[3H]-M-MPEP, a potent, subtype-selective radioligand for the metabotropic glutamate receptor subtype 5

Article abstract of DOI:10.1016/S0960-894X(01)00767-3

The synthesis of a new potent, subtype-selective radioligand [³H]-M-MPEP (2-methyl-6-((3-methoxyphenyl)ethynyl)-pyridine) and its in vitro pharmacological characteristics are described.

Full text of DOI:10.1016/S0960-894X(01)00767-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 407–409
[3H]-M-MPEP, a Potent, Subtype-Selective Radioligand for the
Metabotropic Glutamate Receptor Subtype 5
Fabrizio Gasparini,a,* Hendrik Andres,b Peter Josef Flor,a Micheline Heinrich,a
Werner Inderbitzin,a Kurt Lingenhohl,a Hanspeter Muller,a Veronica Cecilia Munk,a
Kyla Omilusik,a Christine Stierlin,a Natacha Stoehr,a Ivo Vranesica and Rainer Kuhna
aNervous System Research, Novartis Pharma AG, 4002 Basel, Switzerland
bDMPK/Isotope Laboratory, Novartis Pharma AG, 4002 Basel, Switzerland
Received 23 June 2001; revised 23 October 2001; accepted 13 November 2001
Abstract—The synthesis of a new potent, subtype-selective radioligand [3H]-M-MPEP (2-methyl-6-((3-methoxyphenyl)ethynyl)-
pyridine) and its in vitro pharmacological characteristics are described. #2002 Elsevier Science Ltd. All rights reserved.
Introduction
potent and selective ligands has hampered the develop-
l-Glutamate is the main excitatory neurotransmitter in
the mammalian brain and acts through a heterogeneous
family of two major types of receptors: ionotropic
(iGluRs) and metabotropic glutamate receptors
(mGluRs). Ionotropic glutamate receptors are ligand
gated channels, whereas metabotropic receptors are
G-protein coupled receptors (GPCRs) linked to second
messenger pathways. To date eight metabotropic recep-
tor subtypes have been identified and classified into
three groups according to their amino acid identities,
second messenger coupling and pharmacology.1 Group
I mGluRs (1 and 5) are coupled to the phosphoinosi-
tide/Ca2+ cascade. Group II mGluRs (2 and 3) and
Group III mGluRs (4–8) are negatively coupled to ade-
nylate cyclase. mGlu receptors are members of the
superfamily 3 of GPCRs and possess a unique structure
with a large N-terminus involved in the glutamate
recognition site.2À4
ment of radioligands to devise useful receptor binding
assays. The identification of potent and Group II selec-
tive ligands such as DCG-IV, LY354740 and LY341495
opened up the possibility for the radiolabelingand
recent reports described the characterization of [3H]-
LY354740 (1), [3H]-LY341495 (2), [3H]-DCG-IV (3)
and [3H]-quisqualatic acid (4), respectively (Fig. 1).6À9
Recent work by Mutel et al.10 demonstrated that a non-
selective agonist, [3H]-Quisqualate (4) (Fig. 1), could be
a useful tool to label Group I mGlu receptors (1 and 5).
However, this radioligand does not allow us to selec-
tively label each Group I subtype and therefore there is
still a lack of subtype-selective ligands.
Chemistry
Recently, we reported the identification and the char-
acterization of a series of non-competitive mGluR5
selective antagonists illustrated by SIB-1757 [6-methyl-
2-(phenazo)-pyridin-3-ol] (5), SIB-1893 [(E)-2-methyl-6-
(phenylethenyl)-pyridine] (6) and MPEP [2-methyl-6-
(phenylethynyl)-pyridine] (7) (Fig. 2).11,12 In the course
of our optimization program we aimed to identify a
potent and suitable derivative for radiolabelingin view
of the development of a radioligand based binding
assay. Such a radioligand binding technique could be a
useful tool to investigate the receptor distribution and
pharmacology.
mGluR Ligands
The vast majority of known mGluR ligands are amino
acid derivatives and therefore interact with the con-
served glutamate binding site.5 Until recently the lack of
*Correspondingauthor. Tel.: +41-61-696-1412; fax: +41-61-696-
8676; e-mail: fabrizio.gasparini@pharma.novartis.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00767-3

