Discovery of a Selective and CNS Penetrant Negative Allosteric Modulator of Metabotropic Glutamate Receptor Subtype 3 with Antidepressant and Anxiolytic Activity in Rodents

Article abstract of DOI:10.1021/acs.jmedchem.5b01005

Previous preclinical work has demonstrated the therapeutic potential of antagonists of the group II metabotropic glutamate receptors (mGlus). Still, compounds that are selective for the individual group II mGlus (mGlu₂ and mGlu₃) hav

Full text of DOI:10.1021/acs.jmedchem.5b01005

Article
pubs.acs.org/jmc
Discovery of a Selective and CNS Penetrant Negative Allosteric
Modulator of Metabotropic Glutamate Receptor Subtype 3 with
Antidepressant and Anxiolytic Activity in Rodents
Julie L. Engers, Alice L. Rodriguez, Leah C. Konkol, Ryan D. Morrison, Analisa D. Thompson,†
†
†
†
†
†,§
†,∥
†
†,⊥
†
Frank W. Byers, Anna L. Blobaum, Sichen Chang, Daryl F. Venable, Matthew T. Loch,
†
†,∥
†
†
†,‡
Colleen M. Niswender, J. Scott Daniels, Carrie K. Jones, P. Jeffrey Conn, Craig W. Lindsley,
and Kyle A. Emmitte*
,
†,‡,#
†Vanderbilt Center for Neuroscience Drug Discovery, Department of Pharmacology, Vanderbilt University Medical Center, Nashville,
Tennessee 37232, United States
‡
Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States
*
S Supporting Information
ABSTRACT: Previous preclinical work has demonstrated the
therapeutic potential of antagonists of the group II
metabotropic glutamate receptors (mGlus). Still, compounds
that are selective for the individual group II mGlus (mGlu and
2
mGlu ) have been scarce. There remains a need for such
3
compounds with the balance of properties suitable for
convenient use in a wide array of rodent behavioral studies.
We describe here the discovery of a selective mGlu NAM 106
3
(
VU0650786) suitable for in vivo work. Compound 106 is a
member of a series of 5-aryl-6,7-dihydropyrazolo[1,5-a]pyrazine-4(5H)-one compounds originally identified as a mGlu positive
5
allosteric modulator (PAM) chemotype. Its suitability for use in rodent behavioral models has been established by extensive in
vivo PK studies, and the behavioral experiments presented here with compound 106 represent the first examples in which an
mGlu NAM has demonstrated efficacy in models where prior efficacy had previously been noted with nonselective group II
3
antagonists.
INTRODUCTION
(NAMs), also known as noncompetitive antagonists, of the
group II mGlus. Although such compounds are generally
■
Glutamate (L-glutamic acid) is the major excitatory neuro-
transmitter in the mammalian central nervous system (CNS)
and acts on both ionotropic and metabotropic glutamate
receptors (mGlus). While ionotropic glutamate receptors are
ligand-gated ion channels, mGlus are a family of eight G-
protein coupled receptors (GPCRs). Belonging to family C of
the GPCRs, the mGlus possess a seven transmembrane (7TM)
α-helical domain connected via a cysteine-rich region to a large
bilobed extracellular amino-terminal domain containing the
orthosteric binding site. The mGlus have been further
categorized into three groups according to their homology,
preferred signal transduction mechanisms, and pharmacology:
group I (mGlu and mGlu ), group II (mGlu and mGlu ), and
selective versus the other six members of the mGlu family,
6
selectivity between mGlu and mGlu is negligible. Still, the
2
3
use of such compounds in animal models has established a
potential role for mGlu2/3 antagonists in a variety of CNS
disorders. Many of these studies have been carried out with two
orthosteric antagonists, both of which are highly functionalized
7
8
glutamate analogues, 1 (LY341495) and 2 (MGS0039)
Figure 1). For example, work with these compounds has
(
helped establish mGlu2/3 inhibition as a potential therapeutic
9,10
application for obsessive-compulsive disorder (OCD),
anxiety, cognition, and Alzheimer’s disease.
11
12
13−15
Addition-
1
5
2
3
ally, antidepressant efficacy has been demonstrated in
1
−3
group III (mGlu , mGlu , mGlu , and mGlu ).
Both mGlu2
4
6
7
8
numerous rodent models of depression with these com-
and mGlu are primarily located presynaptically in neurons and
3
8,9,11,16−19
pounds,
including those meant to assess treatment-
coupled to Gi/o and the inhibition of adenylyl cyclase activity; in
20
21
resistant depression (TRD), anhedonia, and depression
associated with withdrawal from addictive substances.
addition, mGlu is also expressed in glial cells. The group II
3
22,23
mGlus are widely expressed throughout the CNS, including
regions of the brain associated with emotional states such as the
amygdala, hippocampus, and prefrontal cortex.
Compound 1 has also been used as a tool to establish a
Received: June 26, 2015
4
,5
Researchers have been successful in designing both
orthosteric antagonists and negative allosteric modulators
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Importantly, not only was 7 selective versus mGlu5 but
selectivity versus mGlu was also notable (>15-fold). Further
2
optimization within this chemotype identified a second-
40
generation compound 8 (VU0469942, ML337) (Figure 2)
that was devoid of both mGlu and mGlu activity. While 8 has
2
5
proven quite useful as an in vitro tool compound and can be
41
used in mice at high doses (100 mg/kg), lower CNS
penetration and higher protein binding in rats prevent its utility
in that species. Furthermore, 1,2-diarylethyne chemotypes
similar to this one are prone to bioactivation at the alkyne
moiety and subsequent formation of reactive metabolites that
42,43
can lead to toxicity.
Thus, the discovery of a superior
mGlu NAM from outside the 1,2-diphenylethyne chemotype,
3
with the balance of pharmacology and DMPK properties for
convenient use in both rats and mice, remained a worthy goal
and is the subject of this manuscript.
RESULTS AND DISCUSSION
Lead Identification. The previous success in identification
■
of an mGlu NAM lead with inherently good selectivity versus
3
Figure 1. mGlu2/3 orthosteric antagonist tools 1 and 2, mGlu2/3 NAM
tools 3, 4, and 6, and Roche mGlu2/3 NAM clinical compound 5.
39,40
mGlu from a mGlu PAM chemotype
prompted the
2
5
mining of our internal collection of mGlu PAM chemotypes
5
lacking an alkyne moiety. Utilization of this resource provided
potential utility for mGlu inhibition in the treatment of
glioma.
Research with mGlu2/3 NAMs has been less extensive than
some examples of compounds with evidence of mGlu NAM
2
/3
3
2
4−27
activity amid the available associated cross-screening data versus
44,45
the mGlu family.
We selected many of these interesting
with orthosteric antagonists; however, closely related tools 3
compounds as well as additional closely related analogues to
arrive at approximately 160 compounds for full concentration
response curve (CRC) measurements in our cell-based
2
8
29,30
(
RO4491533) and 4 (RO4432717)
(Figure 1) have been
employed in multiple in vivo assays. In particular, these
compounds have demonstrated efficacy in multiple models of
functional assay for mGlu . This fluorescence-based assay
3
3
1
30,32,33
depression and cognition.
in genetically modified mice with 1 and mGlu2/3 NAM 6
Figure 1) point toward a potential application for group II
antagonists in the treatment of certain autism spectrum
disorders. Finally, one mGlu NAM, 5 (decoglurant,
Furthermore, recent studies
measures calcium mobilization induced by mGlu activation in
3
34
35
a cell line stably expressing rat mGlu and the promiscuous G-
3
(
protein Gα15 and is capable of detecting agonists, PAMs, and
NAMs of mGlu . We have developed similar assays and cell
3
2
/3
lines for rat mGlu and rat mGlu , and both were used
2
5
3
6
RO4995819) (Figure 1), has advanced into human clinical
trials, including a phase II trial in patients with major depressive
disorder (MDD) and resistant to ongoing treatment with
antidepressants (NCT01457677). In spite of the wealth of
preclinical evidence for the potential utility of mGlu2/3
antagonists, identification of compounds with ample selectivity
between the two group II mGlus and the balance of
pharmacology and drug metabolism and pharmacokinetics
throughout this project for assessing selectivity.
One of the most interesting series to emerge from the full
CRC mGlu3 screen outlined above is exemplified by
compounds 9−11 (Figure 3). These compounds are from
3
7
(
DMPK) properties required for use in vivo has been elusive.
Such tools are essential for further validation of the precise
roles of each receptor in the etiology of disease.
Our first foray into this arena of research began by an
observation of occasional weak mGlu NAM activity in a series
3
of 1,2-diphenylethyne mGlu positive allosteric modulators
5
3
8
(
PAMs). An optimization plan for mGlu3 activity that
centered on modification of the functional groups appended
to the two phenyl rings within this scaffold delivered first-
3
9
generation tool 7 (VU0463597, ML289) (Figure 2).
Figure 3. Representative compounds from a new mGlu lead series.
3
within a series of 5-aryl-6,7-dihydropyrazolo[1,5-a]pyrazine-
46
4
(5H)-ones that includes many potent mGlu PAMs. An
5
interesting piece of SAR was noted with respect to the presence
of a chiral methyl group at the seven position of the pyrazine-
4
(5H)-one ring in the (R)-configuration (10). Whereas the
potency of (R)-methyl analogue 10 at mGlu was only 2-fold
5
less than unsubstituted analogue 9, the efficacy of 10 (Glu Max
= 26.5%) was much weaker than that observed with 9 (Glu
Figure 2. mGlu NAMs from the 1,2-diphenylethyne chemotype.
3
B
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of Primary Alcohol Intermediate 20
a
t
Reagents and conditions: (a) Na, EtOH, (EtO C) , 0 °C to rt, 27%; (b) NH NH ·H O, EtOH, 80 °C, 98%; (c) PPh , D BAD, THF, μwave, 120
2
2
2
2
2
3
°
C, 20 min; (d) 4 M HCl in dioxane, then saturated aq NaHCO , 74%, 2 steps; (e) CuI, K CO , 4-fluorobromobenzene, N,N′-
3
2
3
dimethylethylenediamine, PhMe, 120 °C, 83%; (f) BBr , CH Cl , 0 °C to rt; (g) KOAc, DMF, 60 °C, 100%, 2 steps; (h) 1 M aq LiOH, MeOH,
THF, 94%.
3
2
2
a
Scheme 2. Synthesis of New Western Ether Analogues 22−39 and 41−51
a
Reagents and conditions: (a) PS-PPh , THF, DIAD, 7−37%; (b) NEt , MsCl, CH Cl , 63%; (c) Cs CO , DMF, 90 °C, 35−52%; (d) NaH, DMF,
3
3
2
2
2
3
8
−37%.
Table 1. mGlu NAM and mGlu SAR of Western Phenyl Ethers 18, 22−39
3
5
mGlu pIC
mGlu IC
% Glu Max
mGlu5
mGlu pEC
mGlu EC
% Glu Max
b,c
3
50
3
50
5
50
5
50
a
a
a
,
b
c
c
c
no.
R
(± SEM)
(nM)
(± SEM)
activity
(± SEM)
(nM)
(± SEM)
18
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
H
6.57 ± 0.15
6.28 ± 0.20
6.11 ± 0.03
6.34 ± 0.04
6.38 ± 0.22
6.14 ± 0.14
5.84 ± 0.05
5.92 ± 0.02
6.27 ± 0.18
5.79 ± 0.02
5.93 ± 0.01
5.59 ± 0.18
5.89 ± 0.02
5.98 ± 0.02
5.73 ± 0.01
6.08 ± 0.05
6.11 ± 0.12
5.94 ± 0.06
5.39 ± 0.04
267
530
2.21 ± 0.80
2.90 ± 1.05
1.19 ± 0.75
2.09 ± 0.57
1.70 ± 0.35
1.56 ± 0.37
2.35 ± 0.18
1.72 ± 0.55
1.63 ± 0.33
1.96 ± 0.11
1.86 ± 0.52
2.32 ± 1.39
1.78 ± 0.53
1.76 ± 0.39
4.13 ± 0.09
2.29 ± 0.84
2.53 ± 0.70
3.39 ± 0.87
PAM
PAM
PAM
PAM
PAM
PAM
7.34 ± 0.02
6.71 ± 0.04
7.14 ± 0.02
7.03 ± 0.05
6.51 ± 0.05
5.83 ± 0.04
<5.0
46
195
87.7 ± 1.4
89.3 ± 3.0
86.1 ± 2.9
87.0 ± 1.0
82.3 ± 2.7
83.7 ± 2.5
39.7 ± 7.7
92.5 ± 2.6
84.4 ± 2.1
32.8 ± 6.1
81.8 ± 0.3
63.7 ± 3.1
32.4 ± 3.0
40.2 ± 4.8
2-F
3-F
773
73
4-F
462
92
2,4-di-F
2-Me
3-Me
4-Me
2-Cl
417
307
721
1500
>10000
1080
419
d
1440
1190
532
NAM
PAM
PAM
5.97 ± 0.04
6.38 ± 0.01
<5.0
d
3-Cl
1620
1170
2540
1290
1040
1850
830
NAM
PAM
>10000
604
4-Cl
6.22 ± 0.04
<5.0
d
2-OMe
3-OMe
4-OMe
4-CF3
4-Et
PAM
PAM
PAM
>10000
1840
1340
>30000
3210
1940
>30000
>30000
5.74 ± 0.04
5.87 ± 0.16
<4.5
PAM
PAM
5.49 ± 0.03
5.71 ± 0.06
<4.5
50.8 ± 3.7
38.6 ± 4.8
4-CN
4-OEt
4-OCF3
784
1160
4070
2.66 ± 0.34
<4.5
a
b
Calcium mobilization mGlu assay; values are average of n ≥ 3. Amplitude of response in the presence of 30 μM test compound as a percentage of
3
c
d
maximal response (100 μM glutamate); average of n ≥ 3. Calcium mobilization mGlu assay; values are average of n ≥ 3. Weak activity;
concentration response curve (CRC) does not plateau.
