6-Aryl-3-pyrrolidinylpyridines as mGlu5 receptor negative allosteric modulators

Article abstract of DOI:10.1016/j.bmcl.2011.06.014

A series of 6-aryl-3-pyrrolidinylpyridine analogs was explored as structurally novel negative allosteric modulators of the mGlu5 receptor lacking an alkyne or oxadiazole moiety. Compounds in this series were characterized by tractable SAR, good in vitro potencies and brain penetration in rodents.

Full text of DOI:10.1016/j.bmcl.2011.06.014

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4891–4899
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
6-Aryl-3-pyrrolidinylpyridines as mGlu5 receptor negative
allosteric modulators
Jesse M. Weiss a, Hermogenes N. Jimenez a, Guiying Li a, , Myriam April a, Michelle A. Uberti b,
⇑
Maria D. Bacolod a, Robb M. Brodbeck b, Darío Doller a,
⇑
a Chemical & Pharmacokinetic Sciences, Lundbeck Research USA, 215 College Road, Paramus, NJ 07652, USA
b Synaptic Transmission Disease Biology Unit, Lundbeck Research USA, 215 College Road, Paramus, NJ 07652, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 6-aryl-3-pyrrolidinylpyridine analogs was explored as structurally novel negative allosteric
modulators of the mGlu5 receptor lacking an alkyne or oxadiazole moiety. Compounds in this series were
characterized by tractable SAR, good in vitro potencies and brain penetration in rodents.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 24 May 2011
Revised 3 June 2011
Accepted 6 June 2011
Available online 17 June 2011
Keywords:
mGlu5 NAM
Metabotropic glutamate receptors
GPCR
mGluR
CNS drug discovery
L
-Glutamic acid (1) is the major excitatory neurotransmitter in
generating seven a-helical transmembrane domains. GPCRs have
the mammalian central nervous system and is responsible for a
variety of physiological effects acting through a number of recep-
tors.1 These receptors are either ionotropic (iGluRs) or metabotropic
glutamate receptors (mGluRs). iGluRs are ion channels, and have
been divided in three classes based on their selective interactions
with three different ligands: N-methyl-D-aspartic acid (NMDA, 2),
kainic acid (KA, 3) and (S)-2-amino-3-(-3-hydroxy-5-methyl-4-
isoxazolyl)propionic acid (AMPA, 4).
been classified into six distinct classes, identified as A–F.2 How-
ever, from a drug discovery perspective, classes A, B and C have
captured most of the efforts aimed at finding new therapeutic
agents.3
mGluRs belong to class C of GPCRs, together with calcium-
sensing receptors, GABAB receptors, and sweet taste receptors,
among a variety of others. Class C GPCRs are characterized by a
unique molecular pharmacology.4 The so-called orthosteric ligands
HO
CO2H
CO2H
HO2C
CO2H
NH2
CO2H
NHMe
N
O
HO2C
CO2H
NH2
NH
L-Glutamic acid (1)
N-methyl-D-aspartic acid
(NMDA, 2)
Kainic acid (KA, 3)
AMPA (4)
mGluRs, on the other hand, are coupled to a variety of second
messenger systems via G proteins, thus belonging to the G-protein
coupled receptor family (GPCRs). The main structural feature of all
known GPCRs is their extensive amino acid sequence similarities,
of mGlu receptors bind in the extracellular amino-terminal do-
main (Fig. 1). These ligands often suffer from poor selectivity
among mGlu receptor subtypes due to a relatively highly con-
served binding region across the receptor family.5 In the past dec-
ade a number of allosteric sites, those not native to the glutamate
binding site, have been identified for several of the mGlu recep-
tors. In most cases, allosteric binding sites are thought to be in
⇑
Corresponding authors. Tel.: +1 201 350 0341; fax: +1 201 261 0623.
