High-affinity, non-peptide agonists for the ORL1 (Orphanin FQ/nociceptin) receptor

Article abstract of DOI:10.1021/jm991129q

The discovery of 8-(5,8-dichloro-1,2,3,4-tetrahydro-napthalene-2-yl)-1- phenyl-1,3,8-triazaspiro[4.5]decan-4-one, 1a, as a high-affinity ligand for the human ORL1 (orphanin FQ/nociceptin) receptor led to the synthesis of a series of optimized ligands. The

Full text of DOI:10.1021/jm991129q

J . Med. Chem. 2000, 43, 1329-1338
1329
High -Affin ity, Non -P ep tid e Agon ists for th e ORL1 (Or p h a n in F Q/Nocicep tin )
Recep tor
Stephan Ro¨ver,* Geo Adam, Andrea M. Cesura, Guido Galley, Franc¸ois J enck, Frederick J . Monsma, J r.,‡
J u¨rgen Wichmann, and Frank M. Dautzenberg
Pharma Division, Preclinical Research, Nervous System, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland
Received J uly 23, 1999
The discovery of 8-(5,8-dichloro-1,2,3,4-tetrahydro-naphthalen-2-yl)-1-phenyl-1,3,8-triazaspiro-
[4.5]decan-4-one, 1a , as a high-affinity ligand for the human ORL1 (orphanin FQ/nociceptin)
receptor led to the synthesis of a series of optimized ligands. These compounds exhibit high
affinity for the human ORL1 receptor, exhibit moderate to good selectivity versus opioid
receptors, and behave as full agonists in biochemical assays. In this paper we present the
synthesis, structure-activity relationship (SAR), and biochemical characterization of substituted
1-phenyl-1,3,8-triazaspiro[4.5]decan-4-ones culminating in the discovery of 8-(5-methyl-1,2,3,4-
tetrahydro-naphthalen-1-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one, 1p , and 8-acenaphten-
1-yl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one 1q, two high-affinity, potent ORL1 receptor
agonists with good to moderate selectivity versus the other opioid receptors.
Sch em e 1
In tr od u ction
The cloning of G-protein coupled receptors selective
for δ, µ, and κ opioids led several groups1 to the
discovery of a fourth member of the family by homology
cloning. The amino acid sequence of this new receptor,
the ORL1 (OFQ/nociceptin) receptor, is 47% identical
to the opioid receptors overall and 64% identical in the
transmembrane domains. None of the established opioid
ligands, however, with the exception of lofentanil,2 a
high-affinity µ opioid receptor agonist, have appreciable
affinity for this new opioid receptor. Only after the
discovery that the endogenous agonist is a 17 amino acid
neuropeptide, named orphanin FQ or nociceptin,3 has
it been possible to investigate the pharmacological
relevance of this new opioid-like receptor system. The
isolation of orphanin FQ (nociceptin) has led to an
explosion of biochemical, pharmacological, and physi-
ological data on the OFQ/ORL1 receptor system.4 The
primary focus of research has been on the traditional
opiate field of pain and analgesia,5 but additionally
cardiovascular,6 memory,7 and behavioral effects8 have
been observed. Interestingly it has been found that OFQ
(nociceptin) also displays potent anxiolytic and anti-
stress effects.9
Many exciting results have been generated with OFQ
(nociceptin) and peptide analogues including the dis-
covery of [Phe1ψ(CH2-NH)Gly2]-NC(1-13)-NH2, which
has been characterized as an antagonist in the mouse
vas deferens but, probably depending on receptor den-
sity, behaves like a partial agonist in other models.10
The field of research still suffers from limitations
inherent in the use of peptides, namely poor metabolic
stability and the need for intravenous or even intra-
thecal or intracerebroventricular administration in
animals. These limitations may be overcome by the
identification of non-peptide agonists (and antagonists)
suitable for intraperitoneal or oral administration.
These compounds should be potent in vitro and in vivo
and selective versus the other opioid receptors.
Recently, we and groups from Banyu and Pfizer have
independently discovered and patented the first non-
peptide ligands for the ORL1 receptor.11 Herein we
describe the discovery of our series of 1-phenyl-1,3,8-
triazaspiro[4.5]decan-4-ones as ORL1 receptor ligands.
These new compounds as well as the compounds dis-
covered by Banyu and Pfizer bear some family resem-
blance to lofentanil (Scheme 1) and the fentanyl series
of opioid receptor ligands.12
We started with a high-throughput screening hit,
8-(5,8-dichloro-1,2,3,4-tetrahydro-naphthalen-2-yl)-1-
phenyl-1,3,8-triazaspiro[4.5]decan-4-one 1a , which is an
unselective ORL1 receptor ligand, and developed high-
affinity, potent ORL1 receptor ligands with fair to
moderate selectivity against the other opioid receptors.
In a GTPγS assay these compounds behave as agonists
at the ORL1 receptor.
Ch em istr y
Naphthalen-2-one 7a (Scheme 2) was prepared start-
ing from the corresponding inden-1-one13 3 by ring
expansion with ethyl diazoacetate followed by decar-
boxylation to naphthalen-1-one 4. Subsequent reduction,
elimination, and dihydroxylation furnished the diol 6,
which in turn was treated with p-toluenesulfonic acid
to give 7a . The 8-substituted 1-phenyl-1,3,8-triazaspiro-
* Address correspondence to Dr. Stephan Ro¨ver, F. Hoffmann-La
Roche Ltd., PRBC, Bldg. 15/142, CH-4070 Basel, Switzerland. Tel: +41
61 688 5906. Fax: +41 61 688 8714. E-mail: stephan.roever@roche.com.
‡ Present address: Schering-Plough Research Institute, K-15, C303,
3600, 2015 Galloping Hill Road, Kenilworth, NJ 07033.
10.1021/jm991129q CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/17/2000

1330 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Ro¨ver et al.
Sch em e 2a
Sch em e 4a
Reagents and conditions: (a) N2CH2CO2Et, Et3O+BF4-, CH2Cl2,
a
0 °C-rt, 37%; (b) H2O, 240 °C, 17 bar, 75%; (c) NaBH4, EtOH/
H2O, rt - reflux; (d) p-TsOH, toluene, reflux, 89% both steps; (e)
t-BuOH, NMMO, OsO4, rt, 88%; (f) p-TsOH, toluene, reflux, 22%.
Sch em e 3a
a
Reagents and conditions: (a) NaNO2, H2SO4, Et2O, rt; (b)
1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one, MS 4 Å, CH3CN, rt; (c)
NaBH3CN, THF/EtOH, rt; (d) Ti(i-OPr)4, 1-phenyl-1,3,8-tri-
azaspiro[4.5]decan-4-one, THF, rt; (e) NaBH3CN, THF/EtOH, rt.
Sch em e 5a
a
Reagents and conditions: (a) MS 4 Å, toluene, reflux; (b)
NaBH3CN, THF/EtOH, rt; (c) NaH, DMF, 80°, then R′-X, rt.
[4.5]decan-4-ones 1a -f and 1m were synthesized from
the corresponding ketones by condensation with com-
mercially available 1-phenyl-1,3,8-triazaspiro[4.5]decan-
4-one 8 to enamines and subsequent reduction with
sodium cyanoborohydride (Scheme 3). The two-step
procedure worked in our hands somewhat better than
the one-pot reductive alkylation of amines. While this
works quite well for naphthalen-2-one and other steri-
cally unencumbered substrates, 1-indanones and naph-
thalen-1-ones required somewhat more forceful condi-
tions (Scheme 4). Reductive amination of 1-indanone or
acenaphthenone to give 1k and 1q worked well in the
presence of titanium(IV) isopropoxide,14 while for the
synthesis of 1j and 1p the ketones had to be activated
as nitrimines which in turn were prepared from the
corresponding oximes in situ.15
a
Reagents and conditions: (a) DBU, DPPA, toluene, 0 °C - rt;
(b) LiAlH4, THF, rt, 63% both steps; (c) 1-methyl-1-ethyl-piperi-
dinium iodide, K2CO3, EtOH, H2O, reflux; (d) aniline, TMSCN,
acetic acid, 76% both steps; (e) Ac2O, HCO2H, rt; (f) H2O2, t-BuOH,
H2O, NH4OH, rt, 79% both steps; (g) HCONH2, 200 °C; (h) NaBH4,
MeOH, rt, -60 °C, 23% both steps.
(R)-(-)-1,2,3,4-tetrahydro-1-naphthol 10n was con-
verted in good yield to the corresponding (S)-azide by
treatment with diphenylphosphoryl azide (DPPA) and
DBU,16 and the azide in turn was reduced to the (S)-
1,2,3,4-tetrahydro-1-naphthylamine 11n with lithium
aluminum hydride. The naphthylamine 11n was then
heated with 1-methyl-1-ethyl-piperidiniumiodide to fur-
nish the corresponding piperidone in a double Hoffmann
elimination, Michael addition sequence17 and converted
by a Strecker reaction to 12n . Hydrolysis of 12n to the
corresponding amide 13n proved to be relatively tedious,
basic protocols leading to retro-Strecker reaction and
acidic protocols to elimination of dihydro-naphthalene.
