Discovery of isatin and 1H-indazol-3-ol derivatives as D-amino acid oxidase (DAAO) inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.02.004

D-Amino acid oxidase (DAAO) is a potential target in the treatment of schizophrenia as its inhibition increases brain D-serine level and thus contributes to NMDA receptor activation. Inhibitors of DAAO were sought testing [6+5] type heterocycles and identified isatin derivatives as micromolar DAAO inhibitors. A pharmacophore and structure-activity relationship analysis of isatins and reported DAAO inhibitors led us to investigate 1H-indazol-3-ol derivatives and nanomolar inhibitors were identified. The series was further characterized by pK_a and isothermal titration calorimetry measurements. Representative compounds exhibited beneficial properties in in vitro metabolic stability and PAMPA assays. 6-fluoro-1H-indazol-3-ol (37) significantly increased plasma D-serine level in an in vivo study on mice. These results show that the 1H-indazol-3-ol series represents a novel class of DAAO inhibitors with the potential to develop drug candidates.

Full text of DOI:10.1016/j.bmc.2018.02.004

Accepted Manuscript
Discovery of isatin and 1H-indazol-3-ol derivatives as D-amino acid oxidase
(DAAO) inhibitors
Bence Szilágyi, Péter Kovács, György G. Ferenczy, Anita Rácz, Krisztina
Németh, Júlia Visy, Pál Szabó, Janez Ilas, György T. Balogh, Katalin
Monostory, István Vincze, Tamás Tábi, Éva Szökő, György M. Keserű
PII:
S0968-0896(17)32415-X
https://doi.org/10.1016/j.bmc.2018.02.004
BMC 14190
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 December 2017
27 January 2018
3 February 2018
Please cite this article as: Szilágyi, B., Kovács, P., Ferenczy, G.G., Rácz, A., Németh, K., Visy, J., Szabó, P., Ilas,
J., Balogh, G.T., Monostory, K., Vincze, I., Tábi, T., Szökő, E., Keserű, G.M., Discovery of isatin and 1H-indazol-3-
ol derivatives as D-amino acid oxidase (DAAO) inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.02.004
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Discovery of Isatin and 1H-indazol-3-ol Derivatives
as D Amino Acid Oxidase (DAAO) Inhibitors
Bence Szilágyi, Péter Kovács, György G. Ferenczy, Anita Rácz, Krisztina Németh, Júlia Visy, Pál Szabó, Janez
Ilas, György T. Balogh, Katalin Monostory, István Vincze, Tamás Tábi, Éva Szökő, György M. Keserű*
Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2. H-1117
Budapest, Hungary

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Discovery of isatin and 1H-indazol-3-ol derivatives as D amino acid oxidase
(DAAO) inhibitors
Bence Szilágyia, Péter Kovácsa, György G. Ferenczya, Anita Rácza, Krisztina Némethb, Júlia Visya, Pál
Szabóc, Janez Ilasd, György T. Baloghe, Katalin Monostoryf, István Vinczeg, Tamás Tábig, Éva Szökőg,
György M. Keserűa∗
aMedicinal Chemistry Research Group, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2. H-1117 Budapest,
Hungary;
bChemical Biology Research Group, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2. H-1117 Budapest, Hungary
cMS Metabolomics Laboratory, Core Facility, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2. H-1117 Budapest,
Hungary
dUniversity of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia
eCompound Profiling Laboratory, Gedeon Richter plc. Gyömrői út 30-32, H-1103 Budapest, Hungary
fMetabolic Drug-Interactions Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok
krt. 2. H-1117 Budapest, Hungary
gDepartment of Pharmacodynamics, Semmelweis University, Nagyvárad tér 4. Budapest, H-1089, Hungary
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
D-amino acid oxidase (DAAO) is a potential target in the treatment of schizophrenia as its
inhibition increases brain D-serine level and thus contributes to NMDA receptor activation.
Inhibitors of DAAO were sought testing [6+5] type heterocycles and identified isatin
derivatives as micromolar DAAO inhibitors.
A pharmacophore and structure-activity
Available online
relationship analysis of isatins and reported DAAO inhibitors led us to investigate 1H-indazol-3-
ol derivatives and nanomolar inhibitors were identified. The series was further characterized by
pKa and isothermal titration calorimetry measurements. Representative compounds exhibited
beneficial properties in in vitro metabolic stability and PAMPA assays. 6-fluoro-1H-indazol-3-ol
(37) significantly increased plasma D-serine level in an in vivo study on mice. These results
show that the 1H-indazol-3-ol series represents a novel class of DAAO inhibitors with the
potential to develop drug candidates.
Keywords:
D-amino acid oxidase
Optimization
Protonation state
Binding thermodynamics
2009 Elsevier Ltd. All rights reserved
.
———
∗ Corresponding author. Tel.: +36-1-382-6821; e-mail: gy.keseru@ttk.mta.hu

