One-step radiosynthesis of [18F]LBT-999: A selective radioligand for the visualization of the dopamine transporter with PET

Article abstract of DOI:10.1002/jlcr.1412

LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3-beta-p-tolyl-8-aza-bicyclo[3.2.1] octane-2-beta-carboxylicacid methyl ester) is a recently developed cocaine derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands (K_D

Full text of DOI:10.1002/jlcr.1412

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 716–723.
Published online in Wiley InterScience
JLCR
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1412
Research Article
One-step radiosynthesis of [18F]LBT-999: a selective
radioligand for the visualization of the dopamine transporter
with PET
1,
2
1
3,4
3
1
´ ´
´
¨
FREDERIC DOLLE *, JULIE HELFENBEIN , FRANC- OISE HINNEN , SYLVIE MAVEL , ZOIA MINCHEVA , WADAD SABA ,
MARIE-ANNE SCHOLLHORN-PEYRONNEAU , HERIC VALETTE , LUCETTE GARREAU3,4, SYLVIE CHALON3,4, CHRISTER
1
1
¨
5
6
7
1
2
¨
HALLDIN , JEAN-CLAUDE MADELMONT , JEAN-BERNARD DELOYE , MICHEL BOTTLAENDER , JOEL LE GAILLIARD ,
DENIS GUILLOTEAU3,4,8 and PATRICK EMOND3,4
1
´
´
´
´ ´
CEA, Service Hospitalier Frederic Joliot, DSV/Institut d’Imagerie BioMedicale, 4 Place du General Leclerc, F-91401 Orsay, France
2 Orphachem, ZATE INSERM U484, rue Montalembert, F-63005 Clermont-Ferrand, France
3 INSERM U619, F-37000 Tours, France
4
´
Universite Franc- ois Rabelais de Tours, F-37000 Tours, France
5 Karolinska Institutet, Department of Clinical Neuroscience, Karolinska Hospital, S-17176 Stockholm, Sweden
6
´
´
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INSERM U484, Laboratoire Etude Metabolique des Molecules Marquees, rue Montalembert, F-63005 Clermont-Ferrand, France
7
8
ˆ
Laboratoires Cyclopharma, Biopole Clermont Limagne, F-63360 Saint Beauzire, France
´
CHRU Tours, 2 boulevard Tonnelle, F-37044 Tours, France
Received 11 May 2007; Revised 23 May 2007; Accepted 25 May 2007
Abstract: LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3-beta-p-tolyl-8-aza-bicyclo[3.2.1]octane-2-beta-carboxylicacid
methyl ester) is a recently developed cocaine derivative belonging to a new generation of highly selective dopamine
transporter (DAT) ligands (KD: 9 nM for the DAT and IC50 > 1000 nM for the serotonin and norepinephrine transporter).
Initial fluorine-18-labelling of LBT-999 was based on the robust and reliable two-step radiochemical pathway often
reported for such tropane derivatives, involving first the preparation of (E)-1-[18F]fluoro-4-tosyloxybut-2-ene followed
by a N-alkylation reaction with the appropriate nor-tropane moiety. In the present work, a simple one-step fluorine-18-
labelling of LBT-999 is reported, based on a chlorine-for-fluorine nucleophilic aliphatic substitution, facilitating as
expected both automation and final high-performance liquid chromatography (HPLC) purification. The process
involves: (A) reaction of K[18F]F–Kryptofix1222 with the chlorinated precursor (3.5–4.5 mg) at 1658C for 10min in
DMSO (0.6 mL) followed by (B) C-18 PrepSep cartridge pre-purification and finally (C) semi-preparative HPLC
purification on a Waters Symmetry1 C-18. Typically, 3.70–5.92 GBq of [18F]LBT-999 (> 95% chemically and
radiochemically pure) could be obtained with specific radioactivities ranging from 37 to 111 GBq/mmol within
85–90min (HPLC purification and Sep-Pak-based formulation included), starting from a 37.0GBq [18F]fluoride batch
(overall radiochemical yields: 10–16%, non-decay-corrected). Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: fluorine-18; LBT-999; dopamine transporter; tropane
Introduction
boxylic acid methyl ester) belongs to a new generation
of highly selective dopamine transporter (DAT) ligands
(KD: 9 nM for the DAT and IC50 > 1000 nM for the
serotonin and norepinephrine transporter1,2). As
already reported, LBT-999 has been successfully
labelled with the positron-emitter carbon-11 (half-life:
20.38 min) at its methyl ester function from the
corresponding carboxylic acid precursor, allowing pre-
liminary in vivo pharmacological evaluation of this
radiotracer by both biodistributions in rodents and
brain imaging in non-human primates with positron
The cocaine derivative LBT-999 (1, 8-((E)-4-fluoro-but-
2-enyl)-3b-p-tolyl-8-aza-bicyclo[3.2.1]octane-2b-car-
*Correspondence to: Fre´de´ric Dolle´, CEA, Service Hospitalier Fre´de´ric
Joliot, Institut d’Imagerie BioMe´dicale, 4 Place du Ge´ne´ral Leclerc,
F-91401 Orsay, France. E-mail: frederic.dolle@cea.fr
Contract/grant sponsor: Re´seau National de Technologies pour la
Sante´ program; contract/grant number: RNTS 03B243 FLUOPARK
Contract/grant sponsor: Biotechnocentre, Re´gion Centre, France
Contract/grant sponsor:EC-FP6-project DiMI; contract/grant number:
LSHB-CT-2005-512146
Copyright # 2007 John Wiley & Sons, Ltd.

