Design and synthesis of new bifunctional sigma-1 selective ligands with antioxidant activity

Article abstract of DOI:10.1021/jm3017893

Herein we report the synthesis of new bifunctional sigma-1 (σ₁)-selective ligands with antioxidant activity. To achieve this goal, we combined the structure of lipoic acid, a universal antioxidant, with an appropriate sigma aminic moiety. Ligands 14 and 26 displayed high affinity and selectivity for σ₁ receptors (K _iσ₁ = 1.8 and 5.5 nM; K_iσ ₂/σ₁ = 354 and 414, respectively). Compound 26 exhibited in vivo antiopioid effects on kappa opioid (KOP) receptor-mediated analgesia. In rat liver and brain mitochondria (RLM, RBM), this compound significantly reduced the swelling and the oxidation of thiol groups induced by calcium ions. Our results demonstrate that the tested compound has protective effects against oxidative stress.

Full text of DOI:10.1021/jm3017893

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Article
Design and synthesis of new bifunctional
sigma-1 selective ligands with antioxidant activity
Orazio Prezzavento, Emanuela Arena, Carmela Parenti, Lorella Pasquinucci, Giuseppina
Aricò, Giovanna Maria Scoto, Silvia Grancara, Antonio Toninello, and Simone Ronsisvalle
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm3017893 • Publication Date (Web): 08 Mar 2013
Downloaded from http://pubs.acs.org on March 13, 2013
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Design and synthesis of new bifunctional sigma-1
selective ligands with antioxidant activity.
O. Prezzavento,*,§ E. Arena, § C. Parenti,# L. Pasquinucci, § G. Aricò, § G. M. Scoto, # S. Grancara, ‡ A.
Toninello, ‡ S. Ronsisvalle. §
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§ Department of Drug Sciences, Medicinal Chemistry Section, University of Catania, Viale A. Doria 6,
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95125 Catania, Italy. Department of Drug Sciences, Pharmacology and Toxicology Section,
‡
University of Catania, Viale A. Doria 6, 95125 Catania, Italy. Department of Biomedical Sciences,
University of Padua, Viale G. Colombo 3, 35131 Padua, Italy.
*To whom correspondence should be addressed.
Orazio Prezzavento
Department of Drug Sciences, Medicinal Chemistry Section
University of Catania
V.le A.Doria 6, 95125 Catania
Telephone:+39ꢀ095ꢀ7384205
prezzave@unict.it
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Abstract
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Herein we report the synthesis of new bifunctional sigmaꢀ1 (σ1)ꢀselective ligands with antioxidant
activity. To achieve this goal, we combined the structure of lipoic acid, a universal antioxidant, with an
appropriate sigma aminic moiety. Ligands 14 and 26 displayed high affinity and selectivity for σ1
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receptors (Kiσ1 = 1.8 and 5.5 nM; Kiσ2/σ1 = 354 and 414, respectively). Compound 26 exhibited in vivo
antiopioid effects on kappa opioid (KOP) receptorꢀmediated analgesia. In rat liver and brain
mitochondria (RLM, RBM), this compound significantly reduced the swelling and the oxidation of thiol
groups induced by calcium ions. Our results demonstrate that the tested compound has protective effects
against oxidative stress.
Introduction
The sigma receptors (σ), originally postulated by Martin et al. in the midꢀ1970s to be a subtype of
opioid receptors,1 are currently classified as a unique biochemical entity different from other known
neurotransmitter receptors.2 Two different subtypes, designated sigmaꢀ1 (σ1) and sigmaꢀ2 (σ2), have
been identified;3 however, only the σ1 receptor has been cloned from various tissues and species.4ꢀ6 The
σ1 receptor is involved in many cellular functions and biological processes,7 including neuroprotection.8ꢀ
10 Many investigations have documented the involvement of σ receptors in some important pathways
implicated in different neurodegenerative illnesses such as Alzheimer’s,11 Lou Gehrig’s12 and
Parkinson’s13,14 diseases. It has been observed that some general pathways, such as an increase in the
intracellular calcium levels, mitochondrial dysfunction, reactive species production, metal
dyshomeostasis and protein misfolding, participate in the pathogenic cascades of these diseases.15 In
particular, mitochondrial damage increases the production of reactive oxygen species (ROS) and the
susceptibility to cell death, playing a key role in these diseases.16 Recently, the σ1 protein has been
characterized as a chaperone localized to the endoplasmic reticulum (ER) at the mitochondriaꢀassociated
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ER membrane (MAM), where it forms a complex with the ER chaperone called Binding
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immunoglobulin protein (Bip).17 The activation of σ1, induced by the depletion of ER calcium ions or
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by σ ligands, leads to the dissociation of the σ1ꢀBip complex, resulting in prolonged mitochondrial
calcium signaling through the inositol 1,4,5ꢀtrisphosphate receptor (IP3R). These events regulate ERꢀ
mitochondrial interorganellar calcium signaling and cell survival. Some investigations have found that
the σ1 receptors are associated with oxidative stress; for example, ER stress promotes the upregulation
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of σ1 receptors,18 whereas the knockdown of σ1 receptors potentiates ER stressꢀinduced apoptosis.19
Significant increases in the concentration of metabolites, considered markers of oxidative stress, have
been observed in the livers of σ1 receptorꢀknockout mice relative to wildꢀtype liver. This antioxidant
effect is mediated by the activation of the antioxidant response element (ARE), which upregulates the
expression of NAD(P)H:quinone oxidoreductase 1 (NQO1) and superoxide dismutase 1 (SOD1).20
Many other studies have demonstrated that σ1 selective compounds can provide neuroprotection under
cytotoxic conditions.21ꢀ24 These data suggest that σ1 receptors have a critical role in protection against
oxidative stress and neuronal apoptosis. The use of antioxidants is considered a promising approach for
slowing the progression of neurodegenerative diseases;25 however, clinical studies have demonstrated
that treatment with a single antioxidant is not able to prevent or treat these disorders.26 A greater
therapeutic benefit can be achieved with drugs that act on multiple molecular targets, which may be part
of a system or may be involved in different pathways.27 We focused our attention on αꢀlipoic acid 1
(ALA), a natural antioxidant. Compound 1 has amphiphilic properties, crosses the bloodꢀbrain barrier
and is considered an important metabolite for energy production in mitochondria.28,29 Compound 1 can
easily quench radicals and chelate metals.30 Furthermore, it can regenerate endogenous antioxidants and
repair oxidative tissue damage. A large number of studies have documented that the therapeutic effect of
