2-(1-naphthyloxy)ethylamines with enhanced affinity for human 5-HT(1Dβ) (h5-HT(1B)) serotonin receptors

Article abstract of DOI:10.1021/jm970507t

Although the β-adrenergic antagonist propanolol (1) binds at rodent 5- HT(1B) serotonin receptors, it displays low affinity (K(i) > 10 000 nM) for its species homologue 5-HT(1DB) (i.e., h5-HT(1B)) receptors. The structure of propanolol was systematically modified in an attempt to enhance its affinity for the latter population of receptors. Removal of the alkyl hydroxyl group, shortening of the O-alkyl chain from three to two methylene groups, and variation of the terminal amine substituent resulted in compounds, such as N- monomethyl-2-(1-naphthyloxy)-ethylamine (11; K(i) = 26 nM), that display significantly higher h5-HT(1B) affinity than propanolol. Compound 11 was shown to bind equally well at human 5-HT(1Dα) (h5-HT(1D) receptors (K(i) = 34 nM) and was further demonstrated to possess h5-HT(1B) agonist character in an adenylate cyclase assay. It would appear that such (aryloxy)alkylamines may represent a novel class of 5-HT(1D) receptor agonists.

Full text of DOI:10.1021/jm970507t

J . Med. Chem. 1997, 40, 4415-4419
4415
Notes
2
-(1-Na p h th yloxy)eth yla m in es w ith En h a n ced Affin ity for Hu m a n 5-HT1Dâ
(
h 5-HT1B) Ser oton in Recep tor s
†
†
†
‡
‡
‡
Abd M. Ismaiel, Malgorzata Dukat, Ho Law, Rajender Kamboj, Ermi Fan, David K. H. Lee,
‡
‡
§
†
,†
Lucia Mazzocco, Donna Buekschkens, Milt Teitler, Myles E. Pierson, and Richard A. Glennon*
Department of Medicinal Chemistry, School of Pharmacy, Medical College of Virginia/ Virginia Commonwealth University,
Richmond, Virginia 23298, Allelix Biopharmaceuticals, Mississauga, Ontario L4V1V7, Canada, and Department of
Pharmacology, Albany Medical College, Albany, New York 12208
X
Received August 1, 1997
Although the â-adrenergic antagonist propranolol (1) binds at rodent 5-HT1B serotonin receptors,
it displays low affinity (K > 10 000 nM) for its species homologue 5-HT1Dâ (i.e., h5-HT1B)
i
receptors. The structure of propranolol was systematically modified in an attempt to enhance
its affinity for the latter population of receptors. Removal of the alkyl hydroxyl group,
shortening of the O-alkyl chain from three to two methylene groups, and variation of the
terminal amine substituent resulted in compounds, such as N-monomethyl-2-(1-naphthyloxy)-
ethylamine (11; K
i
) 26 nM), that display significantly higher h5-HT1B affinity than propranolol.
Compound 11 was shown to bind equally well at human 5-HT1DR (h5-HT1D) receptors (K
i
) 34
nM) and was further demonstrated to possess h5-HT1B agonist character in an adenylate cyclase
assay. It would appear that such (aryloxy)alkylamines may represent a novel class of 5-HT1D
receptor agonists.
Certain aryloxyalkylamines such as propranolol (1)
and pindolol (2) are recognized as â-adrenergic antago-
nists. However, these and related agents are known to
also bind with nanomolar affinity at certain populations
of serotonin receptors, particularly 5-HT1A and 5-HT1B
receptors (e.g. 1-3). We and others have previously
examined the SAR for the binding of such compounds
at these receptors. Much less is known about the
binding of these agents at other populations of 5-HT
receptors.
difference in affinity has been related to the presence
of a particular amino acid residue; that is, h5-HT1B
receptors possess a threonine at position 355 whereas
r5-HT1B receptors possess an asparagine moiety at the
cognate position. The presence of the asparagine moi-
ety, also found at a corresponding position in 5-HT1A
1
4
1
6
receptors, appears important for the binding of (aryl-
oxy)alkylamines. Although there exist other structural
differences between h5-HT1B and r5-HT1B receptors,
mutant h5-HT1B receptors (i.e., a T355N mutant), in
which threonine 355 has been replaced by an aspar-
agine, display enhanced affinity for propranolol.1
Indeed, mutation of this single amino acid enhances the
binding of (-)-propranolol such that it is now compa-
rable to the affinity of (-)-propranolol at r5-HT1B
7-20
1
7-20
receptors.
