3-[(2-Methyl-1,3-thiazol-4-yl)ethynyl]-pyridine: A potent and highly selective metabotropic glutamate subtype 5 receptor antagonist with anxiolytic activity

Article abstract of DOI:10.1021/jm025570j

2-Methyl-6-(phenylethynyl)pyridine (3), a potent noncompetitive mGlu5 receptor antagonist widely used to characterize the pharmacology of mGlu5 receptors, suffers from a number of shortcomings as a therapeutic agent, including off-target activity and poor

Full text of DOI:10.1021/jm025570j

204
J . Med. Chem. 2003, 46, 204-206
Sch em e 1a
3-[(2-Meth yl-1,3-th ia zol-4-yl)eth yn yl]-
p yr id in e: A P oten t a n d High ly Selective
Meta botr op ic Glu ta m a te Su btyp e 5
Recep tor An ta gon ist w ith An xiolytic
Activity
Nicholas D. P. Cosford,* Lida Tehrani, J effrey Roppe,
Edwin Schweiger, Nicholas D. Smith,
J effrey Anderson, Linda Bristow, J esse Brodkin,
Xiaohui J iang, Ian McDonald, Sara Rao,
Mark Washburn, and Mark A. Varney
a
Reagents and conditions: (a) for X ) H; phenylacetylene,
PdCl2(PPh3)2, CuI, NEt3, DME, 80 °C, 16 h, (60%); for X ) Br;
phenylacetylene, Pd(PPh3)4, CuI, NEt3, DME, 80 °C, 18 h, (86%);
(b) Me4Sn, Pd2(dba)3, P(tBu)3, CsF, 1,4-dioxane, 100 °C, 5 h, (17%).
Merck Research Laboratories, MRLSDB2, 3535,
General Atomics Court, San Diego, California 92121
Sch em e 2a
Received August 9, 2002
Abstr a ct: 2-Methyl-6-(phenylethynyl)pyridine (3), a potent
noncompetitive mGlu5 receptor antagonist widely used to
characterize the pharmacology of mGlu5 receptors, suffers
from
a number of shortcomings as a therapeutic agent,
a
Reagents and conditions: phenylacetylene, Pd(PPh3)4, CuI,
including off-target activity and poor aqueous solubility.
Seeking to improve the properties of 3 led to the synthesis of
compound 9, a highly selective mGlu5 receptor antagonist that
is 5-fold more potent than 3 in the rat fear-potentiated startle
model of anxiety.
NEt3, DME, 80 °C, 5 h, (77%).
Sch em e 3a
In tr od u ction . Glutamate is the principal excitatory
transmitter in the central nervous system acting through
ionotropic glutamate receptors; however, it also plays
a major role in activating modulatory pathways through
G-protein-coupled metabotropic glutamate (mGlu) re-
ceptors.1,2 Group I mGlu receptors include the mGlu1
and mGlu5 subtypes, which are coupled to stimulation
of phospholipase C resulting in phosphoinositide hy-
drolysis and elevation of intracellular Ca2+ levels
([Ca2+]i).3,4 Excessive activation of mGlu5 receptors has
been implicated in several diseases, and selective mGlu5
receptor antagonists may be of therapeutic benefit in
the treatment of various pain states,5 psychiatric dis-
orders such as anxiety and depression,6-11 and other
neurological impairments such as drug addiction and
drug withdrawal.12
a
Reagents and conditions: (a) BTMSA, AlCl3, CH2Cl2, 0 °C, 2
h and then 25 °C, 1 h, (54%); (b) thioacetamide, DMF, 25 °C, 16
h, (72%); (c) for Ar ) Ph; iodobenzene, PdCl2(PPh3)2, PPh3, CuI,
NEt3, TBAF, Bu4NI, DMF, 70 °C, 36 h, (73%); for Ar ) 2-py;
2-bromopyridine, PdCl2(PPh3)2, CuI, NEt3, TBAF, DMF, 70 °C, 30
min, (73%); for Ar ) 3-py; 3-bromopyridine, Pd (PPh3)4, CuI, NEt3,
TBAF, DME, 70 °C, 26 h, (65%); for Ar ) 4-py; 4-bromopyridine
hydrochloride, Pd(PPh3)4, CuI, NEt3, TBAF, DME, 70 °C, 32 h,
(75%).