1. Introduction
Compounds 1-3 are examples of inhibitors that contain a
carboxylic acid moiety similarly to endogenous ligands.
Significant effort has been devoted to identify inhibitors that do
not contain carboxylic acid (see e.g. compounds 4-6) and having
beneficial pharmacokinetic profile. However, the DAAO binding
site prefers small and highly polar ligands and this seriously
limits ligand variability. Moreover, although the binding pocket
features an Arg residue as a key interaction site the incorporation
of non-carboxylic acid Arg binder motifs12 into DAAO inhibitors
did not prove to be a straightforward strategy. Thus, it still
remains a significant challenge to find inhibitors with reasonable
affinity and balanced pharmacokinetic properties.
D-amino acid oxidase (DAAO) is a flavoprotein that catalyses
the oxidative deamination of D-amino acids. Genetic studies
have found association between schizophrenia and single
nucleotide polymorphisms in the DAAO and its regulator (G72)1.
Furthermore, DAAO expression and enzyme activity have been
reported to be increased in post mortem brain tissue samples
from patients with schizophrenia compared to healthy controls2.
Moreover reduced D-serine levels were described in the serum
and cerebrospinal fluid of schizophrenic patients, therefore it has
been suggested that the inhibition of DAAO could ameliorate
schizophrenic symptoms, especially with the co-administration
with D-serine3,4. D-serine, a D-amino acid that is regulated by
DAAO, is a potent, endogenous co-agonist of the N-methyl-D-
aspartic acid (NMDA) receptor5. Because NMDA receptor
dysfunction is thought to be involved in the positive (psychotic),
negative and cognitive symptoms in schizophrenia, there has
been much interest in developing potent and selective DAAO
inhibitors for the treatment of this disease. Until now several
DAAO inhibitors were reported, including 5-methylpirazole-3-
carboxylic acid 16,7, 4H-furo[3,2-b]pyrrole-5-carboxylic acid 28,
4H-thieno[3,2b]pyrrole-5-carboxylic acid 39, 3-hydroxyquinolin-
2(1H)-one 410, 1-hydroxy-1H-benzo[d]imidazol-2(3H)ones 511,
and 6-chlorobenzo[d]isoxazol-3-ol (CBIO) 64 (Figure 1).
In this paper we report novel sets of DAAO inhibitors having
isatin and 1H-indazol-3-ol scaffolds. The latter chemotype was
optimized to nanomolar inhibitors that were further evaluated in
ADME tests and an in vivo proof of concept assay.
2. Results and discussion
Our initial analysis of known DAAO inhibitors revealed that
most of the chemotypes are small and polar mono- or bicyclic
compounds having an acidic functionality (Figure 1).
Consequently, in the first round of searching for new inhibitor
scaffolds we screened [6+5] type heterocycles of this type. .All
compounds contain the pharmacophore elements of the reference
compounds in different arrangements. Table 1 shows the
inhibitory activity of tested compounds together with two
reference inhibitors, namely CBIO (6)4, and BIO (7)4 Most of the
scaffolds showed no activity at 20 µM concentration against
DAAO except for compounds 9, 11 and 16. Since 9 and 16
showed only moderate activity we selected the isatin scaffold
(11) for further evaluation (Table 2).
Figure 1. Chemical structure of DAAO inhibitors.
Table 1 [6+5] type heterocyclic compounds tested for DAAO inhibition.
Compound
Structure
Inhibition % at 20µMa Compound
Structure
Inhibition % at 20µMa
6 (CBIO)
114 (3)
-38 (2)
-42 (1)
7 (BIO)
913
101 (2)
813
26 (1)
46 (6)
1014
1115
H
N
1216
1418
1620
-5 (2)
-1 (1)
26 (1)
1317
1519
1719
1 (0)
-27 (0)
-4 (4)
O
N
H
O
NH
a All measurements were performed n=2; standard deviation in percentage is shown in parenthesis

Table 2. DAAO inhibitory activity of isatin analogues.
IC50 [µM] or %
inhibition at
20µMa
IC50 [µM] or %
inhibition at
20µMa
Compound
Structure
Compound
Structure
1815
1115
1915
2015
19% (1)
46% (6)
13% (2)
-1% (1)
2221
2322
2423
2523
49% (7)
12% (2)
8% (3)
O
O
13.1 (20)
N
H
2115
7.87 (7)
a All measurements were performed n=2; standard deviation in percentage is shown in parenthesis
Haloscan resulted in 21 as the most active derivative that is
behind the reference compounds. Although we got similar
potency for 25 the 7-fluoro derivative (23) was found to be much
active. The SAR inconsistency together with the order of
magnitude higher IC50 values compared to reference compounds
made further optimization of this scaffold less promising. In
order to improve the potency of compound 21 we decided to
modify the pharmacophore elements of the isatin scaffold (Figure
2).
been realized in the case of the 3-hydroxyquinolin-2(1H)-one
scaffold that contains a deprotonable enol instead of the upper
carbonyl group of the isatin scaffold. It is interesting to note that
the corresponding 3-hydroxy-2H-chromen-2-one scaffold was
found to be somewhat less active against DAAO indicating that
the donor adjacent to the aromatic ring might be beneficial.
Comparing the binding modes of these compounds revealed the
formation of an extra H-bond between the NH of the 3-
hydroxyquinolin-2(1H)-one and the backbone carbonyl of
Gly313 (Figure 3).
Figure 3. Experimental binding modes of 3-hydroxyquinolin-
2(1H)-one and 3-hydroxy-2H-chromen-2-one scaffolds.
Figure 2. Design paradigm leading to the 1H-indazol-3-ol
scaffold. Abbreviations: PhP, pharmacophore; exoA – H-bond
acceptor in exo position; A, acceptor; exoD H-bond donor in exo
position; D, donor.
Contracting the six membered ring of these compounds we
obtain two [6+5] type scaffolds as the benzo[d]isoxazol-3-ol (see
e.g. 7) and the 1H-indazol-3-ol (see e.g. 28). Although the former
one turned to be very potent DAAO inhibitors, the latter has not
been investigated yet. Consequently, our chemistry program
aimed at the exploration of the 1H-indazol-3-ol scaffold. To
achieve this goal, we prepared several 1H-indazol-3-ol fragments
(Table 3).
Comparative analysis of the available DAAO-inhibitor co-
crystal structures revealed that all of the known inhibitors bind to
Arg283 forming H-bonding interactions with the guanidine NHs.
Considering the positively charged character of the Arg
headgroup these interactions could be improved significantly
forming a negatively charged moiety on the scaffold. This has

