Novel highly potent and selective σ1 receptor antagonists related to spipethiane

Article abstract of DOI:10.1021/jm901542q

Conservative chemical modifications of the core structure of the lead spipethiane (1) afforded novel potent σ₁ ligands. σ₁ affinity and σ₁/σ₂ selectivity proved to be favored by the introduction of polar functions (oxygen atom or carbonyl group) in position 3 or 4 (4-6) or by the elongation of the distance between the two hydrophobic portions of the molecule with the simultaneous presence of a carbonyl group in position 4 (8 and 9). The observed cytostatic effect against the human breast cancer cell line MCF-7/ADR, highly expressing σ₁ receptors, and not against MCF-7, as well as the enhancement of morphine analgesia highlighted the σ₁ antagonist profile of this series of compounds. In particular, due to its high σ₁ affinity (pK_i = 10.28) and σ₁/ σ₂ selectivity ratio (29510), compound 9 might be a novel valuable tool for σ receptor characterization and a suitable template for the rational design of potential therapeutically useful σ₁ antagonists. 2010 American Chemical Society.

Full text of DOI:10.1021/jm901542q

J. Med. Chem. 2010, 53, 1261–1269 1261
DOI: 10.1021/jm901542q
Novel Highly Potent and Selective σ1 Receptor Antagonists Related to Spipethiane
Alessandro Piergentili,† Consuelo Amantini,‡ Fabio Del Bello,† Mario Giannella,† Laura Mattioli,‡ Maura Palmery,§
Marina Perfumi,‡ Maria Pigini,† Giorgio Santoni,‡ Paolo Tucci, Margherita Zotti, and Wilma Quaglia*,†
†Dipartimento di Scienze Chimiche, Universitaꢀdi Camerino, via S. Agostino 1, 62032 Camerino, Italy, ‡Dipartimento di Medicina Sperimentale e
SanitaꢀPubblica, Universitaꢀdi Camerino, via Madonna delle Carceri 3, 62032 Camerino, Italy, §Dipartimento di Fisiologia e Farmacologia Vittorio
Erspamer, UniversitaꢀLa Sapienza, P.le A. Moro 5, 00185 Roma, Italy, and Dipartimento di Scienze Biomediche, Universitaꢀdi Foggia, via Pinto 1,
71100 Foggia, Italy
Received October 17, 2009
Conservative chemical modifications of the core structure of the lead spipethiane (1) afforded novel
potent σ1 ligands. σ1 affinity and σ1/σ2 selectivity proved to be favored by the introduction of polar
functions (oxygen atom or carbonyl group) in position 3 or 4 (4-6) or by the elongation of the distance
between the two hydrophobic portions of the molecule with the simultaneous presence of a carbonyl
group in position 4 (8 and 9). The observed cytostatic effect against the human breast cancer cell line
MCF-7/ADR, highly expressing σ1 receptors, and not against MCF-7, as well as the enhancement of
morphine analgesia highlighted the σ1 antagonist profile of this series of compounds. In particular, due
to its high σ1 affinity (pKi = 10.28) and σ1/σ2 selectivity ratio (29510), compound 9 might be a novel
valuable tool for σ receptor characterization and a suitable template for the rational design of potential
therapeutically useful σ1 antagonists.
Introduction
remains controversial whether or not sigma receptors are
associated with G-proteins.15,16 Both σ1 and σ2 receptors are
involved in Ca2þ release from endoplasmic reticulum and
mitochondrial membrane.17,18 There is evidence suggesting
that σ1 receptors play a role in the regulation of ion channels
including Ca2þ and Kþ channels19-21 and are involved in the
modulation of glutamatergic,22 dopaminergic,23 and choliner-
gic neurotransmission.24 Because of their widespread expres-
sion in many human tissues and their involvement in several
physiological processes, σ receptors have proved to be highly
attractive pharmacological targets for the treatment of var-
ious pathologies. In particular, σ1 ligands play a potential
role in the treatment of neuropathic pain,25 depression,26
cocaine abuse,27 epilepsy,28 psychosis,29 as well as Alzheimer’s
and Parkinson’s disease.30,31 Moreover, σ1 antagonists and
σ2 agonists may be useful as anticancer agents and radio-
Sigma (σ) receptors were initially classified as opioid
receptor subtypes and subsequently erroneously identified
1
with the phencyclidine (PCP)a site on the N-methyl-D-aspar-
tate (NMDA) receptor channel.2 Further studies demon-
strated that they were distinct from both opioid receptors
and PCP/NMDA receptor complex.3 At present, σ receptors
are considered to be a unique receptor family comprising
at least two pharmacologically distinct subtypes, namely σ1
and σ2,4,5 which are widely distributed in the central nervous
system as well as in some peripheral tissues such as digestive
tract, kidney, liver, lung, heart, and adrenal medulla.6,7 They
are also present in cells and organs of the immune and
endocrine systems.8 Moreover, both σ receptor subtypes are
overexpressed in many human and rodent tumor cell lines.9
While the σ2 receptor has not yet been cloned, the σ1 subtype
has been cloned from various tissues, including guinea pig
liver,10 rat and mouse brain,11,12 as well as from human
placental choriocarcinoma cell lines.13 Only recently, the
hallucinogen N,N-dimethyltryptamine has been hypothesized
to be an endogenous σ1 receptor regulator.14 Although many
efforts have been directed to defining the intracellular path-
ways mediating σ receptor signaling (e.g., calcium, IP3, PKC),
the molecular mechanisms have not fully been worked out. It
labeled ligands as selective tumor imaging agents.32,33
A
variety of structurally unrelated compounds is able to bind
σ receptors.34,35 However, while only few σ2 ligands generally
show a selectivity toward the σ1 subtype,36 several structures
are known to bind selectively the σ1 receptor and a pharma-
cophoric model, including an amine site flanked by two
hydrophobic domains, has been proposed for σ1 ligands.37
In our previous work about σ ligands, the N-benzyl-spiro-
piperidine spipethiane (1) proved to be a highly potent and
selective σ1 ligand.38 This result and the observation that the
bioisosteric replacement of the sulfur atom of 1 with an
oxygen atom (compound 2) (Chart 1) was compatible with
the maintenance of the high σ1 receptor affinity38 prompted us
to improve our knowledge about structure-affinity and
structure-selectivity relationships to better characterize σ
receptors. Therefore, the methylene analogue 3 was prepared
and the bioisosteric substitution with oxygen was extended to
position 3 of 1 and 2 (compounds 4 and 5, respectively).
