18F-labelling of a cyclic pentapeptide inhibitor of the chemokine receptor CXCR4

Article abstract of DOI:10.1016/j.jfluchem.2011.11.003

The chemokine receptor CXCR4 is overexpressed in a variety of cancers including breast, prostate and lung cancer. Expression is also associated with invasion and metastasis. The possibility to image and quantify CXCR4 expression in vivo would be a valuabl

Full text of DOI:10.1016/j.jfluchem.2011.11.003

Journal of Fluorine Chemistry 135 (2012) 200–206
Contents lists available at SciVerse ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
18F-labelling of a cyclic pentapeptide inhibitor of the chemokine receptor CXCR4
˚
*
Ola Aberg , Federica Pisaneschi, Graham Smith, Quang-De Nguyen, Elizabeth Stevens, Eric O. Aboagye
Comprehensive Cancer Imaging Centre, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK
A R T I C L E I N F O
A B S T R A C T
Article history:
The chemokine receptor CXCR4 is overexpressed in a variety of cancers including breast, prostate and
lung cancer. Expression is also associated with invasion and metastasis. The possibility to image and
quantify CXCR4 expression in vivo would be a valuable tool in the clinic to aid treatment regimens and to
potentially understand the underlying biology of metastasis. Herein we describe the synthesis and the
radiolabelling of an 18F-labelled cyclic pentapeptide, [18F]CCIC-0007 designed to bind to the extracellular
domains of CXCR4. Radiolabelling was performed via conjugation of [18F]fluorobenzaldehyde with an
aminooxy functionalised cyclopentapeptide. Typically, starting with 1.10 GBq (30 mCi) of aqueous
Received 15 June 2011
Received in revised form 8 November 2011
Accepted 9 November 2011
Available online 28 November 2011
Keywords:
CXCR4
[
18F]fluoride, 105 MBq (2.85 mCi) of the formulated tracer was obtained within 2.5 h (23 ꢀ 8% dc rcy, 8%
Cyclic pentapeptide
PET imaging
EOS yield). Tissue pharmacokinetic studies in mice demonstrated rapid blood clearance, together with biliary
and renal elimination.
[
18F]fluorobenzaldehyde
ß 2011 Elsevier B.V. All rights reserved.
Radiolabelling
1. Introduction
Positron emission tomography (PET) is a non invasive imaging
modality which uses short lived positron emitting bioprobes to
Malignant cells have the ability to metastasise to distant sites
provoking the spread of cancer through the human body. The
chemokine receptor CXCR4, a member of seven transmembrane
domain G-protein coupled receptors, has been found to play a
crucial role in homing cancer cells to distant sites through binding
with its native ligand CXCL12/SDF-1 (chemokine stromal cell-
derived factor-1) [1]. Even though metastases tend to show higher
expression of CXCR4 than primary tumours, primary lesions with
high expression of CXCR4 are recognised as having an aggressive
phenotype prone to metastasise [2].
Inhibition of CXCR4-CXCL12 signalling through the use of
antagonists of the natural ligand was investigated as potential
therapeutic strategies; efficacy, expressed as reduced metastasis,
was successfully demonstrated in preclinical metastatic models of
breast cancer [3]. CXCR4 inhibitors reported to date are mainly
cyclams [4] or peptides [5], however, an increasing number of other
small molecule inhibitors are appearing in the literature [6].
Moreover, publication of the crystal structure of CXCR4 in late
2010 is likely to speed up the search for more active antagonists [7].
From a diagnostic perspective, the role played by CXCR4 in
invasion and metastasis makes it a valuable biomarker to identify
primary tumours prone to metastasise.
obtain quantitative measures of the biodistribution of the
radioactivity within the body [8]. Among the positron emitting
radionuclides exploited, 18F (t1/2 = 109 min) is attractive because
its relative long half-life allows slower kinetics in vivo to be
monitored when compared to for example 11C (t1/2 = 20.4 min),
15N (t1/2 = 9.97 min) and 15O (t1/2 = 2.04 min). It also permitting
more elaborate radiosynthesis tracer production and 18F-radi-
olabelled tracers can also be potentially transported and used at
hospitals without an on-site cyclotron [9].
A few examples of PET imaging tracers for CXCR4 have recently
been reported in the literature [10] and they are almost exclusively
based on the structure of bicyclam inhibitor Plerixafor (Mozobil,
AMD3100) [10a,b] or of the peptide antagonist T140 [10d,e]. So far,
the only example of 18F radioimaging agent, 4-18F-T140 [10d],
which suffers from pronounced off target binding to red blood cells
when used in tracer concentrations and high specific activity.
In the present paper we report the radiosynthesis of [18F]CCIC-
0007, a [18F]fluorobenzoimino PEG functionalised cyclo(Nal1-Gly2-
D
-Tyr3-Orn4-Arg5) pentapeptide based on FC131 (Scheme 1), as
potential radiotracer for imaging CXCR4 expression. Radiosynthesis
was successfully accomplished by conjugation of the corresponding
aminooxy PEG functionalised cyclo(Nal-Gly-D-Tyr-Orn-Arg) penta-
peptide and 4-[18F]fluorobenzaldehyde ([18F]FBA). The use of a
radiolabelled cyclopentapeptide seemed attractive for its anticipat-
ed lower immunogenicity compared to a larger peptide and the ease
of the radiolabelling strategy chosen to accomplish the radio-
synthesis. The tissue pharmacokinetic profile in mice is also
reported.
