New bifunctional antioxidant/σ1agonist ligands: Preliminary chemico-physical and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2016.05.045

We previously reported bifunctional sigma-1 (σ₁) ligands endowed with antioxidant activity (1 and 2). In the present paper, pure enantiomers (R)-1 and (R)-2 along with the corresponding p-methoxy (6, 11), p-fluoro derivatives (7, 12) were synth

Full text of DOI:10.1016/j.bmc.2016.05.045

Accepted Manuscript
New Bifunctional Antioxidant/σ1 Agonist Ligands: Preliminary Chemico-phys-
ical and Biological Evaluation
Emanuela Arena, Ivana Cacciatore, Laura S. Cerasa, Hasan Turkez, Valeria
Pittalà, Lorella Pasquinucci, Agostino Marrazzo, Carmela Parenti, Antonio Di
Stefano, Orazio Prezzavento
PII:
S0968-0896(16)30375-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.05.045
BMC 13029
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 February 2016
19 May 2016
20 May 2016
Please cite this article as: Arena, E., Cacciatore, I., Cerasa, L.S., Turkez, H., Pittalà, V., Pasquinucci, L., Marrazzo,
A., Parenti, C., Stefano, A.D., Prezzavento, O., New Bifunctional Antioxidant/σ1 Agonist Ligands: Preliminary
Chemico-physical and Biological Evaluation, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.bmc.2016.05.045
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

New Bifunctional Antioxidant/σ Agonist Ligands: Preliminary Chemico-physical
1
and Biological Evaluation
Emanuela Arenaa, Ivana Cacciatoreb, Laura S. Cerasab, HasanTurkezb,c, Valeria Pittalàa, Lorella
Pasquinuccia, Agostino Marrazzoa, Carmela Parentid, Antonio Di Stefanob, and Orazio
Prezzaventoa*
aDepartment of Drug Sciences, Medicinal Chemistry Section, University of Catania, Viale A. Doria
6, 95125 Catania, Italy.
bDepartment of Pharmacy, University G. d’Annunzio, Via dei Vestini 31, 66100 Chieti, Italy.
cDepartment of Molecular Biology and Genetics, Faculty of Science, Erzurum Technical
University, Erzurum, Turkey.
dDepartment of Drug Sciences, Pharmacology Section, University of Catania, Viale A. Doria 6,
95125 Catania, Italy.
* Corresponding author
Tel: +39-095-7384205; E-mail: prezzave@unict.it
Viale A. Doria 6, 95125 Catania, Italy
KEYWORDS. σ1 receptors, oxidative stress, neuroprotection, chemical and enzymatic stabilities,
antioxidants, lipoic acid.
ABSTRACT: We previously reported bifunctional sigma-1 (σ1) ligands endowed with antioxidant
activity (1 and 2). In the present paper, pure enantiomers (R)-1 and (R)-2 along with the
corresponding p-methoxy (6, 11), p-fluoro derivatives (7, 12) were synthesized. σ1 and σ2 affinities,
1

antioxidant properties, and chemico-physical profiles were evaluated. Para derivatives, while
maintaining strong σ1 affinity, displayed improved σ1 selectivity compared to the parent compounds
1 and 2. In vivo evaluation of compounds 1, 2, (R)-1, 7, and 12 showed σ1 agonist pharmacological
profile. Chemico-physical studies revealed that amides 2, 11 and 12 were more stable than
corresponding esters 1, 6 and 7 under our experimental conditions. Antioxidant properties were
exhibited by fluoro derivatives 7 and 12 being able to increase total antioxidant capacity (TAC).
Our results underline that p-substituents have an important role on σ1 selectivity, TAC, chemical
and enzymatic stabilities. In particular, our data suggest that new very selective compounds 7 and
12 could be promising tools to investigate the disorders in which σ1 receptor dysfunction and
oxidative stress are contemporarily involved.
2

1. Introduction
Originally, the sigma (σ) receptors were thought to be a subtype of opioid receptors [1]. However,
further studies reclassified these receptors as a new unique binding site. In the early 90's two
different receptor subtypes were recognized, termed sigma-1 (σ1) and sigma-2 (σ2) [2]. Both of
them are highly expressed in peripheral organs and in the central nervous system, even if with
slightly different distribution. They possess different molecular weight, drug selectivity patterns,
and signaling pathways [2; 3]. Unlike σ2 receptors, the σ1 subtype has been cloned, is well
characterized, and very recently was reported the crystal structure of the human σ receptor [4]. It is
1
located at the mitochondria-associated endoplasmic reticulum membrane (MAM) where, acting as
molecular chaperone, modulates calcium signaling [5-7]. Several reports have documented the
involvement of this subtype in many physiological and pathological pathways such as immune
reactions [8], neuronal plasticity [9], cognitive capacities [10;
11], pain [12;
13],
neuroinflammation [14], psychiatric and neurodegenerative disorders [15-18]. Recent studies have
also suggested that activation of σ1 receptors is implicated in protection against oxidative stress [19-
23]. In this regard, the σ1 agonist PRE084 (2-morpholin-4-ylethyl 1-phenylcyclohexane-1-
carboxylate) increased the levels of cellular antioxidants by activating the NF-kB pathway [24]. It
has been observed that knockdown of σ1 receptors is able to promote an accumulation of reactive
oxygen species (ROS) [19; 20], while their up-regulation or activation by agonists produces a
suppression of oxidative stress [21]. Increased ROS levels and decreased expression of endogenous
antioxidant systems were also found in Müller cells harvested from σ1 receptors-knockout mice
[22]. Even if the mechanism remains to be elucidated, the σ1 modulation of calcium release from
endoplasmic reticulum (ER) and its chaperoning activities seem to play an important role in
counteracting oxidative stress. Moreover, σ1 receptors mediate the activation of the antioxidant
response elements (ARE) which in turn up-regulates the expression of downstream genes, such as
3

NAD(P)H:quinone oxidoreductase 1 and superoxide dismutase 1, able to counterbalance oxidative
stress [23]. Thus, σ1 agonists selective toward the related σ2 receptors could represent potential
therapeutic agents for the treatment of oxidative stress related neurodegenerative diseases.
However, since these disorders mostly involve multiple pathways and are characterized by a
multifactorial etiology arising from the interaction of genetic, epigenetic, environmental, and
endogenous factors, a multi-target approach is preferred.
Therefore, our purpose is the design of bifunctional ligands composed by a σ1 selective portion
linked to a well-known antioxidant moiety. We have already reported a set of novel bifunctional
antioxidants/σ1 ligands (1 and 2, Chart 1) [25].
Chart 1. σ1 Selective ligands containing the (R/S) lipoyl function.
In the present paper, we designed and synthesized novel compounds obtained by linking the strong
natural antioxidant α-lipoic acid (ALA) and substituted phenyl- piperidine or piperazine scaffolds
well known as σ1 pharmacopores. Furthermore, since (R)-α-lipoic acid ((R)-ALA) is the only
naturally occurring enantiomer, the only active form in biological systems, and possesses a
favorable pharmacokinetic profile [26-29] we synthesized (R)-1 and (R)-2 enantiomers. In
particular, it has been reported that for animal and human uses the administration of (R)-ALA is
superior to its racemate inducing a more effective and faster biological response [27]. To determine
4

