Thiophene bioisosteres of spirocyclic σ receptor ligands: Relationships between substitution pattern and σ receptor affinity

Article abstract of DOI:10.1021/jm300302p

On the basis of the 6′,7′-dihydrospiro[piperidine-4,4′- thieno[3,2-c]pyran] framework, a series of more than 30 σ ligands with versatile substituents in 1-, 2′-, and 6′-position has been synthesized and pharmacologically evaluated in order to find novel structure-affinity relationships. It was found that a cyclohexylmethyl residue at the piperidine N-atom instead of a benzyl moiety led to increased σ₂ affinity and therefore to decreased σ₁/ σ₂ selectivity. Small substituents (e.g., OH, OCH₃, CN, CH₂OH) in 6′-position adjacent to the O-atom were well tolerated by the σ₁ receptor. Removal of the substituent in 6′-position resulted in very potent but unselective σ ligands (13). A broad range of substituents with various lipophilic and H-bond forming properties was introduced in 2′-position adjacent to the S-atom without loss of σ₁ affinity. However, very polar and basic substituents in both 2′- and 6′-position decreased the σ₁ affinity considerably. It is postulated that the electron density of the thiophene moiety has a big impact on the σ₁ affinity.

Full text of DOI:10.1021/jm300302p

Article
pubs.acs.org/jmc
Thiophene Bioisosteres of Spirocyclic σ Receptor Ligands:
Relationships between Substitution Pattern and σ Receptor Affinity
Christoph Oberdorf,† Dirk Schepmann,† Jose Miguel Vela,‡ Helmut Buschmann,‡ Jorg Holenz,‡
̈
and Bernhard Wunsch*,†
̈
†Institut fur Pharmazeutische und Medizinische Chemie der Universitat Munster, Hittorfstraße 58-62, D-48149 Munster, Germany
̈
̈
̈
̈
‡Esteve, Av. Mare de Deu de Montserrat 221, 08041 Barcelona, Spain
S
* Supporting Information
ABSTRACT: On the basis of the 6′,7′-dihydrospiro-
[piperidine-4,4′-thieno[3,2-c]pyran] framework, a series of
more than 30 σ ligands with versatile substituents in 1-, 2′-,
and 6′-position has been synthesized and pharmacologically
evaluated in order to find novel structure−affinity relation-
ships. It was found that a cyclohexylmethyl residue at the piperidine N-atom instead of a benzyl moiety led to increased σ2 affinity
and therefore to decreased σ1/σ2 selectivity. Small substituents (e.g., OH, OCH3, CN, CH2OH) in 6′-position adjacent to the O-
atom were well tolerated by the σ1 receptor. Removal of the substituent in 6′-position resulted in very potent but unselective σ
ligands (13). A broad range of substituents with various lipophilic and H-bond forming properties was introduced in 2′-position
adjacent to the S-atom without loss of σ1 affinity. However, very polar and basic substituents in both 2′- and 6′-position decreased
the σ1 affinity considerably. It is postulated that the electron density of the thiophene moiety has a big impact on the σ1 affinity.
available. However, Aydar et al. postulated a σ1 receptor model
consisting of two transmembrane domains and both the amino
and carboxy termini located intracellularly.10 Very recently Pricl
et al. reported on a calculated 3D model of the σ1 receptor
protein analyzing the ligand binding site on the level of amino
acids.11
The σ2 receptor is less characterized than the σ1 receptor. A
very recent report showed the identity of the σ2 receptor and
the progesterone receptor membrane component 1 (pgrmc1),
which has been cloned in 1996. This protein is comprised of
194 amino acids and has a molecular weight of 21.67 kDa.12,13
The intracellular signal transduction pathway after activation
of σ1 receptors is not yet completely elucidated. It has been
shown that σ1 receptors play a crucial role in the regulation of a
variety of ion channels including K+-channels14,15 and Ca2+-
channels.16,17 Moreover, σ1 receptors are involved in the
modulation of various neurotransmitter systems. In particular
the glutamatergic,18 dopaminergic,19 and cholinergic20 neuro-
transmission is influenced by σ1 receptors.
INTRODUCTION
■
The observation of atypical pharmacological effects of typical
opioid receptor ligands of the benzomorphan type led to the
subclassification of opioid receptors into μ-, κ-, and σ-opioid
receptors.1 However, this classification was disproved because
the opioid antagonist naloxone was not able to antagonize σ
ligand mediated effects.2 Later, it was postulated that the σ
receptor and the phencyclidine (PCP) binding site of the
NMDA receptor are identical.3 This hypothesis was also
disproved by the receptor binding profile of the antipsychotic
agent haloperidol showing high affinity toward σ receptors but
no affinity toward the PCP binding site of the NMDA
receptor.4 Today, σ receptors are well established as a
nonopioid, nonphencyclidine, and haloperidol-sensitive recep-
tor family with a characteristic binding profile and widespread
distribution in the central nervous system (CNS) as well as in
endocrine, immune, and some peripheral tissues (e.g., lung,
kidney, heart, liver).5,6
Depending on their different interactions with dextrorotatory
benzomorphans, the class of σ receptors was subdivided into
two subtypes, which were termed σ1 and σ2 receptor. About 15
years ago, the σ1 receptor was cloned from various species and
tissues including guinea pig liver,7 rat brain,8 and human
placental choriocarcinoma cell lines.9 These cloned σ1 receptors
are more than 92% identical and more than 95% similar on the
level of amino acid sequence. The rat gene encodes for a
protein of 223 amino acids with a molecular weight of 25.3
kDa. With the exception of the yeast enzyme sterol-Δ8/Δ7-
isomerase showing a 30% homology to the σ1 receptor protein,
mammalian receptors or even other known proteins do not
reveal any homology with the cloned σ1 receptor. An X-ray
crystal structure of the membrane bound σ1 receptor is not yet
Because σ1 receptors play a crucial role in several
neurological processes, their corresponding ligands represent
promising drug candidates for the treatment of various
neurological and psychiatric disorders. The potential of σ1
ligands for the treatment of anxiety, depression, memory
disorders, Alzheimer’s disease as well as alcohol and cocaine
abuse has been shown in animal models.21−24 Moreover, σ1
receptors are involved in the perception of pain as an
endogenous antiopioid system. Whereas σ1 agonists (e.g.,
(+)-pentazocine) reduce μ-, κ-, and δ-opioid receptor mediated
Received: March 2, 2012
Published: April 19, 2012
© 2012 American Chemical Society
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analgesia, σ1 antagonists (e.g., haloperidol) enhance opioid
induced antinociception.25 The potential of σ1 antagonists for
the treatment of neuropathic pain has been shown by the
successful phase II clinical trial with a pyrazole-based σ1
antagonist.26 The high density of σ1 (and σ2 receptors) in
some human tumor cell lines (e.g., lung, prostate, breast cancer
cells) has stimulated the development of novel σ ligand-based
antitumor drugs and tumor diagnostics.27,28
In Figure 1, structurally diverse drugs in clinical use for
different indications binding with high affinity toward σ1
Figure 2. Development of spirocyclic σ1 receptor ligands 9 with
variations in the 1-, 2′-, and 6′-position.
