Heteroaromatic analogs of 1-[2-(diphenylmethoxy)ethyl]- and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazines (GBR 12935 and GBR 12909) as high-affinity dopamine reuptake inhibitors

A new series of heteroaromatic GBR 12935 [1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine] (I) and GBR 12909 [1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine] (2) analogs was synthesized and evaluated as dopamine transporter (DAT) ligands. Analogs 5-16, in which the benzene ring in the phenylpropyl side chain of the GBR molecule had been replaced with a thiophene, furan, or pyridine ring, exhibited high affinity and selectivity for the DAT vs serotonin transporter (SERT) and stimulated locomotor activity in rats in a manner similar to the parent compound 2. In cocaine and food self-administration studies in rhesus monkeys, both thiophene-containing (6 and 8) and pyridine-containing (14 and 16) derivatives displayed potency comparable to 2 in decreasing the cocaine-maintained responding at the doses tested (0.8, 1.7, and 3 mg/kg). However, these compounds did not produce the degree of separation between food- and cocaine-maintained responding that was seen with 2. Among the bicyclic fused-ring congeners 17-38, the indole-containing analog of 2, 22, showed the greatest affinity for binding to the DAT, with IC50 = 0.7 nM, whereas the corresponding indole-containing derivative of 1, 21, displayed the highest selectivity (over 600-fold) at this site vs the SERT site.     

Development of novel, potent, and selective dopamine reuptake inhibitors through alteration of the piperazine ring of 1-[2-(diphenylmethoxy)ethyl]-and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazines (GBR 12935 and GBR 12909)

The design, synthesis, and biological evaluation of compounds related to the dopamine (DA) uptake inhibitors: 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (1) and 1-[2-[bis-(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (2) (GBR 12395 and GBR 12909, respectively), directed toward the development and identification of new ligands interacting with high potency and selectivity at the dopamine transporter (DAT) is reported. The substitution of the piperazine ring in the GBR structure with other diamine moieties resulted in the retention of the high affinity of new ligands for the DAT. Some of the modified GBR analogs (e.g. 8, 10, (-)-49, or (-)-50) displayed substantially higher selectivity (4736- to 693-fold) for the dopamine (DA) versus the serotonin (5HT) reuptake site than the parent compounds. The bis(p-fluoro) substitution in the (diphenylmethoxy)ethyl fragment slightly increased the affinity of the ligands at the DA reuptake site but reduced their selectivity at this site (e.g. 9 and 8, 11 and 10, or 17 and 16, respectively). Congeners, such as the series of monosubstituted and symmetrically disubstituted piperazines and trans-2,5-dimethylpiperazines, which lack the (diphenylmethoxy)ethyl substituent lost the affinity for the DAT yet exhibited very high potency for binding to the sigma receptors (e.g.28). The chiral pyrrolidine derivatives of 1, (-)-49, and (+)-49, exhibited an enantioselectivity ratio of 181 and 146 for the inhibition of DA reuptake and binding to the DAT, respectively.     

New trends in biological monitoring: application of biomarkers to genetic ecotoxicology

We extended observations on cocaine-induced turning and its interactions with mu-opioid receptor agonists in nigrally-lesioned rats to GBR12909 (1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-[3-phenyl-propyl]-piperazine) , a selective dopamine reuptake inhibitor. GBR12909 produced turning that was potentiated by the mu-opioid receptor agonists morphine and methadone. The effects of these opioids were blocked by the general opioid receptor antagonist naloxone, which did not affect the action of GBR12909. The reuptake inhibitors nisoxetine (norepinephrine) and fluoxetine (serotonin) did not produce turning alone or in combination with morphine. Antagonists selective for each opioid receptor subtype did not alter GBR12909-induced turning. However, naltrexone, another general opioid receptor antagonist, potentiated turning induced by GBR12909. This was blocked by naloxone, suggesting that naltrexone has opioid receptor agonist actions, in contrast to naloxone. These results indicate that cocaine-induced turning and its potentiation by mu-opioid receptor agonists are dependent upon the inhibition of dopamine reuptake.     

