Design, synthesis and pharmacological evaluation of spirocyclic σ1 receptor ligands with exocyclic amino moiety (increased distance 1)

Article abstract of DOI:10.1016/j.bmc.2011.04.002

Various pharmacophore models for potent σ₁ ligands specify a basic amino group flanked by two different hydrophobic regions in defined distances to the basic amine (distance 1 and distance 2, respectively). According to these models distance 1 of the potent spirocyclic σ₁ ligand 1 is too short. In order to find a new class of more potent σ₁ ligands and to verify the distance hypothesis of the pharmacophore models spirocyclic compounds 2 with an exocyclic amino group were designed and synthesized. The secondary amines 8 and 9 with N-benzyl residues are >100-fold less potent than the spirocyclic piperidine 1. However, the tertiary methylamines trans-11 and cis-11 represent potent σ₁ ligands with K_i-values of 43 and 24 nM, respectively. Whereas one large benzyl moiety is required for high σ₁ receptor binding, a second large N-substituent is not tolerated by the σ₁ receptor protein. As a rule, cis-configured diastereomers with a longer distance 1 (predominantly 7.16-7.23 ) show higher σ₁ affinities than their trans-configured counterparts (distance 1 is predominantly 5.88-6.26 ).

Full text of DOI:10.1016/j.bmc.2011.04.002

Bioorganic & Medicinal Chemistry 19 (2011) 3141–3151
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and pharmacological evaluation of spirocyclic r1 receptor
ligands with exocyclic amino moiety (increased distance 1)
Elisabeth Rack a, Roland Fröhlich b, Dirk Schepmann a, Bernhard Wünsch a,
⇑
a Institut für Pharmazeutische und Medizinische Chemie der Universität Münster, Hittorfstraße 58-62, D-48149 Münster, Germany
b Organisch-Chemisches Institut der Westfälischen Wilhelms-Universität Münster, Corrensstraße 40, D-48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Various pharmacophore models for potent
r1 ligands specify a basic amino group flanked by two differ-
Received 13 December 2010
Revised 30 March 2011
Accepted 1 April 2011
Available online 6 April 2011
ent hydrophobic regions in defined distances to the basic amine (distance 1 and distance 2, respectively).
According to these models distance 1 of the potent spirocyclic 1 ligand 1 is too short. In order to find a
new class of more potent 1 ligands and to verify the distance hypothesis of the pharmacophore models
r
r
spirocyclic compounds 2 with an exocyclic amino group were designed and synthesized. The secondary
amines 8 and 9 with N-benzyl residues are >100-fold less potent than the spirocyclic piperidine 1. How-
Keywords:
ever, the tertiary methylamines trans-11 and cis-11 represent potent
24 nM, respectively. Whereas one large benzyl moiety is required for high
r
1 ligands with Ki-values of 43 and
1 receptor binding, a second
r1 Receptor ligands
r1 Pharmacophore models
Spirocyclic r1 ligands
Diastereoselectivity
Conformational analysis
Structure–r1 affinity relationships
X-ray
r
large N-substituent is not tolerated by the r1 receptor protein. As a rule, cis-configured diastereomers
with a longer distance 1 (predominantly 7.16–7.23 Å) show higher r1 affinities than their trans-
configured counterparts (distance 1 is predominantly 5.88–6.26 Å).
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
cally show modulatory properties, for example, modulation of
Ca2+-, K+-, Na+-, Clꢀ-channels as well as NMDA and IP3 receptors.17
Initially the
type.1 The fact that classical opioid receptor antagonists like nalox-
one and naltrexone did not inhibit the effects of ligands led to
classification of
receptors as an independent receptor class.2,3
Differentiation via ligand binding experiments and biochemical
r
receptor was classified as an opioid receptor sub-
Several r1 mediated effects occur only after preactivation of an-
other biological system.18 Some neurosteroids interact with r1
receptors and are therefore assumed to be the endogenous r1 li-
gands.19 In contrast to most known r1 receptor ligands they do
not contain a basic element. Dehydroepiandrosterone and preg-
nenolone sulfate show r1 agonistic and progesterone r1 antago-
nistic activity.20 The steroid levels in the brain are modified
especially in diseases associated with r1 receptors.21,22 Very re-
r
r
analysis defined two
r receptor subtypes, which were termed r1
and r2 receptors.4
The r1 receptor is a 25.3 kDa membrane bound protein, which
has been cloned.5–7 The amino acid sequence does not show any
homology or even similarity with any other known mammalian
protein. On the contrary a 30% homology with the yeast enzyme
cently N,N-dimethyltryptamine with a Ki-value of 15
lM has been
postulated as endogenous r1 receptor ligand.23
Since many known centrally active drugs also display high
affinity to r1 receptors, r1 ligands are considered as useful drugs
for the treatment of several psychiatric disorders.24,25 Pharmaco-
logical analyses lead to potential applications as antidepres-
sants26,27 and anxiolytic agents.28 There is particular interest in
r1 ligands for the treatment of memory disorders,22,29 Alzheimer
disease as well as cocaine abuse.22,28 Moreover, r1 ligands can be
used as neuroprotectants at the eye.13,30 Chen and Pasternak de-
ergosterol-D8 7-isomerase was detected.6 r1 receptors are lo-
/D
cated with high density in the central nervous system (CNS)—espe-
cially in regions, which are associated with motoric, cognitive and
sensoric functions.8,9 Moreover, large amounts of r1 receptors
were discovered in some organs and tissues in the periphery
including the heart,10 liver,11 kidney12 and the eye13 as well as in
some human tumor cell lines.14
The exact r1 mediated signal transduction pathway is not
scribe the
tazocine, a known
mediated analgesia, whilst
antinociception.31 Down regulation of r1 receptors potentiates
opioid mediated analgesia.17 Therefore,
1 antagonists show a high
r
system as an endogenous anti-opioid system. (+)-Pen-
agonist, antagonizes , and d receptor
1 antagonists enhance opioid induced
known yet. But it has been clearly shown that
r
1 receptors are nei-
r
l, j
ther ion channel receptors, soluble receptors, tyrosine kinase
r
receptors nor classical G-protein coupled receptors.15,16 They basi-
r
potential as new analgesics with reduced side effects. Furthermore
the high density of r1 receptors in some human tumor cell lines
⇑
Corresponding author. Tel.: +49 251 8333311; fax: +49 251 8332144.
E-mail address: wuensch@uni-muenster.de (B. Wünsch).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.002

3142
E. Rack et al. / Bioorg. Med. Chem. 19 (2011) 3141–3151
Figure 1. Different pharmacophore models for r1 ligands. (A)
r
Pharmacophore model according to Gilligan et al.46 (B) r1 pharmacophore model according to Glennon
et al.33,47,48 (C) 3D-r1 pharmacophore model according to Langer et al. including calculations of distances and surface of the highly potent r1 receptor model ligand
fenpropimorph.49
will be exploited for the development of novel tumor diagnostics
and antitumor drugs.25,32
The differentiation of the r receptor into r1 and r2 subtypes
required some modifications of the Gilligan model. In 1994
Glennon et al. described the first 2D r1 pharmacophore model.
(Fig. 1B) Based on deconstruction–reconstruction–elobaration
In this article we report on the synthesis and pharmacological
evaluation of spirocyclic compounds bearing an exocyclic amino
moiety. Theoretical considerations taking various pharmacophore
models and the corresponding distances of pharmacophoric ele-
ments into account provided the rationale for the development
of these compounds.
analyses of different flexible
r1 ligands a pharmacophore compris-
ing one basic amine flanked by two ‘hydrophobic regions’ was de-
fined. The distances between the basic amino group and the
‘primary’ and ‘secondary hydrophobic regions’ should be in the
range of 6–10 Å and 2.5–3.9 Å, respectively. The basic group can
be a secondary or tertiary amine, but in the case of a tertiary amine
the third N-substituent should be rather small (e.g., a CH3 moi-
ety).47 The term hydrophobic region is slightly confusing here, be-
cause in pharmacophore models ligand properties are usually
described, but the expression ‘region’ refers better to the properties
of the receptor surface. According to Glennon’s model the binding
2. Pharmacophore models of r1 ligands
Several structural diverse r1 ligands with high affinity are known
today, including benzomorphans, conformationally constrained and
flexible arylalkylamines,33 spirocyclic compounds,34–37 bicyclic
ligands,38,39 guanidines and steroids20 as well as clinically used drugs
like fluvoxamine40 and haloperidol.41
OCH3
Forthedevelopmentof novel
r1 ligandsthestructuralproperties,
OCH3
which are crucial for high 1 receptor affinity were investigated. In
r
O
O
1986 Lloyd and Andrews postulated general requirements for CNS
active compounds—a phenyl ring and a basic amino group.42 Based
on this hypothesis Manallack et al. and Gund et al. superimposed
N
independently different
r ligands and defined a distance between
N
R
the phenyl ring and the basic amino moiety of 5.06 Å (Manallack)
and 5.3–5.7 Å (Gund), respectively.43–45 In 1992 Gilligan et al. pub-
1
2
lished a
and distances of two ‘hydrophobic groups’ and one ‘basic nitrogen
center’ to each other for potent
1 ligands.46 (Fig. 1A)
r pharmacophore model, which defines the orientation
Figure 2. Novel spirocyclic r1 ligands 2 are derived from the potent spirocyclic
piperidine 1.
