Abstract
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Objectives: SB207145 is a novel 5-HT4 receptor antagonist with high selectivity and affinity for the 5-HT4 receptor with an in vitro KD value of about 0.4 nM. Currently, the 5-HT4 antagonist [11C]-SB207145 is the only available PET tracers for imaging 5HT4 receptors in humans.1 We report herein a simplified automated radiosynthesis for efficient production of [11C]-SB207145 using the GE Healthcare Tracerlab FXC synthesis unit which eliminates the need for base during [11C]methylation.
Methods: [11C]-SB207145 was produced on the Tracerlab FXC radiosynthesis unit by methylation of precursor, SB206453 (piperidin-4-ylmethyl 8-amino-7-chloro-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate) with [11C]CH3I. SB206453 (0.5 ± 0.1 mg) was dissolved in 300 µL of DMF: DMSO (2:1) and cooled to 10ºC. [11C]CH3I was slowly bubbled into the reaction mixture. After the transfer was complete, the reaction mixture was heated at 120oC for 4 min, then cooled to 50ºC and diluted with 2 mL of water (Scheme 1, The synthesis of [11C]-SB207145 by methylation of N-desmethyl-SB206453 with 11CH3I under thermal conditions). The crude product was then purified with semi-preparative isocratic RP-HPLC (Waters XBridge C18, 5µ, 10 x 250 mm, 20% EtOH/Phosphate Buffered Saline, pH 7 (PBS), 5 mL/min). The semi-preparative HPLC fraction containing the major radiochemical product (tR = 12 min) was collected, and diluted with 6 mL of PBS solution. The [11C]-SB207145 was then passed through a 0.22 µm PVDF sterilizing filter into a sterile vented vial without additional processes such as trap and release from SPE.
Results: We anticipated two methylation sites on the N-desmethyl-SB206453 molecule, one at the 1-N piperidinyl position and the second at the 8-N primary amine position. The synthesis conditions for 1-N-methylation of N-desmethyl-SB206453 were screened using [11C]CH3I. A previously published synthesis method of [11C]-SB207145 used pentamethylpiperidine as a base and performed the reaction in a HPLC sample loop heated to 80ºC.1 Due to the inability to reproducibly heat the Tracerlab FXC HPLC loop, we screened a number of commonly used bases in radiochemistry. We tested NaOH and TBAOH solutions for their ability to selectively deprotonate either the 1-N or 8-N position of N-desmethyl-SB206453 at room temperature and under thermal conditions in the FXC reactor. We found that both bases did not give the expected N-methylation product (tR=10 min), instead both bases produced two unknown radioactive peaks on analytical HPLC analysis of the crude reaction mixture. The first radioactive peak was at void volume, tR = 2 min, and second radioactive peak was at tR = 3.5 min. However, [11C]-SB207145 was successfully achieved in the presence of mixture of solvents (DMF and DMSO) at 120ºC. Based on the results of our experiments, we identified a novel automated synthesis method using thermal conditions to perform selective piperidinyl N-methylation of N-desmethyl-SB206453 without base. [11C]-SB207145 was successfully synthesized in good yield and high radiochemical purity (n = 6, 21-45% DCY to EOB, >95% RCP). Quality control analysis of the formulated product demonstrated high radiochemical purity (95-99%) and specific activity of 1.7-2.72 Ci/mmol.
Conclusion: This work presents a novel automated radiosynthesis method for [11C]-SB207145 which eliminates the need for the presence of base and a heated HPLC loop during methylation which makes the synthesis feasible on the Tracerlab FXC and other C-11 automated synthesis modules. It also eliminates the need for solid phase extraction (SPE) re-formulation of the purified product. Research Support:
Gee AD, Martarello L, Passchier J, Wishart M, Parker C, Matthews J, Comely R, Hooper R, Gunn J. Synthesis and evaluation of [11C]SB207145 as the first in vivo serotonin 5-HT4 receptor radioligand for PET imaging in man. Curr Radiopharm 2008, 1, 110-114.
Conclusion: Research Supported By: R01MH100350 (PI: Normandin)