General Information
Unless otherwise stated, all chemicals were obtained from commercial sources and used without further purification. The 1H and 13C NMR spectra were taken on Bruker nuclear magnetic resonance spectrometer. Chemical shifts are reported as δ in units of parts per million (ppm) relative to internal standard (1H NMR: SiMe4 = 0.00 ppm). Data for 1H NMR spectra are reported as follows: chemical shifts are reported as δ in units of parts per million (ppm) relative to tetramethylsilane (δ = 0, s); multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), or br (broadened); coupling constants are reported as a J value in Hertz (Hz); the number of protons (n) for a given resonance is indicated nH, and based on the spectral integration values. High-performance liquid chromatography (HPLC) analysis and purification was performed on a waters HPLC system equipped with a SPD UV detector, a LC pump system, and a CBM BUS module. A Lablogic Scan-RAM radio-HPLC detector was used for the radioactive signal. Water containing 0.1% acetic acid/sodium acetate and 0.2 g/L ascorbic acid) was filtered before use as HPLC mobile phase. HPLC purification of 6-[18F]fluoro-L-DOPA was performed on a semi-preparative reversed-phase Phenomenex Gemini column (Phenomenex, Luna, C18(2), 5 µ, ,250 mm ⋅ 10 mm) with method A (flow rate: 5 mL min−1). Analysis of 6-[18F]fluoro-L-DOPA was performed on a reversed-phase analytical Phenomenex column (Phenomenex, Lu, a༌C18(2), 5 µm, 250 mm ⋅ 4.6 mm) with Method B (flow rate: 1 mL min−1).
Synthesis Of Precursor 1b
tert -Butyl (S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate (2)
Under argon atmosphere, to a solution of L-DOPA (2.96 g, 15 mmol) in tert-butyl acetate (30 mL, 225 mmol) at 0 oC was added HClO4 (70% in H2O, 1.9 mL, 22 mmol). The resulting mixture were stirred overnight, allowing the reaction temperature raise to room temperature. Upon completion of the reaction, water was added and the pH of solution was adjusted to 8 with a 10% aqueous solution of K2CO3. The organic phase was separated and the aqueous phase was extracted with DCM (60 mL × 3). The combined organic layers were dried over Na2SO4 and purified by chromatography on a silica gel column to afford titled compound as a yellow liquid (1.62 g, 42%). 1H NMR (400 MHz, CDCl3) δ 6.72 (d, J = 7.9 Hz, 1H), 6.56 (d, J = 2.0 Hz, 1H), 6.55 (dd, J = 8.0, 1.7 Hz, 1H), 4.34 (s, 4H), 3.62 (dd, J = 8.2, 4.7 Hz, 1H), 3.01 (dd, J = 13.8, 4.7 Hz, 1H), 2.72 (dd, J = 13.9, 8.4 Hz, 1H), 1.49 (s, 9H).
tert -Butyl ( S )-2-(( tert -butoxycarbonyl)amino)-3-(3,4-dihydroxyphenyl) propan-oate (3)
Under argon atmosphere, to a solution of 2 (0.77 g, 3 mmol) in EtOH (15 mL) at room temperature was added Boc2O (0.68 g, 3 mmol). The resulting mixture were stirred at room temperature for 5 h. After the solvent was removed via Rota-Vap, the residue was purified by chromatography on a silica gel column (PE/EA = 3/1) to afford titled compound a white solid (0.8 g, 74%). 1H NMR (400 MHz, CDCl3) δ 6.74 (d, J = 6.3 Hz, 1H), 6.70 (s, 1H), 6.56 (d, J = 8.1 Hz, 1H), 6.33 (s, 1H), 6.15 (s, 1H), 5.06 (d, J = 11.8 Hz, 1H), 4.38 (q, J = 14.0, 6.5 Hz, 1H), 2.99 – 2.84 (m, 1H), 1.41 (s, 1H).