r

E. Rack et al. / Bioorg. Med. Chem. 19 (2011) 3141–3151
3143
pocket of the receptor tolerates sterically demanding hydrophobic
substituents at both ends.33,48
phobic interactions supports the hypothesis that r1 ligands
interact with the steroid binding domain like region 1 (SBDL1) of
the r1 receptor protein.51
The first 3D computer-based r1 pharmacophore model was
developed by Langer et al. and consists of four hydrophobic groups
and one positive ionizable group. (Fig. 1C)The postulated distances
between the basic amino moiety and the hydrophobic groups are
4.1, 6.3 and 9.8 Å, respectively. These distances correspond pretty
well with the distances defined in the Glennon model. Compared
with the Glennon model the Langer model defines two additional
hydrophobic groups and the three dimensional orientation to each
other.49
Recently Zampieri et al. developed a computer based model
consisting of five pharmacophoric features—one basic amine, two
hydrophobic aromatic groups, one hydrophobic group and one H-
bond acceptor. The distances of 7.01 and. 8.50 Å, respectively as
well as 3.58 Å are in good accordance with the Glennon and Langer
models. However in this model an additional H-bond acceptor was
postulated, which has been previously postulated in the Gilligan
model.50
3. Theoretical considerations and design of novel r1 ligands
The majority of reported potent r1 receptor ligands are con-
formationally rather flexible. In our group several spirocyclic com-
pounds with high
r
1 affinity have been synthesized.34–37,52–54 The
rigid spirocyclic framework of these compounds reduces the con-
formational flexibility and defines the relative orientation of the
pharmacophoric elements to each other. The spirocyclic 2-
benzopyran 1, which represents a highly potent (Ki = 1.29 nM)
and subtype selective (Ki (
as lead compound for this project. (Fig. 2)
r2) >1000 nM) r
1 ligand,52 was selected
The high r1 affinity of 1 can be explained with the described
pharmacophore models. (see Fig. 1) For the determination of the
distances of the pharmacophoric elements a stochastic conforma-
tional search (MOE—Molecular Operating Environment) was per-
formed with energy cut off of 7 kcal/mol. Since it is assumed that
the basic amino moiety is protonated in physiological milieu, the
corresponding protonated compound 1H+ was included into the
study. Moreover, both enantiomers were included in the calcula-
tions, respectively. The distances between the basic amino moiety
and the two hydrophobic regions were calculated for all energeti-
cally favored conformations using the center of the phenyl rings.
The definitions of distance 1 and distance 2 are given in Figure 3.
Very recently, a pseudoreceptor model for spirocyclic r1 li-
gands has been reported, which simulates the binding characteris-
tics of r1 receptor ligands and visualizes potential structural
features of the receptor surface. In this model an H-bond acceptor
group was postulated on the receptor surface, which interacts with
the protonated amino group of the
r1 ligand. The receptor surface
consists of many hydrophobic particles leading to many hydropho-
bic interactions during ligand binding. The large amount of hydro-
Figure 3. Visualization of distances 1 and 2 for the lead compound 1 and the designed ligands trans-8 and cis-8.

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E. Rack et al. / Bioorg. Med. Chem. 19 (2011) 3141–3151
Generally the spirocyclic compound 1 can adopt two types of
chair conformations, which are termed trans/cis-2-1 and trans/cis-
2-2. (Fig. 4)
conformations: in the first type of conformations 1-1 the oxygen
atom of the benzopyran ring adopts the axial position at the piper-
idine chair (Fig. 4, left), whereas in the second type of conforma-
tions 1-2 the oxygen atom adopts the equatorial position (Fig. 4,
right). Within the energy frame of 7 kcal/mol both types of confor-
mations were found for the free piperidine 1. Nevertheless, the
number of energetically favored conformations of type 1-1 with
the oxygen atom in the axial orientation is much higher. For the
protonated compound 1H+ only conformations of type 1-1 were
observed. (Table 1)
In Table 1 the distances between the N-atom and the centers of
the phenyl moieties are summarized. The distance 2 between the
N-atom and the center of the N-benzyl residue is exactly within
the range, which has been postulated by Gilligan (3 1 Å), Glennon
(2.5–3.9 Å) and Langer (around 4.1 Å). However, distance 1 be-
tween the N-atom and the phenyl moiety of the benzopyran sys-
tem is not matching to the models. The analysis led to two types
of conformers. Whereas distance 1 in the less populated conforma-
tion type 1-2 is 5.09–5.18 Å the conformations of type 1-1 have a
distance 1 of 5.65–5.75 Å. These distances are considerably shorter
than the postulated distances of 6–10 Å (Glennon) and 6.3 Å and
9.5 Å (Langer).
In order to compare the distances 1 and 2 of the designed spiro-
cyclic compounds 2 with those of the spirocyclic piperidine 1 a sto-
chastic conformational search was performed with both
benzylamines trans-8 and cis-8. Again an energy cut off of 7 kcal/
mol was defined and both types of conformers 8-1 (O-atom of
the benzopyran ring axially oriented at the cyclohexane ring) and
8-2 (O-atom in equatorial position) as well as both enantiomers
were considered, respectively.
In case of trans-8 energetically favored conformations of type
trans-8-1 and trans-8-2 were found. However, distances 1 of both
conformation types are very similar 5.88–6.26 Å. In addition to
clear chair conformations of the cyclohexane ring two energetically
allowed (E <7 kcal/mol) distorted chair conformations were found
for the protonated compound trans-8H+ with an increased distance
1 of 6.52–6.56 Å.
The cis-configured compound cis-8 can also adopt both types of
conformations, although the conformations of type cis-8-1 with ax-
ial orientation of the benzopyran-O-atom are slightly preferred.
Whereas distances 1 of type cis-8-2 conformations are in the range
of 6.25–6.30 Å, these distances are considerably longer (7.16–
7.23 Å) for cis-8-1 type conformations.
These results stimulated the idea to increase the distance 1 be-
tween the N-atom and the phenyl moiety of the benzopyran sys-
tem of the spirocyclic r1 ligands in order to improve the r1
affinity and selectivity over other receptor systems. One possibility
to increase distance 1 is the removal of the N-atom from the piper-
idine ring and attach it at the ring system. This modification results
in the spirocyclic compounds 2 with an exocyclic amino moiety at-
tached to the spirocyclic connected cyclohexane ring. (Fig. 2)
Attachment of the amino moiety at the cyclohexane ring provides
two diastereomeric compounds trans-2 and cis-2 reflecting the rel-
ative orientation of N- and O-substituents at the cyclohexane ring.
The central cyclohexane ring of both diastereomers can adopt two
Distance 1 of the spirocyclic ligands with exocyclic amino moi-
ety is increased in the following order: tans-8-1 = trans-8-2 < cis-8-
2 < cis-8-1. These distances are longer than the distances 1 in the
lead compound 1 and fulfill exactly the requirements defined in
the pharmacophore models of Glennon (6–10 Å), Langer (6.3 and
9.5 Å) and Zampieri (7.01 and 8.56 Å). (Fig. 1)
Since in this study only benzylamines were considered, dis-
tances 2 of the lead compound 1 and the cyclohexane derivatives
8 are very similar (3.69–3.85 Å), independently on the position of
the N-atom in or at the ring system, the configuration or the pro-
tonation state. Initially the N-benzyl substituent was not modified,
since the corresponding distances fit well into the described mod-
els of Glennon (2.5–3.9 Å), Laggner (4.1 Å) and Zampieri (3.58 Å).
4. Synthesis
OCH3
The synthesis of the designed spirocyclic r1 ligands of type 2
with an exocyclic amino moiety started with 2-bromobenzalde-
hyde (3). (Scheme 1) Homologation of the aldehyde was performed
via a Wittig reaction with (methoxymethyl)triphenylphosphonium
chloride and KOtBu and subsequent addition of methanol to the
resulting enol ether 4, which gave the dimethyl acetal 5.55
Aryl bromide 5 was treated with n-BuLi at ꢀ78 °C and the
formed aryllithium intermediate was trapped with an excess of
cyclohexane-1,4-dione to afford the hydroxy acetal 6 in 50–60%
yields. During NMR-spectroscopy of the hydroxy acetal 6 in CDCl3
cyclization was observed, which was due to the presence of small
amounts of HCl in CDCl3. Therefore, the intramolecular transacetal-
ization of the hydroxy acetal 6 to the 2-benzopyran 7 was con-
ducted in CHCl3 after addition of catalytic amounts of HCl. After
stirring the reaction mixture for 90 min at room temperature, the
racemic spirocyclic benzopyran 7 was isolated in 81% yield. Prolon-
gation of the reaction time led to reduced yields, since elimination
of methanol occurred as side reaction.
O
N
OCH3
O
N
1-1
1-2
OCH3
O
N
R
OCH3
O
N
R
trans-2-1
trans-2-2
OCH3
R
N
Reductive amination of the spirocyclic ketone 7 with benzyl-
O
56
amine, NaBH(OAc)3 and one equivalent of acetic acid led to the
OCH3
O
R
N
diastereomeric benzylamines trans-8 and cis-8, which were sepa-
rated by flash chromatography and isolated in 35% and 52% yields,
respectively. The slight preference of cis-8 with an equatorially ori-
ented amino moiety is explained by stereoelectronic effects, that is,
the intermediate iminium ion is preferably attacked from the
axial side, which is favored by binding overlap between the
cis-2-1
cis-2-2
and the designed
Figure 4. Conformational analysis of the lead compound
spirocyclic compounds 2 with an exocyclic amino group.
1
occupied
r-molecular orbital of the newly formed C–H bond and

E. Rack et al. / Bioorg. Med. Chem. 19 (2011) 3141–3151
3145
Table 1
Calculated distances 1 and 2 for the energetically most favored conformers of spirocyclic piperidine 1 and the benzylmaines trans-8 and cis-8
Compd
Structure
Config.
