Novel highly potent and selective sigma1 receptor antagonists effectively block the binge eating episode in female rats

Article abstract of DOI:10.1021/acschemneuro.0c00456

In this paper, the benzo-cracking approach was applied to the potent sigma1 (σ1) receptor antagonist 1 to afford the less conformationally constrained 1,3-dioxane derivatives 2 and 3. To evaluate the effect of the increase in the distance between the two hydrophobic structural elements that flank the basic function, the cis and trans diastereomers of 4 and 5 were also prepared and studied. Compounds 2 and 3 showed affinity values at the σ1 receptor significantly higher than that of the lead compound 1. In particular, 3 displayed unprecedented selectivity over the σ2 receptor, the phencyclidine site of the NMDA receptor, and opioid receptor subtypes, as well as over the dopamine transporter. Docking results supported the structure-activity relationship studies. Due to its interesting biological profile, derivative 3, selected for an in vivo study in a validated preclinical model of binge eating, was able to counteract the overeating of palatable food only in binging rats, without affecting palatable food intake in the control group and anxiety-like and depression-related behaviors in female rats. This result strengthened the involvement of the σ1 receptor in the compulsive-like eating behavior and supported the σ1 receptor as a promising target for the management of eating disorders.

Full text of DOI:10.1021/acschemneuro.0c00456

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Article
Novel Highly Potent and Selective Sigma1 Receptor Antagonists
Effectively Blocking the Binge Eating Episode in Female Rats
Carlo Cifani, Emanuela Micioni Di Bonaventura, Luca Botticelli, Fabio Del Bello, Gianfabio
Giorgioni, Pegi Pavleti#, Alessandro Piergentili, Wilma Quaglia, Alessandro Bonifazi, Dirk
Schepmann, Bernhard Wünsch, Giulio Vistoli, and Maria Vittoria Micioni Di Bonaventura
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.0c00456 • Publication Date (Web): 04 Sep 2020
Downloaded from pubs.acs.org on September 5, 2020
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Novel Highly Potent and Selective Sigma1 Receptor Antagonists Effectively Blocking the Binge
Eating Episode in Female Rats
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Carlo Cifani,§ Emanuela Micioni Di Bonaventura,§ Luca Botticelli,§ Fabio Del Bello,*,† Gianfabio
Giorgioni,† Pegi Pavletić,† Alessandro Piergentili,† Wilma Quaglia,*,† Alessandro Bonifazi,†,‡ Dirk
Schepmann,⊥ Bernhard Wünsch,⊥ Giulio Vistoli,∥ and Maria Vittoria Micioni Di Bonaventura §
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§School of Pharmacy, Pharmacology Unit, University of Camerino, Via Madonna delle Carceri 9,
62032 Camerino, Italy
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†School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Via S. Agostino 1, 62032
Camerino, Italy
‡Current Address: Medicinal Chemistry Section, Molecular Targets and Medications Discovery
Branch, National Institute on Drug Abuse – Intramural Research Program, National Institutes of
Health, Baltimore, Maryland, 333 Cassell Drive, Baltimore, Maryland 21224
⊥Institut für Pharmazeutische und Medizinische Chemie, Universität Münster, Corrensstraße 48,
48149 Münster, Germany
∥Department of Pharmaceutical Sciences, University of Milan, Via Mangiagalli 25, 20133 Milano,
Italy
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ABSTRACT
In this paper the benzo-cracking approach was applied to the potent sigma1 (σ1) receptor antagonist
1 to afford the less conformationally constrained 1,3-dioxane derivatives 2 and 3. To evaluate the
effect of the increase of the distance between the two hydrophobic structural elements that flank the
basic function, the cis and trans diastereomers of 4 and 5 were also prepared and studied. Compounds
2 and 3 showed affinity values at σ1 receptor significantly higher than that of the lead compound 1.
In particular, 3 displayed unprecedented selectivity over σ2 receptor, the phencyclidine site of the
NMDA receptor, and opioid receptor subtypes, as well as over dopamine transporter. Docking results
supported the structure-activity relationship studies. Due to its interesting biological profile,
derivative 3, selected for an in vivo study in a validated preclinical model of binge eating, was able
to counteract the overeating of palatable food only in binging-rats, without affecting palatable food
intake in the control group and anxiety-like and depression-related behaviors in female rats. This
result strengthened the involvement of σ1 receptor in the compulsive-like eating behavior and
supported σ1 receptor as a promising target for the management of eating disorders.
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Keywords: selective sigma1 ligands, binge eating episode, highly palatable food, open field test,
forced swimming test.
