Automated commercial synthesis system for preparation of O-(2-[18F]fluoroethyl)-L-tyrosine by direct nucleophilic displacement on a resin column

Article abstract of DOI:10.1002/jlcr.682

A slightly modified automated commercial synthesis system for preparation of O-(2-[18F]fluoroethyl)-L-tyrosine (FET), an amino acid tracer for tumor imaging with positron emission tomography, is described. Direct nucleophilic fluorination of [18F]fluoride

Full text of DOI:10.1002/jlcr.682

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 645–659.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.682
Research Article
Synthesis and evaluation of 5,7-dichloro-4-
(3-{4-[4-(2-[18F]fluoroethyl)-piperazin-1-yl]-
phenyl}-ureido)-1,2,3,4-tetrahydroquinoline-
2-carboxylic acid as a potential NMDA
ligand to study glutamatergic
neurotransmission in vivo
1
Markus Piel , Ralf Schirrmacher , Sabine Hohnemann , Wilhelm
1
1
.
Hamkens1, Beate Kohl3, Michaela Jansen3, Ullrich Schmitt2, Hartmut
2
3
1,
.
.
Luddens , Gerd Dannhardt and Frank Rosch *
1 Institute of Nuclear Chemistry, Fritz-Strassmann-Weg 2, D-55128 Mainz,
Germany
2 Department of Psychiatry, University Mainz, Germany
3 Institute of Pharmacy, University Mainz, Germany
Summary
The neurotransmitter glutamate is thought to be crucially involved in a huge
number of neurological and psychiatric disorders, such as Morbus Parkinson,
Alzheimer’s disease and schizophrenia. Aiming at an improved diagnostic tool
for PET a new [18F]fluorine labelled NMDA receptor ligand was developed
that may potentially allow the in vivo visualization of glutama-
tergic neurotransmission. The 19F-analogue trans-5,7-dichloro-4-(3-{4-[4-(2-
fluoroethyl)-piperazin-1-yl]-phenyl}-ureido)-1,2,3,4-tetrahydro
quinoline-2-
carboxylic acid was synthesised to determine the binding affinity, lipophilicity
and biodistribution of the ligand. This substance exhibits a Ki of 12 nM for the
glycine binding site using [3H]MDL-105,519 assays on pig cortical membranes.
A logD of 1.3 was determined for this compound according to the OECD
.
*Correspondence to: F. Rosch, Institute of Nuclear Chemistry, Fritz-Strassmann-Weg 2, D-55128
Mainz, Germany. E-mail: frank.roesch@uni-mainz.de
Copyright # 2003 John Wiley & Sons, Ltd.
Received 3 May 2002
Revised 3 October 2002
Accepted 5 November 2002

646
M. PIEL ET AL.
guidelines employing the HPLC method. Radiosynthesis of this ligand was
achieved by labelling the precursor trans-5,7-dichloro-4-[3-(4-piperazin-1-yl-
phenyl)-ureido]-1,2,3,4-tetrahydroquinoline-2-carboxylic acid methyl ester
with 2-[18F]fluoroethyltosylate and subsequent cleaving of the methyl ester
moiety, resulting in an overall decay corrected yield of 35% of the final product
trans-5,7-dichloro-4-(3-{4-[4-(2-[18F]fluoroethyl)-piperazin-1-yl]-phenyl}-urei-
do)-1,2,3,4-tetrahydroquinoline-2-carboxylic acid. The biodistribution kinetics
of this compound were determined with Sprague Dawley rats ex vivo for brain,
liver, kidney, and bone. The ligand showed a maximum brain uptake
30 min.p.i. of about 0.1% ID/g. Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: Glutamate; NMDA receptor; Glycine binding site; 18F-fluoroalk-
ylation
Introduction
Glutamate is the most abundant excitatory amino acid (EAA) in the
central nervous system of mammals and plays an important role in
neurodevelopment, synaptic plasticity and neurotoxicity.1,2 Glutamate
is recognized by metabotropic and ionotropic receptors, the latter ones
subdivided into the three subtypes named after their selective agonists:
N-methyl-D-aspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxa-
zole-propionate (AMPA) and kainate.
