[11C]PR04.MZ, a promising DAT ligand for low concentration imaging: Synthesis, efficient 11C-O-methylation and initial small animal PET studies

Article abstract of DOI:10.1016/j.bmcl.2009.05.090

PR04.MZ was designed as a highly selective dopamine transporter inhibitor, derived from natural cocaine. Its binding profile indicates that [¹¹C]PR04.MZ may be suited as a PET radioligand for the non-invasive exploration of striatal and extrastriatal DAT populations. As a key feature, its structural design facilitates both, labelling with fluorine-18 at its terminally fluorinated butynyl moiety and carbon-11 at its methyl ester function. The present report concerns the efficient [¹¹C]MeI mediated synthesis of [¹¹C]PR04.MZ from an O-desmethyl precursor trifluoroacetic acid salt with Rb₂CO₃ in DMF in up to 95 ± 5% labelling yield. A preliminary μPET-experiment demonstrates the reversible, highly specific binding of [¹¹C]PR04.MZ in the brain of a male Sprague-Dawley rat.

Full text of DOI:10.1016/j.bmcl.2009.05.090

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4343–4345
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[11C]PR04.MZ, a promising DAT ligand for low concentration imaging:
Synthesis, efficient 11C-O-methylation and initial small animal PET studies
a
a
a
a
b
Patrick J. Riss a,b, , Jacob M. Hooker , David Alexoff , Sung-Won Kim , Joanna S. Fowler , Frank Rösch
*
a Medical Department, Brookhaven National Laboratory, Upton, NY 11973, USA
b Institute of Nuclear Chemistry, University of Mainz, Fritz-Strassmann-Weg 2, D-55128 Mainz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
PR04.MZ was designed as a highly selective dopamine transporter inhibitor, derived from natural
cocaine. Its binding profile indicates that [11C]PR04.MZ may be suited as a PET radioligand for the non-
invasive exploration of striatal and extrastriatal DAT populations. As a key feature, its structural design
facilitates both, labelling with fluorine-18 at its terminally fluorinated butynyl moiety and carbon-11
at its methyl ester function. The present report concerns the efficient [11C]MeI mediated synthesis of
Received 2 April 2009
Revised 19 May 2009
Accepted 20 May 2009
Available online 28 May 2009
[
11C]PR04.MZ from an O-desmethyl precursor trifluoroacetic acid salt with Rb2CO3 in DMF in up to
Keywords:
Dopamine transporter
MicroPET
95 5% labelling yield. A preliminary
lPET-experiment demonstrates the reversible, highly specific bind-
ing of [11C]PR04.MZ in the brain of a male Sprague–Dawley rat.
Ó 2009 Elsevier Ltd. All rights reserved.
Carbon-11
Test-block
O-methylation
Non-invasive molecular imaging is an emerging interdisciplin-
ary field of research. Among the available techniques for the visu-
alisation of the biochemical and physiological function of
biological tissue approached by suitable imaging probes, positron
emission tomography (PET) provides great potential in terms of
quantification, sensitivity, temporal and lateral resolution.1 The
dopaminergic system and in particular the presynaptic dopamine
transporter (DAT) represents an important biological target for
the development of specific PET radioligands, due to its role in a
variety of pathologies.2
PR04.MZ (2) is a highly potent (IC50(hDAT) = 3 nM) DAT inhibi-
tor based on the phenyltropane lead derived from cocaine. An
inflexible, extended 4-fluorobut-2-yn-1-yl chain has been added
to the tropane nitrogen. Thereby conformational flexibility has
been reduced to increase selectivity among the structurally similar
monoamine transporters. The mammalian DAT represents a 49%
amino acid homology to the mammalian serotonin transporter
(SERT) and a 67% homology to the norepinephrine transporter
(NET). This modification resulted in a good selectivity profile
(DAT/SERT >100, DAT/ NET ꢀ10) compared to earlier examples of
cocaine derivatives for the application as PET-radioligands.3
PR04.MZ is currently under investigation with respect to its suit-
ability as a PET tracer for in vivo imaging of the DAT. Although it
was specifically designed as an 18F-labelled radiopharmaceutical,
one of its key features is the possibility to incorporate two different
radiolabels. In this regard, both, 11C labelling as well as 18F-fluori-
nation provides specific advantages. The shorter half-life of 11C
permits multiple tracer injections into the same subject on the
same day.4 Thereby, a variety of high value protocols for scientific
studies, that is, test–retest, test-block or baseline-challenge, can be
performed in the same subject without altering the orientation
within the gantry of a PET camera. In contrast, fluorine-18 provides
a more convenient half-life with regard to the total duration of the
radio-synthesis, delivery to off-site PET centers and total acquisi-
tion time of clinical PET examinations.5
The present work is concerned with (a) the synthesis of
non-radioactive 2 as reference, (b) the synthesis of the carbon-11
labelling precursor, (c) the highly efficient O-methylation of the car-
boxylic acid function of 3 and (d) an initial, preliminary PET-study
that underlines the feasibility of exploring the DAT using [11C]-2.
