Synthesis, radiosynthesis and in vivo preliminary evaluation of [ 11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET

Article abstract of DOI:10.1016/j.bmc.2005.09.035

LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3β-p-tolyl-8-aza-bicyclo[3.2.1] octane-2β-carboxylic acid methyl ester), a cocaine derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands, and its corresponding carboxylic acid

Full text of DOI:10.1016/j.bmc.2005.09.035

Bioorganic & Medicinal Chemistry 14 (2006) 1115–1125
Synthesis, radiosynthesis and in vivo preliminary evaluation
of [11C]LBT-999, a selective radioligand for the visualisation
of the dopamine transporter with PET
a,
b
b
a
a
*
´ ´
´
´
Frederic Dolle, Patrick Emond, Sylvie Mavel, Stephane Demphel, Franc¸oise Hinnen,
Zo¨ıa Mincheva,b Wadad Saba,a Heric Valette,a Sylvie Chalon,b Christer Halldin,c
Julie Helfenbein,d Joe¨l Legaillard,d Jean-Claude Madelmont,e Jean-Bernard Deloye,f
Michel Bottlaendera and Denis Guilloteaub
a
´ ´
´
´
Service Hospitalier Frederic Joliot, Departement de Recherche Medicale, CEA/DSV,
´ ´
4 Place du General Leclerc, F-91401 Orsay, France
b
´
INSERM U619, CHRU de Tours, Universite, Franc¸ois-Rabelais de Tours, 31 avenue Monge, F-37200 Tours, France
cKarolinska Institutet, Department of Clinical, Neuroscience, Psychiatry Section, Karolinska Hospital, S-17176 Stockholm, Sweden
dOrphachem, ZATE INSERM U484, rue Montalembert, F-63005 Clermont-Ferrand, France
e
´
´
´
INSERM U484, Laboratoire Etude Metabolique des Molecules Marquees, rue Montalembert, F-63005 Clermont-Ferrand, France
f
ˆ
Laboratoires Cyclopharma, Biopole Clermont Limagne, F-63360 Saint Beauzire, France
Received 21 June 2005; revised 5 September 2005; accepted 13 September 2005
Available online 10 October 2005
Abstract—LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3b-p-tolyl-8-aza-bicyclo[3.2.1]octane-2b-carboxylic acid methyl ester), a cocaine
derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands, and its corresponding carboxylic
acid derivative, the latter used as precursor for labelling both with tritium and the positron-emitter carbon-11 (half-life: 20.38 min),
were synthesized from (R)-cocaine. [3H]LBT-999 (>99% radiochemically pure, specific radioactivity of 3.1 TBq/mmol) was prepared
from [3H]methyl iodide, allowing its in vitro pharmacological evaluation (KD: 9 nM for DAT and IC50 > 1000 nM for SERT and
NET). Routine production batches of 4.5–9.0 GBq of iv injectable solutions of [11C]LBT-999 (with specific radioactivities ranging
from 30 to 45 GBq/lmol) were prepared in 25–30 min (HPLC purification and formulation included) using the efficient methylation
reagent [11C]methyl triflate. The preliminary in vivo pharmacological evaluation of [11C]LBT-999, using both biodistributions in rats
and brain imaging in monkeys with positron emission tomography (PET), clearly illustrates that this ligand is an excellent candidate
for quantification with PET of DAT in humans.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
marker of the integrity and number of presynaptic stri-
atal dopamine-producing neurons, considerable efforts
have been spent in recent years on the design and devel-
opment of DAT-selective radioligands as in vivo posi-
tron emission tomography (PET) imaging tools for the
study of these illnesses and disorders as well as the eval-
uation of the degree of success of their treatment.
Several affections, including ParkinsonÕs disease, schizo-
phrenia, attention deficit disorder or drug abuse, are
related to abnormalities within the brainÕs dopamine
system.1 In particular, the neuronal dopamine trans-
porter (DAT),
a membrane-bound presynaptically
located protein, plays a key role in regulating the synap-
tic concentration of dopamine at nerve terminals and
thus dopamine neurotransmission in the brain. Taking
into account that DAT density can be considered as a
Certain tropane-type derivatives,2–66 including co-
caine,67,68 represent today the largest class of molecules
ever developed for imaging the DAT with PET and
therefore labelled with a positron-emitting radioisotope
(Table 1). From a chemical point of view, all chemical
structures present (a) a 2b (exo) ester group (two excep-
tions: NS-2214 and b-CPPIT); (b) a 3b (exo) (often
para-substituted) phenyl ring (exception: cocaine) and
Keywords: Carbon-11; LBT-999; Dopamine transporter; Tropane.
*
Corresponding author. Tel.: +33 1 69 86 77 04; fax: +33 1 69 86 77
49; e-mail: frederic.dolle@cea.fr
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.035

´
F. Dolle et al. / Bioorg. Med. Chem. 14 (2006) 1115–1125
1116
Table 1. Chemical structures of [11C]cocaine and other tropane derivatives showing the positions that have been labelled with the positron-emitters
carbon-11, fluorine-18 and bromine-76
R1
H311C
8 N
N
CO2R2
CO2CH3
O
1
5
2
4
7
3
6
O
[
11C]Cocaine
R3
R5
R4
Code
R1
R2
R3
R4
R5
Ref.
