Discovery of novel positive allosteric modulators of the metabotropic glutamate receptor 5 (mGlu5)

Article abstract of DOI:10.1016/j.bmcl.2011.01.027

Novel in vitro mGlu₅ positive allosteric modulators with good potency, solubility, and low lipophilicity are described. Compounds were identified which did not rely on the phenylacetylene and carbonyl functionalities previously observed to be required for in vitro activity. Investigation of the allosteric binding requirements of a series of dihydroquinolinone analogs led to phenylacetylene azachromanone 4 (EC ₅₀ 11.5 nM). Because of risks associated with potential metabolic and toxicological liabilities of the phenylacetylene, this moiety was successfully replaced with a phenoxymethyl group (27; EC₅₀ 156.3 nM). Derivation of a second-generation of mGlu₅ PAMs lacking a ketone carbonyl resulted in azaindoline (33), azabenzimidazole (36), and N-methyl 8-azaoxazine (39) phenylacetylenes. By scoping nitrogen substituents and phenylacetylene replacements in 39, we identified phenoxymethyl 8-azaoxazine 47 (EC₅₀ 50.1 nM) as a potent and soluble mGlu₅ PAM devoid of both undesirable phenylacetylene and carbonyl functionalities.

Full text of DOI:10.1016/j.bmcl.2011.01.027

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1402–1406
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel positive allosteric modulators of the metabotropic
glutamate receptor 5 (mGlu5)
⇑
Jeffrey G. Varnes , Andrew P. Marcus, Russell C. Mauger, Scott R. Throner, Valerie Hoesch, Megan M. King,
Xia Wang, Linda A. Sygowski, Nathan Spear, Reto Gadient, Dean G. Brown, James B. Campbell
CNS Discovery Research, AstraZeneca Pharmaceuticals, 1800 Concord Pike, Wilmington, DE 19850, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel in vitro mGlu5 positive allosteric modulators with good potency, solubility, and low lipophilicity
are described. Compounds were identified which did not rely on the phenylacetylene and carbonyl func-
tionalities previously observed to be required for in vitro activity. Investigation of the allosteric binding
requirements of a series of dihydroquinolinone analogs led to phenylacetylene azachromanone 4 (EC50
11.5 nM). Because of risks associated with potential metabolic and toxicological liabilities of the phenyl-
acetylene, this moiety was successfully replaced with a phenoxymethyl group (27; EC50 156.3 nM). Der-
ivation of a second-generation of mGlu5 PAMs lacking a ketone carbonyl resulted in azaindoline (33),
azabenzimidazole (36), and N-methyl 8-azaoxazine (39) phenylacetylenes. By scoping nitrogen substitu-
ents and phenylacetylene replacements in 39, we identified phenoxymethyl 8-azaoxazine 47 (EC50
50.1 nM) as a potent and soluble mGlu5 PAM devoid of both undesirable phenylacetylene and carbonyl
functionalities.
Received 11 November 2010
Revised 5 January 2011
Accepted 7 January 2011
Available online 11 January 2011
Keywords:
mGlu5
Metabotropic glutamate receptor 5
Glutamate
NMDAr
N-Methyl-D-aspartate receptor
Positive allosteric modulator
Schizophrenia
Ó 2011 Elsevier Ltd. All rights reserved.
O
Approximately 1% of the world’s population is affected by
schizophrenia. Current therapies, however, are associated with un-
wanted side effects and also do not provide sufficient relief across
O
NH
HBA1
HBA2
Phenylacetylene
N
all symptom domains.1,2 Hypofunction of the N-methyl-
D-aspar-
O
tate receptor (NMDAr) has been implicated in the symptoms of
schizophrenia, which makes activation of GPCRs with the ability
to normalize NMDAr signaling a promising approach for the devel-
opment of antipsychotics.3
1
2
EC50 172 nM
EC50 5.6 nM
Figure 1. Pharmacophore features and reported EC50’s of 1 and 2.
