Synthesis and receptor binding properties of fluoro- and iodo-substituted high affinity sigma receptor ligands: identification of potential PET and SPECT sigma receptor imaging agents.

Unlabeled fluoro- and iodo-substituted ligands exhibiting very high affinity and selectivity for sigma receptors were synthesized based on three different structural classes of sigma receptor ligands. These compounds were evaluated for sigma receptor affinity and specificity in order to assess their potential as PET/SPECT imaging agents. Thus, (+)- and (-)-N-(5-fluoro-1-pentyl)normetazocines [(+)- and (-)-4] based on the (+)-benzomorphan class of sigma ligands were synthesized via N-alkylation of optically pure (+)- and (-)-normetazocine with 5-[(methylsulfonyl)oxy]-1-pentyl fluoride (11). (+)- and (-)-4 displaced [3H](+)-3-PPP with Ki values of 0.29 and 73.6 nM and [3H](+)-pentazocine with Ki values of 10.5 and 38.9 nM, respectively. The second class of PET/SPECT ligands was based upon the N-(arylethyl)-N-alkyl-2-(1-pyrrolidinyl)ethylamine class of sigma ligands; N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-(3-fluoro-1-propyl)-2-(1- pyrrolidinyl)ethylamine (5) was obtained via N-alkylation of N-[2-(3,4-dichlorophenyl)-1-ethyl]-2-(1-pyrrolidinyl)ethylamine (14) with 3-fluoropropyl p-toluenesulfonate. 5 exhibited Ki values of 4.22 and 5.07 nM for displacement of [3H](+)-3-PPP and [3H](+)-pentazocine, respectively, comparable with the parent N-propyl compound. Attempts to synthesize N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-[3- [(methylsulfonyl)oxy]-1-propyl]-2-(1-pyrrolidinyl)ethylamine (26), a precursor to 5 that could conceivably be converted to [18F]-5 by treatment with 18F-, proved unsuccessful. The sequence of regioselective nitration, catalytic hydrogenation, and diazotization followed by NaI quench of N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-methyl-2-(1- pyrrolidinyl)ethylamine (2) afforded the iodinated ethylenediamine N-[2-(2-iodo-4,5-dichlorophenyl)-1-ethyl]-N-methyl-2-(1- pyrrolidinyl)ethylamine (8), a potential SPECT ligand for sigma receptors. This compound showed an affinity of 0.54 nM ([3H](+)-3-PPP) comparable with the parent compound 2 (Ki = 0.34 nM, [3H](+)-3-PPP). Ligand 8 exhibited a similar potency against [3H](+)-pentazocine. The third class of high-affinity sigma receptor ligands was rationalized based on rearrangement of the bonds in ethylenediamine 2 to give 1-[2-(3,4-dichlorophenyl)-1-ethyl]-4-(1-propyl)piperazine (3). This compound exhibited very high affinity (Ki = 0.31 nM, [3H](+)-3-PPP) and selectivity for sigma receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and evaluation of N-substituted cis-N-methyl-2-(1-pyrrolidinyl)cyclohexylamines as high affinity sigma receptor ligands. Identification of a new class of highly potent and selective sigma receptor probes

