Isoxazolopyridone derivatives as allosteric metabotropic glutamate receptor 7 antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.11.070

This Letter describes the synthesis and evaluation of mGluR7 antagonists in the isoxazolopyridone series. In the course of modification in this class, novel solid support synthesis of the isoxazolopyridone scaffold was developed. Subsequent chemical modification led to the identification of several potent derivatives with improved physicochemical properties compared to a hit compound 1. Among these, 2 showed good oral bioavailability and brain penetrability, suggesting that 2 may be useful for in vivo study to elucidate the role of mGluR7.

Full text of DOI:10.1016/j.bmcl.2009.11.070

Bioorganic & Medicinal Chemistry Letters 20 (2010) 726–729
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Isoxazolopyridone derivatives as allosteric metabotropic glutamate receptor
7 antagonists
*
Masayuki Nakamura , Hideki Kurihara, Gentaroh Suzuki, Morihiro Mitsuya, Mitsuru Ohkubo, Hisashi Ohta
Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd, Okubo-3, Tsukuba, Ibaraki 300-2611, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2009
Revised 10 October 2009
Accepted 13 November 2009
Available online 11 December 2009
This Letter describes the synthesis and evaluation of mGluR7 antagonists in the isoxazolopyridone series.
In the course of modification in this class, novel solid support synthesis of the isoxazolopyridone scaffold
was developed. Subsequent chemical modification led to the identification of several potent derivatives
with improved physicochemical properties compared to a hit compound 1. Among these, 2 showed good
oral bioavailability and brain penetrability, suggesting that 2 may be useful for in vivo study to elucidate
the role of mGluR7.
Keywords:
mGluR7
Ó 2009 Elsevier Ltd. All rights reserved.
Antagonist
Solid support synthesis
Isoxazolopyridone
Metabotropic glutamate receptors (mGluRs) belong to a family
of G protein-coupled receptors thought to contribute to the modu-
lation of neuronal excitability and neurotransmitter release. Eight
mGluR subtypes (mGluR1–mGluR8) have been cloned, and have
been classified into three groups based on sequence homology,
pharmacology and signal transduction pathway. Among these,
mGluR7 is widely expressed in the central nervous system and
the characteristics and functionality of mGluR7 have been exten-
sively investigated.1 As with several other members of the mGluR
family, mGluR7 is primarily located on presynaptic terminals
where it is thought to regulate neurotransmitter release.1c,d Of
the presynaptic mGluR subtypes, however, mGluR7 is the most
widely distributed and is present at a broad range of synapses that
are postulated to be critical for both normal CNS function and a
range of human disorders. Furthermore, unlike some presynaptic
mGluR subtypes, mGluR7 is localized directly in the presynaptic
zone of the synaptic cleft of glutamatergic synapses, where it is
thought to act as a traditional autoreceptor that is activated by
the glutamate released from the presynaptic terminal during ac-
tion potentials. mGluR7 has been thought to be a key player in
shaping synaptic responses at glutamatergic synapses as well as
in regulating key aspects of inhibitory GABAergic transmission.
impossible to study the physiological effects of activation of this
receptor without confounding effects induced by activation of
these related mGluR subtype. Despite this lack of pharmacological
tools, anatomical cellular studies, as well as experiments with
mGluR7 KO mice, have led to suggestions that selective ligands
for this receptor may treat a wide variety of neurological and psy-
chiatric disorders.3
Mitsukawa reported the identification of AMN082 3 with selec-
tive mGluR7 agonist activity, and found that the compound is
orally active and penetrates the blood–brain barrier.4 This pharma-
cological tool provided a breakthrough that may ultimately impact
the understanding of the basic roles of mGluR7. Indeed, AMN082 3
induced a robust increase in stress hormone levels that was absent
in mGluR7 knockout animals, providing powerful support to a
growing set of findings that suggest that antagonists of this recep-
tor may be useful in conditions involving chronic stress such as
depression and anxiety disorders.