the agonist or antagonist profile of the most interesting compounds in the series, we also performed
in vivo studies [30].
Herein, we also aimed to assess the cytotoxicity potentials of novel reported compounds in primary
rat cerebral cortex neuron cultures in order to establish their potential use in pharmaceutical
applications. Cell injury was evaluated by measuring cell viability with methylthiazol tetrazolium
(MTT) assay, and by determining the release of intracellular lactate dehydrogenase. Total
antioxidant capacity (TAC) and total oxidative stress (TOS) levels were also determined to evaluate
the oxidative alterations. Finally, the chemical and enzymatic stabilities of compounds 1 and 2, and
their respective p-methoxy and p-fluoro substituted analogs were evaluated by in vitro
pharmacokinetics studies, in rat and human plasma.
2. Materials and methods
2.1. Chemistry
The synthetic procedure to compounds (R)-1, (R)-2, 6-7, and 11-12 is reported in Schemes 1 and 2.
Briefly, condensation of ALA or (R)-ALA with the appropriate 1-(4-substitutedbenzyl)piperidin-4-
ol (3-5) afforded (R)-1, 6, and 7. The reaction was carried out in dry dichloromethane (DCM) with
N,N'-dicyclohexylcarbodiimide
(DCC) and 4-(N,N-dimethylamino)pyridine (DMAP) as a
nucleophilic catalyst (Scheme 1). Compounds 4 and 5 were synthesized as previously reported [31].
Scheme 1. Reagents and conditions: (a) (R)-ALA or ALA, DCC, DCM, DMAP, 3 h at room
temperature (rt).
5

Acylation of commercially available piperazines 8-10 with ALA or (R)-ALA using the above
reported procedure without using 4-(N,N-dimethylamino)pyridine as catalyst gave (R)-2, 11, and 12
(Scheme 2).
Scheme 2. Reagents and conditions: (b) (R)-ALA or ALA, DCC, DCM, 3 h rt.
2.1.1. Experimental
All commercial chemicals used for synthesis were purchased from Sigma-Aldrich Company
(Milan, Italy) and utilized as received without any further purification. Reactions were monitored
by thin-layer chromatography (TLC) on precoated silica gel 60 F254 aluminum sheets (Merck,
Darmstadt, Germany) and the spots were visualized under UV light or in an iodine chamber. Flash
column chromatography was performed using Merck silica gel 60 (230-400 mesh, Merck). Melting
points were determined in open capillary tubes on digital Electrothermal apparatus 9100 (Rochford,
UK) and are reported uncorrected. Optical rotations were determined in MeOH solution with a
1
13
Perkin-Elmer 241 polarimeter. H NMR and C-NMR spectra were recorded with a Varian Inova
200 and 500 MHz spectrometer. Proton chemical shifts were reported in ppm (δ) relative to the
internal standard tetramethylsilane (TMS, δ= 0.00 ppm). Carbon chemical shifts were reported in
ppm (δ) relative to the residual solvent signal (CDCl3, δ= 77.0 ppm, dimethyl sulfoxide-d6, δ= 39.5
ppm or CD3OD-d4, δ= 49.2 ppm). Gas chromatography-mass spectroscopy (GC-MS) analyses were
recorded using a Shimadzu QP500 EI 171 (70 eV). Elemental analyses (C, H, N), performed on an
elemental analyser Carlo Erba Model 1106 (Carlo Erba, Milan, Italy), confirmed that the samples
had a purity equal to or greater than 95%; the analytical results were within ± 0.4% of the
6

theoretical values (Table S2). The radioactive materials (+)-cis-N-2-dimethylallyl-N-normetazocine
([3H]-(+)-Pentazocine; specific activity 29 Ci/mmol) and 1,3-di-(2-tolyl)-guanidine ([3H]-DTG)
(specific activity 53.3 Ci/mmol) were purchased from Perkin Elmer (Milan, Italy). Compounds 4
and 5 were synthesized following procedures described in the literature [31].
2.1.2. 1-Benzylpiperidin-4-yl-5-[(3R)-1,2-dithiolan-3-yl]pentanoatechloride ((R)-1).
To a solution of benzylpiperidin-4-ol (3) (0.185 g, 0.97 mmol), (R)-ALA (0.2 g, 0.97 mmol), and
DMAP(11.85 mg, 0.097 mmol) in 2.43 ml of dry DCM at 0 °C was added 0.2 g of DCC (0.97
mmol) under stirring and nitrogen atmosphere. The crude mixture was concentrated and purified by
flash chromatography using cyclohexane/EtOAc (6:4) as mobile phase. The free base was
converted into corresponding hydrochloride salt with 4 N HCl in dioxane and recrystallized from
diethyl ether/methanol. Yield = 43%; Rf = 0.5 (AcOEt/cyclohexane, 8:2); mp = 173 °C dec; MS m/z
1
[M]+ =379.15;
+ 62° (c 1.0, CH3OH); H NMR (200 MHz, CDCl3) δ 1.37−1.76 (m, 6H),
[ ]2D5
H
1.82−1.93 (m, 4H), 2.17−2.35 (m, 4H), 2.39−2.54 (m, 2H), 2.66-2.71 (m, 2H), 3.05-3.26 (m, 2H),
3.50 (s, 2H), 3.57−3.64 (m, 1H), 4.73-4.86 (m, 1H), 7.26-7.33 (m, 5H);13C NMR (500 MHz,
CD3OD-d4) δ 174.1, 132.6, 131.5, 130.6, 130.3, 65.3, 61.9, 57.7, 51.6, 41.5, 39.5, 35.8, 35.1, 29.9,
29.3, 28.3, 25.8. Anal. (C20H29NO2S2·HCl) C, H, N, S.
2.1.3. 1-(4-Methoxybenzyl)piperidin-4-yl-5-(1,2-dithiolan-3-yl)pentanoate chloride (6).
This compound was synthesized using 1-(4-methoxybenzyl)piperidin-4-ol (4) (0.53 g, 2.42 mmol),
ALA (0.5 g, 2.42 mmol), DMAP (29.57 mg, 0.242 mmol), and DCC (2.42 mmol) in 6 ml of dry
DCM by means of the same procedure above reported for (R)-1. Purification of the crude compound
was performed by flash silica gel chromatography using EtOAc/cyclohexane (5:5) and afforded a
yellow oil. The free base was converted into the corresponding hydrochloride salt with 4 N HCl in
dioxane. Yield = 44.5%; Rf = 0.6 [EtOAc/EtOH, 8:2]; mp = 176.3-177.6 °C; MS m/z [M]+ = 410.7;
1H NMR (200 MHz, CDCl3) δH 1.37−1.74 (m, 6H), 1.83−1.99 (m, 4H), 2.17−2.34 (m, 4H),
2.39−2.54 (m, 2H), 2.62-2.72 (m, 2H), 3.05-3.25 (m, 2H), 3.44 (s, 2H), 3.50−3.64 (m, 1H), 3.80 (s,
7

