Note: Descriptions are shown in the official language in which they were submitted.
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TRICYCLIC PYRAZOLE DERIVATIVES FOR THE TREATMENT OF
INFLAMMATION
FIELD OF THE INVENTION
[001] The present invention in general is in the field of anti-inflammatory
pharmaceutical agents and specifically relates to substituted tricyclic
pyrazole
derivatives, compositions comprising them, and their use as therapeutic agents
in
the treatment of diseases linked to protein kinases, particularly for treating
cancer,
inflammation, and inflammation-associated disorders, such as arthritis.
BACKGROUND OF THE INVENTION
[002] The following description of the background of the invention is provided
to aid in the understanding the invention, but is not admitted to be or
describe prior
art to the invention.
[003] NF-~cB is a ubiquitous transcription factor that plays a prominent role
in
the activation of the immune system and in stress responses by regulating the
transcription of many early, inducible genes including proinflammatory
cytokines,
adhesion molecules, growth factors, enzymes, and receptors (Ghosh S., May, M.
J.,
and Kopp. E (1998) Annu. Rev. Immunol. 16, 115-260; Zandi, E., and Karin, M.
(1999) Mol. Cell. Biol. 19, 4547-4551; Karin, M. (1999) J. Biol. Chena. 274,
27339-
27342). Specificity of gene expression is determined at a cellular level by a
diverse
array of external stimuli such as bacterial products including LPS, as well as
cytokines, most importantly tumor necrosis factor-a (TNFa) and interleukin-/3
(ILl(3). Through the synergistic interaction with other transcription factors,
further
specificity can be achieved while maintaining enormous potential to
coordinately
induce a large number of functionally related genes. NF-oB is composed of homo
and heterodimers of the Rel protein family and is sequestered in an inactive
form in
the cytoplasm by members of the IxB family of inhibitory proteins (Ghosh S.,
May,
M. J., and Kopp. E (1998) ArafZU. Rev. Imnaunol. 16, 115-260; Zandi, E., and
Karin,
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M. (1999) Mol. Cell. Biol. 19, 4547-4551; Karin, M. (1999) J. Biol. Chem. 274,
27339-27342). hcBs mask the nuclear localization signal on NF-xB, preventing
nuclear translocation and hence DNA binding to the promoter regions of
responsive
genes. Stimulation of cells with an agonist that activates NF-oB leads to a
series of
biochemical signals, ultimately resulting in the phosphorylation,
ubiquitinylation,
and degradation of IoBs, thereby releasing NF-oB for nuclear translocation
(Ghosh
S., May, M. J., and Kopp. E (1998) Anrau. Rev. Immunol. 16, 115-260; Zandi,
E.,
and Karin, M. ( 1999) Mol. Cell. Biol. 19, 4547-4551; Karin, M. ( 1999) J.
Biol.
Chem. 274, 27339-27342). Recently, two hcB kinases (IKK1 or TKK_a and IKK2 or
IKK(3), which phosphorylate hcBs and thereby initiate their degradation, have
been
cloned and characterized by a number of laboratories (Ghosh S., May, M. J.,
and
Kopp. E (1998) Annu. Rev. Imrnunol. 16, 115-260; Zandi, E., and Karin, M.
(1999)
Mol. Cell. Biol. 19, 4547-4551; Karin, M. (1999) J. Biol. Clzem. 274, 27339-
27342). The catalytic subunits, IKK1 and IKK2, are similar structurally as
well as
enzymatically and exist as a heterodimer in a large protein complex referred
to as
the IKK signalsome (Regnier, C., Song, H., Gao, X., Goeddel, D., Cao, Z. and
Rothe, M. (1997) Cell 90, 373-383; DiDonato, J.A., Hayakawa, M., Rothwarf,
D.M., Zandi, E. and Karin, M. ( 1997) Nature 388, 548-554; Mercurio, F., Zhu,
H.,
Murray, B.W., Shevchenko, A., Bennett, B.L., Li, J.W., Young, D.B., Barbosa,
M.,
Mann, M., Manning, A. and Roa, A. (1997) Seierzce 278, 860-866; Zandi, E.
Rothwarf, D.M., Delhase, M., Hayadawa, M and Karin, M. (1997) Cell 91, 243-
252; Woronicz, J.D., Gao, X., Cao, Z., Rothe, M. And Goeddel, D.V. (1997)
Science 278, 866-869). A third protein, NEMO (IKKy, IKKAPl), is a regulatory
adapter protein necessary for IKK activation and kinase activity (Yamaoka, S.,
Courtois, G., Bessia, C., Whiteside, S. T., Weil, R., Agou, F., Kirk, H. E.,
Kay, R.
J., and heal, A. (1998) Cell 93, 1231-1240; Rothwarf, D. M., Zandi, E.,
Natoli, G.,
Karin, M. (1998) Nature 395, 297; Mercurio, F., Murray, B. W., Shevchenko, A.,
Bennet, B. L., Young, D. B., Li, J. W., Pascual, G., Motiwala, A., Zhu, H.,
Mann,
M and Manning, A. M. (1999) Mol. Cell. Biol. 2, 1526-1538). IKKl and IKK2 are
co-expressed in most human adult tissues as well as in different developmental
stages of mouse embryos (Regnier, C., Song, H., Gao, X., Goeddel, D., Cao, Z.
and
Rothe, M. (1997) Cell 90, 373-383; DiDonato, J.A., Hayakawa, M., Rothwarf,
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D.M., Zandi, E. and Karin, M. (1997) Nature 388, 548-554; Mercurio, F., Zhu,
H.,
Murray, B.W., Shevchenko, A., Bennett, B.L., Li, J.W., Young, D.B., Barbosa,
M.,
Mann, M., Manning, A. and Roa, A. (1997) Science 278, 860-866; Zandi, E.
Rothwarf, D.M., Delhase, M., Hayadawa, M and Karin, M. (1997) Cell 91, 243-
252; Woronicz, J.D., Gao, X., Cao, Z., Rothe, M. and Goeddel, D.V. (1997)
Science 278, 866-869; Hu, M. C. T., and Wang, Y. (1998) Gene 222, 31-40). This
kinase complex appears to represent a critical, common denominator in the
activation of NF-icB in a number of signal transduction pathways stimulated by
a
variety of agonists including cytokines, such as TNFa and ILl(3, microbial
products
such as LPS and viral proteins such as TAX, as well as phorbol esters,
oxidizing
agents and serine/tyrosine phosphatases (Ghosh S., May, M. J., and Kopp. E
(1998)
Arznu. Rev. In2mufzol. 16, 115-260; Zandi, E., and Karin, M. (1999) Mol. Cell.
Biol.
19, 4547-4551; Karin, M. (1999) J. Biol. Chem. 274, 27339-27342).
[004 IKK1 (also termed IKKa, Regnier, C., Song, H., Gao, X., Goeddel, D.,
Cao, Z. and Rothe, M. (1997) Cell 90, 373-383; DiDonato, J.A., Hayakawa, M.,
Rothwarf, D.M., Zandi, E. and Karin, M. (1997) Nature 388, 548-554; Mercurio,
F.,
Zhu, H., Murray, B.W., Shevchenko, A., Bennett, B.L., Li, J.W., Young, D.B.,
Barbosa, M., Mann, M., Manning, A. And Roa, A. (1997) Science 278, 860-866)
was cloned simultaneously by standard biochemical purification of the IxB
kinase
activity from TNFa stimulated HeLa S3 cells and by its interaction with the
MAP3K, NF-~cB inducing kinase (NIK), in a yeast two-hybrid screen. IKKl was
identified as the previously cloned serine-threonine kinase, CHUK (Connelly,
M.
and Marcu, K. (1995) Cell. Mol. Biol. Res. 41, 537-549). IKKl (also termed
IKKa) is an 85 kDa, 745 amino acid protein that contains an N-terminal
serine/threonine kinase catalytic domain, a leucine zipper-like amphipathic
helix,
and a C-terminal helix-loop-helix domain. IKK2 (also termed IKK~3) was also
cloned by standard biochemical purification, copurifying with IKK1 from TNFa
stimulated HeLa S3 cells as well as by being identified in the public database
from
an EST clone with sequence homology to IKKl (Mercurio, F., Zhu, H., Murray,
B.W., Shevchenko, A., Bennett, B.L., Li, J.W., Young, D.B., Barbosa, M., Mann,
M., Manning, A. and Roa, A. (1997) Science 278, 860-866; Zandi, E. Rothwarf,
3
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D.M., Delhase, M., Hayadawa, M and Karin, M. (1997) Cell 91, 243-252;
Woronicz, J.D., Gao, X., Cao, Z., Rothe, M. And Goeddel, D.V. (1997) Science
278, 866-869). IKK2 is an 87 kDa, 756 amino acid protein with the same over
all
topology as IKKl except for the addition of an 11 amino acid extension at the
C-terminus. IKKl and IKK2 are 52% identical overall with 65% identity in the
kinase domain and 44% identity in the protein interaction domains in the C-
terminus. Data obtained using transient mammalian expression analysis, by in
vitro
translation experiments and by coexpression in a baculoviral system reveals
that
IKKl and IKK2 associate preferentially as a heterodimer through their leucine
zipper motifs. Although homodimers have also been described in these systems,
the
heterodimer is thought to be the physiologic form of the kinase in mammalian
cells
(Zandi, E. Rothwarf, D.M., Delhase, M., Hayadawa, M and Karin, M. (1997) Cell
91, 243-252; Li, J., Peet, G.W., Pullen, S.S., Schembri-King, J., Warren,
T.C.,
Marcu, K.B., Kehry, M.R., Barton, R. and Jakes, S. (1998) J. Biol. Chem. 273,
30736-30741). Finally, NEM~ (also termed IKKy) contains three a-helical
regions
including a leucine zipper, interacts preferentially with IKK2 and is required
for
activation of the heterodimeric kinase complex perhaps by bringing other
proteins
into the signalsome complex (Yamaoka, S., Courtois, G., Bessia, C., Whiteside,
S.
T., Weil, R., Agou, F., Kirk, H. E., Kay, R. J., and heal, A. (1998) Cell 93,
1231-
1240; Rothwarf, D. M., Zandi, E., Natoli, G., Karin, M. (1998) Nature 395,
297;
Mercurio, F., Murray, B. W., Shevchenko, A., Bennet, B. L., Young, D. B., Li,
J.
W., Pascual, G., Motiwala, A., Zhu, H., Mann, M and Manning, A. M. (1999) Mol.
Cell. Biol. 2, 1526-1538).
[005] The kinase activities of IKKl and IKK2 are regulated by
phosphorylation and require an intact leucine zipper (LZ) for dimerization as
well as
an intact helix-loop-helix (HLH) domain, which can exert a positive regulatory
effect on kinase activity even when it is expressed in trans with the
remainder of the
IKK protein (Regnier, C., Song, H., Gao, X., Goeddel, D., Cao, Z. and Rothe,
M.
(1997) Cell 90, 373-383; DiDonato, J.A., Hayakawa, M., Rothwarf, D.M., Zandi,
E.
and Karin, M. (1997) Nature 388, 548-554; Mercurio,F., Zhu, H., Murray, B.W.,
Shevchenko, A., Bennett, B.L., Li, J.W., Young, D.B., Barbosa, M., Mann, M.,
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Manning, A. and Roa, A. (1997) Science 278, 860-866; Zandi, E. Rothwarf, D.M.,
Delhase, M., Hayadawa, M and Karin, M. (1997) Cell 91, 243-252; Woronicz,
J.D.,
Gao, X., Cao, Z., Rothe, M. and Goeddel, D.V. (1997) Science 278, 866-869;
Dehase, M., Hayakawa, M., Chen, Y., and Karin, M. (1999) Science 284, 309-
313).
Both IKK subunits contain a canonical MAPKK activation loop motif near the N-
terminus which is the target for phosphorylation and activation of kinase
activity by
MAP3Ks such as NIK and MEKKl, although the physiologic regulation by these
two upstream kinases awaits further characterization (Zandi, E., and Karin, M.
(1999) Mol. Cell. Biol. 19, 4547-4551; Karin, M. (1999) J. Biol. Chem. 274,
27339-
27342; Karin, M., and Delhase, M. (1998) Proc. Natl. Acad. Sci. USA 95, 9067-
9069). Finally, phosphorylation of serines in the C-terminus of IKK2 results
in a
decrease in IKK activity and it is postulated to be responsible for the
transient
kinase activity seen after stimulation of cells with an agonist (Dehase, M.,
Hayakawa, M., Chen, Y., and Karin, M. (1999) Science 284, 309-313).
[006] IKKZ demonstrates a more potent kinase activity compared to IKKl
using hcBa or IxB[3 as a substrate (Mercurio, F., Zhu, H., Murray, - B.W.,
Shevchenko, A., Bennett, B.L., Li, J.W., Young, D.B., Barbosa, M., Mann, M.,
Manning, A. and Roa, A. (1997) Science 278, 860-866; Zandi, E. Rothwarf, D.M.,
Delhase, M., Hayadawa, M and Karin, M. (1997) Cell 91, 243-252; Woronicz,
J.D.,
Gao, X., Cao, Z., Rothe, M. and Goeddel, D.V. (1997) Science 278, 866-869;
Dehase, M., Hayakawa, M., Chen, Y., and Karin, M. (1999) Science 284, 309-
313).
Mutations of the phospho-acceptor serine residues within the MAPKK activation
loop alters IKK2 kinase activity; the serine to alanine substitutions result
in
decreased kinase activity whereas the serine to glutamic acid substitutions
result in
a constitutively active kinase. Similar alanine mutations in IKKl do not
result in a
decreased stimulation of total IKK activity in response to TNFa or IL1(3
(Dehase,
M., Hayakawa, M., Chen, Y., and Karin, M. (1999) Science 284, 309-313). IKK2
being the dominant kinase activity within the IKK complex is further supported
by
the analysis of fibroblasts from mice deficient in IKK1 or IKK2. Fibroblasts
lacking IKKl retain full IKK activity in response to cytokines and could
activate
NF-xB. In contrast, fibroblasts lacking IKK2 do not exhibit IKK activity when
5
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stimulated with cytokines nor do they activate NF-xB. Furthermore, the
phenotypes
of each IKK knock out is unique with IKKl deficiency resulting in skin and
skeletal
defects and IKK2 knock out being embryonic lethal due to hepatocyte apoptosis
(Li,
Q., Antwerp, D. V., Mercurio, F., Lee, K., and Verma, I. M. (1999) Science
284,
321-325; Takeda, K., Tekeuchi, O., Tsujimura, T., Itami, S., Adachi, O.,
Kawai, T.,
Sanjo, H., Yoshikawa, K., Terada, N, and Akira, S. (1999) Science 284, 313-
316;
Hu, Y., Baud, V., Delhase, M., Zhang, P., Deerinck, T., Ellisman, M., Johnson,
R.,
and Karin, M. (1999) Science 284, 315-320; Li, Q., Lu, Q., Hwang, J. Y.,
Buscher,
D., Lee, K., Izpisua-Belmonte, J. C., and Verma, I. M. (1999) Gene and
Development 13, 1322-132; Tanaka, M., Fuentes, M. E., Yamaguchi, K., Durnin,
M. H., Dalrymple, S. A., Hardy, K. L., and Goeddel, D. V. (1999) I»zmunity 10,
421-429).
[007] It is well-known that NF-KB plays a key role in the regulated expression
of a large number of pro-inflammatory mediators including cytokines such as IL-
6
and IL-8, cell adhesion molecules, such as ICAM and VCAM, and inducible nitric
oxide synthase (iNOS). Such mediators are known to play a role in the
recruitment
of leukocytes at sites of inflammation and in the case of iNOS, may lead to
organ
destruction in some inflammatory and autoimmune diseases. The importance of
NF-~cB in inflammatory disorders is further strengthened by studies of airway
inflammation including asthma in which NF-~cB has been shown to be activated.
This activation may underlie the increased cytokine production and leukocyte
infiltration characteristic of these disorders. In addition, inhaled steroids
are known
to reduce airway hyperresponsiveness and suppress the inflammatory response in
asthmatic airways. In light of the recent findings with regard to
glucocorticoid
inhibition of NF-xB, one may speculate that these effects are mediated through
an
inhibition of NF-oB. Further evidence for a role of NF-xB in inflammatory
disorders comes from studies of rheumatoid synovium. Although NF-xB is
normally present as an inactive cytoplasmic complex, recent
immunohistochemical
studies have indicated that NF-xB is present in the nuclei, and hence active,
in the
cells comprising rheumatoid synovium. Furthermore, NF-xB has been shown to be
activated in human synovial cells in response to stimulation with TNF-oc. Such
a
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distribution may be the underlying mechanism for the increased cytokine and
eicosanoid production characteristic of this tissue. See Roshak, A. K., et
al., J. Biol.
Chem., 271, 31496-31501 (1996).
[008] NF-~cB in inflammatory disorders is further strengthened by studies of
airway inflammation including asthma in which NF-~cB has been shown to be
activated. This activation may underlie the increased cytokine production and
leukocyte infiltration characteristic of these disorders. In addition, inhaled
steroids
axe known to reduce airway hyper responsiveness and suppress the inflammatory
response in asthmatic airways. In light of the recent findings with regard to
glucocorticoid inhibition of NF-xB, one may speculate that these effects are
mediated through an inhibition of NF-xB. Further evidence for a role of NF-xB
in
inflammatory disorders comes from studies of rheumatoid synovium. Although NF-
xB is normally present as an inactive cytoplasmic complex, recent
immunohistochemical studies have indicated that NF-xB is present in the
nuclei,
and hence active, in the cells comprising rheumatoid synovium. Furthermore, NF-
oB has been shown to be activated in human synovial cells in response to
stimulation with TNF-a. Such a distribution may be the underlying mechanism
for
the increased cytokine and eicosanoid production characteristic of this
tissue. See
Roshak, A. K., et al., J. Biol. Chem., 271, 31496-31501 (1996).
[009] The NF-xB/Rel and IxB proteins are also likely to play a key role in
neoplastic transformation. Family members are associated with cell
transformation
in vitro and in vivo because of overexpression, gene amplification, gene
rearrangements, or translocations (Gilmore TD, Trends Gerzet 7:318-322, 1991;
Gillmore TD, Oncogene 18:6925-6937, 1999; Rayet B. et al., Oncogene 18: 6938-
6947, 1991 ). In addition, rearrangement and/or amplification of the genes
encoding
these proteins are seen in 20-25% of certain human lymphoid tumors. In
addition, a
role for NF-oB in the regulation of apoptosis, cell cycle progression,
invasion, and
metastasis has been reported (Bours V. et al., Biochemical Plzarnzacology
60:1085-
1090, 2000) strengthening the role of this transcription factor in the control
of cell
proliferation. The inhibition of NF-xB has been shown to potentiate TNF- and
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cancer therapy through increased apoptosis (Wang C-Y et al., Science 274:784-
787,
1996; Wang C-Y et al., Nat Med 5:412-417, 1999). It, has also been shown that
human T-cell leukemia virus type 1 (HTLV1) infected cells (the etiological
agent of
an aggressive malignancy of activated CD4+ T lymphocytes), IKKoc and IKK(3 are
expressed constitutively, which normally function in a transient manner (Chu Z-
L et
al., J of Biological Cl~ernistry 273:15891-15894, 1998). The HTLV1
transforming
and transactivating protein (Tax) has been shown to bind MEKK1 and increases
the
activity of IKK(3 to enhance phosphorylation of serine residues in IoBoc that
lead to
its degradation.
