Synthesis of 18F-labelled β-lactams by using the Kinugasa reaction

Article abstract of DOI:10.1002/chem.201304056

Owing to their broad spectrum of biological activities and low toxicity, β-lactams are attractive lead structures for the design of novel molecular probes. However, the synthesis of positron emission tomography (PET)-isotope-labelled β-lactams has not yet

Full text of DOI:10.1002/chem.201304056

DOI: 10.1002/chem.201304056
Full Paper
&
Isotopic Labeling
18
Synthesis of F-Labelled b-Lactams by Using the Kinugasa
Reaction
Boris D. Zlatopolskiy,[a, b, c] Philipp Krapf,[a, b] Raphael Richarz,[a, b] Holm Frauendorf,[d]
Felix M. Mottaghy,[c, e] and Bernd Neumaier*[a, b]
Abstract: Owing to their broad spectrum of biological activi-
ties and low toxicity, b-lactams are attractive lead structures
for the design of novel molecular probes. However, the syn-
thesis of positron emission tomography (PET)-isotope-la-
belled b-lactams has not yet been reported. Herein, we de-
scribe the simple preparation of radiofluorinated b-lactams
by using the fast Kinugasa reaction between 18F-labelled ni-
trone [18F]-1 and alkynes of different reactivity. Additionally,
18F-labelled fused b-lactams were obtained through the reac-
tion of a cyclic nitrone 7 with radiofluorinated alkynes [18F]-
6a,b. Radiochemical yields of the Kinugasa reaction products
could be significantly increased by the use of different CuI li-
gands, which additionally allowed a reduction in the amount
of precursor and/or reaction time. Model radiofluorinated b-
lactam-peptide and protein conjugates ([18F]-10 and 18F-la-
belled BSA conjugate) were efficiently obtained in high yield
under mild conditions (aq. MeCN, ambient temperature)
within a short reaction time, demonstrating the suitability of
the developed method for radiolabelling of sensitive mole-
cules such as biopolymers.
Introduction
physiological processes. A prerequisite for the latter is a de-
tailed understanding of the biology underlying normal or dis-
eased states at the molecular level. Molecular probes for PET-
imaging must be labelled with suitable b+-emitting nuclides.
Among the spectrum of easily available radionuclides 18F-fluo-
rine is still the nuclide with the highest impact in PET research.
This is mainly due to the excellent nuclear properties of 18F in
comparison to other cyclotron-produced nuclides. Decay char-
Amongst the available imaging technologies, positron emis-
sion tomography (PET) plays a very important role due to its
outstanding potential to visualize physiological processes at
the molecular level in real time. PET is therefore essential in
clinical diagnostics and has gained major significance in drug
development. Beside technical improvements, PET benefits
from innovations in the field of tracer development, compris-
ing both progress in labelling strategies and an intelligent
design of selective molecular probes with the capability to vis-
ualize molecular targets involved in physiological and patho-
acteristics of 18F [E(b+)=630 keV, abundance: 97%; t = =
1
2
109.8 min] make it an ideal PET-isotope with respect to half-life
and resolution. However, although much effort has been spent
on the development of novel methods for incorporation of 18F
into molecules of interest, radiofluorination methods are still
rather rare in comparison to fluorination methods used in con-
ventional organic chemistry.[1] This is due mainly to the tiny
amount of no-carrier-added (n.c.a.) 18FÀ (subnanomolar range)
as well as to time restrictions and radiation safety measures.
Hence, even promising modern fluorination techniques cannot
easily be transferred from synthetic organic chemistry to radio-
chemistry.[2] Despite this, there are several successful examples
of such translations, including the azide–alkyne “click” reac-
tions[3] and metal-catalyzed fluorination methods, which
enable easy access to otherwise inaccessible novel radiotrac-
ers.[4,5] Furthermore, a simple and efficient metal-free prepara-
tion of 18F-labelled compounds through (3+2) cycloaddition
reactions of radiofluorinated 1,3-dipoles other than azide to
double or triple CÀC bonds has recently been reported.[6]
b-Lactam antibiotics are amongst the most successful thera-
peutic agents developed to date. They exert their activity by
inhibition of bacterial cell wall biosynthesis. The mechanism of
action involves an irreversible inactivation of penicillin binding
proteins (PBPs), which are serine proteases with transglycosy-
[a] Dr. B. D. Zlatopolskiy,+ Dipl.-Chem. P. Krapf,+ Dipl.-Chem. R. Richarz,
Prof. Dr. B. Neumaier
Institute of Radiochemistry and Experimental Molecular Imaging
University Clinic Cologne, Kerpener Strasse 62, 50937 Cologne (Germany)
Fax: (+49)221-478-86851
E-mail: bernd.neumaier@uk-koeln.de
[b] Dr. B. D. Zlatopolskiy,+ Dipl.-Chem. P. Krapf,+ Dipl.-Chem. R. Richarz,
Prof. Dr. B. Neumaier
Max Planck Institute of Neurological Research
Gleueler Strasse 50, 50931 Cologne (Germany)
[c] Dr. B. D. Zlatopolskiy,+ Prof. Dr. F. M. Mottaghy
Clinic of Nuclear Medicine, RWTH Aachen University
Pauwelsstrasse 30, 52074 Aachen (Germany)
[d] Dr. H. Frauendorf
Institute of Organic and Biomolecular Chemistry
Georg-August University, Tammannstrasse 2, 37077 Gçttingen (Germany)
