Design, synthesis, and evaluation of positron emission tomography/fluorescence dual imaging probes for targeting facilitated glucose transporter 1 (GLUT1)

Article abstract of DOI:10.1039/d1ob00199j

Increased energy metabolism followed by enhanced glucose consumption is a hallmark of cancer. Most cancer cells show overexpression of facilitated hexose transporter GLUT1, including breast cancer. GLUT1 is the main transporter for 2-deoxy-2-[¹⁸F]fluoro-d-glucose (2-[¹⁸F]FDG), the gold standard of positron emission tomography (PET) imaging in oncology. The present study's goal was to develop novel glucose-based dual imaging probes for their use in tandem PET and fluorescence (Fl) imaging. A glucosamine scaffold tagged with a fluorophore and an¹⁸F-label should confer selectivity to GLUT1. Out of five different compounds, 2-deoxy-2-((7-sulfonylfluoro-2,1,3-benzoxadiazol-4-yl)amino)-d-glucose (2-FBDG) possessed favorable fluorescent properties and a similar potency as 2-deoxy-2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)-d-glucose (2-NBDG) in competing for GLUT1 transport against2-[¹⁸F]FDGin breast cancer cells. Radiolabeling with¹⁸F was achieved through the synthesis of prosthetic group 7-fluoro-2,1,3-benzoxadiazole-4-sulfonyl [¹⁸F]fluoride ([¹⁸F]FBDF) followed by the reaction with glucosamine. The radiotracer was finally analyzedin vivoin a breast cancer xenograft model and compared to2-[¹⁸F]FDG. Despite favourablein vitrofluorescence imaging properties,2-[¹⁸F]FBDGwas found to lack metabolic stabilityin vivo, resulting in radiodefluorination. Glucose-based2-[¹⁸F]FBDGrepresents a novel dual-probe for GLUT1 imaging using FI and PET with the potential for further structural optimization for improved metabolic stabilityin vivo.

Full text of DOI:10.1039/d1ob00199j

Organic &
Biomolecular Chemistry
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PAPER
Design, synthesis, and evaluation of positron
emission tomography/fluorescence dual
imaging probes for targeting facilitated
glucose transporter 1 (GLUT1)†
Cite this: Org. Biomol. Chem., 2021,
19, 3241
Richard Yuen,a,b Michael Wagner,b Susan Richter,b Jennifer Dufour,b
a,b,c
Melinda Wuest,b,c Frederick G. Westa,c and Frank Wuest
*
Increased energy metabolism followed by enhanced glucose consumption is a hallmark of cancer. Most
cancer cells show overexpression of facilitated hexose transporter GLUT1, including breast cancer. GLUT1
is the main transporter for 2-deoxy-2-[18F]fluoro-D-glucose (2-[18F]FDG), the gold standard of positron
emission tomography (PET) imaging in oncology. The present study’s goal was to develop novel glucose-
based dual imaging probes for their use in tandem PET and fluorescence (Fl) imaging. A glucosamine
scaffold tagged with a fluorophore and an 18F-label should confer selectivity to GLUT1. Out of five
different compounds, 2-deoxy-2-((7-sulfonylfluoro-2,1,3-benzoxadiazol-4-yl)amino)-D-glucose (2-
FBDG) possessed favorable fluorescent properties and a similar potency as 2-deoxy-2-((7-nitro-2,1,3-
benzoxadiazol-4-yl)amino)-D-glucose (2-NBDG) in competing for GLUT1 transport against 2-[18F]FDG in
breast cancer cells. Radiolabeling with 18F was achieved through the synthesis of prosthetic group
7-fluoro-2,1,3-benzoxadiazole-4-sulfonyl [18F]fluoride ([18F]FBDF) followed by the reaction with glucos-
amine. The radiotracer was finally analyzed in vivo in a breast cancer xenograft model and compared to
2-[18F]FDG. Despite favourable in vitro fluorescence imaging properties, 2-[18F]FBDG was found to lack
metabolic stability in vivo, resulting in radiodefluorination. Glucose-based 2-[18F]FBDG represents a novel
dual-probe for GLUT1 imaging using FI and PET with the potential for further structural optimization for
improved metabolic stability in vivo.
Received 2nd February 2021,
Accepted 22nd March 2021
DOI: 10.1039/d1ob00199j
rsc.li/obc
rescence (FI) imaging relies on stable fluorophores, which
allows for longitudinal imaging as PET radiotracers involve the
Introduction
Non-invasive cancer imaging is an essential tool in precision use of radioisotopes with relatively short decay half-lives (18F
medicine for accurate diagnosis and monitoring of treatment t1/2 = 109.8 min). The development of dual-probes capable of
responses.1 In recent years, the combination of different mole- both PET and FI imaging would combine the advantages of
cular imaging modalities and the development of dual-func- both imaging techniques to improve patient care and
tionality imaging probes has significantly advanced the field outcomes.6–8 Utilizing a single imaging agent for both PET
of precision medicine in oncology and beyond.2–4 Positron and FI detection, rather than using two separate probes, will
emission tomography (PET) is a routine nuclear medicine ensure no differences in biodistribution, resulting in a high
technique that offers detection of metabolic and physiological correlation between the two imaging techniques.2 In the
processes with high sensitivity.5 Complementary to PET, fluo- clinic, solid tumour management would benefit from the accu-
rate detection of the tumour with PET, followed by fluo-
rescence-guided surgical resection of the tumour mass. For
practical purposes in the clinic, the patient would first be
administered the radiolabeled dual-probe for PET imaging.
Then, the patient is administered the non-radioactive dual-
probe prior to surgery to enable FI. This two-dose strategy
would usually be required as the radioactive compound is
administered in trace amounts. A first-in-human study in 2018
demonstrated the feasibility of PET and optical dual-modality
image-guided surgery using 68Ga-IRDye800CW-bombesin in
aDepartment of Chemistry, 11227 Saskatchewan Drive University of Alberta,
Edmonton, AB, Canada T6G 2G2. E-mail: wuest@ualberta.ca; Fax: +780-432-8483;
Tel: +780-989-8150
bDepartment of Oncology, Cross Cancer Institute, University of Alberta, Edmonton,
AB, Canada T6G 1Z2
cCancer Research Institute of Northern Alberta, University of Alberta, Edmonton, AB,
Canada T6G 2S2
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1ob00199j
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glioblastoma.9 The unrivalled sensitivity of PET and the capa-
bility to visualize tumour margins through FI will allow for
precise targeting and better patient outcomes by removing
critical tumour margins during tumour resection while mini-
mizing damage to healthy tissue.10 This technique is particu-
larly beneficial for the surgical removal of solid tumours where
visual differentiation between tumour tissue and healthy
tissue is difficult, as frequently observed in breast cancers.10
One of the hallmarks of cancer is the Warburg effect or the
dysregulation of cellular energy metabolism.11 Cancer cells
often require more energy than normal cells as they grow and
divide at a higher rate. In cancer cells, a marked shift from
mitochondrial oxidative phosphorylation to less efficient cyto-
solic glycolysis to synthesize adenosine triphosphate (ATP) is
observed. Consequently, cancer cells are characterized by the
overexpression of various facilitated hexose transporters
known as GLUTs. Glucose transporter 1 (GLUT1), the primary
glucose transporter, is overexpressed in many cancer cells.12
Therefore, targeting GLUT1 as a cancer biomarker will confer
selective cancer cell uptake of the imaging probe compared to
normal cells. A standard method for targeting GLUT1 involves
the functionalization of glucose to obtain specific GLUT1 tar-
geting imaging agents.13,14
Fig. 1 Structures of the different fluorine-containing fluorophores
attached to GLUT1-targeting glucosamine (center) that are presented in
this work, enabling dual-probe capability (top left: diaryl-pyrazoles/pyr-
azolines; bottom left/top right: coumarins; bottom right: benzoxadiazoles).
The non-radioactive 19F-containing fluorophores should be
readily accessible, and the synthetic route to the respective 18F-
labelled fluorophores should be feasible. In addition to these
requirements, the dual-imaging reporter motifs should readily
conjugate to the glucosamine scaffold to generate the desired
bimodal GLUT1 imaging probe. Herein, we have synthesized
and tested novel dual-probes based on diarylpyrazoline/diaryl-
pyrazole, coumarin, and benzoxadiazole fluorophores for their
fluorescent properties as well as their potency to compete with
2-[18F]FDG for transport through GLUT1 (Fig. 1). In a second
step, we have chosen 2-FBDG for radiolabeling with 18F to
form 2-[18F]FBDG. 2-[18F]FBDG was tested in a breast cancer
model in vivo as a potential dual PET/FI imaging agent.
In this line, 2-deoxy-2-[18F]fluoro-D-glucose (2-[18F]FDG) rep-
resents the gold standard radiotracer for clinical PET imaging
of different types of cancer.13 Another glucose derivative,
2-deoxy-2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)-D-glucose
(2-NBDG), has been successfully used for fluorescence (FI)
imaging of GLUT1 expression in cells.14 2-NBDG demonstrated
that glucose modification at the C2 position with a moderately
sizeable aromatic group does not prevent GLUT1 recognition.
The need to expand on the range of available tracers is
evident, given the crucial individual variability seen in cancer
patients as each cancer is characterized by a different gene
and protein expression profile.
