DPA-C5yne, a novel fluorine-18-labelled analogue of DPA-714: Radiosynthesis and preliminary evaluation as a radiotracer for imaging neuroinflammation with PET

Article abstract of DOI:10.1002/jlcr.3199

DPA-C5yne, the lead compound of a novel series of DPA-714 derivatives in which the fluoroethoxy chain linked to the phenylpyrazolopyrimidine scaffold has been replaced by a fluoroalkyn-1-yl moiety, is a high affinity (K_i: 0.35 nM) and selective ligand targeting the translocator protein 18 kDa. In the present work, DPA-C5yne was labelled with no-carrier-added [¹⁸F] fluoride based on a one-step tosyloxy-for-fluorine nucleophilic substitution reaction, purified by cartridge and HPLC, and formulated as an i.v. injectable solution using a TRACERLab FX N Pro synthesizer. Typically, 4.3-5.2 GBq of [¹⁸F]DPA-C5yne, ready-to-use, chemically and radiochemically pure (> 95%), was obtained with specific radioactivities ranging from 55 to 110 GBq/μmol within 50-60 min, starting from a 30 GBq [¹⁸F]fluoride batch (14-17%). LogP and LogD of [¹⁸F]DPA-C5yne were measured using the shake-flask method and values of 2.39 and 2.51 were found, respectively. Autoradiography studies performed on slices of ((R,S)-α-amino-3-hydroxy-5- methyl-4-isoxazolopropionique (AMPA)-lesioned rat brains showed a high target-to-background ratio (1.9 ± 0.3). Selectivity and specificity of the binding for the translocator protein was demonstrated using DPA-C5yne (unlabelled), PK11195 and Flumazenil (central benzodiazepine receptor ligand) as competitors. Furthermore, DPA-C5yne proved to be stable in plasma at 37°C for at least 90 min.

Full text of DOI:10.1002/jlcr.3199

Research Article
Received 28 January 2014,
Revised 17 March 2014,
Accepted 19 March 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3199
18
[ F]DPA-C5yne, a novel fluorine-18-labelled
analogue of DPA-714: radiosynthesis and
preliminary evaluation as a radiotracer for
imaging neuroinflammation with PET
†
†
Vincent Médran-Navarrete,a Nicholas Bernards,a,b Bertrand Kuhnast,a
Annelaure Damont,a Géraldine Pottier,a,b Marie-Anne Peyronneau,a
Michael Kassiou,c,d Frank Marguet,e Frédéric Puech,e
a
Raphaël Boisgard,a,b and Frédéric Dollé
*
DPA-C5yne, the lead compound of a novel series of DPA-714 derivatives in which the fluoroethoxy chain linked to the
phenylpyrazolopyrimidine scaffold has been replaced by a fluoroalkyn-1-yl moiety, is a high affinity (Ki: 0.35 nM) and selective
ligand targeting the translocator protein 18kDa. In the present work, DPA-C5yne was labelled with no-carrier-added [18F]
fluoride based on a one-step tosyloxy-for-fluorine nucleophilic substitution reaction, purified by cartridge and HPLC, and
formulated as an i.v. injectable solution using a TRACERLab FX N Pro synthesizer. Typically, 4.3–5.2 GBq of [18F]DPA-C5yne,
ready-to-use, chemically and radiochemically pure (> 95%), was obtained with specific radioactivities ranging from 55 to
110 GBq/μmol within 50–60min, starting from a 30GBq [18F]fluoride batch (14–17%). LogP and LogD of [18F]DPA-C5yne were
measured using the shake-flask method and values of 2.39 and 2.51 were found, respectively. Autoradiography studies
performed on slices of ((R,S)-α-amino-3-hydroxy-5-methyl-4-isoxazolopropionique (AMPA)-lesioned rat brains showed a high
target-to-background ratio (1.9 0.3). Selectivity and specificity of the binding for the translocator protein was demonstrated
using DPA-C5yne (unlabelled), PK11195 and Flumazenil (central benzodiazepine receptor ligand) as competitors. Furthermore,
DPA-C5yne proved to be stable in plasma at 37°C for at least 90min.
Keywords: fluorine-18; radiosynthesis; DPA-C5yne; DPA-714; TSPO 18 kDa; PBR
approximately equally. Differences in affinity are higher for [11C]
Introduction
PBR28 (50-fold), whereas pyrazolo[1,5-a]pyrimidine-acetamides
show lower differences (only fourfold for [11C]DPA-713).36
Overexpression of the translocator protein (TSPO) 18 kDa (formerly
known as the peripheral benzodiazepine receptor (PBR)1) in the
On the other hand, recent in-house studies have demonstrated
that [18F]DPA-714 is extensively metabolized in vivo in both
brain, during microglial cell activation in response to cerebral
insults, has attracted considerable attention for the development
rodents and non-human primates.38 One metabolic pathway,
of positron emission tomography (PET) radioligands as imaging
which was demonstrated in vitro and was also suggested to occur
markers of neuroinflammation.2,3
in vivo, results from O-dealkylation that leads to the formation
While several radioligands are still underdevelopment,4–9
the pyrazolo[1,5-a]pyrimidine-acetamide [18F]DPA-71410–12 is
currently considered as a major challenger13–23 of the reference
ligand, the isoquinoline-carboxamide [11C]PK11195,24,25 together
with the phenoxypyridin-3-yl-acetamide [11C]PBR2826–32 and its
closely related fluorinated analogue [18F]FEPPA.33,34 However,
the use of radioligands belonging to the latter class faces
difficulties today in human PET imaging because of differences
between individuals linked to the variable expression of two
binding sites (a low and a high affinity) for the TSPO, encoded
by a single polymorphism (rs6971) at the gene level.35–37
Subjects may thus be classified as high-affinity binders or low-
affinity binders (when one single binding site for the TSPO is
expressed with either high or low affinity, but may also be
of fluorine-18-labelled small metabolites such as [18F]
fluoroacetaldehyde, or its oxidation product [18F]fluoroacetic acid.
aCEA, I2BM, Service Hospitalier, Frédéric Joliot, Orsay, France
bInserm, U1023, Université Paris Sud, Orsay, France
cSchool of Chemistry, University of Sydney, Sydney, Australia
dDiscipline of Medical Radiation Sciences, University of Sydney, Sydney, Australia
eExploratory Unit, Sanofi, Paris, France
*Correspondence to: Dr Frédéric Dollé, Service hospitalier Frédéric Joliot, Institut
d’imagerie biomédicale, CEA, 4 place du Général Leclerc, F-91406 Orsay, France.