[0010] Pyrazoles have been described for use in the treatment of inflammation.
U.S. Patent No. 5,134,142 to Matsuo et al describes 1,5-diaryl pyrazoles, and
specifically, 1-(4-fluorophenyl)-5-[4-(methylsulfonyl)phenyl]-3-
trifluoromethyl
pyrazole, as having anti-inflammatory activity.
[0011] U.S. Patent No. 3,940,418 to R. Hamilton describes tricyclic 4,5-
dihydrobenz[g]indazoles as antiinflammatory agents. In addition, R. Hamilton
[J.
Heterocyclic Claem., 13, 545 (1976)] describes tricyclic 4,5-
dihydrobenz[g]indazoles as antiinflammatory agents. U.S. Patent No. 5,134,155
describes fused tricyclic pyrazoles having a saturated ring bridging the
pyrazole and
a phenyl radical as HMG-CoA reductase inhibitors. European publication EP
477,049, published Mar. 25, 1992, describes [4,5-dihydro-1-phenyl-1H-
benz[g]indazol-3-yl]amides as having antipsychotic activity. European
publication
EP. 347,773, published Dec. 27, 1989, describes [4,5-dihydro-1-phenyl-1H-
benz[g]indazol-3-yl]propanamides as immunostimulants. M. Hashem et al [J. Med.
Chem., 19, 229 (1976)] describes fused tricyclic pyrazoles, having a saturated
ring
bridging the pyrazole and a phenyl radical, as antibiotics.
[0012] Certain substituted pyrazolyl-benzenesulfonamides have been described
in the literature as synthetic intermediates. Specifically, 4-[5-(4-
chlorophenyl)-3-
phenyl-1H pyrazol-1-yl]benzenesulfonamide has been prepared from a pyrazoline
compound as an intermediate for compounds having hypoglycemic activity [R.
s
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Soliman et al, J. Plzarnz. Sci., 76, 626 (1987)]. 4-[5-[2-(4-Bromophenyl)-2H
1,2,3-
triazol-4-yl]-3-methyl-1H pyrazol-1-yl]benzenesulfonamide has been prepared
from
a pyrazoline compound and described as potentially having hypoglycemic
activity
[H. Mokhtar, Pak. J. Sci. Irad. Res., 31, 762 (1988)]. Similarly, 4-[4-bromo-5-
[2-(4-
chlorophenyl)-2H 1,2,3-triazol-4-yl]-3-methyl-1H pyrazol-1-
yl]benzenesulfonamide has been prepared [H. Mokhtar et al, Pak. J. Sci. Ind.
Res.,
34, 9 (1991)].
[0013] The phytotoxicity of pyrazole derivatives is described [M. Cocco et al,
Il. Farmaco-Ed. Sci., 40, 272 (1985)], specifically for 1-[4-
(aminosulfonyl)phenyl]-
5-phenyl-1H-pyrazole-3,4-dicarboxylic acid.
[0014] The use of styryl pyrazole esters for antidiabetes drugs is described
[H.
Mokhtar et al, Pharmazie, 33, 649-651 (1978)]. The use of styryl pyrazole
carboxylic acids for antidiabetes drugs is described [R. Soliman et al,
Pharmazie,
33, 184-5 (1978)]. The use of 4-[3,4,5-trisubstituted-pyrazol-1-
yl]benzenesulfonamides as intermediates for sulfonylurea anti-diabetes agents
is
described, and specifically, 1-[4-(aminosulfonyl)phenyl]-3-methyl-5-phenyl-1H-
pyrazole-4-carboxylic acid [R. Soliman et al, J. Pharm. Sci., 72, 1004
(1983)]. A
series of 4-[3-substituted methyl-5-phenyl-1H pyrazol-1-yl]benzenesulfonamides
has been prepared as intermediates for anti-diabetes agents, and more
specifically,
4-[3-methyl-5-phenyl-1H pyrazol-1-yl]benzenesulfonamide [H. Feid-Allah,
Pharmazie, 36, 754 (1981)]. In addition, 1-(4-[aminosulfonyl]phenyl)-5-
phenylpyrazole-3-carboxylic acid has been prepared from the above described 4-
[3-
methyl-5-phenyl-1H pyrazol-1-yl]benzenesulfonamide compound [R. Soliman et al,
J. Plzann. Sci., 70, 602 (1981)].
[0015] WO 00/27822 discloses tricyclic pyrazole derivatives, WO 00/59901
discloses dihydroindeno pyrazoles, WO 95/15315 discloses diphenyl pyrazole
compounds, WO 95/15317 discloses triphenyl pyrazole compounds, WO 95/15318
discloses tri-substituted pyrazole compounds, and WO 96/09293 discloses
Benz[g]indazolyl derivatives.
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[0016] WO 95!15316 discloses substituted pyrazolyl benzenesulfamide
derivatives.
DETAILED DESCRIPTION OF THE INVENTION
[0017] A class of compounds, which are useful in treating cancer,
inflammation, and inflammation related disorders, is defined by Formula I:
R"
wherein
A is selected from the group consisting of: (CH2)m and (CH~)n CH=CH-
(CH2)n; wherein each CH2 may be independently substituted with one or
more substitution selected from the group consisting of: hydroxy, halo,
alkoxy, lower alkyl, amino, aminoalkyl, alkylamino, alkenyl, and alkynyl;
misOto8;
n is independently selected from 0, 1 or 2;
Q is a 5 or 6 membered heteroaryl, or aryl, optionally saturated, or
optionally substituted with Rl, R2, or Rla;
~ is an aromatic heterocyclic;
X is selected from the group consisting of: N and C;
Y and Z are independently selected from the group consisting of: N, C, CH,
CR3, S, and O;
Rl is selected from the group consisting of: hydrido, halogen, alkyl, aryl,
heteroaryl, alkenyl, alkynyl, haloalkyl, CN, NO~, ORS, OCOORS, C02R7,
to
CA 02476665 2004-08-18
WO 03/070706 PCT/US03/04844
CON(R6)R7, CORE, SR6, SORE, S02R6, NR6R7, NR6COR7, NR6CONHR7,
NR6S02R7, NR6SO2NHR7, and S02N(R6)R7 wherein R6 and R7 may be
taken together to form a 3-7 membered carbocyclic ring having 1 to 3
substituted or unsubstituted heteroatoms selected from the group consisting
of: S, SO, 502, O, and NR6; wherein said alkenyl, alkynyl, alkyl, aryl,
heteroaryl or ORS are optional substituted with, hydrido, halogen, alkyl,
hydroxyalkyl, aryl, heteroaryl, haloalkyl, COCF3, CN, N02, ORS, OCOORS,
C02R7, CON(R6)R7, CORE, SR6, SORE, S02R6, NR6R7, NR6COR7,
NR6CONHR7, NR6S02R7, NR6S02NHR7, and S02N(R6)R7 wherein R6 and
R7 may be taken together to form a 3-7 membered carbocyclic ring having 1
to 3 substituted or unsubstituted heteroatoms selected from the group
consisting of: S, SO, 502, O, and NR6;
R2 is selected from the group consisting of: halogen, hydrido, hydroxyalkyl,
alkyl, OR6, CN, N02, SR6, NHR~, CON(R6)R7, NHCONHR6, C02H, and
haloalkyl;
Ri and R2 may be taken together to form a 5 to 7 membered saturated or
unsaturated carbocyclic ring optionally containing 0 to 3 heteroatoms
selected from the group consisting of: N, O, or S, and wherein said ring is
optionally substituted with Rl;
R3 is selected from the group consisting of: substituted or unsubstituted
amidine, alkylamino, aminoalkyl, CONHR16, NH2, NHCOR6, and
CH2NHCOR6;
R4 is selected from the group consisting of: halogen, alkylsulfinyl,
alkylsulfonyl, cyano, alkoxycarbonyl, alkyl, haloalkyl, hydrido,
hydroxyalkyl, haloalkoxy, heterocyclic, nitro, acylamino, aryl, heteroaryl,
and alkenyl, OR13, SRB, S02N(R8)RB~, NHR9, NHCOR9, NR9COR9,
NHCO(OR9), NR9CO(OR9), NR$S02R1°, NHS02N(Rl°)Rio~,
NR6CON(Rl°)Rio~, COR9, C02R8, CON(R8)RB~, wherein R8 and R8~ may
be
taken together to form a 3-7 membered carbocyclic ring having 1 to 3
substituted or unsubstituted heteroatoms selected from S, SO, 502, O, N,
and NR6, and wherein Rl° and Rl°~ may be taken together to form
a 3-7
membered carbocyclic ring having 1 to 3 substituted or unsubstituted
n
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heteroatoms selected from S, SO, 502, O, N, and NR6 wherein said aryl,
heterocyclic, heteroaryl, or alkenyl are optionally substituted with R9;
R5 is selected from the group consisting of: hydrido, alkyl, aryl, arylalkyl,
heteroaryl, heterocyclicalkyl, and heteroarylalkyl, wherein aryl, alkyl,
arylalkyl, heteroaryl, heterocyclicalkyl, or heteroarylalkyl are optionally
substituted with one or more radicals selected from the group consisting of:
OR14, N(R14)R14~, and glycols;
R6 is independently selected from the group consisting of: hydrido, aryl,
heteroaryl, lower alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl,
aminoalkyl, alkylaminoalkyl, alkoxy, alkoxyalkyl, heterocyclicalkyl, and
heterocyclic;
R' is independently selected from the group consisting of: hydrido, aryl,
heteroaryl, lower alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl,
aminoalkyl, alkylaminoalkyl, alkoxy, alkoxyalkyl, heterocyclicalkyl, and
heterocyclic;
R$ is independently selected from the group consisting of: hydrido, aryl,
heteroaryl, arylalkyl, heterocyclic, haloalkyl, arylalkylamino,
alkylaminoalkyl, dialkylaminoalkyl, alkyl, alkenyl, alkynyl, heteroarylalkyl,
and heterocyclicalkyl;
Rg is independently selected from the group consisting of: hydrido, aryl,
heteroaryl, arylalkyl, heterocyclic, haloalkyl, arylalkylamino,
alkylaminoalkyl, dialkylaminoalkyl, alkyl, alkenyl, alkynyl, heteroarylalkyl,
and heterocyclicalkyl;
R9 is independently selected from the group consisting of: hydrido, lower
alkyl, aryl, heteroaryl, arylalkyl, heterocyclic, cycloalkyl,
heterocyclicalkyl,
haloalkyl, arylalkylamino, amino, aminoalkyl, aminoacyl, nitro, azido, and
heteroarylalkyl, wherein alkyl, aryl, heteroaryl, aminoalkyl, or arylalkyl are
optionally substituted with one or more radical selected from the group
consisting of: alkylsulfonamide, sulfamyl, alkyl, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkoxy, halogen,
acyloxy, oxy, formyl, haloalkyl, cyano, haloalkoxy, acyl, carboxyl, hydroxy,
hydroxyalkyloxy, phenoxy, nitro, azido, benzyloxy, dialkylaminoacyl,
12
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thioalkyl, aminoacyloxy, thiocyanate, isothiocyanate, alkyldioxy,
hydroxyalkyl, alkylamino, alkyloxycarbonyl, alkoxyalkyl, alkenylamino,
alkynylamino, alkenyl, alkynyl, dialkylaminoalkyfoxy, and heterocyclic
optionally substituted with alkyl, alkylamino, aminoalkyl, hydroxyalkyl, and
alkylaminoalkyl;
Rl° is independently selected from the group consisting of:
hydrido, lower
alkyl, heteroaryl, heterocyclic, haloalkyl, arylalkylamino, heteroarylalkyl,
aryl, and arylalkyl, wherein aryl, heteroaryl, heterocyclic, or arylalkyl are
optionally substituted with one or more radical selected from alkyl, alkoxy,
halogen, haloalkyl, cyano, haloalkoxy, acyl, carboxyl, hydroxy,
hydroxyalkyloxy, phenoxy, benzyloxy, dialkylaminoalkyloxy, and
heterocyclic,
Ri°~ is independently selected from the group consisting of:
hydrido, lower
alkyl, heteroaryl, heterocyclic, haloalkyl, arylalkylamino, heteroarylalkyl,
aryl, and arylalkyl, wherein aryl, heteroaryl, heterocyclic, or arylalkyl are
optionally substituted with one or more radical selected from alkyl, alkoxy,
halogen, haloalkyl, cyano, haloalkoxy, acyl, carboxyl, hydroxy,
hydroxyalkyloxy, phenoxy, benzyloxy, dialkylaminoalkyloxy, and
heterocyclic,
Ril is selected from the group consisting of: hydrido, halogen, haloalkyl,
CN, C02R5, lower alkyl, lower alkenyl, lower alkynyl, alkoxy, and CONH2;
R12 is selected from the group consisting of: hydrido, halogen, alkyl, and
alkoxy;
R13 is selected from the group consisting of: hydrido, alkyl, aryl, arylalkyl,
heteroaryl, heterocyclicalkyl, and heteroarylalkyl, wherein aryl, alkyl,
arylalkyl, heteroaryl, heterocyclicalkyl, or heteroarylalkyl are optionally
substituted with one or more radicals selected from the group consisting of:
ORlø, N(R14)R14~, and glycols;
R14 is independently selected from the group consisting of: hydrido, and
lower alkyl;
R14~ is independently selected from the group consisting of: hydrido, and
lower alkyl;
13
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Rls is selected from the group consisting of: hydrido, halogen, alkyl,
cycloalkyl, aryl, haloalkyl, heteroaryl, heterocyclic, alkylalkene,
alkylalkyne,
hydroxy, hydroxyalkyl, alkylhydroxy, amino, aminoalkyl, alkylamino,
alkylaminoalkyl, alkylhydroxyalkyl, vitro, cyano, alkylthio, alkylsulfinyl,
and alkylsulfonyl; wherein aryl or arylalkyl are optionally substituted with
one or more radical selected from alkyl, alkoxy, halo, haloalkyl, cyano,
haloalkoxy, acyl, carboxyl, hydroxy, hydroxyalkyloxy, phenoxy, benzyloxy,
dialkylaminoalkyloxy, heterocyclic; and
R16 is independently selected from the group consisting of: hydrido, aryl,
arylalkyl, lower alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl,
alkoxy, and alkoxyalkyl;
or isomers, tautomers, carriers, esters, prodrugs, pharmaceutically acceptable
salts thereof.
