An orally bioavailable positive allosteric modulator of the mGlu 4 receptor with efficacy in an animal model of motor dysfunction

Article abstract of DOI:10.1016/j.bmcl.2010.06.078

A high-throughput screening campaign identified 4-((E)-styryl)-pyrimidin-2- ylamine (11) as a positive allosteric modulator of the metabotropic glutamate (mGlu) receptor subtype 4. An evaluation of the structure-activity relationships (SAR) of 11 is described and the efficacy of this compound in a haloperidol-induced catalepsy rat model following oral administration is presented.

Full text of DOI:10.1016/j.bmcl.2010.06.078

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4901–4905
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
An orally bioavailable positive allosteric modulator of the mGlu4 receptor
with efficacy in an animal model of motor dysfunction
e
f
a
e
Stephen P. East e, , Samantha Bamford , Matthias G. A. Dietz , Christian Eickmeier , Adam Flegg ,
Boris Ferger b, Mark J. Gemkow f, Ralf Heilker d, Bastian Hengerer b, Adrian Kotey e, Pui Loke e,
Gerhard Schänzle c, Hans-Dieter Schubert d, John Scott e, Mark Whittaker e, Mildred Williams e,
*
Przemyslaw Zawadzki e, Kai Gerlach a,
*
a Medicinal Chemistry, Boehringer Ingelheim Pharma GmbH & Co. KG, 88400 Biberach, Germany
b CNS Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, 88400 Biberach, Germany
c Drug Discovery Support, Boehringer Ingelheim Pharma GmbH & Co. KG, 88400 Biberach, Germany
d Lead Generation and Optimisation Support, Boehringer Ingelheim Pharma GmbH & Co. KG, 88400 Biberach, Germany
e Evotec (UK) Ltd, 114 Milton Park, Abingdon, OX14 4SD, UK
f Evotec AG, Schnackenburgallee 114, 22525 Hamburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A high-throughput screening campaign identified 4-((E)-styryl)-pyrimidin-2-ylamine (11) as a positive
allosteric modulator of the metabotropic glutamate (mGlu) receptor subtype 4. An evaluation of the
structure–activity relationships (SAR) of 11 is described and the efficacy of this compound in a haloper-
idol-induced catalepsy rat model following oral administration is presented.
Received 10 May 2010
Revised 11 June 2010
Accepted 11 June 2010
Available online 25 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Allosteric modulator
mGlu4
Parkinson’s disease
Styryl pyrimidinyl amines
The metabotropic glutamate (mGlu) receptors are members of
the Group C class of G-protein coupled receptors that bind the
excitatory neurotransmitter glutamate. To date, there are eight
known mGlu receptors (1–8) and they are sub-divided into three
groups according to their amino acid sequence homology, signal
transduction pathway and pharmacology (Group I: mGlu 1&5;
Group II: mGlu 2&3; Group III: mGlu 4,6,7,8).1–4 These receptors
are distributed throughout the CNS and they are responsible for
the regulation of a variety of physiological processes. Two different
approaches to modulate the biological response of the mGlu recep-
tors have been postulated. One approach is to identify ligands that
compete with glutamate for binding to the orthosteric site and this
can lead to direct activation or inhibition of the receptor. The alter-
native approach is to identify ligands that do not compete with
glutamate but instead bind to an allosteric site. Allosteric ligands
can potentially bind in the presence of glutamate and positively
or negatively modulate the response of the receptor.
The debilitating side effects associated with the current treat-
ments for patients suffering from Parkinson’s disease (PD) coupled
with an ageing population indicate that PD is an area of neurolog-
ical research with a growing unmet medical need.5 Stimulation of
mGlu4 is currently regarded as an attractive non-dopaminergic tar-
get for the palliative treatment of PD which might provide new
drugs without the motor complications (dyskinesias) associated
with the existing frontline therapy
mGlu4 agonists such as
L
-DOPA.6,7 Recently orthosteric
L
-AP-4 or LSP1-21118 and mGlu4 positive
allosteric modulators (PAMs) such as (À)-PHCCC9,10 (1) have dem-
onstrated improvement of motor function in pre-clinical rodent
models of PD. One of the key advantages of positive allosteric mod-
11
ulation of mGlu4 is that the mGlu allosteric sites are likely to be
less well conserved across the different mGlu subtypes and hence
the specificity for mGlu4 over other mGlu receptors should be
achievable. Additionally positive enhancers do not activate the
receptor on their own and thereby minimize interference with
the highly regulated neurotransmission. From the medicinal chem-
istry perspective another advantage of targeting the allosteric site
instead of the orthosteric site is that the allosteric ligands will be
different to glutamate and so they will have markedly different
properties with the potential for improved central nervous system
* Corresponding authors. Tel.: +44 1235 441735 (S.P.E.); tel.: +49 7351 542726
(K.G.).
