Synthesis and biodistribution of new radiolabeled high-affinity choline transporter inhibitors [11C]hemicholinium-3 and [18F]hemicholinium-3

Article abstract of DOI:10.1016/j.bmcl.2007.01.105

The high-affinity choline transporter (CHT1) system is an attractive target for the development of positron emission tomography (PET) biomarkers to probe brain, cardiac, and cancer diseases. An efficient and convenient synthesis of new radiolabeled CHT1 inhibitors [¹¹C]hemicholinium-3 and [¹⁸F]hemicholinium-3 by solid-phase extraction (SPE) technique using a cation-exchange CM Sep-Pak cartridge has been well developed. The preliminary evaluation of both tracers through biodistribution studies in 9L-glioma rats has been performed, and the uptakes in the heart and tumor were observed, while very low brain uptake was seen.

Full text of DOI:10.1016/j.bmcl.2007.01.105

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2220–2224
Synthesis and biodistribution of new radiolabeled high-affinity
choline transporter inhibitors [11C]hemicholinium-3 and
[18F]hemicholinium-3
Qi-Huang Zheng,a,* Mingzhang Gao,a Bruce H. Mock,a Shuyan Wang,a Toshihiko Hara,a
Rachid Nazih,a Michael A. Miller,a Tim J. Receveur,a John C. Lopshire,b
William J. Groh,b Douglas P. Zipes,b Gary D. Hutchinsa and Timothy R. DeGradoa
aDepartment of Radiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
bKrannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
Received 20 December 2006; revised 21 January 2007; accepted 22 January 2007
Available online 4 February 2007
Abstract—The high-affinity choline transporter (CHT1) system is an attractive target for the development of positron emission
tomography (PET) biomarkers to probe brain, cardiac, and cancer diseases. An efficient and convenient synthesis of new radiola-
beled CHT1 inhibitors [11C]hemicholinium-3 and [18F]hemicholinium-3 by solid-phase extraction (SPE) technique using a cation-
exchange CM Sep-Pak cartridge has been well developed. The preliminary evaluation of both tracers through biodistribution studies
in 9L-glioma rats has been performed, and the uptakes in the heart and tumor were observed, while very low brain uptake was seen.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The high-affinity choline uptake (CHT1) system located
in peripheral and central cholinergic nerve terminals
plays a regulating and rate-limiting role in the intraneu-
ronal synthesis of acetylcholine (ACh).1,2 Three ACh-re-
lated proteins are considered specific markers for
cholinergic neurons, including choline acetyltransferase
(ChAT), the vesicular ACh transporter (VAChT), and
the high-affinity choline transporter (CHT1).3 Synthesis
of ACh is catalyzed by ChAT, and ChAT antibodies
have been used to map central cholinergic pathways
and to identify cholinergic nerves in peripheral tis-
sues.4–6 VAChT transfers ACh to storage vesicles in
nerve terminals or varicosities, and VAChT antibodies
have been used to identify cholinergic neurons and nerve
fibers in brain and heart.6–9 The CHT1 protein is highly
expressed at cholinergic varicosities and transports cho-
line into neurons for synthesis of ACh, and CHT1 anti-
bodies have been used for immunohistochemical
detection in multiple species and tissues.10–17 Biomedical
imaging technique positron emission tomography (PET)
studies with carbon-11 and fluorine-18 labeled choline
analogues have shown moderate to high choline tracer
accumulations in several types of primary and metastatic
tumors, which imply choline transporter activity to sup-
port accumulation of tracer in the tumors.18–21 In our
previous work,21 we performed mechanistic studies on
the transport of choline in the PC-3 human prostate can-
cer cell line for clarification of the properties of the cho-
line transporter. Although the uptake of choline
analogues in cultured prostate cancer cells was found
to be mediated by an intermediate affinity transporter
distinct from CHT1, it was found sensitive to inhibition
with the CHT1 inhibitor, hemicholinium-3 (HC-3). Re-
sults of cellular binding of [3H]HC-3 in PC-3 cells dem-
onstrated the importance of the transporter for choline
processing in PC-3 human prostate cancer cells. Favor-
able tumor cell binding and in vivo biodistribution prop-
erties of [3H]HC-3 in a 9L-glioma-bearing rat model
encouraged further characterization and development
of molecular imaging agents that target the choline
transporter in cancer cells.
