Thiophene bioisosteres of spirocyclic σ receptor ligands. 1. N-substituted spiro[piperidine-4,4′-thieno[3,2-c]pyrans]

Article abstract of DOI:10.1021/jm8007739

Herein, the synthesis and pharmacological evaluation of thiophene bioisosteres of the highly potent spirocyclic benzopyran 1 are detailed. The synthesis of 1-benzyl-6′-methoxy-6′,7′- dihydrospiro[piperidine-4,4′-thieno[3.2-c]pyran] (2a) was performed starting with 3-bromothiophene (3). After introduction of the acetaldehyde substructure (7), halogen metal exchange, addition of 1-benzylpiperidin-4-one, and cyclization led to the spirocyclic thienopyran 2a. The removal of the benzyl group afforded the secondary amine 2f, which was substituted with various residues. With respect to σ₁ affinity the N-benzyl derivative 2a, the N-cyclohexylmethyl derivative 2d, and the N-p-fluorobenzyl derivative 2i represent the most potent compounds of this series binding with K_i values of 0.32, 0.29, and 0.62 nM, respectively. Electronic properties of the substituents have only little impact on σ₁ affinity. The most potent σ₁ ligands display high selectivity against σ₂, 5-HT_1A, 5-HT₆, 5-HT₇, α_1A, α₂, and NMDA receptors. The activity of 2a in the mouse capsaicin assay seems to indicate σ₁ antagonistic activity.

Full text of DOI:10.1021/jm8007739

J. Med. Chem. 2008, 51, 6531–6537
6531
Thiophene Bioisosteres of Spirocyclic σ Receptor Ligands. 1. N-Substituted
Spiro[piperidine-4,4′-thieno[3,2-c]pyrans]
Christoph Oberdorf,† Dirk Schepmann,† Jose Miguel Vela,‡ Jose Luis Diaz,‡ Jo¨rg Holenz,‡ and Bernhard Wu¨nsch*,†
Institut fu¨r Pharmazeutische and Medizinische Chemie der UniVersita¨t Mu¨nster, Hittorfstrasse 58-62, D-48149 Mu¨nster, Germany, and EsteVe,
AV. Mare de Deu de Montserrat 221, 08041 Barcelona, Spain
ReceiVed June 25, 2008
Herein, the synthesis and pharmacological evaluation of thiophene bioisosteres of the highly potent spirocyclic
benzopyran 1 are detailed. The synthesis of 1-benzyl-6′-methoxy-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3.2-
c]pyran] (2a) was performed starting with 3-bromothiophene (3). After introduction of the acetaldehyde
substructure (7), halogen metal exchange, addition of 1-benzylpiperidin-4-one, and cyclization led to the
spirocyclic thienopyran 2a. The removal of the benzyl group afforded the secondary amine 2f, which was
substituted with various residues. With respect to σ1 affinity the N-benzyl derivative 2a, the N-
cyclohexylmethyl derivative 2d, and the N-p-fluorobenzyl derivative 2i represent the most potent compounds
of this series binding with Ki values of 0.32, 0.29, and 0.62 nM, respectively. Electronic properties of the
substituents have only little impact on σ1 affinity. The most potent σ1 ligands display high selectivity against
σ2, 5-HT1A, 5-HT6, 5-HT7, R1A, R2, and NMDA receptors. The activity of 2a in the mouse capsaicin assay
seems to indicate σ1 antagonistic activity.
Introduction
The rat brain σ1 gene encodes for a protein consisting of 223
amino acids with a molecular weight of 23 kDa. The membrane
localized σ1 receptor has not been crystallized yet, but in 2002,
Aydar et al. postulated a σ1 receptor model with two trans-
membrane regions and both the C-and N-termini localized
intracellularly.9 In 2003, σ1 knockout mice were generated,
which are viable and fertile but show a significant decrease in
motility.10
About 30 years ago, the σ receptor was first discovered and
classified as an opioid receptor, because typical opioid receptor
ligands of the benzomorphan type showed atypical pharmaco-
logical effects.1 However, this classification was abolished when
further investigations revealed that σ ligand mediated effects
were not antagonized by the opioid antagonist naloxone.2 The
assumption that the σ receptor and the phencyclidine (PCPa)
binding site of the NMDA receptor are identical was also
disproved because the antipsychotic agent haloperidol binds with
high affinity to σ receptors but has no affinity to the PCP binding
site.3 Today, σ receptors are well established as a non-opioid,
non-phencyclidine, and haloperidol-sensitive receptor family
with its own binding profile and a characteristic distribution in
the central nervous system (CNS) as well as in endocrine,
immune, and some peripheral tissues, like kidney, liver, lung,
and heart.4,5
The class of σ receptors comprises two subtypes, termed σ1
and σ2 receptor. The two subtypes have been pharmacologically
characterized. Haloperidol and di-o-tolylguanidine bind with
high affinity to σ1 as well as to σ2 receptors, whereas (+)-
benzomorphans, e.g., (+)-pentazocine, displays high affinity and
selectivity for the σ1 subtype. The σ1 receptor was cloned from
various tissues including guinea pig liver6 and rat brain.7
Recently, the σ1 receptor was also cloned from human placental
choriocarcinoma cell lines.8 The similarity of the amino acid
sequence of the σ1 receptors cloned from various species is
greater than 95%, and the σ1 receptors are more than 92%
identical. Surprisingly, there is no significant homology between
the σ1 receptor protein and any other known mammalian
receptors or even proteins. However, the yeast enzyme ergosterol-
∆8/∆7-isomerase shows a ∼30% homology to the σ1 receptor.
