Synthesis of [11C]PBR06 and [18F]PBR06 as agents for positron emission tomographic (PET) imaging of the translocator protein (TSPO)

Article abstract of DOI:10.1016/j.steroids.2011.06.012

The translocator protein 18 kDa (TSPO) is an attractive target for molecular imaging of neuroinflammation and tumor progression. [ ¹⁸F]PBR06, a fluorine-18 labeled form of PBR06, is a promising PET TSPO radioligand originally developed at NIMH.

Full text of DOI:10.1016/j.steroids.2011.06.012

Steroids 76 (2011) 1331–1340
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of [11C]PBR06 and [18F]PBR06 as agents for positron emission
tomographic (PET) imaging of the translocator protein (TSPO)
Min Wang a, Mingzhang Gao a, Kathy D. Miller b, Qi-Huang Zheng a,
⇑
a Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, L3-202, Indianapolis, IN 46202-2111, USA
b Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202-2111, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The translocator protein 18 kDa (TSPO) is an attractive target for molecular imaging of neuroinflamma-
tion and tumor progression. [18F]PBR06, a fluorine-18 labeled form of PBR06, is a promising PET TSPO
radioligand originally developed at NIMH. [11C]PBR06, a carbon-11 labeled form of PBR06, was designed
and synthesized for the first time. The standard PBR06 was synthesized from 2,5-dimethoxybenzalde-
hyde in three steps with 71% overall chemical yield. The radiolabeling precursor desmethyl-PBR06 was
synthesized from 2-hydroxy-5-methoxybenzaldehyde in five steps with 12% overall chemical yield.
The target tracer [11C]PBR06 was prepared by O-[11C]methylation of desmethyl-PBR06 with [11C]CH3OTf
in CH3CN at 80 °C under basic condition and isolated by HPLC combined with SPE purification with 40–
Received 27 April 2011
Received in revised form 27 June 2011
Accepted 29 June 2011
Available online 3 July 2011
Keywords:
[
[
11C]PBR06
18F]PBR06
60% decay corrected radiochemical yield and 222–740 GBq/lmol specific activity at EOB. On the similar
The translocator protein 18 kDa (TSPO)
Positron emission tomography (PET)
Neuroinflammation imaging
Tumor imaging
grounds, [18F]PBR06 was also designed and synthesized. The previously described Br-PBR06 precursor
was synthesized from 2,5-dimethoxybenzaldehyde in two steps with 78% overall chemical yield. A
new radiolabeling precursor tosyloxy-PBR06, previously undescribed tosylate congener of PBR06, was
designed and synthesized from ethyl 2-hydroxyacetate, 4-methylbenzene-1-sulfonyl chloride, and
N-(2,5-dimethoxybenzyl)-2-phenoxyaniline in four steps with 50% overall chemical yield. [18F]PBR06
was prepared by the nucleophilic substitution of either new tosyloxy-PBR06 precursor or known Br-
PBR06 precursor in DMSO at 140 °C with K[18F]F/Kryptofix 2.2.2 for 15 min and HPLC combined with
SPE purification in 20–60% decay corrected radiochemical yield, >99% radiochemical purity, 87–95%
chemical purity, and 37–222 GBq/l
mol specific activity at EOB. Radiosynthesis of [18F]PBR06 using
new tosylated precursor gave similar radiochemical purity, and higher specific activity, radiochemical
yield and chemical purity in comparison with radiosynthesis using bromine precursor.
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
cholesterol from the outer to the inner mitochondrial membrane
before its transformation by cytochrome P-450 into pregnenolone,
The translocator protein 18 kDa (TSPO), also known as periphe-
ral-type benzodiazepine receptor (PBR), is a protein which has high
affinity cholesterol- and drug-binding protein in the outer mito-
chondrial membrane [1]. TSPO is also found in many tissues such
as lung, liver, heart, spleen, kidney, adrenals, brain, glial cells,
masts cell and macrophages, however, TSPO is expressed at the
highest levels under normal conditions only in those steroid
synthesized tissues [2]. TSPO is involved in a variety of cellular
functions, including cholesterol import into mitochondria (a key
involvement in steroidogenesis), apoptosis, cell proliferation, dif-
ferentiation, anion transport, porphyrin transport, heme synthesis,
and regulation of mitochondrial function [3]. It has been exten-
sively reported that the most characterized functional role of the
TSPO is its ability to regulate the rate-limiting translocation of
which leads to the production of various steroids [4–6]. The over-
expression of TSPO has been linked in multiple disorders, including
cancer, brain injury, neurodegeneration, and ischemia–reperfusion
injury [7]. High-affinity TSPO ligands can stimulate steroid produc-
tion and become promising therapeutic tools [8]. Recent reports
have indicated that TSPO ligand PK11195 can be used as a thera-
peutic agent for neurological and psychiatric disorders, and cancers
[3,9,10]. TSPO is an attractive target for molecular imaging of neur-
oinflammation like in Alzheimer’s disease and in tumor progres-
sion using the biomedical imaging technique positron emission
tomography (PET) [11]. The clinically useful prototypical TSPO-
selective PET radioligand is [11C]PK11195; however, it is reported
to have many limitations such as low uptake and sensitivity [12].
These limitations have motivated investigators to search for new
TSPO PET radioligands. Recently numerous new radioligands have
been developed, and promising candidates progressing to human
PET studies include [11C]DAA1106, [18F]FEDAA1106, [11C]PBR28,
⇑
Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278 9711.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
0039-128X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.06.012

1332
M. Wang et al. / Steroids 76 (2011) 1331–1340
OCH3
OCH3
O
O
O
F
N
O
F
N
O
N
O
N
O11CH3
O11CH3
O
18F
18F]FEDAA1106
[
11C]DAA1106
[
11C]PBR28
[
18F
OCH3
F
OCH3
F
OCH3
O
O
O
OPh
OPh
OPh
O11CH3
11C]PBR06
OCH3
PBR06
OCH3
18F]PBR06
[
[
Fig. 1. Chemical structures of TSPO radioligands.
and
[
18F]PBR06 (N-(2,5-dimethoxybenzyl)-2-[18F]fluoro-N-(2-
uncorrected. 1H NMR spectra were recorded on Bruker Avance II
500 MHz NMR spectrometers using tetramethylsilane (TMS) as
an internal standard. Chemical shift data for the proton resonances
were reported in parts per million (ppm, d scale) relative to inter-
nal standard TMS (d 0.0), and coupling constants (J) were reported
in hertz (Hz). The high resolution mass spectra (HRMS) were ob-
tained using a Thermo Electron Corporation MAT 95XP-Trap spec-
trometer. Chromatographic solvent proportions are indicated as
volume:volume ratio. Thin-layer chromatography (TLC) was run
using Analtech silica gel GF uniplates (5 Â 10 cm2). Plates were
visualized under UV light. Preparative TLC was run using Analtech
silica gel UV 254 plates (20 Â 20 cm2). Normal phase flash column
chromatography was carried out on EM Science silica gel 60
(230–400 mesh) with a forced flow of the indicated solvent system
in the proportions described below. All moisture- and/or air-
sensitive reactions were performed under a positive pressure of
nitrogen maintained by a direct line from a nitrogen source.