3H), 4.72-5.30 (m, 1H), 6.82-6.90 (m, 2H), 7.20-7.27 (m, 2H).13C NMR (200 MHz, CDCl3) δ
171.9, 160.9, 132.9, 119.7, 114.6, 64.3, 60.7, 56.3, 55.3, 47.3, 40.2, 38.5, 34.5, 34.1, 28.7, 26.9,
24.6. Anal. (C21H31NO3S2·HCl) C, H, N, S.
2.1.4. 1-(4-Fluorobenzyl)piperidin-4-yl 5-(1,2-dithiolan-3-yl)pentanoate chloride (7).
This compound was prepared through the procedure described for compound 6, using as starting
material, 1-(4-fluorobenzyl)piperidin-4-ol (0.55 g, 2.63 mmol) (5) and ALA (0.54 g, 2.63 mmol).
The crude compound was purified by flash chromatography on silica gel using EtOAc/cyclohexane
(6:4) and afforded a yellow oil. The free base was converted into the corresponding hydrochloride
salt with 4 N HCl in dioxane. Yield = 49%; Rf = 0.57 [EtOAc]; mp = 175.9-176.9 °C; MS m/z [M]+
= 398.1; 1H NMR (200 MHz, CDCl3) δ 1.41−1.76 (m, 6H), 1.82−2.05 (m, 4H), 2.18−2.35 (m, 4H),
H
2.42−2.51 (m, 2H), 2.61 and 2.76 (m, 2H), 3.10-3.20 (m, 2H), 3.47 (s, 2H), 3.50−3.57 (m, 1H),
13
4.75-4.81 (m, 1H), 6.94-7.04 (m, 2H), 7.23-7.31 (m, 2H); C NMR (200 MHz, CDCl3) δ 173.1,
162.8, 134.0, 131.2, 115.2, 71.7, 62.1, 56.0, 48.3, 40.1, 38.4, 34.7, 34.5, 34.0, 28.7, 25.0. Anal.
(C20H28FNO2S2·HCl) C, H, N, S.
2.1.5. 1-(4-Benzylpiperazin-1-yl)-5-[(3R)-1,2-dithiolan-3-yl]pentan-1-one chloride ((R)-2).
A solution of (R)-ALA (0.2 g, 0.97 mmol) in anhydrous DCM (3.88 ml) was added dropwise to a
solution of DCC (0.2 g, 0.97 mmol) in the same solvent (3.88 ml) under stirring and nitrogen
atmosphere. After 10 min, a solution of 1-benzylpiperazine (8) (0.24 g, 0.97 mmol) in DCM (1.94
ml) was added dropwise, and the resulting mixture was stirred for 3 h. The N,N -
- ass n nn , and the
filtrate was washed with sodium bicarbonate 5% solution (2 × 10 ml) and brine (2 × 10 ml). After
being dried over Na2SO4, the solvent was removed under reduced pressure. The organic phase was
concentrated and the crude product was purified by flash chromatography on silica gel using
EtOAc/cyclohexane 7:3 as mobile phase. The free base was converted into the corresponding
hydrochloride salt with 4 N HCl in dioxane and recrystallized from diethyl ether/methanol to give
(R)-2. Yield = 43.8%; Rf = 0.7 [dichloromethane/CH3OH, 9:1]; mp = 188.6 °C dec; MS m/z [M]+ =
8

1
25
364.30;
+ 26°(c 1.0, CH3OH); H NMR (200 MHz, CDCl3) δ 1.42-1.51 (m, 2H), 1.54-1.76
[ ]D
H
(m, 4H), 1.83-2.00 (m, 1H), 2.28-2.54 (m, 7H), 3.05-3.25 (m, 2H), 3.43-3.65 (m, 7H), 7.32-7.38 (m,
13
5H). C NMR (500 MHz, CD3OD-d4) δ 174.1, 132.6, 131.6, 130.6, 130.0, 61.8, 57.8, 52.7, 43.6,
41.5, 39.7, 39.5, 35.9, 33.5, 30.10, 26.0. Anal. (C19H28N2OS2·HCl) C, H, N, S.
2.1.6. 5-(1,2-Dithiolan-3-yl)-1-[4-(4-methoxybenzyl)piperazin-1-yl]pentan-1-onechloride (11). This
compound was synthesized using ALA (1 g, 4.85 mmol) in DCM (20 ml), a solution of DCC (1.031
g, 5 mmol) in the same solvent (20 ml) and a solution of 1-(4-methoxybenzyl)piperazine (9) (1 g,
4.85 mmol) in DCM (10 ml) with the same procedure above reported for (R)-2. Purification of the
crude compound was performed by flash silica gel chromatography using EtOAc as mobile phase.
The free base was converted into the corresponding hydrochloride salt with 4 N HCl in dioxane.
Yield = 15.5%; Rf = 0.6 [EtOAc /EtOH, 8:2]; mp = 176.6-177.2 °C; MS m/z [M]+ = 349.7; 1H NMR
(200 MHz, CDCl3) δ 1.38−1.54 (m, 2H), 1.58−1.77 (m, 4H), 1.83−2.02 (m, 1H), 2.28−2.54 (m,
H
7H), 3.05−3.25 (m, 2H), 3.42−3.69 (m, 6H), 3.81 (s, 3H), 4.07-4.18 (m, 1H), 6.83-6.90 (m, 2H),
7.20-7.27 (m, 2H). 13C NMR (200 MHz, dimethyl sulfoxide-d6) δ 171.2, 160.5, 133.5, 133.2, 121.6,
114.7, 114.3, 58.5, 56.7, 55.7, 50.3, 42.0, 40.8, 38.5, 34.6, 32.1, 28.8, 24.7. Anal.
(C20H30N2O2S2·HCl) C, H, N, S.
2.1.7. 5-(1,2-Dithiolan-3-yl]-1-[4-(4-fluorobenzyl)piperazin-1-yl]pentan-1-one chloride (12). This
compound was synthesized using 1-(4-fluorobenzyl)piperazine (10) (0.85 g, 4.36 mmol) in
according to the synthetic method described for 11. Purification of the crude compound was
performed by flash silica gel chromatography (EtOAc 10, EtOAc/EtOH 9:1) affording a yellow oil,
that was converted into the corresponding hydrochloride salt with 4 N HCl in dioxane. Yield =
1
44%; Rf = 0.64 [EtOAc]; mp = 189-191.7 °C dec; MS m/z [M]+ = 382.6; H NMR (200 MHz,
CDCl3) δ 1.44−1.51 (m, 2H), 1.54−1.70 (m, 4H), 1.86-1.96 (m, 1H), 2.28−2.51 (m, 7H), 2.76-2.77
H
13
(m, 1H), 3.10−3.20 (m, 1H), 3.43−3.64 (m, 7H), 6.95-7.05 (m, 2H), 7.24-7.31 (m, 2H). C NMR
9

(200 MHz, CDCl3) δ 171.0, 161.2, 133.7, 133.5, 123.3, 116.5, 116.3, 60.0, 56.4, 51.4, 51.0, 42.0,
40.2, 38.5, 38.1, 34.5, 32.5, 28.8, 24.5. Anal. (C19H27FN2OS2·HCl) C, H, N, S.
2.2. Biology
2.2.1. Radioligand Binding Assays.
In vitro σ binding assays were performed as earlier reported [32]. Binding assays were carried out
1
on guinea pig brain membranes in accordance with experimental protocol described by DeHaven et
al. [33]. In brief, 500 μg of membrane protein were suspended in 50 mM Tris-HCl (pH 7.4) and
incubated with 3 nM [3H]-(+)-Pentazocine (29 Ci/mM; the value of the apparent dissociation
constant (Kd) was 14 0.3 nM, n = 3). Test compounds were added at concentrations ranging from
10-5 to 10-11 M. Nonspecific binding was evaluated in the presence of 10 μM of unlabeled
haloperidol. The reaction was performed for 150 min at 37 °C and stopped by filtering the solution
through Whatman GF/B glass fiber filters, presoaked for 1 h in a 0.5% poly(ethylenimine) solution.
Filters were washed twice with 4 ml ice cold incubation buffer. Regarding the σ binding assays
2
[34], the membranes (360 μg/sample) were incubated with 3 nM [3H]DTG (53.3 Ci/mM; Kd = 11
±0.8 nM; n = 3) in the presence of 400 nM (+)-SKF10,047 for blocking sites [2]. Nonspecific
1
binding was assessed with DTG (5μM). Incubation was carried out in 50 mM Tris-HCl (pH 8.0)
for 120 min at rt and terminated by adding ice cold 10 mM Tris-HCl (pH 8.0). Samples were
filtered by a millipore filter apparatus onto Whatman GF/B glass fibers filters, presoaked for 1 h in
a 0.5% poly(ethylenimine) solution and afterward washed with ice cold buffer (2 x 4 ml). Filters
were dried, soaked in 4 ml of “Ultima Gold MV” scintillation cocktail and counted on a Beckman
LS 6500 liquid scintillation counter. Inhibition constants (Ki values) were calculated using the
EBDA/LIGAND program purchased from Elsevier/Biosoft.
2.2.2. Cell Cultures
10

