Copper-mediated nucleophilic radiofluorination of [18F]β-CFT for positron emission tomography imaging of dopamine transporter

Article abstract of DOI:10.1002/jlcr.3905

[¹⁸F]β-CFT is a positron emission tomography (PET) ligand for imaging of dopamine transporter. It was proved to be a sensitive PET marker to detect presynaptic dopaminergic hypofunction in Parkinson's disease. In recent years, copper-mediated ¹⁸F-fluorination of aryl boronic esters has been successful in some molecules containing aromatic groups. In this study, we describe the novel synthetic strategy of [¹⁸F]β-CFT by copper-mediated nucleophilic radiofluorination with pinacol-derived aryl boronic esters upon reaction with [¹⁸F]KF/K₂₂₂ and Cu (OTf)₂(py)₄. The radiolabeling protocol was optimized with [¹⁸F]fluoride elution method and amount of copper catalyst used. [¹⁸F]β-CFT is obtained from boronic ester precursors in 2.2% to 10.6% non-isolated radiochemical yield (RCY). Purified [¹⁸F]β-CFT with >99% radiochemical purity (RCP) and high molar activity was obtained in validation runs. The radiolabeling procedure is straightforward and can easily be adapted for clinical use.

Full text of DOI:10.1002/jlcr.3905

Received: 29 October 2020
DOI: 10.1002/jlcr.3905
Revised: 28 January 2021
Accepted: 29 January 2021
R E S E A R C H A R T I C L E
Copper-mediated nucleophilic radiofluorination of [18F]β-
CFT for positron emission tomography imaging of
dopamine transporter
Hung-Man Yu1
Yu Chang2
| Ching-Yun Li2 | Shiu-Wen Liu2 | Chun-Hung Yang1
|
1Isotope Application Division, Institute of
Nuclear Energy Research, Taoyuan City,
Taiwan
2Chemistry Division, Institute of Nuclear
Energy Research, Taoyuan City, Taiwan
[18F]β-CFT is a positron emission tomography (PET) ligand for imaging of
dopamine transporter. It was proved to be a sensitive PET marker to detect
presynaptic dopaminergic hypofunction in Parkinson's disease. In recent years,
copper-mediated 18F-fluorination of aryl boronic esters has been successful in
some molecules containing aromatic groups. In this study, we describe the
novel synthetic strategy of [18F]β-CFT by copper-mediated nucleophilic radio-
fluorination with pinacol-derived aryl boronic esters upon reaction with [18F]
KF/K222 and Cu (OTf)2(py)4. The radiolabeling protocol was optimized with
[18F]fluoride elution method and amount of copper catalyst used. [18F]β-CFT
is obtained from boronic ester precursors in 2.2% to 10.6% non-isolated radio-
chemical yield (RCY). Purified [18F]β-CFT with >99% radiochemical purity
(RCP) and high molar activity was obtained in validation runs. The radio-
labeling procedure is straightforward and can easily be adapted for
clinical use.
Correspondence
Hung-Man Yu, Isotope Application
Division, Institute of Nuclear Energy
Research, Taoyuan City, Taiwan.
Email: hwyu@iner.gov.tw
Funding information
Atomic Energy Council of Taiwan, Grant/
Award Number: MOST 108-3111-Y-042A-
118
K E Y W O R D S
boronic pinacol esters, copper-mediated, dopamine transporter, fluorine-18, radiochemistry
1 | INTRODUCTION
fluorine in drug design,13–15 PET also has become a pow-
erful and useful tool to facilitate drug discovery and
The first medical imaging applying positron annihilation
radiation1 and the development of the first clinical posi-
tron emission tomography (PET) scanner were mile-
stones in the development of PET imaging.2 Further
technological developments combined with the appear-
ance of radiopharmaceuticals, perhaps most frequently
used 2-[18F]fluoro-2-deoxy-d-glucose,3 quickly established
PET as a non-invasive imaging modality for measure-
ment and quantification of in vivo biochemical processes.