Conform.
Number conform.
Distance 1 arom 1–N [Å]
Distance 2 arom 2–N [Å]
OCH3
OCH3
OCH3
1-1
2-2
1-1
1-2
22
6
22
3
5.65–5.74
5.09–5.18
5.67–5.74
5.10–5.18
(R)
(S)
3.75–3.84
3.75–3.84
O
1
Bn
N
1-1
1-1
1-2
1-1
1-2
17
0
15
0
5.67–5.75
—
5.67–5.75
—
3.78–3.85
—
3.78–3.85
—
(R)
(S)
O
1H+
Bn
N+
H
1-1H+
(R)
(S)
trans-8-1
trans-8-2
trans-8-1
trans-8-2
39
49
5.98–6.15
5.88–6.17
3.69–3.77
3.69–3.77
O
trans-8
N
H
Bn
trans-8-1
OCH3
trans-8-1
trans-8-2
trans-8-1
trans-8-2
(R)
(S)
42
42
5.99–6.26
5.99–6.26
3.70–3.78
3.70–3.77
O
trans-8H+a
+N
H
H
Bn
Bn
trans-8-1H+
OCH3
cis-8-1
cis-8-2
cis-8-1
cis-8-2
36
9
43
11
7.16–7.22
6.25–6.29
7.16–7.22
6.25–6.30
(R)
(S)
3.69–3.80
3.69–3.81
O
H
cis-8
N
cis-8-1
OCH3
cis-8-1
cis-8-2
cis-8-1
cis-8-2
32
9
39
11
7.18–7.23
6.26–6.28
7.18–7.23
6.26–6.29
(R)
(S)
3.70–3.81
3.70–3.80
O
H
N+
cis-8H+
H
Bn
cis-8-1H+
The conformations are grouped into two classes 8-1 and 8-2 according to Figure 4. The calculations were performed for the free bases and the corresponding protonated
species, respectively. Both enantiomers were taken into account.
a
For the protonated isomer trans-8H+ two additional conformations with a distorted geometry were found. Distance 1 of these conformations was 6.52–6.56 Å.
the non-occupied, parallel oriented, antibonding r⁄-molecular
orbitals of the adjacent axially oriented C–H bonds in 30- and
50-position of the cyclohexane ring.57
zylamine cis-8. The ethyl derivative cis-12 was obtained by alkyl-
ation of cis-8 with 1-iodoethane using microwave irradiation.
Alkylation of benzylamine cis-8 with valeraldehyde or benzalde-
hyde using the reducing agent NaBH(OAc)3 provided the pentyl
and benzyl derivatives cis-13 and cis-14, respectively.
The axial orientation of the amino group of trans-8 was shown
by the quintet-type signal at 3.03 ppm (J = 2.7 Hz) for the equatori-
ally oriented proton in 40-position of the cyclohexane ring. The
triplet of triplets at 2.66 ppm with two large (J = 10.5 Hz) and
two small coupling constants (J = 3.8 Hz) is caused by the axially
oriented proton at 40-position of the cyclohexane ring of cis-8. Sim-
ilar signals are also found for trans-9-11 and cis-9-14. However the
arrangement of the amino moiety relative to the benzopyran ring
could not be determined unequivocally, since both diastereomers
trans-8 and cis-8 can adopt conformations with axially (trans-8-1,
cis-8-2) and equatorially oriented (trans-8-2, cis-8-1) amino
substituent.
Reaction of the spirocyclic ketone 7 with p-methoxybenzyl-
amine (PMB-NH2) and NaBH(OAc)3 provided trans-9 and cis-9 in
23% and 35% yield, respectively. Again stereoelectronic effects are
responsible for the production of the cis-configured diastereomer
cis-9 in higher amounts than trans-9. The diastereoselectivity was
increased considerably, when performing the reductive amination
with a secondary amine. After reductive amination of ketone 7
with dimethylamine and NaBH(OAc)3 the diastereomeric tertiary
amines trans-10 and cis-10 were isolated in 18% and 66% yields,
respectively.
In order to investigate the influence of a second substituent at
the amino moiety, benzylamines 8 were converted into various
tertiary amines. (Scheme 2) Reductive methylation of trans-8 and
cis-8 with formalin and NaBH(OAc)3 led to the tertiary amines
trans-11 and cis-11 in 88% and 84% yields, respectively. Since the
cis-configured diastereomers cis-8 and cis-11 revealed higher r1
receptor affinity than their trans-configured analogues trans-8
and trans-11 and, moreover, cis-8 was obtained in higher yields,
further alkyl residues were introduced into the cis-configured ben-
In order to prove the relative configuration unequivocally the
trans-configured diastereomer trans-9 was recrystallized from
ethyl acetate, which led to crystals suitable for X-ray crystal struc-
ture analysis. The X-ray crystal structure shows the amino moiety
and the O-atom of the benzopyran ring on opposite sides of the

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E. Rack et al. / Bioorg. Med. Chem. 19 (2011) 3141–3151
OCH3
OCH3
CH=O
Br
OCH3
(c)
(a)
(b)
Br
Br
3
4
5
OCH3
OCH3
OH
OCH3
(d)
O
O
O
6
7
(e) or (f) or (g)
Figure 5. X-ray crystal structure of the trans-configured p-methoxybenzylamine
OCH3
trans-9.
OCH3
O
O
was employed as radioligand. Homogenates of rat liver served as
R2N
source for
r2 receptors in the r2 assay. Since a r2 selective radio-
R2N
trans-8: NR2 = NHBn
trans-9: NR2 = NHPMB
trans-10: NR2 = N(CH3)2
ligand is not commercially available, the non-selective radioligand
[3H]-1,3-di(o-tolyl)guanidine was employed in the presence of an
excess of non-tritiated (+)-pentazocine, which selectively occupies
r1 receptors.52–54
cis-8: NR2 = NHBn
cis-9: NR2 = NHPMB
cis-10: NR2 = N(CH3)2
In Table 2 the r1 and r2 receptor affinities of the exocyclic
PMB = p-methoxybenzyl
amines 8–14 are compared with the
r affinities of the spirocyclic
Scheme 1. Synthesis of spirocyclic r1 receptor ligands with exocyclic amino
moiety. Reagents and conditions: (a) methoxymethyltriphenylphosphonium chlo-
ride, KOtBu, THF, start at ꢀ10 °C, then 16 h rt, 71%;55 (b) pTosOHꢂH2O, MeOH, 72 h,
reflux, 92%;55 (c) n-BuLi, THF, ꢀ78 °C, 20 min, then cyclohexane-1,4-dione, 2 h,
ꢀ78 °C, 1 h, rt, 56%; (d) CHCl3, HCl, 1.5 h, rt, 81%; (e) benzylamine, THF, HOAc,
NaBH(OAc)3, 2 h, rt, 35% (trans-8), 52% (cis-8); (f) 4-methoxybenzylamine, THF,
HOAc, NaBH(OAc)3, 2.5 h, rt, 23% (trans-9), 35% (cis-9); (g) dimethylamine in EtOH,
THF, HOAc, NaBH(OAc)3, 2.5 h, rt, 18% (trans-10), 66% (cis-10).
piperidine 1 and the reference compounds (+)-pentazocine and
haloperidol. The r1 receptor affinities of the secondary amines
trans-8, cis-8, trans-9, and cis-9 are >100-fold lower than the r1
receptor affinity of the lead piperidine 1. However, transformation
of the secondary amines 8 into methylated tertiary amines trans-
11 and cis-11 led to a considerable increase of the r1 affinity:
the Ki-value of trans-11 is 43 nM the Ki-value of the cis-configured
diastereomer cis-11 is 24 nM. The low r1 affinities of the diaste-
reomeric dimethylamines trans-10 and cis-10 demonstrate that
at least one large substituent (e.g., a benzyl moiety) is necessary
cis-8
(a), (b), (c) or (d)
trans-8
(a)
for high
r1 affinity. However, the size of the second alkyl substitu-
ent is rather limited, since the r1 affinity was decreased with
increasing size of this substituent: cis-11 (Me) > cis-12 (Et) > cis-
13 (Pent) ꢁ cis-14 (Bn). This observation is in good accordance with
the reported pharmacophore models, which postulate a small third
substituent (H, CH3) at the basic N-atom.
A considerable decrease of r1 affinity was observed after shift-
ing the amino group at the cyclohexane ring as demonstrated with
the secondary amines 8 and 9. Only tertiary amines trans-11 and
cis-11 show r1 receptor affinities in the range of reference
OCH3
OCH3
O
O
Bn
N
R
N
Bn
CH3
trans-11
cis-11: R = CH3
cis-12: R = C2H5
cis-13: R = C5H11
cis-14: R = CH2C6H5
Table 2
Scheme 2. Alkylation of secondary amines to obtain tertiary amines. Reagents and
conditions: (a) CH2@O, CH2Cl2, NaBH(OAc)3, 23 h or 3 h, rt, 88% (trans-11), 84% (cis-
11); (b) C2H5I, K2CO3, CH3CN, microwave, 45%; (c) valeraldehyde, CH2Cl2,
NaBH(OAc)3, 23 h, rt, 90%; (d) benzaldehyde, CH2Cl2, NaBH(OAc)3, 23 h, rt, 41%.
r1 and r2 receptor affinities of spirocyclic ligands with an exocyclic amino group
compared with reference compounds.
