Discovery of molecular switches that modulate modes of metabotropic glutamate receptor subtype 5 (mGlu5) pharmacology in vitro and in vivo within a series of functionalized, regioisomeric 2- and 5-(phenylethynyl) pyrimidines

Article abstract of DOI:10.1021/jm900654c

We describe the synthesis and SAR of a series of analogues of the mGlu5 partial antagonist 5-(phenylethynyl)pyrimidine. New molecular switches are identified that modulate the pharmacological activity of the lead compound. Slight structural modifications

Full text of DOI:10.1021/jm900654c

J. Med. Chem. 2009, 52, 4103–4106 4103
DOI: 10.1021/jm900654c
oped based on 7, by the incorporation of a basic heterocycle in
the 3-position of the oxadiazole.14 On the basis of our
experience in the development of allosteric modulators of
mGluRs with a broad range of activities including negative
allosteric modulators, positive allosteric modulators and neu-
tral allosteric site ligands at the allosteric binding site occupied
by 1, together with theoretical models of allosteric function,
we postulated that it might be possible to develop “partial
antagonists”. As envisioned, a “partial antagonist” would
fully occupy the binding site of 1 on the mGlu5 receptor but
only partially block agonist response, resulting in partial
mGlu5 inhibition; moreover, Rodriguez et al. identified sev-
eral mGlu5 partial antagonists.15 In 2008, Sharma et al.
conducted a limited optimization effort focused on the mGlu5
partial antagonist lead 8. Within two 24-member libraries,
SAR elucidated a “molecular switch” to modulate pharma-
cological activity (Figure 1).16 Lead 8, with an unsubstituted
distal phenyl ring, fully occupied the allosteric binding site
of 1, possessed an IC50 of 486 nM, but only afforded partial
response (29% response, 71% partial antagonism), that is,
allosteric partial antagonism. Incorporation of small chemical
moieties in the 3-position of the distal phenyl ring, such as a
3-methyl group, delivered 9, a full noncompetitive mGlu5
antagonist (IC50 = 7.5 nM). When the methyl group was
moved from the 3-position to the 4-position as in 10, an
efficacious (99% of glutamate max) mGlu5 PAM resulted
(EC50=3.3 μM, 4.2-fold shift), which also represented a new
mGlu5 PAM chemotype.16 The observation of a conserved
molecular switch, accessed by toggling between 3- and
4-substitution on the distal phenyl ring, within this chemical
series was unprecedented. These preliminary data encouraged
us to further optimize 8 and survey the impact of incorporat-
ing incorporating substituents on the pyrimidine ring, as well
as examining regioisomeric pyrimidines to develop potent and
Discovery of Molecular Switches That Modulate
Modes of Metabotropic Glutamate Receptor
Subtype 5 (mGlu5) Pharmacology in Vitro and in
Vivo within a Series of Functionalized,
Regioisomeric 2- and 5-(Phenylethynyl)
pyrimidines
Sameer Sharma,† Jeffrey Kedrowski,‡ Jerri M. Rook,‡
Randy L. Smith,‡ Carrie K. Jones,‡,§ Alice L. Rodriguez,‡,§
P. Jeffrey Conn,‡,§ and Craig W. Lindsley*,†,‡,§
†Department of Chemistry, ‡Department of Pharmacology, and
§Vanderbilt Program in Drug Discovery, Vanderbilt Institute of
Chemical Biology, Vanderbilt University Medical Center, Vanderbilt
University, Nashville, Tennessee 37232
Received May 18, 2009
Abstract: We describe the synthesis and SAR of a series of analo-
gues of the mGlu5 partial antagonist 5-(phenylethynyl)pyrimidine.
New molecular switches are identified that modulate the pharma-
cological activity of the lead compound. Slight structural modifica-
tions around the proximal pyrimidine ring change activity of the
partial antagonist lead to that of potent and selective full negative
allosteric modulators and positive allosteric modulators, which
demonstrate in vivo efficacy in rodent models for anxiolytic and
antipsychotic activity, respectively.
