One step radiosynthesis of 6-[18F]fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester ([18F]F-Py-TFP): A new prosthetic group for efficient labeling of biomolecules with fluorine-18

Article abstract of DOI:10.1021/jm9015813

The labeling of biomolecules for positron emission tomography (PET) with no-carrier-added fluorine-18 is almost exclusively accomplished using prosthetic groups in a two step procedure. The inherent complexity of the process renders full automation a challenge and leads to protracted synthesis times. Here we describe a new ¹⁸F-labeled prosthetic group based on, nicotinic acid tetrafiuorophenyl ester. Reaction of [¹⁸F]fluoride at 40°C with the trimethylammonium precursor afforded 6-[¹⁸F]fluoronicotinic acid tetrafiuorophenyl ester ([¹⁸F]F-Py-TFP) directly in 60-70% yield. [¹⁸F]F-Py-TFP was conveniently purified by Sep-Pak cartridge prior to incubation with a peptide containing the RGD sequence. The desired conjugate was formed rapidly and in good yields. An in vitro receptor-binding assay for the integrin α_vβ₃ was established to explore competition with peptide and peptidomlmetic prepared from F-Py-TFP with ¹²⁵I-echlstatin. The nonradioactive conjugates were found to possess high binding affinities with calculated K_i values in the low nanomolar range.

Full text of DOI:10.1021/jm9015813

1732 J. Med. Chem. 2010, 53, 1732–1740
DOI: 10.1021/jm9015813
One Step Radiosynthesis of 6-[18F]Fluoronicotinic Acid 2,3,5,6-Tetrafluorophenyl Ester ([18F]F-Py-TFP):
A New Prosthetic Group for Efficient Labeling of Biomolecules with Fluorine-18
Dag E. Olberg,*,† Joseph M. Arukwe,‡ David Grace,‡ Ole K. Hjelstuen,†,‡ Magne Solbakken,‡ Grete M. Kindberg,‡
and Alan Cuthbertson‡
†Department of Pharmaceutics and Biopharmaceutics, Institute of Pharmacy, University of Tromsø, N-9037 Tromsø, Norway and
‡GE Healthcare MDx R&D, Nycoveien 2, NO-0401 Oslo, Norway
Received October 26, 2009
The labeling of biomolecules for positron emission tomography (PET) with no-carrier-added fluorine-
18 is almost exclusively accomplished using prosthetic groups in a two step procedure. The inherent
complexity of the process renders full automation a challenge and leads to protracted synthesis times.
Here we describe a new 18F-labeled prosthetic group based on nicotinic acid tetrafluorophenyl ester.
Reaction of [18F]fluoride at 40 °C with the trimethylammonium precursor afforded 6-[18F]fluoro-
nicotinic acid tetrafluorophenyl ester ([18F]F-Py-TFP) directly in 60-70% yield. [18F]F-Py-TFP was
conveniently purified by Sep-Pak cartridge prior to incubation with a peptide containing the RGD
sequence. The desired conjugate was formed rapidly and in good yields. An in vitro receptor-binding
assay for the integrin Rvβ3 was established to explore competition with peptide and peptidomimetic
prepared from F-Py-TFP with 125I-echistatin. The nonradioactive conjugates were found to possess
high binding affinities with calculated Ki values in the low nanomolar range.
Introduction
having shown promise for the noninvasive detection of tumors
with PET.13-16
Fluorine-18 is among the radionuclides for positron emis-
sion tomography (PETa) having the most suitable character-
istics for in vivo imaging. This is due to a relatively long half-
life (109.7 min), high positron abundance (97%), and low
positron emission energy (max 635 keV) combined with a well
established production method allowing for multi-Curie
batches through the 18O(p,n)18F reaction.1-4
The automated radiosynthesis of peptide-based PET radio-
pharmaceuticals remains the Achilles’ heel of PET radiopharma-
ceutical manufacture. High yielding reactions for incorporation
of fluorine-18 into complex biomolecules are essential for suc-
cessful commercial adoption because of the high cost of goods of
both peptide and fluoride production.
The labeling of peptides with [18F]fluoride is most com-
monly performed by reaction of an 18F-prosthetic group with
a suitable fuctionalized peptide vector. An example of such a
prosthetic group is N-succinimidyl 4-[18F]fluorobenzoate
([18F]SFB); its radiosynthesis requires two to three steps and
HPLC purification prior to conjugation with peptide.17-19
Furthermore, following conjugation with [18F]SFB an addi-
tional purification step is often required to obtain the 18F-
peptide with sufficient specific activity and purity for in vivo
studies. This complex multistep radiosynthesis complicates
the route to automated production of the final 18F-peptide
drug product. From a radiochemical standpoint the key
challenge is therefore to develop new methodologies that
simplify the manufacturing process.
