Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02464924 2004-04-27
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HETEROAROMATIC CARBOXAMIDE DERIVATIVES FOR THE
TREATMENT OF INFLAMMATION
The present application claims priority under Title 35, United States Code, ~
I 19
to United States Provisional application Serial No. 60/340,816 filed October
30,
2001, which is incorporated by reference in its entirety as if written herein.
FIELD OF THE INVENTION
[001] The present invention in general is in the field of anti-inflammatory
pharmaceutical agents and specifically relates to heteroaromatic carboxamide
derivatives, compositions comprising such, and methods 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-xB 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) A»nu. Rev. Im»aurzol. 16, 115-260; Zandi,
E., and Karin, M. (1999) Mol. Cell. Biol. 19, 4547-4551; Karin, M. (1999) J.
Biol. Chem. 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 (IL.I (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
1
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genes. NF-~cB 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) Annu.
Rev. Irnmunol.16, 115-260; Zandi, E., and Karin, M. (1999) Mol. Cell. Biol.19,
4547-4551; Karin, M. (1999) J. Biol. Chem. 274, 27339-27342). IxBs 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 I2cBs, thereby releasing NF-xB for nuclear translocation (Ghosh
S., May, M. J., and Kopp. E (1998) Annu. Rev. Immunal. 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 IxB kinases (IKKl or IKKa and
IKK2 or IKK(3), which phosphorylate IxBs 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. Immunol. 16, 115-260;
Zandi, E., and Karin, M. (1999) Mol. Cell. Biol. 19, 4547-4551; Karin, M.
(1999) J. Biol. Chenz. 274, 27339-27342). The catalytic subunits, IKKl 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. andRothe, 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) Scierzce 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.,
2
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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, D.M., Zandi, E. and Karin, M. (1997}
Nature 388, S48-554; 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-xB 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) Anrzu.
Rev. Immurzol. 16, 115-260; Zandi, E., and Karin, M. (1999) Mol. Cell. Biol.
19,
4547-4551; Karin, M. (1999) J. Biol. Claenz. 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 a83; 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 IrcB kinase activity from TNFa stimulated HeLa S3 cells and by its
interaction with the MAP3K, NF-xB inducing kinase (NIK), in a yeast two-
hybrid screen. IKK1 was identified as the previously cloned serine-threonine
kinase, CHUK (Connelly, M. and Marcu, K. (1995) Cell. Mol. Biol. Res. 41,
537-549). IKK1 (also termed IKKa) is an 85 kDa, 745 amino acid protein that
contains an N-terminal serine/threonine kinase catalytic domain, a leucine
3
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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
IKKI (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). IKK2 is an 87
IO kDa, 756 amino acid protein with the same over aII topology as IKKl except
for
the addition of an 11 amino acid extension at the C-terminus. IKKl and IKK2
are S2% 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. (2998) J.
Biol. Chem. 273, 30736-30741). Finally, NEMO (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) Natuoe 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, I526-1538).
[005] The kinase activities of IKKI and IKK2 axe regulated by
4
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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 385, 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) Sciefzce 278, 866-869; Debase, 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. Clzem. 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 (Debase, M.,
Hayakawa, M., Chen, Y., and Karin, M. (1999) Science 284, 309-313).
[006] 1KK2 demonstrates a more potent kinase activity compared to IKKl
using IxBa or hcB(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; Debase, M., Hayakawa, M., Chen, Y., and Karin, M. (1999)
Scie~ace 284, 309-313). Mutations of the phospho-acceptor serine residues
within the MAPKK activation loop alters IKK2 kinase activity; the serine to
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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) Scietzce 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 stimulated
with cytokines nor do they activate NF-~cB. Furthermore, the phenotypes of
each
IKK knock out is unique with IKK1 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)
Sciezzce
284, 32I-325; Takeda, K., Tekeuchi, O., Tsujimura, T., Itarni, 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, 1322-1328; Tanaka, M., Fuentes, M. E.,
Yamaguchi, K., Durnin, M. H., Dalrymple, S. A., Hardy, K. L., and Goeddel, D.
V. (1999) Immunity 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
[008] NF-xB in inflammatory disorders is further strengthened by studies
of airway inflammation including asthma in which NF-xB has been shown to be
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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 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-~cB 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-~cB 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 (I996).
[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, Trerzds
Genet7:318-322, 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°10
of certain
human lymphoid tumors. In addition, a role for NF-~cB in the regulation of
apoptosis, cell cycle progression, invasion, and metastasis has been reported
(Bours V. et al., Biochemical Plzezrmac~logy 60:1085-1090, 2000) strengthening
the role of this transcription factor in the control of cell proliferation.
The
inhibition of NF-~cB has been shown to potentiate TNF- and cancer therapy
through increased apoptosis (Wang C-Y et al., Scienee 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), IKKa and IKK(3 are
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expressed constitutively, which normally function in a transient manner (Chu Z-
L et al., J of Biological Claenaistry 273:15891-15894, 1998). The HTLV 1
transforming and transactivating protein (Tax) has been shown to bind MEKKl
and increases the activity of IKK[3 to enhance phosphorylation of serine
residues
in IxBoc that lead to its degradation.
[0010] WO 98/02430 and EP 853 083 disclose various 4-pyridyl
derivatives, and EP 908 456 discloses various 3-pyrazolyl derivatives.
[0011] DE 19725450 discloses various 3-pyridinyl and 5-pyrimidyl
derivatives.
[0012] WO 99/46244, WO 9854166, and EP 202 538 disclose a series of
substituted thienyl compounds said to possess biological activity.
[OOI3] WO 01158890 discloses a series of heteroaromatic carboxamide
derivatives, which allegedly are inhibitors of IKK-2.
DETAILED DESCRIPTION OF THE INVENTION
[0014] A class of compounds, which are useful in treating cancer,
inflammation, and inflammation related disorders, is defined by Formula I:
NH2
wherein
O
8
n~ mu
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A is a 5-membered heteroaromatic ring containing one or two
heteroatoms independently selected from oxygen, nitrogen, or sulfur;
Ri is selected from the group consisting of: hydrogen, halogen, cyano,
nitro, -N(R3)2, -CON(R3)2, -COORS, -NR3COR3, S(O)mR3, -SOZN(R3)2,
-NR3SOZR3, alkyl, trifluoromethyl, trifluoromethoxy, alkenyl, alkynyl,
alkoxy, alkanoyl, substituted or unsubstituted aryl, and a substituted or
unsubstituted 5- to 7-membered heteroaromatic ring containing one to
three heteroatoms independently selected from oxygen, nitrogen, or
sulfur, wherein said substituent(s) are independently selected from the
group consisting of: halogen, cyano, nitro, -N(R4)2, -CON(R4)2, -
COOR4, -NR4COR4, S(O),r,R4, 'S02N(R4~2, -NR4SOZR4, alkyl,
trifluoromethyl, trifluoromethoxy, alkenyl, alkynyl, alkoxy, alkanoyl,
aminoalkyl, and aryl;
RZ is selected from the group consisting of: substituted or unsubstituted
aryl, and a 5- to 7-membered substituted or unsubstituted heteroaromatic
ring containing one to three heteroatoms independently selected from
oxygen, nitrogen, or sulfur, wherein said substituent(s) are
independently selected from the group consisting of: halogen, cyano,
vitro, -N(R4)2, -CON(R4)2, -COOR4, -NR4COR4, S(O)mR4, -SOzN(R4)2,
-NR4S02R4, alkyl, trifluoromethyl, trifluoromethoxy, alkenyl, alkynyl,
alkoxy, alkanoyl, aminoalkyl, and aryl;
RI and R2 can optionally be taken together form a 5 or 6 membered
saturated or unsaturated ring optionally substituted with one or more
substituent selected from the group consisting of: halogen, cyano, vitro,
-N(R3)2, -CON(R3)2, -COORS, -NR3COR3, S(O)n,R3, -SO2N(R3)z, -
NR3SOZR3, alkyl, trifluoromethyl, trifluoromethoxy, alkenyl, alkynyl,
alkoxy, alkanoyl, substituted or unsubstituted aryl, and a substituted or
unsubstituted 5- to 7-membered heteroaromatic ring containing one to
three heteroatoms independently selected from oxygen, nitrogen, or
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
sulfur, wherein said substituent(s) are independently selected from the
group consisting of: halogen, cyano, nitro, -N(R4)Z, -CON(R4)~, -
COOR4, -NR'~COR4, S(O)n,R4, -S02N(R'~)Z, -NR4S02R4, alkyl,
trifluoromethyl, trifluoromethoxy, alkenyl, alkynyl, alkoxy, alkanoyl,
aminoalkyl, and aryl;
15
R3 is selected from the group consisting of: hydrogen or alkyl;
R4 is selected from the group consisting of: hydrogen or alkyl;
m is an integer 0, 1, or 2; and
isomers, tautomers, carriers, prodrugs, pharmaceutically acceptable salts
thereof.