1 is based on its antioxidant properties and have found that this compound has neuroprotective effects.31ꢀ
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Thus, our design strategy is based to the development of σ1 agonists with improved antioxidant
properties. To achieve this goal, we combined the structure of 1 with an appropriate aminic moiety
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based on the previously proposed structureꢀactivity relationship (SAR) for σ ligands.34ꢀ36 In particular,
Gilligan et al.34 identified four regions in their lead compound: 1) a distal aromatic ring (Region A), 2) a
nitrogen heterocycle (Region C), 3) a space between the nitrogen heterocycle and the distal hydrophobic
site (Region B), and 4) a substituent on the basic nitrogen (Region D) (Figure 1). They noted that a
variety of spacer groups could be inserted in Region B and that the replacement of the distal phenyl
group (Region A) with other aryl or heteroaryl nuclei had no appreciable effect on the sigma binding
affinity or selectivity. Furthermore, they indicated that the chemical nature of the Nꢀsubstituent and its
distance from Region C significantly affect the selectivity for σ binding over binding to other receptors
such as those for dopamine (D2) and serotonin (5ꢀHT2). Thus, we synthesized new piperazine and
piperidine derivatives containing the lipoyl function group in Regions A and B and different Nꢀ
substituents in Region D (Figure 1). Particularly, we investigated how the chemical structure of the Nꢀ
substituent and its distance from Region C influence the σ1/σ2 selectivity. To determine the
pharmacological profile of the most interesting compound in the series, we also performed in vivo
studies on the modulation of the analgesic effect induced by the kappa opioid (KOP) agonist transꢀ
(1S,2S)ꢀ3,4ꢀdichloroꢀNꢀmethylꢀNꢀ[2ꢀ(1ꢀpyrrolidinyl)ꢀcyclohexyl]benzeneacetamide 2 [(ꢀ)ꢀU50,488H].37
Preliminary biological studies were also conducted to assess the antioxidant properties in rat liver and
brain mitochondria (RLM, RBM).38
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Chemistry
Commercially available secondary piperazines 3-9 acylated with lipoic acid in dry dichloromethane
(DCM) using N,N'ꢀdicyclohexylcarbodiimide (DCC) at room temperature (rt) provided the final ketones
10-16 (Scheme 1).39 The 1ꢀalkylꢀ4ꢀhydroxypiperidines 21-23-24 were synthesized by the reaction of
piperidinꢀ4ꢀol (20) with the alkyl bromides 17-19 following the experimental procedures reported in the
literature (Scheme 2).40 1ꢀBenzylpiperidinꢀ4ꢀol (22) was purchased from SigmaꢀAldrich and was used
without further purification. The condensation of lipoic acid with secondary alcohols 21-24 was
performed in dry DCM using DCC and 4ꢀ(N,Nꢀdimethylamino)pyridine (DMAP) as a nucleophilic
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catalyst, furnishing the required esters 25-28 (Scheme 2).41
Results and Discussion
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The synthesized piperazine (10-16) and piperidine (25-28) derivatives were evaluated for their affinities
for both σ1 and σ2 receptors (Table 1). Compound 13, in which the aromatic ring is directly bound to the
basic nitrogen center, did not exhibit significant affinity for either σ subtype, whereas the corresponding
Nꢀmethyl derivative 10 exhibited moderate affinity only for the σ1 receptor (Ki = 115 ± 1 nM). A
significant increase in the affinity for both subtypes was observed when a cyclohexyl group was present
on the nitrogen heterocycle (11) (Kiσ1 = 0.4 ± 0.0 nM; Kiσ2 = 11.25 ± 0.55 nM). A comparable σ1/2
binding profile was also found for the cyclohexylmethyl analog 12 (Kiσ1 = 1.25 ± 0.25 nM; Kiσ2 = 35.5
± 2.5 nM). The replacement of the cyclohexylmethyl side chain with the benzyl group led to 14, which
presented approximately 354ꢀfold selectivity for σ1 receptors (Kiσ1 = 1.8 ± 0.2 nM, Kiσ2 = 637 ± 15
nM). Related compounds containing a phenylethyl (15) or phenylpropyl (16) moiety failed to display
σ1ꢀselective binding. In fact, both showed a lower σ1 binding affinity (Ki= 8.4 ± 1 nM and 10 ± 2 nM,
respectively) compared to compound 14, and a moderate σ2 affinity (Ki= 209 ± 11 nM and 91.6 ± 7 nM,
respectively). Comparable results were observed for the piperidine derivatives 25-28. We found that
compound 25 bound with high affinity to both subtypes (Kiσ1 = 1.14 ± 0.8 nM; Kiσ2 = 14 ± 2 nM).
Although piperidine 26 bound with lower affinity to σ1 (Ki = 5.56 ± 0.5 nM) than piperazine analog 14,
it exhibited a notable selectivity for σ1 receptors (Kiσ2/Kiσ1 > 400). The elongation of the alkyl chain
(27 and 28) led to a decreased affinity for the σ1 sites (Ki = 13.5 ± 0.5 nM and 25.7 ± 2 nM,
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respectively). These compounds exhibited considerable affinity for σ2 (Ki = 160.4 ± 8 nM; Ki = 100.8 ±
4 nM) with respect to the benzyl analog. These results suggest that the lipoyl function group is well
tolerated in Regions A and B when a suitable moiety is present on the basic nitrogen center. In
particular, in our compounds the Nꢀbenzyl substituent seems to play a pivotal role in discriminating
between the sigma receptor subtypes. Further binding studies performed by Cerep, Inc (Poitiers,
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France), on compounds 14, 15 and 26 using a different experimental protocol procedure (see the
experimental section) furnished better results in terms of σ1 affinity and selectivity. Compounds 14 and
15 had σ1 affinities similar to our values (Ki = 2.5 and 6.5 nM, respectively) but lower σ2 affinities (Ki =
2500 and 3000 nM, respectively). A considerable increase in the σ1 affinity (Ki = 0.4 nM) and a
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reduction in the σ2 affinity (Ki = 8300 nM) were found for piperidine 26. The different Ki values
obtained by Cerep are attributed to the different experimental conditions. However, all together, these
data confirm the affinity and selectivity for σ1 of our compounds. Given the implication of σ1 receptors
in KOPꢀmediated analgesia,42 we analyzed the ability of compound 26 to modulate the analgesic effect
of the systemically injected kappa agonist, 2. Our results demonstrate that the systemic administration of
26 (1 mg/kg sc) does not modify basal tailꢀflick latency. Preꢀtreatment with compound 26, at the same
dose, significantly decreased the antinociceptive effect of 2 (5 mg/kg sc) from 30 min after opioid
administration up to the entire observation period (Figure 2). This antiopioid effect was prevented by the
σ1
antagonist
4ꢀ[4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxyꢀ1ꢀpiperidyl]ꢀ1ꢀ(4ꢀfluorophenyl)ꢀbutanꢀ1ꢀone
(haloperidol).43 To assess the antioxidant properties of compound 26, we performed in vitro studies in
RLM and RBM.38 In particular, we tested this compound’s effects on calcium ionꢀinduced swelling and
on the percentage of reduced thiol groups. Figure 3 shows that the RLM suspension energized by the
