Novel potent and selective θ ligands: Evaluation of their agonist and antagonist properties

Article abstract of DOI:10.1021/jm200144j

Novel enantiomers and diastereoisomers structurally related to θ ligand (+)-MR200 were synthesized to improve θ₁/θ ₂ subtype selectivity. The selective θ₁ ligand (-)-8 showed an antagonist profile determined by phenytoin differential modulation of binding affinity in vitro, confirmed in vivo by an increase of κ opioid analgesia. The θ₂ ligand (-)-9 displayed agonist properties in an in vitro isolated organ bath assay and antiproliferative effects on LNCaP and PC3 prostate cancer cell lines.

Full text of DOI:10.1021/jm200144j

BRIEF ARTICLE
pubs.acs.org/jmc
Novel Potent and Selective σ Ligands: Evaluation of Their
Agonist and Antagonist Properties
Agostino Marrazzo,*,† Enrique J. Cobos,§ Carmela Parenti,‡ Giuseppina Aricꢀo,‡ Giuseppina Marrazzo,†
Simone Ronsisvalle,† Lorella Pasquinucci,† Orazio Prezzavento,† Nicola A. Colabufo,||
Marialessandra Contino,|| Luis G. Gonzꢁalez,§ Giovanna M. Scoto,‡ and Giuseppe Ronsisvalle†
†Department of Drug Sciences, Medicinal Chemistry Section, and ‡Department of Drug Sciences, Pharmacology and
Toxicology Section, University of Catania, Viale A. Doria 6, 95125 Catania, Italy
§Department of Pharmacology and Institute of Neuroscience, Faculty of Medicine, University of Granada, Granada,
Avenida de Madrid 11, 18012 Granada, Spain
Department of Pharmacochemistry, University of Bari “A. Moro”, Via Orabona 4, 70125 Bari, Italy
S Supporting Information
b
ABSTRACT: Novel enantiomers and diastereoisomers structurally related to σ ligand (þ)-MR200 were synthesized to improve
σ1/σ2 subtype selectivity. The selective σ1 ligand (ꢀ)-8 showed an antagonist profile determined by phenytoin differential
modulation of binding affinity in vitro, confirmed in vivo by an increase of κ opioid analgesia. The σ2 ligand (ꢀ)-9 displayed agonist
properties in an in vitro isolated organ bath assay and antiproliferative effects on LNCaP and PC3 prostate cancer cell lines.
’ INTRODUCTION
The classification of σ receptors into two distinct subtypes,
σ1 and σ2,1 based on molecular weight (25 and 18ꢀ21.5 kDa,
respectively), tissue distribution, and subcellular localization has
prompted interest in investigating the functional roles of these
two receptor sites.
The σ1 receptor is an intracellular chaperone protein associated
with endoplasmic reticulum and mitochondrial membranes.2
Ligands of σ1 receptors have been shown to prevent neuronal
death related to glutamate toxicity,3 play a role in memory
processes,4 and be involved in the development of cocaine-induced
rewarding properties.5 Moreover, several studies have shown a
relationship between σ1 and opioid receptors in pain modulation,
Figure 1. Structures of (þ)-1, haloperidol, and (ꢀ)-2.