NMDA receptors have been extensively characterised since their
discovery in 1977.3 They represent an important target for medicinal
chemistry due to their involvement in Morbus Parkinson, Alzheimer’s
disease and schizophrenia.4 Like other ligand-gated ion channels they
are proposed to have a heteropentameric structure, though their
transmembrane regions are at variance with e.g. GABAA and nicotinic
acetylcholine receptors. They posses a variety of binding sites both in
the extracellular domains of the receptor for glycine, glutamate and
polyamines, and in the ion channel itself for Zn2+, Mg2+ and the use-
dependent psychotropic drug MK 801.5
Glycine has been defined as a co-agonist of NMDA receptors. Thus
for the synthesis of new ligands the glycine binding site is an important
target, especially as these ligands have less side effects compared to
ligands of the glutamate and ion channel binding site. Therefore several
studies have dealt with positron emitter labelled ligands for the in vivo
visualisation of the NMDA receptor using PET.6–8
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS OF A 18F-LABELLED NMDA ANTAGONIST
647
18
F
NH
N
N
N
O
O
Cl HN
N
H
Cl HN
N
H
OH
OH
Cl
N
H
Cl
N
H
O
O
2
1
18
F-labelled derivative
lead structure
3
K = 6.0 nmol ([ H]DCKA)
i
Figure 1. Lead structure 1 and the fluoroethylated derivative trans-5,7-dichloro-
4-(3-{4-[4-(2-[18F]fluoroethyl)-piperazin-1-yl]-phenyl}-ureido)-1,2,3,4-tetrahydro-
quinoline-2-carboxylic acid (2)
Since the discovery of kynurenic acid as an antagonist of the glycine
binding site its derivatives were optimised for improved affinity and
selectivity resulting in the development of the trans-5,7-dichloro 4-
substituted 2-carboxytetrahydroquinolines, providing affinities in the
nanomolar range.9 The [18F]fluorine labelled derivative trans-5,7-
dichloro-4-(3-{4-[4-(2-[18F]fluoroethyl)-piperazin-1-yl]-phenyl}-ureido)-
1,2,3,4-tetrahydroquinoline-2-carboxylic acid (2) of the lead structure 1
was considered for in vivo visualisation of the NMDA receptor. In the
following figures and schemes the 2R,4S-configuration represents the
trans-isomers, whilst the 2S,4S-configuration represents the cis-isomers
(Figure1).
Results and discussion
Chemistry
The aim of this study was the labelling of trans-5,7-dichloro-4-[3-(4-
piperazin-1-yl-phenyl)-ureido]-1,2,3,4-tetrahydroquinoline-2-carboxylic
acid with 2-[18F]fluoroethyltosylate. In a first step the 19F-analogue of
this molecule was synthesised to evaluate the in vitro affinity and
lipophilicity of the ligand.
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M. PIEL ET AL.
The treatment of acryloyl acid chloride with sodium azide afforded
the appropriate acid azide which was rearranged according to Curtius at
1208C. The forming vinyl isocyanate was converted in situ into the
appropriate benzyl vinylcarbamate 5 by addition of benzyl alcohol and
purified by distillation. In a further step 3,5-dichloro aniline (3) was
converted with methyl glyoxylate to the Schiff base and afterwards in
situ with the benzyl vinylcarbamate to form compounds 6c and 6t.
Because this cyclization did not favour one stereochemical route, the
synthesised diastereomers were separated by means of column chroma-
tography. The cis-isomers however, can be converted into the trans-
isomers using catalytic quantities of sodium methylate (Scheme 2). After
removing the benzyloxycarbonyl protecting group using iodotrimethyl
silane, compound 7 was separated as hydrochloride (Scheme 1 and 2).
NH
O
2
O
(a)
+
+
N
H
O
OCH
O
3
Cl
Cl
3
4
5
O
Cl
O
Cl
NH
3
Cl HN
O
O
(b),(c)
OMe
Cl
N
H
OMe
Cl
N
H
6t
7
Scheme 1. (a) sodium sulfate, boron trifluoride diethyl ether complex (b)
iodotrimethyl silane, methanol (c) HCl, ethyl acetate
O
O
Cl HN
O
O
Cl HN
O
O
(a)
OMe
OMe
Cl
N
H
Cl
N
H
6c
6t
Scheme 2. (a) potassium methylate, methanol
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS OF A 18F-LABELLED NMDA ANTAGONIST
649
The synthesis of 12 could be achieved by carrying out the following
reaction steps: 1-(4-nitrophenyl)-piperazine (8) was first reacted with 1-
bromo-2-fluoroethane and subsequently reduced with H2/Pd to obtain
compound 10. This compound was then coupled with 7 using
triphosgene to yield compound 11. After cleavage of the ester moiety
with 1 N LiOH compound 12 was obtained (Scheme 3).