b-CFT (WIN 35,428)
b-CIT (RTI-55)
b-CCT (RTI-31)
b-CMT (RTI-32)
FECNT
[
[
11C]CH3
11C]CH3
[
[
[
[
11C]CH3
11C]CH3
11C]CH3
11C]CH3
[
18F]F
H
H
2–17
18–27
28–30
28,29
31–39
31,40
41
I
Cl
H
H
CH3
CH3
CH2CH2[18F]F
(CH2)2CH2[18F] F
CH3
CH3
H
H
CH3
Cl
H
H
CH3
CH3
H
H
b-CCT-FP (b-FPCT)
b-CBT
b-FECT (b-FE-CCT)
b-FETT (b-FE-CMT)
b-FIPCT (b-FiP-CCT)
b-CFT-FE
Cl
[
H
H
CH3
CH2CH2[18F]F
CH2CH2[18F]F
CH(CH3)CH2[18F]F
76Br]Br
H
H
Cl
CH3
Cl
F
H
H
29,42
29,42
43,44
45,46
45
CH3
H
H
CH3
H
H
CH2CH2[18F]F
(CH2)2CH2[18F] F
CH2CH2F
CH3
CH3
CH3
H
H
b-CFT-FP
b-CBT-FE
F
H
H
[
76Br]Br
H
H
47
48–52
53
b-CIT-FP (FP-CIT)
b-CMT-FP (FP-CMT)
b-CBT-FP (FP-CBT)
b-CDCT
b-IP-CIT (RTI-121)
NS-2214 (BMS-204756)
b-CIT-FE
b-CpFMT
b-CmFMT
b-CoFMT (o-FWIN)
b-CPPIT
FCT
(CH2)2CH2[18F] F
(CH2)2CH2[18F] F
(CH2)2CH2[18F] F
CH3
[
11C]CH3
I
CH3
H
H
CH3
CH3
H
H
[
Cl
76Br]Br
H
Cl
H
47,54
30
23
[
11C]CH3
H
[
[
11C]CH3
11C]CH3
CH(CH3)2
See belowa
I
Cl
H
Cl
H
H
55
CH2CH2F
CH3
CH3
[
11C]CH3
CH3
I
H
H
15,56,57
58,59
58
CH2[18F]F
H
H
CH3
CH3
See belowb
CH2CH3
H
H
Cl
Cl
F
CH2[18F]F
H
CH3
H
H
H
H
H
H
H
CH2[18F]F
59
[
11C]CH3
H
H
H
H
H
H
60,61
62
63
4-[18F]FBn
CH2CH=CHI
CH3
E-IACFT (Altropane)
MCL301
MCL322
[
11C]CH3
CH2CH2[18F]F
CH2CH2[18F]F
CH2CH2[18F]F
I
64,65
64,65
66
CH3
CH3
Br
I
FE@CIT
a
b
N
O
H311C
H311C
N
N
H
N OCH3
Cl
Cl
Cl
(c) a methyl group or a longer alkyl chain at the 8-nitro-
gen atom. From a radiochemical point of view, many of
these derivatives have been labelled with the short-lived
radioisotope carbon-11 (half-life: 20.38 min) at their N-
methyl group or methyl ester function using [11C]methyl
iodide or [11C]methyl triflate (for example, b-CIT or b-
CCT). Also, a relatively large number of these deriva-
tives have been labelled with fluorine-18 (half-life:
109.8 min), today the most widely used positron-emit-
ting radioisotope, by aliphatic nucleophilic fluorination
(for example, FE-CNT or b-FE-CCT) or aromatic elec-
trophilic fluorination (b-CFT). Finally, a few of these
derivatives have been labelled with bromine-76, a longer-
lived positron-emitter (half-life: 16.1 h), by electrophilic
bromination (for example, b-CBT). From an imaging
point of view, many of these derivatives have been more
or less successfully used in pre-clinical and clinical PET
studies for the quantification of the DAT in the brain. A
constant major drawback characterizing these radioli-
gands is the low selectivity for the DAT, as most of these
derivatives have a relatively high affinity for the seroto-
nine transporter (SERT) and/or the norepinephrine
transporter (NET) too. Another limitation, especially
amplified with carbon-11-labelled radiotracers, is that
the uptake of most of these radioligands in the striatum,
the dopamine-rich region, increases continuously and

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F. Dolle et al. / Bioorg. Med. Chem. 14 (2006) 1115–1125
1117
does not reach an equilibrium state, needed for quanti-
fication, within the duration of a PET examination.
The, in this respect, most suited radioligand so far de-
scribed seems to be FECNT,31–39 with the more attrac-
tive kinetic characteristics by achieving a peak striatal
uptake at 60–75 min post-injection. However, recently,
the presence of a polar labelled metabolite, which cross-
es the blood–brain barrier, was reported, which could
possibly compromise the use of this radioligand.69
for the DAT (over SERT and NET) of tropane-type
derivatives was improved with chemical structures pre-
senting both an unsaturated alkyl chain at the 8-nitrogen
atom (R1) and a methyl function at the phenyl ring
(R3 = methyl, instead of the frequently used halogens,
see Table 1).72 Among these compounds, PE2I (Fig. 1)
(as well as its bromo analogue PE2Br (structure not
shown73) were characterized by a moderate in vitro affin-
ity but high selectivity for the DAT.72,74 In initial in vivo
studies, PE2I, labelled with carbon-11 or the SPECT-ra-
dioisotope iodine-123, not only accumulated rapidly and
selectively in the striatum with a favourable kinetic
profile, yielding excellent quality images within 30 min
post-injection, but also afforded a high specific- to non-
specific-binding ratio within 1 h.75–83 These results have
directed us towards the design of a fluorine-containing
structure based on PE2I as the parent molecule (tolyl
moiety) and presenting a fluorobutenyl alkyl chain at
the 8-nitrogen atom, coded LBT-999 ((E)-N-(4-fluoro-
but-2-enyl)-2b-carbomethoxy-3b-(40-tolyl) nortropane
(1), Fig. 1). This new highly DAT-selective analogue
can on the one hand be labelled with both the posi-
tron-emitter fluorine-18 and carbon-11 for in vivo imag-
ing purposes, and on the other hand be readily accessible
to tritium-labelling for in vitro characterization
purposes.
Today, fluorine-18, beyond its adequate physical and
nuclear characteristics, appears as the most attractive
positron-emitting radioisotope for radiopharmaceutical
chemistry and PET imaging, part of this continuous
growing interest probably due to the successful use in
clinical oncology of [18F]FDG, actually the most widely
used PET-radiopharmaceutical.