This problem could be circumvented by formylating 12n
and subsequently hydrolyzing the nitrile with basic
hydrogen peroxide to the amide (concomitantly cleaving
the formamide bond). The procedure yielded 13n in a
fair yield. Compound 13n was then cyclized by treat-
The (R)- and (S)-enantiomers (1h , 1g) of 8-(5,8-
dichloro-1,2,3,4-tetrahydro-naphthalen-2-yl)-1-phenyl-
1,3,8-triazaspiro[4.5]decan-4-one 1a could be obtained
by fractional crystallization of the diastereomeric salts
with chiral 1,1′-binaphthyl-2,2′-diyl hydrogen phos-
phate. Since we were not able to identify a suitable
chiral acid for resolution of 1j, the corresponding enan-
tiomers 1n (S) and 1o (R) were obtained by total
synthesis, starting from commercially available chiral
1,2,3,4-tetrahydro-1-naphthols (Scheme 5).

High-Affinity ORL1 Receptor Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1331
Ta ble 1. Receptor Binding of 1-Phenyl-8-(1,2,3,4-tetrahydro-naphthalen-2-yl)-1,3,8-triazaspiro[4.5]decan-4-onesa
Ki (( SEM) [nM]
compd
1a
1b
1c
1d
1e
1f
1g (S)
1h (R)
2a
2b
2c
2d
2e
2f
R′′
R′
hORL1
µ
κ
δ
5,8-Cl2
5-Cl
6-Cl
7-Cl
8-Cl
H
H
H
H
H
H
H
H
5.6 (1.1)
2.9 (0.8)
218 (55)
5.8 (1.4)
7.3 (2.2)
6.3 (2.1)
2.8 (0.9)
20.7 (7.8)
3.3 (1.1)
6.8 (2.0)
15.3 (6.1)
15.0 (1.5)
7.2 (0.9)
8.5
7.2 (1.7)
8.9 (3.0)
77 (19)
44.2 (8.4)
20.7 (6.7)
149 (41)
16.4 (4.5)
48 (16)
47.1 (9.1)
40.1 (8.0)
47.3 (7.8)
17.0 (2.4)
26.2 (9.4)
52 (14)
680 (160)
450 (80)
1800 (500)
480 (150)
1410 (420)
620 (280)
415 (74)
587 (30)
313 (64)
nd
10.9 (1.6)
16.6 (2.2)
15.1 (5.5)
5.9 (2.6)
8.4 (2.2)
2.6 (0.9)
5.9 (1.8)
19.2 (4.7)
61 (23)
H
5,8-Cl2
5,8-Cl2
5,8-Cl2
5,8-Cl2
5,8-Cl2
8-Cl
Me
allyl
benzyl
146 (34)
510 (190)
285 (58)
240
CH2COPh
CH2CH2OH
CH2OMe
28.8 (7.5)
16.4 (2.4)
17.5
8-Cl
8-Cl
3.3 (1.0)
7.5
lofentanil
24b
0.14b
5.5b
a
b
See Table 2. Data given for lofentanil are literature values.23
Ta ble 2. Receptor Binding of 1-Phenyl-1,3,8-triazaspiro[4.5]decan-4-ones Substituted with Aryl-cycloalkyl Derivatives on the
Piperidine Nitrogena
Ki (( SEM) [nM]
compd
R
hORL1
µ
κ
δ
1f
1j
1k
1m
1n (S)
1o (R)
1p
2-tetralinyl
1-tetralinyl
1-indanyl
6.3 (2.1)
2.1 (0.7)
0.7
15.1 (5.5)
12.8 (1.4)
3.4 (0.8)
26.0 (0.6)
13.3 (4.6)
12.3 (2.6)
31.7 (8.8)
5.9
47.1 (9.1)
10.7 (2.3)
5.3
620 (280)
480 (140)
540 (290)
710
2-indanyl
2.5
161
1-tetralinyl
1-tetralinyl
5-Me-1-tetralinyl
acenaphthenyl
10.2 (3.7)
2.5 (1.3)
1.4 (0.4)
0.52
17 (13)
46.3 (5.2)
44 (11)
26
nd
530 (160)
460 (71)
250
1q
a
The data are the mean of two to three (( SEM) different binding experiments performed in triplicate. The Kd of the radioligands used
were as follows: [3H]-OFQ 70 pM for hORL1 receptors, [3H]-naloxone 1.3 nM for hµ receptors and 2.8 nM for hκ receptors, [3H][Ile5,6]-
deltorphin II 0.36 nM for hδ receptors.
ment with formamide at 200 °C to yield 1n together
with the corresponding unsaturated 1,3,8-triazaspiro-
[4.5]dec-2-en-4-one. Reduction with sodium borohydride
yielded pure 1n .
The lead compound, 1a (Table 1 and Scheme 1), was
discovered by high-throughput screening (HTS) screen-
ing of our compound libraries for hORL1 receptor
ligands. Compound 1a showed high affinity for the
hORL1 receptor but had similar affinity for the hµ
receptor and still significant affinity also for the hκ
receptor. The compound was shown to be an agonist at
the hORL1 receptor in GTPγS (Figure 1, EC50(1a ): 1.5
µM vs 63 nM for OFQ) and cyclase assays (data not
shown). Being interested in the pharmacology of this
new and exciting member of the opioid receptor family,
we set out to improve the biochemical profile of the
newly discovered ligand 1a in a classical medicinal
chemistry program. In this paper we report on the SAR
of simple variations in the lipophilic substituent in the
8-position of the triazaspirodecanone (Tables 1 and 2)
and the effects of different substitutions at the amide
nitrogen (Table 1) on affinity and selectivity toward the
members of the opioid receptor family. Our original lead
A series of substituted amides (2a -f) was prepared
by simple (and unoptimized) alkylation procedures in
moderate yields (Scheme 3).
Resu lts a n d Discu ssion
The compounds described were evaluated in radioli-
gand binding assays as described in the experimental
part using membranes expressing hORL1 receptors
(permanent expression in HEK 293 cells) or membranes
from BHK cells infected with Semliki Forest virus
encoding the cDNAs for either hµ, hκ, or hδ receptors.
Results are given as Ki values calculated according to
Cheng and Prusoff18 and compared to the values given
for lofentanil in the literature.23

1332 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Ro¨ver et al.
1) may serve to illustrate the relatively weak effects one
observes upon substitution at this position. Alkylating
the amide with small substituents such as methyl and
allyl (2a ,b ) leads only to a slight increase in affinity
especially for the hµ and hκ receptors. This indicates
that the amide proton does not participate in any type
of H-bonding interaction with the hORL1 receptor, and
the same is true for binding to the other opioid receptors.
Increasing the substituent size as for 2c and 2d (benzyl
and phenacyl) leads to a 3-fold loss of affinity for the
hORL1 receptor, indicating some minor unfavorable
interaction (probably steric). The opioid receptors in
general, and the hORL1 receptor in particular, tolerate
a wide range of functional groups at the amide position.
Both polar groups like the amide itself or the hydroxy-
ethyl amide (1a , 2e) and lipophilic such groups as
methyl, allyl, and methoxymethyl amides (2a -b, 2e)
are tolerated without strong effects on binding affinity.
As both polar and small lipophilic groups have no
significant impact on binding affinity, we can only
speculate that the microenvironment in this part of the
molecule does not change dramatically upon binding.
The simplest explanation for this might be that this part
of the ligand is exposed to the water layer.
Having discovered that substitution of the amide does
not provide an easy handle to increase selectivity of the
lead compound, we turned our attention back to varia-
tions of the lipophilic 8-substituent of 1a . As evident
from the data (Table 2), the 2-tetralinyl derivative 1f is
actually the least potent member of a family of tetralinyl
and indanyl derivatives (1j-m ).
The 1-indanyl derivative 1k with a Ki of 700 pM is
about 10-fold more potent for the hORL1 receptor than
1f, but this gain in affinity toward hORL1 receptors is
accompanied by a similar gain in affinity for the hµ and
hκ receptors. Compounds 1j and especially 1m , with a
Ki of 2.5 nM at the hORL1 receptor and a 10- and 70-
fold lower affinity versus hµ and hκ receptors, respec-
tively, present a more significant step toward our goal
to develop selective hORL1 receptor ligands. Interest-
ingly, in the pair of tetralinyls (1f, 1j) and also in the
pair of indanyls (1k , 1m ), it is always the 1-substituted
derivative that has higher affinity for the ORL1 receptor
(and the κ receptor). This may reflect the stability gain
of gauche- (1j, 1k ) versus anti-conformers (1f, 1m ) for
the 1-substituted compounds.
F igu r e 1. Stimulation of GTPγS binding by OFQ and 1a or
1q, respectively. Membranes were incubated with increasing
concentrations (0.1 nM-100 µM) of agonists at 22 °C for 60
min. The results are representatives of two independent
experiments performed in triplicates.
1a is highly substituted in the tetralinyl part as well
as a racemate, so we first explored the importance of
the chlorine substituents and stereochemistry of 1a for
binding affinity (Table 1).
As evident from the data of the monochloro and
unsubstituted tetralinyls (1b-e and 1f), all the recep-
tors tolerate lipophilic substituents on the tetralinyl
ring. With the exception of the 6-Cl compound 1c, all
monochloro-substituted tetralinyls are virtually equi-
potent to the original lead on all four receptors tested.
The chloro substitution is tolerated, again with the
exception of 1c, but does not contribute to binding, since
the molecule with the unsubstituted tetralinyl ring (1f)
has a binding profile very similar to 1a . Indeed, the first
progress toward higher affinity and more selective
ligands came from the resolution of 1a into its enanti-
omers. The (S)-enantiomer 1g is about 8 times more
potent on hORL1 receptors than the (R)-enantiomer 1h .