(a), (b)
N
N
O N
2
NH
H N
2
N
F
8
10
O
F
N
N
Cl HN
N
H
(c)
(d)
OMe
Cl
O
N
H
O
11
F
N
N
Cl HN
N
H
OH
Cl
N
H
O
12
Scheme 3. (a) 1-bromo-2-fluoroethane, [1,4]dioxane (b) H2/palladium, methanol
(c) triphosgene, dichloro methane, DIEA, 7 (d) THF, LiOH (1 N)
The precursor 15 was synthesised by reacting FmocCl and 8, followed
by the reduction of the nitro moiety using H2/Pd to obtain 13. After
coupling this compound with 7 using triphosgene, the Fmoc moiety was
removed with piperidine to yield the desired precursor 15 (Scheme 4).
The radiolabelling with 2-[18F]fluoroethyltosylate was performed in
DMSO at a temperature of 1408C within 25 min yielding the desired
product 16 in 55% radiochemical yield (referring to the starting activity
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650
M. PIEL ET AL.
(a), (b)
N
O N
N
NH
H N
N
Fmoc
2
2
8
13
O
N
Fmoc
N
Cl HN
N
H
(c)
(d)
OMe
Cl
O
N
H
O
14
NH
N
Cl HN
N
H
OCH
3
Cl
N
H
O
15
Scheme 4. (a) 9-fluorenylmethyl chloroformate, [1,4]dioxane, NaHCO3 (10%)
(b) H2/palladium, methanol (c) triphosgene, dichloro methane, DIEA, 7 (d)
piperidine, THF
of 2-[18F]fluoroethyltosylate). Purification of the crude product was
accomplished by HPLC. Subsequent quantitative cleavage of the
remaining methyl ester moiety within 5 min gave the final product 2
in an overall radiochemical yield of 35%. The total preparation time
until a solution for the biodistribution studies was obtained was
approximately 110 min (Scheme 5).
Pharmacology
Preliminary pharmacological experiments were performed with com-
pound 12. The lipophilicity of the molecule was determined using the
HPLC method10 and Sorensen buffer as eluent, resulting in a logD of
.
1.3. In [H3]MDL-105,519 binding assays11 the in vitro affinity was
examined, which showed a high affinity of the ligand to the glycine
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS OF A 18F-LABELLED NMDA ANTAGONIST
651
O
NH
N
Cl HN
N
H
(a), (b)
OCH
3
Cl
N
H
O
15
O
18
F
N
N
Cl HN
N
H
OH
Cl
N
H
O
2
Scheme 5. Radioactive labelling of 13 with a) 2-[18F]fluoroethyltosylate, DMSO
and subsequent ester hydrolysis to 1 with b) LiOH (1.25 N)
binding site with a Ki of 12 nM, favourably comparing with the Ki of 6
nM of the parental compound.
The in vitro stability of 2 was measured 8 h after the radiosyn-
thesis with radio TLC and showed 97% intact radioligand.
The maximum brain uptake of trans-5,7-dichloro-4-(3-{4-[4-(2-
[18F]fluoroethyl)-piperazin-1-yl]-phenyl}-ureido)-1,2,3,4-tetrahydroqui-
noline-2-carboxylic acid of about 0.1% ID/g was reached after 30 min
p.i. (Table 1). Excretion was primarily via the urine, while kidney and
liver showed an uptake of up to 8% ID/g and 3% ID/g within
60 min.The low 18F-activity in the bone, with a maximum of 2% ID/g,
indicates a small metabolic hydrolysis of the tracer under release of 18F-
fluoride.
In a preliminary autoradiographic study the biodistribution of the
radioligand, using sagittal brain sections (including cerebellum and
hippocampus) after 30 min past application, was evaluated. According
to previous studies,12 the NMDA receptor density in cortex and
hippocampus is very high, whilst it is very low in the cerebellum. Due to
an ubiquitous low amount of accumulation of the ligand in all brain
structures this distribution could not be detected and therefore no
blocking experiments were undertaken.
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M. PIEL ET AL.
Table 1. Organ uptake of 2 measured ex vivo in Sprague Dawley rats (n=3) at
5, 15, 30 and 60 minutes p.i
Tissue
18F-activity [%ID/g]
5 min p.i.
15 min p.i.
30 min p.i.
60 min p.i.
