Discovery of (1S,2R,3S,4S,5R,6R)-2-Amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid Hydrochloride (LY3020371·HCl): A Potent, Metabotropic Glutamate 2/3 Receptor Antagonist with Antidepressant-Like Activity

Article abstract of DOI:10.1021/acs.jmedchem.6b01119

As part of our ongoing efforts to identify novel ligands for the metabotropic glutamate 2 and 3 (mGlu_2/3) receptors, we have incorporated substitution at the C3 and C4 positions of the (1S,2R,5R,6R)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid scaffold to generate mGlu_2/3 antagonists. Exploration of this structure-activity relationship (SAR) led to the identification of (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid hydrochloride (LY3020371·HCl, 19f), a potent, selective, and maximally efficacious mGlu_2/3 antagonist. Further characterization of compound 19f binding to the human metabotropic 2 glutamate (hmGlu₂) site was established by cocrystallization of this molecule with the amino terminal domain (ATD) of the hmGlu₂ receptor protein. The resulting cocrystal structure revealed the specific ligand-protein interactions, which likely explain the high affinity of 19f for this site and support its functional mGlu₂ antagonist pharmacology. Further characterization of 19f in vivo demonstrated an antidepressant-like signature in the mouse forced-swim test (mFST) assay when brain levels of this compound exceeded the cellular mGlu₂ IC₅₀ value.

Full text of DOI:10.1021/acs.jmedchem.6b01119

Subscriber access provided by University of Otago Library
Article
Discovery of (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-
difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-
dicarboxylic acid hydrochloride (LY3020371.HCl): A Potent, Metabotropic
Glutamate 2/3 Receptor Antagonist with Antidepressant-Like Activity
Mark D. Chappell, Renhua Li, Stephon C. Smith, Bruce A Dressman, Eric G. Tromiczak, Allie E. Tripp,
Maria-Jesus Blanco, Tatiana Vetman, Steven J. Quimby, James Matt, Thomas C. Britton, Adam M.
Fivush, Jeffrey M. Schkeryantz, Daniel Mayhugh, Jon A. Erickson, Mark G. Bures, Carlos Jaramillo,
Mercedes Carpintero, Jose Eugenio de Diego, Mario Barberis, Susana Garcia-Cerrada, Jose F. Soriano,
Stephen Antonysamy, Shane Atwell, Iain MacEwan, Bradley Condon, Christine Sougias, Jing Wang,
Aiping Zhang, Kris Conners, Chris Groshong, Stephen R. Wasserman, John W. Koss, Jeffrey M. Witkin,
Xia Li, Carl Overshiner, Keith A. Wafford, Wesley Seidel, Xu-Shan Wang, Beverly A. Heinz, Steven
Swanson, John T. Catlow, David W. Bedwell, James A Monn, Charles H. Mitch, and Paul L. Ornstein
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01119 • Publication Date (Web): 11 Nov 2016
Downloaded from http://pubs.acs.org on November 14, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Research and Technologies
Atwell, Shane; Eli Lilly and Company, Discovery Chemistry Research and
Technologies
MacEwan, Iain; Eli Lilly and Company, Discovery Chemistry Research and
Technologies
Condon, Bradley; Eli Lilly and Company, Discovery Chemistry Research and
Technologies
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Sougias, Christine; none,
Wang, Jing; Eli Lilly and Company, Discovery Chemistry Research and
Technologies
Zhang, Aiping; Eli Lilly and Company, Discovery Chemistry Research and
Technologies
Conners, Kris; Eli Lilly and Company, Discovery Chemistry Research and
Technologies
Groshong, Chris; Eli Lilly and Company Biotechnology Center San Diego,
Discovery Chemistry Research and Technologies
Wasserman, Stephen; Eli Lilly and Company, Discovery Chemistry
Research and Technologies
Koss, John; Eli Lilly and Company, Discovery Chemistry Research and
Technologies
Witkin, Jeffrey; Eli Lilly and Company
Li, Xia; Eli Lilly and Company, Neuroscience Research
Overshiner, Carl; Eli Lilly and Company, Neuroscience Research
Wafford, Keith; Eli Lilly and Company, Neuroscience Research
Seidel, Wesley; Eli Lilly and Company, Neuroscience Research
Wang, Xu-Shan; Eli Lilly and Company, Discovery Chemistry Research and
Technologies
Heinz, Beverly; Eli Lilly and Company, Lilly Research Laboratories
Swanson, Steven; Eli Lilly and Company, Lilly Research Laboratories
Catlow, John; Eli Lilly and Company, Drug Disposition
Bedwell, David; Eli Lilly and Company, Drug Disposition
Monn, James; Eli Lilly and Company, Discovery Chemistry Research
Mitch, Charles; Lilly Research Laboratories, Discovery Chemistry Research
& Tech.
Ornstein, Paul; Roosevelt University, College of Pharmacy
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Discovery of (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-
difluorophenyl)sulfanylmethyl]-4-hydroxy-
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bicyclo[3.1.0]hexane-2,6-dicarboxylic acid hydrochloride
.
(
LY3020371 HCl): A Potent, Metabotropic Glutamate 2/3
Receptor Antagonist with Antidepressant-Like Activity
,†
†
†
†
Mark D. Chappell,* Renhua Li, Stephon C. Smith, Bruce A. Dressman, Eric G.
†
†
†
†
†
Tromiczak, Allie E. Tripp, MariaꢀJesus Blanco, Tatiana Vetman, Steven J. Quimby,
†
†
†
†
James Matt, Thomas C. Britton, Adam M. Fivush, Jeffrey M. Schkeryantz, Daniel
†
†
†
↑
↑
Mayhugh, Jon A. Erickson, Mark G. Bures, Carlos Jaramillo, Mercedes Carpintero,
↑
↑
↑
↑
José Eugenio de Diego, Mario Barberis, Susana GarciaꢀCerrada, José F. Soriano,
§
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§
Stephen Antonysamy, Shane Atwell, Iain MacEwan, Bradley Condon, Christine
§
§
§
§
§
Sougias, Jing Wang, Aiping Zhang, Kris Conners, Chris Groshong, Stephen R.