[0018] Another class of compounds is defined by formula II
R' ~
wherein
A is selected from the group consisting of: (CH2)m and (CH2)n CH=CH-
(CH2)n; wherein each CHI may be independently substituted with one or
more substitution selected from the group consisting of: hydroxy, halo,
alkoxy, lower alkyl, amino, aminoalkyl, alkylamino, alkenyl, and alkynyl;
m is 0 to 8;
n is independently selected from 0, l, or 2;
14
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Q is a 5 or 6 membered heteroaryl, or aryl, optionally saturated, or
optionally substituted with Rl, R2, or R12;
B is an aromatic heterocyclic;
Rl is selected from the group consisting of: hydrido, halogen, alkyl, aryl,
heteroaryl, alkenyl, alkynyl, haloalkyl, CN, N02, ORS, OCOORS, C02R7,
CON(R6)R7, CORE, SR6, SORE, S02R6, NR6R7, NR6COR7, NR6CONHR7,
NR6S02R7, NR6S02NHR7, and S02N(R6)R7 wherein R6 and R7 may be
taken together to form a 3-7 membered carbocyclic ring having 1 to 3
substituted or unsubstituted heteroatoms selected from the group consisting
of: S, SO, 502, O, and NR6; wherein said alkenyl, alkynyl, alkyl, aryl,
heteroaryl or ORS are optional substituted with, hydrido, halogen, alkyl,
hydroxyalkyl, aryl, heteroaryl, haloalkyl, COCF3, CN, N02, ORS, OCOORS,
C02R7, CON(R6)R7, CORE, SR6, SORE, S02R6, NR6R7, NR6COR7,
NR6CONHR7, NR6S02R7, NR6SO2NHR7, and SO2N(R6)R7 wherein R6 and
R7 may be taken together to form a 3-7 membered carbocyclic ring having 1
to 3 substituted or unsubstituted heteroatoms selected from the group
consisting of: S, SO, 502, O, and NR6;
R2 is selected from the group consisting of: halogen, hydrido, hydroxyalkyl,
alkyl, OR6, CN, NO2, SR6, NHR6, CON(R6)R7, NHCONHR6, C02H, and
haloalkyl;
Ri and R2 may be taken together to form a 5 to 7 membered saturated or
unsaturated carbocyclic ring optionally containing 0 to 3 heteroatoms
selected from the group consisting of: N, O, or S, and wherein said ring is
optionally substituted with Rl;
R3 is selected from the group consisting of: substituted or unsubstituted
amidine, alkylamino, aminoalkyl, CONHRl6, NH2, NHCOR6, and
CH2NHCOR6;
R4 is selected from the group consisting of: halogen, alkylsulfinyl,
alkylsulfonyl, cyano, alkoxycarbonyl, alkyl, haloalkyl, hydrido,
hydroxyalkyl, haloalkoxy, heterocyclic, vitro, acylamino, aryl, heteroaryl,
and alkenyl, OR13, SRB, S02N(R8)RB~, NHR9, NHCOR9, NR9COR9,
NHCO(OR9), NR9C0(OR9), NR$SO2R1°, NHS02N(R1°)Rloy
is
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NR6CON(Rl°)Rlo~, COR9, C02R8, CON(R$)RB~, wherein R8 and R8~ may
be
taken together to form a 3-7 membered carbocyclic ring having 1 to 3
substituted or unsubstituted heteroatoms selected from S, SO, 502, O, N,
and NR6, and wherein Rl° and Rl°~ may be taken together to form
a 3-7
membered carbocyclic ring having 1 to 3 substituted or unsubstituted
heteroatoms selected from S, SO, 502, O, N, and NR6 wherein said aryl,
heterocyclic, heteroaryl, or alkenyl are optionally substituted with R9;
RS is selected from the group consisting of: hydrido, alkyl, aryl, arylalkyl,
heteroaryl, heterocyclicalkyl, and heteroarylalkyl, wherein aryl, alkyl,
arylalkyl, heteroaryl, heterocyclicalkyl, or heteroarylalkyl are optionally
substituted with one or more radicals selected from the group consisting of:
OR14, N(R14)R14~, and glycols;
R6 is independently selected from the group consisting of: hydrido, aryl,
heteroaryl, lower alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl,
aminoalkyl, alkylaminoalkyl, alkoxy, alkoxyalkyl, heterocyclicalkyl, and
heterocyclic;
R' is independently selected from the group consisting of: hydrido, aryl,
heteroaryl, lower alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl,
aminoalkyl, alkylaminoalkyl, alkoxy, alkoxyalkyl, heterocyclicalkyl, and
heterocyclic;
R$ is independently selected from the group consisting of: hydrido, aryl,
heteroaryl, arylalkyl, heterocyclic, haloalkyl, arylalkylamino,
alkylaminoalkyl, dialkylaminoalkyl, alkyl, alkenyl, alkynyl, heteroarylalkyl,
and heterocyclicalkyl;
R8~ is independently selected from the group consisting of: hydrido, aryl,
heteroaryl, arylalkyl, heterocyclic, haloalkyl, arylalkylamino,
alkylaminoalkyl, dialkylaminoalkyl, alkyl, alkenyl, alkynyl, heteroarylalkyl,
and heterocyclicalkyl;
R9 is independently selected from the group consisting of: hydrido, lower
alkyl, aryl, heteroaryl, arylalkyl, heterocyclic, cycloalkyl,
heterocyclicalkyl,
haloalkyl, arylalkylamino, amino, aminoalkyl, aminoacyl, nitro, azido, and
heteroarylalkyl, wherein alkyl, aryl, heteroaryl, aminoalkyl, or arylalkyl are
16
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optionally substituted with one or more radical selected from the group
consisting of: alkylsulfonamide, sulfamyl, alkyl, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkoxy, halogen,
acyloxy, oxy, fonnyl, haloalkyl, cyano, haloalkoxy, acyl, carboxyl, hydroxy,
hydroxyalkyloxy, phenoxy, nitro, azido, benzyloxy, dialkylaminoacyl,
thioalkyl, aminoacyloxy, thiocyanate, isothiocyanate, alkyldioxy,
hydroxyalkyl, alkylamino, alkyloxycarbonyl, alkoxyalkyl, alkenylamino,
alkynylamino, alkenyl, alkynyl, dialkylaminoalkyloxy, and heterocyclic
optionally substituted with alkyl, alkylamino, aminoalkyl, hydroxyalkyl, and
alkylaminoalkyl;
Ri° is independently selected from the group consisting of:
hydrido, lower
alkyl, heteroaryl, heterocyclic, haloalkyl, arylalkylamino, heteroarylalkyl,
aryl, and arylalkyl, wherein aryl, heteroaryl, heterocyclic, or arylalkyl are
optionally substituted with one or more radical selected from alkyl, alkoxy,
halogen, haloalkyl, cyano, haloalkoxy, acyl, carboxyl, hydroxy,
hydroxyalkyloxy, phenoxy, benzyloxy, dialkylaminoalkyloxy, and
heterocyclic,
Ri°~ is independently selected from the group consisting of:
hydrido, lower
alkyl, heteroaryl, heterocyclic, haloalkyl, arylalkylamino, heteroarylalkyl,
aryl, and arylalkyl, wherein aryl, heteroaryl, heterocyclic, or arylalkyl are
optionally substituted with one or more radical selected from alkyl, alkoxy,
halogen, haloalkyl, cyano, haloalkoxy, acyl, carboxyl, hydroxy,
hydroxyalkyloxy, phenoxy, benzyloxy, dialkylaminoalkyloxy, and
heterocyclic,
R11 is selected from the group consisting of: hydrido, halogen, haloalkyl,
CN, C02R5, lower alkyl, lower alkenyl, lower alkynyl, alkoxy, and CONH2;
R12 is selected from the group consisting of: hydrido, halogen, alkyl, and
alkoxy;
R13 is selected from the group consisting of: hydrido, alkyl, aryl, arylalkyl,
heteroaryl, heterocyclicalkyl, and heteroarylalkyl, wherein aryl, alkyl,
arylalkyl, heteroaryl, heterocyclicalkyl, or heteroarylalkyl are optionally
17
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substituted with one or more radicals selected from the group consisting of:
OR14, N(R14)Ri4y and glycols;
R14 is independently selected from the group consisting of: hydrido, and
lower alkyl;
R14~ is independently selected from the group consisting of: hydrido, and
lower alkyl;
Ris is selected from the group consisting of: hydrido, halogen, alkyl,
cycloalkyl, aryl, haloalkyl, heteroaryl, heterocyclic, alkylalkene,
alkylalkyne,
hydroxy, hydroxyalkyl, alkylhydroxy, amino, aminoalkyl, alkylamino,
alkylaminoalkyl, alkylhydroxyalkyl, nitro, cyano, alkylthio, alkylsulfinyl,
and alkylsulfonyl; wherein aryl or arylalkyl are optionally substituted with
one or more radical selected from alkyl, alkoxy, halo, haloalkyl, cyano,
haloalkoxy, acyl, carboxyl, hydroxy, hydroxyalkyloxy, phenoxy, benzyloxy,
dialkylaminoalkyloxy, heterocyclic; and
R16 is independently selected from the group consisting of: hydrido, aryl,
arylalkyl, lower alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl,
alkoxy, and alkoxyalkyl;
or isomers, tautomers, carriers, esters, prodrugs, pharmaceutically acceptable
salts thereof.
[0019] A preferred class of compounds is defined by formula III
R' ~
wherein
ig
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A is (CH2)m, wherein each CH2 may be independently substituted with one
or more substitution selected from the group consisting of: hydroxy, halo,
alkoxy, lower alkyl, amino, aminoalkyl, alkylamino, alkenyl, and alkynyl;
misOto8;
(~ is a 5 or 6 membered heteroaryl, or aryl, optionally saturated, or
optionally substituted with Rl, R2, or R12;
B is an aromatic heterocyclic;
Rl is selected from the group consisting of: hydrido, halogen, alkyl, aryl,
heteroaryl, alkenyl, alkynyl, haloalkyl, CN, N02, ORS, OCOORS, C02R7,
CON(R6)R7, CORE, SR6, SORE, S02R6, NR6R7, NR6COR7, NR6CONHR7,
NR6S02R7, NR6S02NHR7, and S02N(R6)R7 wherein R6 and R7 may be
taken together to form a 3-7 membered carbocyclic ring having 1 to 3
substituted or unsubstituted heteroatoms selected from the group consisting
of: S, SO, 502, O, and NR6; wherein said alkenyl, alkynyl, alkyl, aryl,
heteroaryl or ORS are optional substituted with, hydrido, halogen, alkyl,
hydroxyalkyl, aryl, heteroaryl, haloalkyl, COCF3, CN, N02, ORS, OCOORS,
C02R7, CON(R6)R7, CORE, SR6, SORE, S02R6, NR6R7, NR6COR7,
NR6CONHR7, NR6S02R7, NR6S02NHR7, and SO2N(R6)R7 wherein R6 and
R7 may be taken together to form a 3-7 membered carbocyclic ring having 1
to 3 substituted or unsubstituted heteroatoms selected from the group
consisting of: S, SO, 502, O, and NR6;
R2 is selected from the group consisting of: halogen, hydrido, hydroxyalkyl,
alkyl, OR6, CN, NO2, SR6, NHR6, CON(R6)R7, NHCONHR6, C02H, and
haloalkyl;
Rl and R2 may be taken together to form a 5 to 7 membered saturated or
unsaturated carbocyclic ring optionally containing 0 to 3 heteroatoms
selected from the group consisting of: N, O, or S, and wherein said ring is
optionally substituted with Rl;
R3 is CONHRIS;
R4 is selected from the group consisting of: halogen, alkylsulfinyl,
alkylsulfonyl, cyano, alkoxycarbonyl, alkyl, haloalkyl, hydrido,
hydroxyalkyl, haloalkoxy, heterocyclic, nitro, acylamino, aryl, heteroaryl,
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and alkenyl, OR13, SR8, S02N(R$)RB~, NHR9, NHCOR9, NR9COR9,
NHCO(OR9), NR9C0(OR9), NR8S02R1°, NHS02N(Rl°)Rlo~,
NR6CON(Rl°)Rlo~, COR9, C02R8, CON(R8)RB~, wherein R$ and R8~ may
be
taken together to form a 3-7 membered carbocyclic ring having 1 to 3
substituted or unsubstituted heteroatoms selected from S, SO, 502, O, N,
and NR6, and wherein R1° and Rl°~ may be taken together to form
a 3-7
membered carbocyclic ring having 1 to 3 substituted or unsubstituted
heteroatoms selected from S, SO, 502, O, N, and NR6 wherein said aryl,
heterocyclic, heteroaryl, or alkenyl are optionally substituted with R9;
RS is selected from the group consisting of: hydrido, alkyl, aryl, arylalkyl,
heteroaryl, heterocyclicalkyl, and heteroarylalkyl, wherein aryl, alkyl,
arylalkyl, heteroaryl, heterocyclicalkyl, or heteroarylalkyl are optionally
substituted with one or more radicals selected from the group consisting of:
OR14, N(R14)Ri4~, and glycols;
R6 is independently selected from the group consisting of: hydrido, aryl,
heteroaryl, lower alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl,
aminoalkyl, alkylaminoalkyl, alkoxy, alkoxyalkyl, heterocyclicalkyl, and
heterocyclic;
R' is independently selected from the group consisting of: hydrido, aryl,
heteroaryl, lower alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl,
aminoalkyl, alkylaminoalkyl, alkoxy, alkoxyalkyl, heterocyclicalkyl, and
heterocyclic;
Rg is independently selected from the group consisting of: hydrido, aryl,
heteroaryl, arylalkyl, heterocyclic, haloalkyl, arylalkylamino,
alkylaminoalkyl, dialkylaminoalkyl, alkyl, alkenyl, alkynyl, heteroarylalkyl,
and heterocyclicalkyl;
Rg~ is independently selected from the group consisting of: hydrido, aryl,
heteroaryl, arylalkyl, heterocyclic, haloalkyl, arylalkylamino,
alkylaminoalkyl, dialkylaminoalkyl, alkyl, alkenyl, alkynyl, heteroarylalkyl,
and heterocyclicalkyl;
R9 is independently selected from the group consisting of: hydrido, lower
alkyl, aryl, heteroaryl, arylalkyl, heterocyclic, cycloalkyl,
heterocyclicalkyl,
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haloalkyl, arylalkylamino, amino, aminoalkyl, aminoacyl, nitro, azido, and
heteroarylalkyl, wherein alkyl, aryl, heteroaryl, aminoalkyl, or arylalkyl are
optionally substituted with one or more radical selected from the group
consisting of: alkylsulfonamide, sulfamyl, alkyl, alkylthio, alkylsulfinyl,
alkylsulfonyl, alkylamino, aminoalkyl, alkylaminoalkyl, alkoxy, halogen,
acyloxy, oxy, formyl, haloalkyl, cyano, haloalkoxy, acyl, carboxyl, hydroxy,
hydroxyalkyloxy, phenoxy, nitro, azido, benzyloxy, dialkylaminoacyl,
thioalkyl, aminoacyloxy, thiocyanate, isothiocyanate, alkyldioxy,
hydroxyalkyl, alkylamino, alkyloxycarbonyl, alkoxyalkyl, alkenylamino,
alkynylamino, alkenyl, alkynyl, dialkylaminoalkyloxy, and heterocyclic
optionally substituted with alkyl,~alkylamino, aminoalkyl, hydroxyalkyl, and
alkylaminoalkyl;
Rl° is independently selected from the group consisting of:
hydrido, lower
alkyl, heteroaryl, heterocyclic, haloalkyl, arylalkylamino, heteroarylalkyl,
aryl, and arylalkyl, wherein aryl, heteroaryl, heterocyclic, or arylalkyl are
optionally substituted with one or more radical selected from alkyl, alkoxy,
halogen, haloalkyl, cyano, haloalkoxy, acyl, carboxyl, hydroxy,
hydroxyalkyloxy, phenoxy, benzyloxy, dialkylaminoalkyloxy, and
heterocyclic,
Ri°~ is independently selected from the group consisting of:
hydrido, lower
alkyl, heteroaryl, heterocyclic, haloalkyl, arylalkylamino, heteroarylalkyl,
aryl, and arylalkyl, wherein aryl, heteroaryl, heterocyclic, or arylalkyl are
optionally substituted with one or more radical selected from alkyl, alkoxy,
halogen, haloalkyl, cyano, haloalkoxy, acyl, carboxyl, hydroxy,
hydroxyalkyloxy, phenoxy, benzyloxy, dialkylaminoalkyloxy, and
heterocyclic,
Ril is selected from the group consisting of: hydrido, halogen, haloalkyl,
CN, C02R5, lower alkyl, lower alkenyl, lower alkynyl, alkoxy, and CONH2;
Rl~ is selected from the group consisting of: hydrido, halogen, alkyl, and
alkoxy;
R13 is selected from the group consisting of: hydrido, alkyl, aryl, arylalkyl,
heteroaryl, heterocyclicalkyl, and heteroarylalkyl, wherein aryl, alkyl,
zi
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arylalkyl, heteroaryl, heterocyclicalkyl, or heteroarylalkyl are optionally
substituted with one or more radicals selected from the group consisting of:
OR14, N(R14)R14~, and glycols;
R14 is independently selected from the group consisting of: hydrido, and
lower alkyl;
R14~ is independently selected from the group consisting of: hydrido, and
lower alkyl;
R15 is selected from the group consisting of: hydrido, halogen, alkyl,
cycloalkyl, aryl, haloalkyl, heteroaryl, heterocyclic, alkylalkene,
alkylalkyne,
hydroxy, hydroxyalkyl, alkylhydroxy, amino, aminoalkyl, alkylamino,
alkylaminoalkyl, alkylhydroxyalkyl, nitro, cyano, alkylthio, alkylsulfinyl,
and alkylsulfonyl; wherein aryl or arylalkyl are optionally substituted with
one or more radical selected from alkyl, alkoxy, halo, haloalkyl, cyano,
haloalkoxy, aryl, carboxyl, hydroxy, hydroxyalkyloxy, phenoxy, benzyloxy,
dialkylaminoalkyloxy, heterocyclic; and
R16 is independently selected from the group consisting of: hydrido, aryl,
arylalkyl, lower alkyl, haloalkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl,
alkoxy, and alkoxyalkyl;
or isomers, tautomers, carriers, esters, prodrugs, pharmaceutically acceptable
salts thereof.
Definitions
[0020] The present invention includes the use of all hydrates, solvates,
complexes and prodrugs of the compounds of this invention. Prodrugs are any
covalently bonded compounds, which release the active parent drug according to
Formula I in vivo. If a chiral center or another form of an isomeric center is
present
in a compound of the present invention all forms of such isomer or isomers,
including enantiomers and diastereomers, are intended to be covered herein.
Compounds containing a chiral center may be used as a racernic mixture, an
enantiornerically enriched mixture, or the racemic mixture may be separated
using
22
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well-known techniques and an individual enantiomer may be used alone. In cases
in
which compounds have unsaturated carbon-carbon double bonds, both the cis (Z)
and trans (E) isomers are within the scope of this invention. In cases wherein
compounds may exist in tautomeric forms, such as keto-enol tautomers, each
tautomeric form is contemplated as being included within this invention
whether
existing in equilibrium or predominantly in one form.
[0021] The meaning of any substituent at any one occurrence in Formula I or
any sub-formula thereof is independent of its meaning, or any other
substituents
meaning, at any other occurrence, unless specified otherwise.
[0022] The present invention includes the use of all hydrates, solvates,
complexes and prodrugs of the compounds of this invention. Prodrugs are any
covalently bonded compounds, which releases the active parent drug according
to
Formula I or Formula II in vivo. If a chiral center or another form of an
isomeric
center is present in a compound of the present invention all forms of such
isomer or
isomers, including enantiomers and diastereomers, are intended to be covered
herein. Compounds containing a chiral center may be used as a racemic mixture,
an
enantiornerically enriched mixture, or the racemic mixture may be separated
using
well-known techniques and an individual enantiomer may be used alone. In cases
in
which compounds have unsaturated carbon-carbon double bonds, both the cis (Z)
and trans (E) isomers are within the scope of this invention. In cases wherein
compounds may exist in tautomeric forms, such as keto-enol tautomers, each
tautomeric form is contemplated as being included within this invention
whether
existing in equilibrium or predominantly in one form.
[0023] The meaning of any substituent at any one occurrence in Formula I or
Formula II or any sub-formula thereof is independent of its meaning, or any
other
substituents meaning, at any other occurrence, unless specified otherwise.
[0024] The term "alkyl" is used, either alone or within other terms such as
"haloalkyl" and "alkylsulfonyl"; it embraces linear or branched radicals
having one
23
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to about twenty carbon atoms or, preferably, one to about twelve carbon atoms.
More preferred alkyl radicals are "lower alkyl" radicals having one to about
ten
carbon atoms. Most preferred are lower alkyl radicals having one to about five
carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl,
isopropyl,
n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, hexyl, octyl and
the, like.
The term "hydrido" denotes a single hydrogen atom (H). This hydrido radical
may
be attached, for example, to an oxygen atom to form a hydroxyl radical or two
hydrido radicals may be attached to a carbon atom to form a methylene (-CH2-)
radical. The term "halo" means halogens such as fluorine, chlorine, and
bromine or
iodine atoms. The term "haloalkyl" embraces radicals wherein any one or more
of
the alkyl carbon atoms is substituted with halo as defined above. Specifically
embraced are monohaloalkyl, dihaloalkyl, and polyhaloalkyl radicals. A
monohaloalkyl radical, for one example, may have a bromo, chloro, or a fluoro
atom within the radical. Dihalo radicals may have two or more of the same halo
atoms or a combination of different halo radicals and polyhaloalkyl radicals
may
have more than two of the same halo atoms or a combination of different halo
radicals. The term "hydroxyalkyl" embraces linear or branched alkyl radicals
having
one to about ten carbon atoms any one of which may be substituted with one or
more hydroxylradicals. The terms "alkoxy" and "alkoxyalkyl" embrace linear or
branched oxy-containing radicals each having alkyl portions of one to about
ten
carbon atoms, such as methoxy radical. The term "alkoxyalkyl" also embraces
alkyl
radicals having two or more alkoxy radicals attached to the alkyl radical,
that is, to
form monoalkoxyalkyl and dialkoxyalkyl radicals. The "alkoxy" or "alkoxyalkyl"
radicals may be further substituted with one or more halo atoms, such as
fluoro,
chloro, or bromo, to provide "haloalkoxy" or "haloalkoxyalkyl" radicals.
Examples
of "alkoxy" radicals include methoxy, butoxy, and trifluoromethoxy. The term
"aryl", alone or in combination, means a carbocyclic aromatic system
containing
one, two, or three rings wherein such rings may be attached together in a
pendent
manner or may be fused. The term "aryl" embraces aromatic radicals such as
phenyl, naphthyl, tetrahydronapthyl, indane, and biphenyl. The term
"heterocyclic"
embraces saturated, partially saturated, and unsaturated heteroatom-containing
ring-
shaped radicals, where the heteroatoms may be selected from nitrogen, sulfur
and
24
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WO 03/070706 PCT/US03/04844
oxygen. Examples of saturated heterocyclic radicals include pyrrolidyl and
morpholinyl. The term "heteroaryl" embraces unsaturated heterocyclic radicals.