Brain
Liver
Kidney
Bone
Blood
0.07 Æ 0.01
2.69 Æ 0.99
7.99 Æ 2.01
2.04 Æ 1.43
0.37 Æ 0.10
0.07 Æ 0.01
0.92 Æ 0.09
1.02 Æ 0.04
0.74 Æ 0.01
0.08 Æ 0.01
0.09 Æ 0.02
0.83 Æ 0.65
0.79 Æ 0.63
0.80 Æ 0.74
0.07 Æ 0.03
0.03 Æ 0.01
1.12 Æ 0.99
0.73 Æ 0.71
0.45 Æ 0.38
0.08 Æ 0.04
Materials and methods
General
All reagents were purchased from commercial sources and were used
without further purification. Methyl glyoxylate (4) and benzyl
vinylcarbamate (5) were prepared according to the literature.13,14 Solid
phase columns were purchased from Merck (Lichrolut EN). Analytical
thin layer chromatography (TLC) was performed using plates from
Merck (Silicagel 60 F254). High performance liquid chromatography
(HPLC) was performed with an HPLC system from Sycam S1100, UV
detection was obtained using a UV detector from 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. MS spectra were
obtained on a Finnigan MAT90 spectrometer. Detection of radio-
activity was performed using an instant imager (Packard Canberra) for
radio-TLC and detection of radio-HPLC was performed using a NaI-
radiodetector (Canberra Packard). [18F]Fluoride, produced via the
18O(p,n)18F reaction, was purchased. Purification of the labelling
precursor 2-[18F]fluoroethyltosylate and of the labelled compound 16
was accomplished using a HPLC column (Lichrospher RP18-EC5,
250 Â 10 mm), while for the HPLC analysis of 2 a Lichrospher RP18-
EC5, 250 Â 4 mm HPLC column was used.
Pharmacological procedures
The logD value of 12 was examined according to the OECD-guideline
for testing chemicals using the high performance liquid chromatography
(HPLC) method.10 Determination of the in vitro binding affinity was
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SYNTHESIS OF A 18F-LABELLED NMDA ANTAGONIST
653
performed using [3H]MDL-105,519 binding assay according to the
method of a previous study.11
The animal experiments were carried out in compliance with the
German legal regulation of animal experimentation. About 12 MBq of
the labelled compound 2 were injected intravenously in the tail vein of
12 Sprague-Dawley rats (220-250 g). After 5, 15, 30 and 60 min in each
case 3 rats were anaesthetised using fluothane (Zeneca), and sacrificed.
Brain, liver, kidney and bones were removed and the biodistribution
was measured. Detection of the radioactivity in organ tissue was carried
out using a NaI(TI)-detector. One of the brains removed after 30 min
was rapidly frozen on dry ice and 30 mm sagittal sections cut on a Jung
microtom. The sections were exposed over night to a phospho image
screen and the screens analysed in a Storm 860 (Molecular Dynamics).
Chemistry
Trans-4-benzyloxycarbonylamino-5,7-dichloro-1,2,3,4-tetrahydroquino
line-2-carboxylic acid methyl ester (6t)15. 44.1 g (0.27 mol) 3,5-Di-
chloro-aniline (3) and 44 g (0.31 mol) sodium sulfate were added to 1 l
dry dichloro methane and the resulting suspension stirred at ambient
temperature. After adding rapidly 27.3 g (0.31 mol) methyl glyoxylate
(4) the mixture was stirred for another 30 min at ambient temperature
and then filtrated to separate the sodium sulfate. To this solution 55 g
(0.31 mol) benzyl vinylcarbamate (5) were added under argon atmo-
sphere and the resulting mixture cooled to 108C. Under strong stirring
4.5 ml (0.042 mol) boron trifluoride diethyl ether complex were then
added dropwise to the solution, whereby the mixture turned orange
slowly. The reaction stirred for another hour at ambient temperature
after which the solvent was removed in vacuum and the resulting oil was
purified via column chromatography (dichloro methane/ethyl acetate: 9/
1) to yield 20.3 g (0.05 mol, 18%) of the trans- (6t) and 34.1 g (0.08 mol,
31%) of the cis-4-benzyloxycarbonylamino-5,7-dichloro-1,2,3,4-tetra-
hydroquinoline-2-carboxylic acid methyl ester (6c).
