Radiosynthesis of 3-(3-[18F]fluoropropoxy)-4-(benzyloxy)-N-[(1- dimethylaminocyclopentyl)methyl]-5-methoxybenzamide, a potential PET radiotracer for the glycine transporter GlyT-2

Article abstract of DOI:10.1002/jlcr.1145

The recently described selective and potent GlyT2 antagonist, 4-benzyloxy-3, 5-dimethoxy-N-[(1-dimethylaminocyclopentyl) methyl]benzamide (IC₅₀ = 16 nM) provided an important additional tool to further characterize GlyT2 pharmacology. In order to identify an effective PET radioligand for in vivo assessment of the GlyT-2 transporter, 3-(3-[ ¹⁸F]fluoropropoxy)-4-(benzyloxy)-N-((1-dimethylaminocyclopentyl) methyl)-5-methoxybenzamide ([¹⁸F]3), a novel analog of 4-benzyloxy-3,5-dimethoxy-N-[(1-dimethylaminocyclopentyl) methyl]benzamide was synthesized using a one-pot, two-step method. The NCA radiofluorination of 1,3-propanediol di-p-tosylate in the presence of K₂CO₃ and Kryptofix-222 in acetonitrile gave 81% 3-[¹⁸F]fluoropropyl tosylate, which was subsequently coupled with 4-benzyloxy-3-hydroxy-5-methoxy-N-[(1- dimethylaminocyclopentyl) methyl]-benzamide in the same reaction vessel. Solvent extraction and HPLC (Eclipse XDB-C8 column, 80/20/0.1 MeOH/H ₂O/Et₃N, 3.0 ml/min) gave [¹⁸F]3 in 98.5% radiochemical purity. The radiochemical yield was determined to be 14.0-16.2% at EOS, and the specific activity was 1462 ± 342 GBq/μmol. The time of synthesis and purification was 128 min. The final product was prepared as a sterile saline solution suitable for in vivo use. Copyright

Full text of DOI:10.1002/jlcr.1145

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 1247–1258.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1145
Research Article
Radiosynthesis of 3-(3-[18F]fluoropropoxy)-4-
(benzyloxy)-N-[(1-dimethylaminocyclopentyl)methyl]-
5-methoxybenzamide, a potential PET radiotracer for
the glycine transporter GlyT-2
Haibin Tian, Rebecca Vogel, Louis Amici, Gilles Tamagnan and
Ronald M. Baldwin*
Departments of Psychiatry and Diagnostic Radiology, Yale University School of
Medicine, Yale-VA Positron Emission Tomography Center, Veterans Affairs Medical
Center, West Haven, CT, USA
Summary
The recently described selective and potent GlyT2 antagonist, 4-benzyloxy-3,
5-dimethoxy-N-[(1-dimethylaminocyclopentyl) methyl]benzamide (IC50=16 nM)
provided an important additional tool to further characterize GlyT2 pharmaco-
logy. In order to identify an effective PET radioligand for in vivo assessment of
the GlyT-2 transporter, 3-(3-[18F]fluoropropoxy)-4-(benzyloxy)-N-((1-dimethyl-
aminocyclopentyl) methyl)-5-methoxybenzamide ([18F]3), a novel analog of 4-
benzyloxy-3,5-dimethoxy-N-[(1-dimethylaminocyclopentyl) methyl]benzamide was
synthesized using a one-pot, two-step method. The NCA radiofluorination of
1,3-propanediol di-p-tosylate in the presence of K2CO3 and Kryptofix-222 in
acetonitrile gave 81% 3-[18F]fluoropropyl tosylate, which was subsequently coupled
with 4-benzyloxy-3-hydroxy-5-methoxy-N-[(1-dimethylaminocyclopentyl) methyl]-
benzamide in the same reaction vessel. Solvent extraction and HPLC (Eclipse
XDB-C8 column, 80/20/0.1 MeOH/H2O/Et3N, 3.0 ml/min) gave [18F]3 in
98.5% radiochemical purity. The radiochemical yield was determined to be
14.0–16.2% at EOS, and the specific activity was 1462 Æ 342 GBq/mmol. The
*Correspondence to: Ronald M. Baldwin, Department of Radiology/PET Chemistry, Vanderbilt Uni-
versity School of Medicine, CCC-1118, 1161 21st Ave S, Nashville TN, USA.
