Beyond azide-alkyne click reaction: Easy access to 18F-labelled compounds via nitrile oxide cycloadditions

Article abstract of DOI:10.1039/c2cc31335a

Radiofluorinated 4-fluorobenzonitrile oxide and N-hydroxy-4- fluorobenzimidoyl chloride rapidly react with different alkenes and alkynes under mild conditions. These cycloadditions are suitable for the preparation of low-molecular weight radiopharmaceutic

Full text of DOI:10.1039/c2cc31335a

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Chemical Communications
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Volume 48 | Number 57 | 21 July 2012 | Pages 7105–7220
ISSN 1359-7345
COMMUNICATION
Bernd Neumaier et al.
Beyond azide–alkyne click reaction: easy access to 18F-labelled compounds via nitrile oxide cycloadditions

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18
Beyond azide–alkyne click reaction: easy access to F-labelled
compounds via nitrile oxide cycloadditionsw
b
b
ac
Boris D. Zlatopolskiy,ab Rene Kandler, Diana Kobus, Felix M. Mottaghy and
´
Bernd Neumaier*b
Received 22nd February 2012, Accepted 17th April 2012
DOI: 10.1039/c2cc31335a
Radiofluorinated 4-fluorobenzonitrile oxide and N-hydroxy-4-
fluorobenzimidoyl chloride rapidly react with different alkenes
and alkynes under mild conditions. These cycloadditions are
suitable for the preparation of low-molecular weight radiopharma-
ceuticals and, in a strain-promoted variant, can enable easy
labelling of sensitive biopolymers.
under different reaction conditions and metabolic stability of
the formed 1,2,3-triazoles. Recently, Feringa et al. published
the preparation of an 18F-labelled bombesin analogue via
copper-free strain-promoted cycloaddition between 18F-labelled
azides and aza-dibenzocyclooctyne-modified peptides in DMF.5
This methodology for radiolabelling allows to overcome
problems associated with the application of copper catalysts
such as cytotoxicity, copper-promoted degradation of peptides
and proteins or formation of insoluble copper-acetylide.4c
Until quite recently only azide–alkyne coupling was
exploited for radiolabelling although numerous other [3+2]-
dipolar cycloadditions are known.6 Some of them require only
readily available simple starting materials and afford meta-
bolically and chemically stable cycloadducts. This year, we have
reported on the first example of a successful application of
nitrone–alkene cycloaddition for radiolabelling.7 18F-labelled
isoxazolidines were obtained in high radiochemical yields (rcy)
by rapid reaction of C-(4-[18F]fluorophenyl)-N-phenyl nitrone
([18F]1) with model maleimides. However, due to relatively
high reaction temperatures (80–110 1C) this approach is unsuitable
for radiolabelling of more sensitive substrates like proteins. There-
fore, we turned our attention to other [3+2]-dipolar cyclo-
additions such as fast metal-free cycloaddition reactions between
nitrile oxides and carbon–carbon multiple bonds leading to
metabolically and chemically stable 2-isoxazolines or isoxazoles.
In this communication we describe the in situ generation of
4-[18F]fluorobenzonitrile oxide ([18F]2) and its subsequent
reaction with unsaturated compounds. To demonstrate the
potential of this novel approach, the preparation of radiofluori-
nated COX-2 specific tracers (inflammation markers) is presented.
Finally, the application of the proposed methodology for radio-
labelling of model dipeptides is described.
Positron emission tomography (PET) is a powerful non-
invasive diagnostic molecular imaging tool for quantitative
3D-visualization of physiological and pathological processes
in vivo using biologically active molecules labelled with
b+-emitting nuclides (PET-tracers).z Fluorine-18 (18F) is one
of the most widely used radioisotopes for PET. 18F (in the
form of 18Fꢀ) is easily accessible from H218O via 18O(p,n)18F
nuclear reaction. It decays almost completely by low-energetic
b+-emission [E(b+) = 630 keV, 97%] and, consequently,
gives 3D emission scans with a high intrinsic resolution.