Keywords: High-affinity choline transporter (CHT1); Positron emission
tomography (PET); [11C]hemicholinium-3 ([11C]HC-3); [18F]hemicho-
linium-3 ([18F]HC-3); Biodistribution.
CHT1 is a marker of the functional status of the cholin-
ergic presynaptic terminals and associated with a variety
of diseases, such as brain Parkinson’s disease, and Alz-
heimer’s disease, coronary heart disease and
*
Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278
9711; e-mail: qzheng@iupui.edu
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.105

Q.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2220–2224
2221
cancers.22,23,21 Thus, choline transporter systems provide
Br2
H2
AgOTf
11CO2
11CH3Br
11CH3OTf
11CH4
attractive targets for the development of PET
biomarkers to probe brain, heart, and cancer
diseases. HC-3 [2,2-(4,4-biphenylene)bis(2-hydroxy-4,
4-dimethylmorpholinium bromide)] and its derivatives
represent the most potent known competitive synthetic
CHT1 inhibitors.1 HC-3 is a gold standard for studying
the high-affinity choline transport in vitro.2 Carbon-11
and fluorine-18 labeled HC-3 analogues, [11C]hemicho-
3
11CH3OTf/CH3CN
O
OH
HO
O
([11C]HC-3)
and
[18F]hemicholinium-3
N
N
linium-3
H3C
OTf
11CH3
([18F]HC-3), may serve as in vivo PET agents for the
choline transporter. We present here, for the first time,
our initial investigation on synthesis and biodistribution
of [11C]HC-3 and [18F]HC-3.
CH3
CH3I/EtOH
CM cartridge
O
OH
HO
I
O
N
N
Synthesis of HC-3 precursor and reference standard was
performed using a modification of the literature meth-
od.24 The synthetic approach is outlined in Scheme 1.
The bis-tertiary amine precursor 4,40-bis-(1-methyl-3-
hydroxy-morpholinyl-(3))-biphenyl (3) was synthesized
from 4,40-bis-bromoacetyl-biphenyl and 2-(methylami-
no)ethanol in 80% yield. The reference standard HC-3
(1) was synthesized from 4,40-bis-bromoacetyl-biphenyl
and 2-(dimethylamino)ethanol in 92% yield.
H3C
OTf
11CH3
H3C
CH3
[
11C]1, [11C]HC-3
Scheme 2. Synthesis of [11C]HC-3.
tion technique we used in the radiosynthesis of [11C]HC-
3 is the solid-phase extraction (SPE) method,23 and the
key part in this technique is a CM Sep-Pak cartridge.
Synthesis of [18F]HC-3 ([18F]2) is indicated in Scheme 3.
Using similar methodology, fluorine-18 labeled target
tracer [18F]HC-3 was prepared by primary N-[18F]fluo-
romethylation of the precursor 3 using a new and reac-
tive [18F]fluoromethylating agent, [18F]fluoromethyl
triflate ([18F]FCH2OTf) prepared starting from K18F/
Kryptofix2.2.2 with dibromomethane,27 followed by
secondary N-[12C]methylation using methyl iodide and
purified by the SPE technique using a CM Sep-Pak car-
tridge in an automated multi-purpose 18F-radiosynthesis
(FBM) module20 with 5–10% radiochemical yields based
on K18F, 30–40 min overall synthesis time from EOB,
>99% radiochemical purity, and >1.0 Ci/lmol specific
activity at end of synthesis (EOS).
Synthesis of [11C]HC-3 ([11C]1) is shown in Scheme 2.