In the course of our work on the binding of 1 at
5
-HT1A, r5-HT1B, and bovine 5-HT1D receptors, we found
5
-HT1B receptors represent serotonin autoreceptors in
that the presence of the hydroxyl group of 1 is not
critical for binding, and that replacement of the ether
oxygen atom with a methylene group abolishes affin-
rodents; most other species including humans possess
a species homologue of 5-HT1B receptors (i.e., 5-HT1D
receptors) that seems to serve the same function.
distinct populations of human 5-HT1D receptors have
been identified, 5-HT1DR or h5-HT1D receptors and
5
-9
Two
1
,2,21
ity.
The results of this preliminary study, coupled
2
with increased interest in 5-HT1B/1D receptors, prompted
us to undertake further study of the interaction of
propranolol and related (aryloxy)alkylamines at this
latter population of receptors. Specifically, we wished
to determine the role of the naphthalene ring, the
importance of alkyl chain length, and particularly the
effect of amine substitution, on h5-HT1B binding. Se-
lected compounds were also examined at h5-HT1D
receptors to determine if they possess any selectivity.
5
-HT1Dâ or h5-HT1B receptors (i.e., intraspecies sub-
types). The h5-HT1B receptors are considered a species
homologue of rat (r) 5-HT1B receptors,1
0-15
and agents
that bind at one population typically bind at the other.
Aryloxy)alkylamines are a curious exception. For
example, propranolol (1) binds with high affinity (Ki ≈
(
1
5-60 nM) at r5-HT1B receptors but displays low
3
affinity (Ki > 5000 nM) at h5-HT1B receptors. The
Ch em istr y
†
Richmond.
Ontario.
Albany.
‡
Compounds 4,22 5-10, and 16-20 have been previ-
ously reported. Compounds 11-15, 21, 22, 24, 26, and
1
22
§
X
Abstract published in Advance ACS Abstracts, November 15, 1997.
S0022-2623(97)00507-4 CCC: $14.00 © 1997 American Chemical Society

4
416 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Notes
Ta ble 1. Physicochemical Properties of (Aryloxy)alkylamines
ArO(CH ) NRR′
2
n
Ar
n
R
R′
mp (°C)
RSa
% yield
empirical formulab
1
1
1
1
1
2
2
2
2
2
2
1
2
3
4
5
1
2
4
5
6
7
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
phenyl
2
2
2
2
2
2
2
3
2
3
2
H
H
H
H
H
H
H
Me
Me
H
Me
Et
CH2CH2OH
n-Pr
i-Pr
Me
Me
n-Pr
n-Pr
i-Pr
179-181
155-157
198-200
236-238
228-230g
174-175
169-171
135-138
193-195
164-166
150-152
A
A
B
A
A
C
A
D
E
50
60
72
40
40
40
83
25
40
46
20
C13H15NO‚HClc
C14H17NO‚HCl
d
C14H17NO2‚C2H2O4
d,f
d
C15H19NO‚C2H2O4
C15H19NO‚C2H2O4
2,3-diMe Ph
C11H17NO‚HCl
e
d
2-CPP
2-CPP
C18H29NO‚C2H2O4
C17H27NO‚HCl
e
d
d
4-indolyl
4-indolyl
A
A
C15H20NO‚C2H2O4
C14H18NO‚C2H2O4
H
i-Pr
a
b
Recrystallization solvents: A ) EtOH/Et2O; B ) MeOH; C ) MeCN; D ) EtOAc; E ) 2-butanone; F ) MeOH/Et2O. All compounds
analyzed for C, H, N within 0.4% of theory. Crystallized with 0.25 mol of H2O. d Oxalate salt. e 2-CPP ) 2-(cyclopentyl)phenyl. f Crystallized
with 0.2 mol of H2O. Lit. mp 175-176 °C.