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INTRODUCTION
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Sigma (σ) receptors are scarcely understood transmembrane proteins involved in a large number of
cellular functions.1 Initially, they were classified as subtypes of the opioid receptor family and,
subsequently, it was hypothesized that they corresponded to the phencyclidine (PCP) binding site of
the ionotropic N-methyl-D-aspartate (NMDA) receptor. At present, they are reported as a distinctive
receptor family, comprised of two subtypes (σ1 and σ2 receptors).1 Both subtypes have been cloned2-5
and the crystal structures of σ1 receptor complexed with known agonists and antagonists have recently
been reported.6, 7 σ1 receptors work as molecular chaperones in the mitochondria-associated
endoplasmic reticulum (ER) membrane and play a role in the cellular stress response and
homeostasis.8, 9
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Their wide distribution in the nervous system and their involvement in several physiological and
pathological conditions make σ1 receptors very promising targets for the management of numerous
disorders. In particular, central σ1 receptors are implicated in different neuropsychiatric and
neurodegenerative diseases,10-12 as well as in pain.13 The observation that the σ1 agonist ANAVEX™
(NCT02244541) and the σ1 antagonist E-52862 (EudraCT number: 2012-000400-14) are being
evaluated in clinical trials for the treatment of Alzheimer's disease and neuropathic pain, respectively,
supports the validity of σ1 receptors as clinical targets.14 Moreover, experimental evidence has
demonstrated that the blockade of σ1 receptors can counteract the addictive effects elicited by
psychostimulants15, 16 and ethanol.17-20 While several papers report the involvement of σ1 receptors
in drug abuse, very few studies suggest that this receptor system is implicated in binge eating
behavior, despite many behavioral and brain mechanisms overlapping between food and drug
addiction. In fact, compulsive fast overeating and strong craving, with a consequent withdrawal for
hedonic food and impulsivity, are features correlated with binge eating behavior, similarly to the
substance dependence.21, 22 In a pioneering study, the σ1 antagonist BD-1063 (Figure 1) proved to
reduce binge-like eating and to block compulsive eating in palatable rats, suggesting that the σ1
receptor system might play a role in binge-like eating following to neurobiological adaptations.23
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Moreover, a relationship between food reinforced operant responding and σ1 receptors has been
recently highlighted. Indeed, the potent σ1 antagonist PD144418 (Figure 1) demonstrated to decrease
the motivational effort of a food-reinforced behavior maintaining food palatability.24 Finally, in a
recent study we demonstrated that the spipethiane analog 2-(1-benzylpiperidin-4-yl)thiochroman-4-
one (Figure 1), behaving as a potent σ1 receptor antagonist,25 decreased the binge eating episode in
female rats, supporting the involvement of σ1 receptors in compulsive-like eating disorder.26
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N
CH3
O
N
H3C
CH3
Cl
Cl
N
N
BD-1063
PD144418
O
S
S
N
N
Spipethiane
2-(1-Benzylpiperidin-4-yl)thiochroman-4-one
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Figure 1. Structures of the σ1 antagonists BD-1063, PD144418, spipethiane and 2-(1-
benzylpiperidin-4-yl)thiochroman-4-one.
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Among the analogs of spipethiane, another potent σ1 receptor ligand (pKi = 10.05), endowed with
high σ1/σ2 selectivity (2515), is the 1,3-benzodioxane derivative 1 (Figure 2). Functional assays
performed on MCF-7 and MCF-7/ADR highlighted the σ1 antagonist profile of this compound.25
With the aim to improve the σ1 receptor affinity and selectivity over σ2 subtype, the conformationally
constrained 1,3-benzodioxane moiety of 1 was replaced by the more flexible 1,3-dioxane nucleus by
benzo-cracking approach.27 In particular, derivatives 2 and 3, in which the phenyl substituent is linked
to the positions 4 or 5 of the 1,3-dioxane ring, respectively, were prepared and studied (Figure 2).
Moreover, to evaluate the effect of the distance between the two hydrophobic portions that flank the
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basic function of 2 and 3, the diastereomers 4a/b and 5a/b were also prepared and studied. In these
novel derivatives, the N-benzylpiperidine moiety is spaced from the 1,3-dioxane ring (Figure 2),
resulting in a further increase in the conformational flexibility of the molecule. The separation of the
cis and trans diastereomers of 4 and 5 permitted us to evaluate the role played by the relative
configuration on the σ1 receptor affinity.
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(cis)
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(cis)
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Figure 2. Structures of 2-5, analogs of the potent σ1 ligand 1.
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The novel derivatives 2-5 were tested by radioligand binding assays at σ1 and σ2 receptors. Moreover,
to confirm the involvement of the σ1 receptor system in binge-like eating disorder, the aim of this
work was also the evaluation of the most interesting compound 3 in a female rat model of binge
eating. Finally, the affinities of compounds 2 and 3 were also assessed at the PCP site of the NMDA
receptor, opioid receptors and/or dopamine transporter (DAT), all of which playing a role in binge
eating disorders,28 considering that many σ1 ligands also bind these targets with high affinity.
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RESULTS AND DISCUSSION
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Derivatives 2-5 were synthesized following the synthetic route reported in Scheme 1.
