ChemComm
`
`COMMUNICATION
`
`[18F]–NHC–BF3 adducts as water stable
`radio-prosthetic groups for PET imaging†‡
`
`Kantapat Chansaenpak,§a Mengzhe Wang,§b Zhanhong Wu,b Rehmat Zaman,a
`Zibo Li¶*b and François P. Gabbaı¨¶*a
`
`Received 3rd June 2015,
`Accepted 25th June 2015
`
`DOI: 10.1039/c5cc04545b
`
`www.rsc.org/chemcomm
`
`The radiofluorination of N-heterocyclic carbene (NHC) boron trifluoride
`adducts affords novel [18F]–positron emission tomography probes
`which resist hydrolytic fluoride release. The labelling protocol relies
`on an 18F–19F isotopic exchange reaction promoted by the Lewis acid
`SnCl4. Modification of the NHC backbone with a maleimide function-
`ality provides access to a model peptide conjugate which shows no
`evidence of defluorination when imaged in vivo.
`
`Positron emission tomography (PET) is a rapidly growing imaging
`technique that relies on the use of molecular radiotracers contain-
`ing a positron emitting isotope.1 To date, a great deal of attention
`has been devoted to the use of fluorine-18 (18F), a radionuclide
`that can be easily generated from [18O]–water and whose nuclear
`decay characteristics are ideally suited for applications in PET
`imaging.2 One difficulty faced in the synthesis of 18F-containing
`molecular radiotracers is the short half-life of the isotope (110 min).
`It follows that the best methods to access 18F-containing molecular
`radiotracers should be fast and preferably carried out in the late
`stages of the synthesis of the radiopharmaceutical probe.3 An
`attractive approach that provides a possible solution to these
`challenges is based on molecules containing a boron atom as a
`fluoride binding site.4 This approach was pioneered by Perrin
`who showed that arylboronic acids or esters featuring electron-
`withdrawing groups quickly react with fluoride ions to form the
`corresponding aryltrifluoroborates.5 Over the years, Perrin and
`other groups have investigated a number of backbones designed
`to stabilize the trifluoroborate unit and prevent its decomposi-
`tion in vivo (Chart 1).6 Although the rate of hydrolysis can be
`
`a Department of Chemistry, Texas A&M University, College Station, Texas 77843,
`USA. E-mail: gabbai@mail.chem.tamu.edu
`b Department of Radiology, Biomedical Research Imaging Center, University of
`North Carolina, Chapel Hill 27599, USA. E-mail: ziboli@med.unc.edu
`† This work is dedicated to Manfred Scheer on the occasion of his 60th birthday.
`‡ Electronic supplementary information (ESI) available: Experimental, character-
`ization and imaging data. CCDC 1402916–1402918. For ESI and crystallographic
`data in CIF or other electronic format see DOI: 10.1039/c5cc04545b
`§ Contributed equally to the work.
`¶ Jointly conceived the study.
`
`Chart 1
`
`slowed down drastically, all fluoroborates investigated to date
`are unstable toward hydrolysis. This hydrolysis reaction is
`potentially problematic because the fluoride ions liberated by
`hydrolysis of the radiotracer lead to unwanted background
`signal in particular from the skeleton.
`Recently we introduced a strategy based on the use of zwitter-
`ionic trifluoroborates.6f, g In particular, we found that the trifluoro-
`borate moiety can be significantly stabilized against hydrolysis by a
`proximal cationic functionality such as a phosphonium unit as in
`the case of D and E.6g This approach is further validated by the
`recent work of Perrin who showed that ammonium trifluoroborate
`moieties of type C show sufficient stability for in vivo imaging.6b,c As
`part of our continuing interest in this chemistry, we were drawn by
`the remarkable stability of N-heterocyclic carbene (NHC) boron
`fluoride adducts7 such as 1.8 Compound 1, which can also be
`described as a zwitterionic imidazolium trifluoroborate is highly
`resistant to hydrolysis and can be recrystallized from boiling water.
`Encouraged by these properties, we questioned whether such
`NHC–BF3 adducts could be radiofluorinated and used as prosthetic
`groups for PET imaging. In this paper, we describe the initial
`results that we have obtained while working toward this goal.
`
`This journal is © The Royal Society of Chemistry 2015
`
`Chem. Commun., 2015, 51, 12439--12442 | 12439
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`Cite this: Chem. Commun., 2015,
`51, 12439
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`Published on 26 June 2015. Purchased by kushan@me.com on 11 March 2025.