One receptor with the ability to potentiate NMDAr glutamater-
gic postsynaptic neurotransmission is the metabotropic glutamate
receptor 5 (mGlu5).4 Achieving mGlu receptor subtype selectivity
with orthosteric agonists remains challenging due to highly con-
served extracellular binding sites. As a result, current strategies
to selectively enhance mGlu5 signaling in the presence of its
endogenous ligand glutamate have focused on the discovery of
compounds which bind to the less well conserved allosteric mod-
ulatory site.5
In 2008, Merz Pharmaceuticals and Vanderbilt University re-
ported dihydroquinolinone 16 and phthalimide 27 (Fig. 1), respec-
tively, as mGlu5 positive allosteric modulators (PAMs). Both
compounds possess a phenylacetylene which is also found in well
known mGlu5 negative allosteric modulators such as MPEP and
MTEP.8 Based on published data, we utilized a working hypothesis
that one or both of the oxygen lone pairs in 1 (HBA1 as in Fig. 1)
could possibly serve as a rigid hydrogen-bond acceptor. Further-
more, evidence also suggested that another possible hydrogen-
bond acceptor (HBA2) could be accommodated in the form of
either an aromatic nitrogen (1) or a second carbonyl (2). The sim-
ilarity of these features suggests that both compounds potentially
interact with the same or similar spatial regions of the allosteric
binding pocket. Our efforts to discover novel mGlu5 PAMs have fo-
cused on exploiting these common pharmacophore features using
hypothesis-driven design to enhance our understanding of alloste-
ric binding requirements while also optimizing physiochemical
profiles for in vivo testing.
Compounds were prepared through synthesis or purchase of
heteroaryl halides, alcohols, or triflates, which were then elabo-
rated into final compounds using known literature conditions
(Fig. 2; see Supplementary data).
⇑
Corresponding author. Tel.: +1 781 839 4944.
E-mail address: jeffrey.varnes@astrazeneca.com (J.G. Varnes).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.027

J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1402–1406
1403
Table 1
Core modifications to probe HBA1 and HBA2
Ph
Ar
EC50 (nM) Solb M) clogPc
(l
a
Compd Ar
pEC50
O
1
7.85 0.02
7.94 0.16
14.0
11.6
22
3.7
3.8
(a) Sonogashira/Stille coupling.
(b) Suzuki coupling.
(c) Hydrogenation.
(e) Ullmann coupling/Alkoxide addn.
(f) Suzuki coupling (Molander reagent)
(g) Mitsunobu reaction.
N
O
(d) Alkylation.
(h) Acylation.
NH
2
>600
Figure 2. Installation of phenylacetylene and replacement side chains on hetero-
aromatic/aromatic (Ar) cores.
O
O
4
6
7.94 0.03
7.27 0.13
11.5
53.2
23
16
3.4
3.0
The syntheses of unique heteroaryl cores and their correspond-
ing final compounds are described in Schemes 1 and 2. Azachroma-
none 4 was synthesized in five steps from 2,6-dichloropyridine
(Scheme 1). Lithiation, followed by quenching with TBS-protected
3-hydroxypropanal, exclusively afforded addition at the 3-position
of the pyridyl ring. Silyl cleavage then yielded diol 3, which cy-
clized upon exposure to potassium tert-butoxide in tert-butanol
at elevated temperature. Incorporation of the phenylacetylene
group then proceeded smoothly under either Stille or Sonogashira
conditions. Higher yields were obtained if this coupling was per-
formed prior to (rather than following) Swern oxidation.
N
O
O
N
N
O
7
8
6.29 0.09 518.8
<1
4.4
4.0
O
7.04 0.10
91.9
ntd
To prepare azaquinolizinone 6, commercially available 4-bro-
mo-2-aminopyridine (Scheme 2) was reacted with the methoxym-
ethylene derivative of Meldrum’s acid, followed by heating to
240 °C to form azaquinolizinone bromide 5. Stille coupling condi-
tions were then used to append the phenylacetylene group.
Initial efforts to develop novel mGlu5 PAMs focused on modifi-
cation of the heteroaryl cores of 1 and 2 while maintaining the
putative HBA1 interaction with the allosteric binding pocket (Ta-
ble 1). Specifically, we examined the impact of subtle changes in
ring size, ring saturation, and carbonyl C–O bond polarization to
evaluate the geometric and electronic constraints of this hydrogen
bond. For example, known lactams 127 and 137 were >13-fold
more potent than ketones 7 and 9. While the potential impact of
a newly introduced amide N–H as a hydrogen bond donor should
not be discounted, the observed potency increase supports the
concept of a stronger hydrogen bond between the allosteric bind-
ing pocket and an amide oxygen versus that with a ketone oxygen.