The σ2 receptor has not been cloned yet. The molecular
weight was estimated to be about 21.5 kDa.11 Endogenous
ligands for the σ1 and σ2 receptor have not been identified so
far. However, it has been shown that neuro(active)steroids, e.g.,
pregnenolone, progesterone, and dehydroepiandrosterone (DHEA),
interact with moderate affinity with the σ1 receptor, and
therefore, they are discussed to be its endogenous ligands.12
Although the intracellular signal transduction pathway is not
yet elucidated, σ1 receptors are involved in the modulation of
various systems including the glutamatergic,13 dopaminergic,14
and cholinergic15 neurotransmission. Additionally, the influence
on the regulation and activity of a variety of ion channels
including K+-channels16,17 and Ca2+-channels18,19 is an impor-
tant feature of σ1 receptors.
The σ1 receptors play a role in several physiological and
pathophysiological processes. In particular σ1 antagonists can
be used for the treatment of psychosis, since they do not lead
to the extrapyramidal motoric side effects of typical D2 receptor
antagonists.20 Several neuroleptic drugs like haloperidol bind
not only at dopamine D2 receptors but also at σ receptors.21,22
Furthermore, σ1 ligands represent a new principle for the
treatment of neuropathic pain,23,24 depressions,25,26 cocaine
abuse,27 and epilepsy.28 Neuroprotective and antiamnesic prop-
erties of some σ1 ligands have been explored for the treatment
of diseases associated with the loss of cognitive functions, e.g.,
Alzheimer’s disease and Parkinson’s disease.29,30 Because of
the high expression of σ1 and σ2 receptors in some human tumor
cell lines, radiolabeled σ1 and σ2 ligands could be applied as
selective tumor imaging agents for the diagnosis of tumors and
their metastases.31,32
* To whom correspondence should be addressed. Phone: +49-251-
8333311. Fax: +49-251-8332144. E-mail: wuensch@uni-muenster.de.
†
Universita¨t Mu¨nster.
Esteve.
‡
a Abbreviations: CNS, central nervous system; DHEA, dehydroepi-
androsterone; 5-HT, 5-hydroxytryptamine; NMDA, N-methyl-D-aspartate;
PCP, phencyclidine.
10.1021/jm8007739 CCC: $40.75
2008 American Chemical Society
Published on Web 09/25/2008

6532 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Oberdorf et al.
Scheme 2. N-Deprotection (a) and (b) and Alkylation (c) and
(d)a
Figure 1. Comparison of spirocyclic benzopyran σ1 receptor ligand 1
with the bioisosteric thienopyran derivatives 2.
The structures of σ1 receptor ligands are quite diverse. In
order to gain more information about the σ1 binding site, it is
necessary to develop conformationally restricted ligands with
high σ1 affinity and high selectivity against the σ2 receptor. In
2002, the synthesis of the spirocyclic benzopyran 1 (Figure 1)
with a σ1 affinity of 1.3 nM was reported. Moreover, this
compound displays high selectivity against the σ2 subtype.33,34
Herein, we report on the synthesis of thiophene bioisosteres of
1 with the general structure 2. Thiophene is a common
heterocycle for the bioisosteric benzene replacement in drug
development.35 The size of the thiophene heterocycle is similar
to the size of benzene, but the electronic properties and in
particular the electron density are quite different. The relation-
ship between the N-substituent of spirocyclic thienopyran
derivatives 2 and their σ1 and σ2 affinity is detailed.
a Reagents and conditions: (a) ClCO2CHClCH3, THF, -78 °C, 20 min,
then MeOH, reflux, 40 min, yield 65%; (b) 2 M NaOH, dioxane/H2O, 100
°C, 5 h, yield 54%; (c) 2g,h,j: R-Cl, CH3CN, K2CO3, reflux, yield 2g 38%,
yield 2h 34%, yield 2j 48%; 2d,l,m,o R-Br, CH3CN, K2CO3, reflux, yield
2d 95%, yield 2l 21%, yield 2m 65%, 2o 87%. (d) 2e,i,k,n: 2e
isovaleraldehyde, 2i p-methoxybenzaldehyde, 2k thiophene-2-carbaldehyde;
2n valeraldehyde, NaBH(OAc)3, CH2Cl2, room temp, 2-4 h, yield, 2e 83%,
yield, 2i 92%, yield; 2k 82%, yield; 2n 88%.
p-toluenesulfonic acid (10 mol%) to form the desired dimethyl
acetal 7 in 93% yield. The halogen-metal exchange of the
bromo acetal 7 with n-butyllihtium at -78 °C led to a
thienyllithium intermediate, which was trapped with the 1-sub-
stituted piperidin-4-ones 8a and 8b. The intramolecular transac-
etalization of the resulting hydroxy acetals 9a and 9b was carried
out with catalytic amounts of p-toluenesulfonic acid at room
temperature to provide the spirocyclic thienopyrans 2a and 2b
in 75% and 44% yield, respectively.
In order to get access to further N-substituted spiro[piperidin-
4,4′-thieno[3.2-c]pyrans], the benzyl group of 2a should be split
off to obtain the secondary amine 2f (Scheme 2). Attempts to
remove the N-benzyl moiety of 2a via transfer hydrogenolysis
under reflux conditions with ammonium formate and Pd/C as
catalyst36 failed, even with an excess of the catalyst and very
long reaction times of 3-4 days. Hydrogenolysis with hydrogen
at room temperature in the presence of Pd/C also did not provide
the secondary amine 2f. An explanation for this phenomenon
might be poisoning of the catalyst Pd/C by the thiophene sulfur.
As solution of this problem, a catalyst independent debenzylation
method seemed to be reasonable for synthesizing the secondary
amine 2f. R-Chloroethyl chloroformate is a reagent, which is
able to remove N-substituents without any catalysts. Benzyl
protected tertiary amines react with R-chloroethyl chloroformate
under cleavage of the benzyl group to form an unstable
R-chloroethyl carbamate that can easily be hydrolyzed.37 This
deprotection method provided the secondary amine 2f in 65%
yield (Scheme 2).
