Novel sigma receptor ligands: Synthesis and biological profile

Article abstract of DOI:10.1021/jm0611197

The aim of the present study was to investigate the biological profile of new substituted 1-phenyl-2-cyclopropylmethylamines. High affinity for both σ subtypes was achieved when 4-phenylpiperidin-4-ol (4a-e) and 4-benzylpiperidine moieties were present (5

Full text of DOI:10.1021/jm0611197

J. Med. Chem. 2007, 50, 951-961
951
Novel Sigma Receptor Ligands: Synthesis and Biological Profile
Orazio Prezzavento,† Agata Campisi,‡ Simone Ronsisvalle,† Giovanni Li Volti,§ Agostino Marrazzo,† Vincenzo Bramanti,|
Giuseppe Cannavo`, Luca Vanella,‡ Alfredo Cagnotto,# Tiziana Mennini,# Riccardo Ientile,| and Giuseppe Ronsisvalle*,†
Department of Pharmaceutical Sciences and Department of Biological Chemistry, Medical Chemistry, and Molecular Biology, UniVersity of
Catania, Viale A. Doria 6, 95125 Catania, Italy, Department of Biomedical Sciences and Biotechnology, DiVision of Human Anatomy,
UniVersity of Brescia, Viale Europa, 25123 Brescia, Italy, Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche
Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy, and Department of Biochemical, Physiological, and Nutritional Sciences
and Department of Hygiene, Public Health, and PreVentiVe Medicine, UniVersity of Messina, Via Consolare Valeria, 90100, Messina, Italy
ReceiVed September 26, 2006
The aim of the present study was to investigate the biological profile of new substituted 1-phenyl-2-
cyclopropylmethylamines. High affinity for both σ subtypes was achieved when 4-phenylpiperidin-4-ol (4a-
e) and 4-benzylpiperidine moieties were present (5a-e). (1R,2S/1S,2R)-2-[4-Hydroxy-4-phenylpiperidin-
1-yl)methyl]-1-(4-methylphenyl)cyclopropanecarboxylate (4b) showed high affinity for the σ1 sites (Ki )
1.5 nM) and the most favorable σ1/σ2 selectivity (Ki(σ2)/Ki(σ1) ) 33.9). Binding affinity studies showed
that 4b binding on N-methyl-D-aspartate (NMDA), dopaminergic (D1, D2, D3), muscarinic, histaminergic
H1, adrenergic (R1, R2), serotoninergic (5-HT2A, 5-HT2C, 5-HT3, 5-HT4, 5-HT6), DA (DAT), and 5-HT (SERT)
transporters was not significant. Interestingly, σ ligands differently induced the expression of tissue
transglutaminase (TG-2) in primary astroglial cell cultures. We suggest that 4b may act as a σ1/σ2 agonist
and that the σ ligands may modulate TG-2 differently.
Introduction
healing. In addition, it plays a modulatory role in apoptosis and
cell response to stressors, depending on the types of stimuli
that provoke an increase in the transamidating activity.16
However, TG-2 has additional activities and it has also been
reported to be a protein disulfide isomerase18 and a G-protein-
coupled membrane receptor.17 Although TG-2 is found mostly
in the cytosol, associated with the plasma membranes,18 in the
mitochondria, and in the nucleus, the multiple effects of TG-2
in different cellular pathways may be due to its different cell
localization and its transamidanting and GTPase activities.19
TG-2 is also expressed in the brain, and it is implicated in
numerous and diverse processes of the central and peripheral
nervous systems.15 Several lines of evidence suggest that
aberrant TG-2 activity, although not causative, may contribute
to the inexorable decline associated with several neurodegen-
erative diseases such as Huntington’s, Alzheimer’s, and Par-
kinson’s disease.20 It is well-known that astroglial cells in
primary culture represent a valuable tool for studying the
biochemical mechanism involved during the development and
maturation of these cultured nerve cells.21
The σ receptors have been classified as a unique protein
family different from the opioid1 and phencyclidine (PCP)
receptors.2 At least two different subtypes of σ1 and σ2 receptors
have been documented.3,4 Both subtypes are expressed in
peripheral organs and in the brain, while each type is localized
in different subcellular compartments.5,6 The σ1 receptor was
cloned and sequenced in the mid-1990s,7 and they are clearly
involved in various biological and pharmacological functions.8
Among the most salient ones, σ receptors mediate potassium
conductance9 and calcium signaling,10 and they play neuro-
modulatory roles on N-methyl-D-aspartate (NMDA), DA, and
Ach transmission.11-13 In addition, it has been suggested that σ
receptors are associated with the toxin-sensitive Gi/o and the
choler toxin-sensitive Gs proteins phospholipase C (PLC) and
protein kinase C (PKC).10 Conversely, the σ2 receptors have
not yet been cloned and their activation has induced changes
in cell morphology and apoptosis, producing transient and
sustained increases in calcium ions.14 Increased calcium ions
influx leads to the activation of several calcium-dependent
proteins, such as tissue transglutaminase (TG-2).15 This is a
ubiquitary isoform of a family of transglutaminases (TGase),
catalyzing the formation of ꢀ-(γ-glutamyl)lysine cross-links
between polypeptide chains, resulting in protein polymerization,
the cross-linking of dissimilar proteins, and the incorporation
of diamines and polyamines into proteins.15 Furthermore, TG-2
is implicated in numerous cellular processes, such as cellular
differentiation, signal transduction, cell survival, and wound
In this report we present a new series of 1-methoxycarbonyl-
1-phenyl-2-cyclopropylmethylamines. Here, the (+)-cis-N-
normetazocine nucleus of methyl (1′R,2′S)-2-{[(2R,6R,11R)-
8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-
benzazocin-3(2H)-yl]methyl}-1-phenylcyclopropane-
carboxylate ((+)-MPCB)22 (Figure 1) has been substituted with
different substructures of the benzomorphan system such as
4-phenyl-4-hydroxypiperidine, 4-benzylpiperidine, 1-(pyridin-
2-yl)piperazine, and 2-(piperazin-1-yl)pyrimidine moieties. The
aim was to develop σ ligands with higher affinity and selectivity.
We also explored the importance of the para substituent on the
aromatic ring supported on the cyclopropane ring. All the
compounds were tested as racemates for binding at σ1 and σ2
receptors. In addition, our purpose was to assess the relationship
between TG-2 and the σ receptors in primary rat astroglial cell
cultures. Furthermore, in order to verify the activation of the
σ-mediated apoptotic pathway, we studied caspase-3 cleavage
and DNA fragmentation.
* To whom correspondence should be addressed. Telephone/fax: +39-
095-336722. E-mail address: ggrmedch@unict.it.
† Department of Pharmaceutical Sciences, University of Catania.
‡ Department of Biological Chemistry, Medical Chemistry, and Molecular
Biology, University of Catania.
§ University of Brescia.
| Department of Biochemical, Physiological, and Nutritional Sciences,
University of Messina.
Department of Hygiene, Public Health, and Preventive Medicine,
University of Messina.
# Istituto di Ricerche Farmacologiche Mario Negri.
10.1021/jm0611197 CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/13/2007

952 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
PrezzaVento et al.
Scheme 2. Synthetic Route for 4a-e, 5a-e, 6a-f, and 7a-f
Figure 1. Structure of (+)-(1′R,2′S)-2-{[(2R,6R,11R)-8-hydroxy-6,-
11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-
methyl}-1-phenylcyclopropanecarboxylate ((+)-MPCB).
Scheme 1. Synthetic Route for 1a-f and 2a-f
On the basis of σ2/σ1 selectivity and the calculated log P
value (3.36 ( 0.39), we selected the 4-hydroxy-4-phenylpip-
eridine derivative 4b in order to investigate its binding profile
with respect to the receptors and transporters reported in Table
2. Compared to haloperidol, a prototypical σ ligand,24 4b
displays much lower affinity for the dopamine D1, D2, and
D3 receptors (Ki > 10.000, 1.850, 4.900 nM, respectively),
5-HT2A (Ki ) 3.149 nM), and R1 receptors (Ki ) 605 nM). In
addition, 4b does not bind adrenergic R2, muscarinic, histamin-
ergic H1, and serotonin 5-HT2C, 5-HT3, 5-HT4, and 5-HT6
receptors as well as on DA (DAT) and 5-HT (SERT) transport-
ers. These results are consistent with previous reports show-
ing that the modifications on the butyrophenone moiety are
injurious for dopamine receptors (D2 and D3) but not for the σ
receptors.28
Our results confirm the role of the N-substituent to address
an appropriate amino pharmacophore to σ receptors, at the same
time preventing the interaction with the other receptors. In order
to investigate the relationships between the σ receptors and the
calcium-dependent protein TG-2, on primary rat astroglial cell
cultures we explored the biological profile of 4b and some σ
reference compounds, presenting different activity and selectivity
profiles (Table 3). This in vitro model represents a valuable
tool for studying the biochemical mechanism involved during
the development and maturation of these cultured nerve cells.21
Primary rat astroglial cell cultures were prepared and
characterized by immunofluorescent staining for a specific
marker, such as glial fibrillary acidic protein (GFAP). About
90% of the cells were GFAP-positive, indicating that cultures
were rich in astrocytes.21
Chemistry
Lactones 1a-d were prepared as previously reported by
Casadio et al.23 The reaction of the appropriate lactone with
SOCl2 and CH3OH/HCl (3 N) in benzene gave the cis racemic
halogenated esters 2a-f (Scheme 1). Ester 2e was obtained by
nitration with fuming HNO3 starting from lactone 1a and the
subsequent esterification of 1e. Reduction of 4-nitro lactone 1e
with SnCl2‚2H2O gave the amino lactone 1f. Esterification of
1f with an analogous procedure of 1a-d gave the amino ester
2f. The reaction of the secondary commercially available amines
4-phenylpiperidin-4-ol (3a), 4-benzylpiperidine (3b), 1-(pyridin-
2yl)piperazine (3c), and 2-(piperazin-1yl)pirimidine (3d) with
the suitable cis-(()-halogenated ester derivative gave the final
compounds cis-(()-4a-e, cis-(()-5a-e, cis-(()-6a-f, and cis-
(()-7a-f, respectively (Scheme 2).