E-mail address: dado@Lundbeck.com (D. Doller).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.014

4892
J. M. Weiss et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4891–4899
the seven-transmembrane spanning region of the mGlu receptors,
and designing ligands that target this region is a strategy that has
been demonstrated to produce selective receptor modulation.6
To date, eight different mGluR subtypes (mGlu1-8) have been
cloned and subsequently expressed in various cell lines. The
mGluRs are further subdivided into three groups based on se-
quence homology, second messenger coupling and pharmacology:
Group 1 (mGlu1 and 5), Group 2 (mGlu2 and 3) and Group 3
(mGlu4, 6, 7 and 8). mGlu5 receptors are principally localized post-
synaptically and couple primarily via activation of members of the
Gq/11 family of G-proteins to phospholipase C-b and, hence, have
been reported to lead to elevation of intracellular calcium ion
levels.3
A number of reports in the literature reviewed the potential
clinical use of novel mGlu5 allosteric modulators.6,7 In the late sev-
enties, fenobam (5) demonstrated clinical efficacy as an anxiolytic
operating through a then unknown, non-benzodiazepine mecha-
nism. Eventually, it was found to be a potent mGlu5 receptor
NAM.8 Although the compound did not progress into late stage tri-
als due to poor pharmacokinetics, the efficacy demonstrated in a
clinical setting propelled investigations seeking novel NAMs of
the mGlu5 receptor. Research intensified upon discovery of tool
compounds like MPEP (6) or MTEP (7), which have shown efficacy
In search of novel chemotypes lacking the alkyne moiety as the
central feature, the 6-aryl-3-pyrrolidinylpyridine series was inves-
tigated as a potential starting point (scaffold A, 11). Conceptually,
this scaffold is related to recently reported lead compounds (e.g.,
1215a and 1315b) and evolved through a scaffold hopping exercise
(Fig. 2). We reasoned that a potential advantage of the pyridine
ring over the oxadiazole system is the possibility of adding substit-
uents in the pyridine ring as a strategy leading to optimized drug
candidates through potency improvements or by modulation of
physicochemical/biochemical attributes. A computational overlay
showed that the optimal Ar and/or Y substituents might not be
identical, since they project differently into space (Fig. 3).16
Based on prior SAR knowledge for mGlu5 receptor NAMs,17 the
initial focus was centered on 3-chlorophenyl derivatives. Scheme 1
depicts the reaction sequence leading to analogs without substitu-
tion on C-4 of the pyridine core. Starting with 6-chloropyridine-3-
carboxaldehyde 14a, the procedure reported by Stetter et al. for the
addition of aldehydes to activated double bonds yielded ketoester
15.18 Reductive amination followed by spontaneous cyclization
afforded lactam 16, which was submitted to a standard Suzuki cou-
pling to yield compound 17. Reduction to pyrrolidine 18 was
accomplished in good yield with BH3ꢀTHF complex. Acylation with
an appropriate carboxylic acid in the presence of EDCI and DMAP
provided compounds 11a–11f (Table 1).19
in a variety of preclinical models, including pain, anxiety, L-dopa
induced dyskinesia (LID) and Fragile X Syndrome.9 In addition, a
number of mGlu5 NAMs have entered clinical studies and proven
efficacious in indications such as migraine,10 gastro-esophageal re-
flux disease (GERD),11 Fragile X syndrome,12 and LID.13 Many of
these compounds [e.g., ADX10059 (8), ADX48621 (9), AFQ056
(10)] are characterized by a disubstituted alkyne as a key compo-
nent of the structural motif. The safety and toxicological properties
of this moiety have been questioned for a number of reasons, not
the least of which was the termination of the clinical studies of
ADX10059 for chronic indications due to liver function abnormal-
ities.14 This and our interest in this target and its potential thera-
peutic value motivated us to explore novel mGlu5 chemical
space lacking the alkyne functionality.
Scheme 2 shows the reaction sequence leading to analogs with
4-methyl or 4-methoxy substitution in the pyridine ring. To that
end, the appropriately substituted 6-chloropyridine-3-carboxalde-
hyde 14b or 14c (R1 = Me or OMe, respectively) underwent a Suzu-
ki coupling to establish the biaryl functionality of the scaffold. The
aldehyde group in 19a or 19b was functionalized to the homoallyl
azide 21a or 21b, respectively, via a Grignard addition of allylmag-
nesium bromide, followed by reactions of alcohols 20a or 20b with
diphenylphosphoryl azide (DPPA). Treatment of intermediate
azides 21a or 21b with dicyclohexylborane (prepared in situ) affor-
ded the pyrrolidine 22a or 22b, respectively, in very good overall
Me
H
N
Cl
N
N
N
N
O
HN
S
N
O
Fenobam (5)
MPEP (6)
MTEP (7)
F
N
HO
O
H
N
N
H
OMe
N
F
N
NH2
ADX10059 (8, Addex)
ADX48621 (9, Addex)
AFQ-056 (10, Novartis)
S
R1
O
O
N
N
N
N
N
N
N
Cl
O
R2
O
N
O
S
Cl
Cl
Scaffold A (11)
(12, Gedeon Richter15a
)
(13, Vanderbilt Univ. 15b
)

J. M. Weiss et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4891–4899
4893
O
O
= Orthosteric
= Allosteric
O
A
A
A
A
O
Class A
Class B
Class C
Figure 1. Schematic representations of the architecture of class A, B and C GPCRs, indicating the spatial relationship between the allosteric and the orthosteric binding sites.
R1
being ca. 10-fold shifted (vide infra). Testing of a number of cyclic
N
Ar'
Y'
amide analogs indicated that ring size of 4 or 5 was preferred over
6, with the pyrazine amide 11e being the most potent compound
among the six-membered heteroaryl amides (data not shown).
Among five-membered heterocyclic rings, the 2-thiazolyl or 5-
thiazolyl moieties were particularly potent (11b and 11c, respec-
tively). Polar surface areas (PSA) and c Log P values were consistent
with those required for optimal design of CNS drugs.21 However,
lipophilic ligand efficiency (LLE) values were relatively low, indi-
cating that further potency increases were needed to afford im-
proved compounds. This was accomplished by substitution on
the pyridine ring.