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b)
1
: R = C6H5, R2 = H (71% yield)
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3
: R = H, R = C6H5 (70% yield)
O
O
OH OH
R2
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R2
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: R = C6H5, R2 = H
: R = OCH2CH3, R2 = C6H5
: R = C6H5, R2 = H (69% yield)
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: R = H, R = C6H5 (72% yield)
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(trans): R = C6H5, R2 = H (15% yield)
(cis): R = C6H5, R2 = H (48% yield)
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(trans): R = H, R = C6H5 (44% yield)
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(cis): R = H, R = C6H5 (24% yield)
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Scheme 1. a) LiAlH4, Et2O, r.t. for 2 h b) p-toluenesulfonic acid, toluene, reflux for 5 h.
4
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The commercially available ethyl 3-oxo-3-phenylpropionate (6) and diethyl 2-phenylmalonate (7)
were subjected to a reduction reaction with LiAlH4 to the corresponding diols 8 and 9, respectively.
The condensation of 8 and 9 with the suitable N-benzylpiperidine carbonyl derivatives 10 and 11 in
presence of p-toluenesulfonic acid afforded the desired derivatives 2 and 3 and the mixtures of the
diastereomers 4a/b and 5a/b, respectively. The cis and trans diastereomers of 4 and 5 were separated
by flash chromatography.
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The stereochemical relationship between the N-benzylpiperidine moiety in the position 2 and the
phenyl substituent in the positions 4 or 5 of 4a/b and 5a/b, respectively, was determined by 1H NMR
analysis (NOESY studies). In particular, an evident NOE was observed between the protons in the
positions 2 and 4 (4.48 and 4.65 ppm, respectively) of 4b, highlighting that both the piperidine and
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phenyl rings in the positions 2 and 4, respectively, are equatorially oriented. Therefore, the
stereochemical relationship between the substituents in the positions 2 and 4 is cis in 4b and,
consequently, trans in 4a (Figure 3). Concerning 5a, the axial proton in the position 4 (δ 3.78 ppm)
showed two large coupling constants (J = 10.8 Hz and J = 11.3 Hz), one with the geminal equatorial
proton and the other with the axial proton in the position 5. Consequently, the phenyl ring adopts an
equatorial orientation. Moreover, a clear NOE was observed between the axial protons in the positions
2 and 4 at 4.36 and 3.78 ppm, respectively, evidencing that the N-benzylpiperidine moiety also adopts
an equatorial orientation. Therefore, the relative configuration between the substituents in the
positions 2 and 5 is trans in 5a and, consequently, cis in 5b (Figure 3).
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H
NOE
NOE
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5a
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Figure 3. Structure of compounds 4b and 5a.
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The affinities of compounds 2-5 for σ1 and σ2 receptors were assessed on guinea pig brain and rat
liver membranes, respectively. [3H]-(+)-pentazocine and [3H]-di-o-tolylguanidine in presence of an
excess of (+)-pentazocine were used as radioligands for σ1 and σ2 receptors, respectively.29, 30 The pKi
values are reported in Table 1. The lead compound 1 was included for useful comparison.
Compounds 2 and 3 were also evaluated for their affinity for DAT, the PCP site of the NMDA
receptor as well as µ, κ and δ opioid receptor subtypes. The assays were performed with rat striatal
([3H]-WIN35,428), pig brain cortex ([3H]-(+)-MK-801), guinea pig brain ([3H]-DAMGO), guinea
pig brain ([3H]-U-69593) and rat brain ([3H]-DPDPE) membranes for DAT, NMDA, µ-, κ- and δ-
opioid receptors, respectively.29, 31-33 The pKi values are shown in Table 1.
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Table 1. Affinity values (pKi)a of 1-5 at σ1 and σ2 receptors and of 2 and 3 at DAT, the PCP site of
the NMDA receptor, µ, κ and δ opioid receptor subtypes.
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: R = C6H5, R2 = H
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5a
5b
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(trans): R = C6H5, R2 = H
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4
pKi
Compd
σ1
σ2
DAT
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NMDA
µ
κ
-
δ
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2
10.05±0.08 6.65±0.09
11.00±0.07 6.33±0.11
-
<5
<5
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<5
<5
<5
-
<5
<5
-
8.60±0.14
3
10.89±0.05 6.09±0.07 5.63±0.09
5.82±0.08
4a
4b
5a
5b
8.43±0.07 6.75±0.10
9.62±0.15 7.42±0.08
8.44±0.14 7.25±0.02
8.31±0.06 6.60±0.10
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5
6
7
8
aEquilibrium dissociation constants (Ki) were derived from IC50 values using the Cheng-Prusoff equation.34 The reported
pKi values are the mean ± S.E.M. of three to five independent experiments, each performed in triplicate, according to the
methods described in the S.I.
