Synthesis and σ receptor affinity of regioisomeric spirocyclic furopyridines

Article abstract of DOI:10.1016/j.ejmech.2014.06.073

In order to investigate systematically the effect of the position of the pyridine N-atom on the σ₁ receptor affinity four regioisomeric furopyridines 2a-d were synthesized and pharmacologically evaluated. The key steps of the synthesis comprise bromine/lithium exchange at regioisomeric bromopyridinecarbaldehyde acetals 7a-d, subsequent addition to 1-benzylpiperidin-4-one and cyclization. The regioisomeric acetals 7a-d were obtained either by o-metalation of bromopyridines 5b and 5c or by oxidation of bromopicolines 3a and 3d. In radioligand binding studies the regioisomeric furopyridines 2a-d showed 7- to 12-fold lower σ₁ affinity than the benzofuran analog 1. The reduced σ₁ affinity of the furopyridines 2a-d is explained with the reduced electron density of the pyridine ring. Since the four regioisomeric furopyridines show almost the same σ₁ affinity (K_i = 4.9-10 nM), a directed interaction of the pyridine N-atom with the receptor protein can be excluded.

Full text of DOI:10.1016/j.ejmech.2014.06.073

European Journal of Medicinal Chemistry 83 (2014) 709e716
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and
s
receptor affinity of regioisomeric spirocyclic
furopyridines
,
, c, *
Kengo Miyata a b, Dirk Schepmann a, Bernhard Wünsch a
a
€
€
Institut für Pharmazeutische und Medizinische Chemie der Westfalischen Wilhelms e Universitat Münster, Corrensstraße 48, D-48149 Münster, Germany
b Research Center for Materials Science and Department of Chemistry, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
c Cells-in-Motion Cluster of Excellence (EXC 1003 e CiM), University Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to investigate systematically the effect of the position of the pyridine N-atom on the s1 receptor
affinity four regioisomeric furopyridines 2aed were synthesized and pharmacologically evaluated. The
key steps of the synthesis comprise bromine/lithium exchange at regioisomeric bromopyr-
idinecarbaldehyde acetals 7aed, subsequent addition to 1-benzylpiperidin-4-one and cyclization. The
regioisomeric acetals 7aed were obtained either by o-metalation of bromopyridines 5b and 5c or by
oxidation of bromopicolines 3a and 3d. In radioligand binding studies the regioisomeric furopyridines 2a
ed showed 7- to 12-fold lower s1 affinity than the benzofuran analog 1. The reduced s1 affinity of the
furopyridines 2aed is explained with the reduced electron density of the pyridine ring. Since the four
regioisomeric furopyridines show almost the same s1 affinity (Ki ¼ 4.9e10 nM), a directed interaction of
the pyridine N-atom with the receptor protein can be excluded.
Received 6 May 2014
Received in revised form
23 June 2014
Accepted 29 June 2014
Available online 30 June 2014
Keywords:
s1 receptor ligands
Spirocyclic piperidines
Furopyridines
© 2014 Elsevier Masson SAS. All rights reserved.
Regioisomers
Structure affinity relationships
1. Introduction
well as in peripheral tissues (e.g. liver, kidney, heart, eye) [14e18].
The s1 receptor protein is found predominantly in the endoplasmic
Due to the pharmacological profile of ( )-SKF-10047 the
receptor was originally classified as a new opioid receptor sub-
type [1]. Later, it was supposed that receptors are identical
with the phencyclidine (PCP) binding site of the N-methyl-
aspartate (NMDA) receptor [2]. However, these hypotheses are
disproved [3,4]. Today, the receptor is recognized as a non-
opioid, non-PCP but haloperidol-sensitive receptor. Two re-
s
reticulum. It contains two transmembrane domains and both the
amino and carboxy termini are on the cytoplasmic side, whereas
the loop between the transmembrane domains is located within
the endoplasmic reticulum [19].
It was shown that several very diverse compounds interact with
the s1 receptor. For example, the dextrorotatory benzomorphans
(þ)-SKF10047 and (þ)-pentazocine [20e24], haloperidol and NE-
100 represent prototypical s1 receptor ligands [21,24e26].
s
D
-
s
s
ceptor subtypes, which are termed s1 and s2 receptor, have been
identified.
The s1 receptor was cloned from guinea pig [5], human [6],
mouse [7,8], and rat tissues [9]. The human s1 gene encodes for a
protein consisting of 223 amino acids with a molecular weight of
25.3 kDa. In contrast, the s2 receptor has not been cloned yet.
However, it was postulated that the s2 receptor and the progester-
one receptor membrane component 1 (pgrmc1) are identical [10].
Both
s receptor subtypes are expressed in the central nervous
system [11e13] but also in the endocrine and immune systems, as
* Corresponding author. Institut für Pharmazeutische und Medizinische Chemie
€
€
der Westfalischen Wilhelms e Universitat Münster, Corrensstraße 48, D-48149
Münster, Germany.
Fig. 1. The spirocyclic benzofuran 1 with high s1 receptor affinity serves as the lead
compound for regioisomeric furopyridines 2.
E-mail address: wuensch@uni-muenster.de (B. Wünsch).
http://dx.doi.org/10.1016/j.ejmech.2014.06.073
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

710
K. Miyata et al. / European Journal of Medicinal Chemistry 83 (2014) 709e716
Scheme 1. Synthesis of regioisomeric bromopyridinecarbaldehydes 6aed. Reagents and reaction conditions: (a) NBS, benzoyl peroxide, CCl4, reflux, 3e16 h; 4a, 90%; 4d, 90%; (b)
CaCO3, H2O, reflux, 14e79 h; 6a, 57%; 6d, 78%; (c) DA, THF, ꢀ78 ꢁC, then N-formylpiperidine, ꢀ78 ꢁC, 15e50 min, then rt, 1e1.5 h; 6b, 48%; 6c, 48%.
Neurosteroids such as progesterone and dehydroepiandrosterone
have been postulated to be the endogenous s1 receptor ligands
[27e29]. High affinity s1 receptor ligands have been considered to
play an important role for the treatment of various neurological
disorders, including depression, schizophrenia, neuropathic pain,
and Alzheimer's disease [30e36].
2. Chemistry
The synthesis of furopyridine regioisomers 2 started from
known regioisomeric o-bromopyridinecarbaldehydes 6aed
(Scheme 1). The pyridinecarbaldehydes 6a and 6d with the N-atom
in a- and d-position were prepared from bromopicolines 3a and 3d
[39]. At first, bromopicolines 3a and 3d were converted into
dibromomethyl derivatives 4a and 4d by a radical bromination
using NBS as a bromine source and benzoyl peroxide as a radical
chain initiator. Subsequent hydrolysis of the dibromomethylpyr-
idines 4a and 4d afforded the aldehydes 6a and 6d in 57% and 78%
yield, respectively. The regioisomeric aldehydes 6b and 6c with the
N-atom in b- and c-position were synthesized from bromopyr-
idines 5b and 5c [40,41]. Regioselective metalation of 5b and 5c
with LDA at ꢀ78 ꢁC generated pyridyllithium intermediates, which
were trapped with N-formylpiperidine to obtain the pyr-
idinecarbaldehydes 6b and 6c in 48% yield, respectively.
