Radiosynthesis of [18F]LBT-999, a selective radioligand for the visualization of the dopamine transporter with PET

Article abstract of DOI:10.1002/jlcr.1092

LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3β-p-tolyl-8-aza-bicyclo[3.2.1] octane-2β-carboxylic acid methyl ester) is a cocaine derivative belonging to a new generation of highly selective dopamine transporter ligands (K _D:9 nM). LBT-999 was labelled

Full text of DOI:10.1002/jlcr.1092

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 687–698.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1092
Research Article
Radiosynthesis of [18F]LBT-999, a selective radioligand
for the visualization of the dopamine transporter with
PET
Frede
ric Dolle1,*, Franc-oise Hinnen1, Patrick Emond2, Sylvie Mavel2,
´ ´ ´
Zoıa Mincheva2, Wadad Saba1, Marie-Anne Schollhorn-Peyronneau1,
¨
¨
Heric Valette1, Lucette Garreau2, Sylvie Chalon2, Christer Halldin3,
Julie Helfenbein4, Joel Legaillard4, Jean-Claude Madelmont5,
Jean-Bernard Deloye6, Michel Bottlaender1 and Denis Guilloteau2
¨
1
´ ´
CEA, DSV, DRM, SHFJ, Departement de Recherche Medicale, Service Hospitalier
´ ´ ´ ´
Frederic Joliot, 4 Place du General Leclerc, F-91401 Orsay, France
2
´
INSERM U619, CHRU de Tours, Universite Franc-ois-Rabelais de Tours, 31 avenue
Monge, F-37200 Tours, France
3 Karolinska Institutet, Department of Clinical Neuroscience, Psychiatry Section,
Karolinska Hospital, S-17176 Stockholm, Sweden
4 Orphachem, ZATE INSERM U484, rue Montalembert, F-63005 Clermont-Ferrand,
France
5
´
´
´
INSERM U484, Laboratoire Etude Metabolique des Molecules Marquees, rue
Montalembert, F-63005 Clermont-Ferrand, France
6 Laboratoires Cyclopharma, Biopoˆle Clermont Limagne, F-63360 Saint Beauzire,
France
Summary
LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3b-p-tolyl-8-aza-bicyclo[3.2.1]octane-2b-carboxylic
acid methyl ester) is a cocaine derivative belonging to a new generation of highly
selective dopamine transporter ligands (KD:9 nM). LBT-999 was labelled with
fluorine-18 at its fluoromethylvinyl moiety using the following two-step radiochemical
process: (a) No-carrier-added nucleophilic aliphatic radiofluorination from (E)-1,
4-ditosyloxybut-2-ene and the activated K[18F]F-Kryptofix1222 complex in acetoni-
trile at 708C for 10 min giving (E)-1-[18F]fluoro-4-tosyloxybut-2-ene, followed by (b)
condensation of the latter with 3b-p-tolyl-8-aza-bicyclo[3.2.1]octane-2b-carboxylic
acid methyl ester in N,N-dimethylformamide containing potassium iodide for 20 min
*Corresponding to: Dr Frede
´ ´
Medicale, CEA 4 place du Ge
ric Dolle, Service Hospitalier Frederic Joliot, Departement de Recherche
´ ´ ´ ´
ral Leclerc, F-91401 Orsay, France. E-mail: frederic.dolle@cea.fr
´
´
ne
´
Contract/grant sponsor: EC-FP6-project; contract/grant number: LSHB-CT-2005-512146
Copyright # 2006 John Wiley & Sons, Ltd.

´
F. DOLLE ET AL.
688
at 908C and (c) HPLC purification (SunFireTM C18, eluent H2O/CH3CN/TFA
(72:28:0.1 (v/v/v)). Radiochemically pure (> 95%) [18F]LBT-999 (2.03–2.96 GBq, 37–
111 GBq/mmol) was obtained in 95–100 min starting from a 44.4 GBq [18F]fluoride ion
production batch (4.6–6.7% non-decay-corrected overall yield). Copyright # 2006
John Wiley & Sons, Ltd.
