Application of Palladium-Mediated 18F-Fluorination to PET Radiotracer Development: Overcoming Hurdles to Translation

New chemistry methods for the synthesis of radiolabeled small molecules have the potential to impact clinical positron emission tomography (PET) imaging, if they can be successfully translated. However, progression of modern reactions from the stage of synthetic chemistry development to the preparation of radiotracer doses ready for use in human PET imaging is challenging and rare. Here we describe the process of and the successful translation of a modern palladium-mediated fluorination reaction to non-human primate (NHP) baboon PET imaging–an important milestone on the path to human PET imaging. The method, which transforms [18F]fluoride into an electrophilic fluorination reagent, provides access to aryl–18F bonds that would be challenging to synthesize via conventional radiochemistry methods.


Materials and Methods
All air-and moisture-insensitive reactions were carried out under an ambient atmosphere, magnetically stirred, and monitored by thin layer chromatography (TLC) using EMD TLC plates pre-coated with 250 m thickness silica gel 60 F254 plates and visualized by fluorescence quenching under UV light. Flash chromatography was performed on Dynamic Adsorbents Silica Gel 40-63 m particle size using a forced flow of eluent at 0.3-0.5 bar pressure. All air-and moisture-sensitive manipulations were performed using oven-dried glassware, including standard Schlenk and glovebox techniques under an atmosphere of nitrogen. Methylene chloride was purged with nitrogen, dried by passage through activated alumina, and stored over 3Å molecular sieves. 1 Benzene, benzene-d 6 , diethyl ether, toluene, pentane, dioxane, and THF were distilled from deep purple sodium benzophenone ketyl. Methylene chloride-d 2 was dried over CaH 2 and vacuum-distilled. Acetonitrile and acetonitrile-d 3 were dried over P 2 O 5 and vacuum-distilled. Pyridine was dried over CaH 2 and distilled.
DMSO was distilled from sodium triphenylmethanide and stored over 3Å sieves. 2

Potassium tetra(1H-pyrazol-1-yl)borate (S2)
Based on a reported procedure: 5 As solids, KBH 4 (7.00 g, 0.130 mol, 1.00 equiv) and pyrazole (44.2 g, 0.649 mol, 5.00 equiv) were combined in a round-bottom flask equipped with a reflux condenser under a N 2 atmosphere. This mixture was heated at 250 ºC for 16 hours. The melt was then cooled to 23 °C. The residue was dissolved in methanol (200 mL). The solution was added to diethyl ether (600 mL). A precipitate formed that was isolated by filtration. The precipitate was washed with additional diethyl ether (2 × 100 mL), affording 37.6 g of the title compound as a colorless solid (91% yield).

Synthesis of Pd(IV) fluoride complex 7 Benzo[h]quinolinyl (tetrapyrazolylborate) Pd(IV) fluoride trifluoromethanesulfonate (7)
In a glove box, to benzo[h]quinolinyl (tetrapyrazolylborate) Pd(IV) 4-picoline trifluoromethanesulfonate (6) (284 mg, 0.297 mmol, 1.00 equiv) dissolved in CH 3 CN (15 mL) in a soda lime glass bottle was added KF (17.3 mg, 0.297 mmol, 1.00 equiv) and 18-crown-6 (235 mg, 0.891 mmol, 3.00 equiv) in one portion at 23 ºC. The bottle was sealed, taken out of the glove box, sonicated at 23 °C for 5 minutes, immersed in an oil bath heated at 50 °C for 5 minutes while vigorously stirring the suspension. CH 3 CN (10 mL) was added to the solution, and the solution was filtered through Celite, eluting with additional CH 3 CN (10 mL). The filtrate was concentrated in vacuo. The residue was triturated with THF (3 × 15 mL) and subsequently dried in vacuo to afford 195 mg of the title compound as an orange solid (90%). Thermal stability of 7: 7 was placed in a vial and heated for 24 hours at 100 ºC under dynamic vacuum (10 -4 Torr). The solid was analyzed by 1 H and 19 F NMR spectroscopy, and showed no decomposition.
Tolerance of 7 toward water: 2.4 mg of 7 (3.3 mol) and THF (2.0 L) (internal standard) were dissolved in CD 3 CN (0.55 mL) in an NMR tube. D 2 O (61 L) was added to the solution. The solution was kept at +23 ºC for 3 hours and monitored by 1 H and 19 F NMR spectroscopy, which showed no decomposition ( Figure S1).

