Microwave assisted synthesis of spirocyclic pyrrolidines - σ1 receptor ligands with modified benzene-N-distance

Article abstract of DOI:10.1016/j.ejmech.2012.04.018

Two series of σ₁ ligands with a spiro[[2]benzopyran-1, 3′-pyrrolidine] (3) and a spiro[[2]benzofuran-1,3′-pyrrolidine] (4) framework were synthesized and pharmacologically evaluated. Several reaction steps were considerably improved by microwave irradiation. The σ₁ affinity of the spirocyclic ligands correlates nicely with the benzene-N-distance, i.e. 2 < 3 < 4 < 1. The σ₁ affinity of both compound classes could be increased with large N-substituents (e.g. 2-phenylethyl, octyl). Nevertheless the benzyl derivative 4a represents the most promising σ₁ ligand (K_i = 25 nM) due to its high selectivity against the σ₂ subtype (>40-fold), the NMDA receptor and 5-HT₆ and 5-HT₇ receptors. Moreover, 4a did not inhibit the hERG channel in the heart.

Full text of DOI:10.1016/j.ejmech.2012.04.018

European Journal of Medicinal Chemistry 53 (2012) 327e336
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Microwave assisted synthesis of spirocyclic pyrrolidines e s1 receptor ligands
with modified benzene-N-distance
Annemarie Jasper a, Dirk Schepmann a, Kirstin Lehmkuhl a, Jose Miguel Vela b, Helmut Buschmann b,
,
Jörg Holenz b, Bernhard Wünsch a
*
a Institut für Pharmazeutische und Medizinische Chemie, Hittorfstraße 58-62, 48145 Münster, 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
Two series of s1 ligands with a spiro[[2]benzopyran-1,30-pyrrolidine] (3) and a spiro[[2]benzofuran-
1,30-pyrrolidine] (4) framework were synthesized and pharmacologically evaluated. Several reaction
steps were considerably improved by microwave irradiation. The s1 affinity of the spirocyclic ligands
correlates nicely with the benzene-N-distance, i.e. 2 < 3 < 4 < 1. The s1 affinity of both compound classes
could be increased with large N-substituents (e.g. 2-phenylethyl, octyl). Nevertheless the benzyl deriv-
ative 4a represents the most promising s1 ligand (Ki ¼ 25 nM) due to its high selectivity against the s2
subtype (>40-fold), the NMDA receptor and 5-HT6 and 5-HT7 receptors. Moreover, 4a did not inhibit the
hERG channel in the heart.
Article history:
Received 13 January 2012
Received in revised form
12 April 2012
Accepted 13 April 2012
Available online 21 April 2012
Keywords:
s
Receptor ligands
Ó 2012 Elsevier Masson SAS. All rights reserved.
Spirocyclic pyrrolidines
Microwave irradiation
Receptor binding studies
Structure affinity relationships
hERG channel
1. Introduction
The unique structure of the s1 receptor protein renders the
discovery of the mechanism of signal transduction rather difficult.
After the first postulation of
subtype [1], it has been shown that
s
receptors as opioid receptor
It has been shown that the s1 receptor is involved in the modula-
tion of some neurotransmitter systems including the cholinergic,
dopaminergic and glutamatergic neurotransmission [11]. However,
more importantly the s1 receptor is able to regulate some ion
channels (e.g. Naþ, Kþ, and Ca2þ-channels) [12e15].
s receptors represent a unique,
non-opioid, non-phencyclidine but haloperidol sensitive receptor
family. Receptors have a characteristic distribution in the central
s
nervous system but they are also found in some organs and tissues
in the periphery including lung, liver, kidney and heart. Today two
subtypes are known, which are termed s1 and s2 receptor [2e4].
The amino acid sequence of the s1 receptor subtype was
determined by successful cloning [5e9]. According to the amino
acid sequence (223 amino acids, molecular weight 25.3 kDa), the
membrane-bound s1 receptor containing two transmembrane
domains represents a unique receptor type differing from classical
G-protein coupled receptors, ion channel receptors and tyrosine
kinase receptors. Whereas a similarity to a mammalian protein
could not be detected, a 30% homology to the yeast enzyme sterol-
s1 Receptor ligands are considered as promising drug candidates
for the treatment of various neurological and psychiatric disorders
[15e19]. There is a particular potential of s1 ligands for the treat-
ment of depression [20e22], anxiety [17], memory disorders [23],
Alzheimer’s disease as well as alcohol and cocaine abuse [16,17].
Chen and coworkers have described the s1 system as an endoge-
nous anti-opioid system. The well-known s1 agonist (þ)-pentazo-
cine is able to antagonize m-, k-, and d-opioid receptor mediated
analgesia, whereas s1 antagonists enhance opioid induced anti-
nociception [24]. Furthermore, opioid mediated analgesia was
potentiated by down regulation of s1 receptors [18]. Therefore s1
antagonists possess a high potential as innovative analgesics with
D8 D7 isomerase was found [6]. The s2 receptor subtype is less
/
characterized, it has not been cloned yet, but its molecular weight is
approximately 21.5 kDa [10].
reduced side effects. The development of novel
s ligand-based
antitumor drugs and tumor diagnostics is stimulated by the high
density of s1 (and s2 receptors) in some human tumor cell lines (e.g.
breast, lung, prostate cancer cells) [25,26].
* Corresponding author. Tel.: þ49 251 83 33311; fax: þ49 251 83 32144.
E-mail address: wuensch@uni-muenster.de (B. Wünsch).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.018

328
A. Jasper et al. / European Journal of Medicinal Chemistry 53 (2012) 327e336
Since we are interested in conformationally restricted receptor
benzofuran 4a (R ¼ Bn) this distance is slightly increased to
4.8e5.1 Å (Fig. 2).
ligands, we have developed potent s1 ligands with a spirocyclic
framework [27e41]. Compounds of type 1 containing a spi-
rocyclic piperidine moiety represent a typical example of this
class of s1 ligands (Fig. 1). As an example the N-benzyl derivative
1a (R ¼ Bn) displays a s1 affinity in the low nanomolar range
(Ki ¼ 1.3 nM) [27]. Recently we have shown that shifting of the N-
atom to the adjacent position (2) led to 1000-fold decrease of s1
affinity (2a, R ¼ Bn, Ki > 1400 nM) [30]. The reduced s1 affinity
was explained with a reduced distance between the benzene ring
of the benzopyran ring and the basic N-atom (benzene-N-
distance).
In Fig. 2 the piperidine chair conformations of the spirocyclic
piperidine 1a are shown. Since the energies are very similar, both
conformations 1ae1 and 1ae2 have to be taken into account. The
benzene-N-distance of conformer 1ae1 bearing the phenyl
moiety in equatorial orientation of the piperidine ring amounts
5.7 Å. The corresponding conformer 1ae2 with axial position of
the phenyl moiety shows a considerably shorter distance of 5.1 Å.
A similar situation is observed for the regioisomeric spirocyclic
piperidine 2a. In conformer 2ae1 with equatorially oriented
phenyl ring the benzene-N-distance is about 0.5 Å longer than in
conformer 2ae2.
A comparison of 1a and 2a clearly demonstrates that the
benzene-N-distance of 2a (4.4e4.9 Å) is considerably shorter than
the benzene-N-distance of 1a (5.1e5.7 Å). The explanation of the
reduced s1 affinity of 2a (Ki > 1400 nM) with the reduced benzene-
N-distance is in good accordance with established s1 pharmaco-
phore models [41e46]. In order to learn more about the crucial
benzene-N-distance in this type of spirocyclic s1 ligands, replace-
ment of the piperidine moiety of 1 and/or 2 with a pyrrolidine
ring (compounds 3 and 4 in Fig. 1) was envisaged. Since the
five-membered pyrrolidine ring is more flexible than the piperidine
ring, several energetically favored conformations have to be
considered. Analysis of the conformations of benzopyran 3a
(R ¼ Bn) led to benzene-N-distance in the range of 4.6e5.0 Å. In the
2. Chemistry
Exchange of the bromine atom of the bromoacetal 5 [47]
with n-BuLi at ꢀ78 ꢁC followed by trapping of the resulting
aryllithium intermediate with 1-benzylpyrrolidin-3-one provided
the hydroxyacetal 6 in 33% isolated yield. Treatment of the
hydroxyacetal 6 with 1.2 equivalents of p-toluenesulfonic acid in
methanol led to cyclization via intramolecular transacetalization.