which suggests that a tonically active antiopioid σ1 system markedly
influences the sensitivity toward opioid analgesia, especially
κ-opioid receptor mediated.6,7 Recently, studies have reported that
a nonpolymorphic mutation (c.672*51G) in the 30-untranslated
region of the σ1 receptor gene is involved in frontotemporal lobar
degenerationꢀmotor neuron disease, which is the most common
cause of early onset dementia.8
Although the σ2 receptor subtype has not been cloned,
σ2 agonists have been shown to cause phosphatidylserine
translocation, DNA fragmentation, and chromatin condensation,
which indicates that σ2 receptors induce apoptosis in diverse
tumor cell types.9 The potential role of the σ2 receptor in
regulating cellular proliferation has led to an increased interest
in investigating the biological function of this receptor. Further-
more, σ2 selective ligands could be used as imaging agents for
measuring the proliferative status of breast tumors by positron
emission tomography analysis.3,10
Recently, we reported the synthesis and pharmacological
evaluation of (þ)-methyl (1R,2S)-2-{[4-(4-chlorophenyl)-4-hy-
droxypiperidin-1-yl]methyl}-1-phenylcyclopropanecarboxylate
[(þ)-MR200, (þ)-1]. This new σ ligand, which is structurally
related to haloperidol (Figure 1), has enhanced selectivity to
different transporter and receptor systems.11
In vivo evaluation of the ability of (þ)-1 to increase μ-, δ-, and
κ-opioid receptor analgesia provided evidence of an antagonistic σ1
profile, but as previously reported, the σ1/σ2 selectivity of (þ)-1 was
not very high.7 To obtain compounds with improved selectivity,
we synthesized new enantiomers and diastereoisomers with some
modifications of the amino moiety of (þ)-1. We eliminated the
4-chloro substituent from (þ)-1; moreover, we synthesized tropane
analogues to increase the structural rigidity and steric hindrance
around the basic piperidine nitrogen atom. This last modification has
already been reported for 3-(ω-aminoalkyl)-1H-indole σ ligands,
Because the specific pharmacological roles of the two receptor
subtypes are still being defined, the design of selective σ subtype
receptor ligands is the subject of pharmaceutical studies.
Received: February 9, 2011
Published: April 08, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
BRIEF ARTICLE
Scheme 1a
Table 1. σ1 and σ2 Binding Affinities of Synthesized Com-
pounds, Haloperidol, Haloperidol Metabolite II, and 1,3-Di-
(2-tolyl)guanidine (DTG)
Ki ( SD (nM)a
b
compd
σ1
σ2
σ1/σ2
(þ)-1
3.95 ( 0.56
5.61 ( 1.33
5.02 ( 1.26
7.01 ( 0.37
44.0 ( 4.23
520 ( 8.87
397 ( 5.56
198 ( 4.66
2.20 ( 1.22
5.4 ( 0.67
21.9 ( 2.55
23.4 ( 2.77
28.2 ( 2.55
571 ( 4.77
58.3 ( 4.95
25.2 ( 3.22
943 ( 9.67
565 ( 7.56
16.0 ( 2.68
0.98 ( 0.23
5.5
(ꢀ)-1
4.2
(þ)-8
5.6
(ꢀ)-8
81.4
1.3
a Reagents and conditions: (a) n-BuLi, THF, ꢀ70 °C; (b) 1,2-dichlor-
oethane, 1-chloroethyl chloroformate, reflux, 24 h; (c) CH3COOH,
HCl (37%), reflux, 25 min.
(þ)-9
(ꢀ)-9
0.05
2.4
(þ)-10
(ꢀ)-10
haloperidol
haloperidol
metabolite II
DTG
2.8
Scheme 2a
7.3
0.18
69.4 ( 0.56
23.0 ( 0.56
0.33
a Each value is the mean ( SD of three determinations. b This value is
derived from the Ki for σ2 binding affinity divided by the Ki for σ1
binding affinity. Values of >1 indicate selectivity for σ1 over σ2. Values of
<1 indicate selectivity for σ2 over σ1.
or (()-3-(4-chlorophenyl)-8-azabicyclo[3.2.1]oct-2-ene (6) with
the enantiomers (þ)-(1R,2S) and (ꢀ)-(1S,2R)-methyl 2-(bromo-
methyl)-1-phenylcyclopropanecarboxylate18ꢀ20 (7) provided 8, 9,
or 10, respectively (Scheme 2).
a Reagents and conditions: (a) NaHCO3, DMF, 60 °C.
