All chemicals, reagents, and solvents for the synthesis and analysis were analytical grade. The amino acid precursors H-Glu(OtBu)-OtBu.HCl and H-Lys(Z)-OtBu.HCl were purchased from EMD Millipore. The Bu4N+-HCO3
− solution and Chromafix (30-PS-HCO3, Macherey-Nagel) cartridges were purchased from ABX. All other chemicals and solvents were purchased from Sigma-Aldrich. All solvents were dried and/or distilled prior to utilization. 1H-NMR and 13C-NMR spectra were recorded on an Agilent/Varian Inova two-channel 400-MHz spectrometer, an Agilent/Varian Inova four-channel 500-MHz spectrometer, and an Agilent/Varian VNMRS three-channel 600-MHz spectrometer. Chemical shifts are given in parts per million (ppm) referenced to internal standards (s = singlet, bs = broad singlet, d = doublet, dd = doublet of doublet, ddd = doublet of doublet of doublet, t = triplet, M = multiplet, m = massif). Mass spectra were recorded using a Micromass ZABSpec Hybrid Sector-TOF by positive mode electrospray ionization. High resolution mass spectra (HRMS) were carried out on an Agilent Technologies 6220 oaTOF. Crude reaction mixtures were analyzed by TLC and HPLC. Thin-layer chromatography (TLC) was monitored using HF254 silica gel. HPLC analyses were performed on a semi-preparative Luna C18 column (100 Å, 10 μm, 250 × 10 mm) or Jupiter C12 (100 Å, 10 μm, 250 × 10 mm).
Both columns were connected to their corresponding guard columns (Phenomenex Nucleosil LUNA (II) RP C18 pre-column (5 μm, 50 × 10 mm) and Jupiter C12 pre-column (5 μm, 50 × 10 mm)). UV detection was performed at 210 and 254 nm. Radioactivity detection was achieved using a well-scintillation NaI (Tl) detector.
[18F]Fluoride was produced by the 18O(p,n)18F nuclear reaction through proton irradiation of enriched (98 %) 18O water (3.0 mL, ROTEM, Germany) using a TR19/9 cyclotron (Advanced Cyclotron Systems, Inc., Richmond, BC, Canada).
Chemical synthesis
(S)-Di-tert.-butyl-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)-l-glutamate 2
One hundred fifty milligrams (0.507 μmol) of Glu(OtBu)-OtBu·HCl 1 was dissolved in 2 mL of freshly distilled CH3CN. Seventy microliters of Et3N (0.507 μmol) and 142 mg (0.554 μmol) of N,N′-disuccinimidyl carbonate was added to the solution. The reaction was stirred at 25 °C for 12 h and concentrated under reduced pressure. After re-solubilization in 5 mL of EtOAc and successive washing with 10 mL of 10 % citric acid and 10 mL of brine, the organic layer was dried over Na2SO4 and concentrated under reduced pressure to afford 197 mg of a pale yellow powder. This unpurified powder contained 90 % of desired compound 2 (yield 78 %). TLC: (EtOAc/hexane, 3/1): R
f
= 0.7. 1H-NMR (400 MHz, CDCl3) δ: 1.46 (s, 9H), 1.50 (s, 9H), 1.94–2.14 (m, 1H), 2.10–2.21 (m, 1H), 2.24–2.44 (m, 2H), 2.83 (s, 4H), 4.23 (dt, 1H, J = 5.1 Hz, J = 7.7 Hz), 6.19 (d, 1H, J = 7.7 Hz). 13C-NMR (100.5 MHz, CDCl3) δ: 25.47, 27.59, 27.97, 28.00, 31.15, 54.78, 80.95, 83.09, 151.06, 169.60, 169.76, 171.95.
