D-Amino acid oxidase inhibitors based on the 5-hydroxy-1,2,4-triazin-6(1H)-one scaffold

Article abstract of DOI:10.1016/j.bmcl.2016.02.068

A series of 3-substituted 5-hydroxy-1,2,4-triazin-6(1H)-one derivatives were designed and synthesized as a new class of d-amino acid oxidase (DAAO) inhibitors. Some of the newly synthesized derivatives showed potent inhibitory activity against human DAAO with IC₅₀ values in the nanomolar range. Among them, 6-hydroxy-3-phenethyl-1,2,4-triazin-5(2H)-one 6b and 3-((6-fluoronaphthalen-2-yl)methylthio)-6-hydroxy-1,2,4-triazin-5(2H)-one 6m were found to be metabolically stable in mouse liver microsomes. In addition, compound 6b was found to be orally available in mice and able to enhance plasma d-serine levels following its co-administration with d-serine compared to the oral administration of d-serine alone.

Full text of DOI:10.1016/j.bmcl.2016.02.068

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
D-Amino acid oxidase inhibitors based on the
-hydroxy-1,2,4-triazin-6(1H)-one scaffold
5
Niyada Hin , Bridget Duvall , James F. Berry , Dana V. Ferraris a,c, Rana Rais a,b, Jesse Alt a,
a
a
a,b
a,d
a,b
a,b,
⇑
Camilo Rojas , Barbara S. Slusher , Takashi Tsukamoto
a
Johns Hopkins Drug Discovery Program, Johns Hopkins University, 855 North Wolfe Street, Baltimore, MD 21205, USA
Department of Neurology, Johns Hopkins University, 855 North Wolfe Street, Baltimore, MD 21205, USA
Department of Chemistry, McDaniel College, 2 College Hill, Westminster, MD 21157, USA
b
c
d
Department of Molecular and Comparative Pathobiology, Johns Hopkins University, 855 North Wolfe Street, 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 3-substituted 5-hydroxy-1,2,4-triazin-6(1H)-one derivatives were designed and synthesized
Received 23 December 2015
Revised 19 February 2016
Accepted 22 February 2016
Available online xxxx
as a new class of D-amino acid oxidase (DAAO) inhibitors. Some of the newly synthesized derivatives
showed potent inhibitory activity against human DAAO with IC50 values in the nanomolar range.
Among them, 6-hydroxy-3-phenethyl-1,2,4-triazin-5(2H)-one 6b and 3-((6-fluoronaphthalen-2-yl)
methylthio)-6-hydroxy-1,2,4-triazin-5(2H)-one 6m were found to be metabolically stable in mouse liver
microsomes. In addition, compound 6b was found to be orally available in mice and able to enhance
Keywords:
plasma
D-serine levels following its co-administration with
D
-serine compared to the oral administration
D-Amino acid oxidase
of D-serine alone.
Flavoenzyme
Pharmacokinetics
Schizophrenia
Ó 2016 Elsevier Ltd. All rights reserved.
D-Serine
D
-Amino acid oxidase (DAAO, EC 1.4.3.3) catalyzes the oxidative
binding site.6 2-Substituted 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-
diones such as 3 exhibited not only potent DAAO inhibitory activity
but also improved metabolic stability compared to 1–2 in liver
deamination of neutral -amino acids including -serine, a co-agonist
D
D
at the glycine modulatory site of NMDA receptors. Because of the
implication that hypofunction of NMDA receptors plays an
important role in the pathophysiology of schizophrenia, there has
been considerable efforts in developing DAAO inhibitors as a phar-
7
microsomes. This secondary binding site was also exploited by
8
9
carboxylate-based DAAO inhibitors such as 4 and 5. In the search
for a new class of DAAO inhibitors, we have examined a variety of
scaffolds that may serve as a bioisostere for the carboxylic acid
moiety interacting with the active site of DAAO.