’ RESULTS AND DISCUSSION
Binding affinities for σ1 and σ2 receptors of the newly synthesized
8ꢀ10 and the two enantiomers (þ)-1 and (ꢀ)-1 are reported in
Table 1. Compounds (þ)-8 and (ꢀ)-8 showed σ1 receptor
affinities (Ki of 5.02 and 7.01 nM, respectively) similar to those of
their respective enantiomers of 1 [i.e., (þ)-4-chloro substituted
(þ)-1 (Ki = 3.95 nM) and (ꢀ)-1 (Ki = 5.61 nM)]. Although the
(þ)-8 enantiomer displayed a comparable σ2 receptor affinity with
respect to (þ)-1, the (ꢀ)-8 enantiomer possessed a lower σ2
binding affinity (Ki = 571 nM), which suggested that the (ꢀ)-8
enantiomer had increased σ1 subtype selectivity compared with
(þ)-1 and (ꢀ)-1. Compounds (þ)-9 and (ꢀ)-9, which had
increased structural rigidity and steric hindrance on the amino
moiety, showed reduced σ1 binding affinity (Ki of 44.0 and 520 nM,
respectively). Conversely, for σ2 receptor subtypes, we only ob-
served a slight reduction for the (þ)-9 isomer (Ki = 58.3 nM).
Interestingly, the affinity of the (ꢀ)-9 enantiomer (Ki = 25.2 nM)
did not differ from the affinity of (ꢀ)-1. The elimination of the
hydroxyl group on (þ)-10 and (ꢀ)-10 significantly decreased the
affinity to both σ receptors, which was probably related to an
additional structural rigidity that was not suitable for interactions
with σ receptors. The present binding results revealed that (ꢀ)-8
was selective for the σ1 receptor and (ꢀ)-9was moderately selective
for the σ2 receptor. Therefore, we also investigated the affinity of
these compounds for other neurotransmitter systems (see Support-
ing Information Table 2). Similar to the parent compound (þ)-1,11
(ꢀ)-8 and (ꢀ)-9 showed very low affinity for opioid, dopamine
(D1, D2, D3, D4.2), 5HT2A, and R1 receptors compared with
haloperidol. In addition, they did not show any significant affinities
for other receptors or transporters tested.
which provided a compound with high affinity and selectivity for σ2
subtype receptors.12 All compounds were tested to determine σ1 and
σ2 binding affinity and selectivity, and ligands with desirable values
were also evaluated in other transporter and receptor systems. The
pharmacological activity of the more selective σ1 ligand was assessed
by evaluating the phenytoin differential modulation of bind-
ing affinity in guinea pig brain.13 Moreover, we analyzed the
σ1 analgesic modulation of the κ agonist (ꢀ)-U50,488H
[trans-(1S,2S)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyc-
lohexyl]benzeneacetamide, (ꢀ)-2] in rats.7,14 The activity of the
compound with the best σ2 affinity and selectivity was evaluated
by the inhibition of electrically evoked contractions in guinea pig
ileum with desensitized σ1 receptor.15 In addition, we evaluated
the antiproliferative effects on hormone sensitive LNCaP and
hormone refractory PC3 prostate tumor cell lines, where the
presence of σ2 receptors has been already demonstrated.16,17
’ CHEMISTRY
The synthesis of the amines (3-exo)-3-(4-chlorophenyl)-8-azabi-
cyclo[3.2.1]octan-3-ol (4) and (()-3-(4-chlorophenyl)-8-azabi-
cyclo[3.2.1]oct-2-ene (6) originated from the treatment of commer-
cially available tropine-3-one with 1-bromochlorobenzene and
n-butyllithium at ꢀ70 °C (Scheme 1). Acidic dehydration of alcohol
3with acetic acid and HCl (37%) generated alkene 5. Demethylation
of 3 and 5 with 1-chloroethyl chloroformate provided the inter-
mediate amines (for details, see Supporting Information). The
reaction between commercially available amine 4-phenylpiperidin-
4-ol, (3-exo)-3-(4-chlorophenyl)-8-azabicyclo[3.2.1]octan-3-ol (4),
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BRIEF ARTICLE
Figure 3. Effect of (ꢀ)-8 (1 mg/kg sc) on (ꢀ)-2 (5 mg/kg sc)
analgesia. Results are expressed as MAUC (after the last injection) over
the 60 min testing period. Columns represent the mean ( SD: /, p <
0.05 vs saline-treated rats (n = 8ꢀ10); //, p < 0.05 vs (ꢀ)-2-treated rats
(n = 8ꢀ10).