Compd
NR2
Ki SEM [nM]
r1
r2
cyclohexane ring plain indicating trans-configuration. (Fig. 5) In
the crystals the cyclohexane ring adopts the conformation trans-
8-1 (see Fig. 4) with the amino moiety in axial orientation. Appar-
ently, trans-8-1 represents an energetically favored conformation
in the solid state (X-ray crystal structure) as well as in solution
(compare 1H NMR data).
1
—
NHBn
NHBn
1.29 0.18
538 56
>1000
>1000
>1000#
664 178
142 33
>1000
>1000
>1000
329
666 106
719
>1000
—
trans-8
cis-8
trans-9
cis-9
trans-10
cis-10
trans-11
cis-11
cis-12
cis-13
cis-14
(+)-Pentazocine
Haloperidol
158
5
NHPMB
NHPMB
N(CH3)2
N(CH3)2
NCH3Bn
NCH3Bn
N(C2H5)Bn
N(C5H11)Bn
N(Bn)2
—
169 19
174 12
>1000
>1000
43 18
24 4.7
107 25
>1000
>1000
5.7 2.2
6.3 1.6
5. Receptor affinity
The
r receptor affinities of the spirocyclic compounds 8–14
were determined in competition experiments with radioligands.
—
78 2.3
In the r1 assay homogenates of guinea pig brains were used as
r
1 selective ligand [3H]-(+)-pentazocine
#
receptor material and the
No correlation between dose and receptor affinity.

E. Rack et al. / Bioorg. Med. Chem. 19 (2011) 3141–3151
3147
compounds and the lead compound 1. But despite the enlargement
of distance 1 in cis-11 the 1 receptor affinity is still about 15-fold
lower than the r1 affinity of 1. The reduced r1 affinity of cis-11
may be attributed to entropic factors, since attachment of the ben-
zylamino moiety at the ring leads to increased flexibility.
With exception of the equipotent diastereomeric p-
methoxybenzyl derivatives 9, generally, the cis-configured
diastereomers show higher r1 receptor affinities than the trans-
configured diastereomers. The higher r1 receptor affinity corre-
lates well with the increased distance 1. For the more populated
conformations of cis-8 distance 1 (7.16–7.23 Å) is about 1 Å longer
than distance 1 of the conformations of trans-8 (5.88–6.26 Å).
Almost all compounds show negligible affinity towards r2
receptors indicating high selectivity against the r2 subtype, at
least for the most potent tertiary r1 ligands trans-11 and cis-11.
Only the p-methoxybenzylamine cis-9 and the tertiary amine cis-
IR spectrophotometer 480Plus FT-ATR-IR (Jasco). 1H NMR
(400 MHz), 13C NMR (100 MHz): Mercury-400BB spectrometer
(Varian); d in ppm related to tetramethylsilane; coupling constants
are given with 0.5 Hz resolution. HPLC: Merck Hitachi Equipment;
UV detector: L-7400; autosampler: L-7200; pump: L-7100; degas-
ser: L-7614; Method A: column: LiChrospherÒ 60 RP-select B
r
(5
lm), 250–4 mm; flow rate: 1.00 mL/min; injection volume:
5.0
lL; detection at k = 210 nm; solvents: A: water with 0.05% (v/
v) trifluoroacetic acid; B: acetonitrile with 0.05% (v/v) trifluoroace-
tic acid: gradient elution: (A%): 0–4 min: 90% , 4–29 min: gradient
from 90% to 0%, 29–31 min: 0%, 31–31.5 min: gradient from 0% to
90%, 31.5–40 min: 90%. The purity of all test compounds was great-
er than 95%.
7.2.2. 2-[2-(1-Hydroxy-4-oxocyclohexyl)phenyl]acetaldehyde
dimethyl acetal (6)
13 with an N-pentyl residue are equipotent at both
subtypes.
r
receptor
Under N2 2-(2-bromophenyl)acetaldehyde dimethyl acetal55 (5,
1.03 g, 4.20 mmol) was dissolved in THF abs (32 mL) and cooled
down to ꢀ78 °C. Subsequently, n-BuLi (1.48 M in n-hexane,
3.12 mL, 4.62 mmol) was added slowly. After 20 min a solution
of cyclohexane-1,4-dione (0.946 g, 8.4 mmol in THF, 12 mL) was
added rapidly and the mixture was stirred for 2 h at ꢀ78 °C and
1 h at rt. Then H2O was added (30 mL), after addition of CH2Cl2
the layers were separated and the aqueous layer was extracted
with CH2Cl2 (2x), the combined organic layers were dried (Na2SO4),
concentrated in vacuo and the residue was purified by FC (5 cm,
cyclohexane/ethyl acetate 2:1, 20 cm, 30 mL, Rf = 0.20). Colorless
solid, mp 45 °C, yield 652 mg (56%). C16H22O4 (278.4). MS (EI):
6. Conclusion
Despite an increased distance 1, which fits exactly into various
r1 pharmacophore models, the r1 receptor affinity of the second-
ary amines 8 and 9 is >100-fold reduced compared with the r1
affinity of the spirocyclic piperidine 1 with a shorter distance 1.
In contrast to the established models secondary amines 8 and 9
are less tolerated by the r1 receptor protein than the correspond-
ing tertiary methylamines 11. The affinities of the tertiary amines
10–14 demonstrate that one large N-substituent (e.g., a benzyl
group) is required, but two large substituents (e.g., two benzyl
groups) are not accepted by the receptor protein. As a rule, cis-
configured diastereomers show higher r1 receptor affinities than
their trans-configured counterparts, which leads to cis-11 as the
most potent r1 ligand of this series (Ki = 24 nM). The high r1
affinity of cis-11 represents a promising starting point for the
m/z (%) = 278 [M, 1], 246 [MꢀMeOH, 2], 189 [MꢀMeOH,
–
CH3CH2C@O, 50], 157 [M–2 MeOH, –CH3CH2C@O, 100]. MS (ESIꢀ):
m/z (%) = 555 [(2MꢀH)ꢀ,15], 277[(MꢀH)ꢀ, 100]. IR:
m
~
(cmꢀ1) = 3424 (m, broad, v, OH), 3057 (w, v, C–H, arom), 2938
(s,v, C–H, alkyl), 2833 (m, v, OCH3), 1710 (s, v, C@O), 1443 (m, d,
C–H, alkyl), 760 (s, d, C–H, o-disubst. arom). 1H NMR (DMSO-d6):
d (ppm) = 2.14–2.28 (m, 6H, (CH2CH2)2C@O), 2.84 (‘dt’, J = 14.4/
9.5 Hz, 2H, (CH2CH2)2C@O), 3.30 (s, 6H, Ar-CH2CH(OCH3)2), 3.33
(d, J = 5.3 Hz, 2H, Ar-CH2CH(OCH3)2), 4.67 (t, J = 5.4 Hz, 1H, Ar-
CH2CH(OCH3)2), 5.33 (s, 1H, OH), 7.16–7.24 (m, 2H, Ar-H), 7.30–
7.34 (m, 1H, Ar-H), 7.38–7.42 (m, 1H, Ar-H).
development of novel potent
r1 ligands, since the exocyclic amino
moiety allows fine tuning of the pharmacological properties of this
compound class by modifying two N-substituents.
7.2.3. 3-Methoxy-3,4-dihydrospiro[[2]benzopyran-1,10-
cyclohexan]-40-one (7)
7. Experimental
7.1. Conformational analysis
A solution of 2 M HCl (0.06 mL) and hydroxy acetal 6 (300 mg,
1.08 mmol) in CHCl3 (24 mL) was stirred vigorously at rt for
90 min. Afterwards CH2Cl2 (24 mL) was added, and the organic
layer was washed with 0.2 M NaOH (12 mL) and H2O (12 mL).
The organic layer was dried (Na2SO4), concentrated in vacuo and
the residue was purified by FC (4 cm, cyclohexane/ethyl acetate
4:1, 20 cm, 30 mL, Rf = 0.23). Colorless solid, mp 126.4 °C, yield
214 mg (81%). C15H18O3 (246.3). MS (EI): m/z (%) = 246 [M, 4],
214 [M–MeOH, 4], 189 [M–CH3CH2C⁄=O, 66], 157 [M–MeOH, –
CH3CH2C⁄@O, 100]. MS (ESI): m/z (%) = 515 [2M+Na, 33], 269
~
3D-Structures were generated with MOE (Molecular Operating
Environment), Version 2009.10, Chemical computing group AG
(CCG, Montreal, Canada). Structures were drawn with modul Mol-
ecule Builder. Stochastic conformational search was carried out at
standard conditions. Method: Stochastic, Rejection Limit: 100, Iter-
ation Limit 10,000, RMS Gradient: 0.005, MM Iteration Limit: 500,
RMSD Limit: 0.25, Strain cutoff: 7 kcal/mol, Conformation Limit:
10,000).