Received 28 April 2006; Revised 9 May 2006; Accepted 9 May 2006
Key Words: Fluorine-18; LBT-999; dopamine transporter; tropane
Introduction
LBT-999 (1, 8-((E)-4-fluoro-but-2-enyl)-3b-p-tolyl-8-aza-bicyclo[3.2.1]octane-
2b-carboxylic acid methyl ester, Figure 1) is a 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 serotonine and norepinephrine
transporter).1 Recently, 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 and the methylation reagent
[11C]methyl triflate,2 permitting preliminary in vivo pharmacological evalua-
tion of this radiotracer by both biodistributions in rodents and brain imaging
in non-human primates with positron emission tomography (PET).1,2 Taken
together, the results so far obtained demonstrate that LBT-999 is a highly
promising candidate for in vivo exploration of DAT in humans, using PET.1,2
The chemical structure of LBT-999 permits also its labelling at its
fluoromethylvinyl moiety with fluorine-18 (half-life: 109.8 min), today one of
the most attractive positron-emitting radioisotopes for radiopharmaceutical
chemistry.3–5
18F
N
[ ]
CO2CH3
18F
N
CO2CH3
X
[ ]
X
A
B
C
D
E
F
[
18F]FECNT
Cl
F
-CH2CH2-
-CH2CH2-
β-[18F]CFT-FE
CH3
[
18F]FE-3FPT
-[18F]CIT-FP
-[18F]CMT-FP
-[18F]CBT-FP
3'-furyl -CH2CH2-
I
-CH2CH2CH2-
-CH2CH2CH2-
-CH2CH2CH2-
-CH2CH2CH2-
β
β
β
[18F]LBT-999
Me
Br
F
[18F]-1
β-[18F]CFT-FP
G
H
(E/Z)-[18F]FBClNT
Cl
-CH2(CH=CH)CH2-
Figure 1.
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

RADIOSYNTHESIS OF A SELECTIVE RADIOLIGAND
689
The robust and reliable pathway frequently proposed for the labelling with
fluorine-18 of tropane derivatives is a two-step radiochemical process,
involving first the preparation of an appropriate halogeno or better
sulfonyloxy [18F]reagent. The radiosynthesis of n-bromo-, n-tosyloxy- and
n-mesyloxy 1-[18F]fluoroalkanes (n=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 complex6,7 has been extensively
studied.8 This methodology has been applied with success to the preparation
of several [18F]fluoroethyl-9–20 and [18F]fluoropropyl-9,10,21–26 tropane deriva-
tives (compound A-G, Figure 1), like for example, [18F]FECNT and
b-[18F]CIT-FP. More recently, the radiosynthesis of both (E)- and
(Z)-1-[18F]fluoro-4-tosyloxybut-2-ene has also been briefly described and used
for the first time for the preparation of (E)- and (Z)- [18F]FBClNT (compound
H, Figure 1).27,28
This paper presents the two-step radiochemical synthesis of LBT-999 (1)
with fluorine-18, involving the preparation of (E)-1-[18F]fluoro-4-tosyloxybut-
2-ene followed by its condensation with 3b-p-tolyl-8-aza-bicyclo[3.2.1]octane-
2b-carboxylic acid methyl ester.
Results and discussion
Chemistry
LBT-999 (1) was synthesized from 3b-p-tolyl-8-aza-bicyclo[3.2.1]octane-2b-
carboxylic acid methyl ester (4) and (E)-1-fluoro-4-tosyloxybut-2-ene (toluene-
4-sulfonic acid (E)-4-fluoro-but-2-enyl ester, 3) using already reported
procedures (Scheme 1).2
Briefly, (E)-1-fluoro-4-tosyloxybut-2-ene (3) was prepared in 19% overall
yield from dimethyl fumarate (commercially available) using the following
1. DIBAL-H, PhCH
3
O
-10˚C, 2 hrs
then RT, 16 hrs
Bu NF
4
OMe
OTs
OTs
MeO
TsO
F
THF
reflux, 1 hr
2. TsCl (2eq.)
BnEt NCl / KOH
3
O
3
2
(30%)
dimethylfumarate
(63%)
dioxane
-3˚C, 30 min
then RT, 3 hrs
F
H
N
N
CO2CH3
CO2CH3
3
CH CN
3
RT, 16 hrs
1 (LBT-999)
(50%)
4
CH3
CH3
Scheme 1.