(3R,4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl]chroman-4,7-diol: a conformationally restricted analogue of the NR2B subtype-selective NMDA antagonist (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)- 1-propanol

(1S,2S)-1-(4-Hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP-101,606, 1) is a recently described antagonist of N-methyl-D-aspartate (NMDA) receptors containing the NR2B subunit. In the present study, the optimal orientation of compounds of this structural type for their receptor was explored. Tethering of the pendent methyl group of 1 to the phenolic aromatic ring via an oxygen atom prevents rotation about the central portion of the molecule. Several of the new chromanol compounds have high affinity for the racemic [3H]CP-101,606 binding site on the NMDA receptor and protect against glutamate toxicity in cultured hippocampal neurons. The new ring caused a change in the stereochemical preference of the receptor-cis (erythro) compounds had better affinity for the receptor than the trans isomers. Computational studies suggest that steric interactions between the pendent methyl group and the phenol ring in the acyclic series determine which structures can best fit the receptor. The chromanol analogue, (3R,4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1- yl]chroman-4,7-diol (12a, CP-283,097), was found to possess potency and selectivity comparable to CP-101,606. Thus 12a is a new tool to explore the function of the NR2B-containing NMDA receptors.     

Pharmacological characterization of the novel discriminative stimulus effects of a low dose of cocaine

Twelve rats were trained to press one lever after cocaine injection (3 mg/kg i.p.) and another lever after saline injection. Once rats were reliably discriminating cocaine from saline, other drugs were examined for their efficacies in substituting for cocaine. The dopamine uptake inhibitors WIN 35,428 [2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane-1,5-naphthalene - disulfonate] and GBR 12909 (1-[2-bis(4-fluorophenyl)methoxy]ethyl]-4-[3- phenylpropyl]piperazine dihydrochloride) fully substituted for cocaine (cocaine responding > 80%), whereas the peripherally active cocaine methiodide and the 5-hydroxytryptamine uptake inhibitor fluoxetine did not substitute at all. Pentobarbital also failed to produce any cocaine-appropriate responding. Two selective norepinephrine uptake inhibitors were tested: tomoxetine fully substituted for the 3-mg/kg dose of cocaine and nisoxetine approached full substitution (79.7% cocaine responding). The direct-acting dopamine D-1 agonists SKF 38393 [(+-)-7-bromo-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-be nzazepin e HCl], SKF 77434 [(+-)-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-benzazepine HCl] and SKF 75670 [3-methyl-7,8-dihydroxyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benza zep ine HBr] fully substituted for cocaine, whereas the peripherally active dopamine D-1 agonist fenoldopam did not. Of four dopamine D-2 agonists tested, only quinpirole fully substituted; the others (N-0434 [(+-)-2-(N-propyl-N-phenylethylamino)-5-hydroxytetralin], (-)-NPA [R(-)-propylnorapomorphine HCl] and SDZ 208-912 (N-[(8-)-2,6-dimethylergoline-8-yl]-2,2-dimethyl-propanamide)) produced very limited partial substitution (cocaine responding < 32%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of phentermine on responding maintained under multiple fixed-ratio schedules of food and cocaine presentation in the rhesus monkey

Drugs that decrease drug-maintained responding at doses that do not decrease other behaviors in animals may be suitable candidates for development as medications to treat drug abuse in humans. The present study examined whether this effect could be obtained with phentermine, a drug that has been reported to decrease cocaine intake in humans. Rhesus monkeys were trained under multiple fixed-ratio 30-response schedules of food and i.v. cocaine delivery. Phentermine was always given as a slow, i.v. infusion. Acute treatment with phentermine (0.3-10 mg/kg) decreased cocaine-maintained responding at doses that did not decrease, or decreased less, food-maintained responding for each of three unit doses of cocaine (10-100 microg/kg/injection). Subacute treatment with phentermine (3 or 5.6 mg/kg, daily) also decreased cocaine-maintained responding more than food-maintained responding. After subacute treatment was terminated, rates of cocaine-maintained responding generally recovered to levels comparable to those seen during untreated control sessions. Phentermine (0.3-3 mg/kg) did not generally increase responding associated with a very low (1 microg/kg/injection) unit dose of cocaine, suggesting that the decrease in cocaine-maintained responding at higher unit doses was not the result of a leftward shift in the cocaine unit dose-effect function. Phentermine (0.1-3 mg/kg) decreased responding maintained by 1-[2-[bis(4-fluorophenyl) methoxy]ethyl]-4-[3-phenylpropyl] piperazine (GBR 12909) (30 microg/kg/injection) at doses similar to those that decreased food-maintained responding. These results show that phentermine is effective in decreasing cocaine self-administration and suggest that it may be an effective medication for cocaine abuse.     