408
F. Gasparini et al. / Bioorg. Med. Chem. Lett. 12 (2002) 407–409
Figure 3. Effects of M-MPEP on PI turnover at the human mGluR5a
stably expressed in L(tkÀ) cells (*) and the human mGluR1b stably
expressed in CHO-K1 cells (&). Results are expressed as percentage
(meanÆSEM) of the stimulation obtained with the agonists at the
respective EC80 (quisqualate at 0.3 and 20 mM for human mGluR5a
and mGluR1b, respectively). Percentages were pooled from N=3
independent experiments performed in triplicate.
Figure 1. Structures of currently known tritiated radioligands for
metabotropic glutamate receptors and the corresponding radiolabeling
position.
Table 1. In vitro functional activity and bindingaffinity for the
recombinant human mGlu5 receptor
IC50 (nM)
PI-hydrolysisa
[3H]-M-MPEP
displacementb
13, M-MPEP
7, MPEP
6, SIB-1893
10
36
3000
3.6
20
1100
aData taken from refs 11,12.
bDisplacement of [3H]-M-MPEP from membrane of L(tkÀ) cells
expressingthe human mGluR5.
Figure 2. Structures of non-competitve antagonists.
The lead optimization process of this series started from
the original leads 5 (SIB-1757) and 6 (SIB-1893) (Fig.
2).11 Several lines of chemical modifications were inves-
tigated such as modification of the nature and the length
of the spacer between the two aromatic moieties. These
modifications led to the identification of MPEP [6-
methyl-2-(phenylethynyl)pyridine], the first potent and
selective mGlu5 receptor antagonist.12 Further investi-
gations of the substituents around the phenyl ring
allowed the identification of the methoxy substituted
derivative 2-methyl-6-((3-methoxyphenyl)ethynyl)-pyri-
dine (M-MPEP, 13), a more potent derivative (IC50=10
nM, PI turnover assay; Fig. 3; Table 1) which was con-
sidered for radiolabelingat the methoxy position.
In Vitro Pharmacology
M-MPEP (2-(3-methoxy-phenylethynyl)-6-methyl-pyri-
dine) has been functionally characterized at the human
mGlu receptor subtypes 1, 2, 4, 7 and at the recombi-
nantly expressed ionotropic glutamate hNMDA 1A/2A,
1A/2B, hGlu3 (AMPA) and hGlu6 (kainate) receptors. At
these receptor subtypes M-MPEP did not show activity
up to 10 mM (data not shown). Furthermore, using
standard receptor bindingtechniques M-MPEP was
tested for affinity at a panel of twenty-four G-protein
coupled receptors and did not show any significant affinity
up to 10 mM (data not shown). At the human mGlu5
receptor expressed in L-(tkÀ) cells, M-MPEP inhibits
quisqualate induced PI hydrolysis with an IC50 of 10 nM
(Fig. 3).
The synthesis of the radioligand is shown in Scheme 1
and uses 2,6-dimethyl-pyridine 8 and 3-hydroxy-benz-
aldehyde 9 as startingmaterials. 8 and 9 are condensed
into the styrene derivative 10 in acetic anhydride. This
intermediate was dibrominated and the mixture of
dibromo derivatives 11 eliminated usingpotassium tert-
butylate to deliver 12, which contains the necessary
phenolic function for the methylation. Introduction of
the methyl can be performed either with dimethylsulfate
or with methyliodide as alkylatingagents. The latter is
more suitable for the radiosynthesis since [3H]-methyl-
iodide is commercially available.
The in vitro bindingcharacteristics of [ 3H]-M-MPEP
were studied in membranes of recombinant human
mGluR5 expressingL(tk À) cells (150–200 mgtissue/
protein) and usingthe filtration technique to separate the
bound from the free ligand. Time course association/
dissociation studies were determined and showed that
bindingwas >90% of total bindingat a concentration
of 2 nM of [3H]-M-MPEP, fully reversible and satur-
able. Quantification showed a Bmax 879Æ32 fmol/mg
protein and a Kd=2.0Æ0.3 nM. Displacement studies

