Discovery of VU0431316: A negative allosteric modulator of mGlu5 with activity in a mouse model of anxiety

Article abstract of DOI:10.1016/j.bmcl.2014.06.003

Development of SAR in an aryl ether series of mGlu₅ NAMs leading to the identification of pyrazine analog VU0431316 is described in this Letter. VU0431316 is a potent and selective non-competitive antagonist of mGlu ₅ that binds at a known allosteric binding site. VU0431316 demonstrates an attractive DMPK profile, including moderate clearance and good bioavailability in rats. Intraperitoneal (IP) dosing of VU0431316 in a mouse marble burying model of anxiety, an assay known to be sensitive to mGlu ₅ antagonists and other anxiolytics, produced dose proportional effects.

Full text of DOI:10.1016/j.bmcl.2014.06.003

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3307–3314
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of VU0431316: A negative allosteric modulator
of mGlu5 with activity in a mouse model of anxiety
Brittney S. Bates a,b, Alice L. Rodriguez a,b, Andrew S. Felts a,b, Ryan D. Morrison a,b, Daryl F. Venable a,b
,
Anna L. Blobaum a,b, Frank W. Byers a,b, Kera P. Lawson a,b, J. Scott Daniels a,b, Colleen M. Niswender a,b
,
Carrie K. Jones a,b,d, P. Jeffrey Conn a,b, Craig W. Lindsley a,b,c, Kyle A. Emmitte a,b,c,
⇑
a Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
d Tennessee Valley Healthcare System, U.S. Department of Veterans Affairs, Nashville, TN 37212, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Development of SAR in an aryl ether series of mGlu5 NAMs leading to the identification of pyrazine analog
VU0431316 is described in this Letter. VU0431316 is a potent and selective non-competitive antagonist
of mGlu5 that binds at a known allosteric binding site. VU0431316 demonstrates an attractive DMPK
profile, including moderate clearance and good bioavailability in rats. Intraperitoneal (IP) dosing of
VU0431316 in a mouse marble burying model of anxiety, an assay known to be sensitive to mGlu5
antagonists and other anxiolytics, produced dose proportional effects.
Received 30 April 2014
Revised 30 May 2014
Accepted 2 June 2014
Available online 11 June 2014
Keywords:
Glutamate
CNS
Ó 2014 Elsevier Ltd. All rights reserved.
mGlu5
Allosteric modulator
Anxiety
The metabotropic glutamate receptors (mGlus) comprise a fam-
ily of eight related G-protein-coupled receptors (GPCRs) wherein
each receptor acts through binding glutamate, the major excitatory
transmitter in the mammalian central nervous system (CNS). In
these seven transmembrane spanning (7TM) receptors, the orthos-
teric binding sites are located in the extracellular domain while
known allosteric binding sites are contained in the transmembrane
domain.1 Design of highly selective orthosteric ligands has contin-
ually proven difficult due to the extensive homology of the binding
sites across the mGlu family. In many instances, the development
of allosteric modulators of mGlus has been established as a viable
solution to enhancing selectivity among family members.2 Among
the individual mGlus investigated as potential drug targets, a
substantial portion of that attention has been devoted to the
design of small molecule negative allosteric modulators (NAMs),
or non-competitive antagonists, of mGlu5.3
Efficacy has been reported with these compounds across a number
of different disease models. Examples include pain,6 anxiety,7
gastroesophageal reflux disease (GERD),8 Parkinson’s disease
levodopa induced dyskinesia (PD-LID),9 fragile X syndrome (FXS),10
and other autism spectrum disorders.11 Furthermore, both MPEP
and MTEP have been used effectively in various animal models
of addictive behavior with well-known drugs of abuse, such as
cocaine,12 nicotine,12g,13 methamphetamine,14 morphine,15 and
ethanol.16
Currently, several mGlu5 NAMs have progressed to human tri-
als, and results from studies in patients with GERD,17 FXS,18 and
PD-LID19 have been encouraging. Though structural diversity
among mGlu5 NAMs in the literature has expanded considerably
in recent years, the majority of clinical compounds have been from
the disubstituted alkyne structure class.3a Furthermore, the most
advanced clinical compounds, mavoglurant (AFQ056) and
basimglurant (RG7090, RO4917523), each contain the alkyne moi-
ety (Fig. 1).20 Recently, concerns that such alkyne compounds
might be prone to metabolic activation and resultant toxicities
have proven warranted, at least in one instance. Pfizer has now
disclosed their observation of biliary epithelial hyperplasia in
non-human primate regulatory toxicology studies with the
disubstituted alkyne compound known as GRN-529. Glutathione
The vast majority of the preclinical behavioral work with mGlu5
NAMs has been conducted using one of two structurally related
disubstituted alkyne tool compounds, MPEP4 and MTEP5 (Fig. 1).
