A novel 2-cyanobenzothiazole-based 18F prosthetic group for conjugation to 1,2-aminothiol-bearing targeting vectors

Article abstract of DOI:10.1039/c4ob02637c

In a bid to find an efficient means to radiolabel biomolecules under mild conditions for PET imaging, a bifunctional ¹⁸F prosthetic molecule has been developed. The compound, dubbed [¹⁸F]FPyPEGCBT, consists of a 2-substituted pyridine moiety for [¹⁸F]F^- incorporation and a 2-cyanobenzothiazole moiety for coupling to terminal cysteine residues. The two functionalities are separated by a mini-PEG chain. [¹⁸F]FPyPEGCBT could be prepared from its corresponding 2-trimethylammonium triflate precursor (100 °C, 15 min, MeCN) in preparative yields of 11% ± 2 (decay corrected, n = 3) after HPLC purification. However, because the primary radiochemical impurity of the fluorination reaction will not interact with 1,2-aminothiol functionalities, the ¹⁸F prosthetic could be prepared for bioconjugation reactions by way of partial purification on a molecularly imprinted polymer solid-phase extraction cartridge. [¹⁸F]FPyPEGCBT was used to ¹⁸F-label a cyclo-(RGDfK) analogue which was modified with a terminal cysteine residue (TCEP·HCl, DIPEA, 30 min, 43°C, DMF). Final decay-corrected yields of ¹⁸F peptide were 7% ± 1 (n = 9) from end-of-bombardment. This novel integrin-imaging agent is currently being studied in murine models of cancer. We argue that [¹⁸F]FPyPEGCBT holds significant promise owing to its straightforward preparation, 'click'-like ease of use, and hydrophilic character. Indeed, the water-tolerant radio-bioconjugation protocol reported herein requires only one HPLC step for ¹⁸F peptide purification and can be carried out remotely using a single automated synthesis unit over 124-132 min.

Full text of DOI:10.1039/c4ob02637c

Organic &
Biomolecular Chemistry
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PAPER
A novel 2-cyanobenzothiazole-based
1
8
F prosthetic group for conjugation to
Cite this: Org. Biomol. Chem., 2015,
3, 3667
1
1,2-aminothiol-bearing targeting vectors†
a
b
a
James A. H. Inkster, Didier J. Colin and Yann Seimbille*
In a bid to find an efficient means to radiolabel biomolecules under mild conditions for PET imaging, a
1
8
18
bifunctional F prosthetic molecule has been developed. The compound, dubbed [ F]FPyPEGCBT, con-
sists of a 2-substituted pyridine moiety for [ F]F− incorporation and a 2-cyanobenzothiazole moiety for
18
1
8
coupling to terminal cysteine residues. The two functionalities are separated by a mini-PEG chain. [ F]-
FPyPEGCBT could be prepared from its corresponding 2-trimethylammonium triflate precursor (100 °C,
1
5 min, MeCN) in preparative yields of 11% ± 2 (decay corrected, n = 3) after HPLC purification. However,
because the primary radiochemical impurity of the fluorination reaction will not interact with 1,2-amino-
18
thiol functionalities, the F prosthetic could be prepared for bioconjugation reactions by way of partial
18
purification on a molecularly imprinted polymer solid-phase extraction cartridge. [ F]FPyPEGCBT was
1
8
used to F-label a cyclo-(RGDfK) analogue which was modified with a terminal cysteine residue
18
(
TCEP·HCl, DIPEA, 30 min, 43 °C, DMF). Final decay-corrected yields of F peptide were 7% ± 1 (n = 9)
from end-of-bombardment. This novel integrin-imaging agent is currently being studied in murine
1
8
Received 19th December 2014,
Accepted 3rd February 2015
models of cancer. We argue that [ F]FPyPEGCBT holds significant promise owing to its straightforward
preparation, ‘click’-like ease of use, and hydrophilic character. Indeed, the water-tolerant radio-bioconju-
18
DOI: 10.1039/c4ob02637c
gation protocol reported herein requires only one HPLC step for F peptide purification and can be
www.rsc.org/obc
carried out remotely using a single automated synthesis unit over 124–132 min.
high temperatures and basic conditions typically required to
Introduction
1
8
−
permit the direct incorporation of [ F]F . Thus, the design of
1
8
Positron emission tomography (PET) is a non-invasive nuclear easy-to- F label small bifunctional prosthetic groups which
1
molecular imaging technique which permits the visualization can be selectively coupled to sensitive biomolecules remains
and quantification of biological and pharmacological processes an active area of research.
in living systems. Successful PET research and diagnosis
requires the development of selective and bio-available targeting [ F]F under ‘classical’ S Ar fluorination conditions. Unlike
2-Substituted pyridines are known to efficiently incorporate
1
8
−
2,3
N
molecules which are labelled with positron-emitting radio- their C6 arene counterparts, they do not require an additional
11
64
68
isotopes. Among the available PET isotopes (e.g. C, Cu, Ga), electron-withdrawing functionality to elicit high reaction
18
18
F stands out, owing to its attractive nuclear properties (t1/2
=
yields. As such, a number of 2-[ F]fluoropyridine prosthetic
1
09.8 min; Emax = 635 keV; 97% positron abundance) and ease compounds have been prepared (Fig. 1). These include
18
18
18
18
of production on medical cyclotrons via the O(p,n) F reaction. [ F]FPyME and [ F]FPyBrA for conjugation to free thiol
4
–6
The exquisite affinity and selectivity of certain peptides, groups.
Unfortunately, the radiosynthetic protocols used to
proteins and oligonucleotides for specific bio-molecular furnish bromoacetamide- and maleimide-bearing compounds
1
8
18
targets has made them attractive PET targeting vectors. such as [ F]FPyME and [ F]FPyBrA are typically complex
However, many biological targeting agents will not tolerate the because the functionalities used for bioconjugation are incom-
1
8
patible with standard nucleophilic [ F]fluorination conditions
1
8
(
K[ F]F-K2.2.2./K
2 3
CO ). Therefore, the conjugating moiety must
a
18
University Hospitals of Geneva, Cyclotron Unit, Geneva, Switzerland.