tert -Butyl (S)-3-(3,4-bis(methoxymethoxy)phenyl)-2-(( tert -butoxycarbonyl)amino) propanoate (4)
Under argon atmosphere, to a solution of 3 (562 mg, 1.6 mmol) in DCM (8 mL) at 0 oC were added DIPEA (0.7 ml, 4 mmol) and DMAP (20 mg, 0.16 mmol). The reaction mixture was stirred at the same temperature for 10 min, then MOMCl (0.3 mL, 4 mmol) was added dropwise. The resulting mixture were then heated to reflux for 20 h. After cooling to room temperature, water was then added and the aqueous phase was extracted with DCM. The combined organic layers were washed by a 10% aqueous solution of K2CO3 and dried over Na2SO4 and purified by chromatography on a silica gel column (PE/EA = 10/1) to afford titled compound as a colorless liquid (477 mg, 68%). 1H NMR (400 MHz, CDCl3) δ 7.07 (d, J = 8.3 Hz, 1H), 6.97 (s, 1H), 6.76 (d, J = 8.3 Hz, 1H), 5.21 (s, 4H), 4.98 (d, J = 8.0 Hz, 1H), 4.42 (dd, J = 13.5, 6.1 Hz, 1H), 3.51 (s, 6H), 2.99 (d, J = 4.7 Hz, 2H), 1.42 (s, 18H). 13C NMR (101 MHz, CDCl3) δ 170.96, 155.07, 147.08, 146.15, 130.66, 123.50, 118.06, 116.53, 95.45, 81.99, 79.63, 56.16, 54.70, 37.79, 28.32, 27.96.
tert -Butyl (S)-2-(( tert -butoxycarbonyl)amino)-3-(2-iodo-4,5-bis(methoxymethoxy) phenyl)propanoate (5)
Under argon atmosphere, to a solution of 4 (450 mg, 1.02 mmol) in DCM (anhydrous, 20 mL) at 0 oC were added [Bis(trifluoroacetoxy)iodo]benzene (526 mg, 1.224 mmol) and I2 (260 mg, 1.02 mmol). The reaction mixture was stirred for 3 h, allowing the reaction temperature raised to room temperature. Upon completion of the reaction (monitored by TLC), the reaction was quenched by an aqueous solution of Na2SO3. The organic layer was separated and the aqueous phase was extracted with DCM. The combined organic layers were dried over Na2SO4 and purified by chromatography on a silica gel column (PE/EA = 10/1) to afford titled compound as a colorless liquid (399 mg, 69%). 1H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 7.03 (s, 1H), 5.19 (s, 4H), 5.03 (d, J = 8.7 Hz, 1H), 4.51 (q, J = 14.7, 8.6 Hz, 1H), 3.50 (s, 6H), 3.16 (dd, J = 14.0, 5.6 Hz, 1H), 2.95 (dd, J = 13.9, 9.1 Hz, 1H), 1.43 (s, 9H), 1.38 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 171.06, 155.02, 147.36, 146.56, 133.87, 126.86, 118.24, 95.44, 91.23, 81.99, 79.61, 56.30, 54.08, 42.99, 28.28, 27.96.
tert -Butyl (S)-3-(4,5-bis(methoxymethoxy)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-(( tert -butoxycarbonyl)amino)propanoate (6)
Under argon atmosphere, to a solution of bis(pinacolato)diboron (139.7 mg, 0.55 mmol), Pd(dppf)Cl2.CH2Cl2 (20.4 mg, 0.025 mmol) and CH3COOK (147 mg,1.5 mmol) in DMF (anhydrous, 20 mL) at room temperature was added 5 (284 mg, 0.5 mmol). The reaction mixture was heated to 80 oC for 18 h. After cooling to room temperature, a saturated aqueous solution of NaCl was added, followed by ethyl acetate. The organic layer was separated and the aqueous phase was extracted with ethyl acetate (50 mL×3). The combined organic layers were then dried over Na2SO4 and purified by chromatography on a silica gel column (PE/EA = 15/1) to afford titled compound as a colorless liquid (166 mg, 58%). 1H NMR (400 MHz, CDCl3) δ 7.51 (s, 1H), 7.05 (s, 1H), 5.94 (d, J = 8.2 Hz, 1H), 5.26 (m, 4H), 4.17 (m, 1H), 3.53 (s, 3H), 3.50 (s, 3H), 3.30 – 2.95 (m, 2H), 1.47 (s, 9H), 1.37 (s, 6H), 1.36 (s, 6H), 1.34 (s, 9H).
