[carbonyl-11C]Benzyl acetate: Automated radiosynthesis via Pd-mediated [11C]carbon monoxide chemistry and PET measurement of brain uptake in monkey

Article abstract of DOI:10.1002/jlcr.1779

[carbonyl-¹¹C]Benzyl acetate ([¹¹C]1) has been proposed as a potential agent for imaging glial metabolism of acetate to glutamate and glutamine with positron emission tomography. [¹¹C]1 was synthesized from [¹¹C]carbon monoxide, iodomethane and benzyl alcohol via palladium-mediated chemistry. The radiosynthesis was automated with a modified Synthia platform controlled with in-house developed Labview software. Under production conditions, [¹¹C]1 was obtained in 10% (n = 6) decay-corrected radiochemical yield from [¹¹C]carbon monoxide in >96% radiochemical purity and with an average specific radioactivity of 2415 mCi/μmol. The total radiosynthesis time was about 45 min. Peak uptake of radioactivity in monkey brain (SUV = 3.1) was relatively high and may be amenable to measuring uptake and metabolism of acetate in glial cells of the brain.

Full text of DOI:10.1002/jlcr.1779

Research Article
Received 9 March 2010,
Revised 24 March 2010,
Accepted 24 March 2010
Published online 7 July 2010 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1779
[carbonyl-11C]Benzyl acetate: Automated
radiosynthesis via Pd-mediated [11C]carbon
monoxide chemistry and PET measurement
of brain uptake in monkeyy
Ã
Shuiyu Lu,a Jinsoo Hong,a Tetsuji Itoh,a Masahiro Fujita,a Osamu Inoue,b
Robert B. Innis,a and Victor W. Pikea
[carbonyl-11C]Benzyl acetate ([11C]1) has been proposed as a potential agent for imaging glial metabolism of acetate to
glutamate and glutamine with positron emission tomography. [11C]1 was synthesized from [11C]carbon monoxide,
iodomethane and benzyl alcohol via palladium-mediated chemistry. The radiosynthesis was automated with a modified
Synthia platform controlled with in-house developed Labview software. Under production conditions, [11C]1 was obtained
in 10% (n = 6) decay-corrected radiochemical yield from [11C]carbon monoxide in 496% radiochemical purity and with an
average specific radioactivity of 2415 mCi/mmol. The total radiosynthesis time was about 45 min. Peak uptake of
radioactivity in monkey brain (SUV = 3.1) was relatively high and may be amenable to measuring uptake and metabolism of
acetate in glial cells of the brain.
Keywords: [11C]benzyl acetate; carbon-11; [11C]carbon monoxide; carbonylation; PET
may also vary in reactivity with concentration and age.
Furthermore, such reagents are sensitive to trace moisture and
Introduction
Radiolabeled acetate has been proposed as a potential in vivo
marker of cerebral oxidative metabolism1 and also of glial cell
metabolism,2 specifically the incorporation of acetate into
glutamate and glutamine. However, the brain uptake of
radiolabeled acetate following intravenous administration is
inadequate.3 [carbonyl-14C]Benzyl acetate, among several
14C-labeled aryl acetates, displays much higher brain–blood
barrier permeability and brain retention than [carbonyl-14C]
acetate in rat.4–6 Benzyl acetate (1) is readily hydrolyzed to
acetate within brain. The labeling of 1 in its carbonyl position
with the positron-emitter carbon-11 (t1/2 = 20.4 min) might
therefore provide an agent for the delivery of [carbonyl–11C]
acetate into brain for the purpose of imaging glial metabolism
in vivo with positron emission tomography (PET).
Classically, 11C-labeled esters have been synthesized via the
carboxylation of Grignard7–12 or organolithium reagents13,14 with
cyclotron-produced [11C]carbon dioxide, followed by chlorination
with a suitable reagent, such as thionyl chloride, oxalyl chloride or
phthaloyl chloride, and finally reaction of the resulting 11C-labeled
acid chloride with the appropriate alcohol. On a practical note,
methods using Grignard reagents are demanding because freshly
prepared reagent is usually required to avoid low specific
radioactivity and low radiochemical yield. Any ingress of atmo-
spheric carbon dioxide into the reagent will lower the specific
radioactivity of the reacting [11C]carbon dioxide and therefore of
the radioactive product. Grignard reagents, which are complex
mixtures of species participating in the ‘Schlenk equilibrium’,15
oxygen, so increasing the difficulty of their use in small-scale
automated radiochemistry. Organolithium reagents show similar
handling difficulties and sensitivity to moisture.
Alternatively, [carbonyl-11C]esters have also been prepared via
transition metal-16,17 or radical-mediated18,19 carbonylation
reactions of organo-halides with [11C]carbon monoxide,20
derived from cyclotron-produced [11C]carbon dioxide. The
radiochemical yields are quite low because alcohols are weak
nucleophiles.16 Nevertheless, useful radiochemical yields can still
be obtained through careful choice of appropriate alkyl halides
and alcohols. Transition metal-mediated [11C]carbon monoxide
chemistry does not require freshly prepared reagents and has
low sensitivity to moisture.19 Moreover, the normally very low
level of atmospheric carbon monoxide poses negligible risk for
contaminating [11C]carbon monoxide reagent and thereby for
diluting the specific radioactivity of labeled product. For these
aMolecular Imaging Branch, National Institute of Mental Health, National
Institutes of Health, 10 Center Drive, Bethesda, MD 20892-1003, USA
bDivision of Health Sciences, Graduate School of Medicine, Osaka University, 1-7
Yamadaoka, Suita, Osaka 565-0871, Japan
*Correspondence to: Shuiyu Lu, Molecular Imaging Branch, National Institute of
Mental Health, National Institutes of Health, 10 Center Drive, Bethesda, MD
20892-1003, USA.
E-mail: Shuiyu.Lu@mail.nih.gov
yThis article is a U.S. government work and is in the public domain in the USA.
J. Label Compd. Radiopharm 2010, 53 548–551
Published in 2010 by John Wiley & Sons, Ltd.

