Efficient Radiosynthesis of PET Tracers [11C]WAY-100635 and [11C]-(+)-PHNO

In-loop−¹¹C-carboxylation: 80 cm polyethylene (PE)-tubing was coiled into a loop and equipped with two Luer-fittings. Loops were coated with a mixture of one of the three respective Grignard reagents (500 µL) or diethyl ether (as negative control) in THF (1000 µL) for comparison reasons. Therefore, the respective diluted Grignard solution was pushed through the loop and completely drained by a smooth He-stream (5 mL/min). (note: Grignard reagents were purchased as solutions in diethyl ether). The inlet of the loop was connected immediately after impregnation to the line from the molecular sieve CO₂ trap and the outlet to an Ascarite II trap to collect unreacted [¹¹C]CO₂. Upon delivery from the cyclotron, [¹¹C]CO₂ was transported to the hot cell and trapped on-line within the molecular sieve. Subsequently, it was released by heating the trap to 400 °C and the gas was passed to the previously impregnated loop using a smooth stream of helium (3–5 mL/min). Excess of unreacted [¹¹C]CO₂ (or not combined ¹¹C-intermediates) was retained within the Ascarite II trap. The bound ¹¹C-acylation synthon was swept out of the loop using a mixture of THF (400 µL) and thionyl chloride (5 µL).
[Carbonyl-¹¹C]WAY-100635: Precursor compound, WAY-100634, and reference compound, WAY 100635, were obtained from ABX-Advanced Biochemical Compounds (Radeberg, Germany). Semi-preparative HPLC: column: Phenomenex^® Gemini, 10 µm 110 A, 250×10 mm²; mobile phase: methanol/0.1 M ammonium formate (70/30) (v/v%) plus 3 mL TEA per liter; 8 mL/min; analytical HPLC: column: Waters^® µ-Bondapak C-18 (5 µm, 300×3.9 mm² WAT027324) mobile phase: 0.1 M ammonium formate/ACN (55/45 v/v%); 2 mL/min.
Up-scaled [carbonyl-¹¹C]WAY-100635 production was performed according to Wadsak et al. (2007) with modifications due to the implementation of a new TRACERlab^™ FX C pro synthesizer. Briefly, loop was coated by pushing a cyclohexane magnesium chloride solution (0.5 mL) in THF (1 mL) through the PE-tubing equipped with Luer-fittings. Radioactivity was trapped nearly quantitatively and converted on-line to magnesium chloride cyclohexane [¹¹C]carboxylate. Using a thionyl chloride solution (5 µL in 400 µL THF), the Grignard reaction intermediate was converted to the respective carboxylic acid chloride, swept out the loop and transferred into the reactor vial containing precursor (WAY-100634, 3.4–3.6 mg) in TEA (20 µL) and THF (50 µL). Resulting reaction mixture was heated up to 70 °C for 4 min, cooled down to room temperature and quenched with water (1 mL). Crude reaction mixture was automatically transferred and injected to the semi-preparative HPLC system triggered by a fluid detector. Product peak was collected (6–8 mL) and diluted (80 mL water) within the bulb and passed through an SPE (C-18 plus) column. After complete transfer, the column was washed with water (10 mL) and the purified product was eluted with 1.5 mL ethanol and 5 mL 0.9% saline solution. For final formulation, further 9 mL saline solution 0.9%, 1 mL saline solution 3% and 1 mL phosphate buffer (125 mM) were added, transferred to a lead shielded laminar-air-flow hot cell and sterile-filtered on-line.
[¹¹C]-(+)-PHNO: Precursor compound, (+)-HNO hydrochloride, and reference compound, (+)-PHNO, were obtained from ABX-Advanced Biochemical Compounds (Radeberg, Germany). Semi-preparative HPLC: column: Phenomenex^® Luna C18(2), 10 µm, 250×10 mm²; mobile phase: 25 mM PBS (pH 7.0)/acetonitrile (ACN) (60/40 v/v%); 6 mL/min); analytical HPLC: LichroCART^® Lichrospher 100, RP-18 (5 µm, 4×250 mm²) with LichroCART^® Lichrospher RP-18 guard column (5 µm, 4×4 mm²), mobile phase: 10 mM PBS (pH 7.0)/ACN (60/40 v/v%); 1.5 mL/min).
The [¹¹C]-(+)-PHNO-radiosynthesis is outlined in Scheme 1. The first reaction step in the synthesis sequence, the Grignard reaction, was performed on the basis of the previously described production of [carbonyl-¹¹C]WAY-100635 adopting the loop method. In this case, the PE-loop was coated with ethyl magnesium bromide (500 µL) in THF (1000 µL) for the conversion to magnesium bromide [¹¹C]propionate. Using thionyl chloride solution (5 µL in 400 µL THF), the built [¹¹C]propionic acid chloride was transferred directly into the reaction vessel containing (+)-HNO (2.5 mg) suspended in TEA (50 µL) and THF (400 µL). The resulting reaction mixture was heated to 80 °C for 5 min. After cooling down to −15 °C, LiAlH₄ (120 µL) in THF (400 µL) was added to the reaction intermediate (i.e. [¹¹C]1-((4αR,10βR)-9-hydroxy-5,6-dihydro-2H-naphtho[1,2-β][1,4]oxazin-4(3 H,4αH,10βH)-yl)propan-1-one) and subsequently heated up to 80 °C for 2 min. Subsequently, THF was evaporated completely within 2 min and the reaction mixture was cooled to 45 °C. Crude [¹¹C]-(+)-PHNO was dissolved in 1 M HCl (800 µL) and neutralized with 1 M sodium hydroxide solution (800 µL) prior to injection onto semi-preparative HPLC. The HPLC fraction containing purified [¹¹C]-(+)-PHNO was collected (7–9 mL), diluted with water (70 mL) and passed through a C-18 Sep-Pak^® plus cartridge. Further workup procedure and formulation of the final product were identical to the described [carbonyl–¹¹C]WAY-100635 method (see above).