Whereas a reaction time of 4 d at room temperature was required
to achieve a yield of 67%, microwave irradiation during the cycli-
zation step afforded a yield of 73% within 15 min. However, the best
yield over two steps (41%) was obtained by consecutive conversions
without isolation of the unstable intermediate 6 (Scheme 1).
The N-benzyl moiety of 3a was removed by a transfer hydro-
genolysis using ammonium formate and Pd/C [48]. The resulting
secondary amine 3b was alkylated with various arylalkyl and alkyl
halides to obtain the tertiary amines 3cef. Microwave irradiation
reduced the reaction times of the transformations and increased
the yields of the products 3cef. The spirocyclic pyrrolidines 3 were
formed as 1:1-mixtures of cis- and trans-configured diastereomers.
The stereodescriptors cis/trans refer to the orientation of the OCH3
group and the NeCH2emoiety on the same side/on opposite sides
of the benzopyran plane. For the present the compounds were
tested as 1:1-mixtures.
InordertoinvestigatetheeffectofaringcontractedO-heterocycle
on the s1 receptor affinity, the corresponding spirocyclic benzofuran
derivatives 4 were synthesized in the same manner starting
with 2-bromobenzaldeyhde acetal 7 (Scheme 2). Halogen-metal
exchange followed by reaction with 1-benzylpyrrolidin-3-one led
to hydroxyacetal 8 in 51% yield. The acid-catalyzed cyclization of 8
was supported by microwave irradiation providing the spirocyclic
benzofuran 4a in 92%. The N-benzyl substituent of 4a was exchanged
by transfer hydrogenolysis [48] and subsequent alkylation of the
secondary amine 4b. For the introduction of N-substituents
a consecutive two-step procedure was used consisting of a micro-
wave supported Finkelstein reaction of the corresponding arylalkyl
chlorides or alkyl bromides with NaI in acetone and a microwave
assisted alkylation of the secondary amine 4b with the in situ formed
(aryl)alkyl iodides.
OCH3
OCH3
O
O
N
Reaction of the secondary amine 4b with 1,4-dibromobutane led
to a spirocyclic quaternary ammonium ion, which did not react
with further nucleophiles. Therefore, the secondary amine 4b was
reacted with 1-(4-chlorobutyl)imidazole, which was available by
alkylation of imidazole with 1-bromo-4-chlorobutane, under
microwave irradiation to form the imidazolylbutyl derivative 4g in
65% yield.
R
N
R
2
1
As described for the spirocyclic benzopyrans 3 the spirocyclic
benzofurans 4 were produced and tested as 1:1-mixtures of cis-
and trans-configured diastereomers.
OCH3
O
OCH3
3. Pharmacological evaluation
At first the s1 and s2 receptor affinities of the spirocyclic
pyrrolidines 3 and 4 were determined in competition experiments
with radioligands. In the s1 assay homogenates of guinea pig
brains served as receptor material and the s1 selective ligand
[3H]-(þ)-pentazocine as radioligand. The non-specific binding was
determined in the presence of a large excess of non-labeled
(þ)-pentazocine [27,34]. Rat liver membrane preparations were
the source of s2 receptors in the s2 assay. Since a s2 selective radi-
O
N
N
R
R
3
4
oligand is not commercially available, the non-selective
s ligand
Fig. 1. Development of spirocyclic pyrrolidines 3 and 4 from spirocyclic piperidines
with symmetric (1) and unsymmetrical piperidine connection (2).
[3H]-di(o-tolyl)guanidine was employed and the present s1

A. Jasper et al. / European Journal of Medicinal Chemistry 53 (2012) 327e336
329
OCH3
O
N
OCH3
O
N
3.8 Å
5.1 Å
1a-2
5.7 Å
3.8 Å
1a-1
1: Ki = 1.3 nM
OCH3
OCH3
O
O
N
N
3.8 Å
4.4 Å
4.9 Å
3.8 Å
2a-1
2a-2
1: Ki > 1400 nM
OCH3
OCH3
O
O
N
N
4.6-5.0 Å
4.8-5.1 Å
3.8 Å
3.8 Å
3a-1
4a-1
1: Ki = 73 nM
1: Ki = 25 nM
Fig. 2. Comparison of the crucial benzene-N-distances of spirocyclic compounds 1e4 and correlation with their s1 affinities.
receptors were masked with an excess of the non-radiolabeled s1
4. Results and discussion
selective ligand (þ)-pentazocine. Performing of the s2 assay in the
presence of an excess of non-tritiated 1,3-di(o-tolyl)guanidine
provided the non-specific binding of the radioligand [27,34].
4.1. s1 Receptor affinity
Since the structures of
s
and NMDA receptor ligands are some-
In Table 1 the s1 receptor affinities of the spirocyclic pyrrolidines
3 and 4 are given and compared with those of lead and reference
compounds. The spiro[ [2]benzopyran-1,30-pyrrolidine] 3a with an
N-benzyl residue displays a 55-fold reduced s1 affinity (Ki ¼ 73 nM)
compared with the s1 affinity of the lead compound 1a. Increasing
of the length of the N-residue led to the 2-phenylethyl derivative 3c
with increased s1 affinity (Ki ¼ 25 nM). A further enlargement of
the chain length of the N-residue reduced the s1 affinity to 53 nM
(3d). Replacement of the N-benzyl residue of 3a with the aliphatic
butyl moiety (3e) slightly reduced the s1 affinity. A similar trend
was observed after replacement of the N-benzyl moiety of the lead
compound 1a with an N-butyl residue (1b, Ki ¼ 2.3 nM). The most
times very similar, the affinity of the spirocyclic pyrrolidines 3 and 4
toward the phencyclidine binding site of the NMDA receptor was
recorded with pig brain cortex membrane preparations and [3H]-
(þ)-MK-801 as radioligand [49]. Furthermore, the affinities of 3 and
4 toward the serotonin receptors 5-HT6 and 5-HT7 were recorded.
The hERG (human ether-a-go-go related gene) channel in the
heart represents an antitarget for the development of novel drugs.
In a fluorescence-based assay the blockade of the hERG channel by
the spirocyclic pyrrolidines 3 and 4 was tested [26,50,51]. The
remaining hERG channel activity after incubation with 10
compounds is summarized in Table 1.
mM of test

330
A. Jasper et al. / European Journal of Medicinal Chemistry 53 (2012) 327e336
OCH3
OCH3
OCH3
OCH3
OCH3
(a) or (c)
(b) or (c)
(d)
O
OH
O
Br
N
N
N
Bn
Bn
Bn
5
3a
6
OCH3
OCH3
3
R
(e)
O
c
d
e
f
(CH2)2Ph
(CH2)4Ph
n-Bu
O
N
N
H
n-Octyl
R
3b
3c-f
Scheme 1. Synthesis of spiro[[2]benzopyran-1,30-pyrrolidines] 3. Reagents and reaction conditions: (a) n-BuLi, THF, ꢀ78 ꢁC, 2 h, then rt, 16 h, 33%. (b) p-TolSO3H 1.2 equiv., H3COH,
rt, 4 d, 67%; microwave irradiation 73%. (c) n-BuLi, THF, ꢀ78 ꢁC, 2 h, then rt, 12 h; HCl 5%, rt, 41%. (d) NH4HCO2, Pd/C, CH3OH, 65 ꢁC, 2 h, 82%. (e) ReCl or ReBr, K2CO3, acetonitrile,
microwave irradiation, 45e69%.
potent s1 ligand of this series of compounds is the N-octyl deriva-
tive 3f (Ki ¼ 9.4 nM).
Similar effects of the N-residue on the s1 affinity have
been described for analogous spirocyclic s1 receptor ligands
[27,28,33,34,39].
