Discovery of a potent and brain penetrant mGluR5 positive allosteric modulator

Article abstract of DOI:10.1016/j.bmcl.2009.04.095

This Letter describes the discovery of a novel series of mGluR5 positive allosteric modulators (PAMs). The lead compound, 11c, exhibits excellent potency (EC₅₀ = 30 nM) in vitro, and reaches high brain levels in both rats and mice after oral ad

Full text of DOI:10.1016/j.bmcl.2009.04.095

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3275–3278
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a potent and brain penetrant mGluR5 positive allosteric modulator
a
b
b
c
Andreas Ritzén a, , Rikke Sindet , Morten Hentzer , Nannette Svendsen , Robbin M. Brodbeck ,
*
Christoffer Bundgaard d
a Medicinal Chemistry Research DK, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark
b Molecular Pharmacology, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark
c Drug Discovery Biology, Lundbeck Research USA, Inc., 215 College Road, Paramus, NJ 07652-1431, USA
d Discovery ADME, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark
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 discovery of a novel series of mGluR5 positive allosteric modulators (PAMs). The
lead compound, 11c, exhibits excellent potency (EC50 = 30 nM) in vitro, and reaches high brain levels in
both rats and mice after oral administration.
Received 24 February 2009
Revised 19 April 2009
Accepted 20 April 2009
Available online 24 April 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
mGluR5
Glutamate
Allosteric
Positive allosteric modulator
Rat
Mouse
Glutamate is the principal excitatory neurotransmitter in the
mammalian central nervous system, and it mediates its effects
through interaction with ionotropic and metabotropic receptors.
The metabotropic glutamate receptors (mGluRs) are class C GPCRs
and eight subtypes are known. They are divided into three groups
based on their preferred G protein coupling and pharmacological re-
sponses to various ligands: Group I consisting of mGluR1 and
mGluR5, group II consisting of mGluR2 and mGluR3, and group III
consisting of mGluR4, mGluR6, mGluR7, and mGluR8.1 These recep-
tors contain two distinct domains; a large extracellular domain
which binds glutamate at the orthosteric binding site, and a heptah-
elical transmembrane domain, which has been found to bind a vari-
ety of ligands at one or more allosteric binding sites.2 The effects of
glutamate binding on receptor activation can be modulated either
positively or negatively by the binding of ligands to an allosteric site.
Activation of mGluR5 has been postulated to ameliorate both
positive symptoms and cognitive deficits in schizophrenia.3–9 It
has proven difficult to devise selective agonists at this receptor be-
cause of its close homology with the mGluR1 receptor, particularly
at the orthosteric binding site.10 Recently, positive allosteric mod-
ulators (PAMs) binding to sites in the transmembrane domain have
been reported.11–13 Unlike orthosteric agonists, PAMs can be highly
selective for the mGluR subtype of interest because they bind to
sites with lower levels of homology than the L-Glu binding site.
We now wish to report a new series of mGluR5 PAMs.14 The lead
compound of this series exhibits excellent potency and metabolic
stability, sufficient aqueous solubility to allow in vivo dosing in
DMSO-free vehicles, and high brain exposure after oral administra-
tion to rodents.
A screening campaign of a collection of approximately 50,000
compounds using a functional Ca2+ flux assay yielded a moderately
potent hit 1 (Fig. 1). The assay was set up using transiently trans-
fected BHK cells to detect PAMs by adding the test compound to-
gether with
a low concentration of L-Glu (0.30 lM roughly
corresponding to an EC10 of this agonist).15 Potentiation of this
low response indicates PAM activity of the test compound. In this
assay, screening hit 1 had an EC50 for potentiation of 0.97
potentiated the -Glu signal to 73% of its maximum value at satu-
rating -Glu concentration.
lM and
L
L
The structure of 1 shares the (2-phenylethynyl)aryl motif with
the previously reported mGluR5 PAM 2,16 and also with the nega-
tive allosteric modulator (NAM) MPEP (4),10,17 while the structure
of the PAM CDPPB (3)9,18 is unrelated. We reasoned that one of the
aminal hydrogen bond acceptor nitrogen atoms of 1 might overlap
with the carbonyl hydrogen bond acceptor of 2 and that both of
these compounds would bind similarly to the receptor. Based on
this hypothesis, transformation of the aminal-bearing benzene ring
to a pyridine ring and exploration of alternative hydrogen bond
acceptors to replace the aminal moiety were investigated.
* Corresponding author. Tel.: +45 36433205; fax: +45 36438237.
E-mail address: arit@lundbeck.com (A. Ritzén).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.095