Examples of unsaturated heterocyclic radicals, also termed "heteroaryl"
radicals
include thienyl, pyrrolyl, furyl, pyridyl, pyrimidyl, pyrazinyl, pyrazolyl,
oxazolyl,
isoxazolyl, imidazolyl, thiazolyl, and tetrazolyl. The term also embraces
radicals
where heterocyclic radicals are fused with aryl radicals. Examples of such
fused
bicyclic radicals include benzofuran, benzothiophene, and the like. The term
"heterocyclic alkyl" embraces alkyl attached to the heterocyclic. The term
"sulfonyl", whether used alone or linked to other terms such as alkylsulfonyl,
denotes respectively divalent radicals -S02-. "Alkylsulfonyl", embraces alkyl
radicals attached to a sulfonyl radical, where alkyl is defined as above. The
term
"arylsulfonyl" embraces sulfonyl radicals substituted with an aryl radical.
The terms
"sulfamyl" or "sulfonamidyl", whether alone or used with terms such as "N-
alkylsulfamyl", "N-arylsulfamyl", "N,N-dialkylsulfamyl" and "N-alkyl-N-
arylsulfamyl", denotes a sulfonyl radical substituted with an amine radical,
forming
a sulfonamide (-SOZ-NH2). The terms "N-alkylsulfamyl" and "N,N-
dialkylsulfamyl" denote sulfamyl radicals substituted, respectively, with one
alkyl
radical, a cycloalkyl ring, or two alkyl radicals. The terms "N-arylsulfamyl"
and "N-
alkyl-N-arylsulfamyl" denote sulfamyl radicals substituted, respectively, with
one
aryl radical, and one alkyl and one aryl radical. The terms "carboxy" or
"carboxyl",
whether used alone or with other terms, such as "carboxyalkyl", denotes -C02H.
The term "carboxyalkyl" embraces radicals having a carboxyradical as defined
above, attached to an alkyl radical. The term "carbonyl", whether used alone
or with
other terms, such as "alkylcarbonyl", denotes -(C=O)-. The term
"alkylcarbonyl"
embraces radicals having a carbonyl radical substituted with an alkyl radical.
An
example of an "alkylcarbonyl" radical is CH3-(C=O)-. The term
"alkylcarbonylalkyl" denotes an alkyl radical substituted with an
"alkylcarbonyl"
radical. The term "alkoxycarbonyl" means a radical containing an alkoxy
radical, as
defined above, attached via an oxygen atom to a carbonyl (C=O) radical.
Examples
of such "alkoxycarbonyl" radicals include (CH3)3CO-C=O)- and -(O=)C-OCH3.
The term "alkoxycarbonylalkyl" embraces radicals having "alkoxycarbonyl", as
defined above substituted to an alkyl radical. Examples of such
CA 02476665 2004-08-18
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"alkoxycarbonylalkyl" radicals include (CH3)3COC(=O) (CHZ)2- and -
(CH2)2(O=)COCH3. The term "amido" when used by itself or with other terms such
as "amidoalkyl", "N-monoalkylamido", "N-monoarylamido", "N,N-dialkylamido",
"N-alkyl~N-arylamido", "N-alkyl-N-hydroxyamido" and "N-alkyl-N-
hydroxyamidoalkyl", embraces a carbonyl radical substituted with an amino
radical.
The terms "N-alkylamido" and "N,N-dialkylamido" denote amido groups which
have been substituted with one alkyl radical and with two alkyl radicals,
respectively. The terms "N-monoarylamido" and "N-alkyl-N-arylamido" denote
amido radicals substituted, respectively, with one aryl radical, and one alkyl
and one
aryl radical. The term "N-alkyl-N-hydroxyamido" embraces amido radicals
substituted with a hydroxyl radical and with an alkyl radical. The term "N-
alkyl-N-
hydroxyamidoalkyl" embraces alkyl radicals substituted with an N-alkyl-N-
hydroxyamido radical. The term "amidoalkyl" embraces alkyl radicals
substituted
with amido radicals. The term "aminoalkyl" embraces alkyl radicals substituted
with amino radicals. The term "alkylaminoalkyl" embraces aminoalkyl radicals
having the nitrogen atom substituted with an alkyl radical. The term "amidino"
denotes an -C(=NH)-NH2 radical. The term "cyanoamidino" denotes an -C(=N-
CN)-NH2 radical. The term "heterocycloalkyl" embraces heterocyclic-substituted
alkyl radicals such as pyridylmethyl and thienylmethyl. The term "aralkyl"
embraces
aryl-substituted alkyl radicals such as benzyl, diphenylmethyl,
triphenylmethyl,
phenethyl, and diphenethyl. The terms benzyl and phenylmethyl are
interchangeable. The term "cycloalkyl" embraces radicals having three to ten
carbon
atoms, such as cyclopropyl cyclobutyl, cyclopentyl, cyclohexyl, and
cycloheptyl.
The term "cycloalkenyl" embraces unsaturated radicals having three to ten
carbon
atoms, such as cylopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and
cycloheptenyl. The term "alkylthio" embraces radicals containing a linear or
branched alkyl radical, of one to ten carbon atoms, attached to a divalent
sulfur
atom. An example of "alkylthio" is methylthio, (CH3-S-). The term
"alkylsulfinyl"
embraces radicals containing a linear or branched alkyl radical, of one to ten
carbon
atoms, attached to a divalent -S(=O)- atom. The terms "N-alkylamino" and "N, N-
dialkylamino" denote amino groups which have been substituted with one alkyl
radical and with two alkyl radicals, respectively. The term "acyl", whether
used
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WO 03/070706 PCT/US03/04844
alone, or within a term such as "acylamino", denotes a radical provided by the
residue after removal of hydroxyl from an organic acid. The term "acylamino"
embraces an amino radical substituted with an acyl group. An example of an
"acylamino" radical is acetylamino (CH3C(=O)-NH-).
[0025] Another aspect of the present invention is chemical intermediates in
the
synthesis of the claimed compounds.
[0026] Another aspect of the present invention is methods of syntheses of the
claimed compounds.
[0027] Compounds of Formula I or Formula II would be useful for, but not
limited to, the treatment of inflammation in a subject, and for treatment of
other
inflammation-associated disorders, such as, as an analgesic in the treatment
of pain
and headaches, or as an antipyretic for the treatment of fever. For example,
compounds of Formula I or Formula II would be useful to treat arthritis,
including
but not limited to rheumatoid arthritis, spondylo arthopathies, gouty
arthritis,
osteoarthritis, systemic lupus erythematosus, and juvenile arthritis. Such
compounds of Formula I or Formula II would be useful in the treatment of
asthma,
bronchitis, menstrual cramps, tendinitis, bursitis, and skin related
conditions such as
psoriasis, eczema, burns, and dermatitis. Compounds of Formula I or Formula II
also would be useful to treat gastrointestinal conditions such as inflammatory
bowel
disease, Crohn's disease, gastritis, irritable bowel syndrome, and ulcerative
colitis
and for the prevention of colorectal cancer. Compounds of Formula I or Formula
II
would be useful in treating inflammation in such diseases as vascular diseases
such
as vascularitus, migraine headaches, periarteritis nodosa, thyroiditis,
aplastic
anemia, Hodgkin's disease, sclerodoma, rheumatic fever, type I diabetes,
myasthenia gravis, sarcoidosis, nephrotic syndrome, Behcet's syndrome,
polymyositis, gingivitis, hypersensitivity, conjunctivitis, swelling occurring
after
injury, myocardial ischemia, and the like. The compounds of the present
invention
may also be used for pain. The compounds axe useful as antiinflammatory
agents,
such as for the treatment of arthritis, with the additional benefit of having
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significantly less harmful side effects. The compounds of formula I or II are
useful
as agents for treating cancer or anticancer agents. The compounds of formula I
or II
may be proapoptotic, antiapoptotic, anticell cycle progressive, antiinvasive,
antiproliferative, antiangiogenic, and antimetastatic. The cancer may be
colon,
ovarian, breast, prostate, gastric, B-cell lymphoma, and multiple myeloma.
More
specifically, the compounds of this invention are useful in the treatment of a
variety
of cancers including, but not limited to: carcinoma such as bladder, breast,
colon,
kidney, liver, lung, including small cell lung cancer, esophagus, gall-
bladder, ovary,
pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous
cell
carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute
lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell-,
lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma
and Burkett's lymphoma; hematopoietic tumors of myeloid lineage, including
acute
and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic
leukemia; tumors of mesenchymal origin, including fibrosarcoma and
rhabdomyosarcoma; tumors of the central and peripheral nervous system,
including
astrocytoma, neuroblastoma, glioma and schwannomas; other tumors, including
melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum,
keratoxanthoma, thyroid follicular cancer and Kaposi's sarcoma. Due to the key
role of protein kinases in the regulation of cellular proliferation, these
compounds
are also useful in the treatment of a variety of cell proliferative disorders
such as, for
instance, benign prostate hyperplasia, familial adenomatosis, polyposis, neuro-
fibromatosis, psoriasis, vascular smooth cell proliferation associated with
atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post-
surgical
stenosis and restenosis. The compounds of formula I or lI may be used as an
anitviral agent. The compounds of this invention are useful as inhibitors of
protein
kinases. The compounds of this invention are useful as inhibitors of IKK1
and/or
IKK2, IKKa/IKK(3 heterodimer, TBK or IKKi. The compounds of the invention
may also useful as inhibitors of other protein kinases such as, for instance,
protein
kinase C in different isoforms, cyclin dependent kinase (cdk), Met, PAK-4, PAK-
5,
ZC-1, STLK-2, DDR-2, Aurora l, Aurora 2, Bub-1, PLK, Chkl, Chk2, HER2, rafl,
MEKl, MAPK, EGF-R, PDGF-R, FGF-R, IGF-R, VEGF-R, PI3K, weel kinase,
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Src, Abl, Akt, ILK, MK-2, Cdc7, Nek, and thus be effective in the treatment of
diseases associated with other protein kinases. The present invention
preferably
includes compounds, which selectively inhibit IKK2 over other kinases.
Preferably
the compounds have a selectivity ratio of IKK2 inhibition over other kinase
inhibition of at least 50, and more preferably of at least 100. The present
invention
preferably includes compounds, which selectively inhibit IKK2 over IKKl.
Preferably, the compounds have an IKK2 IC50 of less than 1 ~.M, and have a
selectivity ratio of IKK2 inhibition over IKKl inhibition of at least 50, and
more
preferably of at least 100. Even more preferably, the compounds have an IKKl
IC50 of greater than 10 ~,M, and more preferably of greater than 100 wM. The
compounds of formula may also be used to treat angiogenesis associated
cardiovascular, ophthalmology and osteoporosis disorders. The compounds of the
present invention may also be used for treatment of knee injury such as sport
injuries.
[0028] While it is possible for an active ingredient to be administered alone
as
the raw chemical, it is preferable to present it as a pharmaceutical
formulation. The
present invention comprises a pharmaceutical composition comprising a
therapeutically effective amount of a compound of the present invention in
association with at least one pharmaceutically acceptable carrier, adjuvant,
or
diluent. The present invention also comprises a method of treating
inflammation or
inflammation associated disorders in a subject, the method comprising
administering to the subject having such inflammation or disorders a
therapeutically
effective amount of a compound of the present invention. Also included in the
family of compounds of the present invention are the pharmaceutically
acceptable
salts thereof. The term "pharmaceutically acceptable salts" embraces salts
commonly used to form alkali metal salts and to form addition salts of free
acids or
free bases. The nature of the salt is not critical, provided that it is
pharmaceutically
acceptable. Suitable pharmaceutically acceptable acid addition salts of
compounds
of the present invention may be prepared from an inorganic acid or from an
organic
acid. Examples of such inorganic acids are hydrochloric, hydrobromic,
hydroiodic,
nitric, carbonic, sulfuric, and phosphoric acid. Appropriate organic acids may
be
29
51067-33
CA 02476665 2004-08-18
selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,
carboxylic and sulfonic classes of organic acids, examples of which ate
formic,
acetic, propionic, suecinie, glyeolie, gluconie, lactic, malie, tartaric,
citric, ascorbic,
glucuronic, malefic, fumaric, pyruvic, aspartic, glutamic, benzoic,
anthranilic,
mesylic, salicyclic, salicyclic, phydroxybenzoic, phenylacetic, mandelic,
embonic
(pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,
tolue.nesulfonic, 2-hydroxyethanesulfonic, sulfanilic, stearic,
cyclohexylaminosulfonic, algenic, (3-hydroxybutyric, salicyclic, galactaric
and
galacturonic acid. Suitable pharmaceutically acceptable base addition salts of
compounds of the present invention include metallic salts made from aluminum,
calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made
from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,
ethylenediamine, meglumine (N-methyl-glucamine) and procaine. All of these
salts
may be prepared by conventional means from the corresponding compound of the
present invention by reacting, for example, the appropriate acid or base with
the
compound of the present invention.
[0029] Also embraced within this invention are pharmaceutical compositions
comprising one or more compounds of the present invention in association with
one
or more non-toxic, pharmaceutically acceptable carriers and/or diluents and/or
adjuvants and/or excipient (collectively referred to herein as "carrier"
materials)
and, if desired, other active ingredients. Accordingly, the compounds of the
present
invention may be used in the manufacture of a medicament.
Pharmaceutical compositions of the invention may be
contained in a commercial package, preferably together with
instructions for the use thereof as herein described.
Pharmaceutical compositions of the compounds of the present invention prepared
as
herein before described may be formulated as solutions or lyophilized powders
for
51067-33
CA 02476665 2004-08-18
parenteral administration. Powders may be reconstituted by addition of a
suitable diluent or
other pharmaceutically acceptable carrier prior to use. The liquid formulation
may
be a buffered, isotonic aqueous solution. The compounds of the present
invention
may be administered by any suitable route, preferably in the form of a
phanmaceutical composition adapted to such a route, and in a dose effective
for the
treatment intended. The compounds and composition may, for example, be
administered intravascularly, intraperitoneally, intravenously,
subcutaneously,
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WO 03/070706 PCT/US03/04844
intramuscularly, intramedullary, orally, or topically. For oral
administration, the
pharmaceutical composition may be in the form of, for example, a tablet,
capsule,
suspension, or liquid. The active ingredient may also be administered by
injection
as a composition wherein, for example, normal isotonic saline solution,
standard
5% dextrose in water or buffered sodium or ammonium acetate solution may be
used as a suitable carrier. Such formulation is especially suitable for
parenteral
administration, but may also be used for oral administration or contained in a
metered dose inhaler or nebulizer for insufflation. It may be desirable to add
excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia,
polyethylene glycol, mannitol, sodium chloride, or sodium citrate. The
pharmaceutical composition is preferably made in the form of a dosage unit
containing a particular amount of the active ingredient. Examples of such
dosage
units are tablets or capsules. The amount of therapeutically active compound
that is
administered and the dosage regimen for treating a disease condition with the
compounds and/or compositions of this invention depends on a variety of
factors,
including the age, weight, sex and medical condition of the subject, the
severity of
the disease, the route and frequency of administration, and the particular
compound
employed, and thus may vary widely. The pharmaceutical compositions may
contain active ingredient in the range of about 0.1 to 2000 mg, preferably in
the
range of about 0.5 to 500 mg and most preferably between about 1 and 100 mg. A
daily dose of about 0.01 to 100 mg/kg bodyweight, preferably between about 0.1
and about 50 mg/kg body weight and most preferably between about 1 to 20 mg/kg
bodyweight, may be appropriate. The daily dose can be administered in one to
four
doses per day. For therapeutic purposes, the compounds of this invention are
ordinarily combined with one or more adjuvants appropriate to the indicated
route
of administration. If administered orally, the compounds may be admixed with
lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose
alkyl
esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and
calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium
alginate,
polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or
encapsulated
for convenient administration. Such capsules or tablets may contain a
controlled
release formulation as may be provided in a dispersion of active compound in a
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WO 03/070706 PCT/US03/04844
sustained release material such as glyceryl monostearate, glyceryl distearate,
hydroxypropylmethyl cellulose alone or with a wax. Formulations for parenteral
administration may be in the form of aqueous or non-aqueous isotonic sterile
injection solutions or suspensions. These solutions and suspensions may be
prepared from sterile powders or granules having one or more of the carriers
or
diluents mentioned for use in the formulations for oral administration. The
compounds may be dissolved in water, polyethylene glycol, propylene glycol,
ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol,
sodium
chloride, andlor various buffers. The pharmaceutical preparations are made
following the conventional techniques of pharmacy involving milling, mixing,
granulating, and compressing, when necessary, for tablet forms; or milling,
mixing
and filling for hard gelatin capsule forms. When a liquid carrier is used, the
preparation will be in the form of a syrup, elixir, emulsion, or an aqueous or
non-
aqueous suspension. Such a liquid formulation may be administered orally or
filled
into a soft gelatin capsule. For rectal administration, the compounds of the
present
invention may also be combined with excipients such as cocoa butter, glycerin,
gelatin, or polyethylene glycols and molded into a suppository. The methods of
the
present invention include topical administration of the compounds of the
present
invention. By topical administration is meant non-systemic administration,
including the application of a compound of the invention externally to the
epidermis, to the buccal cavity and instillation of such a compound into the
ear, eye,
and nose, wherein the compound does not significantly enter the blood stream.
By
systemic administration is meant oral, intravenous, intraperitoneal, and
intramuscular administration. The amount of a compound of the present
invention
(hereinafter referred to as the active ingredient) required for therapeutic or
prophylactic effect upon topical administration will, of course, vary with the
compound chosen, the nature and severity of the condition being treated and
the
animal undergoing treatment, and is ultimately at the discretion of the
physician.
[0030] The topical formulations of the present invention, both for veterinary
and for human medical use, comprise an active ingredient together with one or
more
acceptable carriers therefore, and optionally any other therapeutic
ingredients. The
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carrier must be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the recipient thereof.
Formulations suitable for topical administration include liquid or semi-liquid
preparations suitable for penetration through the skin to the site of where
treatment
is required such as: liniments, lotions, creams, ointments or pastes, and
drops
suitable for administration to the eye, ear or nose. The active ingredient may
comprise, for topical administration, from 0.01 to 5.0 wt%. of the
formulation.
[0031] Drops according to the present invention may comprise sterile aqueous
or oily solutions or suspensions and may be prepared by dissolving the active
ingredient in a suitable aqueous solution of a bactericidal and/or fungicidal
agent
and/or any other suitable preservative, and preferably including a surface
active
agent. The resulting solution may then be clarified by filtration, transferred
to a
suitable container, which is then sealed and sterilized by autoclaving, or
maintaining
at 90-100° C for half an hour. Alternatively, the solution may be
sterilized by
filtration and transferred to the container by an aseptic technique. Examples
of
bactericidal and fungicidal agents suitable for inclusion in the drops are
phenylmercuric nitrate or acetate (0.00217c), benzalkonium chloride (0.0 1 %)
and
chlorhexidine acetate (0.0 1 %). Suitable solvents for the preparation of an
oily
solution include glycerol, diluted alcohol, and propylene glycol.