Since the other three opioid receptors do not discrimi-
nate the enantiomers, both enantiomers being virtually
equipotent at the µ, κ, and δ receptors, 1g is about twice
as selective as 1a for hORL1 receptors.
With a view to improve selectivity, the enantiomers
of the 1-tetralinyl derivative 1j were synthesized and
tested. Surprisingly, this time it is the (R)-enantiomer
1o that binds with higher affinity to hORL1 receptors.
Indeed, this leads to a improvement in selectivity,
especially toward κ receptors. The effect is, however,
only small since the enantiomers differ only by a factor
of 4 in affinity toward the ORL1 receptor, so that the
better enantiomer 1o was determined to be about
equipotent to the racemate (the standard error being
actually bigger than the expected improvement by a
factor of 2).
Trying to understand the result that both the (S)-
tetralin-2-yl derivative 1g and the (R)-tetralin-1-yl 1o
are more potent than their respective enantiomers, we
hypothesize that the complementary lipophilic binding
pocket in the hORL1 receptor should be able to accom-
modate even bigger substituents. Indeed this seems to
With a small library of compounds we explored the
relevance of substituents at the amide nitrogen. A small
subset of all the compounds synthesized (2a -f, Table

High-Affinity ORL1 Receptor Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1333
Ta ble 3. Stimulation of GTPγS Bindinga
ties for D1 and 5HT receptors (5HT1DR, 5HT2a, 5HT2c,
5HT6, 5HT7) are low for all these compounds (Ki > 1000
nM).
ORL1
µ
compd
1a
1f
1j
EC50 [nM]
type
full
partial
full
EC50 [nM]
type
full
1500
490
87
310
Con clu sion
We have succeeded in the identification of high-
affinity, partially selective non-peptide agonists for the
fourth member of the opioid receptor family, the ORL1
(OFQ/nociceptin) receptor. Systematic modification of
our original lead, 1a , resulted in the identification of
compounds with improved affinity and high potency in
the GTPγS and cyclase assays. Compounds 1p and 1q
are moderately selective for OFQ versus the µ and κ
receptors and have only low affinity toward δ receptors.
These compounds will serve as starting points for
further inroads into the development of truly selective
non-peptide ORL1 receptor agonists and allow phar-
macological characterization of the OFQ/ORL1-receptor
system. Indeed 1q, or more precisely one of its enanti-
omers, has already been shown to display anxiolytic-
like properties in the elevated plus-maze procedure in
rats.19
1k
63
full
7900
full
1m
1p
1q
500
510
40
partial
full
full
3700
2500
full
full
OFQ
DAMGO
63
full
39
full
a
A compound was considered to be a full agonist when its ability
to stimulate GTPγS binding was >75% in comparison to OFQ
(ORL1) or DAMGO (µ). A partial agonist stimulated GTPγS
binding with efficiency >25% but <75%.
be the case: both the 5-methyl-1-tetralinyl derivative
1p and the acenaphtenyl derivative 1q, which have an
increased steric demand compared to 1j and 1m ,
respectively, are even more potent than their predeces-
sors. Gratifyingly, this increase in potency was paral-
leled by an increase in selectivity. These new, high-
affinity hORL1 receptor ligands are 10- (1q) to 20-fold
(1p ) selective toward hµ receptors in our in vitro binding
assay. Selectivity toward hκ receptors is even better.
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. Melting points were
taken with a Bu¨chi 535 melting point apparatus and are
uncorrected. The 1H spectra were recorded on a Bruker AC250
instrument in DMSO (unless noted otherwise). Low-resolution
EI-MS spectra (EI: 70 eV) were recorded on a MS9 updated
with a VG ZAB console, Finnigan data system SS300, with
direct sample introduction. Microanalysis (C, H, N) were
performed on a Heraeus Vario EL. NMR data are reported in
parts per million (δ) relative to internal tetramethylsilane and
are referenced to the deuterium lock signal from the sample
solvent (DMSO-d6 unless otherwise stated); coupling constants
(J ) are in hertz. All reactions were performed under argon.
Drying of organic solutions was with Na2SO4, evaporation was
in a rotary evaporator at 40 °C in vacuo as appropriate. For
chromatography, Merck silica gel 60 (size 70-230 mesh) was
used. Starting materials were high-grade commercial products
unless stated otherwise.
5,8-Dich lor o-3,4-d ih yd r o-2H-n a p h th a len -1-on e (4). To
a solution of 4,7-dichloro-2,3-dihydro-1H-inden-1-one13 (3, 20
mmol) in dichloromethane (80 mL) cooled to 0 °C was added
triethyloxonium tetrafluoroborate (63 mmol). Diazoethyl ac-
etate (37 mmol) was added drop by drop at a temperature
below 3 °C with stirring. Stirring continued at 0 °C for 2 h
and at room temperature overnight. The mixture was quenched
with saturated sodium carbonate solution (100 mL) and
extracted with dichloromethane (2 × 50 mL). Organic phases
were pooled, and dried with MgSO4, and the solvents were
removed in vacuo. The residue (7.5 g, orange oil) was purified
by chromatography on silica gel (ethyl acetate/hexane, 1:6) to
yield 2.1 g (37%) of the mixture of keto- and enol-tautomers
of 5,8-dichloro-1,2,3,4-tetrahydro-1-oxo-2-naphthalenecarboxy-
lic acid ethyl ester as a reddish oil. A mixture of this oil and
water (20 mL) was heated for 2.5 h to 240 °C (17 bar). After
cooling, the mixture was partitioned between water and
dichloromethane, organic phases were pooled and dried with
MgSO4, and the solvents were removed in vacuo. The residue
(0.75 g, brown oil) was purified by chromatography on silica
gel (ethyl acetate/hexane, 1:6) to yield 1.2 g (28%, both steps)
of the title compound as a light brown oil which solidified on
standing: mp 39-41 °C; 1H NMR (CDCl3) 2.15 (m, 2H, 3-CH2),
2.69 (t, J ) 6.6, 2H, 4-CH2), 3.04 (t, J ) 6.3, 2H, 2-CH2), 7.28
(d, J ) 8.6, 1H, 7-CH), 7.43 (d, J ) 8.6, 1H, 6-CH); MS m/z
214, 216 (M)+. Anal. Calcd (C10H8Cl2O) C, H.
The efficacy of our compounds has been assayed
primarily by their effect on stimulating GTPγS binding
to membranes of HEK 293 cells overexpressing hORL1
receptors. Although this assay has in our hands a high
interassay variability, it is suitable for miniaturization
to a 96-well format. Our lead compound 1a behaves as
an agonist in this assay, despite its potency being 2
orders of magnitude lower than that of OFQ itself
(Figure 1 and Table 3). Results in the GTPγS binding
assay are given in Table 3. ORL1 receptor ligands such
as 1k and 1q with sub-nanomolar affinity to the
receptor had potencies similar to or even surpassing
OFQ itself in the GTPγS assay. Potency expressed as
EC50 for stimulation of GTPγS binding was 63 nM for
1k and 40 nM for 1q with intrinsic activity similar to
that of OFQ as shown for 1q (Figure 1). The compound
1p was not as potent as expected in the GTPγS assay;
consequently 1p and 1q were tested also in a cyclase
assay head to head with OFQ (nociceptin). Both 1p and
1q are full agonists in this assay with EC50’s comparable
to OFQ (1p : 0.14 nM; 1q: 0.28 nM; OFQ: 0.2 ( 0.1
nM).
We were aware that the 1-phenyl-1,3,8-triazaspiro-
[4.5]decan-4-one core is a common substructure of
antipyschotic compounds;12 therefore, we have, in ad-
dition, assayed the affinity of a selection of the com-
pounds described here toward dopamine and serotonin
receptors. While our lead compound 1a (ORL1: Ki )
5.6 nM) indeed shows some affinity for dopamine
receptors (D2: Ki ) 32, D4.4: Ki ) 11 nM), selectivity
toward dopamine receptors already improves for the
1-tetralinyl derivative 1j (ORL1: 2.1, D2: 84, D3: 45,
D4.4: 1000 nM). The 1-indanyl 1k has a selectivity
profile very similar to 1j (ORL1: 0.7, D2: 56, D3: 31,
D4.4: 470 nM). The best selectivity toward dopamine
receptors was generally found for compounds which are
also selective toward the opioid receptors, such as 1q
(ORL1: 0.52, D2: 520, D3: 1210, D4.4: 350 nM). Affini-
5,8-Dich lor o-1,2,3,4-t et r a h yd r o-n a p h t h a len -1-ol (14).
Sodium borohydride (2.7 mmol) was added to a solution of 5,8-
dichloro-3,4-dihydro-2H-naphthalen-1-one (5.4 mmol) in etha-
nol/water (20 mL, 95%) with stirring. The mixture was stirred

1334 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Ro¨ver et al.
for another hour at room temperature and for 0.5 h at reflux
temperature. Ethanol was removed in vacuo and the residue
was partitioned between water and dichloromethane. Organic
phases were pooled and dried with Na2SO4, and the solvent
was concentrated to yield 1.1 g (99%) of the title compound as
beige solid: mp 99-105 °C; 1H NMR (CDCl3) 1.6-1.8 (m, 1H,
3-CH2), 1.8-2.1 (m, 2H, 3-CH2, 2-CH2), 2.1-2.2 (m, 1H, 2-CH2),
2.35 (bs, 1H, OH), 2.4-2.6 (m, 1H, 4-CH2), 3.0 (dd, J ) 18, J
) 4.7, 1H, 4-CH2), 5.07 (bs, 1H, CHOH), 7.15-7.30 (m, 2H,
6,7-CH); MS m/z 216, 218 (M)+. Anal. Calcd (C10H10Cl2O) C,
H.
