[18F]FPyKYNE, a fluoropyridine-based alkyne reagent designed for the fluorine-18 labelling of macromolecules using click chemistry

Article abstract of DOI:10.1002/jlcr.1533

FPyKYNE (2-fluoro-3-pent-4-yn-1-yloxypyridine) is a novel fluoropyridine-based structure, designed for the fluorine-18 labelling of macromolecules using copper-catalysed Huisgen 1,3-dipolar cycloaddition (click chemistry). FPyKYNE (non-labelled as reference), as well as the 2-bromo, 2-nitro and 2-trimethylammonium analogues (as precursors for labelling with fluorine-18), was synthesized in 44, 95, 60 and 41%, respectively, from commercially available 5-chloropent-1-yne and the appropriate 2-substituted-3-hydroxypyridines. [¹⁸F]FPyKYNE was synthesized in one single radiochemical step by reaction of no-carrier-added K[ ¹⁸F]F-Kryptofix 222 (DMSO, 165°C, 3-5 min) followed by C-18 SepPak cartridge pre-purification and finally semi-preparative HPLC purification on a Hewlett Packard SiO₂ Zorbax Rx-SIL. Using the 2-nitropyridine or the pyridin-2-yltrimethylammonium trifluoromethanesulphonate precursor for labelling (30 and 10 μmol, respectively), incorporation yields up to 90% were observed and 7.0-8.9 GBq (190-240 mCi) of [¹⁸F]FPyKYNE ([¹⁸F]-1) could be isolated within 60-70 min (HPLC purification included), starting from a 37.0 GBq (1.0 Ci) [¹⁸F]fluoride batch (overall decay-corrected and isolated yields: 30-35%). Copyright

Full text of DOI:10.1002/jlcr.1533

Research Article
Received 9 June 2008,
Accepted 30 June 2008
Published online in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1533
[18F]FPyKYNE, a fluoropyridine-based alkyne
reagent designed for the fluorine-18 labelling
of macromolecules using click chemistry
Bertrand Kuhnast,a Franc-oise Hinnen,a Bertrand Tavitian,a,b
a
Ã
´ ´
and Frederic Dolle
´
FPyKYNE (2-fluoro-3-pent-4-yn-1-yloxypyridine) is a novel fluoropyridine-based structure, designed for the fluorine-18
labelling of macromolecules using copper-catalysed Huisgen 1,3-dipolar cycloaddition (click chemistry). FPyKYNE (non-
labelled as reference), as well as the 2-bromo, 2-nitro and 2-trimethylammonium analogues (as precursors for labelling with
fluorine-18), was synthesized in 44, 95, 60 and 41%, respectively, from commercially available 5-chloropent-1-yne and the
appropriate 2-substituted-3-hydroxypyridines. [18F]FPyKYNE was synthesized in one single radiochemical step by reaction
of no-carrier-added K[18F]F-Kryptofixs222 (DMSO, 1651C, 3–5 min) followed by C-18 SepPak cartridge pre-purification and
finally semi-preparative HPLC purification on a Hewlett Packard SiO2 Zorbaxs Rx-SIL. Using the 2-nitropyridine or the
pyridin-2-yltrimethylammonium trifluoromethanesulphonate precursor for labelling (30 and 10 lmol, respectively),
incorporation yields up to 90% were observed and 7.0–8.9 GBq (190–240 mCi) of [18F]FPyKYNE ([18F]-1) could be isolated
within 60–70 min (HPLC purification included), starting from a 37.0 GBq (1.0 Ci) [18F]fluoride batch (overall decay-corrected
and isolated yields: 30–35%).
Keywords: fluorine-18; FPyKYNE; macromolecule; click chemistry
with a macromolecule can then be done using milder conditions
needed to preserve the latter’s integrity.7,8 At present, these
Introduction
High-molecular-weight bioactive chemical structures (com-
monly termed as macromolecules), such as oligonucleotides,
aptamers, peptide nucleic acids, peptides, proteins, antibodies,
diabodies and others, are increasingly proposed as
radiopharmaceuticals. Their applications are gaining importance
in nuclear medicine for therapy on one side, but also for
diagnostic purposes with in particular the development of
‘macroprobes’ labelled with positron-emitters for positron
emission tomography.
strategies exploit only a limited number of chemical conjugation
reactions. They often use functions that are naturally provided
by the macromolecule but sometimes also ‘man-added’ ones
and mainly include acylation of an amine function with an
activated carboxylic acid reagent such as the archetype N-
succinimidyl 4-[18F]fluorobenzoate
8–12
(Figure 1, compound A)
and alkylation of a sulphhydryl function with bromoacetamide-
13–22 (Figure 1, compounds B and C) or maleimide 23–26 (Figure
1, compounds D–G) reagents.
Fluorine-18 is often, among the other conventional short-lived
positron-emitters, the isotope of choice for the labelling of
macromolecules. It can be reliably and routinely cyclotron-
produced at the multi-Curie level as [18F]fluoride ion and
efficiently incorporated into various chemical structures using
nucleophilic fluorination reactions. Its half-life (109.8 min) allows
multi-step synthetic approaches1–6 and is long enough to give
access to relatively extended imaging protocols, often required
with macromolecules.
