Potent mGluR5 antagonists: Pyridyl and thiazolyl-ethynyl-3,5-disubstituted- phenyl series

Article abstract of DOI:10.1016/j.bmcl.2011.04.047

We report the synthesis of four series of 3,5-disubstituted-phenyl ligands targeting the metabotropic glutamate receptor subtype 5: (2-methylthiazol-4-yl) ethynyl (1a-j,), (6-methylpyridin-2-yl)ethynyl (2a-j), (5-methylpyridin-2-yl) ethynyl (3a-j,), and (pyridin-2-yl)ethynyl (4a-j,). The compounds were evaluated for antagonism of glutamate-mediated mobilization of internal calcium in an mGluR5 in vitro assay. All compounds were found to be full antagonists and exhibited low nanomolar to subnanomolar activity.

Full text of DOI:10.1016/j.bmcl.2011.04.047

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3243–3247
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent mGluR5 antagonists: Pyridyl
and thiazolyl-ethynyl-3,5-disubstituted-phenyl series
David Alagille a, , Herve DaCosta a, Yelin Chen b, Kamondanai Hemstapat b, Alice Rodriguez b,
⇑
Ronald M. Baldwin c, Jeffrey P. Conn b, Gilles D. Tamagnan a
a Institute for Neurodegenerative Disorders, 60 Temple St., Suite 8A, New Haven, CT 06510, USA
b Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN, USA
c Department of Psychiatry, Yale School of Medicine , New Haven, CT, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis of four series of 3,5-disubstituted-phenyl ligands targeting the metabotropic glu-
tamate receptor subtype 5: (2-methylthiazol-4-yl)ethynyl (1a–j,), (6-methylpyridin-2-yl)ethynyl (2a–j),
(5-methylpyridin-2-yl)ethynyl (3a–j,), and (pyridin-2-yl)ethynyl (4a–j,). The compounds were evaluated
for antagonism of glutamate-mediated mobilization of internal calcium in an mGluR5 in vitro assay. All
compounds were found to be full antagonists and exhibited low nanomolar to subnanomolar activity.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 15 February 2011
Revised 11 April 2011
Accepted 12 April 2011
Available online 20 April 2011
Keywords:
Glutamate
mGluR5
MPEP
Antagonist
It is well established that glutamate is the main excitatory neu-
rotransmitter in the central nervous system (CNS) and acts through
two classes of receptors. Fast excitatory transmission at glutamate
synapses is mediated by the ligand-gated ionotropic receptors
disease (PD), the selective mGluR5 antagonist 2-methyl-6-(phenyl-
ethynyl)-pyridine8 (MPEP, Fig. 1) has been shown to elicit a neuro-
protective effect and attenuates both dyskinesia (L-DOPA-induced)
and akinesia (6-OHDA induced).9–11
(iGluRs), consisting of
propionic acid (AMPA), kainate (KA), and N-methyl-
a
-amino-3-hydroxy-5-methyl-4-isoxazole
-aspartate
The finding that antidepressant treatments change the mGluR5
receptor expression in rat hippocampus12 prompted investigations
to relate mGluR5 and affective disorders (anxiety and depression).
