4-Hydroxypyridazin-3(2 H)-one derivatives as novel d-amino acid oxidase inhibitors

Article abstract of DOI:10.1021/jm400095b

d-Amino acid oxidase (DAAO) catalyzes the oxidation of d-amino acids including d-serine, a coagonist of the N-methyl-d-aspartate receptor. We identified a series of 4-hydroxypyridazin-3(2H)-one derivatives as novel DAAO inhibitors with high potency and substantial cell permeability using fragment-based drug design. Comparisons of complex structures deposited in the Protein Data Bank as well as those determined with in-house fragment hits revealed that a hydrophobic subpocket was formed perpendicular to the flavin ring by flipping Tyr224 in a ligand-dependent manner. We investigated the ability of the initial fragment hit, 3-hydroxy-pyridine-2(1H)-one, to fill this subpocket with the aid of complex structure information. 3-Hydroxy-5-(2- phenylethyl)pyridine-2(1H)-one exhibited the predicted binding mode and demonstrated high inhibitory activity for human DAAO in enzyme-and cell-based assays. We further designed and synthesized 4-hydroxypyridazin-3(2H)-one derivatives, which are equivalent to the 3-hydroxy-pyridine-2(1H)-one series but lack cell toxicity. 6-[2-(3,5-Difluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)- one was found to be effective against MK-801-induced cognitive deficit in the Y-maze.

Full text of DOI:10.1021/jm400095b

Article
pubs.acs.org/jmc
4‑Hydroxypyridazin-3(2H)‑one Derivatives as Novel D‑Amino Acid
Oxidase Inhibitors
Takeshi Hondo,* Masaichi Warizaya, Tatsuya Niimi,* Ichiji Namatame, Tomohiko Yamaguchi,
Keita Nakanishi, Toshihiro Hamajima, Katsuya Harada, Hitoshi Sakashita, Yuzo Matsumoto,
Masaya Orita, and Makoto Takeuchi
Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
S
* Supporting Information
ABSTRACT: D-Amino acid oxidase (DAAO) catalyzes the
oxidation of D-amino acids including D-serine, a coagonist of
the N-methyl-D-aspartate receptor. We identified a series of 4-
hydroxypyridazin-3(2H)-one derivatives as novel DAAO inhib-
itors with high potency and substantial cell permeability using
fragment-based drug design. Comparisons of complex structures
deposited in the Protein Data Bank as well as those determined
with in-house fragment hits revealed that a hydrophobic
subpocket was formed perpendicular to the flavin ring by flipping
Tyr224 in a ligand-dependent manner. We investigated the ability
of the initial fragment hit, 3-hydroxy-pyridine-2(1H)-one, to fill
this subpocket with the aid of complex structure information. 3-
Hydroxy-5-(2-phenylethyl)pyridine-2(1H)-one exhibited the pre-
dicted binding mode and demonstrated high inhibitory activity for human DAAO in enzyme- and cell-based assays. We further
designed and synthesized 4-hydroxypyridazin-3(2H)-one derivatives, which are equivalent to the 3-hydroxy-pyridine-2(1H)-one
series but lack cell toxicity. 6-[2-(3,5-Difluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one was found to be effective against MK-
801-induced cognitive deficit in the Y-maze.
Several compounds have been reported to exert inhibitory
activity against DAAO (Figure 1), including benzoic acid 1,14 5-
methylpyrazole-3-carboxylic acid (AS057278) 2a,15 6-
chlorobenzo[d]isoxazole-3-ol (CBIO) 3,16 4H-thieno[3,2-b]-
pyrrole-5-carboxylic acid 4, and 4H-furo[3,2-b]pyrrole-5-
carboxylic acid 5,17 which are all characterized as aryl carboxylic
acids or corresponding acid-isosteres with small molecular
weight. Although these compounds are reported to exhibit high
inhibitory activity for human DAAO in cell-free systems and
IC50 values in the submicromolar to nanomolar range (except
benzoic acids), their high acidity and low hydrophobicity
hamper cell permeability. Two recently reported inhibitors, 5-
chloro-6-fluoro-3-hydroxy-1,8-naphthyridin-2(1H)-one 618 and
4,6-difuloro-1-hydroxy-1H-benzo[d]imidazol-2(3H)-one 7,19
overcome this drawback; however, their structures leave little
room to introduce new chemical groups to either or both
increase the activity or modify physicochemical properties for
improving the pharmacokinetics profiles.18 While administra-
tion of DAAO inhibitors can ameliorate some NMDAR
antagonist-induced deficits,1,2 such effects have not been
observed across all behaviors related to NMDAR activity.17
Further, although NMDAR-mediated behaviors are indicative
of activity in forebrain regions, the addition of DAAO inhibitors
INTRODUCTION
■
The flavoenzyme D-amino acid oxidase (DAAO) catalyzes the
stereospecific oxidative deamination of D-amino acids to α-keto
acids, NH3, and H2O2. D-Serine, which is metabolized by
DAAO, acts as an endogenous coagonist at the glycine site of
the N-methyl D-aspartate receptor (NMDAR) as well as
glycine. Noncompetitive NMDAR antagonists such as ketamine
or phencyclidine have been shown to induce psychotic and
neurocognitive symptoms similar to those observed in
schizophrenia patients, including positive, negative, and
cognitive symptoms.1,2 Additionally, hypofunction of
NMDAR has been hypothesized to be alleviated by increasing
the D-serine level via DAAO inhibition without excessive
stimulation. Indeed, several factors have shown links between
DAAO, D-serine, and schizophrenia. Decreased levels of D-
serine in CSF and brain have been seen in schizophrenia
patients,3−5 while administration of high doses of D-serine in
combination with antipsychotic drugs was found to reduce
positive, negative, and cognitive symptoms of schizophrenia.6,7
DAAO expression and activity have been reported to be
elevated in schizophrenia patients,8,9 and mutant mice lacking
functional DAAO are resistant to the effects of NMDAR
antagonists.10−12 Furthermore, the G72 gene on chromosome
13q, which is thought as a DAAO activator, has been
significantly associated with schizophrenia.13
Received: January 21, 2013
Published: April 8, 2013
© 2013 American Chemical Society
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Figure 1. Representative inhibitors of DAAO.
increases D-serine levels in the cerebellum but not in the
cortex.17,20 Therefore, compounds with more potent activity
and better pharmacokinetics profiles than known inhibitors are
preferred for testing the therapeutic value of DAAO inhibitors.
Here, we report 4-hydroxypyridazin-3(2H)-one derivatives as
novel DAAO inhibitors using fragment-based drug discovery
(FBDD), a recent and novel approach for the identification of
small-molecular inhibitors which has already helped identify a
number of clinical candidates by taking full advantage of
structural information on a number of complex structures.21
Using X-ray crystallography, we designed and synthesized
compounds that can utilize a subpocket formed by flipping
Tyr224. The subpocket is considered unoccupied in complex
structures with inhibitors 1−7, and using this pocket, we
identified compounds with high potency and substantial cell
permeability.
Scheme 2. Synthesis of 4-Hydroxypyridazin-3(2H)-one
Derivatives 13−20 and 22−32
a
a
Reagents and conditions: (a) R1CHC(R2)B(OH)2 or correspond-
ing pinacol ester, Na2CO3, Pd(PPh3)4, toluene−H2O, 120 °C, or
terminal alkynes, Cs2CO3, PdCl2(PPh3)2, CuI, DMF, 100 °C; (b)
CHEMISTRY
■
22
t
PhCH2OH, BuOK, toluene, 120 °C; (c) 2,4,6-triisopropylbenzene-
sulfonyl hydrazide, DCM, rt; (d) BBr3, DCM, 0 °C; (e) H2, Pd−C,
EtOH, rt.
Compound 12 was prepared as illustrated in Scheme 1. 5-
Bromo-3-hydroxypyridine-2(1H)-one 33 was first converted to
a
Scheme 1. Synthesis of 12
phenol yielded 40. Compound 21 was obtained from 40 in the
same manner as shown in Scheme 2.
RESULTS AND DISCUSSION
■
Fragment Screening and Complex Structure Deter-
mination. Six tertiary structures of human DAAO complexed
with inhibitors or ligand analogues were deposited in the
Protein Data Bank (PDB). The binding pocket, which is packed
between Tyr224 and a flavin ring, appears to accommodate
only small-sized compounds corresponding to a two-fused ring.
Comparisons of all structures revealed that Tyr224 adopts two
conformations (Figure 2). In the first conformation, the phenyl
ring of Tyr224 is oriented toward the side chain of Leu215 so
that the overlap between the phenyl ring of Tyr224 and flavin
ring is minimized (PDB ID: 2DU8, 2E4A; Tyr224-in). In the
second conformation, Tyr224 moves so that a subpocket
surrounded by Gln53, Leu215, His217, and Tyr224 is formed
perpendicular to the original binding pocket (PDB ID: 2E49,
2E82, 3CUK, 3G3E; Tyr224-out). In the complex structure
with substrate analogue imino-DOPA, the catechol moiety fills
this subpocket.23 3-(2-Phenylethyl)-1H-pyrazole-5-carboxylic
acid 2b,24 which has a bulkier side chain than 2a, was also
predicted to stretch into this subpocket in the recent review.25
Because the subpocket faces an entry site and is large enough
to introduce chemical groups, we carried out high-concen-
tration screening of an in-house 3500 fragment library to find
a
Reagents and conditions: (a) CH3OCH2Cl, NaH, DMF, 0 °C; (b)
PhCHCHB(OH)2, K3PO4, Pd(PPh3)4, dioxane−H2O, 100 °C; (c)
H2, Pd−C, EtOH, rt; (d) BBr3, DCM, 0 °C.
the 2,3-bisprotected pyridine 34. Suzuki-coupling reaction
using 2-phenylvinyl boronic acid with 34 and hydrogenation
of the double bond and subsequent deprotection gave 12.
