Radiosynthesis of (±)-(2-((4-(2-[18F]fluoroethoxy)phenyl)bis(4-methoxy- phenyl) methoxy)ethylpiperidine-3-carboxylic acid: A potential GAT-3 PET ligand to study GABAergic neuro-transmission in vivo

Article abstract of DOI:10.1002/jlcr.492

A dysfunction of GABAergic neurotransmission is related to diseases such as epilepsy, Huntington-disease and Parkinson-syndrome. A new ¹⁸F-fluorine labelled GABA transporter ligand for the GABA-transporter subtype GAT-3 was developed which may allow the in vivo visualisation of GABAergic neurotransmission. The precursors ethyl (2-(4-hydroxyphenyl)bis (4-methoxyphenyl)-methoxy)ethyl)-piperidine-3-carboxylate and ethyl(2-((4-(2-tosylethoxy)phenyl)-bis(4-methoxyphenyl)-methoxy) ethyl)piperidine-3-carboxylate were synthesised and labelled by the use of 2-[¹⁸F]fluoroethyltosylate or [¹⁸F]fluoride. Subsequent cleavage of the ester moiety gave the final product (±)-(2-((4-(2-[¹⁸F]fluoroethoxy)phenyl)bis(4-methoxy- phenyl)methoxy)ethyl)-piperidine-3-carboxylic acid in a decay corrected yield of 33-36%. Preliminary biodistribution kinetics were determined with BALB/c mice ex vivo for brain, liver, kidney, spleen, blood and bone. (2-((4-(2-[¹⁸F]fluoroethoxy)-phenyl)bis(4-methoxyphenyl)methoxy)- ethyl) piperidine-3-carboxylic acid showed a maximum brain uptake after 1 h p.i. of about 0.3% ID/g. Copyright

Full text of DOI:10.1002/jlcr.492

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 627–642.
DOI: 10.1002/jlcr.492
Radiosynthesis of (Æ)-(2-((4-(2-[18F]fluoro-
ethoxy)phenyl)bis(4-methoxy-phenyl)
methoxy)ethylpiperidine-3-carboxylic acid:
a potential GAT-3 PET ligand to study
GABAergicneuro-transmission in *i*o
Ralf Schirrmacher1, Wilhelm Hamkens1, Markus Piel1, Ulrich Schmitt2,
2
Hartmut Luddens , Christoph Hiemke and Frank Rosch
2
1,
*
.
.
1 Institute for Nuclear Chemistry, D-55128, Mainz, Germany
2 Department of Psychiatry, Johannes Gutenberg-University Mainz, Germany
Summary
A dysfunction of GABAergic neurotransmission is related to diseases such as
epilepsy, Huntington-disease and Parkinson-syndrome. A new 18F-fluorine
labelled GABA transporter ligand for the GABA-transporter subtype GAT-3
was developed which may allow the in vivo visualisation of GABAergic
neurotransmission.
The
precursors
ethyl
(2-(4-hydroxyphenyl)bis
(4-methoxyphenyl)-methoxy)ethyl)-piperidine-3-carboxylate and ethyl(2-((4-
(2-tosylethoxy)phenyl)-bis(4-methoxyphenyl)-methoxy) ethyl)piperidine-3-car-
boxylate were synthesised and labelled by the use of 2-[18F]fluoroethyltosylate
or [18F]fluoride. Subsequent cleavage of the ester moiety gave the final product
( Æ )-(2-((4-(2-[18F]fluoroethoxy)phenyl)bis(4-methoxy-phenyl)methoxy)ethyl)-
piperidine-3-carboxylic acid in a decay corrected yield of 33–36%. Preliminary
biodistribution kinetics were determined with BALB/c mice ex vivo for brain,
liver, kidney, spleen, blood and bone. (2-((4-(2-[18F]fluoroethoxy)-phenyl)bis(4-
methoxyphenyl)methoxy)-ethyl) piperidine-3-carboxylic acid showed a max-
imum brain uptake after 1 h p.i. of about 0.3% ID/g. Copyright # 2001 John
Wiley & Sons, Ltd.
Key Words: GABA; GABA transporter ligand; [18F]fluoroethyl-derivatives
.
*Correspondence to: F. Rosch, Institute for Nuclear Chemistry, D-55128 Mainz, Germany.
E-mail: frank.roesch@uni-mainz.de.
Contract/grant sponsor: Deutsche Forschungsgemeinschaft; contract/grant number: DFG Ro 985/8-1
Copyright # 2001 John Wiley & Sons, Ltd.
Received 8 November 2000
Revised 27 April 2001
Accepted 19 May 2001
Published online \ \ \ \

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R. SCHIRRMACHER ET AL.
