A novel radiochemical approach to 1-(2'- deoxy-2'-[18F]fluoro-β-D-arabinofuranosyl) cytosine (18F-FAC)

Article abstract of DOI:10.1002/jlcr.3233

¹⁸F-FAC (1-(2'-deoxy-2'-[¹⁸F]fluoro-β-D-arabinofuranosyl)-cytosine) is an important 2'-fluoro-nucleoside-based positron emission tomography (PET) tracer that has been used for in vivo prediction of response to the widely used cancer chemotherapy drug gemcitabine. Previously reported synthetic routes to ¹⁸F-FAC have relied on early introduction of the ¹⁸F radiolabel prior to attachment to protected cytosine base. Considering the ¹⁸F radiochemical half-life (110 min) and the technical challenges of multi-step syntheses on PET radiochemistry modular systems, late-stage radiofluorination is preferred for reproducible and reliable radiosynthesis with in vivo applications. Herein, we report the first late-stage radiosynthesis of ¹⁸F-FAC. Cytidine derivatives with leaving groups at the 2'-position are particularly prone to undergo anhydro side-product formation upon heating because of their electron density at the 2-carbonyl pyrimidone oxygen. Our rationally developed fluorination precursor showed an improved reactivity-to-stability ratio at elevated temperatures. ¹⁸F-FAC was obtained in radiochemical yields of 4.3-5.5% (n = 8, decay-corrected from end of bombardment), with purities ≥98% and specific activities ≥63 GBq/μmol. The synthesis time was 168 min.

Full text of DOI:10.1002/jlcr.3233

Research Article
Received 5 June 2014,
Revised 28 July 2014,
Accepted 8 August 2014
Published online 25 September 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3233
A novel radiochemical approach to 1-(2’-
1
8
deoxy-2’-[ F]fluoro-β-D-arabinofuranosyl)
†
1
8
cytosine ( F-FAC)
a,b
b
a
Jan-Philip Meyer, Katrin C. Probst, Iuni M. L. Trist,
Christopher McGuigan, and Andrew D. Westwell *
a
a
1
8
18
F-FAC (1-(2’-deoxy-2’-[ F]fluoro-β-D-arabinofuranosyl)-cytosine) is an important 2’-fluoro-nucleoside-based positron
emission tomography (PET) tracer that has been used for in vivo prediction of response to the widely used cancer
1
8
chemotherapy drug gemcitabine. Previously reported synthetic routes to F-FAC have relied on early introduction of the
1
8
18
F radiolabel prior to attachment to protected cytosine base. Considering the F radiochemical half-life (110 min) and
the technical challenges of multi-step syntheses on PET radiochemistry modular systems, late-stage radiofluorination is
preferred for reproducible and reliable radiosynthesis with in vivo applications. Herein, we report the first late-stage
1
8
radiosynthesis of F-FAC. Cytidine derivatives with leaving groups at the 2’-position are particularly prone to undergo
anhydro side-product formation upon heating because of their electron density at the 2-carbonyl pyrimidone oxygen. Our
rationally developed fluorination precursor showed an improved reactivity-to-stability ratio at elevated temperatures.
1
8
F-FAC was obtained in radiochemical yields of 4.3–5.5% (n = 8, decay-corrected from end of bombardment), with
purities ≥98% and specific activities ≥63 GBq/μmol. The synthesis time was 168 min.
Keywords: nucleosides; positron emission tomography; radiochemistry; radiopharmaceuticals; computational chemistry
Introduction
kinase (dCK) activating enzyme and rapid deactivating
metabolism by cytidine deaminase (CDA). In addition, serious
Nucleoside derivatives represent an important emerging class
of biomarkers for positron emission tomography (PET)
dose-limiting toxicities are associated with this chemotherapy
8
drug.
Prediction of patient response to gemcitabine
1
imaging. For example, the clinically approved proliferation
chemotherapy would therefore be an important milestone
towards personalized patient chemotherapy in the cancer clinic.
Given the close structural resemblance to gemcitabine,
1
8
18
biomarker
F-FLT (3’-deoxy-3’-[ F]fluoro-L-thymidine) is
accessible via an effective radiochemical synthesis in high
radiochemical yield and purity suitable for human production
18
F-FAC was found to have a comparably high affinity for
2
under Good Manufacturing Practice guidelines. Hence,
deoxycytidine kinase (dCK), a rate-limiting enzyme in the
nucleoside salvage pathway. Hence, in vivo imaging
determined dCK-positive and dCK-negative tumours.