1
trans-isomers. H-NMR (200 MHz, CDCl3) d [ppm]: 7.33 (s, 5 H),
6.73 (d, 1 H), 6.53 (d, 1 H), 5.15 (s, 2 H), 5.05 (m, 1 H), 4.84 (m, 1 H),
4.78 (m, 1 H), 3.98 (dd, 1 H), 3.80 (s, 3 H), 2.65 (dm, 1 H), 1.67 (ddd,
1 H), MS (FD): m/z (% rel. int.) 407.9 (100.0, [M]+), mp 155–1578C.
1
cis-isomers: H-NMR (200 MHz, CDCl3) d [ppm]: 7.31 (s, 5 H), 6.72
(d, 1 H), 6.53 (d, 1 H), 5.12 (s, 2 H), 4.95 (m, 1 H), 4.69 (m, 1 H), 4.60 (m,
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1 H), 4.04 (dd, 1 H), 3.47 (s, 3 H), 2.88 (dm, 1 H), 2.03 (ddd, 1 H), MS
(FD): m/z (% rel. int.) 407.9 (100.0, [M]+), mp 152–1548C.
Isomerization of the diastereomers:. To a stirred solution of 4 g
(9.8 mmol) of the cis-isomers (6c) in 300 ml dry methanol, catalytic
quantities of sodium methylate (3-5 mg) were added and the resulting
mixture was refluxed for 36 h at 858C. After adding a small amount of
sodium hydrogen carbonate the solvent was evaporated and the residue
purified with column chromatography (dichloro methane/ethyl acetate:
9/1) to yield 2.39 g (5,9 mmol, 60%) trans- and 1.39 g (3,4 mmol, 35%)
cis-isomers.
Trans-4-amino-5,7-dichloro-1,2,3,4-tetrahydroquinoline-2-carboxylic acid
methyl ester hydrochloride (7). Following reported methods,16 to a
stirred solution of 4.23 g (10 mmol) 6t in 150 ml dry dichloro methane at
08C under an argon atmosphere were slowly added 2.2 g (11 mmol)
iodotrimethyl silane, whereby the solution clouded. The cooling was
removed and the mixture stirred for 4 h at ambient temperature. After
adding 10 ml methanol and stirring the resulting clear solution for
another 15 min the solvent was removed in vacuum. The product was
purified by precipitation of the corresponding hydrochloride using
150 ml ethyl acetate, saturated with gaseous HCl, to yield 2.4 g
(7.4 mmol, 74%) of 7 as a white solid.1H-NMR (200 MHz, DMSO-
d6) d [ppm]: 8.43 (s, 3 H), 7.22 (s, 1 H), 6.91 (s, 1 H), 6.78 (s, 1 H), 4.61 (s,
1 H), 4.34 (d, 1 H), 3.77 (s, 3 H), 2.53 (d, 1 H), 1.93 (ddd, 1 H), mp
192–1948C (decomposition).
1-(2-Fluoroethyl)-4-(4-nitrophenyl)-piperazine (9). A mixture of 3.1 g
(24 mmol) 1-bromo-2-fluoroethane, 5 g (24 mmol) 1-(4-nitrophenyl)-
piperazine (8), 9.95 g (72 mmol) potassium carbonate and 45 mg sodium
iodide in 30 ml dioxane was heated overnight under reflux. Afterwards
the solvent was evaporated, the residue taken up in ethyl acetate and
washed with water. The organic layer was dried with sodium sulfate,
evaporated and the residue purified via column chromatography
(methanol/chloroform: 1/1) to obtain 4.0 g (16 mmol, 65%) of 9 as a
1
yellow solid. H-NMR (200 MHz, CDCl3) d [ppm]: 8.10 (dd, 2 H), 6.80
(dd, 2 H), 4.60 (dt, 2 H, 48 Hz), 3.43(t, 4 H), 2.74 (dt, 2 H, 29 Hz), 2.68 (t,
4 H), MS (FD): m/z (% rel. int.) 253.5 (100.0, [M]+), mp 1018C.
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SYNTHESIS OF A 18F-LABELLED NMDA ANTAGONIST
655
4 -[4-(2-Fluoroethyl)-piperazin-1-yl]-phenylamine (10). 4.0 g (15.7 mmol)
9 were dissolved in 80 ml methanol, 400 mg palladium (10% on activated
charcoal) were added and the mixture was hydrogenated for 4 h at 08C
and 1 bar hydrogen pressure. The resulting suspension was filtered, the
solvent evaporated and the residue separated through column chromato-
1
graphy (ethyl acetate) to yield 2.7 g (12 mmol, 77%) of 10. H-NMR
(200 MHz, CDCl3) d [ppm]: 6.80 (dd, 2 H), 6.63 (dd, 2 H), 4.60 (dt, 2 H,
48 Hz), 3.07 (t, 4 H), 2.75 (dt, 2 H, 29 Hz), 2.69 (t, 4 H), MS (FD): m/z (%
rel. int.) 222.8 (100.0, [M]+), mp 528C.
