Synthesis and biochemical evaluation of lid-open D-amino acid oxidase inhibitors

Article abstract of DOI:10.3390/molecules24020290

Most of the known inhibitors of D-amino acid oxidase (DAAO) are small polar molecules recognized by the active site of the enzyme. More recently a new class of DAAO inhibitors has been disclosed that interacts with loop 218?224 at the top of the binding pocket. These compounds have a significantly larger size and more beneficial physicochemical properties than most reported DAAO inhibitors, however, their structure-activity relationship is poorly explored. Here we report the synthesis and evaluation of this type of DAAO inhibitors that open the lid over the active site of DAAO. In order to collect relevant SAR data we varied two distinct parts of the inhibitors. A systematic variation of the pendant aromatic substituents according to the Topliss scheme resulted in DAAO inhibitors with low nanomolar activity. The activity showed low sensitivity to the substituents investigated. The variation of the linker connecting the pendant aromatic moiety and the acidic headgroup revealed that the interactions of the linker with the enzyme were crucial for achieving significant inhibitory activity. Structures and activities were analyzed based on available X-ray structures of the complexes. Our findings might support the design of drug-like DAAO inhibitors with advantageous physicochemical properties and ADME profile.

Full text of DOI:10.3390/molecules24020290

molecules
Article
Synthesis and Biochemical Evaluation of Lid-Open
D-Amino Acid Oxidase Inhibitors
Bence Szilágyi 1 , Csilla Hargitai 2, Ádám A. Kelemen 1, Anita Rácz 3 , György G. Ferenczy 1
,
Balázs Volk 2,
*
and György M. Keseru˝
*
1,
1
Medicinal Chemistry Research Group, Research Centre for Natural Sciences, Hungarian Academy of
Sciences, Magyar tudósok krt. 2, H-1117 Budapest, Hungary; szilagyi.bence@ttk.mta.hu (B.S.);
kelemen.adam@ttk.mta.hu (Á.A.K.); ferenczy.gyorgy@ttk.mta.hu (G.G.F.)
Directorate of Drug Substance Development, Egis Pharmaceuticals Plc., P.O. Box 100, H-1475 Budapest,
Hungary; hargitai.csilla@egis.hu
Plasma Chemistry Research Group, Research Centre for Natural Sciences, Hungarian Academy of Sciences,
Magyar tudósok krt. 2, H-1117 Budapest, Hungary; racz.anita@ttk.mta.hu
Correspondence: volk.balazs@egis.hu (B.V.); keseru.gyorgy@ttk.mta.hu (G.M.K.);
Tel.: +36-1-803-5874 (B.V.); +36-1-382-6821 (G.M.K.)
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 8 December 2018; Accepted: 10 January 2019; Published: 14 January 2019
Abstract: Most of the known inhibitors of D-amino acid oxidase (DAAO) are small polar molecules
recognized by the active site of the enzyme. More recently a new class of DAAO inhibitors has
been disclosed that interacts with loop 218−224 at the top of the binding pocket. These compounds
have a significantly larger size and more beneficial physicochemical properties than most reported
DAAO inhibitors, however, their structure-activity relationship is poorly explored. Here we report
the synthesis and evaluation of this type of DAAO inhibitors that open the lid over the active
site of DAAO. In order to collect relevant SAR data we varied two distinct parts of the inhibitors.
A systematic variation of the pendant aromatic substituents according to the Topliss scheme resulted
in DAAO inhibitors with low nanomolar activity. The activity showed low sensitivity to the
substituents investigated. The variation of the linker connecting the pendant aromatic moiety and
the acidic headgroup revealed that the interactions of the linker with the enzyme were crucial for
achieving significant inhibitory activity. Structures and activities were analyzed based on available
X-ray structures of the complexes. Our findings might support the design of drug-like DAAO
inhibitors with advantageous physicochemical properties and ADME profile.
Keywords: D-amino acid oxidase (DAAO); inhibitor; lid-open conformation; Topliss scheme;
structure-activity relationship
1. Introduction
D-Amino acid oxidase (DAAO) is a flavoprotein that catalyzes the oxidative deamination of
D-amino acids. It plays a crucial role in oxidizing D-serine, a co-agonist of the NMDA receptor,
whose hypoactivity is thought to be involved in the positive, negative and cognitive symptoms in
schizophrenia. Therefore, increased DAAO activity results in lower D-serine level [1,2] and lower
NMDA activity. Indeed, it has been shown from post mortem brain tissue samples of patients who
suffered from schizophrenia that DAAO expression and enzyme activity were elevated compared to
healthy controls [
brain D-serine level and may have positive effect on the symptoms of schizophrenia [4].