In Fig. 2 the s1 receptor affinities of the spirocyclic compounds
1ae4a are correlated with the distance of the basic amino group
and the benzene moiety of the benzopyran or benzofuran ring
(benzene-N-distance). The data in Fig. 2 indicate high s1 receptor
A similar relationship between the s1 affinity and the structure
of the N-residues was observed for the spirocyclic benzofuran
derivatives 4, although the level of s1 affinity is considerably higher.
In particular the N-phenylalkyl (4a, 4c, 4d) and the N-octyl deriv-
atives 4f represent very potent s1 receptor ligands interacting in the
low nanomolar range with s1 receptors.
affinity for 1a with
a
long benzene-N-distance (5.1e5.7 Å).
OCH3
OCH3
OH
OCH3
OCH3
(a)
(b)
(c)
OCH3
O
O
Br
N
N
N
Bn
Bn
7
Bn
4a
8
OCH3
O
OCH3
O
4
R
c
d
e
f
(CH2)2Ph
(CH2)4Ph
n-Bu
n-Octyl
(CH2)4imidazolyl
(d) or (e)
N
NH
R
g
4b
4c-g
Scheme 2. Synthesis of spiro[[2]benzofuran-1,30-pyrrolidines] 4. Reagents and reaction conditions: (a) n-BuLi, THF, ꢀ78 ꢁC, 2 h, then rt, 16 h, 51%. (b) p-TolSO3H 1.2 equiv., H3COH,
microwave irradiation, 92%. (c) NH4HCO2, Pd/C, CH3OH, 65 ꢁC, 2 h, 87%. (d) 1. ReCl or ReBr, NaI, acetone, microwave irradiation, filtration; 2. 4b, K2CO3, acetone, microwave
irradiation, 35e75% (4cef). (e) 1-(4-chlorobutyl)imidazole, K2CO3, acetonitrile, microwave irradiation, 65%.

A. Jasper et al. / European Journal of Medicinal Chemistry 53 (2012) 327e336
331
Table 1
assay) and 100 nM (5-HT assays) the inhibition of the radioligand
binding was always lower than 40% indicating rather low affinities
Affinities of the spirocyclic pyrrolidines 3 and 4 toward s1 and s2 receptors and
inhibition of hERG channel activity.
(IC50 > 10
mM for NMDA receptor, IC50 > 100 nM for 5-HT
receptors).
OCH3
( )n
4.3. Inhibition of the hERG channel
O
The hERG (human ether-a-go-go related gene) channel is
a voltage gated Kþ channel in the heart. Blockade of this channel
prolongs the QT interval in the electrocardiogram. Drugs blocking
the hERG channel in the heart increase the probability of ventric-
ular arrhythmia, which may degenerate into ventricular fibrillation
and acute sudden death. Therefore the inhibition of the hERG
channel by new drug candidates should be taken into account at
a very early stage of drug development [52].
N
R
Compd. R
n
s1 Ki ꢂ SEM s2 Ki ꢂ SEM Selectivity s1
/
s2 hERG
(nM)a
(nM)a
(%)b
1a [27] CH2Ph
1b [30] n-Bu
2a [30] CH2Ph
2b [30] n-Bu
e 1.3 ꢂ 0.2
e 2.3 ꢂ 0.8
e >1400
e 1000
3500
1020
2708
443
e
92
87
n.d.
n.d.
93
23
24
98
6
99
99
6
97
25
n.d.
n.d.
n.d.
n.d.
At a concentration of 10 mM only the N-benzyl (3a) and N-butyl
(3e) derivatives of the spirocyclic benzopyran series 3 did not
influence the Kþ-ion permeability through the hERG channel
(Table 1). Compounds with larger N-substituents (3c, 3d, 3f) led to
a reduced permeability of 6e24%.
However, the spirocyclic benzofuran derivatives 4, which are
more potent s1 receptor ligands than the benzopyran derivatives 3,
showed reduced interactions with the hERG channel. With excep-
tion of 4f with the very large N-octyl residue, the spirocyclic
benzofurans 4 did not block the hERG channel permeability at
>1500
>1500
>1000
>1000
428 ꢂ 34
1180
290 ꢂ 30
>1000
>1000
127 ꢂ 38
964
e
3a
3c
3d
3e
3f
4a
4c
4d
4e
4f
CH2Ph
1
1
1
1
1
0
0
0
0
0
73 ꢂ 5.1
25 ꢂ 4.3
53 ꢂ 24
104 ꢂ 30
9.4 ꢂ 3.1
25 ꢂ 3.8
22 ꢂ 4.4
21 ꢂ 13
55 ꢂ 16
8.3 ꢂ 2.6
>14
>40
8
11
31
>40
>45
6
18
10
(CH2)2Ph
(CH2)4Ph
n-Bu
n-Octyl
CH2Ph
(CH2)2Ph
(CH2)4Ph
n-Bu
a concentration of 10 mM.
n-Octyl
84 ꢂ 10
4g
(CH2)4imidazolyl 0 1520
453 ꢂ 167 0.30
(þ)-Pentazocine
4.2 ꢂ 1.1
3.9 ꢂ 1.5
61 ꢂ 18
e
e
5. Conclusion
Haloperidol
78 ꢂ 2.3
42 ꢂ 17
20
0.7
Di-o-tolylguanidine
The s1 affinity of the spirocyclic compounds increases in the
order 2 < 3 < 4 < 1. This order correlates nicely with the distance
between the basic amino moiety and the benzene ring of the
benzopyran/benzofuran system: 2 < 3 < 4 <1. The N-benzyl moiety
represents the most promising N-substituent in terms of s1 affinity,
n.d. ¼ not determined.
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
Remaining hERG channel activity (%) after incubation with 10
mM test
compound.
s1/s2-selectivity and potential hERG channel inhibition. Although
replacement of the benzyl moiety with larger phenylalkyl or linear
aliphatic octyl residue increased the s1 affinity, these modifications
also led to reduced s1/s2-selectivity and to potential heart prob-
Decreasing of the benzene-N-distance to 4.4e4.9 Å in the regioi-
someric piperidine 2a results in almost complete loss of s1 affinity.
Increasing of this distance to 4.6e5.0 Å (3a) and 4.8e5.1 Å (4a) led
to considerably increased s1 affinities with Ki ¼ 73 nM and
Ki ¼ 25 nM, respectively.
lems via hERG channel inhibition. Altogether the spiro[[2]
benzofuran-1,30-pyrrolidine] 4a is a potent s1 ligand (Ki ¼ 25 nM)
with high selectivity against the s2 subtype (>40-fold), the phen-
cyclidine binding site of the NMDA receptor and 5-HT6 and 5-HT7
receptors. Moreover, inhibition of the hERG channel in the heart
was excluded.
4.2. Receptor selectivity
In addition to the s1 affinity the selectivity of this new class of
potent s1 ligands toward related receptor systems was investigated.
At first the s2 affinity was recorded. Table 1 clearly demonstrates
low s2 affinity of the spirocyclic pyrrolidines 3 and 4 indicating high
selectivity for the s1 over the s2 subtype. However, large residues at
the pyrrolidine N-atom like a 4-phenylbutyl (3d, 4d) or octyl (3f, 4f)
residue increased the s2 affinity and thus reduced the s1/s2 selec-
tivity. This tendency is in good accordance with observations made
with analogous spirocyclic s1 ligands [27,28,33,34,39].
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
and cool off time. Thin layer chromatography (tlc): Silica gel 60 F254
The spirocyclic pyrrolidine 4g with a 4-imidazolylbutyl residue
at the pyrrolidine N-atom shows special properties, since it is the
only compound of this series with a preference for the s2 subtype.
Replacement of the phenyl moiety of the 4-phenylbutyl derivative
4d with the heterocyclic imidazolyl residue (4g) led to a slightly
reduced s2 affinity (Ki ¼ 453 nM), but a strongly reduced s1 affinity
(Ki ¼ 1520 nM).
plates (Merck). Flash chromatography (fc): Silica gel 60, 40e64 mm
(Merck); parentheses include: diameter of the column, eluent,
fraction size, Rf value. Elemental analyses: Vario EL (Elementar-
analysesysteme 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):
The affinity of the spirocyclic pyrrolidines 3 and 4 toward the
phencyclidine binding site of the NMDA receptor, the 5-HT6 and 5-
HT7 receptors was recorded in competition experiments with
Mercury-400BB spectrometer (Varian);
d in ppm related to tetra-
radioligands. At test compound concentrations of 10 mM (NMDA
methylsilane; coupling constants are given with 0.5 Hz resolution

332
A. Jasper et al. / European Journal of Medicinal Chemistry 53 (2012) 327e336
and the assignments of 1H and 13C NMR signals were supported by
2D NMR techniques (COSY, HETCOR). Compound 5 was prepared
according to Ref. [47].
purified by fc (4 cm, methanol/ammonia 98:2, 30 mL, Rf ¼ 0.48).
