Novel Sigma Receptor Ligands: Synthesis and Biological Profile

Novel Sigma Receptor Ligands: Synthesis and Biological Profile

Orazio Prezzavento,† Agata Campisi,‡ Simone Ronsisvalle,† Giovanni Li Volti,§ Agostino Marrazzo,† Vincenzo Bramanti,|

Giuseppe Cannavo`,⊥ Luca Vanella,‡ Alfredo Cagnotto,# Tiziana Mennini,# Riccardo Ientile,| and Giuseppe Ronsisvalle*,†

Department of Pharmaceutical Sciences and Department of Biological Chemistry, Medical Chemistry, and Molecular Biology, UniVersity ofCatania, Viale A. Doria 6, 95125 Catania, Italy, Department of Biomedical Sciences and Biotechnology, DiVision of Human Anatomy,UniVersity of Brescia, Viale Europa, 25123 Brescia, Italy, Department of Molecular Biochemistry and Pharmacology, Istituto di RicercheFarmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy, and Department of Biochemical, Physiological, and Nutritional Sciencesand Department of Hygiene, Public Health, and PreVentiVe Medicine, UniVersity of Messina, Via Consolare Valeria, 90100, Messina, Italy

ReceiVed September 26, 2006

The aim of the present study was to investigate the biological profile of new substituted 1-phenyl-2-cyclopropylmethylamines. High affinity for bothσ subtypes was achieved when 4-phenylpiperidin-4-ol (4a-e) and 4-benzylpiperidine moieties were present (5a-e). (1R,2S/1S,2R)-2-[4-Hydroxy-4-phenylpiperidin-1-yl)methyl]-1-(4-methylphenyl)cyclopropanecarboxylate (4b) showed high affinity for theσ1 sites (Ki )1.5 nM) and the most favorableσ1/σ2 selectivity (Ki(σ2)/Ki(σ1) ) 33.9). Binding affinity studies showedthat 4b binding onN-methyl-D-aspartate (NMDA), dopaminergic (D1, D2, D3), muscarinic, histaminergicH1, adrenergic (R1, R2), serotoninergic (5-HT2A, 5-HT2C, 5-HT3, 5-HT4, 5-HT6), DA (DAT), and 5-HT (SERT)transporters was not significant. Interestingly,σ ligands differently induced the expression of tissuetransglutaminase (TG-2) in primary astroglial cell cultures. We suggest that4b may act as aσ1/σ2 agonistand that theσ ligands may modulate TG-2 differently.

Introduction

The σ receptors have been classified as a unique proteinfamily different from the opioid1 and phencyclidine (PCP)receptors.2 At least two different subtypes ofσ1 andσ2 receptorshave been documented.3,4 Both subtypes are expressed inperipheral organs and in the brain, while each type is localizedin different subcellular compartments.5,6 The σ1 receptor wascloned and sequenced in the mid-1990s,7 and they are clearlyinvolved in various biological and pharmacological functions.8

Among the most salient ones,σ receptors mediate potassiumconductance9 and calcium signaling,10 and they play neuro-modulatory roles onN-methyl-D-aspartate (NMDA), DA, andAch transmission.11-13 In addition, it has been suggested thatσreceptors are associated with the toxin-sensitive Gi/o and thecholer toxin-sensitive Gs proteins phospholipase C (PLC) andprotein kinase C (PKC).10 Conversely, theσ2 receptors havenot yet been cloned and their activation has induced changesin cell morphology and apoptosis, producing transient andsustained increases in calcium ions.14 Increased calcium ionsinflux leads to the activation of several calcium-dependentproteins, such as tissue transglutaminase (TG-2).15 This is aubiquitary isoform of a family of transglutaminases (TGase),catalyzing the formation ofε-(γ-glutamyl)lysine cross-linksbetween polypeptide chains, resulting in protein polymerization,the cross-linking of dissimilar proteins, and the incorporationof diamines and polyamines into proteins.15 Furthermore, TG-2is implicated in numerous cellular processes, such as cellulardifferentiation, signal transduction, cell survival, and wound

healing. In addition, it plays a modulatory role in apoptosis andcell response to stressors, depending on the types of stimulithat provoke an increase in the transamidating activity.16

However, TG-2 has additional activities and it has also beenreported to be a protein disulfide isomerase18 and a G-protein-coupled membrane receptor.17 Although TG-2 is found mostlyin the cytosol, associated with the plasma membranes,18 in themitochondria, and in the nucleus, the multiple effects of TG-2in different cellular pathways may be due to its different celllocalization and its transamidanting and GTPase activities.19

TG-2 is also expressed in the brain, and it is implicated innumerous and diverse processes of the central and peripheralnervous systems.15 Several lines of evidence suggest thataberrant TG-2 activity, although not causative, may contributeto the inexorable decline associated with several neurodegen-erative diseases such as Huntington’s, Alzheimer’s, and Par-kinson’s disease.20 It is well-known that astroglial cells inprimary culture represent a valuable tool for studying thebiochemical mechanism involved during the development andmaturation of these cultured nerve cells.21

In this report we present a new series of 1-methoxycarbonyl-1-phenyl-2-cyclopropylmethylamines. Here, the (+)-cis-N-normetazocine nucleus of methyl (1′R,2′S)-2-{[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]methyl}-1-phenylcyclopropane-carboxylate ((+)-MPCB)22 (Figure 1) has been substituted withdifferent substructures of the benzomorphan system such as4-phenyl-4-hydroxypiperidine, 4-benzylpiperidine, 1-(pyridin-2-yl)piperazine, and 2-(piperazin-1-yl)pyrimidine moieties. Theaim was to developσ ligands with higher affinity and selectivity.We also explored the importance of the para substituent on thearomatic ring supported on the cyclopropane ring. All thecompounds were tested as racemates for binding atσ1 andσ2

receptors. In addition, our purpose was to assess the relationshipbetween TG-2 and theσ receptors in primary rat astroglial cellcultures. Furthermore, in order to verify the activation of theσ-mediated apoptotic pathway, we studied caspase-3 cleavageand DNA fragmentation.

* To whom correspondence should be addressed. Telephone/fax:+39-095-336722. E-mail address: [email protected].

† Department of Pharmaceutical Sciences, University of Catania.‡ Department of Biological Chemistry, Medical Chemistry, and Molecular

Biology, University of Catania.§ University of Brescia.| Department of Biochemical, Physiological, and Nutritional Sciences,

University of Messina.⊥ Department of Hygiene, Public Health, and Preventive Medicine,

University of Messina.# Istituto di Ricerche Farmacologiche Mario Negri.

951J. Med. Chem.2007,50, 951-961

10.1021/jm0611197 CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 02/13/2007

Chemistry

Lactones1a-d were prepared as previously reported byCasadio et al.23 The reaction of the appropriate lactone withSOCl2 and CH3OH/HCl (3 N) in benzene gave the cis racemichalogenated esters2a-f (Scheme 1). Ester2ewas obtained bynitration with fuming HNO3 starting from lactone1a and thesubsequent esterification of1e. Reduction of 4-nitro lactone1ewith SnCl2‚2H2O gave the amino lactone1f. Esterification of1f with an analogous procedure of1a-d gave the amino ester2f. The reaction of the secondary commercially available amines4-phenylpiperidin-4-ol (3a), 4-benzylpiperidine (3b), 1-(pyridin-2yl)piperazine (3c), and 2-(piperazin-1yl)pirimidine (3d) withthe suitable cis-(()-halogenated ester derivative gave the finalcompoundscis-(()-4a-e, cis-(()-5a-e, cis-(()-6a-f, andcis-(()-7a-f, respectively (Scheme 2).

Results and Discussion

The synthesized compounds evaluated for theirσ affinityrevealed high affinity for theσ receptors when 4-phenylpiperi-din-4-ol (4a-e) and 4-benzylpiperidine (5a-e) moieties werepresent. Table 1 shows that all the racemates displayed apreference for theσ1 receptors. Compound4b showed the bestprofile of σ2/σ1 selectivity (Ki(σ2)/Ki(σ1) ) 33.9), whereascompound5e exhibited a subnanomolarKi value for theσ1

receptor. However, for the two aforementioned series, a nano-molar affinity was also achieved for theσ2 receptors in a similarway to haloperidol (Ki(σ2)/Ki(σ1) ) 7.3).24 Conversely the1-(pyridin-2-yl)piperazine derivatives6b, 6d, and6e and the2-(piperazin-1-yl)pyrimidine derivatives7b, 7d, and7eshowedonly moderate affinity for theσ1 receptors without appreciableσ2 receptor affinity (Table 1). The differences in bindingaffinities suggest that the presence of 1-phenyl cyclopropyl-methycarboxylate moiety joined to the 1-(pyridin-2-yl)piperazineor 2-(piperazin-1-yl)pyrimidine moiety, both with electron-poorsystems, is an unfavorable combination for theσ1 receptors andis detrimental for theσ2 sites.