Chemistry
The synthesis of σ ligands with the general structure 2 started
with 3-bromothiophene (3) (Scheme 1). Regioselective lithiation
at position 2 of the thiophene ring led to a thienyllithium
intermediate, which was trapped with 1-formylpiperidine to form
3-bromothiophene-2-carbaldehyde (4) in 79% yield. The alde-
hyde 4 reacted with the P-ylide, which had been formed by
deprotonation of the phosphonium salt Ph3P+CH2OCH3Cl- (5)
with KOtBu, to afford the enol ether 6 as a mixture of (E)- and
(Z)-isomers (ratio 1:2). In the next step methanol was added to
the double bond of 6 in the presence of catalytic amounts of
Scheme 1. Synthesis of Spirocyclic σ Receptor Ligands with
Thienopyran Substructurea
Alternatively, the secondary amine 2f was synthesized by
reaction of the bromo acetal 7 with the ethoxycarbonyl protected
piperidone 8c instead of the benzyl protected piperidone 8a.
Subsequent cyclization of the hydroxy acetal 9c and hydrolysis
of the ethyl carbamate 2c upon heating with an excess of 2 M
NaOH under reflux gave the secondary amine 2f in 54% yield.
However, the debenzylation of 2a with R-chloroethyl chloro-
formate gave slightly higher yields than the hydrolysis of 2c
and was also advantageous because of the milder reaction
conditions and shorter reaction times (1 h instead of 5 h).
The secondary amine 2f was transformed either by alkylation
with alkyl halides or by reductive alkylation with aldehydes
a Reagents and conditions: (a) LDA, THF, 0 °C, 0.5 h, then 1-formylpi-
peridine, THF, 0 °C, 2 h, yield 79%; (b) Ph3P+CH2OCH3Cl- (5), KOtBu,
THF, -10 °C, 2 h, yield 83%; (c) p-TosOH·H2O, CH(OCH3)3, MeOH,
reflux, 24 h, yield 93%; (d) n-BuLi, THF, -78 °C, 15 min, then 8a-e,
THF, -78 °C, 1-2 h, yield 9a 68%, yield 9b 8%, yield 9c nd*, yield 9d
46%, yield 9e nd*; (e) p-TosOH·H2O, CH(OCH3)3, MeOH, room temp,
1-3 h, yield 2a 75%, yield 2b 44%, yield 2c 53%,* yield 2d: 68%, yield
2e: 16%*. (/) The hydroxy acetals 9c and 9e were directly cyclized to give
the thienopyrans 2c and 2e. Therefore, the yields of 2c and 2e are calculated
starting from the bromo acetal 7 (two steps).
38
and NaBH(OAc)3 into the substituted spirocycles 2d,e,g-o

Thiophene Bioisosteres
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6533
Table 1. Affinities of the Spiro[piperidine-4,4′-thieno[3,2-c]pyrans] toward σ1 and σ2 Receptors
Ki ( SEM (nM)a
compd
R
σ1 ([3H]-(+)-pentazocine)
σ2 ([3H]di-o-tolylguanidine)
σ1/σ2 selectivity
133
2a
2b
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
1.3 ( 0.2
0.32 ( 0.1
6.0 ( 3.0
0.29 ( 0.1 (n ) 4)
1.0 ( 0.3
602 (n ) 1)
2.4 ( 1.0
7.7 ( 1.2
2.2 ( 0.6
3500 ( 350
1260 (n ) 1)
191 (n ) 1)
25 ( 8.0
2708
3940
32
86
294
CH2C6H5
(CH2)2C6H5
CH2C6H11
(CH2)2CH(CH3)2
H
(CH2)3C6H5
(CH2)4C6H5
CH2C6H4-p-OCH3
CH2C6H4-p-F
CH2-2-Th
CH2CHC(CH3)2
C4H9
285 (n ) 1)
17%b
203 (n ) 1)
99 ( 13
2970 (n ) 1)
425 (n ) 1)
1040 (n ) 1)
162 ( 65
90
13
1325
685
515
169
57
280
238
21
0.62 ( 0.3
2.0 ( 0.4
1.0 ( 0.2
4.3 ( 0.3
246 (n ) 1)
460 (n ) 1)
1200 (n ) 1)
78 ( 2.0
C5H11
C8H17
1.6 ( 0.3
5.1 ( 1.7 (n ) 4)
3.9 ( 1.5 (n ) 5)
61 ( 18 (n ) 5)
haloperidol
di-o-tolylguanidine
42 ( 15 (n ) 7)
1.4
a The Ki values were determined in three independent experiments (n ) 3) unless otherwise noted. b Percent inhibition at a concentration of 1 µM test
compound.
(Scheme 2). Because of the extraordinarily high affinity of the
the equation of Cheng and Prusoff.41 The σ receptor binding
data of the spirocyclic thiophene derivatives 2 are summarized
in Table 1.
N-benzyl substituted compound 2a (Table 1), the homologous
arylalkyl derivatives 2b, 2g, and 2h were synthesized and the
secondary amine 2f was substituted with benzyl bioisosteric
groups, e.g., thienylmethyl (2k) and cyclohexylmethyl (2d)
residues. Furthermore, spirocyclic pyran derivatives with elec-
tron rich (2i) and electron poor (2j) benzyl moieties were
generated. To explore the influence of alkyl residues on the σ
receptor affinity, the secondary amine 2f was substituted with
linear (2m, 2n, 2o) and branched alkyl moieties (2e, 2l).
Discussion
The bioisosteric replacement of the benzene ring of the
spirocyclic benzopyran 1 for a thiophene ring results in a 4-fold
increased σ1 affinity of 0.32 nM for the spirocyclic derivative
2a and, furthermore, an improved σ1/σ2 selectivity (3940-fold).
As already mentioned, the size of the thiophene ring is similar
to the size of the benzene ring, but the electronic properties are
quite different. The replacement of the benzopyran substructure
for the thienopyran substructure leads to a higher electron
density in the aromatic part of 2a (see Figure 2). Furthermore,
the thiophene ring represents a better H-bond acceptor than the
benzene ring. Altogether, these effects might be the reason for
the increased σ1 receptor affinity.