Figure S1. NMR Spectra of 7 in 10% aqueous acetonitrile solution
Supporting Information S11 Alternative synthesis of 7

Bis(pyridinio-1)iodobenzene bis(trifluoroacetate) (S3)
Based on a reported procedure: 7 All manipulations were carried out in a dry box under a N 2 atmosphere. To (diacetoxyiodo)benzene (15.0 g, 46.6 mmol, 1.00 equiv) dissolved in 300 mL CH 2 Cl 2 in a round-bottom flask was added TMSOTf (20.7 g, 93.1 mmol, 2.00 equiv) dropwise over 5 minute at 23 °C. Pyridine (7.37 g, 7.53 mL, 93.1 mmol, 2.00 equiv) in 50 mL CH 2 Cl 2 was added to the solution dropwise to give a white precipitate and the mixture was stirred for 30 min vigorously at 23 °C. The solid was filtered off and washed with 50 mL CH 2 Cl 2 three times and subsequently dried under vacuum to afford 29.9 g of the title compound as a colorless solid (97%).

Benzo[h]quinolinyl (tetrapyrazolylborate) Pd(IV) pyridine trifluoromethanesulfonate (3)
All manipulations were carried out in a dry box under a N 2 atmosphere. To benzo[h]quinolinyl (tetrapyrazolylborate)palladium (3) (10.00 g, 17.7 mmol, 1.00 equiv) in CH 3 CN (50 mL) at 23 ºC was added bis(pyridinio-1)iodobenzene bis(trifluoroacetate) (S3) (12.0 g, 18.1 mmol, 1.02 equiv). After stirring for 20 min the reaction mixture was concentrated in vacuo. The resulting residue was triturated with THF (30 mL) and collected on a frit by filtration as a light brown solid. The collected solid was further washed with THF (30 mL) three times and dried in vacuo. The solid was redissolved in 30 mL CH 3 CN and volatiles including residual THF were removed in vacuo to afford 18.0 g of the title compound as a brown solid (94%).

Benzo[h]quinolinyl (tetrapyrazolylborate) Pd(IV) fluoride trifluoromethanesulfonate (7)
In a glovebox, to benzo[h]quinolinyl (tetrapyrazolylborate) Pd(IV) pyridine trifluoromethanesulfonate (S4) (7.80 g, 8.29 mmol, 1.00 equiv) dissolved in 150 mL CH 3 CN was added KF (0.54 g, 9.26 mmol, 1.12 equiv) and 18-crown-6 (0.16 g, 0.62 mmol, 0.07 equiv) in one portion at room temperature. After the solution was vigorously stirred for 3 days at room temperature and another 350 mL of CH 3 CN was added to the reaction solution. The flask was sealed, taken out of the glove box, immersed in a water bath heated at +50 °C until the turbid solution became clear, which took less than 30 min, and the solution was filtered through Celite eluting with 100 mL of dry CH 3 CN. The filtrate was concentrated in vacuo. The residue was triturated with dry THF (3 × 50 mL), filtered off, and subsequently dried in vacuo to afford 5.80 g of the title compound as an orange solid (94%).

Synthesis of aryl palladium complexes (15 and 20, examples of 9) [{(4-Methoxyphenyl)sulfonyl}imino]phenyliodinane (S5)
Based on a reported procedure: 8,9 To p-methoxybenzenesulfonamide (5.00 g, 26.7 mmol, 1.00 equiv) in a round-bottom flask open to air in methanol (100 mL) at 23 ºC was added potassium hydroxide (3.75 g, 66.8 mmol, 2.50 equiv). The reaction mixture was stirred at 23 ºC for 10 minutes and subsequently cooled to 0 ºC. To the reaction mixture at 0 ºC was added iodobenzene diacetate (8.60 g, 26.7 mmol, 1.00 equiv). The reaction mixture was stirred at 0 ºC for 10 minutes and further stirred at 23 ºC for 2.0 hours. The reaction mixture was poured into cold water (700 mL) and kept at 0 ºC for 4 hours. The suspension was filtered and the filter cake was washed with water (2 × 200 mL) and methanol (2 × 200 mL) to afford 7.90 g of the title compound as a colorless solid (76% yield).

Acetato palladium complex (8)
To chloro palladium complex (S7) (5.00 g, 8.34 mmol, 1.00 equiv) in a round-bottom flask fitted with a reflux condenser open to air in CH 2 Cl 2 (300 mL) at 23 ºC was added AgOAc (4.87 g, 29.2 mmol, 3.50 equiv). The suspension was stirred at 40 ºC for 3 hours. After cooling to 23 ºC, the suspension was filtered through a plug of Celite, eluting with additional CH 2 Cl 2 (50 mL). The filtrate was concentrated in vacuo and the residue was triturated with diethyl ether (100 mL). The solid was collected by filtration and washed with diethyl ether (2 × 50 mL) to afford 5.07 g of the title compound as a yellow solid (95% yield).

