Sex-differences in fine-scale home-range use in an upper-trophic level marine predator.

BACKGROUND: The distribution of prey in the ocean is spatially and temporally patchy. How predators respond to this prey patchiness may have consequences on their foraging success, and thus physical condition. The recent ability to record fine-scale movements of marine animals combined with novel home-range analyses that incorporate the dimension of time should permit a better understanding of how individuals utilise different regions of space and the consequences on their foraging success. METHODS: Over a six-year study, we used T-LoCoH (Time-Local Convex Hull) home-range software to model archival GPS (Global Positioning System) data from 81 grey seals to investigate the fine-scale spatio-temporal use of space and the distribution of apparent foraging effort. Regions of home-ranges were classified according to the frequency of return visits (site fidelity) and duration of visits (intensity of use). Generalized linear mixed -effects models were used to test hypotheses on seasonal changes in foraging distribution and behaviour and the role of space-use and state on determining foraging success. RESULTS: Male grey seals had larger home-ranges and core areas than females, and both sexes showed a contraction in home-range and core area in fall leading up to the breeding season compared with summer. Heavier individuals had smaller core areas than lighter ones, suggesting access to higher quality habitat might be limited to those individuals with greater foraging experience and competitive ability. The size of the home-range or core area was not an important predictor of the rate of mass gain. A fine-scale spatio-temporal analysis of habitat use within the home-range provided evidence of intra-annual site fidelity at presumed foraging locations, suggesting predictably in prey distribution. Neither sex nor season were useful predictors for classifying behaviour. Rather, individual identity explained much of the variation in fine-scale behaviour. CONCLUSIONS: Understanding how upper-trophic level marine predators use space provides opportunities to explore the consequences of variation in foraging tactics and their success on fitness. Having knowledge of the drivers that shape this intraspecific variation can contribute toward predicting how these predators may respond to both natural and man-made environmental forcing.

Foraging behaviour of a continental shelf marine predator, the grey seal (Halichoerus grypus), is associated with in situ, subsurface oceanographic conditions.

BACKGROUND: The heterogeneous oceanographic conditions of continental shelf ecosystems result in a three-dimensionally patchy distribution of prey available to upper-trophic level predators. The association of bio-physical conditions with movement patterns of large marine predators has been demonstrated in diverse taxa. However, obtaining subsurface data that are spatio-temporally relevant to the decisions made by benthically-foraging species can be challenging. METHODS: Between 2009 and 2015, grey seals were captured on Sable Island, Nova Scotia, Canada during summer and fall and instrumented with high-resolution archival GPS tags. These tags recorded location data as well as depth (m), temperature (°C), and light level measurements during dives, until animals returned to the haulout site to breed. Hidden Markov models were used to predict apparent foraging along movement tracks for 79 individuals (59 females, 20 males) every 3 h. In situ measurements were used to estimate chlorophyll-a concentration (mg m- 3) and temperature within the upper-water column (50 m) and temperature and depth at the bottom of dives. As chlorophyll-a could only be estimated from 10:00 to 14:00 AST for dive depths ≥50 m, we formulated two generalized linear mixed-effects models to test the association of predicted grey seal behavioural states with oceanographic conditions and phytoplankton biomass: the first representing conditions of the upper-water column likely to influence primary productivity, and a second model including environmental conditions encountered by grey seals at the bottom of dives, when seals were more likely to be foraging. RESULTS: Predicted grey seal behavioural states were associated with fine-scale chlorophyll-a concentrations and other environmental conditions they encountered across the continental shelf. In the Water Column Model, season had no influence on the probability of observing apparent foraging, but chlorophyll-a, upper-water column temperature, and sex did, with females having a greater probability of foraging than males. In the Bottom Conditions Model, again season had no influence on the probability of apparent foraging, but females were over twice as likely as males to be foraging. CONCLUSIONS: The results of this study highlight the value of in situ measurements of oceanographic properties that can be collected at high temporal resolution by animal-borne data loggers. These data provide insight into how inferred behavioural decisions made by large marine predators, such as the grey seal, may be influenced by fine-scale oceanographic conditions.