Attempts to directly label active ester based prosthetic
groups with 18F have been previously reported in the litera-
ture; however, degradation of the active ester under the harsh
18F-labeling conditions resulted in disappointingly low radio-
chemical yield.20,21 We hypothesized that active esters of
nicotinic acid functionalized with a suitable leaving group at
the 2-position would be highly activated for nucleophilic
aromatic substitution with [18F]fluoride and allow direct
labeling under conditions where the active ester moiety would
be less prone to degradation. The 2-[18F]fluoropyridines can
be produced in high yields with high specific activity and have
Currently, 2-[18F]fluoro-2-deoxy-D-glucose (FDG) is by far
the most widely available PET tracer supported in part by the
cost-effective, high yielding automated production methods.5,6
There are, however, many other biomolecules that show
promise for molecular imaging with PET where efficient
fluorine-18 incorporation still represents a significant chal-
lenge to the radiochemist. Peptide-based 18F-tracers have
already demonstrated clinical utility for imaging various types
of diseases, the most important being in the field of oncology.7,8
Complex peptides can be readily synthesized using solid-phase
chemistry and can be tailored to target the desired tissue/
receptor with high specificity.9,10 In addition, they display fast
excretion kinetics, making them very suitable for in vivo
imaging.11,12 Examples include 18F-conjugates of Arg-Gly-
Asp (RGD) and bombesin peptides, both molecular classes
*To whom correspondence should be addressed. Phone: þ47
41517119. Fax: þ47 23186014. E-mail: dag.erlend.olberg@petsenteret.
no.
a Abbreviations: DCC, N,N’-dicyclohexylcarbodiimide; K222, kryp-
tofix 222; PEG, polyethylene glycol; PET, positron emission tomo-
graphy; RCP, radiochemical purity; RGD, Arg-Gly-Asp; tR, retention
time; [18F]SFB, N-succinimidyl 4-[18F]fluorobenzoate; TBA, tetrabutyl-
ammonium; t-BuOH, 2-methyl-2-propanol; TFP-OH, 2,3,5,6-tetra-
fluorophenol; TMSOTf, trimethylsilyl trifluoromethanesulfonate.
r
pubs.acs.org/jmc
Published on Web 01/20/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1733
Scheme 1. Synthesis of Precursor 2 and Prosthetic Group [18F]3 or Nonradioactive 3a
a Reagents and conditions: (i) TFP-OH, DCC, DCM, overnight; (ii) (1) trimethylamine, THF, 3 h; (2) TMSOTf, DCM; (iii) KF, K222, MeCN, room
temperature, 15 min (nonradioactive 3) or TBA-[18F], t-BuOH/MeCN (8:2), 40 °C, 10 min.
Table 1. Summary of the Main Reaction Components As Identified in NMR Experiments at Different Time Pointsa
a The ratios are derived from the mole ratio from the 1H NMR spectra.
the advantage that they do not require an additional electron-
withdrawing substituent for activation, as is common for the
nucleophilic homoaromatic substitutions.22-24 Furthermore,
they are known to be stable in vivo and therefore represent a
plausible alternative to [18F]fluorohomoaromatic systems.25-27
Herein, we describe the synthesis and application of a novel
prosthetic group based on nicotinic acid tetrafluorophenyl
(TFP) ester. A single radiosynthesis step is required, affording
6-[18F]fluoronicotinic acid tetrafluorophenyl ester ([18F]F-Py-
TFP, [18F]3) in good yields from the trimethylammonium
precursor. The resultingprosthetic groupis readilypurified on
a Sep-Pak cartridge prior to conjugation with the peptide
precursor. By use of this methodology, an 18F-RGD peptide
was synthesized in 20-30% isolated yield (decay corrected)
based on [18F]fluoride within 90 min.
further enhancement was provided through the electron-
withdrawing tetrafluorophenyl ester moiety. By use of con-
ditions similar to those described by Barlin et al.,29 the 2-
trimethylammonium chloride salt of nicotinic acid TFP ester
could be conveniently obtained in high purity as a precipitate
from the reaction of the chloride 1 with trimethylamine in
THF. Because of the poor solubility of the chloride salt of 2
in acetonitrile, the counterion was changed to the trifluoro-
methanesulfonate salt before progressing further. Addition
of silver triflate in acetonitrile to the chloride salt gave target
compound 2; however, this method required chromato-
graphic purification of the product. More conveniently,
addition of trimethylsilyl trifluoromethanesulfonate
(TMSOTf) to a suspension of the chloride salt in dichloro-
methane afforded 2 directly following filtration of solid
material and evaporation in vacuo of the organic phase. The
residue was precipitated and washed well with diethyl ether
to obtain pure 2. This triflate salt provided an inert counter-
ion along with excellent solubility of the precursor in aceto-
nitrile. It was noteworthy that attempts to synthesize an
N-hydroxysuccinimide (NHS) ester analogue proved unsuc-
cessful, as extensive decomposition of the ester occurred on
contact with trimethylamine (data not shown). The precur-
sor 2 was progressed further to investigate its reaction with
fluoride ion. By employment of a standard fluorination
protocol with potassium fluoride/kryptofix 222 (K222) com-
plex in acetonitrile at room temperature (15 min), a white
precipitate was observed in the reaction mixture along
with a distinct odor of trimethylamine. LC-MS analysis
of the reaction mixture revealed a product eluting in the
void volume which was identified as products from precursor
Results and Discussion
Chemistry. The synthesis of precursor 2 from 6-chloro-
nicotinic acid and its fluorination by nucleophilic aromatic
substitution affording nonradioactive 3 are outlined in
Scheme 1. Precursor 2 was obtained in good yields from
6-chloronicotinic acid in three steps. Ester formation using
N,N0-dicyclohexylcarbodiimide (DCC) of the starting ma-
terial with 2,3,5,6-tetrafluorophenol (TFP-OH) produced
intermediate 1 which was readily purified by crystallization
from hexane in good yield. Normally trimethylammonium
precursors are obtained by methylation of substituted di-
methylanilines with strong methylating agents such as meth-
yl trifluoromethanesulfonate.28 In this case we exploited the
fact that the chloro-substituent of intermediate 1 was highly
activated toward nucleophilic aromatic substitution and

1734 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Olberg et al.