Serum leptin and IGF-I levels in cystic fibrosis

(3S,4R)-4-(4-Fluorophenyl)-3-[[3,4-(methylenedioxy)phenoxy]methyl] piperidine [(3S,9R)-3, paroxetine] is a selective serotonin reuptake inhibitor (SSRI) used as an antidepressant in humans. In previous studies, we reported that certain (1R)-3 beta-(substituted phenyl)nortropane-2 beta-carboxylic acid methyl esters (2a) exhibited high affinity and reasonable selectivity for the serotonin transporter (5-HTT). The major structural differences between 2a and (3S,4R)-3 are that 2a possesses a different absolute stereochemistry and has an ethylene bridge not present in 3. In addition, 2a possesses a carbomethoxy substituent adjacent to the aryl ring, whereas (3S,4R)-3 contains a [3,4-(methylenedioxy)phenoxy]methyl group. In this study, we present the synthesis and biological evaluations of six of the possible eight isomers of 3-(4-fluorophenyl)-2-[[3,4-(methylenedioxy)phenoxy]methyl]nortropane+ ++ (4). The data for inhibition of [3H]paroxetine binding show that (1R)-2 beta, 3 alpha-4c, which has the same stereochemistry as paroxetine, has the highest affinity at the 5-HTT. Strikingly, the most potent compounds for inhibition of [3H]WIN-35,428 binding were not the (1R)-2 beta, 3 beta-isomers but rather (1R)-2 beta, 3 alpha-4c and (1S)-2 beta, 3 alpha-4f. Conformational analyses show that these isomers exist in a flattened boat conformation with pseudoequatorial substituents. Thus, the binding data show that this conformation is recognized by the DAT-associated binding site and also suggest that this conformation of paroxetine is recognized by the 5-HTT-associated binding site.     

Studies of the biogenic amine transporters. IV. Demonstration of a multiplicity of binding sites in rat caudate membranes for the cocaine analog [125I]RTI-55

The drug 3 beta-[4'-iodophenyl]tropan-2 beta-carboxylic acid methyl ester (RTI-55) is a cocaine congener with high affinity for the dopamine transporter (Kd < 1 nM). The present study characterized [125I]RTI-55 binding to membranes prepared from rat, monkey and human caudates and COS cells transiently expressing the cloned rat dopamine (DA) transporter. Using the method of binding surface analysis, two binding sites were resolved in rat caudate: a high-capacity binding site (site 1, Bmax = 11,900 fmol/mg of protein) and a low-capacity site (site 2, Bmax = 846 fmol/mg of protein). The Kd (or Ki) values of selected drugs at the two sites were as follows: (Ki for high-capacity site and Ki for low-capacity site, respectively): RTI-55 (0.76 and 0.21 nM), 1-[2-diphenyl-methoxy)ethyl]-4-(3-phenylpropyl)piperazine (0.79 and 358 nM), mazindol (37.6 and 631 nM), 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (45.0 and 540 nM) and cocaine (341 and 129 nM). Nisoxetine, a selective noradrenergic uptake blocker, had low affinity for both sites. Serotonergic uptake blockers had a high degree of selectivity and high affinity for the low-capacity binding site (Ki of citalopram = 0.38 nM; Ki of paroxetine = 0.033 nM). The i.c.v. administration of 5,7-dihydroxytryptamine to rats pretreated with nomifensine (to protect dopaminergic and noradrenergic nerve terminals) selectively decreased the Bmax of site 2, strongly supporting the idea that site 2 is a binding site on the serotonin (5-HT) transporter. This serotonergic lesion also increased the affinity of [125I]RTI-55 for the DA transporter by 10-fold. The ligand selectivity of the caudate 5-HT transporter was different from the [I125]RTI-55 binding site on the 5-HT transporter present in membranes prepared from whole rat brain minus caudate. The [125I]RTI-55 binding to the DA transporter was further resolved into two components, termed sites 1a and 1b, by using human and monkey (Macaca mulatta) caudate membranes but not the membranes prepared from rat caudate or COS cells that transiently expressed the cloned cocaine-sensitive DA transporter complementary DNA. Similar experiments also resolved two components of the caudate 5-HT transporter. Viewed collectively, these data provide evidence that [125I]RTI-55 labels multiple binding sites associated with the DA and 5-HT transporters.     