5
Max = 93.0%). The (S)-methyl analogue 11 proved highly
also inactive up to the top concentration tested (30 μM) in our
preferential for mGlu PAM activity and was only weakly active
mGlu calcium mobilization assay. Given the potential benefits
5
2
at mGlu , inhibiting the glutamate response only at the top
engendered by this (R)-methyl group, we incorporated this
3
concentration tested (30 μM). Gratifyingly, compound 10 was
functional group into the design of future compounds.
C
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 2. mGlu NAM and mGlu SAR of Western 2-Pyridyl Ethers 41−51
3
5
mGlu pIC
mGlu IC
% Glu Max
mGlu5
mGlu pEC
mGlu EC
% Glu Max
b,c
3
50
3
50
5
50
5
50
a
a
a
,
b
c
c
c
no.
R
(± SEM)
(nM)
(± SEM)
activity
(± SEM)
(nM)
(± SEM)
41
42
43
44
45
46
47
48
49
50
51
3-F
5.96 ± 0.02
5.53 ± 0.02
5.17 ± 0.02
5.68 ± 0.01
5.78 ± 0.01
5.43 ± 0.00
6.27 ± 0.02
6.22 ± 0.04
5.62 ± 0.02
5.63 ± 0.02
5.80 ± 0.02
1100
2940
6760
2090
1670
3690
539
2.22 ± 0.84
2.11 ± 0.96
−1.14 ± 2.54
0.86 ± 0.47
1.62 ± 0.42
−0.18 ± 1.39
1.71 ± 0.55
1.84 ± 0.51
1.12 ± 1.08
2.06 ± 0.79
PAM
5.37 ± 0.06
<4.5
4300
>30000
>30000
>10000
>10000
>10000
>10000
2920
78.5 ± 6.1
3-CF3
3-OMe
4-Me
4-CF3
4-OMe
5-F
<4.5
d
NAM
NAM
NAM
NAM
<5.0
46.9 ± 5.9
12.9 ± 2.9
54.7 ± 5.8
46.5 ± 3.4
81.4 ± 2.7
2.47 ± 0.17
d
d
d
<5.0
<5.0
<5.0
5-Cl
605
PAM
NAM
5.53 ± 0.06
5.65 ± 0.07
<4.5
6-Me
6-CF3
6-OMe
2370
2360
1590
2250
>30000
>10000
d
1.94 ± 0.62
PAM
<5.0
31.2 ± 3.1
a
b
Calcium mobilization mGlu assay; values are average of n ≥ 3. Amplitude of response in the presence of 30 μM test compound as a percentage of
3
c
d
maximal response (100 μM glutamate); average of n ≥ 3. Calcium mobilization mGlu assay; values are average of n ≥ 3. Weak activity; CRC does
not plateau.
5
Development of SAR in the Western Region. Our initial
plan centered on the development of structure−activity
relationships (SAR) in the area occupied by the 2-pyridyl
ether of 10, termed the western region of the scaffold.
Intermediate primary alcohol 20 was envisioned as a valuable
intermediate for late stage diversification of this area (Scheme
2-fluoropyridines 40 to facilitate a S Ar reaction and provide
41−51.
The results obtained from preparing and testing unsub-
stituted phenyl ether intermediate 18 revealed a near 6-fold
preference for mGlu5 vs mGlu3 activity, which ultimately
proved a substantial hurdle with this subset of compounds
(Table 1). To determine if substitution of the phenyl group
N
1). The synthesis of 20 began with commercially available
phenoxyacetone 12. The sodium enolate of 12 was prepared
and treated in situ with diethyl carbonate to afford 2,4-diketone
intermediate 13. Reaction of 13 with hydrazine readily
provided 14, which could be N-alkylated via a Mitsunobu
could engender preference for mGlu activity, we systematically
3
installed a variety of functional groups at all positions (22−34).
Most substituents at the 2-position (22, 26, 29) only modestly
affected mGlu3 NAM activity relative to 18; however, 2-
methoxy analogue 32 was nearly 10-fold less potent than 18.
4
7
reaction with commercially available chiral alcohol 15. This
Mitsunobu reaction was carried out with microwave heating
and resulted in inversion of stereochemistry as expected.
Treatment of intermediate 16 with acid resulted in cleavage of
the tert-butylcarbamate protecting group, and subsequent
exposure to aqueous base provided the lactam 17. Chiral
HPLC analysis of 17 showed 98.6% ee for this key
intermediate. N-Arylation of 17 was accomplished by a copper
The mGlu PAM activity was improved relative to 18 but
5
remained suboptimal with these 2-substituted analogues. 2,4-
Difluoro analogue 25 demonstrated modestly enhanced
selectivity versus mGlu5 relative to its monosubstituted
comparators 22 and 24. Although substitution of the 3-position
with fluorine (23) provided little impact on activity at either
receptor, installation of larger substituents (27, 30, 33) resulted
in reductions in potency at both receptors and two instances of
4
8
mediated coupling with 4-fluorobromobenzene to yield 18.
Treatment of 18 with boron tribromide cleaved the phenoxy
ether, affording the corresponding primary bromide, which was
then reacted with potassium acetate under mild heating to
afford acetate ester 19. Hydrolysis of the acetate group was
accomplished with aqueous lithium hydroxide to give the
desired intermediate alcohol 20.
pharmacology mode switching at mGlu (27 and 30). Such
5
“molecular switches” have been noted previously in other
49,50
mGlu chemotypes.
Substitution of the 4-position with
5
fluorine (24) only minimally impacted potency at either
receptor; larger groups (28, 31, 34) reduced potency at both
receptors, although the effect on mGlu was more pronounced.
5
Preliminary mGlu NAM SAR obtained from the testing of
analogues of lead 10 that were included in the original set of
60 compounds indicated a preference for phenyl and 2-pyridyl
Thus, desiring to further examine the effects of substitution at
the 4-position, additional analogues were prepared (35−39).
3
1
The trend toward mGlu -preferring compounds continued with
3
ethers in the western portion of the scaffold. Late stage
conversion of intermediate alcohol 20 to new analogues was
accomplished through one of three methods (Scheme 2). For
the synthesis of substituted phenyl ether analogues 22−39,
alcohol 20 was converted to the corresponding mesylate, which
was then reacted with the desired phenols 21 and cesium
carbonate to afford 22−39. Alternatively, the phenols 21 and
the 4-ethoxyphenyl analogue 38 and 4-trifluoromethoxy
analogue 39, demonstrating no activity versus mGlu up to
5
the highest concentration tested (30 μM).
Given the promising profile of initial lead 10, our hope for
identifying more attractive compounds within the 2-pyridyl
analogues (41−51) remained high; however, this region proved
relatively intolerant of substitution (Table 2). Although a 3-
fluoro substituent (41) only modestly impacted potency, larger
substituents (42, 43) had more deleterious effects. Similarly
modest results were observed with 4-position (44−46) and 6-
47
alcohol 20 were coupled directly via a Mitsunobu reaction.
For synthesis of 2-pyridyl ether targets 41−51, the sodium
alkoxide of 20 was prepared in situ and treated with substituted
D
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 3. DMPK Profiling of Early Analogues
a
b
c
d
d
d
no.
cLogP
LLE
mGlu IC (nM)
fold vs mGlu5
rat plasma Fu
t1/2 (min)
CLplasma (mL/min/kg)
VSS (L/kg)
3
50
2
3
4
4
4
8
7
8
3.96
4.24
3.06
3.57
2.38
1.70
3.21
2.65
462
0.20
>25
0.060
0.005
0.135
0.034
95
32
40
82
82
29
3.5
2.2
6.6
1160
539
>18
88
605
4.8
141
5.2
a
b
c
Calculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/). LLE (ligand-lipophilicity efficiency) = pIC − cLogP. F =
50
u
d
fraction unbound. Rat IV PK results (n = 2); dose = 0.2 mg/kg; solution in 9% EtOH, 37% PEG 400, 54% DMSO (2 mg/mL).
a
Scheme 3. Synthesis of 5-Fluoropyridin-2-yl Ether Analogues 55−72
a
Reagents and conditions: (a) BBr , CH Cl , 0 °C to rt; (b) KOAc, DMF, 60 °C, 54%, 2 steps; (c) 1 M aq LiOH, MeOH, THF, 37%; (d) NaH,
3
2
2
DMF, 2,5-difluoropyridine, 76%; (e) CuI, K CO , aryl or heteroaryl halide, N,N′-dimethylethylenediamine, PhMe, 120 °C, 17−70%.
2
3
a
Scheme 4. Synthesis of 5-Chloropyridin-2-yl Ether Analogues 80−110
a
t
Reagents and conditions: (a) PPh , D BAD, THF, μwave, 120 °C, 20 min; (b) 4 M HCl in dioxane, then saturated aq NaHCO , 89%, 2 steps; (c)
3
3
NaH, 4-methoxybenzyl chloride, DMF, 0 °C to rt, 73%; (d) NaBH , THF, MeOH, 0−60 °C, 84%; (e) NaH, DMF, 5-chloro-2-fluoropyridine, 0 °C
4
to rt, 84%; (f) (NH ) Ce(NO ) , MeCN, H O, 66%; (g) CuI, K CO , aryl or heteroaryl halide, N,N′-dimethylethylenediamine, PhMe, 120 °C,16−
4
2
3
6
2
2
3
9
9%.
position (49−51) analogues. Still, it was encouraging to
identify additional compounds (42, 43, and 50) that displayed
24 exhibited moderate clearance, with a half-life of over 1.5 h.
Not surprisingly, replacement of the fluorine atom (24) with
the more metabolically labile ethoxy group (38) increased
clearance to approximately hepatic blood flow and reduced half-
life. The results obtained with the 2-pyridyl analogues 47 and
48 revealed a profound metabolic difference. Whereas 5-fluoro
analogue 47 was rapidly cleared, 5-chloro analogue 48 had a
moderate clearance and a half-life of approximately 2 h. As
no activity versus mGlu up to the highest concentration tested
5
(30 μM) as well as several analogues with only weak activity at
mGlu . Fortunately, 5-halo analogues 47 and 48 proved an
5
exception to the general trend of modest mGlu potency with
3
these analogues. Furthermore, 5-fluoro analogue 47 demon-
strated enhanced selectivity approaching 20-fold versus mGlu5,
while 5-chloro analogue 48 was more modest with regard to
selectivity at approximately 5-fold.
53
indicated by their ligand-lipophilicity efficiency (LLE) values,
the 2-pyridyl ethers possessed a better balance of potency and
lipophilicity than the phenyl ether compounds. Notably, 4-
ethoxyphenyl ether 38 also exhibited exceedingly high protein
binding to rat plasma. These factors led to the conclusion that
optimization should continue in the context of the 2-pyridyl
With some initial SAR in hand, we further profiled some of
the more promising early analogues (38, 47, 48) to assess other
properties important for the development of a useful in vivo
tool compound (Table 3). In addition to assessing the degree
51
to which the compounds were bound to rat plasma, the
compounds were evaluated in a rat cassette pharmacokinetics
ethers. Superior LLE, selectivity versus mGlu , and plasma
5
52
unbound fraction relative to 48, made 5-fluoro analogue 47 an
attractive starting point for further optimization; however, its
high plasma clearance remained an issue. To avoid focusing
future optimization efforts exclusively in the context of a
(
PK) study using intravenous (IV) dosing to assess their
metabolic stability in vivo. In spite of its poor selectivity profile
versus mGlu , 4-fluorophenyl ether 24 was also included with
5
these compounds as a comparator compound. In fact, analogue
E
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 4. mGlu NAM and mGlu SAR of 5-Fluoropyridin-2-yl Ether Analogues 55−72
3
5
mGlu pIC
mGlu IC
% Glu Max
mGlu5
mGlu pEC
mGlu EC
% Glu Max
b,c
3
50
3
50
5
50
5
50
a
a
a
,
b
c
c
c
no.