Introduction
g-Aminobutyricacid (GABA) is the major inhibitory neurotransmitter
in the mammalian central nervous system.1 For most neurotransmitters
including GABA, neurotransmission is terminated by rapid uptake of
neurotransmitters via specific, high-affinity transporters located in the
2
presynapticterminal and/or surrounding glial cells. Efforts have been
made to examine the inhibitory neurotransmission by directly radio-
labelling GABA with carbon-11.3 These attempts were unsuccessful
because GABA does not permeate the blood–brain barrier. GABA
derivatives such as [11C] methoxy-norchloroprogabidic acid4 were not
able to visualize GABA receptors in vivo. Furthermore, the in vivo
evaluation of the tritiated GABAA/Benzodiazepine receptor ligands
[methyl-3 H]L655,708 and [ethyl-3 H]RY80 as putative PET ligands
showed that both were not suitable candidates for further develop-
ment.5 Alternatively, a promising target for PET might be found in the
GABA transporter ligands.
The approach to utilise neurotransmitter transporters located at the
presynapticreceptor membrane has been demonstrated for other CNS
receptor systems. For the dopaminergic system in particular, there are
labelled dopamine transporter (DAT) ligands available. Individual PET
tracers have been in use for quantifying the presynaptic synthesis of
dopamine as well as for the binding of dopamine and dopamine
derivatives at D2-like receptors which are located at the postsynaptic
membrane. Still, DAT-ligands provide valuable additional tools for
quantifying dopaminergicfunction in vivo.6–11
GABA is synthesised in vivo via enzymaticdeacrboxylation of
glutamate, stored in vesicles and finally released into the synaptic cleft
where it interacts with GABAA and GABAB receptors. Its action is
terminated by high-affinity transporters. GABA transporters (GAT)
can be inhibited by pharmacologic agents, increasing the level of
neurotransmitter in the synapse and thus enhancing GABAergic
transmission. Up to now five subtypes of GABA transporter have been
reported, three GABA transporters, GAT-1 to GAT-3, a GABA/betain
transporter, BGT-1 and the structurally diverse vesicular GABA
transporter, vGAT. The isoforms have been sequenced and cloned.20, 21
Several potential radioligands for GABA uptake sites have been
synthesised,6–9 most of them derivatives of nipecotic acid. However,
pharmacological evaluation of these ligands was either not included or
restricted to the characterisation of GABA uptake inhibition which not
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18F-GABA-TRANSPORTER LIGANDS
629
necessarily is synonymous with the ligands’ affinity to GABA
transporters. 1-[4-[4-[11C]methoxyphenyl)-4-phenyl-but-3-enyl]-piperi-
dine-3-carboxylic acid9 and l-[3,3-bis-(4-[18F]trifluoromethylphenyl)-
propyl]piperidine-3-carboxylic acid6 are very similar in structure to
1-(4,4-diphenyl-but-3-enyl)piperidine-3-carboxylic acid (SK&F 89976A)
and 1-[3,3-bis-(4-trifluoromethylphenyl)propyl]1,2,5,6-tetrahydro-pyri-
dine-3-carboxylic acid (CI-966) which were evaluated for their subtype
specificity.18 Both are potent GAT-1 inhibitors (IC50 values for
inhibition of [3 H]GABA uptake of 0.13 and 0.26 mM) compared to
GAT-3 inhibition (1990 and 333 mM, respectively).18 Until now, no
information is available about the analogous binding characteristics of
the 11C- and 18F-labelled derivatives of SK&F 89976A9 and CI-966.6
However, 11C-methylation of SK&F 89976A9 or the exchange of the
guvacine structure element with nipecotic acid in the case of CI-9666 for
example, not necessarily influence the subtype specificity of the ligands.
Thus, the PET ligands proposed so far for GABA uptake sites bind
predominantly to the human GABA transporter subtype 1.
The regional distribution of the GABA transporter subtypes varies
significantly.15,16,17,20 GAT-1 and GAT-2 are widespread in the human
brain whereas GAT-3 is more concentrated in the hypothalamus and
midline thalamus, making the latter sub-type an interesting target for
PET. In order to develop a regionally specific PET ligand, the GAT-3
system thus seems to be more promising than GAT-1 or GAT-2.
A variety of lipophilictransporter ligands for all subtypes of GABA-
transporters have been synthesised. Starting from one of these studies,
Dhar and co-workers synthesized triaryl-substituted nipecotic acid
derivatives with high affinity and selectivity for the GAT-3 subtype.18
The GAT-3 ligand 1-[2,2,2-tris-(4-methoxy-phenyl)propyl]piperidine-
3-carboxylic acid (SNAP 5114) with an IC50 value of 10 mM for the
inhibition of [3H]GABA uptake18 has the highest potency of all
evaluated compounds and was therefore selected as a lead compound
for the development of 18F- and eventually 11C-labelled derivatives for
potential applications using PET. The 18F-fluorine labelled derivative
(Æ)-(2-((4-(2-[18F]fluoroethoxy)phenyl)-bis(4-methoxyphenyl)methoxy)
ethyl)piperidin-3-carboxylic acid ([18F]fluoroethyl-SNAP-5114) (1)
%
(Figure 1) was considered to be a candidate for measuring the dynamic
changes of GABA concentration in the synaptic cleft.