oxidation of succinate in the presence of rotenone, when treated with Ca2+ and phosphate, exhibited a
strong decrease in the apparent absorbance at 540 nm. This reduction, indicative of largeꢀamplitude
swelling, was strongly inhibited by compound 26 at 25 ꢀM and abolished at 50 ꢀM. The absorbance
decrease was also completely prevented in the presence of cyclosporin A (CsA), adenosine 5 ꢀ
diphosphate (ADP) or dithioerythritol (DTE) (Figure 3). The colloid osmotic swelling of the matrix,
related to the absorbance decrease, is induced by the mitochondrial permeability transition (MPT),
which is associated with the opening of a proteinaceous pore on the membrane of this organelle.44,45 The
observation that two typical inhibitors of the MPT, CsA and ADP, are able to abolish matrix swelling
confirmed the above statement. Furthermore, the decrease in absorbance prevented by the reductant
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DTE indicated that MPT is also related to mitochondrial oxidative stress. To study the protective
mechanism of compound 26, we analyzed the mitochondrial redox state by evaluating the percentage of
the sulfhydryl groups in their reduced state under different experimental conditions. The RLM, in the
presence of Ca2+ plus phosphate, exhibited a 30% reduction in SH groups versus the control (50
nmol/mg prot.) (Figure 4). This percentage of reduced sulfhydryl groups was restored to the control
value when RLM were treated with compound 26 at 25 ꢀM or 50 ꢀM or with the antioxidant DTE
(Figure 4). Although the protective mechanism of compound 26 is not completely understood, our
results suggest that it has antioxidant effects. Nevertheless, some differences have been found between
assays performed with RBM and those performed with RL mitochondria. As shown in Figure 5, the
RBM suspension exhibited a spontaneous drop off in absorbance at 540 nm, which gradually decreased
over time. In the presence of Ca2+ and phosphate, this reduction was further enhanced without reaching
the extent observed in the RLM. CsA completely abolished this decrease, revealing that this effect is a
consequence of MPTꢀinduced mitochondrial swelling. ADP had only a slight protective effect on RBM,
whereas the reductant DTE was almost completely ineffective (Figure 5). Under these experimental
conditions, compound 26 strongly (25 ꢀM) or completely inhibited (50 ꢀM) MPT induction, confirming
that this compound is much more effective than the tested MPT inhibitors. As shown in Figure 6, the
calcium ion in the presence of phosphate induced oxidation and a decrease in sulfhydryl groups
(approximately 10%) with respect to the untreated RBM. This effect was completely prevented by
compound 26 and DTE, suggesting an antioxidantꢀlike effect. Consistent with these findings, compound
26 had a significant protective effect against MPT induction and oxidative stress in both RLM and
RBM. However, it must be noted that in RLM, MPT induction requires the establishment of oxidative
stress; in fact, some typical MPT inhibitors are antioxidant agents (e.g., DTE). According to all of the
above results, compound 26 behaves like these inhibitors. However, the results obtained in the RBM
assays support the hypothesis, proposed in a previous paper,38 that the mechanism of MPT induction is
not correlated only with oxidative stress. Thus, the strong protective effect exhibited by 26 against the
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MPT is due not only to its antioxidant activity but, most likely, also another mechanism involving pore
opening in RBM. This mechanism could involve the interaction of Ca2+ with its critical binding site(s)46
on adenine nucleotide translocase (AdNT), which is considered the main event leading to the MPT.
Therefore, our results indicate that 26 is an MPT inhibitor with antioxidant properties in both RLM and
RBM. Further studies are in progress to better characterize its possible role in some important process
such as protection against intrinsic apoptosis and the maintenance of mitochondrial redox homeostasis.
Conclusions
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In conclusion, we designed a set of novel bifunctional compounds that have both σ1 agonist and
antioxidant activities that act synergistically. To reach our goal, we combined a natural antioxidant
moiety, αꢀlipoic acid, and an appropriate aminic moiety based on the piperidine or piperazine scaffold
following the Gilligan pharmacophore hypothesis of σ ligands. We also investigated how the chemical
structure of the Nꢀsubstituent and its distance from Region C influence σ1/σ2 selectivity. Compounds
with the Nꢀbenzyl moiety, 14 and 26, displayed the highest affinity and selectivity for the σ1 receptors
(Kiσ1 = 1.8 and 5.5 nM; Kiσ2/σ1 = 354 and 414, respectively). Among the newly tested ligands, we
selected compound 26 on the basis of its excellent σ1 selectivity and agonist activity for evaluation of its
possible antioxidant property. Our results indicate that this compound possesses not only antioxidant
activity but also MPT inhibitor activity. Taking into account that the cell death due to intrinsic apoptosis
is mediated by the opening of the transition pore (MPT induction) as the result of an oxidative stressꢀ
dependent (RLM) or oxidative stressꢀindependent (RBM) mechanism, the observation that the σ1
receptor agonist 26 acts as an MPT inhibitor supports its potential protective role against diseases
related to intrinsic apoptosis and makes it a very promising tool for the treatment of neurodegenerative
diseases.
Experimental Section.
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Chemical Materials and Methods. Reagents and reactants used for synthesis were purchased from
Sigma & Aldrich (Milan, Italy) with the exception of the 1ꢀ(3ꢀphenylpropyl)piperazine that was
furnished by Apollo Scientific Ltd (UK). They were utilized as received without further purification.
Reaction courses were monitored by thinꢀlayer chromatography (TLC) on precoated silica gel 60 F254
aluminium sheets (Merck, Darmstadt, Germany) and the spots were visualized under UV light or in an
iodine chamber. Merck silica gel 60, 230-400 mesh was used for flash column chromatography. Melting
points were determined in open capillary tubes on digital Electrothermal apparatus 9100 (Rochford,
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UK) and are reported uncorrected. H NMR and CꢀNMR spectra were recorded with a Varian Inova
200 and 500 MHz or Varian Mercury 300 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). Gas
chromatographyꢀmass spectroscopy (GCꢀMS) analysis was 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), confirm that the samples had a purity equal to or greater than 95%; the
analytical results were within ± 0.4% of the theoretical values (Table S1, see Supporting Information).
The radioactive materials [3H]ꢀ(+)ꢀPentazocine (sa 29 Ci/mmol) and [3H]ꢀDTG [1,3ꢀdiꢀ(2ꢀtolyl)ꢀ
guanidine] (sa 53.3 Ci/mmol) were obtained from Perkin Elmer.