Figure 4. Representative dose-response curve of (ꢀ)-9 in guinea
pig ileum.
Figure 2. Inhibition of [3H](þ)-pentazocine binding by (ꢀ)-8 (A),
(þ)-1 (B), and cocaine (C) in the absence (b) or presence of 250 μM
(O) or 1 mM (9) phenytoin.
Injection of the κ agonist (ꢀ)-2 (5 mg/kg sc) significantly
increased the nociceptive latency following thermal stimulation,
which clearly demonstrated an analgesic effect. Indeed, the percent
change from basal level of TFL, compared with the group of saline-
treated rats, was increased from 1.07% to 75.6%. Pretreatment with
(ꢀ)-8 (1 mg/kg sc) 45 min prior to (ꢀ)-2 (5 mg/kg sc) increased
the antinociceptive effect of the opioid agonist (Figure 3). In
particular, the calculated value of MAUC (118.2%) was signifi-
cantly higher than the corresponding value obtained with (ꢀ)-2
(75.6%). Similar to haloperidol and (þ)-1,6,7 (ꢀ)-8 demonstrated
σ1 antagonist actions, which confirmed the in vitro results obtained
with the phenytoin binding assays.
In our studies, phenytoin (250 μM and 1 mM) did not
significantly modify the inhibition curves of (ꢀ)-8 (Figure 2A).
The ratios of the control Ki with respect to those obtained in the
presence of phenytoin were slightly lower than unity (see Support-
ing Information Table 3), which has previously been reported for
several known σ1 antagonists, including the structurally related
compound haloperidol.13,21 Similar results were obtained with the
σ1 antagonist parent (þ)-17 (Figure 2B). To further validate this
assay, we also tested the putative σ1 agonist cocaine,3,22 and
incubation with phenytoin markedly shifted the inhibition curves
of cocaine to the left in a concentration-dependent manner
(Figure 2C).13,21 Hill analysis of all competition curves yielded
straight lines (r2 = 0.97ꢀ0.99) with slopes or pseudo-Hill coeffi-
cients (n0H) close to unity for all drugs tested (in the presence or
absence of phenytoin; see Supporting Information Table 3). This
confirms the existence of a single population of binding sites with
an n0H that does not change in the presence of phenytoin.
In an ex vivo isolated organ bath experiment, (ꢀ)-9 displayed
σ2 partial agonist activity (R = 0.4) in inhibiting twitch contrac-
tion with an EC50 of 2.3 μM23 (Figure 4).
In LNCaP and PC3 cells, incubation with compound (ꢀ)-9
(100 μM) for 48 h caused an antiproliferative effect that was
more pronounced in LNCaP cells (Figure 5). A similar pattern
was observed for the reference compounds haloperidol and
haloperidol metabolite II. Although LNCaP and PC3 cells have
similar σ1/σ2 receptor density, (ꢀ)-9 did not exert the same
activity in the two cell lines, which could be ascribed to the
presence of some efflux pumps, such as P-glycoprotein (P-gp), in
the hormone refractory PC3 cell line.24
In vivo results showed that (ꢀ)-8 (1 mg/kg sc), which was the
dose used in previous experiments with (þ)-1),7 did not affect
basal tail flick latency (TFL), expressed as the mean area under the
curve (MAUC), during the entire time of observation (60 min).