If the labelled GABA transporter ligand reached the transporter and
the binding was reversible, stimulation of GABA release into the
synaptic cleft would lead to a dissociation of the GAT-3/GAT-3-ligand
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R. SCHIRRMACHER ET AL.
Figure 1. Structures of the original compound SNAP-5114 and the fluoroethy-
lated derivative [18F]fluoroethyl-SNAP-5114 (1)
%
complex. This process could possibly be visualized with PET as
an indirect measure of the GABA concentration in the synaptic cleft,
being useful to indicate illness-related changes in GABAergic neuro-
transmission.
Results and discussions
It was the strategy of these syntheses to label the compound SNAP-5114
with [18F]fluorine in a simple way avoiding too many reaction steps.
Consequently, the two precursors ethyl(2-((4-(2-tosylethoxy)phenyl)-
bis(4-methoxyphenyl)methoxy)-ethyl) piperidine-3-carboxylate (15) and
(2-(4-hydroxyphenyl)bis(4-methoxyphenyl)-methoxy)ethyl)piperidine-3-
carboxylate (16) were synthesized. (16) comprises a phenolic hydroxy
function and could – with regard to the original structure of the GAT-3
ligand SNAP 5114 – be labelled with 2-[18F]fluoroethyltosylate21
in two steps. Compound (15) could be labelled directly with
[18F]fluoride with subsequent cleavage of the nipecotic ester moiety.
The precursor synthesis of (16) was published recently.22 The synthesis
of the standard compound (8) was performed in a similar manner
%
(Scheme 1).
The synthesis of (15) could be achieved by carrying out the following
reaction steps: methyl 4-hydroxybenzoate (2) was first reacted with 2-
%
bromoethanol and subsequently with 3,4-dihydro-2H pyran to obtain
methyl 4-(2-(2-oxanyloxy)ethoxy) benzoate (10). This compound was
then coupled with 4-methoxyphenyl magnesium bromide and reacted
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631
Scheme 1. (a) methyl 4-hydroxybenzoate, 1-bromo-2-fluoroethane, MeOH; (b)
4-methoxyphenyl magnesium bromide, THF; (c) acetylchloride; (d) (6), (5),
%
%
DMAP, THF; (e) LiOH (1 N), methanol
with acetyl chloride to yield 2-(4-(bis(4-methoxyphenyl)chloro-methyl)-
phenoxy)ethyl acetate (12). Ethyl (2-hydroxyethyl)piperidine-3-carbox-
ylate (6) was reacted with (12) in THF. The acetyl group was removed
with sodium methanolate in methanol to yield (14). Finally, the hydroxy
moiety was tosylated with tosyl chloride to yield compound (15) in an
overall yield of 2.6% (Scheme 2).
The final radiolabelling with 2-[18F]fluoroethyltosylate was performed
in DMSO as a solvent at a temperature of 808C within 5 min yielding
the desired product ethyl (2-((4-(2-[18F]fluoroethoxy)phenyl)bis(4-meth-
oxyphenyl)methoxy)ethyl)piperidine-3-carboxylate (17) in a decay
corrected radiochemical yield of 37–40% (Scheme 3). Purification of
the crude product was accomplished by HPLC. Subsequent quantitative
cleavage of the remaining ethyl ester moiety within 5 min gave the final
product [18F]fluoroethyl-SNAP-5114 (1) in 33–36% radiochemical yield
(decay corrected). The total preparation time till a solution for the
biodistribution studies was obtained was approximately 50 min.
Alternatively, a direct labelling approach was performed. The
radiolabelling of (15) with [18F]fluoride was carried out in acetonitrile
at a temperature of 808C within 40 min to yield the 18F-fluorinated
product in 21% radiochemical yield (decay corrected) (Scheme 3).
Further work-up was accomplished as described above. The total
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R. SCHIRRMACHER ET AL.
Scheme 2. (a) 2-bromoethanol, (b) 3,4-dihydro-2H-pyran, (c) 4-methoxyphenyl
magnesium bromide, H2SO4, (d) acetyl chloride, (e) ethyl (2-hydroxyethyl)piper-
idine-3-carboxylate, (f ) sodium methanolate, methanol, (g) 4-toluenesulfonyl
chloride
preparation time was 570 min and the total decay corrected radio-
chemical yield of (1) was 19%.
%
The in vitro stability of (1) was measured 24 h after the radiosynthesis
%
with radio TLC and showed 95% of the radioligand to be intact. The
maximum brain uptake of (Æ)-(2-((4-(2-[18F]fluoroethoxy)phenyl)bis(4-
methoxyphenyl)methoxy)ethyl)-piperidin-3-carboxylic acid in BALB/c
mice was 0.3% ID/g and was reached at 1 h p.i. Excretion was primarily
via the urine (20% ID/g after 60 min). The kidneys showed an
increasing uptake of 3–5% ID/g radioactivity within 60 min. The liver
uptake was 55% after 60 min. These results suggest that further in vitro
comparative studies of the cold standard compound (8) against the
%
original tritiated GAT-3 ligand [3H]SNAP-5114 are warranted. For that
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18F-GABA-TRANSPORTER LIGANDS
633
Scheme 3. Radioactive labelling of (16) with 2-[18F]fluoroethyltosylate (A) and
of (15) with [18F]fluoride (B) to yield (17) followed by subsequent ester hydrolysis
to (1)
%
purpose the tritium labelled original SNAP-5114 was synthesised.20 The
in vitro evaluation of fluoroethylated derivative of SNAP-5114 (8) is
%
under investigation.
