Fully automated radiosynthesis of [1-(2-[18F]fluoroethyl),1H[1, 2,3]triazole 4-ethylene] triphenylphosphonium bromide as a potential positron emission tomography tracer for imaging apoptosis

Article abstract of DOI:10.1002/jlcr.3024

A novel phosphonium salt bearing a fluorine-18 labelled triazole has been designed as a potential imaging agent for apoptosis. The radiosynthesis of [1-(2-[¹⁸F]fluoroethyl),1H[1,2,3]triazole 4-ethylene] triphenylphosphonium bromide ([¹⁸F]MitoPhos-01) has been carried out on a fully automated system in a two-step reaction. Radiolabelling an ethyl azide and then carrying out a copper-mediated 1,3-cycloaddition reaction has allowed for total synthesis time to be slightly more than 1 h from aqueous [¹⁸F]fluoride. After purification by HPLC, the average radiochemical yield was determined to be 9% (not decay corrected); the specific activity was on average 70 GBq/μmol at the end of synthesis, and the radiochemical purity was >99%. A novel phosphonium salt bearing a fluorine-18 labelled triazole has been designed as a potential imaging agent for apoptosis. The radiosynthesis of [1-(2-[¹⁸F]fluoroethyl),1H[1,2,3]triazole 4-ethylene] triphenylphosphonium bromide ([¹⁸F]MitoPhos-01) has been carried out on a fully automated system in a two-step reaction, with the average radiochemical yield determined to be 9% (not decay corrected); the specific activity being around 70 GBq/μmol at the end of synthesis and the radiochemical purity above 99%. Copyright

Full text of DOI:10.1002/jlcr.3024

Research Article
Received 12 October 2012,
Revised 15 November 2012,
Accepted 24 December 2012
Published online 30 January 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3024
Fully automated radiosynthesis of [1-(2-[18F]
fluoroethyl),1H[1,2,3]triazole 4-ethylene]
triphenylphosphonium bromide as a potential
positron emission tomography tracer for
imaging apoptosis
a
b
c
a
*
Anna Haslop, Antony Gee, Christophe Plisson, and Nicholas Long
A novel phosphonium salt bearing a fluorine-18 labelled triazole has been designed as a potential imaging agent for
apoptosis. The radiosynthesis of [1-(2-[18F]fluoroethyl),1H[1,2,3]triazole 4-ethylene] triphenylphosphonium bromide
([18F]MitoPhos_01) has been carried out on a fully automated system in a two-step reaction. Radiolabelling an ethyl azide
and then carrying out a copper-mediated 1,3-cycloaddition reaction has allowed for total synthesis time to be slightly more
than 1 h from aqueous [18F]fluoride. After purification by HPLC, the average radiochemical yield was determined to be 9%
(not decay corrected); the specific activity was on average 70 GBq/mmol at the end of synthesis, and the radiochemical
purity was >99%.
Keywords: apoptosis imaging; phosphonium salts; fluoride; automated synthesis
results in mind, a new design of lipophilic phosphonium salt
Introduction
has been developed with attractive synthetic features.
Apoptosis is the most common form of programmed cell death and
The discovery that Cu (I) is able to catalyse the Huisgen 1,3-dipolar
is found to be a key underlying mechanism in many pathologies
cycloaddition between an alkyne and an azide, colloquially known
such as neurodegenerative disease, Alzheimer’s disease, ischemic
as the ‘Click’ or ‘CuAAC’ reaction, has become increasingly used
heart perfusion, human immunodeficiency virus and cancer.1,2
Being able to understand this mechanism could lead to huge
advances in detection, drug development and treatment. Currently,
there are very few non-invasive techniques in which to quantify
and assess the process of apoptosis in humans.3–6 The discovery that
the mitochondria play an important role in the early stages of
apoptosis and therefore its dysfunction can be directly related to
these pathologies has directed focus to targeting the mitochondria.7
Evidence has shown that alterations to the inner membrane
potential of the mitochondria can be directly related to the
organelles dysfunction and therefore allows a potential process to
image.8,9
Evidence has shown that carcinoma cells exert a higher
membrane potential, and therefore, compounds such as lipophilic
cations can selectively accumulate in cancer cells over healthy
cells.10,11 It is also known that apoptosis is the key mechanism of
action for many anti-cancer drugs, and so, lipophilic cations have
the potential to not only target cancer cells but also could be used
to observe apoptosis during treatment.
in drug discovery.15,16 A simple method to radiolabel an azide
with fluorine-18 developed by Glaser et al. has also meant that
‘click’ chemistry has become widely available for the use in the
development of positron emission tomography tracers.17
The design of this probe utilises the advantages of the ‘click’
reaction to rapidly radiolabel phosphonium cations. Reducing the
number of reaction steps required to yield the final tracer can result
in increased yields, shorter reaction times and increased reliability.