be introduced after the [ F]fluorination step, to the detriment
of the overall radiosynthetic procedure. [ ]FPyKYNE,
F]FPy5yne, and PEG-[ F]FPyKYNE have been introduced
1
8
7
E-mail: Yann.Seimbille@unige.ch
b
University of Geneva, Centre for BioMedical Imaging, Geneva, Switzerland
18
8
18
9
[
†
Electronic supplementary information (ESI) available: Experimental details for
18/19
18/19
for copper-catalyzed azide–alkyne cycloaddition (CuAAC) to
compounds 4, 6, 7 and 15; UPLC co-injections of [
F]-2 and [
F]-16. See
18
DOI: 10.1039/c4ob02637c
azide-modified targeting agents (Fig. 1). Such F compounds
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1
8
18
afforded [ F]-1, which was subsequently used to F-label a
terminal cysteine-modified dimeric c(RGD) peptide (vide infra)
and bioluminescent Renilla luciferase.
nd
18
We envisioned a ‘2 generation’ CBT-based F prosthetic
that marries the rapid, chemoselective bioconjugation poten-
1
8
tial of this functionality with the [ F]fluorination potential of
a 2-substituted pyridine. The following details our attempts to
1
8
synthesize such a bifunctional molecule, dubbed [ F]FPy-
1
8
PEGCBT ([ F]-2). Peptides bearing the arginine-glycine-
aspartic acid (RGD) motif may serve as ligands for certain
1
4,15
cancer-associated integrin receptors.
Integrin cell surface
receptors play a role in the regulation of carcinogenesis, and
these receptors are found upregulated in a variety of tumour
1
6
18
types. As such, a variety of F-labelled RGD analogues have
been designed and tested in vivo as potential integrin imaging
1
8
agents. An incomplete list of F-RGD preparation strategies
1
8
17
includes: oxime couplings with 4-[ F]fluorobenzaldehyde
1
8
18
18
19
and [ F]FDG;
4
acylation couplings with [ F]SFB
-nitrophenyl 2- F-fluoropropionate ([ F]NFP); CuAAC con-
jugations with [ F]FPyKYNE, (N-(4-[ F]fluorophenyl)pent-
-ynamide, and [ F]F-pentyne; chelation of Al[ F]F; and
tetrazine-trans-cyclooctene ligation. We describe herein the
synthesis of a new F peptide ligand of this class, which can
and
1
8
18
20
1
8
21
18
2
2
18
23
18
24
4
1
8
Fig. 1 Some 2-[ F]fluoropyridine-bearing prosthetic groups.
2
5
1
8
1
8
are highly amenable to Cu-irrelevant systems, but are not good be prepared via conjugation of [ F]FPyPEGCBT to a 1,2-amino-
choices when potential outcomes include Cu binding, toxicity thiol-modified cyclo-(RGDfK) peptide.
or metal-catalyzed degradation.
The coupling of 2-cyanobenzothiazole (CBT) and 1,2-amino-
thiols via chemoselective condensation chemistry adheres to
Results
Non-radioactive small molecule synthesis
1
0
‘click’ criteria. The CBT moiety reacts reversibly with free
thiol groups but condenses rapidly and specifically with
1
1
N-terminal cysteine residues. Both aqueous buffer and mix- As 2-nitro and 2-trimethylammonium triflate moieties are
tures of buffer and organic co-solvent may be used as reaction known to serve as good leaving groups in nucleophilic aro-
1
8
matrices. This ligation strategy has been used to label a matic [ F]fluorination reactions, we undertook the non-radio-
diverse field of bioactive compounds with fluorescent tags, active synthesis of precursors 12 and 14 (Scheme 1). First,
including luciferase protein, cyan florescent protein expressed ethylene glycol was activated toward nucleophilic substitution
1
1,12
26,27
on the surface of live cells, and a 90-mer DNA sequence.
In at both ends by conversion to the tosylated di-ester (4).
012, an F-bearing CBT derivative ([ F]-1) was introduced as Base-mediated displacement of one tosyl group by either
1
8
18
2
1
3
a prosthetic group for PET applications (Fig. 2). In this case, 2-nitro-3-hydroxypyridine (5) or 2-dimethylamino-3-hydroxy-
nucleophilic aliphatic [ F]fluorination of a tosylated precursor pyridine (7) at elevated temperature yielded compounds 8 and
0 respectively. A satisfactory yield of 2-nitro-bearing com-
1
8
8
1
pound 8 could not be achieved by this method. A second coup-
ling reaction with 6′-hydroxy-2-cyanobenzothiazole (11) under
similar conditions installed the second functionality. Nearly
1
9
identical chemistry was used to furnish F standard 2, start-
2
8
ing from 2-fluoro-3-hydroxypyridine
(6; Scheme 1). An
additional methylation step was required to prepare trimethyl-
ammonium triflate salt 14 from 2-dimethylamino pyridine 13.
Non-radioactive synthesis of peptides
1
8
The peptide chosen for radiolabelling with [ F]-2 consisted of
a cyclo-(RGDfK) sequence for integrin targeting which was
modified with a terminal cysteine residue [Cys-PEG-c(RGDfK); 15].
The two components are separated by a PEG2 tethering chain.
The synthesis of peptide 15 is described in the ESI.† Non-
1
9
1
8
13 18
radioactive labelling of 15 with 2 (2 equiv.) to afford
F
Fig. 2
F-bearing benzothiazoles described in Jeon et al. ([ F]-1)
1
8
18
and this work ([ F]-2 and [ F]-3).
peptide standard 16 was carried out by mixing of the two com-
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18
19
Scheme 1 Synthesis of F-labelling precursors 12 and 14, along with F standard 2.
pounds in DMSO at room temperature (3 h sonication, 25 h
Table 1, entry 1 represents a starting point for our investi-
standing), followed by semi-preparative HPLC purification gations, using trimethylammonium triflate precursor 14
1
8
(
LC A, Program 1).
(30 µmol K
researchers reported that non-basic crown ether
1,4,7,10,13,16-hexaoxacyclooctadecane (18-Cr-6) was a superior
2 3
CO , 90 °C, 10 min). During the synthesis of [ F]-
1
,
1
8
Optimizing the radiochemical yield of [ F]-2
1
3
alternative to commonly-used Kryptofix® 2.2.2. (K2.2.2.).
Therefore, we chose to use this phase transfer catalyst as well.
F]F was eluted from Sep-pak light QMA anion exchange
cartridges using mixtures of 18-Cr-6 in acetonitrile (2 mL) and
aqueous K CO (0.2 mL).
When perchlorate anion was used to strip [ F]F from the
QMA cartridge, yields of [ F]-2 by radio-TLC decreased slightly
entry 2 vs. entry 1). Employing DMSO as solvent did not
improve yields, and was accompanied by a 2-fold relative
A series of test reactions were carried out in an attempt to find
1
8
optimized conditions for the preparative synthesis of [ F]-2.
