J. Chiba et al. / Bioorg. Med. Chem. Lett. 15 (2005) 41–45
45
medium (Hamꢀs F-12) supplemented with 10% fetal calf
serum, 100U/mL penicillin, 100lg/mL streptomycin, and
2mM L-glutamine (Invitrogen Corporation, Carlsbad,
USA) and 1mg/mL G-418 (Geneticin, Invitrogen Corpo-
ration, Carlsbad, USA). Human VCAM-1/Fc Chimera
(R&D Systems Inc., Minneapolis, USA) was labeled with
Eu as follows. The protein was reconstituted in labeling
buffer (150mM NaCl, 50mM sodium carbonate, pH8.5).
Eu-Labeling Reagent (Perkin–Elmer Inc., Wellesley,
USA) reconstituted with the labeling buffer was added to
the protein solution. The mixture was then incubated at
room temperature for 4days. The Eu-labeled protein was
purified with a PD-10 column (Amersham Biosciences
KK, Tokyo, Japan) and stored at À80ꢁC until use. All
assays were performed in duplicate. Prior to the assay, 4B4
cells were suspended at 3 · 105 cells/mL in F-12 medium
(Hamꢀs F-12) supplemented with 10% fetal calf serum,
100U/mL penicillin, 100lg/mL streptomycin and 2mM
L-glutamine (Invitrogen Corporation, Carlsbad, USA).
One hundred microliters of the 4B4 cell suspension at
3 · 105 cells/mL was distributed into each well of 96 well-
culture plates (Costar, 3599, USA). The plates were
incubated in a 5% CO2 humidified incubator at 37ꢁC
(Themo Forma, model: 3120, Forma Scientific, Inc. USA)
for 2days. The medium was discarded and each well was
washed twice with 300lL of chilled Wash Buffer (25mM
HEPES (pH7.5), 150mM NaCl, 1mM CaCl2, 1mM
MgCl2, 4mM MnCl2). Then 50lL of compound solution
was distributed to the wells, followed by 50lL of 2nM of
Eu-labeled Human VCAM-1/Fc Chimera diluted with the
Wash Buffer (final concentration: 1nM). The plates were
incubated for 60min at room temperature, and the wells
were washed four times with 300lL of chilled Wash
Buffer. Finally 100lL of the enhancement solution (Per-
kin–Elmer Inc., Wellesley, USA) was added to each well.
The plates were placed on a shaker for 5min. Eu
fluorescence was then measured in a time-resolved fluo-
rometer (DELFIA Research fluorometer, model: 1234-
001, Perkin–Elmer Inc., Wellesley, USA). The concentra-
tion of compound required for 50% inhibition in the assay
was determined.
series of 4-piperidinylacetic acid, 1-piperazinylacetic
acid, and 4-aminobenzoic acid derivatives as potent
VLA-4 antagonists with IC50 values of less than
10nM. In addition, a representative compound mor-
pholinyl-4-piperidinylacetic acid derivative 13d demon-
strated efficacy in vivo asthma model by oral
administration. Further pharmacokinetic improvements
and structural modifications of these leading com-
pounds will be presented in forthcoming publications.
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´
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