Figure 1. (Continued on next page).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1735
Figure 1. Structures of peptides and peptidomimetics with their corresponding conjugates.
normal-phase chromatography, the two products were
isolated for their identification by NMR. In addition, an
in-depth NMR study of the fluorination reaction was per-
formed by immediate addition of precursor 2 to the KF/
K222 complex in MeCN-d3. The progress of the reaction was
followed for approximately 25 min acquiring multiple 19F-
1
and H-spectra. NMR spectra of the two major products
confirmed the structure of the 3.54 min peak to be target
compound 3. Interestingly, the 4.81 min peak proved to be a
side product of nicotinic acid TFP ester where the trimethyl-
ammonium moiety of precursor 2 was substituted with a
TFP ether as shown in Table 1, 4. Furthermore, the NMR
mediated monitoring of the reaction at 27 °C showed that
product 3 was formed rapidly (see Supporting Information).
After only 5 min >60% of the precursor 2 had been
converted to product 3, the reaction reaching a plateau at
15 min with around 70% conversion. Increase of reaction
temperature served only to accelerate the formation of 4.
The increased stabilities of TFP esters compared to other
active esters have been documented previously for radio-
iodination reactions.30 Indeed, direct labeling of 2,3,5,6-
tetrafluorophenyl pentafluorobenzoate with TBA-[18F] in
DMSO has been reported by Herman et al.31 In this reaction,
a [18F]fluoride for fluoride exchange in the pentafluoro-
phenyl system resulted in a decay corrected yield of 32%.
However, this method results in an 18F-product with inher-
ently low specific activity due to isotopic dilution. Impor-
tantly, the tetrafluorophenoxy moiety proved not to be
sufficiently activated to allow for isotopic exchange with
[18F]fluoride as demonstrated in the same work by Herman
et al.
Figure 2. Radio-HPLC (system 1) analysis of an aliquot taken
from the crude reaction using TBA-HCO3/t-BuOH/MeCN system
after 10 min at 40 °C: (a) radioactive channel ([18F]3 = 5.0 min
peak); (b) UV channel at 254 nm (4 = 6.2 min peak).
hydrolysis. A small peak corresponding to starting material 2
was also identified with a retention time of 1.75 min,
but interestingly two major new products eluting at 3.54
and 4.81 min in the chromatogram were observed; however,
LC-MS was unable to resolve the identity of these main
products. Following purification of the reaction mixture by
After the successful synthesis and isolation of 3, its utility
as an acylating agent was investigated. Two RGD peptides,
one with a free amine 5 (NC100717)32 and the second
functionalized with an aminooxy moiety 6, were chosen as
model compounds. The aminooxy functionality was selected
in order to investigate whether 3 was capable of reaction with

1736 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Olberg et al.
Table 2. Summary of Tested Conditions for the Radiosynthesis of [18F]3a
recovery
from reaction
vessel (%)e
yield (%)
(TLC ꢀ
precursor
amount (mg)
time
(min)
base/amount
(mg or μL)b
temperature
(°C)
yield (%)
(radio-TLC)d
PTCc
solvent
MeCN
recovery)f
7
10
5
KHCO3/1.6
KHCO3/2.1
KHCO3/3.0
K2CO3/5.0
KHCO3/3.0
KHCO3/1.5
KHCO3/3.0
KHCO3/3.0
40
50
45
40
40
40
40
40
K222
K222
K222
K222
K222
K222
K222
K222
77.6
63.1
19
52.2
45.7
ND
57.2
ND
65
40.5
28.8
ND
22.4
ND
38.4
13.2
26.5
10
7
MeCN
MeCN
THF
10
10
10
10
10
10
8.2
10
7
39.2
trace
59
THF
MeCN
MeCN
7.3
7.6
17.2
30.7
76.9
86.3
MeCN/
t-BuOH (2:8)
DMSO
MeCN
8
10
10
10
10
10
10
TBA-HCO3/30
TBA-HCO3/20
TBA-HCO3/25
TBA-HCO3/30
TBA-HCO3/30
40
40
40
40
40
TBA
TBA
TBA
TBA
TBA
5.1
65.4
70
3.6
45.5
69.5
76.8
73.7
75.9
9
15
MeCN
MeCN
55.0
67.4
66.3 (5.1)g
42.2
49.7
50.3 (3.9)g
9 (n = 4)
MeCN/
t-BuOH (2:8)
a ND = not determined b Indicates base used in reaction; K2CO3/KHCO3 expressed in mg or μL of a 0.8 M aqueous TBA-HCO3 solution. c Phase-
transfer catalyst (PTC) used. d Radiochemical yield by radio-TLC. e Indicates total amount of radioactivity recovered from reactor decay corrected to
start of synthesis. f TLC yield multiplied by total recovered radioactivity. g Standard deviation.