R
(± SEM)
(nM)
(± SEM)
activity
(± SEM)
(nM)
(± SEM)
f
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
2-fluorophenyl
3-fluorophenyl
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
2-methylphenyl
3-methylphenyl
4-methylphenyl
2-methoxyphenyl
3-methoxyphenyl
4-methoxyphenyl
2,3-difluorophenyl
2,5-difluorophenyl
2,6-difluorophenyl
pyridin-2-yl
6.79 ± 0.17
6.72 ± 0.08
6.85 ± 0.16
6.84 ± 0.10
6.71 ± 0.13
6.46 ± 0.06
6.67 ± 0.15
6.57 ± 0.03
6.47 ± 0.10
6.70 ± 0.06
6.49 ± 0.10
6.73 ± 0.16
6.91 ± 0.11
6.74 ± 0.08
6.04 ± 0.05
6.36 ± 0.13
6.05 ± 0.04
6.46 ± 0.07
162
192
141
145
197
346
216
269
339
198
320
184
123
181
915
436
881
349
1.53 ± 0.12
1.44 ± 0.38
1.37 ± 0.72
2.30 ± 0.17
2.17 ± 0.40
1.72 ± 0.16
1.93 ± 0.56
1.04 ± 0.45
1.49 ± 0.60
1.02 ± 0.12
1.23 ± 0.38
2.15 ± 0.33
2.62 ± 0.47
1.49 ± 0.17
0.96 ± 0.63
0.64 ± 0.70
0.77 ± 0.44
1.31 ± 0.11
NAM
<5.0
>10000
2720
1010
1850
364
29.1 ± 7.3
9.71 ± 5.78
8.78 ± 5.29
19.2 ± 10.0
51.6 ± 7.7
NAM
NAM
5.56 ± 0.24
6.00 ± 0.25
5.73 ± 0.24
6.44 ± 0.03
e
NAM
PAM
f
d
d
d
NAM
<5.0
>10000
35.8
f
NAM
<5.0
<5.0
>10000
>10000
301
36.6 ± 8.7
54.8 ± 9.4
3.28 ± 0.45
59.7 ± 6.1
32.3 ± 5.2
21.0 ± 10.6
18.8 ± 6.1
f
NAM
NAM
6.52 ± 0.17
<5.0
f
NAM
>10000
1380
PAM
5.86 ± 0.05
5.68 ± 0.20
<5.0
e
NAM
2110
f
NAM
>10000
e
d
d
d
NAM
5.91
1230
50.8
NAM
5.45 ± 0.04
<5.0
3520
>10000
>30000
9740
3.71 ± 0.47
46.3 ± 2.9
f
pyridin-3-yl
NAM
pyridin-4-yl
<4.5
5-fluoropyridin-2-yl
NAM
5.01 ± 0.13
7.0 ± 2.5
a
b
Calcium mobilization mGlu assay; values are average of n ≥ 3. Amplitude of response in the presence of 30 μM test compound as a percentage of
3
c
d
e
maximal response (100 μM glutamate); average of n ≥ 3. Calcium mobilization mGlu assay; values are average of n ≥ 3. Average of n = 2. Partial
NAM; CRC plateaus above 10% glutamate maximum. Weak activity; CRC does not plateau.
5
f
Table 5. In Vitro DMPK Profiling of Select 5-Fluoropyridin-2-yl Ether Analogues
a
b
c
d
no.
R
cLogP
LLE
mGlu IC (nM)
fold vs mGlu5
rat plasma Fu
rat CLhep (mL/min/kg)
3
50
55
56
57
58
61
66
67
68
2-fluorophenyl
3.06
3.06
3.57
3.57
3.23
3.16
3.16
3.16
3.73
3.66
3.28
3.27
3.44
3.57
3.75
3.58
162
>61
14
0.098
0.094
0.054
0.034
0.051
0.092
0.089
0.064
64.7
53.0
66.5
51.9
58.4
63.6
49.6
3-fluorophenyl
192
141
145
216
184
123
181
2-chlorophenyl
3-chlorophenyl
3-methylphenyl
2,3-difluorophenyl
2,5-difluorophenyl
2,6-difluorophenyl
7.2
13
17
11
>81
6.8
64.6
a
b
c
Calculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/). LLE (ligand-lipophilicity efficiency) = pIC − cLogP. F =
50
u
d
fraction unbound. Predicted hepatic clearance based on intrinsic clearance in rat liver microsomes.
potential PK liability, the decision was made to prepare
analogues of both 47 and 48 in parallel.
enabled a final stage installation of the aryl or heteroaryl eastern
ring according to methods described previously to yield the
desired products 55−72.
Development of SAR in the Eastern Region. To enable
final step diversification and the rapid synthesis of new
analogues, modified syntheses were required for development
of SAR in the eastern portion of the chemotype. Synthesis of
new 5-fluoropyridin-2-yl ether analogues 55−72 was accom-
plished via a reordering of the previously outlined reactions
Because of the poor yield encountered in the synthesis of
intermediate 53, we employed what proved to be a more
scalable and shorter synthesis in the preparation of the 5-
chloropyridin-2-yl ether analogues (Scheme 4). Synthesis of
intermediate 75 from commercially available 73 was accom-
47
(
Scheme 3). Intermediate 17 was treated with boron
plished via an analogous two-step Mitsunobu coupling and
deprotection/cyclization sequence as described previously. The
lactam nitrogen was protected with a 4-methoxybenzyl group to
afford 76. Reduction of the ethyl ester was accomplished with
sodium borohydride to yield 77. Formation of the 5-
tribromide and subsequently potassium acetate to afford
acetate ester 52. Hydrolysis of 52 was accomplished with
lithium hydroxide; however, the yield of alcohol 53 suffered due
to difficulty in isolation of this polar and water-soluble
intermediate. Still, preparation of the 5-fluoropyridin-2-yl
chloropyridin-2-yl ether 78 was carried out via S Ar chemistry
N
ether 54 via S Ar chemistry was readily accomplished, which
as before. Oxidative removal of the 4-methoxybenzyl protecting
N
F
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 6. mGlu NAM and mGlu SAR of 5-Chloropyridin-2-yl Ether Analogues 80−101
3
5
mGlu pIC
mGlu IC
% Glu Max
mGlu5
mGlu pEC
mGlu EC
% Glu Max
b,c
3
50
3
50
5
50
5
50
a
a
a
,
b
c
c
c
no.
R
(± SEM)
(nM)
(± SEM)
activity
(± SEM)
(nM)
(± SEM)
8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
00
01
2-fluorophenyl
6.65 ± 0.04
6.69 ± 0.04
6.48 ± 0.08
6.25 ± 0.07
5.91 ± 0.25
6.24 ± 0.18
6.41 ± 0.05
6.39 ± 0.21
6.16 ± 0.15
6.50 ± 0.05
6.36 ± 0.11
6.37 ± 0.05
6.08 ± 0.26
6.27 ± 0.05
6.07 ± 0.23
6.55 ± 0.08
6.65 ± 0.04
6.38 ± 0.07
6.28 ± 0.06
6.50 ± 0.07
6.48 ± 0.07
6.22 ± 0.10
226
206
328
558
1230
571
392
410
690
313
439
429
825
539
852
280
225
420
529
315
328
608
1.74 ± 0.10
1.38 ± 0.47
1.87 ± 0.29
1.63 ± 0.20
0.94 ± 1.46
1.95 ± 0.70
1.71 ± 0.44
1.39 ± 0.25
1.29 ± 0.18
2.00 ± 0.32
2.07 ± 0.68
1.64 ± 0.04
1.75 ± 0.41
1.84 ± 0.19
0.33 ± 0.71
1.34 ± 0.25
2.06 ± 0.31
2.18 ± 0.19
1.55 ± 0.31
1.92 ± 0.12
1.07 ± 0.04
1.73 ± 0.53
PAM
PAM
PAM
PAM
PAM
PAM
PAM
5.49 ± 0.06
5.22 ± 0.05
5.26 ± 0.03
5.54 ± 0.02
5.48 ± 0.15
5.64 ± 0.03
5.44 ± 0.11
<5.0
3240
5970
57.3 ± 4.6
63.5 ± 0.8
51.0 ± 3.9
65.8 ± 8.8
51.2 ± 10.3
80.7 ± 0.5
69.9 ± 6.5
70.8 ± 3.1
58.5 ± 1.2
78.0 ± 1.8
59.4 ± 7.3
38.1 ± 5.0
32.2 ± 3.7
23.1 ± 6.8
36.1 ± 8.4
70.8 ± 0.8
40.3 ± 5.6
33.1 ± 2.3
8
8
8
8
8
8
8
8
8
9
9
9
9
9
9
9
9
9
9
1
1
3-fluorophenyl
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
2-methylphenyl
3-methylphenyl
4-methylphenyl
2-methoxyphenyl
3-methoxyphenyl
4-methoxyphenyl
2-cyanophenyl
5450
2870
3310
2280
3600
e
PAM
>10000
>10000
5070
e
NAM
PAM
PAM
PAM
<5.0
5.29 ± 0.08
5.36 ± 0.06
5.30 ± 0.10
<5.0
4330
4970
e
3-cyanophenyl
PAM
PAM
PAM
PAM
PAM
>10000
3320
4-cyanophenyl
5.48 ± 0.08
5.17 ± 0.29
5.59 ± 0.07
5.85 ± 0.09
<5.0
2,3-difluorophenyl
2,4-difluorophenyl
2,6-difluorophenyl
3,5-difluorophenyl
2-cyano-5-fluorophenyl
3-cyano-5-fluorophenyl
pyridin-2-yl
6680
2580
1420
e
PAM
>10000
>30000
<4.5
d
d
d
PAM
5.98
1044
28.0
<4.5
<4.5
>30000
>30000
pyridin-3-yl
a
b
Calcium mobilization mGlu assay; values are average of n ≥ 3. Amplitude of response in the presence of 30 μM test compound as a percentage of
3
c
d
e
maximal response (100 μM glutamate); average of n ≥ 3. Calcium mobilization mGlu assay; values are average of n ≥ 3. Average of n = 2. Weak
activity; CRC does not plateau.
5
group was carried out with ceric ammonium nitrate to provide
penultimate intermediate 79. Conversion of 79 into analogues
stability was also assessed in vitro by measuring the intrinsic
clearance of the compound when incubated with rat liver
54
8
0−110 utilized the copper mediate N-arylation methods
microsomes (RLM). While the fraction unbound in rat
plasma was encouraging for most compounds, metabolic
stability was uniformly poor. Unfortunately, on the basis of
their intrinsic clearance in RLM, the compounds were
employed previously.
Testing of new 5-fluoropyridin-2-yl ether analogues 55−72
showed eastern ring modification was a useful strategy for
55
enhancing mGlu NAM activity (Table 4). In the case of
3
predicted to exhibit hepatic clearance near blood flow.
monosubstituted phenyl ethers (55−65), several analogues
Fortunately, testing of new 5-chloropyridin-2-yl ether
exhibited mGlu IC values less than 200 nM. Specifically, at
3
50
analogues 80−101 ultimately provided another path forward
the 2-position, fluorine (55) and chlorine (57) substitution was
preferred to methyl (60) and methoxy (63) substituents, while
for the design of both potent and highly selective mGlu NAMs
3
(
Table 6). Monosubstituted phenyl analogues (80−93)
little difference in mGlu activity was observed with variation of
3
generally exhibited similar activity at mGlu (IC = 200−600
nM) regardless of the position of the substituent on the ring. 4-
3
50
the same substituents at the 3-position (56, 58, 61, 64).
Substitution at the 4-position (59, 62, 65) was slightly less
favorable. Encouragingly, several of these analogues also
Chlorophenyl analogue 84, 2-methoxyphenyl analogue 88, and
3
-cyanophenyl analogue 92 were exceptions to this trend with
demonstrated only weak activity at mGlu . Some difluor-
5
each exhibiting reduced activity at mGlu . Although most of
3
ophenyl analogues (66−68) were also prepared and found to
these monosubstituted phenyl analogues (80−93) demon-
exhibit good potency. Simple pyridyl analogues (69−71) were
strated only modest selectivity versus mGlu , 4-methylphenyl
5
less potent versus mGlu than the majority of the phenyl
3
analogue 87, 2-methoxyphenyl analogue 88, and 3-cyanophenyl
analogue 92 exhibited weak activity at that receptor.
Preparation of disubstituted phenyl analogues (94−99) yielded
analogues; however, selectivity versus mGlu was notable in the
5
case of 70 and 71. Finally, simple fluorine substitution of the
pyridine ring (72) enhanced mGlu3 activity relative to
unsubstituted comparator 69.
additional compounds with improved selectivity versus mGlu .
5
3
,5-Difluorophenyl analogue 97 was a weak mGlu PAM, and
Although several of these new 5-fluoropyridin-2-yl ether
5
2
-cyano-5-fluorophenyl analogue 98 was inactive versus mGlu5
analogues demonstrated improved mGlu3 NAM potency,
53
modest levels of mGlu selectivity, and improved LLE values
up to the highest concentration tested (30 μM). Finally,
encouraging results were observed with unsubstituted pyridyl
analogues 100 and 101, where both proved inactive versus
mGlu while maintaining good mGlu NAM activity.
