Development of Novel, CNS Penetrant Positive Allosteric Modulators for the Metabotropic Glutamate Receptor Subtype 1 (mGlu1), Based on an N-(3-Chloro-4-(1,3-dioxoisoindolin-2-yl)phenyl)-3-methylfuran-2-carboxamide Scaffold, That Potentiate Wild Type and Mutant mGlu1 Receptors Found in Schizophrenics

Article abstract of DOI:10.1021/acs.jmedchem.5b00727

The therapeutic potential of selective mGlu₁ activation is vastly unexplored relative to the other group I mGlu receptor, mGlu₅; therefore, our lab has focused considerable effort toward developing mGlu₁ positive allosteric modulators (PAMs) suitable as in vivo proof of concept tool compounds. Optimization of a series of mGlu₁ PAMs based on an N-(3-chloro-4-(1,3-dioxoisoindolin-2-yl)phenyl)-3-methylfuran-2-carboxamide scaffold provided 17e, a potent (mGlu₁ EC₅₀ = 31.8 nM) and highly CNS penetrant (brain to plasma ratio (K_p) of 1.02) mGlu₁ PAM tool compound, that potentiated not only wild-type human mGlu₁ but also mutant mGlu₁ receptors derived from deleterious GRM1 mutations found in schizophrenic patients. Moreover, both electrophysiological and in vivo studies indicate the mGlu₁ ago-PAMs/PAMs do not possess the same epileptiform adverse effect liability as mGlu₅ ago-PAMs/PAMs and maintain temporal activity suggesting a broader therapeutic window.

Full text of DOI:10.1021/acs.jmedchem.5b00727

Article
pubs.acs.org/jmc
Development of Novel, CNS Penetrant Positive Allosteric Modulators
for the Metabotropic Glutamate Receptor Subtype 1 (mGlu1), Based
on an N‑(3-Chloro-4-(1,3-dioxoisoindolin-2-yl)phenyl)-3-methylfuran-
2-carboxamide Scaffold, That Potentiate Wild Type and Mutant
mGlu1 Receptors Found in Schizophrenics
Pedro M. Garcia-Barrantes,†,‡ Hyekyung P. Cho,†,‡ Colleen M. Niswender,†,‡ Frank W. Byers,†,‡
Charles W. Locuson,†,‡ Anna L. Blobaum,†,‡ Zixiu Xiang,†,‡ Jerri M. Rook,†,‡ P. Jeffrey Conn,†,‡
and Craig W. Lindsley*,†,‡,§
†Vanderbilt Center for Neuroscience Drug Discovery, and ‡Department of Pharmacology, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232-6600, United States
§Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232-6600, United States
S
* Supporting Information
ABSTRACT: The therapeutic potential of selective mGlu1 activation is vastly unexplored relative to the other group I mGlu
receptor, mGlu5; therefore, our lab has focused considerable effort toward developing mGlu1 positive allosteric modulators
(PAMs) suitable as in vivo proof of concept tool compounds. Optimization of a series of mGlu1 PAMs based on an N-(3-chloro-
4-(1,3-dioxoisoindolin-2-yl)phenyl)-3-methylfuran-2-carboxamide scaffold provided 17e, a potent (mGlu1 EC50 = 31.8 nM) and
highly CNS penetrant (brain to plasma ratio (Kp) of 1.02) mGlu1 PAM tool compound, that potentiated not only wild-type
human mGlu1 but also mutant mGlu1 receptors derived from deleterious GRM1 mutations found in schizophrenic patients.
Moreover, both electrophysiological and in vivo studies indicate the mGlu1 ago-PAMs/PAMs do not possess the same
epileptiform adverse effect liability as mGlu5 ago-PAMs/PAMs and maintain temporal activity suggesting a broader therapeutic
window.
modulators (NAMs), for which clinical candidates have been
developed (and clinical proof-of-concept has been shown in the
case of mGlu5 NAMs).5−12 In contrast, a diverse array of mGlu1
NAMs exists,2 but very few mGlu1 PAMs have been reported,
despite being the first PAMs for any mGlu receptor.3 The first
generation mGlu1 PAMs 1−3 and 5 were developed by Roche
(Figure 1) in the early 2000s and were potent (mGlu1 EC50
values of 56−200 nM), but the drug metabolism and
pharmacokinetic (DMPK) profiles were poor and the majority
displayed significant species differences (only 2 and 5 proved to
potentiate both human and rat mGlu1).13−16 While 5 has been
used in the literature to validate the role of mGlu1 in various
CNS disorders,17−20 it is highly protein bound (Fu = 0.01),
CNS penetration is modest (Kp = 0.29), and it possesses a short
half-life (t1/2 = 54 min) in rat, rendering 5, as well as the later
introduced 4, less than ideal as in vivo tool compounds by
INTRODUCTION
■
Since the discovery in the 1980s that glutamate (Glu) activated
metabotropic glutamate (mGlu) receptors, in addition to α-
amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)
and N-methyl-D-aspartate (NMDA) receptors, these class C
GPCRs have been the focus of intense research and drug
discovery efforts.1,2 There are eight subtypes of mGlu receptors,
mGlu1−8, that are classified into three groups (group I, mGlu1,5;
group II, mGlu2,3; group III, mGlu4,6,7,8) based on sequence
homology, pharmacology, and signal transduction mechanism.1
While early efforts to understand the physiological role of
group I mGlu receptors relied on orthosteric, “glutamate-
derived” agonists, excitotoxicity and seizure liability limited
their utility.1−9 Later efforts employed allosteric ligands that
afforded subtype selectivity as well as mitigation of the adverse
pharmacology by mimicking physiological conditions.1−9 Of
the group I mGlus, mGlu5 is by the far the most understood,
due in large part to a diverse array of allosteric ligands, both
positive allosteric modulators (PAMs) and negative allosteric
Received: May 11, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.5b00727
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Structure of the known mGlu1 PAMs 1−5, upon which target validation and therapeutic potential have been based. mGlu1 PAMs 1−3
were the first described, but only 2 and 5 are active at both rat and human mGlu1. In addition to pronounced species differences, all possessed poor
physicochemical and DMPK properties, which limited their utility as in vivo proof of concept tool compounds.
Figure 2. Second generation mGlu1 PAM 8, derived from a series of “molecular switches”, converting a highly selective mGlu4 PAM 6 into an mGlu1
NAM 7 and then, with limited optimization, conversion into a selective mGlu1 PAM with comparable activity at human, rat, and mutant mGlu1
receptors.
today’s standards. However, as the only mGu1 PAM tool
compound with CNS exposure, the field had no other option
than to employ the lone existing tool to validate and
understand the receptor’s role in numerous CNS disor-
ders.17−20 Thus, to provide an mGlu1 PAM in vivo tool
compound with a more desired profile (Fu > 3%, Kp > 0.8, clean
CYP profiles) to ensure robust target engagement and
validation, as well as a distinct chemotype to avoid chemo-
type-related pharmacology, our lab has recently focused on the
development of next generation mGlu1 PAMs and ago-PAMs.
While our interest has historically resided in the development
of mGlu5 PAMs and NAMs for various CNS disorders,5−12 two
recent, independent studies identified 12 rare, deleterious
nonsynonymous single nucleotide polymorphisms (nsSNPS) in
the GRM1 gene, which encodes mGlu1, in schizophrenic
patients.21,22 These findings led us to explore these loss of
function mutant receptors and assess if signaling could be
restored with an mGlu1 PAM.23,24 In lieu of an HTS campaign
to provide the desired mGlu1 tool compounds, we relied on the
propensity of certain allosteric ligands to possess “molecular
switches”.25,26 In this case, an iterative parallel synthesis
optimization effort focused on the mGlu4 PAM 6 (based on
an N-(3-chloro-4-((3aR,4S,7R,7aS)-1,3-dioxo-1,3,3a,4,7,7a-hex-
ahydro-2H-4,7-methanoisoindol-2-yl)phenyl)picolinamide scaf-
fold) provided the highly selective mGlu1 NAM 7 (Figure 2),
which underwent a “double molecular switch” (e.g., a switch for
mGlu receptor selectivity from mGlu4 to mGlu1 as well as a
concomitant switch in the mode of pharmacology from PAM to
NAM). Further optimization resulted in an additional mode of
pharmacology (MoP) “molecular switch” to afford the highly
selective mGlu1 PAM 8 (mGlu1 EC50 = 390 nM) and a
fundamentally new mGlu1 PAM chemotype.23 Both Roche’s 5
and our 8 were able to potentiate (i.e., render glutamate a more
potent agonist) all of the mutant mGlu1 receptors (as well
human WT mGlu1) and to restore efficacy (Glu Max) in a
limited number of the mutant receptors. There are also double
and triple mutants that correlate with even worse disease
prognosis, and those are currently being generated for
evaluation with mGlu1 PAMs.17,18 Across all of our diverse
family A, B, and C GPCR PAM programs, the pharmacological
parameters that predict in vivo efficacy vary greatly.5−8,27,28 In
some case, we observe no effect on Emax, yet see robust in vivo
efficacy, even with a low fold-shift, while for other GPCRs,
increases in either Emax or large fold-shifts are required.5−8,27−37
Thus, the ideal pharmacological profile for an mGlu1 PAM
against these mutant receptors is not clear, and we have yet to
characterize PAMs against the double and triple mGlu1
mutants.21,22 On the basis of the susceptibility of mGlu1 to
change its response to allosteric modulators due to small
changes in its sequence (i.e., the species disconnect observed
between human and rat form of the receptor due to a L757V
change), we were pleased to see that the mutant mGlu1
B
DOI: 10.1021/acs.jmedchem.5b00727
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. Optimization plans for mGlu1 PAM 8 to enhance potency, stability, and CNS penetration via analogs 9.
a
Scheme 1. General Synthetic Route to Access mGlu1 PAMs 9, 10, and 17
a
Reagents and conditions: (a) phthalic anhydride, AcOH, reflux, 88%; (b) SnCl2, HCl, dioxane, rt, 72%; (c) RCO2H or substituted furylcarboxylic
acids, HATU, DIEA, DCM, rt, 43−96%; (d) 3-methyl-2-furanyl chloride, DIEA, DCE, microwave 120 °C, 30 min, 97%; (e) SnCl2, HCl, dioxane, rt,
96%; (f) phthalic anhydrides, AcOH, reflux, 34−92%.
receptors explored could still be modulated with small molecule
PAMs.24 Thus, these preliminary data fueled our interest in
mGlu1 as a potential new schizophrenia target for patients
harboring these mutations (as well as to revisit other
indications for mGlu1 PAMs); however, the DMPK profile of
8, while improved over 5 in terms of Kp (0.84 vs 0.29),23 could
still be further optimized in order to obtain a more suitable tool
for in vivo studies (e.g., improve potency, PK, Fu). This is also
essential for target validation with a PAM.5−8,27,28 Our studies
have shown that for some mGlu receptors, such as mGlu4, free
brain levels of the PAM correlate with efficacy, while for others,
such as mGlu5, total brain levels of the PAM afford the most
robust correlate.5−8,27−37 Thus, to truly validate mGlu1 PAMs/
ago-PAMs across a broad range of CNS disorders, a toolkit of
chemical probes with diverse structures, pharmacology, and
DMPK profiles are required.
anhydride in refluxing acetic acid provided 12 in 88% yield.
