An improved synthesis of [11C]MENET via Suzuki coupling with [11C]methyl iodide

Article abstract of DOI:10.1002/jlcr.3047

[¹¹C]MENET, a promising norepinephrine transporter imaging agent, was prepared by Suzuki cross coupling of 1 mg N-t-Boc pinacolborate precursor with [¹¹C]CH₃I in DMF using palladium complex generated in situ from Pd₂(dba)₃ and (o-CH ₃C₆H₄)₃P together with K ₂CO₃ as the co-catalyst, followed by deprotection with trifluoroacetic acid. This improved radiolabeling method provided [ ¹¹C]MENET in high radiochemical yield at end of synthesis (EOS, 51 ± 3%, decay-corrected from end of ¹¹CH₃I synthesis, n = 6), moderate specific activity (1.5-1.9 Ci/μmol at EOS), and high radiochemical (>98%) and chemical purity (>98%) in a synthesis time of 60 ± 5 min from the end of bombardment. Copyright

Full text of DOI:10.1002/jlcr.3047

Protocols and Methods
Received 13 February 2013,
Revised 8 March 2013,
Accepted 13 March 2013
Published online 24 April 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3047
An improved synthesis of [11C]MENET via
Suzuki coupling with [11C]methyl iodide
*
Fanxing Zeng, Ronald J. Voll, Ronald J. Crowe, Michael S. Waldrep,
Karen B. Dolph, and Mark M. Goodman
[
11C]MENET, a promising norepinephrine transporter imaging agent, was prepared by Suzuki cross coupling of 1 mg N-t-Boc
pinacolborate precursor with [11C]CH3I in DMF using palladium complex generated in situ from Pd2(dba)3 and (o-CH3C6H4)3P
together with K2CO3 as the co-catalyst, followed by deprotection with trifluoroacetic acid. This improved radiolabeling
method provided [11C]MENET in high radiochemical yield at end of synthesis (EOS, 51 Æ 3%, decay-corrected from end of
11CH3I synthesis, n = 6), moderate specific activity (1.5–1.9 Ci/mmol at EOS), and high radiochemical (>98%) and chemical
purity (>98%) in a synthesis time of 60 Æ 5 min from the end of bombardment. Copyright © 2013 John Wiley & Sons, Ltd.
Keywords: [11C]MENET; Suzuki coupling; carbon-11; pinacolborate
Introduction
Experimental
[11C]MENET ([11C](2S,3S)-2-[a-(2-methylphenoxy)phenylmethyl]
morpholine), a carbon-11 labeled reboxetine analogue, is a
promising norepinephrine transporter (NET) imaging agent.1
In vivo microPET imaging studies in rhesus monkeys showed that
[11C]MENET possesses appropriate regional cerebral activity
distributions correlated to known NET density profiles with the
ratio of uptake of [11C]MENET in midbrain to that in caudate
peaked at 1.45 at 45 min, and a specific binding peak equilibrium
was achieved in less than 1 h after injection, a highly favorable
pharmacokinetic property for mapping NET occupancy in the
human brain. [11C]MENET was initially prepared by Stille
coupling of the corresponding aryltrimethyl stannane with [11C]
CH3I. In the process of preparation of [11C]MENET for initial
clinical studies, unfortunately, the specific activity of [11C]MENET
was determined to be 140–200 mCi/mmol at EOS, which was far
below the criteria (must be >300 mCi/mmol at time of patient
administration) suitable for use in clinical PET imaging studies.
Although the mechanism for the formation of low specific
General
(2S,3S)-N-tert-Butoxycarbonyl-2-[a-(2-iodophenoxy)phenylmethyl]
morpholine (1) was synthesized according to the recently reported
procedure.1 All reagents used were obtained from commercially available
sources and were used without further purification. Solvents were
anhydrous grade purchased from Aldrich and used as received. 1H NMR
spectra were recorded on a Varian spectrometer at 300MHz and referenced
to the NMR solvent (chemical shifts in ppm values, J values in Hz). High-
resolution mass spectra were acquired on a VG 70-S double focusing mass
spectrometer using electron ionization. Flash chromatography was carried
out using Merck silica gel 60 (40–63 mm particle size). Glassware used in
the radiolabeling was foil-wrapped and depyrogenated.
Synthesis of the aryl boronate precursor (2)
To a solution of 1 (81 mg, 0.16 mmol) in dioxane (2 mL) was added Pd
(OAc)2 (1.8 mg, 8 mmol), Et3N (92 mL, 0.66 mmol), and (2-biphenyl)
dicyclohexyl phosphine (11.2 mg, 0.032 mmol). The mixture was purged
under Ar for 15 min, and then pinacolborane (70 mL, 0.48 mmol) was
added dropwise. The reaction mixture was heated at 75 ꢀC for 1 h. After
activity has not been fully clarified, transfer of one of the methyl cooling to room temperature, the reaction was quenched by adding a
sat. solution of NH4Cl, and the aqueous phase was extracted with CH2Cl2.
groups in the trimethyl stannane was suggested to contribute to
The extracts were combined and dried over Na2SO4, and the solvent was
the amount of unlabeled product and consequently resulted in
evaporated under reduced pressure. The crude product was purified by
low specific activity.2–4 One possible alternative approach is to
flash chromatography on silica (deactivated with 5% Et3N) eluted with
use tributyl stannane as the precursor. However, probably
because of steric hindrance, none or only trace amount of
tributyltin precursor was obtained in spite of numerous attempts
(data not presented). Recent advances in palladium-catalyzed
methylation of organoboron compounds with [11C]CH3I based
on Suzuki–Miyaura coupling prompted us to pursue a boron
hexane/EtOAc (80:20) to afford 2 as colorless oil (64 mg, 79%). 1H NMR
(CDCl3, 300 MHz), d 7.62 (dd, J = 7.3, 1.7 Hz, 1H), 7.45 (d, J = 6.7 Hz, 2H),
7.17–7.33 (m, 4H), 6.86 (m, 1H), 6.68 (d, J = 8.2 Hz, 1H), 5.22 (d, J = 5.0 Hz,
Department of Radiology and Imaging Sciences, Emory University, 1841 Clifton
precursor as an alternative approach. [11C]MENET was prepared Road NE, Atlanta, GA, 30329, USA
by cross coupling of N-t-Boc arylboronate ester with [11C]CH3I
*Correspondence to: Fanxing Zeng, Department of Radiology and Imaging
under palladium catalysis followed by deprotection with TFA in
high radiochemical yield and moderate specific activity.
Sciences, Emory University, 1841 Clifton Road NE, Atlanta, GA 30329 USA.
E-mail: fzeng@emory.edu
J. Label Compd. Radiopharm 2013, 56 307–309
Copyright © 2013 John Wiley & Sons, Ltd.