Colorless oil, which solidified upon storage at 4 ꢁC, yield 604 mg
(82%). C13H17NO2 (219.3). MS (EI): m/z
¼
219 [Mþ], 188
[Mþ ꢀ OCH3], 159 [Mþ ꢀ OCHOCH3], 145 [Mþ ꢀ OCH3, eNH(CH2)2].
6.2. 2-(1-Benzyl-3-hydroxypyrrolidin-3-yl)phenylacetaldehyde
dimethyl acetal (6)
IR:
n
(cmꢀ1) ¼ 2939/2873 (
n CeH), 1073 (n CeO), 754 (d
CeH). 1H
(ppm) ¼ 2.03e2.41 (m, 2H, CH2CH2N), 2.59 (s, 1H,
~
NMR (CDCl3):
d
CH2NHCH2), 2.88e3.01 (m, 2.5H, CH2CH2N (2H), ArCH2CHOCH3
(0.5H; cis or trans)), 3.13 (d, J ¼ 12.5 Hz, 0.5H, ArCH2CHOCH3 (cis or
trans)), 3.20e3.30 (m, 1H, CH2CH2NCH2), 3.32e3.43 (m, 2H,
ArCH2CHOCH3 (1H; cis or trans), CH2CH2NCH2 (1H)), 3.52/3.53 (2s,
together 3H, OCH3 (cis, trans)), 4.77e4.85 (m, 1H, ArCH2CHOCH3),
7.09e7.26 (m, 4H, arom. H). Ratio of diastereomers 57:43.
Under N2 bromoacetal 5 (252 mg, 1.03 mmol) was dissolved in
THF (4.5 mL) and the solution was cooled to ꢀ78 ꢁC. A 1.6 M
solution of n-BuLi in n-hexane (0.75 mL, 1.2 mmol) was added
slowly. After 20 min at ꢀ78 ꢁC, a solution of 1-benzylpyrrolidin-3-
one (178 mg, 1.1 mmol) in THF (2 mL) was added dropwise and the
mixture was stirred at ꢀ78 ꢁC for 2 h. After stirring at rt for another
16 h a saturated NH4CI solution was added. The organic layer was
separated, washed with NaHSO3 solution (10%), dried (Na2SO4) and
filtered. The solvent was removed in vacuo, and the residue was
purified by fc (2 cm, petroleum ether/ethyl acetate 1:2, 10 mL,
Rf ¼ 0.35). Pale yellow oil, yield 116 mg (33%). C21H27NO3 (341.5).
~
6.5. cis/trans-3-Methoxy-10-(2-phenylethyl)-3,4-dihydrospiro[[2]
benzopyran-1,30-pyrrolidine] (3c)
1-Chloro-2-phenylethane (57.0 mg, 0.41 mmol) and K2CO3
(298 mg, 2.16 mmol) were added to a solution of the secondary
amine 3b (58.6 mg, 0.27 mmol) in acetonitrile (10 mL). The mixture
was filled into a 10 mL-microwave pressure vial and irradiated with
microwaves (program: standard; max. power 180 W; max. pressure
5 bar; temperature 140 ꢁC; time program: 5 min ramp time; 25 min
hold time; 5 min cool off time). The mixture was filtered through
CeliteÒ, washed with CH2Cl2, concentrated in vacuo and the residue
was purified by fc (1 cm, petroleum ether/ethyl acetate 7:3, 5 mL,
Rf ¼ 0.22). Pale yellow oil, yield 40 mg (45%). C21H25NO2 (323.4).
Anal. calcd. C 77.99 H 7.79 N 4.33 found C 77.73 H 7.85 N 4.24. MS
~
MS (EI): m/z ¼ 327 [MHþ eCH3], 248 [Mþ ꢀ OH2, eCH(OCH3)2]. IR:
n
(cmꢀ1) ¼ 2924 (
n
CeH). 1H NMR (CDCl3):
d
(ppm) ¼ 1.52e1.77 (m,
4H, CH2CH2N), 2.45e2.57 (m, 1H, (CH2)2NCH2), 2.90e3.03 (m, 1H,
(CH2)3NCH2), 3.11e3.19 (m, 2H, ArCH2CeH), 3.30 (s, 3H, CHOCH3),
3.34 (s, 3H, CHOCH3), 3.60e3.74 (m, 2H, NCH2Ph), 4.58e4.62 (m,
1H, ArCH2CH), 7.18e7.21 (m, 9H, arom. H). A signal for the OH
proton is not seen in the spectrum.
6.3. cis/trans-10-Benzyl-3-methoxy-3,4-dihydrospiro[[2]
benzopyran-1,30-pyrrolidine] (3a)
(EI): m/z
¼
292 [Mþ
n
ꢀ
OCH3], 232 [Mþ
CeH), 1076 ( CeO), 752/698 (
(ppm) ¼ 2.11e2.20 (m, 0.5H, CH2CH2N
ꢀ
CH2Ph]. IR:
n
Under N2 bromoacetal 5 (1.18 g, 4.82 mmol) was dissolved in
THF (15 mL) and cooled to ꢀ78 ꢁC. A 1.6 M solution of n-BuLi in n-
hexane (3.7 mL, 5.92 mmol) was added slowly. After 20 min
at ꢀ78 ꢁC, a solution of 1-benzylpyrrolidin-3-one (846 mg,
5.3 mmol) in THF (10 mL) was added dropwise and the mixture was
stirred at ꢀ78 ꢁC for 2 h and at rt for 12 h. A saturated NH4Cl
solution was added, the mixture was extracted with Et2O (6ꢃ), and
the Et2O layer was washed with H2O (2ꢃ). The organic layer was
extracted with aqueous HCl (5%, 6ꢃ) and the acidic aqueous layer
was washed with Et2O (2ꢃ). Solid KOH was added to the aqueous
layer (pH > 9). Then it was extracted with Et2O (6ꢃ). The organic
layer was washed with H2O (2ꢃ) and with NaHSO3 solution (10%,
2ꢃ), then it was dried (Na2SO4), filtered, the filtrate was concen-
trated in vacuo and the residue was purified by fc (5 cm,
CH2Cl2:methanol ¼ 98:2, 30 mL, Rf ¼ 0.41). Pale yellow oil, yield
617 mg (41%). C20H23NO2 (309.4). Anal. calcd. C 77.64 H 7.49 N 4.53
found C 77.34 H 7.57 N 4.37. MS (EI): m/z ¼ 309 [Mþ], 278
~
(cmꢀ1) ¼ 2939 (
n
d CeH monosubst.
benzene). 1H NMR (CDCl3):
d
(cis or trans)), 2.29e2.46 (m, 1.5H, CH2CH2N (cis, trans)), 2.77e3.06
(m, 9H, N(CH2)2Ph (4H), CH2CH2NCH2 (4H), ArCH2CHOCH3 (1.5H;
cis, trans)), 3.19e3.24 (m, 0.5H, ArCH2CHOCH3), 3.53/3.54 (2s,
together 3H, OCH3 (cis, trans)), 4.80e4.85 (m, 1H, ArCH2CHOCH3),
7.03e7.08 (m, 1H, arom. H), 7.15e7.39 (m, 8H, arom. H). Ratio of
diastereomers 52:48.