First generation DAAO inhibitors 10] are mostly small polar molecules in accordance
with the properties of the enzyme active site (Figure 1). These compounds, however, tend to have
3]. These findings suggest that the inhibition of DAAO may result in an increase of
1–6 [5–
Molecules 2019, 24, 290; doi:10.3390/molecules24020290
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suboptimal pharmacokinetic properties. In particular, they are characterized by poor absorption and
penetration through the blood-brain barrier.
Figure 1. Known active site DAAO inhibitors in the literature.
In 2014, Terry-Lorenzo et al. [11] reported that during the screening of a computationally
prioritized library, a structurally novel compound (
inhibitory properties in the low nanomolar range. An analogue of
carboxylic acid group to a bioisosteric hydroxypyridazinone moiety to obtain compound
7
) was identified showing competitive D-serine
was synthesized by changing the
(Figure 2).
7
8
Figure 2. Novel DAAO inhibitors that interact with the flexible loop and the structural moieties of the
lid-open type compounds.
Compounds
previous active site inhibitors, these compounds also interact with residues at the entrance of the
binding pocket. X-Ray structures of the complexes of and with DAAO [11] revealed that the pendant
phenyl group interacted with the flexible loop formed by residues 218 224. This loop acts as a lid that
covers the entry of the binding pocket when small compounds are bound, and it remains open in the
complexes of and . Therefore, the compounds in this series can be used to explore the properties
7 and 8 represent a new generation of DAAO inhibitors because, in contrast to
7
8
−
7
8
and optimal interactions of the flexible loop (amino acids 218–224). Moreover, the absorption of this
compound class is expected to be more favorable than that of small, polar compounds.
Targeting active site lids, if available, is a feasible strategy for enzyme inhibition. Since enzymes
with lid-gated active sites operate by an induced fit mechanism [12], here we investigated the impact of
different structural elements on lid opening and stabilization. Compounds
7 and 8 can be divided into
three structural parts (Figure 2). We can identify an aromatic part which is responsible for maintaining
the loop in the open conformation, a linker part which is an aromatic moiety with hydrogen-bond
donors and acceptors, and an acid or acid bioisoster headgroup which interacts with Arg283 close

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to the isoalloxazine ring of flavin adenine dinucleotide (FAD). In this paper we present the design,
synthesis and testing of lid-open type analogues with potential DAAO inhibitory activity.
2. Results and Discussion
We introduced modifications in the linker and in the pendant aromatic part while we used acidic
and acid bioisoster headgroups already described for DAAO inhibitors [11
we explored what kind of interactions could be formed between the flexible loop and the aromatic
part of the compounds, so we have designed derivatives of compound mono-substituted at the
,13–16]. In the first step,
8
aromatic part. The scheme proposed by Topliss [17] has been applied for the stepwise selection of
compounds to be synthesized. This scheme is designed for the systematic optimization of aromatic
substituents by identifying the effects of the Hammet constant (
σ), the hydrophobic substituent
constant ( ) and Taft’s steric constant (Es) on the activity of the compounds. This stepwise process
π
includes the synthesis and testing of a number of compounds in each step and selection of compounds
for the next step based on the observed activities in the previous step. In this way, the variation of a few
substituents and the activity measurement of a limited number of compounds allow the exploration of
the binding site and the optimization of activity.
The prepared compounds were tested as DAAO inhibitors with the KYNA enzyme inhibitory
assay [18]. Although D-serine is a natural substrate of DAAO, its metabolite is not suitable for
fluorometric evaluation. However in the applied assay, D-kynurenine can be metabolized by
DAAO [19] and the metabolite (KYNA) has a favorable fluorescence property. Thus it can be used for
fluorescence measurements [20].
We started with compounds
8 and 9 (Figure 3). Since they exhibited similar activity (Table 1),
we proceeded with the synthesis of 10. This latter compound showed slightly lower activity, therefore
we decided to synthesize 11. The activity of this 3-Cl substituted derivative (11) was lower than
100 nM therefore we synthesized the 3-Me derivative (12). Although the X-ray structure of the complex
of
8 with DAAO (PDB code: 4QFC [11]) suggested that small substituents in the meta position
could be accommodated in the binding pocket, the larger 3-OMe substituent in 13 did not yield
further improvement in the activity. DAAO binding and activity of representative compounds have
been confirmed in orthogonal differential scanning fluorometry (DSF) and the coupled Horseradish
peroxidase (HRP)/Amplex Red assays, respectively (see Supplementary materials). The designed
compounds were docked into the protein structure obtained from the complex of
of the ligand. The docking of compounds 12 resulted in complexes with ligand positions highly
similar to that of and suggested that the meta substituent in 11 and 12 points away from the
hydroxypyridazinone headgroup (Figure 4).