[0015] Another class of compounds is defined by formula II
NH2
O
wherein
Rl is selected from the group consisting of: hydrogen, halogen, cyano,
nitro, -N(R3)~, -CON(R3)?, -COORS, -NR3COR3, S(O)mR3, 'S021~F(R3)2,
-NR3S02R3, alkyl, trifluoromethyl, trifluoromethoxy, alkenyl, alkynyl,
alkoxy, alkanoyl, substituted or unsubstituted aryl, and a substituted or
unsubstituted 5- to 7-membered heteroaromatic ring containing one to
three heteroatoms independently selected from oxygen, nitrogen, or
l0
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
sulfur, Wherein said substituent(s) are independently selected from the
group consisting of: halogen, cyano, nitro, -N(R~)2, -CON(R4)a, -
COOR4, -NR4COR4, S(O)mR4, -SOZN(R4)Z, -NR4S02Ra, alkyl,
trifluoromethyl, trifluoromethoxy, alkenyl, alkynyl, alkoxy, alkanoyl,
aminoalkyl, and aryl;
R2 is selected from the group consisting of: substituted or unsubstituted
aryl, and a S- to 7-membered substituted or unsubstituted heteroaromatic
ring containing one to three heteroatoms independently selected from
oxygen, nitrogen, or sulfur, wherein said substituent(s) are
independently selected from the group consisting of: halogen, cyano,
nitro, -N(R4)2, -CON(R4)Z, -COORS, -NR4COR4, S(O)mR4, -S02N(R4)2,
-NR4S02R4, alkyl, trifluoromethyl, trifluoromethoxy, alkenyl, alkynyl,
alkoxy, alkanoyl, aminoalkyl, and aryl;
Rt and RZ can optionally be taken together form a 5 or 6 membered
saturated or unsaturated ring optionally substituted with one or more
substituent selected from the group consisting of: halogen, cyano, nitro,
-N(R3)2, -CON(R3)4, -COORS, -NR3COR3, S(O)n,R3, -SO~1V(R3)?, -
NR3SOZR3, alkyl, trifluoromethyl, trifluoromethoxy, alkenyl, alkynyl,
alkoxy, alkanoyl, substituted or unsubstituted aryl, and a substituted or
unsubstituted 5- to 7-membered heteroaromatic ring containing one to
three heteroatoms independently selected from oxygen, nitrogen, or
sulfur, wherein said substituent(s) are independently selected from the
group consisting of: halogen, cyano, nitro, -N(R4)2, -CON(R4)2, -
COOR4, -NR4COR'~, S(O)n,R4, -SO2N(R4)2, -NR4SO~R'~, alkyl,
trifluoromethyl, trifluoromethoxy, alkenyl, alkynyl, alkoxy, alkanoyl,
aminoalkyl, and aryl;
R3 is selected from the group consisting of: hydrogen or alkyl;
R4 is selected from the group consisting o~ hydrogen or alkyl;
11
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WO 03/037886 PCT/US02/34801
m is an integer 0, 1, or 2; and
isomers, tautomers, carriers, prodrugs, pharmaceutically acceptable salts
thereof.
Definitions
[0016] The present invention includes the use of all hydrates, solvates,
complexes and prodrugs of the compounds of this invention. Prodrugs are any
covalently banded compounds, which release the active paxent 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 enantiomerically 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 traps (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.
[0017] 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.
[0018] The term "alkyl" is used, either alone or within other terms such as
"haloalkyl" and "alkylsulfonyl"; it embraces linear or branched radicals
having
one to about twenty carbon atoms or, preferably, ape 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
12
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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 oxygen. Examples of saturated heterocyclic
radicals include pyrrolidyl and morpholinyl. The term "heteroaryl" embraces
13
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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 -
SOZ-. "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 (-
S02 NHZ). The terms "N-alkylsulfarnyl" 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 -CO2H.
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 "alkoxycarbonylalkyl" radicals include (CH3)3COC(=O)
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(CHZ)2- and -(CHZ)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)-
NHZ radical. The term "cyanoamidino" denotes an -C(=N-CN)-NHZ 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 terns "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
CA 02464924 2004-04-27
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alkyl radicals, respectively. The term "acyl", whether used 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 examples of an "acylamino"
radical is acetylamino (CH3C(=O)-NH-).
[0019] Compounds of Formula I and 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 and 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 and 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 and
II also would be useful to treat gastrointestinal conditions such as
inflammatory
bowel disease, Crohn's disease, gastritis, irntable bowel syndrome, and
ulcerative colitis and for the prevention of colorectal cancer. Compounds of
Formula I and II would be useful in treating inflammation in such diseases as
vascular diseases, 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 are useful as
antiinflammatory agents, such as for the treatment of arthritis, with the
additional benefit of having significantly less harmful side effects. The
compounds of formula I or II are useful as agents for treating cancer or as an
anticancer agents. The compounds of formula I or II may be proapoptotic,
antiapoptotic, anticell cycle progressive, antiinvasive, and antimetastatic.
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,
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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 II 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 IKKl and/or IKK2, IKKaIIKK(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-l,
STLK-2, DDR-2, Aurora 1, Aurora 2, Bub-l, PLK, Chkl, Chk2, HER2, rafl,
MEKI, MAPK, EGF-R, PDGF-R, FGF-R, IGF-R, VEGF-R, PI3K, weel kinase,
Src, Abl, Akt, ILK, MK-2, IKK-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
IKK1. Preferably, the compounds have an IKK2 IC50 of less than 1 ~M, and
have a selectivity ratio of IKK2 inhibition over IKK1 inhibition of at least
50,
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and more preferably of at least 100. Even more preferably, the compounds have
an IKI~1 IC50 of greater than 10 ~.M, and more preferably of greater than 100
p,M. 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.
[0020] 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
selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,
carboxylic and sulfonic classes of organic acids, examples of which are
formic,
acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric,
citric,
ascorbic, glucuronic, malefic, fumaric, pyruvic, aspartic, glutamic, benzoic,
anthranilic, mesylic, salicyclic, salicyclic, phydroxybenzoic, phenylacetic,
mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic,
pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, stearic,
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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.
[0021] 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 compounds of the present
invention prepared as herein before described may be formulated as solutions
or
lyophilized powders for 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 pharmaceutical
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,
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
n
solution, standard 5% dextrose in water or buffered sodium or ammonium
acetate solution may be used as a suitable carrier. Such formulation is
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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, andlor 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 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
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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.
[0022] 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 Garner 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
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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.
[0023] 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.
[0024] 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 hydrocarbons such as hard, soft or liquid
paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural
origin
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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
[0025] 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).
[0026] The compounds of this invention may be prepared by employing
reactions as shown in the schemes below, in addition to other standard
manipulations as are known in the literature or exemplified in the
experimental
procedures. These schemes, therefore, are not limited by the compounds listed
or by any particular substituents employed for illustrative purposes.
[0027] Synthetic scheme I shows the preparation of thiophenes from starting
material 1. In step 1 of synthetic scheme I, a methyl ketone is treated with
phosphorous trichloride in DMF at a reduced temperature followed by
hydroxylamine hydrochloride to give a chloroacrylonitrile compound. In step 2
of scheme I, the chloroacrylonitrile is dissolved in a solvent such as an
alcohol
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and treated with 2-mercaptoacetamide followed by a base such as sodium
methoxide.
