless
otherwise indicated.
[0031] The phrase "optionally substituted" is used interchangeably
with the phrase "substituted or unsubstituted." Unless otherwise
indicated, an optionally substituted group may have a substituent
at each substitutable position of the group, and each substitution
is independent of the other.
[0032] The term "aliphatic" or "aliphatic group" as used herein
means a straight-chain or branched C.sub.1-C.sub.10 hydrocarbon
chain that is completely saturated or that contains one or more
units of unsaturation, or a monocyclic C.sub.3-C.sub.8 hydrocarbon
or bicyclic C.sub.8-C.sub.12 hydrocarbon that is completely
saturated or that contains one or more units of unsaturation, but
which is not aromatic (also referred to herein as "carbocycle" or
"cycloalkyl"), that has a single point of attachment to the rest of
the molecule wherein any individual ring in said bicyclic ring
system has 3-7 members. For example, suitable aliphatic groups
include substituted or unsubstituted linear or branched alkyl,
alkenyl, or alkynyl groups and hybrids thereof such as
(cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
[0033] The terms "alkyl", "alkoxy", "hydroxyalkyl", "alkoxyalkyl",
and "alkoxycarbonyl", used alone or as part of a larger moiety
include both straight and branched chains containing one to twelve
carbon atoms. The terms "alkenyl" and "alkynyl" used alone or as
part of a larger moiety shall include both straight and branched
chains containing two to twelve carbon atoms.
[0034] The terms "haloalkyl", "haloalkenyl" and "haloalkoxy" means
alkyl, alkenyl or alkoxy, as the case may be, substituted with one
or more halogen atoms. The term "halogen" means F, Cl, Br, or
I.
[0035] The term "heteroatom" means nitrogen, oxygen or sulfur and
includes any oxidized form of nitrogen and sulfur, and the
quaternized form of any basic nitrogen. Also, the term "nitrogen"
includes a substitutable nitrogen of a heterocyclic ring. As an
example, in a saturated or partially unsaturated ring having 0-3
heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen
may be N (as in 3,4-dihydro-2H-pyrrolyl)- , NH (as in pyrrolidinyl)
or NR.sup.+ (as in N-substituted pyrrolidinyl). It is understood
that the compounds of this invention are limited to those that can
exist in nature as stable chemical compounds.
[0036] The term "unsaturated", as used herein, means that a moiety
has one or more units of unsaturation, and includes aryl rings.
[0037] The term "aryl", used alone or as part of a larger moiety as
in "aralkyl", "aralkoxy", or "aryloxyalkyl", refers to monocyclic,
bicyclic and tricyclic ring systems having a total of five to
fourteen ring members, wherein at least one ring in the system is
aromatic and wherein each ring in the system contains 3 to 7 ring
members. The term "aryl" may be used interchangeably with the term
"aryl ring". The term "aryl" also refers to heteroaryl ring systems
as defined hereinbelow.
[0038] The term "heterocycle", "heterocyclyl", or "heterocyclic",
as used herein means non-aromatic, monocyclic, bicyclic, or
tricyclic ring systems having five to fourteen ring members in
which one or more ring members is a heteroatom, wherein each ring
in the system contains 3 to 7 ring members.
[0039] The term "heteroaryl", used alone or as part of a larger
moiety as in "heteroaryalkyl" or "heteroarylalkoxy", refers to
monocyclic, bicyclic and tricyclic ring systems having a total of
five to fourteen ring members, wherein at least one ring in the
system is aromatic, at least one ring in the system contains one or
more heteroatoms, and wherein each ring in the system contains 3 to
7 ring members. The term "heteroaryl" may be used interchangeably
with the term "heteroaryl ring" or the term "heteroaromatic".
[0040] An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the
like) or heteroaryl (including heteroaralkyl and heteroarylalkoxy
and the like) group may contain one or more substituents. Suitable
substituents on the unsaturated carbon atom of an aryl, heteroaryl,
aralkyl, or heteroaralkyl group are independently selected from
halogen, --R.sup.o, --OR.sup.o, --O(CH.sub.2).sub.yR.sup.o,
--SR.sup.o, 1,2-methylene-dioxy, 1,2-ethylenedioxy, phenyl (Ph)
optionally substituted with R.sup.o, --O(Ph) optionally substituted
with R.sup.o, --CH.sub.2(Ph) optionally substituted with R.sup.o,
--CH.sub.2CH.sub.2(Ph) optionally substituted with R.sup.o, 5-8
membered heteroaryl optionally substituted with R.sup.o, 5-8
membered heterocycle optionally substituted with R.sup.o,
--NO.sub.2, --CN, --N(R.sup.o).sub.2, --N(R.sup.o)
(CH.sub.2).sub.yR.sup.o, --NR.sup.oC(O)R.sup.o,
--NR.sup.oC(O)N(R.sup.o).- sub.2, --NR.sup.oCO.sub.2R.sup.o,
--NR.sup.oNR.sup.oC(O)R.sup.o,
--NR.sup.oNR.sup.oC(O)N(R.sup.o).sub.2,
--NR.sup.oNR.sup.oCO.sub.2R.sup.o- , --C(O)C(O)R.sup.o,
--C(O)CH.sub.2C(O)R.sup.o, --CO.sub.2R.sup.o, --C(O)R.sup.o,
--C(O)N(R.sup.o).sub.2, --OC(O)N(R.sup.o).sub.2,
--S(O).sub.2R.sup.o, --SO.sub.2N(R.sup.o).sub.2, --S(O)R.sup.o,
--NR.sup.oSO.sub.2N(R.sup.o).sub.2, --NR.sup.oSO.sub.2R.sup.o,
--C(.dbd.S)N(R.sup.o).sub.2, --C(.dbd.NH)--N(R.sup.o).sub.2, or
--(CH.sub.2).sub.yNHC(O)R.sup.o, wherein each R.sup.o is
independently selected from hydrogen, optionally substituted
Cl.sub.6 aliphatic, phenyl, --O(Ph), or --CH.sub.2(Ph), wherein y
is 0-6. When R.sup.o is a C.sub.1-6 aliphatic group or a phenyl
ring, it may be substituted with one or more substituents selected
from --NH.sub.2, --NH(Cl.sub.4 aliphatic), --N(C.sub.1-4
aliphatic).sub.2, --S(O)(C.sub.1-4 aliphatic), --SO.sub.2(C.sub.1-4
aliphatic), halogen, --(C.sub.1-4 aliphatic), OH, --O(C.sub.1-4
aliphatic), NO.sub.2, CN, CO.sub.2H, --CO.sub.2(C.sub.1-4
aliphatic), --O(halo C.sub.1-4 aliphatic), or -halo(C.sub.1-4
aliphatic); wherein each C.sub.1-4 aliphatic is unsubstituted.
[0041] An aliphatic group or a non-aromatic heterocyclic ring may
contain one or more substituents. A saturated carbon of an
aliphatic group or of a non-aromatic heterocyclic ring may have one
or more substituents. Suitable substituents on the saturated carbon
of an aliphatic group or of a non-aromatic heterocyclic ring are
selected from those listed above for the unsaturated carbon of an
aryl or heteroaryl group as well as the following: .dbd.O, .dbd.S,
.dbd.NNHR*, .dbd.NN(R*).sub.2, .dbd.N--, .dbd.NNHC(O)R*,
.dbd.NNHCO.sub.2(alkyl), .dbd.NNHSO.sub.2(alkyl), or .dbd.NR*,
where each R* is independently selected from hydrogen or an
optionally substituted C.sub.1-6 aliphatic. When R* is C.sub.1-6
aliphatic, it may be substituted with one or more substituents
independently selected from --NH.sub.2, --NH(C.sub.1-4 aliphatic),
--N(C.sub.1-4 aliphatic).sub.2, halogen, C.sub.1-4 aliphatic, OH,
O(C.sub.1-4 aliphatic), NO.sub.2, CN, CO.sub.2H, CO.sub.2(C.sub.1-4
aliphatic), O(halo C.sub.1-4 aliphatic), or halo(C.sub.1-4
aliphatic); wherein each C.sub.1-4 aliphatic is unsubstituted.
