Development of New Benzylpiperazine Derivatives as σ1Receptor Ligands with in Vivo Antinociceptive and Anti-Allodynic Effects

Article abstract of DOI:10.1021/acschemneuro.1c00106

σ-1 receptors (σ1R) modulate nociceptive signaling, driving the search for selective antagonists to take advantage of this promising target to treat pain. In this study, a new series of benzylpiperazinyl derivatives has been designed, synthesized, and characterized for their affinities toward σ1R and selectivity over the σ-2 receptor (σ2R). Notably, 3-cyclohexyl-1-{4-[(4-methoxyphenyl)methyl]piperazin-1-yl}propan-1-one (15) showed the highest σ1R receptor affinity (Ki σ1 = 1.6 nM) among the series with a significant improvement of the σ1R selectivity (Ki σ2/Ki σ1 = 886) compared to the lead compound 8 (Ki σ2/Ki σ1 = 432). Compound 15 was further tested in a mouse formalin assay of inflammatory pain and chronic nerve constriction injury (CCI) of neuropathic pain, where it produced dose-dependent (3-60 mg/kg, i.p.) antinociception and anti-allodynic effects. Moreover, compound 15 demonstrated no significant effects in a rotarod assay, suggesting that this σ1R antagonist did not produce sedation or impair locomotor responses. Overall, these results encourage the further development of our benzylpiperazine-based σ1R antagonists as potential therapeutics for chronic pain.

Full text of DOI:10.1021/acschemneuro.1c00106

pubs.acs.org/chemneuro
Research Article
Development of New Benzylpiperazine Derivatives as σ1 Receptor
Ligands with in Vivo Antinociceptive and Anti-Allodynic Effects
̀
Giuseppe Romeo, Federica Bonanno, Lisa L. Wilson, Emanuela Arena, Maria N. Modica, Valeria Pittala,
Loredana Salerno, Orazio Prezzavento, Jay P. McLaughlin, and Sebastiano Intagliata*
Cite This: ACS Chem. Neurosci. 2021, 12, 2003−2012
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: σ-1 receptors (σ1R) modulate nociceptive signaling, driving the search for selective antagonists to take advantage of
this promising target to treat pain. In this study, a new series of benzylpiperazinyl derivatives has been designed, synthesized, and
characterized for their affinities toward σ1R and selectivity over the σ-2 receptor (σ2R). Notably, 3-cyclohexyl-1-{4-[(4-
methoxyphenyl)methyl]piperazin-1-yl}propan-1-one (15) showed the highest σ1R receptor affinity (Ki σ1 = 1.6 nM) among the
series with a significant improvement of the σ1R selectivity (Ki σ2/Ki σ1 = 886) compared to the lead compound 8 (Ki σ2/Ki σ1 =
432). Compound 15 was further tested in a mouse formalin assay of inflammatory pain and chronic nerve constriction injury (CCI)
of neuropathic pain, where it produced dose-dependent (3−60 mg/kg, i.p.) antinociception and anti-allodynic effects. Moreover,
compound 15 demonstrated no significant effects in a rotarod assay, suggesting that this σ1R antagonist did not produce sedation or
impair locomotor responses. Overall, these results encourage the further development of our benzylpiperazine-based σ1R antagonists
as potential therapeutics for chronic pain.
KEYWORDS: Benzylpiperazines, σ receptors, σ-1 antagonist, analgesia, neuropathic pain
more challenging to define than σ1R, and its crystal structure
has not yet been reported. Indeed, the protein has just recently
been cloned, with a sequence identical to the transmembrane
protein 97 (TMEM97), a protein involved in cholesterol
homeostasis.9 Since then, it is general practice to refer to this
protein as σ2R/TMEM97. Like σ1R, σ2R/TMEM97 was
initially miscategorized and correlated to the progesterone
membrane component 1 (PGRMC1), which was thought to be
the σ2R binding site.10 Finally, further studies clarified that
σ2R/TMEM97 might still interact with PGRMC1 and LDLR
(low-density lipoprotein receptor), forming a ternary complex
that mediates LDL internalization and trafficking.11,12
INTRODUCTION
■
The σ-1 receptor (σ1R) was initially categorized as an opioid
receptor subtype because of the binding with the nonselective
benzomorphan alazocine (SKF10,047).1 Subsequent studies
have proven that naloxone did not possess antagonism at this
receptor,2 and later molecular cloning and the X-ray crystallo-
graphic structure of the human σ1R revealed no homology with
opioid receptors.3,4 Indeed, unlike opioid receptors, which
possess the seven-transmembrane domain structure character-
istics of G protein-coupled receptors, σ1R is a transmembrane
protein that is present in numerous oligomeric states.4 The
protomer contains one transmembrane domain, five α helices,
and 10 β strands forming the ligand-binding pocket.4,5
Therefore, σ1R is now recognized as a unique chaperone
protein mostly expressed at the endoplasmatic reticulum and is
a highly conserved protein among different species with over
90% identical amino acid sequences.6
Received: February 26, 2021
Accepted: May 10, 2021
Published: May 21, 2021
A second σ receptor subtype (named σ2R) was discovered
and differentiated from the first subtype on the basis of size,
tissue distribution, and ligand affinity.7,8 σ2R has been even
© 2021 The Authors. Published by
American Chemical Society
https://doi.org/10.1021/acschemneuro.1c00106
ACS Chem. Neurosci. 2021, 12, 2003−2012
2003

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Figure 1. Structures of σRs ligands with antinociceptive activities: selective σ1R antagonists 1 and 2; selective σ2R ligands 3 and 4; mixed σ1R/σ2R
ligands 5, 6, trans-7, and cis-7.
Figure 2. (A) 2D and 3D structures of lead compound 8. (B) Glennon’s σ1R pharmacophoric features (i.e., primary hydrophobic, blue; basic
nitrogen, green; secondary hydrophonic, red) and the general structure of newly benzylpiperazine derivatives.
Both σ receptor (σR) subtypes are present in several CNS
areas and peripheral tissues such as the spleen and liver, as well
as overexpressed on different human tumors.6,13 σ receptors
are known to be expressed in key areas of pain control in the
CNS such as the locus coeruleus, periaqueductal gray, and
rostroventral medulla.14,15 In the CNS, these receptors are also
very highly expressed in the dorsal root ganglia of the spinal
cord, indicating a key role in the function of peripheral pain
pathways.16,17 In agreement with their anatomical distribution,
σRs modulate a broad range of body functions.18 Conversely,
dysregulation of the physiological activities of σRs has been
observed in several medical conditions, including drug
addiction, neuropsychiatric disorders, cancer, and chronic
pain.18−22 With an improving understanding of σRs, σR ligands
2004
https://doi.org/10.1021/acschemneuro.1c00106
ACS Chem. Neurosci. 2021, 12, 2003−2012

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
a
Scheme 1. Reaction Pathways for Compounds 13−16 and 20−22
a
Reagents and conditions: (a) CDI, dry DCM, RT, then 1-(4-methoxybenzyl)piperazine, 0 °C for 30 min, then RT, 1−2 h, 33−68%; (b) TEA, dry
THF, 0 °C for 30 min, then RT, 1−3 days, 61−67%.
a
Scheme 2. Reaction Pathways for Compounds 23 and 24
a
Reagents and conditions: (a) CDI, dry DCM, room temperature, then piperazine, 0 °C for 30 min, then RT, 1 h, 75%; (b) 4-
(chloromethyl)benzyl alcohol, K2CO3, KI, DCM, MW (150 W), 120 °C, 2 h, 90%.
have recently attracted increased attention from the scientific
community for their potential as new medications to treat
unmet medical needs,23−25 including the novel coronavirus
disease 2019 (COVID-19) as recently reported.26,27 Notably,
novel σR selective ligands are currently under evaluation in
clinical trials as diagnostic agents (i.e., PET radiotracers) and
therapeutic efficacy for some of the diseases mentioned
above.28−30
Consistent with their molecular role as chaperones, the σRs
interact with various proteins, modifying their function.31,32
Concerning their active modulatory activity in pain signaling,
these protein targets include the μ-opioid receptor, ion
channels, and the NMDA receptors.14 Significantly, σ1R
antagonists block the activity of the σ1Rs, producing increased
opioid and decreased NMDA receptor signaling, thereby
enhancing antinociception by opioids and decreasing the
hypersensitivity commonly associated with pathological pain.33
Interestingly, an increased number of ligands possessing
different chemotypes and heterogeneous σRs binding profiles
showed a significant antinociception effect in different
preclinical in vivo pain models (Figure 1).34−41
Continuing our efforts to discover selective σ1R ligands,42,43
in this paper, a set of new benzylpiperazines was synthesized
and pharmacologically characterized for their analgesic effects
in mice models of pain. Previously, we developed a series of
bifunctional σRs ligands with in vitro antioxidant properties.44
These ligands were obtained by combining a preferred σR
cyclic amino moiety, such as benzylpiperazine, with the 1,2-
dithiolan-3-yl moiety belonging to the natural antioxidant
compound α-lipoic acid (Figure 2A). The 4-methoxybenzylpi-
perazinyl derivative 8 was previously identified as a potent and
selective ligand for the σ1R over σ2R/TMEM97 (Figure 2A).
