Sigma Sites Research Articles

Rat brain binding sites for pramipexole, a clinically useful D 3-preferring dopamine agonist

Pramipexole (PPX[ is currently being evaluated for treatment of schizophrenia and Parkinson's disease. In studies with cloned subtypes of the dopamine (DA[ D 2 receptor subfamily, PPX has higher affinity for the D 3 compared to the D 2 and D 4 subtypes; unlike 7-[ 3H]hydroxy- N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT[, it does not bind to sigma sites. Receptor binding autoradiography with [ 3H]PPX (5 nM, 62 Ci/mmol) was used to evaluate the distribution of PPX binding sites within the rat brain. Consistent with its preference for D 3-binding sites, the highest concentrations of [ 3H]PPX binding sites were found in the islets of Calleja (ICj), previously reported to contain D 3 but not D 2 or D 4 mRNA. [ 3H]PPX binding was also high in other mesolimbic areas such as the nucleus accumbens (N. accum), olfactory tubercle, and amygdala. [ 3H]PPX binding was also high in caudate (Cd), although slightly less than in mesolimbic areas. Less [ 3H]PPX binding sites were found in ventral tegmental area (VTA) and substantia nigra, areas rich in cell bodies for DA neurons. Thus, although PPX most potently stimulates DA autoreceptors, PPX binding sites have their highest concentrations in projection areas containing both DA terminal and postsynaptic receptors. Because of PPX's preferential affinity for the D 3 receptor subtype and its resultant high mesolimbic binding, it could have a unique therapeutic profile for treatment of psychiatric and/or neurological diseases.

Neuroprotective sigma ligands interfere with the glutamate‐activated NOS pathway in hippocampal cell culture

We studied neuroprotective properties of 12 structurally different sigma site ligands in primary rat hippocampal cell cultures and analyzed whether they interfere with glutamate-induced activation of the nitric oxide synthase (NOS) pathway. Neurotoxicity was triggered with 1 mM glutamate on day 8 of culture. Cells were treated with various concentrations of the compounds for 7 days before glutamate exposure (prolonged pretreatment), or during glutamate exposure (acute treatment). Protection was seen after prolonged pretreatment (long-term protection) with sabeluzole, opipramole, haloperidol, ifenprodil, fenpropimorph, carbetapentane, and tiospirone, with pIC50s of 7.30, 7.15, 6.87, 6.68, 6.66, 6.39, and 6.34, respectively. There was no protection with PD 128298, 1,3-ortho-di-tolylguanidine, BMY-14802, (+)3-(3-hydroxyphenyl)-N-1(propyl) piperidine, or dextromethorphan. Upon acute treatment, only ifenprodil was protective. Interference of the drugs with glutamate activation of the NOS pathway was determined by measuring glutamate-activated cGMP formation and citrulline levels. Glutamate-activated cGMP formation was reduced by all neuroprotective sigma ligands after prolonged pretreatment but not after acute treatment. Sigma ligands added to cell culture lysate did not reduce citrulline formation, evidence that there was no direct effect on the NOS enzyme. We conclude that some but not all sigma ligands exert long-term protective properties against glutamate-induced neurotoxicity in primary hippocampal cultures, and that this protection is accompanied by attenuation of cGMP formation in the NOS pathway. However, inhibition of cGMP formation by itself appeared not sufficient for obtaining neuroprotective effects, as inhibition of glutamate-activated cGMP formation by N omega-nitro-L-arginine, haemoglobin, or PD128298 did not provide neuroprotection.

Characterization of non-adrenergic [3H]clonidine binding sites in rat stomach: high affinity of imidazolines, guanidines and sigma ligands.