5
(Table 5), their DMPK profiles remained a critical unanswered
question. Thus, selected analogues were profiled in our
51
aforementioned rat protein binding assay, and metabolic
5
3
G
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 7. mGlu NAM and mGlu SAR of Additional 5-Chloropyridin-2-yl Ether Analogues 102−110
3
5
mGlu pIC
mGlu IC
% Glu Max
mGlu5
mGlu pEC
mGlu EC
% Glu Max
b,c
3
50
3
50
5
50
5
50
a
a
a
,
b
c
c
c
no. series
R
(± SEM)
(nM)
(± SEM)
activity
(± SEM)
(nM)
(± SEM)
d
102
103
104
105
106
107
108
109
110
I
I
3-F
6.44 ± 0.03
6.17 ± 0.06
6.59 ± 0.09
6.53 ± 0.08
6.41 ± 0.05
6.32 ± 0.03
6.32 ± 0.03
6.22 ± 0.01
6.39 ± 0.06
362
677
260
294
392
482
481
605
408
1.64 ± 0.36
1.35 ± 0.55
2.44 ± 0.32
1.75 ± 0.19
1.71 ± 0.41
1.45 ± 0.22
1.85 ± 0.32
1.08 ± 0.50
PAM
<5.0
<4.5
<5.0
<5.0
<4.5
<4.5
<4.5
<4.5
<5.0
>10000
>30000
>10000
>10000
>30000
>30000
>30000
>30000
>10000
32.4 ± 4.4
3-CN
5-F
d
I
PAM
47.9 ± 5.1
24.8 ± 5.5
d
I
6-F
NAM
II
II
II
II
II
2-F
4-F
5-F
5-CN
6-F
d
1.45 ± 0.29
PAM
30.6 ± 2.4
a
b
Calcium mobilization mGlu assay; values are average of n ≥ 3. Amplitude of response in the presence of 30 μM test compound as a percentage of
3
c
d
maximal response (100 μM glutamate); average of n ≥ 3. Calcium mobilization mGlu assay; values are average of n ≥ 3. Weak activity; CRC does
not plateau.
5
Table 8. In Vitro DMPK Profiling of Select 5-Chloropyridin-2-yl Ether Analogues
a
b
c
d
no.
R
cLogP
LLE
mGlu IC (nM)
fold vs mGlu5
rat plasma Fu
rat CLhep (mL/min/kg)
3
50
9
9
2-cyano-5-fluorophenyl
pyridin-2-yl
3.29
2.57
2.16
2.67
2.26
2.26
1.88
2.99
3.91
4.06
3.74
4.06
4.06
4.34
529
>56
>91
>49
>76
>62
>62
>49
0.045
0.051
0.118
0.083
0.085
0.092
0.078
47.7
69.5
42.0
36.9
36.0
26.6
100
101
106
107
108
109
328
608
392
482
481
605
pyridin-3-yl
2-fluoropyridin-3-yl
4-fluoropyridin-3-yl
5-fluoropyridin-3-yl
5-cyanopyridin-3-yl
22.8
a
b
c
Calculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/). LLE (ligand-lipophilicity efficiency) = pIC − cLogP. F =
50
u
d
fraction unbound. Predicted hepatic clearance based on intrinsic clearance in rat liver microsomes.
Table 9. In Vivo DMPK Profiling of Select 5-Chloropyridin-2-yl Ether Analogues
b
c,d,e
rat IV PK results
rat IP tissue distribution results
a
a
f
g
no.
rat plasma Fu
rat brain Fu
t1/2 (min)
CLplasma (mL/min/kg)
VSS (L/kg)
plasma conc (μM)
brain conc (μM)
Kp
Kp,uu
9
9
0.045
0.118
0.083
0.092
0.078
0.032
194
21
42
30
59
13
50
37
50
22
2.2
1.2
1.6
1.9
1.4
1.68
9.49
4.52
2.41
1.4
1.7
1.2
1.0
1
1
1
1
01
06
08
09
0.052
0.033
15.85
1.0
5.28
0.49
a
b
c
F = fraction unbound. n = 2; dose = 0.2 mg/kg; solution in 10% EtOH, 38−40% PEG 400, 50−52% DMSO (2 mg/mL). n = 2; time point = 15
u
d
e
min; dose = 10 mg/kg. For 99 and 109, fine homogeneous suspension in 0.1% Tween 80 and 0.5% methyl cellulose in H O (4 mg/mL). For 106,
fine homogeneous suspension in 10% EtOH and 90% PEG400 (4 mg/mL). K = total brain to total plasma ratio. K = unbound brain (brain Fu
2
f
g
p
p,uu
×
total brain) to unbound plasma (plasma F × total plasma) ratio
u
Encouraged by the selectivity profiles seen with pyridyl
Unfortunately, in the pyridin-2-yl set (102−105), the majority
analogues 100 and 101 but suspecting that these unsubstituted
compounds may be prone to rapid metabolism, we immediately
prepared several substituted analogues of each (Table 7).
of these modifications enhanced mGlu activity, albeit only
5
slightly. Cyano analogue 103 was the lone exception; however,
this compound was approximately 2-fold less potent than
H
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 4. Progressive fold-shift of the glutamate CRC by mGlu NAM 106 (A) and mGlu NAM 111 (B); compound concentrations shown in M.
3
2/3
unsubstituted analogue 100. Results with the pyridin-3-yl set
between the compartments and a low probability of the
57
(
106−110) were more encouraging as all new compounds
compounds being substrates for transporters. Compound 109
had a Kp,uu value of 0.49, indicating possible efflux; however,
additional experiments would be required to determine such
conclusively. Compound 106 (VU0650786) was deemed the
most attractive and targeted for extensive profiling.
except 6-fluoro analogue 110 maintained the excellent
selectivity profile versus mGlu observed with 101 without a
5
loss of activity at mGlu . At this point, several compounds with
3
good mGlu NAM activity and devoid of mGlu activity were in
3
5
hand, which set the stage for further profiling in pursuit of
compounds meriting extensive in vivo evaluation.
Profiling of Compound 106. Profiling of compound 106
began with determination of its full selectivity versus other
As before, we moved several promising compounds into
members of the mGlu family. In addition to mGlu , selectivity
5
51
assays to assess protein binding in rat plasma as well as
versus fellow group II receptor subtype, mGlu , was critical to
2
54
metabolic stability in RLM (Table 8). The lone eastern
phenyl analogue 99 was slightly more protein bound than its
eastern pyridyl comparators, which was not surprising given its
higher lipophilicity. A range of predicted hepatic clearance
values based on the intrinsic clearance of the compound in
RLM were observed including one analogue of less than one-
third hepatic blood flow (109) and one analogue near hepatic
blood flow (100). The remaining analogues were predicted to
have moderate clearance in vivo. Somewhat surprisingly,
unsubstituted pyridine-3-yl analogue 101 was more metabol-
ically stable than its regioisomeric comparator 100. Still,
substituted versions of 101 (106−109) did exhibit increased
stability relative to 101.
assess. We evaluated the selectivity of 106 versus rat mGlu2
using full CRC analysis, and the compound was inactive up to
the highest concentration tested (30 μM). Thus, compound
106 has been established to have no functional activity at either
mGlu or mGlu up to a concentration that is more than 75-
2
5
fold over the functional potency at mGlu . The effect of 10 μM
3
106 on the orthosteric agonist CRC was measured in fold-shift
44,45
experiments to evaluate selectivity versus the other mGlus.
No significant effect was found, indicating the compound was
inactive at those receptors as well. We further evaluated the
nature of the interaction between 106 and mGlu by examining
3
the effects of increasing concentrations of 106 on the glutamate
CRC in a progressive fold-shift experiment. If an antagonist acts
via a noncompetitive mechanism, we anticipate increasing
concentrations of antagonist would shift the glutamate curve to
the right and decrease the maximal signal of glutamate. Figure 4
depicts the effects of multiple concentrations of 106 and known
Several interesting analogues were next advanced into rat
52
cassette PK studies using IV dosing to assess their metabolic
stability in vivo (Table 9). As the most promising analogue with
an eastern phenyl group, 99 was selected for these studies.
Unsubstituted eastern pyridyl analogue 101 and several
substituted comparators (106, 108, and 109) were also chosen
based on the totality of data collected to that point.
Distinguishing between analogues 108 and 107 was difficult;
however, compound 108 was ultimately selected as it had a
marginally better in vitro DMPK profile. 5-Cyanopyridin-3-yl
analogue 109 was selected as it was predicted to have the
lowest clearance, and 2-fluoropyridin-3-yl analogue 106 was
chosen as it was the most potent analogue with a moderate
predicted clearance. Compound 99 exhibited a lower clearance
in vivo than expected and had a long half-life in excess of 3 h.
The in vivo clearance for the pyridine-3-yl analogues was
generally well predicted by the RLM experiments, with
analogue 108 being the lone exception. Analogues 99, 106,
and 109 were thus selected for further study in single time
point (15 min) tissue distribution studies at a higher dose (10
58
mGlu2/3 NAM 111 (MNI-137) on the glutamate CRC. As
expected, both compounds exhibited the characteristic right-
ward shift and depressed glutamate maximum typically
observed with NAMs, suggesting that 106 does not bind to
the orthosteric glutamate binding site but instead acts via an
allosteric mechanism.
To evaluate the ancillary pharmacology of the compound, a
commercially available radioligand binding assay panel of 68
clinically relevant GPCRs, ion channels, kinases, and trans-
59
porters was employed, and only a single significant response
60
(5-HT , 65% inhibition) was found at 10 μM 106. Because
2B
5-hydroxytryptamine receptor 2B (5-HT ) agonists are
2
B
61
associated with cardiotoxicity, we followed this result up
with a functional cell-based assay to assess potential agonist or
62
antagonist activity of the compound at 5-HT . Fortunately,
2B
testing up to 10 μM in this assay revealed no functional activity
for 106 at 5-HT . Potential for drug−drug interactions was
5
6
mg/kg). Intraperitoneal (IP) dosing was chosen for these
studies as this route is convenient for use in our planned
behavioral studies. The same three compounds were also
examined in protein binding assays with rat brain homoge-
2B
negligible as assessed in a human liver microsomes (HLM)
cocktail assay with probe substrates for four common P450s
6
3
(Table 10). Finally, permeability and potential for P-
glycoprotein (P-gp) mediated efflux was assessed in Madin−
Darby canine kidney (MDCK) cells transfected with the human
5
1
nates. CNS penetration with each compound was excellent,
with compound 106 exhibiting the highest plasma and brain
levels. Both 99 and 106 exhibited unbound brain to unbound
plasma ratios (Kp,uu) of one, indicating distribution equilibrium
64
MDR1 gene. Not surprisingly, compound 106 was highly
permeable with no evidence of efflux in this assay.
I
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 10. P450 Inhibition and Permeability Profile of
mg/kg IP studies in both rats and mice was at or beyond the
Compound 106
measured functional mGlu NAM activity. With that in mind,
3
evaluation of compound 106 in rodent behavioral models
known to be sensitive to mGlu2/3 antagonists was initiated.
Behavioral Pharmacology of Compound 106. It is well-
known that naive mice will bury foreign objects, such as glass
̈
marbles, in deep bedding. This behavior can be inhibited by
a
b
P450 inhibition
permeability
pretreatment with low doses of certain benzodiazepines, such as
65
45.0 × 10−6 cm/s
49.9 × 10− cm/s
diazepam, as well as certain selective serotonin reuptake
CYP1A2 IC50
CYP2C9 IC50
CYP2D6 IC50
CYP3A4 IC50
>30 μM
25.6 μM
>30 μM
A−B Papp
B−A Papp
efflux ratio
6
6
6
inhibitors (SSRIs), such as fluvoxamine. Likewise, this
1.1
behavior has proven sensitive to a variety of mGlu NAM
5
38,67,68
>30 μM
compounds from diverse chemotypes.
As such, the
a
b
marble burying assay has often been used as a convenient
method for assessing anxiolytic activity. It is worth noting that
recent reports have argued that the assay reflects a repetitive
and perseverative behavior such as OCD as opposed to novelty-
Cocktail assay in HLM. MDR1-MDCK cells.
Confident that 106 possessed a favorable profile with respect
to its pharmacology, in vitro DMPK properties, and preliminary
in vivo DMPK properties, we moved the compound into
several definitive in vivo DMPK studies in rats and mice (Table
69
induced anxiety. For our purposes, the most important fact
was that the mGlu2/3 orthosteric antagonists 1 and 2 have both
9,10
been previously shown to inhibit marble burying.
Thus,
5
6
1
1). Time course studies using IP dosing revealed nearly
examination of the selective mGlu NAM 106 in this assay was
3
identical and rapidly reached Cmax values in both rats and mice.