4-Hydroxypyridazin-3(2H)-one derivatives were prepared as
illustrated in Scheme 2. 3-Chloro[1,4]benzodioxino[2,3-c]-
pyridazine 35 was used as the starting material and converted
by Suzuki or Sonogashira coupling to intermediates 36.
Subsequent reduction of multiple bonds followed by
deprotection gave 4-hydroxypyridazin-3(2H)-one derivatives
13, 14, 16−20, and 22−32. Compound 15 was directly
deprotected after the coupling reaction.
Compound 21, which has an ether linkage, was prepared as
illustrated in Scheme 3. 3-Chloro[1,4]benzodioxino[2,3-c]-
pyridazine 35 was first converted to corresponding ester 38
by CO insertion. After the reduction of the methyl ester and
conversion to the corresponding leaving group, alkylation with
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a
Scheme 3. Synthesis of 21
a
Reagents and conditions: (a) Pd(OAc)2, dppf, CO, Et3N, DMSO−MeOH, 80 °C; (b) NaBH4, THF−MeOH, 0 °C; (c) SOCl2, DCE, 80 °C; (d)
22
t
PhOH, K2CO3, DMF, rt; (e) PhCH2OH, BuOK, toluene, 120 °C; (f) H2, Pd−BaSO4, EtOH, rt.
Tyr224-in conformation. Among the fragment hits, we selected
8 as a starting fragment because it did not contain highly acidic
groups and was found to induce the Tyr224-out conformation,
which helped us access the newly identified subpocket (Figure
3A). Introduction of a flexible linker into the 5-position of 8
Figure 2. Binding sites of human DAAO in (upper) Tyr224-in
conformation (PDB ID: 2DU8) and (lower) Tyr224-out conforma-
tion (PDB ID: 3CUK). Flavin ring of FAD (flavin adenine
dinucleotide) and inhibitors are shown in orange and purple,
respectively. Tyr224 residues are shown in yellow, and other residues
are shown in green. The right panels are views in a different
orientation to highlight the subpocket in Tyr224-out conformation
indicated in dotted circle. The surfaces of the binding sites are
represented in green. Figures were prepared using MOE2011
(Chemical Computing Group Inc., Quebec, Canada).
Figure 3. Binding sites of human DAAO in the complex structures
with 8 (A) and 12 (B). Hydrogen bonds are shown in dotted lines.
Color schemes and the software used to generate the figures are the
same as those in Figure 2.
fragment hits that were expected to be elongated into the
subpocket on the basis of complex structures. Most of the
fragment hits belonged to small aryl carboxylic acids as
predicted. One exception was 3-hydroxy-pyridine-2(1H)-one
8, which is a substructure of 6 (Figure 1). All fragment hits had
a common interaction motif: π−π stacking with re-face of the
flavin ring and the phenyl ring of Tyr224, and an electrostatic
interaction with Arg283. Tyr224 residues adopted two
conformations in the complex structures of fragment hits as
well as those in the PDB. We found that hydrogen-bonding
with Gly313 induces the Tyr224-out conformation. Although
the mechanism of this conformation switch is not clear, this
rule held true for each complex structure (data not shown).
The only exception in the PDB and in-house structure data was
the complex structure (PDB ID: 2E4A), in which the amino
group of o-aminobenzoate makes a suboptimal hydrogen bond
with the carbonyl atom of Gly313 when Tyr224 adopts the
seemed to connect the original pocket and the subpocket
without strain on the basis of the complex structure, indicating
that 8 is an appropriate starting fragment to fill the subpocket
during subsequent elongation. This discovery motivated us to
design and synthesize 3-hydroxy-pyridine-2(1H)-one deriva-
tives.
Synthesis and SAR Analysis of 3-Hydroxypyridine-
2(1H)-one Derivatives. Unsubstituted 3-hydroxy-pyridine-
2(1H)-one compound 8 was much less potent than known
inhibitor 5 in the enzyme inhibitory assay. On the basis of the
complex structure of 8 and DAAO, we speculated that the 5-
position was appropriate to elongate substituents into the
subpocket and that it was difficult to introduce large
substituents to the 4- and 6-positions because there is little
room for elongation.
First, we introduced a chloro and methyl group to the 5-
position. The resulting compounds exhibited significantly
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enhanced activity compared with 8 (Table 1). Quinolone
analogue 11, in which the 5- and 6-positions of 3-
found to induce cell toxicity at high concentrations. In an effort
to identify new compounds with reduced cell toxicity, we
designed compounds that are predicted to conserve the
interaction equivalent to 3-hydroxypyridine-2(1H)-one deriva-
tives: a stacking interaction with flavin and Tyr224, an
electrostatic interaction with Arg283, and hydrogen bonding
with Gly313, which induces the Tyr224-out conformation as
mentioned above. 4-Hydroxy-pyridazin-3(2H)-one derivative
13 provided the expected properties (Figure 4). Compound 13
Table 1. Preliminary SAR Studies of Analogues of Fragment
Hit 3-Hydroxypyridin-2(1H)-one 8 for Human DAAO
Figure 4. Binding sites of human DAAO in the complex structure with
13. Hydrogen bonds are shown in dotted lines. The inter-residue
distance between Tyr224 and Leu215 is shown by the blue dotted line.
Color schemes and the software used to the generate the figures are
the same as those in Figure 2.
retained potent inhibitory activity (Table 2) in both enzymes
(IC50 = 3.8 nM) and cell lines (IC50;human = 2.4 nM, IC50;mouse
=
3.3 nM, IC50;rat = 4.6 nM) without cytotoxicity even at high
a
Effective permeability measured by parallel artificial membrane
b
permeability assay (PAMPA) method. pH of donor buffer. pION
membrane lipid was used. N.T.: not tested. Cytotoxicity was
observed at high concentration conditions.
Table 2. In Vitro Inhibitory Activities of 4-Hydroxy-
pyridazin-3(2H)-one Derivatives 13−21 for Human, Mouse,
and Rat DAAOs
c
d
hydroxypyridin-2(1H)-one were converted to cyclic structures,
showed very strong inhibitory activity as previously reported.17
The improved activity might be due to a π−π stacking effect
between the flavin and quinolone rings, as this effect would
have been improved by the increased π-surface of the bicyclic
structure. To investigate whether or not occupation of the
subpocket increased affinity, we introduced a 2-phenylethyl
group at the 5-position of 3-hydroxypyridin-2(1H)-one. The
inhibitory activity of 12 was found to be more than 30 times as
potent as that of compound 10, which was comparable to that
of 11. The complex structure of DAAO with 12 revealed that
12 was able to bind to DAAO as expected (Figure 3B). The
binding mode of the 3-hydroxypyridine-2(1H)-one scaffold of
12 closely resembled that of 8, in that hydrogen bonds with
Arg283 and π−π stacking with the flavin ring were retained.
The introduced 2-phenylethyl group connected by a flexible
linker occupied the subpocket perpendicular to the pyridin-
2(1H)-one ring, and the benzene ring was involved in T-shaped
stacking with Tyr224. Of note, 12 was much more potent than
5 or 9 when evaluated in cell lines, possibly due to an increase
in lipophilicity and cell permeability. These results demonstrate
that the subpocket provides a preferable region not only for
enhancement of inhibitory affinity but also for modulation of
the molecular properties. To the best of our knowledge, this is
the first finding to raise a new possibility of this subpocket.
Synthesis and SAR Analysis of 4-Hydroxy-pyridazin-
3(2H)-one Derivatives. While 12 was highly potent DAAO
inhibitor in the evaluation of enzymes and cell lines, it was
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concentrations. Because the 4-hydroxypyridazin-3(2H)-one
scaffold is confirmed to be less toxic than that of 3-hydroxy-
pyridine-2(1H)-one, the SAR and substituent tolerance at the 5
position was investigated with this new scaffold.
benzene ring of 13 and Tyr224 not being essential, as
mentioned above. However, the SAR of disubstituted
compounds (30−32) suggests that large groups introduced in
the benzene ring cannot be accommodated by the subpocket.
While compound 30, which is the smallest of the three, is as
potent as 13, the inhibitory activity decreases with increasing
size of the substituent; 32 is 3-fold less potent than 13, and 31,
which is the largest of the three, is 15-fold less potent than 13.
In total, the data in Tables 2 and 3 demonstrate that the
inhibitory activities of enzyme and cell lines can be improved by
the use of the newly identified subpocket. In addition, this
subpocket exhibits broad preference for size, aromaticity, and
hydrophobicity of substituents. Further, amino acid residues
around the subpocket are well-conserved among human,
mouse, and rat DAAOs. Consistent with this, 17 and 22−32
showed comparable activities among three species. Taken
together, these findings suggest the possibility that the physical
properties such as hydrophobicity can be controlled to improve
the pharmacokinetic profile by changing substituents at the 5′-
position of 4-hydroxypyridazine-3(2H)-one without loss of
enzymatic and cellular activities in humans, mice, and rats.
