Thienopyridine Inhibitors of IκB Kinase-â
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2907
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100 µM Na3VO4, 20 mM â-glycerophosphate, 1 mM DTT, 2%
DMSO, 250 nM ATP, 0.4 nM [33P]ATP (specific activity, 3000
Ci/mmol), 53 µg/mL of IκBR substrate, 0.09 µg/mL of IKKâ
enzyme, and the test compound. The reactions were initiated by
adding a solution of IκBR substrate and ATP to polypropylene
plates containing the IKKâ enzyme that was preincubated for 5
min with the test compound. Then, the reaction mixtures were
incubated for 1 h at 25 °C and quenched by the addition of 150 µL
of 10% trichloroacetic acid and 5% disodium pyrophosphate. After
mixing, the entire contents of the quenched reaction mixtures were
transferred to Packard Unifilter filtration plates, aspirated, and
washed six times with 250 µL of doubly distilled H2O using the
Packard Filtermate Harvester. The filtration plates were left to air-
dry, and then 40 µL of microscint 20 scintillation fluid was added
to each well. The plates were briefly shaken, and the 33P-labeled
reaction products were quantitated using the Packard TopCount
scintillation counter. The compounds were tested in duplicate,
starting at a top concentration of 50 µM. The assay was run in
conjunction with a concentration determination: DMSO stock
solutions at 5 mg/mL were diluted 3-fold with the reaction buffer,
and the samples were centrifuged for 10 min. The supernatants were
used in the assay, and the concentration of the supernatants was
determined using HPLC by comparison to a reference solution. The
IC50 data reflects the corrected concentrations, and the values are
an average of at least two data points.
IKKR Dose Response Method. The same method described
above for IKKâ was used with the following exceptions: 245 µg/
mL of IκBR substrate and 3.6 µg/mL of IKKR enzyme were used.
The compound solutions were used directly with no centrifugation
and no concentration determinations.
Solubility in HBSS. Approximately 1 mg of compound was
combined with 10 mL of Hank’s balanced salt solution (HBSS) in
a brown 20.0 mL bottle. The mixture was stirred overnight. The
mixture was then filtered using a 0.45 µm PVDF Whatman filter.
The recovered solution was then analyzed by HPLC. Solubility was
determined using a standard curve.
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(regulated on activation, normal T-cell expressed and secreted);
ICAM (intercellular adhesion molecule); VCAM (vascular cell
adhesion molecule); COX (cyclooxygenase); iNOS (inducible nitric
oxide synthase); MMP (matrix metalloproteinase).
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structural filters.
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Acknowledgment. We thank Ed Pack for providing HPLC
data, Kathy Last-Barney and Tin Htut for providing cellular
data, Ming-Hong Hao for conformational analyses, and John
Proudfoot, Eugene Hickey, and Roman Fleck for discussions
and evaluation of the manuscript.
Supporting Information Available: Additional selectivity data
for compounds 3, 20, 24, and 38, ATP competition data for
compounds 2, 3, 20, 24, 36, and 38, cell data for compounds 2, 3,
and 38, and combustion analyses. This material is available free of
(13) Aminothiophenecarboxamide 2 has been identified and described
recently by others as an inhibitor of IKKâ. See: (a) Baxter, A.;
Brough, S.; Cooper, A.; Floettmann, E.; Foster, S.; Harding, C.;
Kettle, J.; McInally, T.; Martin, C.; Mobbs, M.; Needham, M.;
Newham, P.; Paine, S.; St-Gallay, S.; Salter, S.; Unitt, J.; Xue, Y.
Hit-to-lead studies: The discovery of potent, orally active, thiophen-
ecarboxamide IKK-2 inhibitors. Bioorg. Med. Chem. Lett. 2004, 14,
2817-2822. (b) Kishore, N.; Sommers, C.; Mathialagan, S.; Guzova,
J.; Yao, M.; Hauser, S.; Huynh, K.; Bonar, S.; Mielke, C.; Albee,
L.; Weier, R.; Graneto, M.; Hanau, C.; Perry, T.; Tripp, C. S. A
selective IKK-2 inhibitor blocks NF-κB-dependent gene expression
in interleukin-1â-stimulated synovial fibroblasts. J. Biol. Chem. 2003,
278, 32861-32871. Other series of IKKâ inhibitors have recently
been described. See (c) Bingham, A. H.; Davenport, R. J.; Gowers,
L.; Knight, R. L.; Lowe, C.; Owen, D. A.; Parry, D. M.; Pitt, W. R.
A novel series of potent and selective IKK2 inhibitors. Bioorg. Med.
Chem. Lett. 2004, 14, 409-412. (d) Murata, T.; Shimada, M.;
Sakakibara, S.; Yoshino, T.; Kadono, H.; Masuda, T.; Shimazaki,
M.; Shintani, T.; Fuchikami, K.; Sakai, K.; Inbe, H.; Takeshita, K.;
Toshior, M.; Umeda, M.; Bacon, K. B.; Ziegelbauer, K. B.; Lowinger,
T. B. Discovery of novel and selective IKK-â serine-threonine protein
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K.; Bacon, K. B.; Ziegelbauer, K. B.; Lowinger, T. B. Synthesis and
structure-activity relationships of novel IKK-â inhibitors. Part 2:
Improvement of in vitro activity. Bioorg. Med. Chem. Lett. 2004,
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