Changes in ring size (7 to 9, 8 to 10, and 12 to 13) had very little
O
O
9
6.23 0.10 588.8
<1
1
4.8
4.4
O
10
7.53 0.07
29.9
O
O
11
6.93 0.14 117.5
<1
7
4.0
3.5
3.6
3.5
O
O
127
137
14
7.53 0.08
7.35 0.09
29.5
44.3
NH
O
O
1
NH
NH
6.74 0.01 182.0
<1
a
Data are the average of at least two experiments. For details on pEC50 and EC50
determination, Ref. 9.
b
Equilibrium solubility (pH 7.4).10
Calculated logP.
nt = not tested.
c
d
Scheme 1. Reagents and conditions: (a) LDA, HCOCH2CH2OTBS, THF, À78 °C, 75%;
(b) TBAF, THF, 0 °C, 92%; (c) t-BuOK, t-BuOH, 85 °C, 96%; (d) Bu3SnCCPh, Pd(PPh3)4,
PhMe, 110 °C, 71%; (e) (COCl)2, DMSO, Et3N, CH2Cl2, À78 °C, 84%.
O
effect, while the introduction of unsaturation (10 to 11 and 13 to
14) consistently decreased potency by approximately 4-fold. If it
is assumed that the rigid phenylacetylene occupies the same re-
gion of space for the compounds in question, these results suggest
that some movement of the carbonyl within the allosteric binding
pocket is tolerated. What is not clear is if increased planarity intro-
duced by unsaturation decreases potency due to steric constraints,
decreased movement of the carbonyl (thereby limiting the ability
to form a hydrogen bond) or both. We had expected the vinylogous
O
N
a, b
c
N
N
Br
NH2
R
N
Br
N
6
5
Ph
R =
Scheme 2. Reagents and conditions: (a) (CH3)2C(OCO)2CHCH2OCH3, iPrOH, 82 °C,
85%; (b) Ph2O, 240 °C, 86%; (c) Bu3SnCCPh, Pd(PPh3)4, PhMe, W, 110 °C, 47%.
l

1404
J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1402–1406
Table 2
ester in 11 to increase electron density on the carbonyl oxygen,
thereby making it a better hydrogen bond acceptor. Although 11
is still a reasonably potent compound, this hypothesis did not
translate to dramatic potency shifts, perhaps due to other newly
introduced negative interactions with the added functionality.
We also evaluated the proposed HBA2 interactions of 1 and 2. In
particular, we were keen to determine if the pyridyl nitrogen of 1
and the basal carbonyl of 2 were forming hydrogen bonds of equal
importance, potentially with the same region(s) of the mGlu5 allo-
steric binding pocket. This theory was supported by molecular
overlays but not substantiated by in vitro data. Thus, there is a
>40-fold potency difference between 1 and its des-aza analog 9.
In contrast, there is only a 2- to 3-fold decrease in potency going
from phthalimide 2 to isoindolone 12. These results suggest that
the contributions to potency of the pyridyl nitrogen of 1 and the
basal carbonyl of 2 are not equivalent.
Chromanone and azachromanone phenylacetylene analogs
O
R
X
O
a
#
R
X
pEC50
EC50 (nM)
Solb
(l
M)
cLogPc
10
15
16
17
18
19
20
21
22
4
23
24
25
26
27
28
PhCC
PhCH2O
C
C
C
C
C
C
C
C
C
N
N
N
N
N
N
N
7.53 0.07
6.78 0.25
6.68 0.09
6.59 0.18
6.53 0.18
6.24 0.27
6.11 0.21
6.46 0.18
7.53 0.30
7.94 0.03
6.97 0.20
6.69 0.14
6.62 0.18
6.28 0.36
6.81 0.16
6.34 0.09
29.9
167.9
208.9
285.5
295.1
572.1
776.2
349.4
29.9
1
7
1
2
7
3
5
12
1
23
400
57
14
>480
64
4.4
3.5
4.2
3.7
2.9
4.0
3.7
3.4
4.4
3.4
3.1
3.7
3.5
3.2
3.0
2.5
3-Cl-PhCH2O
3-F-PhCH2O
3-CN-PhCH2O
3-Me-PhCH2O
4-F-PhCH2O
PhOCH2
PhC(O)O
PhCC
C5H9CC
C6H11CC
11.5
107.2
206.5
242.7
524.8
156.3
453.6
Much like the nitrogen of dihydroquinolinone 1, the aryl ether
oxygens of 8 and 10 also contributed significantly to mGlu5 poten-
tiation. Thus, benzofuranone 8 was >5-fold more potent than inde-
none 7. Similarly, chromanone 10 was >19-fold more active than
(E)-PhCHCH
PhCH2CH2
PhCH2O
PhOCH2
100
a-tetralone 9. To determine if the potency contributions exempli-
a
Data are the average of at least two experiments. For details on pEC50 and EC50
fied by the ring heteroatoms of 1 and 10 were additive, we de-
signed azachromanone 4 with a secondary goal of improving
determination, Ref. 9.