The precursor 3 was labeled by a reactive [11C]methylat-
ing agent, [11C]methyl triflate ([11C]CH3OTf)25 prepared
from 11CO2, in acetonitrile through the primary N-
[11C]methylation and trapped on a cation-exchange
CM Sep-Pak cartridge to release the non-reacted bis-ter-
tiary amine precursor with ethanol and to retain the
pure 11C-methylated single-side quaternary amine inter-
mediate on the same CM Sep-Pak. The labeled interme-
diate underwent the secondary N-[12C]methylation by
addition of methyl iodide in ethanol to the same
cartridge. After 2 min, non-reacted methyl iodide was
removed from the cartridge by rinsing with ethanol.
The final bis-quaternary amine carbon-11 labeled
product [11C]HC-3 was then eluted from the cartridge
with saline. The synthesis was performed in an automat-
ed multi-purpose 11C-radiosynthesis module, allowing
measurement of specific activity during synthesis.26
The radiochemical yields were 50–60% based on 11CO2
decay corrected to end of bombardment (EOB), and
specific activity was in a range of 4.0–6.0 Ci/lmol at
EOB. The overall synthesis time was 20–30 min from
EOB. The radiochemical purity was >99%. The purifica-
Both [11C]HC-3 and [18F]HC-3 are radiolabeled bis-qua-
ternary amine salts, therefore, it is not possible for the
tracers to cross the blood-brain barrier (BBB). Thus,
their application appears limited for in vivo imaging
studies of the central nervous system (CNS) diseases in
the brain such as Alzheimer’s disease, for which a dra-
matic loss of ACh and its metabolically related enzymes
from cortex and hippocampus is a characteristic feature
of Alzheimer’s disease.22 Rather, the potential applica-
O
OH
HO
Br
O
(CH3)2NCH2CH2OH
THF
N
N
Br
H3C
H3C
CH3
CH3
1, HC-3 standard
BrCH2OC
COCH2Br
O
OH
HO
O
CH3NHCH2CH2OH
THF
N
N
H3C
CH3
3, HC-3 precursor
Scheme 1. Synthesis of HC-3 precursor and reference standard.

2222
Q.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2220–2224
AgOTf
included normal brain and heart. All animal experi-
ments were performed under a protocol approved by
the Indiana University Institutional Animal Care and
Use Committee (IACUC). The in vivo biodistribution
of [11C]HC-3 was determined in 9L-glioma rats at 5
and 20 min post intravenous (iv) injection of the tracer.
Likewise, the in vivo biodistribution of [18F]HC-3 was
determined in 9L-glioma rats at 5 and 30 min post iv
injection of the tracer. Urine was collected by a syringe
from the bladder. The data are listed in Table 1. The
data represent the average value in three rats. Biodistri-
bution data of [11C]HC-3 at 5 and 20 min time points
and [18F]HC-3 at 5 and 30 min time points in 9L-glioma
rats showed the major organs of uptake to be kidney.
Heart uptake was observed in different regions including
L-atrium, R-atrium, septum, L-ventricle, R-ventricle,
and A–V node. Tumor uptake was moderate, and tu-
mor/muscle ratios were 3.0–5.1 for both [11C]HC-3
and [18F]HC-3 at 5–30 min post-injection. In compari-
son with the uptakes in urine and kidney, the uptake
in brain was very low. The minimal penetration of both
tracers into brain confirmed the lipophobicity30 of the
bis-quaternary amine tracers, which is consistent with
their HPLC retention times. Gliomas are brain tumors
derived from glia,31 and the glioma tumor was implant-
ed in the thigh of the rat, not in the brain. Thus, the
tumor uptakes were able to be seen in 9L-glioma rat
model in biodistribution study, and both radiotracers
had relative good uptake in tumor and good tumor/
background ratios. This study does not address the
question of whether these tracers can cross the blood-
brain barrier when glioma is present. There was little
change in the tissue biodistributions from 5 to 30 min
post-injection, demonstrating that the disposition of
the labeled HC-3 analogues occurs rapidly. Significant
fractions of radioactivity were accumulated in urine
(ꢀ25% at 30 min). Since bone and fat were not the
CH2Br2
K18F/K222
18FCH2Br
18FCH2OTf
3
18FCH2OTf/CH3CN
HO
O
OH
O
N
N
OTf
H3C
CH218F
CH3
CH3I/EtOH
CM cartridge
O
OH
HO
I
O
N
N
OTf
CH218F
H3C
H3C
CH3
[
18F]2, [18F]HC-3
Scheme 3. Synthesis of [18F]HC-3.
