Fully automated synthesis and initial PET evaluation of [11C]PBR28

Article abstract of DOI:10.1016/j.bmcl.2009.08.051

Fully automated synthesis and initial PET evaluation of a TSPO radioligand, [¹¹C]PBR28 (N-(2-[¹¹C]methoxybenzyl)-N-(4-phenoxypyridin-3-yl)acetamide), are reported. These results facilitate the potential preclinical and clinical PET s

Full text of DOI:10.1016/j.bmcl.2009.08.051

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5636–5639
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fully automated synthesis and initial PET evaluation of [11C]PBR28
Min Wang a, Karmen K. Yoder a, Mingzhang Gao a, Bruce H. Mock a, Xiao-Ming Xu b, Andrew J. Saykin a,
Gary D. Hutchins a, Qi-Huang Zheng a,
*
a Department of Radiology, Indiana University School of Medicine, 1345 West 16th Street, Room 202, Indianapolis, IN 46202, USA
b Department of Neurosurgery, Indiana University School of Medicine, 950 West Walnut Street, Room 427, Indianapolis, IN 46202, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Fully automated synthesis and initial PET evaluation of
a TSPO radioligand, [
11C]PBR28 (N-(2-
[
11C]methoxybenzyl)-N-(4-phenoxypyridin-3-yl)acetamide), are reported. These results facilitate the
Received 23 July 2009
Revised 5 August 2009
Accepted 5 August 2009
Available online 15 August 2009
potential preclinical and clinical PET studies of [11C]PBR28 in animals and humans.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Translocator protein (TSPO)
[
11C]PBR28
Radiosynthesis
Positron emission tomography (PET)
Traumatic brain injury (TBI)
Brain imaging
Translocator protein 18 kDa (TSPO, formerly known as the
peripheral benzodiazepine receptor)1 is a protein found in lung, li-
ver, heart, spleen, kidney, adrenals, brain, glial cells, masts cell, and
macrophages, and is implicated in numerous nervous system disor-
ders such as epilepsy, cerebral ischemia, nerve injury and neurode-
generative diseases, and immune system diseases such as cancer.2
Brain TSPO density increases in several neuropathological condi-
tions and after experimental injuries to the central nervous system
as well.3 TSPO is an attractive target for molecular imaging of neur-
oinflammation like Alzheimer’s disease and tumor progression
usingthebiomedicalimagingtechniquepositronemissiontomogra-
phy (PET).4 The prototypical TSPO-selective PET radioligand is
The precursor N-(2-hydroxybenzyl)-N-(4-phenoxypyridin-3-
yl)acetamide (desmethyl-PBR28, 4a) and reference standard N-(2-
methoxybenzyl)-N-(4-phenoxypyridin-3-yl)acetamide (PBR28, 4b)
were prepared according to the procedures outlined in Scheme 1.
The route taken was generally based on the literature methods with
slight modifications.11,14,15 Displacement of 4-chloride by phenol
was readily achieved by treatment of 4-chloro-3-nitropyridine with
phenol in the presence of K2CO3 to give 3-nitro-4-phenoxypyridine
(1) in 97% yield. Reduction of nitro group of compound 1 was per-
formed efficiently with SnCl2 and concentrated HCl instead of 6 N
HCl in MeOH to afford 4-phenoxy-3-pyridinamine (2)16 in 92% yield.
Condensation of compound
2
with o-salicylaldehyde or
[
11C]PK11195; however, it is reported to have many limitations such
o-anisaldehyde in MeOH, followed by reduction with NaBH4 affor-
ded 2-((4-phenoxypyridin-3-ylamino)methyl)phenol (3a) and
N-(2-methoxybenzyl)-4-phenoxypyridin-3-amine (3b) in 91% and
90% yield, respectively. PBR28 (4b) was obtained directly by acetyla-
tion of the amine 3b with acetyl chloride in CH2Cl2 in 84% yield. Acet-
ylation of the amine and phenolic hydroxyl groups of compound 3a
with acetyl chloride, subsequent hydrolysis of its acetate with LiOH
inMeOHprovideddesmethyl-PBR28(4a)in 78%yield. As depictedin
Scheme 2, PBR28 (4b) can be achieved by direct O-methylation of
desmethyl-PBR28 (4a) involving anion formation with NaH, fol-
lowed by CH3I in DMF in 36% yield.
as low brain uptake and low sensitivity.5 These limitations have
motivated investigators to search for new TSPO PET radioligands.