Moreover, previous structure−affinity relationships (SAfiRs)
suggested that the introduction of a para-substituent at the
secondary hydrophobic domain (HYD2) improved the affinity
and selectivity at σ1R.44 On the contrary, little exploration of
2005
https://doi.org/10.1021/acschemneuro.1c00106
ACS Chem. Neurosci. 2021, 12, 2003−2012

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
both the chain linker and the primary hydrophobic domain
(HYD1) was performed. With this in mind, we used
compound 8 as our lead molecule to develop new
benzylpiperazine derivatives as potentially more potent and
selective σ1R ligands over σ2R (Figure 2B).
Table 1. σRs Binding Affinities for 13−16, 20−22, and 24
a
Ki (nM) SD
compound
σ1R
σ2R
Ki σ2/Ki σ1
b
8
5.7 0.1
18.1 0.44
13.3 0.22
1.6 0.05
11.3 0.26
102 0.6
8.8 0.22
145 0.5
6.1 0.1
2,460 85
1,162 40
1,644 37
1,418 18
3,968 130
4,367 33
3,253 40
23,190 146
2,583 54
17.6 0.1
432
64
124
886
351
43
370
160
423
11
Similar to 8, the newly synthesized compounds fulfilled
Glennon’s pharmacophore model in which two distal hydro-
phobic regions and a central positive ionizable nitrogen give
the essential features for the binding at σ1R (Figure 2A,B).45
Due to the promising outcomes obtained with previous
benzylpiperazines, in this new series, we maintained the 4-
methoxybenzylpiperazinyl moiety as the HYD2 and we
modified the other distal hydrophobic region and the linker
portion (i.e., 13−16 and 20−22). Specifically, the substitution
of a phenyl, phenoxy, or cyclohexyl group in place of a lipoyl
one as the HYD1 was carried out. Additionally, to explore the
importance of an additional H-bond donor group for the target
binding, the 4-methoxy substituent was replaced with a
hydroxymethyl one (24).
13
14
15
16
20
21
22
24
haloperidol
1.6 0.1
a
Ki values are expressed as mean
SD of three independent
b
experiments. Data from ref 44.
21, and 22 (i.e., ethylene > vinylene ≃ propylene > butylene ≫
methylene). Indeed, chain shortening from four (13) to two
methylene units (21) led to an increase in σ1R affinity (Ki σ1 =
18.1 and 8.8 nM, respectively); however, a further shortening
to only one methylene unit (22) was detrimental (Ki σ1 = 145
nM). Thus, the length of the ethylene chain in compound 21
produced optimal σ1R affinity (Ki = 8.8 nM) and selectivity
(370-fold) among this set. A phenyl ring instead of a lipoyl one
was tolerated concerning the HYD1 domain, although a loss of
selectivity was observed (8 vs 13), whereas the introduction of
a phenoxy group (20) gave the worst result. Further
replacement with a cyclohexyl ring resulted in a remarkable
improvement of both the affinity and selectivity for σ1R (15 vs
21).
Finally, an additional H-bond donor group (24) in the
secondary hydrophobic domain improved neither the σ1R
affinity nor the selectivity significantly (24 vs 8). Compound
15, which showed the best binding profile among the series (Ki
σ1 = 1.6 nM; Ki σ2 = 1,418 nM; Ki σ2/Ki σ1 = 886), was
selected for a more in-depth in vivo pharmacological
evaluation.
Focally Induced Inflammatory Nociception. Due to the
lack of reliable in vitro assays to establish the agonist/
antagonist properties of σRs ligands, the intrinsic functional
activity of 15 was assessed by using a behavioral model of
nociception. Consistent with the evidence of σ1R modulation
of nociceptive signaling,46 σ1R antagonists ameliorate pain
responses in a focally induced inflammatory nociception model
such as the formalin assay.15,47,48 Compound 15 showed
significant dose-dependent antinociception in this assay
(Figure 3A), consistent with action as a putative σ1R
antagonist.
RESULTS AND DISCUSSION
■
Chemistry. Reagents and conditions for preparing the final
compounds 13−16, 20−22, and 24 are summarized in
Schemes 1 and 2. Precisely, 4-methoxybenzylpiperazinyl
analogues 13−16 and 20−22 were synthesized starting from
the suitable activated acids 9−12 or acyl chlorides 17−19,
according to the two pathways reported in Scheme 1. In the
first case, acids 9−12 were activated by a reaction with 1,1′-
carbonyldiimidazole (CDI) in dry dichloromethane (DCM) to
give acyl imidazole intermediates (not isolated), which were
then reacted with 1-(4-methoxybenzyl)piperazine in a
protective nitrogen atmosphere to afford final amides 13−16
in good yields (36−68%). Amides 20−22 were obtained
directly by the coupling of 1-(4-methoxybenzyl)piperazine
with the corresponding organic halides (17−19) in dry
tetrahydrofuran (THF) and using triethylamine as a base
catalyst.
The final compound 24 was prepared following the two-step
reaction depicted in Scheme 2. The acid derivative 9 was
activated by CDI and converted into amide intermediate 23 by
reaction with piperazine. Subsequently, treatment with the
commercially available 4-(chloromethyl)benzyl alcohol, with
K2CO3 and KI, and using microwaves (MW) irradiation gave
the final benzylpiperazine derivative 24 in excellent yield
(90%).
Final compounds were characterized as a free base and
submitted as such for the in vitro binding assay. Compound 15
has been converted into oxalate salt for in vivo behavioral
studies.
σR Binding Properties and SAfiRs. The affinities of the
newly synthesized benzylpiperazine derivatives (13−16, 20−
22, and 24) for the σ1 and σ2 receptors were evaluated in
radioligand binding assay using [3H]-pentazocine and [3H]-
DTG, respectively, as radioligands in the presence of
haloperidol to determine the nonspecific binding. All tested
compounds displayed higher selectivity ratio values (Ki σ2/Ki
σ1) than the reference ligand, haloperidol (Table 1). Moreover,
compounds 15 and 24 showed improved or similar σ1R
selectivity (Ki σ2/Ki σ1 = 886 and 423, respectively) compared
to the lead compound 8 (Ki σ2/Ki σ1 = 432).
Compound 15 showed a similar efficacy at the highest dose
in reducing time spent licking the injected paw compared to
the positive control CM304 (1), a well-characterized selective
σ1R antagonist (Figure 3B),35 with an ED50 (and 95% C.I.)
value of 12.7 (9.9−16.6) mg/kg, i.p. These results confirm
previous reports from Romero et al. in 2012, Gris et al. in
2014, and Cirino et al. in 2019,35,49,50 where σ1R antagonists
blocked peripheral nociception associated with inflammatory
pain responses.
Focally Induced Neuropathy. On the basis of the
observed antinociceptive effect exerted by 15 in the formalin
assay, we further characterized 15 in a representative model of
neuropathic pain. We selected the chronic constriction injury
(CCI) model as a widely used and validated assay to produce
Regarding the σ1R affinity, a clear trend based on the length
of the linker chain between the distal phenyl ring and the
central amide group can be observed in analogues 13−14, 16,
2006
https://doi.org/10.1021/acschemneuro.1c00106
ACS Chem. Neurosci. 2021, 12, 2003−2012

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Figure 3. Formalin-induced inflammation assay: (A) compound 15 demonstrates a dose-dependent increase in % antinociception and (B)
decreases summed time spent licking of formalin-treated paw in a dose-dependent manner compared to the vehicle control and the selective σ1R
antagonist (CM304). n = 10 for all points.
allodynia.51−54 Compound 15 demonstrated significant dose-
dependent anti-allodynic effects (F(5, 184) = 21.17; p < 0.0001;
two-way ANOVA with Tukey’s post hoc test; Figure 4), with
locomotion or sedation, the effect of compound 15 on elicited
locomotor activity was assessed using the rotarod assay.35 The
positive control and κ-opioid receptor agonist trans-( )-3,4-
dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-
benzeneacetamide hydrochloride (U50,488, 10 mg/kg, i.p.)
significantly impaired evoked locomotor activity compared to
the vehicle control (F(4, 301) = 26.02; p < 0.0001; two-way
ANOVA with Tukey’s post hoc test; Figure 5) up to 60 min
Figure 4. Chronic constriction injury model testing: mechanical
allodynia produced from sciatic nerve constriction were reduced after
compound 15 treatment, similar to the positive control (gabapentin)
with a longer duration of action than the reference compound
CM304. n = 8−13 for all groups. * = significantly different from
vehicle controls; p < 0.05.
