R. J. Mattson et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4245–4248
4247
16. N-(2-Phenoxy-ethyl)-N0-[(1S)-(1-phenyl-ethyl)]-[1,3,5]tri-
azine-2,4,6-triamine 10. Cyanuric chloride (25.0 g, 0.135
mol) was dissolved in THF (500 mL) and diisopropylethyl-
amine (17.5 g, 0.135 mol) in at 0 ꢁC. A solution of (1S)-1-
phenylethylamine (16.4 g, 0.135 mol) in THF (100 mL) was
added dropwise while maintaining the reaction tempera-
ture at or near 0 ꢁC. The mixture was allowed to warm to
ambient temperature and was then concentrated in vacuo.
The residue was dissolved in ethyl acetate (500 mL) and
extracted with 1 N HCl (250 mL), H2O (250 mL), and
brine (250 mL). The organic layer was dried over sodium
sulfate, filtered, and concentrated in vacuo to give (4,6-
dichloro-[1,3,5]triazin-2-yl)-[(1S)-(1-phenyl-ethyl)]-amine
as a white solid (35 g, 96%). MP 146–147 ꢁC, 1H NMR
(CDCl3): d 7.39 (m, 5H), 6.40 (b d, 1H), 5.25 (m, 1H), 1.59
(d, 3H); MS (ESIþ) (Mþ) 269.1 obsd.
dose–response curve for 5-CT (a potent 5-HT7 receptor
agonist) to the right in the manner of an antagonist (Fig.
1).
In conclusion, the present studies have identified 5-HT7
receptor antagonists of a new structural type, the amino-
triazines. Compounds 10 and 17 had high affinity for the
5-HT7 receptor and did not bind to either the 5-HT2C or
5-HT6 receptors. These compounds produce no agonist
effects by themselves and appear to function as potent 5-
HT7 receptor antagonists in vitro. In particular, 17 has
demonstrated selectivity and good bioavailability in rat.
This compound may prove to be a valuable tool in
elucidating the role of the 5-HT7 receptor as a thera-
peutic target.
A solution of the above intermediate in THF (500 mL) and
NH4OH (28%, 50 mL) was stirred for 48 h and then
concentrated in vacuo. The residue was dissolved in
methylene chloride (500 mL) and washed with water
(2 · 250 mL). The organic layer was filtered, dried over
sodium sulfate, and concentrated in vacuo to give 6-
chloro-N-[(1S)-(1-phenyl-ethyl)]-[1,3,5]triazine-2,4-diamine
as a white solid (29.7 g, 90%). MP 163–164 ꢁC, 1H
(CDCl3): d 7.78 (m, 5H), 6.1–6.2 (m, 4H), 1.52 (d, 3H),
MS (ESIþ) (Mþ) 249.7 obsd.
References and notes
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A solution of the above intermediate (15.0 g, 0.056 mol), 2-
phenoxyethylamine (8.6 g, 0.062 mol), diisopropylethyl-
amine (25 mL, 0.178 mol), and THF (500 mL) was heated
to reflux for 24 h, then cooled, and concentrated in vacuo.
The amber residue was dissolved in CH2Cl2 and washed
with 1 N HCl (300 mL). The product precipitated from the
organic layer and was collected by filtration, washed with
water and acetonitrile. The product was triturated in hot
acetonitrile, collected by filtration, and dried to give N-(2-
phenoxy-ethyl)-N0-[(1S)-(1-phenyl-ethyl)]-[1,3,5]triazine-
2,4,6-triamine, 9, as a white solid HCl salt (18.2 g,
88%). MP 207–208 ꢁC, 1H NMR (CDCl3): d 7.19–7.3
(m, 7H), 6.91–6.96 (m, 3H), 5.16–6.08 (b m, 3H), 4.90 (b
s, 2H), 3.32–4.04 (m, 4H), 1.49 (d, 3H); MS (ESIþ)
(M+H)þ 351.2 obsd; C, H, N calcd for C19H22N6OÆHCl:
C, 58.98; H, 5.99; N, 21.72; Found C, 59.02; H, 5.87; N,
21.67.
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18. 5-HT7 binding assay. Membranes are prepared for binding
using the human 5-HT7 receptor expressed in CHO cells.
Cells are collected and ruptured using a dounce homo-
genizer. The cells are spun at 18,000g for 10 min and the
pellet is resuspended in assay buffer, frozen in liquid
nitrogen, and kept at )80 ꢁC until the day of the assay.
The assay is carried out in 96-deep-well plates with a total
of 30 lg protein used per well in an assay buffer of 50 mM
HEPES. The membrane preparation is incubated at 25 ꢁC
for 60 min with 0.1–1000 nM test compound and 1 nM 3H-
5-carboxamidotryptamine. 10 lM serotonin is used as
blocking agent to determine nonspecific binding. The
reaction is terminated by the addition of 1 mL of ice cold
50 mM HEPES buffer and rapid filtration through a
Brandel Cell Harvester using Whatman GF/B filters. The
filter pads are counted in an LKB Trilux liquid scintilla-
tion counter. Ki values are determined using nonlinear
regression by Exel-fit.