⇑
Corresponding author. Tel.: +1 615 936 8401; fax: +1 615 322 8577.
E-mail address: kyle.a.emmitte@vanderbilt.edu (K.A. Emmitte).
http://dx.doi.org/10.1016/j.bmcl.2014.06.003
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

3308
B. S. Bates et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3307–3314
Figure 1. mGlu5 NAM tool and advanced clinical compounds.
conjugation at the alkyne moiety was believed to be related to
these adverse findings.21
Scheme 1. Reagents and conditions: (a) For 2 (Z = N; V = Q = CH), 3 (V = N; Q = Z =
CH), 4 (Q = N; V = Z = CH), and 5 (V = Z = N; Q = CH); RCO2H (R = 3-chlorophenyl),
EDC, DMAP, CH2Cl2, (83–94%); (b) 3-hydroxypyridine, CuI, Cs2CO3, Me2NCH2CO2-
HÁHCl (35–55%); (c) For 14 (W = N; Q = Z = CH; X = F; Y = Br), 15 (Q = W = N; Z = CH;
X = Y = Cl), and 16 (Q = Z = N; W = CH; X = Y = Cl); 3-hydroxypyridine, K2CO3, DMF,
microwave, 150 °C (60–93%); (d) RCONH2 (R = 3-chlorophenyl), NaOtBu, Pd(OAc)2,
Xantphos, PhMe, 100 °C (50–78%).
Our mGlu5 NAM program has long been centered on the identi-
fication and optimization of compounds from chemotypes that do
not contain a disubstituted alkyne motif. The majority of this effort
has been spent on the optimization of hits identified from a func-
tional cell-based high-throughput screen (HTS) of a collection of
160,000 compounds;22 however, rational design approaches23
and a virtual screening approach also produced new non-alkyne
based mGlu5 NAM tool compounds.24 We recently reported on a
lead optimization effort based around hit compound 1 from our
functional HTS (Fig. 2).22a This particular optimization effort,
based on 1, culminated in discovery of the in vivo tool compound
VU0409106.
Concomitant to the recently described work that led to the dis-
covery of VU0409106, we were also pursuing additional analogs of
1. Reasoning that a potential route of metabolism for analogs of 1
might include amide bond cleavage, we immediately sought to
identify compounds that would not produce electron rich anilines
should the amide bond indeed be cleaved in vivo. In the case of
VU0409106 and associated analogs, we achieved this goal by
reversing the orientation of the amide bond; however, the work
described herein centers on the replacement of the phenyl core
with heteroaryl rings. Preparation of the initial heteroaryl ether
analogs of 1 was executed according to one of the two general
methods outlined here (Scheme 1).25
Certain pyridine (10–12) and pyrimidine (13) analogs were
prepared by first coupling the commercial amines 2–5 with
3-chlorobenzoic acid to afford the corresponding amides 6–9
(Route I). Reaction with 3-hydroxypyridine in the presence of cop-
per (I) iodide and dimethylglycine afforded the desired compounds
10–13. Alternatively, pyridine 20, pyrazine 21, and pyrimidine 22
were prepared via a route relying on initial installation of the aryl
ether (Route II). Reaction of the commercial monomers 14–16 with
3-hydroxypyridine in a microwave assisted nucleophilic aromatic
substitution (SNAr) reaction provided heteroaryl halide intermedi-
ates 17–19. The final analogs were prepared directly through a
Buchwald–Hartwig coupling with 3-chlorobenzamide in moderate
to high yields.26
Evaluation of these initial analogs against mGlu5 yielded clear
SAR (Table 1). Our functional assay measures the ability of the
compound to block the mobilization of calcium induced by an
EC80 concentration of glutamate in HEK293A cells expressing rat
mGlu5.27 Among the pyridine analogs, compounds 12 and 20 were
superior to compounds 10 and 11. In fact both 12 and 20 exhibited
potency at a level near hit 1. Pyrimidine analogs 13 and 22 were
weak antagonists; however, pyrazine 21 exhibited the best
potency in this set of analogs. Having established the pyrazine core
as a favorable group for further SAR development, lead optimiza-
tion continued in that area.
