Facile and improved synthesis of [11C]Me-QNB

Article abstract of DOI:10.1002/jlcr.2979

[¹¹C]Me-QNB is a muscarinic acetylcholinergic receptor antagonist that has been used for the assessment of myocardial muscarinic receptors density in different cardiovascular pathologies. In the current technical note, we report a facile, highly efficient and fully automated method for the preparation of this radiotracer. The radiosynthesis was performed by reaction of [¹¹C]CH₃I with the desmethylated precursor (QNB) at room temperature using the captive solvent method. Excellent radiochemical yield (91.1 ± 2.4%, decay-corrected) and radiochemical purity (>99.5%), and good specific activity (137 ± 5 GBq/μmol) were obtained when the purification was performed by reverse phase HPLC in overall synthesis time <31 min. Purification using solid-phase extraction offered lower radiochemical yield (27.6 ± 3.1%, decay-corrected) and radiochemical purity (>95%) but higher specific activity (244 ± 18 GBq/μmol) in shorter reaction times (<21 min). These results, especially concerning radiochemical yield, significantly improve those previously reported in which the reaction was performed in a vial and the purification step was based on ionic chromatography.

Full text of DOI:10.1002/jlcr.2979

Technical Note
Received 20 July 2012,
Revised 17 September 2012,
Accepted 2 October 2012
Published online 25 October 2012 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2979
Facile and improved synthesis of [11C]Me-QNB
*
Vanessa Gómez-Vallejo, Mikel González-Esparza and Jordi Llop
[
11C]Me-QNB is a muscarinic acetylcholinergic receptor antagonist that has been used for the assessment of myocardial
muscarinic receptors density in different cardiovascular pathologies. In the current technical note, we report a facile, highly
efficient and fully automated method for the preparation of this radiotracer. The radiosynthesis was performed by reaction
of [11C]CH3I with the desmethylated precursor (QNB) at room temperature using the captive solvent method. Excellent
radiochemical yield (91.1 Æ 2.4%, decay-corrected) and radiochemical purity (>99.5%), and good specific activity
(137 Æ 5 GBq/mmol) were obtained when the purification was performed by reverse phase HPLC in overall synthesis time
<31 min. Purification using solid-phase extraction offered lower radiochemical yield (27.6 Æ 3.1%, decay-corrected) and
radiochemical purity (>95%) but higher specific activity (244 Æ 18 GBq/mmol) in shorter reaction times (<21 min). These
results, especially concerning radiochemical yield, significantly improve those previously reported in which the reaction was
performed in a vial and the purification step was based on ionic chromatography.
Keywords: Carbon-11; Me-QNB; positron emission tomography; muscarinic receptor
values; however, the whole preparation could be completed in
shorter times, and consequently, higher specific activities were
obtained.
Introduction
Me-QNB (1, N-methyl-quinuclidin-3-yl benzilate, Scheme 1) is a
hydrophobic, non-metabolized and highly specific muscarinic
acetylcholinergic antagonist,1 which has been previously labeled
with Carbon-112,3 and characterized as a radiotracer for the
Materials and methods
in vivo imaging of myocardial muscarinic receptors using
positron emission tomography.2,4,5 This radiotracer has been used
General
for the assessment of myocardial muscarinic receptor density
All chemicals and solvents (unless otherwise specified) were of
and affinity constants in heart transplant patients,6 in chronic
analytic or HPLC grade from Panreac Química (Madrid, Spain)
idiopathic dilated cardiomyopathy patients,7 and in familiar
or Sigma-Aldrich (Spain). C-18 Light Sep-Pak cartridges were
amyloid neuropathy patients.8
obtained from Waters Cromatografía, S.A. (Spain) and were pre-
The radiosynthetic procedure for the preparation of [11C]Me-QNB,
conditioned sequentially with ethanol (5 mL) and water (5 mL).
on the basis of the reaction of the desmethyl precursor (QNB)
Accell Plus CM Sep-Pak cartridges were obtained from Waters
with [11C]methyl iodide ([11C]CH3I), was first developed by
Cromatografía, S.A. (Spain) and used without preconditioning.