Primary rat cerebral cortex neuron cultures were prepared using rat fetuses as described previously
[35]. Briefly, nine newborn Sprague–Dawley rats were used in the study. The rats were decapitated
by making a fracture in the cervical midline, and the cerebral cortex was dissected and removed.
The cerebral cortex was placed into 5 ml of Hank's balanced salt solution, which had already been
placed in a sterile Petri dish and macromerotomy was performed with two lancets. This composition
was pulled into a syringe and treated at 37 °C for 25-30 min as 5 ml Hank's balanced salt solution
plus 2 ml Trypsin– ehylenediaminetetraacetic acid (0.25% trypsin–0.02% ehylenediaminetetraacetic
acid) and chemical decomposition was achieved. Then 8 µl of DNase type 1 (120 U/ml) was added
to this solution, treated for 1-2 min and centrifuged at 800 rpm for 3 min. After having thrown away
the supernatant, 31.5 ml of neurobasal medium and 3.5 ml fetal calf serum were added to the
residue. The single cell, which was obtained after physical and chemical decomposition, was
divided into 3.5 ml samples in each of ten flasks coated with poly-D-lysine formerly dissolved in
phosphate buffered saline. The flasks were left in the incubator including 5% CO2 at 37 °C. The
flasks were then changed with a fresh medium of half of their volumes every three days until the
cells were branched and had reached certain maturity; in vitro experiments were performed eight
days later. This study was conducted at the Medical Experimental Research Center in Atatürk
University (Erzurum, Turkey). The Ethical Committee of Atatürk University approved the study
protocol.
2.2.2.1. Cell Culture Treatments
ALA, (R)-ALA and compounds 1-2, 6-7, and 11-12 were dissolved in dimethyl sulfoxide (0.5%).
The others were dissolved in sterile distilled water. Different aqueous concentrations (1, 10, 25, 50
and 100 µM) of each formulation were added to the cultures. After applications of compounds, the
cultures were incubated for 24 h at 37 °C. Mitomycin C (C15H18N4O5; Sigma-Aldrich, at 10-7 M)
alone added to group was considered as a positive control (control+) for cytotoxicity analysis.
Lik wis , as o bi a id (10 μM) and yd o n p oxid (25 μM) add d o ps w a so s d as
11

the control+ in TAC and TOS analysis, respectively. Each individual rat cerebral cortex culture
without compound was studied as a control group (control). The cell viability and oxidative
alterations were carried out in four independent experiments.
2.2.3. Evaluation of Cell Viability
2.2.3.1. MTT Assay
The viability of cells was assessed by measuring the formation of a formazan from MTT
spectrophotometrically via commercially available kits (Cayman Chemical, USA). At the end of the
experiment, the cells were incubated with 0.7 mg/ ml MTT for 30 min at 37°C. After washing, the
blue formazan was extracted from cells with isopropanol/formic acid (95:5) and photometrically
determined at 560 nm. The density of formazan formed in control cells was taken as indicative of
100% viability.
2.2.3.2. Lactate Dehydrogenase Assay
La tat d yd o nas as d om dama d s in t m di m was q antifi d by sin
lactate dehydrogenase assay kit (Cayman Chemical, USA) and 100 µl of cell medium was used for
lactate dehydrogenase analysis. Released lactate dehydrogenase catalyzed the oxidation of lactate to
pyruvate with simultaneous reduction of NAD+ to NADH. The rate of NAD+ reduction was
measured as an increase in absorbance at 490 nm. The rate of NAD+ reduction was directly
proportional to lactate dehydrogenase activity in the cell medium.
2.2.4. TAC and TOS Analyses
After cells were exposed to each compound for 24 h, the cultures were washed with ice-cold
phoshate buffered saline and homogenized with 0.9% normal saline. Following homogenization,
intracellular levels of TAC and TOS were determined by using commercially available kits (Rel
Assay Diagnostics, Gaziantep, Turkey).
12

2.3. In vivo pharmacology
2.3.1. Animals
Male Swiss CB1 mice (Harlan Laboratories, S.Pietro al Natisone (UD)) weighing 25–30 g were
housed six to a cage. Animals were kept at a constant rt (25 ± 1 °C) under a 12:12 h light and dark
cycle with free access to food and water. Each mouse was used for only one experiment.
Experimental procedures were approved by the Local Ethical Committee (IACUC) and conducted
in accordance with international guidelines as well as European Communities Council Directive and
National Regulations (CEE Council 86/609 and DL 116/92).
2.3.2. Tail-flick test
Nociception was evaluated by the radiant heat tail-flick test, as previously reported [36]. Briefly, it
consisted of irradiation of the lower third of the tail with an IR source (Ugo Basile, Comerio, Italy).
The day before the experiment, mice were habituated to the procedure for measuring nociception
threshold. Experiments were performed at rt (25 ± 1 °C). The basal pre-drug latency was
established between 3 and 4 s and was calculated as the average of the first three measurements,
which were performed at 5 min intervals. A cutoff latency of 15 s was established to minimize
damage to the tail. Post-treatment TFLs were determined at 30, 45, 60, and 90 min after
intraperitoneally (i.p.) injection. TFLs obtained in succeeding tests, after each pharmacological
treatment, were expressed as percentage changes from basal level and we have reported the results
as mean areas under the curve (MAUC) over a 90 min testing session. Data are expressed as the
mean ± SEM. Intergroup comparisons were assessed by analysis of variance (ANOVA), followed
by Post-hoc test (Bonferroni). A p value less than 0.05 (p < 0.05) was considered statistically
significant
2.4 Chemico-physical Evaluation
2.4.1. HPLC–UV Assay
13