Scheme I
o Cl
Rl a ~ -, b ,~ R~ CN
1 Rz 2 Rz
NH2 Rz NHz
HS~
3 0 ~ ~NHz
R1 SS
c O
a) PCl3, DMF; b) NH20H~HCl; c) NaOCH3/CH~OH
[0028 Synthetic scheme II shows the preparation of amino amide
thiophenes from the corresponding ester. In step 1 of synthetic scheme II , an
ester is converted to a carbazide with hydrazine. In step 2, the hydrazide is
converted to the amide by reducing with Raney nickel.
Scheme II
Ra NHz
a
~OR ---
z
S Rl S
0 5 O
b R NHz
~~2
Ri S/ ~'(\
O
a) N2H4; b) raney nickel
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[0029] Synthetic scheme III shows the preparation of amino amide
thiophenes from the corresponding ester. In step 1 of synthetic scheme DI , an
ester is converted to a carbazide with hydrazine. In step 2, the hydrazide is
converted to the amide by reducing with Raney nickel.
Scheme III
a ~a
/ ~oR ---, / \ . r~2
s s \\
4 O 5 0
NHS
/\
s
0
a) N2Hq.; b~ raney nickel
Once the thiophene nucleus has been established, further compounds may be
prepared by applying standard techniques for functional group.
interconversion,
well known in the art.
[0030] 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.
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Compounds were named using ACD/name software. ACD/name generates
IUPAC names rather than CAS names and is not guaranteed to produce the
correct IUPAC name in all cases.
EXAMPLES
[0031] Example 1
NH2
NH2
\O
3-amino-5-(3-methoxyphenyl)thiophene-2-carboxamide
[0032] Step l: Preparation of 3-chloro-3-(3-methoxyphenyl)prop-2-
enenitrile.
A solution of 3'-methoxyacetophenone (10 g, 66.6 mmol) in
dimethylformamide (200 ml) under nitrogen was chilled in an ice-water bath.
To this solution was added phosphorus trichloride ( 11.6 mL, 1~.3 g, 133 mmol)
drop wise. The ice-water bath was removed, and the resulting reaction mixture
was stirred under nitrogen for 2 hours. Hydroxylamine hydrochloride (13.9 g,
200 mmol) was added in small portions resulting in an exothermic reaction.
The reaction mixture was stirred at room temperature overnight. Saturated
aqueous I~aHC03 was added to the reaction mixture, and it was extracted with
ethyl acetate 3 X. The combined organic extracts were washed with brine, dried
over MgS04, filtered, and concentrate in vacuo. This material was used without
further purification.
[0033] Step 2. Preparation of 3-amino-5-(3-methoxyphenyl)thiophene-2-
carboxamide.
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2-mercaptoacetamide (18.2 g, 200 mmol) was added to a solution of the
material from step 1 in methanol (100 mL) under nitrogen. To this mixture was
added a solution of sodium methoxide (7.I9 g, 133 mmol) in methanol (200
mL) drop wise. The resulting reaction mixture was stirred at room temperature
S for 5 houxs. Saturated aqueous NaHC03 was added followed by 1 N HCl until
gas evolution was observed. Water was added and the mixture was extracted 3x
with ethyl acetate, dried over MgS04, and filtered. The filtrate was
concentrated in vacuo, recrystallized from ethyl acetate/ hexane to yield a
white
solid (3 g). This material was taken up in methanol and 1 eq. of sodium
methoxide in methanol was added. The resulting reaction mixture was stirred at
room temperature for 2 hours. The mixture was concentrated. Water and ethyl
acetate were added, and the layers were separated. The organic phase was dried
over MgS04, filtered, and concentrated in vacuo. The product was purified by
chromatography on a silica gel column eluting with 70% ethyl acetate to
hexane. The fractions containing the desired product were combined,
' concentrated, and recrystallized from ethyl acetate/ hexane to afford the
title
compound as a yellowlorange solid. (1.28 g, 7.7 %): mp 113.8-116.6 °C.
1H
NMR (CDCl3/300 MHz) 7.39-7.30 (m, 3H), 7.15 (d, 1H), 7.10 (t, 1H), 6.95-
6.85 (m, 1H), 6.79 (s, 1H), 5.21 (brs, 2H), 3.85 (s, 3H). ES+LRMS m/z 249
(M+H). ES+HRMS m/z 249.0679 (M+H, C12H1aNa0aS requires 294.0697).
Anal Calcd. for C12HI~N20~S: C, 58.05; H, 4.87; N, 11.28. Found: C, 57.85; H,
4.98; N, 11.13.
[0034] Example 2
NH2
CI I ~ NN2
w ,S ~O
3-amino-5-(2-chlorophenyI)thiophene-2-carboxamide
[0035] Step 1. Preparation of 3-chloro-3-(2-chlorophenyl)prop-2-enenitrile.
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A solution of 2'-chloroacetophenone (5 g, 32.3 mmol) in DMF (100 mL) under
nitrogen was chilled in an ice-water bath. To this cool solution was added
drop
wise phosphorus trichloride (8,88 g, 64.7 mmol). Upon complete addition of
the PC13, the ice-water bath was removed, and the resulting reaction mixture
was stirred under nitrogen for 2 hours. Then hydroxylamine hydrochloride
{6.74 g, 97.0 mmol) was added in small portions resulting in an exothermic
reaction. The reaction mixture was stirred at room temperature overnight.
Ethyl acetate was added, and the layers were separated. The organic phase was
washed 3x with brine, dried aver MgS04, filtered, and concentrated in vacuo.
This material was used in the next reaction without further purification.
[0036] Step 2. Preparation of 3-amino-5-(2-chlorophenyl)thiophene-2-
carboxamide.
2-mercaptoacetamide (8.83 g, 97.0 mmol) was added to a solution of the
material from step 1 in methanol (50 mL) under nitrogen. To this mixture was
added sodium methoxide in methanol (0.5 M, 388 mL, 194 mmol) drop wise.
The reaction mixture was stirred at room temperature for 72 h. The reaction
was concentrated in vacuo and water was added. A solid was filtered and
washed with water several times to afford the title compound as solid. (2.24
g,
27 %): mp 129.6-129.7 °C. 1H NMR (CDC13/300 MHz) 7.58-7.45 (m, ZH),
7.38-7.22 (m, 5H), 6.88 (d, 2H). ES+LRMS mlz 253 (M+H). ES+HRMS mlz
253.0204 (M+H, C~ 1H~N~OSCI requires 253.0202). Anal Calcd. for
CI1H9N~OSC1 + HZO: C, 48.80; H, 4.10; N, 10.35. Found: C, 48.62; H, 3.85;
N, 10.29.
[0037] Example 3
NH2
NH2
NC I ~ S
/ C
3-amino-5-(3-cyanophenyl)thiophene-2-carboxamide
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[0038] Step 1. Prepared by using the procedures found in example 2, steel.
[0039] Step 2. Preparation of 3-amino-5-(3-cyanophenyl)thiophene-2-
carboxamide.
2-mercaptoacetamide (5.65 g, 62.1 mmol) was added to a solution of the
material from step 1 (20.7 mmol) in methanol (100 mL) under nitrogen. To this
mixture was added a solution of sodium methoxide in methanol (0.5M, 166 mL,
82.8 rnmol) drop wise. After stirnng at room temperature overnight, the
reaction mixture was concentrated in vacuo to half its volume, and water was
added. A solid was filtered, washed with water several times, and
recrystallized
from ethyl acetatelhexane to afford the title compound as a solid. (2.61 g, 52
%):
mp 193.2-193.5 °C. 1H NMR (CDC13/300 MHz) 7.78-7.66 (m, 2H), 7.55-7.46
(m, 1H), 7.46-7.36 (m, 1H), 6.78 (m, 1H), 3.84 (brs, 4H). ES+LRMS rnlz 244
(M+H). ES+HRMS rnlz 244.0538 (M+H, C12H9N30S requires 244.0545).
Anal. Calcd. for C12H9N3OS: C, 59.24; H, 3.73; N, 17.27. Found: C, 58.27; H,
3.92; N, 17.10.
