Synthesis and SAR of a novel metabotropic glutamate receptor 4 (mGlu 4) antagonist: Unexpected 'molecular switch' from a closely related mGlu4 positive allosteric modulator

Article abstract of DOI:10.1016/j.bmcl.2011.09.131

Herein we report the discovery and SAR of a novel antagonist of metabotropic glutamate receptor 4 (mGlu₄). The antagonist was discovered via a molecular switch from a closely related mGlu₄ positive allosteric modulator (PAM). This antagonist (VU0448383) displays an IC₅₀ value of 8.2 ± 0.4 μM and inhibits an EC₈₀ glutamate response by 63.1 ± 6.6%.

Full text of DOI:10.1016/j.bmcl.2011.09.131

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6955–6959
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of a novel metabotropic glutamate receptor 4 (mGlu4)
antagonist: Unexpected ‘molecular switch’ from a closely related mGlu4
positive allosteric modulator
Thomas Utley a,b, Dustin Haddenham a, James M. Salovich a,b,c, Rocio Zamorano a,b, Paige N. Vinson a,b
,
Craig W. Lindsley a,b,c,d, Corey R. Hopkins a,b,c,d, , , Colleen M. Niswender a,b, ,
⇑
⇑
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN 37232, USA
d Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2011
Revised 28 September 2011
Accepted 30 September 2011
Available online 8 October 2011
Herein we report the discovery and SAR of a novel antagonist of metabotropic glutamate receptor 4
(mGlu4). The antagonist was discovered via a molecular switch from a closely related mGlu4 positive allo-
steric modulator (PAM). This antagonist (VU0448383) displays an IC50 value of 8.2 0.4
an EC80 glutamate response by 63.1 6.6%.
lM and inhibits
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Metabotropic glutamate receptor 4
mGlu4
Molecular switch
Antagonist
G-protein coupled receptor (GPCR) orthosteric ligands currently
represent the largest class of drug molecules. However, over the
past several years, modulation of these targets via a novel mecha-
nism, allosteric modulation, has generated much attention and re-
search focus.1–3 One phenomenon that has been reported recently
is the notion of ‘molecular switching’, referring to allosteric ligands
that switch modes of pharmacology upon subtle modifications of
the chemical scaffold.4 While there have been a number of reports
of molecular switches within the Groups I5,6 and II7 metabotropic
glutamate receptors, to the best of our knowledge, this is the first
report of a molecular switch within allosteric ligands of the Group
III subfamily.
was then evaluated for concentration-dependent activity at M4, a
stage which involved counter screening using four cell lines, one of
which was a cell line co-expressing metabotropic glutamate recep-
tor 4 (mGlu4) and Gqi5.9 The purpose of this counter screen was to
control for nonspecific GPCR or Gqi5-mediated effects of HTS hits.
While VU0219493 failed to confirm as active in these follow-up
screens for muscarinic receptors (IC50 >30 lM for both M4 and
M1), surprisingly, the compound robustly potentiated the mGlu4/
200
OH
O
Br
As part of the Molecular Libraries Production Center Network
(MLPCN),8 we had previously performed a high throughput screen
of the NIH Molecular Libraries Small Molecule Repository (MLSMR)
collection of compounds to identify muscarinic acetylcholine recep-
tor 4 (M4)-specific antagonists using a cell line expressing M4 with a
chimeric G protein, Gqi5, to induce calcium mobilization. During pri-
mary and confirmation screening, VU0219493 was flagged as an M4
O
150
100
50
HO
0
-10
antagonist when screened at a single dose of 10 lM. The compound
-9
-8
-7
-6
-5
-4
Log VU0291493, [M]
⇑
Corresponding authors.
E-mail address: colleen.niswender@vanderbilt.edu (C.M. Niswender).
These authors equally contributed to this work.
Figure 1. VU0219493 (inset structure) potentiates glutamate-induced calcium
stimulation in human mGlu4/Gqi5 cells in a concentration-dependent fashion. Data
represent the mean SEM of three independent experiments performed in triplicate.
1
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.131