Even though these strategies have been used with success for
the fluorine-18 labelling of quite a large number of macro-
molecules, they also present some limitations such as (a) the
multi-step, time- and yield consuming preparation of the
fluorinated prosthetic reagent, (b) the poor chemoselectivity
occasionally observed in the conjugation with the macromole-
cule, (c) the sometimes required protection of other chemically
reactive functions of the macromolecule (especially with
peptides) and (d) the often discussed hazardous stability of
Methods for radiolabelling macromolecules with fluorine-18
have been of interest for several decades. Today, the generally
accepted approach involves a prosthetic group in the form of a
small fluorine-18-labelled reagent that is coupled chemoselec-
tively to an appropriate reactive function of the macromolecule.
This foreign-labelling strategy has the advantage of offering a
flexibility in the choice of chemical routes, including those
requiring drastic chemical conditions for the preparation of the
labelled prosthetic reagent, while the conjugation of the latter
a
´ ´
CEA, I2BM, Service Hospitalier Frederic Joliot, Institut d’Imagerie Biomedicale, 4
place du General Leclerc, F-91401 Orsay, France
´
´ ´
b
´ ´
INSERM, U803, Service Hospitalier Frederic Joliot, F-91401 Orsay, France
´ ´
´
*Correspondence to: Frederic Dolle, CEA, I2BM, Service Hospitalier Frederic Joliot,
Institut d’Imagerie Biomedicale, 4 place du General Leclerc, F-91401 Orsay,
France.
E-mail: frederic.dolle@cea.fr
´ ´
´
´ ´
J. Label Compd. Radiopharm 2008, 51 336–342
Copyright r 2008 John Wiley & Sons, Ltd.

B. Kuhnast et al.
O
O
H
N
O
O
O
O
Br
N
N
Br
N
H
N
H
O
O
O
O
18F
N
18F
18F
18F
A, [18F]SFB
B, [18F]FBnBrA
C, [18F]FPyBrA
D, [18F]FBEM
O
H
O
O
O
O
N
O
H
N
( )n
N
O
18F
N
18F
18F
18F
G, [18F]FPyME
N
O
H, [18F]FBA
E, [18F]FBABM
F, [18F]FBAHM
[
18F]FPyKYNE ([18F]-1)
(n=1)
(n=3)
Figure 1. Selected fluorine-18-labelled reagent for conjugation to macromolecules including FPyKYNE (1).
the linkage between the macromolecule and the prosthetic o-[18F]fluoroalkynes,35 demonstrating that the Cu(I)-catalysed
group. In order to circumvent part of the drawbacks listed 1,3-dipolar cycloaddition is compatible with the half-life of
above, [18F]fluorobenzaldehyde (Figure 1, compound H), a fluorine-18 and the constraints associated with its use. The XVIIth
reagent that can be prepared in only one chemical step, has International Symposium on Radiopharmaceutical Sciences, which
been used in a highly chemoselective oxime formation (a was hosted at Aachen, Germany, in the spring of 2007,
function claimed stable in vivo) with macromolecules provided comprised a full session entitled ‘click-labelling methods’,
with an aminooxy function.27,28 Another emerging promising demonstrating that such strategies can be applied to the
strategy is the use of ‘click chemistry’.
labelling with radioactive isotopes (fluorine-18 especially) of a
‘Click chemistry’ is a chemical concept introduced by Barry large variety of compounds including macromolecules36–41 but
Sharpless in 2001 and implies chemistry for the quick and also highlighting the need for developing new suitable
reliable generation of substance families by way of a tailor-made fluorinated ‘clickable’ reagents.
pair of reactive complementary functional groups providing the
As part of our continuous efforts in the development of
‘click’.29 It is often associated with combinatorial, high-through- [18F]fluoropyridine-based reagents,21,22,26 a novel alkyne re-
put screening and building chemical libraries in order to speed agent, [18F]FPyKYNE ([18F]-1, 2-[18F]fluoro-3-pent-4-yn-1-yloxy-
up new drug discoveries. Sharpless and co-workers defined pyridine, Figure 1), has been designed for the prosthetic
what makes a ‘click-chemistry’ reaction as one that is wide in labelling of macromolecules with the positron-emitter fluorine-
scope and easy to perform. It uses only readily available 18 using 1,3-dipolar cycloadditions, the work of which is
reagents and is insensitive to oxygen and water. Reaction work- reported herein.
up and purification use benign solvents (often water) and try to
avoid chromatography.
Of the reactions comprising the ‘click universe’, the perfect
example is the Cu(I)-catalysed variant of the Huisgen 1,3-dipolar
Results and discussion
Chemistry
cycloaddition of terminal alkynes to organoazides to form 1,4-
disubstituted-1,2,3-triazoles.30,31 The triazole has similarities to The synthesis of FPyKYNE (1, 2-fluoro-3-pent-4-yn-1-yloxypyr-
the ubiquitous amide moiety found in nature, but unlike amides idine) as a reference compound is illustrated in Scheme 1 as well
is not susceptible to cleavage and is nearly impossible to oxidize as the preparation of three precursors for labelling with fluorine-
or reduce. Besides using mild and often fast reaction conditions, 18, the 2-bromopyridine 8, the 2-nitropyridine 9 and the pyridin-
being very efficient and not requiring extended purification in 2-yltrimethylammonium trifluoromethanesulphonate 11.
