Synthesis of σ receptor ligands with unsymmetrical spiro connection of the piperidine moiety

Article abstract of DOI:10.1016/j.ejmech.2009.07.017

The symmetrically connected spiro[[2]benzopyran-1,4′-piperidines] 1 are highly potent and selective σ₁ receptor ligands. Changing the position of the spirocyclic nitrogen atom led to the unsymmetrically connected spiro[[2]benzopyran-1,3′-piperidines] 2 with a reduced distance between the aromatic system and the basic nitrogen atom. The synthesis of 2 was performed by halogen-metal exchange at the aryl bromide 3 followed by addition to the piperidone 5 and intramolecular transacetalization. The yield of 2a was considerably improved by transmetallation of the aryllithium intermediate 4a with CeCl₃ (4c). The cis and trans diastereomers cis-2 and trans-2 were separated and characterized by nuclear Overhauser effect. After removal of the benzyl group, the secondary amine 2b was alkylated with various alkyl and arylalkyl halides. The σ₁ and σ₂ receptor affinity of the spirocyclic piperidines 2 were determined with receptor binding studies. Compared with the spirocyclic piperidines 1, the unsymmetrically connected piperidines 2 show remarkably reduced σ₁ receptor affinities, whereas the selectivity over σ₂ and NMDA receptors was retained. A stereoselective interaction of the σ₁ receptor protein with the cis- or trans-configured spirocyclic compounds 2 was not observed. It was shown that alkyl residues at the N-atom can replace the lipophilic N-arylalkyl groups and interact with the primary hydrophobic binding site of the σ₁ receptor protein.

Full text of DOI:10.1016/j.ejmech.2009.07.017

European Journal of Medicinal Chemistry 44 (2009) 4306–4314
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of
s
receptor ligands with unsymmetrical spiro connection of the
piperidine moiety
Annemarie Jasper a, Dirk Schepmann a, Kirstin Lehmkuhl a, Jose Miguel Vela b, Helmut Buschmann b,
,
Jo¨rg Holenz b, Bernhard Wu¨nsch a
*
a Institut fu¨r Pharmazeutische und Medizinische Chemie, Hittorfstraße 58-62, 48145 Mu¨nster, Germany
b Esteve, Av. Mare de Deu de Montserrat 221, 08041 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
The symmetrically connected spiro[[2]benzopyran-1,40-piperidines] 1 are highly potent and selective s1
receptor ligands. Changing the position of the spirocyclic nitrogen atom led to the unsymmetrically
connected spiro[[2]benzopyran-1,30-piperidines] 2 with a reduced distance between the aromatic system
and the basic nitrogen atom. The synthesis of 2 was performed by halogen–metal exchange at the aryl
bromide 3 followed by addition to the piperidone 5 and intramolecular transacetalization. The yield of 2a
was considerably improved by transmetallation of the aryllithium intermediate 4a with CeCl3 (4c). The
cis and trans diastereomers cis-2 and trans-2 were separated and characterized by nuclear Overhauser
effect. After removal of the benzyl group, the secondary amine 2b was alkylated with various alkyl and
arylalkyl halides. The s1 and s2 receptor affinity of the spirocyclic piperidines 2 were determined with
receptor binding studies. Compared with the spirocyclic piperidines 1, the unsymmetrically connected
piperidines 2 show remarkably reduced s1 receptor affinities, whereas the selectivity over s2 and NMDA
receptors was retained. A stereoselective interaction of the s1 receptor protein with the cis- or trans-
configured spirocyclic compounds 2 was not observed. It was shown that alkyl residues at the N-atom
can replace the lipophilic N-arylalkyl groups and interact with the primary hydrophobic binding site of
the s1 receptor protein.
Article history:
Received 22 January 2009
Received in revised form
13 July 2009
Accepted 16 July 2009
Available online 21 July 2009
Keywords:
s
Receptor ligands
Spirocyclic piperidines
Structure–affinity relationships
Receptor binding studies
Halogen–metal exchange
Transmetallation
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
the yeast ergosterol-D8 D7wisomerase [10]. The s2 receptor is less
/
characterized. So far, it has not been cloned and its molecular weight
is approximately 21.5 kDa [6].
Both subtypes are involved in neuromodulatory processes and,
therefore, s receptors serve as targets for the development of novel
Originally, the
subtype [1]. However, since the first postulation it has been shown
that receptors representan unique classofreceptorswith aspecific
s receptor has been regarded as an opioid receptor
s
ligand binding profile and a characteristic distribution both in the
central nervous system (CNS) and in many peripheral tissues (e.g.
drugs for the treatment of different neurological disorders, e.g.
schizophrenia, depression or dementia [11–13]. Moreover, it has
been shown that various cancer cell lines including breast, lung and
kidney, liver, heart) [2–6]. The
s receptor class can be divided into at
least two subtypes: s1 and s2 receptors. The s1 receptor has already
been cloned. It is a non-metabotropic transmembrane receptor
comprising two transmembrane helices and a large extracellular
loop [7,8]. The rat brain receptor protein consists of 223 amino acids,
which results in a molecular weight of 23 kDa [9]. Although human
and animal s1 receptors show a structural similarity of more than
95%, there is no resemblance to other known mammalian proteins.
However, a 30% homologyexists between the cloned s1 receptorand
prostate cancer cells overexpress s receptors [14,15]. Thus, radio-
labeled s1 and s2 receptor ligands represent attractive tools for
imaging of various cancer cells, e.g. breast cancer cells [16].
Among various classes of s1 receptor ligands, the class of spi-
rocyclic piperidines 1 contains potent s1 receptor ligands with high
affinity and selectivity (Fig. 1). In particular the benzyl substituted
spirocyclic compound 1a (R ¼ benzyl) has a Ki value of 1.29 nM for
the s1 receptor [17]. In order to investigate the effect of the N-
position in the piperidine heterocycle on the
s affinity, we planned
to shift the nitrogen to the adjacent position. This shift leads to the
novel class of spiro[[2]benzopyran-1,30-piperidines] 2 with a modi-
fied distance between the aromatic benzopyran ring and the basic
* Corresponding author. Tel.: þ49 251 83 33311; fax: þ49 251 83 32144.
E-mail address: wuensch@uni-muenster.de (B. Wu¨nsch).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.07.017

A. Jasper et al. / European Journal of Medicinal Chemistry 44 (2009) 4306–4314
4307
nucleophilic addition to the ketone 5. At first, the Grignard reagent
4b was generated by reaction of the aryl bromide 3 with Mg. This
modification provided an increased yield of 26% of 2a [20].
However, due to the difficult preparation of the ortho-substituted
arylmagnesium compound 4b by insertion of Mg metal into the
aryl–C–Br bond, the yields of the spirocyclic piperidine 2a were not
reproducible.
OCH3
OCH3
O
O
N
N
R
R
Instead of using metallic magnesium it is sometimes more effi-
cient to synthesize ortho-substituted arylmagnesium compounds by
a halogen–metal exchange with neopentyl-MgBr [21]. The yield of
the spirocyclic piperidine 2a was slightly improved by this variation
(29% yield).
1
2
Fig. 1. Compounds with symmetrical (1) and unsymmetrical (2) spiro connection of
the piperidine moiety.
nitrogen atom. Furthermore, the new center of chirality of the spiro
atom leads to two diastereomeric pairs of enantiomers of the spi-
rocyclic piperidines cis-2 and trans-2 (see Scheme 2).
The purpose of this work was to find a straightforward access to
spiro[[2]benzopyran-1,30-piperidines] 2 as a new class of
ligands. The focus was on the stereochemistry and the systematic
variation of the residue R at the piperidine nitrogen atom in order
to find an optimal nitrogen substituent (Fig. 1).
For the nucleophilic attack of substrates, susceptible to enolate
formation due to the presence of acidic protons, the oxophilic
organocerium reagents are the reagents of choice [22]. Therefore,
the aryllithium intermediate 4a was transmetallated with CeCl3 to
afford the almost non-basic arylcerium reagent 4c. Subsequent
addition of the arylcerium intermediate 4c to the piperidone 5 gave
the desired spirocyclic piperidine 2a in 30% yield. In conclusion, the
addition of the ortho-substituted aryl metal compounds 4 to the
piperidone 5 was improved from 18% yield using the aryllithium
compound 4a to 30% using the arylcerium compound 4c.
Due to the additional center of chirality in the spirocyclic system
2a, twodiastereomerswereformedalmostintheratio1:1(Scheme 2).
In the cis isomer cis-2a the methoxy group and the 20-CH2 group of
the piperidine ring are on the same side and in the trans isomer
trans-2a these groups are on opposite sides of the benzopyran ring
plane. The diastereomers cis-2a and trans-2a were separated by
flash chromatography, the corresponding structures were deter-
mined by 1H NMR spectroscopy and the relative configuration was
assigned by NOE difference spectroscopy, respectively.