[0042] Substituents on the nitrogen of a non-aromatic heterocyclic
ring are selected from --R.sup.+, --N(R.sup.+).sub.2,
--C(O)R.sup.+, --CO.sub.2R.sup.+, --C(O)C(O)R.sup.+,
--C(O)CH.sub.2C(O)R.sup.+, --SO.sub.2R.sup.+,
--SO.sub.2N(R.sup.+).sub.2, --C(.dbd.S)N(R.sup.+).sub.- 2,
--C(.dbd.NH)--N(R.sup.+).sub.2, or --NR.sup.+SO.sub.2R.sup.+;
wherein each R.sup.+ is independently selected from hydrogen, an
optionally substituted C.sub.1-6 aliphatic, optionally substituted
phenyl, optionally substituted --O(Ph), optionally substituted
--CH.sub.2(Ph), optionally substituted --CH.sub.2CH.sub.2(Ph), or
an unsubstituted 5-6 membered heteroaryl or heterocyclic ring. When
R.sup.+ is a C.sub.1-6 aliphatic group or a phenyl ring, it may be
substituted with one or more substituents selected from --NH.sub.2,
--NH(C.sub.1-4 aliphatic), --N(C.sub.1-4 aliphatic).sub.2, halogen,
C.sub.1-4 aliphatic, OH, O(C.sub.1-4 aliphatic), NO.sub.2, CN,
CO.sub.2H, CO.sub.2(C.sub.14 aliphatic), O(halo C.sub.1-4
aliphatic), or halo (C.sub.1-4 aliphatic); wherein each C.sub.1-4
aliphatic is unsubstituted.
[0043] The term "alkylidene chain" refers to a straight or branched
carbon chain that may be fully saturated or have one or more units
of unsaturation and has two points of attachment to the rest of the
molecule.
[0044] A combination of substituents or variables is permissible
only if such a combination results in a stable or chemically
feasible compound. A stable compound or chemically feasible
compound is one that is not substantially altered when kept at a
temperature of 40.degree. C. or less, in the absence of moisture or
other chemically reactive conditions, for at least a week.
[0045] It will be apparent to one skilled in the art that certain
compounds of this invention may exist in tautomeric forms, all such
tautomeric forms of the compounds being within the scope of the
invention.
[0046] Unless otherwise stated, structures depicted herein are also
meant to include all stereochemical forms of the structure; i.e.,
the R and S configurations for each asymmetric center. Therefore,
single stereochemical isomers as well as enantiomeric and
diastereomeric mixtures of the present compounds are within the
scope of the invention. Unless otherwise stated, structures
depicted herein are also meant to include compounds which differ
only in the presence of one or more isotopically enriched atoms.
For example, compounds having the present structures except for the
replacement of a hydrogen by a deuterium or tritium, or the
replacement of a carbon by a .sup.13C- or .sup.14C-enriched carbon
are within the scope of this invention.
[0047] One embodiment of the present invention relates to compounds
which are 2,1-benzisoxazoles, represented by formula I-A shown
below. Another embodiment of this invention relates to compounds
which are 1,2-benzisoxazoles, represented by formula I-B shown
below: 3
[0048] wherein Ar, T, n, R.sup.1, and R.sup.2 are as described
above for formula I.
[0049] Ar is preferably a substituted or unsubstituted five or
six-membered aromatic ring having zero to two ring heteroatoms
selected from nitrogen, sulfur or oxygen. A more preferred Ar is a
substituted or unsubstituted six-membered aromatic ring having zero
to two ring nitrogens. Most preferably, Ar group is a substituted
or unsubstituted phenyl ring. Preferably, Ar is substituted with
one or more substituents independently selected from C.sub.1-10
aliphatic, C.sub.5-10 aryl, C.sub.6-12 aralkyl, C.sub.3-10
heterocyclyl, C.sub.4-12 heterocyclylalkyl, halo, CN, OR,
N(R).sub.2, SR, C(.dbd.O)R, CO.sub.2R, CONR.sub.2, NRC(.dbd.O)R,
NRCO.sub.2(C.sub.1-6 aliphatic), OC(.dbd.O)R, SO.sub.2R,
S(.dbd.O)R, SO.sub.2NR.sub.2, or NRSO.sub.2(C.sub.1-6 aliphatic),
or two substituents on adjacent positions are optionally taken
together with their intervening atoms to form a fused 5-8 membered
unsaturated or partially unsaturated ring having zero to two
heteroatoms selected from nitrogen, oxygen or sulfur; wherein R is
as described above for formula I.
[0050] R.sup.1 is preferably hydrogen or an aryl ring, such as a
phenyl or pyridyl ring. Optional substituents on R.sup.1 are
independently selected from halogen, --R, --OR, --OH, --SH, --SR,
protected OH (such as acyloxy), --NO.sub.2, --CN, --NH.sub.2,
--NHR, --N(R).sub.2, --NHCOR, --NHCONHR, --NHCON(R).sub.2, --NRCOR,
--NHCO.sub.2R, --CO.sub.2R, --CO.sub.2H, --COR, --CONHR,
--CON(R).sub.2, --S(O).sub.2R, --SO.sub.2NH.sub.2, --S(O)R,
--SO.sub.2NHR, or --NHS(O).sub.2R, where R is a C.sub.1-6 aliphatic
group or a substituted C.sub.1-6 aliphatic group, preferably having
one to three carbons. A particularly preferred substituent on the
C.sub.1-6 aliphatic group is --SO.sub.2NH.sub.2.
[0051] R.sup.2 is preferably hydrogen or a C.sub.1-4 alkyl group,
most preferably hydrogen.
[0052] R.sup.3 is preferably hydrogen, halo, O(C.sub.1-4 alkyl), or
a C.sub.1-4 alkyl group. Most preferably R.sup.3 is hydrogen.
Representative examples of compounds of formula I-A are shown below
in Table 1.
1TABLE 1 Examples of Compounds of formula I-A 4 No. Structure I-A1
5 I-A2 6 I-A3 7 I-A4 8 I-A5 9 I-A6 10 I-A7 11 I-A8 12 I-A9 13 I-A10
14 I-A11 15 I-A12 16 I-A13 17 I-A14 18 I-A15 19 I-A16 20 I-A17 21
I-A18 22 I-A19 23 I-A20 24 I-A21 25 I-A22 26 I-A23 27 I-A24 28
I-A25 29 I-A26 30 I-A27 31 I-A28 32 I-A29 33 I-A30 34 I-A31 35
I-A32 36 I-A33 37 I-A34 38 I-A35 39 I-A36 40 I-A37 41 I-A38 42
I-A39 43 I-A40 44 I-A41 45 I-A42 46 I-A43 47 I-A44 48 I-A45 49
I-A46 50 I-A47 51 I-A48 52 I-A49 53 I-A50 54 I-A51 55 I-A52 56
I-A53 57 I-A54 58 I-A55 59 I-A56 60 I-A57 61 I-A58 62 I-A59 63
I-A60 64 I-A61 65 I-A62 66 I-A63 67 I-A64 68 I-A65 69 I-A66 70
I-A67 71 I-A68 72 I-A69 73 I-A70 74 I-A71 75 I-A72 76 I-A73 77
I-A74 78 I-A75 79 I-A76 80 I-A77 81 I-A78 82 I-A79 83 I-A80 84
I-A81 85 I-A82 86 I-A83 87 I-A84 88 I-A85 89 I-A86 90 I-A87 91
I-A88 92 I-A89 93
[0053] The compounds of this invention may be prepared in general
by methods known to those skilled in the art for analogous
compounds, as illustrated by the general scheme below and by the
preparative examples that follow. 94
[0054] Reagents and conditions: (a) ArCH.sub.2CN, KOH, MeOH, room
temperature (rt); (b) formic acid, rt (c) N,N-dimethylformamide
dimethyl acetal, CH.sub.3CN, 80.degree. C.; (d)
N-phenylguanidine-HCl, CH.sub.3CN, reflux.
[0055] Scheme I above shows a synthetic route for preparing
compounds of the present invention. For various Ar groups, the
intermediate 3 can be obtained commercially or obtained by known
methods as shown in steps (a) and (b) above. See R. B. Davis and L.
C. Pizzini, J. Org. Chem., 1960, 25, 1884-1888. A Mannich reaction
provides intermediate 4, which can be treated with phenylguanidine
to give the desired compounds 5. It will be obvious to one skilled
in the art that phenylguanidine may be replaced with other
arylguanidines, which are readily available, to provide other
compounds of this invention. 95
[0056] Reagents and conditions: (a)
R.sup.1NHC(.dbd.NH)NH.sub.2--HCl, CH.sub.3CN, reflux; (b)
4-Br--C.sub.6H.sub.4--CH.sub.2CN, KOH, MeOH, room temperature (rt);
(c) R.sup.4B(OH).sub.2, Pd(PPh.sub.3).sub.4, Na.sub.2CO.sub.3,
dioxane
[0057] Scheme II above shows an alternative synthetic route where
the pyrimidine ring is constructed before the benzisoxazole ring.