The discovery in these laboratories of the first subtype
selective, noncompetitive mGlu5 receptor antagonists,
1 and 2,13 led to the subsequent identification of
2-methyl-6-(phenylethynyl)pyridine (MPEP, 3), a struc-
turally related compound with similar selectivity but
improved in vitro potency at mGlu5 receptors.14 These
F igu r e 1. [3H]-3-methoxy-5-(pyridin-2-ylethynyl)pyridine.
While compound 3 has proven to be a useful tool for
both in vitro and in vivo studies, it has a number of
drawbacks as a drug. First, compound 3 is not com-
pletely selective for mGlu5 receptors. For example, it
has been shown to block N-methyl-D-aspartate (NMDA)
receptors,16,17 albeit at high concentrations, and our own
studies suggest the presence of other off-target activities
of 3. Second, the compound is poorly water soluble, as
indicated by a log D value of 3.5, which would limit
solubility in CSF and very likely reduce in vivo efficacy.
pharmacological tools have led to a plethora of research
to understand the role of mGlu5 receptors in the brain
and nervous system and hence the viability of mGlu5
receptors as a molecular therapeutic target.15
* To whom correspondence should be addressed. Phone: 858-202-
5299. Fax: 858-202-5752. E-mail: nicholas_cosford@merck.com.
10.1021/jm025570j CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/12/2002

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 205
Ta ble 1. In Vitro Potency Data for mGlu5 Receptor
(pyridin-2-ylethynyl)pyridine (11, Figure 1) from rat
Antagonistsa
cortical membranes.21
The 1,3-thiazole ring is a classical isostere of pyridine,
and therefore, a logical first step was to prepare
analogues in which the pyridyl ring (A) in 3 was
replaced with a thiazole moiety. Replacement of the
6-methylpyridine moiety in 3 with 2- or 4-thiazolyl
units, as in 4 and 5, gave compounds with reduced in
vitro potency at the mGlu5 receptor (Table 1). By
analogy with 3, introduction of a methyl group adjacent
to the thiazole nitrogen in 4 and 5 gave compounds 6
and 7 with greatly enhanced in vitro potency at the
mGlu5 receptor. In particular, 2-methyl-4-(phenylethy-
nyl)-1,3-thiazole (7) inhibited mGlu5 recepor mediated
Ca2+ flux with IC50 ) 6 nM and Ki ) 6 nM in the binding
assay, which is in the same potency range as compound
3 in these assays. However, log D values (Table 1)
indicated that both 3 (log D ) 3.5) and 7 (log D ) 3.3)
are lipophilic molecules. It was hypothesized that
introduction of a second basic nitrogen into the molec-
ular framework of 7 would reduce the log D and increase
aqueous solubility. A nitrogen scan in the phenyl ring
(B) of compound 7 demonstrated that, while the 2-and
4-pyridyl isomers (8 and 10) were less potent than 7,
the 3-pyridyl isomer (9) was potent in vitro (IC50 ) 5
nM in the Ca2+ flux assay, Ki ) 16 nM). Furthermore,
3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP, 9)
has a log D value of 2.1 compared with 3.5 for 3 and
3.3 for 7 (Table 1). On the basis of these data, 9 was
selected for further in vitro and in vivo evaluation.
a
Data are presented as the geometric mean followed in paren-
theses by the standard deviation and the number of replicates.
b Ca2+ flux assay using glutamate as agonist.13,19,20 c Displacement
by test compounds of [3H]-3-methoxy-5-(pyridin-2-ylethynyl)pyri-
dine (11) bound to rat cortical membranes.21 See ref 18.
d
Ta ble 2. In Vitro Selectivity Data for mGlu5 Receptor
Antagonists 3 and 9
mGlu1a IC50 (µM)
NR2Bb IC50 (µM)
MAOAc IC50 (µM)
3
9
>100
>100
18
>300
8 (6, 13)
30 (17, 40)
In Vitr o P r ofiles of Com p ou n d s 3 a n d 9. To
identify any off-target activities, 3 and 9 were profiled
extensively against a battery of in vitro assays. Experi-
ments were performed initially at a drug concentration
of 10 µM. For assays in which inhibition was detected
at the single dose, concentration-response curves were
generated. Like 3, compound 9 is highly selective for
the mGlu5 receptor over the mGlu1 receptor (Table 2).