tion of both tracers would be in heart imaging and can-
cer imaging.23,21
The preliminary biological evaluation of [11C]HC-3 and
[18F]HC-3 was carried out through biodistribution stud-
ies in a subcutaneous 9L-glioma rat model. We chose
against a xenograft mouse model for this study, because
a rat model would allow for more practical collection of
arterial blood samples and previous work with the PC-3
xenografts showed accumulations of choline radiotracers
indicative of poor perfusion of these tumors.19 Since
elevation of choline uptake is found in a wide variety
of tumors including gliomas,28,29 the model serves as a
representative model for evaluating choline-based imag-
ing probes. Moreover, this model is also suitable for
evaluating in vivo biodistribution properties of brain
and heart regions, since our regions-of-interest (ROIs)
Table 1. Biodistribution data of [11C]HC-3 and [18F]HC-3 in 9L-glioma rats (% ID/g)
Organ
[
11C]HC-3 (n = 3, 5 min)
[
11C]HC-3 (n = 3, 20 min)
[
18F]HC-3 (n = 3, 5 min)
[
18F]HC-3 (n = 3, 30 min)
Blood
1.383 0.267
3.648 0.683
0.060 0.038
1.081 0.471
0.848 0.381
7.640 2.782
6.381 6.876
0.749 0.093
1.453 0.383
0.035 0.008
0.590 0.074
1.239 0.369
3.656 0.322
1.467 0.685
3.732 0.680
0.042 0.021
0.944 0.584
1.228 0.662
8.676 4.732
0.378 0.198
1.272 0.536
0.030 0.008
0.533 0.228
1.536 0.585
5.935 5.577
Plasma
Brain
Lung
Liver
Kidney
Urine (% dose)
24.980 6.583
10.188 12.182
27.623 15.663
Heart regions
L-Atrium
R-Atrium
Septum
1.026 0.606
0.963 0.325
0.381 0.115
0.415 0.162
0.401 0.129
1.074 0.964
0.457 0.070
0.398 0.085
0.223 0.011
0.244 0.028
0.234 0.009
0.478 0.243
0.824 0.324
2.023 1.848
0.530 0.390
0.438 0.219
0.734 0.699
5.607 7.484
0.767 0.312
0.999 0.266
0.262 0.155
0.253 0.134
0.243 0.134
0.600 0.211
L-Ventricle
R-Ventricle
A–V node
Solar Plexus
Ske. Musc.
0.522 0.258
0.207 0.061
2.057 1.631
0.159 0.046
1.186 0.143
0.194 0.087
1.009 0.693
0.123 0.070
Tumor
Tumor/blood
Tumor/muscle
0.649 0.176
0.466 0.064
3.199 0.522
0.457 0.146
0.622 0.219
3.094 1.310
0.952 0.400
0.572 0.311
5.085 1.771
0.359 0.165
0.978 0.095
3.024 0.393
The data presented here represented the average value in 3 rats. The time points post iv injection of the tracer were 5 and 20 min for [11C]HC-3, and 5
and 30 min for [18F]HC-3, respectively.