Experimental
1-Bromo-2-fluoroethane (Lancaster), ethyl piperidine-3-carboxylate
(Fluka), 4-bromo anisole (Fluka), methyl 4-hydroxy benzoate (Fluka),
acetyl chloride (Merck), dimethylaminopyridine (DMAP) (Merck) and
Kryptofix 2.2.2. (Merck) were purchased from the companies shown in
brackets. All other reagents were of analytical grade and were used
without further purification. Solid-phase columns were purchased from
Merck (Lichrolut EN). Analytical thin layer chromatography (TLC)
were performed using plates from Merck (Silicagel 60 F254). High-
performance liquid chromatography (HPLC) was performed with a
Sycam S1100 system, UV detection was obtained using a UV detector
Sycam S3200. NMR spectra were recorded using a Bruker 200-MHz-
FT–NMR spectrometer AC 200. Chemical shifts are quoted in d (ppm)
downfield from tetramethylsilane (TMS) as an internal standard. IR
spectra were recorded using a Perkin Elmer FT–IR spectrometer 1760X
with KBr presslings. MS spectra were obtained on a Finnigan MAT90
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R. SCHIRRMACHER ET AL.
spectrometer. Detection of radioactivity was performed using an instant
imager (Packard Canberra) for radio-TLC and detection of radio-
HPLC was performed using a NaI-radiodetector (Canberra Packard).
The HPLC-system was used isocratically for the purification of the ester
protected 18F-fluorinated tracer. [18F]Fluoride as produced via the
18O(p,n)18F reaction was purchased. Purification of the labelling
precursor 2-[18F]fluoroethyltosylate was accomplished using an HPLC
column (Lichrosphere RP18-EC5, 250 Â 10 mm). Purification of the
labelled compound ethyl (2-((4-(2-[18F]fluoroethoxy)phenyl)bis(4-meth-
oxyphenyl)methoxy)ethyl)piperidine-3-carboxylate was achieved using
a HPLC column (Lichrosphere PRP 1-10, 250 Â 4.6 mm).
The preliminary animal experiments were carried out in compliance
with the German legal regulation of animal experimentation. About
1.5 MBq each of (1) were injected intraveneously in the tail vein of eight
%
BALB/c mice and the biodistribution of radioactivity was measured ex
vivo for brain, liver, kidney, muscle, suprarenal body, colon, small
intestine, stomach and heart at 5, 10, 25 and 60 min p.i. Detection of the
radioactivity in organ tissue was carried out using a NaI(TI)-detector.
Ethyl(2-hydroxyethyl)piperidine-3-carboxylate (6)
%
To a solution of ethyl piperidine-3-carboxylate (2.48 ml, 16 mmol) in
1,4-dioxane (20 ml) were subsequently added 2-bromo ethanol (1.13 g,
16 mmol), potassium carbonate (6.63 g, 48 mmol) and a small amount of
sodium iodide. The mixture was refluxed overnight, concentrated under
reduced pressure, extracted with ethyl acetate (30 ml), filtered and dried.
The organic layer was evaporated under reduced pressure. The product
(6) was obtained as a yellow oil after separation using column
%
chromatography (2:1, ethylacetate/hexane) (2.7 g, 13.3 mmol, 83%)
according to a published procedure.4
1H-NMR (200 MHz, CDCl3): (3H, m, 1.2 ppm), (8H, m, 1.5–2.6 ppm),
(2H, t, 2.6ppm), (IH, m, 2.7–2.76 ppm), (lH, m, 2.85–2.9ppm), (2H, t,
3.6 ppm), (2H, q, 4.1ppm).
Elemental Analysis (C10H19NO3) C, H, N.
Methyl 4-(2-fluoroethoxy)benzoate (3)
%
To a sodium methanolate solution (sodium: 0.46 g (0.02 mol), methanol
(30 ml)) methyl 4-hydroxybenzoate (2) (3 g, 0.02 mol) and l-bromo-2-
%
fluoroethane (2.54 g, 0.02 mol) were added. The mixture was refluxed
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635
under exclusion of moisture for 5 h. The reaction mixture was passed
into water and the precipitating product was collected, washed with
water and dried under vacuum. The yield of methyl 4-(2-fluoroethoxy)
benzoate (3) was 69% (2.7 g, 0.013 mol).