ONE-STEP RADIOSYNTHESIS OF [18F]LBT-999 717
emission tomography (PET).1–3 The pharmacological
characteristics so far obtained demonstrate that LBT-
999 is a highly promising candidate for in vivo explora-
tion of the DAT in humans.
(Institut fu¨r Kernchemie, Universitat Mainz, Germany)
¨
presented also a similar one-step radiochemical synth-
esis of this radioligand (in this case, a tosyloxy-for-
fluorine substitution), highlighting the growing interest
for [18F]LBT-999.
LBT-999 had been designed with a fluorine atom in
its structure (Figure 1), allowing ultimate labelling with
fluorine-18, a longer-lived positron-emitter (half-life:
109.8 min) and today one of the most attractive PET
isotopes for radiopharmaceutical chemistry.4–7 The
robust and reliable pathway frequently proposed for
the labelling of tropane derivatives with fluorine-18 is a
two-step radiochemical process, involving first the
preparation of an o-halogeno- or better o-sulphony-
loxyalkyl[18F]fluoride reagent and secondly its coupling
to the appropriate nor-tropane moiety. This methodol-
ogy, which has been applied with success to the
Results and discussion
Chemistry
The target compound LBT-999 (1) was synthesized in
50% yield from 3b-p-tolyl-8-aza-bicyclo[3.2.1]octane-
2b-carboxylic acid methyl ester (5)36 and (E)-1-fluoro-
4-tosyloxy-2-butene (6) according to already reported
procedures1 (Scheme 1B). Its chloro analogue 7 as
precursor for labelling with fluorine-18 was synthe-
sized from the same nor-tropane 5 but using (E)-1-
preparation of several
18F]fluoropropyl-8,9,20–26 derivatives like [18F]FECNT
[
18F]fluoroethyl-8–19 and
[
chloro-4-tosyloxy-2-butene (4). Compound
4 was
and b-[18F]CIT-FP (Figure 1), is based on the well-
established radiosyntheses of n-bromo-, n-tosyloxy-
and n-mesyloxy-1-[18F]fluoroalkanes (n ¼ 1–3) from
the corresponding bifunctional alkanes by nucleophilic
aliphatic substitution with no-carrier-added [18F]fluor-
ide ion as its activated K[18F]F–Kryptofix1222 com-
plex27,28 as originally studied by Block et al.29 Based
on the radiosynthesis of (E)-[18F]FBClNT (Figure 1), to
our knowledge the first 4-[18F]fluoro-2-butenyl deriva-
tive reported,30,31 LBT-999 was successfully labelled
with fluorine-18 in two radiochemical steps, involving
the preparation of (E)-1-[18F]fluoro-4-tosyloxy-2-bu-
tene32,33 permitting further evaluation of this radioli-
gand in non-human primates.
prepared in 20% overall yield (Scheme 1A) from (E)-2-
butene-1,4-diol (2)37,38 using the following two-step
sequence: monochlorination of
2 using 1.1 eq. of
triphenylphosphine in refluxing tetrachloromethane
(27% yield) followed by tosylation of the remaining
alcohol function (using 1.1 eq. of tosyl chloride in
dioxane containing triethylbenzylammonium chloride
and aqueous potassium hydroxyde, 75% yield). Con-
densation of 5 with 4 in acetonitrile gave the chloro
derivative 7 in 18% yield (Scheme 1B).
Radiochemistry
In the present work, a simple one-step fluorine-18-
labelling of LBT-999 is reported, based on a chlorine-
for-fluorine nucleophilic aliphatic substitution, in
order to facilitate both the automation and the final
purification process. These results were presented at
the XVIIth International Symposium on Radiopharma-
ceutical Sciences (Aachen, Germany, 30 April–4th May
2007),34 in the same meeting, Riss and Roesch35
LBT-999 (1) was labelled with fluorine-18 at its 4-
fluoro-2-butenyl moiety from the corresponding chloro
analogue 7 using the one-step radiochemical process
outlined in Scheme 2.