Figure 5. Sedation and evoked, coordinated locomotor function were
assessed using the rotarod apparatus following the administration of
either saline (i.p.), U50,488 (10 mg/kg, ip), CM304 (45 mg/kg, i.p.),
or compound 15 (30 and 60 mg/kg, i.p.). * = significantly different
from baseline response (p < 0.05). n = 8−12.
significant increases in withdrawal thresholds at 20, 60, and 80
min post-injection of a 60 mg/kg, i.p. dose (p < 0.05; Tukey’s
post hoc test). These effects were comparable to the results of
the positive control gabapentin (Figure 4). CM304, the
reference selective σ1R antagonist, demonstrated anti-allodynic
effects that are comparable to those of gabapentin in a time-
dependent manner. The anti-allodynic effects of CM304
peaked at 40 min but began to diminish at 60 min. The
current results are consistent with a mechanistic interpretation
of anti-allodynia through the σ1R antagonism. Notably, CCI
produces a focal injury of the sciatic nerve that has been
demonstrated to enhance the labeling of spinal σ1Rs in a
manner enhancing nociceptive signaling.55 Currently, these
studies with compound 15 verify previous findings stating that
noxious stimuli are attenuated by σ1R antagonists.35,48,56
Induced Locomotor Activity. Treatments for neuro-
pathic pain may be complicated by concordant sedation and
impairment of motor function, as demonstrated by gabapen-
tin.57 To eliminate the potential complication of impaired
after administration. In contrast, at doses proving effective in
the pain assays, compound 15 did not significantly impair
evoked locomotor activity, although the 30 mg/kg, i.p. dose
produced a singular increase in locomotor performance 60 min
post-administration (p = 0.003). Although the mechanism of
σRs involvement in motor coordination and sedation has not
yet been fully defined, the current results confirm the recent
finding by Cirino et al.,35 showing that selective σ1R
antagonists fail to produce sedative effects or impair evoked
locomotor activity in rodents, confirming their analgesic
properties.
CONCLUSIONS
This work has described the design and synthesis of new
benzylpiperazinyl derivatives possessing high affinities for σ1R
■
2007
https://doi.org/10.1021/acschemneuro.1c00106
ACS Chem. Neurosci. 2021, 12, 2003−2012

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
1-{4-[(4-Methoxyphenyl)methyl]piperazin-1-yl}-4-phenylbutan-
1-one (14). The crude was purified by recrystallization from ethanol/
water (1:2, v/v) to afford 14 (0.431 g, 54.5%) as white crystals. Mp:
91.0−93.9 °C. IR (KBr, selected lines): cm−1 3028, 2952, 1640, 1509,
(Ki σ1 = 1.6−145 nM) and selectivity over σ2R (Ki σ2/Ki σ1 =
43−886). Following Glennon’s structural features criteria
necessary for σ1R binding, we discovered compound 15 as a
potent and selective σ1R ligand. Especially, the use of
hydrophobic cyclohexyl or phenyl groups and the 4-
methoxybenzylpiperazinyl moiety (HYD1 and HYD2, respec-
tively) linked by three-carbon units linker (i.e., 15, 16, and 21)
was an excellent combination to obtain optimal σRs binding
profiles. Importantly, behavioral pharmacology studies showed
that 15 produced significant antinociceptive and anti-allodynic
effects in preclinical mouse models of pain without impaired
locomotor activity, supporting the development of benzylpi-
perazine-based σ1R antagonists as potential therapeutics for
chronic pain.
1
1240. H NMR (200 MHz, DMSO-d6): δ 7.37−7.10 (m, 5H + 2H,
aromatic), 6.95−6.80 (m, 2H, aromatic), 3.72 (s, 3H, OCH3), 2.57 (s,
2H, ArCH2N), 3.50−3.20 (m, 4H, piperazine), 3.40 (t, J = 7.2 Hz,
2H, NCOCH2CH2), 2.40−2.20 (m, 4H + 2H, piperazine +
CH2CH2C6H5), 1.85−1.60 (m, 2H, CH2CH2CH2). 13C NMR (50
MHz, DMSO-d6): δ 170.2, 158.4, 141.9, 130.2, 129.6, 128.3, 125.8,
113.6, 61.3, 55.0, 52.8, 52.3, 44.9, 41.0, 34.7, 31.7, 26.8. Anal. calcd for
C22H28N2O2: C, 74.97; H, 8.01; N, 7.95. Found: C, 75.11; H, 8.22; N,
7.83.
3-Cyclohexyl-1-{4-[(4-methoxyphenyl)methyl]piperazin-1-yl}-
propan-1-one (15). Compound 15 was prepared by the general
procedure described for the synthesis of derivatives 13−16 using dry
THF instead of dry DCM as a solvent. The yellow crude oil was
purified by flash column chromatography using ethyl acetate/
methanol (9.7:0.3, v/v) as an eluent to afford 15 (0.360 g, 54.4%)
as a white solid. Mp: 69.6−72.5 °C. IR (KBr, selected lines): cm−1
METHODS
■
Chemistry. Melting points were performed in an IA9200
electrothermal apparatus equipped with a digital thermometer in
glass capillary tubes and are uncorrected. The elemental analyses for
C, H, and N were within 0.4% of the theoretical values and were
recorded on a Carlo Erba elemental analyzer Mod 1108 apparatus.
Infrared spectra were determined in KBr disks (solid samples) or
NaCl plates (oil samples) on a PerkinElmer 1600 Series FT-IR
1
3064, 3029, 2828, 1643, 1277, 1037, 737. H NMR (free base, 200
MHz, DMSO-d6): δ 7.25−7.15 (m, 2H, aromatic), 6.95−6.85 (m,
2H, aromatic), 3.73 (s, 3H, OCH3), 3.50−3.20 (m, 4H, piperazine),
3.40 (s, 2H, ArCH2N), 2.40−2.10 (m, 4H + 2H, piperazine +
NCOCH2CH2), 1.75−1.45 (m, 5H, cyclohexane), 1.40−1.00 (m, 2H
+ 4H, CH2CH2C6H11 + cyclohexane), 1.00−0.70 (m, 2H, cyclo-
hexane). 13C NMR (oxalate salt, 126 MHz, DMSO-d6): δ 171.1,
163.2, 159.4, 131.7, 125.0, 114.0, 59.7, 55.2, 51.5, 51.1, 43.2, 36.8,
32.7, 32.2, 29.7, 26.2, 25.8. Anal. calcd for C21H32N2O2: C, 73.22; H,
9.36; N, 8.13. Found: C, 73.02; H, 9.13; N, 8.32.
spectrometer. The H NMR and 13C NMR spectra of intermediate
1
and final compounds were recorded with a Varian Inova Unity (200
MHz) spectrometer and a Varian Inova Unity (500 MHz)
spectrometer using a DMSO-d6 solution. The chemical shifts are
reported in δ values (ppm), using tetramethylsilane (TMS) as the
internal standard; the coupling constants (J) are given in hertz (Hz).
The signal multiplicities are characterized as s (singlet), d (doublet), t
(triplet), or m (multiplet). Microwave irradiation experiments were
carried out with a CEM Discovery instrument using closed Pyrex glass
tubes with Teflon-coated septa. Thin-layer chromatography (TLC)
on Merck plates (aluminum sheet coated with silica gel 60 F254) was
used to monitor the progress of reactions and to test the purity
(≥95%) of all the synthesized compounds, and spots were visualized
under UV (λ = 254 and 366 nm) or in an iodine chamber. The
purification of synthesized compounds by column chromatography
was performed using Merck silica-gel 60 (230−400 mesh). All
chemicals and solvents were purchased from commercial vendors and
were of reagent grade.
General Procedure for the Synthesis of 4-(Methoxyphenyl)-
methylpiperazine Derivatives 13−16. 1,1′-Carbonyldiimidazole
(1.0 equiv) was mixed to a stirred solution of suitable acid 9−12 (1.0
equiv) in dry DCM (6 mL) at room temperature. Then, after no gas
evolution was observed, the mixture thus obtained was added
dropwise to a stirred solution of 1-(4-methoxybenzyl)piperazine (1.1
equiv) in dry DCM (6 mL) at 0 °C under a nitrogen atmosphere. The
reaction was carried out for 30 min at 0 °C and then for 1−2 h at
room temperature. The mixture was washed with 10% aqueous NaCl
solution (4 × 10 mL) and H2O (2 × 10 mL). The organic layer was
dried over anhydrous sodium sulfate and then evaporated under
reduced pressure to obtain a crude, which was purified as specified for
each final product.