Recently, we reported the discovery of novel mGluR7 antago-
nists and the comprehensive in vitro pharmacological characteriza-
tion of isoxazolopyridone derivatives, such as 1 and 2 (Fig. 1).5 In
this study we showed that these isoxazolopyridone derivatives
were allosteric mGluR7 antagonists and were selective against
mGluR1, mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, and mGluR8
at 1000 nM. However, only in vitro biological data were reported.
In this Letter, we describe the development of a novel solid sup-
port synthesis of the isoxazolopyridone derivatives which led to
the discovery of potent and selective 2 with improved physico-
chemical properties. And we also report on the PK profile and brain
penetrability of 2 in rats to determine if 2 could be useful for
in vivo study.
1d,e
However, mGluR7 has been the most intractable of the mGluR
subtypes in terms of discovery of selective ligands.
Until the discovery of AMN082 3 (Fig. 1), there have been no
selective agonists or antagonists of this receptor2 and it has been
* Corresponding author. Tel.: +81 29 865 4527; fax: +81 29 865 2170.
E-mail address: masayuki_nakamura2@oasis.ocn.ne.jp (M. Nakamura).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.070

M. Nakamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 726–729
727
N
2HCl
H
O
O
N
N
H
5
N
7
N
1
N
N
O
O
4
MeO
AMN082 (3)
2
1
Figure 1.
After screening the in-house chemical collection, 1, which
contains an isoxazolopyridone scaffold, was identified as a hit
compound. Compound 1 displayed mGluR7 antagonistic activity
(IC50 = 20 nM) and had no detectable activity on other mGluRs at
1000 nM.5 However, 1 showed poor metabolic stability (predicted
FH: 34%) on rat hepatocyte assay6 and low aqueous solubility
cally, as with the aryl portions on C-4, we attempted to develop
a new solid support synthesis of the isoxazolopyridone scaffold.
Taken the conventional synthesis shown in Scheme 1, we assumed
that the amide NH in 8 would be connected to an appropriate
resin and eventually released from under acidic conditions to
facilitate spontaneous cyclization of the corresponding 9, thereby
providing the desired products. After optimization of reaction
conditions, a solid support synthesis of the isoxazolopyridone
around the aryl portions at C-4 was developed as shown in
Scheme 2.
Reductive amination of commercially available ArgoGel-MB-
CHO Resin™ 10 with methyl amine and NaBH(OAc)3 gave methyl
amine 11, which was acylated with 5-methyl-3-phenylisoxazole-
4-carboxylic acid using 2-chloro-1,3-dimethylimidazolium
chloride. The resulting amide 12 was exposed to KHMDS in THF–
toluene (1:1) at room temperature, followed by addition of
commercially available esters10 to afford ketone 13. Finally, treat-
ment with 50% TFA in CH2Cl2 furnished the desired derivatives,
which were purified by silica gel chromatography or preparative
HPLC.
The data for substituents on the N-5 and aryl portions at C-7 of
the selected isoxazolopyridone derivatives are summarized in
Table 1. Substitution with larger ethyl and iso-propyl groups on
the N-5 position markedly decreased potency (14 and 15). In the
series of the C-7 derivatives, the methoxy derivatives (16, 17, and
18) have equipotent activity compared to 1 with retaining c log D7.4
value. 4-Aza derivative 19 with reduced lipophilicity slightly
decreased potency, while 3- and 2-aza derivatives (20 and 21)
displayed 12-fold and >50-fold decreases in potency, respectively.
Compound 19 also exhibited improvement of metabolic stability
(predicted FH: 65%) on rat hepatocyte assay. As expected, this result
(0.17 l
g/mL, pH 7.4).7 We assumed that poor metabolic stability
and low aqueous solubility may be due to its high lipophilicity
(c log D7.4: 3.5).8 Therefore, our investigation of this class aimed
to reduce the lipophilicity while retaining mGluR7 potency and
to obtain tool compounds useful for in vivo study.