CHEMISTRY
■
For the synthesis of spirocyclic thiophene based σ1 ligands with
various substituents in the 6′-position, the lactol (intramolecular
hemiacetal) 10a and the nitriles 11 were selected as building
blocks (Scheme 1). The lactol 10a was prepared by hydrolysis
of the methyl acetal 8a with diluted HCl. Although the
elimination product 12a was formed as side product, the
desired lactol 10a was isolated in 64% yield. Treatment of the
methyl acetals 8a and 8b with an excess of trimethylsilyl
cyanide (TMSCN) and 1.6 equiv of BF3·Et2O37 at −20 °C
provided the nitriles 11a and 11b in 79% and 43% yields,
Figure 1. Clinically used drugs with additional σ1 receptor affinity.
receptors are summarized. In addition to dopamine D2 receptor
antagonistic effects, the prototypical butyrophenone antipsy-
chotic haloperidol (1, Ki (σ1) = 3.9 nM) has potent σ1 receptor
antagonistic activity. Whereas racemic pentazocine (( )-2) is
used as strong analgesic, its dextrorotatory enantiomer (+)-2 is
a potent σ1 receptor agonist (Ki (σ1) = 4.2 nM). Both
antidepressants, the selective serotonin reuptake inhibitor
fluvoxamine (3, Ki (σ1) = 36 nM)29 and the tricyclic
antidepressant opipramol (4),30 possess high affinity toward
σ1 receptors. The neuroprotective effects of the acetylcholine
esterase inhibitor donepezil (5), which is clinically used for the
treatment of Alzheimer’s disease, are supported by its σ1
receptor antagonistic activity.31
a
Scheme 1
Our interest has been focused on the development of novel
σ1 receptor ligands with reduced conformational flexibility
giving information on the complementary surface of the σ1
receptor binding site.32,33 The spirocyclic benzofurans 6 and
benzopyrans 7 represent very potent σ1 receptor antagonists
showing high selectivity over the σ2 subtype.34,35 (Figure 2)
Recently, we have reported the synthesis and biological activity
of the corresponding thiophene bioisosteres 8 exceeding the σ1
receptor affinity of the parent benzene derivatives 6 and 7.36
Because of the characteristic chemical features of the thiophene
moiety, the spirocyclic thienopyrans 8 represent an optimal
starting point for broad variations of the substituents of the
spirocyclic system. Herein we report on the synthesis and
pharmacological evaluation of spirocyclic thienopyrans 9 being
modified in the 1-, 2′- and 6′-position. Various polar
substituents allowing the formation of H-bonds and lipophilic
substituents increasing hydrophobic interactions with the
receptor protein are envisaged to study ligand receptor
interactions.
a
Reagents and reaction conditions: (a) 2 M HCl, CH3CN, reflux, 1 h,
64% 10a, 15−20% 12a; (b) TMSCN, BF3.Et2O, CH2Cl2, −20 °C, 30
min, 79% 11a, 18% 12a, 43% 11b, 30%, 12b;(c) H2, Pd/C, CH3OH,
rt, 6−14 h, 85% 13a, 64% 13b.
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a
Scheme 2
a
Reagents and reaction conditions: (a) MeOH or EtOH, H2SO4 conc, reflux, 18−40 h, 56% 14, 71% 15; (b) TiCl4, AcOH, 0 °C, 15 min, then H2O,
rt, 2 h, 60%; (c) LiAlH4, THF, −10 °C, 1 h, 57 b%; (d) PhCHO, NaBH(OAc)3, CH2Cl2, rt, 1 h, 25%; (e) DIBAL, toluene, −78 °C, 45 min, 37%;
(f) NaBH4, MeOH, rt, 1 h, 70%; (g) HN(CH3)2, NaBH(OAc)3, CH2Cl2, rt, 2 h, 46%.
a
Scheme 3
a
Reagents and reaction conditions: (a) Pr4NRuO4 (5 mol %), NMMO (5 equiv), CH2Cl2/CH3CN, molecular sieves 4 Å, rt, 1.5 h, 66%; (b)
(Ph)3PCHCN, Cs2CO3, THF, reflux, 3.5 h, 94%; (c) (Ph)3PCH(CO)CH3, Cs2CO3, THF, reflux, 12 h, 67%; (d) (Ph)3PCHCO2CH3,
Cs2CO3, THF, reflux, 6 h, 70%; (e) (Ph)3PCHCO2C2H5, KOtBu, THF, reflux, 10.5 h, 47%; (f) 26, LiAlH4, THF, −20 °C, 30 min, 80%.
respectively. Higher temperature or lower amounts of TMSCN
led to reduced yields of the nitriles 11 and increased formation
of the elimination products 12. Nevertheless, the elimination
products 12a and 12b were converted into the hydrogenated
thienopyrans 13a and 13b upon treatment with H2 and Pd/C.
The nitrile 11a was employed for the introduction of
substituents with one C-atom in 6′-position (Scheme 2). At
first, the nitrile 11a was converted into the esters 14 and 15
upon heating with concentrated H2SO4 in methanol or ethanol.
The primary amide 16 was obtained by reaction of the nitrile
11a with TiCl4 (5 equiv) at 0 °C and subsequent hydrolysis.38
LiAlH4 reduction of the nitrile 11a39 led to the primary amine
17, which was monobenzylated with benzaldehyde and
NaBH(OAc)3 to form the benzylamine 18. The aldehyde
40
19 was prepared by reduction of the nitrile 11a with
diisobutylaluminum hydride (DIBAL). Reduction of the
aldehyde 19 with NaBH4 yielded the primary alcohol 20,
which can be regarded as homologue of the hemiacetal 10a and
constitutional isomer of the methyl acetal 8a. Reductive
amination40 of the aldehyde 19 with dimethylamine and
NaBH(OAc)3 led to the tertiary amine 21.
The lactol 10a served as starting material for the synthesis of
lactone 22 and derivatives 23−27 with a side chain of two
carbon atoms (Scheme 3). Oxidation of the lactol 10a was
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performed with Pr4NRuO4 (TPAP, 5 mol %) and an excess of
N-methylmorpholine-N-oxide (NMMO, 1.5 equiv)41 to afford
the lactone 22 in 66% yield. The lactol substructure of 10a has
a tetrahedral geometry and can serve as an H-bond donor and
acceptor, whereas the carbonyl moiety of 22 is planar and can
only form an H-bond with another H-bond donating moiety.
For the introduction of C2-side chains in 6′-position the
lactol 10a was reacted with phosphorylides of the type Ph3P
CH-A (A = acceptor group). In this tandem reaction,42 the
hemiacetal 10a was opened to form an hydroxyaldehyde, which
reacted with the Wittig reagents to give an α,β-unsaturated
ketone, nitrile or ester. The intramolecular conjugate addition
of the tertiary alcohol at the electron deficient double bond was
supported by the base Cs2CO3, providing the benzofuran
derivatives 23−25 in 67−94% yields. The yield of the ethyl
ester 26 did not exceed 44%, although KOtBu was used for the
cyclization step. Reduction of the ethyl ester 26 with LiAlH4 led
to the hydroxyethyl derivative 27, representing the next
homologue in the series lactol 10a and hydroxymethyl
derivative 20.
