Synthesis and evaluation of a series of heterobiarylamides that are centrally penetrant metabotropic glutamate receptor 4 (mGluR4) positive allosteric modulators (PAMs)

Article abstract of DOI:10.1021/jm9005065

We report the synthesis and evaluation of a series of heterobiaryl amides as positive allosteric modulators of mGluR4. Compounds 9b and 9c showed submicromolar potency at both human and rat mGluR4. In addition, both 9b and 9c were shown to be centrally pe

Full text of DOI:10.1021/jm9005065

J. Med. Chem. 2009, 52, 4115–4118 4115
DOI: 10.1021/jm9005065
allosteric modulation of mGluR4 (Figure 1).12-16 Unfortu-
nately, all of the disclosed mGluR4 PAMs are deficient in
theirpenetration intothe CNS andtheir effects have only been
demonstrated via intracerebroventricular (icv) injection or
with the use of toxic vehicles such as a 50% DMSO solution.
Herein, we report a class of centrally penetrant mGluR4
PAMs which can be administered in a nontoxic vehicle.
A high-throughput screening(HTS)campaign was initiated
at Vanderbilt to identify novel mGluR4 PAMs.12-14 In addi-
tion to the ligands shown in Figure 1, there were a number of
small arylamide compounds identified as having mGluR4
PAM activity (Figure 2). These were attractive hits because
oftheirfavorablecalculatedproperties(MW <300, cLogP<
3.50, total polar surface area (tPSA) < 40, ligand efficiency
(LE)17 > 0.30). From these lead compounds we initiated an
optimization program in order to further profile this chemical
series as novel mGluR4 PAMs.
Synthesis and Evaluation of a Series of
Heterobiarylamides That Are Centrally
Penetrant Metabotropic Glutamate Receptor 4
(mGluR4) Positive Allosteric Modulators
(PAMs)
Darren W. Engers,†,§ Colleen M. Niswender,†,§
C. David Weaver,†,§,‡ Satyawan Jadhav,†,§ Usha N.
Menon,†,§ Rocio Zamorano,†,§ P. Jeffrey Conn,†,§,‡
Craig W. Lindsley,†,§,‡, and Corey R. Hopkins*,†,§
†Department of Pharmacology, Vanderbilt University Medical
Center, Nashville, Tennessee 37232, and Department of Chemistry,
§Vanderbilt Program in Drug Discovery, and ‡Vanderbilt Institute of
Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232
Received April 21, 2009
Initial effort was directed at the left-side heteroarylamide
portion for which the synthesis is outlined in Scheme 1.
A small library utilizing the furylamide revealed that the
3,4-dichloroaniline displayed good potency and efficacy at
the hmGluR4 receptor. Next, a more detailed SAR evaluation
of this structural class was undertaken.
Abstract: We report the synthesis and evaluation of a series of
heterobiaryl amides as positive allosteric modulators of mGluR4.
Compounds 9b and 9c showed submicromolar potency at both
human and rat mGluR4. In addition, both 9b and 9c were shown to
be centrally penetrant in rats using nontoxic vehicles, a major
advance for the mGluR4 field.
Keeping the right-hand anilineconstant as the 3,4-dichloro-
phenyl, we first evaluated different aryl, cycloalkyl, and
heteroarylamide replacements (Table 1). One issue that has
been observed for many allosteric ligands is the noted intract-
able SAR;14,19,20 thisproperty was also observed for the amide
modifications. While the furylamide, 8a, was active in the
micromolar range with good efficacy (>5 μM, 135% Glu-
Max) and bromo substitution was tolerated (8b, 4.1 μM,
62% GluMax), further modifications led to loss of activity
(see 8c,d). An additional survey of five-membered hetero-
cycles was undertaken, but all compounds were inactive as
mGluR4 PAMs (8e-g). Substituting a cyclohexyl, 8h, or
phenyl, 8i, amide for the furyl also led to inactive compounds.
