Positive allosteric modulators of the metabotropic glutamate receptor subtype 4 (mGluR4). Part II: Challenges in hit-to-lead

Article abstract of DOI:10.1016/j.bmcl.2008.11.104

This Letter describes the synthesis and SAR of two mGluR4 positive allosteric modulator leads, 6 and 7. VU001171 (6) represents the most potent (EC₅₀ = 650 nM), efficacious (141% Glu Max) and largest fold shift (36-fold) of any mGluR4 PAM repor

Full text of DOI:10.1016/j.bmcl.2008.11.104

Bioorganic & Medicinal Chemistry Letters 19 (2009) 962–966
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Positive allosteric modulators of the metabotropic glutamate receptor subtype
4 (mGluR4). Part II: Challenges in hit-to-lead
Richard Williams a,c, , Colleen M. Niswender a,c, , Qingwei Luo a,c, Uyen Le b,c
,
P. Jeffrey Conn a,c, Craig W. Lindsley a,b,c,
*
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Department of Chemistry, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Vanderbilt Program in Drug Discovery, Vanderbilt University Medical Center, 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 two mGluR4 positive allosteric modulator leads, 6 and 7.
VU001171 (6) represents the most potent (EC50 = 650 nM), efficacious (141% Glu Max) and largest fold
shift (36-fold) of any mGluR4 PAM reported to date. However, this work highlights the challenges in
hit-to-lead for mGluR4 PAMs, with multiple confirmed HTS hits displaying little or no tractable SAR.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 5 November 2008
Revised 18 November 2008
Accepted 20 November 2008
Available online 25 December 2008
Keywords:
mGluR4
PAM
Positive allosteric modulator
Parkinson’s Disease
The metabotropic glutamate receptors (mGluRs) are members
of the GPCR family C, characterized by a large extracellular ami-
no-terminal agonist binding domain. To date, eight mGluRs have
been cloned, sequenced and assigned to three groups (Group I:
mGluR1 and mGluR5; Group II: mGluR2 and mGluR3; Group III:
mGluRs 4,6,7,8) based on their sequence homology, pharmacology
and coupling to effector mechanisms.1 The Group III mGluRs are
the least explored and characterized of the mGluRs, but despite
this fact, mGluR4 has garnered a great deal of attention as a ther-
apeutic target for multiple indications.2
sus mGluRs 2, 3, 5, 6, 7, 8.5,6 However, PHCCC is a partial antagonist
(30%) of mGluR1. Despite this, PHCCC has been a very important
proof of concept (POC) compound demonstrating a therapeutic role
(R)n
NH2
CO2H
L-AP4, 1
H2O3P
H2N
CO2H
The reason for the slower pace of development within Group III
mGluRs concerns the availability of selective ligands.2,3 Most phar-
macological studies employ prototypical Group III agonists such as
phenylglycines, 2
L
-(+)-2-amino-4-phosphonobutryic acid, L-AP4, 1 or functionalized
OH
carboxyphenylglycines 2, which have limited CNS penetration
(Fig. 1).4 A major breakthrough in the field occurred when Maj
and co-workers reported on the discovery of N-phenyl-7-(hydrox-
yimino)cyclopropa(b)chromen-1a-carboxamide, ((À)-PHCCC) 3,
the first mGluR4 positive allosteric modulator (PAM), derived from
the mGluR1 negative allosteric modulator (NAM) 7-hydroxyimino-
cyclopropan[b]chromen-1a-carboxylic acid ethyl ester ((À)-CPC-
N
N
N
COOH
H
N
N
Cl
O
N
O
N
HN
O
Cl
COEt).5 PHCCC possesses an EC50 of 4.1
lM, with a 5.5-fold
leftward shift of the glutamate response curve and selectivity ver-
3
4
5
(-)-PHCCC,
VU0080241,
VU0155041,
* Corresponding author. Tel.: +1 615 322 8700; fax: +1 615 343 6532.
E-mail address: craig.lindsley@vanderbilt.edu (C.W. Lindsley).
These authors contributed equally to this work.