On the basis ofσ2/σ1 selectivity and the calculated logPvalue (3.36( 0.39), we selected the 4-hydroxy-4-phenylpip-eridine derivative4b in order to investigate its binding profilewith respect to the receptors and transporters reported in Table2. Compared to haloperidol, a prototypicalσ ligand,24 4bdisplays much lower affinity for the dopamine D1, D2, andD3 receptors (Ki > 10.000, 1.850, 4.900 nM, respectively),5-HT2A (Ki ) 3.149 nM), andR1 receptors (Ki ) 605 nM). Inaddition,4b does not bind adrenergicR2, muscarinic, histamin-ergic H1, and serotonin 5-HT2C, 5-HT3, 5-HT4, and 5-HT6receptors as well as on DA (DAT) and 5-HT (SERT) transport-ers. These results are consistent with previous reports show-ing that the modifications on the butyrophenone moiety areinjurious for dopamine receptors (D2 and D3) but not for theσreceptors.28

Our results confirm the role of the N-substituent to addressan appropriate amino pharmacophore toσ receptors, at the sametime preventing the interaction with the other receptors. In orderto investigate the relationships between theσ receptors and thecalcium-dependent protein TG-2, on primary rat astroglial cellcultures we explored the biological profile of4b and someσreference compounds, presenting different activity and selectivityprofiles (Table 3). This in vitro model represents a valuabletool for studying the biochemical mechanism involved duringthe development and maturation of these cultured nerve cells.21

Primary rat astroglial cell cultures were prepared andcharacterized by immunofluorescent staining for a specificmarker, such as glial fibrillary acidic protein (GFAP). About90% of the cells were GFAP-positive, indicating that cultureswere rich in astrocytes.21

In our preliminary experiments (data not shown), in view ofboth a vitality test and microscopy observations, we establishedthe concentrations and the time of exposure of the cultures toσ ligands. These sets of experiments showed that the nontoxicconcentration of the drugs was 25µM and the optimal time ofexposure was 24 h. At 50µM, astroglial cell cultures appearedactivated, whereas the higher concentration (100µM) inducedcell death (data not shown). Since it has been suggested thatthe multiplicity effects of TG-2 in various cellular pathwaysmay be due to its different cellular localization and itstransamidanting and GTPase activity, we evaluated its expres-

Figure 1. Structure of (+)-(1′R,2′S)-2-{[(2R,6R,11R)-8-hydroxy-6,-11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-methyl}-1-phenylcyclopropanecarboxylate ((+)-MPCB).

Scheme 1.Synthetic Route for1a-f and2a-f

Scheme 2.Synthetic Route for4a-e, 5a-e, 6a-f, and7a-f

952 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 PrezzaVento et al.

sion and its intracellular localization by Western blot andconfocal laser scanning microscopy (CLSM) analysis followingσ ligands exposure.

Figure 2A shows the Western blot and Figure 2B thedensitometric analysis of TG-2 expression in 14 DIV astroglialcell cultures in the absence, or in the presence, ofσ ligands (25µM for 24 h). Specifically, the ibogaine treament, ibogaine beingaσ2 agonist,29 significantly increased the percentage of the TG-2expression (Figure 2Ab and Figure 2Bb). Haloperidol treatment,even if it possesses high affinity for multiple neuroreceptors,24

used as aσ2 agonist andσ1 antagonist,24 presented30 a patternof TG-2 expression similar to the untreated cells (Figure 2Acand Figure 2Bc). We also used BD1047, aσ antagonist,31 eventhough it has been reported to act as a partial agonist.32 Thistreatment did not increase the percentage of TG-2 expression(Figure 2Ad and Figure 2Bd). By contrast, in the4b-treatedcells we observed a significant increase of the percentage ofTG-2 expression (Figure 2Ae and Figure 2Be). The (+)-pentazocine, used as aσ1 agonist, did not induce changes of

TG-2 expression (Figure 2Af and Figure 2Bf). These resultssuggest the existence of a possible link between the TG-2expression andσ receptors.

Figure 3 shows CLSM analysis in the astroglial cell cultures.In untreated astroglial cell cultures TG-2 fluorescence wasdistributed at low levels in the cytosol and in the mitochondria(Figure 3a). The exposure of astrocyte cell cultures to ibogaine(Figure 3b) significantly increased TG-2 expression and redis-tributed the protein prevalently in the cytosol, in the mitochon-dria, in the nuclei, and in the nucleoli. On the other hand,treatment with haloperidol significantly increased TG-2 expres-sion and the protein appeared localized only in the nuclei andin the nucleoli (Figure 3c). In the BD1047-treated astrocyteswe observed a low but significant increase of TG-2 expressionwhen compared to the untreated cells. The protein appearedprevalently distributed in the mitochondria and along thecytosolic projections (Figure 3d). In addition, it was alsoobserved at very low levels in the nuclear compartment, evenif its level was higher than in the control.

Table 1. Structures andσ Binding Affinities of 1-Phenyl-2-cyclopropylmethylamine Derivatives

a Each value is the mean( SD of three determinations.

NoVel σ Receptor Ligands Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5953

In 4b-treated astroglial cell cultures a significant increase ofTG-2 expression was observed (Figure 3e). The fluorescencedistribution showed that the protein was localized in the cytosol,along cytosol projections, in the mitochondria, in the nuclei,and in the nucleoli (Figure 3e). In this case the fluorescenceintensity was higher in the cytosol and in the mitochondria thanin ibogaine-treated astroglial cells, even though the fluorescencelevels in4b-treated astrocytes in both the nuclei and nucleoliwere lower than those observed in ibogaine-treated cells (partsb and e of Figure 3). However, the fluorescence levels observedin the nuclei and nucleoli of4b-treated astrocytes were lowerthan in haloperidol-treated ones (parts c and e of Figure 3). Thetreatment of the cells with (+)-pentazocine induced a lowincrease of TG-2 when compared to the untreated control (Figure

3f). The fluorescence appeared at low levels in the cytosol, inthe cellular projections, in the nuclei, and in the nucleoli (Figure3f).

The mean energy of CLSM analysis further demonstrated thefluorescence intensity of TG-2 in the cytosol and in the nuclearcompartment (Figure 4). The presence of TG-2 in the nuclearcompartment and in the cytosol may be due to its different celllocalization and its transamidanting and GTPase activities.15

Moreover, although TG-2 is found mostly in the cytosol, it isalso present in the nucleus21 and associated with the plasmamembrane.18

Since intracellular localization affects protein function bydetermining which interactions are likely to occur, it is possiblethat with access to different substrates in different intracellularpools, the multiplicity of TG-2 effects in different cellularpathways may be due to its localization and to which enzymaticactivity of TG-2 is favored (transamidating or GTP binding/hydrolyzing).33 In addition, it has recently been demonstratedthat TG-2 possesses a serine/treonine kinase activity and is ableto phosphorylate histone proteins.19 Although it has beenimplicated in the apoptotic pathway, there is evidence that undercertain circumstances TG-2 can also protect against apoptosis.19

Since TG-2 plays a modulatory role in apoptosis and cellresponse to stressors, depending on the types of the stimuli thatprovoke an increase in the transamidating activity,16 we studiedthe involvement of the protein inσ-receptor-mediated apoptosisas measured by caspase-3 cleavage and the terminal deoxy-nucleotidyltransferase-mediated dUTP nick-end-labeling (TUNEL)test.

This set of experiments showed a marked enhancement ofcaspase-3 cleavage in ibogaine-treated astrocytes when com-pared to the untreated control (Figure 5Ab and Figure 5Bb).Furthermore, a slight increase of caspase-3 cleavage in halo-peridol-treated astrocytes was found (Figure 5Ac and Figure5Bc), whereas a decrease in both BD1047 (Figure 5Ad andFigure 5Bd) and (+)-pentazocine-treated astrocytes (Figure 5Afand Figure 5Bf) was observed. Conversely, in4b-treatedastrocytes we observed a significant increase of caspase-3cleavage (Figure 5Ae and Figure 5Be).