Ar
Y
Ar"
Y"
N
O
N
Alkyne Core
Oxadiazole Core
Pyridine Core
Figure 2. Series of scaffold hopping operations leading to series A.
A methyl group on the 4-position of the pyridine ring led to a
three- to four-fold enhancement in binding affinity and 4–13-fold
increase in functional potency (11h vs 11c, and 11i vs 11d). A 4-
methoxy substituent had 5–30-fold increase in binding affinity
(11s vs 11c, and 11t vs 11f), but changes in functional potency were
modest (one to three-fold for the same two pairs of compounds).
There is precedent for such disconnects between functional and
binding affinity for mGlu5 receptor allosteric modulators.15c These
enhancements in binding affinity may reflect increases in basicity
from the 4-unsubstituted core (pKa 3.5) to the more basic 4-methyl
(pKa 4.1) and 4-methoxy (pKa 5.0) derivatives (Table 1). The 4-
hydroxypyridine 11u and the tertiary alcohol 11v were less potent.
Five- and six-membered heterocyclic amides, as well as cyclobutyl
carboxamides (11p and 11t) were preferred substituents. As previ-
ously noted, among five-membered heterocycles, both 2- and 5-
thiazolyl amides were preferred substituents. Alicyclic five-mem-
bered ring systems were explored, but were not tolerated (e.g.,
11q–r), and six-membered heteroaromatic rings were less potent,
with the notable exception of the 2-pyrazinyl derivative 11j.
In spite of having good binding and functional potency at the
target, physicochemical parameters within the optimal range for
CNS drug candidates and, in some cases, LLE values approaching
5, compounds showed high human microsomal intrinsic clearance
and, in some cases, modest aqueous solubility. Thus, our optimiza-
tion efforts turned to other regions in the scaffold as the structure
activity requirements at the amide region were apparently tight.
ca. 4Å
Figure 3. Low energy conformations overlay for pyridine and oxadiazole scaffolds.
Cyclobutylamide analogs are exemplified. The 3-chlorophenyl groups are projected
into slightly different directions, and the chlorine atoms are separated by ca. 4 Å.
yield. Finally, amide formation with the corresponding carboxylic
acid afforded the desired final products in scaffold A.
Early SAR studies focused on the effects of modifying the amide
substituent R2, as well as exploiting the potential impact of an R1
group in the 4-position of the pyridine ring on basicity and overall
shape. Functional activities were measured by the ability of the
compound to inhibit calcium mobilization caused by an EC80 con-
centration of glutamate in HEK293 cells expressing the human
mGlu5 receptor.20a Binding affinities were determined via dis-
placement by a compound of the allosteric radioligand [3H]-
MPEP.20b Values for relevant compounds are summarized in Table
1.
Pyridine analogs unsubstituted at the 4-position (11a–11f)
showed excellent functional potency and potent binding, the later
R1
O
a
b
c
OHC
O
N
H
MeO2C
CO2Me
N
Cl
N
Cl
N
Cl
14a (R1=H)
15
16
R1
O
N
H
N
N
d
e
H
Cl
Cl
R2
Cl
N
N
N
O
17
18
11 (Scaffold A)
Scheme 1. Synthesis of analogs in scaffold A (R1 = H). Reagents and conditions: (a) NaCN, DMF, rt, 93%; (b) NH4OAc, NaCNBH3, MeOH, rt, 42%; (c) m-Cl-PhB(OH)2, Pd(PPh3)4,
Na2CO3, DME, water, 70 °C, 73%; (d) BH3ꢀTHF, reflux, 78%; (e) R2CO2H, EDCI, DMAP, DCM, 70–80%.

4894
J. M. Weiss et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4891–4899
Table 1
Structure–activity relationships and physicochemical properties for a series of scaffold A analogs
R1
N
R2
Cl
N
O
Scaffold A
b
Compd
R1
H
R2
c Log P
PSA (Å2)
33
IC50 (nM)
Ki (nM)
LLEa
3.6
hCLint (L/min)
Solubilityc
24
(lM)
pKa
S
S
11a
4.5
8
5
89
3.0
3.8
1.6
—
3.6
11b
11c
11d
H
H
H
3.5
3.5
3.5
46
46
59
140
110
540
4.8
4.5
3.9
35
210
100
—
N
S
9
3.6
3.5
N
O
N
41
N
11e
H
3.1
59
74
540
4.0
3.3
230
3.4
N
11f
H
3.2
3.9
33
46
23
14
360
19
4.4
4.0
—
190
18
—
—
S
11g
Me
5.2
N
S
11h
11i
Me
Me
3.9
4.0
46
59
2
3
30
4.8
4.5
35
38
170
100
4.2
4.2
N
O
N
N
150
11j
Me
3.1
59
10
120
4.9
3.3
230
4.1
N
N
11k
11l
Me
Me
3.1
3.1
59
59
84
180
290
4.0
3.9
5.3
—
170
240
—
—
N
N
110
N
N
11m
11n
Me
Me
4.1
4.1
46
46
140
140
370
540
2.8
2.8
—
—
160
200
—
—
N
N
11o
11p
11q
Me
Me
Me
4.1
3.7
2.0
46
33
67
68
11
180
15
3.1
4.3
3.7
—
200
140
220
—
—
—
5.3
—
1900
7000
SO2
Me
N
11r
Me
4.0
53
10,000
16,000
1.0
—
240
—
O
S
11s
11t
OMe
OMe
3.9
3.6
55
42
8
8
20
12
4.2
4.5
1.7
—
8.7
54
5.0
—
N

J. M. Weiss et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4891–4899
4895
Table 1 (continued)
b
Compd
R1
R2
c Log P
PSA (Å2)
53
IC50 (nM)
680
Ki (nM)
LLEa
2.7
hCLint (L/min)
Solubilityc
210
(l
M)
pKa
11u
OH
3.5
7800
—
—
2.8
—
OH
S
N
11v
3.3
66
2400
13,000
2.3
12
a
b
c
LLE = pIC50-c Log P.
