A. Kirjavainen et al.: Electrophilic addition of chlorine monofluoride for PET-tracers
Electrophilic addition of chlorine monofluoride for PET-tracers
Anna Kirjavainen, Sarita Forsback, Tove J. Grönroos, Laura Haavisto, Merja Haaparanta
and Olof Solin
Email:
Supplementary Information
Contents: Supplementary Methods
Supplementary Methods
Radiosynthesis
Solvents were purchased from commercial suppliers. All reagents and solvents were used without further purification. [18F]Fluoride was produced through the nuclear reaction 18O(p,n)18F by irradiating 18O-enriched water (18O, >98 atom%, Rotem Industries, Israel) with an 18 MeV proton beam produced with a CC-18/9 cyclotron (Efremov Scientific Research Institute of Electrophysical Apparatus, St Petersburg, Russia). A niobium target was filled with 2.1 ml 18O-enriched water for irradiation. The irradiated water was transferred to the synthesis device through PEEK tubing. After a typical 30 minutes irradiation with 40 µA beam current, the amount of [18F]F- was about 37 GBq at EOB.
The [18F]F2 was produced in an electrical discharge chamber by 18F/19F-exchange reaction. The source of 18F-fluorine was [18F]fluoromethane, which was produced from methyl iodide by a nucleophilic substitution reaction with [18F]F-/Kryptofix K2.2.2-complex in acetonitrile. The [18F]fluoromethane was transferred with low amounts of carrier fluorine (~1000 nmol) in neon (neon gas 99.99 %), 0.5% F2 in neon: Linde AG - Geschäftsbereich Linde Gas, Unterschleissheim, Germany) to a quartz discharge chamber and a high voltage discharge was initiated through this mixture. A detailed description of the [18F]F2 synthesis is presented elsewhere1.
After the [18F]fluoromethane/F2-discharge chlorine gas (1400 nmol, 1.0% Cl2 in neon: AGA, Turku, Finland) was added to the quartz vessel. This gas mixture was then bubbled through a solution of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,3,3-trifluoroallyl)acetamide (EF1,2A) (1.0 - 1.24 mg, 3.8 - 4.7 µmol) dissolved in trifluoroacetic acid (750 µl) at room temperature. Trifluoroacetic acid was evaporated using neon flow with heating at 60 ºC. The residue was dissolved in HPLC-eluent and injected to the HPLC. [18F]EF4Cla, [18F]EF4Clb, [18F]EF5, and EF3Cl2 were separated by a radio-HPLC method described below.
Chromatographic and mass-spectrometric methods
Radio-HPLC was performed using a VWR-Hitachi L-2130 HPLC pump (VWR Hitachi, VWR International GmbH, Darmstadt, Germany) combined with a VWR-Hitachi L-2400 UV-absorption detector (λ=325 nm) and a 2 x 2 inch NaI-crystal for radioactivity detection. A SunFire C18 column (5 µm, 4.6 x 150 mm, Waters Corp., Milford, MA, USA) was used and was eluted with 0.1% formic acid in methanol: water (38:62, v/v) at a flow rate 1.0 ml/min. The same HPLC conditions were used for separation of the radiolabel compounds (Supplementary Fig. 1).
Liquid chromatography mass spectrometry (LC-MS) was used for the identification of the chlorinated isomers, [18F]EF5, and non-radioactive EF3Cl2 by their mass per charge (m/z). Radio-LC-MS(/MS) was performed with a linear ion trap quadrupole mass spectrometer (QTRAP, Applied Biosystems SCIEX, Toronto, Canada) equipped with a turbo ion spray source and an Agilent 1100 series pump (Agilent Technologies, CA, USA). A Rheodyne injector with a 0.5 µl loop was used (Rheodyne, IDEX Health & Science LLC, Rohnert Park, CA, USA). The radioactivity detector was placed between the LC outlet and MS inlet. The radioactivity detector consisted of an approximate 0.1 µl teflon loop embedded in a plastic scintillator (Meltilex®, Wallac, Perkin Elmer, Turku, Finland). Scintillation light from β+-particles interacting with the scintillator was detected by a double cathode PM-tube and this scintillation signal was converted to millivolt signal proportional to the radioactivity concentration eluting from the column. Samples were separated on a Waters SunFire C18 column (2.1 x 100 mm, 5 μm, Waters Corp., Milford MA, USA) using 0.1 ml/min flow rate. The mobile phase consisted of 38% methanol with 0.1% formic acid and 62% water (v/v). The turbo ion spray source was operated in negative ion mode. Selected ion masses (SIM) were monitored; EF1,2A m/z 263 amu, [18F]EF5 m/z 301 amu, [18F]EF4Cla m/z 317 amu, [18F]EF4Clb m/z 317 amu, EF3Cl2 m/z 333 amu. The widths of windows were 0.5 amu in LC/MS identification (Supplementary Fig. 2).