Ongoing research has identified the 5-fluoropyridin-3-yl and
pyrimidin-5-yl ethers as optimal groups in the northern portion
of the chemotype.22a Thus, much of the SAR was developed in
the context of one or both of these moieties. Compounds
containing the pyrimidine moiety are less lipophilic than their
5-fluoropyridine counterparts,28 a feature that can often provide
advantages with respect to drug-like properties. Though the syn-
thesis outlined in Scheme 1 (Route II) was utilized to prepare some
new pyrazine analogs, a new synthetic route allowing for the
preparation of a broader diversity of amides was utilized in most
cases (Scheme 2).29 This route also begins with a similar SNAr
reaction, providing ethers 23–24. A Buchwald–Hartwig coupling
with t-butyl carbamate was employed to afford intermediates
25–26.30 Cleavage of the protecting group was carried out under
acidic conditions to yield amines 27–28. Conversion to the desired
amide products was accomplished using standard coupling
conditions.
Evaluation of various substituted benzamides revealed some
additional potent compounds (Table 2). As anticipated, both the
5-fluoropyridin-3-yl (35) and pyrimidin-5-yl (29) ethers proved
competent replacements for the simple pyridine-3-yl (21) ether.
Furthermore, the importance of the 3-chloro substituent on the
benzamide was established through preparation of unsubstituted
analogs 30 and 36. Many additional 3-substituted analogs were
prepared and tested (31–34 and 36–41); however, only the
3-methyl analogs 32 and 38 demonstrated potency comparable
to 29 and 35. 3-Cyanobenzamides 33 and 39 were approximately
six fold less potent than 3-chlorobenzamide comparators 29 and
35. Additional monosubstituted benzamides demonstrated moder-
ate to weak antagonist activity. Several disubstituted benzamides
were also evaluated (42–50), with only 2-fluoro-5-chlorobenzam-
ide 45 demonstrating an IC50 less than one micromolar.
Figure 2. HTS hit 1 and mGlu5 NAM in vivo tool VU0409106.

B. S. Bates et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3307–3314
3309
Table 1
Core SAR
% Glu Maxa,b
1.3 0.2
( SEM)
a
Compd
Core
mGlu5 pIC50
6.55 0.19
(
SEM)
mGlu5 IC50 (nM)
284
1
10
11
12
13
20
21
6.00 0.21
5.43 0.26
6.44 0.19
5.53 0.23
6.39 0.07
6.89 0.04
<5.0c
994
3700
359
1.8 0.7
À4.9 4.7
2.0 0.7
1.1 0.5
1.1 0.4
1.0 0.3
2930
403
129
22
>10,000
44
8
a
Calcium mobilization mGlu5 assay; values are average of n P 3.
b
c
Amplitude of response in the presence of 30
lM test compound as a percentage of maximal response (100 lM glutamate); average of n P 3.
Concentration response curve (CRC) does not plateau.
Turning our attention from benzamide analogs to analogs with
a heteroaryl amide moiety identified a mixture of weak antagonists
and compounds that were inactive up to the top concentration
tested of 30 lM (Table 3). Recognizing the importance of
substitution that was observed in the case of the benzamide
analogs, particularly at the 3-position, we decided to prepare some
additional analogs of the weak antagonists identified from this
initial set (Table 4). Methyl substitution resulted in modest
potency enhancement in the case of thiophene 70 relative to
55. More dramatic potency improvement was noted with
picolinamides 67 and 75 when compared to 59. Preparation of
additional 4-substituted picolinamides (68, 69, and 76) identified
4-chloropicolinamide 68 (VU0431316) as the most potent mGlu5
NAM in this series.
Further in vitro characterization of 68 (VU0431316) was subse-
quently initiated. Competitive displacement of the established radi-
oligand [3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine31 confirmed
the interaction of the compound with a known mGlu5 allosteric
binding site (mGlu5 Ki = 37 nM (n = 1)).32 Evaluation of 68
(VU0431316) in cell based functional assays against the other seven
Scheme 2. Reagents and conditions: (a) 3-fluoro-5-hydroxypyridine or 5-hydroxy-
pyrimidine, K2CO3, DMF, microwave, 120 °C (51% for 23; 62% for 24); (b)
H2NCO2tBu, NaOtBu, Pd2(dba)3ÁCHCl3, tBuXPhos, PhMe (46% for 25; 64% for 26);
(c) For 25 ? 27, TFA, CH2Cl2 (96%); (d) For 26 ? 28, 4 N HCl in dioxane (100%); (e)
RCO2H, EDC, DMAP, CH2Cl2 or RCO2H, HATU, DIEA, CH2Cl2, DMF or RCO2H, POCl3,
pyridine or RCOCl, DMAP, CH2Cl2 (30–80%).