Results and discussion
Chemistry
Several fluorescent probes targeting GLUT1 have been pub-
lished recently,15–17 and their uptake profiles were correlated
with that of 2-[18F]FDG.18–20 Besides a few reports discussing The synthesis of FI imaging probes Py-GlcN and Pyin-GlcN
bimodal PET/FI probes for in vivo oncologic and neurodegen- containing a diaryl-pyrazoline/diarylpyrazole fluorophore is
erative disease imaging,21–23 to date, no dual PET/FI probes outlined in Scheme 1. 19F-Containing tetrazoles 1 and 2 were
targeting GLUT1 have been described.
subjected to a photo-activated click reaction with acrylamide-
The development of GLUT1-targeting dual-probes that conjugated glucosamine 3.22,26,27 The [3 + 2] dipolar cyclo-
contain both a fluorophore and an 18F reporter is a particular addition between an in situ-generated nitrile imine and 3 gen-
challenge. The fluorophores that are used must be of reason- erated pyrazolines 4 and 5. During the reaction, pyrazoline 4
able size and enable facile incorporation of 18F while preser- was susceptible to oxidation to form pyrazole 6. Thus, oxi-
ving favourable fluorescent properties. Several reports dation with DDQ provided a single characterizable compound.
described the use of large chelator molecules such as NOTA or Tetrazole 2 was more reactive than tetrazole 1 under irradiation
DOTA for the radiolabeling with radiometals like 68Ga and with a UV light meant for visualizing TLC plates (365 nm). The
64Cu in addition to fluorescent tags to prepare bifunctional substituent on the 2-phenyl group had to be sufficiently elec-
peptide and protein imaging agents.22,24,25 However, the large tron-donating to undergo the photo-click reaction as aryl
fluorophore/chelator concept may not be feasible for targeting groups with less electron-donating alkyl groups had unfavour-
GLUT1 as the increasing steric demand would likely result in able reaction kinetics.
the loss of substrate recognition by the transporter. The
However, the more electron-rich tetrazole 2 proved to be
present study aimed to design, synthesize, and screen poten- difficult to synthesize (3% yield). This was likely due to the dia-
tial fluorine-containing fluorophores for substrate recognition zonium salt undergoing electrophilic substitution with an
by GLUT1 in an in vitro assay.
equivalent of the parent aniline, forming the azo dye as a
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Scheme 1 Synthesis of Py-GlcN and Pyin-GlcN: (a) NaNO2, HCl, EtOH, 0 °C, 1 h; (b) pyridine, −15 °C, 1 h, 1: 44%, 2: 3% yield; (c) 365 nm light,
CH3CN, 1–3 days, 5: 89% yield; (d) 365 nm light, CH3CN, air, 6: 35% yield; (e) DDQ, CH2Cl2, overnight; (f) NaOH, MeOH/H2O, 10 min, Pyin-GlcN:
45%, Py-GlcN: 45% yield.
byproduct. When R = Me or tert-Bu, the reaction took several
days and provided incomplete conversion (yields <5%).
Following the formation of 5 and 6, a deacetylation reaction
was performed using NaOH in aqueous methanol. Py-GlcN
and Pyin-GlcN could be synthesized in decent yields (both
45% yield). The yields of the final hydrolysis reaction were
lower than expected likely due to loss during HPLC purifi-
cation. Attempts to obtain the pyrazoline fluorophore of Py-
GlcN failed as the compound readily oxidized to the respective
pyrazole under the reaction conditions. Oxidation of com-
pound 5 with DDQ in CH2Cl2 resulted in a non-fluorescent
pyrazole 7. Hence, only pyrazoline Pyin-GlcN was used for
further evaluation. Pyin-GlcN could be isolated and stored as it
was more stable against oxidation than Py-GlcN. The synthesis
of probes CO-GlcN-1 and CO-GlcN-2 as coumarin-based fluoro-
phores conjugated to glucosamine is depicted in Scheme 2.
The fluorescence of coumarin 7 is quenched due to the
presence of the azide group, a phenomenon previously
described by Sivakumar et al. in similar coumarin deriva-
tives.28 A Cu-catalyzed azide–alkyne click reaction was used to
couple alkyne 8 to azide 7 to create fluorescent triazole-linked
Scheme 2 Synthesis of CO-GlcN-1: (a) CuSO4·5H2O, NaAsc, t-BuOH,
H2O, acetone, 24 h, 68% yield; (b) K2CO3, MeOH/H2O, 30 min, 6% yield.
When the deprotection reaction with K2CO3 in aqueous
compound 9.28,29 The reaction proceeded with 68% yield. MeOH was left overnight, there were several uncharacterized
However, the following deprotection step proved to be a sub- degradation products, none of which corresponded to the
stantial challenge. Several reaction conditions were tested to desired compound.
remove the acetyl protecting groups, but none gave a satisfac-
Upon reacting compound 9 under basic conditions with
tory yield. Using very mild deprotection conditions, stirring 9 LiOH in THF/H2O, or NaOH in MeOH/H2O, interestingly,
with K2CO3 in aqueous MeOH for 30 min provided CO-GlcN-1 amine 10 was formed (Scheme 3). Deprotection with HCl and
in only 6% yield, leaving mostly partially deprotected products. heat also did not yield the desired product.
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Scheme 3 Side reaction from acetyl deprotection under basic conditions.
Scheme 4 Synthesis of CO-GlcN-2: (a) glucosamine·HCl, HBTU, DIPEA, DMF, overnight, 74% yield.
Probe CO-GlcN-2 was prepared via a simple amide-coupling
reaction between carboxylic acid 11 and glucosamine. This
HBTU mediated reaction provided CO-GlcN-2 in 74% yield
after HPLC purification (Scheme 4).
Inspired by the favourable properties of GLUT1-selective
fluorescent glucose compound 2-NBDG, we set up the syn-
thesis of a respective fluorine-containing analog. Starting from
commercially available precursor 12, an electrophilic aromatic
substitution with chlorosulfonic acid afforded sulfonyl chlor-
ide CBDF in 86% yield.30 Next, a halogen exchange was per-
formed on CBDF with KHF2 to give FBDF in quantitative yield
using a modified procedure by Dong et al.31 Finally, FBDF
underwent a nucleophilic aromatic substitution reaction with
glucosamine to furnish 2-FBDG in 99% yield. This rapid syn-
thetic route led to a compound with extremely desirable
optical properties (Table 1) in 85% overall yield over three
steps. The synthesis of 2-FBDG is depicted in Scheme 5.
The original synthetic route towards a benzoxadiazole-
fluorophore involved the formation of a sulfonamide from
CBDF using an amine. However, the addition of glucosamine
to CBDF produced only 2-FBDG. The fluoride anion liberated
from the SNAr reaction reacted with the sulfonyl chloride to
form the thermodynamically more stable sulfonyl fluoride.
Therefore, step (b) of Scheme 5 was not required, but for the
sake of unambiguous product identity, the halogen exchange
was performed. Additionally, synthesis of FBDF was necessary
because it was used for identification purposes as a non-radio-
active reference compound for radiochemistry. The spectral
properties of non-radioactive dual-probes are summarized in
Table 1.
Scheme 5 Synthesis of 2-FBDG: (a) HSO3Cl, CHCl3, 0 °C – reflux, 3 h,
86% yield; (b) KHF2, CH3CN, H2O, rt, 2 h, 99% yield; (c) glucosamine,
NaHCO3, DMF, rt, overnight, 99% yield.
The pyrazole/pyrazoline and coumarin fluorophores absorb
in the UV region and fluoresce between 400–450 nm. The
slight hypsochromic shift in emission wavelength and smaller
Stokes shift of CO-GlcN-2 as compared to CO-GlcN-1 is likely
due to the lack of the triazolyl linker.
2-FBDG absorbs in the visible region and fluoresces at a
wide range of wavelengths with a peak at 570 nm, which is
more red-shifted than 2-NBDG (Fig. 2).
In vitro competition against uptake of 2-[18F]FDG
All novel dual-probes were used in an in vitro competition
assay to test their interaction with GLUT1. 2-[18F]FDG was
used as the known GLUT1-selective radiotracer assay, which
was recently developed by our research group.32 The murine
mammary carcinoma cell line EMT6 used in this assay is
Table 1 Fluorescent properties of GlcN-based dual-probes under known to express high levels of GLUT1.32,33
aqueous conditions (pH 7.4)
Fig. 3 summarizes the effects of increasing concentrations
of all five novel dual-probes, as well as reference compounds
D-glucose and 2-NBDG – both of which are known to be trans-
ported by GLUT1. Fig. 3 also contains the calculated half-
maximum inhibitory concentrations (IC50) for each analyzed
compound. Dual-probes Py-GlcN, Pyin-GlcN, CO-GlcN-2, and
2-FBDG all showed substantial inhibition of 2-[18F]FDG uptake
Compound
λ (excitation)
λ (emission)
Py-GlcN
320
320
340
350
425
440
450
420
400
570
Pyin-GlcN
CO-GlcN-1
CO-GlcN-2
2-FBDG
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dering it more biocompatible than the other candidates.
Another advantage is that the Cl–18F halogen exchange is oper-
ationally simpler than the substitution methods noted above
as it obviates the need for a fluoride-drying step.
Recent work from Brito et al. showed that certain aromatic
N-glucosides could form a nanoscale supramolecular network
around cancer cells expressing GLUT1.34 They reported that
the sterically large N-fluorenylmethyloxycarbonyl-glucosamine-
6-phosphate is recognized by GLUT1 but is not transported.
Instead, the fluorenyl moiety forms π-stacking interactions
with other fluorenyl groups to form a barrier-like structure
around cells. This effect could potentially explain why Py-GlcN
was one order of magnitude more potent than glucose itself,
assuming the diarylpyrazole moiety mimics a fluorenyl group
as in N-fluorenylmethyloxycarbonyl-glucosamine-6-phosphate.