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BRIEF ARTICLE
ether: yield 57.0%; mp 200ꢀ202 °C; [R]20D = þ30 (c 1, MeOH); 1H
NMR (CDCl3) (free base) δ 1.50 (dd, 1H, J = 4.8, 9.0 Hz), 1.66 (dd, 1H,
J = 4.8, 7.0 Hz), 1.70ꢀ1.92 (m, 2H), 1.92ꢀ2.12 (m, 1H), 2.18ꢀ2.42
(m, 2H), 3.12ꢀ3.50 (m, 6H), 3.58 (s, 3H), 6.368 (s, br, 5H), 7.20ꢀ7.55
(m, 10H); 13C NMR (DMSO-d6) (oxalate salts) δ 20.26, 22.65, 33.74,
35.08, 47.95, 48.17, 52.56, 54.21, 68.07, 124.64, 126.85, 127.36, 128.14,
128.28, 129.90, 139.32, 147.97, 164.63, 171.82; Anal. C23H27NO3 0.8
3
C2H2O4 1.2 H2O.
3
Compounds (ꢀ)-(1S,2R)-8, (þ)-(1R,2S)-9, (ꢀ)-(1S,2R)-9, (þ)-
(1R,2S)-10 and (ꢀ)-(1S,2R)-10 were synthesized analogously.
(ꢀ)-Methyl (1S,2R)-2-[(4-Hydroxy-4-phenylpiperidin-1-yl)-
methyl]-1-phenylcyclopropanecarboxylate (8). Yield 55.0%;
mp 199ꢀ201 °C; [R]2D0 ꢀ30 (c 1, MeOH); H NMR (CDCl3) (free
1
Figure 5. Effects of haloperidol, haloperidol metabolite II, and (ꢀ)-9
(100 μM) on LNCaP and PC3 cells after 48 h of exposure: /, p < 0.05 vs
CTRL.
base) δ 1.50 (dd, 1H, J = 4.8, 9.0 Hz), 1.66 (dd, 1H, J = 4.8, 7.0 Hz),
1.70ꢀ1.92 (m, 2H), 1.92ꢀ2.12 (m, 1H), 2.18ꢀ2.42 (m, 2H), 3.12ꢀ
3.50 (m, 6H), 3.58 (s, 3H), 6.368 (s, br, 5H), 7.20ꢀ7.55 (m, 10H); 13C
NMR (DMSO-d6) (oxalate salts) δ 20.26, 22.65, 33.74, 35.08, 47.95,
48.17, 52.56, 54.21, 68.07, 124.64, 126.85, 127.36, 128.14, 128.28, 129.90,
139.32, 147.97, 164.63, 171.82. Anal. C23H27NO3 0.8C2H2O4 1.2H2O.
In summary, we reported the synthesis of new σ ligands that are
structurally related to the σ1 antagonist (þ)-1. Compounds (ꢀ)-8
and (ꢀ)-9 showed the best affinity and selectivity for σ1 and σ2
receptors, respectively, and did not show any significant affinity for
other receptor or transporter systems. In vitro and in vivo pharma-
cological evaluations provided evidence of antagonist profile for
(ꢀ)-8 and agonist activity for (ꢀ)-9. In light of the emerging
implications of σ subtype receptors in drug abuse,25 neuropathic
pain,26 frontotemporal lobar degenerationꢀmotor neuron
disease,8 and anticancer activity,16 the two selective compounds
(ꢀ)-8 and (ꢀ)-9 could be considered as new pharmacological
speculative tools.
3
3
(þ)-Methyl (1R,2S)-2-{[(3-endo)-3-(4-Chlorophenyl)-3-hy-
droxy-8-azabicyclo[3.2.1]oct-8-yl]methyl}-1-phenylcyclopro-
panecarboxylate (9). Yield 52.0%; mp 130ꢀ132 °C; [R]2D0 þ34
(c 1, MeOH); 1H NMR (CDCl3) (free base) δ 1.33 (dd, 1H, J = 5.0, 9.2
Hz), 1.62ꢀ2.45 (m, 10H), 2.64 (dd, 1H, J = 7.6, 13.0 Hz), 2.79 (dd, 1H,
J = 5.6, 13.0 Hz), 3.23ꢀ3.57 (m, 3H), 3.60 (s, 3H), 7.10ꢀ7.50 (m, 9H);
13C NMR (CDCl3) (free base) δ 20.18, 25.85, 26.12, 29.25, 33.71,
46.02, 46.16, 50.23, 52.28, 58.57, 59.38, 73.12, 126.06, 127.05, 128.15,
130.24, 132.26, 140.80, 148.98, 173.06. Anal. C25H28ClNO3 C2H2O4
3
3
0.5H2O.
