Synthesis and resolution of cis -(±)-methyl (1 R,2 S /1 S,2 R)-2-[(4-hydroxy-4-phenylpiperidin-1-yl)methyl]-1-(4-methylphenyl) cyclopropanecarboxylate [(±)-PPCC)]: New σ receptor ligands with neuroprotective effect

Article abstract of DOI:10.1021/jm100116p

The enantiomers of cis-(±)-methyl (1R,2S/1S,2R)-2-[(4-hydroxy-4- phenylpiperidin-1-yl)methyl]-1-(4-methylphenyl)cyclopropanecarboxylate [1, (±)-PPCC], a selective - ligand, were synthesized. The (+)- and (?)-enantiomers bind predominantly to ?₁ receptors and have a reduced ?₂ affinity. Both individually restore the astroglial oxidative status modified by glutamate, counteracting also transglutaminase-2 overexpression. They exhibited in vivo anti-opioid effects on - opioid (KOP) receptor-mediated analgesia. Our findings demonstrate that the enantiomers display mainly ?₁ agonist activity and that they have neuroprotective effects.

Full text of DOI:10.1021/jm100116p

J. Med. Chem. 2010, 53, 5881–5885 5881
DOI: 10.1021/jm100116p
Synthesis and Resolution of cis-(()-Methyl (1R,2S/1S,2R)-2-[(4-Hydroxy-4-phenylpiperidin-1-
yl)methyl]-1-(4-methylphenyl)cyclopropanecarboxylate [(()-PPCC)]: New σ Receptor Ligands
with Neuroprotective Effect
‡
§
†
†
ꢀ §
Orazio Prezzavento,*,† Agata Campisi, Carmela Parenti, Simone Ronsisvalle, Giuseppina Arico, Emanuela Arena,
Marco Pistolozzi, Giovanna M. Scoto,§ Carlo Bertucci, Angelo Vanella,‡ and Giuseppe Ronsisvalle†
†Department of Pharmaceutical Sciences, Medicinal Chemistry Section, ‡Department of Biological Chemistry, Medical Chemistry, and Molecular
Biology, and §Department of Pharmaceutical Sciences, Pharmacology Section, University of Catania, Viale A. Doria 6, 95125 Catania, Italy, and
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy
Received October 29, 2009
The enantiomers of cis-(()-methyl (1R,2S/1S,2R)-2-[(4-hydroxy-4-phenylpiperidin-1-yl)methyl]-1-(4-
methylphenyl)cyclopropanecarboxylate [1, (()-PPCC], a selective σ ligand, were synthesized. The (þ)-
and (-)-enantiomers bind predominantly to σ1 receptors and have a reduced σ2 affinity. Both
individually restore the astroglial oxidative status modified by glutamate, counteracting also transglu-
taminase-2 overexpression. They exhibited in vivo anti-opioid effects on κ opioid (KOP) receptor-
mediated analgesia. Our findings demonstrate that the enantiomers display mainly σ1 agonist activity
and that they have neuroprotective effects.
Introduction
dependentproteins, mitochondrial impairment, anddecreases
in ATP concentration and reactive oxygen species (ROS)
production, which in concert contribute to neuronal cell death.8
It has been demonstrated that the recovery of cellular redox
status achieved by antioxidants is accompanied by a concomitant
and dose-dependent reduction in glutamate-induced transgluta-
minase-2 (TG2) up-regulation.9 TG2 is a calcium-dependent
multifunctional protein implicated in several acute and chronic
neurodegenerative diseases associated with excitotoxicity.10
Inprevious studies, wereported the synthesisand biological
profile of the new σ selective ligand cis-(()-methyl (1R,2S/
1S,2R)-2-[(4-hydroxy-4-phenylpiperidin-1-yl)methyl]-1-(4-
methylphenyl)cyclopropanecarboxylate [1, (()-PPCC]11,12
(Figure 1). We demonstrated that 1 was able to modulate TG2
expression in primary rat astroglial cell cultures.11
Herein, we report the synthesis of the corresponding (þ)-
and (-)-enantiomers in high optical purity and their σ recep-
tor binding affinities. Furthermore, we describe the effects of
1, its enantiomers, (þ)-pentazocine, and ibogaine on primary
rat neocortical astroglial cell cultures in the absence or in the
presence of glutamate on intracellular oxidative status and on
glutamate-induced TG2 overexpression.