(S)-2-[3-((S)-1-tert.-Butylcarboxylate-(5-benzyloxycarbonylpentyl))ureido]-di-tert.-butyl pentanedioate 4
Compounds 4 and 5 and DCFPyL were previously synthesized [15, 16]. Herein, we describe an improved methodology. The yellow powder containing 90 % of compound 2 (197 mg, 90 % purity) was dissolved without further purification in 3 mL of CH2Cl2 containing 100 μL of Et3N and 187 mg (501 μmol) of μs-benzyloxycarbonyl-l-lysine tert.-butyl ester hydrochloride (H-Lys(Z)-OtBu.HCl) 3. The reaction mixture was stirred for 12 h at 25 °C, and progress of the reaction was monitored by TLC (CH2Cl2/MeOH 95/5). Upon completion, the reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography (CH2Cl2/MeOH 95/5) to afford 252 mg (92 %) of desired compound 4 as a clear gel. TLC: (CH2Cl2/MeOH 95/5): R
f
= 0.2. 1H-NMR (600 MHz, CDCl3) δ: 1.16–1.24 (m, 1H), 1.24–1.30 (m, 1H), 1.35 (s, 9H), 1.37 (s, 9H), 1.38 (s, 9H), 1.39–1.47 (m, 2H), 1.48–1.56 (m, 1H), 1.61–1.69 (m, 1H), 1.69–1.77 (m, 1H), 1.93–2.01 (m, 1H), 2.13–2.25 (m, 2H), 3.02–3.15 (m, 2H), 4.22–4.28 (m, 1H), 4.28–4.33 (m, 1H), 4.99 (d, 1H, J = 12.5 Hz), 5.05 (d, 1H, J = 12.5 Hz), 5.37 (d, 1H, J = 8 Hz), 5.44 (s, 1H), 5.45 (d, 1H, J = 8.1 Hz), 7.20–7.24 (m, 1H), 7.24–7.30 (m, 4H). 13C-NMR (150.9 MHz, CDCl3) δ: 21.36, 26.98, 27.00, 27.05, 27.32, 28.33, 30.56, 31.61, 39.69, 51.84, 52.25, 65.46, 79.44, 80.58, 81.14, 126.93, 127.03, 127.43, 135.76, 155.66, 156.10, 171.33, 171.57, 171.89. m/z (ESI) C32H51N3O9 ([M + H+]) calcd. 622.4, found 622.4.
(S)-2-[3-((S)-5-Amino-1-tert.-butoxycarbonylpentyl)ureido]pentanedioic acid di-tert.-butyl ester 5 [15, 16]
A solution of compound 4 (220 mg, 0.354 μmol) in 3 mL of MeOH was stirred with 10 mg of Pd/C under H2 atmosphere for 12 h. The reaction was filtered through celite and concentration under reduced pressure afforded 164 mg (95 %) of compound 5 as a clear gel. 1H-NMR (600 MHz, CD3OD) δ: 1.45–1.47 (m,1H), 1.47 (s, 9H), 1.47–1.49 (m, 1H) 1.49 (s, 9H) 1.50 (s, 9H), 1.57–1.70 (m, 3H), 1.78–1.86 (m, 2H), 2.04–2.10 (m, 1H), 2.28–2.40 (m, 2H), 2.79–2.88 (m, 2H), 4.18 (dd, 1H, J = 5.1 Hz, J = 9.0. Hz), 4.22 (dd, 1H, J = 5.0 Hz, J = 8.9 Hz). m/z (ESI) C24H45N7O7 ([M + H+]) calcd. 488.3, found 488.3.