mGlus showed no detectable activity at 10 l
M.33 Additionally, the
functional activity of 68 (VU0431316) at human mGlu5 was
determined and was essentially identical to that at the rat receptor
(human mGlu5 IC50 = 85 nM (n = 1)).34 Finally, 68 (VU0431316)

3310
B. S. Bates et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3307–3314
Table 2
Benzamide SAR
a
Compd
A
R
mGlu5 pIC50
(
SEM)
mGlu5 IC50 (nM)
% Glu Maxa,b
( SEM)
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
N
N
N
N
N
N
3-Cl
H
3-F
3-Me
3-CN
3-OMe
3-Cl
H
3-F
6.94 0.10
<5.0c
116
>10,000
5140
212
709
4110
118
8650
3820
199
662
1.2 0.0
12 11
1.5 2.7
1.5 0.5
1.5 0.2
À4.3 2.7
2.0 0.3
À9.4 2.3
À0.4 1.7
2.1 0.4
1.2 0.3
À5.6 5.1
52 11
—
5.29 0.17
6.67 0.16
6.15 0.17
5.39 0.15
6.93 0.10
5.06 0.09
5.42 0.09
6.70 0.18
6.18 0.16
5.36 0.17
<5.0c
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
3-Me
3-CN
3-OMe
3-NMe2
2-F, 3-Cl
3-Cl, 4-F
3-Cl, 5-F
2-F, 5-Cl
3,5-Di-F
3,5-Di-Cl
3,5-Di-Me
3,5-Di-OMe
3-CN, 5-F
4400
>10,000
>30,000
4590
>10,000
894
>30,000
>30,000
6600
>30,000
>30,000
<4.5
5.34 0.15
À2.8 6.2
48 10
1.0 0.8
—
<5.0c
6.05 0.13
<4.5
<4.5
5.18 0.18
<4.5
<4.5
—
6.6 2.0
—
—
a
Calcium mobilization mGlu5 assay; values are average of n P 3.
b
c
Amplitude of response in the presence of 30
CRC does not plateau.
lM test compound as a percentage of maximal response (100
lM glutamate); average of n P 3.
Table 3
Heteroaryl amide SAR
a
Compd
A
R
mGlu5 pIC50
<5.0c
(
SEM)
mGlu5 IC50 (nM)
>10,000
% Glu Maxa,b
20d
( SEM)
51
N
52
N
<5.0c
>10,000
15
35
8
6
53
54
55
N
<5.0c
>10,000
6370
CF
CF
5.2 0.11
5.5 0.15
7.8 2.8
0.1 2.4
3450
56
57
CF
CF
<4.5
>30,000
>10,000
—
<5.0c
40
9

B. S. Bates et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3307–3314
3311
Table 3 (continued)
a
Compd
A
R
mGlu5 pIC50
5.07 0.05
(
SEM)
mGlu5 IC50 (nM)
8430
% Glu Maxa,b
22 11
( SEM)
58
59
60
61
62
63
CF
CF
CF
CF
CF
CF
<5.0c
<4.5
<4.5
<4.5
<4.5
>10,000
>30,000
>30,000
>30,000
>30,000
40 15
—
—
—
—
64
CF
<4.5
>30,000
—
a
Calcium mobilization mGlu5 assay; values are average of n P 3.
b
c
Amplitude of response in the presence of 30
CRC does not plateau.
lM test compound as a percentage of maximal response (100
lM glutamate); average of n P 3.
d
Average of n = 2.