(2E)-1-{4-[(4-Methoxyphenyl)methyl]piperazin-1-yl}-3-phenyl-
prop-2-en-1-one (16). The yellow crude oil was purified by flash
column chromatography using ethyl acetate/methanol (9.7:0.3, v/v)
as an eluent to afford 16 (0.139 g, 32.8%) as a light yellow solid. Mp:
120.8−122.6 °C. IR (KBr, selected lines): cm−1 2986, 1651, 1605,
1
1455, 1236. H NMR (200 MHz, DMSO-d6): δ 7.80−7.60 (m, 2H,
aromatic), 7.55−7.31 (m, 3H + 1H, aromatic + COCHCH), 7.30−
7.12 (m, 2H + 1H, aromatic + COCHCH), 6.95−6.80 (m, 2H,
aromatic), 3.74 (s, 3H, OCH3), 3.80−3.25 (m, 4H, piperazine), 3.43
(s, 2H, ArCH2N), 2.55−2.20 (m, 4H, piperazine). 13C NMR (126
MHz, DMSO-d6): δ 164.4, 158.3, 141.5, 135.1, 130.2, 129.6, 129.5,
128.7, 128.0, 118.2, 113.6, 61.2, 55.0, 53.1, 52.2, 45.1, 41.7. Anal.
calcd for C21H24N2O2: C, 74.97; H, 7.19; N, 8.33. Found: C, 74.78;
H, 7.32; N, 8.11.
General Procedure for the Synthesis of 4-(Methoxyphenyl)-
methylpiperazine Derivatives 20−22. A mixture of 1-(4-
methoxybenzyl)piperazine (1.0 equiv) and triethylamine (1.0 equiv)
in dry THF (5 mL) was prepared and left under stirring for 10 min at
0 °C. Subsequently, the appropriate acyl chloride (17−19, 1.0 equiv)
was added to the obtained solution, and the reaction was carried out
at 0 °C for 30 min and then at room temperature for 1−3 days. At the
end of the reaction time, the solvent was evaporated to dryness under
a vacuum. The crude product thus obtained was solubilized in DCM
and then washed with a water solution of Na2CO3 0.1 M (2 × 20 mL)
and NaCl 10% (20 mL). The organic layer was dried over anhydrous
sodium sulfate and evaporated under reduced pressure to obtain a
residue, which was purified as specified for each final product.
1-{4-[(4-Methoxyphenyl)methyl]piperazin-1-yl}-4-phenoxybu-
tan-1-one (20). The yellow crude oil was triturated with light
petroleum ether at 40−60 °C to give a white solid, which was
collected, washed with petroleum ether, and dried. The crude thus
obtained was purified by recrystallization from ethanol/water (1:2, v/
v) to afford 20 (0.277 g, 59.8%) as white crystals. Mp: 88.3−91.3 °C.
IR (KBr, selected lines): cm−1 3058, 2945, 1647, 1252, 757. 1H NMR
(200 MHz, DMSO-d6): δ 7.35−7.15 (m, 2H + 2H, aromatic), 7.00−
6.80 (m, 2H + 3H, aromatic), 3.96 (t, J = 6.4 Hz, 2H, CH2CH2OPh),
3.73 (s, 3H, OCH3), 3.50−3.20 (m, 4H, piperazine), 3.39 (s, 2H,
ArCH2N), 2.45 (t, J = 7.2 Hz, 2H, NCOCH2CH2), 2.35−2.20 (m,
4H, piperazine), 2.00−1.80 (m, 2H, CH2CH2CH2). 13C NMR (50
MHz, DMSO-d6): δ 170.0, 158.5, 158.4, 130.2, 129.6, 129.5, 120.5,
1-{4-[(4-Methoxyphenyl)methyl]piperazin-1-yl}-5-phenylpentan-
1-one (13). The yellow crude oil was purified by flash column
chromatography using ethyl acetate/methanol (9.5:0.5, v/v) as an
eluent to afford 13 (0.547 g, 60.4%) as a colorless oil. IR (neat,
1
selected lines): cm−1 3447, 2945, 1646, 1508, 1458, 1242, 748. H
NMR (200 MHz, DMSO-d6): δ 7.00−7.35 (m, 5H + 2H, aromatic),
6.80−6.95 (m, 2H, aromatic), 3.73 (s, 3H, OCH3), 3.35−3.50 (m, 4H
+ 2H, piperazine + ArCH2N), 2.57 (t, J = 7.0 Hz, 2H,
NCOCH2CH2), 2.10−2.40 (m, 4H
+ 2H, piperazine +
CH2CH2C6H5), 1.35−1.65 (m, 4H, CH2CH2CH2CH2). 13C NMR
(126 MHz, DMSO-d6): δ 170.4, 158.3, 142.1, 130.1, 129.6, 128.3,
128.2, 125.6, 113.6, 61.3, 55.0, 52.7, 52.2, 44.9, 41.0, 34.9, 32.1, 30.6,
24.4. Anal. calcd for C23H30N2O2: C, 75.37; H, 8.25; N, 7.64. Found:
C, 75.15; H, 8.32; N, 7.56.
2008
https://doi.org/10.1021/acschemneuro.1c00106
ACS Chem. Neurosci. 2021, 12, 2003−2012

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
114.4, 113.6, 66.7, 61.3, 55.0, 52.7, 52.3, 44.9, 41.1, 28.6, 24.5. Anal.
calcd for C22H28N2O3: C, 71.71; H, 7.66; N, 7.60. Found: C, 71.94;
H, 7.75; N, 7.73.
Anal. calcd for C23H30N2O2: C, 75.37; H, 8.25; N, 7.64. Found: C,
75.20; H, 8.09; N, 7.50.
σRs Binding Assays. Binding tests were performed following
known reported protocols.39,44 The binding assay for σ1R was carried
out using guinea pig brain membrane homogenates according to
DeHaven-Hudkins et al.,58 while the binding assay for σ2R was
performed following experimental procedures described by Mach et
al.59 Inhibitory constants (Ki) were calculated using the radioligand
binding analysis software EBDA/Ligand (Elsevier/Biosoft).
1-{4-[(4-Methoxyphenyl)methyl]piperazin-1-yl}-3-phenylpro-
pan-1-one (21). The yellow crude oil was purified by flash column
chromatography using ethyl acetate/methanol (9.5:0.5, v/v) as an
eluent to afford 21 (0.289 g, 67.9%) as a colorless oil. IR (neat,
selected lines): cm−1 3482, 2934, 2806, 1637, 1512, 1245, 1032, 999,
1
701. H NMR (200 MHz, DMSO-d6): δ 7.35−7.10 (m, 5H + 2H,
aromatic), 6.95−6.80 (m, 2H, aromatic), 3.73 (s, 3H, OCH3), 3.55−
3.20 (m, 4H, piperazine), 3.38 (s, 2H, ArCH2N), 2.79 (t, J = 7.8 Hz,
2H, NCOCH2CH2), 2.58 (t, J = 7.8 Hz, 2H, CH2CH2C6H5), 2.40−
2.15 (m, 4H, piperazine). 13C NMR (126 MHz, DMSO-d6): δ 169.8,
158.3, 141.4, 130.1, 129.6, 128.4, 128.2, 125.9, 113.6, 61.3, 55.0, 52.6,
52.2, 44.9, 41.1, 33.9, 30.8. Anal. calcd for C21H26N2O2: C, 74.52; H,
7.74; N, 8.28. Found: C, 74.41; H, 7.60; N, 8.10.
1-{4-[(4-Methoxyphenyl)methyl]piperazin-1-yl}-2-phenylethan-
1-one (22). The yellow crude oil was purified by flash column
chromatography using ethyl acetate/methanol (9.5:0.5, v/v) as an
eluent to afford 22 (0.252 g, 61.7%) as a white solid. Mp: 97.6−99.8
°C. IR (KBr, selected lines): cm−1 3032, 3018, 2924, 2802, 1647,
1438, 1235, 1036, 794. 1H NMR (200 MHz, DMSO-d6): δ 7.35−7.10
(m, 5H + 2H, aromatic), 6.93−6.80 (m, 2H, aromatic), 3.73 (s, 3H,
OCH3), 3.69 (s, 2H, NCOCH2C6H5), 3.50−3.30 (m, 4H,
piperazine), 3.38 (s, 2H, ArCH2N), 2.30−2.15 (m, 4H, piperazine).
13C NMR (50 MHz, DMSO-d6): δ 168.8, 158.4, 135.9, 130.2, 129.6,
Behavioral Pharmacology. Animals. Adult male C57BL/6J and
CD-1 mice housed five to a cage (8−12 weeks of age) were used.
