Radiofluorination and reductive amination using a microfluidic device

Article abstract of DOI:10.1002/jlcr.2970

A commercial microfluidic device (NanoTek, Advion) was used as a synthesis platform for the preparation of fluorine-18 labelled tertiary amines in two consecutive steps. Firstly, the nucleophilic radiofluorination of an aromatic aldehyde and secondly, the

Full text of DOI:10.1002/jlcr.2970

Research Article
Received 12 June 2012,
Revised 24 August 2012,
Accepted 8 September 2012
Published online 11 October 2012 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2970
Radiofluorination and reductive amination
using a microfluidic device
a
a,b
Kenneth Dahl, Magnus Schou, and Christer Halldina
*
A commercial microfluidic device (NanoTek, Advion) was used as a synthesis platform for the preparation of fluorine-18
labelled tertiary amines in two consecutive steps. Firstly, the nucleophilic radiofluorination of an aromatic aldehyde and
secondly, the reductive amination to produce the corresponding amine. Fluorine-18 labelled [18F]fluorobenzaldehyde
([18F]2) was obtained in an analytical radiochemical yield (rcy) of 93% and a preparative yield of 60% (decay corrected).
The produced [18F]2 was applied in two model reactions yielding [18F]5 and [18F]6 in analytical rcy 70 and 75%, respectively.
To further test the utility of this methodology, a delta opioid agonist, [18F]8, was also radiolabelled using the same setup in
an analytical rcy of 29%.
In a preparative run, 1050 MBq (28.4 mCi) isolated product ([18F]6) was obtained in a 37.5% decay corrected overall rcy
calculated from [18F]fluoride. The radiochemical purity of [18F]6 was greater than 99% and the specific radioactivity
298 GBq/mmol (8052 Ci/mmol) at end of synthesis.
Keywords: microfluidics; reductive amination; radiofluorination; fluorine-18
amination is particularly important, direct reductive amination
Introduction
of carbonyl compounds, mostly aldehydes or ketons, is one of
Positron emission tomography (PET) is a powerful tool using
the most powerful methods for the synthesis of substituted
amines.20–24
radiolabelled compounds as molecule probes to study biological
processes in vivo. The use of PET in medical applications
We report here the use of a commercially available microfluidic
radiochemistry system as synthetic platform applied in a two-step
and drug discovery has stimulated the development of new radi-
olabelling strategies and tracer molecules labelled with 11C
synthetic preparation of fluorine 18-labelled fluorobenzyl amines,
(t1/2 = 20.3 min) and 18F (t1/2 = 109.7 min) as positron emitting
via the aromatic nucleophilic radiofluorination and followed by
radionuclides.1–3 It has been increasingly recognized that
micro-reactors (microfluidic) may present considerable advan-
tages in the radiochemistry of PET tracer labelled with positron
emitting radionuclides.4,5 The advantages of using microfluidic
reactors for organic chemistry are well-documented and include
the direct reductive amination starting from a mono substituted
benzaldehyde compound. (Scheme 1)
Results and discussion
benefits associated with miniaturization, which offer advantages
over the conventional reaction settings, such as reduced chemi-
cal consumption, high surface-area-to-volume ratios, automa-
Radiosynthesis of [18F]fluorobenzaldehyde ([18F]2) (Scheme 1)
tion, and improved control over mass and heat transfer.6–9 As a model substrate for the reductive amination, we used the
Microfluidic reactors for PET radiosynthesis have generated aromatic aldehyde, [18F]2 as a labelling precursor, a strategy first
considerable interest primarily because miniaturization reaction published by Wilson and co-workers in 1990.25
systems have the potential to address the challenges of increas-
The first step involves the nucleophilic aromatic substitution
ing the speed of labelling reaction and improving the overall of the para-substituted 4-trimethylammoniumbenzaldehyde
efficiency of the radiolabelling reaction process.10–13
triflate precursor.
Throughout the past decennium, several studies on the
application of microfluidics for PET have been reported, mainly
focusing on reactor design and proof-of-principle reactions, as
in the multiple reports on the carbonylation reaction of carbon
11-labelled amides using [11C]carbon monoxide14–16 and the full
radiosynthesis of the well-established PET radioligand [18F]2-
fluoro-2-deoxy-D-glucose.17–19
Amines are an important class of compounds found in many
natural products, pharmaceuticals, and other valuable organic
molecules. Although there are many synthetic methods that
can be used to produce amines, there is still a need for alterna-
aKarolinska Institutet, Department of Clinical Neuroscience, Center for Psychiatric
Research, Karolinska Hospital, S-171 76, Stockholm, Sweden
bAstraZeneca R&D, Innovative Medicines, CNSP iMed, Medicinal Chemistry 151
36, Södertälje, Sweden
*Correspondence to: Kenneth Dahl, Karolinska Institutet, Department of Clinical
Neuroscience, Center for Psychiatric Research, Karolinska Hospital, S-171 76
Stockholm, Sweden.
tive ways that are mild and reproducible. Of these, reductive E-mail: kenneth.dahl@ki.se
J. Label Compd. Radiopharm 2012, 55 455–459
Copyright © 2012 John Wiley & Sons, Ltd.