*To whom correspondence should be addressed. Phone:
þ390737402237. Fax: þ390737637345. E-mail: wilma.quaglia@unicam.it.
aAbbreviations: PCP, phencyclidine; NMDA, N-methyl-D-aspar-
tate; [3H]DTG, [3H]1,3-di(2-tolyl)guanidine; SRB, sulforhodamine B;
GI50, growth inhibition 50; PI, propidium iodide; FACS, fluorescence
activated cell sorting; 5-HT, 5-hydroxytryptamine; PB28, 1-cyclohexyl-
4-[3-(5-methoxy-1,2,3,4,-tetrahydronaphthalen-1-yl)-n-propyl]piperazine;
MPE, maximumpossibleeffect;Ki, inhibition or dissociation constant; PS,
phosphatidylserine; TCA, trichloroacetic acid; MFI, mean fluorescence
intensity
r
2010 American Chemical Society
Published on Web 01/12/2010
pubs.acs.org/jmc

1262 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Piergentili et al.
Moreover, compound 6 was also included in this study for
useful comparison. Compounds 3 and 6 had already been
reported in the literature39,40 but not evaluated for their
σ binding affinity. Finally, the effect of the elongation of the
distance between the two hydrophobic moieties, with a con-
sequent increase of the conformational freedom of the struc-
ture, and the oxidation of the methylene group in position 4
(compounds 7-10) were investigated. The pharmacological
profiles of the compounds were determined by binding and
functional studies.
tives 11 and 12 with LiAlH4 (Scheme 2). Treatment of
2-(pyridin-4-yl)-1-benzopyran-4-one43 with benzyl bromide
afforded the intermediate 13, whose hydrogenation over
PtO2 gave compound 8 (Scheme 3). Condensation of 1-(2-
mercapto-phenyl)-ethanone44 or commercially available
1-(2-hydroxy-phenyl)-ethanone with 1-benzyl-piperidine-
4-carbaldehyde or 1-phenethyl-piperidine-4-carbaldehyde,45
respectively, afforded compounds 9 and 10 (Scheme 4).
Finally, reduction of 9 with borane-dimethyl sulfide complex
in dry diglyme furnished derivative 7 (Scheme 4). Compound
6 was prepared according to the method reported in the
literature.40
Chemistry
Compounds 3-5 and the racemic mixtures 7-10 were synthe-
sized according to the methods reported in Schemes 1-4.
Reduction of 10-benzyl-1,2,3,4-tetrahydrospiro[naphthalene-2,40-
piperidin]-1-one41 with borane-dimethyl sulfide complex in
dry diglyme afforded the tetraline derivative 3 (Scheme 1).
Compounds 4 and 5 were prepared by condensation of
(2-mercapto-phenyl)-methanol42 or commercially available
2-hydroxymethyl-phenol, respectively, with 1-benzoyl-
piperidin-4-one, followed by reduction of the amide deriva-
Biology
Binding Experiments. The pharmacological profiles of com-
pounds 3-10 were evaluated by radio-receptor binding assays
using 1 and haloperidol as reference compounds. [3H](þ)-
pentazocine and [3H]1,3-di(2-tolyl)guanidine ([3H]DTG) in
the presence of 300 nM (þ)-pentazocine were used to label σ1
and σ2 receptors expressed in Jurkat cell and rat cerebral cortex
membranes, respectively.46,47
In Vitro Functional Assays
Chart 1. Chemical Structures of Spipethiane (1) and Its Related
Compounds 2-10
Cell Growth Inhibition. The effects of the novel compounds
4-10 and 1 on cell growth inhibition were evaluated on MCF-7
and MCF-7/ADR human breast cancer cell lines.48 These cell
lines are characterized by low and high σ1 expression, respec-
tively, and they both express high levels of the σ2 subtype.
The in vitro cell growth inhibition was evaluated using the
sulforhodamine B (SRB) assay, according to the National
Cancer Institute protocol.49 The results are expressed as growth
inhibition 50 (GI50), representing the molar concentration of
the compound, which inhibited 50% net of cell growth. Each
quoted value is the mean of triplicate experiments.
Annexin V Staining. The phosphatidylserine (PS) exposure
on MCF-7 and MCF-7/ADR, treated for 48 h with 10 μM of
the selected compounds 1, 8, and 9, was evaluated by
Annexin V staining and fluorescence activated cell sorting
(FACS) analysis.50 Data are the percentage of Annexin Vþ
positive cells ( SEM of three separate experiments.
Scheme 1a
Cell Cycle Analysis. Cell cycle analysis of MCF-7 and
a Reagents: (a) BH3 MeSMe, dry diglyme.
MCF-7/ADR, treated for 48 h with 10 μM of the selected
3
Scheme 2a
a Reagents: (a) HClgas, CH2Cl2; (b) LiAlH4, THF.
Scheme 3a
a Reagents: (a) Δ, CH3CN; (b) H2/PtO2, EtOH.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1263
Scheme 4a
a Reagents: (a) pyrrolidine, MeOH; (b) BH3 MeSMe, dry diglyme.