Human hepatic blood flow = 1.5 L/min.23
Kinetic solubility.24
R1
OH R1
R1
OHC
b
c
OHC
a
Cl
Cl
N
N
N
Cl
14b (R=Me)
14c (R=OMe)
19a (R=Me)
19b (R=OMe)
20a (R=Me)
20b (R=OMe)
N3 R1
R1
N
R1
N
N
H
N
e
d
R2
Cl
Cl
Cl
N
O
21a (R=Me)
21b (R=OMe)
22a (R=Me)
22b (R=OMe)
11 (Scaffold A)
Scheme 2. Synthesis of analogs in scaffold A (R1 = Me or OMe). Reagents and conditions: (a) m-Cl-PhB(OH)2, Pd(PPh3)4, Na2CO3, toluene, water, 50%; (b) CH2@CHCH2MgBr,
THF, 70%; (c) DPPA, DBU, toluene, 90 °C, 85%; (d) BH3–DMS, cyclohexene, THF, 75%; (e) R2CO2H, EDCI, DMAP, DCM, 30–70%.
The effect of varying the aryl group on position 6 of the pyridine
core on analogs within scaffold A was investigated next, and the re-
sults are shown in Table 2. At the outset, our goal was to identify a
less lipophilic substituent than the 3-chlorophenyl group, seeking
to improve the overall physicochemical properties in our target
compounds. The only analogs made were those containing the pre-
ferred amides R2 (vide supra). These studies showed that the 3-
chlorophenyl substituent afforded an optimal balance between
in vitro potency at the target and physicochemical properties. A
simple switch to the fluoro analog showed a 10-fold loss of potency
(23a and 23b). The 3-chloro-5-fluorophenyl disubstituted analogs
23e and 23f were about 20 times less potent than the 3-chloro-
phenyl, but five times more potent than the 3,5-difluorophenyl
substituent, further illustrating the preference for the chloro group.
Aqueous solubilities were not improved comparing with the 3-
chlorophenyl analogs. A number of heterocyclic replacements were
explored. The most potent analogs obtained incorporated a 3-
methyl-2-pyridyl group, albeit these were still 10-fold weaker than
the 3-chlorophenyl. While LLE values approached 5 for compounds
23g and 23h, mainly due to their low c Log P, these compounds had
relatively weak potencies.
Seeking to further optimize these analogs in terms of physico-
chemical properties and improving microsomal stability, we ex-
plored the effects of introducing fluorine or hydroxy substituents
on the pyrrolidine ring, while using optimal fragments on the other
regions. These hydroxy- and fluoro-pyrrolidine derivatives were
prepared via a cyanomorpholine intermediate, an acyl anion equiv-
alent, as shown in Scheme 3.22 The cyanomorpholine was prepared
by reacting the previously described aldehyde 19a (R1 = Me) with
morpholine in the presence of sodium cyanide. This intermediate
24 was alkylated with epibromohydrin yielding the epoxide 25,
which was subsequently treated with 7 N methanolic ammonia
at 60 °C. During the course of this reaction, the ketone intermedi-
ate was liberated and spontaneously cyclized to the pyrroline 26.
Sodium borohydride reduction of 26 followed by amide coupling
produced the hydroxypyrrolidine 27 as a ca. 1:1 mixture of diaste-
reomers. Fluorination of intermediate 27 with DAST followed by
amide coupling gave rise to the fluoropyrrolidine 28, also as a
1:1 mixture of isomers. Amidation of 27 or 28 under standard con-
ditions yielded the final amide analogs 29a–f.
The results obtained with this strategy are shown in Table 3.
Hydroxy substitution rendered compounds 29a–c having c log P
values greater than 1 unit lower than the corresponding unsubsti-
tuted analog, bringing the lipophilicity of these compounds closer
to preferred values for CNS drugs.21 Human microsomal intrinsic
clearance was also improved, albeit at the cost of a 10-fold loss
in potency at the target. Fluoro substituted compounds 29d–f
had c log P values similar to the corresponding unsubstituted ana-
logs and also showed weaker in vitro potencies as mGlu5 NAMs. In
vitro clearance and LLE values did not improve.