Analytical high performance liquid chromatography (HPLC) was
phenoxyphenyl)acetamide) [13–16], as indicated in Fig. 1. The link
between PET imaging of TSPO and the importance of this target for
steroids has been strengthened, and a high-affinity TSPO-specific
PET radioligand may provide significant information for brain neu-
rosteroidogenesis, detecting progression of disease and efficacy of
TSPO ligand therapy [17,18].
[
18F]PBR06, a fluorine-18 labeled form of PBR06, is a promising
PET brain TSPO radioligand originally developed and characterized
by Briard et al. at National Institute of Mental Health (NIMH) [19],
which displayed high PBR (TSPO) binding affinities (Ki = 0.180,
0.318 and 0.997 nM in brain homogenates from rat, monkey and
human, respectively) [19,20]. Wishing to study this compound in
our laboratory, we synthesized the radiolabeling precursors and
standards following the literature methods. However, the pub-
lished production of [18F]PBR06 using the reaction of [18F]fluoride
ion with the corresponding bromide precursor resulted in low
specific activity, radiochemical yield and chemical purity of
[
18F]PBR06 in our hands, due to difficult separation of the fluo-
performed using a Prodigy (Phenomenex) 5 lm C-18 column,
rine-18 labeled product from the bromine precursor, and poor
leaving group of the bromide precursor. While studying the re-
ported methods for [18F]PBR06 production, we designed the fully
automated synthesis of [11C]PBR06 (Fig. 1), a carbon-11 labeled
form of PBR06, for the first time. Compared to fluorine-18 tracers
(half-life 110 min), carbon-11 tracers (half-life 20 min) have some
advantages in back-to-back same-day PET studies such as allowing
a patient to receive multiple C-11 injections to shorten the diag-
nostic process when multiple tracer studies are required, and in
reducing the radiation exposure for both radiopharmaceutical pro-
duction staff and environment [21,22]. We also discovered a new
labeling precursor, the previously undescribed tosylate congener
of PBR06 with better tosyloxy leaving group, and investigated a
fully automated synthesis of [18F]PBR06.
4.6 Â 250 mm; 3:1:1 CH3CN/MeOH/20 mM, pH 6.7 phosphate
(buffer solution) mobile phase; flow rate 1.5 mL/min; and UV
(254 nm) and
HPLC was performed using a YMC-Pack ODS-A, S-5
c
-ray (PIN diode) flow detectors. Semi-preparative
m, 12 nm,
l
10 Â 250 mm C-18 column; 3:1:1 CH3CN/MeOH/20 mM, pH 6.7
phosphate (buffer solution) mobile phase; 4.0 mL/min flow rate;
UV (254 nm) and
Pak cartridges were obtained from Waters Corporation (Milford,
MA). Sterile Millex-FG 0.22 m filter units were obtained from
c-ray (PIN diode) flow detectors. C18 Plus Sep-
l
Millipore Corporation (Bedford, MA).
2.2. 2-(Benzyloxy)-5-methoxybenzaldehyde (1)
To a stirred solution of 2-hydroxy-5-methoxybenzaldehyde
(10.0 g, 65.7 mmol) in DMF (100 mL) was added K2CO3 (10.9 g,
78.9 mmol), followed by benzyl bromide (8.2 mL, 69.0 mmol).
The reaction mixture was stirred at 45 °C for 5 h. After cooling to
room temperature (RT), water was added. The resulting mixture
was acidified to pH 7.0 with ice-cold 3% aqueous HCl and extracted
with EtOAc. The combined organic layers were washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The crude product was purified by silica gel column chromatogra-
phy (10:1 hexanes/EtOAc) to afford 1 (14.33 g, 90%) as a white
solid, mp 46–47 °C (lit [24], 48–49 °C). 1H NMR (CDCl3): d 10.51
(s, 1H, CHO), 7.43–7.38 (m, 4H, ArH), 7.34–7.33 (m, 2H, ArH),
2. Materials and methods
2.1. General
All commercial reagents and solvents were purchased from
Sigma–Aldrich and Fisher Scientific and used without further
purification.
[
11C]methyl triflate ([11C]CH3OTf) was prepared
according to a literature procedure [23]. Melting points were
determined on a MEL-TEMP II capillary tube apparatus and were

M. Wang et al. / Steroids 76 (2011) 1331–1340
1333
7.11 (dd, J = 9.0, 3.5 Hz, 1H, ArH), 7.00 (d, J = 9.0 Hz, 1H, ArH), 5.15
(s, 2H, ArCH2O), 3.80 (s, 3H, OCH3).
poured into ice-water. The resulting mixture was extracted with
EtOAc. The combined organic layers were washed with brine, dried
over anhydrous Na2SO4, filtered and concentrated in vacuo. The
crude product was purified by silica gel column chromatography
(from 5:1 to 3:2 hexanes/EtOAc) to afford 4 (360 mg, 48%) as a pale
yellow oil. 1H NMR (CDCl3): d 7.31–7.28 (m, 3H, ArH), 7.26–7.18
(m, 3H, ArH), 7.15–7.09 (m, 2H, ArH), 7.10 (t, J = 7.5 Hz, 1H, ArH),
7.04 (d, J = 2.5 Hz, 1H, ArH), 6.97–6.91 (m, 2H, ArH), 6.79 (d,
J = 8.0 Hz, 1H, ArH), 6.75–6.69 (m, 4H, ArH), 5.22 (d, J = 14.5 Hz,
1H, NCHH), 4.82–4.64 (m, 5H, NCHH + ArCH2OAr + CH2F), 3.66 (s,
3H, OCH3). HRMS (EI, m/z): calcd for C29H26FNO4 (M+) 471.1840;
found 471.1834.
2.3. N-(2-(Benzyloxy)-5-methoxybenzyl)-2-phenoxyaniline (2)
A suspension of compound 1 (3.0 g, 12.4 mmol) and 2-phenox-
yaniline (2.3 g, 12.4 mmol) in MeOH (20 mL) was stirred at RT for
2 h, and then MeOH was removed in vacuo. After the residue was
heated at 95 °C for 2 h under nitrogen atmosphere, the mixture
was cooled to RT and diluted with MeOH (20 mL). NaBH4 (1.87 g,
49.6 mmol) was added portionwise at 0 °C. The reaction mixture
was allowed to warm to RT and stirred for 1 h. The mixture was
cooled to 0 °C, 5% aqueous acetic acid (100 mL) was added drop-
wise. After stirring at RT for 30 min, the resulting mixture was ex-
tracted with EtOAc. The combined organic layers were washed
with saturated aqueous NaHCO3, brine, dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The crude product
was purified by silica gel column chromatography (12:1 hexanes/
EtOAc) to afford 2 (2.25 g, 49%) as a yellow oil. 1H NMR (CDCl3):
d 7.35–7.21 (m, 7H, ArH), 7.00–6.94 (m, 2H, ArH), 6.91 (d,
J = 8.5 Hz, 2H, ArH), 6.85 (d, J = 2.5 Hz, 1H, ArH), 6.81 (dd, J = 8.0,
1.0 Hz, 1H, ArH), 6.77 (d, J = 8.5 Hz, 1H, ArH), 6.71 (d, J = 8.0 Hz,
1H, ArH), 6.66 (dd, J = 7.5, 3.0 Hz, 1H, ArH), 6.59 (dt, J = 8.0,
1.0 Hz, 1H, ArH), 4.93 (s, 2H, ArCH2O), 4.37 (s, 2H, NCH2), 3.62 (s,
3H, OCH3). HRMS (EI, m/z): calcd for C27H25NO3 (M+) 411.1829;
found 411.1821.
