The precursor used for the preparation of [18F]FMISO, 1-(2′-nitro-1′-imidazolyl)- 2-O-tetra- hydropyranyl-3-O-toluenesulfonylpropane-diol (NITTP) and [18F]FAZA, 1-(2,3-di-O-acetyl-5-O- tosyl-α-D-arabinofuranosyl)-2-nitroimidazole, as well as the reference compounds for FMISO and FAZA were purchased from ABX advanced biochemical compounds (Radeberg, Germany). All reagents and solvents were of analytical reagent grade and were used without further purification unless otherwise specified. (TLC) was performed on precoated plates of silica gel 60 F254 (Merck, Darmstadt, Germany). 1H-NMR and Carbon-13 nuclear magnetic resonance (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 ppm referenced to internal standards (s = singlet, singlet, d = doublet). High-resolution mass spectrometry (HR-MS) was carried out on an Agilent Technologies 6220. Solid-phase extraction cartridges (Oasis HLB (3 cm3) cartridge, Sep-Pak light QMA cartridge, neutral Al2O3 cartridge, and Sep-pak C-18 cartridge) were obtained from Waters (Milford, MA, USA). Radiosynthesis of 4-[18F]fluorobenzylamine [18F]FBA, [18F]FAZA, and [18F]FMISO were performed on a GE TRACERlab™ FX (General Electric Company, Fairfield, Connecticut, United States). The synthesis unit was installed in a hot cell allowing for safe handling of a large amount of activity to be utilized.
High-performance liquid chromatography (HPLC) purification and analysis of the radiolabeled products were performed using the Phenomenex LUNA® C18(2) column (100 Å, 250 × 10 mm, 10 μm) and Agilent C18, 4.6 mm × 250 mm, 5 μ, 100 Å respectively and the HPLC system was from Gilson 321 pump, 171 diode array detector, Berthold Technologies Herm LC. Experiments involving serum from live animals were approved by the Animal Ethics Committee of the Nuclear and Energy Research Institute (IPEN-CNEN/SP), São Paulo, Brazil. (Protocol number: 54/15/CEUA-IPEN/USP).
Synthesis of 2-nitroimidazole acetic acid 3
2-Bromoethyl acetate (216 µL, 1.32 mmol) was added to a solution of 2-nitroimidazole (200 mg, 1.77 mmol) and potassium carbonate (741 mg, 0.69 mmol) in dry acetonitrile (10 mL). The mixture was reacted under constant stirring for 21 h at room temperature, and the resulting precipitate was filtered of and washed with acetone (3×). Evaporation of the filtrate under reduced pressure yielded a yellow oil (310 mg; 1.56 mmol). Analytical TLC revealed a pure product; Rf = 0.4, EtOA/hexane, 7:3). The purified nitroimidazole ethyl acetate 2 (310 mg, 1.56 mmol) was added to a solution of 4 N NaOH (377 µL, 1.56 mmol), water (2 mL), and MeOH (2 mL). The resulting solution was stirred at room temperature for 3 h until no ester derivative was present (TLC analysis: CH2Cl2/MeOH, 9:1; Rf = 0.2). Cation-exchange resin (H + , Bio-Rad, 4 g), which had been conditioned by washing with 1.0 N H2SO4 (10 mL) and distilled water, was used to acidify the solution.
Filtration and drying of the filtrate yielded a dark yellow solid. Column chromatography of the crude product on silica gel [eluent, CH2Cl2/CH3OH (95:5)] yielded a yellow solid of compound 3 (201 mg, 97%): 1H-NMR (400 MHz, CD3OD) δ: 11.91 (s, 1 H), 7.39 (s, 1 H), 7.11 (s, 1 H), 3.31 (s, 2 H), HR-MS (ESI) calculated for C5H5N3O4H−[M + H]−: 170,0207; found, 170,0210.
Synthesis of N-(4-fluorobenzyl)-2-(2-nitro-1H-imidazol-1-yl)acetamide 4 (FBNA)
A mixture of acetic acid 3 (41 mg, 0.238 mmol) and trifluoroacetic anhydride (31.8 µL, 0.24 mmol) in 2 mL of anhydrous THF was mixed at 0 °C for 30 min, then 4-fluorobenzylamine (136.1 uL, 0.24 mmol) was added. The mixture was stirred at room temperature for an additional 30 min. Once the reaction was complete as monitored by TLC (CH2Cl2/MeOH, 9:1; Rf = 0.4), the filtrate was concentrated, and the crude product was purified by column chromatography (CH2Cl2/MeOH, 9:1 v:v) providing reference compound 4. Yield 41.3% (20.1 mg, 0.07 mmol); Rf = 0.3 (DCM: MeOH 9: 1 v: v). 1H-NMR. δH (400 MHz, CD3OD) 8.77 (s, 1 H), 7.58 (dd, J = 1.1 Hz, 2.3 Hz, 1H), 7.25 (ddd, J = 2.6 Hz, 5.1 Hz, 8.0 Hz, 2H,), 7.10 (m, 3 H), 5.19 (s, 2H), 4.23 (d, J = 5.1 Hz, 2H). 13C-NMR. δc (CD3OD, 150 MHz), 166.23, 162.51, 160.91, 135.49, 129.70, 129.29, 127.99, 115.58, 52.04, 42.07. HR-MS (ESI) calculated for C12H11FN4O3[M + H]+: 279.0815, found: 279.0886.