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

´
F. DOLLE ET AL.
690
three-step sequence: reduction of both ester functions using DIBAL-H,
conversion into their tosylates and finally mono tosyl-to-fluoro substitution
using 1.2eq. of tetrabutylammonium fluoride. Condensation of the nortropane
4 with (E)-1-fluoro-4-tosyloxybut-2-ene (3) gave LBT-999 (1) in 50% yield.
Radiochemistry
LBT-999 (1) was labelled with fluorine-18 at its fluoromethylvinyl moiety
using the two-step radiochemical process outlined in Scheme 2.
Introduction of the cyclotron-produced fluorine-18 (step 1) as the, no-
carrier-added, activated K[18F]F-Kryptofix1222 complex6,7 was performed at
708C for 10 min in acetonitrile containing (E)-but-2-ene-1,4-diol ditosylate
(2, about 10–12 mmol) in an unsealed vessel. At this stage, the remaining
radioactivity was measured, which was 97% to over 99% of the initial
radioactivity. The radiochemical yields of fluorine-18 incorporation, calcu-
lated from the TLC-radiochromatogram and defined as the ratio of
radioactivity area of [18F]-3 over total fluorine-18 radioactivity area, were
about 30–50%. A SiO2 Sep-Pak cartridge was then used to rapidly isolate
[18F]-3 (with a radiochemical purity >95%, according to radio-TLC), which
represented 30–45% of the total radioactivity amount engaged in the
fluorination process whereas 55–70% of the total radioactivity was left on
the cartridge, most likely non-reacted [18F]fluoride ion. These values clearly
confirmed the radiochemical yields measured by radio-TLC.
The coupling reaction (step 2, Scheme 2) of [18F]-3 with the nortropane 4
(about 20 mmol) was performed at 908C for 20 min in N,N-dimethylformamide
containing potassium iodide in an unsealed vessel. At this stage, the remaining
radioactivity was measured again, which was 85–95% of the radioactivity
engaged in this second step.
HPLC purification of [18F]LBT-999 ([18F]-1) was performed on a
semipreparative SunFireTM C18 column (HPLC A). Using these conditions,
[18F]-1 (tR:8.0–8.5 min) could be obtained within a short total purification time
(less than 10 min) with a >95% chemically and radiochemical purity,
separated from the non-labelled nortropane 4 (tR:5.0–5.5 min), as well as
18F
N
CO2CH3
18
K[ F]F-K
4 (5 mg)
222
K CO
KI, DMF
2
3
OTs
OTs
2 (4.0-5.0 mg)
18F
TsO
°
90 C, 20 min
CH CN
CH3
3
[
18F]-3
[
[
18F]-1
70˚C, 10 min
followed by
18F]LBT-999
HPLC purification
(SunFire C-18)
followed by
cartridge purification
(Prep-Sep SiO )
2
Scheme 2.
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

RADIOSYNTHESIS OF A SELECTIVE RADIOLIGAND
691
from the fluorine-18-labelled reagent [18F]-3 (tR:2.5–3.0 min) and an uni-
dentified [18F]impurity at tR:6.0–6.5 min. HPLC purification of [18F]-1 proved
to be particularly delicate. For example, the use of a semipreparative
Symmetry1 C18 column (HPLC B) or a semipreparative Zorbax1 SB C-18
column (HPLC C), with the same eluent as described for HPLC A, gave only
moderately high radiochemical and chemical purity (85–90%). A too low
radiochemical purity (585%) was also observed using a Zorbax1 SB C-18
with different eluents (see HPLC D and HPLC E).
The radiochemical yields for the coupling reaction, defined as the ratio of
radioactivity collected for [18F]-1 after HPLC over the total radioactivity
engaged in this chemical step, were about 30–50%. These values correlated
well with the ratio of radioactivity collected for uncoupled [18F]-3 over the
total radioactivity (up to 40%). In all runs, the unidentified [18F]impurity
(HPLC A, tR:6.0–6.5 min) represented less than 10% of the total radioactivity
engaged in this chemical step. Noteworthy, when the coupling reaction was
performed without any potassium iodide, the radiochemical yields of [18F]-1
were systematically lower to those described above. This observation is in
accordance with a recent report, showing that alkali iodide highly efficiently
promotes N-[18F]fluoroethylations when using the bromo or tosyloxy
[18F]fluoroethane reagent.29 Also, increasing the reaction temperature to over
1208C, did not improve the yields, but led to partial decomposition, which
complicated the final HPLC purification.
