Synthesis of kojic acid derivatives as secondary binding site probes of d-amino acid oxidase

Article abstract of DOI:10.1016/j.bmcl.2013.04.062

A series of kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) derivatives were synthesized and tested for their ability to inhibit d-amino acid oxidase (DAAO). Various substituents were incorporated into kojic acid at its 2-hydroxymethyl group. These

Full text of DOI:10.1016/j.bmcl.2013.04.062

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3910–3913
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Bioorganic & Medicinal Chemistry Letters
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Synthesis of kojic acid derivatives as secondary binding site probes
of D-amino acid oxidase
Mithun Raje a, Niyada Hin b, Bridget Duvall b, Dana V. Ferraris b, James F. Berry a, Ajit G. Thomas b,
Jesse Alt b, Camilo Rojas b, Barbara S. Slusher a,b, Takashi Tsukamoto a,b,
⇑
a Department of Neurology, Johns Hopkins University, Baltimore, MD 21205, USA
b Brain Science Institute, Johns Hopkins University, Baltimore, MD 21205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) derivatives were synthesized and
tested for their ability to inhibit -amino acid oxidase (DAAO). Various substituents were incorporated
into kojic acid at its 2-hydroxymethyl group. These analogs serve as useful molecular probes to explore
the secondary binding site, which can be exploited in designing more potent DAAO inhibitors.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 18 March 2013
Revised 17 April 2013
Accepted 22 April 2013
Available online 1 May 2013
D
Keywords:
D-Amino acid oxidase
Flavoenzyme
Kojic acid
Schizophrenia
D
-Amino acid oxidase (DAAO, EC 1.4.3.3) is a flavoenzyme that
critical hydrogen bond with the nitrogen group of imino-DOPA
generated in situ by oxidation of
-DOPA (Fig. 1B).8
catalyzes the oxidative deamination of neutral
D
-amino acids and
D
produces the corresponding
a
-keto acids, ammonia, and hydrogen
In addition to benzoic acid, a variety of structurally diverse
DAAO inhibitors have been discovered to date (Fig. 2).4 Consistent
with the crystal structure of DAAO in complex with benzoic acid,
the majority of DAAO inhibitors share common structural features,
namely an aromatic ring system with a carboxylic acid or its
peroxide.1,2 In humans, DAAO is predominantly expressed in the
liver, kidneys, and some regions of the brain and is primarily
responsible for the metabolism of neutral
D-amino acids including
D-serine, an endogenous agonist at the NMDA receptor glycine
modulatory site. DAAO has gained substantial interest as a thera-
peutic target for disorders associated with NMDA receptor hypo-
function such as schizophrenia3,4 as well as neuropathic pain in
which hydrogen peroxide is believed to act as a key contributor.5
Thus, substantial efforts have been made recently to identify po-
tent and selective DAAO inhibitors as novel therapeutic agents.
Benzoic acid6 is one of the early DAAO inhibitors and has served
as a useful probe to study the mechanism and physiological role of
the enzyme. Indeed, the first crystal structure of human DAAO was
solved as a complex with benzoic acid,7 providing critical insights
into the structural details of the DAAO active site (Fig. 1A). Benzoic
acid binds parallel to the flavin ring on the re face of the cofactor
while it interacts with the side chain of Tyr224, which stacks
against the face of the benzene ring opposite to the cofactor. The
carboxylate group is coplanar with the benzene ring and forms
key hydrogen bonds with Arg283 and Tyr228. Although Gly313 ap-
pears to play a minor role in the binding of benzoic acid to the
DAAO active site, the carbonyl group of Gly313 participates in a
bioisostere. For instance, 6-chlorobenzo[d]isoxazol-3-ol
1
(IC50 = 188 nM),9 3-hydroxyquinolin-2(1H)-one 2a (IC50 = 4 nM),10
and 3-hydroxychromen-2-one 2b (IC50 = 440 nM)11 are considered
benzoic acid derivatives in which the carboxylic acid was replaced
by an isoxazol-3-ol, an a-hydroxylactam, and an a-hydroxylactone,
respectively. 4,6-Difluoro-1-hydroxy-1H-benzo[d]imidazol-2(3H)-
one 3 (IC50 = 80 nM) represents a newly discovered pharmacophore
based on the cyclic N-hydroxyurea scaffold.12 The relatively higher
potency of compound 2a can be attributed to its ability to form a
hydrogen bond with the carbonyl group of Gly313 as shown by
its co-crystal structure with human DAAO.10 In these series, SAR
studies indicated that sterically hindered substituents are not well
tolerated on the benzene ring, which is consistent with the limited
space available at the active site of DAAO.
Fused pyrrole carboxylic acids 4a (IC50 = 141 nM) and 4b
(IC50 = 245 nM),13 pyrrole-2-carboxylic acid 5a (IC50 <10 M),14
l
and 1H-pyrazole-3-carboxylic acid 5b (IC50 <100 l
M)14 represent
additional classes of DAAO inhibitors for which extensive SAR
studies have been conducted. It is conceivable that these com-
pounds bind to the DAAO active site in a manner similar to that
of benzoic acid with an additional interaction between the NH
⇑
Corresponding author. Tel.: +1 410 614 0982; fax: +1 410 614 0659.
E-mail address: ttsukamoto@jhmi.edu (T. Tsukamoto).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.062