The unsubstituted secondary amine 2f showed only low
affinity for both σ receptor subtypes. According to the phar-
macophore model of Glennon et al., two hydrophobic residues
in defined distances to the basic amine are required for high σ1
affinity.42,43 In compound 2f the second hydrophobic residue
is missing, which explains the loss of affinity. Nevertheless,
the thiophene derivative 2f binds with considerably higher
affinity for σ1 receptors (Ki ) 602 nM) than the corresponding
secondary amine with spirocyclic benzopyran structure (Ki )
19 800 nM).33
Replacement of the N-benzyl moiety of 2a by the electron
rich bioisosteric thienylmethyl (2k) and the p-methoxybenzyl
residues (2i) resulted in compounds with high affinity for the
σ1 receptor (Ki ) 2.0 and 2.2 nM) and a selectivity against the
σ2 receptor (see Table 1). Compared with 2a, the electron poor
p-fluorobenzyl derivative 2j shows nearly the same subnano-
molar σ1 receptor affinity but an increased σ2 affinity and thus
a reduced selectivity (2a, Ki(σ2) ) 1260 nM; 2j, Ki(σ2) ) 425
nM). In the case of aromatic substituents, the electronic
properties of the aromatic system seem to influence the σ2
affinity but have only minor effects toward the σ1 affinity.
The cyclohexylmethyl moiety of the spirocyclic thienopyran
2d represents a further bioisosteric replacement of the benzyl
residue, which is nonaromatic and because of its chair confor-
mation somewhat more voluminous. The σ1 affinity of 2d is
extraordinarily high (Ki ) 0.29 nM). However, 2d displays also
In order to shorten the synthesis, the highly potent σ1 ligands
2d and 2e were also synthesized by addition of the appropriate
N-substituted piperidones 8d and 8e to the bromo acetal 7 after
treatment of 7 with n-BuLi. The piperidones 8d and 8e were
prepared by alkylation of piperidin-4-one hydrochloride with
cyclohexylmethyl bromide and isopentyl bromide, respec-
tively.39 Transacetalization of the formed hydroxy acetals 9d
and 9e led to the spirocyclic pyran derivatives 2d and 2e.
σ Receptor Binding Studies
The σ1 and σ2 receptor affinities of the spirocyclic compounds
2a,b,d-o were determined in competition experiments with
radioligands. In the σ1 assay membrane preparations of guinea
pig brains were used as receptor material and [3H]-(+)-
pentazocine was used as radioligand. The nonspecific binding
was determined in the presence of a large excess of nontritiated
(+)-pentazocine. Homogenates of rat liver served as source for
σ2 receptors in the σ2 assay. Since a σ2 selective radioligand is
not commercially available, the nonselective radioligand [3H]-
di-o-tolylguanidine was employed in the presence of an excess
of nonradiolabeled (+)-pentazocine (500 nM) for selective
masking of σ1 receptors. An excess of nontritiated di-o-
tolylguanidine was used for determination of the nonspecific
binding. For highly potent ligands, the affinities were determined
with six different test compound concentrations from 10 µM to
0.1 nM. When the screening with high test compound concen-
trations indicated low affinity, only the inhibition of the
radioligand binding (in %) at a test compound concentration of
1 µM is listed.34,40 The Ki values were calculated according to

6534 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Oberdorf et al.
selectivity toward the phencyclidine binding site of the NMDA
receptor was investigated in receptor binding studies using MK-
801 as radioligand.40 At a concentration of 10 µM, the
spirocyclic compounds 2 did not significantly bind to the NMDA
receptor. Furthermore, the affinity of the highly potent σ1 ligands
toward the 5-HT1A (human), 5-HT6 (human), 5-HT7 (human),
R1 (rat) and R2A (human) receptors, and the 5-HT transporter
was determined. All compounds exhibit a negligible affinity for
the investigated receptor and transporter systems, indicating high
selectivity for the σ1 receptor over these receptors and transporters.
In Vivo Activity. It has been shown that σ1 antagonists are
able to reduce pain responses in the capsaicin assay.23,24
Therefore, σ1 antagonists are interesting compounds for the
treatment of neuropathic pain. The thiophene derivative 2a was
investigated in the capsaicin assay in rats and showed at a dose
of 32 mg/kg body weight (ip) a low analgesic effect of 10%.
This activity indicates the spirocyclic compound 2a could be a
σ1 antagonist.
Conclusion
The presented data indicate that the benzene-thiophene
exchange leads to highly potent σ1 receptor ligands. The
thiophene derivative 2a even exceeds the σ1 affinity of 1 with
benzopyran substructure. A similar increase of σ1 affinity could
be observed for the secondary amine 2f compared to the NH-
benzopyran derivative. An aromatic residue at the basic amine
is not necessary for a high σ1 affinity. Alkyl moieties but even
better cycloalkyl moieties led to extraordinarily high σ1 affinity
but a reduced selectivity toward σ2 receptors. The thiophene
derivatives show high selectivity for the σ1 receptor over
NMDA, 5-HT1A, 5-HT6, 5-HT7, R1A, and R2 receptors and the
5-HT transporter. Most probably, the synthesized compounds
with thienopyran substructure act as antagonists at the σ1
receptor, since 2a reduces pain response in the capsaicin assay.
Figure 2. 3D electrostatic contour plots (Connolly analytic) calculated
with the molecular modeling program MOE (Molecular Operating
Environment) of the AM1 minimized (R)-enantiomers of σ1 ligands 1
and 2a. Blue regions of the surface indicate a positive electrostatic
potential, and red regions indicate a negative electrostatic potential.
rather high σ2 receptor affinity (Ki ) 25 nM) and thus represents
an unselective σ ligand in contrast to the bioisosteric N-benzyl
derivative 2a. The high affinity of 2d indicates that an aromatic
system attached to the basic nitrogen is not essential for high
σ1 affinity. In order to investigate the effect of nonaromatic
moieties on the σ receptor affinities, compounds 2e,l-o with
linear and branched alkyl substituents were examined. Very low
Ki values indicating high σ1 affinity were determined for the
pentyl (2n, Ki ) 1.6 nM), isopentyl (2e, Ki ) 1.0 nM), and
isopentenyl derivatives (2l, Ki ) 1.0 nM). The σ1 affinities of
the N-butyl and N-octyl derivatives 2m and 2o are slightly lower.