Formulation of [18F]LBT-999 ([18F]-1) as 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 (less than 10% of
the total radioactivity engaged in this step was left on the cartridge). The
solution was then sterile-filtered and diluted with physiological saline to an
ethanol concentration below 10%. The radiotracer preparation was a clear
and colorless solution with a measured pH between 5 and 7. As demonstrated
by analytical HPLC analysis (HPLC F, see experimental), the radiotracer
preparation was found to be >95% chemically and radiochemically pure (1,
tR:2.28 min). The preparation was shown to be free of non-radioactive
precursor nortropane 4 (tR:1.26 min) and was chemically and radiochemically
stable for at least 120 min. Specific radioactivities ranged from 1 to 3 Ci/mmol
(37–111 GBq/mmol) at the end of the synthesis.
Conclusion
LBT-999 (1) has been successfully labelled with fluorine-18. Typically,
55–80 mCi of [18F]-1 (2.03–2.96 GBq) could be obtained in 95–100 min of
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

´
F. DOLLE ET AL.
692
radiosynthesis (HPLC purification and formulation included), starting from a
1.2 Ci (44.4 GBq) [18F]fluoride ion production batch (overall yields:4.6–6.7%
non-decay-corrected and 8.3–12.5% decay-corrected). The potential of [18F]-1
for in vivo exploration of the DAT with PET is currently evaluated in non-
human primates.
Experimental
General
Chemicals, Flash chromatography and TLC analysis. Chemicals were pur-
chased from Aldrich-, Fluka- or Sigma France and were used without further
purification. Flash chromatographies were conducted on silica gel
(0.63–0.200 mm, VWR) columns. TLCs were run on pre-coated plates of
silica gel 60F254 (Merck). The compounds 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 a Berthold TraceMaster 20 automatic TLC linear
analyzer.
HPLC analysis. [HPLC A]: Equipment: system equipped with a Waters 600
Pump and a Waters 600 Controller, a Shimadzu SPD10-AVP UV-multi-
wavelength detector and a miniature ionization chamber probe; column:
semipreparative SunFireTM C18, Waters (250 Â 10.0 mm); porosity: 5 mm;
eluent H2O / CH3CN / TFA: 72:28:0.1 (v/v/v); flow rate: 5 ml/min; absorbance
detection at l=220 nm. tR ([18F]-1): 8.0–8.5 min.
[HPLC B]: Equipment: system equipped with a Waters 600 Pump and a
Waters 600 Controller, a Shimadzu SPD10-AVP UV-multi-wavelength
detector and a miniature ionization chamber probe; column: semipreparative
Symmetry1 C18, Waters (300 Â 7.8 mm); porosity: 7 mm; eluent H2O /
CH3CN / TFA: 70:30:0.1 (v/v/v); flow rate: 5 ml/min; absorbance detection
at l=220 nm. tR ([18F]-1): 7.0–7.5 min.
[HPLC C]: Equipment: system equipped with a Waters 600 Pump and a
Waters 600 Controller, a Shimadzu SPD10-AVP UV-multi-wavelength
detector and a miniature ionization chamber probe; column: semipreparative
Zorbax1 SB C-18, Hewlett Packard (250 Â 9.4 mm); porosity: 5 mm; eluent
H2O / CH3CN / TFA: 65:35:0.1 (v/v/v); flow rate: 5 ml/min; absorbance
detection at l=220 nm. tR ([18F]-1): 9.0–9.5 min.
[HPLC D]: Equipment: system equipped with a Waters 600 Pump and a
Waters 600 Controller, a Shimadzu SPD10-AVP UV-multi-wavelength
detector and a miniature ionization chamber probe; column: semipreparative
Zorbax1 SB C-18, Hewlett Packard (250 Â 9.4 mm); porosity: 5 mm; eluent
Copyright # 2006 John Wiley & Sons, Ltd.
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RADIOSYNTHESIS OF A SELECTIVE RADIOLIGAND
693
H2O / CH3CN / TEA: 50:50:0.1 (v/v/v); flow rate: 6 ml/min; absorbance
detection at l=220 nm. tR ([18F]-1): 10.0–10.5 min.