5). Furthermore, the sterically demanding benzyl residue
should be introduced into the thiophene moiety, which
increases the electron density in the thiophene ring and the
lipophilic interactions with the receptor protein but is not able
to form any H-bonds. The direct benzylation of 29 with benzyl
bromide after deprotonation with LDA provided the benzylated
derivative 35 in only 24% yield. Therefore the hydroxybenzyl
derivative 32 was prepared by addition of phenylmagnesium
bromide to the aldehyde 30 (87%) or by addition of
benzaldehyde to the lithiated thiophene derivative 29 (92%).
Finally, the hydroxy group of 32 was removed reductively with
butylsilane (BuSiH3 ) in the presence of tris-
(pentafluorophenyl)borane (B(C6F5)3)43 to obtain the benzy-
lated product 35 in 58% yield.
Next, the spirocyclic piperidines 39−41 were obtained by
performing a halogen-metal exchange of the brominated
thiophene derivatives 33−35 with n-BuLi and subsequent
trapping of the thienyllithium intermediates with 1-benzylpi-
peridin-4-one. Finally, intramolecular transacetalization of 36−
38 was catalyzed by p-toluenesulfonic acid to afford the
spirocyclic piperidines 39−41 (Scheme 5).
To introduce different substituents in 2′-position of the
spirocyclic system, it was planned to modify the thienopyran 8a
after lithiation in α-position. However, deprotonation with
LDA or n-BuLi and reaction with 1-formylpiperidine led
predominantly to base catalyzed elimination of methanol,
resulting in the aldehyde 28 as main product (Scheme 4).
The thiophenecarbaldehyde acetal 39 was exploited for the
introduction of additional substituents in 2′-position. Treat-
ment of 39 with 1 M HCl at room temperature led exclusively
to hydrolysis of the dimethyl acetal, providing the aldehyde 42
in 96% yield (Scheme 6). The high chemoselectivity of this
transformation is explained by the higher reactivity of the
aromatic dimethyl acetal compared with the reactivity of the
aliphatic acetal within the pyran moiety.
a
Scheme 4
Treatment of the aldehyde 42 with LiAlH4 gave the primary
alcohol 43 in 57% yield. Reductive amination with dimethyl-
amine and NaBH(OAc)3 converted the aldehyde 42 into the
tertiary amine 44. Nucleophilic addition of the Grignard
reagent phenylmagnesium bromide to the aldehyde 42
produced the hydroxybenzyl derivative 45, which was oxidized
with TPAP and NMMO to afford the ketone 46. The oxime 47
was obtained by condensation of the aldehyde 42 with
hydroxylamine. Dehydration of the oxime 47 with phthalic
anhydride44 led to the nitrile 48, which was transformed into
the methyl ester 49 upon heating with methanol and
concentrated H2SO4.
a
Reagents and reaction conditions: (a) n-BuLi, THF, 0 °C, 3 min,
then 1-formylpiperidine, 0 °C, 1 h, 21%; (b) LDA, THF, 0 °C, 30 min,
then 1-formylpiperidine, 0 °C, 30 min, 35% 30, 20% 31.
RECEPTOR BINDING STUDIES
■
The σ1 and σ2 receptor affinities of the spirocyclic thiophenes of
the general formula 9 were determined in competition
experiments with radioligands. The highly σ1 selective radio-
ligand [3H]-(+)-pentazocine (compare compound 2 in Figure
1) and homogenates of guinea pig brains were used in the σ1
assay. The nonspecific binding was recorded with a large excess
of nonradiolabeled (+)-pentazocine. In the σ2 assay membrane
preparations of rat liver served as source for σ2 receptors. The
nonselective radioligand [3H]-di-o-tolylguanidine was em-
ployed in the σ2 assay because a σ2 selective radioligand is
not commercially available. To mask the σ1 receptors, an excess
of nontritiated (+)-pentazocine (500 nM) was added to the
assay solution. A high concentration of nontritiated di-o-
tolylguanidine was used to determine the nonspecific binding.
Generally the IC50-values of all ligands were recorded with six
different test compound concentrations ranging from 10 μM to
0.1 nM.34,35,45,46 In the case of very potent ligands (e.g., 8a,
11a), a further dilution step (0.01 nM) was necessary to
improve the accuracy of the nonlinear regression analysis. The
Therefore the substitution was performed at an earlier step of
the synthesis sequence. The brominated thienylacetaldeyhde
acetal 29, which is the starting compound of the spirocyclic
thienopyrans 8,36 was deprotonated with LDA at 0 °C and the
thienyllithium intermediate was trapped with 1-formylpiper-
idine. This reaction led to the aldehydes 30 and 31 in 35% and
20% yields, respectively. Again the base catalyzed the
elimination to produce the enol ether 31. However,
optimization of the reaction conditions (reduction of the
temperature to −45 °C, increase of the amount of LDA to 1.8
equiv, reduction of the reaction time to 30 min after addition of
1-formylpiperidine) led to the aldehyde 30 in 93% yield
without formation of the enol ether 31. The aldehyde 30 was
protected as dimethyl acetal 33 for further transformations
(Scheme 5).
The introduction of an electron withdrawing chlorine atom
in α-position was achieved by an electrophilic aromatic
substitution of 29 with N-chlorosuccinimide (NCS) (Scheme
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a
Scheme 5
a
Reagents and reaction conditions: (a) LDA, THF, −40 °C, 10 min, then N-formylpiperidine, −40 °C, 30 min, 93%; (b) CH3OH, HC(OCH3)3, p-
TolSO3H, 65 °C, 3 h, 92%; (c) NCS, CH2Cl2/CH3OH 1:1, rt, 3 h, 60%; (d) LDA, THF, −45 °C, 10 min, then benzyl bromide, −40 °C, 30 min,
24%; (e) LDA, THF, −40 °C, 10 min, then benzaldehyde, −40 °C, 30 min, 92%; (f) PhMgBr, THF, 0 °C, 10 min, 87%; (g) BuSiH3, B(C6F5)3,
CH2Cl2, rt, 8 h, 58%; (h) n-BuLi, THF, −78 °C, 15 min, then 1-benzylpiperidin-4-one, −78 °C, 1−2 h, rt, 1 h; (i) CH3OH, HC(OCH3)3, p-
TolSO3H, rt, 1−3 H, 32% 39, 33% 40, 26% 41.
a
Scheme 6
a
Reagents and reaction conditions: (a) 1 M HCl, CH3CN, rt, 15 min, 96%; (b) LiAlH4, THF, −40 °C, 30 min, 57%; (c) HN(CH3)2, NaBH(OAc)3,
CH2Cl2, rt, 1.5 h, 77%; (d) PhMgBr, THF, 0 °C, 15 min, 98%; (e) Pr4NRuO4 (5 mol %), NMMO (5 equiv), CH2Cl2, molecular sieves 4 Å, rt, 30
min, 72%; (f) H2NOH HCl, pyridine, 60 °C, 30 min, 70%; (g) phthalic anhydride, pyridine, 90 °C. 1 h, 67%; (i) CH3OH, H2SO4 conc, reflux, 22 h,
60%.
