3
758 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Letters
Table 1. Binding Affinity of 3-Imino-2-indolones for Human GAL3a
(2) Reviews: (a) R o¨ kaeus, Å. Galanin: A Newly Isolated Biologically
Active Neuropeptide. Trends Neurosci. 1987, 10, 158-164. (b)
Crawley, J. N. Biological Actions of Galanin. Regul. Pept. 1995,
59, 1-16. (c) R o¨ kaeus, Å.; Jiang, K.; Spyrou, G.; Waschek, J.
Transcriptional Control of the Galanin Gene: Tissue-Specific Expres-
sion and Induction by NGF, Protein Kinase C, and Estrogen. Ann.
N. Y. Acad. Sci. 1998, 863, 1-13
(
3) (a) Bartfai, T.; H o¨ kfelt, T.; Langel, U¨ . Galanin: A Neuroendocrine
Peptide. Curr. ReV. Neurobiol. 1993, 7, 229-274. (b) Kask, K.;
Langel, U¨ .; Bartfai, T. GalaninsA Neuropeptide with Inhibitory
Actions. Cell. Mol. Neurobiol. 1995, 15, 653-673. (c) Bedecs, B.;
Berthold, M.; Bartfai, T. Int. Galanins10 Years with a Neuroendo-
crine Peptide. Int. J. Biochem. Cell Biol. 1995, 27, 337-349. (d)
Kask, K.; Berthold, M.; Bartfai, T. Galanin Receptors: Involvement
in Feeding, Pain, Depression and Alzheimer’s Disease. Life Sci. 1995,
60, 1523-1533.
compd
R1
R2
GAL3 Ki, nM
2
3
4
5
6
7
8
9
2,3-di-Cl
4-Cl
4-OMe
3-CF3
3,4-di-Cl
3-CF3
3-CF3
H
H
H
H
437
850
>10000
596
150
52
89
allyl
1-ethylpropyl
2-propynyl
Ph
(4) (a) The official nomenclature of the International Union of Pharma-
cology for the three galanin receptors are to be referred to as GAL1,
GAL2, and GAL3, and the respective genes from which they are
derived are referred to as GALR1, GALR2, and GALR3, respectively.
Foord, S. M.; Bonner, T. I.; Neubig, R. R.; Rosser, E. M.; Pin, J.-P.;
Davenport, A. P.; Spedding, M.; Harmar, A. J. International Union
of Pharmacology. XLVI. G Protein-Coupled Receptor List. Phar-
macol. ReV. 2005, 57, 279-288. (b) Review of galanin receptor
subtypes: Branchek, T. A.; Smith, K. E.; Gerald, C.; Walker, M.
W. Galanin Receptor Subtype. Trends Pharmacol. Sci. 2000, 21,
3-CF3
17
a
Binding affinity was determined using a previously described 125I-
galanin displacement assay. Ki values are the averages from two or more
individual determinations: for N ) 2, the mean values are within 3-fold of
their individual determinations; for N > 2, the values for the standard error
of the mean are less than 60% of their mean values.
8
1
09-116.
Table 2. Rat Exposure Levels with Compound 9a
(5) Smith, K. E.; Walker, M. W.; Artymyshyn, R.; Bard, J.; Borowsky,
B.; Tamm, J. A.; Yao, W.-J.; Vaysse, P. J.-J.; Branchek, T. A.; Gerald,
C.; Jones, K. A. Cloned Human and Rat Galanin GALR3 Receptors.
route
dose, mg/kg)
time,
h
plasma level,a
brain level,
(
µg/mL
µg/g
+
Pharmacology and Activation of G-Protein Inwardly Rectifying K
Channel. J. Biol. Chem. 1998, 273, 23321-23326.
pp (10)
sc (10)
ip (30)
ip (30)
2
2
2
4
0.09
0.99
0.78
1.68
NTb
0.35
1.06
1.21
(6) Scott, M. K.; Ross, T. M.; Lee, D. H. S.; Wang, H.-Y.; Shank, R.
P.; Wild, K. D.; Davis, C. B.; Crooke, J. J.; Potocki, A. C.; Reitz, A.
B. 2,3-Dihydrodithiin and -Dithiepine-1,1,4,4-tetroxides: Small
molecule Non-peptide Antagonists of the Human Galanin hGAL-1
Receptor. Bioorg. Med. Chem. 2000, 8, 1383-1391.
a
Values determined from an average of two animals with variability of
b
<
25% around the average value. Not tested.
