Development of an automated, GMP compliant FASTlab radiosynthesis of [18F]GE-179 for the clinical study of activated NMDA receptors

Article abstract of DOI:10.1002/jlcr.3831

N-(2-chloro-5-(S-2-[¹⁸F]fluoroethyl)thiophenyl)-N'-(3-thiomethylphenyl)-N'-methylguanidine, ([¹⁸F]GE-179), has been identified as a promising positron emission tomography (PET) ligand for the intra-channel phencyclidine (PCP) binding

Full text of DOI:10.1002/jlcr.3831

Khan Imtiaz (Orcid ID: 0000-0001-8070-7900)
Development of an automated, GMP compliant FASTlabTM radiosynthesis of [18F]GE-
179 for the clinical study of activated NMDA receptors
Imtiaz Khan,1* Tom Christian Berg,2 Jane Brown,1 Rajiv Bhalla,1 Anthony Wilson,1 Andrew
Black,1 Graeme McRobbie,1 James Nairne,1 Andreas Olsson,2 and William Trigg1
1. Core Imaging R&D, Life Sciences, GE Healthcare, Pollards Wood, Nightingales Lane,
Chalfont St Giles, Bucks, HP8 4SP, UK
2. Core Imaging R&D, Life Sciences, GE Healthcare, Nycoveien 1, PO Box 4220 Nydalen,
0401 Oslo, Norway.
Abstract
N-(2-chloro-5-(S-2-[18F]fluoroethyl)thiophenyl)-N’-(3-thiomethylphenyl)-N’-
methylguanidine, ([18F]GE-179), has been identified as a promising positron emission
tomography (PET) ligand for the intra-channel phencyclidine (PCP) binding site of the N-
methyl-D-aspartate (NMDA) receptor. The radiosynthesis of [18F]GE-179 at low
radioactivity levels has been previously reported.1 However, the manufacture of a GMP
compliant product at high radioactivity levels was required for clinical studies. We describe
the development of a process using the GE FASTlabTM radiosynthesis platform coupled with
HPLC purification. The radiosynthesis is a two-step process, involving the nucleophilic
fluorination of ethylene ditosylate, 11, followed by alkylation to the deprotonated thiol
precursor, N-(2-chloro-5-thiophenol)-N’-(3-thiomethylphenyl)-N’-methyl guanidine, 8.
The crude product was purified by semi-preparative HPLC to give the formulated product in
an activity yield (AY) of 7 ± 2% (n = 15) with a total synthesis time of 120 minutes. The
radioactive concentration (RAC) and radiochemical purity (RCP) were 328 ± 77 MBq/mL
and 96.5 ± 1% respectively and the total chemical content was 2 ± 1 µg. The final
formulation volume was 14 mL.
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doi: 10.1002/jlcr.3831
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The previously described radiosynthesis of [18F]GE-179 was successfully modified to deliver
an process on the FASTlabTM that allows the manufacture of a GMP quality product from
high starting radioactivitity (up to 80 GBq) and delivers a product suitable for clinical use.
Background
N-Methyl-D-aspartate (NMDA) receptors are ligand-gated heteromeric ion channels that
regulate fast neurotransmission by allowing prolonged influx of Ca2+, Na+ and K+ ions, into
the synapse. The NMDA receptor is part of the glutaminergic excitatory pathway involved in
many neurological processes including synaptic transmission and plasticity.2, 3 Abnormalities
in the level of activation of NMDA receptors are known to be involved in the
pathophysiology of many diseases, (e.g. epilepsy, neuropathic pain). It is believed that an
increased level of activation is important to these disease states rather than the up-regulation
of the receptor population.4-9 A tracer that can image activated channels has potential utility
in epilepsy, TBI and other central nervous system (CNS) disorders.
Our group have previously reported the design and evaluation of [18F]GE-179,1 which has
been identified as a lead candidate to progress to clinical studies to assess its potential as a
PET tracer for imaging activated NMDA receptors.10-14 The reported precursor synthesis and
radiosynthesis were appropriate to support a biological screening program for the
identification of the lead compound, however, the manufacture of a GMP quality product was
required for clinical studies.15 The original radiosynthesis1 was suitable for the preparation of
[18F]GE-179 for pre-clinical studies but was not suitable for delivering a GMP quality
product for clinical studies with high starting radioactivity (up to 80 GBq). In order to
deliver an automated process capable of delivering a GMP quality product, a number of steps
needed to be introduced or updated. These included the development of an [18F]fluoride
cartridge trapping and elution regime, and the replacement of the insoluble base with a
soluble base to deprotonate the thiol precursor, 8. Furthermore, the original radiosynthesis1
was a 2-pot process with two semi-preparative HPLC purifications to purify both [18F]9 and
[18F]GE-179. As the FASTlabTM only has one reaction vessel and capacity for one semi-
preparative HPLC purification step, the original radiosynthesis was not compatible and
required modifications in order to automate the radiosynthesis of [18F]GE-179.
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In this work, we describe the development of an improved, scalable precursor synthesis and
the development of a GMP compliant automated radiosynthesis of [18F]GE-179 on the
FASTlabTM synthesiser using a single use, disposable cassette coupled with semi-preparative
HPLC purification.
Methods
Materials and analysis
All reagents and solvents (unless stated otherwise) were purchased from Alfa Aesar
(Massachusetts, U.S), TCI Europe (Zwijndrecht, Belgium), VWR International (Lutterworth,
UK) or Sigma-Aldrich (Gillingham, UK) and used without further purification. FASTlabTM
cassette and consumables were supplied by Clinical Supplies, GE Healthcare (Oslo, Norway).
Saline for injection (0.9% w/v) and dehydrated ethanol BP were purchased from Hameln
Pharmaceuticals (Gloucester, UK) and Martindale Pharmaceuticals (High Wycombe, UK)
respectively. QMA-carbonate-Sep Pak (p/n 186004051) and tC18 SPE (p/n WAT036810)
cartridges were purchased from Waters (Elstree, Hertfordshire, UK). Millex GS, 0.22 µm
filter unit (P/N SLG5V255F) was purchased from Sigma-Aldrich (Gillingham, UK).