IC50-values were transformed into Ki-values by the equation of
Cheng and Prusoff.47
receptor, the 5-HT6, 5-HT7, and α2A receptor were determined
in receptor binding studies.
To investigate the receptor selectivity, the affinity of all
spirocyclic thiophenes toward the phencyclidine binding site of
the NMDA receptor was recorded with pig brain cortex
preparations and the radioligand [3H]-(+)-MK-801.48 More-
over, the affinities of selected compounds toward the human σ1
DISCUSSION
■
The σ receptor binding data of the synthesized spirocyclic
thiophene derivatives are summarized in Tables 1−3. Table 1
shows that the σ1 affinities of the cyclohexylmethyl derivatives
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Table 1. Affinities of the Spiro[piperidine-4,4′-thieno[3,2-c]pyrans] with Different Substituents in Position 1 and 6′ towards σ1
and σ2 Receptors
a
Ki SEM (nM) (n = 3)
compd
R1
R2
σ1
σ2
σ1/σ2 selectivity
b
b
6a34
Bn
Bn
Bn
1.1 0.22
1.3 0.18
0.32 0.1
0.29 0.1
3.6 0.7
1.4 0.2
0.46 0.04
2.0 0.5
1.7 0.1
0.31 0.06
0.66 0.16
3.9 1.5
61 18
1280 137
3500 352
1260
1160
2700
3940
86
7a34
8a36
OCH3
OCH3
OH
8b
CH2C6H11
Bn
25 8.0
391
10a
109
560
57
11a
Bn
CN
783
11b
CH2C6H11
Bn
CN
26 4.0
61 18
45 12
13 2.5
3.3 0.3
78 2.0
42 15
12a
C6′C7′
C6′C7′
H
31
12b
CH2C6H11
Bn
26
13a
42
13b
CH2C6H11
H
5
haloperidol
di-o-tolylguanidine
(+)-pentazocine
20
0.7
4.2 1.1
a
Triplicates were recorded only for high affinity compounds. The affinity of compounds, which did not reduce significantly the radioligand binding
b
in the first assay, were recorded only once. The structures of 6a and 7a are shown in Figure 2.
(b-series) are comparable with the σ1 affinities of the
corresponding benzyl derivatives (a-series). In both series of
compounds, the cyano moiety can be regarded as bioisosteric
replacement of the methoxy group because the corresponding
Ki-values of 8 and 11 are very similar (e.g., 8b, Ki = 0.30 nM;
11b, Ki = 0.46 nM). In addition to the methoxy derivatives 8
and the nitriles 11 the unsubstituted compounds 12 and 13
reveal low nanomolar and even subnanomolar σ1 affinities.
Taking the σ2 affinity into account, two general rules were
derived from this series of compounds. Replacement of the N-
benzyl moiety with the N-cyclohexylmethyl residue led to a
considerable increase of the σ2 affinity and thus to a reduced
σ1/σ2-selectivity (e.g., 11a, σ1/σ2 = 560; 11b, σ1/σ2 = 57).
Second, an increased σ2-affinity and thus reduced σ1/σ2-
selectivity was observed upon removal of the substituent in 6′-
position of the spirocyclic system (e.g., 8a, σ1/σ2 3940; 12a, σ1/
σ2 = 31; 13a, σ1/σ2 = 42). Therefore, in the next series of
compounds, the N-benzyl moiety was retained and the
substituents in 6′-position were varied. (Table 2)
In Table 2, the affinity data of spirocyclic thiophene
derivatives with various substituents in 6′-position are
summarized. Small substituents in 6′-position like a methoxy
group of 8a, a hydroxy group of 10a, and a cyano group of 11a
gave very potent σ1 ligands. However, a carbonyl group (22)
was not tolerated by the σ1 receptor protein. Increasing of the
chain length of the homologous alcohols 10a, 20, and 27 led to
continuous decrease of σ1 affinity, indicating that the size of the
substituent tolerated by the σ1 receptor is limited. Comparing
the σ1 affinities of the homologous nitriles 11a and 23 showed
the same tendency of 50-fold reduced σ1 affinity by extension of
the side chain. Esters 14 and 15 bearing the carbonyl moiety
directly at the spirocyclic system revealed lower σ1 affinities
than the corresponding homologous 24−26 with an additional
CH2-moiety between the spirocyclic system and the carbonyl
moiety. Very polar (e.g., primary amide in 16) and basic
substituents (e.g., primary amine in 17, benzylamine in 18,
dimethylamine in 21) were less tolerated by the σ1 receptor
protein.
It is remarkable that the compounds with substituents in 6′-
position generally show low affinity to the σ2 receptor,
indicating high σ1/σ2 selectivity.
To expand the structure−affinity relationships of these types
of spirocyclic compounds, substituents with various H-bond
forming and lipophilic properties were introduced in 2′-position
adjacent to the S-atom. For this purpose the benzyl substituent
at the piperidine N-atom and the methoxy group in 6′-position
were selected because these structural elements led to the
highest σ1 affinity and σ1/σ2 selectivity in this class of
compounds (compare compound 8a). The σ affinities of the
compounds are depicted in Table 3.
It can be clearly seen that the σ1 receptor accepts a broad
variety of substituents at the 2′-position of the spirocyclic
system. These substituents include the lipophilic benzyl, α-
hydroxybenzyl, and benzoyl moieties of 41 (Ki = 3.3 nM), 45
(Ki = 3.6 nM), and 46 (Ki = 3.7 nM), which cannot form any
H-bonds (41), react as H-bond donor and acceptor (45), or
only as H-bond acceptor (46). Similar high σ1 receptor affinities
were determined for the acetal 39, the chloro derivative 40, the
alcohol 43, and the nitrile 48. Low σ1 affinities for compounds
bearing very polar or basic substituents in 2′-position (e.g.,
oxime 47, tertiary amine 44) were observed to be following the
same trend as the compounds with polar and basic substituents
in the 6′-position.
As discussed before, the N-benzyl moiety and the 6′-methoxy
group are responsible for the preferred interaction of the
ligands with the σ1 receptor. Table 3 clearly shows that
additional substituents in 2′-position do not change this
situation. The σ2 affinity of all 2′-substituted compounds is
very low, leading to high σ1/σ2 selectivity of these compounds.