[HPLC E]: Equipment: system equipped with a Waters 600 Pump and a
Waters 600 Controller, a Shimadzu SPD10-AVP UV-multi-wavelength
detector and a miniature ionization chamber probe; column: semipreparative
Zorbax1 SB C-18, Hewlett Packard (250 Â 9.4 mm); porosity: 5 mm; eluent
0.9% aqueous NaCl / EtOH / AcOH: 71:28:1 (v/v/v); flow rate: 7 ml/min;
absorbance detection at l=220 nm. tR ([18F]-1): 9.0–9.5 min.
[HPLC F]: Equipment: Waters Alliance 2690 (or a Waters binary HPLC
pump 1525) equipped with a UV spectrophotometer (Photodiode Array
Detector, Waters 996) and a Berthold LB509 radioactivity detector; column:
analytical Symmetry-M1 C-18, Waters (50 Â 4.6 mm); porosity: 5.0 mm;
conditions: isocratic elution with solvA / solvB: 55 / 45 (v/v) [solvent A: H2O
containing Low-UV PIC1 B7 reagent (composition (% by weight): methanol
(18–22%), heptane sulfonic acid } sodium salts (4–6%), phosphate buffer
solution (3–7%), water (65–75%), pH 3, Waters), 20 ml for 1000 ml; solvent B:
H2O / CH3CN: 30:70 (v/v) containing Low-UV PIC1 B7 reagent (20 ml for
1000 ml)]; flow rate: 2.0 ml/min; temperature: 308C; UV detection at l: 205 nm.
tR ([18F]-1): 2.28 min.
Spectroscopies and elemental analyses. NMR spectra were recorded on a
Bruker DPX Avance (200 MHz) apparatus using the hydrogenated residue of
the deuterated solvent CDCl3 (d=7.30 ppm) and/or TMS (d=0.00 ppm) as
1
internal standards for H-NMR as well as the deuterated solvent CDCl3
(d=77.0 ppm) and/or TMS 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), ES+, were
measured on a Hewlett-Packard 5989A GC/EI-MS spectrometer. Elemental
analyses were performed by the service d’Analyses du CNRS (Vernaison,
France) and results were within Æ 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, stainless
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 diameter;
1/16 in external diameter), 2.0 bar helium drive pressure, transfer time
3–6 min. Typical production of [18F]Fluoride ion at the end of bombardment
for a 20 mA, 30 min (10 mA h) irradiation: 750–800 mCi (27.7–29.6 GBq).
Copyright # 2006 John Wiley & Sons, Ltd.
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F. DOLLE ET AL.
694
Miscellaneous. Radiosyntheses using fluorine-18, including the HPLC pur-
ifications, were performed in a 7.5-cm-lead shielded cell using a computer-
assisted Zymate robot system (Zymark Corporation, USA).
Chemistry
(E)-But-2-ene-1,4-diol ditosylate (2). Synthesized from dimethyl fumarate
((E)-but-2-ene-1,4-dioic acid dimethyl ester) according to Miller et al. and
Reddy et al.30,31 1H-NMR (CDCl3): d: 2.45 (s, 6 H); 4.47 (dd, 4 H, 3J=3.0 Hz,
4J=1.4 Hz); 5.71–5.75 (m, 2 H); 7.36 (d, 4 H, 3J=8.3 Hz); 7.77 (d, 4 H,
3J=8.3 Hz). 13C-NMR (CDCl3): d: 22.7 (2C); 68.7 (2C); 128.1 (2C); 128.3
(4C); 130.4 (4C); 132.5 (2C); 146.0 (2C).