Promising candidates progressing to human PET studies include
[
11C]DAA1106, [18F]FEDAA1106, and [11C]PBR286–8 as indicated in
Figure 1. [11C]PBR28 (N-(2-[11C]methoxybenzyl)-N-(4-phenoxy-
pyridin-3-yl)acetamide, IC50 0.658 nM) was originally developed
by Innis and Pike et al. at the National Institute of Mental Health
(NIMH).8–11 Wishing to study this compound in our laboratory, we
investigated a fully automated synthesis of [11C]PBR28 using
[
11C]methyl triflate ([11C]CH3OTf)12,13 and performed initial PET
imaging in an animal model of traumatic brain injury (TBI), which
overexpresses TSPO.
Synthesis of the target tracer [11C]PBR28 ([11C]4b) is indicated
in Scheme 3. The phenolic precursor 4a was labeled using
[
11C]CH3OTf12,13 through O-[11C]methylation17 under basic condi-
tions (NaH) and isolated by a semi-preparative HPLC method18 to
* Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278 9711.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
produce the corresponding pure radiolabeled compound [11C]4b
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.051

M. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5636–5639
5637
O
O
N
N
N
11CH3
N
11CH3
Cl
Cl
(S)-[11C]PK11195
(R)-[11C]PK11195
OCH3
OCH3
O
O
O
F
N
O
N
O
F
N
O
N
O11CH3
O11CH3
O
18F
18F]FEDAA1106
[
11C]PBR28
[
11C]DAA1106
[
Figure 1. Chemical structures of PET TSPO radioligands.
NH2
O
O
O
NO2
N
N
N
O
N
O
NO2
Cl
a
b
N
N
[
a
N
O
O11CH3
OH
2
1
11C]PBR28 ([11C]4b)
desmethyl-PBR28 (4a)
c
Scheme 3. Synthesis of [11C]PBR28. Reagents, conditions, and yields: (a) NaH,
11C]CH3OTf, CH3CN, 80 °C, 3 min, 70–80%.
[
O
H
N
N
O
N
N
d
OR
OR
in 70–80% radiochemical yield, decay corrected to end of bombard-
ment (EOB), based on [11C]CO2. In comparison with the results re-
ported in the literature,11 several significant improvements in the
radiosynthesis have been made. [11C]CH3OTf was used as a radio-
labeled precursor, which is a proven methylation reagent with
O
4a: R=H (desmethyl-PBR28)
4b: R=Me (PBR28)
3a: R=H
greater reactivity than commonly used
[
11C]methyl iodide
3b: R=Me
([11C]CH3I).11 NaH was used as a strong base instead of (tBu)4NOH,
and CH3CN was used as the reaction solvent instead of MeOH. The
reaction temperature 80 °C was higher than room temperature,
and the reaction time was only 3 min, shorter than 7 min in the lit-
erature. We also used a ‘vial’ method instead of the reported ‘loop’
method. Therefore, the radiochemical yields for [11C]PBR28 in our
method is much higher than that reported previously (26%).11
The radiosynthesis was performed in an in-house automated mul-
ti-purpose 11C-radiosynthesis module, allowing measurement of
specific radioactivity during synthesis.19,20 The overall synthesis,
purification and formulation time was 25–30 min from EOB. The
Scheme 1. Synthesis of desmethyl-PBR28 and PBR28. Reagents, conditions, and
yields: (a) phenol, K2CO3, DMF, 80 °C, 97%; (b) SnCl2, concd HCl, MeOH, 80 °C, 92%;
(c) for 3a: o-salicylaldehyde, 90 °C, then NaBH4, MeOH, room temperature (rt), 91%;
for 3b: o-anisaldehyde, 100 °C; then NaBH4, MeOH, rt, 90%; (d) for 4a: acetyl
chloride, DMAP, CH2Cl2, rt, then sat. LiOH, MeOH, rt, 84%; for 4b: acetyl chloride,
DMAP, CH2Cl2, rt, 78%.
O
O
N
O
N
O
N
N
specific radioactivity was in a range of 5–15 Ci/lmol at EOB. Chem-
a
OCH3
OH
ical purity and radiochemical purity were determined by analytical
HPLC.21 The chemical purity of the precursor and reference stan-
dard was >96%. The radiochemical purity of the target tracer was
>99% determined by radio-HPLC through
c-ray (PIN diode) flow
detector, and the chemical purity of the target tracer was >93%
determined by reverse-phase HPLC through UV flow detector.
The characterization data for compounds 1–4 and experimental
details for the tracer [11C]4b are given.22
PBR28 (4b)
desmethyl-PBR28 (4a)
Scheme 2. Alternate synthetic approach for PBR28. Reagents, conditions, and
yields: (a) NaH, CH3I, DMF, rt, 36%.