E-mail: ronald.baldwin@vanderbilt.edu
Contract/grant sponsor: Center on Neural Mechanisms and Treatment Response in Depression (VA REAP)
Contract/grant sponsor: VA Schizophrenia Biological Research Center SRC
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time of synthesis and purification was 128 min. The final product was prepared
as a sterile saline solution suitable for in vivo use. Copyright # 2006 John Wiley &
Sons, Ltd.
Received 5 July 2006; Revised 26 September 2006; Accepted 27 September 2006
Key Words: glycine transporter 2(GlyT2); PET; fluorine-18; radiotracer transporter
2(GlyT2)
Introduction
Glycine is an obligatory co-agonist at N-methyl-D-aspartate receptor
(NMDA-R), and recent clinical studies have demonstrated efficacy of
exogenous glycine in treating negative symptoms of psychosis. As extracellular
glycine is regulated by high-affinity glycine transporters, pharmacological
inhibition of such transporters represents a novel mechanism for increasing
glycine within the brain.1 Clinical studies with high dose glycine therapy,
which improves outcome of schizophrenic patients with negative symptoms,
supports the idea that augmenting NMDA transmission by increasing glycine
levels can benefit psychotic patients.2 Glycine transporters have been cloned
and are at present classified into two distinct gene families, glycine transporter
1 (GlyT1) and glycine transporter 2 (GlyT2). These have now been further
divided into three subtypes of GlyT-1 (a, b, and c) and two splice variant
versions of GlyT2 (a and b).3–5 Immunocytochemical studies showed that the
GlyT1 transporter has a wide distribution throughout the central nervous
system (CNS) whereas the GlyT2 transporter has a similar distribution to
strychnine-sensitive glycine receptor (ssGlyR), being confined to the spinal
cord and brain stem.6,7 Alberati D et al.8 reported a novel class of GlyT1
inhibitors. But, the molecules are devoid of activity at the GlyT2 isoform.
Recently, the first potent and selective GlyT2 inhibitor was reported,
4-benzyloxy-3,5-dimethoxy-N-[(1-dimethylaminocyclopentyl)
methyl]benza-
mide 1 (Figure 1), which provides an important tool to further characterize
GlyT2 pharmacology.9,10 This compound had a high affinity for GlyT2
(IC50=16 nM) and negligible affinity (IC50>100 mM) for GlyT1. Haradahira1
et al.,11 reported a synthesis of C-11 labeled 1 as a potential PET radioligand,
however, in vivo studies have not been published with this compound.
Therefore, we synthesized a benzamide analog substituted with a fluoropropyl
group as potential PET GlyT2 radiotracer. Our approach to a radiolabeled
analog was to replace one of the methoxy groups with a fluoroalkyl ether. 3-(3-
[18F]Fluoropropoxy)-4-(benzyloxy)-N-((1-dimethylaminocyclopentyl) methyl)-
5-methoxybenzamide ([18F]3) was synthesized in a one-pot two-step process
by reacting the mono-demethylated phenolic precursor 2 (Figure 1) with
3-[18F]fluoropropyl tosylate.
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RADIOSYNTHESIS OF [18F]3
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Figure 1. Known GlyT2 ligand 4-benzyloxy-3,5-dimethoxy-N-[(1-dimenthyla-
minocyclopentyl) methyl]benzamide 1 and novel analogues 2, 3
Results and discussion
Synthetic chemistry
The fluoropropyl analog 3 was prepared in a convergent synthesis from the
retrosynthetic components 5 and 10 (Figure 1). The key challenge was
generating the unsymmetrically substituted phenolic synthon 10. Initial
attempts to selectively remove one methoxy group were unsuccessful, as the
benzyl ether was more labile to cleavage than the methyl ethers under every
condition tried. We therefore built the molecule starting from methyl gallate
(Scheme 1). Selective 4-O-benzylation of methyl gallate has been reported;12
however, in our hands, only low yields of the desired isomer were obtained.
The successful route converted methyl gallate to the triacetoxy ester 6 with
acetic anhydride and pyridine. Treatment of the ester with potassium
carbonate in the presence of potassium iodide and benzyl chloride in refluxing
acetone resulted in selective hydrolysis and concomitant Williamson alkylation
to give the 4-O-benzyl ester 7 in 72 % yield. Acid hydrolysis at 08C resulted in
preferential cleavage of the aryl acetate esters groups to give methyl 4-O-
benzylgallate 8 in 90% yield. Careful reaction with one molar equivalent of
methyl iodide with K2CO3 in DMF at 08C resulted in the desired
unsymmetrical monomethyl ether 9, separated from the dimethyl byproduct
by flash chromatography. Saponification of the methyl ester 9 gave the
unsymmetrically substituted acid 10.