Production of no-carrier-added (n.c.a.) [18F]fluoride
No-carrier-added (n.c.a.) [18F]fluoride was produced via the 18O(p,n)18F nuclear reaction from [18O]H2O (Rotem Industries Ltd, Hyox oxygen-18 enriched water, min. 98%) on an ACSI TR19/9 Cyclotron (Advanced Cyclotron Systems Inc., Richmond, Canada). Cyclotron-produced [18F] fluoride was trapped on a Waters SepPak® light QMA anion exchange cartridge.
Radiosynthesis of [18F]fluoroazomycin arabinoside ([18F]FAZA), [18F]fluoro-misonidazole ([18F]FMISO) and 4-[18F]fluorobenzyl amine ([18F]FBA)
[18F] FAZA and [18F] FMISO were prepared according to literature procedures (Sorger et al. 2003; Hayashi et al. 2011; Kurihara et al. 2012) with some modifications in a GE TRACERlab™ FX automated synthesis unit. A solution of aminopolyether 2.2.2 (Kryptofix 222) (50 mg) and K2CO3 (1.8 mg) in CH3CH/water (86%, 1 mL) was used to elute the n.c.a. [18F]fluoride (16–22 GBq) from the QMA cartridge into a glassy carbon reactor. CH3CN (1.5 mL) was added to the reactor, and azeotropic drying was performed at 50 °C and 85 °C under a nitrogen flow under a vacuum for 15 min.
For the radiosynthesis of [18F]FAZA, this was followed by the addition of a solution of 1-α-D-(2,3-diacetyl-5-tosyloxy-arabinofuranosyl)-2-nitroimidazole (5 mg, 10.3 µmol) in dimethylsulfoxide (0.8 mL) and the mixture was heated for 10 min at 120 °C. After cooling at room temperature, 0.1 M NaOH (1.0 mL) was added for 3 min at room temperature to ensure complete deprotection. Finally, [18F]FAZA was neutralized with the addition of 0.2 M AcOH (1.5 mL) and the reaction mixture was passed through a Waters SepPak Alumina N Light cartridge into a sealed product vial.
The collected eluent (3.5 mL) was directly injected onto HPLC, using a Phenomenex Luna C18(2) column with an isocratic elution profile of 8% EtOH in water at flow rate of 2 ml/min. [18F]FAZA retention time under this conditions was 31–34 min.
For [18F]FMISO, a solution of 1- (2′-nitro-1′-imidazolyl) -2-O-tetrahydropyranyl-3-O-toluenesulfonylpropane-diol (NITTP, 5 mg) in CH3CH (1.0 mL) was added to the reactor and the mixture was heated at 100 °C for 10 min. After cooling at room temperature, 1 M HCl (1.0 mL) was added for 10 min at 100 °C to ensure complete deprotection. After cooling to room temperature, the reaction was neutralized with 30% w/v sodium acetate solution in water (1.0 mL). The reaction mixture was then passed through a Waters SepPak Alumina N Light cartridge into a sealed product vial. The collected eluent (3.0 mL) was directly injected onto HPLC, using a Phenomenex Luna C18(2) column with an isocratic elution profile of 8% EtOH in water at a flow rate of 2 ml/min. [18F]FMISO retention time under this conditions was 14.5 to 17.4 min.
[18F]FBA was synthesized according to the published automated synthesis procedure (Way and Wuest 2013).
Radiosynthesis of N-(4-[18F]fluorobenzyl)-2-(2-nitro-1H-imidazol-1-yl)acetamide ([18F]FBNA)
The radiosynthesis of [18F]FBNA was performed manually. Compound 3 (5 mg) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (4.5 mg, (1 equiv.) dissolved in DMSO (500 µL) was reacted in a sealed vial with [18F]FBA (~ 740 MBq) in THF (500 µL), and 25 µL (2.6 equiv.) of DIPEA. The reaction was optimized by screening different temperatures (room temperature, 65 °C, 85 °C and 100 °C) in a heating block with agitation. Upon completion, the reaction was allowed to cool for 5 min prior to HPLC purification. The crude reaction mixture was diluted in Mili Q water (1 mL) and injected onto a HPLC column for purification.