(S)-2-[3-((S)-1-Carboxy-5-[3-(6-fluoropyridine)carbonyl)amino)pentyl)ureido]-pentanedioic acid (DCFPyL) [15]
In a flame-dried flask, HBTU (156 mg, 410 μmol), DIPEA (72 μL, 410 μmol), and 6-fluoronicotinic acid 6 (58 mg, 410 μmol) were added to a solution of 100 mg (0.205 μmol) of compound 5 in 4 mL of distilled CH2Cl2. The reaction was stirred at 25 °C for 3 h under nitrogen, and progress of the reaction was monitored by TLC (hexane/ethyl acetate 1/1 v/v). Upon completion, the reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography (CH2Cl2/MeOH 97/3) to afford 97 mg (78 %) of tert.-butyl ester intermediate as a white powder. TLC: (EtOAc/hexane 1/1): R
f
= 0.45. 1H-NMR (600 MHz, CDCl3) δ: 1.39 (s, 9H), 1.41–1.46 (m, 19H), 1.51–1.63 (m, 2H), 1.64–1.71 (m, 1H), 1.73–1.80 (m, 1H), 1.80–1.86 (m, 1H), 1.98–2.08 (m, 1H), 2.25–2.39 (m, 2H), 3.33–3.42 (m, 1H), 3.50–3.59 (m, 1H), 3.69–3.77 (m, 1H), 4.45–4.24 (m, 2H), 5.59 (t, 1H, J = 9.5 Hz), 5.88 (t, 1H, J = 9.8 Hz), 7.00 (d, 1H, J = 8.1Hz), 7.81–7.90 (m, 1H), 8.41 ( t, 1H, J = 7.9 Hz), 8.81 (s, 1H). 13C-NMR (150.9 MHz, CDCl3) δ: 23.36, 27.81, 27.88, 27.95, 28.04, 28.66, 31.51, 32.43, 39.93, 53.12, 53.69, 80.72, 81.64, 82.54, 109.09 (d, J = 37.6 Hz), 128.56 (d, J = 4.3 Hz) 140.96 (d, J = 8.7 Hz), 147.95 (d, J = 13.1 Hz), 157.60, 164.86 164.87 (d, J = 244 Hz), 172.16, 172.31, 173.49. m/z (ESI) C30H47FN4O8 ([M + H+]) calcd. 611.3, found 611.3.
Tert.-butyl ester intermediate was treated with 6 mL of CH2Cl2/TFA (1/1 v/v) for 8 h at 25 °C, concentrated under reduced pressure, and purified by HPLC. HPLC purification was performed on a semi-preparative Jupiter C12 column (100 Å, 10 μm, 250 × 10 mm). The eluting solvent started with a 10/90 CH3CN/(water 0.5 % TFA) gradient for 5 min at a flow rate of 2 mL min−1 followed by a gradient from 10/90 to 70/30, v/v, for 25 min. Compound DCFPyL eluted at 14.5 min, and after solvent removal, 54 mg (67 %) of DCFPyL was isolated as a white powder. 1H-NMR (400 MHz, D2O) δ: 1.42–1.56 (m, 2H), 1.60–1.73 (m, 2H), 1.73–1.83 (m, 1H), 1.85–1.95 (m, 1H), 1.95–2.05 (m, 1H), 2.13–2.24 (dt, 1H, Jd = 20.6 Hz, Jt = 7.5 Hz), 2.51 (t, 2H, J = 7.2 Hz), 3.43 (t, 2H, 6.5 Hz), 4.25 (ddd, 2H, J = 9.4 Hz, J = 5.1 Hz, J = 9.2 Hz), 7.23 (d, 1H, J = 8.2 Hz), 8.31 (dt, 1H Jt = 8.2 Hz, Jd = 2.2 Hz), 8.57 (d, 1H, J = 2.2 Hz).13C-NMR δ (125.7 MHz, D2O): 23.26, 27.11, 28.56, 31.00, 31.55, 40.68, 53.51, 54.07, 109.05 (d, J = 37.8 Hz), 128.47 (d, J = 4.4 Hz) 140.87 (d, J = 8.1 Hz), 147.98 (d, J = 12.9 Hz), 160.26, 164.62 (d, J = 240.1 Hz), 168.16, 177.12, 178.05, 178.19. m/z (HRMS) C18H22FN4O8 ([M − H+]) calcd. 441.1427, found 441.1430.