Indeed, the 4-hydroxy-pyridazin-3(2H)-one derivatives 13−32
take a wide range (0.13−3.35) of ACDlogP values.
Effects on MK-801-Induced Working Memory Deficit
in Mice. For further evaluation, we selected those compounds
from 13−32 expected to be present in high concentrations in
plasma and brain. Because this series of compounds exhibited
low sensitivity in mass spectroscopy, we first selected
compounds using the parallel artificial membrane permeation
assay (PAMPA) for blood−brain barrier as indices of cell
permeability and brain penetration (Supporting Information
Table S1). Brain concentrations of the resulting compounds
were then measured after oral administration (30 mg/kg) to
mice. Compound 30 exhibited the most preferable combination
of in vitro activities and brain concentrations as early as 30 min
(Cbrain, t=0.5h = 460 ng/mL, Cplasma, t=0.5h = 6540 ng/mL). We
next evaluated the effect of 30 on the MK-801-induced
spontaneous alternation deficit using the mouse Y-maze task,
which is a model of cognitive impairment associated with
schizophrenia (CIAS) based upon the NMDA hypothesis.26
The alternation rate and the number of total arm entries
were measured. While 30 significantly increased the alternation
rate at doses of 0.03 and 0.1 mg/kg, po (Figure 5), it did not
affect total number of arm entries in MK801-treated mice (data
not shown), suggesting that 30 ameliorated the cognitive deficit
induced by MK-801. We were unable to reduce the dose range
of compound 30 when determining plasma and brain levels (30
mg/kg, po) to that for the Y-maze study (0.003−0.3 mg/kg,
po) because of the low sensitivity of the mass spectroscopy.
The brain concentration of compound 30 after administration
(0.1 mg/kg, po 0.5 h) was expected to be 6.1 nM, which was
1.4-fold the IC50 determined in the cell-based assay, assuming
linearity between the dose and brain level. This in vivo result
supports the hypothesis that DAAO inhibitors are effective for
the treatment of CIAS. To our knowledge, this is the second
demonstration of efficacy in animal models of schizophrenia by
oral administration of a DAAO inhibitor.15 We also note that
the dose−response curve of 30 was an inverted U-shape in the
Y-maze, a characteristic shared by many other compounds
when investigated in cognitive tests despite different modes of
action.27−29 For instance, previous studies have suggested that
proper cognitive function requires dopamine levels be
Comparison of the data for 13−16 shows that the subpocket
can tolerate a series of one to three methylene linkers and loss
of aromacity. This property clearly indicates that the T-shaped
stacking interaction between the benzene ring of 13 and
Tyr224 observed in the complex structure is not essential for
the high affinity. Compound 17 shows the introduction of a
chloro group at the p-position was acceptable for inhibitory
activity. A significant decrease in the activity of 18 compared to
17 suggests the importance of linker flexibility, as the rigid
linker of 18 may fail to provide the correct twist needed to
allow the benzene ring to locate perpendicular to the pyridazine
ring, instead creating a steric clash. Low potency of 19 in which
the cyclohexyl group is directly bonded to the 5′-position may
be also attributed to a lack of flexibility. According to the
complex structure with 13, the space sandwiched between
Tyr224 and Leu215 of the subpocket is too narrow to accept
bulky substituents. The inter-residue distance between Tyr224
and Leu215 is 7.1 Å for Tyr224 CE1 and Leu215 CD1 and 5.1
Å for Tyr224 HE1 and Leu215 HD1. This narrow space is
consistent with the reduced activity of 20, which has a t-Bu
group instead of a benzene ring. The subpocket can
accommodate not only a lipophilic moiety but also the polar
linkers introduced in 21.
We further examined the effect of substituents on the
benzene ring of compound 13 (Table 3). These data indicate
that the introduction of small groups to the benzene ring is
tolerated regardless of the electron-withdrawing or electron-
donating properties and effect likely due to (i) small
substituents not being expected to interact with DAAO
residues and (ii) T-shaped stacking interactions between the
Table 3. In Vitro Inhibitory Activities of 4-Hydroxy-
pyridazin-3(2H)-one Derivatives 22−32 for Human, Mouse,
and Rat DAAOs
enzyme
cell(h)
cell(m)
cell(r) IC50
(nM)
compd
R1-
IC50 (nM) IC50(nM) IC50 (nM)
−
17
22
23
4-Cl-C6H4
2.7
2.0
8.4
6.3
4.4
1.9
3.6
7.1
2.4
4.9
41
−
2-F-C6H4
2-CF3- −
C6H4
14
−
24
25
3-F-C6H4
1.4
2.4
6.0
8.3
1.9
2.3
2.6
2.2
3-CF3- −
C6H4
26
3-MeO−-
C6H4
2.1
3.1
8.8
7.2
4.4
5.6
27
28
4-F-C6H4
2.5
5.9
2.2
5.0
4-F3C- −
C6H4
12
27
10
7.0
29
30
31
32
4-MeO−-
C6H4
2.9
1.5
3.6
4.4
3.8
16
3,5-diF-
−
C6H3
3,5-diC−F3-
C6H3
63
13
250
25
490
12
1900
19
3,5-diMeO-
−
C6H3
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SARs in a series of DAAO inhibitors that showed no
cytotoxicity in 4-hydroxy-pyridazin-3(2H)-one derivatives.
Among the compounds prepared in this series, compound 30
demonstrated improved DAAO inhibitory activity over 2−7,
showed oral activity and brain penetrability, and ameliorated
MK-801-induced cognitive deficit in mice.
EXPERIMENTAL SECTION
■
Protein Production and Purification. The recombinant human
DAAO was produced and purified using the same procedures
described previously.37 The purified enzyme was dialyzed against
buffer containing 10 mM Na citrate (pH 8.0), 20 μM FAD, and 400
μM Na benzoate at 4 °C and then concentrated to 10 mg/mL.
Cell Line Establishment and Cell Culture. Cell lines and cell
cultures were made using previous methods with some modifica-
tions.38 Briefly, full-length human, rat, and mouse DAAO were cloned
by PCR using first strand cDNA synthesized from human kidney
RNA, rat kidney RNA, and mouse kidney RNA (Clontech. Inc.. Palo
Alto. CA, USA), respectively. Primer sets used were: 5′-AAG CTT
ATG CGT GTG GTG GTG ATT GG-3′ and 5′-GGA TCC TCA
GAG GTG GGA TGG TGG CA-3′ for human DAAO, 5′-AAG CTT
ATG CGC GTG GCC GTG ATT GG-3′ and 5′-GGA TCC TCA
GAG GTG GGA TGG AGG CA-3′ for rat DAAO, and 5′-AAG CTT
ATG CGC GTG GCC GTG ATC GG-3′ and 5′-GGA TCC TCA
GAG GTG GGA GGG AGG CA-3′ for mouse DAAO. The PCR
products were cloned into a pCR2.1-TOPO vector (Invitrogen,
Carlsbad, CA, USA), and the sequences were confirmed. The
confirmed plasmids were then digested with restricted enzymes,
HindIII/BamHI, and the digested products were inserted into a
pcDNA3.1(+) vector (Invitrogen).
Figure 5. Effects of 30 on MK-801-induced working memory deficit in
the mouse Y-maze test. The alternation rate was measured..
Compound 30 was administered orally, followed 10 min later by
intraperitoneal injection of MK-801 (0.15 mg/kg). Then 20 min after
administration of MK-801, each mouse was placed at the end of one
arm and allowed to freely explore the apparatus for 8 min. Each value
shows the mean SEM (n = 8). N, normal group treated with saline;
C, control group treated with MK-801. ##p < 0.01: statistically
significant compared with normal group, as assessed by Student’s t
test. **p < 0.05: statistically significant compared with control group,
as assessed by Dunnett’s multiple comparison test.
optimum, otherwise cognition is impaired.30 Similarly, DAAO
inhibition by agents such as 30 may also have an optimum level.
D-Serine levels in the brain after administration of 30 were
not measured, preventing us from drawing definitive con-
clusions about the mode of action of this compound. Previous
studies indicate that the administration of DAAO inhibitors
increases D-serine levels in the cerebellum and plasma but not
in the frontal cortex.17,20 If 30 had similar effects on D-serine
levels in the cortex, then the mode of action could be explained
by other substrates for DAAO like D-alanine, which is also
known to function as an coagonist of NMDAR.31 In contrast to
D-serine, D-alanine levels were reported to increase across
multiple brain regions not only in the cerebellum in mice
lacking DAAO activity.32,33 Another possibility was that
improvement of MK-801 induced working memory deficit
was mediated by regions other than forebrain regions via an
unknown mechanism. The third possible mode of action
involves off-target of 30. It is noted that 30 exhibited no
significant activity against a panel of 57 receptors, ion channels,
transporters, and enzymes (Supporting Information Table S2).
Detailed analysis using microdialysis is required to address the
mode of action.
