3-(2-Ethoxy-4-{4-[3-hydroxy-2-methyl-4-(3-methylbutanoyl)phenoxy]butoxy} phenyl)propanoic acid: A brain penetrant allosteric potentiator at the metabotropic glutamate receptor 2 (mGluR2)

Article abstract of DOI:10.1016/j.bmcl.2005.02.078

We have identified and synthesized a brain penetrant propanoic acid as an allosteric potentiator of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward improving the potency, level of potentiation and brain penetration led to the discovery of 8 (EC ₅₀ = 1200 nM, 77% potentiation, 119% brain/plasma in rat, 20 mpk ip, brain level of 5700 nM).

Full text of DOI:10.1016/j.bmcl.2005.02.078

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2389–2393
3-(2-Ethoxy-4-{4-[3-hydroxy-2-methyl-4-(3-methylbutanoyl)-
phenoxy]butoxy}phenyl)propanoic acid: a brain penetrant allosteric
potentiator at the metabotropic glutamate receptor 2 (mGluR2)
Rowena V. Cube,a,* Jean-Michel Vernier,a John H. Hutchinson,a
Michael F. Gardner,a Joyce K. James,a Blake A. Rowe,b Herve Schaffhauser,b
Lorrie Daggettb and Anthony B. Pinkertona
aDepartment of Chemistry, Merck Research Laboratories, MRLSDB2, 3535 General Atomics Court, San Diego, CA 92121, USA
bDepartment of Neuropharmacology, Merck Research Laboratories, MRLSDB1, 3535 General Atomics Court, San Diego,
CA 92121, USA
Received 8 November 2004; revised 23 February 2005; accepted 25 February 2005
Available online 28 March 2005
Abstract—We have identified and synthesized a brain penetrant propanoic acid as an allosteric potentiator of the metabotropic glu-
tamate receptor 2. Structure–activity relationship studies directed toward improving the potency, level of potentiation and brain
penetration led to the discovery of 8 (EC50 = 1200 nM, 77% potentiation, 119% brain/plasma in rat, 20 mpkip, brain level of
5700 nM).
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
fluoro-4-oxobicyclo[3.1.0]hexane-2,6-dicarboxylic acid9
in both animal models as well as human clinical
trials.10,11 Both compounds are non-selective mGlu2/3
receptor agonists. Due to the high degree of sequence
homology between group II mGlu receptors, selective
agonists for mGlu2 over mGlu3 have not, as yet, been
discovered. Therefore, another strategy for selectivity in-
volves the discovery of allosteric modulators that do not
bind at the glutamate binding site.12–14 This paper de-
tails the discovery and SAR of a class of selective mGlu2
receptor potentiators.
The neurotransmitter, glutamate, plays an important
role in a wide variety of CNS functions, exerting its ef-
fect on both the ionotropic glutamate receptors, which
are glutamate-gated ion channels, as well as the metabo-
tropic glutamate receptors (mGlu), which are in the class
of G-protein coupled receptors. Eight subtypes of the
mGlu receptors fall into three main groups.1,2 Group I
consists of mGlu1 and ꢀ5, which have mainly been
shown to be stimulatory. Groups II (mGlu2 and ꢀ3)
and III (mGlu 4, ꢀ6, ꢀ7, ꢀ8), however, are often con-
centrated presynaptically and generally inhibit neuro-
transmission. Therefore, agents targeting the mGlu
receptors may have utility in a variety of diseases3–5
including epilepsy, anxiety, and schizophrenia.6 The
physiological importance of group II mGlu receptors
has been shown by the efficacy of a rigid glutamate ana-
logs such as (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane
2,6-dicarboxylic acid7,8 and (1R,2S,5S,6S)-2-amino-6-
Screening15–17 of the Mercksample collection led to the
discovery of tetrazole 1, which displayed moderate activ-
ity as an mGluR2 potentiator. Utilizing tetrazole 1 as
the lead compound, three strategies were employed to
improve the potency, level of potentiation and brain
penetration. The first strategy focused on the modifica-
tion of the acetophenone moiety, leading to the discov-
ery of tetrazole 2.18 The second strategy investigated
different linkers between the acetophenone and the phe-
nyl tetrazole functional groups.18 The third strategy fo-
cused on the replacement of the tetrazole functionality
with the goal of increasing brain penetration and hence
improving the pK profile of the compounds. A variety
of tetrazole replacements were examined, which led to
Keywords: mGluR2; Glutamate; Potentiator; Coumarin; Allosteric
modulator; Tetrazole.
*
Corresponding author. Tel.: +1 858 202 5448; fax: +1 858 202
5752; e-mail: rowena_cube@merck.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.078