6-Trimethylammonium-nicotinic acid 2,3,5,6-tetrafluorophenyl ester triflate salt 8 (adapted from reference [21])
One gram (6.34 mmol) of 6-chloronicotinic acid 7; 1.1 g (6.5 mmol) of 2,3,5,6 tetrafluorophenol; and 1.31 g (6.34 mmol) of N,N′-dicyclohexylcarbodiimide (DCC) were stirred in dioxane (40 mL) for 5 h at 25 °C. Progress of the reaction was monitored by TLC (EtOAc/hexane 4/1). Upon completion, the reaction mixture was filtered and concentrated under reduced pressure and the residue was purified by recrystallization in hot hexane to afford 1.35 g (70 %) of the 6-chloronicotinic acid active ester intermediate as a white powder [18]. TLC: (EtOAc/hexane 4/1): R
f
= 0.28. 1H-NMR (600 MHz, CDCl3) δ 7.11 (tt, 1H, J = 7.1 Hz, J = 9.9 Hz), 7.57 (d, 1H, J = 8.3 Hz), 8.43 (dd, 1H, J = 8.3 Hz, J = 2.7 Hz), 9.21 (d, 1H, J = 2.7Hz). m/z (ESI) C12H4ClF4NO2 ([M + H+]) calcd. 305.0, found 304.9.
6-Chloronicotinic acid active ester intermediate (130 mg) [18] was dissolved in 3 mL of a 1 M Me3N solution in THF and stirred 2 h at 25 °C. After 5 min, a white precipitate was formed. After completion of the reaction, the precipitate was collected by filtration and washed with diethyl ether and cold CH2Cl2. The obtained white powder was suspended in 5 mL of CH2Cl2 containing 2 % TMSOTf and sonicated for 10 min. The reaction mixture was concentrated under reduced pressure and washed with diethyl ether to afford 140 mg (68 % over two steps) of a gray powder after drying. 1H-NMR (600 MHz, CD3CN) δ 7.43 (tt, 1H, J = 7.4 Hz, J = 10.5 Hz), 8.07 (dd, 1H, J = 8.6 Hz, J = 0.8 Hz), 8.85 (dd, 1H, J = 8.6 Hz, J = 2.3 Hz), 9.34 (dd, 1H, J = 2.3 Hz, J = 0.8 Hz). m/z (ESI) C15H13F4N2O2 ([M+]) calcd. 330.1, found 330.0.
(S)-2-[3-((S)1-Carboxy-5-((6-trimethylammonium-pyridine-3-carbonyl)-amino)-pentyl)-ureido]-pentanedioic acid trifluoroacetate salt 9
To a solution of 80 mg (164 μmol) of compound 5 in CH2Cl2 (4 mL) was added compound 8 (100 mg, 209 μmol) and 100 μL of DIPEA (572 μmol). The reaction was stirred for 2 h at 25 °C and then concentrated under reduced pressure.
