[3H]-(R)-NPTS, a radioligand for the type 1 glycine transporter.

Article abstract of DOI:10.1016/S0960-894X(03)00126-4

The synthesis of NPTS, 6, a potent inhibitor of the type 1 glycine transporter (GlyT1) is described, as well as preparation of 6 in optically active and tritiated form for use as a radioligand for affinity displacement assay of GlyT1.

Full text of DOI:10.1016/S0960-894X(03)00126-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1291–1292
[3H]-(R)-NPTS, a Radioligand for the Type 1 Glycine Transporter
John A. Lowe, III,* Susan E. Drozda, Katherine Fisher, Christine Strick, Lorraine Lebel,
Christopher Schmidt, Donna Hiller and Kathleen S. Zandi
Central Research Division, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA
Received 3 November 2002; accepted 18 January 2003
Abstract—The synthesis of NPTS, 6, a potent inhibitor of the type 1 glycine transporter (GlyT1) is described, as well as preparation
of 6 in optically active and tritiated formfor use as a radioligand for affinity displacement assay of GlyT1.
# 2003 Elsevier Science Ltd. All rights reserved.
GlyT1, the type 1 glycine transporter, and its type 2
counterpart GlyT2 mediate the specific reuptake of gly-
cine during synaptic transmission.1 GlyT1 and GlyT2
belong to the Na+/Cl-dependent family of transpor-
ters, which includes transporters for the neuro-
transmitters dopamine, serotonin, adrenaline, and
GABA.2 Glycine is an obligatory co-agonist at the
NMDA class of glutamate receptors, and since a defi-
ciency in NMDA neurotransmission is thought to play a
role in the etiology of schizophrenia, an increase of glycine
levels has been proposed as a novel approach to treating
psychosis.3 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.4 Blockade of GlyT1-medi-
ated reuptake of glycine may provide the same benefit,
as GlyT1 is co-localized with NMDA receptors.5 Recently,
the first potent and selective GlyT1 inhibitor was reported,
(R)-N[3-(40fluorophenyl)-3-(40phenylphenoxy)propyl]-sar-
cosine, ALX 5407.6,7 NFPS, the racemic form of ALX
5407, has been shown to augment downstream effects of
NMDA transmission, supporting the proposed role of
GlyT1 in regulating NMDA transmission.8 We began a
programto explore the role of GlyT1 inhibitors in
treating schizophrenia by developing an analogue of
ALX 5407, (R)-N[3-phenyl-3-(40-(4-toluoyl)phenoxy)-
propyl]sarcosine ((R)-NPTS) as a suitable radioligand
for a GlyT1 binding affinity assay (Fig. 1). We report
herein the SAR and synthetic studies leading to NPTS,
as well as the synthesis of [3H]-(R)-NPTS.
The key step in the synthesis of compound 6 is the pal-
ladium-mediated boronic acid coupling with the acid
chloride 4 to produce the requisite benzophenone deri-
vative 5 (Fig. 2). This step follows a recent literature
report, and afforded comparable yields and similar ver-
satility in our hands.9 As shown in Figure 1, 6 was
somewhat weaker than 1 in a standard rat synaptoso-
mal uptake assay using tritiated glycine. We decided to
use 6 as the basis for a radioligand binding assay with
improved sensitivity for detecting new GlyT1 inhibitors.
Synthesis of a suitable precursor in optically active form
for radiolabeled (R)-NPTS is outlined in Figure 3. The
challenge of preparing the water soluble amino alcohol
8 was solved with the Staudinger reaction.10 The com-
mercially available R-(+)-3-chloro-1-phenyl-1-propa-
nol, 7, was converted to the iodide and then to the azide,
followed by reduction with triphenylphosphine and
water in tetrahydrofuran. Without isolation, the amino
alcohol 8 was reacted with sodiumhydride in NMP and
Figure 1. Structures of NFPS, 1, and NPTS, 6, and their activity
in an assay measuring inhibition of [3H]-glycine uptake into rat
synaptosomes.
*Corresponding author. Fax: +1-860-686-0013; e-mail: john_a_
lowe@groton.pfizer.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00126-4