E-mail: frederic.dolle@cea.fr
classified as mixed affinity binders when both sites are expressed † These two authors equally contributed to the work.
J. Label Compd. Radiopharm 2014
Copyright © 2014 John Wiley & Sons, Ltd.

V. Médran-Navarrete et al.
The main issue is that [18F]fluoroacetate is known to cross the
blood brain barrier at a certain level39–41 and may thus confound
the brain PET signal. Moreover, [18F]fluoroacetate may be
converted to [18F]fluoride ion,42–44 which bind avidly to the
bones, including the skull.45 This accumulation of radioactivity
in the skull is also problematic as far as brain imaging is
concerned because it interferes with the quantification of
the radiotracer due to the partial volume effect.45
Consequently, structural modifications of the DPA-714/DPA-713
chemotype that could prevent O-dealkylation and thus avoid the
formation of [18F]fluoroacetate and [18F]fluoride ions were
explored. While keeping the scaffold of DPA-714, new analogues
have recently been synthesized, in which the oxygen atom
bridging the phenyl ring and the fluoroalkyl chain is replaced with
a carbon atom. Of particular interest is a novel subclass of
compounds featuring an alkyne bond and a short alkane spacer
linking the phenylpyrazolopyrimidine scaffold and the fluorine
atom.46 This series includes four analogues of DPA-714 with a
side-chain length ranging from three to six carbon atoms, which
all exhibit high affinity and selectivity towards the TSPO. Within
this series, DPA-C5yne (1, Figure 1) exhibited the highest affinity
for the TSPO with a Ki value of 0.35 nM (as reference, the Ki
measured for DPA-714 in the same assay was 0.91 nM). No affinity
for the central benzodiazepine receptor (CBR) was observed
(Ki > 1 μM).46 Furthermore, preliminary in vitro metabolism
evaluation of this series using rat microsomal incubations and
liquid chromatography–mass spectrometry (LC–MS) analyses
showed the absence of defluorinated metabolites.46
(diethylamino)-2-oxoethyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-
2-yl)phenyl)pent-4-yn-1-yl) 4-methylbenzenesulfonate) as pre-
cursor for labelling with fluorine-18 is illustrated in Scheme 1.
Briefly, both compounds were synthesized in a single step from
the alkynol 3, the latter being prepared in five chemical steps
from commercially available methyl 4-iodobenzoate.46 Then,
fluorodeoxygenation of 3 was performed using Deoxofluor®
(Sigma-Aldrich, Saint-Quentin Fallavier, France) in dichloromethane
at room temperature for 3 days and afforded the corresponding
fluorinated compound, DPA-C5yne (1), in 24% yield. The tosylate 2
was also prepared from the alkynol 3, by treatment with 4-
toluenesulfonic anhydride in dichloromethane at room temperature
for 2–3 days, and obtained in 54% yield.
Radiochemistry
DPA-C5yne (1) was labelled with fluorine-18 from the tosyloxy
derivative 2, purified by HPLC and formulated as an i.v. injectable
solution by implementing the one-step radiochemical process
outlined in Scheme 2. The whole procedure was performed
using a commercially available TRACERLab FX N Pro synthesizer
(GE Medical Systems, Germany). The positioning of all reagents,
solvents and consumables dedicated to the preparation of [18F]
DPA-C5yne ([18F]-1) on the TRACERLab FX N Pro is illustrated
on the control screen copy displayed in Figure 2. Further details
are given in the Experimental Section.
No-carrier-added K[18F]F-Kryptofix®222 (Sigma-Aldrich, Saint-
Quentin Fallavier, France) complex47,48 was prepared from
cyclotron-produced [18F]fluoride, K2CO3, Kryptofix®222, acetonitrile
and water using a step-by-step sequence (time list) developed by
GEMS and implemented on the synthesizer at its reception on site.
The sequence includes the trapping of [18F]fluoride anions on a
dedicated QMA cartridge, their release using a H2O/ CH3CN
Based on these data, DPA-C5yne (1) was selected for labelling
with the short-lived positron-emitter fluorine-18 (T1/2: 109.8 min).
The present manuscript briefly describes the preparation of the
tosyloxy derivative as precursor for labelling and then focuses
on the radiolabelling with fluorine-18 of 1 on a TRACERLab FX
N Pro module, the measurement of its lipophilicity (LogP/LogD) solution containing both K2CO3 and Kryptofix®222 and a two-step
based on the shake-flask method and its preliminary evaluation concentration-to-dryness process in a vessel (reactor) made from
by autoradiography on brain slices of our in-house-developed glassy carbon. K[18F]F-Kryptofix®222 was obtained in 18 min and
rat model of acute local neuroinflammation.
used without any additional drying sequence.
Radiofluorination was then performed by dilution of the
aforementioned reagent using
a DMSO solution (700 μL)
Results and discussion
containing 7.0–8.7 μmoles (4.0–5.0 mg) of the tosylate 2,
followed by stirring and heating. Initially, the conditions used
were those optimized for the preparation of [18F]DPA-714, for
Chemistry
The preparation of DPA-C5yne (1, N,N-diethyl-2-(2-(4-(3-fluoropent-1- example, 165°C for 5 min.12 Using the latter, [18F]DPA-C5yne
yn-1-yl)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide) ([18F]-1) was formed and could be isolated after cartridge/HPLC
as reference compound and the tosylate
2 (6-(4-(3-(2- purifications (see in the succeeding texts). However, overall
Figure 1. The TSPO radioligands [11C]PK11195, [11C]PBR28, [18F]FEPPA, [11C]DPA-713, [18F]DPA-714 and [18F]DPA-C5yne ([18F]-1, present work).
