18F-FESB: Synthesis and automated radiofluorination of a novel 18F-labeled pet tracer for β-amyloid plaques

Article abstract of DOI:10.1002/jlcr.1011

1-(2′-[¹⁸F]-fluoroethoxy)-2,5-bis(4′-methoxystyryl) benzene (¹⁸F-FESB) was synthesized in ～76% radiochemical yield (specific activity > 58.6 GBq or 1.58 Ci/μmol) in an Advanced Cyclotron Systems' automated synthesis unit by nucleophi

Full text of DOI:10.1002/jlcr.1011

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 983–996.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1011
Research Article
18F-FESB: synthesis and automated radiofluorination
of a novel F-labeled pet tracer for b-amyloid plaques
18
Piyush Kumar1,*, Weizhong Zheng2, Stephen A. McQuarrie1,2, Jack H.
Jhamandas3 and Leonard I. Wiebe2
1 Division of Oncologic Imaging, Cross Cancer Institute, Edmonton, Alta.,
Canada T6G 1Z2
2 Faculty of Pharmacy and Pharmaceutical Sciences, Edmonton, Alta.,
Canada T6G 2N8
3 Department of Medicine (Neurology) and Centre for Alzheimer and
Neurodegenerative Research, University of Alberta, Edmonton, Alta.,
Canada T6G 2S
Summary
1-(20-[18F]-fluoroethoxy)-2,5-bis(40-methoxystyryl)benzene (18F-FESB) was synthe-
sized in ꢀ76% radiochemical yield (specific activity >58.6 GBq or 1.58 Ci/mmol) in
an Advanced Cyclotron Systems’ automated synthesis unit by nucleophilic substitu-
tion of 1-(20-toluenesulfonylethoxy)-2,5-bis(40-methoxystyryl)benzene and purified
using reversed phase column chromatography. When performed in the presence of
ionic fluid, either 1-butyl-3-methylimidazolium tetrafluoroborate (Bmimtetrafluor-
oborate; BmimBF4) or 1-ethyl-3-methylimidazolium trifluoromethanesulfonate
(Emimtriflate; EmimTFMS), radiochemical yields of 18F-FESB ranged from 17 to
76%. The radiochemical yields were consistently lower (ꢀ3–7%) in the absence of
these ionic fluids. Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: 18F-labeled styryl benzene derivatives; automated synthesis; HPLC
purification; PET marker of b-amyloid plaques; ionic fluids
Introduction
In spite of recent advances in the development of new radiopharmaceuticals
for the clinical evaluation of Alzheimer’s disease (AD), positron emission
tomography (PET) and single photon emission computed tomography
(SPECT) are not currently in routine use as diagnostic modalities.1 Structural
*Correspondence to: Piyush Kumar, Division of Oncologic Imaging, Cross Cancer Institute, Edmonton,
Alta., Canada T6G 1Z2. E-mail: piyushku@cancerboard.ab.ca
Contract/grant sponsor: University of Alberta Foundation; contract/grant number: G533000094
Copyright # 2005 John Wiley & Sons, Ltd.
Received 6 August 2005
Revised 10 August 2005
Accepted 25 August 2005

984
P. KUMAR ET AL.
magnetic resonance imaging (MRI) and blood flow studies depend on gross
changes in brain structure and function, and are ‘secondary markers’ since
they diagnose functional alterations that have already occurred in the brain
and may therefore not be ideal for following patients over time.2,3
[18F]fluorodeoxyglucose, a glucose analog, is a PET tracer that is used to
evaluate metabolic disorders and, therefore, is an indirect tool to detect the
presence and progression of AD.4 However, the important roles of amyloid
cascades and neurofibrillary tangles (NFTs) in the pathogenesis of AD
necessitate the development of a biomarker that facilitates early diagnosis of
the disease, allows clinico-pathological correlations of amyloid deposition at
an early stage and is receptor specific so that it serves as a true diagnostic tool
for anti-amyloid therapies.5–7
A recent study suggests that anti-amyloid therapies, when co-investigated in
combination with positron PET or SPECT amyloid-imaging tracers, could
facilitate in vivo evaluation of the efficacy of therapy in the aging human
brain.8 The efficacies of [11C]-labeled Congo Red and [99mTc]-Chrysamine-G
analogs9 as markers of b-amyloid plaques suffer from marginal brain entry10–
13
which consequently makes the detection of the disease difficult. Recently, a
study with 11C-labeled [1,4-bis(40-hydroxystyryl)-2-[11C]methoxybenzene
([11C]-methoxy-XO4), synthesized in ꢀ6.7% radiochemical yield,
11
O
CH
3
OH
HO
11C-Methoxy XO4
Tg 2576APPsw and PS1(M146L) transgenic mice showed that it binds
specifically to b-amyloid plaques.14 Another 11C-labeled analog of (E,E)-1-