8 by the removal
9
–
8
Figure 3. Prepared derivatives of compound 8 monosubstituted at the aromatic part.

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Table 1. Enzyme assay test results of compounds 8–13.
Compound
IC50 [nM] *
8
9
10
11
12
13
112(17)
119(24)
474(12)
69(18)
52(9)
73(11)
* Standard deviations are shown in parenthesis.
Figure 4. Proposed binding pose of compound 12. (
ribbon diagram, ligand in green tube and the isoalloxazine ring of FAD in cyan tube representation.
) Details of interactions are indicated by dashed lines: yellow hydrogen-bond, cyan aromatic–aromatic.
A) Overall structure with protein shown in
(B
(H-bond criterion: heavy atom distance < 2.8 Å; aromatic-aromatic contact criteria: face-to-face distance
< 4.4 Å; face-to edge distance < 5.5 Å).
Following the variation of the aromatic substituents, we next focused on the linker region.
Starting from the original linker used in compounds and 8, we selected 1,5-dihydro-2H-pyrrolo
7
[3,2-d]pyrimidine-2,4(3H)-dione and 1,2,3,4-tetrahydroisoquinoline skeletons that might resemble in
shape and possible substitution pattern (Figure 5).
H
N
O
NH
N
NH
H
O
1,2,3,4-tetrahydroisoquinoline
1,5-dihydro-2H-pyrrolo[3,2-d]
pyrimidine-2,4(3H)-dione
Figure 5. Linkers designed to replace the 7-hydroxy-2H-chromen-2-one moiety used in 7 and 8.
As regards the headgroup, the best option would have been the hydroxypyridazinone moiety
used in the original compound (8), but we failed to produce derivative 14 (Figure 6) that would have
been suitable for the coupling reaction. Therefore, we decided to attach a known acidic headgroup of
the pyrrole-2-carboxylic acid by using derivative 15.
Figure 6. Selected headgroups for the synthesis of lid-open DAAO inhibitors.

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The prepared new compounds 16
–
26 (Figure 7) were tested in the KYNA enzyme inhibitory
assay, and they did not show any inhibitory activity at 20
materials). Comparing these compounds with DAAO inhibitors
acid containing inhibitors known from the literature [16 21] strongly suggests that it is the linker part
of molecules 16 26 that prevents DAAO activity. The replacement of the 7-hydroxy-2H-chromen-2-one
linker of compounds 13 changes the interactions of the linker, in addition it modifies the exit vectors
of both the acidic headgroup and the pendant aromatic moiety in compounds 16 19. An analysis
µM concentration (see Supplementary
8–13 and with pyrrole-2-carboxylic
,
–
7
–
–
of the X-ray structure of complexes of
the loss of linker planarity in compounds 20
planar linker of and
hydrophobic interactions may not be optimally formed with the nonplanar tetrahydroisoquinoline
group. Furthermore, the OH group of the 7-hydroxy-2H-chromen-2-one linker in and is able to form
H-bond with the Gln53 backbone carbonyl and the carbonyl group of the linker forms a water-mediated
H-bond to Tyr224. These H-bonds are clearly missing in potential complexes of 20 26. While the
presence of H-bond to the Gln53 backbone is highly possible in DAAO complexes of 16 19, no H-bond
acceptor to the water-mediated interaction with Tyr224 can be assumed. Perhaps more importantly,
the different orientation of the pendant aromatic group in 16 19 compared to compounds 13 might
be responsible for the decreased activity. This group in derivatives 13 points toward the flexible
loop formed by residues 218 224 and may not be able to adopt a position beneficially interacting with
7
and
8
(PDB codes: 4QFD [11] and 4QFC [11]) suggests that
–
26 is likely to contribute to the loss of activity as the
7
8 beneficially interacts with Tyr55, Leu215 and Tyr224, while these stacking and
7
8
–
–
–
9–
9–
−
the loop. These observations suggest that the linker contributes significantly to DAAO activity both by
its interactions with the enzyme and by properly orienting the pendant aromatic moiety.
Figure 7. Prepared compounds with new linker part.