These results were also confirmed by evaluating DNAfragmentation performed in primary astroglial cell cultures bythe TUNEL test (Figure 6).34 The percentage of the apoptoticcells compared to the nonapoptotic cells is reported in the Table4.

A marked increase of apoptotic cells in ibogaine-treatedastroglial cells (Figure 6b) was found when compared tountreated control ones (Figure 6a). By contrast, haloperidol,BD1047, and (+)-pentazocine did not have any effect on DNAfragmentation (parts c, d, and f of Figure 6). Interestingly, in4b-treated cells (Figure 6e) a significant increase of DNAfragmentation was observed when compared to the control(Figure 6a), even if apoptotic cells appeared lower than inibogaine-treated astrocytes (Figure 6b).

These results suggest that primary astroglial cell cultures areresponsive toσ ligands and that TG-2 is part ofσ receptoractivation pathway. In particular, the prevalent localization ofTG-2 in the cytosol of the ibogaine-treated astroglial cell culturessuggests that the protein may be involved in the activation ofthe σ2-mediated apoptotic pathway. Furthermore, it has beenhypothesized thatσ2 receptors are histone proteins and thatσ2

ligands also bind histone proteins35 that also been shown to bea substrate of TG-2.36 The apparent discrepancy of the resultsobtained with BD1047, with respect to ibogaine, may be relatedto the methods used, since the caspase-3 cleavage and TG-2

Table 2. Binding Affinity Profile of 4b

receptor Ki ( SD,a nM

DAD1 ([3H]SCH 23390) >10000D2 ([3H]spiperone) 1285( 384D3 ([3H]-7OH-DPAT) 4900( 1077

5HT5-HT2A ([3H]ketanserin) 3149( 4405-HT2C ([3H]mesulergine) 4357( 11765-HT3 ([3H]LY278584) >100005-HT4 ([3H]GR13808) >100005-HT6 ([3H]-5HT) >10000

RR1 ([3H]prazosin) 605( 157R2 ([3H]RX821002) 9414( 900

muscarinic ([3H]-QNB) >10000H1 ([3H]pyrilamine) 1422( 327NMDA ([ 3H]CGP 39653) >10000NMDA channel ([3H]MK 801) >10000DAT ([3H]-WIN 35,428) >10000SERT ([3H]citalopram) >10000

a Each value is the mean( SD of three determinations.

Table 3. Activity of Testedσ Receptor Ligands


expression were evaluated by Western blot in the total celllysates, whereas CLSM analysis was performed on a single cell.This is strictly dependent on the different stages of the cells. Infact, it has been demonstrated that the functions and thelocalization of the protein are dependent on the type of thestimuli and the cell response to stressors.33 This hypothesis isfurther substantiated by other observations showing that theactivation ofσ2 receptors mediated apoptotic pathway may becaspase-independent.37 The haloperidol, acting as BD1047 onthe σ receptors and also possessing high affinity for the otherneuroreceptors,24 did not cause a significant increase of TG-2expression by Western blot analysis, caspase-3 cleavage, andDNA fragmentation. In contrast, CLSM analysis performed ona single cell showed the expression and the localization of TG-2in the nuclear compartment that may be associated with theantiapoptotic function of the protein.33 Compound4b showeda pattern similar to that of ibogaine, even though it resulted ina lower TG-2 expression in cytosol than in ibogaine-treated cells.

These differences may be due to the major lypophilic charac-teristic of ibogaine (calculated logP ) 4.04 ( 0.43). On the

Figure 2. (A) Representative immunoblots of TG-2 expression in primary rat astroglial cell cultures of cultured astroglial cells in the (a) absenceor (b-f) presence of 25µM sigma ligands for 24 h: (b) ibogaine, (c) haloperidol, (d) BD1047, (e)4b, (f) (+)-pentazocine. HUVEC cell extractwas used as positive control. (B) Densitometric analysis of the percentage of TG-2 expression in response toσ ligand exposure. Results are expressedas the mean( SD of the values of four experiments: (/) p < 0.05, significant differences versus the controls.

Figure 3. CLSM analysis of TG-2 distribution in primary rat astroglial cell cultures at 14 DIV. Representative pictures of TG-2 fluorescentstaining in the (a) absence or (b-f) presence of 25µM σ ligands for 24 h: (b) ibogaine, (c) haloperidol, (d) BD1047, (e)4b, (f) (+)-pentazocine.Immunostaining for TG-2 in the cytosol (green) and in the nuclei (red) is shown. The photos are from sequential analysis of the same microscopicfield, followed by a merge of different images with specific staining.

Figure 4. Mean energy of CLSM analysis of TG-2 fluorescence presentin the cytosol or in the nuclei of primary rat astroglial cell cultures at14 DIV exposed to 25µM σ ligands for 24 h: (a) untreated cells, (b)ibogaine, (c) haloperidol, (d) BD1047, (e)4b, (f) (+)-pentazocine.


other hand, (+)-pentazocine, inducing a low increase of theintracellular calcium levels,38 did not have any effect on theactivation of the apoptotic pathway and on TG-2 expression.CSLM analysis showed that the protein was localized at verylow levels in the cytosol and also in the nuclear compartment.These results suggested that TG-2 may play a protective roleagainst apoptosis.33

Together, these results show that replacement of the normeta-zocine nucleus of (+)-MPCB with 4-phenyl-4-hydroxypiperi-dine or 4-benzylpiperidine provides high-affinityσ ligands witha preference forσ1 binding sites. Interestingly,4b, the mostselectiveσ1 ligand (Ki(σ2)/Ki(σ1) ) 33.9) of the series was alsovery selective with respect to many receptors and transporter

systems compared to haloperidol. The biological results furthersuggest thatσ receptors may modulate TG-2 expression, itstranslocation into the nuclei and nucleoli, and its different func-tions in primary rat astroglial cell cultures. We also demonstratedthatσ ligands differently induced the activation of the apoptoticpathway that is prevalently related to theσ2 agonist ligands. Inaddition, since4b increased TG-2 expression and its translo-cation into the nuclear compartment and activated the apoptoticpathway, it may act asσ1/σ2 agonist. Furthermore, our resultssuggest that the selectiveσ ligands may represent an interestingtool to modulate intracellular calcium levels and consequentlythe up-regulation of TG-2 typical of several neurodegenerativediseases.15,20 Further studies are now warranted in order to

Figure 5. (A) Representative immunoblots of caspase-3 cleavage in primary rat astroglial cell cultures of cultured astroglial cells in the (a) absenceor (b-f) presence of 25µM σ ligands for 24 h: (b) ibogaine, (c) haloperidol, (d) BD1047, (e)4b, (f) (+)-pentazocine. RSV-3T3 was used aspositive control. (B) Densitometric analysis of the percentage of caspase-3 cleavage in response toσ ligand exposure. Results are expressed as themean( SD of the values of four experiments: (/) p < 0.05, significant differences versus the controls.

Figure 6. Representative pictures of TUNEL assay performed in primary rat astroglial cell cultures at 14 DIV exposed to 25µM σ ligands for 24h, (b) ibogaine, (c) haloperidol, (d) BD1047, (e)4b, (f) (+)-pentazocine, in comparison with (a) the untreated cells. Immunostaining of nonapoptotic(red) and apoptotic (green) cells is shown.


investigate the importance of the stereochemistry of the cyclo-propane ring and to clarify the biochemical mechanismsinvolved in the interactions between TG-2 andσ receptors.

Experimental Section

Radioligand Binding Assays.Theσ binding experiments wereperformed as previously reported.39 Briefly, σ1 binding assays wereperformed on guinea pig brain membranes,32 incubating 500µg ofmembrane protein with 3 nM [3H]-(+)-pentazocine (45 Ci/mM;the value of the apparent dissociation constant (Kd) was 1.2( 0.3nM, n ) 3) in 50 mM Tris-HCl (pH 7.4). Test compounds wereadded at concentrations ranging from 10-5 to 10-11 M. Nonspecificbinding was assessed by the addition of 10µM of haloperidol. Thereaction was performed for 150 min at 37°C and terminated byfiltering the solution through Whatman GF/B glass fiber filters thatwere presoaked for 1 h in a 0.5% poly(ethylenimine) solution.Filters were washed twice with 4 mL of ice-cold buffer.

