Identification of a novel benzoxazolone derivative as a selective, orally active 18 kDa translocator protein (TSPO) ligand

Article abstract of DOI:10.1021/jm401325r

Optimization of the pharmacokinetic properties for a series of benzoxazolone derivatives led to the identification of 9b, which showed anxiolytic effect in a rat model. However, 9b, like known benzodiazepines, induced motor impairment. Investigation into

Full text of DOI:10.1021/jm401325r

Brief Article
pubs.acs.org/jmc
Identification of a Novel Benzoxazolone Derivative as a Selective,
Orally Active 18 kDa Translocator Protein (TSPO) Ligand
Takayuki Fukaya,* Takeo Ishiyama, Satoko Baba, and Shuji Masumoto
Drug Research Division, Dainippon Sumitomo Pharma Co., Ltd., 33-94 Enoki-cho, Suita, Osaka 564-0053, Japan
*
S Supporting Information
ABSTRACT: Optimization of the pharmacokinetic properties for a series of benzoxazolone derivatives led to the identification
of 9b, which showed anxiolytic effect in a rat model. However, 9b, like known benzodiazepines, induced motor impairment.
Investigation into the cause of this unexpected side effect and management of 9b off-target binding affinity led to the
identification of 10d, which showed oral anxiolytic effect in the rat model with improved safety profile.
INTRODUCTION
■
Benzodiazepines such as diazepam are widely used for the
treatment of anxiety owing to their fast onset and potent
efficacy. However most benzodiazepines produce adverse
events, such as drug dependence, development of tolerance,
1
−3
and withdrawal symptoms, which limit their clinical use.
Antidepressants, including selective serotonin reuptake inhib-
itors, are also prescribed in the treatment of anxiety disorders,
although their anxiolytic effect is often delayed by several
3
weeks. Considering these drawbacks, there is great need for
new antianxiety agents with fast onset and potent effect but
minimum unfavorable side effects.
4
The 18 kDa translocator protein (TSPO), which was first
identified as a discrete receptor for diazepam, is known to be
5
Figure 1. Chemical structures of selected TSPO ligands.
functionally and structurally distinct from the central
6,7
benzodiazepine receptor (CBR).
TSPO functions as a
heterotrimeric complex with the 32 kDa voltage-dependent
anion channel (VDAC) and the 30 kDa adenine nucleotide
transporter (ANT) and is mainly distributed in the
4864 (1), including 7a and 10a as potent and selective TSPO
ligands. Benzoxazolone derivatives’ high affinity for TSPO
23
8
was rationalized in light of a pharmacophore model comprising
24,25
mitochondrial membrane of peripheral organs, including the
kidney, heart, and endocrine glands, and in glial cells in the
three lipophilic pockets and a hydrogen-donor group.
Additionally, we have found in a preliminary study on
benzoxazolone derivatives that substitution at the amide part
and the C-5 position in the benzoxazolone ring plays an
important role as hydrophobic group in TSPO binding. Owing
to the necessity of this hydrophobic group in TSPO binding, a
number of benzoxazolone derivatives have poor druglike
properties. Although the introduction of hydrophilic group
into the amide part or the C-5 position in the benzoxazolone
ring seems to be difficult, our structure−activity relationship
9
central nervous system (CNS). Although the physiological role
of TSPO in the CNS remains to be investigated, accumulated
evidence suggests that TSPO in the CNS most likely promotes
1
0−12
the synthesis of neurosteroids.
Indeed, it has been
suggested that stimulation of TSPO increases neurosteroid
concentration in the CNS by activating cholesterol transport
10−12
from the outer to the inner mitochondrial membrane,
which is known as the rate-limiting step of neurosteroids
1
3
(
SAR) survey led to the discovery of compounds bearing a
biosynthesis. As neurosteroids have been reported to have
1
4,15
hydrophilic substituent with acceptable TSPO affinity. Among
these compounds, 10a selectively bound to TSPO and showed
beneficial effects in psychiatric disorders,
TSPO ligands are
expected to be useful in the treatment for such disorders.