3. Materials and Methods
3.1. General Information
Melting points were determined on an OptiMelt SRS instrument (Stanford Research Systems,
Sunnyvale, CA, USA) and are uncorrected. NMR measurements were performed on a System 500 NMR
1
spectrometer (Varian, Palo Alto, CA, USA) or a Varian System 300 spectrometer. H- and 13C-NMR
1
spectra were measured at room temperature (25 ◦C) in an appropriate solvent. H and 13C chemical

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shifts are expressed in parts per million (δ) referenced to TMS or residual solvent signals. Reactions
were monitored with silica gel 60 F254 TLC plates (Merck, Darmstadt, Germany). All chemicals and
solvents were used as purchased. HPLC-MS measurements were performed using a LC-MS-2020
device (Shimadzu, Kyoto, Japan) equipped with a Reprospher 100 C18 (5
µ
m, 100
×
3 mm) column
and positive-negative double ion source (DUIS ) with a quadrupole mass spectrometer in a range of
±
50–1000 m/z. Samples were eluted with gradient elution using eluent A (0.1% formic acid in water) and
eluent B (0.1% formic acid in acetonitrile). Flow rate was set to 1.5 mL/min. The initial condition was
0% B eluent, followed by a linear gradient to 100% B eluent by 2 min, from 2 to 3.75 min 100% B eluent
was retained, and from 3.75 to 4.5 min back to initial condition and retained to 5 min. The column
◦
temperature was kept at 30 C and the injection volume was 1
µL. High resolution mass spectrometric
measurements were performed using a Q-TOF Premier mass spectrometer (Waters, Milford, MA, USA)
in positive electrospray ionization mode. Compound was prepared following the procedure reported
8
in ref. [11]. Preparation of compounds 9–13 and 16–26 is described in the Supplementary materials.
3.2. KYNA Enzyme Inhibition Assay
D-2-Amino-4-(2-aminophenyl)-4-oxobutanoic acid (D-KYN) was used to measure D-amino acid
oxidase activity based on the protocol described in ref. [18,20]. Human DAAO (purchased from
TargetEx Ltd. (Dunakeszi, Hungary)) was used for the measurements. The buffer contained 20 mM
TRIS-HCl and 100 mM NaCl (pH = 8). Flavin adenine dinucleotide (FAD—obtained from Sigma
(St. Louis, MO, USA)) in 5 µM concentration was also included in the assay. Compounds were dissolved
originally in DMSO and the measured samples were diluted with the buffer solution (the final DMSO
concentration was always below 5%). The mixed solution was incubated at 37 ◦C for 1 h. After the
enzymatic reaction, ZnCl2 dissolved in H2O was added and vortex-mixed. Single point measurements
were performed at 20 µm inhibitor concentration. For the IC50 measurements, the inhibitors were used
at 5 nM, 50 nM, 500 nM, 2.5 µM, 5 µM, 10 µM, 50 µM. Measurements were carried out on a Citation3
cell imaging multi-mode microplate reader (BioTek Instruments Inc., Winooski, VT, USA) with 364
well plates. The applied wavelengths were 340 nm and 396 nm.
3.3. Molecular Modeling
Compounds were docked into the DAAO structure (PDB: 4QFC). Protein and ligand preparations
were performed with Schrödinger’s tools [22] with standard settings and Glide [22] was used for
docking and scoring.
4. Conclusions
In the current investigation, we synthesized and tested compounds derived from DAAO inhibitors
that, in contrast to most other reported inhibitors, interact with loop 218 224 at the top of the ligand
−
binding pocket. This loop must be in the open position to allow for substrate binding and typically
closes when the ligand is within the pocket. However, large ligands force the loop to leave the pocket
partially open after ligand binding and this opens up new opportunities in the development of DAAO
inhibitors. We varied two moieties of extended DAAO inhibitors, namely the pendant aromatic group
and the linker that connects the former with the acid or acid mimetic headgroup. The compounds
produced are of synthetic and medicinal chemistry value, as they explore structure-activity relationship
of lid-open DAAO inhibitors. Moreover, a systematic variation of the aromatic substituents according
to the scheme proposed by Topliss resulted in DAAO inhibitors in the 2 3 digit nanomolar affinity
−
range. The activity showed low sensitivity to the substituents investigated. The variation of the linker
part revealed that the interactions of the linker with the enzyme were crucial for achieving significant
inhibitory activity. The analysis of the structures and their activities suggests that a planar linker with
H-bond forming ability at suitable positions is indispensable for DAAO activity. These results may
find use in DAAO inhibitor design since these types of compounds broaden the operational space to
develop inhibitors with advantageous physicochemical properties and ADME profile.