However, upon introduction of the 2-pyridyl group, good
potency and GluMax values were restored (8j, 1.4 μM, 80%
GluMax). The 2-pyrazine compound, 8k, lost activity, while
the 4-pyrimidine, 8l, retained potency, again highlighting this
feature of intractable SAR around allosteric modulators. On
the basis of these results, the 2-pyridylamide was chosen to
further analyze for SAR.21
Starting from the 3,4-dichloroaniline derivative 8j, a num-
ber of halogenated and/or oxygenated compounds were
synthesized and evaluated (Table 2). Compounds 9a-p were
synthesized and tested, and a clear improvement on the HTS
lead was identified. Compounds 9a-c were the best com-
pounds evaluated in this series and presently are some of the
best compounds for mGluR4 PAM activity that have been
disclosed to date. These compounds have excellent potency at
hmGluR4 (240-780 nM) and rmGluR4 (80-370 nM) recep-
tors and exhibit excellent efficacy (hGluMax, 182-235%).
The SAR shows that oxygen in the 3-position is favored;
however, a 3-Cl substituent also imparts activity. As has been
seen with other allosteric modulators, the SAR is very narrow
with very minor modifications, leading to compounds with
reduced or no activity. A number of modifications highlight
this fact; see 9c (340 nM) vs 9d (>5 μM), where a simple
The metabotropic glutamate receptors (mGluRsa) are
members of the GPCR family C, characterized by a large
extracellular amino-terminal binding domain (agonist) along
with a seven-transmembrane spanning (7TM) domain which
is the binding site for most known mGluR allosteric modula-
tors.1-3 The eight cloned mGluRs have been assigned to three
groups (groups I, II, III) based on their structural similarity,
ligand specificity, and preferred coupling mechanisms.4 The
group I subfamily is composed of mGluR1 and mGluR5;
group II receptors include mGluR2 and mGluR3; and the
group III receptors are represented by mGluR4, mGluR6,
mGluR7, and mGluR8. Among the mGluRs, the group III
receptors have thus far received less attention in terms of their
therapeutic potential because of the paucity of selective
ligands. However, recently there have been numerous reports
detailing the potential benefits of mGluR4 activation in
several disease models, most notably rodent models of Par-
kinson’s disease.5,6 It has been shown that activation of
mGluR4 decreases GABAergic transmission at the inhibitory
striato-pallidal synapse with the basal ganglia, a mechanism
that is expected to provide palliative benefit for the treatment
of Parkinson’s disease.7 In addition, there have been recent
reports detailing the neuroprotective effects of an mGluR4
PAM in cultured neurons and in vivo.8,9
The most well characterized mGluR4 PAM for many years
has been the compound PHCCC, 1, a partially selective
mGluR4 potentiator.8,10,11 More recently, our laboratory
and others have expanded the list of novel probes for positive
*To whom correspondence should be addressed. Phone: 615-936-
6892. Fax: 615-322-8577. E-mail: corey.r.hopkins@vanderbilt.edu.
a Abbreviations: mGluR, metabotropic glutamate receptor; PAM, positive
allosteric modulator; HTS, high-throughput screening; tPSA, total polar
surface area; LE, ligand efficiency; HLM, human liver microsomes; RLM,
rat liver microsomes; PPB, protein binding.
r
2009 American Chemical Society
Published on Web 05/27/2009
pubs.acs.org/jmc

4116 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Engers et al.
Table 1. Amide SAR
Figure 1. Chemical structure of (-)-PHCCC, 1, the mGluR4
ago-potentiator VU0155041, 2, and mGluR4 PAMs VU0001171,
3, VU0080241, 4, and VU0092145, 5.
Figure 2. Initial HTS hits.
Scheme 1. Synthesis of Amides 8 and 9a
a EC50 and GluMax are the average of at least three independent
determinations performed in triplicate.
a Reagents and conditions: (a) for acid chloride, DIEA, DMF, 12 h;
(b) for carboxylic acids, EDCI, HOBt, 1,4-dioxane, 50 °C, 12 h.
All library compounds were purified by mass-directed HPLC when
required.18
In addition, the selectivity of a few compounds (9a-c) was
determined among the various mGluR subtypes. Although
these compounds are very active at the human and rat
mGluR4 receptor, they showed little activity against the
other mGluRs, with the exceptions being weak PAM
activity at mGluR5 and mGluR8. For example, 9b and 9c
showed weak PAM activity at mGluR5 and -8 (Supporting
Information, Table 1); however, there is more than a
20-fold separation in potencies compared to mGluR4.