4-(4-Bromophenyl)-1-(4-methoxyphenyl)-5,6-dihydropyridin-2(1H)-one (10)
To 1-(4-bromophenyl)-3-((4-methoxyphenyl)amino)propan-1-one (S8) (19.7 g, 58.8 mmol, 1.00 equiv) suspended in CH 2 Cl 2 (200 mL) in a round-bottom flask open to air was added ethyl 3chloro-3-oxopropanoate (9.32 g, 7.84 mL, 58.8 mmol, 1.00 equiv) and a saturated aqueous solution of Na 2 CO 3 (100 mL). The reaction mixture was stirred at 23 ºC for 1 hour and then poured onto H 2 O (100 mL) in a separatory funnel. The separatory funnel was shaken and the organic phase collected and concentrated in vacuo. The residue was dissolved in CH 2 Cl 2 (200 mL) and ethyl 3-chloro-3-oxopropanoate (4.66 g, 3.92 mL, 29.4 mmol, 0.500 equiv) and a saturated aqueous solution of Na 2 CO 3 (100 mL) were added. The reaction mixture was stirred at 23 ºC for 1 hour and then poured onto H 2 O (100 mL) in a separatory funnel. The separatory funnel was shaken and the organic phase collected. The aqueous phase was extracted from with CH 2 Cl 2 (100 mL). The combined organic phases were washed with brine (100 mL), dried with Na 2 SO 4 , and concentrated in vacuo. The residue was dissolved in EtOH (400 mL). NaOH (9.41 g, 235 mmol, 4.00 equiv) was added, and the reaction mixture was stirred at 23 ºC for 15 minutes and then heated at reflux for 30 minutes. The reaction mixture was cooled to 23 ºC and then poured into an Erlenmeyer flask containing H 2 O (600 mL). The reaction suspension was cooled to 10 ºC in a refrigerator and the precipitate was collected on a frit. Azeotropic removal of water by suspending the solid in benzene and concentration in vacuo (3 × 50 mL) afforded 17.6 g of the title compound as a beige yellow solid (84% yield).

Figure S2. Enantiodiscriminating HPLC trace of S9
HPLC method: Chiralpak IB column with 35% isopropanol/hexanes eluent for racemic S9 and enantioenriched S9. Percent of total integration listed for each peak.

Figure S3. Enantiodiscriminating HPLC trace of 12
HPLC method: Chiralcel OD-H column with 10% isopropanol/hexanes eluent for racemic 12 and enantioenriched 12. Percent of total integration listed for each peak.
The residue was purified by chromatography on silica gel, eluting with 2-7% EtOAc in hexanes (v/v) to afford 1.05 g of the title compound as a colorless solid (83% yield).

S24
To (E)-ethyl 3-(5-bromo-2-(cyclopropylmethoxy)phenyl)acrylate (S12) (3.78 g, 11.6 mmol, 1.00 equiv) in PhMe (30 mL) in a flame-dried round-bottom flask under a N 2 atmosphere at -78 ºC was added a 1.0 M solution of diisobutylaluminum hydride (DIBAL-H) in PhMe (26 mL, 26 mmol, 2.2 equiv) in 6 portions dropwise every 10 minutes for 1 hour. The reaction was warmed to 0 ºC over 2 hours and then warmed to 23 ºC and stirred at this temperature for 1 hour. The reaction mixture was poured onto a concentrated aqueous Rochelle's salt (potassium sodium tartrate) solution (400 mL). EtOAc (400 mL) was added and the mixture was stirred for 3 hour until two liquid phases separated cleanly. The phases were partitioned and the aqueous phase was extracted from with EtOAc (300 mL). The organic phases were combined and washed with brine (200 mL), dried with Na 2 SO 4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel, eluting with a gradient of 10-25% EtOAc in hexanes (v/v) to afford 2.77 g of the title compound as a colorless solid (84% yield).

