Allosteric potentiators of the metabotropic glutamate receptor 2 (mGlu2). Part 2: 4-Thiopyridyl acetophenones as non-tetrazole containing mGlu2 receptor potentiators

Article abstract of DOI:10.1016/j.bmcl.2004.09.028

We have identified and synthesized a series of 4-thiopyridyl acetophenones as positive allosteric potentiators of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward replacement of the tetrazole in the initial lead led to the discovery of 16 (EC₅₀ = 340 nM), which showed improved brain penetration over the initial lead. We have identified and synthesized a series of 4-thiopyridyl acetophenones as positive allosteric potentiators of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward replacement of the tetrazole in the initial lead led to the discovery of 16 (EC₅₀ = 340 nM), which showed improved brain penetration over the initial lead.

Full text of DOI:10.1016/j.bmcl.2004.09.028

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5867–5872
Allosteric potentiators of the metabotropic glutamate
receptor 2 (mGlu2). Part 2: 4-Thiopyridyl acetophenones
as non-tetrazole containing mGlu2 receptor potentiators
Anthony B. Pinkerton,* Rowena V. Cube, John H. Hutchinson, Joyce K. James,
´
Michael F. Gardner, Herve Schaffhauser, Blake A. Rowe, Lorrie P. Daggett and
Jean-Michel Vernier
Merck Research Laboratories, MRLSDB2, 3535 General Atomics Court, San Diego, CA 92121, USA
Received 12 August 2004; revised 13 September 2004; accepted 14 September 2004
Available online 1 October 2004
Abstract—We have identified and synthesized a series of 4-thiopyridyl acetophenones as positive allosteric potentiators of the
metabotropic glutamate receptor 2. Structure–activity relationship studies directed toward replacement of the tetrazole in the initial
lead led to the discovery of 16 (EC50 = 340nM), which showed improved brain penetration over the initial lead.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
animal models as well as human clinical trials.10,11 These
agonists are generally rigid glutamate analogs. However,
compounds selective for mGlu2 over mGlu3 have not
been discovered using this approach. Therefore, another
strategy for selectivity involves the discovery of allo-
steric modulators that do not bind at the glutamate
binding site.12–14 Screening identified a selective mGlu2
receptor potentiator, phenyl-tetrazolyl acetophenone 1
(EC50 = 348nM, 31% potentiation, with potentiation
being defined as the response obtained using the test
compound up to 10lM plus an EC10 of glutamate nor-
malized to the maximal response obtained with gluta-
mate alone).15–17 However, this compound, while
displaying in vivo activity, needed to be dosed icv due
to poor brain penetration. Working on the hypothesis
that the tetrazole, or other acidic functionality, was
Glutamate is the major excitatory neurotransmitter in
the CNS and plays an important role in many CNS
functions. Glutamate receptors are classified into two
main types, ionotropic (iGlu), which are glutamate
mediated ion channels, and metabotropic (mGlu), which
are a class of G-protein coupled receptors.1,2 Currently,
mGlu receptors are divided into eight subtypes and three
main groups (I–III). Group II (mGlu2 and -3) mGlu
receptors are mainly concentrated presynaptically and
generally inhibit neurotransmission. Therefore, agents
targeting group II mGlu receptors may have utility in
a variety of CNS disorders3–5 including epilepsy, anxi-
ety, and schizophrenia.6 Recently, nonselective mGlu2/
3 receptor agonists7–9 have shown activity in numerous
OCH3
O
O
OH
N
O
O
O S O
N
N
CF3
N
2 (LY487379)
1
N
HN
Keywords: GPCRs; Glutamate receptors; Schizophrenia; Allosteric.
*
Corresponding author. Tel.: +1 858 202 5379; fax: +1 858 202 5752; e-mail: anthony_pinkerton@merck.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.028

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A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5867–5872
responsible for the poor brain penetration, we investi-
gated the replacement of the tetrazole with a variety of
groups that would maintain potency. Concurrent to this
work, researchers at Lilly recently disclosed a structur-
ally distinct class of mGlu2 receptor potentiators (e.g.,
2, EC50 = 1700nM, 52% potentiation), which, unlike
compound 1, contains no acidic protons.13 This paper
outlines the discovery of a new class of brain penetrant,
nontetrazole containing mGlu2 receptor potentiators
from the initial tetrazole containing lead 1.
2. SAR studies
Our starting point for the SAR, as outlined in Table 1,
was the previously described lead (3), which displayed
both good activity (229nM) as well as level of potentia-
tion (89%).16 Attempts at replacing the tetrazole with
other 5 membered heterocycles gave mostly inactive or
very weakly active compounds. For example, N-linked
triazole 4 had a 20-fold drop in potency from compound
3. Removing the tetrazole group altogether (5) also led
Table 1. Binding affinities for tetrazole replacements
O
OH
R'
RO
O
Compd
R
R0
hmGlu2
GTPcS binding
% Potentiationb
EC50 (nM)a
1
2
—
—
—
—
348
1700
31
52
N
N
N
HN
3
4
–CH3
–CH3
229
89
18
N
4237
N
N
5
6
–CH3
–CH3
4550
1987
24
30
F
F
F
7
–CH3
–CH3
–CH3
–CH3
–Br
384
3273
1717
3855
1500
558
18
33
46
40
53
88
67
N
8
\
N
9
10
11
12
13
N
F
F
–Br
N
–Br
575
a Value represents mean of two or more experiments.
b Result expressed as a percentage of the maximum glutamate response at 1mM.