E-mail addresses: stephen.east@evotec.com (S.P. East), kai.gerlach@boehringer-
ingelheim.com (K. Gerlach).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.078

4902
S. P. East et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4901–4905
(CNS) exposure. Several literature reports of mGlu4 PAMs that de-
tail structure–activity relationships (SAR) conducted on different
chemical series have emerged recently and selected examples of
compounds 2,12,13 3,14 4,15 5,14 6,16 7,17,18 from these publications
are illustrated in Figure 1.
N
O
OEt
N
N
N
H
O
8
9
Following a high-throughput screening campaign on mGlu4 we
N
identified compound 8 (Fig. 2) which showed an EC50 ꢀ5
lM (82%
glutamate max) in our mGlu4 positive modulator assay.19 Although
8 was a singleton it attracted our attention because of its structural
similarity to SIB 1893 9 and MPEP 10 which have been published
previously as mGlu5 negative allosteric modulators (NAMs) with
weak mGlu4 PAM activity.20,21 During the resynthesis of 8 we pro-
filed the parent aminopyrimidine 11 (the product of a two step
synthesis from (E)-4-phenyl-but-3-en-2-one22) and found that this
compound, and not the oxalate ester 8, was the source of the
N
N
NH2
11
10
Figure 2. mGlu ligands.
Table 1
Modifications to the amino group
mGlu4 PAM activity with an EC50 ꢀ1
lM (106% Glu max). With this
N
result in hand we performed an SAR analysis around 11 in an effort
to improve the potency and to identify compounds suitable for
evaluation in vivo.
N
R
For our SAR exploration we divided compound 11 into four
parts, the amino group, the pyrimidine, the olefin and the phenyl
ring and made conservative modifications in each of these regions
of the molecule.
Changes to the amino moiety on the pyrimidine ring are re-
ported in Table 1. Methylation or dimethylation of 11 to provide
12 and 13 was detrimental to activity. Sulfonylation to give 14 also
gave a compound that showed no activity at the highest concentra-
tion tested. Replacement of the amino group with either a methyl
to give the SIB 1893 analogue 15 or a thiol to give 17 provided
Compound
R
mGlu4 (h) EC50
(
l
M)
% Glu maxa
11
12
13
14
15
16
17
NH2
1.0
106
—
—
—
67
—
NHMe
NMe2
NHSO2Me
Me
Ph
SH
>30
>30
>30
6.9
>30
7.8
115
a
EC50 for potentiation of an EC20 glutamate concentration. All assay signals were
compounds that were ꢀ7–8
l
M but introducing a phenyl ring in
normalised to saturating concentrations of (À)-PHCCC (1) which was set to 100%
Glu max. Accordingly, the % Glu max of test compounds was referenced to 1 at
place of the amino moiety (compound 16) resulted in loss of activ-
ity. The results for 13, 14 and 16 suggest that there might be strin-
gent steric requirements at this region of the binding site.
Additionally it could be postulated that an H-bond donor is respon-
sible for improved potency but it is perhaps inconclusive whether
the amino group is forming a key hydrogen bond with the receptor.
None of the modifications that were made to the amino group rep-
resented an advantage over compound 11 in terms of their positive
modulatory effect on mGlu4.
maximal activation. In the assay described19 1 yielded an average EC50 of 5
lM.
the 5- and 6-position as in compound 20 no synergistic effect could
be seen. Complete loss of activity was induced when a typical
hydrogen donor group such as the amino or a hydroxy group (com-
pounds 21 and 22) were attached in position 4 whereas capping
the hydroxyl in 22 to give the methoxy derivative 23 regained
activity. Introduction of small electron-withdrawing groups (24,
25 and 26) in 11 had minimal effect on the potency in the mGlu4
positive modulation assay. When a nitrogen was removed from
11 to give aminopyridine 27 or when the pyrimidine was replaced
with a pyrazine as shown with compound 28 it had a detrimental
effect on the activity suggesting that the nitrogen in the 1-position
of 11 might be important for binding to mGlu4.