In 2002, we reported the synthesis of the spirocyclic benzofuran
1, which represents a potent s1 receptor antagonist (Fig. 1). In
addition to its high s1 affinity, 1 displays high s1
/s2 selectivity
(Ki(s1) ¼ 1.14 nM, s1 s2 ¼ 1130) [37,38]. Herein, we wish to present
/
the synthesis and pharmacological evaluation of pyridine ana-
logues 2 of the spirocyclic benzofuran 1. Since four CH-moieties of
the benzene ring of benzofuran 1 can be replaced with an N-atom,
four regioisomeric furopyridines 2 will be synthesized and phar-
macologically evaluated. Due to the high electronegativity of the N-
atom, the pyridine ring represents an electron deficient aromatic
system in contrast to the benzene ring. Moreover, the N-atom of the
pyridine ring can form an H-bond with an H-bond donor group of
the s1 receptor binding site. For this purpose, the position of the N-
atom is of great interest.
Acetalization of aldehydes 6aed with methanol in the presence
of p-toluenesulfonic acid led to dimethyl acetals 7aed in 71e90%
yields (Scheme 2). Treatment of bromopyridine acetals 7aed with
Scheme 2. Synthesis of spirocyclic furopyridines 2aed. Reagents and reaction conditions: (a) p-TsOH$H2O, CH(OCH3)3, CH3OH, reflux, 14.5e22 h; 7a, 90%; 7b, 71%; 7c, 89%; 7d, 79%;
(b) n-BuLi, THF, ꢀ78 ꢁC, 2e4 min, then 1-benzylpiperidin-4-one, THF, ꢀ78 ꢁC, 10e15 min, then rt, 2.5e4 h; 8c, 70%; (c) p-TsOH$H2O, CH(OCH3)3, CH3OH, rt, 14e65 h; 2a, 13%; 2b,
35%; 2c, 81%; 2d, 17%. The hydroxy acetals 8a, 8b, and 8d were directly cyclized to give the furopyridines 2a, 2b, and 2d. Therefore the yields of furopyridines 2a, 2b, and 2d are
calculated over two steps starting from bromo acetals 7a, 7b, and 7d.

K. Miyata et al. / European Journal of Medicinal Chemistry 83 (2014) 709e716
711
n-butyllithium at ꢀ78 ꢁC and subsequent reaction with 1-
benzylpiperidin-4-one afforded the hydroxy acetals 8aed. With
the exception of 8c, the hydroxy acetals 8 were not isolated, but
directly cyclized by p-toluenesulfonic acid to yield the spirocyclic
furopyridines 2aed.
pharmacologically evaluated. The regioisomeric furopyridines
2aed represent potent s1 ligands with Ki values between 4.9 and
10 nM. However, the position of the N-atom does not influence the
s1 affinity considerably, which leads to the conclusion that a
directed interaction such as an H-bond is not formed via the pyri-
dine N-atom. The s1 affinity of the furopyridines 2aed is reduced
compared to the analogous benzofuran 1, which is attributed to the
electron deficient pyridine ring in 2 and/or its higher polarity.
3. Receptor affinity
The s1 and s2 receptor affinities of the spirocyclic furopyridines 2
were determined in competition experiments with radioligands.
Guinea pig brain membrane preparations were used as the receptor
material and [3H]-(þ)-pentazocine was used as a highly s1 selective
radioligand. The nonspecific binding was determined in the presence
of a large excess of non-tritiated (þ)-pentazocine. In the s2 assay
membranepreparationsofratliverservedasa sourcefors2 receptors
and [3H]-di-o-tolylguanidine was employed as a radioligand. A large
amount of non-radiolabeled (þ)-pentazocine was added to selec-
tively mask s1 receptors, because di-o-tolylguanidine interacts with
5. Experimental
5.1. Chemistry
5.1.1. General
THF was dried with sodium/benzophenone and was freshly
distilled before use. Thin layer chromatography (tlc): Silica gel 60
F254 plates (Merck). Flash chromatography (fc): Silica gel 60,
40e64 mm (Merck); parentheses include: diameter of the column,
eluent. Melting point: Melting point apparatus SMP 10 (Stuart
Scientific), uncorrected. Nuclear magnetic resonance (NMR) spectra
were recorded on Agilent 600-MR (600 MHz for 1H, 151 MHz for
13C) or Agilent 400-MR spectrometer (400 MHz for 1H, 101 MHz for
s
receptor subtypes. An excess of non-tritiated di-o-tolylguanidine
was used for the determination of the non-specific binding [42e45].
The receptor binding data of the four regioisomeric spirocyclic
s
furopyridines 2 and the benzofuran 1 are summarized in Table 1.
The four regioisomeric furopyridines 2aed show almost the
same high s1 receptor affinity. Their Ki values are in the range of
4.9e10 nM. Obviously, the position of the N-atom does not influ-
ence the s1 affinity to a large extend. The affinity toward the s2
13C);
7.26 ppm (1H NMR) and
2.50 ppm (1H NMR) and
3.31 ppm (1H NMR) and
d in ppm related to (CH3)4Si and measured referring to CHCl3
(d
d
77.2 ppm (13C NMR)), CHD2C(O)CD3 (
39.5 ppm (13C NMR)), and CHD2OD (
d
d
d
d
49.0 ppm (13C NMR)); coupling constants
are given with 0.5 Hz resolution; the assignments of 13C and 1H
NMR signals were supported by 2D NMR techniques. MS:
micrOTOF-Q II (Bruker Daltronics); APCI, atmospheric pressure
chemical ionization. IR: FT-IR spectrophotometer MIRacle 10 (Shi-
madzu) equipped with ATR technique. HPLC: Merck Hitachi
Equipment; UV detector: L-7400; autosampler: L-7200; pump: L-
7100; degasser: L-7614; Method: column: LiChrospher® 60 RP-
subtype is very low (see Table 1), which leads to an excellent s1
selectivity of at least >200.
/s2
The s1 affinity and s1/s2 selectivity of the furopyridines 2 are,
however, lower than those of the analogous benzofuran 1, indi-
cating that an electron deficient aromatic system such as the pyr-
idine ring in 2aed leads to a decreased s1 affinity and selectivity.
The higher polarity of the pyridine ring compared to the polarity of
the benzene ring could also influence the interactions with the s1
receptor protein. Since the position of the N-atom in the pyridine
ring does not influence the s1 affinity, the formation of a directed
interaction via an H-bond of the pyridine ring with an H-bond
donor group of the receptor protein can be excluded. It can be
concluded that the reduced s1 affinity of the furopyridines 2aed is
due to the electron deficient more polar aromatic system.
select B (5
mm), 250e4 mm cartridge; flow rate: 1.00 mL/min; in-
jection volume: 5.0
m
L; detection at
l
¼ 210 nm; solvents: A: water
with 0.05% (v/v) trifluoroacetic acid; B: acetonitrile with 0.05% (v/v)
trifluoroacetic acid: gradient elution: (A %): 0e4 min: 90%,
4e29 min: gradient from 90% to 0%, 29e31 min: 0%, 31e31.5 min:
gradient from 0% to 90%, 31.5e40 min: 90%. The purity of all test
compounds was determined by this method. The purity of all test
compounds is higher than 95%.