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The data reported in Table 1 reveal that the benzo-cracking approach performed on the 1,3-
benzodioxane derivative 1 is favorable for the binding to σ1 receptor, while causes a slight reduction
in σ2 receptor affinity, with a consequent increase in σ1/σ2 selectivity. In fact, both compounds 2 and
3 display very high affinity for σ1 receptor and a remarkable σ1/σ2 selectivity. Several potent σ1
ligands belonging to different chemical classes and highly selective over σ2 receptor have been
discovered.35 Interestingly, 3 shows an impressive σ1/σ2 selectivity ratio (σ1/σ2 = 63096) and, to our
knowledge, is the most selective σ1 ligand reported so far. A significant reduction in affinity for σ1
receptor and an increase in those for σ2 are observed when the benzo-cracking approach is combined
with the further increase in the distance between the two lipophilic moieties of 2 and 3 (compounds
4a/b and 5a/b, respectively). Consequently, the σ1/σ2 affinity ratios displayed by 4a/b and 5a/b are
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significantly lower than those of 2 and 3. Probably, the increase in the conformational freedom and
in the distance between the two lipophilic portions is detrimental for the optimal interaction with σ1
receptor. Stereochemistry appears to play a role in the binding to the σ1 receptor when the phenyl ring
is in the position 4 of the 1,3-dioxane nucleus, with the cis isomer 4b showing an affinity value
significantly higher than that of the trans diastereomer 4a. On the contrary, the trans and cis 5-phenyl
diastereomers 5a and 5b show similar affinity values.
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From the results obtained with the off-targets it emerges that ligand 2 shows negligible affinity for
DAT, NMDA, µ and κ opioid receptors, and high affinity for δ subtype (pKi = 8.60), although it is
251-fold lower than that for σ1. Interestingly, compound 3, which also binds δ receptor with sub-
micromolar affinity, displays a remarkable selectivity for σ1 receptor over all the evaluated targets
(σ1/DAT = 181970, σ1/NMDA > 776247, σ1/µ > 776247, σ1/κ > 776247, σ1/δ = 117490). The binding
profile of 3 is noticeable, given that many potent σ1 ligands also bind to DAT, NMDA and/or opioid
receptors with high affinity.1, 36, 37
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To rationalize the affinity profiles of the proposed ligands at the σ1 receptor, docking simulations
were performed based on the resolved σ1 structure (PDB Id: 5HK1) using the PLANTS software and
following the same recently reported computational protocol.26 As discussed below, the complex
stability is evaluated by calculating the APBS score which is focused on the polar interactions.38
Figure 4 compares the computed putative poses for 1 (Figure 4A) and 2 (Figure 4B) and reveals some
differences which can justify the increase of affinity observed for the latter.
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Figure 4. Main interactions stabilizing the putative complexes for 1 (A) e (R)-2 (B) as computed
using the resolved σ1 receptor structure. The reported scores are calculated by using the APBS
method.
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In detail, Figure 4A highlights the key interactions stabilized by 1 which can be schematized as
follows: (a) the ligand ammonium head stabilizes a clear ion-pair with Glu172 reinforced by a H-
bond with Tyr103; (b) the benzyl moiety is inserted within a hydrophobic subpocket where it mostly
contacts alkyl side-chains plus  stacking with Tyr103 and a Sulfur contact with Met93; (c) the
benzodioxane system is accommodated within a subpocket lined by several aromatic residues while
the O1 oxygen atom is engaged by a H-bond with Tyr120. The enantiomers of 2 afford very similar
putative complexes and attention is here focused on the complex for (R)-2 since it shows a slightly
better APBS score compared to (S)-2 (-42.56 vs. -41.38 kJ/mol). Specifically, Figure 4B emphasizes
that (R)-2 elicits an interaction pattern very similar to that already seen in Figure 4A, even though
some key interactions appear to be enhanced when compared to those elicited by 1. This positive
effect can be seen in the contacts stabilized by: (a) the benzyl moiety which elicits an optimized 
stacking with Tyr103; (b) the dioxane oxygen atoms which better approach Tyr120 and (c) the phenyl
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ring which is engaged by an extended set of  stacking interactions with Phe107, Phe133, His154
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and Trp164. These reinforced contacts are reflected into better complex stability as encoded by the
scores displayed in Figure 4A. As also confirmed by APBS score (-38.91 kJ/mol), compound 3 yields
an in-between docking result, with the two aromatic rings being engaged by enhanced contacts, while
the dioxane ring is unable to conveniently approach Tyr120, as seen in Figure 4B. Finally, the
compounds 4 and 5 reveal computed poses rather similar to those observed for the previous ligands,
even though the free dioxane ring assumes a rather different arrangement which hampers its
interactions with Tyr120. The lack of this contact induces flipped poses of the most hindered ligands
by which the dioxane ring approaches Tyr103.