2.6. 2-Fluoro-N-(2-hydroxy-5-methoxybenzyl)-N-(2-phenoxyphenyl)-
acetamide (desmethyl-PBR06, 5)
A solution of compound 4 (300 mg, 0.64 mmol) in MeOH
(10 mL) was hydrogenated over 10% Pd–C (50 mg) at 60 psi for
6 h. The catalyst was filtered through a layer of Celite, and then
the solvent was removed in vacuo. The crude product was purified
by silica gel column chromatography (2:1 hexanes/CH3COCH3) to
afford 5 (128 mg, 53%) as a colorless oil. 1H NMR (CDCl3): d 8.56
(br s, 1H, OH), 7.36–7.32 (m, 3H, ArH), 7.18–7.15 (m, 1H, ArH),
7.13–7.08 (m, 2H, ArH), 6.91 (dd, J = 7.5, 1.0 Hz, 1H, ArH), 6.86–
6.84 (m, 3H, ArH), 6.76 (dd, J = 9.0, 3.0 Hz, 1H, ArH), 6.24 (d,
J = 3.0 Hz, 1H, ArH), 4.93 (d, J = 14.5 Hz, 1H, NCHH), 4.77 (d,
J = 47.0, 5.5 Hz, 1H, CH2F), 4.53 (d, J = 14.5 Hz, 1H, NCHH), 3.62 (s,
3H, OCH3). HRMS (EI, m/z): calcd for C22H20FNO4 (M+) 381.1371;
found 381.1387.
2.4. N-(2-(Benzyloxy)-5-methoxybenzyl)-2-bromo-N-(2-
phenoxyphenyl)acetamide (3)
To a stirred solution of compound 2 (2.1 g, 5.1 mmol) and NEt3
(0.86 mL, 6.12 mmol) in CH2Cl2 (10 mL) was added bromoacetyl
bromide (0.49 mL, 5.62 mmol) dropwise at 0 °C. The reaction mix-
ture was allowed to warm to RT and stirred for 3 h. The resulting
mixture was poured into water and extracted with CH2Cl2. The
combined organic layers were washed with 0.5 N aqueous HCl, sat-
urated aqueous NaHCO3, brine, dried over anhydrous Na2SO4, fil-
tered and concentrated in vacuo. The crude product was purified
by silica gel column chromatography (5:1 hexanes/EtOAc) to afford
3 (1.42 g, 86%) as a yellow oil. 1H NMR (CDCl3): d 7.32–7.16 (m, 8H,
ArH), 7.12–7.08 (m, 2H, ArH), 7.05 (d, J = 2.5 Hz, 1H, ArH), 6.96 (dt,
J = 7.5, 1.0 Hz, 1H, ArH), 6.81 (dd, J = 8.5, 1.0 Hz, 1H, ArH), 6.78 (d,
J = 7.5 Hz, 2H, ArH), 6.74–6.69 (m, 2H, ArH), 5.23 (d, J = 14.5 Hz,
1H, NCHH), 4.81–4.72 (m, 3H, NCHH + ArCH2OAr), 3.78 (dd,
J = 24.0, 11.0 Hz, 2H, CH2Br), 3.67 (s, 3H, OCH3). HRMS (EI, m/z):
calcd for C29H26BrNO4 (M+) 531.1040; found 531.1024.
2.7. (2-(Benzyloxy)-5-methoxyphenyl)methanol (6)
To a solution of compound 1 (3.0 g, 12.4 mmol) in CH2Cl2 (8 mL)
and MeOH (40 mL) was added NaBH4 (650 mg, 17.2 mmol) por-
tionwise at 0 °C. The reaction mixture was allowed to warm to
RT and stirred for 5 h, and then the solvents were removed in va-
cuo. Water was added and the mixture was extracted with EtOAc.
The combined organic layers were washed with saturated aqueous
NH4Cl, brine, dried over anhydrous Na2SO4, filtered and concen-
trated in vacuo. The crude product was purified by silica gel column
chromatography (2:1 hexanes/EtOAc) to afford 6 (2.90 g, 96%) as a
white solid, mp 48–49 °C. 1H NMR (CDCl3): d 7.41–7.37 (m, 4H,
ArH), 7.35–7.31 (m, 1H, ArH), 6.90 (d, J = 3.0 Hz, 1H, ArH), 6.87
(d, J = 9.0 Hz, 1H, ArH), 6.77 (dd, J = 9.0, 3.0 Hz, 1H, ArH), 5.06 (s,
2H, ArCH2OAr), 4.69 (s, 2H, ArCH2OH), 3.77 (s, 3H, OCH3), 2.17 (s,
1H, CH2OH).
2.5. N-(2-(Benzyloxy)-5-methoxybenzyl)-2-fluoro-N-(2-phenoxyphe-
nyl)acetamide (4)
Method A: A mixture of compound 3 (770 mg, 1.45 mmol) and
dry KF (253 mg, 4.35 mmol) in diethylene glycol (8 mL) was heated
rapidly to 150 °C in a preheated oil-bath. After stirring for 5 h at
150 °C, the reaction mixture was cooled to RT and quenched with
water. The resulting mixture was extracted with EtOAc. The com-
bined organic layers were washed with brine, dried over anhy-
drous Na2SO4, filtered and concentrated in vacuo. The crude
product was purified by silica gel column chromatography (5:1
hexanes/EtOAc) to afford 4 (384 mg, 56%) as a pale yellow oil.
Method B: To a stirred solution of 2-fluoro-N-(2-phenoxyphe-
nyl)acetamide (9) (200 mg, 0.82 mmol) in DMF (3 mL) was added
NaH (60% dispersion in mineral oil, 50 mg, 1.25 mmol) at 0 °C.
After the mixture was allowed to warm to RT and stirred for
30 min, a solution of 1-(benzyloxy)-2-(bromomethyl)-4-methoxy-
benzene (7) (280 mg, 91.4 mmol) in DMF (1 mL) was added drop-
wise. The reaction mixture was stirred at RT for 4 h, and then
2.8. 1-(Benzyloxy)-2-(bromomethyl)-4-methoxybenzene (7)
To a stirred solution of compound 6 (2.2 g, 8.19 mmol) in CH2Cl2
(20 mL) was added a solution of PBr3 (0.77 mL, 8.19 mmol) in
CH2Cl2 (5 mL) dropwise at 0 °C. The reaction mixture was allowed
to warm to RT and stirred for 5 h. The resulting mixture was
poured into ice-water and extracted with CH2Cl2. The combined
organic layers were washed with cold saturated aqueous NaHCO3,
brine, dried over anhydrous Na2SO4, and filtered. The solvent was
removed in vacuo to afford 7 (2.67 g, 97%) as a brown solid, which
was used without further purification. 1H NMR (CDCl3): d 7.47 (d,
J = 7.5 Hz, 2H, ArH), 7.39 (t, J = 7.5 Hz, 2H, ArH), 7.33–7.30 (m,
1H, ArH), 6.92 (d, J = 3.0 Hz, 1H, ArH), 6.85 (d, J = 9.0 Hz, 1H,
ArH), 6.79 (dd, J = 9.0, 3.0 Hz, 1H, ArH), 5.10 (s, 2H, ArCH2OAr),
4.57 (s, 2H, ArCH2Br), 3.76 (s, 3H, OCH3). HRMS (EI, m/z): calcd
for C15H15BrO2 (M+) 306.0250; found 306.0235.