Repeatability in lactation performance and the consequences for maternal reproductive success in gray seals.

In mammals, the most significant maternal effect on offspring growth during lactation is the ability of females to efficiently transfer milk energy to their neonates. However, despite the importance of the transfer of milk energy to both maternal and offspring fitness, nothing is known about the extent to which variation among females may be attributed to differences in individual quality or environmental variation in natural populations. We measured repeatability over multiple lactation periods in components of lactation performance in free-ranging, multiparous gray seal (Halichoerus grypus) females to examine to what extent variation among females in pup weaning mass may be attributed to inherent differences in their physiological capacity to deliver milk energy. Levels of repeatability were high for milk composition (r = 0.38-0.50), daily milk output (r = 0.46), and the duration of lactation (r = 0.57), demonstrating that there are consistent differences among females in these characters across lactations and that the overall capacity of gray seal females to deliver milk energy to their pups is characteristic of individuals. The repeatability in pup weaning mass (r = 0.48) was consistent with the values for the components of total milk energy output and suggests that, over a large proportion of their reproductive life, individual gray seal females will consistently wean pups with greater or lesser probabilities of survival. Our results suggest that inherent differences among females in their physiological capacity to deliver milk energy may be an important component of variation in individual quality and, thus, lifetime reproductive success in mammals. High levels of repeatability also suggest that components of milk energy transfer may have a significant heritable genetic basis.

Sexual segregation of seasonal foraging habitats in a non-migratory marine mammal.

Many animal species segregate by sex. Such segregation may be social in nature, or ecological, or both. Grey seals (Halichoerus grypus), like many large mammals, are sexually size dimorphic. In size dimorphic species, allometric differences in morphology, metabolic rate and reproductive costs are likely. Such differences may require the sexes to use different foraging strategies or different habitats. To investigate sexual segregation of habitat in grey seals, we used satellite tracks from 95 (male 46; female 49) adults breeding at Sable Island, Nova Scotia (44 degrees N, 60 degrees W) collected from 1995 to 2005. Location estimates were made from satellite fixes using a state-space movement model to estimate true locations and regularize them in time. Location estimates were used to calculate home range kernels of male and female habitat use each month. Month by sex kernel home ranges revealed striking differences and dynamics in habitat use between males and females on spatial scales broader than most terrestrial examples and at temporal and spatial resolutions rarely available for marine species. Differences were most pronounced just before (October-December) and immediately after breeding (February-March). During both periods, males primarily used areas along the continental shelf break, while females mainly used mid-shelf regions. Coupled with previously identified sex-specific seasonal patterns of energy storage, diving and diet, our findings suggest that males and females differ profoundly in their spatial foraging strategies. These differences may serve to maximize fitness by reducing intersexual competition during key foraging periods.

Sigma-1 and sigma-2 receptors are expressed in a wide variety of human and rodent tumor cell lines.