(30,40-dihydroxystyryl)-4-(30-methoxy-40-hydroxystyryl)benzene, synthesized in
ꢀ2.3% radiochemical yield, has been evaluated for its specific binding to
amyloid cells and is found to exhibit high brain uptake and selective binding
affinity to Ab(1-40) fibrils in vitro.15 Recently, a fluorine containing 13C-
labeled styrylbenzene derivative has been developed as a contrast agent for
MRI to locate AD pathologies in vivo.16
The first human study of a novel amyloid-imaging PET tracer, named
Pittsburgh Compound-B (PIB), in a limited number of patients diagnosed with
mild AD, further supports the role of a PET AD diagnostic radiotracer in AD
therapy management. PIB typically showed marked retention in the areas
known to contain large amounts of amyloid deposits in AD, most prominently
in frontal cortex (1.94-fold, p=0.0001), parietal (1.71-fold, p=0.0002),
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 983–996

18F-LABELED STYRYL BENZENE DERIVATIVES
985
temporal (1.52-fold, p=0.002), and occipital (1.54-fold, p=0.002) cortex and
the striatum (1.76-fold, p=0.0001) in comparison to healthy patients.17
Low radiochemical yields but promising efficacy and pharmacokinetic
properties of 11C-XO4 and other PET diagnostic agents in providing
quantitative information on amyloid deposits in living subjects support the
development of an 18F-labeled XO4 PET. Such a tracer would be expected to
possess similar binding to b-amyloid proteins and would have a longer
observation window due to the longer decay time. The present work describes
a structural modification of XO4 where radioactive fluorine is incorporated
nucleophilically to produce a high specific activity radiopharmaceutical
without a major alteration in the basic structure of XO4. We now report
the synthesis of 1-(20-fluoroethoxy)-2,5-bis(40-methoxystyryl)benzene, 7, and
its 18F-labeled analog 8.
Experimental
Materials and methods
2,5-Dimethylphenol was purchased from Sigma-Aldrich Chemical Company
Inc., USA. All chemicals were reagent grade and were used without further
purification unless otherwise noted. Emimtriflate (EmimTFMS) and Bmimte-
trafluoroborate (BmimBF4) were purchased from Fluka, Switzerland. Thin-
layer chromatography (TLC) was carried out on Kieselgel 60 F254 (Merck)
silica gel coated micro TLC plates (2.5 ꢁ 7.5 cm). Column chromatography
was performed using silica gel 60 (230–400) Mesh ASTM, 0.040–0.063 mm
supplied by Rose Scientific Ltd. (Edmonton, Canada). IR spectra were
1
recorded on a Perkin-Elmer 700 spectrometer. H, 13C and 19F NMR spectra
were obtained using a Bruker AM-300 spectrometer. The chemical shifts are
quoted in d ppm with respect to tetramethylsilane (1H and 13C) and
hexafluorobenzene (C6F6, as an external standard for 19F) and the coupling
constants (J) are reported in Hertz. 13C-NMR spectral assignments were aided
by the J-MOD technique. All NMR spectra were acquired by preparing the
samples in CDCl3 solution. When necessary, high-resolution mass spectra
(HRMS) were obtained in lieu of elemental analysis and were acquired on a
Kratos MS50 MASPEC system (Manchester, UK).
The automated synthesis unit (ASU) employed for the manufacture of 18F-
FESB was purchased from Advanced Cyclotron Systems (Richmond,
Canada). It performs unit operations including the movement of liquids and
gases (vacuum, pressure), valve actuation and heating systems on a time-
dependent basis through a well-defined flow path, all under computer (CPU)
control. Feedback control, monitoring and diagnostic functions are built into
the operation using various sensor-based systems. The purification of the
synthesized radiopharmaceutical involved the use of a Beckman HPLC system
Copyright # 2005 John Wiley & Sons, Ltd.
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P. KUMAR ET AL.
Table 1. Radiofluorination reaction parameters for 18F-FESB
Reaction #
Ionic fluida
Amount
(ml)
Reaction
time (min)
Purified RCY
(%)
Specific activity
(Ci/mmol)
1
2
3
4
5
6
7
8
BmimBF4
BmimBF4
BmimBF4
EmimTFMS
EmimTFMS
EmimTFMS
None
20
20
50
50
50
50
n/a
n/a
12
8
9
8
8
8
4
8
16.9
43.2
76.9
39.6
25.5
42.5
3.0
>0.45
>2.19
>1.58
>1.57
>0.70
>0.49
NC
None
7.0
NC
a Acetonitrile (2.0 ml) and anhydrous DMF (50 ml) were used as solvent in reactions 1–8. n/a = not
applicable; NC=not calculated.
