Access to 18F-labelled isoxazoles by ruthenium-promoted 1,3-dipolar cycloaddition of 4-[18F]fluoro-N-hydroxybenzimidoyl chloride with alkynes

Article abstract of DOI:10.1002/jlcr.3708

4-[¹⁸F]Fluoro-N-hydroxybenzimidoyl chloride (¹⁸FBIC), an ¹⁸F-labelled aromatic nitrile oxide, was developed as building block for Ru-promoted 1,3-dipolar cycloaddition with alkynes. ¹⁸FBIC is obtained in a one-p

Full text of DOI:10.1002/jlcr.3708

Access to 18F-labelled isoxazoles by Ruthenium-promoted 1,3-dipolar
cycloaddition of 4-[18F]fluoro-N-hydroxybenzimidoyl chloride with alkynes
Silvia Roscales# and Torsten Kniess
Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research,
Bautzner Landstrasse 400, 01328 Dresden, Germany
#) Present address: Technological Institute PET, Manuel Bartolomé Cossio St 10, 28040
Madrid, Spain
Abstract
4-[18F]Fluoro-N-hydroxybenzimidoyl chloride (18FBIC) an 18F-labelled aromatic nitrile oxide was
developed as building block for Ru-promoted 1,3-dipolar cycloaddition with alkynes. 18FBIC is
obtained in a one-pot synthesis in up to 84% radiochemical yield starting from [18F]fluoride with 4-
[18F]fluorobenzaldehyde (18FBA) and 4-[18F]fluorobenzaldehyde oxime (18FBAO) as intermediates, by
reaction of 18FBAO with N-chlorosuccinimide (NCS). 18FBIC was found to be a suitable and stable
synthon to give access to 18F-labelled 3,4-diarylsubstituted isoxazoles by [Cp*RuCl(cod)]-catalyzed
1,3-dipolar cycloaddition with various alkynes. So, the radiosynthesis of a fluorine-18 labelled COX-2
inhibitor [18F]1b, a close derivative of valdecoxib, was performed with 18FBIC and 1-ethynyl-4-
(methylsulfonyl)benzene, providing [18F]1b in up to 40% RCY after purification in 85 min. The
application of 18FBIC as a building block in the synthesis of 18F-labelled heterocycles will generally
extend the portfolio of available PET radiotracers.
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1. Introduction
Positron emission tomography (PET) is a modern imaging technique used for various applications in
nuclear medicine, including diagnosis of disease, monitoring of treatment and post-operative
medical care of the patient. In pre-clinical research PET has an important impact on the
determination of the pharmacokinetics and pharmacodynamics of radiolabelled drugs and on the
development and evaluation of new potential radiotracers. Fluorine-18 is produced by cyclotrons
and is the most widely and routinely used positron-emitting radionuclide for PET, possessing a very
suitable positron energy (0.633 MeV, 97% β+) and a high spatial resolution (approx. 1mm) in PET
images.1 At 109 min, the half-life of fluorine-18 is adequate for performing multi-step radiolabelling
reactions and is sufficient to enable further transport of 18F-labelled radiopharmaceuticals to the
application site.