Glutamate is the major excitatory neurotransmitter in the
mammalian central nervous system and exerts its effects
through both ionotropic and metabotropic glutamate recep-
tors. The metabotropic glutamate receptors (mGluRsa) are
members of the G-protein-coupled recpetor (GPCR) family
C, which are characterized by a large extracellular amino-
terminal agonist-binding domain. To date, eight mGluRs
have been cloned, sequenced, and assigned to three groups
(group I, mGlu1 and mGlu5; group II, mGlu2 and mGlu3;
group III, mGlu4,6,7,8) based on their sequence homology,
pharmacology, and coupling to effector mechanisms.1,2 In
preclinical models, studies with the negative allosteric mod-
ulators (NAMs) 1 (MPEP) and 2 (MTEP) (Chart 1) have
demonstrated that selective antagonism of mGlu5 has ther-
apeutic potential for chronic disorders such as pain, anxiety,
depression, addiction, and fragile X syndrome.3-7 Further-
more, there is direct clinical validation of anxiolytic activity by
allosteric antagonism of mGlu5 in patients with fenobam 3.8
Alternatively receptor activity can be enhanced through
positive allosteric modulators (PAMs) such as 4 (DFB), 5
(CPPHA), 6 (CDPPB), and 7 (ADX-47273), which with the
exception of 5 share the same allosteric binding site as 1.9-13
PAMs 6 and 7, both ago-potentiators, have demonstrated
in vivo proof of concept in preclinical schizophrenia models
in which other known antipsychotics show similar positive
effects.10-13 Recently, pure mGlu5 PAMs have been devel-
Chart 1. mGlu5 Allosteric Ligands
*To whom correspondence should be addressed. Phone: 615-322-
8700. Fax: 615-322-8577. E-mail: craig.lindsley@vanderbilt.edu.
a Abbreviations: mGluR, metabotropic glutamate receptor; NAM,
negative allosteric modulator; PAM, positive allosteric modulator;
GPCR, G-protein-coupled receptor.
r
2009 American Chemical Society
Published on Web 06/19/2009
pubs.acs.org/jmc

4104 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Sharma et al.
selective mGlu5 NAMs and PAMs suitable for in vivo studies
to confirm the observed in vitro pharmacology.
microwave-assisted Sonogashira conditions (Scheme 1) to
provide analogues 16. In parallel, we prepared a small three-
member library employing the regiosiomeric 2-bromopyrimi-
dine 12 and 13-15 to deliver analogues 17.
For the next round of chemical lead optimization, we relied
on an iterative analogue library synthesis approach17 to
rapidly prepare a 24-member library18 in which 2-substi-
tuted-5-bromopyrimidines 11 were treated with phenylacety-
lene 13, 3-methylphenyl acetylene (the NAM “switch”) 14,
or 4-methylphenyl acetylene (the PAM “switch”) 15 under
SAR from this library was “flat”, with few actives (Table 1);
however, unexpected modulation of the mode of mGlu5
pharmacologywas observed. All new analogues 16 containing
the 4-methylphenyl moiety were uniformly inactive, save for
16f, a weak mGlu5 PAM. When R1 was an ethoxy group in
combination with the NAM “switch”, 3-methylphenyl, 16a
resulted, a potent mGlu5 NAM (IC50 = 21 nM). The remain-
ing analogues 16 were inactive or, more surprisingly, potent
mGlu5 PAMs. When an aminomethyl group was incorpo-
rated at the 2-position of the pyrimidine, in conjunction with
an unsubstituted phenyl ring, 16b resulted, which represents
the most potent (EC50 =14.3 nM, 15-fold shift) rat mGlu5
PAM reported to date (10- to 15-fold more potent than 6
and 7). Addition of the NAM “switch” 3-methylphenyl
moiety with the 2-aminomethyl group 16c unexpectedly
afforded a similarly potent mGlu5 PAM (EC50 =21.1 nM,
5.9-fold shift), suggesting the 3-methylphenyl moiety is not a
conserved molecular switch for engendering NAM activity.