Figure 1. Chemical structures of orthosteric mGluR4 agonists
alized phenylglycines (2) and the mGluR4 PAMs (À)-PHCCC (3), VU0080241 (4) and
the mGluR4 ago-potentiator VU0155041 (5).
L
-AP4 (1), function-
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.104

R. Williams et al. / Bioorg. Med. Chem. Lett. 19 (2009) 962–966
963
N
N
250
O
DMSO
N
200
150
100
50
NH
N
1 uM 6
10 uM 6
30 uM 6
N
H
OH
VU0001171, 6
VU0092145, 7
0
Figure 2. Chemical structures of new mGluR4 PAM series VU0001171 (6) and
VU0092145 (7) identified in HTS.
-11 -10 -9 -8 -7 -6 -5 -4 -3 -2
Log glutamate, [M]
for selective mGluR4 activation in Parkinson’s disease,6,7 anxiety,8
depression,9 neuroprotection10 and oncology.11 We recently re-
ported on two new series of mGluR4 PAMs: 4, with improved fold
Figure 4. VU0001171 (6) shifts the glutamate agonist response curves to the left
36-fold at 30 M with an elevation in % Glu Max. Data represents the average of at
least three independent determinations performed in triplicate.
l
shift versus 3 (EC50 = 5 lM, 11.8-fold shift), and 5, a more potent
(EC50 = 750 nM, 6.4-fold shift) mGluR4 PAM than 3, which also dis-
played efficacy in preclinical Parkinson’s disease models.12,13 De-
spite these advances, SAR for 4 was shallow, and, akin to 3 was a
700
mGluR1
mGluR2
mGluR4
mGluR5
mGluR7
mGluR8
600
500
400
300
200
100
0
full antagonist of mGluR1 (IC50 = 2.6 l
M).12 In contrast, 5 was
highly selective for mGluR4 versus mGluR1, 2, 5, 7 and 8 as well
as a full panel of GPCRs, ion channels and transporters. However,
5 possessed significant allosteric agonist activity, and is therefore
more accurately described as an ago-potentiator.13 In order to eval-
uate the in vivo efficacy of selective, pure potentiation of mGluR4,
versus agonist activation, new small molecule tools are required.
In this Letter, we describe the discovery and structure activity
relationship (SAR) of two novel mGluR4 PAMs, VU0001171 (6)
and VU0092145 (7), based on a functionalized benzylidene hydra-
zinyl-3-methylquinazoline scaffold or a bis-2,3-dihydroquinazo-
lin-4(1H)-one core, respectively (Fig. 2). While ‘flat’ SAR is a
staple of allosteric ligands,14–16 we have never encountered such
shallow SAR as in the hit-to-lead campaign from our mGluR4 high
throughput screen (HTS). This report will detail the challenges in
-10
-9
-8
-7
-6
-5
-4
Log VU0001171 (6), [M]
175
150
125
100
75
N
N
N
N
N
N
NH
N
NH
N
NH
N
50
OH
25
0
OH
F
OH
VU0030662, 9
mGluR1 mGluR2 mGluR5 mGluR7 mGluR8
VU0001171, 6
VU0001167, 8
Figure 5. Activation and inhibition responses of VU0001171 (6) in both EC20 and
EC80 assays to determine selectivity versus the other mGluRs. VU0001171 was not
an antagonist of any mGluR and afforded only moderate activation of mGluR1 and
175
150
125
100
75
VU0001171, 6
VU0001167, 8
VU0030662, 9
mGluR8 at 30 lM.
the hit-to-lead process when multiple HTS hits confirm after re-
synthesis, but virtually any structural modification affords a com-
plete loss of mGluR4 PAM activity.