Over the last decades, a broad, ever-widening portfolio of methods has been developed for the
synthesis of 18F-labelled compounds (radiotracers) and 18F-labelled medicinal drugs
(radiopharmaceuticals). This covers both the direct substitution of a leaving group at the target
molecule by the radionuclide (direct approach) as well as the application of so-called building blocks
for radiolabelling (indirect approach). The latter makes use of small reactive 18F-labelled molecules
which are coupled with the (bio)molecule of interest, forming the final radiotracer. Both approaches
have advantages and limitations displayed by such parameters as reaction time and conditions,
yield, regioselectivity, requirement of protective groups and metabolic stability of the product, what
has been discussed recently in excellent reviews.2,3
Within the indirect radiolabelling approach, copper-catalysed Huisgen cycloaddition between an
azide and a terminal alkyne to form 1,4-disubstituted 1,2,3-triazoles (“click-chemistry”) has emerged
as a powerful method of building radiotracers for PET and Single Photon Emission Computed
Tomography (SPECT). 3,4,5
When 1,3-dipolar cycloaddition is performed between nitrile oxides and carbon-carbon double or
triple bonds, 2-isoxazolines and isoxazoles can be obtained, respectively. These reactions, although
well-known in organic chemistry6 have, with one exception, not been further exploited for
radiotracer synthesis. Zlatopolskiy et al. were the first to describe the generation of an 18F-labelled
nitrile oxide in situ and its application in 1,3-dipolar cycloaddition with alkenes and alkynes to form
18F-labelled 2-isoxazolines and 3,5-disubstituted or 3,4,5-trisubstituted isoxazoles.7
On the basis of our ongoing interest in the imaging of functional expression of cyclooxygenase-2
(COX-2) by PET, we aimed to develop novel 18F-radiolabelled COX-2 inhibitors as radioactive probes
for the characterization of inflammatory and tumorigenic lesions.8 Valdecoxib, a 3,4-diaryl-
substituted isoxazole (Scheme 1) with high potency and selectivity to COX-2 (IC50 = 5 nmol) was
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withdrawn from the market owing to cardiovascular side effects. Nevertheless, valdecoxib might
serve as a lead structure for PET radiotracer design because for a PET investigation only a few
nanomoles of the drug are applied to the patient. In the past, radiolabelled valdecoxib derivatives
have been developed; however, these radiotracers suffered from some limitations as in vivo
de[18F]fluorination or short half-life of the used radioisotope .9,10
Ruthenium-catalysed cycloaddition of nitrile oxides and alkynes was published by Grecian and Fokin
as a practicable reaction for building 3,4-diarylsubstituted isoxazoles.11 We recently found that new
fluorine-containing isoxazoles on the basis of a valdecoxib lead structure can be easily formed in one
step by ruthenium-promoted 1,3-dipolar cycloaddition of 4-fluoro-substituted nitrile oxides with
alkynes.12 4-Fluoro-N-hydroxybenzimidoyl chloride turned out to be an ideal synthon whereas the
ruthenium catalyst steers the reaction to only one isomer, the vicinal diaryl-substitution pattern
(Scheme 1). A number of the isoxazoles obtained in this manner exhibited low nanomolar affinity
and high selectivity towards COX-2; remarkably, through the introduction of a fluorine substituent
the high affinity was preserved.12 Altogether, these findings make this reaction an interesting
method for the development of 18F-labelled PET tracers targeting COX-2.
Scheme 1. 18/19F-labelled isoxazoles by Ru-promoted 1,3-dipolar cycloaddition of 18/19F-labelled nitrile
oxides with alkynes.
To explore this reaction in fluorine-18 chemistry we set up a study to evaluate Ru-promoted 1,3-
dipolar cycloaddition with an 18F-labelled aromatic nitrile oxide in analogy to non-radioactive
synthesis. The focus of interest is the establishment of a reliable and robust radiosynthesis of an 18F-
labelled nitrile oxide as a building block and the evaluation of the reaction conditions for 1,3-diplolar
cycloaddition with alkynes (solvent, temperature, catalyst) in order to build 18F-labelled isoxazoles.
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2. Experimental Section
2.1. General
All commercially available chemicals and solvents purchased were used without further purification.
The non-radioactive isoxazoles 1a-f served as reference compounds and the corresponding methyl-
and aminosulfonyl-substituted aryl alkynes were synthesized as described recently.12 4-formyl-
N,N,N,-trimethylanilinium triflate was synthesized according to a published procedure.13 The non-
radioactive 4-fluoro-N-hydroxybenzimidoyl chloride (FBIC) was prepared as previously reported.14
No-carrier-added aqueous [18F]fluoride was produced in a CYCLONE 18/9® cyclotron by irradiation of
[18O]H2O via the 18O(p,n)18F nuclear reaction. Radio-thin layer chromatography was performed on
silica gel F-254 aluminium plates, chromatograms were assessed using a Fuji BAS 2000® scanner
system. Radiochemical yields (RCY) are decay corrected. For SPE-based purification the following
cartridges were used; Sep-Pak light Accell Plus QMA® (130 mg, Waters, Part. Nr. WAT023525), Sep-
Pak Silica Plus Long cartridge (960 mg, Waters, Part. Nr. WAT 020520), Sep-Pak tC18 (400 mg,
Waters, Part. Nr. WAT036810); and Sep-Pak Dry Sodium Sulfate (2.85 g, Waters, Part. Nr.
