Facile synthesis of [11C]edrophonium and its analogues as new potential PET imaging agents for heart acetylcholinesterase

Article abstract of DOI:10.1016/S0960-894X(03)00259-2

[¹¹C]Edrophonium and its analogues have been synthesized for evaluation as new potential positron emission tomography (PET) imaging agents for heart acetylcholinesterase. The tracers were prepared by N-[¹¹C]methylation of precursors

Full text of DOI:10.1016/S0960-894X(03)00259-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1787–1790
11
Facile Synthesis of [ C]Edrophonium and its Analogues as New
Potential PET Imaging Agents for Heart Acetylcholinesterase
Qi-Huang Zheng,* Xuan Liu, Xiangshu Fei, Ji-Quan Wang, Bruce H. Mock,
Barbara E. Glick-Wilson, Michael L. Sullivan and Gary D. Hutchins
Department of Radiology, Indiana University School of Medicine, 975 West Walnut Street, Room 028C,
Indianapolis, IN 46202-5121, USA
Received 17 September 2002; accepted 7 February 2003
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Abstract—[ C]Edrophonium and its analogues have been synthesized for evaluation as new potential positron emission tomo-
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graphy (PET) imaging agents for heart acetylcholinesterase. The tracers were prepared by N-[ C]methylation of precursors using
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1
[
C]methyl triflate and isolated by solid-phase extraction (SPE) purification procedure in 50–65% radiochemical yields.
2003 Elsevier Science Ltd. All rights reserved.
#
Edrophonium and neostigmine are acetylcholinesterase
AChE) inhibitors used to treat a number of heart con-
ditions in humans due to their high affinities to the
PET marker for heart AChE and parasympathetic
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3
(
innervation. As part of our efforts to evaluate poten-
4,15
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tial heart imaging agents,
phonium and its methyl and ethyl analogues.
we synthesized [ C]edro-
1À7
enzyme target. AChE positive neuron fibers make up
a significant portion of the heart’s conduction system
and sensory nerves, and AChE enzyme-based imaging
The reliable routine production, automation and spe-
cific radioactivity of carbon-11 heart imaging agents
require high yields, short reaction times, lower reaction
temperatures and more efficient purification methods.
In order to explore novel methodology for the synthesis
of carbon-11 heart imaging agents, we have developed a
convenient and reliable gas phase production (GPP)
method for the most commonly used radiolabeled pre-
8
agents could be useful in studying parasympathetic
function in the heart. In vivo biomedical imaging tech-
nique positron emission tomography (PET) coupled
with appropriate radiopharmaceuticals has become a
clinically valuable and accepted diagnostic tool to image
9
,10
heart diseases,
since it produces powerful images of
the human body’s biological functions and reveals the
mysteries of health and disease. Considerable efforts
have been devoted to the development of radio-
pharmaceuticals for the in vivo imaging of the AChE,
however, only a limited number of PET studies have
been conducted to image AChE and monitor its
response to AChE inhibitors treatment, because of the
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cursor [ C]methyl triflate from gas phase bromination
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of [ C]methane (Fig. 2), a fast and efficient solid
phase extraction (SPE) method for isolation and pur-
ification of C-radioligands, and a convenient
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18À21
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limited accessibility of radiotracers.
Radiotracer
development, both for labeled drugs and for labeled
tracers, is a key area for advancement of research and
clinical applications of PET in the detection and treat-
1
2
ment of heart diseases. The short-lived positron emit-
ting radionuclide carbon-11 (half-life t1/2 20.4 min)
labeled analogues of edrophonium and neostigmine
might be able for PET to image heart AChE in vivo. We
Figure 1. Synthesis of [11C]neostigmine.
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1
have developed [ C]neostigmine (Fig. 1) as a potential
*
1
Corresponding author. Tel.: +1-317-278-4671; fax: +1-317-278-
072; e-mail: qzheng@iupui.edu
Figure 2. GPP method for [11C]methyl triflate.