K. Dahl et al.
Scheme 1. The two-step synthesis route for two fluorine 18-labelled fluorobenzyl amines, [18F]5 and [18F]6. (a) 18F/K+/K222 in MeCN; (b) piperazine, sodium cyanoboro-
hydride, acidic acid in 1:1 mixture between MeCN/MeOH; (c) 4-phenyl-piperidine, sodium cyanoborohydride, acidic acid in 1:1 mixture between MeCN/MeOH.
Reaction parameter screening (Figure 1)
production of [18F]2, that is, when using a larger reagent volume
for the reaction, 400 mL versus 20 mL. After decreasing the amounts
of potassium carbonate and kryptofix (personal communication,
Shuiyu Lu, NIMH), we did not experience any more plugged reac-
tors. [18F]2 was produced in a decay corrected rcy of 60 Æ 3%
approximately 8 min after start of reaction. The radiochemical
purity was greater than 99%.
A series of reactions was conducted in a 2-meter micro-reactor
(i.d. 100 mm) using acetonitrile as solvent, to establish conditions
where [18F]2 is produced rapidly in high and reproducible yield.
The flow rate and volume of the two reagents, precursor solution
and [18F]fluoride ion solution, were set equal at 50 mL/min and
20mL, respectively. The reaction mixtures leaving the micro-reactor
were collected in a vial containing ethanol before the product
mixture was analyzed by radio-thin layer chromatography (TLC)
to calculate the radiochemical yield (rcy). The influence of
temperature on the rcy was studied at different precursor concen-
trations, the results are displayed in Figure 1. An 80% rcy was
achieved when using a precursor concentration of 3.2mM, which
is about 4–6 times lower than the reported by other groups using
conventional methods.25–27 The reaction/residence time for a par-
ticular liquid plug inside the micro-reactor was 9.4s, thus allowing
for a rapid screening of the reaction parameters as well as decreas-
ing the number of required cyclotron productions. The highest rcy
based on [18F]fluoride, 93%, was obtained at 200ꢀC with a precur-
sor concentration of 31.9 mM. However, because a lower concen-
tration of precursor would be beneficial for the subsequent
reductive amination, we chose to use 4.8 mM at a reaction tem-
perature of 160ꢀC for the continued study. The lower temperature
was selected to increase the stability of the reaction.
Preparation of fluorine-18 labelled [18F]fluorobenzyl amines
(Scheme 1)
Having defined the radiofluorination conditions, we proceeded
to study [18F]2 reactivity in the subsequent reductive amination
reaction.
Direct reductive amination involves the initial formation of an
intermediate hemiaminal that dehydrates to form an imine.
Under acidic condition, the imine is protonated to form an
iminium ion and is subsequently reduced to produce the corre-
sponding alkylated amine. The reduction of the imine is usually
achieved by borohydride reducing agents or catalytic hydroge-
nation. In this study, we utilized sodium cyanoborohydride
as reductant.
Reaction parameter screening (Figure 2)
We conducted a series of reactions using [18F]2 and a secondary
amine mixture including the reducing agent sodium cyanoboro-
Batch production of [18F]2
As opposed to in the reaction parameter screening, we experi- hydride dissolved in methanol. The secondary amine solution
enced several plugged reactors when setting up the full batch was pH adjusted by adding droplets of acetic acid, with a final
pH between 3 and 5, in accordance with earlier publications.22
The crude reaction mixture from the first step was mixed online
with reagent mixture 2 at the inlet of the second micro-reactor
(4 m long). The overall labelling occurred as a single process
without any intermediate purification step.
The influence of temperature and amine concentration on the
rcy was performed using test compounds 3 and 4 by varying the
amine concentration between 10 and140 mM and at a tempera-
ture interval of 100–175ꢀC, whereas the reaction conditions from
the first step were kept constant. For the second step, the flow
rate from respective syringe pump was kept at 30 mL/min, with
a final reaction time of 31.4 s. The reaction mixture leaving the
micro-reactor was collected in a vial containing ethanol before
the product mixture was analyzed by radio-HPLC to assess the
rcy; results are displayed in Figure 2.
Figure 1. Radiochemical yield based on [18F]fluoride is presented as a function of
temperature and precursor concentration for the radiofluorination step, establish
by analytical radio-TLC.
The highest rcy of [18F]5 was 70% and the corresponding
value for [18F]6 was 75%. The reproducibility in the preparation
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Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 455–459