±
±
⊥
⊥
⊥
Wasserman, John W. Koss, Jeffrey M. Witkin, Xia Li, Carl Overshiner, Keith A.
∆
∆
‡
‡
║
Wafford, Wesley Seidel, XuꢀShan Wang, Beverly A. Heinz, Steven Swanson, John
║
║
†
†
†
T. Catlow, David W. Bedwell, James A. Monn, Charles H. Mitch, Paul L. Ornstein
†
‡
Discovery Chemistry Research and Technologies: Medicinal Chemistry, Quantitative
↑
Biology, Eli Lilly and Company, Indianapolis, IN 46285, United States; Discovery
Chemistry Synthesis Group, Centro de Investigación Lilly S.A. Avda. de la Industria, 30
§
AlcobendasꢀMadrid 28108, Spain; Structural Biology, Lilly Biotechnology Center, Eli
±
Lilly and Company, San Diego, CA 92121, United States; Structural Biology, Eli Lilly
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and Company, Advanced Photon Source, Argonne National Laboratory, Building 438A,
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700 S. Cass Ave., Argonne, IL 60439, United States
∆
Neuroscience Research, Eli Lilly and Company, Erl Wood Manor, Sunninghill Road,
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Windlesham, Surrey, UK GU20 6PH
║
⊥
Drug Disposition, Neuroscience Research, Eli Lilly and Company, Indianapolis, IN
4
6285, United States
Abstract
As part of our ongoing efforts to identify novel ligands for the metabotropic glutamate 2
and 3 (mGlu ) receptors, we have incorporated substitution at the C3 and C4 positions
2
/3
of the (1S,2R,5R,6R)ꢀ2ꢀaminoꢀbicyclo[3.1.0]hexaneꢀ2,6ꢀdicarboxylic acid scaffold to
generate mGlu2/3 antagonists. Exploration of this structureꢀactivity relationship (SAR)
lead to the identification of (1S,2R,3S,4S,5R,6R)ꢀ2ꢀaminoꢀ3ꢀ[(3,4ꢀ
difluorophenyl)sulfanylmethyl]ꢀ4ꢀhydroxyꢀbicyclo[3.1.0]hexaneꢀ2,6ꢀdicarboxylic acid
.
hydrochloride (LY3020371 HCl
,
19f), a potent, selective, and maximally efficacious
mGlu2/3 antagonist. Further characterization of compound 19f binding to the human
metabotropic 2 glutamate (hmGlu ) site was established by coꢀcrystallization of this
2
molecule with the amino terminal domain (ATD) of the hmGlu receptor protein. The
2
resulting coꢀcrystal structure revealed the specific ligandꢀprotein interactions, which
likely explain the high affinity of 19f for this site and support its functional mGlu2
antagonist pharmacology. Further characterization of 19f in vivo demonstrated an
antidepressantꢀlike signature in the mouse forcedꢀswim test (mFST) assay when brain
levels of this compound exceeded the cellular mGlu IC value.
2
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Key Words: mGlu2/3 antagonists, LY3020371 HCl, LY341495.
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Introduction
Modern medicines for psychiatric and neurological disorders generally have mechanisms
1
of action that involve the modulation of monoaminergic neurotransmission. In the past
decade, biological, pharmacological, and clinical data have converged upon the
neurotransmitter glutamate and its role in driving aberrant neurotransmission in circuitry
associated with both psychiatric and neurological disorders. Glutamate acts upon
receptors in the CNS that are divided into two main categories: ionotropic and
metabotropic. The ionotropic family of ligand gated ion channels are grouped into αꢀ
aminoꢀ3ꢀhydroxyꢀ5ꢀmethylꢀ4ꢀisoxazolepropionic acid (AMPA), Nꢀmethyl ꢀaspartate
D
2
(NMDA), kainate, and δꢀreceptor classes, while the metabotropic glutamate (mGlu)
receptors are type C Gꢀprotein coupled receptors (GPCRs) consisting of eight known
receptor subtypes that are further divided into three categories: group I (mGlu1, 5),
3
group II (mGlu2, 3), and group III (mGlu4, 6, 7, 8). The metabotropic glutamate
receptors are viable targets for small molecules to engender anxiolytic and antipsychotic
4
5
effects preclinically and clinically, although there are currently no marketed therapies
directly acting on this receptor class.
The Group II mGlu receptors, mGlu and mGlu , demonstrate high sequence homology
2
3
and regulate extracellular glutamate levels; however, they are differentially distributed in
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the CNS and have distinct functions. The mGlu receptors are localized presynapically
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and are involved in acute antipsychotic and antinociceptive responses in rodents, while
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a
9b
mGlu receptors regulate synaptic activity through presynaptic, postsynaptic, and glial
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mechanisms, the latter of which has demonstrated neuroprotective effects. The mGlu
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and mGlu receptors are both expressed in cortical and limbic brain structures, which
3
are relevant regions for the treatment of psychiatric disorders. Recent data suggest that
mGlu2/3 receptor blockade produces antidepressantꢀlike effects in rodents via signaling
1
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mechanisms that overlap with those produced by ketamine, a nonꢀcompetitive NMDA
receptor antagonist that enhances downꢀstream glutamate release and promotes AMPA
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receptor function. Ketamine has been shown to alleviate symptoms in patients suffering
from major depressive disorder (MDD) that do not respond to conventional therapeutics
1
4
(treatmentꢀresistant depression or TRD). Specifically, preclinical studies have
demonstrated that mGlu2/3 antagonists such as (2S)ꢀ2ꢀaminoꢀ2ꢀ[(1S,2S)ꢀ2ꢀ
1
5
carboxycyclopropꢀ1ꢀyl]ꢀ3ꢀ(xanthꢀ9ꢀyl)propanoic acid 1 (LY341495, Figure 1) and
(
1R,2R,3R,5R,6R)ꢀ2ꢀaminoꢀ3ꢀ(3,4ꢀdichlorobenzyloxy)ꢀ6ꢀfluorobicyclo[3.1.0]hexaneꢀ2,6ꢀ
16
dicarboxylic acid 2 (MGS0039, Figure 1) enhance mTOR signaling and elicit AMPA
1
2b,17
receptorꢀdependent antidepressant responses in rodents.