Our mGluR4 PAM HTS identified three 3-methylquinazolines
that afforded a concentration-dependent potentiation of an EC20
of glutamate in human mGluR4/Gqi5 CHO cells (Fig. 3).13 When
HTS stocks were evaluated with full concentration–response
curves, 6, (E)-4-((2-(3-methylquinoxalin-2-yl)hydrazono)methyl)
phenol, was a stand-out compound with an EC50 for potentiation
50
25
0
-10
-9
-8
-7
-6
-5
-4
Log Compound, [M]
of 1.7 lM while having no effect on mGluR4 in this assay in the
absence of glutamate. While 6 was not a particularly attractive
chemical scaffold from a drug discovery perspective, it exhibited
Figure 3. Concentration-dependent potentiation of glutamate in mGluR4/Gqi5
CHO cells by 6, 8 and 9 identified via high throughput screening campaign (HTS). In
the absence of an EC20 of glutamate, 6, 8 and 9 do not elicit receptor activation. Data
represent mean SEM of four independent experiments performed in triplicate.
a 36-fold shift of the glutamate response at 30
lM, the largest fold
shift reported to date for a Class C GPCR (Fig. 4). Additionally, when

964
R. Williams et al. / Bioorg. Med. Chem. Lett. 19 (2009) 962–966
N
N
N
N
a
NH2
N
N
N
N
N
Cl
Ar
H
HCl
10
11
13
N
N
b
N
N
N
N
N
Ar
H
N
N
H
12a-h
O
Scheme 1. Reagents and conditions: (a) NH2NH2, EtOH, MW, 135 °C, 35 min, 95–
98%; (b) ArCHO, EtOH, DIPEA, MW, 135 °C, 35 min, 40–86%.
14
15
Figure 6. Alternative linkers to replace the hydrazinoimine moiety.
Table 1
Potency values for VU0001171 analogs 12
aldehyde to 11 in EtOH followed by microwave irradiation at
135 °C afforded analogs 12a–e (Table 1).17
N
Re-synthesis of the hit compound 6 afforded improved potency
with an EC50 of 650 nM as compared to the HTS deck sample with
an EC50 of 1.7 lM. Walking the hydroxyl group around the phenyl
N
Ar
N
N
H
12
ring to the meta position (12a) gave >3-fold loss in potency, and
the para analog, 12b, afforded a >6-fold loss in potency as well as a
diminished glutamate response. Of the 24-member library that
was synthesized and tested, only the 4-fluorophenyl (12c) and phe-
nyl (12d) analogs, outside of the phenolic congeners, afforded
mGluR4 PAM activity. Overall, a modest SAR was observed with no
improvement in potency. Attention turned to replacing the hydra-
zino imine moiety with a more stable linker. To retain the ‘‘diamino”
linker region, a series of pyrazoles were synthesized (Fig. 6). The
commercially available 3-phenylpyrazoles were deprotonated
using NaH in DMF and reacted with 10 to afford analogs 13; how-
ever, these analogs were devoid of mGluR4 PAM activity.
a
Compound
Ar
EC50
(lM)
% Glu Max
6, VU0001171
1.7
144
141
136
OH
OH
0.65
2.4
12a
12b
OH
4.1
57
Replacing the hydrazine imine moiety with either an acetylene
(14) or an amide (15) linker was also unsuccessful (Fig. 6). As the
labile hydrazino imine linker could not be replaced with a more
stable linker, and coupled with the ‘flat’ SAR, this series was not ad-
vanced further. Still, 6 is an important tool compound, devoid of
mGluR1 antagonist activity. Moreover, 6 is the most potent
mGluR4 PAM reported to date (EC50 = 650 nM, 141% Glu Max)
and possesses the largest fold shift (36-fold shift of the glutamate
CRC) ever reported for a Class C GPCR.
12c
12d
>5
87
F
4.4
129
From the HTS screen an attractive hit was identified, 7, which
has some structural features similar to that of PHCCC (Fig. 7). Com-
O
12e
Inactive
—
pound 7 (VU0092145) has a potency value (EC50 = 1.8
lM) that is
comparable to that of PHCCC (EC50 = 4.1 M), however 7 repre-
l
sents another fundamentally novel mGluR4 chemotype. Unlike 6
with a massive 36-fold shift of the glutamate CRC, 7 possessed only
a 2.7-fold shift of the glutamate response curve.
a
Potency values represent one experimental performed in triplicate.