Steps (a) and (b) are analogous to the corresponding steps shown
above in Scheme I except that they are performed in the opposite
order. Step (c) illustrates one of many ways known to those skilled
in the art in which certain compounds of this invention may be
modified to provide further compounds of this invention. For
example, the bromo substituent of compound 8 may be replaced by
other groups using standard coupling methods. R.sup.4 is preferably
an aryl or heteroaryl ring. It will be obvious to one skilled in
the art that this scheme may be modified to provide other compounds
of this invention. 96
[0058] Reagents and conditions: (a) NaH, DMF/THF 1:1,
R.sup.5C(O)Cl, ambient temp; wherein R.sup.1 is --C(O)R.sup.5; (b)
R.sup.7NCO, DMSO, ambient temp/80.degree. C.; wherein R.sup.1 is
--C(O)NHR.sup.7; (c) [from the p-NO.sub.2-phenyl carbamic esters]
R.sup.7NH.sub.2, DMSO/THF 1:1, 80.degree. C.; wherein R.sup.1 is
--C(O)NHR.sup.7. Alternatively, reagents and conditions for
carbamate formation (not shown): (a) R.sup.6OC(O)Cl, DMSO, DIPEA,
ambient temp; wherein R.sup.1 is --C(O)OR.sup.6.
[0059] Scheme III shows general methods for the preparation of
compounds of Formula I wherein NH--R.sup.1 taken together form an
amide (shown in step (a) above), carbamate (not shown) or a urea
(shown in steps (a) and (c) or step (b) above). Acylation of the
aminopyrimidine with acid chlorides, chloroformates and isocyanates
provides amides, cabamates and ureas respectively. Alternatively,
ureas can be generated by a nucleophilic displacement reaction with
a primary or secondary amine via the corresponding
p-nitrophenylcarbamate. 97
[0060] Reagents and conditions: a) NHR.sup.o.sub.2, Pd(OAc).sub.2,
P-tBu.sub.3, KOtBu, toluene, 90.degree. C.
[0061] Scheme IV shows a general method for obtaining compounds 2
(scheme I) wherein the Ar group is substituted with an amine
functionality as in 2b, and wherein R.sup.o is as described above.
Compounds of type 2b may then be taken forward according to Schemes
I-III.
[0062] The activity of a compound utilized in this invention as an
inhibitor of GSK-3 or JAK kinase may be assayed in vitro, in vivo
or in a cell line according to methods known in the art. In vitro
assays include assays that determine inhibition of either the
phosphorylation activity or ATPase activity of activated GSK-3 or
JAK. Alternate in vitro assays quantitate the ability of the
inhibitor to bind to GSK-3 or JAK. Inhibitor binding may be
measured by radiolabelling the inhibitor prior to binding,
isolating the inhibitor/GSK-3 or inhibitor/JAK complex and
determining the amount of radiolabel bound. Alternatively,
inhibitor binding may be determined by running a competition
experiment where new inhibitors are incubated with GSK-3 or JAK
bound to known radioligands. Detailed conditions for assaying a
compound utilized in this invention as an inhibitor of GSK-3 or JAK
kinase are set forth in the Examples below.
[0063] According to another embodiment, the invention provides a
composition comprising a compound of this invention or a
pharmaceutically acceptable derivative thereof and a
pharmaceutically acceptable carrier, adjuvant, or vehicle. The
amount of compound in the compositions of this invention is such
that is effective to detectably inhibit a protein kinase,
particularly GSK-3 or JAK kinase, in a biological sample or in a
patient. Preferably the composition of this invention is formulated
for administration to a patient in need of such composition. Most
preferably, the composition of this invention is formulated for
oral administration to a patient.
[0064] The term "patient", as used herein, means an animal,
preferably a mammal, and most preferably a human.
[0065] The term "pharmaceutically acceptable carrier, adjuvant, or
vehicle" refers to a non-toxic carrier, adjuvant, or vehicle that
does not destroy the pharmacological activity of the compound with
which it is formulated. Pharmaceutically acceptable carriers,
adjuvants or vehicles that may be used in the compositions of this
invention include, but are not limited to, ion exchangers, alumina,
aluminum stearate, lecithin, serum proteins, such as human serum
albumin, buffer substances such as phosphates, glycine, sorbic
acid, potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wool fat.
[0066] The term "detectably inhibit", as used herein means a
measurable change in GSK-3 or JAK activity between a sample
comprising said composition and a GSK-3 or JAK kinase and an
equivalent sample comprising GSK-3 or JAK kinase in the absence of
said composition.
[0067] As used herein, the term "JAK" is used interchangeably with
the terms "JAK kinase" and "a JAK family kinase". Preferably JAK
refers to JAK3 kinase.
[0068] A "pharmaceutically acceptable derivative" means any
non-toxic salt, ester, salt of an ester or other derivative of a
compound of this invention that, upon administration to a
recipient, is capable of providing, either directly or indirectly,
a compound of this invention or an inhibitorily active metabolite
or residue thereof. As used herein, the term "inhibitorily active
metabolite or residue thereof" means that a metabolite or residue
thereof is also an inhibitor of a GSK-3 or JAK family kinase.
[0069] Pharmaceutically acceptable salts of the compounds of this
invention include those derived from pharmaceutically acceptable
inorganic and organic acids and bases. Examples of suitable acid
salts include acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate, citrate, camphorate,
camphorsulfonate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptanoate, glycerophosphate, glycolate, hemisulfate,
heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,
2-hydroxyethanesulfonate, lactate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate, propionate, salicylate, succinate,
sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other
acids, such as oxalic, while not in themselves pharmaceutically
acceptable, may be employed in the preparation of salts useful as
intermediates in obtaining the compounds of the invention and their
pharmaceutically acceptable acid addition salts.
[0070] Salts derived from appropriate bases include alkali metal
(e.g., sodium and potassium), alkaline earth metal (e.g.,
magnesium), ammonium and N.sup.+(C.sub.1-4 alkyl).sub.4 salts. This
invention also envisions the quaternization of any basic
nitrogen-containing groups of the compounds disclosed herein. Water
or oil-soluble or dispersible products may be obtained by such
quaternization.
[0071] The compositions of the present invention may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion techniques.
Preferably, the compositions are administered orally,
intraperitoneally or intravenously. Sterile injectable forms of the
compositions of this invention may be aqueous or oleaginous
suspension. These suspensions may be formulated according to
techniques known in the art using suitable dispersing or wetting
agents and suspending agents. The sterile injectable preparation
may also be a sterile injectable solution or suspension in a
non-toxic parenterally-acceptable diluent or solvent, for example
as a solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium.
[0072] For this purpose, any bland fixed oil may be employed
including synthetic mono- or di-glycerides. Fatty acids, such as
oleic acid and its glyceride derivatives are useful in the
preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant, such as carboxymethyl cellulose or similar dispersing
agents that are commonly used in the formulation of
pharmaceutically acceptable dosage forms including emulsions and
suspensions. Other commonly used surfactants, such as Tweens, Spans
and other emulsifying agents or bioavailability enhancers which are
commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms may also be used for the
purposes of formulation.
[0073] The pharmaceutically acceptable compositions of this
invention may be orally administered in any orally acceptable
dosage form including, but not limited to, capsules, tablets,
aqueous suspensions or solutions. In the case of tablets for oral
use, carriers commonly used include lactose and corn starch.
Lubricating agents, such as magnesium stearate, are also typically
added. For oral administration in a capsule form, useful diluents
include lactose and dried cornstarch. When aqueous suspensions are
required for oral use, the active ingredient is combined with
emulsifying and suspending agents. If desired, certain sweetening,
flavoring or coloring agents may also be added.
[0074] Alternatively, the pharmaceutically acceptable compositions
of this invention may be administered in the form of suppositories
for rectal administration. These can be prepared by mixing the
agent with a suitable non-irritating excipient that is solid at
room temperature but liquid at rectal temperature and therefore
will melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0075] The pharmaceutically acceptable compositions of this
invention may also be administered topically, especially when the
target of treatment includes areas or organs readily accessible by
topical application, including diseases of the eye, the skin, or
the lower intestinal tract. Suitable topical formulations are
readily prepared for each of these areas or organs.
[0076] Topical application for the lower intestinal tract can be
effected in a rectal suppository formulation (see above) or in a
suitable enema formulation. Topically-transdermal patches may also
be used.
[0077] For topical applications, the pharmaceutically acceptable
compositions may be formulated in a suitable ointment containing
the active component suspended or dissolved in one or more
carriers. Carriers for topical administration of the compounds of
this invention include, but are not limited to, mineral oil, liquid
petrolatum, white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutically acceptable compositions can be
formulated in a suitable lotion or cream containing the active
components suspended or dissolved in one or more pharmaceutically
acceptable carriers. Suitable carriers include, but are not limited
to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl
esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0078] For ophthalmic use, the pharmaceutically acceptable
compositions may be formulated as micronized suspensions in
isotonic, pH adjusted sterile saline, or, preferably, as solutions
in isotonic, pH adjusted sterile saline, either with or without a
preservative such as benzylalkonium chloride. Alternatively, for
ophthalmic uses, the pharmaceutically acceptable compositions may
be formulated in an ointment such as petrolatum.
