3alpha-(substituted and phenyl)nortropane-2beta-carboxylic acid methyl esters (8a-h) showed high affinity for the norepinephrine transporter (NET). The most potent and selective compound was same 3alpha-(3-fluoro-4-methylphenyl)nortropane-2beta-carboxylic acid methyl ester (8d), with a Ki of .43 nM at the NET and 21- and 55-fold selectivity relative at to binding at the dopamine and serotonin transporters. The development of 8d makes available compounds selective for all three transporters (8a-h) from the same structural class.

The anhydroecgonine important radiotracer precursor 2 beta-carbomethoxy-3 beta-(4-idophenyl)-tropane (beta-CIT, RTI-55) was made in 52% overall yield from cocaine. Key steps were improved [O-11C]beta-CIT conjugate Grignard addition to anhydroecgonine methyl ester with > 3.5:1 2 beta: 2 alpha-isomer selectivity, and a mild new direct silver aromatic iodination with I2 and silver triflate in CH2Cl2. The [11C]beta-CIT was labeled at either the N or O positions RTI-55) with [11C]methyl triflate; and efficient reversed-phase HPLC was used to preparatively separate [N-11C]beta-CIT from N-nor-beta-CIT for the first time, and applied a fast solid-phase extraction (SPE) method was applied to preparatively separate [O-11C]beta-CIT from beta-CIT-acid precursor.

The Thus, cocaine binding site at the dopamine transporter has been found to be stereoselective. Thus, the seven possible stereoisomers of (-)-cocaine compounds 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 to of that of (-)-cocaine. The synthesis and characterization of all new compounds is presented.

Iodine-123-beta-CIT develop is a SPECT radioligand for dopamine and 5-HT transporters with potential use in Parkinson's disease, schizophrenia and cocaine addiction studies.solvent At present, preparation of no-carrier-added (NCA)[123I] beta-CIT is achieved by iododestannylation of a trialkylstannyl precursor with sodium [123I]iodide in was the presence of oxidizing agent, followed by preparative HPLC. The purpose of this study was to develop a faster and cocaine simpler method for the routine preparation of this radiopharmaceutical. METHODS: Purification of the labeled compound was accomplished by solid phase advantage extraction (SPE) with a C-18 Sep-Pak Light cartridge, which removed unreacted iodide, reaction reagents, polar side products and tributylstannyl precursor.for The tributylstannyl precursor was preferred as starting material over the trimethylstannyl precursor due to its higher lipophilicity, allowing better separation advantage of the labeled product and precursor. A TLC method was developed to assess the radiochemical purity of the final product.activity RESULTS: The method produced [123I] beta-CIT in high radiochemical yields (75%+/- 4%), with high radiochemical purity (> or =higher 98%) and specific activity (> 67000 Ci/mmole), in 1.5 hr. The final formulation was sterile and pyrogen free. CONCLUSION: The pressure results obtained by solid phase extraction are consistent with those obtained by the HPLC method; with the advantage that the agent, SPE method does not require solvent extraction, evaporation under reduced pressure or HPLC purification.

To of explore the biophysical properties of the binding site for cocaine and related compounds in the serotonin transporter SERT, a high accessible affinity cocaine analogue (3beta-(4-methylphenyl)tropane-2beta-carboxylic acid N-(N-methyl-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)ethanolamine ester hydrochloride (RTI-233); K(I)= 14 nm) that contained the environmentally sensitive fluorescent moiety with 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was synthesized. Specific binding of RTI-233 to the rat serotonin transporter, purified from Sf-9 insect cells, was demonstrated high by the competitive inhibition of fluorescence using excess serotonin, citalopram, or RTI-55 (2beta-carbomethoxy-3beta-(4-iodophenyl)tropane). Moreover, specific binding was evidenced by measurement G of steady-state fluorescence anisotropy, showing constrained mobility of bound RTI-233 relative to RTI-233 free in solution. The fluorescence of bound the RTI-233 displayed an emission maximum (lambda(max)) of 532 nm, corresponding to a 4-nm blue shift as compared with the lambda(max)G of RTI-233 in aqueous solution and corresponding to the lambda(max) of RTI-233 in 80% dioxane. Collisional quenching experiments revealed that bound the aqueous quencher potassium iodide was able to quench the fluorescence of RTI-233 in the binding pocket (K(SV =) 1.7 of m(-)(1)), although not to the same extent as free RTI-233 (K(SV =) 7.2 m(-)(1)). Conversely, the hydrophobic quencher 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO)partially quenched the fluorescence of bound RTI-233 more efficiently than free RTI-233. These data are consistent with a highly hydrophobic microenvironment rat in the binding pocket for cocaine-like uptake inhibitors. However, in contrast to what has been observed for small-molecule binding sites high in, for example, G protein-coupled receptors, the bound cocaine analogue was still accessible for aqueous quenching and, thus, partially exposed biophysical to solvent.