Fluorination with the cyclotron-produced [18F]fluor-
ide as the, no-carrier-added, activated K[18F]F–Krypto-
fix1222 complex27,28 was performed in DMSO using
10–13 mmol of precursor at 1658C for 10 min without
stirring the contents. After cooling the reaction vessel,
96 to over 99% of the initial radioactivity was still
present. The radiochemical yields of fluorine-18 in-
corporation, calculated from the thin-layer chromato-
graphy (TLC)-radiochromatogram and defined as the
ratio of radioactivity area of [18F]-1 over total fluorine-
18 radioactivity area, were about 30–40%. At this stage,
a C-18 PrepSep cartridge was used to rapidly isolate
18F
18F
()
n
N
N
CO2CH3
CO2CH3
R
R
[
18F]-1 (with a radiochemical purity > 90%, according to
R
R
n
1
2
radio-HPLC) from the reaction mixture, which repre-
sented 30–35% of the total radioactivity amount
engaged in the fluorination process whereas most likely
unreacted [18F]fluoride, 65–70% of the total radio-
activity, was eluted off the cartridge as waste. These
CH3 [18F]LBT-999 ([18F]-1)
Cl (E)-[18F]FBClNT
18F]FECNT
Cl
I
[
β-[18F]CIT-FP
Figure
1 Chemical structures of [
18F]LBT-999 and pre-
viously reported [18F]fluorinated tropane-analogues.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 716–723
DOI: 10.1002.jlcr

´
718 F. DOLLE ET AL.
TsCl, BnEt3NCl
aq. 50% KOH
CCl4/PPh3
reflux, 4 hrs
OH
OH
OTs
HO
Cl
Cl
CH2Cl2
0°C, 4 hrs
A
2
3 (27%)
4 (75%)
X
OTs
H
N
R
N
CO2CH3
CO2CH3
4 (R=Cl)
6 (R=F)
7 (X=Cl) (18%)
1 (X=F) LBT-999 (50%)
CH3CN
RT, 16 hrs
5
CH3
CH3
B
Scheme 1
18F
18
Cl
K[ F]F-K
222
K CO , DMSO
N
N
CO2CH3
2
3
CO2CH3
165˚C, 10 min
followed by
cartridge purification
(Prep-SepTM C-18)
and
CH3
CH3
[18F]LBT-999 ([18F]-1)
7 (3.5-4.5 mg)
HPLC purification
®
(Symmetry C-18)
Scheme 2
values clearly confirmed the radiochemical yields
measured by radio-TLC.
tion yields (calculated from the TLC-radiochromato-
gram or after the C-18 PrepSep cartridge purification)
were similar to those described above, with, however,
formation in significantly higher ratio of the unidenti-
fied fluorine-18-labelled side product (about 40–50%
compared to [18F]-1). HPLC purification using HPLC A
system gave nevertheless > 95% chemically and radio-
chemically pure [18F]-1, still well separated from the
side product.
Formulation of [18F]LBT-999 ([18F]-1) as an i.v.
injectable solution was performed using a home-made
Sep-Pak1Plus C18 device. The HPLC-collected fraction
containing the radiotracer was diluted with water and
the resulting solution was passed through a C18 Sep-
Pak1 cartridge. The cartridge was then washed with
water, partially dried with nitrogen and finally eluted
with ethanol followed by physiological saline. The solu-
tion was then sterile-filtered and diluted with physio-
logical saline to an ethanol concentration below 10%.
Quality controls of [18F]LBT-999 ([18F]-1) were per-
formed on an aliquot of the preparation ready for i.v.
injection. The radiotracer preparation was a clear and
colourless solution with a measured pH between 5 and
7. As demonstrated by analytical HPLC analysis (HPLC
C, see Experimental), the radiotracer preparation was
found to be > 95% chemically and radiochemically pure
(1, tR: 2.25 min). The preparation was also shown to be
free of the non-radioactive precursor, the chloro
HPLC purification of
performed on
semi-preparative Symmetry1 C18
[
18F]LBT-999 ([18F]-1) was
a
column (HPLC A, see Experimental), using a mixture
of H2O, CH3CN and TFA as the eluent. Using these
conditions, [18F]-1 (tR: 12.5–13.5 min) could be ob-
tained with > 95% chemical and radiochemical purity
and was completely separated within a short total
purification time (less than 20 min) from the non-
labelled chloro-derivative 7 (tR: 15.5–17.5 min), as well
as from an unidentified fluorine-18-labelled side pro-
duct at tR: 14.0–15.0 min (up to 10% compared to [18F]-1).
At least, two chemical structures can be proposed for
this side product: one resulting from a cis–trans
isomerization (leading to the (Z)-isomer) and the other
resulting from the addition of the fluorine atom to the
double bond with concomitant shift of the latter to the
terminal position (leading to the 2-fluoro-3-butenyl-
isomer). However, no further efforts were made to
identify this side product.