WAT054265). Analytical HPLC was performed on a C18 column (Luna, Phenomenex, 5 µm 250 x 4.6
mm) using Agilent 1200 HPLC: pump G1311A, auto sampler G1329A, column oven G1316A, degasser
G1322A, UV detector G1315D, gamma-detector Gabi Star®; isocratic eluent: MeCN/H2O + 0.1% TFA
55/45 (v/v), 1 mL/min flow rate. The products were monitored at λ = 254 nm using a reference
wavelength λ ref = 360 nm unless otherwise specified. The shift in the retention time of the signal of
UV- and γ-detector originates by the distance between both detectors.
Semi-preparative HPLC and analytical HPLC were performed with a JASCO®-system consisting of a
manual injector, a PU2080 degasser gradient pump and a 2075 UV-detector with 254 nm detection,
controlled by an LC-Net II ADC interface and integrated gamma-detector GABI-Star under the
following conditions: analytical (column Kinetex C18 Phenomenex, 5 µm, 250 x 4.5 mm, isocratic,
eluent MeCN / H2O + 0.1%TFA 50/50 (v/v), 1 mL/min flow rate); semi-preparative (column Discovery
C18, Supelco, 5 µm, 250 x 10 mm, isocratic, MeCN / H2O + 0.1%TFA 50/50 (v/v), 5 mL/min and 4
mL/min flow rate).
2.2. Radiosynthesis and purification of 4-[18F]fluorobenzaldehyde oxime (18FBAO)
Cyclotron-produced aqueous [18F]fluoride solution was loaded on a QMA cartridge, which had been
previously activated by elution with 10 mL of H2O, 5mL of 1M NaHCO3 and 10 mL H2O. The cartridge
was rinsed with 1 mL of anhydrous methanol to remove water, then the [18F]fluoride was eluted in
opposite to the normal elution direction with 0.7 mL of dry methanol containing 8 mg 4-formyl-
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N,N,N,-trimethylanilinium triflate into a vial. The methanol was removed by evaporation in a
nitrogen stream under reduced pressure at 60°C. Afterwards, 0.5 mL anhydrous DMF was added and
the sealed vial was heated for 10 min at 90°C whilst being stirred. Then 14 µL 1 M NaOH was added
•
to a solution of 3 mg NH2OH HCl in 0.2 mL DMF and the released base was added to the crude 4-
[18F]fluorobenzaldehyde (18FBA). The sealed vial was stirred at 45°C for an additional 5 min to yield
crude 18FBAO as used for reactions described in 2.3.
For purification, the solution containing crude 18FBAO was diluted with 8 mL of hexane and eluted
over a Sep-Pak Silica Plus cartridge which had been preconditioned with 20 mL of hexane. The
cartridge was dried with air and the 18FBAO was recovered by elution with 1.5 mL dichloromethane.
A typical experiment starting with 520 MBq [18F]fluoride provided 134 MBq of purified 18FBAO after
40 min (34% RCY).
2.3. Radiosynthesis and purification of 4-[18F]fluoro-N-hydroxybenzimidoyl chloride (18FBIC)
To a vial containing the crude 18FBAO, synthesized as described at 2.2., was added a solution of 13
mg N-chlorosuccinimide in 200 µL of DMF. The vial was sealed and stirred at 45°C for 5 min. After
dilution with 8 mL of water the mixture was passed through a tC18 cartridge, which has been
preconditioned with 5 mL ethanol and 20 mL water. The cartridge was dried by flushing with 20 mL
of air yielding 18FBIC adsorbed on the tC18 cartridge suitable for further reactions as described in
2.4.
For analytical purposes and for the synthesis of [18F]1b, 18FBIC was recovered from the tC18
cartridge by elution with 1.5 mL CHCl3 and passed through a Sep-Pak sodium sulfate drying cartridge.
In a typical experiment, 440 MBq of purified 18FBIC was obtained from 773 MBq of [18F]fluoride
starting activity after 50 min (79% RCY).
2.4. Optimization of Ru-promoted 1,3-dipolar [3+2]cycloaddition of 18FBIC with 4-ethynylbenzene
sulfonamide
18FBIC, adsorbed on a tC18 cartridge as described in 2.3., was eluted with 1.5 mL of solvent as
indicated in Table 1. In the case of entry 16 Table 1 only, the solution was passed through a Sep-Pak
sodium sulfate drying cartridge. In all experiments, the solution was then added to a vial containing
22 µmol of 4-ethynylbenzene sulfonamide and a magnetic stirrer. 6.5 µmol (2.5 mg) of
[Cp*RuCl(cod)] dissolved in 0.3 ml of the corresponding solvent. The closed vial was stirred for 10
min at the temperature given in Table 1. The radiochemical yield of the 18F-labelled cycloaddition
products [18F]1a-f, the content of unreacted 18FBIC and of 18F-labelled by-products was determined
by analytical radio-HPLC.