Cleavage of the benzyl group of 2a was performed with
ammonium formate [23] in the presence of the catalyst Pd/C and
led to the secondary amine 2b. The homologous series of phenyl-
alkyl (2c–2e) and alkyl substituted spirocyclic piperidines (2f–2i)
were synthesized by SN2 reaction of the secondary amine 2b with
various arylalkyl and alkyl halides. Reaction of the secondary amine
2b with the primary halides R–X was performed in refluxing THF,
acetone or acetonitrile in the presence of K2CO3 to afford the
(aryl)alkylated spirocyclic piperidines 2c–2i. In order to improve
s receptor
2. Chemistry
The starting compound for the synthesis of the spi-
ro[[2]benzopyran-1,30-piperidines] 2 was the bromo acetal 3 [18].
Upon treatment with n-butyllithium at ꢀ78 ꢁC 3 was transformed
by a halogen–metal exchange into the aryllithium intermediate 4a.
Subsequent trapping of 4a with 1-benzylpiperidin-3-one (5)
afforded the hydroxy acetal 6. Cyclization of the hydroxy acetal 6
was catalyzed by p-toluenesulfonic acid [19]. In order to avoid
elimination of a second molecule CH3OH to give a cyclic enol ether,
CH3OH was the preferred solvent for the intramolecular trans-
acetalization of isolated hydroxy acetal 6. However, the best yield
(18%) of the spirocyclic piperidine 2a was obtained by direct
cyclization of the unpurified hydroxy acetal 6 with HCl during the
work-up procedure (Scheme 1).
Despite several optimization attempts the yield of the spiro
compound 2a did not exceed 18%. We assume that enolate forma-
tion of 5 was competing with the nucleophilic addition. Therefore,
less basic aryl metal compounds were investigated for the
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
(a) – (c)
(e)
(d)
O
OH
Br
M
N
Bn
3
4a: M = Li
N
4b: M = MgBr
4c: M = CeCl2
6
Bn
5
2
R
c
d
e
f
g
h
i
(CH2)2Ph
(CH2)3Ph
(CH2)4Ph
n-Bu
n-Hex
n-Oct
OCH3
Bn
OCH3
OCH3
(f)
(g)
O
N
O
N
O
NH
R
n-Dec
2a
2b
2c-i
Scheme 1. Synthesis of compounds with an unsymmetrical spiro connection of the piperidine moiety. Reagents and reaction conditions: (a) n-BuLi, ꢀ78 ꢁC, THF, formation of 4a.
(b) Mg, THF, 60 ꢁC, then ꢀ30 ꢁC or neopentyl-MgBr, THF, ꢀ40 ꢁC, formation of 4b. (c) n-BuLi, ꢀ78 ꢁC, THF, 1 h, then addition of CeCl3 in THF, 1 h, ꢀ78 ꢁC, formation of 4c. (d) reaction
of 4a with 5, 2 h, ꢀ78 ꢁC, 16 h, rt, 28%. (e) from 6: p-TosOH, CH3OH, 4 d, rt; from 3: conditions as described under (c) via 4c and work-up with 5% HCl, 30%. (f) NH4HCO2, CH3OH, Pd/C,
2 h, 65 ꢁC, 91%. (g) R-X, THF or CH3CN or acetone, K2CO3.

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A. Jasper et al. / European Journal of Medicinal Chemistry 44 (2009) 4306–4314
3. Receptor binding studies
H3CO
H
H
OCH3
O
O
The s1 and s2 receptor affinities of the novel spirocyclic
compounds 1 and 2 were determined by means of competition
experiments with radioligands. Homogenates of guinea pig brains
were used as receptor material in the s1 receptor assay. The s1
selective ligand [3H]-(þ)-pentazocine served as radioligand, and the
non-specific binding was determined in the presence of a large
excess of non-labeled (þ)-pentazocine [17,25,26]. Homogenates of
rat livers served as source for s2 receptors in the s2 assay. Since a s2
selective radioligand is not commercially available, the non-selective
2‘
N
2‘
N
R
R
4‘
4‘
cis-2a-i
trans-2a-i
Scheme 2. Structures for cis- and trans-configured diastereomers of 2.
the yields and reduce the very long reaction times, most of the
nucleophilic substitution reactions were performed using micro-
wave irradiation [24] instead of heating to reflux. The n-hexyl
residue was introduced with 1-iodohexane which had been
synthesized from 1-chlorohexane by a Finkelstein reaction with NaI
under microwave irradiation.
s
ligand [3H]-di(o-tolyl)guanidine was employed in the presence of
an excess of the non-radiolabeled s1 selective ligand (þ)-pentazo-
cine for selective masking of s1 receptors. Performing of the s2 assay
in the presence of an excess of non-tritiated 1,3-di(o-tolyl)guanidine
led to the non-specific binding of the radioligand [6,26,27].
The diastereomeric spirocyclic piperidines cis-2a and trans-2a
were formed in a ratio of about 1:1. Since the diastereomeric
composition did not change during the hydrogenolytic removal of
the benzyl group and the alkylation step the cis- and trans-
configured spirocyclic piperidines 2c–2i were also obtained in the
ratio of about 1:1. The diastereomers were separated by flash
chromatography, respectively. However, due to very similar Rf
values and strong tailing of the diastereomers the isolation of pure
stereoisomers required more than one (usually 3–5) chromato-
graphic run with considerable loss of yields. The cis and trans
isomers of the phenylbutyl derivative 2e could not be separated by
fc. Therefore the pure diastereomers cis-2b and trans-2b, which
were prepared by hydrogenolytic removal of the benzyl group from
the diastereomerically pure benzyl derivatives cis-2a and trans-2a,
were alkylated with 1-chloro-4-phenylbutane under microwave
irradiation to yield the pure diastereomers cis-2e and trans-2e,
respectively.
In order to prove the configurational stability of the isolated
diastereomers solutions of the pure diastereomers cis-2a and trans-
2a were stored in different aqueous TRIS/HCl-buffers (pH 4.0,
pH ¼ 7.4) over a period of 1 week. Careful tlc analysis showed that
both diastereomers did not isomerize under these conditions
(Scheme 2).
The receptor binding studies revealed that an aromatic residue
within the N-substituent is not crucial for high s1 receptor affinity
since ligands with linear alkyl substituents showed comparable s1
receptor affinities as compounds with arylalkyl moieties (see
Receptor binding studies). In order to confirm this observation,
spirocyclic 1,40-piperidines 1 with alkyl substituents were envis-
aged. The spirocyclic 1,40-piperidine derivative 1b was prepared
according to Ref. [17]. Alkylation of the secondary amine 1b with
butyl bromide and n-octyl bromide provided the spi-
ro[[2]benzopyran-1,40-piperidines] 1f with a short (C4H9) and 1h
with a long (C8H17) alkyl residue, respectively (Scheme 3).
4. Results and discussion
The s1 and s2 receptor affinities of the cis- and trans-configured
spiro[benzopyran-1,30-piperidines] 2a–2i are listed in Table 1
together with the
s
affinities of the spiro[benzopyran-1,40-piperi-
dines] 1a, 1f and 1h. The spiro[benzopyran-1,30-piperidines] 2a and
2c–2e substituted with arylalkyl residues possess s1 receptor
affinities in the low micromolar to high nanomolar range. The
benzyl compounds cis-2a and trans-2a show the lowest s1 receptor
affinity (Ki ¼ 1.72
mM and 1.44 mM, respectively), whereas the
phenylpropyl derivative trans-2d is the most potent s1 ligand
within this series of compounds (Ki ¼ 376 nM). Generally, the trans-
configured derivatives reveal slightly higher s1 affinities than the
corresponding cis-diastereomers, e.g. trans-2d has about threefold
higher s1 receptor affinity than the cis-diastereomer cis-2d. The s1
affinity increases with increasing length of the side chain
(2a < 2c < 2d z 2e) with the trans-configured phenylpropyl and
phenylbutyl derivatives trans-2d and trans-2e being the most active
compounds. Certainly three to four methylene moieties represent
the optimal N-aryl distance in the trans-configured derivatives.
The s1 receptor affinity is retained after removing of the phenyl
moiety in the nitrogen side chain. The alkyl substituted compounds
2f–2i and the analogous arylalkyl substituted compounds 2a, 2c–2e
reveal comparable s1 affinities. The n-hexyl compounds cis-2g and
trans-2g with a medium chain length show the highest s1 receptor
affinities, which even exceeds the s1 affinities of the phenylpropyl
(trans-2d) and phenylbutyl derivatives (trans-2e). Ligands with
a shorter (2f) or a longer (2h, 2i) side chain are less active. In the
spirocyclic benzopyran-1,30-piperidine series of compounds the cis-
configured hexyl derivative cis-2g is the most active s1 ligand with
a Ki value of 159 nM. Thus, aliphatic alkyl chains, in particular alkyl
chains with six C-atoms, are able to replace arylalkyl residues at the
N-atom of potent s1 ligands.
The s2 receptor affinities of the spiro[benzopyran-1,30-piperi-
dines] 2 are considerably lower than their s1 receptor affinities.