3276
A. Ritzén et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3275–3278
Table 1
O
O
Structures and potencies of compounds 7 and 11
N
N
O
O
Y
N
X
11
1
2: MRZ3573
a
Compound
X
Y
EC50 (lM)
11a
7
11b
N
N
N
NH2
OEt
6.6
b
O
N
CN
HNCH2CH2CH(CH3)2
0.51
N
N
N
H
H
N
11c
11d
11e
11f
N
N
N
N
0.030
0.24
1.2
4: MPEP
3: CDPPB
H
N
Figure 1. Original screening hit 1, previously known PAMs 2 and 3, and NAM 4.
H
N
Carboxylic acid esters and amides were selected as suitable
hydrogen bond acceptors, and a flexible synthesis of the target
compounds 7 and 11 was developed (Scheme 1).19 Iodonicotinic
acid 520 was esterified and coupled with phenylacetylene using a
microwave-assisted Sonogashira reaction to afford ethyl ester 7.
This compound could be transformed into the primary amide
11a (Table 1) using NaCN and methanolic ammonia, or could be
hydrolyzed to the acid 8. This acid, or the corresponding benzoic
acid 10 (obtained from oxidation of commercially available alde-
hyde 9) was conveniently coupled with a variety of primary and
secondary amines using solid-supported carbodiimide as a cou-
pling reagent.
By this protocol, 16 compounds were prepared, and their poten-
cies as mGluR5 PAMs were evaluated (Table 1). Small substituents
on the carbonyl yielded very weakly active or inactive compounds,
for example, 7 and 11a, but larger substituents afforded com-
pounds with a range of activities. Both secondary and tertiary
amides were active, and the optimum potency was achieved with
five or six heavy atoms in a five- or six-membered lipophilic ring.
Three pairs of compounds, pair-wise identical except for the aro-
matic CH to N transformation in the amide-bearing ring, where in-
cluded to investigate the effect of this substitution. In all three
cases, the pyridine derivative was more potent than the benzene
one.
NH
NH
O
0.18
S
11g
11h
CH
N
1.2
0.14
11i
N
0.28
N
N
11j
11k
CH
N
0.72
0.13
11l
11m
CH
N
3.1
1.6
N
N
11n
N
N
0.99
N
N
O
S
11o
0.046
The most potent compound, 11c, was selected for further inves-
tigation. First, its allosteric mode of action was confirmed by its
inability to displace the orthosteric agonist [3H]-quisqualic acid
a
EC50 of the test compound for the potentiation of the response to a low con-
-Glu (0.30 M roughly corresponding to an EC10 of this agonist).
No effect at 10 M.
centration of
L
l
b
l
in a radioligand binding assay. At concentrations above 1
lM,
11c actually enhanced binding of [3H]-quisqualic acid slightly
O
O
b
I
N
OR
N
OR
c
5: R = H
6: R = Et
7: R = Et
8: R = H
a
e
d
O
d
O
R
X
N R2
R1
9: R = H
10: R = OH
11
f
Scheme 1. Reagents and conditions: (a) EtOH, H2SO4, 3 h, reflux, 63% yield; (b)
PhC„CH, PdCl2(PPh3)2, CuI, Et3N, 140 °C, 10 min,
lw, 96% yield; (c) LiOH, THF,
Figure 2. Concentration–response curves of
three fixed concentrations of 11c at the human mGlu5 receptor. The EC50 for
glutamate was 3.3 M in the absence of 11c and shifted to 2.1, 1.3, and 0.6 M in
the presence of 11c at 10, 100, and 1000 nM, respectively.
L-glutamate alone and together with
water, rt, 2.5 h, 73% yield; (d) HNR1R2, PS-Carbodiimide, 1,2-dichloroethane, 50 °C,
18 h; (e) NaCN, NH3, MeOH, 50 °C, 16 h; (f) NaClO2, KH2PO4, 2-methyl-2-butene, t-
BuOH, water, rt, 1.5 h, 99% yield.
L-
l
l