HPLC conditions for radiochemistry
Semi-preparative radio-HPLC was carried out on a Knauer Smartline 1000 system equipped
with a Bioscan Flowcount FC3200 sodium iodide/PMT gamma detector (Lablogic, Sheffield,
UK) and a Knauer Smartline UV detector.
Analytical HPLC analysis was carried out on an Agilent 1100 series system equipped with a
Bioscan Flowcount FC3200 Iodide/PMT gamma detector and Laura 3 software (Lablogic,
Sheffield, UK).
Analytical conditions for synthetic chemistry
Nuclear magnetic resonance (1H NMR and 13C NMR) spectra were obtained on either a
Bruker Avance DPX-400 MHz or a Varian 500 MHz NMR spectrometer. Chemical shifts are
reported in parts per million (ppm) with reference to an internal solvent. HPLC was obtained
on an Agilent 1100 series HPLC system with UV detection. The analytical HPLC column
used was an Xbridge shield Reverse Phase 18, particle size 2.5 µM with 4.6 mm id and a
length of 50 mm. Analytical thin layer chromatography (TLC) was carried out using Merck
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TLC Silica gel 60 F254 aluminum plates. Flash column chromatography was carried out using
a Biotage Flash system using pre-packed silica columns or using a glass column packed with
Merck Silica gel 60 (0.040-0.063 mm).
Synthesis of thiol precursor (8) and GE-179 reference standard (10)
Synthesis of N-(2-chloro-5-thiophenol)-N’-(3-thiomethylphenyl)-N’-methyl guanidine (8)
The synthesis of 8 and unlabelled standard GE-179 described in our original publication was
somewhat challenging and not as scalable or robust as is ideal for a GMP manufacture.1 In
particular, the final debenzylation step was difficult to reproduce at multigram scales.
Therefore, an alternative strategy was developed wherein the dimeric molecule 7 was
prepared, negating the need for a protecting group that is difficult to remove. The final step
of the synthesis was a reduction to cleave the disulfide to give the free thiol. The improved
synthetic route is described in Scheme 1 and has been employed to synthesise multigram
quantities of 8 for GMP radiosynthesis.
N-(3-(Methylthio)phenyl)cyanamide (2)
3-(Methylthio)aniline (99.8 g, 0.72 mol) was dissolved in diethyl ether (600 mL) in a flask
equipped with a gas scrubber containing a sodium hypochlorite solution. The mixture was
cooled to 0-5 °C. A solution of cyanogen bromide (38.1 g, 0.36 mol) dissolved in diethyl
ether (500 mL) was added drop-wise to the reaction mixture, while maintaining the reaction
temperature below 5 °C. Upon complete addition, the reaction mixture was slowly allowed to
come to ambient temperature and stirred for a total of 18 h. A solution of hydrochloric acid
(550 mL of a 0.028% v/v solution) was added to quench the reaction. The organic phase was
washed with an additional portion of HCl (550 mL of a 0.028% v/v solution) and dried over
MgSO4, filtered and concentrated in vacuo at 20-25 oC to afford the product as a brown oil
which solidified upon standing (53.0 g, 90%). 1H NMR (400 MHz, CDCl3) δ 7.23 (d, 1H),
6.96 (dm, 1H), 6.87 (t, 1H), 6.75 (dt, 1H), 6.01 (broad, s, 1H), and 2.48 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 141.2, 137.7, 130.1 (1C), 121.7, 112.7, 111.9, 110.5, and 15.4. Purity by
HPLC (Agilent 1100 series, X-Bridge column) 98.6 % area.
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N-Methyl-N’-(3-(methylthio)phenyl)cyanamide (3)
The cyanamide 2 (53 g, 0.32 mol) was dissolved in THF (400 mL) and cooled to 0 oC.
Sodium hydride, 14.9 g of a 60% dispersion in mineral oil, 0.37 mol) was washed with
heptane (2 x 195 mL) and suspended in THF (145 mL). The NaH suspension was added to
o
the reaction mixture portion wise while maintaining the reaction temperature below 20 C.
The resulting mixture was cooled to 0-5 °C and methyl iodide (92.3 g, 0.65 mol, 40.2 mL)
was added dropwise while maintaining the reaction temperature below 5 °C. The reaction
mixture was slowly allowed to come to ambient temperature and stirred for a total of 18.5 h.
THF was removed in vacuo at 20-25 °C to form a thick paste. The thick paste was suspended
in diethyl ether (645 mL), stirred for 45 minutes and filtered. The filtrate was evaporated in
vacuo at 20-25 °C. The crude material was purified using a Biotage flash system with a pre-
packed silica column (400 g silica), using DCM as eluent. The product fractions were
combined and concentrated in vacuo at 20-25 °C to afford the product as a pale-yellow oil
(54.1 g, 94%). 1H NMR (400 MHz, CDCl3) δ 7.26 (t, 1H), 6,96 (m, 1H), 6.95 (m, 1H), 6.82,
(m, 1H), 3.31 (s, 3H), and 2.48 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 140.9 (1C), 129.8
(1C), 121.0 (1C), 115.6 (1C), 113.8 (1C), 112.6 (1C) 111.4 (1C), 36.9 (1C), and 15.5 (1C).
Purity by HPLC (Agilent 1100 series, X-Bridge column) 98.7 % area.
5,5’-disulfanediylbis(2-chloroaniline) HCl salt (6)
Tin(II) chloride (205 g, 1.08 mol) was dissolved in concentratedhydrochloric acid (495 mL)
and added 4-chloro-3-nitrobenzenesulfonyl chloride (25.1 g, 0.01 mol) The resulting mixture
was refluxed for 4 h. The reaction mixture was then allowed to come to ambient temperature
and stirred overnight. The product was isolated by filtration and dried using suction to afford
the aminothiophenol 5 as a pale-yellow solid that was used without further purification. The
aminothiophenol 5 was suspended in water (125 mL) and agitated at ambient temperature. An
aqueous solution of potassium iodide (59.8 g, 0.36 mol) and iodine (61 g, 0.24 mol) in water
(900 mL) was added to the mixture portionwise. The reaction was monitored by HPLC
analysis. Upon complete conversion, the pH in the reaction mixture was adjusted to pH 12 by
addition of sodium hydroxide (6 M). The mixture was extracted using diethyl ether (3 x 1000
mL) and the combined organic phases were dried over MgSO4, filtered and concentrated in
vacuo to approximately 1000 mL. HCl in diethyl ether (65 mL of a 2 M solution) was added
dropwise. The precipitated solid was collected by filtration and further washed with diethyl
ether (100 mL). The solid was dried in vacuo overnight to afford the product as an off-white
solid (16.3 g, 86%). 1H NMR (400 MHz, d6-DMSO) δ 7.35 (broad s, 4H), 7.23 (d, 2H), 7.05
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(d, 2H), and 6.73 (dd, 2H). 13C NMR (100 MHz, d6-DMSO) δ143.8, 134.9, 129.8, 117.3,
116.2, and 113.9. Purity by HPLC (Agilent 1100 series, X-Bridge column) 98.5 % area.