Crude reaction mixtures were assayed by radio-TLC to deter-
mine relative amounts of polar radio-impurities, including
1
8
−
[
1
8
−
18
2
3
[
F]F ; desired product [ F]-2; and non-reactive carboxamide
1
8
−
1
8
side product [ F]-3 (Fig. 3). A description of the general
method can be found in the ESI.† Results are summarized in
Table 1. See Fig. 3 for a representative radio-TLC trace.
1
8
(
increase in impurity 3 (entry 3 vs. entry 1). Also under these
conditions,
90 °C→120 °C) was not found to be beneficial (entry 4 vs.
entry 1). A decrease in base (K CO ) from 30 to 8 µmol was
a
significant
increase
in
temperature
(
2
3
accompanied by a significant increase in product yield, along
with a mitigation of 3 (entry 5 vs. entry 1). An alternative crown
ether was also tested (2,3,11,12-dibenzo-1,4,7,10,13,16-hexaoxa-
1
8
cyclooctadeca-2,11-diene; DB18-Cr-6), with a decrease in [ F]-2
yield observed (entry 6 vs. entry 5). Increasing the concen-
tration of precursor did not appear to have a significant effect
on reaction yield (entry 7 vs. entry 5). When the amount of
K CO employed was increased from 8 to 15 µmol, a signifi-
2
3
1
8
cant decrease in [ F]-2 yield was observed (entry 8 vs. entry 5);
however, this change was deemed essential as [ F]F could
1
8
−
not be efficiently removed from QMA sorbent using the lower
1
8
18
2 3
concentration of K CO .
As expected, the replacement of K2.2.2. for 18-Cr-6 as phase
transfer catalyst resulted in the formation of carboxamide
Fig. 3 Representative radio-TLC of crude
[ F]FPyPEGCBT ([ F]-2)
18
−
18
F F
reaction mixture. Peak 1: [ F]F , R = 0. Peak 3: [ F]-3, R = 0.5. Peak 4:
18
[
F
F]-2, R = 0.6.
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18
a
Table 1 Radio-TLC assessment of trial reactions for the optimization of [ F]-2
1
8
18
18
18
Entry
1
Precursor
PTC
Base (μmol)
T (°C)
Time (min)
[
F]-2 (%)
[
F]-3 (%)
[ F]-2/[ F]-3
14
14
14
14
14
14
14
14
14
14
14
12
12
18-Cr-6
18-Cr-6
18-Cr-6
18-Cr-6
18-Cr-6
DB18-Cr-6
18-Cr-6
18-Cr-6
30
30
30
30
8
8
8
15
15
15
15
14
15
90
90
90
120
90
90
90
90
90
90
10
10
10
10
10
10
10
10
10
15
15
15
15
20
15
20
19
45
23
42
22
8
10
18
45
22
24
9
15
8
70
28
15
1
2.1
0.9
0.5
0.9
1.9
2.6
2.8
2.8
0.1
1.7
3.3
0.5
0.2
b
2
3
c
4
5
6
d
7
8
9
1
1
1
1
K
2.2.2.
0
1
2
3
18-Cr-6
18-Cr-6
18-Cr-6
18-Cr-6
48
49
1
100
100
100
c
3
17
a
Reactions are in MeCN (1 mL) and utilize 3.6–4.0 µmol precursor, 64 μmol 18-Cr-6 as phase transfer catalyst (PTC), and K
2
CO
3
as base, unless
b
c
d
4
noted. KClO used as base. Reaction in DMSO (1 mL). Precursor = 7.3 µmol.
1
8
impurity [ F]-3 as the major product (entry 9 vs. entry 8). For-
1
8
tunately, the radiochemical yield of [ F]-2 in reactions con-
taining 15 µmol K CO could be improved by increasing the
2
3
reaction time from 10 to 15 min (entry 10 vs. entry 8). In the
1
8
end, the highest radiochemical yield and highest ratio of [ F]-
1
8
2
/[ F]-3 was observed when both temperature and time were
increased (entry 11; 15 µmol K CO , 100 °C, 15 min). Attempts
2
3
1
8
were also made to F-label 2-nitro pyridine precursor 12
under these optimized conditions in both MeCN and DMSO
(entries 12 & 13 respectively). In both cases, labelling yields
were very low. In light of these results, and because the separ-
1
8
ation of 12 and [ F]-2 cannot presumably be achieved by solid
phase extraction methods (vide infra), precursor 12 was not
employed for preparative radiosyntheses.
18
18
Preparative synthesis of prosthetic group [ F]FPyPEGCBT ([ F]-2)
18
18
Scheme 2 Preparative synthesis of [ F]FPyPEGCBT ([ F]-2).
1
8
18
18
The preparative syntheses of [ F]-2 and F peptide [ F]-16
were carried out remotely using a Tracerlab FXFN automated
synthesis unit. [ F]FPyPEGCBT was synthesized according to
1
8
18
18
(a) a loss of activity in the form of H[ F]F during [ F]F con-
the conditions as described in Table 1, entry 11, with some centration steps, as a result of minimal added base and the
modifications (Scheme 2). In a bid to further improve the use of suboptimal phase transfer catalyst.
1
8
−
18
efficiency of [ F]F extraction from anion exchange sorbent
(b) the hydrolysis of [ F]-2 or precursor 14 to their carbox-
without increasing K CO mass, QMA light anion exchange amide side-products under the conditions required to facilitate
2
3
1
8
18
columns were replaced with ORTG ‘ F trap-and-release’ [ F]fluorination.
columns, which can be eluted with less eluent due to their
For full radio-bioconjugate syntheses, HPLC purification of
smaller size (12.6 mg resin vs. 130 mg for QMA). The car- [ F]-2 was abandoned in favour of solid-phase extraction on
2
9
18
1
8
tridge was extracted with aqueous K
2 3
CO
alone (0.5 mL), while AffiniMIP SPE F molecularly imprinted polymer cartridges.
the phase transfer catalyst in MeCN (2 mL) was added directly This sorbent has been validated for the separation of 4-fluoro-
1
8
to the reactor. The addition and distillation of a second benzaldehyde and ethyl 4-[ F]fluorobenzoate from their
portion of MeCN (2 mL) was employed to further facilitate respective dimethylamino and phenolic side products, as well
1
8
− 30
anhydrous conditions.
as K2.2.2. and free [ F]F . To our knowledge, this sorbent has
1
8
[
F]FPyPEGCBT could be obtained chemically and radio- not been previously reported for the purification of trimethyl-
chemically pure (>98% by radio-TLC) after HPLC purification ammonium triflate-bearing pyridines. Product application
1
8
(
LC C, Program 3). Product identity was verified by co-injection notes describe the effective elution of AffiniMIP® SPE F car-
1
8
of F prosthetic group with non-radioactive standard (see tridges with MeCN. This solvent is not, unfortunately, amen-
ESI†). Decay-corrected, collected yield was 11% ± 2 (n = 3). able to many bioconjugation reactions, including this one.