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J. Label Compd. Radiopharm 2014

V. Médran-Navarrete et al.
Scheme 1. Synthesis of DPA-C5yne (1, reference compound) and its tosylated analogue (2) as labelling precursor.
Scheme 2. One-step radiosynthesis, purification and formulation of [18F]DPA-C5yne ([18F]-1).
Figure 2. Description of the TRACERLab FX N Pro synthesizer used for the preparation of [18F]DPA-C5yne ([18F]-1) and positioning of all required reagents, solvents
and consumables.
yields were rather low and not reproducible. Moreover, high slightly extending the reaction time (100°C/10 min) provided
chemical and radiochemical purities were not systematically [18F]DPA-C5yne ([18F]-1) in higher and more reproducible yields
reached based on Quality Control (QC) analysis. These without any QC issues.
behaviours were not observed during the preparation of [18F]
The crude reaction mixture was then cooled down to 50°C
DPA-714. Decreasing the reaction temperature together with before being diluted with the HPLC solvent used for the
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V. Médran-Navarrete et al.
purification and then passed through an Al2O3 cartridge (Sep-
Pak® Alumina NTM cartridge, Waters (Guyancourt, France)) in
order to remove (trap) unreacted [18F]fluoride. The left-over
activity in the reactor was then rinsed once with a second
portion of HPLC-solvent and passed through the cartridge too.
The combined fractions were then purified by HPLC on a
semipreparative SymmetryPrep® C-18 column (Waters), using a
mixture of CH3CN, H2O and Trifluoroacetic acid (TFA) as the
eluent (HPLC D, see Experimental Section). Using the latter
conditions, [18F]-1 (tR: 9–10 min) could be obtained with a > 95%
chemical and radiochemical purity and was completely
separated from the remaining tosylate 2 (tR: 15–17 min). Removal
of the HPLC solvents and formulation of [18F]DPA-C5yne ([18F]-1)
as an i.v. injectable solution (0.9% aq NaCl containing EtOH) were
performed using a reverse phase cartridge (Sep-Pak®Plus C18
In vitro stability in rat plasma
Stability of DPA-C5yne (1) was checked in vitro in rat spiked
plasma at 37°C by LC–MS. Analyses of acetonitrile plasma
extracts at different incubation time points (0, 5, 15, 60 and 90
min) showed only one single peak with a retention time
(tR: 12.9 min) and UV-spectrum (λmax = 270 nm) identical to those
of authentic DPA-C5yne. The variability in the peak area
measurement as a function of time was less than 2%.
Furthermore, for each time point, MS exhibited one molecular
ion, at m/z 421 [M + H+], with a mass spectrum and a
fragmentation profile also identical to those of authentic DPA-
C5yne. Thus, DPA-C5yne (1) proved to be stable in plasma at
37°C for at least 90 min because no decomposition product
could be observed.
cartridge). The whole process, including radiofluorination,
cartridge and HPLC purifications, as well as formulation, lasted
between 37 and 42 min.
Autoradiography studies
Typically, 4.3–5.2 GBq of [18F]DPA-C5yne ([18F]-1) ready-to-use
was obtained with specific radioactivities ranging from 55 to
110 GBq/μmol within 50–60 min (HPLC purification and
Sep-Pak®-based formulation included), starting from a 30 GBq
Autoradiography studies were performed on slices of our in-
house-developed rat model of acute local neuroinflammation.
This model is based on an excitotoxic AMPA-mediated brain
lesion in the right striatum of Wistar rats. It has already been
validated for TSPO expression by immunohistochemical
analyses49–51 and used for the evaluation of several TSPO
radioligands, such as [11C]PK11195,49,50 [11C]CLINME,50,52 [11C]
DPA-713,49,51 [18F]PBR111,52 [18F]DPA-71451 and more recently
[11C]SSR180575.53 Briefly, brain slices were incubated with [18F]
DPA-C5yne ([18F]-1, 7.4 nM) alone, and also coincubated with
non-labelled DPA-C5yne (1, 20 μM), or PK11195 (20 μM, the
TSPO ligand of reference) or Flumazenil (20 μM, a CBR ligand
of reference), respectively, in order to confirm the selectivity
and specificity of the binding. As shown in Figure 3(A), a
significantly increased binding of [18F]DPA-C5yne ([18F]-1)
was detected in the lesion when compared to the control
side. The target-to-background ratio (TBR), calculated as the
bound tracer in the lesion versus the bound tracer in the
contralateral side, was relatively high, 1.9 0.3 (p < 0.0001).
Addition of an excess of unlabelled DPA-C5yne or PK11195
(20 μM, 2700-fold mass excess compared to [18F]DPA-C5yne)
fully inhibited the binding (Figure 3(B) and 3(C)) in the
lesioned area (TBR = 1.0 0.1 for both ligands), proving a high
specificity of the binding and selectivity for the TSPO.
However, the addition of a similar excess of Flumazenil did
[18F]fluoride batch. Overall, non-decay corrected, isolated yields
were 14–17% (20–25% decay corrected).
Quality controls of [18F]DPA-C5yne ([18F]-1) were performed
on an aliquot of the ready for i.v. injection batch. The prepared
radiotracer solution was clear and colourless with a pH between
6 and 7. As demonstrated by analytical-HPLC control (HPLC E,
see Experimental Section), the radiotracer preparation was found
to be >95% chemically and radiochemically pure (1, tR:
2.20 min). The preparation was also shown to be free of the
non-radioactive precursor, the tosylate 2 (tR: 3.51 min), and was
chemically and radiochemically stable for at least 120 min.
Lipophilicities
LogP (n-octanol/water partition coefficient) and LogD (n-octanol/
buffer pH 7.4 partition coefficient) of [18F]DPA-C5yne ([18F]-1) were
measured using the shake-flask method, and values of 2.39 and
2.51 were found, respectively. The calculated LogP (cLogP) was
determined using CHEMBIODRAW ULTRA SOFTWARE, and a value of
4.28 was found. These values were all slightly higher than the ones
previously obtained for [18F]DPA-714 (LogP: 1.66, LogD: 1.74 and
cLogP: 3.33) but remained in the recommended range for a
radiotracer dedicated to brain imaging (1 < LogP/LogD < 3). As
with DPA-714, cLogP value was much higher than LogP and
LogD measurements.
not affect the binding of
yielding a calculated TBR of 1.9 0.1, a value identical to
the one found for
18F]DPA-C5yne alone, proving its
selectivity for the TSPO and not for the CBR.