Minor changes to the pyrimidine moiety of 11 were generally
well tolerated as shown for compounds 18–28 in Table 2. Addition
of a methyl group in the 6-position of compound 11 led to the
equipotent compound 18 whereas addition in the 5-position as
in compound 19 led to an improvement in potency. Interestingly,
when trying to combine this SAR by inserting methyl groups in
O
OH
N
N
N
OH
N
NH
N
O
N
N
O
O
N
HN
Cl
N
HN
1
2
3
4
Cl
OH
O
Cl
N
O
N
S
N
N
OMe
N
H
N
N
NH
N
H
H
7
5
6
Figure 1. Literature mGlu4 PAMs.

S. P. East et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4901–4905
4903
Table 2
Table 3
Modifications to the pyrimidine
Modifications to the olefin
R
Compound Structure
mGlu4 (h) EC50
>30
(lM) % Glu max
N
N
29
—
Compound
R
mGlu4 (h) EC50
(
lM)
% Glu max
99
N
N
NH2
NH2
N
18
0.8
30
20
86
N
NH2
NH2
N
19
20
0.2
1.6
94
88
N
N
31
32
>30
4.9
—
N
NH2
NH2
N
N
N
NH2
NH2
NH2
NH2
NH2
N
58
N
O
S
21
22
23
>30
>30
2
—
N
N
OH
33
34
>30
>30
—
—
N
NH2
NH2
N
—
N
N
OMe
N
N
106
N
CF3
Table 4
N
N
24
25
0.6
1.6
95
Modifications to the phenyl ring
NH2
N
N
Cl
Br
Ar
N
NH2
102
N
NH2
Compound
Ar
mGlu4 (h) EC50
(lM)
% Glu max
N
26
27
28
1.3
94
92
72
35
36
37
38
39
40
41
42
43
44
45
46
47
48
o-F Ph
2.9
4.5
4.5
>30
>30
>30
>30
>30
>30
4.1
101
99
106
—
—
—
—
—
—
98
94
92
55
—
N
NH2
m-F Ph
p-F Ph
o-CN Ph
m-CN Ph
p-CN Ph
o-Me Ph
m-Me Ph
p-Me Ph
2-Thienyl
2-Furyl
5.8
N
N
NH2
15.8
N
NH2
9.4
2-Pyridyl
3-Pyridyl
4-Pyridyl
11.7
16.4
>30
The changes to the olefin moiety in 11 are presented in Table 3.
Saturation of the double bond to give 29 resulted in a >30-fold drop
in potency. Replacement of the olefin in 11 with an alkyne to give
30 also resulted in a drop in potency although it was less dramatic
and the compound was active with an EC50 ꢀ20
l
M (86% Glu max).
observed. This was also the case for the methyl analogues (41–
43) perhaps indicating steric rather than electronic influences.
Replacement of the phenyl ring with isosteric heteroaryl groups
(44–48) provided compounds with some activity although the
Cyclopropanation of the double bond to give 31 resulted in loss of
potency, however, imposing additional conformation constraint on
the scaffold as shown with the benzofuranyl derivative 32 was
tolerated. When the size of the bicyclic ring was increased to a ben-
zothiophenyl (33) or naphthyl (34), then no activity was observed
4-pyridyl (48) was inactive up to 30 lM.
The results from our preliminary SAR evaluation of compound
11 suggested that there were limited options for optimisation of
the mGlu4 PAM potency. Whilst several compounds with at least
comparable activity had been identified following small changes
to the pyrimidine ring, we had observed that mGlu4 PAM activity
was lost when minor point modifications were made to the four
regions of the molecule. This observation of limited SAR within
at 30 lM.