many cases, Huisgen 1,3-dipolar cycloaddition also draws its
FPyKYNE (1) was prepared in two chemical steps from
success from the ease with which azides and alkynes can be commercially available 2-amino-3-hydroxypyridine (2) and
introduced into a molecule (macromolecule included). Both obtained in 44% overall yield. Balz–Schiemann reaction of 2 in
functions are chemically relatively stable towards a large variety 70% hydrofluoric acid in pyridine containing sodium nitrite (4
of organic reaction conditions and inert towards aqueous equiv.) gave the 2-fluoropyridine 3 in 57% yield.42 Alkylation of 3
environments and buffers. Thus, these cycloadditions have with commercially available 5-chloropent-1-yne (7, 1.5 equiv.)
been applied with success to the preparation of a large variety was performed in DMF for 15 h at 701C in the presence of
of new compounds, in particular conjugated macromolecules. potassium carbonate (2 equiv.) and sodium iodide (0.1
DNA, for example, has been tagged with fluorescent probes and equiv.),43–45 and afforded FPyKYNE (1) in 77% yield, after flash
proteins and live organisms with rhodamine, fluorescein, biotin chromatography purification.
or nanoparticle dyes.30,32–34 More recently, the scope of these
2-Bromo-3-pent-4-yn-1-yloxypyridine (8) and 2-nitro-3-pent-4-
cycloadditions has been extended to the chemistry field yn-1-yloxypyridine (9) were both synthesized in one chemical
involving radioactive isotopes, not in the least among which step from the corresponding commercially available 2-bromo-
the short-lived positron-emitter fluorine-18. Marik and co- and 2-nitro-3-hydroxypyridines (4 and 5) using the procedure
workers described in 2006 the first fluorine-18 labelling of described above for the preparation of FPyKYNE and were
peptides tagged with a 3-azidopropionic acid moiety using obtained in 95 and 60% yields after purification, respectively.
J. Label Compd. Radiopharm 2008, 51 336–342
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

B. Kuhnast et al.
The pyridin-2-yltrimethylammonium trifluoromethanesulpho- as the temperature were selected for these reactions based on
nate 11 was prepared in three steps from the 2-fluoropyridine literature,49,50 whereas different times of reaction were studied.
3 and obtained in 41% overall yield. Reaction of 3 with A DMSO solution (600 mL) containing the precursor for labelling
dimethylamine hydrochloride (1.3 equiv.) and potassium carbo- (8, 9 or 11) was transferred to 300–600 mCi of the dried K[18F]F-
nate (1 equiv.) in refluxing DMSO/H2O (3/1, v/v) at 1101C for Kryptofixs222 complex (see the Experimental) in a reaction vial
15 h46 gave the 2-dimethylaminopyridine 6 in 45% yield. (Vacutainers tube). The open tube was then placed in a heating
Alkylation of 6 with 5-chloropent-1-yne (7) using the procedure block at 1651C for 1–5 min. Aliquots were withdrawn at 1, 2, 3, 4
described above afforded 10 in 55% yield. Derivative 11 was and 5 min of reaction and analysed by radio-TLC.
then cleanly obtained in 97% yield by methylation of 10 using
For each time point, TLC analyses (SiO2, EtOAc, see the
methyl trifluoromethanesulphonate (1.3 equiv.) in toluene at Experimental) showed only one radioactive peak (Rf: 0.75), co-
room temperature (RT) for 1 h.21,26,46
migrating with FPyKYNE (1), besides unreacted [18F]fluoride (Rf:
0). The radiochemical yields (RCY) of fluorine-18 incorporation
were calculated from the TLC-radiochromatogram and defined
as the ratio of radioactivity area of [18F]-1 over total radioactivity
Radiochemistry
FPyKYNE (1) was labelled with fluorine-18 at its 2-fluoropyridinyl area. The results obtained (each value being the mean of three
moiety using the one-step radiochemical process outlined in independent experiments) using 30 mmol of precursor for
Scheme 2.
labelling are reported in Table 1.
Fluorinations were performed with cyclotron-produced
The nitropyridine 9 as well as the pyridinyltrimethylammo-
[18F]fluoride as the no-carrier-added, activated K[18F]F-Krypto- nium trifluoromethanesulphonate 11 were both highly reactive,
fixs222 complex.47,48 All three precursors for labelling synthe- with incorporation yields increasing with the reaction time and
sized above (derivatives 8, 9 and 11) were tested within a range reaching values of more than 90% from 3 to 5 min of heating.
of 10–30 mmol engaged per run. DMSO as the solvent and 1651C The bromopyridine 8 was almost not reactive with RCYs o5%
H
H
Cl
O
HO
7
K CO , NaI, DMF
70°C, 15 hrs
2
3
X
X
N
X
X
N
NH2
2
1 F (77%, from 3)
8 Br (95%, from 4)
9 NO2 (60%, from 5)
10 NMe2 (55%, from 6)
11 N+Me3.CF3SO3- (97%)
HF, pyridine
NaNO , rt, 2 h
2
3 F (57%)
4 Br
HNMe .HCl, K CO
3
2
2
DMSO/H O
2
NO2
5
6
CF SO Me, Toluene
3
3
110°C, 15 h
0°C, then rt, 1 h
NMe2 (45%)
Scheme 1.
18
K[ F]F-K
222
H
H
K CO , DMSO
165°C, 1-5 min
2
3
O
O
X
followed by
18F
N
X
N
cartridge purification
TM
8 Br
(Prep-Sep C-18)
9 NO2
11 N+Me3.CF3SO3
and
[
18F]FPyKYNE ([18F]-1)
-
HPLC purification
®
(Zorbax RX-Sil)
Scheme 2.