Structurally, 1 can be divided into three parts: the substitution
on the N-5 position of the isoxazolopyridone core and the two aryl
portions at the C-4 and C-7 positions. We envisioned that these
three parts could be modified by introducing more hydrophilic
substituents and/or replacing the aryl with heteroaromatic groups
in order to reduce lipophilicity.
The synthetic route for the representative compound 2 is shown
in Scheme 1. Condensation of 4-pyridinecarboxaldehyde 3 with
hydroxylamine hydrochloride afforded oxime 4 in 94% yield. Oxime
4 was reacted with NCS in the presence of pyridine, followed by
addition of ethyl 3-pyrrolidine-crotonate 5 to give isoxazole 6 in
60% yield, which was hydrolyzed to acid 7 in quantitative yield. Acid
7 was condensed with methylamine to give the corresponding
amide 8 in 67% yield. Lithiation of amide 8 with excess n-BuLi at
ꢀ78 °C followed by addition of methylbenzoate afforded the desired
ketone 9 in 64% yield. Finally, cyclization of ketone 9 under acidic
conditions gave the desired isoxazolopyridone derivative 2 in 60%
yield. Other derivatives were synthesized in a similar manner, with
slight modification of reaction conditions.9
Solid support synthesis is one of the most powerful tools in
modern drug discovery to rapidly determine SAR data. Syntheti-
indicated that
a decrease in lipophilicity was effective for
N
N
a
c
b
N
O
O
N
N
O
HO
EtO
HO
N
N
N
O
O
O
3
4
OEt
6
7
5
N
N
O
N
O
N
O
N
e
d
f
H
O
N
O
N
N
H
O
N
O
MeO
8
9
2
MeO
Scheme 1. Synthesis of isoxazolopyridone 2. Reagents and conditions: (a) NH2OHꢁHCl, MeOH, reflux, 94%; (b) NCS, Py, CHCl3, 50 °C, then 5, Et3N, 60%; (c) NaOHaq, MeOH,
100%; (d) MeNH2, WSC, HOBt, DMF, rt, 67%; (e) n-BuLi, THF, -78 °C, then methyl 4-methoxybenzoate, 64%; (f) TsOH (cat.), toluene, reflux, 60%.

728
M. Nakamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 726–729
CHO
O
O
a
NHMe
b
N
O
N
Me
O
O
10 ArgoGel-MB-CHO ResinTM
11
12
O
O
c
d
N
N
Me
O
N
N
O
O
R3
O
R3
22 - 32
13
Scheme 2. Solid support synthesis of the isoxazolopyridone derivatives. Reagents and conditions: (a) MeNH2, NaBH(OAc)3, AcOH, DMF, rt; (b) 5-methyl-3-phenylisoxazole-
4-carboxylic acid, 2-chloro-1,3-dimethylimidazolium chloride, i-Pr2NEt, CHCl2, rt; (c) KHMDS, THF, rt, then appropriate benzoate esters, rt; (d) TFA, CH2Cl2.
Table 1
SAR for isoxazolopyridone derivatives
R2
O
R1
N
N
R3
O
Compd
R1
R2
R3
mGluR7
IC50 (nM)
c log D7.4
Metabolic stability predicted FH (%)
a
1
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
2
Me
Et
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
20
3.5
3.9
4.5
3.6
3.6
3.6
2.3
2.3
2.4
3.6
4.0
3.8
3.6
4.2
4.9
3.6
4.5
3.5
3.7
4.3
2.4
34
910
>10,000
29
12
21
101
240
1000
22
51
72
140
>10,000
150
31
96
26
210
580
26
i-Pr
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
4-MeO–Ph
3-MeO–Ph
2-MeO–Ph
4-Py
3-Py
2-Py
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
4-Py
65
50
Ph
4-MeO–Ph
4-Me–Ph
4-F–Ph
4-Me2N–Ph
4-i-PrO–Ph
4-n-Pr–Ph
3-Me2N–Ph
3-CF3–Ph
3,4-OCH2O–Ph
3-F–4-MeO–Ph
3-Br–4-MeO–Ph
4-MeO–Ph
76
a
þ
The IC50 values were calculated based on inhibition curves for 0.5 mM L-AP-induced Ca2 mobilization in CHO cells expressing mGluR7 and G
a15 and were the mean of at
least two independent assays.
improvement in metabolic stability in this class. Based on these
results, 4-pyridine moiety was selected as an optimal substituent
at the C-7 particularly due to its reduced lipophilicity.