K. Dahl et al.
Scheme 2. The two-step synthesis route of AZ12439516 ([18F]8) via the reductive
amination of [18F]Fluorobenzaldehyde ([18F]2). (a) step (1) 18F/K+/K222, 4-trimethy-
lammoniumbenzaldehyde triflate in MeCN; step (2) amine precursor (7), sodium
cyanoborohydride, acidic acid in 1:1 mixture between MeCN/MeOH.
Experimental
General procedures
MP-1 anionic rasin cartridges were supplied by ORTG (Tennessee, USA). All
chemicals and solvents were purchased from commercial sources and used
without further purification, except for 4-trimethylammoniumbenzaldehyde
triflate precursor, which was prepared according to the literature.25 High
pressure liquid chromatographic analysis (HPLC) was performed using a
Hitachi L-6200 gradient pump and a Hitachi L-4000 variable wavelength
UV-detector in a series with a Bioscan b+-flow detector. Analytical HPLC
analysis was performed using a reverse phase column (Waters mBondapak,
C18, 10 mm, 3.9 Â 300 mm) eluted with a gradient between acetonitrile (A)
and 10 mM H3PO4 (B). The gradient was linear between 20 and 100% over
10 min at a flow rate of 2ml/min. Thin-Layer Chromatograhpy (TLC) was
performed on a Raytest MiniGita TLC scanner (Raytest, Munich, Germany).
Liquid chromatography - Mass Spectrometry (LC-MS) was performed on
a Waters QTof Premier coupled with a Waters Acquity UPLC system (Waters,
Sollentuna, Sweden). Semi-preparative HPLC purification was performed on
a reversed-phase column (ACE – C18, 5mm, 10 Â 250 mm) eluted with an
isocratic mobile phase consisting of MeCN and 50mM H3PO4 (30:70, v/v)
at a flow rate of 6 ml/min. The eluate from the HPLC column was monitored
for UV absorbance (254 nm) and radioactivity using a Knauer smartline UV-
detector 2600 and an uncalibrated GM-tube, and the signal was recorded
using a flatbed recorder (Kipp & Zonen, Holland). No-carrier-added (NCA)
Figure 2. Radiochemical yields (Rcy) of [18F]5 and [18F]6 based on [18F]2 are pre-
sented as a function of temperature and precursor concentration for the reductive
amination step, establish by analytical radio-HPLC. Rcy values at 70 mM is a mean
from three consecutive runs, n = 3.
of [18F]5 and [18F]6 was also studied by three consecutive
productions cycles at an amine concentration of 70 mM.
Batch production of [18F]6
In a preparative run, 1050 MBq (28.4mCi) isolated product ([18F]6)
was obtained in a 37.5% decay corrected overall rcy from [18F]
fluoride. The radiochemical purity was greater than 99%, and
the specific radioactivity was 298GBq/mmol (8052 Ci/mmol). The
relatively low radioactivity recovery could partly be explained by
[18F]fluoride ion absorption onto inner surfaces, for example,
micro-reactor, tubing, and drying vial. Absorption is a common
phenomenon in reactions of dry NCA [18F]fluoride ion.28
[
18F]fluoride ion was obtained from the 18O(n, p)18F nuclear reaction by
irradiating [18O]water for 2 min with a proton beam (16.4 MeV, 35 mA) gen-
erated with a GEMS PETtrace cyclotron (GE, Uppsala, Sweden). [18F]Fluoride
ion was isolated onto an anion resin (MP-1) cartridge and subsequently
eluted with a solution of K2CO3 (0.45 mg, 3.3mmol) and Kryptofix 2.2.2
(4.5mg, 11.9mmol) in MeCN-H2O (450 ml, 1:1 v/v) into a vial (2ml, Alltech).
The solvents were azeotropically dried at 105ꢀC in 15min. The formed
Radiosynthesis of [18F]AZ12439516 ([18F]8) (Scheme 2)
[
18F]F-/K+/K222 complex was diluted with anhydrous MeCN and loaded onto
storage loop (P3, 400 ml) in the apparatus. The automatic microfluidic
system was either setup for one-step or two-step radiosynthesis (Figure 3).
For more detail description of the apparatus, we refer to earlier studies.10,11
To further test the utility of this methodology, we also radiola-
belled a delta opioid agonist using the optimal conditions estab-
lished for compounds [18F]5 and [18F]6. The analytical rcy of the
crude product was 29%. The rather low rcy could be a conse-
quence of a poor drying step for that particular experiment.
Microfluidic synthesis of [18F]2
Dry
[
18F]F-/K+/K222 solution and 4-trimethylammoniumbenzaldehyde
triflate precursor (0.64–31.9mM), both in anhydrous acetonitrile (400ml),
were loaded into their respective storage loop. The precursor solution
was filtered (filter, 0.45mm, Waters) before use. About 20ml of solution from
each loop was simultaneously dispensed through a coiled silica glass micro-
reactor (100 mm i.d., 2m long; internal volume 15.7ml) using variable
temperature (100–200ꢀC) at a set flow rate (50 ml/min). The product mixture
was analyzed by radio-TLC on silica gel (Merck), developed with CH2Cl2, and
quantified using a radio-TLC scanner. In the full batch production, 400ml
of each solution was dispensed through the micro-reactor at the
optimal conditions established in the test stage. [18F]2 was isolated on a
conditioned (10 ml EtOH, 15ml H2O) reversed phase SPE cartridge
Conclusion
A method for the two-step preparation of 18F-fluorobenzyl
amines using reductive amination in a commercially available
microfluidic radiochemistry system has been developed. The
microfluidic apparatus allowed for a rapid parameter optimiza-
tion and were scalable to produce adequate radioactivities for
PET applications. The method is simple and provides good yields
and was successfully applied in the radiolabelling of a potentially
new radioligand for the delta opioid receptor.
J. Label Compd. Radiopharm 2012, 55 455–459
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