Noteworthy, a poor separation (leading to a too low –
90% – chemical purity) was observed with the HPLC
system previously used for the purification of [18F]-1 in
the old two-step process (HPLC B, see Experimental, tR
([18F]-1): 9.0–10.0 min).33
Introduction of fluorine-18 was also performed using
microwave activation (at 100 W for 2 min). Incorpora-
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 716–723
DOI: 10.1002.jlcr

ONE-STEP RADIOSYNTHESIS OF [18F]LBT-999 719
derivative 7 (tR: 3.45 min) as well as the unidentified
fluorine-18-labelled side product (tR: 2.58 min), and
was chemically and radiochemically stable for at least
120 min. These results were in compliance with our in-
house quality control/assurance specifications.
containing Low-UV PIC1 B7 reagent (20 mL for
1000 mL); solvent B: H2O/CH3CN: 30:70 (v/v) contain-
ing Low-UV PIC1 B7 reagent (20 mL for 1000 mL)]; flow
rate: 2.0 mL/min; temperature: 308C; absorbance
detection at l ¼ 205 nm.
Typically, using the one-step synthesis described
herein, 3.70–5.92 GBq (100–160 mCi) of [18F]LBT-999
([18F]-1) ready to use could be obtained with a specific
radioactivity of 37–111 GBq/mmol (1–3 Ci/mmol) within
85–90 min (HPLC purification and Sep-Pak-based for-
mulation included), starting from a 37.0 GBq (1.0 Ci)
Spectroscopies and elemental analyses. NMR spectra
were recorded on a Bruker (Wissembourg, France) DPX
Avance (200 MHz) apparatus using the hydrogenated
residue of the deuterated solvent CDCl3 (d ¼ 7:30 ppm)
or TMS (d ¼ 0 ppm) as internal standards for 1H-NMR
as well as the deuterated solvent CDCl3 (d ¼ 77:0 ppm)
as internal standard for 13C-NMR. The chemical shifts
are reported in ppm, downfield from TMS (s, d, t, m for
singlet, doublet, triplet and multiplet, respectively). The
mass spectra (MS) were measured on a Hewlett-
Packard (Issy les Moulineaux, France) 5989A GC/EI-
[
18F]fluoride batch (overall non-decay-corrected iso-
lated radiochemical yield: 10–16%).
Experimental
General
MS spectrometer (EI at 70 eV) and on
a Bruker
Chemicals, flash chromatography and TLC analysis.
Chemicals, except (R)-cocaine, were purchased from
Aldrich-, Fluka- or Sigma France and were used
without further purification. Flash chromatographies
were conducted on silica gel or alumina gel (0.63–
0.200 mm, VWR) columns. TLCs were run on pre-
coated plates of silica gel 60F254 (Merck). The com-
pounds were localized (1) when possible at 254 nm
using a UV-lamp and/or (2) by dipping the TLC-plates
in a 1% ethanolic ninhydrin solution and heating on
a hot plate. Radioactive spots were detected using
(Wissembourg, France) Esquire-LC spectrometer
(ESþ). Elemental analyses were performed by the
Service d’Analyses du CNRS (Vernaison, France) and
results were within ꢁ0:4% of theoretical values.
Radioisotope production. No-carrier-added aqueous
[
18F]fluoride ion was produced via the [18O(p,n)18F]
nuclear reaction by irradiation of a 2 mL [18O]water
(> 97%-enriched, CortecNet, Paris, France) target on
an IBA Cyclone-18/9 cyclotron (18 MeV proton
beam) and was transferred to the appropriate hot cell.
Target hardware: commercial, 2-mL, two-port, stain-
a
Berthold TraceMaster 20 automatic TLC linear
analyser.
less steel target holder equipped with
a domed-
end niobium cylinder insert. Target to hot cell liquid-
transfer system: 60 m PTFE line (0.8 mm internal
HPLC analysis. [HPLC A]: Equipment: system equipped
with a Waters 600 pump and Waters 600 Controller, a
Shimadzu SPD10-AVP UV-multi-wavelength detector and
a miniature ionization chamber probe; column: semi-
preparative Symmetry1 C18, Waters (300 ꢀ 7.8 mm);
porosity: 7 mm; eluent H2O/CH3CN/TFA: 72:28:0.1
(v/v/v); flow rate: 5 mL/min; temperature: RT; absor-
bance detection at l ¼ 220 nm. [HPLC B]: Equipment:
system equipped with a Waters 600 pump and Waters
600 Controller, a Shimadzu SPD10-AVP UV-multi-
wavelength detector and a miniature ionization cham-
ber probe; column: semi-preparative SunFireTM C18,
Waters (250 ꢀ 10.0 mm); porosity: 5 mm; eluent H2O/
CH3CN/TFA: 70:30:0.1 (v/v/v); flow rate: 5 mL/min;
temperature: RT; absorbance detection at l ¼ 220 nm.