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Considering its intriguing σ1 affinity and selectivity profile, compound 3 was selected for the in vivo
study, using a validated preclinical animal model of binge eating, to further investigate the function
of σ1 receptor system on compulsive-like eating disorder. Female rats were used in relation to the
higher prevalence of binge eating disorder and bulimia nervosa in women than in men.39 In the binge
eating model,40-42 female rats were randomly separated into four groups: non restricted and not
exposed to stress group (NR + NS); non restricted and exposed to stress group (NR + S); restricted
and not exposed to stress group (R + NS); restricted and exposed to stress group (R + S). The
association of three consecutive food restriction/refeeding periods and acute stress is able to trigger a
strong increase of highly palatable food (HPF) consumption only in R + S rats in a short period of
time (120 min). Stress is induced by placing a coffee cup containing HPF for 15 min, letting the
animal see the cup and smell HPF odor, without the possibility to eat it. Thus, on the binge test day,
NR + NS and R + NS had immediate access to HPF for 120 min, whereas NR + S and R + S had free
access to it only after 15 min of stress. This stressful condition, although mild, has already shown to
enhance the corticosterone blood level in stressed rats.43-46 In line with our previous studies,47, 48 the
ANOVA in the vehicle groups revealed a marked interaction among the three factors (food restriction
x stress x sessions time) [Finteraction (3,72) = 4.8; P < 0.01]. Bonferroni post hoc test revealed a
significant (P < 0.01) increase in HPF consumption in the first 15 min in the R + S group (binging
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group), compared to the other three groups. On the other hand, during the time of the other sessions
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(15–30; 30-60; 60-120 min) no change in HPF intake was observed among all groups (Figure 5, left
panel). At the end of the binge eating test (120 min) one-way ANOVA showed a two-way interaction
(food restriction x stress) [Finteraction (1,24) = 4.3; P < 0.05] and the post hoc analyses (P < 0.01)
revealed that only R + S rats significantly enhanced HPF eating with respect to the other rats (Figure
5, right panel). Thus, the stress exposure induced binge-like behavior only in previously restricted
rats, which consumed a large amount of HPF within 15 min and no compensatory changes during the
remaining 15–120 min were detected.
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Acute intraperitoneal (i.p.) injection of 3, 30 min before giving access to HPF, selectively blocked
the episode of binge eating in a dose-dependent manner in the R + S group, without affecting
consumption in the other experimental groups during 120 min of observation (Figure 5B-E).
Specifically in R + S rats, ANOVA reported a significant effect of treatment at 0–15 min [F(2,20) =
6.7; P < 0.05] and in 0–120 min [F(2,20) = 10.9; P < 0.01]. Post hoc analyses indicated that both used
dosages (3 or 7 mg/kg) significantly decreased HPF consumption in R + S at each time point as
indicated in Figure 5E.
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Figure 5. Administration of 3 blocked the episode of binge eating. A. HPF intake shown in kcal/kg
at different sessions time (0-15, 15-30, 30-60, 60-120 min; left) and at 120 min (right) in the vehicle
(veh) injected rats. **P < 0.01; ***P < 0.001 different from the other three groups. B. HPF eating
(kcal/kg) after 15 min (left) or 120 min (right) to free access to cup containing chocolate paste in veh
or treated rats: NR + NS (B, Non Restricted + Non Stressed), NR + S (C, Non Restricted + Stressed),
R + NS (D, Restricted + Non Stressed), R + S (E, Restricted + Stressed) groups. *P < 0.05; ***P <
0.01 vs R + S veh. Data are expressed as mean ± SEM. N = 6-8 per group.
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In addition, to assess if the systemic injection of 3 may influence different aspects of animal behavior
in the control or binging group, the open field (OF) and forced swimming tests (FST) were performed.
The OF test is a validated test, commonly used for evaluating locomotor activity and anxiety-like
behavior in rodents in an unfamiliar environment,49 while FST is a suitable tool for evaluating a
depressed state.50 The administration of 3 showed to not affect any measured behavioral parameters
in these present tests. In fact, analyzing the locomotor activity in the entire OF arena, ANOVA
showed a significant effect of restriction and stress conditions [Frestriction (1,48) = 7.9; P < 0.01; Fstress
(1,48) = 30.9; P < 0.001] and no effect of the treatment with 3 [Ftreatment (1,48) = 0.6; P > 0.05]. R +
S veh and R + S 3 (7 mg/kg) showed the highest distance travelled compared to the other groups
(Table 2).