All modifications that were made to the phenyl ring (Table 4)
led to at least a twofold drop in activity on mGlu4. Introduction
of fluorine (35–37) in the ortho-, meta-, or para-positions all led
to a slight drop in potency but when a cyano group (38–40) was
introduced in any position
a complete loss of activity was

4904
S. P. East et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4901–4905
Table 5
30 mg/kg dose, the plasma concentration was found to be
11.6 M. Encouragingly, 11 was highly CNS penetrant with a con-
centration in the brain, also at the 30 min time point, measured as
ꢀ33.8 M (brain:plasma ratio ꢀ2.9). The CSF levels were measured
as ꢀ0.7 M which is in line with the predicted free fraction based
on the plasma protein binding data and in the range of the in vitro
mGlu4 EC50
Selectivity profile for compound 11
l
Species Receptor MOA
EC50/IC50
1.0
(
l
M) % Glu max/% antagonism
l
Human mGlu4
Human mGlu4
Human mGlu5
Human mGlu5
Human mGlu5
PAM
106
—
—
—
—
Agonisma >30
l
NAM
PAM
>30
>10
.
Agonisma >10
Although 11 suffered from limited in vitro mGlu4 activity to-
gether with rapid in vivo clearance, we reasoned that the high
CNS levels (CSF levels in the range of the in vitro EC50) that were
achieved in rats following oral administration together with a
selectivity of >10-fold over mGlu5 (in the rat assays) qualified 11
as a potential tool compound for an in vivo proof of concept study.
We selected the haloperidol-induced catalepsy rat model as our
symptomatic model for PD and we administered three doses of
11 (1, 10 and 30 mg/kg po; n = 8 per experimental group) orally
30 min prior to haloperidol (dosed at 2.5 mg/kg ip). Catalepsy
was indicated by duration of time (seconds, y-axis) that the rat
remains with its forelimbs on a bar (height 8 cm). The cut off time
was 60 s.26 From the results illustrated in Figure 3 compound 11
showed dose dependent efficacy with a reduction in catalepsy ob-
served at all doses. The ED50 was estimated as ꢀ1 mg/kg.
Rat
Rat
mGlu4
mGlu5
PAM
NAM
1.0
>10
110
—
MOA, mode of action.
a
mGlu assay was run in the absence of glutamate.
the field of mGlu4 PAMs has been well documented for other
chemical series.13,15
In the absence of a significantly superior compound in terms of
potency on mGlu4 (i.e., >10-fold more potent), we elected to profile
11 in more detail (Table 5). In the rat mGlu4 PAM in vitro assay the
EC50 was 1
man mGlu4 assay was also run in the absence of glutamate and
the EC50 was >30 M suggesting that 11 does not act as an agonist
of mGlu4. For our selectivity profiling of 11, we considered mGlu5
to be the most relevant related target of interest because NAMs
of mGlu5 have been reported to show efficacy in animal models
of PD.4,23 Hence, 11 was screened in rat and human mGlu5 assays.
lM which is comparable to the human assay. The hu-
l
To conclude we have identified the mGlu4 PAM 4-((E)-styryl)-
pyrimidin-2-ylamine 11 from a high-throughput screen. We con-
ducted a preliminary SAR study around 11 and we found that there
was only limited scope to modify the structure and improve or in-
deed maintain the PAM activity on mGlu4. In vivo pharmacokinetic
profiling of 11 revealed it to be highly brain penetrant following oral
administration and subsequent examination of 11 in the haloperi-
dol-induced catalepsy model revealed a dose dependent effect thus
providing further support for stimulation of mGlu4 as a potential
treatment for PD. To our knowledge compound 11 represents the
first disclosure of an orally available mGlu4 PAM displaying efficacy
in a symptomatic PD animal model. Additional in vivo evaluation of
the tool compound 11 will be presented in due course.
In our hands, the IC50 of 11 was >30
lM on human mGlu5 and
>10 M on rat mGlu5 and so the window of activity between
l
mGlu5 NAM and mGlu4 PAM is estimated to be >30-fold and
>10-fold in human and rat, respectively. The EC50 for compound
11 in human mGlu5 PAM or agonist assays was measured as
>10 lM. Additionally, 11 was run in a receptor screening panel of
68 targets and no activity was observed at P50% at 10 lM for
any of the receptors.24
Compound 11 was profiled in several in vitro ADMET assays in
order to determine whether it was a suitable candidate for in vivo
evaluation in pre-clinical PD rodent models. Permeability was as-
sessed in CaCo-2 cells and 11 was found to have good permeability
Acknowledgements
with no apparent efflux issue (A–B 41.9 Â 10À6 cm sÀ1
, B–A
The authors thank Maren Köhler, Ellen Gumz, Dörthe Fust,
Christina Schmidt, Svenja Block and Michaela Pirsch for the biolog-
ical assay results.
