Discovery, synthesis, and structure-activity relationships of 2-aminoquinazoline derivatives as a novel class of metabotropic glutamate receptor 5 negative allosteric modulators

Article abstract of DOI:10.1016/j.bmcl.2013.06.049

A virtual screening approach using various in silico methodologies led to the discovery of 2-(m-tolylamino)-7,8-dihydroquinazolin-5(6H)-one (1) as a moderately active negative allosteric modulator (NAM) of the metabotropic glutamate receptor subtype 5 (mGluR5) showing high selectivity against the subtype mGluR1. Modifications of the parent compound by rational design yielded a series of highly potent derivatives which will serve as valuable starting points for further hit-to-lead optimization efforts toward a suitable drug candidate for the treatment of l-DOPA induced dyskinesia.

Full text of DOI:10.1016/j.bmcl.2013.06.049

Accepted Manuscript
Discovery, synthesis and structure-activity relationships of 2-aminoquinazoline
derivatives as a novel class of metabotropic glutamate receptor 5 negative al‐
losteric modulators
Holger Kubas, Udo Meyer, Bjoern Krueger, Mirko Hechenberger, Maksims
Vanejevs, Ronalds Zemribo, Valerjans Kauss, Raisa Ambartsumova, Ilya
Pyatkin, Alexey I. Polosukhin, Ulrich Abel
PII:
S0960-894X(13)00770-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.06.049
BMCL 20613
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
14 May 2013
14 June 2013
17 June 2013
Please cite this article as: Kubas, H., Meyer, U., Krueger, B., Hechenberger, M., Vanejevs, M., Zemribo, R., Kauss,
V., Ambartsumova, R., Pyatkin, I., Polosukhin, A.I., Abel, U., Discovery, synthesis and structure-activity
relationships of 2-aminoquinazoline derivatives as a novel class of metabotropic glutamate receptor 5 negative
allosteric modulators, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.
2013.06.049
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Graphical Abstract
Discovery, synthesis and structure-activity
relationships of 2-aminoquinazoline
derivatives as a novel class of metabotropic
glutamate receptor 5 negative allosteric
modulators
Leave this area blank for abstract info.
Holger Kubasa, Udo Meyera, Bjoern Kruegera, Mirko Hechenbergera, Maksims Vanejevsb, Ronalds Zemribob,
Valerjans Kaussb, Raisa Ambartsumovac, Ilya Pyatkinc, Alexey I. Polosukhinc, and Ulrich Abela
a Merz Pharmaceuticals GmbH, Eckenheimer Landstrasse 100, 60318 Frankfurt am Main, Germany
b Institute of Organic Synthesis, Aizkraukles iela 21, Riga, LV1006, Latvia
c Asinex Ltd., 20 Geroev Panfilovtzev, Building 1, Moscow 125480, Russia
Hit Optimization
VS Hit

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery, synthesis and structure-activity relationships of 2-aminoquinazoline
derivatives as a novel class of metabotropic glutamate receptor 5 negative
allosteric modulators
Holger Kubasa, Udo Meyera, Bjoern Kruegera, Mirko Hechenbergera, Maksims Vanejevsb, Ronalds
Zemribob, Valerjans Kaussb, Raisa Ambartsumovac, Ilya Pyatkinc, Alexey I. Polosukhinc, and Ulrich
Abela,∗
a Merz Pharmaceuticals GmbH, Eckenheimer Landstrasse 100, 60318 Frankfurt am Main, Germany
b Institute of Organic Synthesis, Aizkraukles iela 21, Riga, LV1006, Latvia
c Asinex Ltd., 20 Geroev Panfilovtzev, Building 1, Moscow 125480, Russia
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A virtual screening approach using various in silico methodologies led to the discovery of 2-(m-
tolylamino)-7,8-dihydroquinazolin-5(6H)-one (1) as a moderately active negative allosteric
modulator (NAM) of the metabotropic glutamate receptor subtype 5 (mGluR5) showing high
selectivity against the subtype mGluR1. Modifications of the parent compound by rational
design yielded a series of highly potent derivatives which will serve as valuable starting points
for further hit-to-lead optimization efforts toward a suitable drug candidate for the treatment of
L-DOPA induced dyskinesia.