A single time point (30 min) tissue distribution study in mice
analogous to the one previously conducted in rats showed
excellent CNS penetration in that species as well. Again, the
Kp,uu value was near one as would be expected for a highly
permeable compound devoid of efflux issues. A definitive rat
IV PK study (1.0 mg/kg) was conducted, and results were
essentially identical to those collected with the prior cassette
study (0.2 mg/kg). Finally, a rat oral PK study (3.0 mg/kg) was
carried out to assess bioavailability, which proved quite good
warranted to determine the contribution of mGlu versus
2
mGlu to this effect (Figure 5). Gratifyingly, dose dependent
3
efficacy was observed in this assay, with statistically significant
effects at all three doses and essentially complete inhibition at
the highest dose (56.6 mg/kg). The positive control for this
57
assay was the well characterized mGlu NAM 3-[(2-methyl-1,3-
5
70
thiazol-4-yl)ethynyl]-pyridine (112). On the basis of the
available DMPK data, it is estimated that CNS unbound
exposure of compound 106 at the 10 mg/kg dose in this study
reached a peak of 564 nM or approximately 1.5-fold the
functional IC50.
(
60%). Taking into account the data summarized herein, it was
estimated that the unbound Cmax in the CNS following the 10
Table 11. Rodent PK Profile of Compound 106
a
b
c
F = fraction unbound. n = 3; fine microsuspension in 0.1% Tween 80 and 0.5% methyl cellulose in H O (4 mg/mL). n = 2; dose = 1.0 mg/kg;
u
2
d
solution in 10% EtOH, 50% PEG 400, 40% saline (1 mg/mL). n = 2; dose = 3.0 mg/kg; fine microsuspension in 0.1% Tween 80 and 0.5% methyl
cellulose in H O (0.3 mg/mL). n = 3; time point = 30 min post dose; fine microsuspension in 0.1% Tween 80 and 0.5% methyl cellulose in H O (1
e
2
2
f
g
mg/mL). K = total brain to total plasma ratio. K = unbound brain (brain F × total brain) to unbound plasma (plasma F × total plasma) ratio.
p
p,uu
u
u
h
n = 3 per time point; fine microsuspension in 0.1% Tween 80 and 0.5% methyl cellulose in H O (1 mg/mL).
2
J
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
pathways in the antidepressant effects of ketamine and mGlu2/3
antagonists, including activation of the α-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid (AMPA) receptor and the
18,19,78−82
mammalian target of rapamycin (mTOR) pathway.
Such studies have raised the possibility that mGlu2/3 antagonists
or selective antagonists of each individual group II mGlu might
represent novel approaches to rapid acting antidepressants
without the side effect profile of ketamine. A selective and CNS
penetrant mGlu NAM compound such as 106 will be a
3
valuable tool in shedding light on such questions.
CONCLUSION
■
A cross screening hit from a nonalkyne mGlu PAM chemotype
served as a successful launching point for the discovery of
5
compound 106, a highly selective mGlu NAM with DMPK
3
Figure 5. Inhibition of marble burying in mice by compound 106. n =
properties that enable its convenient use in rodent models of
psychiatric disorders. In addition to these features, the
compound displays moderate clearance and good bioavailability
in rats. Furthermore, the compound is highly permeable, not a
substrate for P-gp mediated efflux, and possesses an attractive
P450-inhibition profile. The compound has demonstrated
efficacy in two rodent models previously shown to be sensitive
8
−10 male CD-1 mice per treatment group; vehicle = 10% Tween 80
in H O; 15 min pretreatment with compound or vehicle; 30 min
2
burying time; *, p < 0.05 vs vehicle control group. Compound 112 is
the mGlu NAM 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine.
5
Having established its efficacy in an anxiolytic/OCD mouse
model, profiling of compound 106 in a rat model of depression
was considered valuable for illustrating the utility of the
compound. The forced swim test (FST) measures immobility
time in rats placed in a tank of water from which they cannot
escape and is sensitive to many antidepressants including
to mGlu2/3 inhibition. This highly selective mGlu NAM can
3
thus serve as a useful tool for elucidating the role of selective
inhibition of mGlu3 and its potential utility as a novel
therapeutic target. Such studies will constitute the subject of
future communications.
7
1
several SSRIs. Importantly, efficacy with the mGlu2/3
orthosteric antagonists 1 and 2 has been demonstrated in this
8
assay, making the examination of 106 compelling (Figure 6).
EXPERIMENTAL SECTION
Diethyl (R)-1-(1-((tert-Butoxycarbonyl)amino)propan-2-yl)-
H-pyrazole-3,5-dicarboxylate (74). Diethyl 3,5-pyrazoledicarbox-
■
1
ylate 73 (4.24 g, 20 mmol, 1.0 equiv) and tert-butyl (S)-(2-
hydroxypropyl) carbamate 15 (7.01 g, 40 mmol, 2.0 equiv) were
dissolved in THF (100 mL. 0.2 M), and triphenyl phosphine (9.44 g,
3
0
6 mmol, 1.8 equiv) was added. After 5 min, the mixture was cooled to
°C and di-tert-butyl azodicarboxylate (8.29 g, 36 mmol, 1.8 equiv)
was added. The reaction mixture was then subjected to microwave
irradiation for 25 min at 120 °C. The mixture was cooled to room
temperature, and the solvent was removed in vacuo. Purification via
flash chromatography on silica gel provided the title compound as a
semisolid (8.2 g, yield was not determined due to contamination of
t
1
D BAD byproduct, di-tert-butyl hydrazine-1,2-dicarboxylate). H NMR
(
400 MHz, MeOD) δ 7.29 (s, 1H), 5.69−5.01 (m, 1H), 4.41−4.35
(
1
1
m, 4H), 3.51−3.39 (m, 2H), 1.51 (d, J = 6.8 Hz, 3H), 1.41−1.38 (m,
5H). 13C NMR (100 MHz, CDCl ) δ 161.7, 159.0, 155.8, 142.6,
3
Figure 6. Decrease in immobility in the FST in rats by compound 106.
n = 8−10 male Sprague−Dawley rats per treatment group; 106 vehicle
34.2, 114.0, 79.4, 61.4, 61.1, 53.4, 45.1, 28.2 (3C), 18.5, 14.3, 14.2.
+
LCMS (method A): R = 1.052 min, m/z = 314.2 [M + H] . HRMS,
T
=
10% Tween 80 in H O; ketamine vehicle = saline; 30 min
calcd for C H N O [M], 369.1900; found, 369.1899.
2
17 27
3
6
pretreatment with compound or vehicle; 6 min testing session; *, p <
Ethyl (R)-7-Methyl-4-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]-
pyrazine-2-carboxylate (75). Compound 74 (8.2 g, 22.2 mmol, 1.0
equiv) was treated with a solution of 4 N HCl in 1,4-dioxane (78 mL).
Deprotection of the tert-butyl carbamate protecting group was
monitored by LCMS. Once deprotection was complete, the reaction
mixture was carefully basified with saturated aqueous NaHCO3
(verified by pH paper) and was allowed to stir at room temperature
overnight. The mixture was diluted with dichloromethane, and the
aqueous layer was extracted with dichloromethane (3×). The
combined organic layers were washed with brine, dried over
Na SO , and concentrated in vacuo to provide the title compound
0
.05 vs vehicle control group.
In this case, significant effects in decreasing immobility time
were observed at the highest dose (56.6 mg/kg). On the basis
of the available DMPK data and an assumption of dose
linearity, it is estimated that CNS unbound exposure of 106 at
the high dose in these studies reached a peak of 2.3 μM or
approximately 6-fold over the functional IC . The positive
5
0
2
4
control in this assay was the N-methyl-D-aspartate (NMDA)
72
as a white solid (4.4 g, 89% yield over two steps), which was used
receptor antagonist drug ketamine,7 which was introduced to
1
3
without further purification. H NMR (400 MHz, CDCl
H), 7.13 (bs, 1H), 4.68−4.62 (m, 1H), 4.41 (q, J = 7.1 Hz, 2H), 3.86
ddd, J = 15.8, 10.2, 1.4 Hz, 1H), 3.51 (ddd, J = 9.4, 6.4, 3.0 Hz, 1H),
3
) δ 7.35 (s,
the market over 50 years ago. Ketamine has recently
1
demonstrated rapid acting antidepressant efficacy in TRD
(
1
7
4−76
patients.
desirable side effects including psychotomimetic effects.
Preclinical studies implicate common downstream signaling
Unfortunately, ketamine produces several un-
13
.65 (d, J = 6.6 Hz, 3H), 1.39 (t, J = 7.1 Hz, 3H). C NMR (100
77
MHz, CDCl ) δ 161.7, 159.3, 143.8, 134.4, 110.7, 61.3, 53.2, 46.0,
3
17.4, 14.3. LCMS (method A): R = 0.546 min, m/z = 224.2 [M + H].
T
K
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
25
1
HRMS, calcd for C H N O [M], 223.0957; found, 223.0957. [α]
mg, 65% yield) as a pale-yellow solid. H NMR (400 MHz, DMSO-d )
10
13
3
3
D
6
=
−29.9° (c 0.500, CHCl ).
δ 8.26 (d, J = 2.7 Hz, 1H), 8.21 (s, 1H), 7.83 (dd, J = 8.8, 2.7 Hz, 1H),
6.92 (dd, J = 8.8, 0.5 Hz, 1H), 6.77 (s, 1H), 5.29 (s, 2H), 4.51−4.46
(m, 1H), 3.66 (ddd, J = 13.0, 8.7, 8.7 Hz, 1H), 3.34 (ddd, J = 13.1, 7.9,
2.2 Hz, 1H), 1.45 (d, J = 6.5 Hz, 3H). 13C NMR (100 MHz, DMSO-
d6) δ 161.9, 158.7, 147.7, 145.3, 139.7, 135.1, 124.1, 112.9, 107.4, 61.7,
3
Ethyl (R)-5-(4-Methoxybenzyl)-7-methyl-4-oxo-4,5,6,7-
tetrahydropyrazolo[1,5-a]pyrazine-2-carboxylate (76). Com-
pound 75 (2.23 g, 10 mmol, 1.0 equiv) was dissolved in DMF (50
mL, 0.2 M), cooled to 0 °C, and treated with 60% sodium hydride in
mineral oil (480 mg, 12 mmol, 1.2 equiv) in five portions. The
reaction mixture was stirred for 15 min, and 4-methoxybenzyl chloride
52.3, 45.6, 17.0. LCMS (method A): R = 0.804 min, m/z = 293.2 [M
T
+
+ H] . HRMS, calcd for C H ClN O [M], 292.0727; found,
13
13
4
2
292.0727. [α]25 = −38.3° (c 0.442, CHCl ).
(
1.63 mL, 12 mmol, 1.2 equiv) was added. After 16 h, the reaction
D
3
mixture was diluted with water and extracted with EtOAc (3×). The
combined extracts were washed with water and brine, dried over
Na SO , filtered, and concentrated in vacuo. The crude material was
(R)-2-(((5-Chloropyridin-2-yl)oxy)methyl)-5-(2-fluoropyridin-
3-yl)-7-methyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one
(
106). Copper(I) iodide (13.7 mg, 0.072 mmol, 2.1 equiv) was added
2
4
to a suspension of compound 79 (10 mg, 0.035 mmol, 1.0 equiv), 3-
bromo-2-fluoropyridine (7.40 μL, 0.072 mmol, 2.1 equiv), potassium
carbonate (10 mg, 0.072 mmol, 2.1 equiv), and N,N′-dimethylethy-
lenediamine (20.7 μL, 0.19 mmol, 5.5 equiv) in toluene (0.44 mL) in a
sealed reaction vial. The reaction mixture was stirred at 120 °C. After
purified by flash chromatography on silica gel to provide the title
compound (2.51 g, 73% yield) as a pale-yellow solid. H NMR (400
1
MHz, CDCl ) δ 7.37 (s, 1H), 7.23 (d, J = 14.1, 2H), 6.87 (d, J = 14.1,
3
2
H), 4.76 (d, J = 14.5, 1H), 4.59−4.50 (m, 2H), 4.44−4.36 (m, 2H),
3
.79 (s, 3H), 3.69 (dd, J = 13.1, 4.6 Hz, 1H), 3.35 (dd, J = 13.1, 6.3
13
1
6 h, the mixture was diluted with EtOAc, filtered through a Celite pad
Hz, 1H), 1.47 (d, J = 6.6 Hz, 3H), 1.38 (t, J = 7.0 Hz, 3H). C NMR
100 MHz, CDCl ) δ 161.7, 159.5, 137.1, 143.9, 134.8, 129.9 (2C),
which was rinsed with EtOAc (2×), and concentrated in vacuo.
Purification using reserve phase HPLC method 1 with 39−71%
CH CN in H O (0.1% TFA) over 4 min provided the title compound
(
3
1
27.9, 114.3, 110.9 (2C), 61.2, 55.3, 52.9, 50.4, 48.8, 17.5, 14.3. LCMS
+
(
method A): R = 0.955 min, m/z = 344.2 [M + H] . HRMS, calcd for
3
2
T
1
25
(7.2 mg, 53% yield) as a white powder. H NMR (400 MHz, DMSO-
C H N O [M], 343.1532; found, 343.1533. [α] = −8.1° (c 0.157,
CHCl3).