Furthermore, taking into account logistical considerations 18F
has a sufficient half-life of 109.8 min enabling transportation of
18F-labelled radiopharmaceuticals to other clinical sites.
A broad spectrum of direct and indirect methods for
18F-labelling of small molecules and for indirect radiofluori-
nation of biopolymers (e.g. proteins and nucleic acids) was
developed.1 Each of them have their own advantages and
limitations. An important contribution to this area was the
implementation of Cu(I)-catalyzed [3+2]-azide–alkyne Huisgen
cycloaddition (azide–alkyne click reaction)2 for radiolabelling.
Since Marik and Sutcliffe prepared 18F-labelled peptides by
the click reaction between [18F]fluoroalkynes and peptides
bearing an azide moiety3 numerous 18F-labelled tracers were
prepared using this approach.4 Among a variety of advantages
of this approach are: regioselectivity, non-requirement of
protection groups, mild reaction conditions, acceptable yields
Preparation of no-carrier-added [18F]2 started with the
synthesis of 4-[18F]fluorobenzaldehyde ([18F]FB–CHO) according
to Haka et al.8 (Scheme 1). [18F]FB–CHO was prepared in
30–50% rcy (4–10 GBq, no decay corrected) within 50 min.
4-[18F]Fluorobenzaldoxime ([18F]3) was prepared by reacting
[18F]FB–CHO with hydroxylamine in 92.1 ꢁ 2.4% yield (n = 23)
and >90% radiochemical purity after 10 min incubation in
aqueous MeOH at 40 1C. Phenyl iodine bis(trifluoroacetate)
(PIFA) was added to crude oxime [18F]3y to generate nitrile
oxide [18F]2.9 Afterwards, [18F]2 was allowed to react with the
appropriate dipolarophile (Scheme 1 and Table 1).
a Klinik fur Nuklearmedizin, Aachen University, RWTH,
¨
Pauwelsstr. 30, 52074 Aachen, Germany
b Max Planck Institute for Neurological Research, Gleueler Str. 50,
50931 Cologne, Germany. E-mail: bernd.neumaier@nf.mpg.de;
Fax: +49-221-4726-298; Tel: +49-221-4726-270
c Department of Nuclear Medicine, Maastricht University Medical
Center, P. Debyelaan 25, 6229HX Maastricht, The Netherlands
w Electronic supplementary information (ESI) available: Details of the
synthesis of the reference compounds and precursors as well as of
labelling experiments; NMR-spectra and HPLC traces. See DOI:
10.1039/c2cc31335a
c
7134 Chem. Commun., 2012, 48, 7134–7136
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In all cases the respective 18F-labelled 2-isoxazolines/isoxazoles
([18F]4–11)w were obtained in good to excellent yields with
complete regioselectivity already after 10 min of reaction time.
Surprisingly, even with the less reactive dipolarophiles such
as styrene and 4-pentyn-1-ol (entries 5 and 8, respectively) high
yields of cycloaddition products were achieved. Highly strained
(1R,8S,9s)-9-hydroxymethylbicyclo[6.1.0]nonyne (BCN-OH)10
reacted with [18F]2 to give isoxazole [18F]4 in virtually quanti-
tative yield at room temperature (entry 1). Noteworthily,
quantitative cycloaddition of [18F]2 to much less strained
norbornene at room temperature (entry 2) and to moderately
activated methyl N-maleimidoacetate at 40 1C (entry 3) was
also observed. Alkynes other than BCN-OH (entries 6–8) afforded
besides isoxazoles noticeable amounts of by-products (10–35%).