Interestingly, the NAM 16a differs from the PAM 16c by
substitution at the 2-position of the pyrimidine, OEt versus
NHMe, respectively, with equal potency (IC50=21 nM and
EC50=21 nM, respectively) but opposite mode of pharmacol-
ogy. Other groups were tolerated in the 2-position of the
pyrimidine such as SMe 16d and t-Bu 16e and found to
engender mGlu5 PAM activity (EC50 = 120 nM, 11-fold shift
and EC50 = 247 nM, 6-fold shift, respectively) but were
inactive in the presence of the 3- or 4-Me-phenyl moieties.
Overall, 16b represents a 235-fold improvement in potency
over mGlu5 PAM 10, was selective for mGlu5 (>10 μM vs
mGlu1-4,7,8), and warranted further evaluation.
Figure 1. Identification of “molecular switches” that convert an
mGlu5 partial antagonist 8 to a full noncompetitive antagonist
(NAM) 9 or a weak but fully efficacious mGlu5 positive allosteric
modulator (PAM) 10.
Scheme 1. Synthesis of Analogues of 16 and 17a
The PAMs reported here demonstrated no activity in the
absence of glutamate, but in the presence of a subthreshold
concentration of glutamate (EC20), a concentration depen-
dent potentiation of mGlu5 response was observed (Figure 2).
Importantly, 16b is a pure mGlu5 PAM, not an ago-potentia-
tor like 6 and 7. In addition, 16b demonstrated a robust
15-fold leftward shift of the glutamate concentration response
a Reagents and conditions: (a) 10 mol % Pd(PPh3)4, 20 mol % CuI,
20.0 equiv of diethylamine, DMF, microwave, 70 °C, 10 min, 16-95%;
all compounds purified by mass-directed HPLC to >98% purity.19
Table 1. Structures, Activity, and Mode of Pharmacology of Analogues 16 and 17
R1
R2
allosteric activitya
IC50, EC50 (nM)a
antagonism (%)a
fold shifta
8
H
H
H
H
PA
486 ( 28
7.5 ( 1.2
3,300 ( 290
21.1 ( 2.8
14.3 ( 2.3
21.1 ( 1.8
120 ( 25
247 ( 24
704 ( 86
195 ( 65
10.8 ( 2.1
>10000
71
100
N/A
N/A
3.3
9
10
3-Me
4-Me
3-Me
H
NAM
PAM
NAM
PAM
PAM
PAM
PAM
PAM
NAM
NAM
N/A
N/A
100
16a
16b
16c
16d
16e
16f
17a
17b
17c
OEt
N/A
15
NHMe
NHMe
SMe
N/A
N/A
N/A
N/A
N/A
100
3-Me
H
5.9
11
t-Bu
H
6.0
5.7
NHMe
N/A
N/A
4-Me
H
N/A
N/A
N/A
3-Me
4-Me
100
N/A
N/A
a IC50, EC50, antagonism, and fold shift are the average of at least three independent determinations. N/A = not applicable. PA = partial antagonist.
NAM = negative allosteric modualtor. PAM = positive allosteric modulator. Fold shift at 10 μM fixed concentration of compound.

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4105
Figure 3. Reversal of amphetamine-induced hyperlocomotion
with mGlu5 PAM 16b in dose-dependent manner with the nontoxic
vehicle, 10% Tween-80.
Figure 2. Compound 16b potentiates mGlu5 activation by gluta-
mate. In the absence of glutamate, 16b does not activate mGlu5. In
the presence of a subthreshold quantity of glutamate, 16b potenti-
ates mGlu5 in a concentration-dependent manner. Compound 16b’s
potentiation of response to glutamate is manifested as increased
mGlu5 agonist sensitivity. The glutamate EC50 is shifted from 493 to
32 nM, or a 15-fold shift with 10 μM 16b.
curve (EC50 shifts from 493 to 32 nM) with an increase in
glutamate max (Figure 2).
In the regiosiomeric pyrimidine series 17, the 4-Me con-
gener 17c was inactive. The unsubstitiuted phenyl analogue
17a was a moderately potent mGlu5 NAM (IC50=195 ( 65
nM). Unlike series 16, the 3-Me NAM “switch” performed as
expected in series 17, significantly increasing mGlu5 NAM
activity (IC50=10.8 ( 2.7 nM) for 17b. Moreover, 17b was
selective for mGlu5 (>10 μM vs mGlu1-4,7,8).