[0032] Lotions according to the present invention include those suitable for
application to the skin or eye. An eye lotion may comprise a sterile aqueous
solution
optionally containing a bactericide and may be prepared by methods similar to
those
for the preparation of drops. Lotions or liniments for application to the skin
may
also include an agent to hasten drying and to cool the skin, such as an
alcohol or
acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or
arachis
oil. Creams, ointments, or pastes according to the present invention are semi-
solid
formulations of the active ingredient for external application. They may be
made by
mixing the active ingredient in finely divided or powdered form, alone or in
solution or suspension in an aqueous or non-aqueous fluid, with the aid of
suitable
machinery, with a greasy or non-greasy basis. The basis may comprise
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hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a
metallic
soap; a mucilage; an oil of natural origin such as almond, corn, arachis,
castor or
olive oil; wool fat or its derivatives, or a fatty acid such as stearic or
oleic acid
together with an alcohol such as propylene glycol or macrogols. The
formulation
may incorporate any suitable surface-active agent such as an anionic,
cationic, or
non-ionic surface-active agent such as sorbitan esters or polyoxyethylene
derivatives thereof. Suspending agents such as natural gums, cellulose
derivatives
or inorganic materials such as silicaceous silicas, and other ingredients such
as
lanolin may also be included. Other adjuvants and modes of administration are
well
and widely known in the pharmaceutical art. Although this invention has been
described with respect to specific embodiments, the details of these
embodiments
are not to be construed as limitations.
GENERAL SYNTHETIC PROCEDURES
[0033] The starting materials used herein are commercially available or are
prepared by routine methods well known to those of ordinary skill in the art
and can
be found in standard reference books, such as the COMPENDIUM OF ORGANIC
SYNTHETIC METHODS, Vol. I-VI (published by Wiley-Interscience).
[0034] The compounds of the invention can be synthesized according to the
following procedures of Schemes I-XVI, wherein the R1-R7 substituents, linker
A,
are as defined for Formula I and Formula II, above, except where further
noted.
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WO 03/070706 PCT/US03/04844
S CHEME I
HN~NH2 HCl
O OH
~ EtOH or MeOH
~R3 +
or HOAc
Ra ~ ~ O R~ R2 Rs
R4
3
[0035] Synthetic Scheme I illustrates the procedure used to prepare the anti-
inflammatory pyrazoles of the present invention. 1,3-Dicarbonyl compounds such
as 1, or the shown enol form which is in equilibrium with the 1,3-diketone,
are
allowed to react with a substituted hydrazine hydrochloride 2 in warm methanol
or
ethanol or acetic acid to provide the pyrazoles 3 via a condensation reaction.
SCHEME II
O O OH
O R
Ra ~ LHMDS R / a
w ~ + /O O/ v -
A O (or) NaOMe / MeOH
4 1
[0036] Synthetic Scheme II illustrates the procedure for the preparation of
substituted diketones 1. An appropriately substituted ketone, including but
not
limited to; indanones, 3-coumaranones, 3-thiocourmaranones, 3-
azacourmaranones,
1-tetralones, chromanones, thiochromanones, azachromanones, isochromanones,
isothiochromanones, isoazachromanones, 4 is first treated with base, such as
sodium methoxide, lithium bistrimethylsilylamide or lithium diisopropylamide
(LDA), followed by condensation with a suitable acylating agent, such as;
dimethyl
or diethyl oxalate, in an appropriate solvent, such as methanol, diethyl ether
or
tetrahydrofuran, to provide 1,3-dicarbonyl compounds 1 which are suitable for
conversion into anti-inflammatory pyrazoles as illustrated in Scheme 1.
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WO 03/070706 PCT/US03/04844
Alternatively, the dicarbonyl compounds 1 can be directly prepared from
commercially available cyclic ketones 4.
SCHEME III
1. Conc. HCl I ~ Hcl
/ OH ~ Ra ~ / S NH2
2. thiourea
5 6
NaOH l EtOH
CICHzC02H
O
TFA / TFAA R _~ O
/ S 4 I / S v 'OH
S
7
[0037] Synthetic Scheme III illustrates the three step procedure for the
preparation of substituted isothiochromanones. In step one, an appropriately
substituted benzyl alcohol 5 is converted into the corresponding benzyl
chloride by
stirring with concentrated hydrochloric acid and then immediately converted
into a
thiouronium salt 6 upon treatment with thiourea at reflux. In step two, the
thiouronium salt is converted to the free mercaptide, according to the
procedure of
Lumma and Berchtold (J. ~rg. Chem., 34, 1566 (1969), and then trapped with
chloroacetic acid or a related salt to provide the acetic acid derivatives 7.
In step
three, the acids 7 are reacted with trifluoroacetic anhydride (TFAA) in
trifluoroacetic acid (TFA) to give the isothiochromanone products 8. The
thiouronium salts 6 can also be prepared from appropriate commercially
available
benzyl halides.
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WO 03/070706 PCT/US03/04844
SCHEME IV
0
O \ O TFA/TFAA
Ra ~ / + Ra I ~ ~ I
I OH I / S~OH Ra i \
SH ~ /
S
g 10 11
[0038] Synthetic Scheme 1V illustrates a three step procedure used for the
preparation of substituted thiochromanones. In step one, an appropriate
substituted
thiophenol 9 is converted into the corresponding propionic acid derivatives 10
upon
treatment with acrylic acid at a temperature in a range of room temperature to
about
50°C. In step two, the propionic acids 10 are subjected to treatment
with a mixture
of trifluoroacetic anhydride and trifluoroacetic acid to effect intramolecular
Friedel-
Crafts acylation, thus providing thiochromanones 11. Alternatively, the
Friedel-
Crafts acylation can be affected with H2S04. Dicarbonyl compounds 1 can also
be
directly prepared from commercially available thiochromanones 11.
S CHEME V
0
O ~ O TFA/TFAA
Ra i / + Ra I ' I \
NH2 ~ OH I / N~QH Ra lI
H N
H
12 13 14
[0039] Synthetic Scheme V illustrates a three step procedure used for the
preparation of substituted azachromanones. In step one, an appropriate
substituted
aniline 12 is converted into the corresponding propionic acid derivatives 13
upon
treatment with acrylic acid at a temperature in a range of room temperature to
about
50°C. In step two, the propionic acids 13 are subjected to treatment
with a mixture
of H2S04 to effect intramolecular Friedel-Crafts acylation, thus providing
azachromanones 14. Dicarbonyl compounds 1 can also be directly prepared from
commercially available azachromanones 14. Suitable protection of the aza
nitrogen
is effected when necessary using protecting groups such as benzyl, benzoyl,
benzyloxycarbonyl (Cbz), t-butoxycarbonyl (Boc) or sulfonamido groups (mesyl,
Ms or tosyl, Ts).
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SCHEME VI
0
O \ O TFA ! TFAA
/ + ~ R4 i ~ ~ -- i
I H ! / O' v _Ot-I R4 ' \
OH ~ /
O
15 16 17
[0040] Synthetic Scheme VI details the three step prbcedure used to prepare
substituted chromanone derivatives 17. In step one, substituted phenols 15 are
condensed with acrylic acid to afford 3-phenoxypropionic acids 16. In step
two, the
acids 16 are treated with a mixture of trifluoroacetic anhydride, and
trifluoroacetic
acid to affect intramolecular Friedel-Crafts acylation affording selected
chromanones 17. Alternatively, the Friedel-Crafts acylation can be affected
with
H2S04, The dicarbonyl compounds 1 can be directly formed from commercially
available chromanones 17.
SCHEME VII
0
\ 1. NaH R \ O 1. NaOH ' R _ \
/ OH 2. O 4 ~ / O~ 4 ~ / O
O Et 2. TFA / TFAA
~O
lg 19
[0041] Synthetic Scheme VlI illustrates a procedure used to prepare
isochromanone derivatives 19. In step one, selected benzyl alcohol derivatives
5 are
treated with sodium hydride or other suitable base and subsequently treated
with
ethyl bromoacetate to provide the desired ethers 18. In step two, the ester
group of
18 is hydrolyzed with aqueous sodium hydroxide and then treated with a mixture
of
trifluoroacetic acid and trifluoroacetic anhydride to promote intramolecular
.Friedel-
Crafts acylation affording isochromanone 19 derivatives.
38
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SCHEME VIII
0
\ 1. NaH \ O 1. NaOH
O
NH2 2. Ra i / N~OEt 2.TFA/TFAA R4 I / NH
Br~O~
20 21 22
[0042] Synthetic Scheme VII illustrates a procedure used to prepare
isoazachromanone derivatives 22. In step one, selected benzyl amine
derivatives 20
are treated with ethyl bromoacetate and a suitable acid scavenger, such as
triethylamine, to provide the desired amines 21. In step two, the ester group
of 21 is
hydrolyzed with aqueous sodium hydroxide and then treated with a mixture of
trifluoroacetic acid and trifluoroacetic anhydride to promote intramolecular
Friedel-
Crafts acylation affording isochromanone 22 derivatives. Suitable protection
of the
aza nitrogen is effected when necessary using protecting groups such as
benzyl,
benzoyl, benzyloxycarbonyl (Cbz), t-butoxycarbonyl (Boc) or sulfonamido groups
(mesyl, Ms or tosyl, Ts).
SCHEME IX
0
\ 1. NaH \ \
1. NaOH _
O R4 ~ OEt
~O H 2' ~O 2. TFA l TFAA ~ O
Br~O~
15 23 24
[0043] Synthetic Scheme IX illustrates a procedure used to prepare substituted
3-coumaranones 24. Phenols 15 are first treated with a base, such as NaOH,
lithium
diisopropyl amide (LDA) or sodium methoxide followed by condensation with
ethyl bromoacetate in an appropriate solvent such as diethyl ether, ethanol,
or
tetrahydrofuran to provide the phenoxyacetate 23. In step two, the ester group
of 23
is hydrolyzed with aqueous sodium hydroxide and then treated with a mixture of
trifluoroacetic acid and trifluoroacetic anhydride to promote intramolecular
Friedel-
Crafts acylation affording 3-coumaranones derivatives 24.
39
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SCHEME X
'o
0
Br~
R ~ \ O I \ 1. NaOH _ I \
4 ~ / Et N Ra I / OEt Ra lI /
NH2 H~ 2.TFA/TFAA N
II H
O
12 25 26
[0044] Synthetic Scheme X illustrates a procedure used to prepare substituted
3-
azacoumaranones 26. Anilines 12 are treated with a acid scavenger, such as
triethylamine followed by alkylation with ethyl bromoacetate in an appropriate
solvent such as diethyl ether, ethanol, or tetrahydrofuran to provide the
phenoxyacetate 25. In step two, the ester group of 25 is hydrolyzed with
aqueous
sodium hydroxide and then treated with a mixture of trifluoroacetic acid and
trifluoroacetic anhydride to promote intramolecular Friedel-Crafts acylation
affording 3-azacoumaranones derivatives 24. Suitable protection of the aza
nitrogen is effected when necessary using protecting groups such as benzyl,
benzoyl, benzyloxycarbonyl (Cbz), t-butoxycarbonyl (Boc) or sulfonamido groups
(mesyl, Ms or tosyl, Ts).
SCHEME XI
0
\ 1. NaH Ra I \ 1. NaOH _ R4
Ra ~ O
/ SH 2' I / S OEt 2.TFAlTFAA / S
Br~O~
O
27 28
9
[0045] Synthetic Scheme XI illustrates a procedure used to prepare substituted
3-thiocoumaranones 28. Thiophenols 9 are first treated with a base, such as
NaOH,
lithium diisopropyl amide (LDA) or sodium methoxide followed by condensation
with ethyl bromoacetate in an appropriate solvent such as diethyl ether,
ethanol, or
tetrahydrofuran to provide the phenoxyacetate 27. In step two, the ester group
of 27
is hydrolyzed with aqueous sodium hydroxide and then treated with a mixture of
CA 02476665 2004-08-18
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trifluoroacetic acid and trifluoroacetic anhydride to promote intramolecular
Friedel-
Crafts acylation affording 3-coumaranones derivatives 28.
SCHEME XII
0
i \ 1. KO-t-butoxide, ~ \ OH (CH3)2S' BH3 i \ OH
Ra lI Ra lI Ra lI /
n-butyl lithium, -78C /
2. COz (or) LiAlH4
29 30 S
[0046] Synthetic Scheme XII illustrates a two step procedure used for the
preparation of substituted benzyl alcohols 5. In step one, a mixture of
potassium
tert-butoxide and anhydrous tetrahydrofuran, cooled to -78°C and
treated with a 1.6
M solution of n-butyl lithium in hexanes, is added to an appropriate
substituted
benzene 29 the anion thereby generated is reacted with carbon dioxide to yield
the
benzoic acid 30. In step two, the benzoic acid 30 is dissolved in a solvent,
such as
tetrahydrofuran, and treated with a reducing agent, such as borane dimethyl
sulfide
complex, to form the desired benzyl alcohol 5.
SCHEME XBI
O ~p O OH
O ROH/C6H6 Ro i LiHMDS RO / C02Et
reflux ~ (C02Et)2
31 32 33
NHNH2 R R\ ~
R Rv \ y \ Ri r
' ~ , R1 ~ / Me2NCH(OtBu)2 / N-N
N-N \
ACOH O ~ \ GO Et I O ~ COzEt
,N /
34 35
R
cyclizing R ~~ ~ ~\
R~ r
/
-N deprotection aminolysis N-N
agents ' ~ /
N \
\ _ -~ Het \ CONH2
CO~Et
He
36 3~
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[0047] Scheme XIZI describes the synthesis of the pyrazoles with fused
heterocycles such as substituted pyrimdine and pyrazole. In step one, 1,2-
cyclohexanedione (31) was refluxed with alcohols such as methanol or ethanol
in
benzene to provide the desired enone (32). In step two, enone 32 was treated
with a
base such as lithium bistrimethylsilylamide, followed by condensation with
diethyl
oxylate to afford 1,3-diketone (33). In step three, 1,3-diketone was allowed
to react
with a suitably substituted phenylhydrazine to form pyrazole 34. Appropriate
substituents could be; but are not limited to, methyl sulfone or sulfonamide,
which
may be protected. A suitable protecting group for the sulfonamide is 2,5-
dimethylpyrrole. In step four, pyrazole was treated with dimethylformamide di-
tert-
butyl acetal to give enaminone 35. In step five, enaminone was condensed with
cyclizing agents such as hydrazine, guanidine, or thiourea to afford fused
pyrazoles
and pyrimidines 36. In the final step, the ester was converted to amide 37 by
treating with ammonium hydroxide in methanol. For compounds where the
sulfonamide is protected with the 2,5-dimethylpyrrole, deprotection is
achieved by
treatment with refluxing trofluoroacetic acid and water.
SCHEME XIV
Oxidation
[0048] Synthetic Scheme XIV illustrates a procedure used for the preparation
of
the anti-inflammatory oxidized thio-containing fused tricyclic pyrazoles 3.
The
appropriate pyrazole 3 from Scheme 1, where A is S or -(CH2)mS(CH2)n-, is
treated
with an oxidizing agent such as m-chloroperbenzoic acid (MCPBA), hydrogen
peroxide, peracids, or potassium peroxymonosulfate. Compounds having differing
amounts of oxidation (sulfinyls and sulfones) can be prepared by controlling
the
stoichiometry of oxidant to sulfide or separated by chromatography.
42
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SCHEME XV
I \
R4 ~ / O=N-OR R I \
NH2
12 / N°N
H2/ Pd/C
O
EWG \ 1) H2N~H~OR
R4 ~ / N~NH~
+ H
2) H~o ) N2Ha
2
[0049] Scheme XV illustrates a method for the preparation of substituted
arylhydrazines 2. Anilines 12 can be treated with hydroxylamine-O-sulfonic
acid to
generate arylhydrazines 2 (JOC, 14, ~ 13, 1949). Anilines 12 can also be
diazotized
used sodium nitrite, or an alkyl nitrite, followed by catalytic reduction to
generate
arylhydrazines 2. In selected cases, suitably activated aryl rings, such as; 4-
fluoronitrobenzene or 4-fluorophenylmethylsulfone (EWG = electron withdrawing
group; such as nitro or methylsulfone), can be converted to arylhydrazines via
displacement of the fluorine with hydrazine or a carbazate, followed by
hydrolysis
of the protecting group.
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SCHEME XVI
Base, ethanol
direct
aminolysis
amine ( HNR4R5)
DCC or CDI
[0050] Synthetic Scheme XVI shows procedures for preparing anti-
inflammatory agents 3 of Formula l, wherein R3 = OCH3 is converted to OH or
NHR7. The esters 1 R3 = OCH3, which can be prepared as shown in Scheme I, is
dissolved in aqueous methanol and a base such as 10% NaOH is added. The
reaction is stirred at room temperature or heated to reflux to give the acids
3, R3 =
OH. The acids 3, R3 = OH, can be converted to the appropriate amides 3, R3 =
NHR7, by dissolving in methanol and treating with an appropriate amine in the
presence of a condensing agent such as dicyclohexylcarbodiimide (DCC) or
carbonyl diimidazole (CDI). The amides 3, R3 = NHR7 can also be prepared by
direct aminolysis of 3, R3 = OCH3.
[0051] The following examples contain detailed descriptions of the methods of
preparation of compounds of Formula I. These detailed descriptions fall within
the
scope, and serve to exemplify, the above-described General Synthetic
Procedures
that form part of the invention. These detailed descriptions are presented for
44
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illustrative purposes only and are not intended as a restriction on the scope
of the
invention. All parts are by weight and temperatures axe in degrees centigrade
unless
otherwise indicated.
[0052] The compounds of the present invention may also be synthesized
according to the methods of United States Patent 5,547,975.
[0053] The complete content of all publications, patents, and patent
applications
cited in this disclosure are herein incorporated by reference as if each
individual
publication, patent, or patent application were specifically and individually
indicated to incorporated by reference. Although the foregoing invention has
been
described in some detail by way of illustration and example for the purposes
of
clarity of understanding, it will be readily apparent to one skilled in the
art in light
of the teachings of this invention that changes and modifications can be made
without departing from the spirit and scope of the present invention. The
following
examples are provided for exemplification purposes only and are not intended
to
limit the scope of the invention, which has been described in broad terms
above.
EXAMPLES
Example 1
Ethyl 1-{4-[(aminothio)peroxy]phenyl }-8-nitro-4,5-dihydro-1H-benzo[g]indazole-
3-carboxylate
N2NSO2 0
'N-N
p2N / ~ I OEt
~5 I
Step 1
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WO 03/070706 PCT/US03/04844
O OLi
02N / / OEt
O
[0054] To 7-vitro-1-tetralone (4.6 g, 0.024 mol) and ethyl oxalate (3.5 mL,
0.026 mol) in ether (100 mL) was added dropwise lithium
bis(trimethylsilyl)amide
(1M in THF, 26 mL). The slurry was stirred overnight and filtered to give the
product as an olive green solid, 6.2 g (87% yield). 'H NMR (DMSO-d6/ 300 MHz)
8.45 (d, 1H); 8.05 (d of d, 1H'); 7.42 (d, 1H); 4.08 (q, 2H); 2.82-2.72 (m,
2H); 2.51-
2.43 (m, 2H); 1.21 (t, 3H).