Thirteen tumor-derived cell lines of human and nonhuman origin and from various tissues were examined for the presence and density of sigma-1 and sigma-2 receptors. Sigma-1 receptors of a crude membrane fraction were labeled using [3H](+)-pentazocine, and sigma-2 receptors were labeled with [3H]1,3-di-o-tolylguanidine ([3H]DTG); in the presence or absence of dextrallorphan. [3H](+)-Pentazocine-binding sites were heterogeneous. In rodent cell lines (e.g., C6 glioma, N1E-115 neuroblastoma, and NG108-15 neuroblastoma x glioma hybrid), human T47D breast ductal carcinoma, human NCI-H727 lung carcinoid, and human A375 melanoma, [3H](+)-pentazocine bound to high- and low-affinity sites with Kd1 = 0.67-7.0 nM, Bmax1 = 25.5-108 fmol/mg protein, Kd2 = 127-600 nM, and Bmax2 = 942-5431 fmol/mg protein. However, [3H](+)-pentazocine bound to a single site in other cell lines. In human U-138MG glioblastoma, SK-N-SH neuroblastoma, and LNCaP.FGC prostate, Kd = 28-61 nM and Bmax = 975-1196 fmol/mg protein, whereas in ThP-1 leukemia Kd = 146 nM and Bmax = 1411 fmol/mg protein. The sigma-1-like nature of [3H](+)-pentazocine-binding sites was confirmed by competition studies which revealed high affinity for haloperidol and enantioselectivity for (+)-pentazocine over (-)-pentazocine. Interestingly, human MCF-7 breast adenocarcinoma showed little or no specific binding of [3H](+)-pentazocine, suggesting the absence of sigma-1 receptors in this cell line. All cell lines examined expressed a high density of sigma-2 receptors with Kd values for [3H]DTG ranging from 20 to 101 nM and Bmax values of 491 to 7324 fmol/mg protein. Competition studies indicated possible heterogeneity of sigma-2 receptors. While sites labeled by [3H]DTG in all cell lines tested exhibited affinity for haloperidol and preference for (-)-pentazocine over the (+)-enantiomer, human cell lines generally showed 4- to 7-fold lower affinity for haloperidol and approximately 10-fold lower affinity for (-)-pentazocine compared with the rodent cell lines. The high density of sigma-1 and sigma 2-binding sites in these cell lines suggests important cellular functions in cancer, as well as potential diagnostic utility for tumor-imaging agents which target sigma sites. These cell lines may be useful as model systems in which to study the functions of sigma sites in normal tissues, as well as their possible role in tumor biology.

Targeting sigma receptor-binding benzamides as in vivo diagnostic and therapeutic agents for human prostate tumors.

Sigma receptors are known to be expressed in a variety of human tumor cells, including breast, neural, and melanoma tumors. A very high density (1.0-1.5 million receptors/cell) of sigma receptors was also reported in a human androgen-dependent prostate tumor cell line (LNCaP). In this study, we show that a very high density of sigma receptors is also expressed in an androgen-independent human prostate tumor cell line (DU-145). Pharmacological binding studies using the sigma-1-selective ligand [3H](+)-pentazocine showed a high-affinity binding (Kd = 5.80 nM, Bmax = 1800 fmol/mg protein). Similarly, binding studies with [3H]1,3-di-o-tolylguanidine in the presence of dextrallorphan also showed a high-affinity binding (Kd = 15.71 nM, Bmax = 1930 fmol/mg protein). Radioiodinated benzamide N-[2-(1'-piperidinyl)ethyl]-3-[125I]iodo-4-methoxybenzamide ([125I]PIMBA) was also shown to bind DU-145 cells in a dose-dependent manner. Three different radioiodinated benzamides, [125I]PIMBA, 4-[125I]iodo-N-[2-(1'-piperidinyl)ethyl]benzamide, and 2-[125I]-N-(N-benzylpiperidin-4-yl)-2-iodobenzamide, were screened for their potential to image human prostate tumors in nude mice bearing human prostate cells (DU-145) xenografts. All three compounds showed a fast clearance from the blood pool and a high uptake and retention in the tumor. Therapeutic potential of nonradioactive PIMBA was studied using in vitro colonogenic assays. A dose-dependent inhibition of cell colony formation was found in two different human prostate cells. These results demonstrate the potential use of sigma receptor binding ligands in non-invasive diagnostic imaging of prostate cancer and its treatment.

Synthesis and pharmacological characterization of 4-[125I]-N-(N-benzylpiperidin-4-yl)-4-iodobenzamide: a high affinity sigma receptor ligand for potential imaging of breast cancer.