Abbreviations: PET, positron emission tomography; Nal,
Orn,
-ornithine; [18F]FBA, [18F]fluorobenzaldehyde.
L-3-(2-naphthyl)alanine;
L
* Corresponding author. Tel.: +44 020 8383 3759.
˚
E-mail address: o.aberg@imperial.ac.uk (O. Aberg).
0022-1139/$ – see front matter ß 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jfluchem.2011.11.003

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O. Aberg et al. / Journal of Fluorine Chemistry 135 (2012) 200–206
201
Scheme 1. Arg4 is replaced by Orn4 and functionalised with PEG and an aminooxy group for labelling with [18F]FBA.
2. Results and discussion
nitro counterpart of being more easily separated from the labelled
product [15] by solid phase extraction (SPE) ensuring simple faster
purification compared to time consuming HPLC separations [16].
Recently, a procedure involving microwave irradiation allowed the
reaction time to be substantially shortened from tens of minutes
The use of radiolabelled peptides and proteins for receptor
imaging and tumour targeting is now well established [11].
Although examples of direct fluorination for the radiolabelling of
peptides have recently been reported [12], functional group
incompatibilities and the harsh conditions required for the
introduction of [18F]fluoride makes this approach particularly
challenging. The use of prosthetic groups for introduction of
down to seconds [17]. We synthesised
[
18F]FBA from the
trimethylanilinium triflate precursor 1 by 15 s of 50 W microwave
irradiation set at 80 8C with 60 ꢀ 9% dc rcy (n = 15) (Scheme 2).
However, it should be noted that the IR temperature probe measures
the outside of the glass reaction vessel which usually rises from r.t. to
60–70 8C during the reaction. Considering the low volume of solvent
we, therefore, assume the actual temperature of the reaction mixture
to be higher.
[
18F]fluoride is usually preferable because of ease and high yields of
the chemical reactions involved and the high chemoselectivity of
the radiolabelling process. The peptide or protein is normally pre-
labelled with the reactive moiety at a specific position not involved
in a crucial biological role. The radiolabelled prosthetic group is
then linked to the reactive moiety in a chemoselective manner.
Several prosthetic groups such as amino reactive acylation agents
(e.g. N-succinimidyl 4-[18F]fluorobenzoate [18F]SFB), thiol reactive
succinamides (e.g. 4-[18F]fluorobenzaldehyde-O-[6-(2,5-dioxo-
2,5-dihydro-pyrrol-1-yl)-hexyl]oxime [18F]FBAM and 4-[18F]fluor-
obenzaldehyde-O-(2-ethyl)oxime [18F]FBOM) and amino or ami-
nooxy reactive benzaldehydes (e.g. 4-[18F]fluorobenzaldehyde
As previously reported for this reaction, 4-(N,N-dimethylami-
no)benzaldehyde 3 was formed as a by-product [17]. Although the
side reaction has been suggested to occur via
a reverse
Menschutkin reaction with fluoride [18], the amount of 4-(N,N-
dimethylamino)benzaldehyde in this case greatly exceeded the
available amount of fluoride in the reaction mixture. Therefore we
propose that other anions such as triflate or carbonate anions must
be involved. The 4-(N,N-dimethylamino)benzaldehyde 3 could not
be separated from [18F]FBA (2) in the SPE workup (Fig. 1). We tried
to trap the byproduct 3 (estimated pKa ꢁ3.5, i.e. 2 pH units higher
than the pH of the eluent) on a strong cationic exchange cartridge
(Phenomenex SCX) eluting with PBS adjusted to pH 1.5 in order to
protonate the dimethylaniline while keeping the solid phase
bound sulphonic acid (estimated pKa <1) in its deprotonated state.
However, while reducing the amount of byproduct, under these
conditions also 50% of the desired [18F]FBA (2) was trapped and
further attempts were abandoned.
[
18F]FBA) have been reported and extensively used over the last
25 years [13].
In the present study, the [18F]fluorobenzoimino polyethylene-
glycol (PEG) functionalised cyclo(Nal-Gly-
peptide [18F]CCIC-0007 was planned to be radiosynthesised from
D-Tyr-Orn-Arg) penta-
the corresponding aminooxy PEG functionalised cyclo(Nal-Gly-D-
Tyr-Orn-Arg) pentapeptide 4 by conjugation with 4-[18F]fluor-
obenzaldehyde ([18F]FBA, 2).
Design of [18F]CCIC-0007 was based on the potent cyclic
pentapeptide CXCR4 inhibitor FC131 reported by Fujjii et al. in
2003(Scheme 1)[5b]. PeptideFC131inhibited[125I]SDF-1binding to
CXCR4 transfected Chinese hamster ovary (CHO) cells with an IC50 of
4 nM. As Arg4 in the original peptide could be exchanged for several
non-natural amino acids without loss of potency, it also seemed
reasonable to modify that position for radiolabelling. Among the
many cyclic pentapeptide analogues reported we found the short
chained Orn4-analogue (IC50 = 19 nM) especially interesting as it
could easily be extended with a linker and a prosthetic group [14].