Q.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2220–2224
2223
7. Schafer, M. K.; Weihe, E.; Erickson, J. D.; Eiden, L. E.
J. Mol. Neurosci. 1995, 6, 225.
8. Schafer, M. K.; Weihe, E.; Eiden, L. E.; Erickson, J. D.
J. Mol. Neurosci. 1994, 5, 1.
tissues of interest, no bone and fat tissues were excised in
biodistribution study, and no bone and fat accumula-
tion data were reported.
9. Schafer, M. K.; Eiden, L. E.; Weihe, E. Neuroscience 1998,
84, 361.
10. Suszkiw, J. B.; Pilar, G. J. Neurochem. 1976, 26, 1133.
11. Kuhar, M. J.; Sethy, V. H.; Roth, R. H.; Aghajanian, G.
K. J. Neurochem. 1973, 20, 581.
The experimental details and characterization data for
compounds 3 and 1, new tracers [11C]1 and [18F]2, and
biodistribution study are given.32
In summary, an efficient and convenient chemical and
radiochemical synthesis of the precursors, reference,
standards and target tracers has been well developed.
The synthetic methodology of [11C]HC-3 and [18F]HC-
3 employed bis-tertiary amine precursor, featuring
primary N-[11C]methylation with [11C]CH3OTf and N-
[18F]fluoromethylation with [18F]FCH2OTf in a reaction
vessel, purification of 11C-methylated and 18F-fluorome-
thylated single-side quaternary amine intermediates on a
CM Sep-Pak cartridge, followed by secondary N-
[12C]methylation with CH3I and isolated final bis-qua-
ternary amines carbon-11 and fluorine-18 labeled
products [11C]HC-3 and [18F]HC-3 by SPE technique
on the same cartridge. The radiolabeling reactions and
purification are rapid, efficient, and convenient, and
resulting radiolabeled products were shown to have
moderate to excellent radiochemical yields. Preliminary
findings from biodistribution studies of both tracers in
9L-glioma rats indicate that uptakes in the heart and
tumor were observed, while very low brain uptake was
seen. Further study will be to determine the specificity
of binding of HC-3 analogue tracers to choline
transporters, and to explore structural modifications to
provide tracers for higher lipophilicity needed to cross
the blood-brain barrier for the purpose of CNS imaging
studies of CHT1.
12. Apparsundaram, S.; Ferguson, S. M.; Geroge, A. L., Jr.;
Blakely, R. D. Biochem. Biophys. Res. Commun. 2000, 276,
862.
13. Apparsundaram, S.; Ferguson, S. M.; Blakely, R. D.
Biochem. Soc. Trans. 2001, 29, 711.
14. Okuda, T.; Haga, T. FEBS Lett. 2000, 484, 92.
15. Misawa, H.; Nakata, K.; Matsuura, J.; Nagao, M.;
Okuda, T.; Haga, T. Neuroscience 2001, 105, 87.
16. Kus, L.; Borys, E.; Ping, C. Y.; Ferguson, S. M.; Blakely,
R. D.; Emborg, M. E.; Kordower, J. H.; Levey, A. I.;
Mufson, E. J. J. Comp. Neurol. 2003, 463, 341.
17. Ferguson, S. M.; Savchenko, V.; Apparsundaram, S.;
Zwick, M.; Wright, J.; Heilman, C. J.; Levey, A. I.;
Blakely, R. D. J. Neurosci. 2003, 23, 9697.
18. Hara, T.; Kosaka, N.; Shinoura, N.; Kondo, T. J. Nucl.
Med. 1997, 38, 842.
19. DeGrado, T. R.; Coleman, R. E.; Wang, S.; Baldwin, S.
W.; Orr, M. D.; Robertson, C. N.; Polascik, T. J.; Price,
D. T. Cancer Res. 2000, 61, 110.
20. DeGrado, T. R.; Baldwin, S. W.; Wang, S.; Orr, M. D.;
Liao, R. P.; Friedman, H. S.; Reiman, R.; Price, D. T.;
Coleman, R. E. J. Nucl. Med. 2001, 42, 1805.
