Discovery of 5-aryl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-ones as positive allosteric modulators of metabotropic glutamate subtype-2 (mGlu2) receptors with efficacy in a preclinical model of psychosis

Article abstract of DOI:10.1016/j.bmcl.2016.01.021

Optimization of a benzimidazolone template for potency and physical properties revealed 5-aryl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-ones as a key template on which to develop a new series of mGlu2 positive allosteric modulators (PAMs). Systematic invest

Full text of DOI:10.1016/j.bmcl.2016.01.021

Bioorganic & Medicinal Chemistry Letters 26 (2016) 1260–1264
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 5-aryl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-ones as
positive allosteric modulators of metabotropic glutamate subtype-2
(mGlu2) receptors with efficacy in a preclinical model of psychosis
a
a
a
a
Mark E. Layton a, , Alexander J. Reif , Timothy J. Hartingh , Kevin Rodzinak , Vadim Dudkin ,
Cheng Wang a, Ken Arrington a, Michael J. Kelly III a, Robert M. Garbaccio a, Julie A. O’Brien b,
Brian C. Magliaro c, Jason M. Uslaner d, Sarah L. Huszar d, Kerry L. Fillgrove e, Cuyue Tang e,
Yuhsin Kuo e, Marlene A. Jacobson c
⇑
a Department of Medicinal Chemistry, Merck Research Laboratories, P.O. Box 4, Sumneytown Pike, West Point, PA 19486, United States
b Department of In Vitro Sciences, Merck Research Laboratories, P.O. Box 4, Sumneytown Pike, West Point, PA 19486, United States
c Department of Psychiatry Research, Merck Research Laboratories, P.O. Box 4, Sumneytown Pike, West Point, PA 19486, United States
d Department of Central Pharmacology, Merck Research Laboratories, P.O. Box 4, Sumneytown Pike, West Point, PA 19486, United States
e Department of Drug Metabolism, Merck Research Laboratories, P.O. Box 4, Sumneytown Pike, West Point, PA 19486, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Optimization of a benzimidazolone template for potency and physical properties revealed 5-aryl-1,3-dihy-
dro-2H-imidazo[4,5-b]pyridin-2-ones as a key template on which to develop a new series of mGlu2 posi-
tive allosteric modulators (PAMs). Systematic investigation of aryl-SAR led to the identification of
compound 27 as a potent and highly selective mGlu2 PAM with sufficient pharmacokinetics to advance
to preclinical models of psychosis. Gratifyingly, compound 27 showed full efficacy in the PCP- and MK-
801-induced hyperlocomotion assay in rats at CSF concentrations consistent with mGlu2 PAM potency.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 3 December 2015
Revised 7 January 2016
Accepted 8 January 2016
Available online 11 January 2016
Keywords:
Metabotropic glutamate receptor
Positive allosteric modulator
mGlu2 PAM
Schizophrenia
The metabotropic glutamate subtype-2 (mGlu2) receptor is a
Class C G-protein coupled receptor (GPCR) that is activated by glu-
tamate, the major excitatory neurotransmitter in the central ner-
vous system. mGlu2 receptors are expressed presynaptically on
neurons and regulate the release of glutamate in the synapse.
Excessive release of glutamate has been associated with the patho-
physiology of several neuropsychiatric disorders including
schizophrenia. Activation of mGlu2 receptors in the forebrain can
reduce glutamatergic neurotransmission and may represent a
novel approach to the treatment of several neurological disorders.
Of the eight mGlu receptors, both mGlu2 and mGlu3 receptors
developing agonists that are highly selective for mGlu2 and devoid
of mGlu3 activity has remained elusive for small molecules target-
ing the orthosteric binding site.
In stark contrast to the highly conserved orthosteric binding
site, allosteric sites in the 7-transmembrane domain (TMD) of the
GPCR are less conserved between the mGlu receptors.8 Therefore
selective activation of mGlu2 receptors may be attainable with
subtype-selective positive allosteric modulators (PAMs) that bind
in the TMD. In addition to superior selectivity between mGlu sub-
types, this alternate approach to modulating mGlu2 activation
offers several additional potential advantages over orthosteric ago-
nists.9 Since PAMs are pharmacologically active only in the pres-
belong to Group II.