Briefly, fluorine-18 displays simple decay- and emission
properties with a high 97% positron abundance. Com-
pared with the other conventional short-lived positron-
emitting radionuclides carbon-11, nitrogen-13 and
oxygen-15, fluorine-18 has a relatively low positron
energy (maximum 635 keV) and the shortest positron
linear range in tissue (2.3 mm), resulting in the highest
resolution in PET imaging. Its half-life (109.8 min) is
long enough to give access to relatively extended imag-
ing protocols compared to what is possible with
carbon-11, therefore facilitating kinetic studies and
high-quality plasma metabolite analysis. Moreover,
from a chemical point of view, fluorine-18 allows for
multi-step synthetic approaches that can be extended
over hours. Finally, fluorine-18 can be reliably and
routinely produced at the multi-Curie level, using the
well-characterized (p,n) nuclear reaction on an oxygen-
18-enriched water target on widely implemented bio-
medical cyclotrons of a relatively low-energy proton
beam (e.g., 18 MeV). This distinctive advantage, com-
bined with its favourable half-life, permits the transport
and the use of fluorine-18-labelled radiopharmaceuti-
cals (such as the archetype [18F]FDG) at ÔsatelliteÕ
PET units without an on-site cyclotron production
facility.
This paper presents the synthesis of LBT-999 (1) from
(R)-cocaine as well as the synthesis of the corresponding
carboxylic acid derivative as precursor for subsequent
tritium/carbon-11 labelling by methylation. Although
the ultimate goal of this project is fluorine-18-labelling
of this new DAT-ligand, this paper presents a first time
radiolabelling of LBT-999 (1) with the radiochemically
more practical carbon-11. Preliminary in vitro and in vi-
vo pharmacological evaluation, including one selected
PET study, is also presented.
2. Discussion
2.1. Chemistry
The target compound LBT-999 (1) as well as its precur-
sor acid for tritium- and carbon-11-labelling (10) were
both synthesized from (R)-cocaine (2) via the intermedi-
ate tropanamine 5, namely 3b-p-tolyl-8-aza-bicy-
(E)-FBClNT (Fig. 1) is a recently reported tropane deriv-
ative, structurally closely related to FECNT (Table 1) in
which the 8-nitrogen atom is alkylated with an (E)-fluo-
robutenyl group (R1), offering an opportunity for fluo-
rine-18 labelling.70,71 Even though this new derivative
displays a high in vitro affinity for the DAT (Ki =
2.54 nM) and a high accumulation in rat brain areas
dense in DAT, its reported binding to the SERT excludes
high selectivity both in vitro and in vivo. Previous struc-
ture-activity studies have demonstrated that selectivity
clo[3.2.1]octane-2b-carboxylic
acid
methyl
ester
(Schemes 1 and 3). Compound 5 was synthesized in
16% overall yield as shown in Scheme 1. Briefly, (R)-co-
caine (2) was converted to ecgonidine methyl ester (3) in
84% overall yield using procedures previously described
(Scheme 1).72 1–4 Addition involving tolylmagnesium
bromide at ꢀ40 ꢁC afforded preferentially the 2b,3b
F
F
I
N
N
CO2CH3
N
CO2CH3
CO2CH3
PE2I
(E)-FBClNT
LBT-999, 1
Cl
CH3
CH3
Figure 1. Chemical structures of (E)-FBClNT, PE2I and LBT-999 (1).

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F. Dolle et al. / Bioorg. Med. Chem. 14 (2006) 1115–1125
1118
H3C
H3C
1.
Conc. aq. HCl
reflux, 3 hrs
N
CO2CH3
N
CO2CH3
O
O
2. POCl
reflux, 16 hrs
3
3 (84%)
2 (Cocaine)
3. CH OH
3
RT, 1 week
H3C
1.
MgBr
H
H3C
1. Cl CCH OCOCl
120˚C, 2.5 hrs
3
2
N
N
CO2CH3
CO2CH3
Et O, -40˚C, 3 hrs
2
2.
CH COOH / Zn
3
2. TFA, Et O, -80˚C, 1 hr
2
RT, 5 days
5 (50%)
4 (39%)
CH3
CH3
Scheme 1. Preparation of 3b-p-tolyl-8-aza-bicyclo[3.2.1]octane-2b-carboxylic acid methyl ester (5).
O
DIBAL-H
OCH3
OH
7 (76%)
H3CO
HO
PhCH
3
O
6
-10˚C, 2 hrs
then RT, 16 hrs
TsCl (2eq.)
Bu NF
BnEt NCl / KOH
3
4
OTs
OTs
9 (30%)
TsO
F
THF
reflux, 1 hr
dioxane
-3˚C, 30 min
then RT, 3 hrs
8 (83%)
Scheme 2. Preparation of toluene-4-sulfonic acid (E)-4-fluoro-but-2-enyl ester (9).
F
H
N
N
CO2CH3
CO2CH3
OTs
F
9
CH CN
3
5
1 (50%)
(LBT-999)
RT, 16 hrs
CH3
F
CH3
N
CO2H
H O / Dioxane
2
reflux, 5 days
10 (53%)
CH3
Scheme 3. Preparation of LBT-999 (1) and the corresponding carboxylic acid derivative 10 as precursor for labelling.
isomer 4, which was isolated in 39% yield after flash
chromatography purification. N-demethylation was
performed using the following two-step procedure: (a)
treatment of 4 with 2,2,2-trichloroethylchloroformate
at 120 ꢁC for 2.5 h, followed by (b) reduction of the
intermediate carbamate with zinc powder in acetic acid
at room temperature for 5 days, giving the tropanamine
5 in 50% yield.
hydroxyde, and giving the ditosylate 8 in 83% yield,85
and finally (c) mono tosyl-to-fluoro substitution using
tetrabutylammonium fluoride (1.2 equiv) in refluxing
THF, giving the fluorobutenyl tosylate 9 in 30% yield.71
Condensation of the tropanamine 5 with toluene-4-sul-
fonic acid (E)-4-fluoro-but-2-enyl ester (9) in acetonitrile
at room temperature for 16 h gave LBT-999 (1) in 50%
yield (Scheme 3). Hydrolysis of the methyl ester function
was performed in a refluxing 1/1 (v:v) water/dioxane
mixture for 5 days and afforded the carboxylic acid
derivative 10 in 53% yield.