Progress of the reaction was monitored by TLC: (CH2Cl2/MeOH 4/1): R
f
= 0.26. HPLC purification was performed on a semi-preparative Jupiter C12 column (100 Å, 10 μm, 250 × 10 mm). The eluting solvent started with a gradient from 5/95 to 70/30 acetonitrile/(water 0.5 % TFA) for 20 min at a flow rate of 2 mL min−1. Then the eluent was kept at 70/30 acetonitrile/(water 0.5 % TFA) for 10 min to elute the desired compound at 25.8 min. After removal of the solvent under reduced pressure gave 96 mg (77 %) of desired compound 9 as a white powder. 1H-NMR (600 MHz, D2O) δ: 1.32 (s, 9H), 1.34 (s, 9H), 1.35(s, 9H), 1.35–1.39 (m, 2H) 1.55–1.66 (m, 3H), 1.70–1.82 (m, 2H), 1.95–2.03 (m, 1H), 2.30 (M, 2H), 3.36 (t, 2H, J = 6.8 Hz), 3.57 (s, 9H), 4.02 (ddd, 2H, J = 9.5 Hz, J = 8.7 Hz, J = 5.1 Hz), 7.94 (d, 1H, J = 8.8 Hz), 8.35 (dd, 1H Jt = 8.8 Hz, Jd = 2.3 Hz), 8.57 (d, 1H, J = 2.3 Hz). 13C-NMR (125.7 MHz, D2O) δ: 23.35, 27.63, 28.19, 28.20, 28.31, 28.78, 31.92, 32.58, 40.80, 54.34, 54.99, 56.06, 83.47, 84.18, 84.36, 115.48, 118.47, 133.37, 141.14, 148.90, 160.16, 167.46, 174.55, 175.14, 175.33. m/z (HRMS) C33H56NO8 ([M+]) calcd. 650.4123, found 650.4116. Mp = 56 °C.
Radiosynthesis and quality control of [18F]DCFPyL
Radiosynthesis of [18F]DCFPyL was performed on a GE TRACERlabTM FX (GE Healthcare, Mississauga, ON, Canada). The synthesis module was modified in terms of program and hardware (see Fig. 3). The synthesis unit was installed and operated in a shielded hot cell.
Analytical HPLC was carried out using a Gilson HPLC (Mandel Scientific Company Inc.; Guelph, Ontario, Canada) by injection of HPLC-purified [18F]DCFPyL onto a Phenomenex Nucleosil Luna C18 column (10 μm, 250 × 10 mm) and elution with 20 % CH3CN/0.2 % TFA for 5 min at 2 mL min−1, followed by gradient elution from 20 % to 38 % CH3CN for 5 min and from 38 % to 70 % CH3CN for 15 min with isocratic elution at 70 % CH3CN for 15 min. Radio-TLC analysis on silica gel plates gave a R
f
value of 0.6 in 95 % CH3CN/H2O (Additional file 1: Figure S4).
Automated synthesis of [18F]DCFPyL
Radiosynthesis of [18F]DCFPyL was performed on a GE TRACERlabTM FX (GE Healthcare, Mississauga, ON, Canada). The synthesis module was modified in terms of program and hardware (Fig. 3). The synthesis unit was installed and operated in a shielded hot cell.
In vivo tumor models
All animal experiments were carried out in accordance with the guidelines of the Canadian Council on Animal Care (CCAC) and approved by the local animal care committee (Cross Cancer Institute, University of Alberta).
PET imaging experiments were carried out in LNCaP and PC3 tumor-bearing Balb/c nude mice (Charles River Laboratories, Quebec, Canada). Male Balb/c nude mice were housed under standard conditions with free access to standard food and tap water. LNCaP and PC3 cells (5 × 106 cells in 100 μL of PBS) were injected into the upper left flank of the mice (20–24 g). Before injecting LNCaP cells, the mice received a 1.0-mg/pellet containing testosterone in a 60-day release preparation (Innovative Research of America, Sarasota, FL, USA).
The pellet was implanted subcutaneously into the upper right flank in order to provide a constant level of testosterone needed by the androgen receptor positive LNCaP cells. Tumors reached sizes of approximately 1 cm3 which were suitable for all in vivo experiments.
Radiometabolite analysis
Normal BALB/c mice were injected with 10–20 MBq of [18F]DCFPyL. Venous blood samples were collected at 5, 15, 30, and 60 min p.i. via the mouse tail vein and further processed. Blood cells were separated by centrifugation (13,000 rpm × 5 min). Precipitation of proteins in the supernatant was achieved by the addition of 2 volume parts of MeOH, and the samples were centrifuged again (13,000 rpm × 5 min). Fractions of blood cells, proteins, and plasma were measured in a Wizard gamma counter to determine radioactivity per sample. The clear plasma supernatant was injected onto a Shimadzu HPLC system.