[0040] Example 4
/ NH2
Cf S S ~
NH2
O
4-amino-5'-chloro-2,2'-bithiophene-5-carboxamide
The title compound was prepared using the procedures found in example 3: mp
185.6-188.3 °C. IH NMR (CDC13/300 MHz) 7.25 (s, 1H), 6.96 (d, 1H), 6.80
(d,
1H), 6.59 (s, 1H), 3.40 (brs, 3H). ES+LRMS rnlz 259 (M+H). ES+HRMS rnlz
258.9761 (M+H, C9H7N2OSZC1 requires 258.9767). Anal. Calcd. for
C~H7NZOSZC1: C, 41.78; H, 2.73; N,10.83. Found: C, 41.13; H, 2.87; N, 10.68.
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[0041 ] Example 5
4-amino-2',5'-dimethyl-2,3'-bithiophene-5-carbaxamide
The title compound was prepared using the procedures found in example 3: mp
151.4-152.3 °C. 1H NMR (CDC131300 MHz) 7.26 (s, 2H), 6.76 (s, 1H), 6.54
(s,
1H), 5.19 (brs, 2H), 2.52 (s, 3H), 2.42 (s, 3H). FAB+LRSM m/z 253 (M+H}.
ES+HRMS m~z 253.0462 (M+H, C~IHIZN20S2 requires 253.0469). Anal.
Calcd. for CllHrzNzOSz: C, 52.36; H, 4.79; N, 11.10. Found: C, 52.12; H, 4.97;
N, 11.01.
[0042] Example 6
NH2
NH2
S~O
C! ~
S"GI
4-amino-2',5'-dichloro-2,3'-bithiophene-5-carboxamide
The title compound was prepared using the procedures found in example 3: mp
175.2-176.4 °C. ~H NMR (CD30D1300 MHz) 7.21 (s, 1H), 7.05 (s, 1H), 4.90
(brs, 4H). FAB+LRMS mlz 293 (M+H). ES+HRMS mlz 292.9392 (M+H,
C~H6NZOS2Clz requires 292.9397. Anal Calcd. for C9H6NZOSZCIz: C, 36.87; H,
2.06; N, 9.55. Found: C, 36.53; H, 2.15; N, 9.36.
[0043] Example 7
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NH2
NH2
02N I ~ S
O
3-amino-5-(3-nitrophenyl)thiophene-2-carboxamide
The title compound was prepared using the procedures found in example 3: mp
210.5-210.9 °C. 1H NMR (DMSO-d~/300 MHz) 8.31 (t, 1H), 8.23-8.I4 (m,
1H), 8.08-7.98 (m, 1H), 7.79-7.68 (m, 1H), 7.15 (s, 1H), 7.02 (brs, 2H), 6.53
(brs, 2H). FAB+LRSM m/z 264 (M+H). ES+HRMS m/z 264.0456 (M+H,
C11Ti9N3O3S requires 264.0443).
[0044] Example 8
( NH2
g~NH2
I(O
3-amino-5-(2-phenanthryl)thiophene-2-carboxamide
The title compound was prepared using the procedures found in example 3: mp
>300 °C. 1H NMR (DMSO-d~1300 MHz) 8.96-8.77 (m, 2H), 8.21 (s, 1H), 8.06-
7.83 (m, 4H), 7:76-7.60 (m, 2H), ?.14 (s, 1H), 6.97 (brs, 2H), 6.53 (brs, 2H).
ES+LRMS m/z 319 (M+H). ES+HRMS fnlz 319.0909 (M+H, C19Hi4NaOS
requires 319.0905).
[0045] Example 9
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H2N NHZ
~Y ~~O
S
S
4-amino-5'-methyl-2,2'-bithiophene-5-carboxamide
The title compound was prepared using the procedures found in example 3: mp
205.6-206.4 °C. IH NMR (CD3CN/300 MHz) 7.10 (d, 1H), 6.76 (d, 1H), 6.68
(s, 1H), 5.91 (brs, 1H), 5.64 (brs, 2H), 2.49 (s, 3H), 2.20-2.00 (m, 1H).
ES+LRMS m/z 239 (M+H). ES+HRMS m/z 239.0329 (M+H, CloHioNzOS2
requires 239.0313).
. [0046] Examplel0
NH2
N02 ~ ~ NH2
S ~O
3-amino-5-(2-nitrophenyl)thiophene-2-carboxamide
The title compound was prepared using the procedures found in example 3: 1H
NMR (DMSO-db/300 MHz) 7.98-7.88 (m, 1H), 7.77-7.57 (m, 3H), 6.96 (s, 2H),
6.59 (brs, 1H), 6.50 (brs, 2H). ES+LRMS n~/z 264 (M+H). ES+HRMS m/z
264.0435 (M+H, C~ 1H9N3O3S requires 264.0443). Anal Calcd. for
C11H9N3O3S: C, 50.18; H, 3.45; N, 15.96. Found: C, 50.15; H 3.3&; N, 15.99.
[0047] Example 11
NN2
NH2
,S ~O
02N
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3-amino-5-(4-nitrophenyl)thiophene-2-carboxamide
The title compound was prepared using the procedures found in example 3: 1H
NMR (DMSO-d~1300 MHz) 8.28 (d, 2H), 7.84 (d, 2H), 7.15 (s, 1H), 7.05 (brs,
2H), 6.54 (brs, 2H). ES+LRMS rnlz 264 (M+H). ES+HRMS rnlz 264.0434
(M+H, CI1H9N3O3S requires 264.0443).
[0048] Example 12
NH2
NHZ
,S O
N
3-amino-5-pyridin-3-ylthiophene-2-carboxamide
The title compound was prepared using the procedures found in example 3: mp
207.3-207.5 °C. 1H NMR (DMSO-d61300 MHz) 8.80 (d, 1H), 8.53 (m, 1H),
8.03-7.88 (m, 1H), 7.55-7.37 (m, 1H), 7.13-6.84 (m, 3H), 6.51 (brs, 2H).
ES+LRMS m/z 220 (M+H). ES+HRMS m/z 220.0527 (M+H, CloH9N3OS
requires 220.0545).
[0049] Example 13
NH2
NH2
\ 'S O
N
3-amino-5-pyridin-4-ylthiophene-2-carboxamide
The title compound was prepared using the procedures found in example 3: mp
245.8-246.7 °C. 1H NMR (DMSO-d~/300 MHz) 8.62 (m, 2H), 7.55 (m, 2H),
7.19 (s, 1H), 7.07 (brs, 2H), 6.55 (brs, 2H). ES+LRMS m/z 220 (M+H).
ES+HRMS m/z 220.0547 (M+H, CIOH~N30S requires 220.0545). Anal Calcd.
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for C~oH~N30S: C, 54.78; H, 4.14; N, 19.16. Found: C, 54.26; H, 4.04; N,
19.24.
[0050] Example 14
NH2
~~--~~
S S NH2
O
4-amino-3'-methyl-2,2'-bithiophene-5-carboxamide
The title compound was prepared using the procedures found in example 3: mp
161.9-161.9 °C. 1H NMR (DMSO-461300 MHz) 7.47 (d, 1H), 7.09-6.82 (m,
3H), 6.71 (s, 1H), 6.48 (brs, 2H), 2.37 (s, 3H). ES+LRMS m/z 239 (M+H).
ES+HRMS m/z 239.0332 (M+H, CloHioN20Sz requires 239.0313).
[0051] Example 15
NH2
NH2
I ~ \S O
3-amino-5-(4-methylphenyl)thiophene-2-carboxamide
The title compound was prepared using the procedures found in example 3: mp
254.3-254.8 °C. 1H NMR (DMSO-46/300 MHz) 7.49 (d, 2H), 7.26 (d, 2H),
6.88 (m, 3H), 6.46 (s, 2H), 2.33 (s, 3H). ES+LRMS m/z 233 (M+H).
ES+HRMS m/z 233.0765 (M+H, Cl2HiaNaOS requires 233.0749). Anal Calcd.
for C~ZH12N20S: C, 62.04; H, 5.21; N, 12.06. Found: C, 62.09; H, 5.16; N,
11.98.