the aminooxy group at low pH, expanding the scope of the
process to encompass site-specific labeling of molecules
possessing amino groups. In addition, an Rvβ3 receptor
antagonist 7 belonging to the quinoline-4-one class of pepti-
domimetics was also synthesized according to Harris et al.33
Compound 7 was further functionalized with a polyethylene
glycol (PEG10) unit, affording 8. The pharmacophore com-
ponents of the selected peptides and peptidomimetics have
previously been shown to possess affinity for the integrin
Rvβ3 in the low nanomolar range.32,33 Conjugation with 1.3
equiv of 3 with respect to precursors 5, 7, and 8 in a mixture
of 0.6 mL of DMSO/acetonitrile/phosphate buffer pH 9
(1:1:1) was used as the standard reaction conditions.34 As
indicated by LC-MS, the conjugates 9, 10, and 11 formed
rapidly and nearly quantitatively under these conditions with
little hydrolysis of the active ester at room temperature after
30 min. The conjugates were purified by reversed-phase
preparative HPLC, and the desired products were isolated
from the pure fractions by lyophilization. The reaction of 3
with the aminooxy modified peptide 6 at pH 4.3 in a citrate/
phosphate/DMSO system proceeded slowly. Improved re-
action was observed at elevated temperature and extended
reaction time (60 °C;1 h). The conjugate 12 was indeed
formed, although yields were significantly lower compared to
the aminolysis reactions with 3. Conjugate 12 was purified and
isolated in 16% yield with respect to starting peptide. This
strategy may be an alternative for the introduction of active
esters site-selectively at low pH in many biomolecules possess-
ing a multitude of free amino functionalities. The structures of
the peptides and peptidomimetics with their corresponding
conjugates formed with 3 are shown in Figure 1.
as phase-transfer catalyst in acetonitrile/t-BuOH (2:8) as reac-
tion solvent. This protocol gave 66.3% incorporation of
[18F]fluoride after 10 min at 40 °C with an average of 76% of
the total activity recovered from the reaction vessel (n = 4).
A summary of all the conditions tested is given in Table 2.
Interestingly the t-BuOH system gave less precipitation in the
reaction mixture and in general cleaner chromatograms. To our
knowledge this is the first reported use of a tertiary alcohol used
in nucleophilic aromatic substitution reactions with no-carrier-
added [18F]fluoride, originally reported by Kim et al. for SN2-
type reactions.35 The nonradioactive profile observed in the
radioactive experiments proved to be very similar to the initial
findings with the “cold” fluorination experiments: small
amounts of intact precursor (tR = 3.06 min) with the major
impurities being the hydrolyzed product of precursor 2 eluting
close to the void, 2,3,5,6-tetrafluorophenol (tR =4.31min), and
impurity 4 (tR = 6.21 min). A typical chromatogram of the
crude reaction mixture for the synthesis of [18F]F-Py-TFP is
shown in Figure 2.
After establishment of the optimal conditions for the
radiosynthesis of [18F]3, the next goal was to develop a
simple method for purification. By dilution of the crude
reaction mixture with water, [18F]F-Py-TFP could be
trapped efficiently on an Oasis MCX Plus cartridge with
mix-mode reversed-phase and cation-exchange properties.
The column was rinsed with 5 mL of water followed by
elution of [18F]3 with a water/acetonitrile mixture (3.5:6.5,
2.1 mL) in >95% radiochemical purity and 70-80% recov-
ery from SPE cartridge. Positively charged species and the
highly hydrophobic impurity 4 with the intact active ester
were nearly completely retained on the Sep-Pak cartridge, a
prerequisite for efficient labeling in the next step. The specific
activity for the Sep-Pak purified [18F]F-Py-TFP was calcu-
lated from a calibration curve of pure 3 and was calculated to
be 4 GBq/μmol. However, these calculations were done from
an experiment starting with 125 MBq of [18F]fluoride; hence,
higher specific activity of [18F]F-Py-TFP is expected with
higher amounts of [18F]fluoride.
Radiochemistry. Azeotropic drying for the preparation of the
reactive 18F-complex is described in Materials and Methods.
The radiosynthesis of [18F]3 from precursor 2 (9mg, 19 μmol)
was first attempted using K222/K2CO3 in THF for 10 min at
40 °C. These conditions resulted in formation of precipitates in
the reaction mixture and a maximum yield of 39% [18F]3 from
resolubilized [18F]fluoride as evidenced by radio-HPLC and
TLC. The less basic bicarbonate salt and acetonitrile as solvent
increased the radiochemical yield to 77% (radio-TLC) at 40 °C
for 10 min with 52% of the activity recovered from the reaction
vessel. Best conditions were found to be those using TBA-HCO3
Initially, conjugation to peptide precursor 5 in phosphate
buffer/DMSO/acetonitrile (pH 9) was studied by addition
of aliquots of purified [18F]F-Py-TFP (100 μL, 2-3 MBq).
The reaction was studied at various peptide concentrations

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1737
Table 4. Isolated Radiochemical Yields of [18F]9 (n = 4)
Table 3. Radiolabeling of [18F]F-Py-TFP with Peptide Precursor 5 As
Analyzed by Radio-HPLC (n = 1 per Time Point)
peptide
conju-
gate
conjuga-
reaction tion time
total synthesis RCPc
radiolabeling yield (%)
isolated
yielda (%)
temp (°C)
(min)
timeb (min)
(%)
time (min) 0.5 mg/mL peptide 1 mg/mL peptide 3 mg/mL peptide
[
18F]9
40
15
22.5 ( 6.0
85 ( 2
>99
10
20
30
62
92
84
>95
>95
>95
>95
>95
a Yield of isolated [18F]fluoropeptide, based on starting [18F]fluoride
(corrected for decay). b Average time of synthesis measured from
incoming [18F]fluoride to end of purification. c Radiochemical purity
analyzed by radio-HPLC.