K. Dahl et al.
Figure 3. Microfluidic apparatus setup, (P3) 18F-/K+/K222 complex solution; (P1) precursor solution; (P2) amine precursor solution.
(Waters, Sep-pak tC18 PLUS) using the following procedure: The crude flow rate; 30 ml/min). Amine solution (40 ml) and the crude reaction
reaction mixture was diluted with 25 ml H2O before it was pushed through mixture solution from step one (40 ml) was simultaneously dispensed
the SPE. The isolated product ([18F]2) was eluted with 2ml of ethanol. The through a micro-reactor (100 mm i.d., 4 m long; internal volume 31.4 ml),
radiochemical purity was established with radio-HPLC.
the micro-reactor was kept at 175ꢀC and a set flow rate of 30 ml/min.
Radiochemical purities were analyzed by analytical radio-HPLC.
Microfluidic optimization of [18F]5 and [18F]6
Solutions of 4-trimethylammoniumbenzaldehyde triflate precursor
(4.8 mM) in MeCN (400 ml), dry [18F]F-/K+/K222 solution in MeCN (400 ml)
and a solution of amine (10–140 mM, 3 or 4), sodium cyanoborohydride
(8 mg, 127.3 mmol), acetic acid (10 ml, 158.7 mmol) in MeOH (400 ml), was
loaded onto their respective storage loop. The radiolabelling step condi-
tions were kept constant (reagent volume, 20 ml; temperature, 160ꢀC;
syringe flow rate, 30 ml/min). Amine solution (40 ml) and the crude reac-
tion mixture solution from step one (40 ml) was simultaneously dispensed
through a micro-reactor (100mm i.d., 4m long; internal volume 31.4 ml), the
micro-reactor was kept at a fixed temperature (100–175ꢀC) using a set
flow rate of 30 ml/min. Radiochemical purities were analyzed by analytical
radio-HPLC.
Acknowledgements
Thanks to all the members of the PET group at Karolinska Institutet.
Conflict of Interest
The authors did not report any conflict of interest.
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