We have identified and characterized non-adrenergic [3H]clonidine binding sites in rat stomach. The binding of [3H]clonidine was rapid, reversible, partly specific (as defined by cirazoline 0.1 nmol/l), saturable and of high affinity. The specific binding of [3H]clonidine to rat stomach membranes was concentration-dependently inhibited by various imidazolines and guanidines including the sigma site ligand 1,2-di-(2-tolyl)guanidine (DTG), by the butyrophenone derivative (+)-3-PPP[(R)-3-(3-hydroxyphenyl)-N-propylpiperidine]; the latter two compounds are also known to exhibit affinity for sigma sites. In contrast, rauwolscine, histamine, ranitidine and the non-hydrolysable GTP-analogue Gpp(NH)p (5' guanylylimidodiphosphate) did not, or with negligible affinity, inhibit [3H]clonidine binding. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases for a single detectable site) was as follows: cirazoline > idazoxan > or = DTG > (+)-3-PPP > chlonidine > guanabenz > haloperidol. This rank order is not compatible with the pharmacological properties of either I1- or I2-imidazoline binding sites. However, the ability of haloperidol, (+)-3-PPP and DTG to displace [3H]clonidine (the latter two with high affinity) suggests that the [3H] clonidine binding sites in rat stomach may be related to sigma-like sites.

Comparative tissue distribution of conformationally restricted radioiodinated vesamicol receptor ligands

Three conformationally restricted analogs of vesamicol, 1′-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]spirol[1H-indene-1,4′-piperidine] ( 5), 1′-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-3,4-dihydrospiro[indene-1,4′-piperidine] ( 6) and 1′-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl)-3,4-dihydrospiro[naphthalene-1(2H),4′-piperidine] ( 7), were labelled with iodine-125 and evaluated as potential radioligands for mapping vesamicol receptor (VR) density and cholinergic function in vivo. All compounds showed similar kinetics in most tissues. However, differences were observed in the brain. Although comparable levels of each corresponding enantiomeric pair were obtained initially in the brain, the levels of the dextrorotatory enantiomers (+)- 5, (+)- 6 and (+)- 7 were found to decrease by 72–82% over a period of 3 h. In contrast, the brain levels of the corresponding levorotatory isomers were maintained throughout the duration of the experiment. Among the dextrorotatory isomers, (+)- 6 showed the highest brain extraction, while (+)- 7 showed the lowest. In tissue dissection experiments, the levels of (+)- 5, (+)- 6 and (+)- 7 were highest in the striatum and moderate to low in the cortex and cerebellum. Co-administration of haloperidol with (+)- 6 decreased the levels of the latter in the striatum by 27%, while the levels in the cortex and cerebellum were each reduced by 60%. In addition, haloperidol failed to affect the regional distribution of (+)- 7 in the brain. However, both haloperidol and spiperone increased the striatal levels of (+)- 5 by 67 and 76%, respectively, suggesting that the binding of this radioligand is related to cholinergic function. Furthermore, haloperidol reduced the concentration of (+)- 5 in the cortex and cerebellum by 25 and 33%, respectively, thereby implicating the sigma site as a secondary target for this ligand in the cortex.

Vesamicol analogues as sigma ligands: Molecular determinants of selectivity at the vesamicol receptor

The present study compares the affinities of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, 1) and selected analogues of the latter at the vesamicol receptor (VR) with the corresponding affinities at σ1 and σ2 binding sites. For this study, the parent structure 1 was divided into three fragments: A (cyclohexyl), B (piperidyl) and C (phenyl). Vesamicol analogues were then selected to reflect structural modifications in these fragments. Consistent with earlier reports, vesamicol was found to exhibit nanomolar affinities at the VR and σ1 and σ2 sites, resulting in poor selectivity for the VR over the sigma sites. Vesamicol analogues characterized by an acyclic A-fragment showed moderate to low affinities at the VR and moderate to high affinities at σ1 and σ2 sites. As a result, many of these analogues showed poor selectivity for the VR. Replacement of the C4 carbon of 1 with a halobenzyl amine resulted in higher affinities at the VR coupled with moderate to low affinities at σ1 and σ2 sites. The introduction of a benzofused substituent at the C4 and C5 positions of 1 (compound 2) resulted in a 200-fold increase in affinity at the VR accompanied by a 5- to 6-fold decrease in affinity at σ1 and σ2 sites relative to the parent structure. Consequently, compound 2 showed 12,000-fold higher affinity at the VR than at sigma sites. Restricting the rotation of fragment C relative to B (by means of alkyl and alkenyl bridges) generally yielded analogues with subnanomolar affinities at the VR. The corresponding affinities of these spirofused conformationally restricted analogues were moderate to poor at σ1 and σ2 sites when fragment A was preserved. In contrast, the affinities at σ1 and σ2 sites were decreased 3- to 11-fold when fragment A was modified at position C4 and decreased up to 100-fold with benzofusion at the C4 and C5 positions of fragment A. Consequently, the spirofused analogues 15–19 were among the most selective VR ligands examined. Thus, the effect of conformational restriction in fragments A and B-C is to increase affinity at the VR while decreasing affinity at σ1 and σ2 sites, and thereby increasing selectivity for the VR over the sigma sites.