Reduction of the nitro moiety with tin(II) chloride afforded 13
in 72% yield, and a HATU-mediated amide coupling gives
analogs 9 in yields ranging from 43% to 96%, depending on the
heterocyclic carboxylic acid employed. The same sequence of
steps provides the furylamide based analogs 10. Finally, analogs
17 were also prepared in three steps from aniline 14. Here,
microwave-assisted acylation of 14 with 3-methyl-2-furanyl
chloride affords furylamide 15 in 97% yield. Once again, a
tin(II) chloride mediated reduction of the nitro group provided
aniline 16 (96%), which was condensed with phthalic
anhydride in refluxing acetic acid to yield analogs 17 (34−
92%).39
SAR for the initial diversity library of analogs 9 was strikingly
“flat” (Table 1), with only 2 of 14 analogs possessing any
measurable mGlu1 PAM activity. The 2-furylamide congener 9a
was the most active (mGlu1 EC50 = 550 nM, pEC50 = 6.26
0.10, 76 6% Glu Max), while related derivatives, such as the
2-thienyl 9b and 2-pyrrolyl 9c were devoid of mGlu1 activity
(mGlu1 EC50 values of >10 μM). Only the 4-thiazolyl analog 9h
showed modest activity (mGlu1 EC50 = 4.5 μM, pEC50 = 5.35
0.10, 107 7% Glu Max), while the regioisomeric thiazole 9i
was inactive, as were oxazoles (9d and 9e), other aza-
congeners, as well as a tetrahydrofuran 9n. It is worth noting
that the inactive compounds described did not decrease the
glutamate response during the screening, which excludes a
switch of mode of pharmacology to NAMs as it has been
observed in other cases; however, the possibility that these
modifications could generate silent allosteric modulator
(SAMs) or a “molecular switch” that changes the subtype
selectivity toward other mGlu receptor has not been explored
or ruled out.
RESULTS AND DISCUSSION
■
Chemistry. Focused optimization around 8 to enhance
mGlu1 PAM potency and improve the DMPK profile was
plagued by crossover activity at mGlu4, driven in large part by
the picolinamide moiety engendering mGlu4 PAM activity
when subtle electronic and/or steric perturbations were
performed.23,37,38 On the basis of the structure−activity
relationships (SAR) we have previously developed around
this chemotype for mGlu4 PAM activity, our optimization plan
for 8 was modified to focus on a diverse array of five-membered
heterocyclic amides to replace the picolinamide moiety, as well
as key substituents on the phthalimide group, providing analogs
9 (Figure 3).23,37,38 Both structural modifications were designed
to diminish activity at mGlu4, but the impact on mGlu1 PAM
activity, while hopeful, was less certain.
On the basis of these data, we prepared a library of diverse
furylamides 10, surveying a wide range of substituents (Table
2). Like analogs 9, SAR for this series was similarly “flat”;
As shown in Scheme 1, analogs 9 were readily prepared in
three steps from commercial materials and in good overall
yields.39 Starting from aniline 11, condensation with phthalic
C
DOI: 10.1021/acs.jmedchem.5b00727
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 1. Structures and Activities for Selected Five-
Membered Heterocyclic Amides 9
Table 2. Structures and Activities for Selected Furylamides
10
a
Calcium mobilization mGlu1 assays. Values are average of three (n =
3) independent experiments performed in triplicate.
a
Calcium mobilization mGlu1 assays. Values are average of three (n =
3) independent experiments performed in triplicate.
nM, pEC50 = 7.09 0.10, 94 9% Glu Max, 27.7-fold versus
mGlu4); 17e, the 3-F analog (mGlu1 EC50 = 31.8 nM, pEC50 =
7.49 0.15, 98 6% Glu Max, 34.9-fold versus mGlu4); and
however, many of these analogs showed weak PAM activity at
∼10 μM (% Glu Max 30−60%). Again, a single derivative 10j, a
3-methylfuryl congener, emerged with promising activity
(mGlu1 EC50 = 97 nM, pEC50 = 7.01 0.15, 118 3% Glu
Max). Interestingly, substituents in the 5-position (10a−i) were
uniformly inactive, as were fused benzofurans 10m and 10n.
Addition of a methyl group to the 5-position of 10j, providing
the 3,5-dimethyl analog 10l, also resulted in a loss of mGlu1
PAM activity.
The next round of optimization held the 3-methylfurylamide
of 10j constant and surveyed alternative substituents on the
phthalimide moiety delivering analogs 17 (Table 3), providing,
for the first time, robust SAR and a collection of potent and
highly efficacious mGlu1 PAMs (EC50 values of 24.3−98.8 nM).
In this instance, small groups in either the 3- or 4-position of
the phthalimide moiety afforded potent mGlu1 PAMs (17a−f),
and the 4-aza derivative was also tolerated (17g). Efficacy (%
Glu Max) was tightly maintained (86−98%) across all analogs
17. Surprisingly, analogs 17 all retained some residual mGlu4
PAM activity, with selectivity versus mGlu4 ranging from 5.9- to
>100-fold.
17g the 4-aza analog (mGlu1 EC50 = 98.8 nM, pEC50 = 7.01
0.08, 94
4% Glu Max, >100-fold versus mGlu4).39 These
three analogs displayed comparable potency at rat mGlu1 and
enhanced efficacy and represent a 3- to >10-fold improvement
in mGlu1 PAM potency over our initial lead mGlu1 PAM 8
(Figure 4). As shown in Figure 5, all three mGlu1 PAMs
induced a parallel leftward shift of the glutamate concen-
tration−response curve (CRC). 17d induces a maximum 8.3-
fold shift at 10 μM, with modest agonism (<20%) noted at the
highest concentration. 17e induces a maximum 11.4-fold shift
at 10 μM, with higher levels of agonism (∼35%) noted at the
highest concentration, and 17f induces a maximum 10.9-fold
shift at 10 μM, with modest agonism (<20%) noted at the
highest concentration in cells expressing human mGlu1. On the
basis of these profiles, these modulators are best described as
mGlu1 ago-PAMs in this calcium mobilization assay, though the
degree of allosteric agonism varies.23,24,39 As glutamatergic tone
may vary across brain regions, possessing tool compounds with
a range of pure PAM to ago-PAM activity is very attractive and
will be useful in in vivo proof of concept studies and
translation.5−8,27,28 We also assessed selectivity of 17d, 17e,
and 17g versus the other remaining six mGlu receptors in our
Molecular Pharmacology. From this set of analogs 17
(Table 3), three compounds were selected for further
characterization: 17d, the 4-Cl analog (mGlu1 EC50 = 80.9
D
DOI: 10.1021/acs.jmedchem.5b00727
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 3. Structures and Activities for Selected 3-Methyl-2-furylcarboxamide Analogs 17
b
hmGlu1
EC50 (nM)
pEC
% Glu Max
a
hmGlu4
EC50 (nM)
pEC
50 a
% Glu Max
a
fold selective vs
mGlu4
a
50 a
a
compd
R
( SEM)
( SEM)
( SEM)
( SEM)
17a
17b
17c
17d
17e
17f
3-Me
4-Me
3-Cl
4-Cl
3-F
24.3
54.4
33.0
80.9
31.8
24.1
98.8
7.61 0.16
7.26 0.21
7.48 0.32
7.09 0.10
7.49 0.15
7.62 0.07
7.01 0.08
86
86
91
94
98
88
94
3
5
6
9
6
3
4
620
6.21 0.18
6.49 0.26
5.94 0.22
5.64 0.11
5.95 0.16
614 0.30
>5.00
153 11
25.5
5.9
323
69
163 22
89
122 14
6
1140
2250
1110
712
34.5
27.8
34.9
29.7
>100
5
4-F
97
18
7
4
17g
4-Aza
>10 000
a
b
Calcium mobilization mGlu1 assays. Values are average of three (n = 3) independent experiments performed in triplicate. Glu Max is expressed as
% of PHCCC response.
Figure 4. All three mGlu1 PAMs (17d, 17e, and 17g) are equipotent and equiefficacious on human and rat mGlu1 WT receptors. (A)
Concentration−response curves (CRCs) on human mGlu1 WT receptors: 17d, mGlu1 EC50 = 80.9 nM, pEC50 = 7.09 0.10, 94 9% Glu Max;
17e, mGlu1 EC50 = 31.8 nM, pEC50 = 7.49 0.15, 98 6% Glu Max; and 17g mGlu1 EC50 = 98.8 nM, pEC50 = 7.01 0.08, 94 4% Glu Max. (B)
Concentration−response curves (CRCs) on rat mGlu1 WT receptors: 17d, mGlu1 EC50 = 113 nM, pEC50 = 6.95 0.13, 107 4% Glu Max; 17e,
mGlu1 EC50 = 40.7 nM, pEC50 = 7.39 0.18, 103 5% Glu Max; and 17g mGlu1 EC50 = 113 nM, pEC50 = 6.95 0.15, 115 5% Glu Max.