F. Zeng et al.
1H), 3.82–3.95 (m, 4H), 3.52 (dt, J = 2.6, 11.7 Hz, 1H), 2.89 (m, 1H), 2.71
(m, 1H), 1.39 (m, 21H). HRMS [MH]+ Calcd for C28H39BNO6: 496.2865,
Found: 496.2862.
Radiosynthesis of [11C]MENET
No carrier-added [11C]CO2 was produced through the bombardment of 14N2
gas containing 1% 16O2 (dual targets, 55 mA, 80 min) by a Siemens 11 MeV
RDS 111 cyclotron (Knoxville, TN, USA) at Emory University Center for Systems
Imaging through the 14N[p,a]11C reaction. A GE TracerLab FX MeI system was
employed for the conversion of [11C]CO2 to [11C]CH3I. Pd2(dba)3 (1.8 mg,
2.0 mmol), (o-Tol)3P (2.4 mg, 7.7 mmol), and K2CO3 (2.8 mg, 20 mmol) were
placed in a 3-mL dry conical vial, and then the vial was sealed and purged
with Ar for 15 min. A solution of 2 (1.0 mg, 2.0 mmol) in DMF (300 mL) was
added to the vial, and then 11CH3I was bubbled through the solution by a
stream of helium (12 mL/min) at room temperature. When the maximum
delivery of [11C]MeI was reached, the vial was heated at 90 ꢀC for 4min, TFA
(0.22 mL, ~750 equiv) was added, and the solution was heated at 90 ꢀC for
another 6 min, cooled in an ice bath for 40 s, and neutralized by addition of
5M NH4OH(aq) (0.6 mL, ~800 equiv.). The reaction mixture was pressure
filtered through a 0.45-mm Acrodisc filter into a conical vial to remove salts
and palladium residues before loading on a semipreparative HPLC (Water
XTerra Prep RP18 5mm, 19 Â 100 mm) for purification. The column was eluted
with buffered mobile phase consisting 45:55:0.1 v/v/v EtOH/H2O/Et3N at a
9-mL/min flow rate. Under these conditions, the [11C]MENET product had a
retention time of approximately 11 min. The desired fractions eluting at this
time were collected, diluted with a double volume of water, and loaded onto
a Waters tC18 SepPak cartridge, which was then washed with saline (0.9%
NaCl, 40 mL). The radioactive product was eluted from the cartridge by
absolute ethanol (1.0 mL) into a sealed sterile vial containing 9.0 mL of saline.
The resulting solution was then filtered under pressure through a 0.2-mm
Millex-FG filter to give a sterile aqueous solution of [11C]MENET, which was
submitted for quality control testing.
Figure 1. Chromatograms of analytical HPLC of [11C]MENET: (a) radioactivity and
(b) UV absorbance at 280 nm.
Although several methods were reported for the synthesis of
sterically hindered ortho-substituted arylboronic acid or
arylbornate, in our hands, 2 was efficiently synthesized using
the procedure developed by Baudoin et al.5–9 As shown in
Scheme 1, under the catalytic system of Pd(OAc)2 and sterically
hindered phosphine ligand with a ratio of 1:4, pinacolboranate
precursor 2 was obtained in a high isolated yield of 79% by
reaction of iodo compound 1 with pinacolborane in dioxane
using Et3N as the base under relatively mild condition.
Radiochemical and chemical purities were assessed using a Waters
Alliance 2695 HPLC equipped with a Bioscan flow-count radioactivity
detector (Waters Nova-Pak C18 4 mm, 3.9 Â 300 mm; mobile phase:
MeOH/H2O/Et3N = 75:25:0.1; flow rate: 1.0 mL/min; tR = 7.39 min).
A
representative chromatogram is shown in Figure 1. Specific activity was
determined by using the same analytical HPLC system against a
calibration curve prepared with the [12C]MENET standard. Sterility testing
was performed in-house per US Pharmacopeial guidelines. Briefly, culture
tubes of soybean casein and fluid thioglycollate mediums were inoculated
with samples of [11C]MENET and incubated, along with positive and negative
controls, for 14 days with daily visual inspect (excluding weekends and
holidays). All [11C]MENET-doped cultures were clear of any growth of
microorganisms (n= 6). Bacterial endotoxin pyrogen testing was performed
using the Charles River Laboratories Endosafe Portable Testing System and
[11C]MENET was prepared from N-t-Boc pinacolboranate (2) in
a two-step, one-pot radiosynthesis. The first step was a
palladium-catalyzed cross coupling reaction between 2 and
[11C]CH3I using a modified procedure developed by Suzuki et al.10
Palladium-catalyzed cross coupling reactions utilizing organoboron
in accordance with US Pharmacopeia. All results showed an endotoxin compounds, commonly referred to as Suzuki couplings, have
concentration of <5.0 EU/mL (n=6).
recently been shown to be an effective labeling method alternative
to the Stille coupling for the synthesis of compounds containing a
[11C]methylphenyl moiety.10–14
Results and discussion
Reacting 2 (1 mg) with [11C]CH3I at 90 ꢀC using palladium
The N-t-Boc pinacolboranate precursor (2) was prepared by palladium- complex generated in situ from Pd2(dba)3 and (o-CH3C6H4)3P
catalyzed borylation of iodo compound (1)1 with pinacolborane. (1:4) together with K2CO3 as the co-catalyst gave us a high
Scheme 1. Synthesis of N-t-Boc pinacolborane precursor (2) and radiosynthesis of [11C]MENET.
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 307–309