1
8
F-FLT is now commonly used for the early detection o3f
various cancers and the evaluation of treatment response.
Especially in high-grade tumours, it appears to be more
powerful in predicting treatment response and survival than
18
Furthermore, an additional study showed that F-FMAC PET
imaging provides information about cytidine deaminase
1
8
18
F-FDG, as measured by standard uptake values of F-FLT
(
CDA) enzymatic activity in vivo, which is related to
directly corresponding to cell proliferation shown by the
Ki-67 proliferation index.
Cytidine-based radiotracers for PET imaging such as F-FAC
6
gemcitabine resistance.
Together, cytidine-based PET
4
probes enable further conclusions towards personalized
18
18
(1, Figure 1) and 1-(2’-deoxy-2’-[ F]fluoroarabinofuranosyl)-
1
8
a
School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood
5
-methylcytosine ( F-FMAC, 2, Figure 1) have been previously
Building, King Edward VII Avenue, Cardiff, CF10 3NB, Wales, UK
shown to be successful as predictors of response for
nucleoside analogue-based treatment of cancer as well as
5,6
b
Wales Research
& Diagnostic PET Imaging Centre (PETIC), Institute for
7
18
local immune activation in mouse models. As such, F-FAC Translation, Innovation, Methodology & Engagement (TIME), School of Medicine,
Cardiff University, Heath Park, Cardiff, CF14 4XN, Wales, UK
has been used for response prediction to the widely used
chemotherapy drug gemcitabine. Gemcitabine is frequently
used for (combination) chemotherapy of a number of solid
5
*
Correspondence to: Andrew D. Westwell, School of Pharmacy and
Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII
tumours such as pancreatic, non-small cell lung, breast Avenue, Cardiff, CF10 3NB, Wales, UK.
E-mail: WestwellA@cf.ac.uk
and bladder cancer. Importantly, gemcitabine efficacy is
compromised by poor cellular uptake by nucleoside
†
Additional supporting information may be found in the online version of this
transporters, variable activity of the essential deoxycytidine article at the publisher’s web-site.
J. Label Compd. Radiopharm 2014, 57 637–644
Copyright © 2014 John Wiley & Sons, Ltd.

J.-P. Meyer et al.
1
8
18
6
Scheme 1. Heat-mediated intramolecular cyclization towards 12 in d -DMSO.
Figure 1. Chemical structures of the cytidine-based biomarkers F-FAC, F-
FMAC and F-FIAC (1–3), and the anticancer drug gemcitabine.
1
8
molecules containing a leaving group at the 2’-position are
even more prone to undergo anhydro formation especially
chemotherapy that, considering the low (≤20%) response
rates and severe side effects, could lead to significantly
at high temperatures (≥75 °C). Thus,
a
reasonable
8
improved treatment efficacy.
radiofluorination precursor must combine sufficient reactivity
1
8
18
18
2
’-[ F]arabinocytidines such as
F-FAC and
F-FIAC
with stability towards heat as well as basic conditions. In this
(
3, Figure 1) have been synthesized in high purities using
study, we present for the first 18time
a
late-stage
1
8
9
an early stage
F introduction strategy. However, this
radiofluorination approach to the
F-labelled cytidine
synthetic approach limits reliable and reproducible
production on commercially available synthesis modules for
18
derivative F-FAC.