3
Table 1. Affinity Constants, Expressed as pKi, toward σ1 and σ2 Receptors,a and Efficacy, Expressed as IC50, in the Guinea Pig Ileum (σ2)b for
Spipethiane (1), and Compounds 2-10
compd
X
Y
Z
n
pKiσ1
pKiσ2
σ1/σ2c selectivity ratio
IC50 σ2 (μM)
1 (Spipethiane)
2
S
CH2
CH2
CH2
CH2
CH2
CO
CH2
CH2
CH2
O
9.23
9.21
6.40
7.66
7.85
6.65
6.64
6.52
7.24
6.76
5.81
8.08
6.95
676
36
6.3 ( 0.92
NTd
O
CH2
S
3
9.36
32
>10e
4
10.05
10.01
9.68
2512
2344
1445
1000
2630
29512
76
>10e
5
O
O
S
O
CH2
3.9 ( 0.82
7.2 ( 0.62
8.4 ( 0.54
7.5 ( 0.46
4.7 ( 0.71
>10e
6
7
CH2
CO
1
1
1
2
10.24
10.18
10.28
9.96
8
O
S
9
10
CO
CO
O
haloperidol
8.11
15
NTd
a Equilibrium dissociation constants (Ki) were derived from IC50 values using the Cheng-Prusoff equation.64 The affinity estimates were derived from
displacement of [3H](þ)-pentazocine and [3H]DTG in the presence of 300 nM (þ)-pentazocine binding for σ1 and σ2 receptor subtypes, respectively.
Each experiment was performed in triplicate. Ki values were from two to three experiments, which agreed within (10%. b IC50 represents the half-
maximal inhibitory concentration for a drug in a given tissue and was estimated by interpolation from a log-concentration curve which contained 4
concentrations within the range of 20-80% of maximal effect. Values represent the mean ( SEM from six separate experiments. c The σ1/σ2 selectivity
ratio is the antilog of the difference between pKi at σ1 and σ2 receptors. d Not tested. e IC50 not obtained up to 10 μM.
compounds 1, 8, and 9, was evaluated by propidium iodide
(PI) staining and FACS analysis.51 Data are representative
of one out of three separate experiments.
Isolated Guinea Pig Ileum. Compounds 1 and 3-10 were
also tested on guinea pig ileum longitudinal muscle/myenteric
plexus, where σ ligands are known to dose dependently regulate
electrically or 5-hydroxytryptamine (5-HT)-evoked contrac-
tions.52
In Vivo Study. σ1 antagonists are reported to potentiate
opioid analgesia in rats and mice.53,54 Therefore, the selected
compound 9 was evaluated for its ability to modulate
morphine analgesia using the classic algesiometric tail flick
test.
hypothesize that the sulfur atom in position 1 of 1 does not
contribute to σ1 receptor binding by means of hydrogen bond
formation or electronic effects, as a sulfur, or an oxygen atom,
or a methylene group were effective as well. In contrast, these
bioisosteric modifications appeared to favor the σ2 receptor
interaction. A significant increase in σ1 affinity and a main-
tenance in that for σ2 subtype with a consequent improved
selectivity for σ1 subtype were obtained by introducing an
oxygen atom in position 3 of 1 and 2 (compounds 4 and 5,
respectively) or by oxidizing the methylene group in position 4
of 2 (compound 6). The elongation of the distance between the
two hydrophobic portions produced a significant increase of
σ1 affinity and σ1/σ2 selectivity (7 vs 1 and 8 vs 6). Probably,
the improved distance between the two hydrophobic portions,
leading to a conformationally more flexible structure, allowed
a more productive interaction with σ1 receptor sites. More
interestingly, such a modification proved to be highly favor-
able for σ1 selectivity when, simultaneously, the methylene
group in position 4 of 1 was replaced by a carbonyl function.
Infact, the moststriking resultof the present investigation was
the high σ1 affinity and selectivity toward σ2 receptors dis-
played by compound 9; its σ1/σ2 selectivity ratio of 29512 was
resolutely higher than that observed for the lead 1. To our
knowledge, the highly affine compound 9 is the most selective
σ1 ligand reported in the literature to date. The further
Results and Discussion
An analysis of the results reported in Table 1 revealed that
all the compounds showed σ1 affinity values similar or
significantly higher than that of 1. The σ2 affinities were by
far lower than those for σ1 subtype and ranged within at least
3 orders of magnitude. The bioisosteric replacement of the
sulfur atom of 1 by a methylene group, affording 3, produced
the same effect obtained by replacing it with an oxygen atom
(compound 2)38 (Table 1), a maintenance in σ1 affinity, and an
increase in that for σ2 were obtained with a remarkable
decrease of σ1/σ2 selectivity ratio. This result allowed us to

1264 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Piergentili et al.
apoptotic cell death is the loss of phospholipid asymmetry and
the expression of PS on the outer layer of the plasma mem-
brane. Weanalyzedwhetherthe treatment for 48 hwith10μM
of compounds 1, 8, or 9 induced externalization of PS residues
from the inner to the outer leaflet of the plasma membrane in
MCF-7 and MCF-7/ADR cancer cells. To this end, MCF-7-
and MCF-7/ADR-treated cells were stained with annexin
V-FITC and analyzed by flow cytometry.50 As shown in
Figure 2B, the treatment of MCF-7 cells with 10 μM of
compounds 8 and 9 did not induce translocation of PS,
whereas PS exposure was observed in MCF-7/ADR cells,
although at low levels (10.1% and 12.5%, respectively) with
respect to untreated or vehicle-treated cells (data not shown).