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M. Wang et al. / Steroids 76 (2011) 1331–1340
2.9. 2-Bromo-N-(2-phenoxyphenyl)acetamide (8)
11 (1.30 g, 80%) as a clear oil. 1H NMR (CDCl3): d 7.35–7.32 (m, 2H,
ArH), 7.23–7.20 (m, 1H, ArH), 7.16–7.11 (m, 2H, ArH), 6.98 (dt,
J = 7.5, 1.5 Hz, 1H, ArH), 6.95 (d, J = 8.0 Hz, 1H, ArH), 6.91–6.89
(m, 2H, ArH), 6.85 (dd, J = 8.0, 1.0 Hz, 1H, ArH), 6.72 (dd, J = 8.5,
3.0 Hz, 1H, ArH), 6.67 (d, J = 9.0 Hz, 1H, ArH), 5.19 (d, J = 14.5 Hz,
1H, NCHH), 4.69 (d, J = 14.0 Hz, 1H, NCHH), 3.79 (d, J = 7.0 Hz,
2H,CH2Br), 3.67 (s, 3H, OCH3), 3.53 (s, 3H, OCH3).
To a stirred solution of 2-phenoxyaniline (1.0 g, 5.4 mmol) and
NEt3 (0.83 mL, 5.94 mmol) in CH2Cl2 (5 mL) was added bromoace-
tyl bromide (0.52 mL, 5.94 mmol) dropwise at 0 °C. The reaction
mixture was allowed to warm to RT and stirred for 2 h. The result-
ing mixture was poured into water and extracted with CH2Cl2. The
combined organic layers were washed with 0.5 N aqueous HCl, sat-
urated aqueous NaHCO3, brine, dried over anhydrous Na2SO4, fil-
tered and concentrated in vacuo. The crude product was purified
by silica gel column chromatography (1:3 hexanes/CH2Cl2) to af-
ford 8 (1.42 g, 86%) as pale yellow solid, mp 49–51 °C. 1H NMR
(CDCl3): d 8.78 (br s, 1H, NH), 8.39 (dd, J = 8.5, 1.5 Hz, 1H, ArH),
7.38–7.34 (m, 2H, ArH), 7.17–7.13 (m, 2H, ArH), 7.08–7.03 (m,
3H, ArH), 6.90 (dd, J = 8.0, 1.0 Hz, 1H, ArH), 3.99 (s, 2H, CH2Br).
2.13. N-(2,5-Dimethoxybenzyl)-2-fluoro-N-(2-phenoxyphenyl)acet-
amide (PBR06, 12)
A mixture of compound 11 (400 mg, 0.88 mmol) and dry KF
(192 mg, 3.30 mmol) in diethylene glycol (5 mL) was heated rap-
idly to 150 °C in a preheated oil-bath. After the reaction mixture
was stirred at 150 °C for 6 h, it was cooled and diluted with water.
The resulting mixture was extracted with EtOAc. The combined or-
ganic layers were washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The crude product
was purified by silica gel column chromatography (5:1 hexanes/
EtOAc) to afford 12 (315 mg, 91%) as a pale yellow oil. 1H NMR
(CDCl3): d 7.34 (t, J = 8.0 Hz, 2H, ArH), 7.22–7.19 (m, 1H, ArH),
7.15 (t, J = 7.5 Hz, 1H, ArH), 7.03–6.95 (m, 2H, ArH), 6.93 (d,
J = 3.0 Hz, 1H, ArH), 6.87 (d, J = 7.5 Hz, 2H, ArH), 6.83 (dd, J = 8.5,
1.0 Hz, 1H, ArH), 6.72 (dd, J = 9.0, 3.0 Hz, 1H, ArH), 6.66 (d,
J = 8.5 Hz, 1H, ArH), 5.16 (d, J = 14.0 Hz, 1H, NCHH), 4.76 (d,
J = 14.0 Hz, 1H, NCHH), 4.75 (dd, J = 47.0, 3.5 Hz, 2H, CH2F), 3.66
(s, 3H, OCH3), 3.50 (s, 3H, OCH3).
2.10. 2-Fluoro-N-(2-phenoxyphenyl)acetamide (9)
A mixture of compound 8 (1.0 g, 3.28 mmol) and dry KF
(480 mg, 8.26 mmol) in diethylene glycol (10 mL) was heated rap-
idly to 150 °C in a preheated oil-bath. After the reaction mixture
was stirred at 150 °C for 2 h, it was cooled and diluted with water.
The resulting mixture was extracted with CH2Cl2. The combined
organic layers were washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The crude product
was purified by silica gel column chromatography (1:3 hexanes/
CH2Cl2) to afford 9 (384 mg, 48%) as a brown solid, mp 74–76 °C
(lit [11] 75–77 °C). 1H NMR (CDCl3): d 8.59 (br s, 1H, NH), 8.45
(dd, J = 8.5, 1.5 Hz, 1H, ArH), 7.38–7.30 (m, 2H, ArH), 7.17–7.12
(m, 2H, ArH), 7.08–7.03 (m, 3H, ArH), 6.88 (dd, J = 8.5, 1.5 Hz, 1H,
ArH), 4.90 (d, J = 47.0 Hz, 2H, CH2F).
2.14. Ethyl 2-(tosyloxy)acetate (13)
To a stirred solution of ethyl glycolate (5.0 g, 48.0 mmol) and to-
syl chloride (9.15 g, 48.0 mmol) in anhydrous Et2O (40 mL) was
added NEt3 (96.0 mmol, 13.4 mL) dropwise at 0 °C. After the reac-
tion mixture was stirred at 0 °C for 2 h, water was added and the
phase separated. The aqueous phase was extracted with Et2O.
The combined organic layers were washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The crude
product was purified by silica gel column chromatography (5:1
hexanes/EtOAc) to afford 13 (9.33 g, 75%) as a white solid, mp
42–43 °C. 1H NMR (CDCl3): d 7.84 (d, J = 8.0 Hz, 2H, ArH), 7.36 (d,
J = 8.0 Hz, 2H, ArH), 4.58 (s, 2H, OCH2CO), 4.19 (q, J = 7.0 Hz, 2H,
OCH2CH3), 2.46 (s, 3H, ArCH3), 1.24 (t, J = 7.0 Hz, 3H, OCH2CH3).
2.11. N-(2,5-Dimethoxybenzyl)-2-phenoxyaniline (10)
To a stirred solution of 2-phenoxyaniline (5.0 g, 27.0 mmol) in
MeOH (30 mL) was added a solution of 2,5-dimethoxy-benzalde-
hyde (4.93 g, 29.7 mmol) in MeOH (25 mL) at RT. The reaction mix-
ture was heated at reflux for 2 h, and then MeOH was removed in
vacuo. The residue was heated at 120 °C for 1 h under nitrogen
atmosphere, and then it was cooled to RT and diluted with MeOH
(40 mL). NaBH4 (4.5 g, 119.0 mmol) was added portionwise at 0 °C.