Mazière et al.2 After a purification step using HPLC, average
QNB and Me-QNB were purchased from ABX (Avanced Biomedical
decay-corrected radiochemical yields of 23.3 Æ 10% with respect
Compounds, Radeberg, Germany). Purified water (ultrapure, Type I
to [11C]CH3I were obtained. A more efficient strategy based on
water, ISO 3696) was obtained from a Milli-QW Integral system
the use of [11C]methyl triflate ([11C]MeOTf) as labeling agent
(Millipore Iberica S.A.U., Madrid, Spain).
was developed later.9 Decay-corrected radiochemical yields of
48.5 Æ 10% (based on [11C]MeOTf) could be obtained with associated
decay-corrected specific radioactivities of 98 Æ 36 GBq/mmol.
Recently, we have developed methodologies for the preparation
of different 11C-labeled radiotracers10,11 using the captive solvent
method first developed by Wilson and co-workers.12 This method
has been proven to improve radiochemical yields; in addition,
reactions that take place only at high temperature using the
conventional in vial reaction can be performed at room tempera-
ture with equivalent results.11
Production of [11C]CH3I
The synthesis of [11C]CH3I was carried out using a TRACERlab FXC
Pro synthesis module (GE Healthcare, Buckinghamshire, UK)
starting from cyclotron produced [11C]CH4, which was generated
in an IBA Cyclone 18/9 cyclotron by bombardment (target
current = 22 mA, integrated current = 1 mAh) of a gas N2/H2
mixture (99/1, starting pressure = 16 bar) with high energy
(18 MeV) protons.
In this study, we describe the fully automated radiosynthesis
of [11C]Me-QNB using the captive solvent method and [11C]
CH3I as a methylating agent. The reaction was completed in
1 min at room temperature. When the purification step was
performed by HPLC, significantly higher radiochemical yields than
those previously reported2,9 were obtained. Implementation of a
purification step on the basis of solid-phase extraction lowered
Radiochemistry Department, CIC biomaGUNE, Paseo Miramón 182, San Sebastián
20009, Spain
*Correspondence to: Jordi Llop, Radiochemistry Department, CIC biomaGUNE,
Paseo Miramón 182, San Sebastián 20009, Spain.
decay-corrected radiochemical yield and radiochemical purity E-mail: jllop@cicbiomagune.es
J. Label Compd. Radiopharm 2012, 55 470–473
Copyright © 2012 John Wiley & Sons, Ltd.

V. Gómez-Vallejo et al.
Scheme 1. Radiosynthesis of [11C]Me-QNB.
Synthesis of [11C]Me-QNB: purification with HPLC
complete trapping, the solution was pushed with CH3CN (5 mL)
into a vial and the activity was measured in the dose calibrator.
This value was used for the calculation of radiochemical yields
in all syntheses performed within the same day.
[11C]CH3I synthesized as described previously was trapped in a
2-mL stainless steel HPLC loop (AutoLoopTM system) pre-charged
with a solution of QNB (1 mg) in dimethyl sulfoxide (80 mL). After
complete trapping of [11C]CH3I, the reaction was allowed to
occur for 1 min at room temperature (Scheme 1). The reaction
mixture was purified by means of HPLC using a Mediterranean
Sea18 column (250 Â 10 mm, 5 mm; Teknokroma, Barcelona,
Spain) as stationary phase and sodium acetate aqueous solution
(2 g/L) adjusted to pH = 10.0 with 1M NaOH/CH3CN 50/50 as a
mobile phase at a flow rate of 5 mL/min. The purified fraction
(retention time ~8 min, radiometric and UV detection) was
collected and reformulated in the TRACERlab FXC Pro synthesis
module by diluting with water (20 mL), trapping in a C-18
cartridge, rinsing with water, elution with ethanol (1 mL), further
elution with physiologic solution (9 mL) and final filtration
through a 0.22-mm filter (MillexW-GS, Millipore).