Results and discussion
The radiosynthesis of [18F]MitoPhos_01 was carried out in a
two-step reaction shown in Figure 1. The precursor for the
radiosynthesis, 2-azidoethyl-4-tosylate, was first prepared via a
two-step reaction involving the conversion of 2-bromoethanol to
aDepartment of Chemistry, Imperial College London, Exhibition Road, London,
SW7 2AZ, UK
A novel positron emission tomography voltage sensor, 18F-
fluorobenzyl triphenyl phosphonium bromide (18F-FBnTP) has
been shown to be able to target the membrane potential and
provide a viable strategy for the early detection of cancer and
for quantifying the evolution dynamics of apoptosis.12 Work
previously carried out by Min and Elmaleh also supports the
bDivision of Imaging Sciences and Biomedical Engineering, The Rayne Institute,
King’s College London, Lambeth Palace Road, London, SE1 7EH, UK
cImanova Ltd, Hammersmith Hospital, Du Cane Road, London, W12 0NN, UK
*Correspondence to: Nicholas Long, Department of Chemistry, Imperial College
London, Exhibition Road, London, SW7 2AZ, UK.
use of phosphonium salts for tumour imaging.13,14 With these E-mail: n.long@imperial.ac.uk
J. Label Compd. Radiopharm 2013, 56 313–316
Copyright © 2013 John Wiley & Sons, Ltd.

A. Haslop et al.
of the dose. The conversion yield of the azide to the triazole was
found to vary between 54% and 96% (n= 6). Typical synthesis (i.e.
55 mA, 5 min, approximately 10 GBq) provided 850 MBq of purified
product. The total synthesis time from delivery of the [18F]fluoride
was 63 min.
Analytical HPLC analysis revealed that the radiolabelled
product exhibited identical retention time with a fully charac-
terised unlabelled [1-(2-[19F]fluoroethyl),1H[1,2,3]triazole 4-ethylene]
triphenylphosphonium bromide standard (Figure 2). The radioche-
mical purity of [18F]MitoPhos_01 determined by analytical HPLC
was above 99%, chemical purity >95%, and radiochemical yield
was 9% at end of synthesis with specific activity 41–101 GBq/mmol.
The unlabelled [1-(2-[19F]fluoroethyl),1H[1,2,3]triazole 4-ethylene]
triphenylphosphonium bromide standard was synthesised using
similar conditions to those used during the production of the
labelled version. Firstly, the azide was synthesised following a
method described Glaser et al. and then the CuAAC step carried
out using copper sulphate solution, sodium ascorbate to reduce,
TBTA as a stabilising agent and the alkyne.17 The reaction was then
stirred at room temperature in a sealed vial for 4 days. The differ-
ence in reaction kinetics is believed to be due to the difference
in catalyst loadings and also the introduction of dimethylforma-
mide to the reaction mixture. The desired compound was
isolated through various washing to give a brown oil in good
yield (77%).
Figure 1.
[
18F]MitoPhos_01 radiosynthesis.
2-azidoethanol followed by a functional group exchange of the
alcohol to a tosylate. The procedures were reported by Wu and
Sagai, respectively.18,19 2-Azidoethanol was formed by adding an
excess of sodium azide to 2-bromoethanol followed by the
addition of 4-toluenesulfonyl chloride in the presence of a base in
dichloromethane. Moderate yields were seen for both reactions
(67% and 72%, respectively), and further purification was only
required for 2-azidoethyl-4-tosylate in the form of column
chromatography on silica gel (2:1 ethyl acetate : petroleum
benzene) to give a colourless oil. The alkyne precursor (but-3-yne
triphenyl phosphonium bromide) was synthesised via a nucleophilic
addition reaction between triphenyl phosphine and 4-bromo-1-
butyne in dry toluene under reflux. Moderate yields were achieved
after recrystallisation.