[0052] Example 16
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NH2
NH2
\ ~S O
3-amino-5-(3-methylphenyl)thiophene-2-carboxamide
The title compound was prepared using the procedures found in example 3: mp
193.3-194.1 °C. 1H NMR (DMSO-d~/300 MHz) 7.46-7.27 (m, 3H), 7.19 (d,
1H), 6.99-6.80 (m, 3H), 6.47 (s, 2H), 2.35 (s, 3H). ES+LRMS rnlz 233 (M+H).
ES+HRMS m/z 233.0750 (M+H, CIZH~2N20S requires 233.0749). Anal Calcd
for Ct2HtzNzOS + 1l2H20: C, 59.73; H, 5.43; N, 11.61. Found: C, 60.35; H,
4.99; N, 11.73.
[0053] Example 17
NN2
NH2
F3C ' \ S/ 'O
3-amino-5-[3-(trifluoromethyl)phenyl]thiophene-2-carboxamide
The title compound was prepared using the procedures found in example 3: mp
166.6-167.2 °C. IH NMR (DMSO-d61300 MHz) 7.94-7.81 (m, 2H), 7.78-7.63
(m, 2H), 7.11 (s, 1H), 6.97 (s, 2H), 6.50 (s, 2H). ES+LRMS mlz 287 (M+H).
ES+HRMS rnlz 287.0468 (M+H, C~~H9N20F3S requires 287.0466). Anal Calcd
for Ct~,HgN20F3S: C, 50.35; H, 3.17; N, 9.79. Found: C, 50.48. H, 3.05; N,
9.60.
[0054] Example 18
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NH2
NH2
CI I ~ S~ ~
O
CI
3-amino-5-(3,4-dichlorophenyl)thiophene-2-carboxamide
The title compound was prepaxed using the procedures found in example 3: mp
217.0-217.1 °C. 1H NMR (DMSO-d~/300 MHz) 7.83 (s, 1H), 7.70 (d, 1H),
7.63-7.48 (m, 1H), T.12-6.86 (m, 3H), 6.50 (s, 2H). ES+LRMS m/z 287 (M+H).
ES+HRMS m/z 286.9818 (M+H, CtIH$N~OSC12 requires 286.9813). Anal
Calcd for C11H$N20SCh: C, 46.01; H, 2.81; N, 9.76. Found: C, 45.88; H, 2.67;
N, 9.74.
[0055] Example 19
H2
3-amino-5-[4-(methylsulfonyl)phenyl]thiophene-2-carboxamide
The title compound was prepared using the procedures found in example 3: 'H
NMR (DMSO-db/300 MHz) 7.99 (s, 2H), 7.84 (d, 2H), 7.13 (s, 1H), 7.03 (s,
1H), 6.53 (d, 1H), 3.25 (brs, 2H), 3.20 (s, 3H). ES+LRMS m/z 297 (M+H).
ES+HRMS m/z 297.0377 (M+H, CIZHI~N203S~ requires 297.0368} Anal
Calcd for C12H12N2~3S3: C, 48.63; H, 4.08; N, 9.45. Found: C, 48.17; H, 3.83;
N, 9.34.
[0056] Examp1e20
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H2
3-amino-4,5-diphenylthiophene-2-carboxamide
The title compound was prepared using the procedures found in example 3: mp
208.4-208.9 °C. 1H NMR (DMSO-d6/300 MHz) 7.49-6.96 (m, 11H), 5.99 (brs,
2H), 3.32 (brs, 1H). ES+LRMS m/z 295 (M+H). ES+HRMS m1z 295.0913
(M+H, Cl7HiaNaOS requires 295.0905). Anal Calcd for Cl7HiaNaOS: C, 69.36;
H, 4.79; N, 9.52. Found: C, 69.55; H, 4.41; N, 9.39.
[0057] Example 21
NH2
NH2
HEN ( ~ S~
3-amino-5-(3-aminophenyl)thiophene-2-carboxamide
[0058] The product of example 7, 3-amino-5-(3-nitrophenyl)thiophene-2-
carboxamide (1 g, 3.80 mmol), triethylamine (20 mL) and 10 % palladium on
carbon were heated to boiling followed by the drop wise addition of formic
acid
(0.75 g, 16.3 mmol). The resulting mixture was heated at reflux 3 hours. The
reaction was cooled and filtered through a plug of Celite eluting with
methanol.
The filtrate was concentrated in vacuo and purified by silica gel
chromatography
eluting with 5% methanol / dichloromethane. The fractions containing the
desired product were concentrated to yield a yellow solid. The solid was
washed with methanol to yield a yellow solid. (746 Mg): 225.3-229.4 °C.
1H
NMR (DMSO-d6/300 MHz) 7.05 (t, 1H), 6.92-6.66 (m, 5H), 6.60-6.35 (m, 3H),
5.27 (s, 2H). ES+LRMS nzlz 234 (M+H). ES+HRMS rnlz 234.0702 (M+H,
C11HI~N30S requires 234.0701).
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[0059] Example 22
N H2
NH2
~S O
H2N
3-amino-5-(4-aminophenyl)thiophene-2-carboxamide
The title compound was prepared using the procedures found in example 21:
mp 235.8-241.0 °C. 1H NMR (DMSO-db/300 MHz) 7.25 (d, 2H), 6.77-6.51 (m,
5H), 6.40 (brs, 2H), 5.46 (brs, 2H). ES+LRMS m/z 234 (M+H). ES+HRMS
m/z 234.0744 (M+H, C11H~1N30S requires 234.0701).
[0060] Example 23
NH2
NH2 I ~ NH2
~ 'S O
3-amino-5-(2-aminophenyl)thiophene-2-carboxamide
The title compound was prepared using the procedures found in example 21:
mp 149.9-150.3 °C. 1H NMR (DMSO-d6/300 MHz) 7.17-6.9? (m, 2H}, G.89-
6.68 (m, 4H), 6.65-6.52 (m, 1H), 6.42 (brs, 2H), 5.12 (brs, 2H}. ES+LRMS »a/z
234 (M+H). ES+HRMS m/z 234.0700 (M+H, CIIH~ 1N30S requires 234.0701).
[0061] Example 24
NH2
~ I ~ NH2
H2N ~ ~ 'S O
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3-amino-5-[3-(aminocarbonyl)phenyl]thiophene-2-carboxamide
[0062] The product of example 3, 3-amino-5-(3-cyanophenyl)thiophene-2-
carboxamide (200 mg, 0.82 mmol) and sulfuric acid (3 mL) were heated on a
steam bath for 30 minutes. The reaction mixture was poured into ice/water (50
mL) and stirred for 1 hour. After neutralizing with NaHC03, the mixture was
extracted 3x with ethyl acetate. The organic extracts were combined, dried
over
MgS04, filtered, and concentrated in vacuo. The solid material was triturated
with ethyl acetate and filtered to afford the desired product (50 mg): mp
242.0-
242.3 °C. 1H NMR (DMSO-d~/300 MHz) 8.18-7.99 (m, 2H), 7.83 (d, 1H),
7.70
(d, 1H), 7.59-7.33 (m, 2H), 7.11-6.80 (m,3H), 6.49 (brs, 2H). FAB+LRMS m/z
262 (M+H). ES+HRMS m/z 262.0649 (M+H, Ci2HnN30aS requires
262.0650). Anal Calcd for Cl2HlN30~S: C, 55.16; H, 4.24; N, 16.08. Found:
C, 54.59; H, 4.15; N, 15.78.
[0063] Example 25
H2
H2N
3-amino-5-[4-(arninocarbonyl)phenyl]thiophene-2-carboxamide
The title compound was prepared using the procedures found in example 24. 1H
NMR (DMSO-d~/300 MHz) 8.10-7.82 (m, 3H), 7.66 (d, 2H), 7.41 (brs, 1H),
?.13-6.82 (m, 3H), 6.49 (brs, 2H). ES+LRMS mlz 262 (M+H). ES+HRMS m1z
262.0669 (M+H, C12H11N3~2s requires 262.0650). Anal Caled for
C12H11N3O2S: C, 55.16; H, 4.24; N, 16.08. Found: C, 54.83; H 4.29; N, 15.63.