Results and discussion
The synthetic strategy reported here is based on the captive
solvent method first described by Wilson and co-workers.12
Historically, this methodology has offered better results than
the traditional in vial reaction because of three main factors: First,
direct introduction of the reaction crude into the purification
system (usually HPLC) prevents loss of radioactivity derived
from liquid transfers; second, less solvent can be used in the
preparation of the precursor solution, resulting in a higher
concentration, which favors the methylation reaction; third, the
precursor solution is distributed on the loop surface creating a
thin film, which results in improved interaction of the precursor
with the labeling agent with subsequent increase in radiochemical
conversion values. This improved reactivity allows to complete
reactions at room temperature in shorter or equivalent reaction
Synthesis of [11C]Me-QNB: purification with solid-phase
extraction
The production of [11C]CH3I and the methylation reaction were times (as compared with in vial reactions) minimizing thus the
performed as described in this section. The reaction mixture formation of undesired by-products.
was pushed from the loop with purified water (10 mL), and the
Our first syntheses were performed without purification to
resulting eluate was passed through an Accell Plus CM cationic optimize experimental conditions. Dimethyl sulfoxide was used
exchange cartridge. The cartridge was rinsed with ethanol as a solvent, and reaction time was fixed at 5 min. Although
(2 mL) and purified water (3 mL). Finally, the retained radioactive the reaction was carried out at room temperature, radiochemical
product was eluted with 10% ethanol in physiologic saline conversion values (determined by analytical HPLC) close to 100%
solution (total volume = 3 mL) and filtered through a 0.22 mm were obtained. Thus, the reaction time was progressively
filter (MillexW-GS, Millipore).
shortened, and surprisingly, equivalent results were obtained
Irrespective of the purification method, the amount of when the reaction time was fixed at 3 and 1 min. Because of
radioactivity of the final radiotracer was measured in a dose these excellent results, the experimental conditions were not
calibrator (PETDOSE HC, Comecer; Ravenna, Italy), and a sample further optimized.
was submitted to quality control. Chemical and radiochemical
The purification step was performed at first instance by HPLC.
purity as well as specific radioactivity were determined by HPLC, The methodology described by Dolle et al.9 was based on ionic
using an Agilent 1200 Series HPLC system (Madrid, Spain) with a chromatography; thus, a mixture of 0.9% aq. NaCl/EtOH (65/35)
multiple wavelength UV detector (l = 207 nm) and a radiometric was used as a mobile phase, and a semi-preparative SpherisorbW
detector (Gabi, Raytest; Straubenhardt, Germany). An RP-C18 SCX Column was used as stationary phase. Such mobile phase
column (Agilent XDB-C18, 50 Â 4.6 mm, 1.8 mm) was used as has the advantage that no reformulation step is required,
stationary phase, and ammonium formate aqueous solution because dilution of the collected fraction with the appropriate
(3.15 g/L) adjusted to pH = 6.0 with HCOOH/CH3CN 75/25 was volume of physiologic saline solution results directly in an
used as a mobile phase. The retention times for QNB and injectable solution. However, as mentioned in the paper, under
Me-QNB were 3.67 and 3.08 min, respectively.
these chromatographic conditions, the retention times for the
Radiochemical yields were calculated with respect to [11C] precursor and the desired radiotracer were 5.5–6.5 min and
CH3I. For that purpose, one synthesis of this labeling agent was 8.0–9.5 min, respectively; because of this fact, the use of higher
performed each working day between [11C]Me-QNB runs. [11C] amount of precursor was restricted because the separation of
CH3I was collected in AutoLoopTM system, where the loop the radiotracer and the precursor was not successful in all
had been pre-charged with dimethyl sulfoxide (80 mL). After batches. In our case, taking into account that we were using
J. Label Compd. Radiopharm 2012, 55 470–473
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

