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(A2) series, the corresponding pyrido[3,4-d]pyrimidine
analog 28 was significantly less potent (Fig. 2). In light
of the aforementioned X-ray co-crystal structure for
the imidazo[1,5-a]pyrazine analog 24, this result was
surprising since all key interactions of compound 28
with the enzyme appear to be in place for optimal po-
tency. A plausible explanation could be that the H-
bonding trajectory of either the imidazo or pyrazolo
nitrogens toward the available water molecule held by
Asp168/Lys53 is not reproduced when the nitrogen is
in a pyrimidine ring. In the former case, the plane of
interaction is in line with the water (OH) and is about
1.7 A in length (based on the X-ray structure). When
the pyrimidine analog was modeled, the resulting trajec-
tory is significantly off-plane and the distance increases
˚
to 2.1 A. Thus, the pyrimidine core offers a less efficient
H-bonding opportunity to this water molecule.
7. (a) Goldstein, D. M.; Gabriel, T. Curr. Top. Med. Chem.
2005, 5, 1017; (b) Dominguez, C.; Powers, D. A.; Tamayo,
N. Curr. Opin. Drug Discov. Dev. 2005, 8, 421.
˚
8. (a) Liu, C.; Wrobleski, S. T.; Lin, J.; Ahmed, G.; Metzger,
A.; Wityak, J.; Gillooly, K. M.; Shuster, D. J.; McIntyre,
K. W.; Pitt, S.; Shen, D. R.; Zhang, R. F.; Zhang, H.;
Doweyko, A. M.; Diller, D.; Henderson, I.; Barrish, J. C.;
Dodd, J. H.; Schieven, G. L.; Leftheris, K. J. Med. Chem.
2005, 48, 6261; (b) Hynes, J., Jr.; Dyckman, A. D.; Lin, S.;
Wrobleski, S. T.; Wu, H.; Gillooly, K. M.; Lonial, H.;
Loo, D.; McIntyre, K. W.; Pitt, S.; Shen, D. R.; Shuster,
D. J.; Zhang, X.; Behnia, K.; Marathe, P. H.; Doweyko,
A.; Barrish, J.; Dodd, J.; Schieven, G.; Leftheris, K. J.
Med. Chem, submitted for publication.
9. Mavunkel, B. J.; Chakravarty, S.; Perumattam, J. J.;
Luedtke, G. R.; Liang, X.; Lim, D.; Xu, Y.-J.; Laney, M.;
Liu, D. Y.; Schreiner, G. F.; Lewicki, J. A.; Dugar, S.
Biorg. Med. Chem. Lett. 2003, 13, 3087.
10. Dugar, S. 7th World Congress on Inflammation, Mel-
bourne, Australia., August 20–24, 2005.
11. Dugar, S. WO2004/032874.
In conclusion, we have identified novel series of pyrazol-
o[4,3-c]pyridine (A1), imidazo[1,5-a]pyrazine (A2) and
pyrido[3,4-d]pyrimidine (A3)-based p38a inhibitors.
Incorporation of an oxalamide moiety at the C-3 posi-
tion of the indole significantly improved the enzyme po-
tency in these series. One of the compounds was a potent
inhibitor of TNF-a release (compound 21, Table 3,
IC50 = 460 nM) in human peripheral blood mononu-
clear cells (PBMCs).14
12. p38 Enzyme assay: the assays were performed in U-
bottom 384-well plates. The final assay volume was 30 ll
prepared from 15 ll additions of enzyme and substrates
(fluoresceinated peptide FL-IPTSPITTTYFFFKKK-OH
and ATP) and test compounds in assay buffer (100 mM
Hepes, pH 7.2, 10 mM MgCl2, 0.015% Brij35, and 4 mM
DTT). The reaction was initiated by the combination of
bacterially expressed, activated p38 with substrates and
test compounds. The reaction mixture was incubated at
room temperature for 60 min and terminated by adding
30 ll of 35 mM EDTA to each sample. The reaction
mixture was analyzed on the Caliper LabChip 3000 by
electrophoretic separation of the fluorescent substrate and
phosphorylated product. Inhibition data were calculated
by comparison to no enzyme control reactions for 100%
inhibition and vehicle-only reactions for 0% inhibition.
The final concentrations of reagents in the assays are ATP,
20 lM; FL-IPTSPITTTYFFFKKK-OH, 1.5 lM; p38,
6 nM; and DMSO, 0.6%.
Acknowledgments
The authors thank their colleagues Susan E. Kiefer, John
A. Newitt, and Kevin Kish for their efforts in expressing
and purifying p38a protein and producing p38a co-crys-
tals with compound 24 that were used in this study.
References and notes
1. Wagner, G.; Laufer, S. Med. Res. Rev. 2006, 26, 1.
2. (a) Kavanaugh, A. Adv. Ther. 2006, 23, 208; (b) Fleisch-
mann, R. Expert Rev. Clin. Immunol. 2006, 2, 331.
3. Grunke, M.; Kalden, J. R. Expert Rev. Clin. Immunol.
2005, 1, 313.
4. (a) Saleem, B.; Mackie, S.; Emery, P. Expert Rev. Clin.
Immunol. 2006, 2, 193; (b) Cottone, M.; Mocciaro, F.;
Modesto, I. Expert Opin. Biol. Ther. 2006, 6, 401.
5. For reviews of this area see: (a) Hynes, J., Jr.; Leftheris, K.
Curr. Top. Med. Chem. 2005, 5, 967; (b) Goldstein, D. M.;
13. PDB deposition number is 2QD9.
14. Dyckman, A.; Hynes, J.; Leftheris, K.; Liu, C.; Wrobleski,
S. T. WO 090912, 2003, Chem. Abstr. 2003, 139, 292275.