%
1H-NMR (CDCl3): (3H, s, 4.0 ppm), (1H, m, 4.1 ppm), (1H, m,
4.3 ppm), (1H, m, 4.6 ppm), (1H, m, 4.9 ppm), (4H, m, 6.9–8 ppm).
IR: n(CO): 1210, n(C–F): 1180–1325, n(CO): 1730.
(4-(2-Fluoroethoxy)phenyl)bis(4-methoxyphenyl)methanol (4)
%
To a Grignard solution of 4-methoxyphenyl magnesium bromide
(0.024 mol) in THF a solution of methyl 4-(2-fluoroethoxy)benzoate
(3) (2.3 g, 0.012 mol) in THF was slowly added under stirring. The
%
mixture was kept at 48C over night, hydrolysed with saturated ammonia
chloride solution, extracted with ether and the organic layer was dried
over sodium sulphate. The solvent was evaporated under reduced
pressure. The crude product was recrystallised from acetic acid/water
to yield (4-(2-fluoroethoxy)phenyl)bis(4-methoxyphenyl)methanol(4)
%
(4.2 g, 0.01 mol, 95%).
1H-NMR (CDCl3): (6H, s, 3.8 ppm), (1H, m, 4.1 ppm), (1H, m,
4.3 ppm), (1H, m, 4.6 ppm), (1H, m, 4.9 ppm), (12H, m, 6.5–7.5 ppm).
IR: n(Ct–OH): 1000–1200, n(C–F): 1180–1325, n(Ph–O–CH3): 2830.
(4-(2-Fluoroethoxy)phenyl)bis(4-methoxyphenyl)chloride (5)
%
To (4-(2-fluoroethoxy)phenyl)bis(4-methoxyphenyl)methanol (4) (4.2 g,
%
0.01 mol) was added acetyl chloride (50 ml) and the mixture was refluxed
for 2 h. After removing the excess of acetyl chloride under reduced
pressure, product (5) was quantitatively obtained as a red solid.
%
1H-NMR (CDCl3): (6H, s, 3.8 ppm), (1H, m, 4.1 ppm), (1H, m,
4.3 ppm), (1H, m, 4.6 ppm), (1H, m, 4.9 ppm), (12H, m, 6.5–7.5 ppm).
IR: n(C–Cl): 560–630, n(C–F): 1180–1325, n(Ph–O–CH3): 2830.
Ethyl(2-((4-(2-fluoroethoxy)phenyl)bis(4-methoxyphenyl)methoxy)
ethyl)-piperidine-3-carboxylate (7)
%
To a solution of ethyl (2-hydroxyethyl)piperidine-3-carboxylate (6)
%
(1.63 g, 8.1 mmol) in dry THF (10 ml) were subsequently added
triethylamine (2 ml, 18.6 mmol), 2-dimethylaminopyridine (DMAP)
(98 mg) and (4-(2-fluoroethoxy)phenyl)bis(4-methoxyphenyl)chloride
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(5) (3.7 g, 10.5 mmol) and stirred over night at room temperature. The
%
solution was passed into a mixture of dichloromethane (50 ml) and
water (10 ml). The organiclayer was dried, filtered and evaporated. The
crude reaction product was purified using flash-chromatography (ethyl-
acetate/hexane/triethylamine 40/60/+0.1%) on silica gel and (7) was
%
obtained as a yellow oil with a yield of 35%.
1H-NMR (CDCl3): (3H, t, 1.24 ppm), (5H, m, 1.45–2.0 ppm), (1H, t,
2.2 ppm), (1H, m, 2.5–2.55 ppm), (2H, t, 3.2 ppm), (6H, s, 3.7 ppm), (2H,
q, 4.0 ppm), (1H, m, 4.1 ppm), (1H, m, 4.3 ppm), (1H, m, 4.6 ppm), (1H,
m, 4.9 ppm), (12H, m, 7.3–7.9 ppm).
Elemental Analysis (C33H40FNO6) C, H, N.
(2-((4-(Fluoroethoxy)phenyl)bis(4-methoxyphenyl)methoxy)ethyl)
piperidine-3-carboxylic acid (8)
%
To a solution of ethyl (2-((4-(2-fluoroethoxy)phenyl)bis(4-methoxyphe-
nyl)-methoxy)ethyl)piperidine-3-carboxylate (7) (0.61 g, 1.35 mmol) in
%
ethanol (6 ml) LiOH (1N) (2 ml, 2.7 mmol) was added and stirred at
room temperature for 18 h. The reaction mixture was adjusted to pH 6
using a sodium hydrogen phosphate solution (5%) and passed into
mixture of dichloromethane/water (60 ml/10 ml). The organic layer was
dried over sodium sulphate, filtered and separated from the solvent to
yield (8) as a foamy solid (0.46 g, 0.9 mmol, 72%).
%
1H-NMR (CDCl3): (5H, m, 1.45–2.0 ppm), (1H, t, 2.2 ppm), (1H, m,
2.5–2.55 ppm), (2H, t, 3.2 ppm), (6H, s, 3.7 ppm), (1H, m, 4.1 ppm),
(1H, m, 4.3 ppm), (1H, m, 4.6 ppm), (1H, m, 4.9 ppm), (12H, m,
7.3–7.9 ppm).