On the basis of the potency and efficacy of 9a-e, and the
very favorable calculated properties, we next looked at meta-
bolic stability and protein binding (PPB) (Table 3). These
compounds were not stable in human or rat liver microsomes
(HLM, RLM), with three compounds (9a-c) having less than
10% of the parent remaining after the incubation period and
two compounds (9d,e) having less than 25% remaining.
However, it is worth noting that 9d and 9e are significantly
more stable; neither compound possesses the metabolically
unstable methoxy group. All of these compounds (except 9d)
possess favorable % free fraction in human and rat protein
binding experiments. Because of these findings and the po-
tency of these compounds, 9b and 9c were advanced further
for in vivo DMPK analysis.
modification of a difluoromethoxy to a trifluoromethoxy
imparts a >10-fold loss of activity. Substituting the phenyl
ring of the aniline with a pyridine (9n,o) or pyrimidine (9p)
also led to compounds with much reduced activity (9b
(240 nM) vs 9n (>5 μM)) or inactive compounds (9o,p). A
number of compounds were synthesized to examine the 3-
methoxy substitution by making longer alkyl chain substitu-
tions or by cyclization (data not shown). However, each of
these modifications led to inactive or weakly active com-
pounds.
A number of the more active compounds were next
evaluated for their ability to shift the glutamate response
curve to the left. The fold shift for both the human and rat
receptors is shown in Table 2. Many of the compounds that
were analyzed showed a robust shift of the glutamate
response (Figure 3, 9b). The SAR for this series of com-
pounds was also mirrored in the fold shift data. As was
observed in the potency data (Table 2), 9a-c were the most
potent compounds and they also produced the largest fold
shift in the human and rat cell lines. In addition to these
compounds, 9f and 9j also produced robust fold shifts,
giving this series several compounds with fold shifts of >15.
The hydrochloride salts of these compounds (9b,c) were
synthesized and dosed intraperitoneally (10 mg/kg) as an
aqueous microsuspension containing 10% Tween-80, and

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4117
Table 2. SAR of the Aniline
Figure 3. Potency and efficacy of the novel mGluR4 PAM, 9b.
(a) Compound 9b was added in progressively higher concentrations
to cell coexpressing human mGluR4 and the chimeric G protein
Gqi5 (white boxes). After a 2.5 min incubation period, an EC20
concentration of glutamate was added (black boxes) and the change
in fluorescence was monitored using a kinetic imaging plate reader.
Data are three independent determinations performed in triplicate.
(b) DMSO vehicle or 30 μM 9b was incubated with human mGluR4/
Gqi5 cells, and 2.5 min later increasing concentrations of gluta-
mate were added. In this experiment, performed in triplicate and
representative of three independent determinations, 9b shifted the
glutamate concentration-response curve 33-fold to the left.
Table 3. In Vitro Pharmacokinetic Evaluation of 9a-c
HLM
(% remaining)
RLM
(% remaining)
PPB (h)
(% free)
PPB (r)
(% free)
compd
9a
9b
9c
9d
9e
3.0
5.3
2.4
22
6.3
1.8
1.4
20
5.1
2.2
4.0
0.8
1.3
6.5
2.4
5.8
1.6
2.9
a EC50, GluMax, and FS are the average of at least three independent
determinations performed in triplicate.
26
19
Table 4. Rat Pharmacokinetic Data for Compounds 9b and 9c
9b 9c
the amount of compound present in brain and plasma was
determined at 0.5, 1, and 8 h after administration (Table 4).