Figure 5. All three mGlu1 PAMs induce a concentration-dependent, parallel leftward shift of the glutamate concentration−response curve (CRC) in
cells expressing the human mGlu1 WT receptor. (A) 17d induces a ∼8.3-fold shift, with modest (<20%) agonism noted at the 10 μM concentration.
(B) 17e induces a ∼11.4-fold shift, with noted (∼35%) agonism at the 10 μM concentration, and (C) 17g induces a ∼10.9-fold shift, with modest
(<20%) agonism noted at the 10 μM concentration. Thus, all three are more accurately described as mGlu1 ago-PAMs in this assay.
E
DOI: 10.1021/acs.jmedchem.5b00727
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 4. DMPK Characterization of mGlu1 PAMs 5, 8, 17d, 17e, and 17g
parameter
5
8
17d
2.90
17e
17g
167
human CLINT (mL min−1 kg−1
)
262
17.2
3.10
2.70
207
human CLHEP (mL min−1 kg−1
)
19.4
661
9.46
2.54
18.6
405
59.7
rat CLINT (mL min−1 kg−1
)
39.9
22.8
rat CLHEP (mL min−1 kg−1
human fu plasma
rat fu plasma
CYP450 IC50 (μM)
1A2
)
63.3
0.014
0.003
9.46
17.2
52.3
0.051
0.030
unstable
unstable
unstable
unstable
unstable
0.030
13
>30
>30
>30
>30
>30
>30
>30
>30
25.2
6.45
>30
>30
>30
>30
>30
>30
2C9
0.6
2D6
>30
>30
3A4
rat iv PK (0.2 mg/kg)
t1/2 (min)
54
ND
ND
ND
ND
91.1
53.5
33.0
1.76
53.6
64.1
13.3
0.85
5.38
7.85
382
MRT (min)
CLp (mL min−1 kg−1
50
)
35
Vss (L/kg)
1.34
3.00
rat iv PBL (0.25 h, 0.2 mg/kg)
Cn plasma (ng/mL)
Cn brain (ng/mL)
Kp
276
81
336
284
0.84
173
108
0.63
200
204
1.02
12.5
35.2
2.81
0.29
a
ND, not determined.
Figure 6. Ex vivo metabolism of 17d in rat and human plasma. Upon incubation in either rat or human plasma at 37 °C, parent 17d is exclusive
species at t = 0, but by 4 h, no parent remains. Hydrolysis of the phthalimide moiety is complete in both rat and human plasma by 4 h, providing the
regioisomeric phthalamic acids M1 and M2 as major metabolites, as well as the minor M3, the product of amide hydrolysis of M1 and M2.
standard fold-shift assay paradigm at a 10 μM concentra-
tion.30,31,33,36 17d and 17g were selective against mGlu2,3,5,6,7,8
17.2 mL min−1 kg−1), a clean CYP profile (IC50 > 30 μM versus
CYP 1A2, 2C9, 2D6, and 3A4), moderate in vivo clearance
(CLp = 33 mL min−1 kg−1), reasonable half-life (t1/2 = 91 min),
and good CNS penetration (brain/plasma ratio Kp of 0.63).
However, 17d, like 8, was unstable in both human and rat
plasma, though acceptable in vivo. In contrast, 17g, the 4-aza
congener, displayed high hepatic clearance in both human
(CLHEP = 18.6 mL min−1 kg−1) and rat (CLHEP = 59.7 mL
min−1 kg−1), a clean CYP profile (IC50 > 30 μM versus CYP
1A2, 2C9, 2D6, and 3A4), superhepatic in vivo clearance (CLp
= 283 mL min−1 kg−1), exceedingly short half-life (t1/2 = 5.4
min), yet high CNS penetration (brain/plasma ratio Kp of
2.81). Interestingly, 17g was unstable in human plasma but
stable in rat plasma (fu = 0.03). 17e represents a middle ground
between these two extremes of disposition and emerged as the
mGlu1 ago-PAM with the most balanced profile for in vivo
studies. For the first time in this series, 17e was stable in both
human and rat plasma, enabling determination of plasma free
fraction (human fu = 0.05, rat fu = 0.03). 17e displayed low
hepatic clearance in human (CLHEP = 2.70 mL min−1 kg−1) and
high in rat (CLHEP = 59.7 mL min−1 kg−1) and a mixed but
acceptable CYP profile (IC50 > 30 μM versus CYP 2D6 and
3A4, IC50 = 25.2 μM at 1A2, IC50 = 6.45 μM at 2C9).
,
inducing less than a ∼2-fold shift of the glutamate CRC at 10
μM. 17e was selective versus mGlu2,3,8 (less than ∼2-fold shift
of glutamate CRC at 10 μM, a modest 2.7-fold shift at mGlu5,
and larger shifts at mGlu6 (6.7) and mGlu7 (7.0). Thus, the
mGlu1 PAMs are suitable for in vivo studies.
Metabolism and Disposition. A major goal of the
optimization effort was to improve not only mGlu1 PAM
potency but also the DMPK profile relative to the lead 8 (and
5) to provide an in vivo tool compound. A hallmark of the
earlier mGlu1 PAMs was moderate to high clearance in rodent
species, mixed cytochrome P450 (CYP) profiles, poor CNS
penetration (in the case of 5, Kp < 0.3), and in the case of 8,
instability in human and rat plasma, making it impossible to
assess free fraction (Fu) and thus free drug levels.23 Therefore,
we profiled 5, 8, 17d, 17e, and 17g in a battery of in vitro and
in vivo DMPK assays (Table 4) to determine if this second
generation series of mGlu1 PAMs addressed key issues.39 As
shown in Table 4, all three analogs addressed different aspects
of the major limitations (low Kp, high plasma protein binding
and plasma instability). 17d displayed low hepatic clearance in
both human (CLHEP = 2.54 mL min−1 kg−1) and rat (CLHEP
=
F
DOI: 10.1021/acs.jmedchem.5b00727
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Interestingly, there was not a good in vitro/in vivo (IVIVC)
correlation, as 17e possessed low in vivo clearance (CLp = 13.3
mL min−1 kg−1), a comparable half-life (t1/2 = 53.6 min) to 5,
and improved CNS penetration (brain/plasma ratio Kp of
1.02).
receptor cell surface expression and that mGlu1 PAMs 5 and 8
could potentiate calcium signaling induced by activation of
these mutant receptors with glutamate or S-3,5-dihydroxyphe-
nylglycine (DHPG), restoring functionality in cells expressing
mGlu1 possessing K563N and P1015A mutations (i.e., those
which had only a small negative impact on glutamate signaling),
while in other mutants, enhancements in glutamate potency,
but not efficacy, were observed.23 Thus, mGlu1 PAMs may be a
viable therapeutic intervention in patients harboring these
mutations. We selected four of the nine single point mutant
receptors (Figure 7), three in the transmembrane domain
(P729T, A863E, Y632H) and one in the intracellular C-
terminal domain (P1014S), and evaluated the ability of the
three PAMs to restore the reduction in glutamate-mediated
calcium signaling utilizing a fold-shift experiment as shown
previously for human WT mGlu1 (Figure 5). It was pleasing to
see that the SAR driven on human mGlu1 to deliver the second
generation mGlu1 PAMs is retained across WT and the mutant
mGlu1 receptors. Across the four mutants, mGlu1 PAMs 17d,
17e, and 17g induce a parallel-leftward shift of the glutamate
CRC, providing maximum shifts of ∼10-fold at 10 μM
compound concentration and, in some cases, modest increases
in glutamate efficacy (Figure 8). Similar to human WT mGlu1,
17e displayed robust ago-PAM activity across all four mutants
(up to ∼40%), and this was noted at concentrations of
compound above 1 μM, while ago-PAM activity was greatly
diminished for 17d and 17g at these mutants compared to the
WT receptor. As the major deficit in these mutant receptors is
the decrease in the efficacy of the receptor signal, the possibility
of these probes having a therapeutic benefit in schizophrenic
patients carrying these mutations is still an open question. The
increase in sensitivity toward glutamate might be beneficial;
however, in vivo studies in mutant mice harboring these
mutations should be carried out in order to assess if this feature
is sufficient to observe an improvement in the phenotype.
Adverse Effect Liability. While early efforts to understand
the physiological role of group I mGlu receptors relied on
orthosteric, “glutamate-derived” agonists, excitotoxicity, and
seizure liability limited their utility.1−9 For example, the group I
dual mGlu1/5 agonist DHPG induces severe epileptiform
activity,42,43 and this liability weighed heavily on early mGlu5
PAM programs,30,31,42−47 as it was not clear if this was due to
mGlu1, mGlu5, or both; moreover, it was not clear if a pure
mGlu5 PAM would avoid this liability.42−47 Since this field has
matured, multiple mGlu5 PAMs, across diverse chemotypes that
possessed even modest ago-PAM or pure PAMs that in vivo
generated ago-PAM metabolites activity, have been shown to
induce epilepitform activity in rat hippocampal CA3 region in
vitro as well as racine scale (4+) seizures in rodents.30,31,42−47
This severe adverse effect liability has hindered and slowed the
development of this promising antipsychotic target. Thus, with
the development of 17e, we were poised to assess the adverse
effect liability of an mGlu1 ago-PAM/PAM with excellent CNS
penetration and free drug levels. To determine if a selective
mGlu1 ago-PAM/PAM has any effect in inducing the
epileptiform activity, we performed experiments analogous to
those described in the previous studies and recorded
spontaneous extracellular spike activity from CA3 pyramidal
cell body layer in a mouse hippocampal slice preparation.34,43
Bath application of the mGlu1 PAM 17e (10 μM, >300-fold
above the mGlu1 EC50) did not induce epileptiform activity,
reflected by no change in frequency of spontaneous spike
activity following application of the mGlu1 PAM (Figure 9).