The synthetic route to PR04.MZ (2) and the labelling precursor
(3) is illustrated in Scheme 1.
Commercially available cocaine hydrochloride was hydrolysed
to ecgonine using dilute hydrochloric acid. Subsequent 1,2-elimi-
nation in refluxing POCl3 afforded the a,b-unsaturated acid anhy-
droecgonine which was re-esterified under Fischer-conditions to
afford the Michael-acceptor in 80% yield over three steps.7a The
latter was subsequently exposed to a two-fold excess of p-tolyl
magnesium bromide in CH2Cl2/Et2O at low temperature. The use
of p-tolyl lithium via transmetallation to the Gilman-cuprate was
also examined. Careful protonation followed by chromatographic
purification afforded 75% of the desired exo-isomer.7b Demethyla-
tion in refluxing dichloroethane containing 1-chloroethyl chloro-
formate (ACE-Cl) gave nortropane 1 in up to 90% yield. PR04.MZ
(2) was obtained from 1 via alkylation with 4-fluoro-but-2-ynyl
* Corresponding author. Tel.: +49 6131 3925701.
E-mail address: riss@uni-mainz.de (P.J. Riss).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.090

4344
P. J. Riss et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4343–4345
O
O
F
O
F
CH3
CH3
x TFA
OH
O
O
a
b
N
HN
N
3
2
1
Scheme 1. Synthesis route to PR04.MZ (2) and labelling precursor 3; (a) 4 fluoro-but-2-ynyl chloride; DiPEA, MeCN, 70 °C, 12 h, 95%; (b) 1 M HCl, reflux 1 d, 75%.
chloride in a yield of 95%.3 Finally, the labelling precursor 3 was
obtained by aqueous acidic hydrolysis of methyl ester 2 in reflux-
ing dilute hydrochloric acid. HPLC-purification afforded the pure,
slightly hygroscopic precursor in 60% overall yield.8
after the end of [11C]CO2-production (end of bombardment, EOB)
(Scheme 2).10
All further reactions were carried out using system e, rubidium
carbonate in DMF with a precursor concentration of 0.66 mg/ml. In
a typical production run, base and precursor were weighed into a
Several groups have reported low yields of 6–20% when
employing an ammonium salt precursor for O-methylation.6a,b In
particular, the [11C]MeI methylation of a variety of carboxylic acid
functions demanded liberation of the free base prior to labelling.6a
V-shaped borosilicate vial and 450 ll DMF were added. The vol-
In some cases, the highly reactive
11C]methyl triflate ([11C]MeOTf) had to be used.6a–c
Analogue results were found when the initial labelling of
[
11C]methylation source
[
cerebellum
PR04.MZ was conducted with an organic nitrogen base or common
inorganic bases, such as sodium hydroxide or potassium carbonate.