(
7) Our laboratory has presented two posters at a recent American
Chemical Society meeting on another series of GAL3 antagonists:
(
a) Topiwala, U.; Chen, H.; Jimenez, H.; Reitman, M.; Walker, M.;
Han, K.; Boyle, N.; Caputo, G.; Muske, G.; Konkel, M.; Wetzel, J.
-Phenyl-2-indolylcarbohydrazides and their Azo Analogues as Potent
levels in rat by ip, sc, and po administration. The results of
these experiments are shown in Table 2.
In conclusion, 3-imino-2-indolones bind to the GAL3 receptor.
N-Aryl substitution is critical for obtaining high affinity.
Compound 9 is a high-affinity ligand for the GAL3 receptor
3
Antagonists of the GalR3 Receptor. Presented at the 228th National
Meeting of the American Chemical Society, Philadelphia, PA, August
2
2-26, 2004; Paper MEDI 30. (b) Chen, H.; Topiwala, U. P.; Boteju,
L. W.; Eldemenky, E.; Jimenez, H.; Reitman, M.; Walker, M.; Han,
K.; Boyle, N.; Caputo, G.; Muske, G.; Yang, J.; Konkel, M. J.;
Wetzel, J. M. Discovery of 3-Phenyl-2-indolylcarbohydrazides as
Antagonists of the GalR3 Receptor. Presented at the 228th National
Meeting of the American Chemical Society, Philadelphia, PA, August
(Ki ) 17 nM), and it was demonstrated to be an antagonist in
an in vitro functional assay. It was further characterized and
shown to be highly selective for the GAL3 receptor against a
panel of 75 cross-reactivity targets. It was also shown to give
exposure levels to rat in plasma and brain above its Ki. These
properties make it a good tool to further characterize the
pharmacology of galanin receptors. The use of this tool for
elucidating some pharmacological effects of GAL3 receptor
antagonism will be reported shortly.
2
2-26, 2004; Paper MEDI 31.
(
(
8) H o¨ kfelt, T. Galanin and Its Receptors: Introduction to the Third
International Symposium, San Diego, California, USA, 21-22
October 2004. Neuropeptides 2004, 39, 125-142.
9) Borowsky, B.; Walker, M. W.; Huang, L.-Y.; Jones, K. A.; Smith,
K. E.; Bard, J.; Branchek, T. A.; Gerald, C. Cloning and Charac-
terization of the Human Galanin GALR2 Receptor. Peptides 1998,
1
9, 1771-1781.
Acknowledgment. We thank the following people for their
support of this research: Deborah Hurtado, Anastatia Kokki-
nakis, and Tracy Johnson Blake for cell culture and membrane
preparation; Marc Dizon and Kiho Han for radioligand displace-
ment assays and in vitro functional assay; QingPing Han and
John Wetzel for 19F NMR studies and discussion; Rui Li and
Asanthi Pieris-Gunatilaka for the pharmacokinetic studies of
(
10) The screening was carried out at a single concentration of 10 µM in
duplicate. Hits with >50% displacement were followed up with 12
concentration points. Ki determination was done in duplicate.
11) When the human Gal3 receptor is cotransfected with GRz into COS-7
cells, activation by porcine galanin results in suppression of forskolin-
stimulated cAMP accumulation. Pretreatment with increasing con-
centrations of 9 produced a rightward shift in the galanin dose
response curve. Linear regression of the corresponding Schild plot
yields an average slope of 0.92 ( 0.04 (mean ( SEM, n ) 4)
consistent with competitive antagonism. Constraining the slope to
unity generates pKb ) 7.54 ( 0.10, Kb ) 29 nM (n ) 4).
(
9
.
Supporting Information Available: Detailed experimental and
(
12) Less than 50% inhibition in 74 out of 75 assays was observed. The
characterization data for 5 and 7-9 and dosing solutions for the
pharmacokinetic studies of 9. This material is available free of
charge via the Internet at http://pubs.acs.org.
single exception was the human 5-HT serotonin receptor, in which
4
case the binding affinity of 9 (Ki ) 72 nM, n )2) was within a
2-fold range of that for human Gal3. However, when incubated with
intact cells expressing the cloned human 5-HT4 receptor, 9 displayed
no 5-HT4 agonist or antagonist activity at concentrations up to 10
µM.
References
(
1) Langel, U¨ .; Bartfai, T. Chemistry and Molecular Biology of Galanin
Receptor Ligands. Ann. N. Y. Acad. Sci. 1998, 863, 86-93.
JM060001N