[
18F]DPA-C5yne (Figure 3(D)),
[
Figure 3. Autoradiography studies on slices of AMPA-lesioned rat brains incubated with [18F]DPA-C5yne ([18F]-1, 7.4 nM) alone (A) or with non-labelled DPA-C5yne
(20 μM) (B), with PK11195 (20 μM) (C) and with Flumazenil (20 μM) (D), respectively.
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J. Label Compd. Radiopharm 2014

V. Médran-Navarrete et al.
(s, d, t, q, m, b for singlet, doublet, triplet, quadruplet, multiplet and
Conclusions
broad, respectively). The MS were measured on a Thermo Scientific (Les
The pyrazolo[1,5-a]pyrimidine-acetamide [18F]DPA-C5yne is a Ulis, France) Ion Trap LCQ Deca XP + spectrometer (ESI+). The high
resolution MS (HRMS) analyses were performed by Imagif (ICSN-CNRS,
Gif-sur-Yvette, France) by electrospray with positive (ESI+) ionization
mode on a Waters LCT Premier XE (Milford, MA, USA) spectrometer.
novel, fluorine-18-labelled, radioligand targeting the TSPO with
promising in vitro properties. PET imaging studies are currently
being carried out in both acute and chronic animal models of
neurodegeneration in order to evaluate the in vivo potential of
this DPA-714 analogue.
Radioisotope production
No-carrier-added fluorine-18 (half-life: 109.8 min) was produced via the
[
18O(p,n)18F] nuclear reaction by irradiation of 18O]water
a 2 mL [
Experimental
General
(>97%-enriched, Rotem (CortecNet, Paris, France)) target on an IBA
Cyclone-18/9 (IBA, Louvain-la-Neuve, Belgium) cyclotron (18 MeV proton
beam), and the aqueous radioactive solution was then transferred to the
appropriate hot cell. Target hardware: commercial, 2-mL, two-port,
stainless steel target holder equipped with a domed-end niobium
cylinder insert. Target to hot cell liquid-transfer system: 50 m
Polytetrafluoroethylene (PTFE) line (0.8 mm internal diameter; 1/16 inch
external diameter), 2.0 bar helium drive pressure and transfer time
2–3 min. Typical production of [18F]fluoride at the end of bombardment
for a 25 μA, 30 min (12.5 μAh) irradiation: 31.5–32.5 GBq.
Chemicals
Chemicals were purchased from Aldrich, Fluka, Sigma or Cooper (France)
and were used without further purification, unless otherwise stated.
Thin-layer chromatography analysis and flash chromatography
Thin-layer chromatography was run on pre-coated plates of silica gel
60F254 (VWR, Fontenay-sous-Bois, France). The compounds were localized
when possible at 254nm using a UV-lamp and/or by dipping the TLC-
plates in a 1% ethanolic ninhydrin solution, a basic KMnO4 aqueous
Acute neuroinflammatory animal model
Striatal AMPA-mediated excitotoxicity and acute local neuroin-
solution or a 1% MeOH/H2O (1/1, v:v) FeCl3 solution and heating on a flammation was induced in the brain of Wistar rats according to reported
hot plate. Flash chromatographies were conducted on silica gel or alumina procedures.49–51 The protocol used includes a stereotactic injection in
gel (0.63–0.200 mm, VWR) columns.
the right striatum of 0.5 μL of AMPA ((R,S)-α-amino-3-hydroxy-5-methyl-
4-isoxazolopropionique, 15 mM in phosphate buffered saline buffer) in
anaesthesized and normothermically controlled animals followed by a
resting period of 7 days.
High-performance liquid chromatography analysis
[HPLC A]: Equipment: Waters (Guyancourt, France) system equipped with
a 600E System Controller, a Prep LC 3000 pump, a 490E programmable
multiwavelength UV-detector and a Kipp and Zonen (Emerainville, France)
Chemistry
BD12E flatbed recorder; column: preparative Zorbax® RX-SIL, Hewlett
Packard (Les Ulis, France) (250 × 21.2mm); porosity: 5μm; conditions:
eluent: CH2Cl2/ MeOH: 98.5/1.5 [v:v]; flow rate: 10 mL/min; temperature:
room temperature (RT); UV detection at λ: 230 nm. [HPLC B]: Equipment:
N,N-diethyl-2-(2-(4-(4-hydroxypent-1-yn-1-yl)phenyl)-5,7-dimethylp-
yrazolo[1,5-a]pyrimidin-3-yl)acetamide (3)
Synthesized in five steps from commercially available methyl 4-
iodobenzoate according to reference.46 Rf (CH2Cl2/MeOH: 95/5 v:v):
0.21. 1H-NMR (CDCl3, 400 MHz): δ 7.74 (d, 2H, J = 8.0 Hz, Ph), 7.46 (d, 2H,
J = 8.0 Hz, Ph), 6.54 (s, 1H), 3.93 (s, 2H, CH2), 3.81 (t, 2H, J = 6.4 Hz, CH2OH),
3.49 (q, 2H, J = 7.2 Hz, NCH2CH3), 3.40 (q, 2H, J = 7.2 Hz, NCH2CH3), 2.74 (s,
3H, CH3), 2.56 (s, 3H, CH3), 2.55 (t, 2H, J = 6.4 Hz, CH2), 1.86 (q5, 2H,
J = 6.4 Hz, CH2), 1.22 (t, 3H, J = 7.2 Hz, NCH2CH3), 1.10 (t, 3H, J = 7.2 Hz,
NCH2CH3). 13C-NMR (CDCl3, 400 MHz): δ 169.8 [C], 157.6 [C], 154.5 [C],
147.5 [C], 145.0 [C], 133.0 [C], 131.6 [2xCH], 128.4 [2xCH], 123.6 [C],
108.4 [CH], 101.3 [C], 90.2 [C], 81.1 [C], 61.7 [CH2], 42.2 [CH2], 40.6 [CH2],
31.3 [CH2], 28.0 [CH2], 24.5 [CH3], 16.8 [CH3], 16.0 [CH2], 14.3 [CH3], 13.0
[CH3]. MS (ESI+): m/z 419 (M + H)+.
