Synthesis and SAR of a novel positive allosteric modulator (PAM) of the metabotropic glutamate receptor 4 (mGluR4)

Article abstract of DOI:10.1016/j.bmcl.2009.07.072

This Letter describes the synthesis and SAR of the novel positive allosteric modulator, VU0155041, a compound that has shown in vivo efficacy in rodent models of Parkinson's disease. The synthesis takes advantage of an iterative parallel synthesis approach to rapidly synthesize and evaluate a number of analogs of VU0155041.

Full text of DOI:10.1016/j.bmcl.2009.07.072

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4967–4970
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of a novel positive allosteric modulator (PAM)
of the metabotropic glutamate receptor 4 (mGluR4)
Richard Williams a,c, Kari A. Johnson a, Patrick R. Gentry a,c, Colleen M. Niswender a,c, Charles D. Weaver a,c
,
P. Jeffrey Conn a,c, Craig W. Lindsley a,b,c, Corey R. Hopkins a,c,
*
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
c Vanderbilt Program in Drug Discovery, Vanderbilt Institute of Chemical Biology, 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:
This Letter describes the synthesis and SAR of the novel positive allosteric modulator, VU0155041, a com-
pound that has shown in vivo efficacy in rodent models of Parkinson’s disease. The synthesis takes advan-
tage of an iterative parallel synthesis approach to rapidly synthesize and evaluate a number of analogs of
VU0155041.
Received 8 June 2009
Revised 8 July 2009
Accepted 14 July 2009
Available online 18 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
mGluR4
Metabotropic glutamate receptor 4
Allosteric modulator
PAM
Parkinson’s disease
In recent years there has been great progress in the discovery
and clinical development of allosteric modulators of several GPCR
targets.1,2 There are several advantages of allosteric modulators
over traditional orthosteric agonists/antagonists. First, specific
allosteric modulators (i.e., those with no intrinsic agonist activity)
only exert their effect in the presence of the endogenous ligand.
Second, the potential exists for greater receptor selectivity due to
sequence divergence in allosteric binding sites.1 Third, for targets
such as metabotropic glutamate receptors, orthosteric ligands are
most often amino acid analogs with poor brain penetration and
suboptimal pharmacokinetic parameters. Our interest in allosteric
modulation is centered on metabotropic glutamate receptor 4
(mGluR4), a member of the larger mGluR family of class C GPCRs.
mGluR4 has been shown by a number of groups to be a potential
drug target in a variety of disease states, including Parkinson’s dis-
ease (PD).3–7
The first positive allosteric modulator (PAM) of mGluR4,
(ꢀ)-PHCCC, 1,3,8,9 has been studied extensively by a number of
groups and has shown efficacy in a number of PD rodent models.3,5
Despite these initial findings and the promise of mGluR4 activation
as a treatment for PD, until 2008 very few mGluR4 PAMs had been
reported in the literature.10 Recently, our laboratories have
published several reports of mGluR4 PAMs; all compounds were
discovered after a successful high-throughput screening (HTS)
campaign initiated at Vanderbilt University4,11–13 (Fig. 1).
Previously, we disclosed VU0155041, 2, a novel mGluR4 posi-
tive allosteric modulator (PAM) that showed in vivo efficacy in
two anti-Parkinsonian rodent models (haloperidol-induced cata-
lepsy and reserpine-induced akinesia).4 Limited structure–activity
relationship (SAR) was explored in that previous report and was
restricted to the compounds found in the HTS at Vanderbilt as well
as the cis/trans isomers of VU0155041 (the cis compounds were
found to be significantly more active than the trans4) and the enan-
tiomerically pure cis isomers. Herein we report a detailed SAR anal-
ysis surrounding VU015504114 as
allosteric modulator tool compound.4
a
novel mGluR4 positive
OH
N
O
OH
O
O
NH
HN
Cl
O
1
Cl
VU0155041
(−)-PHCCC
2
* Corresponding author. Tel.: +1 615 936 6892; fax: +1 615 343 9332.
E-mail address: corey.r.hopkins@vanderbilt.edu (C.R. Hopkins).
Figure 1. Structures of (ꢀ)-PHCCC, 1, and VU0155041, 2, two mGluR4 PAMs that
show activity in vivo.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.072