When incorporated into molecules, polyethyleneglycol (PEG)
chains are known to enhance hydrophilicity and reduce nonspe-
cific binding and generally also reduces hepatic metabolism. We
hypothesised that the introduction of a PEG chain at Orn4 would
counteract the increased lipophilicity introduced by the prosthetic
group, [18F]FBA. Finally the terminal aminooxy functionality linked
to the PEG chain would allow rapid reaction with [18F]FBA (2) to
form an oxime.
The radiosynthesis of [18F]CCIC-0007 was accomplished by
conjugation of the cyclopentapeptide aminooxy precursor 4 and
[
18F]FBA (Scheme 3).
A
sample of nonradioactive peptide CCIC-0007 was also
synthesised and characterised for analytical purposes. Precursor 4
was reacted with 4-fluorobenzaldehyde in methanol and ammoni-
um formate buffer at pH 2.5. The final peptide CCIC-0007 was
characterised by mass spectrometry and used as reference
compound for the radiosynthesis of [18F]CCIC-0007 (Scheme 3).
[
18F]FBA has been previously synthesised and purified using a
range of protocols largely involving displacement of a nitro group
or of a trimethylammonium group by [18F]fluoride. The charged
trimethylanilinium triflate precursor has the advantage over the
Scheme 2. (a) [18F]KF/[2.2.2.]kryptand/KHCO3, Me2SO, MW 15 s, 50 W, 80 8C.

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O. Aberg et al. / Journal of Fluorine Chemistry 135 (2012) 200–206
202
the biomolecule. This effect, named a-effect, makes the aminooxy
group chemoselective over amino bearing amino acids such as Lys
or Arg. In addition, the resulting oxime is stable under physiologi-
cal conditions. Formation of both the cis and trans oximes is of
minor consequence as this portion of the molecule is not involved
in the binding interaction with CXCR4.
As the radiochemical yield of a bioconjugation usually depends
on temperature, peptide concentration, pH and reaction time, a
focused screening for the best conditions was performed. Different
reaction conditions are reported in Table 1. While mild conditions
are generally preferred in peptide labelling we found that the
precursor was dissolving very slowly at 20 8C in the reaction
mixture. Thus, the optimal conditions were found to be heating in
MeOH and ammonium formate (AMF) buffer (pH = 2.0) at 80 8C for
20 min (entry b, Table 1), giving [18F]CCIC-0007 from [18F]FBA in
satisfactory decay corrected radiochemical yield 23 ꢀ 8% (n = 13).
The identity of the tracer [18F]CCIC-0007 was confirmed by co-elution
with the equivalent 19F-peptide.
It should be noted that the by-product 3 competed with
[
18F]FBA 2 in the conjugation reaction and the conjugated peptide
5 could not be separated from [18F]CCIC-0007 by HPLC (Fig. 2B, UV
trace, tR = 12:50 min) and both products could be detected in the
final formulation by LC–MS (m/z 1012.45 and 1037.50). This
affected the apparent specific activity (ratio of radioactivity to the
total amount of peptide) which varied in the range of 2–
60 GBq
m .
molꢂ1
Fig. 1. Analytical radio and UV chromatograms [10% ! 80% MeCN (0.1% TFA) in
water (0.1% TFA) in 10 min then 80% MeCN (0.1% TFA) in water (0.1% TFA),
1 mL minꢂ1] for the SPE purified [18F]FBA (2).
The major microspecies of [18F]CCIC-0007 at physiological pH is
assumed to be the monoprotonated form where the side chain of
Arg5 is protonated (estimated pKa ꢁ12). The distribution coeffi-
cient at physiological pH (Log D7.4) was assessed by evaluating the
distribution of radioactivity in the biphasic octanol/PBS system
using the ‘‘shake flask’’ method and Log D7.4 was 1.09. While many
successful brain imaging tracers have a Log P of 0.9–2.5 [20], no
such guidelines yet exist for imaging probes for somatic tissue,
When [18F]FBA reacts with an aminooxy moiety to form an
oxime (Scheme 3) [19], the overlap of the lonepairs of the
neighbouring oxygen raises the HOMO energy and renders the
aminooxy moiety more nucleophilic compared to amines within
Scheme 3. (a) 4-[18F]FBA or FBA, MeOH, ammonium formate buffer pH 2.0, 80 8C, 20 min.

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O. Aberg et al. / Journal of Fluorine Chemistry 135 (2012) 200–206
203
Table 1
Conjugation of [18F]FBA with 4.
Entry
Solvents
pH
Temp (8C)
Time (min)
Analytical RCY (%)a
a
b
c
MeOH, AMF buffer
MeOH, AMF buffer
MeOH, AMF buffer
MeOH, AMF buffer
2.5
2.0
2.0
2.0
80
80
20
20
20
20
20
5
61
79
71
47
d
a
Determined by analytical UV–radio–HPLC. [10% ! 80% MeCN (0.1% TFA) in water (0.1% TFA) in 10 min then 80% MeCN (0.1% TFA) in water (0.1% TFA), 1 mL minꢂ1].
