All chemicals and reagents were purchased from Aldrich, Merck and Dorwil. Analytical TLC were performed on silica gel 60F-254 plates and visualized with UV light (254 nm) and p-anisaldehyde in acidic ethanolic solution or iodine vapours. Column chromatography was performed using silica gel (SAI, 63–200 μm). NMR spectra were recorded on a Bruker DPX-400 spectrometer. The assignment of chemical shifts was based on standard NMR experiments (1H, 1H–COSY, HETCOR and 13C–NMR). The chemical shifts values were expressed in ppm relative to tetramethylsilane as internal standard. Mass spectra were determined on a Shimadzu DI-2010 (EI-MS) or Applied Biosystem API 2000 (ESI-MS). IR were obtained using a Shimadzu IR equipment Affinity-1 (Fourier Transform Infrared Spectrophotometer). Materials, instruments, protocols and documents used for precursor synthesis were in agreement with GMP recommendations.
i-a) A mixture of lithium borohydride (0.27 g, 12 mmol) in dry THF (6 mL) was cooled at 0 °C and trimethylsilyl chloride (3.1 mL, 48 mmol) was added subsequently. The ice/water bath was removed and the mixture stirred at room temperature for 20 min. Then, the mixture was again cooled to 0 °C and L-phenylalanine (1 g, 6 mmol) was added. The ice/water bath was removed, and the reaction mixture was stirred at room temperature for 12 h. The reaction mixture was cooled to 0 °C, and methanol (9 mL) was added dropwise, followed by aqueous sodium hydroxide (5 mL, 2.5 M). Finally, the mixture was evaporated in vacuum, and the residue extracted with chloroform (5 × 5 mL). The combined extracts were dried with Na2SO4, filtered, and evaporated in vacuum. The white solid obtained was dried under vacuum for 24 h to yield 1 (0.84 g, 92% yield).
i-b) To a solution of L-Phenylalanine methyl ester hydrochloride (300 mg, 1.39 mmol) in a 1:1 (v/v) mixture of water and ethanol (3.5 mL) was added slowly with stirring a solution of lithium borohydride (103 mg, 4.73 mmol) in the same solvent (3.5 mL) cooled externally in an ice/water bath. When the addition of borohydride was complete the mixture was stirred for 1 h at room temperature. Next, the solution was evaporated under reduced pressure and the residual aqueous solution treated first with sodium hydroxide and then with sodium chloride to saturate the solution before extraction with ethyl acetate (5 × 5 mL). The extract was washed with brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure to yield 1 as white solid (0.172 g, 82% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 7.35–7.31 (m, 2H), 7.27–7.21 (m, 3H), 3.68 (dd, J = 4 Hz, J = 10.4 Hz 1H), 3.44 (dd, J = 7.2 Hz, J = 10.8 Hz, 1H), 3.18–3.12 (m, 1H), 2.84 (dd, J = 5.6 Hz, J = 13.6, 1H), 2.59 (dd, J = 8.8 Hz, J = 13.6 Hz, 1H), 2.02 (bs, 2H). IR (KBr): 3360, 3295, and 1580 cm−1; MS (ESI,) m/z: 152.1 (M+. + H), 134.1 (M+. - 18, H2O), 117.1 (PhCHCHCH2
.+), 91.0 (PhCH2
(S)-tert-Butyl (1-hydroxymethyl-2-phenylethyl)-carbamate (2): To a magnetically stirred suspension of 1 (1.0 g, 6.6 mmol) in water (6.5 mL) was added di-tert-butyl dicarbonate ((Boc)2O, 1.5 g, 9.9 mmol) at room temperature. After stirring for 25 min the reaction mixture, the white solid formed was filtered, washed with water and dried under vacuum for 48 h to yield 2 (1.31 g, 79% yield). 1H–NMR (CDCl3) δ (ppm): 7.35–7.31 (m, 2H), 7.27–7.23 (m, 3H), 4.76 (bs, 1H), 3.89 (bs, 1H), 3.72–3.67 (m, 1H), 3.60–3.55 (m, 1H), 2.87 (d, J = 7.2 Hz, 2H), 2.38 (bs, 1H), 1.44 (bs, 9H). IR (KBr) 3360, 1685, and 1525 cm−1; MS (ESI) m/z: 252 (M+. + H), 235 (M+. – OH, 17), 196 (M+. - tert-butene, 56), 152 (M+. – Boc, 101), 91 (PhCH2
(S)-tert-Butyl (1-iodomethyl-2-phenylethyl)-carbamate (3): A mixture of iodine (1.59 g, 6.28 mmol), imidazole (0.47 g, 6.9 mmol) and triphenylphosphine (1.65 g, 6.26 mmol) in dry dichloromethane (50 mL) was cooled at 0 °C with stirring for 15 min. Next, the mixture was stirred at room temperature for another 15 min, and a solution of 2 (1.44 g, 5.71 mmol) in dry dichloromethane (18 mL) was added dropwise. The mixture was stirred for 15 min at room temperature; the solid formed was filtered and the organic layer washed with diluted aqueous Na2S2O3 and water, dried with Na2SO4 and evaporated in vacuo. After the workup, the crude was purified by column chromatography (SiO2, Hexane/EtOAc (9:1)), yielding derivative 3 as a white solid (1.6 g, 80%). 1H–NMR (CDCl3) δ (ppm): 7.35–7.32 (m, 2H), 7.29–7.25 (m, 3H), 4.72 (d, J = 7.2 Hz, 1H), 3.62 (bs, 1H), 3.44 (dd, J = 3.6 Hz, J = 10 Hz, 1H), 3.20 (dd, J = 4 Hz, J = 10 Hz, 1H), 2.96 (dd, J = 5.6 Hz, J = 13.2 Hz, 1H), 2.82 (dd, J = 8.4 Hz, J = 13.6 Hz, 1H), 1.46 (s, 9H). IR (KBr): 3350, 1690, 1525 cm−1. MS (ESI) m/z: 362.2 (M+. + H) 306.1 (M+. – tert-butene, 56), 105 (PhCHCH3), 91 (PhCH2
.+), 57 (+.C(CH3)3).