However, it was analysed that a system consisting of rubidium car-
bonate (Rb2CO3) as base, combined with dry N,N-dimethylformam-
ide (DMF) as solvent, affords [11C]PR04.MZ in high yield. Contrary
to earlier reports, these conditions facilitate the use of readily
available, high-specific-activity [11C]MeI, together with a stable
and insensitive 2,2,2-trifluoroacetic acid (TFA)-salt precursor. Iso-
lation of the free base can be avoided.
caudate
putamen
harderian
glands
[
11C]Methyl iodide was obtained 14–18 min after EOB from a
common GEÒ 11C]methyl iodide module (GE box). The radioactiv-
Figure 1. PET/MRI-Fusion of brain images of [11C]PR04.MZ in a male Sprague–
Dawley rat. Injected dose: 37 MBq, 67 GBq/
[
lmol (1 mCi, 1.8 Ci/
lmol) (left:
ity was trapped in DMF at room temperature and partitioned over
multiple reaction vessels to facilitate screening.9 Appropriate
labelling conditions were elucidated and verified in duplicate.
Reaction of precursor 3 with [11C]MeI in DMF or a 1:1 mixture of
DMF and dimethylsulfoxide (DMSO) using common bases like so-
dium hydroxide, triethylamine, sodium or potassium carbonate
or potassium hydrogen carbonate did not lead to acceptable radio-
chemical yields (0–7%, Table 1). Following solubility consider-
ations, we examined rubidium carbonate and caesium carbonate
as alternatives. The use of these highly soluble bases strikingly in-
creased the labelling yields for [11C]MeI methylation. Initially,
1 mg of precursor 3 was used. In a typical synthesis for biological
studies, 2.04 GBq (55 10 mCi) of [11C]-2 were obtained 45 min
baseline; right: block; summed images from 20 to 60 min).
3
3
2
2
1
1
0
3
2
1
0
cerebellum
20
striatum
40
Table 1
0
Impact of various bases on the radiochemical yield (RCY) of [11C]MeI-supported
0
60
methylation of precursor 3
time/min
Entry
Base
Solvent
RCYa (%)
0
3
a
b
c
d
e
f
1 M NaOHaq
Na2CO3
K2CO3
Cs2CO3
Rb2CO3
Rb2CO3
DMF
DMF
DMF
DMF
DMF
DMF/DMSO
3
7
1
1
90
95
92
8
5
6
2
1
0
a
Errors are given as one SEM after three independent runs.
F
O
11CH3
O
O
F
x TFA
cerebellum
20
striatum
40
OH
c
0
60
N
N
time/min
[11C]-2
3
Figure 2. Time activity curve for [11C]PR04.MZ in a male Sprague–Dawley rat.
Injected dose: 37 MBq (1 mCi), specific activity: 67 GBq/lmol (1.8 Ci/lmol). Upper
Scheme 2. Carbon-11 labelling, (c) Rb2CO3 (2.1 equiv), DMF, [11C]MeI, 5 min, 75 °C.
curve: baseline scan, lower curve: blocking study.

P. J. Riss et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4343–4345
4345
Table 2
Quantitative uptake of [11C]PR04.MZ and distribution volume ratios for striatum and cerebellum, derived from the preliminary
lPET-study
Aquisition timea (min)
Baseline
Pretreatment
Reductiond (%)
%ID(striatum) (cm3)
Striatum/cerebellumb
BP(striatum)c
%ID(striatum) (cm3)
Striatum/cerebellumb
BP(striatum)c
15
30
60
2.0
2.1
2.1
2.4
3.6
5.0
3.4
4.6
6.0
2.1
1.6
1.2
1.25
1.67
1.61
2.3
2.7
2.6
32
41
57
a
Time-frame of the
Radioactivity–concentration ratio.
Striatal binding potential from simplified reference tissue model.
Effect of pretreatment to BP(striatum).
l
PET-scan.
b
c
d
ume was increased from 300
efficiency. This vial was thoroughly vortexed and connected to the
11C]MeI delivery tubing. [11C]MeI was directly trapped in this
suspension.
Subsequently, the vial was placed in an oil bath and heated to
75 °C for 5 min. The reaction mixture was quenched with HPLC-
eluent, purified by HPLC and concentrated by rotary evaporation.
After evaporation to dryness, the product was dissolved in sterile
sodium chloride solution and passed through a small MilliporeÒ
l
l to 450
l
l to increase the trapping
[
11C]methyl iodide in high yield. In a typical production run start-
ing from ꢀ6.3 GBq (ꢀ170 mCi) of [11C]MeI, 2.04 GBq (55 10 mCi)
of injectable radiotracer were obtained after 45 min.