Fig. 2 Absorbance and fluorescence spectra of 2-FBDG in pH
7.4 Krebs–Ringer buffer. Bottom right shows an image of the solution
irradiated at 365 nm using a UV lamp.
Confocal microscopy experiments
To further analyze and confirm that 2-FBDG is being trans-
ported and taken up into the cells via GLUT1 instead of being
only membrane-bound, qualitative confocal microscopy experi-
ments were performed using breast cancer cell lines EMT6
(murine) and MDA-MB231 (triple-negative human breast
cancer cell line) to visualize intracellular localization of 2-
FBDG.
As shown in Fig. 4, there is evident cytosolic uptake of com-
pound 2-FBDG. Additionally, intracellular uptake of 2-FBDG
was inhibited in the presence of extracellular high levels of
D-glucose, providing further evidence that 2-FBDG is trans-
ported by GLUT1 and results in intracellular uptake in murine
and human breast cancer cells.
Fig. 3 Inhibition of 2-[18F]FDG uptake into EMT6 cells using increasing
concentrations of test dual-probes in comparison to reference com-
pounds D-glucose and 2-NBDG. Data shown as mean
SEM from n
data points out of x experiments. Calculated IC50 values for 2-[18F]FDG
uptake inhibition are shown in the table of Fig. 3.
into EMT6 cells. CO-GlcN-1 did not show any effect even at a
high concentration of 1 mM. Among all compounds tested, Py-
GlcN exhibited the highest potency (IC50 of 71 6 µM; n = 6/2),
providing evidence that Py-GlcN is better recognized by GLUT1
than its natural substrate D-glucose. Compound Pyin-GlcN
resulted in a similar potency to D-glucose (IC50 of 330
120 µM; n = 6/2 vs. 320
100 µM; n = 9/3). Compound
CO-GlcN-2 only showed a trend for similar inhibition as
2-NBDG, but analysis of higher concentrations was impossible
due to its limited solubility. 2-FBDG had a potency between
D-glucose and 2-NBDG (IC50 of 540 9 µM; n = 6/2). The good
potency of compound 2-FBDG combined with the opportunity
to introduce [18F]fluoride make 2-FBDG an interesting candi-
date for radiolabeling and further analysis, including PET
imaging. While the other potential dual-probes could easily be
fluorinated by SNAr or SN2 chemistry of their appropriate pre-
cursors, 2-FBDG was also the only probe that possessed both
Fig. 4 Confocal microscopy images were obtained in two different
breast cancer cell lines: EMT6 and MDA-MB-231. The cells were incu-
bated with 200 µM of 2-FBDG in the absence or presence of 50 mM
emission and excitation wavelengths in the visible region, ren- glucose for 1 h. DAPI was used as a nuclear stain.
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Radiochemistry
lation and reformulation in saline for subsequent in vivo
studies. When starting with 200 µg of CBDF and 234 MBq of
[18F]CsF, the effective molar activity achieved was 96 MBq
µmol−1. The effective molar activity can be improved by
increasing the amount of starting radioactivity and by lowering
the amount of compound 17. This reaction is also advan-
tageous because of the short reaction times and the absence of
a fluoride drying step.
The radiosynthesis of 2-[18F]FBDG was accomplished via pros-
thetic group [18F]FBDF and subsequent SNAr reaction with
glucosamine (Scheme 6).
[18F]FBDF was formed by applying mild sulfonyl [18F]fluor-
ide chemistry, employing aqueous conditions and room temp-
erature, which was introduced by Inkster et al.35 Briefly,
aqueous no-carrier-added (n.c.a) [18F]CsF was added to a solu-
tion of sulfonyl chloride 17 in tert-BuOH, followed by the
addition of pyridine and subsequent incubation at room temp-
erature for 15 min. After several attempts, it became evident
that pyridine was degrading prosthetic group [18F]FBDF. The
rationale for adding pyridine was that it scavenges unreacted
sulfonyl chloride to simplify purification and yields radio-
labeled compounds at high molar activity.35 However, in our
case, the need for this step is negligible as a high molar
activity is not a concern for the radiotracer due to ubiquitous
glucose present in the blood. Additionally, the subsequent
SNAr reaction generates a free fluoride anion which reacts with
the sulfonyl chloride. This consumes the remaining sulfonyl
chloride and ensures high chemical purity.
Animal experiments
Dual-probe 2-[18F]FBDG was evaluated in vivo with PET
imaging experiments in NIH-III mice bearing MDA-MB231
breast cancer xenografts. PET images were collected dynami-
cally over 60 min after the injection of radiotracer 2-[18F]FBDG
(∼5 MBq in saline) into the tail vein.
Fig. 5 summarizes the PET images at selected time points
compared to 2-[18F]FDG in the same mouse. While uptake into
MDA-MB-231 tumours increased after injection of 2-[18F]FDG
over time, this was not observed with 2-[18F]FBDG. Instead,
bone uptake increased over time while background clearance
in reference muscle tissue was detected. Fig. 6 depicts the
kinetic profile of 2-[18F]FBDG accumulation and clearance in
MDA-MB231 tumours, muscle and bone as analyzed from
respective time-activity curves (TACs) over time. Interestingly,
some radioactivity was delivered to the MDA-MB231 tumours
and was selectively trapped as no washout was observed com-
pared to muscle as reference tissue.
The bone TAC showed that after reaching an initial low-
level uptake at 5 min, a systematic and continuous increase in
bone uptake was observed over time. When compared with a
[18F]NaF injection, the bone uptake of 2-[18F]FBDG followed a
similar trend over time, but on a lower level. The latter indi-
cates a metabolic change of 2-[18F]FBDG in vivo represented by
a radiodefluorination process.36 To confirm this observation,
further analysis of the radiotracer’s stability was carried out by
examining mouse blood samples over time.
Following these findings, the addition of pyridine was
omitted, and the formation of [18F]FBDF was achieved in
nearly quantitative yields. The fluorination reaction was
usually complete within 5 minutes.
For further radiosynthesis optimization, some different pre-
cursor concentrations were also tested, showing that as little
as 100–200 µg of precursor CBDF in 200 µL tert-BuOH provided
consistent quantitative yields within a 5 min reaction time.
Lowering the amount of labelling precursor CBDF to 20 μg still
resulted in a 70% 18F incorporation within 15 min.
Following the radiofluorination step, [18F]FBDF was trapped
on a solid-phase cartridge and eluted with DMF into a vial con-
taining glucosamine and NaHCO3 to start the SNAr reaction.
Several different cartridges were tested (Waters Sep-Pak tC18
Plus Light, Waters Sep-Pak tC18 Plus, and Macherey-Nagel
Chromafix C18), but they either had low trapping efficiency or
required too much DMF to elute the product. We found that
directly diluting the initial reaction mixture with 600 µL DMF
and omitting the solid-phase extraction had no detrimental
effect on the SNAr reaction. This procedure simplified the syn-
thetic sequence and vastly improved the final decay-corrected
radiochemical yield after HPLC from 20% to 69 3% (n = 3)
over two steps in <110 min total synthesis time, including iso-
Metabolic stability of 2-[18F]FBDG was assessed in a normal
mouse by analyzing blood samples at 5, 15, 30, and 60 minutes
p.i. The blood samples were centrifuged to remove the blood
cells, and the resulting supernatant was treated with methanol
to precipitate the proteins. Second centrifugation followed by
HPLC analysis of the remaining supernatant provided data on
the plasma’s free radiotracer content. The graph shown in Fig. 7
represents the amounts of intact 2-[18F]FBDG versus [18F]F− as
radiometabolite over the time course of 60 min.
However, the metabolic stability of 2-[18F]FBDG may be
even lower as constant bone uptake of free [18F]F− occured
over time, removing it from the blood and plasma.
The extraction efficiency (the amount of radioactivity recov-
ered) was quite low, which further exemplifies this hypothesis.
This is consistent with the uptake profile in the tumor-bearing
mice where bone uptake was also detected (Fig. 5 and 6). Also,
the full extent of radiodefluorination could potentially be
underestimated as it has been shown that [18F]F− can retain
on reverse-phase silica columns (especially when using sol-
vents with pH < 5), and therefore not be detected by the radio-
Scheme 6 Radiolabeling of 2-FBDG: (a) [18F]CsF, H2O, tert-BuOH, rt,
5 min; (b) glucosamine·HCl, NaHCO3, DMF, H2O, t-BuOH, rt, 20 min.
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Fig. 5 Uptake of 2-[18F]FBDG (top) and 2-[18F]FDG (bottom) in a MDA-MB231 tumor-bearing mouse taken at 5, 20, 30, and 60 min (p.i. = post-
injection, SUV = standardized uptake value).
Fig. 6 Time-activity curves (TACs) of 2-[18F]FBDG in vivo. A: Comparison of SUV between MDA-MB231 tumours and muscle; B: comparison of SUV
in the bone between injection of 2-[18F]FBDG versus injection of [18F]NaF.
detector.37 To mitigate this, we used acid-free distilled water in
The present results are consistent with recent literature con-
our mobile phase. In addition, the stability of 2-[18F]FBDG was cerning the stability of sulfonyl [18F]fluorides.35,38,39 Sulfonyl
evaluated in vitro in saline, and 4% free [18F]F− was observed fluorides are known to act as ‘privileged warheads’ and react
after 10 min (time = 0 min in Fig. 7), increasing to 8% free with protein-based nucleophiles under special conditions.40
[18F]F− after 4.5 hours. This was indicative of the greater The release of free [18F]fluoride from radiotracer 2-[18F]FBDG
benchtop chemical stability of the radiotracer compared to the can also be explained by nucleophilic attacks on the sulfonyl
in vivo stability.