Fig. 2. (A) UV and radiochromatograms for the semipreparative HPLC purification of [18F]CCIC-0007. (B) Analytical UV and radiochromatograms for the determination of
radiochemical purity, concentration and specific activity for [18F]CCIC-0007.
however, it is reasonable to assume that a similar or lower range
would be suitable to permit relatively fast kinetics in vivo in
keeping with the half-life of the radionuclide. The experimentally
determined value is therefore assumed to be within the optimal
range.
blood through both biliary and renal elimination routes. Between 5
and 15 min post-injection, high levels of intestinal radioactivity
were primarily detected within the duodenum, whereas at 30 and
60 min post-injection this had progressed to the jejunum. The high
levels of radioactivity within the gallbladder and very low
accumulation within the stomach appears to confirm the bile as
the entry point for intestinal [18F]CCIC-0007. High levels of
radioactivity present within the urine suggest a rapid clearance
from kidneys. The low levels of radioactivity in bone suggest little
or no defluorination of the radiolabelled compound. Low activity
was also detected within the lungs. The injected dose per gram
An IC50-value of [18F]CCIC-0007 was determined to 0.80
mM in
an 125I-SDF-1 competition assay using a metastatic breast cancer
cell line MDA-MB-231. To ascertain the radiotracer pharmacoki-
netics, an in vivo biodistribution study was performed in female
BALB/c mice. As shown in Fig. 3, the tissue biodistribution were
characterised with rapid clearance of [18F]CCIC-0007 from the
Fig. 3. In vivo biodistribution of [18F]CCIC-0007 in various tissues at 5, 15, 30 and 60 min following 3.7 MBq (100
mCi) i.v. injection via tail cannulae [n = 3]. Error bars denote
SEM.

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O. Aberg et al. / Journal of Fluorine Chemistry 135 (2012) 200–206
204
(%ID/g) values for the analysed tissues are reported in Table S1
(Supplementary information). Compared to other tracers [10d] the
rapid clearance from the blood could translate into a lower level of
nonspecific binding. The low activity in lung is an encouraging
result because it potentially enables the detection of CXCR4 related
metastases in this particular tissue. However, the little evidence of
and (B) acetonitrile. Gradient 20–30% B in 15 min. Flow rate
4.0 mL minꢂ1. Analytical UV-radio-HPLC was carried out as above
but using a Bioscan Flowcount FC3200 sodium iodide/PMT gamma
detector (Lablogic), a Thermo Separation Products Spectra System
UV1000 (wavelength 254 nm), and a Phenomenex Luna C18 3
m
50 mm ꢃ 4.6 mm column with a column guard cartridge. Injection
[
18F]CCIC-0007 uptake in tissues that endogenously express CXCR4
loop 20 mL. Mobile phase (A) 0.1% TFA in water, (B) 0.1% TFA in
such as the spleen and bone marrow [21] is of our concern as this
suggests a non satisfactory level of specific binding.
acetonitrile. Gradient 5–50% B in 15 min. Flow rate 1 mL minꢂ1
.
4.2. Chemistry
3. Conclusions
4.2.1. (4-Formylphenyl)trimethylammonium
We have successfully radiosynthesised [18F]fluorobenzoimino
trifluoromethansulfonate 1
PEG functionalised cyclo(Nal-Gly-
D
-Tyr-Orn-Arg) pentapeptide
(4-Formylphenyl)trimethylammonium trifluoromethansulfo-
nate 1 [15] was synthesised as reported in the literature with
minor modifications. Methyl trifluoro-methanesulfonate (1.81 g,
11.0 mmol) was added to a to a septum-equipped vial containing a
ice-cold solution of N,N-dimethylamino benzaldehyde (1.48 g,
10.0 mmol) in dry dichloromethane (10 mL). The reaction mixture
was stirred under N2 and allowed to warm to r.t. and after approx.
90 min a white precipitate was formed. After 3 h the precipitate
was filtered off and rinsed repeatedly with cold dry dichlor-
omethane and dried under vacuum to yield the crude product as a
white solid material. The crude product was repeatedly triturated
in refluxing dichloromethane until the essentially pure product
was obtained as a white powder (0.923 g, 29%). The product was
kept in the dark at 4 8C until used for further experiments. 1H NMR
([18F]CCIC-0007), potential tracer for CXCR4. The radiosynthesis
was accomplished by bioconjugation of the aminooxy PEG
functionalised cyclo(Nal-Gly-D-Tyr-Orn-Arg) pentapeptide 4 with
[
18F]FBA in a satisfactory 23 ꢀ 8% dc rcy and 8% EOS yield and took
about 2.5 h from aqueous [18F]fluoride. The initial in vivo biodistribu-
tion of [18F]CCIC-0007 at various time points showed high accumula-
tionof radioactivityinthe eliminationtissuesand anencouraging rapid
blood clearance. The low accumulation of the tracer in lungs could
potentially enable the visualisation of CXCR4 related lung metastases.
However, the little evidence of uptake in tissues that endogenously
express CXCR4 is of our concern because it suggests a low level of
specific binding. In conclusion, the cyclopentapeptidic core structure
appears to be a suitable platform for the potential visualisation of
CXCR4 in vivo, but structural modifications to improve the receptor
binding are required to improve the performance of the tracer and they
are in development in our laboratories.