The synthesis of [125I]-N-(N-benzylpiperidin-4-yl)-4-iodobenzamide (4-[125I]BP), a novel radiopharmaceutical that possesses high affinity for both sigma-1 and sigma-2 receptor subtypes, and its binding characteristics to MCF-7 breast cancer cells are described. To obtain high yields (with high specific activity) of radioiodinated ligand, (N-benzylpiperidin-4-yl)-4-tri-butylstannyl benzamide was synthesized. Radiolabeled 4-[125I]BP was prepared from tri-butylstannyl precursor with the use of chloramine-T or hydrogen peroxide as an oxidizing agent in high yields (71-86%). The competition binding studies of 4-[125I]BP in MCF-7 breast tumor cells with haloperidol and DTG (known sigma ligands) showed a dose-dependent displacement and high affinity binding (Ki = 4.6 and 56 nM, respectively), demonstrating that sigma receptors are expressed in MCF-7 breast tumor cells. Scatchard analysis of 4-[125I]BP binding in MCF-7 cells revealed saturable binding, with a Kd = 26 nM and a Bmax = 4000 fmol/mg protein. Furthermore, the Scatchard analysis of [3H]DTG binding in MCF-7 cells gave a Kd of 24.5 nM and a Bmax of 2071 fmol/mg of protein. The biodistribution and clearance of 4-[125I]BP was studied in rats. The radiopharmaceutical cleared quickly from the blood pool but rather slowly from the hepatobiliary system. The in vivo specificity was demonstrated by blocking the receptor binding in the presence of haloperidol. A decrease of 55, 63, 43, and 68% was found at 1 h postinjection in brain, kidney, heart, and lung, respectively. These results demonstrate that a high density of sigma receptors are expressed in MCF-7 cells and that radioiodinated 4-IBP may be useful for imaging breast cancer by targeting sigma sites.

Synthesis and evaluation of optically pure [3H]-(+)-pentazocine, a highly potent and selective radioligand for sigma receptors.

Tritium-labeled (+)-pentazocine ([3H]-1b) of specific activity 26.6 Ci/mmol was synthesized in 3 steps starting with (+)-normetazocine (2) of defined optical purity. [3H]-1b has been characterized as a highly selective ligand for labeling of sigma receptors. Competition data revealed that [3H]-1b could be displaced from guinea pig brain membrane preparations with a number of commonly used sigma receptor ligands. [3H]-1b exhibited saturable, enantioselective binding with a Kd of 5.13 +/- 0.97 nM and a Bmax of 1146 +/- 122 fmol/mg protein. Phencyclidine (PCP) displaced [3H]-1b with low affinity while MK-801 was inactive, thus indicating insignificant activity at the PCP-binding site; apomorphine failed to displace [3H]-1b indicating lack of dopamine receptor cross-reactivity. Since the affinity of [3H]-1b is about 6 times that of the two commonly employed sigma ligands ((+)-3-[3H]PPP and [3H]DTG) and since it is more selective for sigma receptors than the benzomorphan [3H]SKF-10,047, it represents the first example of a highly selective benzomorphan based sigma receptor ligand. [3H]-1b should prove useful for further study of the structure and function of sigma receptors.

Evidence for a role of haloperidol-sensitive sigma-'opiate' receptors in the motor effects of antipsychotic drugs.

Haloperidol exhibits a high affinity for a subclass of sigma- "opiate" binding sites which have a unique anatomic distribution and a unique drug selectivity pattern. These binding sites differ from phencyclidine-sensitive sigma-receptors and are found in many brain areas involved in the control of movement. 1,3-Di-o-tolylguanidine (DTG), a highly selective ligand for the haloperidol-sensitive sigma-receptor, produced marked dystonia in rats after microinjection into the red nucleus, a motor area rich in this receptor. Haloperidol and another sigma-ligand [(+)-SKF 10,047] produced similar effects. On the other hand, clozapine, an antipsychotic drug which fails to bind to sigma-receptors and fails to induce movement disorders in humans, failed to induce these dystonic reactions in rats. Phencyclidine was also without effect, as were injections of the active compounds in sites distant to the red nucleus. Microinjections of DTG in the substantia nigra produced vigorous contralateral circling behavior at extremely low doses. These findings suggest that sigma-binding sites represent biologically functional receptors that are active in the neural control of movement. Since haloperidol (and many other antipsychotic drugs) exhibit an affinity for sigma-receptors which is at least equal to its affinity for dopamine receptors, these data raise the further possibility that sigma-receptors are involved in the motor side effects of antipsychotic drugs.

Rimcazole analogs attenuate the convulsive effects of cocaine: correlation with binding to sigma receptors rather than dopamine transporters.