6956
T. Utley et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6955–6959
Table 2
SAR of the pyridyl group
Gqi5 calcium response with a potency of 1.4 0.4
activity of this hit was also verified in cells co-expressing rat mGlu4
with GIRK potassium channels10 (EC50 = 1.1
M) and a 30 M con-
lM (Fig. 1). The
l
l
O
Br
centration of the compound shifted a concentration-response curve
for glutamate ninefold to the left (data not shown).
O
R
With a verified positive allosteric modulator (PAM) hit in
hand,11 we began a chemical optimization program around this
scaffold. SAR exploration was begun by reacting commercially
3a-e
Compd
R
hmGlu4 IC50
(l
M)a
% Inhibition
63.1 6.6
available a-bromoketones with 2-bromophenol to yield the desired
compounds. Methylation of the phenolic groups or replacing the
phenols with fluorine led to inactive compounds (2b and 2c). The
2,4-dihydrophenyl group could be replaced with 2-pyridyl (2d;
∗
2e
8.2 0.4
Cl
Cl
N
4.6 1.4 lM); however, a significant loss of potency was observed.
∗
In our screening program, we routinely assess compound activ-
ity in the presence of glutamate concentrations that generate a re-
sponse that is either 20% (to detect potentiators) or 80% (to identify
antagonists) of a maximal agonist response. Using this strategy, we
found that inclusion of a 4-chloro-3-pyridyl group engendered a
‘molecular switch’ within the scaffold, resulting in a compound
3a
Inactive
À3.1 1.3
∗
3b
3c
3d
Inactive
Inactive
Inactive
À6.9 3.0
À4.0 6.1
À1.5 1.5
∗
∗
with mGlu4 antagonist activity (2e, 7.8 lM, blockade of the EC80
glutamate response by approximately 75%, Table 1).
Based on the activity of 2e, a brief SAR around the left-hand pyr-
idyl moiety was undertaken (Table 2). Removal of the pyridine
nitrogen led to inactive compounds (3a, 3b, and 3e). In addition,
moving the nitrogen around the ring also was unproductive and
led to inactive compounds (3c and 3d). Interestingly, the 2-pyridyl
compound was an activator (2d), but other regioisomers were
inactive.
N
N
Cl
∗
3e
Inactive
2.3 1.6
Cl
a
Antagonist activity at mGlu4 is assessed by measuring the ability of a com-
Table 1
pound to concentration-dependently inhibit an EC80 glutamate response. IC50 and %
inhibition values are the average of at least three independent determinations
(mean SEM shown in table). Compounds were ranked as active if they inhibited
the EC80 response by at least 20%.
Initial SAR for mGlu4 PAM VU0219493
O
Br
2-bromophenol, K2CO3,
DMF
O
O
R
Br
R
1
2a-e
A more extensive SAR optimization was undertaken to further
evaluate compounds as antagonists of the mGlu4 receptor
(Table 3). The initial antagonist hit was re-synthesized and evalu-
ated in antagonist mode where potency and efficacy were con-
firmed (8.2 0.4 lM; 63.1% inhibition). Further evaluation by
displacement of the 4-chloro, however, was not productive as all
Compd
R
hmGlu4 EC50
1.4 0.4
(l
M)a
Efficacy %PHCCC
79.0 3.1
OH
O
∗
compounds were inactive (4a–h and 4j–m) or very weak in their
activity (4i, >10 lM, 25.5% inhibition).
2a
HO
In contrast to the 4-chloro-3-pyridyl portion of the molecule,
there was somewhat more tractable SAR on the right-hand aryl
portion of the scaffold (Table 4). These compounds were either
made in an analogous fashion as shown in Table 1, or, for the biaryl
compounds, they were synthesized from compound 2e via a palla-
dium-mediated Suzuki cross coupling.12 The ether could not be re-
placed by a thio-ether (5a) or an unsubstituted phenyl derivative
(5b); however, the 2-bromo could be substituted with a 3-bromo
∗
2b
2c
Inactive
12.1 3.1
12.2 4.7
O
F
∗
Inactive
4.6 1.4
without significant loss in potency or efficacy (5d, 7.7 0.8 lM;
F
58.5% inhibition). 2-fluoro (5c), 3-methoxy (5e), 3-dimethyl amine
(5f), 4-methyl (5h), 3-methyl ester (5g) and 4-methoxy (5i) groups
were not tolerated. In contrast, 4-trifluoromethyl was similar in
N
∗
2d
2e
81.9 2.7
potency to the original antagonist (5j, 4.7 0.5 lM; 59.8% inhibi-
∗
tion). The SAR of the 2-substituted phenyl derivatives was not
predictive and rather shallow. For example, the 2-phenyl and 2-
(2-pyridyl) derivatives were active with weak inhibition (5n,
Antagonistb(IC50 = 7.8)
%Inhibition 74.4
Cl
N
a
Potentiation EC50 and efficacy (% PHCCC) are the average of at least three
>10 lM; 31.1% inhibition and 5o, 8.0 0.9 lM; 60.0% inhibition,
independent determinations performed in triplicate (mean SEM shown in table).
The maximal response generated in mGlu4 CHO cells in the presence of mGlu4
PAMs varies slightly in each experiment; therefore, PAM data are normalized to a
control PAM, PHCCC, response obtained in each day’s run.
respectively). In contrast, the other two pyridyl regioisomers (5p
and 5q), as well as a cyclohexyl or other replacements ( 5s, 5u,
5v, and 5x), were inactive. Several other compounds evaluated
b
exhibited weak antagonist activity (5t; >10
5w; >10 M; 36.5% inhibition).
lM, 56.2% inhibition,
For 2e, potency and efficacy (% inhibition) data are for blockade of an EC80
glutamate response.
l

