Synthesis and biological evaluation of D-amino acid oxidase inhibitors

Article abstract of DOI:10.1021/jm800200u

D-Amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids including D-serine, a full agonist at the glycine site of the NMDA receptor. A series of benzo[d]isoxazol-3-ol derivatives were synthesized and evaluated as DAAO inhibitors. Among them,

Full text of DOI:10.1021/jm800200u

J. Med. Chem. 2008, 51, 3357–3359
3357
Synthesis and Biological Evaluation of
D-Amino Acid Oxidase Inhibitors
Dana Ferraris,† Bridget Duvall,† Yao-Sen Ko,†
Ajit G. Thomas,† Camilo Rojas,† Pavel Majer,†
Kenji Hashimoto,‡ and Takashi Tsukamoto*,†
MGI Pharma, Inc., 6611 Tributary Street,
Baltimore, Maryland 21224, and DiVision of Clinical Neuroscience,
Chiba UniVersity Center for Forensic Mental Health, 1-8-1 Inohana,
Chiba 260-8670, Japan
ReceiVed February 25, 2008
Abstract: D-Amino acid oxidase (DAAO) catalyzes the oxidation of
D-amino acids including D-serine, a full agonist at the glycine site of
the NMDA receptor. A series of benzo[d]isoxazol-3-ol derivatives were
synthesized and evaluated as DAAO inhibitors. Among them, 5-chloro-
benzo[d]isoxazol-3-ol (CBIO) potently inhibited DAAO with an IC50
in the submicromolar range. Oral administration of CBIO in conjunction
with D-serine enhanced the plasma and brain levels of D-serine in rats
compared to the oral administration of D-serine alone.
We tested other structurally related compounds, muscimol,
(S)-AMPA, benzo[d]isoxazol-3-ol (BIO), 6-chloro-3-methoxy-
benzo[d]isoxazole (CMBI), and 4,5,6,7-tetrahydrobenzo[d]is-
oxazol-3-ol (THBIO). All of these compounds except BIO
exhibited negligible DAAO inhibition.
These findings led us to speculate that CBIO inhibits DAAO
in a mode similar to that of benzoic acid, a prototype competitive
DAAO inhibitor (Ki ≈ 16 µM).10 Figure 1A illustrates a crystal
structure of DAAO complexed with benzoic acid.11 As il-
lustrated in Figure 1B, we propose that the isoxazole group of
CBIO plays a role similar to the carboxylate group of benzoic
acid, forming a critical hydrogen bond network with Arg283
and Tyr228. This explains the loss of DAAO inhibitory potency
observed in CMBI. The benzene ring of CBIO is believed to
lay parallel to the flavin ring with Tyr224 on the opposite side
of the benzene ring, leading to strong π-π stacking interactions.
This may explain the negligible DAAO inhibition achieved by
a saturated analogue, THBIO, due to its inability to form these
interactions. These preliminary biological results prompted the
synthesis of a variety of benzo[d]isoxazol-3-ols to evaluate
structure-activity relationships within this class of compounds.
Our initial attempt to synthesize benzo[d]isoxazol-3-ol de-
rivatives 3 involved the formation of N-hydroxysalicylamides
2 from salicylic acid methyl esters 1 (Scheme 1). Cylization of
2 by the treatment with thionyl chloride (step b)12 or carbonyl
diimidazole (step c),13 however, afforded undesired product 4
or 5, respectively, as major products, providing 3 in low yields.
To circumvent the formation of these byproducts, we
developed an alternative method that utilizes 2-fluorobenzoyl
chlorides 6 and N-(2,4,6-trimethoxybenzyl)hyroxylamine 7
(Scheme 2). Coupling of 6 and 7 afforded N-hydroxybenzamide
derivatives 8. Subsequent treatment with potassium carbonate
followed by deprotection yielded benzo[d]isoxazol-3-ol deriva-
tives 3.