(RS)-8-(5,8-Dich lor o-1,2,3,4-tetr a h yd r o-n a p h th a len -2-
yl)-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]d eca n -4-on e h yd r och lo-
r id e (1a ): (4.1 g, 47%) of a colorless solid from diethyl ether/
1
ethanol (1:1); mp 290-293 °C; H NMR 1.91 (m, 3H), 2.4 (b,
1H), 2.8 (m, 1H), 2.9-3.2 (m, 4H), 3.3-4.0 (m, 6H), 4.65 (s,
2H), 6.80 (t, 1H), 7.09 (d, 2H), 7.24 (t, 2H), 7.42 (s, 2H), 9.07
(s, 1H), 10.8 (bs, 1H); MS m/z 430.4 (MH)+. Anal. Calcd (C23H25
Cl2N3O‚HCl) C, H, N.
-
(RS)-8-(5-Ch lor o-1,2,3,4-t et r a h yd r o-n a p h t h a len -2-yl)-
1-p h en yl-1,3,8-t r ia za sp ir o[4.5]d eca n -4-on e h yd r och lo-
r id e (1b): (0.8 g, 74%) of a colorless solid from ethyl acetate/
1
ethanol (1:1); mp 283-285 °C dec; H NMR 1.90 (m, 3H), 2.4
5,8-Dich lor o-1,2-d ih yd r o-n a p h th a len e (5). A catalytic
amount of p-toluenesulfonic acid was added to a solution of
5,8-dichloro-1,2,3,4-tetrahydro-naphthalen-1-ol (5.1 mmol) in
toluene (25 mL), and the mixture was boiled overnight with
separation of water. The mixture was then washed with
saturated sodium bicarbonate solution that in turn was
extracted with toluene. Organic phases were pooled and dried
with Na2SO4, and the solvents were removed in vacuo. 5,8-
Dichloro-1,2-dihydro-naphthalene was isolated as brown oil
(0.97 g, 96%) which was used without further purification in
(b, 1H), 2.7 (m, 1H), 3.0-3.9 (m, 10H), 4.64 (s, 2H, 2-CH2),
6.78 (t, J ) 7, 1H, p-ArH), 7.1-7.3 (m, 7H), 9.07 (s, 1H, 3-NH),
11.6 (bs, 1H, NH+); MS m/z 396.4, 398.4 (MH)+. Anal. Calcd
(C23H26ClN3O‚HCl) C, H, N.
(RS)-8-(6-Ch lor o-1,2,3,4-t et r a h yd r o-n a p h t h a len -2-yl)-
1-p h en yl-1,3,8-t r ia za sp ir o[4.5]d eca n -4-on e h yd r och lo-
r id e (1c): (0.44 g, 41%) of a colorless solid from chloroform/
methanol (1:1); mp 288-290 °C; 1H NMR 1.8-2.0 (m, 3H), 2.35
(m, 1H), 2.8-3.9 (m, 11H), 4.64 (s, 2H), 6.79 (t, 1H), 7.10 (d,
2H), 7.21 (m, 5H), 9.06 (s, 1H), 10.8 (bs, 1H); MS m/z 396.2,
398.2 (MH)+. Anal. Calcd (C23H26ClN3O‚HCl) C, H, N.
1
the next steps. H NMR (CDCl3) 2.34 (m, 2H, 3-CH2), 2.91 (t,
2H, 4-CH2), 6.22 (m, 1H, 2-CH), 6.84 (“d”, 1H, 1-CH), 7.11 (s,
2H, 6,7-CH); MS m/z 198, 200 (M)+.
(RS)-8-(7-Ch lor o-1,2,3,4-t et r a h yd r o-n a p h t h a len -2-yl)-
1-p h en yl-1,3,8-t r ia za sp ir o[4.5]d eca n -4-on e h yd r och lo-
r id e (1d ): (0.32 g, 30%) of a colorless solid from ethyl acetate/
5,8-Dich lor o-1,2,3,4-tetr ah ydr o-1,2-n aph th alen ediol (6).
5,8-Dichloro-1,2-dihydro-naphthalene (4.9 mmol) was dissolved
in acetone (1 mL) and tert-butyl alcohol (1 mL). This solution
was added to a preformed mixture of N-methylmorpholine
N-oxide (5.4 mmol) and osmium tetroxide (0.25 mL of 2.5%
solution) in water (4 mL), acetone (2.4 mL), and tert-butyl
alcohol (0.7 mL). This mixture was then stirred for 20 h at
room temperature and after that partitioned between brine
(50 mL) and ethyl acetate (3 × 30 mL). Organic phases were
washed with sodium hydrogensulfite (40 mL, 2%) and satu-
rated sodium bicarbonate solution. Organic phases were pooled
and dried with Na2SO4 and the solvents evaporated. The
residue was boiled with hexane (20 mL) and yielded upon
cooling the title compound (1.0 g, 88%) as a colorless solid:
1
ethanol (8:1); mp 287-289 °C dec; H NMR 1.8-2.0 (m, 3H),
2.35 (m, 1H), 2.7-3.8 (m, 11H), 4.64 (s, 2H, 2-CH2), 6.77 (t, J
) 7, 1H, p-ArH), 7.1-7.3 (m, 7H), 9.07 (s, 1H, 3-NH), 11.6 (bs,
1H, NH+); MS m/z 396.2, 398.2 (MH)+. Anal. Calcd (C23H26
ClN3O‚HCl) C, H, N.
-
(RS)-8-(8-Ch lor o-1,2,3,4-t et r a h yd r o-n a p h t h a len -2-yl)-
1-p h en yl-1,3,8-t r ia za sp ir o[4.5]d eca n -4-on e h yd r och lo-
r id e (1e): (0.59 g, 30%) of a colorless solid from ethanol; mp
286-289 °C; 1H NMR 1.8-2.0 (m, 3H), 2.35 (m, 1H), 2.8-3.2
(m, 5H), 3.4-4.0 (m, 6H), 4.65 (s, 2H), 6.80 (t, 1H), 7.0-7.4
(m, 7H), 9.07 (s, 1H), 10.8 (bs, 1H); MS m/z 396.2, 398.2 (MH)+.
Anal. Calcd (C23H26ClN3O‚HCl) C, H, N.
(RS)-8-(1,2,3,4-Tetr a h yd r o-n a p h th a len -2-yl)-1-p h en yl-
1,3,8-tr iazaspir o[4.5]decan -4-on e h ydr och lor ide (1f): (0.45
g, 46%) of a colorless solid from ethyl acetate/ethanol (10:1);
mp >265 °C dec; 1H NMR 1.8-2.0 (m, 3H), 2.35 (m, 1H), 2.8-
4.0 (m, 11H), 4.64 (s, 2H), 6.78 (t, 1H), 7.1-7.3 (m, 8H), 9.06
(s, 1H), 11.4 (bs, 1H); MS m/z 362.3 (MH)+. Anal. Calcd
(C23H27N3O‚HCl) C, H, N.
8-In d a n -2-yl-1-p h en yl-1,3,8-t r ia za sp ir o[4.5]d eca n -4-
on e (1m ): (0.60 g, 62%) of a colorless solid from ethyl acetate/
ethanol (5:3); mp 270 °C dec; 1H NMR 1.90 (d, J ) 14, 2H,
6,10-CH2, eq), 3.06 (td, J ) 14, J ) 4, 2H, 6,10-CH2, ax), 3.3-
3.7 (m, 8H, 7,9-CH2, 1′,3′-CH2), 4.12 (m, J ) 7.5, 1H, 2′-CH),
4.64 (s, 2H, 2-CH2), 6.78 (t, 1H, p-ArH), 7.0-7.3 (m, 8H,
4,′5′,6′,7′-ArH, o,m-ArH), 9.04 (s, 1H, 3-NH), 11.4 (bs, 1H,
NH+); MS m/z 348.4 (MH)+. Anal. Calcd (C22H25N3O‚HCl) C,
H, N.
1
mp 148-149 °C; H NMR (CDCl3) 2.01 (m, 2H, 3-CH2), 2.6-
2.8 (m, 3H, 4-CH2, OH(2×)), 3.15 (dt, J ) 18, J ) 4, 1H, 4-CH2),
3.8 (m, 1H, 2-CH), 5.03 (bs, 1H, 1-CH), 7.27 (m, 2H, 6,7-CH);
MS m/z 232, 234 (M)+. Anal. Calcd (C10H10Cl2O2) C, H.