19.6 Â 10À6 cm sÀ1). Additionally the plasma protein binding in
rats was measured as 90% bound. The metabolic stability of 11
was assessed in rat and human microsomes and found to be 62%
and 83% hepatic blood flow (%Qh) in these species.25 The limited
stability translated into a high in vivo clearance in rats of 75 mL/
min/kg (107% Qh) and 11 had a moderate volume of distribution
(2.7 L/kg) with a short mean residence time (0.6 h) when dosed
at 2 mg/kg via intravenous injection. Compound 11 was orally bio-
available (F = 51%) and 30 min following administration of a
References
1. Knöpfel, T.; Kuhn, R.; Allgeier, H. J. Med. Chem. 1995, 38, 1417.
2. Lavreysen, H.; Dautzenberg, F. M. Curr. Med. Chem. 2008, 15, 671.
3. Niswender, C. M.; Conn, P. J. Annu. Rev. Pharmacol. Toxicol. 2010, 50, 295.
4. Conn, P. J.; Battaglia, G.; Marino, M. J.; Nicoletti, F. Nat. Rev. Neurosci. 2005, 6,
787.
5. Schapira, A. V. H.; Bezard, E.; Brotchie, J.; Calon, F.; Collingridge, G. L.; Ferger, B.;
Hengerer, B.; Hirsch, E.; Jenner, P.; Le Novère, N.; Obeso, J. A.; Schwarzschild, M.
A.; Spampinato, U.; Davidai, G. Nat. Rev. Drug Disc. 2006, 5, 845.
6. Cuomo, D.; Martella, G.; Barabino, E.; Platania, P.; Vita, D.; Madeo, G.; Selvam,
C.; Goudet, C.; Oueslati, N.; Pin, J.-P.; Acher, F.; Pisani, A.; Beurrier, C.; Melon, C.;
Kerkerian-Le Goff, L.; Gubellini, P. J. Neurochem. 2009, 109, 1096.
7. Hopkins, C. R.; Lindsley, C. W.; Niswender, C. M. Future Med. Chem. 2009, 1, 501.
8. Beurrier, C.; Lopez, S.; Révy, D.; Selvam, C.; Goudet, C.; Lhérondel, M.; Gubellini,
P.; Kerkerian-Le Goff, L.; Acher, F.; Pin, J.-P.; Amalric, M. FASEB 2009, 23, 3619.
9. 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.
60
40
vehicle
10. Battaglia, G.; Busceti, C. L.; Molinaro, G.; Biagioni, F.; Traficante, A.; Nicoletti, F.;
Bruno, V. J. Neurosci. 2006, 26, 7222.
20
compound 11 (1 mg/kg)
11. Conn, P. J.; Christopoulos, A.; Lindsley, C. W. Nat. Rev. Drug Disc. 2009, 8, 41.
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. Mol. Pharmacol. 2008, 74, 1345.
13. Williams, R.; Johnson, K. A.; Gentry, P. R.; Niswender, C. M.; Weaver, C. D.;
Conn, P. J.; Lindsley, C. W.; Hopkins, C. R. Bioorg. Med. Chem. Lett. 2009, 19,
4967.
compound 11 (10 mg/kg)
compound 11 (30 mg/kg)
0
0
30
60
90
120
150
180
time after haloperidol (min)
14. Williams, R.; Niswender, C. M.; Luo, Q.; Le, U.; Conn, P. J.; Lindsley, C. W. Bioorg.
Med. Chem. Lett. 2009, 19, 962.
Figure 3. Dose dependent effect of compound 11 in
catalepsy rat model.
a haloperidol-induced

S. P. East et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4901–4905
4905
15. Niswender, C. M.; Lebois, E. P.; Luo, Q.; Kim, K.; Muchalski, H.; Yin, H.; Conn, P.
J.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2008, 18, 5626.
16. Engers, D. W.; Niswender, C. M.; Weaver, C. D.; Jadhav, S.; Menon, U. N.;
Zamorano, R.; Conn, P. J.; Lindsley, C. W.; Hopkins, C. R. J. Med. Chem. 2009, 52,
4115.
y = A + (B À A)/(1 + (10^((C À x) Ã D))); C ? log (EC50) value; D ? slope factor)
and values for EC50 and Hill slope were calculated. Parameters A (top) and B
(bottom) were fixed.
