Synthesis and pharmacology of site-specific cocaine abuse treatment agents: 2-substituted-6-amino-5-phenylbicyclo[2.2.2]octanes

A series of 2-substituted-6-amino-5-phenylbicyclo[2.2.2]octanes was synthesized and tested for inhibitor potency in [(3)H]WIN 35,428 (WIN) binding at the dopamine (DA) transporter and [(3)H]DA uptake assays. To demonstrate transporter selectivity for the compounds, inhibitor potency was also tested using [(3)H]nisoxetine and [(3)H]paroxetine binding assays for the norepinephrine (NE) and serotonin (5-HT) transporters, respectively. Synthesis was accomplished by bisannulation of the enamine derived from phenylacetaldehyde and dimethylamine with 2-cyclohexenone to give a mixture of endo- and exo-trans-6-amino-5-phenylbicyclo[2.2. 2]octan-2-ones. The separated ketones were reduced to the four diastereomeric alcohols which were converted to acetyl and benzoyl esters. The ketones, alcohols, and acetyl esters generally have low affinity for the three transporters and do not effectively inhibit the uptake of [(3)H]DA. In all cases, the benzoates show significantly greater inhibition of WIN binding compared to the corresponding ketones, alcohols, or acetate esters. One compound, (1R/S,4R/S)-6R/S-(N,N-dimethylamino)-5R/S-phenylbicyclo[2.2. 2]oct-2S/R-yl benzoate, is almost as potent as cocaine in binding to the DA transporter (IC(50) = 270 nM versus 159 nM for cocaine). The C-2 epimer, (1R/S,4R/S)-6R/S-(N, N-dimethylamino)-5R/S-phenylbicyclo[2.2.2]oct-2R/S-yl benzoate, was selective and potent in binding to the 5-HT transporter (IC(50) = 53 nM versus 1050 nM for cocaine) as compared to the DA transporter (IC(50) = 358 nM). A preliminary molecular modeling study of the benzoyl esters indicates that their relative potencies in the WIN binding assay are not correlated to the nitrogen to benzoate phenyl (centroid) distance or to the deviation of the nitrogen from the plane defined by the benzoate ring.     

Synthesis and pharmacology of site specific cocaine abuse treatment agents: 8-substituted isotropane (3-azabicyclo[3.2.1]octane) dopamine uptake inhibitors

A series of 8-substituted-3-azabicyclo[3.2.1]octanes (isotropanes) were synthesized and tested for inhibitor potency using [(3)H]WIN 35,428 binding at the dopamine (DA) transporter, [(3)H]citalopram binding at the serotonin (5-HT) transporter, and [(3)H]DA uptake assays. The synthesis started with a Mannich condensation of cyclopentanone, benzylamine, and fomaldehyde to afford N-benzyl-3-azabicyclo[3.2.1]octan-8-one (6). The 8-phenyl group was introduced by Grignard addition to ketone 6 or nucleophilic displacement via a triflate of the corresponding alcohol 7a. The 8beta-phenyl-8alpha-alcohols from Grignard addition generally have low affinity for the two transporters and do not effectively inhibit the uptake of [(3)H]DA. The 8beta-phenyl compound (14) without the hydroxyl group at C-8 was much more potent (22-fold) for [(3)H]WIN 35,428 binding inhibition than the corresponding 8beta-phenyl-8alpha-hydroxy compound (7a). The 8alpha-phenyl compound 8a was almost as potent as cocaine in binding to the DA transporter (IC(50) = 234 nM vs 159 nM for cocaine), whereas the C-8 epimer, compound 14, was somewhat less potent (IC(50) = 785 nM). The lower potency of 14 (beta-orientation of 8-phenyl group) as compared to 8a (alpha-orientation) was unexpected, based on modeling studies comparing the new compounds to WIN 35,065-2, an analogue of cocaine. The benzhydryl ethers at C-8 (17), analogous to the benztropines, had better selectivity than the corresponding phenyl compounds, 8a and 14, for the DA transporter as compared to the 5-HT transporter. The isotropane and benzisotropine analogues seem to bind in a manner that is more similar to that of the benztropine compounds 5 rather than those of cocaine and WIN 35,065-2.     