At the present time, PET is used routinely for clinical
diagnosis of cancers,4–6 neurological disorders,7–9 and
cardiovascular diseases.10–12 Due to the favorable proper-
ties of 18F as a positron emitter and the prevalence of
development.16–18 These utilizations and applications
have created a large demand for new radiochemistry
focusing on 18F-radiofluorination.19–21
In the past two decades, the number of methods for
incorporating fluorine into aromatic hydrocarbons has
continuously increased as these motifs are commonly
found in pharmaceuticals to impact drug potency.22–24
On the other hand, the synthesis of 18F-labeled aromatic
hydrocarbons that are not accomplishable by aromatic
nucleophilic substitution (SNAr) with [18F]fluoride
remains strenuous.25,26 A number of metal-mediated
reactions have recently emerged for the direct 18F fluori-
nation of aromatic hydrocarbons not suitable for
228
© 2021 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jlcr
J Label Compd Radiopharm. 2021;64:228–236.

YU ET AL.
229
SNAr.27–31 However, these metal-based precursors and
complexes are generally not commercially available and
are not easy to prepare by non-chemist specialists. More-
over, development of automated synthetic procedures is
required in compliance with good manufacturing prac-
tice (GMP). These characteristics and situations encour-
aged the development of easier and faster direct
nucleophilic 18F fluorination of aromatic hydrocarbons
from more applicable and stable materials.
[3H]β-CFT binding sites in the striata of brains of patients
with Parkinson's disease,41 studies of β-CFT were focused
on in vivo PET imaging of dopamine transporter.
After β-CFT had been successfully labeled with
11C,42,43 preclinical animal44–46 and clinical human45,47
PET studies have been evaluated with [11C]β-CFT. Never-
theless, these studies showed that no equilibrium was
reached between target and non-target tissue during PET
imaging due to the main disadvantage of short half-life
(20.4 min) of 11C. These results of [11C]β-CFT encouraged
the further studies to labeled β-CFT with 18F for PET
imaging of dopamine transporter.48 PET studies of [18F]β-
CFT showed that [18F]β-CFT is a sensitive ligand of pre-
synaptic dopaminergic hypofunction in Parkinson's dis-
ease and can be used in the diagnosis of disease severity
of patients.49–53
Recently, a novel method of nucleophilic 18F fluorina-
tion of pinacol-derived aryl boronic esters with [18F]KF/
Kryptofix 222 in the presence of the commercially avail-
able copper complex [Cu (OTf)2(py)4] (OTf = trifluoro
methanesulfonate, py = pyridine) was reported.32–34 This
method is characterized by high tolerance to a broad
range of substrates (electron-poor/rich arenes and vari-
ous functional groups) and its direct synthesis of radio-
pharmaceuticals from [18F]KF, and stable and easily
prepared precursors.
A series of cocaine analogs lacking the ester group at
the C-3 carbon atom and containing para substitutions
on the phenyl ring was synthesized by Clarke et al. in
1973.35 Two of these compounds had higher binding
affinity to the dopamine reuptake site than cocaine itself.
Spealman et al. showed that 2-β-carbomethoxy-3-β-
(4-fluorophenyl)tropane (β-CFT, WIN 35,428) had potent
cocaine like activity in vivo in further studies.36 Later, the
specific binding ratio of β-CFT was measured with the
3H-labeled β-CFT.37–39 [3H]β-CFT was considered to be a
promising radioligand to study the dopamine reuptake
site due to its high affinity (IC50 = 11 nM for dopamine)
and its good selectivity for the dopamine transporter
(affinity ratio of 5HTDA = 151).40,41 Since postmortem
studies showed that there is an apparent decrease of
The conventional synthesis of [18F]β-CFT involves an
electrophilic substitution with [l8F]acetylhypofluorite as
the fluorinating agent derived from 18F2 gas (Scheme 1).
The difficulty of generating and handling 18F2 gas and
the large amount of 19F-β-CFT carrier that is formed dur-
ing the electrophilic reaction are major disadvantages.
These situations led us to develop nucleophilic substitu-
tion protocols for the production of [18F]β-CFT using no-
carrier-added [18F]fluoride. In this article, we presented
the radiolabeling of [18F]β-CFT from a boronic ester pre-
cursor (Scheme 1).