Cocaine interacts with dopamine transporters and sigma receptors at concentrations that are achievable in vivo, suggesting that they may both be viable targets for the development of anti-cocaine agents. Rimcazole binds to both of these targets and also attenuates cocaine-induced locomotor activity and sensitization. To further characterize the mechanism(s) underlying the attenuation of cocaine-induced convulsions and lethality, rimcazole and three analogs (SH3/24, SH2/21, SH1/57), with a range of affinities for dopamine transporters and sigma receptors, were evaluated. The highly selective and potent sigma receptor ligand LR176 was used as a reference. Competition binding studies confirmed that the rank order of the compounds at dopamine transporters vs. sigma receptors differed, thus enabling a correlation between the relative anti-cocaine activities of the compounds in behavioral studies and their affinities for dopamine transporters vs. sigma receptors. In behavioral studies, male Swiss Webster mice were pre-treated with one of the compounds (0-60 mg/kg, i.p.), then challenged 15 min later with either a convulsive (60 mg/kg, i.p.) or lethal (125 mg/kg, i.p.) dose of cocaine. When the compounds were ranked according to their protective effect, there was a significant correlation between their anticonvulsant actions and their affinities for sigma receptors, but not dopamine transporters. Although the rimcazole analogs were ineffective against the lethal effects of cocaine, the selective sigma receptor ligand LR176 provided significant protection. These data thus suggest that sigma receptors may mediate some of the toxic effects associated with cocaine and that sigma receptor antagonists may be developed as pharmacotherapeutic agents for this application.

Wash-resistant inhibition of phencyclidine- and haloperidol-sensitive sigma receptor sites in guinea pig brain by putative affinity ligands: determination of selectivity.

Several putative affinity ligands, based on the structures of phencyclidine etoxadrol, 5-methyl-10,11-dihydro-5H-dibenzo[a,d] cycloheptene-5,10-imine (MK801) and 1,3-di-(2-methylphenyl)guanidine (DTG) were evaluated in vitro for their ability to produce a wash-resistant inhibition of phencyclidine and sigma receptor sites in homogenates of the brain of the guinea pig. All the phencyclidine-based ligands, including 1-[1-(3-isothiocyanatophenyl)cyclohexyl]piperidine (Metaphit) and (+/-)-N-(2-isothiocyanatoethyl) MK801 [(+/-)-MK801-NCS], produced a wash-resistant inhibition of binding sites for phencyclidine, labelled by [3H]-1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) and sigma binding sites, labelled by [3H]DTG. The DTG-based ligands, 1-(4-isothiocyanato-2-methylphenyl)-3-(2-methylphenyl)guanidine (DIGIT) and 1-(4-[2-(2-isothiocyanatoethoxy)ethoxy]-2-methyl-phenyl)-3-(2- methylphenyl)guanidine (DIGIE), produced a wash-resistant inhibition of sigma sites, at concentrations as small as 1 microM and also inhibited binding sites for phencyclidine at larger concentrations (100 microM). Both 1-(3-isothiocyanatophenyl)-1-ethyl-4-(2-piperidyl)-1,3-dioxolane (ETOX-NCS) and 1-[1-(3-bromoacetyloxyphenyl)cyclohexyl]-1,2,3,6-tetrahydropyri din e (Bromoacetyl-PCP) were the most potent and selective inhibitors of the binding of [3H]TCP, while DIGIT was the most selective inhibitor of the binding of [3H]DTG. Future studies will examine the selectivity of these agents in vivo after intracerebroventricular administration.

Synthesis and evaluation of aryl-substituted N-(arylethyl)-N-methyl-2-(1-pyrrolidinyl)ethylamines and corresponding arylacetamides for sigma receptor affinity.

A series of aryl-monosubstituted arylacetamides (4-9) and arylethylenediamine (10-18) compounds were synthesized based on the structure of the high-affinity sigma ligand N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (2). These compounds were prepared to evaluate the effect of aromatic substitution patterns on sigma-1 and sigma-2 receptor binding affinity and selectivity. The data indicate that 10-18 possessed higher affinity than 4-9 for both sigma sites, especially when substituted with an electron-withdrawing group. The diamine compounds 10-18 were selective for sigma-1 binding sites, whereas the arylacetamide compounds 4-9 generally exhibited an increased selectivity for sigma-2 sites compared to sigma-1. No clear pattern between the orientation of aromatic substituents and the sigma binding activity was observed.