In addition to orthosteric
antagonists, mGlu and mGlu negative allosteric modulators (NAMs) have also been
2
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investigated. Given the wellꢀestablished rapid, robust, and relatively longꢀlasting
antidepressant effect mediated by a single dose of ketamine, and the putative analogous
signaling mechanisms between ketamine and the mGlu2/3 antagonists, there is
considerable interest in the mGlu antagonists as novel therapeutic agents for alleviating
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MDD with the possibility of avoiding the known liabilities associated with ketamine.
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Figure 1. Historical aminobicyclo[3.1.0]hexaneꢀ2,6ꢀdicarboxylate mGlu2/3 receptor
agonists and antagonists.
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The aminobicyclo[3.1.0]hexaneꢀ2,6ꢀdicarboxylate scaffold has successfully delivered
multiple mGlu2/3 receptor agonists [(1S,2S,5R,6S)ꢀ2ꢀaminobicyclo[3.1.0]hexaneꢀ2,6ꢀ
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dicarboxylic acid 3 (LY354740), (−)ꢀ(1R,4S,5S,6S)ꢀ4ꢀaminoꢀ2ꢀ
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sulfonylbicyclo[3.1.0]hexaneꢀ4,6ꢀdicarboxylic acid 4 (LY404039,) (Figure 1)] that
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show efficacy in rodent models of anxiety, psychosis, and pain while oral prodrugs of
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these agents demonstrate clinical efficacy in patients with generalized anxiety disorder
2
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and schizophrenia, respectively. The first clinical candidate from this series (3) is
unsubstituted at the C3/C4ꢀpositions and exhibits potent, maximally efficacious mGlu2/3
receptor agonist activity (Figure 2). Incorporating hydroxyl substitution at the C4
position of 3 in either the S (5) or R (6) stereochemical configuration maintained mGlu2/3
2
4
agonist activity and potency
.
However, substitution of a benzyl group at the C3 position
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caused a functional switch to moderately potent antagonists such as 7. The present
discovery effort explored the structureꢀactivity relationship (SAR) of combining
substituents at the C3 and C4ꢀpositions of the aminobicyclo[3.1.0]hexaneꢀ2,6ꢀ
dicarboxylate scaffold with the goal of better understanding the molecular drivers of
functional mGlu receptor responses for compounds in this series.
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Figure 2. Functional effects of C3 and C4 substitution on the
aminobicyclo[3.1.0]hexaneꢀ2,6 dicarboxylate scaffold.
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Agonist
hmGlu EC = 7.0 ± 2.3 nM
hmGlu EC = 27.9 ± 6.0 nM
Agonist
hmGlu EC = 22.6 ± 3.7 nM
hmGlu EC = 6.3 ± 4.3 nM
Agonist
hmGlu EC = 8.6 ± 0.6 nM
hmGlu EC = 11.2 ± 6.6 nM
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C4
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Optimize functional antagonist activity
through C3/C4 disubstitution
Antagonist
hmGlu IC = 408 ± 142 nM
hmGlu IC = 110 ± 74.8 nM
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Chemistry
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The previously described nonꢀracemic bicyclic ketone 8 was utilized as the starting
material for the synthesis of the C3,C4ꢀdisubstituted structures (Scheme 1). The
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sequence began with condensation of ketone 8 utilizing Bredereck’s reagent to provide
enamine 9 in 97% yield, which was subsequently reduced with one equivalent of DIBALꢀ
H to give enone 10 (84%). The conjugate addition of thiophenols was explored in this
study owing to the ease in accessing a diverse array of thiol reagents and overall synthetic
feasibility. The Michael addition of thiophenol initially proved to be a challenging step
as no products were observed with strong bases (K CO or NaH). After screening
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Scheme 1. Preparation of C3-phenyl-thioether-C4-hydroxy-
aminobicyclo[3.1.0]hexane-2,6-dicarboxylates.
a)
b)
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15b-j
f)
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19a (R=Ph)
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◦
Reagents and conditions: (a) [(CH ) N] CH(OtꢀBu), PhMe, 80 C, 97%; (b) DIBALꢀH,
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.
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4%; (c) PhSH, Et N (cat.), Et O, 50°C; (d) SꢀCBS, BH (CH ) S, Et O, ꢀ20°C, 13: 13%,
3 2 3 3 2 2
.
14: 64%; (e) RꢀCBS, BH (CH ) S, Et O, ꢀ20°C, 15a: 19%, 15b-j: 32ꢀ75%, 16: 42%; (f)
3
3 2
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Procedure A: 4N HCl in 1,4ꢀdioxane, H O; Procedure B: 4N HCl in 1,4ꢀdioxane, 70°C,
2
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h; or 50% AcOH, 140°C microwave, 15 min. for 19b; 17: 45%; 18: 42%; 19a: 20%;
9b-j: 71ꢀ100%, 20: 40%. See Table 1 for the definition of R for 19bꢀ19j.
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numerous reaction conditions, thiophenol in the presence of catalytic triethylamine
efficiently added in a 1,4ꢀconjugate manner to the enone and provided the intermediate
ketones 11 and 12 as an inseparable mixture of diastereomers (1.4:1 ratio). Reduction of
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the ketone diastereomers utilizing Corey’s (S)ꢀCBS method provided excellent
stereocontrol and afforded only compounds 13 and 14 possessing the (R)ꢀC4ꢀhydroxyl
configuration. Likewise, using the (R)ꢀCBS reagent lead to the (S)ꢀC4 hydroxyl isomers
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5a and 16. Structural confirmation of all diastereomers was established by H NMR
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nuclear Overhauser effect (NOE) experiments. After chromatographic separation, each
analog was exhaustively deprotected under acidic conditions to provide the final amino
dicarboxylic acids 17-20. This synthesis has proven general for the addition of various
alkyl and substituted aryl thiols and effectively provided the 3S, 4S diastereomeric series
1
9a-j.