Apart from intrinsic losses due to HPLC, low preparative yields However, we observed no evidence of precursor 14 in samples
were also attributed to:
analyzed by UPLC-MS when a ‘0.7 mL’ size column was eluted
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with 1 mL DMF. Extraction efficiency of radioactivity was 60% DMF in the presence of TCEP·HCl (2 equiv.) and DIPEA
decay-corrected), which is consistent with the removal of all (12 equiv.). Choice of base, solvent and reducing agent were
(
1
8
non-polar F species from the cartridge, as estimated by radio- based on a non-radioactive 2-cyanobenzothiazole/1,2-amino-
3
1
TLC. The advantages realized through the use of AffiniMIP® thiol condensation reaction reported earlier. Non-automated,
1
8
−1
SPE F technology are significant in this case-namely, the trial reactions at room temperature with 1 mg mL precursor
obviation of a time-consuming HPLC step and a significant sim- peptide 15 afforded near-total bioconjugation yields (30 min,
plification of the overall radiochemical protocol.
sonication). The assumption in this case is that the major
1
8
radiochemical impurity ([ F]-3), which does not contain a 2-
1
8
1
8
18
cyano moiety, will not interfere with the coupling of [ F]-2
and 15. Indeed, analytical radio-UPLC of such a reaction
mixture before and after addition of peptide precursor suggests
Preparative synthesis of F peptide [ F]-16
1
8
18
18
F peptide [ F]-16 was obtained via mixture of F prosthetic
1
8
group [ F]-2 and precursor peptide Cys-PEG-c(RGDfK) (15) in
1
8
that [ F]-3 and other radio-impurities are chemically irrele-
vant under these conditions (Fig. 4).
In a bid to minimize the use of costly peptide precursor
and use techniques more amenable to automated synthesis,
preparative syntheses were carried out with 0.6 mg of 15 in
.4 mL DMF (0.43 mg mL− ) at 43 °C (Scheme 3). Mixing
1
1
was accomplished via intermittent bubbling with argon.
However, under these conditions, bioconjugation yields were
1
8
not quantitative (Fig. 5). Nevertheless, [ F]-16 could be easily
separated from precursor 15 and radioactive impurities using
semi-preparative HPLC (LC C, Program 4). Immobilization and
elution from tC18 sorbent afforded the product peptide in a
final formulation of 10% EtOH in isotonic saline. Product
1
8
identity was verified by co-injection of the F peptide with
non-radioactive standard (see ESI†). Full automated synthesis
of [ F]-16 was reproducibly achieved in a decay-corrected yield
of 7% ± 1% (n = 9) from end-of-bombardment (EOB). Total
1
8
Fig. 4 Radio-UPLC traces of partially purified [ F]-2 before (top) and
1
8
−1
after (bottom) incubation with 15 (1 mg mL of peptide, sonication,
0 min). LC B, Program 2. [ F]-2 = 4.4 min. [ F]-3 = 3.6 min. [ F]-16 =
18
18
18
3
3
18
18
.3 min. Percent yield by HPLC of [ F]-16 relative to intact F prosthetic synthesis time was 124–132 min from EOB. The effective
1
8
is 97% (63% relative to total radioactivity).
specific activity of [ F]-16 prepared at our site was estimated
18
Scheme 3 Radiosynthesis of cyclo-(RGDfK) peptide analogue [ F]-16.
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1
8
18
18
18
Fig. 5 Radio-HPLC trace of [ F]-16 reaction mixture. LC C, Program 4. [ F]-16 = 22.5 min. [ F]-3 = 25.2 min. [ F]-2 = 27.6 min. % yield by
1
8
18
HPLC of [ F]-16 relative to intact [ F]FPyPEGCBT is 74% (40% relative to total radioactivity).
to be 4–12 GBq µmol− (n = 8) based on the amount of UV-
absorbing material (320 nm) co-eluting with radio-product as
1
Conclusion
1
8
determined by mass standard curve. It is hypothesized that 2-Cyanobenzothiazole-bearing [ F]FPyPEGCBT was invented
1
8
effective specific activities and radio-bioconjugation yields for the F-labelling of potential biological PET imaging agents
could be improved by decreasing the mass of precursor 14 by way of a mild, water-compatible ‘click’ conjugation reaction
1
8
18
used for the preparative synthesis of [ F]FPyPEGCBT (6 mg in with 1,2-aminothiol groups. This novel F prosthetic was pre-
2
1
8
mL MeCN), as this would presumably decrease the amount pared in a single radiochemical step via [ F]fluorination of a
of 2-dimethylamino- and 2-hydroxy-pyridine impurities gener- 2-trimethylammonium pyridine-bearing precursor. Unfortu-
ated during radio-fluorination reactions. nately, the 2-cyanobenzothiazole moiety is sensitive to hydro-
Lipophilicity is thought to have a profound effect on radio- lytic degradation and necessitates the need to use suboptimal
1
8
tracer biodistribution and uptake in vivo; in some cases, the [ F]fluorination conditions, to the detriment of preparative
introduction of a non-polar tag can enhance clearance through radiochemical yields. However, this disadvantage can be par-
3
2
the undesirable hepatobiliary pathway. In the hopes of miti- tially overcome through the use of molecularly imprinted
1
8
18
gating this effect, [ F]-16 was designed with two short ethyl- polymer cartridges to partially purify [ F]FPyPEGCBT prior to
ene glycol (mini-PEG) chains, a modification which has been bioconjugation in an efficient fashion. In this way, a terminal
1
8
reported to reduce overall bio-tracer lipophilicity and improve cysteine-modified c(RGDfK) analogue was labelled with F for
biodistribution profiles for other RGD-based F imaging integrin-based PET imaging. The bioconjugate exhibited
agents. The distribution coefficient (log D7.4) of [ F]-16 in favourable hydrophilicity as required for in vivo imaging appli-
1
8
3
3
18
1
4
-octanol and PBS (pH 7.4) was found to be −1.22 ± 0.02 (n = cations. In vitro receptor binding affinity assays, integrin speci-
1
8
), suggesting that the F peptide is relatively hydrophilic.
ficity assays, and µPET evaluation of this new radiotracer using
We obtained HPLC-purified [ F]FPyPEGCBT in preparative brain and ovarian cancer cell lines are currently underway. As
decay-corrected yields which are inferior to prosthetic group this protocol requires no manual handling, it can be easily
1
8
1
8
18
[
F]-1 (11% vs. ∼20% from EOB respectively). It should be scaled up to produce larger quantities of F radiopharmaceu-
noted however that such a comparison may not be representa- tical if required. The production of proteins and oligo-
1
8
tive of the overall utility of [ F]-2, as this labelling agent can nucleotides bearing terminal cysteine residues has been well
be obtained in functionally useful form after rapid AffiniMIP® established, in large part because such constructs can be used
1
8
34,35
18
SPE purification. By other metrics, [ F]-2 offers advantages in native chemical ligation reactions.
