A. Alanine et al. / Bioorg. Med. Chem. Lett. 14 (2004) 817–821
821
Table 4. Recombinant hA2A, rA2A and hA1 receptor affinities of
compounds 38, 40, 42, 45 [variation of R], determined by displacement
of [3H]SCH-58261 [A2A] or [3H] DPCPX [A1] radioligand binding7
Ferre, S.; Karcz-Kubicha, M.; Hope, B. T.; Popoli, P.;
Burgueno, J.; Gutierrez, M. A.; Casado, V.; Fuxe, K.;
Goldberg, S. R.; Lluis, C.; Franco, R.; Ciruela, F. Proc.
Natl. Acad. Sci. 2002, 99, 11940.
4. Yacoubi, M. E.; Ledent, C.; Parmentier, M.; Bertorelli,
R.; Ongini, E.; Costentin, J.; Vaugeois, J.-M. Brit. J.
Pharmacol. 2001, 134, 68.
5. Richardson, P. J.; Kase, H.; Jenner, P. G. Trends Phar-
macol. Sci. 1997, 18, 338.
6. The original hit was identified as the cyclic version and
was later confirmed to be the open chain form.
Compd
R
hA2A Ki hA1 Ki sel. rA2A Ki PAMPA8
[mM]a
[mM]a
[mM]a
10À6 s/cm
38
40
42
45
0.01
0.1
7
51
69
20
0.04
1.9 (high)
7. hA2A, hA1 and rA2A receptor affinities were determined in
a 96 well plate assay, using [3H]SCH-58261 (hA2A and
rA2A; final concentration 1 and 0.4 nM, respectively)
and [3H] DPCPX (final concentration 0.6 nM) to radi-
olabel the receptors in the presence of 10 concentrations
of competing compound or buffer. Non-specific binding
was determined using 2 mM Xanthine amine congener
(XAC). Assay buffer consisted of Tris–HCl (50 mM, pH
0.02
0.02
0.1
0.9
1.7
2.5
0.04
0.09
0.4
3.8 (high)
0.9 (high)
0.3 (med.)
.
7.4), NaCl 120 mM, KCl 5 mM, CaCl2 2H2O 2 mM,
.
MgCl2 6H2O 10 mM. Membrane preparations of recep-
tors (approximately 2 mg/well in a 96-well plate) were
mixed with adenosine deaminase (10 mg/mL) and scintil-
lation proximity beads (SPA; 0.5 mg/well; Amersham)
and added to the wells to initiate the incubation for 60
min (hA2A and hA1) or 90 min (rA2A) at room temper-
ature. The assay was terminated by centrifugation (3000
rpm, 3 min) and counted in a scintillation counter (Top-
count, Packard). All assays were performed in duplicate
in at least two separate experiments. Graphs were plotted
with the % specific binding using the iterative curve fitting
program XL-fit.
References and notes
1. Fredholm, B. B.; Ijzerman, A. P.; Jacobson, K. A.; Klotz,
K.-N.; Linden, J. Pharmcol. Rev. 2001, 53, 527.
2. (a) Svenningsson, P.; Le Moine, C.; Fisone, G.; Fred-
holm, B. B. Progress in Neurobiology 1999, 59, 355. (b)
Hettinger, B. D.; Lee, A.; Linden, J.; Rosin, D. L. J.
Comp. Neurology 2001, 431, 331.
3. (a) Hillion, J.; Canalx, M.; Torvinen, M.; Casado, V.;
Scott, R.; Terasmaa, A.; Hansson, A.; Watson, S.; Olah,
M. E.; Mallol, J.; Canela, E. I.; Zoli, M.; Agnati, L. F.;
Ibanez, C. F.; Lluis, C.; Franco, R.; Ferre, S.; Fuxe, K. J.
Biol. Chem. 2002, 277, 18091. (b) Johansson, B.; Geor-
giev, V.; Fredholm, B. B. Neuroscience 1997, 80, 1187. (c)
8. PAMPA=Parallel Artificial Membrane Permeation
Assay: Kansy, M.; Senner, F.; Gubernator, K. J. Med.
Chem. 1998, 41, 1007.
9. Solubility determined in 0.05 M phosphate buffer at pH
6.5.