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2.5. Radiosynthesis and purification of 3-(4-[18F]fluorophenyl)-4-(4-(methylsulfonyl)phenyl)-isoxazole
[18F]1b
18FBIC, adsorbed on a tC18 cartridge as described in 2.3., was eluted with 1.5 mL CHCl3 and passed
through a sodium sulfate drying cartridge into a vial containing 22 µmol (4 mg) 1-ethynyl-4-
(methylsulfonyl)benzene. Cycloaddition was initiated by addition of 6.5 µmol (2.5 mg) of
[Cp*RuCl(cod)] dissolved in 0.3 ml CHCl3 and stirred at 45°C for 10 min. Then the CHCl3 was
evaporated under reduced pressure and a stream of N2. The residue was dissolved in 0.6 mL of HPLC
eluent (MeCN/H2O + 0.1%TFA 50/50 (v/v)), passed through a syringe filter and injected into semi-
preparative HPLC (MeCN/H2O + 0.1%TFA 50/50 (v/v), isocratic flow, 4 mL/min). The product fraction
of [18F]1b, eluting from 9.4 and 10.2 min, was collected and analyzed by radio-HPLC. In a typical
experiment 140 MBq of [18F]1b were isolated from 595 MBq of [18F]fluoride starting activity within
85 min total synthesis time (40% RCY).
3. Results and discussion
3.1 Radiosynthesis of 4-[18F]fluorobenzaldehyde oxime (18FBAO) and 4-[18F]fluoro-N-
hydroxybenzimidoyl chloride (18FBIC)
A reliable and reproducible radiosynthesis of an 18F-labelled nitrile oxide building block is an
essential precondition for performing Ru-catalyzed 1,3-dipolar cycloaddition with alkynes.
4-[18F]Fluorobenzaldehyde oxime (18FBAO) seems to be an ideal candidate since its radiosynthesis
was described recently.7 We applied the reported synthesis sequence starting with the synthesis of
4-[18F]fluorobenzaldehyde (18FBA) as displayed in Scheme 2. In contrast to the majority of published
radiosyntheses of 18FBA, where azeotropic drying of [18F]fluoride is mandatory, we applied so-called
“minimalist approach” which was recently published by Neumaier and coworkers which obviates the
need of azeotropic drying.15 Following the “minimalist approach”, by elution of the [18F]fluoride from
the QMA cartridge with methanol containing 4-formyl-N,N,N,-trimethylanilinium triflate precursor,
evaporation of the solvent and subsequent 18F-fluorination for 10 min at 90°C in DMF, we were able
to perform 18FBA synthesis within 25 min with 72-84 % (n=20) yield as determined by radio thin layer
chromatography (radio-TLC). DMF was the solvent of our choice since it was found to be the most
suitable solvent for the subsequent steps.
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Scheme 2. Approaches for 18FBIC synthesis and Ru-promoted 1,3-dipolar cycloaddition
The synthesis of 18FBAO was performed at 45°C in DMF by reacting the crude 18FBA with
hydroxylamine hydrochloride and 1 M NaOH solution as described by Zlatopolskiy et al.7, providing
the crude 18FBAO in 59-69% yield (n=3) after two steps as determined by radio-TLC. Initially we used
a solvent mixture of MeOH/DMF for 18FBAO synthesis to enable the subsequent reaction with
phenyl iodine bis(trifluoroacetate) (PIFA) followed by cycloaddition with the alkyne as reported.7,16
The key step of this reaction sequence is the instant oxidation of 18FBAO with PIFA to 4-
[18F]fluorobenzonitrile oxide (18FBO), which is unstable and can only be obtained in situ. (Scheme 2,
Approach A)
Since our first attempts at PIFA-supported cycloaddition of alkyne with the crude 18FBAO failed, we
focused on an intermediate purification of 18FBAO via SPE-based methods. By testing several types
of SPE-cartridges based on reverse-phase (C18) as well as on normal phase (silica), we found that all
methods were accompanied with considerable losses of the 18F-labelled oxime. Finally, it was found
that 18FBAO could be purified successfully with a silica gel-based SPE cartridge, providing the
compound in 21-34% RCY (n=5). Purified 18FBAO was applied to a series of Ru-promoted
cycloaddition reactions in the presence of PIFA, alkyne and several solvents (methanol/water, DMF,
dichloromethane, THF and dichloroethane). In summary, and contrary to our expectations, we were
not able to detect any cycloaddition product by radio-HPLC in these PIFA-supported reactions.