Table 4
Substituted heteroaryl amide SAR
a
Compd
A
R
mGlu5 pIC50
<4.5
(
SEM)
mGlu5 IC50 (nM)
>30,000
% Glu Maxa,b
—
( SEM)
65
N
66
67
68
69
N
N
N
N
<4.5
>30,000
193
—
6.72 0.22
7.20 0.06
7.00 0.29
2.1 0.8
1.6 0.1
2.3 0.6
62.4
100
70
71
72
CF
CF
CF
6.00 0.04
<5.0c
990
1.1 0.75
20 10
>10,000
4640
5.33 0.17
19
6
73
74
CF
CF
<4.5
<4.5
>30,000
>30,000
—
—
(continued on next page)

3312
B. S. Bates et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3307–3314
Table 4 (continued)
a
Compd
A
R
mGlu5 pIC50
6.51 0.15
(
SEM)
mGlu5 IC50 (nM)
311
% Glu Maxa,b
1.7 0.4
( SEM)
75
CF
76
CF
6.95 0.29
111
2.0 0.3
a
Calcium mobilization mGlu5 assay; values are average of n P 3.
b
c
Amplitude of response in the presence of 30
CRC does not plateau.
lM test compound as a percentage of maximal response (100
lM glutamate); average of n P 3.
Table 5
In vitro DMPK Profile for 68 (VU0431316)
Protein bindinga,b (Fu)
MDCK-WT permeability
Rat PPB
0.179
0.167
0.120
0.054
Papp (A-B)
Papp (B-A)
Efflux ratio
50.4 Â 10À6 cm/s
Cynomolgus PPB
Human PPB
Mouse BHB
45.0 Â 10À6 cm/s
0.89
c
CYP inhibition IC50
(l
M)
Caco-2 permeability
3A4
>30
2D6
>30
2C9
>30
1A2
0.6
Papp (A-B)
Papp (B-A)
Efflux ratio
66.8 Â 10À6 cm/s
71.4 Â 10À6 cm/s
1.07
a
b
c
Fu = fraction unbound.
PPB = plasma protein binding; BHB = brain homogenate binding.
Assayed in pooled HLM in the presence of NADPH.
Table 6
Rat PK Results for 68 (VU0431316)
Figure 3. Dose dependent inhibition of marble burying with 68 (VU0431316);
n = 7–8 per dose; ⁄P < 0.05 versus vehicle control group, Dunnett’s test. Bars denote
marbles buried. Vehicle = 10% Tween 80.
IVa,b
POa,c
Half-life
Clearance
VSS
193 min
30.2 mL/min/kg
2.7 L/kg
Plasma Cmax
Plasma Tmax
AUC
F
1.12
240 min
8.37
48%
lM
lM h
AUC
1.73 lM h
were calculated from an IV dosing of 68 (VU0431316) to male
Sprague-Dawley rats; hepatic clearance was moderate with an
approximate three hour half-life. Bioavailability from a single oral
(PO) dose was also encouraging, approaching fifty percent. A satel-
lite tissue distribution study was conducted one hour after a
10 mg/kg PO dose. Seventy percent of the compound was detected
in plasma relative to the hepatic portal vein, indicative of a low
first-pass effect and consistent with the previously observed clear-
ance. CNS penetration was also excellent with a brain to plasma
ratio (B/P) of 1.6.41
It is recognized that naïve mice will bury foreign objects such as
glass marbles in deep bedding. Pretreating such mice with low
doses of anxiolytic benzodiazepines will consistently inhibit this
behavior.42 Additionally, the well-known and thoroughly charac-
terized mGlu5 NAMs MPEP and fenobam each inhibit marble bury-
ing in this model.7a,d Furthermore, we have used this model
successfully to evaluate several of our own novel mGlu5 NAM tool
compounds.22a,23b,24 More recent reports have raised the possibil-
ity that marble burying reflects a repetitive and perseverative
behavior as opposed to novelty-induced anxiety.43 Still, given the
convenience and reliability of the marble burying assay, it has
served as the frontline behavioral assay for our mGlu5 NAM discov-
ery program.44 Thus, a dose response study with 68 (VU0431316)
using a 15 minute pretreatment time following intraperitoneal
(IP) administration was carried out in this model (Fig. 3).45 Statis-
tically significant inhibition of marble burying was noted at all
doses greater than or equal to 10 mg/kg. A satellite tissue distribu-
tion experiment in mice at the top dose of 30 mg/kg (IP) showed
considerable concentrations of compound in the brain.46 Using
the results from this study to extrapolate exposures at 10 mg/kg
PO tissue distribution studya,c
Time
60 min
Plasma
0.92
HPV
1.31
Brain
B/P ratio
1.6
l
M
lM
1.45
l
M
a
b
c
Male Sprague-Dawley rats (n = 2 per time point).
1 mg/kg; vehicle = 10% ethanol, 90% PEG400.