The preclinical and clinical evidence supporting the role of
NMDAa receptor hypofunction in schizophrenia has prompted
clinical trials of agents that enhance NMDA receptor function.1
For example, schizophrenic patients receiving D-serine, a full
agonist at the glycine site of the NMDA receptor, with
concomitant neuroleptic therapy have shown significant im-
provements in their positive, negative, and cognitive symptoms.2
Furthermore, reduced D-serine levels were reported in the serum
and cerebrospinal fluid of schizophrenic patients.3,4
In animals, however, D-serine is believed to be metabolized
substantially by D-amino acid oxidase (DAAO), diminishing its
oral bioavailability.5 In addition, at high doses, D-serine is
reported to cause selective necrosis to the pars recta region of
the renal proximal tubules in the rat.6 The mechanism of
D-serine-induced nephrotoxicity is believed to be associated with
oxidative stress caused by hydrogen peroxide, a byproduct of
DAAO-mediated metabolism of D-serine.7
These findings prompted us to identify small molecule DAAO
inhibitors that can be coadministered with D-serine to minimize
its metabolism by DAAO. DAAO inhibition should not only
improve bioavailability of D-serine but also reduce its nephro-
toxic effects. This paper describes the synthesis of a series of
small molecule DAAO inhibitors based on a benzo[d]isoxazol-
3-ol core structure and the in vivo effects of DAAO inhibition
on D-serine pharmacokinetics.
Using a fluorescence-based DAAO assay,8 we screened a
large number of compounds for their ability to inhibit DAAO
and found that 6-chlorobenzo[d]isoxazol-3-ol (CBIO) potently
inhibits DAAO with an IC50 of 188 nM.9 Subsequent kinetic
studies showed that CBIO is a competitive inhibitor with respect
to D-serine with a Ki of 100 nM.
* To whom correspondence should be addressed. Phone: 410-631-6762.
Fax: 410-631-6804 . E-mail: takashi.tsukamoto@mgipharma.com.
†
MGI Pharma, Inc.
Chiba University Center for Forensic Mental Health.
‡
a Abbreviations: NMDA, N-methyl-D-aspartate; DAAO, D-amino acid
oxidase; CBIO, 6-chlorobenzo[d]isoxazol-3-ol; AMPA, R-amino-3-hydroxy-
5-methyl-4-isoxazolepropionic acid; BIO, benzo[d]isoxazol-3-ol; CMBI,
6-chloro-3-methoxybenzo[d]isoxazole; THBIO, 4,5,6,7-tetrahydrobenzo[d-
]isoxazol-3-ol.
Figure 1. (A) Schematic illustration of the active site of DAAO in
complex with benzoic acid. (B) Proposed model of CBIO bound to
DAAO.
10.1021/jm800200u CCC: $40.75
2008 American Chemical Society
Published on Web 05/29/2008

3358 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Scheme 1. Synthesis of Benzo[d]isoxazol-3-olsa
Letters
a Reagents and conditions: (a) hydroxylamine hydrochloride, KOH,
methanol, room temp, 20-95%; (b) pyridine, thionyl chloride, THF, room
temp, 27-37%; (c) carbonyl diimidazole, reflux, 1-33%.
Scheme 2. Synthesis of Benzo[d]isoxazol-3-olsa
Figure 2. Effects of CBIO on rat plasma levels of D-serine following
oral administration of D-serine. Male SD rats were administered D-serine
(30 mg/kg, po) or D-serine (30 mg/kg, po) and CBIO (30 mg/kg, po).
Each point shown is the mean ( SD (n ) 4).
a Reagents and conditions: (a) triethylamine, dichloromethane, room
temp, 45-60%; (b) potassium carbonate, DMF, 120 °C, 47-99%; (c)
triisopropylsilane, TFA, dichloromethane, room temp, 12-61%.