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.

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.

Asymmetric synthesis of 9-alkyl-2-benzyl-6,7-benzomorphans: characterization as novel sigma receptor ligands.

A convenient enantioselective synthesis of (1R,5R,9R)- and (1S,5S, 9S)-9-alkyl-2-benzyl-6,7-benzomorphans (2a-c) which starts with naphthaldehyde is described. These compounds were designed to gain additional information on the structure-sigma binding relationship of the 6,7-benzomorphan class of sigma ligands. In contrast to pentazocine and most 6,7-benzomorphans, the (1R,5R,9R)-isomers of 2a-c showed greater affinity for the sigma(1) receptor than the (1S, 5S,9S)-isomers. Despite reversal of enantioselectivity at the sigma(1) sites, moderate affinity and enantioselectivity at the sigma(2) sites [greater affinity for (1R,5R,9R)-isomers than (1S,5S, 9S)-isomers] were maintained. A comparison of the binding affinities of 2a-c to the more conformationally flexible trans-2-alkyl-1-benzaminoethyl-1,2-dihydronaphthalenes (10a-c) suggested that the relatively rigid structure of 2a-c played an important part in their sigma(1) binding properties. These compounds, particularly (1R,5R,9R)-2-benzyl-9-methyl-6,7-benzomorphan [(-)-2a], which has a K(i) value of 0.96 nM, will be useful in further characterization of the sigma(1) receptor.

Characterization of novel N,N'-disubstituted piperazines as sigma receptor ligands.

sigma Receptors have been the focus of extensive studies because of their potential functional role in several important physiological and biochemical processes. To further evaluate the properties of sigma receptors, especially sigma-1 and sigma-2 subtypes, we have synthesized a series of N,N'-disubstituted piperazine compounds (1-32). The design of these compounds was based upon the early structure-activity relationship (SAR) studies of the minimum structural requirements of a molecule necessary to elicit sigma receptor binding activity. In the N-(3-phenylpropyl)piperazine series, compounds with the ethylenediamine moiety (8-11, 15-17) showed 6-20-fold higher affinity for sigma-1 and 2-40-fold higher affinity for sigma-2 relative to their corresponding amides (1-7). The (m-nitrophenethyl)piperazine 10 exhibits a subnanomolar affinity for the sigma-1 site, whereas the corresponding o-nitro compound 9 shows the highest affinity for the sigma-2 site (Ki = 4.9 nM). Compounds with a free amino terminus were designed as precursors for use as bioconjugated affinity compounds. Some of these compounds displayed high affinity for sigma-1 and moderate affinity for sigma-2 sites and are currently used for the purification and characterization of the receptor subtypes.

A new approach to the design of sigma-2-selective ligands: synthesis and evaluation of N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1- pyrrolidinyl)ethylamine-related polyamines at sigma-1 and sigma-2 receptor subtypes.