[18F]fluoride in vivo by plasma proteins.
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and clearance profile. This work may include fluorophores
with more stable C–F bonds, or the relatively new and mostly
unexplored pentafluorosulfanyl group.38
Experimental
Chemistry
General methods. All reagents were purchased from com-
mercial sources and used without further purification. NMR
spectra were recorded using the Agilent DD2 400, Varian Inova
500, Varian VNMRS 500, Varian VNMRS 600, Bruker Avance III
1
600, or the Agilent VNMRS 700. H and 13C NMR spectra were
referenced to the residual signals of deuterated solvents as
internal standards. Coupling constants (J) are reported in
hertz (Hz). Low resolution mass spectra were acquired using
an Agilent 6130 Mass Spectrometer coupled with an Agilent
1260 HPLC instrument. High resolution mass spectra were
acquired using the Agilent Technologies 6220 oaTOF (ESI) or
the Kratos MS50G (EI). Absorbance and fluorescence spectra
were acquired using a BioTek Synergy H1 Multi Detection
Microplate Reader. Column chromatography was performed
using 230–400 mesh silica gel. TLC analyses were completed
on silica gel 60 F254 aluminum plates, purchased from
Millipore-Sigma. Photo-click reactions were performed using a
tabletop TLC UV lamp (23 W). Semi-preparative HPLC was per-
formed on a Gilson system (Mandel Scientific, Guelph, ON,
Canada) with a 321 pump and a 155 dual-wavelength detector
(using 210 and 254 nm) installed with a Phenomenex Jupiter
10u Proteo 90 Å, 250 × 10 mm, 4.5 μm C12 column. Analytical
Fig. 7 Metabolic stability of 2-[18F]FBDG in vivo. Blood samples were
taken at 5, 15, 30, and 60 min, processed and subjected to analytical
radio-HPLC analysis to determine the amounts of intact 2-[18F]FBDG.
Additionally, the S–F bond of sulfonyl fluorides have been
shown to be more stable as steric bulk increases.39 This
characteristic is also described in the well established [18F]SiFA
motif, where the Si–F bond benefits from steric bulk around
the respective functional group to protect against radiodefluor-
ination.41 The electronic nature of the benzoxadiazole ring
may also be contributing to the instability of the sulfonyl fluor-
ide. We first postulated that the electron-rich aromatic system
would discourage nucleophilic attack and/or hydrolysis of the
sulfonyl fluoride. While 2-[18F]FBDG did exhibit stability to
water, the in vivo stability was not satisfactory. Analagous to
[18F]sulfonyl fluorides, [18F]trifluoroaryl borates also initially
suffered from radiodefluorination in vivo before it was found
that ortho electron-withdrawing groups stabilize the B–F
bond.42 Interestingly, it was shown that radiolabeled aryl [18F]
fluorosulfates were more stable than sulfonyl fluorides, and no
radiodefluorination was observed in vivo.43 Indeed, the stabi-
lity of these S–F bonds vary greatly, and further investigation
into the stereoelectronic effects governing the stability of the
S–F bond in sulfonyl fluorides could produce more promising
iterations of 2-[18F]FBDG.
HPLC was performed on
a Shimadzu system (Mandel
Scientific, Guelph, ON, Canada) equipped with a DGU-20A5
degasser, a SIL-20A HT autosampler, a LC-20AT pump, a
SPD-M20A photodiodearray detector, and a Ramona Raytest
radiodetector using a Phenomenex Luna 10u C18(2) 100A, 250
× 4.6 mm column.
5-(4-Fluorophenyl)-2-(4-methoxyphenyl)-2H-tetrazole (1).
The tetrazole was synthesized according to a published pro-
cedure,44 purified using flash column chromatography
(10 : 1 hexanes/EtOAc), and two recrystallization steps
(hexanes/EtOAc) – this produced a white solid 430 mg,
1.59 mmol, 44% yield. Rf (10 : 1 hexane/EtOAc) = 0.32; mp =
139 °C; 1H NMR (600.27 MHz, CDCl3) δ 3.90 (s, 3 H), 7.06/8.09
(AA′BB′, 4 H), 7.21/8.23 (AA′BB′, 4 H); 13C NMR (150.94 MHz,
2
CDCl3) δ 55.6, 114.7, 116.1 (d, JC–F = 22.1 Hz), 121.4, 123.5
4
3
(d, JC–F = 3.3 Hz), 129.0 (d, JC–F = 8.3 Hz), 130.4, 160.5, 164.1
Conclusion
1
(d, JC–F = 250.0 Hz), 164.9; 19F NMR (564.82 MHz, CDCl3) δ
In this study, we have introduced various synthesis pathways −109.82 (tt); LRMS (ESI) m/z: [M + H]+ calcd for C14H12FN4O
for the preparation of different glucose-based dual PET/Fl 271.1: found 271.1; UV/Vis (CH3CN): absorbance λmax (ε):
imaging probes for targeting facilitated hexose transporter 290 nm (18 800 L mol−1 cm−1), 275 nm (18 300 L mol−1 cm−1).
GLUT1. Compound 2-FBDG shows excellent potential as an
1,3,4,6-Tetra-O-acetyl-2-acrylamido-2-deoxy-D-glucose (3). The
in vitro fluorescent probe with retained affinity to GLUT1. product was synthesized according to a published procedure.45
Dual-probe 2-[18F]FBDG interacts with GLUT1 and is trans- The crude reaction mixture was concentrated, then purified by
ported by GLUT1 into murine and human breast cancer cells. flash column chromatography using a gradient of 50% EtOAc/
Further research is required to address its in vivo metabolic hexanes to 75% EtOAc/hexanes. A pure white powder was
stability concerns to improve the radiotracer’s tumor uptake obtained, 158 mg, 0.393 mmol, 99% yield. Rf (1 : 1 EtOAc/
3248 | Org. Biomol. Chem., 2021, 19, 3241–3254
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1
hexanes) = 0.26; H NMR (600.27 MHz, CDCl3) δ 2.02 (s, 3 H), NaOH, extracted with CH2Cl2, and concentrated under
2.04 (s, 3 H), 2.1 (d, J = 1.9 Hz, 6 H), 3.83 (m, 1 H), 4.14 (dd, J = vacuum. The crude compound was purified using a silica gel
2.3, 12.6 Hz, 1 H), 4.28 (dd, J = 4.7, 12.4 Hz, 1 H), 4.41 (m, column (with 10 : 1 hexane/EtOAc) and a recrystallization step
1 H), 5.17 (m, 2 H), 5.70 (m, 3 H), 6.01 (dd, J = 10.4, 16.9 Hz, (hexanes/toluene) – this produced yellow crystals, 110 mg,
1 H), 6.25 (d, J = 16.6 Hz, 1 H); 13C NMR (150.94 MHz, CDCl3) 0.39 mmol, 3% yield. Rf (10 : 1 hexanes/EtOAc) = 0.27; 1H NMR
δ 20.58, 20.59, 20.7, 20.8, 53.0, 61.6, 67.6, 72.5, 73.1, 92.7, (600.27 MHz, CDCl3) δ 3.74 (s, 6 H), 6.81/8.01 (AA′BB′, 4 H),
127.8, 130.0, 165.4, 169.2, 169.6, 170.7, 171.3. LRMS (ESI) m/z: 7.21/8.24 (AA′BB′, 4 H); 13C NMR (150.94 MHz, CDCl3) δ 40.4,
[M + Cl−]− calcd for C17H23ClNO10 436.1: found 436.1.
Compound 6. Tetrazole 1 (0.136 mmol) and 3 (0.177 mmol) 126.6, 128.9 (d, JC–F = 7.7 Hz), 151.1, 163.8, 164.0 (d, JC–F
111.9, 116.0 (d, 2JC–F = 22.1 Hz), 121.1, 123.9 (d, 4JC–F = 3.3 Hz),
3
1
=
were dissolved in acetonitrile. This solution was irradiated at 249.9 Hz); 19F NMR (564.82 MHz, CDCl3) δ −110.28 (tt); LRMS
365 nm for several days. The pyrazoline 4 oxidizes to the pyra- (ESI) m/z: [M + H]+ calcd for C15H15FN5 284.1: found 284.2;
zole under ambient conditions over the course of the reaction. UV/Vis (CH3CN): absorbance λmax (ε): 330 nm (22 000 L mol−1
The acetonitrile was removed under reduced pressure, and the cm−1), 230 nm (19 000 L mol−1 cm−1).