Toluene-4-sulfonic acid (E)-4-fluoro-but-2-enyl ester (9,
Scheme 2) was prepared in 19% overall yield from di-
methylfumarate (6) using the following three-step se-
quence: (a) reduction of both ester functions using
DIBAL-H in toluene at ꢀ10 ꢁC for 2 h, giving the diol
7 in 76% yield,84 followed by (b) conversion of the two
alcohol functions into their tosylates, using two equiva-
lents of tosyl chloride in dioxane containing triethyl-
benzylammonium chloride and aqueous potassium
2.2. Radiochemistry
LBT-999 (1) was labelled with carbon-11 at its methyl
ester function from the corresponding carboxylic acid
derivative 10 and the highly efficient methylation

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F. Dolle et al. / Bioorg. Med. Chem. 14 (2006) 1115–1125
1119
F
F
11
[
C]CH OTf
3
N
N
CO2H
aq. NaOH, acetone
- 10˚C, 2-3 min
CO211CH3
followed by
concentration to dryness
(110˚C, 1-2 min, N flow)
2
[
[
11C]-1
10
(Nor-LBT-999)
CH3
CH3
11C]LBT-999
and
HPLC purification
(SymmetryPrep® C-18)
Scheme 4. Preparation of [11C]LBT-999 ([11C]-1).
reagent [11C]methyl triflate. [11C]Methyl triflate was pre-
pared according to literature procedure from
radiochemical yield of [11C]-1, based on starting
[11C]CO2, was 19–45% (n = 10). No attempts were made
to further optimize these reactions, the yields obtained
being sufficient for our purposes. The specific radioactiv-
ity measured at the end of the radiosynthesis ranged
from 30 to 45 GBq/lmol).
a
[11C]methyl iodide using silver triflate.86 [11C]Methyl io-
dide was prepared from cyclotron-produced [11C]carbon
dioxide using the well-known two-step protocol, consist-
ing of the trapping of [11C]CO2 and conversion into
[11C]methanol (LiAlH4) followed by iodination using
aqueous HI giving [11C]methyl iodide.87 On average,
about 25 GBq of [11C]CH3OTf was routinely obtained
in 7–8 min after the end of bombardment (EOB) in
70% decay-corrected yield, based on starting [11C]CO2.
Concerning quality controls, the radiopharmaceutical
preparation was a clear and colourless solution and its
pH was between 5 and 7. As demonstrated by the HPLC
analysis, the radiopharmaceutical preparation was
found to be >99% chemically and radiochemically pure
and the preparation was shown to be free of non-radio-
active precursor (derivative 10) and was radiochemically
stable for at least 60 min. Administration to animals was
performed within 10 min following the end of the syn-
thesis. These results were in compliance with our
in-house quality control/assurance specifications.
Esterification of carboxylic acid 10 with [11C]methyl tri-
flate (Scheme 4), employing the standard conditions that
are now used in our laboratory for the routine radiosyn-
thesis of several radiotracers,88–91 including [11C]PE2I,80
gave excellent yields for the preparation of [11C]LBT-
999 ([11C]-1, Scheme 4). The conditions used were the
following: (1) trapping at ꢀ10 ꢁC of [11C]methyl triflate
in 400 lL acetone containing 0.6–0.8 mg of precursor 10
(1.9–2.5 lmol) and 3 lL of aqueous NaOH (3 N) (about
4 equiv); (2) concentration to dryness of the reaction
mixture (at 110 ꢁC, using a nitrogen stream for 1–
2 min); (3) taking up the residue in 0.5 mL of the HPLC
mobile phase (H2O/CH3CN/TFA: 35/65/0.1 (v/v/v)) and
(4) purification using a semi-preparative reversed-phase
HPLC.
LBT-999 (1) was labelled with tritium at its methyl ester
function from the corresponding carboxylic acid deriva-
tive 10 and commercially available [3H]methyl iodide
(Scheme 5).
Esterification of carboxylic acid 10 with [3H]methyl
iodide (Scheme 5) employed the following standard con-
ditions: (1) heating at 90 ꢁC for 15 min a DMF/toluene
solution (300 lL/150 lL) containing [3H]methyl iodide
and 0.7 mg of precursor 10 (2.2 lmol); (2) purification
using semi-preparative HPLC. [3H]LBT-999 ([3H]-1,
>99% radiochemically pure) was obtained with a specific
radioactivity of 3.1 TBq/mmol.
Formulation of LBT-999 ([11C]-1) for iv injection was
carried out as follows: The HPLC-collected fraction
containing the radiotracer was diluted with water and
the resulting solution was passed through a C18 Sep-
pakꢂ 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 further diluted with
physiological saline.
2.3. PET imaging and preliminary in vitro/in vivo
pharmacological evaluation
In vitro binding experiments on rat striatal membranes
showed that [3H]LBT-999 bound to a single site with a
Kd of 9 nM (Bmax: 17 pmol/mg protein) and displayed
a very high selectivity for the DAT (IC50 versus GBR
12909—another DAT-selective inhibitor—and PE2I:
2.4 and 18 nM, respectively; IC50 versus paroxetine,
Typically, starting from 50 to 60 GBq of a [11C]CO2 pro-
duction batch, 4.5–9.0 GBq of [11C]-1 was obtained
within 25–30 min of radiosynthesis, including HPLC
purification and formulation. The total decay-corrected
3
F
F
[ H]CH I
3
CO2C3H3
N
N
CO2H
DMF
90˚C, 15 min
followed by
HPLC purification
(µ-Bondapak® C-18)
[3H]-1
10
CH3
CH3
[3H]LBT-999
(Nor-LBT-999)
Scheme 5. Preparation of [3H]LBT-999 ([3H]-1).