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`Scheme 1 Synthesis of 2.
`
`As a starting point for these studies, we synthesized the
`carbene–BF3 adduct 2 as a model compound. Using the method
`recently employed by for the monoethyl analog (1),8 compound
`2 was obtained by thermolysis of 1,3-dimethyl-1H-imidazolium
`tetrafluoroborate under reduced pressure (Scheme 1).8 The presence
`of the trifluoroborate moiety is confirmed by the detection of a
`quartet in both the 11B NMR spectrum (0.21 ppm, JB–F = 37.0 Hz)
`and the 19F NMR spectrum (139.2 ppm, JB–F = 37.0 Hz). The
`1H NMR spectrum shows two singlets at 3.85 ppm and 7.11 ppm
`corresponding to the methyl and the methine groups, respectively.
`The structure of this compound has also been studied by single
`crystal X-ray diffraction (Fig. 1). The B(1)–C(1) bond connecting the
`NHC ligand to the boron center (1.641(3) Å) is comparable to the
`boron–carbon bond of 1 (1.644(3) Å),8 indicating a strong coordina-
`tion of the NHC ligand to the boron atom.
`Next, we turned our attention toward the synthesis of a NHC–BF3
`adduct that could be easily conjugated with biomolecules for
`targeted disease imaging. After reviewing different functionalization
`possibilities, we decided to synthesize the amino-substituted deriva-
`tive 5 (Scheme 2). We successfully accessed this new derivative by
`reaction of the known nitrocarbene–AgI complex 39 with BF3–OEt2.
`This reaction afforded the nitrocarbene–BF3 adduct 4 as a white
`solid in 83% yield. Hydrogenation of 4 over palladium afforded 5 in
`a 78% yield. The 1H NMR spectrum of 4 and 5 display two singlets
`(3.94 ppm and 4.16 ppm for 4 and 3.61 and 3.73 ppm for 5)
`corresponding to the methyl group and a singlet (8.21 ppm for 4 and
`6.37 ppm for 5) corresponding to the methine proton. The presence
`of an amino group in 5 is confirmed by the detection of a broad
`signal at 4.02 ppm. As in the case of 2, quartets are observed
`in the 11B NMR and 19F NMR spectra of 4 and 5 (11B NMR:
`0.13 ppm, JB–F = 33.5 Hz for 4 and 0.27 ppm, JB–F = 37.2 Hz for 5;
`19F NMR: 137.9 ppm, JB–F = 33.5 Hz for 4 and 138.0 ppm,
`JB–F = 37.2 Hz for 5). The crystal structure of 4 has also been
`determined. The carbene–BF3 moiety is essentially analogous
`to that in 2 (Fig. 1). The only notable difference is observed in
`the B(1)–C(1) separation (1.657(2) Å) which is slightly longer than
`in 2 (1.637(5) Å). This elongation is assigned to the electron
`
`Fig. 1 Crystal structures of the Arduengo carbene borane adducts 2, 4,
`and 7. Ellipsoids are scaled to the 50% probability level and hydrogen
`atoms have been omitted for clarity.
`
`Scheme 2 Synthesis of the maleimide derivative 7.
`
`withdrawing properties of the nitro group and the associated
`weaker donor properties of the carbene–carbon atom.
`Compound 5 can be easily converted into the maleimide
`derivative 7 in two steps as illustrated in Scheme 2. The spectro-
`scopic properties of 7 are close to those of 5. The methine signal is
`observed at 7.08 ppm. The trifluoroborate moiety gives rise to a
`quartet at 0.29 ppm in the 11B NMR spectrum ( JB–F = 35.6 Hz) as
`well as a quartet at 138.5 ppm in the 19F NMR spectrum ( JB–F =
`35.6 Hz). The structure of this derivative has also been confirmed
`by X-ray diffraction (Fig. 1). The B(1)–C(1) separation (1.656(9) Å) is
`close to that in 4, a characteristic consistent with the electron
`withdrawing properties of the maleimide functional group.