M. Raje et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3910–3913
3911
Figure 1. (A) Benzoic acid (cyan) bound to the active site of DAAO (2DU8). Key residues and FAD (flavin adenine dinucleotide) are shown in green. (B) Imino-DOPA (white)
bound to the active site of DAAO (2E82). Key residues and FAD are shown in orange. Tyr224 residue of 2DU8 (green) is superimposed to highlight the repositioning (red
arrow) of this residue upon binding of imino-DOPA.
DAAO and serves as an attachment point for various substituents
that may allow us to perform systematic SAR studies exploring
the secondary binding site. Herein, we report the synthesis and
SAR of kojic acid derivatives as molecular probes for the secondary
binding site of DAAO. These new DAAO inhibitors should provide
new insights into the molecular features enabling the additional
interaction at the secondary binding site.
As shown in Scheme 1, 2-(hydroxymethyl)-5-((4-methoxyben-
zyl)oxy)-4H-pyran-4-one 7 and its derivatives 10 and 11 served
as starting materials16 for the synthesis of the O-alkylated deriva-
tives of kojic acid. Reactive organic halides can be directly coupled
with compound 7 through its 2-hydroxymethyl group to give com-
pounds 8. Subsequently, the p-methoxybenzyl group of 8 can be
removed by TFA to give the desired products 9. Either mesylate
OH
N
OH
N
OH
O
F
O
X
N
H
O
Cl
2a (X = NH)
2b (X = O)
1
F 3
6
R
X
OR
O
5
O
X
HO
CO2H
CO2H
N
H
N
H
2
3
5a
5b
5c
5d
6a
(R = H)
4a (X = O)
4b (X = S)
(X = CH, R = H)
6b
(X = N, R = H)
(R = CH3)
(X = CH, R = PhCH2CH2)
(X = N, R = PhCH2CH2)
Figure 2. Representative inhibitors of DAAO.
(of the pyrrole or pyrazole ring) and Gly313. One unique aspect of
the pyrrole and pyrazole carboxylates lies in their ability to gain
affinity to DAAO by incorporating a side chain. For instance, when
the 4-position of 5a was substituted by a phenethyl group, the IC50
value is in the submicromolar range as exemplified by 5c (IC50
O
O
O
O
O
O
OPMB
OPMB
b
OH
a
HO
RO
RO
7
9a-9k
8a-8k
<1
l
M). Similarly, substituted compound 5d also exhibited im-
proved potency (IC50 <1
l
M) over the parent compound 5b.14
c
Given the limited space available at the active site on the re face
of the flavin ring, the side chains of 5c and 5d likely stretch into an-
other hydrophobic pocket adjacent to the active site in order to
gain additional binding affinity. On a related note, human DAAO
in complex with imino-DOPA (2E82) shows its catechol moiety
O
O
O
O
O
O
OPMB
OPMB
e
OH
d
X
RS
RS
13a-13e
10
12a-12e
(X = OMs)
taking
a position nearly perpendicular to the flavin ring
11 (X = Br)
(Fig. 1B).8 This mode of binding was enabled by the repositioning
of Tyr224, which swings away from the active site. The Tyr224
shift results in the widening of the substrate entry path of DAAO
by 2–3 Å compared to the 2DU8 structure and creates space for
the catechol moiety of imino-DOPA.15 We hypothesized that the
side chains of compounds 5c and 5d stretched into the same
hydrophobic cavity occupied by the catechol moiety of imino-
DOPA. This hypothesis prompted us to design new DAAO inhibitors
exploiting this secondary binding site.
Scheme 1. Reagents and conditions: (a) NaH, DMF, RI or RBr, 43–75%; (b) TFA,
dichloromethane, rt, 4 h, 36–96%; (c) ROH, NaH, DMF, 0 °C to rt, 4 h, 26–93%; (d)
RSH, NaH, DMF, 0 °C to rt, 4 h, 16–56%; (e) TFA, dichloromethane, rt, 4 h, 30–78%.
O
O
O
O
O
O
OBn
OBn
OBn
OCH3
P
O
a
b
Br
H3CO
O
15
16
OH
14
Kojic acid 6a, 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one,
represents another class of DAAO inhibitors with a distinct phar-
macophore. Kojic acid 6a was originally identified as an inhibitor
OH
O
OBn
c
d
of porcine DAAO with a Ki value of 21 l
M.6 Given its structural
O
N
O
similarity to compound 2a, we speculate that the 5-hydroxy-4H-
pyran-4-one moiety acts as a carboxylic acid surrogate and binds
to the active site of DAAO. Indeed, as described later, 5-methoxy
derivative 6b was found to be completely devoid of any inhibitory
activity. However, we speculate that the 2-hydroxymethyl group of
kojic acid plays an insignificant role in binding of kojic acid to
O
17
18
19
Scheme 2. Reagents and conditions: (a) P(OMe)3, benzene, reflux, 24h, 90%; (b)
NaH, THF, 0 °C, then benzaldehyde, 12 h, 0 °C to rt, 66%; (c) 6 N HCl/HOAc, TFA,
reflux, 48 h then H2 (1 atm), Pd/C (5%), rt, 1 h, 50%; (d) MeOH, phenethylamine,
72 h, rt then H2 (1 atm), 5% Pd/C (5%), EtOAc, 1 h, 50%.