Keywords:
Metabotropic glutamate receptor
mGluR5
2013 Elsevier Ltd. All rights reserved.
Allosteric modulator
Virtual screening
L-DOPA induced dyskinesia
Glutamate is the major excitatory neurotransmitter in the brain
and fundamental for memory formation, regulation and learning.1
It acts via ionotropic glutamate receptors (NMDA, AMPA, and
Kainate), which are ligand-gated ion channels, and via G-protein
coupled metabotropic glutamate receptors (mGluR). The mGluRs
belong to the GPCR family C, members of which are structurally
composed by a seven transmembrane (7TM) α-helical domain,
and by a large extracellular N-terminal domain. The bilobed
venus flytrap structure of the N-terminus accommodates the
orthosteric binding site, whereas an allosteric binding site is
situated in the transmembrane region.2 Functional mGluRs are
homodimers, which are covalently linked by a disulfide bridge,
where each flytrap motif may independently adopt a resting state
conformation or an active state conformation, which is stabilized
upon binding of orthosteric agonists.
C, which upon activation leads to the formation of inositol-1,4,5-
trisphosphate and finally triggers the release of intracellular
calcium.3 In contrast, group II and group III mGluRs bind to
Gi/G0 and are negatively coupled to cAMP as second messenger.4
Group I mGluRs are located postsynaptically, where they can act
as regulators of co-localized NMDA receptors.5 The mGluR5 is
centrally expressed in the limbic cortex, hippocampus, amygdala,
basal ganglia, and thalamus, as well as peripherally in the skin
and the vagal nerve system,6 which reflects the multitude of
pharmacological effects anticipated for mGluR5 modulators.
Early mGluR antagonists were designed as glutamate analogs
stabilizing the orthosteric binding site in the resting state.
However, their use was limited by a high compound polarity,
leading to poor bioavailability and low brain penetration.
Moreover, the orthosteric site is highly conserved among mGluR
subtypes, further complicating the medicinal chemistry way to
selective drug candidates.7 In contrast, negative allosteric
modulators (NAMs) are in general more lipophilic, since they
were optimized to bind to the lipophilic transmembrane region,
and mGluR5 NAMs were shown to exhibit properties, which are
in accordance with the use as CNS-acting compounds. Since
The mGluR family of GPCRs clusters into three groups based
on their structural similarity, signal transduction pathway, and
main synaptic localization. Group I comprises mGluR1 and
mGluR5, group II comprises mGluR2 and mGluR3, and mGlu
receptor subtypes 4, 6, 7, and 8 are assigned to group III. Group I
receptors bind to Gq and are positively coupled to phospholipase
———
∗ Corresponding author. Tel.: +49-(0)69-1503-8156; fax: +49-(0)69-1503-188; e-mail: ulrich.abel@merz.de

NAMs exert their modulatory effect in a non-competitive way,
the tempo-spatial signaling pattern of endogenous glutamate is
maintained and modulated rather than being fully suppressed.
containing the calculated conformers of the respective known
actives (groups 1 and 2) and inactives (groups 3, 4 and 5) of
mGluR5 and mGluR1, respectively, were created by Schrödinger
Phase and Schrödinger Macromodel. In Schrödinger Phase an
alignment of 19 mGluR5-active in-house compounds and
literature compounds was performed, and a set of 30 different
pharmacophores was created, each being defined by four to seven
pharmacophoric features. Each pharmacophore was analyzed
against the evaluation databases in terms of the ability to
distinguish between known actives and decoys using early
enrichment factors (EF1% and EF2.5%) and ROC-AUC.24 The
eight best performing pharmacophores were additionally tested to
ensure an at least 10-fold selectivity against mGluR1 activity.
Finally, two four-point-pharmacophore models performing best
according to these criteria were selected (Figure 2).
mGluR5 NAMs have been clinically tested in L-DOPA
induced dyskinesia (LID),8 anxiety,6 Fragile-X syndrome (FXS),9
gastro-esophageal reflux disease (GERD),10 migraine,11 and
chorea in Huntington´s disease.12 Moreover, compounds were
evaluated preclinically in models of chronic pain13 and drug
addiction.14 Most advanced clinical candidates are Novartis´
Mavoglurant (AFQ-056, I),15 Addex´ Dipraglurant (ADX48621,
II),16 and Roche´s RG-7090 (III),17 all of which have been tested
in phase II clinical trials (Figure 1). Fenobam (IV) was
developed by McNeil Laboratories in the 1970s as an anxiolytic
agent and was later found to be a potent mGluR5 NAM.18
Eventually, it was developed by Neuropharm Group as an orphan
drug and evaluated in FXS patients in a phase 2 clinical trial.
However, its development appeared to be discontinued. In
addition, several lead compounds are currently under preclinical
development such as Novartis’ compound 16m (V, Figure 1)19,
which interestingly is structurally very similar to hit compound 1
independently found by a virtual screening approach described in
this article.
A
B
Figure 2. Pharmacophore models A and B.