1
0
18
human application. For instance, multiple steps after
F
introduction with an additional purification step due to the
formation of the D- and L-isomers, as well as the use of toxic
Experimental section
substances such as hydrogen bromide, make the translation Radiosynthetic procedures
to automated synthesizers difficult. These challenges go hand
4
18
N -bis-Boc-3’,5’-O-bis-tetrahydropyranyl-2’-deoxy-2’-[ F]
in hand with reproducibility issues when it comes to quality
control in clinical practice according to the British/European/
American Pharmacopeia. Furthermore, as the number and
fluoroarabinocytidine (25)
1
8
[
F]fluoride was trapped on a QMA cartridge (equilibrated with 5%
space in hot cells is limited, multiple-step radiosyntheses NaHCO
should preferably be replaced by shorter and less complex an aqueous solution (0.7 mL) of KHCO
3
solution (5 mL) and water (5 mL)) before it was eluted with
(22.6 mg/mL) and kryptofix
3
1
8
K
3
222 (22.6 mg/mL) in MeCN. The resulting F/KHCO /K222-complex
synthetic procedures. Lazari et al. recently presented a newly
was dried by co-evaporation under reduced pressure and a stream
of nitrogen. The drying process was repeated three times (3 × 1 mL
MeCN). A solution of precursor (23) (10 mg, 14.5 μmol) in DMF
designed radiosynthesis module system that furnished the
1
8
18
nucleoside analogues F-FMAU and F-FAC in good overall
radiochemical yields (46% and 31%, respectively, decay-
corrected) and with acceptable reproducibility following early
(
1
0.3 mL) was then added, and the resulting mixture was stirred at
10 °C for 20 min. The crude mixture was either analysed directly
1
1
stage fluoride introduction. Overall synthesis times were
18
for determination of F-incorporation (radio-TLC) or passed through
an Alumina-Sep-Pak cartridge (equilibrated with water (5 mL)) for
reported to be 165–170 min in each case. The flexible
1
8
radiosynthesizers developed are described as being suitable characterization (radio-HPLC). The
F-labelled intermediate was
for both the development and production of different eluted with EtOAc (2 mL), and an aliquot (0.2 mL) of the mixture
radiotracers for applications in PET.
The aim of this study was to develop a novel and
straightforward synthetic approach towards
was analysed by analytical HPLC using co-elution with the non-
radioactive reference compound (24). R = 17.2 min, MeCN/H O 9:1,
1 mL/min, ≥87%, radio-TLC, R = 0.6 (4% MeOH/DCM).
t
2
1
8
f
F-FAC that
could be applied to the radiosynthesis of related cytidine-
based radiotracers. A more straightforward radiosynthesis
18
F-FAC (1)
1
8
similar to the
F-FDG synthesis in combination with
1
8
After elution of the F-labelled intermediate (25) with EtOAc into a
new reaction V-vial, the solvent was removed under reduced
pressure and a stream of nitrogen at 80 °C. 2N HCl solution (0.3 mL)
was added to the dried residue followed by stirring at 90 °C for
innovative radiosynthesizers would enable a faster transition
from tracer development to clinical application. Late-stage
1
8
F-introduction at the 2’-arabino position of an intact
nucleoside towards nucleoside-based radiotracers was
1
5 min. The solution was then neutralized with 2N NaOH solution
1
2
previously considered as difficult. However, Alauddin and (0.3 mL). The resulting mixture was loaded onto a semi-preparative
co-workers recently developed a novel radiosynthesis of the HPLC column via a 10-mL injection loop. The fraction containing
1
8
18
uridine analogue
F-FMAU based on
F-introduction approach in order to increase possibilities
for making nucleoside-based PET tracer suitable for clinical
a
late-stage
2
F-FAC was collected after 22.5 min with 3% MeCN in H O and a
1
8
flow rate of 3.5 mL/min. The appropriate fraction was flushed through
a sterile filter and concentrated via evaporation on the module (100 °
C under a stream of nitrogen for 10 min). The residue was taken up
in sterile saline. An aliquot (0.2 mL) of the mixture was analysed by
analytical HPLC using co-elution with the non-radioactive reference
1
0,12
applications.
The key towards a stable precursor that
would be less likely to undergo anhydro side-product
formation (e.g. Scheme 1) due to neighbouring group
participation was the introduction of a good electron-
withdrawing N-protecting group(s). Because the amino group
compound 27,
t 2
R = 13.8 min, 3% MeCN/H O, 1 mL/min, ≥98%. A
carrier-added synthesis was performed according to the previous
procedure, and the product F/ F-FAC was saved for decay for 20
1
9
18
at the 4-position of cytidine derivatives likely enhances the half-lives (≈2 days) and subsequently analysed by mass spectrometry;
+
+
nucleophilicity of the 2-carbonyl oxygen, potential precursor MS (70 eV): m/z (%): 245.4 (5) [(M + H) ], 267.7 (95) [(M + Na) ].
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J. Label Compd. Radiopharm 2014, 57 637–644

J.-P. Meyer et al.