S. Lu et al.
reasons along with our interest in exploring the carbonylation of methyl triflate towards benzyl alcohol. The Pd ligand was
chemistry, we chose to use palladium-mediated [11C]carbon generally used at 1–4% molar equivalent to the methyl iodide.
monoxide chemistry to produce [11C]1 in adequate activity for (4-Dimethylamino)pyridine has previously been found useful for
evaluation in monkey as a potential PET radiotracer. A Synthia the syntheses of 11C-labeled esters from [11C]carbon monoxide,16
apparatus,21 equipped with a high pressure micro-autoclave22 but here its use as an acylation catalyst did not improve the
and controlled with in-house developed software, was used to radiochemical yield of [11C]1 (Table 1 entry 3). Use of a larger
prepare [11C]1 automatically from [11C]carbon monoxide within quantity of any of the reagents or longer reaction time, increased
a lead-shielded hot-cell (Figure 1). The brain uptake of [11C]1 in a the yield only marginally. However, excess reagents, especially
rhesus monkey was measured with PET.
benzyl alcohol, complicated the subsequent HPLC purification of
[11C]1 because benzyl alcohol (tR = 13.2–14.2 min) eluted very
near [11C]1 (tR = 14.3–15.0 min). The conditions listed in entry 12
were selected as optimal for the production of [11C]1 for
intravenous injection. The weak nucleophilicity of benzyl alcohol
accounts for the overall low radiochemical yields.18
Although the boiling point of benzyl acetate is 2061C, we
observed heavy loss of NCA [11C]1 during its formulation for
intravenous injection, where acetonitrile must be removed from
the HPLC fraction under reduced pressure. The pressure,
temperature and time had to be carefully controlled to conserve
[11C]1 (Table 2). Removal of acetonitrile at 351C and at reduced
pressure (32 Pa) for 30 s achieved a good balance between
retaining most (ꢀ70%) of the radioactive product and removing
solvent to an acceptably safe level (20 mg/kg)23,24 for intrave-
nous injection.
Results and discussion
[11C]1 was successfully produced for intravenous injection into
monkey from the Pd-mediated 11C-carbonylation of methyl
iodide in the presence of benzyl alcohol (Table 1). No [11C]1 was
obtained from the use of methyl triflate in place of methyl iodide
(Table 1, entry 1), possibly because of the greater direct reactivity
For production runs, formulated NCA [11C]1 was obtained in
10% (n = 6) decay-corrected radiochemical yield (RCY) from
[11C]carbon monoxide in 496% radiochemical purity and with
an average specific radioactivity of 2415mCi/mmol (n = 6). The
total radiosynthesis time was about 45min from the end of
radionuclide production. The specific radioactivity value for [11C]1
is at the lower end of the range achieved in our laboratory for the
Figure 1. Synthesis of [carbonyl-11C]benzyl acetate via Pd-mediated [11C]carbon
monoxide chemistry.
Table 1. Synthesis of [11C]1 with Pd-mediated [11C]carbon monoxide chemistry under various conditions
Entry
Pd catalyst (mmol)
MeI (mmol)
C6H5CH2OH (mmol)
Temp. (1C)
Time (min)
RCY of [11C]1a (%)
1
2
3
4
5
6
7
8
2.4
2.8
2.9
1.7
1.6
1.8
0.8
0.9–1.3
1.3
1.3
1.2
1.4
62 (MeOTf)
112
112
161
161
161
80
193
193
193
193
193
193
48
96
48
48
77
130
130
130
130
140
140
140
140
140
140
140
140
4
4
4
4
2
6
2
2
2
2
2
2
0
12.1
9.6b
14.2
6.7
13.5
5.7
9.6 (n = 4)
2.6
4.3
12.1
10.1 (n = 6)
80
32
48
48
9
10
11
12
80
77
aDecay-corrected radiochemical yield from trapped [11C]carbon monoxide.
bWith DMAP (22 mmol).
Table 2. Formulation efficiency for four production runs
[11C]1 HPLC
fraction (mCi)
Rotary evaporation
conditions (1C) ꢁ (s)
Recovery
efficiency (%)
MeCN
(mg/mL)a
Entry
Final dose (mCi)
1
2
3
4
3.60
13.8
0.80
12.2
80 ꢁ 30
70 ꢁ 52
35 ꢁ 30
35 ꢁ 35
1.33
1.36
0.42
5.70
45
12
70
59
0.395
0.034
9.32
5.50
aMeasured by GC using PrCN as external standard.
J. Label Compd. Radiopharm 2010, 53 548–551
Published in 2010 by John Wiley & Sons, Ltd.
www.jlcr.org