Isothiocyanate derivatives of cocaine: irreversible inhibition of ligand binding at the dopamine transporter

Isothiocyanate derivatives of (-)-cocaine were prepared and tested for inhibitory potency at the cocaine receptor in rat striatal membranes. Coincubation with m-isothiocyanatobenzoylecgonine methyl ester (m-ISOCOC), p-isothiocyanatobenzoylecgonine methyl ester (p-ISOCOC), and 3 beta-(4-isothiocyanatophenyl)tropane-2-carboxylic acid methyl ester (ISOWIN) resulted in inhibition of [3H]WIN 35,428 binding, but the compounds were about 10-fold weaker than (-)-cocaine. However, p-ISOCOC was approximately 3-fold more potent than metaphit, an isothiocyanate derivative of phencyclidine. p-ISOCOC was equipotent at the serotonin transporter but was much less potent at the norepinephrine transporter and was inactive at the D2 dopamine receptor at 1000 microM concentration. The IC50 value for m-ISOCOC and p-ISOCOC varied with tissue concentration, suggesting irreversible inhibition of binding. Preincubation with m-ISOCOC and p-ISOCOC resulted in inhibition of [3H]WIN 35,428 binding that could not be removed by washing of the membranes; in contrast, preincubation with (-)-cocaine caused inhibition that was readily removed by washing. Preincubation with 1 microM concentrations of p-ISOCOC resulted in a large reduction in Bmax of the high affinity binding site for [3H]WIN 35,428. Preincubation with 100 microM p-ISOCOC eliminated the high affinity site and apparently reduced the affinity at the low affinity site. Coincubation of 10 microM p-ISOCOC with 100 microM cocaine prevented the total loss of [3H]WIN 35,428 binding. The uptake of [3H]dopamine was inhibited by p-ISOCOC with an IC50 comparable to that of cocaine. Additionally, preincubation of rat striatal synaptosomes with 10 microM p-ISOCOC reduced the Vmax of [3H]dopamine uptake after washing. These data suggest that m-ISOCOC and p-ISOCOC are useful irreversible acylators of (-)-cocaine binding sites at the dopamine transporter.     

Binding domains for blockers and substrates on the dopamine transporter in rat striatal membranes studied by protection against N-ethylmaleimide-induced reduction of [3H]WIN 35,428 binding

Binding sites for 2β-carbomethoxy-3β-(4-fluorophenyl)[³H]tropane ([³H]WIN 35,428) on rat striatal membranes were alkylated with N-ethylmaleimide (NEM), and the protective potency was measured of the blockers cocaine, N[1-(2-benzo[b]thiophenyl) cyclohexyl]piperidine (BTCP), benztropine, WIN 35,428, and nomifensine, and of the substrates dopamine, norepinephrine, S(+)-amphetamine, tyramine, and metaraminol. In general, the protective potency was lower (at least 3 times) than the potency in inhibiting [³H]WIN 35,428 binding with the compounds present under the same experimental conditions used for the NEM-induced alkylation. However, the disparity was substantially greater for all substrates tested (10- to 93-fold) than for the blockers (2- to 6-fold), especially cocaine and BTCP (3-fold). [³H]WIN 35,428 binding was best described by a 1-site model under the present conditions. The results are discussed in terms of models involving blocker-induced conformational changes and overlapping nonidentical binding domains for blockers and substrates. Received: 14 May 1996 / Accepted: 11 September 1996     

New, potent cocaine analogs: ligand binding and transport studies in rat striatum

Two potent cocaine analogs have been developed that have the highest known affinities for the cocaine binding site in rat striatum. Both 3 beta-(4-chlorophenyl)- (RTI-COC-31) and 3 beta-(4-methylphenyl)-tropane-2-carboxylic acid methyl ester (RTI-COC-32) compete for [3H]WIN 35,428 and [3H]mazindol binding with a IC50 that is 100 times more potent than that of (-) cocaine. Additionally, these compounds inhibit [3H]dopamine uptake with a similar, high potency. These results may lead to the development of high affinity probes for the cocaine binding site.     

Expansion of structure-activity studies of piperidine analogues of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (GBR 12935) compounds by altering substitutions in the N-benzyl moiety and behavioral pharmacology of selected molecules.