Table 1. Inhibition of DAAO by Benzo[d]isoxazol-3-ol Derivatives
compd
R4
R5
R6
R7
IC50 (µM)a
BIO
3a
3b
3c
3d
3e
3f
CBIO
3g
3h
3i
3j
3k
3l
H
CF3
F
H
H
H
H
H
H
H
F
Cl
CH3
OMe
OEt
NO2
CF3
CF3
H
H
1.88 ( 0.00
>100
H
H
Br
I
H
H
H
H
H
H
H
H
H
H
H
H
F
7.82 ( 0.96
0.599 ( 0.006
1.51 ( 0.12
2.95 ( 0.15
0.444 ( 0.032
0.188 ( 0.001b
0.269 ( 0.013
2.57 ( 0.27
>100
0.722 ( 0.024
5.00 ( 0.24
>100
23.0 ( 0.3
>100
Figure 3. Effects of CBIO on rat brain levels of D-serine following
oral administration of D-serine. Male SD rats were administered D-serine
(30 mg/kg, po), CBIO (30 mg/kg, po), or D-serine (30 mg/kg, po) and
CBIO (30 mg/kg, po) at t ) 0 min. D-Serine levels are expressed as
percent of the basal level. Each point shown is the mean ( SD (n )
4).
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
H
H
H
H
H
H
H
H
H
H
and only accommodates substrates or inhibitors of moderate size.
This may pose a challenge to further optimization of benzo[d]
isoxazol-3-ol-based DAAO inhibitors because minor structural
modifications are unlikely to generate an additional hydrophobic
interaction at the active site that could contribute to a higher
affinity for DAAO.
3m
3n
F
CH3
H
a Values are the mean ( SE of duplicate experiments. b n ) 4.
To assess the effects of DAAO inhibition on plasma levels
of D-serine in rats, D-serine was given orally with or without
CBIO, and plasma samples were analyzed for D-serine levels
using an enantiospecific bioanalytical method.15
As shown in Figure 2, coadministration of CBIO (30 mg/
kg) markedly enhanced the plasma levels of D-serine in rats
compared to D-serine alone treatment. The results demonstrate
that an orally administered DAAO inhibitor can enhance the
oral bioavailability of coadministered D-serine.
To assess whether the increased plasma concentrations of
D-serine would reflect the extracellular D-serine levels, we treated
rats with oral D-serine +/- CBIO and analyzed microdialysis
samples from the prefrontal cortex for D-serine concentrations.15
As shown in Figure 3, oral D-serine increased the extracellular
D-serine levels by 25% compared to the basal levels. At some
A large number of benzo[d]isoxazol-3-ols were synthesized
using one of the three methods described above and tested for
their ability to inhibit DAAO. Table 1 summarizes in vitro
DAAO inhibitory data of these compounds.
In general, larger substituents on the benzene ring caused a
significant increase in IC50 values. Smaller groups are better
tolerated especially at positions 5 and 6. Compounds with similar
potency to CBIO have small substituents at one of these
positions as represented by 3c, 3f, 3g, and 3j.
These observations are in a good agreement with DAAO’s
preferred substrate specificity for D-amino acids bearing hy-
drophobic side chains up to four carbon atoms long or compact
ring systems.14 Our SAR analysis coupled with previous findings
suggests that the active site pocket of DAAO is of limited space

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3359
receptor hypofunction hypothesis of schizophrenia. Arch. Gen. Psy-
chiatry 2003, 60, 572–576.
(4) Hashimoto, K.; Engberg, G.; Shimizu, E.; Nordin, C.; Lindstrom, L. H.;
Iyo, M. Reduced D-serine to total serine ratio in the cerebrospinal fluid
of drug naive schizophrenic patients. Prog. Neuropsychopharmacol.
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(5) Curti, B.; Ronchi, S.; Simonetta, P. M. D- and L-Amino Acid Oxidases.
In Chemistry and Biochemistry of FlaVoenzyme; Mu¨ller, F., Ed.; CRC
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Crespo, I. Direct calcium binding results in activation of brain serine
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(9) Simultaneous to our work on this series, two patent applications were
published disclosing the same series of DAAO inhibitors. (a) Kennis,
L. E. J.; Vanhoof, G. C. P.; Bongartz, J.-P. A. M.; Luyckx, M. G. M.;
Minke, W. E. Benzisoxazoles. PCT Int. Appl. WO 2005/089753, 2005.
(b) Fang, Q. K.; Hopkins, S.; Jones, S. Benzo[d]isoxazol-3-ol DAAO
Inhibitors. U.S. Pat. Appl. 20050143434, 2005.