[0079] The pharmaceutically acceptable compositions of this
invention may also be administered by nasal aerosol or inhalation.
Such compositions are prepared according to techniques well-known
in the art of pharmaceutical formulation and may be prepared as
solutions in saline, employing benzyl alcohol or other suitable
preservatives, absorption promoters to enhance bioavailability,
fluorocarbons, and/or other conventional solubilizing or dispersing
agents.
[0080] Most preferably, the pharmaceutically acceptable
compositions of this invention are formulated for oral
administration.
[0081] The amount of the compounds of the present invention that
may be combined with the carrier materials to produce a composition
in a single dosage form will vary depending upon the host treated,
the particular mode of administration. Preferably, the compositions
should be formulated so that a dosage of between 0.01-100 mg/kg
body weight/day of the inhibitor can be administered to a patient
receiving these compositions.
[0082] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician and the severity of the
particular disease being treated. The amount of a compound of the
present invention in the composition will also depend upon the
particular compound in the composition.
[0083] Depending upon the particular condition, or disease, to be
treated or prevented, additional therapeutic agents, which are
normally administered to treat or prevent that condition, may also
be present in the compositions of this invention. As used herein,
additional therapeutic agents that are normally administered to
treat or prevent a particular disease, or condition, are known as
"appropriate for the disease, or condition, being treated".
[0084] For example, chemotherapeutic agents or other
anti-proliferative agents may be combined with the compounds of
this invention to treat proliferative diseases and cancer. Examples
of known chemotherapeutic agents include, but are not limited to,
Gleevec.TM., adriamycin, dexamethasone, vincristine,
cyclophosphamide, fluorouracil, topotecan, taxol, interferons, and
platinum derivatives.
[0085] Other examples of agents the inhibitors of this invention
may also be combined with include, without limitation: treatments
for Alzheimer's Disease such as Aricept.RTM. and Excelon.RTM.;
treatments for Parkinson's Disease such as L-DOPA/carbidopa,
entacapone, ropinrole, pramipexole, bromocriptine, pergolide,
trihexephendyl, and amantadine; agents for treating Multiple
Sclerosis (MS) such as beta interferon (e.g., Avonex.RTM. and
Rebif.RTM.), Copaxone.RTM., and mitoxantrone; treatments for asthma
such as albuterol and Singulair.RTM.; agents for treating
schizophrenia such as zyprexa, risperdal, seroquel, and
haloperidol; anti-inflammatory agents such as corticosteroids, TNF
blockers, IL-1 RA, azathioprine, cyclophosphamide, and
sulfasalazine; immunomodulatory and immunosuppressive agents such
as cyclosporin, tacrolimus, rapamycin, mycophenolate mofetil,
interferons, corticosteroids, cyclophophamide, azathioprine, and
sulfasalazine; neurotrophic factors such as acetylcholinesterase
inhibitors, MAO inhibitors, interferons, anti-convulsants, ion
channel blockers, riluzole, and anti-Parkinsonian agents; agents
for treating cardiovascular disease such as beta-blockers, ACE
inhibitors, diuretics, nitrates, calcium channel blockers, and
statins; agents for treating liver disease such as corticosteroids,
cholestyramine, interferons, and anti-viral agents; agents for
treating blood disorders such as corticosteroids, anti-leukemic
agents, and growth factors; and agents for treating
immunodeficiency disorders such as gamma globulin.
[0086] The amount of additional therapeutic agent present in the
compositions of this invention will be no more than the amount that
would normally be administered in a composition comprising that
therapeutic agent as the only active agent. Preferably the amount
of additional therapeutic agent in the presently disclosed
compositions will range from about 50% to about 100% of the amount
normally present in a composition comprising that agent as the only
therapeutically active agent.
[0087] According to another embodiment, the invention relates to a
method of inhibiting GSK-3 or JAK kinase activity in a biological
sample comprising the step of contacting said biological sample
with a compound of this invention, or a composition comprising said
compound.
[0088] The term "biological sample", as used herein, includes,
without limitation, cell cultures or extracts thereof; biopsied
material obtained from a mammal or extracts thereof; and blood,
saliva, urine, feces, semen, tears, or other body fluids or
extracts thereof.
[0089] Inhibition of GSK-3 or JAK kinase activity in a biological
sample is useful for a variety of purposes that are known to one of
skill in the art. Examples of such purposes include, but are not
limited to, blood transfusion, organ-transplantation, biological
specimen storage, and biological assays.
[0090] According to another embodiment, the invention provides a
method for treating or lessening the severity of a GSK-3-mediated
disease or condition in a patient comprising the step of
administering to said patient a composition according to the
present invention.
[0091] The term "GSK-3-mediated condition", as used herein means
any disease or other deleterious condition in which GSK-3, is known
to play a role. Such diseases or conditions include, without
limitation, diabetes, Alzheimer's disease, Huntington's,
Parkinson's, AIDS associated dementia, amyotrophic lateral
sclerosis (AML), multiple sclerosis (MS), schizophrenia,
cardiomycete hypertrophy, ischemia/reperfusionand baldness.
[0092] According to another embodiment, the invention provides a
method for treating or lessening the severity of a JAK-mediated
disease or condition in a patient comprising the step of
administering to said patient a composition according to the
present invention.
[0093] The term "JAK-mediated disease", as used herein means any
disease or other deleterious condition in which a JAK family
kinase, in particular JAK3, is known to play a role. Such
conditions include, without limitation, immune responses such as
allergic or type I hypersensitivity reactions, asthma, autoimmune
diseases such as transplant rejection, graft versus host disease,
rheumatoid arthritis, amyotrophic lateral sclerosis, and multiple
sclerosis, neurodegenerative disorders such as Familial amyotrophic
lateral sclerosis (FALS), as well as in solid and hematologic
malignancies such as leukemias and lymphomas.
[0094] In an alternate embodiment, the methods of this invention
that utilize compositions that do not contain an additional
therapeutic agent, comprise the additional step of separately
administering to said patient an additional therapeutic agent. When
these additional therapeutic agents are administered separately
they may be administered to the patient prior to, sequentially with
or following administration of the compositions of this
invention.
[0095] The compounds of this invention or pharmaceutically
acceptable compositions thereof may also be incorporated into
compositions for coating an implantable medical device, such as
prostheses, artificial valves, vascular grafts, stents and
catheters. Vascular stents, for example, have been used to overcome
restenosis (re-narrowing of the vessel wall after injury). However,
patients using stents or other implantable devices risk clot
formation or platelet activation. These unwanted effects may be
prevented or mitigated by pre-coating the device with a
pharmaceutically acceptable composition comprising a kinase
inhibitor. Suitable coatings and the general preparation of coated
implantable devices are described in U.S. Pat. Nos. 6,099,562;
5,886,026; and 5,304,121. The coatings are typically biocompatible
polymeric materials such as a hydrogel polymer,
polymethyldisiloxane, polycaprolactone, polyethylene glycol,
polylactic acid, ethylene vinyl acetate, and mixtures thereof. The
coatings may optionally be further covered by a suitable topcoat of
fluorosilicone, polysaccarides, polyethylene glycol, phospholipids
or combinations thereof to impart controlled release
characteristics in the composition. Implantable devices coated with
a compound of this invention are another embodiment of the present
invention.
[0096] In order that the invention described herein may be more
fully understood, the following examples are set forth. It should
be understood that these examples are for illustrative purposes
only and are not to be construed as limiting this invention in any
manner.
SYNTHETIC EXAMPLES
Example 1
N-phenylguanidine
[0097] Aniline (30 mmol, 1 equiv.), cyanamide (1.3 g, 31 mmol, 1.03
equiv.), and 4N hydrogen chloride in dioxane (8 mL, 32 mmol) was
stirred for 10 minutes at room temperature and heated to 80.degree.
C. for 18 hours. The mixture was diluted with water (30 mL) and
diethyl ether (50 mL). The aqueous layer was washed with ether (30
mL) and the organic layers were discarded. The aqueous layer was
neutralized with 6N aqueous HCl (6 mL) and diluted with ethyl
acetate (50 mL). The aqueous layer was extracted with ethyl acetate
(50 mL) four times. The combined organic layers were concentrated
under reduced pressure to afford a solid compound. The solid was
washed with diethyl ether (30 mL) to provide pale yellow title
compound. The compound was characterized by LC/MS and HPLC.
[0098] The following arylguanidine intermediates were prepared by
the procedure described above in Example 1 except the aniline was
replaced with the appropriate arylamine:
N-(4-fluoro-phenyl)-guanidine; N-(6-chloro-pyridin-3-yl)-guanidine;
N-(3-chloro-phenyl)-guanidine; N-(3-methoxy-phenyl)-guanidine;
N-(3-benzyloxy-phenyl)-guanidine; 4-guanidino-benzenesulfonamide;
3-guanidino-benzenesulfonamide.