resulting solids were purified by flash column chromatography
Compound 5. Tetrazole
2
(0.0688 mmol) and
3
(30% EtOAc/hexanes to 50% EtOAc/hexanes). A white solid was (0.0977 mmol) were dissolved in acetonitrile. This solution
isolated, 30.6 mg, 0.0477 mmol, 34.7% yield. Rf (1 : 1 EtOAc/ was irradiated at 365 nm for 3 days. The acetonitrile was
1
hexanes) = 0.45; H NMR (600.27 MHz, CDCl3) δ 2.03 (s, 3 H), removed under reduced pressure, and the resulting solids were
2.06 (s, 3 H), 2.10 (d, 6 H), 3.81 (m, 1 H), 3.86 (s, 3 H), 4.14 (dd, purified by flash chromatography (50% EtOAc/hexanes to 66%
J = 2.3, 12.5 Hz, 1 H), 4.27 (dd, J = 4.6, 12.5 Hz, 1 H), 4.35 (m, EtOAc/hexanes). A yellow-green solid was isolated, 35.4 mg,
1 H), 5.16 (m, 2 H), 5.71 (d, 1 H), 6.04 (d, J = 8.8 Hz, 1 H), 6.91 0.054 mmol, 89% yield. Rf (1 : 1 EtOAc/hexanes) = 0.18;
(s, 1 H), 7.37/6.97 (AA′BB′, 4 H), 7.81/7.12 (AA′BB′, 4 H); 1H NMR (600.27 MHz, (CD3)2CO) δ 1.96–2.03 (d, 12 H), 2.85 (s,
13C NMR (150.94 MHz, CD3Cl) δ 20.56, 20.63, 20.69, 20.84, 6 H), 3.12 (dq, 1 H), 3.77–4.65 (m, 9 H), 5.03 (dt, 2 H), 5.36 (dt,
53.3, 55.6, 61.6, 67.5, 72.3, 73.0, 92.5, 105.5, 114.1, 115.7 (d, 1 H), 5.48 (m, 1 H), 5.59 (m, 1 H), 5.92 (ddd, 1 H), 6.07 (dd, 0.5
3
2JC–F = 22.1 Hz), 126.8, 127.5 (d, JC–F = 7.7 Hz), 128.27 (d, H), 6.15 (s, 0.5 H), 6.16 (d, 0.5 H), 6.75/6.94 (AA′BB′, 4 H), 7.20/
4JC–F = 3.2 Hz), 132.7, 137.3, 150.4, 159.0, 159.8, 162.9 (d, 7.98 (AA′BB′, 4 H) [contains additional signals due to epimers
1JC–F = 247.7 Hz), 169.2, 169.3, 170.1, 171.2; LRMS (ESI) m/z: and oxidized product]; 13C NMR (150.94 MHz, (CD3)2CO) δ
[M + H]+ calcd for C31H33FN3O11 642.2: found 642.2.
20.62, 20.68 (t), 20.8 (d, J = 11.9 Hz), 20.9 (d, J = 3.9 Hz), 40.0
Py-GlcN. Compound 6 (0.027 mmol) was dissolved in MeOH (d, J = 17.7 Hz), 41.6 (d, J = 4.2 Hz), 53.7 (d, J = 5.8 Hz), 54.1,
(4 mL) and 0.125 M NaOH (1 mL). The solution was stirred at 62.6 (d, J = 3.9 Hz), 67.0 (d, J = 11.3 Hz), 69.4 (d, J = 3.0 Hz),
room temperature for 10 minutes. The solution was then neu- 69.6, 72.4, 73.4 (m), 92.7, 92.9, 93.1, 115.1 (d, J = 10.0 Hz),
2
tralized with 1 M HCl. Excess solvent was removed under 115.7 (d, J = 11.6 Hz), 116.4 (dd, JC–F = 21.8 Hz), 126.5, 128.8
reduced pressure. The resulting solids were purified by (dd, 3JC–F = 8.3 Hz), 130.0 (d, J = 3.0 Hz), 132.1, 138.7 (d, J = 3.6
1
reverse-phase HPLC using a flow rate of 2 mL min−1 with the Hz), 147.0 (d, J = 7.5 Hz), 163.9 (dd, JC–F = 247.4 Hz), 169.4,
following gradient where H2O (0.2% TFA) is solvent A and 169.5, 170.1, 170.4, 170.6, 170.7, 170.8 (d, J = 3.0 Hz), 172.7,
CH3CN is solvent B: 0–5 min 20% B, 10 min 35% B, 20 min 172.9 [mixture of epimers and oxidized product]; LRMS (ESI)
50% B, 32 min 84% B, then lyophilized. A white solid was m/z: [M + H]+ calcd for C32H38FN4O10 657.3: found 657.2.
recovered, 5.7 mg, 0.012 mmol, 45% yield. 1H NMR
Pyin-GlcN. Compound 5 (0.046 mmol) was dissolved in
(498.12 MHz, CD3OD) δ 3.31–3.95 (m, 9 H), 4.70 (d, β-anomer MeOH (4 mL) and 0.120 M NaOH (1 mL). The solution was
proton, J = 8.3 Hz), 5.16 (d, α-anomer proton, J = 3.5 Hz), 7.20 stirred at room temperature for 10 minutes. The solution was
(s, 1 H), 7.00/7.47 (AA′BB′, 4 H), 7.15/7.87 (AA′BB′, 4 H); 13C then neutralized with 1 M HCl. Excess solvent was removed
NMR (125.69 MHz, CD3OD) mixture of α/β anomers δ 56.02, under reduced pressure. The resulting solids were purified by
56.05, 56.3, 59.0, 62.8, 62.9, 72.3, 72.5, 72.6, 73.2, 75.9, 78.2, reverse-phase HPLC using a flow rate of 2 mL min−1 with the
2
92.4, 96.9, 106.4, 106.8, 115.0, 115.1, 116.6 (d, JC–F = 21.6 Hz), following gradient where H2O (0.2% TFA) is solvent A and
3
3
127.0, 127.3, 128.74 (d, JC–F = 8.2 Hz), 128.77 (d, JC–F = 8.2 CH3CN is solvent B: 0–5 min 20% B, 10 min 35% B, 20 min
Hz), 130.17, 130.20, 134.40, 134.46, 139.96, 140.03, 151.64, 50% B, 32 min 84% B, then lyophilized. A pale colorless solid
1
1
151.68, 162.0 (d, JC–F = 245.9 Hz), 161.1, 163.0, 164.3 (d, JC–F was recovered, 10.7 mg, 0.022 mmol, 45% yield. 1H NMR
= 245.9 Hz); 19F NMR (468.64 MHz, CD3OD) −77.04 (s, TFA), (600.27 MHz, HDO + H2O suppression) δ 3.22/3.32 (s, 6 H),
−115.88 (m); HRMS (ESI) m/z: [M
+
Na]+ calcd for 3.36–3.90 (m, 6 H), 4.64 (d, J = 8.4 Hz, 1 H), 4.89 (m, 2 H), 5.16
C23H24FN3O7Na 496.1490: found 496.1494; UV/Vis (1 : 1 (α-anomeric proton, d, J = 3.6 Hz, 1 H), 7.15 (m, 4 H), 7.46 (m,
[CH3CN] : [pH 7.4 PBS]): absorbance λmax (ε): 230 nm (7900 L 2 H), 7.73 (m, 2 H); 13C NMR (150.94 MHz, D2O) δ 39.2, 46.4,
mol−1 cm−1); fluorescence (1 : 1 [CH3CN] : [pH 7.4 PBS]): λmax
440 nm (320 nm excitation).
=
54.1/54.2, 56.6, 60.5/60.6, 63.16/62.50, 70.0, 70.2, 70.5, 71.51/
71.56, 73.43/73.59, 75.9, 90.7, 94.7, 113.8, 115.8 (d, JC–F = 21.8
2
4-(5-(4-Fluorophenyl)-2H-tetrazol-2-yl)-N,N-dimethylaniline Hz), 121.3, 121.5, 126.5 (d, J = 7.7 Hz), 127.1, 127.9, 128.3,
1
(2). The tetrazole was synthesized according to a published 133.8, 145.4, 151.2, 163.4 (d, JC–F = 245.0 Hz), 173.7 (different
procedure with slight modifications.44 Instead of quenching protonation states); LRMS (ESI) m/z: [M + H]+ calcd for
the pyridine with HCl, the reaction mixture was washed with C24H30FN4O5 489.2: found 489.2. UV/Vis (pH 7.4 PBS): absor-
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bance λmax (ε): 250 nm (12 640 L mol−1 cm−1), 320 nm 5.21 (dd, 0.4 H), 5.53 (d, J = 0.6 Hz, 8.5 Hz), 6.40 (d, J = 3.6 Hz,
(9300 L mol−1 cm−1); fluorescence (pH 7.4 PBS): λmax = 450 nm 0.4 H); 13C NMR (150.94 MHz, CDCl3) δ 20.6, 20.72, 20.74,
(320 nm excitation).
20.93, 20.95, 20.97, 21.1, 36.8, 37.3, 58.7, 60.0, 61.7, 61.8, 68.1,
3-Azido-7-(2-fluoroethoxy)-2H-chromen-2-one (7). 3-Azido-7- 68.2, 69.5, 71.4, 72.0, 72.4, 73.8, 90.7, 95.3, 168.98, 169.01,
hydroxy-2H-chromen-2-one, which was synthesized according 169.6, 170.7, 171.0, 171.3; LRMS (ESI) m/z: [M + H]+ calcd for
to a published procedure (0.900 mmol),28 was stirred with C17H24NO9 386.1: found 386.1.
