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their potency in whole blood. Based on the enzyme
activity of the above-mentioned analogues, it was clear
that the 7-substituent brought about increased potency
primarily by steric rigidity and hydrophobic interac-
tions. In order to explore a potential salt forming inter-
action with the proximal Asp-168, a polar amine was
extended from the piperazine. The marginal increase in
potency observed for 29 and 30 was not conclusive with
formation of a salt bridge. In order for a transparent
carryover of in vitro enzyme activity to functional effi-
cacy, it was observed that a basic amine was required in
all cases.
enson, M. E.; Smietana, J. M.; Hall, R. F.; Garigipati, R. S.;
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This new class of compounds when tested against a
variety of other closely related kinases including JNK2,
ERK1 and ZAP70 did not show any appreciable activ-
ity. Insights gained in the development of this class of
p38 inhibitors will aid in the design of second and third
generation of analogues with further improved potency
and functional activity. A comprehensive study encom-
passing the development of good pharmacokinetic pro-
files for this series is underway and will be the subject of
a future publication.
7. Dumas, J.; Sibley, R.; Reidl, B.; Monahan, M. K.; Lee, W.;
Lowinger, T. B.; Redman, A. M.; Johnson, J. S.; Kingery-
Wood, J.; Scott, W. J.; Smith, R. A.; Bobko, M.; Schoenleber,
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10. Enzyme activity was measured by two methods: The filter-
binding assay is described in ref 5b. A SPA-bead based assay
was carried out using mouse p38. Compounds were serially
diluted into a 96 well plate containing a MOPS based p38
assay buffer. The assay was initiated by addition of cold ATP,
33P ATP (gamma) and biotin labeled GST-ATF2 substrate
(4 mM). After incubation at 30 ꢀC for 3 h, the reaction was
stopped by addition of a PBS based quench buffer with 2Â
moles of SPA beads over the amount of substrate used. The
extent of phosphorylation of GST-ATF2 was measured using
a topcount reader and subtracted from background. IC50s are
means of two experiments.
11. Anti human TNF-a was coated on immulon 4 plates.
THP-1 cells (density=2.5 Â 106/mL) were suspended into
96-well plates containing a PBS based medium. Compound
was added as solution in DMSOfollowed by addition of LPS.
The reaction was incubated for 4 h at 37 ꢀC under CO2. TNF-
a release was measured in the supernatants by ELISA.
Reported IC50s are means from three measurements.
12. Wang, Z.; Canagarajah, B. J.; Boehm, J. C.; Kassisa, S.;
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Acknowledgements
The authors would like to thank Drs. Malcolm Mac-
Coss, Arthur A. Patchet and James V. Heck for useful
discussions.
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