The σ2 binding assays were carried out on guinea pig brainmembranes prepared as previously described.29 Briefly, the mem-branes were incubated with 3 nM [3H]DTG (1,3-di-(2-tolyl)-guanidine) (31 Ci/mM,Kd ) 9.9( 0.8 nM,n ) 3) in the presenceof 400 nM (+)-SKF10,047 to blockσ1 sites. Incubation was carriedout in 50 mM Tris-HCl (pH 8.0) for 120 min at room temperature,and assays were terminated by the addition of ice-cold 10 mM Tris-HCl, pH 8.0, followed by filtration through Whatman GF/B glassfibers filters that were presoaked for 1 h in a 0.5% poly-(ethylenimine) solution. Filters were washed twice with 4 mL ofice-cold buffer. Nonspecific binding was evaluated in the presenceof 5 µM DTG. Various rat brain regions were dissected to assessthe following binding assays; rat striatum for D1 and D2 receptorsand DAT; rat olfactory tubercle for D3 receptors; rat cortex formuscarinic, 5-HT2A, 5-HT3, R1, R2, H1, and NMDA receptors andSERT; guinea pig cortex for 5-HT4 receptors. 5-HT2C binding assayswere carried out on pig brains (frontal cerebral cortex) obtainedfrom a local slaughterhouse and immediately placed on ice untiluse. Binding experiments on 5-HT6 receptors were performed usinga rat-cloned serotonin receptor subtype 6 in HEK-293 cells, asreported by Boess et al.40 Tissue preparations and binding assayswere carried out according to Gobbi et al.41 and Mennini et al.42

After incubation the samples were filtered through Whatman GF/Bor GF/C glass fiber filters using a Brandel apparatus (model M-48R)or Millipore filter apparatus. The filters were washed three timeswith 4 mL of ice-cold buffer. Radioactivity was counted in 4 mLof “Ultima Gold MV” or Filter Count Cocktail (Packard) in a DSA1409 (Wallac) or 1414 Winspectral Perkin-Elmer Wallac liquidscintillation counter. For inhibition experiments, the drugs wereadded to the binding mixture at seven to nine different concentra-tions. Inhibition constants (Ki values) were calculated using the“Allfit” program or the EBDA/LIGAND program purchased fromElsevier/Biosoft.

Materials and Methods. Reagents used for synthesis werepurchased from Sigma-Aldrich (Milan, Italy) unless otherwisespecified. The course of the reaction was monitored by thin-layerchromatography (TLC) on precoated silica gel 60 F254 aluminum

sheets (Merck, Darmstadt, Germany). Visualization was providedunder UV light or in an iodine chamber. Merck silica gel 60, 230-400 mesh, was used for flash column chromatography. Meltingpoints were obtained in open capillary tubes with a Bu¨chi 530apparatus (Bu¨chi Italia, Assago, Italy) and are uncorrected.1H NMRspectra were recorded with a Varian Inova 200 MHz spectrometer(Varian, Leini, Italy). Elemental analyses (C, H, N) were determinedon an elemental analyzer, Carlo Erba model 1106 (Carlo Erba,Milan, Italy), and the results were within(0.4% of the theoreticalvalues. The logP values of the examined compounds werecalculated using the ACD/LogP DB software (version 4.55).

The following radioactive materials were used: [3H]-(+)-pentazocine (45 Ci/mmol), [3H]DTG [1,3-di-(2-tolyl)guanidine] (31Ci/mmol), obtained from Perkin-Elmer. [3H]SCH 23390 (75.5 Ci/mmol), [3H]spiperone (20 Ci/mmol), [3H]QNB (42 Ci/mmol), [3H]-ketanserin (63.3 Ci/mmol), [3H]-5HT (80 Ci/mmol), [3H]prazosin(83.8 Ci/mmol), [3H]pyrilamine (20 Ci/mmol), [3H]CGP 39653 (40Ci/mmol), [3H]WIN 35,428 (86 Ci/mmol), and [3H]MK 801 (30Ci/mmol) were obtained from NEN, and [3H]-7OH-DPAT (139 Ci/mmol), [3H]mesulergine (86 Ci/mmol), [3H]LY 278584 (84 Ci/mmol), [3H]GR 113808 (81 Ci/mmol), [3H]citalopram (85 Ci/mmol), and [3H]RX 821002 (49 Ci/mmol) were obtained fromAmersham (U.K.). Ibogaine was a kind gift from the Slater & Frith(U.K.).

Cell culture media and sera were from Invitrogen (Milan, Italy).FITC and TRITC-conjugated anti-IgG and other chemicals ofanalytical grade were from Sigma-Aldrich (Milan, Italy). Mousemonoclonal antibody against TG-2 was from NeoMarkers (Fremont,CA). Mouse monoclonal antibody against caspase-3 was fromBecton-Dickinson (Milan, Italy). The horseradish peroxidase-conjugated anti-mouse IgG and ECL (enhanced chemiluminescence)developing system for immunoblots were purchased from Amer-sham Pharmacia Biotech (Milan, Italy). The kit for the TUNELtest was from Clonteck.

Compound Syntheses.Compounds1a-d were synthesized aspreviously reported by Casadio and co-workers.23

(1R,5S/1S,5R)-1-(4-Nitrophenyl)-3-oxabicyclo[3.1.0]hexan-2-one (1e).To 1 g (5.74 mmol) of lactone1a we added, dropwise,16.5 mL (-40 °C) of fuming HNO3. After being stirred for 2 min,the mixture was poured into ice and the crystalline product wasfiltered and washed with water until neutral pH was reached. Afterdrying, the resultant crude product was crystallized twice frommethanol to give 0.60 g of the desired product. Yield 48%; mp142-143 °C; 1H NMR (CDCl3) δ 1.55 (dd,J ) 5.0, 5.2 Hz, 1H),1.72 (dd,J ) 5.0, 7.8 Hz, 1H), 2.65-2.85(m, 1H), 4.35 (d,J )9.2 Hz, 1H), 4.51 (dd,J ) 4.4, 9.2 Hz, 1H), 7.63 (d,J ) 8.8 Hz,2H), 8.20 (d,J ) 9.0 Hz, 2H). Anal. (C11H9NO4) C, H, N.

(1R,5S/1S,5R)-1-(4-Aminophenyl)-3-oxabicyclo[3.1.0]hexan-2-one (1f).A mixture of 1 g (4.5 mmol) of lactone1e, 5.1 g (22.78mmol) of SnCl2 dihydrate, and 10 mL of EtOH/EtOAc (2:1) wasstirred under reflux for 30 min. The cooled solution was combinedwith ice, and 5% NaHCO3 was added up to pH 7-8. The resultingmixture was extracted twice with EtOAc (2× 70 mL). The organiclayer was washed with water, dried over Na2SO4, and filtered. Thefiltrate was evaporated in vacuo and gave 840 mg of product. Yield98.7%; mp 96-102 °C; 1H NMR (CDCl3) δ 1.27 (dd,J ) 4.6,4.8, Hz, 1H), 1.58 (dd,J ) 4.8, 7.8 Hz, 1H), 2.35-2.55 (m, 1H),3.70 (br s, 2H), 4.26 (d,J ) 9,2 Hz, 1H), 4.52 (dd,J ) 4.6, 9.2Hz, 1H), 6.60-7.35 (m, 4H). Anal. (C11H11NO2) C, H, N.

Methyl (1R,2S/1S,2R)-2-(Chloromethyl)-1-phenylcyclopro-panecarboxylate (2a).Thionyl chloride (0.98 mL, 13.42 mmol)was added dropwise to 500 mg (2.80 mmol) of (()-1a in 8.3 mLof anhydrous benzene at 0°C and was heated to reflux for 5 h.The mixture was cooled to 0°C, and a solution of 3 N CH3OH/HCl (3.02 mL) was added dropwise. The reaction mixture was thenallowed to stand at room temperature overnight. The mixture wasevaporated under reduced pressure, and the residue was dissolvedin Et2O and extracted with a saturated solution of NaHCO3. Theorganic phase was dried (Na2SO4) and evaporated in vacuo to give322 mg of the desired ester. Yield 51%; mp 77-80 °C; 1H NMR(CDCl3) δ 1.50 (dd,J ) 4.8, 8.8 Hz, 1H), 1.82 (dd,J ) 4.8, 7.0

Table 4. TUNEL Testa

treatment % of apoptotic cells

control 0ibogaine 95( 5(+)-pentazocine 0BD1047 7( 1haloperidol 4( 0.54b 65 ( 4.5

a Percentage of the apoptotic cells compared to the nonapoptotic cellsfor each microscopic field. The ratio is expressed as a percentage, takingthe total number of cells as 100 and comparing 10 random microscopic foreach dish. The DNA fragmentation assay incorporates fluorescein-dUTPat the free 3′-hydroxyl ends of the fragmented DNA using TUNEL.Apoptotic cells appear green, while nonapoptotic cells appear red: (/) p <0.05, significant differences versus the controls.


