2250
Y. Wang et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2247±2250
that the M2 antagonists upon oral administration,
stimulate brain acetylcholine release in functional
microdialysis assay. A detailed structure±activity rela-
tionship study of this class of compounds, as well as their
in vivo ecacy in animal models of cognition, will be
published in the future.
golo, N. Life Sci. 1999, 64, 535. (b) Doller, D.; Chacka-
lamannil, S.; Czarniecki, M.; McQuade, R.; Ruperto, V.
Bioorg. Med. Chem. Lett. 1999, 9, 901. (c) Engel, W. E.;
Eberlein, W. G.; Mihm, G.; Hammer, R.; Trummlitz, G. J.
Med. Chem. 1989, 32, 1718. (d) Malaska, M. J.; Fauq, A. H.;
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7. Billard, W.; Binch, H., III; Bratzler, K.; Chen, L. Y.;
Crosby, G.; Duy, R. A.; Dugar, S.; Lachowicz, J.; McQuade,
R.; Pushpavanam, P.; Ruperto, V. B.; Taylor, L. A.; Clader, J.
W. submitted together with this letter for publication.
8. Pine, S. H.; Shen, G. S.; Hoang, H. Synthesis 1991, 165.
9. Shimazaki, M.; Takahashi, M.; Komatsu, H.; Ohta, A.;
Kajiii, K.; Kodama, Y. Synthesis 1992, 555.
Acknowledgements
The authors would like to thank Mr. Yao H. Ing and
Dr. P. Das for mass spectral data, and Dr. Murgolo for
molecular modeling studies.
10. All of the target compounds showed satisfactory result in
the analyses of NMR, MS, LC/MS, and HRMS.
11. For radioligand binding analysis, each muscarinic recep-
tor subtype was stably expressed in CHO-K1 cells. Clonal cell
lines were selected which expressed receptors at levels between
1 and 9 pmol/mg protein. The Kd of QNB (l-quinuclidinyl
benzilate) at each receptor subtype was determined by satura-
tion binding using 5±2500 pM [3H]QNB in 10 mM potassium
phosphate buer, pH 7.4. Protein concentrations were adjus-
ted for each assay to achieve between 700 and 1500 cpm spe-
ci®c binding. Competition binding experiments were
performed using 180 pM [3H] QNB. All binding experiments
were performed in the presence of 1% DMSO and 0.4%
methylcellulose. Non-speci®c binding was de®ned by 0.5 mM
atropine. After equilibrium was reached (120 min incubation
at room temperature), bound and free radioactivity were
separated by ®ltration using Whatman GF-C ®lters. Investi-
gation of M2 receptor antagonist activity was performed on
related compounds by measuring the eects of the compounds
adenylyl cyclase inhibition mediated by oxotremorine M (see
6a for details).
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