Revisit to Radiomethylation of [11C]MeNER: Optimization and Improved automated loop radiosynthesis of PET radiopharmaceutical [11C]MeNER for norepinephrine transporters imaging

668 Objectives: (S,S)-[11C]MeNER also known as (S,S)-[11C]methylreboxetine or (S,S)-[11C]MRB, has been considered a candidate radioligand for imaging brain norepinephrine transporters (NETs) in the living human brain with PET. We report herein the synthesis conditions for (S,S)-[13C]MeNER of (S,S)-desethylreboxetine were screened using sub-stoichiometric [13C]CH3I in order to preoptimize for radiochemistry with [11C]CH3I and simplified HPLC loop radiomethylation for an efficient production of [11C]MeNER using the GE Healthcare Tracerlab FXC-pro synthesis unit in the presence of mild aqueous base during [11C]methylation. Methods: [11C]MeNER was produced on the Tracerlab FXC radiosynthesis unit by methylation of precursor, (S,S)-desethylreboxetine, 1 with [11C]CH3I (Scheme 1). (S,S)-Desethylreboxetine, 1 (~0.5 mg) was dissolved in DMF (100 µL), 3 µ L of 0.5N aq. NaOH solution was added and votex for a minute under nitrogen atmosphere. The precursor solution was then preloaded into the HPLC loop for radiomethylation. [11C]CH3I stream was passing through the loop for 4 minutes, followed by semi-preparative isocratic RP-HPLC purification (Waters XSelect HSS T3 column, 5µ, 10 x 250 mm, flow at 5 mL/min with 30:70 ACN: 50mM Sodium Phosphate pH 4.5) and the major radiochemical product (tR ~12 min, i.e. [11C]MeNER) (Figure 1) was collected into a HPLC fraction dilution vessel containing 24 mL water, and transfer to SPE cartridge and eluted with 1mL of EtOH and 10 mL 0.9% sodium chloride. The [11C]MeNER was then passed through a 0.22µ PES sterilizing filter into a sterile vial. Results: We anticipated two methylation sites on the precursor (S,S)-desethylreboxetine, one at the morpholine position and the second at the phenolic position. An excess of base is often used in radiochemistry, and under these conditions we observed deprotonation at the phenolic hydroxy as well as morpholine amine position of precursor. However, in our hands, the desired product, [11C]MeNER was never successfully obtained by the published procedures (Schou et al. 2003, Lin and Ding et al. 2004). Firstly, when use of excess of 1N NaOH with precursor, DMF-d7 and substoichiometric [13C]CH3I at 25oC resulted in the formation of the [13C]MeNER product and an undesired N-[13C]-methyl desethylreboxetine by-product in a 1:1 ratio. Secondly, when use of 0.5N aq. NaOH added to precursor solution in DMF-d7 and sub-stoichiometirc [13C]CH3I at 25oC was furnished [13C]MeNER as the major product. The respective position of 13C-labeling was also confirmed by 13C NMR. Based on the results of our 13C NMR experiments, the optimized reaction conditions were subsequently applied to carbon-11 radiochemistry. [11C]MeNER was successfully validated using the HPLC loop in good yield as described above (n = 35, 8±4 % DCY) for human use. Quality control analysis of the formulated product demonstrated high radiochemical purity (95-99%) (Figure 2) and specific activity ranging from 2907 to 12573 mCi/µmol at end of synthesis. Conclusion: Gratifyingly, our lab method was able to control the reaction to the desired [11C]MeNER as major product, only in the presence mild aqueous base during [11C]methylation and have a good semi-preparative isocratic RP-HPLC purification method to separate two radioactive peaks, such as (S,S)-N-[11C]-methyl desethylreboxetine, 3 and (S,S)-O-[11C]-methyl desethylreboxetine namely, (S,S)-[11C]MeNER, 2. Thus, (S,S)-[11C]MeNER was validated at Massachusetts General Hospital Gordon PET Core for human use with a GE TRACERlab FXC-Pro radiosynthesis module (Figure 3) according to USP and FDA requirements. Acknowledgements We thank Tim Beaudoin, John A. Correia, Steven J. Gallo, Peter Khom, David F. Lee Jr., Tiffany V. L’Heureux, Hamid Sabet, and Gary A. Siwruk of the Massachusetts General Hospital for isotope production, routine synthesis, quality control and technical support.