R)-2-(Hydroxymethyl)-5-(4-methoxybenzyl)-7-methyl-6,7-
1
8
21
3
4
D
d6) δ 8.27 (dd, J = 2.7, 0.5 Hz, 1H), 8.23 (ddd, J = 4.8, 2.7, 2.7 Hz,
1
H), 8.09 (ddd, J = 9.6, 7.7, 1.8 Hz, 1H), 7.84 (dd, J = 8.8, 2.7 Hz,
(
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (77). Sodium borohy-
dride (1.16 g, 30.6 mmol, 5.0 equiv) was added slowly to a solution of
compound 76 (2.1 g, 6.11 mmol, 1.0 equiv) in THF (20 mL) and
MeOH (5.0 mL) at 0 °C. The reaction was heated to 60 °C, and after
1H), 7.50 (ddd, J = 9.0, 4.9, 1.3 Hz, 1H) 6.95 (s, 1H), 6.92 (d, J = 0.5
Hz, 1H), 5.35 (s, 2H), 4.82−4.75 (m, 1H), 4.27 (dd, J = 12.8, 4.3 Hz,
1
3
1H), 3.98 (dd, J = 12.8, 7.2 Hz, 1H) 1.55 (d, J = 6.5 Hz, 3H).
C
NMR (100 MHz, DMSO-d
) δ 161.9, 158.2 (d, JC,F = 239 Hz), 156.6,
48.4, 146.4 (d, JC,F = 14 Hz), 145.3, 133.9 (d, JC,F = 13 Hz), 139.8,
34.0, 124.4 (d, JC,F = 28 Hz), 124.2, 123.2 (d, JC,F = 4 Hz), 112.9,
08.7, 61.6, 54.0, 52.7, 17.1. LCMS (method A): R = 0.944 min, m/z
6
1
1
1
3
0 min at that temperature, the reaction mixture was diluted with
water and extracted with dichloromethane. The aqueous layer was
acidified with a 1 M aqueous HCl solution and extracted with
dichloromethane (2×). The combined extracts were dried over
Na SO and concentrated in vacuo. Purification by flash chromatog-
T
+
=
388.2 [M + H] . HRMS, calcd for C H ClFN O [M], 387.0898;
18 15 5 2
25
found, 387.0899. [α] = −23.6° (c 0.100, DMSO).
D
2
4
raphy on silica gel provided the title compound as a viscous oil (1.55 g,
1
8
6
4% yield). H NMR (400 MHz, CDCl ) δ 7.23−7.21 (m, 2H), 6.87−
3
ASSOCIATED CONTENT
■
.84 (m, 3H), 4.72 (d, J = 14.5 Hz, 1H), 4.68 (s, 2H), 4.59 (d, J = 14.4
Hz, 1H), 4.43−4.35 (m, 1H), 3.78 (s, 3H), 3.60 (dd, J = 13.0, 4.6 Hz,
H), 3.31 (dd, J = 13.0, 7.4 Hz, 1H), 1.42 (d, J = 6.5 Hz, 3H). 13
NMR (100 MHz, CDCl ) δ 159.4, 157.8, 152.6, 134.5, 129.8 (2C),
*
S
Supporting Information
1
C
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01005.
3
1
28.2, 114.3 (2C), 106.5, 58.7, 55.3, 52.0, 50.8, 48.7, 17.2. LCMS
+
(
method A): R = 0.680 min, m/z = 302.2 [M + H] . HRMS, calcd for
T
25
C H N O [M], 301.1426; found, 301.1428. [α] = −5.3° (c 0.98,
Experimental procedures and spectroscopic data for
additional compounds, molecular pharmacology meth-
ods, DMPK methods, behavioral pharmacology methods,
and the ancillary pharmacology profile details of 106
(PDF)
1
6
19
3
3
D
CHCl3).
R)-2-(((5-Chloropyridin-2-yl)oxy)methyl)-5-(4-methoxyben-
zyl)-7-methyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one
78). To a solution of compound 77 (1.5 g, 4.98 mmol, 1.0 equiv) in
DMF (25 mL, 0.2 M) at 0 °C was added NaH (300 mg, 12.44 mmol,
(
(
2
2
.5 equiv). The resulting mixture was stirred for 15 min, and 5-chloro-
-fluoropyridine (1.25 mL, 12.44 mmol, 2.5 equiv) was added. The
Molecular formula strings (CSV)
mixture was stirred overnight and extracted with EtOAc (3×). The
combined extracts were concentrated in vacuo. Purification by flash
AUTHOR INFORMATION
■
chromatography on silica gel afforded the title compound (1.72 g, 84%
1
Corresponding Author
yield) as a viscous oil. H NMR (400 MHz, CDCl ) δ 8.13 (d, J = 2.6
3
Hz, 1H), 7.54 (dd, J = 8.8, 2.6 Hz, 1H), 7.25 (d, J = 8.5 Hz, 2H), 6.98
*Phone: 817-735-0241. Fax: 817-735-2603. E-mail: kyle.
emmitte@unthsc.edu.
(
2
(
1
s, 1H), 6.88 (d, J = 8.5 Hz, 2H), 6.75 (d, J = 8.8 Hz, 1H), 5.39 (s,
H), 4.75 (d, J = 14.5 Hz, 1H), 4.63 (d, J = 14.5 Hz, 1H), 4.50−4.42
m, 1H), 3.81 (s, 3H), 3.64 (dd, J = 13.0, 4.6 Hz, 1H), 3.35 (dd, J =
Present Addresses
§
3.0, 7.4 Hz, 1H), 1.48 (d, J = 6.5 Hz, 3H). 1 C NMR (100 MHz,
3
Incyte Corporation, Experimental Station, E336/131A, Route
CDCl ) δ 161.6, 159.4, 157.7, 148.9, 145.1, 138.6, 134.5, 129.8 (2C),
141 and Henry Clay Road, Wilmington, Delaware 19880,
United States.
3
1
28.2, 124.4, 114.2 (2C), 112.3, 108.2, 61.6, 55.3, 52.1, 50.8, 48.8, 17.2.
+
∥
LCMS (method A): R = 1.080 min, m/z = 413.2 [M + H] . HRMS,
calcd for C H ClN O [M], 412.1302; found, 412.1305. [α]
Sano Informed Prescribing, Cool Springs Life Sciences Center,
T
25
=
21
21
4
3
D
393 Nichol Mill Lane, Suite 34, Franklin, Tennessee 37067,
United States.
−
10.3° (c 1.512, CHCl3).
(
R)-2-(((5-Chloropyridin-2-yl)oxy)methyl)-7-methyl-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one (79). Compound 78
1.65 mg, 4.0 mmol, 1.0 equiv) was dissolved in MeCN (40 mL, 0.1
⊥
Covance Inc., 671 South Meridian Road, Greenfield, Indiana
4
6140, United States.
(
#
Department of Pharmaceutical Sciences, UNT System College
of Pharmacy, University of North Texas Health Science Center,
3500 Camp Bowie Boulevard, Fort Worth, Texas 76107,
United States.
M). and a solution of ceric ammonium nitrate (6.57 g, 12 mmol, 4.0
equiv) in water (12 mL) was added. After 30 min at room
temperature, solvents were removed in vacuo. Purification using
flash chromatography on silica gel provided the title compound (764
L
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Author Contributions
(6) Cel
́
anire, S.; Sebhat, I.; Wichmann, J.; Mayer, S.; Schann, S.;
Drs. Emmitte and Lindsley directed and designed the
chemistry. Drs. Engers and Konkol performed the medicinal
chemistry. Drs. Conn and Niswender directed and designed the
molecular pharmacology experiments. Dr. Rodriguez directed
and performed molecular pharmacology experiments. Mr.
Venable and Mr. Loch performed molecular pharmacology
experiments. Dr. Daniels directed and designed the DMPK
experiments. Dr. Blobaum directed DMPK experiments and
performed bioanalytical work. Mr. Morrison performed
bioanalytical work. Mr. Chang performed in vitro DMPK
work. Mr. Byers performed in vivo DMPK work. Dr. Jones
directed and designed the behavioral experiments. Dr.
Thompson performed the behavioral experiments.
Gatti, S. Novel metabotropic glutamate receptor 2/3 antagonists and
their therapeutic applications: a patent review (2005 − present).
Expert Opin. Ther. Patents 2015, 25, 69−90.
(7) Ornstein, P. L.; Bleisch, T. J.; Arnold, M. B.; Kennedy, J. H.;
Wright, R. A.; Johnson, B. G.; Tizzano, J. P.; Helton, D. R.; Kallman,
M. J.; Schoepp, D. D.; Herin, M. 2-Substituted (2SR)-2-amino-2-
(1SR,2SR)-2-carboxycycloprop-1-yl)glycines as potent and selective
́
(
antagonists of group II metabotropic glutamate receptors. 2. Effects of
aromatic substitution, pharmacological characterization, and bioavail-
ability. J. Med. Chem. 1998, 41, 358−378.
(8) Chaki, S.; Yoshikawa, R.; Hirota, S.; Shimazaki, T.; Maeda, M.;
Kawashima, N.; Yoshimizu, T.; Yasuhara, A.; Sakagami, K.; Okuyama,
S.; Nakanishi, S.; Nakazato, A. MGS0039: a potent and selective group
II metabotropic glutamate receptor antagonist with antidepressant-like
activity. Neuropharmacology 2004, 46, 457−467.
Notes
(9) Bespalov, A. Y.; van Gaalen, M. M.; Sukhotina, I. A.; Wicke, K.;
The authors declare no competing financial interest.
Mezler, M.; Schoemaker, H.; Gross, G. Behavioral characterization of
the mGlu group II/III receptor antagonist, LY-341495, in animal
models of anxiety and depression. Eur. J. Pharmacol. 2008, 592, 96−
ACKNOWLEDGMENTS
■
We gratefully acknowledge the generous support of the
National Institute of Mental Health for the funding of this
work, NIMH R01MH099269 (K.A.E) and U54MH084659
1
(
02.
10) Shimazaki, T.; Iijima, M.; Chaki, S. Anxiolytic-like activity of
MGS0039, a potent group II metabotropic glutamate receptor
(
C.W.L.).
antagonist, in a marble-burying behavior test. Eur. J. Pharmacol.
2
004, 501, 121−125.
ABBREVIATIONS USED
(11) Yoshimizu, T.; Shimazaki, T.; Ito, A.; Chaki, S. An mGluR2/3
antagonist, MGS0039, exerts antidepressant and anxiolytic effects in
behavioral models in rats. Psychopharmacology 2006, 186, 587−593.
■
Ac, acetate; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole-
propionic acid; AUC, area under the curve; C, concentration;
CL, clearance; CNS, central nervous system; CRC, concen-
tration response curve; DIAD, di-iso-propyl azodicarboxylate;
DMF, N,N-dimethylformamide; DMPK, drug metabolism and
(12) Higgins, G. A.; Ballard, T. M.; Kew, J. N.; Richards, J. G.; Kemp,
J. A.; Adam, G.; Woltering, T.; Nakanishi, S.; Mutel, V.
Pharmacological manipulation of mGlu2 receptors influences cognitive
performance in the rodent. Neuropharmacology 2004, 46, 907−917.
t
pharmacokinetics; DMSO, dimethyl sulfoxide; D BAD, di-tert-
butyl azodicarboxylate; Et, ethyl; FST, forced swim test; F,
bioavailability; F , fraction unbound; GPCR, G-protein-coupled
receptors; HLM, human liver microsomes; IP, intraperitoneal;
(13) Kim, S. H.; Steele, J. W.; Lee, S. W.; Clemenson, G. D.; Carter,
T. A.; Treuner, K.; Gadient, R.; Wedel, P.; Glabe, C.; Barlow, C.;
Ehrlich, M. E.; Gage, F. H.; Gandy, S. Proneurogenic group II mGluR
antagonist improves learning and reduces anxiety in Alzheimer Aβ
oligomer mouse. Mol. Psychiatry 2014, 19, 1235−1242.
u
IV, intravenous; K , brain to plasma ratio; Kp,uu, unbound brain
p
(14) Kim, S. H.; Fraser, P. E.; Westaway, D.; St. George-Hyslop, P.
to unbound plasma ratio; LLE, ligand-lipophilicity efficiency;
max, maximum; MDCK, Madin−Darby canine kidney; MDD,
major depressive disorder; Me, methyl; mGlu, metabotropic
glutamate receptor; Ms, methanesulfonyl; mTOR, mammalian
target of rapamycin; NAM, negative allosteric modulator;
NMDA, N-methyl-D-aspartate; OCD, obsessive-compulsive
disorder; PAM, positive allosteric modulator; PEG, poly-
ethylene glycol; Ph, phenyl; PK, pharmacokinetics; PO, oral;
PS, polystyrene; RLM, rat liver microsomes; SAR, structure−
activity relationships; THF, tetrahydrofuran; TRD, treatment-
resistant depression; T, time; t , half-life; V , volume of
H.; Ehrlich, M. E.; Gandy, S. Group II metabotropic glutamate
receptor stimulation triggers production and release of Alzheimer’s
amyloid β42 from isolated intact nerve terminals. J. Neurosci. 2010, 30,
3
(
870−3875.
15) Yoshimizu, T.; Chaki, S. Increased cell proliferation in the adult
mouse hippocampus following chronic administration of group II
metabotropic glutamate receptor antagonist, MGS0039. Biochem.
Biophys. Res. Commun. 2004, 315, 493−496.