[0064] Example 26
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NH2
NH2
H2N I W ,S O
3-amino-5-[3-(aminomethyl)phenyl]thiophene-2-carboxamide
A par reaction bottle was charged with 3-amino-5-(3-cyanophenyl}thiophene-2-
carboxamide (1 g, 4.12 mmol), 10 % palladium on carbon, and methanol (40
mL). The par bottle was pressurized to 40 psi and stirred at room temperature
for 3 days. 'The mixture was filtered through a plug of Celite eluting with
methanol. The filtrate was concentrated in vacuo and recrystallized with ethyl
acetate/ hexane to give the title compound as a yellow solid (168 mg): mp
182.1-185.0 °C. 1H NMR (CD30D1300 MHz) ?.63 (s, 1H), 7.58-7.47 (m, 1H),
7.43-7.29 (m, 2H), 6.92 (m, 1H), 4.84 (brs, 6H), 3.80 (s, 2H). ES+LRMS rrzlz
248 (M+H). ES+HRMS rnlz 248.0856 (M+H, C1zH13~305 requires 248.0858).
[0065] Example 27
NH2
S NH2
~ \O
S
ii
o I/
3-amino-5-{ 3-[(ethylsulfonyl)amino]phenyl }thiophene-2-carboxamide
The product of example 21, 3-amino-5-(3-aminophenyl)thiophene-2-
carboxamide, (1.16 g, 4.98 mmol), dichloromethane (20 mL), and pyridine (20
mL) were stirred at room temperature. To this mixture was added ethylsulfonyI
chloride (500 ltM, 0.67 g, 5.23 mmol) drop wise. After stirring at room
temperature for 3 hours, water was added, and the mixture was extracted 3x
with ethyl acetate. The organic extracts were combined, dried over MgSO4,
filtered, and concentrated in vacuo. This material was chromatographed on a
silica gel column eluting with ethyl acetate. The fractions containing the
desired
product were combined and concentrated in vacuo to afford a solid (17.1 mg)
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mp 240.2-240.7 °C. 1H NMR (DMSO-dG/300 MHz) 9.96 (s, 1H), 7.58-7.11 (m,
4H), 6.91 (d, 3H), 6.50 (s, 2H), 3.13 (q, 2H), 1.20 (q, 3H). ES+LRMS fWz 326
(M+H). ES+HRMS m/z 326.0626 (M+H, C13H15N303S2 requires 326.0633).
[0066] Example 28
NH2
N I \ . NH2
\ S~ ~~O
O'
5~[3-(acetylamino)phenyl]-3-aminothiophene-2-carboxamide
The title compound was prepared using the procedures found in Example 27:
mp 230.9-233.4 °C. 1H NMR (DMSO-d6/300 MHz) 10.06 (s, 1H), 7.94 (s,
1H),
7.54-7.17 (m, 3H), 6.87 (d, 3H), 6.47 (s, 2H), 2.05 (s, 3H). ES+LRMS YnJz 276
(M+H). ES+HRMS m/z 276.0776 (M+H, C13H13N3~2s requires 276.0807).
[0067] Example 29
~2
$ ~ ~ 'S~~$
NHZ
3-amino-5-(3-thienyl)thiophene-2-carboxamide
3-acetylthiophene (100 mmol, 12.62 g) and N, N'-dimethylformamide (200 mL)
were stirred at 0° C: Phosphorous trichloride (200 mmol, 27.47 g) was
added
drop-wise maintaining the temperahzre below 20° C. After the addition
was
complete, the mixture was allowed to warm to room temperature and stir for
three hours. After re-cooling to 0° C, hydroxylamine hydrochloride (300
mmol,
20.85 g) was added in small portions maintaining the temperature below
20° C.
The mixture was allowed to warm to room temperature and stir for one hour.
After concentrating to 50 mL, ethyl acetate (200 mL) was added and the mixture
washed with brine. Saturated aqueous sodium bicarbonate solution was added
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until gas evolution ceased. The layers were separated and the organics were
washed with brine two times. After drying over magnesium sulfate, the mixture
was concentrated and dissolved in methanol (250 mL). 2-mercaptoacetamide
(300 mmol, 27.34 g) was added, followed by sodium methoxide (25- wt%, 200
mmol, 43.22 g) drop-wise. The mixture was allowed to stir until all of the
materials dissolved. Saturated aqueous sodium bicarbonate (100 mL) was
added followed by hydrochloric acid (1 N) until gas evolution was observed.
Water was added to a total volume on 1L and the mixture was allowed to stand
16 h. A light brown solid was collected by filtration. (14.3 g, 64%): mp 208.5-
210.9 °C. 1H NMR (DMSO-d6/300 MHz) 7.7 (m, 1H), 7.61 (m, .1H), 7.3 (m,
1H), 6.8 (bs, 2H), 6.7 (s, 1H), 6.4 (bs, 2H). ESHRMS m!z 225.016 (M+H,
C9H$NZOS requires 225.016).
[0068] Example 30
NHZ
/ ~o
~'s
i
NC
3-amino-5-(4-cyanophenyl)thiophene-2-carboxamide
[0069] Step l: Preparation of 4-[1-chloro-2-cyanoethenyl]benzonitrile.
4-acetylbenzonitrile (10 mmol, 1.45 g) was stirred in N, N'-dimethylformamide
(10 mL) at room temperature. Phosphorous trichloride was added drop-wise
with a large exotherm. After stirring for one hour, hydroxylamine
hydrochloride
was added in small portions. After stirring for 16 h, ethyl acetate (100 mL)
was
added and the mixture washed with brine, saturated aqueous sodium bicarbonate
and brine 2 X. After drying over magnesium sulfate and concentrating, a tan
solid was obtained by crystallization fiom ethyl acetate l hexanes. (1.6 g,
85%):
mp 157.5-159.0 °C. 1H NMR (DMSO-d6/300 MHz) 7.97 (m, 4H), 7.67 (dd,
1H), 7.10 (s, 1H).
42
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
[0070] Step 2: Preparation of 3-amino-5-(4-cyanophenyl)thiophene-2-
carboxamide.
4-[1-chloro-2-cyanoethenyl]benzonitrile (2 mmol, 377 Mg) was stirred at room
temperature in methanol (25 mL). Sodium methoxide (25 wt%, 4 mmol, 864
Mg) was added followed immediately by 2-mercaptoacetamide (6 mmol, 547
Mg). After one hour, water (100 mL) was added and a yellow-orange solid was
filtered. (230 Mg, 47%): mp 250.4-251.6 °C. 1H NMR (DMSO-d6/300 MHz)
7.86 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 7.08 (s, 1H), 7.00 (bs,
2H),
6.48 (bd, 2H). ESHRMS m/z 244.057 (C12H~N30S requires 244.054).
[0071 ] Example 31
NHZ
~O
s '~S
/ NHZ
OCHg
3-amino-5-(2-anisyl)-thiophene-2-carboxamide
2'-methoxyacetophenone (10 mmol, 1.5 g) was stirred in N, N'-
dimethylformamide (15 mL) at room temperature. Phosphorous trichloride (20
mmol, 2.75 g) was added drop-wise. After stirring for two hours,
hydroxylamine hydrochloride (30 mmol, 2.08 g) was added in small portions.
After one hour, the mixture was concentrated and ethyl acetate (100 mL} was
added. After washing successively with brine, saturated aqueous sodium
bicarbonate, and brine, the mixture was dried over magnesium sulfate and
concentrated. Methanol (50 mL) was added followed by 2-mercaptoacetamide
(30 mmol, 2.73 g). This mixture was stirred at room temperature when sodium
methoxide (25 wt%, 20 mmol, 4.32 g) was added drop-wise. After two hours,
saturated aqueous sodium bicarbonate was added followed by 1 N hydrochloric
acid until gas evolution was observed. Water was added to a total volume of
500 mL and the mixture was allowed to stand for 16 hours. A yellow solid was
filtered and recrystallized from N, N'-dimethylformamide l water. (1.1 g,
44%):
43
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
mp 155.2-155.3 °C. 1H NMR (DMSO-d6/300 MHz) 7.55 (dd, 1H), 7.31 (m,
1H), 7.11 (d, 1H), 7.00 (s, 1H), 6.~8 (d, 1H), 6.81 (bs, 2H), 6.33 (bd, 2H),
3.86
(s, 3H). ESHRMS rnlz 249.070 (Cl2HiaNaOaS requires 249.068).
