Synthesis of [3H]fenobam, a radioligand for the mGlu5 receptor

Article abstract of DOI:10.1002/jlcr.1470

Fenobam is a clinically efficacious anxiolytic that acts as metabotropic glutamate receptor 5 (mGlu5) antagonist by binding to an allosteric site. Other mGlu5 receptor antagonists such as MPEP and MTEP bind to the same allosteric site and are efficacious

Full text of DOI:10.1002/jlcr.1470

Note
Received 13 August 2007,
Accepted 7 October 2007
Published online 2 January 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1470
Synthesis of [3H]fenobam, a radioligand for
the mGlu5 receptor
a
Ã
b
b
¨
Thomas Hartung, Jens-Uwe Peters, Jurgen Wichmann,
Christian Hubschwerlen,c and Georg Jaeschkeb
Fenobam is a clinically efficacious anxiolytic that acts as metabotropic glutamate receptor 5 (mGlu5) antagonist by binding
to an allosteric site. Other mGlu5 receptor antagonists such as MPEP and MTEP bind to the same allosteric site and are
efficacious in preclinical models of anxiety and depression. Consequently, the allosteric-binding site of the mGlu5 receptor
is an attractive target for the discovery of novel psychiatric therapies. Radioligands of this binding site can be used for
in vitro and in vivo pharmacodynamic studies. We report here a short synthesis of such a radioligand for the allosteric
mGlu5 receptor-binding site, [3H]fenobam.
Keywords: radioligand; fenobam; mGlu5; allosteric binding; tritium
Introduction
Experimental
The G-protein-coupled metabotropic glutamate receptor
5
All reagents were of commercial quality unless otherwise stated.
(mGlu5) is highly expressed in the limbic areas of the brain, 3-Chloro-[2,6-3H]aniline hydrochloride (23 Ci/mmol, 851 GBq/mmol)
which suggests a potential role of this receptor in psychiatric was prepared by the Tritium Custom Preparations Group, Amersham
disorders, such as anxiety and depression.1–6 Indeed, the Biosciences, Cardiff, and was supplied as ethanolic solution.
selective mGlu5 antagonists MPEP and MTEP (Figure 1) have
Radioactivity measurements were carried out using a Wallac
been shown to be active in a broad range of preclinical tests.7 liquid scintillation counter 1414 WinSpectralTM instrument and
Recently, it was found that the clinically efficacious anxiolytic Optiphase Hisafe 3 as scintillant. Analytical HPLC analyses were
fenobam binds selectively and with high affinity (Ki 561nM) to conducted using
the same allosteric site of the mGlu5 receptor as do MPEP and (250 Â 4 mm) with
a
Nucleosils 100-5 C18 HD column
mobile phase system composed of
a
MTEP.8–11 This finding makes fenobam an interesting pharmaco- 30% acetonitrile in water (v/v) to 70% acetonitrile in water
logical tool for preclinical studies, even if its clinical development (v/v) over 10 min at 0.9 ml/min on an Agilent 1100 Series HPLC
was discontinued due to a highly variable PK profile in humans system equipped with quaternary pump, degasser, Rheodyne
and unwanted side effects.12 The recently published data about 7725i manual injector, temperature controller (301C), multi-
the identification of potent, metabolically stable fenobam wavelength detector (250 nm), and a Raytest Ramona 2000
analogs and their structure–activity relationship emphasized the radioactivity flow detector with a solid scintillation cell (glass,
current interest in the field of mGlu5 antagonists.13–15 [14C]Feno- 100 ml, 32–45 mm). Flash column chromatography was per-
bam has been used for metabolism studies.12 A preparation of formed using Merck silica gel 0.04–0.063 mm. Thin-layer
[3H]fenobam has not yet been described, although it might chromatography (TLC) was run on Merck Kieselgel silica gel 60
be
a valuable tool for mGlu5 radioligand-binding assays F254 glass plates. Radio-TLC was carried out on a Berthold TLC-
or for mGlu5 distribution studies. We therefore devised a Linear Analyzer Tracemaster 20 and on a Raytest Rita-Star
short synthesis of tritium-labeled fenobam, which is reported instrument.
here.
(1-Methyl-4-oxo-imidazolin-2-yl)-carbamic acid 2,5-dioxo-pyrro-
lidin-1-yl ester, 3: Under an atmosphere of nitrogen, a suspension
Results and discussions
aPharma Research Basel, Technical Sciences, F. Hoffmann-La Roche Ltd.,
CH-4070 Basel, Switzerland
(1-Methyl-4-oxo-imidazolin-2-yl)-carbamic acid 2,5-dioxo-pyrroli-
din-1-yl ester (3) as a precursor for [3H]fenobam was obtained in
analogy to the method of Takeda et al.16 from creatinine and
di(N-succinimidyl) carbonate (Scheme 1).
bPharma Research Basel, Discovery Chemistry, F. Hoffmann-La Roche Ltd.,
CH-4070 Basel, Switzerland
cActelion Pharmaceuticals Ltd., Gewerbestrasse 16, CH-4123 Allschwil,
Switzerland
Precursor 3 was then reacted with commercially available
3-chloro-[2,6-3H]aniline hydrochloride (4) according to Scheme 2.
After high-performance liquid chromatography (HPLC) purifica-
tion, [3H]fenobam 5 was obtained in a satisfactory yield and
high radiochemical purity.
*Correspondence to: Thomas Hartung, F. Hoffmann-La Roche Ltd., Building 90/
620B, CH-4070 Basel, Switzerland.
E-mail: thomas.hartung@roche.com
J. Label Compd. Radiopharm 2008, 51 77–79
Copyright r 2008 John Wiley & Sons, Ltd.