All N-alkyl substituted thienopyrans display a considerably
reduced σ1/σ2 selectivity compared to the N-benzyl compound
2a.
In order to explore the influence of the distance between the
aromatic system and the basic amine on the σ1 and σ2 receptor
affinity and selectivity, the homologous N-arylalkyl derivatives
2b, 2g, and 2h were synthesized. The N-phenylethyl and
N-phenylbutyl derivatives 2b and 2h show a 20- to 25-fold
reduced σ1 affinity compared to 2a. Surprisingly, the phenyl-
propyl derivative 2g possesses a higher σ1 affinity (Ki ) 2.4
nM) than 2b and 2h. The reduced but still high σ1 affinity of
the three homologous arylalkyl derivatives 2b, 2g, 2h demon-
strates the tolerance of the σ1 binding site for bulky and highly
flexible moieties at the basic amino group. In contrast to the
slightly decreased σ1 affinity of the arylalkyl compounds 2b,
2g, and 2h the σ2 affinity increases with further elongation of
the nitrogen-phenyl distance up to the phenylbutyl derivative
2h with a Ki value of 99 nM. Thus, the phenylbutyl derivative
2h shows the lowest σ1/σ2-selectivity (factor 13) of all inves-
tigated compounds.
Experimental Section
Chemistry. General. For glash chromatography (fc), silica gel
60, 40-64 µm (Merck), was used. Parameters in parentheses
include diameter of the column, eluent, Rf value. For 1H NMR (400
MHz) and 13C NMR (100 MHz), a mercury 400BB spectrometer
(Varian) was used. The reported δ in ppm is referenced to
tetramethylsilane, and coupling constants are given with 0.5 Hz
resolution. The assignments of 13C and 1H NMR signals were
supported by 2D NMR techniques.
(E/Z)-3-Bromo-2-(2-methoxyvinyl)thiophene (6). Under N2, the
Wittig reagent 5 (13.1 g, 38.3 mmol) was suspended in THF (100
mL) at -10 °C and a solution of KOtBu (1 M in THF, 38.3 mL,
38.3 mmol) was added. After 5 min 4 (5.64 g, 29.5 mmol) was
added and the mixture was stirred for 2 h at -10 °C. Then H2O
(80 mL) was added, and the mixture was extracted with Et2O (3×).
The organic layer was dried (Na2SO4), filtered, and concentrated
in vacuo. The precipitate of triphenylphosphane oxide was washed
with a mixture of cyclohexane/EtOAc, 1:1, and the organic layer
was again concentrated in vacuo. The residue was purified by
distillation. Colorless liquid, bp 72 °C (5.6 × 10-2 mbar), yield
1
5.39 g (83%). C7H7BrOS (219.1). H NMR (CDCl3): δ 3.64 (s, 1
H, 6-(E) OCH3), 3.77 (s, 2 H, 6-(Z) OCH3), 5.70 (dd, J ) 6.6/1.9
Hz, 0.67 H, 6-(Z) ThCHdCH), 5.89 (d, J ) 12.9 Hz, 0.33 H, 6-(E)
ThCHdCH), 6.18 (d, J ) 6.6 Hz, 0.67 H, 6-(Z) ThCHdCH), 6.82
(d, J ) 5.4 Hz, 0.33 H, 6-(E) 4-H-Th), 6.85 (d, J ) 5.4 Hz, 0.67
H, 6-(Z) 4-H-Th), 6.89 (d, J ) 5.4 Hz, 0.33 H, 6-(E) 5-H-Th),
6.97 (d, J ) 12.9 Hz, 0.33 H, 6-(E) ThCHdC H), 7.09 (dd, J )
5.4/0.9 Hz, 0.67 H, 6-(Z) 5-H-Th).
2-(3-Bromothiophen-2-yl)acetaldehyde Dimethyl Acetal (7). The
enol ether 6 (5.39 g, 24.6 mmol), p-toluenesulfonic acid monohy-
drate (470 mg, 2.46 mmol), and trimethyl orthoformate (5 mL) were
dissolved in MeOH. The solution was heated to reflux for 24 h.
Receptor Selectivity. In order to get information about the
selectivity of the spirocyclic thiophene derivatives 2, the

Thiophene Bioisosteres
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6535
After neutralization with NaOH (2 M, 5 mL), H2O was added and
the mixture was extracted with CH2Cl2 (3×). The organic layer
was dried (Na2SO4) and concentrated in vacuo, and the residue was
purified by distillation. Colorless liquid, bp 78 °C (5.6 × 10-2
mbar), yield 5.73 g (93%). C8H11BrO2S (251.1). 1H NMR (CDCl3):
δ 3.03 (d, J ) 5.6 Hz, 2 H, ThCH2CH), 3.31 (s, 6 H, CH(OCH3)2),
4.47 (t, J ) 5.6 Hz, 1 H, ThCH2CH), 6.84 (d, J ) 5.4 Hz, 1 H,
4-H-Th), 7.15 (d, J ) 5.4 Hz, 1 H, 5-H-Th).
1-Benzyl-6′-methoxy-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3.2-
c]pyran] (2a). Synthesis was according to general procedure A.