[0099] The following synthetic intermediates were obtained
commercially (from Bionet):
1-[3-phenyl-benzo[c]isoxazol-5-yl]-ethanone;
1-[3-(4-fluoro-phenyl)-benzo[c]isoxazol-5-yl]-ethanone;
1-[3-(4-chloro-phenyl)-benzo[c]isoxazol-5-yl]-ethanone;
3-dimethylamino-1-(3-phenyl-benzo[c]isoxazol-5-yl)-propenone;
3-dimethylamino-1-[3-(4-fluoro-phenyl)-benzo[c]isoxazol-5-yl]-propenone;
3-dimethylamino-1-[3-(4-chloro-phenyl)-benzo[c]isoxazol-5-yl]-propenone;
and 1-(4-nitro-phenyl)-3-dimethylamino-propenone.
Example 2
Phenyl-[4-(3-phenyl-benzo[c]isoxazol-5-yl)-pyrimidin-2-yl]-amine
(Compound I-A1)
[0100] 98
[0101]
3-Dimethylamino-1-(5-methyl-3-methylsulfanyl-1-phenyl-1H-pyrazol-4--
yl)-propenone (30 mg, 0.1 mmol) and N-phenylguanidine (15 mg, 1.1
equiv.) were slurried in acetonitrile (0.5 mL) and heated at
100.degree. C. for 24 hours. The mixture was diluted with methanol
(2 mL) and heated briefly and cooled. The resulting solid was
filtered and washed with methanol (1 mL). The solid was dried under
reduced pressure to afford the title compound. The compound was
characterized by LC/MS and HPLC.
Example 3
(4-Fluoro-phenyl)-[4-(3-phenyl-benzo[c]isoxazol-5-yl)-pyrimidin-2-yl]-amin-
e (Compound I-A2)
[0102] 99
[0103] Compound I-A2 was prepared according to the procedure
described above in Example 2 except that N-phenylguanidine was
replaced by N-(4-fluoro-phenyl)-guanidine.
Example 4
(6-Chloro-pyridin-3-yl)-[4-(3-phenyl-benzo[c]isoxazol-5-yl)-pyrimidin-2-yl-
]-amine (Compound I-A3)
[0104] 100
[0105] Compound I-A3 was prepared according to the procedure
described above in Example 2 except that N-phenylguanidine was
replaced by N-(6-chloro-pyridin-3-yl)-guanidine.
Example 5
(3-Chloro-phenyl)-[4-(3-phenyl-benzo[c]isoxazol-5-yl)-pyrimidin-2-yl]-amin-
e (Compound I-A4)
[0106] 101
[0107] Compound I-A4 was prepared according to the procedure
described above in Example 2 except that N-phenylguanidine was
replaced by N-(3-chloro-phenyl)-guanidine.
Example 6
4-[4-(3-Phenyl-benzo
[c]isoxazol-5-yl)-pyrimidin-2-ylamino]-benzenesulfona- mide
(Compound I-A19)
[0108] 102
[0109] Compound I-A19 was prepared according to the procedure
described above in Example 2 except that N-phenylguanidine was
replaced by 4-guanidino-benzenesulfonamide.
Example 7
N-{4-[3-(4-Chorophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-yl}-acetamide
(1-A22)
[0110] 103
[0111] Step A.
4-[3-(4-Chlorophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-yl-
amine.
[0112] To a mixture of sodium pellets (14 mg, 0.609 mmol) in
methanol (1 mL) at room temperature, was added guanidine
hydrochloride (10 mg, 0.105 mmol) and commercially available
1-[3-(4-chlorophenyl)-benzo[c]isoxazole--
5-yl]-3-dimethylamino-propenone (50 mg, 0.153 mmol). The reaction
mixture was heated at 80.degree. C. for 18 hours. The mixture was
cooled to room temperature and diluted with water (6 mL). The
granular precipitate was filtered, dissolved in dichloromethane,
then dried over magnesium sulfate. Purification by silica gel
chromatography (4:1 ethyl acetate/hexane) gave
4-[3-(4-chlorophenyl)-benzo[c]isoxazol-5-yl]-pyrimid- in-2-ylamine
as a yellow solid (35 mg, 98% yield). .sup.1H NMR (500 MHz,
d.sub.6-DMSO) .delta. 8.68 (s, 1H), 8.35 (d, 1H), 8.25-8.19 (m,
3H), 7,82-7.80 (m, 1H), 7.78-7.72 (m, 2H), 7.4 (d, 1H), 6.79 (s,
1H) ppm. LC-MS (ES+) m/e=323.04 (M+H).
[0113] Step B.
N-{4-[3-(4-Chorophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2--
yl}-acetamide.
[0114] To a suspension of
4-[3-(4-chlorophenyl)-benzo[c]isoxazol-5-yl]-pyr- imidin-2-ylamine
in toluene (1.5 mL) at room temperature, was added acetic anhydride
(0.5 mL). The mixture was heated at 100.degree. C. for 3 hours. The
reaction mixture was diluted with water (6 mL) and the precipitate
filtered then washed with toluene (2.times.6 mL). Purification was
achieved by silica gel chromatography (4:1 ethyl acetate/hexane
then 2% methanol/dichloromethane), followed by a 5% aqueous sodium
bicarbonate wash (1.times.50 mL) to give the title compound as a
yellow solid (12 mg, 30% yield). .sup.1H NMR (500 MHz,
d.sub.6-DMSO) .delta. 10.62 (s, 1H), 8.85 (s, 1H), 8.75 (d, 1H),
8.31 (d, 1H), 8.25 (d, 2H), 8.02 (d, 1H), 7.85 (d, 1H), 7.75 (d,
2H), 2.3 (s, 3H) ppm. LC-MS (ES+) m/e=365.13 (M+H).
Example 8
{4-[3-(4-Chlorophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-yl}-methylamine
(I-A23)
[0115] 104
[0116] This compound was prepared in an analogous manner to that
described in Example 2 using 1-methylguanidine hydrochloride to
yield the title compound as a yellow solid (30 mg, 98% yield).
.sup.1H NMR (500 MHz, d.sub.6-DMSO) .delta. 8.7 (s, 1H) 8.41 (s,
1H), 8.31-8.2 (m, 3H), 7.82 (d, 1H), 7.72 (d, 2H), 7.38 (d, 1H),
7.25-7.2 (m, 1H), 2.95-2.85 (m, 3H) ppm. LC-MS (ES+) m/e=337.04
(M+H).
Example 9
3-(4-Chlorophenyl)-5-(2-morpholin-4-yl-pyrimidin-4-yl)-benzo[c]isoxazole
(I-A24)
[0117] 105
[0118] This compound was prepared according to the procedure
described in Example 13, Step E, except using morpholinoformamidine
hydrobromide to yield
3-(4-chlorophenyl)-5-(2-morpholin-4-yl-pyrimidin-4-yl)-benzo[c]isox-
azole as a yellow solid (30 mg, 98% yield). .sup.1H NMR (500 MHz,
d.sub.6-DMSO) .delta. 8.7 (S, 1H), 8.5 (d, 1H), 8.3-8.22 (m, 3H),
7.82 (s, 1H), 7.75 (d, 2H), 7.55 (d, 1H), 3.85-3.8 (m, 4H),
3.75-3.68 (m, 4H) ppm. LC-MS (ES+) m/e=393.13 (M+H).
Example 10
4-[3-(4-Piperidin-1-yl-phenyl)-benzo[c]isoxazol-5-yl]pyrimidin-2-ylamine
(I-A32)
[0119] 106
[0120] Step A.
5-(2-Methyl-[1,3]dioxolan-2-yl)-3-(4-piperidin-1-yl-phenyl)-
benzo[c]isoxazole
[0121] This compound was prepared in a manner analogous to that
described in Example 13, Step B except starting with piperidine and
a reaction duration of 2.5 h, giving the title compound, after
purification, as a bright yellow solid (174 mg, 69% yield). .sup.1H
NMR (500 MHz, CDCl.sub.3) .delta. 8.02-7.81 (m, 3H), 7.53 (d,
J=9.25 Hz, 1H), 7.10-6.92 (m, 2H), 4.15-3.96 (m, 2H), 3.94-3.71 (m,
2H), 3.47-3.23 (m, 4H), 1.83-1.60 (m, 9H). LC-MS (ES+) m/e=365.19
(M+H).
[0122] Step B.