T. Hartung et al.
H
H
N
N
Cl
N
N
N
N
O
S
N
O
MPEP
MTEP
fenobam
Figure 1. The anxiolytics MPEP, MTEP and fenobam bind to the same allosteric-binding site of the mGlu5
receptor.
O
O
O
H
N
H2N
N
O
DMAP, acetonitrile
O
N
+
O
N
N
N
O
N
r.t. overnight
89%
O
O
O
N
O
O
O
1
2
3
Scheme 1
T
T
O
H
N
H
N
H
N
+
NEt3
Cl
NH
O
O
3 Cl
N
, acetone
Cl
N
O
N
+
O
60˚C, 4h
23%
O
N
N
O
T
T
4
3
5
[3H]fenobam
Schme 2
of creatinine (500 mg, 4 mmol) in acetonitrile (10 ml) was added purity:499% (by analytical radio-HPLC analysis); specific activity
slowly and dropwise over a period of 1.5 h to a solution of di(N- (by MS): 23.5 Ci/mmol (869 GBq/mmol).
succinimidyl)-carbonate (2.26 g, 9 mmol) and DMAP (54 mg,
0.4 mmol) in acetonitrile (15 ml). The light brown reaction Conclusion
mixture was stirred at room temperature overnight. The
Tritium-labeled fenobam as a radioligand for the allosteric
solvent was evaporated, and the residue was suspended in
fenobam/MPEP-binding site of the mGlu5 receptor can be
dichloromethane, and stirred for 30 min. Filtration then provided
the title compound (1.00 g, 89%). 1H NMR (300 MHz, d6 DMSO) d
11.3 (1H, bs), 4.12 (2H, s), 2.98 (3H, s), 2.77 (4H, s).
obtained easily from the commercially available tritiated
precursor 4 and the carbamic acid ester reagent 3 in excellent
radiochemical purity and sufficient high specific activity.
1-(3-Chloro-[2,6-3H]phenyl)-3-(1-methyl-4-oxo-2-imidazolin-2-yl)
urea, [3H]fenobam, 5: A solution of 150 mCi (5.55 GBq) of
3-chloro-[2,6-3H]aniline hydrochloride (6.5 mmol, 1.08 mg) in
8 ml of ethanol was evaporated to dryness at 401C under
reduced pressure. After addition of 0.2 ml of toluene the solvent
was removed again in vacuo to get rid of residual ethanol. The
References
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solution was transferred into a heavy-wall pressure tube
containing 5.2 mg (20.5 mmol) of (1-methyl-4-oxo-imidazolin-2-
yl)-carbamic acid 2,5-dioxo-pyrrolidin-1-yl ester (3) and 3 ml
(20.1 mmol) of triethylamine. The pressure tube was closed and
the reaction mixture was heated at 601C for 4 h. The product
formation was confirmed by TLC analysis (silica, dichloro-
methane/methanol 95:5, Rf 5 0.56). Subsequently, the reaction
mixture was evaporated under reduced pressure and the
remaining residue was purified by silica gel column chromato-
graphy with dichloromethane/methanol (98:2 to 97:3) as eluent.
Radiochemically pure fractions were combined, the solvent was
removed in vacuo and the resulting tritiated fenobam was
dissolved in 25 ml of degassed tert-butyl methylether/toluene
1:1. Yield: 34 mCi (23%). Product specifications: radiochemical
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