equipped with guard column, reversed phase C-18 (10ODS3; 25 ꢁ 0.9 cm)
Whatman column, UV detector (wavelength 370 and 254 Zm) and a
radiodetector. Impure radiolabeled mixture was purified using ethanol–water
(75:25, v/v) as an eluent at a flow rate of 1.5 ml/min. The purified 18F-FESB
was collected using a sterile manifold that was attached to the terminal HPLC
outlet and equipped with a sterile neutral alumina cartridge (to remove any
traces of unreacted fluoride) and a Millipore LG (0.45 mm pore size filter). The
specific activity was interpolated from a calibration curve plotting peak area as
a function of cold FESB (1 mg–10 Zg). The minimum detectable amount for
FESB peak under present chromatographic conditions was 10 Zg. The
radiochemical yield of 18F-FESB reached to ꢀ77% (specific activity
>58.6 GBq or 1.58 Ci/mmol) when Bmimtetrafluoroborate (50 ml) was used
in the radiosynthesis (Table 1). However, the radiochemical yield of 18F-FESB
varied between 16.9 and 76.9% depending on the nature and amount of the
ionic fluid used and the labeling time.
Chemistry
2-[(Tert-butyldiphenylsilyl)oxy]-1,4-dimethylphenol 1
2,5-Dimethylphenol (3.73 g, 30.5 mmol) and imidazole (2.1 g, 30.9 mmol) were
added to anhydrous dimethylformamide (DMF) (18 ml) under stirring at
228C. After 10 min, tert-butyldiphenylsilyl chloride (TBDPS chloride, 9.3 ml,
36.3 mmol) was added to this solution and stirring was continued for an
additional 16 h at this temperature. The TLC at this time showed complete
disappearance of the starting phenol and a new product appeared at a higher
Rf value. The reaction mixture was poured into water (200 ml) and the water
layer was extracted with methylene chloride (150 ml ꢁ 3). The combined
organic phase was washed with water (50 ml), dried over anhydrous sodium
sulfate (Na2SO4), filtered and evaporated. The impure residue was purified by
Copyright # 2005 John Wiley & Sons, Ltd.
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18F-LABELED STYRYL BENZENE DERIVATIVES
987
silica gel column chromatography using hexanes–ethyl acetate (20:1, v/v) as
eluent to afford colorless crystals (9.67 g, 87.9%) of 1; mp 47–488C; 1H-NMR
(CDCl3) d 7.79–7.76 (m, 4H, ArH of TBDPS), 7.50–7.41 (m, 6H, ArH of
TBDPS), 7.07 (d, J=7.6 Hz, 1H, H-500 of benzene), 6.64 (d, J=7.6 Hz, 1H, H-
600 of benzene), 6.28 (s, 1H, H-300 of benzene), 2.40 and 1.99 (two s, 6H, each
for 3H of two CH3), 1.16 (s, 9H, C(CH3)3); 13C-NMR (CDCl3) d 153.5, 136.0,
135.4, 133.2, 130.4, 129.7, 127.7, 125.1, 121.4, 119.3, 26.7 (s, C(CH3)3), 20.9
(s, CH3Ph), 19.7(s, C(CH3)3), 16.8 (s, CH3Ph); analytically calculated for
C24H28OSi: C, 79.95; H, 7.83. Found: C, 79.36; H, 8.02.
1-[(Tert-butyldiphenylsilyl)oxy]-2,5-bis(bromomethyl)benzene 2
Benzoyl peroxide (23.4 mg) was added to a solution of 1 (4.37 g, 11.7 mmol)
and N-bromosuccinimide (4.6 g, 25.8 mmol) in carbon tetrachloride (CCl4)
(26 ml) and the mixture was heated under reflux for 110 min. Afterwards, the
reaction mixture was cooled, filtered and the solvent was evaporated on a
rotary evaporator. The impure viscous residue was purified on a silica gel
column using hexanes–methylene chloride (100:3; v/v) as eluent to give a white
solid (3.0 g, 48.2%); mp 91–928C; 1H-NMR (CDCl3) d 7.77–7.72 (m, 4H, ArH
of TBDPS), 7.53–7.27 (m, 6H, ArH of TBDPS), 7.30 (d, J= 7.6 Hz, 1H, H-500
of benzene), 6.86 (d, J=7.6 Hz, 1H, H-600 of benzene), 6.40 (s, 1H, H-300 of
benzene), 4.70 (s, 2H, BrCH2), 4.05 (s, 2H, BrCH2), 1.17 (s, 9H, C(CH3)3);
13C-NMR (CDCl3) d 153.7, 139.2, 135.4, 135.3, 131.8, 131.6, 127.9, 121.6,
119.8, 118.6, 118.0, 32.6, 28.9, 26.5, 19.6; analytically calculated for
C24H26Br2OSi: C, 55.61; H, 5.06. Found: C, 53.95; H, 4.70.