c
g
25
Sch em e 1a
amines may bind with higher affinity than tertiary
amines. Chain lengthening of 5 by one methylene unit
(i.e., 9, Ki ) 470 nM) halved affinity, whereas chain
shortening by a single methylene (i.e., 16, Ki ) 94 nM)
doubled affinity. Consequently, we continued by exam-
ining the effect of various amine substituents in ad-
ditional naphthalene-containing chain-shortened ether
analogues (i.e., 10-20).
a
(a) ClCH2CH2OH, NaOH; (b) TsCl/pyridine, 0-10 °C; (c)
NH2CH3, K2CO3, ∆.
The primary amine 10 (Ki ) 100 nM) binds with much
higher affinity than propranolol. The secondary amines
11, 12, and 14, where the amine substituent is varied
from methyl to ethyl to n-propyl (Ki ) 26, 66, and 200
nM), reveal a small but consistent trend of reduction in
affinity as substituent length is increased. The hy-
droxyethyl derivative 13 and isopropyl derivative 15 (Ki
) 107 and 120 nM, respectively) bind with the same
affinity as the primary amine. In general, the secondary
amines bind with relatively little variation in affinity.
N-Monomethylation of 11 and 14, to give the tertiary
amines 16 and 17, results in a 4-fold reduction in
affinity. The tertiary amines 16-20 bind, but there
seems to be limited bulk tolerance; that is, apparently,
only a certain amount of bulk is tolerated around the
amine group, and the N-monomethyl derivative 11 was
identified as the highest affinity agent in this series.
2
7 were prepared in a simple three-step process as
exemplified for 11 in Scheme 1. In general, the ap-
propriate hydroxy compound (i.e., naphthol, phenol, or
-hydroxyindole) was O-alkylated with 2-chloroethanol
or 3-chloropropanol, and the resulting alcohol was
converted to a tosylate and allowed to react with an
amine (see Table 1); synthetic detail is provided for 11.
Compound 25 was prepared in a slightly different
manner from the remainder of the series; 2-cyclopen-
tylphenol was alkylated with 1,2-dibromoethane, and
the resultant product was allowed to react with N-n-
propyl-N-methylamine.
4
Resu lts a n d Discu ssion
For purpose of comparison, we determined or rede-
termined the h5-HT1B affinities of (()-propranolol (1)
Ki > 10 000 nM), (-)-propranolol (Ki ) 4100 nM), (()-
Having established that the shorter chain compounds
(e.g. compare 4 with 10, and 5 with 16) bind with
(
severalfold higher affinity than those with an O-alkyl
substituent length corresponding to that found in pro-
pranolol, and that an N-monomethylamine is optimal,
we reexamined the role of the unsubstituted benzene
ring. The chain-shortened monocyclic derivative 21 (Ki
pindolol (2) (Ki > 10 000 nM), and (-)-pindolol (Ki )
600 nM) (see Table 2). As previously reported, none
2
of these agents displayed high affinity for h5-HT1B
receptors. We have already demonstrated that deshy-
droxypropranolol (3) (Ki ) 7660 nM) binds with low
affinity, but at least as well as racemic propranolol, and
with an affinity comparable to (-)-propranolol at h5-
)
5960 nM) binds, as expected, with low affinity; its
affinity is >200-fold lower than that of 11. Evidently,
however, the intact naphthalene nucleus is not essential
for binding; for example, the 2,3-dimethyl derivative (22;
Ki ) 316 nM) binds with enhanced affinity relative to
21. Although the naphthyl ring still seems optimal, the
methyl substituents of 22 may be sufficient to mimic,
at least in part, the binding properties of the unsubsti-
tuted benzene ring of 11. Nevertheless, 22 still binds
with 10-fold lower affinity than 11.