F. Zeng et al.
coupling yield. The N-Boc group was cleaved under acidic deprotection provided [11C]MENET in high radiochemical yield
conditions, and the reaction mixture was then neutralized before and improved specific activity. Imaging studies using [11C]
HPLC purification.
MENET in humans are now in progress.
The total synthesis time (including purification and formulation)
of [11C]MENET was 60 Æ 5 min from the end of bombardment. The
dose of [11C]MENET was a clear, colorless, and particulate-free
solution with pH = 5.
Conflict of Interest
The authors did not report any conflict of interest.
Starting with 950–1100 mCi of [11C]methyl iodide, typical
syntheses provided 104–140 mCi (uncorrected) of [11C]MENET at
EOS in an average radiochemical yield of 51 Æ 3% (n=6, decay-
corrected from end of 11CH3I synthesis). The radiochemical and
chemical purity of [11C]MENET were consistently >98%, and the
specific activities were in the range of 1.5–1.9 Ci/mmol at EOS (n=6).
It is worthy to point out that, to the best of our knowledge,
this is the first report that showed successful radiolabeling using
only 1 mg precursor in the Suzuki coupling or Stille coupling with
[11C]CH3I, whereas all other previous reports used 2 mg or more
precursor in the coupling reaction.
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In this study, an improved method for the preparation of the NET
imaging agent [11C]MENET has been reported. The method
based on the Suzuki cross-coupling of N-t-Boc pinacolboranate
precursor with [11C]CH3I under palladium catalysis followed by
J. Label Compd. Radiopharm 2013, 56 307–309
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org