A series of substituted N-benzyl analogues of the dopamine transporter (DAT) specific compound, 4-[2-(diphenylmethoxy)ethyl]-1-benzylpiperidine were synthesized and biologically characterized. Different 4'-alkyl, 4'-alkenyl, and 4'-alkynyl substituents were introduced in the phenyl ring of the benzyl moiety along with the replacement of the same phenyl ring by the isomeric alpha- and beta-naphthyl groups. Different polar substitutions at the 3'- and 4'-position were also introduced. Novel compounds were tested for their binding affinity at the dopamine, serotonin, and norepinephrine transporter systems in the brain by competing for [(3)H]WIN 35 428, [(3)H]citalopram, and [(3)H]nisoxetine, respectively. Selected compounds were also evaluated for their activity in inhibiting the uptake of [(3)H]dopamine. Binding results demonstrated that alkenyl and alkynyl substitutions at the 4'-position produced potent compounds in which compound 6 with a vinyl substitution was the most potent. In vivo evaluation of three selected compounds indicated that despite their high potency at the DAT, these compounds stimulated locomotor activity (LMA) less than cocaine when tested across similar dose ranges. In a drug discrimination study procedure, none of these three compounds generalized from cocaine in mice trained to discriminate 10 mg/kg cocaine from vehicle. In a 4 h time course LMA experiment, one of our previous lead piperidine derivatives (1a) showed considerable prolonged action. Thus, in this report, we describe a structure-activity relationship study of novel piperidine analogues assessed by both in vitro transporter assays and in vivo behavioral activity measurements.     

Probes for the cocaine receptor. Potentially irreversible ligands for the dopamine transporter.

Several potentially irreversible ligands (i.e., wash-resistant binding inhibitors) for the cocaine receptor site on the dopamine transporter, derived from (-)-cocaine or 3 beta-phenyltropan-2 beta-carboxylic acid methyl ester (WIN 35,065-2), were prepared and shown to produce wash-resistant inhibition of [3H]-3 beta-(p-fluorophenyl)tropan-2 beta-carboxylic acid methyl ester ([3H]WIN 35,428) binding. All the compounds prepared had the same absolute configuration as cocaine; they include analogues possessing chemically reactive groups such as the isothiocyanato and bromoacetamido as well as photoactive azido groups. The potentially irreversible ligands, as well as all the intermediates prepared in this study, were evaluated for their ability to inhibit the binding of [3H]WIN 35,428 in coincubation experiments. Of the potentially irreversible ligands, 3 beta-(p-chlorophenyl)tropan-2 beta-carboxylic acid 2-[p-(bromoacetamido)phenyl]ethyl ester (6c) had the highest apparent potency. The potentially irreversible ligands were also preincubated, and inhibition of [3H]WIN 35,428 binding was determined both before and after washing the ligand-exposed tissues. The most effective ligands in this regard were 3 beta-(3-iodo-4-azidophenyl)tropan-2 beta-carboxylic acid methyl ester (5) and 3 beta-(p-chlorophenyl)tropan-2 beta-carboxylic acid 2-(3-iodo-4-azidophenyl)ethyl ester (6d). The structure-activity relationships of these data are discussed.     

Synthesis and receptor binding of N-substituted tropane derivatives. High-affinity ligands for the cocaine receptor

The synthesis and pharmacological characterization of a series of N-substituted 3-(4-fluorophenyl)tropane derivatives is reported. The compounds displayed binding characteristics that paralleled those of cocaine, and several had substantially higher affinity at cocaine recognition sites. Conjugate addition of 4-fluorophenyl magnesium bromide to anhydroecgonine methyl ester gave 2 beta-(carbomethoxy)-3 beta-(4-fluorophenyl)tropane (4a, designated CFT, also known as WIN 35,428) after flash chromatography. N demethylation of 4a was effected by Zn/HOAc reduction of the corresponding 2,2,2-trichloroethyl carbamate to give 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)nortropane (5), which was alkylated with allyl bromide to afford the N-allyl analogue, 6. The N-propyl analogue, 7, was prepared by catalytic reduction (Pd/C) of 6. The most potent analogue, 4a, was tritiated at a specific activity of 81.3 Ci/mmol. [3H]4a bound rapidly and reversibly to caudate putamen membranes; the two-component binding curve typical of cocaine analogues was observed. Equilibrium was achieved within 2 h and was stable for at least 4 h. High- and low-affinity Kd values observed for [3H]4a (4.7 and 60 nM, respectively) were more than 4 times lower than those for [3H]cocaine, and the density of binding sites (Bmax = 50 pmol/g, high, and 290 pmol/g, low) for the two drugs were comparable. Nonspecific binding of [3H]4a was 5-10% of total binding.     