With a potent mGlu5 PAM 16b and a potent mGlu5 NAM
17b, we were poised to determine if the modes of mGlu5
modulation observed in our in vitro cellular assays would be
mirrored in standard in vivo behavioral paradigms. To eval-
uate the PAM 16b, we chose to study the ability of 16b to
reverse amphetamine-induced hyperlocomotion in rats, as 6
and 7 displayed robust efficacy in this preclinical model where
other known antipsychotic agents show similar positive re-
sults.10-13 In the event, 16b was dosed ip at 3, 10, or 30 mg/kg
30minpriorto sc administration of 1 mg/kg amphetamine. As
shown in Figure 3, a modest dose response is observed with
16b, with significant reversal noted at the 30 mg/kg dose, and
no effect (i.e, sedation) of 16b/vehicle alone. Thus, the mGlu5
PAM activity observed incell-based invitro assays is mirrored
in vivo with 16b and comparable to the effects seen with 6
and 7.10-13 Moreover, the reversal of amphetamine-induced
hyperlocomotion with 16b is important, as 16b lacks the
intrinsic agonism of the ago-potentiators 6 and 7, suggesting
for the first time that positive allosteric modulation alone is
sufficient for an antipsychotic profile in this preclinical model.
Previously, mGlu5 NAMs such as 1 and 2 have demon-
strated anxiolytic activity in numerous preclinical models.
Figure 4. Dose-response curves for the effects of 17b on punished
(upper panel) and unpunished (lower panel) responding. The data
are the mean number of punished and unpunished responses that
animals made when tested on 1, 3, 10, 30 mg/kg 17b and vehicle.
Each value represents the mean ( SEM for 18 animals. For
punished responding animals tested on 10 and 30 mg/kg 17b made
significantly greater number of responses than animals tested on
vehicle (p < 0.05). Unpunished responding did not change signifi-
cantly at any of the doses tested.
Therefore, 17b was tested ina modifiedGeller-Seifterconflict
model wherein an increase in punished responding is consis-
tent with an anxiolytic-like profile.20 As seen in Figure 4, 17b
produced a significant dose-dependent increase in punished
responding with the 30 mg/kg dose approaching a 300%
increase in response rate [F(4,17) = 22.69, p < 0.0001] (upper
panel) with no significant effect on unpunished responding
(lower panel). Post hoc analysis indicated that the 10 and
30 mg/kg doses in the punished component of the schedule
differed significantly from vehicle ( p<0.05, Newman-Keul).

4106 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Sharma et al.
Therefore, the NAM activity observed in cell-based in vitro
assays was again paralleled in a standard anxiolytic in vivo
assay where classical mGlu5 NAMs display similar positive
results.3-6,20
by the mGluR5 antagonist MPEP. Neuropharmacology 2005, 49,
1053–1066.
(8) Porter, R. H. P.; Jaeschke, G.; Spooren, W.; Ballard, T. M.;
Buttelmann, B.; Kolczewski, S.; Peters, J. U.; Prinseen, E.;
Wichman, J.; Vieira, E.; Muhlemann, A.; Gatti, S.; Mutel, V.;
Malherbe, P. Fenobam: a clinically validated nonbenzodiazepine
anxiolytic is a potent, selective, and noncompetitive mGlu5 recep-
tor antagonist with inverse agonist activity. J. Pharmacol. Exp.
Ther. 2005, 315, 711–721.
(9) O’Brien, J. A.; Lemaire, W.; Chen, T. B.; Chang, R. S. L.; Jacobson,
M. A.; Ha, S. N.; Lindsley, C. W.; Sur, C.; Pettibone, D. J.; Conn,
P. J.; Williams, D. L. A family of highly selective allosteric mod-
ulators of the metabotropic glutamate subtype 5. Mol. Pharmacol.
2003, 64, 731–740.