In order to gain further insight on the structure–activity rela-
tionships of the compounds made, an examination of the pIC50 ver-
sus pKi was undertaken (Fig. 4). A linear relationship between the
functional and binding potencies of these analogs was observed,
showing that under the assay conditions used for these in vitro
screens the binding affinity Ki was consistently shifted ca. one
log unit to the right (weaker values) compared to IC50 values. High
potency was achieved with compounds having c Log P values in the
3.5–4.5 range. Compounds with LLEs near five were obtained with
c Log P values more appropriate for CNS drugs, albeit their potency
was weak. This indicated the lead optimization within this series
required the need for a compromise between potency and lipophil-
icity, and limited the potential scope of this scaffold.
Based on the argument above and the balanced properties
shown for racemic 11g (Table 1), we selected this compound for
resolution and further in vivo brain and plasma exposure studies

4896
J. M. Weiss et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4891–4899
Table 2
Structure–activity relationships and physicochemical properties for a series of scaffold A analogs
N
R2
Ar
N
O
Compound
Ar
R2
c Log P
PSA (Å2)
46
IC50 (nM)
30
Ki (nM)
LLEa
4.1
Solubilityb
100
(lM)
F
S
23a
3.4
110
N
N
F
F
N
23b
23c
2.6
3.5
59
46
120
570
1100
360
4.3
2.7
52
69
N
S
F
F
F
F
N
N
23d
23e
23f
2.7
4.1
3.3
59
46
59
2000
74
2400
150
3.0
3.0
3.1
77
—
F
S
N
N
Cl
Cl
N
N
390
800
54
S
23g
23h
2.3
1.5
59
72
120
370
840
—
4.6
4.9
—
N
N
N
N
170
a
LLE = pIC50-c Log P.
b
Kinetic solubility.24
O
O
N
CHO
N
c
a
b
O
Cl
Cl
CN
N
CN
Cl
Cl
N
N
19a
24
25
R1
N
HN
OH
R1
d
f
N
Cl
R2
Cl
N
N
N
O
27: R1 = OH
26
29a-f
e
1
28:
R = F
Scheme 3. Synthesis of substituted pyrrolidine analogs. Reagents and conditions: (a) morpholine, NaCN, PTSA, THF, 90%; (b) NaH, epibromohydrin, DMF, 60%; (c) NH3, MeOH,
reflux, 40%; (d) NaBH4, EtOH, H2O, 99%; (e) DAST, DCM, 26–58%; (f) R2CO2H, EDCI, DMAP, DCM, 30–50%.

J. M. Weiss et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4891–4899
4897
Table 3
Structure–activity relationships and physicochemical properties for a series of substituted pyrrolidines
R1
N
R2
Cl
N
O
b
Compd
29a
R1
R2
c Log P
2.7
PSA (A2)
66
IC50 (nM)
80
Ki (nM)
230
LLEa
4.4
hCLint (L/min)
S
N
OH
OH
0.9
—
29b
2.8
53
320
570
3.7
N
29c
OH
1.9
79
320
3000
4.6
1.6
N
S
29d
29e
F
F
3.9
3.7
46
33
94
100
130
3.1
3.2
7.1
—
N
140
N
N
29f
F
3.1
59
120
260
3.8
2.6
a
LLE = pIC50-c Log P.
Human hepatic blood flow = 1.5 L/min.23
b
on the preferred enantiomer, once identified. Upon resolution, we
found that mGlu5 activity resided essentially in one enantiomer,
the absolute configuration of which has been arbitrarily assigned.
The eutomer obtained from the chiral chromatographic separation
(compound 30) showed an eudismic ratio of 46 and was potent in
both binding and functional mGlu5 assays (Ki = 12 nM and
IC50 = 17 nM, respectively), had good aqueous solubility at pH 7.4
eral or central drug exposure profiles to support once daily oral
administration in human. The strategy of exploring alternate im-
proved scaffolds is ongoing.
In summary, while several mGlu5 NAMs have achieved clinical
proof-of-concept for a number of indications, the discovery of no-
vel chemotypes lacking the alkyne functionality remains a chal-
lenge. We discovered a series of 6-aryl-3-pyrrolidinylpyridines as
structurally novel, non-alkyne negative allosteric modulators of
the mGlu5 receptor. Functional and binding potency correlate well
in the in vitro screens used, SAR was found to be tractable, and the
switch to either positive or silent (PAMs or SAMs, respectively)
allosteric modulators reported in other series has not been ob-
served in this chemotype.15b,c At the amide moiety, five-membered
heterocycles are preferred, while the 3-chlorophenyl group ap-
pears to be preferred and difficult to replace. Substitution in the
4-position of the pyridine core with a methyl or methoxy group
yielded compounds with superior in vitro potency, and reasonable
CNS partition profiles and physicochemical properties. Further-
more, the SAR observed in these pyridine derivatives matches
(44 lg/mL), and reasonable passive membrane permeability (PAPP
of 15 ꢁ 10ꢂ6 cm/s, estimated using a PAMPA assay). Experimental
Log D7.4 was 4.3, in line with high rat and human plasma protein
binding (rPPB = 99.5% and hPPB = 99.6%, respectively) and a low
rat brain free fraction (UBBR = 0.7%). A single dose–exposure study
in Sprague–Dawley rats revealed that 1 h after an oral dose of
10 mg/kg (formulated in 20% w/v aqueous b-hydroxypropylcyclod-
extrin), 30 had moderate plasma and brain concentrations, and a
brain/plasma ratio of 0.8. Free drug concentrations of 30 in plasma
and brain (both 1 nM) were similar to its CSF concentration (3 nM),
albeit below the mGlu5 NAM IC50 or Ki values at this oral dose, a
likely consequence of extensive hepatic metabolism.