21. Hara, T.; Bansal, A.; DeGrado, T. R. Mol. Imaging 2006,
5, 498.
22. Mulholland, K. K.; Wieland, D. M.; Kilbourn, M. R.;
Frey, K. A.; Sherman, P. S.; Carey, J. E.; Kuhl, D. E.
Synapse 1998, 30, 263.
23. Gao, M.; Miller, M. A.; DeGrado, T. R.; Mock, B. H.;
Lopshire, J. C.; Rosenberger, J. G.; Dusa, C.; Das, M. K.;
Groh, W. J.; Zipes, D. P.; Hutchins, G. D.; Zheng, Q.-H.
Bioorg. Med. Chem. 2007, 15, 1289.
24. Haarstad, V. B.; Domer, F. R.; Chihal, D. M.; Rege, A.
B.; Charles, H. C. J. Med. Chem. 1976, 19, 760.
25. Mock, B. H.; Mulholland, G. K.; Vavrek, M. T. Nucl.
Med. Biol. 1999, 26, 467.
26. Mock, B. H.; Zheng, Q.-H.; DeGrado, T. R. J. Labelled
Compd. Radiopharm. 2005, 48, S225.
27. Iwata, R.; Pascali, C.; Bogni, A.; Furumoto, S.; Terasaki,
K.; Yanai, K. Appl. Radiat. Isot. 2002, 57, 347.
28. Gillis, R. J.; Barry, J. A.; Ross, B. D. Magn. Reson. Med.
1994, 32, 310.
Acknowledgments
This work was partially supported by the Donald W.
Reynolds Foundation, the Susan G. Komen Breast Can-
cer Foundation BCTR0504022, NIH R01CA108620, and
Indiana Genomics Initiative (INGEN) of Indiana
University, which is supported in part by Lilly
Endowment Inc. The authors would like to thank
Barbara E. Glick-Wilson and Michael L. Sullivan for
their assistance in production of 11CH3OTf and K18F/
Kryptofix2.2.2. The referees’ criticisms and editor’s
comments for the revision of the manuscript are greatly
appreciated.
29. Walum, E. Cell Mol. Neurobiol. 1981, 1, 389.
30. Liu, X.; Wang, J.-Q.; Zheng, Q.-H. Biomed. Chromatogr.
2005, 19, 379.
31. Barinaga, M. Science 1999, 278, 1226.
32. Experimental details and characterization data.
(a) General: all commercial reagents and solvents were
used without further purification. 11CH3OTf was made
according to a literature procedure.25 18FCH2OTf was
made according to a modification of the literature
method.27 Melting points were determined on a MEL-
TEMP II apparatus and are uncorrected. 1H NMR
spectra were recorded on a Bruker QE 300 FT NMR
spectrometer 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
internal standard TMS (d 0.0), and coupling constants (J)
are reported in hertz (Hz). The low resolution mass spectra
(LRMS) were obtained using an Agilent 1100 series LC/
MSD mass spectrometer. Thin layer chromatography
References and notes
1. Smart, L. A. J. Med. Chem. 1983, 26, 104.
2. Gilissen, C.; de Groot, T.; Bronfman, F.; van Leuven, F.;
Verbruggen, A. M.; Bormans, G. M. J. Nucl. Med. 2003,
44, 269.
3. Hoover, D. B.; Ganote, C. E.; Ferguson, S. M.; Blakely,
R. D.; Parsons, R. L. Cardiovasc. Res. 2004, 62, 112.
4. Woolf, N. J. Prog. Neurobiol. 1991, 37, 475.
5. Crick, S. J.; Sheppard, M. N.; Anderson, R. H.; Polak, J.
M.; Wharton, J. J. Anat. 1996, 188, 403.
6. Arvidsson, U.; Riedl, M.; Elde, R.; Meister, B. J. Comp.
Neurol. 1997, 378, 454.