Radioligand Binding Assays. In vitro σꢀBinding experiments were carried out as previously reported.47
σ1 binding assays were performed on guinea pig brain membranes according to experimental protocol
described by DeHaven et al.48 Briefly, 500 ꢀg of membrane protein were incubated with 3 nM [3H]ꢀ(+)ꢀ
pentazocine (29 Ci/mM; the value of the apparent dissociation constant (Kd) was 14 ± 0.3 nM, n = 3) in
50 mM TrisꢀHCl (pH 7.4). Test compounds were added in concentration ranging from 10ꢀ5 to 10ꢀ11 M.
Nonspecific binding was assessed in the presence of 10 ꢀM of unlabeled haloperidol. The reaction was
performed for 150 min at 37 °C and terminated by filtering the solution through Whatman GF/B glass
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fiber filters which were presoaked for 1 h in a 0.5% poly(ethylenimine) solution. Filters were washed
with ice cold buffer (2 x 4 mL). In the matter of σ2 binding assays49 the membranes 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 so as
to mask σ1 sites. Nonspecific binding was evaluated with DTG (5 ꢀM). Incubation was carried out in 50
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mM TrisꢀHCl (pH 8.0) for 120 min at room temperature and assays were terminated by the addition of
iceꢀcold 10 mM TrisꢀHCl (pH 8.0). Each sample was filtered through Whatman GF/B glass fibers
filters, which were presoaked for 1 h in a 0.5% poly(ethylenimine) solution, using a millipore filter
apparatus. Filters were washed twice with 4 mL of ice cold buffer. Radioactivity was counted in 4 mL
of “Ultima Gold MV” in a 1414 Winspectral PerkinElmer Wallac liquid scintillation counter. Inhibition
constants (Ki values) were calculated using the EBDA/LIGAND program purchased from
Elsevier/Biosoft. Further σ receptor binding studies were performed by Cerep on compounds 12, 13 and
24 as described by Ganapathy et al.50 and Bowen et al.51 Briefly, the σ1 binding assays were conducted
by incubating Jurkat cell membranes with 15 nM [3H]ꢀ(+)ꢀpentazocine (Kd = 37 nM) in 5 nM TrisꢀHCl
buffer (pH 7.4). After addition of test compounds in concentration ranging from 10ꢀ5 to 10ꢀ11 M, the
reaction was conducted for 2 h at 22 °C. Nonspecific binding was evaluated in the presence of unlabeled
haloperidol (10 ꢀM). σ2 Receptors binding assays were performed on rat cerebral cortex membranes
that were incubated with 5 nM [3H](+)ꢀDTG (Kd = 32 nM) in the presence of (+)pentazocine (300 nM)
for masking σ1 sites binding. Test compounds were added in concentration ranging from 10ꢀ5 to 10ꢀ11
M
in 5 nM TrisꢀHCl buffer (pH 7.4). The reaction was performed at 22 °C for 120 min. Nonspecific
binding was defined by the addition of haloperidol (10 ꢀM). The data were obtained using a software
developed at Cerep (Hill software) and validated by comparison with data generated by the commercial
software SigmaPlotꢀ4.0 for Windows.
In vivo studies
Animals. Male SpragueꢀDawley rats (Morini, S. Polo d'Enza, Reggio Emilia, Italy), weighing 180–200
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g, were used. Animals were kept at a constant room temperature (25±1 °C) under a 12:12 h light and
dark cycle with free access to food and water. Each rat 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).
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Nociceptive test. Nociception was evaluated by the radiant heat tailꢀflick test52. Briefly, it consisted of
irradiation of the lower third of the tail with an infrared source (Ugo Basile, Comerio, Italy). The day
before the experiment, rats were habituated to the procedure for measuring nociception threshold.
Experiments were performed at room temperature (25±1 °C). The basal preꢀdrug 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 cutꢀoff latency of 10 s was established to minimize damage to the
tail. Postꢀtreatment tail flick latencies (TFLs) were determined 30, 45, 60, 75 and 90 min after
subcutaneous (sc) injection.
Statistical analysis. All values are presented as means ± SD. Intergroup comparisons were assessed
using an initial twoꢀway analysis of variance (ANOVA) followed by Duncan's multiple range postꢀhoc
test. Differences were considered significant when *p<0.05.
In vitro biological studies
Isolation of RLM and RBM. Rat liver homogenized in isolation medium (pH 7.4) composed of 250
mM sucrose, 5 mM 4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazineethanesulfonic acid (HEPES) and 0.5 mM ethylene
glycol tetraacetic acid (EGTA) was centrifuged at 900 x g for 5 min. The resulting supernatant was reꢀ
centrifuged at 12,000 x g for 10 min to separate the crude mitochondrial pellets; the obtained pellets
were suspended in isolation medium lacking EGTA.53 RBM were isolated using a conventional
differential centrifugation method and purified by the Ficoll gradient method essentially according to
Nicholls54 but with some modifications. Briefly, rat brain (cerebral cortex) was homogenized in
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isolation medium (320 mM sucrose, 5 mM HEPES, 0.5 mM EDTA, pH 7.4) supplemented with 0.3%
bovine serum albumin (BSA) and centrifuged (900 x g) for 5 min. The supernatant was then centrifuged
at 17,000 x g for 10 min to precipitate the crude mitochondrial pellets. These pellets were resuspended
in isolation medium supplemented with 1 mM ATP and layered on top of a discontinuous gradient
composed of 2 ml of isolation medium containing 12% (w/v) Ficoll and 3 ml each of isolation medium
containing 9% and 6% (w/v) Ficoll. The mitochondrial suspension and gradient were centrifuged for 30
min at 75,000 x g. The mitochondrial pellets were suspended in isolation medium and centrifuged for 10
min at 17,000 x g. The resulting pellets were then resuspended in isolation medium without
ethylenediaminetetraacetic acid (EDTA). The protein content was measured by the biuret method using
BSA as the standard.55 These studies were performed in accordance with the guiding principles for the
care and use of animals and were approved by the Italian Ministry of Health.
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Standard incubation conditions for RLM and RBM. RBM and RLM (1 mg protein/ml) were
incubated in a waterꢀjacketed cell at 20 °C. The standard medium contained 200 mM sucrose, 10 mM
HEPES (pH 7.4), 5 mM sodium succinate, 1 mM sodium phosphate, and 1.25 ꢁM rotenone. Ca2+ at 50
or 100 ꢁM was added for the assays using RLM or RBM, respectively. Variations and/or other additions
are described in the sections for the specific experiments.