The
synthesis
of
the
regioisomeric
bromopyr-
4. Conclusion
idinecarbaldehydes 6 has already been described [38e40]. How-
ever, the procedures are given here in detail, because we have
performed some important modifications leading to increased
yields and, furthermore, some additional spectroscopic data are
given for the aldehydes 6.
In this paper the effect of the position of the pyridine N-atom on
the s1 affinity was investigated. For this purpose, four regioisomeric
spirocyclic
furopyridines
2aed
were
prepared
and
Table 1
ð receptor affinity of spirocyclic pyridines compared with lead and reference
compounds.
5.1.2. 3-Bromo-2-(dibromomethyl)pyridine (4a) [39]
NBS (2.85 g, 16.0 mmol) and benzoyl peroxide (282 mg,
0.872 mmol) were added to a solution of 3a (1.25 g, 7.27 mmol) in
CCl4 (15 mL). The mixture was heated to reflux for 4 h. Then,
another portion of NBS (1.0 g, 5.62 mmol) and benzoyl peroxide
(100 mg, 0.310 mmol) were added and the reaction mixture was
heated to reflux for another 12 h. The reaction mixture was filtered
to remove the insoluble materials, and concentrated in vacuo. The
crude product was purified by fc (4 cm, EtOAc:cyclohexane 1:10).
Pale yellow oil (EtOAc:cyclohexane 1:4, Rf ¼ 0.30), yield 2.16 g
Compd.
Ki SEM (nM) (n ¼ 3)a
s1/s2 selectivity
s1 ([3H]-(þ)-
s2 ([3H]di-o-
pentazocine)
tolylguanidine)
1 [37]
2a
2b
2c
1.1 0.22
4.9 2.1
8.2 1.4
10 1.2
6.5 3.8
6.3 1.6
89 29
1280 137
30%b
1130
e
1690a
12%b
206
e
2d
1850a
78 2.3
58 18
e
284
12
0.7
e
haloperidol
di-o-tolyguanidine
(þ)-pentazocine
(90%). 1H NMR (400 MHz, CDCl3):
d
(ppm) ¼ 7.12e7.21 (m, 2H, 5-H-
Py, PyCH), 7.86 (dd, J ¼ 8.1/1.5 Hz, 1H, 4-H-Py), 8.68 (dd, J ¼ 4.5/
5.7 2.2
1.4 Hz, 1H, 6-H-Py).
a
Triplicates were recorded only for high affinity compounds.
b
The affinity of compounds, which did not significantly reduce the radioligand
binding in the first experiment, was recorded only once. In these cases, the per-
centage of inhibition of the radioligand binding at a concentration of 10 M is given,
indicating an IC50-value >10 M.
5.1.3. 2-Bromo-3-(dibromomethyl)pyridine (4d) [39]
NBS (2.85 g, 16.0 mmol) and benzoyl peroxide (282 mg,
0.872 mmol) were added to a solution of 3d (1.25 g, 7.27 mmol) in
m
m

712
K. Miyata et al. / European Journal of Medicinal Chemistry 83 (2014) 709e716
CCl4 (15 mL). The mixture was heated to reflux for 4 h. The reaction
mixture was filtered to remove the insoluble materials, and
concentrated in vacuo. The crude product was purified by fc (4 cm,
EtOAc:cyclohexane 1:10). Pale yellow oil (EtOAc:cyclohexane 1:4,
Rf ¼ 0.41), yield 2.16 g (90%). 1H NMR (400 MHz, DMSO-d6):
d
(ppm) ¼ 7.22 (d, J ¼ 4.9 Hz, 1H, 5-H-Py), 8.72 (d, J ¼ 4.9 Hz, 1H, 6-
H-Py), 8.92 (s, 1H, 2-H-Py), 10.37 (s, 1H, CHO).
5.1.7. 2-Bromopyridine-3-carbaldehyde (6d) [39]
A mixture of 4d (2.15 g, 6.52 mmol) and CaCO3 (1.44 g,
14.3 mmol) in water (35 mL) was heated at reflux for 14 h. The
reaction mixture was extracted with EtOAc (3 ꢂ 20 mL). The
combined organic layers were washed with water (50 mL), brine
(50 mL) and dried over Na2SO4. Filtration and evaporation afforded
crude product, which was purified by fc (4 cm, EtOAc:cyclohexane
1:5). Colorless solid (EtOAc:cyclohexane 1:3, Rf ¼ 0.24), yield
d
(ppm) ¼ 7.27 (s, 1H, PyCH), 7.61 (dd, J ¼ 7.8/4.7 Hz, 1H, 5-H-Py),
8.42e8.33 (m, 2H, 4-H-Py, 6-H-Py).
5.1.4. 3-Bromopyridine-2-carbaldehyde (6a) [39]
A mixture of 4a (2.15 g, 6.52 mmol) and CaCO3 (1.44 g,
14.3 mmol) in water (35 mL) was heated to reflux for 79 h. The
reaction mixture was extracted with EtOAc (4 ꢂ 20 mL). The
combined organic layers were washed with water (60 mL), brine
(60 mL) and dried over Na2SO4. Filtration and evaporation afforded
crude product, which was purified by fc (4 cm, EtOAc:cyclohexane
1:10 to EtOAc:cyclohexane 1:3). Colorless solid (EtOAc:cyclohexane
1:3, Rf ¼ 0.24), yield 690 mg (57%). 1H NMR (400 MHz, CDCl3):
943 mg (78%). 1H NMR (400 MHz, DMSO-d6):
d
(ppm) ¼ 7.64 (dd,
J ¼ 7.6/4.7 Hz, 1H, 5-H-Py), 8.18 (dd, J ¼ 7.6/2.1 Hz, 1H, 4-H-Py), 8.63
(dd, J ¼ 4.7/2.1 Hz, 1H, 6-H-Py), 10.16 (s, 1H, CHO).
5.1.8. 3-Bromopyridine-2-carbaldehyde dimethyl acetal (7a)
A solution of 6a (685 mg, 3.68 mmol), p-TsOH$H2O (771 mg,
4.05 mmol) and trimethyl orthoformate (3 mL) in CH3OH (25 mL)
was heated to reflux for 22 h. After concentration of the mixture in
vacuo, the residue was partitioned between satd. NaHCO3 solution
(45 mL) and EtOAc (30 mL). The aqueous layer was extracted with
EtOAc (2 ꢂ 20 mL) and the combined organic layers were dried over
Na2SO4. Filtration and evaporation afforded crude product, which
was purified by fc (4 cm, EtOAc:cyclohexane 1:2 to EtOAc:cyclo-
hexane 1:1). Pale yellow oil (EtOAc:cyclohexane 1:1, Rf ¼ 0.26),
d
(ppm) ¼ 7.37 (dd, J ¼ 8.1/4.5 Hz, 1H, 5-H-Py), 8.05 (dd, J ¼ 8.1/
1.4 Hz,1H, 4-H-Py), 8.77 (dd, J ¼ 4.5/1.4 Hz,1H, 6-H-Py),10.25 (s,1H,
CHO).