MS (FD): m=z (% relative intensity) 537.3 (100.0, [M]+), 491,2 (6.63,
[M-C2H4F]+).
Methyl 4-(2-hydroxyethoxy)benzoate (9)
%
To a solution of methyl (4-hydroxy)benzoate sodium salt (3.8 g,
22.19 mmol prepared as described for (3)) in methanol (20 ml) were
%
added 2-bromo ethanol (2.72 g, 22 mmol) and a small amount of sodium
iodide. This mixture was stirred at 608C for 18 h, the precipitating
sodium bromide was removed by suction, the remaining solution was
evaporated to dryness and the crude product was purified via flash-
chromatography on silica gel (ethylacetate/n-hexane 1:1). The product
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637
methyl 4-(2-hydroxyethoxy)benzoate (9) was obtained as a white solid
%
(1.5 g, 7.6 mmol, 35%).
1H-NMR (CDCl3): (2H, t, 2.7 ppm), (2H, t, 3.3 ppm), (3H, s,
3.8 ppm), (4H, m, 6.2–8.0 ppm).
MS (FD): m=z (% relative intensity) 181.231 (100.0, [M+1]).
Methyl 4-(2-(2-oxanyloxy)ethoxy)benzoate (10)
To a solution of (9) (1.5 g, 7.6 mmol) and 3,4-dihydro-2H pyrane (2.5 g,
%
30 mmol) in dichloromethane (ca 5 ml) few drops of iodo trimethylsilane
were added and stirred at 258C for 30 min (TLC control). The mixture
was evaporated to dryness and purified via flash-chromatography on
silica gel (ethylacetate/hexane, 70:30) to yield (10) (1.95 g, 6.9 mmol,
90%).
1H-NMR (CDCl3): (6H, m, 1.4–1.8 ppm), (2H, t, 2.7 ppm), (2H, t,
3.3 ppm), (1H, m, 3.5 ppm), (3H, s, 3.8 ppm), (1H, m, 3.8 ppm), (1H, m,
4.3 ppm), (4H, m, 6.2–8.0 ppm).
2-(4-(bis(4-methoxyphenyl)hydroxymethyl)phenoxy)ethan-1-ol (11)
To a Grignard solution of 4-methoxyphenyl magnesium bromide
(0.014 mol) was added a solution of (10) (1.7 g, 6 mmol) in THF
(10 ml) under vigorous stirring within 10 min. The solution was kept at
48C over night and was hydrolysed with 1 N H2SO4, extracted with
ether, dried over sodium sulphate and the organiclayer was evaporated
under reduced pressure. The crude product was recrystallised from
acetic acid/water to yield the product (11) as red crystals (1.8 g,
4.9 mmol, 85%).
1H-NMR (CDCl3): (2H, t, 2.7 ppm), (2H, t, 3.3 ppm), (6H, s,
3.8 ppm), (12H, m, 6.5–7.5 ppm).
MS (FD): m=z (% relative intensity) 440.8 (100.0, [M+]), 442.5 (15.6,
[M+2]+).
2-(4-(bis(4-methoxyphenyl)chloromethyl)phenoxy)ethyl acetate (12)
2-(4-(Hydroxy bis(4-methoxyphenyl)methyl)phenyl)ethanol (11) (1.8 g,
4.9 mmol) were added to acetyl chloride (30 ml) and refluxed for 2 h. The
acetylchloride was removed under reduced pressure and the crude
product was recrystallised from acetic acid/water (2.0 g, 4.75 mmol,
98%).
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1H-NMR (CDCl3): (3H, s, 2.1), (2H, t, 2.7 ppm), (2H, t, 3.3 ppm),
(6H, s, 3.8 ppm), (12H, m, 6.3–7.3 ppm).
IR n(1/cm): n(C–Cl): 560–630, n(CO): 1210, n(CO): 1730, n(Ph–O–
CH3): 2830.
2-(4-(bis(4-methoxyphenyl)(2-(3-(ethoxycarbonyl)piperidyl)ethoxy)-
methyl)-phenoxy)ethyl acetate (13)
To a solution of ethyl (2-hydroxyethyl)piperidine-3-carboxylate (6)
%
(0.8 g, 4 mmol) in dry THF (5 ml) were added subsequently triethyl-
amine (1 ml, 9.3 mmol), 4-dimethyl aminopyridine (50 mg) and 2-(4-
(bis(4-methoxyphenyl)chloromethyl)-phenoxy)ethyl acetate (12) (1.72 g,
4 mmol). The reaction mixture was stirred overnight at room
temperature and passed into a dichloro methane/water (5:1) mixture
(50 ml). The organiclayer was dried over sodium sulphate, filtered,
concentrated in vacuum and purified via flash-chromatography
(ethylacetate/n-hexane/triethylamine, 1:1:0.1%) to yield the product
(13) as a yellow oil (0.55 g, 0.88 mmol, 22%).