(Scheme 2, Approach A)
18FBIC was described as a stable precursor for nitrile oxide formation, used for cycloaddition with
alkenes and alkynes7 and also for labelling of biomolecules.17,18 Hence, we focused on the
radiosynthesis of this building block which can be generated by reaction of 18FBAO with N-chloro
tosylamide sodium salt (Chloramine T), (Scheme 2, Approach B).7 However, after reaction of purified
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18FBAO with Chloramine T in aqueous methanol we were unable to detect 18FBIC by means of radio-
HPLC. To investigate if Chloramine T-produced 18FBIC was at least generated in situ and hence
principally available for further reactions, alkyne and Ru-catalyst were added to the crude reaction
mixture, but this likewise did not result in the formation of a cycloaddition product.
Since the non-radioactive FBIC can be prepared easily by reaction of 4-fluorobenzaldehyde oxime
with N-chlorosuccinimide (NCS)14, we adapted this route for the synthesis of 18FBIC. To the best of
our knowledge, this approach has not been practised in fluorine-18 chemistry yet and we were very
pleased to find by radio-HPLC that after reaction of crude 18FBAO with NCS in DMF for 5 min at 45°C
the 18FBIC was quantitatively formed. (Scheme 2, Approach D)) Because a subsequent cycloaddition
of crude 18FBIC with alkynes was unsuccessful (Scheme 2, Approach C), we performed a purification
of the synthon by SPE with a tC18 cartridge. Notably, the strategy of 18FBIC purification offered two
major advantages; in contrast to 18FBAO purification, we observed a significantly higher yield of
isolated purified product and the synthon obtained was of high purity, suitable for subsequent
cycloaddition in a solvent of choice.
In summary, 18FBIC is accessible from 4-formyl-N,N,N,-trimethylanilinium triflate via three steps
covering i) nucleophilic substitution with [18F]fluoride using the ‘minimalist’ approach to form 18FBA,
ii) reaction with NH2OH.HCl to form 18FBAO, iii) chlorination with NCS followed by SPE purification.
This three-step procedure performed in one pot provides 18FBIC in 55-84% isolated RCY (n>15)
within 50 min synthesis time starting from [18F]fluoride.
3.2. Ru-promoted 1,3-dipolar [3+2]cycloaddition of 18FBIC with alkynes
After having developed a reliable radiosynthesis of 18FBIC, this synthon was evaluated in a series of
experiments (Table 1) to find the most suitable solvent and best reaction temperature for the Ru-
promoted 1,3-diploar cycloadditon with alkynes. For that purpose, purified 18FBIC was eluted from
the SPE cartridge with the solvent as indicated (THF, DMF, EtOH, MeCN, DCE, DCM, CCl4, CHCl3) into
a reaction vial containing the alkyne (4-ethynylbenzene sulfonamide) and 10 mol% of
[Cp*RuCl(cod)]. Triethylamine (TEA) was added in specific cases and the mixture was stirred for the
stated time and temperature. The conversion of 18FBIC to cycloaddition product 4-(3-(4-
[18F]fluorophenyl)isoxazol-4-yl)benzenesulfonamide [18F]1a and the content of 18F-radiolabelled by-
products was determined by means of radio-HPLC; the results are displayed in Table 1.
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Table 1. Impact of solvent and reaction temperature on Ru-promoted 1,3-dipolar cycloaddition of
18FBIC with 4-ethynylbenzene sulfonamide
reaction
temperature time
reaction
ratio of compounds
#
solvent
additive
18FBIC
92 %
7 %
[18F]1a
[18F]by-products
1
2
3
4
5
6
7
8
9
THF
-
r.t.
10 min
7 %
-
THF
-
60°C
r.t.