A new series of DAAO inhibitors reported after the
submission of this manuscript has a chemical structure similar
to that of 3034 and is therefore expected to show high affinity
and good cell permeability. The compounds in that series as
well as 30 could prove useful in evaluating the importance of
DAAO inhibitors. Furthermore, such compounds are also
expected to be useful in studying the effects of coadministration
of D-serine and a DAAO inhibitor, which has been reported to
be effective even in cases when DAAO inhibitors alone did not
exert efficacy, potentially representing new therapeutic
possibilities.35,36
A human embryonic kidney (HEK)-293 cell line was cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with
10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL
streptomycin at 37 °C, 5% CO2. To obtain human, rat, or mouse
DAAO-overexpressing HEK293 cell lines, we transfected the vectors
into HEK293 cell lines using Lipofectoamine 2000 (Invitrogen) and
maintained the transfected cell lines in the above medium with 800
μg/mL of Geneticin (G418).
Crystallography. Small crystals of human DAAO were obtained
using the sitting-drop vapor-diffusion method under previously
reported conditions.37 After optimization of the conditions, ligand-
free holoenzyme crystals suitable for X-ray diffraction experiments
were grown at 293 K using the small crystals as seeds. Proteins were
mixed with an equal volume of reservoir solution including 10−15%
(w/v) PEG 4000, 0.1 M sodium citrate, pH 8.0, 0.2 M ammonium
dihydrogen phosphate, and 10% (v/v) glycerol. Plate-shaped yellow
crystals appeared within a day and reached average dimensions of 0.2
× 0.2 × 0.05 mm3 after 3 days. Crystals in complex with inhibitors
were prepared by the soaking method. Soaking solutions were
prepared by mixing stabilization solution (20% PEG 4000, 0.1 M
sodium citrate pH 8.0, 0.2 M ammonium dihydrogen phosphate, and
20% (v/v) glycerol) with 100 mM compound solution dissolved in
DMSO at a ratio of 10 to 1. The ligand-free holoenzyme crystal was
soaked in the soaking solution for 12 h at 293 K. X-ray diffraction data
were collected using a synchrotron beamline AR-NE3A at the Photon
Factory. All data sets were processed and scaled with HKL2000.39
Structure solution and model refinement were carried out using CCP4
suite.40 The structures were solved by molecular replacement using a
previously reported human DAAO structure (PDB ID: 2DU8) as a
model. Automated ligand fitting into initial fo−fc map were performed
using AFFIT-CL,41 followed by model rebuilding using COOT.42 Data
collection and refinement statistics are presented in Supporting
Information.
Animals. Mice were housed in groups of 10 in temperature- and
humidity-controlled colony rooms (23 1 °C and 55 5%) under a
12 h light/dark cycle with water and laboratory chow supplied ad
libitum. All experiments were conducted in accordance with the
Astellas Pharma Inc. guidelines for the care and use of animals and
CONCLUSION
■
We identified a series of novel DAAO inhibitors, 4-hydroxy-
pyridazin-3(2H)-one derivatives, using FBDD from fragment 3-
hydroxypyridine-2(1H)-one 1 and successfully established
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under approved protocols from the Institutional Animal Care and Use
Committee of Astellas Pharma Inc.
respectively) converging at equal angles. Ten minutes after oral
administration of 30 or vehicle, 0.15 mg/kg of MK-801 ((+)-MK-801
hydrogen maleate; Sigma Aldrich, St. Louis, MO, USA) was
administered intraperitoneally to each animal. Normal animals were
administered with vehicle and saline instead of 30 and MK-801,
respectively. Twenty minutes after administration of MK-801 or saline,
each mouse was placed at the end of one arm and allowed to explore
the apparatus freely for 8 min. Alternation was defined as entries into
all three arms on consecutive occasions. The alternation percentage
was calculated using the following formula:
Human DAAO Inhibition Enzyme Assay. All compounds were
screened in human DAAO inhibition assays as described below.
Amplex Red reagent (catalgue code: A-12222, Invitrogen) was used to
measure H2O2 produced by the DAAO reaction. Briefly, a reaction
mixture containing 50 mM potassium phosphate buffer (pH 7.5),
0.02% CHAPS, 5 nM DAAO, 6 μM FAD, 0.75 mM D-alanine, 0.5 U/
mL HRP, and 50 μM Amplex Red in the presence or absence of the
compounds was incubated at room temperature for 30 min. The
fluorescence signal was then detected in a plate reader (Envision,
Perkin-Elmer Inc., MA, USA) with excitation at 544 nm and emission
at 590 nm. Each IC50 value was comprised of at least two
measurements of inhibition per data point.
alternation(%) = 100 × number of alternations
/(number of total arm entries − 2)
DAAO Inhibition Cell Assays. DAAO inhibition cell assays were
conducted as previously described with some modification.38 Briefly,
the reaction mixture containing 50 mM potassium phosphate buffer
(pH 7.5), 0.02% CHAPS, HEK293/human (mouse, rat) DAAO cells
(20000 cells/well), 6 μM FAD, 50 mM D-alanine, 0.5 U/mL HRP, and
80 μM Amplex Red in the presence or absence of compounds was
incubated at room temperature for 30 min. The fluorescence signal
was then detected in a plate reader (Envision, Perkin-Elmer Inc.) with
excitation at 544 nm and emission at 590 nm. Each IC50 value was
comprised of at least two measurements of inhibition per data point.
Cytotoxicity Evaluation. HEK293/human DAAO cells viability
in the presence of the compounds was measured using alamar Blue
(Wako Pure Chemical Industries, Ltd., Osaka, Japan).
All values are given as the mean
standard error of the mean.
Statistical analysis of the normal (vehicle + saline) group vs control
(vehicle + MK-801) group was conducted using the unpaired
Student’s t-test. Control (vehicle + MK-801) vs drug + MK-801
groups were compared using one-way ANOVA followed by Dunnett’s
multiple comparison test. Groups contained eight animals each.
Chemistry. Unless otherwise noted, all materials were obtained
1
from commercial suppliers and used without further purification. H
NMR spectra were recorded on a Varian 400 MHz, BRUKER 400
MHz, or JEOL 400 MHz spectrometer, and the chemical shifts were
expressed in δ (ppm) values with trimethylsilane as an internal
reference (s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, and br = broad peak). Mass spectra were recorded on an
Agilent 6140 Quadrupole LC/MS, Waters UPLC/SQD-LC/MS
system, or JEOL JMS-LX2000 spectrometer. High-resolution mass
spectroscopy (HRMS) values were recorded on a Waters Q-Tof
Premier or Waters LCT Premier XE. The purities of the tested
compounds were found to be above 95%, as determined using the
Waters UPLC LC/MS system and UPLC HSS T3 2.1 mm × 50 mm
column, 1.8 μm, 5−95% MeOH in H2O with 0.1% formic acid for 3
min at 0.7 mL/min and UV detection at 254 nm.
Parallel Artificial Membrane Permeability Assay (PAMPA).
The PAMPA Evolution instrument from pION Inc. (Woburn, MA,
USA) was used in this study. In PAMPA, a “sandwich” is formed from
a 96-well microtiter plate (PN 110163; pION Inc.) and a 96-well filter
plate (IPVH; Milipore, Bedford, MA, USA), such that each composite
well is divided into two chambers: a donor at the bottom and an
acceptor at the top. The two were separated by a 125 μm thick
microfilter disc (0.45 μm diameter pores) and coated with a 20% (w/
v) dodecane solution of a lecithin mixture (PN 110669; pION Inc.).
Drug samples were introduced as 10 mM DMSO stock solutions in a
96-well polypropylene microtiter plate. The robotic liquid handling
system drew 5 μL aliquots of the DMSO stock solution and mixed it
into an aqueous buffer solution including 10% (v/v) of DMSO to
bring the final sample concentration to 50 μM. The drug solutions
were filtered using a 96-well filter plate (PVDF, Corning) and added to
the donor compartments. The donor solutions were adjusted in pH
5.0 and 6.5 (NaOH-treated universal buffer, PN 110151; pION Inc.),
while the acceptor solutions were kept at pH 7.4 (PN 110139; pION
Inc.). The plate sandwich was formed and allowed to incubate at 25
°C for 2 h in a humidity-saturated atmosphere. Upon completion of
the prescribed incubation time, the sandwich plates were separated,
and both the donor and acceptor compartments were assayed to
determine the amount of material present using UV spectra (270−400
nm) obtained from reference standards. Mass balance was used to
determine the amount of material remaining in the membrane barrier,
and permeability (Pe) was calculated using PAMPA Evolution
software (pION Inc.). The blood−brain barrier permeability by
PAMPA (PAMPA-BBB) was measured as described previously.43
Determination of Plasma and Brain Levels in Mice. The
pharmacokinetic characterization of test compounds was conducted in
6-week-old male ddY mice (Japan SLC, Inc., Hamamatsu, Japan). Test
compounds were orally administrated as 0.5% methylcellulose
suspensions at a dose of 30 mg/kg. Blood samples for the
determination of the test compound concentration were obtained at
0.5 and 2 h after single dose administration of the compound.
Concentrations of the unchanged compound were analyzed using
liquid chromatography/tandem mass spectrometry (LC-MS/MS).
Groups contained three animals each.
5, 8, and 9 are commercially available and were obtained from
MAYBRIDGE (catalogue code: CC39001DA), Aldrich (catalogue
code: 122505) and WAKO (catalogue code: 325-72092), respectively.