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J. A. Lowe, III et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1291–1292
Figure 4. Scatchard plot of [3H]-(R)-NPTS binding to HEK 293 cells
expressing the GlyT1c transporter.
saturable binding by Scatchard analysis (Fig. 4). Using
tissue fromrat hippocampus, [ 3H]-(R)-NPTS gave a Kd
value of 0.8 nM, while compound 1 in the same tissue
gave a Ki value of 1.6 nM.
Figure 2. Preparation of NPTS in racemic form.
The use of [3H]-NPTS in a radioligand binding assay
provides a higher level of sensitivity than the standard
tritiated glycine uptake assay, which should facilitate
SAR efforts in this new area.
References and Notes
1. Lopez-Corcuera, B.; Geerlings, A.; Aragon, C. Mol. Mem.
Biol. 2001, 18, 13.
2. Gadea, A.; Lopez-Colome, A. M. J. Neurosci. Res. 2001,
64, 218.
3. Heresco-Levy, U.; Javitt, D. C. Eur. J. Neuropsycho-
pharmacol. 1998, 8, 141.
4. Heresco-Levy, U.; Javitt, D. C.; Ermilov, M.; Mordel, C.;
Silipo, G.; Lichtenstein, M. Arch. Gen. Psychiatry 1999, 56, 29.
5. Supplison, S.; Bergmann, C. J. Neurosci. 1997, 17, 4580.
6. Atkinson, B. N.; Bell, S. C.; De Vivo, M.; Kowalski, L. R.;
Lechner, S. M.; Ognyanov, V. I.; Tham, C.-S.; Tsai, C.; Jia, J.;
Ashton, D.; Klitencik, M. A. Mol. Pharmacol. 2001, 60, 1414.
7. Aubrey, K. R.; Vandenberg, R. J. Br. J. Pharmacol. 2001,
134, 1429.
Figure 3. Synthesis of (R)-NPTS.
8. Bergeron, R.; Meyer, T. M.; Coyle, J. T.; Greene, R. W.
Proc. Natl. Acad. Sci. 1998, 95, 15730.
9. Haddach, M.; McCarthy, J. R. Tetrahedron Lett. 1999, 40,
3109.
10. Staudinger, H.; Meyer, J. Helv. Chim. Acta 1919, 2, 635.
11. Robertson, D. W.; Krushinski, J. H.; Fuller, R. W.;
Leander, J. D. J. Med. Chem. 1988, 31, 1412.
12. (R)-NPTS: [a]D=ꢀ1.72ꢁ. 13C NMR (d, CDCl3): 21.80,
33.32, 41.65, 54.18, 56.73, 77.52, 115.68, 126.04, 128.54,
129.11, 129.23, 130.19, 131.07, 132.35, 135.27, 139.70, 143.06,
160.92, 167.88, 195.46. [3H]-(R)-NPTS was purified by HPLC
using an Inertsil ODS 10ꢂ250 mm column and a 20 mM aq
potassiumphosphate buffer, pH=3 gradient with acetonitrile.
We thank ChemSyn Laboratories in Lenexa, KS for carrying
out the tritiation and purification procedures.
p-fluoro(40-methyl)benzophenone, in overall 58% yield
to this point, in analogy with earlier work on the
synthesis of fluoxetine.11 The final steps are carried out
as before, and the opposite enantiomer was prepared
fromthe corresponding S-(ꢀ)-alcohol. [3H]-(R)-NPTS
was prepared by substituting tritiated methyl iodide in
the penultimate step, affording after purification mate-
rial of >98% purity and 71 Ci/mmol.12
In studies of binding to the cloned human GlyT1c sub-
type of the GlyT1 transporter expressed in HEK 293
cells, [3H]-(R)-NPTS showed a Kd value of 1 nM with