>95
ranging from 0.5 to 3.0 mg/mL peptide at 40 °C. The
conversion of [18F]3 to [18F]9 was monitored by radio-
HPLC, and the yields were calculated from the percentage
of radioactivity peak area of the desired product [18F]9
relative to starting product [18F]F-Py-TFP.
Table 5. In Vitro Assay Results for Conjugates 9, 10, 11 and 12 in the
Rvβ3 Ea-Hy 926 Assay
conjugate
Rvβ3 in vitro assay Ki (nM)a
After 10 min >95% of the labeling reagent [18F]F-Py-TFP
had been converted into [18F]9 at 3 mg/mL of peptide 5. At
peptide concentrations of 1 and 0.5 mg/mL conversion was
incomplete after 10 min, reaction times of 20 and 30 min,
respectively, were required to achieve conjugation yields
of >95%. This clearly demonstrates the dependency of
peptide concentration on radiochemical yield.36,37 The re-
sults are summarized in Table 3. The one step protocol and
short synthesis time (10 min) of [18F]F-Py-TFP in good yields
(50% decay corrected) compare favorably to the normal
three step synthesis of [18F]SFB requiring 35-100 min with
yields around 25-60% and serve to demonstrate the promise
of [18F]F-Py-TFP as a new 18F-prosthetic group for the mild
and efficient conjugation of amino groups.12,18 Moreover, by
omission of the time-consuming HPLC purification step
customarily employed for [18F]SFB, [18F]F-Py-TFP allows
conjugation to biomolecules bearing a free amine under mild
conditions (phosphate buffer/organic solvent, pH 9, 40 °C)
within 10-30 min in excellent yields (>90%).
9
2.9 ( 0.4
10
11
12
19.1 ( 12.2b
14.8 ( 0.3
2.0 ( 0.9
a n>3 experiments. b Poor solubility of conjugate 10 in the test
medium.
the four nonradioactive conjugates.33 Compounds 9, 10, 11,
and 12 were found to compete with 125I-echistatin with
calculated Ki values in the low nanomolar range (as shown
in Table 5), indicating that the 2-fluoronicotinate moiety did
not interfere with the binding properties of the pharmaco-
phores.
The nonradioactive conjugate 9 was investigated in hu-
man plasma to acquire preliminary stability data. The pep-
tide conjugate was incubated in phosphate buffered saline
(PBS) and in freshly collected human plasma at 37 °C for
60 min followed by analysis by LC-MS. In both test vehicles
no degradation of conjugate 9 was observed. These results
are in good agreement with the published in vivo data
conducted with other 2-[18F]fluoropyridines systems with
particular relevance to defluorination.26,27
Finally, to assess the efficiency of [18F]F-Py-TFP as a
labeling agent in a more realistic setting, the process for
radiosynthesis of [18F]9 was performed semiautomatically
where only the transfer of reaction mixture to the HPLC
injection loop after peptide labeling was handled manually.
The synthesis and purification of [18F]F-Py-TFP were per-
formed under the optimal conditions described above. After
elution of [18F]F-Py-TFP from the Sep-Pak cartridge back to
the reaction vessel, 2 mg of peptide 5 in 1 mL of phosphate
buffer/DMSO (1:1), pH 9, was added from a prefilled vial,
giving a total volume of 3.1 mL and a final peptide concen-
tration of 0.65 mg/mL (0.5 mM). The reaction mixture was
heated to 40 °C for 15 min followed by dilution with water
(1.0 mL) and purification by semipreparative reversed-phase
HPLC. The fraction containing the desired radioactive
product was measured in a dose calibrator and analyzed by
radio-HPLC. With this protocol [18F]9 could be isolated in
up to 30% yield based on [18F]fluoride and excellent radio-
chemical purity (>99%). In a typical set of experiments the
decay corrected isolated yields calculated from [18F]fluoride
averaged around 22 ( 6% (n = 4). The overall synthesis time
including purification was 85 ( 2 min and is likely to be
amendable to even shorter synthesis time in a fully optimized
automated process. The radiochemical yields compare favorably
with other protocols for synthesis of 18F-peptides.7,38-40
The promising overall radiochemical yield and simplification
to the process could therefore represent an alternative
methodology for the fully automated production of 18F-
peptides. The results of the experiments are summarized in
Table 4.
Conclusion
The starting tetrafluorophenol ester precursor 2 was
synthesized in three steps from readily available reagents with
no chromatographic purification steps required. An opti-
mized method for the fluorination of the active ester was
developed giving good yields of [18F]F-Py-TFP in a single step
followed by a convenient purification protocol using an SPE
cartridge. The simplification of the radiosynthesis and puri-
fication of this prosthetic group was amenable to automation
on a commercially available synthesis platform. Conjugation
of[18F]F-Py-TFPtoanRGD-peptide was foundtoproceed in
good yields and in excellent radiochemical purity.