10 (CAS Registry no.: 856952-50-6) was prepared in a similar manner
to 12, described below. 1H NMR (DMSO-d6) δ 1.95 (s, 3H), 6.57 (s,
1H), 6.61 (s, 1H), 8.84 (br, 1H), 11.39 (br, 1H). MS (ESI/APCI+) m/
z = 126 (MH+). 11 (CAS Registry no.: 26386-86-7) was prepared
from 2-aminobenzaldehyde as described in the literature:44 1H NMR
(DMSO-d6) δ 7.08−7.14 (m, 2H), 7.24−7.48 (m, 2H), 7.49 (d, J = 7.6
Hz, 1H), 9.44 (br, 1H), 12.00 (br, 1H). MS (ESI+) m/z = 162 (MH+).
3-Hydroxy-5-(2-phenylethyl)pyridin-2(1H)-one (12). Step 1:
To a suspension of NaH (55% with oil, 253 mg, 5.80 mmol) in DMF
was added 5-bromo-2,3-dihydroxypyridine (500 mg, 2.63 mmol) at 0
°C. The mixture was stirred for 1 h at room temperature, and then
chloromethyl methyl ether (0.44 mL, 5.85 mmol) was added at 0 °C.
The mixture was stirred for 3 h at room temperature and poured into
water (20 mL). The aqueous layer was extracted with AcOEt, and the
organic layer was dried over MgSO4 and concentrated in vacuo.
Purification via column chromatography on silica gel (CHCl3/MeOH)
gave 5-bromo-2,3-bis(methoxymethoxy)pyridine 34 (149 mg, 20%) as
a colorless oil. 1H NMR (CDCl3) δ 3.52 (s, 3H), 3.54 (s, 3H), 5.24 (s,
2H), 5.57 (s, 2H), 7.53 (d, J = 2.4 Hz, 1H), 7.88 (d, J = 2.4 Hz, 1H).
MS ((ESI/APCI+) m/z = 278, 280 (MH+) along with 5-bromo-3-
(methoxymethoxy)-1-(methoxymethyl)pyridin-2(1H)-one (363 mg,
1
50%) as a colorless oil. H NMR (CDCl3) δ 3.41 (s, 3H), 3.50 (s,
3H), 5.23 (s, 2H), 5.33 (s, 2H), 7.07 (d, J = 2.4 Hz, 1H), 7.22 (d, J =
2.4 Hz, 1H). MS (ESI/APCI+) m/z = 278, 280 (MH+).
Step 2: To a solution of 5-bromo-2,3-bis(methoxymethoxy)pyridine
34 (149 mg, 0.536 mmol) in dioxane (10 mL) and water (2 mL) was
added [(E)-2-phenylvinyl]boronic acid (119 mg, 0.804 mmol), K3PO4
(341 mg, 1.61 mmol), and Pd(PPh3)4 (62 mg, 0.054 mmol), and the
mixture was stirred at 100 °C for 6 h under an argon atmosphere. The
reaction mixture was cooled to room temperature, the residue
partitioned between AcOEt and water, and the organic layer was
washed with brine, dried over MgSO4, and concentrated in vacuo.
MK-801-Induced Working Memory Deficit in the Mouse Y-
Maze Test. The mouse Y-maze test was performed as previously
described,26 and male ddY mice (5 weeks old; Japan SLC, Inc.) were
used. The maze was made of gray polyvinyl chloride with arms (40 cm
long, 13 cm tall, and 3 and 10 cm wide at the bottom and top,
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Purification via column chromatography on silica gel (Hex/AcOEt)
gave 2,3-bis(methoxymethoxy)-5-[(E)-2-phenylvinyl]pyridine (114
mg, 71%) as a pale-yellow oil. H NMR (CDCl3) δ 3.56 (s, 6H),
5.31 (s, 2H), 5.62 (s, 2H), 6.99 (d, J = 16.4 Hz, 1H), 7.02 (d, J = 16.4
Hz, 1H), 7.24−7.29 (m, 1H), 7.36 (t, J = 7.6 Hz, 2H), 7.50 (d, J = 7.6
Hz, 2H), 7.61 (d, J = 2.0 Hz, 1H), 7.91 (d, J = 2.0 Hz, 1H). MS (ESI/
APCI+) m/z = 258 (MH+ − CH2OCH3).
boronic acid in place of [(E)-2-phenylvinyl]boronic acid in 15% yield
(3 steps) as a white solid. 1H NMR (DMSO-d6) δ 1.48 (d, J = 7.2 Hz,
3H), 4.00 (q, J = 7.2 Hz, 1H), 6.41 (s, 1H), 7.18−7.35 (m, 5H), 10.72
(br, 1H), 12.74 (s, 1H). MS (ESI+) m/z = 217 (MH+). HRMS (ESI+)
calcd for [C12H12N2O2 + H]+, 217.0977; found, 217.0981.
4-Hydroxy-6-(3-phenylpropyl)pyridazin-3(2H)-one (15). The
title compound was prepared in a manner similar to that used to
prepare 13, with the substitution of starting material [(1E)-3-
phenylprop-1-en-1-yl]boronic acid in place of [(E)-2-phenylvinyl]-
boronic acid in 10% yield (3 steps) as a white solid. 1H NMR (DMSO-
d6) δ 1.80−1.92 (m, 2H), 2.42−2.52 (m, 2H), 2.55−2.62 (m, 2H),
6.55 (s, 1H), 7.14−7.23 (m, 3H), 7.24−7.31 (m, 2H), 10.69 (br, 1H),
12.66 (s, 1H). MS (ESI+) m/z = 231 (MH+). HRMS (ESI+) calcd for
[C13H14N2O2 + H]+, 231.1134; found, 231.1141.
1
Step 3: To a solution of 2,3-bis(methoxymethoxy)-5-[(E)-2-
phenylvinyl]pyridine (114 mg, 0.378 mmol) in EtOH (10 mL) was
added 10% Pd−C (50 mg) with EtOH (10 mL). The mixture was
stirred at room temperature for 4 h under a hydrogen atmosphere and
then passed through a Celite pad, where the filtrate was concentrated
in vacuo to give 3-(methoxymethoxy)-5-(2-phenylethyl)pyridin-
1
2(1H)-one (94 mg, 96%) as a colorless oil. H NMR (CDCl3) δ
2.69 (t, J = 7.6 Hz, 2H), 2.83 (t, J = 7.6 Hz, 2H), 3.49 (s, 3H), 5.19 (s,
2H), 6.81 (s, 1H), 6.96 (s, 1H), 7.10−7.29 (m, 5H), 13.24 (br, 1H).
MS (ESI/APCI+) m/z = 260 (MH+).
6-(2-Cyclohexylethyl)-4-hydroxypyridazin-3(2H)-one (16).
The title compound was prepared in a manner similar to that used
to prepare 13, with the substitution of starting material [(E)-2-
cyclohexylvinyl]boronic acid in place of [(E)-2-phenylvinyl]boronic
Step 4: To a solution of 3-(methoxymethoxy)-5-(2-phenylethyl)-
pyridin-2(1H)-one (94 mg, 0.363 mmol) in CH2Cl2 (5.0 mL) was
added 1 M BBr3 in CH2Cl2 (5.0 mL, 5.0 mmol) at 0 °C. The mixture
was stirred at room temperature for 4 h and concentrated in vacuo,
and the residue was washed with water and purified by column
chromatography on silica gel (CHCl3/MeOH) to give a solid. The
solid was recrystallized from EtOH to give 3-hydroxy-5-(2-
phenylethyl)pyridin-2(1H)-one (38 mg, 48%) as a white solid. 1H
NMR (DMSO-d6) δ 2.53−2.61 (m, 2H), 2.73−2.80 (m, 2H), 6.58 (s,
1H), 6.68 (s, 1H), 7.14−7.33 (m, 5H), 8.83 (s, 1H), 11.39 (br, 1H).
MS (ESI+) m/z = 216 (MH+). HRMS (ESI+) calcd for [C13H13NO2 +
H]+, 216.1025; found, 216.1019.
4-Hydroxy-6-(2-phenylethyl)pyridazin-3(2H)-one (13) (Typi-
cal Procedure). Step 1: To a solution of 3-chloro[1,4]benzodioxino-
[2,3-c]pyridazine 35 (500 mg, 2.27 mmol) in dimethoxyethane (18
mL) was added [(E)-2-phenylvinyl]boronic acid (402 mg, 2.72
mmol), 2 M Na2CO3 aq (1 mL), and Pd(PPh3)4 (266 mg, 0.230
mmol), and the mixture was stirred at 120 °C for 1 h under microwave
irradiation. The reaction mixture was cooled to room temperature, the
residue partitioned between CHCl3 and water, and the organic layer
was washed with brine, dried over Na2SO4, and concentrated in vacuo.
Purification using column chromatography on silica gel (CHCl3/
MeOH) gave 3-[(E)-2-phenylvinyl][1,4]benzodioxino[2,3-c]-
pyridazine (554 mg, 85%) as a dark solid. 1H NMR (CDCl3) δ
6.95−7.16 (m, 4H), 7.27−7.49 (m, 4H), 7.53−7.59 (m, 2H), 7.64−
7.70 (m, 2H). MS (ESI/APCI+) m/z = 289 (MH+).