C57BL/6J mice were used for evoked locomotor rotarod and formalin
assays.35,60,61 Antinociception was confirmed with the use of CD-1
mice in the CCI nerve assay. The CD-1 strain has been well-validated
for antinociceptive62 and mechanical anti-allodynic testing.63,64 All
test compounds were administered using the intraperitoneal (i.p.)
route. All animal studies reported herein adhere to ARRIVE
guidelines.65 Animals were randomly assigned, and researchers were
blinded to group treatments. Animals were housed on a 12:12 h light/
dark cycle (lights off at 7:00 pm) with ad libitum access to food and
water except during experimental sessions. All procedures were
preapproved by the Institutional Animal Care and Use Committee
(University of Florida) and conducted in accordance with the 2011
NIH Guide for the Care and Use of Laboratory Animals.
Formalin Test. The efficacy of compound 15 to ameliorate
inflammatory nociception was achieved with the use of C57BL/6J
mice in the formalin assay as previously described.53 After a 10 min
pretreatment (i.p.) of vehicle control (saline), CM304 (3−30 mg/kg,
i.p.), or compound 15 (3−30 mg/kg, i.p.), an intraplantar (i.pl.)
injection of 5% formalin (2.5 μg in 15 μL) was administered into the
right hind paw. Time spent licking the right hind paw was recorded in
5 min intervals for 60 min following injection. The last 55 min of
assessment was used to determine the inflammatory response
stimulus. Data were analyzed as the summed duration of licking
hind paw.
CCI Assay. CCI was introduced in CD-1 mice that were first
anesthetized with isoflurane as described by Hoot et al.66 and Cirino
et al.35 to induce mechanical allodynia.51−54 After anesthetization,
mice were subjected to surgery where an incision was made along the
surface of the biceps femoris of the right hind paw.66 Blunt forceps
were used to split the muscle and expose the right sciatic nerve. The
tips of two 0.1−10 μL pipet tips facing opposite directions were
passed under the sciatic nerve to allow for easy passing of two sutures
under the nerve, 1 mm apart. The sutures were tied loosely around
the nerve and knotted twice, and the skin was closed with 29 mm skin
staples. Mice were given a 7 day recovery period prior to the baseline
von Frey testing as described below to confirm the induction of
hyperalgesia in each mouse. Animals demonstrating allodynia or a
response to lower pressure were deemed to have neuropathic pain.
Allodynic mice were then administered (i.p.) either vehicle (saline),
morphine (10 mg/kg, i.p.), gabapentin (50 mg/kg, i.p.), CM304 (45
mg/kg, i.p.), or compound 15 (10−60 mg/kg, i.p.). Note that
gabapentin was tested 1 h postinjection to circumvent known sedative
effects that may confound the assay.67 Each mouse was then tested for
the modulation of tactile allodynia every 20 min up until 80 min post-
injection with the use of von Frey testing. The assessment of
mechanical allodynia was performed to measure compound 15’s
efficacy against CCI-induced allodynia as described.51−54 Mice were
habituated on a mesh platform for 1 h prior to testing. Filaments of
increasing pressure (0.4−6 g) were applied and then held to the
plantar surface of both the injured and uninjured hind paws of mice
for approximately 1−2 s prior to drug administration to record
baseline responses to a peripheral stimulus. The filaments were
applied with increasing strengths, and threshold responses were
defined as two hind paw responses per trial of the same filament
strength.
129.0, 128.4, 126.4, 113.6, 61.3, 55.0, 52.7, 52.1, 45.5, 41.3. Anal.
calcd for C20H24N2O2: C, 74.04; H, 7.46; N, 8.64. Found: C, 73.87;
H, 7.27; N, 8.55.
5-Phenyl-1-(piperazin-1-yl)pentan-1-one (23). 1,1′-Carbon-
yldiimidazole (0.910 g, 5.61 mmol) was added to a solution of 5-
phenyl-valeric acid (9) (0.80 g, 4.49 mmol) in dry DCM (8 mL) at
room temperature. Then, after no gas evolution was observed, the
mixture thus obtained was added dropwise to a stirred solution of
piperazine (1.93 g, 22.44 mmol) in dry DCM (10 mL) at 0 °C, under
a nitrogen atmosphere. The reaction was carried out for 30 min at 0
°C and for 1 h at room temperature. The mixture was washed with
10% aqueous NaCl solution (4 × 10 mL) and H2O (2 × 10 mL). The
organic layer was dried over anhydrous sodium sulfate and evaporated
to dryness under reduced pressure to obtain 23 (0.82 g, 73.8%) as a
pure yellow oil and used for the next step without further purification.
IR (KBr, selected lines): cm−1 3460, 2936, 1652, 1455, 701. 1H NMR
(200 MHz, DMSO-d6): δ 7.34−7.09 (m, 5H, aromatic), 3.60−2.80
(m, 4H, piperazine), 2.68−2.46 (m, 4H + 2H, piperazine +
NCOCH2CH2), 2.28 (t, J = 7.2 Hz, 2H, CH2CH2C6H5), 1.68−1.48
(m, 4H, CH2CH2CH2CH2). Anal. calcd for C15H22N2O: C, 73.13; H,
9.00; N, 11.37. Found: C, 73.00; H, 9.15; N, 11.17.
1-(4-{[4-(Hydroxymethyl)phenyl]methyl}piperazin-1-yl)-5-
phenylpentan-1-one (24). A mixture of compound 16 (0.765 g,
2.98 mmol), K2CO3 (0.619 g, 4.48 mmol), a catalytic amount of KI,
and [4-(chloromethyl)phenyl]methanol (0.146 g, 3.58 mmol) in
DCM (2 mL) was placed in a 10 mL Pyrex glass tube, sealed with a
Teflon-coated septum. The mixture was heated and stirred at 120 °C
under microwave irradiations for 2 h (run time 2 min, microwave max
power 150W, max pressure 150 Psi). Subsequently, the reaction
mixture was washed with 10% aqueous NaCl solution (4 × 10 mL)
and H2O (2 × 10 mL). The organic layer was dried over anhydrous
sodium sulfate and evaporated to dryness under reduced pressure to
obtain a yellow oil. The purification of the crude product was
performed by flash column chromatography using ethyl acetate/
methanol (9:1, v/v) as an eluent to afford 24 (0.54 g, 91.7%) as a
light yellow oil. IR (KBr, selected lines): cm−1 3420, 3024, 2933,
1636, 1458, 1346, 1231, 1000, 701. 1H NMR (200 MHz, DMSO-d6):
δ 7.35−7.10 (m, 5H + 4H, aromatic), 5.16 (t, J = 5.7 Hz, 1H,
CH2OH), 4.47 (d, J = 5.7 Hz, 2H, CH2OH), 3.50−3.15 (m, 4H,
piperazine), 3.44 (s, 2H, ArCH2N), 2.57 (t, J = 7.2 Hz, 2H,
NCOCH2CH2), 2.40−2.10 (m, 2H + 4H, CH2CH2C6H5
+
Control or test compounds were administered (i.p.), and paw-
withdrawal thresholds were again recorded from 20 to 80 min
postinjection. Each hind paw was tested in a counterbalanced manner.
Each time was measured in triplicate and then averaged.
piperazine), 1.70−1.35 (m, 4H, CH2CH2CH2CH2). 13C NMR (126
MHz, DMSO-d6): δ 170.4, 142.1, 141.3, 136.1, 128.7, 128.3, 128.2,
126.4, 125.6, 62.7, 61.7, 52.9, 52.3, 44.9, 41.0, 34.9, 32.1, 30.5, 24.4.
2009
https://doi.org/10.1021/acschemneuro.1c00106
ACS Chem. Neurosci. 2021, 12, 2003−2012

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Responsiveness was a clear withdrawal, shaking, or licking of the paw.
To account for variability between mice, data are presented as the
percent of baseline paw withdrawal thresholds following filament
stimulation of the ipsilateral hind paw. The following equation was
used: % anti-allodynia = 100 × ([mean paw withdrawal force {g} in
control group − paw withdrawal force {g} of each mouse]/mean paw
withdrawal force [g] in control group).