2 | MATERIALS AND METHODS
All chemicals and solvents were purchased from Sigma-
Aldrich (St. Louis, MO, USA), Acros Organic (Geel,
Belgium), Alfa Aesar (Ward Hill, MA, USA), Mallinckrodt
SCHEME 1 Synthesis of β-CBT, β-CFT,
and [18F]β-CFT

230
YU ET AL.
Chemicals (St. Louis, MO, USA), or Merck (Darmstadt,
Germany) and used without further purification. nuclear
magnetic resonance (NMR) spectra were recorded on a
Varian Mercury Plus 300 MHz Spectrometer. High resolu-
tion mass spectra (HRMS) were recorded on a Thermo
LTQ Orbitrap XL® ETD Hybrid Ion Trap-Orbitrap mass
spectrometer (San Jose, CA, USA). Infrared spectra were
recorded using a Varian FTS 800 FT-IR spectrometer. [18F]
fluoride was obtained from a 30 MeV TR30/15 cyclotron at
Institute of Nuclear Energy Research (INER). For quality
control of [18F]β-CFT, a reversed-phase high-performance
liquid chromatographic (RP-HPLC) system was used,
including a Thermo UltiMate® 3000 pump, a Thermo
UltiMate® 3000 variable wavelength UV/visible detector
operating at 254 nm, and a LabLogic Flow-RAM NaI/PMT
radioactivity detector. The radioactivity was measured
with a dose calibrator (CRC®-25R, Capintec). An imaging
scanner (AR-2000; Bioscan, Washington, DC, USA) was
used to scan thin-layer chromatography plates (aluminum
silica gel 60G F254 plate, MERCK) and to analyze the
chromatograms.
concentrated under reduced pressure to afford 14.44 g
(80.3 mmol) of (R)-(-)-anhydroecgonine methyl ester 2,
(93% yield). IR (KBr): ν 1730 (C=O) cm−1 1H NMR
.
(CDCl3): δ 6.81 (m, 1H), 3.98 (d, J = 5.6 Hz, 1H), 3.94 (s,
3H, OCH3), 3.45 (m, 1H), 2.83 (d, br, J = 19.8 Hz, 1H),
2.54 (s, 3H, NCH3), 2.36 (m, 2H), 2.04 (m, 2H), 1.72 (m,
1H). 13C NMR (CDCl3): δ 173.1 (C=O) 166.1, 135.8, 58.1,
56.3, 51.1 (OCH3), 36.0 (NCH3), 34.0, 31.7, 29.9.
To a solution containing 4-chlorophenyl magnesium
bromide 1 M solution/diethyl ether (Grignard reagent,
160 ml, 160 mmol) in anhydrous dichloromethane
(950 ml), compound 2 (14 g, 78 mmol) in anhydrous dic-
hloromethane (50 ml) was added dropwise at −50ꢀC
under nitrogen atmosphere; the mixture was stirred for
3 h and then cool to −78ꢀC. Trifluoroacetic acid (12.3 ml,
0.160 mol) and anhydrous dichloromethane (25 ml) were
added and stirred for 30 min. The mixture was return to
room temperature after stirring. Pure water (about 1 L)
was added and the mixture was acidified with
hydrochloric acid to pH 1–2. The pH of aqueous phase
was adjusted to 11–12 with saturated sodium hydroxide
solution (3 × 200 ml). Then the aqueous phase was
extracted with ethyl acetate (3 × 1 L). The organic phase
was dried with anhydrous sodium sulfate and concen-
trated under reduced pressure to obtain the crude prod-
uct which was then purified by liquid column
2.1 | Synthesis of 2-carbomethoxy-
3-(4-pinacolborylphenyl)tropane 4 (β-CBT)
Cocaine hydrochloride (30 g, 89 mmol) in 0.8 N HCl