Both MPEP and 3-[(2-methyl-1,3-thiazol-4yl)-ethynyl]-pyridine
(MTEP, Fig. 1), a more selective and potent mGluR5 antagonist,
show antidepressant-like activity in mouse and rat forced swim
D
(NMDA) receptors. iGluRs are permeable to both potassium and so-
dium, and in the case of NMDA receptors and some AMPA receptors,
calcium ions.1,2 In contrast, metabotropic glutamate receptors
(mGluRs) mediate slower responses through G-proteins, coupled
to various transduction cascades.1 Group I mGluRs (mGluR1 and
5) are positively associated with phospholipase C via Gq/11 proteins,
leading to phosphoinositide hydrolysis and formation of two intra-
cellular second messengers: inositol triphosphate (IP3), which in-
duces intracellular Ca2+ release, and diacylglycerol (DAG), which
can activate protein kinase C. The mGluR subtypes belonging to
Group II (mGluR2 and 3) and group III (mGluR4, 6, 7 and 8) are neg-
atively coupled to adenylate cyclase via Gi/o proteins and will there-
fore decrease the intracellular concentration of cAMP.3,4 In addition,
glutamate can, through the mGluRs, modulate excitatory (AMPA
and NMDA) and inhibitory (GABA) signaling pathways as well as
other ion channel receptors.5–7 Modulation of mGluR5 through
the action of specific antagonists has potential for treatment of
various CNS related diseases. In rodent models of Parkinson’s
S
N
N
MTEP
MPEP
N
F
S
N
N
CN
CN
F-MTEB
F-PEB
F
4d
⇑
Corresponding author. Tel.: +1 203 401 4310; fax: +1 203 401 4301.
E-mail addresses: dalagille@indd.org, dalagille@mnimaging.com (D. Alagille).
Figure 1. Selected mGluR5 antagonists described in the literature.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.047

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D. Alagille et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3243–3247
test, with an interesting synergistic effect with the tricyclic antide-
pressant imiprimine.13,14
Both peripheral and central mGluR5 receptors are implicated in
pain transmission15 and MPEP was recently shown to dose-depen-
dently attenuate pain in a rat post-operative model of pain (MPEP
ED50 = 15 mg/kg ip; morphine ED50 = 1.3 mg/kg sc).16
from 21 nM in the hypothalamus to 83 nM in caudate/putamen27
(fmol/mg tissue = nM, assuming a specific gravity of 1 for the tissue
and homogeneous distribution in the region of interest). It is gen-
erally accepted that a ratio Bmax/Kd >>1 is required to obtain a good
imaging agent, which leads in the case of mGluR5 to compounds
with an affinity of at least 2–5 nM. Low non specific binding is also
a criterion for good radiotracer and is often associated with low
lipophilicity; as a general rule, a value of Log P<3 usually leads to
low non specific binding (higher values are acceptable for tracers
with high Bmax/Kd ratio). Log P values <1 are usually too polar to
penetrate the blood–brain-barrier and therefore render the tracer
useless for brain imaging. Similarly, compounds that are substrates
of the brain p-glycoprotein efflux pump (P-gp) will not achieve suf-
ficient brain levels for imaging.
The same synthetic methodology was used in the preparation of
the four different structural series and is exemplified in Scheme 1.
The pyridylethynyltrimethylsilyl derivatives 6–8 were prepared
by Sonogashira coupling reaction between the desired bromopyri-
dine and trimethylsilylacetylene using bis(triphenylphosphine)pal-
ladium(II) chloride as catalyst and triethylamine as both base and
solvent. The thiazole analog 5 was prepared from chloroacetyl chlo-
ride as previously reported.28 The 3,5-disubstituted phenyl bromide
or iodide derivatives 9a–j were either commercially available (9i
and j) or were synthesized using two different approaches (Scheme
2). The first one involves the introduction of a cyano group by dehy-
dration of the corresponding amide with thionyl chloride, in which
case the starting materials were the commercially available benzoic
acids (9a,d,e,f). The second approach involves the introduction of
one or two halogens using the ortho-directing effect of commercial
aniline, followed by deamination via the diazonium salt (9b,c,g,h).
