Two-electrode voltage-clamp electrophysiology. For expression
in Xenopus oocytes, rat NR1-1a (GenBank U11418) cDNA was
subcloned into a pCI-IRES-neo vector and rat NR2A (GenBank
D13211), NR2B (GenBank M91562), NR2C (GenBank D13212),
and NR2D (GenBank D13214) cDNAs were subcloned into
a pCI-IRES-bla vector containing a T7 site upstream from
the 5ꢀ untranslated region. Constructs used for expression in
Xenopus oocytes were linearized by restriction enzymes in order
to produce cRNAs, using mMessage mMachine kits.37 Oocytes
were surgically removed from mature female Xenopus laevis as
previously described27 and co-injected with cRNA encoding NR1-
1a and NR2B at a 1 : 1 ratio and maintained at 18 ◦C in Barth’s
solution (in mM: 88 NaCl, 1.0 KCl, 2.4 NaHCO3, 0.41 CaCl2,
0.82 MgSO4, 0.3 Ca(NO3)2, and 15 HEPES pH 7.6) supplemented
with 100 IU mL−1 penicillin and 100 lg mL−1 streptomycin
(Invitrogen, Carlsbad, CA). The electrophysiological recordings
and data analysis were performed as previously described.30
for determining the pKa values. This work was supported by
the Danish Medical Research Council and the Novo Nordisk
Foundation.
References
1 M. L. Mayer, Curr. Opin. Neurobiol., 2005, 15, 282–288.
2 J. N. C. Kew and J. A. Kemp, Psychopharmacology, 2005, 179, 4–29.
3 J. P. Pin, T. Galvez and L. Prezeau, Pharmacol. Ther., 2003, 98, 325–354.
4 H. Bra¨uner-Osborne, J. Egebjerg, E. Ø. Nielsen, U. Madsen and P.
Krogsgaard-Larsen, J. Med. Chem., 2000, 43, 2609–2645.
5 S. Schorge and D. Colquhoun, J. Neurosci., 2003, 23, 1151–1158; S.
Cull-Candy, S. Brickley and M. Farrant, Curr. Opin. Neurobiol., 2001,
11, 327–335.
6 R. Dingledine, K. Borges, K. Bowie and S. F. Traynelis, Pharmacol.
Rev., 1999, 51, 7–61.
7 C. H. Eugster, G. F. R. Mu¨ller and R. Good, Tetrahedron Lett., 1965,
23, 1813–1815; D. Michelot and L. M. Melendez-Howell, Mycol. Res.,
2003, 107, 131–146; C. Li and N. H. Oberlies, Life Sci., 2005, 78, 532–
538.
8 M. B. Hermit, J. R. Greenwood, B. Nielsen, L. Bunch, C. G. Jørgensen,
H. T. Vestergaard, T. B. Stensbøl, C. Sanchez, P. Krogsgaard-Larsen,
U. Madsen and H. Bra¨uner-Osborne, Eur. J. Pharmacol., 2004, 486,
241–250.
9 H. Bra¨uner-Osborne, B. Nielsen and P. Krogsgaard-Larsen,
Eur. J. Pharmacol., 1998, 350, 311–316.
10 L. Bunch, P. Krogsgaard-Larsen and U. Madsen, J. Org. Chem., 2002,
67, 2375–2377.
11 P. L. Ornstein, T. J. Bleisch, M. B. Arnold, R. A. Wright, B. G. Johnson
and D. D. Schoepp, J. Med. Chem., 1998, 41, 346–357.
12 H. T. Vestergaard, S. B. Vogensen, U. Madsen and B. Ebert, Eur. J. Phar-
macol., 2004, 488, 101–109.
mGluR activity. Chinese hamster ovary (CHO) cell lines stably
expressing rat mGluR1a, mGluR2 and mGluR4a were prepared as
previously described38 (see also Clausen et al.27 for further details).
Measurement of intracellular Ca2+ levels and cyclic AMP
formation: pharmacological activity at mGluR1a was assessed by
measurement of intracellular Ca2+ levels as previously described.13
Pharmacological activity at mGluR2 and mGluR4a was assessed
by measuring intracellular cAMP levels as previously described39
(see also Clausen et al.27 for further details).
13 E. J. Bjerrum, A. S. Kristensen, D. S. Pickering, J. R. Greenwood,
B. Nielsen, T. Liljefors, A. Schousboe, H. Bra¨uner-Osborne and U.
Madsen, J. Med. Chem., 2003, 46, 2246–2249.
Molecular modeling
14 P. Cal´ı and M. Begtrup, Synthesis, 2002, 63–66.
A homology model of the agonized ligand binding domain of
NR2B was constructed using the crystal structure of the soluble
NR2A–S1S2 construct in complex with Glu (Protein Data Bank
code 2A5S) as a template. The sequence of NR2B was aligned with
NR2A and truncated so that a sequence consisting of residues
D404–R540 from S1, followed by the GT linker and residues
Q662–H826 from S2 forms a virtual NR2B–S1S2 construct.