1-[(Tert-butyldiphenylsilyl)oxy]-2,5-bis(diethylphosphonatomethyl)benzene 3
A mixture of 2 (1.0 g, 1.89 mmol) and triethylphosphite (0.63 g, 3.8 mmol) was
stirred and heated at 1608C for 4 h. The reaction mixture was cooled to 228C
and excess triethylphosphite was evaporated under reduced pressure. The
residue was purified by silica gel column chromatography using dichlor-
omethane–methanol (100:2; v/v) as eluent that afforded 3 as a yellow oil
1
(0.74 g, 61.0%); H-NMR (CDCl3d)- 7.72–7.68 (m, 4H, ArH of TBDPS),
7.44–7.31 (m, 6H of ArH of TBDPS), 7.29 (d, J=7.8 Hz, 1H, H-500 of
benzene), 6.79 (d, J=8.0 Hz, 1H, H-600 of benzene), 6.40 (s, 1H, H-300 of
benzene), 4.12–3.94 (m, 4H, 2 ꢁ OCH2), 3.79–3.58 (m, 4H, 2 ꢁ OCH2), 3.39 (d,
Jgem=22 Hz, 2H, PCH2), 2.67 (d, Jgem=22Hz, 2H, PCH2), 1.24 (t, J=6.9 Hz,
6H, 2 ꢁ CH2CH3),1.08 (s, 9H, C(CH3)3), 1.02 (t, J=6.9 Hz, 6H, 2 ꢁ CH2CH3);
13C-NMR (CDCl3d)- 153.0–153.2 (m), 135.4, 132.3, 131.2–131.1 (m), 129.9,
127.7, 122.4, 120.4, 120.2, 120.1, 61.8, ꢂ61.7, 33.27 (d, J=137.33 Hz, CH2P), 26.84
(d, J=140.62Hz, CH2P), 26.57 (CH3 of t-butyl)19.5, 16.49–16.26 (m); analytically
calculated for C32H49O7P2Si: C, 60.74; H, 7.33. Found: C, 59.56; H, 7.29.
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988
P. KUMAR ET AL.
2,5-Bis(40-methoxystyryl)phenol 4
Sodium hydride (0.92 g, 23 mmol, 60% in mineral oil) was added to a solution
of 3 (3.0 g, 4.65 mmol) in anhydrous diomethoxyethyl ether (10 ml) at 08C.
After stirring the mixture at 228C for 30 min, p-anisaldehyde (1.26 g,
9.26 mmol) was added to it and the stirring was continued for an additional
16 h at 858C. The reaction mixture was cooled and then poured into 50 ml of
water. The product was extracted in dichloromethane (150 ml ꢁ 3) and the
combined organic phase was washed with water (50 ml), dried over anhydrous
Na2SO4, filtered and evaporated. The impure product, so obtained, was
purified on a silica gel column using hexanes–ethyl acetate(8:1) as eluent that
1
gave 4 as a yellow solid (1.21 g, 73.8%); mp 238–2408C; H-NMR (CDCl3d)-
9.76 (br, 1H, OH), 7.81 (d, J=8.3 Hz, 2H, aromatics), 7.54 (d, J=8.2 Hz, 1H,
H-500 of benzene), 7.49–7.45 (two d, J=8.4Hz, 4H, ArH), 7.35 (s, 1H, H-300 of
benzene), 7.24 (d, J=8.0 Hz, 1H, H-600 of benzene), 7.21 (d, J=16.3, 2H,
CH ¼ CH), 7.11–6.92 (m, 6H, 4H, aromatics and 2H of CH ¼ CH), 3.77 (s,
3H, OCH3) and 3.40 (s, 3H, OCH3); 13C-NMR (CDCl3d)- 158.9, 154.7, 137.2,
130.4, 129.6, 127.1–127.8 (m), 126.3, 126.0, 123.4, 121.1, 117.6, 114.4, 113.2,
55.2; HR-MS (M+) m/z calculated for C24H22O3 358.15689, measured
358.15755.
1-(20-Bromoethoxy)-2,5-bis(40-methoxystyryl)benzene 5
Sodium hydride (22 mg, 0.55 mmol, 60% in mineral oil) was added to a
solution of 4 (100 mg, 0.28 mmol) in anhydrous DMF (1 ml) at 08C. The
temperature of the reaction mixture was slowly brought up to 228C and
stirring was continued for an additional 10 min. 1-Bromoethyltoluenesulfo-
nate18 (114 mg, 0.36 mmol) was added to this reaction mixture at this time and
the mixture was allowed to stir at this temperature for 48 h. Afterwards, the
reaction mixture was poured into cold water (15 ml) and the solution was
extracted with dichloromethane (20 ml ꢁ 3). The combined organic phase was
washed with water (10 ml), dried over anhydrous Na2SO4 and filtered. The
solvent was removed in vacuo and the residue was purified by silica gel column
chromatography using hexanes–ethyl acetate (9:1) as eluent to afford 5 as a
1
yellow solid (55 mg, 42.2%); mp 137–1408C; H-NMR (CDCl3)- 7.61 (d,
J=7.9 Hz, 1H, H-500 of benzene), 7.51 (two d, J=8.5 and 8.8 Hz, 4H, H-30 and
H-50 of two anisyl moieties), 7.35 (d, J=16.8 Hz, 1H, vinyl H), 7.30 (s, 1H, H-
300 of benzene), 7.26 (d, J=17.5 Hz, 1H, vinyl H), 7.22 (d, J=8.0 Hz, 1H, H-600
of benzene), 7.14 (d, J=15.9 Hz, 1H, vinyl H), 7.05 (d, J=16.5 Hz, 1H, vinyl
H), 6.95 (d, J=8.5 Hz, 4H, phenyl), 4.46 (t, J=5.1 Hz, 2H, CH2), 3.94 (t,
J=5.2 Hz, 2H, CH2), 3.77 (s, 6H, 2 ꢁ OCH3); HS-MS (M+) m/z calculated for
C26H25BrO3 464.098706, measured 464.09644.