[M+Na, 62], 264 [M+NH4, 100]. IR:
m
(cmꢀ1) = 2948 (s, v, C–H, al-
7.2. Experimental, chemistry
kyl), 2905 (m, v, C–H, alkyl), 2872 (m, v, OCH3), 1717 (s, v, C@O),
773 (s, d, C–H, o-disubst. arom). 1H NMR (CDCl3): d (ppm) = 2.12
(‘td’, J = 14.0/4.4 Hz, 1H, (CH2CH2)2C@O, 2.22–2.42 (m, 5H,
(CH2CH2)2C@O), 2.82–2.92 (m, 1H, (CH2CH2)2C@O), 2.92–3.06 (m,
3H, (CH2CH2)2C@O (1H), ArCH2CHOCH3 (2H)), 3.58 (s, 3H, OCH3),
4.99 (dd, J = 6.8/3.5 Hz, 1H, ArCH2CHOCH3), 7.08–7.11 (m, 1H, Ar-
H), 7.12–7.16 (m, 1H, Ar-H), 7.19 - 7.22 (m, 2H, Ar-H). 13C NMR
(CDCl3): d (ppm) = 35.5 (1C, CH2CHOCH3), 37.4 (1C, (CH2CH2)2O),
37.7 (1C, (CH2CH2)2C@O), 37.8 (1C, (CH2CH2)2C@O), 39.3 (1C,
(CH2CH2)2C@O), 56.6 (1C, OCH3), 75.6 (1C, spiro-C), 97.4 (1C,
CH2CHOCH3), 124.5 (1C, arom), 127.1 (1C, arom), 127.5 (1C, aro-
ma), 129.7 (1C, arom), 131.6 (1C, arom), 139.9 (1C, arom), 211.8
(1C, C@O). Purity (HPLC): 99.6%, tR = 18.87 min.
7.2.1. General
Unless otherwise noted, moisture sensitive reactions were con-
ducted under dry nitrogen. THF was dried with sodium/benzophe-
none and was freshly distilled before use. Thin layer
chromatography (TLC): Silica Gel 60 F254 plates (Merck). Flash
chromatography (FC): Silica Gel 60, 40–64 lm (Merck); parenthe-
ses include: diameter of the column, length of the column, eluent,
fraction size, Rf value. Melting point: Melting point apparatus SMP
3 (Stuart Scientific), uncorrected. MS: MAT GCQ (Thermo-Finni-
gan); EI = electron impact; Thermo Finnigan LCQÒ ion trap mass
spectrometer with an ESI = electrospray ionization interface. IR:

3148
E. Rack et al. / Bioorg. Med. Chem. 19 (2011) 3141–3151
7.2.4. trans-N-Benzyl-3-methoxy-3,4-dihydrospiro-
[[2]benzopyran-1,10-cyclohexan]-40-amine (trans-8) and
cis-N-Benzyl-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,10-
cyclohexan]-40-amine (cis-8)
Under N2 ketone 7 (145 mg, 0.588 mmol) was dissolved in THF
(7 mL). Benzylamine (64 lL, 0.588 mmol), acetic acid (34 lL,
(K2CO3), concentrated in vacuo and the residue was purified by
FC (2 cm, CH2Cl2/cyclohexane 1:1 + 2% N,N-dimethylethanamine,
23 cm, 10 mL). After concentration in vacuo NaOH (1 M, 4 mL)
was added to the separated and purified compounds and the mix-
ture was extracted with CH2Cl2 (4ꢃ). The organic layers were dried
(K2CO3) and concentrated in vacuo.
0.588 mmol) and NaBH(OAc)3 (95%, 175 mg, 0.784 mmol) were
added and the mixture was stirred for 2 h at rt. Subsequently
1 M NaOH (8 mL) was added and the mixture was extracted with
CH2Cl2 (4 ꢃ 8 mL), the combined organic layers were dried
(K2CO3), concentrated in vacuo and the residue was purified by
FC (3 cm, cyclohexane : ethyl acetate 9:1 + 2% N,N-dimethylethan-
amine, 20 cm, 10 mL).
trans-9 (Rf = 0.23): Colorless solid, mp 84 °C, yield 26.3 mg
(23%). C23H29NO3 (367.5). MS (ESI): m/z (%) = 368 [MH,100]. FT-
~
IR:
m
(cmꢀ1) = 3061 (m, v, C–H, arom), 2993, 2926 (s, v, C–H, alkyl),
2833 (s, v, OCH3), 1611,1510 (w, C@C, arom), 1442 (m, d, C–H, al-
kyl), 823 (s, d, C–H, p-disubst. arom), 754 (s, d, C–H, o-disubst.
1
arom). H NMR (CDCl3): d (ppm) = 1.61–1.72 (m, 3H, 20-He, 30-He,
50-He), 1.80 (ddd, J = 13.0/5.3/2.7 Hz, 1H, 60-He), 1.98 (‘tt’, J = 13.4/
3.2 Hz, 1H, 50-Ha), 2.02–2.14 (m, 2H, 30-Ha, 60-Ha), 2.32 (‘td’,
J = 13.7/4.2 Hz, 1H, 20-Ha), 2.89 (ddd, J = 15.6/6.8/0.9 Hz, 1H, ArCH2-
CHOCH3), 2.94 (ddd, J = 15.7/4.1/0.7 Hz, 1H, ArCH2CHOCH3), 3.02
(‘quint’, J = 2.8 Hz, 1H, 40-He), 3.57 (s, 3H, OCH3), 3.75 (s, 2H,
ArCH2NH), 3.82 (s, 3H, ArOCH3), 4.86 (dd, J = 6.7/4.2 Hz,1H, ArCH2-
CHOCH3), 5.30 (s, 1H, NH), 6.88–6.92 (m, 2H, Ar-H), 7.06–7.10 (m,
1H, Ar-H), 7.14–7.19 (m, 1H, Ar-H), 7.20–7.23 (m, 2H, Ar-H), 7.29–
7.33 (m, 2H, Ar-H). Purity (HPLC): 99.4%, tR = 19.20 min. A sample
was recrystallized from ethyl acetate to obtain crystals, which
were suitable for X-ray crystal structure analysis.
trans-8 (Rf = 0.31): Colorless solid, mp 96 °C, yield 70 mg (35%).
~
22H27NO2 (337.5). MS (ESI): m/z (%) = 338 [MH,100]. IR: m
C
(cmꢀ1) = 3043 (m, v, C–H, arom), 2928, 2870 (s, v, C–H, alkyl),
1604, 1486 (w, C@C, arom), 1441 (m, d, C–H, alkyl), 757 (s, d, C–
H, o-disubst. arom), 737, 699 (s, d, C–H, monosubst. arom). 1H
NMR (CDCl3): 1.61–1.73 (m, 3H, 20-He, 30-He, 50-He), 1.80 (ddd,
J = 13.1/5.6/3.0 Hz, 1H, 60-He) ,1.99 (‘tt’, J = 13.3/ 3.1 Hz, 1H, 50-
Ha), 2.04–2.16 (m, 2H, 30-Ha, 60-Ha), 2.34 (‘td’, J = 13.4/4.0 Hz, 1H,
20-Ha), 2.89 (dd, J = 15.6/6.8 Hz, 1H, ArCH2CHOCH3), 2.94 (dd,
J = 15.6/4.2 Hz, 1H, ArCH2CHOCH3), 3.03 (‘quint’, J = 2.7 Hz, 1H,
CH2CH-NH), 3.57 (s, 3H, OCH3), 3.83 (s, 2H, Ar-CH2-NH), 4.87 (dd,
J = 6.7/4.2 Hz, 1H, ArCH2CHOCH3), 7.07–7.11 (m, 1H, Ar-H), 7.14–
7.20 (m, 1H, Ar-H), 7.21–7.25 (m, 2H, Ar-H), 7.26–7.31 (m, 1H,
Ar-H), 7.34–7.43 (m, 4H, Ar-H), a signal for the NH-proton is not
X-ray crystal structure analysis for trans-9: formula
C
23H29NO3ꢂH2O, M = 385.49, colorless crystal 0.35 ꢃ 0.30 ꢃ 0.20
mm, a = 12.0010(4), b = 13.0086(5), c = 13.8753(1) Å, b = 105.890
(2)°, V = 2083.39(14) Å3, calcd = 1.229 g cmꢀ3 = 0.668 mmꢀ1
empirical absorption correction (0.800 6 T 6 0.878), Z = 4, mon-
clinic, space group P21/c (No. 14), k = 1.54178 Å, T = 223 K, and
scans, 15594 reflections collected ( h, k, l), [(sin h)/
k] = 0.60 Åꢀ1, 3672 independent (Rint = 0.039) and 3419 observed
q
,
l
,
13
seen in the spectrum. C NMR (CDCl3): d (ppm) = 26.2 (1C, 30-C
or 50-C), 26.5 (1C; 30-C or 50-C), 30.7 (1C, 60-C), 33.5 (1C, 20-C),
35.7 (1C, CH2CHOCH3), 50.6 (1C, 40-C), 52.0 (1C, NHCH2Ar), 56.4
(1C, OCH3), 77.5 (1C, spiro-C), 96.6 (1C, CH2CHOCH3), 125.4 (1C,
arom), 126.7 (1C, arom), 126.8 (1C, arom), 127.2 (1C, arom),
128.4 (2C, arom), 128.7 (2C, arom), 129.4 (1C, arom), 131.6 (1C,
arom), 141.6 (1C, arom), 142.7 (1C, arom). Purity (HPLC): 99.4%,
tR = 18.81 min.
x
u
reflections [I P 2r
(I)], 267 refined parameters, R = 0.043, wR2 =
0.116, max. residual electron density 0.27 (ꢀ0.17) e Åꢀ3, hydrogen
atoms at N and water from difference fourier calculations, others
calculated and refined as riding atoms.
cis-8 (Rf = 0.12): Colorless oil, yield 102 mg (52%). C22H27NO2
~
Data set was collected with a Nonius KappaCCD diffractometer.