c
Compd Structure
IC50 (nM) Ki (nM) LLEa Brain (nM) Plasma (nM) CSF (nM) rPPBb (%) rUBBR (%)
N
S
30
17
12
3.9
133
162
3
99.5
0.7
Cl
N
O
N
a
b
c
LLE = pIC50-c Log P.
Rat plasma protein binding.25
Rat brain free fraction.25
Additional exploration of pharmacokinetic properties of multi-
ple derivatives in this series did not afford the appropriate periph-
observations recently reported for related five-membered hetero-
cyclic ring systems (e.g., oxadiazoles and tetrazoles).15a,b Follow

4898
J. M. Weiss et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4891–4899
14. Development of ADX10059 Ended for Long-term Use. http://www.add-
expharma.com/press-releases/press-release-details/article/development-of-ad
x10059-ended-for-long-term-use/ accessed on October 15, 2010.
15. (a) Wágner, G.; Wéber, C.; Nyéki, O.; Nógrádi, K.; Bielik, A.; Molnár, L.; Bobok,
A.; Horváth, A.; Kiss, B.; Kolok, S.; Nagy, J.; Kurkó, D.; Gál, K.; Greiner, I.;
}
Szombathelyi, Z.; Keseru, G. M.; Domány, G. Bioorg. Med. Chem. Lett. 2010, 20,
3737; (b) Lamb, J. P.; Engers, D. W.; Niswender, C. M.; Rodriguez, A. L.; Venable,
D. F.; Conn, P. J.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2011, 21, 2711; (c)
Graven Sams, A.; Mikkelsen, G. K.; Brodbeck, R. M.; Pu, X.; Ritzén, A. Bioorg.
Med. Chem. Lett. 2011. doi:10.1016/j.bmcl.2011.03.103.
16. Geometry optimizations were conducted using ChemBio3D Ultra 12.0,
interfaced to GAMESS, using the HF method, basis set 6–31G, wave function
R-closed shell and Pople exponent, which is distributed by CambridgeSoft of
100 Cambridge Park Drive, Cambridge, MA 02140.
17. Jimenez, H. N.; Li, G.; Doller, D.; Grenon, M.; White, A. D.; Guo, M.; Ma, G. WO
2010011570.
18. Stetter, H.; Schreckenberg, M.; Wiemann, K. Ber. 1976, 109, 541.
19. The structures of all final compounds made were confirmed using low
resolution mass spectrometry. For representative examples of final analogs
within a series, structures were confirmed using nuclear magnetic resonance
(NMR), as well as low resolution mass spectrometry. Purity of all final products
was determined to be >95% based on their LC–MS trace using ELSD or UV
detection in the range of 240–400 nm, as well as at
a single 254 nm
wavelength. Purifications were carried out either on a preparative thin layer
chromatography plate or on a reversed phase liquid chromatography/mass
spectrometry (RP-HPLC/MS) purification system. Flow rate: 100 mL/min.
Mobile phase additive: 48 mM of ammonium formate. Column: InertsilÒ C18,
Figure 4. Representation of pIC50 versus pKi for analogs in Tables 1–3.
30 ꢁ 50 mm, 5
lm particle size.
20. (a) The cDNA for human metabotropic glutamate receptor 5 was a generous
gift from S. Nakanishi (Kyoto University, Kyoto, Japan). The hmGluR5 was
stably expressed in a HEK 293 cell line and grown in Dulbecco’s Modified Eagle
Medium (DMEM) (Invitrogen, Carlsbad, CA) with supplements (10% bovine calf
up efforts expanding the six-membered heterocyclic central ring to
diverse heterocyclic systems will be reported in future
communications.
serum, 4 mM glutamine, 100 units/mL penicillin, 100lg/mL streptomycin and
0.75 mM G1418) at 37 °C, 5% CO2. Twenty-four hours prior to assay, cells were
seeded into 384-well black wall microtiter plates coated with poly-Dlysine.