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R. V. Cube et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2389–2393
the discovery of 4-thiopyridyl acetophenone (3) as a
potent mGluR2 potentiator.19 During these efforts,
7-hydroxycoumarin (4) was also found to be a suitable
tetrazole replacement. (EC50 1400 nM, 45% potentia-
tion, with potentiation being defined as the response
obtained using the test compound up to 10 lM plus
an EC10 of glutamate normalized to the maximal
response obtained with glutamate alone.) Compound 4
and the other analogs described below only showed
activity at mGluR2 with no activity observed for
mGluR3 as well as the other mGluRs. Herein we
describe the detailed efforts on the replacement of the
tetrazole with 7-hydroxycoumarin (4), chromanone,
and propanoic acid functionalities.
sequent SAR study focused on two subseries: (1) the
simple propionic acid analogs and (2) the fused six-
membered ring ethers. The first subseries studied how
the four atom chain affected the activity without the
phenol or ether moiety present. The second subseries fo-
cused on cyclic ethers and simple phenol analogs.
Removal of the phenol moiety as in acid 11 when com-
pared to the phenol–acid 6 gave mixed results: a loss in
potency (801–1450 nM), but a slight increase in potenti-
ation level (66–82%). Comparison of the methyl esters
12 and 7 showed a decrease in potency (478–3619 nM)
and no change in potentiation (56–54%). These results
indicate the roles of the phenol and the carboxylic
OH
O
OH
O
O
O
O
O
N
N
N
NH
N
N
NH
N
1
2
OH
O
OH
O
O
O
O
S
O
O
N
3
4
2. Biology
acid/ester are additive for binding affinity, but not neces-
sarily additive for level of potentiation. Minimal activity
in both potency and level of potentiation were observed
in the propanol analog 13 and the 2-butanone com-
pound 14. Likewise, modification to the cyclic ethers
15 and 16 gave weakly potent compounds. Surprisingly,
the simple phenol 17, although showing a weakbinding
affinity of 1580 nM, gave a decent level of potentiation
of 69%. From these results, an acidic proton either from
a phenol or carboxylic acid is required to maintain a
moderate level of potentiation (>50%).
Table 1 illustrates the SAR modifications of lead com-
pound 4. Reduction of the 7-hydroxy-coumarin moiety
to lactone 5 gave a favorable result of enhancing both
the binding affinity (406 nM) and level of potentiation
of 67% as compared to 4. Reduction and hydrolysis of
the 7- hydroxycoumarin moiety to the carboxylic acid–
phenol 6 also led to an improvement in potency when
compared to lead 4 (1400–801 nM), but unfortunately
no substantial increase in level of potentiation (45–
66%) was observed. The same result was again seen
for methyl ester 7 with an increase in binding affinity
of 478 nM, but no substantial increase in level of poten-
tiation at 56%. Ethers 8 and 9 did not give more potent
compounds than the lead 4 (1180 and 2200 nM, respec-
tively), but their high levels of potentiation (77% and
173%, respectively), justified further study. Replacement
of the methyl group with a bromide at the acetophenone
portion of the molecule as in compound 10 gave a three-
fold increase in binding (1400–360 nM) and an almost
twofold increase in level of potentiation (45–73%) as
compared to lead 4. These positive results encouraged
us to further investigate the modifications of the 7-
hydroxycoumarin moiety.
Subsequent testing of all compounds listed in Table 1
for
mGluR3
potency
showed
no
activity
(>10,000 nM), further providing evidence that potenti-
ators can be used to enhance selectivity for mGluR2
over mGluR3.
Pharmacokinetic data of the tetrazole lead 2 and se-
lected coumarin analogs (6, 8, and 10) are shown in
Table 2. Both tetrazole 2 and phenol/carboxylic acid 6
showed a brain to plasma ratio of less than 3%. Capping
of the phenol as in ethyl ether 820 gratifyingly increased
the brain to plasma ratio to 1.19 with a brain level of
5700 nM, the highest level of brain penetration of the
series. At this point it is unclear what the causes for
the improved brain penetration for compound 8 are.
None of the compounds tested (2, 6, 8, 10) were found
To single out which specific groups in the 7-hydroxy-
coumarin functionality were required for potency, a sub-