Prevention of nimodipine-induced impairment of learning by the selective sigma ligand PRE-084.

The high selectivity of the phencyclidine derivative PRE-084 for sigma (sigma) sites is demonstrated. We previously reported that this compound is able to markedly attenuate the impairment of learning induced in mice by the non-competitive NMDA antagonist MK-801, and the cholinergic nicotinic antagonist mecamylamine. In this study, we examined the effect of PRE-084 on the impairment of learning induced by acute administration of the calcium channel antagonist nimodipine. Nimodipine (0.3 mg/kg i.p.) impaired the spontaneous alternation behaviour in a Y-maze, decreased the step-down latency (SDL) in a passive avoidance task, and altered place learning and retention in a water-maze paradigm, with no marked effect on the motility observed using an open-field test. Preadministration of PRE-084 resulted in an attenuation of the impairment of alternation, in the 0.3-1 mg/kg s.c. range, in a marked increase in SDL, at 1-3 mg/kg, and improved place learning and retention in the water-maze, at 1 mg/kg. The effects on alternation behaviour and passive avoidance were completely prevented by co-administration of the purported sigma antagonist BMY-14802 (10 mg/kg i.p.), implicating the sigma sites. These results confirm the beneficial effect of the sigma ligand PRE-084 on pharmacological models of learning impairments, and indicate that sigma sites may modulate Ca2+ fluxes through VDCC, which may in turn bear some as yet unknown relationship to the previously described interaction with neurotransmitter systems.

Pharmacological evidence for the involvement of sigma sites in DTG-induced contralateral circling in rats

The central distribution of sigma sites labelled by di- o -tolylguanidine (DTG), a compound which has specific affinity for sigma sites, and its ability to produce postural movements, are consistent with the hypothesis that sigma sites may play a functional role in the regulation of movement. The aim of the present study was to evaluate the specificity of the circling behaviour induced by unilateral intranigral injection of DTG in rats. As previously described, DTG produced dose-dependent unilateral rotations (2.5–20 nmol/rat). A similar dose-dependent circling behaviour was observed with DMTG and (+) NANM (3–40 nmol/rat), compounds which bind to both sigma and PCP sites, and with haloperidol (3–20 nmol/rat) whereas raclopride and d,l-sulpiride did not elicit any circling (10 nmol/rat). DTG-induced circling after intranigral injection (10 nmol/rat) was decreased in a dose-dependent manner by rimcazole (20–40 mg/kg, i.p.), a selective ligand for sigma sites, and by BMY 14802 (3, 10, 30mg/kg, i.p.), ifenprodil and eliprodil (1, 3, 10 mg/kg, i.p.), non-selective sigma ligands. In contrast, naloxone (1 mg/kg, s.c.) and CGS 19755 (1, 3, 10 mg/kg, i.p.) did not change the DTG-induced circling. Eliprodil failed to inhibit circling produced by compounds devoid of any affinity for sigma sites such as APV, dizocilpine or muscimol, indicating the specificity of the inhibition observed with eliprodil on the DTG-induced circling. These results suggest that circling behaviour produced by unilateral intranigral injection of DTG may be a useful model for studying the interaction of new drugs with sigma sites and might also be used to investigate further the potential involvement of these sites in the regulation of the activity of the nigro-striatal dopaminergic pathways.