(Me2SO-d6, 25 8C, 400 MHz) d
10.10 (s, 1 H, CHO), 8.20 (AA0BB0, 2 H,
aromatic), 8.15 (AA0BB0, 2 H, aromatic), 3.65 (s, 3 ꢃ CH3) ppm. 13C
NMR (Me2SO-d6, 25 8C, 100 MHz) 192.3 (CHO), 151.1, 136.9,
131.0, 121.8, 120.7 (q, JCF = 320, CF3), 111.2, 56.5 (3 ꢃ CH3) ppm.
19F NMR (Me2SO-d6, 25 8C, 376 MHz) ꢂ77.79 ppm. Analytical
HPLC, product 1 tR 1:41 min, 4-N,N-dimethylamino benzaldehyde
tR 11:00 min.
4. Materials and methods
4.1. General
The aminooxyfuctionalised peptide 4 (>95% pure by HPLC,
= 230 nm) was purchased from Cambridge Research Biochem-
4.2.2. Cyclo-[Nal-Gly-(
CCIC-0007.
D-Tyr)-Orn(PEG2–O–N55CH-4-F-Ph)-Arg],
l
icals (Billingham, UK). Dichloromethane was dried using
a
Cyclo-[Nal-Gly-(
was dissolved in methanol (400
(0.68 mg, 5.47 mol) was added as a solution in methanol (50
and ammonium formate 10 mM pH 2.5 (450 L) was added to a
D
-Tyr)-Orn(PEG2–O–NH2) (5.5 mg, 6.07
L) and 4-fluorobenzaldehyde
L)
mmol)
PureSolv drying system (Innovative Technology). All other
reagents and solvents were purchased from Sigma–Aldrich
(Gillingham, United Kingdom) and used without further purifica-
tion. 1H NMR spectra were obtained on a Bruker 400 MHz NMR
machine and spectra were referenced to residual solvent (for
CHCl3, 1H 7.26 ppm and 13C 77.16 ppm, for Me2SO-dn 1H 2.50 ppm
and 13C 39.50 ppm). Coupling constants (J) are given in Hertz (Hz).
Mass spectra were obtained in positive electrospray ionisation
mode on a Micromass LCT Premier equipped with a Waters
m
m
m
m
reaction vial and heated to 80 8C for 20 min. The product was
purified using semi-preparative HPLC. The combined fractions
were diluted with water and immobilised on a Waters SepPak tC18
Light cartridge (preconditioned with 5 mL ethanol followed by
10 mL water) and eluted with 5 mL EtOH. Freeze drying yielded the
title compound as a white solid (4.10 mg, 74%). TOF MS ES+ m/z
(rel. intensities) for C50H63FN11O11 [M+H]+: 1012.45 (100%),
1013.45 (58%), 1014.45 (12%). HRMS ES+ [M+H]+m/z calcd for
Atlantis C18 3
m
column 2.1 mm ꢃ 30 mm. Mobile phase (A) water
(0.1% formic acid), (B) acetonitrile. A Metler Toledo pH meter was
used when pH of the ammonium formate solution was buffered
with formic acid. No carrier-added [18F]fluoride was prepared by
the 18O(p,n)18F nuclear reaction on a GE PETtrace cyclotron (GE
Medical Systems, Uppsala, Sweden) with 16.4 MeV proton
irradiation of an enriched [18O]H2O target. For microwave heating
a Resonance Instruments Model 521 (Skokie, IL, USA) equipped
with a single mode transverse electric (TE) cavity and an infrared
thermometer measuring the outside temperature of the reaction
vial was used. For conventional heating a heating block was used
and the temperature was measured in the heating block using a
mercury thermometer. Preparative UV-radio-HPLC was carried out
using a Beckman Pump 127 and Laura 3 software (Lablogic,
Sheffield, UK) equipped with a linear UV-106 detector (wavelength
C
50H63FN11O11 1012.4693, found 1012.4657. 19F NMR (Me2SO-d6,
25 8C) ꢂ110.20 ppm.
4.3. Radiochemistry
4.3.1. 4-[18F]Fluorobenzaldehyde 2
4-[18F]Fluorobenzaldehyde 2 was synthesised according to a
reported method with minor modifications [17]. A Wheaton vial
(3 mL) was charged with KryptofixTM (4,7,13,16,21,24-hexaoxa-
1,10-diazabicyclo[8.8.8]-hexacosane, 5.0 mg, 13
m
mol), potassium
L),
hydrogen carbonate (0.7 mg, 7.2 mol) dissolved in water (50
m
m
acetonitrile (0.50 mL), and 18F–water (0.10–0.40 mL, typically
0.74–1.11GBq, 20–30 mCi). The water was removed azeotropically
at 100 8C using a nitrogen stream (100 mL minꢂ1) for 10–25 min.
Anhydrous acetonitrile (3 ꢃ 0.5 mL) was added and sequentially
evaporated under previous conditions, approximately 3 ꢃ 5 min.
The vial was cooled to room temperature by immersion in a water
254 nm),
(Lablogic) and a Phenomenex Luna C18 5
HPLC column. Injection loop 1000 L. Mobile phase (A) ammoni-
um formate in water (10 mM adjusted to pH 3.5 with formic acid)
a
Bioscan Flowcount FC-3400 PIN diode detector
m
100 mm ꢃ 10 mm
m

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O. Aberg et al. / Journal of Fluorine Chemistry 135 (2012) 200–206
205
bath. A freshly prepared solution of (4-formylphenyl)trimethylam-
monium trifluoromethansulfonate 1 (2.0 mg, 6.4 mol) in anhy-
gamma counter and analysed for 2 min each and the decay
corrected the logarithm of the octanol/water ratio was calculated.