Step 2: To a solution of benzyl alcohol (0.50 mL, 4.83 mmol) in
toluene (10 mL) was added t-BuOK (540 mg, 4.82 mmol) at 0 °C. A
solution of 3-[(E)-2-phenylvinyl][1,4]benzodioxino[2,3-c]pyridazine
(554 mg, 1.92 mmol) in DMF (10 mL) and toluene (5 mL) was
added dropwise to the above reaction mixture at 0 °C, and reaction
mixture was heated to 120 °C for 4.5 h. The reaction mixture was
cooled to room temperature, the residue was partitioned between
CHCl3 and water, and the organic layer was washed with brine, dried
over MgSO4, and concentrated in vacuo. Purification by column
chromatography on silica gel (Hex/AcOEt) gave 3,4-bis(benzyloxy)-6-
[(E)-2-phenylvinyl]pyridazine (274 mg, 36%) as a pale-yellow gum.
1H NMR (DMSO-d6) δ 5.33 (s, 2H), 5.56 (s, 2H), 7.30−7.50 (m,
1
acid in 26% yield (3 steps) as a white solid. H NMR (DMSO-d6) δ
0.81−0.97 (m, 2H), 1.05−1.29 (m, 4H), 1.39−1.50 (m, 2H), 1.55−
1.77 (m, 5H), 2.41−2.48 (m, 2H), 6.52 (s, 1H), 10.67 (br, 1H), 12.63
(s, 1H). MS (ESI+) m/z = 223 (MH+). HRMS (ESI+) calcd for
[C12H18N2O2 + H]+, 223.1447; found, 223.1450.
6-[2-(4-Chlorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one
(17). Steps 1 and 2: 3,4-Bis(benzyloxy)-6-[(E)-2-(4-chlorophenyl)-
vinyl]pyridazine was prepared by a method similar to steps 1 and 2
described in the synthesis of 13 in 25% yield (2 steps) as a gray solid.
1H NMR (DMSO-d6) δ 5.32 (s, 2H), 5.56 (s, 2H), 7.32−7.52 (m,
13H), 7.60−7.71 (m, 4H). MS (ESI+) m/z = 429, 431 (MH+).
Step 3: To a solution of 3,4-bis(benzyloxy)-6-[(E)-2-(4-
chlorophenyl)vinyl]pyridazine (200 mg, 0.47 mmol) and triethylamine
(0.20 mL, 1.43 mmol) in dimethoxyethane (10 mL) was added 2,4,6-
triisopropylbenzenesulfonohydrazide (700 mg, 2.35 mmol), and the
mixture was stirred at room temperature for 40 h. Triethylamine (0.20
mL, 1.43 mmol) and 2,4,6-triisopropylbenzenesulfonohydrazide (700
mg, 2.35 mmol) were added, and the mixture was stirred again at room
temperature for 24 h. The reaction mixture was partitioned between
CHCl3 and water, and the organic layer was washed with brine, dried
over MgSO4, and concentrated in vacuo. Purification via column
chromatography on silica gel (Hex/AcOEt) gave 3,4-bis(benzyloxy)-6-
[2-(4-chlorophenyl)ethyl]pyridazine (70 mg, 35%) as a white solid. 1H
NMR (DMSO-d6) δ 2.96−3.07 (m, 4H), 5.21 (s, 2H), 5.48 (s, 2H),
7.20−7.24 (m, 3H), 7.29−7.47 (m, 12H). MS (ESI+) m/z = 431, 433
(MH+). The title compound was prepared in a manner similar to that
used to prepare 12 in step 4 in 73% yield as an orange solid. 1H NMR
(DMSO-d6) δ 2.71−2.74 (m, 2H), 2.85−2.89 (m, 2H), 6.52 (s, 1H),
7.23 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 10.73 (br, 1H),
12.63 (s, 1H). MS (ESI+) m/z = 251, 253 (MH+). HRMS (ESI+) calcd
for [C12H11ClN2O2 + H]+, 251.0587; found, 251.0582.
6-[(E)-2-(4-Chlorophenyl)vinyl]-4-hydroxypyridazin-3(2H)-
one (18). The title compound was prepared in a manner similar to
that used to prepare 12 in step 4, with the substitution of starting
material 3,4-bis(benzyloxy)-6-[(E)-2-(4-chlorophenyl)vinyl]pyridazine
in place of 3-(methoxymethoxy)-5-(2-phenylethyl)pyridin-2(1H)-one
1
in 67% yield as an orange solid. H NMR (DMSO-d6) δ 7.00 (d, J =
14H), 7.60−7.69 (m, 4H). MS (ESI/APCI+) m/z = 395 (MH+).
Step 3: To a solution of 3,4-bis(benzyloxy)-6-[(E)-2-phenylvinyl]-
pyridazine (272 mg, 0.69 mmol) in EtOH (8 mL) was added 10%
Pd−C (90 mg). The mixture was stirred at room temperature for 4 h
under an hydrogen atmosphere. The reaction mixture was passed
through a Celite pad, and the filtrate was concentrated in vacuo to give
a solid. The solid was recrystallized from CHCl3−AcOEt to give 4-
hydroxy-6-(2-phenylethyl)pyridazin-3(2H)-one 13 (60 mg, 40%) as a
pale-gray solid. 1H NMR (DMSO-d6) δ 2.73−2.77(m, 2H), 2.85−2.89
(m, 2H), 6.57 (s, 1H), 7.15−7.29 (m, 5H), 12.7 (s, 1H). MS (ESI+)
m/z = 217 (MH+). HRMS (ESI+) calcd for [C12H12N2O2 + H]+,
217.0977; found, 217.0973.
16.8 Hz, 1H), 7.11 (s, 1H), 7.28 (d, J = 16.8 Hz, 1H), 7.44 (d, J = 8.0
Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H), 10.90 (s, 1H), 13.00 (s, 1H). MS
(ESI+) m/z = 249, 251 (MH+). HRMS (ESI+) calcd for
[C12H9ClN2O2 + H]+, 249.0431; found, 249.0426.
6-Cyclohexyl-4-hydroxypyridazin-3(2H)-one (19). The title
compound was prepared in a manner similar to that used to prepare
13, with the substitution of starting material cyclohex-1-en-1-ylboronic
acid in place of [(E)-2-phenylvinyl]boronic acid in 14% yield (3 steps)
1
as a white solid. H NMR (DMSO-d6) δ 1.11−1.41 (m, 5H), 1.61−
1.84 (m, 5H), 2.32−2.45 (m, 1H), 6.57 (s, 1H), 10.64 (br, 1H), 12.65
(s, 1H). MS (ESI+) m/z = 195 (MH+). HRMS (ESI+) calcd for
[C10H14N2O2 + H]+, 195.1134; found, 195.1136.
4-Hydroxy-6-(1-phenylethyl)pyridazin-3(2H)-one (14). The
title compound was prepared in a manner similar to that used to
prepare 13, with the substitution of starting material (1-phenylvinyl)-
6-(3,3-Dimethylbutyl)-4-hydroxypyridazin-3(2H)-one (20).
The title compound was prepared in a manner similar to that used
to prepare 13, with the substitution of starting material [(1E)-3,3-
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dimethylbut-1-en-1-yl]boronic acid in place of [(E)-2-phenylvinyl]-
boronic acid in 13% yield (3 steps) as a white solid. 1H NMR (DMSO-
d6) δ 0.90 (s, 9H), 1.39−1.48 (m, 2H), 2.37−2.44 (m, 2H), 6.53 (s,
1H), 10.66 (br, 1H), 12.63 (s, 1H). MS (ESI+) m/z = 197 (MH+).
HRMS (ESI+) calcd for [C10H17N2O2 + H]+, 197.1290; found,
197.1292.
Cs2CO3 (580 mg, 1.78 mmol), PdCl2(PPh3)2 (360 mg, 0.51 mmol),
and DMF (5 mL) was added 1-ethynyl-2-(trifluoromethyl)benzene
(1.00 g, 5.88 mmol), and the mixture was stirred at 100 °C for 3 h
under an argon atmosphere. The reaction mixture was cooled to room
temperature and passed through a Celite pad, and the filtrate was
partitioned between AcOEt and water. The organic layer was washed
with brine, dried over Na2SO4, and concentrated in vacuo. Purification
by column chromatography on silica gel (Hex/AcOEt) gave 3-{[2-
(trifluoromethyl)phenyl]ethynyl}[1,4]benzodioxino[2,3-c]pyridazine
(450 mg, 61%). MS (ESI+) m/z = 355 (MH+). 23 was prepared by a
method similar to steps 2 and 3 described in the synthesis of 13 in 8%
yield (2 steps) as a white solid. 1H NMR (DMSO-d6) δ 2.73−2.81 (m,
2H), 3.00−3.08 (m, 2H), 6.59 (s, 1H), 7.39−7.46 (m, 1H), 7.49−7.54
(m, 1H), 7.58−7.64 (m, 1H), 7.65−7.71 (m, 1H), 10.78 (br, 1H),
12.70 (s, 1H). MS (ESI+) m/z = 285 (MH+). HRMS (ESI+) calcd for
[C13H11N2O2F3 + H]+, 285.0851; found, 285.0852.