A series of polyamines based on the high affinity sigma receptor ligand N-[2-(3,4-dichlorophenyl)-ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (3) were developed and evaluated for their binding characteristics at sigma-1 and sigma-2 receptor subtypes. The data indicated that a considerable degree of structural variation is possible while still retaining nanomolar affinity at sigma receptors. As the structure of the polyamines was varied, their binding at sigma-1 and sigma-2 subtypes showed quite different and in some cases opposite trends, supporting the belief that these are pharmacologically distinct entities. Polyamines containing two nitrogen atoms showed optimal binding at both sigma-1 and sigma-2 receptor subtypes. Although additional nitrogen atoms resulted in decreased affinity at sigma-1 and sigma-2 subtypes, an increase in selectivity for sigma-2 subtypes was evident; the parent 3 showed greater selectivity for sigma-1 subtypes. Internitrogen spacings had a large effect on binding affinity and subtype selectivity. For example, the difference between N-[3-(1-pyrrolidinyl)propyl]-N'-(3,4-dichlorobenzyl)-N,N'- dimethylethylenediamine (8) [K(i) = 29.9 nM at sigma-1 receptor and 18.3 nM at sigma-2 receptor] to N-[3-(1-pyrrolidinyl)propyl]-N'-(3,4-dichlorobenzyl)- N,N'-dimethylethylenediamine (10) [K(i) = 1.49 nM at sigma-1 receptor and 12.1 nM at sigma-2 receptor] illustrates the importance of internitrogen spacing. Triamines 11 and 13 [Ki(sigma-2)/K(i)(sigma-1) = 0.19 and 0.10, respectively] containing the N-N-N-Ar spacings 3-3-2 and 4-4-2, proved to be the most sigma-2 subtype selective of the 15 polyamines examined in this study. The N-N-N spacings appear to be an important factor in their sigma-2 subtype selectivity. These compounds will serve as templates in the design of still further sigma-2 subtype selective ligands. The pyrrolidine ring (present in most of the polyamines tested in this series) proved to be an important recognition site for sigma receptor binding activity. Furthermore, alkyl substitution also appears to be important since the stripped down polyamines N-[2-(3,4-dichlorophenyl)ethyl]ethylenediamine (15) and N1-[2-(3,4-dichlorophenyl)ethyl]diethylenetriamine (16) exhibited relatively low binding affinity.

(+)-cis-N-(para-, meta-, and ortho-substituted benzyl)-N-normetazocines: synthesis and binding affinity at the [3H]-(+)-pentazocine-labeled (sigma 1) site and quantitative structure-affinity relationship studies.

sigma 1 receptor ligands have potential pharmacological significance as antipsychotic drugs, as tools in the study of drug-induced motor function disorders, and as radiopharmaceutical imaging agents for the noninvasive imaging of malignant tumors in human subjects. A series of substituted N-benzyl-N-normetazocines were synthesized and their binding affinity at the sigma 1 receptor evaluated in order to examine the details of the structure--affinity relationships (SAR) of a previously determined high-affinity lead compound, (+)-cis-N-benzyl-N-normetazocine (Ki = 0.67 nM). Variation in the benzyl substituents of these compounds produced a 1590-fold range in affinity at the sigma 1 receptor from the unsubstituted benzyl analog to the lowest affinity p-tert-butylbenzyl analog (Ki = 1066 nM). The nanomolar binding affinity for the sigma 1 receptor of (+)-cis-N-(4-fluorobenzyl)-N-normetzocine suggests that this analog may be a useful PET imaging agent.

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)

Adrenergic agents. 4. Substituted phenoxypropanolamine derivatives as potential beta-adrenergic agonists.

A series of 1-(substituted phenoxy)-3-(tert-butylamino)-2-propanols in which the ring substituents were 3,4-dihydroxy (6f), 3- and 4-hydroxy (6g and 6h, respectively), 3-hydroxy-4-methylsulfonamido (6i), its 3,4-transposed isomer (6j), and 4-methylsulfonylmethyl (6k) was prepared and examined for beta-adrenergic agonist and/or antagonist properties. Two of these compounds, 6f and 6j, were potent beta-adrenoreceptor agonists in in vitro tests that measure a compound's ability to relax guinea pig tracheal smooth muscle and to increase the rate of contraction of guinea pig right atria. Several compounds had a dose-dependent effect. Although they produced potent beta-adrenergic agonist activity at low concentrations, 6g, 6h, and 6j antagonized the effects of a standard beta-adrenoreceptor agonist at higher concentrations. The methylsulfonylmethyl derivative 6k produced beta-adrenergic blocking effects as demonstrated by attenuation of isoproterenol-induced increases in the rate of contraction of an isolated rabbit heart preparation. On the basis of these pharmacological results, coupled with NMR spectral data, it appears that the previous suggestion that aryloxypropanolamines interact with beta-adrenocreceptors as a consequence of their ability to assume an orientation in which the benzene ring the ethanolamine moieties can be superimposed on those of corresponding adrenergic phenylethanolamines is invalid. An alternative "bicyclic" rigid conformation involving two intramolecular hydrogen bonds in the protonated form of the aryloxypropanolamines is suggested to account for the similar beta-adrenoreceptor activity of these compounds and related phenylethanolamines.