T. Utley et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6955–6959
6957
Table 3
SAR of substituted pyridine compounds
O
Br
O
Br
R-XH, DIEA or NaH,
O
O
DMSO
R
Cl
N
X
N
2e
4a-m
Compd
R–X
hmGlu4 IC50
8.2 0.4
(
l
M)a
% Inhibition
2e
Cl
63.1 6.6
∗
N
4a
Inactive
À5.4 6.1
∗
N
4b
4c
Inactive
Inactive
À7.3 3.9
À7.2 4.8
N
N
O
4d
Inactive
À7.2 2.3
∗
N
H
4e
4f
Inactive
Inactive
À9.9 4.9
À2.9 5.8
∗
N
H
∗
N
O
N
∗
N
4g
4h
Inactive
Inactive
À15.8 6.1
N
O
∗
N
H
2.2 6.0
4i
>10
25.5 9.2
∗
N
H
∗
4j
Inactive
Inactive
À4.8 3.0
N
H
∗
∗
N
H
4k
0.9 5.4
4l
Inactive
Inactive
14.0 15.5
4.3 13.9
O
Cl
N
N
∗
4m
N
H
a
Antagonist activity at mGlu4 is assessed by measuring the ability of a compound to concentration-dependently inhibit an EC80 glutamate response. IC50 and % inhibition
values are the average of at least three independent determinations performed in triplicate (mean SEM shown in table). Compounds were ranked as active if they inhibited
the EC80 response by at least 20%.
To confirm that these compounds were specific to mGlu4, we
counter screened the entire set against the original screening cell
line which contains the human M4 muscarinic receptor co-ex-
pressed with Gqi5. No compounds significantly affected an EC20
response to the M4 agonist acetylcholine; 5r and 5s, which were
inactive at mGlu4, weakly inhibited EC80 responses to ACh by
26% and 23%, respectively. As a further validation that these com-
pounds were inhibitors of mGlu4, the lead antagonist for this ser-
ies, VU0448383, was confirmed to be active in the rat mGlu4
thallium flux assay (Fig. 2). Although this antagonist, and the ser-
ies in general, is of low potency, it is important to note that in
our previous reports exploring the structure activity relationships
of other mGlu4 PAM scaffolds (representing approximately 5,000
compounds), we have never observed a similar pharmacology
‘switch’ for mGlu4, suggesting that this compound represents a
major advance as a tool compound for the study of mGlu4
biology.
In conclusion, we have shown that slight structural modifica-
tions to a novel mGlu4 PAM scaffold lead to a ‘molecular switch’,
resulting in a novel mGlu4 antagonist. Specifically, changing the

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T. Utley et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6955–6959
Table 4
Table 4 (continued)
Compd
R1
hmGlu4 EC50
Inactive
(l
M)a
% Inhibition
O
R
N
Cl
N
5q
À4.8 6.4
5a-x
O
O
O
O
∗
Compd
R1
hmGlu4 EC50
(
l
M)a
% Inhibition
12.0 12.2
Br
F
N
S
5a
5b
5c
Inactive
∗
∗
5r
5s
5t
Inactive
Inactive
>10
7.8 3.3
∗
∗
∗
O
Inactive
Inactive
À8.7 4.2
O
O
O
À10.7 10.2
7.4 5.2
∗
∗
∗
Br
O
5d
5e
7.7 0.8
Inactive
58.5 16.3
N
O
N
56.2 12.7
À2.8 5.0
O
N
O
5f
Inactive
Inactive
4.4 9.9
∗
5u
Inactive
À10.4 9.4
O
O
O
5g
12.9 13.2
∗
∗
∗
O
O
O
∗
∗
5h
5i
Inactive
Inactive
16.9 11.0
5v
Inactive
À0.1 4.6
À18.3 12.0
O
N N
O
O
∗
∗
5j
4.7 0.5
59.8 14.6
5w
>10
36.5 13.7
CF3
O
O
∗
∗
5l
Inactive
Inactive
À17.0 3.6
À14.8 8.7
S
N
N
5x
Inactive
À7.1 9.2
5m
N
O
∗
∗
a
IC50 and % EC80 values are the average of at least three independent determi-
nations performed in triplicate (mean SEM shown in table). Compounds were
ranked as active if they inhibited the EC80 response by at least 20%.
5n
5o
>10
31.1 15.8
N
2,4-dihydroxyphenol to a 4-chloro-3-pyridyl moiety resulted in
the switch from PAM to antagonist (2e). Further modifications of
the 4-chloro-3-pyridyl moiety were not tolerated suggesting a
shallow binding pocket for that portion of the molecule. The far
right side of the molecule did allow for some modification; how-
ever, all compounds were shown to be equipotent with 2e.
Although ‘molecular switches’ have been reported for other mGlu
receptors, to our knowledge this is the first report for a Group III
mGlu receptor, namely, mGlu4. Further studies are planned and
will be reported in due course.
8.0 0.9
60.0 9.4
O
O
∗
∗
N
5p
Inactive
À3.4 2.4