b
Equilibrium solubility (pH 7.4).10
Calculated logP.
physiochemical properties (Sol >10
l
M; clogP 6 3.5). While a slight
c
increase in potency was observed compared to 10, azachromanone
4 was equipotent with dihydroquinolinone 1. One hypothesis that
might explain these results is that both 1 and 10 are participating
in the same HBA2 interaction, which exists as a bifurcated hydro-
gen bond for azachromanone 4 (Fig. 3).
for all azachromanones relative to their chromanone counterparts.
It was also found that positive modulator activity persisted with
(E)-styrenyl (25) and phenethyl (26) derivatives, which was not
observed for the chromanone core (data not shown).
Despite our success with the compounds of Table 2, we had
concerns about the inherent reactivity of a ketone carbonyl and
its in vivo disposition. In agreement with literature results for 1,6
Due to the perceived potential metabolic and toxicological lia-
bilities of the acetylene linker, we chose to proactively develop a
strategy to identify an acetylene surrogate rather than run the risk
of retaining an intractable series flaw in the drug discovery pro-
cess. Using a matrix approach, acetylene replacements (e.g., styre-
nyl, phenethyl, 3-phenylazetidine, benzyloxy, phenoxymethyl,
3-phenylbicyclo[1.1.1]pentyl, etc.) were systematically surveyed
across multiple cores from Table 1.11 While many cores lost most,
if not all, activity once the acetylene was removed, the chromanone
core of 10 maintained good potency for several acetylene surro-
gates (Table 2). Thus, both benzyloxy (15) and phenoxymethyl
(21) chromanones exhibited good mGlu5 potentiation and had im-
proved solubility and lower clogPs compared to 10. Additionally,
both solubility and clogP could be further modulated through sub-
stitution of the terminal aromatic ring of 15 without appreciable
loss of activity (16–18). We were also intrigued to find that po-
tency could be maintained if the phenylacetylene of chromanone
10 was replaced with a simple benzoate moiety (22). Due to the
potential liability of the phenyl ester in vivo, we regard 22 as a tool
compound that has helped reinforce the possibility of finding
acetylene surrogates.
the racemic alcohol analog of 4 (EC50 3.0 lM) was much less active
than 4 itself. We also discovered that the azachromanone core had
moderate stability under acidic conditions, poor stability under ba-
sic and photolytic conditions, and significant microsomal degrada-
tion in vitro.12 While not prohibitive, taken together these factors
did adversely impact our ability to interpret in vitro/in vivo data
and effectively advance this series in a suitable time frame.
To develop a second generation of non-phenylacetylene, non-
carbonyl containing mGlu5 PAMs, we investigated a number of po-
tent pyrimidine phenylacetylenes disclosed by Vanderbilt Univer-
sity.13 One example is pyrimidine 29 (Table 3), which compares
favorably with both compounds of Figure 1 but lacks a carbonyl
(HBA1) and hydrogen bond acceptor directly analogous to the
HBA2 of either 4 or 10. We modified the pyrimidine core of 29 to
determine the scope of allosteric binding requirements and the tol-
erance for changes in physiochemical properties. Solubility was a
property we were particularly keen to improve. Conversion of
the pyrimidine ring to a pyridazine (32) or pyridine (not shown)
decreased or abolished activity, respectively. In contrast, cycliza-
tion of the aniline nitrogen to form azaindoline 33 improved po-
tency by twofold while subsequent alkylation (34) weakened
potency by >6-fold. Interestingly, benzimidazole 35 also exhibited
good solubility and potentiation of mGlu5 but had a higher than
desired clogP. These properties could be improved through re-
introduction of a pyridine nitrogen analogous to that of 33 to afford
36. The observed potency improvement is consistent with the pro-
posed role of the pyridine nitrogen as a hydrogen bond acceptor. Of
the pyrimidine, azaindoline, and azabenzimidazole series, very lit-
tle tolerance for phenylacetylene modification was observed (31,
37).