6.6. cis/trans-3-Methoxy-10-(4-phenylbutyl)-3,4-dihydrospiro[[2]
benzopyran-1,30-pyrrolidine] (3d)
A mixture of the secondary amine 3b (58.0 mg, 0.26 mmol), 1-
chloro-4-phenylbutane (60.7 mg, 0.36 mmol), K2CO3 (298 mg,
2.16 mmol) and acetonitrile (2 mL) was filled into a 10 mL-micro-
wave pressure vial and irradiated with microwaves (program:
standard; max. power 180 W; max. pressure 5 bar; temperature
140 ꢁC; time program: 5 min ramp time; 25 min hold time; 5 min
cool off time). The mixture was filtered through CeliteÒ, washed
with CH2Cl2, concentrated in vacuo and the residue was purified by
fc (1 cm, petroleum ether/ethyl acetate 6:4, 5 mL, Rf ¼ 0.38). Pale
yellow oil, yield 60 mg (56%). C23H29NO2 (351.5). Anal. calcd. C
78.60 H 8.32 N 3.98 found C 78.13 H 8.36 N 3.95. MS: m/z ¼ 351
[Mþ], 320 [Mþ ꢀ OCH3], 232 [Mþ ꢀ (CH2)3Ph], 201 [Mþ ꢀ OCH3,
~
[Mþ ꢀ OCH3], 249 [Mþ ꢀ OCHOCH3]. IR:
n
(cmꢀ1) ¼ 2910 (
n CeH),
1075 (
(CDCl3):
n CeO), 754, 697 (d
CeH monosubst. benzene). 1H NMR
d
(ppm) ¼ 2.13e2.24 (m, 0.5H, CH2CH2N (cis or trans)),
2.32e2.45 (m, 1.5H, CH2CH2N (cis, trans)), 2.71e2.80 (q, J ¼ 7.2 Hz,
0.5H, CH2CH2NCH2 (cis or trans)), 2.87e3.05 (m, 5H, CH2CH2NCH2
(1.5H; cis, trans), CH2CH2NCH2 (2H), ArCH2CHOCH3 (1.5H; cis,
trans)), 3.10e3.18 (m, 0.5H, ArCH2CHOCH3 (cis or trans)), 3.50, 3.54
(2s, together 3H, OCH3 (cis, trans)), 3.69e3.80 (m, 2H, NCH2Ph),
4.73, 4.82 (2 t, J ¼ 5.2 Hz, J ¼ 3.3 Hz, together 1H, CH2CHOCH3 (cis,
trans)), 7.03 (d, J ¼ 7.2 Hz, 1H, arom. H), 7.13e7.48 (m, 8H, arom. H).
Ratio of diastereomers 52:48.
e(CH2)3Ph]. IR:
n
(cmꢀ1) ¼ 2933 (
n
CeH), 1076 (
n CeO), 748/698 (d
CeH monosubst. benzene). 1H NMR (CDCl3):
d
(ppm) ¼ 1.49e1.53
(m, 2H, NCH2CH2CH2CH2Ph (2H)), 1.62e1.66 (m, 2H,
NCH2CH2CH2CH2Ph (2H)), 2.03e2.10 (m, 0.5H, CH2CH2N (cis or
trans)), 2.20e2.38 (m, 1.5H, CH2CH2N (cis, trans)), 2.47e2.51 (m, 2H,
N(CH2)3CH2Ph), 2.55e2.59 (m, 2H, CH2CH2N), 2.61e2.67 (m, 0.5H,
NCH2(CH2)3Ph (cis or trans)), 2.75e2.92 (m, 5H, NCH2(CH2)3Ph
(1.5H; cis, trans), ArCH2CHOCH3 (2H), CH2CH2NCH2 (1.5H; cis,
trans)), 3.05e3.09 (m, 0.5H, CH2CH2NCH2 (cis or trans)), 3.46 (s, 3H,
OCH3), 4.70e4.78 (m, 1H, ArCH2CHOCH3), 6.95e7.02 (m, 1H, arom.
H), 7.08e7.38 (m, 8H, arom. H). Ratio of diastereomers 54:46.
6.4. cis/trans-3-Methoxy-3,4-dihydrospiro[[2]benzopyran-1,30-
pyrrolidine] (3b)
Under N2 a mixture of 3a (1.04 g, 3.38 mmol), ammonium
formate (1.09 g, 17.3 mmol), Pd/C (10%, 213 mg) and methanol
(20 mL) was heated to reflux for 2 h. Then it was filtered through
CeliteÒ, the solvent was removed in vacuo and the residue was

A. Jasper et al. / European Journal of Medicinal Chemistry 53 (2012) 327e336
333
6.7. cis/trans-10-Butyl-3-methoxy-3,4-dihydrospiro[[2]benzopyran-
NaHSO3 solution (10%, 2ꢃ), dried (Na2SO4), filtered, concentrated in
vacuo and the residue was purified by fc (6 cm, petroleum ether/
ethyl acetate ¼ 1:2, 30 mL, Rf ¼ 0.38). Pale yellow oil, yield 1.65 g
(51%). C20H25NO3 (327.4). MS (EI): m/z ¼ 327 [Mþ], 312 [Mþ ꢀ CH3],
~
1,30-pyrrolidine] (3e)
1-Bromobutane (88.0 mg, 0.64 mmol) and K2CO3 (483 mg,
3.50 mmol) were added to a solution of the secondary amine 3b
(92.3 mg, 0.42 mmol) in acetonitrile (3 mL). The mixture was filled
into a 10 mL-microwave pressure vial and irradiated with micro-
waves (program: standard; max. power 180 W; max. pressure
5 bar; temperature 140 ꢁC; time program: 5 min ramp time; 40 min
hold time; 5 min cool off time). The mixture was filtered through
CeliteÒ, washed with CH2Cl2, concentrated in vacuo and the residue
was purified by fc (1 cm, petroleum ether/ethyl acetate 7:3, 5 mL,
Rf ¼ 0.31). Pale yellow oil, yield 76 mg (69%). C17H25NO2 (275.4).
Anal. calcd. C 74.14 H 9.15 N 5.09 found C 74.10 H 9.60 N 4.60. MS
(EI): m/z ¼ 275 [Mþ], 244 [Mþ ꢀ OCH3], 232 [Mþ ꢀ (CH2)2CH3], 201
~
296 [Mþ ꢀ OCH3]. IR:
n
(cmꢀ1) ¼ 3437 (
n eOH), 2929/2799 (n CeH),
1070 (n CeO), 752/698 (d
CeH monosubst. benzene). 1H NMR
(CDCl3):
d
(ppm) ¼ 2.28e2.42 (m 2H, CH2CH2N), 2.60e2.68 (m, 1H,
CH2CH2NCH2), 3.00e3.09 (m, 3H, CH2CH2NCH2 (2H), CH2CH2NCH2
(1H)), 3.33 (s, 3H, CH(OCH3)2), 3.37 (s, 3H, CH(OCH3)2), 3.77 (s, 2H,
NCH2Ph), 5.98 (s, 1H, ArCH(OCH3)2), 7.26e7.39 (m, 8H, arom. H),
7.68e7.71 (m, 1H, arom. H). A signal for the OH proton is not seen in
the spectrum.
6.10. cis/trans-10-Benzyl-3-methoxy-3H-spiro[[2]benzofuran-1,30-
pyrrolidine] (4a)
[Mþ ꢀ OCH3, e(CH2)2CH3]. IR:
n
(cmꢀ1) ¼ 2929 (
n CeH), 1076 (n
(ppm) ¼ 0.85, 0.87 (2 t,
CeO), 758 (
d
CeH). 1H NMR (CDCl3):
d
Hydroxyacetal 8 (476 mg,1.45 mmol) and p-toluenesulfonic acid
(360 mg, 1.89 mmol) were dissolved in methanol (3 mL). The
solution was filled into a 10 mL-microwave pressure vial and irra-
diated with microwaves (program: standard; max. power 220 W;
max. pressure 2 bar; temperature 80 ꢁC; time program: 5 min ramp
time; 15 min hold time; 5 min cool off time). Then CH2Cl2 was
added and the mixture was washed with 2 M NaOH. The organic
layer was dried (Na2SO4), filtered, concentrated in vacuo and the
residue was purified by fc (3 cm, petroleum ether/ethyl acetate 3:1,
20 mL, Rf ¼ 0.31). Pale yellow oil, yield 380 mg (92%). C19H21NO2
(295.4). Anal. calcd. C 77.26 H 7.17 N 4.64 found C 77.03 H 7.11 N
4.74. MS (EI): m/z ¼ 295 [Mþ], 264 [Mþ ꢀ OCH3], 131 [Mþ ꢀ OCH3,
~
J ¼ 7.2 Hz, together 3H, N(CH2)3CH3 (cis, trans)), 1.31 (m, 2H,
N(CH2)2CH2CH3), 1.43e1.47 (m, 2H, NCH2CH2CH2CH3), 2.03e2.12
(m, 0.5H, CH2CH2N (cis or trans)), 2.21e2.36 (m, together 1.5H,
CH2CH2N (cis, trans)), 2.44e2.51 (m, 2H, CH2CH2N), 2.62e2.70 (m,
0.5H, NCH2(CH2)2CH3), 2.78e2.95 (m, together 5H, NCH2(CH2)2CH3
(1.5H; cis, trans), ArCH2CHOCH3 (2H), CH2CH2NCH2 (1.5H; cis,
trans)), 3.08e3.13 (m, 0.5H, CH2CH2NCH2 (cis or trans)), 3.47 (s, 3H,
OCH3), 4.72e4.76 (m, 1H, ArCH2CHOCH3), 6.98e7.00 (m, 1H, arom.