Table 1. Yields of fluorine-18 incorporation at 1651C using 30 mmol of precursor for labelling: influence of the reaction time
and the leaving group
Precursor used (30 mmol)
Reaction time (in min)
Structure
X
Code
1 (%)
2 (%)
3 (%)
4 (%)
5 (%)
H
Br
NO2
NþMe3 ꢀ CF3SOꢁ3
8
9
11
5
22
38
4
49
80
5
490
490
6
490
490
16
490
490
O
X
N
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Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 336–342

B. Kuhnast et al.
from 1 to 3 min of reaction (RCY of 16% at 5 min). The ranking 11 (or the nitropyridine 9) as a precursor for labelling,
order of reactivity observed, NMe3 ꢂ NO2 ꢃ Br, is fully in 7.0–8.9 GBq (190–240 mCi) of [18F]FPyKYNE ([18F]-1) could be
accordance with results previously obtained for a simple model isolated within 60–70 min (HPLC purification included), starting
structure – the radiosynthesis of 2-[18F]fluoropyridine49,50 – as from a 37.0 GBq (1.0 Ci) [18F]fluoride batch.
well as for the preparation of a number of [2-18F-fluoropyridi-
Quality control of [18F]FPyKYNE ([18F]-1) was performed on an
nyl]epibatidine analogues.51–53 Worth mentioning is that the acetonitrile-diluted aliquot of the fraction collected after semi-
RCYs observed in this series are higher and within shorter time preparative HPLC purification. As demonstrated by analytical
of reaction than those reported in the examples cited above. A HPLC analysis (HPLC B, see the Experimental), [18F]FPyKYNE
similar observation was already made54,55 for the radiofluorina- ([18F]-1, tR: 2.60 min) was found to be 495% chemically and
tion of other 3-alkoxy-substituted pyridines such as, for radiochemically pure and was obtained with a specific radio-
example, the known azetidine 2-[18F]F-A-85380
42,46
and the activity of 150–300 GBq/mmol (4–8 Ci/mmol).
two prosthetic reagents [18F]FPyBrA21,22 and [18F]FPyME26
(Figure 1).
Experimental
At the end of the heating period mentioned above, the
reaction vessel was then cooled using an ice–water bath and the
remaining radioactivity was measured: 96% to over 99% of the
initial radioactivity was still present. At this stage, a C-18
PrepSepTM cartridge was used to rapidly trap and isolate [18F]-1
(with a radiochemical purity 495%, according to radio-TLC)
from the reaction mixture (with unreacted [18F]fluoride most
likely eluted off the cartridge as waste). The fraction trapped on
the cartridge represented 15, 75 and 75% of the total
radioactivity amount engaged in the fluorination process for
derivatives 8, 9 and 11, respectively, confirming the RCYs
measured by radio-TLC. Noteworthy, 2–5% of the total radio-
activity amount engaged in the fluorination process was left
behind in the initial fluorination reactor tube.
Decreasing the amount of starting labelling precursor
(20 mmol and down to 10 mmol) gave lower incorporation yields
(calculated from the TLC-radiochromatogram or after the C-18
PrepSepTM cartridge purification) when considering the nitro-
pyridine 9 and the bromopyridine 8, whereas similar yields were
observed with the pyridin-2-yltrimethylammonium trifluoro-
methanesulphonate 11.
[18F]FPyKYNE ([18F]-1) was then eluted from the C-18
PrepSepTM cartridge with CH2Cl2 (about 5% remaining trapped
and being lost on the cartridge) and purified by HPLC on a semi-
preparative SiO2 Zorbax Rx-SIL column (HPLC A, see the
Experimental). A series of conditions (combination of solvent
and flow rate) were tested and gave the following results. When
using a 90/10 mixture of heptane and ethyl acetate (condition 1,
see the Experimental), [18F]-1 (tR: 9.5–10.0 min) could be
obtained with a 495% chemical and radiochemical purity and
was well separated from the remaining precursors for labelling
(tR: 8: 12–12.5 min; 9 or 11: 420 min). Substituting heptane by
pentane (condition 2) gave similar results (tR: 1: 9.5–10 min; 8:
12.5–13 min; 9 or 11: 420 min). However, when using a 95/5
mixture of dichloromethane and diethylether (condition 3), [18F]-
1 (tR: 9.5–10.0 min) could only be obtained with a high (495%)
chemical and radiochemical purity if the precursor for labelling
used is the pyridin-2-yltrimethylammonium trifluoromethane-
sulphonate 11 (tR420 min), a co-elution of [18F]-1 with the
bromopyridine 8 or the nitropyridine 9 being observed in these
conditions. The average isolated yield of [18F]-1 after HPLC
purification and based on injected radioactivity onto the system
ranged from 60 to 80%. Noteworthy, the HPLC-radiochromato-
grams showed one minor radioactive by-product, well separated
from [18F]-1 (tR: 7.5–8.0 min, representing less than 10% of the
injected radioactivity) and this independently of the HPLC
conditions or precursor for labelling used.
General
Chemicals and flash chromatographies
Chemicals were purchased from Aldrich, Fluka or Sigma France
and were used without further purification, unless otherwise
stated. Flash chromatographies were conducted on silica gel
(0.63–0.200 mm, VWR) columns.
TLC and HPLC analyses
TLCs were run on pre-coated plates of silica gel 60F254 (VWR).