Step 2
[0055] The material of step 1 (6.2 g, 0.021 mol) and 4-
sulfonamidophenylhydrazine hydrochloride (5.1 g, 0.023 mol) were stirred in
methanol (100 mL) overnight. Conc HCl (2 mL) was added to the thick slurry and
the contents were heated on a steam bath for 1 hour. Contents were allowed to
cool
and filtered to give an off white solid, 6.9 g. NMR and LC/MS analysis show
the
solid to contain two components, the desired, and the hydrated pyrazole. TFA
(60
mL) and TFAA (20 mL) were added to the solid and heated on a steam bath for 1
hour. Contents were concentrated in vacu~ leaving the product as a solid, 6.4
g
(69% yield). FABHRMS m/z 443.1020 (M+H, C~oH,9N406S requires 443.1025). 'H
NMR (DMSO-d6/ 300 MHz) 8.10 (d of d, 1H); 8.03 (d, 2H); 7.82 (d, 2H); 7.70 (d,
1H); 7.62 (s, 1H); 7.50 (d, 1H); 4.33 (q, 2H); 3.20-2.95 (m, 4H); 1.33 (t,
3H).
Anal. Calcd for CZOH,8N406S: C, 54.29; H, 4.10; N, 12.66. Found: C, 54.49; H,
4.00; N, 12.52.
Example 2
1-{4-[(aminothio)peroxy]phenyl }-8-vitro-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide
46
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WO 03/070706 PCT/US03/04844
H2NS02\~
N-N
02N ~ ~ ~ NH2
O
[0056] The final product of Example 1 (718 mg, 0.0016 mol), conc. ammonium
hydroxide (30 mL),, and methanol (15 mL) were stirred in a stoppered flask for
72
hours. Contents were filtered to give a light amber solid (606 mg). The solid
was
recrystallized from acetonitrile to give the product as a light amber solid ,
450 mg
(68% yield). FABHRMS m/z 414.0902 (M+H, C,$H,6NSOSS requires 414.0872). 'H
NMR (DMSO-d6l 300 MHz) 8.15 - 7.95 (m, 3H); 7.83 (d, 2H); 7.80-7.40 (m, 6H);
3.20-2.95 (m, 4H).
Anal. Calcd for C,BH,SNSOsS: C, 52.30; H, 3.66; N, 16.94. Found: C, 52.04; H,
3:64; N, 16.61.
Example 3
ethyl8-amino-1-[4-(aminosulfonyl)phenyl]-4,5-dihydro-1H-benzo[g]indazole-3-
carboxylate
H2NS02\~
N-N
H2N / ~ I OEt
O
[0057] The final product of Example 1 (2.0 g) and 10% Pd/C (350 mg) in DMF
(20 mL) were shaken at 55 PSI hydrogen for 3 hours. Contents were filtered and
the filtrate was concentrated in vacuo leaving an amber wax. The wax was
triturated with methanol and filtered to give the product as a light amber
solid, 1.6 g
(86% yield). FABHRMS m/z 413.1293 (M+H, C~oHZ,N404S requires 413.1284). 1H
NMR (DMSO-d6l 300 MHz) 8.00 (d, 2H); 7.73 (d, 2H); 7.50 (s, 2H); 7.01 (d, 1H);
6.43 (d of d, 1H); 6.00 (d, 1H); 4.83 (br s, 2H); 4.30 (q, 2H); 2.85-2.70 (m,
4H);
1.31 (t, 3H).
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Anal. Calcd for CZOH2oN4O4S (0.25 H20): C, 57.61; H, 4.96; N, 13.44. Found: C,
57.62; H, 5.11; N, 13.15.
Example 4
8-amino-1-{4-[(aminothio)peroxy]phenyl)-4,5-dihydro-1H-benzo[g]indazole-3-
carboxamide
H2NS02~
N-N
H2N / ~ I NH2
O
[0058] Example 4 was prepared similarly to Example 2 in 70 % yield.
FABHRMS m/z 384.1136 (M+H, C,$H,$N503S requires 384.1130). 'H NMR
(DMSO-el6/ 300 MHz) 7.95 (d, 2H); 7.75 (d, 2H); 7.53 (br s, 1H); 7.43 (br s,
1H);
7.32 (br s, 1H); 7.01 (d, 1H); 6.44 (d of d, 1H); 6.03 (s, 1H); 4.81 (s, 2H);
2.93-2.65
(m, 4H).
[0059] Anal. Calcd for C,$H,~N503S: C, 56.38; H, 4.47; N, 18.27. Found: C,
56.31; H, 4.42; N, 18.31.
Example 5
0 0
s OEt
HO \ I O
[0060] To 6-hydroxy-1-tetralone (10.4 g, 0.064 mol) and ethyl oxalate (17.4
mL, 0.128 mol) in THF ( 100 mL) was added dropwise lithium
bis(trimethylsilyl)amide (1M in THF, 130 mL). The slurry was stirred overnight
and a solid was filtered. The solid was dissolved in water and made acidic to
pH
2.5 with 3 N HCI, precipitating a waxy solid. The waxy solid was extracted
into
EtOAc, dried (MgS04) and concentrated in vacuo leaving a dark solid (15.7 g).
The
48
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solid was purified by chromatography on silica gel, eluting with 15%
EtOAc/hexanes to give a yellow solid (5.9 g). The solid was recrystallized
from
EtOAc/hexanes to give the product as a yellow solid, 3.7 g (22% yield).
FABHRMS m/z 263.0925 (M+H, C14H1505 requires 263.0919): 'H NMR (CDC13 /
300 MHz) 7.93 (d, 1H); 6.80 (d of d, 1H); 6.68 (s, 1H); 5.72 (s, 1H); 4.39 (q,
2H);
3.00-2.75 (m, 4H); 1.40 (t, 3H).
Anal. Calcd for C,QH,4O5: C, 64.12; H, 5.38. Found: C, 63.79; H, 5.35.
Example 6
ethyl 1-[4-(aminosulfonyl)phenyl]-7-hydroxy-4,5-dihydro-1 H-benzo [g]indazole-
3-
carboxylate
H2NS02\~
N-N
OEt
HO \ I °
[0061] The material prepared in Example 5 (2.0 g, 0.0076 mol) and 4-
sulfonamidophenylhydrazine hydrochloride (1.9 g, 0.0085) were stirred in
glacial
acetic acid (25 mL) for 96 hours. Contents were heated at 55°C for 5
hours,
allowed to cool, diluted with water (75 mL), and filtered to give the product
as a
white solid, 3.1 g (90% yield). FABHRMS m/z 414.1146 (M+H, CZOH2oN3O5S
requires 414.1124). 'H NMR (DMSO-d6 / 300 MHz) 9.72 (s, 1H); 8.00 (d, 2H);
7.73 (d, 2H); 7.53 (s, 1H); 6.80 (s, 1H); 6.60-6.40 (m, 2H); 4.30 (q, 2H);
2.90 (s,
4H); 1.30 (t, 3H).
Anal. Calcd for CZOH,9N3O5S (0.2 HzO): C, 57.60; H, 4.69; N, 10.08. Found: C,
57.72; H, 4.91; N, 9.68.
Example 7
ethyl 1-[4-(aminosulfonyl)phenyl]-6-benzyl-1,4,5,6-tetrahydropyrrolo[2,3-
g]indazole-3-carboxylate
49
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Step 1
0
~I
~ N
[0062] To 1,5,6,7-tetrahydro-4H-indol-4-one (5.0 g, 0.037 mol) in DMF (20
mL) was added benzyl chloride (4.4 mL, 0.038 mol) and potassium carbonate (5.3
g, 0.038 mol). Contents were heated at 70°C overnight. Contents were
allowed to
cool and partitioned between EtOAc and water. The EtOAc layer was dried
(MgS04) and concentrated ifa vacuo leaving an amber oil, 7.9g. The oil was
purified by chromatography on silica gel eluting with 25% EtOAc/hexanes to
give
the desired as a white solid, 7.1 g (85%o yield). FABHRMS m/z 226.1213 (M+H,
C,SH,6N0 requires 226.1232). 'H NMR (CDC13 / 300 MHz) 7.40-7.28 (m, 3H);
7.10-7.00 (m, 2H); 6.63-6.53 (m, 2H); 5.02 (s, 2H); 2.70-2.60 (m, 2H); 2.50-
2.40
(m, 2H); 2.10-2.06 (m, 2H).
Anal. Calcd for C,SH,SNO: C, 79.97; H, 6.71; N, 6.22. Found: C, 79.90; H,
6.65; N,
6.09.
Step 2
0 0
~~ %~OEt
O
~ N
[0063] The product of step 2 was prepared similarly to Example 5 starting with
the material of step 1 in 67% yield. FABHRMS mlz 326.1393 (M+H, C,9H~oN04
so
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WO 03/070706 PCT/US03/04844
requires 326.1392). 'H NMR (CDCl3/ 300 MHz) 7.40-7.26 (m, 3H); 7.10-7.00 (m,
2H); 6.70-6.60 (m, 2H); 5.06 (s, 2H); 4.33 (q, 2H); 3.20-3.08 (m, 2H); 2.75-
2.60
(m, 2H); 1.40 (t, 3H).
Anal. Calcd for C,9H19NO4: C, 70.14; H,~5.89; N, 4.31. Found: C, 70.09; H,
5.75;
N, 4.08.
Step 3
[0064] The final product of Example 7 was prepared similarly to Example 6
starting with the product of step 2 in 37% yield. FABHRMS m/z 477.1609 (M+H,
C25H25N404S requires 477.1596). 'H NMR (DMSO-d6 / 300 MHz) 8.00 (d, 2H);
7.80 (d, 2H); 7.50 (s, 1H); 7.40-7.20 (m, 3H); 7.20-7.05 (m, 2H); 6.80 (s,
1H); 5.62
(s, 1H); 5.13 (s, 2H); 4.25 (q, 2H); 3.10-2.92 (m, 2H); 2.89-2.70 (m, 2H);
1.30 (t,
3H).
Anal. Calcd for Cz5H24NøO~S: C, 63.01; H, 5.08; N, 11.76. Found: C, 62.95; H,
5.02; N, 11.76.
Example 8
1-[4-(aminosulfonyl)phenyl]-6-benzyl-1,4,5,6-tetrahydropyrrolo[2,3-g]indazole-
3-
carboxamide
NH2
[0065] Example 8 was prepared similarly to Example 2 starting with the
compound of Example 7 in 75% yield. FABHRMS m/z 448.1477 (M+H,
Cz3HZZN50sS requires 448.1443). 'H NMR (DMSO-d6 / 300 MHz) 8.00 (d, 2H);
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WO 03/070706 PCT/US03/04844
7.80 (d, 2H); 7.60-7.15 (m, 3H); 7.10 (d, 2H); 6.79 (d, 1H); 5.65 (d, 1H);
5.12 (s,
2H); 3.10-2.93 (m, 2H); 2.82-2.68 (m, 2H).
Anal. Calcd for C23HZ,NSO3S: C, 61.73; H, 4.73; N, 15.65. Found: C, 61.33; H,
4.52; N, 15.43.
Example 9
ethyl 1-[4-(aminosulfonyl)phenyl]-6-benzyl-1,4,5,6-tetrahydropyrazolo[3,4-
e]indazole-3-carboxylate
H2NS02~
N-N
OEt
N~ ~ O
N
Step 1
0 0
OEt
N~ ~ O
~ N
[0066] The product of step 1 was prepared similarly to Example 5 starting with
1,5,6,7-tetrahydro-1-(phenylmethyl)indazol-4-one [Fleterocycles, 32 (1) 41-72
(1991)] in 89% yield. FABHRMS m/z 327.1347 (M+H, Cl$H19N2O4 requires
327.1345). 'H NMR (CDCl3 l 300 MHz) 8.00 (s, 1H); 7.40-7.30 (m, 3H); 7.13 (d,
2H); 5.30 (s, 2H); 4.31 (q, 2H); 3.19-3.03 (m, 2H); 2.80-2.68 (m, 2H); 1.38
(t, 3H).
Anal. Calcd for C,8H,8Nz04: C, 66.25; H, 5.56; N, 8.58. Found: C, 66.35; H,
5.47;
N, 8.78.
Step 2
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[0067] The final product of Example 9 was prepared similarly to Example 6
starting with the compound of step 1 in 64% yield. FABHRMS m/z 478.1549
(M+H, C2dHz4N5O4S requires 478.1551). 'H NMR (DMSO-d6 / 300 MHz) 8.02 (d,
2H); 7.82 (d, 2H); 7.51 (s, 1H); 7.40-7.22 (m, 3H); 7.21-7.13 (m, 2H); 7.10
(s, 1H);
5.40 (s, 2H); 4.30 (q, 2H); 3.20-2.90 (m, 4H); 1.30 (t, 3H).
Anal. Calcd for Cz4H23N504S: C, 60.36; H, 4.85; N, 14.67. Found: C, 60.60; H,
4.86; N, 14.71.
Example 10
1-[4-(aminosulfonyl)phenyl]-6-benzyl-1,4,5,6-tetrahydropyrazolo[3,4-a]indazole-
3-
carboxamide
NH2
[0068] Example 10 was prepared similarly to Example 2 starting with the
product of Example 9 in 79% yield. FABHRMS m/z 449.1399 (M+H, CZ,Hz,N6O3S
requires 449.1396). 'H NMR (DMSO-d6 / 300 MHz) 8.00 (d, 2H); 7.84 (d, 2H);
7.60-7.40 (m, 2H); 7.40-7.22 (m, 3H); 7.25-7.10 (m, 2H); 5.37 (s, 2H); 3.20-
2.90
(m, 4H).
Anal. Calcd for C~ZHZON603S (0.8 HZO): C, 57.08; H, 4.70; N, 18.15. Found: C,
57.54; H, 4.56; N, 17.77.
Example 11
ethyl 1-[4-(aminosulfonyl)phenyl]-1,4,5,6-tetrahydropyrazolo[3,4-a]indazole-3-
carboxylate
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H2NS02~
N-N
OEt
N~ ~ O
N
H
[0069] The material of Example 9 (4.2 g, 0.009 mol), DMF (40 mL), glacial
acetic acid (20 mL), and Pearlman's catalyst (1.5 g) were shaken at 55 psi
hydrogen
for 96 hours. Contents were filtered through celite and the filtrate was
concentrated
in vacuo leaving the product as a gray solid, 2.4 g (70% yield). FABHRMS m/z
388.1124 (M+H, C"H,8N504S requires 388.1080). 'H NMR (DMSO-d6/ 300 MHz)
8.02 (d, 2H); 7.80 (d, 2H); 7.60 (br s, 1H); 7.30 (s, 1H); 4.35 (q, 2H); 3.15-
3.00 (m,
2H); 3.00-2.80 (m, 2H); 1.32 (t, 3H).
Anal. Calcd for C,~H"N504S: C, 52.70; H, 4.42; N, 18.08. Found: C, 52.47; H,
4.18; N, 17.89.
Example 12
1-[4-(aminosulfonyl)phenyl]-1,4,5,6-tetrahydropyrazolo[3,4-a]indazole-3-
carboxamide
H2NS02\~
N-N
NH2
N~ ~ O
N
H
[0070] Example 12 was prepared similarly to Example 2 starting with the
material of llin 86% yield. FABHRMS m/z 359.0939 (M+H, C,SH,SN6O3S
requires 359.0926). 'H NMR (DMSO-d6 / 300 MHz) 8.02 (d, 2H); 7.87 (d, 2H);
7.56 9s, 1H); 7.50 (br s, 2H); 7.35 (s, 2H); 3.20-3.00 (m, 2H); 2.95-2.80 (m,
2H).
[0071] Anal. Calcd for CISH14N6O3S' C~ 50.27; H, 3.94; N, 23.45. Found: C,
50.07; H, 3.73; N, 23.08.
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Example 13
ethyl 1-[4-(aminosulfonyl)phenyl]-4,5-dihydro-1H-thieno[2,3-g]indazole-3-
carboxylate
H2NS02~
N-N
OEt
O
s
Step 1
0
~I
s
[0072] 4-(2-Thienyl)butyric acid (9.7 g, 0.057 mol), acetic anhydride (12 mL),
and phosphoric acid (85%, 0.25 mL) were refluxed for 3 hours. Contents were
allowed to cool and partitioned between EtOAc and water. The EtOAc layer was
dried (MgS04) and concentrated iiz vacuo leaving a dark oil, 9.0 g. The oil
was
distilled on a kugelrohr apparatus at 50°C (0.1 mm) to give a white
solid, 5.0 g
(58% yield). 'H NMR (CDCl3/ 300 MHz) 7.37 (d, 1H); 7.02 (d, 1H); 3.08-2.95
(m, 2H); 2.60-2.50 (m, 2H); 2.30-2.10 (m, 2H).
Step 2
0 0
0
0
[0073] The product of step 2 was prepared similarly to step 1 of Example 1
using the material of step 1 in 77% yield. 'H NMR (DMSO-d6 / 300 MHz) 7.32-
7.05 (m, 2H); 4.05 (q, 2H); 2.85-2.50 (m, 4H); 1.20 (t, 3H).
Step 3
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[0074] The final product of Example 13 was prepared similarly to Example 6
starting with the material of step 2 in 86% yield. FABHRMS mlz 404.0702 (M+H,
C,BH,$N3OaS2 requires 404.0739). 'H ~NMR (DMSO-d6l 300 MHz) 8.05 (d, 2H);
7.70 (d, 2H); 7.59 (s, 1H); 7.39 (d, 1H); 6.40 (d, 1H); 4.35 (q, 2H); 3.10 (s,
4H);
1.35 (t, 3H).
Anal. Calcd for C,8H1~N30~Sz: C, 53.58; H, 4.25; N, 10.41. Found: C, 53.51; H,
4.02; N, 10.45.
Example 14
1-[4-(aminosulfonyl)phenyl]-4,5-dihydro-1H-thieno[2,3-g]indazole-3-carboxamide
' H2NS02\~
N-N
~~~NH2
O
[0075] Example 14 was prepared similarly to Example 2 staring with the
compound of Example 13 in 85% yield. FABHRMS m/z 375.0601 (M+H,
~16H15N4~3~2 requires 375.0586). 'H NMR (DMSO-d6l 300 MHz) 8.05 (d, 2H);
7.70 (d, 2H); 7.59 (s, 1H); 7.55 (s, 2H); 6.47 (d, 1H); 3.10 (s, 4H).
Anal. Calcd for C16H14N4u3S2' C~ 56.06; H, 4.70; N, 13.08. Found: C, 56.49; H,
4.74; N, 13.21.