Table 3. DAAO inhibitory activity of 1H-indazol-3-ol fragments.
IC50 [µM] or %
IC50 [µM] or %
inhibition at
20µMa
Compound
Structure
inhibition at
20µMa
Compound
Structure
2624
0% (0)
66% (2)
19% (8)
27
29
not measurableb
28
34% (0)
not measurableb
0.15 (13)
not measurableb
not measurableb
0.12 (42)
30
32
34
31
33
35
1.20 (25)
10.1 (13)
36
37
a All measurements were performed n=2; standard deviation in percentage is shown in parenthesis
b Strong autofluorescence
Although the 1H-pyrazol-3-ol (26) showed no activity both
the phenetyl (27) and the condensed 1H-indazol-3-ol derivatives
were found to be inhibitors of the target. 1H-indazol-3-ols
substituted in position 6 or 7 had significant activity, of which 6-
chloro (34) and 6-fluoro-1H-indazol-3-ol (35) exhibited near to
low nanomolar inhibition. An analysis of reported potent DAAO
inhibitors suggests that they are all able to deprotonate under
slightly basic conditions (pH~8) where DAAO works optimally.
In order to better characterize the 1H-indazol-3-ol series we
determined the pKa value for several representative compounds
(Table 4).
The pKa values of the investigated compounds are typically
between 7 and 8 suggesting that they are at least partially
deprotonated at slightly basic pH. This appears to be a common
property of DAAO inhibitors and also of the endogenous ligand.
Whether the compounds bind to the enzyme in a deprotonated
form depends on the local environment within the binding
pocket, however, our free energy perturbation calculations (data
not shown) strongly suggest that the bound compounds of the
1H-indazol-3-ol series are deprotonated and are carrying a
negative charge. This finding, in fact, explains the reduced
affinity of isatins (the measured pKa value of 21 is 9.44 and it has
a low fraction of ionized microspecies at pH 7.4). We also note,
that the substituents of the benzole ring of the 1H-indazol-3-ol
series affect pKa on one hand, and influences affinity via its
interactions with DAAO, on the other hand.
Table 4. Measured pKa values of representative compounds
% of deprotonated % of deprotonated
Cmpd pKa
microspeciesa
(pH=7.4)
microspeciesa
(pH=8)
SD
9.44 0.106
0.004
Table 5. Dissociation constants (Kd) from ITC and inhibitory
activity (IC50) from D-kynurenine assay.
21
3.96
14.1
IC50 [µM]a
Cmpd
Kd porcine ITC [µM]
0.15 (0.03)
21.5
38.7
35.7
25.0
21.7
19.3
32.8
22.0
52.2
71.6
68.8
57.0
52.5
48.8
64.7
52.9
8.16
28
29
30
31
34
35
36
37
7.51 0.004
7.51 0.002
7.53 0.006
4
CBIO(6)
BIO (7)
25
0.027 (36)
0.87 (0.03)
0.418 (9.0)
0.44 (0.14)
0.009
7.59
13.140 (13)
8.23 (1.46)
7.84 0.001
6.52 0.005
7.28 0.007
-
-
-
4.43 (1.63)
28
0.78 (0.24)
31
a calculated by the ChemAxon pKa plugin
3.29 (0.81)
32