(16) Gleason, S. D.; Li, X.; Smith, I. A.; Ephlin, J. D.; Wang, X. S.;
Heinz, B. A.; Carter, J. H.; Baez, M.; Yu, J.; Bender, D. M.; Witkin, J.
M. mGlu2/3 agonist-induced hyperthermia: An in vivo assay for
detection of mGlu2/3 receptor antagonism and its relation to
antidepressant-like efficacy in mice. CNS Neurol. Disord.: Drug Targets
1
/2
SS
distribution at steady-state; 5-HT , 5-hydroxytryptamine
receptor 2B; 7TM, seven transmembrane
2
B
2
013, 12, 554−566.
REFERENCES
(17) Koike, H.; Fukumoto, K.; Iijima, M.; Chaki, S. Role of BDNF/
TrkB signaling in antidepressant-like effects of a group II metabotropic
glutamate receptor antagonist in animal models of depression. Behav.
Brain Res. 2013, 238, 48−52.
■
(
1) Niswender, C. M.; Conn, P. J. Metabotropic glutamate receptors:
Physiology, pharmacology, and disease. Annu. Rev. Pharmacol. Toxicol.
2
010, 50, 295−322.
(18) Koike, H.; Iijima, M.; Chaki, S. Involvement of the mammalian
(
2) Schoepp, D. D.; Jane, D. E.; Monn, J. A. Pharmacological agents
target of rapamycin signaling in the antidepressant-like effect of group
II metabotropic glutamate receptor antagonists. Neuropharmacology
acting at subtypes of metabotropic glutamate receptors. Neuro-
pharmacology 1999, 38, 1431−1476.
2
(
011, 61, 1419−1423.
(
3) Conn, P. J.; Pin, J.-P. Pharmacology and functions of
19) Karasawa, J.; Shimazaki, T.; Kawashima, N.; Chaki, S. AMPA
metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol.
997, 37, 205−237.
4) Chaki, S.; Ago, Y.; Palucha-Paniewiera, A.; Matrisciano, F.; Pilc,
1
receptor stimulation mediates the antidepressant-like effect of a group
II metabotropic glutamate receptor antagonist. Brain Res. 2005, 1042,
92−98.
(20) Ago, Y.; Yano, K.; Araki, R.; Hiramatsu, N.; Kita, Y.; Kawasaki,
T.; Onoe, H.; Chaki, S.; Nakazato, A.; Hashimoto, H.; Baba, A.;
Takuma, K.; Matsuda, T. Metabotropic glutamate 2/3 receptor
antagonists improve behavioral and prefrontal dopaminergic alter-
(
A. mGlu2/3 and mGlu5 receptors: Potential targets for novel
antidepressants. Neuropharmacology 2013, 66, 40−52.
(
5) Palucha, A.; Pilc, A. Metabotropic glutamate receptor ligands as
possible anxiolytic and antidepressant drugs. Pharmacol. Ther. 2007,
1
15, 116−147.
M
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
ations in the chronic corticosterone-induced depression model in
Consolidation and Reconsolidation: Molecular Mechanisms II; Proceed-
ings of the 40th Annual Meeting of the Society for Neuroscience, San
Diego, CA, Nov 13−17, 2010; Society for Neuroscience: Washington,
DC, 2010; 406.9/MMM57.
mice. Neuropharmacology 2013, 65, 29−38.
(
21) Dwyer, J. M.; Lepack, A. E.; Duman, R. S. mGluR2/3 blockade
produces rapid and long-lasting reversal of anhedonia caused by
chronic stress exposure. J. Mol. Psychiatry 2013, 1, 15.
(34) Choi, C. H.; Schoenfeld, B. P.; Bell, A. J.; Hinchey, P.; Kollaros,
M.; Gertner, M. J.; Woo, N. H.; Tranfaglia, M. R.; Bear, M. F.; Zukin,
R. S.; McDonald, T. V.; Jongens, T. A.; McBride, S. M.
Pharmacological reversal of synaptic plasticity deficits in the mouse
model of fragile X syndrome by group II mGluR antagonist or lithium
treatment. Brain Res. 2011, 1380, 106−119.
(
22) Iijima, M.; Koike, H.; Chaki, S. Effect of an mGlu2/3 receptor
antagonist on depressive behavior induced by withdrawal from chronic
treatment with methamphetamine. Behav. Brain Res. 2013, 246, 24−
2
8.
(
23) Markou, A. Metabotropic glutamate receptor antagonists: Novel
therapeutics for nicotine dependence and depression? Biol. Psychiatry
007, 61, 17−22.
24) Ciceroni, C.; Bonelli, M.; Mastrantoni, E.; Niccolini, C.;
(
35) Gatti McArthur, S.; Saxe, M.; Wichmann, J.; Woltering, T.
2
(
mGlu2/3 antagonists for the treatment of autistic disorders. PCT Int.
Pat. Appl. WO 2014/064028 A1, May 1, 2014.
Laurenza, M.; Larocca, L. M.; Pallini, R.; Traficante, A.; Spinsanti, P.;
Ricci-Vitiani, L.; Arcella, A.; De Maria, R.; Nicoletti, F.; Battaglia, G.;
Melchiorri, D. Type-3 metabotropic glutamate receptors regulate
chemoresistance in glioma stem cells, and their levels are inversely
related to survival in patients with malignant gliomas. Cell Death Differ.
(
36) Structure of decoglurant disclosed in Recommended Interna-
tional Nonproprietary Names (INN). WHO Drug Information; World
Health Organization: Geneva, Switzerland, 2013; Vol. 27, no. 3, p 150.
(
depressive disorder and inadequate response to ongoing antidepres-
sant treatment. ClinicalTrials.gov; U.S. National Institutes of Health:
Bethesda, MD, 2011; https://www.clinicaltrials.gov/ct2/show/
NCT01457677 (accessed August 12, 2015).
37) ARTDeCo Study: A study of RO4995819 in patients with major
2
(
013, 20, 396−407.
25) Ciceroni, C.; Arcella, A.; Mosillo, P.; Battaglia, G.; Mastrantoni,
E.; Oliva, M. A.; Carpinelli, G.; Santoro, F.; Sale, P.; Ricci-Vitiani, L.;
De Maria, R.; Pallini, R.; Giangaspero, F.; Nicoletti, F.; Melchiorri, D.
Type-3 metabotropic glutamate receptors negatively modulate bone
morphogenetic protein receptor signaling and support the tumouri-
genic potential of glioma-initiating cells. Neuropharmacology 2008, 55,
(
38) Rodriguez, A. L.; Grier, M. D.; Jones, C. K.; Herman, E. J.; Kane,
A. S.; Smith, R. L.; Williams, R.; Zhou, Y.; Marlo, J. E.; Days, E. L.;
Blatt, T. N.; Jadhav, S.; Menon, U. N.; Vinson, P. N.; Rook, J. M.;
Stauffer, S. R.; Niswender, C. M.; Lindsley, C. W.; Weaver, C. D.;
Conn, P. J. Discovery of novel allosteric modulators of metabotropic
glutamate receptor subtype 5 reveals chemical and functional diversity
and in vivo activity in rat behavioral models of anxiolytic and
antipsychotic activity. Mol. Pharmacol. 2010, 78, 1105−1123.
5
(
68−576.
26) Arcella, A.; Carpinelli, G.; Battaglia, G.; D’Onofrio, M.; Santoro,
F.; Ngomba, R. T.; Bruno, V.; Casolini, P.; Giangaspero, F.; Nicoletti,
F. Pharmacological blockade of group II metabotropic glutamate
receptors reduces the growth of glioma cells in vivo. Neuro-Oncology
(
39) Sheffler, D. J.; Wenthur, C. J.; Bruner, J. A.; Carrington, S. J. S.;
2
(
005, 7, 236−245.
Vinson, P. N.; Gogi, K. K.; Blobaum, A. L.; Morrison, R. D.; Vamos,
M.; Cosford, N. D. P.; Stauffer, S. R.; Daniels, J. S.; Niswender, C. M.;
Conn, P. J.; Lindsley, C. W. Development of a novel, CNS-penetrant,
metabotropic glutamate receptor 3 (mGlu ) NAM probe (ML289)
derived from a closely related mGlu PAM. Bioorg. Med. Chem. Lett.
27) D'Onofrio, M.; Arcella, A.; Bruno, V.; Ngomba, R. T.; Battaglia,
G.; Lombari, V.; Ragona, G.; Calogero, A.; Nicoletti, F. Pharmaco-
logical blockade of mGlu2/3 metabotropic glutamate receptors
reduces cell proliferation in cultured human glioma cells. J. Neurochem.
3
5
2
(
003, 84, 1288−1295.
2
(
012, 22, 3921−3925.
28) Campo, B.; Kalinichev, M.; Lambeng, N.; Yacoubi, M. E.;
40) Wenthur, C. J.; Morrison, R.; Felts, A. S.; Smith, K. A.; Engers, J.
Royer-Urios, I.; Schneider, M.; Legrand, C.; Parron, D.; Girard, F.;
Bessif, A.; Poli, S.; Vaugeois, J.-M.; Le Poul, E.; Celanire, S.
Characterization of an mGluR2/3 negative allosteric modulator in
rodent models of depression. J. Neurogenet. 2011, 25, 152−166.
L.; Byers, F. W.; Daniels, J. S.; Emmitte, K. A.; Conn, P. J.; Lindsley, C.
W. Discovery of (R)−−(2-fluoro-4-((−4-methoxyphenyl)ethynyl)-
phenyl(3-hydroxypiperidin-1-yl)methanone (ML337), An mGlu
3
selective and CNS penetrant negative allosteric modulator (NAM).
(
29) Pritchett, D.; Jagannath, A.; Brown, L. A.; Tam, S. K. E.; Hasan,
J. Med. Chem. 2013, 56, 5208−5212.
S.; Gatti, S.; Harrison, P. J.; Bannerman, D. M.; Foster, R. G.; Peirson,
S. N. Deletion of metabotropic glutamate receptors 2 and 3 (mGlu2 &
mGlu3) in mice disrupts sleep and wheel-running activity, and
increases the sensitivity of the circadian system to light. PLoS One
(41) Walker, A. G.; Wenthur, C. J.; Xiang, Z.; Rook, J. M.; Emmitte,
K. A.; Niswender, C. M.; Lindsley, C. W.; Conn, P. J. Metabotropic
glutamate receptor 3 activation is required for long-term depression in
medial prefrontal cortex and fear extinction. Proc. Natl. Acad. Sci. U. S.
A. 2015, 112, 1196−1201.
2
015, 10, e0125523.
30) Woltering, T. J.; Wichmann, J.; Goetschi, E.; Knoflach, F.;
Ballard, T. M.; Huwyler, J.; Gatti, S. Synthesis and characterization of
,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives: Part 4. In vivo
(
(
42) Zhang, L.; Balan, G.; Barreiro, G.; Boscoe, B. P.; Chenard, L. K.;
Cianfrogna, J.; Claffey, M. M.; Chen, L.; Coffman, K. J.; Drozda, S. E.;
Dunetz, J. R.; Fonseca, K. R.; Galatsis, P.; Grimwood, S.; Lazzaro, J. T.;
Mancuso, J. Y.; Miller, E. L.; Reese, M. R.; Rogers, B. N.; Sakurada, I.;
Skaddan, M.; Smith, D. L.; Stepan, A. F.; Trapa, P.; Tuttle, J. B.;
Verhoest, P. R.; Walker, D. P.; Wright, A. S.; Zaleska, M. M.; Zasadny,
K.; Shaffer, C. L. Discovery and preclinical characterization of 1-
methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazo-
lo-[3,4-b]pyrazine (PF470): a highly potent, selective, and efficacious
metabotropic glutamate receptor 5 (mGluR5) negative allosteric
modulator. J. Med. Chem. 2014, 57, 861−877.
1
active potent and selective non-competitive metabotropic glutamate
receptor 2/3 antagonists. Bioorg. Med. Chem. Lett. 2010, 20, 6969−
6
974.
(
́
31) Yacoubi, M. E.; Vaugeois, J.-M.; Kalinichev, M.; Celanire, S.;
Parron, D.; Le Poul, E.; Campo, B. Effects of a mGluR2/3 negative
allosteric modulator and a reference mGluR2/3 orthosteric antagonist
in a genetic mouse model of depression. In Behavioral Studies of Mood
Disorders; Proceedings of the 40th Annual Meeting of the Society for
Neuroscience, San Diego, CA, Nov 13−17, 2010; Society for
Neuroscience: Washington, DC, 2010; 886.14/VV7.
(
43) Zhuo, X.; Huang, X. S.; Degnan, A. P.; Snyder, L. B.; Yang, F.;
(
32) Goeldner, C.; Ballard, T. M.; Knoflach, F.; Wichmann, J.; Gatti,
Huang, H.; Shu, Y.-Z.; Johnson, B. M. Identification of glutathione
conjugates of acetylene-containing positive allosteric modulators of
metabotropic glutamate receptor subtype 5. Drug Metab. Dispos. 2015,
43, 578−589.