F. Gasparini et al. / Bioorg. Med. Chem. Lett. 12 (2002) 407–409
409
Scheme 1. Reagents and conditions: (a) Ac2O, 165 ꢀC, 3.5 h (53%); (b) Br2, CCl4, RT (dark), 1.5 h (80%); (c) KOH, KOtBu, crown ether 18C6,
THF, 50 ꢀC, 1.5 h (59%); (d) (cold) KOH, dimethyl sulfate, dioxane, 40 ꢀC, 7 h (78%); (d) (radioactive): [3H]-MeI (0.5 equivalent), K2CO3, DMF,
100 ꢀC/180 min, purification on reversed-phase chromatography. Radiochemical purity 98%, specific activity 86 Ci/mmol.
Displacement studies of [3H]-M-MPEP with the non-
labeled M-MPEP and two known antagonists MPEP
and SIB-1893 led to IC50 values of the same order of
magnitude as in the functional assay and the same rank
order of potency.
In summary, the characterization, in vitro, of [3H]-M-
MPEP showed that this new radioligand is a very useful
tool to label the mGlu5 receptor subtype.
References and Notes
1. Conn, P. J.; Pin, J. P. Annu. Rev. Pharmacol. Toxicol. 1997,
37, 205.
2. Takahishi, K.; Tsuchida, K.; Tanabe, Y.; Masayuki, M.;
Figure 4. Displacement of [3H]-M-MPEP by M-MPEP (*), MPEP
(^) and SIB-1893 (Á). Values are presented as percent of specific
Nakanishi, S. J. Biol. Chem. 1993, 268, 19341.
3. O’Hara, L. J.; Sheppard, P. O.; Thogersen, H.; Venezia, D.;
bindingin the absence of compound tested and represent mean ÆSEM
Haldema, B. A.; McGrane, V.; Houamed, K. M.; Thomsen,
of N=3 independent experiments performed in triplicate.
C.; Gilbert, T. L.; Mulvihill, E. R. Neuron 1993, 11, 41.
4. Pin, J. P.; De Colle, C.; Bessis, A. S.; Acher, F. Eur. J.
Pharmacol. 1999, 375, 277.
using[ 3H]-M-MPEP and membranes of hmGluR5
expressingcells showed the followingrank order of
5. Schoepp, D. D.; Jane, D. E.; Monn, J. A. Neuropharma-
cology 1999, 38, 1431.
6. Schaffhauser, H.; Grayson Richards, J.; Cartmell, J.; Cha-
affinity: M-MPEP>MPEP>> SIB-1893 (Fig. 4), which
is in full accordance with the rank order of potency
determined usingfunctional assays such as the PI
hydrolysis (Table 1).11,12
boz, S.; Kemp, J. A.; Klingelschmidt, A.; Messer, J.; Stadler,
H.; Woltering, T.; Mutel, V. Mol. Pharmacol. 1998, 53, 228.
7. Johnson, B. G.; Wright, R. A.; Arnold, M. V.; Wheeler,
W. J.; Ornstein, P. L.; Schoepp, D. D. Neuropharmacology
1999, 38, 1519.
8. Ornstein, P. L.; Arnold, M. B.; Bleisch, T. J.; Wright, R. A.;
Wheeler, W. J.; Schoepp, D. D. Bioorg. Med. Chem. Lett.
1998, 8, 1919.
9. Cartmell, J.; Adam, G.; Chaboz, S.; Henningsen, R.;
Kemp, J. A.; Klingelschmidt, A.; Metzler, V.; Monsma, F.;
Schaffauser, H.; Wichmann, J.; Mutel, V. Br. J. Pharmacol.
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10. Mutel, V.; Ellis, G. J.; Adam, G.; Chaboz, S.; Nilly, A.;
Messer, J.; Bleuel, Z.; Metzler, V.; Malherbe, P.; Schlaeger,
H.-J.; Roughley, B. S.; Faull, R. L. M.; Grayson Richards, J.
J. Neurochem. 2000, 75, 2590.
11. Varney, M. A.; Cosford, N. D. P.; Jachec, C.; Rao, S. P.;
Sacaan, A.; Lin, F.-F.; Bleicher, L.; Santori, E. M.; Flor, P. J.;
Allgeier, H.; Gasparini, F.; Kuhn, R.; Hess, S. D.; Velicelebi,
G.; Johnson, E. C. J. Pharmacol. Exp. Ther. 1999, 290, 170.
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Discussion
Our results show that the introduction of a methoxy
moiety in the 3-position of the phenyl ringof MPEP
allowed the identification of a more potent derivative
with the same functional efficacy and subtype selectivity
for the metabotropic glutamate receptor 5. Further-
more, this new derivative was radiolabeled at the methoxy
position usinga phenolic precursor ( 12) and tritiated
methyliodide. The labelingcan be achieved at the last step
of the synthesis and leads to a radioligand with high
specific activity and radiochemical purity.
The characterization of this novel radioligand using
membranes of cells recombinantly expressingthe human
mGlu5 receptor showed that the ligand has a high
affinity (Kd=2 nM) and a high percentage of specific
binding >90% over the 1–10 nM concentration range.
Hess, S. D.; Rao, S. P.; Sacaan, A. I.; Santori, E. M.; Velic
lebi, G.; Kuhn, R. Neuropharmacology 1999, 38, 1493.
¸ e-