1,1'-(Disulfanediylbis(6-chloro-1,3-phenylene))bis(3-methyl-3-(3-
(methylthio)phenyl)guanidine) (7)
The cyanamide 3 (21.1 g, 0.12 mol) was heated to 140-145 °C in a reaction flask equipped
with a mechanical stirrer. The aniline hydrochloride 6 (11.6 g, 0.03 mol) was added in
portions to the melt.. Upon complete addition, the resulting melt was stirred for 5 h, in
process HPLC analysis confirmed that the reaction had reached completion. The reaction
melt was cooled to ambient temperature and the residue was re-dissolved in chloroform
(1055 mL) and washed with hydrochloric acid (4 x 1055 mL of a 0.1% v/v solution). The pH
of the combined aqueous phases was adjusted to pH 9 using sodium hydroxide (2M). The
aqueous phase was then back-extracted with diethyl ether (4 x 950 mL), and the combined
organic phases were dried over MgSO4, filtered and evaporated to afford a yellow oil. The oil
was purified by flash chromatographic purification, using silica gel eluting with chloroform
added methanol (0-15%) afford the product as a foamy yellow solid (15.27 g, 78%). 1H NMR
(400 MHz, CDCl3) δ 7.30 (d, 2H), 7.29 (d, 2H), 7.17 (t, 2H), 7.13 (m, 2H), 7.11 (d, 2H), 7.06
(m, 2H), 7.04 (m, 2H), 3.89 (broad s, 4H), 3.34 (s, 6H), and 2.49 (s, 6H). 13C NMR (100
MHz, CDCl3) δ150.7, 147.7, 144.9, 140.6, 135.9, 130.5, 129.9, 127.3, 124.5, 124.3, 124.1,
123.3, 122.6, 38.8, and 15.6. Purity by HPLC (Agilent 1100 series, X-Bridge column) 95.2 %
area.
N-(2-Chloro-5-mercaptophenyl)-N’- N’-methyl -N’-(3-thiomethylphenyl) guanidine (8)
All solvents were degassed with argon prior to use. The disulfide 7 (5.1 g, 7.6 mmol)
dissolved in ethanol (100 mL) was treated with sodium borohydride (954.7 mg, 25.2 mmol)
by portion wise addition. The reaction progress was monitored by in-process HPLC analysis.
Upon complete reaction, the reaction mixture was concentrated in vacuo at 20-25 °C to afford
a foamy pale yellow solid. The solid was purified by filtration through celite using
chloroform as eluent. The collected product fractions were acidified to pH 1 by addition of
hydrogen chloride in diethyl ether (2 M) and concentrated in vacuo at 20-25 °C to afford the
product as a foamy pale-brown solid (4.8 g, 95%). 1H NMR (500 MHz, CDCl3) δ 9.68 (broad
s, 1H), 8.53 (broad s, 1H), 7.13 (t, 1H), 7.03 (d, 1H), 7.02 (m, 2H), 6.92 (s, 1H), 6.89 (d, 1H),
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6.86 (d, 1H), 3.74 (s, 1H), 3.66 (s, 3H), and 2.46 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
150.7, 147.7, 144.9 , 140.6, 135.9, 130.5, 129.9, 127.3, 124.5,124.3, 124.1, 123.3, 122.6,
38.8, and 15.6. Purity by HPLC (Agilent 1100 series, X-Bridge column) 93.3 % area.
3-(2-Chloro-5-(2-fluoroethylthio)phenyl)-N’-methyl-N’-(3-
(methylthio)phenyl)guanidine, GE-179, (10)
The route described in Scheme 2 was employed to synthesise GE-179 for analytical reference
standard or cold competition studies.
The disulfide 7 (3.0 g, 4.5 mmol) was dissolved in ethanol (120 mL) and 2-fluoroethyl
tosylate, 9, (2.1 g, 9.8 mmol) was added. The reaction mixture was heated to 60°C under an
inert atmosphere and sodium borohydride (0.8 g, 22.3 mmol) was added portionwise. The
resulting mixture was stirred for 45 minutes before being evaporated to dryness in vacuo. The
crude product was purified by flash column chromatographic purification over silica gel,
eluting with chloroform added methanol (2%) to afford the product as a clear oil that
crystalised on standing (2.2g, 5.6 mmol, 62.9%).
1H NMR (300MHz, CDCl3): δ 7.32 (t, 1H), 7.30 (d, 1H), 7.21 (t, 1H), 7.14 (ddd, 1H), 7.09
(ddd, 1H), 7.07 (d, 1H), 6.95 (dd, 1H), 4.53 (dt, 2H), 3.90 (broad s, 2H), 3.41 (s, 3H), 3.18
(dt, 2H), and 2.51 (s, 3H). 13C NMR (75 MHz, CDCl3) δC 150.7, 147.7, 145.0, 140.6, 133.9,
130.4, 130.0, 126.6, 126.2, 124.7, 124.6, 124.4, 123.4, 81.6 38.8, 33.8, and 15.5. Purity by
HPLC (Agilent 1100 series, X-Bridge column) 96.1 %.
Radiosynthesis of [18F]GE-179
Preparation of reagents and FASTlabTM cassette for the radiosynthesis of [18F]GE-179
The reagents and consumables for the radiosynthesis of [18F]GE-179 were prepared as
described in Table 1 and were contained in sealed vials or bottles in the cassette positions
according to Figure 1. The tC18 Sep-Pak cartridge was pre-conditioned with ethanol (2 mL)
followed by water (10 mL) before being placed on the FASTlabTM cassette.