[
The value [11C]PR04.MZ as a highly selective probe for the pre-
synaptic DAT has been assessed by an preliminary rat study. These
results indicate highly selective binding to rat dopamine transport-
ers. The reversibility of the highly selective DAT-ligand was dem-
onstrated by pre-treatment with a structurally non-analogous
DAT-inhibitor (GBR12909).
sterile filter (0.22 lm) into a multi-injection vial (MIV). This simple
process afforded [11C]-7 in a total, non-decay corrected yield of 20%
and a radiochemical purity of greater than 98% after 45 min of total
synthesis duration. The specific activity of [11C]PR04.MZ at this
References and notes
1. (a) Lasne, M.-C.; Perrio, C.; Rouden, J.; Barré, L.; Roeda, D.; Dolle, F.; Crouzel, C.
Top. Curr. Chem. 2002, 222, 201; (b) Cai, L.; Lu, S.; Pike, V. W. Eur. J. Org. Chem.
2008, 2853.
2. (a) Fowler, J. S.; Volkow, N. D.; Wolf, A. P.; Dewey, S. L.; Schlyer, D. J.;
Macgregor, R. R.; Hitzemann, R.; Logan, J.; Bendriem, B.; Gatley, S. J., et al
Synapse 1989, 4, 371; (b) Volkow, N. D.; Fowler, J. S.; Gatley, S. J.; Logan, J.;
Wang, G.-J.; Ding, Y.-S.; Dewey, S. J. Nucl. Med. 1996, 37, 1242; (c) Rinne, J. O.;
Bergman, J.; Ruottinen, H.; Haaparanta, M.; Eronen, E.; Oikonen, V.; Sonninen,
P.; Solin, O. Synapse 1999, 31, 119.
3. P. J. Riss, R. Hummerich, P. Schloss, Org. Biomol. Chem, in press. doi:10.1039/
B902863C.
4. Antoni, G.; Kihlberg, T.; Langstrom, B.. In Handbook of Nuclear Chemistry;
Kluwer Academic: Dordrecht, 2003; Vol. 4, pp 119–165.
point exceeded 67 GBq/
A 300 g male adult Sprague–Dawley rat was used for a preli-
minary Imager. In a test-block
PET-study in a CTIÒ Focus 120
lmol (1.8 Ci/lmol).
l
l
experiment, the animal was anaesthetised with ketamine/xylazine
(90/10, 100 mg/kg), and injected with approximately 37 MBq
(1 mCi) of the radiotracer, directly into the tail vene. A full-dy-
namic scan was performed for 60 min. Subsequently, the same ani-
mal was pretreated with 1.5 mg/kg of GBR12909, 45 min prior to
the injection of a second 37 MBq dose of [11C]PR04.MZ. Data acqui-
sition was started simultaneous to the injection of tracer and con-
tinued for 60 min. Image reconstruction and corregistration was
performed using the commercial image processing software
PMOD (www.pmod.com). Binding potentials (BP) were calculated
using the simplified reference tissue model (SRTM).
5. Wester, H. J.. In Handbook of Nuclear Chemistry; Kluwer Academic: Dordrecht,
2003; Vol. 4, pp 166–202.
6. (a) Dolle, F.; Bottlaender, M.; Demphel, S.; Emond, P.; Fuseau, C.; Coulon, C.;
Ottaviani, M.; Valette, H.; Loc’h, C.; Halldin, C.; Mauclaire, L.; Guilloteau, D.;
Maziere, B.; Crouzel, C. J. Labelled Compd. Radiopharm. 2000, 43, 997; (b)
Lundkvist, C.; Sandell, J.; Nagren, K.; Pike, V. W.; Halldin, C. J. Labelled Compd.
Radiopharm. 1998, 41, 545; (c) Någren, K.; Halldin, C.; Müller, L.; Swahn, C.-G.;
Lehikoinen, P. Nucl. Med. Biol. 1995, 22, 965.
