T. Sato et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4323–4326
4325
Figure 1. Evaluation of ( )-, (R), and (S)-Ki16425 in the cell migration assay.
10. Heasley, B. H.; Jarosz, R.; Carter, K. M.; Van, S. J.; Lynch, K. R.; Macdonald, T. L.
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enantiomers of Ki16425 were synthesized, respectively, according
to the synthetic route we established.
Biological activity of both enantiomers of Ki16425 was evalu-
33
ated by both the cell migration assay with PC-3 cells
TGF
and the
a
shedding assay.34 PC-3 cells are sensitive to LPA-dependent
cell migration and Ki16425 inhibits the migratory response of
PC-3 cells, as described previously.35 In this assay, PC-3 cells mi-
grated in response to LPA (Fig. 1A). When PC-3 cells preincubated
with Ki16425 were stimulated with 10 nM LPA, Ki16425 showed
antagonistic activity against cell migration (Fig. 1B). The rank order
13. Gududuru, V.; Zeng, K.; Tsukahara, R.; Makarova, N.; Fujiwara, Y.; Pigg, K. R.;
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of antagonistic activity was (R)- > racemic > (S)-Ki16425. AP-TGF
a
release assay confirmed that (R)-Ki16425 was more potent in ana-
tagonizing LPA1 and LPA3 than (S)-Ki16425, and the rank order was
(R)- > racemic > (S)-Ki16425.
In conclusion, we have established a synthesis of Ki16425 via
1,3-dipolar cycloaddition of nitrile oxide as the key step. By utiliing
the synthetic route, both enantiomers of Ki16425 were synthesized
through Noyori’s catalytic asymmetric reduction. In comparison to
the known synthetic route to 1,19 our 1,3-dipolar cycloaddition
strategy should be advantageous since facile preparation of isoxaz-
ole core possessing a various substituted pattern is possible simply
by switching nitroalkanes and substituted propiolates. Evaluation
of LPA antagonistic activity of both enantiomers of Ki16425 re-
vealed that (R)-isomer possessed higher activity.38
22. Touaux, B.; Klein, B.; TexierBoullet, F.; Hamelin, J. J. Chem. Res., Synop. 1994,
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Acknowledgments
This work was supported by the Targeted Proteins Research
Program and the Funding Program for Next Generation World-
Leading Researchers (LS008), the Cabinet Office, Government of
Japan.
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Supplementary data
32. Mitsunobu, O. Synthesis 1981, 1.
Supplementary data associated with this article can be found, in
33. Chemotaxis assay: PC-3 (prostate cancer) cells were cultured in RPMI 1640
(Nissui, Japan) supplemented with 5% fetal bovine serum. Cell migration was
determined by a modified Boyden chamber assays as described previously.36 In
brief, polycarbonate filters with 8-lm pores (Neuro Probe, Inc.) were coated
with 0.001% of fibronectin (Sigma). PC-3 cells (1 Â 105 cells in 200
lL/well)
References and notes
were loaded into upper chambers and incubated at 37 °C for 3 h to allow
migration. The cell migration to the bottom side of the filter was evaluated by
measuring optical densities at 594 nm. For Ki16425 treatment, cells were
preincubated with each concentration of Ki16425 for 30 min.
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transforming growth factor-a (AP-TGF-a) and for LPA receptor (human LPA1 or
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a released from cells or remained in cells were
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determined by measuring the AP activity using p-nitrophenyl phosphate (p-
NPP) as a substrate. Activation of each LPA receptor was expressed as % release
of AP activity using the following formula: (AP activity in the culture
supernatant)/(AP activity in the culture supernatant + AP activity remained
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