Analytical HPLC measurements were run on a Waters 600 HPLC pump (Waters Corporation,
Milford, MA, USA). The column was a Phenomenex Kinetex RP-C8 (4.6 x 150 mm, 5 µm). The
mobile phase was a mixture of KH2PO4/ CH3OH (pH 2.5) using isocratic conditions and a flow rate
of 1 ml/min. The chromatograms were recorded at 220 nm using the Waters 2996 photodiode array
detector. Stock solutions of all compounds were prepared by dissolving them in acetonitrile in order
to obtain a final concentration of 10-4 M.
2.4.2. Kinetics of Chemical Hydrolysis
Briefly, a 0.02 M HCl buffer at pH 1.3, as non-enzymatic simulated gastric fluid, and a 0.02 M
phosphate buffer at pH 7.4 were used for these experiments. Reactions were initiated by adding 1
ml of 10-4 M stock solution in acetonitrile of the compound to 10 ml of the properly thermostated
(37 ± 0.5 °C) aqueous buffer solution, containing 20% acetonitrile. At appropriate time intervals,
samples of 20 µL were withdrawn and analyzed by HPLC. The experiments were run in triplicate
and the mean values of the rate constants were calculated [37].
2.4.3. Kinetics of Enzymatic Hydrolysis.
Human and rat plasma were obtained by centrifugation of blood samples containing 0.3% citric acid
at 3000 × g for 15-20 min. Plasma fractions (4 ml) were diluted with 0.02 M phosphate buffer (pH
7.4) to give a final volume of 5 ml (80% plasma). Incubation was performed at 37 ± 0.5 °C using a
shaking water bath. The reaction was initiated by adding 200 µL of a stock solution of compound (1
mg/ml in acetonitrile) to 5 ml of preheated plasma. Aliquots (100 µL) were taken at various times
and deproteinized by mixing with 200 µL of 0.01 M HCl in CH3OH. After centrifugation for 5 min
at 5000 × g, 10 µL of the supernatant layers were analyzed by chromatography as described above.
The amounts of remaining intact compound were plotted as a function of incubation time [38].
2.4.4. Drug Stability Testing in Intestinal Fluids
14

To assess enzymatic stability, hydrochloric buffer with pepsin (10 or 40 mg/ml) and phosphate
buffer with pancreatin (10 or 40 mg/ml) were used. Buffer solution (250 µL) was preincubated at 37
°C and 50 µL of drug stock solution [50 µM in ethanol and Fasted State Intestinal Fluid 1:9] was
added and shaken at 37 °C and 650 rpm. Samples of 100 µL were withdrawn at various times and
100 µL of ice-cold acetonitrile containing 0.5 vol.% formic acid was added to stop the enzymatic
activity [38]. The samples were vortexed and centrifuged at 2 °C and 10000 rpm, for 10 min. The
drug content in the supernatant was analyzed by HPLC.
2.4.5. Data Analyses
The data are expressed as the mean ± standard derivation (SD) of four repetitions. One-way analysis
o va ian was s d to d t min t si nifi ant di n s b tw n t o ps o ow d by a
D nn tt’s t t st o m tip ompa isons. A p obabi ity < 0.05 was onsid d as si nifi ant. A
analyses were performed using SPSS version 15.0 (USA).
3. Results and Discussion
Binding affinity of newly synthesized compounds was evaluated at σ1 and σ2 receptors by
radioligand binding assays (Table 1). Compound (R)-1 and (R)-2 when compared to the
corresponding racemic analogues showed only a slight decrease in σ1 affinities and selectivity
towards σ2 receptors (Table 1). On the contrary, phenyl-substituted piperidines 6 and 7 when
compared to compound 1 showed improved σ1 affinity (Ki = 3.1 ± 0.1 and 4.1 ± 0.2 nM,
respectively) and selectivity toward σ2 receptors (Kiσ2/Kiσ1 = 484 and 1,585, respectively). Thus,
the introduction of a substituent on the phenyl ring of compound 1, even if presenting opposite σ
and π parameters, seems to improve the σ1 binding profile. Phenyl-substituted piperazines 11 and
12, even if they displayed a negligible loss in σ1 affinity when compared to 2 (Kiσ1= 5.7 ± 0.1 and
2.9 ± 0.1 nM, respectively) showed an improvement in selectivity (Kiσ2/Kiσ1 = 432 and 448,
15

respectively). These binding data suggest that the introduction of a substituent on the phenyl ring of
compounds 1 and 2 slightly improved σ1 affinity and selectivity. Among synthesized compounds, 7
stands out for its improvement in σ2/ σ1selectivity ratio (Kiσ2/Kiσ1 = 1,585).
Table 1. σ-Binding affinities of novel compounds (R)-1, (R)-2, 6-7, 11-12. Each value is the means
± SD of three determinations.
σ Binding affinities (Ki, nM ± SD)
Compound
σ1
[3H]-Pentazocine
17.7 ± 1.5
6.1 ± 0.4
σ2
Kiσ2/Kiσ1
[3H]-DTG
1,870 ± 50
1,245 ± 35
1,500 ± 42
6,500 ± 127
2,460 ± 85
1,300 ± 37
17.0 ± 1.5
105.6
204.1
484
1,585
432
(R)-1
(R)-2
6
7
11
12
3.1 ± 0.1
4.1 ± 0.2
5.7 ± 0.1
2.9 ± 0.1
448
6.3
2.7 ± 0.5
Haloperidol
Given the implication of σ receptors in opioid-mediated analgesia [30], we analyzed the ability of
1
compounds 1, 2, (R)-1, 7, and 12 to modulate the analgesic effect of the systemically injected
morphine (M). In vivo results showed that 1, 2, (R)-1, 7, and 12 (1 mg/kg i.p.), (dose used in
previous experiments with 1 [25]) did not affect basal tail flick latency (TFL), expressed as MAUC,
during the entire time of observation (data not shown). Injection of M (4 mg/kg i.p.) significantly
increased the nociceptive latency following thermal stimulation, clearly demonstrating its analgesic
effect. Indeed, the percent change from basal level of TFL, compared with the group of saline
treated rats, was increased up to 84,54%. Pretreatment with 1, 2, (R)-1, 7, and 12 (1 mg/kg i.p.) 45
min prior to M (4 mg/kg i.p.) significantly decreased the antinociceptive effect of the opioid agonist
(Figure 1). In particular, the calculated value of MAUC was significantly lower than the
corresponding value obtained with M. Similarly to 1 σ agonist profile [25], also evaluated in the
1
16

present experimental conditions (Figure 1), the in vivo results confirmed the σ agonist profile also
1
for 2, (R)-1, 7, and 12 compounds.
Figure 1. Effect of 1, 2, (R)-1, 7, 12 (1 mg/kg i.p.) on morphine (M) (4 mg/kg i.p.) analgesia.
Results are expressed as MAUC (after the last injection) over 90 min testing period. Columns
represent the mean ±SD; #, p <0.05 vs saline-treated rats (n = 8-10); *, p < 0.05 vs (M) treated rats
(n = 8-10).
In order to assess the potential cytotoxicity of our compounds, ALA, and (R)-ALA, we evaluated
cell injury in primary rat cerebral cortex neuron cultures. Thus, we measured cell viability with
MTT assay and determined the release of intracellular lactate dehydrogenase (Table S1, supporting
information). MTT abso ban ’s va was 46.1 ± 2.9% w n t rat cerebral cortex cells were
17