Synthesis and ligand binding studies of 4'-iodobenzoyl esters of tropanes and piperidines at the dopamine transporter

Four analogs and two homologs of cocaine, designed as potent cocaine antagonists, were synthesized. The SN2 reaction between ecgonine methyl ester (13) or appropriately substituted piperidinol (19, 21) and appropriately substituted 4-iodobenzoyl chloride gave 4-iodobenzoyl esters of tropanes and piperidines (5-8). 2'-Hydroxycocaine (9) was obtained from 2'-acetoxycocaine (12) by selective transesterification with MeOH saturated with dry HCl gas. 2'-Acetoxycocaine (12) was synthesized from acetylsalicyloyl chloride (23) and ecgonine methyl ester (13). The binding affinities of these compounds were determined at the dopamine transporter for the displacement of [3H]WIN-35428. An iodo group substitution at the 4'-position of cocaine decreased dopamine transporter binding potency, while a hydroxy or acetoxy group at the 2'-position exhibited increased binding potency for the dopamine transporter compared to cocaine (10- and 3.58-fold, respectively). 2'-Hydroxylation also enhanced the bidning potency of 4'-iodococaine (5) by 10-fold. Replacement of the tropane ring with piperidine led to poor binding affinities.     

Synthesis, structure, dopamine transporter affinity, and dopamine uptake inhibition of 6-alkyl-3-benzyl-2-[(methoxycarbonyl)methyl]tropane derivatives

A series of 6-alkyl-3 beta-benzyl-2-[(methoxycarbonyl)methyl]tropane analogues were synthesized and evaluated as cocaine binding site ligands at the dopamine transporter (DAT). The in vitro affinity (Ki) for the DAT of the 6-alkyl-3 beta-benzyl-2-[(methoxycarbonyl) methyl]tropane analogues was determined by inhibition of [3H]WIN 35,428 in rat caudate putamen tissue. The inhibition of dopamine uptake (IC50) was also measured for selected compounds which demonstrated moderate affinity for the dopamine transporter. The unsubstituted enantiopure analogues (-)-19a (Ki = 33 nM) and surprisingly (+)-20a (Ki = 60 nM) were found to be almost equipotent with the high-affinity binding components of cocaine and WIN 35,065-2 and exhibited slightly more potent dopamine uptake inhibition than both cocaine and WIN 35,065-2. In general, substitution at the 6-position of racemic 19a and 20a with alkyl groups was found to result in decreased activity relative to increased chain length of the substituent. The 3 beta-benzyl-2 beta-[(methoxycarbonyl)methyl]-6 beta-methyltropane (21b; Ki = 57 nM) was the only 6-alkyl derivative to exhibit moderately potent activity. The 6 beta-isomer 21b was 4-fold more potent than the 6 alpha-isomer 19b (Ki = 211 nM) and was nearly equipotent with (-)-19a and (+)-20a as well as with cocaine and WIN 35,065-2. The results of this study further demonstrate the steric constraints associated with the C(6)-C(7) methylene bridge of the tropane ring system for molecular recognition of cocaine analogues at the cocaine binding site(s) on the DAT.     

