CB2-SelectiVe Cannabinoid Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5483
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cDNA sequences encoding either the human CB1 or the human
CB2 cannabinoid receptors were kindly provided by Dr. M.
Detheux and Dr. P. Nokin, respectively (Euroscreen s.a., Gosselies,
Belgium). Cell cultures and membrane preparation were reported
previously.19
Competition Binding Assay. [3H]-SR-141716A (52 Ci/mol) was
purchased from Amersham (Roosendaal, The Netherlands) and
[3H]-CP-55,940 (101 Ci/mol) from NEN Life Science (Zaventem,
Belgium). Binding assay procedure was previously reported,19 under
those conditions, using [3H]-SR-141716A, the Bmax value was 57
pmoles/mg protein and the Kd value was 1.13 ( 0.13 nM for the
hCB1 cannabinoid receptor, and using [3H]-CP-55,940, the Bmax
value was 194 pmoles/mg protein and the Kd value was 4.3 ( 0.13
nM for the hCB2 cannabinoid receptor. The results are expressed
as mean ( SEM of at least three experiments performed in
duplicate.
[35S]GTPγS Assay. [35S]-GTPγS (1173 Ci/mmol) was obtained
from Amersham (Roosendaal, The Netherlands). The experiments
were performed as previously described.19 Gpp(NH)p 100 µM
was used to determine the nonspecific binding. The results are
expressed as mean ( SEM of at least three experiments performed
in duplicate and are reported for a concentration of ligand of
10 µM.
Data Analysis. IC50 values were determined by nonlinear
regression analysis performed using the GraphPad prism 4.0
program (GraphPad Software, San Diego). The Ki values were
calculated from the IC50, based on the Cheng-Prusoff equation:
Ki ) IC50/(1 + L/Kd). The statistical significance of [35S]-GTPγS
assay results was assessed using a one-way ANOVA followed by
a Dunett post-test.
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Acknowledgment. Dr. Laurence Goossens is acknowledged
for her contribution to the chiral preparative HPLC separation
of 24, 25, and 59-62. GGM is a postdoctoral researcher from
the National Fund for Scientific Research (FNRS, Belgium).
This work was partially supported by Charcot Foundation
(Bruxelles, Belgium) and by the National Fund for Scientific
Research (FNRS, Cre´dit aux chercheurs. Belgium).
Supporting Information Available: Elemental analysis of
compounds 10-25, 29-35, 38-40, 42-44, 47, 52-58, 67, 68,
71, 76, and 80-98, spectroscopic data and melting point for all
compounds, and procedures for the synthesis of intermediates.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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