5.1.5. 4-Bromopyridine-3-carbaldehyde (6b) [41]
Under an N2 atmosphere, a 1.6 M solution of n-butyllithium in
hexane (3.53 mL, 5.65 mmol) was added slowly to a cooled (ꢀ78 ꢁC)
solution of diisopropylamine (624 mg, 0.864 mL, 6.17 mmol) in THF
(15 mL). After 30 min stirring at 0 ꢁC, the mixture was cooled
to ꢀ78 ꢁC. Meanwhile, 4-bromopyridine hydrochloride (5b$HCl)
(1.00 g, 5.14 mmol) was dissolved in satd. NaHCO3 solution. This
was extracted with diethyl ether (3 ꢂ 15 mL). The combined organic
layers were washed with brine (25 mL) and dried over Na2SO4.
After filtration, the resulting solution was concentrated under
reduced pressure to approximately 5 mL and diluted with THF
(5 mL). This solution was added to the reaction mixture. The
mixture was stirred at ꢀ78 ꢁC for 1.5 h, and then a solution of N-
formylpiperidine (2.62 g, 2.57 mL, 23.1 mmol) was added slowly.
The solution was stirred at ꢀ78 ꢁC for 15 min. Then it was allowed
to warm to room temperature and it was stirred for another 1 h. A
satd. NH4Cl solution (20 mL) was added. The aqueous layer was
extracted with EtOAc (3 ꢂ 15 mL). The combined organic layers
were washed with brine (30 mL) and dried over Na2SO4. Filtration
and evaporation afforded crude product, which was purified by fc
(4 cm, EtOAc:cyclohexane 1:10 to EtOAc:cyclohexane 1:5). Yellow
solid (EtOAc:cyclohexane 3:1, Rf ¼ 0.44), yield 459 mg (48%). 1H
yield 766 mg (90%). 1H NMR (400 MHz, CD3OD):
d
(ppm) ¼ 3.43 (s,
6H, OCH3), 5.69 (s, 1H, PyCH), 7.33 (dd, J ¼ 8.1/4.7 Hz, 1H, 5-H-Py),
8.08 (dd, J ¼ 8.1/1.4 Hz, 1H, 4-H-Py), 8.53 (dd, J ¼ 4.7/1.4 Hz, 1H, 6-
H-Py). 13C NMR (101 MHz, CD3OD):
d
(ppm) ¼ 54.9 (2C, OCH3),
103.5 (1C, PyCH), 121.7 (1C, C-3-Py),126.6 (1C, C-5-Py), 143.0 (1C, C-
4-Py), 148.4 (1C, C-6-Py), 154.9 (1C, C-2-Py). C8H10BrNO2 (231.0).
Exact mass (APCI): m/z ¼ 231.9983 (calcd. 231.9968 for C8H11BrNO2
[MþHþ]). IR (neat): v (cmꢀ1) ¼ 2932 (CeH), 2827 (CeH), 1570
(CeH), 1057 (CeO).
5.1.9. 4-Bromopyridine-3-carbaldehyde dimethyl acetal (7b)
A solution of 6b (450 mg, 2.42 mmol), p-TsOH$H2O (506 mg,
2.66 mmol) and trimethyl orthoformate (2 mL) in CH3OH (20 mL)
was heated to reflux for 16 h. After concentration of the mixture in
vacuo, the residue was partitioned between satd. NaHCO3 solution
(25 mL) and EtOAc (20 mL). The aqueous layer was extracted with
EtOAc (2 ꢂ 15 mL) and the combined organic layers were dried over
Na2SO4. Filtration and evaporation afforded crude product, which
was purified by fc (2 cm, EtOAc:cyclohexane 1:6 to EtOAc:cyclo-
hexane 1:3). Colorless oil (EtOAc:cyclohexane 3:1, Rf ¼ 0.46), yield
NMR (400 MHz, CDCl3):
d
(ppm) ¼ 7.62 (d, J ¼ 5.4 Hz, 1H, 5-H-Py),
396 mg (71%). 1H NMR (400 MHz, CD3OD):
d
(ppm) ¼ 3.39 (s, 6H,
8.56 (d, J ¼ 5.4 Hz, 1H, 6-H-Py), 9.00 (s, 1H, 2-H-Py), 10.38 (s, 1H,
CHO).
OCH3), 5.60 (s, 1H, PyCH), 7.70 (d, J ¼ 5.4 Hz, 1H, 5-H-Py), 8.34 (d,
J ¼ 5.4 Hz, 1H, 6-H-Py), 8.63 (s, 1H, 2-H-Py). 13C NMR (101 MHz,
5.1.6. 3-Bromopyridine-4-carbaldehyde (6c) [39,40]
CD3OD):
d
(ppm) ¼ 54.4 (2C, OCH3), 103.2 (1C, PyCH), 129.8 (1C, C-
Under an N2 atmosphere, a 1.6 M solution of n-butyllithium in
hexane (7.06 mL, 11.3 mmol) was added slowly to a cooled (ꢀ78 ꢁC)
solution of diisopropylamine (1.25 g, 1.73 mL, 12.4 mmol) in THF
(25 mL). After 30 min stirring at 0 ꢁC, the mixture was cooled
to ꢀ78 ꢁC and a solution of 3-bromopyridine 5c (1.63 g, 1.00 mL,
10.3 mmol) in THF (5 mmol) was added slowly. The mixture was
stirred at ꢀ78 ꢁC for 15 min, and then a solution of N-for-
mylpiperidine (5.24 g, 5.14 mL, 46.4 mmol) was added slowly. The
solution was stirred at ꢀ78 ꢁC for 50 min. Then it was allowed to
warm to room temperature and it was stirred for another 1.5 h. A
satd. NH4Cl solution (40 mL) was added. The aqueous layer was
extracted with EtOAc (3 ꢂ 30 mL). The combined organic layers
were washed with brine (60 mL) and dried over Na2SO4. Filtration
and evaporation afforded crude product, which was purified by fc
(5.5 cm, EtOAc:cyclohexane 1:4). Yellow solid (EtOAc:cyclohexane
2:1, Rf ¼ 0.55), yield 914 mg (48%). 1H NMR (600 MHz, CDCl3):
5-Py), 135.0 (1C, C-3-Py), 135.2 (1C, C-4-Py), 150.0 (1C, C-2-Py),
151.0 (1C, C-6-Py). C8H10BrNO2 (231.0). Exact mass (APCI): m/
z ¼ 231.9962 (calcd. 231.9968 for C8H11BrNO2 [MþHþ]). IR (neat): v
(cmꢀ1) ¼ 2932 (CeH), 2828 (CeH), 1551 (CeH), 1057 (CeO).