Programs used: data collection COLLECT (Nonius B.V., 1998), data
reduction Denzo-SMN,58 absorption correction Denzo,59 structure
(337.5). MS (ESI): m/z (%) = 338 [MH,100]. IR:
m
(cmꢀ1) = 3027 (m,
v, C–H, arom), 2927, 2855 (s, v, C–H, arom), 1603, 1491 (w, C@C,
arom), 1451 (m, d, C–H, alkyl), 754 (s, d, C–H, o-disubst. arom),
730, 698 (s, d, C–H, mono-subst. arom). 1H NMR (CDCl3): d
(ppm) = 1.58–1.74 (m, 2H, (CH2CH2)2CHN), 1.74–1.99 (m, 5H,
(CH2CH2)2CHN), 2.09 (ddd, J = 13.2/5.2/2.6 Hz, 1H, 20-He), 2.66
(‘tt’, J = 10.5/3.8 Hz, 1H, 40-Ha), 2.89 (dd, J = 15.3/6.2 Hz, 1H, ArCH2-
CHOCH3), 2.94 (dd, J = 15.4/3.7 Hz, 1H, ArCH2CHOCH3), 3.57 (s, 3H,
OCH3), 3.89 (s, 2H, Ar-CH2-NH), 4.86 (dd, J = 6.7/3.9 Hz, 1H, ArCH2-
CHOCH3), 7.06–7.10 (m, 2H, Ar-H), 7.13–7.20 (m, 2H, Ar-H), 7.25–
7.30 (m, 2H, Ar-H), 7.33–7.38 (m, 3H, Ar-H), a signal for the NH-
solution SHELXS-97,60 structure refinement SHELXL-97,61 graphics
62
SCHAKAL
.
CCDC 804299 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge at
www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cam-
bridge Crystallographic Data Centre, 12 Union Road, Cambridge
CB2 1EZ, UK; fax: (internat.) +44 (1223)336 033, E-mail:
deposit@ccdc.cam.ac.uk].
cis-9 (Rf = 0.16): Colorless solid, mp 91 °C, yield 41 mg (35%).
~
C23H29NO3 (367.5). MS (ESI): m/z (%) = 368 [MH, 100]. FT-IR: m
proton is not seen in the spectrum. 13C NMR (CDCl3):
d
(ppm) = 29.3 (1C, 30-C or 50-C or 60-C), 29.4 (1C, 30-C or 50-C or 60-
C), 36.6 (1C, ArCH2CHOCH3), 36.1 (1C, 20-C), 38.5 (1C, 30-C or 50-C
or 60-C), 51.4 (1C, NHCH2Ar), 55.9 (1C, 40-C), 56.5 (1C, OCH3), 76.6
(1C, spiro-C), 96.7 (1C, ArCH2CHOCH3), 124.9 (1C, arom), 126.7
(1C, arom), 126.9 (1C, arom), 127.2 (1C, arom), 128.4 (2C, arom),
128.8 (2C, arom), 129.5 (1C, arom), 131.9 (1C, arom), 141.1 (1C,
arom), 141.9 (1C, arom). Purity (HPLC): 99.9%, tR = 18.64 min.
(cmꢀ1) = 3067, 3029 (m, v, C–H, arom), 2993, 2930 (s, v, C–H, arom),
2833 (s, v, OCH3), 1610, 1510 (w, C@C, arom), 1442 (m, d, C–H, alkyl),
811 (s, d, C–H, p-disubst. arom), 755 (s, d, C–H, o-disubst. arom). 1H
NMR (CDCl3): d (ppm) = 1.58–1.72 (m, 2H, (CH2CH2)2CHN), 1.76–
1.98 (m, 5H, (CH2CH2)2CHN), 2.05–2.12 (m, 1H, 20-He), 2.64 (‘tt’,
J = 10.5/3.9 Hz, 1H, 40-Ha), 2.89 (dd, J = 15.3/6.5 Hz, 1H, ArCH2-
CHOCH3), 2.94 (dd, J = 15.5/3.7 Hz, 1H, ArCH2CHOCH3), 3.57 (s, 3H,
OCH3), 3.81 (s, 3H, ArOCH3), 3.82 (s, 2H, ArCH2NH), 4.86 (dd,
J = 6.7/3.9 Hz, 1H, ArCH2CHOCH3), 5.80 (s, 1H, NH), 6.86–6.91
(m,2H, Ar-H), 7.06–7.11 (m,2H, Ar-H), 7.13–7.21 (m,2H, Ar-H),
7.24–7.29 (m,2H, Ar-H). Purity (HPLC): 98.7%, tR = 19.01 min.
7.2.5. trans-N-(p-Methoxybenzyl)-3-methoxy-3,4-dihydrospiro-
[[2]benzopyran-1,10-cyclohexan]-40-amine (trans-9) and
cis-N-(p-Methoxybenzyl)-3-methoxy-3,4-dihydrospiro-
[[2]benzopyran-1,10-cyclohexan]-40-amine (cis-9)
Under N2 ketone 7 (79 mg, 0.32 mmol) was dissolved in THF
7.2.6. trans-3-Methoxy-N,N-dimethyl-3,4-dihydrospiro-
[[2]benzopyran-1,10-cyclohexan]-40-amine (trans-10) and
cis-3-Methoxy-N,N-dimethyl-3,4-dihydrospiro-
(4 mL), 4-methoxybenzylamine (46
lL, 0.35 mmol), acetic acid
(18 L, 0.32 mmol) and NaBH(OAc)3 (95%, 95 mg, 0.43 mmol) were
l
added and the mixture was stirred for 2.5 h at rt. Subsequently 1 M
NaOH (5 mL) was added and the mixture was extracted with
CH2Cl2 (4 ꢃ 5 mL), the combined organic layers were dried
[[2]benzopyran-1,10-cyclohexan]-40-amine (cis-10)
Under N2 ketone 7 (70.6 mg, 0.287 mmol) was dissolved in THF
(4 mL). A solution of dimethylamine in ethanol (2 M, 158 lL,

E. Rack et al. / Bioorg. Med. Chem. 19 (2011) 3141–3151
3149
0.32 mmol), acetic acid (16
l
L, 0.287 mmol) and NaBH(OAc)3 (95%,
the residue was purified by FC (2 cm, cyclohexane + 1% N,N-
dimethylethanamine, 20 cm, 5 mL, Rf = 0.10). Colorless solid, mp
85 mg, 381 mmol) were added and the mixture was stirred for
2.5 h at rt. Then, 1 M NaOH (5 mL) was added and the mixture
was extracted with CH2Cl2 (4 ꢃ 5 mL), the combined organic layers
were dried (K2CO3), concentrated in vacuo and the residue was
purified by FC (2 cm, cyclohexane/ethyl acetate 9/1 + 2% N,N-
dimethylethanamine, 20 cm, 5 mL).
91 °C, yield 47 mg (84%).
(%) = 352 [MH, 100]. FT-IR:
C
~
m
23H29NO2 (351.5). MS (ESI): m/z
(cmꢀ1) = 3024 (m, v, C–H, arom),
2928 (s, v, C–H, alkyl), 2839 (s, v, OCH3), 2788 (m, v, N-CH3)
1603, 1492 (w, v, C@C, arom), 754 (s, d, C–H, o-disubst. arom),
732, 698 (s, d, C–H, mono-subst. arom). 1H NMR (CDCl3): d
(ppm) = 1.64 (‘td’, J = 14.0/3.9 Hz, 1H, 20-Ha), 1.75–1.84 (m, 2H,
(CH2CH2)2CHN), 1.84–1.95 (m, 2H, (CH2CH2)2CHN), 1.96 - 2.09
(m, 2H, (CH2CH2)2CHN), 2.11 (ddd, J = 14.1/6.4/3.2 Hz, 1H, 20-He),
2.28 (s, 3H, NCH3), 2.67 (‘tt’, J = 11.6/3.6 Hz, 1H, 40-Ha), 2.90 (dd,
J = 15.3/6.7 Hz, 1H, ArCH2CHOCH3), 2.95 (dd, J = 15.4/3.5 Hz, 1H,
ArCH2CHOCH3), 3.59 (s, 3H, OCH3), 3.65 (s, 2H, NHCH2Ar), 4.88
(dd, J = 6.9/3.9 Hz, 1H, Ar-CH2-CH–OCH3), 7.07–7.12 (m, 2H, Ar-
H), 7.13–7.22 (m, 2H, Ar-H), 7.22–7.29 (m, 1H, Ar-H), 7.30–7.39
(m, 4H, Ar-H). Purity (HPLC): 99.8%, tR = 17.68 min.
trans-10 (Rf = 0.38): Colorless solid, mp 67 °C, yield 13.8 mg
(18%). C17H25 NO2 (275.4). MS (ESI): m/z (%) = 276 [MH, 100]. IR:
~
m
(cmꢀ1) = 2959, 2926 (s, v, C–H, alkyl), 2854 (s, v, OCH3), 2805,
2760 (m, v, N-CH3), 1449 (s, d, C–H, alkyl), 759 (s, d, C–H, o-disubst.
arom).1H NMR (CDCl3): d (ppm) = 1.57–1.65 (m, 1H, 20-He), 1.76
(ddd, J = 13.4/5.8/2.9 Hz, 1H, 60-He), 1.84–1.92 (m, 3H, 30-He, 50-
Ha, 50-He), 1.92–2.05 (m, 2H, 30-Ha, 60-Ha), 2.10 (‘quint’, J = 2.8 Hz,
1H, 40-He), 2.15 (‘td’, J = 13.3/3.7 Hz, 1H, 20-Ha), 2.28 (s, 6H,
N(CH3)2), 2.89 (dd, J = 15.6/6.9 Hz, 1H, ArCH2CHOCH3), 2.94 (dd,
J = 15.6/3.9 Hz, 1H, ArCH2CHOCH3), 3.57 (s, 3H, OCH3), 4.86 (dd,
J = 6.8/4.1 Hz, 1H, ArCH2CHOCH3), 7.05–7.09 (m, 1H, Ar-H), 7.15
(‘td’, J = 7.3/1.6 Hz, 1H, Ar-H), 7.20 (‘td’, J = 7.4/1.6 Hz, 1H, Ar-H),
7.26–7.29 (m, 1H, Ar-H). Purity (HPLC): 99.6%, tR = 15.09 min.
cis-10 (Rf = 0.07): Colorless solid, mp 83 °C, yield 51.9 mg (66%).