A conformer library of commercially available compounds as
described above was screened using the two selected
pharmacophore model scoring functions. Data fusion of 6,000
best scoring virtual hits from the two VS approaches was
applied.25 As a next step, the number of unique Bemis-Murcko
(BM) scaffolds26 was determined by means of the Pipeline Pilot
“generate fragments” component, and a k-nearest neighbor
clustering algorithm as implemented in Pipeline Pilot (version 7,
Accelrys Inc., 10188 Telesis Court, San Diego, CA 92121, USA)
was applied utilizing the previously determined number of
different BM scaffolds as cluster count. The VS hits were
combined into 312 clusters corresponding to their BM scaffolds.
Visual inspection of each cluster suggested 336 virtual hits in
total, which were finally purchased and tested for their in vitro
activities on mGluR5 and mGluR1, respectively.
Figure 1. Selected mGlu5 receptor negative allosteric modulators.
Many classical scaffolds described as potent mGluR5
allosteric modulator series are based on acetylenic chemotypes
structurally derived from well-known ligands like MPEP or
21
MTEP.20,
However, the triple bond motif has often been
associated with unfavorable properties like, e.g., cytochrome
P450 enzyme interactions, formation of chemically reactive
metabolites, and protein adduct formation, which may cause
undesired events in vivo.22 Therefore, our goal was to identify a
novel scaffold devoid of this potentially detrimental structural
feature. As a result of a virtual screening campaign applying two
different pharmacophore methodologies 2-aminoquinazolinone
derivative 1 (Figure 1) was discovered as a new, moderately
active mGluR5 negative allosteric modulator, serving as
chemical starting point for further optimization.
The pharmacological screening yielded eight novel scaffolds
(hitrate: 2.4%) with sufficient activity and selectivity, i.e.,
mGluR5 IC50-values below 10 µM and an at least 10-fold
selectivity against mGluR1. These scaffolds contained a variety
of non-acetylenic linker types such as, e.g., an amino group in
4,5,6,7-tetrahydrobenzo[d]thiazol derivative
0
(Figure 3)
discovered by means of pharmacophore A. Compound 0 showed
an IC50 value of 2.57 µM on mGluR5, which was determined
with a functional assay using the human mGlu receptor subtype
5, and a good selectivity against mGluR1 (~25-fold), and was
therefore chosen as initial starting point for further investigations.
Unfortunately, potency on target could not be improved
substantially in an initial medicinal chemistry optimization
round. Numerous modifications of the substitution pattern of
both the partially saturated benzothiazol ring system and the 4-
fluorophenyl residue did not result in IC50 values below 1 µM. In
order to identify additional scaffolds of the same or closely
related connection types, the database of commercially available
screening compounds was subsequently screened by a 2D-
similarity screening using FCFP-4 fingerprints.27 This screening
yielded a new mGluR5 chemotype represented by compound 1
(Figure 3), showing an IC50 value of 0.496 µM. In addition to the
submicromolar activity on target, compound 1 presented an
excellent selectivity toward the mGluR subtype 1 (est. IC50 >100
µM, selectivity >200-fold), which highlighted its suitability to
serve as a structural template for further optimization. This
In a first step toward a suitable virtual screening (VS) process
a screening database was generated from an in-house VS
compound library comprising 6.5 million unique commercially
available compounds, and up to 250 conformers per compound
were calculated using Schrödinger Phase (version 3.4,
Schrödinger, LLC, New York, NY, USA, 2009)23 and
Macromodel (version 9.7, Schrödinger, LLC, New York, NY,
USA, 2009). In the validation phase, a number of different
scoring functions were tested with respect to their ability to
identify known mGluR5 negative modulators (definition group 1:
IC50 (mGluR5) <125 nM and group 2: IC50 (mGluR5) <1 µM)
from decoy molecules (definition group 3: not active on mGluR)
as well as in-house and literature known inactives (definition
group 4: IC50 (mGluR5) >1 µM). It was also tested whether the
scoring function developed allowed a discrimination against
mGluR1 active compounds (group 5: IC50 (mGluR1) <125 nM)
as the major antitarget. For each group, evaluation databases

presumption was further proven by only subtle structural
modifications of compound 1 that led to a significant increase of
in vitro potencies, as described below.
employed for the synthesis of 3a–3e guanidine 9c was made from
4-difluoromethoxyaniline in the presence of cyanamide and nitric
acid in ethanol to yield the respective nitrate salt. In the case of
pyridylguanidines 9d and 9e the two-step procedure via the Boc-
protected intermediates 8c and 8d was carried out (Scheme 2).
Deprotection was accomplished with trifluoroacetic acid to form
corresponding TFA salts. In case of the cycloalkylguanidine 9f a
third strategy was applied using 1H-pyrazole-1-carboximidamide
hydrochloride instead of cyanamide.34
F
F
F
F
Figure 3. Virtual screening hits.