[e] Prof. Dr. F. M. Mottaghy
Department of Nuclear Medicine, Maastricht University Medical Center
P. Debyelaan 25, 6229HX Maastricht (The Netherlands)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201304056.
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lase, dd-transpeptidase and dd-carboxypeptidase activities,
that act through acylation of the nucleophilic serine residue at
the enzyme active site.[7] Beside b-lactam antibiotics, numerous
b-lactams with nonantibiotic activities have been discovered.[8]
They exert their effects by inhibition of further serine protei-
nases such as thrombin, elastases, prostate specific antigen
(PSA), human cytomegalovirus (HCV) protease as well as matrix
metalloproteinases, cysteine proteases, and tubulin polymeri-
zation. Consequently, radiolabelled b-lactams can potentially
be used for PET-imaging of bacterial and viral infections as
well as thrombosis, emphysema, and tumors. However, no PET-
tracers with a b-lactam structural motif have been published
to date.
nugasa reaction between the easily accessible 18F-labelled C-4-
fluorophenyl-N-phenyl nitrone ([18F]-1)[6b] and a range of termi-
nal alkynes. Furthermore, a model cyclic nitrone 7 was reacted
with radiolabelled fluorophenylacetylenes [18F]-6a,b under Ki-
nugasa conditions to give the corresponding labelled bicyclic
b-lactams [18F]-8a,b. Finally, a study was carried out on the ap-
plicability of the radio-Kinugasa reaction for labelling of pep-
tides and biomolecules, using H-bAla-Phe-OMe and bovine
serum albumin (BSA) as model peptide and protein, respective-
ly.
Results and Discussion
The Kinugasa reaction[9] (Scheme 1) is a valuable tool in
preparative organic chemistry, offering easy access to b-lac-
[18F]-1 was prepared by acid-catalyzed condensation of 4-[18F]-
fluorobenzaldehyde ([18F]-FBA) with N-phenylhydroxylamine, as
described earlier,[6b] in
a radiochemical yield of 89–92%
(Scheme 2). The 18F-labelled synthon was allowed to react with
Scheme 1. Proposed mechanisms of the Kinugasa reaction. Path a: via oxa-
ziridinium intermediate B;[9b, 10b] path b: via b-aminoketene intermediate
C.[10c]
tams through copper(I)-catalyzed reaction between nitrones
and terminal alkynes. Two plausible mechanisms of the Kinuga-
sa reaction have been proposed. According to both, the trans-
formation begins with the formation of a copper acetylide in
situ (in the initial report[9a] preformed copper acetylides were
used) (Scheme 1). Subsequent 1,3-cycloaddition of the latter to
the nitrone gives 5-isoxazolinyl cuprate A. According to Ding
and Irwing (path a),[9b,10a,b] the initially formed cuprate A rear-
ranges into the highly strained, extremely unstable intermedi-
ate bicyclic oxaziridinium salt B, which undergoes spontaneous
opening of the oxaziridinium ring to give (after protonation)
the corresponding b-lactam as a mixture of cis/trans isomers.
In an alternative mechanism, proposed by Ye et al.[10c] (path b),
diastereomeric 2-azetidinones are obtained from cuprate A
through ring-opening fragmentation followed by recyclization
and protonation. Although the Kinugasa reaction usually gives
b-lactams as mixtures of racemic diastereoisomers, numerous
stereoselective variants of this transformation using the appro-
priate chiral CuI ligands or chiral auxiliaries have been repor-
ted.[10d–f] Application of the Kinugasa reaction to radiochemistry
could allow simple access to radiolabelled 2-azetidinones.