Dopamine transporter ligand binding domains. Structural and functional properties revealed by limited proteolysis

Dopamine transporters (DATs) are members of the Na+- and Cl--dependent neurotransmitter and amino acid transporter family predicted by hydrophobicity analysis to have 12 transmembrane-spanning helices. The structure of DAT was studied using the photoaffinity compounds [125I]1-[2-(diphenylmethoxy)-ethyl]-4-[2-(4-azido-3-iodophenyl) ethyl] piperazine ([125I]DEEP), a 1-(2-diphenylmethoxy)-ethyl-4-(3-phenyl propyl)piperazine (GBR analog), and [125I]-3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82), a cocaine analog, which had been shown in a previous study to become incorporated into different regions of the DAT primary sequence. The proximity of the photolabeled binding sites to integral membrane structures was investigated by subjecting photolabeled membrane suspensions to limited proteolysis with trypsin and separately analyzing the resulting membranes and supernatants for the presence of photolabeled DAT fragments. Trypsin treatment of [125I] DEEP-labeled membranes generated labeled 45- and 14-kDa DAT fragments that immunoprecipitated with an epitope-specific antiserum generated against amino acids 42-59 near the first putative transmembrane domain, whereas [125I]RTI 82 was found in 32- and 16-kDa tryptic fragments that precipitated with an antiserum directed against a sequence near transmembrane domain 4 (amino acids 225-238). All of the photolabeled fragments were recovered in the protease-treated membranes, indicating that they possess integral membrane structures that prevent their release from the membrane as soluble forms. The size of the two smallest fragments in conjunction with their retention in the membrane suggests that incorporation of the photoaffinity ligands occurs in or near membrane spanning regions and delineates the maximum possible distance between the transmembrane structures, incorporated photolabel, and antibody epitopes. Carbohydrate analysis of the fragments identified sialic acids and N-linked oligosaccharides exclusively on the 45-kDa [125I]DEEP-labeled fragment, which, based on size, would be expected to contain four consensus glycosylation sites between putative transmembrane domains 3 and 4. Photoaffinity labeling after trypsin treatment of membranes showed that the larger but not the smaller fragments retain binding capacity, as the 45- and 32-kDa fragments were capable of becoming photolabeled. Binding of photoaffinity ligands at these fragments was displaced with the same pharmacology as that of intact DATs. These results verify numerous aspects of DAT structure and topology heretofore only predicted from theoretical considerations and extend our knowledge of DAT structure-function properties.     

Investigation of the N-substituent conformation governing potency and mu receptor subtype-selectivity in (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonists

A study of the binding site requirements associated with the N-substituent of (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) derivatives was undertaken using a set of rigid vs flexible N-substituents. The study showed that compounds 7-9 bearing the trans-cinnamyl N-substituent most closely reproduced the potency at the opioid receptor of the flexible N-propylphenyl or N-propylcyclohexyl analogues previously reported. Neither the N-substituted cis-cinnamyl nor the cis-phenylcyclopropylmethyl compounds 10 and 11, respectively, showed high affinity for the opioid receptor. However, the N-trans-phenylcyclopropylmethyl compound 12 closely approximated the affinity of compounds 7-9. Additionally, we found that free rotation of the phenyl ring is necessary for high affinity binding and mu receptor subtype selectivity as the planar N-substituted thianaphthylmethyl and benzofuranylmethyl compounds 13 and 14 had significantly lower binding affinities. Altogether, these findings suggest that the high binding affinity, selectivity, and antagonist potency of N-propylphenyl or N-propylcyclohexyl analogues of (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) are achieved via a conformation wherein the connecting chain of the N-substituents is extended away from piperidine nitrogen with the appended ring system rotated out-of-plane relative to the connecting chain atoms. This conformation is quite similar to that observed in the solid state for 5, as determined by single crystal X-ray analysis. Additionally, it was found that, unlike naltrexone, N-substituents bearing secondary carbons attached directly to the piperidine nitrogen of 4 suffer dramatic losses of potency vs analogues not substituted in this manner. Using a functional assay which measured stimulation or inhibition of [35S]GTP-gamma-S binding, we show that the trans-cinnamyl analogues of (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) retain opioid pure antagonist activity and possess picomolar antagonist potency at the mu receptor.     