(S)-tert-Butyl (1-methyl-2-phenylethyl)-carbamate (4):
A mixture of 3 (1.53 g, 4.24 mmol) in anhydrous tetrahydrofuran (32 mL) was cooled to −10 °C under nitrogen atmosphere. Next, a solution of sodium tri-sec-butylborohydride (N-Selectride) 1 M in tetrahydrofuran (6.36 mL, 6.36 mmol) was added dropwise and the resulting mixture was stirred at 0–5 °C for about 2 h. The reaction was quenched by the slow addition of water (3.0 mL) followed by the dropwise addition of a solution made by combining 45 mL of H2O, 3.0 g of K2CO3, and 23 mL of 10% H2O2. The reaction mixture was stirred at room temperature for 1 h. The THF was evaporated under reduced pressure, and the product was extracted with dichloromethane (4 × 15 mL). The organic layers were dried with Na2SO4 and the solvent evaporated in vacuo. After the workup the crude was purified by column chromatography (SiO2, Hexane/EtOAc (9:1)), yielding derivative 4 as a white solid (0.92 g, 93%). 1H–NMR (DMSO-d6) δ (ppm): 7.29–7.25 (m, 2H), 7.20–7.16 (m, 3H), 6.79 (d, J = 8.0 Hz, 1H), 3.68–3.61 (m, 1H), 2.75 (dd, J = 7.2 Hz, J = 13.2 Hz, 1H), 2.58 (dd, J = 7.2 Hz, J = 13.2 Hz, 1H), 1.34 (bs, 9H), 1.00 (d, J = 6.4, 3H). IR (KBr): 3360, 1687, 1520 cm−1. MS (ESI) m/z: 236 (M+. + H), 180 (M+. – tert-butene, 56), 119 (180 – NH3, OH, CO2), 91 (PhCH2
(1,1-d2)-2-propyn-1-ol (5): A 1 M solution of LiAlD4 (29.0 mL, 29.0 mmol) in ether was cooled to −55 °C under nitrogen atmosphere in a two neck round-bottomed flask. Next, a solution of methyl propiolate (2.7 mL, 30 mmol) in anhydrous ether (10 mL) was added dropwise, over a period of about 60 min. The reaction mixture was stirred for another 90 min at −30 °C and was then allowed to warm to room temperature over a period of about 3 h and stirred overnight. Finally, the mixture was cooled to about 0 °C and quenched by the slow addition of water (1.5 mL) followed by the dropwise addition of a solution of NaOH (0.11 g in 0.75 mL) and 1 mL of H2O. The solid was allowed to settle and decanted. The solid formed was filtered, washed with ether (2 × 25 mL), the organic layers dried with Na2SO4 and the ether was evaporated under vacuum. d2-Propargyl alcohol was obtained as an oil (∼50% by 1H NMR signals) and was used in the next reaction without further purification. 1H–NMR (CDCl3) δ (ppm): 3.4 (s, 1H, OH), 2.4 (s, 1H, CH). 13C–NMR (CDCl3) δ (ppm): 60.4, 73.7, 81.0.