3912
M. Raje et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3910–3913
Table 1
mediated removal of the benzyl group followed by catalytic
Inhibition of human DAAO by kojic acid derivatives
hydrogenation at atmospheric pressure. We also synthesized 3-hy-
droxy-1-phenethyl-4(1H)-pyridinone 19 by reacting 3-(phenyl-
methoxy)-4H-pyran-4-one 1817 with phenethylamine followed
by catalytic hydrogenation (Scheme 2).
Inhibitory potencies of the kojic acid derivatives were measured
using recombinant human DAAO as previously reported.9 Table 1
summarizes the in vitro DAAO inhibitory data.
a
Compd
Structure
IC50 (lM)
O
O
HO
6a
2
>100
50
OH
O
HO
In our assay, kojic acid 6a inhibited human DAAO with an IC50
value of 2.0 lM. As mentioned earlier, methylation of the 5-hydro-
6b
9a
9b
9c
9d
9e
9f
OCH3
O
xy group of kojic acid resulted in complete loss of potency as in
compound 6b presumably due to its inability to form hydrogen
bonds with Tyr228 and Arg283. 2-Alkoxymethyl derivatives of
kojic acid exhibited a wide range of DAAO inhibitory potencies
(compounds 9a–9k) depending on the substituent. Saturated
groups were ineffective at this position as compounds 9a, 9b,
and 9k were significantly less potent than kojic acid. On the other
hand, O-phenyl derivative 9c and its close analogs (compounds
9d–9i) exhibited potency comparable or superior to that of kojic
acid. Insertion of one methylene unit, however, resulted in dra-
matic loss of potency as in compound 9j. These findings indicate
that the secondary binding pocket has a preference for an aromatic
side chain containing a two-atom linker to the core pyran ring.
Subsequently, we examined compounds containing other two-
atom linkers (Table 2). Sulfide-based inhibitors 13a–13d were
found to be slightly more potent than the corresponding ether
derivatives. Among them, 13d was the most potent DAAO inhibitor
within the kojic acid series with an IC50 value of 100 nM. Phenethyl
derivative 17 was slightly more potent than the ether counterpart
9c. Interestingly, 4(1H)-pyridinone-based derivative 19 containing
a phenethyl branch exhibited no inhibitory activity, underscoring
the unique interaction formed by the 5-hydroxy-4H-pyran-4-one
pharmacophore with the key residues of the DAAO active site.
An attempt to dock compound 13d to the active site of DAAO
(Fig. 3) was only successful with the crystal structure containing
O
H3CO
C2H5O
OH
O
O
>100
OH
O
O
O
0.9
0.8
0.7
0.5
3
OH
O
O
O
O
F
O
OH
O
O
Cl
Cl
O
OH
O
OH
Cl
N
O
O
O
9g
OH
O
O
O
9h
9i
0.9
0.8
OH
Table 2
O
Inhibition of human DAAO by kojic acid derivatives
O
a
O
Compd
Structure
IC50
(l
M)
OH
O
O
O
S
13a
0.2
0.3
0.2
0.1
4
OH
O
O
O
O
9j
20
OH
OH
O
F
S
13b
13c
13d
13e
OH
O
O
9k
>100
O
O
O
O
Cl
Cl
S
OH
a
Assay methods are described in the Supplementary data.
O
S
OH
10 or bromide 11 was utilized for the reaction with alcohols to ob-
tain compounds 8, which were subsequently converted into 9.
Compounds 10 and 11 were also coupled with thiols to give 12.
Deprotection with TFA provided sulfide-based analogs 13.
Synthesis of 5-hydroxy-2-phenethyl-4H-pyran-4-one 17 is
illustrated in Scheme 2. Reaction of 2-(bromomethyl)-5-(phenyl-
methoxy)-4H-pyran-4-one 1416 with trimethyl phosphite afforded
phosphonate derivative 15. Horner–Wadsworth–Emmons reaction
of 15 with benzaldehyde gave styryl derivative 16. Our attempt to
remove the benzyl group and reduce the olefin in one step via cat-
alytic hydrogenation (30 psi) resulted in the formation of multiple
by-products presumably due to the concurrent reduction of the
pyran ring. Thus, the desired product 17 was obtained via acid-
Cl
N
O
O
S
N
OH
OH
O
O
O
17
19
0.5
OH
>100
N
O
a
Assay methods are described in the Supplementary data.