Development of FUB 181, a selective histamine H3-receptor antagonist of high oral in vivo potency with 4-(omega-(arylalkyloxy)alkyl)-1H-imidazole structure

A series of 4-(omega-(arylalkyloxy)alkyl)-1H-imidazoles and related sulphur-containing compounds have been prepared and evaluated for their histamine H3-autoreceptor antagonist in vitro potency in an assay on synaptosomes of rat cerebral cortex. In addition, the in vivo potency has been determined from the changes in N tau-methylhistamine levels in brain after p.o. administration to mice. Compounds with different alkyl chains and various aryl moities have been synthesized and tested to explore structure-activity relationships. Within this series of novel antagonists, (1H-imid-azol-4-yl)methyl and 2-(1H-imidazol-4-yl)ethyl ether derivatives showed low to moderate H3-receptor antagonist potency, whereas the corresponding allyl and propyl derivatives were compounds with high antagonist in vitro potency. Corresponding thioether or sulphoxide derivatives also showed antagonists activity. Additionally, some ether derivatives possessed high in vivo potency as well. The most active ether derivatives under in vivo conditions were 4-(3-(3-(4-fluorophenyl)propyloxy)propyl)-1H-imidazole (11b) and the corresponding chloro compound 11c (FUB 181) with ED50 values of 0.76 and 0.80 mg/kg, respectively. On the other hand, all compounds tested showed weak activity at histamine H1 or H2 receptors. Furthermore, the most promising ether FUB 181 exhibited low activity at adrenergic alpha 1, beta 1/2, serotonergic 5-HT2A, 5-HT3, and muscarinic M3 receptors. Time-course investigations of FUB 181 in mice showed a rapid mode of action with the highest value 3 h after p.o. application. Thus, FUB 181 appears to block histamine H3 receptors potently and selectively.     

Ranitidine effervescent and famotidine wafer in the relief of episodic symptoms of gastro-oesophageal reflux disease

Radioligands that specifically target dopamine uptake sites can provide a means of determining dopamine fiber loss at intrastriatal mesencephalic grafts in Parkinsonian patients, using Positron Emission Tomography (PET). The BTCP derivative, 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-(2-hydroxyethyl)-piperazine, shows in vitro high affinity and selectivity for the dopamine transporter. To evaluate the potential of such a compound as a potential dopaminergic PET tracer the positron-emitting analogues, 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-(2-[18F]fluoroethyl)-piperazine and 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-[11C]methylpiperazine, were synthesized. Radiofluorination was carried out by the reaction of 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-(2-chloroethyl)-piperazine with cyclotron-produced n.c.a. 18F-(half life 109.9 min) obtained by the (p,n) reaction on 18O-enriched water. Labelling with carbon-11 (half life 20.4 min) was achieved by 11C methylation of 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-piperazine with [11C]methyl iodide. After intravenous administration to rats these two compounds enter the brain, but despite their high in vitro affinity they display a high non specific binding in vivo which greatly limits their use as PET radioligands.     

Identification of an opioid kappa receptor subtype-selective N-substituent for (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine

A three-component library of compounds was prepared in parallel using multiple simultaneous solution-phase synthetic methodology. The compounds were biased toward opioid receptor antagonist activity by incorporating (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (a potent, nonselective opioid pure antagonist) as one of the monomers. The other two monomers, which included N-substituted or unsubstituted Boc-protected amino acids and a range of substituted aryl carboxylic acids, were selected to add chemical diversity. Screening of these compounds in competitive binding experiments with the kappa opioid receptor selective ligand [3H]U69,593 led to the discovery of a novel kappa opioid receptor selective ligand, N-?(2'S)-[3-(4-hydroxyphenyl)propanamido]-3'-methylbutyl?-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (8, RTI-5989-29). Additional structure-activity relationship studies suggested that 8 possesses lipophilic and hydrogen-bonding sites that are important to its opioid receptor potency and selectivity. These sites appear to exist predominantly within the kappa receptor since the selectivity arises from a 530-fold loss of affinity of 8 for the mu receptor and an 18-fold increase in affinity for the kappa receptor relative to the mu-selective ligand, (+)-N-[trans-4-phenyl-2-butenyl]-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (5a). The degree of selectivity observed in the radioligand binding experiments was not observed in the functional assay. According to its ability to inhibit agonist stimulated binding of [35S]GTPgammaS at all three opioid receptors, compound 8 behaves as a mu/kappa opioid receptor pure antagonist with negligible affinity for the delta receptor.     