0
960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00259-2

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Q.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1787–1790
labeling and isolation method for the preparation of
1
onto the SiO Sep-Pak cartridge by gas pressure. The
2
1
11
[
ation of their precursors,
C-methyl]quaternary amines through N-[ C]methyl-
2
cartridge was washed with ethanol to conveniently
remove non-reacted tertiary amine precursor, reaction
2,23
in which the similar
1
1
11
method for the production of [ C-methyl]quaternary
1
solvent, and non-reacted [ C]methyl triflate, which was
decomposed by ethanol, therefore, there was no coelu-
tion with the product, then the final labeled product
1
24
amine [ C]choline was also reported by Pascali et al.
We used these new methodologies to label these novel
agents.
1
1
[ C-methyl]quaternary amine was eluted with an aqu-
eous solution of 2% acetic acid, which can also contain
The synthetic approach for 11C-labeled edrophonium and
analogues was shown in Scheme 1. The commercially
available starting material 3-(ethylamino)phenol (4) was
methylated by methyl iodide to give the precursor
up to 8% ethanol to enhance recovery of some [ C-
methyl]quaternary cations. The presence of acid appears
11
1
1
+
3
to be crucial for efficient release of the R N– CH
3
from the silica. Only a minor pH adjustment with 2 M
NaOH and 150 mM NaH PO mixed solution and
2
5,26
3-(ethylmethylamino)phenol (5) for radiolabeling.
precursor 5 was labeled by [ C]methyl triflate through
The
2
4
1
1
sterile filtration are required to make the product ready
for injection. To be sure the final product was not con-
1
1
22À24
and isolated by SPE purifica-
N-[ C]methylation
tion to produce pure target compound [ C]edrophonium,
1
1
11
taminated with [ C]methyl triflate or derivatives, a
simple experiment was performed in light of the sug-
1
1
[ C]dimethylethyl-(3-hydroxyphenyl)ammonium
The commercially available precursor 3-(diethylamino)-
phenol (6) was N-[ C]methylated and SPE-purified to
provide [ C]edrophonium ethyl analogue, [ C]diethyl-
methyl-(3-hydroxyphenyl)ammonium (2). Another
commercially available precursor 3-(dimethylamino)-
(1).
1
1
gestions made by a reviewer. [ C]Methyl triflate was
submitted directly, without reacting it with a precursor,
to the Sep-Pak procedure, and then the eluates were
analyzed on HPLC. The radio-HPLC chromatogram of
aqueous eluate unequivocally proved the final product
1
1
1
1
11
1
1
11
phenol (7) was N-[ C]methylated and SPE-purified to
1
was not contaminated with [ C]methyl triflate or deri-
vatives. The principle of radiolabeling and SPE isolation
1
11
provide [ C]edrophonium methyl analogue, [ C]tri-
methyl-(3-hydroxyphenyl)ammonium (3).
1
1
of [ C-methyl]quaternary amines was illustrated in
Figure 3. The schematic diagram of combined
1
1
A simple technique for convenient labeling and isolation
1
N-[ C]methylation system apparatus was configured as
indicated in Figure 4.
1
11
of [ C-methyl]quaternary amines by N-[ C]methyla-
2
2À24
tion method
was employed in the radiosynthesis of
C-labeled edrophonium and analogues. The key part
in this technique is a SiO Sep-Pak cartridge containing
General
1
method
for
C]edrophonium and its ethyl and methyl analogues (1–3)
the
radiosynthesis
of
1
1
1
[
2
1
1
14
11
0
.5–2 g of adsorbent, which was obtained from Waters
CO was produced by the N(p,a) C nuclear reac-
3
2
Corporate Headquarters, Milford, MA. The large
polarity difference between the tertiary amine precursor,
tion in small volume (12.3 cm ) aluminum gas target
(CTI) from 11 MeV proton cyclotron on research purity
nitrogen (+3% O ) in a Siemens radionuclide delivery
1
1
11
[
C]methyl triflate and the labeled [ C-methyl]qua-
2
ternary amine product permitted the use of SPE tech-
nique for fast purification of radiotracer from the
radiolabeling reaction mixture, and warranted the final
product cannot be contaminated with [ C]methyl tri-
flate or derivatives. The reaction mixture was loaded
system (RDS-112). The precursor (5, 6, or 7) (1–2 mg)
was dissolved in acetonitrile (250 mL). The mixture was
transferred to a small volume, three-neck reaction tube.