Compounds 2 show s2 affinities in the low micromolar range. The
phenylpropyl compound trans-2d possesses the highest s2 affinity
OCH3
OCH3
(Ki ¼ 1.11
mM) within this series. The determined Ki values indicate
(a)
O
O
that the s2 receptor protein does not differentiate between
compounds with arylalkyl and alkyl substituents and between cis
and trans isomers.
N
R
N
H
Generally, most of the new spirocyclic ligands show a slight
preference for the s1 receptor. Compounds with the highest s1
affinity reveal the highest s1/s2 selectivity. For example the phe-
nylpropyl derivative trans-2d and the n-hexyl derivative cis-2g have
selectivity factors of 3.0 and 7.7, respectively.
1b
1f: R = n-Bu
1h: R = n-Oct
Scheme 3. Synthesis of alkyl substituted compounds with
a symmetrical spiro
Surprisingly, the trans-configured n-butyl derivative trans-2f
was chemically unstable. Over a period of 2 weeks the s1 receptor
connection. Reagents and reaction conditions: (a) n-BuBr or n-Oct-Br, CH3CN, K2CO3,
30 h, 81 ꢁC, 37% (1f) and 68% (1h).

A. Jasper et al. / European Journal of Medicinal Chemistry 44 (2009) 4306–4314
4309
Table 1
Affinities of the spirocyclic piperidines 1 and 2 towards s1 and s2 receptors.
Compound
R
Ki ꢂ SEM (nM)a
s1 ([3H]-(þ)-pentazocine)
s1
/s2 Selectivity
s2 ([3H]-di-o-tolylguanidine)
1a [17]
1f
1h
cis-2a
trans-2a
cis-2c
trans-2c
cis-2d
trans-2d
cis-2e
trans-2e
cis-2f
trans-2f
cis-2g
trans-2g
cis-2h
trans-2h
cis-2i
trans-2i
Haloperidol
(þ)-Pentazocine
Ditolylguanidine
Bn
n-Bu
n-Oct
Bn
1.3 ꢂ 0.2
3500 ꢂ 350
1020
534
2708
443
55
2.3
9.8
1720
1440
470 ꢂ 87
725 ꢂ 94
1220
0%b
n.d.
1.3
n.d.
2.0
2.4
3.0
1.1
3.3
n.d.
n.d.
7.7
5.1
6.8
0.5
2.5
0.9
20
Bn
1900
13%b
1450
2940
1110
1340
1300
0%b
–(CH2)2Ph
–(CH2)2Ph
–(CH2)3Ph
–(CH2)3Ph
–(CH2)4Ph
–(CH4)2Ph
n-Bu
376 ꢂ 62
1170
392 ꢂ 106
984
(535)
159 ꢂ 27
288 ꢂ 256
1070
2980
2080
4360
n-Bu
0%b
n-Hex
n-Hex
n-Oct
n-Oct
n-Dec
1230
1480
7230
1390
5280
4110
78 ꢂ 2.3
–
n-Dec
3.9 ꢂ 1.5
4.2 ꢂ 1.1
61 ꢂ 18
n.d.
0.7
42 ꢂ 17
n.d. ¼ not determined due to the non-specified s2 affinity.
The Ki value of trans-2f is given in brackets, since the compound was not stable.
a
The standard error of the mean (SEM) is given, when three independent experiments have been performed. For compounds with low affinity (Ki > 1000 nM) the Ki value
was recorded only once and, then, a SEM is not given.
b
Inhibition of radioligand binding at a test compound concentration of 1 mM.
affinity was determined three times. The Ki values changed from
535 nM to 2.0 M, to 3.4 M. This variation was due to compound
distance of compounds 2 with an unsymmetrical spiro connection
might explain the reduced s1 receptor affinity.
m
m
decomposition in the test solution (DMSO, TRIS buffer). Thus, the s1
affinity of the pure compound is in the nanomolar range, a standard
error of the mean (SEM) could however not be determined.
The promising s1 affinity of the N-alkyl derivatives, in particular
the N-butyl (2f) and N-hexyl (2g) derivatives, prompted us to
synthesize and evaluate pharmacologically the corresponding N-
alkyl derivatives 1 derived from the symmetrical spiro piperidine
ring connection. The N-butyl (1f) and N-octyl (1h) derivatives
interact with high affinity with s1 receptors (Table 1) indicating
that the s1 receptor protein accepts an aliphatic residue at the
piperidine N-atom instead of an arylalkyl chain. With a Ki value of
2.3 nM the butyl derivative 1f is almost as potent as the benzyl
derivative 1a. Compared with 1a (2708) the s1/s2 selectivity of 1f
(443) and 1g (55) is reduced due to their slightly increased s2
receptor affinities.
A comparison of the s1 receptor affinities of the N-benzyl (1a/
2a), N-butyl (1f/2f) and N-octyl (1h/2h) pairs clearly shows that
5. Conclusion
A series of spirocyclic s1 receptor ligands with an unsymmet-
rical spiro connection of the piperidine moiety has been synthe-
sized. Since the molecules contain two centers of chirality two
diastereomeric pairs of enantiomers are possible. The s1 affinity of
most of the cis- and trans isomers is very similar, although the s1
receptor protein has a small preference for some trans-configured
N-arylalkyl derivatives (e.g. trans-2d, trans-2e). Compared with the
symmetrically connected spiro compounds 1 the unsymmetrically
connected spiro derivatives 2 are considerably less active. This may
be due to the reduced distance between the basic N-atom and the
benzene moiety of the benzofuran system (4.95 Å). According to
the pharmacophore model of Glennon, a distance of 6–10 Å
between the basic N-atom and the primary hydrophobic region is
required for high s1 receptor affinity. It was shown that N-alkyl
derivatives (e.g. 2f, 2g) are also potent s1 receptor ligands with
even higher s1 affinity than the corresponding N-arylalkyl deriva-
tives. This result was transferred to the symmetrically connected
spirocyclic piperidines 1 and it was shown that N-alkyl derivatives
(e.g. 1f, 1h) are very potent s1 receptor ligands.
compounds
2 with an unsymmetrical spiro piperidine ring
connection are considerably less potent than the corresponding
symmetrical spirocyclic piperidines 1. The reduced s1 affinity of the
unsymmetrical spirocyclic piperidines is explained by the phar-
macophore model of Glennon et al. [28,29]. According to this model
the distance between the basic N-atom and the primary hydro-
phobic region should be 6–10 Å to achieve high s1 receptor affinity.
The piperidine ring of the spirocyclic piperidines 1 and 2 can
adopt two chair conformations with the phenyl moiety of the
benzopyran ring in the equatorial or axial orientation. In both types
of compounds the conformation with equatorial orientation of the
phenyl ring is energetically favored. In the favored conformations
the distance between the basic N-atom and the center of the
benzopyran phenyl moiety is 5.68 Å (1) and 4.95 Å (2), respectively.
The obtained Ki values support the pharmacophore model of
Glennon [28,29], which requires a distance of 6–10 Å between the
basic N-atom and the primary hydrophobic region. The shorter
6. Experimental
6.1. Chemistry, general methods
Unless otherwise noted, moisture sensitive reactions were
conducted under dry nitrogen. THF was distilled from sodium/
benzophenone ketyl prior to use. CH3OH was distilled from
magnesium/iodine prior to use. Microwave irradiation: Discover
synthesis microwave apparatus (CEM); the following parameters
are given in parenthesis: program; max. power; max. pressure;
temperature; the reaction time is divided into ramp time, hold time

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A. Jasper et al. / European Journal of Medicinal Chemistry 44 (2009) 4306–4314
and cool off time. Thin layer chromatography (tlc): Silica gel 60 F254
plates (Merck). Flash chromatography (fc): Silica gel 60, 40–64
(cmꢀ1) ¼ 2923 (C–H), 1076, 1034 (C–O). 1H NMR (CDC13):
mm
d
(ppm) ¼ 1.60–1.66 (m, 2H, CH2CH2CH2N), 1.81–1.87 (m,
(Merck); parentheses include: diameter of the column, eluent,
fraction size, Rf value. Melting point: Melting point apparatus SMP 3
(Stuart Scientific), uncorrected. Elemental analyses: Vario EL (Ele-
mentaranalysesysteme GmbH). MS: MAT GCQ (Thermo-Finnigan);
mode of ionization: electron impact (EI). IR: IR spectrophotometer
480Plus FT-ATR-IR (Jasco). 1H NMR (400 MHz), 13C NMR (100 MHz):
1H, CH2CH2CH2N), 1.92–1.98 (m, 1H, CH2CH2CH2N), 2.00–2.04
(m,1H, CH2CH2CH2N), 2.10–2.15 (m,1H, (CH2)3NCH2), 2.85–3.06 (m,
4H, CH2CH2CH2NCH2), ArCH2CH). 3.40 (d. J ¼ 11.2 Hz, 1H, NCH2Ph),
3.70 (d, J ¼ 13.3 Hz, 1H, NCH2Ph), 3.58 (s, 3 H. CHOCH3), 4.79 (t,
J ¼ 3.9 Hz, ArCH2CH), 7.05–7.33 (m, 9H, arom). Anal. calcd. for
C21H25NO2: C 77.99 H 7.79 N 4.33 found C 77.89 H 8.06 N 4.04.