macological approach for increasing
D-serine and facilitating
NMDA receptor-mediated neurotransmission. DAAO has been also
implicated as a potential therapeutic target for the treatment of
chronic pain since hydrogen peroxide, a reactive oxygen species
co-generated by DAAO, is believed to contribute to pain hypersen-
We found that the 5-hydroxy-1,2,4-triazin-6(1H)-one moiety
can serve as an effective carboxylate isostere and that its 3-posi-
tion can be utilized to attach a branched chain that extends into
the secondary binding site of DAAO. These derivatives of 6 were
found to potently inhibit human DAAO with IC50 values in the
nanomolar range. In this Letter, we describe the SAR for this new
class of DAAO inhibitors as well as the pharmacological properties
of selected compounds, including metabolic stability, oral
1
sitivity. In the past decade, a wide variety of DAAO inhibitors have
been identified by several research groups.2 As shown in Figure 1,
newer scaffolds of DAAO inhibitors contain a branched chain,
which occupies the secondary pocket adjacent to the active site
of DAAO recognized by the crystal structure of human DAAO in
,3
4
complex with imino-DOPA. For example, our group exploited this
secondary binding site using kojic acid derivatives represented by
compound 1. Similarly, a group at Astellas reported potent DAAO
inhibitors including 2a–b based on a 4-hydroxypyridazin-3(2H)-
pharmacokinetics, and effects on plasma levels of
following oral co-administration.
D-serine in mice
5
Synthetic methods of 3-substituted 5-hydroxy-1,2,4-triazin-6
(1H)-one derivatives 6a–o are illustrated in Scheme 1. 3-Arylalkyl
derivatives 6a–c were prepared by first treating nitriles 7a–c with
hydrogen chloride gas to form the corresponding acetimidate
one scaffold with a phenethyl group extending to the secondary
1
0
⇑
Corresponding author. Tel.: +1 410 614 0982; fax: +1 410 614 0659.
E-mail address: ttsukamoto@jhmi.edu (T. Tsukamoto).
8a–c. Subsequent reaction with methyl chlorooxoacetate in the
presence of DIEA, followed by treatment with hydrazine afforded
http://dx.doi.org/10.1016/j.bmcl.2016.02.068
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
0
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2
N. Hin et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
X
Table 1
Cl
Cl
Inhibition of human DAAO by 3-substituted 5-hydroxy-1,2,4-triazin-6(1H)-one
derivatives 6a–o
O
OH
O
N
OH
O
S
X
N
Compd
Structure
IC50a
(lM)
O
N
OH
2a (X = H)
2b (X = F)
N
H
O
N
H
1
4
3
OH
OH
N
6a
0.07
Cl
4
N
CO H
O
2
3
5
OH
O
N NH
N
CO H
O
2
N
H
6
OH
2
N
H
N
N
O
5
6
6
6
b
c
0.06
4.0
O
NH
OH
Figure 1. Representative known DAAO inhibitors 1–5 and 3-substituted
-hydroxy-1,2,4-triazin-6(1H)-one derivatives 6.
N
N
5
O
NH
OH
the desired products 6a–c. 3-Arylalkylthio derivatives 6d–f were
prepared from thiosemicarbazide 9, which was condensed with
glyoxylic acid and subsequently reacted with arylalkyl bromide
N
6d
0.1
0.3
S
O
N
NH
11
to afford 3-arylalkylthio-1,2,4-triazin-5(4H)-ones 10d–f. Oxida-
OH
tion of the 6-carbon with bromine12 provided the final products
N
6
6
e
f
S
O
6
d–f. The final oxidation step, however, produced sulfoxide deriva-
N
OH
NH
tives as by-products that often made the purification of the desired
products difficult. To this end, we developed an alternative route to
N
N
S
O
40.0
3
-arylalkylthio derivatives, devoid of an oxidation step.
Thiosemicarbazide 9 was condensed with diethyl oxalate to form
-hydroxy-3-mercapto-1,2,4-triazin-6(1H)-one 11 in situ, which
was reacted with alkyl bromide to obtain the final products
g–o. Although the yields of the new synthetic procedure were
NH
OH
5
Cl
N
N
6g
0.03
0.05
0.05
0.09
0.7
S
O
NH
OH
6
poor across all the derivatives (8–21%), the one-pot preparative
method was technically simpler and generated no inseparable
by-products.