O
O
HNO3 NH
a
b
H2N
N
H2N
Initial structural modifications consisted in the methylation of
different positions of the bicyclic system as well as in systematic
variations of the tolyl residue by replacement with various aryl,
heteroaryl and cycloalkyl groups. In addition, the importance of
the carbonyl group was examined by designing derivatives
devoid of this functional group.
H
9c
CF3COOH
NH
Boc
R
R
R
N
Boc
N
N
N
c
N
H
N
H
H2N
N
H
H2N
8c: R=Cl
8d: R=Me
9d: R=Cl
9e: R=Me
Derivatives and analogs of hit structure 1, which could not be
purchased via commercial sources, were synthesized as outlined
in Schemes 1–7.28
HCl NH
H2N
d
N
H
H2N
9f
Scheme 3. Reagents and conditions: (a) H2NCN, HNO3,
O
O
HNO3 NH
H2N
R2
EtOH, reflux, 12 h; (b) (BocNH)2C=S, HgCl2, Et3N, DCM, 0
N
N
R2
oC to rt, 16–20 h; (c) TFA, DCM, rt, 5–20 h; (d) 1H-pyrazole-
R1
R1
R1
R1
N
H
a
N
N
H
O
1-carboximidamide*HCl, DMF, DIPEA, 60 oC, 13–15 h.
R1=H, Me
3a: R2=3-F
3b: R2=2-Cl
3c: R2=3-Cl
3d: R2=4-Cl
3e: R2=4-CN
4: R1=H, R2=3-F
5: R1=H, R2=2-Cl
6: R1=H, R2=3-Cl
7: R1=H, R2=4-Cl
13: R1=Me, R2=4-Cl
14: R1=Me, R2=4-CN
Preparation of the chiral 7-mono-methylated 2-aminoquinolin-
5-one derivatives shown in Scheme
4 started with the
dimethylaminomethylation of symmetric 5-methylcyclohexane-
1,3-dione by means of N,N-dimethylformamide dimethyl acetal
(DMF-DMA) to form intermediate 18 which was either
condensed with corresponding substituted guanidines to obtain
final compounds 19–22, 26, and 28–30, or with unsubstituted
guanidine to yield 2-amino-7-methyl-7,8-dihydroquinazolin-
5(6H)-one (23). The subsequent palladium-catalyzed Buchwald-
Hartwig coupling of intermediate 23 with an appropriate aryl
halide provided target compounds 24 and 27.
Scheme 1. Reagents and conditions: (a) Et3N, EtOH, reflux,
3–24 h.
Diversely substituted 2-aminoquinoline-5-one derivatives 4–7,
10–11, and 13–14 were prepared via direct condensation of 2-
((dimethylamino)methylene)cyclohexane-1,3-dione or its 5,5-
dimethylated derivative29 with the corresponding arylguanidines
3a–3e or 9a–9b at elevated temperature (Schemes 1 and 2).
While the synthesis of 3a–3e was successfully performed
following literature procedures,30-32 a different strategy was
employed for 3-cyanophenyl derivatives 9a and 9b. The
appropriate aniline was first reacted with di-Boc-protected
thiourea in the presence of mercury dichloride to form
intermediates 8a and 8b, respectively, which were subsequently
deprotected under acidic conditions to furnish corresponding
guanidines (Scheme 2).33 Final compounds 10 and 11 were made
according to the condensation procedure described above.
Scheme 4. Reagents and conditions: (a) DMF-DMA, EtOAc,
rt, overnight; (b) Et3N, EtOH, reflux, 3–24 h; (c)
guanidine*HCl, Et3N, EtOH, reflux, 10 h; (d) 4-bomo-2-
fluorobenzonitrile, Pd(OAc)2, XantPhos, Cs2CO3, dioxane,
argon, MWI, 80 oC, 2 h; (e) 5-chloro-2-iodopyridine,
Pd(OAc)2, XanthPhos, Cs2CO3, dioxane, argon, 90 oC, 6 h.
The corresponding 7-ethyl (32) and 7-propyl (33) homologs of
compound 21 (Scheme 5) as well as the 6,6- and 8,8-
dimethylated analogs 35–38 (Scheme 6) were made in close
analogy to the procedure described above. 5-Ethylcyclohexane-
1,3-dione and 5-propylcyclohexane-1,3-dione, respectively,
served as starting materials to prepare final products 32 and 33
(Scheme 5). The reaction of 4,4-dimethylcyclohexane-1,3-dione
with corresponding guanidines 3f35 and 3g32 initially produced
pairs of regioisomers, 35 and 37 or 36 and 38, which were then
separated by means of column chromatography (Scheme 6).
Scheme 2. Reagents and conditions: (a) (BocNH)2C=S,
HgCl2, Et3N, DCM, 0 oC, 15 min, rt, 1 h, reflux, 4 h; (b) 1.