Colorless solid, mp 78 °C, yield 199 mg (75%), Rf ) 0.21
(cyclohexane/EtOAc, 7:3). Anal. (C19H23NO2S) C, H, N. 1H NMR
(CDCl3): δ 1.76 (dd, J ) 13.5/2.1 Hz, 1 H, N(CH2CH2)2),
1.80-1.93 (m, 2 H, N(CH2CH2)2), 2.04 (td, J ) 13.4/4.5 Hz, 1 H,
N(CH2CH2)2), 2.37 (td, J ) 11.6/3.7 Hz, 1 H, N(CH2CH2)2), 2.45
(td, J ) 12.4/2.6 Hz, 1 H, N(CH2CH2)2), 2.65-2.73 (m, 2 H,
N(CH2CH2)2), 2.77 (dd, J ) 15.6/7.5 Hz, 1 H, ThCH2CH), 2.92
(dd, J ) 15.6/3.3 Hz, 1 H, ThCH2CH), 3.48 (d, J ) 13.0 Hz, 1 H,
NCH2Ph), 3.49 (s, 3 H, OCH3), 3.54 (d, J ) 13.0 Hz, 1 H,
NCH2Ph), 4.81 (dd, J ) 7.2/3.3 Hz, 1 H, ThCH2CH), 6.73 (d, J )
5.4 Hz, 1 H, 3′-H-Th), 7.02 (d, J ) 5.4 Hz, 1 H, 2′-H-Th),
7.15-7.31 (m, 5 H, Ph-H). 13C NMR (CDCl3): δ 31.7 (1 C,
ThCH2CH), 35.7 (1 C, N(CH2CH2)2), 38.8 (1 C, N(CH2CH2)2), 49.4
(1 C, N(CH2CH2)2), 49.5 (1 C, N(CH2CH2)2), 56.8 (1 C, OCH3),
63.7 (1 C, NCH2Ph), 74.7 (1 C, ThCO), 96.9 (1 C, ThCH2CH),
123.4 (1 C, C-2′-Th), 124.0 (1 C, C-3′-Th), 127.2 (1 C, para-C-
Ph), 128.4 (2 C, meta-C-Ph), 129.5 (2 C, ortho-C-Ph), 130.7 (1 C,
C-7a′-Th), 138.7 (1 C, Ph-C), 140.8 (1 C, C-3a′-Th). (329.5).
1-(Cyclohexylmethyl)-6′-methoxy-6′,7′-dihydrospiro[piperidine-
4,4′-thieno[3.2-c]pyran] (2d). Synthesis was according to general
procedure A. Colorless solid, mp 98 °C, yield 490 mg (68%), Rf )
2-[3-(1-Benzyl-4-hydroxypiperidin-4-yl)-2-thienyl]acetalde-
hyde Dimethyl Acetal (9a). A solution of n-BuLi (1.35 M in
n-hexane, 8.3 mL, 11.3 mmol) was added dropwise over 2-3 min
to a cooled (-78 °C) solution of dimethyl acetal 7 (2.18 g, 8.7
mmol) in THF (100 mL). After the mixture was stirred for 15 min,
a solution of 8a (1.81 g, 9.57 mmol) in THF (1.5 mL) was added
at -78 °C, and the mixture was stirred for 1 h at -78 °C and for
2 h at room temperature. After addition of H2O (20 mL) and a
solution of NaHSO3 (10 mL, 10%) the mixture was extracted with
CH2Cl2 (3×). The organic layer was dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by fc (L ) 5 cm,
cyclohexane/EtOAc, 7:3, Rf ) 0.28). Pale-yellow oil, yield 2.14 g
1
(68%). C20H27NO3S (361.5). H NMR (CDCl3): δ 1.67 (dd, J )
14.0/2.5 Hz, 2 H, N(CH2CH2)2), 2.04 (td, J ) 13.2/4.5 Hz, 2 H,
N(CH2CH2)2), 2.45 (td, J ) 12.3/2.1 Hz, 2 H, N(CH2CH2)2),
2.62-2.71 (m, 2 H, N(CH2CH2)2), 3.30 (s, 6 H, CH(OCH3)2, 3.38
(d, J ) 5.4 Hz, 2 H, ThCH2CH), 3.50 (s, 2 H, NCH2Ph), 3.78 (s,
1 H, OH), 4.44 (t, J ) 5.4 Hz, 1 H, ThCH2CH), 6.83 (d, J ) 5.4
Hz, 1 H, 4-H-Th), 6.99 (d, J ) 5.4 Hz, 1 H, 5-H-Th), 7.12-7.23
(m, 5 H, Ph-H).
1
0.45 (cyclohexane/EtOAc, 7:3). Anal. (C19H29NO2S) C, H, N. H
NMR (CDCl3): δ 0.82-0.87 (m, 2 H, cHex-H), 1.12-1.20 (m, 2
H, cHex-H), 1.41-1.51 (m, 1 H, cHex-H), 1.56-1.86 (m, 9 H
N(CH2CH2)2 and cHex-H), 2.05 (td, J ) 13.2/4.5 Hz, 1 H,
N(CH2CH2)2), 2.12 (d, J ) 7.0 Hz, 2 H, CH2cHex), 2.27 (td, J )
12.1/3.3 Hz, 1 H, N(CH2CH2)2), 2.34 (td, J ) 12.5/2.3 Hz, 1 H,
N(CH2CH2)2), 2.64-2.70 (m, 2 H, N(CH2CH2)2), 2.78 (dd, J )
16.0/7.1 Hz, 1 H, ThCH2CH), 2.92 (dd, J ) 15.6/3.1 Hz, 1 H,
ThCH2CH), 3.51 (s, 3 H, OCH3), 4.81 (dd, J ) 7.4/3.1 Hz, 1 H,
ThCH2CH), 6.72 (d, J ) 5.1 Hz, 1 H, 3′-H-Th), 7.02 (d, J ) 5.5
Hz, 1 H, 2′-H-Th). The synthesis of 2d was also performed
according to general procedure B using cyclohexylmethyl bromide
(see Table 3). Colorless solid, mp 98 °C, yield 118 mg (95%).
6′-Methoxy-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]py-
ran] (2f). Method 1. To a solution of carbamate 2c (0.92 g, 2.96
mmol) in dioxane/H2O, 1:1 (50 mL), NaOH (2 M, 100 mL, 70
equiv) was added, and the mixture was heated to reflux for 5 h.