The σ a receptors are a distinct class of proteins with wide-
spreaddistribution inthe centralnervous system (CNS) and in
peripheral tissues.1 Biochemical and pharmacological studies
have suggested that there are at least two types of σ receptors
(σ1 and σ2)2 which areinvolved innumerous physiologicaland
pharmacological functions.3,4 σ2 receptor agonists produce
transient and sustained increases in intracellular calcium ion
concentration [Ca2þ]i, and have been implicated in the induc-
tion of apoptosis.4
The activation of cloned σ1 receptor,5 in vitro and in vivo,
prevents the neuronal death associated with glutamate
excitotoxicity.3,6 It is well-known that hyperstimulation of
glutamate receptors leads to the neuronal loss associated with
many neurological diseases.7 In fact, briefly exposing differ-
entiated astrocytes to glutamate causes an increase in [Ca2þ]i,
glutathione (GSH) depletion, the activation of several calcium-
*To whom correspondence should be addressed. Phone: þ39-095-
738-4205. Fax: þ39-095-222-239. E-mail: prezzave@unict.it.
a Abbreviations: ATP, adenosine triphosphate; CD, circular dichro-
ism; DCFDA, 2,7-dichlorofluorescein diacetate; DIV, days in vitro;
DMEM, Dulbecco’s modified Eagle’s medium; DMF, N,N-dimethyl-
formamide; DTG, 1,3-di-(2-tolyl)guanidine; ECL, enhanced chemilu-
minescence; EDTA, ethylenediaminetetraacetic acid; ee, enantiomeric
excess; EGTA, ethylene glycol tetraacetic acid; FBS, fetal bovine serum;
GFAP, glial fibrillary acidic protein; GSH, glutathione; GSSG, glu-
tathione disulfide; haloperidol, 4-[4-(4-chlorophenyl)-4-hydroxypiperi-
din-1-yl]-1-(4-fluorophenyl)butan-1-one; HPLC, high performance
liquid chromatography; HRP, horseradish peroxidase; ibogaine, 12-
methoxyibogamine; IgG, immunoglobulin G; LDH, lactate dehydro-
genase; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide;
NMR, nuclear magnetic resonance; PBS, phosphate buffered saline;
(þ)-pentazocine, (2S,6S,11S)-6,11-dimethyl-3-(3-methylbut-2-en-1-yl)-
1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocin-8-ol; PMSF, phenyl-
methylsulfonyl fluoride; ROS, reactive oxygen species; sc, subcuta-
neous; SD, standard deviation; SDS, sodium dodecyl sulfate; σ, sigma
receptor; TFLs, tail flick latencies; TG2, transglutaminase-2; TLC, thin-
layer chromatography; UV, ultraviolet.
Because σ1 receptor agonists attenuate KOP-induced an-
algesia while σ1 receptor antagonists potentiate KOP-induced
analgesia,12-14 we also performed in vivo studies to determine
whether the enantiomers were able to modulate the analgesic
effect induced by the KOP agonist trans-(1S,2S)-3,4-dichloro-
N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide
[2, (-)-U50,488H]15,16 in rats.
Chemistry
Treatment of cis-lactone (()-3, which was prepared as pre-
viously described,11,17 with R-(þ)-R-methylbenzylamine pro-
vided two diastereoisomeric amides, (þ)-4and (-)-4(Scheme1;
r
2010 American Chemical Society
Published on Web 07/15/2010
pubs.acs.org/jmc

5882 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Prezzavento et al.