HCl/dioxane, rt, 24 h; 2. K2CO3; (c) Et3N, EtOH, reflux, 3–24
h.
Methods used to prepare variously substituted guanidines are
summarized in Scheme 3. In analogy to the literature procedure

N
N
N
+
a
O
O
b
c
O
N
39
R
N
N
H
Cl
Scheme 5. Reagents and conditions: (a) DMF-DMA,
benzene, reflux, 4–6 h; (b) 3d (as free base), EtOH, reflux, 8–
24 h.
N
40: R=3-Cl
41: R=4-Cl
+
N
Br
H2N
Scheme 7. (a) argon, rt, 24 h, 110 oC, 1 h; (b) 3c, Et3N, EtOH,
reflux, 6 h; (c) Cs2CO3, PdCl2(dppf)*DCM, toluene, 100 oC,
16 h.
All the compounds were tested for activities at the human
mGlu5 receptor (expressed in CHO cells) in a functional assay
based on the detection of intracellularly mobilized calcium by use
of a calcium sensitive dye (for details see supplementary
material). For all compounds which were found to be active
(%inhibition >50%) at 10 µM test concentration follow-up
measurements were conducted as concentration response curves
to determine their functional potencies. Most active compounds
were further characterized on mGluR5 in primary cells (rat
primary astrocytes, calcium readout as described above),
confirming their mGluR5 NAM activities with good agreement
of potencies at rat and human mGluR5.
Scheme 6. Reagents and conditions: (a) DMF-DMA, MeCN,
rt, overnight; (b) Et3N, EtOH, reflux, 6 h.
Compounds devoid of a carbonyl group were prepared
following the synthesis routes depicted in Scheme 7. Synthesis of
the intermediate 39 was performed using cyclohexanone and 1-
tert-butoxy-N,N,N',N'-tetramethylmethanediamine instead of
DMF-DMA.36 Since all attempts to isolate pure 39 failed the
crude intermediate was directly reacted with the corresponding
arylguanidine 3c to yield final compound 40. For the preparation
of 4-chlorophenyl analog 41 an alternative route was applied by
Surprisingly, compound 35 potentiated rather than inhibited
the mGluR5 agonist signal and was therefore characterized as
positive allosteric modulator (PAM). Consequently, we
determined an EC50 value for the potentiation of a basal agonist
signal. Preincubation of 10 µM of this compound shifted an
agonist concentration curve to the left and led to a 2-fold higher
potency compared to the agonist potency in the presence of
vehicle alone. In control experiments compound 36 and 24
additional examples from the presented series had no significant
effect on mGluR1-mediated Ca-signaling. However, in a first
selectivity screen against several unrelated pharmacological
targets monoamine oxidase B (MAO-B) was found to be a
significantly affected off-target (Table 1).
coupling
commercially
available
2-bromo-5,6,7,8-
tetrahydroquinazoline with 4-chloroaniline using 1,1'-
bis(diphenylphosphino)ferrocene-palladium(II)dichloride
dichloromethane complex as a suitable catalyst.

Table 1. mGluR5 and MAO-B in vitro potency data
6
7
8
Human mGluR5 (CHO)
IC50 (µM)a
Rat mGluR5 (rpA)
IC50 (µM)a
Compound ID
X
Y
R
MAO-B IC50 (µM)b
1
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
H
3-Me-Ph
0.496 0.12
>100
0.194
nt
2
H
3,5-di-Me-Ph
3-F-Ph
nt
nt
4
H
2.23
nt
nt
5
H
2-Cl-Ph
>100
nt
nt
6
H
3-Cl-Ph
0.120 0.034
0.722
nt
0.0718
7
H
4-Cl-Ph
nt
nt
10
11
12
13
14
15
16
17
19
20
21
22
24
25
26
27
28
29
H
3-CN-Ph
3-CN-5-F-Ph
4-Me-Ph
4-Cl-Ph
0.272 0.054
11.0
nt
0.927
H
nt
nt
7,7-di-Me
7,7-di-Me
7,7-di-Me
7-Me (rac)
7-Me (E1)
7-Me (E2)
7-Me (rac)
7-Me (rac)
7-Me (rac)
7-Me (rac)
7-Me (rac)
7-Me (rac)
7-Me (rac)
7-Me (rac)
7-Me (rac)
7-Me (rac)
0.492
0.356
nt
0.245 0.0078
0.463
nt
0.0344
4-CN-Ph
4-Me-Ph
4-Me-Ph
4-Me-Ph
2-Cl-Ph
nt
nt
0.0936 0.019
0.085 0.0080
0.170 0.025
>100
0.0578
0.00798
nt
(53 % inh. at 10 nM)
nt
(72 % inh. at 10 nM)
nt
nt
3-Cl-Ph
6.16
nt
nt
4-Cl-Ph
0.0343 0.0033
0.0831 0.012
0.157 0.043
0.598
nt
(87 % inh. at 10 nM)
4-CN-Ph
4-CN-3-F-Ph
4-OMe-Ph