This new methodology is considered useful for labeling of
biomolecules where [18F]SFB is currently employed offering
the advantage of a much simpler and rapid radiosynthesis
process. In addition, in comparison to the more commonly
used18F-homoaromaticsthe 2-[18F]fluoropyridines havebeen
shown to produce conjugates with lower log P values poten-
tially resulting in more favorable biodistribution character-
istics of the labeled conjugates.41
Materials and Methods
Chemicals and solvents of reagent grade obtained commer-
cially were of a purity of g95% and were used without further
purification. Compounds synthesized were analyzed by HPLC
confirming g95% purity, unless otherwise stated. All anhydrous
chemicals were purchased from Sigma-Aldrich (Norway). HPLC
solvents were obtained from Merck KGaA (VWR). Solvents for
In Vitro Affinity and Stability. A competitive binding assay
was used to measure the binding affinity to integrin Rvβ3 of

1738 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Olberg et al.
all analytic and preparative HPLC were water/0.1% TFA
(solvent A) and acetonitrile/0.1% TFA (solvent B). For complete
analytical and preparative conditions see Supporting Informa-
tion.
Synthesis of N,N,N-Trimethyl-5-((2,3,5,6-tetrafluorophenoxy)-
carbonyl)pyridin-2-aminium Trifluoromethanesulfonate (2).
6-Chloronicotinic acid 2,3,5,6-tetrafluorophenyl ester (1.0 g,
3.3 mmol) was dissolved in dry THF (15 mL). The solution
was filtered into a 35 mL vial and capped with a rubber septum.
A steady stream of trimethylamine gas at room temperature was
passed through the filtrate under vigorous stirring, allowing
excess trimethylamine to escape through a venting needle. After
5 min a white precipitate started to form and the reaction was
allowed to proceed for 3 h while maintaining trimethylamine
flow. The solid material was filtered off and washed with diethyl
ether (100 mL) and cold dichloromethane (50 mL). The solid
material (0.53 g, 1.5 mmol) (N,N,N-trimethyl-5-((2,3,5,6-tetra-
fluorophenoxy)carbonyl)pyridin-2-aminium chloride) was sus-
pended in dichloromethane (50 mL) under an argon atmosphere
by means of ultrasonification. To the vigorously stirred suspen-
sion was added trimethylsilyl trifluoromethanesulfonate (0.78 mL,
4.4 mmol) over 5 min. The solution was filtered, and volatile
components were removed under reduced pressure. The dry
residue was washed with diethyl ether (3 ꢀ 50 mL) and dried
under vacuum, affording N,N,N-trimethyl-5-((2,3,5,6-tetra-
fluorophenoxy)carbonyl)pyridin-2-aminium trifluoromethane-
sulfonate (2) as a white fluffy solid (0.5 g, 32%). 1H NMR
(500 MHz, CD3CN): δ 9.34 (dd, J1 = 0.8 Hz, J2 = 2.3 Hz, 1H),
δ 8.84 (dd, J1 = 2.3 Hz, J2 = 8.7 Hz, 1H), δ 8.07 (dd, J1 = 0.8 Hz,
J2 = 8.7 Hz, 1H), δ 7.43 (tt, J1 = 7.3 Hz, J2 = 10.6 Hz, 1H), δ
3.60 (s, 9H). 19F NMR (470 MHz, CD3CN): δ -79.72 (s, 3F),
δ -140.74 (m, 2F), δ -154.77 (m, 2F). Purity (HPLC): 98%,
tR= 1.76 min. HRMS-TOF (m/z): found, 329.1253 [M]þ, calcd
for C15H13F4N2O2 329.0913.
NMR spectra were run on a Varian Unity Inova 500 spectro-
meter equipped with a 5 mm 1H-broadband PFG indirect
detection probe and on a Bruker Avance III 400 spectrometer
equipped with a 5 mm BBFO PFG probe. Mass spectra and
analytical HPLC were recorded on a LCQ DECA XP MAX
instrument using electrospray ionization (ESI) operated in
positive mode at 4.5 kV and scan rate 5500 Da/s coupled to a
Finnigan Surveyor PDA chromatography system. High resolu-
tion MS of compounds 1, 2, and 3 were recorded on a Agilent
1110 series HPLC system coupled to a Agilent MS-TOF
(1969A) with a Zorbax SB C18 (50 mm ꢀ 4.6 mm, 1.8 μm)
using a gradient of 20-90% acetonitrile in 10 mM ammonium
acetate over 7.2 min with a flow rate of 1.5 mL/min. Thin layer
chromatography (TLC) was run on precoated plates of silica gel
60F254 (Merck) developed in hexane/ethyl acetate (1:1). Pre-
parative HPLC purifications of nonradioactive reference stan-
dards were performed on a Shimadzu system with a Pheno-
menex Luna C18(2) column (250 mm ꢀ 21.2 mm, 5 μm) at a flow
rate of 10 mL/min over 60 min with detection at 214 or 254 nm
(see Supporting Information). Accurate mass measurements for
conjugates 9, 10, 11, and 12 were carried out on a QToF-micro
orthogonal acceleration time-of-flight mass spectrometer
(Micromass UK Ltd., Manchester, U.K.) coupled with an Agilent
1100 chromatography system (Agilent Technology, Stockport,
U.K.) operating in the positive ion ESI mode. Prior to analysis,
the instrument was calibrated over a mass range of m/z
100-2000 using sodium iodide. An acquisition rate of 1 spec-
trum per second was used for all data analyses. The reference
compound was iodixanol (3,30,5,50-tetrakis(2,3-dihydroxypropyl-
carbamoyl)-2,20,4,40,6,60-hexaiodo-N,N0-(2-hydroxypropane-
1,3-diyl)-diacetanilide) and was provided by GE Healthcare
(Oslo, Norway). Data acquisition and processing were per-
formed using the Masslynx 4.0.