Hz, 1H), 1.90-2.20 (m, 1H), 3.64 (s, 3H), 3.80 (dd,J ) 10.2,10.4 Hz, 1H), 4.00 (dd,J ) 5.6, 10.2 Hz, 1H), 7.20-7.45 (m, 5H).Anal. (C12H13ClO2) C, H, N.

Compounds2b-f were synthesized analogously.Methyl (1R,2S/1S,2R)-2-(Chloromethyl)-1-(4-methylphenyl)-

cyclopropanecarboxylate (2b).This compound was purified bysilica gel flash column chromatography using cyclohexane/ethylacetate (8:2). Yield 88%; mp 58-61 °C; 1H NMR (CDCl3) δ 1.47(dd,J ) 4.8, 8.8 Hz, 1H), 1.80 (dd,J ) 4.8, 7.0 Hz, 1H), 1.99 (m,1H), 2.34 (s, 3H), 3.65 (s, 3H), 3.80 (dd,J ) 10.0, 11.4 Hz, 1H),4.00 (dd,J ) 5.6, 11.4 Hz, 1H), 7.13 (d,J ) 8.4 Hz, 2H), 7.27 (d,J ) 7.8 Hz, 2H). Anal. (C13H15ClO2) C, H, N.

Methyl (1R,2S/1S,2R)-2-(Chloromethyl)-1-(4-methoxyophe-nyl)cyclopropanecarboxylate (2c). Yield 90%; oil; 1H NMR(CDCl3) δ 1.46 (dd,J ) 4.8, 8.6 Hz, 1H), 1.80 (dd,J ) 4.8, 7.0Hz, 1H), 1.85-2.10 (m, 1H), 3.54 (s, 3H), 3.72 (s, 3H), 3.75 (dd,J ) 10.0, 11.2 Hz, 1H), 4.00 (dd,J ) 5.6, 11.2 Hz, 1H), 6.80-7.38 (m, 4H). Anal. (C13H15ClO3‚0.3Et2O) C, H, N.

Methyl (1R,2S/1S,2R)-2-(Chloromethyl)-1-(4-chlorophenyl)-cyclopropanecarboxylate (2d).The crude product was purifiedby chromatography on silica gel (cyclohexane/ethyl acetate, 9:1).Yield 80%; mp 47-51 °C; 1H NMR (CDCl3) δ 1.47 (dd,J ) 4.8,9.0 Hz, 1H), 1.83 (dd,J ) 4.8, 6.8 Hz, 1H), 1.90-2.10 (m, 1H),3.62 (s, 3H), 3.77 (dd,J ) 6.2, 11.4 Hz, 1H), 4.00 (dd,J ) 5.6,11.4 Hz, 1H), 7.20-7.40 (m, 4H). Anal. (C12H12Cl2O2) C, H, N.

Methyl (1R,2S/1S,2R)-2-(Chloromethyl)-1-(4-nitrophenyl)cy-clopropanecarboxylate (2e).Thionyl chloride (2.5 mL, 34.25mmol) was added dropwise to 1.5 g (6.84 mmol) of racemic lactone1e in 20 mL of anhydrous benzene at 0°C and was heated to refluxfor 5 h. The mixture was cooled to 0°C, and a solution of 3 NCH3OH/HCl (7.3 mL) was added dropwise. The reaction mixturewas then allowed to stand at room temperature overnight. Themixture was evaporated under reduced pressure, and the residuewas dissolved in Et2O and washed with a saturated solution ofNaHCO3. The organic phase was dried (Na2SO4) and evaporatedin vacuo to give the desired ester, which was used without furtherpurification.

Methyl (1R,2S/1S,2R)-1-(4-Aminophenyl)-2-(chloromethyl)-cyclopropanecarboxylate (2f).Thionyl chloride (1.57 mL, 21.51mmol) was added dropwise to 836 mg (4.42 mmol) of1f in 13.13mL of anhydrous benzene at 0°C and was heated to reflux for 5h. The mixture was cooled to 0°C, and a solution of 3 N CH3-OH/HCl (3.02 mL) was added dropwise. The reaction mixture wasthen allowed to stand at room temperature overnight. The mixturewas evaporated under reduced pressure, and the residue wasdissolved in Et2O and extracted with a saturated solution ofNaHCO3. The organic phase was dried over Na2SO4 and evaporatedin vacuo and the resultant crude product was purified by chroma-tography on silica gel (cyclohexane/ethyl acetate, 6.5:3.5) to obtain500 mg of product. Yield 65.8%; mp, decomposes;1H NMR(CDCl3) δ 1.43 (dd,J ) 4.8, 9.0 Hz, 1H), 1.75 (dd,J ) 4.8, 6.8Hz, 1H), 1.80-2.05 (m, 1H), 3.51 (br s, 2H), 3.64 (s, 3H), 3.75 (d,J ) 11.4 Hz, 1H), 4.12 (dd,J ) 5.6, 11.4 Hz, 1H), 6.63 (d,J )8.4 Hz, 2H), 7.15 (d,J ) 8.4 Hz, 2H). Anal. (C12H14NO2Cl) C, H,N.

Methyl (1R,2S/1S,2R)-2-[(4-Hydroxy-4-phenylpiperidin-1-yl)-methyl]-1-phenylcyclopropanecarboxylate Oxalate (4a).A mix-ture of 4-phenylpiperidin-4-ol 200 mg (1.13 mmol), methyl (1R,2S/1S,2R)-2-(chloromethyl)-1-phenylcyclopropanecarboxylate (2a, 253mg, 1.13 mmol), and NaHCO3 (190 mg, 2.26 mmol) in dry DMF(10 mL) was heated to 60°C under a nitrogen atmosphere for 24h. The solvent was removed under reduced pressure, and the mixturewas extracted with CHCl3. The combined organic layers werewashed with water and with 5% aqueous NaHCO3 and dried overanhydrous Na2SO4. The crude product was purified by silica gelflash column chromatography using ethyl acetate/cyclohexane/ethanol (6:2:2). The resulting colorless oil was dissolved in diethylether and treated with a solution of oxalic acid dihydrate in diethylether to give 127 mg of colorless solid. Yield 27%; mp 201-202°C; 1H NMR (DMSO-d6) δ 1.40 (dd,J ) 4.6, 9.0 Hz, 1H), 1.58(dd,J ) 4.8, 6.8 Hz, 1H), 1.66-1.78 (m, 2H), 1.85-1.97 (m, 1H),

2.04-2.20 (m, 2H), 2.80-3.20 (m 7H), 3.56 (s, 3H), 5.80 (br s,1H), 7.16-7.52 (m, 10H). Anal. (C23H27NO3‚0.5C2H2O4) C, H, N.

Compounds4b-e were synthesized analogously.Methyl (1R,2S/1S,2R)-2-[(4-Hydroxy-4-phenylpiperidin-1-yl)-

methyl]-1-(4-methylphenyl)cyclopropanecarboxylate Oxalate (4b).Yield 39%; mp 197-198 °C; 1H NMR NMR (DMSO-d6) δ 1.37(dd, J ) 4.6, 8.8 Hz, 1H), 1.56 (dd,J ) 4.6, 6.8 Hz, 1H), 1.63-1.78 (m, 2H), 1.80-1.88 (m, 1H), 2.0-2.21 (m, 2H), 2.27 (s, 3H),2.82-3.20 (m, 7H), 3.55 (s, 3H), 5.65 (br s, 1H), 7.03-7.52 (m,9H). Anal. (C24H29NO3‚0.5C2H2O4) C, H, N.