Certain benzeneacetamides [(-)- and (+)-cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]benzeneacetamide] were recently reported to be potent sigma receptor ligands. In order to determine whether efficacy for the sigma receptor could be improved, a series of compounds related to the benzeneacetamides, N-substituted cis-2-(1-pyrrolidinyl)-N-methylcyclohexylamines, were synthesized and their structure-activity requirements were determined. The compounds were synthesized by starting with the previously reported (+/-)-, 1S,2R-(+)-, and 1R,2S-(-)-cis-2-(1-pyrrolidinyl)-N-methylcyclohexylamines. Analysis of sigma ([3H](+)-3-PPP), kappa ([3H]bremazocine and [3H]U69,593), dopamine-d2 ([3H](-)-sulpiride), and phencyclidine (PCP) ([3H]TCP) receptor binding in guinea pig brain revealed a number of highly potent and selective sigma receptor ligands. Notably, 1S,2R-cis-(-)-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-(2-naphthyl) acetamide [(-)-29] (Ki = 8.66 +/- 0.35 nM), (+/-)-cis-2-amino-4,5-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide [(+/-)-17] (Ki = 11 +/- 3 nM), 1S,2R-(-)-cis-N-methyl-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl ) cyclohexylamine [(-)-44] (Ki = 1.3 +/- 0.3 nM), and 1R,2S-(+)-cis-N-methyl-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl ) cyclohexylamine. [(+)-44] (Ki = 6 +/- 3 nM) exhibited very high affinity at sigma receptors, by displacement of [3H]-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [( 3H]-(+)-3-PPP). These compounds showed insignificant affinity for kappa, dopamine, or PCP receptors, making them valuable tools for the study of sigma receptors. Furthermore, these compounds also exhibited enantioselectivity ranging from 5-fold for (+)- and (-)-44 to 160-fold for (+)- and (-)-29. Several other compounds showed equivalent selectivity but displayed lower sigma receptor affinity.     

Synthesis and biological evaluation of conformationally restricted 2-(1-pyrrolidinyl)-N-[2-(3,4-dichlorophenyl)ethyl]-N-methylethylenediam ines as sigma receptor ligands. 1. Pyrrolidine, piperidine, homopiperidine, and tetrahydroisoquinoline classes.

The synthesis and sigma receptor affinity of a series of conformationally restricted derivatives of 2-(1-pyrrolidinyl)-N-[2-(3,4-dichlorophenyl)ethyl]-N-methylethylenedi amine (1) is described. The pyrrolidinyl (or N,N-dialkyl),ethylenediamine,N-alkyl, and phenylethyl portions of this sigma receptor pharmacophore were restricted by its incorporation into 1,2-cyclohexanediamine-, pyrrolidine-, piperidine-, homopiperidine-, and tetrahydroisoquinoline-containing ligands. The sigma receptor binding affinities of these compounds were determined using [3H](+)-pentazocine in guinea pig brain homogenates. The synthesis of all but one class was achieved by acylation and alane reduction of the appropriate diamine precursors whose synthesis is also reported. sigma receptor affinities ranged from 1.34 nM for 6,7-dichloro-2-[2-(1-pyrrolidinyl)ethyl]tetrahydroisoquinoline (12) to 455 nM for (1R,2R)-trans-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2- (1-pyrrolidinyl)cyclohexylamine [(-)-4]. In this displacement assay, (+)-pentazocine exhibited a Ki of 3.1 nM while DTG and haloperidol showed Ki values of 27.7 and 3.7 nM, respectively. The conformationally free parent compound 1 exhibited a Ki value of 2.1 nM. Comparison of both the sigma receptor affinities and nitrogen atom geometry of the compounds revealed that a gauche relation of the nitrogen atoms of cis-1,2-cyclohexanediamines is not imperative for high affinity as we had previously thought. It is highly likely that nitrogen lone pair orientations and steric factors on the aliphatic portions of these ligands play a major role in the sigma receptor binding of this pharmacophore.     

N-alkyl substituted analogs of the sigma receptor ligand BD1008 and traditional sigma receptor ligands affect cocaine-induced convulsions and lethality in mice

Cocaine binds to sigma receptors with comparable affinity to its well-established interaction with dopamine transporters. Previous studies have shown BD1008 (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine) to have high affinity and selectivity for sigma receptors, and to additionally attenuate the locomotor stimulatory effects of cocaine. Therefore, in the present study, three N-alkyl substituted analogs of BD1008 were characterized in receptor binding and behavioral studies: BD1060 (N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl)ethylamine), BD1067 (N-[2-(3,4-dichlorophenyl)ethyl]-N-ethyl-2-(1-pyrrolidinyl)ethylamine), and BD1052 (N-[2-(3,4-dichlorophenyl)ethyl]-N-allyl-2-(1-pyrrolidinyl)ethylamine). Similarly to BD1008, all three analogs exhibited high affinity and selectivity for sigma receptors. In behavioral studies, BD1008, BD1060 or BD1067 attenuated cocaine-induced convulsions and lethality in Swiss Webster mice. The protective effects appear to be mediated through sigma receptor antagonism because traditional sigma receptor antagonists with high to moderate affinity for these receptors also attenuated the behavioral toxicity of cocaine. In contrast, traditional and novel sigma receptor agonists such as di-o-tolylguanidine and BD1052 worsened the behavioral toxicity of cocaine. To further characterize the actions of the N-alkyl substituted compounds, they were microinjected into the rat red nucleus, a functional assay of sigma receptor activity, where they produced agonist vs. antagonist actions that were consistent with their effects on cocaine-induced behaviors. Together, the data demonstrate that BD1008, BD1060 or BD1067 can attenuate the behavioral toxicity of cocaine, most likely through functional antagonism of sigma receptors.     

