F. Medda et al. / Bioorg. Med. Chem. Lett. 23 (2013) 528–531
531
5. Greenhough, A.; Smartt, H. J. M.; Moore, A. E.; Roberts, H. R.; Williams, A. C.;
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reflux for 0.5 h. After cooling down to room temperature, aqueous NaOH (aq)
was added at once and the product 5 precipitated as a white microcrystalline
solid which was isolated by suction filtration and used in the next reaction
without further purification (1.72 g, 6.05 mmol, 80%). 5 (200 mg, 0.70 mmol,
1 equiv) was dissolved in dioxane-H2O (4:1, 5 mL) and phenylboronic acid
(171 mg, 1.40 mmol, 2 equiv), Bis(triphenylphosphine)palladium(II) dichloride
(25 mg, 0.03 mmol, 0.05 equiv) and K2CO3 (193 mg, 1.39 mmol, 2 equiv) were
added. The reaction was subjected to microwave irradiation for 1 h at 100 °C.
After filtering through celite, H2O (50 mL) was added and the aqueous layer
extracted with DCM (3 Â 50 mL). The organic layers were collected, washed
with brine (50 mL), dried (MgSO4) and the solvent removed under reduced
pressure. The final product 2xxiv was purified by silica-gel column
chromatography (hexane/EtOAc, 0–50%) using an ISCOTM system and
obtained as a yellow microcrystalline powder (50 mg, 0.33 mmol, 48%). Mp:
210–213 °C; 1H NMR (400 MHz, DMSO-d6) d 9.11 (br s, 1H), 8.61 (d, J = 8.3 Hz,
1H), 7.97 (ddd, J = 8.3, 6.8, 1.5 Hz, 1H), 7.89 (ddd, J = 8.0, 6.8, 1.1 Hz, 1H), 7.86–
7.77 (m, 3H), 7.66–7.60 (m, 2H), 7.58–7.47 (m, 3H), 6.98–6.92 (m, 2H), 3.75 (s,
3H). 13C NMR (101 MHz, DMSO-d6) d 155.4, 153.3, 152.4, 137.4, 134.0, 132.4,
131.9, 130.1, 128.9, 128.8, 126.1, 126.1, 123.6, 123.0, 118.7, 114.1, 55.7. LC–MS
[MH]+ 328.00. Purity (LC–MS): >99% (ELSD-LT); >99% (254 nm); 90% (214 nm).
14. PGE2 production assay: Cells were seeded in 6-well plates and incubated
overnight in DMEM/10% FBS. They were serum starved for the next 18 h. Cells
were then treated with 10 ng/ml IL-1b and increasing concentration of
compounds (dissolved in DMSO) in 1 mL serum-free medium. After 72 h of
incubation, the supernatants were collected for PGE2 level detection using the
PGE2 EIA kit (R&D Systems).
9. Moon, J. T.; Jeon, J. Y.; Park, H. A.; Noh, Y.-S.; Lee, K.-T.; Kim, J.; Choo, D. J.; Lee, J.
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Meuillet, E. J.; Hulme, C. Bioorg. Med. Chem. 2012, 22, 3567; (b) Chang, H. H.;
Song, Z.; Wisner, L.; Tripp, T.; Gokhale, V.; Meuillet, E. J. Invest. New Drugs.
2012, 1865, 30.
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Piatnitski Chekler, E. L.; Katoch-Rouse, R.; Sherman, D.; Wong, W. C.; Smith, L.
M., II; Kawakami, J. K.; Kiselyov, A. S.; Milligan, D. L.; Balagtas, C.; Hadari, Y. R.;
Wang, Y.; Patel, S. N.; Rolster, R. L.; Tonra, J. R.; Surguladze, D.; Mitelman, S.;
Kussie, P.; Bohlen, P.; Doody, J. F. Bioorg. Med. Chem. 2009, 17, 731.
13. General procedure for the preparation of analog 2xxiv: 1,4-Dichlorophthalazine
(3, 1.50 g, 7.57 mmol, 1 equiv) and para-anisidine (4, 940 mg, 7.57 mmol,
1 equiv) were mixed together in ethanol (10 mL) and the reaction stirred at
15. COX-2 cell-free assay: COX-2 activity was measured by a COX Fluorescent
inhibitor screen assay kit following the manufacturer’s instructions (Cayman