A wide variety of TSPO ligands have already been reported
26
anxiolytic effect in the rat Vogel conflict model. Although 10a
showed acceptable anxiolytic effect in rats, there was room for
improvement of its PK profile. On the basis of the results of a
preliminary SAR study of benzoxazolone derivatives, optimiza-
tion of 10a PK profile led to the identification of 9b as a
potential anxiolytic worthy of further development. However,
(
(
Figure 1). In addition to the classical TSPO ligands Ro5-4854
1) and PK11195 (2), several other TSPO ligands have
1
6
17
been identified. Among them are the indole derivative FGIN-1-
1
8
19,20
2
7 (3), the 8-oxopurine derivative AC-5216 (4),
the 2-
2
1
phenylindolglyoxylamide (5), and the pyrazolo[3,4-b]-
quinoline derivative (6).
22
We have recently reported a series of benzoxazolone
Received: May 5, 2013
derivatives designed by opening of the diazepine ring of Ro5-
Published: September 19, 2013
©
2013 American Chemical Society
8191
dx.doi.org/10.1021/jm401325r | J. Med. Chem. 2013, 56, 8191−8195

Journal of Medicinal Chemistry
Brief Article
regardless of its high selectivity for TSPO over CBR, 9b, like
conventional benzodiazepines, showed motor impairment.
In this paper, we describe our investigation into the cause of
moderate affinity for TSPO with improved solubility (45 μg/
mL at pH 7.4). On the basis of this finding, we selected the 3-
pyridyl unit as the amide part and prepared 8b and 9b. As 9b
exhibited improved TSPO affinity and metabolic stability, it was
selected for further PK examination. Both 9b and its HCl salt
(9b·HCl) showed acceptable solubility (20 and 23 μg/mL) in a
pH 6.8 buffer with bile acids, suggesting good bioavailability
after oral administration. Accordingly, we evaluated the PK
profile of 9b·HCl in rats after oral administration at 10 mg/kg.
As shown in Table S1 in the Supporting Information, 9b·HCl
displayed good oral absorption with low clearance and good
CNS penetration (brain/blood ratio of 1.3). Encouraged by
these results, we evaluated the anxiolytic effect of 9b·HCl in the
rat Vogel conflict model. As shown in Figure S1 in the
Supporting Information, oral administration of 9b·HCl (3.0
mg/kg) significantly produced an anxiolytic effect in the rat
model.
9
b unexpected side effects on motor coordination, and
management of 9b’s off-target binding affinity. Our efforts led
to the identification of 10d, which showed high binding affinity
and good selectivity for TSPO and exhibited anxiolytic effect in
the rat Vogel conflict model, producing few side effects
associated with benzodiazepines.
CHEMISTRY
■
The target compounds in this study were synthesized as
illustrated in Scheme 1. Compounds 16a−d were prepared via
a
Scheme 1. Synthesis of Benzoxazolone Derivatives
We examined the safety profile of 9b. As mentioned in the
Introduction, benzodiazepines are known to cause adverse
effects by allosterically modulating the action of GABA via
CBR. On the other hand, recent studies have shown that
selective TSPO ligands can have anxiolytic effect without
19,20
causing the side effects associated with benzodiazepines.
However, contrary to our expectation, 9b·HCl, like conven-
tional benzodiazepines, induced motor impairment in the
horizontal rotarod test at a dose of 300 mg/kg (Table S3 in the
Supporting Information). Investigation of the 9b·HCl
pharmacokinetics profile revealed that 9b·HCl’s concentration
in the rats does not correlate with the dose administered, i.e., 2-
fold increase in concentration for a 30-fold increase in dose
a
Reagents and conditions: (a) H , 5% Rh-C, THF, rt; (b) CDI, THF,
2
rt; (c) tert-butyl bromoacetate, K CO , DMF, rt; (d) ArB(OH) ,
Pd(PPh ) , K CO , 1,4-dioxane, H O, reflux; (e) HCl/dioxane,
AcOH, 50 °C; (f) amine, BOPCl, Et N, 1-hydroxybenzotriazole,
DMF, rt; (g) amine, EDCI, DMF, 50 °C.