Computational methods
group (cytidines) impacts the electric charge density at the
-carbonyl oxygen. The N-substituents were chosen with
2
MOE 2010.10 software was used to design each compound and to
explore possible conformations. The searching method applied was
LowModMD that generates conformations using a short run of
molecular dynamics at constant temperature followed by an all-atom
energy minimization. Amongst the conformations, 16 of them
regard to both their synthetic accessibility and deprotection
mechanisms that would be suitable for radiochemical
syntheses in terms of subsequent efficient removal. The
3
N -nitro-2’-(methanesulfonyl)uridine (11) was considered as a
3
were selected for each compound for quantum mechanics (QM) standard value as
a
N -nitro-2’-(trifluoromethanesulfonyl)
evaluation. QM calculations were executed for the selected uridine derivative
conformations with the GAMESS software. A geometry optimization successfully synthesized and thermal stable. However, the
was
previously
reported
as
13
14
was performed in vacuo, using analytical energy gradients with the
radiofluorination method could not be applied to
N -nitro-uridine-based precursor.
a
restricted Hartree–Fock wave function. The 3-21G split valence basis
set was used, and diffuse sp shell was added to heavy atoms. The
initial hessian was guessed, and the convergence gradient tolerance
3
The results shown later (Table 1) indicate that the electron
density at the 2-carbonyl oxygen is reduced when
H-substitution at the N (N for uridine derivatives) with an
electron-withdrawing group is performed. Furthermore, it
shows that the stronger the electron-withdrawing effect of
h
was set to 0.0005 E /bohr. The electrostatic potential was calculated
4
3
at points determined by Michael Connolly algorithm on the
surface of van der Waals fused spheres. Atomic charges were
consequently fitted to this potential. The partial net charges of atoms
of interest were gathered from each molecule conformation, and the substituent(s), the lower the calculated electron density
4
values were averaged.
Ω at the 2-carbonyl oxygen. For instance, double N -Boc-
protection (7) decreases the electron density more than
4
4
single N -Boc-protection (6) and N -monoacetylation (5),
respectively.
Results and discussion
Compounds 5 and 7 were synthesized (see Synthesis
section and Supporting information) and subsequently tested
Precursor design
In order to develop a new precursor for radiofluorination of for their ability to undergo heat-mediated decomposition by
the 2’-arabino position of cytidine derivatives, the idea of forming the appropriate anhydro compounds 12 and 13.
radiofluorination of an intact nucleoside moiety was applied. First, an NMR sample of compound 5 in d -DMSO was heated
6
1
The main challenge of this new approach is the rational up to 50 °C. H-NMR spectra were recorded hourly. Figure 3
design of precursors that combine reactivity and stability in shows that almost full conversion to anhydro compound 12
1
8
a way that the F-nucleophile can be incorporated at high occurred after 2 h (Scheme 1).
temperatures resulting in acceptable conversion rates whilst
Chemical shifts of the amino proton (11.5 to 12.25 ppm)
heat-mediated anhydro formation is kept to a minimum. A and the ribose protons clearly show the conversion into the
quantitative method for the characterization of precursor cyclic nucleoside compound (Figure 3). Furthermore, the
stabilities was desirable prior to synthesis. The electric charge vanishing signal of the mesyl group at 3.5 ppm also indicates
density of the 2-carbonyl oxygen of the pyrimidone moiety decomposition as the free mesylate is expected to have a
was determined using theoretical methods for the potential different chemical shift that indeed could be observed at
to undergo an intramolecular nucleophilic attack at the around 2.3 ppm next to the methyl-signal of the acetamide.
1
3
2
’-position. Hence, the net electric charge of the 2-carbonyl
C-NMR data after 1-h incubation showed 24 instead of 12
4
oxygen of
a family containing N -substituted cytidine signals of pure compound 5. Additionally, ESI-MS was
derivatives (4–8, Figure 2) was calculated using quantum performed in order to show the change in the molecular
+
chemical calculations with restricted Hartree–Fock wave mass. The m/z-signal 268 [M + H]
further confirmed
function and the 3-21G basis set.
Additionally, selected uridine derivatives (9–11, Figure 2)
generation of anhydro compound 12.