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Supplementary Materials: The following are available online. Scheme S1. Synthesis of the derivatives of 8
substituted on the benzene ring of the aromatic part, Scheme S2. Preparation of compounds 16 26, Scheme S3.
–
Preparation of linkers containing a substituted aromatic moiety, Scheme S4. Synthesis of the headgroup, results
from differential scanning fluorimetry (DSF) measurements, results from the coupled HRP/Amplex Red assay for
compound, limit dose (20 µM) screening results obtained for compounds 8–13 and 16–26.
Author Contributions: B.S., Á.A.K. and C.H. performed the reactions. A.R. tested the key compounds in the
activity assay. G.G.F. carried out molecular modeling and compiled the relevant parts of the manuscript. B.S., B.V.
and G.M.K. wrote and finalized the manuscript. G.M.K. conducted the project.
Funding: This research was supported by the National Brain Research Program (2017–1.2.1-NKP-2017-00002) of
the National Research, Development and Innovation Office, Hungary.
Acknowledgments: The authors are grateful to Ágnes Gömöry, Pál Szabó and Tímea Imre for the
HRMS measurements.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Hashimoto, K.; Fukushima, T.; Shimizu, E.; Komatsu, N.; Watanabe, H.; Shinoda, N.; Nakazato, M.;
Kumakiri, C.; Okada, S.I.; Hasegawa, H.; et al. Decreased serum levels of D-serine in patients with
schizophrenia: Evidence in support of the N-methyl-D-aspartate receptor hypofunction hypothesis of
schizophrenia. Arch. Gen. Psychiatry 2003, 60, 572–576. [CrossRef] [PubMed]
2.
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. Schizophr. Res. 2007, 90, 41–51.
[CrossRef]
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.
[CrossRef] [PubMed]
4.
5.
Smith, S.M.; Uslaner, J.M.; Hutson, P.H. The Therapeutic Potential of D-Amino Acid Oxidase (DAAO)
Inhibitors. Open Med. Chem. J. 2010, 4, 3–9. [CrossRef] [PubMed]
Adage, T.; Trillat, A.C.; Quattropani, A.; Perrin, D.; Cavarec, L.; Shaw, J.; Guerassimenko, O.; Giachetti, C.;
Gréco, B.; Chumakov, I.; et al. In vitro and in vivo pharmacological profile of AS057278, a selective D-amino
acid oxidase inhibitor with potential anti-psychotic properties. Eur. Neuropsychopharmacol. 2008, 18, 200–214.
[CrossRef]
6.
7.
8.
Katane, M.; Osaka, N.; Matsuda, S.; Maeda, K.; Kawata, T.; Saitoh, Y.; Sekine, M.; Furuchi, T.; Doi, I.;
Hirono, S.; et al. Identification of novel D-amino acid oxidase inhibitors by in silico screening and their
functional characterization in vitro. J. Med. Chem. 2013, 56, 1894–1907. [CrossRef]
Sparey, T.; Abeywickrema, P.; Almond, S.; Brandon, N.; Byrne, N.; Campbell, A.; Hutson, P.H.; Jacobson, M.;
Jones, B.; Munshi, S.; et al. The discovery of fused pyrrole carboxylic acids as novel, potent D-amino acid
oxidase (DAO) inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 3386–3391. [CrossRef] [PubMed]
Duplantier, A.J.; Becker, S.L.; Bohanon, M.J.; Borzilleri, K.A.; Chrunyk, B.A.; Downs, J.T.; Hu, L.-Y.;
El-Kattan, A.; James, L.C.; Liu, S.; et al. Discovery, SAR, and Pharmacokinetics of a Novel
3-Hydroxyquinolin-2(1H)-one Series of Potent D-Amino Acid Oxidase (DAAO) Inhibitors. J. Med. Chem.
2009, 2, 3576–3585. [CrossRef]
9.
Chumakov, I.; Blumenfeld, M.; Guerassimenko, O.; Cavarec, L.; Palicio, M.; Abderrahim, H.; Bougueleret, L.;
Barry, C.; Tanaka, H.; La Rosa, P.; et al. Genetic and physiological data implicating the new human gene G72
and the gene for D-amino acid oxidase in schizophrenia. Proc. Natl. Acad. Sci. USA 2002, 99, 13675–13680.
