Allosteric potentiators of the metabotropic glutamate receptor 2 (mGlu2). Part 1: Identification and synthesis of phenyl-tetrazolyl acetophenones

Article abstract of DOI:10.1016/j.bmcl.2004.08.020

We have identified and synthesized a series of aryl-tetrazoyl acetophenones as positive allosteric potentiators of the metabotropic glutamate receptor 2. Structure activity relationship studies directed toward improving the potency and level of potentiation led to the discovery of 22 (EC₅₀ = 93 nM, 128% potentiation). We have identified and synthesized a series of aryl-tetrazoyl acetophenones as positive allosteric potentiators of the metabotropic glutamate receptor 2. Structure activity relationship studies directed toward improving the potency and level of potentiation led to the discovery of 22 (EC₅₀ = 93 nM, 128% potentiation).

Full text of DOI:10.1016/j.bmcl.2004.08.020

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5329–5332
Allosteric potentiators of the metabotropic glutamate
receptor 2 (mGlu2). Part 1: Identification and
synthesis of phenyl-tetrazolyl acetophenones
Anthony B. Pinkerton,* Rowena V. Cube, John H. Hutchinson, Blake A. Rowe,
´
Herve Schaffhauser, Xiumin Zhao, Lorrie P. Daggett and Jean-Michel Vernier
Merck Research Laboratories, MRLSDB2, 3535 General Atomics Court, San Diego, CA 92121, USA
Received 25 June 2004; revised 7 August 2004; accepted 9 August 2004
Available online 3 September 2004
Abstract—We have identified and synthesized a series of aryl-tetrazoyl acetophenones as positive allosteric potentiators of the
metabotropic glutamate receptor 2. Structure activity relationship studies directed toward improving the potency and level of poten-
tiation led to the discovery of 22 (EC50 = 93nM, 128% potentiation).
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
OCH3
O
HO2C
H
HO2C
F
Glutamate is the transmitter of the large majority of fast
excitatory synapses in the CNS and plays an important
role in a wide variety of CNS functions. The actions of
glutamate are mediated by the activation of both iono-
tropic glutamate receptors (iGlu), which are glutamate-
gated ion channels, as well as the metabotropic
glutamate receptors (mGlu), which are a class of G-pro-
tein coupled receptors. Eight subtypes of the mGlu
receptors have been identified, which fall into three main
groups.1,2 Group I consists of mGlu1 and -5, which are
primarily stimulatory. Groups II (mGlu2 and -3) and III
(mGlu4, -6, -7, -8), however, are presynaptic and gener-
ally inhibit neurotransmission. Therefore, agents target-
ing mGlu receptors may have utility in a variety of
clinical conditions3–5 including epilepsy, anxiety, and
schizophrenia.6 The physiological importance of group
II mGlu receptors has been shown by the efficacy
of rigid glutamate analogs such as (1S,2S,5R,6S)-2-
aminobicyclo[3.1.0]hexane 2,6-dicarboxylic acid (1,
LY354740)7,8 and (1R,2S,5S,6S)-2-amino-6-fluoro-4-
oxobicyclo[3.1.0]hexane 2,6-dicarboxylic acid (2,
MGS0028)9 in both animal models as well as human
clinical trials.10,11 Both compounds are non-selective
mGlu2/3 receptor agonists.
H
H
N
S
H
O
NH2
NH2
O
O
CO2H
CO2H
N
CF3
1 (LY354740)
2 (MGS0028)
3 (LY487379)
Agonists selective for mGlu2 over mGlu3 have not, as
yet, been discovered, presumably due to the high
degree of sequence homology between group II mGlu
receptors. Therefore, another strategy for selectivity in-
volves the discovery of allosteric modulators that do
not bind at the glutamate binding site.12 Researchers
at Lilly have recently disclosed 3-pyridyl sulfonamides
such as 3 (LY487379) as allosteric modulators of the
mGlu2 receptor, which do not bind at the glutamate
binding site.13,14 This paper details the discovery and
SAR of a new class of mGlu2 receptor selective
potentiators.
O
OH
O
O
N
Keywords: Metabotropic glutamate receptors; GPCRs.
*
N
Corresponding author. Tel.: +1 858 202 5379; fax: +1 858 202
5752; e-mail: anthony_pinkerton@merck.com
4
N
HN
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.020