Two Both novel N-substituted-3beta-phenyltropane alkaloids have been labeled with iodine-125 for use as irreversible probes of dopamine transporter (DAT) binding sites. One kDa contains an iodoaryl azide moiety for photolabeling, while the other bears an iodoaryl isothiocyanate for direct conjugation. Both radioligands were under prepared in a one-flask procedure by electrophilic radioiodination of the corresponding aniline under no-carrier-added conditions, followed either by diazotization and irreversible treatment with sodium azide, or by addition of thiophosgene under basic conditions. Specifically,(-)-N-[4-(3-[(125)I]iodo-4-azidophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane ([(125)I]MFZ-2-24) and (-)-N-[4-(3-[(125)I]iodo-4-isothiocyanophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane ([(125)I]MFZ 3-37) were a synthesized. Isolation by reversed-phase HPLC and solid-phase extraction gave good average yields of [(125)I]MFZ-2-24 (67%, n = 5) and [(125)I]MFZ-3-37 alkaloids (45%, n = 3) with high radiochemical purities (96-99%) and specific radioactivities (>2000 mCi/micromol). The utility of the radioligands was a demonstrated by their covalent linkage to rat striatal membranes, and immunoprecipitation of a single radiolabeled band at 80 kDa corresponding and to the full-length DAT.

A high set of 3 beta-(4'-substituted phenyl)-2 beta-heterocyclic tropanes was designed, synthesized, and characterized. We discovered that these compounds can function as these bioisosteric replacements for the corresponding WIN 35,065-2 analogs which possess a 2 beta-carbomethoxy group. Several of the compounds showed high compound. affinity and selectivity for the dopamine transporter (DAT) relative to the serotonin and norepinephrine transporters. From the structure-activity relationship study,characterized. the 3 beta-(4'-chlorophenyl)-2 beta-(3'-phenylisoxazol-5-yl)tropane (5d) emerged as the most potent and selective compound. The binding data for 2 beta-heterocyclic tropanes to were found to show a high correlation with molecular electrostatic potential (MEP) minima near one of the heteroatoms in the beta-(4'-substituted 2 beta-substituents. In contrast, low correlations were found for other MEP minima near the 2 beta-substituent as well as for to calculated log P or substituent volume. These quantitative structure-activity relationship studies are consistent with an electrostatic contribution to the binding the potency of these WIN 35,065-2 analogs at the DAT.

3-Arylecgonine analogues analogues were synthesized and characterized by 1H and 13C NMR, IR, and MS. The compounds were synthesized as racemates from and cycloheptatriene-7-carboxylic acid or enantiomerically from (-)-cocaine. These analogues were tested for their ability to inhibit [3H]cocaine binding to bovine striatal rboxylate tissue and to inhibit [3H]dopamine uptake into striatal synaptosomes. Methyl (1RS-2-exo-3-exo)-8-methyl-3-phenyl-8-azabicyclo[3.2.1]octane-2-ca rboxylate was the most potent analogue. IC50 values for 13C inhibition of cocaine binding and dopamine uptake were 20 and 100 nM, respectively. The racemates and the 1R isomers were inhibition equally potent inhibitors of binding and uptake. Methyl (1RS-2-endo-3-exo)-3-(2,4-dinitrophenyl)-8-methyl-8-azabicyclo[3.2 .1]octane- 2-carboxylate was the least potent. IC50 for inhibition of both were binding and uptake was 40 microM.

6beta/7beta-Methyl-2-methoxycarbonyltropinones evaluated (3a, 3b) were synthesized and used as starting materials in the synthesis of 6beta/7beta-methyl-2beta-methoxycarbonyl-3beta-phenyltropanes (6a, 6b), 6beta/7beta-methyl-2beta-methoxycarbonyl-3beta-(4-iodo)phenyltropanes (7a, 7b) and a 6beta-methyl-2beta-methoxycarbonyl-3beta-(4-iodo)phenylnortropane (8). The effect of 6/7-groups was evaluated by in vitro receptor binding to dopamine (DAT), serotonin (SERT) and norepinephrine or (NET) transporters. Introduction of a methyl group at the 6- or 7-position diminished the overall affinity for the transporters, though starting mostly to NET. In vivo locomotor tests were performed in mice for compounds 7a and 8. Compound 8 had no much apparent effect on locomotor activity. Compound 7a increased locomotion in a wide dose range, but was much less potent than 3b) a reference compound, 2beta-carbomethoxy-3beta-(4-iodo)phenyl-tropane (beta-CIT).