A. Ritzén et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3275–3278
3277
Figure 3. Plasma (ng/mL) and brain (ng/g) concentration–time courses of 11c following oral administration of 5 mg/kg (C57/6J mice) and 2.5 mg/kg (Lister Hooded rats). Each
data point represents an average of n = 3 SEM.
(ca. 30%, see Supplementary data), clearly demonstrating that 11c
does not bind to the orthosteric site. Because of its structural sim-
ilarity with MPEP (4), it was expected that 11c would displace
[3H]-MPEP or the structurally similar [3H]-methoxy-PEPy2,21 in a
radioligand binding assay. It has been shown that some PAMs,
for example, CDPPB (3) do displace [3H]-methoxy-PEPy,9 while at
least CPPHA does not.22 Interestingly, for CDPPB (3) binding affinity
versus [3H]-methoxy-PEPy was two orders of magnitude lower
than functional efficacy. Indeed 11c was found to displace [3H]-
ness of 11c as an in vivo tool compound. Exposure studies with
compound 11c showed it to be orally absorbed with high plasma
concentrations following administration of 5 mg/kg in mice and
2.5 mg/kg in rats (Fig. 3). A vehicle consisting of PEG400/Cremo-
phor (80/20 v/v) dosed in a volume of 5 mL/kg was used for all
experiments.15 The ability of compound 11c to permeate the
blood-brain barrier was verified from analysis of brain homogenate
samples. Brain-plasma distribution ratios of 0.5 and 0.3 were found
in mice and rats, respectively, over the entire duration of the con-
centration–time courses. The elimination half-lives were approxi-
mately 1 and 4 h in mice and rats with similar half-lives in
plasma and brain within the species. These pharmacokinetic prop-
erties make compound 11c useful as tool compound for in vivo
pharmacological investigations in rodents.
MPEP with Ki = 1.8
the same site as MPEP (4) and CDPPB (3). Next, concentration–re-
sponse curves (CRCs) of -Glu were recorded in the presence of
three fixed concentrations of 11c. As expected, a concentration-
dependent leftward shift of the -Glu CRC was observed, but at
the highest tested concentration of 11c, 0.60 M, a clear depres-
sion of the maximum response of the mGluR5 receptor to -Glu
lM, suggesting that this compound binds to
L
L
l
In conclusion, a potent mGluR5 PAM, 11c, has been discovered,
and the adequate physicochemical properties and good metabolic
stability of this lead should make it useful as an in vivo pharmaco-
logical tool for investigating the mGluR5 receptor.
L
was detected. At this concentration, an eightfold shift of the CRC
was found. Because other mGluR5 PAMs, for example, CDPPB
(3),9,18 DFB23 and CPPHA22 were not reported to exhibit this
depression of the maximum response, we suspected it to be an
artifact of our assay.24 To test this hypothesis, a new assay was
developed using stably transfected BHK cells. In this new assay,
Acknowledgment
The authors thank Mr. Jude Alfred for technical assistance.
L
-Glu EC10 was 1.0
the response to this
Glu were again recorded in the presence of three fixed concentra-
tions of 11c (Fig. 2). As with the old assay, a leftward shift of the
l
M and the EC50 of 11c for the potentiation of
L
-Glu concentration was 0.90 M. CRCs of
l
L-
Supplementary data
L
-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.04.095.
Glu CRC was observed, but no depression of the maximum re-
sponse was seen. Also, under these assay conditions, no depression
of the maximum response was observed for the PAM reference
References and notes
compound CPPHA (see Supplementary data). At 1.0
est tested concentration of 11c, a 5.5-fold shift of the CRC was
found, as the -Glu EC50 shifted from 3.3 to 0.6 M.
The selectivity of compound 11c was evaluated against a panel
of 63 receptors and seven enzymes using binding assays. At 10 M,
11c caused less than 50% inhibition of radioligand binding in all
cases. In a second evaluation against a panel of 34 GPCRs using
functional assays designed to detect both agonism and antago-
nism, 11c caused less than 30% activation or inhibition at 10 lM
in all cases. A full panel of mGlu receptors was not available to
us, but at mGluR1, 11c showed no agonism, antagonism or alloste-
ric modulation at 10 lM.
lM, the high-
1. Schoepp, D. D.; Jane, D. E.; Monn, J. A. Neuropharmacology 1999, 38, 1431.
2. Pin, J. P.; Galvez, T.; Prezeau, L. Pharmacol. Ther. 2003, 98, 325.
3. Chavez-Noriega, L. E.; Marino, M. J.; Schaffhauser, H.; Campbell, U. C.; Conn, P. J.
Curr. Neuropharmacol. 2005, 3, 9.
4. Homayoun, H.; Stefani, M. R.; Adams, B. W.; Tamagan, G. D.; Moghaddam, B.
Neuropsychopharmacology 2004, 29, 1259.
5. Kinney, G. G.; Burno, M.; Campbell, U. C.; Hernandez, L. M.; Rodriguez, D.;
Bristow, L. J.; Conn, P. J. J. Pharmacol. Exp. Ther. 2003, 306, 116.
6. Lecourtier, L.; Homayoun, H.; Tamagnan, G.; Moghaddam, B. Biol. Psychiatry
2007, 62, 739.
7. Liu, F.; Grauer, S.; Kelley, C.; Navarra, R.; Graf, R.; Zhang, G.; Atkinson, P. J.;
Popiolek, M.; Wantuch, C.; Khawaja, X.; Smith, D.; Olsen, M.; Kouranova, E.; Lai,
M.; Pruthi, F.; Pulicicchio, C.; Day, M.; Gilbert, A.; Pausch, M. H.; Brandon, N. J.;
Beyer, C. E.; Comery, T. A.; Logue, S.; Rosenzweig-Lipson, S.; Marquis, K. L. J.
Pharmacol. Exp. Ther. 2008, 327, 827.
L
l
l
Low aqueous solubility has hampered in vivo experiments with
previous mGluR5 PAMs,18 or in the case of CDPPB (3), necessitated
the use of rather harsh vehicles containing significant fractions of
DMSO.6,9 The solubility of compound 11c in aqueous pH 7.4 buffer
8. Darrah, J. M.; Stefani, M. R.; Moghaddam, B. Behav. Pharmacol. 2008, 19, 225.
9. Kinney, G. G.; O’Brien, J. A.; Lemaire, W.; Burno, M.; Bickel, D. J.; Clements, M.
K.; Chen, T. B.; Wisnoski, D. D.; Lindsley, C. W.; Tiller, P. R.; Smith, S.; Jacobson,
M. A.; Sur, C.; Duggan, M. E.; Pettibone, D. J.; Conn, P. J.; Williams, D. L., Jr. J.
Pharmacol. Exp. Ther. 2005, 313, 199.
was found to be modest, approximately 0.5
to 1.7 M. Because this is >50-fold above the EC50 value, and signif-
icantly above the highest concentration tested in the -Glu CRC
shift described above (Fig. 2), solubility should not limit the useful-
l
g/mL corresponding
10. Spooren, W. P. J. M.; Gasparini, F.; Salt, T. E.; Kuhn, R. Trends Pharmacol. Sci.
2001, 22, 331.
11. Chen, Y.; Conn, P. J. Drugs Future 2008, 33, 355.
12. Gasparini, F.; Spooren, W. Current Neuropharmacology 2007, 5, 187.
13. Rodriguez, A. L.; Williams, R. Curr. Opin. Drug Disc. 2007, 10, 715.
l
L