S. Lu et al.
in-house. Cyclotron-produced NCA [11C]carbon dioxide was first
collected in a 5-loop stainless steel tube trap filled with
molecular sieves (13X) at room temperature. The trap was
purged with helium at 90 mL/min for 2 min to remove oxygen.
[11C]Carbon dioxide was released in a helium stream (16 mL/
min) at 3601C into a cryogenic trap filled with silica. [11C]Carbon
monoxide was obtained by single pass conversion of the
[11C]carbon dioxide over a mixture of molybdenum wire26
(99.97%, 0.05 mm diameter, Strem Chemicals, Newburyport, MA)
and powder (99.95%, 100 mesh, Strem Chemicals) in a quartz
tube (22 cm length, 0.7 cm i.d.) heated at 8451C. [11C]Carbon
monoxide was first collected on silica gel in a stainless steel tube
cooled with liquid nitrogen and then released into the
autoclave22 for reaction by warming the trap with a lamp.
Iodomethane (80mmol), benzyl alcohol (80 mmol) and Pd(PPh3)4
(1.4mmol) were premixed in THF (150mL) and loaded into the
autoclave. [11C]Carbon monoxide was released from the cryo-
genic trap and directed into the autoclave at room temperature.
The autoclave was sealed and heated at 1401C for 2 min in a
heating well. The reaction mixture was then flushed out of the
autoclave with THF (0.7 mL), diluted with water (3mL) and then
injected onto a semi-preparative size reverse phase column (Luna
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Cerebral cortices
Cerebellum
0
0.5
1
1.5
2
Time after injection (h)
Figure 2. Brain region time–activity curves after intravenous injection of [11C]1
into a rhesus monkey.
production of other 11C-labeled radiotracers from [11C]carbon
dioxide under similar cyclotron irradiation conditions, and
indicates that contamination of the radiosynthetic mixture by
extraneous carbon monoxide is not greatly problematic.
After administration of NCA [11C]1 to monkey, radioactivity
entered brain quickly and reached quite high levels of 3.1 and 2.1
SUV in cerebral cortices and cerebellum, respectively, at 1.75min
(Figure 2). Thereafter, radioactivity inside brain declined slowly.
˚
C18, 5 m 100 A, 250 ꢁ 10mm i.d.; Phenomenex, Torrance, CA)
eluted at 4 mL/min with a linear gradient of water (A)-MeCN (B),
starting with 30% B for 4 min and increasing to 60% B in 2 min.
Eluate was monitored for radioactivity and absorbance at 210 nm.
The product fraction eluting between 14.3 and 15.0 min was
collected. Acetonitrile was removed at 351C under reduced
pressure (32 Pa). [11C]1 was formulated in saline containing
ethanol (10% w/v) and filtered through a Millex-MP sterile filter
(0.22 mm; Millipore, Carrigtwohill, Ireland). RCY was calculated
from trapped [11C]carbon monoxide. The starting radioactivity of
[11C]carbon monoxide was measured separately in six identical
production runs for the computation of average RCY.
Experimental
General procedures
LC-MS analysis was performed on a Surveyor LC system
equipped with a Quest LC QDECA ESI probe (Thermo Electron
Corp.; San Jose, CA). GC-MS was performed on a Trace gas
chromatograph (Thermo Finnigan; San Jose, CA) equipped with
Polaris Q mass spectrometer. Acetonitrile residue was deter-
mined with gas chromatography on a 6850 Network GC
instrument (Agilent Technologies, Foster City, CA) equipped
