Accelerating preclinical PET-screening: Reductive amination with [ 11C]methoxybenzaldehydes

Article abstract of DOI:10.1039/c4ra02506g

We report, herein, a simple and efficient labelling strategy for multiple PET tracer preparation using a common intermediate, which has the potential to accelerate preclinical PET radiotracer screening. This procedure was applied to and compared with a previously published labelling strategy illustrating the advantages of this newly developed combinatorial approach. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra02506g

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Accelerating preclinical PET-screening: reductive
amination with [11C]methoxybenzaldehydes†
abc
Cite this: RSC Adv., 2014, 4, 21347
Sebastian Leth-Petersen,b Szabolcs Lehel,a Martin Hansen,b
*
Matthias M. Herth,
Gitte M. Knudsen,c Nic Gillings,a Jacob Madsena and Jesper L. Kristensenb
Received 21st March 2014
Accepted 29th April 2014
DOI: 10.1039/c4ra02506g
www.rsc.org/advances
We report, herein, a simple and efficient labelling strategy for multiple thought to be inferior due the short half-life of carbon-11 and
PET tracer preparation using a common intermediate, which has the thus lower radiochemical yield (RCY).
potential to accelerate preclinical PET radiotracer screening. This
procedure was applied to and compared with a previously published 5-HT2A receptor.4 During the development phase, we screened 12
We recently developed a carbon-11 labelled radiotracer for the
labelling strategy illustrating the advantages of this newly developed ligands synthesised from a classical labelling approach of ani-
combinatorial approach.
sols. This required the synthesis of specic precursors for each
tracer resulting in 24 additional precursor synthesis steps
(Scheme 1A). Therefore, we became interested in developing a
new approach that would provide ready access to this class of
tracers and circumvent the need for arduous precursor synthesis.
Inspired by the use of 18F-labelled benzaldehydes for 18F-label-
ling of related compounds (Scheme 1B),5 we aimed to develop a
similar procedure based on an early-stage 11C-labelling of a
simple fragment, which could subsequently be coupled to any
amine via a reductive amination step (Scheme 1C).
Positron emission tomography (PET) is a powerful non-invasive
tool to characterize e.g., receptors, enzymes and other targets in
vivo. For example, it can be used to quantify receptor binding or
receptor occupancy of a given drug at a specic target. For drug
discovery processes or disease diagnosis, these outcomes are
extremely useful to inform drug development or treatment
decisions.1
PET tracer development is usually guided by medicinal
chemistry structure activity relationship (SAR) studies, where
affinity, selectivity towards a certain target and lipophilicity
is optimized. Based on these, a few compounds are selected,
subsequently labelled and nally evaluated.1d Carbon-11
(t1/2 ¼ 20.4 min) is oen the chosen nuclide for development
research due to the possibility to conduct repeated PET
studies in the same subject within hours.2 Traditionally, a
last-stage 11C-methylation strategy is applied,3 whereas
multi-step syntheses involving 11C-labelled intermediates are
We believe that such a 2-step radiosynthesis is advantageous
compared to late-stage labelling – in particular in the preclinical
aPET and Cyclotron Unit, Copenhagen University Hospital Rigshospitalet, Blegdamsvej
9, DK-2100 Copenhagen, Denmark. E-mail: matthias.herth@nru.dk; Fax: +45 3545
3545; Tel: +45 35458621
bDepartment of Drug Design and Pharmacology, Faculty of Health and Medical
Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen,
Denmark. Fax: +45 35 33 60 41; Tel: +45 353 36487
cCenter for Integrated Molecular Brain Imaging, Rigshospitalet and University of
Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Fax: +45 3545 6713;
Tel: +45 3545 6711
† Electronic supplementary information (ESI) available: Experimental procedures,
analytical HPLC conditions, semi-prep HPLC conditions, GMP compliant
radiosynthesis for Cimbi-36, spectroscopic data for selected compounds. See
DOI: 10.1039/c4ra02506g
Scheme 1 (A) Classical approach to labelling of anisole derivatives
requiring protection of amines. (B) 18F-labelling via formation of
18F-benzaldehydes follow by reductive amination of a primary amine.
(C) Proposed fusion of the former protocols.
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screening phase, because necessary precursor synthesis steps unreliable,1d,7 and the development of new PET-tracers is still
are minimized since the needed amines already have been largely a “trial-and-error game”.
prepared during the synthesis of the target compounds.