(ꢀ)-Methyl (1S,2R)-2-{[(3-endo)-3-(4-Chlorophenyl)-3-hy-
droxy-8-azabicyclo[3.2.1]oct-8-yl]methyl}-1-phenylcyclopro-
panecarboxylate (9). Yield 53.7%; mp 130ꢀ132 °C; [R]2D0 ꢀ35 (c 1,
MeOH); 1H NMR (CDCl3) (free base) δ 1.33 (dd, 1H, J = 5.0, 9.2 Hz),
1.62ꢀ2.45 (m, 10H), 2.64 (dd, 1H, J = 7.6, 13.0 Hz), 2.79 (dd, 1H, J =
5.6, 13.0 Hz), 3.23ꢀ3.57 (m, 3H), 3.60 (s, 3H), 7.10ꢀ7.50 (m, 9H); 13C
NMR (CDCl3) (free base) δ 20.18, 25.85, 26.12, 29.25, 33.71, 46.02,
46.16, 50.23, 52.28, 58.57, 59.38, 73.12, 126.06, 127.05, 128.15, 130.24,
132.26, 140.80, 148.98, 173.06. Anal. C25H28ClNO3 C2H2O4 0.4H2O.
’ EXPERIMENTAL SECTION
For details, see the Supporting Information.
General Details. Reagents used for synthesis were purchased from
Sigma-Aldrich (Milan, Italy) unless otherwise specified. The course of
the reaction was monitored by thin layer chromatography (TLC) on
precoated silica gel 60 F254 aluminum sheets (Merck, Darmstadt,
Germany). Visualization was performed under ultraviolet (UV) light
or in an iodine chamber. Merck silica gel 60, 230ꢀ400 mesh, was used
for flash column chromatography. Melting points were obtained in open
capillary tubes with a B€uchi 530 apparatus (B€uchi Italia, Assago, Italy)
3
3
(þ)-Methyl (1R,2S)-2-{[(1R,5S/1S,5R)-3-(4-Chlorophenyl)-
8-azabicyclo[3.2.1]oct-2-en-8-yl]methyl}-1-phenylcyclopro-
panecarboxylate (10). Diastereoisomeric mixture was obtained
from (()-3-(4-chlorophenyl)-8-azabicyclo[3.2.1]octan-2-ene (6) and
(þ)-methyl (1R,2S)-2-(bromomethyl)-1-phenylcyclopropanecarboxy-
1
and are uncorrected. H and 13C nuclear magnetic resonance (NMR)
spectra were recorded with a Varian Inova 200 MHz spectrometer
(Varian, Leini, Italy). Chemical shifts are reported in δ values (ppm)
relative to an internal standard of tetramethylsilane. Optical rotations
were determined in MeOH (c = 1) with a Perkin-Elmer 241 polarimeter.
Elemental analyses (C, H, N) were determined on an elemental
analyzer, Carlo Erba model 1106 (Carlo Erba, Milan, Italy), and the
results were within 0.4% of the theoretical values (purities of tested
compound were g99%).