exposed to mitomycin C (10 μM), indicating that mitomycin C caused cell death. Likewise,
mitomycin C-induced neurological damage was clearly evidenced by seven fold increases in the
activity of lactate dehydrogenase (2.23 ± 0.18) compared with the observations of control groups
(Table S1). On the contrary, cultured primary rat cerebral cortical neurons treated with synthesized
compounds did not show any significant alterations in cell viability. MTT and lactate
dehydrogenase assays were performed at different concentrations (0.1, 1, 10, 25, 50 and 100 µM) of
the tested compounds (Table S1). Only the highest concentrations of 6 (100 µM) and 11 (50 and
100 µM) caused cell death or injury cytotoxicity (Table S1). Furthermore, TAC and TOS
parameters were determined in rat cerebral cortex cell cultures to assess the H2O2-induced oxidative
alterations (Table 2). As shown in Table 2, all treatments with p-methoxy derivatives 6 and 11, and
compound (R)-1 did not alter TAC and TOS levels, while the other tested derivatives increased
TAC levels without changing TOS levels, likewise to ALA and (R)-ALA (Table 2).
Table 2. Total antioxidant capacity (TAC) and total oxidative stress (TOS) levels in rat cerebral
cortex cells maintained 24 h in the presence of tested compounds in vitro. Values are means ±
standard deviation (n = 4);* symbol represents a statistically significant difference from control- (p<
0.05); ▲triangles indicate statistically important increases of TAC or TOS levels.
TAC
(mmolTrolo
x
Equiv./l)
10.7±2.1
26.9±2.8*▲
TAC
(mmolTrolo
x
Equiv./l)
10.7±2.1
26.9±2.8*▲
TOS
TOS
(µmol H2O2
Equiv./l)
Groups
(µmol H2O2 Groups
Equiv./l)
Control
Ascorbic acid 10
0.5±0.1
--
Control
Ascorbic acid 10 μM
0.5±0.1
--
μM
--
--
2.3±0.3*▲
2.3±0.3*▲
H2O2 25 μM
0,1 µM
1 µM
H2O2 25 μM
0,1 µM
1 µM
10.7±2.4
10.5±2.1
10.9±2.6
11.8±2.0*▲
12.1±2.5*▲
14.2±2.6*▲
10.8±2.4
0.4±0.1
0.5±0.1
0.4±0.1
0.5±0.1
0.5±0.1
0.5±0.1
0.5±0.1
10.7±2.4
10.9±1.9
11.0±2.4
11.2±2.2
12.7±2.4*▲
13.5±2.3*▲
10.9±2.7
0.4±0.1
0.5±0.1
0.4±0.1
0.4±0.1
0.5±0.1
0.5±0.1
0.4±0.1
10 µM
25 µM
50 µM
100 µM
10 µM
25 µM
ALA
(R)-ALA
50 µM
100 µM
0,1 µM
0,1 µM
1
(R)-1
18

1 µM
10.9±2.2
11.4±3.0
13.8±2.7*▲
13.6±2.6*▲
12.5±2.4*▲
10.4±2.0
0.5±0.1
0.4±0.1
0.5±0.1
0.4±0.1
0.5±0.1
0.5±0.1
0.5±0.1
0.4±0.1
0.5±0.1
0.5±0.1
0.5±0.1
0.5±0.1
0.4±0.1
0.4±0.1
0.5±0.1
0.5±0.1
0.5±0.1
0.4±0.1
0.4±0.1
0.4±0.1
0.4±0.1
0.5±0.1
0.5±0.1
1 µM
10.7±2.1
10.9±2.5
11.3±2.8
10.9±2.6
11.0±2.7
10.2±2.6
10.5±2.8
10.8±2.3
13.0±2.6*▲
12.8±2.1*▲
11.2±2.4
10.5±2.9
11.2±2.9
10.9±3.2
11.3±2.7
11.0±2.6
10.8±2.9
10.9±3.1
11.2±2.8
12.6±3.1*▲
13.9±3.4*▲
11.4±2.7
11.1±3.0
0.4±0.1
0.4±0.1
0.5±0.1
0.5±0.1
0.5±0.1
0.5±0.1
0.5±0.1
0.4±0.1
0.4±0.1
0.4±0.1
0.5±0.1
0.4±0.1
0.5±0.1
0.5±0.1
0.5±0.1
0.5±0.1
0.5±0.1
0.4±0.1
0.5±0.1
0.4±0.1
0.5±0.1
0.5±0.1
0.5±0.1
10 µM
25 µM
50 µM
100 µM
0,1 µM
1 µM
10 µM
25 µM
50 µM
100 µM
0,1 µM
1 µM
10 µM
25 µM
50 µM
100 µM
0,1 µM
1 µM
10 µM
25 µM
50 µM
100 µM
10 µM
25 µM
50 µM
100 µM
0,1 µM
1 µM
10 µM
25 µM
50 µM
100 µM
0,1 µM
1 µM
10 µM
25 µM
50 µM
100 µM
0,1 µM
1 µM
10 µM
25 µM
50 µM
100 µM
11.1±2.4
12.6±2.7*▲
12.9±3.0*▲
14.8±2.8*▲
14.6±2.5*▲
10.8±3.1
11.3±2.5
11.0±3.0
11.4±2.9
10.9±3.1
10.7±2.7
11.0±3.1
11.2±2.7
11.1±3.2
12.6±2.7*▲
13.5±2.8*▲
12.4±3.0*▲
2
6
7
(R)-2
11
12
Finally, we decided to investigate the chemico-physical profile of compounds 1, 2, and the
correspondent p-methoxy and p-fluoro-substituted analogs (6-7, 11-12). We evaluated their
chemical and enzymatic stabilities in human and rat plasma, using two buffers (pH 1.3 and 7.4), in
simulated gastric and intestinal fluid with different concentrations (10 mg/ml and 40 mg/ml) of
enzymes (pepsin and pancreatin) [37; 39]. As reported in Table 3, all compounds are quite stable at
pH 1.3 (t > 47 h) and pH 7.4 (t > 37 h). In the presence of increasing concentrations of enzymes,
½
½
the rate of hydrolysis of all compounds was significantly faster at pH 7.4 than 1.3; however, in the
presence of pepsin (10 and 40 mg/ml) at pH 1.3, the most stable compounds were the amides 2, 11
and 12 with t superior to 23 h. The high stability of these compounds at low pH is an advantage for
½
oral administration since they may remain for a longer period in the stomach without undergoing
metabolism. On the other hand, in the presence of 40 mg/ml of pancreatin at pH 7.4, ester derivative
7 was immediately hydrolyzed, while 1 and 6 degraded with t of 0.09 and 2.83 h, respectively. In
½
the same experimental conditions, derivatives 2, 11 and 12 resulted in more stable than the
19

corresponding ester ones (t >7 h). Outcomes also suggested that compound 2 was 2.8-fold (pH 1.3)
½
and 105-fold (pH 7.4) more stable than ester 1 in the presence of 40 mg/ml of pepsin or pancreatin,
respectively. A similar behavior was observed for the couple of compounds 6, 11 and 7, 12.
Compounds 1 and 2 were immediately degraded in rat plasma. The most stable compound was 11
(t = 4.13 h) followed by fluoro derivative 12. The rate of hydrolysis in 80% human plasma was
½
slower when compared to rat plasma, thus suggesting that the latter may have more selective
enzymes
for
hydrolysis.
20