10 min
10 min
10 min
10 min
10 min
10 min
10 min
10 min
10 min
10 min
10 min
10 min
5 min
19 %
-
73 %
5 %
THF
TEA
94 %
77 %
33 %
53 %
47 %
81 %
84 %
62 %
32 %
98 %
12 %
-
DMF
DMF
DMF
EtOH
MeCN
DCE
-
r.t.
8 %
14 %
9 %
-
80°C
r.t.
57 %
15 %
17 %
-
TEA
-
31 %
35 %
18 %
-
r.t.
r.t.
r.t.
15 %
21 %
57 %
-
10 DCE
11 DCM
12 CCl4
13 CHCl3
14 CHCl3
15 CHCl3
60°C
r.t.
16 %
10 %
2 %
-
r.t.
r.t.
69 %
-
18 %
99 %
13 %
-
TEA
r.t.
-
r.t.
30 min
10 min
13 %
-
73 %
96-99 %
#
16 CHCl3
(n=3)
#
-
r.t.
18FBIC, adsorbed on a tC18 cartridge as described in 2.3., was eluted with 1.5 mL CHCl3 and passed through sodium
sulfate drying cartridge into the vial for cycloaddition.
As demonstrated in Table 1, the solvent had a significant impact on the Ru-promoted 1,3-dipolar
cycloaddition of 18FBIC with an alkyne. When using THF at r.t., initially only 7% cycloaddition product
[18F]1a was formed, leaving 92% of 18FBIC unreacted (entry 1). Increased reaction temperature
(60°C) resulted, on the one hand, in higher conversion to [18F]1a (19%) and nearly complete
consumption of 18FBIC, but at the same time to increased formation of unidentified 18F-labelled by-
products (73%). At room temperature, DMF showed cycloaddition yields comparable to THF, but the
yield of [18F]1a increased to 57% when the temperature was raised to 80°C (entries 4 and 5).
As described in the literature, 4-fluorobenzonitrile oxide (FBO) is the active reagent for 1,3-dipolar
11,12,19
cycloaddition, and usually is generated in situ from the stable FBIC by addition of TEA.
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Consequently, we added TEA to the cycloaddition reactions in order to increase the radiochemical
yields (entries 3, 6). However, we noticed that addition of TEA did not lead to an improved yield of
[18F]1a but instead to the growing formation of 18F-labelled side products; one of them might
correspond to 18FBO. We therefore performed further cycloadditions without addition of TEA. This is
notable since in the majority of cases in non-radioactive chemistry addition of TEA is essential to
form the nitrile oxide moiety. It is supposed that the [Cp*RuCl(cod)] complex acts as a transition
metal Lewis acid, catalyzing the cycloaddition. The same effect was observed for an acetone-
cyclopentadienyl-BIPHOP based ruthenium complex which supported the [3+2] dipolar cycloaddition
reaction between nitrile oxides and α,β-unsaturated aldehydes.20
After having tested ethanol and acetonitrile as solvents, albeit with minor success, we proceeded
with the halogen-containing solvents 1,2-dichloroethane (DCE), dichloromethane (DCM), and carbon
tetrachloride. These halogen-containing solvents, especially DCE, are commonly used for Ru-
catalyzed 1,3-dipolar cycloaddition11 but show limited reactivity when containing traces of water.
Specifically, DCE gave only low radiochemical yields of [18F]1a when reacted at r.t. and at 60°C
(entries 9 and 10). In DCM the yield of cycloaddition product was significantly higher (57%) at room
temperature; this could not be further increased due to the solvent’s low boiling point (entry 11).
Carbon tetrachloride turned out to be inappropriate as a solvent, since it left the 18FBIC unreacted
(entry 12). Finally, we performed the cycloaddition in chloroform and achieved 53-69% yield of
[18F]1a at r.t. The yield was increased to 73% by extending the reaction time to 30 min; the content
of 18F-labelled by-products was low (13-18%) (entries 13 and 15). For comparison, an experiment
with addition of TEA in CHCl3 was performed, but in line with former findings we observed complete
decomposition of 18FBIC and detected no cycloaddition product (entry 14). For further optimization
we performed a drying step by passing the 18FBIC/CHCl3 solution through a Na2SO4 cartridge. This
increased the yield of cycloaddition to 96-99% without formation of side products (entry 16).