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R. Williams et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4967–4970
Table 1
O
2.
SAR evaluation of the amide substitution, 4a–i
M) hmGluR44,a % Glu Max
127
OH
O
Compound
R
EC50
(l
3.
2
3,5-Dichlorophenyl
0.740
4a
4b
4c
4d
4e
4f
4g
4h
4i
3,5-Difluorophenyl
3,5-Dimethylphenyl
3,5-Di(trifluoromethyl)phenyl Inactive
3,4-Dimethylphenyl
3-Chloro-5-fluorophenyl
3-Bromophenyl
3,4-Difluorophenyl
Phenyl
Inactive
Inactive
HN
Cl
1.
Inactive
2.0
Inactive
Inactive
>10
Cl
VU0155041, 2
138
39
Figure 2. Areas of SAR exploration of VU0155041, 2.
Benzyl
Inactive
∗
N
N
The SAR surrounding compound 2 centered around three struc-
tural motifs: 1, exploring the 3,5-dichloroamide; 2, exploring the
carboxylic acid; and 3, exploring the cyclohexyl ring (Fig. 2).
Starting from the commercially available cis-1,2-cyclohexanedi-
carboxylic anhydride, the carboxylic acid amide was generated
after ring opening with the appropriate amine (24–80%) (Scheme
1). The 3,5-dichlorophenyl, 2, was the original hit previously re-
ported (740 nM, GluMax = 127%).4 A number of compounds were
synthesized to explore substitution around the phenyl amide 4a–
n (Table 1). Similarly substituted mono- and di-substituted haloge-
nated phenyl analogs were evaluated, and only the 3-chloro-5-flu-
4j
Inactive
N
4k
4l
4m
4n
2-Pyridyl
Inactive
Inactive
Inactive
Inactive
Morpholine
Cyclohexyl
Cyclobutyl
a
Represents the average of at least one experiment performed in triplicate.
O
O
H
H
H
H
NRR1
O
oro, 4e, displayed any activity (2.0
l
M, GluMax = 138%).15 The
M,
OH
O
a
unsubstituted phenyl, 4h, was found to be a weak PAM (>10
l
GluMax = 39%); whereas the benzyl analog, 4i, was inactive. Two
heteroaryl compounds were also evaluated (1,3-dimethyl-1H-
1,2,4-triazole, 4j, 2-pyridyl, 4k); however, these compounds were
also inactive. Lastly, replacements for the phenyl group including
morpholino, 4l, cyclohexyl, 4m, and cyclobutyl, 4n, were synthe-
sized and found to be inactive. This rather ‘shallow’ SAR is a hall-
mark of allosteric modulators.13,16
Cl
HN
Cl
HN
5a-h
2
Cl
Cl
Scheme 2. Library synthesis of 5a–i. Reagents and conditions: (a) RR0NH, EDC,
HOBt, DIPEA, DMF (18–56%). EDC = 1-ethyl-3-(30-dimethylaminopropyl)carbodiim-
ide, HOBt = hydroxybenzotriazole, DIPEA = diisopropyl ethylamine. All library
compounds were purified by mass-directed prep LC where required.17,18
We next turned our attention to finding a suitable replacement
for the carboxylic acid moiety as this group often possesses low
membrane permeability across the blood–brain barrier.19–21
A
variety of amide compounds were made via activation of the acid
with EDC, HOBt and then coupling with the appropriate amine
(Scheme 2, Table 2). The primary carboxamide, 5a, gave similar
potency to the carboxylic acid 2 (950 nM vs 740 nM). All the other
secondary or tertiary amides that were evaluated were either inac-
tive or gave a >10-fold loss in potency (5b–i). A major byproduct of
the reaction, due to the proximity of the activated acid to the
Table 2
SAR evaluation of carboxylic acid replacements, 5a–h
Compound
R1
R
EC50
(
l
M) hmGluR44,a
% Glu Max
5a
5b
5c
5d
5e
5f
H
H
H
H
H
H
H
H
Me
DiMe
Propyl
Cyclopropyl
Cyclopentyl
Cyclopropylmethyl
Morpholine
0.95
10
Inactive
9.6
Inactive
Inactive
10
104
106
57
80
amide, is cyclization to form the cyclic imide, 5i, (3.3 lM,
GluMax = 152%). Although this analog is more active than many
of the other acid replacements; it still shows a fivefold loss of activ-
ity versus 2, and a threefold loss versus 5a.
Three other acid replacements were evaluated (Table 3). The
synthesis of the nitrile 7a, tetrazole 7b, and thiadiazolone 7c, is
shown in Scheme 3. The nitrile compound 7a was active (2 lM,
GluMax = 55%); however, the GluMax was much less than either
the carboxylic acid or carboxamide. The tetrazole mimetic 7b also
5g
5h
Inactive
O
Cl
H
H
5i
N
3.3
152
O
Cl
a
Represents the average of at least one experiment performed in triplicate.
was active as an mGluR4 PAM (>10 lM, GluMax = 152%); however,
this substitution led to a 10-fold loss in potency. The other known
carboxylic acid bioisostere 7c (thiadiazolone) was inactive.20,21
Table 3
SAR evaluation of carboxylic acid mimetics, 7a–c
Compound
EC50
(
l
M)mGluR44,a
% Glu Max
O
O
H
H
7a
7b
7c
2.0
>10
Inactive
55
152
a
OH
O
O
H
H
a
O
HN
Represents the average of at least one experiment performed in triplicate.
R
3
4a-n
Lastly, the cyclohexyl moiety was modified in order to deter-
mine the optimal orientation of the acid and amide. Starting from
the commercially available cyclic anhydrides 8a–k (Table 4), the
Scheme 1. Library synthesis of 4a–n. Reagents and conditions: (a) RR0NH, THF,
55 °C, (24–80%). All library compounds were purified by mass-directed prep LC
where required.17,18

