R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5979–5983
5983
4. Tynebor, R. M.; Chen, M-H; Natarajan, S. R.; O’Neill, E. A.; Thompson, J. E.;
Fitzgerald, C. E.; O’Keefe, S. J.; Doherty, J. B. Bioorg. Med. Chem. Lett. 2010, 20, 2765.
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which was immediately reacted with either hydrazine to yield
hydrazone 28 or an amine to yield amides 18–19. Crude hydrazone
28 was then heated in trimethyl orthoacetate to generate oxadiaz-
ole 20 with a 3 step yield of 58% from acid 26.
In summary, our research efforts completed SAR in the pyrido-
pyridazin-6-one series and led to the discovery of 2-methoxy, 5(-5-
methyl-1,3,4-oxadiazol-2-yl) phenyl as the optimal A-ring for the
pyridopyridazin-6-one scaffold. As a result of our efforts, com-
(b) 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
pound 20 possessed subnanomolar p38
a activity with moderate
labeled GST-ATF2 substrate (4 lM). After incubation at 30 °C for 3 h, the
p38b isoform selectivity while significantly improving cellular/
functional potency and efficacy in the rat LPS model over lead 1.
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.
References and notes
6. 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 DMSO followed by addition
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of LPS. The reaction was incubated for 4 h at 37 °C under CO2. TNFa release was
measured in the supernatants by ELISA. Reported IC50s are means from three
measurements.
7. Natarajan, S. R.; Chen, M.-H.; Heller, S. T.; Tynebor, R. M.; Crawford, E. M.;
Minxiang, C.; Kaizheng, H.; Dong, J.; Hu, B.; Hao, W.; Chen, S.-H. Tetrahedron Lett.
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Fitzgerald, C. E.; O’Keefe, S. J.; Doherty, J. B. Bioorg. Med. Chem. Lett. 2011, 21, 411.