(10) Lindsley, C. W.; Wisnoski, D. D.; Leister, W. H.; O’Brien, J. A.;
Lemiare, W.; Williams, D. L.; Burno, M.; Sur, C.; Kinney, G. G.;
Pettibone, D. J.; Tiller, P. R.; Smith, S.; Duggan, M. E.; Hartman,
G. D.; Conn, P. J.; Huff, J. R. Discovery of positive allosteric
modulators for the metabotropic glutamate receptor subtype 5
from a series of N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides that
potentiate receptor function in vivo. J. Med. Chem. 2004, 47, 5825–
5828.
(11) Kinney, G. G.; O’Brien, J. A.; Lemaire, W.; Burno, M.; Bickel, D.
J.; Clements, M. K.; Wisnoski, D. D.; Lindsley, C. W.; Tiller, P. R.;
Smith, S.; Jacobson, M. A.; Sur, C.; Duggan, M. E.; Pettibone, D.
J.; Williams, D. W. A novel selective positive allosteric modulator
of metabotropic glutamate receptor subtype 5 has in vivo activity
and antipsychotic-like effects in rat behavioral models. J. Pharma-
col. Exp. Ther. 2005, 313, 199–206.
In summary, slight structural changes to an mGlu5 allosteric
partial antagonist lead resulted in a shift in activity from partial
antagonist to potent full antagonist to potent positive allosteric
modulator. Two new molecular switches were elucidated
through these changes. A regiosiomeric pyrimidine congener
17b resulted in full NAM activity in vitro and in vivo. The
incorporation of an amino methyl group into the 2-position of
the pyrimidine core resulted in PAM activity, and this new
molecular “switch” was able to override previously identified
NAM molecular “switches”. In this series, 16b represents the
most potent mGlu5 PAM reported to date and the first
example of in vivo efficacy of a pure mGlu5 PAM in reversing
amphetamine-induced hyperlocomotion. The resulting mGlu5
NAM 17b and PAM 16b showed in vivo efficacy in rodent
models of anxiety and schizophrenia, respectively, which
mirrored the observed in vitro mode of pharmacology. With
such subtle structural modifications capable of fully reversing
modes of pharmacology, lead optimization campaigns focused
on ligands that bind to the allosteric site occupied by 1 are
especially challenging. Further work in this area is in progress
and will be reported in due course.
(12) O’Brien, J. A.; Lemaire, W.; Wittman, M.; Jacobson, M. A.; Ha,
S. N.; Wisnoski, D. D.; Lindsley, C. W.; Wisnoski, D. D.; Schaffhauser,
H. J.; Sur, C.; Duggan, M. E.; Pettibone, D. J.; Conn, P. J.;
Williams, D. L. A novel selective allosteric modulator potenti-
ates the activity of native metabotropic glutamate receptor
subtype 5 in rat forebrain. J. Pharmacol. Exp. Ther. 2004, 309,
568–577.
Acknowledgment. The authors thank the NIH and NIDA
(Grant RO1DA023947-01) for support of our work.
(13) (a) Farina, M.; Gagliardi, S.; Le Poul, E.; Mutel, V.; Palombi, G.;
Poli, S. M.; Rocher, J.-P. Novel Oxadiazole Derivatives and Their
Use as Positive Allosteric Modulators of Metabotropic Glutamate
Receptors and Their Preparation, Pharmaceutical Compositions
and Use in the Teatment of Central and Peripheral Nervous System
Disorders. WO123255, 2006.(b) Epping-Jordan, M.; Le Poul, M.;
Rocher, J. P. Allosteric modulation: a novel approach to drug discovery.
Innovations Pharm.Technol. 2007, 24, 24–26.
Supporting Information Available: Experimental procedure
and analytical data for 16a-f and 17a-c; details of the in vitro
and in vivo assays. This material is available free of charge via
the Internet at http://pubs.acs.org.