Synthesis and receptor binding of enantiomeric N-substituted cis-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl)cyclohexylamines as high-affinity sigma receptor ligands.

N-Alkyl-substituted derivatives of (+)- and (-)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl)cyclohexylamin e have been synthesized in nine steps in a stereospecific manner starting from cyclohexene oxide. The key step in the reaction sequence involved catalytic hydrogenation of oxime 8 in the presence of PtO2 and AcOH to give the cis diamine (+/-)-7. Most of the compounds in this series exhibited very high affinity at sigma receptors when tested against [3H]-(+)-3-PPP, and in general it was observed that the 1R,2S enantiomers bound more potently to sigma receptors than their corresponding 1S,2R enantiomers. The most potent sigma ligand found in this class was the unsubstituted derivative (1R,2S)-(-)-4, which exhibited an affinity constant of 0.49 nM. This compound was also found to be very selective for sigma receptors. It exhibited little or no affinity for kappa opioid, PCP, and dopamine-D2 receptors. It was also demonstrated that the cis configuration as opposed to the trans configuration of (+)- and (-)-5 was necessary for a higher sigma receptor affinity.     

Alterations in the stereochemistry of the kappa-selective opioid agonist U50,488 result in high-affinity sigma ligands

The synthesis and in vitro sigma receptor activity of the two diastereomers of U50,488 [(+/-)-2], namely, (1R,2S)-(+)- cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacet ami de [(+)-1] and (1S,2R)-(-)-cis-3,4-dichloro- N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide [(-)-1], are described. (+)-1 and (-)-1 were synthesized from (+/-)-trans-N-methyl-2-aminocyclohexanol [(+/-)-3]. Pyridinium chlorochromate (PCC) oxidation of the N-t-Boc-protected derivative of (+/-)-3 afforded (+/-)-2-[N- [(tert-butyloxy)carbonyl]-N-methylamino]cyclohexanone [(+/-)-5]. The sequence of enamine formation with pyrrolidine, catalytic reduction, N-deprotection, and optical resolution afforded (1R,2S)-(-)-cis-2-pyrrolidinyl-N-methylcyclohexylamine [(-)-10] and (1S,2R)-(+)-cis-2-pyrrolidinyl-N-methylcyclohexylamine [(+)-10]. The optical purity (greater than 99.5%) of (-)-10 and (+)-10 was determined by HPLC analysis of the diastereomeric ureas formed by reaction with optically pure (R)-alpha-methylbenzyl isocyanate. The absolute configuration of (-)-10 and (+)-10 was determined by single-crystal X-ray diffractometry of the bis-(R)-mandelate salt. Condensation of optically pure (-)-10 and (+)-10 with 3,4-dichlorophenylacetic acid furnished (+)-1 and (-)-1, respectively. Compounds (+)-1, (-)-1, (-)-2, and (+)-2 were compared for their binding affinities at kappa opioid, sigma, D2-dopamine, and phencyclidine (PCP) receptors in competitive binding assays using [3H]bremazocine ([3H]BREM) or [3H]U69,593, [3H]-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [[3H]-(+)-3-PPP], or [3H]-1,3-di(o-tolyl)guanidine ([3H]DTG), [3H]-(-)-sulpiride [[3H]-(-)SULP], and [3H]-1- [1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP), respectively. In the systems examined, (-)-2 exhibited the highest affinity for kappa receptors, with a Ki of 44 +/- 8 nM. However, (-)-2 also showed moderate affinity for sigma receptors, with a Ki of 594 +/- 3 nM [[3H]-(+)-3-PPP]. The (1R,2R)-(+)-enantiomer, (+)-2, had low affinity for both kappa and sigma receptors, exhibiting Ki values of 1298 +/- 49 nM at kappa ([3H]BREM) and 1270 +/- 168 nM at sigma [[3H]-(+)-3-PPP]. In contrast, the chiral cis compounds (+)-1 and (-)-1 showed high affinity for sigma receptors and negligible affinity for kappa opioid receptors in the [3H]BREM assay. Compound (-)-1 exhibited a Ki of 81 +/- 13 nM at sigma receptors [[3H]-(+)-3-PPP] and 250 +/- 8 nM ([3H]DTG).(ABSTRACT TRUNCATED AT 400 WORDS)     