Methyl (1R,2S/1S,2R)-2-[(4-Hydroxy-4-phenylpiperidin-1-yl)-methyl]-1-(4-methoxyphenyl)cyclopropanecarboxylate Oxalate(4c). Yield 20%; mp 185-186 °C; 1H NMR (DMSO-d6) δ 1.37(dd,J ) 4.6, 8.8, Hz, 1H), 1.43-2.0 (m, 4H), 2.05-2.13 (m, 2H),2.80-3.20 (m, 7H), 3.54 (s, 3H), 3.72 (s, 3H), 5.30 (br s, 1H),6.80-7.42 (m, 9H). Anal. (C24H29NO4‚0.5C2H2O4‚0.75H2O) C, H,N.

Methyl (1R,2S/1S,2R)-1-(4-Chlorophenyl)-2-[(4-hydroxy-4-phenylpiperidin-1-yl)methyl]cyclopropanecarboxylate (4d).Yield68%; mp 125-126 °C; 1H NMR NMR (DMSO-d6) δ 1.21 (dd,J) 4.4, 8.8 Hz, 1H), 1.37-1.87 (m, 6H), 2.27-2.77 (m, 7H), 3.44(s, 3H), 4.69 (s, 1H), 7.0-7.42 (m, 9H). Anal. (C23H26NClO3‚0.4H2O) C, H, N.

Methyl (1R,2S/1S,2R)-2-[(4-Hydroxy-4-phenylpiperidin-1-yl)-methyl]-1-(4-nitrophenyl)cyclopropanecarboxylate Oxalate (4e).This compound was purified by silica gel flash column chroma-tography using ethyl acetate/cyclohexane (2:1). Yield 43%; mp204-205°C; 1H NMR NMR (DMSO-d6) δ 1.60 (dd,J ) 4.6, 9.0Hz, 1H), 1.70 (m, 3H), 1.94-2.23 (m, 3H), 2.84-3.27 (m, 7H),3.58 (s, 3H), 5.03 (br s, 1H), 7.18-8.23 (m, 9H). Anal. (C23H26N2O5‚0.5C2H2O4‚0.25H2O) C, H, N.

Methyl (1R,2S/1S,2R)-2-[(4-Benzylpiperidin-1-yl)methyl]-1-phenylcyclopropanecarboxylate Oxalate (5a).A mixture of4-benzylpiperidine (0.20 mL, 1.11 mmol), (1R,2S/1S,2R)-2-(chlo-romethyl)-1-phenylcyclopropanecarboxylate (2a, 250 mg, 1.11mmol), and NaHCO3 (200 mg, 2.22 mmol) in dry DMF (10 mL)was heated to 60°C under a nitrogen atmosphere for 24 h. Thesolvent was removed under reduced pressure, and the mixture wasextracted with CHCl3. The combined organic layers were washedwith water and dried over anhydrous Na2SO4. The crude productwas purified by silica gel flash column chromatography using ethylacetate/cyclohexane (6:4). The resulting colorless oil was dissolvedin diethyl ether and treated with a solution of oxalic acid dihydratein diethyl ether to give 220 mg of the title compound. Yield 44%;mp 141-142 °C; 1H NMR (DMSO-d6) δ 1.30-2.10 (m, 8H),2.42-3.52 (m, 8 H), 3.56 (s, 3H), 7.10-7.40 (m, 10H), 7.80 (br s,3H). Anal. (C24H29NO2‚0.8C2H2O4‚H2O) C, H, N.

Compounds5b-f were synthesized analogously.Methyl (1R,2S/1S,2R)-2-[(4-Benzylpiperidin-1-yl)methyl]-1-

(4-methylphenyl)cyclopropanecarboxylate Oxalate (5b).Yield20%; mp 149-150°C; 1H NMR (CDCl3) (free base)δ 1.25-2.10(m, 10H), 2.32 (s, 3H), 2.30-2.70 (m, 4H), 2.90-3.10 (m, 2H),3.58 (s, 3H), 7.05-7.35 (m, 9H). Anal. (C25H31NO2‚1.25C2H2O4‚3H2O) C, H, N.

Methyl (1R,2S/1S,2R)-2-[(4-Benzylpiperidin-1-yl)methyl]-1-(4-methoxyphenyl)cyclopropanecarboxylate Oxalate (5c).Yield37%; mp 144-145 °C; 1H NMR NMR (DMSO-d6) δ 1.30-2.00(m, 8H), 2.42-3.60 (m, 8 H), 3.61 (s, 3H), 3.73 (s, 3H), 6.78-7.40 (m, 9H), 7.90 (br s, 1H). Anal. (C25H31NO3‚0.5C2H2O4‚1.75H2O) C, H, N.

Methyl (1R,2S/1S,2R)-2-[(4-Benzylpiperidin-1-yl)methyl]-1-(4-chlorophenyl)cyclopropanecarboxylate Oxalate (5d).Yield34%; mp 86-87 °C; 1H NMR NMR (DMSO-d6) δ 1.30-2.05 (m,8H), 2.42-3.51 (m, 8 H), 3.55 (s, 3H), 7.12-7.42 (m, 9H), 8.0 (brs, 4H). Anal. (C24H28ClNO2‚0.5C2H2O4‚1.6H2O) C, H, N.

Methyl (1R,2S/1S,2R)-2-[(4-Benzylpiperidin-1-yl)methyl]-1-(4-nitrophenyl)cyclopropanecarboxylate Oxalate (5e).Yield44%; mp 99-100 °C; 1H NMR NMR (DMSO-d6) δ 1.30-2.10(m, 8H), 2.42-3.52 (m, 8 H), 3.58 (s, 3H), 6.80 (br s, 4H), 7.05-8.22 (m, 9H). Anal. (C24H28N2O4‚0.5C2H2O4‚2H2O) C, H, N.


The following compounds were prepared using the aboveprocedure.

Methyl (1R,2S/1S,2R)-1-Phenyl-2-[(4-pyridin-2-ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (6a).This com-pound was purified by silica gel flash column chromatography usingchloroform as eluent. Yield 19%; mp 205-207 °C; 1H NMR(DMSO-d6) (free base)δ 1.45 (dd,J ) 4.8, 8.8 Hz, 1H), 1.59 (dd,J ) 4.8, 6.8 Hz, 1H), 1.90-2.07 (m, 1H), 3.07-3.19 (m, 5H), 3.24(dd, J ) 6.4, 13.2 Hz, 1H), 3.56 (s, 3H), 3.62-3.80 (m, 4H), 6.70(dd, J ) 5.2, 6.8 Hz, 1H), 6.90 (d,J ) 8.6 Hz, 1H), 7.22-7.37(m, 5H), 7.53 (dt,J ) 1.6, 8.6 Hz, 1H), 8.14 (dd,J ) 1.6, 4.6 Hz,1H). Anal. (C21H25N3O2‚0.7H2C2O4‚1.3H2O) C, H, N.

Methyl (1R,2S/1S,2R)-1-(4-Methylphenyl)-2-[(4-pyridin-2-ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (6b).Compound6b was purified by silica gel flash column chromatog-raphy using chloroform/ethanol (9.8:0.2). Yield 42%; mp 202-205 °C; 1H NMR (DMSO-d6) (free base)δ 1.42 (dd,J ) 4.6, 8.4Hz, 1H), 1.57 (dd,J ) 4.6, 6.8 Hz, 1H), 1.80-2.05 (m, 1H), 2.27(s, 3H), 3.0-3.20 (m, 5H), 3.28 (dd, 6.0, 13.2 Hz, 1H), 3.55 (s,3H), 3.60-3.72 (m, 4H), 6.70 (dd,J ) 4.8, 6.6 Hz, 1H), 6.91 (d,J ) 8.6 Hz, 1H), 7.16 (d,J ) 8.2 Hz, 2H), 7.22 (d,J ) 8.2 Hz,2H), 7.60 (dt,J ) 1.6, 6.6 Hz, 1H), 8.14 (dd,J ) 1.6, 4.2 Hz, 1H).Anal. (C22H27N3O2‚0.7H2C2O4‚2H2O) C, H, N.