The reaction mixture was allowed to warm to RT and stirred for
1 h. The mixture was cooled to 0 °C, 5% aqueous acetic acid
(100 mL) was added dropwise. After stirring at RT for 30 min, the
resulting mixture was extracted with EtOAc. The combined organic
layers were washed with saturated aqueous NaHCO3, brine, dried
over anhydrous Na2SO4, filtered and concentrated in vacuo. The
crude product was purified by silica gel column chromatography
(12:1 hexanes/EtOAc) to afford 10 (8.87 g, 98%) as a pale yellow
solid, mp 48–49 °C. 1H NMR (CDCl3): d 7.30–7.25 (m, 2H, ArH),
7.05–6.98 (m, 2H, ArH), 6.94–6.92 (m, 2H, ArH), 6.85–6.83 (m,
2H, ArH), 6.78–6.70 (m, 3H, ArH), 6.64 (dt, J = 7.5, 1.0 Hz, 1H,
ArH), 4.34 (s, 2H, NCH2), 3.70 (s, 3H, OCH3), 3.69 (s, 3H, OCH3).
2.15. 2-(Tosyloxy)acetic acid (14)
To a stirred suspension of compound 13 (5.0 g, 19.4 mmol) in
EtOH (20 mL) was added 5% aqueous NaOH (16 mL). After the reac-
tion mixture was stirred at RT for 5 h, EtOH was removed in vacuo,
and the residue was cooled with ice-water and acidified with 5%
aqueous HCl (25 mL). The solid was collected by filtration, rinsed
with water. The crude product was recrystallized from EtOAc/hex-
anes to afford 14 (3.49 g, 78%) as a white solid, mp 136–138 °C. 1H
NMR (CD3OD): d 7.82 (d, J = 8.0 Hz, 2H, ArH), 7.44 (d, J = 8.0 Hz, 2H,
ArH), 4.59 (s, 2H, OCH2CO), 2.45 (s, 3H, ArCH3).
2.12. 2-Bromo-N-(2,5-dimethoxybenzyl)-N-(2-phenoxyphenyl)acet-
amide (Br-PBR06, 11)
To a stirred solution of compound 10 (1.2 g, 3.58 mmol) and
NEt3 (0.55 mL, 3.94 mmol) in CH2Cl2 (5 mL) was added bromoace-
tyl bromide (0.34 mL, 3.94 mmol) dropwise at 0 °C. The reaction
mixture was allowed to warm to RT and stirred for 2 h. The result-
ing mixture was poured into water and extracted with CH2Cl2. The
combined organic layers were washed with 0.5 N aqueous HCl, sat-
urated aqueous NaHCO3, brine, dried over anhydrous Na2SO4, fil-
tered and concentrated in vacuo. The crude product was purified
by silica gel column chromatography (5:1 hexanes/EtOAc) to afford
2.16. 2-Chloro-2-oxoethyl 4-methylbenzenesulfonate (15)
A mixture of compound 14 (3.0 g, 13.0 mmol) and thionyl chlo-
ride (10 mL) was heated at reflux for 2 h. The excess thionyl chlo-
ride was removed in vacuo, and the residue was dried under high
vacuum to afford 15 (2.97 g, 92%) as pale yellow oil, which was
used without further purification. 1H NMR (CD3Cl): d 7.82 (d,
J = 8.5 Hz, 2H, ArH), 7.38 (d, J = 8.5 Hz, 2H, ArH), 4.90 (s, 2H, OCH2CO),
2.47 (s, 3H, CH3).

M. Wang et al. / Steroids 76 (2011) 1331–1340
1335
2.17. 2-((2,5-Dimethoxybenzyl)(2-phenoxyphenyl)amino)-2-oxoethyl
4-methylbenzenesulfonate (tosyloxy-PBR06, 16)
at 140 °C for 15 min to affect radiofluorination. After cooling to
ꢀ90 °C, the contents of the reaction vial were diluted with NaHCO3
(1 mL, 0.1 M), and injected onto the semi-preparative HPLC column
with 3 mL injection loop for purification. The product fraction was
collected in a recovery vial containing 30 mL water. The diluted
tracer solution was then passed through a C-18 Sep-Pak Plus car-
tridge, and washed with water (5 mL Â 5). The cartridge was eluted
with EtOH (1 mL Â 2) to release [18F]PBR06 ([18F]12). The eluted
To a stirred solution of compound 10 (500 mg, 1.49 mmol) and
NEt3 (0.24 mL, 1.72 mmol) in CH2Cl2 (2 mL) was added a solution
of compound 15 (415 mg, 1.67 mmol) in CH2Cl2 (2 mL) dropwise
at 0 °C. After the reaction mixture was allowed to warm to RT
and stirred for 3 h, water was added and the phases were sepa-
rated. The aqueous phase was extracted with CH2Cl2. The com-
bined organic layers were washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The crude
product was purified by silica gel column chromatography (1.5:1
hexanes/EtOAc) to afford 16 (747 mg, 92%) as a pale yellow solid,
mp 109–110 °C. 1H NMR (CD3Cl): d 7.80 (d, J = 8.0 Hz, 2H, ArH),
7.34–7.29 (m, 4H, ArH), 7.22–7.18 (m, 1H, ArH), 7.15 (t,
J = 7.5 Hz, 1H, ArH), 7.01–6.95 (m, 2H, ArH), 6.87 (d, J = 3.0 Hz,
1H, ArH), 6.83 (d, J = 7.5 Hz, 2H, ArH), 6.79 (dd, J = 8.5, 1.0 Hz, 1H,
ArH), 6.71 (dd, J = 9.0, 3.0 Hz, 1H, ArH), 6.64 (d, J = 9.0 Hz, 1H,
ArH), 5.05 (d, J = 14.5 Hz, 1H, CONHH), 4.71 (d, J = 14.5 Hz, 1H,
CONHH), 4.57 (s, 2H, OCH2CO), 3.64 (s, 3H, OCH3), 3.48 (s, 3H,
OCH3), 2.42 (s, 3H, CH3). HRMS (EI, m/z): calcd for C30H29NO7S
(M+) 547.1659; found 547.1654.
product was then sterile-filtered through a Millex-FG 0.22 lm
membrane into a sterile vial and formulated with 10 mL saline. To-
tal radioactivity was assayed and total volume was noted for tracer
dose dispensing. Retention times in the semi-preparative HPLC
system were: tR 11 = 8.18 min, tR 16 = 9.43 min, tR 12 = 7.12 min,
tR
tem were: tR 11 = 5.70 min, tR 16 = 7.31 min, tR 12 = 4.48 min, tR
11C]12 = 4.48 min.