Scheme 2. Preparation of C4-(S)-substituted 3-(S)-(3-methyl-4-fluoro)
phenylthioether-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates.
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a)
b)
c)
1
0
21
22
d)
0
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7
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9
0
e)
25
24
23
f)
f)
f)
2
7
26
28
Reagents and conditions: (a) (i) 3ꢀmethyl, 4ꢀfluorophenylthiol, cat. Et N (10 mol%),
3
Et O, 50°C; (ii) SꢀCBS, 92% (2 steps); (b) MsCl, Et N, THF, 0°C to RT, 99%; (c) (i)
2
3
NaN , K CO , DMF, 90°C, 58%; (ii) 1,3ꢀpropanedithiol, Et N, MeOH, rt, 66%; (d) 1Hꢀ
3
2
3
3
1
,2,4ꢀtriazoleꢀ3ꢀthiol, Cs CO , DMF, 65°C, 13%; (e) AcCl, Et N, CH Cl , 62%; (f) 4N
2 3 3 2 2
HCl, 1,4ꢀdioxane, rt, 26: 86%, 27: 93%, 28: quant.
The 3(S)ꢀ(3ꢀmethyl, 4ꢀfluoro) phenyl, 4(R)ꢀhydroxy derivative 21 was synthesized
directly from enone 10 by treatment with the corresponding thiophenol and catalytic
triethylamine followed by (S)ꢀCBS mediated reduction (Scheme 2). The hydroxyl group
served as a convenient handle for the incorporation of structural diversity at the C4
ꢀ
position. Alcohol 21 was initially activated by conversion to mesylate 22. The mesylate
was then displaced with either 1Hꢀ1,2,4ꢀtriazoleꢀ3ꢀthiol to afford intermediate 23, or
sodium azide followed by 1,3ꢀpropanedithiol mediated reduction to provide C4ꢀamino
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analog 24 which was acetylated to give 25. Finally, exhaustive deprotection provided
C4ꢀamino (26), acetamido (27), and triazoleꢀthioether (28) analogs, respectively.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
hmGlu2/3 Receptor Binding and Function:
Newly prepared compounds 17, 18, 19a, and 20 were evaluated in their ability to displace
the tritiated mGlu2/3 receptor agonist 1R,2S,4R,5R,6R)ꢀ2ꢀaminoꢀ4ꢀ
3
3
31
fluorobicyclo[3.1.0]hexaneꢀ2,6ꢀdicarboxylic acid ([ H]ꢀLY459477, [ H]ꢀ29) from cell
membranes expressing mGlu or mGlu receptors, and to block agonistꢀinhibited
2
3
forskolinꢀstimulated cAMP production in these cells (Table 1). All compounds tested
demonstrated competitive displacement of the orthosteric radioligand and were
maximally efficacious antagonists with no agonist activity in the functional assays. The
data on diastereomers 17, 18, 19a, and 20 support the conclusion that C3ꢀsubstitution
leads to consistent mGlu2/3 receptor antagonist pharmacology with varying degrees of
potency. Compounds 17 and 19a, which possess the phenylthiomethylene substituent in
the 3ꢀ(S) configuration, display increased potency relative to the corresponding 3ꢀ(R)
isomers 18 and 20 across the mGlu2/3 receptor binding and functional assays. All four
diastereomers demonstrate a rightꢀshift in mGlu and mGlu functional antagonist activity
2
3
relative to binding affinity (5ꢀ21x). Within the more active 3ꢀ(S) configuration, the 4ꢀ(S)ꢀ
hydroxy stereoisomer 19a demonstrated comparable binding affinity to stereoisomer 17
Table 1: Binding affinities and functional effects of C3-thioether-C4-hydroxy-
aminobicyclo[3.1.0]hexane-2,6-dicarboxylates at recombinant hmGlu2/3 receptors:
effects of C3 stereochemical changes on activity.
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5
5
6
3
Inhibition of [ H]-29 binding to Functional antagonist activity
human mGlu receptors
in cells expressing recombinant
human mGlu receptors
IC (nM) ± SEM
K (nM) ± SEM
i
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
n)
5
0
a
(% efficacy, n)
No.
hmGlu2
hmGlu3
hmGlu2
hmGlu3
3
0.9 ± 3.5
26.0 ± 1.6
(9)
335 ± 70.0
(101, 6)
547 ± 133
(80.7, 6)
(9)
17
271 ± 21.5
9)
238 ± 32.1
(9)
2050 ± 411
(95.9, 6)
2160 ± 240
(71.4, 6)
(
18
2
1.4 ± 4.7
19.2 ± 2.4
(12)
114 ± 14.5
(103, 9)
202 ± 27.3
(96.0, 12)
(12)
1
9a
2
68 ± 60.7
251 ± 34.3
(12)
13 ± 163 (104,
10)
2410 ± 549
(91.0, 12)
(12)
2
0
a
Maximum reversal of the inhibition of cAMP production, relative to an EC90 of
glutamate, in cells expressing recombinant human mGlu receptors.
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though exhibiting a modest increase in functional antagonist potency over 17 at both
mGlu and mGlu . Additional SAR at the C3ꢀ and C4ꢀpositions of the more active
2
3
diastereomer 19a was thus explored to further optimize mGlu2/3 receptor activity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A variety of thiomethylene substituents were examined at C3 of the [3.1.0]bicyclic
system holding the C4ꢀ(S)ꢀOH substituent constant. The C3 isopropylthioether
substituent in 19b, where the steric volume of the thiophenyl group is reduced and the
ability to form πꢀstacking interactions absent, led to significantly decreased affinity and
functional antagonist activity (Table 2). Hence, the focus remained on aryl analogs
where fluoroꢀsubstitution was first examined in detail. The orthoꢀfluoro analog 19c was
consistently less potent across all assays, while metaꢀfluoro compound 19d was
equivalent to unsubstituted analog 19a. In contrast, the paraꢀ fluoro compound 19e
displayed increased binding affinity (hmGlu K = 10.4 ± 1.9 nM, hmGlu K = 6.8 ± 0.6
2
i
3
i
nM) and functional activity (hmGlu IC = 20.2 ± 0.9 nM, hmGlu IC = 41.9 ± 19.0
2
50
3
50
nM). Monoꢀfluoro substitution at all three isomeric positions brought greater alignment
between the mGlu binding affinity and functional activity (~2x difference). The ortho
2
fluoro analog 19c also showed only a 2.6x difference between mGlu binding affinity and
3
functional activity; however, a larger separation in the mGlu activities was observed for
3
the meta (19d, 11x) and para (19e, 6x) isomers, respectively.