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Production of [18F]Fluoride
No carrier added [18F]fluoride (1.5 mL, 40-80 GBq) was produced via an 18O(p,n)18F nuclear
reaction on a GE PETtrace cyclotron by irradiation of an enriched [18O]H2O target using 16.4
MeV protons. The aqueous [18F]fluoride solution was transferred through a Teflon line by
helium overpressure of the target from the GE PETtrace cyclotron directly into the collection
funnel at position 6 on the FASTlabTM cassette (Figure 1).
Quality Control
Quality control of [18F]GE-179 was carried out in accordance with the European
Pharmacopeia.16
Visual Inspection and pH
Doses were visually examined to be clear, colourless and free of particulate matter.
ColourpHast 2.0-9.0 pH-indicator strips (Fisher Scientific, Loughborough, UK) were used to
confirm the pH by comparison with the scale provided.
Radionuclidic Identity
Radionuclidic identity was confirmed by taking three activity measurements at 5 minute
intervals using the Capintec CRC-15R radioisotope dose calibrator. The half-life was
calculated from the generated calibration and compared with the known half-life of
[18F]fluorine (109.8 mins).
Bioburden, Sterility and Endotoxin Testing
Bioburden and sterility was performed by international Laboratory Services (Shardlow, UK).
Samples prior to aseptic dispensing were analysed for total yeast, mould and total aerobic
microbial count. Post aseptic sample were incubated for 14 days and tested by EP/USP
membrane filtration, absence of culture growth after 14 days was indicative of sterility.
Endotoxin content was analysed using a Charles River Laboratories Endosafe Portable
Testing System according to European Pharmacopeia guidelines.16
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Aseptic Dispensing and Filter Integrity Testing
Aseptic dispensing was performed via a single-use arrangement of tubing, filter and needles
provided as a sterile (gamma-irradiated) pre-assembled kit from Pall (Portsmouth, UK).
Integrity testing was achieved by bubble point analysis performed in situ by Pall Palltronic
Flowstar II integrity test instrument.
Residual solvent and Kryptofix®222 analysis
Residual solvent analysis was determined by using an Agilent gas chromatography (GC)
system, equipped with headspace autoinjector, split/splitless inlet, flame ionisation detector
(FID) and a Restek column (Stabilwax 30 m x 0.25 mm, 0.25 µm, G16 stationary phase).
Kryptofix®222 levels were analysed using the established spot test method.17, 18
Specification for the release of formulated [18F]GE-179
[18F]GE-179 for clinical use was analysed in accordance with European Pharmacopeia
guidelines for radiopharmaceuticals.16 Table 3 summarises the specification for the release of
[18F]GE-179 for clinical use with a maximum injected dose of 10 mL.
Results and Discussion
Radiosynthesis of [18F]GE-179
The radiosynthesis of [18F]GE-179 (Scheme 3) is a two-step process involving the
nucleophilic fluorination of 11 to produce 2-[18F]fluoroethyl tosylate, [18F]9, which is a
common intermediate for the addition of a [18F]fluoroethyl group.19-21 The second step is the
alkylation of the thiolate, 12. The crude product is purified by semi-preparative HPLC
followed by reformulation on a tC18 Sep-Pak cartridge. In our original synthesis, a 2-pot-2-
HPLC purification process was employed which was both not compatible with the
FASTlabTM and low yielding. In this work, we have developed a one-pot-one-HPLC
purification process which results in a higher yielding radiosynthesis.
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Development of the automated FASTlabTM radiosynthesis of [18F]GE-179
The radiosynthesis of [18F]GE-179 for pre-clinical studies has previously been reported.1 In
order to deliver [18F]GE-179 for clinical studies, a GMP compliant automated radiosynthesis
was required. However, the original radiosynthetic route was not compatible with
automation on FASTlabTM for three reasons:
1) [18F]Fluoride needs to be introduced directly from the cyclotron. Therefore, an
automated and reproducible [18F]fluoride trapping and elution process at high
radioactive levels was required
2) Cesium carbonate is not soluble in either anhydrous acetonitrile or ethanol. Therefore,
an alternative soluble base to deprotonate 8 was required
3) A 2-pot-2-semi-preparative HPLC process is not compatible with the FASTlabTM,
due to the restricted number of cassette positions. Therefore, a one-pot-one-HPLC
purification process was required
The original radiosynthesis involved drying a mixture of [18F]fluoride in target [18O]H2O
(200-300 µL), Kryptofix®222 (5 mg, 13 µmol) and potassium carbonate (1 mg, 7 µmol) in
anhydrous acetonitrile (800 µL) and water (200 µL).1 Automated processes require
[18F]fluoride to be trapped on a QMA-carbonate Sep-Pak cartridge followed by elution using
a fixed volume of eluent (e.g. Kryptofix®222 and potassium carbonate solution). This allows
for a fixed [18F]fluoride drying process. Secondly, we previously used cesium carbonate as
the base to deprotonate 8 to create the thiolate, 12. This base is not suitable for use on the
FASTlabTM as it is not soluble in either anhydrous acetonitrile or ethanol. Furthermore, the
introduction of a second base, in addition to the QMA eluent, increased the number of
reagent vials on the cassette and limited the number of vial positions for other reagents on the
FASTlabTM cassette. Therefore, the Kryptofix®222 and potassium carbonate solution was
investigated as the base for the deprotonation of 8.
The QMA eluent vial for the commercial synthesis of [18F]FDG on FASTlabTM contains
Kryptofix®222 (44 mg, 117 µmol), and potassium carbonate (7.8 mg, 57 µmol) in acetonitrile
(660 µL) and water (165 µL). To enable the use of this higher concentration FASTlabTM vial,
we had to use a smaller volume (70 µL) which contained the same amount of Kryptofix®222
and base as the original radiosynthesis. This was diluted with water (500 µL) and used to
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elute the trapped [18F]fluoride from the QMA-carbonate Sep-Pak cartridge into the reaction
vessel. After the [18F]fluoride solution was evaporated to dryness, ethylene ditosylate in
anhydrous acetonitrile was added and the reaction mixture was heated at 100 °C for 10
minutes. These conditions delivered crude [18F]9 with a radiochemical conversion of > 80%
(Figure 2).