Methyl (1R,2S/1S,2R)-1-(4-Methoxyphenyl)-2-[(4-pyridin-2-ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (6c).Yield 31%; mp 194-195°C; 1H NMR (CDCl3) (free base)δ 1.34(dd,J ) 4.2, 8.6 Hz, 1H), 1.61-1.85 (m, 2H), 2.58-2.70 (m, 6H),3.45-3.75 (m, 7H), 3.80 (s, 3H), 6.58-6.78 (m, 2H), 6.84 (d,J )8.8 Hz, 2H), 7.27 (d,J ) 9.0 Hz, 2H), 7.50 (dt,J ) 1.6, 6.6 Hz,1H), 8.14 (dd,J ) 1.6, 4.0 Hz, 1H). Anal. (C22H27N3O3‚H2C2O4‚H2O) C, H, N.

Methyl (1R,2S/1S,2R)-1-(4-Chlorophenyl)-2-[(4-pyridin-2-ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (6d).Yield 36%; mp 198-200 °C; 1H NMR (DMSO-d6) (free base)δ1.47 (dd,J ) 4.8, 9.0 Hz, 1H), 1.59 (dd,J ) 4.8, 7.0 Hz, 1H),1.92 (m, 1H), 3.05-3.28 (m, 6H), 3.56 (s, 3H), 3.62-3.78 (m,4H), 6.70 (dd,J ) 5.0, 6.6 Hz, 1H), 6.90 (d,J ) 8.8 Hz, 1H), 7.38(s, 4H), 7.58 (dt,J ) 1.6, 6.6 Hz, 1H), 8.13 (dd,J ) 1.6, 5.0 Hz,1H). Anal. (C21H24ClN3O2‚0.6H2C2O4‚2H2O) C, H, N.

Methyl (1R,2S/1S,2R)-1-(4-Nitrophenyl)-2-[(4-pyridin-2-ylpip-erazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (6e).Com-pound6ewas purified by silica gel flash column chromatographyusing chloroform/ethanol (9.8:0.2). Yield 52%; mp 197-201 °C;1H NMR (CDCl3) (free base)δ 1.25-1.27 (m, 1H), 1.81-1.89(m, 2H), 2.61-2.96 (m, 6H), 3.47-3.70 (m, 7H), 6.60-6.72 (m,2H), 7.41-7.60 (m, 4H), 7.96-8.0 (m, 1H), 8.13-8.21 (m, 1H).Anal. (C21H24N4O4‚0.9H2C2O4‚0.3H2O) C, H, N.

Methyl (1R,2S/1S,2R)-1-(4-Aminophenyl)-2-[(4-pyridin-2-ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (6f).Compound6f was purified by silica gel flash column chromatog-raphy using chloroform/ethanol (9.8:0.2). Yield 37%; mp 187-190°C; 1H NMR (CDCl3) (free base)δ 1.31 (dd,J ) 4.6, 8.8 Hz,1H), 1.59-1.79 (m, 2H), 2.65-2.72 (m, 6H), 3.54-3.61 (m, 7H),4.40 (br s, 2H), 6.58-6.72 (m, 4H), 7.13 (d,J ) 8.6 Hz, 2H), 7.52(dt, J ) 1.6, 8.6 Hz 1H), 8.19 (dd,J ) 1.6, 5.0 Hz, 1H). Anal.(C21H26N4O2‚0.9H2C2O4‚1.6H2O) C, H, N.

Methyl (1R,2S/1S,2R)-1-Phenyl-2-[(4-pyrimidin-2-ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (7a).A 1.32 g(5.94 mmol) mixture ofcis-(()-halogenated ester2a, 2-(piperazin-1-yl)pyrimidine (0.88 g, 5.4 mmol), and 0.9 g (10.8 mmol) ofNaHCO3 in DMF (30 mL) was heated to 70°C for 24 h. The solventwas evaporated at decreased pressure, and the crude product wasdissolved with CHCl3 and washed twice with water. The organicphase was separated, dried over Na2SO4, and filtered, and thesolvent was removed in vacuo. The residual product was purifiedby chromatography on silica gel, CHCl3/EtOH (9.8/0.2), and thecolorless oil was dissolved in diethyl ether and treated with asolution of oxalic acid dihydrate in diethyl ether to give the oxalatesalts as a colorless solid. Yield 39% as free base; mp 228-230°C; 1H NMR (DMSO-d6) (free base)δ 1.44 (dd,J ) 4.6, 9.0 Hz,1H), 1.59 (dd,J ) 4.6, 7.0 Hz, 1H), 1.84-2.04 (m, 1H), 2.94-

3.12 (m, 5H), 3.19 (dd,J ) 6.4, 13.2 Hz, 1H), 3.55 (s, 3H), 3.80-4.0 (m, 4H), 6.70 (t,J ) 4.8, 9.6 Hz, 1H), 7.20-7.40 (m, 5H),8.40 (d,J ) 4.8 Hz, 2H). Anal. (C20H24N4O2‚0.5H2C2O4‚2H2O‚0.2(C2H5)2O) C, H, N.

Methyl (1R,2S/1S,2R)-1-(4-Methylphenyl)-2-[(4-pyrimidin-2-ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (7b).Yield 37%; mp 222-225°C; 1H NMR (CDCl3) (free base)δ 1.34(dd, J ) 4.4, 8.8 Hz, 1H), 1.65 (dd,J ) 4.4, 7.2 Hz, 1H), 1.72-1.90 (m, 1H), 2.34 (s, 3H), 2.50-2.82 (m, 6H), 3.62 (s, 3H), 3.90(m, 4H), 6.49 (t,J ) 4.8 Hz, 1H), 7.12 (d,J ) 8.0 Hz, 2H), 7.24(d, J ) 8.4 Hz, 2H), 8.31 (d,J ) 4.8 Hz, 2H). Anal. (C21H26N4O2‚0.3H2C2O4‚2.5H2O‚0.3(C2H5)2O) C, H, N.

Methyl (1R,2S/1S,2R)-1-(4-Methoxyphenyl)-2-[(4-pyrimidin-2-ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (7c).Yield 36%; mp 218-219 °C; 1H NMR (DMSO-d6) (free base)δ1.40 (dd,J ) 4.6, 8.8 Hz, 1H), 1.55 (dd,J ) 4.6, 7.0 Hz, 1H),1.73-1.80 (m, 1H), 2.80-3.20 (m, 6H), 3.58 (s, 3H), 3.76 (s, 3H),3.80-3.94 (m, 4H), 6.74 (t,J ) 4.8, 9.4 Hz, 1H), 6.91 (d,J ) 8.8Hz, 2H), 7.29 (d,J ) 8.6 Hz, 2H), 8.43 (d,J ) 4.8 Hz, 2H). Anal.(C21H26N4O3‚H2C2O4‚H2O‚(C2H5)2O) C, H, N.

Methyl (1R,2S/1S,2R)-1-(4-Chlorophenyl)-2-[(4-pyrimidin-2-ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (7d).This compound was purified by silica gel flash column chroma-tography using chloroform as eluent. Yield 26%; mp 201-204°C;1H NMR (DMSO-d6) (free base)δ 1.46 (dd,J ) 4.6, 9.0 Hz, 1H),1.59 (dd,J ) 4.6, 6.6 Hz, 1H), 1.84-2.0 (m, 1H), 2.88-3.10 (m,5H), 3.17 (dd,J ) 6.4, 12.6 Hz, 1H), 3.56 (s, 3H), 3.80-4.0 (m,4H), 6.70 (t,J ) 4.8 Hz, 1H), 7.30-7.42 (m, 4H), 8.40 (d,J ) 4.8Hz, 2H). Anal. (C20H23ClN4O2‚0.6H2C2O4‚1.7H2O‚0.2(C2H5)2O) C,H, N.

Methyl (1R,2S/1S,2R)-(4-Nitrophenyl)-2-[(4-pyrimidin-2-ylpip-erazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (7e).Yield22%; mp 205-207 °C; 1H NMR (DMSO-d6) (free base)δ 1.56(dd, J ) 4.6, 9.0 Hz, 1H), 1.59 (dd,J ) 4.6, 7.2 Hz, 1H), 1.94-2.10 (m, 1H), 2.88-3.10 (m, 5H), 3.17 (dd,J ) 6.6, 13.4 Hz, 1H),3.57 (s, 3H), 3.80-4.0 (m, 4H), 6.69 (t,J ) 4.6, Hz, 1H), 7.65 (d,J ) 8.8 Hz, 2H), 8.17 (d,J ) 8.8 Hz, 2H), 8.39 (d,J ) 4.6 Hz,2H). Anal. (C20H23N5O4‚0.5H2C2O4‚1.2H2O) C, H, N.