(44) Noetzel, M. J.; Rook, J. M.; Vinson, P. N.; Cho, H.; Days, E.;
Zhou, Y.; Rodriguez, A. L.; Lavreysen, H.; Stauffer, S. R.; Niswender,
C. M.; Xiang, Z.; Daniels, J. S.; Jones, C. K.; Lindsley, C. W.; Weaver,
C. D.; Conn, P. J. Functional impact of allosteric agonist activity of
selective positive allosteric modulators of metabotropic glutamate
S.; Umbricht, D. Cognitive impairment in major depression and the
mGlu2 receptor as a therapeutic target. Neuropharmacology 2013, 64,
3
(
37−346.
33) Kalinichev, M.; Campo, B.; Lambeng, N.; Cel
́
anire, S.;
Schneider, M.; Bessif, A.; Royer-Urios, I.; Parron, D.; Legrand, C.;
Mahious, N.; Girard, F.; Le Poul, E. An mGluR2/3 negative allosteric
modulator improves recognition memory assessed by natural
forgetting in the novel object recognition test in rats. In Memory
N
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
receptor subtype 5 in regulating central nervous system function. Mol.
Pharmacol. 2012, 81, 120−133.
(62) Serotonin (5-Hydroxytryptamine) 5-HT , IP ; (catalogue no.
355260); Eurofins Panlabs, Inc.: Redmond, WA, (www.
eurofinspanlabs.com).
2B
1
(
45) Niswender, C. M.; Johnson, K. A.; Luo, Q.; Ayala, J. E.; Kim, C.;
Conn, P. J.; Weaver, C. D. A novel assay of Gi/o-linked G protein-
coupled receptor coupling to potassium channels provides new
insights into the pharmacology of the group III metabotropic
glutamate receptors. Mol. Pharmacol. 2008, 73, 1213−1224.
(63) Zientek, M.; Miller, H.; Smith, D.; Dunklee, M. B.; Heinle, L.;
Thurston, A.; Lee, C.; Hyland, R.; Fahmi, O.; Burdette, D.
Development of an in vitro drug-drug interaction assay to
simultaneously monitor five cytochrome P450 isoforms and perform-
ance assessment using drug library compounds. J. Pharmacol. Toxicol.
Methods 2008, 58, 206−214.
(64) Wang, Q.; Rager, J. D.; Weinstein, K.; Kardos, P. S.; Dobson, G.
L.; Li, J.; Hidalgo, I. J. Evaluation of the MDR-MDCK cell line as a
permeability screen for the blood−brain barrier. Int. J. Pharm. 2005,
(
46) Conn, P. J.; Lindsley, C. W.; Stauffer, S. L.; Bartolome-Nebreda,
J. M.; Conde-Ceide, S.; MacDonald, G. J.; Tong, H. M.; Jones, C. K.;
Alcazar-Vaca, J. M.; Andres-Gil, J. I.; Malosh, C. Bicyclic triazole and
pyrazole lactams as allosteric modulators of mGluR5 receptors. PCT
Int. Pat. Appl. WO 2012/083224 A1, June 21, 2012.
(
47) But, T. Y. S.; Toy, P. H. The Mitsunobu reaction: Origin,
mechanism, improvements, and applications. Chem. - Asian J. 2007, 2,
340−1355.
48) Klapars, A.; Huang, X.; Buchwald, S. L. A general and efficient
copper catalyst for the amidation of aryl halides. J. Am. Chem. Soc.
002, 124, 7421−7428.
49) Gregory, K. J.; Nguyen, E. D.; Reiff, S. D.; Squire, E. F.; Stauffer,
2
(
88, 349−359.
65) Njung'e, K.; Handley, S. L. Evaluation of marble-burying
behavior as a model of anxiety. Pharmacol., Biochem. Behav. 1991, 38,
3−67.
66) Njung'e, K.; Handley, S. L. Effects of 5-HT uptake inhibitors,
1
(
6
(
2
(
agonists and antagonists on the burying of harmless objects by mice; a
putative test for anxiolytic agents. Br. J. Pharmacol. 1991, 104, 105−
S. R.; Lindsley, C. W.; Meiler, J.; Conn, P. J. Probing the metabotropic
1
12.
glutamate receptor 5 (mGlu ) positive allosteric modulator (PAM)
5
(67) Nicolas, L. B.; Kolb, Y.; Prinssen, E. P. A combined marble
binding pocket: discovery of point mutations that engender a
burying-locomotor activity test in mice: a practical screening test with
sensitivity to different classes of anxiolytics and antidepressants. Eur. J.
Pharmacol. 2006, 547, 106−115.
“
8
molecular switch” in PAM pharmacology. Mol. Pharmacol. 2013,
3, 991−1006.
50) Lamb, J. P.; Engers, D. W.; Niswender, C. M.; Rodriguez, A. L.;
(
(68) Felts, A. S.; Rodriguez, A. L.; Morrison, R. D.; Venable, D. F.;
Venable, D. F.; Conn, P. J.; Lindsley, C. W. Discovery of molecular
Manka, J. T.; Bates, B. S.; Blobaum, A. L.; Byers, F. W.; Daniels, J. S.;
Niswender, C. M.; Jones, C. K.; Conn, P. J.; Lindsley, C. W.; Emmitte,
K. A. Discovery of VU0409106: A negative allosteric modulator of
mGlu5 with activity in a mouse model of anxiety. Bioorg. Med. Chem.
Lett. 2013, 23, 5779−5785.
switches within the ADX-47273 mGlu PAM scaffold that modulate
5
modes of pharmacology to afford potent mGlu NAMs, PAMs and
5
partial antagonists. Bioorg. Med. Chem. Lett. 2011, 21, 2711−2714.
(
51) Kalvass, J. C.; Maurer, T. S. Influence of nonspecific brain and
plasma binding on CNS exposure: implications for rational drug
(69) Thomas, A.; Burant, A.; Bui, N.; Graham, D.; Yuva-Paylor, L. A.;
discovery. Biopharm. Drug Dispos. 2002, 23, 327−338.
Paylor, R. Marble burying reflects a repetitive and perseverative
behavior more than novelty-induced anxiety. Psychopharmacology
(
52) Bridges, T. M.; Morrison, R. D.; Byers, F. W.; Luo, S.; Daniels, J.
S. Use of a novel rapid and resource-efficient cassette dosing approach
to determine the pharmacokinetics and CNS distribution of small
molecule 7-transmembrane receptor allosteric modulators in rat.
Pharmacol. Res. Perspect. 2014, 2, e00077.
2
(
009, 204, 361−373.
70) Cosford, N. D.; Tehrani, L.; Roppe, J.; Schweiger, E.; Smith, N.
D.; Anderson, J.; Bristow, L.; Brodkin, J.; Jiang, X.; McDonald, I.; Rao,
S.; Washburn, M.; Varney, M. A. 3-[(2-Methyl-1,3-thiazol-4-yl)-
ethynyl]-pyridine: a potent and highly selective metabotropic
glutamate subtype 5 receptor antagonist with anxiolytic activity. J.
Med. Chem. 2003, 46, 204−206.
(
53) Leeson, P. D.; Springthorpe, B. The influence of drug-like
concepts on decision-making in medicinal chemistry. Nat. Rev. Drug
Discovery 2007, 6, 881−890.
(
54) Obach, R. S. Prediction of human clearance of twenty-nine
(71) Detke, M. J.; Rickels, M.; Lucki, I. Active behaviors in the rat
drugs from hepatic microsomal intrinsic clearance data: An
examination of in vitro half-life approach and nonspecific binding to
microsomes. Drug Metab. Dispos. 1999, 27, 1350−1359.
forced swimming test differentially produced by serotonergic and
noradrenergic antidepressants. Psychopharmacology (Berl) 1995, 121,
6
(
6−72.
(
55) Davies, B.; Morris, T. Physiological parameters in laboratory
72) Garcia, L. S.; Comim, C. M.; Valvassori, S. S.; Reus, G. Z.;
́
animals and humans. Pharm. Res. 1993, 10, 1093−1095.
Barbosa, L. M.; Andreazza, A. C.; Stertz, L.; Fries, G. R.; Gavioli, E. C.;
Kapczinski, F.; Quevedo, J. Acute administration of ketamine induces
antidepressant-like effects in the forced swimming test and increases
BDNF levels in the rat hippocampus. Prog. Neuro-Psychopharmacol.
Biol. Psychiatry 2008, 32, 140−144.
(
56) Bridges, T. M.; Rook, J. M.; Noetzel, M. J.; Morrison, R. D.;
Zhou, Y.; Gogliotti, R. D.; Vinson, P. N.; Xiang, Z.; Jones, C. K.;
Niswender, C. M.; Lindsley, C. W.; Stauffer, S. L.; Conn, P. J.; Daniels,
J. S. Biotransformation of a novel positive allosteric modulator of
metabotropic glutamate receptor subtype 5 contributes to seizure-like
adverse events in rats involving a receptor agonism-dependent
mechanism. Drug Metab. Dispos. 2013, 41, 1703−1714.
(
73) Kohrs, R.; Durieux, M. E. Ketamine: Teaching an old drug new
tricks. Anesth. Analg. 1998, 87, 1186−1193.
74) Murrough, J. W.; Iosifescu, D. V.; Chang, L. C.; Al Jurdi, R. K.;
(
(
57) Di, L.; Rong, H.; Feng, B. Demystifying brain penetration in
central nervous system drug discovery. J. Med. Chem. 2013, 56, 2−12.
58) Hemstapat, K.; Da Costa, H.; Nong, Y.; Brady, A. E.; Luo, Q.;
Green, C. E.; Perez, A. M.; Iqbal, S.; Pillemer, S.; Foulkes, A.; Shah, A.;
Charney, D. S.; Mathew, S. J. Antidepressant efficacy of ketamine in
treatment-resistant major depression: a two-site randomized con-
trolled trial. Am. J. Psychiatry 2013, 170, 1134−1142.
(75) Browne, C. A.; Lucki, I. Antidepressant effects of ketamine:
mechanisms underlying fast-acting novel antidepressants. Front.
Pharmacol. 2013, 4, DOI: 10.3389/fphar.2013.00161.
(76) aan het Rot, M.; Collins, K. A.; Murrough, J. W.; Perez, A. M.;
Reich, D. L.; Charney, D. S.; Mathew, S. J. Safety and efficacy of
repeated-dose intravenous ketamine for treatment-resistant depression.
Biol. Psychiatry 2010, 67, 139−145.
(
Niswender, C. M.; Tamagnan, G. D.; Conn, P. J. A novel family of
potent negative allosteric modulators of group II metabotropic
glutamate receptors. J. Pharmacol. Exp. Ther. 2007, 322, 254−264.
(
59) LeadProfilingScreen; (catalogue no. 68) Eurofins Panlabs, Inc.:
Redmond, WA (www.eurofinspanlabs.com).
60) Significant responses are defined as those that inhibited more
than 50% of radioligand binding.
61) Fitzgerald, L. W.; Burn, T. C.; Brown, B. S.; Patterson, J. P.;
(
(
Corjay, M. H.; Valentine, P. A.; Sun, J. H.; Link, J. R.; Abbaszade, I.;
Hollis, J. M.; Largent, B. L.; Hartig, P. R.; Hollis, G. F.; Meunier, P. C.;
Robichaud, A. J.; Robertson, D. W. Possible role of valvular serotonin
(77) Morgan, C. J. A.; Curran, H. V. Ketamine use: A review.
Addiction 2012, 107, 27−38.
(78) Zhou, W.; Wang, N.; Yang, C.; Li, X.-M.; Zhou, Z.-Q.; Yang, J.-J.
Ketamine-induced antidepressant effects are associated with AMPA
5
-HT(2B) receptors in the cardiopathy associated with fenfluramine.
Mol. Pharmacol. 2000, 57, 75−81.
O
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
receptors-mediated upregulation of mTOR and BDNF in rat
hippocampus and prefrontal cortex. Eur. Psychiatry 2014, 29, 419−423.
(
79) Koike, H.; Chaki, S. Requirement of AMPA receptor stimulation
for the sustained antidepressant activity of ketamine and LY341495
during the forced swim test in rats. Behav. Brain Res. 2014, 271, 111−
1
15.
(
80) Duman, R. S.; Li, N.; Liu, R.-J.; Duric, V.; Aghajanian, G.
Signaling pathways underlying the rapid antidepressant actions of
ketamine. Neuropharmacology 2012, 62, 35−41.
(
81) Dwyer, J. M.; Lepack, A. E.; Duman, R. S. mTOR activation is
required for the antidepressant effects of mGluR2/3 blockade. Int. J.
Neuropsychopharmacol. 2012, 15, 429−434.
(
82) Li, N.; Lee, B.; Liu, R. J.; Banasr, M.; Dwyer, J. M.; Iwata, M.; Li,
X. Y.; Aghajanian, G.; Duman, R. S. mTOR-dependent synapse
formation underlies the rapid antidepressant effects of NMDA
antagonists. Science 2010, 329, 959−964.
P
DOI: 10.1021/acs.jmedchem.5b01005
J. Med. Chem. XXXX, XXX, XXX−XXX