A New Method for Radiosynthesis of 11C-Labeled Carbamate Groups and its Application for a Highly Efficient Synthesis of the Kappa-Opioid Receptor Tracer [11C]GR103545

11C-labeled carbamates can be obtained in a three-component coupling reaction of primary or secondary amines with CO2 and 11C-methylation reagents. [11C]Methyl-triflate mediated methylation of carbamino adducts provides the corresponding 11C-labeled carbamate groups in excellent yields under mild conditions (temperatures ≤ 40°C, 2 min reaction time). The utility of the method has been demonstrated by a highly efficient radiosynthesis of [11C]GR103545.


INTRODUCTION
In vivo quantification of opioid receptors (ORs) and changes in their distribution and availability in the central nervous system (CNS) following neurodegenerative diseases, substance abuse and nociceptive signaling is crucial for precise diagnosis and therapy monitoring (for review see: Henriksen and Willoch, 2008 [1]). Three well-defined subtypes of cerebral ORs with different functional properties are known: μ-, -and -OR (Knapp et al., 1995) [2], Connor and Christie, 1999) [3]. A number of tracers have been developed for imaging of opioid receptors with positron emission tomography (PET) (Henriksen and Willoch) [1], Henriksen et al., 2006 [4], however only the -OR-selective [ 11 Cmethyl]naltrindole and the μ-OR-selective [ 11 C]carfentanil depict single subclasses of the ORs in humans.
11C-labeled carbamate (-)-4-methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[ (3,4-dichlorophenyl)acetyl-piperidine ([ 11 C] GR103545) is a promising PET tracer for imaging of the -OR. Dynamic studies with [ 11 C]GR103545 in baboons demonstrated high -OR affinity, excellent brain penetration, rapid uptake and wash-out kinetics, and low degree of nonspecific binding (Talbot et al., 2005 [5] Here we report a new method for radiosynthesis of 11 Ccarbamate groups based on in situ reaction of amines with carbon dioxide and subsequent 11 C-methylation of the carbamino adduct. As the method only requires a single radioactive step, the entire process of labeling, purification and product formulation can readily be carried out using an automated synthesis module. In the initial part of the study, the carbamation of benzylamine 1 was formed by reaction with CO 2 in the presence of Cs 2 CO 3 and tetrabutyl ammonium iodide (TBAI The reduced chemoselectivity of the reaction at elevated temperature may be due to decomposition of the carbamino adduct and subsequent N-alkylation of the free amine as outlined in Scheme 1. a Determined by analytical radio-HPLC b Results represent the mean ± sd, n = 4. c To a solution of benzylamine (1) (2 mg, 18.6 μmol) in anhydrous DMF (500 μl) was added 3 molar equivalents of the TBA-reagent and 3 molar equivalents of Cs2CO3. CO2 gas (20 ml/min) was bubbled through the suspension for 1 h at room temperature. The 11 C-alkylating agent was swept trapped in the reaction vial and allowed to react for the desired period of time.

Synthesis of 11 C-Carbamates
Because of the apparent instability of the carbamino adduct at higher temperature, methylation with the more reactive [ 11 C]methyl-triflate ([ 11 C]CH 3 OTf) [11] was evaluated. Initial experiments with [ 11 C]CH 3 OTf were performed under identical conditions to those used for alkylation with [ 11 C]CH 3 I. Also in these experiments, the N-methylated side product 3 was observed when the reaction was carried out at elevated temperatures. We speculated that the large excess of iodide in the reaction mixture, resulting from addition of TBAI, may react with [ 11 C]CH 3 OTf to form [ 11 C]CH 3 I in situ. Substituting the phase-transfer catalyst with tetrabutyl ammonium triflate (TBAOTf) resulted in RCYs in the range of 69 ± 8 to 91 ± 5% within 2 min at 25 and 40°C, respectively (Table 1). Notably, even at 40°C the reaction provided the [ 11 C]carbamate 2 exclusively with no formation of N-[ 11 C-methyl]-benzylamine (3). Thus, the results suggest that the high reactivity of [ 11 C]CH 3 OTf enables alkylation to proceed to completion prior to decomposition of the carbamino adduct. Cs2CO3. CO2 gas (20 ml/min) was bubbled through the suspension for 1 h at room temperature. Subsequently, the 11 C-alkylating agent was swept trapped in the reaction vial and allowed to react for the desired period of time.
Using the optimized conditions for 11 C-carbamate formation, des-carbamate-GR103545 [12] was converted to [ 11 C]GR103545 in up to 91 ± 5% RCY ( Table 2). The carbamino adduct solution in DMF was found to effectively trap [ 11 C]CH 3 OTf, with a volume of 100 μl sufficient to retain > 90% of the total activity introduced. Using the reduced volume (100 μl) of the precursor solution in preparative runs [13], [ 11 C]GR103545 was obtained in 85 ± 6% isolated RCY, with a specific activity of 1792 ± 312 mCi/μmol and radiochemical purity of > 98%. The total synthesis time, including purification and formulation, was < 25 min after end-of-bombardment (n = 8). The short synthesis time, high specific activity and excellent RCY achieved with this method should facilitate evaluation of the -OR tracer [ 11 C]GR103545 in clinical trials.

CONCLUSION
[ 11 C]MeOTf has been shown to rapidly methylate carbamino adducts of primary and secondary amines, providing the corresponding 11 C-carbamate groups in excellent yields under mild conditions (temperatures 40°C, 2 min reaction time). The utility of the method has been demonstrated by a highly efficient radiosynthesis of [ 11 C]GR103545. The method is suitable for automated routine production using synthesis modules compatible with good manufacturing practice. To a solution of benzylamine (1) (2 mg, 18.6 μmol) in anhydrous DMF (500 μl) was added 3 molar equivalents of the TBA-reagent and 3 molar equivalents of Cs2CO3. CO2 gas (20 ml/min) was bubbled through the suspension for 1 h at room temperature. The 11 Calkylating agent was swept trapped in the reaction vial and allowed to react for the desired period of time. Aliquots were drawn and analyzed by analytical HPLC, which was performed using either a Chromolith  3 mmol) in tetrahydrofuran-water 1:1 (%) (40 ml) and concentrated hydrochloric acid (4 ml) was hydrogenolysed under heterogenous catalytic conditions (atmospheric pressure, room temperature) in the presence of 10% palladium on charcoal (780 mg) for 5 h. The catalyst was removed by filtration and the filtrate was concentrated. Water (60 ml) was added to the residue and the solution was basified with 2M sodium carbonate (80 ml). The suspension was extracted with dichloromethane (5 x 100 ml). The organic layer was dried (Na2SO4) and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (160 g) eluting with chloroform-methanol-NH4OH 9:1:0.1 (v/v/v). Yield: [13] Preparative HPLC was performed using a Chromolith RP18 10 100 mm reverse phase column (Merck) eluted with acetonitrile / 0.1 M ammonium formate (27.5:72.5, V /V) at 10 ml min. In-line HPLC detectors included a UV detector (Sykam) set at 254 nm and a -ray detector (Bioscan Flow-Count fitted with a PIN detector). The fraction containing the product (tr = 2.8 min) was diluted with water and applied to a Sep-Pak C18 solid phase extraction cartridge (Waters). The cartridge was washed with 10 ml of water before eluting the product with EtOH. The labeled product co-eluted with an authentic standard of GR103.545 on the two analytical HPLC systems [9] (system A: tr = 1.8 min; system B: tr = 4.2 min).