The plasma instability of 8, 17d, and 17g was not entirely
unexpected based on literature precedent with reported
hydrolysis of phthalimide-containing drugs in plasma.40,41 To
assess this as the source of plasma instability, we evaluated 17d
in rat and human plasma at a 10 μM incubation concentration
at 0 and 4 h time points at 37 °C (Figure 6) and analyzed it by
mass spectrometry to identify metabolites.39 In both rat and
human plasma, the parent compound is clearly present at t = 0,
but by 4 h, no parent 17d remains, but three metabolites are
formed: M1 (major), M2 (major), and M3 (minor). As
predicted from the literature, M1 and M2 correspond to
hydrolysis of the phthalimide to afford the two regioisomeric
phthalamic acids, M1 and M2, and the aniline M3, resulting
from amide hydrolysis of M1 and M2. However, the presence
of the electronegative fluorine atom in 17e, presumably from
inductive effects, stabilizes the phthalimide moiety to enable
stability in plasma such that Fu can be determined.
Activity at GRM1 Mutants. Though selective mGlu1
activation has been suggested as a mechanism for therapeutic
intervention in a variety of CNS disorders, our interest
primarily resides in schizophrenia and the presence of mutant
mGlu1 receptors derived from deleterious GRM1 mutations
found in schizophrenic patients (Figure 7).21,22 Recently we
reported that we developed stable HEK-293 cell lines
expressing 9 of the 12 mGlu1 mutant receptors, confirmed
that they were loss-of-function mutations and not due to loss of
Figure 7. Cartoon representation of monomeric of human mGlu1,
indicating nine of the nonsynonymous single nucleotide poly-
morphisms (nsSNPs) in the mGlu1 receptor found in schizophrenic
patients. Circled in blue are the four single point points in this study,
three in the transmembrane domain (P729T, A863E, Y632H), and
one in the intracellular, C-terminal domain (P1014S).
G
DOI: 10.1021/acs.jmedchem.5b00727
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 8. All three mGlu1 PAMs 17d, 17e, and 17g induce a parallel leftward shift of the glutamate concentration−response-curve (CRC) in cells
stably expressing human mGlu1 mutant receptors (P729T (A), A683E (B), Y632H (C), P1014S(D)) found in schizophrenics. As in WT human
mGlu1 receptors, ago-PAM activity is noted, with 17e showing robust ago-PAM activity across the four schizophrenia mutants evaluated.
The frequency of spike activity after application of 10 μM 17e
was 1.16 0.44 Hz, which is not significantly different from the
baseline control (0.99 0.38 Hz; p = 0.38, n = 6, paired t test),
despite possessing ∼35% agonism of mGlu1 at this concen-
tration. As a positive control, we applied the group I mGlu
agonist DHPG, which induced marked epileptiform activity, as
evidenced by a robust increase in frequency of spontaneous
activity following application of 50 μM DHPG (13.8 3.0 Hz,
compared to 0.94 0.06 Hz in baseline control; p = 0.0084, n
= 6, paired t test).39
Previous reports have demonstrated that intracerebroven-
tricular injections of the group I mGlu agonist DHPG can
induce behavioral convulsions in rats.39,42,43 DHPG activates
both mGlu1 and mGlu5 receptors. Our group has previously
shown that mGlu5 ago-PAM (R)-5-((3-fluorophenyl)ethynyl)-
Figure 9. mGlu1 ago-PAM 17e does not induce epileptiform activity
in CA3 region of the hippocampus. (A) Sample traces of extracellular
recordings from CA3 pyramidal cell body layer in hippocampal slices
in control, during application and after washout of 10 μM 17e (A1)
and 50 μM DHPG (A2). (B) Average time courses of frequency of
spontaneous activity before, during, and after application of 17e (n =
6) and DHPG (n = 6), respectively. The gray bar indicates the time
point at which the data were taken to make statistical comparison to
the control levels. (C) Bar graphs summarizing the effects of 10 μM
17e (left) and 50 μM DHPG (right) on the frequency of spontaneous
activity in CA3 region of the hippocampus: (n.s.) p = 0.36; (∗∗) p =
0.0084; paired t test.
N-(3-hydroxy-3-methylbutan-2-yl)picolinamide (VU0424465,
18) causes seizure activity and behavioral convulsions in
rats.44 Therefore, to assess the potential contributions of mGlu1
activation in the induction of seizure activity, we evaluated the
behavioral effects of selective mGlu1 PAMs, 17d, 17e, and 17g
alongside the selective mGlu5 ago-PAM 18 in mice.39 Male
C57Bl/6 mice (25−30g) were administered mGlu5 ago-PAM
18 (1 or 3 mg/kg ip) or mGlu1 ago-PAMs 17d, 17e, or 17g
(100 mg/kg ip), and behavioral responses were monitored
continuously for 3 h. Behavioral convulsions were measured
using a modified Racine score (0−5). Systemic administration
of 18 induced severe, dose-dependent behavioral manifestations
of seizure activity with peak responses observed approximately
H
DOI: 10.1021/acs.jmedchem.5b00727
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
5 min after administration (Figure 10) in accord with previous
data in rats.44 In contrast, administration of a much higher dose
EC50 = 31.8 nM) and highly CNS penetrant (brain to plasma
ratio (Kp) of 1.02) mGlu1 PAM emerged as a new in vivo
proof-of-concept tool compound, along with 17d and 17g, with
a balanced DMPK profile and overcoming key liabilities of its
predecessors 5 and 8. Moreover, we showed for the first time
that mGlu1 ago-PAMs do not induce epileptiform activity in the
CA3 region of the hippocampus and do not induce seizures in
vivo at drug concentrations far above the mGlu1 PAM EC50,
suggesting that the adverse effect liability of group I agonists,
such as DHPG, is solely mediated by agonism at mGlu5.
Further in vivo studies and continuing efforts around this
scaffold are in progress and will be reported in due course.
EXPERIMENTAL SECTION
■
Chemistry. All reactions were carried out employing standard
chemical techniques under inert atmosphere. Solvents used for
extraction, washing, and chromatography were HPLC grade. Unless
otherwise noted, all reagents were purchased from Aldrich Chemical
Co. and were used without further purification. Analytical thin layer
chromatography was performed on 250 μm silica gel plates from
Sorbent Technologies. Analytical HPLC was performed on an Agilent
1200 LCMS with UV detection at 215 and 254 nm along with ELSD
detection and electrospray ionization, with all final compounds
showing >95% purity and a parent mass ion consistent with the
desired structure. All NMR spectra were recorded on a 400 MHz
Figure 10. Metabotropic glutamate receptor subtype 1 positive
allosteric modulators do not induce behavioral convulsions in mice.
Severe, dose-dependent behavioral convulsions were observed in mice
administered the mGlu5 ago-PAM 18 (1 and 3 mg/kg, 10% Tween 80,
10 mL/kg) beginning 5 min following intraperitoneal dosing as
measured by the modified Racine scale. In contrast, mGlu1 ago-PAMs/
PAMs 5, 17d, 17e, and 17g induce no adverse effects in mice at doses
that reach brain concentration significantly higher than the
compound’s EC50 at mGlu1 (100 mg/kg, 10% Tween 80, 10 mL/
kg). As two distinct mGlu1 PAM chemotypes afford the same effect,
confidence is high that mGlu1 differentiates from mGlu5 adverse effect
liability. Data represent the mean SEM, n = 3.
Bruker AV-400 instrument. 1H chemical shifts are reported as δ values
̈
in ppm relative to the residual solvent peak (DMSO-d6 = 2.50, CDCl3
= 7.26). Data are reported as follows: chemical shift, multiplicity (br =
broad, s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet,
m = multiplet), coupling constant (Hz), and integration. 13C chemical
shifts are reported as δ values in ppm relative to the residual solvent
peak (DMSO-d6 = 39.52, CDCl3 = 77.16). Low resolution mass
spectra were obtained on an Agilent 1200 LCMS with electrospray
ionization. High resolution mass spectra were recorded on a Waters
QToF-API-US plus Acquity system with electrospray ionization.
Automated flash column chromatography was performed on a
Teledyne ISCO Combiflash Rf system. Preparative purification of
library compounds was performed on a Gilson 215 preparative LC
system. Purity for all final compounds was >95%, and each showed a
parent mass ion consistent with the desired structure (LCMS).
2-(2-Chloro-4-nitrophenyl)isoindoline-1,3-dione (12). In a
flask, 200 mg (1.622 mmol, 1.2 equiv) of 2-chloro-4-nitroaniline and
280 mg (1.350 mmol, 1.0 equiv) of phthalic anhydride were added and
dissolved in 5 mL of acetic acid. The mixture was refluxed for 24 h.
After cooling the solution a precipitate is formed. The solid was
filtered and carefully triturated with cold methanol to obtain a light
of the mGlu1 ago-PAM 17d, 17e, or 17g induced no
measurable behavioral disturbances at any time point (Figure
10).39 Of note, 17e achieved total brain exposure of 9.2 μM,
∼288-fold above the mGlu1 PAM EC50 (31.8 nM) and >10-fold
when corrected for free drug levels (rBHB Fu = 0.04). Similarly,
17d and 17g achieved total brain levels of 1.4 and 2.52 μM,
respectively, both an order of magnitude above their respective
mGlu1 EC50 values. Therefore, selective mGlu1 activation did
not induce observable seizure activity or behavioral convulsions
despite achieving high brain concentrations that are signifi-
cantly higher than those required for maximum potentiation of
mGlu1 activation, and these in vivo data are supported by the
electrophysiology data (Figure 9). To ensure this was not a
chemotype driven effect, we also assessed Roche’s 5 in this
model at 100 mg/kg ip. For the first time, these data suggest
that the epileptiform activity induced by the group I dual
mGlu1/5 agonist is solely mediated by agonism at mGlu5, and
both in vitro electrophysiology data and in vivo data support
this conclusion; moreover, these findings suggest that mGlu1
ago-PAMs/PAMs may have a larger therapeutic window than
mGlu5 ago-PAMs/PAMs and avoid this key adverse effect
liability.