A dual mGlu2/3 orthosteric agonist
LY2140023 (Fig. 1), which is an oral prodrug of LY404039 (1),
showed mixed results in schizophrenic patients1–4 and was
recently discontinued from clinical trials.5 Preclinically, mGlu2/3
agonists have been reported to reduce glutamate release from
presynaptic terminals, as well as modulate behavioral responses
in rodent models predictive of antipsychotic activity.6,7 However,
ence of the orthosteric ligand glutamate, there is a tuned
physiologic effect that aligns with synaptic glutamate levels both
spatially and temporally. In addition, the lack of mGlu2 activation
in the absence of glutamate reduces the potential for receptor
desensitization commonly seen with agonists. Given the perceived
advantages of mGlu2 PAMs over agonists, there has been substan-
tial interest in developing highly potent and subtype-selective
mGlu2 PAMs.10,11 In recent years, there have been two reports of
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2016.01.021
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

M. E. Layton et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1260–1264
1261
O
O
O
CN
H
N
N
S
CH3
O
O
HO2C
H3C
O
HO2C
O
CO2H
H
NH
H3C
H3C
SMe
N
CH3
NH2
1 (LY2140023)
2 (BINA)
3
Figure 1. Structures of mGlu2/3 agonist and historical mGlu2 PAMs.
mGlu2 PAMs advancing to clinical trials. AZD8529 advanced to a
Phase 2 study in schizophrenics, however further development
was halted in 2011 due to a lack of efficacy.12 On the other hand,
ADX71149 (also referred to as JNJ-40411813) may have demon-
strated an effect as add-on therapy to antipsychotics in patients
with residual negative symptoms in an exploratory schizophrenia
trial.13–17
At the start of our efforts, few PAMs targeting mGlu2 receptors
had been reported. Early efforts from the Merck labs had resulted
in the discovery of lipophilic acids including BINA (2)18 as well as
a tricyclic series of oxazolobenzimidazoles (3) (Fig. 1).19,20 Our goal
was to develop a structurally-diverse series of PAMs with sufficient
pharmacokinetic properties to demonstrate robust activity in pre-
clinical models of psychosis following oral dosing. Herein we
report the discovery of a novel series of aryl-linked 1,3-dihydro-
2H-imidazo[4,5-b]pyridin-2-ones that were sufficiently optimized
to provide a tool molecule with robust activity in a MK-801-
induced hyperlocomotion model of psychosis in rats following oral
dosing.
Compound 4, which was derived from an HTS hit, displayed
excellent mGlu2 PAM potency in vitro21 and we believed 4 would
serve as a good starting point to develop a novel series of mGlu2
PAM’s with oral efficacy in preclinical model of psychosis (Fig. 2).
We recognized lead compound 4 had structural elements related
to previous series developed at Merck, and we were intrigued to
define the minimum pharmacophore for mGlu2 PAM activity.
Removal of the biaryl-carboxylic acid in 4 to give the 5-methoxy-
benzimidazolone 5 resulted in a marked reduction in potency,
which initially seemed like an unfavorable result. However further
inspection revealed that the reduced potency was more than com-
pensated by reduced molecular weight and overall resulted in an
improved ligand efficiency (LE) value from 0.32 to 0.37. It was clear
from the improved LE data that the minimum pharmacophore for
mGlu2 PAM activity included the benzimidazolone and we should
focus our efforts on improving the mGlu2 PAM potency of this ring
system. Our medicinal chemistry strategy was to investigate small
changes to the core without growing the molecule substantially
and to do so in an efficient manner. Gratifyingly, we quickly found
incorporation of a 5-phenyl ring (6) in place of the 5-methoxy moi-
ety resulted in a substantial boost in both mGlu2 PAM potency
(EC50 = 31 nM) and ligand efficiency (LE = 0.47). Although incorpo-
ration of the biaryl-ring system resulted in increased lipophilicity,
we were encouraged by the relative balance between mGlu2 PAM
potency and molecular weight compared to the original lead 4.
While compound 6 provided a promising early lead, further
characterization revealed several concerns with the overall drug-
like profile of this structure. Aside from improving LE from 5 to
6, we recognized that 6 was still quite lipophilic with a clogP of
5.2. In addition, 6 suffered from poor solubility at pH7, potent
hERG blockade22 and no measurable fraction unbound (Fu) in the
presence of rat plasma. Since unbound pharmacokinetic (PK)
parameters could not be interpreted, we could not even consider
advancing 6 into in vivo PK studies. We believed the high clogP
was a major contributor to all of these liabilities. Therefore, we
focused on decreasing lipophilicity by incorporating polarity in
the central portion of the molecule. To that end, incorporation of
the aza-benzimidazolone core (7, formally a 1,3-dihydro-2H-imi-
dazo[4,5-b]pyridin-2-one) was not only well tolerated in terms of
mGlu2 PAM potency, it also lowered the clogP by almost a unit.