After having found the best reaction conditions for Ru-promoted 1,3-dipolar cycloaddition of 18FBIC
with 4-ethynylbenzene sulfonamide, we were able to evaluate a series of other alkynes in terms of
reactivity; the results are displayed in Table 2. For all cycloadditions, CHCl3 was used as a solvent and
the 18FBIC solution was passed through the Na2SO4 drying cartridge; the reaction time was 10 min,
the ratio of 18F-labelled cycloaddition product and of non-consumed 18FBIC was determined by
means of radio-HPLC.
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Table 2. Results on Ru-promoted 1,3-dipolar cycloaddition of 18FBIC with various alkynes
#
alkyne
R1
reaction
temperature
ratio of compounds
R2
18F-isoxazole
[18F]1a
97 %
18FBIC
18F-byproducts
1
NH2
CH3
CH3
NH2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
r.t.
−
−
2
H
r.t.
[18F]1b 75 %
[18F]1b 94 %
14 %
−
−
3
H
45°C
r.t.
5 %
−
4
CH3
Cl
−
−
−
98 %
78 %
64 %
74 %
39 %
−
5
r.t.
−
22 %
35 %
13 %
30 %
64 %
23 %
87 %
6
Cl
45°C
r.t.
−
−
7
COCH3
COCH3
CF3
[18F]1c
[18F]1c
12 %
8
45°C
45°C
30 %
[18F]1d 35 %
9
10
11.
COOCH3 45°C
COPh 45°C
[18F]1e
[18F]1f
50 %
10 %
26 %
2 %
Three analytical HPLC chromatograms are displayed as examples. Figure 1 shows the γ-HPLC trace of
18FBIC (tR = 6.10 min) recorded after cartridge purification. Figure 2 displays the γ-HPLC trace of the
reaction mixture of 1,3-dipolar cycloaddition with methyl 3-(4-(methylsulfonyl)phenyl)prop-2-ynoate
after 10 min at 45°C to form [18F]1e (see Table 2, entry 10). It is obvious that the majority of 18FBIC
has been consumed and the 18F-labelled isoxazole [18F]1e (tR = 8.24 min) was formed as a
cycloaddition product in 50% yield. Besides this, two 18F-radiolabelled byproducts were detected (tR
= 3.34 min tR = 9.69 min). The HPLC chromatogram of the non-radioactive reference compound 1e
(methyl 3-(4-fluorophenyl)-4-(4-(methylsulfonyl)phenyl)isoxazole-5-carboxylate), recorded at 254
nm (tR = 7.70 min) is shown in Figure 3.
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Figure 1. Radio-HPLC chromatogram of purified 18FBIC; semi-preparative column, 50% MeCN/50%
0.1% TFA (v/v), flow 5 mL/min
Figure 2. Radio HPLC chromatogram of reaction mixture of Ru-promoted 1,3-dipolar cycloaddition of
18FBIC with methyl 3-(4-(methylsulfonyl)phenyl)prop-2-ynoate, 10 min, 45°C; semi-preparative
column, 50% MeCN/50% 0.1% TFA (v/v), flow 5 mL/min
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Figure 3. HPLC chromatogram (UV-signal, 254 nm) of reference compound 1e; semi-preparative
column, 50% MeCN/50% 0.1% TFA (v/v), flow 5 mL/min
To summarise the results of Ru-promoted 1,3-cycloaddition of 18FBIC with various alkynes (Table 2),
we found that the unsubstituted alkynes are the most reactive partners, showing nearly quantitative
cycloaddition within 10 min at r.t. or 45°C, respectively (entries 1-3). With non-activated alkynes,
such as methyl- and chloro-substituted alkynes, no cycloaddition products were formed at r.t.;
application of higher reaction temperature resulted in decomposition of 18FBIC (entry 4-6). This is
consistent with our findings from non-radioactive chemistry where we were also unable to
synthesize the corresponding methyl- and chloro-substituted isoxazoles via this route.12 Alkynes
carrying a more bulky substituent underwent 1,3-dipolar cycloaddition in moderate yields (10-50%);
however, elevated temperatures (45°C) were required, resulting also in higher amounts of 18F-
labelled byproducts (entries 7-11).