1
yellow powder (359 mg, 88%). H NMR (400.1 MHz, CDCl3) δ
(ppm): 8.45 (1H, d, J = 2.4 Hz), 8.28 (1H, dd, J = 8.7 Hz, J = 2.5 Hz),
8.00 (2H, m), 7.86 (2H, m), 7.58 (1H, d, J = 8.6 Hz). 13C NMR
(100.6 MHz, CDCl3) δ (ppm): 165.6, 148.2, 135.6, 134.9, 134.6,
131.6, 131.4, 125.7, 124.2, 122.6.
2-(4-Amino-2-chlorophenyl)isoindoline-1,3-dione (13). In a
vial, 300 mg (0.993 mmol, 1.0 equiv) of 12 were resuspended in
dioxane. The suspension was cold in an ice bath and purged with
argon. A previously prepared solution of tin(II) chloride (847 mg,
4.469 mmol, 4.5 equiv) in concentrated hydrochloric acid (5 M
concentration of SnCl2) was added dropwise to the suspension. After 2
h of stirring at room temperature, the reaction was neutralized
carefully with aqueous potassium carbonate 20%, filtered, and
extracted with diethyl ether. The organic phase was dried with
magnesium sulfate, filtered and the volatiles were eliminated in vacuo
to yield a yellow powder (196 mg, 72%). 1H NMR (400.1 MHz,
DMSO-d6) δ (ppm): 7.97 (2H, m), 7.90 (2H, m), 7.14 (1H, d, J = 8.5
Hz), 6.76 (1H, d, J = 2.4 Hz), 6.60 (1H, dd, J = 8.6 Hz, J = 2.4 Hz),
5.74 (2H, s, -NH2). 13C NMR (100.6 MHz, DMSO-d6) δ (ppm):
167.5, 151.5, 135.3, 132.7, 131.78, 131.76, 123.9, 116.5, 113.6, 113.0.
General Synthesis of N-(3-Chloro-4-(1,3-dioxoisoindolin-2-
yl)phenyl)amides (9 and 10). In a vial, 0.088 mmol (1.2 equiv) of
CONCLUSION
■
In summary, we optimized a series of mGlu1 PAMs based on an
N-(3-chloro-4-(1,3-dioxoisoindolin-2-yl)phenyl)-3-methylfur-
an-2-carboxamide scaffold, driven by multidimensional, iterative
parallel synthesis to provide a second generation of potent and
CNS penetrant mGlu1 PAMs (17d, 17e, and 17g) with DMPK
profiles to enable in vivo proof of concept studies. All three
PAMs potentiated not only wild-type human mGlu1 but also
mutant mGlu1 receptors derived from deleterious GRM1
mutations found in schizophrenic patients, which will enable
translational studies. From this effort, 17e, a potent (mGlu1
I
DOI: 10.1021/acs.jmedchem.5b00727
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
the carboxylic acid was added and dissolved in 0.5 mL mixture of
DCM/DIEA (9:1), then an amount of 41 mg (0.110 mmol, 1.5 equiv)
of HATU was added. The mixture was stirred for 10 min, and an
amount of 20 mg (0.073 mmol, 1.0 equiv) of 13 dissolved in 0.5 mL of
DCM/DIEA (9:1) was added, followed by 3 drops of DMF. The
reaction was stirred for 24 h at room temperature. After this time, the
reaction was quenched with the addition of water and was worked up
by extraction with DCM (2 mL, thrice). The organic phased was
filtered through a phase separator, volatiles were evaporated, the crude
was dissolved in DMSO and purified by preparative HPLC.
N-(3-Chloro-4-(1,3-dioxoisoindolin-2-yl)phenyl)-3-methyl-
furan-2-carboxamide (10j). Cream powder. 1H NMR (400.1 MHz,
CDCl3) δ (ppm): 8.14 (1H, s), 8.06 (1H, d, J = 2.4 Hz), 7.97 (2H, m),
7.81 (2H, m), 7.64 (1H, dd, J = 8.6 Hz, J = 2.4 Hz), 7.40 (1H, d, J =
1.5 Hz), 7.32 (1H, d, J = 8.6 Hz), 6.43 (1H, d, J = 1.5 Hz), 2.47 (3H,
s). 13C NMR (100.6 MHz, CDCl3) δ (ppm): 166.7, 157.1, 142.8,
141.4, 139.6, 134.4, 133.7, 131.8, 130.8, 129.9, 124.8, 123.9, 120.8,
118.3, 116.0, 11.2. HRMS (TOF, ES+) C20H14ClN2O4 [M + H]+ calcd
mass 381.0642, found 381.0644.
N-(2-Chloro-4-nitrophenyl)-3-methylfuran-2-carboxamide
(15). In a microwave vial, an amount of 912 mg (5.28 mmol, 1.2
equiv) of 2-chloro-4-nitroaniline was added and dissolved in 20 mL of
DCE/DIEA (9:1), followed by addition of 500 μL (4.40 mmol, 1.0
equiv) of the 3-methylfurancarbonyl chloride. The reaction was heated
in the microwave at 120 °C for 30 min. The reaction was cooled to
room temperature and water was added, causing the precipitation of
the product. The crude was filtrated in vacuo and titrated with
methanol to give a cream solid (1195 mg, 97% yield). 1H NMR (400.1
MHz, DMSO-d6) δ (ppm): 10.71 (1H, s), 8.27 (1H, d, J = 2.2 Hz),
8.14 (1H, d, J = 9.1 Hz), 7.99 (1H, dd, J = 9.1 Hz, J = 2.2 Hz), 7.88
(1H, d, J = 1.5 Hz), 6.66 (1H, d, J = 1.5 Hz), 2.37 (3H, s). 13C NMR
(100.6 MHz, DMSO-d6) δ (ppm): 158.1, 144.9, 144.3, 141.9, 141.4,
130.3, 127.7, 126.9, 121.7, 119.0, 116.4, 11.5. HRMS (TOF, ES+)
C12H9ClN2O4 [M+] calcd mass 280.0251, found 280.0254.
(400.1 MHz, CDCl3) δ (ppm): 8.15 (1H, s), 8.05 (1H, d, J = 2.3 Hz),
7.84 (1H, d, J = 7.7 Hz), 7.77 (1H, d, J = 7.4 Hz), 7.62 (1H, dd, J = 8.6
Hz, J = 2.4 Hz), 7.59 (1H, d, J = 7.7 Hz), 7.38 (1H, d, J = 1.5 Hz), 7.30
(1H, d, J = 8.6 Hz), 6.42 (1H, d, J = 1.5 Hz), 2.56 (3H, s), 2.46 (3H,
s). 13C NMR (100.6 MHz, CDCl3) δ (ppm): 166.9, 166.8, 157.1,
145.8, 142.8, 141.4, 139.6, 135.0, 133.8, 132.2, 130.8, 129.9, 129.2,
124.9, 124.4, 123.8, 120.8, 118.3, 116.0, 22.0, 11.2. HRMS (TOF, ES
+) C21H16ClN2O4 [M + H]+ calcd mass 395.0799, found 395.0798.
N-(3-Chloro-4-(4-chloro-1,3-dioxoisoindolin-2-yl)phenyl)-3-
methylfuran-2-carboxamide (17c). Cream powder 1H NMR
(400.1 MHz, CDCl3) δ (ppm): 8.16 (1H, s), 8.06 (1H, d, J = 2.3
Hz), 7.88 (1H, m) 7.74 (2H, m), 7.63 (1H, dd, J = 8.6 Hz, J = 2.3 Hz),
7.39 (1H, d, J = 1.2 Hz), 7.29 (1H, d, J = 8.6 Hz), 6.42 (1H, d, J = 1.2
Hz), 2.46 (3H, s). 13C NMR (100.6 MHz, CDCl3) δ (ppm): 165.2,
164.3, 157.2, 142.9, 141.4, 139.8, 136.1, 135.2, 133.9, 133.7, 132.0,
130.7, 130.0, 127.6, 124.4, 122.3, 120.8, 118.3, 116.1, 11.2. HRMS
(TOF, ES+) C20H13Cl2N2O4 [M + H]+ calcd mass 415.0252, found
415.0254.
N-(3-Chloro-4-(5-chloro-1,3-dioxoisoindolin-2-yl)phenyl)-3-
methylfuran-2-carboxamide (17d). Cream powder. 1H NMR
(400.1 MHz, CDCl3) δ (ppm): 8.16 (1H, s), 8.06 (1H, d, J = 2.3 Hz),
7.94 (1H, d, J = 1.7 Hz), 7.90 (1H, d, J = 8.0 Hz), 7.77 (1H, dd, J = 8.0
Hz, J = 1.7 Hz), 7.63 (1H, dd, J = 8.6 Hz, J = 2.3 Hz), 7.40 (1H, d, J =
1.4 Hz), 7.30 (1H, d, J = 8.6 Hz), 6.43 (1H, d, J = 1.4 Hz), 2.46 (3H,
s). 13C NMR (100.6 MHz, CDCl3) δ (ppm): 165.8, 165.4, 157.1,
142.9, 141.4, 141.2, 139.8, 134.5, 133.6, 133.5, 130.7, 130.0, 129.8,
125.1, 124.4, 124.3, 120.8, 118.3, 116.1, 11.2. HRMS (TOF, ES+)
C20H13Cl2N2O4 [M + H]+ calcd mass 415.0252, found 415.0251.