(500 ml) was refluxed at 105ꢀC for 20 h. Then the mixture
was stirred at 0ꢀC for 30 m and was extracted with ether
(3 × 500 ml). The aqueous phase was concentrated under
reduced pressure at 80ꢀC, and then washed with CH2Cl2
and filtered to afford 19.26 g (87.6 mmol) of ecgonine
hydrochloride 1 as white solid product (98% yield). IR
chromatography (SiO2, EtOAc) to afford 10.16 g
(34.7 mmol) of 2β-carbomethoxy-3β-(4-chloropenyl)
1
tropane 3 (44% yield). IR (KBr) ν 1732 (C=O) cm−1. H
NMR (CDCl3): δ 1.64 (m, 4H, two–CH2), 2.20 (t, 2H,
N=C-CH2), 3.10 (t, 2H, C=N-CH2), 3.35 (dd, 2H, CH2),
3.82 (s, 3H, OCH3), 3.95 (t, 2H, CH2), 4.00 (dd, 2H, CH2),
4.12 (t, 2H, CH2), 6.62 (s, 1H), 6.75 (d, 1H), 7.55 (d, 1H),
7.70 (dd, 1H), 8.15 (d, 1H), 8.63 (d, 1H), 9.15 (d, 2H). 13C
NMR (CD3OD): δ 173.5, 166.5, 163.1, 151.0, 148.9, 146.1,
145.5, 134.2, 127.8, 127.3, 117.3, 108.1, 99.7, 71.3, 61.4,
56.7, 45.9, 25.4, 20.8.
(KBr): ν 3284 (O-H), 1701 (C=O) cm−1 1H NMR
.
(CD3OD): δ 4.35 (m, 1H), 4.10 (d, 1H), 3.88 (m, 1H), 3.15
(dd, 1H), 2.82 (s, 3H, NCH3), 2.36 (m, 2H), 2.10 (m, 3H).
13C NMR (CD3OD): δ 176.8 (C=O), 65.8, 64.7, 61.5, 49.0,
39.2 (NCH3), 36.9, 24.9, 24.2.
Compound 3 (4 g, 13.65 mmol), bis (pinacolato)
Ecgonine hydrochloride 1 (19 g, 86.4 mmol) was
added to phosphoryl chloride (100 ml) and heat to reflux
at 110ꢀC for 3 h. Excess POCl3 was evaporated under
reduced pressure, then the mixture was dried under vac-
uum for 24 h. The dried intermediate product was added
to anhydrous CH3OH (100 ml) in ice bath. The mixture
was stirred at room temperature for 5 h. Excess CH3OH
was then evaporated under reduced pressure. The resid-
ual solid was dissolved with water (100 ml) and the pH
was adjusted to 7 with 1 N sodium hydroxide solution.
The mixture was extracted with ether (3 × 100 ml). The
pH of resulting aqueous phase was adjusted to 10–12
with 1 N sodium hydroxide solution. The aqueous phase
was extracted with ether again (3 × 100 ml). The organic
phase was dried with anhydrous sodium sulfate and was
diboron
(B2Pin2,
10
g,
39.38
mmol),
tris
(dibenzylideneacetone)dipalladium(0) (Pd2dba3, 0.25 g,
0.25 mmol), 2-dicyclohexylphosphino-20,40,60-triisopropyl
biphenyl (XPhos, 0.26 g, 0.55 mmol) and potassium ace-
tate (4 g, 40.95 mmol), were dissolved in anhydrous
1,4-dioxyl biphenyl (30 ml). The reaction mixture was
refluxed for 3 h at 110ꢀC and then concentrated under
reduced pressure. The concentrated solution was dis-
solved in dichloromethane, and then filtered with celite.
The filtrate was washed with water 3 times. The organic
phase was dried with anhydrous sodium sulfate and con-
centrated under reduced pressure to obtain the crude
product which was then purified by liquid column chro-
matography (SiO2, DCM/EA = 5/1) to obtain 0.56 g
(7.34 mmol) of pinacol boronate ester 4 (β-CBT, 54%

YU ET AL.