The target compounds 1–4 were obtained using a one-pot modified
Sonogashira cross-coupling reaction, in which the trimethylsilyl-
The absence of reinforcing and locomotor stimulant effects of
cocaine in mGluR5 null mutant mice was pivotal to link mGluR5
and addiction.17 Since then, MPEP has been shown to attenuate co-
caine self-administration, cocaine-induced reinstatement of drug
seeking, and the discriminative stimulus effects of cocaine at doses
that did not markedly impair motor function or operant behav-
ior.18 In the same manner, MPEP seems to decrease the reinforcing
effects of ethanol in self-administrated mice19 and rats20, but
divergent results have been obtained in models of nicotine and
morphine addiction.21–23
These reports highlight the need for potent mGluR5 antagonists
as therapeutic drugs and as radiotracers for clinical imaging stud-
ies. Among the radiotracers available to image mGluR5 receptors
in vivo, the most studied is [18F]F-PEB ([18F]4d), which has shown
excellent results in non human primates;24,25 however, this tracer
suffers from the relatively short half-life of 18F (110 min), limiting
its use to centers near a cyclotron facility. Other radiohalogens,
such as 123I (T1/2 13.2 h), 76Br (T1/2 16.2 h) and 77Br (T1/2 55.9 h),
have longer physical half lives, but because of limitations in avail-
ability and matching of radiation properties to imaging equipment,
radioiodine is favored over radiobromine.26 In our efforts to devel-
op new iodinated radiotracers that can be used with the 123I, we
explored the structure–activity relationship between four groups
of pyridyl and thiazolyl-ethynyl-3,5-disubstituted-phenyl deriva-
tives and their mGluR5 antagonist properties. Ex vivo saturation
experiments in rat reveal a density of mGluR5 receptors ranging
O
Cl
Cl
N
Br
N
Br
N
Br
c
a
a
a
S
N
N
N
N
SiMe3
SiMe3
b
SiMe3
X
SiMe3
5
6
7
8
Br or I
9a-j
Y
Ar
X
Y
S
Ar =
N
N
N
N
1a b c,d e,f g h i j 2a b c d f
3a b c d e
, , , ,
4a,b,c,d,f,g,h
, ,
,
, , , ,
, , , ,
a
b
c
d
e
f
g
h
i
j
X
Y
CN NO2 CN CN CN CN NO2 NO2 Br Br
NO2 Br
Cl
F
Br
I
F
I
F
Br
Scheme 1. Synthesis of compounds 1, 2, 3, and 4. Reagents and conditions: (a) trimethylsilylacetylene, PdCl2(PPh3)2, Et3N; (b) 9a–j, PdCl2(PPh3)2, Et3N, TBAF, DMF, 60 °C or rt;
(c) see Ref. 20.

D. Alagille et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3243–3247
3245
A
A
A
CO2H
CONH2
CN
a
b
9a A=Br Y=NO2
9d A=Br Y=F
9e A=Y=Br
Y
Y
Y
A=Y=I
9f
NH2
NH2
X
Br
X
X
Br
X
X
c
d
d
9c
X=CN Y=Cl
9g X=NO2 Y=F
Y
Y
Y
NH2
NH2
X
X
9b
9h
c
X=Br
X=I
NO2
NO2
NO2
Scheme 2. Synthesis of compounds 9a–h. Reagents and conditions: (a) (i) SOCl2, reflux, (ii) NH4OH; (b) SOCl2, reflux; (c) Br2, AcOH, 50 °C; (d) isoamyl nitrite, DMF, 50 °C.
Table 1
Inhibition of mGluR5 glutamate-mediated calcium mobilizationa, c Log Pb, and Log Dc
a (CN/NO2)
b (NO2/Br)
c (CN/Cl)
d (CN/F)
e (CN/Br)
1
IC50
c Log P
2
2.63 0.26
3.47
0.73 0.22
4.85
1.61 0.49
4.40
1.89 0.8
4.00
1.06 0.36
4.67
IC50
c Log P
3
3.68 1.66
3.18
0.63 0.25
4.57
1.22 0.44
4.36
1.22 0.14
3.96
IC50
c Log P
4
2.64 0.38
3.14
7.36 4.17
4.52
1.22 0.22
4.14
0.32 0.01
3.74
1.02 0.61
4.41
IC50
c Log P
1.32 0.03
2.72
0.39 0.07
4.10
1.03 0.88
3.65
0.66 0.22
3.25
f (CN/I)
g (NO2/F)
h (NO2/I)
i (Br/F)
j (Br/Br)
1
IC50
c Log P
2
1.05 0.24
5.20
0.61 0.22
4.10
1.75 0.33
5.26
1.11 0.19
4.80
1.45 0.32
5.47
IC50
c Log P
Log Dc
4
0.71 0.16
5.16
2.30
IC50
c Log P
0.66 0.31
4.45
0.66 0.32
3.35
2.09 0.22
4.51
Literature values
MPEP
MTEP
F-PEB
F-MTEB
IC50
c Log P
2.024
3.06
5.024
2.47
0.66 0.228
3.25
1.45 0.528
4.00
a
b
c
IC50 values are expressed as mean S.E.M (nM) of at least three independent experiments. In this assay MPEP IC50 = 2.93 nM.