1H-NMR (CDCl3): (3H, t, 1.2 ppm), (11H, m, 1.3–3.0 ppm), (3H, s,
2.1), (2H, t, 2.6 ppm), (2H, t, 2.7 ppm), (2H, t, 3.1 ppm), (2H, t,
3.3 ppm), (6H, s, 3.7 ppm), (6H, s, 3.8 ppm), (2H, q, 4.0 ppm), (12H, m,
6.5–7.5 ppm).
Elemental Analysis (C33H43NO8) C, H, N.
Ethyl(2-((4-(2-hydroxyethoxy)phenyl)bis(4-methoxyphenyl)methoxy)-
ethyl)piperidine-3-carboxylate (14)
2-(4-(Bis(4-methoxyphenyl)(2-(3-(ethoxycarbonyl)piperidyl)ethoxy)methyl)-
phenoxy)ethyl acetate (13) (0.4 g, 0.64 mmol) was dissolved in methanol
and the pH value was adjusted to 8.5 with a 5% sodium methanolate
solution in methanol (indicator: wet pH paper). This mixture was stirred
overnight, water was added slowly, the precipitating solid was filtered
off and dried in vacuum to yield ethyl (2-((4-(2-hydroxyethoxy)phe-
nyl)bis(4-methoxyphenyl)methoxy)ethyl)piperidine-3-carboxylate (14)
as a yellow solid (0.28 g, 0.5 mmol, 78%).
1H-NMR (CDCl3):(3H, t, 1.2 ppm), (11H, m, 1.3–3.0), (2H, t,
2.6 ppm), (2H, t, 2.7 ppm), (2H, t, 3.1 ppm) (6H, s, 3.7 ppm), (6H, s,
3.8), (2H, q, 4.0 ppm), (12H, m, 6.5–7.5 ppm).
IR n(1/cm): n(OH): 1010–1080, n(OH): 3650.
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639
Ethyl(2-((4-(2-tosylethoxy)phenyl)bis(4-methoxyphenyl)methoxy)-
ethyl)piperidine-3-carboxylate (15)
To a solution of p-toluene sulphonyl chloride (95 mg, 0.5 mmol) and
ethyl (2-((4-(2-hydroxyethoxy)phenyl)bis(4-methoxyphenyl)methox-
y)ethyl)piperidine-3-carboxylate (14) (0.2 g, 0.36 mmol) in chloroform
at 08C, pyridine (79 mg, 1 mmol) was added under stirring. The mixture
was stirred for 1 h and concentrated in vacuum. The crude product was
purified via chromatography (silica gel Si 60) (ethyl acetate/n-hexane/
triethylamine, 30/70/0.1%) to yield the product (15) as a yellow oil
(0.15 g, 0.2 mmol, 58%).
1H-NMR (CDCl3): (4H, m, 7.3–7.7 ppm), (12H, m, 6.8–7.3 ppm),
(2H, t, 3.8 ppm), (3H, s, 2.4 ppm), (2H, t, 2.2 ppm), (3H, t, 1.1–1.3 ppm),
(11H, m, 1.4–2.7 ppm), (4H, dt, 3–3.2 ppm), (6H, s, 3.7 ppm), (2H, q,
4.1 ppm).
MS (FD): m=z (% relative intensity) 545.7 (100.0, [M+–C7H7SO3]+),
717.8 (2.0 [M+l]+).
Elemental Analysis (C40H47NO9S) C, H, N.
2-18F]fluoroethyltosylate
No-carrier-added (NCA) aqueous [18F]fluoride (540–1100 MBq) pre-
pared by the 18O(p,n)18F nuclear reaction on an enriched water (95%)
target was added to a solution of K2CO3 (15 ml (1 N))/Kryptofix 2.2.2.
(10–15 mg) in CH3CN (800 ml). The water was removed using a stream
of nitrogen at 808C and coevaporated to dryness with CH3CN
(2 Â 1 ml).
To the dried Kryptofix 2.2.2./[18F]fluoride (380–750 MBq) complex in
acetonitrile (1 ml) ethyleneglycol-1,2-ditosylate (8–10 mg, 20–25 mmol)
was added and heated under stirring in a sealed vial for 3 min.
Purification of the crude product was achieved using HPLC (acetoni-
trile/water, 50:50, flow rate: 5 ml/mm tr: 8 min). After diluting the HPLC
fraction containing the 2-[18F]fluoroethyltosylate with water, the
product was loaded on a 18C-Sepac column, dried with nitrogen gas
and eluated with 1 ml of tempered (40–508C) DMF to yield a product
activity of 300–600 MBq 2-[18F]fluoroethyltosylate.