V. Gómez-Vallejo et al.
1 mg of precursor and that most of the tracers, which are radioactivity of 137 Æ 5 GBq/mmol in overall synthesis time of
routinely produced in our laboratory, are purified using RP-C18 30–31 min (Table 1, Entry 3). The presence of the precursor in
columns, the development of a new method on the basis the final solution could not be detected by analytical HPLC (limit
of reverse phase chromatography in which the retention time of detection: 0.05 mg/mL), and no other peaks were detected in
of the precursor was longer than the retention time of the the UV chromatogram. The radiochemical purity was above
radiotracer was anticipated to be more convenient. Although 99.5% in all cases (Table 1). These results are significantly better
9
the radiotracer is a cation at any pH < 10.5, pKa calculations than those previously reported by Dollé et al. especially in
showed that the neutral form of the precursor is the major specie terms of radiochemical yield. The slight improvement in specific
present in solution at 8.8 < pH < 11.0. By adjusting the pH at radioactivity values (137 Æ 5 vs. 98.4 Æ 36) might be due to the
10.0 and using a reverse phase C-18 column, we found that fact that Dollé and co-workers used cyclotron produced [11C]
the elution order could thus be easily reversed. As can be CO2, whereas in our case, the primary labeling agent was [11C]
seen in Figure 1, under optimized chromatographic conditions, CH4, which is known to provide radiotracers with higher specific
the retention time of the radiotracer was ~8 min, whereas radioactivities.13
the retention time of the precursor was 9–12 min. Despite the
The cationic nature of the radiotracer led us to consider the
broadness of the latter peak and the severe tailing effect, the implementation of a purification step using solid-phase extrac-
purified fraction could be collected and reformulated, yielding tion cartridges. After reaction, the crude was eluted from the
a radiotracer with a radiochemical yield of 91.1 Æ 2.4% (n = 5) loop with purified water (10 mL), and the resulting solution was
with respect to [11C]CH3I (decay-corrected) and specific eluted through an Accell Plus CM cartridge. The cartridge was
rinsed with ethanol (2 mL) to remove the unreacted precursor,
with purified water (3 mL) to remove the residual ethanol, and
the retained radiotracer was eluted into a vial using 10% ethanol
in physiologic saline solution (total volume = 3 mL). As can
be seen in Table 1 (Entry 4), the synthesis could be completed
in 20–21 min. Decay-corrected radiochemical yield was
considerably lower than the one obtained using HPLC
(27.6 Æ 3.1% vs. 91.1 Æ 2.4%), probably due to partial elution of
the radiotracer from the cartridge while removing the unreacted
precursor. However, higher specific radioactivity (244 Æ 8GBq/mmol
vs. 137 Æ 5 GBq/mmol, decay uncorrected) values were obtained,
and the amount of radioactivity should be sufficient to approach
preclinical and clinical studies. The latter strategy could be
particularly useful when high specific activities are
required or no HPLC system is available. Moreover, shorter
preparation times should increase the throughput in the
synthetic systems.
Figure 1. Chromatographic profiles corresponding to the purification of [11C]Me-
QNB. Radiometric detector (dotted line) and ultraviolet (UV) detector (solid line)
profiles are shown.
Table 1. Experimental conditions, radiochemical yield, specific activity, and chemical and radiochemical purity for the preparation
of [11C]Me-QNB
Entry
QNB
Rt (min)
RT (ꢀC)
[11C]Me-QNB (GBq)
RCY (%)
AS (GBq/mmol)
ST (min)
RCP (%)
CP
1a
2e
3
0.64 mg
0.64 mg
1.0 mg
1.0 mg
2
1
1
1
200
100
30
4.8 Æ 1.2b
34.4 Æ 10c
48.5 Æ 10f
91.1 Æ 2.4c
27.6 Æ 3.1c
3.7–74d
98.4 Æ 36g
137 Æ 5j
<30
27–28
30–31
20–21
–
–
6.8 Æ 1.4b
>99
>99.5
>95
>95%h
NDk
0.81 Æ 0.12i
0.39 Æ 0.05i
4
30
243 Æ 18j
NDk
aObtained from Syrota et al.
1
bStarting from 44.4 GBq of [11C]CO2 (integrated current in target not specified).
cBased on [11C]CH3I, decay-corrected.
dIn the original publication, the authors did not specify whether values were decay-corrected or not.
9
eObtained from Dollé et al.
fBased on [11C]CH3OTf, decay-corrected.
gDecay corrected.
hObtained from chromatographic profile.
iAmount of [11C]CH4 not measured, irradiation conditions as detailed in materials and methods.
jDecay-uncorrected.
kno other peaks apart from Me-QNB detected in the UV chromatogram.