10 mg/kg; vehicle = 10% Tween 80 in 0.5% MC.
was tested in a commercially available radioligand binding assay
panel of 68 clinically relevant GPCRs, ion channels, kinases, and
transporters,35 and no significant responses were found at 10
lM
compound.36
The pharmacological profile of 68 (VU0431316) warranted
further evaluation of its drug-likeness and potential utility as an
in vivo tool (Table 5). Evaluation of the compound’s propensity to
bind to plasma proteins revealed similar results across multiple
species. Nonspecific binding to mouse brain homogenates was
also evaluated to enable the estimation of the fraction unbound
in the CNS.37 Bidirectional permeability was assessed in both
Madin Darby canine kidney (MDCK) and human intestinal
epithelial (Caco-2) cells, and permeability was high with no evi-
dence of efflux.38 Determination of the cytochrome P450 (CYP)
inhibition profile of 68 (VU0431316) indicated potent inhibition
of CYP1A2 with no inhibition of other isoforms up to the top con-
centration tested of 30 l
M.39 As was the case with the related tool
compound VU0409106, the common pyrimidine ether moiety
resulted in a major non-P450 mediated metabolic pathway for 68
(VU0431316).40
The in vitro DMPK profile of 68 (VU0431316) was deemed sup-
portive of in vivo evaluation (Table 6). Pharmacokinetic parameters

B. S. Bates et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3307–3314
3313
14. Gass, J. T.; Osborne, M. P. H.; Watson, N. L.; Brown, J. L.; Olive, M. F.
Neuropsychopharmacology 2009, 34, 820.
15. Kotlinska, J.; Bochenski, M. Eur. J. Pharmacol. 2007, 558, 113.
along with the aforementioned brain homogenate binding data
indicates that unbound compound concentrations in the brain
were likely near the functional IC50 at the minimum effective dose.
In conclusion, a second in vivo tool compound has been
developed from HTS hit 1. VU0431316 offers an advantage to
VU0409106 in that it is orally bioavailable in rats. VU0431316 is
a potent and selective mGlu5 NAM binding to a known allosteric
site. CNS exposure in both mice and rats is quite good and efficacy
has been established in a proven anxiolytic model. A host of addi-
tional behavioral models associated with mGlu5 are either planned
or already underway with VU0431316 and will be the subject of
future communications.
16. (a) Adams, C. L.; Short, J. L.; Lawrence, A. J. Br. J. Pharmacol. 2010, 159, 534; (b)
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Acknowledgments
We thank NIDA (R01 DA023947) and Seaside Therapeutics
(VUMC33842) for their support of our programs in the
development of non-competitive antagonist of mGlu5. We also
thank Tammy S. Santomango for technical contributions with the
protein binding assays.
References and notes
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27. HEK293A cells expressing rat mGlu5 were cultured and plated. The cells were
loaded with a Ca2+ sensitive fluorescent dye and the plates were washed and
placed in the Functional Drug Screening System (Hamamatsu). Compounds
(10 mM in DMSO) were serially diluted 1:3 into 10 point CRC (30 lM to 1 nM
9. Morin, N.; Grégoire, L.; Gomez-Mancilla, B.; Gasparini, F.; Di Paolo, T.
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final) and transferred to daughter plates. Compounds were diluted into assay
buffer and applied to cells 3 s after baseline readings were taken. Cells were
incubated with the test compounds for 140 s and then stimulated with an EC20
concentration of glutamate; 60 s later an EC80 concentration of agonist was
added and readings taken for an additional 40 s. Allosteric modulation by the
compounds was measured by comparing the amplitude of the responses at the
time of glutamate addition plus and minus test compound. For a more detailed
description of the assay, see Ref. 22b.
11. Silverman, J. L.; Tolu, S. S.; Barkan, C. L.; Crawley, J. N.
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28. According to the cLogP calculator developed by ADRIANA.Code
(www.molecular-networks.com) 5-fluoropyridin-3-yl ether compounds are
ꢀ1.2 units more lipophilic than their pyrimidin-5-yl ether counterparts in this
chemotype (e.g., 76 cLogP = 3.72; 68 cLogP = 2.51).