trans-2a (Rf ¼ 0.20): Pale yellow oil. C21H25NO2 (323.4). MS: m/
Mercury-400BB spectrometer (Varian);
d in ppm related to tetra-
methylsilane; coupling constants are given with 0.5 Hz resolution
and the assignments of 1H and 13C NMR signals were supported by
2D NMR techniques (COSY, HETCOR). Compound 3 was prepared
according to Ref. [17].
z ¼ 323 [Mþ], 292 [Mþ ꢀ OCH3], 263 [Mþ ꢀ OCHOCH3]. IR (neat):
n
(cmꢀ1) ¼ 2921 (C–H), 1076 (C–O). 1H NMR CDC13):
d
(ppm) ¼ 1.55–
1.67 (m, 2H, CH2CH2CH2N), 1.91–2.18 (m, 3H, CH2CH2CH2N), 2.44–
2.48 (m, 1H, (CH2)3NCH2), 2.83–3.04 (m, 4H, CH2NCH2, ArCH2CH (2
H)). 3.44 (s, 3 H. CHOCH3), 3.50 (d, J ¼ 13.2 Hz, 1H, NCH2Ph), 3.63 (d,
J ¼ 13.2 Hz, 1H, NCH2Ph), 4.98 (t, J ¼ 3.5 Hz, 1H, ArCH2CH), 7.07–7.39
(m, 9H, arom). Anal. calcd. for C21H25NO2: C 77.99 H 7.79 N 4.33
found C 77.73 H 8.04 N 4.10.
6.2. 2-(1-Benzyl-3-hydroxypiperidin-3-yl)phenylacetaldehyde
dimethyl Acetal (6)
Under N2 the aryl bromide 3 (1.24 g, 5.06 mmol) was dissolved in
THF (10 mL) and cooled to ꢀ78 ꢁC. A 1.6 M solution of n-BuLi in
hexane (3.75 mL, 6.0 mmol) was slowly added. After 20 min
6.4. cis- and trans-3-Methoxy-3,4-dihydrospiro[[2]benzopyran-
1,30-piperidine] (cis-2b and trans-2b)
at ꢀ78 ꢁC,
a solution of 1-benzylpiperidin-3-one (5, 1.24 g,
5.08 mmol) in THF (4 mL) was added dropwise and the mixture was
stirred at ꢀ78 ꢁC for 2 h. After stirring at rt for another 16 h a satu-
rated NH4CI solution was added, and the mixture was extracted with
CH2C12. The organic layer was washed with a NaHSO3 solution (10%),
dried (Na2SO4) and filtered. The solvent was removed in vacuo, and
the crude product was purified by fc (6 cm, petroleum ether/ethyl
acetate 1:2, 30 mL, Rf ¼ 0.43). Pale yellow oil, yield 515 mg
(28%). C22H29NO3 (355.5). MS: m/z ¼ 340 [Mþ ꢀ CH3], 263
Under N2 the benzylamine 2a (734 mg, 2.27 mmol, mixture of
cis-2a and trans-2a) was dissolved in dry methanol (12 mL).
Ammonium formate (716 mg, 11.3 mmol) and 10% Pd/C (145 mg)
were added. The mixture was heated to reflux for 2 h. Then it was
filtered through Celite, the solvent was removed in vacuo and the
residue was purified by fc (3 cm, methanol/ammonia 98:2, 20 mL,
Rf ¼ 0.69). Colorless needles (Et2O), mp 54–58 ꢁC, yield 480 mg
(91%). C14H19NO2 (233.3). MS: m/z ¼ 233 [Mþ], 202 [Mþ ꢀ OCH3],
[Mþ ꢀ OH ꢀ CH(OCH3)2]. IR (neat).
n
(cmꢀ1) ¼ 2922 (C–H). 1H NMR
190 [Mþ ꢀ NH(CH2)2]. IR (neat):
n
(cmꢀ1) ¼ 2933 (C–H), 1055 (C–O).
(CDC13):
d
(ppm) ¼ 1.58–1.70 (m, 3H, CH2CH2CH2N), 1.96–2.12 (in,
1H NMR (CDCI3):
d
(ppm) ¼ 1.54–1.58 (m, 1H, CH2CH2CH2N),
3H, CH2CH2CH2N), 2.42–2.49 (m,1H, (CH2)3NCH2), 2.84–2.99 (m, 2H,
(CH2)3NCH2, ArCH2CH), 3.11–3.19 (in, 1 H. ArCH2CH), 3.28 (s, 3H,
OCH3), 3.32 (s, 3H, OCH3), 3.57–3.65 (m, 2H, NCH2Ph), 4.79
(t, J ¼ 5.2 Hz, 1H, ArCH2CH), 7.14–7.34 (m, 9H, arom).
1.79–2.04 (m, 4H, CH2CH2CH2N), 2.15–2.19 (m, 1H, (CH2)2CH2N),
2.66–2.78 (m, 1H, (CH2)3NCH2), 2.86–2.98 (m, 2 H (CH2)3NCH2,
ArCH2CH), 3.05–3.16 (m, 1H, ArCH2CH), 3,55/3.60 (2 s, together 3H,
CHOCH3 (cis-2b, trans-2b)), 4.87–4.96 (2 t, J ¼ 3.3 Hz each, together
1H, ArCH2CH), 7.09–7.26 (m, 4H, arom).
6.3. cis-10-Benzyl-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,30-
piperidine] (cis-2a) and trans-10-benzyl-3-methoxy-3,4-
dihydrospiro[[2]benzopyran-1,30-piperidine] (trans-2a)
6.5. cis-3-Methoxy-10-(2-phenylethyl)-3,4-
dihydrospiro[[2]benzopyran-1,30-piperidine] (cis-2c) and trans-3-
methoxy-10-(2-phenylethyl)-3,4-dihydrospiro[[2]benzopyran-1,30-
piperidine] (trans-2c)
Under N2 CeCl3 (138 mg, 0.56 mmol) was suspended in THF
(3 mL) and stirred at -78 ꢁC for 2 h. Every 30 min the suspension
was treated with ultrasonic waves for 5 min. In a second flask the
aryl bromide 3 (130 mg, 0.53 mmol) was dissolved in THF (3 mL)
and cooled to ꢀ78 ꢁC. A 1.6 M solution of n-BuLi in hexane (0.34 mL,
0.55 mmol) was slowly added, and the mixture was stirred at
ꢀ78 ꢁC for 1 h. With a transfer needle and N2 pressure the solution
of the formed aryllithium intermediate 4a was transferred to the
CeCl3 suspension and the needle was washed with THF (2 mL).
After stirring for 1 h at ꢀ78 ꢁC, a solution of the piperidone 5
(94.6 mg, 0.50 mmol) in THF (2 mL) was added dropwise to the
solution of the arylcerium intermediate 4c, and the mixture was
stirred for another 1 h at ꢀ78 ꢁC. A saturated NH4Cl solution was
added, the mixture was extracted with Et2O (6x), and the Et2O layer
was washed with H2O (2x). Afterwards, the organic layer was
extracted with aqueous HCl (5%, 6x) and the acidic aqueous layer
was washed with Et2O (2x). Solid KOH was added to the aqueous
layer (pH > 9) which was extracted with Et2O (6x). The organic
layer was dried (Na2SO4), filtered, the solvent was removed in
vacuo and the residue was purified by fc (2 cm, cyclohexane/ethyl
acetate 7:3, 5 mL). Due to tailing only small amounts of the pure
diastereomers were obtained after several chromatographic runs.
Total yield 48 mg (30%).
Method 1: 1-Chloro-2-phenylethane (93.2 mg, 0.66 mmol) and
K2CO3 (334 mg, 2.42 mmol) were added to a mixture of the
secondary amine 2b (96.2 mg, 0.41 mmol) in THF (5 mL) and the
mixture was heated to reflux for 19 h. Then, the mixture was
filtered through Celite, the solvent was removed in vacuo and the
residue was purified by fc (1 cm, cyclohexane/ethyl acetate 7:3,
5 mL). Pale yellow oil, total yield 49 mg (35%).