Orazio Prezzavento − Department of Drug and Health
Sciences, University of Catania, 95125 Catania, Italy;
orcid.org/0000-0002-3521-264X
Jay P. McLaughlin − Department of Pharmacodynamics,
College of Pharmacy, University of Florida, Gainesville,
Florida 32610, United States
Rotarod Assay. The rotarod coordination assay was used to assess
effects on evoked locomotor activity in C57BL/6J mice administered
vehicle (saline, i.p.), morphine (10 mg/kg, i.p.), U50,488 (10 mg/kg,
i.p.), CM304 (45 mg/kg, i.p.), or compound 15 (30−60 mg/kg, i.p.)
using methods described previously.68,69 Seven habituation trials were
performed where the last habituation trial was used as an initial
baseline of performance. The mice were administered (i.p.) test
agents and then evaluated every 10 min in accelerated speed trials
(180 s max latency at 0−20 rpm) over a 60 min period. The latency
to fall was measured in seconds. Data are reported as the mean
percent change from each mouse’s initial baseline latency to fall.
Decreased latencies to fall in the rotarod test indicate impaired motor
coordination or sedation
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.1c00106
Author Contributions
G.R., J.P.M., and S.I. participated in research design. G.R., F.B.,
and V.P. synthesized, purified, and characterized all com-
pounds. E.A. conducted in vitro binding experiments. L.L.W.
conducted in vivo pharmacology experiments. M.N.M. and L.S.
contributed reagents, materials, and analysis tools. M.N.M.,
O.P., L.W., and J.P.M. performed data analysis. S.I. wrote the
original draft. G.R., L.L.W., J.P.M., and S.I. wrote and
contributed to the writing of the manuscript. All authors
reviewed and approved the final version of the manuscript.
Statistical Analysis. All data are presented as mean
SEM.
Significance is indicated as *p < 0.05 and was analyzed using two-way
ANOVA with Tukey’s post hoc analysis. Statistical analysis was
performed with the use of GraphPad Prism 9.0 software. Dose
response lines were analyzed by linear or nonlinear regression
modeling and ED50 values (dose yielding 50% effect) along with 95%
confidence limits using each individual data points. The rotarod data
are expressed as the % change from baseline performance for each
animal’s baseline response.
Funding
This work was supported, in part, by grants from PON R&I
funds 2014−2020, CUP: E66C18001320007, AIM1872330,
activity 1 (S.I.) and from the Department of Defense CMRMP
PR161310/P1 (J.P.M.). Opinions, interpretations, conclusions,
and recommendations are those of the authors and are not
necessarily endorsed by the Department of Defense.
ASSOCIATED CONTENT
Notes
■
sı
The authors declare no competing financial interest.
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.1c00106.
ACKNOWLEDGMENTS
■
Figures of 1H and 13C NMR spectra of compounds 13−
16, 20−22, 23, and 24 (PDF)
The authors would like to thank Dr. Christopher R. McCurdy
for providing the selective σ1R antagonist CM304.
REFERENCES
■
AUTHOR INFORMATION
■
(1) Gilbert, P. E., and Martin, W. R. (1976) The effects of morphine
and nalorphine-like drugs in the nondependent, morphine-dependent
and cyclazocine-dependent chronic spinal dog. J. Pharmacol. Exp.
Ther. 198, 66−82.
(2) Vaupel, D. B. (1983) Naltrexone fails to antagonize the sigma
effects of PCP and SKF 10,047 in the dog. Eur. J. Pharmacol. 92, 269−
274.
Corresponding Author
Sebastiano Intagliata − Department of Drug and Health
Sciences, University of Catania, 95125 Catania, Italy;
orcid.org/0000-0002-0201-1745; Phone: +39-095-738-
4053; Email: s.intagliata@unict.it
(3) Hanner, M., Moebius, F. F., Flandorfer, A., Knaus, H. G.,
Striessnig, J., Kempner, E., and Glossmann, H. (1996) Purification,
molecular cloning, and expression of the mammalian sigma1-binding
site. Proc. Natl. Acad. Sci. U. S. A. 93, 8072−8077.
(4) Schmidt, H. R., Zheng, S., Gurpinar, E., Koehl, A., Manglik, A.,
and Kruse, A. C. (2016) Crystal structure of the human σ1 receptor.
Nature 532, 527−530.
(5) Schmidt, H. R., and Kruse, A. C. (2019) The Molecular
Function of σ Receptors: Past, Present, and Future. Trends Pharmacol.
Sci. 40, 636−654.
Authors
Giuseppe Romeo − Department of Drug and Health Sciences,
University of Catania, 95125 Catania, Italy; orcid.org/
0000-0003-2160-4164
Federica Bonanno − Department of Drug and Health
Sciences, University of Catania, 95125 Catania, Italy
Lisa L. Wilson − Department of Pharmacodynamics, College
of Pharmacy, University of Florida, Gainesville, Florida
32610, United States
Emanuela Arena − Department of Drug and Health Sciences,
University of Catania, 95125 Catania, Italy
Maria N. Modica − Department of Drug and Health Sciences,
University of Catania, 95125 Catania, Italy; orcid.org/
0000-0002-6350-931X
(6) Kekuda, R., Prasad, P. D., Fei, Y. J., Leibach, F. H., and
Ganapathy, V. (1996) Cloning and functional expression of the
human type 1 sigma receptor (hSigmaR1). Biochem. Biophys. Res.
Commun. 229, 553−5588.
(7) Bowen, W. D., Hellewell, S. B., and McGarry, K. A. (1989)
Evidence for a multi-site model of the rat brain sigma receptor. Eur. J.
Pharmacol. 163, 309−318.
̀
Valeria Pittala − Department of Drug and Health Sciences,
University of Catania, 95125 Catania, Italy; orcid.org/
0000-0003-1856-0308
(8) Hellewell, S. B., and Bowen, W. D. (1990) A sigma-like binding
site in rat pheochromocytoma (PC12) cells: decreased affinity for
(+)-benzomorphans and lower molecular weight suggest a different
sigma receptor form from that of guinea pig brain. Brain Res. 527,
244−253.
Loredana Salerno − Department of Drug and Health Sciences,
University of Catania, 95125 Catania, Italy; orcid.org/
0000-0001-6458-3717
2010
https://doi.org/10.1021/acschemneuro.1c00106
ACS Chem. Neurosci. 2021, 12, 2003−2012

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
(9) Alon, A., Schmidt, H. R., Wood, M. D., Sahn, J. J., Martin, S. F.,
and Kruse, A. C. (2017) Identification of the gene that codes for the
σ(2) receptor. Proc. Natl. Acad. Sci. U. S. A. 114, 7160−7165.
(10) Xu, J., Zeng, C., Chu, W., Pan, F., Rothfuss, J. M., Zhang, F.,
Tu, Z., Zhou, D., Zeng, D., Vangveravong, S. H., et al. (2011)
Identification of the PGRMC1 protein complex as the putative sigma-
2 receptor binding site. Nat. Commun. 2, 380.
(11) Riad, A., Zeng, C., Weng, C. C., Winters, H., Xu, K., Makvandi,
M., Metz, T., Carlin, S., and Mach, R. H. (2018) Sigma-2 Receptor/
TMEM97 and PGRMC-1 Increase the rate of internalization of LDL
by LDL receptor through the formation of a ternary complex. Sci. Rep.
8, 16845.
L., et al. (2020) A SARS-CoV-2 protein interaction map reveals
targets for drug repurposing. Nature 583, 459−468.
(27) Gordon, D. E., Hiatt, J., Bouhaddou, M., Rezelj, V. V., Ulferts,
S., Braberg, H., Jureka, A. S., Obernier, K., Guo, J. Z., Batra, J., et al.
(2020) Comparative host-coronavirus protein interaction networks
reveal pan-viral disease mechanisms. Science 370, No. eabe9403.
(28) Agha, H., and McCurdy, C. R. (2021) In vitro and in vivo
sigma 1 receptor imaging studies in different disease states. RSC Med.
Chem. 12, 154−177.
(29) Zeng, C., Riad, A., and Mach, R. H. (2020) The biological
function of sigma-2 receptor/TMEM97 and its utility in PET imaging
studies in cancer. Cancers 12, 1877.
(12) Chu, U. B., Mavlyutov, T. A., Chu, M. L., Yang, H., Schulman,
A., Mesangeau, C., McCurdy, C. R., Guo, L. W., and Ruoho, A. E.
(2015) The sigma-2 receptor and progesterone receptor membrane
component 1 are different binding sites derived from independent
genes. EBioMedicine 2, 1806−1813.
(30) Volz, H. P., and Stoll, K. D. (2004) Clinical trials with sigma
ligands. Pharmacopsychiatry 37 (Suppl 3), 214−220.
(31) Penke, B., Fulop, L., Szucs, M., and Frecska, E. (2017) The role
of sigma-1 receptor, an intracellular chaperone in neurodegenerative
diseases. Curr. Neuropharmacol. 16 (1), 97−116.
(13) Vilner, B. J., John, C. S., and Bowen, W. D. (1995) Sigma-1 and
sigma-2 receptors are expressed in a wide variety of human and rodent
tumor cell lines. Cancer Res. 55, 408−413.
