3-Cyano-5-fluoro-N-arylbenzamides as negative allosteric modulators of mGlu5: Identification of easily prepared tool compounds with CNS exposure in rats

Article abstract of DOI:10.1016/j.bmcl.2010.06.064

Development of SAR in a 3-cyano-5-fluoro-N-arylbenzamide series of non-competitive antagonists of mGlu₅ using a functional cell-based assay is described in this Letter. Further characterization of selected potent compounds in in vitro assays designed to measure their metabolic stability and protein binding is also presented. Subsequent evaluation of two new compounds in pharmacokinetic studies using intraperitoneal dosing in rats demonstrated good exposure in both plasma and brain samples.

Full text of DOI:10.1016/j.bmcl.2010.06.064

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4390–4394
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Cyano-5-fluoro-N-arylbenzamides as negative allosteric modulators
of mGlu5: Identification of easily prepared tool compounds with CNS
exposure in rats
Andrew S. Felts a,b, Stacey R. Lindsley a,b, Jeffrey P. Lamb a,b, Alice L. Rodriguez a,b, Usha N. Menon a,b
,
Satyawan Jadhav a,b, Carrie K. Jones a,b,c, P. Jeffrey Conn a,b, Craig W. Lindsley a,b,d, Kyle A. Emmitte a,b,
*
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Vanderbilt Program in Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Tennesse Valley Healthcare System, U.S. Department of Veterans Affairs, Nashville, TN, 37212, USA
d Department of Chemistry, Vanderbilt University, Nashville, TN 37232, 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 a 3-cyano-5-fluoro-N-arylbenzamide series of non-competitive antagonists of
mGlu5 using a functional cell-based assay is described in this Letter. Further characterization of selected
potent compounds in in vitro assays designed to measure their metabolic stability and protein binding is
also presented. Subsequent evaluation of two new compounds in pharmacokinetic studies using intra-
peritoneal dosing in rats demonstrated good exposure in both plasma and brain samples.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 10 May 2010
Revised 8 June 2010
Accepted 10 June 2010
Available online 15 June 2010
Keywords:
mGlu5
GPCR
Allosteric modulator
CNS
GERD
Fragile X syndrome
Addiction
PD-LID
Glutamate is the major excitatory transmitter in the mammalian
CNS, exerting its effects through both ionotropic and metabotropic
glutamate receptors. The metabotropic glutamate receptors
(mGlus) belong to family C of the G-protein-coupled receptors
(GPCRs). The eight mGlus discovered to date have been further di-
vided according to their structure, preferred signal transduction
mechanisms and pharmacology (Group I: mGlu1 and mGlu5; Group
II: mGlu2 and mGlu3; Group III: mGlu4, mGlu6, mGlu7, and mGlu8).1
Selectively targeting a specific mGlu through the use of an orthoster-
ic ligand can be difficult due to the highly conserved nature of that
binding site. A strategy that has proven effective for achieving selec-
tivity has been the design and use of allosteric modulators of the
target.2
strated efficacy in numerous preclinical models of disease,
including pain,5 anxiety,6 gastroesophageal reflux disease (GERD),7
and fragile X syndrome.8 In addition, recent extensive work with
these compounds has established their utility in numerous animal
models of drug addiction. Attenuation of various cocaine seeking
behaviors in mice,9 rats,10 and squirrel monkeys11 has been re-
ported with these compounds. Further work established their effi-
cacy in animal models with other drugs of abuse, including
nicotine,10f morphine,12 methamphetamine,13 and alcohol.14
Recent years have seen growing clinical evidence of the poten-
tial utility for antagonists of mGlu5. Addex Pharmaceuticals has
disclosed positive data from phase II clinical studies with the
mGlu5 NAM ADX10059 in GERD15 and acute migraine.16 FRAXA Re-
search Foundation and Neuropharm have been exploring the po-
tential of fenobam in treating fragile X syndrome and early
results from these studies in patients have been positive.17 Novar-
tis has reported on efforts with their mGlu5 antagonist AFQ056 di-
rected toward identification of the first approved treatment for
Parkinson’s disease levodopa-induced dyskinesia (PD-LID), which
is a complication that arises following dopamine-replacement
The non-competitive mGlu5 antagonists, also known as nega-
tive allosteric modulators (NAMs), 2-methyl-6-(phenylethynyl)
pyridine (MPEP)3 and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyri-
dine (MTEP)4 are important tool compounds and have demon-
* Corresponding author. Tel.: +1 615 936 8401; fax: +1 615 322 8577.