The second half of the convergent synthesis proceeded in a straightforward
sequence from cyclopentanone. Condensation with potassium cyanide and
dimethylamine gave the amino nitrile 4, which was then reduced with in situ
generated aluminum hydride to give the diamine 5. Final assembly of the
ligand was accomplished by Williamson reaction between phenol 9 and
3-fluoropropyl tosylate (synthesized in two steps from 3-chloro-1-propanol by
reaction with potassium fluoride in ethylene glycol followed by esterification
with p-toluenesulfonyl chloride in pyridine) in DMF using NaH as base to give
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Scheme 1. Chemical Synthesis of fluoro analog 3 and phenolic precursor 2
the ester 14. The methyl ester was hydrolyzed (acid 15), then converted to the
acid chloride 16 and reacted with diamine 5 to give the target ligand 3. For
synthesis of the phenolic precursor 2, acid 10 was converted to the acid
chloride with thionyl chloride 11 and reacted with diamine 5 in THF overnight
at room temperature.
Radiochemistry
[18F]3 was synthesized in a two-step one-pot process as shown in Scheme 2.
The labeling intermediate 3-[18F]fluoropropyl tosylate was prepared by the
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RADIOSYNTHESIS OF [18F]3
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Scheme 2. Radiosynthesis of [18F]3
Figure 2. Radio-HPCL of reaction mixtures of [18F]fluoropropyl tosylate (a)
and [18F]3 (b)
reaction of [18F]FÀ with 1,3-propanediol di-p-tosylate in the presence of
K2CO3 and Kryptofix-222, and provided 3-[18F]fluoropropyl tosylate in 81%
labeling yield. HPLC analysis (Figure 2(a)) indicated that 3-[18F]fluoropropyl
tosylate was stable in acetonitrile for up to 60 min. For preparation of [18F]3,
the labeled radioactive intermediate was not isolated but was subjected directly
to the alkylation reaction in the same pot after evaporation of CH3CN.
Alkylation was carried out by heating the 3-[18F]fluoropropyl tosylate with
2 in DMF in the presence of NaH at 1408C for 30 min. HPLC analysis of the
reaction mixture showed two radioactive peaks (tR 4.6 and 7.8 min),
corresponding to the retention times of 3-[18F]fluoropropyl tosylate and
[18F]3, respectively (Figure 2(b)). Sampling at intermediate time points
indicated initial rapid rate of alkylation followed by slow increase up to
30 min (Figure 3).
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Figure 3. Rate of alkylation of precursor 2 with 3-[18F]fluoropropyl tosylate
(decay-corrected)
Two pre-HPLC purification methods were examined: solid phase extraction
(SPE) and liquid–liquid extraction (LLE). For SPE, the reaction mixture was
added to water and the aqueous mixture was passed through a C-18 Sep-Pak
cartridge. The Sep-Pak was washed with water and the radioactive products
were eluted with methanol. The results showed poor recovery, with the
radioactivity in the methanol extract less than that in the water. On the other
hand, ethyl acetate extraction of [18F]3 from the aqueous reaction mixture
provided 40% recovery. After evaporating ethyl acetate, the residue was
dissolved in methanol and the crude product was purified by semi-preparative
HPLC. The overall radiochemical yield of [18F]3 was 14–16% with a total
synthesis time of about 128 min. The radiochemical purity of [18F]3 was
>98% and the specific activity was 1462 Æ 342 GBq/mmol as determined by
HPLC UV analysis; the stability test showed it was stable during 60 min.