Toluene-4-sulfonic acid (E)-4-fluoro-but-2-enyl ester (3). Synthesized from
(E)-but-2-ene-1,4-diol ditosylate (2) according to Dolle et al. and Goodman
´
and Chen.2,28 1H-NMR (CDCl3): d: 2.45 (s, 3 H); 4.58–4.62 (m, 2 H); 4.85
2
(dd, 2 H, JHÀF=46.4 Hz, 3J=4.8 Hz); 5.81–5.92 (m, 2 H); 7.37 (d, 2 H,
3
3J=8.3 Hz); 7.81 (d, 2 H, J=8.3 Hz). 13C-NMR (CD2Cl2): d: 21.7; 69.9; 82.3
(d, JCÀF = 162.7 Hz); 125.8 (d, JCÀF=12.2 Hz); 128.2 (2C); 130.3 (2C);
1
3
2
130.8 (d, JCÀF=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
(4). Synthesized from (R)-cocaine hydrochloride according to Emond
et al.32 1H-NMR (CDCl3): d: 1.60–1.85 (m, 3 H); 1.93–2.15 (m, 2 H); 2.33 (s,
3 H); 2.43 (td, 1 H, 2J=13.1 Hz, 3J=2.7 Hz); 2.75 (dd, 1 H, 3J=5.9 Hz,
3J=1.9 Hz); 2.95 (s, 1 H); 3.19 (dt, 1 H, 3J=13.1 Hz, 3J=5.5 Hz); 3.41 (s, 3 H);
3.74 (m, 2 H); 7.11 (s, 4 H). 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 (M+; 22); 228 (3); 200 (5); 170 (5); 141 (10); 115 (14); 84 (70); 83 (93); 69
(80); 68 (100).
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 toluene-4-sulfonic acid
(E)-4-fluoro-but-2-enyl ester (3) and 3b-p-tolyl-8-aza-bicyclo[3.2.1]octane-
2b-carboxylic acid methyl ester (4) according to Dolle
´
et al.2 1H-NMR
(CDCl3): d: 1.57–1.68 (m, 3 H); 1.90–2.00 (m, 2 H); 2.22 (s, 3 H); 2.65 (td, 1 H,
3
3J=2J=12.4 Hz, J=2.6 Hz); 2.92–3.05 (m, 4 H); 3.46 (m, 1 H); 3.53 (s, 3 H);
2
3
3.70 (m, 1 H); 4.86 (dd, 2 H, JHÀF=47.2 Hz, J=4.6 Hz); 5.80–5.85 (m, 2 H);
7.10 (d, 2 H, 3J=8.0 Hz); 7.20 (d, 2 H, 3J=8.0 Hz). 13C-NMR (CDCl3):
d: 20.9; 25.8; 26.0; 33.7; 34.0; 50.8; 52.6; 54.8; 61.3; 62.2; 83.0
1
2
(d, JCÀF=161.0 Hz); 126.1 (d, JCÀF=17.1 Hz); 127.1 (2C); 128.5 (2C);
3
134.1 (d, JCÀF=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
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RADIOSYNTHESIS OF A SELECTIVE RADIOLIGAND
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(33); 53 (35). Elemental analyses: Calculated C20H26FNO2 : C, 72.48; H, 7.91;
N, 4.23. Found: C, 72.26; H, 7.94; N, 4.21.
Radiochemistry
Preparation of the K[18F]F-K222-complex. In order to recover and recycle the
[18O]water target, the 2 ml of aqueous [18F]fluoride ion from the target holder
were passed through an anion exchange resin (Sep-Pak1 Light Waters
AccellTM Plus QMA cartridge (chloride form, beforehand washed with
aqueous 1 M NaHCO3 (2 ml) and rinsed with water (20 ml) and CH3CN
(10 ml)) by helium pressure (1.5–2.0 bar). Helium was blown through the
column to maximally extract [18O]water. The [18F]fluoride ion was then eluted
from the resin, using an aqueous 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-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. This complex was further dried twice by
azeotropic removal of water traces by addition of 0.4 ml of CH3CN followed
by concentration to dryness at 145–1508C under a gentle nitrogen stream
for 30 s.