K2CO3 (1.35 mmol) in dry DMF (4 mL) for 10 min at 50 °C
Compound 9. Azide 7 (0.437 mmol) and alkyne 8
under N2. Then 1-fluoro-2-iodoethane (1.35 mmol) was added (0.428 mmol) were dissolved in a solution of 1 mL t-BuOH,
dropwise, and was allowed to react for 1.5 h. The DMF was 4 mL acetone, and 1 mL water. CuSO4·5H2O (1.126 mmol) was
removed by reduced pressure, and the residue was resus- dissolved in 2 mL water, and sodium ascorbate (5.463 mmol)
pended in a solution of 1 : 2 EtOH to 6 M HCl. The product was dissolved in 4 mL water. The Na-ascorbate solution was
precipitated, which was filtered off and washed with water. added dropwise to the Cu(II) solution. When the solution
This provided a fine red powder, 140.3 mg, 0.563 mmol, 63% turned a pale orange colour, 230 µL of this suspension was
yield. Rf (50% EtOAc/hexanes) = 0.78; 1H NMR (600.27 MHz, added to the reaction mixture. The reaction vessel was then
(CD3)2CO) δ 4.42 (m, J = 29.4 Hz, 2 H), 4.82 (m, J = 47.8 Hz, 2 flushed with N2, and the reaction was stirred for 24 h. The
H), 7.00 (s, 1 H), 7.01 (m, 1 H), 7.48 (s, 1 H), 7.58 (d, J = 8.9 Hz, reaction mixture was filtered over Celite and washed with
2
1 H); 13C NMR (150.94 MHz, (CD3)2CO) δ 69.0 (d, JC–F = 19.6 50 mL of acetone. The filtrate was concentrated, and the crude
Hz), 82.8 (d, 1JC–F = 168.5 Hz), 102.1, 114.1, 114.4, 124.1, 127.7, was purified by column chromatography (66% EtOAc/hexanes
129.8, 154.1, 158.2, 161.8; 19F NMR (564.82 MHz, (CD3)2CO) δ to 50% EtOAc/40% hexanes/10% acetone to 50% EtOAc/25%
−224.40 (m). LRMS (ESI) m/z [M–N2 + H]+ calcd for C11H9FNO3 hexanes/25% acetone). This provided
a
pale yellow oil,
222.0: found 222.0.
185.3 mg, 0.292 mmol, 68% yield. Rf (2 : 1 : 1 EtOAc/hexanes/
1,3,4,6-Tetra-O-acetyl-N-(3-trimethylsilyl)propargyl-D-glucos- acetone) = 0.42/0.65 (–OAc); 1H NMR (600.27 MHz, CDCl3) δ
amine. 1,3,4,6-Tetra-O-acetyl-β-D-glucosamine hydrochloride 2.03 (s, 3 H), 2.09 (d, 6 H), 2.21 (s, 3 H), 3.00 (s, 1 H), 3.79 (m,
(1.0 mmol) and 3-(trimethylsilyl)-2-propynal (1.4 mmol) were 1 H), 4.08 (m, 1 H), 4.29 (dd, 2 H), 4.33 (m, J = 27.6 Hz, 2 H),
dissolved in MeOH (10 mL) and stirred at 40 °C for 30 min, 4.83 (m, J = 47.3 Hz, 2 H), 5.07 (dt, 1 H), 5.59 (d, J = 8.5 Hz,
until the solution became clear. NaCNBH3 (1.1 mmol) was 1 H), 6.95 (d, J = 2.4 Hz, 1 H), 7.03 (dd, J = 2.4 Hz, 8.7 Hz, 1 H),
added and the solution was stirred overnight at room temp- 7.60 (d, J = 8.7 Hz, 1 H), 8.51 (d, J = 25.0 Hz, 1 H); 13C NMR
erature. The crude reaction mixture was extracted with (150.94 MHz, CDCl3) δ 14.2, 20.6, 20.7, 20.9, 21.0, 21.2, 29.7,
2
CH2Cl2 then concentrated under vacuum. The crude oil was 42.7, 43.0, 59.8, 60.4, 61.7, 67.7 (d, JC–F = 20.5 Hz), 68.2, 72.5,
1
purified by flash column chromatography (20–50% EtOAc/ 73.6, 81.3 (d, JC–F = 172.2 Hz), 95.1, 101.4, 112.0, 114.3, 122.7,
hexanes) to provide a clear, colorless, viscous oil, 201.3 mg, 130.0, 133.4, 154.5, 156.0, 162.4, 169.1, 169.6, 170.7, 171.1;
0.440 mmol, 44% yield. Rf (1 : 1 EtOAc/hexanes) = 0.61; 1H 19F NMR (564.82 MHz, CDCl3) δ −223.73 (m); LRMS (ESI) m/z:
NMR (600.27 MHz, CDCl3) δ 0.18 (s, 9 H), 2.04 (s, 3 H), 2.09 [M + H]+ calcd for C28H32FN4O12 635.2: found 635.1.
(d, J = 5.1 Hz, 6 H), 2.17 (s, 3 H), 3.08 (dd, 1 H), 3.47 (q,
CO-GlcN-1. The acetylated glucosamine derivative 9 was
2 H), 3.78 (dq, 1 H), 4.08 (dd, 1 H), 4.30 (dd, 1 H), 5.06 (dt, stirred in 75% MeOH in H2O with 4.5 eq. of K2CO3 for 30 min.
2 H), 5.52 (d, J = 8.5 Hz, 1 H); 13C NMR (150.94 MHz, The base was neutralized with Dowex 50WX8 H+, filtered and
CDCl3) δ −0.02, 14.2, 20.6, 20.7, 20.9, 21.1, 38.2, 59.4, 60.4, rinsed with MeOH, and the MeOH was removed under
61.8, 68.2, 72.4, 73.8, 88.0, 95.3, 104.1, 168.9, 169.6, 170.7, reduced pressure. The resulting residue was purified by
171.1; LRMS (ESI) m/z: [M + H]+ calcd for C20H32NO9Si reverse-phase HPLC using a flow rate of 2 mL min−1 with the
458.2: found 458.1.
following gradient where H2O (0.2% TFA) is solvent A and
1,3,4,6-Tetra-O-acetyl-N-propargyl-D-glucosamine (8). 1,3,4,6- CH3CN is solvent B: 0–5 min 20% B, 15 min 35% B, 25 min
Tetra-O-acetyl-N-(3-trimethyl-silyl)propargyl-β-D-glucosamine 40% B, 35 min 85% B, providing the compound as the hydro
(0.312 mmol) was dissolved in 6 mL dry THF at room tempera- trifluoroacetate (contains mixture of α and β anomers), 6.3 mg,
1
ture and flushed with N2. TBAF (1 M in THF) was added 0.0109 mg, 6% yield. H NMR (600.27 MHz, D2O) δ 3.14 (dd,
(0.38 mmol) and the reaction was mixture was stirred for 2 h. J = 8.3, 10.9 Hz, 0.2 H), 3.39 (dd, J = 3.4, 10.5 Hz, 0.8 H), 3.48
The reaction was quenched by adding sat. NH4Cl and diethyl (dd, J = 9.0, 10.2 Hz, 1 H), 3.53 (ddd, J = 1.9, 5.6, 9.8 Hz, 0.2 H),
ether. The product was extracted with diethyl ether, washed 3.76 (m, 1 H), 3.82–3.94 (m, 2 H), 4.03 (dd, J = 9.0, 10.5 Hz, 0.8
with NH4Cl, water, then brine. The organic layer was concen- H), 4.23–4.40 (m, 1 H), 4.40–4.54 (m, 1 H), 4.86 (m, 2 H), 5.13
trated, and the residue was purified by column chromato- (d, J = 8.3 Hz, 0.2 H), 5.68 (d, J = 3.8 Hz, 0.8 H), 6.93–7.09 (m, 2
graphy (50% EtOAc/hexanes). This provided a pale-yellow oil as H), 7.71 (d, J = 8.7 Hz, 1 H), 8.47 (s, 1 H), 8.58–8.73 (m, 1 H);
a mixture of α/β anomers 73.2 mg, 0.190 mmol, 60% yield. Rf 13C NMR (150.94 MHz, D2O) δ 39.7, 41.0, 59.4, 60.2, 60.4, 61.6,
2
(1 : 1 EtOAc/hexanes) = 0.37/0.44; 1H NMR (600.27 MHz, 68.1 (d, JC–F = 18.6 Hz), 69.5, 69.7, 70.0, 71.3, 76.1, 82.3 (d,
CDCl3) δ 2.04 (d, J = 0.75 Hz, 3 H), 2.09 (m, 6 H), 2.18 (d, J = 1JC–F = 164.6 Hz), 87.9, 92.4, 101.4, 111.8, 114.4, 115.3, 117.3,
10.8 Hz, 3 H), 2.23 (dt, 1 H), 3.08 (dd, 0.6 H), 3.20 (dd, 0.4 H), 119.5, 127.2, 127.4, 130.9, 137.9, 138.0, 138.30, 138.33, 154.6,
3.41 (m, 1 H), 3.48 (dt, 1 H), 3.78 (m, 0.6 H), 4.02 (m, 0.4 H), 158.5, 162.8, 163.1; 19F NMR (564.82 MHz, D2O) δ −78.5 (TFA),
4.07 (m, 1 H), 4.31 (m, 1 H), 5.02 (dd, 0.6 H), 5.12 (dt, 1 H), −226.30 (ttt); HRMS (ESI) m/z: [M + H]+ calcd for C20H23FN4O8
3250 | Org. Biomol. Chem., 2021, 19, 3241–3254
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467.1573: found 467.1575; UV/Vis (PBS pH 7.4): absorbance 19F NMR (376.318 MHz, CD3OD) δ −77.09 (s, TFA), −225.60
λmax (ε): 340 nm (12 000 L mol−1 cm−1); fluorescence λmax
420 nm (340 nm excitation).
:
(tt); HRMS (ESI) m/z: [M + Na]+ calcd for NaC18H20O9NF
436.1014: found 436.1013; UV/Vis (0.1% DMSO in Krebs–
Ethyl
Ethyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (0.545 mmol), L mol−1 cm−1); fluorescence λmax: 400 nm (350 nm excitation).
which was synthesized according to a literature procedure,46
7-Fluoro-2,1,3-benzoxadiazole-4-sulfonyl chloride (CBDF).