Results and Discussion
The synthesized compounds evaluated for their σ affinity
revealed high affinity for the σ receptors when 4-phenylpiperi-
din-4-ol (4a-e) and 4-benzylpiperidine (5a-e) moieties were
present. Table 1 shows that all the racemates displayed a
preference for the σ1 receptors. Compound 4b showed the best
profile of σ2/σ1 selectivity (Ki(σ2)/Ki(σ1) ) 33.9), whereas
compound 5e exhibited a subnanomolar Ki value for the σ1
receptor. However, for the two aforementioned series, a nano-
molar affinity was also achieved for the σ2 receptors in a similar
way to haloperidol (Ki(σ2)/Ki(σ1) ) 7.3).24 Conversely the
1-(pyridin-2-yl)piperazine derivatives 6b, 6d, and 6e and the
2-(piperazin-1-yl)pyrimidine derivatives 7b, 7d, and 7e showed
only moderate affinity for the σ1 receptors without appreciable
σ2 receptor affinity (Table 1). The differences in binding
affinities suggest that the presence of 1-phenyl cyclopropyl-
methycarboxylate moiety joined to the 1-(pyridin-2-yl)piperazine
or 2-(piperazin-1-yl)pyrimidine moiety, both with electron-poor
systems, is an unfavorable combination for the σ1 receptors and
is detrimental for the σ2 sites.
In our preliminary experiments (data not shown), in view of
both a vitality test and microscopy observations, we established
the concentrations and the time of exposure of the cultures to
σ ligands. These sets of experiments showed that the nontoxic
concentration of the drugs was 25 µM and the optimal time of
exposure was 24 h. At 50 µM, astroglial cell cultures appeared
activated, whereas the higher concentration (100 µM) induced
cell death (data not shown). Since it has been suggested that
the multiplicity effects of TG-2 in various cellular pathways
may be due to its different cellular localization and its
transamidanting and GTPase activity, we evaluated its expres-

NoVel σ Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 953
Table 1. Structures and σ Binding Affinities of 1-Phenyl-2-cyclopropylmethylamine Derivatives
a Each value is the mean ( SD of three determinations.
sion and its intracellular localization by Western blot and
confocal laser scanning microscopy (CLSM) analysis following
σ ligands exposure.
TG-2 expression (Figure 2Af and Figure 2Bf). These results
suggest the existence of a possible link between the TG-2
expression and σ receptors.
Figure 2A shows the Western blot and Figure 2B the
densitometric analysis of TG-2 expression in 14 DIV astroglial
cell cultures in the absence, or in the presence, of σ ligands (25
µM for 24 h). Specifically, the ibogaine treament, ibogaine being
a σ2 agonist,29 significantly increased the percentage of the TG-2
expression (Figure 2Ab and Figure 2Bb). Haloperidol treatment,
even if it possesses high affinity for multiple neuroreceptors,24
used as a σ2 agonist and σ1 antagonist,24 presented30 a pattern
of TG-2 expression similar to the untreated cells (Figure 2Ac
and Figure 2Bc). We also used BD1047, a σ antagonist,31 even
though it has been reported to act as a partial agonist.32 This
treatment did not increase the percentage of TG-2 expression
(Figure 2Ad and Figure 2Bd). By contrast, in the 4b-treated
cells we observed a significant increase of the percentage of
TG-2 expression (Figure 2Ae and Figure 2Be). The (+)-
pentazocine, used as a σ1 agonist, did not induce changes of
Figure 3 shows CLSM analysis in the astroglial cell cultures.
In untreated astroglial cell cultures TG-2 fluorescence was
distributed at low levels in the cytosol and in the mitochondria
(Figure 3a). The exposure of astrocyte cell cultures to ibogaine
(Figure 3b) significantly increased TG-2 expression and redis-
tributed the protein prevalently in the cytosol, in the mitochon-
dria, in the nuclei, and in the nucleoli. On the other hand,
treatment with haloperidol significantly increased TG-2 expres-
sion and the protein appeared localized only in the nuclei and
in the nucleoli (Figure 3c). In the BD1047-treated astrocytes
we observed a low but significant increase of TG-2 expression
when compared to the untreated cells. The protein appeared
prevalently distributed in the mitochondria and along the
cytosolic projections (Figure 3d). In addition, it was also
observed at very low levels in the nuclear compartment, even
if its level was higher than in the control.

954 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
Table 2. Binding Affinity Profile of 4b
PrezzaVento et al.
3f). The fluorescence appeared at low levels in the cytosol, in
the cellular projections, in the nuclei, and in the nucleoli (Figure
3f).
The mean energy of CLSM analysis further demonstrated the
fluorescence intensity of TG-2 in the cytosol and in the nuclear
compartment (Figure 4). The presence of TG-2 in the nuclear
compartment and in the cytosol may be due to its different cell
localization and its transamidanting and GTPase activities.15
Moreover, although TG-2 is found mostly in the cytosol, it is
also present in the nucleus21 and associated with the plasma
membrane.18
Since intracellular localization affects protein function by
determining which interactions are likely to occur, it is possible
that with access to different substrates in different intracellular
pools, the multiplicity of TG-2 effects in different cellular
pathways may be due to its localization and to which enzymatic
activity of TG-2 is favored (transamidating or GTP binding/
hydrolyzing).33 In addition, it has recently been demonstrated
that TG-2 possesses a serine/treonine kinase activity and is able
to phosphorylate histone proteins.19 Although it has been
implicated in the apoptotic pathway, there is evidence that under
certain circumstances TG-2 can also protect against apoptosis.19
Since TG-2 plays a modulatory role in apoptosis and cell
response to stressors, depending on the types of the stimuli that
provoke an increase in the transamidating activity,16 we studied
the involvement of the protein in σ-receptor-mediated apoptosis
as measured by caspase-3 cleavage and the terminal deoxy-
nucleotidyltransferase-mediated dUTP nick-end-labeling (TUNEL)
test.
receptor
Ki ( SD,a nM
DA
D1 ([3H]SCH 23390)
D2 ([3H]spiperone)
D3 ([3H]-7OH-DPAT)
>10000
1285 ( 384
4900 ( 1077
5HT
5-HT2A ([3H]ketanserin)
5-HT2C ([3H]mesulergine)
5-HT3 ([3H]LY278584)
5-HT4 ([3H]GR13808)
5-HT6 ([3H]-5HT)
3149 ( 440
4357 ( 1176
>10000
>10000
>10000
R
R1 ([3H]prazosin)
605 ( 157
9414 ( 900
>10000
R2 ([3H]RX821002)
muscarinic ([3H]-QNB)
H1 ([3H]pyrilamine)
1422 ( 327
>10000
NMDA ([3H]CGP 39653)
NMDA channel ([3H]MK 801)
DAT ([3H]-WIN 35,428)
SERT ([3H]citalopram)
>10000
>10000
>10000
a Each value is the mean ( SD of three determinations.
Table 3. Activity of Tested σ Receptor Ligands
This set of experiments showed a marked enhancement of
caspase-3 cleavage in ibogaine-treated astrocytes when com-
pared to the untreated control (Figure 5Ab and Figure 5Bb).
Furthermore, a slight increase of caspase-3 cleavage in halo-
peridol-treated astrocytes was found (Figure 5Ac and Figure
5Bc), whereas a decrease in both BD1047 (Figure 5Ad and
Figure 5Bd) and (+)-pentazocine-treated astrocytes (Figure 5Af
and Figure 5Bf) was observed. Conversely, in 4b-treated
astrocytes we observed a significant increase of caspase-3
cleavage (Figure 5Ae and Figure 5Be).
These results were also confirmed by evaluating DNA
fragmentation performed in primary astroglial cell cultures by
the TUNEL test (Figure 6).34 The percentage of the apoptotic
cells compared to the nonapoptotic cells is reported in the Table
4.
A marked increase of apoptotic cells in ibogaine-treated
astroglial cells (Figure 6b) was found when compared to
untreated control ones (Figure 6a). By contrast, haloperidol,
BD1047, and (+)-pentazocine did not have any effect on DNA
fragmentation (parts c, d, and f of Figure 6). Interestingly, in
4b-treated cells (Figure 6e) a significant increase of DNA
fragmentation was observed when compared to the control
(Figure 6a), even if apoptotic cells appeared lower than in
ibogaine-treated astrocytes (Figure 6b).
These results suggest that primary astroglial cell cultures are
responsive to σ ligands and that TG-2 is part of σ receptor
activation pathway. In particular, the prevalent localization of
TG-2 in the cytosol of the ibogaine-treated astroglial cell cultures
suggests that the protein may be involved in the activation of
the σ2-mediated apoptotic pathway. Furthermore, it has been
hypothesized that σ2 receptors are histone proteins and that σ2
ligands also bind histone proteins35 that also been shown to be
a substrate of TG-2.36 The apparent discrepancy of the results
obtained with BD1047, with respect to ibogaine, may be related
to the methods used, since the caspase-3 cleavage and TG-2
In 4b-treated astroglial cell cultures a significant increase of
TG-2 expression was observed (Figure 3e). The fluorescence
distribution showed that the protein was localized in the cytosol,
along cytosol projections, in the mitochondria, in the nuclei,
and in the nucleoli (Figure 3e). In this case the fluorescence
intensity was higher in the cytosol and in the mitochondria than
in ibogaine-treated astroglial cells, even though the fluorescence
levels in 4b-treated astrocytes in both the nuclei and nucleoli
were lower than those observed in ibogaine-treated cells (parts
b and e of Figure 3). However, the fluorescence levels observed
in the nuclei and nucleoli of 4b-treated astrocytes were lower
than in haloperidol-treated ones (parts c and e of Figure 3). The
treatment of the cells with (+)-pentazocine induced a low
increase of TG-2 when compared to the untreated control (Figure

NoVel σ Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 955
Figure 2. (A) Representative immunoblots of TG-2 expression in primary rat astroglial cell cultures of cultured astroglial cells in the (a) absence
or (b-f) presence of 25 µM sigma ligands for 24 h: (b) ibogaine, (c) haloperidol, (d) BD1047, (e) 4b, (f) (+)-pentazocine. HUVEC cell extract
was used as positive control. (B) Densitometric analysis of the percentage of TG-2 expression in response to σ ligand exposure. Results are expressed
as the mean ( SD of the values of four experiments: (/) p < 0.05, significant differences versus the controls.