R. V. Cube et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2389–2393
Table 1. Binding affinities for tetrazole replacements
2391
OH
O
R'
O
R
O
Compounds
R
R0
GTPcS binding EC50 (nM)a
% Potentiationb
2
3
—
—
—
—
229
340
89
33
O
O
O
O
4
5
–CH3
–CH3
1400
406
45
67
HO
HO
6
7
–CH3
–CH3
801
478
66
56
O
HO
O
O
O
HO
8
–CH3
–CH3
–Br
1180
2200
360
77
173
73
O
O
HO
O
9
O
O
10
HO
11
12
13
–CH3
–CH3
–CH3
1450
3619
3200
82
54
39
O
O
O
HO
14
–CH3
1100
28
O
O
15
16
17
–CH3
–CH3
–CH3
869
1090
1580
34
29
69
O
O
HO
a Value represents mean of two or more experiments.
b Result expressed as a percentage of the maximum glutamate response at 1 mM.
to be Pgp substrates. It should be noted that the aqueous
solubility for compound 8 was considerably higher than
for phenol/acid 6 and tetrazole 2; thus a higher plasma
level was obtained for compound 8 compared to com-
pound 6. Interestingly, this was not the case for tetrazole
2, which had high plasma levels but essentially no brain

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R. V. Cube et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2389–2393
Table 2. Rat PK profile comparisons between tetrazole 2 and coumarin analogs 6, 8 and 10
Compound
Cla (mL/min/kg)
Vd (L/kg)
t1/2 (h)
%F a
Plasmab (nM)
Brainb (nM)
Brain/plasmab
2
15
0.5
1.5
63
8200
1380
4789
66
<LOQd
<LOQd
5700
<0.03
<0.03
1.19
6
NDc
NDc
18
NDc
NDc
1.81
NDc
NDc
2.4
NDc
NDc
0
8
10
<LOQd
<0.03
a Dosed 2 mpkiv ( n = 2) and 10 mpkpo ( n = 3) in Sprague-Dawley rats.
b Dosed 20 mpkip ( n = 3) in Sprague-Dawley rats, animals sacrificed at 2 h and brain and plasma levels analyzed.
c Not determined.
d Limit of quantification.
penetration. This effect was observed for the other tetra-
zole containing compounds.18,19 One potential explana-
tion for the high brain levels for 8 is an unidentified
transporter. The most potent coumarin analog, bromide
10, unfortunately showed poor pK characteristics pre-
sumably due to its very poor aqueous solubility.
The synthesis of compounds 15 and 16 began with a
reaction of resorcinol with 3-chloropropionic acid to
give the chloride 22 (Scheme 2).22 Intramolecular cycliz-
ation of chloride 22 afforded ketone 23, which was then
alkylated with bromide 20 to afford ketone 15. Hydroge-
nation of intermediate 23 produced the cyclic ether 24 as
the only product. Compound 24 was then further alkyl-
ated with bromide 20 to give compound 16.
3. Chemistry
The synthesis for the 7-hydroxycoumarin compound 4
and its analogs described herein is outlined in Schemes
1 and 2.21 The acetophenone derivatives 18 and 19, were
alkylated with 1,4-dibromobutane, using potassium car-
bonate in acetone to afford the precursors 20 and 21,
respectively. 7-Hydroxycoumarin was alkylated by these
precursors to give compounds 4 and 10. Compound 4
was further hydrogenated in methanol to afford the
methyl ester 7. Hydrolysis of 7 with lithium hydroxide
produced the carboxylic acid 6, which was then lacton-