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Table 2. Affinities of the Spiro[piperidine-4,4′-thieno[3,2-
c]pyrans] with Different Substituents in Position 6′ towards
σ1 and σ2 Receptors
Table 3. Affinities of the Spiro[piperidine-4,4′-thieno[3,2-
c]pyrans] with Different Substituents in Position 2′ towards
σ1 and σ2 Receptors
a
a
Ki SEM (nM) (n = 3)
Ki SEM (nM) (n = 3)
compd
R3
σ1
σ2
σ1/σ2 selectivity
σ1/σ2
compd
R2
σ1
σ2
selectivity
8a36
39
40
41
43
44
45
46
47
48
49
H
0.32 0.10
2.3 0.4
1.3 0.1
3.3 0.7
5.8 1.2
17 0.6
3.6 1.5
3.7 1.4
39 2.7
1.1 0.1
11 1.3
3.9 1.5
61 18
1260
>1000
3940
>430
>770
125
>170
53
8a36
10a
11a
14
15
16
17
18
20
21
22
23
24
25
26
27
OCH3
OH
0.32 0.10
3.6 0.7
1.4 0.20
29 3.2
144
1260
391
3940
109
559
>34
>7.0
>2.8
>10
3.1
CH(OCH3)2
Cl
>1000
CN
783
CH2C6H5
CH2OH
411
CO2CH3
>1000
>1000
>1000
>1000
621
>1000
CO2C2H5
CH2N(CH3)2
CH(OH)C6H5
(CO)C6H5
CHNOH
CN
898
CONH2
350
882 17
427 163
>1000
245
115
>25
>900
>90
20
CH2NH2
98
CH2NHBn
CH2OH
196 24
5.4 1.5
63
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
78 2.0
42 15
>185
>16
>3
>1000
CH2N(CH3)2
O
CO2CH3
>1000
296
haloperidol
78 2.0
42 15
CH2CN
64 16
9.4 4.9
12 0.7
20 2.8
27 4.3
3.9 1.5
61 18
4.2 1.1
>15
>106
>83
>50
>37
20
di-o-tolylguanidine
(+)-pentazocine
0.7
CH2C(O)CH3
CH2CO2CH3
CH2CO2C2H5
CH2CH2OH
4.2 1.1
a
Triplicates were recorded only for high affinity compounds. The
affinity of compounds, which did not reduce significantly the
radioligand binding in the first assay, were recorded only once.
haloperidol
di-o-tolylguanidine
(+)-pentazocine
0.7
affinity close to the optimum. The reduced negative electro-
static potential (red color) of the thiophene ring of 40 and 48
compared with 8a is displayed in Figure 3.
a
Triplicates were recorded only for high affinity compounds. The
affinity of compounds, which did not reduce significantly the
radioligand binding in the first assay, were recorded only once.
CONCLUSION
■
A flexible synthetic approach allows the introduction of
versatile substituents in various positions of the spirocyclic
system 9. The large series of compounds represents the basis
for the establishment of structure−affinity relationships. To
achieve high σ1 affinity and high selectivity against the σ2
subtype, a benzyl moiety should be attached to the piperidine
N-atom and a small substituent (e.g., OH, CN, CH2OH)
should be introduced into the6′-position. Whereas compounds
without a substituent in the 6′-position represent potent but
unselective σ ligands, compounds with larger substituents in the
6′-position show reduced σ1 affinity. The σ1 receptor tolerates
well substituents in the 2′-position with different lipophilic and
H-bond forming properties. However, it does not accept very
polar and basic substituents in the 2′- and 6′-positions. The
similar Ki-values obtained for 11a and 13a in σ1 assays using
guinea pig and human σ1 receptors show the good correlation
between guinea pig brain and human σ1 receptors.
Next, the affinity of the nitrile 11a and the unsubstituted
compound 13a at the human σ1 receptor was recorded in
addition to their affinities toward the guinea pig brain σ1
receptor. The resulting Ki-values of 4.5 0.5 nM (11a) and
2.1 0.4 nM (13a) are in good accordance with the Ki-values
obtained in the guinea pig brain assay (see Table 1).
Because of the historical correlation of the σ receptor and the
phencyclidine binding site of the NMDA receptor (see
Introduction) and, moreover, the interaction of these receptors
with similar compounds,49,50 the NMDA receptor affinity of all
synthesized spirocyclic compounds was also recorded. Up to a
test compound concentration of 1 μM, significant interactions
with the NMDA receptor were not observed, indicating high
selectivity of the spirocyclic thiophenes over this receptor.
Additionally, the nitrile 11a and the unsubstituted compound
13a did not interact with the 5-HT6, 5-HT7, and α2A receptors.
Replacement of the benzene moiety of the benzopyran 7a
with the thiophene moiety (8a) led to a 4-fold increase of the
σ1 receptor affinity. Introduction of a chloro (40) or cyano
group (48) in 2′-position adjacent to the S-atom resulted in σ1
affinities, which are very similar to the σ1 affinities of the
benzene derivative 7a. As the introduction of a chloro or cyano
substituent into the thiophene system decreases the electron
density of the thiophene ring, it can be assumed that the
electron density of the aromatic system has a significant impact
on the interaction with the σ1 receptor. Compound 8a with the
unsubstituted electron rich thiophene ring has a σ1 receptor
EXPERIMENTAL SECTION
■
Chemistry, General. Unless otherwise noted, moisture sensitive
reactions were conducted under dry nitrogen. Flash chromatography
(fc): silica gel 60, 40−64 μm (Merck). Parentheses include: diameter
of the column, eluent, Rf value. 1H NMR (400 MHz): Mercury-400BB
spectrometer (Varian); δ in ppm related to tetramethylsilane; coupling
constants are given with 0.5 Hz resolution; the assignments of 13C and
1H NMR signals were supported by 2D NMR techniques. Elemental
analysis: CHN-Rapid Analysator (Fons-Heraeus). The purity of all test
compounds was determined by HPLC analysis (purity >95%).
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Article
Figure 3. 3D electrostatic contour plots (Connolly Analytic) calculated with the molecular modeling software MOE (Molecular Operating
Environment) for the AM1 minimized (R)-enantiomers of σ1 ligands 8a, 40, and 48. Blue regions of the surface indicate a positive, red regions a
negative electrostatic potential.
Additionally elemental analyses were performed for key compounds;
all values are within 0.4%.
(200 mg, 0.67 mmol) in CH3OH (20 mL). The mixture was shaken
under H2 pressure (4.5 bar) for 14 h at rt. After filtration, the solvent
was evaporated in vacuo. The residue was purified by fc (2 cm,
cyclohexane:EtOAc, 7:3, Rf = 0.2). Colorless oil, yield 172 mg (85%),
1-Benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-
6′-ol (10a). HCl (2 M, 1 mL) was added to a solution of 8a (74 mg,
0.22 mmol) in CH3CN (4 mL). The mixture was heated to reflux for 1
h. After addition of 2 M NaOH (pH > 9), the mixture was extracted
with Et2O (3 × 5 mL). The organic layer was dried (Na2SO4), filtered,
and concentrated in vacuo, and the residue was purified by
recrystallization with CH3CN. Colorless solid, mp 184 °C, yield 45
mg (64%), C18H21NO2S (315.2). 1H NMR (CDCl3): δ (ppm) = 1.76
(dd, J = 13.6/2.3 Hz, 1 H, N(CH2CH2)2), 1.80−1.89 (m, 2 H,
N(CH2CH2)2), 2.04 (td, J = 13.2/4.4 Hz, 1 H, N(CH2CH2)2), 2.37
(td, J = 11.7/4.5 Hz, 1 H, N(CH2CH2)2), 2.43 (td, J = 11.7/2.5 Hz, 1
H, N(CH2CH2)2), 2.62−2.72 (m, 2 H, N(CH2CH2)2), 2.74 (dd, J =
15.5/7.3 Hz, 1 H, ThCH2CH), 2.91 (s, 1 H, OH), 3.00 (dd, J = 15.5/
3.1 Hz, 1 H, ThCH2CH), 3.48 (d, J = 14.2 Hz, 1 H, NCH2Ph), 3.51
(d, J = 14.2 Hz, 1 H, NCH2Ph), 5.25 (dd, J = 7.3/3.1 Hz, 1 H,
ThCH2CH), 6.74 (d, J = 5.2 Hz, 1 H, 3′-H-Th), 7.04 (d, J = 5.2 Hz, 1
H, 2′-H-Th), 7.16−7.29 (m, 5 H, Ph-H).