c Log P values are calculated using ChemDraw Ultra 10.0.
Log D is the partition coefficient of [123I]2f in octanol/phosphate buffer pH 7.4.
acetylenes 5–8 were desilylated in situ with tetrabutylammonium
fluoride (TBAF) and underwent cross-coupling with the phenyl bro-
mide or iodide 9a–j. This reaction is usually carried out at 60 °C, but
in the case of the di- or tri-halogenophenyl derivatives (9b,h,i,j) the
reaction was performed at room temperature in order to limit for-
mation of bis coupling adduct.
5-methylpyridyl series 3, and pyridyl series 4. The choice of
3,5-substitution was dictated by our previous work showing
good antagonist activity for compounds monosubstituted at posi-
tion 3 bearing a nitro, halo or cyano group.29,30 Compounds were
evaluated for antagonism of glutamate-mediated mobilization of
intracellular calcium31 in cultured human embryonic kidney cells
(HEK-293) expressing rat mGluR5 receptors (Table 1). All com-
pounds had full antagonist properties at the mGluR5 receptor,
with good to excellent activity (3b = 7.3 nM to 3d = 0.32 nM),
indicating tolerance for the different 3,5-disubstitution patterns.
The combination of cyano and nitro (group a) as 3,5-disubsti-
tents seems to be less favorable, with values in all four series
averaging 2.5 nM, followed by iodo/nitro (group h) in series 1
and 4 averaging 1.9 nM, followed by cyano/chloro (group c)
The recently disclosed compounds F-MTEB and F-PEB24,25,28
(Fig. 1) represented a major step in the development of highly
potent and selective mGluR5 antagonists. We hypothesized that
the increase in activity of those compounds could be attributed
to the favorable presence of the 3,5-disubstitution on the phenyl
ring. In order to test this hypothesis, we synthesized disubsti-
tuted compounds in four series previously shown to give potent
mGluR5 antagonist: thiazolyl series 1, 6-methylpyridyl series 2,

3246
D. Alagille et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3243–3247
Table 2
Supplementary data
Competition binding affinity (Ki, nM) of compounds 1a–j using [3H]methoxy-PEPY.
Values are expressed as mean S.E.M. (nM) of at least three independent
experimentsa
Supplementary data (experimental details for the synthesis and
characterization of 7; 8; 9a–j; 1a,b,c,d,e,g,h,i,j; 2a,b,c,d,f; 3a,b,c,d,e
and 4a,b,c,d,f,g,h) associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2011.04.047.
1
1
a (CN/NO2) b (NO2/Br) c (CN/Cl)
0.36 0.01
f (CN/I)
d (CN/F)
e (CN/Br)
0.93 0.02 0.127 0.038 0.36 0.09
0.106 0.023
j (Br/Br)
g (NO2/F)
h (NO2/I)
i (Br/F)
2.14 0.82
0.30 0.02 1.84 0.47
1.06 0.90
0.69 0.28
References and notes
Literature
values
MPEP
1224
MTEP
1624
F-PEB
F-MTEB
1. Kew, J. N. C.; Kemp, J. A. Psychopharmacology 2005, 179, 4.
2. Oswald, R. E.; Ahmed, A.; Fenwick, M. K.; Loh, A. P. Curr. Drug Targets 2007, 8,
573.