Compounds were localized (1) when possible at 254 nm using a
UV-lamp and/or (2) by dipping the TLC-plates in a 1% ethanolic
ninhydrin solution and heating on a hot plate. Radioactive spots
were detected using a Berthold TraceMaster 20 automatic TLC
linear analyser. HPLC analyses were run as follows: (HPLC A):
Equipment: system equipped with a Waters 501 pump, a
Shimadzu SPD10-AVP UV-multi-wavelength detector and a
miniature ionisation chamber probe; column: semi-preparative
SiO2 Zorbax Rx-SIL, Hewlett Packard (250 ꢄ 9.4 mm; porosity:
5 mm); condition 1: eluent: heptane/EtOAc 90/10 (v/v), flow rate:
5 mL/min – condition 2: eluent: pentane/EtOAc 90/10, flow
rate: 5 mL/min – condition 3: eluent: CH2Cl2/Et2O 95/5 (v/v), flow
rate: 4 mL/min; temperature: RT; absorbance detection
at l = 270 nm. (HPLC B): Equipment: Waters Alliance 2690
equipped with a UV spectrophotometer (Photodiode Array
Detector, Waters 996) and a Berthold LB509 radioactivity
detector; column: analytical Symmetry-Ms C-18, Waters
(50 ꢄ 4.6 mm); porosity: 5.0 mm; conditions: isocratic elution
with solvA/solvB: 50/50 (v/v) (solvent A: H2O containing Low-
UV PICs B7 reagent (20 mL for 1000 mL); solvent B: H2O/CH3CN:
30:70 (v/v) containing Low-UV PICs B7 reagent (20 mL for
1000 mL)); flow rate: 2.0 mL/min; temperature: RT; absorbance
detection at l = 270 nm.
Spectroscopies
NMR spectra were recorded on a Bruker Avance 400 MHz
apparatus (Wissembourg, France) using the hydro-
genated residue of the deuterated solvents (CD2Cl2
(d = 5.32 ppm) or DMSO-d6 (d = 2.50 ppm)) or TMS (d = 0.0 ppm)
as internal standards for 1H-NMR as well as the deuterated
solvents (CD2Cl2 (d = 54.0 ppm) or DMSO-d6 (d = 39.5 ppm))
as an internal standard for 13C-NMR. The chemical shifts
are reported in ppm, downfield from TMS (s, d, t, q, m, b for
singlet, doublet, triplet, quadruplet, multiplet, and broad
respectively). The mass spectra (MS) were measured on a
Thermo Electron Ion Trap LCQ Deca XP1 spectrometer (ESI1)
(Les Ulis, France).
Typically, using the one-step synthesis described herein and
the pyridin-2-yltrimethylammonium trifluoromethanesulphonate
J. Label Compd. Radiopharm 2008, 51 336–342
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

B. Kuhnast et al.
Radioisotope production
General procedure for the preparation of the 2-substituted-3-pent-
4-yn-1-yloxypyridines
No-carrier-added fluorine-18 (half-life: 109.8 min) was
produced via the [18O(p,n)18F] nuclear reaction by irradiation
of a 2-mL [18O]water (497% enriched, Rotem (CortecNet,
Paris, France)) target on an IBA Cyclone-18/9 (IBA, Louvain-
la-Neuve, Belgium) cyclotron (18 MeV proton beam) and
the aqueous radioactive solution was then transferred to the
appropriate hot cell. Target hardware: commercial, 2-mL, two-
port, stainless steel target holder equipped with a domed-end
niobium cylinder insert. Target to hot cell liquid-transfer system:
The appropriate 2-substituted-3-hydroxypyridine (3, 4, 5, 6, 1.0g
scale, 5–10 mmol), finely divided K2CO3 (2 equiv.), NaI (0.1 equiv.)
and 5-chloropent-1-yne (7, 1.5 equiv.) were successively dissolved
in DMF (15 mL). The mixture was then stirred overnight at 701C,
cooled to RT and diluted with water (100 mL). The resulting
solution was extracted twice with EtOAc (250 mL). The organic
layers were combined, washed with brine, dried over Na2SO4 and
concentrated to dryness. The residue was purified by flash
chromatography on silica gel to afford the expected 2-substi-
tuted-3-pent-4-yn-1-yloxypyridine (1, 8, 9 and 10, respectively).
1
60 m PTFE line (0.8 mm internal diameter; 16 in external
diameter), 2.0 bar helium drive pressure, transfer time 3–6 min.
Typical production of [18F]fluoride at the end of bombardment
2-Fluoro-3-pent-4-yn-1-yloxypyridine (1)
for a 20 mA, 30 min (10 mA h) irradiation: 27.7–29.6 GBq
(750–800 mCi).
The procedure described above was performed with compound 3
(8.8 mmol). Purification by flash chromatography on silica gel
(heptane/EtOAc: 70/30 (v/v)) gave 1 (1.2 g, 77%) as a white powder.
Rf (heptane/EtOAc: 60/40): 0.6. Rf (EtOAc): 0.75. tR (HPLC A):
9.5–10.0 min. tR (HPLC B): 2.60 min. 1H-NMR (CD2Cl2, 298K):
d: 7.72 (dt, J= 4.8 and 1.6 Hz, 1H); 7.35 (ddd, J=9.6 and 8.0Hz,
1H); 7.15 (dd, J= 8.0 and 4.8 Hz, 1H); 4.17 (t, J= 6.4 Hz, 2H); 2.44 (td,
J= 7.2 and 2.8 Hz, 2H); 2.05 (q5, J=6.4Hz, 2H); 2.05 (t, J=2.8Hz, 1H).