Thus [ F]FPy-
1
8
18
over [ F]-1, including a fully automated labelling protocol on a PEGCBT could prove useful for the F-labelling of these PET
single synthesis unit that includes only one HPLC purification. agent classes as well.
1
8
Finally, we anticipate that the low lipophilicity of [ F]FPy-
PEGCBT might favourably ameliorate biodistribution out-
1
8
comes of F bioconjugates prepared by way of CBT/1,2-
Experimental
Chemicals and media
aminothiol condensation reactions. When Jeon et al. used
1
8
18
[
F]-1 to radiolabel an RGD-based dipeptide ([ F]CBTRGD )
2
for murine PET imaging of human glioblastoma tumours Unless otherwise noted, reagents and solvents were purchased
U87MG), they found increased levels of convoluting radioac- from Sigma-Aldrich (Basel, Switzerland) or VWR (Nyon, Switzer-
(
1
8
tivity in non-target organs relative to an analogous F peptide land) and were used without further purification. Silica gel
labelled with [ F]NFP. The authors suggest that the lipo- (40–63 µm) for flash chromatography was obtained from Silicycle
philic nature of [ F]-1 was responsible for this effect.
1
8
13
1
8
18
(Quebec City, Canada). ‘ F trap-and-release’ SPE anion-exchange
3672 | Org. Biomol. Chem., 2015, 13, 3667–3676
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Paper
cartridges and QMA Plus light anion exchange cartridges were were obtained on an ESI/nanoESI-IT Esquire 3000 plus instrument
obtained from ORTG (Oakdale, USA) and Waters (Baden-Dättwil, (Bruker; Fällanden, Switzerland).
Switzerland) respectively. tC18 light cartridges were purchased
18
Non-radioactive synthesis
from Waters. AffiniMIP® SPE F cartridges (‘0.7 mL’ size) were
obtained from PolyIntell (Val-de-Reuil, France).
2-(2-((2-Nitropyridin-3-yl)oxy)ethoxy)ethyl
tosylate
(8).
2
-Nitro-3-hydroxypyridine (7; 499 mg, 3.56 mmol), diethylene
Chromatography
glycol di(p-toluenesulfonate) (4; 2.96 g, 7.14 mmol), and pow-
TLC was performed on pre-coated silica gel 60F254 aluminum dered K CO (500 mg, 3.62 mmol) were added to a dry round-
2
3
sheets from VWR. The compounds were visualized under ultra- bottomed flask as powders. Dry MeCN (40 mL) was added, and
violet light at 254 nm or 365 nm, or by brief immersion in nin- the resulting slurry was refluxed under Ar for 1.5 h. After
hydrin-collidine, followed by heating. A Cyclone Plus Phosphor cooling to RT, the reaction mixture was poured into 0.05 M
Imager (PerkinElmer, Waltham, USA) with OptiQuant software HCl (100 mL) and extracted three times into ethyl acetate. The
was used for radioactive detection.
organic portions were washed once with brine (30 mL), dried
Liquid chromatography (LC) A. HPLC for semi-preparative over Na SO , and concentrated. The product, an oil, was par-
2
4
use. UltiMate 3000 Rapid Separation LC system (Dionex, Basel, tially extracted from insoluble powders by addition of 6 : 4
Switzerland). The UV detector was set at 220 nm, 254 nm, and ethyl acetate–hexanes. After removal and concentration of the
3
20 nm. The column used was a Phenomenex Luna 5 μm C18 solvent phase, the crude residue was purified by silica gel flash
PFP(2) 100 Å (250 × 10 mm). Program 1: flow rate = 3 mL chromatography (6 : 4 ethyl acetate–hexanes) to yield 253 mg
−
1
1
min . Gradient elution: 5% MeCN in water containing 0.1% (19%) of 8. H NMR (300 MHz, DMSO-d
trifluoroacetic acid (TFA) to 65% MeCN in water containing 3.58–3.65 (m, 2H), 3.65–3.73 (m, 2H), 4.06–4.15 (m, 2H),
.1% TFA over 20 min, then 100% MeCN for 10 min. 4.24–4.33 (m, 2H), 7.38–7.48 (m, 2H), 7.71–7.80 (m, 3H), 7.95
LC B. Ultra performance liquid chromatography (UPLC) for (dd, J = 8.5, 1.1 Hz, 1H), 8.11 (dd, J = 4.5, 1.1 Hz, 1H). C NMR
6
) δ 2.38 (s, 3H),
0
1
3
liquid
chromatography-mass
spectroscopy
(LC-MS). (75 MHz, DMSO-d ) δ 21.09 [CH ], 68.07 [CH ], 68.42 [CH ],
6
3
2
2
ACQUITY® UPLC, H Class (Waters, Baden-Dättwil, Switzer- 69.22 [CH
2
2
], 69.91 [CH ], 125.42 [CH], 127.61 [2 × CH], 129.42
land). The UV detector was set at 220 nm, 254 nm, and [CH], 130.09 [2 × CH], 132.34 [C], 139.34 [CH], 144.89 [C],
20 nm. Radioactivity was detected with a Flow-Ram Radio- 146.07 [C], 148.52 [C]. HR-MS calcd for C H N O S: 383.0908
3
1
6
19 2 7
+
HPLC detector (LabLogic, Sheffield, UK). In-line low resolution [M + H] . Found: 383.0909.
electrospray ionization mass spectroscopy (ESI-MS) was 6-(2-(2-((2-Nitropyridin-3-yl)oxy)ethoxy)ethoxy)benzo[d]thi-
obtained using a Waters ACQUITY® TQ detector. The UPLC azole-2-carbonitrile (12). Tosylated compound 8 (232 mg,
column used was an ACQUITY UPLC HSS T3 1.8 µm (2.1 × 0.607 mmol) and 2-cyano-6-hydroxybenzothiazole (11, 129 mg,
−
1
5
00 mm). Program 2: flow rate = 0.4 mL min . Gradient 0.733 mmol) were added together in a RBF and partially dis-
elution: 5% MeCN in water containing 0.1% formic acid to solved in anhydrous MeCN (25 mL). To this mixture was added
1
1
00% MeCN containing 0.1% formic acid over 5 min, then
00% MeCN containing 0.1% formic acid for 5 min.