The Kryptofix®222 and potassium carbonate solution was found to be a suitable base for the
deprotonation of 8 (Experiment 1, Table 2). Therfore, a series of experiments were performed
with different amounts of eluent from the commercially available FASTlabTM eluent vial
(Experiments 2-6, Table 4). Analysis of the crude reaction mixtures showed that a
conversion of [18F]9 to [18F]GE-179 was >90% for the experiment using 350 µL of the
FASTlabTM eluent and confirmed the FASTlab eluent to be a suitable base required to
deprotonate the thiol precursor 8, prior to the alkylation reaction.
Due to the position of the eluent vial (P2) and the QMA-carbonate Sep-Pak cartridge (P4) on
the FASTlabTM cassette, the volume of eluent in the reagent vial (825 µL) was sufficient to
deliver 70 µL of eluent to elute the [18F]fluoride from the QMA-carbonate Sep-Pak cartridge,
however, due tothe position of the thiol precursor 8 vial (P16) , this volume was not sufficient
to deliver 350 µL of base to the thiol precursor vial at P16. The volume within the
FASTlabTM manifold between P2 and P16 is ca. 500 µL , therefore, to deliver the required
amount of base, the volume of the QMA eluent solution was increased to 1.5 mL while
maintaining the same concentration of reagents [i.e. Kryptofix®222 (79 mg, 210 µmol),
potassium carbonate (14.2 mg, 103 µmol) in anhydrous acetonitrile (1300 µL) and water (200
µL)].
Semi-preparative HPLC purification of crude [18F]GE-179 proved to be a challenge due to
the presence of a close eluting impurity (Figure 3). It was postulated that the close eluting
peak was the acetate, 13. Formation of the acetate was thought to derive from the reaction of
acetonitrile with the hydroxyethylsulfide (Scheme 4), followed by partial hydrolysis of the
imidate.(Scheme 4). Based on this hypothesis, the formation of impurity 14 could be avoided
by replacing acetonitrile with another solvent (or removal of 11 by HPLC purification).
Therefore, a series of experiments were performed to test this hypothesis.
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In the first experiment, both the synthon labelling and alkylation reactions were performed in
acetonitrile. In the second experiment, acetonitrile from the labelling reaction was removed
by evaporation prior to the alkylation and ethanol was used as the solvent for the alkylation
reaction and in the third experiment, HPLC purified [18F]9 in acetonitrile was added to 8,
which was also dissolved in acetonitrile.
The crude reaction material from each of these experiments were analysed by analytical
HPLC and the chromatograms are shown in Figures 3, 4 and 5 respectively. Figure 3 shows
the close eluting peak when using acetonitrile for the alkylation reaction, whereas Figure 4
shows the result of removing acetonitrile by evaporation and using ethanol as the alkylation
solvent. Thus, performing the alkylation reaction in ethanol prevented the formation of the
impurity, 14. Figure 5 shows that by removing unreacted 11 by HPLC purification of [18F]9
and performing the alkylation reaction in acetonitrile also prevented the formation of 14.
Therefore, the data presented in Figures 3-5 support the hypothesis that the close eluting
impurity is 14. Figure 6 shows the synthesised reference standard 14, co-elutes with the
close eluting peak 14, present in the crude reaction.
As a consequence of these results, a change to the composition of the eluent vial and removal
of acetonitrile from the [18F]9 labelling reaction was required. Therefore, the eluent vial was
prepared with ethanol instead of acetonitrile. Over 95% of the [18F]fluoride was eluted from
the QMA cartridge using the modified eluent vial and there was no impact on the
radiochemical yield of [18F]9. After formation of crude [18F]9 in acetonitrile, the reaction
was evaporated to dryness by heating at 100 °C under a nitrogen flow and also by applying a
vacuum to the reaction vessel. The subsequent alkylation reaction was performed in ethanol.
The FASTlabTM only has one reaction vessel and the capacity for one HPLC purification
step. Therefore, the automated process was modified so that both the labelling and alkylation
reactions were performed in the same reaction vessel. Furthermore, as a single HPLC
purification step was required for the purification of [18F]GE-179, it was necessary to remove
the the HPLC purification of [18F]9.
A consequence of using crude [18F]9 was the presence of ethylene ditosylate, 11, in the
alkylation reaction which competes with the reaction of [18F]9 with 8. When using HPLC
purified [18F]9, ethylene ditosylate, 11, would not be present in the alkylation reaction and as
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a result this competing reation would not be present. This would allow the use of lower
amounts of 8, however, in the case of using crude [18F]9, it was necessary to increase the
amount 8. It was observed that when using 3 mg of 11 and 4 mg of 8, the conversion of
[18F]9 to [18F]GE-179 was ca. 40%. However, increasing the amount 8 to 10 mg increased
the conversion of [18F]9 to [18F]GE-179 to ca. 80% (Figure 7).
Automated FASTlabTM radiosynthesis of [18F]GE-179
The incoming [18F]fluoride solution was trapped on a Waters QMA cartridge and eluted into
the reaction vessel. The eluent was prepared in syringe 1 (P3) by diluting the QMA eluent
solution with water (500 µL). The [18F]fluoride solution was then evaporated to dryness with
a combination of nitrogen flow and vacuum at 100 °C for 20 minutes.
Following the [18F]fluoride evaporation, 11 (2.5 mg, 6.7 µmol) in anhydrous acetonitrile (1
mL) was added. The radiolabelling reaction was conducted at 86 °C for 15 minutes to
produce [18F]9. Subsequently, the crude reaction mixture was evaporated to dryness with a
combination of nitrogen flow and vacuum at 80 °C for 15 minutes.