Rt: Reaction time; RT: Reaction temperature; RCY: Radiochemical yield; AS: Specific radioactivity; ST: total synthesis time (including
purification); RCP: Radiochemical purity; CP: Chemical purity. Mean Æ standard deviation values are reported for [11C]Me-QNB
amount of radioactivity, radiochemical yield and specific radioactivity. For entries 3–5, n = 5.
www.jlcr.org
Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 470–473

V. Gómez-Vallejo et al.
Conclusions
References
[1] A. Syrota, P. Merlet, J. Delforge. The Heart, chapter 37, Section 2.
In Cardiac Neurotransmission in Principles of Nuclear Medicine, 2nd
Ed. (Ed.: H. N. Wagner), W. B. Saunders Company: Philadelphia –
London – Toronto – Montreal – Sydney – Tokyo, 1995, pp. 759–773.
[2] M. Mazière, D. Comar, J. M. Godot, P. H. Collard, C. Cepedat,
R. Naquet, Life Sci. 1981, 29, 2391.
[3] M. Mazière, G. Berger, J. M. Godot, C. Prenant, J. Sastre, D. J. Comar,
Radioanal. Chem. 1983, 76, 305.
[4] A. Syrota, G. Paillotin, J. M. Davy, M. C. Aumont, Life Sci. 1984, 35(9),
937.
[5] A. Syrota, D. Comar, G. Paillotin, J. M. Davy, M. C. Aumont, O. Stulzaft,
B. Mazière, Proc. Natl. Acad. Sci. US. 1985, 82, 584.
[6] D. Le Guludec, J. Delforge, A. Syrota, M. Desruennes, H. Valette,
I. Gandjbakhnch, P. Merle, Circulation 1994, 90, 172.
[7] D. Le Guludec, A. Cohen-Solal, J. Delforge, N. Delahaye, A. Syrota,
P. Merlet, Circulation 1997, 96, 3416.
[8] N. Delahaye, S. Dinanian, M. Slama, H. Mzabi, D. Samuel, D. Adams,
P. Merlet, D. Le Guludec, Eur. J. Nucl. Med. 1999, 26, 416.
[9] F. Dollé, F. Hinnen, F. Vaufrey, S. Demphel, Y. Bramoulle, D. Fournier,
M. Ponchant, H. Valette, C. Crouzel, J. Labelled Compd. Radiopharm.
2001, 44, 337.
In this study, an improved method for the preparation of the mus-
carinic receptor antagonist [11C]Me-QNB has been reported. The
methodology based on the reaction of QNB with [11C]CH3I at room
temperature using the captive solvent method considerably
improves the previously reported results in terms of decay-
corrected radiochemical yield (91.1Æ 2.4% vs. 48.5 Æ 10%). The
implementation of a purification step using reverse phase semi-
preparative HPLC reverses the elution order of the precursor and
the radiotracer; thus, higher amounts of precursor can be used
without affecting the chemical purity of the final radiotracer.
Alternatively, a purification method using solid-phase extraction
can be applied. As a result, lower radiochemical yield and radio-
chemical purity values are obtained, but overall preparation time
could be shortened from 30–31 min to 20–21min.
Acknowledgements
[10] V. Gómez, J. D. Gispert, V. Amador, J. Llop, J. Labelled Compd.
Radiopharm. 2008, 51(1), 83.
[11] V. Gómez-Vallejo, J. Llop, Nucl. Med. Commun. 2011, 32(11), 1011.
[12] A. A. Wilson, A. Garcia, L. Jin, S. Houle, Nucl. Med. Biol. 2000,
27, 529.
The authors would like to thank the Departamento de Industria,
Comercio y Turismo of the Basque Government for financial
support.
Conflict of Interest
[13] V. Gómez-Vallejo, V. Gaja, J. Koziorowski, J. Llop. Positron Emission
Tomography – Current Clinical and Research Aspects. Chapter 7.
Intech Ed: Croatia, Europe, 2012, pp. 183–210.
The authors did not report any conflict of interest.
J. Label Compd. Radiopharm 2012, 55 470–473
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org