3278
A. Ritzén et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3275–3278
14. During the preparation of this Letter, a patent application describing a similar
compound series was published; see Conn, P. Jeffrey et al. WO-2008151184.
15. Experimental details are provided as Supplementary data.
16. Parsons, C. G. Allosteric modulation of group I mGluRs. Presented at the 5th
International meeting on metabotropic glutamate receptors, Taormina, Italy,
Sep 18-23, 2005.
17. Gasparini, F.; Lingenhöhl, 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.
Neuropharmacology 1999, 38, 1493.
19. Experimental procedures and analytical data for all new compounds are
provided as Supplementary data.
20. Newkome, G. R.; Moorfield, C. N.; Sabbaghian, B. J. Org. Chem. 1986, 51, 953.
21. Cosford, N. D. P.; Roppe, J.; Tehrani, L.; Schweiger, E. J.; Seiders, T. J.; Chaudary,
A.; Rao, S.; Varney, M. A. Bioorg. Med. Chem. Lett. 2003, 13, 351.
22. O’Brien, J. A.; Wei, L.; Wittmann, M.; Jacobson, M. A.; Ha, S. N.;
Wisnoski, D. D.; Lindsley, C. W.; Schaffhauser, H. J.; Rowe, B.; Sur, C.;
Duggan, M. E.; Pettibone, D. J.; Conn, P. J.; Williams, D. L., Jr. J.
Pharmacol. Exp. Ther. 2004, 309, 568.
23. O’Brien, J. A.; Lemaire, W.; Chen, T. B.; Chang, R. S. L.; Jacobson, M. A.; Ha, S. N.;
Lindsley, C. W.; Schaffhauser, H. J.; Sur, C.; Pettibone, D. J.; Conn, P. J.; Williams,
D. L., Jr. Mol. Pharmacol. 2003, 64, 731.
18. 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. J. Med. Chem.
2004, 47, 5825.
24. Measured EC50 values for these compounds using this assay were in
accordance with those reported in the literature.