4-OCHF2-Ph
5-Cl-2-Py
6-Cl-3-Py
6-Me-3-Py
0.0346 0.006
0.00450
nt
nt
0.481
nt
0.125 0.038
0.721
nt
nt
nt
nt
0.0432 0.0079
0.111 0.020
0.0392 0.012
0.0831 0.012
(101 % inh. at 10 µM)
0.0650
30
C=O
7-Me (rac)
1.13
nt
nt
32
33
C=O
C=O
7-Et (rac)
7-Pr (rac)
4-Cl-Ph
4-Cl-Ph
1.21
nt
nt
nt
nt
>100
(EC50: 0.694 0.13;
35
C=O
6,6-di-Me
3-Me-Ph
nt
nt
left shift: 1.95 0.21)c
36
37
38
40
41
C=O
C=O
C=O
CH2
CH2
6,6-di-Me
8,8-di-Me
8,8-di-Me
H
4-Me-Ph
3-Me-Ph
4-Me-Ph
3-Cl-Ph
4-Cl-Ph
>100
nt
nt
nt
nt
nt
nt
1.04 0.18
>100
nt
nt
0.452 0.043
4.71
0.0779
nt
H
a IC50 values are given as geometric means SEM of at least three independent experiments, each performed in quadruplicate (no SEM-values indicate a lower
number of independent experiments);
b MAO-B IC50 values are given from one experiment performed in duplicate. Values in brackets are results with a concentration range of insufficient coverage to
get a four-parameter fit: % inhibition at the lowest concentration tested;
c EC50-value is given as the geometric mean of three independent replicates, each performed in quadruplicate. The left shift represents the arithmetic mean SEM
of three independent shifting experiments, each performed in quadruplicate with 10 µM compound concentration vs. vehicle.

Structure-activity relationships with respect to mGluR5 in
vitro activity are summarized in Table 1. Starting from the
moderately active virtual screening hit 1 (human IC50: 0.496 µM,
rat IC50: 0.194 µM) systematic modifications were carried out in
order to explore the active chemical space around the 2-
aminoquinazolin-5-one scaffold. At first, various substituted
phenyl groups were introduced as residue R. Interestingly,
commercially available 3,5-dimethylated analog 2 led to a total
loss of activity indicating that only subtle changes could have a
fundamental impact on receptor binding, a presage which was
further confirmed by subsequent variations. Within the halogen
series a small electron-withdrawing fluoro substituent in 3-
position turned out to be detrimental to in vitro potency
(compound 4, hIC50 = 2.23 µM) compared to hit compound 1,
whereas the larger and more lipophilic chlorine atom in
compound 6 significantly improved the potency of 1 6-fold.
Results obtained for respective 2-chloro isomer 5 (inactive at 10
µM) and 4-chloro analog 7 (hIC50 = 0.722 µM) showed that the
3-position of the phenyl ring was favorable. While 3-cyano
derivative 10 (hIC50 = 0.272 µM) was found to be reasonably
active, an additional fluoro substituent led to a dramatic drop of
potency (compound 11, IC50 = 11.0 µM), although the 3-CN-5-F-
phenyl group has often been successfully employed in mGluR5
NAM ligand design in recent years.14 The potency of the so far
most active compound 6 could not be increased by further
modifications of residue R suggesting that the variability at this
part of the molecule is spatially limited and requires a certain
lipophilicity (data not shown).
range, as shown before. However, monomethylation in 7-position
still proved to be advantageous highlighted by a nearly 4-fold
higher activity of compound 21 as compared to compound 6. In
analogy to compound 5, 2-substitution at the phenyl residue was
again shown to be unfavorable demonstrated by compound 19
(inactive at 10 µM).
Additional variations of the phenyl residue of 7,7-
dimethylated compound 13, e.g., replacement of the chloro
substituent by a methyl group (12) or by a cyano group (14) did
not improve the potency further, marking the 4-chlorophenyl
moiety to be the most effective substitution pattern within this
series. All attempts to replace the substituted phenyl by a
heteroaryl group such as pyridine or triazole resulted in a
dramatic drop or even a complete loss of activity (data not
shown).