Additionally 9 has no effect when tested against other
mGlu receptor subtypes (e.g., mGlu1, mGlu2, mGlu7)
or against ionotropic glutamate receptors, including
AMPA and kainate subtypes (e.g., Glu1, Glu3, Glu5,
Glu6). It should be noted that 3 and 9 were not tested
against glutamate transporters or against enzymes for
which glutamate is a substrate. Since it had been
reported that 3 is an antagonist of NMDA NR2B-
containing receptors in vitro,16,17 3 and 9 were tested
for their ability to block NMDA/glycine-evoked increases
in intracellular calcium in a cell line stably expressing
recombinant human NMDA receptors. In this assay, 3
inhibited NR1A/2B receptors (IC50 ) 18 µM, Table 2)
while 9 produced only 19% inhibition at a concentration
of 300 µM. Further profiling revealed that 3 displaced
a
b
Ca2+ flux assay, tested as agonist and antagonist. Ca2+ flux
assay; data generated from a six-point CRC, n ) 8-22 cells/
concentration tested. c Data are presented as the geometric
mean IC50 followed in parentheses by the upper and lower SE (n
) 3).
We therefore sought mGlu5 receptor antagonists with
improved selectivity, a lower log D value, and good
efficacy in vivo. With this goal in mind, we set out to
explore the SAR of 3 in order to identify a compound
possessing all of these desirable properties.
Med icin a l Ch em istr y a n d SAR. The Sonogoshira
cross-coupling methodology employed for the synthesis
of the alkyne derivatives described herein is sum-
marized in Schemes 1-3 (yields are not optimized). Two
in vitro assays were employed in the initial research
phase in addition to the determination of log D for
compounds using an HPLC method.18 The functional
potency of compounds was assessed using an automated
assay employing Ltk cells stably expressing human
recombinant mGlu5 receptors. This cell-based assay
measures changes in cytosolic Ca2+ concentrations
([Ca2+]i) by fluorescence detection using the Ca2+
-
[
125I]methyl (1R,2S,3S,5S)-3-(4-iodophenyl)-8-methyl-8-
sensitive dye fura-2.13,19,20 Binding to native mGlu5
receptors in vitro was determined by measuring the
displacement by test compounds of [3H]-3-methoxy-5-
azabicyclo[3.2.1]octane-2-carboxylate ([125I]RTI-55) from
MAOA with IC50 ) 8 µM compared with IC50 ) 30 µM
Ta ble 3. In Vivo Data for mGlu5 Receptor Antagonists 3 and 9a
b,g
Occ ED50 b,c (mg/kg ip)
plasma levelsd,e (µM)
brain levelsd,e (µM)
CSF levelsd,f (µM)
FPS ED50
(mg/kg ip)
3
9
2.1 (1.1, 3.4)
1 (0.6, 1.2)
0.39 ( 0.02 (7)
1.2 ( 0.2 (6)
0.83 ( 0.05 (7)
1.4 ( 0.2 (6)
0.21 ( 0.3 (5)
1.0 ( 0.27 (5)
5 (3.7, 6.66)
1 (0.65, 2.0)
a
b
All in vivo measurements taken at 1 h after administration. Data presented as mean followed in parentheses by ED50 values from
individual experiments (95% confidence interval). c n ) 4-6 rats/group. Data presented as mean ( SEM (n), where n ) number of
d
rats/group. e Dose ) 3 mg/kg ip. f Dose ) 30 mg/kg po. n ) 8 rats/group.
g

206 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Letters
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for 9. Together, these results indicate that 9 exhibits
fewer off-target effects than 3 and thus greater specific-
ity for the mGlu5 receptor.
