C. Carmi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4021–4025
4025
7. EGFR kinase assay was performed in 100 lL reaction
mixtures containing kinase buffer (10 mM Tris–HCl, pH
7.4, 1 mM MgCl2, 2 mM MnCl2, 10 lM Na3VO4 and
0.1 mg/mL BSA), 4 U EGFR (Sigma Chemicals, MO,
USA), 10 lL of 66 nM EGF (Sigma Chemicals, MO,
USA), 10 lL of 5 mg/ml polyGAT peptide (Sigma Chem-
icals, MO, USA). Compounds (1 lL) dissolved in DMSO
and conveniently diluted were added and the reactions
initiated by adding 1 lL of 100 lM ATP, 2 lCi of
[c-32P]ATP. After 60 min at 30 °C, the reactions were
stopped by adding 900 lL TCA 11% and stored at 4 °C for
3 h. Afterwards, each reaction solution was spotted onto a
phosphocellulose disc (B2.5 cm, Whatman, P81), previ-
ously equilibrated in 1 mM ATP. Then, each disc was
washed three times with TCA 5% at rt, plunged in TCA
5% for 40 min, dried, and incorporated 32P was counted in
a scintillation counter. Gefitinib 1 nM was used as an
internal standard in each experiment. Percent inhibition of
compounds was calculated by comparison with DMSO-
treated controls.
8. Procedures for protein extraction, solubilization, and
analysis by 1-D PAGE were described in Petronini, P.
G.; Alfieri, R.; De Angelis, E.; Campanini, C.; Borghetti,
A. F.; Wheeler, K. P. Br. J. Cancer 1993, 67, 493, A mouse
anti-phospho-EGF receptor (Tyr1173) monoclonal anti-
body at 1:2500 dilution (Upstate, Cell Signaling Solution,
Lake Placid, NY, USA) and a horseradish peroxidase
(HRP)-secondary antibody at 1:20,000 dilution (Amer-
sham Pharmacia Biotech, Buckinghamshire, UK) were
used.
indicates that access to the biophase is not a main
drawback for these compounds.
Finally, the easy and flexible synthesis and workup for
the 5-exo-methylene-substituted derivatives, which can
be exploited to introduce different substituents at posi-
tions 1 and 5, allowing easy exploration of physico-
chemical space, highlight the potential of this scaffold
to generate new kinase inhibitors.
Acknowledgments
`
The Centro Interfacolta Misure and Centro di Calcolo
Elettronico of the University of Parma are gratefully
acknowledged for the use of NMR instrumentation
and software licenses. This investigation was supported
by grants from Regione Emilia Romagna, Associazione
Chiara Tassoni, Parma, and from Associazione Davide
Rodella, Montichiari.
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