in inhibitory potency comparable to those of 6-substituted
compounds.
-
1.20 (0.50)
2.00 (0.40)
1.00 (0.10)
4.59 (1.73)
3.31 (0.54)
33
34
35
36
37
0.156 (13)
0.123 (42)
1.205 (25)
10.139 (13)
It is remarkable that although 3-hydroxyquinolin-2(1H)-one with
an extra H-bond donor with respect to 3-hydroxy-2H-chromen-2-
one is more active (cf. Figure 2) benzo[d]isoxazol-3-ol
derivatives appear to be less active than 1H-indazol-3-ol
derivatives although the former compounds have an extra H-
donor in a similar position (cf. e.g. compounds 28 and 6). An
explanation for this observation is proposed by the comparison of
the complex structure of docked 28 with the X-ray structure of 3-
hydroxyquinolin-2(1H)-one (PDB: 3G3E). The arginine binding
moieties (NCO- in 28 and OCCO- in 3G3E) are slightly shifted
resulting in larger separation between Arg283 and N in 28 versus
Arg283 and O in 3G3E. Moreover, there is also a slight shift in
the position of the ring NH-groups resulting in a suboptimal H-
bond between 28 and Gly313 backbone carbonyl.
aAll measurements were performed n=2; standard deviation in
percentage is shown in parenthesis
Some compounds, although potentially active based on structural
similarity to those having significant DAAO inhibitory potency
could not be measured owing to strong autofluorescense. Here it
is worth noting that the D-kynurenine test25 was selected for
activity measurements because of the assay interference26
observed for the 1H-indazol-3-ol compounds with the HRP27 test
commonly applied to measure DAAO inhibitory activity.
Compounds that could not be measured in the D-kynurenine test
either owing to autofluorescence were subjected to isothermal
titration calorimetric (ITC) affinity determination (Table 5).
Moreover, compounds that exhibited significant inhibitory
activity in the D-kynurenine test were also measured with ITC so
that relation between in vitro activity and ITC affinity scales
could be established.
The analysis of the thermodynamic profiles (Figure 4) showed
that the binding of 34 is more entropically driven as compared to
35 and also to CBIO (6). In fact, the thermodynamic profiles of
35 and 6 are very similar. Interestingly, the compound equipped
with the six membered ring (4) was found to be the only enthalpy
driven DAAO inhibitor in this set of compounds. The high
entropy content of binding supports that these compounds bind in
deprotonated form, since the desolvation of charged species upon
binding has increased advantageous entropic contribution to the
binding free energy.28 Contracting the six membered ring as in
compounds 34 and 35 resulted in some decrease in affinity and
also significant improvements in entropic contribution of 34.
The advantageous in vitro potency of several 1H-indazol-3-ol
compounds prompted us to further investigate their properties
relevant for advanced leads. Compounds 34 and 35 with
IC50=156 nM and 123 nM, respectively were selected for
PAMPA test to characterize absorption properties and
microsomal/hepatocyte stability to characterize metabolic
stability. According to the PAMPA test the 6-Cl derivative (34)
shows high and the 6-F derivative (35) shows medium
permeability. These data demonstrate that these compounds, and
more generally, appropriately substituted derivatives in the 1H-
indazol-3-ol series have potentially advantageous intestinal
absorption. Metabolic stability of compounds 34 and 35 in mouse
and human hepatic microsomes as well as in mouse and human
hepatocytes was also investigated.
Figure 4 Binding thermodynamic profiles of 1H-indazol-3-ols
(34, 35) and reference DAAO inhibitors (1, 4 and 6).
Dissociation constants (Kd) and inhibitory activities in Table 5
change parallel and compounds 31 and 33 that were not
measurable in the D-kynurenine test show Kd values similar to
compounds exhibiting inhibitory activity lower than 1
µM IC50.
This finding shows that 5-substitution is advantageous and results
Table 6 Effective permeability (Pe*10-6cm/s) of representative compounds of the 1H-indazol-3-ol series
grad-pH 5.5-7.4
grad-pH 6.5-7.4
Pe SD MR%
iso-pH 7.4
Pe SD MR%
4.47
0.72
Compound
Permeability
category
High
Pe
SD
MR%
6.7
SD
SD
0.3
1.5
SD
1.1
1.5
34
35
9.53 0.34
1.20 0.01
0.5 7.43
1.3 0.97
0.90
0.12
9.7
7.1
0.29
0.13
2.5
5.8
3.9
Medium
Data in Table 7
Table 7 show that these compounds are stable against first pass metabolism and predict highly favourable bioavailability.
Table 7 Microsomal and hepatocyte stability and predicted bioavailability of representative compounds of the 1H-indazol-3-ol series
34
35