For example, we recently demonstrated that kinetics, non-
Although this labelling approach has been reported in the specic binding and ultimately the binding potential of 9 struc-
literature, it has not been applied to the synthesis of a library of turally related phenethylamines differed quite dramatically, even
PET-ligands as detailed herein.6
though the in vitro proles were comparable.4 Others have also
Computational approaches to predict the in vivo behaviour found that small molecular changes to the lead compound have
of radiolabelled compounds based on in vitro characteristics are profound effects on the compounds behaviour as a PET-ligand.8
Thus, rather than just labelling the ligand from a compound
series with the best in vitro prole, one should investigate several
representatives from the same compound class. Ready access to
structurally similar PET-ligands with similar pharmacological in
vitro proles greatly increases the chance of success.
Only a limited number of studies have been published con-
ducting combinatorial-like multi-step approaches to create PET
tracer libraries.9 For example, Langstrom and coworkers
utilized such a strategy by applying palladium mediated 11C-
carbonylations.9a–c,10 This is surprising since a fast and efficient
PET tracer access would facilitate the preclinical evaluation
process and probably increase the success rate of novel tracers
reaching the clinic.
˚
¨
Scheme 2 This combinatorial labelling approach could provide easy
access to a PET tracer library for in vivo preclinical evaluation studies by
circumventing precursor synthesis for every single PET tracer.
Therefore, our main goal was to extend the existing combi-
natorial-like strategy to
a
2-step key motif 11C-labelling
approach (Scheme 2). Two prerequisites have to be fullled for
such a procedure.
(1) The labelling of the key motif as well as the subsequent
coupling has to be fast and efficient (<10 min, RCY > 50%).
(2) The purication and formulation must be efficient and
rapid (<15 min).
Consequently, the total synthesis time should not exceed 40
min and an isolated yield > 300 MBq has to be achievable.
Reductive aminations are fast, efficient and can be con-
ducted chemoselectively.11 Furthermore, many drugs contain
amines and thus a broad variety of target compounds exist
which can be synthesized applying this strategy.12
To develop this approach, we chose to focus on the labelling
of [11C]Cimbi-36, a new PET-tracer for the visualisation of the
5-HT2A receptor in the CNS.4 The reason for that was twofold:
rstly, procedures for radiolabelling methoxybenzaldehydes
(MB-CHO) in high radiochemical yields (>80%) and short
reaction time (<5 min) are well described.13
Scheme 3 Comparison of the direct labelling approach (a) with the
established labelling procedure via (b) and c). (a) NaBH3CN, AcOH,
DMSO–MeOH, 130 ꢀC, 5 min. (b) [11C]CH3OTf, MeCN–acetone,
NaOH, 40 ꢀC, 30 s, (c) TFA, MeCN, 80 ꢀC, 5 min.
Table 1 Tested radiolabelling conditions of the novel reductive amination approach for the synthesis of [11C]Cimbi-36
2nd key motif coupling (0.6 mL MeOH,
Total yielda
1st key motif labelling (0.3 mL DMSO, [11C]MeOTf)
5 mL AcOH, 130 ꢀC)
2-HBA [mg]
2M NaOH [mL]
Time [s]
Temp. [ꢀC]
2C–B [mg]
NaBH3CN [mg]
Time [min]
Isolated product [MBq]
2
20
3
1
1
1
1
1
1
30
30
30
25
25
25
25
40
60
25
25
25
6
4
4
4
4
4
4
25
6
4
4
4
4
4
4
5
5
5
5
5
5
1
2.5
931
752
815
1684
226
230
20
0.3
0.1
0.1
0.1
0.1
0.1
0.1
60
120
120
30
30
43
a
Isolated yield was determined at end of synthesis, see ESI.