(32) Hayashi, T., and Su, T. P. (2007) Sigma-1 receptor chaperones
at the ER-mitochondrion interface regulate Ca(2+) signaling and cell
survival. Cell 131, 596−610.
(14) Cobos, E. J., Entrena, J. M., Nieto, F. R., Cendán, C. M., and
Pozo, E. (2008) Pharmacology and therapeutic potential of sigma(1)
receptor ligands. Curr. Neuropharmacol. 6, 344−366.
(33) Rodríguez-Munoz, M., Sánchez-Blázquez, P., Herrero-
̃
Labrador, R., Martínez-Murillo, R., Merlos, M., Vela, J. M., and
Garzón, J. (2015) The σ1 receptor engages the redox-regulated
HINT1 protein to bring opioid analgesia under NMDA receptor
negative control. Antioxid. Redox Signaling 22, 799−818.
(15) Sánchez-Fernández, C., Entrena, J. M., Baeyens, J. M., and
Cobos, E. J. (2017) Sigma-1 Receptor antagonists: a new class of
neuromodulatory analgesics. Adv. Exp. Med. Biol. 964, 109−132.
(34) Díaz, J. L., Cuberes, R., Berrocal, J., Contijoch, M., Christmann,
U., Fernández, A., Port, A., Holenz, J., Buschmann, H., Laggner, C.,
et al. (2012) Synthesis and biological evaluation of the 1-arylpyrazole
class of σ(1) receptor antagonists: identification of 4-{2-[5-methyl-1-
(naphthalen-2-yl)-1H-pyrazol-3-yloxy]ethyl}morpholine (S1RA, E-
52862). J. Med. Chem. 55, 8211−8224.
(35) Cirino, T. J., Eans, S. O., Medina, J. M., Wilson, L. L.,
Mottinelli, M., Intagliata, S., McCurdy, C. R., and McLaughlin, J. P.
(2019) Characterization of sigma 1 receptor antagonist CM-304 and
its analog, AZ-66: novel therapeutics against allodynia and induced
pain. Front. Pharmacol. 10, 678.
́
(16) Sánchez-Fernández, C., Montilla-García, A., González-Cano, R.,
Nieto, F. R., Romero, L., Artacho-Cordón, A., Montes, R., Fernández-
Pastor, B., Merlos, M., Baeyens, J. M., Entrena, J. M., and Cobos, E. J.
(2014) Modulation of peripheral μ-opioid analgesia by σ1 receptors. J.
Pharmacol. Exp. Ther. 348, 32−45.
(17) Bravo-Caparrós, I., Ruiz-Cantero, M. C., Perazzoli, G., Cronin,
S. J. F., Vela, J. M., Hamed, M. F., Penninger, J. M., Baeyens, J. M.,
Cobos, E. J., and Nieto, F. R. (2020) Sigma-1 receptors control
neuropathic pain and macrophage infiltration into the dorsal root
ganglion after peripheral nerve injury. FASEB J. 34, 5951−5966.
(18) Rousseaux, C. G., and Greene, S. F. (2016) Sigma receptors
[σRs]: biology in normal and diseased states. J. Recept. Signal
Transduction Res. 36, 327−388.
(19) Sambo, D. O., Lebowitz, J. J., and Khoshbouei, H. (2018) The
sigma-1 receptor as a regulator of dopamine neurotransmission: a
potential therapeutic target for methamphetamine addiction.
Pharmacol. Ther. 186, 152−167.
(20) Huang, Y. S., Lu, H. L., Zhang, L. J., and Wu, Z. (2014) Sigma-
2 receptor ligands and their perspectives in cancer diagnosis and
therapy. Med. Res. Rev. 34, 532−566.
(21) Jia, J., Cheng, J., Wang, C., and Zhen, X. (2018) Sigma-1
receptor-modulated neuroinflammation in neurological diseases.
Front. Cell. Neurosci. 12, 314.
(22) Merlos, M., Romero, L., Zamanillo, D., Plata-Salamán, C., and
Vela, J. M. (2017) Sigma-1 receptor and pain. Handb. Exp. Pharmacol.
244, 131−161.
(23) Intagliata, S., Alsharif, W. F., Mesangeau, C., Fazio, N.,
Seminerio, M., Xu, Y.-T., Matsumoto, R. R., and McCurdy, C. R.
(2019) Benzimidazolone-based selective σ2 receptor ligands: Syn-
thesis and pharmacological evaluation. Eur. J. Med. Chem. 165, 250−
257.
(24) van Waarde, A., Rybczynska, A. A., Ramakrishnan, N. K.,
Ishiwata, K., Elsinga, P. H., and Dierckx, R. A. (2015) Potential
applications for sigma receptor ligands in cancer diagnosis and
therapy. Biochim. Biophys. Acta, Biomembr. 1848, 2703−2714.
(25) Nicholson, H. E., Alsharif, W. F., Comeau, A. B., Mesangeau,
C., Intagliata, S., Mottinelli, M., McCurdy, C. R., and Bowen, W. D.
(2019) Divergent cytotoxic and metabolically stimulative functions of
sigma-2 receptors: structure-activity relationships of 6-acetyl-3-(4-(4-
(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one
(SN79) derivatives. J. Pharmacol. Exp. Ther. 368, 272−281.
(26) Gordon, D. E., Jang, G. M., Bouhaddou, M., Xu, J., Obernier,
K., White, K. M., O’Meara, M. J., Rezelj, V. V., Guo, J. Z., Swaney, D.
(36) Déciga-Campos, M., Melo-Hernández, L. A., Torres-Gómez,
H., Wu
Juárez, J., López-Mun
̈
nsch, B., Schepmann, D., González-Trujano, M. E., Espinosa-
oz, F. J., and Navarrete-Vázquez, G. (2020)
̃
Design and synthesis of N-(benzylpiperidinyl)-4-fluorobenzamide: a
haloperidol analog that reduces neuropathic nociception via σ(1)
receptor antagonism. Life Sci. 245, 117348.
(37) Intagliata, S., Sharma, A., King, T. I., Mesangeau, C., Seminerio,
M., Chin, F. T., Wilson, L. L., Matsumoto, R. R., McLaughlin, J. P.,
Avery, B. A., and McCurdy, C. R. (2020) Discovery of a highly
selective sigma-2 receptor ligand, 1-(4-(6,7-dimethoxy-3,4-dihydroi-
soquinolin-2(1H)-yl)butyl)-3-methyl-1H-benzo[d]imidazol-2(3H)-
one (CM398), with drug-like properties and antinociceptive effects in
vivo. AAPS J. 22, 94.
(38) Sahn, J. J., Mejia, G. L., Ray, P. R., Martin, S. F., and Price, T. J.
(2017) Sigma 2 receptor/Tmem97 agonists produce long lasting
antineuropathic pain effects in mice. ACS Chem. Neurosci. 8, 1801−
1811.
̀
(39) Romeo, G., Prezzavento, O., Intagliata, S., Pittala, V., Modica,
M. N., Marrazzo, A., Turnaturi, R., Parenti, C., Chiechio, S., Arena, E.,
et al. (2019) Synthesis, in vitro and in vivo characterization of new
benzoxazole and benzothiazole-based sigma receptor ligands. Eur. J.
Med. Chem. 174, 226−235.
(40) Bergkemper, M., Kronenberg, E., Thum, S., Börgel, F., Daniliuc,
C., Schepmann, D., Nieto, F. R., Brust, P., Reinoso, R. F., Alvarez, I.,
̈
and Wunsch, B. (2018) Synthesis, receptor affinity, and antiallodynic
activity of spirocyclic σ receptor ligands with exocyclic amino moiety.
J. Med. Chem. 61, 9666−9690.
(41) Prezzavento, O., Arena, E., Sánchez-Fernández, C., Turnaturi,
R., Parenti, C., Marrazzo, A., Catalano, R., Amata, E., Pasquinucci, L.,
and Cobos, E. J. (2017) (+)-and (−)-Phenazocine enantiomers:
Evaluation of their dual opioid agonist/σ(1) antagonist properties and
antinociceptive effects. Eur. J. Med. Chem. 125, 603−610.
2011
https://doi.org/10.1021/acschemneuro.1c00106
ACS Chem. Neurosci. 2021, 12, 2003−2012

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
(42) Intagliata, S., Agha, H., Kopajtic, T. A., Katz, J. L., Kamble, S.
H., Sharma, A., Avery, B. A., and McCurdy, C. R. (2020) Exploring 1-
adamantanamine as an alternative amine moiety for metabolically
labile azepane ring in newly synthesized benzo[d]thiazol-2(3H)one σ
receptor ligands. Med. Chem. Res. 29, 1697−1706.