3-[18F]fluoropropyl tosylate was selected to react with compound 2, the
reason for this selection was that 3-[18F]fluoropropyl tosylate has been
developed as a useful alkylating reagent for introducing F-18 into substrates
containing amine, phenol and amide functional groups. In previous work,13
in one-pot method an alkylating agent, [18F]fluoropropyl tosylate, was
used without intermediate purification. We chose 12 mg 1,3-propanediol
di-p-tosylate as F-18 labeling precursor based on Teija Koivula et al.’s
report,14 the best radiochemical yield in the preparation of [18F]fluoropropyl
tosylate can be achieved using 10–15 mg of 1,3-propanediol-di-p-tosylate
precursor. Other reason we select excess 1,3-propanediol di-p-tosylate was that
the success of the ‘one-pot’ synthesis of [18F]FEM-IMPY and [18F]FPM-
IMPY,15 where excess 1,3-propanediol bistosylate does not interfere with the
radiosynthesis. We also hope that an excess of the fluoropropyl tosylate was
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RADIOSYNTHESIS OF [18F]3
1253
used for the alkylation to avoid the presence of any unreacted compound 2 in
the crude product.
In this work we did not optimize mole ratio of ditosylate and compound 2, a
lower relative yield of [18F]3 from 3-[18F]fluoropropyl tosylate was achieved
(Figure 3), HPLC analysis elutes the product accompanied with impure
byproduct, these results imply that excess ditosylate may compete with the
3-[18F]fluoropropyl tosylate for the precursor and which may have caused a
lower radiochemical yield of [18F]3.
Experimental
General
Melting points (m.p.) were uncorrected; 1H-NMR spectra were recorded on a
Bruker 400 or 500 MHz spectrometer with tetramethylsilane as an internal
standard. 13C NMR spectra were taken on a Bruker 400 or 500 MHz
spectrometer. Elemental analysis was performed at Atlantic Microlab, Inc.
(Norcross, GA). Flash chromatography was done on Merck Kieselgel gel 60
F254. Reagents were purchased from Sigma-Aldrich, St. Louis, MO.
Analytical HPLC was performed on Waters Nova-Pak1C18 4 mm,
4.6 Â 250 mm. (Waters Inc., USA); mobile phase, MeOH/H2O/Et3N (80/20/
0.1, v/v/v), flow rate, 1.0 ml/min. The labeled product was separated by
semi-preparative HPLC using Eclipse XDB-C8 9.4 Â 250 mm. column
(Agilent Technologies Inc. USA), with the same mobile phase at a flow rate
of 3.0 ml/min. HPLC analysis and purification were performed with a HPLC
pump (Varian Prostar 210 solvent delivery module, Varian Associates. Inc.,
USA), an in-line Varian Prostar 320 UV-detector (254 nm), and a G-M
radioactivity detector (Ludlum Model 2600 Spectrometer, Ludlum Measure-
ments. Inc., USA). HPLC data were recorded by a ProStar/Dynamax System
dual channel control/interface module (Varian. Inc., USA) connected to a
microcomputer with Star Chromatography Workstation Version 5 software.
Synthetic chemistry
1-(Dimethylamino)cyclopentanecarbonitrile (4). A solution of KCN (6.5 g,
0.1 mol) in 50 ml H2O was added over 10 min to a stirred, cooled suspension of
HNMe2 ꢀ HCl (8.15 g, 0.1 mol) and cyclopentanone (8.4 g, 0.1 mol). The
mixture was stirred overnight at room temperature and extracted with Et2O.
The organic layer was washed with H2O, dried over MgSO4 and then
evaporated under reduced pressure to give a colorless oil 4 (7.15 g, 52%, lit.
87%). 16 1H NMR(CDCl3) d: 2.27 (s, 6H, CH3 Â 2), 2.1 (m, 2H), 1.8–1.6
(m, 6H); 13C NMR (CDCl3) d: 119.6 (CN), 69.4 (C1), 41.9(CH3 Â 2), 38.4
(C2 and C5), 23.2(C3 and C4).
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1-Aminomethyl-1-dimethylamino-cyclopentane (5). To a stirred suspension of
LiAlH4 (12.4 g, 0.32 mol) in 200 ml dry THF, cooled to 08C under N2, was
added dropwise a solution of H2SO4 (8.2 ml, 0.16 mol) in dry THF(30 ml). The
mixture was stirred for 2 h and then allowed to rest overnight at room
temperature. To this suspension, a solution of 4 (13.8 g, 0.10 mol) in 80 ml
THF was added dropwise at 08C. The mixture was warmed at 40–508C for 3 h,
then cooled and quenched with H2O. The mixture was filtered and the filtrate
was concentrated under reduced pressure. The oily residue was purified by
bulb-to-bulb distillation to give 5 (6.31 g, 45%, lit. 56%);16 1H NMR (CDCl3)
d: 2.6 (s, 2H, NCH2), 2.2 (s, 6H, CH3 x 2), 1.8–1.3 (m, 8H, cyclopentyl); 13C
NMR (CDCl3) d: 70.1 (C1), 48.6 (CNH2), 39.6 (CH3 Â 2), 30.1 (C2), 30.9 (C5),
25.9 (C3 and C4).