Both benzyloxy and phenoxymethyl azachromanones (27 and
28) also maintained good activity with respect to mGlu5. As seen
when comparing 4 and 10, solubilities and clogPs were improved
A chemotype that also exhibited potent positive modulation of
mGlu5 was N-methyl 8-azaoxazine 39. While initially designed
Figure 3. Proposed HBA2 interactions for compounds 1, 4, and 10.

J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1402–1406
1405
Table 3
O
O
O
N
Pyrimidine-derived mGlu5 PAMs
N
N
O
Ph
X Ar
#
X-Ar
pEC50
EC50 (nM) Solb M) clogPc
(l
a
N
R
N
R
R
H
N
N
N
N
39
39b
4
2913
7.51 0.18
30.7
<1
3.1
N
N
Figure 4. Rotation about the aryl-phenylacetylene bond of 39.
N
ether oxygen of 39b is potentially analogous to that of 4. It is also
clear that a carbonyl oxygen (HBA1) is not required for strong
mGlu5 allosteric modulation within this series.
30
31
32
6.95 0.07 113.1
6.25 0.33 566.7
6.40 0.14 401.2
<1
3.2
3.3
3.0
As demonstrated with chromanone 10, we hypothesized that
shifting the position of the pyridine nitrogen to afford 5-azaox-
azine 40 might improve potency (Table 4). Gratifyingly, 40 main-
tained roughly equivalent potency to 39 and also had
significantly improved solubility. However, when the des-methyl
analogs were compared, 8-azaoxazine 41 maintained activity but
5-azaoxazine 42 did not. Analysis in a membrane binding assay
in the presence of a proprietary mGlu5 NAM radioligand competi-
tive with MPEP revealed that 42 binds only weakly to the allosteric
site (data not shown). The discrepancy in activity between 41 and
42 underscores the potential difference in binding mode between
two very similar scaffolds. Acylation of 42 restored potency (43),
and the resulting acetamide also met our criteria for solubility
and clogP, prompting us to examine phenylacetylene replace-
ments. Of the surrogates examined, benzyloxy 44 maintained
moderate potency with respect to mGlu5.
N
N
ntd
ntd
N
N
N
NH
N
33
34
35
7.80 0.13
15.8
ntd
ntd
10
3.9
4.3
4.2
N
N
6.97 0.02 108.4
Table 4
N
Oxazine analogs as mGlu5 PAMs
N
N
N
EC50(nM) Solb
(l
M) cLogPc
a
#
Structure
pEC50
7.43 0.14
7.76 0.09
37.2
17.4
N
N
O
39
7.52 0.20 30.2
4
4.5
N
N
Ph
36
37
42
3.1
2.5
N
N
O
40
41
42
7.25 0.15 56.7
7.69 0.07 20.3
100
ntd
ntd
4.5
4.1
4.1
N
N
O
6.67 0.24 216.3
6.51 0.10 307.8
>550
ntd
Ph
Ph
N
N
H
N
O
O
38
39
4.0
4.5
H
N
O
O
4.60
>25,000
N
N
N
O
N
Ph
Ph
7.52 0.20
30.2
4
O
O
N
N
O
43
44
7.21 0.28 61.7
210
2.7
2.3
a
Data are the average of at least two experiments. For details on pEC50 and EC50
determination, Ref. 9.
Equilibrium solubility (pH 7.4).10
b
c
Calculated logP.
nt = not tested.
d
N
O
6.13 0.08 749.9
>570
Ph
O
because of structural similarity to pyrimidine 29, it also shares
common features with azachromanone 4. As with several of the
cores in Table 3, rotation about the aryl-phenylacetylene bond of
39 is expected to be facile due to a low energy barrier to rotation.
Thus, as shown in Figure 4, it can easily be observed that the aryl
a
Data are the average of at least two experiments. For details on pEC50 and EC50
determination, Ref. 9.
b
Equilibrium solubility (pH 7.4).10
Calculated logP.
nt = not tested.
c
d

1406
J. G. Varnes et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1402–1406
Table 5
previously proposed as being critical for potent activity. Lastly,
Potentiation of 3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazines
through the scoping of nitrogen substituents and phenylacetylene
replacements for 39, we identified 8-azaoxazine 47 as a potent
(EC50 50.1 nM) and soluble mGlu5 PAM with low lipophilicity
(clogP 6 3.5) devoid of both undesirable phenylacetylene and car-
bonyl functionalities.