E-mail address: kyle.a.emmitte@Vanderbilt.edu (K.A. Emmitte).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.064

A. S. Felts et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4390–4394
4391
therapy.18 The link between mGlu5 antagonism and PD-LID was
Table 2
Heteroaryl amides
further bolstered by Addex’s recent communication describing
the efficacy of both ADX10059 as well as their second generation
mGlu5 antagonist ADX48621 in a non-human primate model of
PD-LID.19,20
Such preclinical and now clinical validation of mGlu5 antago-
nists makes it an attractive area for further research. We have been
interested in the identification of new chemotypes for the design of
mGlu5 non-competitive antagonists and have recently reported
some of the results from this effort.21 This previously described
work was based on the development of hits identified using a func-
tional cell-based high-throughput screen. We have also focused a
portion of our mGlu5 NAM effort on rational design and scaffold
hopping approaches. One such area centered on the development
of SAR in a 3-cyano-5-fluoro-N-arylbenzamide series and is the
subject of this Letter.
% Glu maxa,b
a
Entry
1
R
mGlu5 IC50 (nM)
3010 510
>30,000
5.4 0.6
2
3
—
—
>30,000
Numerous research groups have devoted significant effort to-
ward the discovery of new and improved mGlu5 NAMs in recent
years.22 An examination of some of the primary literature describ-
ing the SAR of various mGlu5 NAM chemotypes revealed some
common structural features. One such feature was the presence
of a 3-cyano-5-fluorophenyl ring in several of the most potent ana-
logs across multiple chemical series (Table 1). We developed a
chemical plan in order to build around this common structural mo-
tif. Significant effort has been detailed by the aforementioned re-
search groups around the phenyl portion of their respective
templates. Our plan was to fix this phenyl ring in the form of a
3-cyano-5-fluorobenzamide in order to expand the SAR around
the amine portion of such a template. One of the advantages of
such an approach was that analogs could be prepared in a single
step through the coupling of commercially available 3-cyano-5-
fluorobenzoic acid with amines under standard conditions. Using
such an approach with a high-throughput preparative LC/MS sys-
tem27 allowed for the rapid generation of libraries.28
4
5
6
>10,000c
>10,000c
>30,000
37.4 0.8
46.7 1.4
—
7
8
65 23
59 14
1.2 0.7
1.0 0.3
a
Calcium mobilization mGlu5 assay; values are average of n P 3.
Amplitude of response in the presence of 30
of maximal response (100
b
l
M test compound as a percentage
lM glutamate); average of n P 3.
c
CRC does not plateau.
Our initial focus was the evaluation of new heteroaryl amines
(Table 2). Compounds were evaluated in our functional assay, which
measures the ability of the compound to block the mobilization of
derivative 1 was moderately potent. Thiazole 4 and pyrazole 5 were
weak antagonists, while isoxazole 6 was inactive. Appendage of a 6-
methyl group (7) onto 1 yielded a near 50-fold improvement in po-
tency.30 A similar modification resulted in an even more dramatic
improvement in the case of 4-methylthiazole 8, which is more than
150 times potent than 4. Such extreme shifts in potency due to rela-
tively minorstructural modificationsare quite commonwhenwork-
ing with allosteric modulators of GPCRs.
calcium by glutamate in HEK293A cells expressing rat mGlu5.29
A
survey of pyridyl amines (1–3) revealed that only 2-aminopyridine
Table 1
mGlu5 NAMs with a common 3-cyano-5-fluorophenyl ring
Organization
Merck & Co.
