J. P. Powers et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2842–2845
2845
aryl ring via substituent replacement demonstrated
that some changes were allowed in this region,
although the 3-nitro substituent remained the best
substitution in terms of potency. Modification of the
benzimidazole ring by substituent studies showed that
improvements in potency resulted from modification
of the 5-position, which may be important for future
studies to optimize ADME and potency parameters.
The most effective modifications resulted from optimi-
zation of the N-alkyl substituent, which gave >10-fold
improvements in potency, resulting in the potent
IRAK-4 inhibitors 41 (IC50 = 150 nM) and 46
(IC50 = 200 nM). The acyl-2-aminobenzimidazole scaf-
fold reported herein represents a unique kinase bind-
ing motif, previously unreported in the literature,
and further studies with this scaffold and its utility
in the inhibition of IRAK-4 will be reported in subse-
quent publications.
Figure 2. Proposed binding of acylbenzimidazole tautomeric forms to
IRAK-4.
References and notes
1. Cao, Z.; Henzel, W. J.; Gao, X. Science 1996, 271, 1128.
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T. W.; Yeh, W. C. Nature 2002, 416, 750.
groups (i.e., 41, 46) in the N-alkyl substituent, it is likely
that this group is pointing toward solvent or the phos-
phate binding loop in the kinase domain, although the
equipotence of both acidic (i.e., 43) and basic (i.e., 44)
functionalities when placed at the same distance from
the benzimidazole ring would appear to indicate the
former. When these results are combined together, they
may be used to form a rough model of the binding of
these inhibitors to IRAK-4 (Fig. 2).
6. Lye, E.; Mirtsos, C.; Suzuki, N.; Suzuki, S.; Yeh, W. C.
J. Biol. Chem. 2004, 279, 40653.
7. Medvedev, A. E.; Lentschat, A.; Kuhns, D. B.; Blanco, J.
C.; Salkowski, C.; Zhang, S.; Arditi, M.; Gallin, J. I.;
Vogel, S. N. J. Exp. Med. 2003, 198, 521.
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C.; Elbim, C.; Hitchcock, R.; Lammas, D.; Davies, G.; Al-
Ghonaium, A.; Al-Rayes, H.; Al-Jumaah, S.; Al-Hajjar,
S.; Al-Mohsen, I. Z.; Frayha, H. H.; Rucker, R.; Hawn, T.
R.; Aderem, A.; Tufenkeji, H.; Haraguchi, S.; Day, N. K.;
Good, R. A.; Gougerot-Pocidalo, M. A.; Cassanova, J. L.
Science 2003, 299, 2076.
Having identified a series of potent inhibitors of IRAK-
4, several of the compounds were profiled for selectivity
against other kinases. Inhibitors 36 (IRAK-4
IC50 = 0.4 lM) and 46 (IRAK-4 IC50 = 0.2 lM) were
found to have IC50’s greater than the highest concentra-
tion tested (10 lM) against a panel of 27 other kinases,
including the most closely homologous (outside of the
IRAK family) Lck and pp60SRC. Additionally, com-
pounds 36 and 46 did not show any signs of cytotoxicity
in
a
72 h proliferation assay in HeLa cells
(ED50 > 30 lM). Significant inhibition of IRAK-1 was
observed with both compounds (IRAK-1 IC50 = 0.75
and 0.3 lM, respectively), not unexpectedly, given the
high homology between these two kinases.
9. Cohen, S. B.; Rubbert, A. Rheumatology 2003, 42(Suppl.
2), ii36.
10. All compounds gave satisfactory 1H NMR, HPLC, and
MS data in full agreement with their proposed structures.
11. Zeiger, A. V.; Joullie, M. M. J. Org. Chem. 1977, 42, 542.
12. IRAK-4 enzyme activity was determined by its ability to
phosphorylate a biotinylated synthetic peptide substrate
(RRRVTSPARRS, sequence derived from GFAP) in a
chemiluminescent ELISA. Assays were performed in
streptavidin-coated 96-well plate format utilizing recom-
binant full length IRAK-4 and 10 lM ATP. IC50 values
for inhibition of IRAK-4 were calculated from a minimum
of two separate determinations each performed in
duplicate.
In summary, screening of a small-molecule library
resulted in the discovery of a series of novel acyl-2-
aminobenzimidazole inhibitors of IRAK-4. SAR stud-
ies resulted in significant improvement of potency, and
established the key pharmacophore of the series. The
amide functionality was found to be important for
IRAK-4 inhibition, as was the benzimidazole ring,
indicating the probable kinase binding motif as de-
scribed in Figure 2. Modification of the benzamide