Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4797
inhibition at 10 μM for only eight targets (see Supporting
Information for details). For all active targets assessed, the
subsequent IC50 values were greater than 5 μM, highlighting
that 1a does not exhibit significant off-target activity.20
HG005033 and X01 MH077633, and by an award from the
Global CLL Research Foundation (to J.C.R.).
Note Added after ASAP Publication. This paper was pub-
lished on May 18, 2010 with a typographical error in the Introduc-
tion. The revised version was published on May 21, 2010.
Conclusions
A series of substituted 2-aminobenzimidazoles has been
identified that serve as promising research tools as pathway-
selective chemical inhibitors of NF-κB activation induced by
activators of PKC signaling through B and T cell antigen
receptors. Interestingly, the MLPCN probe candidate (1a)
was identified directly from the screening hit set and only
lesser or equipotent analogues have been prepared, indicating
a limited SAR for the series. Compound 1a was found to be
noncytotoxic and inhibited IL-8 production induced by PKC
activators in HEK293 cells (IC50<0.1 μM), while failing to
inhibit NF-κB reporter gene activation by agonists of the
other NF-κB activation pathways. Compound 1a attenuated
CD3/CD28andPMA/ionomycin-induced productionofIL-2
by Jurkat T-cells (IC50 <5 μM) and anti-IgM-stimulated pro-
liferation of murine B-lymphocytes (IC50 ∼2 μM). We have used
the SAR data to generate the azide and alkynyl containing
derivatives 1o and 1p. These have been utilized in the synthesis
of biotinylated conjugates directed toward affinity-based target
identification and future mode of action studies that will be
reported separately. In addition, the equipotent 2-benzimid-
azolone analogues (19) provide a modified scaffold with an
additional N3-site for future exploration. Compound 1a and its
analogues represent new chemical tools that may be useful toward
further development of therapeutically useful pathway selective
modulators of NF-κB activity that may find application in disease
models of inflammation, cancer and other autoimmune diseases.
Supporting Information Available: Detailed experimental
procedures for the biology, pharmacology, and chemistry sections
andspectroscopiccharacterizationof compounds. This material is
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General Procedure (Method B, Scheme 1) of N1-Alkylation of
2-Aminobenzimidazoles for the Synthesis 1a, 1b, 1d, 1 e, 1g-n,
17a, 17b, 17d, and 17e as Illustrated by Synthesis of 1-(3,5-Di-
tert-butyl-4-hydroxyphenyl)-2-(2-(3-hydroxypropylamino)-5,6-
dimethyl-1H-benzo[d]imidazol-1-yl)ethanone, 1a. To a 20 dram
vial equipped with a stir bar was added 2-aminobenzimidazole
14a (0.37 g, 1.6 mmol) followed by corresponding 2-bromoace-
tophenone 15a (0.62 g, 1.9 mmol). To the mixture was added
10 mL of methanol and solid NaHCO3 (3 equiv). The mixture
was stirred at 23 °C for 3 days, after which the solvent was
evaporated to a suitable volume and subsequent purification by
silica gel flash column chromatography (hexanes to 1:4 hexanes/
ethyl acetate) resulted in a yellow film, which on lyophilization
from a 2:8 acetone/water mixture afforded 1a as white solid
(0.61 g, 78%). 1H NMR (400 MHz, acetone-d6) δ 7.87 (s, 2H),
6.95 (s, 1H), 6.77 (s, 1H), 5.94 (s, 1H), 5.52 (s, 2H), 3.55-3.47 (m,
2H), 3.42 (t, J = 5.6 Hz, 2H), 2.14 (s, 2H), 2.12 (s, 3H), 1.56 (p,
J = 6.0 Hz, 2H), 1.40 (s, 18H). 13C NMR (100 MHz, acetone-d6)
δ 191.58, 159.66, 156.73, 141.17, 137.58, 134.43, 128.85, 127.29,
127.19, 126.16, 116.74, 108.72, 57.65, 48.47, 39.19, 34.90, 34.67,
19.77, 19.74. LRMS (ESI): calcd for C28H39N3O3 [M þ H] =
466.3, obsd [MþH]=466.1. HRMS (ESI): calcd for C28H39-
N3O3 [M þ H] = 466.3070, obsd [M þ H] = 466.3091.
Acknowledgment. We thank Diana Velosa for assistance
with synthetic intermediate preparation. We also thank
Dr. Ted Gauthier (Department of Chemistry, University of
South Florida, Tampa, FL) and Dr. Vasudha Sharma (H. Lee
Moffitt Cancer Center, Tampa, FL) for acquiring the high
resolution mass spectra. This research was supported by the
Molecular Libraries Initiative of the National Institutes of
Health Roadmap for Medical Research NIH grants 5U54