Evaluation of mitochondrial swelling. The extent of mitochondrial swelling was determined by
measuring the apparent absorbance change of the mitochondrial suspensions at a wavelength of 540 nm
using a Kontron Uvikon model 922 spectrophotometer equipped with a thermostatic control.
Analysis of mitochondrial function. The analysis of protein sulfhydryl groups was performed with
aliquots of mitochondrial suspensions taken from the incubations used to evaluate mitochondrial
swelling. In brief, at the end of the incubation period (15 min), the total suspension (1 mg/ml) was
placed in an Eppendorf 4515c tube and centrifuged for 1 min at 12,000 x g. Then, the supernatant was
discarded, and the pellet used for both measurements. A mitochondrial thiol oxidation assay was
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performed after solubilization of the pellet with 1 mg of solubilization medium (pH = 8.3) composed of
10 mM EDTA, 0.2 M TrisꢀHCl and 1% sodium dodecyl sulfate (SDS) and supplemented with Ellman's
reagent [5,5'ꢀdithiobisꢀ(2ꢀnitrobenzoic acid) or DTNB]. The analysis was performed at 412 nm using a
Kontron Uvikon Model 922 spectrophotometer according to the method of Santos et al.56 Variations
between samples were analyzed by ANOVA, and significance was determined using Student’s tꢀtest.
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Compound Syntheses.
General procedure for the preparation of ketones 10-16
5-(1,2-Dithiolan-3-yl)-1-(4-methylpiperazin-1-yl)pentan-1-one (10)
A solution of lipoic acid (1 g, 5 mmol) in DCM (20 mL) was added dropwise to a solution of DCC
(1.031 g, 5 mmol) in anhydrous DCM (20 mL) under stirring and nitrogen atmosphere. After 10 minutes
a solution of 1ꢀmethylpiperazine (3) (0.5 g, 5 mmol) in DCM (10 mL) was added dropwise and the
resulting mixture was stirred for 3 h. The N,N′ꢀdicyclohexylurea (DCU) that has precipitated is removed
by filtration through a frittedꢀglass Büchner funnel, and the filtrate is washed with sodium bicarbonate
solution (5%) (2 x 10 mL) and brine (2 x 10 mL). After dried over Na2SO4, the solvent was removed
under reduced pressure. During this procedure the additional precipitated DCU was removed by several
filtrations. The organic phase was concentrated and the obtained oil was purified by flash
chromatography using CH2Cl2, CH2Cl2/CH3OH 9,75:0,25 and 9.5/0.5 as mobile phase. The free base
was converted into the corresponding hydrochloride salt with 2 N HCl in diethyl ether and recrystallized
from methanol to give 10 as yellow solid. Yield = 20%; Rf = 0.52 [CH2Cl2/CH3OH, 8:2]; mp = 141.7 °C
1
dec; MS m/z [M]+ = 288.1; IR (NaCl) 2924, 2852, 2792, 1642, 1440; H NMR (500 MHz, CDCl3) δH
1.43ꢀ1.52 (m, 2H), 1.60ꢀ1.74 (m, 4H), 1.87ꢀ1.94 (m, 1H), 2.30ꢀ2.34 (m, 5H), 2.40ꢀ2.48 (m, 5H), 3.08ꢀ
3.10 (m, 1H), 3.11ꢀ3.20 (m, 1H), 3.44ꢀ3.47 (m, 2H), 3.49ꢀ3.50 (m, 1H), 3.54ꢀ3.65 (broad t, 2H). 13C
NMR (50 MHz, CDCl3) δ 171.1, 56.4, 55.0, 54.6, 45.8, 45.2, 41.2, 40.2, 38.5, 34.7, 32.9, 29.1, 24.9.
Anal. (C13H24N2OS2 HCl) C, H, N, S.
1-(4-Cyclohexylpiperazin-1-yl)-5-(1,2-dithiolan-3-yl)pentan-1-one (11)
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This compound was synthesized with the same procedure reported above using the 1ꢀ
cyclohexylpiperazine (4) (0.787 g, 4.84 mmol). The crude product was purified by flash
chromatography on silica gel (EtOAc/cyclohexane 8:2, EtOAc 10, EtOAc/EtOH 9.5:0.5). The free base
was converted into the corresponding hydrochloride salt with 2 N HCl in diethyl ether and recrystallized
from methanol. Yield = 44.1%; Rf = 0.55 [CH2Cl2/MeOH, 9:1]; mp = 173.1 °C dec; MS m/z [M]+ =
1
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1
356; IR (KBr) 2933, 2850, 2809, 1634, 1433. H NMR (200 MHz, CDCl3) δH 1.11ꢀ1.30 (m, 4H), 1.42ꢀ
13
1.97 (m, 11H), 2.29ꢀ2.72 (m, 9H), 3.05ꢀ3.26 (m, 2H), 3.46ꢀ3.78 (m, 6H). C NMR (125 MHz, CDCl3)
δ 171.0, 63.6, 56.4, 49.3, 48.7, 46.1, 42.0, 40.2, 38.5, 34.8, 32.9, 29.1, 28.9, 26.2, 25.8, 25.0. Anal.
(C18H32N2OS2 HCl) C, H, N, S.
1-(4-Cyclohexylmethylpiperazin-1-yl)-5-(1,2-dithiolan-3-yl)pentan-1-one (12)
This compound was prepared by using the 1ꢀ(cyclohexylmethyl)piperazine (5) (0.91 g, 5 mmol)
according to the synthetic method described above. The crude product was purified by flash
chromatography on silica gel using ethyl acetate/ cyclohexane 8:2 as eluting system, converted into the
corresponding hydrochloride salt with 2 N HCl in diethyl ether and recrystallized from methanol. Yield
= 31.2%; Rf = 0.47 [AcOEt/EtOH, 9:1]; mp = 202 °C dec; MS m/z [M]+ = 370.3; IR (NaCl) 2922, 2850,
1643, 1444, 1004. 1H NMR (500 MHz, CDCl3) δH 0.76ꢀ0.83 (m, 2H), 1.17ꢀ1.20 (m, 2H), 1.37ꢀ1.45 (m,
3H), 1.54ꢀ1.70 (m, 9H), 1.81ꢀ1.88 (m, 1H), 2.06ꢀ2.10 (d, 2H), 2.23ꢀ2.30 (m, 6H), 2.36ꢀ2.43 (m, 1H),
13
3.02ꢀ3.14 (m, 2H), 3.36ꢀ3.43 (m, 3H), 3.49ꢀ3.54 (m, 3H). C NMR (50 MHz, CDCl3) δ 171.1, 65.4,
56.4, 53.9, 53.2, 45.6, 41.5, 40.2, 38.4, 34.9, 34.7, 32.9, 31.8, 29.1, 26.7, 26.0, 25.0. Anal.