13C-NMR (CD2Cl2, 298K): d: 154.2 (C, JC1ꢁF ¼ 235 Hz); 142.7 (C,
JC2ꢁF ¼ 26 Hz); 137.5 (CH, JC3ꢁF ¼ 13 Hz); 123.1 (CH, JC3ꢁF ¼ 5 Hz);
122.3 (CH, JC4ꢁF ¼ 4 Hz); 83.5 (C); 69.7 (CH); 68.0 (CH2); 28.4 (CH2);
15.4 (CH2). MS: C10H10FNO: 180 [M1H]1.
Miscellaneous
Radiosyntheses using fluorine-18, including the HPLC purifica-
tions, were performed in a shielded cell (7.5 cm lead) using a
computer-assisted Zymate-XP robot system (Zymark corpora-
tion, USA).
Chemistry
2-Fluoro-3-hydroxypyridine (3)
To a stirred and cooled (01C) solution of hydrofluoric acid
pyridine complex (70% HF in pyridine, 100 mL) were successively
added commercially available 2-amino-3-hydroxypyridine (2,
3.5 g, 31.8 mmol) and NaNO2 (7.5 g, 120 mmol, 4 equiv.). The
reaction mixture was stirred for 2 h at RT and then neutralized
using a 3 N aqueous NaOH solution (100 mL). The resulting
solution was extracted twice with EtOAc (250 mL). The organic
layers were combined, washed with brine and dried over
Na2SO4. Concentration to dryness gave 3 (2.0 g, 57%) as a brown
solid that was further used without additional purification. Rf
(EtOAc/heptane: 80/20): 0.65. 1H-NMR (DMSO-d6, 298 K): d: 10.35
(s, 1H); 7.58 (td, J = 4.8 and 1.6 Hz, 1H); 7.36 (ddd, J = 10.4, 7.6 and
1.6 Hz, 1H); 7.10 (ddd, J = 7.6, 4.8 and 1.2 Hz, 1H). 13C-NMR
(DMSO-d6, 298 K): d: 152.8 (C, JC1ꢁF ¼ 232 Hz); 140.2 (C, JC2ꢁF
¼ 27 Hz); 135.6 (CH, JC3ꢁF ¼ 13 Hz); 126.2 (CH, JC3ꢁF ¼ 5 Hz); 122.6
(CH, JC4ꢁF ¼ 4 Hz). MS: C5H4FNO: 114 [M1H]1.
2-Bromo-3-pent-4-yn-1-yloxypyridine (8)
The procedure described above was performed with commer-
cially available 2-bromo-3-hydroxypyridine (4, 5.7 mmol). Pur-
ification by flash chromatography on silica gel (heptane/EtOAc:
70/30 (v/v)) gave 8 (1.3 g, 95%) as a white powder. Rf (heptane/
1
EtOAc: 60/40): 0.65. H-NMR (CD2Cl2, 298 K): d: 7.93 (dd, J = 4.4
and 1.6 Hz, 1H); 7.21 (dd, J = 8.0 and 4.4 Hz, 1H); 7.17 (dd, J = 8.0
and 1.6 Hz, 1H); 4.14 (t, J = 6.0 Hz, 2H); 2.46 (td, J = 7.2 and 2.8 Hz,
2H); 2.04 (q5, J = 6.8 Hz, 2H); 2.01 (t, J = 2.4 Hz, 1H). 13C-NMR
(CD2Cl2, 298 K): d: 152.7 (C); 141.7 (CH); 133.4 (C); 124.0 (CH);
120.2 (CH); 83.6 (C); 69.5 (CH); 68.0 (CH2); 28.4 (CH2); 15.5 (CH2).
MS: C10H10BrNO: 242 [M1H]1, 240 [M1H]1.
2-Nitro-3-pent-4-yn-1-yloxypyridine (9)
The procedure described above was performed with commer-
cially available 2-nitro-3-hydroxypyridine (5, 7.1 mmol). Purifica-
tion by flash chromatography on silica gel (heptane/EtOAc: 70/
30 (v/v)) gave 9 (0.9 g, 60%) as a yellow powder. Rf (heptane/
EtOAc: 50/50): 0.6. 1H-NMR (CD2Cl2, 298 K): d: 8.05 (dd, J = 4.0 and
2.4 Hz, 1H); 7.56–7.54 (m, 2H); 4.24 (t, J = 6.0 Hz, 2H); 2.40 (td,
J = 7.2 and 2.4 Hz, 2H); 2.02 (q5, J = 6.4 Hz, 2H); 2.02 (t, J = 2.8 Hz,
1H). 13C-NMR (CD2Cl2, 298 K): d: 149.5 (C); 147.4 (C); 139.6 (CH);
129.2 (CH); 124.1 (CH); 83.2 (C); 69.6 (CH); 68.5 (CH2); 28.2 (CH2);
15.3 (CH2). MS: C10H10N2O3: 207 [M1H]1.
2-Dimethylamino-3-hydroxypyridine (6)
Compound 3 (1.0 g, 8.84 mmol), finely divided K2CO3 (1.22 g,
8.85 mmol, 1 equiv.) and Me2NH ꢀ HCl (0.94 g, 11.5 mmol, 1.3
equiv) were successively dissolved in a mixture of DMSO (75 mL)
and water (15 mL). The mixture was then stirred overnight
at 1101C, cooled to RT and diluted with water (100 mL). The
resulting solution was extracted twice with EtOAc (250 mL).