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F. Dolle et al. / Bioorg. Med. Chem. 14 (2006) 1115–1125
1120
B
Part 1
Part 2
20.0
18.0
16.0
14.0
12.0
10.0
8.0
A
PE2I (1 mg/kg)
Putamen
Thalamus
Cerebellum
Caudate
6.0
4.0
2.0
0.0
0
20
40
60
80
100
120
Time (minutes)
Figure 2. PET imaging of [11C]LBT-999 ([11C]-1) in a Papio anubis baboon. An adult baboon (10 kg, anaesthetized with 1% isoflurane and 33%/66%
O2/N2O) was iv injected with 160 MBq of [11C]LBT-999 ([11C]-1) and imaged for 80 min on an HR+ Exact positron tomograph (CTI PET Systems,
Knoxville, TN, USA). At 80 min, PE2I (1 mg/kg) was iv injected and PET data were acquired for another 40 min. For data analysis, MRI-based
regions of interest (ROI) were placed on the putamen, the caudate, the thalamus and the cerebellum. The radioactivity measured in each ROI was
corrected for attenuation and carbon-11-decay and expressed as percent of injected dose per 100 mL of tissue (% ID/100 mL).
citalopram, MADAM, nisoxetine and desipramine
>1 lM). Ex vivo cerebral biodistribution in rats per-
formed with [11C]LBT-999 showed a striatum/cerebel-
lum radioactivity ratio of 18 and 25 at 30 and 60 min
post-injection, respectively. Moreover, the radiotracer
accumulation was strongly prevented by a pre-injection
of GBR 12909 (a DAT-selective inhibitor), whereas par-
oxetine (a SERT-selective inhibitor) and nisoxetine
(a NET-selective inhibitor) had no effect.
At 80 min p.i., these ratios further increased to 23.6
and 28.9. These results clearly show that LBT-999 rapid-
ly enters the brain and binds in cerebral regions known
to contain dopaminergic terminals. Moreover, early
equilibrium states (around 40 min) obtained in caudate
and putamen would allow DAT quantifying protocols
within a PET examination even using the carbon-11-la-
belled compound. Figure 2B (part 2: 80–120 min) shows
the effect of PE2I (1 mg/kg, iv administered 80 min after
the radiotracer injection). A rapid wash-out of the
radioactivity in the caudate nucleus, the putamen, and
the thalamus was observed. In these structures, the
radioactivity decreased by 74%, 81%, and 81%, respec-
tively. In the cerebellum, no displacement of radioactiv-
ity was observed. These results confirm the high
selectivity profile of LBT-999 in vivo and clearly demon-
strate that the high levels of LBT-999 binding in caudate
and putamen can be attributed to a DAT selective
labelling.
Based on these encouraging results, PET studies of the
brain distribution of [11C]LBT-999 ([11C]-1) were carried
out in primates (Papio anubis baboon). Only one select-
ed single PET experiment, performed on an adult male
baboon, is described here to illustrate this section. The
full characterization of [11C]LBT-999 as a PET radio-
tracer will be published elsewhere. Figure 2A illustrates
the uptake of [11C]LBT-999 in the baboon brain at 30–
60 min, following iv injection of 160 MBq of [11C]LBT-
999 ([11C]-1). Figure 2B (part 1: 0–80 min) shows the
corresponding time–activity curves obtained in selected
brain regions (putamen, caudate, thalamus, known to
contain DAT, and cerebellum, used as reference area
for non-specific binding). After injection of the radio-
tracer, there was a rapid appearance of radioactivity in
the whole of the brain. The time course of radioactivity
showed a rapid accumulation (within 20 min) in the cau-
date nucleus and putamen, reaching a value of 12.6%
and 15.4% of injected dose per 100 mL of tissue
(% ID/100 mL), respectively, 40 min p.i. At 80 min p.i.,
these values remained high: 12.0% and 14.7% ID/
100 mL, respectively. In the thalamus, radioactivity con-
centrations were maximal at 8 min p.i.: 7.8% ID/100 mL
and then decreased progressively. In the cerebellum,
radioactivity concentration peaked at 4 min (5.9% ID/
100 mL) and then decreased relatively rapidly. At 40
and 80 min p.i., the uptake in the cerebellum was 1.2
and 0.5 ID/100 mL, respectively. Therefore, ratios of
radioactivity caudate nucleus/cerebellum and putamen/
cerebellum were 10.2 and 12.5, respectively at 40 min p.i.
3. Conclusion
LBT-999 (1) and the corresponding carboxylic acid
derivative (10) as precursor for labelling were synthe-
sized from (R)-cocaine. The key step in these syntheses
was the 1–4 Michael addition of 4-tolylmagnesium bro-
mide to ecgonidine methyl ester, affording, after N-de-
methylation, the needed 2b-3b nortropane isomer.
LBT-999 was labelled with tritium at its methylester
function using [3H]methyl iodide as well as labelled at
the same function using the efficient methylation reagent
[11C]methyl triflate, permitting its preliminary in vitro
and in vivo pharmacological evaluation. Taken togeth-
er, the results so far obtained demonstrate that
LBT-999 is a highly promising candidate for in vivo
exploration and quantification in humans of DAT,
using PET. Labelling of LBT-999 with fluorine-18
(half-life: 109.8 min), the most widely used positron-
emitting radiohalogen, is currently in progress.

´
F. Dolle et al. / Bioorg. Med. Chem. 14 (2006) 1115–1125
1121
4. Experimental
tal analyses were performed by the service dÕanalyses du
CNRS (Vernaison, France) and results were within
0.4% of theoretical values.
4.1. General
4.1.1. Chemicals, TLCs, and flash chromatographies.
Chemicals, except (R)-cocaine, were purchased from
Aldrich, Fluka or Sigma France and were used without
further purification. Flash chromatographies were con-
ducted on silica gel (230–400 Mesh, VWR) columns.
TLCs were run on pre-coated plates of silica gel 60F254
(VWR). 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 followed by
heating on a hot plate.
4.1.4. Radioisotope availability/production. [11C]CO2 was
produced via the 14N[p,a]11C nuclear reaction by irradi-
ation of an ultrapure Air Liquide 99.5/0.5 mixture of N2/
O2 target with an 18 MeV proton beam (at 25 lA) on an
IBA Cyclone-18/9 (Louvain-la-Neuve, Belgium) cyclo-
tron (8.5 lA h in about 20 min). At the end of the bom-
bardment, the target contents were transferred to the
5-cm-lead shielded hot cell and passed through a glass
P2O5-guard (70 mm length, 3 mm internal diameter) in
order to remove moisture. [11C]CO2 was then separated
from the target gas by trapping in an empty stainless-
steel coil (150 mm length, 0.51 mm internal diameter),
cooled at ꢀ186 ꢁC using liquid argon. On average,
about 45 GBq (EOB) of [11C]CO2 is routinely obtained
in our laboratory for the irradiations described above.