Several combinations of metaꢀ, paraꢀdisubstitution were then examined: mꢀF, pꢀF (19f),
mꢀCH , pꢀF (19g), mꢀCH , pꢀCH (19h), and mꢀCl, pꢀCl (19i). The difluoro analog 19f
3
3
3
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2: Effects of C3 phenyl substitution on mGlu2/3 binding affinities and
functional activity.
3
Inhibition of [ H]-29
binding to human mGlu
receptors
Functional antagonist
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
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8
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2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
activity in cells expressing
recombinant human mGlu
receptors
K (nM) ± SEM
i
(n)
IC (nM) ± SEM
5
0
(% efficacy, n)
No.
R
hmGlu2
hmGlu3
hmGlu2
hmGlu3
1
9a
21.4 ± 4.7
19.2 ± 2.4
114 ± 14.5
(103, 9)
202 ± 27.3
(
12)
1200 ± 83.6
3)
728 ± 57.5
3)
(12)
614 ± 127
(3)
(96.0, 12)
a
1
9b
2280 ± 195 4560 ± 1400
(
(101, 4)
1440 ± 333
(103, 3)
(82.3, 4)
967 ± 532
(95.1, 3)
272 ± 125
(109, 5)
19c
369 ± 41.9
(3)
(
F
19d
43.7 ± 4.8
(3)
25.1 ± 1.6
(3)
76.2 ± 8.0
(100, 8)
1
9e
10.4 ± 1.9
6.8 ± 0.6
(6)
20.2 ± 0.9
(102, 6)
41.9 ± 19.0
(85.6, 5)
6.2 ± 2.1
(93.2, 3)
27.0 ± 6.0
(97.0, 3)
(
6)
5.3 ± 1.4
3)
8.9 ± 1.1
7)
1
9f
2.5 ± 0.3
(3)
16.2 ± 1.6
(114, 6)
(
19g
5.9 ± 0.8
(7)
17.2 ± 3.8
(103, 4)
(
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1
9h
14.2 ± 1.7
6)
8.4 ± 0.9
3)
20.5 ± 4.9
7)
26.0 ± 1.8
(6)
27.6 ± 4.0
(102, 4)
61.1 ± 17.4
(100, 4)
(
1
9i
9.4 ± 1.4
(3)
9.8 ± 2.3
(105, 10)
49.4 ± 10.3
(99.0, 4)
56.5 ± 11.7
(101, 5)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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45
46
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48
49
50
51
52
53
54
55
56
57
58
59
60
(
19j
16.3 ± 3.1
(7)
62.3 ± 14.1
(99.8, 5)
(
a
Free base
was identified as the most potent compound in the series across both membrane binding
and whole cell functional assays (hmGlu K = 5.3 ± 1.4 nM, hmGlu K = 2.5 ± 0.3 nM,
2
i
3
i
hmGlu IC = 16.2 ± 1.6 nM, hmGlu IC = 6.2 ± 2.1 nM). Changing the metaꢀfluoro
2
50
3
50
substituent to methyl as in 19g led to a modest loss in mGlu antagonist potency (hmGlu
3
3
IC = 27.0 ± 6.0 nM). The dimethyl compound 19h demonstrated decreased potency
5
0
relative to 19f across each of the mGlu2/3 assays (hmGlu K = 14.2 ± 1.7 nM, hmGlu
3
2
i
K = 26.0 ± 1.8 nM, hmGlu IC = 27.6 ± 4.0 nM, hmGlu IC = 61.1 ± 17.4 nM), while
i
2
50
3
50
the dichloro analog 19i was less potent in mGlu binding (hmGlu K = 9.4 ± 1.4 nM) and
3
3
i
functional activity (hmGlu IC = 56.5 ± 11.7 nM) relative to compound 19f. Finally,
3
50
one carbon homologation of the 3,4ꢀdichlorophenyl substituent as in 19j, exhibited
comparable mGlu activity, but decreased mGlu binding affinity and functional potency
3
2
relative to 19i, with the most pronounced impact being on mGlu receptor antagonist
2
potency (mGlu IC = 49.4 nM ± 10.3 for 19j compared to 9.8 ± 2.3 nM for 19i). Each
2
50
of the disubstituted phenyl analogs showed more balanced mGlu2/3 binding affinity
relative to functional potency with the largest difference between these attributes
observed when comparing mGlu binding and function for 19g (4.6x) and 19i (6.0x).
3
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Ultimately, small electron withdrawing substituents at the meta and para positions on the
thiophenyl moiety improved the binding and functional activity profile for this series of
compounds.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3: Effects of C4-(S)-substitution on mGlu2/3 binding affinity and whole cell
functional activity.
Functional antagonist
3
Inhibition of [ H]-29
activity in cells expressing
binding to human mGlu
recombinant human
receptors
mGlu receptors
K (nM) ± SEM
i
IC (nM) ± SEM
5
0
(n)
(% efficacy, n)
No.