2
3
HT1B receptors. Because the N-isopropyl substituent
of 1 is considered an important feature for â-adrenergic
binding, and because N-mono- and N,N-dimethyl sub-
1
stitution is tolerated by r5-HT1B receptors, we began
our investigation with simpler N-alkyl substituents.
Initially, we found that replacement of the N-isopropyl-
amine of 3 with a primary amine or with N,N-dimethyl-
amine resulted in enhanced affinity (i.e., 4 and 5, Ki )
2
90 and 255 nM, respectively; Table 2). Removal of the
The naphthyl ring of propranolol (1) is known to be
important for high affinity at 5-HT1A and r5-HT1B
receptors; that is, replacement of the naphthyl group
with an unsubstituted phenyl ring results in a dramatic
decrease in affinity at these populations of receptors.1
It is also known, however, that certain substituted-
unsubstituted benzene ring of 5 (i.e., 6) abolished
affinity. Replacement of the ether oxygen atom of 6
with a nitrogen atom (i.e., 7) resulted in an inactive
compound. However, monodemethylation of 6 (i.e., 8)
resulted in enhanced affinity, suggesting that secondary

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4417
Ta ble 2. Results of Radioligand Binding Studies with
(Aryloxy)alkylamines
modest affinity at h5-HT1B receptors (Table 2), but 25
binds with an affinity comparable to that of naphthyl
derivative 17.
Finally, in an effort to determine if the affinity-
enhancing effect of chain shortening was specific for the
propranolol series or whether it could be generalized to
other (aryloxy)alkylamines, the affinity of racemic pin-
dolol (2; Ki > 10 000 nM) and deshydroxypindolol (26;
Ki > 10 000 nM) was compared with that of compound
2
7. Compound 27 (Ki ) 1120 nM) was found to bind
with enhanced affinity, but with 10-fold lower affinity
than its corresponding propranolol analogue 15. Ap-
parently, the affinity-enhancing effect of chain shorten-
ing is not limited solely to derivatives of propranolol.
h 5-HT1D (5-HT1DR) Bin d in g. To determine if some
of the present compounds display any selectivity for h5-
HT1B versus h5-HT1D receptors, several were examined
in a h5-HT1D binding assay (see footnote to Table 2).
None of the compounds possessed appreciable selectiv-
ity.
F u n ction a l Assa y. (Aryloxy)alkylamines such as
propranolol (1) and pindolol (2) typically behave as 5-HT
antagonists (or as very weak partial agonists).24 It
would seem likely that compound 11, being an (aryloxy)-
alkylamine, might also behave as a 5-HT1D antagonist.
However, with a shorter side chain and different
terminal amine substituent than that found in 1 and 2,
it would be difficult to predict the functional activity of
this type of compound a priori. Thus, 11 was examined
in a functional assay in order to determine if it is a
5
-HT1D agonist or antagonist. Compound 11 did not
function as an antagonist of forskolin-stimulated inhibi-
-5
tion of adenylate cyclase at concentrations of up to 10
M. In contrast, a nearly full agonist effect was observed
(
(
Figure 1). Hence, 11 behaves as a h5-HT1B agonist
EC50 ) 216 nM) relative to 5-HT itself (EC50 ) 5.5 nM).
Su m m a r y. Human 5-HT1Dâ or h5-HT1B receptors
represent a species homologue of rodent 5-HT1B recep-
tors. The (aryloxy)alkylamines propranolol and pindolol
are rather unique in that they bind with high affinity
at r5-HT1B receptors but with low affinity at h5-HT1B
receptors. On the basis of some preliminary investiga-
tions from our laboratory suggesting it should be pos-
sible to enhance the affinity of (aryloxy)alkylamines for
h5-HT1B receptors if the appropriate structural changes
are made, we undertook the present structure-affinity
investigation. Relative to racemic propranolol (1; Ki >
10 000 nM), deshydroxy derivatives (a) with a two-
rather than three-methylene spacer between the ether
oxygen atom and the terminal amine, and (b) that
possess a primary or secondary amine with small alkyl
groups, bind at h5-HT1B receptors with affinities of e100
a
Standard error not determined where Ki > 10 000 nM.
b
Affinities for h5-HT1D (i.e., 5-HT1DR) receptors were determined
for selected compounds for purpose of comparison; Ki values
(
(SEM) are as follows: 11, 34(9); 13, 68(7); 17, 103(12); 18,
5
90(162); 19, 112(8); 21, 1800(340); 24, 143(20); 25, 82(12) nM.
c
Ki value previously reported.23
phenyl derivatives retain affinity for 5-HT1A/1B receptors.