K
2
CO
3
(169 mg, 1.22 mmol). A condenser was affixed and the
reaction was heated to reflux, under argon. After 3 h, the reac-
LC C. Radio-HPLC for preparative use. PU-2089 Plus Qua- tion was cooled to RT and diluted with ethyl acetate (50 mL).
ternary Pump (JASCO, Schlieren, Switzerland). The UV detector The reaction mixture was filtered and the filtered solids were
Knauer WellChrom K-2001 Filter Photometer; Basel, Switzer- washed generously with ethyl acetate. The filtrate was concen-
land) was set to 254 nm. The native TracerLab FXFN NaI detec- trated and the residue purified on a flash column of silica
tor and software was used. The column used was (7 : 3 ethyl acetate–hexanes) to afford 12 (184 mg, 78%) as an
(
a
1
Phenomenex Luna 5 μm C18 100 Å (250 × 10 mm). Program 3: off-white powder. M.P. = 111–112 °C. H NMR (300 MHz,
flow rate: 3 mL min− . Isocratic elution: 65 : 35 MeCN–H O. DMSO-d ) δ 3.78–3.91 (m, 4H), 4.16–4.26 (m, 2H), 4.34–4.44
1
2
6
−
1
Program 4: flow rate: 3 mL min . Gradient elution: 5% MeCN (m, 2H), 7.29 (dd, J = 9.1, 2.5 Hz, 1H), 7.73 (dd, J = 8.5, 4.5 Hz,
in water containing 0.1% TFA to 65% MeCN in water contain- 1H), 7.86 (d, J = 2.5 Hz, 1H), 7.98 (dd, J = 8.5, 1.2 Hz, 1H), 8.09
ing 0.1% TFA over 20 min, then 100% MeCN for 10 min.
13
(dd, J = 4.5, 1.2 Hz, 1H), 8.12 (d, J = 9.1 Hz, 1H). C NMR
75 MHz, DMSO-d ) δ 68.02 [CH ], 68.61 [CH ], 68.83 [CH ],
9.35 [CH ], 105.13 [CH], 113.62 [C], 118.80 [CH], 125.28 [CH],
(
6
6
2
2
2
NMR
2
NMR spectra were recorded with a Varian Gemini 2000 NMR 125.46 [CH], 129.39 [CH], 133.67 [C], 137.54 [C], 139.31 [CH],
Spectrometer with a 300 MHz Oxford magnet (Oxfordshire, 146.11 [C], 146.22 [C], 148.58 [C], 159.03 [C]. HR-MS calcd for
+
UK). NMR solvents were obtained from Cambridge Isotope
C
17
H
15
N
4
O
5
S: 387.0758 [M + H] . Found: 387.0755.
Laboratories (Burgdorf, Switzerland). Chemical shifts (δ) are
2-(2-((2-Fluoropyridin-3-yl)oxy)ethoxy)ethyl
tosylate
(9).
reported in ppm relative to the hydrogenated residue of the 2-Fluoro-3-hydroxypyridine (6; 1.01 g, 8.90 mmol) and diethy-
deuterated solvents.
lene glycol di(p-toluenesulfonate) (4; 3.66 g, 8.83 mmol) were
added to a dry flask as powders, under Ar. The compounds
were dissolved in dry MeCN (50 mL) and powdered K CO
2 3
Mass spectroscopy (MS)
Low-resolution MS was carried out on the UPLC-MS apparatus (2.42 g, 17.5 mmol) was added. The mixture was refluxed
described above (see LC B, Program 2). High resolution MS spectra under Ar for 2.5 h, then cooled to RT and filtered. The filtrate
This journal is © The Royal Society of Chemistry 2015
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was concentrated and purified on a flash column of silica gel
6-(2-(2-((2-(Dimethylamino)pyridin-3-yl)oxy)ethoxy)ethoxy)-
(
9 : 1 CH Cl –ethyl acetate, then 4 : 1 CH Cl –ethyl acetate) to benzo[d]thiazole-2-carbonitrile (13). 6-Hydroxybenzothiazole-
2 2 2 2
1
afford 1.62 g (52%) of 9 as an oil. H NMR (300 MHz, DMSO- 2-carbonitrile (11, 399 mg, 2.27 mmol) was partially dissolved
d
5
0
1
6
) δ 2.38 (s, 3H), 3.65 (dd, J = 5.2, 3.6 Hz, 2H), 3.70 (dd, J = in a solution of 2-(2-((2-dimethylaminopyridin-3-yl)oxy)ethoxy)-
.2, 3.6 Hz, 2H), 4.20–4.09 (m, 4H), 7.28 (ddd, J = 8.0, 4.8, ethyl tosylate (10; 656 mg, 1.72 mmol) in dry MeCN (45 mL).
.8 Hz, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.63 (ddd, J = 10.6, 8.0, The flask was charged with Ar and powdered K CO (308 mg,
.5 Hz, 1H), 7.75–7.71 (m, 1H), 7.80–7.75 (m, 2H). C NMR 2.23 mmol) was added. The flask was affixed with a condenser
2
3
1
3
(
75 MHz, DMSO-d ) δ 21.09 [CH ], 68.02 [CH ], 68.23 [CH ], and the reaction mixture was heated to reflux, under Ar. The
6
3
2
2
6
8.55 [CH
2
2
], 69.95 [CH ], 122.68 [d, J = 3.8 Hz, CH], 123.71 [d, reaction mixture became bright yellow and insolubles formed.
J = 4.2 Hz, CH], 127.63 [2 × CH], 130.10 [2 × CH], 132.35 [C], After 4 h, the reaction was cooled to RT and filtered through a
36.87 [d, J = 13.5 Hz, CH], 141.55 [d, J = 25.4 Hz, C], 144.89 short plug of Celite, washing with MeCN (80 mL). The concen-
C], 152.68 [d, J = 235.3 Hz, C]). HR-MS calcd for C16 19FNO S: trated residue was purified on a flash column of silica gel (7 : 3
1
[
3
H
5
+
1
56.0963 [M + H] . Found: 356.0965.