Fluorination of aryl palladium complexes (3S,4R)-t-Butyl 3-((benzo[d][1,3]dioxol-5-yloxy)methyl)-4-(4-fluorophenyl)piperidine-1carboxylate (21)
In a glove box under a N 2 atmosphere, palladium aryl complex 15 (135 mg, 141 µmol, 1.00 equiv) was dissolved in acetone (6 mL) and added to a soda lime glass bottle charged with Pd(IV)-F complex 7 (103 mg, 141 µmol, 1.00 equiv). The bottle was sealed, taken out of the glove box, and immersed in an oil bath heated at 85 ºC for 10 minutes. The reaction mixture was cooled and concentrated in vacuo. The residue was purified by chromatography on silica gel, eluting with a gradient of 10-20% EtOAc in hexanes to afford 42.7 mg of the title compound as a colorless solid (71% yield). The enantiomeric excess of the title compound remained >99% ee as determined on a Chiracel OD-H column with 3% isopropanol/hexanes eluent. The absolute configuration was identical to a sample of the title compound prepared from commercially available paroxetine (see Figure S6). Rf = 0.25 (hexanes/EtOAc 6:1 (v/v)).  HPLC method: Chiralcel OD-H column with 3% isopropanol/hexanes eluent for 90% ee 21 and for >99% ee 21 and 21 synthesized from commercially available paroxetine. Percent of total integration listed for each peak.

t-Butyl(((1S,2S)-2-(2-(cyclopropylmethoxy)-5-fluorophenyl)cyclopropyl)methyl)carbamate (22)
In a glove box under a N 2 atmosphere, palladium aryl complex 20 (25.0 mg, 29.4 umol, 1.20 equiv) was dissolved in acetone (3 mL) and added to a soda lime glass bottle charged with Pd(IV)-F complex 7 (18.0 mg, 24.5 umol, 1.00 equiv). The bottle was sealed, taken out of the glove box, and immersed in an oil bath heated at 85 ºC for 10 minutes. The reaction was cooled and concentrated in vacuo. The residue was purified by chromatography on silica gel, eluting with a gradient of 10-15% EtOAc in hexanes to afford 5.9 mg of the title compound as a colorless solid (72% yield). The solution was stirred at 23 ºC for 10 minutes. The solution was then dripped into saturated aqueous NaHCO 3 solution (20 mL). The aqueous layer was extracted with EtOAc (3 × 10 mL). The combined organic phases were washed with brine (15 mL), dried with Na 2 SO 4 , and concentrated in vacuo. The residue was purified by chromatography on silica gel that had been washed with Et 3 N, eluting with 0-10% MeOH in CH 2 Cl 2 (v/v) to afford the amine base. The title compound was isolated by adding a 10% CF 3 CO 2 H in CH 2 Cl 2 solution (1 mL) to the amine base. The solution was concentrated in vacuo and heated in vacuo at 50 ºC to provide 65.5 mg of the title compound as its CF 3 CO 2 H salt (96% yield).  to the vial. The vial was sonicated and then the reaction mixure was allowed to stir at 23 °C for 10 minutes. During this time, the orange/brown clear solution became opaque. At the end of 10 minutes, the vial was opened and the suspension was loaded with a glass pipette into another glass pipette containing 10 mg of cotton and 25 mg of JandaJel™-polypyridine that had been suspended in 0.3 mL of acetone for 15 minutes (to swell the JandaJel™-polypyridine) and then drained prior to loading the reaction suspension. The conical vial was washed with 0.5 mL of acetone and the acetone wash was added onto the JandaJel™-polypyridine in the glass pipette. At this point the combined reaction suspension and acetone wash were fully pushed through the JandaJel™-polypyridine and cotton with air into a new 1 dram vial equipped with a magnetic stir bar. An additional 0.5 mL of acetone was used to wash the conical vial. The acetone wash was added onto the JandaJel™-polypyridine in the glass pipette and pushed through with air into the 1 dram vial.
Second step: To the 1.5 mL acetone solution was added 10. mg of the Pd(II) aryl complex 15 or 20. The vial was capped securely, and the mixture heated at 85 °C. After 10 minutes the solution was cooled. A capillary tube was used to spot the solution on a silica gel TLC plate. The TLC plate was emerged in an appropriate organic solvent mixture. The TLC plate was scanned with a Bioscan AR-2000 Radio TLC Imaging Scanner.