T. Utley et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6955–6959
6959
4. Wood, M. R.; Hopkins, C. R.; Brogan, J. T.; Conn, P. J.; Lindsley, C. W. Biochemistry
2011, 50, 2403.
5. Sharma, S.; Kedrowski, J.; Rook, J. M.; Smith, R. L.; Jones, C. K.; Rodriguez, A. L.;
Conn, P. J.; Lindsley, C. W. J. Med. Chem. 2009, 52, 4103.
6. Lamb, J. P.; Engers, D. W.; Niswender, C. M.; Rodriguez, A. L.; Venable, D. F.;
Conn, P. J.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2011, 21, 2711.
7. Schann, S.; Mayer, S.; Franchet, C.; Frauli, M.; Steinberg, E.; Thomas, M.; Baron,
L.; Neuville, P. J. Med. Chem. 2010, 53, 8775.
8. For information on the Molecular Libraries Probe Production Centers Network
(MLPCN,) see: http://mli.nih.gov/mli/.
VU0448383
100
75
50
25
0
Calcium
Thallium
9. Calcium mobilization assays. FBS, 20 mM HEPES, 100 units/ml penicillin/
streptomycin, and 1 mM sodium pyruvate (Plating Medium). The cells were
grown overnight at 37 °C in the presence of 5% CO2. During the day of assay, the
-10
-9
-8
-7
-6
-5
-4
medium was replaced with 20 lL of 1 lM Fluo-4, AM (Invitrogen, Carlsbad, CA)
prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% (w/v)
pluronic acid F-127 and diluted in Assay Buffer (Hank’s balanced salt solution,
20 mM HEPES and 2.5 mM Probenecid (Sigma–Aldrich, St. Louis, MO)) for
Log compound, [M]
Figure 2. VU0448383 antagonizes glutamate induced responses in human mGlu4/
Gqi5 cells (calcium assay) and rat mGlu4/GIRK cells (thallium assay) in
concentration-dependent fashion.
45 min at 37 °C. Dye was removed and replaced with 20 lL of Assay Buffer. For
a
concentration-response curve experiments, compounds were serially diluted
1:3 into 10 point concentration response curves and were transferred to
daughter plates using the Echo. Test compounds were again applied and
followed by EC20 concentrations of glutamate. Test compounds were
transferred to daughter plates using an Echo acoustic plate reformatter
(Labcyte, Sunnyvale, CA) and then diluted into Assay Buffer. Ca2+ flux was
measured using the Functional Drug Screening System 6000 (FDSS6000,
Hamamatsu, Japan). Baseline readings were taken (10 images at 1 Hz,
Acknowledgments
The authors acknowledge C. David Weaver, Emily Days, and
Christopher Farmer of the Vanderbilt High Throughput Screening
facility. This work was supported by Grants from the NIH and
Michael J. Fox Foundation.
excitation, 470 20 nm, emission, 540 30 nm) and then 20 ll/well test
compounds were added using the FDSS’s integrated pipettor. Cells were
incubated with compounds for approximately 2.5 min and then an EC20
concentration of glutamate was applied. Curves were fitted using a four point
logistical equation using Microsoft XLfit (IDBS, Bridgewater, NJ). Thallium flux
9
assays have been described in detail in Ref.
.
References and notes
10. Niswender, C. M.; Johnson, K. A.; Luo, Q.; Ayala, J. E.; Kim, C.; Conn, P. J.;
Weaver, C. D. Mol. Pharmacol. 2008, 73, 1213.
1. Conn, P. J.; Christopoulos, A.; Lindsley, C. W. Nat. Rev. Drug Disc. 2009, 8, 41.
2. Conn, P. J.; Lindsley, C. W.; Jones, C. Trends Pharm. Sci. 2009, 30, 25.
3. Hopkins, C. R.; Lindsley, C. W.; Niswender, C. M. Future Med. Chem. 2009, 1, 501.
11. All new compounds were characterized by LCMS and/or 1H NMR and found to
be in agreement with their structures.
12. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.