Cocaine receptors labeled by [3H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane

The potent cocaine analog 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)-tropane (CFT, also designated WIN 35,428) was tritiated and evaluated as a molecular probe for cocaine receptors in caudate putamen membranes of cynomolgus monkeys. Kinetic, saturation, and competition experiments indicated that [3H]CFT, like [3H]cocaine, bound to at least two components. Association and dissociation of the radioligand at 0-4 degrees occurred in two phases; the t 1/2 for dissociation of the fast and slow components was 2.5 and 23 min, respectively. Saturation analysis revealed high and low affinity binding components with affinities (Kd) of 4.7 +/- 1.2 and 60 +/- 12 nM (means +/- SE) and densities (Bmax) of 50 +/- 18 and 290 +/- 20 pmol/g of tissue, respectively. [3H]CFT was displaced stereoselectively by the enantiomers of cocaine and by the diastereoisomers of the phenyltropane analog of cocaine. Most congeners displaced [3H]CFT fully, with shallow competition curves (nH, 0.69-0.81). In contrast, several monoamine uptake inhibitors structurally unrelated to cocaine (GBR 12909, Lu 19-005, and mazindol) displaced a maximum of about 90% specifically bound [3H]CFT, with steeper competition curves (nH, 0.89-1.3), suggesting that these drugs bind to a subpopulation of [3H]CFT-labeled sites. The rank order of potency observed in the present study is identical to the rank order of potency at binding sites labeled by [3H]cocaine: Lu 19-005 greater than mazindol greater than CFT greater than GBR 12909 greater than (-)-cocaine greater than bupropion greater than WIN 35,140 greater than (+)-cocaine. Moreover, there is a high positive correlation (r, 0.99, p less than 0.001) between the affinities of drugs at sites labeled by [3H]CFT and [3H]cocaine. The results show that [3H]CFT and [3H]cocaine bind to a similar spectrum of sites in monkey caudate putamen. Because of its higher affinity and slower dissociation rate, [3H]CFT appears to be a superior radioligand probe for these sites.     

Synthesis and biological evaluation at nicotinic acetylcholine receptors of N-arylalkyl- and N-aryl-7-azabicyclo[2.2.1]heptanes

A new series of N-arylalkyl-substituted 7-azabicyclo[2.2.1]heptanes and N-aryl-substituted 7-azabicyclo[2.2.1]heptanes were synthesized and evaluated as potential ligands for neuronal nicotinic acetylcholine receptors. The in vitro binding affinities (K(i)) of the 7-azabicyclo[2.2.1]heptane derivatives were measured by inhibition of [(3)H]cytisine binding to rat brain tissue. The most potent ligand of the series was found to be N-(3-pyridylmethyl)-7-azabicyclo[2.2.1]heptane (5b, K(i) = 98 nM). The chloro analogue (5a, K(i) = 245 nM) 5a and epibatidine (1) produced dose-dependent analgesia in both hotplate and tail-flick tests when administered subcutaneously. However, when compounds 1 and 5a,b were administered intrathecally, all produced analgesia in the tail-flick test but only 5a produced analgesia in the hotplate test.     

Structure-activity relationship studies of 4-[2-(diphenylmethoxy)ethyl]-1-benzylpiperidine derivatives and their N-analogues: evaluation of O-and N-analogues and their binding to monoamine transporters

In our effort to develop a pharmacotherapy for the treatment of cocaine addiction, we embarked on synthesizing novel molecules targeting the dopamine transporter (DAT) molecule in the brain as DAT has been implicated strongly in the reinforcing effect of cocaine. Our previously developed DAT-selective piperidine analogue, 4-[2-(diphenylmethoxy)ethyl]-1-benzylpiperidine, was the basis for our current structure-activity relationship (SAR) studies exploring the significance of the contribution of the benzhydryl O- and N-atoms in these molecules in interacting with the DAT. Thus, we replaced the benzhydryl O-atom with an N-atom, altered the location of the benzhydryl N-atom to an adjacent position, and in one other occasion converted the benzhydryl O-ether linkage into an oxime-type derivative. Furthermore, we also evaluated the important contribution of the piperidine N-atom to binding by altering its pK(a) value chemically. Novel analogues were tested for potency in inhibiting [3H]WIN 35,428, [3H]citalopram, and [3H]nisoxetine binding at the DAT, serotonin transporter (SERT), and norepinepherine transporter (NET). [3H]DA was used to measure DA reuptake inhibition. The results indicated that the benzhydryl O- and N-atoms are exchangeable for the most part. On the other hand, an enhanced interaction with the SERT was observed when the benzhydryl N-atom moved to an adjacent position (21a; DAT (IC(50)) = 19.7, SERT (IC(50)) = 137 nM, NET (IC(50)) = 1111 nM). In either cases, further alkylation of the N-atom reduced the activity for the transporter. The presence of a powerful electron-withdrawing cyano group in compound 5d expectedly produced the most potent and selective ligand for the DAT (DAT (IC(50)) = 3.7 nM, DAT/SERT = 615). Selected compounds were further analyzed in the dopamine reuptake inhibition assay. Preliminary behavioral assessment of some of the selected compounds in mice indicated that these compounds are much less stimulating when compared with cocaine at comparable doses. In drug-discrimination studies these selected compounds incompletely generalized from the cocaine stimulus in mice trained to discriminate 10 mg/kg cocaine from vehicle.     