5638
M. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5636–5639
Figure 2. [11C]PBR28-PET images in 2 TBI and 1 SHM rats.
Initial PET evaluation of [11C]PBR28 was performed in rats.
(2 TBI, 1 SHM), SUV values were 16.9 2.23% higher in the left
ROI relative to the right. [11C]PBR28-PET images in 2 TBI and 1
SHM rats are shown in Figure 2.
In conclusion, an efficient and convenient automated synthesis
of [11C]PBR28 was developed, and PET evaluation of [11C]PBR28
was performed in a rat model of TBI. These results warrant that
the automated preparation of [11C]PBR28 is suitable for preclinical
and clinical studies in animals and humans using PET.
Three female Sprague-Dawley rats were imaged. Two animals
received a moderate controlled cortical impact (2 mm deforma-
tion) to the left parietal cortex (TBI). The third animal received a
sham surgery (SHM; incision, skull preparation with no impact).
Animals were scanned seven days after surgery. Animals were
anesthetized with isoflurane and placed on a stereotaxic-like
head-holder.23 The PET scan was performed right after an intrave-
nous (IV) tail vein injection of 0.31 0.07 mCi
[
11C]PBR28
(0.58 0.35 nmol/kg). Dynamic data were acquired for 90 min on
the IndyPET III scanner, a small animal PET scanner designed and
developed in the Department of Radiology at Indiana University
School of Medicine.24–26 The raw data collected from 40 to
90 min was used to generate the images for analysis. Standardized
uptake value (SUV) images were created by normalizing intensity
values at each voxel by body weight and injected dose. Regions
of interest (ROIs) indicated by the green arrows, approximating
the left and right parietal cortices, were drawn on each SUV image.
An ellipse (superior–inferior radius = 3 mm; anterior–posterior ra-
dius = 4.5 mm) was placed on the approximate anterior–posterior
location of the parietal cortex. The ellipse was placed on sequential
sagittal slices so that the final ROI spanned the entire lateral to
medial extent of each hemisphere. Across all three animals
Acknowledgments
This work was supported in part by the Indiana Genomics Ini-
tiative (INGEN) of Indiana University, as well as by grants from
the NIH 5R01AG019771, 3P30AG010133-18S1, 5P50NS052606,
and Indiana Economic Development Corporation (IEDC 87884).
We gratefully acknowledge Dr. Robert B. Innis at the NIMH for
his assistance. The referees’ criticisms and editor’s comments for
the revision of the manuscript are greatly appreciated.
References and notes
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7.41–7.37 (m, 2H), 7.32–7.31 (m, 1H), 7.28–7.24 (m, 1H), 7.22–7.19 (m, 1H),
7.07–7.04 (m, 2H), 6.94–6.88 (m, 2H), 6.54 (d, J = 5.5 Hz, 1H), 4.73 (t, J = 5.5 Hz,
1H), 4.45 (d, J = 6.0 Hz, 2H), 3.83 (s, 3H). LC/MS (m/z, ESI): [M+H]+ calcd for
C19H19N2O2 307.1, found 307.1. Compound 4a, a white solid, mp 123–124 °C
(lit.10 123–125 °C). 1H NMR (CDCl3, 500 MHz): d 9.07 (br s, 1H), 8.48 (d,
J = 5.5 Hz, 1H), 8.43 (s, 1H), 7.41 (t, J = 8.0 Hz, 2H), 7.30 (t, J = 7.5 Hz, 1H), 7.23–
7.20 (m, 1H), 6.94 (d, J = 8.0 Hz, 1H), 6.79–6.72 (m, 5H), 4.94 (d, J = 14.5 Hz, 1H),
4.79 (d, J = 15.0 Hz, 1H), 2.05 (s, 3H). LC/MS (m/z, ESI): [M+H]+ calcd for
C20H19N2O3 335.1, found 335.1. Compound 4b, a white solid, mp 84–85 °C (lit.9
89–91 °C). 1H NMR (CDCl3, 500 MHz): d 8.28 (d, J = 5.5 Hz, 1H), 8.21 (s, 1H),
7.44–7.40 (m, 2H), 7.39–7.37 (m, 1H), 7.29–7.28 (m, 1H), 7.24–7.20 (m, 1H),