Figure 3. CLSM analysis of TG-2 distribution in primary rat astroglial cell cultures at 14 DIV. Representative pictures of TG-2 fluorescent
staining in the (a) absence or (b-f) presence of 25 µM σ ligands for 24 h: (b) ibogaine, (c) haloperidol, (d) BD1047, (e) 4b, (f) (+)-pentazocine.
Immunostaining for TG-2 in the cytosol (green) and in the nuclei (red) is shown. The photos are from sequential analysis of the same microscopic
field, followed by a merge of different images with specific staining.
expression were evaluated by Western blot in the total cell
lysates, whereas CLSM analysis was performed on a single cell.
This is strictly dependent on the different stages of the cells. In
fact, it has been demonstrated that the functions and the
localization of the protein are dependent on the type of the
stimuli and the cell response to stressors.33 This hypothesis is
further substantiated by other observations showing that the
activation of σ2 receptors mediated apoptotic pathway may be
caspase-independent.37 The haloperidol, acting as BD1047 on
the σ receptors and also possessing high affinity for the other
neuroreceptors,24 did not cause a significant increase of TG-2
expression by Western blot analysis, caspase-3 cleavage, and
DNA fragmentation. In contrast, CLSM analysis performed on
Figure 4. Mean energy of CLSM analysis of TG-2 fluorescence present
in the cytosol or in the nuclei of primary rat astroglial cell cultures at
a single cell showed the expression and the localization of TG-2
in the nuclear compartment that may be associated with the
antiapoptotic function of the protein.33 Compound 4b showed
a pattern similar to that of ibogaine, even though it resulted in
a lower TG-2 expression in cytosol than in ibogaine-treated cells.
14 DIV exposed to 25 µM σ ligands for 24 h: (a) untreated cells, (b)
ibogaine, (c) haloperidol, (d) BD1047, (e) 4b, (f) (+)-pentazocine.
These differences may be due to the major lypophilic charac-
teristic of ibogaine (calculated log P ) 4.04 ( 0.43). On the

956 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
PrezzaVento et al.
Figure 5. (A) Representative immunoblots of caspase-3 cleavage in primary rat astroglial cell cultures of cultured astroglial cells in the (a) absence
or (b-f) presence of 25 µM σ ligands for 24 h: (b) ibogaine, (c) haloperidol, (d) BD1047, (e) 4b, (f) (+)-pentazocine. RSV-3T3 was used as
positive control. (B) Densitometric analysis of the percentage of caspase-3 cleavage in response to σ ligand exposure. Results are expressed as the
mean ( SD of the values of four experiments: (/) p < 0.05, significant differences versus the controls.
Figure 6. Representative pictures of TUNEL assay performed in primary rat astroglial cell cultures at 14 DIV exposed to 25 µM σ ligands for 24
h, (b) ibogaine, (c) haloperidol, (d) BD1047, (e) 4b, (f) (+)-pentazocine, in comparison with (a) the untreated cells. Immunostaining of nonapoptotic
(red) and apoptotic (green) cells is shown.
other hand, (+)-pentazocine, inducing a low increase of the
intracellular calcium levels,38 did not have any effect on the
activation of the apoptotic pathway and on TG-2 expression.
CSLM analysis showed that the protein was localized at very
low levels in the cytosol and also in the nuclear compartment.
These results suggested that TG-2 may play a protective role
against apoptosis.33
systems compared to haloperidol. The biological results further
suggest that σ receptors may modulate TG-2 expression, its
translocation into the nuclei and nucleoli, and its different func-
tions in primary rat astroglial cell cultures. We also demonstrated
that σ ligands differently induced the activation of the apoptotic
pathway that is prevalently related to the σ2 agonist ligands. In
addition, since 4b increased TG-2 expression and its translo-
cation into the nuclear compartment and activated the apoptotic
pathway, it may act as σ1/σ2 agonist. Furthermore, our results
suggest that the selective σ ligands may represent an interesting
tool to modulate intracellular calcium levels and consequently
the up-regulation of TG-2 typical of several neurodegenerative
diseases.15,20 Further studies are now warranted in order to
Together, these results show that replacement of the normeta-
zocine nucleus of (+)-MPCB with 4-phenyl-4-hydroxypiperi-
dine or 4-benzylpiperidine provides high-affinity σ ligands with
a preference for σ1 binding sites. Interestingly, 4b, the most
selective σ1 ligand (Ki(σ2)/Ki(σ1) ) 33.9) of the series was also
very selective with respect to many receptors and transporter

NoVel σ Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 957
Table 4. TUNEL Testa
sheets (Merck, Darmstadt, Germany). Visualization was provided
under UV light or in an iodine chamber. Merck silica gel 60, 230-
400 mesh, was used for flash column chromatography. Melting
points were obtained in open capillary tubes with a Bu¨chi 530
apparatus (Bu¨chi Italia, Assago, Italy) and are uncorrected. 1H NMR
spectra were recorded with a Varian Inova 200 MHz spectrometer
(Varian, Leini, Italy). Elemental analyses (C, H, N) were determined
on an elemental analyzer, Carlo Erba model 1106 (Carlo Erba,
Milan, Italy), and the results were within (0.4% of the theoretical
values. The log P values of the examined compounds were
calculated using the ACD/LogP DB software (version 4.55).
The following radioactive materials were used: [3H]-(+)-
pentazocine (45 Ci/mmol), [3H]DTG [1,3-di-(2-tolyl)guanidine] (31
Ci/mmol), obtained from Perkin-Elmer. [3H]SCH 23390 (75.5 Ci/
mmol), [3H]spiperone (20 Ci/mmol), [3H]QNB (42 Ci/mmol), [3H]-
ketanserin (63.3 Ci/mmol), [3H]-5HT (80 Ci/mmol), [3H]prazosin
(83.8 Ci/mmol), [3H]pyrilamine (20 Ci/mmol), [3H]CGP 39653 (40
Ci/mmol), [3H]WIN 35,428 (86 Ci/mmol), and [3H]MK 801 (30
Ci/mmol) were obtained from NEN, and [3H]-7OH-DPAT (139 Ci/
mmol), [3H]mesulergine (86 Ci/mmol), [3H]LY 278584 (84 Ci/
mmol), [3H]GR 113808 (81 Ci/mmol), [3H]citalopram (85 Ci/
mmol), and [3H]RX 821002 (49 Ci/mmol) were obtained from
Amersham (U.K.). Ibogaine was a kind gift from the Slater & Frith
(U.K.).
Cell culture media and sera were from Invitrogen (Milan, Italy).
FITC and TRITC-conjugated anti-IgG and other chemicals of
analytical grade were from Sigma-Aldrich (Milan, Italy). Mouse
monoclonal antibody against TG-2 was from NeoMarkers (Fremont,
CA). Mouse monoclonal antibody against caspase-3 was from
Becton-Dickinson (Milan, Italy). The horseradish peroxidase-
conjugated anti-mouse IgG and ECL (enhanced chemiluminescence)
developing system for immunoblots were purchased from Amer-
sham Pharmacia Biotech (Milan, Italy). The kit for the TUNEL
test was from Clonteck.
treatment
% of apoptotic cells
control
0
ibogaine
(+)-pentazocine
BD1047
haloperidol
4b
95 ( 5
0
7 ( 1
4 ( 0.5
65 ( 4.5
a Percentage of the apoptotic cells compared to the nonapoptotic cells
for each microscopic field. The ratio is expressed as a percentage, taking
the total number of cells as 100 and comparing 10 random microscopic for
each dish. The DNA fragmentation assay incorporates fluorescein-dUTP
at the free 3′-hydroxyl ends of the fragmented DNA using TUNEL.
Apoptotic cells appear green, while nonapoptotic cells appear red: (/) p <
0.05, significant differences versus the controls.
investigate the importance of the stereochemistry of the cyclo-
propane ring and to clarify the biochemical mechanisms
involved in the interactions between TG-2 and σ receptors.
Experimental Section
Radioligand Binding Assays. The σ binding experiments were
performed as previously reported.39 Briefly, σ1 binding assays were
performed on guinea pig brain membranes,32 incubating 500 µg of
membrane protein with 3 nM [3H]-(+)-pentazocine (45 Ci/mM;
the value of the apparent dissociation constant (Kd) was 1.2 ( 0.3
nM, n ) 3) in 50 mM Tris-HCl (pH 7.4). Test compounds were
added at concentrations ranging from 10-5 to 10-11 M. Nonspecific
binding was assessed by the addition of 10 µM of haloperidol. The
reaction was performed for 150 min at 37 °C and terminated by
filtering the solution through Whatman GF/B glass fiber filters that
were presoaked for 1 h in a 0.5% poly(ethylenimine) solution.
Filters were washed twice with 4 mL of ice-cold buffer.