Copyright # 2005 John Wiley & Sons, Ltd.
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18F-LABELED STYRYL BENZENE DERIVATIVES
989
1-(20-Toluenesulfonylethoxy)-2,5-bis(40-methoxystyryl)benzene 6
Sodium hydride (0.22 g, 5.5 mmol, 60% in mineral oil) was added to a solution
of 4 (0.95 g, 2.7 mmol) in anhydrous DMF (8 ml) at 08C. The temperature of
the reaction was slowly raised to 228C over a duration of 30 min and, then, 1,2-
ditoluenesulfonylethane19 (2.42 g, 6.5 mmol) was added to this reaction
mixture. The stirring was continued at this temperature for 4 days, and then
the reaction mixture was poured into cold water (100 ml). The water layer was
extracted with dichloromethane (150 ml ꢁ 3), the combined organic phase was
washed with water (100 ml) and dried over anhydrous Na2SO4. The solution
was filtered and the solvent was removed on a rotary evaporator. The impure
product was purified by silica gel column chromatography using hexanes–ethyl
acetate (9:1) as eluent that afforded a yellow solid. Recrystallization from
1
dichloromethane–hexanes gave 0.73 g (48.2%) of pure 6; mp 156–1598C; H-
NMR (CDCl3d)- 7.82 (d, J=8.2 Hz, 2H, H-2 and H-6 of toluene), 7.55 (d,
J=8.2 Hz, 1H, H-500 of benzene), 7.47–7.49 (m, 4H, aromatic), 7.26 (s, 1H, H-
300 of benzene), 7.24 (d, J=15.9 Hz, 1H, vinyl H), 7.11 (d, J=9.4 Hz, 1H, H-600
of benzene), 7.08 (d, J=16.8 Hz, 1H, vinyl H), 7.04 (d, J=16.8 Hz, 1H, vinyl
H), 6.92 (d, J=16.2 Hz, 1H, vinyl H), 6.94–6.89 (m, 6H, 4H0of anisyl and H-3
and H-5 of toluene) 4.48 (t, J=4.3Hz, 2H, CH2), 4.30 (t, J=4.3 Hz, 2H, CH2),
3.85, 3.83 (two s, 6H, 2 ꢁ OCH3), 2.37 (s, 3H, ArCH3), 2.37 (s, 3H, tolyl CH3);
13C-NMR (CDCl3d)- 159.3, 159.1, 155.3, 145.2, 137.7, 132.5, 132.3, 130.7,
129.9, 127.9, 126.4, 124.9, 121.0, 120.0, 118.9, 113.9, 110.0, 102.7, 66.7, 55.3,
21.7; LR-MS (MNa+) m/z calculated for C33H32O6S 579.18, measured 579.20.
1-(20-Fluoroethoxy)-2 5-bis(40-methoxystyryl)benzene 7
A solution of 6 (70 mg; 0.14 mmol) in anhydrous DMF (700 ml), methylene
chloride (700 ml) and acetonitrile (2 ml) was heated to 358C gently under
stirring and an inert atmosphere, and at this point, 1-ethyl-3-methylimidazo-
lium trifluoromethanesulfonate (700 ml; 3.7 mmol) was added to the reaction
mixture. The contents were stirred and transferred to the reaction vial
containing vacuum dried tetra-butyl ammonium fluoride (175 mg; 0.7 mmol).
The reaction flask was flushed with argon and the temperature of the reaction
vessel was raised to 908C. The heating was continued for 60 min and, then, the
solvent was removed by rotary evaporation. The impure product was
subjected to flash column chromatography using gradients of hexanes: ethyl
acetate, (starting with 3.5:1 and ending with 2:1 ratios) as eluent to obtain
4.6 mg (9%) of pure 7 as a semi solid along with unreacted precursor 6 (35 mg);