A
slightly different trend was observed for the 7-
monomethylated compound series. In addition to
‘conventionally’ substituted phenyl derivatives like compounds
15 (4-Me, hIC50 = 0.0936 µM), 21 (4-Cl, hIC50 = 0.0343 µM), 22
(4-CN, hIC50 = 0.0831 µM), and the markedly less active 25 (4-
OMe, hIC50 = 0.598 µM) also substituted pyridines were
tolerated here (27–29). A 6-chloropyridin-3-yl residue turned out
to be the most successful heteroaryl substitution leading to an
IC50 value of 0.0432 µM (28). Interestingly, the potency of 25
could be markedly improved 5-fold by replacing the methoxy
group by a difluoromethoxy moiety (26). Moreover, it was
figured that aromaticity in this part of the molecule was
mandatory for reaching high potencies evinced by the 12-fold
weaker potency of 4-methylcyclohexyl derivative 30 compared to
its aromatic analog 15.
In a next step, the bicyclic ring system was modified by
appending one or two methyl groups at distinct positions of the
saturated part of the quinazolin-5-one scaffold, i.e., 6-, 7-, or 8-
position. This approach has already been successfully applied in
previous SAR studies on acetylenic mGlu5 receptor ligands to
gain antagonistic activity (Figure 4).37 Back then we observed
that within the 3-ethinylquinolinone series methylation in 7-
position caused a molecular switch from a PAM (Example 125)
to potent NAMs (Examples 1 and 106, respectively). This
phenomenon as a result of a subtle structural change has already
been reported for other mGluR5 ligand series, in particular in the
context of acetylenic scaffolds.38, 39
Beside the increase of in vitro potency the incorporation of a
single methyl group in 7-position also generated
a new
stereocenter and therefore two possible enantiomers. In order to
investigate the impact of stereochemistry on potency, the racemic
mixture of tolyl derivative 15 was separated into its enantiomers
16 and 17. In vitro activity data revealed that the difference
between stereoisomers 16 and 17 was only 2-fold, which could
be explained by the small size of the methyl group and a certain
flexibility of the partly saturated region of the ring system. The
more active enantiomer 16, which absolute configuration has not
been determined, showed an IC50 of 0.0850 µM.
O
O
O
molecular
switch
Investigations with respect to the most appropriate alkyl
length in 7-position revealed that methyl as the smallest group
was favored since in vitro potencies dropped rapidly with
extension to ethyl (32, hIC50 = 1.23 µM) and propyl (33, inactive
at 10 µM).
PAM
N
N
N
Example 125 (in ref. [37])
Example 1 (in ref. [37])
Example 106 (in ref. [37])
hEC50 = 0.0307 µM
hIC50 = 0.0684 µM
hIC50 = 0.0278 µM
O
O
O
Cl
Cl
Cl
N
N
N
N
N
N
N
N
N
Interestingly, the addition of two methyl groups to the 6-
instead of the 7-position of the bicyclic ring system resulted in a
molecular switch from negative (1, hIC50 = 0.496 µM) to positive
(35, hEC50 = 0.694 µM, left shift = 2.0-fold) allosteric
modulation (Figure 5). The reverse case was observed during an
earlier study on the acetylenic mGluR5 ligand series around
compounds MRZ-3573 and MRZ-8676 where methylation led to
a switch from a PAM to a NAM.40 The agonistic activity of
compound 35, however, was lost when the N-substitution was
altered from meta- to para-tolyl. The resulting compound 36 was
found to be neither a PAM nor a NAM.
H
H
H
7
13
hIC50 = 0.245 µM
21
hIC50 = 0.0343 µM
hIC50 = 0.722 µM
Figure 4. Impact of methyl substituents on mGluR5 negative allosteric
modulatory activity.
In the present study we compared unmethylated compound 7
with its 7-mono- and 7,7-dimethylated analogs. As a result, in
vitro potency on target was increased almost 3-fold by adding
two methyl groups (13) and even 20-fold when only one methyl
group was appended (21). One obvious reason for this gain in
activity was the fact that the preferred position at the phenyl
residue switched from meta to para after methylation of the 7-
position. 3-Chlorophenyl substituted analog 20 showed only a
weak antagonistic activity with an IC50 value of 6.16 µM whereas
its unmethylated counterpart 6 was active in the submicromolar

finding is in line with the calculated ALogP41 values (3.32, 3.39,
3.57, and 3.71, respectively). However, solubility results were
markedly improved by introducing pyridine-containing analogs
such as compound 29 (solubility in assay medium >250 µM,
ALogP = 2.03), revealing that the solubility issue is likely to be
solved within the next optimization round. Notably, respective
melting points were found to be in the same range for each of the
five compounds (205–238 oC). This suggested that the distinction
between the solubilities determined for phenyl- and pyridyl-
containing compounds were not driven by different crystallinity
states.