Table 1. GSH and ROS Levels in Astroglial Cell Culturesa
ROS levels (nmol of
dichlorofluoresceine
produced per mg
protein/30 min)
GSH levels
(nmol/mg
protein)
treatment
control
glutamate
ibogaine
1
14.61 ( 2.1
9.08 ( 0.9*
9.95 ( 1.1*
11.80 ( 0.5*
7.95 ( 1.1*
14.45 ( 0.3
14.15 ( 0.4**
8.05 ( 0.8*
14.87 ( 0.9
13.44 ( 0.7**
13.99 ( 0.9
14.87 ( 0.2**
3.16 ( 0.41
4.45 ( 0.51*
4.99 ( 0.25*
4.25 ( 0.31*
5.82 ( 0.76*
3.68 ( 0.33
3.46 ( 0.44**
5.51 ( 0.81*
3.88 ( 0.19
3.05 ( 0.29**
3.39 ( 0.24
3.67 ( 0.31**
Figure 1
Scheme 1a
glutamate þ ibogaine
(þ)-pentazocine
glutamate þ (þ)-pentazocine
glutamate þ 1
(þ)-7
glutamate þ (þ)-7
(-)-7
glutamate þ (-)-7
a (/) p < 0.05 vs control; (//) p < 0.001 vs control.
Cell cultures were prepared and characterized by immuno-
fluorescent staining for a specific marker, such as glial fibril-
lary acidic protein (GFAP). About 90-95% of the cells were
GFAP-positive, indicating that the cultures were enriched in
astrocytes. The effects of racemate and its enantiomers were
compared with (þ)-pentazocine, a putative σ1 agonist,13 and
ibogaine, a σ2 agonist.20 Specifically, we selected ibogaine
because, even though it interacts with numerous biological
systems in the CNS, it is known to possess a higher affinity for
σ2 receptors than for any other known neurotransmitter
receptors.21 In preliminary experiments, we established the
optimal concentrations of the σ ligands and their optimal
exposure times to the cultures in the absence or in the presence
of glutamate (500 μM).8 This set of experiments showed that
25 μM was a nontoxic concentration of the various drugs for
the cells and that the optimal exposure time was 24 h (data not
shown). It has been reported that ibogaine at concentrations
up to 130 μM does not affect glutamate uptake or release by
rat astrocyte cultures.22
To evaluate the σ ligands’ ability to modify the intracellular
oxidative status of the astroglial cell cultures, we tested GSH
and ROS levels. Significant increases in GSH depletion and
ROS production were observed in glutamate-exposed cell
cultures when compared with the untreated control cultures
(Table 1). Ibogaine had the same effect as glutamate, but its
effect was more evident when the cell cultures were co-treated
with the neurotransmitter. Compound 1 induced a significant
increase in GSH depletion and ROS production with respect
to the controls. Nevertheless, the racemate’s effect was less
pronounced than that of ibogaine or treatment with gluta-
mate. The combination of 1 and glutamate treatment caused a
further increase in GSH depletion and ROS production
(Table 1). These data demonstrate that 1 and ibogaine induce
oxidative stress, which leads to astroglial death by activating
the apoptotic pathway, as previous reported.11,23
a Reagents and conditions: (a) 2-hydroxypyridine, toluene, 70 °C,
24 h; (b) separation by flash chromatography; (c) 1 N H2SO4, dioxane/H2O
(1:1), 5 h, 85 °C; (d) SOBr2, CH3OH, room temp, 24 h; (e) 4-phenylpi-
peridin-4-ol, DMF, NaHCO3, 60 °C, 8 h.
also see Supporting Information). Separation by flash chro-
matography and subsequent hydrolysis of the amides gave
lactones (þ)-5 and (-)-5. Enantiomeric bromides (þ)-6 and
(-)-6 were obtained by esterification of the intermediate
lactones with methanol and thionyl bromide. The alkylation
of commercially available 4-phenylpiperidin-4-ol with the
appropriate enantiomer of the bromoester gave the two
desired enantiomers (þ)-7 and (-)-7.