Just prior to assay, media was aspirated and cells dye-loaded (25
lL/well) with
3
l
M 20 Fluo-4/ 0.01% pluronic acid in assay buffer (Hank’s Balanced Saline
Acknowledgements
Solution (HBSS)): 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, plus
20 mM N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid (HEPES), pH 7.4,
0.1% bovine serum albumin (BSA) and 2.5 mM probenecid) for 1 h in 5% CO2 at
37 °C. After excess dye was discarded, cells were washed in assay buffer and
We are grateful to Drs. Mark Hayward, Qing-Ping Han, Chi
Zhang and Xu Zhang, in the CPS Department, for determinations
of physicochemical properties and the resolution of compound
11g, to Christina L. Bonvicino and Xiaosui Pu for in vitro pharma-
cology determinations and to Dr. Albert J. Robichaud for his sup-
port during this work and for helpful comments to this manuscript.
layered with
a final volume equal to 25 lL/well. Basal fluorescence is
monitored in a fluorometric imaging plate reader (FLIPR, Molecular Devices,
Sunnyvale, CA) with an excitation wavelength of 488 nm and an emission
range of 500–560 nm. Laser excitation energy was adjusted so that basal
fluorescence readings were approximately 10,000 relative fluorescent units.
Cells were stimulated with an EC20 or an EC80 concentration of glutamate in the
presence of a compound to be tested, both diluted in assay buffer, and relative
fluorescent units were measured at defined intervals (exposure = 0.6 s) over a
3 min period at room temperature. Basal readings derived from negative
controls were subtracted from all samples. Maximum change in fluorescence
was calculated for each well. Concentration–response curves derived from the
maximum change in fluorescence were analyzed by nonlinear regression (Hill
equation). A negative modulator can be identified from these concentration–
response curves if a compound produces a concentration dependent inhibition
of the EC80 glutamate response.; (b) Binding assays were performed as
described in [O’Brien, J. A.; et al. Mol Pharmacol. 2003, 64, 731] with slight
modifications. Briefly, after thawing, the membrane homogenates were
resuspended in 50 mM Tris–HCl, 0.9% NaCl binding buffer at pH 7.4 to a final
References and notes
1. Stahl, S. M. Stahl’s Essential Psychopharmacology, 3rd ed.; Cambridge University
press: 2008; Watkins, J. C. Biochem. Soc. Trans. 2000, 28, 297.
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Spedding, M.; Harmar, A. J. Pharmacol. Rev. 2005, 57, 279.
3. G Protein-Coupled Receptors in Drug Discovery. In Taylor & Francis; Lundstrom,
K. H., Chiu, M. L., Eds.; CRC Press, 2006; Behan, D. P.; Chalmers, D. T. Nat. Rev.
Drug Discov. 2002, 1, 599.
4. (a) Schoepp, D. D.; Conn, P. J. Trends Pharmacol. Sci. 1993, 14, 13; (b) Schoepp, D.
D. Neurochem. Int. 1994, 24, 439; (c) Bordi, F.; Ugolini, A. Prog. Neurobiol. 1999,
59, 55.
5. Topiol, S.; Sabio, M.; Uberti, M. Neuropharmacology 2011, 60, 93.
6. Conn, P. J.; Christopoulos, A.; Lindsley, C. W. Nat. Rev. Drug Discovery 2009, 8,
41.
7. (a) Ritzen, A.; Mathiesen, J. M.; Thomsen, C. Basic Clin. Pharmacol. Toxicol. 2005,
97, 202; (b) Kew, J. N. Pharmacol. Ther. 2004, 104, 233.
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Kolczewski, S.; Peters, J.-U.; Prinssen, E.; Wichmann, J.; Vieira, E.; Mühlemann,
A.; Gatti, S.; Mutel1, V.; Malherbe, P. J. Pharmacol. Exp. Ther. 2005, 315, 711.
9. (a) Dekundy, A.; Gravius, A.; Hechenberger, M.; Pietraszek, M.; Nagel, J.; Tober,
C.; van der Elst, M.; Mela, F.; Parsons, C. G.; Danysz, W. J. Neural. Transm. 2010.
doi:10.1007/s00702-010-0526-0. Published online on December 16, 2010; (b)
Phan, K. L.; Fitzgerald, D. A.; Cortese, B. M.; Seraji-Bozorgzad, N.; Tancer, M. E.;
Moore, G. J. Neuroreport 2005, 16, 183; (c) Mathew, S. J.; Price, R. B.; Charney, D.
S. Am. J. Med. Genet. C Semin. Med. Genet. 2008, 148C, 89; (d) Breysse, N.; Baunez,
C.; Spooren, W.; Gasparini, F.; Amalric, M. J. Neurosci. 2002, 22, 5569.