9g
R
hmGlu2
hmGlu3
5.9 ± 0.8
(7)
hmGlu2
17.2 ± 3.8
(103, 4)
hmGlu3
27.0 ± 6.0
(97.0, 3)
163 ± 33.4
(92.8, 8)
12.4 ± 4.2
(102, 5)
1
OH
8.9 ± 1.1
(
7)
24.0 ± 5.4
3)
9.2 ± 1.8
3)
26
NH
2
36.1 ± 5.0
(3)
45.4 ± 8.6
(101, 5)
(
2
7
NHAc
7.7 ± 0.6
(3)
26.2 ± 5.3
(94.9, 3)
(
5
.2 ± 0.7
4.5 ± 0.5
(3)
6.6 ± 1.6
16.1 ± 10.8
(91.2, 5)
28
(3)
(96.8, 10)
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The effect of nonꢀhydroxyl substituents at the C4 position bearing 4(S)ꢀstereochemistry
was explored on the 3(S)ꢀ(3ꢀmethylꢀ4ꢀfluoro) phenyl scaffold in order to evaluate the
structureꢀactivity relationship at this position (Table 3). This scaffold provided a potent
starting point for this research and available quantities of 4(R)ꢀhydroxyl intermediate 21
facilitated the synthesis of analogs 26-28. The 4ꢀamino compound 26 demonstrated
moderate decreases in mGlu2/3 binding affinity and functional antagonist potency
compared to hydroxyl counterpart 19g, the greatest activity difference being an observed
6ꢀfold decrease in mGlu affinity and antagonist potency (26: hmGlu K = 36.1 ± 5.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
3
i
nM, hmGlu IC = 163 ± 33.4 nM; 19g: hmGlu K = 5.9 ± 0.8 nM , hmGlu IC = 27.0
3
50
3
i
3
50
±
6.0 nM). However, acetamide 27 and thiotriazole 28 were similar in both affinity and
antagonist potency to 19g, suggesting that the receptor space adjacent to this position of
the bound ligand can tolerate substituents possessing considerable steric and electronic
diversity. The thiotriazole moiety was previously explored at this position in the absence
of C3 substitution and produced an analog that displayed predominately mGlu agonist
2
32b
activity, but with mixed agonist/antagonist activity at the mGlu receptor.
3
There exists a strong correlation between the binding affinity and functional activity for
2
2
the hmGlu (Figure 3a, R = 0.907) and hmGlu receptors (Figure 3b, R = 0.893). This
2
3
is consistent with the very high protein sequence homology between these receptors in
33
the ligand binding domain. Furthermore, there is an excellent correlation observed for
2
hmGlu versus hmGlu when comparing binding affinity (Figure 3c, R = 0.954) or
2
3
2
functional antagonist potency (Figure 3d, R = 0.874). In each case, there is observed a
right shift in whole cell antagonist potency compared to affinity. This is consistent with
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 3: Correlations between mGlu and mGlu binding affinity and functional
2
3
activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
a)
b)
c)
d)
there being receptor reserve in these cell lines which may be greater in hmGlu cells
3
compared to hmGlu cells, a finding also observed in previous studies focused on agonist
2
24,32
SAR.
Due to the outstanding potency and relatively short synthetic sequence, compound 19f
was identified for further profiling. Compound 19f was evaluated against the historical
3
mGlu2/3 antagonist reference standard 1 by comparing inhibition of [ H]ꢀ29 binding
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affinity, functional activity in the ability to inhibit cAMP production in forskolinꢀ
2
+
stimulated cells, and functional activity in a Ca ꢀbased FLIPR assay (Table 4).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4: Comparison of recombinant hmGlu2/3 receptor-related activity for
compound 19f versus reference standard 1.
3
Inhibition of [ H]-
Functional antagonist activity in cells
2
9 binding to
expressing recombinant human mGlu
receptors
human mGlu
receptors
IC (nM) ± SEM
50
K (nM) ± SEM
i
Cmpd
mGlu2
mGlu3
mGlu2
mGlu3
2
+
2+
Ca
Ca
cAMP
cAMP
FLIPR
FLIPR
1
9f
5.3 ± 1.4 2.5 ± 0.3 16.2 ± 1.6 4.1 ± 0.5 6.2 ± 2.1 2.5 ± 0.5
4.3 ± 1.3 1.3 ± 0.2 26.7 ± 0.7 7.8 ± 0.9 7.7 ± 0.4 2.8 ± 0.5
1
Compound 19f demonstrated similar binding and FLIPR functional response against 1
with all values within 2x of the reference standard, thus essentially equaling the potency
of the primary tool for investigating mGlu2/3 receptor blockade.
Compound 19f was further characterized to determine selectivity for mGlu2/3 versus other
hmGlu receptor subtypes (Table 5). No activity was observed at the highest
concentration tested in agonist mode across the other mGlu receptor subtypes. Compound
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
9f exhibited micromolar functional antagonist (cAMP) activity at mGlu (4,010 ±
6
1
100nM), mGlu (1,260 ± 260nM), and mGlu (1770 ± 289nM), while demonstrating no
7 8
measurable activity at mGlu1, 4, or 5 (>12.5 ꢀM). The minimal selectivity ratios for
0
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3
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mGlu therefore are calculated to be 988x (hmGlu cAMP IC / hmGlu FLIPR ) and
2
6
50
2
248x (hmGlu cAMP IC / hmGlu cAMP ) versus mGlu , 310x (hmGlu FLIPR IC /
6
50
2
6
7
50
hmGlu FLIPR IC ) and 78x (hmGlu FLIPR IC / hmGlu cAMP IC ) versus mGlu ,
2
50
7
50
2
50
7
and 436x (hmGlu FLIPR IC / hmGlu FLIPR IC ) and 109x (hmGlu FLIPR IC /
8
50
2
50
8
50
Table 5: Functional agonist, antagonist, and potentiator responses of 19f in cells
expressing recombinant human mGlu receptor subtypes.