~
7.2.9. cis-N-Benzyl-N-ethyl-3-methoxy-3,4-dihydrospiro-
[[2]benzopyran-1,10-cyclohexan]-40-amine (cis-12)
Benzylamine cis-8 (54 mg, 0.16 mmol), 1-iodoethane (18 lL,
0.226 mmol) and K2CO3 were dissolved and suspended in acetoni-
trile (3 mL) in a microwave tube (10 mL) and irradiated with
microwaves. Reaction parameter: Program: Standard, power:
max. 220 W, pressure: max. 4 bar, temperature: 100 °C, reaction
time: 5 min ramp time, 40 min hold time, 5 min cool off time.
The reaction mixture was filtered and concentrated in vacuo.
H2O (4 mL) was added and the mixture was extracted with CH2Cl2.
The organic layers were dried (K2CO3) concentrated in vacuo and
the residue was purified by FC (2 cm, cyclohexane/ethyl acetate
9:1 + 2% N,N-dimethylethanamine, 15 cm, 5 mL, Rf = 0.55). Color-
less solid, mp 74 °C, yield 26.1 mg (45%). C24H31NO2 (365.6). MS
~
C17H25 NO2 (275.4). MS (ESI): m/z (%) = 276 [MH, 100]. IR: m
(cmꢀ1) = 2979, 2946, 2921, 2857 (s, v, C–H, alkyl), 2824, (m, v,
OCH3), 2774 (m, v, N-CH3), 1446 (s, d, C–H, alkyl), 767 (s, d, C–H,
o-disubst. arom). 1H NMR (CDCl3): d (ppm) = 1.61–1.67 (m, 1H,
(CH2CH2)2CHN), 1.74–1.96 (m, 5H, (CH2CH2)2CHN), 1.96–2.02 (m,
1H, (CH2CH2)2CHN), 2.14 (ddd, J = 13.9/6.2/3.1 Hz, 1H,
(CH2CH2)2CHN), 2.35 (s, 6H, N(CH3)2), 2.32–2.40 (m, 1H, 40-Ha),
2.90 (dd, J = 15.3/6.5 Hz, 1H, ArCH2CHOCH3), 2.94 (dd, J = 15.7/
3.5 Hz, 1H, ArCH2CHOCH3), 3.58 (s, 3H, OCH3), 4.85 (dd, J = 6.9/
3.9 Hz, 1H, ArCH2CHOCH3), 7.07–7.12 (m, 2H, Ar-H), 7.14–7.22
(m, 2H, Ar-H). Purity (HPLC): 95.4%, tR = 15.35 min.
(ESI): m/z (%) = 366 [MH,100]. FT-IR:
m
(cmꢀ1) = 3021 (m, v, C–H,
arom), 2946, 2919 2860 (s, v, C–H, alkyl), 2807 (–H2C–N–), 1602,
1491 (w, v, C@C, arom), 768 (s, d, C–H, o-disubst. arom), 732, 696
(s, d, C–H, mono-subst. arom). 1H NMR (CDCl3): d (ppm) = 1.05 (t,
J = 7.1 Hz, 3H, NCH2CH3), 1.60 (‘td’, J = 13.7/4.0 Hz, 1H, 20-Ha),
1.69–1.79 (m, 2H, (CH2CH2)2CHN), 1.81–1.89 (m, 2H,
(CH2CH2)2CHN), 1.91–2.05 (m, 2H, (CH2CH2)2CHN), 2.13 (ddd,
J = 14.1/6.1/3.1 Hz, 1H, 20He), 2.57–2.66 (m, 2H, N-CH2CH3), 2.74
(‘tt’, J = 11.7/3.7 Hz, 1H, 40-Ha), 2.88 (dd, J = 15.5/6.5 Hz, 1H, Ar-
CH2CHOCH3), 2.94 (dd, J = 15.5/3.6 Hz, 1H, Ar-CH2CHOCH3), 3.58
(s, 3H, OCH3), 3.68 (d, J = 14.6 Hz, 1H, N–CH2–Ar), 3.72 (d,
J = 14.7 Hz, 1H, N-CH2-Ar), 4.86 (dd, J = 6.9/3.9 Hz, 1H, ArCH2-
CHOCH3), 7.06–7.11 (m, 2H, Ar-H), 7.13–7.19 (m, 2H, Ar-H),
7.19–7.25 (m, 1H, Ar-H), 7.29–7.34 (m, 2H, Ar-H), 7.37–7.43 (m,
2H, Ar-H). Purity (HPLC): 97.2%, tR = 19.43 min.
7.2.7. trans-N-Benzyl-3-methoxy-N-methyl-3,4-dihydrospiro-
[[2]benzopyran-1,10-cyclohexan]-40-amine (trans-11)
Under N2 benzylamine trans-8 (56.6 mg, 0.168 mmol) was dis-
solved in CH2Cl2 (2.5 mL) and formalin 37% (250
lL, 3.36 mmol)
and NaBH(OAc)3 (95%, 57 mg, 0.256 mmol) were added. The reac-
tion mixture was stirred for 23 h at rt. Subsequently H2O (10 mL)
was added and the mixture was extracted with CH2Cl2
(4x20 mL). The combined organic layers were dried (K2CO3), con-
centrated in vacuo and the residue was purified by FC (2 cm, cyclo-
hexane + 1% N,N-dimethylethanamine, 20 cm, 10 mL, Rf = 0.25).
Colorless solid, mp 62 °C, yield 51.8 mg (88%).
C
23H29 NO2
~
(351.5). MS (ESI): m/z (%) = 352 [MH, 100]. FT-IR:
m
(cmꢀ1) = 3024
(m, v, C–H, arom), 2952 (s, v, C–H, alkyl), 2834 (s, v, OCH3), 2784
(m, v, N–CH3), 1604, 1494 (w, v, C@C, arom), 1494 (s, d, C–H, alkyl),
752 (s, d, C–H, o-disubst. arom), 732, 697 (s, d, C–H, mono-subst.
arom). 1H NMR (CDCl3): d (ppm) = 1.66 (ddd, J = 13.5/5.8/3.0 Hz,
1H, 20-He), 1.81 (ddd, J = 13.6/5.8/3.0 Hz, 1H, 60-He), 1.94–2.22 (m,
5H, 30-He, 30-Ha, 50-Ha, 50-He, 60-Ha), 2.17 (s, 3H, NCH3), 2.39 (‘td’,
J = 13.2/4.1 Hz, 1H, 20-Ha), 2.46 (‘quint’, J = 3.0 Hz, 1H, 40-He), 2.91
(dd, J = 15.6/6.9 Hz, 1H, ArCH2CHOCH3), 2.96 (dd, J = 15.7/4.1 Hz,
1H, ArCH2CHOCH3), 3.59 (s, 3H, OCH3), 3.60 (s, 2H,NHCH2Ar),
4.89 (dd, J = 6.7/4.2 Hz, 1H, Ar-CH2-CH-OCH3), 7.07–7.11 (m, 1H,
Ar-H), 7.16–7.20 (m, 1H, Ar-H), 7.22–7.30 (m, 3H, Ar-H), 7.33–
7.42 (m, 4H, Ar-H). Purity (HPLC) 98.6%, tR = 18.88 min.
7.2.10. cis-N-Benzyl-3-methoxy-N-pentyl-3,4-dihydrospiro-
[[2]benzopyran-1,10-cyclohexan]-40-amine (cis-13)
Under N2 benzylamine cis-8 (46.5 mg, 0.138 mmol) was dis-
solved in CH2Cl2 (5 mL). Valeraldehyde (18 lL, 0.169 mmol) and
NaBH(OAc)3 (95%, 45 mg, 0.213 mmol) were added and the reac-
tion mixture was stirred for 23 h at rt. Then H2O (10 mL) was
added and the mixture was extracted with CH2Cl2 (4 ꢃ 20 mL)
the combined organic layers were dried (K2CO3), concentrated in
vacuo and the residue was purified by FC (2 cm, cyclohexane + 2%
N,N-dimethylethanamine, 20 cm, 10 mL, Rf = 0.37). Colorless oil,
yield 50.3 mg (90%). C27H37NO2 (407.6). MS (ESI): m/z (%) = 408
~
7.2.8. cis-N-Benzyl-3-methoxy-N-methyl-3,4-dihydrospiro-
[[2]benzopyran-1,10-cyclohexan]-40-amine (cis-11)
Under N2 benzylamine cis-8 (54 mg, 0.16 mmol) was dissolved
in CH2Cl2 (2.5 mL) and formalin 37% (238 lL, 3.2 mmol) and NaB-
H(OAc)3 (95%, 57 mg, 0.256 mmol) were added. The reaction mix-
ture was stirred for 3 h at rt. Subsequently H2O (10 mL) was added
and the mixture was extracted with CH2Cl2 (4 ꢃ 20 mL). The com-
bined organic layers were dried (K2CO3), concentrated in vacuo and
[MH,100]. IR: m
(cmꢀ1) = 3062, 3022 (m, v, C–H, arom), 2926,
2858 (s, v, C–H, alkyl), 1603, 1492 (w, v, C@C, arom), 1451 (m, d,
C–H, alkyl), 754 (s, d, C–H, o-disubst. arom), 731, 697 (s, d, C–H,
monosubst. arom). 1H NMR (CDCl3): d (ppm) = 0.86 (t, J = 7.0 Hz,
3H, NCH2CH2CH2CH2CH3), 1.24–1.27 (m, 4H, NCH2CH2CH2CH2CH3),
1.39–1.46 (m, 2H, NCH2CH2CH2CH2CH3), 1.57 (‘td’, J = 13.7/3.9 Hz,
1H, 20-Ha), 1.67–1.77 (m, 2H, (CH2CH2)2CHN), 1.78–1.91 (m, 2H,
(CH2CH2)2CHN), 1.92–2.04 (m, 2H, (CH2CH2)2CHN), 2.09 (ddd,

3150
E. Rack et al. / Bioorg. Med. Chem. 19 (2011) 3141–3151
J = 14.0/6.1/3.0 Hz, 1H, 20-He), 2.53 (m, 2H, NCH2CH2CH2CH2CH3),
2.65–2.72 (‘tt’, J = 11.7/3.7 Hz, 1H, 40-Ha), 2.88 (dd, J = 15.6/6.6 Hz,
1H, ArCH2CHOCH3), 2.93 (dd, J = 15.4/3.8 Hz, 1H, ArCH2CHOCH3),
3.58 (s, 3H, OCH3), 3.69 (s, 2H, NHCH2Ar), 4.85 (dd, J = 6.9/4.0 Hz,
1H, Ar-CH2–CH–OCH3), 7.06–7.09 (m, 2H, Ar-H), 7012–7.19 (m,
2H, Ar-H), 7.20–7.25 (m, 1H, Ar-H), 7.28–7.34 (m, 2H, Ar-H),
7.37–7.41 (m, 2H, Ar-H). Purity (HPLC): 98.3%, tR = 22.33 min.