Consistent with the poor in vitro microsomal stability, these
compounds showed high clearance and low plasma expo-
sure in rats. However, the brain levels for 9b and 9c were
significant when compared to the total amount of com-
pound, indicative of good brain penetration for these
amides.
parameters
plasma
brain
plasma
brain
Cmax (ng/mL or g)
Tmax (h)
AUC (ng h/mL or g)
154
0.5
1584
0.5
188
0.5
1679
0.5
173
1.9
705
0.15
99
976
0.39
3
T1/2 (h)
clearance/F ((mL/min)/kg)
AUC0-8h, rain/AUC0-8h,plasma
0.26
1419
894
4.1
9.9
In summary, we report a series of small molecule mGluR4
positive allosteric modulators. These compounds represent a
series of 2-pyridylamide compounds that possess excellent
calculated properties, making them ideal candidates for tool
compounds. In addition, a number of compounds have
excellent in vitro potency and efficacy at both the human
and rat mGluR4 receptor, and many possess the ability to
robustly shift the glutamate response to the left (>15). Se-
lected compounds were further profiled for selectivity, in vitro
PK, and ultimately in vivo PK. Two compounds, 9b and 9c,
although possessing less than ideal in vitro PK parameters,
show sufficient brain penetration to enable further evaluation
in anti-Parkinsonian in vivo rodent models, which will be
reported in due course.
Acknowledgment. The authors thank the assistance of
members of the Vanderbilt HTS facility, Miranda Nolan for
the in vitro PK, and Matt Mulder, Chris Denicola, and
Sichen Chang for the purification of compounds utilizing
the mass-directed HPLC system. This work was supported
bythe National InstituteofMental Health, the MichaelJ. Fox
Foundation, the Vanderbilt Department of Pharmacology
and the Vanderbilt Institute of Chemical Biology.

4118 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Engers et al.
Supporting Information Available: Experimental procedures,
spectroscopic data, and NMR data for selected compounds and
biological procedures. This material is available free of charge
via the Internet at http://pubs.acs.org.
(12) Niswender, C. M.; Johnson, K. A.; Weaver, C. D.; Jones, C. K.;
Xiang, Z.; Luo, Q.; Rodriguez, A. L.; Marlo, J. E.; de Paulis, T.;
Thompson, A. D.; Days, E. L.; Nalywajko, T.; Austin, C. A.;
Williams, M. B.; Ayala, J. E.; Williams, R.; Lindsley, C. W.; Conn,
P. J. Discovery, characterization, and antiparkinsonian effect of
novel positive allosteric modulators of metabotropic glutamate
receptor 4. Mol. Pharmacol. 2008, 74 (5), 1345–1358.
(13) Niswender, C. M.; Lebois, E. P.; Luo, Q.; Kim, K.; Muchalski, H.;
Yin, H.; Conn, P. J.; Lindsley, C. W. Positive allosteric modulators
of the metabotropic glutamate receptor subtype 4 (mGluR4): Part I.
Discovery of pyrazolo[3,4-d]pyrimidines as novel mGluR4 posi-
tive allosteric modulators. Bioorg. Med. Chem. Lett. 2008, 18 (20),
5626–5630.
(14) Williams, R.; Niswender, C. M.; Luo, Q.; Le, U.; Conn, P. J.;
Lindsley, C. W. Positive allosteric modulators of the metabotropic
glutamate receptor subtype 4 (mGluR4). Part II: Challenges in hit-
to-lead. Bioorg. Med. Chem. Lett. 2009, 19 (3), 962–966.
(15) Reynolds, I. J. Metabotropic Glutamate Receptors As Therapeutic
Targets in Parkinson’s Disease. Presented at the 6th International
Meeting on Metabotropic Glutamate Receptors, Taormina, Sicily,
Italy, 14 September 14-19, 2008.
(16) Ortuno, D.; Cheng, C.; Weiss, M.; Bergeron, M.; Shanker, Y.
Identification and Characterization of a Potent and Selective
Positive Allosteric Modulator of mGluR4. Presented at the Society
for Neuroscience, Washington, DC, November 15-19, 2008.
(17) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a useful
metric for lead selection. Drug Discovery Today 2004, 9 (10),
430–431.
(18) Leister, W.; Strauss, K.; Wisnoski, D.; Zhao, Z.; Lindsley, C.
Development of a custom high-throughput preparative liquid
chromatography/mass spectrometer platform for the preparative
purification and analytical analysis of compound libraries.