Cl
Cl
N
N
6h
S
O
NH
OH
The inhibitory potency of the synthesized compounds were
determined using recombinant human DAAO as previously
I
N
N
1
3
6i
reported. In vitro DAAO inhibitory data are summarized in
Table 1. 5-Hydroxy-1,2,4-triazin-6(1H)-one derivatives containing
a variety of substituents at the 3-position displayed varying
degrees of inhibitory potency against DAAO. All compounds but
S
O
NH
OH
Ph
N
N
6j
S
O
NH
OH
6
f were found to be potent DAAO inhibitors with IC50 values in
the nanomolar range. In general, the SAR trends are similar to
N
those of 4-hydroxypyridazin-3(2H)-one and 6-hydroxy-1,2,4-tri-
6k
S
O
azine-3,5(2H,4H)-dione derivatives.6 Two-unit chains, –CH
,7
2
2
CH –
Ph
N
NH
OH
2
and –CH S– appear to be very effective linkers connecting the aryl
N
group and the 5-hydroxy-1,2,4-triazin-6(1H)-one moiety, provid-
ing some of the most potent DAAO inhibitors within the series.
Many of the derivatives containing a benzylthio group at the
6
6
l
0.6
S
O
N
NH
OH
3-position were particularly potent DAAO inhibitors (IC50 < 100 nM)
N
m
0.03
0.2
F
S
O
N
NH
O
O
N
OH
OH
N
NH·HCl
OMe
N
N
6n
6o
a
b
S
O
1
R1
R1
R CN
O
N
NH
7
a-c
NH
8
a-c
6a-c
OH
N
N
O
OH
O
S
0.06
S
O
HN
N
N
N
NH2
c
2
d
2
NH
H N
N
R S
R S
O
2
H
N
NH
6d-f
a
Assay methods are described in Supporting information.
9
10d-f
e
OH
OH
N
N
N
N
2
HS
O
R S
O
as represented by 6g–j, 6m, and 6o. Substitutions at the phenyl
ring of these compounds are generally well tolerated, reflecting
the adequate space available at the secondary binding site. There
seems to be a preference for linear substituents at this binding site.
For instance, para-phenyl derivative 6j was 8-fold more potent
than the corresponding meta-phenyl derivative 6k. Further, incor-
poration of a fused ring such as naphthyl (6m) benzisoxazole (6n),
or 1,3-benzodioxole (6o) also produced potent DAAO inhibitors,
NH
NH
1
1
6g-o
Scheme 1. Synthesis of 3-substituted 5-hydroxy-1,2,4-triazin-6(1H)-one deriva-
tives 6a–o. Reagents and conditions: (a) HCl (gas), MeOH/hexanes, 0 °C to rt,
6–97%; (b) (i) methyl chlorooxoacetate, DIEA, THF, 0 °C, (ii) NH
3–96%; (c) (i) 80% aq EtOH, 70 °C, (ii) glyoxylic acid, 80% aq EtOH, 70 °C, (iii) R Br,
, (3 drops), H O, rt, 9–16%; (e) (i) NaOMe (25% soln in
MeOH), diethyl oxalate, 65 °C, (ii) R Br, rt, 50 °C, 8–21%.
8
1
2
NH
2
ÁH
2
O, 0 °C,
2
NaOH, 80 °C, 23–32%; (d) Br
2
2
2
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N. Hin et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
further underscoring the high steric tolerance at the secondary
binding site.
The proposed binding mode of 6b (white) to the active site of
Table 2
Metabolic stability of DAAO inhibitors in mouse liver microsomes
Compd
tPSAa (Å)
% remaining after 60 min
w/UDPGA w/NADPH
DAAO is illustrated in Figure 2 using the crystal structure of DAAO
6
in complex with 2a (cyan) as a template. A key hydrogen bond is
1
47
62
82
74
74
5
NDb
92
observed between Tyr2 and Arg
28
283
and the a-hydroxycarbonyl
2
3
b
59
>99
98
95
moiety of 6b. An additional interaction is observed with the nitro-
>99
79
gen at the 1-position of the 5-hydroxy-1,2,4-triazin-6(1H)-one
6b
6m
moiety and the carbonyl of Gly313. A similar interaction exists in
91
the crystal structure of human DAAO with 2a. The high inhibitory
potency retained by compounds 6j and 6m–o bearing a sterically
hindered substituent at the 3-position can be explained by the
a
tPSA of the carboxylate bioisostere moiety.
Not determined.
b
2
24
spacious pocket created as a result of the movement of Tyr
away
from the active site seen in the crystal structure of DAAO in
6
complex with 2a.
We have previously reported that some DAAO inhibitors pos-
sessing an
a-hydroxycarbonyl moiety as a carboxylate bioisostere
are prone to glucuronidation in liver microsomes in the presence of
1
0
7
,14
UDGPA.