(C19H34N2OS2 HCl) C, H, N, S.
5-(1,2-Dithiolan-3-yl)-1-(4-phenylpiperazin-1-yl)pentan-1-one (13)
This compound was synthesized by the same procedure reported above using the 1ꢀphenylpiperazine (6)
(0.811 g, 5.0 mmol). The crude product was purified by flash chromatography on silica gel using ethyl
acetate, converted into the corresponding hydrochloride salt with 2 N HCl in diethyl ether and
recrystallized from methanol. Yield = 49.2%; Rf = 0.64 [AcOEt/C2H5OH), 9:1]; mp = 136.2 °C dec; MS
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m/z [M]+ = 350.1; IR (KBr) 3042, 2932, 2853, 1642, 1599, 1578.1H NMR (500 MHz, CDCl3) δH 1.47ꢀ
155 (m, 2H), 1.64ꢀ1.78 (m, 4H), 1.89ꢀ1.95 (m, 1H), 2.38 (t, J = 7.5 Hz, 2H), 2.44ꢀ2.50 (m, 1H), 3.10ꢀ
3.22 (m, 6H), 3.58ꢀ3.64 (m, 3H), 3.78 (t, J = 5 Hz, 2H), 6.89ꢀ6.94 (m, 3H), 7.29 (t, J = 7.5 Hz, 2H). 13C
NMR (50 MHz, CDCl3) δ 171.2, 150.9, 129.2, 120.6, 116.6, 56.4, 49.8, 49.4, 45.5, 41.5, 40.2, 38.5,
34.8, 33.0, 29.1, 25.0. Anal. (C18H26N2OS2 HCl) C, H, N, S.
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1-(4-Benzylpiperazin-1-yl)-5-(1,2-dithiolan-3-yl)pentan-1-one (14)
Compound 12 was prepared by use of the 1ꢀbenzylpiperazine (7) (0.33 g, 1.34 mmol) in agreement with
the procedure reported above. Purification of the crude product was carried out by flash chromatography
on silica gel eluting with ethyl acetate. The free base was converted to the corresponding hydrochloride
salt using 4 M HCl in dioxane and recrystallized from diethyl ether and methanol. Yield = 93%; Rf = 0.3
[CH2Cl2/CH3OH 9:1 ]; mp = 200ꢀ201.3 °C; MS m/z [M]+ = 364.2; 1H NMR (300 MHz, CDCl3) δH 1.39ꢀ
1.55 (m, 2H), 1.61ꢀ1.76 (m, 4H), 1.82ꢀ1.94 (m, 1H), 2.23ꢀ2.50 (m, 7H), 3.03ꢀ3.20 (m, 2H), 3.42ꢀ3.62
(m, 7H), 7.20ꢀ7.35 (m, 5H). 13C NMR (75 MHz, CDCl3) δ 171.3, 138.0, 129.4, 128.6, 127.5, 63.1, 56.7,
53.4, 53.0, 45.7, 41.7, 40.5, 38.7, 35.0, 33.2, 29.4, 25.2. Anal. (C19H28N2OS2 HCl) C, H, N, S.
5-(1,2-Dithiolan-3-yl)-1-(4-phenethylpiperazin-1-yl)pentan-1-one (15)
This compound was synthesized via the same procedure detailed above using the 1ꢀ(2ꢀ
phenethyl)piperazine (8) (0.19 g; 1mmol). The crude product was purified by flash chromatography on
silica gel using CH2Cl2/CH3OH 9.5:0.5 as mobile phase. The free base was converted to its
hydrochloride salt with 4 M HCl in dioxane and recrystallized from diethyl ether and methanol. Yield =
1
45%; Rf = 0.3 [CH2Cl2/CH3OH 9.5:0.5 ]; mp = 160ꢀ161.5 °C; MS m/z [M]+ = 378.2; H NMR (300
MHz, CDCl3) δH 1.40ꢀ1.53 (m, 2H), 1.58ꢀ1.77 (m, 4H), 1.80ꢀ1.92 (m, 1H), 2.23ꢀ2.64 (m, 7H), 2.72ꢀ2.81
(m, 2H), 3.01ꢀ3.19 (m, 2H), 3.43ꢀ3.65 (m, 7H), 7.15ꢀ7.29 (m, 5H). 13C NMR (75 MHz, CDCl3) δ 171.4,
140.0, 129.0, 128.7, 126.4, 60.3, 56.7, 53.5, 53.0, 45.5, 41.5, 40.5, 38.7, 35.0, 33.4, 33.2, 29.3, 25.2.
Anal. (C20H30N2OS2 HCl) C, H, N, S.
5-(1,2-Dithiolan-3-yl)-1-(4-(3-phenylpropyl)piperazin-1-yl)pentan-1-one (16)
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This compound was obtained from the 1ꢀ(3ꢀphenylpropyl)piperazine (9) (1.02 g, 5 mmol) in the same
manner as described above and was purified using the procedure described for compound 12. The oil
obtained was converted into the corresponding hydrochloride salt with 2 N diethyl ether and
recrystallized from methanol. Yield = 31.2%; Rf = 0.36 [AcOEt/EtOH, 8:2], mp = 142.3 °C dec; MS m/z
1
2
3
4
5
6
7
8
9
1
[M]+ = 392.2; IR (NaCl) 3024, 2931, 1643, 1440, 748, 702. H NMR (500 MHz, CDCl3) δH 1.44ꢀ1.54
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(m, 2H), 1.66ꢀ1.76 (m, 3H), 1.80ꢀ1.86 (m, 2H), 1.89ꢀ1.94 (m, 1H), 2.60 (t, J = 7.5 Hz, 2H), 2.36ꢀ2.50
(m, 8H), 2.66 (t, J = 7.5 Hz, 2H), 3.10ꢀ3.21 (m, 2H), 3.46ꢀ3.48 (m, 2H), 3.56ꢀ3.64 (m, 3H), 7.18ꢀ7.21
(m, 3H), 7.29 (t, J = 7.5 Hz, 2H). 13C NMR (50 MHz, CDCl3) δ 171.1, 142.0, 128.3, 125.8, 57.7, 56.4,
53.4, 52.8, 45.6, 41.5, 40.2, 38.5, 34.8, 33.5, 32.9, 29.1, 28.4, 25.0. Anal. (C21H32N2OS2 HCl) C, H, N,
S.
The synthesis of alcohols 21, 23 and 24 has already been reported.40
General procedure for the preparation of esters 25-28.