The net result was a slight diminution of LE that was offset by a
lower clogP. The aza-core also showed a marked improvement in
hERG liability along with a modest improvement in pH7 solubility
and plasma free fraction. Unfortunately compound 7 showed poor
oral exposure in rats and only gave a maximal concentration of
0.07 lM when dosed orally at 10 mpk. Collectively the aza-core
represented a much better template than the parent benzoxazoli-
none on which to optimize.
We next turned our attention to interrogating substituents on
the pendant aryl ring. The aryl SAR was readily investigated and
rapidly explored using the chemistry outlined in Scheme 1. Reac-
tion of methylamine with 2,6-dichloro-3-nitropyridine (8), fol-
lowed by reduction of the nitro group with tin chloride, gave
intermediate 10. Cyclization with CDI and alkylation with neopen-
tyl iodide gave the aza-benzimidazolone template 12 that was
readily functionalized with a variety of aryl boronic acids and bor-
onate esters using palladium-catalyzed Suzuki couplings to give
compounds 7 and 13–28.
Our goal was to systematically investigate SAR on the aryl ring
in order to identify groups that would improve both mGlu2 PAM
potency and pH7 solubility (Table 1). The small electron-with-
O
N
O
N
N
N
N
N
O
N
N
O
HO
O
O
O
N
F
7
5
6
4
mGlu2 PAM EC50 = 12870 nM
MW = 248
mGlu2 PAM EC50 = 31 nM
MW = 294
mGlu2 PAM EC50 = 70 nM
MW = 295
mGlu2 PAM EC50 = 15 nM
MW = 462
LE = 0.37
LE = 0.32
LE = 0.47
LE = 0.45
cLogP = 3.3
cLogP = 5.2
cLogP = 5.2
cLogP = 4.3
Soly (pH7) = 0 µM
hERG IP = 0.7 µM
Plasma Fu = 0.0% (rat)
Soly (pH7) = 12 µM
hERG IP = 12 µM
Plasma Fu = 1.6% (rat)
Figure 2. Optimization of ligand efficiency leads to development of 1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one core as a new template on which to develop oral mGlu2
PAMs.21

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M. E. Layton et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1260–1264
H
N
H
Cl
Cl
N
Cl
a
b
c
Cl
N
Cl
N
N
NO2
NO2
NH2
8
9
10
Cl
N
N
N
N
d
e
N
N
N
O
R
O
O
N
N
H
11
12
7, 13 - 28
Scheme 1. General synthesis of 5-aryl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one derivatives. Reagents and conditions: (a) MeNH2, Na2CO3, EtOH, rt, 3 h, 82%; (b) SnCl2,
HCl, reflux, 18 h, 85%; (c) CDI, DMF, 80 °C, 18 h, 95%; (d) neopentyl iodide, Cs2CO3, NMP, 90 °C, 15 h, 78%; (e) aryl-B(OH)2 or Aryl-Bpin, Pd(OAc)2, S-Phos, K3PO4, THF, H2O,
66 °C, 35–80%.