N-(3-Chloro-4-(4-fluoro-1,3-dioxoisoindolin-2-yl)phenyl)-3-
methylfuran-2-carboxamide (17e). Cream powder. 1H NMR
(400.1 MHz, CDCl3) δ (ppm): 8.16 (1H, s), 8.06 (1H, d, J = 2.3
Hz), 7.80 (2H, m), 7.65 (1H, dd, J = 8.6 Hz, J = 2.4 Hz), 7.49 (1H,
m), 7.39 (1H, d, J = 1.4 Hz), 7.30 (1H, d, J = 8.6 Hz), 6.42 (1H, d, J =
1.4 Hz), 2.46 (3H, s). 13C NMR (100.6 MHz, CDCl3) δ (ppm): 165.5,
163.3, 157.9 (1JCF, d, J = 266 Hz), 157.2, 142.9, 142.7, 141.4, 139.8,
137.0 (3JCF, d, J = 7.6 Hz), 133.8 ((2JCF, d, J = 22.6 Hz), 130.8, 130.0,
124.3, 122.7 (2JCF, d, J = 19.6 Hz), 120.8, 120.1 (4JCF, d, J = 3.2 Hz),
118.7, 118.3,116.1, 11.2. HRMS (TOF, ES+) C20H13ClFN2O4 [M +
H]+ calcd mass 399.0548, found 399.0547.
N-(4-Amino-2-chlorophenyl)-3-methylfuran-2-carboxamide
(16). In a flask, an amount of 480 mg (1.71 mmol, 1.0 equiv) of 15
was suspended in dioxane. The suspension was cold in an ice bath and
purged with argon. A previously prepared solution of tin(II) chloride
(1.45 g, 7.70 mmol, 4.5 equiv) in concentrated hydrochloric acid (5 M
concentration of SnCl2) was added dropwise to the suspension. After 2
h of stirring at room temperature, the reaction was neutralized
carefully with aqueous potassium carbonate 20%, filtered, and
extracted with diethyl ether. The organic phase was dried with
magnesium sulfate, filtered and the volatiles were eliminated in vacuo
N-(3-Chloro-4-(5-fluoro-1,3-dioxoisoindolin-2-yl)phenyl)-3-
methylfuran-2-carboxamide (17f). Cream powder. 1H NMR
(400.1 MHz, CDCl3) δ (ppm): 8.16 (1H, s), 8.06 (1H, d, J = 2.3
Hz), 7.97 (1H, dd, J = 8.2 Hz, J = 4.5 Hz), 7.64 (2H, m), 7.47 (1H, td,
J = 8.5 Hz, J = 2.2 Hz), 7.40 (1H, d, J = 1.5 Hz), 7.30 (1H, d, J = 8.6
Hz), 6.43 (1H, d, J = 1.5 Hz), 2.47 (3H, s). 13C NMR (100.6 MHz,
1
CDCl3) δ (ppm): 165.6, 165.3, 157.1, 142.9, 141.4, 139.8, 134.6 (3JCF
,
to yield a cream solid (410 mg, 96% yield). H NMR (400.1 MHz,
d, J = 9.7 Hz), 133.7, 130.7, 130.0, 127.6 (4JCF, d, J = 2.9 Hz), 126.3
(3JCF, d, J = 9.1 Hz), 124.5, 121.5 (2JCF, d, J = 22.9 Hz), 120.8, 118.3,
116.1, 111.6 (2JCF, d, J = 25.0 Hz), 11.2. HRMS (TOF, ES+)
C20H13ClFN2O4 [M + H]+ calcd mass 399.0548, found 399.0546.
N-(3-Chloro-4-(1,3-dioxo-1,3-dihydro-2H-pyrrolo[3,4-c]-
pyridin-2-yl)phenyl)-3-methylfuran-2-carboxamide (17g).
DMSO-d6) δ (ppm): 9.87 (1H, s), 7.76 (1H, d, J = 1.6 Hz), 7.74 (1H,
d, J = 2.3 Hz), 7.41 (1H, dd, J = 8.7 Hz, J = 2.3 Hz), 6.82 (1H, d, J =
8.7 Hz), 6.58 (1H, d, J = 1.6 Hz), 5.51 (2H, br), 2.32 (3H, s). 13C
NMR (100.6 MHz, DMSO-d6) δ (ppm): 157.5, 143.8, 142.3, 139.8,
129.9, 127.7, 121.8, 121.2, 117.8, 116.4, 116.0, 11.4. HRMS (TOF, ES
+) C12H11ClN2O2 [M+] calcd mass 250.0509, found 250.0513.
General Synthesis of Substituted N-Acyl-N-(4-aminophen-
yl)-3-methylfuran-2-carboxamides (17). In a vial, 30 mg (0.12
mmol, 1.0 equiv) of the aniline and 0.18 mmol (1.5 equiv) of the
phthalic anhydride were added and dissolved in 1 mL of acetic acid.
The mixture was heated at 110 °C while stirring for 4 h. After this
time, volatiles were evaporated, the crude was dissolved in DMSO and
resolved by preparative HPLC.
N-(3-Chloro-4-(4-methyl-1,3-dioxoisoindolin-2-yl)phenyl)-3-
methylfuran-2-carboxamide (17a). Cream powder. 1H NMR
(400.1 MHz, CDCl3) δ (ppm): 8.15 (1H, s), 8.05 (1H, d, J = 2.4
Hz), 7.79 (1H, d, J = 7.4 Hz), 7.65 (2H,m), 7.55 (1H, d, J = 7.7 Hz),
7.39 (1H, d, J = 1.5 Hz), 7.29 (1H, d, J = 8.6 Hz), 6.43 (1H, d, J = 1.6
Hz), 2.47 (3H, s), 2.46 (3H, s). 13C NMR (100.6 MHz, CDCl3) δ
(ppm): 167.5, 166.8, 157.1, 142.8, 141.4, 139.5, 138.6, 136.7, 133.9,
133.8, 132.3, 130.8, 129.9, 128.6, 125.0, 121.5, 120.8, 118.2, 116.0,
17.7, 11.2. HRMS (TOF, ES+) C21H16ClN2O4 [M + H]+ calcd mass
395.0799, found 395.0798.
1
Cream powder. H NMR (400.1 MHz, DMSO-d6) δ (ppm): 10.47
(1H, s), 9.31 (1H, s), 9.21 (1H, d, J = 4.8 Hz), 8.21 (1H, d, J = 2.2
Hz), 8.06 (1H, d, J = 4.8 Hz), 7.92 (1H, dd, J = 8.7 Hz, J = 2.2 Hz),
7.86 (1H, d, J = 1.4 Hz), 7.56 (1H, d, J = 8.7 Hz), 6.65 (1H, d, J = 1.4
Hz), 2.37 (1H, s). 13C NMR (100.6 MHz, DMSO-d6) δ (ppm): 166.2,
165.9, 158.0, 156.8, 145.1, 144.5, 141.7, 141.4, 139.3, 132.3, 131.5,
129.4, 125.9, 124.1, 121.1, 120.0, 117.8, 116.3, 11.5. HRMS (TOF, ES
+) C19H12ClN3O4 [M]+ calcd mass 381.0516, found 381.0519.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b00727.
Detailed pharmacology, DMPK, electrophysiology, and
in vivo behavioral methods (PDF)
Molecular formula strings (CSV)
N-(3-Chloro-4-(5-methyl-1,3-dioxoisoindolin-2-yl)phenyl)-3-
methylfuran-2-carboxamide (17b). Cream powder. 1H NMR
J
DOI: 10.1021/acs.jmedchem.5b00727
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(10) Stauffer, S. R. Progress toward positive allosteric modulators of
the metabotropic glutamate receptor subtype 5 (mGlu5). ACS Chem.
Neurosci. 2011, 2, 450−470.
(11) Lindsley, C. W.; Stauffer, S. R. Metabotropic glutamate receptor
5 (mGlu5) positive allosteric modulators (PAMs) for the treatment of
schizophrenia: An historical perspective and a review of the patent
literature. Pharm. Pat. Anal. 2013, 2, 93−108.
(12) Emmitte, K. A. Recent advances in the design and development
of novel negative allosteric modulators of mGlu5. ACS Chem. Neurosci.
2011, 2, 411−432.
(13) Knoflach, F.; Mutel, V.; Jolidon, S.; Kew, J. N.; Malherbe, P.;
Vieira, E.; Wichmann, J.; Kemp, J. A. Positive allosteric modulators of
metabotropic glutamate 1 receptor: characterization, mechanism of
action and binding site. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 13402−
13407.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: craig.lindsley@vanderbilt.edu. Phone: 615-322-8700.
Fax: 615-343-3088.
Author Contributions
P.M.G.-B. performed chemical synthesis and molecular
pharmacology. H.P.C. performed molecular pharmacology
and mutagenesis. C.M.N., P.J.C., and C.W.L. oversaw the
molecular pharmacology and mutagenesis. F.W.B. did all animal
dosing and drawing. C.W.L. and A.L.B. performed and analyzed
all DMPK studies. Z.X. performed the electrophysiology, and
J.M.R. performed the in vivo behavioral studies. C.W.L.
oversaw all aspects of the work (medicinal chemistry,
pharmacology, DMPK) and wrote the manuscript.
(14) Vieira, E.; Huwyler, J.; Jolidon, S.; Knoflach, F.; Mutel, V.;
Wichmann, J. 9H-Xanthene-9-carboxylic acid [1,2,4]oxadiazol-3-yl and
(2H-tertazol-5-yl)-amides as potent, orally available mGlu1 enhancers.
Bioorg. Med. Chem. Lett. 2005, 15, 4628−4631.
Notes
The authors declare no competing financial interest.