with a DB-WAX capillary column (30 m length, 0.25 mm i.d.).
Radioactivity was measured with a calibrated AtomlabTM 300
dose calibrator (Biodex Medical Systems; Shirley, NY), and
corrected for background and physical decay of carbon-11.
No-carrier-added (NCA) [11C]carbon dioxide was prepared by the
14N(p,a)11C nuclear reaction by bombarding a nitrogen gas target
(1% oxygen, pressure 300 p.s.i.) with a beam of protons (16 MeV,
25 mA) from a cyclotron (PETrace; GE) for 40 min. Semi-preparative
scale and analytical HPLC were performed on two different systems
comprising a System Gold 126 solvent module (Beckman Coulter;
Fullerton, CA) coupled with a 166 UV absorbance detector (plus a
Flow-count radioactivity detector (diode for semi-preparative or
PMT for QC analysis; Bioscan, Washington, DC).
[11C]1 (tR = 5.4 min) was shown to co-elute with reference benzyl
˚
acetate from a Luna C18 column (5 m 100 A, 250ꢁ 4.6 mm i.d.;
Phenomenex, Torrance, CA) eluted with acetonitrile-25 mM ammo-
nium formate solution (60:40, v/v) at 1.2 mL/min and monitored for
absorbance at 210 nm and radioactivity. GC-MS analysis of [11C]1
showed identical retention time (tR = 10.69 min) and mass spectrum
(for M1 m/z= 150) compared with non-radioactive standard.
PET imaging
A rhesus monkey (11.28 kg) was anesthetized with 1.5% isoflurane
and injected intravenously with a bolus of NCA [11C]1 (5.99 mCi;
0.43mg of carrier). Serial dynamic images were recorded on a
High Resolution Research Tomography (Siemens/CPS, Knoxville,
TN) PET camera over 120 min. Decay-corrected time–activity
curves were obtained for irregular volumes of interest, selected
from cerebral cortex (10.9 cm3) and cerebellum (3.2cm3). Radio-
activity was normalized for injected dose and monkey weight by
expression as standardized uptake value (SUV):
Animal procedures
Animal procedures were performed in strict accordance with the
National Institutes of Health Guide for Care and Use of Laboratory
Animals25 and were approved by the National Institute of Mental
Health (NIMH) Animal Care and Use Committee.
Injected dose ðmCiÞ per g tissue
ꢁ Body weight ðgÞ
Injected radioactivity ðmCiÞ
Conclusions
Radiochemistry
The procedure was fully automated using a modified Synthia21
module under control with Labview-based software developed
Palladium-mediated [11C]carbonylation chemistry provided a
practically convenient route to [11C]1, avoiding air-sensitive
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Published in 2010 by John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 548–551

S. Lu et al.
reagents. It is envisaged that the automated process could be
used to produce other 11C-labeled esters, as may be required.
[11C]1 has acceptably high brain uptake and radioactivity is well
retained. Further study is needed to assess the suitability [11C]1
as a PET radiotracer of brain glial metabolism.
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This work was supported by the Intramural Research Program of
the National Institutes of Health (National Institute of Mental
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J. Label Compd. Radiopharm 2010, 53 548–551
Published in 2010 by John Wiley & Sons, Ltd.
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