[HPLC C]: Equipment: Waters Alliance 2690 (or a
Waters binary HPLC pump 1525) equipped with a UV
spectrophotometer (Photodiode Array Detector, Waters
1
diameter; 16 in external diameter), 2.0 bar helium drive
pressure, transfer time 3–6 min. Typical production of
[
18F]fluoride at the end of bombardment for a 20 mA,
30 min (10 mA h) irradiation: 27.7–29.6 GBq (750–
800 mCi).
Miscellaneous. Radiosyntheses using fluorine-18, in-
cluding the HPLC purifications, were performed in a
7.5-cm-lead shielded cell using a computer-assisted
Zymate-XP robot system (Zymark corporation, USA).
Microwave activation was performed with a MicroWell
10 oven (2.45 GHz), Labwell AB, Sweden.
Chemistry
(E)-1-Chloro-4-tosyloxy-2-butene (4). Step 1. To a CCl4
solution (100 mL) containing (E)-2-butene-1,4-diol37,38
(2, 10.0 g, 113 mmol), triphenylphosphine (33.3 g,
127 mmol, 1.1 eq.) was added . The resulting mixture
was vigorously stirred and refluxed for 4 h. The mixture
was then cooled to room temperature and diluted with
996) and
a Berthold LB509 radioactivity detector;
column: analytical Symmetry-M1 C-18, Waters
(50 ꢀ 4.6 mm); porosity: 5.0 mm; conditions: isocratic
elution with solvA/solvB: 55/45 (v/v) [solvent A: H2O
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 716–723
DOI: 10.1002.jlcr

´
720 F. DOLLE ET AL.
CH2Cl2 (200 mL). The organic layer was washed with
H2O (2 ꢀ 100 mL), dried over MgSO4 and concentrated
to dryness under reduced pressure. Purification by
flash chromatography (CH2Cl2) gave pure (E)-1-chloro-
4-hydroxy-2-butene (3, 3.3 g, 27%). 1H-NMR (CDCl3): d:
1.69 (b, 1H); 4.06 (dd, 3J ¼ 4:8 Hz, 4J ¼ 1:0 Hz, 2H);
4.16 (d, J3 ¼ 3:6 Hz, 2H); 5.79–6.01 (m, 2H). 13C-NMR
(CDCl3): d: 44.4; 62.5; 127.0; 133.8. Step 2. To a
CH2Cl2 solution (40 mL) containing (E)-1-chloro-4-
hydroxy-2-butene (3, 2.0 g, 18.8 mmol) and benzyl-
triethylammonium chloride (220 mg, 0.96 mmol,
0.05 eq.) was added an aq. 50% KOH solution (40 mL).
The mixture was cooled to 08C, then a CH2Cl2 solution
(40 mL) containing p-toluenesulphonyl chloride (3.93 g,
20.6 mmol, 1.1 eq.) was added dropwise while main-
taining the temperature below 08C. The reaction
mixture was stirred for another 4 h at 08C and then
diluted with H2O (40 mL). The aqueous layer was
separated and extracted with CH2Cl2 (2 ꢀ 80 mL). The
organic layers were combined, washed with brine
(2 ꢀ 30 mL), dried over MgSO4 and concentrated to
dryness under reduced pressure to give pure (E)-1-
chloro-4-tosyloxy-2-butene (4, 3.65 g, 75%) which was
further used without purification. 1H-NMR (CDCl3): d:
2.45 (s, 3H); 3.99 (d, 2H, 3J ¼ 5:6 Hz); 4.55 (d, 2H,
3J ¼ 5:1 Hz); 5.82–5.90 (m, 2H); 7.39 (d, 2H,
3J ¼ 8:2 Hz); 7.79 (d, 2H, 3J ¼ 8:2 Hz). 13C-NMR
(CDCl3): d: 21.7; 43.5; 69.4; 126.4; 128.0 (2C); 130.1
(2C); 131.7; 133.2; 145.1.
8-((E)-4-Fluoro-but-2-enyl)-3b-p-tolyl-8-aza-bicyclo
[3.2.1]octane-2b-carboxylic acid methyl ester (1, LBT-
999). Synthesized
from
3b-p-tolyl-8-aza-bicy-
clo[3.2.1]octane-2b-carboxylic acid methyl ester (5)
and (E)-1-fluoro-4-tosyloxy-2-butene (6) according to
Dolle´ et al.1 1H-NMR (CDCl3): d: 1.57–1.68 (m, 3H);
1.90–2.00 (m, 2H); 2.22 (s, 3H); 2.65 (td, 1H,
2
3J ¼ J ¼ 12:4 Hz, 3J ¼ 2:6 Hz); 2.92–3.05 (m, 4H);
3.46 (m, 1H); 3.53 (s, 3H); 3.70 (m, 1H); 4.86 (dd, 2H,
2JH2F ¼ 47:2 Hz, 3J ¼ 4:6 Hz); 5.80–5.85 (m, 2H); 7.10
(d, 2H, 3J ¼ 8:0 Hz); 7.20 (d, 2H, 3J ¼ 8:0 Hz). 13C-NMR
(CDCl3): d: 20.9; 25.8; 26.0; 33.7; 34.0; 50.8; 52.6;
1
54.8; 61.3; 62.2; 83.0 (d, JC2F ¼ 161:0 Hz); 126.1 (d,
2JC2F ¼ 17:1 Hz); 127.1 (2C); 128.5 (2C); 134.1 (d,
3JC2F ¼ 11:6 Hz); 135.1; 139.8; 171.9. MS: m/e (%):
331 (M+, 24); 272 (13); 258 (6); 180 (7); 154 (46); 141
(97); 140 (100); 122 (66); 108 (60); 68 (33); 53 (35).