2
3
2
3
4
2
3
2
(
10−300/kg) (Table S2 in the Supporting Information). As
similar concentration was observed (at 10 mg/kg, po) in mice,
b·HCl would have the potential to induce motor impairment
3
9
at concentrations relatively close to the concentration needed
for anxiolytic effect.
2
3
Suzuki−Miyaura coupling reaction of 15 with a selected
boronic acid. Deprotection of 16a−d with hydrogen chloride
To identify the mechanism by which 9b·HCl induces motor
impairment, a profiling of 9b using a broad package of in vitro
radioligand binding assays was conducted. In all assays, 9b was
tested in duplicate at 10 μM. The results of these assays showed
that 9b has potent activity at the rat brain site 2 sodium channel
(108% inhibition at 10 μM, IC = 0.70 μM) without significant
(
HCl) provided the key intermediate acid derivatives 17a−d.
Condensation of 17a−d with a selected amine was carried out
in the presence of phosphoric acid bis(2-oxooxazolidide)
chloride (BOPCl) or by combination of 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCI)
and 1-hydroxybenzotriazole to afford the corresponding
amide derivatives 8b, 9b, 10b−e, and 11b−e (Table 1).
Compounds 7a−d, 8a, 9a, 10a, and 11a were obtained as
50
inhibitory effect on other receptors (Table S7 in the Supporting
Information). As compounds that have affinity at the site 2
sodium channel are reported to induce failure of motor
23
27−29
indicated in our previous report.
coordination,
we decided to evaluate the affinity of
previously reported benzoxazolone derivatives (7a,c, 9a, 10a,
and 11a) at the rat site 2 sodium channel. As shown in Table 1,
a substitution at the C-5 position mainly affects site 2 sodium
channel binding. Among the tested compounds, 10a and 11a
showed negligible effect on the rat site 2 sodium channel and
were therefore selected for further modifications at the amide
part.
RESULTS AND DISCUSSION
■
The affinity of the prepared compounds for TSPO and for CBR
was evaluated by measuring each compound’s ability to displace
3
3
[
H]PK11195 and [ H]flumazenil binding to membranes
prepared from rat kidney and rat cerebral cortex. All tested
compounds had negligible affinity for CBR (<50% inhibition at
1
1
0 μM).
As shown in Table 1, 10b with R converted to an ethyl
23
In a previous paper, we have shown that incorporation of a
showed 2-fold increase in TSPO affinity compared to 10a,
albeit reduced metabolic stability. As introduction of a
substituent into the phenyl of the amide part was expected to
increase affinity for TSPO by preliminary study results, we
prepared 10c−e with substitutions at the phenyl ring of the
amide moiety. Contrary to our expectation, introduction of a
methoxy group (10c) or a trifluoromethoxy group (10e)
decreased the affinity for TSPO. On the other hand, a better
overall in vitro profile was identified with 10d having a p-
trifluoromethylphenyl as the amide moiety. Compound 10d
para electron-withdrawing substituent onto the phenyl ring at
the C-5 position of the benzoxazolone (8a and 9a) led to an
increase in TSPO binding affinity and metabolic stability (Table
1
). However, the obtained compounds 8a and 9a suffered from
poor solubility (<1.0 μg/mL at pH 7.4 and pH 2.5). To
improve the aqueous solubility of these compounds, we
decided to modify the amide part of selected compounds.