`Next, we decided to investigate the rates of hydrolysis of these
`new NHC–BF3 adducts (2, 4, 5, 7). This hydrolysis reaction, which is
`expected to produce the corresponding boronic acid according to a
`first order rate process (n = kobs[NHC–BF3]), was monitored by 19F
`NMR spectroscopy in D2O/CD3CN (8/2 vol) at pH 7.5 ([phosphate
`buffer] = 500 mM, [NHC–BF3] = 20 mM).4d,10 Surprisingly, we found
`that the hydrolysis of the adducts was extremely slow. After a
`week, we did not observe any free fluoride for 4 and 7 indicating
`that these two derivatives are essentially immortal. Their stability
`is assigned to the electron withdrawing nature of the nitro or
`maleimide functionality which increases the Lewis acidity of
`the boron center thereby preventing fluoride anion dissociation.
`Compounds 2 and 5 are also surprisingly stable and only show a
`trace amount of free fluoride after a week in D2O/CD3CN (8/2 vol)
`at pH 7.5. By extending this experiment to a longer timescale,
`we have been able to calculate the rate of hydrolysis for these
`two compounds. These rates, which are respectively equal to kobs =
`1.2  106 min1 for 2 and 1.1  106 for 5 are lower than those
`measured under the same conditions for the phosphonium borane
`D. Altogether, these results illustrate the remarkable resistance of
`NHC–BF3 adducts to hydrolysis and suggest that they could be used
`as prosthetic groups for PET imaging.
`Employing the approach developed by our group for the
`preparation of [18F]BODIPY dyes,11 we decided to investigate the
`radiofluorination of these NHC–BF3 adducts via 18F–19F isotopic
`exchange using SnCl4 as a Lewis acid promoter. We first tested
`this approach with the non-functionalized NHC–BF3 adduct 2
`which was mixed with SnCl4 (5–15 eq.) in MeCN and combined
`with a solution of [18F]–fluoride (as the tetra-n-butylammonium
`salt) in MeCN (Scheme 3 and Table 1). The reaction mixture was
`
`12440 | Chem. Commun., 2015, 51, 12439--12442
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`This journal is © The Royal Society of Chemistry 2015
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`Published on 26 June 2015. Purchased by kushan@me.com on 11 March 2025.
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`Scheme 3 Scheme showing the radiolabeling of 2 via SnCl4 assisted
`isotopic 18F–19F exchange.
`
`Table 1 Radiosynthetic results for [18F]2
`
`Entry
`
`[2]
`(mM)
`
`SnCl4
`(equiv.)
`
`Temp.
`(1C)
`
`Time
`(min)
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`
`15
`30
`60
`30
`30
`30
`30
`30
`30
`
`5
`5
`5
`10
`15
`5
`5
`5
`5
`
`25
`25
`25
`25
`25
`40
`60
`25
`25
`
`10
`10
`10
`10
`10
`10
`10
`20
`30
`
`SAa
`(mCi mmol1)
`26.4
`40.6
`40.8
`47.5
`47.8
`45.5
`49.5
`53.5
`36.8
`
`RCYb
`(%)
`
`35.5
`42.1
`47.3
`48.4
`48.1
`56.4
`53.4
`49.9
`39.9
`
`a Specific activity is determined by dividing the product activity by the
`amount of the product (based on the integration of UV-HPLC and compare
`with the UV chromatogram of the standard). b RCY = activity of the
`isolated product/starting 18F activity. All yields are decay corrected.
`
`then shaken for 10 min before being quenched by addition of
`water. The radiolabeled compound ([18F]2) was immobilized on a
`Sep-Pak cartridge (Sep-Pak Plus tC18) and washed with water.
`[18F]2 was eluted off the cartridge with MeCN. An aliquot of the
`resulting MeCN solution was subjected to HPLC analysis.
`The radiochemical yield (RCY) was calculated based on the
`radio-activity of the isolated product and the starting radio-activity.
`As shown in Table 1, the RCY ranges from 35–56% for different
`reaction conditions. It was found that increasing the concentration
`of precursor leads to higher isolation yield (entries 1–3). Interest-
`ingly, variation in the concentration of the Lewis acid promoter
`(entries 3–5) or in the temperature (entries 6 and 7) of the reaction
`had little impact on the RCY. When a long reaction time was
`employed as in entries 8 and 9, a decreased isolation yield was
`observed due to product decomposition. The highest specific
`activity of the final product obtained in this experiment was
`calculated to be 53.5 mCi mmol1 (entry 8).