mGlu1
FLIPR
mGlu4
FLIPR
(nM)
mGlu5
FLIPR
(nM)
mGlu6
cAMP
(nM)
mGlu7
FLIPR
(nM)
mGlu8
FLIPR
(nM)
Mode
(
nM)
Agonist (EC50)
>25,000
>12,500
>25,000
>12,500
>25,000
>12,500
>25,000
>16,700
>25,000
4
,010 ±
100
1,260 ±
260
1770 ±
289
Antagonist (IC )
50
1
Antagonist Selectivity Ratio
>
>
3078
5000
>3078
>5000
>771
>3078
>5000
>771
988
1604
248
310
504
78
436
708
109
285
vs. mGlu FLIPR
2
Antagonist Selectivity Ratio
vs. mGlu FLIPR
3
Antagonist Selectivity Ratio
>
771
vs. mGlu cAMP
2
Antagonist Selectivity Ratio
>
2016
>2016
>2016
647
203
vs. mGlu cAMP
3
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hmGlu cAMP IC ) versus mGlu . The minimal selectivity ratios relative to mGlu are
2
50
8
3
comparable to the mGlu ratios at 1604x (hmGlu cAMP IC / hmGlu FLIPR ) and
2
6
50
3
6
47x (hmGlu cAMP IC / hmGlu cAMP ) versus mGlu , 504x (hmGlu FLIPR IC /
6 50 3 6 7 50
10
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hmGlu cAMP IC ) and 203x (hmGlu FLIPR IC / hmGlu cAMP IC ) versus mGlu ,
3
50
7
50
3
50
7
and 708x (hmGlu FLIPR IC / hmGlu FLIPR IC ) and 285x (hmGlu FLIPR IC /
8
50
3
50
8
50
hmGlu cAMP IC ) versus mGlu , respectively. The selectivity profile for compound
3
50
8
1
5b
1
9f compares well to that reported for 1, which exhibits only 6ꢀfold selectivity for
mGlu vs. mGlu , 19ꢀfold selectivity for mGlu vs. mGlu and 13ꢀfold selectivity for
2
7
3
7
mGlu vs. mGlu . Both mGlu and mGlu regulate glutamate and GABA transmission in
2
8
7
8
multiple brain structures and play roles in animal models of epilepsy, pain, anxiety and
3
4
addiction, thus the improved selectivity of 19f compared to 1 may render it a useful tool
for the in vivo evaluation of mGlu2/3 receptor antagonism.
Compound 19f was also evaluated against a panel of CNS receptors, transporters, ion
35
channels, and the hERG channel at a screening concentration of 10 ꢀM (CEREP, Table
S1, Supporting Information). No significant activity was observed for this molecule
against any of these targets.
Studies with the amino terminal domain (ATD) of recombinant hmGlu2.
Cocrystallization studies were performed with compound 19f and the hmGlu ATD
2
3
6,37
domain
to determine the molecular interactions associated with receptor binding
(
Figure 4). Compound 19f is bound within the hinge domain of the mGlu ATD (Figure
2
4
a, 2.8 Å resolution), which is consistent with previously published structures of mGlu2
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agonists and mGlu antagonists.
The antagonist complex displays a relatively open
3
topology with an angle of 45.7° between the upper (LB1) and lower (LB2) domains
3
9
which is highly analogous to the mGlu antagonist bound structure (45.1°), but in
3
0
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0
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contrast to mGlu ATDꢀagonist bound structures (24.7°ꢀ27.5°). Within the binding site,
2
compound 19f is involved in several key protein Hꢀbond interactions (Figure 4bꢀd). The
C2 carboxylic acid moiety interacts with the amide on LB1ꢀassociated serine (Ser145),
the C2 amino substituent shares a hydrogen bond with the sidechain oxygen in the
threonine (Thr 168) residue, and the C6 carboxylate interacts through a salt bridge with
arginine (Arg61, Figure 4c). Finally, perhaps the most striking observation is the πꢀ
stacking interaction between the C3 3,4ꢀdifluorothiophenyl aromatic substituent and
Figure 4: hmGlu ATDꢀ19f cocrystal structure
2
a)
b)
c)
d)
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Figure 4: (a) 2.8 Å hmGlu ATDꢀ19f bound crystal structure visualized in MOE with
2
open angle of approximately 46°. The open angle of the mGlu ATD is defined by points
2
associated with the C atoms of Gly451 (hinge), Tyr144 (LB1) and Arg271 (LB2). (bꢀd)
α
41
Active site views of hmGlu ATDꢀ19f bound crystal structure viewed in MOE.
2
tyrosine (Tyr216 , Figure 4d), which may explain large potency differences observed
between analogs within the SAR. Aliphatic thioether analog 19b is 20ꢀfold less active
across mGlu2/3 binding affinity and functional activity compared to the phenyl analog
1
9a. This analog lacks the πꢀsystem necessary to undergo the stacking interaction and
may account for the substantial decrease in potency. Additionally, ortho fluoro phenyl
analog 19c is considerably less potent (>10x) than paraꢀfluoro compound 19f. The
inclusion of ortho substitution may induce a conformational ring twist that prevents
proper alignment with Tyr216 for a productive πꢀstacking interaction.
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Compounds 19a and 20 were modeled in the hmGlu active site to explore the impact of
2
C3 stereochemistry on key molecular interactions (Figure 5). Compound 19a was
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Figure 5: Compounds 19a and 20 modelled in the mGlu binding site
2
a)
b)
Figure 5: (a) Compound 19a (yellow) modeled in the mGlu binding site to mimic Xꢀ
2
ray structure of 19f (b) Compound 20 (green) modeled to mimic Xꢀray structure of 19f
with energy minimization of the C3 sidechain to decrease steric interactions with Thr168.
modeled to mimic the conformation of 19f and maintain the key C2/C6 hydrogen
bonding interactions, as well as the πꢀstacking interaction with Tyr216 (Figure 5a).
Compound 20 also maintains a similar conformation of the [3.1.0]ꢀbicyclic system
relative to 19f; however, energy minimization was performed on the C3 sidechain to
reduce steric interactions resulting from the 3ꢀ(R) stereochemistry placing the thiophenyl
substituent in close proximity to Thr168 (Figure 5b). The minimized conformation
causes the chain to twist away from Thr168 and places the phenyl in an unoptimal
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position to form a πꢀstacking interaction with Try216. The lack of this interaction may
account for the consistent 10x decrease in potency for 20 relative to 19a across the
mGlu2/3 binding and functional assays.