Method 2: A mixture of the secondary amine 2b (187 mg,
0.80 mmol), 1-chloro-2-phenylethane (141 mg, 1.00 mmol), K2CO3
(663 g. 4.80 mmol) and acetonitrile (3 mL) was filled into a 10 mL-
microwave pressure vial. The mixture was irradiated with micro-
waves (program: standard; max. power 220 W; max. pressure 4
bar; temperature 100 ꢁC; time program: 5 min ramp time; 40 min
hold time; 5 min cool off time). Afterwards, the mixture was
filtered through Celite, the solvent was removed under reduced
pressure and the residue was purified by fc (2 cm, cyclohexane/
ethyl acetate 7:3, 10 mL). Due to strong tailing only small amounts
of the pure diastereomers were obtained after several chromato-
graphic runs. Total yield 125 mg (46%).
cis-2c (Rf ¼ 0.36): Pale yellow oil. C72H27NO2 (337.4). MS (EI): m/
z ¼ 306 [Mþ ꢀ OCH3], 246 [Mþ ꢀ CH2Ph]. IR (neat):
n
cis-2a (Rf ¼ 0.38): Pale yellow oil. C21H25NO2 (323.4). MS: m/
(crnꢀ1) ¼ 2924 (C–H), 1076, 1034 (C–O). 1H NMR (CDC13):
z ¼ 323 [Mþ]. 292 [Mþ ꢀ OCH3], 263 [Mþ ꢀ OCHOCH3]. IR (neat):
n
d
(ppm) ¼ 1.68–1.75 (m, 1H, CH2CH2CH2N), 1.80–1.89 (m, 1H,

A. Jasper et al. / European Journal of Medicinal Chemistry 44 (2009) 4306–4314
4311
CH2CH2CH2N), 1.97–2.06 (m, 1H, CH2CH2CH2N), 2.22 (m, 3H,
CH2CH2CH2N), 2.52–2.63 (m, 1H, NCH2CH2Ph), 2.70–2.79 (m, 1H,
NCH2CH2Ph), 2.80–2.87 (m, 2H, NCH2CH2Ph), 2.86–2.93 (m, 1H,
ArCH2CH), 3.05–3.10 (m. 3H, ArCH2CH, (CH2)3NCH2), 3.46 (s, 3H,
OCH3), 5.12 (t, J ¼ 3.5 Hz, 1H, ArCH2CH), 7.13–7.29 (m, 9H, arom).
Anal. calcd. for C22H27NO2: C 78.30 H 8.06 N 4.15 found C 78.18 H
8.37 N 4.08.
Method 2 (cis-2e): A solution of the pure diastereomer cis-2b
(51.3 mg, 0.22 mmol) and 1-chloro-4-phenylbutane (50.4 mg,
0.30 mmol) in acetonitrile (3 mL) was filled into a 10 mL-microwave
pressure vial. K2CO3 (240 mg, 1.74 mmol) was added, and the
mixture was irradiated with microwaves (program: standard; max.
power 220 W; max. pressure 4 bar; temperature 100 ꢁC; time
program: 5 min ramp time; 40 min hold time; 5 min cool off time).
The mixture was filtered through Celite, concentrated in vacuo and
the residue was purified by fc (1 cm, petroleum ether/ethyl acetate
7:3, 5 mL, Rf ¼ 0.18). Pale yellow oil (cis-2e), yield 20 mg (25%).
C24H31NO2 (365.4). MS (EI): m/z ¼ 365 [Mþ], 334 [Mþ ꢀ OCH3], 246
trans-2c (Rf ¼ 0.30): Pale yellow oil. C22H27NO2 (337.4). MS (EI):
m/z ¼ 306 [Mþ ꢀ OCH3], 246 [Mþ ꢀ CH2Ph]. IR (neat):
n
(cmꢀ1) ¼ 2923 (C–H), 1077 (C–O). 1H NMR (CDC13):
(ppm) ¼ 1.53–
d
1.74 (m, 4H, CH2CH2CH2N), 1.90–2.17 (m, 2H, CH2CH2CH2N), 2.21–
2.26 (m,1H, NCH2CH2Ph), 2.43–2.52 (m,1H, NCH2CH2Ph), 2.60–2.69
(m, 1H, NCH2CH2Ph), 2.77–2.99 (m, 3H, ArCH2CH, NCH2CH2Ph),
3.00–3.13 (m, 2H, (CH2)3NCH2), 3.50 (s, 3H, OCH3), 4.93 (t, J ¼ 3.5 Hz,
1H, ArCH2CH), 7.01–7.27 (m, 9H, arom). Anal. calcd. for C22H27NO2: C
78.30 H 8.06 N 4.15 found C 78.55 H 8.00 N 3.84.
[Mþ ꢀ (CH2)3Ph]. IR (neat):
n
(cmꢀ1) ¼ 2927 (C–H), 1076 (C–O). 1H
NMR (CDC13):
d
(ppm) ¼ 1.45–1.63 (m, 6H, CH2(CH2)2CH2Ph,
CH2CH2CH2N), 1.69–1.78 (m, 1H, CH2CH2CH2N), 1.89–1.97 (m, 3H,
CH2CH2CH2N), 2.22–2.40 (m, 2H, CH2(CH2)3Ph), 2.50–2.58 (m, 2H,
(CH2)3CH2Ph), 2.78–2.98 (m, 4H, ArCH2CH, (CH2)3NCH2), 3.40 (s, 3H,
OCH3), 5.00–5.06 (m, 1H, ArCH2CH), 7.04–7.20 (m, 9H, arom). Anal.
calcd. for C24H31NO2: C 78.86 H 8.55 N 3.83 found C 78.54 H 8.94 N
3.64.
6.6. cis-3-Methoxy-10-(3-phenylpropyl)-3,4-
dihydrospiro[[2]benzopyran-1,30-piperidine] (cis-2d) and trans-3-
methoxy-10-(3-phenylpropyl)-3,4-dihydrospiro[[2]benzopyran-1,30-
piperidine] (trans-2d)
Method 3 (trans-2e): As described in Method 2 the diastereomer
trans-2b (70.0 mg, 0.30 mmol) was reacted with 1-chloro-4-phenyl-
butane (69.0 mg, 0.41 mmol), the mixture was worked up and the
product was isolated by fc (1 cm, petroleum ether/ethyl acetate 7:3,
5 mL, Rf ¼ 0.13). Pale yellow oil (trans-2e), yield 28 mg (26%). C24H31NO2
(365.4). MS (EI): m/z ¼ 365 [Mþ], 334 [Mþ ꢀ OCH3], 246
1-Bromo-3-phenylpropane (126 mg, 0.63 mmol) and K2CO3
(554 mg, 4.01 mmol) were added to a mixture of the secondary
amine 2b (135 mg, 0.58 mmol) in acetonitrile (12 mL). The mixture
was heated to reflux for 5 d. Then it was filtered through Celite,
concentrated under reduced pressure and the residue was purified
by fc (2 cm, petroleum ether/ethyl acetate 8:2, 10 mL). Due to
strong tailing only small amounts of the pure diastereomers were
obtained after several chromatographic runs. Total yield 166 mg
(82%).
[Mþ ꢀ (CH2)3Ph]. IR (neat):
n
(cmꢀ1) ¼ 2925 (C–H),1077 (C–O). 1H NMR
(CDC13):
d
(ppm) ¼ 1.64–1.70 (m, 6H, CH2(CH2)2CH2Ph, CH2CH2CH2N).
1.88–1.91 (m,1H, CH2CH2CH2N), 2.07–2,19 (m, 3H, CH2CH2CH2N), 2.42–
2.58 (m, 2H, CH2(CH2)3Ph), 2.71–2.75 (m, 2H, (CH2)3CH2Ph), 2.99–3.15
(m, 4H, ArCH2CH, (CH2)3NCH2), 3.64 (s, 3H, OCH3), 5.08–5.11 (m, 1H,
ArCH2CH), 7.23–7.44 (m, 9H, arom). Anal. calcd. for C24H31NO2: C 78.86
H 8.55 N 3.83 found C 78.20 H 8.72 N 3.86.
cis-2d (Rf ¼ 0.30): Pale yellow oil. C23H29NO2 (351.3). MS (EI):
m/z ¼ 351 [Mþ], 320 [Mþ ꢀ OCH3], 246 [Mþ ꢀ (CH2)2Ph]. IR
(neat):
(CDC13):
n
(cmꢀ1) ¼ 2943 (C–H), 1077, 1033 (C–O). 1H NMR
(ppm) ¼ 1.52–1.63 (m, 1H, CH2CH2CH2N), 1.72–1.76
d
6.8. cis-10-Butyl-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,30-
piperidinej (cis-2f) and trans-10-butyl-3-methoxy-3,4-
dihydrospiro[[2]benzopyran-1,30-piperidinej (trans-2f)
(m, 3H, CH2CH2CH2N (1 H), CH2CH2Ph (2 H)), 1.85–2.20 (m, 3H,
CH2CH2CH2N), 2.08–2.17 (m, 1H, CH2CH2CH2N), 2.21–2.28
(m, 1H, NCH2(CH2)2Ph), 2.37–2.45 (m, 1H, NCH2(CH2)2Ph), 2.53
(t, J ¼ 7.8 Hz, 2H, CH2CH2Ph), 2.78–2.98 (m, 4H, ArCH2CH,
(CH2)3NCH2), 3.42 (s, 3H, OCH3), 5.00 (t, J ¼ 4.1 Hz, 1H, ArCH2CH),
7.02–7.19 (m, 9H, arom). Anal. calcd. for C23H29NO2: C 78.59 H
8.32 N 3.99 found C 78.10 H 8.39 N 3.75.
1-Bromobutane (108 mg, 0.79 mmol) and K2CO3 (482 mg,
3.49 mmol) were added to a mixture of the secondary amine 2b
(119 mg, 0.51 mmol) in acetonitrile (13 mL). The mixture was
heated to reflux for 30 h. After filtration through Celite the solvent
was removed in vacuo and the residue was purified by fc (2 cm,
petroleum ether/ethyl acetate 7:3,10 mL). Due to strong tailing only
small amounts of the pure diastereomers were obtained after
several chromatographic runs. Total yield 129 mg (77%).