5,8-Dich lor o-3,4-d ih yd r o-1H-n a p h th a len -2-on e (7a ). p-
Toluenesulfonic acid (150 mg) was added to a solution of 5,8-
dichloro-1,2,3,4-tetrahydro-1,2-naphthalenediol (4.3 mmol) in
toluene (30 mL). This mixture was boiled for 3 h with
separation of water, cooled, and partitioned between saturated
sodium bicarbonate solution and ethyl acetate. Organic phases
were pooled and dried with MgSO4 and the solvents evapo-
rated. The brown oily residue (1.0 g) was purified by chroma-
tography on silica gel (ethyl acetate/hexane, 1:7) to yield 0.2 g
1
(22%) of the title compound as beige solid: mp 83-86 °C; H
NMR (CDCl3) 2.61 (t, J ) 6.9, 2H, 3-CH2), 3.24 (t, J ) 6.9,
2H, 4-CH2), 3.66 (s, 2H 1-CH2), 7.25 (s, 2H, 6,7-CH); MS m/z
214, 216 (M)+. Anal. Calcd (C10H8Cl2O) C, H.
(S)-8-(5,8-Dich lor o-1,2,3,4-t et r a h yd r o-n a p h t h a len -2-
yl)-1-p h en yl-1,3,8-t r ia za sp ir o[4.5]d eca n -4-on e H yd r o-
ch lor id e (1g). A hot solution of (S)-(+)-1.1′-binaphthyl-2,2′-
diyl hydrogen phosphate (0.87 g, 2.5 mmol) in ethanol (40 mL)
was added to a solution of (1a ) (1.8 g, 4.2 mmol) in ethanol
(30 mL). The salt was recrystallized 5 times from ethanol to
afford (1g) (0.48 g) as the phosphate salt. The free base was
liberated and subsequently crystallized as the hydrochloride
salt from ethanol to provide a white crystalline solid: mp 273-
P r epar ation of 8-Su bstitu ted 1-P h en yl-1,3,8-tr iazaspir o-
[4.5]d eca n -4-on es. Gen er a l P r oced u r es. A mixture of the
appropriately substituted 1,2,3,4-tetrahydro-naphthalen-2-one
(7a -f or 7m , 2.5 mmol),20 1-phenyl-1,3,8-triazaspiro[4.5]decan-
4-one (2.5 mmol), and molecular sieves (3 g, 4 Å) in toluene
(50 mL) was boiled overnight. Molecular sieves were filtered
off while hot and washed with toluene, and the filtrate was
evaporated to dryness. The residue was dissolved in a mixture
of THF and ethanol (9:1, 30 mL). Sodium cyanoborohydride
(2.8 mmol) was added with stirring and the pH adjusted to
4.5 with HCl in ethanol. Stirring at room temperature was
continued for 16 h. The mixture was poured into ice-water
(100 g) and saturated potassium carbonate solution (50 mL)
and extracted three times with dichloromethane (50 mL each).
Organic phases were pooled, washed with brine, and dried with
Na2SO4. The solvent was evaporated, and the residue was
purified by chromatography on silica gel (dichloromethane/
methanol 2-5%) and crystallized as hydrochloride salt.
275 °C; [R]20 -54.9 (c 0.9, CH3OH); 1H NMR 1.91 (m, 3H),
D
2.4 (b, 1H), 2.8 (m, 1H), 2.9-3.2 (m, 4H), 3.3-4.0 (m, 6H), 4.65
(s, 2H, 2-CH2), 6.80 (t, J ) 7, 1H, p-ArH), 7.09 (d, J ) 8, 2H,
o-ArH), 7.24 (“t”, J ) 7.5, 2H, m-ArH), 7.42 (s, 2H, 6′,7′-ArH),
9.07 (s, 1H, 3-NH), 10.8 (bs, 1H, NH+). Anal. Calcd (C23H25
-
Cl2N3O‚HCl) C, H, N. Chiral HPLC on a Chiralpak AS column
(hexane/0.4% triethylamine in hexane/ethanol (85:5:10), flow
rate 1.5 mL/min) showed 1g to have 93% ee. A typical retention
time for the S-isomer (1g) was 7.1 min and for the R-isomer
(1h ) 6.3 min.

High-Affinity ORL1 Receptor Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1335
(R)-8-(5,8-Dich lor o-1,2,3,4-t et r a h yd r o-n a p h t h a len -2-
yl)-1-p h en yl-1,3,8-t r ia za sp ir o[4.5]d eca n -4-on e H yd r o-
ch lor id e (1h ). A hot solution of (R)-(-)-1.1′-binaphthyl-2,2′-
diyl hydrogen phosphate (0.66 g, 1.9 mmol) in ethanol (30 mL)
was added to a solution of (1a ) (0.82 g, 1.9 mmol, isolated from
the mother liquors of 1g in ethanol (20 mL). The salt was
recrystallized 3 times from ethanol to afford 1h (0.54 g) as
the phosphate salt. The free base was liberated and subse-
quently crystallized as the hydrochloride salt from ethanol to
provide a white crystalline solid: mp 274-276 °C; [R]20D +53.2
(c 0.9, CH3OH); the absolute configuration was determined by
X-ray crystallography. Anal. Calcd (C23H25Cl2N3O‚HCl) C, H,
N.
P r ep a r a tion of 3-Su bstitu ted 8-(1,2,3,4-Tetr a h yd r o-
n a p h th a len -2-yl)-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]d eca n -4-
on es. Gen er a l P r oced u r e. The appropriately substituted
8-(1,2,3,4-tetrahydro-naphthalen-2-yl)-1-phenyl-1,3,8-triazaspiro-
[4.5]decan-4-one hydrochloride (0.4 mmol) was suspended in
N,N-dimethylformamide (10 mL). Sodium hydride dispersion
(1.5 mmol) was added, and the mixture was stirred for 30 min
at room temperature and for 1 h at 80 °C. The mixture was
cooled to room temperature, and the appropriate alkylating
agent (1.5 mmol) was added. Stirring commenced at 80 °C till
the educt was consumed as evidenced by TLC (dichloromethane/
methanol, 9:1). The reaction mixture was poured into satu-
rated sodium bicarbonate solution (100 mL) and extracted with
ethyl acetate (3 × 50 mL). Organic phases were pooled, washed
with brine (100 mL), and dried with Na2SO4. The solvent was
evaporated, and the residue was purified by chromatography
on silica gel (dichloromethane/methanol 2-5%) and crystal-
lized as hydrochloride salt.
4-on e Hyd r och lor id e (2e). Compound 1e and 2-chloroethoxy-
trimethylsilane yielded a colorless solid (150 mg, 73%) from
ethyl acetatesthe trimethylsilyl protecting was removed
quantitatively during the chromatography: mp >244 °C dec;
1H NMR 1.8-2.1 (m, 3H), 2.35 (m, 1H), 2.8-3.2 (m, 5H), 3.3-
4.0 (m, 10H), 4.78 (s, 2H), 4.95 (t, 1H), 6.83 (t, 1H), 7.1-7.4
(m, 7H), 11.1 (bs, 1H); MS (high res) mass calcd for C25H30
-
ClN3O2 (MH+) ) 440.2105, obsd m/z at 440.2107, deviation
from theoretical ) 0.2 mmu.
(RS)-8-(8-Ch lor o-1,2,3,4-t et r a h yd r o-n a p h t h a len -2-yl)-
3-m et h oxym et h yl-1-p h en yl-1,3,8-t r ia za sp ir o[4.5]d eca n -
4-on e Hyd r och lor id e (2f). Compound 1e and chloromethyl
methyl ether yielded a colorless solid (110 mg, 54%) from ethyl
acetate: mp >227-229 °C dec; 1H NMR 1.8-2.1 (m, 3H), 2.35
(m, 1H), 2.8-3.2 (m, 5H), 3.29 (s, 3H), 3.3-4.0 (m, 6H), 4.80
(s, 2H), 4.81 (s, 2H), 6.86 (t, 1H), 7.1-7.4 (m, 7H), 11.1 (bs,
1H); MS (high res) mass calcd for C25H30ClN3O2 (MH+) )
440.2105, obsd m/z at 440.2109, deviation from theoretical )
0.4 mmu.
(RS)-1-P h en yl-8-(1,2,3,4-tetr a h yd r o-n a p h th a len -1-yl)-
1,3,8-tr ia za sp ir o[4.5]d eca n -4-on e Hyd r och lor id e (1j). So-
dium nitrite (75 mmol) in water (35 mL) was added to a
solution of 3,4-dihydro-1(2H)-naphthalenone oxime (44 mmol)
in diethyl ether (100 mL). Sulfuric acid (1 N, 75 mL) was added
slowly with stirring at room temperature to this mixture.
Stirring continued for 4 h after the addition was complete. The
phases were separated, the water phase was extracted once
with diethyl ether (100 mL), and organic phases were pooled,
dried with Na2SO4, and concentrated. The red oily residue (7.6
g) was dissolved in acetonitrile (50 mL) and added slowly to a
suspension of 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (133
mmol) and molecular sieves (32 g, 4 Å) in acetonitrile (500
mL). The mixture was stirred at room temperature for 36 h,
and molecular sieves were filtered off and washed with
dichloromethane/methanol (9:1, 400 mL). The filtrate was
concentrated and the residue dissolved in THF/ethanol (9:1,
500 mL). Sodium cyanoborohydride (32 mmol) was added with
stirring and the pH adjusted to 4.5 with HCl in ethanol.