[3H]CH3I (toluene solution containing copper metal
scavenger, 370 MBq/mL, 3.1 TBq/mmol) was purchased
from Amersham (Sweden).
4.1.2. HPLCs. [HPLC 1] Equipment: Beckman (Paris,
France) system equipped with a 126P-Solvent module,
a 166-NMP pump; column: semipreparative Stabilityꢂ
C-18, CL Cluzeau (250 mm · 10 mm); conditions: elu-
ent: MeOH/H2O: 50/50 (v/v); flow rate: 2.5 mL/min; tem-
perature: RT; UV detection at k: 254 nm. [HPLC 2]
Equipment: Waters (St-Quentin en-Yvelines, France)
systems equipped with a 510 pump, 440 UV detector or
Shimadzu SPD-10A UV-multiwavelength detector and
4.2. Chemistry
a Geiger–Muller detector; column: semipreparative
¨
SymmetryPrepꢂ C-18, Waters (300 mm · 7.8 mm); con-
ditions: eluent: H2O/CH3CN/CF3CO2H: 35/65/0.1
(v/v/v); flow rate: 5 mL/min; temperature: RT; UV detec-
tion at k: 220 nm. [HPLC 3] Equipment: Waters (St-
Quentin en-Yvelines, France) Alliance 2690 equipped
with a UV spectrophotometer (Photodiode Array Detec-
tor, Waters 996) and a Berthold (Thoiry, France) LB509
radioactivity detector; column: analytical Symmetry-Mꢂ
C-18, Waters (50 mm · 4.6 mm); conditions: isocratic
elution with solvA/solvB: 40/60 (v/v) [solvA: H2O con-
taining Low-UV PICꢂ B7 reagent (% by weight: metha-
nol (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; solvB: H2O/
CH3CN: 50/50 (v/v) containing Low-UV PICꢂ B7 re-
agent (Waters), 20 mL for 1000 mL]; flow rate: 2.0 mL/
min; temperature: 30 ꢁC; UV detection at k: 220 nm.
[HPLC 4] Equipment: System equipped with a Hitachi
(Tokio, Japan) L-7100 pump, a Hitachi L-7400 UV
detector and a Packard (Paris, France) Radiomatic
150TR radioactivity detector equipped with a tritium-
cell (550 lL cell); column: semipreparative l-Bondapakꢂ
C-18, Waters (300 mm · 7.8 mm); conditions: eluent:
H2O/CH3CN/TEA: 65/35/0.1 (v/v/v); flow rate: 6 mL/
min; temperature: RT; UV detection at k: 254 nm.
4.2.1. 3b-p-Tolyl-8-aza-bicyclo[3.2.1]octane-2b-carboxyl-
ic acid methylester (5). Synthesized from (R)-cocaine
hydrochloride according to Ref. 72. H NMR (CDCl3):
1
d: 1.60–1.85 (m, 3H); 1.93–2.15 (m, 2H); 2.33 (s, 3H);
2.43 (td, 1H, J = 13.1 Hz, J = 2.7 Hz); 2.75 (dd, 1H,
2
3
3J = 5.9 Hz, 3J = 1.9 Hz); 2.95 (s, 1H); 3.19 (dt, 1H,
3
3J = 13.1 Hz, J = 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 (M+; 22); 228 (3);
200 (5); 170 (5); 141 (10); 115 (14); 84 (70); 83 (93); 69
(80); 68 (100).
4.2.2. Toluene-4-sulfonic acid (E)-4-fluoro-but-2-enylester
(9). To a solution of (E)-but-2-enediol ditosylate (8, 0.5 g,
1.26 mmol, synthesized from dimethyl fumarate (6, (E)-
but-2-enedioic acid dimethyl ester) according to Refs.
84,85) dissolved in THF (20 mL) tetrabutylammonium
fluoride (1.57 mL, 1 M in THF, 1.57 mmol) was added
dropwise and the mixture was heated under reflux for
1 h. The solvent was evaporated, and the resulting solid
was purified by flash chromatography (petroleum ether/
Et2O: 8/2). Derivative 9 was obtained as a yellow liquid
in 30% yield (91 mg). 1H NMR (CDCl3): d: 2.45 (s, 3H);
2
4.58–4.62 (m, 2H); 4.85 (dd, 2H, JH–F = 46.4 Hz,
3J = 4.8 Hz); 5.81–5.92 (m, 2H); 7.37 (d, 2H,
3J = 8.3 Hz); 7.81 (d, 2H, 3J = 8.3 Hz). 13C NMR
(CD2Cl2): d: 21.7; 69.9; 82.3 (d, 1JC–F = 162.7 Hz); 125.8
4.1.3. Spectroscopies and elemental analyses. NMR spec-
tra were recorded on a Bruker (Wissembourg, France)
DPX Avance (200 MHz) apparatus using the hydroge-
nated residue of the deuterated solvent CDCl3
(d = 7.30 ppm) or TMS (d = 0 ppm) as internal stan-
3
(d, JC–F = 12.2 Hz); 128.2 (2C); 130.3 (2C); 130.8 (d,
2JC–F = 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).
1
dards for H NMR as well as the deuterated solvent
CDCl3 (d = 77.0 ppm) as internal standard for 13C
NMR. The chemical shifts are reported in ppm, down-
field 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-MS spectrometer (ES+). Elemen-
4.2.3. 8-((E)-4-Fluoro-but-2-enyl)-3b-p-tolyl-8-aza-bicy-
clo[3.2.1]octane-2b-carboxylic acid methyl ester (1, LBT-
999). To a solution of toluene-4-sulfonic acid 4-fluoro-
but-2-enyl ester (9, 100 mg, 0.41 mmol) in acetonitrile
(5 mL) was added dropwise 3b-p-tolyl-8-aza-bicy-

´
F. Dolle et al. / Bioorg. Med. Chem. 14 (2006) 1115–1125
1122
clo[3.2.1]octane-2b-carboxylic acid methyl ester (5,
100 mg, 0.38 mmol) dissolved in acetonitrile (5 mL).