The averaged value with the standard deviation is reported
1.09 ꢀ 0.02 (n = 4).
m
drous Me2SO (0.20 mL) was added immediately before microwave
heating (15 s, 50 W, set temperature = 80 8C). The reaction mixture
was immediately quenched by the addition of water (2 mL) and the
reaction vial was rinsed (3 ꢃ 2 mL). The combined fractions were
taken up into a syringe and loaded onto a Waters C18 SepPak-plus
cartridge (preconditioned with 5 mL methanol followed by 10 mL
water). By this rinsing procedure the remaining radioactivity in the
reaction vial was kept <4%. The cartridge was washed with water
(5 mL) and partially dried with N2 (100 mL minꢂ1) for 2 min. The
cartridge was eluted with methanol, the first 0.50 mL was discarded
and the product fraction was collected in 0.40–0.50 mL methanol.
The recovery of immobilised radioactivity was 85–95%. The decay-
corrected radiochemical yield of 2 were in the range of 42–77%
(61 ꢀ 9%, n = 14, typically 0.40 GBq, 11 mCi), and the procedure was
completed within 60 min including the drying of the 18F-fluoride.
Analytical HPLC, product tR 10:35 min.
4.6. CXCR4 receptor binding assay
Competition binding experiments were performed in MDA-MB-
231 human breast carcinoma cells expressing endogenous levels of
CXCR4. Briefly, cells were harvested using a non-enzymatic cell
dissociation solution and resuspended in binding buffer (PBS with
5 mM MgCl2, 1 mM CaCl2, 0.25% BSA, pH = 7.4). Incubation was
conducted in a final volume of 200
m
L per sample containing
2 ꢃ 105 cells, 0.15 nM [125I]SDF1
a (Perkin Elmer, Cambridge, UK)
and 0–25
mM of the non-radioactive isotopologue CCIC-0007, for
60 min at room temperature. After incubation, cells were spun
three times through 10% sucrose at 5000 rpm-3 min, and
transferred into counting tubes. Cell bound radioactivity was
measured using a gamma counter. Binding results were expressed
as counts/min, and IC50 values were calculated using Prism
software (GraphPad, La Jolla, CA).
4.3.2. Cyclo-[Nal-Gly-(
Arg] [18F]CCIC-0007
D
-Tyr)-Orn(PEG2–O–N55CH-4-[18F]fluoro-Ph)-
The Waters SepPak C18Plus immobilised [18F]fluorobenzalde-
hyde 2 was eluted with methanol (0.40–0.50 mL) directly to a
Wheaton vial (3 mL) containing a freshly prepared suspension of the
4.7. Biodistribution in mice
aminooxy functionalised peptide (1.3 mg, 1.3
mmol) in 100 mM
Female BALB/c mice (Charles River, UK) aged 6–8 weeks were
ammonium formate buffer pH 2.0 and the reaction mixture was
heated undera septum for20 minat 80 8C. The reaction mixturewas
cooled to r.t. in a water bath before venting and diluting with
ammonium formate buffer pH 3.5 (0.35 mL) and injecting to a semi-
preparative HPLC. The purified fraction (tR = 20:15–21:15 min) was
diluted with water (8 mL) and >99% of the radioactivity was
immobilised on a Waters SepPak tC18 Light cartridge (precondi-
tioned with 5 mL ethanol followed by 10 mL water). The cartridge
was washed with water (5 mL) and dried using a stream of nitrogen
(100 mL minꢂ1) for 2 min before the product was eluted with 5 mM
HClin ethanol in 8 ꢃ 0.10 mL fractions and >90% of the radioactivity
was collected in 3 fractions. The decay-corrected radiochemical
yield was 11–35% (22 ꢀ 8%, n = 12, typically 0.11 Gbq, 3 mCi)
calculated from aqueous [18F]fluoride. The labelled product was stable
in the acidic ethanolic solution for at least 5 h at r.t. Before further use
anaesthetised with 2.5% isoflurane and injected by i.v. tail vein
cannulae with ꢁ100
mL of PBS containing 3.7 MBq (100 mCi) of
[
18F]CCIC-0007. The animals were sacrificed at 5, 15, 30 and 60 min
post-injection by cardiac puncture, and the extracted blood
centrifuged at 15,000 rpm for 5 min to separate the plasma from
the cellular blood fraction (n = 3). The animals were then dissected
to remove the tissues (Fig. 3). Urine samples were also collected.
For the measurement of radioactivity in the intestine, a small
section of the jejunum was excised and emptied of its content by
running PBS flows in the lumen. The jejunum epithelium section
was then rapidly dried off using absorbing paper and placed in
tubes for gamma counting.
The decay-corrected counts per minute within the tissues were
measured using a Packard Cobra II gamma counter (Perkin Elmer,
UK) for 1 min, and the counts corrected for sample weight. All
animal experiments were done by licensed investigators in
accordance with the United Kingdom Home Office Guidance on
the Operation of the Animal (Scientific Procedures) Act 1986
(HMSO, London, United Kingdom, 1990) and within guidelines set
out by the United Kingdom National Cancer Research Institute
Committee on Welfare of Animals in Cancer Research [22].