Mouse
Mouse
Human
Human
Mouse
Mouse
Human
Human
microsomes hepatocytes microsomes hepatocytes microsomes hepatocytes microsomes hepatocytes
t1/2a (min)
56.51
11.67
52.21
68.19
215.90
0.76
450.38
2.54
79.52
8.29
178.32
19.96
182.34
0.90
411.51
2.78
b
Clint
(mL/min/kg)
c
ClH
9.504
29.275
81.459
0.714
2.075
7.137
14.371
90.898
0.835
2.232
(mL/min/kg)
Fd (%)
93.981
97.966
94.087
95.480
97.619
93.637
a elimination half-life; b intrinsic clearance; c hepatic clearance; dbioavailability
Based on the beneficial in vitro DAAO inhibitory activity,
good predicted absorption and metabolic stability we selected
compound 35 for measuring its in vivo effect on mice D-serine
level (Figure 5). It was found that the co-administration of 35 and
D-serine results in a significant (p<0.05) increase of the D-serine
level compared to the administration of D-serine alone (33%
increase after 10 minutes and 27% increase after 60 minutes with
corresponding blood levels of 13.1 µM and 0.77 µM of 35,
respectively).
1H-indazol-3-ol (34) and 6-fluoro-1H-indazol-3-ol (35) exhibited
good permeability in PAMPA assay and good stability in mouse
and human microsomes as well as in mouse and human
hepatocytes. In an in vivo study, 6-fluoro-1H-indazol-3-ol (35)
significantly increased plasma D-serine level in mice. These
results show that the 1H-indazol-3-ol series represents a novel
class of DAAO inhibitors with the potential to serve as a basis for
developing drug candidates.
4. Experimental
6
4
4.1. Chemistry
4.1.1. General information
Melting points were determined on an Optimelt SRS (Standard
Research Systems) and are uncorrected. NMR measurements
were performed on Varian NMR System 500 spectrometer, 1H
and 13C NMR spectra were measured at room temperature (30
°C) in an appropriate solvent. 1H and 13C chemical shifts are
expressed in parts per million (δ) referenced to TMS or residual
solvent signals. Compounds 6, 7, 10 were purchased from Sigma-
Aldrich, compounds 11, 18, 19, 20, 21, 22, 23, 24, 25 were
purchased from Combi-Blocks. Reactions were monitored with
Merck silica gel 60 F254 TLC plates (0.25 mm thickness). All
the chemicals and solvents were used as supplied. High
resolution mass spectrometric measurements were performed
using a Q-TOF Premier mass spectrometer (Waters Corporation,
Milford, MA, USA) in positive electrospray ionization mode.
2
0
-2
10 min
60 min
120 min
Figure 5 Difference in mice D-serine level in the presence and
absence of compound 35.
3. Conclusion
4.1.2. 5-phenethyl-1H-pyrazol-3-ol (27)
Inhibitors of D-amino acid oxidase were sought using a fragment
based approach. Based on the structure of reported DAAO
inhibitors [6+5] type heterocyclic compounds were selected for
measuring their DAAO inhibitory potency. This led to the
identification of the isatin scaffold based compounds as weak
DAAO inhibitors. Structure-activity studies of isatins including a
haloscan resulted in compounds at most around 8 µM activity.
Therefore, we decided to change the scaffold using
pharmacophore and structure-activity analysis of DAAO
inhibitors and we arrived at the 1H-indazol-3-ol scaffold not yet
tested against DAAO. Several 1H-indazol-3-ol derivatives were
prepared and nanomolar inhibitors were identified. pKa
measurements of several representatives show that although these
compounds are less acidic than the majority of known DAAO
inhibitors they are at least partially deprotonated at pH=8 that
corresponds to the optimal activity of DAAO. Compounds whose
activity could not be measured with the applied enzymatic assay
due to strong autofluorescense were subjected to isothermal
titration calorimetry (ITC) measurements to determine their
affinity towards DAAO. ITC measurements identified
compounds with comparable affinity to the best inhibitors found
by the enzymatic assay. Two representative compounds 6-chloro-
560 mL (11.5 mmol) hydrazine monohydrate was added
dropwise to a stirred solution of 2.5 g (11.3 mmol) ethyl 3-oxo-5-
phenylpentanoate in 25 mL of ethanol. Stirring was continued for
90 min more. Solvent was removed and diethyl ether (20 mL)
was added, then the syrup was crystallized and filtrated. 1,33 g
(6.89 mmol, 61%) white powder was obtained, mp: 167-169°C.
1H NMR (500 MHz, DMSO-d6) δ 2.70 – 2.79 (m, 2H), 2.82 –
2.89 (m, 2H), 5.24 (s, 1H), 7.14 – 7.23 (m, 3H), 7.27 (t, J = 7.4
Hz, 2H), 10.37 (brs, 2H). 13C NMR (125 MHz, DMSO-d6) 27.5,
34.5, 88.2, 125.9, 128.3, 141.1, 143.7, 160.8; HRMS (ESI):
(M+H)+ calcd for C11H13N2O+, 189.1028; found, 189.1028
4.1.3. General procedure for synthesis of 1H-
indazol-3-ols
The solution of sodium nitrite was added dropwise to a
continuously stirred and ice cooled suspension of anthranilic acid
in water and conc. hydrochloric acid, at such a rate that the
temperature remains below 5°C. After the sodium nitrite was all
been added, stirring was continued for 30 min more and a
solution of sodium sulfite in water was added all at once. After
2h conc. hydrochloric acid was added and the mixture was stirred