After the mixture was cooled to room temperature, the aqueous
solution was extracted with Et2O (3×). The organic layer was dried
(Na2SO4), filtered, and concentrated in vacuo. The residue was
purified by fc (L ) 3 cm, MeOH/NH3, 98:2, Rf ) 0.25). Colorless
solid, mp 103 °C, yield 0.385 g (54%).
2-{3-[1-(Cyclohexylmethyl)-4-hydroxypiperidin-4-yl]-2-
thienyl}acetaldehyde Dimethyl Acetal (9d). A solution of n-BuLi
(1.5 M in n-hexane, 5.2 mL, 7.0 mmol) was added dropwise over
2-3 min to a cooled (-78 °C) solution of dimethyl acetal 7 (1.18
g, 4.7 mmol) in THF (100 mL). After the mixture was stirred for
15 min, a solution of 8d (1.0 g, 5.2 mmol) in THF (2 mL) was
added at -78 °C and the mixture was stirred for 1.5 h at -78 °C
and for 2 h at room temperature. After addition of H2O (20 mL)
the mixture was extracted with CH2Cl2 (3×). The organic layer
was dried (Na2SO4), filtered and concentrated in vacuo. The residue
was purified by fc (L ) 4 cm, cyclohexane/EtOAc, 1:1, Rf ) 0.21).
1
Colorless oil, yield 0.80 g (46%). C20H33NO3S (367.5). H NMR
(CDCl3): δ 0.77-0.87 (m, 2 H, cHex-H), 1.07-1.20 (m, 3 H, cHex-
H), 1.40-1.48 (m, 1 H, cHex-H), 1.55-1.75 (m, 5 H, cHex-H),
1.66 (dd, J ) 13.7/2.6 Hz, 2 H, N(CH2CH2)2), 2.03 (td, J ) 12.6/
3.3 Hz, 2 H, N(CH2CH2)2), 2.11 (d, J ) 7.4 Hz, 2 H, NCH2cHex),
2.31 (t, J ) 11.7 Hz, 2 H, N(CH2CH2)2), 2.59-2.65 (m, 2 H,
N(CH2CH2)2), 3.29 (s, 6 H, (OCH3)2), 3.87 (d, J ) 4.2 Hz, 2 H,
ThCH2CH), 3.69 (s, 1 H, OH), 4.44 (t, J ) 4.2 Hz, 1 H, ThCH2CH),
6.83 (d, J ) 5.2 Hz, 1 H, 4-H-Th), 7.00 (d, J ) 5.2 Hz, 1 H, 5-H-
Th).
Method 2. The N-benzyl protected compound 2a (100 mg, 0.31
mmol) was dissolved in THF (8 mL), and R-chloroethyl chloro-
formate (45 µL, 0.40 mmol) was added slowly at -78 °C. After
stirring for 20 min, the mixture was warmed up to room temperature
and THF was removed in vacuo. The residue was dissolved in
MeOH (10 mL), and the solution was heated to reflux for 40 min.
Then MeOH was evaporated in vacuo and the residue was purified
by fc (L ) 0.7 cm, EtOAc/MeOH/NH3, 90:10:2, Rf ) 0.25 (MeOH/
NH3, 98:2)). Colorless solid, mp 103 °C, yield 48 mg (65%).
General Procedure A for the Cyclization of 9a-e to give
2a-e (Table 2). The corresponding hydroxy acetal 9a-e was
dissolved in MeOH. Trimethyl orthoformate and 1.2 equiv of
p-toluenesulfonic acid monohydrate were added, and the solution
was stirred at room temperature for 1-24 h ). Then NaOH (2 M,
5-10 mL) and H2O (10-20 mL) were added and the mixture was
extracted with CH2Cl2 (3×). The organic layer was dried (Na2SO4),
filtered, and concentrated in vacuo. The residue was purified by
fc.
1
C12H17NO2S (239.3). H NMR (CDCl3): δ 1.68 (td, J ) 14.0/4.7
Hz, 1 H, N(CH2CH2)2), 1.76 (dd, J ) 14.0/2.7 Hz, 1 H,
N(CH2CH2)2), 1.84-1.96 (m, 2 H, N(CH2CH2)2), 2.78 (dd, J )
15.6/7.5 Hz, 1 H, ThCH2CH), 2.84-2.88 (m, 1 H, N(CH2CH2)2),
2.88-2.93 (m, 1 H, N(CH2CH2)2), 2.92 (dd, J ) 15.6/3.3 Hz, 1 H,
Table 3. Alkylation of 2f: Reagents and Conditions
Table 2. Cyclization of 9a-e: Reagents and Conditions
amount of CH3CN
alkyl halide,
fc La
t (h) (cm)
hydroxyacetal,
amount (g (mmol))
MeOH
(mL)
TMOFa
(mL)
fc Lb
(cm)
product 2f (mg)
(mL)
amount (mg (mmol))
product
t (h)
2d
2g
2h
2j
2l
2m
2o
90 (0.37)
120 (0.5)
89 (0.36)
90 (0.37)
59 (0.24)
90 (0.37)
180 (0.75)
8
15
10
8
5
8
BrCH2C6H11, 80 (0.45)
Cl(CH2)3C6H5, 119 (0.6)
Cl(CH2)4C6H5, 76 (0.45)
ClCH2C6H4-p-F, 65 (0.6)
BrCH2CHC(CH3)2, 45 (0.3) 20
BrC4H9, 79 (0.57)
BrC8H17, 174 (0.9)
24
16
24
20
1.5
2.0
1.5
2.0
1.0
1.0
2.0
2a
2b
2c
2d
2e
9a, 0.29 (0.81)
9b, 0.055 (0.14)
9c, 1.49c
15
5
50
30
40
2
1
5
3
4
24
24
1
3
2
2
1
3
2.5
4
9d, 0.80 (2.2)
9e, 1.20c
21
16
20
a Trimethyl orthoformate. b fc solvent: cyclohexane/EtOAc, 7:3. c Amount
that includes a small amount of ketone 8c and 8e.
a fc solvent: cyclohexane/EtOAc, 7:3.