In summary, we have identified and evaluated a new non-
acetylenic chemotype comprising active, predominantly negative
allosteric modulators of mGlu receptor subtype 5 which were
developed based on a virtual screening hit. Most potent
compounds show IC50 values in the low nanomolar range and an
excellent selectivity against mGluR subtype 1. By means of
distinct modifications of the scaffold structure a molecular switch
to positive allosteric modulation was accomplished, an event
which has rarely been described for mGluR5 ligand series devoid
of a triple bond linker motif. In further optimization steps toward
a suitable drug candidate for the treatment of L-DOPA induced
dyskinesia the relatively poor solubility in aqueous media as well
as the strong inhibitory effect on the enzyme MAO-B will have
to be addressed, and respective results will be reported in due
course.
Figure 5. Molecular switches through addition of methyl groups.
Eventually, compounds bearing two methyl groups in 8-
position were evaluated regarding their pharmacological in vitro
profile. As already spotted for the unmethylated and the 6,6-
dimethylated analogs, this species also preferred 3-substitution at
the phenyl residue as shown by the compound pair 37 (m-tolyl,
IC50 = 1.04 µM) and 38 (4-tolyl, inactive at 10 µM). However,
IC50 values obtained for compounds of this subseries did not
reach the nanomolar range.
It is worth noting that respective unsubstituted phenyl
derivatives of the presented compound subseries were found to
be only weakly active (e.g., the analog of compound 1 showed an
IC50 of ~25 µM, details not shown). As ‘zero point’ for our SAR
investigations we used the m-tolyl substitution pattern of virtual
screening hit 1 instead.
Acknowledgments
The authors warmly thank Andrea Baude, Sabine Falk and
Christiane Reinbach for expert technical assistance in in vitro
activity experiments, Dr. Barbara Valastro for providing the
MAO-B data, and Jens Neubauer and Kathleen Wolff for
solubility determinations.
The question on how much influence the carbonyl group
exerts on receptor binding was addressed by means of
compounds 40 (hIC50 = 0.452 µM) and 41 (hIC50 = 4.71 µM).
Compared to their carbonyl-containing counterparts 6 and 7 these
‘reduced’ products displayed a 4–5 times weaker potency on
mGluR5. Although the differences were not that huge, the polar
C=O moiety was deemed to be a more beneficial structural
feature as compared with a lipophilic methylene group.
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Supplementary Material
Supplementary data (experimental details for the synthesis and
characterization of intermediate and final products) associated
with this article can be found, in the online version, at http://...

Captions
Figure 1. Selected mGlu5 receptor negative allosteric modulators.
Figure 2. Pharmacophore models A and B.
Figure 3. Virtual screening hits.
Figure 4. Impact of methyl substituents on mGluR5 negative allosteric
modulatory activity.
Figure 5. Molecular switches through addition of methyl groups.
Scheme 1. Reagents and conditions: (a) Et3N, EtOH, reflux,
3–24 h.
Scheme 2. Reagents and conditions: (a) (BocNH)2C=S,
HgCl2, Et3N, DCM, 0 oC, 15 min, rt, 1 h, reflux, 4 h; (b) 1.
HCl/dioxane, rt, 24 h; 2. K2CO3; (c) Et3N, EtOH, reflux, 3–24
h.
Scheme 3. Reagents and conditions: (a) H2NCN, HNO3,
EtOH, reflux, 12 h; (b) (BocNH)2C=S, HgCl2, Et3N, DCM, 0
oC to rt, 16–20 h; (c) TFA, DCM, rt, 5–20 h; (d) 1H-pyrazole-
1-carboximidamide*HCl, DMF, DIPEA, 60 oC, 13–15 h.
Scheme 4. Reagents and conditions: (a) DMF-DMA, EtOAc,
rt, overnight; (b) Et3N, EtOH, reflux, 3–24 h; (c)
guanidine*HCl, Et3N, EtOH, reflux, 10 h; (d) 4-bomo-2-
fluorobenzonitrile, Pd(OAc)2, XantPhos, Cs2CO3, dioxane,
argon, MWI, 80 oC, 2 h; (e) 5-chloro-2-iodopyridine,
Pd(OAc)2, XanthPhos, Cs2CO3, dioxane, argon, 90 oC, 6 h.
Scheme 5. Reagents and conditions: (a) DMF-DMA,
benzene, reflux, 4–6 h; (b) 3d (as free base), EtOH, reflux, 8–
24 h.
Scheme 6. Reagents and conditions: (a) DMF-DMA, MeCN,
rt, overnight; (b) Et3N, EtOH, reflux, 6 h.
Scheme 7. (a) argon, rt, 24 h, 110 oC, 1 h; (b) 3c, Et3N, EtOH,
reflux, 6 h; (c) Cs2CO3, PdCl2(dppf)*DCM, toluene, 100 oC,
16 h.