2 2
CH Cl –ethyl acetate) to afford 13 (628 mg, 95%). H NMR
6
-(2-(2-((2-Fluoropyridin-3-yl)oxy)ethoxy)ethoxy)benzo[d]thi- (300 MHz, CD Cl ) δ 2.95 (s, 6H), 4.00–3.90 (m, 4H), 4.27–4.19
2
2
azole-2-carbonitrile (2). 6-Hydroxybenzothiazole-2-carbonitrile (m, 2H), 4.17–4.08 (m, 2H), 6.70 (dd, J = 7.8, 4.9 Hz, 1H), 7.01
11, 936 mg, 5.31 mmol) was added to a round-bottomed flask (dd, J = 7.8, 1.5 Hz, 1H), 7.25 (dd, J = 9.2, 2.4 Hz, 1H), 7.40 (d,
containing 2-(2-((2-fluoropyridin-3-yl)oxy)ethoxy)ethyl tosylate J = 2.4 Hz, 1H), 7.79 (dd, J = 4.9, 1.5 Hz, 1H), 8.07 (d, J = 9.2 Hz,
(
13
(
9; 1.57 g, 4.41 mmol) and the reagents were partially dissolved 1H). C NMR (75 MHz, DMSO-d
in MeCN (80 mL). The flask was charged with argon and pow- 68.06 [CH ], 68.69 [CH ], 68.97 [CH
dered K CO (1.22 g, 8.80 mmol) was added. The flask was 115.23 [CH], 118.77 [CH], 118.96 [CH], 125.31 [CH], 133.68
6
) δ 40.37 [2 × CH
3 2
], 67.37 [CH ],
2
2
2
], 105.14 [CH], 113.61 [C],
2
3
affixed with a condenser and the reaction mixture was heated [C], 137.56 [C], 138.15 [CH], 144.88 [C], 146.21 [C], 152.22 [C],
+
to reflux, under argon, for 3 h. The reaction was cooled to RT 159.08 [C]. HR-MS calcd for C19
and filtered, washing generously with MeCN. The concentrated Found: 385.1332.
21 4 3
H N O S: 385.1329 [M + H] .
residue was purified on a flash column of silica gel (6 : 4 ethyl
3-(2-(2-((2-Cyanobenzo[d]thiazol-6-yl)oxy)ethoxy)ethoxy)-
trifluoromethane-
(14). 6-(2-(2-((2-(Dimethylamino)pyridin-3-yl)oxy)-
m, 4H), 4.30–4.19 (m, 4H), 7.26 (ddd, J = 8.0, 4.8, 0.6 Hz, 1H), ethoxy)ethoxy)benzo[d]thiazole-2-carbonitrile (13; 619 mg,
acetate–hexanes) to afford 2 (1.26 g, 79%) as a white powder. N,N,N-trimethylpyridin-2-aminium
1
M.P. = 112–114 °C. H NMR (300 MHz, DMSO-d ) δ 3.92–3.82 sulfonate
6
(
7
1
8
δ
1
1
1
2
.31 (dd, J = 9.1, 2.5 Hz, 1H), 7.65 (ddd, J = 10.5, 8.0, 1.5 Hz, 1.61 mmol) was dissolved in anhydrous toluene (5 mL) and
H), 7.71 (dt, J = 4.8, 1.5 Hz, 1H), 7.87 (d, J = 2.5 Hz, 1H), the flask was charged with argon. The reaction was cooled to
1
3
.11 (d, J = 9.1 Hz, 1H). C NMR (75 MHz, DMSO-d °C, then methyl trifluoromethanesulfonate (0.22 mL,
6
)
0
68.02 [CH ], 68.38 [CH ], 68.77 [CH ], 68.81 [CH ], 1.94 mmol) was added dropwise via syringe. After a few
2
2
2
2
05.16 [CH], 113.61 [C], 118.78 [CH], 122.64 [d, J = 4.2 Hz, CH], moments, a yellow gel precipitated out of solution. The reac-
23.71 [d, J = 4.3 Hz, CH], 125.30 [d, J = 3.2 Hz, CH], tion was stirred at 0 °C for 15 min total, after which stirring
33.66 [C], 136.83 [d, J = 13.3 Hz, CH], 137.54 [C], 141.59 [d, J = was stopped and the reaction solvent was removed by glass
5.6 Hz, C], 146.21 [C], 152.70 [d, J = 235.2 Hz, C], 159.04 [C]. pipette. The round-bottomed flask was held at 0 °C while the
+
HR-MS calcd for C17
60.0813.
H
15FN
3
O
3
S: 360.0813 [M + H] . Found: precipitate was triturated with two additional portions of dry
3
toluene (2 × 5 mL). Upon removal of solvent in vacuo, the
1
2
-(2-((2-Dimethylaminopyridin-3-yl)oxy)ethoxy)ethyl tosylate product salt solidified (14; 812 mg, 92%). H NMR (300 MHz,
(
3
2
10). 2-Dimethylamino-3-hydroxypyridine
(5;
.08 mmol) and diethylene glycol di(p-toluenesulfonate) (4; 4.28–4.19 (m, 2H), 4.52–4.43 (m, 2H), 7.30 (dd, J = 9.1, 2.5 Hz,
.56 g, 6.17 mmol) were added to a dry flask as powders, 1H), 7.73 (dd, J = 8.3, 4.5 Hz, 1H), 7.87 (d, J = 2.5 Hz, 1H), 7.97
426
6
mg, DMSO-d ) δ 3.62 (s, 9H), 3.93–3.81 (m, 2H), 4.02–3.93 (m, 2H),
under Ar. The compounds were dissolved in dry MeCN (dd, J = 8.3, 1.1 Hz, 1H), 8.14 (d, J = 9.1 Hz, 1H), 8.17 (dd, J =
1
3
(40 mL) and powdered K
2 3 6
CO (423 mg, 3.06 mmol) was added. 4.5, 1.1 Hz, 1H). C NMR (75 MHz, DMSO-d ) δ 53.33 [3 ×
The slurry was refluxed under Ar for 2 h 20 min, then cooled CH ], 68.02 [CH ], 68.14 [CH ], 68.46 [CH ], 68.83 [CH ],
3
2
2
2
2
to RT and filtered through a short plug of Celite. The filtrate 105.13 [CH], 113.62 [C], 118.69 [CH], 125.38 [CH], 125.45 [CH],
was concentrated and purified on a flash column of silica gel 128.37 [CH], 133.80 [C], 137.60 [C], 138.58 [CH], 142.89 [C],
(
7 : 3 CH Cl –ethyl acetate) to afford 698 mg (59%) of 10, as a clear 146.25 [C], 146.98 [C], 158.99 [C]. HR-MS calcd for
2 2
1
+
oil. H NMR (300 MHz, DMSO-d
3
4
6
) δ 2.38 (s, 3H), 2.85 (s, 6H),
.67–3.59 (m, 2H), 3.75–3.67 (m, 2H), 4.05–3.96 (m, 2H),
.17–4.08 (m, 2H), 6.75 (dd, J = 7.8, 4.8 Hz, 1H), 7.14 (d, J = 7.8
20 23 3 4
C H O N S: 399.1485 [M] . Found: 399.1487.