(1,1-d2)Propargyl p-toluenesulphonate (6): A mixture of 5 (crude mixture of the reduction process) and p-toluenesulfonyl chloride (5.8 g, 30 mmol) in anhydrous ether (70 mL) was cooled a − 10 °C under nitrogen atmosphere. Next, KOH (8.50 g, 152 mmol) was added and the mixture was allowed to warm to room temperature, over a period of about 1 h, and then stirred for 2 h. The solid decanted was filtered, washed with ether (20 mL) and the organic layer washed with brine, dried with Na2SO4 and evaporated in vacuo. After the workup the crude was purified by column chromatography (SiO2, Hexane/EtOAc (9:1)), yielding derivative 6 as a yellow oil (2.45 g, 40% two steps). 1H–NMR (CDCl3) δ (ppm): 7.85 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 2.49 (s, 1H), 2.48 (s, 3H). 13C–NMR (CDCl3) δ (ppm): 21.6, 57.1, 75.3, 77.3, 129.8, 130.1, 132.8, 145.1; MS (ESI) m/z: 235.1 (M+. + Na).
: To a solution of 4 (117 mg; 0.50 mmol) in dichloromethane (1.0 mL) was added trifluoroacetic acid (0.25 mL) and stirred at room temperature for 2 h. The volatile components were removed under reduced pressure. Then, anhydrous DMF (5 mL), potassium carbonate (138 mg, 1.0 mmol) and d2-propargyl tosylate 6 (110 mg, 0.5 mmol) were added at room temperature. The resulting mixture was stirred at ambient temperature for about 24 h. The mixture was then diluted with water (20 mL) and extracted with diethyl ether (3 × 10 mL). The organic layers were combined, washed with brine, dried, and concentrated in vacuo. The resulting residue was then purified by flash column chromatography (hexane/ ethyl acetate (7:3)) to give the desired product (53 mg; 61%). 1H–NMR (CDCl3) δ (ppm): 7.35–7.30 (m, 2H), 7.26–7.22 (m, 3H), 3.24–3.16 (m, 1H), 2.76–2.64 (m, 2H), 2.19 (s, 1H), 1.65 (bs, 1H), 1.1 (d, J = 6.0 Hz, 3H). 13C–NMR (CDCl3) δ (ppm): 139.8, 129.3, 128.7, 126.2, 81.9, 71.1, 52.7, 43.0, 35.1, 19.5 MS (ESI) m/z: 198.2 (M+. + Na), 176.2 (M+. + H), 119.1 (PhCHCH2CH3
+.), 91 (PhCH2
+.), 58 (CHC-CD2-NH3
Radiosynthesis and quality control (QC) of [11C]L-deprenyl-D2
[11C]L-deprenyl-D2 was synthesized from [11C]MeOTf using a method previously described by our group . Briefly, cyclotron produced [11C]CO2 is reduced to [11C]CH4, and further converted in [11C]MeOTf, using the commercial platform TRACERlab® FX C PRO (General Electric). [11C]MeOTf is transferred under helium stream to a small reactor where a solution of L-nordeprenyl-D2 (1.0 ± 0.2) mg in anhydrous MEK (Merck, 0.35 mL). Once the radioactivity in the reactor reached a plateau, solution was heated to 80 °C for 1 min. Crude [11C]L-deprenyl-D2 was separated from its precursor, the solvent and other minor radiochemical impurities using semipreparative reverse-phase HPLC (Nucleosil C18ec, 250 × 10, Macherey-Nagel; CH3COONH4 0.1 M:MeCN 40:60, flow rate 6 mL/min, UV and gamma detection). The fraction containing the [11C]L-deprenyl-D2 was diluted in water (50 mL) for injection, passed through a SPE cartridge (Sep-pak C18 light), and eluted with EtOH (1 mL). [11C]L-deprenyl-D2 was formulated with saline (9 mL) and subjected to sterilizing filtration (0.22 μ).
Chemical and radiochemical impurities were detected and quantified using radio-HPLC: a mixture of TFA 0.1% and acetonitrile (75:25; v/v) was used as the mobile phase at a flow rate of 1.5 mL/min on a Nucleodur C18-ec 100–5 250 × 4.6 column (Macherey-Nagel). The whole HPLC analysis was completed within 10 min. The retention times of the L-nordeprenyl-D2 and L-deprenyl-D2 4.4 ± 0.3 min and 5.4 ± 0.3 min, respectively. The chemical identity of [11C]L-deprenyl-D2 was determined by comparing the retention time of the unlabelled reference compound. The radiochemical purity was calculated considering the portion of [11C]L-deprenyl-D2 in relation to total radioactivity. The specific activity was determined considering total radiopharmaceutical activity and the amount of the unlabelled product.
The residual solvents (such as acetone, MEK and acetonitrile) and ethanol were analysed by gas chromatography (GC) in accordance with USP general chapter <467>. The appearance of the solution was checked by visual inspection, and pH was determined using a calibrated pH-meter. Radionuclidic purity was assessed by recording the corresponding gamma spectrum and radionuclidic identity by measuring the physical half-life.
Sterility and concentration of bacterial endotoxins were tested in accordance with USP general chapters <71>and <85>, respectively.