Trans-5,7-dichloro-4-(3-{4-[4-(2-fluoroethyl)-piperazin-1-yl]-phenyl}-
ureido)-1,2,3,4-tetrahydroquinoline-2-carboxylic acid methyl ester
(11). To a stirred solution of 110 mg (0.37 mmol) triphosgene in 4 ml
dry dichloro methane at 08C under argon atmosphere was slowly added,
over a period of 30 minutes, a solution of 223 mg (1 mmol) 10 and 189 ml
(1.1 mmol) N-ethyldiisopropylamine in 3.5 ml dry dichloro methane.17
After further 15 min stirring a solution of 312 mg (1 mmol) 7 and 378 ml
(2.2 mmol) N-ethyldiisopropylamine in 2 ml dry dichloro methane were
quickly added, the mixture was warmed to ambient temperature and
stirred for another 20 min.The solvent was evaporated and the residue
was taken up in 30 ml ethyl acetate and washed with water. The organic
solvent was removed in vacuum and the remaining crude product
separated by column chromatography (methanol/chloroform: 1/9) to
1
yield 460 mg (0.88 mmol, 88%) of 11. H-NMR (200 MHz, DMSO-d6)
d [ppm]: 7.91 (s, 1 H), 7.23 (d, 2 H), 6.91 (s, 1 H), 6.87 (d,), 6.83 (d, 2 H),
6.72 (d, 1 H), 6.42 (d, 1 H), 4.89(m, 1 H), 4.56 (dt, 2 H, 48 Hz), 3.99 (dm,
1 H), 3.72 (s, 3 H), 3,02 (t, 4 H), 2.65 (dt, 2 H, 28 Hz), 2.58 (t, 4 H),
2.32 (dm, 1 H), 1.65 (ddd, 1 H), MS (FD): m/z (% rel. int.) 523.2
(100.0, [M]+).
Trans-5,7-dichloro-4-(3-{4-[4-(2-fluoroethyl)-piperazin-1-yl]-phenyl}-
ureido)-1,2,3,4-tetrahydroquinoline-2-carboxylic acid (12). 200 mg
(0.38 mmol) 11 were dissolved in 4 ml tetrahydrofurane, diluted with
1 ml water and 400 ml of an aqueous 1 N lithium hydroxide solution.
The mixture was stirred for 6 h at ambient temperature (TLC control),
diluted with 10 ml ethyl acetate and 10 ml water and the two phases
allowed to separate. The aqueous phase was filtered and acidified with 2
N hydrochloric acid until the solution clouded and a precipitate formed.
The precipitate was filtered off to obtain 140 mg (0.27 mmol, 73%) of
12. 1H-NMR (200 MHz, DMSO-d6) d [ppm]: 7.93 (s, 1 H), 7.23 (d, 2 H)
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6.87 (d, 1 H), 6.83 (d, 2 H), 6.67 (d, 1 H), 6.41 (s, 1 H), 4.92 (m, 1 H), 4.56
(dt, 2 H, 48 Hz), 3.85 (dm, 1 H), 3,03 (t, 4 H), 2.64 (dt, 2 H, 28 Hz), 2.58
(t, 4 H), 2.30 (dm, 1 H), 1.59 (ddd, 1 H), MS (FD): m/z (% rel. int.) 509.9
(28.8, [M]+), 465.9 (18.0, [M-CO2]+), 266.7 (100.0 [M-
C10H7NO2Cl2]+), elemental analysis (C23H26Cl2FN5O3) C, H, N, mp
227-2298C (decomposition).