The radiosynthesis of the desired probe was carried out on a
fully automated system. The labelled azide was synthesised
by nucleophilic substitution reaction of 2-azidoethyl-4-tosylate
and no carrier added [18F]fluoride in the presence of Kryptotfix
222 and potassium carbonate in acetonitrile. This step was
carried out on an Advion NanoTekW system before being
distilled at 130 ꢀC under a stream of helium (20 mL/min) into a
sealed vial containing all the components required for
the cycloaddition step (see Experimental). This was then left to
mix at room temperature for 10 min to give the triazole
phosphonium salt that was then transferred into a vial
containing 1 mL of water and then passed through a filter to
eliminate any undissolved tris[(1-benzyl-1H-1,2,3-triazol-4-yl)
methyl]amine (TBTA) and possible salt precipitation before being
injected onto the semi-preparative HPLC column. The retention
time of [18F]fluroethyl azide and [18F]MitoPhos_01 were 5.18
and 6.67 min, respectively, which made the separation of these
two species feasible in a relatively short time; moreover, the mobile
phase was chosen to allow the direct use of the radiotracer in
Experimental
The fully automated synthesis of [18F]MitoPhos_01 was carried out using
the Advion Nanotek platform followed by an in-house developed
automated system consisting of an eight two-way valve tower system,
a mass flow controller, oven and a six-port/two-way HPLC valve. 18O
enriched water, >98% 18O atom, was purchased from Rotem. All other
reagents were from Sigma-Aldrich Chemical Co. Semi-preparative HPLC
used to isolate the radiolabelled phosphonium salt was carried out on a
Gilson 121 model and QC HPLC on a QC Agilent 1100 series (quaternary
pump with diode array UV detector). NMR spectra were recorded on a
Bruker AV 400 MHz spectrometer. ESI mass spectra were recorded on a
Micromass LCT Premier spectrometer by John Barton. Elemental
analyses were completed by Stephen Boyer at the Science Centre,
London Metropolitan University.
Synthesis of but-3-yne triphenyl phosphonium bromide20
4-Bromo-1-butyne (2.46 g, 18.5 mmol) and triphenylphosphine (7.35 g,
in vitro assay or in vivo experiment following a simple dilution 27.5 mmol) were dissolved in dry toluene (25 mL). The mixture was
Figure 2. (A) UV-HPLC chromatogram of [18F]MitoPhos_01 co-injected with cold standard. (B) Radio-HPLC chromatogram of [18F]MitoPhos_01.
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 313–316

A. Haslop et al.
heated to reflux for 24 h when two layers were formed. After
being left to cool to room temperature, the bottom layer became
solid. This was filtered off and washed with toluene (25 mL) to remove
any unchanged starting material and then dried under vacuum. A
white solid was obtained after carrying out a recrystallisation in
acetonitrile (3.95 g, 54%). mp 153–156 ꢀC. 1H NMR (DMSO), d, 2.58
extracted with diethyl ether (5 Â 30 mL) and then the organic phase
dried over magnesium sulphate. After filtration and removal of the
solvent, 2-azidoethanol was obtained as colourless liquid (2.33 g, 67%).
1H NMR (CDCl3), d, 3.46 (t, 3JHH = 5.2 Hz, 2H, CH2), 3.78–3.81 (2H, CH2).
2-Azidoethyl-4-tosylate
4
(m, 2H, PCH2), 3.01 (t, JHH = 2.6 Hz, 1H, –CΞCH), 3.92 (m, 2H, CH2) and
7.76–7.94 (15H, PPh3), 31P NMR (DMSO), d, ppm: 23.62, MS (ES+) m/z:
315.13 (100%, M+), Elemental analysis (calc %): C (66.85), H (5.10),
(actual %): C (66.83), H (5.10)
The following synthesis was carried out with modifications to a
procedure described by Sagai et al.19 2-Azidoethanol (2 g, 23 mmol,
1 eq), triethylamine (4.65 g, 46 mmol, 2 eq) in dichloromethane (25 mL)
were cooled to 0 ꢀC. To this solution, 4-toluenesulfonyl chloride
(5.47 g, 28.7 mmol, 1.2 eq) was added slowly over 20 min before being
stirred at room temperature for 3 h. The mixture was then washed with
HCl (3 M, 3 Â 30 mL) and then brine (3 Â 30 mL) before being dried over
magnesium sulphate and placed under vacuum to remove the solvent.