ized using p-toluenesulfonic acid in refluxing benzene
to give the reduced coumarin analog 5. The alkylation
of phenol 7 with iodoethane and subsequent ester
hydrolysis afforded compound 8.20 To synthesize com-
pounds 9, 11–14, and 18, intermediate 20 was alkylated
with the appropriate phenols utilizing conditions similar
to those outlined in Scheme 1.
4. Conclusion
In summary, we have disclosed a new series of allosteric
potentiators at the mGlu receptor 2 by replacing the tetr-
azole moiety of the lead compound 2 with 7-hydroxy-
coumarin type analogs. SAR strategies led to
compounds with moderate potency, high levels of poten-
tiation and good brain penetration. Although the lead
tetrazole 2 showed good potency of 229 nM and a high
level of potentiation of 89%, this compound was not
brain penetrant. From this SAR, we have found a highly
brain penetrant compound in ether 8 with a brain to
plasma ratio of 1.19 with brain levels at 5700 nM. Ether
8, although less potent at 1180 nM, shows a high level of
potentiation of 77%. This led us to further analyze 8 and
other coumarin type analogs for full pK and in vivo
profiles.
OH
O
OH
O
OH
O
R
O
R
R
O
O
O
O
Br
ii
i
HO
4 R=CH3
10 R= Br
18 R= CH3
19 R= Br
20 R= CH3
21 R= Br
OH
O
OH
O
HO
O
O
HO
O
O
HO
iii
O
v
6
7
O
O
vi
iv
OH
O
O
OH
O
O
O
O
O
O
O
HO
5
8
O
Scheme 1. Reagents and conditions: (i) dibromobutane, K2CO3, acetone, 40 °C, 75%; (ii) 7-hydroxycoumarin, K2CO3, acetone 40°C, 75%; (iii) 10%
Pd/C, MeOH, H2, 1 atm, 8 h, 97%; (iv) (a) iodoethane, K2CO3, acetone, 40°C, 42%, (b) 2.5 N lithium hydroxide, dioxane/water, 4 h, 96%; (v) 2.5 N
lithium hydroxide, dioxane, rt, 4 h, 98%; (vi) p-TsOH, benzene, reflux, 2 h, 65%.

R. V. Cube et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2389–2393
2393
OH
OH
O
HO
O
OH
O
O
OH
OH
HO
O
O
O
O
Cl
iii
ii
i
22
23
15
O
iv
O
O
O
OH
O
v
24
16
Scheme 2. Reagents and conditions: (i) 3-chloropropionic acid, triflic acid, 80 °C, 60%; (ii) 2.0 N sodium hydroxide, 87%; (iii) 20, K2CO3, acetone,
40 °C, 42%; (iv) 10% Pd/C, ethyl acetate, H2, 1 atm, 12 h, 100%; (v) 20, K2CO3, acetone, 40 °C, 46%.
promiscuous G-protein (Ga16). Receptor activity was
detected by changes in [Ca2+], measured using the
fluorescent, Ca2+ sensitive dye fura-2. For further infor-
mation, see: Varney, M. A.; Cosford, N. D.; Jachec, C.;
Rao, S. P.; Sacaan, A.; Lin, F. F.; Bleicher, L.; Santori, E.
M.; Flor, P. J.; Allgeier, H.; Gasparini, F.; Kuhn, R.;
Hess, S. D.; Velicelebi, G.; Johnson, E. C. J. Pharmacol.
Exp. Ther. 1999, 290, 170.
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structure. 1H NMR (CDCl3, 300 MHz) d 13.03 (s, 1H),
7.63–7.61 (d, 1H), 7.07–7.05 (d, 1H), 6.46–6.40 (m, 3H),
4.16–4.12 (m, 2H), 4.06–4.00 (m, 4H), 2.92–2.89 (m, 2H),
2.79–2.78 (d, 2H), 2.68–2.64 (m, 2H), 2.30–2.28 (m, 1H),
2.13 (s, 3H), 2.07–2.00 (m, 4H), 1.44–1.41 (t, 3H), 1.03–1.02
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>95%).
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