After stirring overnight, the mixture was treated with
Et2O (5 mL), H2O (15 mL) and HCl 1 M to pH 1. The
aqueous phase was separated, basified with Na2CO3
and the compound was extracted with Et2O
(5 · 10 mL). The residue after evaporation of solvent
was finally purified by flash chromatography (Et2O/
Et3N: 99/1) to give 1 (LBT-999) as a white solid (55 mg,
lime/P2O5-guard (35 mm length each, 3 mm internal
diameter) and converted into [11C]CH3OTf by passing
through a glass column (33 cm length, 5 mm internal
diameter), heated at 200 ꢁC and containing silver-tri-
flate-impregnated graphitized carbon (200 mg).
4.3.2. Preparation of [11C]LBT-999 ([11C]-1). [11C]CH3
OTf, carried by a flow of nitrogen gas (40 mL/min), was
trapped (bubbling through) at ꢀ10 ꢁC (EtOH–ice bath)
in a reaction vessel containing 0.6–0.8 mg of the carboxyl-
ic acid derivative 10 (1.9–2.5 lmol) dissolved in 400 lL
acetone and 3 lL of aq NaOH (3 N) (9 lmol). Trapping
of [11C]CH3OTf was monitored using an ionization-
chamber probe. When the reading had reached its maxi-
mum (2–3 min), the reaction mixture was concentrated
to dryness at 110 ꢁC (heating block), using a nitrogen
stream for 1–2 min. The reaction vessel was then cooled
(EtOH–ice bath) and the reaction mixture was diluted
with 0.5 mL of the HPLC mobile phase and was injected
onto the column. HPLC-purification (HPLC 2) gave
radiochemically pure [11C]-1 (Rt: 6.5–7.0 min), well sepa-
rated from the unlabelled precursor 10 (Rt: 4.0–5.0 min).
1
50%). H NMR (CDCl3): d: 1.57–1.68 (m, 3H); 1.90–
2.00 (m, 2H); 2.22 (s, 3H); 2.65 (td, 1H, 3J = 2J =
12.4 Hz, J = 2.6 Hz); 2.92–3.05 (m, 4H); 3.46 (m, 1H);
3
2
3.53 (s, 3H); 3.70 (m, 1H); 4.86 (dd, 2H, JH–F
=
3
47.2 Hz, J = 4.6 Hz); 5.80–5.85 (m, 2H); 7.10 (d, 2H,
3J = 8.0 Hz); 7.20 (d, 2H, 3J = 8.0 Hz). 13C NMR
(CDCl3): d: 20.9; 25.8; 26.0; 33.7; 34.0; 50.8; 52.6; 54.8;
1
2
61.3; 62.2; 83.0 (d, JC–F = 161.0 Hz); 126.1 (d, JC–F
=
=
3
17.1 Hz); 127.1 (2C); 128.5 (2C); 134.1 (d, JC–F
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.
4.3.3. Formulation of [11C]LBT-999 ([11C]-1). Formula-
tion of the labelled product for iv 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-pakꢂPlus C18 car-
tridge (Waters, washed with 2 mL EtOH and then rinsed
with 10 mL of water prior to use). The cartridge was
washed with 7 mL water and partially dried by applying
a nitrogen stream for 10 s. The radiotracer was eluted with
2 mL EtOH followed by 8 mL of physiological saline (less
than 10% of the total radioactivity was left on the car-
tridge) and filtered on a 0.22 lm GS-Millipore filter (vent-
ed). 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.92
4.2.4. 8-((E)-4-Fluoro-but-2-enyl)-3b-p-tolyl-8-aza-bicy-
clo[3.2.1]octane-2b-carboxylic acid (10). Thirty milli-
grams of 8-((E)-4-fluoro-but-2-enyl)-3b-p-tolyl-8-aza-
bicyclo[3.2.1]octane-2b-carboxylic acid methyl ester (1,
0.09 mmol) was dissolved in a 1/1 mixture of dioxane
and water (5 mL). The resulting solution was refluxed
for 5 days. The solvents were removed under vacuum
and the residue was purified by semipreparative re-
versed-phase HPLC (HPLC 1, Rt: 11 min) to give 10
1
as a white solid (15 mg, 53%). H NMR (CDCl3): d:
1.77–2.14 (m, 3H); 2.19–2.25 (m, 2H); 2.32 (s, 3H);
3
3
2.54 (td, 1H, J = 2J = 13.3 Hz, J = 2.6 Hz); 2.70–2.73
(m, 1H); 3.15–3.27 (m, 3H); 3.68 (s, 2H); 4.92 (dd, 2H,
2JH–F = 46.5 Hz, J = 3.9 Hz); 5.92–5.96 (m, 2H); 7.15
3
(s, 4H). 13C NMR (CDCl3): d: 21.0; 25.0; 25.6; 34.6;
35.3; 52.6; 53.5; 59.4; 62.0; 82.1 (d, JC–F = 164.5 Hz);
1
4.3.4. Quality control of [11C]LBT-999 ([11C]-1). The radi-
otracer preparation was visually inspected for clarity, ab-
sence of colour and particulates. An aliquot of the
preparation was evaluated for pH using standard pH-
paper. Chemical and radiochemical purities were also
assessed on this aliquot by HPLC, with a sample of
authentic LBT-999 (HPLC 3; Rt: 2.75 min). Finally, spe-
cific radioactivity of the radiotracer was calculated from
three consecutive HPLC analyses (average) and deter-
mined as follows: the area of the UV absorbance peak cor-
responding to the radiolabelled product was measured
(integrated) on the HPLC chromatogram and compared
to a standard curve relating mass to UV absorbance.
3
127.3 (2C); 127.9 (d, JC–F = 12.0 Hz); 129.3 (2C);
2
130.3 (d, JC–F = 17.1 Hz); 136.7 (2C); 174.4. MS: m/e
(%): 317 (M+, 32); 273 (55); 272 (19); 244 (11); 199
(11); 170 (13); 168 (13); 154 (85); 141 (89); 140 (100);
129 (17); 128 (23); 122 (98); 108 (59); 82 (56); 80 (36);
68 (39); 53 (32).