Synthesis of 6-Fluoronicotinic Acid 2,3,5,6-Tetrafluorophenyl
Ester (3). A mixture of potassium fluoride (7.3 mg, 0.12 mmol)
and Kryptofix 222 (59 mg, 0.16 mmol) in dry acetonitrile (1.0 mL)
was stirred for 5 min. A solution of N,N,N-trimethyl-5-((2,3,5,6-
tetrafluorophenoxy)carbonyl)pyridin-2-aminium trifluorome-
thanesulfonate (2) (50 mg, 0.10 mmol) in dry acetonitrile (0.5
mL was added, and the resulting mixture was stirred for 15 min at
room temperature. The reaction mixture was diluted with 2.0 mL
water/0.1% TFA, filtered, and purified by reversed-phase pre-
parative chromatography (Phenomenex Luna C18(2) column (250
mm ꢀ 21.2 mm, 5 μm), flow rate of 10 mL/min, gradient of
40-80% solvent B over 60 min). The collected fractions were
pooled, and acetonitrile was removed under reduced pressure. The
aqueous phase was extracted with dichloromethane (3ꢀ10mL). The
combined organic phases were washed with water (10 mL), brine
(10 mL) and dried (Na2SO4). The organic phase was removed in
vacuo affording 6-fluoronicotinic acid 2,3,5,6-tetrafluorophe-
Radiochemistry. Radiochemical syntheses of [18F]3 and [18F]9
were performed using the TRACERlab FX F-N (GE Medical
Systems) with manual interventions when required. Isocratic
reversed-phased preparative HPLC was run on a Phenomenex
Luna C18(2) (150 mm ꢀ 10 mm, 5 μm), flow 5 mL/min, using
21% solvent B for 20 min with UV detection at 254 nm using the
in-built system of the TRACERlab FX F-N. Analytical HPLC
was performed on an Agilent system (1100 series) with UV
detection in series with a γ-detector (Bioscan flow-count) (see
Supporting Information). Instant Imager (Packard BioScience)
was used to measure the Radio-TLC scan. [18F]Fluoride was
produced by a cyclotron (GE PETtrace 6) using 18O(p,n)18F
nuclear reaction with a 16.5 MeV proton irradiation of an
enriched [18O]H2O target.
Synthesis of 6-Chloronicotinic Acid 2,3,5,6-Tetrafluorophenyl
Ester (1). A solution of 6-chloronicotinic acid (2.0 g, 13 mmol),
tetrafluorophenol (TFP) (2.2 g, 13 mmol), and N,N0-dicyclo-
hexylcarbodiimide (DCC) (2.60 g, 12.5 mmol) in dioxane
(70 mL) was stirred overnight. Dicyclohexylurea (DCU) was
filtered off and the solvent removed in vacuo. The residue was
dissolved in a minimal volume of hot hexane and immediately
filtered. After a short period of time white crystals started to
form. To complete the crystallization, the filtrate was stored at
4 °C overnight. The crystals were filtered off and washed twice
with ice-cold hexane (2 ꢀ 50 mL), affording 6-chloronicotinic
acid 2,3,5,6-tetrafluorophenyl ester (1) as a white fluffy solid (3.0 g,
79%). 1H NMR (500 MHz, CDCl3): δ 9.19 (dd, J1 = 0.6 Hz, J2 =
2.4 Hz, 1H), δ 8.41 (dd, J1 = 2.4 Hz, J2 = 8.3 Hz, 1H), δ 8.07
(dd, J1 = 0.8 Hz, J2 = 8.7 Hz, 1H), δ 7.56 (dd, J1 = 0.7 Hz, J2 =
8.3 Hz, 1H), δ 7.09 (tt, J1 = 7.0 Hz, J2 = 9.8 Hz, 1H). 19F NMR
(470 MHz, CDCl3): δ -139.46 (m, 2F), δ -153.65 (m, 2F). Purity
(HPLC): 95%, tR=3.79min. HRMS-TOF(m/z): found, 305.9985
[M þ H]þ, calcd for C12H4ClF4NO2 305.9940.
1
nyl ester (3) as a waxy off-white solid (10 mg, 37%). H NMR
(500 MHz, CDCl3): δ 9.10 (dt, Jt = 0.5 Hz, Jd = 2.5 Hz, 1H), δ
8.57 (ddd, J1 = 2.5 Hz, J2 = 7.4 Hz, J3 = 8.6 Hz, 1H), δ 7.13 (ddd,
J1 = 0.5 Hz, J2 = 3.0 Hz, J3 = 8.6 Hz, 1H), δ 7.09 (tt, J1 = 7.1 Hz,
J2 = 9.9 Hz, 1H). 19F NMR (470 MHz, CDCl3): δ -58.31 (dd,
J1 = 2.5 Hz, J2 = 7.4 Hz 1F), δ -138.75 (m, 2F), δ -152.95 (m,
2F). Purity (HPLC): 99%, tR= 3.54 min. HRMS-TOF (m/z):
found, 290.0249 [M þ H]þ, calcd for C12H4F5NO2 290.0235.