5.1.10. 3-Bromopyridine-4-carbaldehyde dimethyl acetal (7c) [46]
A solution of 6c (233 mg, 1.25 mmol), p-TsOH$H2O (262 mg,
1.38 mmol) and trimethyl orthoformate (0.15 mL) in CH3OH (10 mL)
was heated to reflux for 14.5 h. After concentration of the mixture
in vacuo, the residue was partitioned between satd. NaHCO3 solu-
tion (20 mL) and EtOAc (20 mL). The aqueous layer was extracted
with EtOAc (3 ꢂ 20 mL) and the combined organic layers were
dried over Na2SO4. Filtration and evaporation afforded crude
product, which was purified by fc (2 cm, EtOAc:cyclohexane 1:2).
Colorless oil (EtOAc:cyclohexane 2:1, Rf ¼ 0.54), yield 259 mg (89%).
1H NMR (600 MHz, CD3OD):
d
(ppm) ¼ 3.40 (s, 6H, OCH3), 5.51 (s,

K. Miyata et al. / European Journal of Medicinal Chemistry 83 (2014) 709e716
713
1H, PyCH), 7.61 (d, J ¼ 5.0 Hz, 1H, 5-H-Py), 8.56 (d, J ¼ 5.0 Hz, 1H, 6-
(590 mg). The crude product was dissolved in CH3OH (10 mL), and
p-TsOH$H2O (651 mg, 3.42 mmol) and trimethyl orthoformate
(1.5 mL) were added. This reaction mixture was stirred at room
temperature for 65 h. After concentration of the mixture in vacuo,
the residue was partitioned between satd. NaHCO3 solution (20 mL)
and EtOAc (20 mL). The aqueous layer was extracted with EtOAc
(2 ꢂ 15 mL). The combined organic layers were washed with brine
(30 mL) and dried over Na2SO4. Filtration and evaporation afforded
crude product, which was purified two times by fc (1. 2 cm,
EtOAc:CH3OH 50:1 to EtOAc:CH3OH 30:1, 2. 2 cm, CH2Cl2:CH3OH
50:1 to CH2Cl2:CH3OH 30:1). Yellow oil (EtOAc:CH3OH 10:1,
Rf ¼ 0.28), yield 45.2 mg (13%). 1H NMR (600 MHz, CD3OD):
H-Py), 8.75 (s, 1H, 2-H-Py).
5.1.11. 2-Bromopyridine-3-carbaldehyde dimethyl acetal (7d)
A solution of 6d (940 mg, 5.05 mmol), p-TsOH$H2O (1.06 g,
5.56 mmol) and trimethyl orthoformate (4 mL) in CH3OH (30 mL)
was heated to reflux for 17 h. After concentration of the mixture in
vacuo, the residue was partitioned between satd. NaHCO3 solution
(40 mL) and EtOAc (30 mL). The aqueous layer was extracted with
EtOAc (2 ꢂ 30 mL) and the combined organic layers were dried over
Na2SO4. Filtration and evaporation afforded crude product, which
was purified by fc (4 cm, EtOAc:cyclohexane 1:4). Colorless oil
(EtOAc:cyclohexane 1:1, Rf ¼ 0.53), yield 922 mg (79%). 1H NMR
d
(ppm) ¼ 1.65 (ddd, J ¼ 13.7/5.4/2.7 Hz,1H, N(CH2CH2)2), 1.80 (ddd,
(400 MHz, CD3OD):
d
(ppm) ¼ 3.39 (s, 6H, OCH3), 5.51 (s, 1H, PyCH),
J ¼ 13.7/5.5/2.7 Hz, 1H, N(CH2CH2)2), 2.04 (ddd, J ¼ 13.1/13.1/4.4 Hz,
1H, N(CH2CH2)2), 2.15 (ddd, J ¼ 13.2/13.2/4.5 Hz, 1H, N(CH2CH2)2),
2.60 (ddd, J ¼ 12.6/12.6/2.7 Hz, 2H, N(CH2CH2)2), 2.86e2.93 (m, 2H,
N(CH2CH2)2), 3.52 (s, 3H, OCH3), 3.65 (s, 2H, NCH2Ph), 5.92 (s, 1H,
PyCH), 7.26e7.30 (m, 1H, Ph-H), 7.32e7.41 (m, 4H, Ph-H), 7.43 (dd,
J ¼ 7.7/4.8 Hz, 1H, 3-H-Py), 7.80 (dd, J ¼ 7.7/1.2 Hz, 1H, 4-H-Py), 8.51
(dd, J ¼ 4.8/1.2 Hz, 1H, 2-H-Py). 13C NMR (151 MHz, CD3OD):
7.44 (dd, J ¼ 7.7/4.8 Hz,1H, 5-H-Py), 7.96 (dd, J ¼ 7.7/2.0 Hz,1H, 4-H-
Py), 8.31 (dd, J ¼ 4.8/2.0 Hz, 1H, 6-H-Py). 13C NMR (101 MHz,
CD3OD):
d
(ppm) ¼ 54.6 (2C, OCH3), 103.6 (1C, PyCH), 124.3 (1C, C-
5-Py),136.2 (1C, C-3-Py),138.7 (1C, C-4-Py),143.1 (1C, C-2-Py),151.0
(1C, C-6-Py). C8H10BrNO2 (231.0). Exact mass (APCI): m/
z ¼ 232.0011 (calcd. 231.9968 for C8H11BrNO2 [MþHþ]). IR (neat): v
(cmꢀ1) ¼ 2932 (CeH), 2828 (CeH), 1562 (CeH), 1049 (CeO).
d
(ppm) ¼ 37.4 (1C, N(CH2CH2)2), 39.1 (1C, N(CH2CH2)2), 50.4 (1C,
N(CH2CH2)2), 50.8 (1C, N(CH2CH2)2), 55.8 (1C, OCH3), 64.1 (1C,
NCH2Ph), 84.4 (1C, PyCO), 105.5 (1C, PyCH), 125.7 (1C, C-3-Py),
128.6 (1C, C-4-Ph), 129.4 (2C, C-3-Ph/C-5-Ph), 130.8 (2C, C-2-Ph/C-
6-Ph), 131.8 (1C, C-4-Py), 138.3 (1C, C-1-Ph), 141.7 (1C, C-4a-Py),
150.5 (1C, C-2-Py), 158.3 (1C, C-7a-Py). C19H22N2O2 (310.2). Exact
mass (APCI): m/z ¼ 311.1736 (calcd. 311.1754 for C19H23N2O2
[MþHþ]). IR (neat): v (cmꢀ1) ¼ 2943 (CeH), 2913 (CeH), 2808
(CeH), 1589 (CeH), 1088 (CeO), 1002 (CeN), 741 (CeH), 698 (CeH).