1-(Cyclohexylmethyl)piperidin-4-yl 5(1,2-dithiolan-3-yl)pentanoate (25)
To a solution of 1ꢀ(Cyclohexylmethyl)piperidinꢀ4ꢀol (21) (0.43 g, 2.20 mmol), αꢀlipoic acid (0.45 g,
42.20 mmol) and DMAP (0.22 mmol, 0.026 mg) in 5.4 mL of dry CH2Cl2 at 0 °C was added 0.45 g of
DCC (2.20 mmol) under stirring and nitrogen atmosphere. After 10 minutes the reaction temperature
was slowly raised to room temperature and the reaction was stirred for over 3 h. The dicyclohexylurea
(DCU) that has precipitated is removed by filtration through a fritted Büchner funnel. The filtrate was
washed twice with 10ꢀmL of sodium bicarbonate solution (5%) and twice with 10ꢀmL of brine solution.
After dried (Na2SO4) the solvent was removed under reduced pressure. During this procedure the
additional precipitated DCU was removed by several filtrations. The organic phase was concentrated
and purified by flash chromatography using ethyl acetate/cyclohexane (7:3). The free base was
converted into the corresponding hydrochloride salt with 4 N HCl in dioxane and recrystallized from
methanol. Yield = 39.6%; Rf = 0.55 (AcOEt); mp = 174.1 °C dec; MS m/z [M]+ = 385.2; IR (NaCl)
1
2921, 2850, 2803, 1730, 1449, 1179, 1034; H NMR (200 MHz, CDCl3) δH 0.83ꢀ0.93 (m, 2H), 1.11ꢀ
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1.99 (m, 19H), 2.05ꢀ2.21 (m, 4H), 2.31 (t, J = 7.4 Hz, 2H), 2.39ꢀ2.54 (m, 1H), 2.61ꢀ2.79 (m, 3H), 3.05ꢀ
1
2
3
4
13
3.25 (m, 2H), 3.50ꢀ3.64 (m, 1H), 4.70ꢀ4.87 (m, 1H). C NMR (50 MHz, CDCl3) δ 172.9, 70.5, 65.5,
5
6
7
8
56.3, 51.3, 40.2, 38.4, 35.3, 34.5, 34.4, 31.9, 30.8, 28.7, 27.7, 26.1, 24.7. Anal. (C20H35NO2S2 HCl) C,
H, N, S.
9
10
11
12
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19
20
21
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1-Benzylpiperidin-4-yl 5-(1,2-dithiolan-3-yl)pentanoate (26)
This compound was prepared from commercially available 1ꢀbenzylpiperidinꢀ4ꢀol (22) (0.19 g, 1mmol)
according to previously reported procedure for the compound 25. The crude product was purified by
flash chromatography on silica gel using cyclohexane/ethyl acetate (7:3) as eluting system. The oil
obtained was converted to its hydrochloride salt with 4 M HCl in dioxane and recrystallized from
isopropanol. Yield = 78%; Rf = 0.6 (AcOEt/cyclohexane 7:3); mp = 191.8–193.1 °C; MS m/z [M]+ =
1
379.58; H NMR (200 MHz, CDCl3) δH 1.44ꢀ1.99 (m, 11H), 2.21ꢀ2.68 (m, 7H), 3.05ꢀ3.25 (m, 2H),
13
3.50ꢀ3.63 (m, 3H), 4.75ꢀ4.85 (m, 1H), 7.26ꢀ7.32 (m, 5H). C NMR (75 MHz, CDCl3) δ 173.2, 138.6,
129.3, 128.4, 127.3, 70.5, 63.2, 56.6, 51.0, 40.4, 38.7, 34.8, 34.6, 31.1, 29.0, 25.0. Anal. (C20H29NO2S2
HCl) C, H, N, S.
1-Phenethylpiperidin-4-yl 5-(1,2-dithiolan-3-yl)pentanoate (27)
This compound was synthesized from the 1ꢀphenethylꢀpiperidinꢀ4ꢀol (23) (0.995 g, 4.85 mmol) by the
general via reported above. The crude product was purified using the procedure described for 25 and the
obtained oil was converted to its hydrochloride salt with 4 N HCl in dioxane and recrystallized from
methanol. Yield = 69.5%; Rf = 0.5 [AcOEt/CH3OH, 8:2]; mp = 155.8 °C dec; MS m/z [M]+ = 394.3; IR
1
(NaCl) 3026, 2932, 2808, 1728, 1603, 1548; H NMR (200 MHz, CDCl3) δH 1.46ꢀ1.57 (m, 2H), 1.60ꢀ
2.0 (m, 8H), 2.29ꢀ2.61 (m, 8H), 2.72ꢀ2.85 (m, 4H), 3.05ꢀ3.26 (m, 2H), 3.51ꢀ3.64 (m, 1H), 4.75ꢀ4.87 (m,
1H), 7.16ꢀ7.34 (m, 5H). 13C NMR (50 MHz, CDCl3) δ 172.9, 140.3, 128.6, 128.3, 125.9, 70.1, 60.4,
56.3, 50.8, 40.2, 38.4, 34.6, 34.4, 33.8, 30.8, 28.7, 24.7. Anal. (C21H31NO2S2 HCl) C, H, N, S.
1-(3-Phenylpropyl)piperidin-4-yl 5-(1,2-dithiolan-3-yl)pentanoate (28)
This compound was prepared by using the 1ꢀ(3ꢀphenylpropyl)ꢀpiperidinꢀ4ꢀol (24) (1.06 g, 4.85 mmol)
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by the same procedure previously employed for 25ꢀ27. The crude product was purified by flash
chromatography and eluted with ethyl acetate/cyclohexane ethyl (7:3). The free base was converted to
its hydrochloride salt with 4 N HCl in dioxane and recrystallized from methanol. Yield = 76.2%; Rf =
0.53 [AcOEt/CH3OH, 8:2]; mp = 121.4 °C dec; MS m/z [M]+ = 407.15; IR (NaCl) 3024, 2929, 2856,
1729, 1453; 1H NMR (200 MHz, CDCl3) δH 1.43ꢀ1.99 (m, 13H), 2.16ꢀ2.59 (m, 7H), 2.63ꢀ2.74 (m, 4H),
3.08ꢀ3.25 (m, 2H), 3.46ꢀ3.64 (m, 1H), 4.72ꢀ4.84 (m, 1H), 7.13ꢀ7.33 (m, 5H). 13C NMR (50 MHz,
CDCl3) δ 172.9, 142.1, 128.3, 128.2, 125.7, 70.2, 57.9, 56.3, 50.8, 40.2, 38.4, 34.6, 34.3, 33.7, 30.8,
28.8, 28.7, 24.7. Anal. (C22H33NO2S2 HCl) C, H, N, S.
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Supporting Information Available: Elemental analysis results. This material is available free of
charge via the Internet at http://pubs.acs.org.