3.3. Radiosynthesis of an 18F-labelled valdecoxib derivative [18F]1b by Ru-promoted 1,3-dipolar
cycloaddition with 18FBIC
The successful application of 18FBIC in PET radiotracer synthesis is demonstrated by the
cycloaddition-based formation of 3-(4-[18F]fluorophenyl)-4-(4-(methylsulfonyl)phenyl)isoxazole
[18F]1b. The non-radioactive reference compound 1b is structurally strongly related to valdecoxib
and shows a comparably high affinity and selectivity towards COX-2 (IC50 = 0.06 µM).12 In brief,
purified 18FBIC was reacted with 1-ethynyl-4-(methylsulfonyl)benzene and [Cp*RuCl(cod)] in CHCl3 at
45°C for 10 min, the solvent was evaporated and the mixture was redissolved in eluent. Purification
of the crude product by semi-preparative HPLC provided the isoxazole [18F]1b in up to 40% RCY,
calculated from the starting activity of [18F]fluoride. The radiochemical purity of [18F]1b was
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determined by analytical γ-HPLC and was found to be > 99 % as shown in Figure 4. The identity of
the radiotracer [18F]1b with the non-radioactive compound was demonstrated by the retention time
of 1b monitored by the UV-signal at 254 nm. (Figure 5).
Figure 4. Radio HPLC chromatogram of purified [18F]1b; analytical column, 50% MeCN/50% 0.1% TFA
(v/v), flow 1 mL/min
Figure 5. HPLC chromatogram (UV-signal, 254 nm) of reference compound 1b; analytical column,
50% MeCN/50% 0.1% TFA (v/v), flow 1 mL/min
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4. Conclusion
We found recently that Ru-catalyzed 1,3-dipolar cycloaddition of 4-fluoro-substituted nitrile oxides
with alkynes gives access to fluorine-substituted isoxazoles, i.e. to novel derivatives of valdecoxib.12
We hypothesized that by application of this method to 18F-labelled nitrile oxides and corresponding
alkynes 18F-labelled isoxazole derivatives would be available and might serve as potential PET probes
for visualization of COX-2. Consequently, we have focused on the development of a reliable high-
yielding synthesis of an 18F-labelled nitrile oxide as a suitable building block.
We have developed the one-pot radiosynthesis of a stable nitrile oxide precursor: 4-[18F]fluoro-N-
hydroxybenzimidoyl chloride (18FBIC) by optimizing the approach of Zlatopolskiy et al.7 We
performed the synthesis and purification of the compound in a three-step procedure covering the
synthesis of 4-[18F]fluorobenzaldehyde and subsequent reaction with hydroxylamine and NCS, and
this is characterised by high radiochemical yields and simple SPE-based purification. The 18FBIC
synthesized and purified in this manner was obtained in 55-84% RCY starting from [18F]fluoride
within 50 min in a non-automated experimental set-up.
The reactivity of 18FBIC was explored in Ru-promoted 1,3-dipolar cycloaddition with alkynes in
various solvents and at different temperatures, and the highest yield of cycloaddition products was
observed in chloroform. Monosubstituted alkynes proved to be the best partners in 1,3-dipolar
cycloaddition.
Finally, the potential of 18FBIC in PET radiotracer synthesis was successfully demonstrated in the
four-step radiosynthesis of the valdecoxib derivative [18F]1b. In a typical non-automated procedure,
the 18F-labelled isoxazole [18F]1b was isolated in up to 40% RCY starting from [18F]fluoride after semi-
preparative purification within 85 min synthesis time.
In summary, we were able to show that 18FBIC is a suitable and stable building block in fluorine-18
chemistry. 18F-labelled isoxazoles can be easily formed via Ru-promoted 1,3-dipolar cycloaddition of
this synthon with various alkynes. The radiosynthesis provides 18FBIC in high yields and might be
further improved by transformation of the process into an automated system, covering a three-step
reaction in one pot in a single solvent (DMF). The SPE-based purification of 18FBIC might likewise be
easily integrated into conventional automated synthesizers.
The extension of the chemistry of 18FBIC for the synthesis of other 18F-labelled heterocycles, e.g. by
reaction with alkenes, nitriles and other activated aromatic systems, is large, as can be reasoned
from non-radioactive chemistry. This potential has to be further evaluated and should extend the
portfolio regarding the design of 18F-radiotracers.
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Acknowledgements
Silvia Roscales is grateful for a post-doctoral fellowship from Ramón Areces Fondation (2015-2016).
Stephan Preusche and Tilow Krauß are acknowledged for production and contribution of
[18F]fluoride respectively. The authors wish to thank Markus Laube and Jörg Steinbach for fruitful
scientific discussions and assistance in the finalisation of the manuscript.
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Graphical Abstract
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