A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5867–5872
5869
to a 20-fold drop in potency. This result led us to inves-
tigate a large number of simple substitutions on the aro-
matic ring (halogen, methyl, methoxy), which gave
compounds that were either inactive or similar in activ-
ity to phenyl derivative 5 (data not shown). The most
potent compounds to arise from these permutations
were the mono fluorophenyl derivative 6 and the 2,3-
difluorophenyl derivative 7. In the case of compound 7
good potency was observed (384 vs 229nM for the
tetrazole compound 3) albeit with low potentiation.
The disclosure from researchers at Lilly of pyridine
derivatives such as compound 2 led us to investigate
the incorporation of pyridine in hope of accessing an
additional binding site. Both 2- and 4-pyridyl derivatives
8 and 10 did not show improved potency. However, 3-
and derivative 9 showed a 2-fold increase in potency
and also a modest (46%) level of potentiation compared
to the simple phenyl derivative 5. Previous work16
showed that replacement of the methyl substituent on
the phenyl ring with a bromine led to an increase in po-
tency and potentiation. Gratifyingly, when an acetophe-
none moiety containing a bromine in the place of a
methyl was examined, even better potencies and levels
of potentiation were obtained. This was the case both
for the simple phenyl derivative 11, and, more impor-
tantly, the 3-pyridyl compound 13, which displayed po-
tency and potentiation approaching that of the tetrazole
containing lead 3. Likewise, 2,3-difluorophenyl deriva-
tive 12 showed good potency and high levels of potenti-
ation. However, compound 12, along with compound 7
were not pursued further due to poor rat PK properties
(see Table 4).
3-, and 4-pyridyl derivatives were increased for 14–16
although the level of potentiation was lower. For exam-
ple, 3-pyridyl O-linked potentiator 9 displayed potency
of 1717 versus 1093nM for 15, but the relative potenti-
ation was 26% for 9 and 46% for compound 15. The
implications of this disparity are not understood at the
present time. Importantly, 4-thiopyridyl derivative 16
was found to have potency of 340nM, which is similar
potency to the more active tetrazole containing leads.
It was also soluble in water as the HCl salt. Compound
16, therefore, became a compound suitable for further
investigation (vide infra). Contrary to previous results,
incorporating the bromine into the acetophenone moi-
ety for this compound gave a compound (17) that was
less potent than 16, albeit with considerably higher
potentiation (82% vs 33%). Oxidation of the sulfur in
compound 16 to the sulfoxide and sulfone gave com-
pounds that displayed much diminished potency. Like-
wise the N-oxide of compound 16 had decreased
potency (data not shown).
With the result of compound 16 in hand, we then sought
to further optimize the activity of the 4-thiopyridine
compounds through modification of the linker (Table
3). When the linker was either shortened (18) or length-
ened (19) by one carbon, inactive compounds were ob-
tained, which initially seemed to indicate a very tight
SAR. However, we were subsequently pleased to find
that a number of aryl linkers could be incorporated to
give active compounds. For example, bis-benzylic linked
compounds 20 and 21 showed both good potency and
potentiation. In particular, compound 20, with activity
of 819nM and 68% potentiation was investigated fur-
ther for PK in rats. Likewise, mono-benzylic com-
pounds 22–24 showed similar levels of potency.
Incorporating a t-butyl group a to the ketone in
place of the isopropyl group gave a compound, 25, that
Due to relatively poor aqueous solubility for compound
13, we turned our attention to examining sulfur linked
pyridines in place of oxygen linked pyridines (Table 2).
Interestingly, with a sulfur linkage, the potency of 2-,
Table 2. Activity of thiopyridines
O
OH
R'
RS
O
Compd
R
R0
huGlu2
GTPcS binding EC50 (nM)a
% Potentiationb
21
N
14
–CH3
1532
N
15
16
17
–CH3
–CH3
–Br
1093
340
26
33
82
N
N
676
a Value represents mean of two or more experiments.
b Result expressed as a percentage of the maximum glutamate response at 1mM.