Table 3. Chemico-physical data at 37 °C of compounds 1, 2, p-methoxy (6-11) and p-fluoro derivatives (7-12). Values are means of three experiments; standard
deviation is given in parentheses.
Compounds
Condition
pH 1.3
1
2
6
7
11
12
52 (± 1)
47 (± 1)
t (h)
Kobs (h-1)
58.71 (± 2.29)
0.012 (± 0.001)
40.45 (± 1.25)
0.017 (± 0.001)
24.51 (± 0.74)
0.028 (± 0.001)
23.06 (± 0.97)
0.028 (± 0.001)
0.29 (± 0.01)
2.389 (± 0.062)
92.97 (± 4.56)
0.007 (± 0.001)
53.80 (± 2.21)
0.013 (± 0.001)
69 (± 2)
240 (± 5)
88 (± 2)
½
0.013 (± 0.001)
37 (± 1)
0.015 (± 0.001)
38 (± 1)
0.003 (± 0.001)
238 (± 6)
0.008 (± 0.001)
81 (± 2)
pH 7.4
t (h)
½
Kobs (h-1)
0.019 (±0.001)
39.15 (± 0.94)
0.018 (± 0.001)
22.16 (± 0.53)
0.031 (± 0.001)
2.78 (± 0.06)
0.249(± 0.006)
0.019 (± 0.001)
3.77(± 0.09)
0.184 (± 0.005)
0.68 (± 0.02)
1.019 (± 0.023)
0.003 (± 0.001)
112 (± 2)
0.009 (± 0.001)
106.89 (± 3.10)
0.007 (± 0.001)
37.19 (± 1.15)
0.019 (± 0.001)
75.08 (± 1.88)
0.009 (± 0.001)
pH 1.3 Pepsin 10mg/ml
pH 1.3 Pepsin 40mg/ml
pH 7.4 Pancreatin 10mg/ml
t (h)
½
Kobs (h-1)
0.010 (± 0.001)
66.16 (± 2.32)
0.010 (± 0.001)
22.23 (± 0.82)
0.031 (± 0.012)
0.006 (± 0.001)
23.68 (± 0.66)
0.029 (± 0.001)
t (h)
½
kobs (h-1)
immediate hydrolysis 60.87 (± 1.71)
t (h)
½
kobs(h-1)
-
0.011 (± 0.001)
pH 7.4 Pancreatin
40mg/ml
0.09 (± 0.01)
9.58 (± 0.33)
2.83 (± 0.07)
immediate hydrolysis 41.64 (± 0.96)
32.31 (± 0.81)
t (h)
½
kobs(h-1)
0.245 (± 0.005)
0.61 (± 0.01)
1.14 (± 0.03)
-
7.615 (± 0.223)
0.43 (± 0.02)
1.61 (± 0.06)
immediate
hydrolysis
-
0.072 (± 0.013)
1.76 (± 0.05)
0.39 (± 0.01)
immediate
hydrolysis
-
0.017 (± 0.001)
5.28 (± 0.14)
0.13 (± 0.01)
0.021 (± 0.001)
4.21 (± 0.11)
0.81 (± 0.02)
0.856 (± 0.03)
Human plasma
Rat plasma
t (h)
½
kobs (h-1)
0.165 (± 0.004)
0.46 (± 0.01)
0.56 (± 0.01)
4.13 (± 0.12)
2.92 (± 0.05)
t (h)
½
kobs (h-1)
1.506 (± 0.042)
1.238 (± 0.034)
0.168(± 0.004)
0.237 (± 0.006)
21

In particular, 11 and 12 displayed a better t (t (h) 5.28 ± 0.14 h and 4.21 ± 0.11, respectively) in
½
½
contrast to compound 2 (t = 1.76 ± 0.05 h).
½
Taken together, these results suggest that the ester derivatives are more susceptible to hydrolysis
both in acidic and physiological environments compared to the amide derivatives. Moreover, the
faster hydrolyses of ester derivatives, observed in plasma samples, confirmed previously
reported data suggesting that well known action of plasma esterases [34]. Notably, 11 and 12 are
the most stable compounds in all experimental tested conditions.
4. Conclusions
In the present paper, we reported the synthesis of the p-methoxy- and p-fluoro-substituted
analogs of compounds 1 and 2 and the corresponding (R)-1 and (R)-2 enantiomers. Our results
point out that p-methoxy (6 and 11) and p-fluoro (7 and 12) derivatives showed an improvement
of σ1 selectivity towards σ2 related receptors. Moreover, in vivo studies displayed σ1 agonist
profile for compounds 1, 2, (R)-1, 7, and 12. Derivatives 7 and 12 also displayed antioxidant
properties by increasing the TAC. Chemico-physical studies revealed that compound 2 was
found to be more stable than corresponding ester 1 under acid and physiological conditions, in
simulated gastric fluid and simulated intestinal fluid and, even if it is degraded in human plasma,
its hydrolysis is slower than 1. Further improvement of the stability at physiological pH in
simulated gastric and intestinal fluid was shown by compounds 11 and 12.
In conclusion, our results highlight that the presence of p-substituents has an important role on σ1
selectivity, TAC, chemical and enzymatic stabilities. In particular, our data suggest that
compounds 7 and 12 are promising tools to investigate the disorders related to σ1 receptors and
22

oxidative stress whereas they can be converted into the corresponding [18F] radiotracers for PET
studies. Preclinical in vivo studies are in progress and very promising results will be guaranteed.
Supporting Information: MTT and LDH studies, their detailed experimental procedures, and
elemental analysis results.
Funding Sources
This work was supported by Research Funding for University (FIR) 2014.
Conflict of interest
The authors declare no conflict of interest.
References
[1] Martin, W. R.; Eades, C. G.; Thompson, J. A.; Huppler, R. E.; Gilbert, P. E. J. Pharmacol.
Exp. Ther. 197 (1976) 517-532.
[2] Quirion, R.; Bowen, W. D.; Itzhak, Y.; Junien, J. L.; Musacchio, J. M.; Rothman, R. B.; Su,
T. P.; Tam, S. W.; Taylor, D. P. Trends Pharmacol. Sci. 13 (1992) 85-86.
[3] Rousseaux, C. G.; Greene, S. F. J. Recept. Sign. Transduct. Res. 1 (2015) 1-62.
[4] Schmidt, H. R.; Zheng, S.; Gurpinar, E.; Koehl, A.; Manglik, A.; Kruse, A. C. Nature 532
(2016) 527-530.
[5] Brune, S.; Pricl, S.; Wunsch, B. J. Med. Chem. 56 (2013) 9809-9819.
[6] Mori, T.; Hayashi, T.; Hayashi, E.; Su, T. P. PloSone 8 (2013) e76941.
[7] Chu, U. B.; Ruoho, A. E. Mol. Pharmacol. 89 (2016) 142-153.
[8] Zhu, L. X.; Sharma, S.; Gardner, B.; Escuadro, B.; Atianzar, K.; Tashkin, D. P.; Dubinett, S.
M. J. Immunol. 170 (2003) 3585-3591.
[9] Maurice, T. Behav. Brain Res. 296 (2016) 270-278.
[10] Antonini, V.; Prezzavento, O.; Coradazzi, M.; Marrazzo, A.; Ronsisvalle, S.; Arena, E.;
Leanza, G. J. Neurochem. 109 (2009) 744-754.
[11] Antonini, V.; Marrazzo, A.; Kleiner, G.; Coradazzi, M.; Ronsisvalle, S.; Prezzavento, O.;
Ronsisvalle, G.; Leanza, G. J. Alzheimers Dis. 24 (2011) 569-586.
[12] Prezzavento, O.; Parenti, C.; Marrazzo, A.; Ronsisvalle, S.; Vittorio, F.; Arico, G.; Scoto, G.
M.; Ronsisvalle, G. Life Sci. 82 (2008) 549-553.
[13] Almansa, C.; Vela, J. M. Future Med. Chem. 6 (2014) 1179-1199.
[14] Wu, Z.; Li, L.; Zheng, L. T.; Xu, Z.; Guo, L.; Zhen, X. J. Neurochem. 134 (2015) 904-914.
[15] Tsai, S. Y.; Pokrass, M. J.; Klauer, N. R.; De Credico, N. E.; Su, T. P. Exp. Opin. Ther.
Targ. 18 (2014) 1461-1476.
23