231
yield). IR (KBr) ν 1746 (C=O) cm−1. H NMR (CDCl3): δ
7.78 (d, 2H, C6H4), 7.32 (d, 2H, C6H4), 3.59 (m, 1H,
NCH), 3.43 (s, 3H, OCH3), 3.35 (m, 1H, NCH), 3.01 (td,
1H, CHC6H4), 2.96 (dd, 1H, CHCO2CH3), 2.58 (td, 1H,
CH2CHC6H4), 2.21 (s, 3H, NCH3), 2.20–1.96 (m, 2H,
CH2CH2), 1.78–1.48 (m, 3H, CH2CH2), 1.34 (s, 12H, BOC
(CH3)2). 13C NMR (CDCl3) δ 172.8 (C=O), 147.2, 135.2,
127.3 and 84.3 (C6H4), 66.0 and 62.9 (NCH), 53.4
(CHCO2), 51.9 (CO2CH3), 42.1 (NCH3), 34.5 (CHC6H4),
30.4 (CH2CHC6H4), 26.6 and 25.8 (CH2CH2), 25.6 (BOC
(CH3)2), 25.5 (BOC (CH3)2). HRMS-ESI: m/z calcd. For
C22H32BNO4 [M + H] 386.24., found 386.22.
enriched [18O]water target (95% enrichment) using a
TR30/15 cyclotron at INER. [18F]fluoride was trapped in
an 18F anion exchange cartridge (Chromabond® PS-
HCO3−, Macherey-Nagel) from a sealed glass vial con-
taining the aqueous [18F]fluoride (3.7 GBq, 1.0 ml). The
activity was eluted to a glass vial with 1 ml 85% acetoni-
trile containing 8.0 mg (21.25 μmol) of Kryptofix 222 and
2.6 mg (18.81 μmol) of potassium carbonate. Portion of
[18F]fluoride solution (0.37 GBq, 0.1 ml) was transferred
to a glass reaction vial. The solvent was evaporated under
a stream of nitrogen at 90ꢀC. Then azeotropic drying was
repeated three times with 1 ml of anhydrous acetonitrile
under the same conditions. After complete drying of [18F]
fluoride complex, 6 mg (15.6 μmol) of boronic ester pre-
cursor and various amounts of Cu (OTf)2(py)4 (3.6 mg,
5.3 μmol) in 1.0 ml of anhydrous dimethylformamide
(DMF) was added to the reaction vial by using syringe
and heated at 110ꢀC for 20 min. Then H2O was added
and the reaction mixture was cooled to room
temperature.
1
2.2 | Synthesis of 2β-Carbomethoxy-3β-
(4-fluorophenyl)tropane 5 (β-CFT)
To a solution containing 4-fluorophenyl magnesium bro-
mide 1 M solution/diethyl ether (Grignard reagent,
5.8 ml, 30 mmol) in anhydrous dichloromethane
(250 ml), compound 2 (2.75 g, 15 mmol) in anhydrous
dichloromethane (50 ml) was added dropwise at −50ꢀC
under nitrogen atmosphere; the mixture was stirred for
3 h and then cool to −78ꢀC. Trifluoroacetic acid (2.42 ml,
30 mmol) and anhydrous dichloromethane (10 ml) were
added and stirred for 30 min. The mixture was return to
room temperature after stirring. Pure water (about 1 L)
was added and the mixture was acidified with
hydrochloric acid to pH 1–2. The pH of aqueous phase
was adjusted to 11–12 with saturated sodium hydroxide
solution (3 × 200 ml). Then the aqueous phase was
extracted with ethyl acetate (3 × 1 L). The organic phase
was dried with anhydrous sodium sulfate and concen-
trated under reduced pressure to obtain the crude prod-
uct which was then purified by liquid column
chromatography (SiO2, EtOAc/CH3OH = 95/5) to afford
1.1 g of compound 5 (36% yield). IR (KBr): ν 1732
(CO) cm−1. 1H NMR (CDCl3): δ 7.17 (dd, 2H, C6H4), 7.32
(dd, 2H, C6H4), 3.56 (m, 1H, C3-H), 3.50 (s, 3H, C16-H3),
3.35 (m, 1H, C6-H), 2.96 (m, 1H, C8-H), 2.87 (m, 1H, C2-
H), 2.55 (m, 1H, C7-Ha), 2.22 (s, 3H, C15-H3), 2.15 (m,
2H, C4-Ha and C5-Ha), 1.63 (m, 3H, C4-Hb, C5-Hb and C7-
Hb). 13C NMR (CDCl3): δ 172.7 (CO), 163.3, 160.13, 139.2,
129.5 (C6H4) and 115.4 (CF), 65.9, 62.9, 53.5, 51.8
(OCH3), 42.6 (NCH3), 34.8, 33.9, 26.55, 25.8. HRMS-ESI:
m/z calcd. For C16H20FNO2 [M+] 277.15, found 277.32.