Example 15
1-[4-(aminosulfonyl)phenyl]-4,5-dihydro-1H-pyrazolo[3,4-f]isoquinoline-3-
carboxamide
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H2
Step 1
OLi O
I \ \ Ow
N / O
[0076] To 7,8-Dihydroisoquinolin-5(6H)-one (1.17 gm, 8 mmol) in diethyl
ether (50 mL) was added 1M LHMDS (8 mL, 8 mmol) dropwise over several
minutes (cf. Lardenois, P.; et al. Synthetic Communications 26(12), 2305-8,
1996 . A precipitate slowly formed and the reaction became light yellow. After
about 15 minutes, dimethyl oxalate (944 mg, 8 mmol) was added as a solid and
the
reaction stirred at room temperature for 72 hours. The resulting precipitate
was
collected by suction filtration and washed extensively with diethyl ether. A
bright
yellow solid was obtained. Yield; 1.7 gm (89%). 'H-NMR (d6 DMSO) 2.41 (t,
2H); 2.63 (t, 2H); 3.56 (s, 3H); 7.51 (d, 1H); 8.37 (s, 1H); 8.40 (d, 1H).
[0077] Step 2
[0078] The enolate from step 1 (717 mg, 3 mmol) was combined with 4-
sulfonamidophenyl-hydrazine hydrochloride ( 669 mg, 2 mmol) in methanol ( 10
mL) and stirred at ambient temperature for 72 hours, then the resulting
precipitate
was collected by suction filtration and washed with methanol. The resulting
hydrated pyrazole (502 mg) was re-suspended in methanol and concentrated HCl
s7
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(0.5 mL) was added. The reaction was stirred at ambient temperature for two
hours,
then concentrated to a yellow solid in-vacuo. The solid was triturated with
methanol and collected by suction filtration. Yield 250 mg (22 %). 'H-NMR (db
DMSO) 3.09 (s, 4H); 3.87 (s, 3H); 6.91 (d, 1H); 7.60 (s, 2H); 7.84 (d, 2H);
8.03 (d,
2H); 8.50 (d, 1H); 8.80 (s, 1H). FABHRMS m/z 385.0987 (M+H, C,BH"N40~S
requires 385.0971).
[0079] Step 3
The methyl ester from step 2 (240 mg, 0.625 mmol) was suspended in methanol (
10
mL) and concentrated ammonium hydroxide (2 mL) was added. The reaction was
heated to 95 C in a sealed tube for 16 hours. The reaction was cooled and
concentrated to a reddish solid, which was triturated with methanol. 'H-NMR
suggested some carboxylic acid may be present, so the solid was dissolved in
methanol (l5mL) and 1 N HCl (3 mL). The homogeneous solution was then made
basic with saturated NaHC03 solution to pH = 8. After concentrating the
solution
under a stream of nitrogen, a brown solid was collected (100 mg). This solid
was
filtered through a plug of silica gel (10 gm) using 10%
methanol/dichloromethane
to obtain an off white solid. Yield: 90 mg (39 %). FABHRMS m/z 370.0966
(M+H, C,~H,6N503S requires 370.0974).
'H-NMR (d4 MeOH + TFA) 3.25 (s, 4H); 7.25 (d, 1H); 7.83 (d, 2H); 8.14 (d, 2H);
8.49 (d, 1H); 8.79 (s, 1H).
Example 16
1-[4-(aminosulfonyl)phenyl]-1,4,5, 8-tetrahydropyrazolo [4, 3-g]indazole-3-
carboxamide
H2N02S
N-N
I
HN N\ CONH2
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Step 1
[0080] A mixture of 1,2-cyclohexanedione (26.0 g) and ethanol (100 mL) in
~ 500 mL of benzene was heated at reflex with a Dean-Stark trap overnight.
After the
removal of solvent, the residue was purified by chromatography on silica gel
(ethyl
acetate/hexane, 2:8) to give 15.3 g of the desired 2-ethoxy-2-cyclohexen-1-one
as a
light yellow oil (47% yield). To 120 mL 1.0 M solution of LiHMDS in THF was
added a solution of the above compound ( 15.3 g, 0.11 mol) in 100 mL of ether
at -
78°C. After the addition, the dark brown mixture was stirred at this
temperature for
1/z h, a solution of diethyl oxylate ( 17.5 g, 0.12 mol) in 30 mL of ether was
added in
one portion. The reaction was allowed to warm up to room temperature over 18
h.
Water was added and acidified to pH = 4 with 1 N HCI. The aqueous phase was
extracted with ethyl acetate and the organic layer was washed with brine,
dried over
MgS04, and filtered. The filtrate was concentrated to afford 21.5 g of product
as a
dark brown liquid that was used without further purification.
Step 2
[0081] A mixture of the crude from step 1 (20.0 g, 0.083 mol) and 1-(4-
hydrazinophenylsulfonyl)-2,5-dimethylpyrrole (22.0 g, 0.083 mol) in 400 mL of
acetic acid was stirred at room temperature overnight. The solvent was removed
and the residue was partitioned between ethyl acetate and conc. ammonium
hydroxide. The organic layer was washed with brine, dried over MgS04, and
filtered. The filtrate was concentrated and the residue was purified by
chromatography on silica gel (ethyl acetate/ hexane, 3:7) to give 16.2 g of
pure
product as a yellow solid,
Step 3
[0082] A mixture of the product from step 2 (3.1 g, 0.0062 mol) and N, N-
dimethylformamide di-tert-butyl acetal (10.2 g, 0.062 mol) was heated at
reflux
overnight. After cooling, excess reagent was removed under vacuum and the
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residue was triturated with cold ethanol to give 2.3 g of pure product as a
yellow
solid (77% yield); mp: 230-231°C; Anal. Calcd. for CZSHzBNøOSS: C,
60.47; H, 5.68;
N, 11.28; S, 6.46. Found: C, 59.98; H, 5.42; N, 10.93; S, 6.11.
Step 4
[0083] To a suspension of the product from step 3 (0.45 g, 0.0009 mol) in 10
mL of ethanol was added hydrazine (0.03 mL, 0.0009 mol) and the mixture was
stirred at reflux overnight. Solvent was removed and the residue was purified
by
chromatography on silica gel (ethyl acetate/hexane, 2:8) to give 0.38 g of
product as
a yellow solid (93% yield); mp: 134-136°C; Anal. Calcd. for
C23H23NSO4S: C, 59.34;
H, 4.98; N, 15.04; S, 6.89. Found: C, 59.28; H, 4.95; N, 14.76; S, 6.93.
Step 5
[0084] The product from step 4 (0.35 g, 0.00075 mol) was treated with a
mixture of TFA (15 mL) and water (5 mL) and the dark brown solution was
refluxed under nitrogen for 2 h. Cooled and basified with conc. ammonium
hydroxide to precipitate out 0.18 g of crude product as a pale yellow solid. A
suspension of this solid in a mixture of conc. ammonium hydroxide (15 mL) and
methanol (5 mL) was stirred at RT for 3 days. Solvent was removed to half
volume
and the solid was filtered to afford 0.1 g of product was white powder (50%
yield
for two steps); mp: 347°C (decomp); Anal. Calcd. for C,SH14N6O3S: C,
50.27; H,
3.94; N, 23.45; S, 8.95. Found: C, 49.65; H, 3.81; N, 22.78; S, 8.77.
Example 17
8-amino-1-[4-(aminosulfonyl)phenyl]-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-
3-carboxamide
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H2N02S /
N-N
I
H2NYN\ \ CONH2
N /
Step 1
[0085] To a suspension of the product from step 3 of Example 16 (8.7 g, 0.018
mol) and guanidine hydrochloride (1.7 g, 0.018 mol) in 250 mL of ethanol, was
added sodium ethoxide ( 1.22 g, 0.018 mol) and the mixture was refluxed under
nitrogen overnight. After the removal of solvent, the residue was partitioned
between ethyl acetate and water. The organic layer was washed with brine,
dried
over magnesium sulfate, and filtered. The filtrate was concentrated and the
cruae
was purified by chromatography on silica gel (ethyl acetate/hexane, 3:7) to
give 5,2
g of product as brown solid (60% yield); mp: 111-112°C; Anal. Calcd.
for
Cz4H24N6C4S: C, 58.52; H, 4.91; N, 17.06; S, 6.51. Found: C, 58.24; H, 4.84;
N,
16.80; S, 6.68.
Step 2
[0086] This compound was synthesized by following the same procedure as
step 5 in Example 1 in 52% yield; mp: 332°C (decomp); Anal. Calcd. for
2O C1gH15N7~3S: C, 49.86; H, 3.92; N, 25.44; S, 8.32. Found: C, 49.49; H,
'3.86; N,
25.52; S, 7.93.
[0087] Examples 18, and 19 were prepared according to Scheme XVIB.
Related compounds such as A, B and C shown in Scheme XVBI can be prepared in
a similar manner using the appropriate R4 group.
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SCHEME XVIB
o-
N CI N
CN / II O HO N ~ CI N ,+
OII
O~ OH O O O
CI N CI N CI N ~ CI N OEt
\ ~ O
38 39
OMe
F Are
Ar\N-N \ / ~ / HZN N \ N NHZ
OEt _ -------
CI N ~ \ -~ N N N N NH -' \ I O
\ p i \
Ar = p-FC6H4 Meo ~ ~ \ ~ 0 Ar = p-FC6H4
40 Example 19
Are
R°HN N ~ N NHz O O
\w o R4 = CI CI cl
Ar = p-FC6H4 N I N I N
~N~ F
F ~ \
\ / N-N
N.-N O N N ~ I NH2
O N N \ I NHx
CI / \ I O
CI / \ I 0
N
N N
C
A
Example 18
8-Chloro-1-(4-fluorophenyl)-4,5-dihydro-1H-pyrazolo[4,3-h]quinoline-3-
carboxamide
F
N-N O
CI
N\ ~ NH2
Step 1
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Ethyl (2-chloro-8-oxo-5,6,7,8-tetrahydroquinolin-7-yl)(oxo)acetate
0 0 0
CI
OEt
[0088] To a suspension of 10.8 g of 2-chloro-6,7-dihydroquinolin-8(5H)-one 38
in 30 ml of anhydrous ethanol, cooled to about 10° C, 24.4 ml of a 21
wt% solution
of sodium ethoxide (65.46 mmol) in anhydrous ethanol (24.4 ml) was added
dropwise with stirring under nitrogen atmosphere for 10 minutes. After 5
minutes
8.82 g of diethyl oxalate (59.54 mmol) was added over 2 minutes. The reaction
mixture was stirred at 10° C for 30 minutes, then 1 hour at room
temperature. To
the reaction mixture, cooled to about 0° C, 66 ml of a 1M solution of
HCl in ethanol
was added dropwise for 10 minutes. A white precipitate was separated by
filtration
and washed with 30 ml of chloroform. The organic portions were combined and
solvents were removed to give 16.5 g (99% yield) of the crude desired product
39,
which was used in following synthesis without additional purification. 1H NMR
(300 MHz, 8, DCCl3): 1.45 (t, 3H, J = 7.15 Hz), 2.88-3.02 (m, 4H), 4.44 (q,
2H, J =
7.15 Hz), 7.43 (d, 1 H, J = 8.05 Hz), 7.62 (d, 1 H, J = 8.05 Hz), 13 .6-14.2 (
1 H,
broad). ESI mass spectrum for (C13H1aC1N04 + 1)''~: 282.1
Step 2
Ethyl 8-chloro-1-(4-fluorophenyl)-4,5-dihydro-1H-pyrazolo[4,3-h]quinoline-3-
carboxylate
CI
Et
[0089] The mixture of ethyl (2-chloro-8-oxo-5,6,7,8-tetrahydroquinolin-7-
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yl)(oxo)acetate 39 from step 1 (13 g, 46.15 mmol) and 4-fluorophenylhydrazine
hydrochloride (7.51 g, 46.15 mmol) in 500 ml of 1M solution of HCl in ethanol
was
placed in 1 L flask with a condenser under nitrogen atmosphere and heated at
reflux
for 1 hour. Then, it was cooled to room temperature and placed in refrigerator
overnight at -5° C. The white crystalline precipitate was filtered and
washed with
ether to give 12.3 g of ethyl 8-chloro-1-(4-fluorophenyl)-4,5-dihydro-1H-
pyrazolo[4,3-h]quinoline-3-carboxylate 40 (71% yield). It was used in
following
synthesis without additional purification. 1H NMR (300 MHz, ~, DCC13): 1.48
(t,
3H, J = 7.15 Hz), 3.01-3.25 (m, 4H), 4.49 (q, 2H, J = 7.15 Hz), 7.10-7.24 (m,
3H),
7.50-7.62 (m, 3H). ESI mass spectrum for (C19H15C1FN302 + 1)+: 372.1
Step 3
8-Chloro-1-(4-fluorophenyl)-4,5-dihydro-1 H-pyrazolo [4,3-h] quinoline-3-
carboxamide
Hz
[0090] A mixture of 1 g of ethyl 8-chloro-1-(4-fluorophenyl)-4,5-dihydro-1H-
pyrazolo[4,3-h]quinoline-3-carboxylate 40 from step 2, 25 ml of methanol, and
25
ml of liquid ammonia were placed in an autoclave and stirred overnight at
130° C.
After cooling to room temperature and removing ammonia, a suspension of gray
crystalline precipitate in methanol was obtained. The solid was isolated by
filtration
to give 0.8 g (87% yield) of 8-chloro-1-(4-fluorophenyl)-4,5-dihydro-1H-
pyrazolo[4,3-h]quinoline-3-carboxamide. 1H NMR (400 MHz, ~, DCC13): 3.01-
3.25 (m, 4H), 5.38 (s, 1H), 6.81 (s, 1H), 7.09 (d, 1H, J = 7.9 Hz) 7.11-7.19
(m, 2H),
7.45-7.56 (m, 3H). ESI mass spectrum for (Cl7HiaC1FN4O + 1)+: 343.1
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Example 19
1-(4-Fluorophenyl)-N-(4-methoxybenzyl)-8-[(4-methoxybenzyl)amino]-4,5-
dihydro-1 H-pyrazolo [4, 3-h] quinoline-3-c arboxamide
HN
H
H3C0
H3C0
[0091] Ethyl8-chloro-1-(4-fluorophenyl)-4,5-dihydro-1H-pyrazolo[4,3-
h]quinoline-3-carboxylate 40 (3.8 g, 10.22 mmol) in 35 ml of 4-
methoxybenzylamine was heated at 185° C for 26 hours. The reaction
mixture
contained the title compound as a major product. It was isolated by thin layer
silica
chromatography (ethyl acetate-hexane 60:40). 1H NMR (400 MHz, ~, DCC13):
2.86-292 (m, 2H), 3.14-3.21 (m, 2H), 3.76-3.78 (m, 6H), (3.95 (d, 2H~ J = 5.9
Hz),
4.28 (t, 1H, J = 5.8 Hz), 4.54 (d, 2H, J = 5.9 Hz), 6.20 (d, 1H, J = 8.2 Hz),
6.78-6.86
(m, 4H), 6.93-7.02 (m, 2H), 7.01-7.06 (m, 2H), 7.20 (t, 1H, J = 5.64), 7.26-
7.30 (m,
3H), 7.42-7.48 (m, 2H). ESI mass spectrum for (C33H3°FNSO3 + 1)+: 564.2
[0092] Table 1 shows the bioactivity for the exemplified compounds as
measured in the IKK heterodimer Resin Enzyme Assay expressed as LC50.
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TABLE 1
COMPOUND STRUCTURE EXAMPLE HetD
ethyl 1-[4- H2NS02\~ Example >200
1' 7
I
(aminosulfonyl)phenyl]-6-~~ !~M
N-N
benzyl-1,4,5,6- ~ ~ oEt
tetrahydropyrrolo[2,3- /
I o
N
g]indazole-3-carboxylate/ \
1-[4-(aminosulfonyl)phenyl]-6-H2NS02 / I Example >200
8
benzyl-1,4,5,6- ' ~ N-N
tetrahydropyrrolo[2,3- ~ ~ NH2
g]indazole-3-carboxamideN I o
/ \
ethyl 1-[4- H2NS02 ~ I Example >200
9
(aminosulfonyl)phenyl]-6-~ N-N I~M
benzyl-1,4,5,6- ~ ~ ~ oEt
tetrahydropyrazolo[3,4- N.N I o
a]indazole-3-carboxylate/ \
1-[4-(aminosulfonyl)phenyl]-6-H2NS02 , I Example >200
10
benzyl-1,4,5,6- ~ N-N ~,M
tetrahydropyrazolo[3,4- ~ ~ I NH2
a]indazole-3-carboxamideN.N I o
/ \
ethyl 1-[4- H2NS02\~ Example >200
I 11
(aminosulfonyl)phenyl]-1,4,5,6-~~ !~M
N-N
tetrahydropyrazolo[3,4- ~ ~ ~ oEt
a]indazole-3-carboxylateN I o
N
H
1-[4-(aminosulfonyl)phenyl]-H2NS02 / I Example 0.67
12
1,4,5,6-tetrahydropyrazolo[3,4-~ N-N
a]indazole-3-carboxamide~ ~ NH2
N~ I o
N
H
ethyl 1-[4- H2NS02 / , Example >200
I 13
(aminosulfonyl)phenyl]-4,5-~ !~M
N-N
dihydro-1 H-thieno [2,3-~ oEt
g]indazole-3-carboxylate<
s I o
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TABLE 1 cont
COMPOUND STRUCTURE EXAMPLE HetD
1-[4-(aminosulfonyl)phenyl]-H2NS02 , I Example 14 3.2
4,5-dihydro-1H-thieno[2,3-~ N-N !~M
g]indazole-3-carboxamide/ I ~ ~ NH2
S
1-[4-(aminosulfonyl)phenyl]-os~ Example 15 8.21
4,5-dihydro-1H-pyrazolo[3,4-H2N \
f]isoquinoline-3-carboxamide_
N
N NH2
N
\
/
1-[4-(aminosulfonyl)phenyl]-H2N2S / Example 16 0.96
1,4,5,8-tetrahydropyrazolo[4,3-~ ~ I~M
g]indazole-3-carboxamideN
'CONH2
HN
8-amino-1-[4- H2N2S / Example 17 2.3
(aminosulfonyl)phenyl]-4,5-
dihydro-1H-pyrazolo[4,3-N2N N
'
h]quinazoline-3-carboxamideCONH2
N %
[0093] In a likewise manner the following compounds of Table 2 could also be
prepared.