Structure
Reference
23
Having observed the dramatic impact of substitution on po-
tency, we decided to explore this further in the context of pyridine
1 and thiazole 4 (Table 3). Methyl substitution at the 3-position (9)
of the pyridine ring produced an inactive compound, while the 4-
methyl analog 10 was a weak antagonist. Moreover, 4,6-dimethyl
analog 11 also lacked potency, indicating that addition of a 6-
methyl group to 10 failed to rescue activity. Choosing to investi-
gate alternative substituents at the 6-position, we found that 6-
chloropyridine 12 and 6-ethylpyridine 15 were only slightly less
potent than 7. While 6-trifluoromethylpyridine 13 was only a weak
antagonist, 6-methoxypyridine 14 was a moderate to weak partial
antagonist. In the case of such compounds, the CRC clearly plateaus
at approximately 20% of the glutamate maximum. A similar phe-
nomenon was observed in the case of 5-methylthiazole 16. Such
partial antagonists have been noted and characterized in other
mGlu5 NAM chemotypes.31 4,5-Dimethylthiazole 17 was a full
antagonist, albeit 10-fold less potent than 8. 4-Cyclopropylthiazole
18 demonstrated a 15-fold reduction in potency compared to 8;
conversely, the loss of potency was even more severe in the case
of 4-tert-butylthiazole 19.
National Institute on Drug
Abuse
24
25
26
Pfizer
Merck & Co.
We were also interested in understanding the tolerance for
amides without heteroaryl groups and turned our attention to that

4392
A. S. Felts et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4390–4394
Table 3
Substituted heteroaryl amides
of the heteroaryl amides, the thorough evaluation of substituted
phenyl amides was a logical next step.
An iterative examination of all positions of the phenyl ring with
small substituents of varying electronic character quickly estab-
lished some clear SAR trends (Table 4). First, substitution at the
2- and 4-positions of the phenyl ring led to reductions in potency
relative to 22. Second, substitution at the 3-position with a chloro
(27) or methyl (30) group improved potency by more than 40-fold.
3-Fluoro (24) substitution yielded a moderately potent partial
antagonist, while 3-methoxy (33) substitution was disfavored.
Having established a preference for substitution at the 3-position,
we focused on that location for additional SAR development. Both
trifluoromethyl (35) and cyano (36) analogs were potent antago-
nists, although less effective than 27 and 30. While a terminal al-
kyne (37) group gave a compound similar in potency to 35 and
36, larger alkyl groups such as ethyl (38) and tert-butyl (39) were
not well tolerated. Introduction of a polar methylsulfone (40)
group was also not tolerated. In order to understand the potential
options that would be available for future optimization of the met-
abolic stability of compounds within this series, we decided to fix
the 3-chloro substituent and examine the effects of fluorination at
all other positions on the phenyl ring. Fluorination at the 2-posi-
tion (41) decreased potency over sevenfold relative to 27. Interest-
ingly, 4-fluoro regioisomer 42 demonstrated near identical potency
to 41, but was a clear partial antagonist. The remaining analogs 43
and 44 were also partial antagonists; however, their potency was
reduced relative to 42.
a
Entry
Series
R
mGlu5 IC50 (nM)
% Glu maxa,b
7
9
A
A
A
A
A
A
A
A
B
B
B
B
B
6-Me
3-Me
4-Me
4,6-di-Me
6-Cl
6-CF3
6-OMe
6-Et
4-Me
5-Me
65 23
1.2 0.7
—
38.6 3.1
—
2.2 0.2
19.6 2.8
17.3 0.4
1.7 0.5
1.0 0.3
17.5 6.8
2.4 0.4
1.5 0.5
29.7 9.3
>30,000
10
11
12
13
14
15
8
16
17
18
19
>10,000c
>30,000
250 40
>10,000c
1510 215
505 145
59 14
863 149
594 102
900 269
>10,000c
4,5-Di-Me
4-cyc-Pr
4-tert-Bu
a
Calcium mobilization mGlu5 assay; values are average of n P 3.
b
Amplitude of response in the presence of 30
of maximal response (100
l
M test compound as a percentage
lM glutamate); average of n P 3.
c
CRC does not plateau.
area next (Table 4). A survey of several simple cylcoalkyl amides
yielded only inactive compounds such as 20. Only adamantyl
amide 21 was a weak antagonist. More interesting was phenyl
amide 22, which was also a relatively weak antagonist. Still, given
the aforementioned impact observed with substitution in the case
Having some compounds with excellent potency in our func-
tional assay, we decided to further evaluate them in a radioligand
binding affinity assay measuring the ability of compound to dis-
place [3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine,
a
close
structural analog of MPEP (Table 5).32 The binding affinity Ki values
for these compounds were generally in good agreement (within
fivefold) with their potency in the functional assay. The affinity
data was also consistent with the allosteric nature of these ligands
and confirms their interaction with the MPEP binding site. The
same four compounds were also tested for their activity in
mGlu1–4 and mGlu7–8 assays and were found to be inactive when
Table 4
Alkyl and phenyl amide SAR
tested at 10 lM against those receptors.