Our preliminary research on the amide part of benzoxazolone
derivatives revealed that 7c with a 3-pyridyl substituent has
8
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Journal of Medicinal Chemistry
Brief Article
Table 1. In Vitro Profile of Benzoxazolone Derivatives
solubility
d
(μg/mL)
R1
R2
TSPO K (nM)
a
CBR inhibition (%)
b
metabolic stability pH 7.4 pH 2.5 Na+ channel (site 2) IC50 (μM)
c
e
compd
Ar
i
7
a
Ph
Ph
Ph
Ph
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
Ph
1.6
23
5
1
<1.0
1.0
1.0
3.0
180
>1.0
f
7
7
7
8
8
9
9
1
1
1
1
1
1
1
1
1
1
b
2-Py
3-Py
4-Py
Ph
9
0
NT
c
28
29
2
0
45
>1.0
f
f
f
f
d
270
0.68
9.8
0.65
4.9
11
NT
26
24
67
32
24
0
NT
NT
<1.0
<1.0
<1.0
4.0
NT
f
a
p-CF
p-CF
p-CF
3
3
3
-Ph
0
<1.0
<1.0
<1.0
<1.0
5.0
NT
f
b
-Ph
3-Py
Ph
6
NT
a
O-Ph
0
0.19
0.70
>1.0
b
p-CF O-Ph
3-Py
Ph
0
3
0a
0b
0c
0d
0e
1a
1b
1c
1d
1e
3-Py
3-Py
3-Py
3-Py
3-Py
4-Py
4-Py
4-Py
4-Py
4-Py
14
21
0
440
f
f
f
Ph
4.4
19
NT
NT
NT
f
f
f
Me
Me
Me
Me
Et
p-MeO-Ph
9
NT
NT
NT
p-CF -Ph
8.6
26
0
38
NT
27
24
47
35
NT
1.0
420
>10
>1.0
>10
3
f
f
f
p-CF O-Ph
0
NT
NT
3
Ph
5.3
3.0
17
8
1.0
730
f
Ph
5
2.0
860
NT
f
Me
Me
Me
p-MeO-Ph
2
<1.0
130
NT
f
p-CF -Ph
6.6
23
0
3.0
>1000
NT
3
f
f
f
f
f
p-CF O-Ph
NT
NT
NT
NT
3
PK11195
1.7
a
b
K values represent the mean of one to three separate experiments run in duplicate using four concentrations of each compound. Percent inhibition
i
3
c
of [ H]flumazenil specific binding at 10 μM compound. Metabolic stability data refer to percent of compound remaining after incubation with rat
liver S-9 fraction (∼2.0 mg·protein/mL) and NADPH (∼3.0 mM) for 30 min at 37 °C. The initial concentration of each compound was 1.0 μM.
d
The solubility was determined by HPLC using the supernatant obtained after shaking of the buffered solution (pH 7.4 and pH 2.5) (0.4 mL)
e
3
containing 1 mg of tested compound, followed by centrifugation. IC represents displacement of [ H]batrachotoxin (5.0 nM) binding to rat brain
by each compound. Not tested.
50
f
showed slightly increased TSPO affinity (K = 8.6 nM) and
metabolic stability (38%) without affinity for the rat site 2
sodium channel. The same modification was carried out in the
significantly increased the number of shocks in this model
(Figure S2 in Supporting Information). As shown in Tables
S3−S6 in the Supporting Information, diazepam produced
motor impairment in the horizontal rotarod test, memory
impairment in the passive avoidance test, decrease in locomotor
activity, and elongation of hexobarbital-induced sleep at doses
close to those that produced anxiolytic effect. On the other
hand, 10d·HCl did not induce motor impairment in the
horizontal rotarod test, memory impairment, and decrease of
locomotor even at the high dose (Tables S3, S4, and S6 in
Supporting Information). Although 10d·HCl slightly prolonged
hexobarbital-induced sleep at 300 mg/kg, the extent of this
effect was much less than that of diazepam (Table S5 in
Supporting Information). These findings are consistent with
i
4-pyridyl derivative 11a, and a trend similar to that of 10a was
observed for TSPO affinity. Among the 4-pyridyl derivatives
prepared, 11b and 11d showed high affinity for TSPO (11b Ki
=
3.0 nM, 11d K = 6.6 nM) with acceptable metabolic stability
i
(11b, 24%; 11d, 35%). However, further profiling of the 4-
pyridyl derivatives 11b and 11d revealed that these compounds
at 1.0 μM significantly inhibit cytochrome P450 in isozyme
2C19 (11b, 71%; 11d, 73%), which could translate into
potential drug−drug interaction. Although not reported here,
introduction of a substituent in the adjacent position of the 4-
pyridine nitrogen resulted in a decrease of metabolic stability.