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B. Limited proteolysis and X-ray crystallography reveal the origin of
substrate specificity and of the rate-limiting product release during
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oxidase. Biochemistry 1997, 36, 5624–5632.
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(12) Kalkote, U. R.; Goswami, D. D. New synthesis of 1,2-benzisoxazole
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points, the extracellular levels of D-serine following coadmin-
istration of CBIO (30 mg/kg) increased by more than 60%
compared to the basal levels.
Figure 3 also shows that oral administration of CBIO alone
did not affect the extracellular levels of D-serine. Therefore, the
enhancement of D-serine levels in the prefrontal cortex following
coadministration of D-serine and CBIO is likely due to increased
systemic D-serine levels because D-serine is capable of penetrat-
ing the blood-brain barrier.17 One could attribute CBIO’s
inability to autonomously enhance D-serine levels in the brain
to its poor blood-brain barrier (BBB) permeability.
Interestingly, a recent report from Dr. Adage’s group showed
a slight increase in D-serine levels in rat cortex and midbrain
following intravenous administration of a pyrazole-3-carboxylate
based DAAO inhibitor alone.16 This effect may be due to its
superior ability to penetrate the blood-brain barrier. It has been
reported, however, that there is little overlap in distribution of
DAAO and NMDA receptors in the brain.18 Therefore, even a
brain-penetrable DAAO inhibitor may not be able to signifi-
cantly enhance NMDA receptor-mediated neurotransmission by
itself.
Our study indicates that coadministration of D-serine and a
DAAO inhibitor represents a more effective approach for
delivering D-serine to the site of its action. Inhibition of DAAO
should also reduce oxidative stress and reduce nephrotoxicity.7
Pharmacological evaluation of D-serine/CBIO coadministration
is currently underway using animal models of schizophrenia.
(13) Geffken, D. The cyclisation of salicylamides and salicylohydroxamic
acids with 1,1′-carbonyldiimidazole. Liebigs Ann. Chem. 1981, 1513–
1514.
(14) Dixon, M.; Kleppe, K. D-Amino acid oxidase II. Specificity, competi-
tive inhibition and reaction sequence. Biochim. Biophys. Acta 1965,
96, 368–382.
Acknowledgment. We thank Yuko Fujita for conducting
in vivo experiments in rats. This work is partly supported by a
grant from the Ministry of Education, Culture, Sports, Science
and Technology of Japan (Grant 18053004 to K.H.).
(15) Fukushima, T.; Kawai, J.; Imai, K.; Toyo’oka, T. Simultaneous
determination of D- and L-serine in rat brain microdialysis sample using
a column-switching HPLC with fluorimetric detection. Biomed.
Chromatogr. 2004, 18, 813–819.
(16) Adage, T.; Trillat, A. C.; Quattropani, A.; Perrin, D.; Cavarec, L.;
Shaw, J.; Guerassimenko, O.; Giachetti, C.; Greco, B.; Chumakov,
I.; Halazy, S.; Roach, A.; Zaratin, P. In vitro and in vivo pharmaco-
logical profile of AS057278, a selective D-amino acid oxidase inhibitor
with potential anti-psychotic properties. Eur. Neuropsychopharmacol.
2008, 18, 200–214.
(17) Bauer, D.; Hamacher, K.; Broer, S.; Pauleit, D.; Palm, C.; Zilles, K.;
Coenen, H. H.; Langen, K. J. Preferred stereoselective brain uptake
of D-serine, a modulator of glutamatergic neurotransmission. Nucl.
Med. Biol. 2005, 32, 793–797.
(18) Horiike, K.; Tojo, H.; Arai, R.; Nozaki, M.; Maeda, T. D-Amino-acid
oxidase is confined to the lower brain stem and cerebellum in rat brain:
regional differentiation of astrocytes. Brain Res. 1994, 652, 297–303.
Supporting Information Available: Synthetic procedures for
CMBI and 3a-n, elemental analysis data, in vitro D-amino acid
oxidase assay method, and in vivo experimental procedures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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JM800200U