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6. The EGFR was purchased from Upstate (Active EGFR,
Cat. No. 14-531) and used directly without
further purification. Kinase assay experiments were per-
formed according to the procedure as described in the
booklet.
7. (a) Bridges, A. J.; Zhou, H.; Cody, D. R.; Rewcastle, G.
W.; McMichael, A.; Showalter, H. D. H.; Fry, D. W.;
Kraker, A. J.; Denny, W. A. J. Med. Chem. 1996, 39, 267;
(b) Barker, A. J.; Gibson, K. H.; Grundy, W.; Godfrey, A.
A.; Barlow, J. J. Bioorg. Med. Chem. Lett. 2001, 11, 1911;
(c) Petrov, K. G.; Zhang, Y.-M.; Carter, M.; Cockerill, G.
S.; Dickerson, S.; Gauthier, C. A.; Guo, Y.; Mook, R. A.,
Jr.; Rusnak, D. W.; Walker, A. L.; Wood, E. R.; Lackey,
K. E. Bioorg. Med. Chem. Lett. 2006, 16, 4686.
8. Some cases of C-30 ethynyl-substituted 4-anilinoquinazo-
line derivatives, see: Waterson, A. G.; Stevens, K. L.;
Reno, M. J.; Zhang, Y.-M.; Boros, E. E.; Bouvier, F.;
Rastagar, A.; Uehling, D. E.; Dickerson, S. H.; Reep, B.;
McDonald, O. B.; Wood, E. R.; Rusnak, D. W.; Alligood,
K. J.; Rudolph, S. K. Bioorg. Med. Chem. Lett. 2006, 14,
2419.
Figure 1. Interacting poses of Tarceva (colored in red), compounds 5e
(colored in yellow), and 6b (colored in green) in EGFR (pdb code 2ito)
from docking modeling.11 The molecular surface colored by atom
partial charges is plotted to show the binding pocket of EGFR. The
distance of the C-30 ethynyl end of Tarceva (also compound 6b) is only
˚
2.8 A to the backbone carbonyl oxygen atom of Leu788. The C-6
hydroxy group in 5e has an additional hydrogen bonding with the
backbone carbonyl oxygen atom of Leu718.
very potent EGFR kinase inhibitor with an IC50 of
14 nM.
9. All of the new compounds were characterized. Preparation
of compound 5e is described herein. A solution of
compound 4, propargyl alcohol, copper(I) iodide
(0.05 equiv), bis(triphenylphosphine)palladium chloride
(0.05 equiv), and triethylamine (2 equiv) in tetrahydrofu-
ran was stirred and refluxed under nitrogen overnight.
After the reaction was completed, the mixture was filtered
through silica gel and eluted with ethyl acetate. The
solvent was evaporated, and the mixture was purified by
chromatography with hexane/ethyl acetate to give the
desired product. 1H NMR (500 MHz, DMSO-d6) d 9.99 (s,
1H), 8.72 (s, 1H), 8.65 (s, 1H), 8.23 (dd, J = 6.8, 2.3 Hz,
1H), 7.87–7.82 (m, 2H), 7.77 (m, 1H), 7.46 (t, J = 9.1 Hz),
5.44 (t, J = 5.6 Hz, 1H), 4.39 (d, J = 5.2 Hz, 2H). 13C
NMR (125 MHz, DMSO-d6) d 157.0, 154.9, 153.4, 149.1,
136.3, 135.3, 128.3, 126.2, 123.5, 122.3, 120.3, 118.8, 116.7,
114.9, 91.3, 83.1, 49.5. MS (EI) m/z 328 MH+.
10. The kinase activity of EGFR was measured by the
incorporated radiolabeled cATP into a tyrosine-contain-
ing peptide. In brief, 5 ll of testing compound was mixed
well with 5 ll of 5· reaction buffer, 2.5 ll of EGFR
(active, 25 ng), 1.25 ll of PolyGlu:Tyr (4/1, 5 lg), 1.25 ll
of manganese chloride (10 mM), 5 ll of ammonium sulfate
(0.8 M), and 5 ll of [c-33P] MgAc/ATP cocktail containing
magnesium acetate (50 mM) and ATP (500 lM). The
reaction mixture of a total volume of 25 ll was incubated
at 30 ꢁC for 30 min. Five microliters of phosphoric acid
(3%) was added to stop the reaction. An aliquot of 20 ll
from reaction was transferred onto the center of a
2 cm · 2 cm P81 paper and allow the radiolabeled sub-
strate to bind to the filter paper for 30 s. Wash the assay
squares five times for 5 min with water. Transfer the assay
squares to vial and add 5 ml scintillation cocktail. Read in
scintillation counter by Beckman LS-6500 counter.
Acknowledgments
The authors thank the Ministry of Economy Affairs of
ROC (Taiwan) for financially supporting this research.
Ms. M.H. Lee is appreciated for assistance with the
NMR analysis.
References and notes
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