Novel analogs of the sigma receptor ligand BD1008 attenuate cocaine-induced toxicity in mice

Previous studies have shown that BD1008 (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine) and related analogs attenuate the toxicity and stimulant effects of cocaine through antagonism of sigma receptors. In the present study, six analogs of BD1008 (UMB 98-103) were synthesized and evaluated in receptor binding and behavioral studies. Competition binding studies confirmed that all six compounds have high affinity for sigma1 receptors, moderate affinity for sigma2 receptors, and low to negligible affinity for monoamine transporters, opioid, N-methyl-D-aspartate, dopamine, and 5-HT receptors. In behavioral pharmacological studies, pretreatment of mice with UMB 100, UMB 101, or UMB 103 significantly attenuated cocaine-induced convulsions and lethality. Together with earlier studies, the data suggest that analogs of BD1008 are promising medication development leads for reducing the toxicity of cocaine.     

sigma(2) Receptors regulate changes in sphingolipid levels in breast tumor cells

sigma(2) Receptors induce apoptosis in various cell types. The sphingolipid, ceramide as well as the sphingoid bases are involved in cell proliferation. Sphingolipids of MCF-7/Adr- and T47D breast tumor cells were metabolically radiolabeled. The sigma(2) receptor agonists (+)-1R,5R-E-8-(3,4-dichlorobenzylidene)-5-(3-hydroxyphenyl)-2-methylmorphan-7-one (CB-184) and 1S,2R-(--)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)-cyclohexylamine (BD737) caused dose-dependent increases in [(3)H]ceramide, with concomitant decreases in [(3)H]sphingomyelin. Both effects were attenuated by the novel sigma(2) receptor antagonist, N-phenethylpiperidine oxalate (AC927). sigma(2) Receptors may produce effects on cell growth and apoptosis by regulating the sphingolipid pathway.     

Mitochondrial respiratory chain as a new target for anti-ischemic molecules

In human neuroblastoma SH-SY5Y cell preparations, sigma(1) receptor agonists (+)-pentazocine and 1S,2R-(-)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)cyclohexylamine (BD737) competed for [3H]haloperidol binding with K(i) values of 67+/-10 and 14+/-10 nM, respectively. (+)-Pentazocine or BD737 up to 10 microM did not affect basal levels of intracellular Ca(2+) concentration ([Ca(2+)](i)) in these cells, but they significantly reduced muscarine-induced [Ca(2+)](i) changes in a dose-related manner. However, the reduction by (+)-pentazocine was not reversed by the sigma(1) receptor antagonist haloperidol. Further studies showed (+)-pentazocine, BD737 and haloperidol could compete for [3H]quinuclidinyl benzilate binding in SH-SY5Y cells with K(i) values of 0.51+/-0.06, 0.32+/-0.07 and 4.4+/-2.3 microM, respectively. Thus, the inhibitory effects on muscarine-induced [Ca(2+)](i) changes by (+)-pentazocine and BD737 in SH-SY5Y cells were likely due to blockade of muscarinic receptors, not due to sigma(1) receptor activation by these ligands.     