The σ2 binding assays were carried out on guinea pig brain
membranes prepared as previously described.29 Briefly, the mem-
branes were incubated with 3 nM [3H]DTG (1,3-di-(2-tolyl)-
guanidine) (31 Ci/mM, Kd ) 9.9 ( 0.8 nM, n ) 3) in the presence
of 400 nM (+)-SKF10,047 to block σ1 sites. Incubation was carried
out in 50 mM Tris-HCl (pH 8.0) for 120 min at room temperature,
and assays were terminated by the addition of ice-cold 10 mM Tris-
HCl, pH 8.0, followed by filtration through Whatman GF/B glass
fibers filters that were presoaked for 1 h in a 0.5% poly-
(ethylenimine) solution. Filters were washed twice with 4 mL of
ice-cold buffer. Nonspecific binding was evaluated in the presence
of 5 µM DTG. Various rat brain regions were dissected to assess
the following binding assays; rat striatum for D1 and D2 receptors
and DAT; rat olfactory tubercle for D3 receptors; rat cortex for
muscarinic, 5-HT2A, 5-HT3, R1, R2, H1, and NMDA receptors and
SERT; guinea pig cortex for 5-HT4 receptors. 5-HT2C binding assays
were carried out on pig brains (frontal cerebral cortex) obtained
from a local slaughterhouse and immediately placed on ice until
use. Binding experiments on 5-HT6 receptors were performed using
a rat-cloned serotonin receptor subtype 6 in HEK-293 cells, as
reported by Boess et al.40 Tissue preparations and binding assays
were carried out according to Gobbi et al.41 and Mennini et al.42
After incubation the samples were filtered through Whatman GF/B
or GF/C glass fiber filters using a Brandel apparatus (model M-48R)
or Millipore filter apparatus. The filters were washed three times
with 4 mL of ice-cold buffer. Radioactivity was counted in 4 mL
of “Ultima Gold MV” or Filter Count Cocktail (Packard) in a DSA
1409 (Wallac) or 1414 Winspectral Perkin-Elmer Wallac liquid
scintillation counter. For inhibition experiments, the drugs were
added to the binding mixture at seven to nine different concentra-
tions. Inhibition constants (Ki values) were calculated using the
“Allfit” program or the EBDA/LIGAND program purchased from
Elsevier/Biosoft.
Compound Syntheses. Compounds 1a-d were synthesized as
previously reported by Casadio and co-workers.23
(1R,5S/1S,5R)-1-(4-Nitrophenyl)-3-oxabicyclo[3.1.0]hexan-2-
one (1e). To 1 g (5.74 mmol) of lactone 1a we added, dropwise,
16.5 mL (-40 °C) of fuming HNO3. After being stirred for 2 min,
the mixture was poured into ice and the crystalline product was
filtered and washed with water until neutral pH was reached. After
drying, the resultant crude product was crystallized twice from
methanol to give 0.60 g of the desired product. Yield 48%; mp
142-143 °C; 1H NMR (CDCl3) δ 1.55 (dd, J ) 5.0, 5.2 Hz, 1H),
1.72 (dd, J ) 5.0, 7.8 Hz, 1H), 2.65-2.85(m, 1H), 4.35 (d, J )
9.2 Hz, 1H), 4.51 (dd, J ) 4.4, 9.2 Hz, 1H), 7.63 (d, J ) 8.8 Hz,
2H), 8.20 (d, J ) 9.0 Hz, 2H). Anal. (C11H9NO4) C, H, N.
(1R,5S/1S,5R)-1-(4-Aminophenyl)-3-oxabicyclo[3.1.0]hexan-
2-one (1f). A mixture of 1 g (4.5 mmol) of lactone 1e, 5.1 g (22.78
mmol) of SnCl2 dihydrate, and 10 mL of EtOH/EtOAc (2:1) was
stirred under reflux for 30 min. The cooled solution was combined
with ice, and 5% NaHCO3 was added up to pH 7-8. The resulting
mixture was extracted twice with EtOAc (2 × 70 mL). The organic
layer was washed with water, dried over Na2SO4, and filtered. The
filtrate was evaporated in vacuo and gave 840 mg of product. Yield
1
98.7%; mp 96-102 °C; H NMR (CDCl3) δ 1.27 (dd, J ) 4.6,
4.8, Hz, 1H), 1.58 (dd, J ) 4.8, 7.8 Hz, 1H), 2.35-2.55 (m, 1H),
3.70 (br s, 2H), 4.26 (d, J ) 9,2 Hz, 1H), 4.52 (dd, J ) 4.6, 9.2
Hz, 1H), 6.60-7.35 (m, 4H). Anal. (C11H11NO2) C, H, N.
Methyl (1R,2S/1S,2R)-2-(Chloromethyl)-1-phenylcyclopro-
panecarboxylate (2a). Thionyl chloride (0.98 mL, 13.42 mmol)
was added dropwise to 500 mg (2.80 mmol) of (()-1a in 8.3 mL
of anhydrous benzene at 0 °C and was heated to reflux for 5 h.
The mixture was cooled to 0 °C, and a solution of 3 N CH3OH/
HCl (3.02 mL) was added dropwise. The reaction mixture was then
allowed to stand at room temperature overnight. The mixture was
evaporated under reduced pressure, and the residue was dissolved
in Et2O and extracted with a saturated solution of NaHCO3. The
organic phase was dried (Na2SO4) and evaporated in vacuo to give
Materials and Methods. Reagents used for synthesis were
purchased from Sigma-Aldrich (Milan, Italy) unless otherwise
specified. The course of the reaction was monitored by thin-layer
chromatography (TLC) on precoated silica gel 60 F254 aluminum
1
322 mg of the desired ester. Yield 51%; mp 77-80 °C; H NMR
(CDCl3) δ 1.50 (dd, J ) 4.8, 8.8 Hz, 1H), 1.82 (dd, J ) 4.8, 7.0

958 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
PrezzaVento et al.
Hz, 1H), 1.90-2.20 (m, 1H), 3.64 (s, 3H), 3.80 (dd, J ) 10.2,
10.4 Hz, 1H), 4.00 (dd, J ) 5.6, 10.2 Hz, 1H), 7.20-7.45 (m, 5H).
Anal. (C12H13ClO2) C, H, N.
2.04-2.20 (m, 2H), 2.80-3.20 (m 7H), 3.56 (s, 3H), 5.80 (br s,
1H), 7.16-7.52 (m, 10H). Anal. (C23H27NO3‚0.5C2H2O4) C, H, N.
Compounds 4b-e were synthesized analogously.
Compounds 2b-f were synthesized analogously.
Methyl (1R,2S/1S,2R)-2-[(4-Hydroxy-4-phenylpiperidin-1-yl)-
methyl]-1-(4-methylphenyl)cyclopropanecarboxylate Oxalate (4b).
Methyl (1R,2S/1S,2R)-2-(Chloromethyl)-1-(4-methylphenyl)-
cyclopropanecarboxylate (2b). This compound was purified by
silica gel flash column chromatography using cyclohexane/ethyl
acetate (8:2). Yield 88%; mp 58-61 °C; 1H NMR (CDCl3) δ 1.47
(dd, J ) 4.8, 8.8 Hz, 1H), 1.80 (dd, J ) 4.8, 7.0 Hz, 1H), 1.99 (m,
1H), 2.34 (s, 3H), 3.65 (s, 3H), 3.80 (dd, J ) 10.0, 11.4 Hz, 1H),
4.00 (dd, J ) 5.6, 11.4 Hz, 1H), 7.13 (d, J ) 8.4 Hz, 2H), 7.27 (d,
J ) 7.8 Hz, 2H). Anal. (C13H15ClO2) C, H, N.
1
Yield 39%; mp 197-198 °C; H NMR NMR (DMSO-d6) δ 1.37
(dd, J ) 4.6, 8.8 Hz, 1H), 1.56 (dd, J ) 4.6, 6.8 Hz, 1H), 1.63-
1.78 (m, 2H), 1.80-1.88 (m, 1H), 2.0-2.21 (m, 2H), 2.27 (s, 3H),
2.82-3.20 (m, 7H), 3.55 (s, 3H), 5.65 (br s, 1H), 7.03-7.52 (m,
9H). Anal. (C24H29NO3‚0.5C2H2O4) C, H, N.
Methyl (1R,2S/1S,2R)-2-[(4-Hydroxy-4-phenylpiperidin-1-yl)-
methyl]-1-(4-methoxyphenyl)cyclopropanecarboxylate Oxalate
1
Methyl (1R,2S/1S,2R)-2-(Chloromethyl)-1-(4-methoxyophe-
nyl)cyclopropanecarboxylate (2c). Yield 90%; oil; 1H NMR
(CDCl3) δ 1.46 (dd, J ) 4.8, 8.6 Hz, 1H), 1.80 (dd, J ) 4.8, 7.0
Hz, 1H), 1.85-2.10 (m, 1H), 3.54 (s, 3H), 3.72 (s, 3H), 3.75 (dd,
J ) 10.0, 11.2 Hz, 1H), 4.00 (dd, J ) 5.6, 11.2 Hz, 1H), 6.80-
7.38 (m, 4H). Anal. (C13H15ClO3‚0.3Et2O) C, H, N.
(4c). Yield 20%; mp 185-186 °C; H NMR (DMSO-d6) δ 1.37
(dd, J ) 4.6, 8.8, Hz, 1H), 1.43-2.0 (m, 4H), 2.05-2.13 (m, 2H),
2.80-3.20 (m, 7H), 3.54 (s, 3H), 3.72 (s, 3H), 5.30 (br s, 1H),
6.80-7.42 (m, 9H). Anal. (C24H29NO4‚0.5C2H2O4‚0.75H2O) C, H,
N.
Methyl (1R,2S/1S,2R)-1-(4-Chlorophenyl)-2-[(4-hydroxy-4-
phenylpiperidin-1-yl)methyl]cyclopropanecarboxylate (4d). Yield
Methyl (1R,2S/1S,2R)-2-(Chloromethyl)-1-(4-chlorophenyl)-
cyclopropanecarboxylate (2d). The crude product was purified
by chromatography on silica gel (cyclohexane/ethyl acetate, 9:1).
Yield 80%; mp 47-51 °C; 1H NMR (CDCl3) δ 1.47 (dd, J ) 4.8,
9.0 Hz, 1H), 1.83 (dd, J ) 4.8, 6.8 Hz, 1H), 1.90-2.10 (m, 1H),
3.62 (s, 3H), 3.77 (dd, J ) 6.2, 11.4 Hz, 1H), 4.00 (dd, J ) 5.6,
11.4 Hz, 1H), 7.20-7.40 (m, 4H). Anal. (C12H12Cl2O2) C, H, N.
Methyl (1R,2S/1S,2R)-2-(Chloromethyl)-1-(4-nitrophenyl)cy-
clopropanecarboxylate (2e). Thionyl chloride (2.5 mL, 34.25
mmol) was added dropwise to 1.5 g (6.84 mmol) of racemic lactone
1e in 20 mL of anhydrous benzene at 0 °C and was heated to reflux
for 5 h. The mixture was cooled to 0 °C, and a solution of 3 N
CH3OH/HCl (7.3 mL) was added dropwise. The reaction mixture
was then allowed to stand at room temperature overnight. The
mixture was evaporated under reduced pressure, and the residue
was dissolved in Et2O and washed with a saturated solution of
NaHCO3. The organic phase was dried (Na2SO4) and evaporated
in vacuo to give the desired ester, which was used without further
purification.