(15) Vieira, E.; Huwyler, J.; Jolidon, S.; Knoflach, F.; Mutel, V.;
Wichmann, J. Fluorinated 9H-Xanthene-9-carboxylic acid oxazol-2-yl
amides as potent, orally available mGlu1 enhancers. Bioorg. Med. Chem.
Lett. 2009, 19, 1666−1669.
(16) Hemstapat, K.; dePaulis, T.; Chen, Y.; Brady, A. E.; Grover, V.
K.; Alagille, D.; Tamagnan, G. D.; Conn, P. J. A novel class of positive
allosteric modulators of metabotropic glutamate receptor subtype 1
interact with a site distinct from that of negative allosteric modulators.
Mol. Pharmacol. 2006, 70, 616−626.
(17) Fazio, F.; Notartomaso, S.; Aronica, E.; Storto, M.; Battaglia, G.;
Vieira, E.; Gatti, S.; Bruno, V.; Biagioni, F.; Gradini, R.; Nicoletti, F.;
Di Marco, R. Switch in the expression of mGlu1 and mGlu5
metabotropic glutamate receptors in the cerebellum of mice
developing experimental autoimmune encephalomyelitis and in
autoptic cerebellear samples from patients with multiple sclerosis.
Neuropharmacology 2008, 55, 491−499.
(18) Ngomba, R. T.; Santolini, I.; Biagioni, F.; Molinaro, G.; Simonyi,
A.; van Rijn, C. M.; D’Amore, V.; Mastroiacovo, F.; Olivieri, G.;
Gradini, R.; Ferraguti, F.; Battaglia, G.; Bruno, V.; Puliti, A.; van
Luijtelaar, G.; Nicoletti, F. Protective role for type-1 metabotropic
glutamate receptors against spike and wave discharges in in the WAG/
Rij model of absence epilepsy. Neuropharmacology 2011, 60, 1281−
1291.
(19) D'Amore, V.; Santolini, I.; Celli, R.; Lionetto, L.; De Fusco, A.;
Simmaco, M.; van Rijn, C. M.; Vieira, E.; Stauffer, S. R.; Conn, P. J.;
Bosco, P.; Nicoletti, F.; van Luijtelaar, G.; Ngomba, R. T. Head-to-
head comparison of mGlu1 and mGlu5 receptor activation in chronic
treatment of absence epilepsy in WAG/Rij rats. Neuropharmacology
2014, 85, 91−103.
(20) Notartomaso, S.; Zappulla, C.; Biagioni, F.; Cannella, M.; Bucci,
D.; Mascio, G.; Scarselli, P.; Fazio, F.; Weisz, F.; Lionetto, L.;
Simmaco, M.; Gradini, R.; Battaglia, G.; Signore, M.; Puliti, A.;
Nicoletti, F. Pharmacological enhancement of mGlu1 metabotropic
glutamate receptors causes a prolonged symptomatic benefit in a
mouse model of spinocerebellar ataxia type 1. Mol. Brain 2013, 6, 48.
(21) Frank, R. A. W.; McRae, A. F.; Pocklington, A. J.; van de
Lagemaat, L. N.; Navarro, P.; Croning, M. D. R.; Komiyama, N. H.;
Bradley, S. J.; Challiss, R. A. J.; Armstrong, J. D.; Finn, R. D.; Malloy,
M. P.; MacLean, A. W.; Harris, S. E.; Starr, J. M.; Bhaskar, S. S.;
Howard, E. K.; Hunt, S. E.; Coffey, A. J.; Ranganath, V.; Deloukas, P.;
Rogers, J.; Muir, W. J.; Deary, I. J.; Blackwood, D. H.; Visscher, P. M.;
Grant, S. G. N. Clustered coding variants in the glutamate receptor
complexes of individuals with schizophrenia and bipolar disorder. PLoS
One 2011, 6, e19011.
ACKNOWLEDGMENTS
■
This work was generously supported by the Warren Family and
William K. Warren Foundation (who also funded with William
K. Warren, Jr., Chair in Medicine to C.W.L.) and Grant NIH/
MLPCN U54 MH084659 (C.W.L.). P.M.G.-B. acknowledge
support from the Vanderbilt International Scholar Program
(VISP).
ABBREVIATIONS USED
■
mGlu1, metabotropic glutamate receptor subtype 1; nsSNP,
nonsynonymous single nucleotide polymorphism; DMPK, drug
metabolism and pharmacokinetics; PAM, positive allosteric
modulator; NAM, negative allosteric modulator; Glu, glutamate
REFERENCES
■
(1) Conn, P. J.; Pin, J.-P. Pharmacology and functions of
metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol.
1997, 37, 205−237.
(2) Owen, D. R. Recent advances in the medicinal chemistry of the
metabotropic glutamate receptor 1 (mGlu1). ACS Chem. Neurosci.
2011, 2, 394−401.
(3) Urwyler, S. Allosteric modulation of Family C G-Protein-
Coupled receptors: from molecular insights to therapeutic perspec-
tives. Pharmacol. Rev. 2011, 63, 59−126.
(4) Emery, A. C.; DiRaddo, J. O.; Miller, E.; Hathaway, H. A.;
Pshenichkin, S.; Takoudjou, G. R.; Grajkowska, E.; Yasuda, R. P.;
Wolfe, B. B.; Wroblewski, J. T. Ligand bias at metabotropic glutamate
1a receptors: molecular determinants that distinguish β-arrestin-
mediated from G protein-mediate signaling. Mol. Pharmacol. 2012, 82,
291−301.
(5) Engers, D. W.; Lindsley, C. W. Allosteric modulation of Class C
GPCRs: A novel approach for the treatment of CNS disorders. Drug
Discovery Today: Technol. 2013, 10, e269−e276.
(6) Lindsley, C. W. 2013 Philip S. Portoghese Medicinal Chemistry
Lectureship: Drug discovery targeting allosteric sites. J. Med. Chem.
2014, 57, 7485−7498.
(7) Conn, P. J.; Lindsley, C. W.; Meiler, J.; Niswender, C. M.
Opportunities and challenges in the discovery of allosteric modulators
of GPCRs for the treatment of CNS disorders. Nat. Rev. Drug
Discovery 2014, 13, 692−708.
(8) Wenthur, C. J.; Gentry, P. R.; Mathews, T. P.; Lindsley, C. W.
Drugs for allosteric sites on receptors. Annu. Rev. Pharmacol. Toxicol.
2014, 54, 165−184.
(9) Conn, P. J.; Lindsley, C. W.; Jones, C. Allosteric modulation of
metabotropic glutamate receptors as a novel approach for the
treatment of schizophrenia. Trends Pharmacol. Sci. 2009, 30, 25−31.
(22) Ayoub, M. A.; Angelicheva, D.; Vile, D.; Chandler, D.; Morar,
B.; Cavanaugh, J. A.; Visscher, P. M.; Jablensky, A.; Pfleger, K. D. G.;
Kalaydjieva, L. Deleterious GRM1 mutations in schizophrenia. PLoS
One 2012, 7, e32849.
(23) Cho, H. P.; Garcia-Barrantes, P. M.; Brogan, J. T.; Hopkins, C.
R.; Niswender, C. M.; Rodriguez, A. L.; Venable, D.; Morrison, R. D.;
K
DOI: 10.1021/acs.jmedchem.5b00727
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Bubser, M.; Daniels, J. S.; Jones, C. K.; Conn, P. J.; Lindsley, C. W.
Chemical modulation of mutant mGlu1 receptors derived from
deleterious GRM1 mutations found in schizophrenics. ACS Chem.
Biol. 2014, 9, 2334−2346.
(24) Cho, H. P.; Engers, D. W.; Venable, D. F.; Niswender, C. M.;
Lindsley, C. W.; Conn, P. J.; Emmitte, K. A.; Rodriguez, A. L. A novel
class of succinimide-derived negative allosteric modulators of
metabotropic glutamate receptor subtype 1 provides insight into a
disconnect in activity between rat and human receptors’. ACS Chem.
Neurosci. 2014, 5, 597−610.
(25) Sharma, S.; Kedrowski, J.; Rook, J. M.; Smith, J. M.; Jones, C. K.;
Rodriguez, A. L.; Conn, P. J.; Lindsley, C. W. Discovery of molecular
switches that modulate modes of mGluR5 pharmacology in vitro and in
vivo within a series of functionalized 5-(phenylethynyl)pyrimidines. J.
Med. Chem. 2009, 52, 4103−4106.
(26) Wood, M. R.; Hopkins, C. R.; Brogan, J. T.; Conn, P. J.;
Lindsley, C. W. ’Molecular switches’ on allosteric ligands that
modulate modes of pharmacology. Biochemistry 2011, 50, 2403−2410.
(27) Menniti, F. S.; Lindsley, C. W.; Conn, P. J.; Pandit, J.; Zagouras,
P.; Volkmann, R. A. Allosteric modulation for the treatment of
schizophrenia: Targeting glutamatergic networks. Curr. Top. Med.
Chem. 2013, 13, 26−54.
(28) Melancon, B. J.; Hopkins, C. R.; Wood, M. R.; Emmitte, K. A.;
Niswender, C. M.; Christopoulos, A.; Conn, P. J.; Lindsley, C. W.
Allosteric modulation of 7 transmembrane spanning receptors:
Theory, practice and opportunities for CNS drug discovery. J. Med.
Chem. 2012, 55, 1445−1464.
(29) Bubser, M.; Bridges, T. M.; Dencker, D.; Gould, R. W.;
Grannan, M.; Noetzel, M. J.; Niswender, C. M.; Daniels, J. S.;
Melancon, B. J.; Tarr, J. C.; Wess, J.; Duggan, M. E.; Brandon, N. J.;
Dunlop, J.; Wood, M. W.; Wood, M. R.; Lindsley, C. W.; Conn, P. J.;
Jones, C. K. Selective activation of M4 muscarinic acetylcholine
receptors reverses MK-801-induced behavioral impairments and
enhances associative learning in rodents. ACS Chem. Neurosci. 2014,
5, 920−942.
