676
C. Zhang et al. / Bioorganic & Medicinal Chemistry Letters 21 (2011) 670–676
7. Hausenloy, D. J. Clin. Lipidol. 2009, 4, 279.
8. Mitch, C. H.; Leander, J. D.; Mendelsohn, L. G.; Shaw, W. N.; Wong, D. T.;
Cantrell, B. E.; Johnson, B. G.; Reel, J. K.; Snoddy, J. D.; Takemori, A. E.;
Zimmerman, D. M. J. Med. Chem. 1993, 36, 2842.
N
a
N
9. Shaw, W. N. Pharmacol. Biochem. Behav. 1993, 46, 653.
10. Neary, P.; Delaney, C. P. Expert Opin. Investig. Drugs. 2005, 14, 479.
11. Portoghese, P. S. J. Med. Chem. 1965, 8, 609.
12. Fries, D. S. Opioid analgesics. In Principles of Medicinal Chemistry; Foye, W. O.,
Lemke, T. L., Williams, D. A., Eds.; Williams & Wilkins: Baltimore, 1995; p 453.
13. Wentland, M. P.; Lou, R.; Ye, Y.; Cohen, D. J.; Richardson, G. P.; Bidlack, J. M.
Bioorg. Med. Chem. Lett. 2001, 11, 623.
14. Pedregal-Tercero, C.; Siegel, M. G.; Stucky, R. D.; Takeuchi, K. WO2004080968,
2004.
HN
CHO
3
22
15. Blanco-Pillado, M.; Chappell, M. D.; Garcia De la Torre, M.; Diaz Buezo, N.; Fritz,
J. E.; Holloway, W. G.; Matt, Jr., J. E.; Mitch, C. H.; Pedregal-Tercero, C.; Quimby,
S. J.; Siegel, M. G.; Smith, D. R.; Stucky, R. D.; Takeuchi, K.; Thomas, E. M.; Wolfe,
C. N. WO2004026305, 2004.
O
O
OH
16. Pingali, S. R. K.; Madhav, M.; Jursic, B. S. Tetrahedron Lett. 2010, 5, 1383.
17. Balczewski, P.; Joule, J. A. Synth. Commun. 1990, 20, 2453.
OH
N
18. [
35S]GTPgammaS binding assay measured by LEADseeker SPA (384 well):
b
Dilute GTPgS35 1:900 in assay buffer in half of required final assay volume
(volume A). Add the corresponding standard agonist, Met-Enkephalin (hOPRD),
Dynorphin A (hOPRK) or DAMGO (hOPRM) to give a solution concentration of
8ꢁ [EC50], for a final assay concentration of 4ꢁ [EC50] to volume A. Re-suspend
LEADSeeker beads in assay buffer in order to generate a 40 mg/mL stock
N
11
23
HN
solution. GDP is dissolved in assay buffer at 1 mM. Add beads (100
final) to assay buffer containing saponin (60 g/mL) in half of final assay
volume (volume B). Mix well by vortexing. Add opioid membranes to each
respective volume B, for a final assay concentration of 1.5 g/well (hOPRD),
lg/well
CHO
l
l
1.0 lg/well (hOPRK), and 1.5 lg/well (hOPRM). Continuously mix the bead/
membrane solution (volume B) for 30 min prior to adding to the GTPgS35
solution (volume A) in a 1:1 ratio using a stir plate. Just prior to adding bead/
Scheme 6. Reagents and conditions: (a) and (b) 2-(1-cyclohexen-1-yl)ethanamine,
membrane solution to the GTPgS35 solution, add GDP to volume B at 20
lM
(10
l
M final assay concentration). Add the bead/membrane solution to the
NaBH(OAc)3, MeOH, rt.
GTPgS35 solution in a 1:1 ratio. Add 10
l
L of the bead/membrane/GTPgS35 mix
to the assay plate using a Multidrop (Titertek). Agitation of the solution is
needed to prevent the beads/membrane from settling at the bottom. Plates are
sealed, spun at 1000 rpm for 2 min, tapped on side to agitate and incubated at
room temperature for 5 h. Plates are then imaged using a Viewlux Plus Imager
(Perkin–Elmer).
References and notes
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