The LONI (www.loni.ucla.edu/ratdata/Rat.html) anatomical
magnetic resonance imaging (MRI)-atlas of the rat brain was used
for PET/MRI-fusion and the identification of brain regions. Regions
of interest were drawn onto a MRI-template and copied into the
7. (a) Meltzer, P. C.; Liang, A. Y.; Brownell, A. L.; Elmaleh, D. R.; Madras, B. K. J. Med.
Chem. 1993, 36, 855; (b) Xu, L.; Trudell, M. J. Heterocycl. Chem. 1996, 33, 2037.
8. 1H NMR (CDCl3, 300 MHz) d in ppm: 10.09 (br s, 1H), 4.95 (d, JHF = 47.5 Hz, 2H),
4.34–4.18 (m, 2H), 3.81–3.66 (m, 1H), 3..36–3.24 (m, 1H), 3.16–2.98 (m, 2H),
2.78 (dt, J = 12.5 Hz, J = 2.9 Hz), 2.39–2.29 (m, 2H), 2.27 (s, 3H), 1.98–2.18 (m,
3H), 1.96–1.84 (m, 2H). 13C NMR (CDCl3, 100 MHz) d in ppm: 177.0, 137.6,
134.7, 129.5, 127.1, 84.4, 78.8, 71.4, 69.1, 62.8, 62.0, 49.9, 45.8, 41.4, 34.2,
32.824.4, 24.1, 20.9, 8.5. HRMS (ESI): 316.1723 (M+) C19H23FNO2 requires
co-registered lPET-datasets.
The results are shown in Figure 1. Peak uptake of 2.22% of the in-
jected dose per cm3 of tissue (%ID/cm3) was achieved in the baseline
scan 4 min post injection. The results clearly indicate the highly spe-
cific binding of [11C]PR04.MZ to striatal DAT-binding sites in the rat
brain (Fig. 2). Figure 1 further illustrates the effect of GBR12909 pre-
treatment in a test-block study. In comparison to the baseline scan, a
significant reduction in striatal radioactivity concentration can be
observed within the blocking study. In contrast, the radioactivity
accumulation in the harderian glands is increased in the pretreated
animal compared to the baseline scan. Furthermore, the radioactiv-
ity concentration ratios between the striatum and the cerebellum
after pretreatment are significantly lower (cf. Table 2). Pre-treat-
ment with the highly selective DAT-inhibitor leads to a significant
decrease in the total activity accumulation in the basal ganglia.
These findings may indicate the highly selective binding of PR04.MZ
to rat dopamine transporters (rDAT).
316.1713. HPLC: phenomenex LunaÒ RP18
(10 Â 250 mm, eluent: 40% MeOH in 0.05% TFAaq).
9. Base screening experiments were conducted as follows: standard 5 ml screw-cap
vials were charged with base (2 equiv), 1.4–4.6 mol) and 3 was added (0.3–
1 mg, 0.7–2.3 mol). The solids were suspended in either DMF (0.15 ml, for
5l semipreparative column
l
l
reactions in pure DMF) or DMSO (0.15 ml, for reactions in a mixture of DMSO
and DMF). These vials were placed in a self-made multi-vial holder and placed
above an oil bath, prior to the addition of 150 l
l of DMF, containing [11C]MeI
(37–74 MBq; 1–2 mCi). The oil bath was elevated and the vials were heated for
5 min at 75 °C. The heat-source was removed subsequently, and the reaction
was immediately quenched by the addition of HPLC-eluent (1 ml). At this point,
the activity inside the vial was measured and an aliquote was withdrawn to
determine the radiochemical yield by HPLC and TLC. The results are
summarised in Table 1.
10. HPLC-purification was performed using
a
PhenomenexÒ LunaÒ RP 18
semipreparative HPLC-column (dimensions 10 Â 250 mm) as stationary
phase. The mobile phase consisted of 36% 0.1 M ammonium formate
solution (v:v) in acetonitrile. At
a flowrate of 4.7 ml/min, the product
In conclusion, an acid ammonium salt labelling precursor for
eluted after a purification time of tr ([11C]-7) = 13 1 min, in a radiochemical
[
11C]PR04.MZ has been prepared and successfully labelled with
purity of >98%.