In addition to the binding assays, the same four compounds
were evaluated for their metabolic stability33 and propensity to
bind plasma proteins34 (Table 6). Only thiazole 8 demonstrated
good stability in both rat and human liver microsomes. The
remaining compounds were notably less stable in human liver
a
Entry
R
mGlu5 IC50 (nM)
% Glu maxa,b
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
Cyclohexyl
Adamantyl
Phenyl
2-Fluorophenyl
3-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
3-Chlorophenyl
>30,000
—
>10,000c
5440 1480
>30,000
34.7 6.2
12.5 4.7
—
22.1 1.1
69.2 2.2
—
0.6 0.2
—
—
2.1 0.2
—
—
35.5 5.6
—
2160 35
>10,000c
>30,000
45 21
>30,000
>30,000
122 29
>30,000
Table 5
Binding affinity of selected compounds
a
Compound
mGlu5 IC50 (nM)
mGlu5 Kib (nM)
4-Chlorophenyl
2-Methylphenyl
3-Methylphenyl
4-Methylphenyl
2-Methoxyphenyl
3-Methoxyphenyl
4-Methoxyphenyl
3-(Trifluoromethyl)phenyl
3-Cyanophenyl
7
8
27
30
65
59
45
157
102
206
100
>30,000
122
>10,000c
>30,000
a
IC50 values are an average of n P 3.
Ki values are an average of n = 2.
b
543 60
489 70
331 70
4940 1250
>30,000
0.9 0.3
1.4 0.3
2.3 0.3
2.4 0.2
—
3-Ethynylphenyl
3-Ethylphenyl
Table 6
In vitro DMPK
3-tert-Butylphenyl
3-(Methylsulfonyl)phenyl
3-Chloro-2-fluorophenyl
3-Chloro-4-fluorophenyl
3-Chloro-5-fluorophenyl
5-Chloro-2-fluorophenyl
Compound
Liver microsomesa
Plasma protein bindingb
>30,000
—
347 80
377 43
1830 446
3780 488
2.5 0.5
43.0 8.0
38.5 5.2
29.8 6.6
Rat
Human
Rat
Human
7
8
27
30
46.5
89.9
96.8
69.7
7.0
70.3
18.0
19.5
84.0
92.4
93.7
95.3
89.9
98.4
97.1
98.3
a
Calcium mobilization mGlu5 assay; values are average of n P 3.
b
Amplitude of response in the presence of 30
lM test compound as a percentage
a
Liver microsomes expressed as percent parent remaining at t = 15 min.
Plasma protein binding expressed as percent bound.
of maximal response (100
lM glutamate); average of n P 3.
b
c
CRC does not plateau.

A. S. Felts et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4390–4394
4393
Table 7
Chojnacka-Wójcik, E.; Nowak, G.; Cosford, N. D. P.; Pilc, A. Neuropharmacology
2004, 47, 342; (d) (d) Spooren, W. P. J. M.; Vassout, A.; Neijt, H. C.; Kuhn, R.;
Gasparini, F.; Roux, S.; Porsolt, R. D.; Gentsch, C. J. Pharmacol. Exp. Ther. 2000,
295, 1267.
PK of 8 and 27 following IP dosing in rats (10 mg/kg)
8
27
7. (a) Jensen, J.; Lehmann, A.; Uvebrant, A.; Carlsson, A.; Jerndal, G.; Nilsson, K.;
Frisby, C.; Blackshaw, L. A.; Mattsson, J. P. Eur. J. Pharmacol. 2005, 519, 154; (b)
Frisby, C. L.; Mattsson, J. P.; Jensen, J. M.; Lehmann, A.; Dent, J.; Blackshaw, L. A.