For example, penbutolol (23) binds at both populations
of receptors with 15-20 times the affinity of propran-
3
,4
olol.
Accordingly, we examined the cyclopentyl de-
rivatives 24 and 25. Both compounds bind only with

4
418 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Notes
A mixture of 2-(1-naphthyloxy)ethanol-p-toluenesulfonate
0.34 g, 1 mmol), an ethanolic solution of N-methylamine (5%,
(
2
.0 mL), and anhydrous K
2 3
CO (0.14 g, 1 mmol) in dioxane
(20 mL) was heated at reflux for 3 h. The solvent was
evaporated under reduced pressure, and the residue was
treated with 10% NaOH solution (10 mL) and extracted with
Et
with H
evaporated. The product was separated by column chromo-
tography using silica gel with CHCl /MeOH (9:1) as eluent.
The HCl salt was prepared and recrystallized from absolute
EtOH/Et O to give 0.12 g (50%) of white crystals: mp 179-
2
O (3 × 20 mL). The combined Et
2
O extract was washed
2
O (3 × 20 mL) and dried (MgSO
4
), and the solvent was
3
2
1
1
4
1
81 °C; H NMR (DMSO-d
.4 (t, 2H, CH ), 7.0 (dd, 1H, Ar-H), 7.5 (m, 4H, Ar-H), 7.8 (m,
H, Ar-H), 8.4 (m, 1H, Ar-H), 9.2 (bs, N H
O) C, H, N.
6
) δ 2.6 (s, 3H, CH
3 2
), 3.4 (t, 2H, CH ),
2
+
2
15
). Anal. (C13H -
NO‚HCl‚0.25H
2
N-Meth yl-N-n -p r op yl-2-(2-cyclop en tylp h en oxy)eth yl-
a m in e Hyd r och lor id e (25). NaOMe in MeOH (25% solution,
0
.7 g, 3.2 mmol) was added to 2-cyclopentylphenol (0.4 g, 2.5
mmol) in MeOH (5 mL), and the reaction mixture was allowed
to stand at room temperature for 0.5 h. After removal of the
solvent in vacuo, CH
3
CN was added to the solid. The sodium
CN was added in a dropwise
2
-cyclopentylphenolate in CH
3
manner to 1,2-dibromoethane (2.3 g, 12.0 mmol) in a screw-
top test tube; the tube was sealed and heated at 100 °C for 12
h and then at 130 °C for an additional 12 h. The solvent was
removed in vacuo, and petroleum ether (bp 60-90 °C) (5 mL)
and aqueous NaOH (2 N, 3 mL) were added to the residue.
The aqueous layer was removed, and the organic portion was
extracted once more with aqueous NaOH (3 mL). The petro-
F igu r e 1. Agonist effect of compound 11 and 5-HT on cAMP
levels in cells stably expressing human 5-HT1Dâ (h5-HT1B
)
serotonin receptors.
nM. Although the naphthyl moiety appears optimal for
binding, it can be replaced with certain substituted
phenyl groups (e.g. 22, 25) or with a 4-indolyl group (e.g.
4
leum ether portion was dried (MgSO ) and the solvent was
removed in vacuo to yield an oil. Kugelrhor distillation of the
oil (bp 52-70 °C, 0.05 mmHg) yielded 0.23 g (35%) of 1-bromo-
2-(2-cyclopentylphenoxy)ethane as a clear oil. A solution of
the above bromide (0.23 g, 0.85 mmol) and N-methylpropyl-
2
7). Several compounds were examined at h5-HT1D
receptors but none showed much more than 10-fold
selectivity for one population of 5-HT1D receptors over
the other. One compound in particular, 11, was exam-
ined for its ability to act as a h5-HT1B agonist or
antagonist using an adenylate cyclase assay. Com-
pound 11 was found to be devoid of antagonist charac-
ter; in contrast, it behaved as an agonist. Additional
work is obviously required on these types of compounds.