(Aminoalkoxy)chromones. Selective sigma receptor ligands.

A series of (aminoalkoxy)chromones has been prepared, members of which bind potently (16-100 nM) at the sigma binding site and bind weakly (greater than 1000 nM) at the dopamine D2 receptor and 33 other receptors, second messenger systems, and ion channels. At the sigma receptor, the preferred position of attachment for the aminoalkoxy side chain to the chromone ring followed the rank order: 7-position greater than 5-position greater than 6-position. Chromones that contained a 2-substituent that was not coplanar with the chromone ring system showed improved binding over compounds with coplanar substituents. The most potent compound at the sigma site, 7-[[7-(4-hydroxypiperidyl)heptyl]oxy]-2-phenylchromone (74), had receptor affinities (IC50) of 16 nM at the [3H]DTG site, 19 nM at the [3H]-(+)-3-PPP site, and 4000 nM (Ki) at the dopamine D2 receptor. The most selective compound examined, 6-[[6-(4-hydroxypiperidyl)hexyl]-oxy]-2-cyclopentylchromone (58), exhibited IC50s of 51 nM at the [3H]DTG site, 55 nM at the [3H]-(+)-3-PPP site, and 21,000 nM (Ki) at the dopamine D2 receptor. Compound 44 (6-[[6-(4-hydroxypiperidyl)hexyl]oxy]-3-methylflavone, NPC 16377) was systemically effective (ip and po) in two behavioral models predictive of antipsychotic compounds and systemically active in animal models of ischemia.     

Trishomocubanes: novel sigma-receptor ligands modulate amphetamine-stimulated [3H]dopamine release.

Several trishomocubane analogues of the type 4-azahexacyclo [5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecane exhibited moderate to high affinity at sigma-receptor subtypes and low or negligible affinity at dopamine and serotonin transporters (SERT). Selected compounds were examined for their effects on amphetamine-stimulated [3H]dopamine release from striatal slices in vitro. Compounds 1, 2, 3 and 4 significantly enhanced amphetamine-stimulated release in a concentration-dependent manner. Compound 4, with the highest affinity and selectivity for the sigma(2)-receptor subtype, displayed the greatest potency. The enhancement produced by 1 and 2 was fully reversed by the selective sigma(2) antagonists 1'-[4-[1-(4-fluorophenyl)-1-H-indol-3-yl]-1-butyl]spiro[iso-benzofuran-1(3H), 4'piperidine] (Lu28-179), endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dihydro-(1-methyl)ethyl-2-oxo-1-H-benzimidazole-1-carboxyamidehydrochloride (BIMU-8) and the non-subtype selective antagonist N-[2-(3,4-dichlorophenyl)-ethyl]-N-methyl-2-pyrrolidinyl)ethylamine (BD1008). These data suggested a potential role for compounds 1 through 4 as sigma(2)-receptor agonists in functional studies. In addition, a D(3)-trishomocubane compound 5 displayed low affinity at sigma receptors (K(i)=3 microM) and moderate affinity at dopamine transporters (K(i)=623 nM). Compound 5 significantly inhibited the potentiation mediated by compound 2, presumably through sigma(2)-receptor antagonism, or a direct action on dopamine transporters.     

Enantiomeric N-substituted N-normetazocines: a comparative study of affinities at sigma, PCP, and mu opioid receptors.

The optical antipodes of N-allyl-N-normetazocine (2; SKF 10047, NANM) were the original compounds used for the classification of the sigma receptor as distinct from other receptors such as the PCP (NMDA), opioid, and dopamine receptors. Later studies showed that (+)-N-(dimethylallyl)-N-normetazocine [(+)-4, (+)-pentazocine] was more potent and selective for the sigma receptor. In order to gain additional structure-activity relationship information, several N-substituted N-normetazocine analogs were prepared and evaluated for their sigma-1 ([3H]-(+)-3-PPP or [3H]-(+)-pentazocine), PCP ([3H]TCP), and mu opioid ([3H]DAMGO) receptor binding affinities. (+)-N-Benzyl-N-normetazocine [(+)-10)] possessed subnanomolar affinities for the sigma site, Ki = 0.67. The analog (+)-10 showed greater than 14,000- and 2400-fold selectivity, respectively, for the sigma receptor relative to the PCP and mu opioid receptors. The N-substituted N-normetazocines were enantioselective for the sigma site. The (+)-N-benzyl analog, (+)-10, showed a 55-fold selectivity relative to (-)-10. Analysis of the data also revealed that (+)-normetazocine [(+)-1] [Ki = 30 nM] possessed the highest affinity for the PCP receptor. However, (+)-metazocine [(+)-5] (Ki = 41 nM) was the most selective compound for the PCP receptor relative to the sigma (51-fold) and mu opioid (greater than 200-fold) sites.     