NE-100, a novel sigma ligand: effects on [3H]TCP binding to intact primary cultured neuronal cells.

N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100), a potent and highly selective sigma ligand, haloperidol, (+)pentazocine, 1-(cyclopropyl-methyl)-4-(2'-(4"-fluorophenyl)-2'-oxoethyl) piperidine HBr (DuP 734) and 4-(2'-(4"-cyanophenyl)-2'-oxoethyl) 1-(cyclopropylmethyl)piperidine (XJ 448) inhibited carbachol-induced inositol 1,4,5-triphosphate (IP3) formation in a dose-dependent manner. The rank order of potency of the tested drugs for inhibition was: haloperidol > or = (+)pentazocine = NE-100 > DuP 734 = XJ 448. In addition, the effects of NE-100, DuP 734 and XJ 448 upon [3H]TCP binding were examined using primary cultured neuronal cells derived from the fetal rat telencephalon. These drugs inhibited [3H]TCP binding to intact cells. The ability of the test drugs to inhibit [3H]TCP binding to primary cultured neuronal cells was in the order: NE-100 > DuP 734 > XJ 448. These observations suggest that NE-100 indirectly modulates the N-methyl-D-aspartate (NMDA)/phencyclidine (PCP) receptor ion channel complex (NMDA receptor-ion channel), presumably through sigma-1 sites.

Quantitative autoradiographic localization of [ 3H]3-OH-PCP (1-(1(3-Hydroxyphenyl)cyclohexyl)piperidine) binding sites in rat brain

Binding of a novel radioligand, [ 3H]3-OH-PCP (1-(1(3-hydroxyphenyl)cyclohexyl)piperidine), to N-methyl- d-aspartate (NMDA) receptor-coupled and -uncoupled PCP sites was investigated in the rat brain. The highest densities of [ 3H]3-OH-PCP binding were observed in the hippocampal formation, notably in the stratum radiatum and oriens of CA1 region, and dentate gyrus. There were relatively high levels of binding in the olfactory system, superficial layer of cortices, the amygdala and the thalamus. In contrast, lower levels of binding were found in the globus pallidus, cerebellum, and brain stem, except for the superior colliculus. These findings demonstrate that [ 3H]3-OH-PCP binds to discrete regions within the rat brain. Its distribution is consistent with autoradiographic localization of [ 3H]TCP and [ 3H]MK-801 binding sites in the rat brain, suggesting that [ 3H]3-OH-PCP binds to NMDA/PCP ion-channel complexes in preference to sigma sites.

NE-100, a novel sigma ligand: Effects on [ 3H]TCP binding to intact primary cultured neuronal cells

N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100), a potent and highly selective sigma ligand, haloperidol, (+)pentazocine, 1-(cyclopropylmethyl)-4-(2′-(4″-fluorophenyl)-2′-oxoethyl) piperidine HBr (DuP 734) and 4-(2′-(4″-cyanophenyl)-2′-oxoethyl)1-(cyclopropylmethyl)piperidine (XJ 448) inhibited carbachol-induced inositol 1,4,5-triphosphate (IP 3) formation in a dose-dependent manner. The rank order of potency of the tested drugs for inhibition was: haloperidol ≥ (+) pentazocine = NE−100 > DuP 734 = XJ 448 . In addition, the effects of NE-100, DuP 734 and XJ 448 upon [ 3H]TCP binding were examined using primary cultured neuronal cells derived from the fetal rat telencephalon. These drugs inhibited [ 3H]TCP binding to intact cells. The ability of the test drugs to inhibit [ 3H]TCP binding to primary cultured neuronal cells was in the order: NE-100 > DuP 734 > XJ 448 . Thes observations suggest that NE-100 indirectly modulates the N-methyl-D-aspartate (NMDA)/phencyclidine (PCP) receptor ion channel complex (NMDA receptor-ion channel), presumably through sigma-1 sites.