overnight. Then it was slowly heated to 80°C and kept at this
temperature for 2h. After cooling to room temperature, the pH
was adjusted to 5.5 with 1M sodium hydroxide. The precipitate
was filtered and triturated with small amount of ethanol.
and a total of 13 points were acquired per curve (excluding the
first injection of 0.4 µL). The stirring rate was set to 600 rpm and
the time between injections was 200 s.
4.1.4. 4-chloro-1H-indazol-3-ol (29)
4.5. Metabolic stability test
1.89 g (11.0 mmol) 2-amino-6-chlorobenzoic acid in 10 mL
water – 2.2 mL conc. hydrochloric acid; 760 mg (11.0 mmol)
NaNO2 in 1.7 mL water; 3.75 g Na2SO3 (29.7 mmol) in 16.5 mL
water; 3.3 mL conc. hydrochloric acid; 447 mg (24%) solid
4.5.1. Isolation of hepatocytes and microsomes
Mouse and human primary hepatocytes or hepatic microsomes
were used in in vitro pharmacokinetic experiments. The
hepatocytes or microsomes were pooled from 6 mice or from 3
human subjects. Male NMRI mice were purchased from Toxi-
Coop Toxicological Research Center (Budapest, Hungary),
whereas human liver tissues were obtained from organ-transplant
donors at the Department of Transplantation and Surgery,
Semmelweis University (Budapest, Hungary). The liver cells
were isolated using collagenase perfusion method of Bayliss and
Skett.30] Briefly: The liver tissues were perfused through the
portal vein with Ca2+-free medium (Earle’s balanced salt
solution) containing EGTA (0.5 mM) and then with the same
medium without EGTA, finally with the perfusate containing
collagenase (Type IV, 0.25 mg/mL) and Ca2+ at physiological
concentration (2 mM). The perfusion was carried out at pH 7.4
and at 37°C. Softened liver tissue was gently minced and
suspended in ice-cold hepatocyte dispersal buffer. Hepatocytes
were filtered and isolated by low-speed centrifugation (50xg),
and washed three times. The yield and percent of cell viability
according to the trypan blue exclusion test were determined.31
For pharmacokinetic studies, the hepatocytes were suspended at
2x106 cells/mL concentration in culture medium.32. Hepatic
microsomal fractions were prepared from pooled hepatocytes.
The cells were homogenized in 0.1 M Tris-HCl buffer (pH 7.4)
containing 1 mM EDTA and 154 mM KCl. Microsomes were
prepared by differential centrifugation as described by van der
Hoeven and Coon.33 All procedures of preparation were
performed at 0-4°C. Microsomal protein content was determined
by the method of Lowry et al.34 with bovine serum albumin as the
standard.
1
product, mp: 188-191°C. H NMR (500 MHz, DMSO-d6) δ 6.95
(dd, J = 5.3, 2.7 Hz, 1H), 7.21 – 7.25 (m, 2H), 11.78 (s, 1H) ; 13
C
NMR (125 MHz, DMSO-d6) 109.2, 109.3, 118.9, 125.6, 127.8,
143.2, 154.6; HRMS (ESI): (M+H)+ calcd for C7H6N2OCl+,
169.0169; found, 169.0167
4.1.5. 7-chloro-1H-indazol-3-ol (37)
1.5 g (8.7 mmol) 2-amino-3-chlorobenzoic acid in 8 mL water –
1.8 mL conc. hydrochloric acid; 603 mg (8.7 mmol) NaNO2 in
1.4 mL water; 2.97 g Na2SO3 (23.5 mmol) in 13.1 mL water; 2.6
mL conc. hydrochloric acid; 36 mg (2.4%) solid product, mp:
1
150-153°C. H NMR (500 MHz, DMSO-d6) δ 6.98 (t, J = 7.7
Hz, 1H), 7.39 (d, J = 7.4 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 10.87
(s, 1H), 11.96 (s, 1H) ; 13C NMR (125 MHz, DMSO-d6) 114.3,
114.7, 119.0, 119.5, 126.2, 139.0, 155.9; HRMS (ESI): (M+H)+
calcd for C7H6N2OCl+, 169.0169; found, 169.0169
4.2. Enzyme inhibition assay
D-2-Amino-4-(2-aminophenyl)-4-oxobutanoic
acid
(D-
kynurenine) was used to measure D-amino acid oxidase activity
according to the protocol described in ref. 25. The 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. The buffer contained 20 mM TRIS-HCl and 100
mM NaCl. Human DAAO was purchased from TargetEx Ltd.
(Dunakeszi, Hungary) and kept in 1 µg/10 µL aliquots at -80 °C.
4.5.2. In vitro pharmacokinetics of 34 and 35
Time courses of the unchanged pharmacons (34 and 35) in
hepatocytes and in hepatic microsomes were obtained. Each
compound was incubated with cell suspension (2×106 cells/mL)
or with microsomes (4 mg/mL) at 37°C in a humid atmosphere
containing 5% CO2. The parent compounds were added directly
to the cell culture medium or to 0.1 M Tris-HCl buffer (pH 7.4)
at the final concentration of 1 µM. The microsomal incubation
mixtures contained an NADPH-regenerating system (1 mM
NADPH, 10 mM glucose 6-phosphate, 5 mM MgCl2 and 2
units/mL glucose 6-phosphate dehydrogenase). At various time
points (at 0, 15, 30, 45, 60, 90, 120, 180, 240 min for hepatocyte
incubations; at 0, 15, 30, 45, 60, 90 min for microsomal
incubations), the incubation mixtures were sampled (aliquots:
0.25 mL) and terminated by the addition of 0.17 mL ice-cold
acetonitrile containing the internal standard, carbamazepine (0.13
µM). The cell and microsomal debris was separated by
centrifugation, and the supernatant was analysed by LC-MS/MS
for quantitation of the parent compound.
4.3. Molecular modeling
Crystal structures of hDAAO were retrieved from the Protein
Data Bank and were processed with Schrödinger’s protein
preparation wizard29. Ligands were prepared with Ligprep29 and
docked by Glide29. Best scored complexes were visually
inspected to select compounds for in vitro enzyme inhibition
assay.
4.4. Isothermal titration calorimetry (ITC)
ITC measurements were carried out against porcine DAAO
(purchased from Calzyme Laboratories, Inc., San Luis Obispo,
CA, USA) on a MicroCal ITC200 microcalorimeter (Malvern
Instruments, Worcestershire, UK). The buffer contained 100 mM
TRIS-HCl, 150 mM NaCl, 0.25 mM TCEP, 50 µM FAD, 1%
V/V DMSO and 0.02% V/V Tween 20. After being dissolved in
the buffer at a concentration of 100 µM (verified on a NanoDrop
1000 spectrophotometer), the protein was kept in 300 µL aliquots
at -20°C. Prior to the ITC measurements, the protein solution was
thawed, the ligand was dissolved in the same buffer at a
concentration of 1 mM, and both solutions were thoroughly
degassed. The DAAO solution was loaded into the sample cell
and was titrated at 25°C with the ligand solution. Injection
volumes were between 0.8 and 2.8 µL during the measurements
4.5.3. Estimation of pharmacokinetic parameters
The
intrinsic
clearance
(Clint)
for
hepatocytes
[mL/(min×2x106cells)] and for microsomes [mL/(min×4mg)]
was calculated from the decrease in the concentration of the
parent compound as follows35:
D
Clint
=
AUC