The hit 7 was re-ordered from a commercial vendor and tested
side by side with in-house synthesized compound (VU0092145-2)
(Fig. 7) employing a novel, microwave-assisted three component
coupling reaction cascade (Scheme 2). With the re-synthesis,
screened across the mGluRs, 6 was observed to be highly selective
for mGluR4 in both activation (EC20 glutamate potentiation) and
inhibition (depression of EC80) assays (Fig. 5). This was a marked
improvement over both PHCCC (3) and our previous reported
mGluR4 PAM, 4 which had significant mGluR1 antagonist activ-
ity.5,16 It is possible that 6 binds at a second allosteric site, distinct
from the PHCCC (3 and 4) allosteric site, which appears to be con-
served with mGluR1.
With a bona fide novel mGluR4 PAM hit in hand, we employed
an iterative analog library synthesis approach to rapidly evaluate
SAR for 6. First, we altered the aromatic group at the hydrazino
imine terminus to determine what functional groups would be tol-
erated. From commercially available 10, a microwave-assisted
SNAr reaction was performed to displace chlorine with hydrazine
to afford 11 (Scheme 1). Imine formation by the addition of an
roughly equivalent potency was observed (EC50 = 3.0
lM vs
EC50 = 1.8 M); however glutamate response differed (129% Glu
l
Max for commercial vs 80% Glu Max for the re-synthesized
material).
The microwave-assisted organic synthesis chemistry was highly
amendable to rapid SAR analysis, and two 24-member libraries
were synthesized were either the 4-methylaniline or the 2-phenyl-
propanal were held constant.18 Of the 48 compounds that were
tested only three compounds, re-synthesized
(6.25%) exhibited any mGluR4 PAM activity (EC50s > 10
7
and 17a–b,
M with
l
increases in % Glu Max), and all with lower potencies than the ori-
ginal hit 7 (Table 2). This was disappointing, but not at all that sur-
prising. There are some structural similarities to that of PHCCC,

R. Williams et al. / Bioorg. Med. Chem. Lett. 19 (2009) 962–966
965
Table 2
O
Potency values for VU0092145 analogs 17
N
O
R
N
H
N
N
H
17
VU0092145, 7
a
Compound
R
EC50
3.0
(
lM)
% Glu Max
129
80
70
60
50
40
30
20
10
0
VU0092145-1
VU0092145-2
7, VU0092145
17a
>10
>10
87
69
F
17b
a
Represent the average of one experiment performed in triplicate.
-10
-9
-8
-7
-6
-5
4
the most potent (EC50 = 650 nM), efficacious (141% of Glu Max) hu-
man mGluR4 PAM reported to date. Moreover, VU0001171 (6)
afforded a significant 36-fold shift of the glutamate response, the
largest reported to date, and the compound did not exhibit mGluR1
antagonist activity. Unfortunately, due to the ‘flat’ SAR and the
inability to replace labile, non drug-like moieties, both the
VU0001171 (6) and VU0092415 (7) scaffolds were discontinued.
However, both series highlight a major challenge in the develop-
ment of mGluR4 allosteric ligands—the common theme of HTS hits
confirming upon re-synthesis, but displaying little to no SAR (typ-
ically less than 7% actives) upon the slightest structural modifica-
tions. Clearly, ligands that potentiate mGluR4, and their allosteric
binding sites, represent significant challenges in the hit-to-lead
stage of drug discovery.
Log VU0092145 (7), [M]
120
100
80
60
40
20
0
DMSO
VU0092145-1
VU0092145-2
-10
-9
-8
-7
-6
-5
-4
-3
2
Acknowledgments
Log Glutamate, [M]
The authors thank C. David Weaver, Emily L. Days, Tasha Naly-
wajko, Cheryl A. Austin and Michael Baxter Williams for their crit-
ical contributions to the HTS portion of the project as well as the
National Institute of Mental Health, the Michael J. Fox Foundation,
the Vanderbilt Department of Pharmacology and the Vanderbilt
Institute of Chemical Biology for support of this research.