Nevertheless, it would appear that 2-(1-aryloxy)ethyl-
amines may represent a novel class of 5-HT1D receptor
ligands.
amine (1.0 g, 14.0 mmol) were combined in CH
heated in a sealed tube at 60 °C for 24 h. The reaction mixture
was allowed to cool, and the N-methylpropylamine and CH
3
CN (1 mL) and
3
-
CN were recovered by distillation. An ethereal solution of the
resulting oil was washed with 1 N NaOH (3 × 1 mL) and then
extracted with HCl (3 × 10 mL). The combined acidic fractions
were basified (to pH 12) with a saturated solution of KOH and
extracted with Et
were dried (MgSO
An ethereal solution of the free base was treated with HCl in
Et O until salt formation ceased. The salt was collected by
filtration and recrystallized from 2-butanone to yield 0.1 g
2
O (3 × 15 mL). The combined Et
2
O fractions
), and the solvent was evaporated in vacuo.
4
2
Exp er im en ta l Section
1
(
40%) of a white solid: mp 193-195 °C; H NMR (CDCl
3
, free
N, J
O, J ) 3 Hz),
Syn th esis. Chemicals were purchased from Aldrich Chemi-
cals (Milwaukee, WI). Elemental analyses was performed by
Atlantic Microlab, Inc. (Norcross, GA); results are within 0.4%
of theory. Melting points were determined on a Thomas-
Hoover melting point apparatus and are uncorrected. Proton
NMR spectra were recorded on a GE QE-300 FT NMR;
chemical shifts are reported as ppm. Tetramethylsilane was
used as an internal standard. Thin-layer chromatography was
performed on precoated silica gel (60F254, Merck) glass plates.
N-Met h yl-2-(1-n a p h t h yloxy)et h yla m in e H yd r och lo-
r id e (11). A mixture of 1-naphthol (2.88 g, 20 mmol) and
base) δ 0.9 (t, 3H, CH ), 1.0-2.5 (m, 12H), 2.8 (t, 2H, CH
3
2
) 3 Hz), 3.0-3.5 (m, 1H, CH), 4.1 (t, 2H, CH
2
6.8-7.2 (m, 4H, ArH). Anal. (C17
H
2
27NO‚HCl) C, H, N.
4
Ra d ioliga n d Bin d in g Assa ys.
Binding studies were
performed in triplicate using 96-well polypropylene microtiter
plates with a reaction volume of 500 µL. Test compounds were
initially assayed at 1 and 0.1 µM concentrations. Compounds
showing <50% inhibition at 1 µM are reported as binding with
K
i
> 10 000 nM.
i
K values were determined for those
3
compounds showing >50% inhibition at 1 µM. [ H]-5-HT
trifluoroacetate (100 Ci/mmol, Amersham) was used as radio-
ligand, 2.5 nM final concentration; nonspecific binding was
defined using 20 µM 5-HT creatinine sulfate (Research Bio-
chemicals Inc.). The incubation buffer was composed of 50 mM
2
-chloroethanol (1.82 g, 20 mmol) in 2 N NaOH (0.80 g in 10
mL H
O) was heated at reflux for 2 h and then allowed to stir
at room temperature overnight. The reaction mixture was
diluted with H O (2 mL) and extracted with CH Cl
(3 × 30
mL), washed with NaOH 10% (50 mL) and with H
O (3 × 30
mL), dried (MgSO ), and evaporated to give 3.00 g (80%) of
-(1-naphthyloxy)ethanol as an oil. p-Toluenesulfonyl chloride
0.99 g, 5 mmol) was added in a portionwise manner to a
2
2
2
2
4
Tris, 10 mM MgSO , 0.5 mM EDTA, 10 µM pargyline, and
2
0.1% (w/V) ascorbic acid, pH 7.4 at 22 °C. Incubation was
started by the addition of membrane homogenate (0.1 mg of
protein per well); plates were vortexed for 20 s and then
incubated at room temperature for 60 min. The binding
reaction was stopped by filtration with the use of a Packard
harvester under vacuum over GF/B Unifilters. Each reaction
plate was washed six times with 1 mL of cold Tris buffer.