Purity (HPLC): 99.2% (tR ¼ 12.2 min).
5.1.12. 3-(1-Benzyl-4-hydroxypiperidin-4-yl)pyridine-4-
carbaldehyde dimethyl acetal (8c)
Under an N2 atmosphere, a 1.6 M solution of n-butyllithium in
hexane (0.743 mL, 1.19 mmol) was added slowly to a cooled
(ꢀ78 ꢁC) solution of 7c (250 mg, 1.08 mmol) in THF (8 mL). After
2 min stirring at ꢀ78 ꢁC, 1-benzylpiperidin-4-one (612 mg,
3.23 mmol) in THF (2 mL) was added slowly. The solution was
stirred at ꢀ78 ꢁC for 10 min. Then it was allowed to warm to room
temperature and it was stirred for another 2.5 h. Then water
(10 mL) was added, the aqueous layer was extracted with EtOAc
(4 ꢂ 10 mL). The combined organic layers were washed with brine
(20 mL) and dried over Na2SO4. Filtration and evaporation afforded
crude product, which was purified by fc (2 cm, EtOAc to EtOAc:-
CH3OH 5:1). Yellow solid (EtOAc:CH3OH 4:1, Rf ¼ 0.26), yield
260 mg (70%). Melting point: 128 ꢁC. 1H NMR (400 MHz, CD3OD):
5.1.14. 10-Benzyl-3-methoxy-3H-spiro[furo[3,4-c]pyridine-1,40-
piperidine] (2b)
Under an N2 atmosphere, a 1.6 M solution of n-butyllithium in
hexane (0.670 mL, 1.07 mmol) was added slowly to a cooled
(ꢀ78 ꢁC) solution of 7b (226 mg, 0.974 mmol) in THF (10 mL). After
4 min stirring at ꢀ78 ꢁC, 1-benzylpiperidin-4-one (276 mg,
1.46 mmol) in THF (2 mL) was added slowly. The solution was
stirred at ꢀ78 ꢁC for 15 min. Then it was allowed to warm to room
temperature and it was stirred for another 4 h. The reaction mixture
was partitioned between water (20 mL) and EtOAc (20 mL). The
aqueous layer was extracted with EtOAc (2 ꢂ 15 mL). The combined
organic layers were washed with brine (30 mL) and dried over
Na2SO4. Filtration and evaporation afforded crude product
(509 mg). The crude product was dissolved in CH3OH (6 mL), and p-
TsOH$H2O (556 mg, 2.92 mmol) and trimethyl orthoformate
(1.5 mL) were added. This reaction mixture was stirred at room
temperature for 15.5 h. After concentration of the mixture in vacuo,
the residue was partitioned between saturated. NaHCO3 solution
(20 mL) and EtOAc (20 mL). The aqueous layer was extracted with
EtOAc (2 ꢂ 15 mL). The combined organic layers were washed with
brine (30 mL) and dried over Na2SO4. Filtration and evaporation
afforded crude product, which was purified by fc (2 cm, EtOAc:-
CH3OH 30:1 to EtOAc:CH3OH 10:1). Yellow oil (EtOAc:CH3OH 10:1,
Rf ¼ 0.27), yield 106 mg (35%). 1H NMR (400 MHz, CD3OD):
d
(ppm) ¼ 1.95e1.99 (m, 2H, N(CH2CH2)2), 2.15e2.23 (m, 2H,
N(CH2CH2)2), 2.65e2.71 (m, 2H, N(CH2CH2)2), 2.78e2.82 (m, 2H,
N(CH2CH2)2), 3.39 (s, 6H, OCH3), 3.63 (s, 2H, NCH2Ph), 6.35 (s, 1H,
PyCH), 7.25e7.39 (m, 5H, Ph-H), 7.66 (d, J ¼ 5.1 Hz, 1H, 5-H-Py), 8.45
(d, J ¼ 5.1 Hz, 1H, 6-H-Py), 8.61 (s, 1H, 2-H-Py). 13C NMR (100 MHz,
CD3OD):
d
(ppm) ¼ 38.6 (2C, N(CH2CH2)2), 49.9 (2C, N(CH2CH2)2),
55.2 (2C, OCH3), 64.1 (1C, NCH2Ph), 71.5 (1C, PyCOH), 101.9 (1C,
PyCH), 123.9 (1C, C-5-Py),128.5 (1C, C-4-Ph), 129.4 (2C, C-3-Ph/C-5-
Ph), 130.9 (2C, C-2-Ph/C-6-Ph), 138.3 (1C, C-1-Ph), 142.7 (1C, C-3-
Py), 147.4 (1C, C-2-Py), 148.8 (1C, C-4-Py), 148.9 (1C, C-6-Py).
C
20H26N2O3 (342.2). Exact mass (APCI): m/z ¼ 343.2042 (calcd.
343.2016 for C20H27N2O3 [MþHþ]). IR (neat): v (cmꢀ1) ¼ 3171
(OeH), 2924 (CeH), 2824 (CeH), 1597 (CeH), 1099 (CeO), 1072
(CeN), 737 (CeH), 698 (CeH).
5.1.13. 10-Benzyl-7-methoxy-7H-spiro[furo[3,4-b]pyridine-5,40-
piperidine] (2a)
Under an N2 atmosphere, a 1.6 M solution of n-butyllithium in
hexane (0.784 mL, 1.25 mmol) was added slowly to a cooled
(ꢀ78 ꢁC) solution of 7a (265 mg, 1.14 mmol) in THF (12 mL). After
3 min stirring at ꢀ78 ꢁC, 1-benzylpiperidin-4-one (324 mg,
1.71 mmol) in THF (3 mL) was added slowly. The solution was
stirred at ꢀ78 ꢁC for 15 min. Then it was allowed to warm to room
temperature and it was stirred for another 3 h. The reaction mixture
was partitioned between water (20 mL) and EtOAc (20 mL). The
aqueous layer was extracted with EtOAc (2 ꢂ 15 mL). The combined
organic layers were washed with brine (40 mL) and dried over
Na2SO4. Filtration and evaporation afforded crude product
d
(ppm) ¼ 1.62 (ddd, J ¼ 13.6/5.5/2.8 Hz, 1H, N(CH2CH2)2), 1.76 (ddd,
J ¼ 13.6/5.5/2.8 Hz, 1H, N(CH2CH2)2), 2.03 (ddd, J ¼ 12.9/12.9/4.4 Hz,
1H, N(CH2CH2)2), 2.13 (ddd, J ¼ 13.1/13.1/4.4 Hz, 1H, N(CH2CH2)2),
2.53 (ddd, J ¼ 11.9/11.9/2.6 Hz, 2H, N(CH2CH2)2), 2.86e2.90 (m, 2H,
N(CH2CH2)2), 3.51 (s, 3H, OCH3), 3.62 (s, 2H, NCH2Ph), 6.14 (s, 1H,
PyCH), 7.25e7.29 (m, 1H, Ph-H), 7.31e7.40 (m, 5H, Ph-H, 7-H-Py),
8.54 (d, J ¼ 5.2 Hz, 1H, 6-H-Py), 8.57 (s, 1H, 4-H-Py). 13C NMR
(101 MHz, CD3OD):
N(CH2CH2)2), 50.5 (1C, N(CH2CH2)2), 50.9 (1C, N(CH2CH2)2), 55.7
d
(ppm) ¼ 37.0 (1C, N(CH2CH2)2), 38.9 (1C,
(1C, OCH3), 64.2 (1C, NCH2Ph), 85.9 (1C, PyCO), 106.1 (1C, PyCH),

714
K. Miyata et al. / European Journal of Medicinal Chemistry 83 (2014) 709e716
118.0 (1C, C-7-Py), 128.5 (1C, C-4-Ph), 129.4 (2C, C-3-Ph/C-5-Ph),
130.8 (2C, C-2-Ph/C-6-Ph), 136.0 (1C, C-3a-Py), 138.6 (1C, C-1-Ph),
145.7 (1C, C-4-Py), 150.6 (1C, C-6-Py), 157.9 (1C, C-7a-Py).