Methyl (1R,2S/1S,2R)-1-(4-Aminophenyl)-2-(chloromethyl)-
cyclopropanecarboxylate (2f). Thionyl chloride (1.57 mL, 21.51
mmol) was added dropwise to 836 mg (4.42 mmol) of 1f in 13.13
mL of anhydrous benzene at 0 °C and was heated to reflux for 5
h. The mixture was cooled to 0 °C, and a solution of 3 N CH3-
OH/HCl (3.02 mL) was added dropwise. The reaction mixture was
then allowed to stand at room temperature overnight. The mixture
was evaporated under reduced pressure, and the residue was
dissolved in Et2O and extracted with a saturated solution of
NaHCO3. The organic phase was dried over Na2SO4 and evaporated
in vacuo and the resultant crude product was purified by chroma-
tography on silica gel (cyclohexane/ethyl acetate, 6.5:3.5) to obtain
500 mg of product. Yield 65.8%; mp, decomposes; 1H NMR
(CDCl3) δ 1.43 (dd, J ) 4.8, 9.0 Hz, 1H), 1.75 (dd, J ) 4.8, 6.8
Hz, 1H), 1.80-2.05 (m, 1H), 3.51 (br s, 2H), 3.64 (s, 3H), 3.75 (d,
J ) 11.4 Hz, 1H), 4.12 (dd, J ) 5.6, 11.4 Hz, 1H), 6.63 (d, J )
8.4 Hz, 2H), 7.15 (d, J ) 8.4 Hz, 2H). Anal. (C12H14NO2Cl) C, H,
N.
1
68%; mp 125-126 °C; H NMR NMR (DMSO-d6) δ 1.21 (dd, J
) 4.4, 8.8 Hz, 1H), 1.37-1.87 (m, 6H), 2.27-2.77 (m, 7H), 3.44
(s, 3H), 4.69 (s, 1H), 7.0-7.42 (m, 9H). Anal. (C23H26NClO3‚
0.4H2O) C, H, N.
Methyl (1R,2S/1S,2R)-2-[(4-Hydroxy-4-phenylpiperidin-1-yl)-
methyl]-1-(4-nitrophenyl)cyclopropanecarboxylate Oxalate (4e).
This compound was purified by silica gel flash column chroma-
tography using ethyl acetate/cyclohexane (2:1). Yield 43%; mp
204-205 °C; 1H NMR NMR (DMSO-d6) δ 1.60 (dd, J ) 4.6, 9.0
Hz, 1H), 1.70 (m, 3H), 1.94-2.23 (m, 3H), 2.84-3.27 (m, 7H),
3.58 (s, 3H), 5.03 (br s, 1H), 7.18-8.23 (m, 9H). Anal. (C23H26N2O5‚
0.5C2H2O4‚0.25H2O) C, H, N.
Methyl (1R,2S/1S,2R)-2-[(4-Benzylpiperidin-1-yl)methyl]-1-
phenylcyclopropanecarboxylate Oxalate (5a). A mixture of
4-benzylpiperidine (0.20 mL, 1.11 mmol), (1R,2S/1S,2R)-2-(chlo-
romethyl)-1-phenylcyclopropanecarboxylate (2a, 250 mg, 1.11
mmol), and NaHCO3 (200 mg, 2.22 mmol) in dry DMF (10 mL)
was heated to 60 °C under a nitrogen atmosphere for 24 h. The
solvent was removed under reduced pressure, and the mixture was
extracted with CHCl3. The combined organic layers were washed
with water and dried over anhydrous Na2SO4. The crude product
was purified by silica gel flash column chromatography using ethyl
acetate/cyclohexane (6:4). The resulting colorless oil was dissolved
in diethyl ether and treated with a solution of oxalic acid dihydrate
in diethyl ether to give 220 mg of the title compound. Yield 44%;
mp 141-142 °C; 1H NMR (DMSO-d6) δ 1.30-2.10 (m, 8H),
2.42-3.52 (m, 8 H), 3.56 (s, 3H), 7.10-7.40 (m, 10H), 7.80 (br s,
3H). Anal. (C24H29NO2‚0.8C2H2O4‚H2O) C, H, N.
Compounds 5b-f were synthesized analogously.
Methyl (1R,2S/1S,2R)-2-[(4-Benzylpiperidin-1-yl)methyl]-1-
(4-methylphenyl)cyclopropanecarboxylate Oxalate (5b). Yield
20%; mp 149-150 °C; 1H NMR (CDCl3) (free base) δ 1.25-2.10
(m, 10H), 2.32 (s, 3H), 2.30-2.70 (m, 4H), 2.90-3.10 (m, 2H),
3.58 (s, 3H), 7.05-7.35 (m, 9H). Anal. (C25H31NO2‚1.25C2H2O4‚
3H2O) C, H, N.
Methyl (1R,2S/1S,2R)-2-[(4-Benzylpiperidin-1-yl)methyl]-1-
(4-methoxyphenyl)cyclopropanecarboxylate Oxalate (5c). Yield
Methyl (1R,2S/1S,2R)-2-[(4-Hydroxy-4-phenylpiperidin-1-yl)-
methyl]-1-phenylcyclopropanecarboxylate Oxalate (4a). A mix-
ture of 4-phenylpiperidin-4-ol 200 mg (1.13 mmol), methyl (1R,2S/
1S,2R)-2-(chloromethyl)-1-phenylcyclopropanecarboxylate (2a, 253
mg, 1.13 mmol), and NaHCO3 (190 mg, 2.26 mmol) in dry DMF
(10 mL) was heated to 60 °C under a nitrogen atmosphere for 24
h. The solvent was removed under reduced pressure, and the mixture
was extracted with CHCl3. The combined organic layers were
washed with water and with 5% aqueous NaHCO3 and dried over
anhydrous Na2SO4. The crude product was purified by silica gel
flash column chromatography using ethyl acetate/cyclohexane/
ethanol (6:2:2). The resulting colorless oil was dissolved in diethyl
ether and treated with a solution of oxalic acid dihydrate in diethyl
ether to give 127 mg of colorless solid. Yield 27%; mp 201-202
1
37%; mp 144-145 °C; H NMR NMR (DMSO-d6) δ 1.30-2.00
(m, 8H), 2.42-3.60 (m, 8 H), 3.61 (s, 3H), 3.73 (s, 3H), 6.78-
7.40 (m, 9H), 7.90 (br s, 1H). Anal. (C25H31NO3‚0.5C2H2O4‚
1.75H2O) C, H, N.
Methyl (1R,2S/1S,2R)-2-[(4-Benzylpiperidin-1-yl)methyl]-1-
(4-chlorophenyl)cyclopropanecarboxylate Oxalate (5d). Yield
34%; mp 86-87 °C; 1H NMR NMR (DMSO-d6) δ 1.30-2.05 (m,
8H), 2.42-3.51 (m, 8 H), 3.55 (s, 3H), 7.12-7.42 (m, 9H), 8.0 (br
s, 4H). Anal. (C24H28ClNO2‚0.5C2H2O4‚1.6H2O) C, H, N.
Methyl (1R,2S/1S,2R)-2-[(4-Benzylpiperidin-1-yl)methyl]-1-
(4-nitrophenyl)cyclopropanecarboxylate Oxalate (5e). Yield
1
44%; mp 99-100 °C; H NMR NMR (DMSO-d6) δ 1.30-2.10
1
°C; H NMR (DMSO-d6) δ 1.40 (dd, J ) 4.6, 9.0 Hz, 1H), 1.58
(m, 8H), 2.42-3.52 (m, 8 H), 3.58 (s, 3H), 6.80 (br s, 4H), 7.05-
8.22 (m, 9H). Anal. (C24H28N2O4‚0.5C2H2O4‚2H2O) C, H, N.
(dd, J ) 4.8, 6.8 Hz, 1H), 1.66-1.78 (m, 2H), 1.85-1.97 (m, 1H),

NoVel σ Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 959
The following compounds were prepared using the above
procedure.
3.12 (m, 5H), 3.19 (dd, J ) 6.4, 13.2 Hz, 1H), 3.55 (s, 3H), 3.80-
4.0 (m, 4H), 6.70 (t, J ) 4.8, 9.6 Hz, 1H), 7.20-7.40 (m, 5H),
8.40 (d, J ) 4.8 Hz, 2H). Anal. (C20H24N4O2‚0.5H2C2O4‚2H2O‚
0.2(C2H5)2O) C, H, N.
Methyl (1R,2S/1S,2R)-1-(4-Methylphenyl)-2-[(4-pyrimidin-2-
ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (7b).
Yield 37%; mp 222-225 °C; 1H NMR (CDCl3) (free base) δ 1.34
(dd, J ) 4.4, 8.8 Hz, 1H), 1.65 (dd, J ) 4.4, 7.2 Hz, 1H), 1.72-
1.90 (m, 1H), 2.34 (s, 3H), 2.50-2.82 (m, 6H), 3.62 (s, 3H), 3.90
(m, 4H), 6.49 (t, J ) 4.8 Hz, 1H), 7.12 (d, J ) 8.0 Hz, 2H), 7.24
(d, J ) 8.4 Hz, 2H), 8.31 (d, J ) 4.8 Hz, 2H). Anal. (C21H26N4O2‚
0.3H2C2O4‚2.5H2O‚0.3(C2H5)2O) C, H, N.