Anal. Calcd C20H26FNO2: C, 72.48; H, 7.91; N, 4.23;
found: C, 72.26; H, 7.94; N, 4.21.
8-((E)-4-Chloro-but-2-enyl)-3b-p-tolyl-8-aza-bicyclo
[3.2.1]octane-2b-carboxylic acid methyl ester (7). To a
08C solution of (E)-1-chloro-4-tosyloxy-2-butene (4,
1.5 g, 5.8 mmol, 1.5 eq.) and triethylamine (800 mL,
5.8 mmol, 1.5 eq.) in acetonitrile (10 mL) was added
dropwise 3b-p-tolyl-8-aza-bicyclo[3.2.1]octane-2b-car-
boxylic acid methyl ester (5, 1.0 g, 3.86 mmol, 1.0 eq.)
dissolved in acetonitrile (20 mL). The resulting reaction
mixture was stirred at room temperature overnight and
then concentrated to dryness under reduced pressure.
The residue was finally purified by flash chromatogra-
phy on alumina gel (pentane/CH2Cl2: 50/50 to 0/100)
to give 8-((E)-4-chloro-but-2-enyl)-3b-p-tolyl-8-aza-bi-
cyclo[3.2.1]octane-2b-carboxylic acid methyl ester (7)
as a white solid (250 mg, 18%). 1H-NMR (CDCl3): d:
1.58–1.69 (m, 3H); 1.95–2.05 (m, 2H); 2.29 (s, 3H);
(E)-1-Fluoro-4-tosyloxy-2-butene (6). Synthesized from
(E)-2-butene-1,4-diol (2)37,38 according to Dolle´ et al.1
1H-NMR (CDCl3): d: 2.45 (s, 3 H); 4.58–4.62 (m, 2 H);
4.85 (dd, 2 H, JH2F ¼ 46:4 Hz, 3J ¼ 4:8 Hz); 5.81–5.92
2
(m, 2 H); 7.37 (d, 2H, 3J ¼ 8:3 Hz); 7.81 (d, 2H,
3J ¼ 8:3 Hz). 13C-NMR (CD2Cl2): d: 21.7; 69.9; 82.3 (d,
2
2.60 (td, 1H, 3J ¼ J ¼ 12:6 Hz, 3J ¼ 2:6 Hz); 2.90–2.96
3
1JC2F ¼ 162:7 Hz); 125.8 (d, JC2F ¼ 12:2 Hz); 128.2
(m, 4H); 3.41 (m, 1H); 3.51 (s, 3H); 3.66 (1H); 4.05 (d,
2H, 3J ¼ 3:1 Hz); 5.70–5.75 (m, 2H); 7.08 (d, 2H,
3J ¼ 8:2 Hz); 7.16 (d, 2H, 3J ¼ 8:2 Hz). 13C-NMR
(CDCl3): d: 21.1; 26.0; 26.1; 34.0; 34.2; 44.9; 51.1;
52.9; 54.9; 61.5; 62.4; 127.3 (2C); 127.6; 128.8 (2C);
133.7; 135.3; 140.0; 172.1. MS: ES [MHþ] Calcd:
348.17; found: 348.32.
2
(2C); 130.3 (2C); 130.8 (d, JC2F ¼ 17:3 Hz); 133.4;
145.6. MS: m/e (%): 225 (3); 224 (7); 155 (32); 92 (37);
91 (100); 89 (19); 72 (62); 65 (56); 39 (52).