Purification to remove tosyl azide was carried out using flash column
chromatography on silica gel (2:1 ethyl acetate : petroleum benzene)
to give a colourless oil (4.04 g, 72%). 1H NMR (CDCl3), d, 2.47 (s, 3H,
2-Fluoroethyltosylate21
2-Fluoroethanol (1g, 15.6mmol) was dissolved in dry pyridine (15mL)
under nitrogen. The solution was stirred at 0 ꢀC, and p-toluene sulfonyl
chloride (6.5g, 34.1mmol) was added slowly to the solution over a period
of 30 min, keeping the temperature below 5ꢀC. The solution was then
stirred at 0 ꢀC for another 4 h before quenching by slow addition of ice
(15g) and then water (20mL). Ethyl acetate (50 mL) was added; the organic
layer was separated and washed with water. Excess pyridine was removed
by washing the organic layer with a 1M HCl solution until the aqueous layer
became acidic. The excess tosyl chloride was removed by washing the
organic layer with an aqueous solution of Na2CO3 (pHꢁ 10). The organic
layer was then washed with brine, dried over MgSO4 and concentrated
under vacuum to obtain 2-fluoroethyltosylate (2.78g, 81%). 1H NMR (CDCl3),
d, 2.44 (s, 3H, CH3), 4.23 (m, 2H, CH2), 4.54 (m, 2H, CH3), 7.35 (d, 3JHH = 8.2Hz,
2H, Ar), 7.78 (d, 3JHH = 8.3Hz, 2H, Ar); MS(CI+): 218 (M + 1) (48), 155 (80), 108
(100), 91 (74).
3
3
CH3), 3.50 (t, JHH = 5.1 Hz, 2H, CH2), 4.17 (t, JHH = 5.1 Hz, 2H, CH3), 7.39
(d, JHH = 8.1 Hz, 2H, Ar), 7.77–7.88 (2H, Ar); MS(CI+): 242 (M + 1) (68),
3
228 (40), 199 (50), 155(100).
[
18F]Fluoroethyl azide17
Aqueous [18F]fluoride (approximately 2 mL) was passed through a resin
cartridge (QMA) where the [18F]fluoride was trapped. It is then eluted
using a mixture of Kryptofix-222 (5 mg, 13.3 mmol), potassium carbonate
(1 mg, 7.2 mmol, dissolved in 50 mL water), and acetonitrile (1 mL). The solvent
was removed by heating at 80 ꢀC under a stream of nitrogen (100 mL/min).
Afterwards, acetonitrile (0.5 mL) was added, and the distillation was contin-
ued. This procedure was repeated twice. After cooling to room temperature,
a solution of 2-azidoethyl-4-tosylate (2 mL, 10 mmol) in acetonitrile (0.5 mL)
was added. The reaction mixture was stirred for 15 min at 80 ꢀC. After addition
of acetonitrile (0.2 mL), [18F]fluoroethyl azide was distilled at 130 ꢀC under a
flow of nitrogen into a trapping vial.
2-Fluoroethyl azide17
To a solution of 2-fluoroethyl-4-toluenesulfonate (128 mg, 0.586 mmol) in
dry dimethylformamide (10mL) was added sodium azide (114 mg,
1.76mmol), and the resulting mixture was stirred at ambient temperature
for 48 h. The reaction mixture was filtered, and the filtrate containing the
title compound was used without isolation for subsequent reactions.
WARNING: Attempts to isolate neat 2-fluoroethylazide may result in an
explosion.