Waters system equipped with a 510 pump and a Shimadzu SPD-10A UV-
multiwavelength detector. Column: semipreparative SymmetryPrep®
C-18, Waters (300 × 7.8 mm); porosity: 7 μm; conditions: eluent:
CH3CN/H2O/TFA: 50/50/0.1 (v:v:v); flow rate: 5 mL/min; temperature:
RT; UV detection at λ: 254 nm. [HPLC C]: Equipment: Waters system
equipped with
a 510 pump and a Shimadzu SPD-10A UV-
multiwavelength detector. Column: semipreparative SymmetryPrep®
C-18, Waters (300 × 7.8 mm); porosity: 7 μm; conditions: eluent:
CH3CN/H2O/TFA: 60/40/0.1 (v:v:v); flow rate: 5 mL/min; temperature:
RT; UV detection at λ: 254 nm. [HPLC D]: Equipment: TRACERLab FX N
Pro (GE Medical Systems, Germany) integrated system, equipped with
a Sykam S1122 Solvent Delivery System (pump), a Knauer K-2501 UV-
multiwavelength detector and a miniaturized gamma-radioactivity
detector. Column: semipreparative SymmetryPrep® C-18, Waters
(300 × 7.8 mm); porosity: 7 μm; conditions: eluent: CH3CN/H2O/TFA: 60/
40/0.1 (v:v:v); flow rate: 5 mL/min; temperature: RT; UV detection at λ:
254 nm. [HPLC E]: Equipment: Waters Alliance 2690 (or a Waters binary
HPLC Pump 1525) equipped with a Waters 996 UV photodiode array
detector and a Berthold LB509 radioactivity detector; column: analytical
Symmetry-M® C-18, Waters (50 × 4.6 mm); porosity: 3.5 μm; conditions:
eluent: solvA/solvB: 20/80 (v:v) [solvA: H2O containing Low-UV PIC® B7
reagent (Waters), 20 mL for 1000 mL; solvB: H2O/CH3CN: 30/70 (v:v)
N,N-diethyl-2-(2-(4-(3-fluoropent-1-yn-1-yl)phenyl)-5,7-dimethylpyr-
azolo[1,5-a]pyrimidin-3-yl)acetamide (1, DPA-C5yne)
To a solution of compound 3 (84 mg, 0.201 mmol) in dry CH2Cl2 (4mL) was
added a 50% Deoxofluor® solution in toluene (0.5mL, excess). The
reaction mixture was stirred for 3days at RT, then diluted with MeOH
(0.5mL) and concentrated to dryness. Finally, the residue was purified by
flash chromatography on silica gel (heptane/acetone: 4/1 (v:v)) to afford
1 (20 mg, 24%) as a yellow solid. For analytical purposes only, compound
1 was further purified using preparative HPLC [HPLC A] and obtained, after
solvents removal, as a light yellow solid. Rf (heptane/acetone: 1/1 (v:v)):
0.35. tR: 30 min [HPLC A], 10.5 min [HPLC B], 9.5min [HPLC C], 9.5min [HPLC
D] and 2.20 min [HPLC E]. 1H-NMR (CDCl3, 400 MHz): δ 7.76 (d, 2H, J = 8.0Hz,
Ph), 7.47 (d, 2H, J = 8.0Hz, Ph), 6.54 (s, 1H), 4.62 (dt, 2H, J2H-F = 47.2 Hz, JH3-
H = 6.0Hz, CH2F), 3.97 (s, 2H, CH2), 3.50 (q, 2H, J = 7.2Hz, NCH2CH3), 3.41
containing Low-UV PIC® B7 reagent (Waters), 20 mL for 1000 mL]; flow
rate: 2.0 mL/min; temperature: 30°C (or RT); UV detection at λ: 280 nm.
Spectroscopies
Nuclear magnetic resonance spectra (1H/13C) were recorded on a Bruker (q, 2H, J = 7.2Hz, NCH2CH3), 2.76 (s, 3H, CH3), 2.59 (t, 2H, J = 7.2Hz, CH2),
(Wissembourg, France) Avance (400 MHz) apparatus using the 2.58 (s, 3H, CH3), 2.01 (dq5, 2H, JH3-F = 25.6 Hz, J3H-H = 6.0 Hz, CH2), 1.23 (t,
hydrogenated residue of the deuterated solvent CDCl3 (δ = 7.23 ppm) as
internal standard for 1H-NMR as well as the deuterated solvent CDCl3
(δ = 77.0 ppm) as internal standard for 13C-NMR. The chemical shifts are
reported in parts per million, downfield from Tetramethylsilane (TMS)
3H, J = 7.2Hz, NCH2CH3), 1.12 (t, 3H, J = 7.2Hz, NCH2CH3). 13C-NMR (CDCl3,
400 MHz): δ 169.7 [C], 157.6 [C], 154.5 [C], 147.4 [C], 145.2 [C], 133.0 [C],
131.6 [2xCH], 128.4 [2xCH], 123.5 [C], 108.4 [CH], 101.3 [C], 89.3 [C], 82.5
[d, J1C-F = 164 Hz, CH2], 81.2 [C], 42.2 [CH2], 40.6 [CH2], 29.5 [d, J2C-F = 20 Hz,
J. Label Compd. Radiopharm 2014
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V. Médran-Navarrete et al.
CH2], 28.0 [CH2], 24.3 [CH3], 16.9 [CH3], 15.4 [d, J3C-F = 4 Hz, CH2], 14.3 [CH3],
13.0 [CH3]. MS (ESI+): m/z: 421 (M + H)+. HRMS (m/z, ESI) calculated for
acetone and ethanol to further clean the synthesizer but also to
dry all reservoirs [Reservoir 1–14], both reactors [Reactor 1–2],
the tube dedicated to the HPLC injection [HPLC tube], the flask
allowing the dilution of the HPLC-collected fraction prior to
cartridge formulation [Dilution flask] and the flask required for
receiving at last the batch of formulated product [Product vial]
as well as all tubings connecting these elements.