1-Benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-
6′-carbonitrile (11a) and 1-Benzylspiro[piperidine-4,4′-thieno[3,2-
c]pyran] (12a). Trimethylsilyl cyanide (TMSCN, 0.88 mL, 7 mmol)
and BF3·Et2O (0.16 mL, 1.6 mmol) were added to a cooled solution
(−20 °C) of 8a (290 mg, 0.88 mmol) in CH2Cl2 (3.5 mL). At first, the
reaction mixture was stirred for 30 min at −20 °C, then for 15 min at 0
°C. Then MeOH (3 mL) and 2 M NaOH (3 mL) were added (pH >
9) and the mixture was extracted with CH2Cl2 (3 × 5 mL). The
organic layer was dried (Na2SO4), filtered, concentrated in vacuo, and
the residue was purified by fc (2 cm, cyclohexane:EtOAc, 7:3, Rf (11a)
= 0.34), Rf (12a) = 0.42).
1
C18H21NOS (299.4). H NMR (CDCl3): δ (ppm) = 1.77 (dd, J =
14.4/2.3 Hz, 2 H, N(CH2CH2)2), 1.91 (td, J = 13.4/4.5 Hz, 2 H,
N(CH2CH2)2), 2.33 (td, J = 12.6/2.5 Hz, 2 H, N(CH2CH2)2), 2.64−
2.67 (m, 2 H, N(CH2CH2)2), 2.75 (t, J = 5.4 Hz, 2 H, ThCH2CH2),
3.49 (s, 2 H, NCH2Ph), 3.85 (t, J = 5.4 Hz, 2 H, ThCH2CH2), 6.74 (d,
J = 5.5 Hz, 1 H, 3′-H-Th), 6.98 (d, J = 5.5 Hz, 1 H, 2′-H-Th), 7.19−
7.30 (m, 5 H, Ph-H).
1-(Cyclohexylmethyl)-6′,7′-dihydrospiro[piperidine-4,4′-thieno-
[3,2-c]pyran] (13b). Pd/C (15 mg, 10% (m/m)) was added to a
solution of 12b (60 mg, 0.2 mmol) in CH3OH (8 mL). The mixture
was shaken under H2 pressure (4.5 bar) for 6 h at rt. After filtration,
the solvent was evaporated in vacuo. The residue was purified by fc
(0.7 cm, cyclohexane:EtOAc, 9:1, Rf = 0.2). Colorless solid, mp 77 °C,
yield 31 mg (51%), C18H27NOS (305.5). 1H NMR (CDCl3): δ (ppm)
= 0.83−0.95 (m, 2 H, cHex-H), 1.13−1.30 (m, 3 H, cHex-H), 1.45−
1.55 (m, 1 H, NCH2-CH(CH2)5), 1.63−1.74 (m, 3 H, cHex-H),
1.75−1.83 (m, 2 H, cHex-H), 1.83 (dd, J = 14.4/2.8 Hz, 2 H,
N(CH2CH2)2), 1.96 (td, J = 13.6/4.5 Hz, 2 H, N(CH2CH2)2), 2.17
(d, J = 7.2 Hz, 2 H, NCH2cHex), 2.27 (td, J = 12.8/2.8 Hz, 2 H,
N(CH2CH2)2), 2.65−2.71 (m, 2 H, N(CH2CH2)2), 2.82 (t, J = 5.6
Hz,2 H, ThCH2CH2), 3.92 (t, J = 5.6 Hz, 2 H, ThCH2CH2), 6.81 (d, J
= 5.2 Hz, 1 H, 3′-H -Th), 7.06 (d, J = 5.2 Hz, 1 H, 2′-H-TH). Anal.
(C18H27NOS) C, H, N.
(1-Benzyl-6′,7′-dihyrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-
6′-yl)methanol (20). NaBH4 (20 mg, 0.52 mmol) was added to a
solution of 19 (85 mg, 0.26 mmol) in CH3OH (3 mL) at 0 °C. After
15 min stirring at 0 °C, the mixture was stirred at rt for 45 min. Then,
brine was added and the mixture was extracted with CH2Cl2 (3 × 5
mL). The organic layer was dried (Na2SO4), filtered, and concentrated
in vacuo. The residue was purified by fc (1 cm, cyclohexane:EtOAc,
7:3, Rf = 0.08). Colorless solid, mp 108 °C, yield 60 mg (70%),
11a: Colorless solid, mp 123 °C, yield 228 mg (79%), C19H20N2OS
1
(324.4). H NMR (CDCl3): δ (ppm) = 1.77 (dd, J = 13.8/2.7 Hz, 1
H, N(CH2CH2)2), 1.80−1.89 (m, 2 H, N(CH2CH2)2), 2.03 (td, J =
13.2/4.5 Hz, 1 H, N(CH2CH2)2), 2.33 (td, J = 11.8/3.1 Hz, 1 H,
N(CH2CH2)2), 2.43 (td, J = 12.6/2.7 Hz, 1 H, N(CH2CH2)2), 2.64−
2.75 (m, 2 H, N(CH2CH2)2), 3.04 (dd, J = 15.6/3.9 Hz, 1 H,
ThCH2CH), 3.16 (dd, J = 15.6/9.0 Hz, 1 H, ThCH2CH), 3.48 (d, J =
13.0 Hz, 1 H, NCH2‑Ph), 3.53 (d, J = 13.0 Hz, 1 H, NCH2Ph), 4.65
(dd, J = 9.0/3.3 Hz, 1 H, ThCH2CH), 6.75 (d, J = 5.1 Hz, 1 H, 3′-H-
Th), 7.09 (d, J = 5.1 Hz, 1 H, 2′-H-Th), 7.19−7.31 (m, 5 H, Ph-H).
Anal. (C19H20N2OS) C, H, N.
12a: Colorless solid, mp 71 °C, yield 47 mg (18%), C18H19NOS
(297.4). 1H NMR (CDCl3): δ (ppm) = 1.84 (td, J = 13.4/4.6 Hz, 2 H,
N(CH2CH2)2), 2.16 (dd, J = 14.3/2.1 Hz, 2 H, N(CH2CH2)2), 2.37
(td, J = 12.5/2.4 Hz, 2 H, N(CH2CH2)2), 2.68 (m, 2 H,
N(CH2CH2)2), 3.50 (s, 2 H, N-CH2-Ph), 5.71 (d, J = 5.9 Hz, 1 H,
Th-CHCH), 6.38 (d, J = 5.9 Hz, 1 H, Th-CHCH), 6.71 (d, J =
5.1 Hz, 1 H, 3′-H-Th), 6.97 (d, J = 5.1 Hz, 1 H, 2′-H-Th), 7.19−7.31
(m, 5 H, Ph-H).