(43) Popa, R., Kamble, S. H., Kanumuri, R. S., King, T. I., Berthold,
E. C., Intagliata, S., Sharma, A., and McCurdy, C. R. (2020)
Bioanalytical method development and pharmacokinetics of MCI-92,
a sigma-1 receptor ligand. J. Pharm. Biomed. Anal. 191, 113610.
(59) Mach, R. H., Smith, C. R., and Childers, S. R. (1995) Ibogaine
possesses a selective affinity for σ2 receptors. Life Sci. 57, PL57−PL62.
(60) Mogil, J. S., Kest, B., Sadowski, B., and Belknap, J. K. (1996)
Differential genetic mediation of sensitivity to morphine in genetic
models of opiate antinociception: influence of nociceptive assay. J.
Pharmacol. Exp. Ther. 276 (2), 532−544.
(61) Wilson, S. G., Smith, S. B., Chesler, E. J., Melton, K. A., Haas, J.
J., Mitton, B., Strasburg, K., Hubert, L., Rodriguez-Zas, S. L., and
Mogil, J. S. (2003) The heritability of antinociception: common
pharmacogenetic mediation of five neurochemically distinct analge-
sics. J. Pharmacol. Exp. Ther. 304, 547−559.
(62) Mogil, J. S., Smith, S. B., O’Reilly, M. K., and Plourde, G.
(2005) Influence of nociception and stress-induced antinociception
on genetic variation in isoflurane anesthetic potency among mouse
strains. Anesthesiology 103, 751−8.
(63) LaCroix-Fralish, M. L., Rutkowski, M. D., Weinstein, J. N.,
Mogil, J. S., and Deleo, J. A. (2005) The magnitude of mechanical
allodynia in a rodent model of lumbar radiculopathy is dependent on
strain and sex. Spine 30, 1821−1827.
(64) Feehan, A. K., Morgenweck, J., Zhang, X., Amgott-Kwan, A. T.,
and Zadina, J. E. (2017) Novel endomorphin analogs are more potent
and longer-lasting analgesics in neuropathic, inflammatory, post-
operative, and visceral pain relative to morphine. J. Pain 18, 1526−
1541.
(65) Kilkenny, C., Browne, W. J., Cuthill, I., Emerson, M., and
Altman, D. G. (2010) Improving bioscience research reporting: the
ARRIVE guidelines for reporting animal research. J. Pharmacol.
Pharmacother. 1 (2), 94−99.
̀
(44) Arena, E., Cacciatore, I., Cerasa, L. S., Turkez, H., Pittala, V.,
Pasquinucci, L., Marrazzo, A., Parenti, C., Di Stefano, A., and
Prezzavento, O. (2016) New bifunctional antioxidant/σ1 agonist
ligands: Preliminary chemico-physical and biological evaluation.
Bioorg. Med. Chem. 24, 3149−56.
(45) Glennon, R. A., Ablordeppey, S. Y., Ismaiel, A. M., el-Ashmawy,
M. B., Fischer, J. B., and Howie, K. B. (1994) Structural features
important for sigma 1 receptor binding. J. Med. Chem. 37, 1214−1219.
(46) Cendán, C. M., Pujalte, J. M., Portillo-Salido, E., Montoliu, L.,
and Baeyens, J. M. (2005) Formalin-induced pain is reduced in
sigma(1) receptor knockout mice. Eur. J. Pharmacol. 511, 73−74.
(47) Cendán, C. M., Pujalte, J. M., Portillo-Salido, E., and Baeyens, J.
M. (2005) Antinociceptive effects of haloperidol and its metabolites
in the formalin test in mice. Psychopharmacology 182, 485−93.
(48) Vidal-Torres, A., Fernández-Pastor, B., Carceller, A., Vela, J. M.,
Merlos, M., and Zamanillo, D. (2014) Effects of the selective sigma-1
receptor antagonist S1RA on formalin-induced pain behavior and
neurotransmitter release in the spinal cord in rats. J. Neurochem. 129,
484−94.
(66) Hoot, M. R., Sim-Selley, L. J., Poklis, J. L., Abdullah, R. A.,
Scoggins, K. L., Selley, D. E., and Dewey, W. L. (2010) Chronic
constriction injury reduces cannabinoid receptor 1 activity in the
rostral anterior cingulate cortex of mice. Brain Res. 1339, 18−25.
(67) Ahmad, N., Subhan, F., Islam, N. U., Shahid, M., Rahman, F.
U., and Sewell, R. D. E. (2017) Gabapentin and its salicylaldehyde
derivative alleviate allodynia and hypoalgesia in a cisplatin-induced
neuropathic pain model. Eur. J. Pharmacol. 814, 302−312.
(68) Eans, S. O., Ganno, M. L., Mizrachi, E., Houghten, R. A.,
Dooley, C. T., McLaughlin, J. P., and Nefzi, A. (2015) Parallel
synthesis of hexahydrodiimidazodiazepines heterocyclic peptidomi-
metics and their in vitro and in vivo activities at μ (MOR), δ (DOR),
and κ (KOR) opioid receptors. J. Med. Chem. 58, 4905−4917.
(69) Reilley, K. J., Giulianotti, M., Dooley, C. T., Nefzi, A.,
McLaughlin, J. P., and Houghten, R. A. (2010) Identification of two
novel, potent, low-liability antinociceptive compounds from the direct
in vivo screening of a large mixture-based combinatorial library. AAPS
J. 12, 318−329.
(49) Romero, L., Zamanillo, D., Nadal, X., Sánchez-Arroyos, R.,
Rivera-Arconada, I., Dordal, A., Montero, A., Muro, A., Bura, A.,
Segalés, C., et al. (2012) Pharmacological properties of S1RA, a new
sigma-1 receptor antagonist that inhibits neuropathic pain and
activity-induced spinal sensitization. Br. J. Pharmacol. 166, 2289−306.
(50) Gris, G., Merlos, M., Vela, J. M., Zamanillo, D., and Portillo-
Salido, E. (2014) S1RA, a selective sigma-1 receptor antagonist,
inhibits inflammatory pain in the carrageenan and complete Freund’s
adjuvant models in mice. Behav. Pharmacol. 25, 226−235.
(51) Bennett, G. J., and Xie, Y. K. (1988) A peripheral
mononeuropathy in rat that produces disorders of pain sensation
like those seen in man. Pain 33, 87−107.
(52) Pitcher, G. M., Ritchie, J., and Henry, J. L. (1999) Nerve
constriction in the rat: model of neuropathic, surgical and central
pain. Pain 83, 37−46.
(53) Cheng, H. Y., Pitcher, G. M., Laviolette, S. R., Whishaw, I. Q.,
Tong, K. I., Kockeritz, L. K., Wada, T., Joza, N. A., Crackower, M.,
Goncalves, J., et al. (2002) DREAM is a critical transcriptional
repressor for pain modulation. Cell 108, 31−43.
(54) Xu, M., Petraschka, M., McLaughlin, J. P., Westenbroek, R. E.,
Caron, M. G., Lefkowitz, R. J., Czyzyk, T. A., Pintar, J. E., Terman, G.
W., and Chavkin, C. (2004) Neuropathic pain activates the
endogenous kappa opioid system in mouse spinal cord and induces
opioid receptor tolerance. J. Neurosci. 24, 4576−4584.
(55) Roh, D. H., Kim, H. W., Yoon, S. Y., Seo, H. S., Kwon, Y. B.,
Kim, K. W., Han, H. J., Beitz, A. J., Na, H. S., and Lee, J. H. (2008)
Intrathecal injection of the sigma(1) receptor antagonist BD1047
blocks both mechanical allodynia and increases in spinal NR1
expression during the induction phase of rodent neuropathic pain.
Anesthesiology 109, 879−89.
(56) Bravo-Caparrós, I., Perazzoli, G., Yeste, S., Cikes, D., Baeyens, J.
M., Cobos, E. J., and Nieto, F. R. (2019) Sigma-1 receptor inhibition
reduces neuropathic pain induced by partial sciatic nerve transection
in mice by opioid-dependent and -independent mechanisms. Front.
Pharmacol. 10, 613.
(57) Evoy, K. E., Morrison, M. D., and Saklad, S. R. (2017) Abuse
and misuse of pregabalin and gabapentin. Drugs 77, 403−426.
(58) DeHaven-Hudkins, D. L., Fleissner, L. C., and Ford-Rice, F. Y.
(1992) Characterization of the binding of [3H](+)-pentazocine to σ
recognition sites in guinea pig brain. Eur. J. Pharmacol., Mol.
Pharmacol. Sect. 227, 371−378.
2012
https://doi.org/10.1021/acschemneuro.1c00106
ACS Chem. Neurosci. 2021, 12, 2003−2012