The samples were analyzed using a Phenomenex Luna 10u C18 [2] 100 A, 250 × 4.6 mm column at a constant flow rate of 1 mL min−1 and the following gradient with water/0.2 % TFA as solvent A and CH3CN as solvent B: 0–7.5 min 20 % B, 7.5–15 min gradient to 90 % B, 15–20 min 90 % B.
Dynamic PET imaging
General anesthesia of tumor-bearing mice was induced with inhalation of isoflurane in 40 % oxygen/60 % nitrogen (gas flow = 1 mL min−1), and the mice were subsequently fixed in prone position. The body temperature was kept constant at 37 °C for the entire experiment. The mice were positioned in a prone position into the center of the field of view. A transmission scan for attenuation correction was not acquired. The mice were injected with 2–10 MBq of [18F]DCFPyL (60–150 ng) in 100–200 μL of isotonic NaCl solution (0.9 %) through a tail vein catheter. For blocking studies, the animals were pre-dosed with 300 μg of DCFPyL in 50 μL saline about 10 min prior to radiotracer injection. Data acquisition was performed over 60 min in a 3D list mode. The dynamic list mode data were sorted into sinograms with 53 time frames (10 × 2, 8 × 5, 6 × 10, 6 × 20, 8 × 60, 10 × 120, 5 × 300 s). The frames were reconstructed using maximum a posteriori (MAP) as reconstruction mode. The pixel size was 0.085 × 0.085 × 0.121 mm3 (256 × 256 × 63), and the resolution in the center of the field of view was 1.8 mm. No correction for partial volume effects was applied. The image files were processed using the ROVER v 2.0.51 software (ABX GmbH, Radeberg, Germany). Masks defining 3D regions of interest (ROI) were set and the ROIs were defined by thresholding.
Mean standardized uptake values [SUV]mean = (activity/mL tissue)/(injected activity/body weight), mL/g, were calculated for each ROI. Time-activity curves (TACs) were generated for the dynamic scans only. All semi-quantified PET data are presented as means ± SEM. Statistical difference for the blocking study was tested by unpaired Student’s t test and was considered significant for P < 0.05.
Internalization experiments
105 LNCaP or PC3 cells were seeded in poly-d-lysine-coated 12-well plates 24–48 h before the assay so that cells could reach 95 % confluency. The medium was removed 1 h before the assay, and the cells were rinsed twice with PBS. After the addition of Krebs buffer (1 mL) to each well, the cells were incubated at 37 °C. Krebs buffer was aspirated, and the cells were incubated with 300 μL of [18F]DCFPyL in 0.9 % NaCl (0.1 MBq ) for 60 min at 37 °C. Cellular uptake was stopped by removing incubation media from the cells and washing the wells twice with ice-cold PBS buffer (1 mL). Surface-bound radioactivity was removed from the cells through incubating the cells twice with 0.5 mL glycine-HCl in PBS (50 mM, pH 2.5) for 5 min at 37 °C. Cells were washed again with ice-cold PBS before the addition of radio-immunoprecipitation assay (RIPA) buffer (400 μL) to lyse the cells. Cells were returned into the incubator for 10 min, and cell lysates were collected for counting. Radioactivity of surface-bound and internalized fraction was measured in a WIZARD2 Automatic gamma counter (Perkin Elmer, Waltham, MA, USA). Total protein concentration in the samples was determined by the bicinchoninic acid method (BCA; Pierce, Thermo Scientific 23227) using bovine serum albumin (800, 600, 400, 300, 200, 100, 50 μg/mL, blank) as protein standard. Data are expressed as percent of total uptake per 1 mg protein (% of total uptake/mg protein).
Tracer kinetic analysis
Tracer kinetic analysis was performed using a two-tissue compartmental model using dynamically acquired PET imaging data. Full details on tracer kinetic analysis are given in the Additional file 1.