Effects of proposed treatments for cocaine addiction on hemodynamic responsiveness to cocaine in conscious rats

Several agents may treat cocaine addiction and toxicity including bromocriptine, desipramine, GBR 12909 [1-(2-(bis(4-fluorphenyl)-methoxy)-ethyl)-4-(3-phenyl-propyl) piperazine], diazepam, buprenorphine and dizocilpine. In this study, we sought to determine whether these specific therapeutic agents alter cardiovascular responses to cocaine in conscious rats. Arterial pressure responses to cocaine (5 mg/kg, i.v.) were similar in all rats whereas cardiac output responses varied widely. In 26 of 33 rats (named vascular responders), cocaine induced a decrease in cardiac output of 8% or more. The remaining rats with little change or an increase in cardiac output were classified as mixed responders. Pretreatment with bromocriptine (0.1 mg/kg) or desipramine (1 mg/kg) increased cardiac output in mixed responders and increased systemic vascular resistance in vascular responders similar to the differential effects noted with cocaine. GBR 12909 (0.5-10 mg/kg) elicited a decrease in cardiac output at higher doses. Diazepam (0.1 and 0.5 mg/kg) had small, short-lasting effects on cardiovascular parameters. Buprenorphine (0.3 mg/kg) or the NMDA (N-methyl-D-aspartic acid) receptor antagonist, dizocilpine (0.05 mg/kg), increased arterial pressure, heart rate and cardiac output in vascular responders. Bromocriptine and desipramine prevented the difference in cardiac output responses in vascular and mixed responders by reducing the cocaine-induced decrease in cardiac output in vascular responders. Pretreatment with GBR 12909 (1 mg/kg) had little effect on cardiovascular responses to cocaine except to depress the increase in cardiac output noted in mixed responders. Buprenorphine selectively enhanced the increase in systemic vascular resistance whereas dizocilpine enhanced the pressor response. These data suggest that several treatment regimens for cocaine addiction alter the cardiovascular responses to cocaine and that dopamine D2 receptor activation may be necessary for the decrease in cardiac output noted in vascular responders.     

The vesicular monoamine transporter, in contrast to the dopamine transporter, is not altered by chronic cocaine self-administration in the rat

Although much evidence suggests that the brain dopamine transporter (DAT) is susceptible to dopaminergic regulation, only limited information is available for the vesicular monoamine transporter (VMAT2). In the present investigation, we used a chronic, unlimited-access, cocaine self-administration paradigm to determine whether brain levels of VMAT2, as estimated using [3H]dihydrotetrabenazine (DTBZ) binding, are altered by chronic exposure to a dopamine uptake blocker. Previously, we showed that striatal and nucleus accumbens DAT levels, as estimated by [3H]WIN 35,428 and [3H]GBR 12,935 binding, are altered markedly using this animal model (Wilson et al., 1994). However, in sequential sections from the same animals, [3H]DTBZ binding was normal throughout the entire rostrocaudal extent of the basal ganglia (including striatum and nucleus accumbens), cerebral cortex, and diencephalon, as well as in midbrain and brainstem monoamine cell body regions, both on the last day of cocaine access and after 3 weeks of drug withdrawal. These data provide additional evidence that VMAT2, unlike DAT, is resistant to dopaminergic regulation.     