Conformationally restricted analogs of BD1008 and an antisense oligodeoxynucleotide targeting sigma1 receptors produce anti-cocaine effects in mice.

Cocaine's ability to interact with sigma receptors suggests that these proteins mediate some of its behavioral effects. Therefore, three novel sigma receptor ligands with antagonist activity were evaluated in Swiss Webster mice: BD1018 (3S-1-[2-(3,4-dichlorophenyl)ethyl]-1,4-diazabicyclo[4.3.0]nonane), BD1063 (1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine), and LR132 (1R,2S-(+)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl)cyclohexylamine). Competition binding assays demonstrated that all three compounds have high affinities for sigma1 receptors. The three compounds vary in their affinities for sigma2 receptors and exhibit negligible affinities for dopamine, opioid, GABA(A) and NMDA receptors. In behavioral studies, pre-treatment of mice with BD1018, BD1063, or LR132 significantly attenuated cocaine-induced convulsions and lethality. Moreover, post-treatment with LR132 prevented cocaine-induced lethality in a significant proportion of animals. In contrast to the protection provided by the putative antagonists, the well-characterized sigma receptor agonist di-o-tolylguanidine (DTG) and the novel sigma receptor agonist BD1031 (3R-1-[2-(3,4-dichlorophenyl)ethyl]-1,4-diazabicyclo[4.3.0]nonane) each worsened the behavioral toxicity of cocaine. At doses where alone, they produced no significant effects on locomotion, BD1018, BD1063 and LR132 significantly attenuated the locomotor stimulatory effects of cocaine. To further validate the hypothesis that the anti-cocaine effects of the novel ligands involved antagonism of sigma receptors, an antisense oligodeoxynucleotide against sigma1 receptors was also shown to significantly attenuate the convulsive and locomotor stimulatory effects of cocaine. Together, the data suggests that functional antagonism of sigma receptors is capable of attenuating a number of cocaine-induced behaviors.     

Multiple affinity binding states of the sigma receptor: effect of GTP-binding protein-modifying agents

The sigma receptor, which is labeled with (+)-[3H]3-(3-hydroxyphenyl)-N- 1-(propyl)piperidine [(+)-[3H]3-PPP], is a site that binds several psychotomimetic opiate benzomorphans and certain antipsychotics, such as haloperidol. In order to elucidate the mechanisms involved in sigma receptor ligand binding, equilibrium binding analysis and kinetics of association and dissociation of the relatively selective sigma receptor ligand (+)-[3H]3-PPP were determined in rat brain membranes in the absence and presence of 5'-guanylylimidodiphosphate [Gpp(NH)p]. In the absence of Gpp(NH)p, (+)-3-PPP, cyclazocine, pentazocine, and (+)-SKF 10047 bind to high and low affinity sites (KH = 1.3-7.5 nM; KL = 84-500 nM), as determined by computer assisted analysis of the inhibition of (+)-[3H]3-PPP binding by the sigma ligands. The antipsychotics haloperidol and chlorpromazine inhibit (+)-[3H]3-PPP binding in a manner indicating interaction with a single state of the receptor. Gpp(NH)p (0.1 mM) abolished the high affinity binding component of the sigma agonist-like compounds tested but had no effect on the affinities of the antipsychotics for the receptor. Gpp(NH)p decreased the association rate of (+)-[3H]3-PPP binding 5-fold and also converted the biexponential dissociation kinetics of the ligand, observed in the absence of Gpp(NH)p, to a rapid monophasic dissociation process. Pretreatment of membranes with N-ethylmaleimide and pertussis toxin inhibited (+)-[3H]3-PPP binding and abolished the effect of Gpp(NH)p on the sigma ligand binding. These findings indicate of the sigma receptor is capable of existing in two discrete states, having high and low affinity for sigma agonist-like drugs. The regulation of the high affinity binding state by GTP-binding protein-modifying agents suggests its coupling to GTP-binding protein(s).     