Methyl 3,4,5-triacetoxybenzoate (6). Methyl 3,4,5-triacetoxybenzoate was
prepared as previously described12 with minor modifications. A mixture of
methyl gallate (5.00 g, 27.15 mmol) and acetic anhydride (9.98 g, 97.7 mmol) in
pyridine (25 ml) was stirred 2 h at 08C to room temperature. The reaction
mixture was then poured into 1.2 M HCl and extracted with EtOAc
(3 Â 75 ml). The combined EtOAc extracts were washed with aqueous
NaHCO3 until the wash solution was basic, washed with brine (50 ml), dried
(MgSO4,), and evaporated in vacuo. Recrystallization (EtOH) gave 7.90 g
(93%) of 6 as a white solid, m.p. 119–1218C (lit 126.5–1288C).17
Methyl 3,5-diacetoxy-4-benzyloxybenzoate (7)
A mixture of methyl 3,4,5-triacetoxybenzoate 6 (4.00 g, 12.90 mmol), K2CO3
(5.40 g, 38.70 mol), KI (0.330 g, 2.0 mmol), and benzyl chloride (3.26 g,
25.80 mmol) was heated in acetone (200 ml) under reflux for 18 h. The reaction
mixture was then cooled, poured into water (300 ml), and extracted with Et2O
(3 Â 100 ml). The combined Et2O extracts were washed with brine (3 Â 100 ml),
dried (MgSO4), and evaporated in vacuo. Recrystallization (EtOH) gave 3.98 g
(72%) of 7, m.p. 104–1068C (lit 94–968C).17 1H NMR (CDCl3) d: 7.7 (s, 2H),
7.4 (m, 5H), 5.1 (s, 2H), 3.8 (s, 3H), 2.1 (6H); 1H NMR (CDCl3) d: 7.69 (2H, s,
H2, H6), 7.36 (5H, s, CH2Ph, ArH); 5.05 (2H, s, CH2); 3.89 (3H, s, CO2Me),
2.20(6H, s, 3-OAc, 5OAc).
Methyl 4-benzyloxy-3,5-dihydroxybenzoate (8). To a solution of 7 (1.60 g,
4.46 mmol) in MeOH (80 ml) was added a solution of K2CO3 (2.80 g) in water
(40 ml) at 08C in 10 min. After being stirred at room temperature for 20 min,
the solvent was evaporated and the residue was acidified (pH=2) with 1 M
HC1, then extracted with EtOAc (3 Â 100 ml). The combined EtOAc extracts
were washed with brine (2 Â 100 ml) and H2O (1 Â 100 ml), dried (MgSO4),
and evaporated in vacuo. Recrystallization (CHC13/hexane) gave 1.06 g (90%)
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RADIOSYNTHESIS OF [18F]3
1255
of 8, m.p. 127–1298C (lit. m.p.:133–1348C).17 1H NMR (CDCl3) d: 7.3
(m, 5H), 7.2 (s, 2H), 5.6 (s, 1H), 5.1 (s, 2H), 3.8 (s, 3H).
Methyl 4-benzyloxy-3-hydroxy-5-methoxybenzoate (9). Methyl 4-benzyloxy-
3,5-dihydroxybenzoate (8) (2.02 g; 7.4 mmol) and 1.96 g K2CO3 were
suspended in 10 ml DMF. Then, 0.994 g (7.00 mmol) CH3I was added at
08C and the resulting mixture was stirred at RT for 5 h. After removing the
solvent, the residue was treated with H2O and extracted with Et2O. The Et2O
extract was dried (MgSO4) and the solvent removed on the rotary evaporator.
The residue was purified by chromatography (ether/hexane 1/1) to afford
1.43 g (yield 67.5%) of white solid, m.p. 124–126 8C. 1H NMR (CDCl3) d: 7.3
(m, 5H), 7.2 (m, 2H), 5.8 (s, 1H), 5.1 (s, 2H), 3.8 (s, 3H), 3.7(s, 3H).