(14) Engers, D. W.; Rodriguez, A. L.; Oluwatola, O.; Hammond, A. S.;
Venable, D. F.; Williams, R.; Sulikowski, G. A.; Conn, P. J.;
Lindsley, C. W. Synthesis, SAR and unanticipated pharmacologi-
cal profiles of analogs of the mGluR5 ago-potentiator ADX-
47273. ChemMedChem 2009, 4, 505–511.
(15) Rodriguez, A. L.; Nong, Y.; Sekaran, N. K.; Alagille, D.; Tamagnan,
G. D.; Conn, P. J. A close structural analog of 2-methyl-6-(pheny-
lethynyl)-pyridine acts a neutral allosteric site ligandonmetabotropic
glutamate receptor subtype 5 and blocks the effects of multiple
allosteric modulators. Mol. Pharmacol. 2005, 68, 1793–1802.
(16) Sharma, S.; Rodriguez, A. L.; Conn, P. J.; Lindsley, C. W.
Synthesis and SAR of a mGlu5 allosteric partial antagonist lead:
unexpected modulation of pharmacology with slight structural
modifications to a 5-(phenylethynyl)pyrimidine scaffold. Bioorg.
Med. Chem. Lett. 2008, 18, 4098–5101.
References
(1) Schoepp, D. D.; Jane, D. E.; Monn, J. A. Pharmacological agents
acting at subtypes of metabotropic glutamate receptors. Neuro-
pharmacology 1999, 38, 1431–1476.
(2) Conn, P. J.; Pin, J. P. Pharmacology and functions of metabotropic
glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 1997, 37, 205–
237.
(3) 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. 2-Methyl-6-(pheny-
lethynyl)-pyrimidine (MPEP), a potent, selective and systemically
active mGluR5 receptor antagonist. Neuropharmacology 1999, 38,
1493–1503.
(4) Roppe, J. R.; Wang, B.; Huang, D.; Tehrani, L.; Kamenecka, T.;
Schweiger, E. J.; Anderson, J. J.; Brodkin, J.; Jiang, X.; Cramer,
M.; Chung, J.; Reyes-Manalo, G.; Munoz, B.; Cosford, N. D. P.
5-[(2-Methyl-1,3-thiazol-4-yl)ethynyl]-2,30-bipyridine: a highly po-
tent, orally active metabotropic glutamate subtype 5 (mGluR5)
receptor antagonist with anxiolytic activity. Bioorg. Med. Chem.
Lett. 2004, 14, 3993–3996.
(5) Lea, P. M.IV; Faden, A. I. Metabotropic glutamate receptor
subtype 5 antagonists MPEP and MTEP. CNS Drug Rev. 2006,
12, 149–166.
(6) Tatarczynska, E.; Klodzinska, A.; Chojnacka,-Wojcik, E.; Palucha,
A.; Gasparini, F.; Kuhn, R.; Pilc, A. Potential anxiolytic- and
antidepressant-like effects of MPEP, a potent, selective and systemi-
cally active mGluR5 receptor antagonist. Br. J. Pharmacol. 2001,
132, 1423–1430.
(17) Kennedy, J. P.; Williams, L.; Bridges, T. M.; Daniels, R. N.;
Weaver, D.; Lindsley, C. W. Application of combinatorial chem-
istry science on modern drug discovery. J. Comb. Chem. 2008, 10,
345–354.
(18) For full description of the 24-member library, see Supporting
Information.
(19) Leister, W.; Strauss, K.; Wisnoski, D.; Zhao, Z.; Lindsley, C.
Development of a custom high-throughput preparative liquid
chromaotgraphy/mass spectrometer platform for the preparative
purification and anlaytical analysis of comound libraries. J. Comb.
Chem. 2003, 5, 322–329.
(20) Busse, C. S.; Brodkin, J.; Tattersall, D.; Anderson, J. J.; Warren,
N.; Tehrani, L.; Bristow, L. J.; Varney, M. A.; Cosford, N. D. P.
The behavioral profile of the potent and selective mGluR5 receptor
anatgonist 3-[(2- methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP)
in rodent models of anxiety. Neuropsychopharmacology 2004, 29,
1971–1979.
(7) Yan, Q. J.; Rammal, M.; Tranfaglia, M.; Bauchwitz, R. P. Sup-
pression of two major fragile X syndrome mouse model phenotypes