[1-(2-[18F]Fluoroethyl),1H[1,2,3]triazole 4-ethylene]
triphenylphosphonium bromide
The [18F]fluoroethyl azide was distilled directly into a vial containing a
premade mixture of CuSO4.5H2O (56mL, 0.4 mM), TBTA (10 mg) and sodium
ascorbate (40 mg) in water (100 mL) and ethanol (50 mL) that had been shaken
until all reagents had mixed. To this, 3-butynyl triphenyl phosphonium
bromide (1 mg) in ethanol (50 mL) was added. Following the distillation of
[18f]fluoroethyl azide into this vial, it was left to mix under nitrogen at room
temperature for 10 min. Water (1 mL) was then added and the entire mixture
passed through a filter (Agilent Tech, polypropylene filter with pore size
0.45 mm, diameter 30 mm) before being injected into the semi-preparative
HPLC (Eclipse C18 250_10 mm). The flow rate was 7 mL/min, with the mobile
phase starting from 10% solvent B (ethanol) and 90% solvent A (50 mM
dihydrogen sodium phosphate buffer pH = 5.5) to 50% ethanol over 7 min.
The radioactive peaks for [18F]fluoroethyl azide and [18F]MitoPhos_01 were
collected at 5.18 min and 6.67 min, respectively. The product was found to
be >99% radiochemically and >95% chemically pure as determined by
analytical HPLC (4.6*250 mm Eclipse C18 column; isocratic 0–6min 70%
50 mM AMF (ammonium formate), pH 4/30% acetonitrile; gradient 6–15 min:
20% 50mM AMF, pH4/80% acetonitrile, flow rate: 2.0mL/min).
[1-(2-Fluoroethyl),1H[1,2,3]triazole 4-ethylene]
triphenylphosphonium bromide
This synthesis was carried out with modifications to a procedure
described by Chan et al.22 To a vial fitted with a screw cap and a stirrer
bar, fluoroethyl azide (17 mg, 0.125 mmol), corresponding but-3yne
triphenyl phosphonium bromide (53 mg, 0.1325 mmol), CuSO4 (2 mg,
0.00265 mmol, 2 mol%), sodium ascorbate (20 mg, 0.108 mmol), TBTA
(4 mg, 0.0076 mmol) and water (0.25 mL) were loaded. The reaction
mixture was left to stir at room temperature for 4 days. All the solvent
was then removed under vacuum with heating and then methanol
added. Any sediment was filtered off and then the methanol removed.
Chloroform was then added and again any solid removed via filtration
and then the solvent removed to yield a dark drown oil-like compound
(47 mg, 77%). 1H NMR (DMSO), d, 3.16 (m, 2H, CH2), 4.03 (m, 2H, CH2),
3
3
4.58 (dt, 2H, JHF = 25.6 Hz, CH2) 4.74 (dt, 2H, JHF = 46.8 Hz, CH2F),
7.66–7.82 (15H PPh3), 8.62 (s, 1H, CH triazole). 31P{1H} NMR (DMSO),
d, 23.40 (s). 19F NMR (DMSO), d, À222.26 (m). MS (ES+) m/z: 404 (M+).
Elemental analysis (calc %): C (59.51), H (4.99), N (8.68), (actual %): C
(59.67), H (5.01), N (8.78).
Conclusions
2-Azidoethanol
A fully automated method has been used in the synthesis of
[1-(2-[18F]fluoroethyl),1H[1,2,3]triazole 4-ethylene] triphenylphospho-
nium bromide ([18F]MitoPhos_1). This two-step approach utilises
the rapid efficiency of the ‘click’ reaction and allows the probe to
be obtained in moderate radiochemical yields under 1 h. This novel
radiotracer is currently being evaluated in cancer cells as biomarker
This synthesis was carried out with modifications to a procedure
described by Wu et al.18 2-Bromoethanol (5 g, 40 mmol) was added to
ice (approximately 100 g) and stirred for 10 min. To this, sodium azide
(3.9 g, 60 mmol) was added slowly over 10 min. The reaction mixture
was left to warm to room temperature and stirred for 6 h. Sodium azide
(2.9 g, 45 mmol) was added and then the mixture heated to 80 ꢀC for
18 h before being cooled to room temperature. The mixture was then for cell death following chemotherapy treatment.
J. Label Compd. Radiopharm 2013, 56 313–316
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org

A. Haslop et al.
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Biochemistry-Moscow 2005, 70, 222–230.
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Acknowledgements
We would like to thank the BBSRC, GSK and Imanova for funding
this research.
[11] L. E. Bakeeva, L. L. Grinius, A. A. Jasaitis, V. V. Kuliene, D. O. Levitsky,
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Conflict of Interest
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The authors did not report any conflict of interest.
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J. Label Compd. Radiopharm 2013, 56 313–316