2224
Q.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2220–2224
(TLC) was run using Analtech silica gel GF uniplates
ethanol and to retain the pure N-11C-methylated single-side
quaternary amine intermediate on the same CM Sep-Pak.
Then, the labeled intermediate underwent the secondary N-
[12C]methylation by addition of CH3I in ethanol to the same
cartridge. After 2 min, the Sep-Pak cartridge was washed
with ethanol (5 mL) and water (2 mL) to remove non-
reacted CH3I, and the washing solution was discarded to a
waste bottle. The final product [11C]1 was eluted from the
CM Sep-Pak with saline (2–4 mL) and sterile-filtered
through a 0.22 lm cellulose acetate membrane and collected
into a sterile vial. Total radioactivity was assayed and the
total volume was noted. The overall synthesis time was 20–
30 min from EOB. The radiochemical yields decay correct-
ed to EOB, from 11CO2, were 50–60%, the radiochemical
purity was >99%, and the chemical purity of the target
tracer was >95% measured by analytical HPLC. Retention
times in the analytical HPLC system were: tR3 = 3.45 min,
tR1 = 2.36 min, tR[11C]1 = 2.36 min.
(5 · 10 cm2). Chromatographic solvent proportions are
expressed on a volume:volume basis. Plates were visual-
ized by UV light. 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 HPLC was performed using a Prodigy
(Phenomenex) 5 lm C18 column, 4.6 · 250 mm; 3:1:1
CH3CN/MeOH/20 mM, pH 6.7 KHPO4ꢁ (buffer solution)
mobile phase, flow rate 1.5 mL/min, and UV (270 nm) and
c-ray (NaI) flow detectors. Semi-prep cation-exchange
CM Sep-Pak cartridges were obtained from Waters
Corporate Headquarters, Milford, MA. Sterile vented
Millex-GS 0.22 lm filter unit was obtained from Millipore
Corporation, Bedford, MA.
(b) 4,40-bis-(1-methyl-3-hydroxy-morpholinyl-(3))-biphe-
nyl (3). To a solution of 4,40-bis-bromoacetyl-1-biphenyl
(396 mg, 1.0 mmol) dissolved in 50 mL THF was added a
solution of 2-(methylamino)ethanol (330 mg, 4.4 mmol)
dissolved in 10 mL of THF. The mixture was stirred at
room temperature for 3 h and heated to reflux for 2 h. The
mixture solution was evaporated under reduced pressure,
added with dichloromethane (120 mL), decanted or fil-
tered from the precipitated N-substituted ethanolamine
hydrobromide, washed with water and brine. Then the
organic solution was extracted with 1N HCl solution, and
the pH of HCl solution was adjusted with ammonium
hydroxide to 9, aqueous solution was again extracted with
dichloromethane, organic solution was dried over magne-
sium sulfate, filtered, and concentrated to give compound3
(306 mg, 87%) as a light yellow solid. Mp: 45–46 ꢁC;
Rf = 0.26 (1:9, MeOH/CH2Cl2). 1H NMR (300 MHz,
DMSO-d6): d 1.99–2.29 (m, 2H), 2.16 (s, 6H, CH3), 2.56–
2.73 (m, 4H, CH2), 3.51–3.91 (m, 4H, CH2), 4.10 (dt,
J = 2.2, 11.0 Hz, 2H, CH2), 6.26 (s, 2H, OH), 7.55–7.70 (m,
8H, Ph–H). MS (ESI): 385 ([M+H]+, 30%), 384 (100%).