1H-NMR (CDCl3d)- 7.58 (d, J=7.9 Hz, 1H, ArH, H-500of benzene), 7.45 (two
d, J=7.9 Hz, 4H, ArH0 of anisyl moiety), 7.40 (d, J=16.5 Hz, 1H, vinyl H),
7.38 (s, 1H, H-300 of benzene), 7.14 (d, J=16.8 Hz, 1H, vinyl H), 7.07 (d,
J=16.5 Hz, 1H, vinyl H), 7.04 (d, J=9.2 Hz, 1H, H-600 of benzene), 7.02
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990
P. KUMAR ET AL.
(d, J=16.8 Hz, 1H, vinyl H), 6.91 (d, 4H, aromatic), 4.83 (d, JFꢂCH ¼ 47:3 Hz,
2
of d, Jgem=10.0 Hz, of t, JCH ꢂCH ¼ 4:3 Hz, 1H, CH2F), 4.58 (d,
2
2
JFꢂCH ¼ 47:3 Hz, of d, Jgem=10.0 Hz, of t, JCH ꢂCH ¼ 3:7 Hz, 1H, CH2F),
2
2
2
4.36 (d, JFꢂOCH ¼ 29:3 Hz, of d, Jgem=10.0 Hz, of t, JCH ꢂCH ¼ 3:7 Hz,
2
2
2
1H, OCH2), 4.28 (d, JFꢂOCH ¼ 23:9 Hz, of d, Jgem=10.0 Hz, of t,
2
JCH ꢂCH ¼ 4:0 Hz, 2H, OCH2), 3.85 (s, 6H, 2 ꢁ OCH3); 19F NMR
2
2
(CDCl3d+C6F6)- 25.58 (dddd, JCH ꢂF ¼ 47:3 Hz, JOCH ꢂF ¼ 24:0 and
2
2
29.3 Hz); 13C-NMR (CDCl3d)- 159.3, 159.1, 155.3, 145.2, 137.7, 132.5, 132.3,
130.7, 129.9, 127.9, 126.4, 124.9, 121.0, 120.0, 118.9, 113.9, 110.0, 102.7, 80.5
(d, JFꢂCH ¼ 173:6 Hz, CH2F), 68.36 (d, JFꢂOCH ¼ 20:9 Hz, OCH2), 66.7,
2
2
55.3, 21.7; EI-MS (M+) m/z calculated for C26H25FO3 404.1787, measured
404.1776 (100%).
Radiochemistry: automated radiosynthesis of 1-(20-[18F]-fluoroethoxy)-
2,5-bis(40-methoxystyryl) benzene [18F]-FESB
The synthesis of 18F-FESB was carried out in the ASU housed in a
commercially available (Comecer, Castelbolognese, Italy) radioisotope iso-
lator unit designed to confer a Class 100 operating environment during ASU
operation. The inlet and exhaust lines of the ASU are attached to nitrogen.
The inlet pressure of nitrogen is regulated at 5–8 psi during the synthesis that
in conjunction with the evacuation system controls the movement of reagents
and maintains inert atmosphere during the synthesis. Time-controlled selective
valves are operated by the Lookout for Win32 Version 4.5.1 software that is
provided by National Instruments, USA. All required reagents were installed
onto the ASU prior to the start of the synthesis. Acetonitrile, eluent (K-2.2.2/
K2CO3, 22 mg/7 mg dissolved in 0.8 ml, 56:44, v/v, acetonitrile/water solution),
tosylate precursor (3.8–5.0 mg, 6.8–9 mmol) pre-dissolved in 2 ml acetonitrile
and a solution of ethanol/water (50:50, 1.5 ml) were prepared in color coded
crimped vials that were then placed at their respective points of attachment in
the ASU. The reactor vial was placed on the heating block and the vacuum
pump and the trap were attached to the ASU. The synthesis was initiated after
confirming all necessary prerequisites for the radiosynthesis.
The currently employed radiochemical method for the synthesis of 18F-
FESB was optimized and adapted for automated radiopharmaceutical
processing. No-carrier-added (NCA) radiofluoride was first adsorbed from
its H128O target solution onto the (K2CO3 conditioned) anion exchange resin
(QMA) and H128O (ꢀ98%) was recovered for potential future reprocessing
and re-use. This was followed by adding eluent (K-2.2.2, 22 mg and potassium
carbonate, 7 mg in 0.45 ml acetonitrile and 0.35 ml water) onto the QMA
cartridge to elute K18F from the resin rapidly, at room temperature, into the
reaction vial. Using preset heat (958C), vacuum (0.8 bar) conditions and
nitrogen carrier gas flow (5–8 psi, 20–30 ml/min), the solvent was removed.
Copyright # 2005 John Wiley & Sons, Ltd.
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18F-LABELED STYRYL BENZENE DERIVATIVES
991
Two azeotropic distillations using repeated additions of dry acetonitrile (1 ml
each) were used to remove the residual traces of water. At the end of this
process, tosylate precursor, pre-dissolved in anhydrous acetonitrile (2 ml) and
BmimBF4 (20–50 ml) or EmimTFMS (50 ml) was added to the reaction vial.