Table 2
Table 1
Combinations of aryl SAR to give disubstituted 5-aryl-1,3-dihydro-2H-imidazo[4,5-b]
In vitro mGlu2 PAM potency and pH7 solubility for substituted 1,3-dihydro-2H-
pyridin-2-ones with improved profiles21
imidazo[4,5-b]pyridin-2-ones21
R2
R1
N
N
N
HO
R1 R1
N
N
N
O
O
Cmpd
R1
mGlu2 PAM EC50 (nM)
Solubility pH7 (lM)
Cmpd R1
R2
H
mGlu2 PAM
EC50 (nM)
Solubility
CYP2C9
M)
PXR (lM)/max
% activation
pH7 (lM)
(l
7
H
2-CN
3-CN
4-CN
2-CH3
3-CH3
4-CH3
2-F
2-OCH3
2-SO2CH3
3-SO2CH3
4-SO2CH3
3-CH2OH
4-CH2OH
70
26
80
192
57
103
313
81
184
4624
191
379
112
181
12
45
0
13
14
15
16
17
18
19
20
21
22
23
24
25
24
26
27
28
H
H
112
182
183
147
NA
8
10.8/129%
>30/13%
>30/22%
5.6/65%
CH3 86
15
33
7
0
CH3 CH3 61
CH3 CN 49
19
<2
0
0
7
131
67
7
182
30
primary hydroxymethyl. As a result, we decided to incorporate a
tertiary alcohol in place of the primary hydroxymethyl. Gratify-
ingly, the mGlu2 PAM potency and CYP2C9 liability of 26 was fur-
ther improved with the tertiary alcohol 27, while the other positive
attributes of the di-substituted aromatic ring were maintained
with respect to pH7 solubility and CYP3A4 PXR activation. In hopes
of further improving mGlu2 PAM potency with 2,5-disubstitution,
we next combined the ortho cyano group from 13 with the tertiary
alcohol from 27 to give 28. Indeed, the mGlu2 PAM potency of 28
was improved relative to 27, however both CYP2C9 inhibition and
CYP3A4 PXR activation reached unacceptable levels. Overall com-
pound 27 had the best profile and warranted further
characterization.
Compound 27 represented a promising lead molecule with
good mGlu2 PAM activity, low molecular weight (367), reasonable
ligand efficiency (LE = 0.34) and lipophilicity (clogP = 4.2). Further
in vitro characterization revealed that 27 was more potent as a
PAM on the rat mGlu2 receptor (EC50 = 25 nM) than human
(EC50 = 61 nM) using a FLIPR assay.21 A glutamate shift assay,
which measures the fold-shift in the glutamate EC50 curve in the
presence of varying concentrations of PAM, was used to assess
the effectiveness of PAM 27 at making the mGlu2 receptor more
sensitive to glutamate. PAM 27 showed ascending shifts in gluta-
drawing cyano group was preferred in the ortho-position (13) over
the meta- or para-positions (14 and 15, respectively) in terms of
both mGlu2 PAM potency and pH7 solubility. Similarly, an ortho
methyl group (16) showed a modest improvement in both mGlu2
PAM potency and pH7 solubility, while the meta- and para-
positions (17 and 18, respectively) were detrimental to both. Halogens
(19) and alkoxy groups (20) offered no improvements at the ortho-
position. Shifting our attention to more polar groups, we found a
methyl-sulfone in the meta position (22) resulted in only a modest
loss in potency and yielded a significant improvement in solubility.
Incorporation of a 3-hydroxymethyl (24) resulted in the best bal-
ance between mGlu2 PAM potency and improved pH7 solubility.
We reasoned that combinations of aryl SAR would result in a
compound with a superior overall profile (Table 2). Indeed, com-
bining the potency enhancing ortho methyl group from 16 with
the solubility enhancing meta hydroxymethyl from 24 resulted in
a potent 2,5-disubstituted mGlu2 PAM with excellent pH7 solubil-
ity (26). Another important component of profiling leads in an
early discovery program is determining the potential for drug-drug
interactions. To that end, profiling 26 in reversible CYP2C9 inhibi-
tion as well as PXR activation of CYP3A4 expression revealed that
incorporation of both substituents on the phenyl ring resulted in
an unexpected improvement over the mono-substituted 24 for
both reversible CYP2C9 inhibition and CYP3A4 PXR activation.
Encouraged by the combination aryl SAR, we next became
concerned about the potential for oxidative metabolism on the
mate EC50 values ranging from 4-fold at 0.1
M 27 to 19-fold at 10 M 27. In addition to a FLIPR-based assay
format, we also evaluated the potency and efficiency of 27 using a
GTP S protocol. Compound 27 was approximately 10-fold less
lM 27, 12-fold at
1
l
l
c
potent as an mGlu2 PAM for both human (EC50 = 600 nM) and rat
(EC50 = 213 nM) mGlu2 receptors with similar glutamate shifts
(11-fold at 1 lM 27 and 2-fold at 0.1 lM 27). Finally, PAM 27
showed weak partial agonist activity on human and rat mGlu2
receptors, with maximum agonist activation of 7% and 37% at
10 lM, respectively. We believed a PAM mode of action would

M. E. Layton et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1260–1264
1263
were gratified to see that pretreatment with 30 mpk PO 27 fully
attenuated locomotor response to both PCP and MK-801 in rats
(Fig. 3). Compound 27 also reduced spontaneous activity, consis-
tent with what is observed following administration of clinically
approved antipsychotics such as olanzapine and haloperidol, and
also as reported for mGluR2/3 agonists such as LY379268 and
mGluR2 PAMs such as LY487379.6,24 Plasma and CSF levels of 27
were measured from each of the animals in this study (Table 3).