In parallel to the [18F]9 labelling reaction, the QMA eluent (350 µL) was added to the vial
containing 8 (15 mg, 44 µmol) dissolved in ethanol (1.8 mL). The resulting ethanolic thiolate
solution (1.5 mL, 10 mg, 30 µmol) was added to the reaction vessel containing dried crude
[18F]9. The alkylation reaction was carried out at 80 °C for 2 minutes followed by 100 °C for
10 minutes. Crude [18F]GE-179 was then diluted with water (2 mL) and 0.1 M hydrochloric
acid (1 mL) and loaded onto a 10 mL HPLC loop. [18F]GE-179 was purified using reverse
phase isocratic semi-preparative HPLC process on a Hichrom ACE C18 (100 x 10 mm, 5
µm) column with a flow rate of 3 mL/min with mobile phase A (0.8% Triethylamine (pH
adjusted to ca. pH 7-7.3 with 85% H3PO4) and mobile phase B (100% acetontrile). The
isocratic method was 60% (B) for 60 mins.
Purified [18F]GE-179 was collected into an external vial containing water (50 mL) and then
trapped on to a tC18 cartridge on the FASTlab™ cassette. After trapping, the tC18 cartridge
was washed with water (20 mL) and dried under a stream of nitrogen. Finally, [18F]GE-179
was eluted into an external formulation vial with ethanol (1 mL). The formulated product
consisted of saline (0.9% w/v, 11.5 mL) and ascorbic acid (1 mg/mL in water, 1.5 mL) to
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give a final formulation in a volume of 14 mL. Quality control was performed using a
reverse phase isocratic analytical HPLC process on a Primesep B2, (250 x 4.6 mm, 5 µm)
HPLC column at a flow rate of 1.6 mL/min with mobile phase A (200 mM Ammonium
acetate) and mobile phase B (100% Acetonitrile). The isocratic method was 44% (B) for 35
mins.
Clinical production of [18F]GE-179 using the FASTlabTM synthesiser
The FASTlabTM synthesiser was developed by GE Healthcare for the automated production
of [18F]fluorine radiotracers. The synthesiser was designed to aid PET radiopharmaceutical
manufacturers to comply with the GMP requirements for PET radiopharmaceuticals.22-33 The
disposable, single-use cassette, removes the need to validate a cleaning protocol as the
disposable fluid path removes any potential for cross contamination between productions.
The radiosynthesis of [18F]GE-179 described above resulted in a process that delivered an
AY of 7 ± 2% (n = 15) with a radioactive concentration (RAC) of 289 ± 48 MBq/mL. The
total synthesis time, including purification, was 120 minutes. Quality control was carried out
in accordance with the European Pharmacopeia.16 The formulated product was clear,
colourless, pH neutral and free of particulate matter. The product successfully passed the
yeast, mould count and total aerobic microbial count from the bioburden placed on the
aseptic dispensing filter. Absence of culture growth following 14 days of incubation
demonstrated the sample sterility post filtration. All samples were pyrogen free.
Analytical radio-HPLC established the radiochemical purity was 96 ± 1% and remained
greater than 95% up to at least 6 hours. The molar activity and total chemical content was
calculated to be 627 ± 416 GBq/µmol and 2 ± 1 µg respectively. The final formulation
volume was 14 mL.
Residual solvent analysis showed the levels of acetonitrile to be within the permitted daily
exposure (PDE) of < 410 ppm.34 Formulation of the product in 0.9% saline (11.5 mL),
ascorbic acid solution (1 mg/mL, 1.5 mL) and ethanol (1 mL) gave a formulation volume of
14 mL with an ethanol content of 7%. Ethanol content is acceptable up to a maximum of 10
% when added as an excipient.34 The formulated product contained less than 50 µg/mL of
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16
Kryptofix® 222 by visual comparison with positive and negative controls.15,
The
specifications for the release of [18F]GE-179 for clinical use are shown in Table 3.
Conclusion
An improved, robust and scalable precursor synthesis has been developed which enabled the
preparation of multigram quantities of the thiol precursor to GMP standard. A successful
transfer of the previously reported 2-pot-2-HPLC radiosynthesis of [18F]GE-179 to an
automated process using FASTlabTM has been achieved. The new process has been
implemented to GMP standard at up to 80 GBq starting activity and has enabled the delivery
of [18F]GE-179 for clinical use. The radiosynthesis delivered an AY of 7 ± 2%, with an RAC
of 289 ± 48 MBq/mL and molar activity of 627 ± 416 GBq/µmol. The RCP was 96 ± 1% and
remained over 95% up to at least 6 hours. The total chemical content was 2 ± 1 µg in a
formulation volume of 14 mL.
Abbreviations
Activity Yield; AY, COC: Cyclic Olefin Copolymer; FDG: Fluorodeoxyglucose; GMP:
Good manufacturing practice;; HPLC: High Performance Liquid Chromatography; NMDA:
N-Methyl-D-aspartate; NMR: Nuclear Magnetic Resonance; ODS: Octadecyl-silica; PET:
Position Emission Tomography; QC: Quality control; QMA: Quaternary Methylammonium;
RAC: Radioactive Concentration; RCP: Radiochemical Purity; RV: Reaction vessel; TLC:
Thin Layer Chromatography ; TEA: Triethylamine; UV: Ultraviolet
Acknowledgements
The authors would like to thank, and for their contribution and also Maria Antalffy and John
Woodcraft at Hammersmith Imanet for the delivery of the clinical preparations.
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References
1.
2.
E. G. Robins, Y. Zhao, I. Khan, A. Wilson, S. K. Luthra and E. Årstad, Bioorg. Med.
Chem. Lett., 2010, 20, 1749.
R. Dingledine, K. Borges, D. Bowie and S. F. Traynelis, Pharmacological Reviews,
1999, 51, 7-61.
3.
4.
5.
6.
C. G. Lau and R. S. Zukin, Nature Reviews Neuroscience, 2007, 8, 413-426.
N. A. Cavara and M. Hollmann, Molecular Neurobiology, 2008, 38, 16-26.
E. Gamzu, Eur. Neuropsychopharm., 1995, 5, 171.
K. Muir, D. Grosset, E. Gamzu and K. Lees, British Journal of Clinical
Pharmacology, 1994, 38, 33-38.
7.
8.
P. Paoletti and J. Neyton, Current Opinion in Pharmacology, 2007, 7, 39-47.
M. R. Walters, A. P. J. Bradford, J. Fischer and K. R. Lees, British Journal of Clinical
Pharmacology, 2002, 53, 305-311.