Characterization of binding sites for [125I]R(+)trans-7-OH-PIPAT in rat brain

Binding characteristics of a novel radioiodinated ligand, [125I]R(+)trans-7-hydroxy-2-(N-n-propyl-N-3'-iodo-2'-propenyl) aminotetralin ([125I]R(+)trans-7-OH-PIPAT), were evaluated using homogenate binding and autoradiographic techniques in rat brain. [125I]R(+)trans-7-OH-PIPAT bound to sites (dopamine receptors) in homogenates of rat basal forebrain (including caudate putamen, nucleus accumbens and olfactory tubercle) with a high affinity (Kd = 0.42 nM). A majority (70%) of the sites labeled by [125I]R(+)trans-7-OH-PIPAT in basal forebrain were GTP-sensitive. In rat hippocampal homogenates, specific and saturable binding of [125I]R(+)trans-7-OH-PIPAT to 5-HT1A receptors, with a Kd value of 1.4 nM and a Bmax value of 210 fmol/mg protein, was observed. Binding of [125I]R(+)trans-7-OH-PIPAT to sigma sites was also demonstrated in rat cerebellar homogenates. In the presence of GTP (to inhibit binding to D2 and 5-HT1A receptors) and DTG (to inhibit binding to sigma sites), dopamine D3 receptors could be selectively labeled with [125I]R(+)trans-7-OH-PIPAT. [125I]R(+)trans-7-OH-PIPAT offers several unique advantages, including high specific activity and high affinity binding, which make it an excellent probe for the investigation and characterization of the distribution of dopamine D3 receptors.

Discriminative stimulus effects of dextromethorphan in the rat

This study was performed to characterize pharmacologically the discriminative stimulus effects of dextromethorphan, an antitussive that binds with high affinity to a subtype of sigma site in the brain. Dextrorphan, a metabolite of dextromethorphan, has phencyclidine (PCP)-like effects. Therefore, training was conducted with dextromethorphan injected by the SC route, which minimizes dextrorphan formation compared to the IP route. The training dose used, 30 mg/kg, by the SC route did not occasion selection of the PCP-appropriate choice lever in rats discriminating IP injections of 2.0 mg/kg PCP from saline. (In contrast, by the IP route the ED50 of dextromethorphan for PCP-appropriate lever selection was 21.7 mg/kg). In rats discriminating 30 mg/kg (SC) of dextromethorphan from distilled water, dextromethorphan was slightly more potent SC than it was IP (ED50s for dextromethorphan-appropriate lever selection: 8.5 and 14.9 mg/kg, respectively). These animals generalized dose-dependently and completely to PCP and to other PCP-receptor ligands, but selected the vehicle-appropriate choice lever when tested with sigma-site ligands, mu-opioid agonists, and naltrexone. Concurrent administration of naltrexone or sigma-site ligands with 30 mg/kg dextromethorphan did not block dextromethorphan-appropriate responding. These results show that the discriminative effects of SC dextromethorphan are PCP-like and are not mediated by the high-affinity dextromethorphan binding site or by the mu-opioid receptor. Because little dextrorphan is formed when dextromethorphan is given SC and because dextromethorphan itself has low affinity for the PCP receptor, the discriminative effects of SC dextromethorphan probably are mediated by a recognition site related closely to but different from the PCP receptor.

In vitro and in vivo expression of opioid and sigma receptors in rat C6 glioma and mouse N18TG2 neuroblastoma cells.