R2
N
N
R1
O
a
#
R1
R2
pEC50
EC50(nM) Solb(
l
M) clogPc
ntd
ntd
4
130
18
4.1
3.2
4.5
3.8
3.5
4.0
3.4
2.2
2.3
1.8
4.5
Supplementary data
41 PhCC
45 PhOCH2
39 PhCC
H
H
7.69 0.07
6.43 0.08 371.5
20.3
Me
Me
Me
CH2CH3
7.52 0.20
7.91 0.03
7.30 0.20
7.22 0.12
30.2
12.3
50.1
60.3
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.027.
46 PhC(O)O
47 PhOCH2
48 PhOCH2
49 PhOCH2
50 PhOCH2
51 PhOCH2
52 PhOCH2
53 (E)-
169
(CH2)2OMe 6.34 0.20 460.6
ntd
References and notes
C(O)OMe
SO2Me
C(O)Me
Me
6.32 0.13 475.0
6.22 0.18 609.5
6.15 0.14 713.4
6.16 0.18 696.0
>520
10
40
1. Conn, J. P.; Lindsley, C. W.; Jones, C. K. Trends Pharmacol. Sci. 2009, 30, 25.
2. Meltzer, H. Y. Biol. Psychiatry 1999, 46, 1321.
3. Conn, J. P.; Tamminga, C.; Schoepp, D. D.; Lindsley, C. Mol. Interventions 2008, 8,
99.
<1
PhCHCH
4. For recent reviews and leading references, see: (a) Lindsley, C. W.; Shipe, W. D.;
Wolkenberg, S. E.; Theberge, C. R.; Williams, D. L.; Sur, C.; Kinney, G. G. Curr.
Top. Med. Chem. 2006, 6, 771; (b) Lindsley, C. W.; Emmitte, K. A. Curr. Opin. Drug
Discovery Dev. 2009, 12, 446.
5. For a more recent short summary of prototypical mGlu5 PAMs and related
references, see: Zhou, Y.; Manka, J. T.; Rodriguez, A. L.; Weaver, C. D.; Days, E. L.;
Vinson, P. N.; Jadhav, S.; Hermann, E. J.; Jones, C. K.; Conn, P. J.; Lindsley, C. W.;
Stauffer, S. R. ACS Med. Chem. Lett. 2010, 1, 433.
54 CyOCH2
Me
6.08 0.04 831.8
>440
3.5
a
Data are the average of at least two experiments. For details on pEC50 and EC50
determination, see Ref. 9.
Equilibrium solubility (pH 7.4).10
b
c
Calculated logP.
nt = not tested.
d
6. Vanejevs, M.; Jatzke, C.; Renner, S.; Müller, S.; Hechenberger, M.; Bauer, T.;
Klochkova, A.; Pyatkin, I.; Kazyulkin, D.; Aksenova, E.; Shulepin, S.; Timonina,
O.; Haasis, A.; Gutcaits, A.; Parsons, C. G.; Kauss, V.; Weil, T. J. Med. Chem. 2008,
51, 634.
We revisited 3,4-dihydropyrido-oxazines 39 and 41 with the
goal of investigating acetylene replacements and decreasing series
lipophilicity (Table 5). In contrast to previous efforts, benzyloxy
surrogates were weakly active or completely inactive (data not
shown). For des-methyl analogs (R2 = H), only the phenoxymethyl
side chain maintained any kind of potent positive modulation of
mGlu5. Similar to chromanone 21 in Table 2, benzoate 46 was
the most potent non-phenylacetylene derivative of Table 5. Weak-
er activity was exhibited by styrenyl and cyclohexyloxymethyl
oxazines 53 and 54, respectively. When N–H oxazine 45 was alkyl-
ated to afford tertiary amine 47, potency improved (>6-fold).
Extending the alkyl side chain (48) maintained activity and in-
creased solubility at the cost of increased lipophilicity. Attenuating
the capacity for electron donation (49–52) decreased activity, sug-
gesting that the electronic character of the oxazine nitrogen is
important or that the increased steric bulk is not well tolerated.
Whether this nitrogen plays a role similar to the carbonyl of com-
pounds in Table 1 remains to be seen.