To explore the new radiofluorination method for tracer
development under ‘‘field conditions’’ we prepared three
18F-labelled indomethacin derivatives ([18F]12–14). These indo-
methacin derivatives were developed to target cyclooxygenase 2
(COX-2) and potentially enable visualization of COX-2
expression in inflammation and cancer (Scheme 2 and Table 1,
entries 9–11). Indomethacin diamides with a sufficiently long
spacer retain a high affinity to COX-2 even in the case of a
bulky second acyl moiety. For example, compound 15 con-
taining a large 5-carboxy-X-rhodamine residue was the first
tracer suitable for optical imaging of COX-2 expression
in vivo.11 The 18F-labelled conjugates [18F]12 and [18F]13 were
prepared from 16 and 17, respectively, in high radiochemical
yields using PIFA as an oxidant. In contrast, the rcy of [18F]14
was quite low due to the relatively low dipolarophilic reactivity
and extremely low solubility of precursor 18. We assumed that
in the case of low cycloaddition rates once formed [18F]2 will
be subjected to acid-promoted decomposition, solvolysis or
react with minor contaminants in the reaction medium. There-
fore, labelling yield will be improved by slow generation of
[18F]2. Consequently, [bis(acetoxy)iodo]benzene (BAIB) which
is a weaker oxidant as PIFA and produces nitrile oxides much
slower as PIFA9,12 was used. Furthermore, during oxidation
by BAIB weak acetic acid instead of strong trifluoroacetic acid
is liberated. With this milder oxidant the desired conjugate
[18F]14 was obtained in 35% rcy. The conjugation products
[18F]12–14 were purified by HPLC and formulated in 20% ethanol.
Scheme 1 Preparation of
[
18F]2 and its reaction with different
F]ꢀ, DMSO, 85 1C, 10 min; (ii)
dipolarophiles. (i) [KC2.2.2]+ 18
[
NH2OHꢂHCl, 1 M NaOH, MeOH, 40 1C, 10 min; (iii) PIFA, 5–10 s;
(iv) dipolarophile.
Table 1 Cycloaddition of [18F]2 to different dipolarophilesa,b
Entry Dipolarophile
Product
Rcy ꢁ SD (%) [T/1C]
95.1 ꢁ 4.7 (rt)
1
2
3
> 95 (rt)
> 95 (40)
4
5
Methyl acrylate
Styrene
91.3 ꢁ 3.9 (80)
78.3 ꢁ 3.4 (80)
6
DMAD
69.5 ꢁ 5.0 (40)
7
8
Methyl propiolate
4-Pentyn-1-ol
57.7 ꢁ 1.6 (80)
57.0 ꢁ 3.6 (80)
9c
10c
11c,d,e 18
16
17
81.4 ꢁ 2.0 (rt)
54.8 ꢁ 1.3 (40)
35.3 ꢁ 4.7 (60)
a
Conditions: a solution of [18F]3 (100–150 MBq) and the corre-
sponding dipolarophile (10 mmol) in 80% MeOH (450 mL) was treated
with PIFA (3.45 mg, 8.23 mmol) in MeOH (50 mL) at rt and the
reaction mixture was stirred at the given temperature for 10 min.
b
c
Each experiment was carried out in triplicate. 1.2–2.5 GBq [18F]3
was used. A solution of [18F]2 and oxidant in MeOH (30 mL) was
added to a preheated solution of 18 in DMF (35 mL). BAIB (8 mmol)
was used instead of PIFA. rcy = radiochemical yield; SD = standard
deviation; rt = room temperature.
d
e
Scheme 2 Indomethacin conjugates: [18F]12–14 prepared via [3+2]-
nitrile oxide–alkene/alkyne cycloaddition; 15—first COX-2 specific
ligand for optical imaging in vivo; 16–18—precursors for radiosynthesis.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 7134–7136 7135

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and of the norbornene-substituted precursor 20 to 50 nmol
(47% rcy of [18F]23) without significant drop in yields.
In summary, we demonstrated for the first time that [3+2]-
nitrile oxide–alkene/alkyne cycloaddition can be applied for
[18F]-radiolabelling. Novel labelling precursors, nitrile oxide
[18F]2 and imidoyl chloride [18F]21, can be efficiently generated
in situ from easily accessible oxime [18F]3 using different
oxidants. [18F]2 and [18F]21 are valuable building blocks
for rapid and high-yielding preparation of 18F-labelled
compounds under mild reaction conditions (10 min, r80 1C,
aqueous media, no catalyst necessary). The proposed method
is a possible alternative to radiofluorination via copper-
catalyzed/copper-free azide–alkyne ‘‘click’’ reaction. Additionally,
radiofluorination via fast strain-promoted [3+2]-nitrile oxide–
alkyne cycloaddition can be carried out in aqueous media at
room temperature with low amounts of dipolarophile. There-
fore, it should be well suited for fast and simple preparation of
radiolabelled biopolymers.