6.95–8.86 (m, 3H), 6.74 (d, J = 8.0 Hz, 1H), 6.57 (d, J = 5.5 Hz, 1H), 5.14 (d,
J = 14.5 Hz, 1H), 4.89 (d, J = 14 Hz, 1H), 3.58 (s, 3H), 1.99 (s, 3H). LC/MS (m/z,
ESI): [M+H]+ calcd for C21H21N2O3 349.1, found 349.0. (b) Production of the
tracer [11C]PBR28 ([11C]4b). [11C]CO2 was produced by the 14N(p,
reaction in small volume (9.5 cm3) aluminum gas target (CTI) from 11 MeV
proton cyclotron on research purity nitrogen (+1% O2) in Siemens
radionuclide delivery system (Eclipse RDS-111). In small reaction vial
(5 mL), the precursor 4a (0.5–1.0 mg) was dissolved in CH3CN (300 L). To this
a)
11C nuclear
a
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solution was added NaH (1 mg). No carrier-added (high specific activity)
[
[
11C]CH3OTf that was produced by the gas-phase production method13 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 reacted 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 2 mL injection loop for
purification, which we used
10 Â 250 mm id C-18 column; 52% CH3CN/H2O mobile phase; flow rate
5.0 mL/min; and UV (254 nm) and -ray (PIN diode) flow detectors. The
a Prodigy (Phenomenex), S-5 lm, 12 nm,
c
product fraction was collected, the solvent was removed by rotatory
18. Wang, M.; Lacy, G.; Gao, M.; Miller, K. D.; Sledge, G. W.; Zheng, Q.-H. Bioorg.
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evaporation under vacuum, and the final product, [11C]PBR28 ([11C]4b), was
formulated in saline, sterile-filtered through
a sterile vented Millex-GS
19. Mock, B. H.; Zheng, Q.-H.; DeGrado, T. R. J. Label. Compd. Radiopharm. 2005, 48,
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0.22 m cellulose acetate membrane, and collected into a sterile vial. Total
radioactivity was assayed and total volume was noted for dose dispensing. The
overall synthesis, purification, and formulation time was 25–30 min from EOB.
l
Retention times in the analytical HPLC, which we used
(Phenomenex) 5
m C-18 column, 4.6 Â 250 mm; 52% CH3CN/H2O mobile
phase; flow rate 1.0 mL/min; and UV (254 nm) and -ray (PIN diode) flow
detectors, were: tR 4a = 6.51 min, tR 4b = 7.57 min, tR
11C]4b = 7.57 min.
Retention times in the semi-preparative HPLC were tR 4a = 6.50 min, tR
a Prodigy
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22. (a) Compound 1, a pale yellow solid, mp 70–71 °C (lit.10 75 °C). 1H NMR (CDCl3,
500 MHz): d 9.14 (s, 1H), 8.55 (d, J = 6.0 Hz, 1H), 7.52–7.48 (m, 2H), 7.37–7.34
(m, 1H), 7.17–7.15 (m, 2H), 6.79 (d, J = 6.0 Hz, 1H). Compound 2, a red oil. 1H
NMR (CDCl3, 500 MHz): d 8.17 (s, 1H), 7.89 (d, J = 5.5 Hz, 1H), 7.43–7.39 (m,
2H), 7.24–7.21 (m, 1H), 7.10–7.07 (m, 2H), 6.58 (d, J = 5.5 Hz, 1H), 3.95 (br s,
2H). Compound 3a, a white solid, mp 193–194 °C (lit.10 180–182 °C). 1H NMR
(DMSO-d6, 500 MHz): d 9.62 (br s, 1H), 7.86 (s, 1H), 7.70 (d, J = 5.5 Hz, 1H),
7.47–7.44 (m, 2H), 7.23 (t, J = 7.5 Hz, 1H), 7.19 (d, J = 7.0 Hz, 1H), 7.12 (d,
J = 8.0 Hz, 2H), 7.07–7.04 (m, 1H), 6.83 (d, J = 7.5 Hz, 1H), 6.74 (t, J = 7.5 Hz, 1H),
6.54 (d, J = 5.5 Hz, 1H), 5.93 (t, J = 6.5 Hz, 1H), 4.35 (d, J = 6.5 Hz, 2H). LC/MS (m/
z, ESI): [M+H]+ calcd for C18H17N2O2 293.1, found 293.0. Compound 3b, a pale
yellow oil. 1H NMR (CDCl3, 500 MHz): d 8.09 (s, 1H), 7.84 (d, J = 5.5 Hz, 1H),
l
c
[
4b = 7.98 min, tR [
11C]4b = 7.98 min. The radiochemical yields were 70–80%
decay corrected to EOB, based on [11C]CO2.
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