In summary we have described the discovery of several novel
in vitro mGlu5 positive allosteric modulators with good potency,
solubility, and low lipophilicity. Investigation of the allosteric
binding requirements of a series of dihydroquinolinone analogs
disclosed by Merz Pharmaceuticals led to phenylacetylene aza-
chromanone 4 (EC50 12 nM; 110% effect). Because of the perceived
liability of the acetylene side chain, we investigated replacing this
moiety. This was accomplished with a number of different func-
tional groups, of which phenoxymethyl (27; EC50 156.3 nM) was
the most potent. To derive a second-generation mGlu5 PAM that
did not contain a ketone carbonyl and offered better stability, we
also explored the SAR of a number of pyrimidine-derived com-
pounds resulting in azaindoline (33), azabenzimidazole (36), and
N-methyl 8-azaoxazine (39) phenylacetylenes as potent mGlu5 po-
sitive allosteric modulators. Oxazine 39 (EC50 30.2 nM) was ob-
served to be very similar to azachromanone 4, leading to the
discovery of 5-azaoxazines 40 and 43. Both 40 and 43 exemplify
7. Conn, P. J.; Lindsley, C. W.; Weaver, C. D.; Rodriguez, A. L.; Niswender, C. M.;
Jones, Carrie K.; Williams, R. PCT Int. Appl. WO 151184, 2008.
8. (a) 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.; Veliçelebi, G.; Kuhn, R.
Neuropharmacology 1999, 38, 1493; (b) Cosford, N. D.; Roppe, J.; Tehrani, L.;
Schweiger, E. J.; Seiders, T. J.; Chaudary, A.; Rao, S.; Varney, M. A. Bioorg. Med.
Chem. Lett. 2003, 13, 351.
9. mGlu5 FLIPR Assay: A Ca2+ Flux FLIPR assay similar to that described by
Andreas Ritzén et al. (Discovery of a potent and brain penetrant mGluR5
positive allosteric modulator. Bioorg. Med. Chem. Lett. 2009, 19, 3275) was used
to detect mGlu5 positive allosteric modulator (PAM) activity in an HEK293 cell
line stably expressing the
assessed by measuring potentiation of the EC20 response to
d
isoform of human mGlu5. PAM activity was
-glutamate in the
L
presence of test compound. Procedure: The day before the experiment 25,000
cells/well were plated in DMEM containing 10% dialyzed FBS (Hyclone) in 384
well poly-D-lysine coated plates (Becton Dickinson). After removal of the
plating medium the following day, cells were labeled for 1 h at 37 °C in 4.3
l
M
Fluo-4 AM (In Vitrogen) containing 10% Pluronic F-127 in assay buffer (HBSS
(CellGro), 20 mM HEPES, 1 mM Probenecid (Sigma), pH 7.4). Cells were washed
at rt to remove excess dye prior to addition of test compounds serially diluted
from 10 mM in 100% DMSO into assay buffer. Compounds were assayed for any
underlying agonist activity by addition of test compounds to the cells on the
FLIPR instrument (first addition: 13 lL test compound to 25 lL assay buffer/
well) and the response was recorded for 1 min. 11 different concentrations
were tested for each compound. Fifteen minutes after the 1st addition, positive
modulator activity was assayed by challenge with EC20 (200–300 nM)
glutamate (second addition: 14 L (740–1100 nM), final concentration 200–
300 nM -glutamate) and the response was recorded for 1 min. Positive
L-
l
L
modulator activity was calculated from the fluorescence max–min data
normalized to yield responses for no modulation (EC20 response) and full
stimulation (10 lM L-glutamate) as 0% and 100% modulation, respectively.
Concentration–response data were fitted to the four-parameter logistic
equation to estimate compound potency (EC50) and efficacy (Emax).
10. Alelyunas, Y. W.; Liu, R.; Pelosi-Kilby, L.; Shen, C. Eur. J. Pharm. Sci. 2009, 37,
172.
11. Phthalimide intermediates exhibitied low stability when subjected to the more
rigorous, basic conditions of Figure 2 and were not extensively examined.
12. Azachromanone 4: t1/2 (37 °C) = 11.6 days at pH 1; t1/2 (37 °C) = 1.0 days at pH
10; 0% remaining after 1 h photolysis. Human liver microsomal intrinsic
clearance (hCLint) was measured as
standard liver microsomal stability assay protocol: hMics Clint (
120; rMics Clint L/min/mg): 95.
l
L/min/mg protein according to the
lL/min/mg):
(
l
13. 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.
dihydroquinolinone analogs that do not contain
a carbonyl