20. Mathiesen, J. M.; Svendsen, N.; Bräuner-Osborne, H.; Thomsen, C.; Ramirez, M.
T. Br. J. Pharmacol. 2003, 138, 1026.
17. Bolea, C.; Celanire, S. WO2009010455, 2009.
21. Recently compounds structurally related to SIB-1893, MTEP and 11 have been
described as mGlu5 ligands that can switch between positive, negative and
partial modulators. See: 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.
18. East, S. P.; Gerlach, K. Expert Opin. Ther. Patents 2010, 20, 441.
19. A CHO-K1 cell line that was engineered to over-express human mGlu4 proteins
was used. Activation of mGlu4 in the respective cell line leads to a decrease of
intracellular free cAMP due to coupling of these receptors to Gi-proteins and
subsequent inhibition of cellular adenylate cyclases. The respective
concentration of intracellular cAMP was quantified in order to assess the
potency of the compounds of interest. Intracellular cAMP concentrations were
determined with the commercially available HitHunter™ cAMP XS+ assay kit
(DiscoverRx) with a luminescence read-out. This cAMP assay was performed
according to the manufacturer’s protocol with slight modifications. Briefly, on
the day of the assay culture medium was removed and the cells washed once
with Dulbecco’s phosphate buffered saline (PBS, PAA). Subsequently, all wash
22. Synthesis of compound 11:
N
DMF-DMA
O
O
115 ºC, 3h
solution was removed and 15 lL of the test compound at the required
concentration in an assay ‘specific compound solution’ (see below) was added
to each well of the 384-well assay plate. After incubation for 30 min at 37 °C,
NH
5
l
L of XS+ Antibody Reagent and 20
ED/LYSIS/CL Substrate Working Solution were added (all kit components)
resulting in a volume of 40 L. This was followed by an incubation of the assay
plate for 1 h at room temperature. Subsequently, 20 L of XS+ EA Reagent (kit
component) were added to each well followed by an incubation of 3 h at room
temperature. Finally, luminescence was read on standard luminescence
l
L of a freshly prepared mixture of XS+
.H2CO3
H2N
NH2
N
2
l
l
N
NH2
MW irradiation,
150°C, 40 min
a
11
reader (Novostar, BMG Labtech). In case the potential to positively modulate
mGlu4 (positive modulator assay) was assayed the ‘specific compound
solution’ consisted of compound at the concentration of interest,
5 lM
23. mGlu5 Antagonists have been shown to have efficacy in anti-Parkinsonian
animal models. See, for example: (a) Dekundy, A.; Pietraszek, M.; Schaefer, D.;
Cenci, M. A.; Danysz, W. Brain Res. Bull. 2006, 69, 318; (b) Rylander, D.;
Iderberg, H.; Li, Q.; Dekundy, A.; Zhang, J.; Li, H.; Baishen, R.; Danysz, W.;
Bezard, E.; Cenci, M. A. Neurobiol. Dis. 2010. doi:10.1016/j.nbd.2010.05.001.
Forskolin and 15 M Glutamate (equivalent to EC20 in this assay) in PBS. In
l
case the pure stimulation of mGlu4 was assayed (agonist assay) the ‘specific
compound solution’ consisted of compound at the concentration of interest
and 5 lM Forskolin in PBS. Test compounds and positive controls were assayed
for dose–response at eight concentrations in triplicate. Serial dilutions in 100%
DMSO were made (at 100-fold of final concentration) and starting and final
concentrations covered a concentration range of three log units. In order to
generate the required assay concentrations in the ‘specific compound
solutions’ the 100% DMSO solutions were diluted in two steps down to 1%
24. The receptor panel included adenosine A2A, 5HT1A and adrenergic a2A
receptors which have all been reported to be potential targets for PD therapy.
25. Compound 11 did not inhibit cytochrome P450 enzymes (3A4, 2D6, 2C9, 2C19)
up to a concentration of 50 lM.
26. Ferger, B.; Buck, K.; Shimasaki, M.; Koros, E.; Voehringer, P.; Buerger, E. Synapse
2010, 64, 533.
DMSO.
A valid EC50 value was calculated from a minimum of three
independent compound dilutions. Dose–response curves were evaluated
according to a ‘one binding site’ equation in XLfit 4.1 software (Model 204;