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8. (a) de Vrij, F. M. S.; Levenga, J.; van der Linde, H. C.; Koekkoek, S. K.; De Zeeuw,
C. I.; Nelson, D. L.; Oostra, B. A.; Willemsen, R. Neurobiol. Dis. 2008, 31, 127; (b)
Yan, Q. J.; Rammal, M.; Tranfaglia, M.; Bauchwitz, R. P. Neuropharmacology
2005, 49, 1053.
Plasma
Brain
Plasma
Brain
Cmax
Tmax (h)
AUC ( g h/mL or
(
l
g/mL or
l
g/g)
7.65
0.25
17.82
4.1:1
28.77
0.25
72.96
0.97
0.25
1.20
1.4:1
1.09
0.25
1.71
l
l
g h/g)a
Brain to plasma ratio
a
AUC measured from 0 to 4 h.
9. (a) McGeehan, A. J.; Olive, M. F. Synapse 2003, 47, 240; (b) Chiamulera, C.;
Epping-Jordan, M. P.; Zocchi, A.; Marcon, C.; Cottiny, C.; Tacconi, S.; Corsi, M.;
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microsomes than in rat liver microsomes. Still, the stability of 8
and 27 in rat liver microsomes was supportive of their further
study. Free fraction was greatest with pyridine 7, although free
fraction in rat plasma proteins was considerable with the other
three compounds. All compounds were more highly bound to hu-
man plasma proteins than to rat plasma proteins.
Evaluation of the in vitro DMPK data indicated that 8 and 27
would both be potentially interesting compounds for evaluation
in vivo. As such, both compounds were studied in rat PK experi-
ments using intraperitoneal dosing (Table 7).35 Exposure of 27
was good in both plasma and brain with a brain to plasma ratio
greater than 1 to 1. Impressive exposure was observed with 8,
which showed excellent levels in both plasma and particularly
brain. In fact, the brain to plasma ratio for 8 was greater than 4
to 1.
In summary, we have discovered and characterized two new
mGlu5 NAM in vivo tool compounds using a rational drug design
approach based on common features of known antagonists. Com-
pounds 8 and 27 potently inhibited the mobilization of calcium
by an EC80 concentration of glutamate in HEK293A cells expressing
rat mGlu5. Their interaction with the known allosteric binding site
was confirmed with a radioligand binding assay, and selectivity
over other mGlus was established. Both compounds can be pre-
pared in a single, simple synthetic step from inexpensive, readily
available starting materials. Furthermore, these compounds are
distinct from the 1,2-diarylalkyne chemotype that has been em-
ployed in the bulk of published preclinical in vivo studies to date.
Our current plans include evaluation of these compounds in vari-
ous rat models of diseases relevant to mGlu5 and will be the sub-
ject of future communications.
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Press Release (2009) November 24.
21. (a) Felts, A. S.; Saleh, S. A.; Le, U.; Rodriguez, A. L.; Weaver, C. D.; Conn, P. J.;
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Acknowledgments
22. (a) Lindsley, C. W.; Emmitte, K. A. Curr. Opin. Drug Discov. Dev. 2009, 12, 446;
(b) Gasparini, F.; Bilbe, G.; Gomez-Mancilla, B.; Spooren, W. Curr. Opin. Drug
Discov. Dev. 2008, 11, 655; (c) Jaeschke, G.; Wettstein, J. G.; Nordquist, R. E.;
Spooren, W. Expert Opin. Ther. Pat. 2008, 18, 123.
23. Hamill, T. G.; Krause, S.; Ryan, C.; Bonnefous, C.; Govek, S.; Seiders, T. J.;
Cosford, N. D. P.; Roppe, J.; Kamenecka, T.; Patel, S.; Gibson, R. E.; Sanabria, S.;
Riffel, K.; Eng, W.; King, C.; Yang, X.; Green, M. D.; O’Malley, S. S.; Hargreaves,
R.; Burns, H. D. Synapse 2005, 56, 205.
We thank NIDA (RO1 DA023947-01) and Seaside Therapeutics
(VUMC33842) for their support of our programs in the develop-
ment of non-competitive antagonist of mGlu5. Matt Mulder, Chris
Denicola, and Sichen Chang are also thanked for the purification
of compounds using the mass-directed HPLC system.