[
11C]12 = 7.12 min. Retention times in the analytical HPLC sys-
[
3. Results and discussion
3.1. Chemistry
The synthetic approach designed for the preparation of the
desmethyl-PBR06 precursor 5 for carbon-11 labeling is outlined
in Scheme 1. The phenolic hydroxyl group of 2-hydroxy-5-
methoxybenzaldehyde was protected as benzyl ether by reacting
with benzyl bromide in DMF in the presence of K2CO3 to afford
benzaldehyde 1 in 90% yield [24]. Condensation of 2-phenoxyani-
line with 1 followed by reduction with NaBH4 in MeOH achieved
2 in 49% yield, which was bromoacetylated with bromoacetyl bro-
mide in CH2Cl2 in the presence of NEt3 to give acetamide 3 in 94%
yield. Acetamide 4 was obtained by nucleophilic substitution of 3
with dry KF in diethylene glycol in 56% yield. The benzyl group
of 4 was removed by catalytic hydrogenation with 10% Pd/C in
MeOH to provide the precursor 5 in 53% yield.
Compound 4 could also be achieved by another approach as
shown in Scheme 2. Reduction of 1 to alcohol 6 was performed
with NaBH4 in a mixture of CH2Cl2 and MeOH in 96% yield. Benzyl
alcohol 6 was converted to benzyl bromide 7 with PBr3 in CH2Cl2 in
97% yield. Bromoacetylation of 2-phenoxyaniline with bromoace-
tyl bromide in CH2Cl2 in the presence of NEt3 generated N-bromo-
acetyl aniline 8 in 86% yield, which was then treated with dry KF in
diethylene glycol to afford N-fluoroacetyl aniline 9 in 48% yield
[19]. Coupling reaction of 7 with 9 in DMF in the presence of
NaH gave 4 in 94% yield.
2.18. 2-Fluoro-N-(2-[11C]methoxy-5-methoxybenzyl)-N-(2-
phenoxyphenyl)acetamide ([11C]PBR06, [11C]12)
[
11C]CO2 was produced by the 14N(p, 11C nuclear reaction in
a)
small volume (9.5 cm3) aluminum gas target (CTI) from 11 MeV
proton cyclotron on research purity nitrogen (+1% O2) in a Siemens
radionuclide delivery system (Eclipse RDS-111). In a small reaction
vial (5 mL), the desmethyl-PBR06 precursor 5 (0.5–1.0 mg) was
dissolved in CH3CN (400 lL). To this solution was added NaH
(ꢀ1 mg) with a microspatula. No carrier-added (high specific activ-
ity) [11C]CH3OTf that was produced by the gas-phase production
method [23] from [11C]CO2 through [11C]CH4 and [11C]CH3Br with
silver triflate (AgOTf) column was passed into the reaction vial at
RT, until radioactivity reached a maximum (ꢀ2 min), and then
the reaction vial was isolated and heated at 80 °C for 3 min. The
contents of the reaction vial were diluted with NaHCO3 (1 mL,
0.1 M), and injected onto the semi-preparative HPLC column with
3 mL injection loop for purification. The product fraction was
collected in a recovery vial containing 30 mL water. The diluted
tracer solution was then passed through a C-18 Sep-Pak Plus car-
tridge, and washed with water (5 mL Â 5). The cartridge was eluted
with EtOH (1 mL Â 2) to release [11C]PBR06 ([11C]12). The eluted
product was then sterile-filtered through a Millex-FG 0.22
l
m
The PBR06 standard 12 was synthesized according to the liter-
ature method with modification [19]. As indicated in Scheme 3,
2-phenoxyaniline was condensed with 2,5-dimethoxybenzalde-
hyde followed by reduction with NaBH4 in MeOH to give phenox-
yaniline 10 in 98% yield. Higher yield at shorter reaction time was
achieved for the condensation performed in MeOH at reflux ini-
tially for 2 h, followed by solvent removal and then further heated
to 120 °C for 1 h. Bromoacetylation of 10 yielded the Br-PBR06 pre-
cursor 11 for fluorine-18 labeling in 80% yield. Fluorination of 11
using dry KF in diethylene glycol provided the reference standard
12 in 91% yield.
membrane into a sterile vial and formulated with 10 mL saline. To-
tal radioactivity was assayed and total volume was noted for
tracer dose dispensing. Retention times in the semi-preparative
HPLC system were: tR 5 = 5.35 min, tR 12 = 7.12 min, tR
7.12 min. Retention times in the analytical HPLC system were: tR
5 = 3.28 min, tR 12 = 4.49 min, tR
11C]12 = 4.49 min.
[
11C]12 =
[
2.19. N-(2,5-Dimethoxybenzyl)-2-[18F]fluoro-N-(2-phenoxyphenyl)-
acetamide ([18F]PBR06, [18F]12)
No-carrier-added (NCA) aqueous H[18F]F was produced by
18O(p,n) 18F nuclear reaction using a Siemens Eclipse RDS-111
cyclotron by irradiation of H218O (2.5 mL). H[18F]F (7.4–18.5 GBq)
in [18O]water plus 0.1 mL K2CO3 solution (1.7 mg) and Kryptofix
2.2.2 (10 mg) in 1.0 mL CH3CN with additional 1 mL CH3CN were
placed in the fluorination reaction vial (10-mL V-vial) and repeated
azeotropic distillation (17 min) was performed at 110 °C to remove
water and to form the anhydrous K[18F]F-Kryptofix 2.2.2 complex.
The precursor Br-PBR06 11 or tosyl-PBR06 16 (1 mg) dissolved in
DMSO (1.0 mL) was introduced to the reaction vessel and heated
To improve the radiochemical yield of 18F-labeling reaction and
chemical purity of [18F]fluorinated product, it became necessary
to design and synthesize the appropriate radiolabeling precursor
bearing suitable leaving group for nucleophilic substitution of
[
18F]FÀ, and possessing suitable polarity difference to labeled
product for separation and purification process. Therefore, we
turned our attention to developing a new tosylate precursor
for [18F]-labeled PBR06. The tosyloxy-PBR06 precursor 16 was
prepared by a four-step sequence of reactions delineated in
Scheme 4. Coupling reaction of ethyl glycolate with tosyl chloride

1336
M. Wang et al. / Steroids 76 (2011) 1331–1340
OMe
OBn
OH
OPh
O
CHO
CHO
OPh
a)
NH
2 /MeOH
BnBr, K2CO3
DMF
BrCCH2Br
NEt3, CH2Cl2
H
N
b) NaBH4
OBn
OPh
OMe
1, 90%
OMe
2, 49 %
OMe
F
OMe
Br
OMe
F
O
O
O
C
KF
C
N
OPh
H2, Pd/C
MeOH
C
N
OPh
HOCH2CH2OCH2CH2OH
N
OH
5 (desmethyl-PBR06), 53%
OBn
OBn
3, 94%
4, 56%
Scheme 1. Synthesis of desmethyl-PBR06 (5).
OBn
OBn
OBn
CHO
CH2Br
CH2OH
NaBH4
MeOH
PBr3
CH2Cl2
OMe
OBn
F
OMe
OMe
7, 97%
O
OMe
6, 96%
C
N
OPh
NaH
DMF
1
O
O
Br
F
O
4, 94%
NH
NH
NH
2 BrCCH2Br
KF
HOCH2CH2OCH2CH2OH
OPh
NEt3, CH2Cl2
OPh
OPh
9, 48%
8, 86%
Scheme 2. Synthesis of an intermediate 4.
OMe
Br
OMe
OMe
OMe
OPh
O
BrCCH2Br
NEt3,CH2Cl2
O
CHO
C
N
OPh
OPh
a)
NH
2 /MeOH
H
N
b) NaBH4
OMe
OMe
F
10, 98%
11 (Br-PBR06), 80%
OMe
O
KF
HOCH2CH2OCH2CH2OH
C
N
OPh
OMe
12 (PBR06), 91%
Scheme 3. Synthesis of Br-PBR06 (11) and PBR06 (12).