No Annexin Vþ cells were found in both MCF-7 and MCF-7/
ADR cells treated with 1. Our findings indicated that σ1
receptor antagonists 8 and 9 inhibited cell growth, arrested
cell cycle at G0/G1 phase and induced apoptosis of MCF-7/
ADR cells. The potency of 8 and 9, with respect to 1, to inhibit
cell proliferation correlated withσ1 receptor affinity (pKi σ1 =
10.18, 10.28, and 9.23, respectively). In addition, the signifi-
cant differences in the susceptibility of MCF-7 and MCF-7/
ADR to σ1 receptor-mediated effects observed in our experi-
ments were likely caused by different levels of wild-type σ1
receptors expressed in MCF-7 with respect to MCF-7/ADR
(MCF7 showed four time less expression of σ1 receptors
than did MCF7/ADR).48 The differences in the MCF-7-
and MCF-7/ADR-susceptibility to σ1 receptor antagonist-
mediated effects may also be related to the proliferation status
of tumor cells. Because σ1 receptor expression positively
correlates with the proliferative status of breast cancer cells,59
and MCF-7/ADR cells show high proliferative status with
respect to MCF-7 cells (doubling time 24 h vs 48 h, re-
spectively),48 the increased sensitivity of MCF-7/ADR, with
respect to MCF-7, to 8 and 9 treatment may also depend on
the increased proliferation of σ1 receptor-positive MCF-7/
ADR. However, it cannot be excluded that differences in
σ1 receptor expression of MCF-7 and MCF-7/ADR, resulting
in diverse sensitivity to σ1 receptor antagonist-dependent
cytostatic effects, may also depend on changes in signal
transduction triggering (e.g., rise in [Ca2þ]i levels).57 Finally,
because the σ1 receptor antagonists we tested selectively
inhibited the growth of MCF-7/ADR cells harboring muta-
tion of p53 tumor suppressor gene, frequently mutated during
tumor progression60 and also involved in the chemotherapeu-
tic drug resistance of breast cancer,61 we suggest a potential
use of these compounds in the therapy of advanced breast
cancer. Overall, the consistent accumulation of MCF-7/ADR
cancer cells, highly expressing σ1 receptors, in G0/G1 phase
after σ1 receptor antagonists 8 and 9 exposure might be
responsible for the σ1 receptor-dependent cell growth inhibi-
tion and induction of apoptotic cell death.
Figure 1. Cell growth inhibition of 1, and compounds 4-10 on
MCF-7 and MCF-7/ADR cell lines. Cell growth inhibition was
evaluated by sulforhodamine B (SRB) assay in MCF-7 and MCF-7/
ADR cell lines, treated for 48 h with 10 μM of σ1 receptor
compounds. Data are the mean ( SEM of three different experi-
ments. Statistical analysis was performed comparing treated MCF-
7 with MCF-7/ADR cells. *p < 0.01.
increase of the distance obtained by replacing the N-benzyl
substituent of 8 by a phenethyl moiety, affording its higher
homologue 10, did not seem to favor the σ1/σ2 selectivity ratio
because such a modification caused a significant increase in
affinity for the σ2 subtype. This result confirmed what has
previously been reported in the literature for other N-benzyl-
spiropiperidine analogues,55 where it has been demonstrated
that σ1 binding site tolerates groups bulkier and more flexible
than the benzyl substituent at the basic nitrogen, but such a
modification is especially favorable for σ2 binding site.
σ1 and σ2 receptors are known to be involved in the
modulation of cell proliferation and death of MCF-7 and
MCF-7/ADR human breast cancer cell lines by inducing cell
cycle arrest and apoptosis, respectively.48 To evaluate the
functional behavior of the ligands of the present study, cell
growth inhibition induced by lead 1 and derivatives 4-10 was
determined on these cell lines and their percent inhibition is
reported in Figure 1. All compounds specifically inhibited the
growth of MCF-7/ADR, the most active being 6 and 8, with
GI50 values in a micromolar range order (10.0 and 7.7 μM,
respectively). No significant growth inhibition was observed
in low σ1 receptor expressing MCF-7. Unlike the σ2 agonist
and σ1 antagonist 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4,-tetra-
hydronaphthalen-1-yl)-n-propyl]piperazine (PB28), which in-
hibits the growth of both cell lines,48 the compounds of the
present study showed potent cytostatic effect only against
MCF-7/ADR, highly expressing σ1 receptors. Therefore, their
biological response may be ascribed to a σ1 antagonist effect.
Several findings indicate that σ1 receptor antagonists inhibit
cell growth and induce apoptosis.48,56-58 We initially evalu-
ated the effects of 10 μM of the lead 1 and of compounds 8 and
9, selected on the basis of the highest cytostatic activity and σ1/
σ2 selectivity ratio, respectively, in the cell cycle progression of
both MCF-7 and MCF-7/ADR cells. Cell cycle analysis
revealed that 8 and 9 increased the number of cells in G0/
G1 (30.4% and 27.8%, respectively) and decreased those in S
phase (46.4% and 46.7%, respectively), in a σ1-dependent
manner, as evidenced by the ability of these compounds to
selectively affect the high σ1 receptor expressing MCF-7/ADR
but not the low σ1 receptor expressing MCF-7 cells (Figure 2A).
Compound 1 did not significantly affect cell cycle in MCF-7/
ADR cells, while it slightly increased (16%) the G0/G1 cell
number in MCF-7 cells (Figure 2A). A typical feature of
Data to evaluate the functional activity of all the com-
pounds in the in vitro guinea pig isolated ileum demonstrated
that all the derivatives showed μM IC50 values, except for
compounds 3, 4, and 10, which failed to inhibit 5-HT-evoked
contractions (Table 1). This result along with the observation
that all the compounds showed no cytotoxic activity both in
MCF-7 and MCF-7/ADR, highly expressing σ2 receptors,
indicated that they are not σ2 agonists.52
Previous studies have demonstrated the involvement of
σ1 receptors in opioid analgesia. Particularly, it has been
reported that the σ1 selective agonist (þ)-pentazocine anta-
gonizes systemic spinal and supraspinal morphine analgesia,54
while nonselective σ1 antagonists, such as haloperidol62 and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1265
Figure 2. (A) Selected compounds induce G0/G1 cell growth arrest. Cell cycle analysis of MCF-7 and MCF-7/ADR, treated with 10 μM of
compounds 1, 8, and 9, was performed by PI staining. Cell percentage relative to different cycle phases are indicated. Data are representative of
three different experiments. (B) PS exposure in MCF and MCF-7/ADR. The PS exposure on MCF-7 and MCF-7/ADR, treated for 48 h with
10 μM of compounds 1, 8, and 9, was evaluated by Annexin V staining and FACS analysis. Data are the percentage of Annexin Vþ positive
cells ( SEM of three separate experiments. Statistical analysis was performed comparing vehicle treated with selected compound-treated cells.