Ki
0.20 0.0128 0.08 0.0228
a
3. Pin, J. P.; Duvoisin, R. Neuropharmacology 1995, 34, 1.
4. Conn, P. J.; Pin, J. P. Annu. Rev. Pharmacol. Toxicol. 1997, 37, 205.
5. Chen, N.; Luo, T.; Raymond, L. A. J. Neurosci. 1999, 19, 6844.
6. Snyder, E. M.; Philpot, B. D.; Huber, K. M.; Dong, X.; Fallon, J. R.; Bear, M. F. Nat.
Neurosci. 2001, 4, 1079.
7. Yu, S. P.; Yeh, C. H.; Sensi, S. L.; Gwag, B. J.; Canzoniero, L. M.; Farhangrazi, Z. S.;
Ying, H. S.; Tian, M.; Dugan, L. L.; Choi, D. W. Science 1997, 273, 114.
8. Gasparini, F.; Lingenhohl, K.; Stoehr, N.; Flor, P. J.; Heinrich, M.; Vranesic, I.;
Biollaz, M.; Allgeier, H.; Heckendorn, R.; Urwyler, S.; Varney, M. A.; Johnson, E.
C.; Hess, S. D.; Rao, S. P.; Sacaan, A. I.; Santori, E. M.; Velicelebi, G.; Kuhn, R.
Neuropharmacology 1999, 38, 1493.
9. Aguirre, J. A.; Kehr, J.; Yoshitake, T.; Liu, F. L.; Rivera, A.; Fernandez-Espinola, S.;
Andbjer, B.; Leo, G.; Medhurst, A. D.; Agnati, L. F.; Fuxe, K. Brain Res. 2005, 1033,
216.
10. Breysse, N.; Amalric, M.; Salin, P. J. Neurosci. 2003, 23, 8302.
11. Mela, F.; Marti, M.; Dekundy, A.; Danysz, W.; Morari, M.; Cenci, M. A. J.
Neurochem. 2007, 101, 484.
In this assay MPEP Ki = 18.7 nM.
averaging 1.27 nM in all four series. No simple trend appeared in
the four series; the average activities gave the following group
ranking:
4 (0.97 nM) > 1 (1.42 nM) P 2 (1.49 nM) > 3
(2.51 nM). However the classification of group 4 the best and 3
the worst is not totally satisfying, since series 3 contained both
the most potent antagonist (3d, IC50 = 0.32 nM) and the least po-
tent (3b IC50 = 7.36 nM). Comparison of groups b versus e (NO2/
Br vs CN/Br), g versus d (NO2/F vs CN/F) as well as h versus f
(NO2/I vs CN/I) indicate the feasibility of substitution of the cy-
ano group by a nitro, leading to compounds with generally bet-
ter or equal activity (except 4h and 3b). Compounds of series 1
were further evaluated in a competition binding assay32 with
[3H]methoxy-PEPY on rat mGluR5-HEK293A cells membrane (Ta-
ble 2). All tested compounds had binding affinity in the low
nanomolar to subnanomolar range. The best compounds in this
series bore a cyano group (1c Ki = 0.12 nM and 1e Ki = 0.10 nM)
but subnanomolar binding affinity was also observed without
the presence of the cyano group, as exemplified by compounds
1b, 1g, and 1j (Ki = 0.3–0.9 nM); all those compounds have suffi-
cient affinity for potential application as mGluR5 brain imaging
agents (defined as Bmax/Kd >>1). Substitution with an iodo group
led in both cases (CN/I 1f, NO2/I 1h) to compounds with the
lowest binding affinity. The calculated lipophilicity of those com-
pound is relatively high, ranging from c Log P = 2.72 (4a) to 5.47
(1j), however those values are only estimates of the lipophilicity;
the experimental value of compound 2a Log D = 2.30 is well be-
low its calculated value c Log P = 5.16 and we therefore estimate
that most of these compounds exhibit a lipophilicity appropriate
for brain imaging application. None of the compounds have been
evaluated as P-gp substrate but their structural resemblance to
MPEP, MTEP, F-PEB, and F-MTEB (which are not P-gp substrates)
is a good indicator of low chances of being a P-gp substrate for
the new compounds.