3b-p-Tolyl-8-aza-bicyclo[3.2.1]octane-2b-carboxylic
acid methyl ester (5). Synthesized from (R)-cocaine
hydrochloride according to Emond et al.36 1H-NMR
(CDCl3): d: 1.60–1.85 (m, 3H); 1.93–2.15 (m, 2H); 2.33
(s, 3H); 2.43 (td, 1H, 2J ¼ 13:1 Hz, 3J ¼ 2:7 Hz); 2.75
(dd, 1H, 3J ¼ 5:9, 1.9 Hz); 2.95 (s, 1H); 3.19 (dt, 1H,
3J ¼ 13:1, 5.5 Hz); 3.41 (s, 3H); 3.74 (m, 2H); 7.11 (s,
4H). 13C-NMR (CDCl3): d: 21.4; 27.6; 29.5; 34.3; 35.7;
51.5; 51.6; 54.1; 56.7; 127.6 (2C); 129.3 (2C); 136.3;
139.6; 174.4. MS: m/e (%): 259 (Mþ; 22); 228 (3); 200
(5); 170 (5); 141 (10); 115 (14); 84 (70); 83 (93); 69 (80);
68 (100).
Radiochemistry
Preparation of the K[18F]F–K222 complex. In order to
recover and recycle the [18O]water target, 2 mL of
aqueous
[
18F]fluoride from the target holder was
passed through an anion-exchange resin (Sep-Pak1
Light Waters AccellTM Plus QMA cartridge, chloride
form, conditioned by washing with aq. 1 M NaHCO3
(2 mL) and rinsing with water (20 mL) and CH3CN
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 716–723
DOI: 10.1002.jlcr

ONE-STEP RADIOSYNTHESIS OF [18F]LBT-999 721
(10 mL)) by helium pressure (1.5–2.0 bar). Helium was
blown through the cartridge to maximally extract the
last traces of [18O]water. The [18F]fluoride ion was then
eluted from the resin, using an aq. K2CO3 solution
(1.0 mL of a 1.0 mg/mL solution), into a Vacutainer1
tube containing Kryptofix1222 (K222: 4,7,13,16,21,24-
nitrogen stream for 3–5 min. Finally, the residue was
redissolved in the HPLC solvent used for purification
(1.0 mL) and the crude was injected onto HPLC (HPLC
A). Isocratic elution gave pure
[
18F]-1 (tR: 12.5–
13.5 min), well separated from unlabelled 7 (tR: 15.5–
17.5 min) and the unidentified fluorine-18-labelled side
product (tR: 14.0–15.0 min). B. Procedure using micro-
wave activation. Same as procedure A, but instead of
the heating block a dedicated microwave oven (at
100 W, for 2 min) was used.
hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane,
12.0–
15.0 mg). The resulting solution was then gently
concentrated to dryness at 145–1508C under a nitrogen
stream for 10 min to give no-carrier-added K[18F]F–
K222 complex as a white semi-solid residue.
Formulation of [18F]LBT-999 ([18F]-1). Formulation of the
labelled product for i.v. injection was effected as
follows: the HPLC-collected fraction containing the
radiotracer was diluted with water (50 mL). The result-
ing solution was passed through a Sep-Pak1Plus C18
cartridge (Waters, washed with 2 mL of EtOH and then
rinsed with 10 mL of water prior to use). The cartridge
was washed with water (10 mL) and partially dried by
applying a nitrogen stream for 10 s. The radiotracer
was eluted with 2 mL of EtOH (less than 10% of the
total radioactivity was left on the cartridge) followed by
8 mL of physiological saline and filtered on a 0.22 mm
GS-Millipore filter (vented). Finally, physiological saline
was added to take the EtOH concentration below 10%.
This whole process was performed using a remote-
controlled dedicated home-made device based on a
literature procedure.40
Preparation of 8-((E)-4-[18F]fluoro-but-2-enyl)-3b-p-to-
lyl-8-aza-bicyclo[3.2.1]octane-2b-carboxylic acid
methyl ester ([18F]-1, [18F]LBT-999). A. Procedure using
conventional heating. DMSO (600 mL) containing 8-
((E)-4-chloro-but-2-enyl)-3b-p-tolyl-8-aza-bicyclo[3.2.1]
octane-2b-carboxylic acid methyl ester (7, 3.5–4.5 mg,
10.0–12.9 mmol) was added into the Vacutainer1 tube
containing the dried K[18F]F–K222 complex. The tube
(not sealed) was thoroughly vortexed (30 s) and then
placed in a heating block (at 1658C, for 10 min) without
stirring the contents. The reaction vessel was then
cooled using an ice-water bath, the remaining radio-
activity was measured (which should be >96% of the
initial radioactivity) and the reaction mixture was
analysed by radio-TLC. The reaction yield was calcu-
lated from the TLC-radiochromatogram and defined as
the ratio of radioactivity area of [18F]LBT-999 ([18F]-1)
over total fluorine-18 radioactivity area (SiO2-TLC
(EtOAc):Rf:[18F]-1:0.5 and Rf:[18F]fluoride ion:0.0). The
reaction mixture was diluted with water (1 mL) and
transferred onto a C18 cartridge (PrepSepTM R-C18
Extraction Column, Fisher Scientific, activated before-
hand with EtOH (2 mL) and then rinsed with water
(10 mL)), pre-filled with water (2 mL). The tube was
rinsed twice with water (1 mL), which was also trans-
ferred and added to the diluted reaction mixture on top
of the cartridge (0.5–1.5% of the total radioactivity
amount engaged in the fluorination process was lost in
the initial tube). An additional portion of water (2 mL)
was further added to the diluted reaction mixture on
top of the cartridge. The whole mixture was then
passed through the cartridge, which was then washed
with water (3 mL) and partially dried for 0.5 min by
applying a nitrogen stream. [18F]-1 was eluted from the
cartridge with CH2Cl2 (3 mL) into an empty 5-mL
reaction vial. Elution was repeated twice with 1 mL of
CH2Cl2 for maximal transfer of [18F]-1 (only 1–2% was
left and lost on the cartridge). The incorporation yield
was estimated after the C18 cartridge elution by the
CH2Cl2 over the total eluted radioactivity (DMSO/
H2OþCH2Cl2) ratio. The eluted CH2Cl2 solution was
concentrated to dryness at 65–758C under a gentle
Quality control of [18F]LBT-999 ([18F]-1). The radiotracer
preparation was visually inspected for clarity, absence
of colour and particulates. An aliquot of the prepara-
tion was removed for determination of pH using
standard pH paper. Chemical and radiochemical pu-
rities were also assessed on this aliquot by HPLC (HPLC
C), with
a sample of authentic 1 (tR: 2.25 min).