7-(2-fluoroethoxy)-2-oxo-2H-chromene-3-carboxylate. Ringer buffer pH 7.4): absorbance λmax (ε): 350 nm (26 000
and K2CO3 (0.818 mmol) was suspended in DMF (1.2 mL) and Synthesized from 4-fluoro-2,1,3-benzoxadiazole (16) according
flushed with N2. This was stirred at 70 °C for 10 min. Then to the literature procedure with slight modifications.30 The
1-fluoro-2-iodoethane (0.818 mmol) was added dropwise, and crude was purified by flash column chromatography using 1 : 1
the reaction was stirred at 70 °C for 6 h. The reaction mixture toluene/hexanes as eluent, providing an off-white solid,
was diluted with EtOAc and washed with sat. NaHCO3, and 384 mg, 1.63 mmol, 86% yield. Rf (1 : 1 toluene/hexanes) =
1
brine. The organic phase was concentrated, and the crude was 0.32; H NMR (498.12 MHz, (CH3)2CO) δ 7.70 (dd, J = 8.0, 9.0
recrystallized from EtOAc/hexane to provide white crystalline Hz, 1 H), 8.60 (dd, J = 4.0, 8.0 Hz, 1 H); 13C NMR (125.69 MHz,
2
3
needles, 103.7 mg, 0.370 mmol, 68% yield. 1H NMR (CH3)2CO) δ 114.5 (d, JC–F = 18.9 Hz), 128.7 (d, JC–F = 5.7 Hz),
3
2
(399.978 MHz, CDCl3) δ 1.40 (t, J = 7.1 Hz, 3 H), 4.24–4.29 (m, 139.9 (d, JC–F = 9.3 Hz), 144.6 (d, JC–F = 20.4 Hz), 146.7 (d, J =
1 H), 4.31–4.36 (m, 1 H), 4.40 (q, J = 7.1 Hz, 2 H), 4.71–4.77 (m, 3.1 Hz), 155.9 (d, JC–F = 276.0 Hz); 19F NMR (468.64 MHz,
1
1 H), 4.84–4.89 (m, 1 H), 6.83 (d, J = 2.4 Hz, 1 H), 6.94 (dd, J = (CH3)2CO) δ −107.71 (dd, J = 3.7, 8.9 Hz); HRMS (EI) m/z: [M]+
2.4, 8.7 Hz, 1 H), 7.52 (d, J = 8.7 Hz, 1 H), 8.50 (d, J = 0.6 Hz, calcd for C6H2ClFN2O3S 235.9459: found 235.9459.
1 H).13C NMR (175.976 MHz, CDCl3) δ 14.2, 61.7, 67.8 (d, 2JC–F
=
7-Fluoro-2,1,3-benzoxadiazole-4-sulfonyl fluoride (FBDF).
20.9 Hz), 81.3 (d, JC–F = 171.9 Hz), 101.0, 112.0, 113.9, 114.6, Synthesis was adapted from Dong et al.31 A solution of KHF2
130.8, 148.8, 157.0, 157.4, 163.3, 163.7. 19F NMR (376.318 MHz, (29.8 mg, 0.381 mmol) in H2O (0.16 mL) was added to a solu-
CDCl3) δ −223.84 (tt); LRMS (ESI) m/z: [M + H]+ calcd for tion of 17 (39.2 mg, 0.166 mmol) dissolved in CH3CN
1
C14H14O5F 281.1; found 281.1.
(0.16 mL). This was then vigorously stirred for 2 h at rt. The
7-(2-Fluoroethoxy)-2-oxo-2H-chromene-3-carboxylic acid (11). reaction was then extracted with CH2Cl2 and washed with
Ethyl
7-(2-fluoroethoxy)-2-oxo-2H-chromene-3-carboxylate water then brine. The organic layer was dried with Na2SO4, fil-
(0.187 mmol) was suspended in 0.3 mL of 2 : 1 EtOH/H2O tered, and concentrated to provide pale white/yellow crystals,
(v/v), and NaOH (0.845 mmol) was added. This was immedi- 36.5 mg, 0.166 mmol, >99% yield. Rf (1 : 1 EtOAc/hexanes) =
ately refluxed for 15 min, then cooled to rt. The reaction 0.65; 1H NMR (498.12 MHz, CDCl3) δ 7.32 (ddd, J = 1.0, 7.8, 8.5
mixture was added dropwise to 0.6 mL of 20% HCl (∼6.7 M) Hz, 1 H), 8.33 (ddd, J = 0.8, 3.9, 7.8 Hz, 1 H); 13C NMR
2
and cooled to 0 °C. The precipitate was filtered and washed (125.69 MHz, CDCl3) δ 112.5 (d, JC–F = 18.3 Hz), 118.7 (dd,
with cold water, providing a pale yellow solid, 36.1 mg, JC–F = 6.2 Hz, 32.5 Hz), 139.0 (dd, JC–F = 2.1, 8.5 Hz), 142.8 (d,
1
0.143 mmol, 77% yield. 1H NMR (399.980 MHz, (CD3)2SO) δ JC–F = 19.8 Hz), 146.1 (d, JC–F = 1.2, 2.8 Hz), 155.3 (d, JC–F
=
4.40 (dt, J = 3.8, 30.0 Hz, 2 H), 4.68–4.87 (m, 2 H), 7.01–7.10 281.1 Hz); 19F NMR (468.64 MHz, CDCl3) δ −102.99 (dd, J =
(m, 2 H), 7.84 (d, J = 8.6 Hz, 1 H), 8.72 (s, 1 H); 13C NMR 3.7, 8.4 Hz), 63.97 (s); HRMS (EI) m/z: [M]+ calcd for
2
(175.976 MHz, (CD3)2SO) δ 68.1 (d, JC–F = 18.4 Hz), 81.8 (d, C6H2F2N2O3S 219.9754: found 219.9753.
1JC–F = 167.4 Hz), 100.9, 111.9, 113.6, 114.1, 131.7, 149.0,
Compound 2-FBDG. FBDF (13 mg, 0.059 mmol),
156.8, 157.2, 163.4, 164.1; 19F NMR (376.318 MHz, (CD3)2SO) D-glucosamine (free base, 16 mg, 0.089 mmol), and NaHCO3
δ −222.35 (tt); LRMS (ESI) m/z: [M + H]+ calcd for C12H10O5F (7.5 mg, 0.089 mmol) was dissolved in DMF (0.5 mL) and
253.1; found 253.1.
stirred at rt overnight. The solvent was removed by reduced
CO-GlcN-2. Compound 11 (0.0649 mmol), glucosamine·HCl pressure evaporation. The crude was purified by reverse-phase
(0.0974 mmol), and DIPEA (0.1974 mmol) was dissolved in HPLC using a flow rate of 2 mL min−1 with the following gradi-
DMF (0.22 mL). HBTU (0.0974 mmol) was added and the flask ent where H2O (0.2% TFA) is solvent A and CH3CN is solvent
was flushed with N2. The reaction was stirred overnight at rt. B: 0–5 min 20% B, 10 min 35% B, 20 min 50% B, 32 min 84%
The crude mixture was diluted with 5.78 mL of 50/50 CH3CN/ B, then lyophilized, providing
a yellow solid, 22 mg,
1
0.2% TFA water, purified by reverse-phase HPLC using a flow 0.058 mmol, 99% yield. Rf (20% MeOH/EtOAc) = 0.57/0.65; H
rate of 2 mL min−1 with the following gradient where H2O NMR (599.93 MHz, CD3CN/D2O) mixture of α/β δ 3.32–3.88 (m,
(0.2% TFA) is solvent A and CH3CN is solvent B: 0–9 min 1% 6 H), 4.78 (d, J = 7.8 Hz, 0.4 H), 5.26 (d, J = 2.9 Hz, 0.6 H), 6.58
B, 25 min 60% B, 32 min 75% B, then lyophilized to give a and 6.60 (d, J = 8.5 Hz, 1 H), 6.99 (br s, 1 H), 7.46 (br d, J = 8.8
1
fluffy white solid, 19.8 mg, 0.0479 mmol, 74% yield. H NMR Hz), 8.13 and 8.14 (d, J = 8.5 Hz, 1 H); 13C NMR (125.69 MHz,
(399.978 MHz, CD3OD) δ 3.37–4.12 (m, 6 H), 4.31–4.36 (m, 1 CD3CN) δ 62.6/62.7, 71.8, 72.9, 73.7, 76.0, 77.3, 91.5, 96.7,
2
H), 4.38–4.44 (m, 1 H), 4.67–4.74 (m, 1 H, second proton 101.2 (d, JC–F = 29.3 Hz), 101.6, 144.2, 145.4/145.6, 146.7/
signal obscured by HDO) 5.19 (d, J = 3.5 Hz, 1 H), 7.02–7.09 146.9; 19F NMR (468.64 MHz, CD3CN) δ 64.21 (s), 64.28 (s);
(m, 2 H), 7.77 (d, J = 8.6 Hz, 1 H), 8.83 (d, J = 0.7 Hz, 1 H); HRMS (ESI) m/z: [M − H]− calcd for C12H13FN3O8S 378.0413:
2
13C NMR (175.976 MHz, CD3OD) δ 56.2, 62.8, 69.6 (d, JC–F
=
found 378.0411; UV/Vis (Krebs–Ringer buffer pH 7.4): absor-
19.8 Hz), 72.3, 73.3, 73.6, 82.8 (d, 1JC–F = 169.5 Hz), 92.6, 102.0, bance λmax (ε): 425 nm (16 400 L mol−1 cm−1); fluorescence:
114.0, 115.4, 115.8, 132.7, 149.6, 158.1, 162.9, 164.4, 165.6; λmax = 570 nm (425 nm excitation).