Consistent with our expectations from the permeability and Pgp
assay, compound 27 showed excellent CNS penetration in rats with
CSF levels that aligned well with the unbound drug in the plasma.
In addition, we observed full efficacy at CSF concentrations that
were approximately 10-fold above the FLIPR mGlu2 PAM potency
(EC50 = 61 nM) of 27 and equal to the GTP
27 in rats (EC50 = 213 nM).
cS mGlu2 PAM EC50 of
Figure 3. Locomotor response to PCP or MK-801 as a function of treatment with 27
in rats (p < 0.001 compared to v-MK801 or v-PCP; p > 0.05 compared to v–v).25
In summary, a 5-substituted benzimidazolone (5) was identi-
fied as the minimum pharmacophore for efficient mGlu2 PAM
activity. Optimization of lipophilicity identified the aza core
1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one as the key template
on which to further improve potency and solubility. Systematic
investigation of aryl-SAR led to the identification of compound
27 as a potent and highly selective mGlu2 PAM with sufficient
pharmacokinetics to advance to preclinical models of psychosis.
Gratifyingly, compound 27 showed full efficacy in the PCP- and
MK-801-induced hyperlocomotion assay in rats at CSF concentra-
tions consistent with mGlu2 PAM potency. Approaches to further
reduce the exposure and dose required for efficacy in the
MK-801-induced hyperlocomotion assay will be described in
future publications.
Table 3
Plasma and CSF exposures of 27 following oral dosing in PCP- and MK801-induced
hyperlocomotion assays
Dose of 27
(mpk, PO)
Stimulant Total plasma
M, 2 h)
Unbound plasma
M, 2 h)
CSF
(nM)
(l
(
l
10
30
100
10
30
PCP
PCP
PCP
1.8
8.5
25
54
26
255
750
42
201
660
399
906
21
211
711
MK-801
MK-801
MK-801
1.4
6.7
22
100
Supplementary data
primarily drive any pharmacology observed with 27 in vivo when
unbound CNS exposures were below 1 M.
In terms of off-target activities, compound 27 showed excel-
lent selectivity over the highly homologous mGlu3 receptor
l
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.01.
021. These data include MOL files and InChiKeys of the most
important compounds described in this article.
(EC50 > 30
l
M), as well as several other mGlu receptors including
M). Consistent with our
mGlu4, mGlu5 and mGlu6 (all EC50 > 30
l
early results with the aza-benzimidazolone 7, compound 27 did
not carry a significant hERG liability based on a very weak hERG
References and notes
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Ki = 1.9 uM), neither of which should impact efficacy at low
unbound concentrations in preclinical models of psychosis.
l
M). In a broad Panlabs screen of 155
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lM
The overall profile of compound 27 made it an excellent candi-
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25. Locomotor response to PCP (5.0 mpk; sc) or MK-801 (0.233 mpk; sc) over the
90 min session as a function of treatment with the mGluR2 PAM 27 (10, 30 or
100 mpk; p.o.) or vehicle (20% VitE TPGS) in pair housed male Sprague Dawley
rats (250–300 g; n = 8/group). Rats were placed in activity monitors and after
30 min habituation, given either vehicle or compound 27 and placed back in
activity monitors. After an additional 30 min animals were given an injection
of saline, PCP, or MK-801. Distance traveled over the following 90 min was
summed. Pretreatment with 30 mpk or 100 mpk 27 completely inhibited
stimulant-induced hyperactivity (p < 0.001 compared to v-MK801 or v-PCP;
p > 0.05 compared to v–v). * indicates significant increase from V–V.
a
indicates
21. The in vitro mGlu2 PAM potency of all compounds is reported as an EC50 from a
concentration-response curve generated in the presence of a submaximal
concentration (EC20) of glutamate in a fluorescent imaging plate reader (FLIPR)
significant decrease from V-PCP.
v-MK801.
b indicates significant decrease from