In summary, we synthesized 3,5-disubstituted phenylethynyl
compounds in four series. All compounds are potent mGluR5 full
antagonists. We demonstrated the apparent equivalency between
the cyano and nitro group as one of the 3,5-substituents. Specific
trends are difficult to draw since each series seems to lead to dif-
ferent favorable 3,5-substitutents profile (compounds 1g, 2b, 3d,
and 4b were the best ligands in each series). The high-affinity com-
pound 1e might find application for imaging with 77Br or 76Br, but
this would not be as widely applicable as a radioiodinated ligand.
Among the compounds bearing an iodine 1h, 1f, 2f, 4f have prom-
ising in vitro potency, but the preliminary binding results (1f, 1h)
shows a relatively low affinity which might impair their use as
SPECT imaging agents.
12. Wieronska, J. M.; Branski, P.; Szewczyk, B.; Palucha, A.; Papp, M.; Gruca, P.;
Moryl, E.; Pilc, A. Pol. J. Pharmacol. 2001, 53, 659.
13. Li, X.; Need, A. B.; Baez, M.; Witkin, J. M. J. Pharmacol. Exp. Ther. 2006, 319, 254.
14. Belozertseva, I. V.; Kos, T.; Popik, P.; Danysz, W.; Bespalov, A. Y. Eur.
Neuropsychopharmacol. 2007, 17, 172.
15. Neugebauer, V. Pain 2002, 98, 1.
16. Zhu, C. Z.; Hsieh, G.; El-Kouhen, O.; Wilson, S. G.; Mikusa, J. P.; Hollingsworth, P.
R.; Chang, R.; Moreland, R. B.; Brioni, J. D.; Decker, M. W.; Honore, P. Pain 2005,
114, 195.
17. Chiamulera, C.; Epping-Jordan, M. P.; Zocchi, A.; Marcon, C.; Cottiny, C.;
Tacconi, S.; Corsi, M.; Orzi, F.; Conquet, F. Nat. Neurosci. 2001, 4, 873.
18. Lee, B.; Platt, D. M.; Rowlett, J. K.; Adewale, A. S.; Spealman, R. D. J. Pharmacol.
Exp. Ther. 2005, 312, 1232.
19. Hodge, C. W.; Miles, M. F.; Sharko, A. C.; Stevenson, R. A.; Hillmann, J. R.;
Lepoutre, V.; Besheer, J.; Schroeder, J. P. Psychopharmacology 2006, 183, 429.
20. Besheer, J.; Stevenson, R. A.; Hodge, C. W. Eur. J. Pharmacol. 2006, 551, 71.
21. McGeehan, A. J.; Olive, M. F. Synapse 2003, 47, 240.
22. Paterson, N. E.; Semenova, S.; Gasparini, F.; Markou, A. Psychopharmacology
2003, 167, 257.
23. Popik, P.; Wrobel, M. Neuropharmacology 2002, 43, 1210.
24. Hamill, T. G.; Krause, S.; Ryan, C.; Bonnefous, C.; Govek, S.; Seiders, T. J.;
Cosford, N. D. P.; Roppe, J.; Kamenecka, T.; Patel, S.; Gibson, R. E.; Sanabria, S.;
Riffel, K.; Eng, W.; King, C.; Yang, X.; Green, M. D.; O’Malley, S. S.; Hargreaves,
R.; Burns, H. D. Synapse 2005, 56, 205.
25. Wang, J.-Q.; Tueckmantel, W.; Zhu, A.; Pellegrino, D.; Brownell, A. L. Synapse
2007, 61, 951.