6536 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Oberdorf et al.
ThCH2CH), 3.04 (td, J ) 12.3/2.7 Hz, 1 H, N(CH2CH2)2), 3.15
(td, J ) 12.3/3.0 Hz, 1 H, N(CH2CH2)2), 3.52 (s, 3 H, OCH3),
4.83 (dd, J ) 7.2/3.3 Hz, 1 H, ThCH2CH), 6.72 (d, J ) 5.1 Hz, 1
H, 3′-H-Th), 7.03 (d, J ) 5.1 Hz, 1 H, 2′-H-Th). A signal for the
N-H proton is not seen.
Genaral Procedure B for the Alkylation of 2f with Alkyl
Halides (Table 3). The secondary amine 2f was dissolved in
CH3CN, and the alkyl halide and 6 equiv of K2CO3 were added.
The mixture was heated to reflux for 16-24 h. K2CO3 was removed
by filtration, and the solution was transferred into a separation
funnel. After addition of brine (10-20 mL), the aqueous solution
was extracted with Et2O (3×). The organic layer was dried
(Na2SO4), filtered, and concentrated in vacuo. The residue was
purified by fc.
General Procedure C for the Reductive Alkylation of 2f
(Table 4). The secondary amine 2f was dissolved in CH2Cl2, and
the corresponding aldehyde and NaBH(OAc)3 were added. After
the mixture was stirred for 2-4 h at room temperature, saturated
saturated solution of NaHCO3 (10 mL) was added and the aqueous
layer was extracted with CH2Cl2 (3×). The organic layer was dried
(Na2SO4), filtered, and concentrated in vacuo. The residue was
purified by fc (L ) 2 cm, cyclohexane/EtOAc, 7:3).
scintillator was allowed to solidify at room temperature. 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]-(+)-pentazocine is 2.9 nM.45
Membrane Preparation for the σ2 Assay.33,40 Two rat livers
were cut into small pieces and homogenized with a potter
(500-800 rpm, 10 up-and-down strokes) in 6 volumes of cold
0.32 M sucrose. The suspension was centrifuged at 1200g for
10 min at 4 °C. The supernatant was separated and centrifuged
at 31000g for 20 min at 4 °C. The pellet was resuspended in
buffer (50 mM Tris, pH 8.0) and incubated at room temperature
for 30 min. After the incubation, the suspension was centrifuged
again at 31000g for 20 min at 4 °C. The final pellet was
resuspended in buffer, the protein concentration was determined
according to the method of Bradford44 using bovine serum
albumin as standard, and subsequently the preparation was frozen
(-80 °C) in 1.5 mL portions containing about 2 mg of protein/
mL.
Performing of the σ2-Assay.33,40 The test was performed with
the radioligand [3H]-di-o-tolylguanidine (50 Ci/mmol, ARC). The
thawed membrane preparation (about 100 µg of the protein) was
incubated with various concentrations of test compounds, 3 nM
[3H]-di-o-tolylguanidine, 500 nM (+)-pentazocine, and buffer
(50 mM Tris, pH 8.0) in a total volume of 200 µL for 180 min
at room temperature. The incubation was terminated by rapid
filtration through the presoaked filtermats using a cell harvester.
After each well was washed five times with 300 µL of water,
the filtermats were dried at 95 °C. Subsequently, the solid
scintillator was placed on the filtermat and melted at 95 °C. After
5 min, the solid scintillator was allowed to solidify at room
temperature. The bound radioactivity trapped on the filters was
counted in the scintillation analyzer. The nonspecific binding
was determined with 10 µM unlabeled ditolylguanidine. The Kd
value of the radioligand [3H]ditolylguanidine is 17.9 nM.46
NMDA Assay. The preparation of the receptor material and the
assay were performed according to ref 40.
Data Analysis. All experiments were carried out in triplicate
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
Prism 3.0 (GraphPad Software) by nonlinear regression analysis.
The Ki values were calculated according to Cheng and Prusoff.41
The Ki values are given as mean values +/-SEM from three
independent experiments.
Table 4. Reductive Alkylation of 2f: Reagents and Conditions
amount of
amount of
2f (mg
NaBH(OAc)3
product (mmol)) aldehyde, amount (mg (mmol)) (mg (mmol)) t (h)
2e
2i
2k
2n
58 (0.24) OdCHCH2CH(CH3)2, 22 (0.25) 80 (0.38)
70 (0.29) OdCHC6H4-p-OCH3, 45 (0.33) 75 (0.35)
4
2
58 (0.24) OdCH-2-Th, 31 (0.28)
80 (0.38)
80 (0.38)
62 (0.26) OdCH(CH2)3CH3, 24 (0.28)
2
Receptor Binding Studies. Materials and General Proce-
dures. Guinea pig brains and rat livers were commercially available
(Harlan-Winkelmann, Germany). The homogenizer was an Elve-
hjem Potter (B. Braun Biotech International). The centrifuge was
a high-speed cooling model Sorvall RC-5C Plus (Thermo Finnigan).
The filter was printed filtermat type B (Perkin-Elmer) presoaked
in 0.5% aqueous polyethylenimine for 2 h at room temperature
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 room temperature, the scintillation was measured using a
MicroBeta Trilux scintillation analyzer (Perkin-Elmer). The count-
ing efficiency was 20%.
Supporting Information Available: Physical and spectroscopic
data of all new compounds, purity data, and general chemistry
methods. This material is available free of charge via the Internet
at http://pubs.acs.org.
Membrane Preparation for the σ1 Assay.33,40 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 Bradford44 using
bovine serum albumin as standard, and subsequently the prepara-
tion was frozen (-80 °C) in 1.5 mL portions containing about
1.5 mg of protein/mL.
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