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Secondly, this would allow us to compare the new approach than sufficient to conduct preclinical imaging since usually
with a known protocol. In Scheme 3, the established procedure about 300 MBq is required for an in vivo PET scan in larger
for 11C-labelling via alkylation of a Boc-protected precursor along animals. In regards to a tracer library labelling approach, the
with the proposed reductive amination approach is outlined.
synthesis fullled the prerequisites and thus, we tested this
The short half-live of carbon-11 puts severe restrictions on method on other structurally related phenethylamine struc-
the number and kind of transformations one can conduct aer tures. Several ortho-, meta- and para-HBA moieties were
the label has been introduced. Ackermann et al. showed that a successfully labelled and subsequently coupled to 2C–B
2-step reductive amination strategy of a 11C-labelled aniline (Scheme 4A).
derivative could be successfully carried out.6a,14 They utilized
Moreover, four diverse primary phenethylamines were
this strategy to circumvent side-product formation otherwise coupled to 2-[11C]MB-CHO (Scheme 4B). Similar reaction
observed using a traditional last-step labelling approach. parameters were observed for all conducted experiments and
Furthermore, successful reductive aminations of [18F]uo- therefore, we believe that this novel combinational 2-compo-
robenzaldehyde ([18F]FB-CHO) with various amines have been nent reductive 11C-amination approach is generally applicable
performed in 10–15 min using sodium cyanoborohydride
(NaBH3CN).5 Thus, the proposed reductive amination approach
should be feasible for a broad spectrum of amines in the short
reaction times required for carbon-11 chemistry (<10 min).
Therefore, we tried to combine the known labelling proce-
dure of 2-[11C]methoxybenzaldehyde (2-[11C]MB-CHO) with the
reductive amination conditions for [18F]FB-CHO and applied
them to primary phenethylamines,5,13 which was successful at
rst. However, several parameters needed optimization: rstly,
a 15–20 minute reaction time was detrimental to the RCY and
secondly, since relatively large amounts of the precursors were
used (25 mg of 2C–B and 2 mg of 2-hydroxybenzaldehyde
(2-HBA)), formation of signicant amounts of non-radioactive
side-products complicated the subsequent purication of the
tracer. Therefore, we aimed to optimize and improve these in
the next phase.
No inuence of precursor concentration on the radiochem-
ical yield of the rst labelling step could be detected. Thus, we
could successfully lower the amount of precursor to 5% of the
original reported procedure. Furthermore, the amount of 2C–B
and NaBH3CN could be reduced, greatly simplifying the nal
purication. Thus, a successful separation aer a 2-step, one-
pot reductive amination was achievable (Table 1).
It is worthwhile to mention that the use of glacial acetic acid
(AcOH) was essential for the 2nd reaction step and that we were
not able to reduce the reaction time for the reductive amination
to below 5 min. Insufficient imine and subsequently amine
conversion was otherwise observed. Moreover, the use of the
less toxic reducing agent sodium triacetoxyhydroborate failed.
Finally, we optimized the RCY for the 1st labelling reaction,
where the use of [11C]CH3OTf instead of [11C]CH3I decreased
the reaction time from 5 min to 60 s. Eventually, the optimal
conditions were found to be: 1st labelling step: [11C]CH3OTf,
1 mmol 2-HBA, 2 mmol 2N NaOH, 300 mL DMSO, 25 ꢀC and 60 s;
2nd labelling step: 15 mmol 2C–B, 63 mmol NaBH3CN, 87.5 mmol
AcOH, 0.6 mL MeOH, 130 ꢀC, 300 s. Table 1 summarizes the
experiments conducted.
In summary, [11C]Cimbi-36 could be labelled in a simple and
efficient 2-step, one-pot synthesis within 40 min (yield: 1.6 GBq,
specic radioactivity: 60–146 GB mmolꢁ1, radiochemical purity:
Scheme 4 Standard reductive 11C-amination conditions (NaBH3CN,
appropriate R–NH2, CH3COOH, DMSO–MeOH, see ESI†) which
>99%). Compared with our previous optimized classical 2-step
11C-labelling strategy, the new method usually resulted in ꢂ10–
resulted in an easy access to 8 PET tracers. (A) Coupling of MB-CHO
derivatives to 2C–B; (B) coupling of 2-[11C]MB-CHO to different
20% lower isolated yield, whereas other parameters were
similar. Despite the lowered yield, this novel strategy is more phenethylamine derivatives.
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to all kinds of key motif reductive amination approaches and
leads, indeed, to an easy tracer library access.
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In conclusion, a library of eight phenethylamines was
labelled without the need for time consuming precursor
synthesis. Thus, we have shown that this novel synthetic library
approach, via a key radiolabelling intermediate, facilitates
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Acknowledgements
The authors wish to thank the staff at the PET and Cyclotron
unit for expert technical assistance. Financial support by the
Faculty of Health and Medical Sciences at the University of
Copenhagen and the Lundbeck Foundation are gratefully
acknowledged.
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