HPLC purification was completed on the Gilson HPLC systems (HPLC Column is a Phenomenex LUNA® C18(2) column (100 Å, 250 × 10 mm, 10 µm) using gradient elution (A: water; B: CH3CN; 3 mL/min, 0 min 5% B, 3.5 min to 23 min gradient to 90% B, 25 min 90% B). The retention time of [18F]FBNA was 20–21 min as confirmed by the co-injection with reference compound. The collected HPLC fraction was concentrated, and the solvent was removed at 50 °C under reduced pressure. Purified radiotracer [18F]FBNA was then reconstituted in saline containing 8% of EtOH.
Aliquots of purified radiotracer [18F]FBNA and reference compound were analyzed by HPLC to determine chemical purity, radiochemical purity, and confirm chemical identity. HPLC analysis involved the following conditions: Stationary phase: Agilent C18, 4.6 mm × 250 mm, 5 μ, 100 Å; -Mobile phase: Solvent (A) Water Milli Q + 0.1% TFA, Solvent (B) 100% CH3CN; Isocratic elution: 70% (A), 30% B for 30 min; flow rate: 1 mL/min; Detection wave length: 280 and 300 nm. The pH of the final solution was determined using pH test strips; an aliquot of the sample was deposited in the test region of the indicator strip and a colorimetric comparison was made with the standard provided by the manufacturer. The chemical identity of radiotracer [18F]FBNA was determined by co-injection with the reference compound.
The molar activity of radiotracers [18F]FBNA, [18F]FAZA and [18F]FMISO was determined using HPLC analysis and calculated at the end of the radiosynthesis using a calibration curve (nmol of the respective reference compound versus area under the curve recorded in the analytical HPLC profile at 300 nm). To calculate the molar activity, the injected activity of the respective radiotracer was divided by the quantities in nmol of the reference compound that was extrapolated from the calibration curve.
In vitro stability studies
The stability of radiotracer [18F]FBNA was studied at room temperature in physiological saline solution (0.9% NaCl) and pH = 7.0. The samples (~ 3.7 MBq) had a final volume of 500 µL. The stability of the compound was monitored via HPLC analysis over a time course of 6 h. The stability of radiotracer [18F]FBNA was also studied in mouse serum to assess its suitability in vivo experiments. 50 µL (3.7 MBq) aliquots of the radiotracer was added to 450 µL of freshly separated mouse serum, and the mixture was incubated at 37° C for up to 6 h. 20 µL aliquots were removed at different time points (0 h, 1 h, 3 h and 6 h). CH3CN (20 µL) was added for the precipitation of serum proteins. After centrifugation (5000× g, for 10 min), the supernatant was analyzed by HPLC as described in the previous section. All experiments were performed in triplicate.
Determination of lipophilicity
The shake-flask method was used to determine the lipophilicity of [18F]FBNA (Wilson et al. 2001). The partition coefficient of [18F]FBNA was measured using n-octanol as the organic phase and PBS (pH 7.4) as the aqueous layer. Then, 500 µL of each layer was added to a LoBind Eppendorf tube, to which 50 µL (< 1 MBq) of [18F]FBNA was added, and the mixture was shaken vigorously for 5 min. The mixture was then centrifuged at 2000 rpm for 2 min to allow the layers to separate. Aliquots of 100 μL were removed from each layer, and the amount of [18F]FBNA present in each layer was measured in a Wizard gamma counter. Experiments were performed in triplicate.
Cellular uptake studies
MKN45 cells (American Type Culture Collection, Manassas, VALLC (Tamura et al. 1996) and AGS cells (ATCC CRL-1739) were cultured in RPMI (Gibco, Life technologies, MD, USA). Both cell lines were supplemented with 10% fetal bovine serum (Gibco, Life technologies, MD, USA) and 50 µg/mL of gentamicin (Gibco, Life technologies, MD, USA). Mycoplasma contamination in cultured cells was excluded using Lonza Mycoplasma Detection Kit.
For cell uptake experiments, 5 × 105 cells were seeded in two 6-well plates and grown for 24 h at 37 °C and 5% CO2 and 21% O2. After this period, one plate remained under normoxic conditions (21% of O2). The other plate was cultured in the H35 Hypoxystation (Don Whitley Scientific Ltd, Shipley) under hypoxic conditions (37 °C, 5% CO2 and 1% of O2) for 48 h. Cells were then incubated with 0.37 MBq of [18F]FBNA or [18F]FAZA (3 wells per plate) for 1 h at 37 °C. At the end of the experiment, cells were washed 5 times with ice-cold PBS and 1.0 M NaOH was added to lyse the cells and remove them into a gamma-counter tube. The radioactivity in each tube was counted in a Cobra II gamma counter (Packard, EUA). The experiments were done three times (n = 3) and GraphPad Prism 7 Software (San Diego, CA, USA) was used to analyze the data.