*p < 0.01.
structurally related compounds,53 enhance it in mice and rats.
In this study, we provided evidence that compound 9, selected
on the basis of its highest affinity and selectivity for the
σ1 receptor, was able to induce a significant increase in
morphine-induced analgesia. In fact, as shown in Figure 3, 9
had no analgesic effect when given alone but significantly
increased the analgesic response induced by morphine at all
times, as evidenced by increased reaction latencies expressed
as %MPE in the tail flick test. The maximum analgesic effect
of morphine (5 mg/kg, sc) was observed after 30 min, and then
it progressively decreased. Pretreatment with 9 (1 mg/kg)
significantly increased the antinociceptive effect produced
by morphine over the entire time course starting at 60 min.
The ANOVA confirmed a statistically significant treatment
Figure 3. Effect of 9 (1 mg/kg, sc) pretreatment on morphine (5 mg/
kg, sc) analgesia in the tail flick test. The reaction latencies were
expressed as a percent of the maximum possible effect (%MPE).
Values are mean ( SEM of 8 mice. **p < 0.01 compared to
morphine group.
effect [F(2.20) = 48.545; p < 0.001]. Therefore, according to
the literature, our data provided evidence of a σ1 antagonist
profile of compound 9.53,62
In conclusion, our lead spipethiane (1) proved to be a
suitable template for the design of novel σ ligands. Indeed,
conservative chemical modifications of its structure such as
(i) the introduction of polar functions (oxygen atom or carbonyl
group) in position 3 or 4 (4-6), or (ii) the elongation of the
distance between the two hydrophobic portions of the mole-
cule with the simultaneous presence of a carbonyl group in
position 4 (8 and 9), provided efficient σ1 ligands endowed

1266 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Piergentili et al.
with high σ1/σ2 selectivity ratio. To our knowledge, com-
pound 9 is the most selective σ1 ligand reported to date (σ1/σ2
selectivity ratio = 29510). The observed cytostatic effect only
against the humanbreast cancer cellline MCF-7/ADR, highly
expressing σ1 receptors, and the enhancement of morphine
analgesia highlighted the σ1 antagonist profile of this series of
compounds. Moreover, from our study it also emerged that
the σ2 affinity appeared significantly favored by the bioiso-
steric substitution of the sulfur atom with an oxygen atom or a
methylene group (2 and 3, respectively) as well as by the
replacement of the N-benzyl with the N-phenethyl substituent
and the simultaneous presence of a carbonyl group in position
4 (10). These results are the basis for promising future work
directed to the characterization of σ receptors and to provide
potential therapeutical agents.
column chromatography. Eluting with cyclohexane/EtOAc
(8:2) afforded 11 as a solid: 2.6 g; 66% yield; mp 84-85 °C.
1H NMR (CDCl3): δ 1.90-2.29 (m, 4H, 30 and 50-CH2),
3.44-4.15 (m, 4H, 20 and 60-CH2), 4.85 (s, 2H, 4-CH2),
7.04-7.43 (m, 9H, ArH).
10-Benzoyl-spiro[4H-1,3-benzodioxin-2,40-piperidine] (12).
This was synthesized from 2-hydroxymethyl-phenol following
the procedure described for 11. Eluting with cyclohexane/
EtOAc (7.5:2.5) afforded 12 as an oil: 11% yield. 1H NMR
(CDCl3): δ 1.68-2.17 (m, 4H, 30 and 50-CH2), 3.36-4.14 (m, 4H,
20 and 60-CH2), 4.86 (s, 2H, 4-CH2), 6.83-7.44 (m, 9H, ArH).
10-Benzyl-spiro[4H-3,1-benzoxathiin-2,40-piperidine] Oxalate
(4). A solution of 11 (1.0 g; 3.07 mmol) in dry Et2O (70 mL)
was added dropwise to a cooled (0 °C) suspension of LiAlH4
(0.35 g; 9.22 mmol) in dry Et2O (65 mL). The reaction was left at
room temperature for 24 h and then heated to reflux for 5 h.
After cooling, H2O (0.35 mL), 5N NaOH (0.35 mL), and again
H2O (1.75 mL) were sequentially added. After filtration, the
solid was washed with ethyl acetate. Removal of dried solvents
gave a residue, which was purified by column chromatography.
Eluting with cyclohexane/EtOAc (8:2) afforded 4 as the free
base: 0.93 g; 97% yield; mp 75-77 °C. 1H NMR (CDCl3): δ 2.12
(m, 4H, 30 and 50-CH2), 2.59 (m, 4H, 20 and 60-CH2), 3.55 (s, 2H,
CH2Ar), 4.83 (s, 2H, 4-CH2), 7.00-7.38 (m, 9H, ArH). The free
base was transformed into the oxalate salt, which was recrys-
Experimental Section
Chemistry. Melting points were taken in glass capillary tubes
€
on a Buchi SMP-20 apparatus and are uncorrected. IR and
NMR spectra were recorded on Perkin-Elmer 297 and Varian
Gemini 200 instruments, respectively. Chemical shifts are re-
ported in parts per million (ppm) relative to tetramethylsilane
(TMS), and spin multiplicities are given as s (singlet), d (doub-
let), dd (double doublet), ddd (doublet of double doublets),
t (triplet), q (quartet), or m (multiplet). IR spectral data (not
shown because of the lack of unusual features) were obtained for
all compounds reported and are consistent with the assigned
structures. The microanalyses were performed by the Micro-
analytical Laboratory of our department. The elemental com-
position of the compounds agreed to within (0.4% of the
calculated value. Chromatographic separations were performed
on silica gel columns (Kieselgel 40, 0.040-0.063 mm, Merck) by
flash chromatography. The term “dried” refers to the use of
anhydrous sodium sulfate. Compounds were named following
IUPAC rules as applied by Beilstein-Institut AutoNom (version
2.1), a software for systematic names in organic chemistry. The
purity of the new compounds was determined by combustion
analysis and was g95%.
tallized from EtOH: mp 206-207 °C. Anal. (C19H21NOS
C2H2O4) C, H, N, S.