1
C19H23NO2S (329.5). H NMR (CDCl3): δ (ppm) = 1.69 (dd, J =
13.6/2.8 Hz, 1 H, N(CH2CH2)2), 1.83 (td, J = 12.8/4.4 Hz, 1 H,
N(CH2CH2)2), 2.02 (dd, J = 13.6/2.8 Hz, 1 H, N(CH2CH2)2), 2.15
(td, J = 13.2/4.8 Hz, 1 H, N(CH2CH2)2), 2.30 (td, J = 12.8/2.4 Hz, 1
H, N(CH2CH2)2), 2.44 (td, J = 12.0/2.4 Hz, 1 H, N(CH2CH2)2), 2.64
(dd, J = 16.0/4.0 Hz, 1 H, ThCH2), 2.67−2.78 (m, 3 H
(N(CH2CH2)2) (ThCH2)), 3.55 (s, 2 H, NCH2Ph), 3.70 (dd, J =
11.0/7.3 Hz, 1 H, ThCH2CHCH2), 3.78−3.84 (m, 1 H,
ThCH2CHCH2), 3.90−3.97 (m, 1 H, ThCH2CHCH2), 6.81 (d, J =
5.2 Hz, 1 H, 3′-H-Th), 7.08 (d, J = 5.2 Hz, 1 H, 2′-H-Th), 7.23−7.34
(m, 5 H, Ph-H). A signal for the O−H proton is not seen.
1-(1-Benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]-
pyran]-6′-yl)acetone (24). Acetylmethylenetriphenylphosphorane (91
mg, 0.28 mmol) and Cs2CO3 (67 mg, 0.20 mmol) were added to a
solution of 10a (60 mg, 0.19 mmol) in THF (4 mL). The mixture was
stirred under reflux for 12 h. Then H2O (10 mL) was added and the
1-Benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]
(13a). Pd/C (50 mg, 10% (m/m)) was added to a solution of 12a
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mixture was extracted with CH2Cl2 (3 × 5 mL). The organic layer was
dried (Na2SO4), filtered, and concentrated in vacuo. The residue was
purified by fc (1 cm, ccyclohexane:EtOAc, 7:3, Rf = 0.25). Colorless
2.74−2.83 (m, 2 H, N(CH2CH2)2), 2.87 (dd, J = 16.4/6.4 Hz, 1 H,
ThCH2CH), 3.10 (dd, J = 16.4/3.6 Hz, 1 H, ThCH2CH), 3.56 (s, 3 H,
OCH3), 3.57 (d, J = 13.2 Hz, 1 H, NCH2Ph), 3.59 (d, J = 13.2 Hz, 1
H, NCH2Ph), 4.92 (dd, J = 6.4/3.6 Hz, 1 H, ThCH2CH), 7.33 (s, 1 H,
3′-H-Th), 7.20−7.38 (m, 5 H, Ph-H). Anal. (C20H22N2O2S) C, H, N.
Receptor Binding Studies. Materials and General Procedures.
guinea pig brains and rat livers were commercially available (Harlan-
Winkelmann, Germany). Homogenizer: Elvehjem potter (B. Braun
Biotech International). Centrifuge: high-speed cooling centrifuge
model Sorvall RC-5C plus (Thermo Finnigan). Filter: Printed
Filtermat Type A (Perkin-Elmer), presoaked in 0.5% aqueous
polyethylenimine for 2 h at rt before use. The filtration was carried
out with a MicroBeta FilterMate-96 harvester (Perkin-Elmer). The
scintillation analysis was performed using Meltilex (Type A) solid
scintillator (Perkin-Elmer). The radioactivity bound to the filter was
measured using a MicroBeta Trilux scintillation analyzer (Perkin-
Elmer). The overall counting efficiency was 20%.
1
oil, yield 45 mg (67%), C21H25NO2S (355.5). H NMR (CDCl3): δ
(ppm) = 1.58 (dd, J = 14.2/5.2 Hz, 1 H, N(CH2CH2)2), 1.73 (td, J =
12.9/4.4 Hz, 1 H, N(CH2CH2)2), 1.94 (dd, J = 14.2/5.2 Hz, 1 H,
N(CH2CH2)2), 2.04 (td, J = 13.2/4.2 Hz, 1 H, N(CH2CH2)2), 2.17
(td, J = 11.8/2.4 Hz, 1 H, N(CH2CH2)2), 2.18 (s, 3 H, CH3), 2.30 (td,
J = 11.8/2.4 Hz, 1 H, N(CH2CH2)2), 2.49 (dd, J = 15.0/4.0 Hz, 1 H,
ThCH2CHCH2), 2.55 (dd, J = 15.6/10.2 Hz, 1 H, ThCH2CHCH2),
2.58−2.67 (m, 2 H, N(CH2CH2)2), 2.70 (dd, J = 15.6/3.1 Hz, 1 H,
ThCH2CHCH2) 2.82 (dd, J = 15.0/8.5 Hz, 1 H, ThCH2CHCH2),
3.45 (d, J = 13.0 Hz, 1 H, NCH2Ph), 3.48 (d, J = 13.0 Hz, 1 H,
NCH2Ph), 4.18 (m, 1 H, ThCH2CHCH2), 6.72 (d, J = 5.2 Hz, 1 H, 3′-
H-Th), 6.99 (d, J = 5.2 Hz, 1 H, 2′-H-Th), 7.17−7.24 (m, 5 H, Ph-H).
(1-Benzyl-6′-methoxy-6′,7′-dihydrospiro[piperidine-4,4′-thieno-
[3.2-c]pyran]-2′-carb-aldehyde Dimethyl Acetal (39). Trimethyl
orthoformate (2 mL) and p-toluenesulfonic acid monohydrate (260
mg, 1.37 mmol) were added to a solution of 36 (460 mg, unpurified)
in MeOH (15 mL). The mixture was stirred for 45 min at rt. After
addition of 2 M NaOH (5 mL), the mixture was extracted with
CH2Cl2 (3 × 5 mL). The organic layer was dried (Na2SO4), filtered,
and concentrated in vacuo. The residue was purified by fc (3 cm,
cyclohexane:EtOAc, 7:3, Rf = 0.23 (cyclohexane:EtOAc, 1:1)).