(c) 2,2-(4,4-biphenylene)bis(2-hydroxy-4,4-dimethylmor-
pholinium bromide) (HC-3, 1). 4,40-bis-bromoacetyl-1-
biphenyl (396 mg, 1.0 mmol) and 2-dimethylaminoethanol
(178 mg, 2.0 mmol) were added into anhydrous THF
(40 mL), stirred at room temperature for 24 h. A white
solid was precipitated, filtered, and washed with dry THF to
(e) [18F]HC-3 ([18F]2). Precursor 3 (0.6–1.0 mg) was dis-
solved in acetone (0.6 mL), and the mixture was placed in a
sealed reaction vessel. No carrier-added (high specific
activity) [18F]fluoromethyl bromide prepared by a literature
20
method
was passed through a silver triflate column at
20 ꢁC to form [18F]FCH2OTf. [18F]FCH2OTf was trapped
in the reaction solution, and then the reaction mixture was
heated at 40 ꢁC for 10 min. The reaction vessel was
connected to a CM Sep-Pak cartridge. The labeled product
mixture solution was passed onto the Sep-Pak cartridge to
release the non-reacted precursor with ethanol and to retain
the pure N-18F-fluoromethylated single-side quaternary
amine intermediate on the same CM Sep-Pak. Then, the
labeled intermediate underwent the secondary N-
[12C]methylation by addition of CH3I in ethanol to the
same cartridge. After 2 min, the Sep-Pak cartridge was
washed with ethanol (5 mL) and water (2 mL) to remove
non-reacted CH3I, and the washing solution was discarded
to a waste bottle. The final product [18F]2 was eluted from
the CM Sep-Pak with saline (2–4 mL) and sterile-filtered
through a 0.22 lm cellulose acetate membrane and collected
into a sterile vial. Total radioactivity was assayed and the
total volume was noted. The overall synthesis time was 30–
40 min from EOB. The radiochemical yields decay correct-
ed to EOB, from K18F, were 5–10%, the radiochemical
purity was >99%, and the chemical purity of the target
tracer was >95% measured by analytical HPLC. Retention
times in the analytical HPLC system were: tR3 = 3.45 min,
tR [18F]2 = 2.42 min.
1
obtain compound 5 (614 mg, 92%). Mp: 178–180 ꢁC. H
NMR (300 MHz, DMSO-d6): d 3.19 (s, 6H, CH3), 3.31–3.39
(m, 2H, CH2), 3.46 (s, 6H, CH3), 3.60 (d, J = 5.1 Hz, 4H,
CH2), 3.66 (d, J = 13.2 Hz, 2H, CHH), 4.08.
(d) J = 13.2 Hz, 2H, CHH, 4.51–4.49 (m, 2H, CH2), 7.39 (d,
J = 1.4 Hz, 2H, OH), 7.64 (d, J = 8.1 Hz, 4H, Ph–H), 7.77
(d, J = 8.1 Hz, 4H, Ph–H). MS (ESI): 414 (M+, 32%), 413
(100%).
(f) Biodistribution study. The 9L-glioma rats (200–300 g)
were injected intravenously with sub-pharmacologic doses
(1–3 mCi) of [11C]HC-3 and/or [18F]HC-3 via the tail vein
while under conscious restraint. At 5 and 20 min post-
injection of [11C]HC-3 or at 5 and 30 min post-injection of
[18F]HC-3, rats were sacrificed by decapitation under
halothane anesthesia, their tissues quickly excised, weighed,
and the decay-corrected radioactive content measured using
a Wallac 1470 gamma counter. The results are expressed as
percentage of injected dose (%ID) for urine and as percent-
age of injected dose per mass of tissue (%ID/g) for all other
tissues. For calculation of total blood activity, blood mass
was assumed to be 7% of the body mass.
(d) [11C]HC-3 ([11C]1). Bis-tertiary amine precursor 3 (0.1–
0.2 mg) was dissolved in acetonitrile (300 lL). The mixture
was placed in a sealed reaction vessel. No carrier-added
(high specific activity) [11C]CH3OTf was passed through the
reaction solution, which was cooled at ꢀ 0 ꢁC, until
radioactivity reached a maximum (ꢀ3 min), and then the
reaction mixture was heated at 80 ꢁC for 2 min. The reaction
vessel was connected to a CM Sep-Pak cartridge. The
labeled product mixture solution was passed onto the Sep-
Pak cartridge to release the non-reacted precursor with