Mouse N18TG2 neuroblastoma and rat C6 glioma cell lines were injected into male nude mice, and the tumors were passaged serially. At each generation, tumors were analyzed for delta opioid binding using [3H][D-Ala2,D-Leu5]enkephalin and for sigma 1 and sigma 2 binding with 1,3-[3H]di-o-tolylguanidine in the presence and absence of 1 microM pentazocine. Receptor density (Bmax) and affinity (KD) were estimated by homologous competition binding assays. Opioid and sigma Bmax values in the solid tumors were significantly lower than their original levels in vitro. KD values for opioid/sigma ligands were similar in vitro and in vivo. With successive passages in the murine host, delta opioid and sigma 1 binding of the neuroblastoma-derived solid tumors became undetectable. In contrast, sigma 2 receptor Bmax values were unchanged with successive passages of the neuroblastoma-derived tumors and doubled in the nude mouse-borne gliomas. When neuroblastoma-derived solid tumors that were devoid of delta opioid binding were returned to culture, opioid receptors appeared to be up-regulated as compared with their original in vitro levels. Serial passaging of these recultured cells in vivo again resulted in a rapid decline in opioid receptor content. The opioid data are consistent with our prior findings on opioid binding diminution in human brain tumors. The pattern of change for sigma binding was more complex, with the sigma 2 response in late passages of the glioma being reminiscent of the formerly observed increase in number of sigma sites in transformed human meninges, kidney, and colon tissue.

Sigma-1 and sigma-2 sites in rat brain: comparison of regional, ontogenetic, and subcellular patterns.

Radioligand binding assay conditions were established for the selective labeling of sigma-1 and sigma-2 sites in membrane homogenates of rat brain. Selective sigma-1 assays were conducted using 5 nM(+)[3H]SKF-10,047 in the presence of 300 nM dizocilpine (MK-801). Selective sigma-2 assays were conducted using 5 nM [3H]DTG in the presence of 1 microM (+)SKF-10,047. Distributions of sigma-1 and sigma-2 binding among brain regions were found to differ. While the brain stem yields the highest level of sigma-1 binding, it yields among the lowest levels of sigma-2 binding. The reverse is true in hippocampal membranes. Different ontogenetic patterns were also observed. Sigma-2 binding decreases substantially during brain development, whereas sigma-1 binding does not vary significantly. Patterns of distribution among subcellular fractions of rat brain homogenates were found to be similar. Both sigma-1 and sigma-2 sites are most enriched in microsomal fractions, and neither is enriched in synaptosomal or mitochondrial fractions. The present results suggest that sigma-1 and sigma-2 sites are distinct entities; they do not appear to be located on a common macromolecule, and they do not represent two different affinity states of a single type of binding site. While the precise subcellular locations of sigma-1 and sigma-2 sites remain to be determined, we conclude that localization of either type of binding site to synaptic regions of plasma membrane or to mitochondria is highly unlikely.

Allosteric modulation of peripheral sigma binding sites by a new selective ligand: SR 31747

The interactions of a new compound SR 31747 with sigma sites were examined in rat spleen membranes and in human peripheral blood leukocytes (PBL). Nanomolar concentrations of SR 31747 selectively inhibited in a non-competitive manner the binding of the prototypic sigma ligands [3H](+)-pentazocine, [3H](+)-3PPP and [3H]DTG on rat spleen membranes. Characterization of SR 31747 binding sites using [3H]SR 31747 as a ligand showed that this compound binds reversibly, with high affinity to one class of sites on rat spleen membranes (Kd 0.66 nM, Bmax 5646 fmol/mg protein). The pharmacological profile of [3H]SR 31747 binding sites was consistent with the presence of specific sites distinct from classical sigma 1 and sigma 2 receptor subtypes strongly suggesting an allosteric modulation of sigma sites by SR 31747. Similarly, [3H]SR 31747 binding sites were demonstrated on human PBL and also on purified subpopulations of human mononuclear cells (granulocytes, NK cells, T4, T8 and B lymphocytes). Administered to mice by i.p. or oral route 30 min before sacrifice, SR 31747 strongly inhibited the binding of [3H](+)-3PPP to mice spleen membranes with ED50 values of 0.18 and 1.43 mg/kg, respectively. Taken together these results could suggest a potential immunological activity of SR 31747 either directly or through allosteric modulation of peripheral sigma sites.