Cocaine displaces [3H]WIN 35,428 binding to dopamine uptake sites in vivo more rapidly than mazindol or GBR 12909

Previous studies have shown that [3H] WIN 35,428 binds preferentially to striatal cocaine receptors at the dopamine transporter after in vivo injection. In vivo binding competition studies were carried out to assess the relative rates of entry and occupancy of cocaine receptors by (-)-cocaine, mazindol and GBR 12909. After i.v. injection, (-)-cocaine occupied receptors relatively more rapidly than GBR 12,909 while mazindol was the slowest.     

Chronic administration of the selective dopamine uptake inhibitor GBR 12909, but not cocaine, produces marked decreases in dopamine transporter density

The chronic continuous infusion of cocaine produces partial behavioral tolerance to cocaine and tolerance to the inhibition of dopamine uptake by cocaine, without changing dopamine transporter binding. In order to examine more closely the dopaminergic contribution to this effect, the selective dopamine uptake inhibitor GBR 12909 (30mg/kg/day), cocaine (50mg/kg/day), or vehicle, were continuously infused via osmotic minipump, and their effects on the dopamine transporter examined. Drug and vehicle pumps were implanted into male Sprague-Dawley rats and removed after seven days. [³H]WIN 35,428 binding and [³H]dopamine uptake were measured in caudate putamen and nucleus accumbens at varying intervals after pump removal. The B _max for [³H]WIN 35,428 binding was decreased by approximately 75% in the caudate putamen and by 40% in the nucleus accumbens of GBR 12909-treated rats both 1 and 4 days after pump removal, and was still significantly decreased after 10 days, but had returned to normal by 20 days post-treatment. In contrast, cocaine did not significantly alter [³H]WIN 35,428 binding. GBR 12909 produced both tolerance to the inhibition of [³H]dopamine uptake by cocaine, and a decrease in total uptake of dopamine, in the caudate putamen, with no change in the nucleus accumbens. The persistent reduction of [³H]WIN 35,428 binding following continuous GBR 12909 does not appear to result from residual drug binding. These findings suggest that GBR 12909 and cocaine may bind to and regulate the dopamine transporter in different ways.     

Synthesis and serotonin binding site studies of some conformationally restricted indolylethylamine analogues based on 2-amino-3-(3'-indolyl)bicyclo[2.2.2]octane

The bicycloannulation reaction between cyclohexenone and indolyl enamines yields trans-3-(cyclic amino)-2-(3'-indolyl)bicyclo[2.2.2]octan-5-ones, and these adducts are conformationally restricted analogues of indolylethylamine (tryptamine) which exhibit structure-dependent affinity for the serotonin 5HT2 and 5HT1a receptors. The stereochemistry of the isomeric endo and exo adducts obtained is assigned from the 1H NMR spectra of the specifically deuterated alkenes prepared from the ketones by the Bamford-Stevens reaction. Molecular mechanics calculations indicate that the conformational flexibility of the amino and indolyl groups is restricted through van der Waals interactions with the bridges of the bicyclic unit. These compounds inhibit the binding of [3H]ketanserin to 5HT2 sites in mouse cerebrocortical membranes, and the binding of [3H]-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OH-DPAT) to 5HT1a sites in mouse hippocampal membranes. The endo compounds are the most potent, and molecular mechanics calculations indicate that these isomers have a less bulky bicyclo bridge proximate to the amine group and more conformational freedom about the C alpha-C beta-N(+)-H dihedral angle (tau 3). In the 5HT2 assay, endo-trans-3-(N-piperidinyl)-2-(3'-indolyl)bicyclo[2.2.2]octan-5-one (10a) is the most potent, and endo-trans-3-(N-pyrrolidinyl)-2-(3'-indolyl)bicyclo[2.2.2]oct-5-ene (12a) is the most potent in the 5HT1a assay. A phenyl-substituted adduct shows the least affinity in these two assays. These data provide insight into the structural differences between the 5HT1a and 5HT2 receptor sites.     