The absolute configuration of the cyclopropane ring was
established by comparison with reported 1H NOE NMR
data18 and by X-ray diffraction analysis.19 Circular dichroism
(CD) spectra of (þ)-7 and (-)-7 are reported in Figure 6 (see
Supporting Information).
Results and Discussion
The target compounds were evaluated with respect to their
σ
1/2 receptor binding affinities. (þ)-7 and (-)-7 display a high
(þ)-Pentazocine did not modify GSH or ROS levels in
untreated cells. However, it was able to completely counteract
the effect of glutamate. Exposure to (þ)-7 or (-)-7 alone or in
glutamate-exposed cells showed a pattern similar to that of
(þ)-pentazocine (Table 1). These data demonstrate that both
enantiomers and (þ)-pentazocine are able to restore oxidative
status after it has been modified by glutamate.
Figures 2 and 3 respectively show immunoblots (A) and
densitometric (B) analyses of TG2 expression in astroglial cell
cultures exposed to σ ligands in the absence or in the presence
of glutamate. A significant increase in the percentage of TG2
affinity for the σ1 subtype (Ki = 1.9 ( 0.1 and 13 ( 1.2 nM,
respectively). In these experiments 1 gave slightly different Ki
values (Ki(σ1) = 1.8 ( 0.08 nM, Ki(σ2) = 20.8 ( 1.8 nM) than
in our previous experiments (Ki(σ1) = 1.5 ( 0.06 nM, Ki(σ2) =
50.8 ( 3.0 nM).11 The (þ)-7 and (-)-7 exhibit moderate σ2
binding affinities (Ki = 50.2 ( 5 and 103 ( 4 nM, respecti-
vely).
To assess the biological effects of the σ ligands, we used
primary rat neocortical astroglial cell cultures that were 14 days
in vitro (DIV) in the absence or in the presence of glutamate.8

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5883
than the control (Figure 2). This σ1 agonist was able to
completely counteract glutamate-induced TG2 up-regulation
(Figure 3). A slight enhancement of TG2 expression was
caused by (þ)-7, even though it produced a down-regulation
of TG2 in the presence of glutamate (Figures 2 and 3). The
other enantiomer, (-)-7, did not exert any effect on TG2
expression (Figure 2). However, it was able to totally counter-
act the effect of glutamate exposure on TG2 expression.
Densitometric analysis showed that (-)-7 had a pattern
similar to that of (þ)-pentazocine on TG2 expression
(Figure 3). Our findings demonstrate that the individual
enantiomers of 1 modulate TG2 expression differently in the
absence or in the presence of glutamate. In particular, we
observed that (-)-7 has more effect on TG2 expression than
(þ)-7. Taken together, our data suggest that (þ)-7 and (-)-7,
at least under our experimental conditions, act mainly as σ1
agonists. The different effects of racemate may be due to a
concentration dependent effect or to an interaction with other
protein targets. Moreover, given the involvement of σ receptors
in different forms of calcium signaling in neuronal cells,24-26
the obtained results could also be justified by the effects of our
σ ligands on [Ca2þ]i. However, the pharmacological charac-
terization of the interaction between σ receptors and calcium
channels has not yet been well elucidated.27 In fact, some
findings demonstrated that there is no correlation between the
potency of the tested compounds and their σ binding affini-
ties,28 while in other experiments, σ1 agonists and σ1 antago-
nists produced the same effects, which might also be due to the
involvement of σ2 receptors.29 Furthermore, the selective σ1
agonists (þ)-pentazocine and 2-(4-morpholino)ethyl-1-phe-
nylcyclohexane-1-carboxylate (PRE 084) caused opposite
effects on the increase in [Ca2þ]i.30 On the basis of these
literature data,27-30 we hypothesize that in our results the
racemate or the single enantiomers may differently modulate
the [Ca2þ]i and consequently TG2 expression levels and
functions.