C25H30FN4O (M+ 1), 421.2404; found, 421.2404.
6-(4-(3-(2-(diethylamino)-2-oxoethyl)-5,7-dimethylpyrazolo[1,5-a]pyri-
midin-2-yl)phenyl)pent-4-yn-1-yl)4-methylbenzenesulfonate (2)
To a solution of compound 3 (200 mg, 0.478 mmol) in dry CH2Cl2 (5 mL)
was added Et3N (80 μL, 0.578 mmol, 1.2 eq) and DMAP (6 mg,
0.049 mmol, 0.1 eq). The flask was degased and flushed with argon
(two cycles), then cooled to 0°C. 4-toluenesulfonic anhydride (170 mg,
0.521 mmol, 1.1 eq.) was slowly added to the previous solution, and the
reaction mixture stirred at 0°C for 15 min, then at RT overnight. 4-
toluenesulfonic anhydride (50 mg, 0.153 mmol, 0.3 eq) and Et3N (100 μL,
0.71 mmol, 1.5 eq) were added once again, and the mixture stirred
another 2 days at RT. The reaction mixture was then evaporated under
reduced pressure, and the resulting brownish oil was portioned between
EtOAc (20 mL) and aq 0.05 M HCl (15 mL). The organic layer was
separated, washed with water (10 mL), brine (10 mL), dried over Na2SO4,
filtered and concentrated to dryness. Finally, the resulting residue was
purified by flash chromatography on silica gel (pure CH2Cl2 to CH2Cl2/
MeOH: 98/2 (v:v)) to afford 2 (145 mg, 54%) as a light yellow solid. Rf
(CH2Cl2/MeOH: 93/7 v:v): 0.58. tR: 21.5 min [HPLC B], 15.5 min [HPLC C],
15.3–15.9 min [HPLC D], 3.51 min [HPLC E]. 1H-NMR (CDCl3, 400 MHz): δ
7.80 (d, 2H, J = 8.0 Hz, Ph), 7.76 (d, 2H, J = 8.4 Hz, Ph), 7.36 (d, 2H,
J = 8.4 Hz), 7.31 (d, 2H, J = 8.0 Hz, Ph), 6.53 (s, 1H), 4.21 (t, 2H, J = 5.6 Hz,
CH2OTs), 3.94 (s, 2H, CH2), 3.51 (q, 2H, J = 7.2 Hz, NCH2CH3), 3.40 (q, 2H,
J = 7.2 Hz, NCH2CH3), 2.74 (s, 3H, CH3), 2.55 (s, 3H, CH3), 2.49 (t, 2H,
J = 6.8 Hz, CH2), 2.39 (s, 3H, CH3), 1.94 (q5, 2H, J = 6.8 Hz, CH2), 1.23
(t, 3H, J = 7.2 Hz, NCH2CH3), 1.11 (t, 3H, J = 7.2 Hz, NCH2CH3). 13C-NMR
(CDCl3, 400 MHz): δ 169.7 [C], 157.6 [C], 154.4 [C], 147.0 [C], 145.0 [C],
144.8 [C], 133.1 [C], 132.8 [C], 131.6 [2xCH], 129.8 [2xCH], 128.4 [2xCH],
127.8 [2xCH], 123.3 [C], 108.4 [CH], 101.3 [C], 88.4 [C], 81.7 [C], 68.9
[CH2], 42.2 [CH2], 40.6 [CH2], 28.0 [CH2], 27.9 [CH2], 24.3 [CH3], 21.6
[CH3], 16.8 [CH3], 15.7 [CH2], 14.3 [CH3], 13.0 [CH3]. MS (ESI+): m/z: 573
(M + H)+. HRMS (m/z, ESI) calculated for C32H37N4O4S (M + 1), 573.2536;
found, 573.2524.
Synthesizer configuration
Valves VZ1 and VZ2 were both turned in up-position for the
preparation of [18F]DPA-C5yne ([18F]-1), therefore by-passing
most of the added functionalities of the TRACERLab FX N
Pro synthesizer when compared to the TRACERLab FX FN one
(e.g. the second reactor [Reactor 2] and its connected reservoirs
[Reservoir 7–11] and associated tubings). Note that valves VX3 and
VX4 as well as the associated option of using an additional
cartridge purification unit were also not requested for this process.
Synthesizer preparation
Prior to [18F]DPA-C5yne ([18F]-1) production, the following
reservoirs and vessels were filled with the solutions described
here: [Reservoir 1]: a mixture of deionized water and acetonitrile
(30/70 v/v, 1 mL) containing 1.5 mg of K2CO3 and 12–15 mg of
Kryptofix®222; [Reservoir 3]: a DMSO solution (0.7 mL) containing
the tosylate 2 (4.0–5.0 mg, 7.0–8.7 μmol), as the labelling
precursor; [Reservoir 4]: HPLC solvent (2 mL, see [HPLC D]);
[Reservoir 5]: HPLC solvent (2 mL, see [HPLC D]; [Reservoir 12]:
deionized water (10 mL); [Reservoir 13]: ethanol (1.5 mL);
[Reservoir 14]: aq 0.9% NaCl (8.5 mL); [Dilution flask]: deionized
water (20 mL). Note that [Reservoir 2], [Reservoir 6], [Reservoir
7–11], [Reactor 1], [Reactor 2], [Product vial], [HPLC tube], [[18O]
Water collect], [Water waste] and all other [WASTE] were left
empty. Additionally, all cartridges requested for the preparation
of [18F]DPA-C5yne ([18F]-1) were, before being mounted on the
synthesizer, conditioned as described here: [QMA cartridge]:
Sep-Pak® Light AccellTM Plus QMA cartridge (Waters), hydroxide
form, generated from the chloride form by washing with aq 1M
NaHCO3 (2 mL) and rinsed with deionized water (20 mL) and
CH3CN (10 mL); [Alumina N cartridge]: Sep-Pak® Alumina NTM
cartridge (Waters), washed with deionized water (10 mL); [C-18
cartridge]: Sep-Pak®Plus C18 cartridge (Waters), washed with
EtOH (2 mL) and then rinsed with deionized water (10 mL).