Novel 1-phenylcycloalkanecarboxylic acid derivatives are potent and selective sigma 1 ligands.

Carbetapentane (1, 2-[2-(diethylamino)ethoxy]ethyl 1-phenyl-1-cyclopentanecarboxylate) binds with high affinity to sigma sites and is a potent antitussive, anticonvulsant, and spasmolytic agent. However, carbetapentane interacts at muscarinic binding sites as well, and it is not clear whether either of these receptor systems is involved in the mechanism(s) of action(s) of this drug. In an attempt to determine whether these psychoactivities can be attributed to interaction at sigma sites, a series of carbetapentane analogs were prepared. Phenyl ring substitution; contraction, expansion, and replacement with a methyl group of the cyclopentyl ring; replacement of the carboxylate function with an amide, methyl ether, and methylamine; and replacement of the N,N-diethyl substituent with a morpholino or piperidino moiety were investigated. All of these novel analogs were evaluated for binding to sigma 1 and sigma 2 sites, and comparison of binding at muscarinic m1 and m2 and PCP (1-(1-phenylcyclohexyl)piperidine) receptors was performed. All of the compounds were selective for sigma 1 over sigma 2 sites, with the three most selective analogs being compounds 34 (65-fold), 35 (78-fold), and 39 (51-fold). None of the compounds were active at PCP sites, and chemical modification including (1) replacing the ester function, (2) replacing the cyclopentyl ring with a smaller ring system (cyclopropyl) or a methyl group, and (3) replacing the diethylamino moiety with a morpholino group resulted in > 220-fold selectivity over muscarinic receptor binding. Therefore, several of these novel compounds are potent, sigma 1-selective ligands which can now be investigated as potential antitussive, anticonvulsant, and antiischemic agents. These studies may reveal whether sigma 1 sites play a role in the pharmacological actions of these drugs.

Novel (4-phenylpiperidinyl)- and (4-phenylpiperazinyl)alkyl-spaced esters of 1-phenylcyclopentanecarboxylic acids as potent sigma-selective compounds.

A series of novel 4-phenylpiperidinyl and (4-phenylpiperazinyl)alkyl 1-phenylcyclopentanecarboxylates was synthesized and evaluated for affinity at sigma 1 and sigma 2 sites by inhibition of [3H]-(+)-pentazocine (PENT) and [3H]-1,3-di(2-tolyl)guanidine (DTG) binding in guinea pig brain. The phenylpiperidines were more potent sigma ligands than the corresponding piperazines. Structural modifications varying the optimal spatial distance between the piperidine nitrogen and ester functions led to the identification of the propyl compound 24 ([3H]PENT Ki = 0.50 nM; [3H]DTG Ki = 1.17 nM) and the butyl derivative 32 ([3H]PENT Ki = 0.51 nM; [3H]DTG Ki = 0.69 nM) as novel high-affinity sigma-selective agents. An ethylene spacer was optimum with para-substituted analogs. A notable finding was the discovery of 2-(4-phenylpiperidinyl)ethyl 1-(4-nitrophenyl)-cyclopentanecarboxylate hydrochloride (15) (RLH-033), which demonstrated potent affinity for the [3H]PENT-defined sigma site with a Ki of 50 pM, selectivity for sigma 1 over muscarinic M1 (> 17,600-fold), M2 (> 34,200-fold), dopamine D1 (> 58,000-fold), and D2 (> 7000-fold) receptors, and inactivity at phencyclidine, NMDA, and opioid receptors. RLH-033 is a valuable tool which will aid further in understanding the biology of the sigma recognition site. Information from this research has further defined the topography of the sigma recognition site, which may provide an explanation for the diverse structures which bind with relatively high affinity.