24. Kulkarni, S. S.; Zou, M.-F.; Cao, J.; Deschamps, J. R.; Rodriguez, A. L.; Conn, P. J.;
Newman, A. H. J. Med. Chem. 2009, 52, 3563.
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28. Prior to biological testing, all compounds were analyzed by LCMS and
determined to be P95% pure, and selected compounds were further
characterized by proton NMR. For a large scale synthesis, compounds can
also be purified via flash chromatography on silica gel. For example, synthesis
and characterization of 8 was as follows: 2-amino-4-methylthiazole (5.00 g,
44 mmol), 3-cyano-5-fluorobenzoic acid (7.23 g, 44 mmol), 1-ethyl-3-(3-
dimethylaminopropyl)
carbodiimide
(8.4 g,
44 mmol)
and
4-
dimethylaminopyridine (0.535 g, 4.4 mmol) were dissolved in CH2Cl2
(100 ml) and stirred at rt overnight. Water was added and the layers were
separated. The organic layer was dried (MgSO4), filtered, and concentrated in

4394
A. S. Felts et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4390–4394
vacuo. Purification by flash chromatography on silica gel afforded 10.18 g (89%)
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. 31a.
30. Compound 7 has also been disclosed by the group at NIDA (Ref. 24). Functional
activity in our assay is in good agreement with their published data.
31. (a) Sharma, S.; Rodriguez, A. L.; Conn, P. J.; Lindsley, C. W. Bioorg. Med. Chem.
Lett. 2008, 18, 4098; (b) Rodriguez, A. L.; Nong, Y.; Sekaran, N. K.; Alagille, D.;
Tamagnan, G. D.; Conn, P. J. Mol. Pharmacol. 2005, 68, 1793.
of 8 as a white solid: 1H NMR (400 MHz, DMSO-d6) d 8.36 (s, 1H), 8.20 (d,
J = 9.3 Hz, 1H), 8.12 (d, J = 8.12 Hz, 1H), 6.84 (s, 1H), 2.30 (s, 3H); HRMS calcd
for C12H8FN3OS (M+H+), found 262.0450. Synthesis and characterization of 27
was as follows: 3-chloroaniline (5.00 g, 39 mmol), 3-cyano-5-fluorobenzoic
acid (7.23 g, 41 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(7.89 g, 41 mmol) and 4-dimethylaminopyridine (503 mg, 4.1 mmol) were
dissolved in CH2Cl2 (300 ml) and stirred at rt overnight. Water was added and
the layers were separated. The organic layer was dried (MgSO4), filtered, and
concentrated in vacuo. Purification by flash chromatography on silica gel
afforded 9.80 g (91%) of the 27 as a white solid: 1H NMR (400 MHz, DMSO-d6) d
10.58 (s, 1H), 8.27 (t, J = 1.2 Hz, 1H), 8.14–8.08 (m, 2H), 7.92 (t, J = 2.0 Hz, 1H),
7.68–7.66 (m, 1H), 7.42 (t, J = 8.1 Hz, 1H), 7.21–7.19 (m, 1H); HRMS calcd for
32. 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. For a detailed
description of the radioligand binding assay see Ref. 31a.
33. The test compounds (1 lM) were incubated for 15 min at 37 °C with shaking,
in medium containing human/rat liver microsomes, phosphate buffer, and the
cofactor NADPH.
C
14H8ClFN2O (M+H+), found 275.0385.
29. HEK293A cells expressing rat mGlu5 were cultured and plated as previously
described. The cells were loaded with a Ca2+-sensitive fluorescent dye and the
plates were washed and placed in the Functional Drug Screening System
(Hamamatsu). Test compound was 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
34. A 96-well rapid equilibrium dialysis (RED) apparatus (Thermo Scientific) was
used to determine the free fraction in rat and human plasma for compound.
35. Compounds were dosed at 10 mg/kg intraperitoneally as microsuspensions in
10% tween 80 in male Sprague Dawley rats. Brain and plasma samples were
collected at 0.25, 0.5, 1, 2, and 4 h post dose. IP dosing was chosen as a
convenient route to help maximize exposure.