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Table 2. Behavioral parameters in female rats performing the open field and forced swimming tests
OPEN FIELD TEST
Parameters
NR + NS
NR + S
R + NS
R + S
Veh
3 (7 mg/kg)
2492.3±225.3
93.7±5
Veh
3 (7 mg/kg)
3467.2±411.8
125.9±5.4
Veh
3 (7 mg/kg)
3132.4±331.6
94.3±6.6
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3 (7 mg/kg)
4461.5±346.3*
126.6±4.5
2305.3±357.3
99±4
3602.9±269.3
123.9±6.1
2626.2±242.5
87±3.3
4335.9±419.3*
131.7±4.3
Tot. dist. trav. (cm)
Tot. vert. counts
Jump counts
104.6±4.8
2367.1±116.9
101.2±25.6
26.4±4.6
113±17.1
137.1±2.1
152±32.5
97.7±6.3
119.5±9.1
2516.7±79.2
96.4±6
172±20.6
170.4±19
2500.3±89.3
112.7±7.1
28.1±2.7
2477.4±161.4
140.3±30.1
34.5±4.1
2270.4±137.5
148.1±13.1
34±6.8
2030.5±280.8
84.3±17.1
18.7±2.8
2305.1±79.7
179.9±24.8
50.4±2.4
2401±119.6
177.5±37.5
47.2±10
Stereot. counts
Cent. dist. trav. (cm)
Cent. zone entries
22.5±2.1
FORCED SWIMMING TEST
Parameters
NR + NS
NR + S
R + NS
R + S
Veh
3 (7 mg/kg)
Veh
3 (7 mg/kg)
Veh
3 (7 mg/kg)
Veh
3 (7 mg/kg)
100.4±12.94
106.7±11.2
92.9±13.5
111.1±11.2
99.6±10.4
94.8±9.2
158.6±10*
163.5±22.5*
Immobility time (s)
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In the entire open field arena: Tot. dist. trav. (cm): total distance travelled; Tot. vert. counts: total vertical counts; Jump
counts; Stereot. counts: stereotypic counts. In the central zone of the open field box: Cent. dist. trav. (cm): distance
travelled in the center; Cent. zone entries: number of entrances in the central zone. Data are the mean ± SEM. *p < 0.05
vs the other groups. N = 6-8 per group.
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Regarding the other parameters, jump and total vertical counts were significantly affected only by
stress [Fstress (1,48) = 11.9; P < 0.01] and [Fstress (1,48) = 86.7; P < 0.001], respectively, but not by
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restriction or treatment conditions. As shown in Table 2, the stress procedure appeared to increase
the general arousal and this effect was confirmed by the significant gain in vehicle or treated stressed
rats (NR + S and R + S) on distance travelled in the central zone [Fstress (1,48) = 19.7; P < 0.001] and
on zone entries [Fstress (1,48) = 39.2; P < 0.001] into the central zone. In particular, the latest finding
also suggested that stress does not influence anxiety-like behavior in stressed rats. Notably, the
reduction of distance travelled or low numbers of entries into the central zone of the OF, marked an
increased emotionality and anxiety in rodents.51
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Finally, no difference in stereotypic counts was found among the groups [Frestriction (1,48) = 0,7; P >
0.05; Fstress (1,48) = 0.02; P > 0.05; Ftreatment (1,48) = 1.6; P > 0.05].
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In addition, using the FST, ANOVA revealed that the immobility time was significantly impacted by
restriction [Frestriction (1,49) = 8,2; P < 0.01], stress [Fstress (1,49) = 11.5; P < 0.01] and by the interaction
between these two factors [Finteraction (1,49) = 12.7, P < 0.01], while compound 3 [Ftreatment (1,48) =
1.6; P > 0.05] did not change the swimming/floating behavior.
Post hoc test exhibited a significantly longer immobility time in vehicle or treated R + S rats compared
with the other groups, revealing that the cycle of food restriction plus stress may increase depression-
like behaviors in female rats.
In summary the stressed rats, particularly R + S, showed an increase of spontaneous locomotor and
exploratory activity, including the central zone of OF test and the vehicle binging rats revealed the
longest immobility time in FST. In this context, 3 pretreatment did not impact the anxiety and
depression-like behaviors in the control groups (NR + NS or NR + S or R + NS) and did not alter the
emotional state detected in the binging rats.
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CONCLUSIONS
The replacement of the conformationally constrained 1,3-benzodioxane structure of 1 with the more
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flexible 1,3-dioxane ring by benzo-cracking approach led to derivatives 2 and 3, which show very
high affinity for σ1 receptor and a remarkable selectivity over σ2 subtype. Docking studies rationalized
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the affinity profiles of the proposed ligands on the σ1 receptor and gave useful information about the
binding mode of this class of compounds. Showing significant affinity also for δ opioid subtype, 2
might be considered a dual σ1/δ receptor ligand. Interestingly, compound 3 displays an uncommon
selectivity for σ1 receptor over all the other evaluated targets. In in vivo studies, it was able to
counteract the overeating of HPF only in binging-rats, without affecting HPF intake in the control
group and anxiety-like and depression-related behaviors in female rats. These findings reinforce the
potential use of σ1 receptor antagonism to selectively block compulsive eating in binging rats,
suggesting σ1 receptor antagonists as promising candidates to treat binge episode, and are noteworthy,
considering that, at present, the treatment approaches to manage pathological feeding behavior are
limited.
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Chemistry
General
Instruments used for the synthesis and characterization of compounds 2-9 are reported in the
Supporting Information.