The separated products and precursor were fragmented using LC-MS/MS system described above and the LC/MS/MS spectra of the three 18F-labeled compounds showed clean splitting patterns (Supplementary Fig. 3). Fragmentation conditions varied for different compounds. [18F]EF4Cla, [18F]EF4Clb, [18F]EF5 and EF3Cl2 were fragmented using 15 kV collision energy. In fragmentation of the precursor, 5 kV collision energy was used.
The mass peaks m/z 96, 112, 196 and 214 amu (1-4, Supplementary Fig. 3) were common to [18F]EF4Cla, [18F]EF4Clb and [18F]EF5. The mass peaks at m/z 270 amu for [18F]EF4Cla and [18F]EF4Clb were identified as the parent ion lacking the NO2-group. The corresponding mass peak for [18F]EF5 was observed at m/z 254 amu. The breakage of the terminal carbon bond of the halogenated tail in [18F]EF4Cla, [18F]EF4Clb and [18F]EF5 was seen in the formation of the mass peaks at m/z 250 amu for [18F]EF4Cla (parent minus -CF3), at m/z 234 for [18F]EF4Clb (parent minus -CClF2) and at m/z 230 for [18F]EF5 (parent minus -CF3). For both [18F]EF5 and [18F]EF4Clb a product was observed at m/z 186 amu, identified as 1-(2-((1,1-difluoroethan-1-id-2-yl)amino)-2-oxoethyl)-1H-imidazol-2-ide. In this ion both the terminal carbohalogen-group as well as the NO2-group were detached. The corresponding product for [18F]EF4Cla at m/z 202 amu was observed in only minute amounts. Furthermore, in the splitting pattern of [18F]EF4Clb ions at m/z 281 amu (parent minus -Cl) and at m/z 261 amu (parent minus -Cl and -F) were observed.
Lipophilicity measurements and calculations
The lipophilicities of [18F]EF4Cla, [18F]EF4Clb and [18F]EF5 were determined at physiological conditions (logD) and ClogP values were calculated using two different commercially available codes. For comparison, HPLC retention volumes of the compounds normalized to that of EF3Cl2 are presented in Table 1a. The apparent partition coefficients (LogD) for [18F]EF4Cla, [18F]EF4Clb and [18F]EF5 were determined using the shake flask method2. Radiolabeled compound (3 - 5 MBq) was shaken in a saturated mixture of n-octanol (10 ml) and 0.1 M phosphate buffer (pH 7.4, 10 ml) for one hour at room temperature. The radioactivity concentration of both phases was measured and logD was calculated from three replicate syntheses for [18F]EF4Cla, [18F]EF4Clb and [18F]EF5. Computed logP (ClogP) values were calculated using ChemDraw©, version 11 (CambridgeSoft Corporation, Cambridge, MA, USA) and ACD/ChemSketch Freeware, version 12.0 (Advanced Chemistry Development, Inc., Toronto, ON, Canada, www.acdlabs.com, 2009). Retention volumes of [18F]EF4Cla, [18F]EF4Clb and [18F]EF5 were calculated from the HPLC-chromatograms and normalized to the retention volume of EF3Cl2 (RV(N)).
The specific radioactivities (SRA) of [18F]EF4Cla, [18F]EF4Clb and [18F]EF5 were determined using authentic EF5 and assuming that the UV absorptions of [18F]EF4Cla and [18F]EF4Clb were equal to that of EF5. The SRAs were corrected to the end of synthesis (EOS) (Table 1b).