broad, J ¼ 13.6/1.9 Hz, 1H, N(CH2CH2)2), 2.16 (ddd, J ¼ 13.2/13.2/
4.6 Hz, 1H, N(CH2CH2)2), 2.27 (ddd, J ¼ 13.2/13.2/4.6 Hz, 1H,
N(CH2CH2)2), 2.58 (t broad, J ¼ 12.1 Hz, 2H, N(CH2CH2)2), 2.85e2.98
(m, 2H, N(CH2CH2)2), 3.50 (s, 3H, OCH3), 3.64 (s, 2H, NCH2Ph), 6.06
(s, 1H, PyCH), 7.25e7.28 (m, 1H, Ph-H), 7.31e7.39 (m, 5H, Ph-H, 3-H-
Py), 7.81 (d, J ¼ 7.6 Hz, 1H, 4-H-Py), 8.52 (d, J ¼ 4.9 Hz, 1H, 2-H-Py).
C
19H22N2O2 (310.2). Exact mass (APCI): m/z ¼ 311.1754 (calcd.
311.1754 for C19H23N2O2 [MþHþ]). IR (neat): v (cmꢀ1) ¼ 2943
(CeH), 2916 (CeH), 2808 (CeH), 1605 (CeH), 1084 (CeO), 995
(CeN), 741 (CeH), 698 (CeH). Purity (HPLC): 96.8% (tR ¼ 7.3 min).
13C NMR (101 MHz, CD3OD):
d
(ppm) ¼ 34.6 (1C, N(CH2CH2)2), 36.3
(1C, N(CH2CH2)2), 49.0 (1C, N(CH2CH2)2), 49.3 (1C, N(CH2CH2)2),
54.0 (1C, OCH3), 62.5 (1C, NCH2Ph), 83.0 (1C, PyCO), 103.9 (1C,
PyCH), 122.9 (1C, C-3-Py), 127.1 (1C, C-4-Ph), 127.9 (2C, C-3-Ph/C-5-
Ph), 129.3 (2C, C-2-Ph/C-6-Ph), 131.4 (1C, C-4a-Ph), 132.3 (1C, C-4-
Py), 137.1 (1C, C-1-Ph), 150.2 (1C, C-2-Py), 165.1 (1C, C-7a-Py).
5.1.15. 10-Benzyl-1-methoxy-1H-spiro[furo[3,4-c]pyridine-3,40-
piperidine] (2c)
A solution of 8c (200 mg, 0.584 mmol), p-TsOH$H2O (444 mg,
2.34 mmol) and trimethyl orthoformate (1 mL) in CH3OH (5 mL)
was stirred at room temperature for 22.5 h. After concentration of
the mixture in vacuo, the residue was partitioned between satd.
NaHCO3 solution (15 mL) and EtOAc (15 mL). The aqueous layer was
extracted with EtOAc (2 ꢂ 15 mL). The combined organic layers
were washed with brine (15 mL) and dried over Na2SO4. Filtration
and evaporation afforded crude product, which was purified by fc
(2 cm, EtOAc:CH3OH 10:1). Yellow oil (EtOAc:CH3OH 5:1, Rf ¼ 0.35),
C
19H22N2O2 (310.2). Exact mass (APCI): m/z ¼ 311.1732 (calcd.
311.1754 for C19H23N2O2 [MþHþ]). IR (neat): v (cmꢀ1) ¼ 2943
(CeH), 2920 (CeH), 2808 (CeH), 1601 (CeH), 1084 (CeO), 995
(CeN), 737 (CeH), 698 (CeH). Purity (HPLC): 97.1% (tR ¼ 13.5 min).
5.2. Receptor binding studies [42e45]
yield 147 mg (81%). 1H NMR (600 MHz, CD3OD):
d
(ppm) ¼ 1.67
5.2.1. Materials
(ddd, J ¼ 13.6/5.6/2.8 Hz, 1H, N(CH2CH2)2), 1.81 (ddd, J ¼ 13.7/5.6/
2.9 Hz, 1H, N(CH2CH2)2), 2.08 (ddd, J ¼ 13.1/13.1/4.4 Hz, 1H,
N(CH2CH2)2), 2.18 (ddd, J ¼ 13.1/13.1/4.4 Hz, 1H, N(CH2CH2)2),
2.53e2.58 (m, 2H, N(CH2CH2)2), 2.88e2.91 (m, 2H, N(CH2CH2)2),
3.50 (s, 3H, OCH3), 3.64 (s, 2H, NCH2Ph), 6.08 (s, 1H, PyCH),
7.27e7.29 (m, 1H, Ph-H), 7.33e7.36 (m, 2H, Ph-H), 7.38e7.39 (m, 2H,
Ph-H), 7.46 (d, J ¼ 5.1 Hz, 1H, 7-H-Py), 8.54 (d, J ¼ 5.1 Hz, 1H, 6-H-
Py), 8.55 (s, 1H, 4-H-Py). 13C NMR (101 MHz, CD3OD):
The guinea pig brains and rat liver for the s1 and s2 receptor
binding assays were commercially available (Harlan-Winkelmann,
Borchen, Germany). Homogenizer: Elvehjem Potter (B. Braun
Biotech International, Melsungen, Germany). Cooling centrifuge
model Rotina 35R (Hettich, Tuttlingen, Germany) and High-speed
cooling centrifuge model Sorvall RC-5C plus (Thermo Fisher Sci-
entific, Langenselbold, Germany). Multiplates: standard 96-well
multiplates (Diagonal, Muenster, Germany). Shaker: self-made
device with adjustable temperature and tumbling speed (scienti-
fic workshop of the institute). Vortexer: Vortex Genie 2 (Thermo
Fisher Scientific, Langenselbold, Germany). Harvester: MicroBeta
FilterMate-96 Harvester. Filter: Printed Filtermat Typ A and B.