4.3. Carbon-11 radiochemistry
4.3.1. Preparation of [11C]CH3OTf. [11C]CO2 was re-
leased from the stainless-steel coil trap (see above) by
raising its temperature to ambient, swept away by a flow
of nitrogen gas (40 mL/min) and trapped at ꢀ10 ꢁC
(EtOH-ice bath) in a mixture of THF (60 lL) and lithi-
um aluminium hydride/THF (8 lL, 1.0 M). Concentra-
tion to dryness (evaporation of solvent at 155 ꢁC using
a stream of nitrogen) followed by hydrolysis (100 lL
of deionized water) of the formed aluminium complex
afforded [11C]CH3OH, which was distilled, using a flow
of nitrogen gas, into 0.8 mL of an aqueous 57% HI solu-
tion (heating block at 155 ꢁC). The [11C]CH3I thus syn-
thesized was continuously swept away by the flow of
nitrogen gas, passed through a combined 1/1 (v:v) soda
4.4. Tritium radiochemistry
4.4.1. Preparation of [3H]LBT-999 ([3H]-1). One hundred
fifty microliters of a toluene solution of [3H]methyl io-
dide (370 MBq/ml, 3.1 TBq/mmol) was added to a solu-
tion of 0.7 mg of the carboxylic acid derivative 10
(2.20 lmol) in 300 ll of N,N-dimethylformamide. The
resulting solution was heated for 15 min at 90 ꢁC. The
reaction mixture was then diluted with 300 lL CH3CN

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F. Dolle et al. / Bioorg. Med. Chem. 14 (2006) 1115–1125
1123
13. Ouchi, Y.; Kanno, T.; Okada, H.; Yoshikawa, E.;
Futatsubashi, M.; Nobezawa, S.; Torizuka, T.; Tanaka,
K. Brain 2001, 124, 784.
14. Sekine, Y.; Iyo, M.; Ouchi, Y.; Matsunaga, T.; Tsukada,
H.; Okada, H.; Yoshikawa, E.; Futatsubashi, M.; Takei,
N.; Mori, N. Am. J. Psychiatry 2001, 158, 1206.
15. Tsukada, H.; Nishiyama, S.; Kakiuchi, T.; Ohba, H.;
Sato, K.; Harada, N. Synapse 2001, 42, 273.
and was injected onto the HPLC column. HPLC-purifi-
cation (HPLC 4) gave radiochemically pure [3H]-1 (Rt:
5.2 min), well separated from the unlabelled precursor
10 (Rt: 2.5–3.5 min) and non-reacted [3H]methyl iodide
(Rt: 2.3 min).
4.4.2. Formulation of [3H]LBT-999 ([3H]-1). Formula-
tion of the labelled product was effected as follows:
The HPLC-collected fraction containing the radiotracer
was gently concentrated to dryness. The residue was
taken up in 100 lL of aq 75% EtOH.
16. Rinne, O. J.; Nurmi, E.; Ruottinen, H. M.; Bergman, J.;
Eskola, O.; Solin, O. Synapse 2001, 40, 193.
17. Nurmi, E.; Bergman, J.; Eskola, O.; Solin, O.; Vahlberg,
T.; Sonninen, P.; Rinne, J. O. Synapse 2003, 48, 109.
18. Muller, L.; Halldin, C.; Farde, L.; Karlsson, P.; Hall, H.;
Swahn, C. G.; Neumeyer, J.; Gao, Y.; Milius, R. Nucl.
Med. Biol. 1993, 20, 249.
4.4.3. Quality control of [3H]LBT-999 ([3H]-1). Radio-
chemical puritywas assessed on an aliquot by HPLC, with
a sample of authentic LBT-999 (HPLC 4, Rt: 5.2 min).
19. Farde, L.; Halldin, C.; Muller, L.; Suhara, T.; Karlsson,
P.; Hall, H. Synapse 1994, 16, 93.
20. Laruelle, M.; Wallace, E.; Seibyl, J. P.; Baldwin, R. M.;
Zea-Ponce, Y.; Zoghbi, S. S.; Neumeyer, J. L.; Charney,
D. S.; Hoffer, P. B.; Innis, R. B. J. Cereb. Blood Flow
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Acknowledgments
21. Seibyl, J. P.; Marek, K. L.; Quinlan, D.; Sheff, K.; Zoghbi,
S.; Zea-Ponce, Y.; Baldwin, R. M.; Fussell, B.; Smith, E.
O.; Charney, D. S., et al. Ann. Neurol. 1995, 38, 589.
The authors wish to thank the cyclotron operators Mr.
Daniel Gouel, Mr. Christophe Peronne, and Mr. Chris-
ˆ
tophe Lechene for performing the irradiations and Mr.
˚
22. Nagren, K.; Muller, L.; Halldin, C.; Swahn, C. G.;
Arsalan Amir for expert technical assistance. The authors
thank Dr. Dirk Roeda for proofreading the manuscript
and suggesting linguistic corrections. This work was sup-
Lehikoinen, P. Nucl. Med. Biol. 1995, 22, 235.
23. Hume, S. P.; Luthra, S. K.; Brown, D. J.; Opacka-Juffry, J.;
Osman, S.; Ashworth, S.; Myers, R.; Brady, F.; Carroll, F.
I.; Kuhar, M. J.; Brooks, D. J. Nucl. Med. Biol. 1996, 23,
377.
´
ported by the ÔReseau National de Technologies pour
´
la SanteÕ under the RNTS 03B243 FLUOPARK pro-
24. Zheng, Q. H.; Mulholland, G. K. Nucl. Med. Biol. 1996,
23, 981.
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gramme and by a grant from Biotechnocentre (Region
Centre—France).
25. Ginovart, N.; Lundin, A.; Farde, L.; Halldin, C.; Back-
man, L.; Swahn, C. G.; Pauli, S.; Sedvall, G. Brain 1997,
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