In vivo experiments
Experimental tumors were achieved by subcutaneous injections of 1x106 MDA-MD-231 (human adenocarcinoma from mammary gland) cells in MatrigelTM at both flanks of three male nude mice (mice I, II and III) (athymic nu/nu, Harlan, the Netherlands). The mice were maintained under controlled pathogen-free environmental conditions (21 ºC, humidity 55 ± 5%, and lights on from 6:00 a.m. to 6:00 p.m.) with free access to tap water and standard food. The experiment procedures were reviewed by the local Ethics Committee on Animal Experimentation at the University of Turku and approved by the local Provincial State Office of Southern Finland.
The PET scans were carried out using the Inveon multimodality PET/CT scanner (Siemens Medical Solutions USA, Knoxville, TN). Mice were anesthetized with 2% isoflurane in oxygen and the body temperature was maintained using a heating pad. Following a transmission scan for attenuation correction using the CT modality, an emission scan was acquired in 3D list mode with an energy window of 350-650 keV.
[18F]EF4Cla, [18F]EF4Clb or [18F]EF5 formulated in a propylene glycol/ethanol-solution (70%/30%) were injected (13.0 ± 0.7 MBq) into a tail vein of the mouse approximately 3 weeks after tumor induction. Initially, on day 1, all three mice were imaged with [18F]EF5 in order to confirm hypoxic volumes in the tumors. The following days (days 2 and 3) mouse I was imaged with [18F]EF5 in order to ensure that the hypoxia volume inside the tumors did not change over the 3-day period, whereas mice II and III received [18F]EF4Cla or [18F]EF4Clb, on day 2 and vice versa on day 3 (Supplementary Table 1).
On days 1 and 2, a 20-min long static scan was acquired 60-min after injection of radiotracers, whereas on day 3, dynamic 80-min long scans were acquired (Supplementary Fig. 4). List mode data was stored in 3D sinograms, which were then Fourier-rebinned into 2D sinograms (25 frames: 7x10 s, 4x 15 s, 2x30 s, 2x120 s, 1x180 s, 6x300 s and 3x600 s). Reconstruction was performed with an OSEM 2D iterative algorithm with four iterations and 16 subsets as the parameters. Normalization, dead time, randomity, attenuation, and scatter corrections were applied.
Volumes of interest (VOIs) were drawn over the tumors and muscle in images between 60-80 min post injection using the Inveon Research Workplace Image Analysis software (Siemens Medical Solutions USA, Knoxville, TN) using the CT template as an anatomical reference. Analyses of dynamic scans confirmed that the time interval 60-80 minutes in proper for analysis of tracer uptake in tumor and muscle.
The uptake of radioactivity derived from [18F]EF5, [18F]EF4Cla or [18F]EF4Clb injections were expressed as tumor-to-muscle (T/M) ratios.
References
1. Bergman J and Solin O (1997) Fluorine-18-Labeled Fluorine Gas for Synthesis of Tracer Molecules. Nucl Med Biol 24:677-683.
2. OECD Guideline for the testing of chemicals, Partition Coefficient (n-octanol/water): Shake method, Adopted by Council on 1995, Test No.107, ISSN: 2074-5753, (online), DOI 10.1787/20745753.
Supplementary figure 1. RadioHPLC chromatogram of crude reaction mixture after radiofluorination.
Supplementary figure 2. Radio-LC/MS chromatograms of separated fractions analyzed by selected ion masses, m/z 301 for [18F]EF5 and m/z 317 for [18F]EF4Cla,b
1 (m/z 96 amu) 2 (m/z 112 amu) 3 (m/z 196 amu) 4 (m/z 214 amu)
Supplementary figure 3. LC-MS/MS spectra of separated [18F]EF5, [18F]EF4Cla and [18F]EF4Clb fractions. Parent is assigned the symbol M. Signals marked 1-4 represent common structures for all molecules and are depicted below the figure.
Supplementary figure 4. Experimental protocol for evaluation of radiolabeled compounds in male nude mice bearing induced tumors. On days 1 and 2, a 20-min long static scan was acquired 60-min after injection of radiotracers, whereas on day 3 dynamic 80-min long scans were acquired.
Supplementary Table 1. Imaging schedule
Mouse I / [18F]EF5 / [18F]EF5 / [18F]EF5
Mouse II / [18F]EF5 / [18F]EF4Cla / [18F]EF4Clb
Mouse III / [18F]EF5 / [18F]EF4Clb / [18F]EF4Cla
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