H), 7.09e7.35 (m, 3H, arom. H). Ratio of diastereomers 52:48.
6.8. cis/trans-3-Methoxy-10-octyl-3,4-dihydrospiro[[2]benzopyran-
1,30-pyrrolidine] (3f)
e(CH2)2NBn]. IR:
n
(cmꢀ1) ¼ 2905 (
n
CeH), 1081 (
n CeO), 750/697 (d
CeH monosubst. benzene). 1H NMR (CDCl3):
d
(ppm) ¼ 2.24e2.35
The secondary amine 3b (91.0 mg, 0.42 mmol) was dissolved in
acetonitrile (3 mL). 1-Bromooctane (111 mg, 0.57 mmol) and K2CO3
(452 mg, 3.28 mmol) were added, the mixture was filled into
a 10 mL-microwave pressure vial and the mixture was irradiated
with microwaves (program: standard; max. power 180 W; max.
pressure 5 bar; temperature 140 ꢁC; time program: 5 min ramp
time; 40 min hold time; 5 min cool off time). The mixture was
filtered through CeliteÒ, washed with CH2Cl2, concentrated in
vacuo and the residue was purified by fc (1 cm, petroleum ether/
ethyl acetate 6:4, 5 mL, Rf ¼ 0.33). Pale yellow oil, yield 85 mg (64%).
C21H33NO2 (331.5). Anal. calcd, C 76.09 H 10.03 N 4.23 found C 76.42
H 10.05 N 4.17. MS (EI): m/z ¼ 331 [Mþ], 300 [Mþ ꢀ OCH3], 232
~
(m, 2H, CH2CH2N), 2.78e2.98 (m, 4H, CH2CH2NCH2), 3.43/3.46
(2s, together 3H, OCH3 (cis, trans)), 3.66e3.77 (m, 2H, NCH2Ph),
6.06/6.07 (2s, together 1H, ArCHOCH3 (cis, trans)), 7.23e7.40 (m, 9H,
arom. H). Ratio of diastereomers 51:49.
6.11. cis/trans-3-Methoxy-3H-spiro[[2]benzofuran-1,30-pyrrolidine]
(4b)
Under N2 a mixture of 4a (856 mg, 2.90 mmol), ammonium
formate (914 mg, 14.5 mmol), 10% Pd/C (178 g) and methanol
(45 mL) was heated to reflux for 2 h. Then it was filtered through
CeliteÒ, the solvent was removed in vacuo and the residue was
purified by fc (3 cm, methanol/ammonia 98:2, 20 mL, Rf ¼ 0.48).
Colorless oil, which solidified upon storage at 4 ꢁC, yield 520 mg
[Mþ
ꢀ
(CH2)6CH3], 201 [Mþ
ꢀ
OCH3, e(CH2)6CH3]. IR:
n
(cmꢀ1) ¼ 2924 (
n
CeH), 1077 (
n
CeO), 757 (
d
CeH). 1H NMR (CDCl3):
d
(ppm) ¼ 0.86, 0.89 (2 t, J ¼ 6.6 Hz, together 3H, CH2CH3 (cis,
(87%). C12H15NO2 (205.3). MS (EI): m/z
¼
205 [Mþ], 174
[Mþ ꢀ OCH3], 131 [Mþ ꢀ OCH3, e(CH2)2NH]. IR:
(cmꢀ1) ¼ 2932/
~
n
trans)), 1.22e1.40 (m, 10H, CH2(CH2)5CH3), 1.48e1.58 (m, 2H,
CH2(CH2)5CH3), 2.10e2.19 (m, 0.5H, CH2CH2N (cis or trans)),
2.28e2.43 (m, 1.5H, CH2CH2N (cis, trans)), 2.53e2.55 (m, 2H,
CH2CH2N), 2.69e2.77 (m, 0.5H, NCH2(CH2)6CH3), 2.83e3.02 (m, 5H,
NCH2(CH2)6CH3 (1.5H), ArCH2CHOCH3 (2H), CH2CH2NCH2 (1.5H)),
3.14e3.19 (m, 0.5H, CH2CH2NCH2 (cis or trans)), 3.54 (s, 3H, OCH3),
4.79e4.83 (m, 1H, ArCH2CHOCH3), 7.03e7.08 (m, 1H, arom. H),
7.16e7.42 (m, 3H, arom. H). Ratio of diastereomers 51:49.
2878 (
n CeH), 1081 (n CeO), 750 (d
CeH). 1H NMR (CDCl3):
d
(ppm) ¼ 2.12e2.33 (m, 2H, CH2CH2N), 3.00e3.50 (m, 5H,
CH2NHCH2), 3.43/3.48 (2s, together 3H, OCH3 (cis, trans)), 6.05/6.10
(2s, together 1H, ArCHOCH3 (cis, trans)), 7.20e7.27 (2 d, J ¼ 7.4 Hz,
together 1H, arom. H (cis, trans)), 7.32e7.43 (m, 3H, arom. H). Ratio
of diastereomers 52:48.
6.12. cis/trans-3-Methoxy-10-(2-Phenylethyl)-3H-spiro[[2]
benzofuran-1,30-pyrrolidine] (4c)
6.9. 2-(1-Benzyl-3-hydroxypyrrolidin-3-yl)benzaldehyde dimethyl
acetal (8)
A solution of 1-chloro-2-phenylethane (210 mg, 1.50 mmol) and
NaI (152 mg, 1.80 mmol) in acetone (3 mL) was filled into a 10 mL-
microwave pressure vial and irradiated with microwaves (program:
standard; max. power 180 W; max. pressure 4 bar; temperature
90 ꢁC; time program: 5 min ramp time; 10 min hold time; 5 min
cool off time). The precipitate (NaCl) was filtered off and the clear
solution containing 1-iodo-2-phenylethane was treated with 4b
(205 mg, 1.0 mmol), K2CO3 (1.10 g, 8.0 mmol) and a small amount of
acetone (1 mL). The mixture was irradiated with microwaves
Under N2 2-bromobenzaldehyde dimethyl acetal (7, 2.31 g,
10.0 mmol) was dissolved in THF (22 mL) and cooled to ꢀ78 ꢁC. A
1.6 M solution of n-BuLi in n-hexane (7.5 mL, 12.0 mmol) was added
slowly and the mixture was stirred at ꢀ78 ꢁC for 20 min. A solution
of 1-benzylpyrrolidin-3-one (1.75 g, 5.0 mmol) in THF (12 mL) was
added dropwise and the mixture was stirred at ꢀ78 ꢁC for 2 h and
at rt for 16 h. After addition of water, the mixture was extracted
with CH2Cl2. The combined organic layer was extracted with

334
A. Jasper et al. / European Journal of Medicinal Chemistry 53 (2012) 327e336
(program: standard; max. power 180 W; max. pressure 4 bar;
temperature 90 ꢁC; time program: 5 min ramp time; 30 min hold
time; 5 min cool off time). After filtration through CeliteÒ (CH2Cl2),
the solvent was removed in vacuo and the residue was purified by
fc (3 cm, cyclohexane/ethyl acetate 7:3, 20 mL, Rf ¼ 0.28). Pale
yellow oil, yield 171 mg (55%). C20H23NO2 (309.4). Anal. calcd. C
77.24 H 7.56 N 4.47 found C 77.64 H 7.49 N 4.53. MS (EI): m/z ¼ 278
~
1.44e1.60 (m, 2H, CH2CH2CH3), 2.27 (t, J ¼ 7.0 Hz, 2H, CH2CH2N),
2.50e2.60 (m, 2H, CH2CH2NCH2), 2.75e2.87 (m, 1H,
NCH2(CH2)2CH3), 2.93e3.03 (m, 3H, NCH2(CH2)2CH3 (1H),
CH2CH2NCH2 (2H)), 3.46/3.48 (2s, together 3H, OCH3 (cis, trans)),
6.05/6.07 (2s, together 1H, ArCHOCH3 (cis, trans)), 7.32e7.39 (m, 4H,
arom. H). Ratio of diastereomers 52:48.