For example, as shown in Table 2, compound 1 was
extensively glucuronidated within 60 min. In contrast, compound
2
3
b exhibited some degree of metabolic stability while compound
displayed complete resistance to glucuronidation. We tested
the metabolic stability of two 5-hydroxy-1,2,4-triazin-6(1H)-one
derivatives 6b and 6m in liver microsomes. As shown in Table 2,
both DAAO inhibitors were found to be resistant to glucuronida-
tion. This is in good agreement with the previous findings that that
increased topological polar surface area (tPSA) of the carboxylate
bioisostere moiety contributes to higher resistance to glucuronida-
1
0
2
4
6
Time (h)
7
,14
tion.
The susceptibility to glucuronidation, however, may also
Figure 3. Plasma levels of 6b following oral administration of (30 mg/kg, po), in
depend on other factors such as the extent of electron delocaliza-
tion and keto–enol tautomerism. Both 6b and 6m were also stable
in mouse liver microsomes in the presence of NADPH, a cofactor for
CYP-dependent oxidation.
mice.
D-Serine
The plasma levels of 6b over tine following oral administration
(
30 mg/kg) in mice are shown in Figure 3. Compound 6b was
D-Serine + 6b
quickly absorbed and achieved its Cmax at 30 min after administra-
tion. The overall exposure calculated from the area under the curve
1
00
10
(
AUC0–6h) was 8.07 lg*h/mL. Binding of compound 6b to mouse
plasma protein was found to be 74%. Considering the high inhibi-
tory potency of 6b, sufficient unbound drug appears to be available
for inhibition of peripheral DAAO at least for the first 3 h.
Given the reasonable plasma exposure by the oral route, we
subsequently evaluated the effects of 6b on
D-serine plasma phar-
macokinetics following oral co-administration (both at 30 mg/kg,
po). As shown in Figure 4, the plasma clearance of D-serine was
0
2
4
6
Time (h)
Figure 4. Effect of compound 6b (30 mg/kg, po) on plasma pharmacokinetics of oral
-serine (30 mg/kg, po) in mice.
D
nearly arrested for the first 3 h. This is consistent with the plasma
pharmacokinetics of 6b in mice. After 3 h, the plasma -serine
levels started to decline with a clearance rate similar to that of
-serine alone treatment, presumably due to the insufficient free
fraction of 6b available for peripheral DAAO inhibition. Overall,
co-administration of -serine with DAAO inhibitor 6b resulted in
a 1.7 fold increase in the -serine AUC0–6h (74.2 g*h/ml) com-
pared with -serine treatment alone (43.3 g*h/ml). Given the
previously reported cross-species variation in IC50 values between
Arg283
D
Tyr228
D
Gly313
D
Tyr224
D
l
D
l
1
5
human and rat forms of DAAO, however, caution needs to be
taken in establishing the PK/PD relationship in mice as we have
not measured the inhibitory potency of 6b in mouse DAAO.
The 3-substituted derivatives of 5-hydroxy-1,2,4-triazin-6(1H)-
one represent a new class of DAAO inhibitors with potent
inhibitory activity and substantial microsomal metabolic stability.
Figure 2. Proposed binding mode of 6b (white) to the active site of DAAO (PDB:
W4K). Key residues and FAD are shown in green and yellow, respectively.
Hydrogen-bonding interactions between 6b and the key residues are shown as gray
dashed lines. Compound 2a (cyan) of 3W4K is also shown.
3
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N. Hin et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Additionally, we have demonstrated the oral co-administration of a
new DAAO inhibitor 6b with -serine can improve plasma expo-
sure to -serine compared with -serine administration alone.
The degree of enhancement in plasma -serine levels (1.7-fold)
was, however, lower than that of compound 3 based on a
-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione scaffold (2.2-fold) pre-
Supplementary data
D
D
D
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.02.068.
D
6
References and notes
7
viously reported. This is in a good agreement with the slightly
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g*h/mL).7 Nevertheless, the
tion relative to that of 3 (12.9
enhancement of plasma -serine levels achieved by 6b should be
sufficient to produce pharmacological effects of -serine in the
CNS as a similar plasma -serine exposure enhancing effect of
another DAAO inhibitor, 6-chloro-1,2-benzisoxazol-3(2H)-one
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Acknowledgments
1
This work was in part supported by the National Institutes of
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