Methyl (1R,2S/1S,2R)-1-Phenyl-2-[(4-pyridin-2-ylpiperazin-
1-yl)methyl]cyclopropanecarboxylate Oxalate (6a). This com-
pound was purified by silica gel flash column chromatography using
chloroform as eluent. Yield 19%; mp 205-207 °C; 1H NMR
(DMSO-d6) (free base) δ 1.45 (dd, J ) 4.8, 8.8 Hz, 1H), 1.59 (dd,
J ) 4.8, 6.8 Hz, 1H), 1.90-2.07 (m, 1H), 3.07-3.19 (m, 5H), 3.24
(dd, J ) 6.4, 13.2 Hz, 1H), 3.56 (s, 3H), 3.62-3.80 (m, 4H), 6.70
(dd, J ) 5.2, 6.8 Hz, 1H), 6.90 (d, J ) 8.6 Hz, 1H), 7.22-7.37
(m, 5H), 7.53 (dt, J ) 1.6, 8.6 Hz, 1H), 8.14 (dd, J ) 1.6, 4.6 Hz,
1H). Anal. (C21H25N3O2‚0.7H2C2O4‚1.3H2O) C, H, N.
Methyl (1R,2S/1S,2R)-1-(4-Methylphenyl)-2-[(4-pyridin-2-
ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (6b).
Compound 6b was purified by silica gel flash column chromatog-
raphy using chloroform/ethanol (9.8:0.2). Yield 42%; mp 202-
Methyl (1R,2S/1S,2R)-1-(4-Methoxyphenyl)-2-[(4-pyrimidin-
2-ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (7c).
Yield 36%; mp 218-219 °C; H NMR (DMSO-d6) (free base) δ
1
1.40 (dd, J ) 4.6, 8.8 Hz, 1H), 1.55 (dd, J ) 4.6, 7.0 Hz, 1H),
1.73-1.80 (m, 1H), 2.80-3.20 (m, 6H), 3.58 (s, 3H), 3.76 (s, 3H),
3.80-3.94 (m, 4H), 6.74 (t, J ) 4.8, 9.4 Hz, 1H), 6.91 (d, J ) 8.8
Hz, 2H), 7.29 (d, J ) 8.6 Hz, 2H), 8.43 (d, J ) 4.8 Hz, 2H). Anal.
(C21H26N4O3‚H2C2O4‚H2O‚(C2H5)2O) C, H, N.
Methyl (1R,2S/1S,2R)-1-(4-Chlorophenyl)-2-[(4-pyrimidin-2-
ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (7d).
This compound was purified by silica gel flash column chroma-
tography using chloroform as eluent. Yield 26%; mp 201-204 °C;
1H NMR (DMSO-d6) (free base) δ 1.46 (dd, J ) 4.6, 9.0 Hz, 1H),
1.59 (dd, J ) 4.6, 6.6 Hz, 1H), 1.84-2.0 (m, 1H), 2.88-3.10 (m,
5H), 3.17 (dd, J ) 6.4, 12.6 Hz, 1H), 3.56 (s, 3H), 3.80-4.0 (m,
4H), 6.70 (t, J ) 4.8 Hz, 1H), 7.30-7.42 (m, 4H), 8.40 (d, J ) 4.8
Hz, 2H). Anal. (C20H23ClN4O2‚0.6H2C2O4‚1.7H2O‚0.2(C2H5)2O) C,
H, N.
Methyl (1R,2S/1S,2R)-(4-Nitrophenyl)-2-[(4-pyrimidin-2-ylpip-
erazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (7e). Yield
22%; mp 205-207 °C; 1H NMR (DMSO-d6) (free base) δ 1.56
(dd, J ) 4.6, 9.0 Hz, 1H), 1.59 (dd, J ) 4.6, 7.2 Hz, 1H), 1.94-
2.10 (m, 1H), 2.88-3.10 (m, 5H), 3.17 (dd, J ) 6.6, 13.4 Hz, 1H),
3.57 (s, 3H), 3.80-4.0 (m, 4H), 6.69 (t, J ) 4.6, Hz, 1H), 7.65 (d,
J ) 8.8 Hz, 2H), 8.17 (d, J ) 8.8 Hz, 2H), 8.39 (d, J ) 4.6 Hz,
2H). Anal. (C20H23N5O4‚0.5H2C2O4‚1.2H2O) C, H, N.
1
205 °C; H NMR (DMSO-d6) (free base) δ 1.42 (dd, J ) 4.6, 8.4
Hz, 1H), 1.57 (dd, J ) 4.6, 6.8 Hz, 1H), 1.80-2.05 (m, 1H), 2.27
(s, 3H), 3.0-3.20 (m, 5H), 3.28 (dd, 6.0, 13.2 Hz, 1H), 3.55 (s,
3H), 3.60-3.72 (m, 4H), 6.70 (dd, J ) 4.8, 6.6 Hz, 1H), 6.91 (d,
J ) 8.6 Hz, 1H), 7.16 (d, J ) 8.2 Hz, 2H), 7.22 (d, J ) 8.2 Hz,
2H), 7.60 (dt, J ) 1.6, 6.6 Hz, 1H), 8.14 (dd, J ) 1.6, 4.2 Hz, 1H).
Anal. (C22H27N3O2‚0.7H2C2O4‚2H2O) C, H, N.
Methyl (1R,2S/1S,2R)-1-(4-Methoxyphenyl)-2-[(4-pyridin-2-
ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (6c).
Yield 31%; mp 194-195 °C; 1H NMR (CDCl3) (free base) δ 1.34
(dd, J ) 4.2, 8.6 Hz, 1H), 1.61-1.85 (m, 2H), 2.58-2.70 (m, 6H),
3.45-3.75 (m, 7H), 3.80 (s, 3H), 6.58-6.78 (m, 2H), 6.84 (d, J )
8.8 Hz, 2H), 7.27 (d, J ) 9.0 Hz, 2H), 7.50 (dt, J ) 1.6, 6.6 Hz,
1H), 8.14 (dd, J ) 1.6, 4.0 Hz, 1H). Anal. (C22H27N3O3‚H2C2O4‚
H2O) C, H, N.
Methyl (1R,2S/1S,2R)-1-(4-Chlorophenyl)-2-[(4-pyridin-2-
ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (6d).
1
Yield 36%; mp 198-200 °C; H NMR (DMSO-d6) (free base) δ
1.47 (dd, J ) 4.8, 9.0 Hz, 1H), 1.59 (dd, J ) 4.8, 7.0 Hz, 1H),
1.92 (m, 1H), 3.05-3.28 (m, 6H), 3.56 (s, 3H), 3.62-3.78 (m,
4H), 6.70 (dd, J ) 5.0, 6.6 Hz, 1H), 6.90 (d, J ) 8.8 Hz, 1H), 7.38
(s, 4H), 7.58 (dt, J ) 1.6, 6.6 Hz, 1H), 8.13 (dd, J ) 1.6, 5.0 Hz,
1H). Anal. (C21H24ClN3O2‚0.6H2C2O4‚2H2O) C, H, N.
Methyl (1R,2S/1S,2R)-1-(4-Aminophenyl)-2-[(4-pyrimidin-2-
ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (7f).
Methyl (1R,2S/1S,2R)-1-(4-Nitrophenyl)-2-[(4-pyridin-2-ylpip-
erazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (6e). Com-
pound 6e was purified by silica gel flash column chromatography
using chloroform/ethanol (9.8:0.2). Yield 52%; mp 197-201 °C;
1H NMR (CDCl3) (free base) δ 1.25-1.27 (m, 1H), 1.81-1.89
(m, 2H), 2.61-2.96 (m, 6H), 3.47-3.70 (m, 7H), 6.60-6.72 (m,
2H), 7.41-7.60 (m, 4H), 7.96-8.0 (m, 1H), 8.13-8.21 (m, 1H).
Anal. (C21H24N4O4‚0.9H2C2O4‚0.3H2O) C, H, N.
Methyl (1R,2S/1S,2R)-1-(4-Aminophenyl)-2-[(4-pyridin-2-
ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (6f).
Compound 6f was purified by silica gel flash column chromatog-
raphy using chloroform/ethanol (9.8:0.2). Yield 37%; mp 187-
190 °C; 1H NMR (CDCl3) (free base) δ 1.31 (dd, J ) 4.6, 8.8 Hz,
1H), 1.59-1.79 (m, 2H), 2.65-2.72 (m, 6H), 3.54-3.61 (m, 7H),
4.40 (br s, 2H), 6.58-6.72 (m, 4H), 7.13 (d, J ) 8.6 Hz, 2H), 7.52
(dt, J ) 1.6, 8.6 Hz 1H), 8.19 (dd, J ) 1.6, 5.0 Hz, 1H). Anal.
(C21H26N4O2‚0.9H2C2O4‚1.6H2O) C, H, N.
1
Yield 19%; mp 180-184 °C; H NMR (DMSO-d6) (free base) δ
1.34 (dd, J ) 4.8, 8.6 Hz, 1H), 1.45-190 (m, 4H), 3.05-3.23 (m,
5H), 3.32 (dd, J ) 5.6, 13.6 Hz, 1H), 3.54 (s, 3H), 3.95-4.05 (m,
4H), 6.47 (d, J ) 8.2 Hz, 2H), 6.72 (t, J ) 4.8, Hz, 1H), 6.97 (d,
J ) 8.2 Hz, 2H), 8.41 (d, J ) 4,8 Hz, 2H). Anal. (C20H25N5O2‚
0.9H2C2O4‚1.8H2O)
Biological Studies. Cell Cultures. Primary cultures of astrocytes
were prepared from newborn albino rat brains (1-2 day-old Wistar
strain) as previously described.37 Briefly, cerebral tissues, after
dissection and careful removal of the meninges, were mechanically
dissociated through sterile meshes of 82 µm pore size (Nitex).
Isolated cells were suspended in Dulbecco’s modified Eagle’s
medium (DMEM), supplemented with 20% (V/V) heat-inactivated
fetal bovine serum (FBS), 2 mM glutamine, streptomycin (50 mg/
mL), and penicillin (50 U/mL), and plated at a density of 3 × 106
cells per 100 mm dish and 0.5 × 105 cells/chamber in Lab-Tek II
multichambered slides. Cells were maintained at 37 °C in a 5%
CO2-95% air humidified atmosphere for 2 weeks, and the medium
was replaced every 3 days. The purity of cell cultures was evaluated
by the immunostaining for GFAP, an astroglial cell marker of
growth, maturation, and differentiation.21,43 Under these experi-
mental conditions, the presence of both σ1 and σ2 has been
demonstrated.44 All efforts were made to minimize both the
suffering and number of animals used. All experiments were
performed according to the guidelines of the Ethical Committee of
University of Catania, Italy.