Stereoisomers of N-[1-hydroxy-(2-phenylethyl)-3-methyl-4-piperidyl]- N-phenylpropanamide: synthesis, stereochemistry, analgesic activity, and opioid receptor binding characteristics

N-[1-(2-Hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N-phenylpropanamide (ohmefentanyl,1) is an extremely potent analgesic agent with high affinity and selectivity for opioid mu receptors. There are three chiral carbons in 1, so eight optically active isomers are possible. Respective reaction of optically active 3-methyl-N-phenyl-4 -piperidinamines (5a-d) with (R)- or (S)-styrene oxide produced eight optically active intermediates which were subsequently converted to eight optically active isomers of 1 (1a-h). The absolute configurations of 1a-h were determined by X-ray analysis of (3R,4S,2'R)-(-)-cis-1a and (3R,4R,2'S)-(-)-trans-1g. The analgesic activity (mice, ip, hot plate) revealed their extreme stereodifferences; the ED50 values of (3R,4S,2'R)-(-)-cis-1a and (3R,4S,2'S)-(+)-cis-1b, which are the most potent isomers among eight isomers, were 0.004 65 (2990 times that of morphine) and 0.001 06 mg/kg (13 100 times that of morphine), respectively, while the corresponding antipodes 1d,c were the least potent compounds among the eight isomers. In agreement with pharmacological results, both 1a,b also had the highest receptor affinity and selectivity for the opioid mu receptor. The ratio of K(i)(DPDPE)&K(i)(DAMGO) was 22 800 for 1a and 22 500 for 1b. All isomers except 1c,d strongly inhibited the electrically evoked smooth muscle contraction of GPI and MVD but not that of RVD, and the inhibitory effects could be reversed by naloxone, which indicated that these compounds were potent mu agonists in GPI and MVD. There was a good linear correlation between the analgesic potencies (ED50) and the receptor affinities (K(i)(DAMGO)) or inhibitory effects (IC50) to GPI and MVD. These results suggested that the analgesic effects of ohmefentanyl are mediated by interaction between the agents and opioid mu receptors in the central nervous system and the 3R,4S configuration at the piperidine 3- and 4-carbon atoms and the S configuration at the phenylethyl 2-carbon atom are beneficial for analgesic potency and inhibitory effects in GPI and MVD and the same for an S or R configuration at the phenylethyl 2-carbon atom besides the 3R,4S configuration for receptor mu affinity and selectivity.     

Response requirements and unit dose modify the effects of GBR 12909 on cocaine-maintained behavior.

Previous studies found that GBR 12909 can decrease cocaine-maintained responding at doses that do not affect food-maintained responding. In this study, the effects of GBR 12909 (0.3–3.0 mg/kg) were further examined by varying the response requirement and unit dose of cocaine. Rhesus monkeys earned food or cocaine under a multiple fixed-ratio (FR) schedule. The FR for food was always 30, but the FR for cocaine was varied from 10–130 and the unit dose was varied from 5.6–56.0 μg/kg per injection. Doses of GBR 12909 were tested in an ascending order, for 5 consecutive sessions each. GBR 12909 selectively decreased cocaine maintained responding in all monkeys in at least 1 condition. These effects were enhanced with large response requirements and/or small unit doses. The results demonstrate that environmental variables can influence the selectivity of GBR 12909's effects and contribute to a growing debate concerning the evaluation of potential pharmacotherapies for drug abuse. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Self-administration of GBR 12909 on a fixed ratio and progressive ratio schedule in rats

Intravenous self-administration of GBR 12909, an indirect dopamine agonist, was examined on a Fixed Ratio (FR 1) and a Progressive Ratio (PR) schedule of reinforcement in rats. Subjects were first trained to self-administer cocaine (1.5 mg/kg/inj) during daily 5 h sessions, after which GBR 12909 (0.187-1.5 mg/kg/inj) was substituted. On the FR 1 schedule, the inter-infusion interval for GBR 12909 self-administration was directly related to dose and was approximately three times longer than that established for equivalent doses of cocaine. Breaking points on the PR schedule were comparable for GBR 12909 and cocaine self-administration. The data indicate that, compared to cocaine, GBR 12909 has a longer duration of action and a similar reinforcing efficacy.