Herein, we describe for the first time a method for the prepara-
tion of radiofluorinated monocyclic b-lactams through the Ki-
Scheme 2. Preparation of 18F-labelled b-lactam [18F]-2 under an inert atmos-
phere.
methyl propiolate in the presence of CuI and Et3N in MeCN at
ambient temperature. The reaction was carried out under Ar to
avoid the consumption of alkyne through Glaser coupling. The
corresponding radiolabelled b-lactam [18F]-2 was formed after
only 10 min reaction time in a radiochemical yield (RCY) of
65%, as a mixture of diastereomers. The thermodynamically fa-
vored trans-isomer was the predominant isomer obtained
(trans/cis=4:1). A prolonged reaction time of 20 min resulted
in a slightly improved radiochemical yield of 67%. The main
impurity (up to 25%) was identified as N-4-[18F]-fluorobenzyli-
dene aniline ([18F]-FBAN).
These promising initial results prompted us to optimize the
Kinugasa reaction with respect to precursor amount, reaction
time, solvent, CuI-stabilizing ligands and temperature.
In initial experiments, 10 mmol methyl propiolate precursor
was used. Normally, this is sufficiently low for labelling of small
molecules. However, this precursor amount is unacceptably
high, for example in the case of peptides and proteins for
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which precursors are generally difficult to separate from the la-
belled products. High precursor content can impair the quality
of PET-images and/or even cause undesirable effects in pa-
tients, impeding the clinical application of the tracer.
To optimize the Kinugasa reaction with respect to precursor
amount, nitrone [18F]-1 was allowed to react with different
amounts of methyl propiolate [0.05–10 mmol at ambient tem-
perature and 10 or 20 min reaction time (Figure 1)]. Reducing
Figure 3. Radiochemical yield of [18F]-2 in different solvents.
The Kinugasa reaction between [18F]-1 and methyl propiolate
was tested in different solvents (Figure 3). In dimethyl sulfoxide
(DMSO) and N,N-dimethylformamide (DMF) formation of [18F]-2
was significantly lower than that observed in MeCN (21 and
16% vs. 31%, respectively). When the radiosynthesis was car-
ried out in pyridine only traces of the product were formed
(ca. 2%), presumably due to almost instantaneous consump-
tion of methyl propiolate in a Michael reaction with pyridine.[11]
Miura et al.[10b] found that application of particular nitrogen
ligands accelerated the Kinugasa reaction due to efficient sta-
bilization of the reactive monomeric copper acetylide, prevent-
ing CuI oxidation and/or disproportionation to Cu0 and CuII.[12]
Accordingly, in the presence of pyridine, a moderate increase
of radiochemical yield (from 31 to 43%) was observed
(Figure 4). A more efficient ligand was 1,10-phenanthroline
Figure 1. Radiochemical yield of [18F]-2 as a function of precursor amount.
the amount of the alkyne precursor from 10 to 0.8 mmol
caused a gradual decrease of RCYs from 65 and 67% to 31
and 55% after 10 and 20 min, respectively. By further reducing
the precursor amount to 0.2 mmol, the RCYs of [18F]-2 de-
creased to 11 and 19% after 10 and 20 min, respectively. All
further optimization experiments were carried out with
0.8 mmol methyl propiolate, at a reaction time of 10 min at am-
bient temperature.
Formation of 18F-labelled b-lactam [18F]-2 was dependent on
temperature (Figure 2). Increasing the temperature from 25 to
608C improved the yield of the product from 31 to 68%
within 10 min. Simultaneously, competitive formation of the
side product [18F]-FBAN (up to 30%) was observed. Further ele-
vation of temperature did not increase RCY.
Figure 4. Influence of CuI-stabilizing ligands on the radiochemical yield of
[18F]-2.
(Scheme 3); in the presence of this ligand a maximum radio-
chemical yield of 75% was observed for [18F]-2.
Having established the optimized reaction conditions for the
radio-Kinugasa reaction with highly reactive alkynes, attention
was focused on less reactive alkynes. Propargyl alcohol was
chosen as a representative less reactive alkyne (Scheme 4). Pre-
liminary experiments had shown that higher reaction tempera-
Figure 2. Dependence of radiochemical yield of [18F]-2 on temperature.
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chemical yield of 65%. In contrast, only traces of [18F]-4 could
be detected when pyridine was used. Instead of the expected
product, two hydrophobic 18F-labelled byproducts were ob-
served in the reaction mixture. This unexpected result can
probably be attributed to elimination of the nucleobase from
the nucleotide analogue [18F]-4 to give the corresponding 3-
methylene-substituted b-lactam.[14] The latter could react fur-
ther with pyridine or 1-propargyluracil under formation of the
respective aza-Michael adducts.