[0072] Example 32
HEN
CI
I ~ I S ~ H2N
O
3-amino-5-(3-chlorophenyl)thiophene-2-carboxamide
3'-chloroacetophenone (10 mmol, 1.5 g) was stirred in N, N'-
dimethylformamide (15 mL) at 0° C. Phosphorous trichloride (20 mmol,
2.75
g) was added drop-wise. After removing the cooling bath and stirring for two
hours, the mixture was again cooled to 0° C and hydroxylamine
hydrochloride
(30 mmol, 2.08 g) was added in small portions. After stirring for 16 hours at
room temperature, the mixture was concentrated and ethyl acetate (150 mL) was
added. After washing with brine 3 X, the mixture was dried over magnesium
sulfate and concentrated. Methanol (50 mL) was added followed by 2-
mercaptoacetamide (30 mmol, 2.73 g). This mixture was stirred at room
temperature when sodium methoxide (25 wt%, 20 mmol, 4.32 g) was added
drop-wise. When all of the starting materials dissolved, saturated aqueous
sodium bicarbonate was added followed by 1 N hydrochloric acid until gas
evolution was observed. Water was added to a total volume of 400 mL and the
mixture was allowed to stand for 16 hours. A tan solid was filtered. (1.6 g,
63%): mp 168.4-171.3 °C. 'H NMR (DM50-d~/300 MHz) 7.58 (m, 1H), 7.50
(m, 1H), 7.41 (m, 2H), 6.98 (s, 1H), 6.81 (bs, 2H), 6.44 (bd, 2H). ESHRMS rnlz
253.021 (C11H~N20~SC1 requires 253.020).
[0073] Example 33
44
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
~z
~o
/ '~s
~z
3-Amino-5-phenyl thiophene-2-carboxamide
[0074] Step 1: Preparation of 3-amino-5-phenylthiophene-2-
carbohydrazide.
Methyl-3-amino-5-phenylthiophene-2-carboxylate (0.250 g, 1.07 mmol) was
added to hydrazine monohydrate (5 mL) and heated at 80 °C for three
hours.
The reaction mixture was cooled to room temperature, and the resulting solid
was filtered and washed with water (0.195 g, 78%). 1H nmr (DMSO l 300 MHz)
7.56-7.55 (m, 2H); 7.44-7.31 (m, 3H); 6.92 (s, 1H); 6.44 (s, br, 2H). ESHRMS
(tolz) 234.0718 (M+H, CtIHIIN3OS requires 234.0701).
[0075] Step 2: Preparation of 3-Amino-5-phenyl thiophene-2-carboxamide.
3-amino-5-phenylthiophene-2-carbohydrazide (0.150 g, 0.643 mmol) was
dissolved in hot ethanol (10 mL). Raney Nickel (0.5 mL, of a 50/50 wt% slurry
in water) was added. After heating at reflux for 16 hours, the mixture was
filtered while hot through celite, and rinsed with ethanol and concentrated.
(10
mL) 24 mg (17%). 'H nmr (DMSO / 300 MHz) 7.60-7.58 (m, 2H); 7.47-7.34
(m, 3H); 6.94 (s, 1H); 6.91 (s, 2H). ESHRMS (m/z) 219.0575 (M+H
C11H1oN2OS requires 219.0592).
[0076] Example 34
~z
~o
~ '~s
~z
3-Aminobenzo[b]thiophene-2-carboxamide
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
The title compound was prepared using the procedures found in example 33.
Steps 1 and 2: lHnmr (DMSO (300 MHz) 8.02 (d, 1H, J = 7.65 Hz); 7.82 (d,
1H, J = 7.85 Hz); 7.49-7.35 (rn 2H); 7.05 (s, 2H).ESHRMS (m/z) 193.0347
(M+H C9H8NZOS requires 193.0346).
Table 1 shows the bioactivity of exemplified compounds in the heterodimer
assay expressed as ICSO
TABLE 1
Example Compound Structure ICSo
#
Example 3-amino-5-(3- NH2 1 <_
1 10
methoxyphenyl)thiophene-2-I ~ NH2 M
p
carboxarnide ,O
~ S
I
Example 3-amino-5-(2- NH2 10 <_
2 100
chlorophenyl)thiophene-2-C~ I ~ NH2 ~M
carboxamide
O
S
Example 3-amino-5-(3- NH2 1 510
3
cyanophenyl)thiophene-2-I ~ NH2 M
~- p
carboxamide NC
-~
S D
Example 4-amino-5'-chloro-2,2'-~ ~ ~ NHS 1 <_
4 10
bithiophene-5-carboxamideC~ S
S N~
NH2
O
Example 4-amino-2',5'-dimethyl-2,3'-H2N NH2 1 <
5 10
bithiophene-5-carboxamide~ O ~M
i
S
S
46
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
Example Compound Structure ICso
#
Example 4-amino-2',5'-dichloro-2,3'-NH2 10 <
6 100
bithiophene-5-carboxamideNH2 _
wM
S O
CI
S CI
Example 3-amino-5-(3- NH2 1 <
7 10
nitrophenyl)thiophene-2-~ NHz
carboxamide ~ M
02N ~
S
Example 3-amino-5-(2- _ - NH2 >100
8 pM
phenanthryl)thiophene-2-~ \ / ~
\ ~
carboxamide s NHz
O
Example 4-amino-5'-methyl-2,2'-H2N NH2 10 <_
9 100
bithiophene-5-carboxamide~
O
S
S
Example 3-amino-5-(2- NH2 10 S
100
nitrophenyl)thiophene-2-NO2
NH2
~ ~ pM
carboxamide
O
S
Example 3-amino-5-(4- NH2 >100
11 p.M
nitrophenyl)thiophene-2-~ NHS
carboxamide ~
~
S
02N
Example 3-amino-5-pyridin-3- NH2 10 _<
12 100
ylthiophene-2-carboxamideNH2
~ ~
~S v0
i
N
Example 3-amino-5-pyridin-4- NH2 10 <_
13 100
ylthiophene-2-carboxamideNH2
~ \ pM
'S O
N
47
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
Example Compound Structure ICSp
# J
Example 4-amino-3'-methyl-2,2'-NH 10 <_
14 z 100
bithiophene-5-carboxamideJ ~ ~ I wM
S S NHz
O
Example 3-amino-5-(4- NHz >100
15 p.M
methylphenyl)thiophene-2-\ NHz
carboxamide ~
~
S
O
Example 3-amino-5-(3- NHz >100
16 ~M
methylphenyl)thiophene-2-\ NHz
carboxamide ~
~
S
O
Example 3-amino-5-[3- NHz. >100
17 E.tM
(trifluoromethyl)phenyl]thiophe' \ NHz
ne-2-carboxamide F3C
~ S O
I
Example 3-amino-5-(3,4- NHz >100
18 N.M
dichloxophenyl)thiophene-2-\ NHz
carboxamide ~
C~
4 ~ S
CI
Example 3-amino-5-[4- NHz 10 S
I9 100
(methylsulfonyl)phenyl]thiopheI \ NH2 p,M
ne-2-carboxamide
I ~ S
O
O
\
O
Example 3-amino-4,5-diphenyIthiophene-~ >100
20 EcM
2-carboxamide ~ , S O
/ NHz
w
NH2
f
Example 3-amino-5-(3- NHz 1 <_
21 10
aminophenyl)thiophene-2-~ \ NH2 pM
carboxamide HzN
~ S
I
O
48
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
Example Compound Structure ICso
#
Example 3-amino-5-(4- NHz >100NM
22
aminophenyl)thiophene-2-~ NHz
carboxamide 1
/-\'\
I ~ S
O
HzN r
Example 3-amino-5-(2- NHz >100pM
23
aminophenyl)thiophene-2-~ NHz
NHz
carboxamide ~
O
Example 3-amino-5-[3- NHz 10 _<
24 100
(aminocarbonyl)phenyl]thiopheO ~ \ NH2 M
p
ne-2-carboxamide HzN I ~ S~O
i
Example 3-amino-5-[4- NHz >100
25 ltM
(aminocarbonyl)phenyl]thiophe~ \ NHz
ne-2-carboxamide
H2N
O
Example 3-amino-5-[3- NHz 10 <
26 100
(aminomethyl)phenyl]thiophene\ NHz
~ M
-2-carboxamide H p
N
~ g o
z
I
i
Example 3-amino-5-(3- NHz 10 <_
27 100
[(ethylsulfonyl)amino]phenyl}tho H ~ \ NHz
N pM
iophene-2-carboxamide~g'
~ S o
i
Example 5-[3-(acetylamino}phenyl]-3-NHS 10 5100
28
aminothiophene-2-carboxamideH ~ \ NHz
N \ S~ ~~
4
Example 3-amino-5-(3-thienyl)thiophene-~z 1 S 10
29
2-carboxamide / o
i
/ s
s
~2
Example 3-amino-5-(4- ~z >100pM
30
cyanophenyl)thiophene-2-/
carboxamide I ~ / ~'s
~z
NC
49
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
Example Compound Structure ICSo
#
Example 3-amino-5-(2-anisyl)-thiophene-~2 10 <-100
31
2-carboxamide \ / ~o
s M
I
/ NHz
OCH3
Example 3-amino-5-(3- H2N 1 <_
32 10
chlorophenyl)thiophene-2-
/ ~M
\
carboxamide ~ ~
S
H2N
O
Example 3-Amino-5-phenyl thiophene-2-~z 10 S
33 100
carboxamide ~ / ~o
's
/ ~2
Example 3-Aminobenzo[b]thiophene-2-~ ~2 10 <_
34 100
carboxamide ~ ~ \ o
s pM
~2
BIOLOGICAL EVALUATION
[0077] Materials
SAM2 ~ 9G 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 Ol 11:B4), and dithiothreitol were obtained from Sigma
Chemicals.