Stirring at room temperature was continued for 2 h. The
mixture was poured into ice-water (200 g) and saturated
potassium carbonate solution (100 mL) and was extracted
three times with dichloromethane (200 mL each). Organic
phases were pooled, washed with brine, and dried with Na2-
SO4. The solvent was evaporated, and the residue was purified
by chromatography on silica gel (dichloromethane/methanol
2-4%) and decolorized with Norite to yield a beige solid (3.0
g, 19%) which was crystallized as the colorless hydrochloride
salt from ethyl acetate/ethanol (2:3): mp 259 °C; 1H NMR 1.67
(m, 1H), 1.86 (t, J ) 14.7, 2H), 1.9-2.3 (m, 3H), 2.6-3.1 (m,
4H, 4′-CH2, 7,9-CH2, eq), 3.25 (m, 1H), 3.40 (m, 1H, 7,9-CH2,
eq), 3.7 (m, 1H, 7,9-CH2, ax), 3.9 (m, 1H, 7,9-CH2, ax), 4.62
(m, 2H, 2-CH2), 4.86 (m, 1H, 1′-CH), 6.80 (t, J ) 7.1, 1H,
p-ArH), 7.11 (d, J ) 8.1, 2H, o-ArH), 7.2-7.4 (m, 5H, 5′,6′,7′-
ArH, m-ArH), 8.04 (m, 1H, 8′-ArH), 9.04 (s, 1H, 3-NH), 10.4
(bs, 1H, NH+); MS m/z 362.2 (MH)+. Anal. Calcd (C23H27N3O‚
HCl) C, H, N.
(RS)-8-(5,8-Dich lor o-1,2,3,4-tetr a h yd r o-n a p h th a len -2-
yl)-3-m et h yl-1-p h en yl-1,3,8-t r ia za sp ir o[4.5]d eca n -4-on e
Hyd r och lor id e (2a ). Compound 1a and methyl iodide yielded
a colorless solid (80 mg, 39%) from ethyl acetate: mp 256-
1
259 °C dec; H NMR 1.8-2.0 (m, 3H), 2.4 (m, 1H), 2.75 (m,
1H), 2.92 (s, 3H), 3.0-3.2 (m, 4H), 3.3-3.9 (m, 6H), 4.71 (s,
2H), 6.80 (t, 1H), 7.10 (d, 2H), 7.25 (t, 2H), 7.41 (s, 2H), 11.1
(bs, 1H); MS (high res) mass calcd for C24H27Cl2N3O (MH+) )
444.1609, obsd m/z at 444.1614, deviation from theoretical )
0.5 mmu.
(RS)-3-Allyl-8-(5,8-d ich lor o-1,2,3,4-t et r a h yd r o-n a p h -
th alen -2-yl)-1-ph en yl-1,3,8-tr iazaspir o[4.5]decan -4-on e Hy-
d r och lor id e (2b). Compound 1a and allyl bromide yielded a
colorless solid (95 mg, 44%) from ethyl acetat: mp 231-235
°C dec; 1H NMR 1.8-2.0 (m, 3H), 2.4 (m, 1H), 2.8 (m, 1H),
3.0-3.2 (m, 4H), 3.3-4.0 (m, 6H), 4.02 (d, J ) 5, 2H, 1′′-CH2),
4.70 (s, 2H, 2-CH2), 5.2-5.3 (m, 2H, 3′′CH), 5.75-5.95 (m, 1H,
2′′-CH), 6.82 (t, J ) 7, 1H, p-ArH), 7.13 (d, J ) 8, 2H, o-ArH),
7.22 (t, J ) 7.5, 2H, m-ArH), 7.41 (s, 2H, 6′,7′-ArH), 11.2 (bs,
1H, NH+); MS (high res) mass calcd for C26H29Cl2N3O (MH+)
) 470.1766, obsd m/z at 470.1767, deviation from theoretical
) 0.1 mmu.
(RS)-3-Ben zyl-8-(5,8-d ich lor o-1,2,3,4-tetr a h yd r o-n a p h -
th alen -2-yl)-1-ph en yl-1,3,8-tr iazaspir o[4.5]decan -4-on e Hy-
d r och lor id e (2c). Compound 1a and benzyl bromide yielded
a colorless solid (150 mg, 63%) from ethyl acetate; mp 263-
266 °C dec; 1H NMR 1.8-2.0 (m, 3H), 2.4 (m, 1H), 2.8 (m, 1H),
3.0-3.2 (m, 4H), 3.3-4.0 (m, 6H), 4.60 (s, 2H), 4.66 (s, 2H),
6.80 (t, 1H), 7.10 (d, 2H), 7.22 (t, 2H), 7.3-7.4 (m, 5H), 7.41
(s, 2H), 11.2 (bs, 1H); MS m/z 520.3, 522.3 (MH)+. Anal. Calcd
(C30H31Cl2N3O‚HCl) C, H, N.
(RS)-8-In d a n -1-yl-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]d eca n -
4-on e Hyd r och lor id e (1k ). A solution of indan-1-one (113
mmol), 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (113 mmol),
and titanium(IV) isopropoxide (142 mmol) in THF (100 mL)
was stirred for 18 h at room temperature. The solvent was
removed and the residue dissolved in THF/ethanol (1:2, 150
mL). Sodium cyanoborohydride (76 mmol) was added with
stirring, and stirring at room temperature was continued for
20 h. Water (30 mL) was added, and the mixture was filtered
through Celite. The filter cake was washed thoroughly with
ethanol, the filtrate evaporated, and the residue purified by
chromatography on silica gel (dichloromethane/methanol
2-10%) to yield a beige solid (17.7 g, 45%) which was
crystallized as the colorless hydrochloride salt from ethyl
(RS)-8-(8-Ch lor o-1,2,3,4-t et r a h yd r o-n a p h t h a len -2-yl)-
3-(2-oxo-2-p h en yl-eth yl)-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]-
d eca n -4-on e Hyd r och lor id e (2d ). Compound 1e and phen-
acyl bromide yielded a colorless solid (60 mg, 24%) from ethyl
1
acetate; mp 272 °C; H NMR 1.7-2.1 (m, 3H), 2.35 (m, 1H),
2.8-3.2 (m, 5H), 3.3-4.0 (m, 6H), 4.75 (s, 2H), 5.04 (s, 2H),
6.86 (t, 1H), 7.1-7.4 (m, 7H), 7.60 (t, 2H), 7.72 (t, 1H), 8.06
(d, 2H), 10.7 (bs, 1H); MS (high res) mass calcd for C31H32
-
ClN3O2 (MH+) ) 514.2261; obsd m/z at 514.2258, deviation
from theoretical ) 0.3 mmu.
1
acetate/ethanol (3:1): mp 270 °C; H NMR 1.82 (d, J ) 14.1,
2H, 2′-CH2), 2.8-3.2 (m, 6H, 6,10-CH2, 3′-CH2), 3.3-3.7 (m,
(RS)-8-(8-Ch lor o-1,2,3,4-t et r a h yd r o-n a p h t h a len -2-yl)-
3-(2-h yd r oxy-eth yl)-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]d eca n -
3H, 7,9-CH2, ax, eq), 3.8 (m, 1H, 7,9-CH2, ax), 4.60 (s, 2H,

1336 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Ro¨ver et al.
2-CH2), 5.03 (m, 1H, 1′-CH), 6.77 (t, J ) 7.3, 1H, p-ArH), 7.11
(d, J ) 8.1, 2H, o-ArH), 7.19 (t, J ) 7.2, 2H, m-ArH), 7.3-7.5
(m, 3H, 4′,5′,6′-ArH), 7.90 (d, 1H, 7′-ArH), 8.98 (s, 1H, 3-NH),
11.3 (bs, 1H, NH+); MS m/z 348.4 (MH)+. Anal. Calcd
(C22H25N3O‚HCl) C, H, N.
(sodium perchlorate (0.5 M)/acetonitrile (30:70), flow rate 0.8
mL/min) showed 12o to have 98% ee.
(S)-4-P h en yla m in o-1-(1,2,3,4-t et r a h yd r o-n a p h t h a len -
1-yl)-p ip er id in e-4-ca r boxylic Acid Am id e (13n ). A mixture
of acetic anhydride (30 mL), formic acid (30 mL), and 12n (17
mmol) was stirred at room temperature for 3 d, poured onto
ice-water (600 g), made basic with sodium hydroxide solution
(28%), and extracted with dichloromethane (2 × 200 mL).
Organic phases were pooled, dried with Na2SO4, and concen-
trated. The residue (6.2 g) was dissolved in tert-butyl alcohol
(120 mL). Water (10 mL) and ammonia (25%, 10 mL) were
added, and then hydrogen peroxide solution (33%, 20 mL) was
slowly added at room temperature. The mixture was stirred
16 h, water (200 mL) was added, and the solvent was removed
in vacuo. The residue was stirred with water (200 mL) and
the precipitate collected and dried to afford 13n (4.7 g, 79%)
as an off white solid: mp 186-187 °C; 1H NMR 1.61 (m, 2H),
1.9-2.8 (m, 12H), 3.74 (m, 1H), 5.47 (s, 1H), 6.5-6.6 (m, 3H),
7.95-7.20 (m, 7H), 7.60 (d, 1H); MS m/z 350.4 (MH)+. Anal.
Calcd (C22H27N3O) C, H, N. Chiral HPLC on a Chiralcel OJ -R
column (sodium perchlorate (0.5 M)/acetonitrile (65:35), flow
rate 0.8 mL/min) showed 13n to have >99.5% ee. A typical
retention time for the S-isomer 13n was 25.9 min and for the
R-isomer 13o 28.4 min.
