3alpha-(substituted phenyl)nortropane-2beta-carboxylic acid methyl esters (8a-h) showed high affinity for the norepinephrine transporter (NET). The most potent and selective compound was 3alpha-(3-fluoro-4-methylphenyl)nortropane-2beta-carboxylic acid methyl ester (8d), with a Ki of 0.43 nM at the NET and 21- and 55-fold selectivity relative to binding at the dopamine and serotonin transporters. The development of 8d makes available compounds selective for all three transporters from the same structural class.

[11C]Methyl triflate was compared with [11C]methyl iodide as a labelled precursor in the synthesis of some commonly used PET radioligands, L-[11C]deprenyl,[11C]m-hydroxyephedrine (MHED),[11C] beta-CIT,[11C] beta-CFT and [11C]SCH 39166 which have been prepared previously in comparatively low yields from [11C]methyl iodide. A new dopamine reuptake radioligand,[11C] alpha-CIT, was also prepared. The results demonstrate that higher yields are obtained with shorter reaction times, lower reaction temperatures and smaller amounts of precursors with [11C]methyl triflate.

The 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 conjugate Grignard addition to anhydroecgonine methyl ester with > 3.5:1 2 beta: 2 alpha-isomer selectivity, and a mild new direct aromatic iodination with I2 and silver triflate in CH2Cl2. The [11C]beta-CIT was labeled at either the N or O positions 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 a fast solid-phase extraction (SPE) method was applied to preparatively separate [O-11C]beta-CIT from beta-CIT-acid precursor.

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.

One aspect of immunopharmacotherapy for cocaine abuse involves the use of a catalytic monoclonal antibody (mAb) to degrade cocaine via hydrolysis of the benzoate ester. A cocaine benzoylthio ester analogue provides a means to implement high-throughput selection strategies to potentially isolate mAbs with high activity. The required analogue was synthesized starting from (-)-cocaine hydrochloride and possessed the cocaine absolute configuration. Key points in the preparation were the introduction of the sulfur atom at C-3 via a bromomagnesium thiolate addition to the exo face of anhydroecgonine, separation of C-2 diastereomers, recycling of a C-2 thio ester byproduct, and formation of the necessary C-2 methyl and C-3 benzoylthio esters. Effects resulting from the lower electronegativity and greater hydrophobicity of sulfur compared to oxygen were observed. These characteristics could result in interesting drug properties. Furthermore, the analogue was found to be a substrate for catalytic mAbs that hydrolyze cocaine as monitored by HPLC and also spectrophotometry by coupling cleavage of the benzoylthio ester to the disulfide exchange with Ellman's reagent. Screening antibody libraries with the new cocaine analogue using the spectroscopic assay provides an avenue for the high-throughput identification of catalysts that efficiently breakdown cocaine.

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

To explore the biophysical properties of the binding site for cocaine and related compounds in the serotonin transporter SERT, a high 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 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 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 of steady-state fluorescence anisotropy, showing constrained mobility of bound RTI-233 relative to RTI-233 free in solution. The fluorescence of bound RTI-233 displayed an emission maximum (lambda(max)) of 532 nm, corresponding to a 4-nm blue shift as compared with the lambda(max) of RTI-233 in aqueous solution and corresponding to the lambda(max) of RTI-233 in 80% dioxane. Collisional quenching experiments revealed that the aqueous quencher potassium iodide was able to quench the fluorescence of RTI-233 in the binding pocket (K(SV =) 1.7 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) quenched the fluorescence of bound RTI-233 more efficiently than free RTI-233. These data are consistent with a highly hydrophobic microenvironment in the binding pocket for cocaine-like uptake inhibitors. However, in contrast to what has been observed for small-molecule binding sites in, for example, G protein-coupled receptors, the bound cocaine analogue was still accessible for aqueous quenching and, thus, partially exposed to solvent.

Two fluorinated congeners of cocaine, 2'-fluoroethyl (1R-2-exo-3-exo)-8-methyl-3-(4-methylphenyl)-8-azabicyclo[3.2.1]octane- 2-carboxylate (FETT) and its 4-chlorophenyl analogue (FECT) were synthesized. Radiolabelling with 18F was achieved by O-[18F]fluoroalkylation of the corresponding carboxylic acid salts with distilled 2-[18F]fluoroethyl bromide in DMF. After HPLC purification, yields of radiochemically pure, formulated products were 22-30%(not corrected for decay) in a synthesis time of 60-70 min. The use of distilled 2-[18F]fluoroethyl bromide was indispensable for the reliable production of pure products.

Two novel N-substituted-3beta-phenyltropane alkaloids have been labeled with iodine-125 for use as irreversible probes of dopamine transporter (DAT) binding sites. One contains an iodoaryl azide moiety for photolabeling, while the other bears an iodoaryl isothiocyanate for direct conjugation. Both radioligands were prepared in a one-flask procedure by electrophilic radioiodination of the corresponding aniline under no-carrier-added conditions, followed either by diazotization and 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 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 (45%, n = 3) with high radiochemical purities (96-99%) and specific radioactivities (>2000 mCi/micromol). The utility of the radioligands was demonstrated by their covalent linkage to rat striatal membranes, and immunoprecipitation of a single radiolabeled band at 80 kDa corresponding to the full-length DAT.