Scintillant (Microscint 0, 35 µL) was added to the dried
Unifilters, and the sealed plates were counted by liquid
scintillation spectrometry (Packard TopCount). Binding dpm
in the presence of test compounds were expressed as a percent
of binding dpm in the absence of compounds. A percent-
binding versus concentration curve was constructed from
4
2
(
solution of 2-(1-naphthyloxy)ethanol (0.94 g, 5 mmol) in
pyridine (10 mL) at 0 °C. The reaction mixture was kept in a
refrigerator for 2 days, after which time the reaction mixture
2
was poured into ice-water (60 mL) and extracted with Et O
(
3 × 25 mL); the Et
2
O extract was washed with 5% HCl (30
O (30 mL). The Et O extract was dried (MgSO )
2 4
mL) and H
2
and solvent evaporated. The solid residue was crystallized
from Et O/petroleum ether (bp 60-80 °C) to give 1.50 g of the
-(1-naphthyloxy)ethanol-p-toluenesulfonate; mp 103-105 °C.
2
2

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4419
which the IC50 value (concentration giving rise to 50% inhibi-
tion) was determined. K values were then calculated from
i
(11) Weinshank, R. L.; Zgombick, J . M.; Macchi, M. J .; Branchek,
T.; Hartig, P. R. Human serotonin-1D receptor is encoded by a
subfamily of two distinct genes: 5-HT1DR and 5-HT1Dâ. Proc. Nat.
Acad. Sci. U.S.A. 1992, 89, 3630-3633.
12) Hamblin, M. W.; Metcalf, M. A. Primary structure and functional
characterization of a human 5-HT1D-type serotonin receptor. Mol.
Pharmacol. 1991, 40, 143-148.
(13) Demchyshyn, L.; Sunahara, R. K.; Miller, K.; Teitler, M.;
Hoffman, B. J .; Kennedy, J . L.; Seeman, P.; van Tol, H. H. M.;
Niznik, H. B. A human serotonin 1D receptor variant (5HT1Dâ)
encoded by an intronless gene on chromosome 6. Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 5522-5526.
the IC50 value using the Cheng and Prusoff transformation.
Ad en yla te Cycla se Assa y. A CHO Pro 5 cell line was
stably tranfected with human recombinant 5-HT1Dâ receptors.
Drugs were screened for agonist activity on the basis of their
ability to inhibit the cAMP production by this cell line in the
presence of 10 µM forskolin and 0.5 mM IBMX. Test com-
pound was added to the culture media, and the cells were
incubated for 30 min at 37 °C. Following incubation, the
reaction mixture was terminated by the addition of cold
ethanolic 5 mM EDTA (2:1 v/v) to extract the cAMP. The
amounts of cAMP were determined by the Enzyme Immuno
Assay kit from Amersham. Serotonin was used for compari-
son. To test for antagonist activity, test compound was
examined in the presence of 10 µM forskolin, 0.5 mM IBMX,
(
(
14) J in, H.; Oksenberg, D.; Ashkenazi, A.; Peroutka, S. J .; Duncan,
A. M. V.; Rozmahel, R.; Yang, Y.; Mengod, G.; Palacios, J . M.;
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bling that of the 5-HT1D subtype. J . Biol. Chem. 1992, 267,
-5
and 10 M serotonin. Results are expressed as mean ( SEM
from three or more experiments.
7
553-7562.
(
(
(
16) Guan, X.-M.; Peroutka, S. J .; Kobilka, B. K. Identification of a
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Ack n ow led gm en t. This work was supported by
funds from Allelix Biopharmaceuticals and from the
Technology Development Center/Virginia Center for
Innovative Technology.
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