Particular attention was paid to the absence of non-
radioactive precursor 7 (tR: 3.45 min). Chemical and
radiochemical stability of the entire preparation was
tested by HPLC (HPLC C) at regular 15-min intervals
during 120 min. Specific radioactivity of the radiotracer
was calculated from three consecutive HPLC (HPLC C)
analyses (average) and determined as follows: the area
of the UV absorbance peak corresponding to the
radiolabelled product was measured (integrated) on
the HPLC chromatogram and compared to a standard
curve relating mass to UV absorbance.
Conclusion
The highly selective dopamine transporter ligand LBT-
999 (1) has been labelled in one radiochemical step
with fluorine-18 and its synthesis represents to our
knowledge the first example of a direct nucleophilic
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 716–723
DOI: 10.1002.jlcr

´
722 F. DOLLE ET AL.
fluorination with [18F]fluoride leading to a 4-fluoro-2-
butenyl-containing PET ligand. The one-step process
described herein gives improved overall isolated radio-
chemical yields and now replaces our former two-step
preparation32,33 of [18F]LBT-999 ([18F]-1). Moreover,
this process is 10 min shorter and appears to be more
reliable (> 20 radiosyntheses performed to date).39
7. Schubiger PA, Lehmann L, Friebe M. In PET
Chemistry: The Driving Force in Molecular Imaging,
Ernst Schering Research Foundation (ed.). Springer:
Berlin, 2007; 1–339.
8. Goodman MM, Kabalka GW, Kung MP, Kung HF,
Meyer MA. J Label Compd Radiopharm 1994; 34:
488–490.
[
18F]LBT-999 is currently under evaluation and its
9. Firnau G, Chen JJ, Murthy D. J Label Compd
Radiopharm 1995; 37: 55–56.
in vivo pharmacological profile will be compared to that
of [11C]LBT-999.
10. Goodman MM, Kilts CD, Keil R, Shi B, Martarello L,
Xing D, Votaw J, Ely TD, Lambert P, Owens MJ,
Camp VM, Malveaux E, Hoffman JM. Nucl Med Biol
2000; 27: 1–12.
Acknowledgements
The authors wish to thank the cyclotron operators Mr
Daniel Gouel, Mr Christophe Peronne and Mr Chris-
tophe Lecheˆne for performing the irradiations. The
authors also wish to thank Dr Dirk Roeda for proof-
reading the manuscript and suggesting linguistic
corrections. This work was supported in part by the
EC-FP6-project DiMI (LSHB-CT-2005-512146), the
‘Re´seau National de Technologies pour la Sante´’
program (RNTS 03B243 FLUOPARK) and a grant from
Biotechnocentre, Re´gion Centre, France.
11. Deterding TA, Votaw JR, Wang CK, Eshima D,
Eshima L, Keil R, Malveaux E, Kilts CD, Goodman
MM, Hoffman JM. J Nucl Med 2001; 42: 376–381.
12. Votaw JR, Howell LL, Martarello L, Hoffman JM,
Kilts CD, Lindsey KP, Goodman MM. Synapse
2002; 44: 203–210.
13. Wilcox KM, Lindsey KP, Votaw JR, Goodman MM,
Martarello L, Carroll FI, Howell LL. Synapse 2002;
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14. Davis MR, Votaw JR, Bremner JD, Byas-Smith
MG, Faber TL, Voll RJ, Hoffman JM, Grafton ST,
Kilts CD, Goodman MM. J Nucl Med 2003; 44:
855–861.
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