4-Benzyloxy-3-hydroxy-5-methoxybenzoic acid (10). Methyl 4-benzyloxy-3-
hydroxy-5-methoxybenzoate 9 (405 mg, 1.4 mmol) was heated at reflux in
10 ml of 30% aqueous KOH and methanol for 1 h. Excess solvent was
removed on the rotary evaporator and 20 ml H2O was added. 12 M HCl was
added dropwise to the mixture to pH 1. The white precipitate was washed with
H2O and dried to give 317 mg (82.4%) 10 as a white solid, m.p. 133–1358C. 1H
NMR (CDCl3) d 7.2–7.3 (m, 7H), 5.2 (s, 1H), 5.1 (s, 2H), 3.8 (s, 3H).
4-Benzyloxy-3-hydroxy-5-methoxybenzoyl chloride (11). A mixture of acid 10
(317 mg, 1.15 mmol) and thionyl chloride (1.19 g, 10 mmol) in toluene (5 ml)
was heated at reflux until no more gas was given off (2 h), then the reaction
mixture was concentrated under reduced pressure. To remove excess of thionyl
chloride, the residue was repeatedly dissolved in toluene (2 Â 10 ml) and
evaporated to dryness to give acid chloride 11 as an oil, which was used
directly in subsequent reactions.
3-Fluoro-1-propanol (12). Compound 12 was prepared from 3-chloro-1-
propanol as described.18 A mixture of 18.9 g. (0.2 mol) of 3-chloro-1-propanol,
23.3 g. (0.4 mol) of KF, and 30 g of ethylene glycol was heated at 175–1808C
with vigorous stirring. During the reaction the product was continuously
distilled off between 130 and 1608C. After 3 h, 8.17 g of a colorless liquid was
collected. The crude 3-fluoro-1-propanol was distilled twice. Yield 8.17 g
1
(54%). H NMR(CDCl3) d 1.8 (m, 2H), 3.3 (m, 1H), 3.7 (m, 2H), 4.4–4.5
(m, 2H).
3-Fluoropropyl p-to1uenesulfonate (13). A mixture of p-toluenesulfonyl
chloride (9.8 g, 50.2 mmol) and pyridine (12 g) was stirred at 08C while
3-fluoro-1-propanol (3.92 g, 50 mmol) was added slowly. Stirring was
continued for 1 h more, resulting in a white precipitate. The mixture was
diluted with H2O, 12 M HCl was added until no pyridine odor was perceptible,
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and the resultant mixture was extracted several times with Et2O. The
combined extracts were washed successively with H2O, aqueous NaHCO3
and finally with H2O again. After drying (MgSO4) and removal of Et2O,
distillation of the residue yielded 3-fluoropropyl p-to1uenesulfonate (7.63 g,
65.9%,), as a colorless liquid. 1H NMR(CDCl3) d 1.8 (m, 2H), 2.3 (s, 3H), 4.0
(m, 2H), 4.2 (m, 2H), 7.2(m, 2H), 7.6(m, 2H).
Methyl 3-(3-fluoropropoxy)-4-(benzyloxy)-5-methoxybenzoate (14). A mix-
ture of compound 9 (560 mg, 1.9 mmol), 928 mg (4 mmol) 3-fluoropropyl
p-toluenesulfonate and NaH (50 mg, 4 mmol) in 10 ml DMF was stirred
at room temperature overnight. The solvent was removed under vacuum,
then the residue was dissolved in DMF and purified by chromatography
(ether/hexane, 1/1), to give 425 mg (50%) oil. 1H NMR (CDCl3) d 2.0 (m, 2H),
3.8 (s, 6H), 4.0 (m, 2H), 4.3 (m, 2H), 5.0 (s, 2H), 7.3 (m, 5H), 7.4(m, 2H).
3-(3-Fluoropropoxy)-4-(benzyloxy)-5-methoxybenzoic acid (15). This com-
pound was synthesized from 570 mg (1.64 mmol) compound 14 according to
the method described for 10 to give 370 mg (68%) oil. 1H NMR(CDCl3) d 2.0
(m, 2H), 3.7 (s, 3H), 3.9 (m, 2H), 4.4–4.5 (m, 2H), 5.0 (s, 2H), 7.3 (m, 5H),
7.4(m, 2H).