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TABLE 2
Ethyl 1-{4- H~
[(methylamino)sulfonyl]phenyl}- o
1,4,5,6-tetrahydropyrazolo[3,4- \ /
a] indazole-3-carboxylate
H
Ethyl 1-[4- ~ o
~'w
(anilinosulfonyl)phenyl]-1,4,5,6- ~ ~ -
tetrahydropyrazolo[3,4-a]indazole-
3-carboxylate i I \
H
Ethyl 1-{4- H
but lamino sulfon 1] henyl}
[( Y ) Y p o
1,4,5,6-tetrahydropyrazolo[3,4- \ /
a]indazole-3-carboxylate
0
H
ethyl 1-{4- /
~~ io
[(dimethylamino)sulfonyl]phenyl}- o
1,4,5,6-tetrahydropyrazolo[3,4- \ /
a]indazole-3-carboxylate
0
H
ethyl l-(4-methoxyphenyl)-1,4,5,6-
tetrahydropyrazolo[3,4-a]indazole-
3-carboxylate
0
'\H NN
68
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TABLE 2 cont
Ethyl 1-[4- HZ
O
(aminocarbonyl)phenyl]-1,4,5,6-
tetrahydropyrazolo[3,4-a]indazole- \
3-carboxylate
N/ / \ I ~~/
O
1-[4-(anilinosulfonyl)phenyl]-
I
1,4,5,6-tetrahydropyrazolo[3,4-
8N~
a]indazole-3-carboxamide o ~ I
\Ngz
i
I
8N
1-{4-
[(methylamino)sulfonyl]phenyl}-
o
1,4,5,6-tetrahydropyrazolo[3,4- \
a]indazole-3-carboxamide H
2
N HN' V O
1-{4-
but lamino sulfon 1]phenyl}-
[( Y ) Y o
1,4,5,6-tetrahydropyrazolo[3,4- \ /
a]indazole-3-carboxamide
~I
1-{4-
[(dimethylamino)sulfonyl]phenyl}- o~ i z
H
1,4,5,6-tetrahydropyrazolo[3,4- ~ N
a]indazole-3-carboxamide w
N~\
vH
69
CA 02476665 2004-08-18
WO 03/070706 PCT/US03/04844
TABLE 2 cont
1-[4-(aminocarbonyl)phenyl]- HZ
1,4,5, 6-tetrahydropyrazolo [3,4-
e]indazole-3-carboxamide
'NHZ
H
1-(4-methoxyphenyl)-1,4,5,6-
tetrahydropyrazolo [3,4-e] indazole-
3-carboxamide
H2
N~
1-[4-(aminosulfonyl)phenyl]-1,6-
dihydropyrazolo [3,4-e] indazole-3-
_/
carboxamide
H2
w
O
H
1-{4-
[(butylamino)sulfonyl]phenyl}-
Hx~,
O H2
1,6-dihydropyrazolo[3,4-
a]indazole-3-carboxamide °°
I
H
1-{4-
[(methylamino)sulfonyl]phenyl}- o
1,6-dihydropyrazolo[3,4-
e]indazole-3-carboxamide ~ ~ HZ
H
CA 02476665 2004-08-18
WO 03/070706 PCT/US03/04844
TABLE 2 cont
1-{4- /
~~ io
[(dimethylamino)sulfonyl]phenyl}- o S i I HZ
1,6-dihydropyrazolo[3,4- ~ N
_°
a]indazole-3-carboxamide
N
~H
Ethyl 1-phenyl-1,6-
dihydropyrazolo[3,4-a]indazole-3-
carboxylate ~ ~ °
0
N~
H
4-(4-Sulfamoyl-phenyl)-4,7- H2N~ ~°
°
dihydro-1 H-cyclopental[ 1,2-c;3,4-
c']dipyrazole-6-carboxylic acid
ethyl ester
IO
H
BIOLOGICAL EVALUATION
Materials
[0094] SAMZ ~ 96 Biotin capture plates were from Promega. Anti-FLAG
affinity resin, FLAG-peptide, NP-40 (Nonidet P-40), BSA, ATP, ADP, AMP, LPS
(E. coli serotype 0111:B4), and dithiothreitol were obtained from Sigma
Chemicals.
Antibodies specific for NEMO (IKK~y) (FL-419), IKKl (H-744), IKK2(H-470) and
IxBoc(C-21) were purchased from Santa Cruz Biotechnology. Ni-NTA resin was
purchased from Qiagen. Peptides were purchased from American Peptide
Company. Protease inhibitor cocktail tablets were from Boehringer Mannheim.
Sephacryl S-300 column was from Pharmacia LKB Biotechnology. Centriprep-10
concentrators with a molecular weight cutoff of 10 kDa and membranes with
7i
CA 02476665 2004-08-18
WO 03/070706 PCT/US03/04844
molecular weight cut-off of 30 kDa were obtained from Amicon. [Y'-33P] ATP
(2500 Ci/mmol) and [Y'-3aP] ATP (6000 Ci/mmol) were purchased from Amersham.
The other reagents used were of the highest grade commercially available.
Cloning and Expression
[0095] cDNAs of human IKKl and IKK2 were amplified by reverse
transcriptase-polymerase chain reaction from human placental RNA (Clonetech).
hIKKl was subcloned into pFastBac HTa (Life Technologies) and expressed as N-
terminal His6-tagged fusion protein. The hIKK2 cDNA was amplified using a
reverse oligonucleotide primer which incorporated the peptide sequence for a
FLAG-epitope tag at the C-terminus of the IKK2 coding region (DYKDDDDKD).
The hIKK2:FLAG cDNA was subcloned into the baculovirus vector pFastBac. The
rhIKK2 (S177S, E177E) mutant was constructed in the same vector used for wild
type rhIKK2 using a QuikChangeTM mutagenesis kit (Stratagene). Viral stocks of
each construct were used to infect insect cells grown in 40L suspension
culture. The
cells were lysed at a time that maximal expression and rhIKK activity were
demonstrated. Cell lysates were stored at -80 °C until purification of
the
recombinant proteins was undertaken as described below.
Enzyj~ze Isolation
[0096] All purification procedures were carried out at 4 °C unless
otherwise
noted. Buffers used are: buffer A: 20 mM Tris-HCI, pH 7.6, containing 50 mM
NaCI, 20 mM NaF, 20 mM (3-Glycerophosphate, 500 uM sodiumortho-vanadate,
2.5 mM metabisulfite, 5 mM benzamidine, 1 mM EDTA, 0.5 mM EGTA, 10%
glycerol, 1 mM DTT, 1X CompleteTM protease inhibitors; buffer B: same as
buffer
A, except 150 mM NaCI, and buffer C: same as buffer A, except 500 mM NaCI.
Isolation of rhll~K1 hornodirner
[0097] Cells from an 8 liter fermentation of baculovirus-expressed IKKl tagged
with His peptide were centrifuged and the cell pellet (MOI 0.1, I=72 hr) was
re-
suspended in 100 ml of buffer C. The cells were microfluidized and centrifuged
at
100,000 X g for 45 min. The supernatant was collected, imidazole added to the
72
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WO 03/070706 PCT/US03/04844
final concentration of 10 mM and incubated with 25 ml of Ni-NTA resin for 2
hrs.
The suspension was poured into a 25 ml column and washed with 250 ml of buffer
C and then with 125 rnl of 50 mM imidazole in buffer C. rhIKK1 homodimer was
eluted using 300 mM imidazole in buffer C. BSA and NP-40 were added to the
enzyme fractions to the final concentration of 0.1 %. The enzyme was dialyzed
against buffer B, aliquoted and stored at -80 °C.
Isolation of rlalKK2 homodimer
[0098] A 10 liter culture of baculovirus-expressing IKK2 tagged with FLAG
peptide was centrifuged and the cell pellet (MOI=0.1 and I=72 hrs) was re-
suspended in buffer A. These cells were microfluidized, and centrifuged at
100,000
X g for 45 min. Supernatant was passed over a G-25 column equilibrated with
Buffer A. Protein peak was collected and incubated with anti-FLAG affinity
resin
on a rotator overnight in buffer B. The resin was washed in batch with 10-15
bed
volumes of buffer C. Washed resin was poured into a column and rhIKK2
homodimer was eluted using 5 bed volumes of buffer B containing FLAG peptide.
5 mM DTT, 0.1 % NP-40 and BSA (concentrated to 0.1 % in final amount) was
added to the eluted enzyme before concentrating in using an Amicon membrane
with a molecular weight cut-off of 30 kDa. Enzyme was aliquoted and stored at -
80
°C.
Isolatiozz of rhIKKllIKK2 heterodimer
[0099] The heterodimer enzyme was produced by coinfection in a baculovirus
system (FLAG IKK2/IKKl His; MOI=0.1 and I=72 hrs). Infected cells were
centrifuged and the cell pellet (10.0 g) was suspended in 50 ml of buffer A.
The
protein suspension was microfluidized and centrifuged at 100,000 X g for 45
min.
Imidazole was added to the supernatant to a final concentration of 10 mM. The
protein was allowed to bind 25 ml of Ni-NTA resin by mixing for 2 hrs. The
protein-resin slurry was poured into a 25 ml column and washed with 250 ml of
buffer A containing 10 mM imidazole followed by 125 ml of buffer A containing
50 mM imidazole. Buffer A, containing 300 mM imidazole, was then used to elute
the protein. A 75 ml pool was collected and NP-40 was added to a final
73
CA 02476665 2004-08-18
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concentration of 0.1 %. The protein solution was then dialyzed against buffer
B.
The dialyzed heterodimer enzyme was then allowed to bind to 25 ml of anti-FLAG
M2 agarose affinity gel overnight with constant mixing. The protein-resin
slurry
was then centrifuged for 5 min at 2,000 rpm. ,The supernatant was collected
and the
resin re-suspended in 100 ml of buffer C containing 0.1 % NP-40. The resin was
washed with 375 ml of buffer C containing 0.1 % NP-40. The protein-resin was
poured into a 25 ml column and the enzyme eluted using buffer B containing
FLAG
peptide. Enzyme fractions (100 ml) were collected and concentrated to 20 ml
using
an Amicon membrane with molecular weight ~ut-off of 30 kDa. Bovine serum
albumin was added to the concentrated enzyme to final concentration of 0.1 %.
The
enzyme was then aliquoted and stored at -80 °C.
Cell Culture
[00100] The wild type (wt) human pre-B cell line, 702/3, and its mutant,
1.3E2,
were generously provided by Dr. Carol Sibley. Wt 70213 and 1.3E2 cells were
grown in RPMI 1640 (Gibco) supplemented with 7 % defined bovine serum
(Hyclone) and 50 ~.M 2-mercaptoethanol. Human monocytic leukemia THP-1 cells,
obtained from ATCC, were cultured in RPMI 1640 supplemented with 10% defined
bovine serum, 10 mM HEPES, 1.0 mM sodium pyruvate and 50 ~M 2-
mercaptoethanol. For experiments, cells were plated in 6 well plates at 1x106
cells/ml in fresh media. Pre-B cells were stimulated by the addition of 10
~.g/ml
LPS for varying lengths of time ranging from 0-4 hr. THP-1 cells were
stimulated
by the addition of 1 ~.g/ml LPS for 45 minutes. Cells were pelleted, washed
with
cold 50 mM sodium phosphate buffer, pH 7.4 containing 0.15 M NaCl and lysed at
4 °C in 20 mM Hepes buffer, pH 7.6 containing 50 mM NaCI, 1 mM EDTA, 1
mM
EGTA, 1 mM sodium orthovanadate, 10 mM (3-glycerophosphate, 1 mM NaF, 1
mM PMSF, 1 mM DTT and 0.5 % NP40 (lysis buffer). The cytosolic fractions
obtained following centrifugation at 10,000 X g were stored at -80° C
until used.
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hnmunoprecipitation and Western Blotting
[00101] SF9 cells paste containing rhIKKs were centrifuged ( 100,000 X g, 10
min) to remove debris. rhIKKs were immunoprecipitated (100 ~,g of cell paste)
from the cell supernatant using 3 ~.g of anti-NEMO antibody ( FL-419),
followed by
coupling to protein A sepharose beads. rhIKKs were also immunoprecipitated
from
affinity chromatography purified protein preparations (1 ~,g) using anti-FLAG,
anti-
His or anti-NEMO antibodies (1-4 ~,g) followed by protein A sepharose
coupling.
The native, human IKK complex was immunoprecipitated from THP-1 cell
homogenates (300 ~,g/condition) using the anti-NEMO antibody. Immune
complexes were pelleted and washed 3 times with 1 ml cold lysis buffer.
Immunoprecipitated rhIKKs were chromatographed by SDS-PAGE (8% Tris-
glycine) and transferred to nitrocellulose membranes (Novex) and detected by
chemiluminescense (SuperSignal) using specific anti-IKK antibodies (IKK2 H-
470,
IKKl H-744). Native IKK2, I~Ba and NEMO proteins from cytosolic lysates (20-
80 ~,g) were separated by SDS-PAGE and visualized by chemiluminescense using
specific antibodies.
Phosphatase Treatment
[00102] Immunoprecipitated rhIKKs were washed 2 times in 50 mM Tris-HCI,
pH 8.2 containing 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF and 2 mM MnCl2 and
resuspended in 50 ~,1. Phosphatase (~,PPase, 1000 LT) was pre-diluted in the
same
buffer and added to the IKK samples. Following an incubation at room
temperature
for 30 minutes with intermittent mixing, cold lysis buffer was added to the
tubes to
stop the reaction. After several washes, 10 % of the beads were removed for
Western analysis, and the remaining material was pelleted and resuspended in
.100
~,1 of the buffer used for the in vitro kinase assay.
IKI~e~SAM Enzyjne Assay
[00103] IKKoc kinase activity was measured using a biotinylated IKBoc peptide
(Gly-Leu-Lys-Lys-Glu-Arg-Leu-Leu-Asp-Asp-Arg-His-Asp-Ser32-Gly-Leu-Asp-
Ser36-Met-Lys-Asp-Glu-Glu), a SAMZ TM 96 Biotin capture plate, and a vacuum
CA 02476665 2004-08-18
WO 03/070706 PCT/US03/04844
system. The standard reaction mixture contained 5 ~.M biotinylated hcBoc
peptide, 1
~ [,~ 33P] ATP (about 1 X 105 cpm), 1 mM DTT, 50 mM KCI, 2 mM MgCl2, 2
mM MnCl2, 10 mM NaF, 25 mM Hepes buffer, pH. 7.6 and enzyme solution (1-10
~,1) in a final volume of 50 ~.1. After incubation at 25 °C for 30 min,
25 ~.l of the
reaction mixture was withdrawn and added to a SAM2 TM 96 Biotin capture 96-
well
plate. Each well was then washed successively with 800 ~,1 2 M NaCI, 1.2 ml of
NaCI containing 1% H3P04, 400 ~,l H20, and 200 ~,1 95% ethanol. The plate was
allowed to dry in a hood at 25 °C for 1 hr and then 25 ~.l of
scintillation fluid
(Microscint 20) was added to each well. Incorporation of ['y 33P] ATP was
measured using a Top-Count NXT (Packard). Under each assay condition, the
degree of phosphorylation of IXBoc peptide substrate was linear with time and
concentration for all purified enzymes. Results from the biotinylated peptide
assay
were confirmed by SDS-PAGE analysis of kinase reaction utilizing a GST-
IycBal_s4
and ['y 3'P] ATP. The resulting radiolabeled substrate was quantitated by
Phosphoimager (Molecular Dynamics). An ion exchange resin assay was also
employed using [y 33P] ATP and GST-I~Bocl_s4 fusion protein as the substrates.
Each assay system yielded consistent results in regard to Km and specific
activities
for each of the purified kinase isoforms. One unit of enzyme activity was
defined as
the amount required to catalyze the transfer of 1 nmole of phosphate from ATP
to
IKBoc peptide per min. Specific activity was expressed as units per mg of
protein.
For experiments related to Km determination of purified enzymes, various
concentrations of ATP or I~Ba peptide were used in the assay at either a fixed
I7cBa
or ATP concentration. For IKBoc peptide Km, assays were carried out with 0.1
~g of
enzyme, 5 p.M ATP and I7cBa peptide from 0.5 to 20 ~M. For ATP Km, assays
were carried out with 0.1 ~,g of enzyme, 10 ~.M I~Ba peptide and ATP from 0.1
to
10 p,M. For Km determination of rhIKKl homodimer, due to its low activity and
higher Km for It~Boc peptide, rhIKKl homodimer (0.3 ~,g) was assayed with 125
~,M
IxBoc peptide and a 5-fold higher specific activity of ATP (from 0.1 to 10
p.M) for
ATP Km experiments and a 5-fold higher specific activity of 5 ~,M ATP and
I~Boc
peptide (from 5 to 200 p.M) for IKBa peptide Km experiments.
76
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WO 03/070706 PCT/US03/04844
IKK,l3Resirz Erzzyme Assay
[00104] IKK(3 kinase activity was measured using a biotinylated I~Boc peptide
(Gly-Leu-Lys-Lys-Glu-Arg-Leu-Leu-Asp-Asp-Arg-His-Asp-Ser32-Gly-Leu-Asp-
Ser36-Met-Lys-Asp-Glu-Glu) (American Peptide Co.). 20 ul of the standard
reaction mixture contained 5 ~.M biotinylated IxBoc peptide, 0.1 ~,Ci/reaction
['y 33P]
ATP (Amersham) (about 1 X 10~ cpm), 1 ~,M ATP (Sigma), 1 mM DTT (Sigma), 2
mM MgCl2 (Sigma), 2 mM MnCl2 (Sigma), 10 mM NaF (Sigma), 25 mM Hepes
(Sigma) buffer, pH 7.6 and 20 ~,l enzyme solution and 10 ul inhibitor in a
final
volume of 50 ~.1. After incubation at 25 °C for 30 min, 150 ~,l resin
(Dowex anion-
exchange resin AG1X8 200-400 mesh) in 900 mM formate, pH 3.0 was added to
each well to stop the reaction. Resin was allowed to settle for one hour and
50 ul of
supernatant was removed to a Micolite-2 flat bottom plate (Dynex). 150 ~.1 of
scintillation fluid (Microscint 40) (Packard) was added to each well.
Incorporation
of ['y 33P] ATP was measured using a Top-Count NXT (Packard).
II~I~ lzeterodimer Resin Enzyme Assay
[00105] IKK heterodimer kinase activity was measured using a biotinylated IKBa
peptide (Gly-Leu-Lys-Lys-Glu-Arg-Leu-Leu-Asp-Asp-Arg-His-Asp-Ser32-Gly-Leu-
Asp-Ser36-Met-Lys-Asp-Glu-Glu) (American Peptide Co.). 20 ul of the standard
reaction mixture contained 5 ~.M biotinylated hcBoc peptide, 0.1 ~.Ci/reaction
['y 33P]
ATP (Amersham) (about 1 X 105 cpm), 1 ~,M ATP (Sigma), 1 mM DTT (Sigma), 2
mM MgCl2 (Sigma), 2 mM MnCl2 (Sigma), 10 mM NaF (Sigma), 25 mM Hepes
(Sigma) buffer, pH 7.6 and 20 ~,l enzyme solution and 10 pl inhibitor in a
final
volume of 50 ~,1. After incubation at 25 °C for 30 min, 150 ~.1 resin
(Dowex anion-
exchange resin AG1X8 200-400 mesh) in 900 mM formate, pH 3.0 was added to
each well to stop the reaction. Resin was allowed to settle for one hour and
50 ul of
supernatant was removed to a Micolite-2 flat bottom plate (Dynex). 150 ~.1 of
scintillation fluid (Microscint 40) (Packard) was added to each well.
Incorporation
of ['y 33P] ATP was measured using a Top-Count NXT (Packard).
77