Synthesis and ligand binding of cocaine isomers at the cocaine receptor

The cocaine binding site at the dopamine transporter has been found to be stereoselective. Thus, the seven possible stereoisomers of (-)-cocaine have been synthesized and found to inhibit [3H]-2 beta-carbomethoxy-3 beta-(4-fluoro-phenyl)tropane [( 3H]WIN 35,428) with potencies ranging from 1/60 to 1/600 of that of (-)-cocaine. The synthesis and characterization of all new compounds is presented.     

Benzo- and cyclohexanomazindol analogues as potential inhibitors of the cocaine binding site at the dopamine transporter

A series of mazindol (1), homomazindol (2), and bishomomazindol (3) derivatives with a benzo or cyclohexano ring fused at various sites were prepared as part of an SAR study to determine the effect of increased aliphatic and aromatic lipophilicity on selected in vitro assays used to identify potential cocaine-like and cocaine antagonism activity. Very good (IC(50) = 2-3 nM) inhibition of [(3)H] WIN 35,428 and [(125)I] RTI-55 binding on rat or guinea pig striatal membranes and HEK cells expressing cDNA for the human dopamine transporter (HEK-hDAT) was shown by the 8,9-benzomazindol 25 and 9,10-benzohomomazindol 28. All new compounds were weaker inhibitors of [(3)H] DA uptake in HEK-hDAT cells than 1 and 2. No improvement in the binding selectivity ratio (SERT/DAT and NET/DAT) was found when compared to 2. Compounds 25and 28 showed a considerable increase versus 1 in uptake/binding discrimination ratios at the DAT (311.0 and 182.1 vs 0.9), SERT (33.6 and 127.3 vs 1.9), and NET (7.3 and 10.0 vs 0.3).     

Translocation of dopamine and binding of WIN 35,428 measured under identical conditions in cells expressing the cloned human dopamine transporter

Translocation of [³H]dopamine and binding of [³H]WIN 35,428 were measured in intact C6 glioma cells expressing the cloned human dopamine transporter (hDAT) under identical conditions of assay buffer (phosphate-Krebs) and temperature (25°C) with uptake at initial velocity and binding at equilibrium. In the intact cells, [³H]dopamine uptake was a one-component process; in contrast, [³H]WIN 35,428 binding included both a high-affinity component, inhibitable by micromolar concentrations of dopamine, and a low-affinity component only partially inhibited by millimolar concentrations of dopamine. Binding (high-affinity) over uptake Ki ratios were on the average 2.3 for the inhibitors WIN 35,428, cocaine, GBR 12909, and BTCP. The potency of dopamine in inhibiting its own translocation was close to that in inhibiting [³H]WIN 35,428 binding consonant with a more rapid reorientation step of the DAT in the C6-hDAT system than in rat striatal synaptosomes. The similarity in turnover values of the DAT estimated in the current experiments with the C6-hDAT system and in our previous study on rat striatal synaptosomes, performed under comparable conditions, suggest that all DAT's inserted into the C6 cell membrane are functionally active.     

Novel synthesis of convulsants with a unique ring system: dihydro-1,4-ethano-1H,3H-thieno[3,4-c]thiophene-3,6(4H)-dione

Dihydro-1,4-ethano-1H,3H-thieno[3,4-c]thiophene-3,6(4H)-dione (1) was obtained in 28% yield by an unusual rearrangement reaction on addition of 4 equiv of BBr3 to O,O-diethyl endo,exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarbothioate. The 5-methyl, 1,4-dimethyl, 5-n-butyl, and 5,6-benzo analogues of 1 were synthesized by similar procedures. Dihydro-1,4-ethano-1H,3H-furo[3,4c]furan-3,6(4H)-dione (6) and its 1,4-dimethyl analogue were obtained by acid-catalyzed cyclization of the appropriate endo,exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acids. Lawesson's reagent was used to convert 1 and 6 to their monothiono and dithiono derivatives. The exo,exo isomers of several of the starting oxabicycloheptanedicarboxylic acids and their anhydrides are potent vesicants and hepatotoxins interacting with the cantharidin binding site of liver cytosol whereas some of the new bis-thiololactones and their thiono analogues are convulsants that probably act in the brain as GABAA receptor antagonists.     