(30) Conde-Ceide, S.; Martin-Martin, M. L.; Alcazar, J.; Manka, T.;
Tong, H. M.; Garcia-Barrantes, P. M.; Lavreysen, H.; Mackie, C.;
Vinson, P. N.; Daniels, S. J.; Menges, A.; Niswender, C. M.; Jones, C.
K.; Macdonald, G. J.; Steckler, T.; Conn, P. J.; Stauffer, S. R.;
Bartolome-Nebreda, J. M.; Lindsley, C. W. Discovery of VU0409551/
JNJ-46778212: An mGlu5 positive allosteric modulator clinical
candidate targeting schizophrenia. ACS Med. Chem. Lett. 2015, 6,
716−720.
(31) Rook, J. M.; Xiang, Z.; Lv, X.; Ghosal, A.; Dickerson, J.; Bridges,
T. M.; Johnson, K. A.; Bubser, M.; Gregory, K. J.; Vinson, P. N.; Byun,
N.; Stauffer, S. R.; Daniels, J. S.; Niswender, C. M.; Lavreysen, H.;
Mackie, C.; Conde-Ceide, S.; Alcazar, J.; Bartolome, J. M.;
Macdondald, G. J.; Steckler, T.; Jones, C. K.; Lindsley, C. W.;
Conn, P. J. Biased mGlu5 positive allosteric modulators provide in vivo
efficacy without potentiating mGlu5 modulation of NMDAR currents.
Neuron 2015, 86, 1029−1040.
(VU0453379): a novel, CNS penetrant GLP-1 positive allosteric
modulator (PAM). J. Med. Chem. 2014, 57, 10192−10197.
(35) Byun, N. E.; Grannan, M.; Bubser, M.; Barry, R. L.; Thompson,
A.; Rosanelli, J.; Gowrishankar, R.; Kelm, N. D.; Damon, S.; Bridges,
T. M.; Melancon, B. J.; Tarr, J. C.; Brogan, J. T.; Avison, M. J.; Deutch,
A. Y.; Wess, J.; Wood, M. R.; Lindsley, C. W.; Gore, J. C.; Conn, P. J.;
Jones, C. K. Antipsychotic drug-like effects of the selective M4
muscarinic acetylcholine receptor positive allosteric modulator
VU0152100. Neuropsychopharmacology 2014, 39, 1578−1593.
(36) Jones, C. K.; Bubser, M.; Thompson, A. D.; Dickerson, J. W.;
Blobaum, A. L.; Bridges, T. M.; Morrsion, R. D.; Daniels, S. J.; Jadhav,
S.; Engers, D. W.; Italiano, K.; Bode, J.; Daniels, J. S.; Lindsley, C. W.;
Hopkins, C. R.; Conn, P. J.; Niswender, C. M. The mGlu4 positive
allosteric modulator VU0364770 produces efficacy alone and in
combination with L-DOPA or an adenosine A2A antagonist in
preclinical rodent models of Parkinson’s disease. J. Pharmacol. Exp.
Ther. 2012, 340, 404−421.
(37) Williams, R.; Zhou, Y.; Niswender, C. M.; Luo, Q.; Conn, P. J.;
Lindsley, C. W.; Hopkins, C. R. Re-exploration of the PHCCC
scaffold: Discovery of improved positive allosteric modulators of
mGluR4. ACS Chem. Neurosci. 2010, 1, 411−419.
(38) Jones, C. K.; Engers, D. W.; Thompson, A. D.; Field, J. R.;
Blobaum, A. L.; Lindsley, S. R.; Zhou, Y.; Gogliotti, R. D.; Jadhav, S.;
Zamorano, R.; Daniels, J. S.; Morrison, R.; Weaver, C. D.; Conn, P. J.;
Lindsley, C. W.; Niswender, C. M.; Hopkins, C. R. Discovery,
synthesis, sar development of a series of n-4-(2,5-dioxopyrrolidin-1-yl)-
phenylpicolinamides: characterization of VU0195 (ML182) as a
centrally active positive allosteric modulator of metabotropic glutamate
receptor 4 (mGlu4) with oral efficacy in an anti-parkinsonian animal
model. J. Med. Chem. 2011, 54, 7639−7647.
(39) See Supporting Information.
(40) Hoffmann, M.; Kasserra, C.; Reyes, J.; Schafer, P.; Kosek, J.;
Capone, L.; Parton, A.; Kim-Kang, H.; Surapaneni, S.; Kumar, G.
Absorption, metabolism and excretion of [14C]pomalidomide in
humans following oral administration. Cancer Chemother. Pharmacol.
2013, 71, 489−501.
(41) Barbosa, M. L. D.; Ramos, T. J. F.; de Arantes, A. C. S.; Martins,
M. A.; e Silva, P. M. R.; Barreiro, E. J.; Lima, L. M. Synthesis and
pharmacological evaluation of novel phenyl sulfonamide derivatives
designed as modulators of pulmonary inflammatory response.
Molecules 2012, 17, 14651−14672.
(42) Tizzano, J. P.; Griffey, K. I.; Schoepp, D. D. Receptor subtypes
linked to metabotropic glutamate receptor agonist-mdeiated limbic
seizures in mice. Ann. N. Y. Acad. Sci. 1995, 765, 230−241.
(43) Wong, R. K.; Bianchi, R.; Chuang, S. C.; Merlin, L. R. Group I
mGluR-induced epileptogenesis: distinct and overlapping roles of
mGlu1 and mGlu5 and implications for antieplileptic drug design.
Epilepsy Curr. 2005, 5, 63−74.
(44) Rook, J. M.; Noetzel, M. J.; Pouliot, W. A.; Bridges, T. M.;
Vinson, P. N.; Cho, H. P.; Zhou, Y.; Gogliotti, R. D.; Manka, J. T.;
Gregory, K. J.; Stauffer, S. R.; Dudek, F. E.; Xiang, Z.; Niswender, C.
M.; Daniels, J. S.; Jones, J. K.; Lindsley, C. W.; Conn, P. J. Unique
signaling profiles of positive allosteric modulators of mGlu5 determine
differences in in vivo activity. Biol. Psychiatry 2013, 73, 501−509.
(45) Turlington, M. L.; Noetzel, M. J.; Chun, A.; Zhou, Y.; Gogliotti,
R. D.; Nguyen, E. D.; Gregory, K. J.; Vinson, P. N.; Rook, J. M.; Gogi,
K. K.; Xiang, Z.; Bridges, T. M.; Daniels, J. S.; Jones, C. K.; Niswender,
C. M.; Meiler, J.; Conn, P. J.; Lindsley, C. W.; Stauffer, S. R.
Exploration of allosteric agonism structure-activity-relationships within
an acetylene series of metabotropic glutamate receptor 5 (mGlu5)
positive allosteric modulators (PAMs): discovery of 5-((3-
fluorophenyl)ethynyl)-N-(3-methyloxetan-3-yl)picolinamide
(ML254). J. Med. Chem. 2013, 56, 7976−7996.
(32) Iderberg, H.; Maslava, N.; Thompson, A. D.; Bubser, M.;
Niswender, C. M.; Hopkins, C. R.; Lindsley, C. W.; Conn, P. J.; Jones,
C. K.; Cenci, M. A. Pharmacological stimulation of metabotropic
glutamate receptor type 4 in a rat model of Parkinson’s disease and L-
DOPA-induced dyskinesia: comparison between a positive allosteric
modulator and an orthosteric agonist. Neuropharmacology 2015, 95,
121−129.
(33) Rook, J. M.; Tantawy, M. N.; Ansari, M. S.; Felts, A. S.; Stauffer,
S. R.; Emmitte, K. A.; Kessler, R. M.; Niswender, C. M.; Daniels, J. S.;
Jones, C. K.; Lindsley, C. W.; Conn, P. J. Relationship between in vivo
receptor occupancy and efficacy of metabotropic glutamate receptor
subtype 5 allosteric modulators with different in vitro binding profiles.
Neuropsychopharmacology 2015, 40, 755−765.
(46) Noetzel, M. J.; Rook, J. M.; Vinson, P. N.; Cho, K. P.; Days, E.;
Zhou, Y.; Rodriguez, A. L.; Lavreysen, H.; Stauffer, S. R.; Niswender,
C. M.; Xiang, Z.; Daniels, J. S.; Lindsley, C. W.; Weaver, C. D.; Conn,
P. J. Functional impact of allosteric agonist activity of selective
allosteric modulators of mGlu5 in regulating CNS function. Mol.
Pharmacol. 2012, 81, 120−133.
(34) Morris, L. C.; Nance, K. D.; Gentry, P. R.; Days, E. L.; Weaver,
C. D.; Niswender, C. M.; Thompson, A. D.; Jones, C. K.; Locuson, C.
W.; Morrison, R. D.; Daniels, J. S.; Niswender, K. D.; Lindsley, C. W.
Discovery of (S)-2-cyclopentyl-N-((1-isopropylpyrrolidin2-yl)-9-meth-
yl-1-oxo-2,9-dihydro-1H-pyrrido[3,4-b]indole-4-carboxamide
L
DOI: 10.1021/acs.jmedchem.5b00727
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(47) Bridges, T. M.; Rook, J. M.; Noetzel, M. J.; Morrison, R. D.;
Zhou, Y.; Gogliotti, R. D.; Vinson, P. N.; Jones, C. K.; Niswender, C.
M.; Lindsley, C. W.; Stauffer, S. R.; Conn, P. J.; Daniels, J. S.
Biotransformation of a novel positive allosteric modulator of
metabotropic glutamate receptor subtype 5 contributes to seizures in
rats involving a receptor agonism-dependent mechanism. Drug Metab.
Dispos. 2013, 41, 1703−1714.
M
DOI: 10.1021/acs.jmedchem.5b00727
J. Med. Chem. XXXX, XXX, XXX−XXX