Preparation of 8-((E)-4-[18F]fluoro-but-2-enyl)-3b-p-tolyl-8-aza-bicyclo[3.2.1]
octane-2b-carboxylic acid methyl ester ([18F]-1, [18F]LBT-999). Step 1:
CH3CN (600 ml) containing (E)-but-2-ene-1,4-diol ditosylate (2, 4.0–5.0 mg,
10.1–12.6 mmol) was directly 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 708C, for 10 min) without stirring
the contents. The reaction vessel was then cooled using an ice-water bath and
the remaining radioactivity was measured. The resulting, often colored
reaction mixture was then analyzed by radio-TLC. The reaction yield was
calculated from the TLC-radiochromatogram and defined as the ratio of
radioactivity area of (E)-4-[18F]fluoro-but-2-enyl ester ([18F]-3) over total
fluorine-18 radioactivity area (SiO2-TLC: Rf: [18F]-3 : 0.35 (heptane/EtOAc:
80/20), 0.45 (heptane/EtOAc: 70/30), 0.60 (heptane/EtOAc: 50/50) and Rf:
[18F]fluoride ion: 0.00 (heptane/EtOAc: 80/20 to 50/50)). The reaction mixture
was diluted with Et2O (1 ml) and transferred onto a SiO2 Sep-Pak cartridge
(PrepSepTM Si Extraction Column, Fisher Scientific, activated beforehand
with Et2O (5 ml)). The tube was rinsed twice with Et2O (1 ml), which was also
transferred and added to the diluted reaction mixture on top of the cartridge
(6–10% of the total radioactivity amount engaged in the fluorination process
usually stays behind in the initial tube). The whole was then passed through
the cartridge and [18F]-3 was directly eluted into a pre-heated (708C) 5 ml
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

´
F. DOLLE ET AL.
696
reaction vial. Another 1 ml of Et2O was used to wash the cartridge for
maximal transfer of [18F]-3 (30–45% of the total radioactivity amount engaged
in the fluorination process usually elutes and is >95% pure [18F]-3, according
to radio-TLC). In addition to the above TLC analysis, the incorporation yield
was also calculated after the SiO2 Sep-Pak cartridge elution by the CH3CN/
Et2O (containing >95% of [18F]-3), over starting fluorine-18 radioactivity
ratio. It was confirmed by the SiO2 Sep-Pak cartridge (most likely non-reacted
[18F]fluoride ion), over starting fluorine-18 radioactivity ratio. The resulting
CH3CN/Et2O solution was concentrated to dryness at 65–758C under a gentle
nitrogen stream for 2–3 min (10–15% of the radioactivity usually is lost most
likely due to the volatility of [18F]-3).
Step 2: To the above-mentioned residue, were successively added DMF
(100 ml) containing KI (2 mg, 12.0 mmol) and DMF (400 ml) containing 3b-p-
tolyl-8-aza-bicyclo[3.2.1]octane-2b-carboxylic acid methyl ester (4, 5 mg,
19.3 mmol). The vial (not sealed) was placed in a heating block (at 908C, for
20 min) without stirring the contents. The reaction vessel was then cooled
using an ice-water bath and the remaining radioactivity was measured. Finally,
the reaction mixture was diluted with 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:8.0–8.5 min), well separated from unlabelled 4 (tR:
5.0–6.0 min) and starting [18F]-3 (tR:2.5–3.0 min).
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 resulting
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
followed by 8 ml of physiological saline and filtered on a 0.22 mm GS-Millipore
filter (vented). Finally, physiological saline was added to lower the EtOH
concentration below 10%. This whole process was performed using a remote-
controlled dedicated home-made device based on a literature procedure.33
Quality control of [18F]LBT-999 ([18F]-1). The radiotracer preparation was
visually inspected for clarity, absence of color and particulates. An aliquot of
the preparation was removed for determination of pH using standard pH-
paper. Chemical and radiochemical purities were also assessed on this aliquot
by HPLC (HPLC F), with a sample of authentic 1 (tR:2.28 min). Particular
attention was paid to the absence of non-radioactive precursor (nortropane 4 :
tR:1.26 min). Chemical and radiochemical stability of the entire preparation
were tested by HPLC (HPLC F) at regular 15-min intervals during 120 min.
Copyright # 2006 John Wiley & Sons, Ltd.
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RADIOSYNTHESIS OF A SELECTIVE RADIOLIGAND
697
Specific radioactivity of the radiotracer was calculated from three consecutive
HPLC (HPLC F) 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.
Acknowledgements
The authors wish to thank the cyclotron operators Mr Daniel Gouel,
Mr Christophe Peronne and Mr Christophe Lechene for performing the
irradiations. The authors also wish to thank Dr Dirk Roeda for proof-reading
the manuscript and suggesting linguistic corrections. This study was funded in
part by the EC - FP6-project DiMI (LSHB-CT-2005-512146).
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