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A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5867–5872
Table 3. Effect of different linkers
O
OH
R"
N
S
Linker
R00
O
Compd
Linker
huGlu2
GTPcS binding EC50 (nM)a
% Potentiationb
33
16
18
19
–H
–H
–H
340
NAc
NAc
—
—
20
21
–H
–H
819
770
68
78
m
p
22
23
24
–H
–H
–H
677
890
38
58
38
o
m
p
1060
25
–CH3
578
79
a Value represents mean of two or more experiments.
b Result expressed as a percentage of the maximum glutamate response at 1mM.
c NA denotes not active <10lM concentration.
displayed improved potency and level of potentiation
(79%). This compound was also examined for its rat
PK properties.
brain/plasma ratios for tetrazole compounds 1 and 3
are included. As shown in Table 4 the brain to plasma
ratios for tetrazoles 1 and 3 were extremely low (<5%).
LY487379 (2) displayed modest brain/plasma ratios,
although considerably better than tetrazole leads 1 and
3. In contrast, thiopyridine 16 displayed an excellent
brain/plasma ratio after sc dosing. Gratifyingly, the
brain/plasma ratios for compounds 20 and 25 were also
much improved, giving brain levels close to or exceeding
the EC50 at 2h post dosing.
3. PK properties
With a number of potent thiopyridine compounds in
hand, we then examined the brain/plasma ratio in rats
for several of these compounds. For reference, the
Table 4. Selected rat PK parameters and brain/plasma ratios
4. Chemistry
Compd Cla (mL/min/kg) %Fa Brain/plasmab Brain levelb
(nM)
The compounds described in Tables 1 and 2 were syn-
thesized as described below (Scheme 1). The synthesis
began with acetophenone derivatives 26,15,16 which were
first alkylated selectively with 1,4-dibromobutane. Com-
pounds 4–17 were then made via alkylation of the
appropriate phenol or thiophenol in good yields.18
1
33
70
63
<1
59
0
0.01
0.15
0.03
NDc
1.10
—
2
10
3
15
75
—
12
16
20
25
NDc
400
330
340
33
3
NDc
NDc
NDc 1.20
NDc 0.21
The compounds in Table 3 were synthesized in a manner
exemplified by the example of compound 20 (Scheme 2).
Beginning from commercially available 3-(bromo-
a Dosed 2mpk iv and 10mpk po.
b Dosed 20mpk ip, levels at 2h.
c Not determined.
O
OH
O
OH
R
O
a,b
R
RX
X= O, S
HO
26
4-17
Scheme 1. Reagents and conditions: (a) 1,4-dibromobutane, K2CO3, acetone, 45°C; (b) RXH, K2CO3, acetone, 45°C.

A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5867–5872
5871
O
OH
N
CO2CH3
a,b
+
Br
S
OH
28
HO
27
29
O
OH
c
N
S
O
20
Scheme 2. Reagents and conditions: (a) 4-mercaptopyridine, K2CO3, acetone, 45°C, 85%; (b) LiAlH4, THF, 0°C, 80%; (c) DTAD, PPh3, THF, 43%.
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5. Conclusion
In summary we have described herein the discovery of a
new class of allosteric modulators of the mGlu2 recep-
tor, in which the phenyl tetrazolyl group of the lead
structure has been replaced by a 4-thiopyridine group.
This advancement has increased our understanding of
the structural variations allowed for mGlu2 receptor
activity and has led to a brain penetrant class of poten-
tiators. The lead compounds from this series, 16 and 20
showed brain/plasma levels > 1 and had potencies of 340
and 819nM with potentiation levels of 33% and 68%,
respectively. The compounds, therefore, had potency
comparable to the original tetrazole containing lead as
well as greater potency compared to 2 (LY487379,
EC50 = 1700nM, 52% potentiation). These compounds,
along with the other compounds outlined herein, were
selective for mGlu2 over mGlu3 as well as the other
mGlu receptors. Further work will focus on continued
optimization as well as use of these potentiators in vivo
models.
Acknowledgements
We thank Merryl Cramer for performing the PK and
brain/plasma ratio studies.
References and notes
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A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5867–5872
16. Pinkerton, A. B.; Cube, R. V.; Hutchinson, J. H.; Rowe,
B. A.; Schaffhauser, H.; Zhao, X.; Daggett, L. P.; Vernier,
J.-M. Bioorg. Med. Chem. Lett. 2004, 14, 5329–5332.
17. The effect of these compounds was characterized in the
[35S]-GTPcS binding assay using a cell line expressing
human mGlu2 receptor. See Ref. 14 for a detailed
description of this assay. First, an EC10 (1lM) of
glutamate was added to the cell line followed immediately
by the test compound at varying concentrations. The
response was then compared to a response using a
saturating amount of glutamate (1mM) to give both an
EC50 and a percent potentiation (the response normalized
to the maximum response of glutamate alone). The same
experiment was carried out in the absence of glutamate to
test if the compound was truly a positive allosteric
modulator. Nonspecific binding was determined by the
addition of 10lM unlabeled GTPcS.
18. All final compounds displayed spectral data (NMR, MS)
that were consistent with the assigned structure.
19. Hughes, D. L. Org. Prep. Proc. Int. 1996, 28, 127–164.