2.3.2 | Low base procedure
[18F]fluoride was trapped in an anion exchange resin car-
tridge (Waters Sep-Pak Accell Plus QMA cartridge,
WAT023525, pre-conditioned with 10 ml 1 N sodium
bicarbonate and 10 ml H2O) from a sealed glass vial con-
taining the aqueous [18F]fluoride (3.7–18.5 GBq, 1–2 ml).
The activity was eluted to a glass vial with 1 ml 80% ace-
tonitrile containing 0.57 mg (1.51 μmol) of Kryptofix
222 and 0.12 mg (0.87 μmol) of potassium carbonate. Por-
tion of [18F]fluoride solution (0.37–5.55 GBq, 0.2 ml) was
transferred to a glass reaction vial. The solvent was evap-
orated under a stream of nitrogen at 90ꢀC. Then
azeotropic drying was repeated three times with 1 ml of
anhydrous acetonitrile under the same conditions. After
complete drying of [18F]fluoride complex, 6/12 mg
(15.6/31.2 μmol) of boronic ester precursor and various
amounts of Cu (OTf)2(py)4 (1.5–5.9 mg, 2.2–8.7 μmol) in
0.5 or 1.0 ml of anhydrous DMF was added to the reac-
tion vial by using syringe and heated at 110ꢀC for 20 min.
Then H2O was added and the reaction mixture was
cooled to room temperature.
2.4 | Purification and analysis of
[18F]β-CFT
2.3 | Radiosynthesis of [18F]β-CFT
2.3.1 | High base procedure
The quenched reaction mixture was used to determine
non-isolated RCY. Non-isolated RCYs were determined
by integrating the area under the curve in the radio-TLC
chromatogram. Radiochemical yield of the prepared
No-carrier-added [18F]F− was produced via the 18O
(p,n)18F reaction by bombardment of an isotopically

232
YU ET AL.
[18F]β-CFT was estimated by radio-TLC using ethyl ace-
tate and methanol (9/1, v/v) as mobile phase. In valida-
tion runs (entry 10), the crude mixture was purified by
radio-HPLC on an semi-preparative C-18 reverse phase
column (Phenomenex Luna® 5 μm, 100 Å, 250 × 10 mm)
using water containing 0.1% trifluoroacetic acid (solvent
A) and acetonitrile (solvent B) in a gradient mode of elu-
tion (0–5 min 10% B, 5–20 min 10–90% B) at a flow rate
of 3 ml/min.. Further identification of [18F]β-CFT was
carried out by radio-HPLC on an analytical C-18 reverse
phase column (Phenomenex Luna® 5 μm, 100 Å,
250 × 5 mm) using water containing 0.1% trifluoroacetic
acid (solvent A) and acetonitrile (solvent B) in a gradient
mode of elution (0–5 min 10% B, 5–20 min 10–90% B) at
a flow rate of 1 ml/min.
was 2.2%. By using low base procedure, the non-isolated
RCY was varied between 2.5% and 10.6%, depending on
the amounts of precursor and Cu (OTf)2(py)4, volume of
DMF used. The retention times of authentic β-CFT and
[18F]β-CFT in HPLC chromatograph were 14.37 and
14.68 min, respectively (Figure 1). In scale-up validation
runs (entry 10), starting from 5.55 GBq of [18F]fluoride,
the radiosynthesis was achieved with an overall decay-
corrected RCY of isolated [18F]β-CFT of 7.2 2.6%, >99%
RCP and 118 35 GBq/μmol molar activity.