Figure 7. Concentration-dependent potentiation of glutamate in mGluR4/Gqi5
CHO cells by one commercial lot of 7 (VU0092415-1) and a re-synthesized lot
(VU0092415-2) along with fold shift of glutamate at 30 lM. Data represent
mean SEM of three independent experiments performed in triplicate.
O
O
References and notes
N
a
O
1. (a) Schoepp, D. D.; Jane, D. E.; Monn, J. A. Neuropharmacology 1999, 38, 1431;
(b) Conn, P. J.; Pin, J.-P. Annu. Rev. Pharmacol. Toxicol. 1997, 37, 205.
2. (a) Marino, M. J.; Hess, J. F.; Liverton, N. Curr. Top. Med. Chem. 2005, 5, 885; (b)
Marino, M. J.; Conn, P. J. Curr. Opin. Pharmacol. 2006, 8, 98.
N
H
N
H
O
3. (a) Yang, Z.-Q. Curr. Top. Med. Chem. 2005, 5, 913; (b) Niswender, C. M.; Jones, J.
K.; Conn, P. J. Curr. Top. Med. Chem. 2005, 5, 847.
16
7
4. (a) Thomsen, C.; Kristensen, P.; Mulvihill, E.; Haldeman, B.; Suzdak, P. D.
Eur. J. Pharmacol. 1992, 227, 361; (b) Gasparini, F.; Bruno, V.; Battaglia, G.;
Lukic, S.; Leonhardt, T.; Inderbitzen, W.; Laurie, D.; Sommer, B.; Varney, M.
A.; Hess, S. D.; Johnson, E. C.; Kuhn, R.; Urwyler, S.; Sauer, D.; Poret, C.;
Schmutz, M.; Nicoletti, F.; Flor, P. J. J. Pharmacol. Exp. Ther. 1999, 289,
1678.
Scheme 2. Reagent and condition: 4-Methylaniline, 2-phenylpropanal, p-TSA, H2O,
MW, 150 °C, 35 min, 95–98%.
which has also reported to have a very flat SAR. One explanation
for the ‘flat’ SAR is that the allosteric binding sites which both 6
(VU0001171) and 7 (VU0092145) as well as PHCCC occupy are very
shallow, similar to the second, non-MPEP, allosteric binding site on
mGluR5 that N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-
2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) occupies.14,15
Whilst the SAR of both VU0001171 (6) and VU0092145 (7) are
flat, they represent the third and fourth literature disclosure of po-
sitive allosteric modulators of mGluR4. VU0001171 (6) represents
5. 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.
Neuropharmacology 2003, 45, 895.
6. Marino, M. J.; Williams, D. L., Jr.; O’Brien, J. A.; Valenti, O.; McDonald, T. P.;
Clements, M. K.; Wang, R.; DiLella, A. G.; Hess, J. F.; Kinney, G. G.; Conn, P. J.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13668.
7. Battaglia, G.; Busceti, C. L.; Molinaro, G.; Biagioni, F.; Traficante, A.; Nicoletti, F.;
Bruno, V. J. Neurosci. 2006, 26, 7222.
8. Stachowicz, K.; Klak, K.; Klodzinska, A.; Chojnacka-Wojcik, E.; Pilc, A. Eur. J.
Pharmacol. 2004, 498, 153.
9. Klak, K.; Palucha, A.; Branski, P.; Pilc, A. Amino Acids 2007, 32, 169.

966
R. Williams et al. / Bioorg. Med. Chem. Lett. 19 (2009) 962–966
10. Canudas, A. M.; Di Giorgi-Gerevini, V.; Iacovelli, L.; Nano, G.; D’Onofrio, M.;
Arcella, A.; Giangaspero, F.; Busceti, C.; Ricci-Vitiani, L.; Battaglia, G.; Nicoletti,
F.; Melchiorri, D. J. Neurosci. 2004, 24, 10343.
Smith, S.; Duggan, M. E.; Hartman, G. D.; Conn, P. J.; Huff, J. R. J. Med. Chem.
2004, 47, 5825.