J. Comb. Chem. 2003, 5 (3), 322–329.
(19) Zhao, Z.; Wisnoski, D. D.; O’Brien, J. A.; Lemaire, W.; Williams,
D. L. Jr.; Jacobsen, M. A.; Wittman, M.; Ha, S. N.; Schaffhauser,
H.; Sur, C.; Pettibone, D. J.; Duggan, M. E.; Conn, P. J.; Hart-
mann, G. D.; Lindsley, C. W. Challenges in the development of
mGluR5 positive allosteric modulators: the discovery of CPPHA.
Bioorg. Med. Chem. Lett. 2007, 17 (5), 1386–1391.
(20) Lindsley, C. W.; Wisnoski, D. D.; Leister, W. H.; O’Brien, J. A.;
Lemaire, W.; Williams, D. L. Jr.; 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 subtype 5
References
(1) Conn, P. J.; Pin, J.-P. Pharmacology and functions of metabotro-
pic glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 1997, 37,
205–237.
(2) Marino, M. J.; Conn, P. J. Glutamate-based therapeutic ap-
proaches: allosteric modulators of metabotropic glutamate recep-
tors. Curr. Opin. Pharmacol. 2006, 6 (1), 98–102.
(3) Conn, P. J.; Christopoulos, A.; Lindsley, C. W. Allosteric mod-
ulators of GPCRs: a novel approach for the treatment of CNS
disorders. Nat. Rev. Drug Discovery 2009, 8 (1), 41–54.
(4) Schoepp, D. D.; Jane, D. E.; Monn, J. A. Pharmacological agents
acting at subtypes of metabotropic glutamate receptors. Neuro-
pharmacology 1999, 38 (10), 1431–1476.
(5) Hopkins, C. R.; Lindsley, C. W.; Niswender, C. M. mGluR4
positive allosteric modulation as potential treatment of Parkin-
son’s disease. Future Med. Chem., in press.
(6) Hefti, F. F. Parkinson’s Disease. Drug Discovery for Nervous
System Diseases; Wiley-Interscience: Hoboken, NJ, 2005; pp 183-
204.
(7) Valenti, O.; Marino, M. J.; Wittmann, M.; Lis, E.; DiLella, A. G.;
Kinney, G. G.; Conn, P. J. Group III metabotropic glutamate
receptor-mediated modulation of the striatopallidal synapse.
J. Neurosci. 2003, 23 (18), 7218–7226.
(8) Maj, M.; Bruno, V.; Dragic, Z.; Yamamoto, R.; Battaglia, G.;
Inderbitzin, W.; Stoehr, N.; Stein, T.; Gasparini, F.; Vranesic, I.;
Kuhn, R.; Nicoletti, F.; Flor, P. J. (-)-PHCCC, a positive allosteric
modulator of mGluR4: characterization, mechanism of action, and
neuroprotection. Neuropharmacology 2003, 45 (7), 895–906.
(9) Battaglia, G.; Busceti, C. L.; Molinaro, G.; Giagioni, F.;
Traficante, A.; Nicoletti, F.; Bruno, V. Pharmacological activation
of mGluR4 metabotropicglutamate receptors reduces nigrostriatal
degeneration in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetra-
hydropyridine. J. Neurosci. 2006, 26 (27), 7222–7229.
(10) Annoura, H.; Fukunaga, A.; Uesugi, M. A novel class of antago-
nists for metabotropic glutamate receptors, 7-(hydroxyimino)cy-
clopropa[b]chromen-1a-carboxylates. Bioorg. Med. Chem. Lett.
1996, 6 (7), 763–766.
(11) Marino, M. J.; Williams, D. L., Jr.; O’Brien, J. A.; Valenti, O.;
McDonald, T. P.; Clements, M. K.; Wang, R.; DiLella, A. G.;
Kinney, G. G.; Conn, P. J. Allosteric modulation of group III
metabotropic glutamate receptor 4: a potential approach to
Parkinson’s disease treatment. Proc. Natl. Acad. Sci. U.S.A.
2003, 100 (23), 13668-13673.
from a series of N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides
that potentiate function in vivo. J. Med. Chem. 2004, 47 (24),
5825–5828.
(21) Bolea, C. Amido Derivatives and Their Use as Positive Allosteric
Modulators of Metabotropic Glutamate Receptors. PCT Int.
Appl. WO2009/010454, 2009; 67 pp.