R. Williams et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4967–4970
4969
Table 4
N
O
N
O
c
a,b
Compound
R,R0
EC50
0.75
Inactive
Inactive
Inactive
Inactive
(l
M) hmGluR44,a
% Glu Max
127
O
O
2
–(CH2)4–
H,H
–(CH2)2–
–(CH2)3–
–(CH(CH3)2)–
HN
7a
Cl
OH
9a
9b
9c
9d
O
6
3
N
N
Cl
N
N
H
O
d
∗
9e
9f
2.7
150
HN
Cl
O
∗
∗
7b
Inactive
Inactive
Inactive
Cl
∗
∗
S
N
9g
9h
N
∗
∗
H
O
e,f
6a
HN
7c
Cl
∗
O
OH
Cl
Scheme 3. Synthesis of the nitrile 7a, tetrazole 7b and thiadiazolone 7c. Reagents
and conditions: (a) NH4OH, 88% (b) DIC, pyridine, carried on w/o purification (c)
9i
Inactive
O
NH
3,5-dichloroaniline, EDC, HOBt, DMF, 60–73% (d) Bu3SnN3, toluene,
lW, 30 min,
160 °C, 10%, (e) hydroxylamineꢁHCl, DMSO, DEA, (f) thiocarbonyl diimidazole, THF,
rt 15% (two steps). DIC = N,N0-carbonyl diimidazole, DEA = diethylamine.
Cl
Cl
O
OH
desired target compounds were synthesized by ring opening of the
anhydride with 3,5-dichloroaniline (THF, 55 °C, 15–56%) (Scheme
4). All of the cyclohexyl replacements that were evaluated were
uniformly inactive, with the exception of the cyclohexene analog,
N
9j
Inactive
O
NH
9e (2.7 lM, GluMax = 150%). However, substitution on the cyclo-
hexene ring system again produced an inactive compound, 9f.
Moving the carboxylic acid and the amide moieties one carbon
away (1,3-orientation) also led to an inactive compound, 9i. An
additional ring system that was evaluated was the piperidine-3-
carboxylic acid, 9j. This series was synthesized by reacting the
commercially available 3-piperidine carboxylic acid with 3,5-
dichlorophenyl isocyanate. A number of compounds were synthe-
sized; however, all compounds were inactive. Lastly, having shown
the primary carboxamide was an active carboxyclic acid replace-
ment, 9k was synthesized and evaluated. The primary carboxam-
ide cyclohexene was also active and was equipotent with the
Cl
Cl
O
NH2
O
9k
3.1
94
HN
Cl
Cl
a
Data for inactive compounds represents the average of at least one experiment
performed in triplicate; active compounds were assessed in three independent
experiments in triplicate.
acid analog (3.1 lM, GluMax = 94%).
As was reported in our initial publication,4 the two enantiomers
of VU0155041 (1R,2S) and (1S,2R) were of equal potency and effi-
cacy (1.1 lM, 6.7-fold shift and 1.1 lM, 8.6-fold shift, respectively).
To determine if these new analogs displayed enantioselective
receptor activation; the cyclohexyl carboxamide 5a, cyclohexene
carboxylic acid 9e, and the cyclohexene carboxamide, 9k, were re-
solved into pure enantiomers by chiral HPLC. For both 5a and 9e,
the resolved enantiomers were of equivalent potency and efficacy
O
R
O
OH
O
R
a
R1
O
HN
Cl
R1
O
8a-k
9a-k
(for 5a, >5
2.5 M, GluMax = 76% and 2.7
However the (+)-isomer of 9k was 15-fold more potent than the
l
M, 5.4-fold shift; 2.4
lM, 4.4-fold shift and for 9e,
Cl
l
lM, GluMax = 72%, respectively).
Scheme 4. Library synthesis of 9a–k. Reagents and conditions: (a) 3,5-dichloroan-
iline, THF, 55 °C (15–56%). All library compounds were purified by mass-directed
prep LC where required.17,18
(ꢀ)-isomer (2.1
lM, 5.5-fold shift and >30 lM, 3.6-fold shift,
respectively).22
In conclusion, we report the SAR evaluation of a novel mGluR4
positive allosteric modulator, 2, that was recently shown to be
active in two anti-Parkinsonian in vivo rodent models.4 The SAR
around this scaffold proved to be very challenging in that limited
modification of the original hit was tolerated. It was found that
only substituted phenyl amides were tolerated with the optimal
substitution being the 3,5-dichlorophenyl amide. In addition,
carboxylic acid replacements were also evaluated with only the
primary carboxamide having activity similar to the acid. Utilizing
known carboxylic acid mimetics proved unfruitful in this scaffold.
Lastly, modification of the cyclohexyl ring system allowed only
minor modifications. In fact, only the cyclohexene derivative was
active, and that compound exhibited a threefold loss of activity