Preparation of Peptide and Peptidomimetic Precursors (5, 6, 7,
and 8). Detailed syntheses of peptide precursors 5 and 6 have
been described previously.33 Quinolin-4-one peptidomimetic 7
was synthesized as described by Harris et al.32
Detailed synthesis of PEG10 modified peptidomimetic 8 from
7 can be found in Supporting Information.
General Synthesis of 19F-Reference Standards (9, 10, 11, and
12). Complete reaction conditions and analytical data are
deposited in Supporting Information.
Synthesis of 6-[18F]Fluoronicotinic Acid 2,3,5,6-Tetrafluoro-
phenyl Ester ([18F]3). Aqueous [18F]fluoride (1 mL, 100-370 MBq)
was passed through an anion-exchange resin (Chromafix 30-PS-
HCO3, Machanery-Nagel). The [18F]fluoride was eluted off the
resin to the TRACERlab reactor vessel using a mixture of 30 μL of
0.8 M aqueous solution of tetrabutylammonium bicarbonate

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1739
(TBA-HCO3) in water (300 μL) and acetonitrile (300 μL). The
solution was concentrated to dryness by heating at 100 °C under
reduced pressure and a flow of nitrogen for 2 min. Acetonitrile
(0.8 mL) was added twice and evaporated off as above. To the
dried TBA-[18F] complex was added 2 (9.0 mg, 19 μmol) dissolved
in 1 mL of acetonitrile/tert-butanol (2:8). The sealed reaction vessel
was heated to 40 °C for 10 min and then analyzed by radio-TLC
and HPLC.
(4) Le Bars, D. Fluorine-18 and medical imaging: radiopharmaceuti-
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SPECT. J. Labelled Compd. Radiopharm. 2002, 45, 167–180.
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Morelle, J. L.; Monclus, M.; Naemen, J. V.; Mulleneers, E.; Aerts,
J.; Plenevaux, A.; Brihaye, C.; Luxen, A. Fast [18F]FDG synthesis
by alkaline hydrolysis on a low polarity solid phase support.
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Synthesis of Peptide Conjugate [18F]9. The crude reaction
mixture containing [18F]3 was diluted with 2.5 mL of water
and loaded onto a preconditioned Oasis MCX Plus Sep-Pak
(Waters). The cartridge was rinsed with 5 mL of water, and
purified [18F]3 was eluted back to the reaction vessel using 2.1 mL
of water/acetonitrile (3.5:6.5). Peptide 5 (2.0 mg, 1.6 μmol) dis-
solved in 0.2 M phosphate buffer (pH 9)/DMSO (1:1), 1 mL, was
added to the acetonitrile/water solution of purified [18F]3, giving a
total volume of 3.1 mL. The reaction mixture was heated to 40 °C
for 15 min, diluted with water, and purified with preparative
HPLC. The fraction containing [18F]9 was collected, measured in
a dose calibrator, and analyzed with radio-HPLC. [18F]9 coeluted
with its authentic reference standard 9. (HPLC conditions and
analytical data are deposited in Supporting Information.)
In Vitro Binding Assay. Membranes from the human endo-
thelial adenocarcinoma cell line EA-Hy926 were prepared,
and Kd for 125I-echistatin was determined in the purified mem-
brane fraction. A competitive binding assay was established to
measure inhibition constants for each test substance without the
need for labeling of the substance itself.33 125I-Echistatin was
used as the labeled ligand and nonradioactive echistatin as a
reference standard. A total of 16 dilutions of test compound
(either echistatin or test substance) were prepared and mixed
with a combination of the radioactive tracer and membrane
prior to incubation for 1 h at 37 °C. Following washing, the
bound material was harvested on a filter using a Skatron
microharvester. The filter spots were finally excised and counted
in a Packard γ-counter. Ki values were calculated from the
binding curves using Prism software.
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comparison with the corresponding 4-[18F]fluorobenzoyl- and
2-[18F]fluoropropionyl-peptides. J. Med. Chem. 2008, 51, 5901–
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In Vitro Plasma Stability. A solution of 9 (2 mg) in 5 μL of
DMSO was added to either 0.4 mL of phosphate buffer saline
(PBS), pH 7.4, or freshly collected humane plasma. The peptide
was allowed to incubate for 1 h at 37 °C. The two solutions were
diluted 1:1 with PBS and ultrafiltered (Ultrafree filter unit 5000
NMWL, Millipore) at 14 000 rpm for 15 min. The resulting
filtrate was diluted with water/0.1% TFA and analyzed by
LC-MS. Two control runs with blank plasma and PBS were
also done as described above.
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emission tomography. Nucl. Med. Biol. 2008, 35, 725–731.
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Acknowledgment. We thank Emma Bjurgert for the excel-
lent synthesis of the parent peptidomimetic along with Erlend
Hvattum and Marianne Fresvig for recording of the HRMS
ꢀ
spectra. Finally, thanks are extended to Frederic Dolle for
supplying information on log P values of 2-fluoropyridines vs
˚
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Supporting Information Available: HPLC conditions, ex-
tended radio-HPLC analytical data, synthesis conditions, affi-
nity measurement, high resolution LC-MS, HPLC spectra, and
NMR spectrum of 4. This material is available free of charge via
the Internet at http://pubs.acs.org.
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ꢀ
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