Arch. Pharm. Chem. Life Sci. 2012, 000, 1–7
Opioid-Active Thiazolo- and Thiazinomorphinans
7
5 mL of ice-cold 50 mM Tris–HCl buffer (pH 7.4). Non-specific
binding was determined in the presence of 10 mM naloxone. The
bound radioactivity was measured by liquid scintillation count-
ing using a Beckman CoulterTM LS6500 (Beckman Coulter Inc.,
Fullerton, CA, USA).
Spetea, M. Koch, R. Meditz, E. Greiner, R. B. Rothman, H.
Schmidhammer, J. Med. Chem. 2002, 45, 5378–5383; (d) P. S.
Portoghese, H. Nagase, K. E. Maloney Huss, C. E. Lin, A. E.
Takemori, J. Med. Chem. 1991, 34, 1715–1720; (e) F. Farouz-
Grant, P. S. Portoghese, J. Med. Chem. 1997, 40, 1977–1981.
Inhibition constant (Ki) values were calculated from compe-
tition binding curves using the nonlinear least-square curve
fitting by GraphPad Prism software (v3; GraphPad Software
Inc., San Diego, CA, USA). All experiments were performed in
duplicate and repeated two to six times.
[5] (a) K. Go¨rlitzer, R. Schumann, Pharmazie 1992, 47, 893–897;
(b) K. Go¨rlitzer, R. Schumann, Pharmazie 1993, 48, 30–33.
[6] A. Zhang, F. Li, C. Ding, Q. Yao, B. I. Knapp, J. M. Bidlack, J. L.
Neumeyer, J. Med. Chem. 2007, 50, 2747–2751.
[7] F. Li, C. Yin, J. Chen, J. Liu, X. Xie, A. Zhang, Chem. Med. Chem.
2009, 4, 2103–2110.
[35S]GTPgS (guanosine-50-O-(3-[35S]thio)-triphosphate)
binding assay
´
´
[8] (a) M. Toth, Zs. Gyulai, S. Berenyi, A. Sipos, Lett. Org. Chem.
2007, 4, 539–543; (b) A. Sipos, L. Giran, H. Mittendorfer,
´
Rat brain membranes (10 mg of protein) were incubated for
60 min at 308C in Tris–EGTA buffer (50 mM Tris–HCl buffer,
3 mM MgCl2, 1 mM EGTA, 100 nM NaCl, pH 7.4) containing
30 mM GDP, 0.05 nM [35S]GTPgS and appropriate concentrations
of test compound in a final volume of 1 mL. Non-specific binding
was measured in the presence of 100 mM unlabeled GTPgS.
Reactions were terminated by vacuum filtration through
Whatman GF/B glass fiber filters and bound [35S]GTPgS retained
on the filters was determined as described for opioid receptor
binding assays. Stimulation of [35S]GTPgS binding produced by
the test compound is given as percentage of the basal activity
(defined as 100%, measured in the absence of test compound).
The EC50 (nM, concentration of ligand to elicit half-maximal
effect) and Emax (%, maximum stimulation) were calculated using
nonlinear regression analysis and sigmoidal curve fitting with
the GraphPad Prism software. All experiments were performed
in triplicate and repeated at least three times.
´
H. Schmidhammer, S. Berenyi, Tetrahedron 2008, 64, 1023–
1028.
[9] A. Coop, W. J. Janetka, J. W. Lewis, K. C. Rice, J. Org. Chem.
1998, 63, 4392–4396.
´
´
[10] S. Berenyi, Cs. Csutoras, A. Sipos, Curr. Med. Chem. 2009, 16,
3215–3242.
[11] P. Osei-Gyimah, S. Archer, J. Med. Chem. 1980, 23, 162–
166.
[12] H. Conroy, J. Am. Chem. Soc. 1955, 77, 5960–5966.
[13] M. Spetea, T. Friedmann, P. Riba, J. Schu¨tz, G. Wunder,
T. Langer, H. Schmidhammer, S. Fu¨rst, Eur. J. Pharmacol.
2004, 483, 301–308.
¨
´
¨
´
[14] M. Spetea, F. Toth, J. Schutz, F. Otvo¨s, G. Toth, S. Benyhe,
A. Borsodi, H. Schmidhammer, Eur. J. Neurosci. 2003, 18, 290–
295.
[15] B. E. Kane, B. Svenson, D. M. Ferguson, AAPS J. 2006, 8, E126–
E137.
Computational procedure
[16] (a) A. D. Becke, J. Chem. Phys. 1993, 98, 5648–5652; (b) A. D.
Becke, Phys. Rev. A 1998, 38, 3098–3100; (c) C. Lee, R. G. Parr,
W. Yang, Phys. Rev. B 1988, 37, 785–789; (d) S. H. Vosko,
L. Wilk, M. Nusair, Can. J. Phys. 1980, 58, 1200–1211.
We carried out the geometry optimization at Becke’s three
parameter hybrid (B3LYP) [16] levels in the DFT with the basis
set 6–31G(d,p) using Gaussian 03 [17]. The solvent effect was not
considered.
[17] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Robb, J. R. Cheeseman, J. A. Montgomery, Jr. T. Vreven, K. N.
Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi,
V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A.
Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota,
R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda,
O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian,
J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts,
R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi,
C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma,
G. A. Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzewski,
S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick,
A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz,
Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov,
G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J.
Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara,
M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W.
Wong, C. Gonzalez, J. A. Pople Gaussian 03, Revision B.05,
Gaussian Inc., Wallingford CT 2003.
Supported by the Hungarian Research Fund (K81701) and the Austrian
Science Fund (FWF: 15481 and TRP 19-B18).
The author have declared no conflict of interest.
References
[1] M. Eguchi, Med. Res. Rev. 2004, 24, 182–212.
[2] J. V. Aldrich, S. C. Vigil-Cruz, Narcotic Analgesics. In Burger’s
Medicinal Chemistry and Drug Discovery, John Wiley & Sons,
New York 2003, p. 329.
[3] P. S. Portoghese, J. Med. Chem. 2001, 44, 2259–2269.
[4] (a) A. E. Takemori, P. S. Portoghese, Ann. Rev. Pharmacol.
Toxicol. 1992, 32, 239–269; (b) S. K. Sharma, R. Jones, T. G.
Metzger, D. M. Ferguson, P. S. Portoghese, Med. J. Chem. 2001,
44, 2073–2079; (c) J. Schu¨tz, C. M. Dersch, R. Horel, M.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com