Finally, the HPLC-column and system was also conditioned with
the appropriate solvent (see [HPLC D]), and DPA-C5yne (1), as
standard, was injected once.
Radiochemistry
The TRACERLab FX N Pro synthesizer: description,
configuration, preparation and sequences used
Synthesizer description and sequences used
Fluorine-18-labelling, purification with HPLC and formulation of
[18F]DPA-C5yne ([18F]-1) as an i.v. injectable solution was
performed using a commercially available TRACERLab FX N Pro
synthesizer (GE Medical Systems, Germany), placed in a 5.0-cm-
lead shielded and ventilated cell. The whole process was thus
fully automated and used a dedicated method supported by
three generic ‘time list’, written according to the programming
mode of General Electric synthesis modules, and named,
respectively, ‘Time list 1: K[18F]F-K222 preparation’, ‘Time list 2:
radiofluorination and pre-purification’ and ‘Time list 3:
purification and formulation’.12 Before each [18F]DPA-C5yne
Radiosynthesis, purification and formulation
of [18F]DPA-C5yne ([18F]-1)
K[18F]F-K222 preparation (Time list 1)
([18F]-1) batch production, the synthesizer was cleaned out (e.g. The target content (oxygen-18-enriched water containing [18F]
used-cartridge removal), purged from any residual chemicals or fluorine-18) was transferred to the TRACERLab FX N Pro
solvents left-over (reservoirs, reactors and tubings) from a synthesiser using helium pressure (2 bars) and was first collected
previous radiotracer production and dried using another in- in the [[18O]-water collect] vial, then sucked through the [QMA
house programmed method, based on the use of two generic cartridge]. At this stage, fluorine-18 was trapped, as [18F]fluoride,
‘time list’, named, respectively, ‘Time list: clean’ and ‘Time list: in the cartridge, and the oxygen-18-enriched water was
clean and dry’.12 The first cleaning procedure used deionized collected in the [Water waste] vial. [18F]fluoride anions were then
water to rinse both reactors [Reactor 1–2], the flask collecting released from the cartridge using the solution stored in
the oxygen-18-enriched water [Water collect] and the tube [Reservoir 1] and eluted into [Reactor 1]. The mixture was gently
dedicated to the HPLC injection [HPLC tube] and most of concentrated to dryness using the following two heating steps
the connecting tubings. The second procedure used both sequentially: (i) 60°C, 7 min at a pressure between 30 and
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J. Label Compd. Radiopharm 2014

V. Médran-Navarrete et al.
35 kPa, then (ii) 120°C, 5 min under vacuum. Additional dilution preparation (5–20 μL) was injected onto HPLC [HPLC E]. The
with CH3CN followed by azeotropic distillation was not chemical identity was determined by UV-spectrometric analysis,
performed. The expected K[18F]F-K222 complex, ready for the by comparison of the UV-profile, λmax and retention time recorded
fluorination step, was obtained within 18 min.
with those observed for the corresponding reference compound
(1) in the same HPLC system and conditions. Chemical purity
was also assessed on the UV-chromatogram based on the relative
integration of the peak areas. Radiochemical identity was also
confirmed by combined radioactive and UV-spectrometric
analysis, by comparison of the retention time of the radioactive
compound with the corresponding non-radioactive reference
compound (1). Radiochemical purity was assessed on the
radioactive chromatogram based on the relative integration of
the peak area corresponding to the radiotracer.
Fluorine-18 incorporation and pre-purification (Time list 2)
The content of the [Reservoir 3] (e.g. 4–5 mg of 2 as precursor for
labelling in solution in 700 μL of DMSO) was added to the
[Reactor 1] (the latter containing the aforementioned K[18F]F-
K222 complex). The [Reactor 1] was then heated at 100°C for
10 min and then cooled down to 50°C. The content of [Reservoir
4] (HPLC solvent, 2 mL) was added to the [Reactor 1]. The diluted
content of [Reactor 1] was then transferred under pressure
through the [Alumina cartridge] and collected in the [HPLC
tube]. The content of [Reservoir 5] (HPLC solvent, 2 mL) was
further added to the [Reactor 1] and the novel content of
[Reactor 1] was then analogously transferred (under pressure)
through the [Alumina cartridge] and collected in the [HPLC tube]
too. The part of the process described here lasted about 17 min.
Specific radioactivity determination
Specific radioactivity of the radiotracer was calculated from three
consecutive HPLC analyses. For this, an aliquot of the radiotracer
preparation (5–20 μL) was injected onto HPLC [HPLC E], and for
each injection, the area of the UV absorbance peak
corresponding to the product was measured (integrated) on
the HPLC chromatogram and compared to a standard curve
relating mass to UV absorbance, allowing the determination of
the corresponding mass. The HPLC fraction corresponding to
the peak was also collected in a vial, and its radioactivity
measured in an ionization chamber (Capintec, Ramsey, NJ,
USA). The specific radioactivity, as a mean of three independent
experiments, was calculated by dividing the counted
radioactivity by the found mass (triplicate).
High-performance liquid chromatography purification and
formulation (Time list 3)
The cartridge-purified solution (about 4.7 mL) collected in the
[HPLC tube] was transferred under pressure into the HPLC
injection loop (5 mL). HPLC purification started at the signal
given by the fluid detector, the latter switching the loop-valve
from the ‘load’ position to the ‘inject’ position. HPLC elution
was followed by both UV (254 nm) and radioactivity detection. On-shelf stability test
The fraction containing [18F]DPA-C5yne ([18F]-1) was collected
Chemical and radiochemical stability of the entire preparation
separately, on the operator’s actions (clicking on the start-collect
and end-collect icons), in the [Dilution flask], prefilled with water
(20 mL). Final formulation of the radiotracer was performed
automatically using a Sep-Pak®Plus C18-based system. For this,
the content of the [Dilution flask] was first passed through the
[C-18 cartridge] (trapping the radiotracer), the latter being then
washed with water (10 mL, stored in the [Reservoir 12]. Elution
of the cartridge with EtOH (1.5 mL, stored in the [Reservoir 13])
afforded [18F]DPA-C5yne ([18F]-1), which was recovered in the
[Product flask]. Addition of 0.9% aq NaCl (8.5 mL, stored in the
[Reservoir 14]) through the [C-18 cartridge] was performed at
last to complete the formulation step. HPLC purification and
formulation steps lasted about 20–25 min.
was checked by HPLC. For this, aliquots of the radiotracer
preparation (20 μL) were injected onto HPLC [HPLC E] at regular
15-min intervals during 120 min. For each injection, the chemical
and radiochemical purity was determined (see in the preceding
text). Moreover, for each injection, the mass associated to the
peak corresponding to the product was calculated (using the
standard curve relating mass to UV absorbance), and its
radioactivity was also measured, before being then corrected
for the decay of fluorine-18. Values of both mass and decay-
corrected radioactivity per sample were plotted against time
and fitted for linearity.