Figure 2. Representative immunoblot (A) and densitometric
(B) analyses of TG2 expression in astroglial cell cultures: (/) p < 0.05
vs control.
The in vivo studies were performed with the aim of better
understanding the pharmacological profile of the individual
enantiomers. Systemic administration of (þ)-7 or (-)-7 at a
dose of 1 mg/kg sc (this dosage was chosen from the previous
evaluation of racemate’s dose-response curve)12 did not mod-
ify the basal tail flick latency in rats, whereas the enantiomers
were able to prevent the analgesic effect induced by the KOP
agonist 2. However, pretreatment with (-)-7 significantly
decreased analgesic effect of 2 about 30 min after opioid
administration, while thedextroisomer’seffectwas significant
over the entire observation period (see Supporting Informa-
tion Figure 4A,B). This anti-opioid effect was prevented, at
least at the dose utilized, by pretreatment with the putative σ1
antagonist haloperidol (data not shown).14 Because it has
been reported that σ1 agonists are able to antagonize KOP-
mediated analgesic response, wehave further confirmed the σ1
agonist activity of (þ)-7 and (-)-7 through their anti-opioid
effect.
In conclusion, our data demonstrate that (þ)-7 and (-)-
7, different from the racemate, are able to restore GSH
and ROS levels to control values in glutamate-exposed
astroglial cell cultures. Furthermore, both enantiomers,
when administered individually, counteract the glutamate
induced TG2 overexpression. Thus, our findings show
that (þ)-7 and (-)-7 possess neuroprotective effects.
Studies are now in progress to clarify the astrocytes’
different response to the single enantiomers and to the
racemate.
Figure 3. Representative immunoblot (A) and densitometric
(B) analyses of TG2 expression in astroglial cell cultures exposed
to glutamate: (/) p < 0.05 vs control; (//) p < 0.05 vs glutamate.
expression was observed in glutamate-exposed cells when
compared with the control cultures (Figure 3). Ibogaine
produced a marked up-regulation of the protein (Figure 2).
The presence of glutamate and ibogaine induced a further
increase in TG2 expression (Figure 3). Compound 1 alone or
in the presence of glutamate also caused a significant up-
regulation in TG2 expression (Figures 2 and 3). However, the
effect appeared less pronounced than that induced by ibo-
gaine (Figures 2). In contrast, in the presence of glutamate,
they showed a similar pattern (Figure 3). (þ)-Pentazocine did
not induce TG2 expression. In fact, cells exposed to (þ)-
pentazocine showed lower levels of the protein expression

5884 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Prezzavento et al.
Experimental Section
Acknowledgment. This work was supported by grants
(FIRB 2003 (Grant No. RBNE03FH5Y) and PRIN 2005
and 2007 (Grant No. 2005032713)) from the MIUR (Rome).
We also thank Slater and Frith for the gift of ibogaine.
For complete details, refer to the Supporting Information.
General Experimental Details. All commercial reagents used
for synthesis were purchased from Sigma-Aldrich (Milan, Italy)
unless otherwise specified. Flash column chromatography was
1
Supporting Information Available: Analgesic effects, elemen-
tal analysis results, chiral HPLC traces, CD and UV spectra, and
details of compound synthesis and biological experiments. This
material is available free of charge via the Internet at http://
pubs.acs.org.
carried out on Merck silica gel 60 (230-400 mesh). H NMR
spectra (δ, ppm) were recorded with a Varian Inova 200 MHz
spectrometer (Varian, Leini, Italy). HPLC analysis was per-
formed at room temperature on a Jasco HPLC system using a
CHIRALCEL AD, 250 mm ꢀ 4.6 mm, 5 μm particle-size HPLC
column (Daicel Chemical Industries LTD) and hexane/2-pro-
panol (97:3) containing 0.1% (v/v) diethylamine (Fluka) as an
eluent. CD spectra were recorded with a Jasco J-810 spectro-
polarimeter (Jasco, Tokyo). Elemental analyses (C, H, N) were
determined on an elemental analyzer Carlo Erba model 1106
(Carlo Erba, Milan, Italy) and were within (0.4% of the
theoretical values. Purities of tested compounds were deter-
mined by elemental analysis and HPLC and were g99%.