M. Raje et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3910–3913
3913
tion exploring a wide variety of substituents may lead to DAAO
inhibitors with greater structural diversity. Furthermore, the sec-
ondary binding site can be exploited by other series of DAAO inhib-
itors, particularly those capable of interacting with the Gly313
residue of the active site to maximize interaction with each of
the two binding sites.
Acknowledgments
This work was in part supported by National Institutes of
Health (R01MH091387 to T.T.) and the Johns Hopkins Brain Sci-
ence Institute NeuroTranslational Drug Discovery program.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.
062.
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Figure 3. Proposed binding mode of 13d (white) to the active site of DAAO (2E82).
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the repositioned Tyr224 residue (2E82). The inability of compound
13d to fit the active site of 2DU8, in which Tyr224 is obstructing
the secondary binding site, also supports the hypothesis that the
phenylthiomethyl group of 13d is occupying the space created by
the Tyr224 movement. Surprisingly, recent efforts to screen 4 mil-
lion commercially available compounds in silico did not identify
any DAAO inhibitors extending to the secondary binding site.11
This could be at least partially due to the concentrated use of the
2DU8 structure and/or docking grid box that failed to cover the en-
tire secondary binding site.
The lack of a nitrogen atom in the core ring of kojic acid pre-
cludes hydrogen bonding with the Gly313 residue, which explains
the relatively weak inhibitory potency of the kojic acid-based
inhibitors compared to the series represented by compounds 2
and 3. Indeed, it was recently reported that substituted derivatives
of 3-hydroxy-pyridine-2(1H)-one and 3-hydroxy-pyridazine-
4(1H)-one can potently inhibit DAAO.18–20 These compounds are
presumably gaining increased affinity through hydrogen bonding
to the Gly313 residue in addition to the interaction with the sec-
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In summary, we tested a series of kojic acid derivatives for their
ability to inhibit human DAAO. These compounds likely occupy
both the active site and the secondary binding site adjacent to
the active site. Since the secondary binding site is a part of the fun-
nel-shaped entrance to the active site, further structural optimiza-
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