The double N -Boc-protected compound (7), however,
4
were included in this study, in order to obtain both a showed significantly increased thermal stability as shown in
reference value as well as a measurement of how the Scheme 2 and Figure 4. Even though a tiny set of additional
substitution of the 4-carbonyl group (uridines) by an amino
Table 1. Calculated electron density Ω of selected cytidine
(4–8) and uridine derivatives (9–11)
1
2
3
a
No.
R
R
R
[Ω ± %]
4
5
6
7
8
9
H
H
H
Boc
H
Ac
Boc
Boc
À0.911 ± 0.09
À0.901 ± 0.05
À0.860 ± 0.01
À0.825 ± 0.06
À0.814 ± 0.08
À0.838 ± 0.02
À0.770 ± 0.03
À0.589 ± 0.10
Phthalimide Phthalimide
H
Boc
NO2
1
1
0
1
a
Quantum chemically calculated net charge at the 2-carbonyl
oxygen.
Figure 2. Selected cytidine-based and uridine-based nucleosides for electric
charge density calculations at 2-carbonyl oxygen.
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J.-P. Meyer et al.
1
Figure 3. Heat-mediated anhydro formation of 12. After only 2 h at 50 °C, nearly full conversion could be observed by H NMR analysis.
peaks appeared after 2 h at 80 °C, the observed stability of
18
compound 7 looked promising with regard to F-radiolabelling
reactions.
Hence, the calculated and experimental data lead to three
major conclusions: (1.) The electron density at the 2-carbonyl
oxygen of the pyrimidine moiety can be used as a measurement
for its ability to undergo intramolecular cyclization resulting
in the appropriate 2,2’-anhydro compounds. (2.) Electron-
withdrawing groups have a measurable/quantifiable effect on
the electron density at the 2-carbonyl oxygen. (3.) Depending on
Scheme 2. Increased thermal stability of 7. No significant formation of side
4
3
product (13) could be observed (in d
4
-MeOD).
the strength of its electron-withdrawing nature, a N (N )-protecting
1
Figure 4. Recorded H-NMR spectra of purified compound (7) after 0, 60 and 120 min at 80 °C. The absence of additional peaks indicate no or minimal anhydro formation
through increased thermal stability.
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J.-P. Meyer et al.
2
TIPDSCl in pyridine yielding compound 15 in good yield,
which was consecutively mesylated at the 2’-position using
an excess of MsCl and TEA in DCM furnishing compound
1
6. Subsequent removal of the silica-based protecting group
using TBAF in anhydrous THF at low temperatures gave test
compound 5 that was purified immediately and characterized
because of its high ability to undergo anhydro formation
(
Scheme 3).
The synthesis of precursor (23) started with commercially
available cytidine (17), which was first converted into the
’- and 5’-protected nucleoside 18 using TIPDSCl and
3
2
8
pyridine at room temperature as previously described.
Subsequent TMS-protection furnished 19 that was followed
4
by double N -Boc-protection using excess of Boc
2
O (5–6 eq.)
and DMAP that gave compound 20 in good yield. Selective
removal of the TMS-group using p-TsOH was performed at
À5 °C to give intermediate 21. Subsequent mesylation of
the free 2’-hydroxy group was performed in high yield to
form compound 22. TBAF-mediated removal of the
bidentate-protecting group had to be carried out at lower
temperatures (approximately À10 °C) in order to avoid
significant decrease in yield probably because of single Boc-
group removal. Purified compound 7 was then converted into
2
Scheme 3. Reagents and conditions: (a) TIPDSCl , pyridine, 0 °C – room
temperature (RT), 12 h, 87%; (b) MsCl, TEA, DCM, 0 °C – RT, 2 h, 81%; (c) TBAF,
THF, À10 °C, 30 min, 53%.
group influences the thermal stability of the nucleoside fluorination precursor 23 using 3,4-dihydro-6H-pyran in
analogue. Finally, an N,N-diBoc fluorination precursor was excess. Highly purified precursor 23 was obtained in 9%
selected for radiofluorination studies.
overall yield from 17 (Scheme 4).
The choice of the leaving group as well as the protecting
1
0
group is based on a previous study that found that 3’/5’-
THP protection of uridine derivatives gave the best
Synthesis
The synthesis of test compound 5 was carried out with the radiochemical yields and a 2’-mesyl group an appropriate
selective 3’- and 5’-protection of N-acetylcytidine (14) using balance between stability and leaving group abilities.