4-(4-Aminophenyl)-piperazine-1-carboxylic acid 9h-fluoren-9-ylmethyl
ester (13). To a solution of 4.0 g (21.7 mmol) 1-(4-nitrophenyl)-
piperazine (8) in 80 ml dioxane was added a sodium hydrogen carbonate
solution (10%, 40 ml). The mixture was rigorously stirred, cooled to 08C
and a solution of 5.0 g (19.3 mmol) 9-fluorenylmethyl chloroformate in
40 ml dry dioxane was slowly added. Afterwards the cooling was
removed and the mixture was stirred for another 10 h at ambient
temperature. The reaction mixture was diluted with 80 ml brine and
extracted several times with 200 ml ethyl acetate. The organic phases
were dried, the solvent evaporated and the residue was taken up in
160 ml methanol. After adding 800 mg palladium (10% on activated
charcoal) the mixture was hydrogenated for 4 h (08C, 1 bar hydrogen
pressure), the catalyst was filtered off and the solvent evaporated. The
crude product was separated by column chromatography (ethyl acetate)
1
to yield 5.9 g (15 mmol, 76%) of 13. H-NMR (200 MHz, CDCl3) d
[ppm]: 7.76 (d, 2 H), 7.58 (d, 2 H), 7.34 (m, 4 H), 6.80 (d, 2 H), 6.65 (d,
2 H), 4.45 (d, 2 H), 4.25 (t, 1 H), 3.60(s, 4 H), 3.47 (s, 2 H), 2.94 (s, 4 H),
MS (FD): m/z (% rel. int.): 399.0 (100.0, [M]+), mp 1268C.
Trans-5,7-dichloro-4-(3-{4-[4-(9H-fluoren-9-ylmethoxycarbonyl)-piper-
azin-1-yl]-phenyl}-ureido)-1,2,3,4-tetrahydroquinoline-2-carboxylic acid
methyl ester (14). To a stirred solution of 110 mg (0.37 mmol)
triphosgene in 4 ml dry dichloro methane at 08C under argon
atmosphere were slowly added, over a period of 30 min, a solution of
400 mg (1 mmol) 13 and 189 ml (1.1 mmol) N-ethyldiisopropylamine in
3.5 ml dry dichloro methane. After further 15 min stirring a solution of
312 mg (1 mmol) 7 and 378 ml (2.2 mmol) N-ethyldiisopropylamine in
2 ml dry dichloro methane were quickly added, the mixture was warmed
to ambient temperature and stirred for another 20 min.The solvent was
evaporated, the residue taken up in 30 ml ethyl acetate and washed 2
times with hydrochloric acid (3%), saturated sodium hydrogen
carbonate solution and saturated sodium chloride solution. The organic
layer was removed in vacuo and the remaining crude product separated
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 645–659

SYNTHESIS OF A 18F-LABELLED NMDA ANTAGONIST
657
by column chromatography (ethyl acetate/chloroform: 1/1) to yield
1
530 mg (0.76 mmol, 76%) of 14. H-NMR (200 MHz, DMSO-d6) d
[ppm]: 7.92 (s, 1H), 7.88 (d, 2H), 7.63 (d, 2H), 7.36 (m, 4H), 7.25 (d,
2H), 6.91 (m, 1H), 6.86 (d, 1H), 6.83 (d, 2H), 6.71 (d, 1H), 6.43 (d, 1H),
4.91 (m, 1H), 4.40 (s, 2H), 4.27 (t, 1H), 3.98 (dm, 1H), 3.70 (s, 3H), 3,41
(s, 4H), 2,89 (t, 4H), 2.31 (dm, 1H), 1.64 (ddd, 1H), MS (FD): m/z (%
rel. int.) 477.0 (100.0, [M-Fmoc]+), 699.3 (31.4, [M]+).
Trans-5,7-dichloro-4-[3-(4-piperazin-1-yl-phenyl)-ureido]-1,2,3,4-tetra-
hydroquinoline-2-carboxylic acid methyl ester (15). To a solution of
350 mg (0.5 mmol) 14 in 2.7 ml in dry tetrahydrofurane were added 300
ml piperidine, which led to a rapid formation of carbon dioxide. The
solution was stirred for 12 h, the solvent was evaporated, and the residue
was taken up in 10 ml dry diethyl ether and treated for 10 min in the
ultrasonic bath. The mixture was filtered and the solid washed with 5 ml
diethyl ether. This crude product was purified by column chromato-
graphy (methanol + 3% N-ethyldiisopropylamine) to yield 210 mg
1
(0.44 mmol, 88%) of 15 as a white solid. H-NMR (200 MHz, DMSO-
d6) d [ppm]: 8.13 (s, 1H), 7.18 (d, 2H), 7.10 (d, 1H), 6.80 (d, 2H), 6.74 (d,
1H), 6.71 (d, 1H), 5.57 (d, 1H), 4.76 (m, 1H), 4.17 (m, 1H), 3.53 (s, 3H,),
2,95 (t, 4H), 2,86 (s, 4H), 2.75 (dm, 1H), 1.84 (ddd, 1H), MS (FD): m/z
(% rel. Int.) 477.0 (100.0, [M]+).