On the basis of these findings, 10d was selected as the most
promising compound for further evaluation.
Next we evaluated the pharmacokinetic properties of the
HCl salt of 10d (10d·HCl) in rats after intravenous and oral
administration at doses of 1 mg/kg iv and 10 mg/kg po,
respectively (Table S1 in Supporting Information). Compound
19
those from previous reports on TSPO ligand.
Finally, 10d profiling in various receptor and transporter
assays using proteins involved in the commonly observed
adverse events in the central nervous, cardiovascular, and
pulmonary systems was conducted. In all assays, 10d was tested
in duplicate at 10 μM. No significant affinity for any of the
tested receptors or transporters was observed (Table S7 in
Supporting Information). Profiling of 10d across species
revealed that 10d has high affinity for human TSPO with a
10d·HCl showed moderate bioavailability and good CNS
penetration (brain/blood = 1.2). Furthermore, 10d·HCl
showed a dose-dependent increase in serum concentration in
rats, presumably due to improved aqueous solubility. Addition-
ally, we confirmed serum concentration of 10d·HCl after oral
administration (10 mg/kg) in mice which was similar to that in
rats (Table S2 in Supporting Information). Then we assessed
the anxiolytic effect of 10d·HCl in the rat Vogel-type conflict
model and evaluated its potential for benzodiazepines side
effects. Oral administration of 10d·HCl (1.0 mg/kg)
K of 8.0 nM and good metabolic stability in human liver S-9
i
(
84% remaining after 30 min). Further pharmacological
investigation of 10d, including its effects on the synthesis of
neurosteroids, is underway.
8
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Journal of Medicinal Chemistry
Brief Article
CONCLUSION
REFERENCES
■
■
We report here the optimization of a series of benzoxazolone
derivatives as a novel selective TSPO ligand. Our efforts led to
the identification of 10d as a potent and highly selective ligand
for TSPO. Compound 10d·HCl exhibited oral anxiolytic effect
in the rat model with few side effects associated with
conventional benzodiazepines. Compound 10d·HCl also
showed satisfactory pharmacokinetics profile, brain exposure,
and preliminary safety profile and has now progressed to
toxicity studies. Further detailed pharmacological evaluation of
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EXPERIMENTAL SECTION
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ASSOCIATED CONTENT
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■
*
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AUTHOR INFORMATION
Corresponding Author
Phone: +81-6-6337-5865. Fax: +81-6-6337-6047. E-mail:
takayuki-fukaya@ds-pharma.co.jp.
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Notes
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ACKNOWLEDGMENTS
■
(
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We appreciate Satoko Toma and Hitomi Oki for technical
assistance in biological evaluations. We thank Keiko Bando for
performing the elemental analysis and Makiko Homma for
recording high-resolution mass spectra.
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ABBREVIATIONS USED
■
TSPO, 18 kDa translocator protein; CBR, central benzodiaze-
pine receptor; VDAC, voltage-dependent anion channel; ANT,
adenine nucleotide transporter; CNS, central nervous system;
CDI, 1,1′-carbonyldiimidazole; BOPCl, phosphoric acid bis(2-
oxooxazolidide) chloride; EDCI, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride; SEM,
standard error of the mean
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V.; Puia, G.; Costa, E.; Guidotti, A. 2-AryI-3-Indoleacetamides (FGIN-
): a new class of potent and specific ligands for the mitochondrial
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Journal of Medicinal Chemistry
Brief Article
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