Binding of uptake blockers to the neuronal dopamine transporter: further investigation about cationic and anionic requirements

Effects of ions on the binding of uptake blockers to the rat dopamine transporter (rDAT) labelled with [3H]WIN 35,428 [2beta-carbomethoxy-3beta-(4-fluorophenyl)-[3H] tropane] and [3H]mazindol were studied at 20 degrees C. [3H]WIN 35,428 binding increased with Na+ concentrations of up to 10-60 mM and decreased at higher concentrations. At pH 7.4, incubation media containing NaCl and/or Na2HPO4/NaH2PO4 were less stimulant than an NaHCO3/NaH2PO4 medium and they shifted maximal binding values to higher ionic concentrations. In an NaHCO3/NaH2PO4-buffered medium, Na+ concentrations >10 mM decreased the binding of 0.2 nM [3H]WIN 35,428, but an increase of the radioligand concentration shifted this decrease to the right. [3H]Mazindol binding was stimulated by Na+ concentrations < or =10 mM and was rather unaffected at higher concentrations. The inhibition of [3H]WIN 35,428 binding produced by 130 mM Na+ was independent of the nature of the anion; in contrast, isothionate and H2PO4-/HCO3 produced a more pronounced inhibition of the [3H]mazindol binding than Cl- and Br-, whereas I- tended to be a stimulant. Ca2+ and Mg2+ more potently inhibited the [3H]WIN 35,428 binding than K+. All these cations recognize a site which is not mutually exclusive with that of the radioligand since they induced the dissociation of the [3H]WIN 35,428-rDAT complex, an effect which was reduced (K+) or modified (Ca2+) when the Na+ concentration was increased. This site is likely to be the Na+ site by which low Na+ concentrations allosterically stimulate the uptake blocker binding. However, the intensity of the cation-induced dissociations was moderate and the main component of the binding inhibition that these cations produced results from the occupancy of a cation site, mutually exclusive with that of the radioligand. Thus, the WIN 35,428 binding inhibition produced by Ca2+, K+ and Na+ was competitive, and Na+ reduced the inhibitory potency of Ca2+ and K+. This reduction was more intense for Ca2+ and Mg2+ than for K+, suggesting that occupancy of the cation site by a divalent cation activated a strong negative allosteric interaction between this site and the Na+ site. Decrease in the Na+ concentration from 10 mM to 5 mM, or replacement of 5 mM HCO3-/H2PO4- by an equimolar concentration of isethionate or Cl- did not modify [3H]WIN 35,428 binding dissociation. Level(s) at which anions stimulate and inhibit the binding of uptake blockers remain uncertain and could be specific for each radioligand.     

Derivatives of (-)-7-methyl-2-(5-(pyridinyl)pyridin-3-yl)-7-azabicyclo[2.2.1]heptane are potential ligands for positron emission tomography imaging of extrathalamic nicotinic acetylcholine receptors

A series of novel racemic 7-methyl-2-(5-(pyridinyl)pyridin-3-yl)-7-azabicyclo[2.2.1]heptane derivatives with picomolar in vitro binding affinity at nicotinic acetylcholine receptors (nAChRs) were synthesized and their enantiomers were resolved by semipreparative chiral HPLC. The (-)-enantiomers showed substantially greater in vitro inhibition binding affinity than the corresponding (+)-enantiomers. The compounds with best binding affinities have been radiolabeled with positron emitting isotopes 11C and 18F as potential radioligands for positron emission tomography imaging of the nAChR. In vivo enantioselectivity of the radiolabeled (-)-7-methyl-2-(5-(pyridinyl)pyridin-3-yl)-7-azabicyclo[2.2.1]heptane derivatives was observed in biodistribution studies in rodents and baboon. One of the radiolabeled compounds, (-)-7-methyl-2-exo-[3'-(2-[18F]fluoropyridin-5-yl))-5'-pyridinyl]-7-azabicyclo[2.2.1]heptane, exhibited good properties as a first practical PET radioligand for imaging of extrathalamic nAChR in baboon brain and holds promise for further investigation for human studies.