J. Das et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2145–2149
2149
in Src by Ser 320 and Lys321, respectively, and in Fyn
by Asn320 and Lys321, respectively.
Chem. Lett. 2002, 12, 3153. (c) Snow, R. J.; Cardozo, M. G.;
Morwick, T. M.; Busacca, C. A.; Dong, Y.; Eckner, R. J.;
Jacober, S.; Jakes, S.; Kapadia, S.; Lukas, S.; Panzenbeck, M.;
Peet, G. W.; Peterson, J. D.; Prokopowicz, A. S., III; Sellati,
R.; Tolbert, R. M.; Tschantz, M. A.; Moss, N. J. Med. Chem.
2002, 45, 3394. (d) Arnold, L. D.; Calderwood, D. J.; Dixon,
R. W.; Johnston, D. N.; Kamens, J. S.; Munschauer, R.;
Rafferty, P.; Ratnofsky, S. E. Bioorg. Med. Chem. Lett. 2000,
10, 2167. (e) Burchat, A. F.; Calderwood, D. J.; Hirst, G. C.;
Holman, N. J.; Johnston, D. N.; Munschauer, R.; Rafferty, P.;
Tometzki, G. B. Bioorg. Med. Chem. Lett. 2000, 10, 2171.
5. Dave, A. M.; Bhatt, K. N.; Undavia, N. K.; Trivedi, P. B.
J. Ind. Chem. Soc. 1988, 65, 365.
In summary, we have developed a novel series of
benzothiazole Lck inhibitors based on our initial thia-
zole lead 1. SAR studies identified several carboxamide
and urea analogues as potent and selective inhibitors.
BMS-243117 (2) was shown to be a potent, and selective
lck inhibitor with good potencyin a T-cell proliferation
assay.
6. (a) Lck enzyme assay: Recombinant Lck expressed as a His-
tagged protein in insect cells using a baculovirus expression
system and purified by nickel affinity chromatography was
incubated in kinase buffer (20 mM MOPS, pH 7, 10 mM
MgCl2) in presence of the test compound. The reaction was
initiated byaddition of substrates to the final concentration of
1 mM ATP, 3.3 mCi/mL [33P}g-APT, and 0.1 mg/mL acid
denatured enolase, and stopped after 10 min byaddition of
10% trichloroacetic acid, 100 mM sodium pyrophosphate fol-
lowed by2 mg/mL bovine serum albumin. The labeled enolase
protein substrate was precipitated at 4 ꢀC, harvested onto
Packard Unifilter plates and counted in a Topcount scintilla-
tion counter. (b) Enzyme assays for Src, Fyn, Hck, Blk, Lyn,
and Fgr were run under the same conditions as for Lck. (c)
PBL proliferation assay: A 96-well plate was coated with
monoclonal antibodyto CD3 (G19-4), the antibodywas
allowed to bind, and the plate was washed. Normal human
peripheral blood T-cells were added to the wells along with the
test compound and anti-CD28 (E.3) antibody. After 3 days,
[3H]-thymidine was added to the cells, after further incubation,
the cells were harvested and counted in a scintillation counter.
7. (a) Two crystal structures of the catalytic domain of Lck
(Tyr394 phosphorylated) were published. See: Yamaguchi, H.;
Hendrickson, W. A. Nature 1996, 384, 484. (b) Zhu, X.; Kim,
J. L.; Newcomb, J. R.; Rose, P. E.; Stover, D. R.; Toledo,
L. M.; Zhao, H.; Morgenstern, K. A. Structure 1999, 7, 651.
Protein Data Bank (1QPC pdb, Research Collaboratoryfor
Structural Bioinformatics.
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