(S)-(-)-1,2,3,4-Tet r a h yd r o-1-n a p h t h yla m in e H yd r o-
ch lor id e (11n ). (R)-(-)-1,2,3,4-Tetrahydro-1-naphthol (10n ,
35 mmol, [R]20 -33 (c 2.5, CHCl3)) and diphenylphosphoryl
D
azide (42 mmol) were dissolved in toluene (80 mL) and cooled
to 0 °C. 1,8-Diazabicyclo[5.4.0]undec-7-ene (42 mmol) dissolved
in toluene (20 mL) was added slowly with cooling, and stirring
was continued for 2 h at 0 °C and 16 h at room temperature.
Filtration with ethyl acetate/hexane (1:6) through silica gel
yielded a mixture of azide and 1,2-dihydro-naphthalene as
colorless oil (5.9 g). This mixture was dissolved in THF (60
mL) and added slowly to a suspension of lithium aluminum
hydride (42 mmol) in THF (50 mL) at room temperature. The
reaction mixture was stirred for 1 h at room temperature and
for another 1 h refluxed. After cooling, the mixture was
hydrolyzed by subsequent careful addition of water (1.6 mL),
sodium hydroxide solution (15%, 3.2 mL), and water (4.8 mL).
The suspension was then aged and filtered through Celite. The
filtrate was concentrated, dissolved in ethyl acetate (120 mL),
and precipitated as the hydrochloride (4.9 g, 63%) after
addition of hydrogen chloride in ethanol: mp 236-237 °C.
(R)-4-P h en yla m in o-1-(1,2,3,4-tetr a h yd r o-n a p h th a len -
1-yl)-p ip er id in e-4-ca r boxylic Acid Am id e (13o). This com-
pound was prepared from 12o (8 mmol). Compound 13o (2.1
g, 75%) was isolated as an colorless solid: mp 182-184 °C.
Anal. Calcd (C22H27N3O) C, H, N. Chiral HPLC on a Chiralcel
OJ -R column (sodium perchlorate (0.5 M)/acetonitrile (65:35),
flow rate 0.8 mL/min) showed 13o to have >99.5% ee.
Anal. Calcd (C10H13N‚HCl) C, H, N. Chiral HPLC on
a
Crownpak CR(-) column (perchloric acid (pH 2)/acetonitrile
(89:11), flow rate 0.9 mL/min) showed 11n to have 92% ee. A
typical retention time for the S-isomer 11n was 8.1 min and
for the R-isomer (11o) 6.7 min.
(R)-(+)-1,2,3,4-Tet r a h yd r o-1-n a p h t h yla m in e H yd r o-
ch lor id e (11o). This compound was prepared from (S)-(+)-
(S)-1-P h en yl-8-(1,2,3,4-t et r a h yd r o-n a p h t h a len -1-yl)-
1,3,8-tr ia za sp ir o[4.5]d eca n -4-on e Hyd r och lor id e (1n ). A
suspension of 13n (12 mmol) in formamide (65 mL) was heated
for 2 h to 200 °C. The mixture was cooled, poured onto ice-
water (800 g), and extracted with dichloromethane (3 × 400
mL). Organic phases were pooled, washed with brine, dried
with Na2SO4, and concentrated. The residue (4.9 g) was
dissolved in methanol (200 mL). Sodium borohydride (18
mmol) was added, and the mixture was stirred for 1 h at room
temperature and for another hour at 60 °C. The solvent was
removed in vacuo, and the residue was partitioned between
ammonia (150 mL, 12%) and dichloromethane (3 × 150 mL).
Organic phases were pooled and dried with Na2SO4. The
solvent was evaporated, and the residue was purified by
chromatography on silica gel (ethyl acetate/hexane 1:1) and
crystallized as the hydrochloride salt from ethyl acetate/
ethanol to afford 1n (1.0 g, 23%) as a colorless solid: mp 269-
271 °C; [R]20D -30.4 (c 1, CH3OH); 1H NMR 1.67 (m, 1H), 1.86
(t, 2H), 1.9-2.3 (m, 3H), 2.6-3.1 (m, 4H), 3.25 (td, 1H), 3.3-
3.5 (m, 1H), 3.7 (m, 1H), 3.9 (m, 1H), 4.61 (m, 2H), 4.86 (m,
1H), 6.79 (t, 1H), 7.11 (d, 2H), 7.2-7.4 (m, 5H), 8.13 (m, 1H),
9.04 (s, 1H), 10.5 (bs, 1H); MS m/z 362.2 (MH)+. Anal. Calcd
(C23H27N3O‚HCl) C, H, N. Chiral HPLC on a Chiralcel OC
column (hexane/10% ethanol in hexane/0.4% triethylamine in
hexane (40:40:20), flow rate 1.5 mL/min) showed 1n to have
>99.5% ee. A typical retention time for the S-isomer 1n was
14.1 min and for the R-isomer 1o 16.7 min.
1,2,3,4-tetrahydro-1-naphthol (27 mmol, [R]20 +33 (c 2.5,
D
CHCl3)). Compound 11o was isolated as the hydrochloride (3.0
g, 60%): mp 236-237 °C. Anal. Calcd (C10H13N‚HCl) C, H, N.
Chiral HPLC on a Crownpak CR(-) column (perchloric acid
(pH 2)/acetonitrile (89:11), flow rate 0.9 mL/min) showed 11o
to have 98% ee.
(S)-4-P h en yla m in o-1-(1,2,3,4-t et r a h yd r o-n a p h t h a len -
1-yl)-p ip er id in e-4-ca r bon itr ile (12n ). To a boiling suspen-
sion of 11n (26 mmol) and potassium carbonate (15 mmol) in
ethanol (50 mL) was added 1-methyl-1-ethyl-4-oxopiperidinium
iodide16 (39 mmol) dissolved in water (17 mL). The reaction
mixture was boiled for another hour, cooled, and diluted with
water (50 mL). Ethanol was removed in vacuo and the residue
was partitioned between sodium hydroxide solution (1 N, 100
mL) and ethyl acetate (3 × 100 mL). Organic phases were
pooled, dried with Na2SO4, and concentrated. The residue was
filtered with ethyl acetate/hexane (1:1) through silica gel to
yield the intermediate 1-(1,2,3,4-tetrahydro-naphthalen-1-yl)-
piperidin-4-one (5.3 g). To a solution of this raw material in
acetic acid (20 mL) were added aniline (25 mmol) and tri-
methylsilyl cyanide (23 mmol) at room temperature. The
mixture was stirred for 90 min after which it was poured onto
a mixture of ice (100 g) and ammonia (25%, 60 mL) and
extracted with dichloromethane (3 × 200 mL). Organic phases
were pooled, dried with Na2SO4, and concentrated to yield a
yellow solid. This solid was digested with diethyl ether to
afford 12n (5.8 g, 76%) as an off white solid, mp 152-153 °C:
1H NMR (CDCl3) 1.70 (m, 2H), 1.85 (m, 1H), 1.97 (m, 3H), 2.27
(m, 1H), 2.40 (m, 1H), 2.55 (m, 1H), 2.6-3.0 (m, 5H), 3.65 (s,
1H), 3.88 (m, 1H), 6.8-6.95 (m, 3H), 7.05-7.20 (m, 3H), 7.25
(t, 2H), 7.62 (m, 1H); MS m/z 332.2 (MH)+. Anal. Calcd
(C22H25N3) C, H, N. Chiral HPLC on a Chiralcel OJ -R column
(sodium perchlorate (0.5 M)/acetonitrile (30:70), flow rate 0.8
mL/min) showed 12n to have 96% ee. A typical retention time
for the S-isomer 12n was 7.8 min and for the R-isomer 12o
6.7 min.
(R)-1-P h en yl-8-(1,2,3,4-t et r a h yd r o-n a p h t h a len -1-yl)-
1,3,8-tr iazaspir o[4.5]decan -4-on e Hydr och lor ide (1o). This
compound was prepared from 13o (5 mmol). Compound 1o (0.2
g, 11%) was isolated as an colorless solid: mp 269-270 °C.
[R]20 +31.3 (c 1, CH3OH); 1H NMR 1.67 (m, 1H), 1.83 (t, 2H),
D
1.9-2.3 (m, 3H), 2.6-3.1 (m, 4H), 3.2-3.5 (m, 2H), 3.7 (m, 1H),
3.9 (m, 1H), 4.61 (m, 2H), 4.85 (m, 1H), 6.78 (t, 1H), 7.11 (d,
2H), 7.2-7.4 (m, 5H), 8.13 (m, 1H), 9.04 (s, 1H), 10.5 (bs, 1H);
MS m/z 362.2 (MH)+. Anal. Calcd (C23H27N3O‚HCl) C, H, N.
Chiral HPLC on a Chiralcel OC column (hexane/10% ethanol
in hexane/0.4% triethylamine in hexane (40:40:20), flow rate
1.5 mL/min) showed 1o to have >99.5% ee.