(þ)-Methyl (1R,2S)-2-[(4-Hydroxy-4-phenylpiperidin-1-yl)-
methyl]-1-(4-nitrophenyl)cyclopropanecarboxylate Oxalate [(þ)-7].
A stirred mixture of 4-phenylpiperidin-4-ol (170 mg, 0.96 mmol),
(þ)-methyl (1R,2S)-2-(bromomethyl)-1-(4-methylphenyl)cyclo-
propanecarboxylate (þ)-6 (300 mg, 1.05 mmol), and NaHCO3
(162 mg, 1.92 mmol) in dry DMF (10 mL) was heated to 60 °C
under a nitrogen atmosphere for 8 h. After cooling, the mixture
was evaporated to dryness and CH2Cl2 was added to the residue.
The organic phase was washed with aqueous NaHCO3 (5%),
dried over anhydrous Na2SO4, and evaporated under reduced
pressure. The residue was purified on a silica gel column
(cyclohexane/ethyl acetate, 6:3) to afford a colorless oil in
80% yield, which was converted to its oxalate salt. The enantio-
meric excess was determined to be 99.5% on a chiral HPLC with
a Chiralpak AD chiral column (25 cm ꢀ 0.46 cm i.d.) using
hexane/2-propanol/triethylamine (97:3:0.1, 1 mL/min) as the
eluent. Mp 162-163 °C (dec). 1H NMR (CDCl3): δ 1.25 (t, 1H,
J = 7.0 Hz), 1.33 (dd, 1H, J = 4.4, 9.0 Hz,), 1.66 (dd, 1H, J =
4.4, 7.0 Hz), 1.79 (m, 2H), 2.22 (m, 2H), 2.33 (s, 3H), 2.59 (m,
2H), 2.78 (m, 2H), 2.96 (m, 2H), 3.63 (s, 3H), 7.09-7.55 (m, 9H).
Rf = 0.30 (cyclohexane:ethyl acetate, 6:3). [R]2D0 þ44° (1% p/v,
MeOH). Anal. (C24H29NO3 C2H2O4) C, H, N.
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1
excess = 97.4%. Mp 156-157 °C (dec). H NMR (CDCl3): δ
1.26 (t, 1H, J = 7.0 Hz), 1.33 (dd, 1H, J = 4.4, 9.0 Hz,), 1.67 (dd,
1H, J = 4.4, 7.0 Hz), 1.78 (m, 2H), 2.22 (m, 2H), 2.33 (s, 3H),
2.59 (m, 2H), 2.78 (m, 2H), 2.96 (m, 2H), 3.63 (s, 3H), 7.07-7.55
(m, 9H). Rf = 0.29 (cyclohexane/ethyl acetate, 6:3); [R]2D0 -46°,
(1% p/v, MeOH). Anal. (C24H29NO3 C2H2O4) C, H, N.
3
The purity of (þ)-7 and (-)-7 was determined by reversed-
phase HPLC assays. The mobile phase consisted of Tris-HCl
(50 mM, pH 7.4)/acetonitrile (60:40, v/v). Flow rate of 2 mL/min
was employed. These chromatographic conditions allowed elut-
ing and separating all the impurities contained into the samples
within 10 min. Chromatograms were recorded setting the de-
tector at 205 nm. Stock solutions were prepared as 1 mg/mL in
acetonitrile and diluted to 0.5 mg/mL with Tris-HCl buffer
before the injection. An amount of 20 μL of the solutions
was injected for each run. The retention time for (þ)-7 and (-)-
7 free bases were approximately 2.5 min, while oxalate ap-
peared at the solvent front. The purity was calculated as the
percentage of the main peak area over the sum of the areas of
all the peaks in the chromatograms. The two samples showed a
purity of g99%.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5885
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