Scintillator: Meltilex (Typ A or B) solid state scintillator. Scintilla-
tion analyzer: MicroBeta Trilux (all Perkin Elmer LAS, Rodgau-
Jügesheim, Germany). Chemicals and reagents were purchased
from different commercial sources and of analytical grade.
d
(ppm) ¼ 37.6 (1C, N(CH2CH2)2), 39.4 (1C, N(CH2CH2)2), 50.6 (1C,
N(CH2CH2)2), 51.0 (1C, N(CH2CH2)2), 55.7 (1C, OCH3), 64.1 (1C,
NCH2Ph), 85.3 (1C, PyCO),106.3 (1C, PyCH),119.9 (1C, C-7-Py),128.5
(1C, C-4-Ph), 129.4 (2C, C-3-Ph/C-5-Ph), 130.8 (2C, C-2-Ph/C-6-Ph),
138.6 (1C, C-1-Ph),143.9 (1C, C-4-Py),144.0 (1C, C-3a-Py),148.9 (1C,
C-7a-Py),149.7 (1C, C-6-Py). C19H22N2O2 (310.2). Exact mass (APCI):
m/z ¼ 311.1746 (calcd. 311.1754 for C19H23N2O2 [MþHþ]). IR (neat):
v (cmꢀ1) ¼ 2943 (CeH), 2913 (CeH), 2808 (CeH), 1585 (CeH), 1088
(CeO), 1057 (CeN), 741 (CeH), 698 (CeH). Purity (HPLC): 95.1%
(tR ¼ 7.7 min).
5.2.2. Preparation of membrane homogenates from guinea pig
brain
5.1.16. 10-Benzyl-5-methoxy-5H-spiro[furo[3,4-b]pyridine-7,40-
piperidine] (2d)
5 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 1200ꢂ g for
10 min at 4 ꢁC. The supernatant was separated and centrifuged at
23,500ꢂ 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
23,500ꢂ g (20 min, 4 ꢁC). This procedure was repeated twice. The
final pellet was resuspended in 5e6 volumes of buffer and frozen
(ꢀ80 ꢁC) in 1.5 mL portions containing about 1.5 mg protein/mL.
Under an N2 atmosphere, a 1.6 M solution of n-butyllithium in
hexane (0.741 mL, 1.18 mmol) was added slowly to a cooled
(ꢀ78 ꢁC) solution of 7d (250 mg, 1.08 mmol) in THF (12 mL). After
4 min stirring at ꢀ78 ꢁC, 1-benzylpiperidin-4-one (307 mg,
1.62 mmol) in THF (3 mL) was added slowly. The solution was
stirred at ꢀ78 ꢁC for 15 min. Then it was allowed to warm to room
temperature and it was stirred for another 2.5 h. The reaction
mixture was partitioned between water (20 mL) and EtOAc (20 mL).
The aqueous layer was extracted with EtOAc (2 ꢂ 15 mL). The
combined organic layers were washed with brine (40 mL) and dried
over Na2SO4. Filtration and evaporation afforded crude product
(512 mg). The crude product was dissolved in CH3OH (8 mL), and p-
TsOH$H2O (616 mg, 3.24 mmol) and trimethyl orthoformate
(1.5 mL) were added. This reaction mixture was stirred at room
temperature for 14 h. After concentration of the mixture in vacuo,
the residue was partitioned between satd. NaHCO3 solution (20 mL)
and EtOAc (20 mL). The aqueous layer was extracted with EtOAc
(2 ꢂ 15 mL). The combined organic layers were washed with brine
(30 mL) and dried over Na2SO4. Filtration and evaporation afforded
crude product, which was purified two times by fc (1. 2 cm,
EtOAc:CH3OH 50:1 to EtOAc:CH3OH 30:1, 2. 2 cm, CH2Cl2:CH3OH
30:1 to CH2Cl2:CH3OH 10:1). Yellow oil (EtOAc:CH3OH 10:1,
Rf ¼ 0.33), yield 58.0 mg (17%). 1H NMR (400 MHz, CD3OD):
5.2.3. Preparation of membrane homogenates from rat liver
Two rat livers were cut into small pieces and 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 1200ꢂ g for
10 min at 4 ꢁC. The supernatant was separated and centrifuged at
31,000ꢂ g for 20 min at 4 ꢁC. The pellet was resuspended in 5e6
volumes of buffer (50 mM TRIS, pH 8.0) and incubated at room
temperature for 30 min. After the incubation, the suspension was
centrifuged again at 31,000ꢂ g for 20 min at 4 ꢁC. The final pellet
was resuspended in 5e6 volumes of buffer and stored at ꢀ80,ꢁC in
1.5 mL portions containing about 2 mg protein/mL.
5.2.4. Protein determination
The protein concentration was determined by the method of
Bradford [47], modified by Stoscheck [48]. The Bradford solution
was prepared by dissolving 5 mg of Coomassie Brilliant Blue G 250
d
(ppm) ¼ 1.56 (dd broad, J ¼ 13.6/1.5 Hz, 1H, N(CH2CH2)2), 1.71 (dd

K. Miyata et al. / European Journal of Medicinal Chemistry 83 (2014) 709e716
715
in 2.5 mL of EtOH (95%, v/v). 10 mL deionized H2O and 5 mL
Acknowledgment
phosphoric acid (85%, m/v) were added to this solution, the mixture
was stirred and filled to a total volume of 50.0 mL with deionized
water. The calibration was carried out using bovine serum albumin
as a standard in 9 concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0
This work was performed within the framework of the Japa-
neseeGerman Graduate Externship Partnership between the
‘University of Münster and Nagoya University in Educational
Research of Chemistry’ in collaboration with the Nagoya University.
Financial support of this project by the Japan Society for Promotion
of Science (Nagoya University) and the Deutsche For-
schungsgemeinschaft (University of Münster) is gratefully
acknowledged.
and 4.0 mg/mL). In a 96-well standard multiplate, 10
calibration solution or 10 L of the membrane receptor preparation
were mixed with 190 L of the Bradford solution, respectively. After
5 min, the UV absorption of the protein-dye complex at
mL of the
m
m
l
¼ 595 nm
was measured with a platereader (Tecan Genios, Tecan, Crailsheim,
Germany).
Appendix A. Supplementary data
5.2.5. General protocol for the binding assays
The test compound solutions were prepared by dissolving
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.073.
approximately 10
mmol (usually 2e4 mg) of test compound in
DMSO so that a 10 mM stock solution was obtained. To obtain the
required test solutions for the assay, the DMSO stock solution was
diluted with the respective assay buffer. The filtermats were pre-
soaked in 0.5% aqueous polyethylenimine solution for 2 h at room
temperature before use. All binding experiments were carried out
in duplicates in 96-well multiplates. The concentrations given are
the final concentrations in the assay. Generally, the assays were
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5.2.6. Protocol of the s1 receptor binding assay
The assay was performed with the radioligand [3H]-
(þ)-Pentazocine (22.0 Ci/mmol; Perkin Elmer). The thawed mem-
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