4 | DISCUSSION
The radiolabeling strategy used to produce [18F]β-CFT in
this study was starting from the aryl boronic derivative 4.
Initially, the radiolabeling experiment was performed by
eluting anion exchange cartridge with K222/K2CO3
(21.25 μmol/18.81 μmol, high base), and then reacting
with 4 and Cu (OTf)2(py)4 in 1 ml DMF at 110ꢀC for
20 min. The RCY of [18F]β-CFT under these conditions
was very low (2.2%, entry 1, Table 1).
An efficient strategy to increase the RCY of copper-
mediated aromatic radiofluorination using a “low base”
protocol has been reported by previous study.32,56,57 When
small amount of K222/K2CO3 (1.51 μmol/0.87 μmol) was
used to elute the anion exchange cartridge, and then
reacted with 4 and Cu (OTf)2(py)4 in 1 ml DMF, an ele-
vated RCY (8.6 2.9%) was observed (entry 6, Table 1). As
this “low base” protocol gave better results, subsequent
radiofluorination studies were performed using the same
amount of K2CO3 and K222 for eluting anion exchange
resin and radiolabeling reaction. The precursor: Cu
(OTf)2(py)4 ratio is another key parameter for
3 | RESULTS
3.1 | Chemistry
The pinacol boronate ester precursor β-CBT 4 was syn-
thesized through four steps from cocaine hydrochloride
as a starting material. The cocaine hydrochloride is
hydrolyzed in hydrochloric acid solution to obtain com-
pound 1. Then compound 2 was obtained after chlorina-
tion, methyl esterification and dehydration. Under the
action of Grignard reagent, compound 3 is formed.
Finally, the boron oxidation reaction is carried out with
B2Pin to obtain β-CBT 454–56, and the overall yield is
21.9%. The non-radioactive reference standard β-CFT
5 can be synthesized from cocaine hydrochloride as a
starting material through three steps. In the last step,
under the action of Grignard reagent, β-CFT 5 was
formed and the overall yield is currently 31.3%.
For both β-CBT and β-CFT, there are two asymmetric
carbons, so theoretically 22 or 4 stereoisomers can be pro-
duced. When compound 2 reacted with Grignard
reagents, due to the steric effect on carbon 3 in tropane
structure, chlorobenzene or fluorobenzene could only be
attached to the β position. For the other asymmetric cen-
ter (carbon 2), two stereoisomers were formed. Therefore,
only two stereoisomers (2α,3β; 2β,3β) were actually pro-
duced. These two stereoisomers can be separated by silica
gel column chromatography with ethyl acetate as mobile
phase. The purity of 2β,3β form was confirmed by NMR
spectroscopy. This is necessary because only 2β,3β form is
effective in PET imaging41.
3.2 | Radiosynthesis of [18F]β-CFT
FIGURE 1 Quality control of the [18F]β-CFT after HPLC
purification. The retention times of authentic β-CBT (blue line) and
[18F]β-CFT (black line) were 14.37 and 14.68 min, respectively
The non-isolated RCY for [18F]β-CFT using high base
procedure with precursor: [Cu (OTf)2(Py)4] ratio = 3:1

YU ET AL.
233
TABLE 1 Radiofluorination of [18F]β-CFT
Cartridge eluentb
Radiofluorination
K222
K2CO3
(μmol)
Precursor
(μmol)
Precursor:[Cu
(OTf)2(Py)4] ratio
DMF
volume (ml)
Non-isolated
RCY (%)c
Isolated
RCY (%)d
Entrya (μmol)
1
21.25
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
18.81
0.87
0.87
0.87
0.87
0.87
0.87
0.87
0.87
0.87
15.6
15.6
15.6
15.6
15.6
15.6
15.6
15.6
31.2
15.6
3:1
1:1
2:1
2:1
3:1
3:1
6:1
6:1
6:1
3:1
1.0
1.0
0.5
1.0
0.5
1.0
0.5
1.0
1.0
1.0
2.2
2.5
n.d.
2
n.d.
3
3.0
n.d.
4
4.1
n.d.
5
4.2
n.d.
6
8.6 2.9
6.3
n.d.