[CrossRef] [PubMed]
10. 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. [CrossRef]
[PubMed]
11. Terry-Lorenzo, R.T.T.; Chun, L.E.E.; Brown, S.P.P.; Heffernan, M.L.L.R.; Fang, Q.K.K.; Orsini, M.A.A.;
Pollegioni, L.; Hardy, L.W.W.; Spear, K.L.L.; Large, T.H.H. Novel human D-amino acid oxidase inhibitors
stabilize an active-site lid-open conformation. Biosci. Rep. 2014, 34, 487–499. [CrossRef] [PubMed]

Molecules 2019, 24, 290
8 of 8
12. Sullivan, S.M.; Holyoak, T. Enzymes with lid-gated active sites must operate by an induced fit mechanism
instead of conformational selection. Proc. Natl. Acad. Sci. USA 2008, 105, 13829–13834. [CrossRef]
13. Hin, N.; Duvall, B.; Berry, J.F.; Ferraris, D.V.; Rais, R.; Alt, J.; Rojas, C.; Slusher, B.S.; Tsukamoto, T. D-Amino
acid oxidase inhibitors based on the 5-hydroxy-1,2,4-triazin-6(1H)-one scaffold. Bioorg. Med. Chem. Lett.
2016, 26, 2088–2091. [CrossRef] [PubMed]
14. Hin, N.; Duvall, B.; Ferraris, D.; Alt, J.; Thomas, A.G.; Rais, R.; Rojas, C.; Wu, Y.; Wozniak, K.M.;
Slusher, B.S.; et al. 6-Hydroxy-1,2,4-triazine-3,5(2H,4H)-dione Derivatives as Novel D-Amino Acid Oxidase
Inhibitors. J. Med. Chem. 2015, 58, 7258–7272. [CrossRef] [PubMed]
15. Hondo, T.; Warizaya, M.; Niimi, T.; Namatame, I.; Yamaguchi, T.; Nakanishi, K.; Hamajima, T.; Harada, K.;
Sakashita, H.; Matsumoto, Y.; et al. 4-Hydroxypyridazin-3(2H)-one Derivatives as Novel D-Amino Acid
Oxidase Inhibitors. J. Med. Chem. 2013, 56, 3582–3592. [CrossRef] [PubMed]
16. Raje, M.; Hin, N.; Duvall, B.; Ferraris, D.V.; Berry, J.F.; Thomas, A.G.; Alt, J.; Rojas, C.; Slusher, B.S.;
Tsukamoto, T. Synthesis of kojic acid derivatives as secondary binding site probes of D-amino acid oxidase.
Bioorg. Med. Chem. Lett. 2013, 23, 3910–3913. [CrossRef] [PubMed]
17. Topliss, J.G. Utilization of Operational Schemes for Analog Synthesis in Drug Design. J. Med. Chem. 1972, 15,
1006–1011. [CrossRef] [PubMed]
18. Kozaki, A.; Iwasa, S.; Hosoda, S.; Nishiguchi, Y.; Nakayama, M.; Ichiba, H.; Fukushima, T. Fluorimetric assay
for D-amino acid oxidase activity in rat brain homogenate by using D-kynurenine as a substrate. Biosci. Trends
2012, 6, 241–247. [CrossRef]
19. Fukushima, T.; Sone, Y.; Mitsuhashi, S.; Tomiya, M.; Toyo’oka, T. Alteration of kynurenic acid concentration in
rat plasma following optically pure kynurenine administration: A comparative study between enantiomers.
Chirality 2009, 21, 468–472. [CrossRef]
20. Song, Z.; Ogaya, T.; Ishii, K.; Ichiba, H.; Iizuka, H.; Fukushima, T. Utilization of Kynurenic Acid Produced
from D-kynurenine in an in Vitro Assay of D-Amino Acid Oxidase Activity. J. Health Sci. 2010, 56, 341–346.
[CrossRef]
21. Hopkins, S.C.; Heffernan, M.L.R.; Saraswat, L.D.; Bowen, C.A.; Melnick, L.; Hardy, L.W.; Orsini, M.A.;
Allen, M.S.; Koch, P.; Spear, K.L.; et al. Structural, Kinetic, and Pharmacodynamic Mechanisms of D-Amino
Acid Oxidase Inhibition by Small Molecules. J. Med. Chem. 2013, 56, 3710–3724. [CrossRef] [PubMed]
22. Schrödinger Suite, version 2018–1; Schrödinger, LLC: New York, NY, USA, 2018.
Sample Availability: Samples of the compounds are not available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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