3-(3-Fluoropropoxy)-4-(benzyloxy)-5-methoxybenzoyl chloride (16). This
compound was synthesized from compound 15 according to the method
described for 11 as oil, used directly in the next reaction.
3-(3-Fluoropropoxy)-4-(benzyloxy)-N-((1-(dimethylamino) cyclopentyl) methyl)-
5-methoxybenzamide (3). A solution of 3-(3-fluoropropoxy)-4-(benzyloxy)-5-
methoxybenzoyl chloride 16 (409 mg, 1.16 mmol) in anhydrous THF(10 ml)
was added dropwise over 15 min to a solution of 1-aminomethyl-1-
dimethylamino-cyclopentane 5 (210 mg, 1.5 mmol) and Et3N (151 mg,
1.5 mmol) in THF (5 ml) at 08C. The reaction was allowed to warm to room
temperature and stirred overnight. The precipitate was removed and the
residue was purified by chromatography (EtOAc/MeOH, 1/9), to give 174 mg
(34.3%) oil. It was dissolved in MeOH, then excess HCl in Et2O was added.
1
Evaporation to dryness yielded the hydrochloride 3 as a gum. H NMR
(CDCl3) d 1.4–1.6 (m, 8H), 1.9(m, 2H), 2.1 (s, 6H), 3.4(s, 2H), 3.7 (s, 3H),
4.0(m, 2H), 4.4–4.5(m, 2H), 5.0 (s, 2H), 7.1–7.3 (m, 7H).
4-Benzyloxy-3-hydroxy-5-methoxy-N-[(1-dimethylaminocyclopentyl)methyl]-
benzamide (2)
This compound was synthesized from 5 (168 mg, 1.2 mmol) and 11 (2.46 g,
8.5 mmol) according to the method described for 3. Yield 188 mg (41.0%) as
oil; HCl salt: gum. 1H NMR(CDCl3) d 1.3 (m, 2H), 1.5 (m, 4H), 1.7 (m, 2H),
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RADIOSYNTHESIS OF [18F]3
1257
2.2 (s, 6H), 3.2(s, 2H), 3.8 (s, 3H), 5.2 (s, 2H), 5.7 (s, 1H), 6.9 (m, 2H), 7.3–7.4
(m, 5H). Analytically Found C, 65.90; H, 7.39; N, 6.22. Calculated for
C23H31ClN2O4: C, 63.51; H, 7.18; N, 6.44.
Radiochemistry
Two-step/one-pot radiofluorination and alkylation. [18F]Fluoride was produced
with an RDS-112 11 MeV negative ion cyclotron by the reaction 18O(p,n)
18F using 150 ml [18O]H2O (97.2 at% 18O) in a silver target. The aqueous
solution of [18F]fluoride was transferred to a 12-ml test tube (red-top
Vacutainer1, BD) containing a mixture of K2CO3 (4 mg) and kryptofix-222
(20 mg) in CH3CN/H2O, then dried by azeotropic distillation with aceto-
nitrile (4 Â 0.5 ml) at 1208C (oil bath) under a stream of Ar(g). After cooling
to room temperature, the residue was dissolved in 0.5 ml CH3CN, 12 mg
1,3-propanediol di-p-tosylate was added, the tube was sealed, and heated
at 858C for 15 min. Aliquots (10 ml) were analyzed by analytical HPLC.
The radiofluorination labeling yield was calculated from the area%
of the radioactive peak at tR 4.6 min, the proof of identity of the products
in the HPLC is by comparison with reference compounds. Solvent was
evaporated under a stream of Ar(g). This residue was used directly for
the alkylation.
A solution of 4-benzyloxy-3-hydroxy-5-methoxy-N-[(1-dimethylamino-
cyclopentyl) methyl]benzamide HCl salt (2) (5 mg) in N,N-dimethyl-
formamide (DMF, 0.5 ml) and NaH (1.3 mg) was added, the tube was
capped, then heated to 1408C. Aliquots (10 ml) were analyzed by analytical
HPLC. The alkylation yield was calculated from the area% of the radioactive
peak at tR 7.8 min. Upon completion of alkylation (after 30 min of reaction),
DMF was evaporated in vacuo at 808C. The residue was dissolved in ethyl
acetate (2 ml) and extracted with H2O (3 ml), and then the organic layer
was passed through a plug filled with Na2SO4. The solvents were removed
and the residue was re-dissolved in 300 ml of MeOH and purified by
semi-preparative HPLC, collecting the fraction corresponding to [18F]3.
