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)     

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.     

1,3-Di(2-[5-3H]tolyl)guanidine labels more than one site in rat forebrain.

Studies of 1,3-di-(2-[5-3H]tolyl)guanidine ([3H]DTG) binding to rat brain membranes revealed that [3H]DTG binds to a high and a low affinity site with Kd values of 19.8 nM and 1.31 microM (corresponding Bmax values 291 fmol/mg protein and 8.68 pmol/mg protein). The order of potency of competitors for [3H]DTG binding revealed a binding profile typical of sigma site ligands. Several sigma ligands such as the enantiomers of 3-PPP (3-(3-hydroxyphenyl)-N- (n-propyl)piperidine) and (+/-)-pentazocine exhibited biphasic competition profiles for [3H]DTG binding, whereas other sigma ligands such as haloperidol displayed monotonic competition curves. Neither phenytoin nor carbamazepine were observed to enhance [3H]DTG binding. These data support the hypothesis that multiple sigma binding sites exist. The lack of phenytoin and carbamazepine modulation of [3H]DTG binding are in agreement with the proposed greater density of sigma site 2 in the rat, since allosteric modulation has been ascribed to the DM1/sigma 1 site.     

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.     

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).     

Interaction of 1,3-di(2-[5-3H]tolyl) guanidine with sigma 2 binding sites in rat heart membrane preparations.

Membrane preparations of rat hearts displayed specific binding activity for the prototypic sigma (sigma) receptor ligand, 1,3-di(2-[5-3H]tolyl) guanidine [( 3H]DTG), but not for the phencyclidine (PCP) receptor ligand, [3H]MK-801. Scatchard plot analysis of [3H]DTG binding revealed the presence of one high affinity saturable binding site with a KD of 8.7 nM and a Bmax of 100 pmol/g protein. The drug specificity profile of the receptor correlated with that of the sigma receptor with the following order of potency: DTG greater than haloperidol greater than (-)-pentazocine greater than (-)-butaclamol greater than (+)-butaclamol greater than (-) SKF-10047 greater than (+)pentazocine greater than PCP greater than TCP greater than MK-801 greater than (+)SKF-10047. [3H]DTG binding was sensitive to the Ca2+ channel blocker, verapamil (Ki 202 nM) but not to the K+ channel blocker, 4-aminopyridine. The reverse stereoselectivity of [3H]DTG binding for (-)-SKF-10047 and (-)-pentazocine (Ki of 1289 and 140 nM as compared with 17,582 and 2190 nM for (+)-SKF-10047 and (+)-pentazocine, respectively) indicated that the heart contains sigma receptors with characteristics of the sigma 2 subtype.     

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.     

Synthesis, characterization, and biological evaluation of a novel class of N-(arylethyl)-N-alkyl-2-(1-pyrrolidinyl)ethylamines: structural requirements and binding affinity at the sigma receptor.

By synthesizing and testing a part-structure, N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (3), derived from our previously reported high affinity sigma receptor ligands (1S,2R)-(-)-N-[2-(3,4-dichlorophenyl)-ethyl]-N-methyl-2-(1- pyrrolidinyl)cyclohexylamine [(-)-2] and (+)-2, we have identified a novel class of superpotent (subnanomolar affinity) sigma ligands specific for the sigma receptor labeled by [3H]-(+)-3-PPP. When 3 was tested for its capacity to displace [3H]-(+)-3-PPP from guinea pig brain membranes, it exhibited a Ki of 0.34 nM, which is better than either of its parent compounds (-)-2 (Ki = 1.3 nM) and (+)-2 (Ki = 6.0 nM). Other compounds related to 3 such as N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-homopiperidinyl)ethy lamine (19) exhibited Ki = 0.17 nM [( 3H]-(+)-3-PPP). The determinants for high sigma receptor affinity of 3 were examined by manipulation of this structure in a number of different ways. The high efficacy of these compounds for the sigma receptor, their relative chemical simplicity and ease of synthesis, and their high degree of selective identifies N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (3) and related compounds as a highly promising base for determination of the functional role of sigma receptors as well as the development of novel therapeutic agents.     