2-[18F]Fluoroethyltosylate. To an aqueous [18F]fluoride solution (1400-
1600 MBq) were added Kryptofix 2.2.2. (10 mg, 25 mmol), 12.5 ml
potassium carbonate (1N) and 1 ml acetonitrile.18 The mixture was
dried in a stream of nitrogen at 808C. The drying procedure was
repeated three times until the reaction mixture was absolutely dry. The
dried Kryptofix 2.2.2./[18F]fluoride-complex was then dissolved in 1 ml
acetonitrile and 4 mg (10 mmol) ethylenglycol-1,2-ditosylate were added
and heated under stirring in a sealed vial for 3 min. Purification of the
crude product was accomplished using HPLC (acetonitrile/water 50:50,
flow rate: 5 ml/min, rt: 8 min). After diluting the HPLC fraction
containing the 2-[18F]fluoroethyltosylate with water, the product was
loaded on a Sep-Pak C18 column, dried with nitrogen and eluated with
1 ml of tempered (40-508C) DMSO (760-890 MBq)
Trans-5,7-dichloro-4-(3-{4-[4-(2-[18F]fluoroethyl)-piperazin-1-yl]-
phenyl}-ureido)-1,2,3,4-tetrahydroquinoline-2-carboxylic acid methyl es-
ter (16). To 3 mg (6.3 mmol) 15 were added 0.5 ml DMSO and the
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 645–659

658
M. PIEL ET AL.
resulting solution tempered for 5 min at 1408C. A solution of 2-
[18F]fluoroethyltosylate (480-560 MBq) in DMSO (500 ml) were added
and stirred in a sealed reaction vessel at 1408C for 25 min.The product
was diluted with 1 ml 0.1% trifluoro acetic acid in water and purified
with HPLC (acetonitrile/water 35:65 + 0.05% trifluoro acetic acid, flow
rate: 5 ml/min, tr: 22.9 min). After diluting the HPLC fraction contain-
ing the product with water, it was loaded on a solid phase column
(LiChrolut EN, Merck), dried with nitrogen and eluated with 2 ml
methanol to yield 180-250 MBq of 16.
Trans-5,7-dichloro-4-(3-{4-[4-(2-[18F]fluoroethyl)-piperazin-1-yl]-
phenyl}-ureido)-1,2,3,4-tetrahydroquinoline-2-carboxylic acid (2). To
the methanolic solution containing 16 were added a LiOH solution
(1.25N, 200 ml) and stirred at ambient temperature for 5 min. The
solution was then diluted with a ten-fold excess of water and passed on a
solid phase column (LiChrolut EN, Merck), dried with nitrogen, eluated
with 1 ml ethanol and diluted with physiological saline solution (160-200
MBq). HPLC analysis (acetonitrile/0.05 M Na2HPO4 buffer 30:70, flow
rate: 1 ml/min, rt: 6.1 min) showed that the radiochemical purity was
>99%, while the specific activity (determined via UV-calibration curve)
was between 50 and 75 GBq/mmol.
Conclusion
The synthesis of the radioligand trans-5,7-dichloro-4-(3-{4-[4-(2-
[18F]fluoroethyl)-piperazin-1-yl]-phenyl}-ureido)-1,2,3,4-tetrahydroqui-
noline-2-carboxylic acid (2) for the glycine binding site of the NMDA
receptor was carried out in an overall radiochemical yield of 35% by
labelling the precursor with 2-[18F]fluoroethyltosylate.
The lipophilicity and the in vitro affinity of compound 12 were
determined, showing that the ligand has a moderate lipophilicity (logD
= 1.3) and a high affinity (Ki = 12 nM) for the glycine binding site.
Biodistribution studies resulted in a maximum uptake of 0.1% ID/g
in rat brain, while urinary excretion was predominating liver uptake.
The relatively low brain uptake may be due to the deprotonated acid
function, which might indicate that an increased brain uptake of this
high-affinity tracer could be achieved by further heightening the
lipophilicity.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 645–659

SYNTHESIS OF A 18F-LABELLED NMDA ANTAGONIST
659
Acknowledgements
Financial support by the Deutsche Forschungsgemeinschaft (DFG Ro
985 / 12-1) and the Fonds der Chemischen Industrie is gratefully
acknowledged.
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Copyright # 2003 John Wiley & Sons, Ltd.
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