Corresponding Author Information: *Phone +39ꢀ095ꢀ7384205; eꢀmail; prezzave@unict.it.
Abbreviations Used
AdNT, adenine nucleotide translocase; ADP, adenosine 5 ꢀdiphosphate; ALA, alpha lipoic acid; ARE,
antioxidant response element; ATP, adenosine 5 ꢀtriphosphate; Bip, Binding immunoglobulin protein;
BSA, bovine serum albumin; CsA, cyclosporin A; DCC, N,N′ꢀdicyclohexylcarbodiimide; DCM,
dichloromethane; DCU, N,N′ꢀdicyclohexylurea; DMAP, 4ꢀ(N,Nꢀdimethylamino)pyridine; DTE,
dithioerythriol; DTG, 1,3ꢀdiꢀ(2ꢀtolyl)ꢀguanidine; DTNB, 5,5'ꢀdithiobisꢀ(2ꢀnitrobenzoic acid); EDTA,
ethylenediaminetetraacetic acid; EGTA, ethylene glycol tetraacetic acid; ER, endoplasmic reticulum;
GCꢀMS, gas chromatographyꢀmass spectrometry; haloperidol, 4ꢀ[4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀhydroxyꢀ1ꢀ
piperidyl]ꢀ1ꢀ(4ꢀfluorophenyl)ꢀbutanꢀ1ꢀone; HEPES, 4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazineethanesulfonic
acid; IACUC, Institutional Animal Care and Use Committee; IP3R, inositol 1,4,5ꢀtrisphosphate
receptor; IR, infrared; Ki, inhibition constant; KOP, kappa opioid; M+, parent molecular ion; MAM,
mitochondriaꢀassociated ER membrane; mp, melting point; MPT, mitochondrial permeability transition;
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Journal of Medicinal Chemistry
MS, mass spectrometry; m/z, massꢀtoꢀcharge ratio (not m/e); NMR, nuclear magnetic resonance; NQO1,
NAD(P)H:quinone oxidoreductase 1; (+)ꢀpentazocine, (2S,6S,11S)ꢀ6,11ꢀdimethylꢀ3ꢀ(3ꢀmethylbutꢀ2ꢀenꢀ
1ꢀyl)ꢀ1,2,3,4,5,6ꢀhexahydroꢀ2,6ꢀmethanoꢀ3ꢀbenzazocinꢀ8ꢀol; RBM, rat brain mitochondria; Rf, retention
factor (in chromatography); RLM, rat liver mitochondria; ROS, reactive oxygen species; Rotenone;
(2R,6aS,12aS)ꢀ1,2,6,6a,12,12aꢀ hexahydroꢀ2ꢀisopropenylꢀ8,9ꢀ dimethoxychromeno[3,4ꢀb] furo(2,3ꢀ
h)chromenꢀ6ꢀone; rt, room temperature; SAR, structureꢀactivity relationship; sc, subcutaneous; SD,
standard deviation; SDS, sodium dodecyl sulphate;σ, sigma receptor; SOD1, superoxide dismutase 1;
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TFLs,
tail
flick
latencies;
TLC,
thinꢀlayer
chromatography;
TrisꢀHCl,
Tris(hydroxymethyl)aminomethane hydrochloride; UV, ultraviolet.
References
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Table 1. σ Binding affinities of piperidines 10-16 and piperazines 25-28
σ Binding affinities (Ki, nM ± SD)a
Page 26 of 37
1
2
3
4
5
6
7
8
Cmpd
σ1
σ2
Kiσ2/Kiσ1
[3H]-Pentazocine
115 ± 1
[3H]-DTG
21000 ± 400
11.25 ± 0.55
35.5 ± 2.5
10600 ± 300
182.6
28
28.4
6.23
10
11
12
13
0.4 ± 0.0
1.25 ± 0.25
1700 ± 100
9
10
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60
1.8 ± 0.2
2.5*
8.4 ± 1
6.5*
637 ± 15
2500*
209 ±11
3000*
91.6 ± 7
14 ± 2
2302 ± 45
8300*
353.8
1000
24.8
461
9.16
12.3
414
>20000
11.8
3.9
14
15
10 ± 2
16
25
26
1.14 ± 0.8
5.56 ± 0.5
0.4*
13.5 ± 0.5
25.7 ± 2
160.4 ± 8
100.8 ± 4
27
28
a Each value is the means ± SD of three determinations. *Data obtained from Cerep laboratories were
determined by single experiments.
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Figure Legends
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Figure 1. The four pharmacophore regions proposed by Gilligan et al., matched with our piperazine and
piperidine derivatives containing the lipoyl function, are represented by a distal aromatic ring (Region
A), a space between the nitrogen heterocycle and the distal hydrophobic site (Region B), a nitrogen
heterocycle (Region C), and a substituent on the basic nitrogen (Region D).
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Figure 2. Effect of 26 on the antinociception induced by 2. Haloperidol reversed the antiopioid effect of
26. The results are expressed as the mean ± SD. *p<0.05 vs 2-treated rats (n= 8−10). #p<0.05 vs 26 + 2-
treated rats (n= 8−10).
Figure 3. Liver mitochondria swelling induced by Ca2+. Prevention of swelling by compound 26, CsA,
ADP and DTE. Where indicated, 1 ꢂM CsA, 0.5 mM ADP, or 3 mM DTE was present. 26 was present
at the indicated concentrations (ꢂM). A downward deflection indicates a decrease in absorbance. The
assays were performed five times with comparable results.
Figure 4. Effect of 26 or DTE on sulfhydryl group oxidation induced by Ca2+ in RLM. 26 was present
at the indicated concentrations (ꢂM). DTE was present at a concentration of 3 mM. Data are expressed
as the percentage of reduced thiol groups and represent average ± mean SD from five independent
experiments.
Figure 5. Brain mitochondria swelling induced by Ca2+. Prevention of swelling by compound 26, CsA,
ADP and DTE. The RBM incubation conditions, compound concentrations, meaning of the downward
deflection, and statistics were as described for Figure 3.
Figure 6. Effect of 26 and DTE on the sulfhydryl group oxidation induced by Ca2+ in RBM. The RBM
incubation conditions compound concentrations, representation of the data, and statistics were as
described in Figure 4. Where indicated, 50 ꢂM 26 or 3 mM DTE was present.
Scheme Legends
Scheme 1.a
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a Reagents and conditions: a) lipoic acid, DCC, DCM, 3 h rt.
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Scheme 2.a
a Reagents and conditions: a) Toluene, K2CO3, reflux 24 h; b) lipoic acid, DCC, DMAP, DCM, 3 h rt.
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Journal of Medicinal Chemistry
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