trans-2d (Rf ¼ 0.23): Pale yellow oil. C23H29NO2 (351.3). MS (EI): m/
z ¼ 351 [Mþ], 320 [Mþ ꢀ OCH3], 246 [Mþ ꢀ (CH2)2Ph]. IR (neat):
n
(cmꢀ1) ¼ 2923 (C–H), 1077. (C–O). 1H NMR (CDCI3):
d
(ppm) ¼ 1.53–
1.58 (m, 2H, CH2CH2CH2Ph),1.66–1.70 (m, 2H, CH2CH2CH2N),1.78–1.83
(m, 2H, CH2CH2CH2N), 1.90–2.03 (m, 2H, CH2CH2CH2N), 2.27–2.80 (m,
2H, NCH2(CH2)2Ph), 2.52 (t, J ¼ 7.2 Hz, 2H, (CH2)2CH2Ph), 2.80–2.96 (m,
4H, ArCH2CH, (CH2)3NCH2), 3.43 (s, 3H, OCH3), 4.88 (t, J ¼ 3.9 Hz, 1H,
ArCH2CH), 6.98–7.21 (m, 9H, arom). Anal. calcd. for C23H29NO2: C 78.59
H 8.32 N 3.99 found C 78.31 H 8.27 N 3.81.
cis-2f (Rf ¼ 0.40): Pale yellow oil. C18H27NO2 (289.3). MS (EI): m/
z ¼ 289 [Mþ], 258 [Mþ ꢀ OCH3], 246 [Mþ ꢀ (CH2)2CH3]. IR (neat):
n
(cmꢀ1) ¼ 2928 (C–H), 1077 (C–O). 1H NMR (CDCI3):
d
(ppm) ¼ 0.88
(t, J ¼ 7.4 Hz, 3H, (CH2)3CH3), 1.30 (sext, J ¼ 7.4 Hz, 2H,
(CH2)2CH2CH3), 1.42–1.50 (m, 2H, CH2CH2CH2CH3), 1.61–1.68 (m,
1H, CH2CH2CH2N), 1.77–1.83 (m, 1H, CH2CH2CH2N), 1.95–2.08 (m,
6.7. cis-3-Methoxy-10-(4-phenylbutyl)-3,4-
3H,
CH2CH2CH2N),
2.12–2.30
(m,
2H,
CH2CH2CH2N,
dihydrospiro[[2]benzopyran-1,30-piperidine] (cis-2e) and trans-3-
methoxy-10-(4-phenylbutyl)-3,4-dihydrospiro[[2]benzopyran-1,30-
piperidine] (trans-2e)
NCH2(CH2)2CH3), 2.38–2.46 (m, 1H, NCH2(CH2)2CH3), 2.85–3.06 (m,
4H, (CH2)3NCH2, ArCH2CH), 3.50 (s, 3H, OCH3), 5.08 (t. J ¼ 3.9 Hz,1H,
ArCH2CH), 7.07–7.19 (m, 4H, arom). Anal. calcd. for C18H27NO2: C
74.70 H 9.40 N 4.84 found C 74.48 H 9.47 N 4.71.
Method 1: 1-Chloro-4-phenylbutane (53.8 mg, 0.32 mmol) and
K2CO3 (243 mg, 1.76 mmol) were added to a solution of the
secondary amine 2b (59.3 mg, 0.25 mmol) in acetonitrile (8 mL)
and the mixture was heated to reflux for 40 h. It was filtered
through Celite, concentrated in vacuo and the residue was purified
by fc (2 cm, petroleum ether/ethyl acetate 7:3, 10 mL). Due to
tailing, a fc separation of the diastereomers cis-2e and trans-2e was
not possible. Pale yellow oil, total yield 33 mg (36%).
trans-2f: Rf ¼ 0.30): Pale yellow oil. C18H27NO2 (289.3). MS (EI):
m/z ¼ 289 [Mþ], 258 [Mþ ꢀ OCH3], 246 [Mþ ꢀ (CH2)2CH3]. IR (neat):
n
(cmꢀ1) ¼ 2930 (C–H), 1078 (C–O). 1H NMR (CDCl3):
(ppm) ¼ 0.89 (t,
d
J ¼ 7.4 Hz, 3H, (CH2)3CH3), 1.29 (sext, J ¼ 7.4 Hz, 2H, (CH7)2CH2CH3),
1.50 (quint, J ¼ 7.4 Hz, 2H, CH2CH2CH2CH3), 1.57–1.66 (m, 2H,
CH2CH7CH2N), 1.94–1.97 (m, 1 H. CH2CH2CH2N), 1.97–2.10 (m, 2H,
CH2CH2CH2N), 2.31–2.45 (m, 3H, CH2CH,CH2N, NCH2(CH2)2CH3),
2.91–3.01 (m, 4H, (CH2)3NCH2, ArCH2CH), 3.54 (s, 3H, OCH3), 4.98 (t,

4312
A. Jasper et al. / European Journal of Medicinal Chemistry 44 (2009) 4306–4314
J ¼ 3.9 Hz, 1H, ArCH2CH), 7.09–7.31 (m, 4H, arom). Anal. calcd. for
the pure diastereomers were obtained after several chromato-
graphic runs. Total yield 183 mg (95%).
C18H27NO2: C 74.70 H 9.40 N 4.84 found C 75.10 H 9.60 N 4.60.
cis-2h (Rf ¼ 0.24): Pale yellow oil. C22H35NO2 (345.5). MS (EI): m/
z ¼ 345 [Mþ], 314 [Mþ ꢀ OCH3], 246 [Mþ ꢀ (CH2)6CH3]. IR (neat):
6.9. cis-10-Hexyl-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,30-
piperidine] (cis-2g) and trans-10-hexyl-3-methoxy-3,4.-
dihydrospiro[[2]benzopyran-1,30-piperidine] (trans-2g)
n
d
(cmꢀ1) ¼ 2924 (C–H), 1078 (C–O). 1H NMR (CDC13):
(ppm) ¼ 0.84–0.88 (m, 3H, (CH2)7CH3), 1.21–1.30 (m, 10H,
CH2CH2(CH2)5CH3), 1.42–1.53 (m, 2H, CH2CH2(dH2)5CH3), 1.62–1.68
(m, 1H, CH2CH2CH2N), 1.76–1.84 (m, 1H, CH2CH2CH2N), 1.95–2.10
(m, 3H, CH2CH2CH2N), 2.16–2.30 (m, 2H, CH2CH2CH2N,
NCH2(CH2)6CH3), 2.39–2.48 (m, 1H, NCH2(CH2)6CH3), 2.85–3.06 (m,
4H, (CH2)3NCH2, ArCH2CH), 3.49 (s, 3H, OCH3), 5.08 (t, J ¼ 3.9 Hz,
1H, ArCH2CH), 7.10–7.26 (m, 4H, arom). Anal. calcd. for C22H35NO2:
C 76.48 H 10.21 N 4.05 found C 76.55 H 10.17 N 3.88.
Method 1: The secondary amine 2b (91.3 mg, 0.39 mmol) and 1-
chlorohexane (63.7 mg, 0.53 mmol) were dissolved in acetonitrile
(9 mL), K2CO3 (334 mg, 2.42 mmol) was added and the mixture was
heated to reflux for 30 h. After filtration through Celite the solvent
was removed in vacuo and the residue was purified by fc (2 cm,
petroleum ether/ethyl acetate 8:2, 10 mL). Pale yellow oil, total
yield 28 mg (23%).
trans-2h (Rf ¼ 0.14): Pale yellow oil. C22H35NO2 (345.5). MS (EI):
m/z ¼ 345 [Mþ], 314 [Mþ ꢀ OCH3], 246 [Mþ ꢀ (CH2)6CH3]. IR (neat):
Method 2: 1-Chlorohexane (183 mg, 1.52 mmol) and NaI
(271 mg, 1.81 mmol) were dissolved in acetone (3 mL),the solution
was filled into a 10 mL-microwave pressure vial and irradiated with
microwaves (program: standard; max. power 180 W; max. pressure
2 bar; temperature 80 ꢁC; time program: 5 min ramp time; 10 min
hold time; 5 min cool off time). The precipitated NaCl was removed
by filtration and the filtrate (1-iodohexane in acetone) was used for
further reaction. The secondary amine 2b (230 mg, 0.99 mmol) and
K2CO3 (1.10 g. 8.0 mmol) along with acetone (1 mL) were added to
the solution of the produced 1-iodohexane. The mixture was irra-
diated with microwaves (program: standard; max. power 180 W;
max. pressure 5 bar; temperature 100 ꢁC; time program: 5 min
ramp time; 40 min hold time; 5 min cool off time). After filtration
through Celite and washing with CH2C12 the solvent was removed
under reduced pressure and the residue was purified by fc (2 cm,
petroleum ether/ethyl acetate 8:2, 10 mL). Due to tailing only small
amounts of the pure diastereomers were isolated after several
chromatographic runs. Total yield 276 mg (88%).