Antibodies specific for NEMO (IKKY) (FL-419), IKKl(H-744), IKK2(H-470)
and IoBa(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 molecular weight cut-off of 30 kDa were obtained from
Amicon. [7('-33P] ATP (2500 Ci/mmol) and [Y'-32P] ATP (6000 Ci/mmol) were
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
purchased from Amersham. The other reagents used were of the highest grade
commercially available.
[0078] Clorzirzg and Expression
cDNAs of human IKK1 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 Hiss-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, E1T7E} mutant was
constructed in the same vector used for wild type rhIKK2 using a
QuikChange~ 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.
[0079] Enzyrrze Isolation
AlI 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.
[0080] Isolation of rhIKKl homodimer
51
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
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 final concentration of 10 mM and incubated with 2S ml of Ni-NTA resin for
2 hrs. The suspension was poured into a 2S ml column and washed with 250 ml
of buffer C and then with 125 ml of 50 mM imidazole in buffer C. rhIKKl
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 -SO °C.
[008I] Isolation of rhIKK2 homodirner
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. S 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.
[0082] Isolation of rhIKKIlIKK2 heterodimer
The heterodimer enzyme was produced by coinfection in a baculovirus system
(FLAG TKK?/IKK1 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
52
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
min. Imidazole was added to the supernatant to a final concentration of IO 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 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 cut-off of 30 kDa. Bovine serum albumin was added to
the concentrated enzyme to final concentration of O.I %. The enzyme was then
aliquoted and stored at -80 °C.
[0083a Cell Culture
The wild type (wt) human pre-B cell line, 70Z/3, and its mutant, 1.3E2, were
generously provided by Dr. Carol Sibley. Wt 70Z/3 and 1.3E2 cells were grown
in RPMI 1640 (Gibco) supplemented with 7 % defined bovine serum (Hyclone)
arid 50 ~t.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 ,uM 2-
mercaptoethanol. For experiments, cells were plated in 6 well plates at Ix106
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 O.IS M
53
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
NaCI and lysed at 4 °C in 20 mM Hepes buffer, pH 7.6 containing 50
mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM ~i-
glycerophosphate, 1 xnM NaF, I 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.
[0084] Immunoprecipitation and Western Blotting
SF9 cells paste containing rhIKKs were centrifuged (140,000 X g, 10 min) to
remove debris. rhIKKs were immunoprecipitated (100 ,ug of cell paste) from
the cell supernatant using 3 ~,g of anti-MEMO antibody ( FL-419), followed by
coupling to protein A sepharose beads. rhIKKs were also immunoprecipitated
from affinity chromatography purified protein preparations (1 p,g) using anti-
FLAG, anti-His or anti-NEMO antibodies (1-4 p,g) followed by protein A
sepharose coupling. The native, human IKK complex was immunoprecipitated
from THP-1 cell homogenates (300 ~tg/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, IKKI H-744). Native IKK2, ItsBa, and NEMO
proteins from cytosolic lysates (20-80 pg) were separated by SDS-PAGE and
visualized by chemiluminescense using specific antibodies.
[0085] Pho~phatase Treatment
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 MnClz and
resuspended in 50 ~,1. Phosphatase (~,PPase, 1000 U) 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
54
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
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 ~,l of the buffer used for the i~a vitro kinase assay.
[0086] IKKc~SAMEnzymeAssay
IKKa kinase activity was measured using a biotinylated I~Ba 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 SAME ~ 96 Biotin capture plate, and a vacuum
system. The standard reaction mixture contained 5 LtM biotinylated I~Ba
peptide, 1 LvM ['y-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 ~,l) in a final volume of 50 I,d. After incubation at 25
°C
for 30 min, 25 ~,1 of the reaction mixture was withdrawn and added to a SAM2
~ 96 Biotin capture 96-well plate. Each well was then washed successively
with 800 ~,l 2 M NaCI, 1.2 ml of NaCI containing 1 % H3P04, 400 ~,1 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 IKBa 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-IKBccI_sa and ['y-32P~ 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-IoBal_sa 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 IKBa
peptide per min. Specific activity was expressed as units per mg of protein.
For
experiments related to Km determination of purified enzymes, various
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
concentrations of ATP or IxBa peptide were used in the assay at either a fixed
IxBa or ATP concentration. For IxBoc peptide Km, assays were carned out with
0.1 ~tg of enzyme, 5 i,~M ATP and IxBa peptide from 0.5 to 20 ~.M. For ATP
Km, assays were carried out with 0.1 ~,g of enzyme, 10 l.tM IKBoc peptide and
ATP from 0.1 to 10 ~.M. For Km determination of rhIKKl homodimer, due to
its low activity and higher Km for IKBa peptide, rhIKK1 homodimer (0.3 ~.g)
was assayed with 125 ~M IKBa peptide and a 5-fold higher specific activity of
ATP (from 0.1 to 10 ~M) for ATP Km experiments and a 5-fold higher specific
activity of 5 ltM ATP and I~Boc peptide (from 5 to 200 ~tlVl} for IxBa peptide
Km experiments.
[0087] IKK,~3Resin Enzyme Assay
IKK(3 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 IKBcc peptide, 0.1 [uCi/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 ~,1 enzyme solution and 10 ul
inhibitor in a final volume of 50 ~l. 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 ~,l of scintillation fluid (Microscint 40) (Packard}
was added to each well. Incorporation of ['y-33P] ATP was measured using a
Top-Count NXT (Packard).
[0088] IKK heterodimer Resin Enzyme Assay
56
CA 02464924 2004-04-27
WO 03/037886 PCT/US02/34801
IKK heterodimer kinase activity was measured using a biotinylated hcBa
peptide (Gly-Leu-Lys-Lys-Glu-Arg-Leu-Leu-Asp-Asp-Arg-His-Asp-Ser32-Gly-
Leu-Asp-Ser3G-Met-Lys-Asp-Glu-Glu) (American Peptide Co.). 20 ul of the
standard reaction mixture contained 5 ltM biotinylated ItsBa peptide, 0.1
~,Cilreaction ['y-33P~ ATP (Amersham) (about 1 X 105 cpm), 1 pM 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 ~1 enzyme
solution and 10 ~l inhibitor in a final volume of 50 p,l. 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).
57