Evidence for a multi-site model of the rat brain sigma receptor

Irradiation of rat brain membranes with light of 254 nm, a treatment which modifies ultra-violet absorbing residues in proteins, decreased binding of both [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([ 3H](+)-3-PPP) and [3H]1,3-di-o-tolylguanidine ([3H]DTG) to sigma receptors. For [3H](+)-3-PPP, this was due to a decreased Bmax. In contrast, irradiation markedly increased binding of [3H](+)-N-allylnormetazocine ([3H](+)-SKF 10,047) due to a decrease in the Kd. Both unlabeled DTG and haloperidol were competitive inhibitors of [3H](+)-3-PPP binding to untreated membranes, causing an increase in the Kd and no change in the Bmax. The benzomorphans, (+)-SKF 10,047 and (+)-pentazocine, were uncompetitive inhibitors, causing a decrease in both the Kd and Bmax for [3H](+)-3-PPP. Finally, the ability of DTG and (+)-3-PPP to inhibit binding of [3H](+)-SKF 10,047 was markedly reduced by ultra-violet irradiation, whereas irradiation had little effect on the potency of unlabeled (+)-SKF 10,047 and (+)-pentazocine. These data suggest that sigma-related (+)-benzomorphans and non-benzomorphans interact either with distinct, allosterically coupled sites on the same sigma receptor macromolecule or with different populations of sigma receptor types.     

Leukotriene B4 receptor antagonists: the LY255283 series of hydroxyacetophenones.

A series of hydroxyacetophenones was prepared for evaluation as leukotriene B4 (LTB4) receptor antagonists, culminating in 1-[5-ethyl-2-hydroxy-4-[[6-methyl-6-(1H-tetrazol-5- yl)heptyl]oxy]phenyl]ethanone (compound 35, LY255283). Using an assay for inhibition of specific [3H]LTB4 binding to human PMN, we found that substitution of a nonpolar substituent in the 5-position was required for activity. Best activity was realized with hydrogen in the 3-position, hydroxyl in the 2-position, short chain alkyl ketone in the 1-position, and a six- or eight-carbon chain linking the oxygen in the 4-position with an unsaturated terminal function. Compound 35, having an IC50 of 87 nM in the binding assay, was chosen for further preclinical evaluation.     

Up-regulation of rat cortical sigma receptors after subchronic administration of rimcazole and 1,3-di(2-tolyl)guanidine

The effects of subchronic administration of rimcazole and 1,3-di(2-tolyl)guanidine (DTG) on the central R(+)[3H]-3-[3-hydroxyphenyl]-N-(1-propyl)piperidine (R(+)[3H]-3-PPP) binding site were investigated in the rat. Subchronic treatment with rimcazole was characterized by a 30% increase in the density and a two-fold decrease in the affinity of R(+)[3H]-3-PPP binding sites relative to saline-treated animals. DTG, a more potent sigma ligand, produced a similar alteration to the affinity but a 130% increase in the density of R(+)[3H]-3-PPP binding sites. These data thus provide evidence for the functional involvement of sigma receptors in the central nervous system.     

Effects of sigma receptor ligands on schedule-controlled behavior of rats: relation to sigma and PCP receptor binding affinity

Eleven drugs were examined for their ability to inhibit sigma and phencyclidine (PCP) receptor binding, as labelled by (+)[³H]-R-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP), [³H]ditolylguanidine (DTG), (+)[³H]N-allylnormetazocine (NANM) and [³H]1-(1-(2-thienyl)cyclohexyl)piperidine (TCP), in membrane preparations from whole rat brain. The same drugs were studied for their effects under a fixed-ratio (FR) schedule of food reinforcement in rats. The relative potency order of the drugs for decreasing FR responding was: haloperidol>(+)-3-PPP>(-)NANM>BMY 14802> PCP>(+)NANM>DTG>rimcazole> JO 1783>JO1784>(-)butaclamol.The binding affinities of all 11 drugs for either the [³H]DTG, (+)[³H]-3-PPP, (+)[³H]NANM or [³H]TCP site did not correlate significantly with the potenties of the same drugs for decreasing FR behavior. Rimcazole, (+)-3-PPP and haloperidol, at behaviorally inactive doses, were studied for their effects as antagonists of the rate-decreasing effects of JO 1784, DTG and (+)NANM: rimcazole attenuated the effects of DTG and (+)NANM but not JO 1784; (+)-3-PPP attenuated the effects of (+)NANM but not JO 1784 and DTG; and haloperidol was devoid of antagonistic actions. Moreover, BMY 14802 did not attenuate the rate-decreasing effects of (+)-3-PPP. These results further indicate that it is difficult to distinguish between purported sigma agonist and antagonist drugs.     