5330
A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5329–5332
Screening of the Merck sample collection led to the dis-
covery of compound 4,15 which displayed moderate
activity as an mGlu2 receptor potentiator, with an
EC50 of 348nM and a 31% level of potentiation. Percent
potentiation is defined as the response obtained using
the test compound plus an EC10 of glutamate normal-
ized to the maximal response obtained with glutamate
alone.17 In the absence of glutamate, 4 showed no activ-
ity at the mGlu2 receptor, confirming that the com-
pound is a positive allosteric potentiator. Compound 4
displayed a similar profile to the Lilly potentiator 3,
which in our assays had an EC50 of 1.7lM with 52%
potentiation. Compound 4, as well as all of the other
compounds described below showed no activity at the
human mGlu3 receptor in the presence or absence of
glutamate, as well as at the other mGlu receptors (-1,
-4, -5, -7, -8) (data not shown). Therefore, our initial
goal was to improve both the potency and especially
the level of potentiation. Three strategies were
employed: (1) investigation of different linkers between
the acetophenone and the phenyl tetrazole, (2) modifica-
tion of the acetophenone moiety, and (3) examination of
various tetrazole replacements.
We initially examined the modification of the linker
between the aryl tetrazole and the acetophenone to in
an attempt to ascertain the requirements for activity
and potentiation. These efforts are summarized in Table
1 in which a range of different linkers were explored
starting from the initial lead 4. When the linker was
either shortened (5) or lengthened (6) by one atom, po-
tency decreased dramatically, which initially indicated a
very tight SAR. Interestingly, additions of methyl
groups either adjacent to the acetophenone (7) tetrazole
(8) or both (9) gave a modest increase in the level of
potentiation, and in the case of compound 9 a small po-
tency boost also compared to compound 4. It should
be noted that compound 9 is a racemic mixture of dia-
stereomers. This indicates that relatively small modifica-
tions of the linker could improve activity. However,
other simple modifications of the linker were not benefi-
cial. The inclusion of nitrogen (10 and 11) as well as
incorporation of an amide (12) or ester (13) gave signif-
icantly lower activity. Whether this is due to an elec-
tronic effect or due to conformation is unclear.
The effects of the modification of the acetophenone moi-
ety are detailed in Table 2. From the initial lead 4, it was
found that removing the propyl group at position 2 (14)
led to loss of activity. Incorporation of a methyl group
in the same position resulted in a 10-fold loss of potency
(15). As with smaller groups such as methyl, larger
groups were also not tolerated at this position. For
example, a pentyl group (16) led to loss of activity.
Switching to a bromo substituent (17) regained some
of the lost potency and also dramatically increased the
potentiation (31–98%). The loss in potency shown with
a methyl group in position 2 could be compensated for
by substituting the ketone with a larger lipophilic group
(R2). Indeed, potency increased from methyl (15) to
ethyl (18) to propyl (19) to isobutyryl (20). Interestingly,
with two larger lipophilic groups (21) potency dropped
off, indicating that too much steric hindrance around
the acetophenone was not optimal. Drawing on the re-
sult for compound 17, wherein a bromine on the aceto-
phenone increases the level of potentiation, a bromo
substituent was reincorporated on the aryl ring with
the isobutyryl group. We were pleased to find this effect
additive, with compound 22 showing an EC50 of 93nM
with greater than 100% potentiation. This represents
one of the most potent compounds in this series. Further
examination of the acetophenone moiety indicated that
in contrast to position 2, substitution at position 6 on
the phenyl ring (23) led to a loss of activity. Likewise,
incorporation of cycloalkyl groups on the ketone (R2)
led to compounds that were considerably less potent
that compound 22 (data not shown).
Table 1. Effect of different linkers
O
OH
N
N
HN
N
Linker
Compd Linker
hmGlu2
GTPcS binding % Potentia-
EC50 (nM)a
tionb
3
4
5
6
––
1700
52
O
O
348
1418
3313
31
41
75
O
O
O
O
CH3
7
918
495
76
67
52
35
98
O
O
O
O
8
CH3
CH3
CH3
O
9
241
O
dr=1:1
H
N
10
11
12
13
939
O
CH3
N
2013
NAc
7517
O
O
H
N
Lastly, we investigated potential replacements for the
tetrazole in compound 4. As shown in Table 3, a num-
ber of other acidic groups could substitute for the tetraz-
ole. In general, acidic groups with a similar pKa to the
tetrazole were active, albeit with diminished potency.
For example, switching to a carboxylic acid (24) led to
a fivefold drop in potency. Likewise, an acyl sulfon-
amide (25) showed lowered activity compared to com-
pound 4. Attempts to improve activity by adding
O
O
O
30
O
a Value represents mean of two or more experiments.
b Result expressed as a percentage of the maximal glutamate response
at 1mM.
c NA = denotes not active @ 10lM concentration.