ration (about 75
concentrations of test compounds, 2 nM [3H]-(+)-pentazocine,
and buffer (50 mM TRIS, pH 7.4) in a total volume of 200 L for
180 min at 37 °C. The incubation was terminated by rapid filtration
through the presoaked filtermats by using the cell harvester. After
lg of the protein) was incubated with various
l
washing each well five times with 300 lL of water, the filtermats
were dried at 95 °C. Subsequently, the solid scintillator was put
on the filtermat and melted at 95 °C. After 5 min, the solid scintil-
lator was allowed to solidify at rt. The bound radioactivity trapped
on the filters was counted in the scintillation analyzer. The non-
7.2.11. cis-N,N-Dibenzyl-3-methoxy-3,4-dihydrospiro-
[[2]benzopyran-1,10-cyclohexan]-40-amine (cis-14)
Under N2 benzylamine cis-8 (65 mg, 0.193 mmol) was dissolved
specific binding was determined with 10 lM unlabeled (+)-pentaz-
in CH2Cl2 (5 mL). Benzaldehyd (23.5
lL, 0.232 mmol) and NaB-
ocine. The Kd-value of the radioligand [3H]-(+)-pentazocine is
H(OAc)3 (95%, 65 mg, 0.291 mmol) were added and the reaction
mixture was stirred for 23 h at rt. Then H2O (10 mL) was added
and the mixture was extracted with CH2Cl2 (4 ꢃ 20 mL), the com-
bined organic layers were dried (K2CO3), concentrated in vacuo and
the residue was purified by FC (2 cm, cyclohexane + 2% N,N-
dimethylethanamine, 20 cm, 10 mL, Rf = 0.39). Colorless solid, mp
136 °C, yield 34 mg (41%). C29H33NO2 (427.6). MS (ESI): m/z
~
2.9 nM.64
7.3.4. Membrane preparation for the r2 assay52,54
Two rat livers were cut into small pieces and homogenized with
a potter (500–800 rpm, 10 up-and-down strokes) in 6 volumes of
cold 0.32 M sucrose. The suspension was centrifuged at 1200g for
10 min at 4 °C. The supernatant was separated and centrifuged at
31,000g for 20 min at 4 °C. The pellet was resuspended in buffer
(50 mM TRIS, pH 8.0) and incubated at rt for 30 min. After the incu-
bation, the suspension was centrifuged again at 31,000g for 20 min
at 4 °C. The final pellet was resuspended in buffer, the protein con-
centration was determined according to the method of Bradford63
using bovine serum albumin as standard, and subsequently the
preparation was frozen (ꢀ80 °C) in 1.5 mL portions containing
about 2 mg protein/mL.
(%) = 428 [MH,100]. IR:
m
(cmꢀ1) = 3061, 3028 (m, v, C–H, arom),
2942, 2921, 2857 (s, v, C–H, alkyl), 1601, 1491 (w, v, C@C, arom),
1451 (m, d, C–H, alkyl), 761 (s, d, C–H, o-disubst. arom), 745, 697
(s, d, C–H, monosubst. arom). 1H NMR (CDCl3): d (ppm) = 1.54
(‘td’, J = 13.6/4.1 Hz, 1H, 20-Ha), 1.74–1.87 (m, 3H, (CH2CH2)2CHN),
1.90–2.03 (m, 2H, (CH2CH2)2CHN), 2.03–2.15 (m, 2H,
(CH2CH2)2CHN), 2.71 (‘tt’, J = 11.9/3.6 Hz, 1H, CH2-CH-NH, 40-Ha),
2.88 (dd, J = 15.4/6.4 Hz, 1H, ArCH2CHOCH3), 2.93 (dd, J = 15.4/
3.6 Hz, 1H, ArCH2CHOCH3), 3.57 (s, 3H, OCH3), 3.70 (d,
J = 14.7 Hz, 2H, N(CH2-Ph)2), 3.73 (d, J = 14.7 Hz, 2H, N(CH2-Ph)2),
4.86 (dd, J = 6.9/3.9 Hz, 1H, ArCH2CHOCH3), 7.00–7.09 (m, 2H, Ar-
H), 7.12–7.17 (m, 2H, Ar-H), 7.20–7.25 (m, 2H, Ar-H), 7.28–7.34
(m, 4H, Ar-H), 7.40–7.44 (m, 4H, Ar-H). Purity (HPLC): 97.7%,
tR = 21.79 min.
7.3.5. Protocol of the r2 assay52,54
The test was performed with the radioligand [3H]-di-o-
tolylguanidine (50 Ci/mmol; ARC). The thawed membrane prepa-
ration (about 100 lg of the protein) was incubated with various
concentrations of test compounds, 3 nM [3H]-di-o-tolylguanidine,
500 nM (+)-pentazocine and buffer (50 mM TRIS, pH 8.0) in a total
7.3. Receptor binding studies
volume of 200
by rapid filtration through the presoaked filtermats using a cell
harvester. After washing each well five times with 300 L of water,
lL for 180 min at rt. The incubation was terminated
7.3.1. Materials and general procedures
l
Guinea pig brains and rat livers were commercially available
(Harlan-Winkelmann, Germany). Homogenizer: Elvehjem Potter
(B. Braun Biotech International). Centrifuge: High-speed cooling
centrifuge model Sorvall RC-5C plus (Thermo Finnigan). Filter:
Printed Filtermat Type A (Perkin Elmer), presoaked in 0.5% aqueous
polyethylenimine for 2 h at rt before use. The filtration was carried
out with a MicroBeta FilterMate-96 Harvester (Perkin Elmer). The
scintillation analysis was performed using Meltilex (Type A) solid
scintillator (Perkin Elmer). The radioactivity bound to the filter
was measured using a MicroBeta Trilux scintillation analyzer (Per-
kin Elmer). The overall counting efficiency was 20%.
the filtermats were dried at 95 °C. Subsequently, the solid scintilla-
tor was put on the filtermat and melted at 95 °C. After 5 min, the
solid scintillator was allowed to solidify at rt. The bound radio-
activity trapped on the filters was counted in the scintillation
analyzer. The non-specific binding was determined with 10 lM
unlabeled ditolylguanidine. The Kd-value of the radioligand [3H]-
ditolylguanidine is 17.9 nM.65
7.3.6. Data analysis
Usually, all experiments were carried out in triplicates using
standard 96-well-multiplates (Diagonal). The IC50-values were
determined in competition experiments with six concentrations
of the test compounds and were calculated with the program
GraphPad PrismÒ 3.0 (GraphPad Software) by non-linear regres-
sion analysis. The Ki-values were calculated according to Cheng
and Prusoff.66 The Ki-values of potent compounds are given as
mean values + SEM from three independent experiments.
7.3.2. Membrane preparation for the r1 assay52,54
Five guinea pig brains were homogenized with the potter (500–
800 rpm, 10 up-and-down strokes) in 6 volumes of cold 0.32 M su-
crose. The suspension was centrifuged at 1200g for 10 min at 4 °C.
The supernatant was separated and centrifuged at 23,500g for
20 min at 4 °C. The pellet was resuspended in 5–6 volumes of buf-
fer (50 mM TRIS, pH 7.4) and centrifuged again at 23,500g (20 min,
4 °C). This procedure was repeated twice. The final pellet was
resuspended in 5–6 volumes of buffer, the protein concentration
was determined according to the method of Bradford63 using bo-
vine serum albumin as standard, and subsequently the preparation
was frozen (ꢀ80 °C) in 1.5 mL portions containing about 1.5 mg
protein/mL.
Acknowledgement
This work was supported by the Deutsche Forschungsgemeins-
chaft, which is gratefully acknowledged.
References and notes
7.3.3. Protocol of the r1 assay52,54
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