9.
R. N. Waterhouse, Nucl. Med. Biol., 2003, 30, 869-878.
10.
F. López-Picón, A. Snellman, O. Shatillo, P. Lehtiniemi, T. J. Grönroos, P.
Marjamäki, W. Trigg, P. A. Jones, O. Solin, A. Pitkänen and M. Haaparanta-Solin, J.
Nucl. Med., 2016, 57, 1442-1447.
11.
12.
13.
C. J. McGinnity, A. Hammers, D. A. R. Barros, S. K. Luthra, P. A. Jones, W. Trigg,
C. Micallef, M. R. Symms, D. J. Brooks, M. J. Koepp and J. S. Duncan, J. Nucl.
Med., 2014, 55, 423-430.
C. J. McGinnity, M. J. Koepp, A. Hammers, D. A. Riaño Barros, R. M. Pressler, S.
Luthra, P. A. Jones, W. Trigg, C. Micallef, M. R. Symms, D. J. Brooks and J. S.
Duncan, Journal of Neurology, Neurosurgery & Psychiatry, 2015, 86, 1150-1157.
Schoenberger M, Schroeder F.A, Placzek M.S, Carter R.L, Rosen B.R, Hooker J.M,
Sander C.Y, In Vivo [18F]GE-179 Brain Signal Does Not Show NMDA-Specific
Modulation with Drug Challenges in Rodents and Nonhuman Primates ACS Chem
Neuroscience, 2018, 9(2):298-305
14.
McGinnity C.J, Årstad E, Beck K, Brooks D.J, Coles J.P, Duncan J.S, Galovic M,
Hinz R, Hirani E, Howes O.D, Jones P.A, Koepp M.J, Luo F, Riaño Barros D.A,
Singh N, Trigg W, Hammers A, Comment on " In Vivo [18F]GE-179 Brain Signal
Does Not Show NMDA-Specific Modulation with Drug Challenges in Rodents and
Nonhuman Primates, , ACS Chem Neuroscience, 2019, 10(1):768-772.
European Association of Nuclear Medicine Radiopharmacy Committee: Guidelines
on current good radiopharmacy practice (cGRPP) in the preparation of
radiopharmaceuticals, version 2., 2007.
15.
16.
European Pharmacopoeia, Council of Europe, EDQM (European Directorate for the
Quality of Medicines), 2008.
17.
18.
19.
B. H. Mock, W. Winkle and M. T. Vavrek, Nucl. Med. Biol., 24, 193-195.
P. J. H. Scott and M. R. Kilbourn, Appl. Radiat. Isot., 2007, 65, 1359-1362.
A. Bauman, M. Piel, R. Schirrmacher and F. Rösch, Tetrahedron Letters, 2003, 44,
97-99.
20.
21.
T. Kniess, M. Laube, P. Brust and J. Steinbach, MedChemComm, 2015, 6, 1714-1754.
T. R. Neal, S. Apana and M. S. Berridge, Journal of Labelled Compounds and
Radiopharmaceuticals, 2005, 48, 557-568.
22.
S. R. Dubash, N. Keat, P. Mapelli, F. Twyman, L. Carroll, K. Kozlowski, A. Al-
Nahhas, A. Saleem, M. Huiban, R. Janisch, A. Frilling, R. Sharma and E. O.
Aboagye, J. Nucl. Med., 2016, 57, 1207-1213.
This article is protected by copyright. All rights reserved.

23.
24.
25.
26.
R. Fortt, G. Smith, R. O. Awais, S. K. Luthra and E. O. Aboagye, Nucl. Med. Biol.,
2012, 39, 1000-1005.
J. Henrottin, C. Lemaire, D. Egrise, A. Zervosen, B. van Den Eynde, A. Plenevaux,
X. Franci, S. Goldman and A. Luxen, Nucl. Med. Biol., 2016, 43, 379-389.
Y.-Y. Huang, S. Taylor, J. Koziorowski, Y.-N. Chang, W.-H. Kao, K.-Y. Tzen and
C.-Y. Shiue, Ann. Nucl. Med., 2016, DOI: 10.1007/s12149-016-1097-4, 1-9.
A. Jackson, M. R. Battle, D. M. O'Shea, W. F. Chau, A. Gaeta, S. L. Brown, A. L.
Ewan, C. L. Jones, P. A. Jones, J. L. Woodcraft, D. R. Bouvet, B. B. Guilbert and W.
Trigg, Nucl. Med. Biol., 2014, 41, 196-202.
27.
A. Jackson, B. B. Guilbert, S. D. Plant, J. Goggi, M. R. Battle, J. L. Woodcraft, A.
Gaeta, C. L. Jones, D. R. Bouvet, P. A. Jones, D. M. O'Shea, P. H. Zheng, S. L.
Brown, A. L. Ewan and W. Trigg, Bioorganic and Medicinal Chemistry Letters,
2013, 23, 821-826.
28.
29.
R. Krasikova, Curr. Org. Chem., 2013, 17, 2097-2107.
C. Lemaire, L. Libert, X. Franci, J. L. Genon, S. Kuci, F. Giacomelli and A. Luxen,
Journal of Labelled Compounds and Radiopharmaceuticals, 2015, DOI:
10.1002/jlcr.3291, n/a-n/a.
30.
C. Lemaire, L. Libert, X. Franci, J. L. Genon, S. Kuci, F. Giacomelli and A. Luxen,
Journal of Labelled Compounds and Radiopharmaceuticals, 2015, 58, 281-290.
J. Z. Long, M. S. Jacobson and J. C. Hung, J. Nucl. Med. Technol., 2013, 41, 32-34.
D. Thonon, D. Goblet, E. Goukens, G. Kaisin, J. Paris, J. Aerts, S. Lignon, X. Franci,
R. Hustinx and A. Luxen, Mol. Imaging Biol., 2011, 13, 1088-1095.
T. Wickstrøm, A. Clarke, I. Gausemel, E. Horn, K. Jørgensen, I. Khan, D. Mantzilas,
T. Rajanayagam, D. J. In 'T Veld and W. Trigg, Journal of Labelled Compounds and
Radiopharmaceuticals, 2014, 57, 42-48.