A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5329–5332
Table 2. Binding affinities for acetophenone modifications
5331
O
OH
R1
O
R2
2
O
6
R3
N
N
N
HN
Compd
R1
R2
R3
hmGlu2
GTPcS binding EC50 (nM)a
% Potentiationb
4
–CH2CH2CH3
–H
–CH3
–CH3
–CH3
–CH3
–CH3
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
348
NAc
3365
NAc
878
31
52
14
15
16
17
18
19
20
21
22
23
–CH3
–CH2(CH2)3CH3
–Br
99
60
–CH3
–CH3
–CH3
–CH2CH2CH3
–Br
–CH2CH3
380
304
–CH2CH2CH3
–CH2CH(CH3)2
–CH2CH(CH3)2
–CH2CH(CH3)2
–CH3
100
89
229
421
74
128
93
NAc
–H
–CH2CH2CH3
a Value represents mean of two or more experiments.
b Result expressed as a percentage of the maximal glutamate response at 1mM.
c NA = denotes not active @ 10lM concentration.
Table 3. Tetrazole replacements
2. Chemistry
O
OH
The compounds described in Tables 1 and 2 and were
synthesized in a straightforward manner with the repre-
sentative method shown below (Scheme 1). The synthe-
sis began with 2-substituted resorcinols (28), which were
reacted with an acid chloride in the presence of alumi-
num trichloride overnight to give acetophenone deriva-
tives 29.18 These were then alkylated selectively using
alkyl bromide 30 with potassium carbonate in acetone
to give the cyano precursor to the desired compounds.
The final products19 were accessed via a tin catalyzed
tetrazole formation using trimethylsilyl azide in reflux-
ing toluene in generally good yields. Compounds 24–
27 were prepared using a similar alkylation strategy,
with a hydrolysis of an ester to give the acid 24 instead
of tetrazole formation. Compounds 5–13 were synthe-
sized in a similar fashion as outlined in Scheme 1 using
the appropriate alkyl bromide equivalent of compound
30.
O
O
R4
Compd R4
hmGlu2
GTPcS binding % Potentiationb
EC50 (nM)a
N
NH
N
4
348
31
54
N
–CO2H
24
1823
O
25
SO2CH3 1041
29
N
H
26
27
–SO2NH2
NAc
NAc
––
––
–CONH2
a Value represents mean of two or more experiments.
b Result expressed as a percentage of the maximal glutamate response
at 1mM.
c NA = denotes not active @ 10lM concentration.
3. Conclusion
In summary we have described herein the discovery of
phenyl-tetrazolyl acetophenones as potent positive allo-
steric modulators of the mGlu2 receptor, which have no
intrinsic activity in the absence of glutamate. These
studies provided us with some understanding of the
structural requirements necessary for mGlu2 receptor
potentiator activity, and led to potent compounds with
high levels of potentiation, for example, compound 22
(EC50 = 93nM, 128% potentiation). This represents a
significant improvement over the initial lead 4 as well
as compound 3, which, when tested in house in our
various substituents on the phenyl tetrazole that would
modulate the pKa were unsuccessful (data not shown).
Also, the meta-substituted phenyl tetrazole gave similar
activity, although the ortho tetrazole was less active
(data not shown). Lastly, compounds without acidic
protons were generally not active. For example, sulfon-
amides (26) and simple amides (27) had no activity com-
pared to the parent tetrazole. Further work examining
tetrazole replacements will be discussed in a subsequent
paper.

5332
A. B. Pinkerton et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5329–5332
OH
O
OH
O
R1
R1
Br
R2
a
+
NC
b,c
HO
30
HO
28
29
O
OH
R1
O
R2
O
N
4, 14-22
N
N
HN
Scheme 1. Reagents and conditions: (a) R2COCl, AlCl3, CH2Cl2; (b) K2CO3, acetone, 45°C; (c) TMS-N3, Bu2SnO, toluene, reflux.
13. (a) Johnson, M. P.; Baez, M.; Jagdmann, G. E., Jr.;
Britton, T. C.; Large, T. H.; Callagaro, D. O.; Tizzano, J.
assays, displayed a potency of 1.7lM with 52% potenti-
ation.14 Further exploration of this series of compounds
is in progress and will be disclosed in due course.
P.; Monn, J. A.; Schoepp, D. D. J. Med. Chem. 2003, 46,
3189; (b) Barda, D. A.; Wang, Z.; Britton, T. C.; Henry, S.
S.; Jagdmann, G. E.; Coleman, D. S.; Johnson, M. P.;
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15. Screening of compounds was carried out using a Ca2+ flux
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the [35S]-GTPcS binding assay using a cell line expressing
human mGlu2 receptor. See Ref. 14 for a detailed
description of this assay. Compounds displayed a similar
profile and potency in both assays.
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17. For the GTPcS assay, 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. Non-
specific binding was determined by addition of 10lM
unlabeled GTPcS.
18. For some similar reactions, see: Sprenger, R. D.; Ruoff, P.
M.; Frazer, A. H. J. Am. Chem. Soc. 1950, 72, 2874–2876;
Ahluwalia, V. K.; Singh, R. P.; Tripathi, R. P. Gazz.
Chim. Ital. 1984, 114(7–8), 359–361.
19. All final compounds displayed spectral data (NMR, MS)
that was consistent with the assigned structure.
11. (a) Grillon, C.; Cordova, J.; Levine, L. R.; Morgan, C. A.,
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