3
10-Benzyl-spiro[4H-1,3-benzodioxin-2,40-piperidine] Oxalate
(5). This was synthesized from 12 following the procedure
described for 4. Eluting with cyclohexane/EtOAc (8.5:1.5) af-
forded 5 as the free base: 69% yield. 1H NMR (CDCl3): δ 1.97
(m, 4H, 30 and 50-CH2), 2.57 (t, J = 8.11 Hz, 4H, 20 and 60-CH2),
3.57 (s, 2H, CH2Ar), 4.85 (s, 2H, 4-CH2), 6.83-7.39 (m, 9H,
ArH). The free base was transformed into the oxalate salt, which
was recrystallized from 2-PrOH: mp 188-189 °C. Anal. (C19-
H21NO2 C2H2O4) C, H, N.
3
(()-1-Benzyl-4-(1-benzopyran-4-one-2-yl)pyridinium Bromide
(13). A solution of 2-(pyridin-4-yl)-1-benzopyran-4-one43 (1.0 g,
4.44 mmol) and benzyl bromide (1.08 mL, 8.88 mmol) in
CH3CN (14 mL) was stirred at reflux for 3 h. After cooling to
room temperature, diethyl ether was added and the solid was
filtered and crystallized twice from EtOH: 1.2 g; 68% yield; mp
10-Benzyl-1,2,3,4-tetrahydrospiro[naphthalene-2,40-piperidine]
Oxalate (3). A solution of 10 M BH3 Me2S (0.52 mL) in dry
3
1
diglyme (1 mL) was added dropwise at room temperature to a
solution of 10-benzyl-1,2,3,4-tetrahydrospiro[naphthalene-2,40-
piperidin]-1-one41 (0.55 g, 1.80 mmol) in dry diglyme (10 mL)
with stirring under a stream of dry nitrogen with exclusion of
moisture. When the addition was completed, the reaction
mixture was heated at 120 °C for 8 h. After cooling to 0 °C,
excess borane was destroyed by cautious dropwise addition of
MeOH (10 mL). The resulting mixture was left to stand at room
temperature, cooled to 0 °C, treated with HCl gas for 15 min,
then heated to 120 °C for 4 h. Removal of the solvent under
reduced pressure gave a residue, which was dissolved in water.
The aqueous solution was basified with NaOH pellets and
extracted with CHCl3. Removal of dried solvents gave a residue,
which was purified by column chromatography. Eluting with
cyclohexane/EtOAc (8:2) afforded 3 as the free base: 0.4 g; 76%
yield. 1H NMR (CDCl3): δ 1.30-1.75 (m, 6H, 3, 30 and 50-CH2),
2.32-2.83 (m, 8H, 1, 4, 20 and 60-CH2), 3.52 (s, 2H, CH2Ar),
7.0-7.43 (m, 9H, ArH). The free base was transformed into the
oxalate salt, which was recrystallized from EtOH: mp 219-221 °C.
Anal. (C21H25N C2H2O4 0.25H2O) C, H, N.
243-245 °C dec. H NMR (CD3OD): δ 3.11 (m, 2H, 3-CH2),
5.89 (s, 2H, CH2Ar), 5.99 (dd, J = 7.42, 14.2 Hz, 1H, 2-CH),
7.11-9.20 (m, 13H, ArH).
(()-2-(1-Benzyl-piperidin-4-yl)-1-benzopyran-4-one Oxalate
(8). Compound 13 (0.87 g, 2.20 mmol) in EtOH (20 mL) was
hydrogenated over PtO2 (0.10 g) at room temperature for 35 min
and 30 psi of pressure. The mixture was filtered through celite,
and the solvent was removed in vacuo. The residue was treated
with 1N NaOH and extracted with EtOAc. Removal of dried
solvents gave a residue, which was purified by column chroma-
tography. Eluting with CHCl3/MeOH (9.8:0.2) afforded 8 as the
1
free base: 0.22 g; 31% yield. H NMR (CDCl3): δ 1.41-2.12
(m, 7H, 30 and 50-CH2, 40-CH, 20 and 60-CHax), 2.70 (m, 2H,
3-CH2), 2.99 (m, 2H, 20 and 60-CHeq), 3.53 (s, 2H, CH2Ar), 4.24
(dt, J = 8.92, 13.4 Hz, 1H, 2-CH), 6.92-7.92 (m, 9H, ArH). The
free base was transformed into the oxalate salt, which was
recrystallizedfrom2-PrOH:mp 195-196 °C. Anal. (C21H23NO2
C2H2O4) C, H, N.
3
(()-2-(1-Benzyl-piperidin-4-yl)-1-benzothiopyran-4-one Oxa-
late (9). A solution of 1-(2-mercapto-phenyl)-ethanone44 (1.24 g,
8.15 mmol), 1-benzyl-piperidine-4-carbaldehyde (1.66 g, 8.15
mmol), and pyrrolidine (1.16 g, 16.3 mmol) in MeOH (42 mL)
was refluxed for 2 h. After cooling, the solvent was evaporated
and the residue was dissolved in NaHCO3 saturated solution
and extracted with EtOAc. Removal of dried solvents gave
a residue, which was purified by column chromatography.
Eluting with EtOAc afforded 9 as the free base: 2.0 g; 73%
yield; mp 80-82 °C. 1H NMR (CDCl3): δ 1.38-2.02 (m, 7H, 30,
3
3
10-Benzoyl-spiro[4H-3,1-benzoxathiin-2,40-piperidine] (11).