29. The synthesis of VU0431316 (68) is representative: (i) 5-((6-chloropyrazin-2-
yl)oxy)pyrimidine (23). A mixture of 2,6-dichloropyrazine (1.00 g, 6.71 mmol,
1.00 equiv), pyrimidin-5-ol (645 mg, 6.71 mmol, 1.00 equiv), and potassium
carbonate (1.39 g, 10.1 mmol, 1.50 equiv) in DMF (20 mL) was heated via
microwave irradiation at 120 °C for 10 min. The reaction was cooled and
diluted with EtOAc and washed with H2O (3Â) and brine (1Â). The organic
layer was dried (MgSO4), filtered, and concentrated in vacuo. Purification by
flash chromatography on silica gel afforded 710 mg (51%) of the title
13. Tronci, V.; Vronskaya, S.; Montgomery, N.; Mura, D.; Balfour, D. J. K.
Psychopharmacology 2010, 211, 33.

3314
B. S. Bates et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3307–3314
compound. 1H NMR (400 MHz, DMSO-d6) d 9.15 (s, 1H), 8.93 (s, 2H), 8.73 (s,
37. Binding to plasma and brain homogenates were measured using equilibrium
dialysis according to methods similar to those described in Kalvass, J. C.;
Maurer, T. S. Biopharm. Drug Dispos. 2002, 23, 327.
38. Bidirectional permeability was carried out according to methods described in
Wang, Q.; Rager, J. D.; Weinstein, K.; Kardos, P. S.; Dobson, G. L.; Li, J.; Hidalgo, I.
J. Int. J. Pharm. 2008, 288, 349.
39. CYP3A4 inhibition assay was carried out according to methods described in (a)
Zientek, M.; Miller, H.; Smith, D.; Dunklee, M. B.; Heinle, L.; Thurston, A.; Lee,
C.; Hyland, R.; Fahmi, O.; Burdette, D. J. Pharmacol. Toxicol. Methods 2008, 58,
206; (b) Kuresh, A. Y.; Lyons, R.; Payne, L.; Jones, B. C.; Saunders, K. J. Pharm.
Biomed. Anal. 2008, 48, 92.
40. Morrison, R. D.; Blobaum, A. L.; Byers, F. W.; Santomango, T. S.; Bridges, T. M.;
Stec, D.; Brewer, K. A.; Sanchez-Ponce, R.; Corlew, M. M.; Rush, R.; Felts, A. S.;
Manka, J.; Bates, B. S.; Venable, D. F.; Rodriguez, A. L.; Jones, C. K.; Niswender, C.
M.; Conn, P. J.; Lindsley, C. W.; Emmitte, K. A.; Daniels, J. S. Drug Metab. Dispos.
2012, 40, 1834.
41. For the IV study, the blood samples were collected at 2, 7, 15, 30, 60, 120,
240, 420, and 1440 min after dose administration. For the PO study, blood
samples were collected at 15, 30, 60, 120, 240, 420, and 1440 min after
dose administration. For the tissue distribution study, rats were euthanized
and decapitated at 60 min after dose administration and blood, hepatic
portal vein, and brain samples were collected. Following protein
precipitation, the supernatants of all plasma and brain homogenate
samples were analyzed by means of LC–MS/MS. PK studies were
approved by the Vanderbilt University Medical Center Institutional Animal
Care and Use Committee.
42. (a) Njung’e, K.; Handley, S. L. Br. J. Pharmacol. 1991, 104, 105; (b) Broekkamp, C.
L.; Rijk, H. W.; Joly-Gelouin, D.; Lloyd, K. L. Eur. J. Pharmacol. 1986, 126,
223.
43. Thomas, A.; Burant, A.; Bui, N.; Graham, D.; Yuva-Paylor, L. A.; Paylor, R.
Psychopharmacology 2009, 204, 361.