Scheme 3 Labelling of model dipeptides 19 and 20 using the
radiofluorinated N-hydroxyimidoyl chloride [18F]21. Conditions: (i)
Chloramine T, aq. EtOH, rt, 4 min, then 19 or 20, rt, 10 min. [18F]23
was obtained as a mixture of two regioisomers (ca. 1.5 : 1).
Table 2 Labelling of model dipeptides 19 and 20 using [18F]2 and the
radiofluorinated N-hydroxyimidoyl chloride [18F]21. Dependence on
precursor amounta
This work was supported by EFRE-ZIEL2 program (North
Rhine-Westphalia, Germany).
Precursor amount/nmol
Rcy of [18F]22 (%)
Rcy of [18F]23 (%)
104b
103b
103c
500c
100c
50c
25c
10c
5c
88.3 ꢁ 1.0
36.7 ꢁ 3.0
83.4 ꢁ 2.6
83.7 ꢁ 1.4
76.7 ꢁ 1.8
70.1 ꢁ 12.2
64.3 ꢁ 12.6
—
52.6 ꢁ 1.6
7.6 ꢁ 2.3
82.3 ꢁ 6.5
71.6 ꢁ 2.7
55.9 ꢁ 5.3
46.5 ꢁ 2.1
26.1 ꢁ 8.5
13.4 ꢁ 3.3
5.9 ꢁ 0.5
—
Notes and references
z An important exception is blood flow imaging, for which labelled
small penetrant molecules and ions such as [13N]NH3, [15O]H2O or
82Rb+ are used.
y The crude [18F]3 can be purified, e.g. by RP-SPE extraction.
However, in majority of cases, the purification step did not improve
yields of the following cycloaddition reactions.
55.8 ꢁ 2.3
1c
2.8 ꢁ 1.4
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a
b
Each experiment was carried out in triplicate. With PIFA as an
c
oxidant, see caption of Table 1. With chloramine T as a chlorination
agent: [18F]3 (25–150 MBq) in EtOH (10 mL) was treated at rt with a
solution of chloramine T (0.23 mg, 1 mmol) in H2O (20 mL) followed
after 4 min by the dipolarophile in EtOH (5–100 mL) and the reaction
mixture was stirred at rt for 10 min.
A biological study of [18F]12–14 is in progress and will be
reported in due course.
The amounts of dipolarophiles used (10 mmol, Table 1) are
acceptably low for labelling of small molecules. However,
these amounts are too high, e.g. in the case of peptides and
proteins where precursors are expensive and generally difficult
to separate from the labelled products. Furthermore, high
precursor content can impair the quality of PET-images and
potentially cause adverse effects in patients. Consequently, the
dependency of the labelling yield on the precursor amount was
studied. BCN- and norbornene-substituted b-Ala-Phe-OMe
(19 and 20, respectively) were chosen as model dipolarophiles
(Scheme 3 and Table 2). If [18F]2 was generated by the
oxidation by PIFA or BAIB acceptable rcy of cycloaddition
products could be obtained only with micromolar amounts of
dipolarophiles. Therefore, radiofluorinated N-hydroxy-4-
fluorobenzimidoyl chloride ([18F]21), a more stable synthon
of [18F]2, was used in these experiments. It was prepared in situ
by chlorination of [18F]3 with chloramine T (Scheme 3). This
modification allowed us to minimize the amount of the BCN-
substituted dipolarophile 19 to 5 nmol (56% rcy of [18F]22)
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c
7136 Chem. Commun., 2012, 48, 7134–7136
This journal is The Royal Society of Chemistry 2012