Furthermore, our pilot studies showed that the appropriate
tosyl-containing and nosyl-containing counterparts already
decomposed during purification. Furthermore, even though
double THP protection makes NMR analysis complex
(
mixture of diastereomers), the later removal of these groups
was straightforward.
Scheme 5. Reagents and conditions: (a) 1N TBAF/THF, DMF, 100 °C, 100 min, 19%.
Scheme 4. Reaction conditions: (a) TIPDSCl
TMSCl, TEA, DCM, 0 °C – RT, 2 h, 83%; (c) Boc
TsOH, THF, À5 °C, 66–78%; (e) MsCl, TEA, DCM, 0 °C, 90%; (f) TBAF, THF, À10 °C,
0–80%; and (g) DHP, pTsOH, DCM, 0 °C – RT, 6–8 h, 45–56%.
2
, pyridine, 0 °C – RT, 18 h, 81%; (b)
2
O, DMAP, DCM, RT, 5 h, 77%; (d) p-
Scheme 6. Radiofluorination was performed using different reaction conditions
as outlined in the table later.
7
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J.-P. Meyer et al.
Firstly, cold fluorination reactions were carried out to assess was eluted from the QMA cartridge using a K222/KHCO
fluoride incorporation for precursor (23). Amongst solution. Table 2 shows the calculated incorporation data,
3
1
8
fluorination conditions tested, the best incorporation yield and Figure 5 shows F-incorporation plotted against the
was found at 100 °C for 100 min in DMF using freshly reaction temperature run in dry DMF as solvent. The highest
prepared 1N TBAF/THF-solution (Scheme 5). The fluorinated incorporation values were obtained using DMF at 110 °C for
intermediate (24) was purified by column chromatography 20 min. Although MeCN or tBuOH as solvent failed to show
on SiO
2
, and its identity and purity (≥97%) confirmed by any conversion, the use of an alternative base such as TBA–
in MeCN or tBuOH might provide an
1
19
H/ F-NMR, HR-MS and9 analytical HPLC (Supporting OH or TBA–CO
3
1
information). Notably, the F-spectrum showed a set of four alternative strategy. However, higher 1temperatures and
8
signals due to the four diastereomers that the THP protection longer reaction times led to decreased F-incorporation in
yields.
the arabino position because of the formation of the
1
8
The obtained intermediate 24 was used as a cold reference anhydro intermediate. F-incorporation was calculated by
standard for co-elution on HPLC in order to identify the hot peak integration of the crude reaction mixture after
intermediate.
radiofluorination using radio-TLC (Supporting information).
1
8
The F-fluorinated intermediate (25) was confirmed by
1
9
1
8
radio-HPLC (Figure 6) and co-injection of the F-containing
Radiosynthesis of F-FAC
reference standard (24) (Supporting information) after
1
8
1
8
The F-incorporation to form intermediate (25) was carried
out using an Eckert Ziegler modular system and
trapping the majority of the unreacted F-fluoride on a
&
Sep-Pak alumina cartridge. The intermediate was eluted with
appropriate software for radiosynthesis. The reaction
parameters of solvent, temperature and reaction time were
2
.5 mL EtOAc into a new reaction vial.
The solvent was then removed at 90 °C under a stream of
1
8
varied in order to determine optimal
conditions, using KHCO3 as base (Scheme 6). [ F]Fluoride
F-incorporation
nitrogen. 2N HCl solution was added to the dried residue
(25), which was then stirred at 95 °C for 20 min (Scheme 7).
A shorter deprotection time was found to lead to decreased
final yields of the product F-FAC.
After neutralization with 2N NaOH solution, the mixture
was loaded onto a semi-preparative HPLC column via a
1
8
1
8
1
8
Table 2. [ F]-incorporation results obtained under different
conditions
1
8
1
0-mL injection loop. [ F]-FAC was eluted after 22.5 min at
1
8
a
No.