3H]N-[4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl]-2-methoxy-5-methylbenzamide: a novel sigma-2 receptor probe

N-[4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl]-2-methoxy-5-methyl-benzamide (RHM-1) and N-[2-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)ethyl]-2-methoxy-5-methylbenzamide (RHM-2), two conformationally flexible benzamide analogues, were radiolabeled with tritium (specific activity=80 Ci/mmol) and the binding of [3H]RHM-1 and [3H]RHM-2 to sigma-2 (sigma2) receptors was evaluated in vitro. [3H]RHM-1 was found to have a higher affinity for sigma2 receptors compared to [3H]RHM-2 and [3H]1,3-di-o-tolylguanidine ([3H]DTG). [3H]RHM-1 had a dissociation constant (Kd) of 0.66+/-0.12 nM in rat liver membrane homogenates, which was 30-fold higher than that of [3H]RHM-2 (Kd=19.48+/-0.51 nM). The lower affinity of [3H]RHM-2 can be attributed to its faster K(off) rate since both radioligands have similar K(on) rates. Competitive binding assays were also conducted using a panel of compounds with known affinity for sigma2 receptors. The pharmacologic profile of [3H]RHM-1 was in agreement with that of [3H]DTG. The results of this study indicate that [3H]RHM-1 is a useful ligand for studying sigma2 receptors in vitro.     

Labeling by [3H]1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands.

Equilibrium binding studies with the sigma receptor ligand [3H]1,3-di(2-tolyl)guanidine ([3H]DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. [Life Sci. 45:1721-1732 (1989)]. Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that [3H]DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of [3H]DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of [3H]DTG from site 2, suggesting an association of this binding site with calcium channels.     

Involvement of the sigma(1) receptor in cocaine-induced conditioned place preference: possible dependence on dopamine uptake blockade.

The involvement of the sigma(1) receptor on the rewarding effects of cocaine was examined using the conditioned place preference (CPP) procedure in C57BL/6 mice. Acquisition or expression of cocaine (20 mg/kg i.p.)-induced CPP was significantly decreased by pre-treatment with the selective sigma(1) receptor antagonists N,N-dipropyl-2-(4-methoxy-3-(2-phenylethoxy)phenyl)ethylamine (NE-100) or N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD1047), 1-10 mg/kg, i.p. The sigma(1) receptor agonists igmesine or 2-(4-morpholinoethyl-1-phenylcyclohexane-1-carboxylate hydrochloride (PRE-084) failed to induce CPP when injected alone. Moreover, the CPP induced by N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine (BTCP), a selective dopamine reuptake inhibitor, was blocked by treatments with the sigma(1) receptor antagonists as was similarly observed with cocaine. In addition, the repetitive treatment with cocaine during conditioning increased sigma(1) receptor mRNA expression in the nucleus accumbens, but not in the caudate putamen, prefrontal cortex or cerebellum. These data show that the sigma(1) receptor is not only necessary for acquisition of the cocaine-induced CPP, but that it is also implicated in its expression, confirming that activation of the sigma(1) receptor is induced during cocaine's early effects. The sigma(1) receptor is activated consequently to dopamine reuptake blockade and is not sufficient to induce CPP by itself. The mechanism of the sigma(1) receptor involvement in CPP and the selectivity toward the CPP-inducing drug remains however to be determined. These results show that strategies targeting the sigma(1) receptor with selective antagonists may allow effective attenuation of cocaine's rewarding properties and, in turn, offer new treatment strategies against drug addiction.     