Nucleophilic substitution of the tosylate by radiofluoride (potassium–
Kryptofix–radiofluoride complex) in anhydrous acetonitrile at 858C, depend-
ing on the nature and amount of the ionic fluid and the reaction time, afforded
radiofluorinated FESB (18F-FESB) in 17–76% radiochemical yield
(Scheme 1). The impure 18F-FESB reaction mixture was dissolved in 1.0 ml
OH
CH3
OTBDPS
CH3
OTBDPS
CH2Br
ii
i
H3C
CH3
BrH2C
1
3
2
iii
OH
o
o
(C2H5O)2PH2C
CH2P(OC2H5)2
iv
OH
H CO
OCH3
3
4
v
vi
OCH2CH2OTs
OCH2CH2Br
H3CO
OCH3
OCH3
H3CO
5
6
vii
viii
OCH2CH218F
OCH2CH2F
H3CO
OCH3
OCH3
H3CO
8
7
i=TBDPSCl, Imidazole; ii=NBS/CCl4;
H ) ; iv=NaH/p-anisaldehyde;
5 3
iii=P(OC2
v=NaH/p-O2NC6H4O2SOCH2CH2Br;
Bu4NF/90 C/60 min and viii = ionic fluid/K F/K-2.2.2./85 C/8−12 min
vi=(CH2OSO2C6H4CH3)2;
vii=Emimtriflate/
18
°
°
Scheme 1. Synthesis route to 1-(20-fluoroethoxy)-2,5-bis(40-methoxystyryl)ben-
zene (FESB), 7, and 18F-FESB, 8
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 983–996

992
P. KUMAR ET AL.
of 50% aqueous ethanol and then dispensed from the ASU into a shielded
product vial.
This radiofluorinated reaction mixture solution, dispensed from the ASU,
was injected onto HPLC and purified using ethanol–water (75:25, v/v) as an
eluent at a flow rate of 1.5 ml/min. Pure 18F-FESB, eluted from the HPLC
column at a retention time of 19.5 min (>90% purity; specific activity 16.5–
81.2 GBq or 0.45–2.19 Ci/mmol) and was collected in a sterile multi-dose vial
(20 ml) containing sterile saline (10 ml) after passing through a sterile neutral
alumina cartridge. Depending on the nature of ionic fluid and the reaction
time used in the fluorination 18F-FESB was isolated with specific activity
ranging between 16.5 and 81.2 GBq/mmol. The results are presented in Table 1.
Results and discussion
The synthesis of 1-(20-fluoroethoxy)-2,5-bis(40-methoxystyryl)benzene 7 and its
tosylate precursor started with 1-O-tert-butyldiphenylsilyl-2,5-dimethylphenol
1 (Scheme 1). 2,5-Dimethylphenol was silylated with tert-butyldiphenylsilane
in anhydrous pyridine since it withstands the hard acidic conditions that exist
during the bromination step. Bromination of the methyl groups at C-2 and C-
5, using N-bromosuccinimide, proceeded through free a radical mechanism
that was assisted by benzoyl peroxide, to afford 1-[(tert-butyldiphenylsily-
l)oxy]-2,5-bis(bromomethyl)benzene 2 in 48% yield. Reaction of the bromo-
methyl product
2
at 858C, with triethylphosphite gave 1-[(tert-
butyldiphenylsilyl)oxy]-2,5-bis(diethylphosphonatomethyl)benzene 3 in satis-
factory yield (61%). The formation of 3 was supported by observing a strong
phosphorous coupling (JPꢂCH ¼ 137:32140:6 Hz) with methylenic carbons in
2
the 13C NMR spectrum. The conversion of this diethylphosphonate derivative
3 to the corresponding 2,5-bis(4-methoxystyryl)phenol, 4, progressed smoothly
(74% yield). This reaction required strongly basic conditions and, therefore,
led to desilylation of the molecule, rendering the hydroxyl group at C-1
aromatic position of the benzene unprotected and free for substitution with an
alkyl chain. The formation of the 2,5-bis styryl linkage was confirmed by the
resonances from the newly formed vinyl protons. The introduction of the
toluene sulfonyloxyethyl chain at the 10-OH group was initially attempted by
condensing 4 with 1-bromoethyltoluenesulfonate in the presence of sodium
hydride, but this led to condensation between toluenesulfonyl and the OH
moiety, resulting in the formation of 1-(20-bromoethoxy)-2,5-bis(40-methox-
ystyryl)benzene 5 (42%). To overcome the formation of this unexpected
product, 1,2-bis-toluenesulfonyloxyethane was used as tosylating reagent.
Tosylation of the free hydroxyl group using 1,2-bis-toluenesulfonyloxyethane
in anhydrous DMF afforded 1-(20-toluenesulfonylethoxy)-2,5-bis(40-methox-
ystyryl)benzene 6 as a yellow solid in 48% yield.
Copyright # 2005 John Wiley & Sons, Ltd.