n
d
(cmꢀ1) ¼ 2923 (C–H), 1079 (C–O). 1H NMR (CDC13):
(ppm) ¼ 0.85–0.89 (m, 3H, (CH2)7CH3), 1.21–1.30 (m, 10H,
CH2CH2(CH2)5CH3), 1.45–1.53 (m, 2H, CH2CH2(CH2)5CH3), 1.57–1.63
(m, 2H, CH2CH2CH2N), 1.92–2.12 (m, 3H, CH2CH2CH2N), 2.32–2.42
(m, 3H, CH2CH2CH2N, NCH2(CH2)6CH3), 2.87–3.02 (m, 4H,
(CH2)3NCH2, ArCH2CH), 3.54 (s, 3H, OCH3), 4.98 (t, J ¼ 3.5 Hz, 1H,
ArCH2CH), 7.08–7.31 (m, 4H, arom). Anal. calcd. for C22H35NO2: C
76.48 H 10.21 N 4.05 found C 76.52 H 10.48 N 4.24.
6.11. cis-10-Decyl-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,30-
piperidine] (cis-2i) and trans-10-decyl-3-methoxy-3,4-
dihydrospiro[[2]benzopyran-1,30-piperidine] (trans-2i)
1-Bromodecane (156 mg, 0.71 mmol) and K2CO3 (486 mg,
3.52 mmol) were added to a solution of the secondary amine 2b
(127 mg. 0.55 mmol) in acetonitrile (14 mL). The reaction mixture
was heated to reflux for 26 h. Then it was filtered through Celite,
the solvent was removed in vacuo and the residue was purified by
fc (3 cm, petroleum ether/ethyl acetate 9:1, 20 mL). Due to tailing
only small amounts of the pure diastereomers were obtained after
several chromatographic runs. Total yield 173 mg (85%).
cis-2g (Rf ¼ 0.28): Pale yellow oil. C20H31NO2 (317.5). MS (EI): m/
z ¼ 317 [Mþ], 286 [Mþ ꢀ OCH3], 246 [Mþ ꢀ (CH2)4 CH3}. IR (neat):
n
(cmꢀ1) ¼ 2925 (C–H), 1077 (C–O). 1H NMR (CDC13):
d
(ppm) ¼ 0.84–0.87 (m. 3H, (CH2)5CH3), 1.20–1.32 (m, 6H,
cis-2i (Rf ¼ 0.21): Pale yellow oil. C24H39NO2 (373.6). MS (EI): m/
CH2CH2(CH2)3CH3). 1.42–1.51 (m, 2H, CH2CH2(CH2)3CH3), 1.63–
1.66 (m, 1H, CH2CH2CH2N), 1.76–1.84 (m, 1H, CH2CH2CH2N),
1.95 ꢀ 2.10 (m, 3H, CH2CH2CH2N), 2.15–2.29 (m, 2H, CH2CH2CH2N,
NCH2(CH2)4CH3), 2.39–2.47 (m, 1H, NCH2(CH2)4CH3), 2.85–3.06
(m, 4H, (CH2)3NCH2, ArCH2CH), 3.49 (s, 3H, OCH3), 5.08 (t,
J ¼ 3.9 Hz. 1H, ArCH2CH), 7.09–7.21 (m, 4H, arom). Anal. calcd. for
C20H31NO2: C 75.67 H 9.84 N 4.41 found C 75.71 H 9.93 N 4.28.
trans-2g (Rf ¼ 0.23): Pale yellow oil. C20H31NO2 (317.5). MS (EI):
m/z ¼ 317 [Mþ], 286 [Mþ ꢀ OCH3], 246 [Mþ ꢀ (CH2)4CH3]. IR (neat):
z ¼ 373 [Mþ], 342 [Mþ ꢀ OCH3], 246 [Mþ ꢀ (CH2)8CH3]. IR (neat):
n
d
(cmꢀ1) ¼ 2923 (C–H), 1078 (C–O). 1H NMR (CDCl3):
(ppm) ¼ 0.85–0.89 (t, J ¼ 7.0 Hz, 3H, (CH2)9CH3),1.19–1.25 (m,14H,
CH2CH2(CH2)7CH3), 1.42–1.51 (m, 2H, CH2CH2(CH2)7CH3), 1.76–1.84
(m, 1H, CH2CH2CH2N), 1.76–1.84 (m, 1H, CH2CH2CH2N), 1.94–2.07
(m, 3H, CH2CH2CH2N), 2.15–2.28 (m, 2H, CH2CH2CH2N,
NCH2(CH2)8CH3), 2.37–2.45 (m, 1H, NCH2(CH2)8CH3), 2.85–3.06 (m,
4 H. (CH2)3NCH2, ArCH2CH), 3.49 (s, 3H, OCH3), 5.08 (t, J ¼ 3.9 Hz,
1H, ArCH2CH), 7.09–7.22 (m. 4H, arom). Anal. calcd. for C24H39NO2:
C 77.16 H 10.52 N 3.75 found C 76.70 H 10.63 N 3.50.
n
d
(cmꢀ1) ¼ 2926 (C–H), 1078 (C–O). 1H NMR (CDC13):
(ppm) ¼ 0.78–0.84 (m, 3H, (CH2)5CH3), 1.15–1.24 (m, 6H,
trans-2i (Rf ¼ 0.14): Pale yellow oil. C24H39NO2 (373.6). MS (EI):
CH2CH2(CH2)3CH3), 1.39–1.47 (m, 2H, CH2CH2(CH2)3CH3), 1.50–1.59
(m, 2H, CH2CH2CH2N), 1.84–2.05 (m, 3H, CH2CH2CH2N), 2.21–2.35
(m, 3H, CH2CH2CH2N, NCH2(CH2)4CH3), 2.84–2.92 (m. 4H,
(CH2)3NCH2, ArCH2CH), 3.46 (s, 3H, OCH3), 493 (t, J ¼ 3.5 Hz, 1H,
ArCH2CH), 7.02–7.23 (m, 4H, arom). Anal. calcd. for C20H31NO2: C
75.67 H 9.84 N 4.41 found C 75.41 H 10.00 N 4.41.
m/z ¼ 373 [Mþ], 342 [Mþ ꢀ OCH3], 246 [Mþ ꢀ (CH2)8CH3]. IR (neat):
n
d
(cmꢀ1) ¼ 2922 (C–H), 1079 (C–O). 1H NMR (CDC13):