[Mþ ꢀ OCH3], 218 [Mþ ꢀ CH2Ph]. IR:
n
(cmꢀ1) ¼ 2927 (
n
CeH), 1081
6.15. cis/trans-3-Methoxy-10-octyl-3H-spiro[[2]benzofuran-1,30-
pyrrolidine] (4f)
(n
CeO), 749/698 (
d
CeH monosubst. benzene). 1H NMR (CDCl3):
d
(ppm) ¼ 2.29e2.36 (m, 2H, CH2CH2N), 2.86e3.03 (m, 4H,
CH2CH2NCH2), 3.03e3.12 (m, 4H, NCH2CH2Ph), 3.48/3.49 (2s,
together 3H, OCH3 (cis, trans)), 6.07/6.09 (2s, together 1H, ArCH-
OCH3 (cis, trans)), 7.20e7.40 (m, 9H, arom. H). Ratio of diastereo-
mers 53:47.
A solution of 1-bromooctane (289 mg, 1.50 mmol) and NaI
(152 mg, 1.80 mmol) in acetone (3 mL) was filled into a 10 mL-
microwave pressure vial and irradiated with microwaves (program:
standard; max. power 180 W; max. pressure 4 bar; temperature
90 ꢁC; time program: 5 min ramp time; 10 min hold time; 5 min
cool off time). The precipitate (NaBr) was filtered off and the clear
solution containing 1-iodooctane was treated with 4b (205 mg,
1.0 mmol), K2CO3 (1.10 g, 8.0 mmol) and acetone (1 mL). The
mixture was irradiated with microwaves (program: standard; max.
power 180 W; max. pressure 4 bar; temperature 90 ꢁC; time
program: 5 min ramp time; 30 min hold time; 5 min cool off time).
After filtration through CeliteÒ (CH2Cl2), the solvent was removed
in vacuo and the residue was purified by fc (3 cm, cyclohexane/
ethyl acetate 7:3, 20 mL, Rf ¼ 0.36). Pale yellow oil, yield 111 mg
(35%). C20H31NO2 (317.5). Anal. calcd. C 75.67 H 9.84 N 4.41 found C
75.03 H 10.01 N 4.26. MS (EI): m/z ¼ 317 [Mþ], 286 [Mþ ꢀ OCH3],
~
6.13. cis/trans-3-Methoxy-10-(4-phenylbutyl)-3H-spiro[[2]
benzofuran-1,30-pyrrolidine] (4d)
A solution of 1-chloro-4-phenylbutane (253 mg, 1.50 mmol) and
NaI (152 mg, 1.80 mmol) in acetone (3 mL) was filled into a 10 mL-
microwave pressure vial and irradiated with microwaves (program:
standard; max. power 180 W; max. pressure 4 bar; temperature
90 ꢁC; time program: 5 min ramp time; 10 min hold time; 5 min
cool off time). The precipitate (NaCl) was filtered off and the clear
solution containing 1-iodo-4-phenylbutane was treated with 4b
(205 mg, 1.0 mmol) and K2CO3 (1.10 g, 8.0 mmol) and a small
amount of additional acetone (1 mL). The mixture was irradiated
with microwaves (program: standard; max. power 180 W; max.
pressure 4 bar; temperature 90 ꢁC; time program: 5 min ramp time;
30 min hold time; 5 min cool off time). After filtration through
CeliteÒ (CH2Cl2), the solvent was removed in vacuo and the residue
was purified by (3 cm, cyclohexane/ethyl acetate 7:3, 20 mL,
Rf ¼ 0.29). Pale yellow oil, yield 253 mg (75%). C22H27NO2 (337.5).
Anal. calcd. C 78.30 H 8.06 N 4.15 found C 77.92 H 8.12 N 4.02. MS
~
218 [Mþ ꢀ (CH2)6CH3]. IR:
n
(cmꢀ1) ¼ 2925 (
n CeH), 1082 (n CeO),
752 (
together
d
CeH). 1H NMR (CDCl3):
d
(ppm) ¼ 0.85e0.89 (2 t, J ¼ 6.7 Hz,
3 H, CH2CH3 (cis, trans)), 1.22e1.38 (m, 10 H,
CH2(CH2)5CH3), 1.50e1.60 (m, 2H, CH2(CH2)5CH3), 2.22e2.28 (m,
2H, CH2CH2N), 2.49e2.60 (m, 2H, CH2CH2NCH2), 2.72e2.83 (m, 1H,
NCH2(CH2)6CH3), 2.90e3.03 (m, 3H, NCH2(CH2)6CH3 (1H),
CH2CH2NCH2 (2H)), 3.46/3.48 (2s, together 3H, OCH3 (cis, trans)),
6.05/6.07 (2s, together 1H, ArCHOCH3 (cis, trans)), 7.32e7.39 (m, 4H,
arom. H). Ratio of diastereomers 57:43.
(EI): m/z ¼ 337 [Mþ], 306 [Mþ ꢀ OCH3], 218 [Mþ ꢀ (CH2)3Ph]. IR:
n
(cmꢀ1) ¼ 2932 (
n
CeH), 1082 (
n
CeO), 747/698 (
d CeH monosubst.
benzene). 1H NMR (CDCl3):
d
(ppm)
¼
1.51e1.53 (m, 2H,
6.16. 1-(4-Chlorobutyl)imidazole
NCH2CH2(CH2)2Ph), 1.60e1.63 (m, 2H, N(CH2)2CH2CH2Ph),
2.15e2.30 (m, 2H, CH2CH2N), 2.49e2.59 (m, 4H, CH2CH2NCH2),
2.60e2.91 (m, 4H, NCH2(CH2)2CH2Ph), 3.39/3.40 (2s, together 3H,
OCH3 (cis, trans)), 5.98/6.00 (2s, together 1H, ArCHOCH3 (cis, trans)),
7.10e7.32 (m, 9H, arom. H). Ratio of diastereomers 51:49.
NaH (60% dispersion, 804 mg, 20.1 mmol) was added to a solu-
tion of imidazole (683 mg, 10.0 mmol) in acetone (40 mL). The
mixture was stirred at rt for 2 h, then a solution of 1-bromo-4-
chlorobutane (1.72 g, 10.0 mmol) in acetone (10 mL) was added
dropwise and the reaction mixture was stirred for 6 h at 70 ꢁC. The
solvent was removed in vacuo, the residue was dissolved in CH2Cl2
(100 mL) and washed with H2O. The combined organic layer was
dried (Na2SO4), filtered, concentrated in vacuo and the residue was
purified by fc (5 cm, ethyl acetate:methanol ¼ 9:1, 30 mL, Rf ¼ 0.31).
Pale yellow oil, yield 1.31 g (83%). C7H11ClN2 (158.6). MS (EI): m/z
(%) ¼ 159 [Mþ], 131 [Mþ ꢀ CHN], 123 [Mþ ꢀ Cl], 82 [Mþ ꢀ (CH2)3Cl].