Scheme 3. CuI-stabilizing ligands used in this work.
Having developed a method for the preparation of radio-
fluorinated monocyclic b-lactams through the radio-Kinugasa
reaction, the application of the
Kinugasa reaction in the synthe-
sis of 18F-labelled fused b-lac-
tams was studied. In this case,
easily accessible o- and p-[18F]-
fluorophenyl acetylenes ([18F]-6a
and [18F]-6b, respectively) were
used as radiolabelled building
blocks (Scheme 5). In the pres-
ence of 1,10-phenanthroline
these compounds reacted with
the model nitrone 3,6-dihydro-
2H-1,4-oxazine-4-oxide (7)[9b] to
give the corresponding radio-
fluorinated bicyclic b-lactams
[18F]-8a and [18F]-8b in radio-
chemical yields of 52 and 41%,
Scheme 4. Synthesis of labelled b-lactam alcohol [18F]-3 and b-lactam-nucleobase chimera [18F]-4.
respectively, within 10 min.
tures were mandatory to obtain [18F]-3 in reasonable radio-
chemical yields within a time-frame that was compatible with
the half-life of 18F. Reaction of [18F]-1 with 10 mmol propargyl al-
cohol in the presence of 1,10-phenanthroline at 1208C for
30 min in MeCN produced [18F]-3 in a radiochemical yield of
60%. Similar results were obtained by using tris[(1-benzyl-1H-
1,2,3-triazol-4-yl)methyl]amine (TBTA),[12] which is widely used
for ligand-promoted acceleration of the copper-catalyzed
Scheme 5. Preparation of the labelled bicyclic b-lactams [18F]-8a and [18F]-
8b.
azide-alkyne cycloaddition reactions. In contrast, a radiochemi-
cal yield of 82% of the desired product was obtained at 508C
in pyridine without ligand. In this case, the preferential forma-
tion of the kinetically favored cis-isomer (trans/cis ratio=1:5)
was observed. The main side product detected in the reaction
mixture was [18F]-FBA, originating from decomposition of [18F]-
1. In MeCN solvent, the radiochemical yields decreased rapidly
with decreasing alkyne amount. When 1 mmol precursor was
used, only 10% [18F]-3 was formed in the reaction mixture. In
contrast, using 1 mmol precursor in pyridine, radiofluorinated
b-lactam was obtained in a satisfactory yield of 50%.
Given the rapid kinetics of the Kinugasa reaction between
C,N-diaryl nitrones and activated alkynes at ambient tempera-
ture, we investigated whether this approach might also be
suitable for the radiofluorination of peptides and biopolymers.
The original Kinugasa reaction should be carried out under an
inert atmosphere in anhydrous organic solvents. This is a signif-
icant drawback with respect to labelling of the unprotected
peptides and proteins because they are often not readily solu-
ble in organic solvents such as acetonitrile. Basak et al.[15] ex-
ploited CuI generation in situ through reduction of CuSO4 with
sodium ascorbate as is usual for the azide-alkyne click reac-
tion.[3] They were able to prepare several b-lactams in moder-
ate yields in aqueous MeCN (or DMF) under an inert atmos-
phere by using Et3N as a base.
With optimized conditions for the Kinugasa reaction be-
tween radiofluorinated nitrone [18F]-1 and moderately activat-
ed alkynes to hand, we tried to prepare 18F-labelled b-lactam-
nucleobase chimera [18F]-4 (Scheme 4). Radiofluorinated com-
pounds of this type are of significant potential for the imaging
of bacterial infections. To this end, [18F]-1 was allowed to react
with 1-propargyl uracyl (5)[13] in MeCN to give the uracyl conju-
gate [18F]-4 as a mixture of cis/trans isomers (4:1) in a radio-
In preliminary experiments we tested the “cold” Kinugasa re-
action between 1 and methyl propiolate under “Basak” condi-
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We next studied the labelling of propiolyl-substituted bAla-
Phe-OMe 9 through conjugation with [18F]-1 (Scheme 6). Radio-
fluorinated depsipeptide [18F]-10 (cis/trans=3:1) was obtained
in a radiochemical yield of 58% within 10 min. A prolonged re-
action time of 20 min was necessary to obtain the maximal ra-
diochemical yields of 68–70%.