Multiple sigma binding sites in guinea-pig and rat brain membranes: G-protein interactions.

1. Evidence is accumulating for multiple sigma (sigma) sites in the mammalian CNS. 2. We have addressed this problem and have examined sigma site - G-protein coupling in guinea-pig and rat brain membranes. 3. Ditolylorthoguanidine (DTG), (+)-3-(3-hydroxyphenyl)-N-1-(propyl)piperidine (3PPP) and dextromethorphan displaced [3H]-DTG (3.4 nM) with low Hill slopes of 0.5, 0.6 and 0.6, respectively in guinea-pig brain membranes. 4. In the presence of 5'-guanylylimidodiphosphate (Gpp(NH)p; 100 microM), the specific binding of [3H]-DTG was reduced by 36.7%, the Hill slope of 3PPP was increased to near unity, the ability of dextromethorphan to displace DTG was virtually abolished and the Hill slope for DTG remained low (0.7), indicating the presence of at least two binding sites. These data indicate that although Gpp(NH)p removes a dextromethorphan high affinity site, two DTG selective sites remain in the presence of Gpp(NH)p. 5. The present study suggests that DTG binds to at least three sites in guinea-pig brain membranes, at least one of which is G-protein linked. 6. In rat brain membranes, DTG displaced itself (3.4 nM) with a Hill slope near 1. 3PPP displacement of [3H]-DTG was comparable with the guinea-pig (Hill slope 0.5) and displaced from more than 1 site. Dextromethorphan did not displace [3H]-DTG at concentrations below 10 microM. 7. The heterogeneity of sigma sites appears to be less in rat than in guinea-pig brain membranes.     

Selective sigma receptor agonist and antagonist affect dopamine neuronal activity

Extracellular recording techniques were used to study the effects of the selective sigma receptor agonist (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP) and selective sigma receptor antagonist BMY 14802 on dopamine (DA) neurons of the substantia nigra. Intravenous administration of (+)-3-PPP produced a dose-dependent inhibition of DA neuron firing rate. Complete inhibition of DA neurons produced by (+)-3-PPP could be completely reversed by administration of BMY 14802. Also, pretreatment with BMY 14802 shifted the (+)-3-PPP dose response curve to the right. These data demonstrate a relationship of the sigma receptor with the dopamine system and further suggest a model system to study agonist/antagonist interactions of sigma ligands.     

Computer-assisted modeling of multiple dextromethorphan and sigma binding sites in guinea pig brain.

Computer-assisted, simultaneous analysis of self- and cross-displacement experiments demonstrated the existence of several binding sites in guinea pig brain for dextromethorphan, (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP), and 1,3-di-o-tolyl guanidine (DTG). Dextromethorphan binds with high affinity to two sites (R1 Kd 50-83 and R2 Kd 8-19 nM) and with low affinity to two additional sites (R3 and R4). (+)-3-PPP binds to one high-affinity (R1 Kd 24-36 nM), to one intermediate-affinity (R3 Kd 210-320 nM), and to two (R2 and R4) low-affinity sites. DTG binds with almost identical high affinity to two different sites (R1 Kd 22-24 and R3 Kd 13-16 nM). These results confirm that dextromethorphan, (+)-3-PPP, and DTG bind to the common DM1/sigma 1 site (R1). The binding of DTG to two different sites with identical affinities precludes the use of this compound as a specific marker for sigma receptors. Besides, haloperidol displaces labeled ligands from both high-affinity DTG sites (R1 and R3) with high affinity. Thus, haloperidol sensitivity should not be used as the single criterion to identify a putative receptor. The resolution of these novel sites also may provide new insights into the multiple effects of antipsychotic drugs. In addition, this investigation has important implications regarding the methods that must be applied to characterize multiple binding sites and their relations with putative receptors.     