Methyl (1R,2S/1S,2R)-1-Phenyl-2-[(4-pyrimidin-2-ylpiperazin-
1-yl)methyl]cyclopropanecarboxylate Oxalate (7a). A 1.32 g
(5.94 mmol) mixture of cis-(()-halogenated ester 2a, 2-(piperazin-
1-yl)pyrimidine (0.88 g, 5.4 mmol), and 0.9 g (10.8 mmol) of
NaHCO3 in DMF (30 mL) was heated to 70 °C for 24 h. The solvent
was evaporated at decreased pressure, and the crude product was
dissolved with CHCl3 and washed twice with water. The organic
phase was separated, dried over Na2SO4, and filtered, and the
solvent was removed in vacuo. The residual product was purified
by chromatography on silica gel, CHCl3/EtOH (9.8/0.2), and the
colorless oil was dissolved in diethyl ether and treated with a
solution of oxalic acid dihydrate in diethyl ether to give the oxalate
salts as a colorless solid. Yield 39% as free base; mp 228-230
Treatment of σ Ligands. In previous experiments, on the basis
of both microscopy observations and MTT assays, we established
the nontoxic concentrations and the time of exposure of the cultures
to the σ ligands for astroglial cell cultures. The time of exposure
to the drugs was selected when the TG-2 expression reached the
1
°C; H NMR (DMSO-d6) (free base) δ 1.44 (dd, J ) 4.6, 9.0 Hz,
1H), 1.59 (dd, J ) 4.6, 7.0 Hz, 1H), 1.84-2.04 (m, 1H), 2.94-

960 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
PrezzaVento et al.
maximum levels of activity and expression. We observed that the
optimal time of exposure to σ ligands was 24 h.
Primary rat astroglial cell cultures at 14 DIV for 24 h were treated
with ibogaine, haloperidol, BD1047, 4b, and (+)-pentazocine, in
the range 25-100 µM. The nontoxic concentration of the exposure
of the astroglial cell cultures to the σ ligands was 25 µM. Four
replicates were performed for each sample.
tion assay incorporates fluorescein-dUTP at the free 3′-hydroxyl
ends of the fragmented DNA using TUNEL and was performed
according to the user’s manual. According to the user’s manual,
apoptotic cells appear green while nonapoptotic cells appear red.
We focused on 10 random microscopic fields for each dish. In each
microscopic field we counted the number of apoptotic cells and
we compared this number with all the nonapoptotic cells visualized
in the same microscopic field; the ratio is expressed as a percentage.
Primary rat astroglial cell cultures, untreated and treated for 24 h
with the σ ligands, were made up according to the user’s manual.
Cells were mounted and visualized directly by fluorescence
microscopy (Leika, Germany) with either a propidium iodide (PI)
filter alone or a FITC filter alone. According to the user’s manual,
apoptotic cells appear green with the FITC filter alone while
nonapoptotic cells appear red under the dual-pass FITC/PI filter
set.
Western Blot. Control and treated primary astroglial cell cultures
were harvested using cell lysis buffer as previously described.23
After treatment, cells were harvested in cold PBS, collected by
centrifugation, resuspended in a homogenizing buffer (50 mM Tris-
HCl, pH 6,8, 150 mM NaCl, 1 mM EDTA, 0.1 mM phenylmeth-
ylsulfonyl fluoride, and a 10 µg/mL each of aprotinin, leupeptin,
and pepstatin), and sonicated on ice. Protein concentration of the
homogenates was diluted to 1 mg/mL with 2× reducing stop buffer
(0.25 M Tris-HCl, pH 6.8, 5 mM EGTA, 25 mM dithiothreitol
(DTT), 2% SDS, and 10% glycerol with bromophenol blue as the
tracking dye). Proteins (30 µg) were separated on 10% SDS-
polyacrylamide gels and transferred to nitrocellulose membranes.
Blots were blocked overnight at 4 °C with 5% nonfat dry milk
dissolved in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Tween-
20 (TBST) buffer at 4 °C. After being washed with TBST, the
membranes were incubated with anti-TG-2 monoclonal antibody
(diluted 1:1.000) or with anti-caspase-3 monoclonal antibody
(diluted 1:1.000) for 1 h at room temperature with constant shaking.
The filters were then washed and subsequently probed with
horseradish peroxidase-conjugated antimouse IgG at a dilution of
1:2000.
Statistical Analysis. All values are presented as the mean (
SEM of five experiments. A one-way ANOVA test, followed by a
Newman-Keuls multiple range test, was used to analyze the time
course of TGase activity, and the Student’s t test was used to
compare values of densitometric analysis of immunoblots.
Acknowledgment. This work was supported by FIRB 2003
(Grant No. RBNE03FH5Y) and PRIN 2005 (Grant No.
2005032713) from MIUR (Rome). We thank Slater & Frith for
the gift of ibogaine. We also thank Professor Isa Picerno and
her colleagues of the Department of Hygiene, Public Health,
and Preventive Medicine, University of Messina, Italy, for their
useful collaboration.
The TG-2 expression and caspase-3 cleavage were visualized
by chemiluminescence with the ECL kit after autoradiography film
exposure. Blots were scanned and quantified with a AlphaImager
1200 system (Alpha Innotech, San Leandro, CA). The results are
expressed as the percentage of TG-2 or caspase-3 cleavage increase
or decrease versus control.
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
Expression and Localization of TG-2 by Confocal Laser
Scanning Microscopy (CLSM). Expression and the cellular
localization of TG-2 in subcellular compartments of fixed control
and σ ligand astroglial cell cultures were determined by CLSM.
The cultures of untreated and treated astrocytes at 14 days in culture
(DIV) were fixed for 20 min with 4% paraformaldehyde in PBS,
washed three times with PBS, incubated for 1 h with 1% normal
blocking serum in PBS to block nonspecific sites, and then
incubated overnight with antimouse monoclonal antibody against
TG-2 (1:500). The slides were then washed and incubated for 2 h
with FITC-conjugated anti-IgG polyclonal antibody (1:64 in PBS)
and for 2 h with TRITC-conjugated anti-IgG polyclonal antibody
(1:64 in PBS). Finally, the slides were extensively washed with
PBS, mounted with 90% glycerol, and observed with CLSM (Leika,
Germany).
Analysis by CLSM. The optical fields were visualized using a
40 × 1.0 NA oil immersion objective in a Leica DM IRB inverted
fluorescence microscope (Leica Microsystems, Heidelberg, Man-
nheim, Germany). At the same time, the slides were examined on
a Leica TCS SP2 CLSM by green (FITC) and red (TRITC)
fluorophore excitation at 488 and 543 nm, respectively, utilizing
Ar/Kr and He/Ne laser sources. The fluorescence emitted from the
specimens was sent through a Leica AOTF mechanism (acoustic
optical turnable filter) and pinhole to two different photomultiplier
tubes (PMTs). Image outputs electronically generated by the same
parameter settings (frame scanning, pinhole aperture, gain voltage,
pixel and spatial resolution) were measured and compared for
intensity (treated vs control) after nonspecific fluorescence subtrac-
tion. Sequential optical sections, obtained using the CLSM z-axis
stepping capability, were combined to form an extended depth of
focus image, and standard image processing was performed to
enhance brightness and contrast, using Leica Confocal Software
(version 2.0, build 0585).
References
(1) Su, T. P. Evidence for sigma opioid receptor: binding of [3H]SKF-
10047 to etorphine-inaccessible sites in guinea-pig brain. J. Phar-
macol. Exp. Ther. 1982, 223, 284-290.
(2) Quirion, R.; Chicheportiche, R.; Contreras, P. C.; Johnson, K. M.;
Lodge, D.; Tam, S. W.; Woods, J. H.; Zukin, S. R. Classification
and nomenclature of phencyclidine and sigma receptors sites. Trends
Neurosci. 1987, 10, 444-446.
(3) Quirion, R.; Bowen, W. D.; Itzhak, Y.; Junien, J. L.; Musacchio, J.
M.; Rothman, R. B.; Su, T. P.; Tam, S. W.; Taylor, D. P. A proposal
for the classification of sigma binding sites. Trends Pharmacol. Sci.
1992, 13, 85-86.
(4) Hellewell, S. B.; Bowen, W. D. A sigma-like binding site in rat
pheochromocytoma (PC12) cells: decreased affinity for (+)-benzo-
morphans and lower molecular weight suggest a different sigma
receptor form from that of guinea pig brain. Brain Res. 1990, 527,
244-253.
(5) Walker, J. M.; Bowen, W. D.; Walker, F. O.; Matsumoto, R. R.; De
Costa, B.; Rice, K. C. Sigma receptors: biology and function.
Pharmacol. ReV. 1990, 42, 355-402.
(6) McCann, D. J.; Weissman, A. D.; Su, T. P. Sigma-1 and sigma-2
sites in rat brain: comparison of regional, ontogenetic, and subcellular
patterns. Synapse 1994, 17, 182-189.
(7) Hanner, M.; Moebius, F. F.; Flandorfer, A.; Knaus, H. G.; Striessnig,
J.; Kempner, E.; Glossmann, H. Purification, molecular cloning, and
expression of the mammalian sigma1-binding site. Proc. Natl. Acad.
Sci. U.S.A. 1996, 93, 8072-8077.
(8) Su, T. P.; Hayashi, T. Understanding the molecular mechanism of
sigma-1 receptors: towards a hypothesis that sigma-1 receptors are
intracellular amplifiers for signal transduction. Curr. Med. Chem.
2003, 10, 2073-2080.
(9) Zhang, H.; Cuevas, J. Sigma receptor activation blocks potassium
channels and depresses neuroexcitability in rat intracardiac neurons.