The dependency of radiochemical yields of [18F]-2 and [18F]-
10 on the amount of alkyne precursor was studied under click
conditions after 10 and 20 min reaction time, respectively
(Figure 5). The maximum radiochemical yield of [18F]-2 (80%)
was already achieved at 0.4 mmol, whereas for [18F]-10 (69%)
the use of 1 mmol precursor was necessary. Minimization of the
amount of alkyne to 100 nmol still produced the radiolabelled
b-lactams [18F]-2 and [18F]-10 in moderate radiochemical yields
of 31 and 40%, respectively.
Scheme 6. Conjugation of nitrone [18F]-1 with methyl propiolate and the
model dipeptide 9 under click conditions.
tions at ambient temperature and were able to detect only
traces of b-lactam 2 in the reaction mixture. In contrast to the
published results, we observed formation of the desired prod-
uct in suitable yields within 10 min without base addition.
Moreover, it should be pointed out that this modification of
the Kinugasa reaction works equally well under air. Nevertheless,
reasonable yields of [18F]-2 from [18F]-1 could only be achieved
with alkyne amounts of more than 50 mmol (Scheme 6). To
reduce the precursor amount, we tested the influence of the
CuI-stabilizing ligands TBTA,[12] bathophenanthroline disodium
disulfonate (BTS)[16] and l-histidine[17] (Scheme 3), which are
known from click chemistry, on the yields of the radio-Kinugasa
reaction. In the presence of each of the three ligands only
0.4 mmol methyl propiolate was necessary to obtain [18F]-2 in
80% radiochemical yields (Figure 5). Nontoxic and inexpensive
Once the protocol for the radio-Kinugasa reaction under
click conditions had been established, we turned to the devel-
opment of a simplified variant of this radiosynthesis. After re-
action of [18F]-FBA with N-phenylhydroxylamine at ambient
temperature for 10 min in the presence of traces of HCl and
subsequent addition of the reaction mixture to an aqueous so-
lution containing the alkyne precursor (1 mmol) and the other
.
reagents (CuSO4 5H2O, AscONa, l-histidine), [18F]-2 and [18F]-10
were obtained in excellent radiochemical yields of 89 and
85%, respectively (Scheme 6).
The hydrolytic and serum stability of [18F]-10 was briefly
studied. No decomposition of [18F]-10 was observed within
a pH range from 1 to 9 or in human blood serum at 378C for
at least 3 h.
The results detailed above raised the question of whether
the radio-Kinugasa reaction might be suitable for the radiolab-
elling of propiolated proteins. To this end, bovine serum albu-
min (BSA; Mr =66.5 kDa), chosen as a prototypical substrate,
was acylated with chloroformate 12 in 0.15m KHCO3 for 5 min
at ambient temperature (Scheme 7). Compound 12 was pre-
pared as follows: propiolic acid was coupled with 3-aminopro-
panol-1 by using 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquino-
line (EEDQ) to give 3-propionamidopropanol-1 (11) in moder-
ate yield. The latter was treated with triphosgene in the pres-
ence of 2,4,6-trimethylpyridine (TMP) in tetrahydrofuran (THF)
to give acylating agent 12, which was immediately used for
protein modification. The crude functionalized protein was pu-
rified by ultrafiltration. Propiolated BSA (19 nmol) was labelled
with [18F]-1 (400–500 MBq) under click conditions in aqueous
MeCN for 10 min at ambient temperature. Finally, the radio-
fluorinated BSA conjugate was isolated by simple filtration
through a PD10 desalting cartridge to give the desired product
in 32% radiochemical yield and in excellent radiochemical and
chemical purity (Figure 6). In a control experiment, native BSA
was treated with [18F]-1. In this case radiolabelling of protein
did not take place.
Figure 5. Dependence of radiochemical yields of [18F]-2 and [18F]-10 on the
amount of alkyne precursors (methyl propiolate and dipeptide 9, respective-
ly) under click conditions.
l-histidine was chosen for further experiments. In contrast to
the preferential formation of the thermodynamically more
stable trans-isomer under basic “classical” Kinugasa reaction
conditions (see above), the kinetically favored cis-isomer (cis/
trans=3:2) was preferentially formed under weak acidic click
conditions. In this case, [18F]-FBA (up to 20%), formed through
hydrolysis of [18F]-1, was identified as the main side product.
Conclusion
We have demonstrated that the Kinugasa reaction is an effi-
cient tool that can be used for the simple and high-yielding
preparation of 18F-labelled b-lactams. The rapid kinetics of the
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Acknowledgements
This work was supported in part
by EFRE-ZIEL2 program (North
Rhine-Westphalia, Germany). We
thank F. Wharton for her careful
proofreading of the manuscript.
Keywords: click chemistry
imaging agents isotopic
labelling b-lactams
radiopharmaceuticals
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Published online on March 11, 2014
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