1-Benzoyl-piperidin-4-one (2.47 g, 12.13 mmol) was added
to an ice-cooled solution of (2-mercapto-phenyl)-methanol42
(1.7 g, 12.13 mmol) in dry CH2Cl2 (62 mL). Anhydrous HCl gas
was bubbled until saturation, followed by the addition of
Na2SO4 (3 g). After stirring at room temperature for 24 h the
solution was filtered, washed with H2O, 2N NaOH, and H2O.
Removal of dried solvents gave an oil, which was purified by

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1267
and 50-CH2, 40-CH, 20 and 60-CHax), 2.80-3.12 (m, 4H, 3-CH2,
20 and 60-CHeq), 3.35 (m, 1H, 2-CH), 3.50 (s, 2H, CH2Ar),
7.10-8.11 (m, 9H, ArH). The free base was transformed into the
oxalate salt, which was recrystallized from MeOH: mp 218-220 °C.
Holland) were stained by 0.4% SRB (Sigma-Aldrich, Milan,
Italy), dissolved in 1% acetic acid. Bound stain was subsequently
solubilized with 10 mM Trizma (Sigma-Aldrich, Milan, Italy), and
the absorbance read on the microplate reader Dynatech model
MR 700 at a wavelength of 520 nm. The cytotoxic activity was
evaluated by measuring the drug concentration resulting in a
50% reduction in the net protein increase (as measured by SRB
staining) in control cells during the drug incubation (GI50). The
percentage of growth inhibition was calculated as: [(Ti - Tz)/(C -
Tz)] ꢀ 100 for concentration for which Ti gTz;[(Ti - Tz)/Tz] ꢀ 100
for concentration for which Ti < Tz, where Tz = absorbance time
zero, C = absorbance in the presence of vehicle, and Ti =
absorbance in the presence of drug at different concentrations.
GI50, determined by software, was obtained by interpolation in a
graph % of growth versus Log(M). Each quoted value represents
the mean of triplicate.
Anal. (C21H23NOS C2H2O4) C, H, N, S.
3
(()-2-(1-Phenethyl-piperidin-4-yl)-1-benzopyran-4-one Oxalate
(10). This was synthesized from 1-(2-hydroxy-phenyl)-ethanone
and 1-phenethyl-piperidine-4-carbaldehyde45 following the
procedure described for 9. Eluting with EtOAc afforded 10 as
1
the free base: 69% yield; mp 96-98 °C. H NMR (CDCl3): δ
1.43-2.18 (m, 7H, 30, and 50-CH2, 40-CH, 20 and 60-CHax),
2.52-3.20 (m, 8H, 3-CH2, 20, 60-CHeq and NCH2CH2Ar), 4.23
(ddd, J = 8.12, 4.27, 11.11 Hz, 1H, 2-CH), 6.92-7.92 (m, 9H,
ArH). The free base was transformed into the oxalate salt, which
was recrystallized from EtOH: mp 216-217 °C. Anal.
(C22H25NO2 C2H2O4) C, H, N.
3
(()-4-1-Benzothiopyran-2-yl-1-benzyl-piperidine Oxalate (7).
This was synthesized from 9 following the procedure described
for 3. Eluting with EtOAc afforded 7 as the free base: 56% yield;
mp 90-92 °C. 1H NMR (CDCl3): δ 1.32-2.32 (m, 9H, 3, 30, and
50-CH2, 40-CH, 20 and 60-CHax), 2.71-3.70 (m, 5H, 2-CH,
4-CH2, 20 and 60-CHeq), 3.49 (s, 2H, CH2Ar), 6.92-7.40
(m, 9H, ArH). The free base was transformed into the oxalate
salt, which was crystallized from EtOH: mp 198-200 °C. Anal.
(C21H25NS C2H2O4 0.5H2O) C, H, N, S.
Biology. Radioligand Binding Assays to σ Receptors. The
σ binding assays were performed by CEREP (Paris, France)
according to Ganapathy et al.46 for σ1 binding and Bowen
et al.47 for σ2 binding. In brief, the σ1 binding assay was
performed by incubating Jurkat cell membranes (100-200 μg
protein per tube) with [3H](þ)-pentazocine (8 nM) and a range
of concentrations of test compounds, at 22 °C for 2 h, in 5 mM
Tris/HCl buffer, pH = 7.4. The σ2 binding assay was performed
by incubating rat cerebral cortex membranes (100-200 μg
protein per tube) with [3H]DTG (5 nM), in the presence of
(þ)-pentazocine (300 nM) to saturate σ1 sites, and a range of
concentrations of test compounds, at 22 °C for 2 h, in 5 mM
Tris/HCl buffer, pH = 7.4. The final assay volume was 0.5 mL.
Nonspecific binding was defined, in both assays, as that remain-
ing in the presence of 10 μM haloperidol. The reaction was
terminated by rapid filtration through Whatman GF/B filters,
which were then washed with 5 ꢀ 1 mL ice-cold 0.9% NaCl
(saline) solution and allowed to dry before bound radioactivity
was measured using liquid scintillation counting. The protein
concentration in the homogenates was determined using the
method of Bradford.63
Cell Line Culture. The two breast cancer cell lines of human
origin, MCF-7 adriamycin-sensitive (p53 wild-type) and MCF-
7/ADR-adriamycin-resistant (p53 mutated), characterized by
low and high σ1 receptor expression,48 respectively, were kindly
provided by Prof. G. Zupi (IRE, Rome, Italy). The cells were
routinely cultured in RPMI 1640 supplemented with 10% FCS,
2 mmol/L-glutamine, 50000 units/L-penicillin, and 80 μmol/
L-streptomycin in a humidified incubator at 37 °C with 5%
CO2 atmosphere.