(þ)-Methyl (1R,2S)-2-[(4-Hydroxy-4-phenylpiperidin-1-yl)-
methyl]-1-phenylcyclopropanecarboxylate (8). A mixture of
4-phenyl-4-hydroxypiperidine (300 mg, 1.69 mmol), methyl (þ)-(1R,2S)-
2-(bromomethyl)-1-phenylcyclopropanecarboxylate (7) (455 mg, 1.69
mmol), and NaHCO3 (213 mg, 2.53 mmol) in dry dimethylformamide
(DMF) (15 mL) was heated at 60 °C for 6 h. The solvent was removed
under reduced pressure, and the residue was dissolved in CHCl3 and
washed with a solution of 4% NaHCO3. The organic layers were dried
over anhydrous Na2SO4, and after evaporation of the solvent, the crude
product was purified by flash column chromatography using cyclohex-
ane/ethyl acetate (1:1) as eluant. The resulting colorless oil (355 mg)
was dissolved in diethyl ether and treated with a solution of oxalic acid in
diethyl ether to give the oxalate salts as a white solid. The analytically
pure sample was obtained by crystallization from methanol/diethyl
late (7): yield 67.0%; mp 126ꢀ128 °C; [R]2D0 þ40 (c 1, MeOH); H
1
NMR (CDCl3) (free base) δ 1.00ꢀ2.30 (m, 8H), 2.50ꢀ2.95 (m, 3H),
3.48 (s, 3H), 3.55ꢀ3.65 (m, 2H), 6.20 (d, 1H, J = 5.4), 7.00ꢀ7.50 (m,
9H); 13C NMR (CDCl3) (free base) δ 19.74, 20.65, 28.58, 28.88, 29.50,
29.57, 33.42, 33.61, 33.97, 47.19, 47.34, 52.21, 52.26, 53.40, 56.06, 56.51,
56.98, 57.65, 125.91, 127.02, 127.06, 127.89, 128.13, 128.15, 128.31,
130.13, 130.17, 131.53, 131.80, 132.66, 138.55, 140.69, 172.81, 172.91.
Anal. C25H26ClNO2 C2H2O4 0.7H2O.
3
3
(ꢀ)-Methyl (1S,2R)-2-{[(1R,5S/1S,5R)-3-(4-Chlorophenyl)-
8-azabicyclo[3.2.1]oct-2-en-8-yl]methyl}-1-phenylcyclopro-
panecarboxylate (10). Diastereoisomeric mixture was obtained
from (()-3-(4-chlorophenyl)-8-azabicyclo[3.2.1]octan-2-ene (6) and
(ꢀ)-methyl (1S,2R)-2-(bromomethyl)-1-phenylcyclopropanecarboxy-
late (7): yield 65.0%; mp 127ꢀ129 °C; [R]2D0 ꢀ39 (c 1, MeOH); H
1
NMR (CDCl3) (free base) δ 1.00ꢀ2.30 (m, 8H), 2.50ꢀ2.95 (m, 3H),
3.48 (s, 3H), 3.55ꢀ3.65 (m, 2H), 6.20 (d, 1H, J = 5.4), 7.00ꢀ7.50 (m,
9H); 13C NMR (CDCl3) (free base) δ 19.74, 20.65, 28.58, 28.88, 29.50,
29.57, 33.42, 33.61, 33.97, 47.19, 47.34, 52.21, 52.26, 53.40, 56.06, 56.51,
56.98, 57.65, 125.91, 127.02, 127.06, 127.89, 128.13, 128.15, 128.31,
130.13, 130.17, 131.53, 131.80, 132.66, 138.55, 140.69, 172.81, 172.91.
Anal. C25H26ClNO2 C2H2O4 0.7H2O.
3
3
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Journal of Medicinal Chemistry
BRIEF ARTICLE
’ ASSOCIATED CONTENT
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S
Supporting Information. Synthesis of 3ꢀ6, elemental
b
analysis results, binding affinity data, binding parameters, and
biological experiments. This material is available free of charge
via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ39-095-7384019. Fax: þ39-095-222239. E-mail:
marrazzo@unict.it.
’ ACKNOWLEDGMENT
This work was supported by grants (PRIN 2007, No.
2005032713) from MIUR (Rome). E.J.C. was supported by
the MICINN/Fulbright program.
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’ ABBREVIATIONS USED
HPLC, high performance liquid chromatography; MAUC, mean
area under the curve; NMR, nuclear magnetic resonance; PET,
positron emission tomography; sc, subcutaneous; SD, standard
deviation; TFL, tail flick latency; TLC, thin-layer chromatogra-
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