1H), 8.62 (s, 1H); ES-MS [M+H]+: 209.1. (ii) tert-Butyl (6-(pyrimidin-5-
yloxy)pyrazin-2-yl)carbamate (25). Compound 23 (760 mg, 3.64 mmol,
1.00 equiv), tert-butyl carbamate (117 mg, 4.37 mmol, 1.20 equiv),
Pd2(dba)3ÁCHCl3 (113 mg, 0.110 mmol, 0.030 equiv), NaOtBu (490 mg,
5.10 mmol, 1.40 equiv), and tBuXPhos (164 mg, 0.330 mmol, 0.090 equiv)
were stirred in toluene (14 mL) at rt overnight. The mixture was filtered
through celite and washed with 5% MeOH in CH2Cl2. The filtrate was collected
and concentrated in vacuo. Purification by flash chromatography on silica gel
afforded 480 mg (46%) of the title compound. 1H NMR (400 MHz, DMSO-d6) d
10.14 (s, 1H), 9.09 (s, 1H), 8.91 (s, 2H), 8.82 (s, 1H), 8.25 (s, 1H), 1.45 (s, 9H); ES-
MS [M+H]+: 290.1. (iii) 6-(pyrimidin-5-yloxy)pyrazin-2-amine (27). Compound
25 (480 mg, 1.66 mmol, 1.00 equiv) was stirred in 4:1 CH2Cl2:TFA (2 mL) at rt
overnight. The reaction mixture was concentrated in vacuo and diluted in
EtOAc. The organic layer was washed with saturated NaHCO3 (3Â) and brine
(1Â), dried (MgSO4), filtered and concentrated in vacuo to give 300 mg (96%) of
the title compound. 1H NMR (400 MHz, DMSO-d6) d 9.04 (s, 1H), 8.79 (s, 2H),
7.65 (s, 1H), 7.60 (s, 1H), 6.64 (s, 2H); ES-MS [M+H]+: 190.1. (iv) 4-Chloro-N-(6-
(pyrimidin-5-yloxy)pyrazin-2-yl)picolinamide (68, VU0431316). Compound
27 (150 mg, 0.790 mmol, 1.00 equiv) and 4-chloropicolinic acid (125 mg,
0.790 mmol, 1.00 equiv) were dissolved in pyridine (10 mL) under argon and
cooled to -15 °C with stirring. POCl3 (0.080 mL, 0.870 mmol, 1.10 equiv) was
added dropwise. The resulting reaction mixture was stirred at À15 °C for
30 min. The reaction was then warmed to rt and quenched with ice water. The
mixture was extracted with CH2Cl2. The organics were washed with water (3Â)
and brine (1Â). The organic layer was dried (MgSO4), filtered, and concentrated
in vacuo. The resulting residue was purified by flash chromatography on silica
gel to provide 125 mg (48%) of the desired product as a white solid. 1H NMR
(400 MHz, DMSO-d6) d 10.64 (s, 1H), 9.24 (s, 1H), 9.14 (s, 1H), 8.99 (s, 2H), 8.71
(d, J = 5.3 Hz 1H) 8.50 (s, 1H), 8.19 (d, J = 1.8 Hz, 1H), 7.89 (dd, J = 5.3, 2.0 Hz,
1H); ES-MS [M+H]+: 329.1.
30. Bhagwanth, S.; Waterson, A. G.; Adjabeng, G. M.; Hornberger, K. R. J. Org. Chem.
2009, 74, 4634.
31. Cosford, N. D. P.; Roppe, J.; Tehrani, L.; Schweiger, E. J.; Seiders, T. J.; Chaudary,
A.; Rao, S.; Varney, M. A. Bioorg. Med. Chem. Lett. 2003, 13, 351.
32. For a detailed description of the mGlu5 radioligand binding assay see Ref. 23b.
33. mGlu selectivity assays are described in 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.; Lindsley, C. W.; Weaver, C. D.; Conn,
P. J. Mol. Pharmacol. 2012, 81, 120.
44. Marble burying experiments were conducted in accordance with the National
Institute of Health regulations of animal care covered in Principles of
Laboratory Animal Care and were approved by the Vanderbilt University
Medical Center Animal Care and Use Committee. For a detailed experimental
procedure for the marble burying assay see Ref. 23b.
45. IP dosing has proven over time to be a convenient and consistent route of
administration for our behavioral studies in mice.
46. Mice were euthanized and decapitated at predetermined time-points after
dose administration and blood and brain samples were collected. Following
protein precipitation, the supernatants of all plasma and brain homogenate
samples were analyzed by means of LC–MS/MS. PK studies were approved by
the Vanderbilt University Medical Center Institutional Animal Care and Use
34. Analogous to Ref. 27 with the exception that HEK293A cells expressing human
mGlu5 were used.
35. LeadProfilingScreenÒ, Eurofins Panlabs, Inc. (http://www.eurofinspanlabs.com).
36. Significant responses are defined as those that inhibited more than 50% of
radioligand binding. In the case of VU0431316, no inhibition greater than 23%
was observed.
Committee. Average plasma concentrations: 3.99
(30 min), and 1.69 (60 min). Average brain concentrations: 6.21
(15 min), 3.69 M (30 min), and 2.80 M (60 min).
l
M
(15 min), 3.00
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