In Vitro Cytotoxicity Assay. Human MCF-7 and MCF-7/
ADR cell lines were used for cytotoxicity testing in vitro using
the SRB (Sulforhodamine B) assay.49 Cells were maintained as
stocks in DMEM (Gibco) supplemented with 10% fetal bovine
serum (Gibco), 2 mM L-glutamine (Gibco). Cell cultures were
passaged twice weekly using trypsin-EDTA to detach the cells
from their culture flasks. The rapidly growing cells were harvested,
counted, and seeded under the appropriate concentrations (1 ꢀ
104 cells/well) in 96-well microtiter plates. After 24 h, different
doses of compound (from 10 nM to 10 μM) dissolved in culture
medium were applied to the culture wells in quadruplicate and
incubated for 48 h at 37 °C in a 5% CO2 atmosphere and 95%
relative humidity. At the same time, a plate was tested to value the
cell population before the drug addition (Tz). Culture fixed with
cold trichloroacetic acid (TCA) (J. T. Baker B.V., Deventer,
Annexin-V Staining. Phosphatidylserine (PS) exposure on
MCF-7 and MCF-7/ADR cells was detected by Annexin V
staining and cytofluorimetric analysis.50 Briefly, 3 ꢀ 105/mL
MCF-7 and MCF-7/ADR cells were treated with 10 μM of
tested compound for 48 h at 37 °C, 5% CO2, in a 24 well plate.
After treatment, cells were stained with Annexin V-FITC (1:40)
for 10 min in binding buffer (10 mM Hepes/NaOH, pH 7.4, 140
mM NaCl, 2.5 mM CaCl2) at room temperature, detached from
the plate, and washed once with binding buffer. Samples were
then analyzed by a FACScan cytofluorimeter using the Cell-
Quest software and data were expressed as mean fluorescence
intensity (MFI).
3
3
Cell Cycle Analysis. First, 3 ꢀ 105/mL MCF-7 and MCF-7/
ADR cells were grown with compounds 1, 8, and 9 (10 μM) for
48 h at 37 °C and 5% CO2. After washing in PBS, cells were
suspended in 1 mL of PBS, fixed by adding 2.5 mL of 70% cold
ethanol, and incubated on ice for 15 min. Then cells were
centrifuged in order to discard ethanol, stained for 40 min at
37 °C with 0.5 mL of PI solution (50 μg/mL PI, 0.1 mg/mL
RNase A, 0.05% Triton-X-100 in PBS), and finally analyzed by
flow cytometry. The percentage of positive cells determined over
10000 events was analyzed on a FACScan cytofluorimeter using
the CellQuest software (Becton Dickinson) and the Cyflogic
software (Cyflo LTD).
Guinea Pig Ileum Functional Assays. The experimental pro-
cedure was essentially that previously described.52 Male guinea
pig weighing 300-400 g (Harlan, Italy) were housed four per
cage in a room with controlled temperature (22 ( 1 °C) and light
(12 h per day) for at least 4 days before being used. Animals were
cared for in accordance with the principles and guidelines of the
Italian Ethics governing these experiments.
Guinea pigs were killed by a blow on the neck. Three to six
segments, 2-3 cm long, of ileum were excised from the same
animal, discarding the 10 cm nearest the cecum. The segments
were cleaned with Tyrode’s solution and set up under 1 g tension
in 10 mL organ bath containing Tyrode’s solution, maintained
at 37 °C, and gassed with 95% O2 and 5% CO2. Changes in
tension were recorded under isotonic conditions (1 g) by a
transducer connected to a recorder (Ugo Basile, Italy) and
calibrated before each experiment.
In experiments, 1 μM ketanserin was included in the Krebs
buffer during the entire experiment. Following the 90 min
equilibration period, 3 noncumulative concentration-response
curves for 5-hydroxytryptamine (5-HT) were obtained, the first
and third in Krebs buffer and the second in Krebs buffer
containing a concentration of a test compound. 5-HT was added
to each organ bath by microliter syringe and remained in contact
with the strips for 1 min. The maximum height of the resulting
contraction was recorded for each concentration of 5-HT. An
interval of 20 min between 5-HT applications was allowed,
during which the strips were washed extensively; preliminary
experiments determined that this procedure avoided tachyphyl-
axis of 5-HT responses. Only strips for which the maximum

1268 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Piergentili et al.
twitch heights obtained during the first and third concentra-
tion-response curves differed by 10% were used.
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Nociceptive Test. Male CD-1 mice (Harlan SRC, Milan,
Italy), weighing 25-35 g, were used. Animals were kept in a
room with a 12:12 h light/dark cycle (lights on at 9:00 a.m.), a
temperature of 20-22 °C, and a humidity of 45-55%. They
were offered free access to tap water and food pellets (4RF18,
Mucedola, Settimo Milanese, Italy). Animal testing was carried
out according to the European Community Council Directive of
24 November 1986 (86/609/EEC).
Nociception was evaluated by the radiant heat tail flick test:
briefly, it consists of the irradiation of the lower third of the tail
with an IR source (Ugo Basile, Comerio, Italy). The basal
predrug latency, ranged between 2 and 3 s, was calculated as
the mean of two trials performed at 30 min interval. Then mice
received 9 (1 mg/kg, sc) or related vehicle (DMSO 1%), 30 min
before morphine (5.0 mg/kg, sc) administration or saline. The
antinociceptive activity was evaluated 30, 60, 90, and 120 min
after morphine injection. A cutoff latency of 12 s was established
to minimize tissue damage.
Statistical Analysis. Antinociceptive effect was expressed as a
percent of the maximum possible effect (MPE) according to the
following formula: %MPE = (measured latency-basal latency)/
(cutoff time - basal latency) ꢀ 100%. Data are reported as means
(SEM. Statistical evaluation of data was carried out by analysis of
variance (ANOVA) and sequential differences among means
according to Student-Newman-Keuls test. Statistical signifi-
cance was set at p < 0.05.
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Acknowledgment. We thank the MIUR (Rome), the Uni-
versity of Camerino, the Monti dei Paschi di Siena Founda-
tion, and the Servier for financial support.
Supporting Information Available: Elemental analyses for
compounds 3-10. This material is available free of charge via
the Internet at http://pubs.acs.org.
ꢁ
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