[
18F]CCIC-0007 was neutralised with PBS to <33% ethanol. Analytical
HPLC, tR = 13:09 min, showed a radiochemical purity >99%. A sample
spiked with the reference compound CCIC-0007 was used to confirm
the identity of the radio peak. TOF MS ES+ m/z for C50H62FN11O11
[M+H]+: 1012.45 (100%), 1013.45 (60%), 1014.45(12%). TOF MS ES+ m/z
for 5 C52H68N12O11 [M+H]+: 1037.50 (100%), 1038.50 (62%), 1039.50
(22%), 1040.51 (5%).
Acknowledgments
4.4. Specific activity
This work was funded by Cancer Research UK-Engineering and
Physical Sciences Research Council grant (in association with the
Medical Research Council and Department of Health (England))
grant C2536/A10337. E.O.A.’s laboratory receives core funding
from the UK Medical Research Council (MC_US_A652_0030).
The non-radioactive isotopologue CCIC-0007 was used for a 4
point HPLC–UV-calibration curve 1–16
mM (l = 254 nm). Injection
loop = 20
m
L. [18F]CCIC-0007 was obtained with an apparent
specific activity of 21 ꢀ 18 GBq mmolꢂ1
.
4.5. Log D measurements
Appendix A. Supplementary data
50
18F]CCIC-0007 was diluted with PBS (750
solution (50 L), PBS (450 L) and octanol (500 m
each of 4 Eppendorf tubes and then vortexed for 1 min before
centrifugation at 5000 rpm for 5 min. From the upper octanol layer,
m
L of purified and formulated (5 mM HCl in ethanol)
L). Diluted radioactive
L) was added to
[
m
m
m
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jfluchem.2011.11.003.
300
pipette. The bottom aqueous layer was transferred to a second
Eppendorf tube using a 2.5 mL syringe before pipetting 300 L to a
gamma counting tube. The gamma counting tubes were placed in a
mL was carefully transferred to a gamma counting tube using a
References
m
[1] S. Liekens, D. Schols, S. Hatse, Curr. Pharm. Des. 16 (2010) 3903–39220.
[2] D. Wong, W. Korz, Clin. Cancer Res. 14 (2008) 7975–7980.

˚
O. Aberg et al. / Journal of Fluorine Chemistry 135 (2012) 200–206
206
[3] (a) Z. Liang, T. Wu, H. Lou, X. Yu, R.S. Taichman, S.K. Lau, S. Nie, J. Umbreit, H. Shim,
(b) S. Nimmagadda, M. Pullambhatla, K. Stone, G. Green, Z.M. Bhujwalla, M.G.
Pomper, Cancer Res. 70 (2010) 3935–3944;
Cancer Res. 64 (2004) 4302–4308;
(b) Z. Liang, Y. Yoon, J. Votaw, M.M. Goodman, L. Williams, H. Shim, Cancer Res. 65
(2005) 967–971.
(c) N. Koglin, M. Anton, A. Hauser, D. Saur, H. Algul, R. Schmid, B. Gansbacher, M.
Schwaiger, H.-J. Wester, J. Nucl. Med. 47 (2006) 505P;
[4] (a) G.J. Bridger, R.T. Skerlj, D. Thornton, S. Padmanabhan, S.A. Martellucci, G.W.
Henson, M.J. Abrams, N. Yamamoto, K.D. Vreese, J. Med. Chem. 38 (1995) 366–378;
(b) A. Khan, G. Nicholson, J. Greenman, L. Madden, G. McRobbie, C. Pannecouque, E.
De Clercq, R. Ullom, D.L. Maples, R.D. Maples, J.D. Silversides, T.J. Hubin, S.J.
Archibald, J. Am. Chem. Soc. 131 (2009) 3416–3417;
(d) O. Jacobson, I.D. Weiss, D.O. Kiesewetter, J.M. Farber, X. Chen, J. Nucl. Med. 51
(2010) 1796–1804;
(e) O. Jacobson, I.D. Weiss, L.P. Szajek, G. Niu, Y. Ma, D.O. Kiesewetter, J.M. Farber,
X. Chen, Theranostics 1 (2011) 251–262.
[11] M. Schottelius, H.J. Wester, Methods 48 (2009) 161–177.
[12] J. Becaud, L. Mu, M.n. Karramkam, P.A. Schubiger, S.M. Ametamey, K. Graham, T.
Stellfeld, L. Lehmann, S. Borkowski, D. Berndorff, L. Dinkelborg, A. Srinivasan, R.
Smits, B. Koksch, Bioconjug. Chem. 20 (2009) 2254–2261.
[13] (a) F. Wuest, L. Ko¨hler, M. Berndt, J. Pietzsch, Amino Acids 36 (2009) 283–295;
(b) D.E. Olberg, O.K. Hjelstuen, Curr. Top. Med. Chem. 10 (2010) 1669–1679;
(c) B. Kuhnast, F. Dolle, Curr. Radiopharm. 3 (2010) 174–201.
[14] H. Tamamura, T. Araki, S. Ueda, Z.X. Wang, S. Oishi, A. Esaka, J.O. Trent, H.