The organic layers were combined, washed with brine, dried
over Na2SO4 and concentrated to dryness. The residue (a green
oil) was purified by flash chromatography on silica gel (heptane/
EtOAc: 80/20 (v/v)) to afford 6 (0.6 g, 45%) as a yellow solid.
Rf (heptane/EtOAc: 80/20): 0.3. 1H-NMR (DMSO-d6, 298 K):
d: 9.53 (bs, w1/2 = 18 Hz, 1H); 7.64 (dd, J = 4.8 and 1.6 Hz, 1H);
6.97 (dd, J = 7.6 and 1.6 Hz, 1H); 6.66 (dd, J = 7.6 and 4.8 Hz, 1H);
2.86 (s, 6H). 13C-NMR (DMSO-d6, 298 K): d: 151.7 (C); 143.7 (C);
137.1 (CH); 121.2 (CH); 115.8 (CH); 40.2 (2 ꢄ CH3). MS: C7H10N2O:
139 [M1H]1.
2-Dimethylamino-3-pent-4-yn-1-yloxypyridine (10)
The procedure described above was performed with compound
6 (7.2 mmol). Purification by flash chromatography on silica gel
(heptane/EtOAc: 85/15 (v/v)) gave 10 (0.8 g, 55%) as a yellow oil.
1
Rf (heptane/EtOAc: 60/40): 0.4. H-NMR (CD2Cl2, 298 K): d: 7.77
(dd, J = 4.8 and 1.4 Hz, 1H); 7.01 (dd, J = 7.8 and 1.4 Hz, 1H); 6.70
(dd, J = 7.8 and 4.8 Hz, 1H); 4.05 (t, J = 6.0 Hz, 2H); 2.95 (s, 6H);
www.jlcr.org
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 336–342

B. Kuhnast et al.
2.43 (td, J = 7.0 and 2.6 Hz, 2H); 2.03 (q5, J = 7.0 Hz, 2H); 2.02 activated beforehand with EtOH (2 mL) and then rinsed with
(t, J = 2.0 Hz, 1H). 13C-NMR (CD2Cl2, 298 K): d: 153.7 (C); 146.1 (C); water (10 mL)), pre-filled with water (2 mL). The tube was rinsed
139.2 (CH); 119.0 (CH); 115.8 (CH); 83.8 (C); 69.4 (CH); twice with water (1 mL), which was also transferred and added
67.3 (CH2); 41.2 (2 ꢄ CH3); 28.8 (CH2); 15.9 (CH2). MS: to the diluted reaction mixture on top of the cartridge. An
C12H16N2O: 205 [M1H]1.
additional portion of water (2 mL) was further added to the
diluted reaction mixture on top of the cartridge. The whole was
then passed through the cartridge, which was then washed with
water (3 mL) and partially dried for 0.5 min by applying a
nitrogen stream. [18F]-1 was eluted from the cartridge with
CH2Cl2 (3 mL) into an empty 5-mL reaction vial. Elution was
repeated twice with 1 mL of CH2Cl2 for maximal transfer of [18F]-
1. The final incorporation yield was also estimated after the C-18
(3-Pent-4-yn-1-yloxypyridine-2-yl)trimethylammonium
trifluoromethanesulphonate (11)
To a stirred and cooled (01C) solution of compound 10 (0.35 g,
1.71 mmol) in toluene (8 mL) was added methyl trifluorometha-
nesulphonate (230 mL, d: 1.45, 334 mg, 2.04 mmol, 1.3 equiv.).
The mixture was stirred at RT for 1 h and then concentrated to
dryness. Derivative 11 was obtained as a brown solid (0.6 g,
ꢁ
cartridge elution by the ratio CH2Cl2 over total radioactivity
amount engaged in the fluorination process.
1
97%). H-NMR (DMSO-d6, 298 K): d: 8.17 (dd, J = 4.4 and 1.6 Hz,
1H); 7.97 (dd, J = 7.6 and o1.5 Hz, 1H); 7.73 (dd, J = 8.0 and
4.4 Hz, 1H); 4.35 (t, J = 6.0 Hz, 2H); 3.61 (s, 9H); 2.89 (t, J = 2.4 Hz,
1H); 2.40 (td, J = 7.2 and 2.8 Hz, 2H); 2.06 (q5, J = 6.8 Hz, 2H). 13C-
NMR (DMSO-d6, 298 K): d: 147.0 (C); 142.5 (C); 138.4 (CH); 128.4
(CH); 125.0 (CH); 121.1 (q4, JC1ꢁF ¼ 320 Hz, CF3); 83.3 (C); 72.1
(CH); 68.6 (CH2); 53.4 (3 ꢄ CH3); 27.0 (CH2); 14.9 (CH2). MS:
C14H19F3N2SO4: 369 [M1H]1.
Purifications
The eluted CH2Cl2 solution containing [18F]-1 was partly
concentrated at 65–751C under a gentle nitrogen stream for
1–2 min. The residue was then diluted with the HPLC solvent
used for purification (0.5 mL) and the crude was injected onto
HPLC (HPLC A, condition 1, 2 or 3).