J. Pharmacol. Exp. Ther. 2005, 313, 1387-1396.
(10) Monnet, F. P. Sigma-1 receptor as regulator of neuronal intracellular
Ca2+: clinical and therapeutic relevance. Biol. Cell 2005, 97, 873-
883.
(11) Monnet, F. P.; Debonnel, G.; Junien, J. L.; De Montigny, C.
N-Methyl-D-aspartate-induced neuronal activation is selectively
modulated by sigma receptors. Eur. J. Pharmacol. 1990, 179, 441-
445.
Terminal Deoxynucleotidyltransferase dUTP Nick End-
Labeling (TUNEL) Assay. The ApoAlert DNA fragmentation
assay kit (Clontech) detecting nuclear DNA fragmentation, a
hallmark of apoptosis, was used.31 The ApoAlert DNA fragmenta-

NoVel σ Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 961
(12) Peeters, M.; Romieu, P.; Maurice, T.; Su, T. P.; Maloteaux, J. M.;
Hermans, E. Involvement of the sigma 1 receptor in the modulation
of dopaminergic transmission by amantadine. Eur. J. Neurosci. 2004,
19, 2212-2220.
(30) Colabufo, N. A.; Berardi, F.; Contino, M.; Niso, M.; Abate, C.;
Perrone, R.; Tortorella, V. Antiproliferative and cytotoxic effects of
some sigma2 agonists and sigma1 antagonists in tumour cell lines.
Naunyn-Schmiedeberg’s Arch. Pharmacol. 2004, 370, 106-113.
(31) Zambon, A. C.; De Costa, B. R.; Kanthasamy, A. G.; Nguyen, B.
Q.; Matsumoto, R. R. Subchronic administration of N-[2-(3,4-
dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD1047)
alters sigma 1 receptor binding. Eur. J. Pharmacol. 1997, 324, 39-
47.
(32) Matsumoto, R. R.; Bowen, W. D.; Tom, M. A.; Vo, V. N.; Truong,
D. D.; De Costa, B. R. Characterization of two novel sigma receptor
ligands: antidystonic effects in rats suggest sigma receptor antago-
nism. Eur. J. Pharmacol. 1995, 280, 301-310.
(33) Milakovic, T.; Tucholski, J.; McCoy, E.; Johnson, G. V. Intracellular
localization and activity state of tissue transglutaminase differentially
impacts cell death. J. Biol. Chem. 2004, 279, 8715-8722.
(34) Gavrieli, Y.; Sherman, Y.; Ben Sasson, S. A. Identification of
programmed cell death in situ via specific labeling of nuclear DNA
fragmentation. J. Cell Biol. 1992, 119, 493-501.
(35) Colabufo, N. A.; Berardi, F.; Abate, C.; Contino, M.; Niso, M.;
Perrone, R. Is the sigma2 receptor a histone binding protein? J. Med.
Chem. 2006, 49, 4153-4158.
(36) Kim, J. H.; Nam, K. H.; Kwon, O. S.; Kim, I. G.; Bustin, M.; Choy,
H. E.; Park, S. C. Histone cross-linking by transglutaminase. Biochem.
Biophys. Res. Commun. 2002, 293, 1453-1457.
(37) Crawford, K. W.; Bowen, W. D. Sigma-2 receptor agonists activate
a novel apoptotic pathway and potentiate antineoplastic drugs in
breast tumor cell lines. Cancer Res. 2002, 62, 313-322.
(38) Vilner, B. J.; Bowen, W. D. Modulation of cellular calcium by
sigma-2 receptors: release from intracellular stores in human SK-
N-SH neuroblastoma cells. J. Pharmacol. Exp. Ther. 2000, 292, 900-
911.
(39) DeHaven-Hudkins, D. L.; Fleissner, L. C.; Ford-Rice, F. Y.
Characterization of the binding of [3H](+)-pentazocine to sigma
recognition sites in guinea pig brain. Eur. J. Pharmacol. 1992, 227,
371-378.
(40) Boess, F. G.; Monsma, F. J., Jr.; Carolo, C.; Meyer, V.; Rudler, A.;
Zwingelstein, C.; Sleight, A. J. Functional and radioligand binding
characterization of rat 5-HT6 receptors stably expressed in HEK293
cells. Neuropharmacology 1997, 36, 713-720.
(41) Gobbi, M.; Crespi, D.; Foddi, M. C.; Fracasso, C.; Mancini, L.;
Parotti, L.; Mennini, T. Effects of chronic treatment with fluoxetine
and citalopram on 5-HT uptake, 5-HT1B autoreceptors, 5-HT3 and
5-HT4 receptors in rats. Naunyn-Schmiedeberg’s Arch. Pharmacol.
1997, 356, 22-28.
(42) Mennini, T.; Bernasconi, P.; Fiori, M. G. Neurotoxicity of nonionic
low-osmolar contrast media. A receptor binding study. InVest. Radiol.
1993, 28, 821-827.
(43) Campisi, A.; Renis, M.; Russo, A.; Sorrenti, V.; Di Giacomo, C.;
Castorina, C.; Vanella, A. Transglutaminase activity in primary and
subcultured rat astroglial cells. Neurochem. Res. 1992, 17, 1201-
1205.
(44) Klouz, A.; Tillement, J. P.; Boussard, M. F.; Wierzbicki, M.;
Berezowski, V.; Cecchelli, R.; Labidalle, S.; Onteniente, B.; Morin,
D. [3H]BHDP as a novel and selective ligand for sigma1 receptors
in liver mitochondria and brain synaptosomes of the rat. FEBS Lett.
2003, 553, 157-162.
(13) Kobayashi, T.; Matsuno, K.; Mita, S. Regional differences of the
effect of sigma receptor ligands on the acetylcholine release in the
rat brain. J. Neural Transm. 1996, 103, 661-669.
(14) Bowen, W. D. Sigma receptors: recent advances and new clinical
potentials. Pharm. Acta HelV. 2000, 74, 211-218.
(15) Lesort, M.; Tucholski, J.; Miller, M. L.; Johnson, G. V. Tissue
transglutaminase: a possible role in neurodegenerative diseases. Prog.
Neurobiol. 2000, 61, 439-463.
(16) Tucholski, J.; Johnson, G. V. Tissue transglutaminase differentially
modulates apoptosis in a stimuli-dependent manner. J. Neurochem.
2002, 81, 780-791.
(17) Nakaoka, H.; Perez, D. M.; Baek, K. J.; Das, T.; Husain, A.; Misono,
K.; Im, M. J.; Graham, R. M. Gh: a GTP-binding protein with
transglutaminase activity and receptor signaling function. Science
1994, 264, 1593-1596.
(18) Slife, C. W.; Dorsett, M. D.; Bouquett, G. T.; Register, A.; Taylor,
E.; Conroy, S. Subcellular localization of a membrane-associated
transglutaminase activity in rat liver. Arch. Biochem. Biophys. 1985,
241, 329-336.
(19) Mishra, S.; Murphy, L. J. The p53 oncoprotein is a substrate for
tissue transglutaminase kinase activity. Biochem. Biophys. Res.
Commun. 2006, 339, 726-730.
(20) Gentile, V.; Cooper, A. J. Transglutaminases, possible drug targets
in human diseases. Curr. Drug Targets: CNS Neurol. Disord. 2004,
3, 99-104.
(21) Campisi, A.; Caccamo, D.; Raciti, G.; Cannavo, G.; Macaione, V.;
Curro, M.; Macaione, S.; Vanella, A.; Ientile, R. Glutamate-induced
increases in transglutaminase activity in primary cultures of astroglial
cells. Brain Res. 2003, 978, 24-30.
(22) Ronsisvalle, G.; Prezzavento, O.; Pasquinucci, L.; Pappalardo, M.
S.; Marrazzo, A.; Vittorio, F.; Carboni, L.; Spampinato, S. Synthesis
of (+)-(1′R,2′S) and (1′S,2′R)-6,11-dimethyl-1,2,3,4,5,6-hexahydro-
3-[[2′-(alkoxycarbonyl)-2′-phenylcyclopropyl]methyl]-2,6-methano-
3-benzazocin-8-ol. Comparison of the affinities for sigma 1 and opioid
receptors within the diastereoisomeric MPCB and CCB. Farmaco
1997, 52, 471-476.
(23) Casadio, S.; Cousse, H.; Mouzin, G.; Stenger, A.; Charveron, M.;
Vilain, P.; Lauressergues, H. New beta-phenethylamine derivatives:
Synthesis and pharmacologic properties. Boll. Chim. Farm. 1978,
117, 83-89.
(24) Schaus, J. M.; Bymaster, F. P. Dopaminergic approches to anthipsy-
chotic agents. Annu. Rep. Med. Chem. 1998, 33, 1-10.
(25) Glennon, R. A.; Ablordeppey, S. Y.; Ismaiel, A. M.; el Ashmawy,
M. B.; Fischer, J. B.; Howie, K. B. Structural features important for
sigma 1 receptor binding. J. Med. Chem. 1994, 37, 1214-1219.
(26) Bowen, W. D.; Vilner, B. J.; Williams, W.; Bertha, C. M.; Kuehne,
M. E.; Jacobson, A. E. Ibogaine and its congeners are sigma 2
receptor-selective ligands with moderate affinity. Eur. J. Pharmacol.
1995, 279, R1-R3.
(27) Danso-Danquah, R.; Bai, X.; Zhang, X.; Mascarella, S. W.; Williams,
W.; Sine, B.; Bowen, W. D.; Carroll, F. I. Synthesis and sigma
binding properties of 2′-substituted 5,9-alpha-dimethyl-6,7-benzo-
morphans. J. Med. Chem. 1995, 38, 2978-2985.
(28) Jaen, J. C.; Caprathe, B. W.; Pugsley, T. A.; Wise, L. D.; Akunne,
H. Evaluation of the effects of the enantiomers of reduced haloperidol,
azaperol, and related 4-amino-1-arylbutanols on dopamine and sigma
receptors. J. Med. Chem. 1993, 36, 3929-3936.
(29) Mach, R. H.; Smith, C. R.; Childers, S. R. Ibogaine possesses a
selective affinity for sigma 2 receptors. Life Sci. 1995, 57, L57-
L62.
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