(predicted FH: 76%). And aqueous solubility (3.3
was also improved compared to 1.
lg/mL, pH 7.4)
In order to determine if 2 could be useful for in vivo study, we
evaluated for PK profile11 and brain permeability12 in rats. The
physicochemical and pharmacokinetic profiles of derivative 2 are
given in Table 2. Compound 2 displayed good oral bioavailability
with low plasma clearance in rats. We then examined brain pene-
trability (Table 3). Compound 2 was orally administrated to rats at
10 mg/kg. Plasma levels were then measured at 0.5 and 2 h after
dosing, and brain penetrability (B/P) was also examined at 2 h
post-dosing. The results showed that 2 had moderate plasma expo-
sure and good brain penetrability.
SAR data for the C-4 aryl portion is also depicted in Table 1,
although the information was very limited due to synthetic diffi-
culty.10 Generally, electron donating 4-methoxy, 3-dimethylamino
and 3,4-methylenedioxy groups (22, 28, and 30) had tendency for
maintaining mGluR7 potency. Compounds with larger substituents
(26 and 27) and di-substituted compounds (31 and 32) were not
acceptable, suggesting that bulkiness around the C-4 aryl portion
was restricted.
Next, we combined the 4-pyridine group at the C-7 with the
4-methoxy at the C-4 based on the SAR above mentioned, provid-
ing compound 2. As expected, 2 showed retained potency, reduced
lipophilicity (c log D7.4: 2.4) and improved metabolic stability
In summary, synthesis and evaluation of a novel isoxazolopyri-
done class were performed. In the course of modification, novel so-
lid support synthesis of this scaffold was developed. As a result, we

M. Nakamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 726–729
729
Table 2
Compared profiles of 1 and 2 and pharmacokinetic parameters in SD ratsa
Compd
mGluR7
c log D7.4
Solubility, pH 7.4 (
lM/mL)
Predicted FH (%)
Fb (%)
AUC (lM h)
CLpc (mL/min/kg)
IC50 (nM)
1
2
20
26
3.5
2.4
0.17
3.3
34
76
NT
65
NT
0.71
NT
1.0
NT, not tested.
a
The values represent the mean for n = 3, iv (1 mg/kg) and po (3 mg/kg).
Oral bioavailability.
Plasma clearance.
b
c
Callaerts, P. F.; Cryan, J. F.; Molnar, E.; D’Hooge, R. J. Neurosci. 2006, 26, 6573; (f)
Mitsukawa, K.; Mombereau, C.; Lotscher, E.; Uzunov, D. P.; van der Putten, H.;
Flor, P. J.; Cryan, J. F. Neuropsychopharmacology 2006, 31, 1112.
Table 3
Brain penetration of 2
4. Mitsukawa, K.; Yamamoto, R.; Ofner, S.; Nozulak, J.; Pescott, O.; Lukic, S.;
Stoehr, N.; Mombereau, C.; Kuhn, R.; McAllister, K. H.; Putten, H.; Cryan, J. F.;
Flor, P. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 18712.
Compound
Rat plasma (
l
M) levela
2 h
B/P ratiob
0.5 h
5.1
5. Suziki, G.; Tsukamoto, N.; Fushiki, H.; Kawagishi, A.; Nakamura, M.; Kurihara,
H.; Mitsuya, M.; Ohkubo, M.; Ohta, H. J. Pharmacol. Exp. Ther. 2007, 323, 147.