7
n.d.
8
3.3
n.d.
9
10e
10.6
8.3 2.2
n.d.
7.2 2.6
aReacted in anhydrous DMF (0.5/1.0 ml) at 110ꢀC for 20 min; radioactivity for fluorination (entry 1-9): ꢁ 0.37 GBq.
bEntry 1: Macherey-Nagel Chromabond PS-HCO3− cartridge, entry 2-10: Waters Sep-Pak Accell QMA cartridge.
cEstimated by radio-TLC of the crude product (% SEM). Entry 6, 10: n = 3; other entries: n = 1.
dCalculated from isolated [18F]β-CFT to the initial [18F]fluoride radioactivity trapped in the Waters Sep-Pak Accell QMA cartridge (% SEM).
eScale-up validation runs: radioactivity for fluorination: ꢁ 5.55 GBq.
18F-fluorination of aryl boronic esters.32,58,59 Different
amounts of Cu (OTf)2(py)4 and precursor 4 were added to
study their influence on the RCY of [18F]β-CFT. For entry
2-8 (Table 1), there was a trend of increase in RCY with
decreasing amount of Cu catalyst,56 and the optimized
RCY was achieved with precursor: Cu (OTf)2(py)4
ratio = 3:1 and DMF volume = 1.0 ml (8.6 2.9%). We also
tried to optimize the radiolabeling procedure with respect
to reaction volume. Decreasing the volume of DMF from
1.0 ml (entries 4, 6, 8) to 0.5 ml (entries 3, 5, 7) with differ-
ent precursor: Cu (OTf)2(py)4 ratio, however the RCY of
[18F]β-CFT was increased only with precursor: Cu
(OTf)2(py)4 ratio = 6:1 (3.3% to 6.3%). The results of the lit-
erature and this study showed that independent refine-
ment of quantity of copper complex used is necessary for
each aryl boronic precursor. Improved RCY of [18F]β-CFT
(entry 8, 3.3% to entry 9, 10.6%) could also be achieved by
doubling the amount of precursor used. Experimental vali-
dation runs (entry 10) starting with high radioactivity
showed consistent RCY results, and [18F]β-CFT with high
RCP and high molar activity was obtained after HPLC
purification.
dimethylacetamide (DMA) was used for replacement of
DMF.58,60,61 The reasons were proposed to be its higher
boiling point and base resistant property. The importance
of addition order of various reactants/reagents and disso-
lution of dried [18F]KF were also demonstrated in previ-
ous study.62
5 | CONCLUSION
In this study, [18F]β-CFT can be prepared from boronic
ester precursor in a one-step radiofluorination procedure
by nucleophilic reaction. 18F-Fluorination of aryl boronic
ester derivatives was optimized with [18F]fluoride elution
and precursor: Cu (OTf)2(py)4 ratio. A low base protocol
is crucial for effective [18F]β-CFT labeling. Due to the
variety of labeling procedures, precursors and results
reported in the literature, a concordant conclusion is not
easily to reach. Performing optimization for each aryl
boronic reagent to be radiolabeled seems to be the sensi-
ble approach at present stage. The availability of boronic
ester precursor could provide a convenient and a reliable
method for the facile preparation of [18F]β-CFT for use in
a clinical practice. As the feasibility of the radiolabeling
method has been confirmed, future work will focus on
automation of the experimental protocol.
Additional modification of reaction protocol is
believed to be able to further improve the RCY. Previous
studies have showed that the presence of oxygen is bene-
ficial for the labeling reaction.32,58 Their results indicated
that aryl-Cu (III) species could be formed by oxidation.
This high-valent species is favorable for 18F–C bond for-
mation and converting pinacol-derived aryl boronic
esters into 18F-aryl fluorides. For reaction solvent, better
conversion efficacies have been reported when
ACKNOWLEDGEMENTS
The present study was supported by Atomic Energy
Council of Taiwan (grant number: MOST 108-3111-Y-
042A-118).

234
YU ET AL.
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How to cite this article: Yu H-M, Li C-Y,
Liu S-W, Yang C-H, Chang Y. Copper-mediated
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