O
O
O
Me
O
SO2Cl
5% NaOH aqueous
TsO
TsO
HO
OEt
13, 75%
EtOH
OH
NEt3, Et2O
OMe
OEt
14, 78%
OPh
H
N
OMe
OTs
O
OMe
C
N
OPh
SOCl2
10
TsO
Cl
NEt3, CH2Cl2
15, 92%
OMe
16 (tosyloxy-PBR06), 92%
Scheme 4. Synthesis of tosyloxy-PBR06 (16).

M. Wang et al. / Steroids 76 (2011) 1331–1340
1337
OMe
F
[
11C]CH3OTf [23,26] through O-[11C]methylation [27,28] at 80 °C
OMe
OH
F
[
11C]CH3OTf, CH3CN
NaH, 80 oC
O
under basic condition (NaH) and isolated by a semi-preparative
HPLC method (C-18 column) and a solid-phase extraction (SPE)
method (C-18 Plus Sep-Pak cartridge) (a second purification or iso-
lation process) [29] to produce the corresponding pure radiola-
beled compound [11C]12 in 40–60% radiochemical yield, decay
corrected to end of bombardment (EOB), based on [11C]CO2.
O
C
N
OPh
C
N
OPh
O11CH3
11C]12 ([11C]PBR06), 40-60%
[
5 (desmethyl-PBR06)
[
11C]CH3OTf is a proven methylation reagent with greater reactiv-
Scheme 5. Synthesis of [11C]PBR06 ([11C]12).
ity than commonly used [11C]methyl iodide ([11C]CH3I) [27,30],
and thus, the radiochemical yields for [11C]PBR06 is relatively high.
Addition of NaHCO3 to quench the radiolabeling reaction and to di-
lute the radiolabeling mixture prior to the injection onto the semi-
preparative HPLC column for purification gave better separation of
in Et2O in the presence of NEt3 afforded compound 13 in 75% yield.
Ethyl ester 13 was hydrolyzed with 5% aqueous NaOH in EtOH to
give compound 14 in 78% yield, which was converted to acyl halide
15 with thionyl chloride [25] in 92% yield. The desired tosylate pre-
cursor 16 was achieved by coupling reaction of 10 with 15 in
CH2Cl2 in the presence of NEt3 in 92% yield.
[
11C]PBR06 from its phenolic precursor [29,31]. The radiosynthesis
was performed in a home-built automated multi-purpose [11C]-
radiosynthesis module, allowing measurement of specific radioac-
tivity during synthesis [32,33]. The overall synthesis, purification
and formulation time was 30–40 min from EOB. The specific radio-
3.2. Radiochemistry
activity was in a range of 222–740 GBq/
purity and radiochemical purity were determined by analytical
lmol at EOB. Chemical
Synthesis of the target tracer [11C]PBR06 ([11C]12) is indicated
in Scheme 5. The desmethyl-PBR06 precursor 5 was labeled by
A
400.00
300.00
200.00
100.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
Minutes
B
0.08
0.06
0.04
0.02
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
6.00
6.00
8.00
9.00
10.00
11.00
12.00
Minutes
C
1000.00
500.00
0.00
1.00
2.00
3.00
4.00
5.00
7.00
8.00
9.00
10.00
Minutes
D
0.15
0.10
0.05
0.00
1.00
2.00
3.00
4.00
5.00
7.00
8.00
9.00
10.00
Minutes
Fig. 2. A representative analytical HPLC chromatographic profile for the tracer [11C]PBR06 produced with either Sep-Pak purification or solvent evaporation. Analytical
radioactive (A) and UV (B) HPLC traces for [11C]PBR06 produced with Sep-Pak purification, tR 11C]PBR06 = 4.49 and 4.33 min, respectively. Analytical radioactive (C) and UV
(D) HPLC traces for [11C]PBR06 produced with solvent evaporation, tR 11C]PBR06 = 4.31 and 4.24 min, respectively.
[
[

1338
M. Wang et al. / Steroids 76 (2011) 1331–1340
radio-HPLC through c-ray (PIN diode) flow detector, and the chem-
OMe
Br
OMe
OMe
18F
ical purity of [11C]PBR06 was >95% determined by reverse-phase
HPLC through UV flow detector. A C-18 Plus Sep-Pak cartridge
was used to significantly improve the chemical purity of the tracer
solution. Initial HPLC purification was employed to separate the
labeled product from its un-reacted excess precursor and other
labeled by-products. The second SPE purification with Sep-Pak
[29,34] was employed, instead of rotary evaporator, to remove po-
tential impurities from the HPLC co-elution with the precursor and
from the residual solvents including HPLC mobile phase solvents
and module set-up cleaning solvents. Rotary evaporation was un-
able to perform in this regard. Moreover, it could result in the
decomposition of the labeled product such as desmethylation dur-
ing the heating. The chemical purity of the [11C]PBR06 tracer solu-
tion with Sep-Pak purification was increased higher 10–20% than
that without Sep-Pak purification. Representative analytical HPLC
chromatograms for [11C]PBR06 produced with either Sep-Pak puri-
fication or solvent evaporation are shown in Fig. 2.
K[18F]F/Kryptofix 2.2.2
DMSO, 140 oC
O
O
C
N
OPh
C
N
OPh
OMe
11 (Br-PBR06)
[
18F]12 ([18F]PBR06), 20-40%
OMe
OTs
OMe
18F
K[18F]F/Kryptofix 2.2.2
DMSO, 140 oC
O
O
C
N
OPh
C
N
OPh
OMe
OMe
16 (tosyloxy-PBR06)
[
18F]12 ([18F]PBR06), 30-60%
Scheme 6. Synthesis of [18F]PBR06 ([18F]12).
Synthesis of the target tracer [18F]PBR06 ([18F]12) is outlined in
Scheme 6. The Br-PBR06 11 or tosyloxy-PBR06 16 precursor was
labeled with K[18F]F/Kryptofix 2.2.2 through nucleophilic
HPLC [34]. The chemical purity of the precursor desmethyl-PBR06
and reference standard PBR06 was >96%. The radiochemical purity
of the target tracer
[
11C]PBR06 was >99% determined by
A
300.00
200.00
100.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
Minutes
0.050
B
0.040
0.030
0.020
0.010
0.000
1.00
2.00
3.00
4.00
5.00
6.00
7.00
6.00
6.00
8.00
9.00
10.00
11.00
12.00
Minutes
C 1000.00
500.00
0.00
1.00
2.00
3.00
4.00
5.00
7.00
8.00
9.00
10.00
Minutes
D
0.10
0.00
1.00
2.00
3.00
4.00
5.00
7.00
8.00
9.00
10.00
Minutes
Fig. 3. A representative analytical HPLC chromatographic profile for the tracer [18F]PBR06 produced by both tosyloxy-PBR06 and Br-PBR06. Analytical radioactive (A) and UV
(B) HPLC traces for [18F]PBR06 produced using tosyloxy-PBR06, tR 18F]PBR06 = 4.48 and 4.32 min, respectively. Analytical radioactive (C) and UV (D) HPLC traces for
18F]PBR06 produced using Br-PBR06, tR 18F]PBR06 = 4.30 and 4.23 min, respectively.