1-[3-(4-Piperidin-1-yl-phenyl)-benzo[c]isoxazol-5-yl)ethano- ne
[0123] This compound was prepared in a manner analogous to that
described in Experiment 17, Step C giving the title compound as an
orange oil (42.6 mg, 97% yield). .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 8.57-8.47 (m, 1H), 8.01-7.91 (m, 2H), 7.88 (dd, J=1.5, 9.4
Hz, 1H), 7.55 (dd, 0.85, 9.4 Hz, 1H), 7.08-6.94 (m, 2H), 3.46-3.30
(m, 4H), 2.66 (s, 3H), 1.82-1.59 (m, 6H). LC-MS (ES+) m/e=321.1
(M+H).
[0124] Step C. 4-[3-(4-Piperidin-1-yl-phenyl)-benzo
[c]isoxazol-5-yl]pyrimidin-2-ylamine (I-A32)
[0125] This compound was prepared in a manner analogous to that
described in Experiment 17, Steps D & E giving the title
compound as an orange solid (30 mg, 70% yield from
1-[3-(4-piperidin-1-yl-phenyl)-benzo[c]isoxa- zol-5-yl)ethanone).
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.58 (s, 1H), 8.38 (d,
J=5.25 Hz, 1H), 8.06-7.85 (m, 3H), 7.62 (d, J=9.4 Hz, 1H),
7.16-6.92 (m, 3H), 5.19-4.91 (br s, 2H), 3.45-3.25 (m, 4H),
1.82-1.61 (m, 6H). HPLC (cyano column) 14.26 min. LC-MS (ES+)
m/e=372.2 (M+H).
Example 11
4-[3-(3-Piperidin-1-yl-phenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine
(I-A33)
[0126] 107
[0127] Step A.
3-(3-Bromophenyl)-5-(2-methyl-[1,3]dioxolan-2-yl)benzo[c]is-
oxazole
[0128] To a solution of KOH (58 g, 1.03 mol) in MeOH (200 mL) at
room temperature was added a solution of
2-methyl-2-(4-nitro-phenyl)-[1,3]diox- olane (10.7 g, 0.051 mol)
and 3-bromophenylacetonitrile (11.34 g, 0.058 mol) in MeOH (100
mL). The mixture was stirred at room temperature under a stream of
nitrogen for 4 days. The product was isolated according to the
procedure given in Example 15 Step A (8.5 g, 46% yield). .sup.1H
NMR (500 MHz, CDCl.sub.3) .delta. 8.19-8.15 (m, 1H), 7.97 (d, 4.0
Hz, 1H), 7.90 (s, 1H), 7.67-7.59 (m, 2H), 7.50-7.40 (m, 2H),
4.15-4.03 (m, 2H), 1.71 (s, 3H). LC-MS ES+) m/e=361.96 (M+H).
[0129] Step B.
3-Dimethylamino-1-[3-(3-piperidin-1-yl-phenyl)-benzo[c]isox-
azol-5-yl]-propanone
[0130] This was prepared according to the procedure described in
Example 13 to give the title compound as a brown solid (141 mg, 48%
yield from
3-(3-bromophenyl)-5-(2-methyl-[1,3]dioxolan-2-yl)benzo[c]isoxazole).
.sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 8.50 (s, 1H), 8.09-7.89
(m, 1H), 7.89-7.64 (m, 2H), 7.63-7.45 (m, 3H), 7.42-7.13 (m, 1H),
6.01 (d, J=12.2 Hz, 1H), 3.51-3.27 (m, 4H), 3.26-3.07 (m, 3H),
3.06-2.80 (m, 3H), 1.84-1.42 (m, 6H). LC-MS ES+) m/e=371.31 (M+H).
HPLC (cyano column) 14.13 minutes.
[0131] Step C.
4-[3-(3-Piperidin-1-yl-phenyl)-benzo[c]isoxazol-5-yl]-pyrim-
idin-2-ylamine (1-A33)
[0132] This compound was prepared in a manner analogous to that
described in Experiment 17, Step E.
[0133] The title compound was isolated as a yellow/brown solid (97
mg, 69%). .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.56 (S, 1H),
8.39 (D, J=5.2 Hz, 1H), 7.97 (dd, J=1.3, 9.4 Hz, 1H), 7.69 (d, 9.5
Hz, 1H), 7.63-7.53 (m, 1H), 7.52-7.37 (m, 2H), 7.17-7.02 (m, 2H),
5.16 (br s, 2H), 3.39-3.19 (m, 4H), 1.86-1.53 (m, 6H). LC-MS ES+)
m/e=361.96 (M+H). HPLC (cyano column) 12.01 minutes.
Example 12
4-[4-(4-Nitro-phenyl)-pyrimidin-2-ylamino]-benzenesulfonamide
[0134] 108
[0135] 1-(4-Nitro-phenyl)-3-dimethylamino-propenone (3 mmol) and
4-guanidino-benzenesulfonamide (3.3 mmol) in acetonitrile (1 mL)
was refluxed for 36 hours. The mixture was diluted with methanol (5
mL) and cooled to room temperature. The yellow solid was filtered
and washed with methanol (3 mL) and dried under reduced pressure to
afford title compound. The compound was characterized by LC/MS and
HPLC.
Example 13
4-[3-(4-Morpholin-4-yl-phenyl)benzo[c]isoxazol-5-yl]pyrimidin-2-yl
amine (I-A34)
[0136] 109
[0137] Step A.
3-(4-Bromo-phenyl)-5-(2-methyl-(1,3]-dioxolan-2-yl)-benzo
[cisoxazole
[0138] To solution of KOH (28.46 g, 508 mmol) in MeOH (50 mL) at
0-10.degree. C. was added a solution of 4-bromophenylacetonitrile
(6.32 g, 32.2 mmol) and 2-methyl-2-(4-nitro-phenyl)-(1,3]-dioxolane
(I) (5.35 g, 25.6 mmol) in MeOH (15 mL). The mixture was stirred at
room temperature under nitrogen for 18 hours giving a thick slurry.
Water (100 mL) was added and the precipitate was filtered, and was
washed with water (2.times.75 mL). The solid was dissolved in hot
CH.sub.2Cl.sub.2, filtered and evaporated to give a brown solid.
Repeated triturations with Et.sub.2O gave the product as a bright
orange solid (5.19 g, 56% yield). .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 7.99-7.68 (m, 1H), 7.79-7.68 (m, 2H), 7.66-7.54 (m, 1H),
7.52-7.40 (m, 1H), 4.17-4.04 (m, 2H), 3.92-3.78 (m, 2H), 1.70 (s,
3H) ppm. LC-MS (ES+) m/e=361.9 (M+H).
[0139] Step B.
5-(2-Methyl-[1,3'-dioxolan-2-yl)-3-(4-morpholin-4-yl-phenyl-
)-benzo[c]isoxazole
[0140] A flame dried, argon flushed flask was charged with
3-(4-bromo-phenyl)-5-(2-methyl-[1,3]-dioxolan-2-yl)-benzo[c]isoxazole
(199.6 mg, 0.56 mmol), Pd(OAc).sub.2 (5 mg, 0.02 mmol),
P(tBu).sub.3 (30 .mu.L of 10% solution in toluene, 0.012 mmol),
NaOtBu (78.8 mg, 0.82 mmol) and morpholine (150 .mu.L, 1.72 mmol)
in anhydrous toluene (1 mL). The mixture was heated at 80.degree.
C. under Argon for 3 hours. The solvent was evaporated, and
purification by flash chromatography (SiO.sub.2) eluting initially
with 1:9 EtOAc:hexanes to 3:7 EtOAc:hexanes provided the title
compound as bright yellow solid (49 mg, 24% yield). .sup.1H NMR
(500 MHz, CDCl.sub.3) .delta. 7.96 (d, 2H), 7.91 (s, 1H), 7.55 (d,
J=9.35 Hz, J=8.9 Hz, 1H), 7.47-7.34 (m, 1H), 7.04 (d, J=8.95 Hz,
2H), 4.17-4.01 (m, 2H), 3.95-3.76 (m, 6H), 3.31 (t, J=5 Hz, 4H),
1.7 (s, 3H). HPLC (cyano column) 8.61 minutes
[0141] Step C.
1-[3-(4-Morpholin-4-yl-phenyl)benzo[c]isoxazol-5-yl]ethanon- e
[0142] A solution of
5-(2-methyl-[1,3]-dioxolan-2-yl)-3-(4-morpholin-4-yl--
phenyl)-benzo[c]isoxazole (37 mg, 0.10 mmol) in formic acid (88%
solution, 1.5 mL) was stirred at room temperature for 70 minutes.
The formic acid was removed in vacuo, and the resultant solid was
dissolved in CH.sub.2Cl.sub.2, dried over sodium sulfate, filtered
and evaporated to give the product as an orange solid (1.42 g, 87%
yield). .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 8.51 (s, 1H),
7.99 (d, J=8.9 Hz, 2H), 7.88 (d, J=1.0 Hz, 1H), 7.58 (d, 9.4 Hz,
1H), 3.90 (t, J=4.8 Hz, 4H), 3.35 (t, J=5.0 Hz, 4H), 2.66 (s, 3H).