26. Adam, M. J.; Wilbur, D. S. Chem. Soc. Rev. 2005, 34, 153.
27. Patel, S.; Krause, S. M.; Hamill, T.; Chaudharyb, A.; Burns, H. D.; Gibson, R. E. Life
Sci. 2003, 27, 371.
28. Cosford, N. D. P.; Tehrani, L.; Roppe, J.; Schweiger, E.; Smith, N. D.; Anderson, J.;
Bristow, L.; Brodkin, J.; Jiang, X.; McDonald, I.; Rao, S.; Washburn, M.; Varney,
M. A. J. Med. Chem. 2003, 46, 204.
29. Alagille, D.; Baldwin, R. M.; Roth, B. L.; Wroblewski, J. T.; Grajkowska, E.;
Tamagnan, G. D. Bioorg. Med. Chem. Lett. 2005, 15, 945.
30. Alagille, D.; Baldwin, R. M.; Roth, B. L.; Wroblewski, J. T.; Grajkowska, E.;
Tamagnan, G. D. Bioorg. Med. Chem. 2005, 13, 197.
31. Rat mGluR5-HEK293A cells were loaded with calcium-sensitive dye according
to the manufacturer’s instructions (Calcium
3 kit; Molecular Devices,
Sunnyvale, CA) after incubation in glutamate/glutamine-free Dulbecco’s
Modified Eagle Medium (DMEM; Invitrogen) containing 10% dialyzed fetal
bovine serum for 5 h. Compound A (1 ml) from Calcium 3 kit was dissolved in
20 ml of 1Â Hanks’ balanced salt solution (HBSS; Invitrogen) containing
2.5 mM probenecid (Sigma), adjusted to pH 7.4. Cells were loaded for 50 min at
37 °C with 5% carbon dioxide. Dye was then carefully removed and cells were
washed with HBSS containing probenecid. Cells were maintained in the same
buffer at room temperature for the following assay. Test compound was added
5 min before the manual addition of glutamate. Glutamate was added at a
speed of 52
(Molecular Devices) at 25 °C. All of the peaks of the calcium response were
normalized to the maximum response to saturated dose of glutamate
(10 M). 1 M glutamate was used to generate EC80 response, allowing for a
lL/s and calcium flux was measured using a Flexstation II
a
Acknowledgment
l
l
response varying from 70% to 90% of the maximum. EC50 values were
calculated from nonlinear curve fitting using GraphPad Prism v. 4.01 and of
This work was supported by a grant from the National Institutes
of Health (DA16180) and a grant from NARSAD to G.D.T.

D. Alagille et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3243–3247
3247
three separate experiments, presented as the mean standard error of the
mean (SEM).
(Unifilter-96 GF/B; PerkinElmer Life and Analytical Sciences, Boston, MA) and
washed three times with binding buffer (Brandel Cell Harvester; Brandel Inc.,
32. mGluR5 binding assay was determined by measuring the displacement by test
compounds of [3H]methoxy-PEPY from rat mGluR5 receptor on membranes
prepared from stable rat mGluR5-HEK293A cells. 2 nM [3H]methoxyPEPY was
Gaithersburg, MD). Scintillation fluid (30 lL) was added to each well and the
membrane-bound radioactivity was measured by liquid scintillation counting
(TopCount; PerkinElmer Life and Analytical Sciences). Nonspecific binding was
incubated with membrane (10
lg/well) in the binding buffer (50 mM Tris/0.9%
estimated using 5 lM MPEP. The inhibition constant Ki was calculated from
NaCl, pH 7.4) with the presence or absence of test compound at room
temperature for 1 h with shaking. The membrane-bound ligand was separated
from free ligand by filtration through 96-well glass–fiber filter plates
IC50, which was obtained from nonlinear curve fitting by GraphPad Prism v.
4.01 from three separate experiments in duplicates.