[
[
[

M. Wang et al. / Steroids 76 (2011) 1331–1340
1339
substitution and isolated by a semi-preparative HPLC method with
C-18 column and a SPE method with a C-18 Plus Sep-Pak cartridge
(a second purification or isolation process) [29] to produce the
corresponding pure radiolabeled compound [18F]12 in 20–40%
and 30–60% decay-corrected radiochemical yield from K[18F]F,
respectively. Likewise, addition of NaHCO3 to quench the radiola-
beling reaction and to dilute the radiolabeling mixture prior to
the injection onto the semi-preparative HPLC column for purifica-
tion gave better separation of [18F]PBR06 from its Br-PBR06 or
tosyloxy-PBR06 precursor [29,31]. The radiosynthesis was per-
formed using a self-designed automated multi-purpose [18F]-
radiosynthesis module. The overall synthesis, purification and for-
mulation time was 50–60 min from EOB. The specific radioactivity
[
18F]PBR06 produced by both tosyloxy-PBR06 and Br-PBR06 are
shown in Fig. 3.
4. Conclusions
Improved and efficient syntheses of PBR06 and its Br-PBR06
precursor (for F-18 labeling) have been developed. New desmeth-
yl-PBR06 precursor (for C-11 labeling) and tosyloxy-PBR06 precur-
sor (for F-18 labeling) have been designed and synthesized for the
first time. Desmethyl-PBR06 was labeled with [11C]CH3OTf, and
isolated by semi-preparative HPLC combined with SPE purification
to provide [11C]PBR06, a carbon-11 labeled form of PBR06, for the
first time, in high radiochemical yield with excellent specific activ-
ity and shorter reaction times. Previously undescribed tosyloxy-
PBR06 precursor was labeled with K[18F]F/Kryptofix 2.2.2 through
nucleophilic substitution, and isolated by semi-preparative HPLC
combined with SPE purification to produce [18F]PBR06, a fluo-
rine-18 labeled form of PBR06, in higher radiochemical yield,
chemical purity and specific activity, compared to previously de-
scribed Br-PBR06 precursor. An automated self-designed multi-
purpose [11C]- and [18F]-radiosynthesis module for the synthesis
of [11C]PBR06 and [18F]PBR06 has been built, featuring the mea-
surement of specific activity by the on-the-fly technique. New
and improved results in the synthetic methodology, radiolabeling,
preparative separation and analytical details for PBR06, desmethyl-
PBR06, Br-PBR06 and tosyloxy-PBR06 precursors, [11C]PBR06 and
was 37–222 GBq/l
mol at EOB. No-carrier-added [18F]fluoride ion
in [18O]water was trapped with or without a QMA cartridge. If
the cyclotron-produced [18F]fluoride ion was trapped without the
use of a cartridge [29,35], it would significantly increase the spe-
cific activity of the prepared [18F]PBR06. The reason was that there
was a low-level contamination of QMA anionic resins with fluoride
ion [35]. The amounts of bromide or tosylated precursor used were
ꢀ1 mg. A large amount of precursor would increase the radio-
chemical yield of [18F]PBR06, but decrease the chemical purity of
the [18F]PBR06 tracer solution due to precursor contamination,
especially for Br-PBR06 precursor. The reaction solvent and tem-
perature were either CH3CN/120 °C or DMSO/140 °C. Radiolabeling
procedure with DMSO at 140 °C resulted in higher radiochemical
yield [36]. Chemical purity and radiochemical purity were
determined by analytical HPLC [34]. The chemical purity of the
Br-PBR06 and tosyloxy-PBR06 precursors and reference standard
PBR06 was >96%. The radiochemical purity of the target tracer
[
18F]PBR06 have been addressed. These methods are efficient and
convenient. It is anticipated that the approaches for the design,
synthesis and automation of new tracer and radiolabeling precur-
sors, and improvements to increase radiochemical yield, chemical
purity and specific activity of the tracers described here can be ap-
plied with advantage to the synthesis of other 11C- and 18F-radio-
ligands for PET imaging. These chemistry results warrant future
preclinical and clinical PET studies of [11C]PBR06 and [18F]PBR06
in animals and humans to image cancer and neuroinflammation.
This work will provide useful information for other investigators
who will perform in vitro and in vivo biological evaluations of these
[
18F]PBR06 was >99%, and the chemical purity of [18F]PBR06 was
87–95% determined by HPLC methods. Likewise, a C-18 Plus
Sep-Pak cartridge was used to significantly improve the chemical
purity of the tracer solution as aforementioned. The chemical pur-
ity of the [18F]PBR06 tracer solution with Sep-Pak purification was
increased higher 10–20% than that without Sep-Pak purification.
We noticed Briard et al. [19] have reported an experimental radio-
synthesis of [18F]PBR06 using 18-crown-6 with KHCO3 in 97%
radiochemical yield, and they have previously shown for a model
compound that 18-crown-6 is preferred to replace Kryptofix
2.2.2 in 18F-labeling reaction because the precursor alkylates
Kryptofix 2.2.2 [37]. However, this method is not practical and use-
ful for the tracer routine production, since the starting [18F]fluoride
ion was <74 mBq (2 mCi), and it is difficult to scale up to and to re-
peat in automated production of 18F-tracers. An automated pro-
duction of [18F]PBR06 using Kryptofix 2.2.2 with K2CO3 and a
commercial module (TRACERlab EXF-N) was also reported by Briard
et al. in the same paper, but it only gave 12% isolated radiochemical
yield [19]. Furthermore, their Investigational New Drug (IND)
Application 23195 for [18F]PBR06 at NIMH submitted to the US
Food and Drug Administration (FDA) (a copy is available at:
http://pdsp.med.unc.edu/snidd/) shows they actually adopt auto-
mated production of [18F]PBR06 (12% using Kryptofix 2.2.2) in their
CMC ([18F]PBR for injection: Chemistry, Manufacturing and
Controls). This method is comparable to our radiosynthesis per-
formed in a self-designed automated [18F]-radiosynthesis module.
Obviously, the radiochemical yield of [18F]PBR06 was significantly
increased in our method, especially for new tosylated precursor
(30–60% isolated radiochemical yield) we developed. Specific
activity of [18F]PBR06 produced by tosylated precursor was higher
than that by the bromide precursor, because there might be a pseu-
do carrier in the bromide precursor. In summary, the radiosynthe-
sis of [18F]PBR06 using new tosylated precursor gave similar
radiochemical purity, and higher specific activity, radiochemical
yield and chemical purity in comparison with the bromide
precursor. Representative analytical HPLC chromatograms for
[
11C] and [18F] tracers and develop new PET tracers for imaging the
TSPO in vivo.
Acknowledgments
This work was partially supported by the Breast Cancer Re-
search Foundation. 1H NMR spectra were recorded on a Bruker
Avance II 500 MHz NMR spectrometer in the Department of Chem-
istry and Chemical Biology at Indiana University Purdue University
Indianapolis (IUPUI), which is supported by a NSF-MRI Grant CHE-
0619254. The referees’ criticisms and editors’ comments for the
revision of the manuscript are greatly appreciated.
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