LC-MS (ES+) m/e=323.09 (M+H).
[0143] Step D.
3-Dimethylamino-1-[3-(4-morpholino-4-yl-phenyl)-benzo[c]iso-
xazol-5-yl]propenone
[0144] A solution of 1-(3-(4-morpholin-4-yl-phenyl)
benzo[c]isoxazol-5-yl]ethanone (25 mg, 0.08 mmol) in DMF (2.5 mL)
was treated with DMF-DMA (50 .mu.L, 0.37 mmol) and was heated at
90.degree. C. for 36 hours and for 100.degree. C. for a further 18
hours. The solvent was evaporated to give the crude product as
brown oil (35.2 mg) which was used directly in the next step
without purification. LC-MS (ES+) m/e=378.2 (M+H).
[0145] Step E.
4-[3-(4-Morpholin-4-yl-phenyl)benzo[c]isoxazol-5-yl]pyrimid-
in-2-yl amine (I-A34)
[0146] To a solution of sodium (spheres, 25 mg, 1.08 mmol) in MeOH
(0.7 mL) at room temperature under nitrogen was added guanidine
hydrochloride (10 mg, 0.105 mmol) and a solution of
3-dimethylamino-1-[3-(4-morpholino--
4-yl-phenyl)-benzo[c]isoxazol-5-yl]propenone (0.08 mmol) in MeOH
(1.5 mL) and the reaction was heated to 90.degree. C. for 18 hours.
The resulting precipitate was filtered to give the product as an
orange solid (25 mg, 84% yield). .sup.1H NMR
[0147] MHz, DMSO-d.sub.6) .delta. 8.61 (s, 1H), 8.33 (d, 1H),
7.98-8.10 (m, 3H), 7.23-6.98 (m, 3H), 3.96-3.75 (m, 4H), 3.43-3.30
(m, 4H), 2.62-2.49 (m, 2H). LC-MS (ES+) m/e=374.18 (M+H), HPLC
(cyano column) 9.42 minutes.
Example 14
4-{4-[3-(3,4-Dimethoxy-phenyl)-benzo
[cisoxazol-5-yl]-pyrimidin-2-ylamino}- -benzenesulfonamide
(Compound I-A35)
[0148] 110
[0149] A mixture of
4-[(4-(4-Nitro-phenyl)-pyrimidin-2-ylamino]-benzenesul- fonamide
(0.2 mmol) and 3,4-dimethoxy-phenylacetonitrile (0.4 mmol) in
dimethyl sulfoxide (2 mL) was treated with 20% sodium ethoxide in
ethanol (0.5 mL) at ice bath temperature. The mixture was stirred
at room temperature for 18 hours and diluted with methanol (2 mL).
Solid was collected and redissolved in methanol (3 mL) and heated
10 minutes at 80.degree. C. and cooled to room temperature. The
solid was recrystallized twice in methanol to afford yellow title
compound. The compound was characterized by LC/MS and HPLC.
Example 15
4-[3-(4-Bromophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine
(1-A36)
[0150] 111
[0151] Step A.
1-[3-(4-Bromophenyl)-benzo[c]isoxazol-5-yl]-ethanone
[0152] A solution of
3-(4-bromo-phenyl)-5-(2-methyl-(1,3]-dioxolan-2-yl)-b-
enzo[c]isoxazole (Example 1, Step A) (2.13 g, 5.93 mmol) in formic
acid (88% solution, 50 ml) was stirred at room temperature for 30
minutes, affording a thick yellow precipitate. The formic acid was
removed in vacuo, and the resultant solid was dissolved in
CH.sub.2Cl.sub.2, dried over sodium sulfate, filtered and
evaporated to give the product as an orange solid (1.42 g, 76%
yield). .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.47 (s, 1H),
8.04-7.85 (m, 3H), 7.85-7.71 (m, 2H), 7.72-7.57 (m, 1H), 2.68 (s,
3H). HPLC (cyano column) 17.68 minutes
[0153] Step B.
1-[3-(4-Bromo-phenyl)-benzo[c]isoxazol-5-yl]-3-dimethylamin-
o-propenone
[0154] This compound was prepared from
[3-(4-bromophenyl)-benzo[c]isoxazol- -5-yl]-ethanone in an
analogous manner to Experiment 15, Step D except that the reaction
duration was 18 hours. The product was isolated as a brown solid
and was used in the next step without purification (1.61 g, 97%
yield). .sup.1H NMR (500 MHz, CDCl.sub.3) d 8.84 (s, 1H), 7.98-7.79
(m, 4H), 7.77-7.67 (m, 2H), 7.66-7.51 (m, 1H), 5.67 (d, J=12.2 Hz,
1H), 3.31-2.78 (m, 6H).
[0155] Step C.
4-[3-(4-Bromophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-yla-
mine
[0156] This compound was prepared in an analogous manner to
4-[3-(4-chlorophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine
(see Example 13). Purification was achieved by trituration with
dichloromethane to yield 4-[3-(4-bromophenyl)-benzo
[c]isoxazol-5-yl]-pyrimidin-2-ylamine as a yellow solid (559 mg,
49% yield). .sup.1H NMR (500 MHz, d.sub.6-DMSO) .delta. 8.67 (s,
1H), 8.36 (d, 1H), 8.2-8.13 (m, 3H), 7.88 (d, 2H), 7.82 (d, 1H),
7.39 (d, 1H), 6.78 (s, 1H) ppm. LC-MS (ES+) m/e=367 (M+H).
Example 16
3-[4-(3-Phenyl-benzo[c]isoxazol-5-yl)-pyrimidin-2-ylamino]-benzenesulfonam-
ide (Compound I-A37)
[0157] 112
[0158] Compound I-A37 was prepared according to the procedure
described above in Example 2 except that N-phenylguanidine was
replaced by 3-guanidino-benzenesulfonamide.
Example 17
N-(4-{3-[3-(2,5-Dimethoxy-pyrimidin-4-yl)-phenyl]-benzo[c]isoxazol-5-yl}-p-
yrimidin-2-yl)-acetamide (Compound I-A50)
[0159] 113
[0160] Step A:
N-{4-[3-(3-Bromophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2--
yl}-acetamide
[0161] Compound I-A50 was prepared according to the procedure
described as above in Example 7 step B utilizing
4-[3-(3-bromophenyl)-benzo[c]isoxazol- -5-yl]-pyrimidin-2-ylamine
instead of 4-[3-(4-Chlorophenyl)-benzo
(cisoxazol-5-yl]-pyrimidin-2-ylamine. Material was isolated, by
removal of the solvent under reduced pressure and trituration with
dichloromethane, as a yellow powder (430 mg, 77% yield). .sup.1H
NMR (500 MHz TFA-d) .delta. 9.15 (s, 1H), 8.85 (d, 1H), 8.41 (d,
1H), 8.38 (s, 1H), 8.32 (d, 1H), 8.17 (d, 1H), 8.05 (d, 1H), 7.94
(d, 1H), 7.64 (dd, 1H), 2.67 (s, 3H) in ppm. LC-MS (ES+) m/e=409
(M+H).
[0162] Step B:
N-(4-{3-[3-(2,5-Dimethoxy-pyrimidin-4-yl)-phenyl]-benzo[c]i-
soxazol-5-yl}-pyrimidin-2-yl)-acetamide
[0163] A flask was charged with
N-{4-[3-(3-bromophenyl)-benzo[c]isoxazol-5-
-yl]-pyrimidin-2-yl}-acetamide (100 mg, 0.272 mmol), cesium
carbonate (97.7 mg, 0.328 mmol), and
2,5-dimethoxypyrimidine-6-boronic acid (55.0 mg, 0.3 mmol). The
flask was evacuated and back-filled with nitrogen 5-7 times before
adding 5 mL of degassed p-dioxane and 1 mL of degassed DMF. To this
stirring solution/suspension was added, 125 .mu.L of a 10% w/v
benzene solution of tri-tertbutylphosphine followed by the addition
of Pd.sub.2(dba).sub.3 (25 mg, 0.0272 mmol) slurred in 1 mL of
degassed DMF. The reaction was stirred under nitrogen atmosphere,
at 80.degree. C. Reaction was followed by HPLC and deemed to be
complete in 4 hours. The reaction mixture was suction filtered hot
through a pad of diatomaceous earth and washed the precipitate with
DMF and acetonitrile. The filtrate was reduced to an oil under
reduced pressure and the crude material purified via HPLC utilizing
acetonitrile/water/TFA as the eluent. The material was isolated as
a bright yellow powder (15 mg, 13% yield). .sup.1H NMR (500 MHz
DMSO-d.sub.6) .delta. 8.93 (s, 1H), 8.6 (s, 1H), 8.31 (s, 1H), 8.29
(d, 1H), 8.25 (d, 1H), 8.07 (d, 1H), 7.87 (d, 1H), 7.81 (d, 1H),
7.77 (m, 1H), 4.02 (2 close sing, 6H) in ppm. LC-MS (ES+) m/e=469
(M+H)
Example 18
(4-(3-(3-Bromo-phenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-yl}-carbamic
acid ethyl ester (Compound I-A55)
[0164] 114
[0165] To a stirring solution of
4-[3-(3-bromophenyl)-benzo[c]isoxazol-5-y- l]-pyrimidin-2-ylamine
(75 mg; 0.205 mmol) in 1 mL of p-dioxane and 0.5 mL of DMSO, was
added 40 .mu.L (45.6 mg, 0.42 mmol) of ethyl chloroformate followed
by 73 .mu.L (54.3 mg, 0.42 mmol) of diisopropylethylamine. The
reaction was stirred at 50.degree. C., in a sealed vessel, for 8
hours. The solvents were removed under vacuo and the crude material
was purified via HPLC with acetonitrile/water/TFA as the eluent.