Characterization of opioid, sigma, and phencyclidine receptors in the neuroblastoma-brain hybrid cell line NCB-20

Opioid, sigma, and phencyclidine (PCP) receptors were characterized in the mouse neuroblastoma--Chinese hamster brain hybrid cell line NCB-20. Quantitative receptor assays under equilibrium binding conditions with highly specific radioligands demonstrated the presence of delta, but not mu or kappa, opioid receptors on NCB-20 cell membranes. NCB-20 cells were shown to possess two distinct sites specific for sigma opioids and PCP derivatives. One site was labeled by (+)-[3H]N-allylnormetazocine [(+)-[3H]SKF-10,047] (Kd = 69 nM; Bmax = 4100 fmol/mg of protein). The rank order of potency of drugs at this site was (+)-3-(3-hydroxy-phenyl)-N-(1-propyl)piperidine [(+)-3-PPP] greater than haloperidol greater than (+)-SKF-10,047 greater than (+/-)-ethylketocyclazocine greater than (+/-)-bremazocine greater than N-[1-(2-thienyl) cyclohexyl]piperidine (TCP) greater than dexoxadrol. This site is similar in its ligand selectivity to the haloperidol-sensitive sigma receptor of rat brain. The other site was labeled by the potent phencyclidine derivative [3H]TCP (Kd = 335 nM; Bmax = 9300 fmol/mg of protein). This density is equivalent to approximately 60,000 sites/cell. The rank order of potency of drugs at this site was TCP greater than (+)-3-PPP greater than PCP greater than dexoxadrol greater than haloperidol greater than cyclazocine greater than levoxadrol greater than (+)-SKF-10,047; mu and delta ligands were inactive. This site is similar to the rat brain PCP receptor. The NCB-20 cell line is the only cultured cell line that has been demonstrated to have PCP receptors.     

Binding of [3H]U-101958 to sigma1 receptor-like sites in human cerebellum and neuroblastoma cells.

1-Benzyl-4-[N-(3-isopropoxy-2-pyridinyl)-N-methyl]-amino-piperidine ([3H]U-101958), a dopamine D4 receptor ligand, was found to bind to a large sigma1 receptor-like component in human cerebellum and SK-N-MC neuroblastoma cells with high affinity (2-4 nM Kd). By contrast, binding to dopamine D4 receptors represented 10% or less of the sigma1 receptor-like site. Considering that U-101958 has been characterized as either a dopamine D4 receptor agonist or antagonist, depending on the system under study, the observation that U-101958 also binds to sigma1 receptor-like sites is important for accurate interpretation of the pharmacological actions of this compound. [3H]U-101958 may be a useful radioligand for sigma1 rather than dopamine D4 receptor sites.     

Benz[f]isoquinoline analogues as high-affinity sigma ligands.

This paper describes the synthesis of some conformationally restricted 4-phenylpiperidine analogues and their affinities for the guinea pig cerebellum sigma recognition site ([3H]-DTG) and the rat striatum dopamine D2 receptor ([3H]-(-)-sulpiride) in order to develop potent selective sigma ligands as tools in the investigation of this site in psychosis. It was found that both hexa- and octahydrobenz[f]isoquinolines with lipophilic N-substituents had high affinities for the sigma site. Notably, trans-3-cyclohexyl-1,2,3,4,4a,5,6,10b-octahydrobenz[f]isoquinoline (26) had an affinity of 0.25 nM making it the highest affinity sigma ligand reported to date. Moreover, it is at least 10,000-fold selective over the D2 receptor and could prove to be a valuable tool in the study of sigma sites. Other analogues such as 1H-indeno[2,1-c]pyridines and 1H-benzo[3,4]cyclohepta[1,2-c]pyridines also displayed high sigma site affinity.     

Spiropiperidines as high-affinity, selective sigma ligands.

A variety of achiral conformationally restricted spirocyclic piperidines have been prepared in an attempt to investigate the functional role of the central sigma recognition site. All the compounds possessed a lipophilic N-substituent incorporating either a tetralin, indan, or benzocycloheptane skeleton. Their in vitro affinity at the sigma site was assessed in radioligand displacement experiments with guinea pig cerebellum homogenates using the sigma-specific radioligand [3H]-N,N'-di-o-tolylguanidine ([3H]-DTG, [3H]-6). A study of the structure-activity relationships identified the N-butyl and N-dimethylallyl substituents as the optimum groups for high affinity and selectivity at the sigma site (e.g., 3,4-dihydro-1'-(3-methylbut-2-enyl)spiro[1H-indene-1,4'-piperidine ] (48), pIC50 = 8.9 vs [3H]-6 and greater than 10,000-fold selective over the dopamine D2 receptor). Such compounds are amongst the highest affinity sigma ligands reported to date, with excellent selectivity over the dopamine D2 receptor, and may serve as a useful tool for exploring the physiological role of the sigma site.