where the dose (D) was the target 1 nmol (in 1 mL) and





 

(VA ⋅VD )
VA +VD
1− MR ⋅VD
CD (t)
CD (0)
− 2,303


 


P =
⋅
⋅lg 1−
⋅

e
 
 
B
A⋅(t −τSS ) (VA +VD )
(
)



AUC =
β
where Pe is the effective permeability coefficient (cm/s), A is the
filter area (0.3 cm2), VD and VA are the volumes in the donor (0.15
mL) and acceptor phase (0.3 mL), t is the incubation time (s), τSS
is the time (s) to reach steady-state, CD(t) is the concentration (M)
of the compound in the donor phase at time t, CD(0) is the
concentration (M) of the compound in the donor phase at time 0,
MR is the membrane retention factor:
The concentration at 0 min (B) was 1 µM (1 nmol/mL) and β
was determined by fitting exponential using the measured drug-
candidate disappearance. Since in our case D was numerically
equal to B, and Clint was numerically equal to β
ln 2
Clint = β =
t1/2


For scaling up the Clint value to obtain Clint per whole liver (g)/bw (kg)
,
CD (t) VA CA (t)
−



MR = 1−


the cell or microsomal protein concentration in the liver (cell
number in mouse liver: 1.35x108cells/g liver, in human liver:
1.39x108cells/g liver; microsomal protein concentration in mouse
liver: 50 mg/g liver, in human liver: 40 mg/g liver), the ratio of
the average liver weight and average body weight parameters (for
mouse: 76.1 g/kg; for human: 23.7 g/kg) were used. The value
for predicted hepatic clearance (ClH) was calculated as
CD (0) VD CD (0)

In the case of gradient-pH conditions (donor: 5.5 → acceptor: 7.4
→ acceptor: 7.4) the effective permeability of
and donor: 6.5
compounds was calculated by the following gradient-pH
equation:
follows36,37
:




1 + ra
C D (t)
C D (0)
− 2,303
1










P =
⋅
⋅ lg − ra
+
⋅
e



A ⋅ (t − τ SS
)
1 + ra
1 − MR



Clint liver / bw * fu *Qplasma
ClH
=
ra is the sink asymmetry ratio (gradient-pH-induced), defined as
(Clint liver / bw * fu) + Qplasma
(
A→D
)
 Pe

VD
where the flow rate of Qplasma=QH*plasma/blood ratio. To
calculate ClH, the hepatic flow rate (for mouse: 90 mL/min/kg;
for human: 20 mL/min/kg), plasma/blood ratio=0.57 and fu=1
values were used38. (QH is the hepatic blood flow, while fu is the
unbound fraction of the compound.) The bioavailability (%) was
determined by using the equation39:

ra =
(
)
D→A
VA



Pe
.
Quantitative chromatographic analysis for PAMPA was
performed on a SHIMADZU Prominence Modular HPLC system
(Shimadzu Corporation, Japan), equipped with
a vacuum
degasser, a binary pump, a Nexera SIL-30ACMP Multiplate
Autosampler, a column oven and controlled with LabSolutions
software (Version 5.53 SP2). Chromatographic analysis was
performed at 40 °C on a Kinetex 2.6 µm C18 100A (30 x 3.0
mm) column with a mobile phase flow rate of 1.1 mL/min.
Composition of mobile phase A was 0.1% (v/v) formic acid in
water, mobile phase B was the mixture of acetonitrile and water
95/5 (v/v) with 0.1% (v/v) formic acid. The following linear
gradient program was applied: 0 min 0% B, 0.3 min 0% B, 1.8
min 100% B, 2.4 min 100% B, 2.41 min 0%. This was followed
by a 1.2 min equilibration period prior to the next injection
corresponding to approximately 3.6 min total analysis time per
sample. Chromatograms were recorded at 220 4 nm and the
applied injection volume was 4 µL.
F=(1-E)*100.
where E is the hepatic extraction ratio36,40
:
E=ClH*QH
4.6. Parallel artificial membrane permeability assay (PAMPA)
method
A slightly modified version of the parallel artificial membrane
permeability assay41 was used to determine the effective
permeability (Pe, cm/s). A 96-well acceptor plate (Multiscreen
Acceptor Plate, MSSACCEPTOR; Merck-Millipore) and a 96-
well filter plate (PVDF, MultiscreenTM-IP, MAIPNTR10, pore
size 0.45µm; Merck-Millipore) were assembled into a sandwich.
The hydrophobic filter material of the 96 well filter plate was
coated with 5 µl of a 4 % (w/v) n-dodecane solution of
4.7. pKa measurement
phosphatidylcholine:cholesterol (2:1).
Subsequently, the
All pKa determinations were carried out in aqueous medium. The
proton-dissociation constants were determined by UV-
spectrophotometric titration method using D-PAS technique
(Sirius Analytical Instruments Ltd., Forest Row, UK; attached to
a Sirius T3 instrument.43,44 The pKa values were calculated by
Refinement Pro™ software. Absorbancies in the spectral region
of 250-450 nm were used in the analysis. All measurements were
performed in solutions of 0.15M KCl under nitrogen atmosphere,
at 25.0 0.5 oC. All pKa values were measured in 6 replicates.
acceptor wells at the bottom of the sandwich were filled with 300
µL of 10 mM PBS solution adjusted to pH 7.4. The donor wells
at the top of the sandwich were hydrated with 150 µL of test
compound solution. The test compound solution was prepared by
diluting x100 from a 10 mM stock solution in DMSO using PBS
solution at pH 5.5, pH 6.5 or pH 7.4 followed by filtration
through a MultiScreen Solubility filter plate. The resulting
sandwich was then incubated at 37 oC for 4h. After the
incubation, PAMPA sandwich plates were separated and
compound concentrations in donor and acceptor solutions were
determined by HPLC-DAD.
4.8. In vivo pharmacology
4.8.1. Animal studies
The effective permeability and membrane retention of molecules
were calculated by the following equations42:
Male NMRI mice of 25-30 g were purchased from Toxi-Coop
Toxicological Research Center (Budapest, Hungary). The
animals were housed in an air-conditioned (22 +/- 2 °C) animal
room with 12 h light and dark cycle. They had free access to
standard laboratory rodent chow and water. Five mice per group

were treated intraperitoneally with 30 mg/kg D-serine in the
absence or presence of 30 mg/kg 35 dissolved in 5% DMSO
containing saline. The test compound and D-serine were
administered simultaneously. After 10, 20, 30, 60, 120, 240 and
360 minutes the mice were anesthetized by carbon dioxide,
decapitated and their blood was collected using EDTA as
anticoagulant. Plasma was obtained by centrifugation at 2000 g,
4 °C for 5 minutes and the samples were stored frozen at -80 °C
until analysis.
Bassissi, F.; de Bruin, N. M. W. J.; Besten, C. den; de Beer, S. B. A.;
Oostenbrink, C.; Markova, N.; Kruse, C. G. Eur. J. Med. Chem. 2011, 46
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Sparey, T.; Abeywickrema, P.; Almond, S.; Brandon, N.; Byrne, N.;
Campbell, A.; Hutson, P. H.; Jacobson, M.; Jones, B.; Munshi, S.;
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8
9
4.8.2. Det ermination of D-serine plasma level
We used
a chiral capillary electrophoresis laser induced
fluorescence (CE-LIF) method developed in our laboratory for
D-serine measurement45. Briefly, plasma was deproteinated by
mixing with 3 volumes of ice cold acetonitrile followed by
centrifugation at 3000 g, 4 °C for 20 min. The supernatant was
spiked with 10-6 M L-cysteic acid as internal standard. 7-fluoro-
10 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.;
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4-nitro-2,1,3-benzoxadiazole (NBD-F,
2
mg/mL final
concentration) was used for sample derivatization in pH 8.5
borate buffer at 65 °C for 20 min. Derivatized samples were
diluted with 2 volumes of distilled water and injected to a
Beckman P/ACE MDQ CE system controlled by 32 Karat
software version 5.0 (Beckman Coulter, Brea, CA, USA)
equipped with a LIF detector containing an Argon-ion laser
source, with excitation and emission wavelengths of 488 and 520
nm, respectively. Separations were performed in fused silica
capillary coated with linear polyacrylamide (75 µm id, 30/40 cm
effective/total length) using 50 mM HEPES pH 6.5 separation
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buffer
containing
6
mM
6-monodeoxy-6-mono(3-
hydroxy)propylamino-β-CD (HPA-β-CD) as chiral selector.
Injection was accomplished with pressure (3447 Pa for 5 s). The
voltage applied was constant -28 kV. Temperature of the liquid
capillary coolant was set at 15 °C. Before each run the capillary
was rinsed with water and separation buffer.
1861
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Acknowledgments
This study was supported by the National Brain Research
Program grant (project KTIA NAP_13). Celesztina Domonkos is
acknowledged for help in biological measurements, Katalin Tóth
and Máté Déri for performing in vitro pharmacokinetic studies
and Judit Müller for the performing PAMPA tests.
26 1H-indazol-3-ols are oxidized in the presence of horseradish peroxidase
and H2O2.
27 Cook S.P., Galve-Roperh I., Martinez del Pozo A., Rodriguez-Crespo I.:.
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Supplementary Material
1H and 13C NMR spectra of compounds 27-33 are available as
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