6. The rank orders of predicted hepatic availability (FH %) using rat hepatocytes:
Shibata, Y.; Takahashi, H.; Ishii, Y. Drug Metab. Dispos. 2000, 28, 1518.
7. Yamashita, T.; Dohta, Y.; Nakamura, T.; Fukami, T. J. Chromatogr. 2008, 1182, 72.
8. Calculated log D7.4 values of compounds were expressed as c log D7.4 by
utilizing PALLAS prologD, version 3.0, CompuDrug international Inc., CA, US.
9. Nakamura, M.; Kurihara, H.; Ohkubo, M.; Tsukamoto, N. PCT Application,
WO02102807, 2002. Analytical data of compound 2: 1H NMR (300 MHz, CDCl3)
ppm: 3.45 (s, 3H), 3.90 (s, 3H), 6.56 (s, 1H), 7.03 (d, J = 8.9 Hz, 2H), 7.34 (d,
J = 8.9 Hz, 2H), 8.30 (d, J = 6.2 Hz, 2H), 8.80 (d, J = 6.2 Hz, 2H). ESI-MS: m/z 334
(M+1)+.
10. There was some limitation in this library. ortho-Substituted and pyridyl esters
were unreactive, and the desired derivatives could not be obtained.
11. Pharmacokinetics. Pharmacokinetic characterizations were conducted in male
SD rats following single oral and single intravenous administration. Single
doses of 2 were administered intravenously in a vehicle of PEG400/EtOH/
2
3.9
1.0
a
Compound 2 (10 mg/kg) was orally administered to rats (n = 3), and the plasma
levels were measured at 0.5 and 2 h.
Compound 2 (10 mg/kg) was orally administered to rats (n = 3), and the plasma
brain levels were measured at 2 h. B/P means the ratio of the brain level (nmol/g) to
b
plasma level (lM) of 2.
identified some isoxazolopyridone derivatives with potent mGluR7
antagonistic activity and metabolic stability compared to a hit
compound 1. Furthermore, 2 with improved physicochemical
properties and metabolic stability showed good oral bioavailability
and brain penetrability in rats. It is expected that 2 would be a
good pharmacological tool for elucidating the role of mGluR7 on
CNS functions in rodents and evaluation of 2 for efficacy in vivo
will be reported in due course.
H2O = 50:10:40 or orally by gavage in
a vehicle of 0.5% methylcellulose
aqueous suspension. Blood samples for the determination of drug plasma
concentrations were obtained at multiple time points up to 8 h after
administration. Blood samples were centrifuged to separate the plasma, and
the plasma samples were deproteinized with ethanol containing an internal
standard. Compound 2 and the internal standard were detected by LC–MS/MS
in a positive ionization mode using the electrospray ionization probe, and the
precursor to product ion combinations were monitored in multiple reaction
monitoring mode.
References and notes
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methylcellulose and orally administered to male Sprague-Dawley rats (7–
10 weeks old, 200–400 g) at 10 mg/kg. At designated time after administration,
blood was collected with a heparinized syringes from the abdominal aorta
under isoflurane anesthesia. Then, the head skin was cut open, and a dental 30
G needle was inserted between the cervical vertebrae, and it was further
inserted into the cavum subarachnoideale. After 50–100 lM cerebrospinal
fluid had been collected by a 1 mL syringe through a tube connected to the
needle, the brain was extracted. The blood sample was centrifuged (40 °C,
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deproteinized with threefold amount of ethanol containing an internal
standard. The brain sample was homogenized with 2.0 mL of water by
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threefold amount of ethanol containing an internal standard. Theses samples
were allowed to stand at ꢀ20 °C for 20 min and then centrifuged (4 °C, 12000g,
10 min). The supernatant was analyzed by LC–MS/MS, and the concentration of
the test compounds in the plasma, brain, and cerebrospinal fluid were
measured by the method using a relative calibration curve.
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