The material was isolated as a yellow powder (30 mg, 32% yield).
.sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 10.5 (br s, 1H), 8.9
(s, 1H), 8.75 (d, 1H), 8.35 (m, 3H), 8.3(s, 1H), 8.0 (d, 1H), 7.89
(t, 2H), 7.65 (t, 1H), 4.2 (q, 2H), 1.26 (t, 3H) in ppm. LC-MS
(ES+) m/e=439/441 (M+H)
Example 19
Thiophene-2-carboxylic acid
{4-(3-(3-bromo-phenyl)-benzo[c]isoxazol-5-yl]--
pyrimidin-2-yl}-amide (Compound I-A 56)
[0166] 115
[0167]
4-[3-(3-bromophenyl)-benzo[c]isoxazol-5-yl]-pyrimidin-2-ylamine
(100 mg; 0.272 mmol) was dissolved in 3 mL of a mixture (2:1) of
dry DMF/THF and stirred under a nitrogen atmosphere at ambient
temperature. Sodium hydride (15 mg, 0.375 mmol, 60% oil dispersion)
was added To the reaction and stirred for 30 minutes.
Thiophenecarbonylchloride (32 .mu.L; 43.7 mg; 0.299 mmol) in 500
.mu.L of dry DMF was added dropwise over 2 minutes and the reaction
was stirred for 18 hours at ambient temperature. Workup was
affected by removing the solvents under reduced pressure and the
resulting residue was triturated with methyltertbutyl ether. The
crude solid was purified via silica column chromatography with 5%
ethanol in methylenechloride to yield 32 mg of a tan powder; 24%
yield. .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.2 (s, 1H),
8.92 (s, 1H), 8.88 (d, 1H), 8.39 (d, 1H), 8.35 (s, 1H), 8.3 (d,
1H), 8.23 (d, 1H), 8.15 (d, 1H), 7.95 (d, 1H), 7.9 (d, 1H), 7.87
(d, 1H), 7.65 (t, 1H), 7.24 (t, 1H) in ppm. LC-MS (ES+) m/e=477/479
(M+H).
Biological Methods
[0168] IC.sub.50 Determination for the Inhibition of GSK-3
[0169] Compounds were screened for their ability to inhibit
GSK-3.beta. (Amino Acids 1-420) activity using a standard coupled
enzyme system (Fox et al. (1998) Protein Sci. 7, 2249). Reactions
were carried out in a solution containing 100 mM HEPES (pH 7.5), 10
mM MgCl.sub.2, 25 mM NaCl, 300 .mu.M NADH, 1 mM DTT and 1.5% DMSO.
Final substrate concentrations in the assay were 10 .mu.M ATP
(Sigma Chemicals, St Louis, Mo.) and 300 .mu.M peptide
(HSSPHQS(PO.sub.3H.sub.2) EDEEE, American Peptide, Sunnyvale,
Calif.). Reactions were carried out at 30.degree. C. and 60 nM
GSK-3.beta.. Final concentrations of the components of the coupled
enzyme system were 2.5 mM phosphoenolpyruvate, 300 .mu.M NADH, 30
.mu.g/ml pyruvate kinase and 10 .mu.g/ml lactate dehydrogenase.
[0170] An assay stock buffer solution was prepared containing all
of the reagents listed above with the exception of ATP and the test
compound of interest. 59 .mu.l of the test reaction was placed in a
96 well 1/2 diameter plate (Corning, Corning, N.Y.) then treated
with 1 .mu.l of a 2 mM DMSO stock containing the test compound
(final compound concentration 30 .mu.M). The plate was incubated
for about 10 minutes at 30.degree. C. then the reaction initiated
by addition of 7 .mu.l of ATP (final concentration 10 .mu.M). Rates
of reaction were obtained using a Molecular Devices Spectramax
plate reader (Sunnyvale, Calif.) over a 5 minute read time at
30.degree. C. Compounds showing greater than 50% inhibition versus
standard wells containing DMSO, but no compound, were titrated and
IC.sub.50 values were determined using a similar protocol in
standard 96 well plates with the assay scaled to a final volume of
200 .mu.l.
[0171] In the GSK-3 inhibition assay described above, many of the
compounds of this invention that were tested were found to provide
an IC.sub.50 value below one micromolar.
[0172] K.sub.i Determination for the Inhibition of GSK-3
[0173] Compounds were screened for their ability to inhibit
GSK-3.beta. (Amino Acids 1-420) activity using a standard coupled
enzyme system (Fox et al. (1998) Protein Sci. 7, 2249). Reactions
were carried out in a solution containing 100 mM HEPES (pH 7.5), 10
mM MgCl.sub.2, 25 mM NaCl, 300 .mu.M NADH, 1 mM DTT and 1.5% DMSO.
Final substrate concentrations in the assay were 20 .mu.M ATP
(Sigma Chemicals, St Louis, Mo.) and 300 .mu.M peptide
(HSSPHQS(PO.sub.3H.sub.2) EDEEE, American Peptide, Sunnyvale,
Calif.). Reactions were carried out at 30.degree. C. and 20 nM
GSK-3.beta.. Final concentrations of the components of the coupled
enzyme system were 2.5 mM phosphoenolpyruvate, 300 .mu.M NADH, 30
.mu.g/ml pyruvate kinase and 10 .mu.g/ml lactate dehydrogenase.
[0174] An assay stock buffer solution was prepared containing all
of the reagents listed above with the exception of ATP and the test
compound of interest. The assay stock buffer solution (175 .mu.l)
was incubated in a 96 well plate with 5 .mu.l of the test compound
of interest at final concentrations spanning 0.002 .mu.M to 30
.mu.M at 30.degree. C. for 10 minutes. Typically, a 12 point
titration was conducted by preparing serial dilutions (from 10 mM
compound stocks) with DMSO of the test compounds in daughter
plates. The reaction was initiated by the addition of 20 .mu.l of
ATP (final concentration 20 .mu.M). Rates of reaction were obtained
using a Molecular Devices Spectramax plate reader (Sunnyvale,
Calif.) over 10 min at 30.degree. C. The K.sub.i values were
determined from the rate data as a function of inhibitor
concentration.
[0175] In the GSK-3 inhibition assay described above, many of the
compounds of this invention that were tested were found to provide
a K.sub.i value below one micromolar.
[0176] JAK Inhibition Assay
[0177] Compound inhibition of JAK were assayed by the method
described by G. R. Brown, et al, Bioorg. Med. Chem. Lett. 2000,
vol. 10, pp 575-579 in the following manner. Into Maxisorb plates,
previously coated at 4.degree. C. with Poly (Glu, Ala, Tyr) 6:3:1
then washed with phosphate buffered saline 0.05% and Tween (PBST),
was added 2 .mu.M ATP, 5 mM MgCl.sub.2, and a solution of compound
in DMSO. The reaction was started with JAK enzyme and the plates
incubated for 60 minutes at 30.degree. C. The plates were then
washed with PBST, 100 .mu.L HRP-Conjugated 4G10 antibody was added,
and the plate incubated for 90 minutes at 30.degree. C. The plate
was again washed with PBST, 100 .mu.L TMB solution is added, and
the plates were incubated for another 30 minutes at 30.degree. C.
Sulfuric acid (100 .mu.L of 1M) was added to stop the reaction and
the plate is read at 450 nm to obtain the optical densities for
analysis to determine IC.sub.50 values.
[0178] While we have described a number of embodiments of this
invention, it is apparent that our basic examples may be altered to
provide other embodiments which utilize the compounds and methods
of this invention. Therefore, it will be appreciated that the scope
of this invention is to be defined by the appended claims rather
than by the specific embodiments which have been represented by way
of example.
* * * * *