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An apo mGlu ATD 2.8 Å structure was obtained to examine the receptor conformational
2
state in the absence of bound ligand (Figure 6a). The apoꢀhmGlu LB1ꢀLB2 angle of
2
Figure 6: hmGlu ATD apo crystal structure
2
a)
b)
Figure 6: (a) 2.8 Å hmGlu ATD native crystal structure with open angle of
2
approximately 53°. (b) hmGlu ATDꢀ19f bound Xꢀray crystal structure (white) overlaid
2
on native (cyan) Xꢀray crystal structure . Superposition of the Nꢀterminal lobe highlights
movement associated with the binding of compound 19f. (Figure prepared using
4
1
MOE.)
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3.0° closely resembles the antagonist bound structure, suggesting that antagonists exert
their pharmacological effect by stabilizing this open state rather than by alternatively
stabilizing a unique conformation of the amino terminal domain. An overlay of the apo
(cyan) and antagonistꢀbound (white) structures in Figure 6b demonstrates the
conformational similarities between unligated and ligated proteins in the crystalline state.
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Pharmacokinetic Attributes of 19f.
Owing to its optimized mGlu2/3 receptor antagonist potency and selectivity profile,
compound 19f was chosen for further assessment and evaluated in vitro in assays that can
impact in vivo pharmacokinetic properties (Table 6). Compound 19f demonstrated high
aqueous solubility (>100 mM @ pH 7.4), no measurable microsomal
Table 6. In vitro solubility, permeability, metabolism and protein
binding of 19f.
Assay
Result
> 100 ꢀM
Aqueous Solubility (pH 7.4)
DMSO Solubility
> 10 mM
a
%
Loss Hepatocytes (mouse, rat, dog, human)
< 15% for each
%
Inhibition Human Cyp Isoforms
<
10% for each
> 60%
b
(3A4, 2D6, 2C9)
Plasma Unbound Fraction (rat, human)
a
b
tested at 2 ꢀM; tested at 10 ꢀM
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metabolism across species (<15% of each), no significant cytochrome P450 enzyme
inhibition, and high plasma unbound fraction (>60%) across multiple species.
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The in vivo pharmacokinetic attributes of 19f were evaluated in mouse and the plasma
pharmacokinetic parameters are presented in Table 7. Intraperitoneal (i.p.) dosing led to
2
2
approximately dose proportional increases in AUC (r = 0.999) and C (r = 0.919)
max
(Figure S18, Supporting Information) over the range of 1.1ꢀ11 mg/kg (actual dose of HCl
salt) with maximal plasma concentrations occurring within 15 min. in the range of 4.5
ꢀM (1.1 mg/kg) ꢀ42 ꢀM (11 mg/kg). Administered by the i.p. route in the mouse,
compound 19f exhibited a short halfꢀlife ranging from 0.5ꢀ1 h across all three doses,
despite displaying low mouse microsomal metabolism. The highly polar nature of 19f
could lead to high renal clearance with low passive reabsorption; however, this was not
studied. Nevertheless, while a detailed investigation of mouse PK was not performed, rat
PK parameters following i.v. administration indicate low clearance (5.83ꢀ8.13
42
mL/min/kg) across a 0.3ꢀ10 mg/kg dose range for 19f.
Table 7: Mean plasma pharmacokinetic parameters for 19f following single
intraperitoneal dosing in mice.
Actual Dose of Salt (mg/kg) (i.p. route)
(Active Potency Equivalent (mg/kg))
Parameter
Units
1.1
3.3
11.0
(1.0)
(3.0)
(10.0)
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Plasma
Plasma
15184 ± 1920
Plasma
AUC0ꢀ24hr
Cmax
nM*Hours
nM
5028 ± 303
4169 ± 879
0.25
45476 ± 3966
20616 ± 2413 38402 ± 11596
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Tmax
Hours
0.25
0.50
4601
0.25
1.07
4134
T1/2
Hours
0.70
AUC/Dose
nM*Hours/mg/kg
4571
In a separate experiment, the unbound brain and plasma concentrations of compound 19f
were evaluated across the same potency equivalent dose range (1ꢀ10 mg/kg) at the 30
min. timepoint (Table 8). The unbound brain concentration increased proportionally
Table 8: Mean unbound brain concentrations for 19f (free base) following a single
intraperitoneal dose in mice and calculated target engagement ratios.
Dose (mg/kg) (i.p. route)
Parameter
Units
1.0
8.1
3.0
47.3
3956
2.9x
7.6x
10.0
151.9
7731
9.4x
[
Br]u
Pl]u
nM
nM
[
840
0.5x
1.3x
mGlu TER
2
mGlu TER
24.5x
3
TER estimated target engagement ratio ([Br]u / hmGlu IC or hmGlu IC
=
2 50 3 50
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across the dose range with values ranging from 8 nM (1 mg/kg) to 152 nM (10 mg/kg)
and ranging from 1ꢀ2% of the unbound plasma levels, thus demonstrating low brain
penetration for 19f. Estimated target engagement ratios (TERs) were calculated using the
human mGlu and mGlu IC values owing to the identical active site sequence
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50
4
3
homology between the human and mouse receptors. The relatively low brain
concentration at 1 mg/kg led to an estimated target engagement ratios of 0.5x (mGlu2),
1
.3x (mGlu ), while estimated TERs of 2.9x (mGlu ), 7.6x (mGlu ) and 9.4x (mGlu ),
3 2 3 2
24.5x (mGlu ) were achieved at the 3 mg/kg and 10 mg/kg doses, respectively.
3
Antidepressant-like response of 19f in mice.
The antiꢀdepressant activity of compound 19f was evaluated in the mouse forced swim
test (mFST), a preclinical model suggestive of antiꢀdepressive effects (Figure 7). In this
model mice are pretreated with compound and then placed in an inescapable tank filled
with water. The escapeꢀrelated mobility behavior is measured and subtracted from the
total measuring time to provide an immobility time; lower immobility times are reflective
Figure 7: Effect of compound 19f on immobility time in the mouse forced swim test
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