9-Benzyl-2-phenyl-1,5-dioxa-9-azaspiro[5.5]undecane (2)
A mixture of 8 (1.7 g, 11.16 mmol), 10 (2.11 g, 11.16 mmol) and p-toluenesulfonic acid (0.85 g, 4.85)
in toluene (50 mL) was heated at reflux for 5 h. After the mixture was cooled, water was added. The
aqueous phase was basified with 2N NaOH and extracted three times with CHCl3. The organic phase
was dried (Na2SO4) and evaporated. The residue was purified by flash chromatography. Eluting with
cyclohexane/EtOAc (7:3) afforded an oil (71% yield). 1H NMR (CDCl3) δ 1.61-2.60 (m, 10H, CH2
and piperidine), 3.51 (s, 2H, NCH2Ar) 3.90 (m, 1H, CH2O), 4.13 (m, 1H, CH2O), 4.98 (dd, 1H,
ArCHO), 7.21-7.42 (m, 10H, ArH). ESI/MS: m/z 324.2 [M + H]+. The free base was transformed
into the oxalate salt that was crystallized from EtOH: mp 202-204 °C. Anal. Calcd for
C21H25NO2·H2C2O4: C, 66.81%; H, 6.58%; N, 3.39%. Found: C, 67.05%; H, 6.42%; N, 3.50%.
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9-Benzyl-3-phenyl-1,5-dioxa-9-azaspiro[5.5]undecane (3)
This compound was synthesized from 9 and 10 according to the procedure described for 2: an oil was
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obtained (70% yield). H NMR (CDCl3) δ 1.82 (m, 2H, piperidine), 2.18 (m, 2H, piperidine), 2.50
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(m, 4H, piperidine), 3.18 (m, 1H, CHAr), 3.53 (s, 2H, NCH2Ar) 3.99 (m, 4H, 2 x CH2O), 7.21-7.39
(m, 10H, ArH). ESI/MS: m/z 324.2 [M + H]+. The free base was transformed into the oxalate salt that
was crystallized from EtOH: mp 211-212 °C. Anal. Calcd for C21H25NO2·H2C2O4: C, 66.81%; H,
6.58%; N, 3.39%. Found: C, 66.59%; H, 6.40%; N, 3.19%.
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1-Benzyl-4-(4-phenyl-1,3-dioxan-2-yl)piperidine (4)
9
This compound was synthesized from 8 and 11 according to the procedure described for 2, to give a
mixture of the diastereomers 4a and 4b, that were separated by flash chromatography, eluting with
cyclohexane/EtOAc (95:5).
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The isomer 4a eluted first as an oil (15% yield). 1H NMR (CDCl3) δ 1.28-2.42 (m, 9H, piperidine),
2.94 (m, 2H, piperidine), 3.49 (s, 2H, NCH2Ar), 3.92 (m, 1H, CH2O), 4.16 (m, 1H, CH2O), 4.42 (d,
1H, J = 6.5 Hz, OCHO), 5.19 (m, 1H, ArCHO), 7.20-7.42 (m, 10H, ArH). ESI/MS: m/z 338.2 [M +
H]+. The free base was transformed into the oxalate salt that was crystallized from 2-PrOH: mp 101-
102 °C. Anal. Calcd for C22H27NO2·H2C2O4: C, 67.43%; H, 6.84%; N, 3.28%. Found: C, 67.27%, H,
6.96%; N, 3.50%.
1
The second fraction was the isomer 4b (48% yield). H NMR (CDCl3) δ 1.19-1.94 (m, 9H,
piperidine), 2.92 (m, 2H, piperidine), 3.50 (s, 2H, NCH2Ar), 3.89 (m, 1H, CH2O), 4.20 (m, 1H,
CH2O), 4.48 (d, 1H, J = 5.6 Hz, OCHO), 4.65 (dd, 1H, J = 11.3, 2.3 Hz, ArCHO), 7.20-7.42 (m, 10H,
ArH). ESI/MS: m/z 338.2 [M + H]+. The free base was transformed into the oxalate salt that was
crystallized from EtOH: mp 161-162 °C. Anal. Calcd for C22H27NO2·H2C2O4: C, 67.43%; H, 6.84%;
N, 3.28%. Found: C, 67.70%, H, 6.98%; N, 3.05%.
1-Benzyl-4-(5-phenyl-1,3-dioxan-2-yl)piperidine (5)
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This compound was synthesized from 9 and 11 according to the procedure described for 2, to give a
mixture of the diastereomers 5a and 5b, that were separated by flash chromatography eluting with
cyclohexane/EtOAc (95:5).
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The isomer 5a eluted first as an oil (44% yield). 1H NMR (CDCl3) δ 1.40-1.98 (m, 7H, piperidine),
2.88 (m, 2H, piperidine), 3.18 (m, 1H, CHAr), 3.50 (s, 2H, NCH2Ar) 3.78 (dd, 1H, J = 11.3, 10.8 Hz,
CH2O), 4.17 (dd, 1H, J = 11.3, 4.5 Hz, CH2O), 4.36 (d, 1H, J = 4.9 Hz, OCHO), 7.12-7.38 (m, 10H,
ArH). ESI/MS: m/z 338.2 [M + H]+. The free base was transformed into the oxalate salt that was
crystallized from 2-PrOH: mp 158-160 °C. Anal. Calcd for C22H27NO2·H2C2O4: C, 67.43%; H,
6.84%; N, 3.28%. Found: C, 67.55%, H, 6.70%; N, 3.48%.