Ethyl(2-((4-(2-[18F]fluoroethoxy)phenyl)bis(4-methoxyphenyl)meth-
oxy)-ethyl)piperidine-3-carboxylate (17) via 2-[18F]fluoroethyltosylate
To ethyl (2-(4-hydroxyphenyl)bis(4-methoxyphenyl)methoxy)ethyl)pi-
peridine-3-carboxylate (16) (8 mg, 0.0154 mol) dissolved in DMF
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640
R. SCHIRRMACHER ET AL.
(0.5 ml), a sodium hydroxide solution (5 N, 2.5 ml) was added and
tempered for at least 5 min at 808C. A solution of 2-[18F]fluoroethylto-
sylate (300–600 MBq) in DMF (300–500 ml) was added and stirred in a
sealed reaction vessel at 808C for 5 min. The product was purified
with HPLC (methanol/water, 80:20+0.1% triethylamine, flow rate:
4 ml/min, tr: 12.3 min). After diluting the HPLC fraction containing the
product with water, it was loaded on a solid-phase column (LiChrolut
EN, Merck), dried with nitrogen and eluated with 1 ml methanol to
yield 200–450 MBq of the product (17). HPLC analysis showed a
radiochemical purity of >99%.
(2-((4-([18F]fluoroethoxy)phenyl)bis(4-methoxyphenyl)methoxy)eth-
yl)-piperidine-3-carboxylic acid (1)
%
To the methanolicsolution ocntaining 200–450 MBq ( 17), a NaOH
solution (1.25 N, 200 ml) was added and stirred at 808C for 5 min. The
solution was then diluted with a five fold excess of water and passed
over a solid-phase column (Li-Chrolut EN, Merck), dried with nitrogen,
eluated with 1 ml ethanol and diluted with physiological saline solution
(10 ml) (180–400 MBq). HPLC analysis (methanol/water, 80 : 20+0.1%
triethylamine, flow rate: 4 ml/min, tr: 4.1 min) showed that the radio-
chemical purity was >99%. The specific activity of (1) was between 0.3
%
and 0.5 Ci/mmol as determined via a UV-calibration curve.
Ethyl(2-((4-(2-[18F]fluoroethoxy)phenyl)bis(4-methoxyphenyl)meth-
oxy)-ethyl)piperidine-3-carboxylate (17) via [18F]fluoride and ester
cleavage to (1)
%
No-carrier-added (NCA) aqueous [18F]fluoride (280–1100 MBq) pre-
pared by the 18O(p,n)18F nuclear reaction on an enriched water (95%)
target was added to a solution of K2CO3 (15 ml (1 N))/Kryptofix 2.2.2.
(10–15 mg) in CH3CN (800 ml). The water was removed using a stream
of nitrogen at 808C and coevaporated to dryness with CH3CN
(2 Â 1 ml).
To the dried Kryptofix 2.2.2/[18F]fluoride complex (200–800 MBq) in
acetonitrile (0.5 ml) (15) (15 mg, 0.02 mmol) in acetonitrile (0.5 ml) was
added and heated at 808C for 40 min. The product was purified with
HPLC (methanol/water, 80:20+0.1% triethylamine, flow rate: 4 ml/mm,
tr: 12.3 min). After diluting the HPLC fraction containing the
product with water, it was loaded on a solid-phase column (Li-Chrolut
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18F-GABA-TRANSPORTER LIGANDS
641
EN, Merck), dried with nitrogen and eluated with 1 ml methanol to
yield 60–240 MBq of product. HPLC analysis indicated a radiochemical
purity of >99%. Ester hydrolysis of (17) was achieved as described
above to yield (1) (54–215 MBq) in a decay corrected yield of 19% with
%
a radiochemical purity >99% (HPLC). The specific activity of (1) was
%
determined via a UV-calibration curve and found to be between 0.3 and
0.5 Ci/mmol.
Conclusion
The radiosynthesis of a potential GAT-3-PET radioligand (Æ)-(2-((4-
([18F]fluoroethoxy)phenyl)bis(4-methoxyphenyl)methoxy)ethyl)piperi-
dine-3-carboxylic acid (1) for the human GABA transporter subtype
%
GAT-3 was carried out in an overall decay corrected radiochemical
yield of 70% in the case of 2-[18F]fluoroethyltosylate as the labelling
precursor. The direct labelling approach has been proven to be less
efficient for two reasons. Firstly, the overall yield of the precursor
synthesis of (15) was only 2.6% in comparison to 27% in the case of
precursor synthesis (16) and secondly, the labelling with [18F]fluoride
could be achieved with 32% radiochemical yield after 40 min only
(decay corrected).
By labelling the precursor (16) with 2-[18F]fluoroethyltosylate, (1)
could be obtained in an injectable ethanolic physiological saline solution
which was stable up to 95% for 24 h. Preliminary biodistribution studies
indicated that the uptake of the radioactivity was 0.3% ID/g in mice
brain which requires further comparisons of (1) with the tritiated
%
original GAT-3 ligand [3H]SNAP-5114.
Acknowledgements
The financial support by the Deutsche Forschungsgemeinschaft (DFG
Ro 985/8-1) is gratefully acknowledged.
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Copyright # 2001 John Wiley & Sons, Ltd.
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