Residues are numbered according to the sequence of total NR2B
protein. From this sequence, a model structure of NR2B was
generated using Prime 3.5.32 Glu and water molecules 6 and 48
from 2A5S were added to the resulting structure and submitted
to the standard preparation and refinement procedure in Glide
3.532 to assign charges, add hydrogens, and perform a series of
15 S. N. Lewis, G. A. Miller, M. Hausman and E. C. Szamborski,
J. Heterocycl. Chem., 1971, 8, 571–580.
16 N. R. A. Beeley, L. M. Harwood and P. C. Hedger, J. Chem. Soc.,
Perkin Trans. 1, 1994, 1, 2245–2251.
17 K. Taubert, S. Kraus and B. Schulze, Sulfur Rep., 2002, 23, 79–121.
18 E. D. Weiler, M. Hausman and G. A. Miller, J. Heterocycl. Chem.,
1977, 14, 725–728; E. D. Weiler, M. Hausman and G. A. Miller, US
Pat., 3 957 808, 1976.
19 E. J. Corey and K. Achiwa, J. Org. Chem., 1969, 34, 3667–3668.
20 Y. Torisawa, T. Nishi and J. Minamikawa, Bioorg. Med. Chem., 2002,
10, 2583–2587.
21 H. M. Bell, C. W. Vanderslice and A. Spehar, J. Org. Chem., 1969, 34,
3923–3926.
22 G. W. Kabalka and J. D. Baker, J. Org. Chem., 1975, 40, 1834–1835.
23 G. A. Molander and C. S. Yun, Tetrahedron, 2002, 58, 1465–1470;
G. A. Molander, C. S. Yun, M. Ribagorda and B. Biolatto, J. Org.
Chem., 2003, 68, 5534–5539.
24 C. H. Eugster, Prog. Chem. Org. Nat. Prod., 1969, 27, 261–321.
25 K. Frydenvang, L. Matzen, P. Norrby, F. A. Sløk, T. Liljefors, P.
Krogsgaard-Larsen and J. W. Jaroszewski, J. Chem. Soc., Perkin Trans.
2, 1997, 2, 1783–1791.
26 T. N. Johansen, Y. L. Janin, B. Nielsen, K. Frydenvang, H. Bra¨uner-
Osborne, T. B. Stensbøl, S. B. Vogensen, U. Madsen and P. Krogsgaard-
Larsen, Bioorg. Med. Chem., 2002, 10, 2259–2266.
27 R. P. Clausen, K. B. Hansen, P. Cal´ı, B. Nielsen, J. R. Greenwood,
M. Begtrup, J. Egebjerg and H. Bra¨uner-Osborne, Eur. J. Pharmacol.,
2004, 499, 35–44.
28 B. Laube, R. Schemm and H. Betz, Neuropharmacology, 2004, 47, 994–
1007; P. E. Chen, M. T. Geballe, P. J. Stansfeld, A. R. Johnston, H.
Yuan, A. L. Jacob, J. P. Snyder, S. F. Traynelis and D. J. A. Wyllie,
Mol. Pharmacol., 2005, 67, 1470–1484; L. Kinarsky, B. Feng, D. A.
Skifter, R. M. Morley, S. Sherman, D. E. Jane and D. T. Monaghan,
J. Pharmacol. Exp. Ther., 2005, 313, 1066–1074.
constrained minimizations (OPLS-AA force field). On this final
model, Van der Waals and electrostatic grids within a 20 A
3
˚
box around the ligand were calculated using Glide 3.5, and these
grids were then used for ligand docking. The ligands 2a–2d were
submitted to a conformational search (Monte Carlo) in tri-ionized
forms using the MMFFs force field and water as solvent in
Macromodel 9.032 The lowest energy conformations were docked
with Glide 3.5 to the agonist binding site of the NR2B–S1S2
model using the automated flexible procedure. Default parameters
were used. All figures of the models were prepared using PyMol
software.32
Acknowledgements
29 N. Armstrong and E. Gouaux, Neuron, 2000, 28, 165–181.
30 K. B. Hansen, R. P. Clausen, E. J. Bjerrum, C. Bechmann, J. R.
Greenwood, C. Christensen, J. L. Kristensen, J. Egebjerg and H.
Bra¨uner-Osborne, Mol. Pharmacol., 2005, 68, 1510–1523.
The authors wish to acknowledge Preben Friis Hansen, H. Lund-
beck A/S, for assistance in up-scaling of the synthesis of 3-
benzyloxyisothiazole and Erling B. Jørgensen, H. Lundbeck A/S,
470 | Org. Biomol. Chem., 2007, 5, 463–471
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