1
3
Radiochemistry
], No-carrier-added [ F]F
7.93 [CH ], 68.77 [CH ], 69.99 [CH ], 115.27 [CH], 118.88 (200–300 μA min) of 2 mL of O-enriched water (>97% pure;
Hz, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.81–7.71 (m, 3H). C NMR
1
8
−
(75 MHz, DMSO-d
6
) δ 21.09 [CH
3
], 40.35 [2 × CH
3
], 67.19 [CH
2
was produced by irradiation
1
8
6
2
2
2
[CH], 127.63 [2 × CH], 130.11 [2 × CH], 132.34 [C], 138.35 [CH], Marshall Isotopes; Tel-Aviv, Israel) using an IBA Cyclone 18
1
44.83 [C], 144.90 [C], 152.31 [C]. HR-MS calcd for MeV cyclotron (Louvain-la-Neuve, Belgium). Total activity pro-
+
C H N O S: 381.1479 [M + H] . Found: 381.1488.
duced was ∼12–18 GBq. Preparative syntheses were carried out
1
8
25 2 5
3674 | Org. Biomol. Chem., 2015, 13, 3667–3676
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Paper
remotely using a Tracerlab FXFN automated synthesis unit (GE
Healthcare, Münster, Germany).
References
1
2
C. Nahmias, Can. Assoc. Radiol., J., 2002, 53, 255–257.
L. Dolci, F. Dolle, S. Jubeau, F. Vaufrey and C. Crouzel,
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1
8
18
Example of preparative radiosynthesis of [ F]FPyPEGCBT ([ F]-2)
1
8
F]F− was extracted from [ O]H
18
[
‘
2
O via immobilization on an
F trap-and-release’ anion-exchange column, then eluted
from the sorbent with a mixture of potassium carbonate
2.1 mg) in water (0.5 mL), into a reactor containing 18-Cr-6
16.8 mg) in dry acetonitrile (2 mL; ABX, Radeberg, Germany).
Solvent was removed by way of azeotropic distillation at 110 °C
under an Ar stream. After 4 min, the reactor was cooled to
0 °C and an additional portion of dry acetonitrile (1 mL) was
added. A second evaporation step was carried out at 110 °C for
.5 min, then the temperature was briefly raised to 120 °C, fol-
lowed by cooling to 40 °C. A solution of precursor 14 (6.4 mg,
1.7 µmol) in dry acetonitrile (2 mL) was added to the reactor
1
8
3 M. Karramkam, F. Hinnen, F. Vaufrey and F. Dolle,
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sides, Nucleotides Nucleic Acids, 2009, 28, 1131–1143.
(
(
6
3
1
and heated to 100 °C for 15 minutes. After cooling to 40 °C,
the reaction was quenched with 0.02 M HCl (10 mL) and
1
8
passed through an AffinaMIP® SPE F cartridge (‘0.7 mL’
size), which was activated previously with acetonitrile (2.5 mL).
The cartridge was washed with an 8 : 2 mixture of H O–MeCN
2
1
8
(
2.5 mL), then [ F]-2 was eluted off with a solution of 10 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem.,
TCEP·HCl (1 mM) in DMF (1.3 mL) for further use.
Int. Ed., 2001, 40, 2004–2021.
1
1 H. J. Ren, F. Xiao, K. Zhan, Y. P. Kim, H. X. Xie, Z. Y. Xia
and J. Rao, Angew. Chem., Int. Ed., 2009, 48, 9658–9662.
1
8
18
Example of preparative radiosynthesis of F peptide [ F]-16
1
8
18
12 Y. Cheng, H. Peng, W. Chen, N. Ni, B. Ke, C. Dai and
F prosthetic group [ F]-2 was prepared as described above
B. Wang, Chem. – Eur. J., 2013, 19, 4036–4042.
and transferred to a 5 mL conical vial. N,N-Diisopropylethyl
amine (39 µM) in DMF (0.1 mL) was added and the reactor was
heated to 43 °C for 30 min. In lieu of mechanical stirring, the
reaction mixture was sparged with argon for 2 seconds every
minute. The bioconjugation reaction was quenched with the
addition 0.02 M HCl (3 mL) and transferred onto a semi-pre-
parative HPLC column (LC C, Program 4). F-labelled peptide
analogue [ F]-16 was identified based on its radioactive HPLC
signal (R = 22.2 min), and collected into a round-bottomed
1
1
1
3 J. Jeon, B. Shen, L. Xiong, Z. Miao, K. H. Lee, J. Rao and
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5 M. Barczyk, S. Carracedo and D. Gullberg, Cell Tissue Res.,
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1
8
1
8
16 J. S. Desgrosellier and D. A. Cheresh, Nat. Rev. Cancer,
010, 10, 9–22.
2
t
1
7 T. Poethko, M. Schottelius, G. Thumshim, U. Hersel,
M. Herz, G. Henriksen, H. Kessler, M. Schwaiger and
H. J. Wester, J. Nucl. Med., 2004, 45, 892–902.
flask containing water (40 mL). The diluted radio-product was
immobilized on a tC18 light solid-phase extraction column,
which was activated previously with MeOH (3 mL) and water
1
8
18 C. Hultsch, M. Schottelius, J. Auernheimer, A. Alke and
(6 mL). The cartridge was washed with water (3 mL), then [ F]-
H. J. Wester, Eur. J. Nucl. Med. Mol. Imaging, 2009, 36,
1
6 was eluted off the column with EtOH (0.5 mL). Upon
1469–1474.
dilution with isotonic saline (4.5 mL), the final formulation
was obtained. Collected activity was 459.1 MBq (8% decay-cor-
rected from EOB). Total synthesis time was 132 min from EOB.
1
9 X. Y. Chen, R. Park, A. H. Shahinian, M. Tohme,
V. Khankaldyyan, M. H. Bozorgzadeh, J. R. Bading,
R. Moats, W. E. Laug and P. S. Conti, Nucl. Med. Biol., 2004,
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from Intrace Medical and the Leenaards Foundation.
J. B. Stubbs, M. G. Stabin, E. D. Hostetler, R. K. Alpaugh,
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