Synthesis and biological evaluation of iodine‐125 iodocaramiphen. A potential M1 muscarinic imaging agent for SPECT

AbstractIodocaramiphen (2) is a selective muscarinic antagonist which binds in vitro with high affinity and selectivity to the M1 subtype of the muscarinic receptor. We report the synthesis of iodine‐125 labeled iodocaramiphen ([125I]‐2) via a tributylstannyl intermediate (3) in 50% radiochemical yield with a specific activity greater than 1000 mCi/μmol. Biodistribution studies in female Fischer rats demonstrated that [125I]‐2 had significant cerebral localization (0.7% injected dose/gram) at 60 minutes post injection. The uptake of activity washed out rapidly from the brain, however, and did not demonstrate specific uptake in those cerebral regions rich in muscarinic receptors. In addition, preinjection with (±)‐QNB (5 mg/kg) blocked uptake of approximately 25% of the injected radiolabel in the brain at 2 hours. The non‐selectivity of 2 toward muscarinic receptors in vivo may result from the metabolism of 2 by various esterases or the affinity of 2 for sigma sites in the brain.

3H]-(+)-pentazocine binding to sigma recognition sites in human cerebellum

The binding characteristics of the sigma-1 selective benzomorphan [ 3H]-(+)-pentazocine were determined in human cerebellar membranes. Saturation binding analysis revealed two affinity sites with a K DH of 1.4 ± 0.7 nM and a K DL of 33.6 ± 11.9 nM. Kinetic studies performed at 25°C demonstrated reversible binding with association with association and dissociation rate constants determined for two classes of sites. In saturation binding studies, the addition of (+)-SKF 10,047 occluded binding of [ 3H]-(+)-pentazocine to high affinity sigma binding sites. The affinity profile of ligands displacing [ 3H]-(+)-pentazocine was consistent with the labeling of sigma-1 recognition sites with haloperidol > (+)-pentazocine > (+)-SKF 10,047 > (+)-3-PPP > DTG > (−)-pentazocine > (−)-SKF 10,047. The potency of the putative D 3 receptor-selective ligand (±)-7-OH-DPAT was close to that measured for (+)-pentazocine in displacement experiments. These data suggest that [ 3H]-(+)-pentazocine labels sigma-1 sites in human cerebellum under appropriate assay conditions.

125I](S)-trans-7-OH-PIPAT: A potential spect imaging agent for sigma binding sites

[ 125I](S)- trans-y-hydroxy-2-[(N-n-propyl-N-(3′-iodo-2′-propenyl)]aminotetralin ([ 125I](S)- trans-7-OH-PIPAT) has been prepared as an iodinated radioligand for studying the sigma binding site. [ 125I](S)- trans-7-OH-PIPAT binds to rat cerebellar membranes with a K d=1.67 ± 0.07 nM and B max=240 ± 72 fmol/mg of protein (determined in the presence of 15 nM spiperone). This new ligand appears to bind to only one site with Hill coefficients close to unity. Inhibition constants for competing ligands determined in the cerebellar tissue homogenates (in the presence of 15 nM spiperone) are closely comparable to inhibition constants determined in the whole brain tissue homogenates (in the absence of spiperone). Furthermore, these inhibition constants are consistent with the values reported for typical sigma ligands. In vivo uptake of [ 125I](S)- trans-7-OH-PIPAT in the rat brain is initially high (2.52% dose/organ at 2 min post i.v. injection) and displays a rapid washout from the brain (0.8% dose/organ at 30 min post i.v. injection). Uptake of [ 125I](S)- trans-7-OH-PIPAT shows moderate target to non-target ratios at 30 minutes (1.54, 1.66 and 1.92 for cerebellar, hypothalamic and hindbrain uptake over striatal uptake, respectively). Pre-injection with haloperidol reduced these ratios to unity suggesting that the ligand binds specifically to haloperidol-sensitive sites in vivo. The selectivity and affinity of [ 125I](S)- trans-7-OH-PIPAT suggest that this new iodinated ligand may be useful for in vitro studies of the sigma sites and can be used in vivo as a potential SPECT imaging agent.