1
1
11
17
[ C]methyl triflate was produced by the GPP method
1
1
from CO as illustrated in Figure 2 and passed into
2
ꢀ
air-cooled reaction tube at À15 to À20 C, which was
generated by a Venturi cooling device powered with 100
psi compressed air, until radioactivity in solution
reached a maximum (2–3 min), then reaction tube was
ꢀ
isolated and heated at 70–80 C for 2–3 min. The reac-
tion tube was connected to the Silica Sep-Pak. The
product solution was passed onto the Silica Sep-Pak for
SPE purification by gas pressure. The reaction tube and
Sep-Pak were washed with ethanol (5 mLÂ2), and the
Figure 3. The principle of radiolabeling and SPE isolation of [11C-
methyl]quaternary amines.
Scheme 1. Synthesis of 11C-labeled edrophonium and analogues (1–3).

Q.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1787–1790
1789
Figure 4. Radiosynthesis and SPE purification system of 11C-labeled edrophonium and analogues.
washing solution was discarded to a waste bottle. The
product was eluted from the Sep-Pak with
Acknowledgements
9
0:8:2H O:EtOH:HOAc (9.2 mL) and sterile-filtered
2
This work was partially supported by the Indiana 21st
Century Research and Technology Fund and the Indi-
ana Genomics Initiative (INGEN) of Indiana Uni-
versity, which is supported in part by Lilly Endowment
Inc. The reviewers’ criticisms and editor’s comments for
the revision of the manuscript are greatly appreciated.
through a Millex-GS 0.22 mm cellulose acetate mem-
brane (Millipore Corporation, Bedford, MA) and col-
lected into a sterile vial. The pH was adjusted to 5.5–7.0
with 2 M NaOH and 150 mM NaH PO mixed solution
2
4
(
0.8 mL). Total radioactivity (200–280 mCi) was
assayed and the total volume (10 mL) was noted. The
overall synthesis time was 10–15 min. The radio-
1
1
chemical yield of [ C]methyl triflate is 70–75% based
1
References and Notes
1
17
on CO2. The radiochemical yields for target radio-
1
1
tracers 1–3 were 50–65%, based on CO , decay cor-
2
1. Katz, R. L. Anesthesiology 1967, 28, 327.
2
rected to end of bombardment (EOB). Chemical purity,
radiochemical purity, and specific radioactivity were
determined by analytical HPLC method, which
employed a Prodigy (Phenomenex) 5 mm C-18 column,
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6
4
.6Â250 mm; 3:1:3 CH CN: MeOH: 20 mM, pH 6.7
3
À
KHPO mobile phase, 1.5 mL/min flow rate, and UV
4
5
(
240 nm) and g-ray (NaI) flow detectors. Retention
times in the analytical HPLC system were: RT5=4.14
min, RT6=5.62 min, RT7=3.35 min; RT1=2.12 min,
RT2=2.06 min, RT3=2.06 min. The chemical purities
of the precursors 5–7 were >98%. The radiochemical
purities of the target radiotracers 1–3 were >99%, and
the chemical purities of the target tracers 1–3 were
6
>
95%. The average (n=5–8) specific radioactivity of
9
1
5
1
1
2
1
. Bergmann, S. R. Semin. Nucl. Med. 1998, 28, 320.
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the target radiotracers 1–3 was >1 Ci/mmol at end of
synthesis (EOS).
In summary, the novel methodology based upon the
1
1
[
C]methyl triflate GPP method and the SPE purifica-
1
1
tion method for the production of C-PET radio-
tracers works very well for the routine synthesis of
heart imaging agents C-labeled edrophonium and
analogues.