~
6.14. cis/trans-10-Butyl-3-methoxy-3H-spiro[[2]benzofuran-1,30-
pyrrolidine] (4e)
A solution of 1-bromobutane (205 mg, 1.50 mmol) and NaI
(152 mg, 1.80 mmol) in acetone (3 mL) was filled into a 10 mL-
microwave pressure vial and irradiated with microwaves (program:
standard; max. power 180 W; max. pressure 4 bar; temperature
90 ꢁC; time program: 5 min ramp time; 10 min hold time; 5 min
cool off time). The precipitate (NaBr) was filtered off and the clear
solution containing 1-iodobutane was treated with 4b (205 mg,
1.0 mmol), K2CO3 (1.10 g, 8.0 mmol) and acetone (1 mL). The
mixture was irradiated with microwaves (program: standard; max.
power 180 W; max. pressure 4 bar; temperature 90 ꢁC; time
program: 5 min ramp time; 30 min hold time; 5 min cool off time).
After filtration through CeliteÒ (CH2Cl2), the solvent was removed
in vacuo and the residue was purified by fc (3 cm, cyclohexane/
ethyl acetate 5:5, 20 mL, Rf ¼ 0.19). Pale yellow oil, yield 128 mg
(49%). C16H23NO2 (261.4). Anal. calcd. C 73.66 H 8.89 N 5.04 found C
73.53 H 8.87 N 5.36. MS (EI): m/z ¼ 261 [Mþ], 230 [Mþ ꢀ OCH3], 218
~
IR:
n
(cmꢀ1) ¼ 2945 (
n CeH), 1699 (n C]N), 737 (n
CeCl). 1H NMR
(ppm) ¼ 1.72e1.78 (m, 2H, NCH2CH2CH2CH2Cl),1.91e1.96
(CDCl3):
d
(m, 2H, NCH2CH2CH2CH2Cl), 3.52 (t, J ¼ 6.3 Hz, 2H, CH2Cl), 3.98 (t,
J ¼ 7.0 Hz, 2H, NCH2), 6.90 (s, 1H, CH2NCHCH), 7.05 (s, 1H,
CH2NCHCH), 7.50 (s, 1H, NCHN).
6.17. cis/trans-10-[4-(Imidazol-1-yl)butyl]-3-methoxy-3H-spiro[[2]
benzofuran-1,30-pyrrolidine] (4g)
A mixture of 4b (227 mg, 1.11 mmol), 1-(4-chlorobutyl)imid-
azole (167 mg, 1.05 mmol), K2CO3 (319 mg, 2.32 mmol), a catalytic
amount of KI and acetonitrile (4 mL) was filled into a 10 mL-
microwave pressure vial and 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). After filtration through CeliteÒ (CH2Cl2), the solvent
[Mþ ꢀ (CH2)2CH3]. IR:
n
(cmꢀ1) ¼ 2929 (
n CeH),1085 (n CeO), 754 (d
(ppm) ¼ 0.92/0.93 (2 t, J ¼ 3.5 Hz, together
CeH). 1H NMR (CDCl3):
d
3H, CH2CH3 (cis, trans)), 1.25e1.41 (m, 2H, CH2CH2CH3 (cis, trans)),

A. Jasper et al. / European Journal of Medicinal Chemistry 53 (2012) 327e336
335
was removed in vacuo and the residue was purified by fc (3 cm,
7.4. Membrane preparation for the s2 assay [27,34]
methanol:ammonia ¼ 98:2, 10 mL, Rf ¼ 0.41). Colorless oil, yield
223 mg (65%). C19H25N3O2 (327.4). Anal. calcd. C 69.70 H 7.70 N
12.83 found C 69.60 H 7.82 N 11.79. MS (EI): m/z ¼ 327 [Mþ], 296
~
Two rat livers were cut into small pieces and homogenized with
a potter (500e800 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
31000ꢃ 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 31000ꢃ 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 [53] 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], 218 [Mþ ꢀ (CH2)3 eC3H3N2]. IR:
n
(cmꢀ1) ¼ 2935 (
n
CeH), 1080
(n
CeO), 756
(d
CeH). 1H NMR (CDCl3):
d
(ppm) ¼ 1.49e1.58 (m, 2H, CH2(CH2)2-imid), 1.81e1.92 (m, 2H,
CH2CH2-imid), 2.18e2.35 (m, 2H, CH2CH2N), 2.53e2.57 (m, 2H,
CH2CH2N), 2.66e2.78 (m, 1H, CH2(CH2)3-imid), 2.82e2.95 (m, 3H,
CH2(CH2)3-imid (1H), CH2CH2NCH2 (2H)), 3.45/3.47 (2s, together
3H, OCH3 (cis, trans)), 3.92e3.99 (m, 2H, (CH2)3CH2-imid), 6.04/6.07
(2s, together 1H, ArCHOCH3 (cis, trans)), 6.91 (s, 1H, CH2NCHCH),
7.04 (s, 1H, CH2NCHCH), 7.25e7.46 (m, 4H, arom. H), 7.50 (s, 1H,
NCHN).
7.5. Performing of the s2 assay [27,34]
7. Pharmacological evaluation
The test was performed with the radioligand [3H]-di-o-tol-
ylguanidine (50 Ci/mmol; ARC). The thawed membrane prepara-
7.1. Receptor binding studies, materials and general procedures
tion (about 100
mg of the protein) was incubated with various
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%.
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
volume of 200 mL for 180 min at rt. 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 [55].
7.6. Affinity at the phencyclidine binding site of the NMDA receptor
This assay was performed as described in Ref. [49].
7.7. Data analysis
7.2. Membrane preparation for the s1 assay [27,34]
Five guinea pig brains were homogenized with the potter
(500e800 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
23500ꢃ g for 20 min at 4 ꢁC. The pellet was resuspended in 5e6
volumes of buffer (50 mM TRIS, pH 7.4) and centrifuged again at
23500ꢃ g (20 min, 4 ꢁC). This procedure was repeated twice. The
final pellet was resuspended in 5e6 volumes of buffer, the protein
concentration was determined according to the method of Bradford
[53] using bovine serum albumin as standard, and subsequently the
preparation was frozen (ꢀ80 ꢁC) in 1.5 mL portions containing
about 1.5 mg protein/mL.
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 [56]. For potent compounds the Ki-values are given as mean
values
þ
SEM from three independent experiments. For
M) the Ki value is
compounds with low affinity (Ki value > 1
m
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
7.3. Performing of the s1 assay [27,34]
The test was performed with the radioligand [3H]-(þ)-pentaz-
compound concentration of 1 mM is given.
ocine (42.5 Ci/mmol; Perkin Elmer). The thawed membrane prep-
aration (about 75
m
g of the protein) was incubated with various
7.8. Interaction with the hERG channel [36]
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
The effects of spirocyclic pyrrolidines 3 and 4 on hERG-potassium
channel activity were investigated using a fluorescence assay. The
study was done by evotec AG (Schnackenburgallee 114, D-22525
Hamburg, Germany). The fluorescence-based microplate assay was
performed using CHO-cells stably transfected with the hERG-
potassium channel, and wild-type CHO-cells for control. The assay
is based on the observation that inhibition of potassium channel
activity is correlated to a positive shift in membrane potential.
Changes of the membrane potential were measured using potenti-
ometric dyes. Fluorescence read-out was performed with a Tecan
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
the radioligand [3H]-(þ)-pentazocine is 2.9 nM [54].

336
A. Jasper et al. / European Journal of Medicinal Chemistry 53 (2012) 327e336
[22] K. Hashimoto, Centr. Nerv. Syst. Agents Med. Chem. 9 (2009) 197e204.
[23] J. Meunier, M. Gue, M. Recasens, T. Maurice, Br. J. Pharmacol. 142 (2004)
689e700.
Safire-fluorescencereader(Tecan, Crailsheim, Germany)on384-well
plates. Spirocyclic pyrrolidines 3 and4 were tested at a concentration
of 10
mM and mean fluorescence increase was correlated to
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Acknowledgment
[31] E. Rack, R. Fröhlich, D. Schepmann, B. Wünsch, Bioorg. Med. Chem. 19 (2011)
3141e3151.
[32] T. Schläger, D. Schepmann, E.U. Würthwein, B. Wünsch, Bioorg. Med. Chem.
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Financial support of this project by the Deutsche For-
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