(R)-4-P h en yla m in o-1-(1,2,3,4-tetr a h yd r o-n a p h th a len -
1-yl)-p ip er id in e-4-ca r bon itr ile (12o). This compound was
prepared from 11o (15 mmol). Compound 12o (2.9 g, 58%) was
isolated as an off white solid: mp 152-153 °C. Anal. Calcd
(C22H25N3) C, H, N. Chiral HPLC on a Chiralcel OJ -R column
(RS)-8-(5-Meth yl-1,2,3,4-tetr a h yd r o-n a p h th a len -1-yl)-
1-p h en yl-1,3,8-t r ia za sp ir o[4.5]d eca n -4-on e H yd r och lo-
r id e (1p ). Sodium nitrite (33 mmol) in water (20 mL) was
added to a solution of 3,4-dihydro-5-methyl-1(2H)-naphthale-

High-Affinity ORL1 Receptor Agonists
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1337
none oxime21 (20 mmol) in diethyl ether (60 mL). Sulfuric acid
(1 N, 35 mL) was added slowly with stirring at room temper-
ature to this mixture. Stirring continued for 4 h after the
addition was complete. The phases were separated, the water
phase was extracted once with diethyl ether (60 mL), and
organic phases were pooled, dried with Na2SO4, and concen-
trated. The orange-red, solid residue (3.6 g) was dissolved in
acetonitrile (20 mL) and added slowly to a suspension of
1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (40 mmol) and mo-
lecular sieves (11 g, 4 Å) in acetonitrile (150 mL). The mixture
was stirred at room temperature for 36 h, and molecular sieves
were filtered off. The filtrate was concentrated, and the residue
was dissolved in THF/ethanol (9:1, 500 mL). Sodium cy-
anoborohydride (20 mmol) was added with stirring and the
pH adjusted to 4.5 with HCl in ethanol. Stirring at room
temperature was continued for 16 h. The mixture was poured
into ice-water (200 g) and saturated potassium carbonate
solution (100 mL) and was extracted three times with dichlo-
romethane (200 mL each). Organic phases were pooled, washed
with brine, and dried with Na2SO4. The solvent was evapo-
rated, and the residue was purified by chromatography on
silica gel (ethyl acetate/hexane 1:1) to yield a yellowish solid
(1.5 g, 19%) which was crystallized as the colorless hydrochlo-
siently expressing hµ, hκ or hδ receptors (10 µg of membrane
protein each) and 1.5 nM (hµ) and 3 nM (hκ) [3H]-naloxone or
0.3 nM [3H][Ile5,6]-deltorphin II (hδ).
Reactions were performed in 1 mL of binding buffer (50 mM
Tris-HCl, pH 7.8; 1 mM EGTA; 5 mM MgCl2; 0.1% BSA) for
60 min at 22 °C. At the end of the incubation, the samples
were filtered through GF/C-filters (Amersham), which had
been precoated (0.3% polyethyleneimine plus 0.1% BSA).
Filters were then washed 3 times with 0.5 mL of cold wash
buffer (50 mM Tris-HCl, pH 7.5). Finally, 60 µL of scintillation
fluid (Micro Scint, Canberra Packard) was added, and samples
were counted in a Top Counter (Packard). Nonspecific binding
was defined in the presence of 1 µM unlabeled OFQ (hORL1
receptor), 10 µM naloxone (hµ and hκ receptors), or 10 µM
deltorphin II (hδ receptors).
GTP γS Bin din g Assay. Agonist-mediated binding of GTPγS
was investigated in 96-well plates using a scintillation proxim-
ity assay (SPA) and membranes prepared from cells expressing
hOFQ, hµ, hκ, or hδ receptors. Binding was performed in 200
µL of 20 mM HEPES-buffer (pH 7.4, plus 6 mM MgCl2 and
100 mM NaCl), supplemented with 20 µM GDP and 0.3 nM
GTP[γ35]S (1130 Ci/mmol). Twenty micrograms of membranes,
1 mg of wheatgerm agglutinin SPA beads (Amersham, Little
Chalfont, U.K.), and either OFQ (10-5 M to 10-10 M) or
synthetic compounds (10-4 M to 10-10 M) were added. Non-
specific binding was determined in the presence of 10 µM
unlabeled GTPγS.
1
ride salt from ethyl acetate/ethanol (1:1): mp 259 °C; H NMR
1.6-1.9 (m, 3H), 2.12 (m, 3H), 2.22 (s, 3H, CH3), 2.6-3.5 (m,
6H), 3.7 (m, 1H, 7,9-CH2, ax), 3.9 (m, 1H, 7,9-CH2, ax), 4.61
(m, 2H, 2-CH2), 4.78 (m, 1H, 1′-CH), 6.78 (t, J ) 7.5, 1H,
p-ArH), 7.11 (d, J ) 7.6, 2H o-ArH), 7.2 (m, 4H, 6′,7′-ArH,
m-ArH), 7.86 (m, 1H, 8′-ArH), 9.04 (s, 1H, 3-NH), 10.3 (bs,
1H, NH+); MS m/z 376.3 (MH)+. Anal. Calcd (C24H29N3O‚HCl)
C, H, N.
The reaction mixture was incubated on a shaker for 60 min
at 22 °C and then centrifuged for 5 min at 1500 rpm in an
Eppendorf 5403 centrifuge. Finally the plates were read in a
Top counter (Packard).
(RS)-8-Acen a p h ten -1-yl-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]-
d eca n -4-on e Hyd r och lor id e (1q). Reaction of acenaphten-
1-one (6.7 mmol), 1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one,
and titanium(IV) isopropoxide (8.4 mmol) in THF (15 mL) and
subsequent reduction with sodium cyanoborohydride (4.5
mmol) in EtOH (15 mL) as described for 1k yielded, after
column chromatography on silica gel (ethyl acetate/hexane
4:1), a pale brown foam (0.56 g, 22%) which was crystallized
as the yellowish hydrochloride salt from diethyl ether/
cAMP Assa y. The inhibition of forskolin-mediated cAMP
accumulation by OFQ and synthetic OFQ agonists was deter-
mined in 96-well plates. Briefly, 20 000 cells were incubated
in Krebs-Ringer-HEPES-buffered solution (KHR; 124 mM
NaCl, 5 mM KCl, 1.25 mM MgSO4, 1.5 mM CaCl2, 1.25 mM
KH2PO4, 25 mM HEPES, pH 7.4) in the presence of 100 µM
Rolipram and 1 µM forskolin (both from Sigma) with increas-
ing concentrations of agonists (10-11 to 10-7 M) for 15 min at
37 °C. Reactions were stopped by the addition of 0.12 mL of
ice-cold ethanol, and mixtures were stored at -80 °C for at
least 4 h. The cAMP content was determined from the
supernatant using the Biotrak nonradioactive cAMP kit (Am-
ersham, Little Chalfont, U.K.) according to the manufacturer’s
instructions.
1
methanol (3:1): mp 263 °C; H NMR 1.82 (d, J ) 13.5, 2H),
2.89-3.25 (m, 3H), 3.29-3.97 (m, 5H), 4.59 (s, 2H, 2-CH2), 5.67
(m, 1H, 1′-CH), 6.78 (t, J ) 8.5, 1H, p-ArH), 7.11 (d, J ) 8.5,
2H, m-ArH), 7.22 (t, J ) 8.5, 2H, o-ArH), 7.50 (d, J ) 7.5, 1H),
7.59 (t, J ) 7.5, 1H), 7.69 (t, J ) 7.5, 1H), 7.79 (d, J ) 7.5,
1H), 7.95 (d, J ) 7.5, 1H), 8.11 (d, J ) 7.5, 1H), 8.99 (s, 1H,
3-NH), 10.74 (bs, 1H, NH+); MS m/z 384.3 (MH)+. Anal. Calcd
(C25H25N3O‚HCl) C, H, N.
Ack n ow led gm en t. The skillful assistance of B. Frei,
R. Augsburger, P. Oberli, D. Wehrli, R. Henningsen, J .
Higelin, C. Kuhn, G. Py, and I. Vogel is gratefully
acknowledged. The authors thank W. Arnold, M. Hen-
nig, W. Vetter, W. Meister, V. Meduna, and G. Oester-
helt for spectroscopic analysis, K. Lundstrom, P. Mal-
herbe, and S. Hankel for cells expressing hOFQ and
opioid receptors, and M. Graf and A. Sleight for binding
data on dopamine and serotonin receptors.
Bioch em istr y. Cell Cu ltu r e a n d Mem br a n e P r ep a r a -
tion . The cDNAs encoding hµ, hκ and hδ receptors were
subcloned into the pSFV2gen vector, and recombinant Semliki
Forest Virus stocks were generated as described previously.22
Baby hamster kidney (BHK) cells were infected and harvested
for membrane preparation 24 h after infection. The cDNA
encoding the hORL1 receptor was inserted into the pcDNA3
vector and stably transfected into human embryonic kidney
293 (HEK293) cells. One cell clone was selected for pharma-
cological characterization.
Membrane fractions were prepared by homogenization of
cells followed by a 30 min centrifugation at 39000g using a
Beckman J A-20 rotor. The resulting membrane pellet was
resuspended in 50 mM Tris, pH 7.8, 1 mM EDTA, 6 mM MgCl2
buffer at a concentration of ∼2 × 107 cells/mL and frozen at
-80 °C.
Ra d ioliga n d s. The radioligands [3H]-OFQ (specific activity
150 Ci/mmol), [3H]-naloxone (specific activity 54.5 Ci/mmol),
and [3H][Ile5,6]-deltorphin II (specific activity 72 Ci/mmol) were
from Amersham (Little Chalfont, U.K.).
Su p p or tin g In for m a tion Ava ila ble: Elemental analysis.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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