[16] Nguyen, L.; Lucke-Wold, B. P.; Mookerjee, S. A.; Cavendish, J. Z.; Robson, M. J.;
Scandinaro, A. L.; Matsumoto, R. R. J. Pharmacol. Sci. 127 (2015) 17-29.
[17] Ruscher, K.; Wieloch, T. J. Pharmacol. Sci. 127 (2015) 30-35.
[18] Fisher, A.; Bezprozvanny, I.; Wu, L.; Ryskamp, D. A.; Bar-Ner, N.; Natan, N.; Brandeis,
R.; Elkon, H.; Nahum, V.; Gershonov, E.; LaFerla, F. M.; Medeiros, R. Neurodegener. Dis. 16
(2016) 95-110.
[19] Wang, L.; Eldred, J. A.; Sidaway, P.; Sanderson, J.; Smith, A. J.; Bowater, R. P.; Reddan, J.
R.; Wormstone, I. M. Mech. Ageing Dev. 133 (2012) 665-674.
[20] Meunier, J.; Hayashi, T. J. Pharmacol. Exp. Ther. 332 (2010) 388-397.
[21] Tuerxun, T.; Numakawa, T.; Adachi, N.; Kumamaru, E.; Kitazawa, H.; Kudo, M.; Kunugi,
H. Neurosci. Lett. 469 (2010) 303-308.
[22] Wang, J.; Shanmugam, A.; Markand, S.; Zorrilla, E.; Ganapathy, V.; Smith, S. B. Free
Radic. Biol. Med. 86 (2015) 25-36.
[23] Pal, A.; Fontanilla, D.; Gopalakrishnan, A.; Chae, Y. K.; Markley, J. L.; Ruoho, A. E. Eur.
J. Pharmacol. 682 (2012) 12-20.
[24] Hyrskyluoto, A.; Pulli, I.; Tornqvist, K.; Ho, T. H.; Korhonen, L.; Lindholm, D. Cell Death
Dis. 4 (2013) e646.
[25] Prezzavento, O.; Arena, E.; Parenti, C.; Pasquinucci, L.; Arico, G.; Scoto, G. M.; Grancara,
S.; Toninello, A.; Ronsisvalle, S. J. Med. Chem. 56 (2013) 2447-2455.
[26] Hagen, T. M.; Ingersoll, R. T.; Lykkesfeldt, J.; Liu, J.; Wehr, C. M.; Vinarsky, V.;
Bartholomew, J. C.; Ames, A. B. FASEB J. 13 (1999) 411-418.
[27] Carlson, D. A.; Smith, A. R.; Fischer, S. J.; Young, K. L.; Packer, L. Altern. Med. Rev. 12
(2007) 343-351.
[28] Brufani, M.; Figliola, R. Acta Biomed. 85 (2014) 108-115.
[29] Nasole, E.; Nicoletti, C.; Yang, Z. J.; Girelli, A.; Rubini, A.; Giuffreda, F.; Di Tano, A.;
Camporesi, E.; Bosco, G. J. Enz. Inhib. Med. Chem. 29 (2014) 297-302.
[30] Mei, J. F.; Pasternak, G. W. J. Pharmacol. Exp. Ther. 300 (2002) 1070-1074.
[31] Weis, R.; Faist, J.; di Vora, U.; Schweiger, K.; Brandner, B.; Kungl, A. J.; Seebacher, W.
Eur. J. Med. Chem. 43 (2008) 872-879.
[32] Matsumoto, R. R.; Bowen, W. D.; Tom, M. A.; Vo, V. N.; Truong, D. D.; De Costa, B. R.
Eur. J. Pharmacol. 280 (1995) 301-310.
[33] DeHaven-Hudkins, D. L.; Fleissner, L. C.; Ford-Rice, F. Y. Eur. J. Pharmacol. 227 (1992)
371-378.
[34] Mach, R. H.; Smith, C. R.; Childers, S. R. Life Sci. 57 (1995) PL57-62.
[35] Ban, J. Y.; Jeon, S. Y.; Nguyen, T. T.; Bae, K.; Song, K. S.; Seong, Y. H. Biol. Pharm. Bull.
29 (2006) 2419-2424.
[36] Parenti, C.; Turnaturi, R.; Arico, G.; Marrazzo, A.; Prezzavento, O.; Ronsisvalle, S.; Scoto,
G. M.; Ronsisvalle, G.; Pasquinucci, L. Life Sci. 90 (2012) 957-961.
[37] Cacciatore, I.; Cornacchia, C.; Fornasari, E.; Baldassarre, L.; Pinnen, F.; Sozio, P.; Di
Stefano, A.; Marinelli, L.; Dean, A.; Fulle, S.; Di Filippo, E. S.; La Rovere, R. M.; Patruno, A.;
Ferrone, A.; Di Marco, V. ChemMedChem 8 (2013) 1818-1829.
[38] Borde, A. S.; Karlsson, E. M.; Andersson, K.; Bjorhall, K.; Lennernas, H.; Abrahamsson, B.
Eur. J. Pharm. Biopharm. 80 (2012) 630-637.
[39] Cacciatore, I.; Baldassarre, L.; Fornasari, E.; Cornacchia, C.; Di Stefano, A.; Sozio, P.;
Cerasa, L. S.; Fontana, A.; Fulle, S.; Di Filippo, E. S.; La Rovere, R. M.; Pinnen, F.
ChemMedChem 7 (2012) 2021-2029.
24

Legends
Chart 1. σ1 Selective ligands containing the (R/S) lipoyl function.
Scheme 1. Reagents and conditions: (a) (R)-ALA or ALA, N,N'-dicyclohexylcarbodiimide,
dichloromethane, 4-(N,N-dimethylamino)pyridine, 3 h rt.
Scheme 2. Reagents and conditions: (b) (R)-ALA or ALA, N,N'-dicyclohexylcarbodiimide,
dichloromethane, 3 h rt.
Figure 1. Effect of 1, 2, (R)-1, 7, 12 (1 mg/kg i.p.) on morphine (M) (4 mg/kg i.p.) analgesia.
Results are expressed as MAUC (after the last injection) over 90 min testing period. Columns
represent the mean ±SD; #, p <0.05 vs saline-treated rats (n = 8-10); *, p < 0.05 vs (M) treated
rats (n = 8-10).
Table 1. σ-Binding affinities of novel compounds (R)-1, (R)-2, 6-7, 11-12. Each value is the
means ± SD of three determinations.
Table 2. Total antioxidant capacity (TAC) and total oxidative stress (TOS) levels in rat cerebral
cortex cells maintained 24 h in the presence of tested compounds in vitro. Values are means ±
standard deviation (n = 4);* symbol represents a statistically significant difference from control-
(p< 0.05); ▲triangles indicate statistically important increases of TAC or TOS levels.
Table 3. Chemico-physical data at 37 °C of compounds 1, 2, p-methoxy (6-11) and p-fluoro
derivatives (7-12). Values are means of three experiments; standard deviation is given in
parentheses.
26

Graphical Abstract
S
F
Y
S
X
N
O
1 Receptor pharmacophore Natural antioxidant ALA or (R)-ALA
Y = O;
Y = --;
7 X = C;
Ki1 = 4.1 ± 0.2 nM Ki = 6,500 ± 127 nM
Ki1 = 2.9 ± 0.1 nM Ki = 1,300 ± 37 nM
12 X = N;