3. Mulholland, G. K.; Zheng, Q.-H.; Mock, B. H.; Carlson,
K. A.; Giger, S.; Vavrek, M. T.; Fain, R.; Hutchins, G. D. J.
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14. Hutchins, G. D.; Chen, T.; Carlson, K. A.; Fain, R. L.;
1
1

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26. Experimental procedure: (A) General: all commercial
reagents and solvents were used without further purification.
H NMR spectra were recorded on a Bruker QE 300 NMR
1
spectrometer using tetramethylsilane (TMS) as an internal
standard. Chemical shift data for the proton resonances were
reported in parts per million (d) relative to internal standard
TMS (d 0.0). Chromatographic solvent proportions are
expressed on a volume: volume basis. Thin layer chromato-
graphy was run using Analtech silica gel GF uniplates (5Â10
Zipes, D. P.; Hutchins, G. Circulation 1998, 98, 2608.
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1
1
7. Mock, B. H.; Mulholland, G. K.; Vavrek, M. T. Nucl.
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8. Mulholland, G. K.; Zheng, Q.-H. Abstracts of Papers of
the American Chemical Society 1996, 212 76-NUCL.
1
2
cm ). Plates were visualized by UV light. Normal phase flash
1
2
2
9. Zheng, Q.-H.; Mulholland, G. K. Nucl. Med. Biol. 1996,
3, 981.
0. Fei, X. S.; Zheng, Q-H.; Hutchins, G. D.; Liu, X.; Stone,
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. The reaction was
performed under a positive pressure of nitrogen maintained by
a direct line from a nitrogen source. (B) 3-(Ethylmethylami-
no)phenol (5): a mixture of 3-ethylaminophenol (4, 1.37 g, 10
mmol), potassium carbonate (2.07 g, 15 mmol), methyl iodide
K. L.; Carlson, K. A.; Mock, B. H.; Winkle, L.; Glick-Wilson,
B. E.; Miller, K. D.; Fife, R. S.; Sledge, G. W.; Sun, H. B.;
Carr, R. E. J. Labelled Compd. Radiopharm. 2002, 45, 449.
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Mock, B. H.; Stone, K. L.; Martinez, T. D.; Miller, K. D.;
3
(1.42 g, 10 mmol), and CH CN (50 mL) was stirred at room
Sledge, G. W.; Hutchins, G. D. Nucl. Med. Biol. 2002, 29, 761.
temperature under nitrogen overnight. The suspension was
filtered and the filtrate was taken down to dryness under
vacuum. The residue was adsorbed onto a silica gel column
and eluted with 1:6 EtOAc:hexane to give the title compound
2
2. Mulholland, G. K.; Zheng, Q.-H.; Mock, B. H.; Vavrek,
M. T. J. Labelled Compd. Radiopharm. 1999, 42 (Suppl. 1), S459.
3. Zheng, Q.-H.; Stone, K. L.; Mock, B. H.; Miller, K. D.;
2
Fei, X.; Liu, X.; Wang, J.-Q.; Glick-Wilson, B. E.; Sledge,
G. W.; Hutchins, G. D. Nucl. Med. Biol. 2002, 29, 803.
f
5 (450 mg, 29.8%) as a yellow oil, R =0.60 (1:3 EtOAc:hex-
1
ane). H NMR (300 MHz, CDCl ): d 1.05 (t, 3H, J =7.4 Hz,
3
1
2
4. Pascali, C.; Bongi, A.; Iwata, R.; Cambie, M.; Bombar-
dieri, E. J. Labelled Compd. Radiopharm. 2000, 43, 195.
5. Zheng, Q.-H.; Hutchins, G. D.; Mock, B. H. J. Labelled
Compd. Radiopharm. 2001, 44 (Suppl. 1), S410.
J
2
=6.6 Hz, NCH
2
CH
2H, J =7.4 Hz, J =6.6 Hz, NCH CH ), 5.70 (brs, 1H, OH),
3
), 2.80 (s, 3H, NCH
3
), 3.26–3.33 (dd,
1
2 2 3
2
6.17–6.19 (m, 2H, ArH), 6.30–6.32 (m, 1H, ArH), 7.02–7.07
(m, 1H, ArH).