Radiochemistry
Optimized conditions
DMSO (600 mL) containing 9 (6.1 mg, 30 mmol) or 11 (3.7 mg,
10 mmol) was added into the Vacutainers tube containing the
dried K[18F]F-K222 complex. The tube (open) was thoroughly
vortexed (30 s) and then placed in a heating block (at 1651C, for
3 min) without stirring the contents. The reaction vessel was
then cooled using an ice–water bath and the reaction mixture
diluted with water (1 mL) and transferred onto a C-18 cartridge
(PrepSepTM R-C-18 Extraction Column, Fisher Scientific, activated
beforehand as described above). The cartridge pre-purification
procedure was performed as mentioned above and [18F]-1 was
HPLC-purified (HPLC A, condition 1 or 3 when using 9 and 11,
respectively).
Preparation of the K[18F]F-K222 complex
In order to recover and recycle the [18O]water target, 2 mL of
aqueous [18F]fluoride from the target holder was passed
through an anion exchange resin (SepPaks Light Waters
AccellTM Plus QMA cartridge, chloride form, conditioned by
washing with 1 M aqueous NaHCO3 (2 mL) and rinsing with
water (20 mL)) by helium pressure (1.5–2.0 bar). Helium was
blown through the cartridge to maximally extract the last traces
of [18O]water. The [18F]fluoride ion was then eluted from the
resin, using an aqueous K2CO3 solution (1.0 mL of a 4.5 mg/mL
solution), into a Vacutainers tube containing Kryptofixs222
(K222
sane, 12.0–15.0 mg). The resulting solution was then gently
:
4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexaco-
Quality controls
concentrated to dryness at 145–1501C under a nitrogen stream Final chemical identification of [18F]-1 was performed on an
for 10 min to give no-carrier-added K[18F]F-K222 complex as a acetonitrile-diluted aliquot of the HPLC-collected fraction by
white semi-solid residue.
analytical HPLC (HPLC B) with a sample of authentic 1. Chemical
and radiochemical purities of [18F]-1 were also assessed on this
aliquot using the same analytical HPLC (HPLC B). Specific
radioactivity of [18F]-1 was calculated from three consecutive
HPLC (HPLC B) analyses (average) and determined as follows:
the area of the UV absorbance peak corresponding to the
radiolabelled product was measured (integrated) on the HPLC-
chromatogram and compared with a standard curve relating
mass to UV absorbance. The specific radioactivity follows from
the found mass and the associated collected radioactivity.
Fluorine-18 incorporation studies
DMSO (600 mL) containing 10–30 mmol of the appropriate
precursor for labelling (8 (2.4–7.2 mg), 9 (2.0–6.1 mg) or 11
(3.7–11.0 mg)) was added into the Vacutainers tube containing
the dried K[18F]F-K222 complex. The tube (open) was thoroughly
vortexed (30 s) and then placed in a heating block (at 1651C, for
1–5 min) without stirring the contents. Aliquots were withdrawn
at regular time intervals in order to study the reaction kinetics
and analysed by radio-TLC. Identity of [18F]-1 was verified by co-
migration with non-labelled 1 (reference). Incorporation yields
Conclusion
were calculated from the TLC-radiochromatogram and defined FPyKYNE (1) is a novel fluoropyridine-based structure, designed
as the ratio of radioactivity area of [18F]-1 over total fluorine-18 for the fluorine-18 labelling of macromolecules using the so-
radioactivity area (SiO2-TLC (EtOAc): Rf: [18F]-1: 0.75 – Rf: called ‘click-chemistry’ approach. FPyKYNE has been labelled
[18F]fluoride ion: 0.0). At the end of the heating period, the with fluorine-18 in 19–24% overall non-decay-corrected and
reaction vessel was cooled using an ice–water bath and the isolated yields (30–35% decay-corrected), using a simple but
remaining radioactivity was measured. The reaction mixture was highly efficient one-step process (a nucleophilic heteroaromatic
then diluted with water (1 mL) and transferred onto a C-18 substitution), the latter being fully automatized on our Zymate-
cartridge (PrepSepTM R-C-18 Extraction Column, Fisher Scientific, XP robotic system. These results were first presented at the
J. Label Compd. Radiopharm 2008, 51 336–342
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

B. Kuhnast et al.
second annual meeting of the European Society of Molecular
Imaging (Naples, Italy, 14–15 June 2007).56 A few weeks earlier,
Inkster, Adam and Ruth (Triumf, Vancouver, Canada) presented
at the XVIIth International Symposium on Radiopharmaceutical
Sciences (Aachen, Germany, 30 April 30–4 May 2007) another
´
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hex-5-yn-1-yloxypyridine).41 This coincidence highlights the
[26] B. de Bruin, B. Kuhnast, F. Hinnen, L. Yaouancq, M. Amessou, L.
growing interest for the development of these methodologies.
Huisgen 1,3-dipolar cycloadditions involving our reagent and
macromolecules are in progress.
´
Johannes, A. Samson, R. Boisgard, B. Tavitian, F. Dolle, Bioconj.
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Acknowledgement
The authors wish to thank the cyclotron operators Mr Daniel
ˆ
[29] C. Hartmuth, H. C. Kolb, K. Finn, K. B. Sharpless, Angew. Chem. Int.
Ed. 2001, 40, 2004–2021.
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Gouel, Mr Christophe Peronne and Mr Christophe Lechene for
performing the irradiations. The authors also wish to thank Dr
Dirk Roeda for proof reading the manuscript and suggesting
linguistic corrections. This work was supported in part by the EC-
FP6-project EMIL (LSH-2004-503569).
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