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The second fraction was the isomer 5b (24% yield). 1.42-1.97 (m, 7H, piperidine), 2.61 (m, 1H,
CHAr), 2.92 (m, 2H, piperidine), 3.50 (s, 2H, NCH2Ar) 4.18 (m, 4H, 2 x CH2O), 4.42 (d, 1H, J = 5.2
Hz, OCHO), 7.18-7.59 (m, 10H, ArH). ESI/MS: m/z 338.2 [M + H]+. The free base was transformed
into the oxalate salt that was crystallized from 2-PrOH: mp 111-112 °C. Anal. Calcd for
C22H27NO2·H2C2O4: C, 67.43%; H, 6.84%; N, 3.28%. Found: C, 67.61%, H, 6.97%; N, 3.41%.
1-Phenylpropane-1,3-diol (8)
A solution of 6 (Aldrich) (1 g, 4.23 mmol) in dry Et2O (3 mL) was added dropwise to a suspension
of LiAlH4 (0.17 g, 4.5 mmol) in dry Et2O (5 mL) at 0° C under a nitrogen atmosphere. The mixture
was stirred for 2 h at room temperature, then it was poured onto ice and 2.5 M NaOH (12.65 mL) was
added. After the precipitate was filtered off over Celite, the organic phase was dried (Na2SO4). The
evaporation of the solvent afforded a residue that was purified by flash chromatography. Eluting with
cycloexane/EtOAc (75:25) gave an oil (69% yield). 1H NMR (CDCl3) δ 1.86 (m, 2H, CH2), 3.24 (br
s, 2H, exchangeable with D2O, 2 x OH), 3.79 (m, 2H, CH2O), 4.88 (dd, 1H, CHO), 7.25-7.36 (m, 5H,
ArH).
2-Phenylpropane-1,3-diol (9)
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This compound was synthesized from 7 (Aldrich) according to the procedure described for 8: a white
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solid was obtained (72% yield). M.p. 49-50 °C. H NMR (CDCl3) δ 1.95 (br s, 2H, exchangeable
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with D2O, 2 x OH), 3.08 (m, 1H, CHAr), 3.96-4.03 (m, 4H, 2 x CH2O), 7.34-7.47 (m, 5H, ArH).
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Radioligand binding studies
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The experimental details of the binding studies at σ1, σ2, NMDA, opioid receptors and DAT are
reported in the Supporting Information.
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In vivo studies
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Female Sprague-Dawley rats (Charles River, Italy), fifty two days old were submitted to the binge
eating protocol as described in previous works52 and in the Supporting information.
OF test was performed to evaluate locomotor activity, exploration, and anxiety-like behavior in
rodents as described in previous studies.53, 54 FST is a validated tool, previously described50 to assess
the depression-like behavior in rodents.
Compound 3 was dissolved in 5% solution of DMSO in distilled water and administered i.p. (2 ml /
kg) at 3 or 7 mg / kg doses. On feeding test, 3 or the vehicle were injected 30 min before allowing
access to HPF. For more detailed information see the Supporting Information.
All rats in the estrous phase were excluded from the results, since binge eating episodes did not occur
during this stage in female rats in the same animal model.55-57
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
http://pubs.acs.org.
Instruments used for the synthesis and characterization of compounds 2-9, experimental details of the
binding studies at σ1, σ2, NMDA, opioid receptors and DAT and binge eating experimental procedure.
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AUTHOR INFORMATION
Corresponding authors
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F.D.B.: Phone +390737402265, e-mail fabio.delbello@unicam.it; W. Q.: Phone +390737402237, e-
mail wilma.quaglia@unicam.it.
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Author Contributions
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F.D.B., G.G., P.P., A.P. and W.Q. designed, synthesized and characterized the new compounds. They
wrote the associated chemical sections. A.B., B.W. and D.S. performed the binding experiments.
G.V. performed the docking experiments. L.B., C.C., E.M.D.B. and M.V.M.D.B. performed the in
vivo experiments and described the relative results and discussion. C.C., F.D.B., M.V.M.D.B. and
W.Q. drafted the main text of the manuscript. All authors critically read and approved the final version
of the manuscript.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The work was supported by grant from the University of Camerino (Fondo di Ateneo per la Ricerca
2018 and Fondo di Ateneo per la Ricerca 2019) and by the Italian Ministry of Education, University
and Research: PRIN2015KP7T2Y to CC.
ABBREVIATIONS USED
PCP, phencyclidine; NMDA, N-methyl-D-aspartate; DAT, dopamine transporter; HPF, highly
palatable food; i.p., intraperitoneal; veh, vehicle.
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