Lipophilicity determination
Quality control of formulated [18F]DPA-C5yne
([18F]-1)
LogP (n-octanol/water partition coefficient)
[18F]DPA-C5yne ([18F]-1, 1–5 kBq in 50 μL of water) was added to
a two-layer system of n-octanol (500 μL) and water (450 μL) in an
Eppendorf cap (VWR, Fontenay-sous-Bois, France). The vessel
was strongly vortexed for 3 min and then centrifuged at
3000 rpm for 2 min. An aliquot of each layer (100 μL) was
assessed for radioactivity in a cross-calibrated Perkin-Elmer
Cobra Quantum γ-counter (Les Ulis, France).
Appearance test
The radiotracer preparation was visually inspected behind a lead-
shielded glass window for clarity and absence of particulates.
pH test
The pH was determined using standard pH paper using an
aliquot of the radiotracer preparation.
LogD (n-octanol/buffer pH 7.4 partition coefficient)
Identification and chemical and radiochemical purity test
The procedure described earlier was repeated by replacing water
Identification as well as chemical and radiochemical purity by 0.1 M phosphate buffered saline pH 7.4 (450 μL). The partition
determinations were assessed by separate analytical-HPLC coefficients (LogP and LogD) were calculated as the decimal
analysis, using a sample of authentic 1 as standard (particular logarithm of the ratio between the counted radioactivity in
attention was paid to the absence of non-radioactive precursor the n-octanol phase and the counted radioactivity in the
2 (tosyloxy derivative)). For this, an aliquot of the radiotracer aqueous phase.
J. Label Compd. Radiopharm 2014
Copyright © 2014 John Wiley & Sons, Ltd.
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V. Médran-Navarrete et al.
CLogP (calculated LogP)
DPA-C5yne and DPA-C5yne (20 μM) as well as [18F]DPA-C5yne
and Flumazenil (20 μM). Brain sections were then washed
two times for 2 min and once for 10 s with cold (4°C) buffer,
then exposed on a Phosphor-Imager (Storm 860, Molecular
Dynamics, Pharmacia, GE Medical systems, Germany) screen
overnight. Autoradiograms were scanned and then analysed
using the IMAGEJ software (developed by the National Institutes
of Health). A region of interest was manually drawn around the
core of the lesion, and an identical area was copy-pasted
symmetrically into the contralateral hemisphere. Binding in the
region of interest was then expressed as the number of counts
per surface unit. The TBR was calculated as the ratio of the
binding in the lesioned versus the contralateral hemisphere.
CLogP values were determined using CHEMBIODRAW ULTRA 11.0.1
software (Cambridge Soft., Perkin-Elmer, Waltham, MA, USA).
In vitro stability in rat plasma
Sample preparation
Rat plasma (4 mL, prepared from whole blood by centrifugation
(5 min, 3000 g and at 4°C)) was spiked with 40 μL of a 10 mM
DPA-C5yne (1) solution in CH3CN (final concentration: 100 μM,
maximum 1% of organic solvent). Two samples of 200 μL were
first removed, and an equal volume of CH3CN (200 μL) was
added. For each sample, the mixture was centrifuged (4°C,
3500 rpm, 5 min) and the supernatant was collected. 20 μL of
each extract were then injected onto the LC–MS system, and
the peak area corresponding to DPA-C5yne was measured (time
zero). The remainder of the spiked plasma was then incubated at
37°C for 5, 15, 30, 60 and 90 min. For each time point, two
samples were removed and processed as indicated earlier.
Acknowledgements
The authors wish to thank the cyclotron operators Mr Daniel
Gouel, Mr Christophe Lechêne and Mr Tony Catarina for
performing the irradiations. This work was supported by the
CEA-I2BM intramural programmes, as well as the European
Union’s Seventh Framework Programme [FP7/2007–2013]
INMiND (Grant agreement no HEALTH-F2-2011-278850). PhD
students Vincent Médran-Navarrete and Nicholas Bernards were
in part supported by a CEA Irtelis doctoral grant and the EU FP7
INMiND programme (see in the preceding text), respectively.
LC–MS analyses
Liquid chromatography–mass spectrometry analyses were
performed using an Ion Trap LCQ Deca XP + mass
spectrophotometer equipped with an electrospray source
(Thermo Scientific, Les Ulis, France). Pressurized nitrogen was
used as sheath gas with a flow rate of 25 units (arbitrary units
for sheath gas pressure as defined by the manufacturer). The
source voltage for ESI was 4.5 kV and the capillary voltage was
38 V. The capillary temperature was 275°C. The HPLC system
(Thermo Scientific) interfaced with the mass spectrophotometer
consisted of a Surveyor pump (54949 series), a Surveyor
autosampler (55989 series), a photodiode array detector (56470
series) and an analytical Atlantis C18 (Waters) column
(2.1 × 150 mm, porosity 5 μm). The mobile phase consisted of A:
H2O containing 0.05% formic acid and B: CH3CN containing
0.05% formic acid. A linear gradient from 40 to 80% of B in
10 min was applied to the column at a flow rate of 200 μL/min
and returns to the initial conditions within 5 min. The whole
output of the LC column was passed through the photodiode
array detector (190–600nm) before ESI probe of the mass
spectrometer (operated in the positive mode). In the full scan MS
acquisition mode, the instrument method was set up to detect
ions in the range of m/z 50–500. Data acquisition and processing
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