tert -Butyl (S)-3-(4,5-bis(methoxymethoxy)-2-(4,4,5,5-tetramethyl-1,3,2-dioxa borolan-2-yl)phenyl)-2-((di tert -butoxycarbonyl)amino)propanoate (1b)
Under argon atmosphere, to a solution of 6 (61 mg, 0.11 mmol) in MeCN (anhydrous, 0.8 mL) at room temperature were added Boc2O (240 mg, 1.1 mmol), DMAP (13.7 mg, 0.11 mmol) and Et3N (46 µl, 0.33 mmol). The reaction mixture were heated to 40 oC for 24 h. After cooling to room temperature, the solvent was removed via Rota-Vap and ethyl acetate (100 mL) was added. The organic phase was then washed by a saturated aqueous solution of NH4Cl, dried over Na2SO4 and purified by chromatography on a silica gel column (PE/EA = 15/1) to afford titled compound as a yellow liquid (56 mg, 78%). 1H NMR (400 MHz, CDCl3) δ 7.49 (s, 1H), 6.88 (s, 1H), 5.28 – 5.11 (m, 5H), 3.93 (dd, J = 13.4, 3.7 Hz, 1H), 3.50 (s, 3H), 3.46 (s, 3H), 3.03 (t, 1H), 1.50 (s, 9H), 1.36 (s, 18H), 1.33 (s, 6H), 1.31 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 169.46, 151.98, 149.44, 144.56, 140.59, 124.05, 118.88, 95.39, 95.11, 83.42, 82.14, 80.82, 60.86, 56.09, 35.02, 27.73, 24.90.
Radiosynthesis Of 6-[f]fluoro-l-dopa
Noncarrier-added [18F]fluoride was obtained via the 18O(p,n)18F nuclear reaction on a RDS111 cyclotron using enriched H218O. A QMA cartridge was eluted to a reaction vessel with an aqueous solution of Kryptofix 222 and K2C2O2 and the solvent was dried azeotropically at 110 ℃ under N2. Acetonitrile (anhydrous, 2 mL) was added and dried at 110℃ under N2. A solution of 1b (20 mg) and Cu(OTf)2(py)4 (20 mg) in DMF (0.8 mL) were added and the mixture were heated to 120 ℃ for 20 minutes. Then the reaction mixture were cooled down and diluted with water (8 mL). The resulting mixture were transfer to a C18 cartridge and washed with water (8 mL). The compounds in the C18 cartridge were thus eluted with acetone (3 mL) to another reaction vessel and the volatile solvent was removed by heating to 110℃ under N2. HCl (aq. 6 M) was then added and the reaction solution was heated to 120 ℃ for 20 minutes. Then was reaction mixture was cooled down and diluted with a NaOH (aq. 0.1M, 5 mL). 6-[18F]fluoro-L-DOPA was obtained after purification by HPLC on a C18 column with water containing 0.1% acetic acid/sodium acetate and 0.2 g/L ascorbic acid as eluent (flow rate = 5 mL/min, tR = 8.9 min).
Pet/ct Scanning
This study was approved by the Institutional Review Board of Huashan Hospital (HIRB), Fudan University, China. All patients were administered intravenously 0.08 to 0.16 mCi/kg of [18F]fluoro-L-DOPA was administered intravenously. A 10 min/bed abdominal static emission scan was acquired 60-70 minutes after injection with a PET/CT scanner (Siemens Biograph 64 HD PET/CT, Siemens, Germany). Attenuation correction was performed using a low-dose CT (30-40mAs, 120 kV, Acq. 32×1.2 mm) before the emission scan. Following corrections for scatter, dead time, and random coincidences, PET images were reconstructed by TrueX+TOF with 4 iterations and 21 subsets, a Gaussian Filter (FWHM 4.0 mm).