158
J. Chang-Fong et al. / Bioorg. Med. Chem. Lett. 12 (2002) 155–158
assay conditions that match the literature conditions as
closely as possible. The h5-HT2A binding results were
8. Glennon, R. A.; Jacyno, J. M.; Young, R.; McKenney,
J. D.; Nelson, D. J. Med. Chem. 1984, 27, 41.
9. Bos, M.; Jenck, F.; Martin, J. R.; Moreau, J. L.; Sleight,
¨
A. J.; Wichmann, J.; Widmer, U. J. Med. Chem. 1997, 40, 2762.
3
confirmed using a second agonist radioligand ([ H]5-
3
HT), as were the h5-HT2C results [using ([ H]DOB). In
fact, under the latter conditions, compound 10 dis-
played only 3-fold selectivity for 5-HT2C receptors.
1
1
1
1
0. Glennon, R. A.; Bartyzel, P.; Teitler, M. Med. Chem. Res.
991, 1, 201.
1. Rydon, H. N.; Siddappa, S. J. Chem. Soc. 1951, 2462.
2. Rajur, S. B.; Merwade, A. Y.; Hendi, S. B.; Basanagou-
In conclusion, none of the examined pyrazinoindoles
nor isotryptamines displayed substantial selectivity for
dar, L. D. Indian J. Chem. 1989, 28B, 1065.
3. von Schmutz, J.; Kunzle, F. Helv. Chim. Acta 1956, 39, 1144.
14. Cell culture and transfection (for human 5HT2A assays):
1
5
-HT versus 5-HT2A receptors. As reported by Bo
2
¨
s et
C
6
al., the presence of a 10-methoxy substituent on the
pyrazinoindole nucleus might be necessary for enhanced
COS-7 cells were grown in DMEM (Life Technologies) with
10% fetal bovine serum (Life Technologies) in 5% CO at 37
2
C and subcultured 1:12 twice a week. Twenty-four hours
before transfection, cells were seeded at 80% confluence in
5
-HT2C affinity. In contrast, we were unable to confirm
the selective nature of compound 10. As a consequence,
caution is advised when interpreting results of pharma-
cological studies using 10 as a selective 5-HT2C agonist.
1
00-mm dishes. Cells were transfected with human 5-HT2A
DNA by Lipofectamine (Life Technologies). This was accom-
plished by combining 20 mL Lipofectamine with 5 mg of plas-
mid per dish. Transfections were performed in serum-free
ꢁ
DMEM for 4.5 h at 37 C. Radioligand binding: Thirty-six
hours after transfection, membranes were prepared from COS-
Acknowledgements
7
0.5 mM EDTA, 5 mM MgCl , pH 7.4 (tissue buffer) and cen-
cells (for human 5-HT2A) by scraping in 50 mM Tris–HCl,
This work was supported in part by DA 01642.
2
trifugation at 10,000g for 30 min. Membranes were re-sus-
pended in tissue buffer, homogenized and centrifuged again.
For human 5-HT2C, NIH3T3 cells stably transfected with
human 5-HT2C INI receptors were grown to confluence and
prepared in the same manner as the COS-7 cells. After re-sus-
pension in assay buffer (tissue buffer+0.1% ascorbate), 0.5-
mL membrane aliquots were added to each assay tube con-
References and Notes
1
. Glennon, R. A.; Dukat, M.; Westkaemper, R. B. Serotonin
Receptor Subtypes and Ligands. In Psychopharmacology, a
Generation of Progress. CD ROM Version, 1999.
. Serotonin Receptors and their Ligands, Olivier, B., van
Wijngaarden, I., Soudin, W., Eds. Elsevier: Amsterdam, 1997.
. Issac, M. Drugs Future 2001, 26, 383.
. Isaac, M.; Slassi, A.; O’Brien, A.; Edwards, L.; MacLean,
N.; Bueschkens, D.; Lee, D. K. H.; McCallum, K.; De Lan-
noy, I.; Demchyshyn, L.; Kamboj, R. Bioorg. Med. Chem.
Lett. 2000, 10, 919.
. Kennett, G. Curr. Opin. Invest. Drugs 1993, 2, 317.
. Bos, M.; Jenck, F.; Martin, J. R.; Moreau, J. L.; Mutel, V.;
Sleight, A. J.; Widmer, U. Eur. J. Med. Chem. 1997, 32, 253.
. Mokrosz, J.; Boksa, J.; Bojarski, A. J.; Charakchieva-
Minol, S. Med. Chem. Res. 1993, 3, 240.
3
3
taining either 2 nM [ H]DOB or 0.4 nM [ H]ketanserin for 5-
3
3
2
HT2A or 2 nM [ H]5-HT or 1 nM [ H]mesulergine for 5-HT
2C
and varying concentrations of competing drug in a final
volume of 1 mL mianserin (10 mM) was used to define non-
specific binding. Samples were incubated at room temperature
3
4
ꢁ
for 30 min (5-HT2A) or at 37 C for 30 min (5-HT2C), filtered
through glass fiber filters (presoaked in 0.3% poly-
ethyleneimine) on a Brandel cell harvester, and counted in
Ecoscint cocktail in a liquid scintillation counter (Beckman,
Berkeley, CA, USA) at 40% efficiency. Data analyses: Data
analyses were performed using Prism software (GraphPad,
San Diego, CA, USA). The Cheng-Prusoff equation was used
to calculate K values from the IC values.
5
6
¨
7
i
50