Nakashima, N. Yamamoto, S.C. Peiper, A. Otaka, N. Fujii, J. Med. Chem. 48
(2005) 3280–3289.
[15] M.S. Haka, M.R. Kilbourn, G.L. Watkins, S.A. Toorongian, J. Labelled Compd.
Radiopharm. 27 (1989) 823–833.
[16] A. Speranza, G. Ortosecco, E. Castaldi, A. Nardelli, L. Pace, M. Salvatore, Appl.
Radiat. Isot. 67 (2009) 1664–1669.
[17] M. Glaser, E. Arstad, S.K. Luthra, E.G. Robins, J. Labelled Compd. Radiopharm. 52
(2009) 327–330.
[18] H.R. Sun, S.G. DiMagno, J. Fluorine Chem. 128 (2007) 806–812.
[19] (a) M. Namavari, O.P. De Jesus, Z. Cheng, A. De, E. Kovacs, J. Levi, R. Zhang, J.K.
Hoerner, H. Grade, F.A. Syud, S.S. Gambhir, Mol. Imaging Biol. 10 (2008) 177–181;
(b) M. Glaser, M. Solbakken, D.R. Turton, R. Pettitt, J. Barnett, J. Arukwe, H.
Karlsen, A. Cuthbertson, S.K. Luthra, E. Arstad, Amino Acids 37 (2009) 717–724.
[20] (a) M. Laruelle, M. Slifstein, Y. Huang, Mol. Imaging Biol. 5 (2003) 363–375;
(b) R.N. Waterhouse, Mol. Imaging Biol. 5 (2003) 376–389.
[21] B. Furusato, A. Mohamed, M. Uhle´n, J.S. Rhim, Pathol. Int. 60 (2010) 497–505.
[22] P. Workman, E.O. Aboagye, F. Balkwill, A. Balmain, G. Bruder, D.J. Chaplin, J.A.
Double, J. Everitt, D.A.H. Farningham, M.J. Glennie, L.R. Kelland, V. Robinson, I.J.
Stratford, G.M. Tozer, S. Watson, S.R. Wedge, S.A. Eccles, V. Navaratnam, S. Ryder,
Br. J. Cancer 102 (2010) 1555–1577.
(c) E. De Clercq, Nat. Rev. Drug Discov. 2 (2003) 581–587;
(d) V. Bodart, V. Anastassov, M.C. Darkes, S.R. Idzan, J. Labrecque, G. Lau, R.M. Mosi,
K.S. Neff, K.L. Nelson, M.C. Ruzek, K. Patel, Z. Santucci, R. Scarborough, R.S.Y. Wong,
G.J. Bridger, R.T. MacFarland, S.P. Fricker, Biochem. Pharmacol. 78 (2009) 993–1000.
[5] (a) H. Tamamura, M. Imai, T. Ishihara, M. Masuda, H. Funakoshi, H. Oyake, T.
Murakami, R. Arakaki, H. Nakashima, A. Otaka, T. Ibuka, M. Waki, A. Matsumoto, N.
Yamamoto, N. Fujii, Bioorg. Med. Chem. 6 (1998) 1033–1041;
(b) N. Fujii, S. Oishi, K. Hiramatsu, T. Araki, S. Ueda, H. Tamamura, A. Otaka, S.
Kusano, S. Terakubo, H. Nakashima, J.A. Broach, J.O. Trent, Z.X. Wang, S.C. Peiper,
Angew. Chem. Int. Ed. 42 (2003) 3251–3253;
(c) S.J. DeMarco, H. Henze, A. Lederer, K. Moehle, R. Mukherjee, B. Romagnoli, J.A.
Robinson, F. Brianza, F.O. Gombert, S. Lociuro, C. Ludin, J.W. Vrijbloed, J. Zum-
brunn, J.-P. Obrecht, D. Obrecht, V. Brondani, F. Hamy, T. Klimkait, Bioorg. Med.
Chem. 14 (2006) 8396–8404.
[6] (a) C.A. Mosley, L.J. Wilson, J.M. Wiseman, J.W. Skudlarek, D.C. Liotta, Expert Opin.
Ther. Pat. 19 (2009) 23–38;
(b) R. Skerlj, G. Bridger, E. McEachern, C. Harwig, C. Smith, T. Wilson, D. Veale, H.
Yee, J. Crawford, K. Skupinska, R. Wauthy, W. Yang, Y. Zhu, D. Bogucki, M. Di Fluri,
J. Langille, D. Huskens, E. De Clercq, D. Schols, Bioorg. Med. Chem. Lett. 21 (2011)
262–266.
[7] B. Wu, E.Y.T. Chien, C.D. Mol, G. Fenalti, W. Liu, V. Katritch, R. Abagyan, A. Brooun,
P. Wells, F.C. Bi, D.J. Hamel, P. Kuhn, T.M. Handel, V. Cherezov, R.C. Stevens, Science
330 (2010) 1066–1071.
[8] E.M. Rohren, T.G. Turkington, R.E. Coleman, Radiology 231 (2004) 305–332.
[9] D. Le Bars, J. Fluorine Chem. 127 (2006) 1488–1493.
[10] (a) I. Weiss, O. Jacobson, D. Kiesewetter, J. Jacobus, L. Szajek, X. Chen, J.M. Farber,
Mol. Imaging Biol. (2011) 1–9;