Solvent
T [°C]
t [min]
[ F] [%]
a flow rate of 3.5 mL/min using 3% MeCN in H O as the
2
mobile phase. Following concentration on the module (100 °C
1
2
3
4
5
6
7
8
9
MeCN
tBuOH
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
90
95
90
20
20
20
20
20
20
20
30
20
20
20
20
n/a
n/a
1.3 ± 0.3
2.6 ± 0.2
4.9 ± 0.6
7.7 ± 0.8
9.4 ± 0.8
7.9 ± 0.6
6.9 ± 0.5
4.1 ± 0.4
2.3 ± 0.2
1.8 ± 0.2
95
100
105
110
110
115
120
125
130
1
1
1
0
1
2
a 18
[
F]-incorporation was calculated using radio-TLC. Each
reaction was repeated three times under identical conditions.
Figure 6. Radio-HPLC was performed in order to confirm the formation of 25. The
fluoridation product was co-eluted with reference compound 24 after 17.2 min
(
2
MeCN/H O 10:1, flow rate of 1 mL/min). The profile of the radio-HPLC diagram is
consistent with the radio-TLC diagram shown earlier.
Scheme 7. Subsequent deprotection of the hot intermediate 25 using (a) 2N HCl
at 95 °C for 20 min. A shorter deprotection time results in decreased final yields of
the product F-FAC (1).
1
8
Figure 5.
[ F]-incorporation plotted against the reaction temperature run in dry
1
8
DMF as solvent.
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J.-P. Meyer et al.
under a stream of nitrogen for 10 min), the sample was taken
up in sterile saline, and an aliquot was co-eluted on an
analytical HPLC in combination with both possible
19
2
’- F-isomers FC (26) and FAC (27) as references (Figures 7
1
8
and 8). F-FAC was confirmed, and its purity calculated to
98% with specific activity of ≥63 GBq/μmol. The total
synthesis time was 168 min, including the concentration and
≥
18
saline formulation time. Finally, 0.75–0.86 GBq of F-FAC was
obtained resulting in radiochemical yields of 4.3–5.5% (n = 8,
decay-corrected from end of bombardment).
Additionally, a temperature study was performed to reveal why
1
8
the F-incorporation decreases when the reaction temperature
is increased. According to the mechanism, intramolecular
anhydro formation is expected at higher temperatures leading
to a lower yield of the correct stereoisomer. Figure 9 shows
radio-HPLC traces that were recorded after radiofluorination at
Figure 7. Radio-HPLC chromatogram of the purified fraction. Co-elution with
1
9
both cold 2’-[ F]-isomers 26 and 27 confirms that the correct stereoisomer was
obtained.
Figure 8. Chemical structures of the two different ‘cold’ 2’-fluoro-cytidines FC (26)
and FAC (27).
Figure 9. Radio-HPLC traces recorded after radiofluorinations performed at
different reaction temperatures.
J. Label Compd. Radiopharm 2014, 57 637–644
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

J.-P. Meyer et al.
1
10, 120 and 130 °C (20 and 40 min, bottom to top panel Acknowledgements
respectively) and co-elution with both 2’-fluoro stereoisomers FC
and FAC (bottom panel). The results show that higher This work was supported by Cancer Research Wales through PhD
temperatures do lead to anhydro formation and radiofluorination studentship funding (to J.-P. M.). We thank Dr Andrea Brancale
18
18
of the anhydro intermediate results in F-FC. The F-FAC/FC ratio (Cardiff University, School of Pharmacy and Pharmaceutical
Sciences) for the advice and support with DFT calculations. We
acknowledge the EPSRC National Mass Spectrometry Facility
decreases as the reaction temperature and time increase.
(Swansea, Wales, UK) for the provision of HR-MS determinations.
Conclusions
Conflict of Interest
1
8
A novel radiochemical approach to F-FAC is described. For the
1
8
The authors did not report any conflict of interest.
first time,
F-FAC was synthesized via late-stage
radiofluorination of the intact pyrimidine nucleoside. Prior to
synthesis, a novel fluorination precursor was designed using in
silico methods and stability studies monitored by NMR. Precursor
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Furthermore, it was shown that long reaction times and high
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18
identified by the detection of F-FC. This study shows that
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a quick and reproducible two-step route towards F-FAC
18
comparable with the radiosynthesis of F-FDG. However, Supporting Information
further optimization is required in order to improve the
radiochemical yield and prepare for transfer to production of Additional supporting information may be found in the online
clinical grade radiotracer.
version of this article at the publisher’s web-site.
www.jlcr.org
Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 637–644