4-Hydroxy-6-(phenoxymethyl)pyridazin-3(2H)-one (21). Step
1: The mixture of 3-chloro[1,4]benzodioxino[2,3-c]pyridazine 35
(2.21 g, 10.0 mmol), Pd(OAc)2 (449 mg, 2.00 mmol), DPPF (2.22 g,
4.00 mmol), MeOH (30 mL), DMSO (30 mL), and Et3N (2.8 mL,
20.1 mmol) was stirred at 80 °C for 19 h under a CO atmosphere. The
reaction mixture was cooled to room temperature and partitioned
between AcOEt and water. The organic layer was washed with brine,
dried over MgSO4, and concentrated in vacuo. Purification via column
chromatography on silica gel (Hex/AcOEt) gave methyl [1,4]-
benzodioxino[2,3-c]pyridazine-3-carboxylate (1.46 g, 60%) as a white
solid. 1H NMR (DMSO-d6) δ 3.92 (s, 3H), 7.09−7.14 (m, 3H), 7.18−
7.22 (m, 1H), 7.65 (s, 1H). MS (ESI/APCI+) m/z = 245 (MH+).
Step 2: To a mixture of methyl [1,4]benzodioxino[2,3-c]pyridazine-
3-carboxylate (1.46 g, 5.97 mmol), MeOH (15 mL), and THF (30
mL) was added sodium borohydride (677 mg, 17.9 mmol) at 0 °C,
and the mixture was stirred at room temperature for 30 min. The
reaction mixture was partitioned between AcOEt and water. The
organic layer was washed with brine, dried over MgSO4, and
concentrated in vacuo. Purification via column chromatography on
silica gel (Hex/AcOEt) gave [1,4]benzodioxino[2,3-c]pyridazin-3-
6-[2-(3-Fluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one
(24). The title compound was prepared in a manner similar to that
used to prepare 13, with the substitution of starting material [(E)-2-(3-
fluorophenyl)vinyl]boronic acid in place of [(E)-2-phenylvinyl]-
boronic acid in 35% yield (3 steps) as a white solid. 1H NMR
1
(DMSO-d6) δ 2.73−2.80 (m, 2H), 2.86−2.94 (m, 2H), 6.59 (s, H),
6.96−7.11 (m, 3H), 7.26−7.34 (m, 1H), 10.72 (br, 1H), 12.67 (s,
1H). MS (ESI+) m/z = 235 (MH+). HRMS (ESI+) calcd for
[C12H11N2O2F + H]+, 235.0883; found, 235.0892.
4-Hydroxy-6-{2-[3-(trifluoromethyl)phenyl]ethyl}pyridazin-
3(2H)-one (25). The title compound was prepared in a manner
similar to that used to prepare 13, with the substitution of starting
material {(E)-2-[3-(trifluoromethyl)phenyl]vinyl}boronic acid in place
of [(E)-2-phenylvinyl]boronic acid in 9% yield (3 steps) as a white
1
ylmethanol (1.13 g, 88%) as a pale-yellow solid. H NMR (DMSO-
d6) δ 4.59 (d, J = 6.0 Hz, 2H), 5.63 (t, J = 6.0 Hz, 1H), 7.03−7.13 (m,
3H), 7.14−7.19 (m, 1H), 7.20 (s, 1H). MS (ESI/APCI+) m/z = 217
(MH+).
1
solid. H NMR (DMSO-d6) δ 2.75−2.83 (m, 2H), 2.94−3.03 (m,
Step 3: The mixture of [1,4]benzodioxino[2,3-c]pyridazin-3-
ylmethanol (8.10 g, 37.5 mmol), 1,2-dichloroethane (80 mL), and
thionyl bromide (38.9 g, 186 mmol) was stirred at 80 °C for 2 h. The
reaction mixture was diluted with chloroform (500 mL), and the
organic layer was neutralized with satd sodium bicarbonate aq (250
mL). The organic layer was dried over MgSO4 and concentrated in
vacuo. The residue was triturated with diethyl ether to give 3-
2H), 6.62 (s, 1H), 7.47−7.61 (m, 4H), 10.72 (br, 1H), 12.68 (s, 1H).
MS (ESI+) m/z = 285 (MH+). HRMS (ESI+) calcd for
[C13H11N2O2F3 + H]+, 285.0851; found, 285.0850.
4-Hydroxy-6-[2-(3-methoxyphenyl)ethyl]pyridazin-3(2H)-
one (26). The title compound was prepared in a manner similar to
that used to prepare 13, with the substitution of starting material [(E)-
2-(3-methoxyphenyl)vinyl]boronic acid in place of [(E)-2-
1
(bromomethyl)[1,4]benzodioxino[2,3-c]pyridazine (8.0 g). H NMR
1
phenylvinyl]boronic acid in 18% yield (3 steps) as a white solid. H
(DMSO-d6) δ 4.73 (s, 2H), 7.06−7.19 (m, 4H), 7.44 (s, 1H). MS
(ESI+) m/z = 279, 281 (MH+).
NMR (DMSO-d6) δ 2.70−2.79 (m, 2H), 2.80−2.88 (m, 2H), 3.72 (s,
3H), 6.59 (s, 1H), 6.71−6.80 (m, 3H), 7.14−7.21 (m, 1H), 10.69 (br,
1H), 12.67 (s, 1H). MS (ESI+) m/z = 247 (MH+). HRMS (ESI+)
calcd for [C13H14N2O3 + H]+, 247.1083; found, 247.1090.
6-[2-(4-Fluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one
(27). The title compound was prepared in a manner similar to that
used to prepare 13, with the substitution of starting material [(E)-2-(4-
fluorophenyl)vinyl]boronic acid in place of [(E)-2-phenylvinyl]-
boronic acid in 4% yield (3 steps) as a white solid. 1H NMR
(DMSO-d6) δ 2.69−2.78 (m, 2H), 2.82−2.91 (m, 2H), 6.58 (s, 1H),
7.04−7.13 (m, 2H), 7.20−7.28 (m, 2H), 10.71 (br, 1H), 12.67 (s,
1H). MS (ESI+) m/z = 235 (MH+). HRMS (ESI+) calcd for
[C12H11N2O2F + H]+, 235.0883; found, 235.0887.
Step 4: The mixture of 3-(bromomethyl)[1,4]benzodioxino[2,3-
c]pyridazine (500 mg, 1.79 mmol), DMF (10 mL), potassium iodide
(30 mg, 181 mmol), K2CO3 (620 mg, 4.49 mmol), and phenol (250
mg, 2.66 mmol) was stirred at room temperature for 24 h. The
reaction mixture was partitioned between AcOEt and water. The
organic layer was washed with 1N NaOH aq and brine, dried over
Na2SO4, and concentrated in vacuo. The residue was triturated with
diethyl ether to give crude 3-(phenoxymethyl)[1,4]benzodioxino[2,3-
c]pyridazine (370 mg, ca. 1.27 mmol). MS (ESI+) m/z = 293 (MH+).
21 was prepared in a manner similar to that used to prepare 13 in
steps 2 and 3, with substitutions of starting material 3-
(phenoxymethyl)[1,4]benzodioxino[2,3-c]pyridazine in place of 3-
[(E)-2-phenylvinyl][1,4]benzodioxino[2,3-c]pyridazine and catalyst
Pd−BaSO4 in place of Pd−C in 29% yield as a white solid. 1H
NMR (DMSO-d6) δ 4.88 (s, 2H), 6.68 (s, 1H), 6.93−7.01 (m, 3H),
7.27−7.32 (m, 2H), 12.93 (s, 1H). MS (ESI+) m/z = 219 (MH+).
HRMS (ESI+) calcd for [C11H10N2O3 + H]+, 219.0770; found,
219.0768.
4-Hydroxy-6-{2-[4-(trifluoromethyl)phenyl]ethyl}pyridazin-
3(2H)-one (28). The title compound was prepared in a manner
similar to that used to prepare 13, with the substitution of starting
material {(E)-2-[4-(trifluoromethyl)phenyl]vinyl}boronic acid in place
of [(E)-2-phenylvinyl]boronic acid in 6% yield (3 steps) as a white
1
solid. H NMR (DMSO-d6) δ 2.75−2.83 (m, 2H), 2.94−3.02 (m,
2H), 6.61 (s, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H),
10.74 (br, 1H), 12.67 (s, 1H). MS (ESI+) m/z = 285 (MH+). HRMS
(ESI+) calcd for [C13H11N2O2F3 + H]+, 285.0851; found, 285.0848.
4-Hydroxy-6-[2-(4-methoxyphenyl)ethyl]pyridazin-3(2H)-
one (29). The title compound was prepared in a manner similar to
that used to prepare 13, with the substitution of starting material [(E)-
2-(4-methoxyphenyl)vinyl]boronic acid in place of [(E)-2-
6-[2-(2-Fluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one
(22). The title compound was prepared in a manner similar to that
used to prepare 13, with the substitution of starting material [(E)-2-(2-
fluorophenyl)vinyl]boronic acid in place of [(E)-2-phenylvinyl]-
boronic acid in 10% yield (3 steps) as a white solid. 1H NMR
(DMSO-d6) δ 2.71−2.78 (m, 2H), 2.86−2.95 (m, 2H), 6.57 (s, 1H),
7.05−7.16 (m, 2H), 7.19−7.31 (m, 2H), 10.71 (br, 1H), 12.66 (s,
1H). MS (ESI+) m/z = 235 (MH+). HRMS (ESI+) calcd for
[C12H11N2O2F + H]+, 235.0883; found, 235.0878.