In Vivo P r ofiles of Com p ou n d s 3 a n d 9. To
evaluate the brain penetration of 3 and 9 and to
correlate affinity at the mGlu5 receptor with in vivo
efficacy, an in vivo receptor occupancy assay was
employed.22 Briefly, at time zero rats were dosed with
the test compound intraperitoneally and at 59 min. [3H]-
3-methoxy-5-(pyridin-2-ylethynyl)pyridine (11)21 was
administered via tail vein injection. One minute later,
the animals were sacrificed and brain binding was
measured. With this paradigm, dose-response relation-
ships were determined for binding to the mGlu5 recep-
tor in vivo and it was found that 9 is twice as potent as
3 in this assay (Table 3). In other experiments, plasma,
brain (hippocampus), and CSF levels for 3 and 9 were
measured following dosing in rats. Interestingly, while
both 3 and 9 exhibited similar drug levels in the
hippocampus (Table 3), the concentration of 9 in CSF
(1 µM) was approximately 5-fold higher than the
concentration of 3 (0.21 µM). This may be a consequence
of the lower log D value for 9 and therefore greater
aqueous solubility compared with 3 (Table 1).
(10) Klodzinska, A.; Tatarczynska, E.; Chojnacka-Wojcik, E.; Pilc, A.
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There is growing evidence of a role for mGlu5 recep-
tors in the modulation of mood disorders including
depression and anxiety.6-11 For example, 3 is reported
to be active in the rodent Geller-Seifter model,6,7
ultrasonic vocalization,6 elevated plus maze,7,9 social
exploration test,7 marble burying test,7 conflict drinking
test,9 and four-plate test.9 Compound 3 also reduces
stress-induced hyperthermia in mice.8 Recently 3 was
also shown to block fear-conditioning in rats as deter-
mined in the fear-potentiated startle (FPS) model of
anxiety.6,11 The FPS model was therefore selected to
assess the relative potencies of 3 and 9 in a rodent model
of anxiety. Compounds 3 and 9 were administered
intraperitoneally to rats, and both compounds were
found to block the expression of fear in this paradigm.
However, the ED50 for 9 was calculated to be 1 mg/kg
compared with 5 mg/kg for 3 (Table 3), indicating that
9 is 5-fold more potent than 3 in this animal model.
Con clu sion . Exploration of the SAR around 3 re-
sulted in the discovery of compound 9, a potent and
selective mGlu5 receptor antagonist with fewer off-
target effects than 3. Furthermore, 9 is more potent
than 3 in vivo (rats) in both a receptor occupancy assay
and in the fear-potentiated startle model of anxiety.
Further details of the SAR and pharmacological profile
of 9 and analogues will be reported in due course.
(11) Schulz, B.; Fendt, M.; Gasparini, F.; Lingenhohl, K.; Kuhn, R.;
et al. The metabotropic glutamate receptor antagonist 2-methyl-
6-(phenylethynyl)-pyridine (MPEP) blocks fear conditioning in
rats. Neuropharmacology 2001, 41, 1-7.
(12) Chiamulera, C.; Epping-J ordan, M. P.; Zocchi, A.; Marcon, C.;
Cottiny, C.; et al. Reinforcing and locomotor stimulant effects
of cocaine are absent in mGluR5 null mutant mice. Nat.
Neurosci. 2001, 4, 873-874.
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A.; et al. SIB-1757 and SIB-1893: selective, noncompetitive
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M.; et al. 2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent,
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mGluR5 antagonists MPEP and SIB-1893 decrease NMDA or
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(18) See Supporting Information.
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(21) Cosford, N. D. P.; Roppe, J .; Tehrani, L.; Seiders, T. J .; Sch-
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thiazol-4-yl)ethynyl]pyridine [3H]-methoxymethyl-MTEP) and
[3H]-3-methoxy-5-(pyridin-2-ylethynyl)pyridine [3H]-methoxy-
PEPy): Potent and selective radioligands for the metabotropic
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in press.
Ack n ow led gm en t. The authors thank Bill Bray,
J anice Chung, Darlene Giracello, Greg Holtz, and
Rodger Pasieczny for expert technical assistance.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails for the preparation of compounds 4-10 and log D
determination using HPLC. This material is available free of
charge via the Internet at http://pubs.acs.org.
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J M025570J