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Organic & Biomolecular Chemistry
Radiochemistry
fluency. 1 h before the assay, the cell media was removed, and
the cells were rinsed with pH 7.4 PBS twice. The cells were
then incubated at 37 °C with 1 mL of Krebs buffer. The buffer
was aspirated, and the cells were treated with 1 mL of either
200 µM 2-FBDG or 200 µM 2-FBDG + 50 µM D-glucose (in pH
7.4 Krebs buffer) for 1 h at 37 °C. The treatment was aspirated,
then the cells were washed with ice-cold PBS twice. The cells
were then incubated for 7 min at rt with 1 mL of 3.5% PFA in
PBS. After PFA removal, the cells were permeabilized using
1 mL of 0.02% Triton X-100 in PBS for 5 min. The permeabiliz-
ing solution was removed and the cells were washed with PBS.
Then the cells were treated with 1 mL of 0.3 µmol mL−1 DAPI
in PBS for 15 min. The DAPI solution was removed and the
cells were rinsed with PBS. The coverslips were then mounted
onto microscopy slides using 15 µL of Mowiol. The slides were
then imaged using the same settings on the Leica SP8 Falcon
STED.
N.c.a. [18F]F− was produced via the 18O(p,n)18F nuclear reac-
tion from [18O]H2O (Rotem Industries Ltd, Hyox 18O-enriched
water, min. 98%) on an ACSI TR19/9 Cyclotron (Advanced
Cyclotron Systems Inc., Richmond, Canada). Cyclotron-pro-
duced [18F]F− (∼1 GBq) was trapped on a Waters SepPak® light
QMA anion exchange cartridge. [18F]CsF was eluted from the
anion exchange cartridge using 300 µL of aqueous Cs2CO3
(10 mg mL−1). 100 µL of the [18F]CsF solution was added to a
solution of CBDF (0.2 mg) in t-BuOH (100 µL) in an Eppendorf
tube, and incubated for 5 min to form [18F]FBDF. The reaction
was added to an Eppendorf tube containing a solution of
glucosamine (free base, 1.5 mg) and NaHCO3 (1.5 mg) in DMF
(500 µL). The vial was rinsed with an additional 100 µL of
DMF to ensure complete transfer of [18F]FBDF. The reaction is
shaken vigorously at rt for 20 min, gradually turning a bright
yellow color. The crude 2-[18F]FBDG was then diluted with
200 µL of distilled H2O and purified by reverse-phase HPLC at
a constant flow rate of 3 mL min−1 using the following gradi-
ent where H2O is solvent A and CH3CN is solvent B:
0–4.25 min 20% B, 7.5 min 35% B, 13 min 50% B, 20 min 84%
B, tR = 15.0 and 15.7 min (α and β anomer). 2-[18F]FBDG
accounted for 83 9% of the crude reaction mixture. The 2-
[18F]FBDG peak was fractionated, isolated using a rotary evap-
orator, and reformulated in 200 µL saline (60–80 MBq 2-[18F]
FBDG final activity). RCY was 69 3% (n = 3) d.c. over 2 steps.
Quality control was performed using thin layer chromato-
graphy (TLC) on silica plates using 10% EtOH/EtOAc as mobile
phase (Rf ([18F]FBDG) = 0.47, 100% radiochemical purity) and
analytical Radio-HPLC using a gradient of MeCN (B)/H2O (A)
0–3 min 10% B, 10 min 30% B, 17 min 50% B, 23 min 70% B,
27–30 min 90% B, 1 mL min−1 (tR = 17.0 and 17.8 min, 95.8%
purity).
Dynamic PET imaging experiments
All animal experiments were carried out in accordance with
guidelines of the Canadian Council on Animal Care and
approved by the local animal care committee of the Cross
Cancer Institute. The procedure was performed on
MDA-MB-231 tumor-bearing BALB/c mice according to Wuest
et al.32 PET imaging of 2-[18F]FBDG was performed on an
INVEON PET/CT scanner (Siemens Preclinical Solutions,
Knoxville, TN, USA). Prior to radiotracer injection, mice were
anesthetized through inhalation of isoflurane in 40% oxygen/
60% nitrogen (gas flow 1 L min−1), and body temperature was
kept constant at 37 °C. Mice were placed in a prone position
into the center of the field of view. A transmission scan for
attenuation correction was not acquired. Mice were injected
with 5 MBq of 2-[18F]FBDG or 5 MBq of 2-[18F]FDG in 150 μL
of isotonic saline solution (0.9% w/v of NaCl) through a tail
vein catheter. Data acquisition was performed over 60 min in
3D list mode. The dynamic list mode data were sorted into
sinograms with 54 time frames (10 × 2, 8 × 5, 6 × 10, 6 × 20, 8 ×
60, 10 × 120, 6 × 300 s). The frames were reconstructed using
maximum a posteriori (MAP) as reconstruction mode. No cor-
rection for partial volume effects was applied. The image files
were processed using the ROVER v2.0.51 software (ABX GmbH,
Radeberg, Germany). Masks defining 3D regions of interest
(ROI) were set, and the ROIs were defined by thresholding.
ROIs covered all visible tumor mass of the subcutaneous
tumors, and the thresholds were defined by 50% of the
In vitro 2-[18F]FDG competition assay
EMT6 cells were seeded in 12-well plates at 100 000 cells per well
24 hours before the competition assay. 1 h before the assay, the
cell media was removed, and the cells were rinsed with pH 7.4
PBS twice. The cells were then incubated at 37 °C with 1 mL of
Krebs buffer. Then the buffer was aspirated, and the cells were
treated with various concentrations of the non-radioactive dual-
probes as well as a constant amount of 2-[18F]FDG per well
(∼0.111 MBq per well) – the total treatment volume was 300 µL
(in Krebs buffer (pH 7.4)). The cells were incubated for 1 h at
37 °C. After incubation, the treatment was removed, and the
cells were rinsed with ice-cold PBS twice. 400 µL of RIPA was maximum radioactivity uptake level. Mean standardized
added to each well to lyse the cells for at least 10 min. After cell uptake values [SUVmean = (activity/mL tissue)/(injected activity/
lysis, 300 µL of the cell lysate was pipetted into scintillation vials body weight), mL g−1] were calculated for each ROI, and time-
for activity using a 2480 automatic gamma counter WIZARD2
(PerkinElmer, Waltham, MA, USA). These experiments were also
repeated using D-glucose and 2-NBDG as reference compounds.
activity curves (TAC) were generated. All semiquantified PET
data are presented as means SEM. Statistical differences
were tested by Student’s t test and were considered significant
for P < 0.05.
Confocal microscopy experiments with 2-FBDG in EMT6 and
MDA-MB231 cells
Metabolic stability studies in vivo
The procedure was adapted from Soueidan et al.47 EMT6 or The procedure was performed according to Richter et al.48 For
MDA-MB231 cells were grown on coverslips to ∼90% con- metabolic stability studies in vivo, normal BALB/c mice were
3252 | Org. Biomol. Chem., 2021, 19, 3241–3254
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Paper
anesthetized through inhalation of isoflurane in 40% oxygen/
60% nitrogen (gas flow 1 L min−1) prior to i.v. injection of 25
MBq [18F]FBDG via the tail vein. Venous blood samples were
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the contained proteins were precipitated by addition of 2 12 R. S. Brown and R. L. Wahl, Cancer, 1993, 72, 2979–2985.
volume parts of methanol (2 vol of MeOH/1 vol of sample).
Another centrifugation step (13 000 rpm × 5 min) was per-
formed to obtain the plasma in the supernatant. The clear
13 R. Paul, R. Johansson, P. L. Kellokumpu-Lehtinen,
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plasma supernatant was injected onto a Shimadzu HPLC 14 L. Speizer, R. Haugland and H. Kutchai, Biochim. Biophys.
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Conflicts of interest
There are no conflicts to declare.
19 B. L. Cox, T. R. Mackie and K. W. Eliceiri, Am. J. Nucl. Med.
Mol. Imaging, 2014, 5, 1–13.
20 X. Tong, A. Garofalakis, A. Dubois, R. Boisgard,
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21 Y.-D. Kwon, Y. Byun and H.-K. Kim, Nucl. Med. Biol., 2021,
93, 22–36.
22 L. Sun, J. Ding, W. Xing, Y. Gai, J. Sheng and D. Zeng,
Bioconjugate Chem., 2016, 27, 1200–1204.
23 V. G. Ostapchenko, J. Snir, M. Suchy, J. Fan, M. R. Cobb,
B. A. Chronik, M. Kovacs, V. F. Prado, R. H. E. Hudson,
S. H. Pasternak, M. A. M. Prado and R. Bartha, Contrast
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Acknowledgements
The authors thank Floyd Baker, David Clendening, and Blake
Lazurko from the Edmonton Radiopharmaceutical Center for
18F production on a TR-19 cyclotron (Advanced Cyclotron
Systems Inc, Vancover, BC, Canada). And Joanne D. Smith
(Dept. of Oncology, University of Alberta) for her assistance
with the confocal imaging. The authors are also grateful to
Dan McGinn (Vivarium of the Cross Cancer Institute,
Edmonton, AB, Canada) for supporting the animal work and
Dr Hans-Soenke Jans (University of Alberta) for technical help
and support of the preclinical PET facility as well as the
Dianne and Irving Kipnes Foundation for supporting this
work.
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