`Using conditions from entry 7, we have also been able to prepare
`[18F]7 with a specific activity of 51.3 mCi mmol1 (RCY = 54%,
`Scheme 4). The identity of [18F]7 was confirmed by co-injection with
`the non-radiolabeled standard (Fig. 2). This radiofluorinated NHC–
`BF3 adduct could be conveniently conjugated with the model peptide
`H–Cys–Phe–OH via a thiol-Michael addition reaction. This synthesis
`was carried out by mixing a solution of [18F]7 in MeCN with an
`aqueous solution of H–Cys–Phe–OH (400 mg, 1.5 mmol) (Scheme 4).
`After shaking for 10 min at room temperature, a portion of the
`reaction mixture (0.01 mCi) was loaded onto the HPLC for purification
`affording [18F]7–H–Cys–Phe–OH with a 95.7% purity. The identity of
`[18F]7–H–Cys–Phe–OH peptide was confirmed by its mass spectrum
`(Fig. S11, ESI‡) as well as by co-injection with the independently
`synthesized non-radiolabeled standard (Fig. 3). The specific activity of
`[18F]7–H–Cys–Phe–OH peptide was calculated as 40.8 mCi mmol1.
`
`Scheme 4 Scheme showing the preparation of the [18F]7–H–Cys–Phe–
`OH conjugate.
`
`Fig. 2 Left: UV trace of 7 as the standard reference. Right: Crude radio-
`HPLC profile for the 18F-labeling of 7.
`
`Fig. 3 Left: UV traces of [18F]7–H–Cys–Phe–OH as the standard refer-
`ence. Right: Crude radio-HPLC profile for the 18F-labeling of [18F]7–H–
`Cys–Phe–OH.
`
`Encouraged by these radiofluorination and conjugation
`results, the stability of [18F]7–H–Cys–Phe–OH was investigated
`in vivo. As a prelude to these studies, we first tested the stability
`of the conjugate in a 1 PBS buffer at 37 1C (Fig. S14, ESI‡).
`Even after 2 hours, the conjugate is not compromised as shown
`by the fact that its purity remains 490% pure. In vivo PET/CT
`imaging in a normal nude mouse afford consistent results. The
`microPET/CT images collected 1 h, 2 h, and 4 h post injection
`show liver and urinary track clearance of the conjugate. More
`importantly, no bone uptake is observed even 4 h post injec-
`tions. Indicating that [18F]–fluoride release by the radiofluori-
`nated carbene unit is negligible (Fig. 4).
`In summary, we have identified a new boron-based fluoride
`captor with an unusually high resistance to hydrolytic fluoride
`release. The stability of this new probe is ascribed to its
`zwitterionic nature, with the cationic charge of the imidazo-
`lium unit acting as an electrostatic anchor for the boron-bound
`fluoride anions. These NHC–BF3 fluoride captors are a new
`
`This journal is © The Royal Society of Chemistry 2015
`
`Chem. Commun., 2015, 51, 12439--12442 | 12441
`
`Published on 26 June 2015. Purchased by kushan@me.com on 11 March 2025.
`
`View Article Online
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`Petitioner GE Healthcare – Ex. 1043, p. 12441
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`Communication
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`ChemComm
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`Fig. 4 Decay-corrected whole-body microPET/CT sagittal images of a
`nude mice from a static scan at 1, 2 h and 4 h after injection of [18F]7–H–
`Cys–Phe–OH.
`
`incarnation of the concepts underlying the stability of the
`phosphonium trifluoroborates of type D and E developed by
`us or ammonium trifluoroborates of type C recently reported by
`the Perrin group.
`This work was supported by the Cancer Prevention Research
`Institute of Texas (RP130604), the National Institute of Biomedical
`Imaging and Bioengineering (1R01EB014354-01A1), the National
`Cancer Institute (P30-CA016086-35-37), and the Biomedical Research
`Imaging Center, University of North Carolina at Chapel Hill. K.C.
`gratefully acknowledges financial support from the Development
`and Promotion of Science and Technology (DPST) program
`administered by the Royal Thai Government.
`
`Notes and references
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`
`12442 | Chem. Commun., 2015, 51, 12439--12442
`
`This journal is © The Royal Society of Chemistry 2015
`
`Published on 26 June 2015. Purchased by kushan@me.com on 11 March 2025.
`
`View Article Online
`
`Petitioner GE Healthcare – Ex. 1043, p. 12442
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