1
phenylvinyl]boronic acid in 19% yield (3 steps) as a white solid. H
NMR (DMSO-d6) δ 2.67−2.74 (m, 2H), 2.76−2.84 (m, 2H), 3.71 (s,
3H), 6.57 (s, 1H), 6.83 (d, J = 8.8 Hz, 2H), 7.11 (d, J = 8.8 Hz, 2H),
10.68 (br, 1H), 12.65 (s, 1H). MS (ESI+) m/z = 247 (MH+). HRMS
(ESI+) calcd for [C13H14N2O3 + H]+, 247.1083; found, 247.1090.
4-Hydroxy-6-{2-[2-(trifluoromethyl)phenyl]ethyl}pyridazin-
3(2H)-one (23). To a mixture of 3-chloro[1,4]benzodioxino[2,3-
c]pyridazine 35 (460 mg, 2.09 mmol), CuI (170 mg, 0.89 mmol),
3590
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Journal of Medicinal Chemistry
Article
6-[2-(3,5-Difluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-
one (30). The title compound was prepared in a manner similar to
that used to prepare 13, with the substitution of starting material 2-
[(E)-2-(3,5-difluorophenyl)vinyl]-4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lane in place of [(E)-2-phenylvinyl]boronic acid in 26% yield (3 steps)
Subanesthetic Effects of the Noncompetitive NMDA Antagonist,
Ketamine, in Humans: Psychotomimetic, Perceptual, Cognitive, and
Neuroendocrine Responses. Arch. Gen. Psychiatry 1994, 51, 199−214.
(3) Bendikov, I.; Nadri, C.; Amar, S.; Panizzutti, R.; De Miranda, J.;
Wolosker, H.; Agam, G. A CSF and postmortem brain study of D-
serine metabolic parameters in schizophrenia. Schizophrenia Res. 2007,
90, 41−51.
1
as a white solid. H NMR (DMSO-d6) δ 2.72−2.81 (m, 2H), 2.87−
2.95 (m, 2H), 6.59 (s, 1H), 6.94−7.06 (m, 3H), 10.74 (br, 1H), 12.68
(s, 1H). MS (ESI+) m/z = 253 (MH+). HRMS (ESI+) calcd for
[C12H10N2O2F2 + H]+, 253.0789; found, 253.0784.
(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. Biol. Psychiatry 2005, 29, 767−769.
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Shigeno, Y.; Toyoshima, M.; Okuno, A.; Takao, H.; Toyota, T.;
Minabe, Y.; Nakamura, K.; Shimizu, E.; Itokawa, M.; Mori, N.; Iyo, M.;
Yoshikawa, T. Identification of Multiple Serine Racemase (SRR)
mRNA Isoforms and Genetic Analyses of SRR and DAO in
Schizophrenia and D-Serine Levels. Biol. Psychiatry 2005, 57, 1493−
1503.
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P.; Bar, G.; Catinari, S.; Ermilov, M. D-Serine efficacy as add-on
pharmacotherapy to risperidone and olanzapine for treatment-
refractory schizophrenia. Biol. Psychiatry 2005, 57, 577−585.
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added to antipsychotics for the treatment of schizophrenia. Biol.
Psychiatry 1998, 44, 1081−1089.
(8) Burnet, P. W. J.; Eastwood, S. L.; Bristow, G. C.; Godlewska, B.
R.; Sikka, P.; Walker, M.; Harrison, P. J. D-Amino acid oxidase activity
and expression are increased in schizophrenia. Mol. Psychiatry 2008,
13, 658−660.
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Panizzutti, R. Increased brain D-amino acid oxidase (DAAO) activity in
schizophrenia. Schizophrenia Res. 2008, 101, 76−83.
6-{2-[3,5-Bis(trifluoromethyl)phenyl]ethyl}-4-hydroxypyri-
dazin-3(2H)-one (31). The title compound was prepared in a
manner similar to that used to prepare 13, with the substitution of
starting material {(E)-2-[3,5-bis(trifluoromethyl)phenyl]vinyl}boronic
acid in place of [(E)-2-phenylvinyl]boronic acid in 6% yield (3 steps)
1
as a white solid. H NMR (DMSO-d6) δ 2.79−2.87 (m, 2H), 3.05−
3.14 (m, 2H), 6.63 (s, 1H), 7.90 (s, 1H), 7.96 (s, 2H), 10.75 (br, 1H),
12.68 (s, 1H). MS (ESI+) m/z = 353 (MH+). HRMS (ESI+) calcd for
[C14H10N2O2F6 + H]+, 353.0725; found, 353.0717.
6-[2-(3,5-Dimethoxyphenyl)ethyl]-4-hydroxypyridazin-
3(2H)-one (32). The title compound was prepared in a manner
similar to that used to prepare 13, with the substitution of starting
material [(E)-2-(3,5-dimethoxyphenyl)vinyl]boronic acid in place of
[(E)-2-phenylvinyl]boronic acid in 18% yield (3 steps) as a white
solid. 1H NMR (DMSO-d6) δ 2.69−2.84 (m, 4H), 3.70 (s, 6H), 6.28−
6.32 (m, 1H), 6.35−6.40 (m, 2H), 6.59 (s, 1H), 10.70 (br, 1H), 12.67
(s, 1H); MS (ESI+) m/z = 277 (MH+). HRMS (ESI+) calcd for
[C14H16N2O4 + H]+, 277.1188; found, 277.1181.
ASSOCIATED CONTENT
* Supporting Information
■
S
Permeability of compounds 13−32 measured by PAMPA and
panel screening of compound 30 for 57 targets. This material is
available free of charge via the Internet at http://pubs.acs.org.
(10) Almond, S. L.; Fradley, R. L.; Armstrong, E. J.; Heavens, R. B.;
Rutter, A. R.; Newman, R. J.; Chiu, C. S.; Konno, R.; Hutson, P. H.;
Brandon, N. J. Behavioral and biochemical characterization of a mutant
mouse strain lacking D-amino acid oxidase activity and its implications
for schizophrenia. Mol. Cell. Neurosci. 2006, 32, 324−334.
(11) Hashimoto, A.; Konno, R.; Yano, H.; Yoshikawa, M.; Tamaki,
R.; Matsumoto, H.; Kobayashi, H. Mice lacking D-amino acid oxidase
activity exhibit marked reduction of methamphetamine-induced
stereotypy. Eur. J. Pharmacol. 2008, 586, 221−225.
Accession Codes
All coordinates have been deposited in the PDB with accession
codes 3W4I (8), 3W4J (12), and 3W4K (13).
AUTHOR INFORMATION
Corresponding Author
■
*For T.H.: phone, +81-29-863-6728; fax, +81-29-854-1519; E-
mail, takeshi.hondo@astellas.com. For T.N.: phone, +81-29-
863-6767; fax, +81-29-854-1519; E-mail, tatsuya.niimi@astellas.
com.
(12) Hashimoto, A.; Yoshikawa, M.; Niwa, A.; Konno, R. Mice
lacking D-amino acid oxidase activity display marked attenuation of
stereotypy and ataxia induced by MK-801. Brain Res. 2005, 1033,
210−215.
Notes
(13) Detera-Wadleigh, S. D.; McMahon, F. J. G72/G30 in
Schizophrenia and Bipolar Disorder: Review and Meta-analysis. Biol.
Psychiatry 2006, 60, 106−114.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(14) Frisell, W. R.; Lowe, H. J.; Hellerman, L. Flavoenzyme catalysis.
Substrate-competitive inhibition of D-amino acid oxidase. J. Biol. Chem.
1956, 223, 75−83.
We thank Katsuhiko Gato, Masahiro Fujii, and Mitsuru Hosaka
for evaluating the physical chemistries, drug metabolisms, and
pharmacokinetics. We also thank Dr. Atsushi Suzuki and Dr.
Takahiko Tobe for helpful discussions.
(15) 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
pharmacological profile of AS057278, a selective D-amino acid oxidase
inhibitor with potential antipsychotic properties. Eur. Neuropsycho-
pharmacol. 2008, 18, 200−214.
(16) Ferraris, D.; Duvall, B.; Ko, Y.-S.; Thomas, A. G.; Rojas, C.;
Majer, P.; Hashimoto, K.; Tsukamoto, T. Synthesis and Biological
Evaluation of D-Amino Acid Oxidase Inhibitors. J. Med. Chem. 2008,
51, 3357−3359.
(17) Smith, S. M.; Uslaner, J. M.; Yao, L.; Mullins, C. M.; Surles, N.
O.; Huszar, S. L.; McNaughton, C. H.; Pascarella, D. M.; Kandebo, M.;
Hinchliffe, R. M.; Sparey, T.; Brandon, N. J.; Jones, B.; Venkatraman,
S.; Young, M. B.; Sachs, N.; Jacobson, M. A.; Hutson, P. H. The
Behavioral and Neurochemical Effects of a Novel D-Amino Acid
Oxidase Inhibitor Compound 8[4H-Thieno[3,2-b]pyrrole-5-carboxylic
Acid] and D-Serine. J. Pharmacol. Exp. Ther. 2009, 328, 921−930.
ABBREVIATIONS USED
■
DAAO, D-amino acid oxidase; NMDA, N-methyl-D-aspartate;
CBIO, 6-chlorobenzo[d]isoxazol-3-ol; FBDD, fragment-based
drug design; PDB, Protein Data Bank; CIAS, cognitive
impairment associated with schizophrenia; PAMPA, parallel
artificial membrane permeability assay; BBB, blood−brain
barrier
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