3740
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6. (a) Ikeda, H.; Yatomi, Y.; Yanase, M.; Satoh, H.;
cyano-3-hydroxy-acrylic acid with overnight treatment of
0.1 M NaOH aq/MeOH. Similar ring opening reaction was
known in anti-inflammatory drug leflunomide kalgutkar,
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15. Measurement of LPA1 receptor inhibitory activity was as
follows: CHO-K1 cells stably transfected with LPA1
receptor were cultured in aMEM/F12 (a-minimum essen-
tial medium:Ham’s F12) 1:1 mixture containing 400 lg/ml
Zeocin, 7% FBS (fetal bovine serum) and Penicillin-
Streptomycin. The cells were loaded with the Ca2+
indicator dye Fluo-3,AM (Biotium) in assay buffer
(20 mM Hepes, 1 · HBSS, 2.5 mM Probenecid) for 1 h
at 37 ꢁC. Cells were washed with assay buffer and
resuspended in assay buffer containing 0.1% BSA and
seeded into 384-well plates (24,000 cells/well). After cells
were attached to the bottom of the well, the plates were
placed into a FLIPR (Fluorometric Imaging Plate Reader,
7. Tangkijvanich, P.; Melton, A. C.; Santiskulvong, C.; Yee,
H. F., Jr. J. Biomed. Sci. 2003, 10, 352.
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2003, 114, 64.
9. (a) Takuwa, Y.; Takuwa, N.; Sugimoto, N. J. Biochem.
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Heasley, B. H.; Mukhin, Y. V.; Macdonald, T. L.; Lynch,
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Molecular
Devices).
Cell
fluorescence
(excita-
tion = 488 nm, emission = 540 nm) was monitored before
and after the addition of test compounds in the presence
or absence of 1 lM LPA. Ki16425 and compound 15 were
used as
(IC50 = 510 150 nM,
a
positive control throughout the assay
n = 25 for Ki16625 and
220 70 nM, n = 29 for 15, respectively) and IC50 values
represent the mean of not less than two different runs.
16. Measurement of inhibitory activity against LPA stimula-
tion with rat hepatic stellate (rHSC) cells was as follows: A
stellate cell fraction was obtained from the liver of male
Wister rat according to a conventional method Kawada,
N.; Tran-Thi, T. A.; Klein, H.; Decker, K. Eur. J. Biochem.
1993, 213(2), 815, The cells were seeded overnight in a 384
well plate at 24,000 cells/well and the medium was
removed. An assay buffer (0.1% BSA, 20 mM Hepes,
1 · HBSS, 2.5 mM Probenecid) containing 4 lM of Fluo-
3, AM was added, and the cells were stained at 37 ꢁC for
1 h. Following the buffer containing the dye reagent was
removed, and an assay buffer was added, intracellular
Ca2+ concentration was measured with FLIPR. Adding a
test substance and LPA at the final concentration of 5 lM,
the inhibitory action of the test substance on the increase
in the intracellular Ca2+ concentration by LPA was
examined. The increase in the intracellular Ca2+ concen-
tration by LPA addition without test substance was taken
as 100%, that without the addition of LPA was taken as
0%, and the substrate’s concentration (IC50) at inhibiting
increase in the intracellular Ca2+ concentration by 50%
was determined. Ki16425 and compound 15 were used as a
positive control throughout the assay (IC50 = 160
33 nM, n = 13 for Ki16625 and 49 12 nM, n = 5 for 15,
respectively) and IC50 values represent the mean of not less
than two different runs.
11. Okusa, M. D.; Ye, H.; Huang, L.; Sigismund, L.;
Macdonald, T.; Lynch, K. R. Am. J. Physiol. Renal
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14. 5-(4-Methyl-phenyl)-isoxazole-4-carboxylic acid methyl
ester, model compound of the ester of isoxazole-4-carbox-
ylate, was easily decomposed into 3-(4-methyl-phenyl)-2-