Structure-affinity relationship study on N-[4-(4-arylpiperazin-1-yl)butyl]arylcarboxamides as potent and selective dopamine D(3) receptor ligands

The benzamide PB12 (N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide) (1), already reported as potent and selective dopamine D(4) receptor ligand, has been modified searching for structural features that could lead to D(3) receptor affinity. Changes in the aromatic ring linked to N-1 piperazine ring led to the identification of 2-methoxyphenyl and 2,3-dichlorophenyl derivatives (compounds 6 and 13) displaying moderate D(3) affinity (K(i) = 145 and 31 nM, respectively). Intermediate alkyl chain elongation in compounds 1, 6, and 13 improved binding affinity for the D(3) receptor and decreased the D(4) affinity (compounds 18-26). Among these latter compounds, the N-[4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butyl]-3-methoxybenzamide (19) was further modified with the replacement or of the 2,3-dichlorophenyl moiety (compounds 27-30) or of the 3-methoxyphenyl ring (compounds 31-41). In this way, we identified several high-affinity D(3) ligands (0.13 nM < K(i)'s < 4.97 nM) endowed with high selectivity over D(2), D(4), 5-HT(1A), and alpha(1) receptors. In addition, N-[4-[4-(2,3-dimethylphenyl)piperazin-1-yl]butyl]-3-methoxybenzamide (27) and N-[4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butyl]-7-methoxy-2-benzofurancarboxamide (41) appear to be valuable candidates for positron emission tomography (PET) because of their affinity values, lipophilicity properties, and liability of (11)C labeling in the O-methyl position.     

Evidence that the sigma(1) receptor is not directly coupled to G proteins

Sigma (sigma) receptors have been implicated in psychosis, cognition, neuroprotection, and locomotion in the central nervous system. The signal transduction mechanisms for sigma receptors have not been fully elucidated. In this study, we examined the possible coupling between sigma(1) receptors and heterotrimeric guanine nucleotide-binding proteins (G proteins) in rodent brain. In sigma(1) receptor-rich cerebellar membrane preparations, the competitive binding curves of two sigma(1) agonists, (+)pentazocine and 1S,2R-(-)-cis-N-[2-(3, 4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)cyclohexylamine (BD737), were unaffected by the addition of 10 microM guanosine-5'-O-(gamma-thio)-triphosphate (GTPgammaS). Neither (+)pentazocine (1-100 microM) nor BD737 (0.01-10 microM) stimulated GTPase activities significantly above basal levels in agonist-stimulated GTPase activity assays in cerebellar membranes. Furthermore, when using the method of agonist-stimulated [35S]GTPgammaS binding as assessed by autoradiography, we did not observe significant stimulation of [35S]GTPgammaS binding in rat brain sections by either (+)pentazocine or BD737. The above results demonstrate that the sigma(1) receptor is not likely be directly coupled to G proteins.     

Involvement of the sigma 1 receptor in the motivational effects of ethanol in mice

In the present study, we examined the involvement of the sigma(1) (sigma(1)) receptor in several behavioral manifestations of ethanol addiction. Administration of ethanol (0.5, 1, and 2 g/kg) in Swiss mice dose-dependently induced locomotor stimulation, conditioned place preference, and conditioned taste aversion, which are considered as behavioral index of drug-induced reward. Pretreatment with the selective sigma(1) receptor antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD1047, 3-30 mg/kg) dose-dependently blocked ethanol (1 g/kg)-induced hyperlocomotion and taste aversion and ethanol (2 g/kg)-induced place preference. Pretreatment with the selective sigma(1) receptor agonist 2-(4-morpholino)ethyl 1-phenylcyclohexane-1-carboxylate (PRE-084, 1-10 mg/kg) before ethanol (0.5 g/kg) failed to affect the resulting locomotor stimulation, but dose-dependently enhanced the conditioned place preference. Each sigma(1) receptor ligand was devoid of behavioral effect by itself. These observations show that activation of the sigma(1) receptor is a necessary component of ethanol-induced motivational effects and suggest a new pharmacological target for alleviating ethanol addiction.