Methyl (1R,2S/1S,2R)-1-(4-Aminophenyl)-2-[(4-pyrimidin-2-ylpiperazin-1-yl)methyl]cyclopropanecarboxylate Oxalate (7f).Yield 19%; mp 180-184 °C; 1H NMR (DMSO-d6) (free base)δ1.34 (dd,J ) 4.8, 8.6 Hz, 1H), 1.45-190 (m, 4H), 3.05-3.23 (m,5H), 3.32 (dd,J ) 5.6, 13.6 Hz, 1H), 3.54 (s, 3H), 3.95-4.05 (m,4H), 6.47 (d,J ) 8.2 Hz, 2H), 6.72 (t,J ) 4.8, Hz, 1H), 6.97 (d,J ) 8.2 Hz, 2H), 8.41 (d,J ) 4,8 Hz, 2H). Anal. (C20H25N5O2‚0.9H2C2O4‚1.8H2O)

Biological Studies. Cell Cultures.Primary cultures of astrocyteswere prepared from newborn albino rat brains (1-2 day-old Wistarstrain) as previously described.37 Briefly, cerebral tissues, afterdissection and careful removal of the meninges, were mechanicallydissociated through sterile meshes of 82µm pore size (Nitex).Isolated cells were suspended in Dulbecco’s modified Eagle’smedium (DMEM), supplemented with 20% (V/V) heat-inactivatedfetal bovine serum (FBS), 2 mM glutamine, streptomycin (50 mg/mL), and penicillin (50 U/mL), and plated at a density of 3× 106

cells per 100 mm dish and 0.5× 105 cells/chamber in Lab-Tek IImultichambered slides. Cells were maintained at 37°C in a 5%CO2-95% air humidified atmosphere for 2 weeks, and the mediumwas replaced every 3 days. The purity of cell cultures was evaluatedby the immunostaining for GFAP, an astroglial cell marker ofgrowth, maturation, and differentiation.21,43 Under these experi-mental conditions, the presence of bothσ1 and σ2 has beendemonstrated.44 All efforts were made to minimize both thesuffering and number of animals used. All experiments wereperformed according to the guidelines of the Ethical Committee ofUniversity of Catania, Italy.

Treatment of σ Ligands. In previous experiments, on the basisof both microscopy observations and MTT assays, we establishedthe nontoxic concentrations and the time of exposure of the culturesto theσ ligands for astroglial cell cultures. The time of exposureto the drugs was selected when the TG-2 expression reached the


maximum levels of activity and expression. We observed that theoptimal time of exposure toσ ligands was 24 h.

Primary rat astroglial cell cultures at 14 DIV for 24 h were treatedwith ibogaine, haloperidol, BD1047,4b, and (+)-pentazocine, inthe range 25-100µM. The nontoxic concentration of the exposureof the astroglial cell cultures to theσ ligands was 25µM. Fourreplicates were performed for each sample.

Western Blot. Control and treated primary astroglial cell cultureswere harvested using cell lysis buffer as previously described.23

After treatment, cells were harvested in cold PBS, collected bycentrifugation, resuspended in a homogenizing buffer (50 mM Tris-HCl, pH 6,8, 150 mM NaCl, 1 mM EDTA, 0.1 mM phenylmeth-ylsulfonyl fluoride, and a 10µg/mL each of aprotinin, leupeptin,and pepstatin), and sonicated on ice. Protein concentration of thehomogenates was diluted to 1 mg/mL with 2× reducing stop buffer(0.25 M Tris-HCl, pH 6.8, 5 mM EGTA, 25 mM dithiothreitol(DTT), 2% SDS, and 10% glycerol with bromophenol blue as thetracking dye). Proteins (30µg) were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes.Blots were blocked overnight at 4°C with 5% nonfat dry milkdissolved in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Tween-20 (TBST) buffer at 4°C. After being washed with TBST, themembranes were incubated with anti-TG-2 monoclonal antibody(diluted 1:1.000) or with anti-caspase-3 monoclonal antibody(diluted 1:1.000) for 1 h atroom temperature with constant shaking.The filters were then washed and subsequently probed withhorseradish peroxidase-conjugated antimouse IgG at a dilution of1:2000.

The TG-2 expression and caspase-3 cleavage were visualizedby chemiluminescence with the ECL kit after autoradiography filmexposure. Blots were scanned and quantified with a AlphaImager1200 system (Alpha Innotech, San Leandro, CA). The results areexpressed as the percentage of TG-2 or caspase-3 cleavage increaseor decrease versus control.

Expression and Localization of TG-2 by Confocal LaserScanning Microscopy (CLSM). Expression and the cellularlocalization of TG-2 in subcellular compartments of fixed controland σ ligand astroglial cell cultures were determined by CLSM.The cultures of untreated and treated astrocytes at 14 days in culture(DIV) were fixed for 20 min with 4% paraformaldehyde in PBS,washed three times with PBS, incubated for 1 h with 1% normalblocking serum in PBS to block nonspecific sites, and thenincubated overnight with antimouse monoclonal antibody againstTG-2 (1:500). The slides were then washed and incubated for 2 hwith FITC-conjugated anti-IgG polyclonal antibody (1:64 in PBS)and for 2 h with TRITC-conjugated anti-IgG polyclonal antibody(1:64 in PBS). Finally, the slides were extensively washed withPBS, mounted with 90% glycerol, and observed with CLSM (Leika,Germany).

Analysis by CLSM. The optical fields were visualized using a40 × 1.0 NA oil immersion objective in a Leica DM IRB invertedfluorescence microscope (Leica Microsystems, Heidelberg, Man-nheim, Germany). At the same time, the slides were examined ona Leica TCS SP2 CLSM by green (FITC) and red (TRITC)fluorophore excitation at 488 and 543 nm, respectively, utilizingAr/Kr and He/Ne laser sources. The fluorescence emitted from thespecimens was sent through a Leica AOTF mechanism (acousticoptical turnable filter) and pinhole to two different photomultipliertubes (PMTs). Image outputs electronically generated by the sameparameter settings (frame scanning, pinhole aperture, gain voltage,pixel and spatial resolution) were measured and compared forintensity (treated vs control) after nonspecific fluorescence subtrac-tion. Sequential optical sections, obtained using the CLSMz-axisstepping capability, were combined to form an extended depth offocus image, and standard image processing was performed toenhance brightness and contrast, using Leica Confocal Software(version 2.0, build 0585).

Terminal Deoxynucleotidyltransferase dUTP Nick End-Labeling (TUNEL) Assay. The ApoAlert DNA fragmentationassay kit (Clontech) detecting nuclear DNA fragmentation, ahallmark of apoptosis, was used.31 The ApoAlert DNA fragmenta-

tion assay incorporates fluorescein-dUTP at the free 3′-hydroxylends of the fragmented DNA using TUNEL and was performedaccording to the user’s manual. According to the user’s manual,apoptotic cells appear green while nonapoptotic cells appear red.We focused on 10 random microscopic fields for each dish. In eachmicroscopic field we counted the number of apoptotic cells andwe compared this number with all the nonapoptotic cells visualizedin the same microscopic field; the ratio is expressed as a percentage.Primary rat astroglial cell cultures, untreated and treated for 24 hwith the σ ligands, were made up according to the user’s manual.Cells were mounted and visualized directly by fluorescencemicroscopy (Leika, Germany) with either a propidium iodide (PI)filter alone or a FITC filter alone. According to the user’s manual,apoptotic cells appear green with the FITC filter alone whilenonapoptotic cells appear red under the dual-pass FITC/PI filterset.

Statistical Analysis. All values are presented as the mean(SEM of five experiments. A one-way ANOVA test, followed by aNewman-Keuls multiple range test, was used to analyze the timecourse of TGase activity, and the Student’st test was used tocompare values of densitometric analysis of immunoblots.

Acknowledgment. This work was supported by FIRB 2003(Grant No. RBNE03FH5Y) and PRIN 2005 (Grant No.2005032713) from MIUR (Rome). We thank Slater & Frith forthe gift of ibogaine. We also thank Professor Isa Picerno andher colleagues of the Department of Hygiene, Public Health,and Preventive Medicine, University of Messina, Italy, for theiruseful collaboration.

Supporting Information Available: Results from elementalanalysis. This material is available free of charge via the Internetat http://pubs.acs.org.

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Novel Sigma Receptor Ligands: Synthesis and Biological Profile

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