After removing solvent on a rotary evaporator, the residue was dissolved
in EtOH and diluted with normal saline to give a final solution of 10%
ethanol-saline.
Conclusion
To develop a PET radiotracer for imaging glycine transporter 2(GlyT2), a
novel benzamide analog substituted with a fluoropropyl group for GlyT2 was
synthesized. The PET radiotracer [18F]3 was synthesized by the alkylation of
the precursor 2 with 3-[18F]fluoropropyl tosylate, the radioligand was obtained
with high specific activity and radiochemical purity and in sufficient
radiochemical yield for PET studies with animals.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1247–1258
DOI: 10.1002/jlcr

1258
H. TIAN ET AL.
Acknowledgements
The authors express their thanks to Dr Eric Brenner, Dr David Alagille,
Dr David Cheng, and Ms Nina Sheung for their helpful support and
discussion. This work was supported in part by Veterans Affairs Research
Enhancement Award Program (REAP) Center on ‘Neural Mechanisms and
Treatment Response in Depression’ and VA Schizophrenia Biological
Research Center SRC.
References
1. Heresco-Levy U, Javitt DC, Ermilov M, Silipo G, Shimoni J. Int
J Neuropsychopharmcol 1998; 1: 131–135.
2. Heresco-Levy U, Javitt DC, Ermilov M, Mordel C, Silipo G, Lichtenstein M.
Arch Gen Psychiatry 1999; 56: 29–36.
3. Guastella J, Brecha N, Weigmann C, Lester HA, Davidson N. Proc Natl Acad Sci
USA 1992; 89: 7189–7193.
4. Liu QR, Nelson H, Mandiyan S, Lopez-Corcuera B, Nelson N. FEBS Lett 1992;
305: 110–114.
5. Smith KE, Borden LA, Hartig PR, Branchek T, Weinshank RL. Neuron 1992; 8:
927–935.
6. Jursky F, Nelson N. J Neurochem 1995; 64: 1026–1033.
7. Zafra F, Aragon C, Gimenez C. Mol Neurobiol 1997; 14: 117–142.
8. Alberati D, Ceccarelli SM, Jolidon S, Krafft EA, Kurt A, Maier A, Pinard E,
Stalder H, Studer D, Thomas AW, Zimmerli D. Bioorg Med Chem Lett 2006; 16:
4305–4310.
9. Coghlan MJ, Kym PR, Elmore SW, Wang AX, Luly JR, Wilcox D, Stashko M,
Lin C-W, Miner J, Tyree C, Nakane M, Jacobson P, Lane BC. J Med Chem 2001;
44: 2879–2885.
10. Caulfield WL, Collie IT, Dickins RS, Epemolu O, McGuire R, Hill DR, McVey
G, Morphy JR, Rankovic Z, Sundaram H. J Med Chem 2001; 44: 2679–2682.
11. Haradahira T, Arai1 T, Igarashi N, Okauchi T, Maeda J, Nagai Y, Suzuki K,
Suhara T. J Label Compd Radiopharm 2003; 46: S226.
12. Elsohly HN, Ma GE, Turner CE, Elsohly MA. J Nat Prod 1984; 47: 445–452.
13. Kazumata K, Dhawan V, Chaly T, Antonini A, Margouleff C, Belakhlef A,
Neumeyer J, Eidelberg D. J Nucl Med 1998; 39: 1521–1530.
14. Koivula1 T, Perhola1 O, Eeva-Liisa Kamarainen EL, Lipponen T, Vepsalainen J,
Solin O. J Label Compd Radiopharm 2005; 48: 463–471.
15. Cai L, Chin FT, Pike VW, Toyama H, Liow JS, Zoghbi SS, Modell K, Briard E,
Shetty HU, Sinclair K, Donohue S, Tipre D, Kung MP, Dagostin C, Widdowson
DA, Green M, Gao W, Herman MM, Ichise M, Innis RB. J Med Chem 2004; 47:
2208–2218.
16. Yang D, Bremont B, Shen S, Kefi S, Langlois M. Eur J Med Chem 1996; 31:
231–239.
17. Pearson. AJ, Bruhn PR. J Org Chem 1991; 56: 7092–7097.
18. Hoffmann FW. J Org Chem 1950; 15: 425–434.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1247–1258
DOI: 10.1002/jlcr