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R. Williams et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4967–4970
14. Compound 2 (VU0155041) is now available from TOCRIS bioscience (Cat. No.
3248).
compared to the original hit. However, the cyclohexene ring sys-
tem with the primary carboxamide showed specificity for one
enantiomer ((+)-isomer) allowing for the identification of a com-
pound with activity similar to the original lead (506 nM vs
740 nM). The limited SAR in scaffolds that have been identified
as allosteric modulators is a well-documented phenomenon in this
area.11–13,16,23 Even though the SAR was limited around this scaf-
fold, compound 2 has been shown to be efficacious in preclinical
PD models further validating mGluR4 as a potential therapeutic
treatment for alleviating PD symptoms.
15. Assay details: Cell culture-human mGluR4/Gqi5/CHO line. Human mGluR4
(hmGluR4)/CHO cells stably transfected expressing the chimeric G protein
Gqi5 in pIRESneo3 (Invitrogen, C., CA) were cultured in 90% Dulbecco’s
Modified Eagle Media (DMEM), 10% dialyzed fetal bovine serum (FBS),
100 units/ml penicillin/streptomycin, 20 mM HEPES (pH 7.3), 1 mM sodium
pyruvate, 2 mM glutamine, 400
VA) and 5 nM methotrexate (Calbiochem, EMD Chemicals, Gibbstown, NJ).;
(30,000 cells/20 l/well) were plated in black-walled, clear-bottomed, TC
lg/ml G418 sufate (Mediatech, Inc., Herndon,
l
treated, 384 well plates (Greiner Bio-One, Monroe, North Carolina) in DMEM
containing 10% dialyzed 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
medium was replaced with 20 lL of 1 lM Fluo-4, AM (Invitrogen, Carlsbad, CA)
Acknowledgments
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
The authors thank Qingwei Luo and Rocio Zamorano for techni-
cal assistance with pharmacology assays and Emily L. Days, Tasha
Nalywajko, Cheryl A. Austin and Michael Baxter Williams for their
critical contributions to the HTS portion of the project and Matt
Mulder, Chris Denicola and Sichen Chang for the purification of
compounds utilizing the mass-directed HPLC system. This work
was supported by the National Institute of Mental Health, the Mi-
chael J. Fox Foundation, the Vanderbilt Department of Pharmacol-
ogy and the Vanderbilt Institute of Chemical Biology.
45 min at 37 °C. Dye was removed and replaced with 20 lL of Assay Buffer.
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, 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; 2 min later an EC80 concentration of
glutamate was added. For 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. For fold shift experiments, compounds were added at 2ꢂ their final
concentration and then increasing concentrations of glutamate were added in
the presence of vehicle or the appropriate concentration of test compound.
Curves were fitted using a four point logistical equation using Microsoft XLfit
(IDBS, Bridgewater, NJ).
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solvent, 6% isopropanol/hexanes, 120 mL/min; 220 nM. RT = 12.7 min, (+)-
isomer, [
MeOH).
a
]
D
+52.9 (c 1, MeOH); RT = 13.8 min, (ꢀ)-isomer, [
a
]
D
ꢀ47.1 (c 1,
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