17. Experimental for the synthesis of 12a: To
a solution of 2-chloro-3-
11. Iacovelli, L.; Arcella, A.; Battaglia, G.; Pazzaglia, S.; Aronica, E.; Spinsanti, P.;
Caruso, A.; De Smaele, E.; Saran, A.; Gulino, A.; D’Onofrio, M.; Giangaspero, F.;
Nicoletti, F. J. Neurosci. 2006, 26, 8388.
12. Niswender, C. M.; LeBois, E. P.; Luo, C.; Kim, K.; Muchalski, H.; Yin, H.; Conn, J.
P.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2008, 18, 5626.
13. Niswender, C. M.; Johnson, K. A.; Weaver, C. D.; Jones, C. J.; Luo, Q.; Rodriguez,
A. L.; Marlo, J.; de Paulis, T.; Thompson, A.; Days, E.; Nalywajko, N. T.; Austin, C.;
Williams, M.; Ayala, J. E.; Williams, R.; Lindsley, C. W.; Conn, P. J. Mol.
Pharmacol. 2008, 74, 1345.
14. Zhao, Z.; Wisnoski, D. D.; O’Brien, J. A.; Lemiare, W.; Williams, D. L., Jr.;
Jacobson, M. A.; Wittman, M.; Ha, S.; Schaffhauser, H.; Sur, C.; Pettibone, D. J.;
Duggan, M. E.; Conn, P. J.; Hartman, G. D.; Lindsley, C. W. Bioorg. Med. Chem.
Lett. 2007, 17, 1386.
methylquinoxaline (100 mg, 0.56 mmol) in EtOH (1 mL) was added
hydrazine (18 mg, 0.56 mmol), sealed and heated in microwave at 135 °C for
35 min. The reaction mixture was cooled to room temperature. The solvent
was removed under vacuum to afford 11 as a light brown solid (117 mg, 99%);
1H NMR (400 MHz, d6-DMSO) d 8.45 (br s, 1H), 7.71 (br s, 1H), 7.49 (s, 1H), 7.31
(br s, 1H), 2.43 (s, 3H); LC–MS, single peak, 2.45 min, m/e, 175.2 (M+1); a
solution of 11 (110 mg, 0.52 mmol), DIPEA (67 mg, 0.52 mmol) and 3-
hydroxybenzaldehyde (63 mg, 0.52 mmol) in EtOH (1 mL) was irradiated at
135 °C for 35 min in a microwave. The reaction mixture was cooled to room
temperature, concentrated under vacuum and purified by mass directed
preparative HPLC to afford 12a as a pale yellow solid; 1H NMR (400 MHz, d6-
DMSO) d 9.63 (br s, 1H), 8.49 (s, 1H), 7.71 (br s, 2H), 7.50 (br s, 1H), 7.41 (br s,
1H), 7.38–7.32 (m, 1H), 7.30–7.26 (m, 2H), 6.84 (d, J = 13.0 Hz, 1H), 2.55 (s, 3H);
LC–MS, 4:1 E/Z, 2.18 and 2.29 min, m/e, 279.1; HRMS calc. 279.1246 (M+H),
C16H15N4O, found 279.1246, C16H15N4O.
15. O’Brien, J. A.; Lemaire, W.; Wittmann, M.; Jacobson, M. A.; Ha, S. N.; Wisnoski,
D. D.; Lindsley, C. W.; Schaffhauser, H. J.; Sur, C.; Duggan, M. E.; Pettibone, D. J.;
Conn, J.; Williams, D. L. J. Pharmacol. Exp. Ther. 2004, 309, 568.
16. Lindsley, C. W.; Wisnoski, D. D.; Leister, W. H.; O’Brien, J. A.; Lemiare, W.;
Williams, D. L., Jr.; Burno, M.; Sur, C.; Kinney, G. G.; Pettibone, D. J.; Tiller, P. R.;
18. Thermal one pot synthesis of bis-2,3-dihydroquinazolin-4(1H)-ones;
Baghbanzadeh, M.; Salehi, P.; Dabiri, M.; Kozehgary, G. Synthesis 2005,
344.