Colorless solid, mp 71 °C, yield 287 mg (32% related to 33 over
two steps), C22H29NO4S (403.5). 1H NMR (CDCl3): δ (ppm) = 1.81
(dd, J = 13.6/2.8 Hz, 1 H, N(CH2CH2)2), 1.85 (td, J = 12.0/4.0 Hz, 1
H, N(CH2CH2)2), 1.94 (dd, J = 13.6/2.8 Hz, 1 H, N(CH2CH2)2),
2.08 (td, J = 13.6/4.8 Hz, 1 H, N(CH2CH2)2), 2.42 (td, J = 12.0/3.2
Hz, 1 H, N(CH2CH2)2), 2.51 (td, J = 12.0/2.8 Hz, 1 H,
N(CH2CH2)2), 2.72−2.79 (m, 2 H, N(CH2CH2)2), 2.74 (dd, J =
15.6/7.2 Hz, 1 H, ThCH2CH), 2.94 (dd, J = 15.6/3.6 Hz, 1 H,
ThCH2CH), 3.35 (s, 6 H, CH(OCH3)2), 3.55 (s, 3 H,
ThCH2CHOCH3), 3.57 (d, J = 16.0 Hz, 1 H, NCH2Ph), 3.59 (d, J
= 16.0 Hz, 1 H, NCH2Ph), 4.86 (dd, J = 7.2/3.6 Hz, 1 H, ThCH2CH),
5.51 (s, 1 H, ThCH), 6.79 (s, 1 H, 3′-H-Th), 7.19−7.32 (m, 5 H, Ph-
H).
Membrane Preparation for the σ1 Sssay.34,46 Five guinea pig
brains were homogenized with the potter (500−800 rpm, 10 up-and-
down strokes) in 6 volumes of cold 0.32 M sucrose. The suspension
was centrifuged at 1200g for 10 min at 4 °C. The supernatant was
separated and centrifuged at 23500g for 20 min at 4 °C. The pellet was
resuspended in 5−6 volumes of buffer (50 mM TRIS, pH 7.4) and
centrifuged again at 23500g (20 min, 4 °C). This procedure was
repeated twice. The final pellet was resuspended in 5−6 volumes of
buffer, the protein concentration was determined according to the
method of Bradford51 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.
Performing of the σ1 Assay.31−34,46 The test was performed with
the radioligand [3H]-(+)-pentazocine (22 Ci/mmol; Perkin-Elmer).
The thawed membrane preparation (about 75 μg of the protein) was
incubated with various concentrations of test compounds, 2 nM [3H]-
(+)-pentazocine, and buffer (50 mM TRIS, pH 7.4) in a total volume
of 200 μL for 120 min at 37 °C. The incubation was terminated by
rapid filtration through the presoaked filtermats by using the cell
harvester. After washing each well five times with 300 μL 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
nonspecific binding was determined with 10 μM unlabeled
(+)-pentazocine. The Kd-value of the radioligand [3H]-(+)-pentazo-
cine is 2.9 nM.52
Data Analysis. Usually, all experiments were carried out in
triplicates using standard 96-well-multiplates (Diagonal). The IC50-
values were determined in competition experiments with at least six
concentrations of the test compounds and were calculated with the
program GraphPad Prism 3.0 (GraphPad Software) by nonlinear
regression analysis. The Ki-values were calculated according to Cheng
and Prusoff.47 The Ki-values of highly affine compounds are given as
mean values SEM from three independent experiments.
1-Benzyl-2′-chloro-6′-methoxy-6′,7′-dihydrospiro[piperidine-
4,4′-thieno[3.2-c]-pyran] (40). Trimethyl orthoformate (0.5 mL) and
p-toluenesulfonic acid monohydrate (120 mg, 0.63 mmol) were added
to a solution of 37 (120 mg, unpurified) in MeOH (6 mL). The
mixture was stirred for 3 h at rt. After addition of 2 M NaOH (5 mL),
the mixture was extracted with CH2Cl2 (3 × 5 mL). The organic layer
was dried (Na2SO4), filtered, and concentrated in vacuo. The residue
was purified by fc (2 cm, cyclohexane:EtOAc, 7:3, Rf = 0.36). Colorless
solid, mp 117 °C, yield 77 mg (33% related to 34 over two steps),
1
C19H22ClNO2S (363.6). H NMR (CDCl3): δ (ppm) = 1.81 (dd, J =
13.2/2.8 Hz, 1 H, N(CH2CH2)2), 1.83 (td, J = 13.6/4.4 Hz, 1 H,
N(CH2CH2)2), 1.93 (dd, J = 14.0/2.8 Hz, 1 H, N(CH2CH2)2), 2.01
(td, J = 13.6/4.4 Hz, 1 H, N(CH2CH2)2), 2.41 (td, J = 11.6/2.8 Hz, 1
H, N(CH2CH2)2), 2.50 (td, J = 11.6/2.8 Hz, 1 H, N(CH2CH2)2),
2.72−2.79 (m, 2 H, N(CH2CH2)2), 2.74 (dd, J = 15.6/7.2 Hz, 1 H,
ThCH2CH), 2.86 (dd, J = 15.6/3.2 Hz, 1 H, ThCH2CH), 3.54 (s, 3 H,
OCH3), 3.54 (d, J = 12.8 Hz, 1 H, NCH2Ph), 3.58 (d, J = 12.8 Hz, 1
H, NCH2Ph), 4.86 (dd, J = 7.2/3.2 Hz, 1 H, ThCH2CH), 6.62 (s, 1 H,
3′-H -Th), 7.20−7.35 (m, 5 H, Ph-H).
1-Benzyl-6′-methoxy-6′,7′-dihydrospiro[piperidine-4,4′-thieno-
[3,2-c]pyran]-2′-carbo-nitrile (48). Hydroxylamine hydrochloride (50
mg, 0.72 mmol) was added to a solution of 42 (150 mg, 0.42 mmol) in
pyridine (4 mL). After stirring the mixture for 30 min at 60 °C,
phthalic acid anhydride (240 mg, 1.6 mmol) was added and the
mixture stirring was continued for 60 min at 90 °C. Then brine was
added, and the mixture was extracted with CH2Cl2 (3 × 5 mL). The
organic layer was dried (Na2SO4), filtered, and concentrated in vacuo.
The residue was purified by fc (3 cm, cyclohexane:EtOAc; 4:6, Rf =
0.37). Colorless solid, mp 154 °C, yield 68 mg (46%), C20H22N2O2S
(354.4). 1H NMR (CDCl3): δ (ppm) = 1.80−1.88 (m, 2 H,
N(CH2CH2)2), 1.92 (dd, J = 14.0/2.8 Hz, 1 H, N(CH2CH2)2), 2.03
(td, J = 12.8/4.8 Hz, 1 H, N(CH2CH2)2), 2.42 (td, J = 11.6/2.8 Hz, 1
H, N(CH2CH2)2), 2.51 (td, J = 11.8/2.8 Hz, 1 H, N(CH2CH2)2),
Experimental details for the σ2 assay: see refs 34,46.
Experimental details for the NMDA assay: see ref 48.
ASSOCIATED CONTENT
■
S
* Supporting Information
Physical and spectroscopic data of all new compounds. Purity
data. General chemistry methods. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49-251-8333311. Fax: +49-251-8332144. E-mail:
wuensch@uni-muenster.de.
Notes
The authors declare no competing financial interest.
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Article
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ACKNOWLEDGMENTS
■
Thanks are due to the Deutsche Forschungsgemeinschaft for
financial support of this project.
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PCP, 1-(1-phenylcyclohexyl)piperidine (phencyclidine);
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TMSCN, trimethylsilyl cyanide; DIBAL, diisobutylaluminum
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