(ppm) ¼ 0.85–0.89 (t, J ¼ 7.0 Hz, 3H, (CH2)9CH3), 1.20–1.33 (m,
14H, CH2CH2(CH2)7CH3), 1.46–1.55 (m, 2H, CH2CH2(CH2)7CH3),
1.55–1.65 (m, 2H, CH2CH2CH2N), 1.94–2.12 (m, 3H, CH2CH2CH2N),
2.30–2.42 (m. 3H, CH2CH2CH2N, NCH2(CH2)8CH3), 2.87–3.02 (m,
4H, (CH2)3NCH2, ArCH2CH), 3.54 (s, 3H, OCH3), 4.98 (t, J ¼ 3.5 Hz,1H,
ArCH2CH), 7.09–7.31 (m, 4H, arom). Anal. calcd. for C24H39NO2: C
77.16 H 10.52 N 3.75 found C 76.75 H 10.80 N 3.83.
6.10. cis-3-Methoxy-10-octyl-3,4-dihydrospiro[[2]benzopyran-1,30-
piperidine] (cis-2h) and trans-3-methoxy-10-octyl-3,4-
dihydrospiro[[2]benzopyran-1,30-piperidine] (trans-2h)
6.12. 10-Butyl-3-methoxy-3,4-dihydrospiro [[2]benzopyran-1,40-
1-Bromooctane (137 mg, 0.71 mmol) and K2CO3 (481 mg,
3.48 mmol) were added to a solution of the secondary amine 2b
(136 mg, 0.56 mmol) in acetonitrile (14 mL). The mixture was
heated to reflux for 26 h, then filtered through Celite and concen-
trated in vacuo. The residue was purified by fc (3 cm, petroleum
ether/ethyl acetate 8:2, 20 mL). Due to tailing only small amounts of
piperidine] (1f)
1-Bromobutane (70.1 mg, 0.51 mmol) and K2CO3 (599 mg,
2.57 mmol) were added to a solution of the secondary amine 1b
[17] (100 mg, 0.43 mmol) in acetonitrile (12 mL) and the mixture
was heated to reflux for 30 h. It was filtered through Celite, the

A. Jasper et al. / European Journal of Medicinal Chemistry 44 (2009) 4306–4314
4313
solvent was removed in vacuo and the residue was purified by fc
6.16. Performing of the s1 assay [17,26]
(2 cm, petroleum ether/ethyl acetate 7:3, 5 mL, Rf ¼ 0.31). Pale
yellow oil, yield 46 mg (37%). C18H27NO2 (289.2). MS (EI): m/z ¼ 289
The test was performed with the radioligand [3H]-(þ)-pentaz-
[Mþ], 258 [Mþ ꢀ OCH3], 246 [Mþ ꢀ (CH2)2CH3]. IR (neat):
n
ocine (42.5 Ci/mmol; Perkin Elmer). The thawed membrane prep-
(cmꢀ1) ¼ 2935 (C–H), 1076 (C–O). 1H NMR (CDCl3):
d
(ppm) ¼ 0.94
aration (about 75 mg of the protein) was incubated with various
(t, J ¼ 7.0 Hz, 3H, (CH2)3CH3), 1.38 (sext, J ¼ 7.0 Hz, 2H,
(CH2)2CH2CH3), 1.51–1.58 (m, 2H, CH2CH2CH3), 1.83–2.04 (m, 3H,
N(CH2CH2)2), 2.21 (td, J ¼ 13.3/4.3 Hz, 1H, N(CH2CH2)2), 2.39–2.54
(m, 4H, NCH2(CH2)2CH3, N(CH2CH2)2), 2.83–2.88 (m, 2H,
N(CH2CH2)2), 2.90–2.93 (m, 2H, ArCH2CH), 3.57 (s, 3H, OCH3), 2.87
(dd, J ¼ 3.9/2.7 Hz, 1H, ArCH2CHOCH3), 7.15–7.22 (m, 4H, arom).
Anal. calcd. for C18H27NO2: C 74.70 H 9.40 N 4.84 found C 74.62 H
9.28 N 4.93.
concentrations of test compounds, 2 nM [3H]-(þ)-pentazocine, and
buffer (50 mM TRIS, pH 7.4) in a total volume of 200 mL for 180 min
at 37 ꢁC. The incubation was terminated by rapid filtration through
the presoaked filtermats by using the cell harvester. After washing
each well five times with 300 mL 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 scintillator was allowed
to solidify at rt. The bound radioactivity trapped on the filters was
counted in the scintillation analyzer. The non-specific binding was
determined with 10
m
M unlabeled (þ)-pentazocine. The Kd-value of
6.13. 3-Methoxy-10-octyl-3,4-dihydrospiro [[2]benzopyran-1,40-
piperidine] (1h)
the radioligand [3H]-(þ)-pentazocine is 2.9 nM [25].
6.17. Membrane preparation for the s2 assay [17,26]
1-Bromooctane (121 mg. 0.51 mmol) and K2CO3 (599 mg,
2.57 mmol) were added to a solution of the secondary amine 1b [17]
(100 mg, 0.43 mmol) in acetonitrile (12 mL) and the mixture was
heated to reflux for 30 h. Then the mixture was filtered through
Celite, concentratedinvacuoandthe residuewaspurified byfc(2 cm,
petroleum ether/ethyl acetate 7:3, 5 mL, Rf ¼ 0.22). Pale yellow oil,
yield 101 mg (68%). C22H35NO2 (345.2). MS (EI): m/z ¼ 345 [Mþ], 314
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 1200ꢃ g for
10 min at 4 ꢁC. The supernatant was separated and centrifuged at
31,000ꢃ g 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
incubation, the suspension was centrifuged again at 31,000ꢃ g for
20 min at 4 ꢁC. The final pellet was resuspended in buffer, the
protein concentration was determined according to the method of
Bradford [30] 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.
[Mþ ꢀ OCH3], 246 [Mþ ꢀ (CH2)6CH3]. IR (neat):
n
(cmꢀ1) ¼ 2935
(C–H). 1075 (C–O). 1H NMR (CDCl3):
d
(ppm) ¼ 0.89 (t, J ¼ 6.1 Hz, 3H,
(CH2)7CH3), 1.23–1.32 (m, 10H, (CH2)5CH3), 1.53–1.57 (m, 2H,
CH2CH2(CH2)5CH3),1.84–2.00 (m, 3H, N(CH2CH2)2), 2.21 (td, J ¼ 12.9/
5.7 Hz, 1H, N(CH2CH2)2), 2.39–2.54 (m.
4 H. NCH2(CH2)6CH3,
N(CH2CH2)2), 2.82–2.85 (m, 2H, N(CH2CH2)2), 2.90–2.93 (m, 2H,
ArCH2CH), 3.57 (s, 3H, OCH3), 2.87 (dd, J ¼ 3.9/2.7 Hz, 1H, ArCH2
-
CHOCH3), 7.15–7.22 (m, 4H, arom). Anal. calcd. for C22H35NO2: C 76.47
6.18. Performing of the s2 assay [17,26]
H 10.21 N 4.05 found C 76.59 H 9.97 N 4.22.
The test was performed with the radioligand [3H]-di-o-tol-
ylguanidine (50 Ci/mmol; ARC). The thawed membrane prepara-
6.14. Receptor binding studies, materials and general procedures
tion (about 100 mg of the protein) was incubated with various
concentrations of test compounds, 3 nM [3H]-di-o-tolylguanidine,
500 nM (þ)-pentazocine and buffer (50 mM TRIS, pH 8.0) in a total
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 solid scintillator was melted on the
filtermat at a temperature of 95 ꢁC for 5 min. After solidification of
the scintillator at rt, the scintillation was measured using
a MicroBeta Trilux scintillation analyzer (Perkin Elmer). The
counting efficiency was 20%.
volume of 200 mL for 180 min at r.t. The incubation was terminated
by rapid filtration through the presoaked filtermats using a cell
harvester. After washing each well five times with 300 L of water,
m
the filtermats were dried at 95 ꢁC. Subsequently, the solid scintil-
lator was put on the filtermat and melted at 95 ꢁC. After 5 min, the
solid scintillator was allowed to solidify at rt. The bound radioac-
tivity trapped on the filters was counted in the scintillation
analyzer. The non-specific binding was determined with 10 mM
unlabeled ditolylguanidine. The Kd-value of the radioligand [3H]-
ditolylguanidine is 17.9 nM [27].
6.19. Data analysis
6.15. Membrane preparation for the s1 assay [17,26]
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 regression
analysis. The Ki values were calculated according to Cheng and
Prusoff [31]. For potent compounds the Ki values are given as mean
values þ SEM from three independent experiments. For
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 sucrose. The suspension was centrifuged at 1200ꢃ g for
10 min at 4 ꢁC. The supernatant was separated and centrifuged at
23,500ꢃ g for 20 min at 4 ꢁC. The pellet was resuspended in 5-6
volumes of buffer (50 mM TRIS, pH 7.4) and centrifuged again at
23,500ꢃ g (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 Brad-
ford [30] using bovine serum albumin as standard, and subse-
quently the preparation was frozen (-80 ꢁC) in 1.5 mL portions
containing about 1.5 mg protein/mL.
compounds with low affinity (Ki value > 1 mM) the Ki value is
recorded only once. When the competition curve does not provide
a clear correlation between the test compound concentration and
the inhibition of radioligand binding only the inhibition at a test
compound concentration of 1 mM is given.

4314
A. Jasper et al. / European Journal of Medicinal Chemistry 44 (2009) 4306–4314
[12] A. Urani, P. Romieu, F. Roman, K. Yamada, Y. Noda, et al., Eur. J. Pharmacol. 486
Acknowledgement
(2004) 15–16.
[13] A. Marrazzo, F. Caraci, E.T. Salinaro, T.P. Su, A. Copani, et al., Neuroreport 16
(2005) 1223–1226.
[14] C.S. John, B.B. Lim, B.C. Geyer, B.J. Vilner, W.D. Bowen, Bioconjug. Chem. 8
(1997) 304–309.
[15] B.J. Vilner, C.S. John, W.D. Bowen, Cancer Res. 55 (1995) 408–413.
[16] V. Caveliers, H. Everaert, C.S. John, T. Lahoutte, A. Bossuyt, J. Nucl. Med. 43
(2002) 1647–1649.
Financial support of this project by the Deutsche For-
schungsgemeinschaft is gratefully acknowledged. Thanks are also
due to the Degussa AG, Janssen-Cilag GmbH, and Bristol-Myers
Squibb for donation of chemicals and reference compounds.
[17] C.A. Maier, B. Wu¨nsch, J. Med. Chem. 45 (2002) 438–448.
[18] B. Wu¨ nsch, Arch. Pharm. 323 (1990) 493–499.
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[20] B. Macchia, M. Macchia, C. Manera, E. Martinotti, S. Nencetti, et al., Eur. J. Med.
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