10. Marin, J. C.; Goadsby, P. J. Expert Opin. Investig. Drugs 2010, 19, 555.
11. Zerbib, F.; Keywood, C.; Strabach, G. Neurogastroenterol. Motil. 2010, 22, 859.
12. Jacquemont, S.; Curie, A.; des Portes, V.; Berry-Kravis, E.; Hagerman, R. J.;
Ramos, F.; Cornish, K.; He, Y.; Paulding, C.; Torrioli, M. G.; Neri, G.; Chen, F.;
Hadjikhani, N.; Martinet, D.; Meyer, J.; Beckmann, J. S.; Delange, K.; Brun, A.;
Bussy, G.; Gasparini, F.; Hilse, T.; Floesser, A.; Branson, J.; Bilbe, G.; Johns, D.;
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assay concentration of 40 l
g protein/well for [3H] 2-methyl-6-phenylethynyl-
pyridine ([3H] MPEP, American Radiolabeled Chemicals, Inc., St. Louis, MO)
filtration binding. Incubations included 5 nM [3H]-MPEP, membranes and
either buffer or varying concentrations of compound. Samples were incubated
for 60 min at room temperature with shaking. Non-specific binding was
defined with 10 lM MPEP. After incubation, samples were filtered over a GF/C
filter (presoaked in 0.25% polyethyleneimine (PEI)) and then washed four times
using a TomtecÒ Harvester 96Ò Mach III cell harvester (Tomtec, Hamden, CT)
with 0.5 mL ice-cold 50 mM Tris–HCl (pH 7.4). IC50 values were derived from
the inhibition curve and Ki values were calculated according to the Cheng and
Prusoff equation of Ki = IC50/(1 + [L]/Kd) described in [Cheng, Y.; Prusoff, W.H.
Biochem. Pharmacol. 1973, 22, 3099] where [L] is the concentration of
radioligand and Kd is its dissociation constant at the receptor, derived from
the saturation isotherm.
21. Shaffer, C. L. Annu. Rep. Med. Chem. 2010, 43, 55. and references cited therein.
22. Hecht, S. S.; Chen, C. B.; Hoffmann, B. J. Med. Chem. 1980, 23, 1175.
23. Determination of metabolic stability in liver microsomes. This study was
conducted by incubating 1 lM concentration of test compounds at 37 °C in
pooled male rat or pooled human liver microsomes (0.5 mg/mL) in 0.1 M
potassium phosphate buffer (pH 7.4) supplemented with NADPH-regenerating
system (1.3 mM NADP+, 3 mM MgCl2, 3.5 mM glucose-6-phosphate, and
4 units glucose-6-phosphate dehydrogenase). Aliquots were taken at 0.25, 5,
15, 30 and 60-minute time points. The aliquots were added to a 96-well plate
containing an equal volume of acetonitrile in order to terminate the reaction.
The samples were vortexed and centrifuged at 3000 rpm for 15 min. A known
volume of internal standard was added to the supernatant. The samples were
injected in an LC–MS/MS system to monitor the disappearance of the parent
13. Berg, D.; Godau, J.; Trenkwalder, C.; Eggert, A.; Csoti, I.; Storch, A.; Gasparini, F.;
Hariry, S.; Vandermeulebroecke, M.; Johns, D.; Gomez-Mancilla, B. Mov. Disord.
2010, 25(Suppl. 2), S290.

J. M. Weiss et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4891–4899
4899
compound. The half-life was measured by plotting the log of the remaining
compound versus time. The in vitro half-life data were used to calculate the
intrinsic clearance (CLint), according to the equation shown below:
phosphate buffer in
supernatant was analyzed by using a Waters Acquity UPLC system (Column:
Acquity UPLC BEH C-18, 1.7
254 nm for quantitation) with a gradient (mobile phase A: 2% w/v ammonium
formate and 20% acetonitrile in water, mobile phase B: 100% acetonitrile) for
2 min.
a
Titer plate shaker at
a
speed of 1.8 for 4 h. The
l
m 2.1 ꢁ 50 mm column and PDA detector at
1
g liver weight
mL incubation
CLint ¼ 0:693 ꢁ
ꢁ
ꢁ
T1=2ðminÞ kg body weight mg microsomal protein
mg microsomal protein
25. Human and rat protein binding studies, and rat brain homogenate free fraction
studies were determined by using
(HTD96b, Catalog# 1006, www.htdialysis.com). Test compounds (10
a
96-well format equilibrium dialyzer
M) were
ꢁ
ꢁ kg body weight
l
g liver weight
spiked into serum from human or rat and dialyzed against phosphate buffered
saline (pH 7.4) via a >5000 molecular weight dialysis membrane for 2.5 h in an
incubator set to maintain 37 °C with 5% CO2. The dialysis block was placed on
an orbital shake. After 2.5 h incubation, buffer and serum samples (or brain
homogenate samples) were collected and transferred into a 96-well matrix
plate with tube inserts. Internal standards were added and the samples were
vortexed and mixed, and analyzed by LC–MS/MS.
where, liver weight is 20 g/kg in human and 45 g/kg in rat, and body weight is 70 kg
for human and 250 g in rat. In this equation, it was assumed that 1 g of liver contains
45 mg of microsomal protein and the binding of the drug to microsomal proteins
was assumed to be zero, hence the unbound drug fraction, fu,mic = 1.
24. Kinetic solubilities were measured from 10 mM DMSO stock solution of the
test compound (300
lL) diluted with 2 ꢁ 200 lL of 25 mM potassium