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18F-LABELED STYRYL BENZENE DERIVATIVES
993
The synthesis of 1-(20-fluoroethoxy)-2, 5-bis(40-methoxystyryl)benzene 7 and
its radiofluorinated 18F-analog 8 started from 6 and involved nucleophilic
substitution of the toluenesulfonyl group in 6 with fluoride. The tosylate 6, on
reaction with tetrabutyl ammonium fluoride at 1008C, gave 1-(20-fluor-
oethoxy)-2,5-bis(40-methoxystyryl)benzene 7 (9%). It was observed that more
than half of the tosylated precursor did not undergo fluorination and was
recovered during the chromatographic purification of 7. The relatively low
chemical yield for 7 may also partially be due to the formation of 1-
fluoroethyltoluenesulfonate that was present as a secondary fluorinated
product. The formation of this side product could be due to competitive
fluorination at a-ethyl carbon of the ethylsulfonate chain. Continuous elec-
tronic delocalization resonances associated with 30-O would exert a strong –I
effect and might be a potential factor for this secondary fluorination.
Substitution of fluorine in 7 is further evident by F–H (47.3 Hz) and F–C
1
(173.6 Hz) coupling constants in its H and 13C NMR spectra. 19F NMR
spectra also displayed a dddd at a chemical shift of d 25.58 with desired F–H
coupling constants that collapsed upon proton decoupling. Fluorine coupling
with 3-ethoxy protons and carbon was also observed in related spectra.
The synthesis of 18F-labeled 8 was initially attempted using the conventional
Kryptofix 2.2.2/K18F complex at elevated temperatures, but this provided only
partial success. Recent reports on ionic fluid-assisted radiofluorination20,21
indicate that a significant increase in the reactivity of fluoride ion could be
expected due to unmasking of the fluoride ion that is most likely a factor for
poor labeling efficiency in radiofluorination reactions. Incorporation of an
appropriate ionic fluid in our radiofluorination reactions supported this
observation and a significant improvement in the labeling efficiency of 18F-
FESB was achieved (Table 1). The radiochromatogram of radiofluorinated
mixture (Figure 1) featured a number of radioactive peaks that eluted between
7 and 10 min, in addition to 18F-FESB at 19.5 min. The retention times for
Emimtriflate and Bmimtetrafluoroborate are 7.3 and 6.9 min, respectively,
therefore, the radioactive peaks appearing between 7 and 10 min in the
radiochromatogram of the labeled 18F-FESB mixture correspond, most likely,
to unreacted fluoride, and radiofluorinated BmimBF4 or EmimTFMS salts
formed due to the exchange of radioactive fluorine with ionic fluid. An
uncharacterized radioactive component appearing between 11 and 18 min is
visible as a hump in the chromatogram (Figure 1) but the purity of 18F-FESB
is not compromised during HPLC purification of the reaction mixture by
collecting 18F-FESB after 30 s of the peak start. Normally, fluoride ion acts as
a strong base that initiates elimination of the leaving (tosylate) group, and is
‘masked’, and therefore is a weak nucleophile.22 The competition of these
competing reactions results in relatively low radiochemical yield of the desired
product. We observed that the radiofluorination of 6 to form 8 proceeded in
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 983–996

994
P. KUMAR ET AL.
0.20
0.15
0.10
0.05
0.00
Retention Time
Area Percent
5
10
15
20
25
30
0
Minutes
Figure 1. HPLC elution profiles of 18F-FESB on a Partisil 10 ODS-3 reversed
phase column using ethanol:water (75:25; 1.5 ml/min) as eluent. 18F-FESB
appears at a retention time of 19.5 min
low radiochemical yield (3–7%), but the use of BmimBF4 or EmimTFMS as
ionic solvent during radiofluorination increased the radiochemical yield to
76%. It was also observed that 18F-FESB was obtained in better radio-
chemical yields with higher specific activity when the radiofluorination was
assisted by BmimBF4 (reactions 2 and 3 compared with reactions 4–6, Table 1)
indicating that during the radiofluorination BmimBF4 presumably undergoes
less radiolysis in comparison to EmimTFMS. This radiolysis is further
supported by the fact that use of a larger amount of ionic fluid led to 18F-
FESB with lower specific activities (compare reaction 2 with 3–6). Also, an
optimum labeling time was found to be 8 min for this product since a longer
reaction time led to possible decomposition resulting in to lower radiochemical
yield (reaction 1, Table 1).
Conclusion
The synthesis of 18F-FESB, a fluorinated Congo Red derivative, has been
developed using an ASU. It was observed that use of ‘ionic fluids’ in the
radiochemical synthesis significantly enhanced the labeling efficiency of the
product. An attempt to correlate radiochemical yield of 8 to varying amounts
of the ionic fluids BmimBF4 or EmimTFMS were inconclusive. The biological
properties of 18F-FESB are currently under investigation.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 983–996

18F-LABELED STYRYL BENZENE DERIVATIVES
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Acknowledgements
The authors sincerely acknowledge the financial support from University of
Alberta Foundation, Edmonton, Canada (grant # G533000094) and opera-
tional spectral help from Dr V.V. Somayaji. Synthesis and technical help in the
operation of the ASU by Mr Courtney Doerkson and Ms Ingrid Koslowski is
highly appreciated.
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