Automated syntheses of [ 18 F]-1 and [ 18 F]-2 using high specific activity [ 18 F]fluoride
Automated syntheses were accomplished using Eckert and Ziegler automated synthesis modules and Modular-Lab in a hot cell. Reaction procedures were identical to the described "by hand" method from the preceding section with the following exceptions: 1) The initial azeotrope contained 0.8 mL MeCN and 0.2 mL H 2 O instead of 1.3 mL MeCN and 0.2 mL H 2 O, 2) the first step of the reaction was stopped after 7.5 minutes instead of 10 minutes, and 3) 1.0 mL of 2butanone was used to wash the vial and JandaJel™-polypyridine, so that the second step of the reaction proceeded in a 1.5 mL solution of 2:1 2-butanone:acetone. After the reaction, the solution was filtered through preconditioned SepPak® Plus Waters Accell™ Plus QMA cartridge (to capture leftover [ 18 F]fluoride). The second vial and anion exchange cartridge were washed with 1 mL acetone. The combined organic solutions were taken out of the hot cell.
Using manual manipulations, the reaction mixture was concentrated, and the residue was suspended in a solution of EtOAc:hexanes (1.0 mL, 1:1 (v/v)) and filtered through a 1 inch plug of silica gel in a glass pipette using an addition 2.0 mL as eluent. The reaction mixture was concentrated to dryness and the residue was dissolved in a solution of trifluoroacetic acid:CH 2 Cl 2 (1.0 mL, 1:1 (v/v)). The solution was immediately concentrated to dryness and the residue was dissolved in a solution of MeCN:H 2 O (0.10 mL, 1:3 (v/v)). The sample was then purified by preparative HPLC on a Macherey-Nagel VP 250/10 Nucleosil 100-5 C18 Nautilus column. (For [ 18 F]-1: Method: 29% MeCN/H 2 O with 0.1% CF 3 CO 2 H, 5 mL/min, elution time: 13.5-14.5 min, (see Figure S7); For [ 18 F]-2: Method: 32% MeCN/H 2 O with 0.1% CF 3 CO 2 H, 5 mL/min, elution time: 10.7-11.7 min (see Figure S8)) The purified product in HPLC solvent was diluted to 25 mL using a basic aqueous buffer (0.1 M K 2 CO 3, 0.1 M KHCO 3 ) was loaded on to a Grace Extract-Clean™ SPE 50mg/1.5mL column. The column was washed with H 2 O (10 mL). The product was eluted from the column using EtOH (0.5 mL), diluted with pH 5 0.1 M NaOAc solution (1 mL) and sterile saline solution (3.5-8.5 mL), and finally filtered through a sterile filter (Millex® Syringe-Driven Filter Unit LG 0.2 µm 25mm). For analytical HPLC of final products compared to authentic reference samples, see Figure S9 for [ 18 F]-1 and Figure S10   Radioactivity trace (top) and 280 nm UV trace (bottom) demonstrating preparatory HPLC purification. Sample [ 18 F]-1 collected at 13.5-14.5 min corresponding to large (saturated) radioactivity peak.

Characterization of 18 F-labeled molecules
18 F-labeled molecules were characterized by comparing the radioactivity HPLC trace of the reaction mixture to the HPLC UV trace of authentic reference sample. An Agilent Eclipse XDB-C18, 5 µm, 4.6 x 150 mm HPLC column was used for analytical HPLC analysis. Note: radioactivity chromatographs have been offset (-0.125 min) to account for the delay volume (time) between the diode array detector and the radioactivity detector.

Determination of specific activity of [ 18 F]-1and [ 18 F]-2
Specific activity of [ 18 F]-1 and [ 18 F]-2 were determined by measuring the UV absorbance of a known amount of radioactivity and comparing to a standard curve of UV absorbance vs amount of unlabeled 1 and 2. For 89 µCi of [ 18 F]-1 a UV absorbance could not be detected. The smallest amount that has been detected corresponded to 7 pmol for a specific activity of at least 13 Ci/µmol at time of injection (TOI). For 122 µCi of [ 18 F]-2 a UV absorbance of 5.1 was measured corresponding to 120 pmol for a specific activity of 1.0 Ci/µmol at time of injection (TOI). The standard curves were generated by integration of the UV absorbance signal (at 280 nm) of at least 5 different known amounts of 1 and 2 in triplicate (see Tables S1 and S2 and Figures S11 and S12).

Determination of palladium content in purified sample of [ 18 F]-2
At the end of the synthesis and reformulation of [ 18 F]-2, A portion of the sample was analyzed using an Agilent 7500a ICP-MS to determine palladium content. An average of 2 samples (HPLC fraction:ICP diluent 1:99 (m/m)) were compared to a standard curve of relative ion count versus palladium concentration. The standard curve was generated by creating a dilution series of known palladium concentrations (from 0.020 ppb to 50.0 ppb) and an internal lutetium control (see Table S3 and Figure S13). The samples averaged less than 0.02 ppb palladium. The final palladium content of the reformulated sample was 2 ppb palladium.