31.
32.
33.
34.
PMP/ICH/283/95 ICH guidelines: ICH topic Q3C impurities: residual solvents
European Medicines Agency, 1998.
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Table 1: FASTlabTM reagents, components and vial composition
CASSETTE POSITION
SILICONE LINE COMPONENT
LENGTH
P1
P2
14 cm
O-18 water Recovery vial
Kryptofix®222 (79 mg, 210 µmol) and
potassium carbonate (14.2 mg, 103 µmol)
Solvent: [water (200 µL) and ethanol (1.3
mL)]
(11 mm glass vial)
Syringe 1
P3
P4-P5
P6
14 cm
QMA-carbonate Sep-Pak cartridge
O-18 water Collection Funnel
COC Reaction vessel
COC Reaction vessel centre
Empty
P7
14 cm
14 cm
P8
P9
P10
P11
P12
Empty
Syringe 2
Ethylene ditosylate, 11, (4.0 mg, 11 µmol)
in anhydrous acetonitrile (1.6 mL)
(11 mm glass vial)
P13
P14
Ethanol (4.0 mL)
(13 mm glass vial)
0.1 M HCl (4.0 mL)
(13 mm glass vial)
P15
P16
Water bag spike and water bag (100 mL)
Thiol precursor 8 (15 mg, 40 µmol) in
ethanol (1.8 mL)
(13 mm glass vial)
Line to HPLC
P17
42 cm
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Table 2: Effect of the amount of FASTlabTM eluent on the conversion of [18F]9 to [18F]GE-
179
Volume of eluent Kryptofix®222
(µL)
K2CO3
8
K2CO3 : 8
Conversion of [18F]9
to [ F]GE-179 (%)
18
1
2
3
4
5
6
50
2.7 mg, 7 µmol
8 mg, 21.2 µmol
8 mg, 21.2 µmol
0.5 mg, 3.6 µmol
10 mg, 30 µmol
1:8
12
70
69
89
91
90
150
150
350
350
700
1.4 mg, 10.3 µmol
1.4 mg, 10.3 µmol
10 mg, 30 µmol
10 mg, 30 µmol
10 mg, 30 µmol
10 mg, 30 µmol
10 mg, 30 µmol
1:3
1:3
18.7
49.7µmol
mg, 3.3 mg, 24.0 µmol
1:1
18.7 mg, 49.7 3.3 mg, 24.0 µmol
µmol
1:1
37.3 mg, 99 6.6 mg, 48.0 µmol
µmol
1.6:1
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Table 3: Specification for the release of formulated [18F]GE-179*
Parameter
Acceptance criteria
Clear, colourless and particulate free
4-7
Appearance
pH
< 17.5 EU/mL
< 16 µg/dose
< 1.5 µg/dose
< 5 µg/dose
≤ 50 µg/mL
≤ 410 ppm
Bacterial endotoxin
Amount of [19F]GE179
Amount of any individual unknown impurity
Amount of total chemical impurities
Kryptofix® 222
Residual solvent: Acetonitrile
Residual solvent: Ethanol
RCP
≤ 10 %
≥ 95%
Pass
Sterility
≥ 3.45 bar
Filter Integrity
* Based on a maximum patient injection of 10 mL
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Table 4: Summary of results from the clinical preparations of [18F]GE-179 at Hammersmith
Imanet
Starting
Activity Yield
(AY) (%)
Formulated
RAC
GE-179 only
(µg)
Total Chemical
Content (µg)
RCP (%)
Molar Activity
(GBq/µmol)
Activity (MBq)
product (MBq)
(MBq/mL)
59108
27956
57123
47862
65514
69650
61663
70765
78492
47823
63197
66495
53319
52180
66479
7.4
13.7
7.0
10.3
6.2
6.2
5.6
5.0
5.8
5.4
6.7
8.2
7.4
7.2
5.4
4385.1
3816.6
4018.6
4931.1
4038.7
4328.7
3426.3
3569.3
4573.0
2568.1
4223.6
5455.5
3966.6
3735.5
3582.4
313.2
272.6
287.0
352.2
288.5
309.2
244.7
255.0
326.6
183.4
301.7
389.7
283.3
266.8
255.9
0.9
0.6
2.0
0.7
0.6
0.5
0.7
0.2
0.5
1.1
0.6
2.7
2.2
1.7
0.6
0.9
2.1
4.6
1.8
0.6
3.2
2.3
1.0
1.0
1.8
1.4
3.1
3.5
3.4
0.6
95.3
97.8
96.1
95.6
96.5
96.7
97.2
97.1
95.7
97.9
95.3
96.1
95.8
95.9
96.1
1177.9
498.6
194.7
601.3
1409.0
308.0
293.8
788.3
1043.5
318.9
689.4
358.5
230.2
234.5
1265.1
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Scheme 1: Synthesis of N-(2-chloro-5-thiophenol)-N’-(3-thiomethylphenyl)-N’-methyl
guanidine (8)
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Scheme 2: Synthesis of N-(2-chloro-5-(2-fluoroethylthio)phenyl)-N’-methyl-N’-(3-
(methylthio)phenyl)guanidine, GE-179.
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Scheme 3: Radiosynthesis of [18F]GE-179
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Scheme 4: Postulated mechanism of the formation of 13 and 14
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Figure 1: FASTlabTM cassette for [18F]GE-179 radiosynthesis
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Figure 2: Analytical HPLC chromatogram of crude [18F]9 (radiochemical in red, UV in blue)
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Figure 3: Analytical HPLC chromatogram of crude [18F]GE-179 (radiochemical in red, UV
in blue) showing the close eluting impurity, 13, when the alkylation reaction was performed
using unpurified [18F]9 in acetonitrile.
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Figure 4: Analytical HPLC chromatogram of crude [18F]GE-179 (radiochemical in red, UV
in blue) showing that the close eluting impurity was not present when the alkylation reaction
was performed using unpurified [18
F]9 in ethanol.
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Figure 5: Analytical HPLC chromatogram of crude [18F]GE-179 (radiochemical in red, UV
in blue) when the alkylation reaction was performed using HPLC purified [18F]9 in
acetonitrile.
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